RU2854591C2 - ANTIBODY-DRUG CONJUGATES OF Mcl-1 INHIBITOR AND METHODS OF THEIR USE - Google Patents

Abstract

FIELD: medicine; oncology.

SUBSTANCE: invention relates to an antibody-drug conjugate which binds to human oncology targets. The antibody-drug conjugate contains a drug group representing an Mcl-1 inhibitor of formula III. The invention further relates to methods and compositions for treating cancer by administering the antibody-drug conjugates.

EFFECT: linker-drug conjugates containing an Mcl-1 inhibitor drug group.

87 cl, 23 dwg, 39 tbl, 20 ex

Description

RELATED APPLICATION

This application claims the benefit and priority of the filing date under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62/850,098, filed May 20, 2019, the entire contents of which are incorporated herein by reference.

AREA OF TECHNOLOGY

The present disclosure relates to antibody-drug conjugates (ADCs) comprising an Mcl-1 inhibitor and an antibody or antigen-binding fragment thereof that binds an antigenic target, such as an antigen expressed on a tumor or other cancer cell. The disclosure also relates to methods and compositions useful for treating and/or diagnosing cancer that express the target antigen and/or are treatable by modulating the expression and/or activity of Mcl-1, as well as methods for producing these compositions. Linker-drug conjugates comprising the drug moiety of an Mcl-1 inhibitor, and methods for producing the same, are also disclosed.

LEVEL OF TECHNOLOGY

Apoptosis, or programmed cell death, is a physiological process that is critical for embryonic development and the maintenance of tissue homeostasis. Apoptotic cell death typically involves morphological changes, such as nuclear condensation and DNA fragmentation, as well as biochemical alterations, such as caspase activation, which can cause damage to key cellular structural components. The regulation of apoptosis is complex and typically involves the activation or repression of multiple intracellular signaling pathways (Cory et al. (2002) Nature Review Cancer 2:647-656).

Dysregulation of apoptosis is associated with certain pathologies. For example, increased apoptosis is associated with neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and ischemia. Conversely, a deficiency in apoptosis may play a role in the development of cancer and chemoresistance, autoimmune diseases, inflammatory diseases, and viral infections. The absence of apoptosis is one of the phenotypic hallmarks of cancer (Hanahan et al. (2000) Cell 100:57-70). Anti-apoptotic proteins of the Bcl-2 family are associated with numerous types of cancer, such as colon cancer, breast cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, prostate cancer, chronic lymphocytic leukemia, lymphoma, myeloma, and pancreatic cancer.

Myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the Bcl-2 family, is a regulator of cell survival. Mcl-1 gene amplification and/or Mcl-1 protein overexpression are observed in several types of cancer and are commonly involved in tumor progression (Beroukhim et al. (2010) Nature 463(7283):899–905). Mcl-1 is one of the most frequently amplified genes in human cancer and is a critical survival factor that has been shown to mediate drug resistance to multiple anticancer agents.

Mcl-1 is believed to promote cell survival by binding to and neutralizing the death-inducing activity of proapoptotic proteins such as Bim, Noxa, Bak, and Bax. Inhibition of Mcl-1 releases these proapoptotic proteins, often leading to the induction of apoptosis in tumor cells dependent on Mcl-1 for survival. Therefore, therapeutic targeting of Mcl-1 or proteins upstream and/or downstream of it in the apoptotic signaling pathway may represent promising strategies for the treatment of various malignancies and overcoming drug resistance in certain human cancers.

ESSENCE OF THE INVENTION

In some embodiments, the present disclosure relates in part to novel antibody-drug conjugate (ADC) compounds with biological activity against cancer cells. The compounds can slow, inhibit, and/or reverse tumor growth in mammals and/or may be useful for treating human cancer patients. The present disclosure more particularly relates, in some embodiments, to ADC compounds that are capable of binding to and killing cancer cells. In some embodiments, the ADC compounds described herein comprise a linker that attaches an Mcl-1 inhibitor to a full-length antibody or antigen-binding fragment. In some embodiments, the ADC compounds are also capable of internalization into a target cell after binding.

In some embodiments, the ADC compounds may be represented by Formula (1):

Ab-(LD) _p → (1)

where Ab is an antibody or its antigen-binding fragment;

D is an inhibitor of Mcl-1;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 16. In some embodiments, Ab is an antibody or antigen-binding fragment thereof that targets a cancer cell.

In some embodiments, p is an integer from 1 to 8. In some embodiments, p is an integer from 1 to 5. In some embodiments, p is an integer from 2 to 4. In some embodiments, p is 2. In some embodiments, p is 4. In some embodiments, p is determined by liquid chromatography-mass spectrometry (LC-MS).

In some embodiments, the linker (L) comprises an attached group, at least one spacer group, and at least one cleavable group. In some cases, the cleavable group comprises a pyrophosphate group and/or a self-destructive group. In specific embodiments, L comprises an attached group; at least one bridging spacer group; and at least one cleavable group comprising a pyrophosphate group and/or a self-destructive group.

In some embodiments, the antibody-drug conjugate comprises a linker-drug (or “linker-payload”) moiety -(L-D) having the formula (A):

(A),

where R ¹ is the attached group, L ₁ is the bridging spacer group and E is the cleavable group.

In some embodiments, the cleavable group comprises a pyrophosphate group. In some embodiments, the cleavable group comprises:

.

In some embodiments, the bridging spacer group comprises a polyoxyethylene (PEG) group. In some cases, the PEG group can be selected from PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14 and PEG15. In some embodiments, the bridging spacer group can comprise: -CO- _CH2 - _CH2- PEG12-. In other embodiments, the bridging spacer group comprises a butanoyl, pentanoyl, hexanoyl, heptanoyl or octanoyl group. In some embodiments, the bridging spacer group comprises a hexanoyl group.

In some embodiments, the attached group is formed from at least one reactive group selected from a maleimide group, a thiol group, a cyclooctyne group, and an azido group. For example, the maleimide group may have the structure:

.

The azido group can have the structure: -N=N ⁺ =N ^- .

The cyclooctyne group can have the structure:

or and where is a link to the antibody.

In some cases, the cyclooctyne group has the structure: and where is a link to the antibody.

In some embodiments, the attached group has a formula comprising or and where is a link to the antibody.

In some embodiments, the antibody is connected to a linker (L) by an attached group selected from: And , Where is a link to the antibody and where is a bond with a bridging spacer group.

In some embodiments, the bridging spacer group is connected to the cleavable group.

In some embodiments, the bridging spacer group is -CO-CH ₂ -CH ₂ -PEG12-.

In some embodiments, the cleavable group is -pyrophosphate-CH ₂ -CH ₂ -NH ₂ -.

In some embodiments, the cleavable group is linked to an Mcl-1 inhibitor (D).

In some embodiments, the cleavable group is linked to the Mcl-1 inhibitor (D) group via a phenylpyrimidinyl group.

In some embodiments, the linker comprises: an attached group, at least one bridging spacer group, a peptide group, and at least one cleavable group.

In some embodiments, the antibody-drug conjugate comprises a linker-drug moiety, -(L-D) of formula (B):

(B),

where R ¹ is the attached group, L ₁ is a bridging spacer, Lp is a peptide moiety containing 1-6 amino acid residues, E is a cleavable group, L ₂ is a bridging spacer, m is 0 or 1; and D is an Mcl-1 inhibitor. In some cases, m is 1 and the bridging spacer contains:

.

In some embodiments, at least one bridging spacer comprises a PEG group. In some cases, the PEG group is selected from PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, and PEG15. In some cases, at least one bridging spacer is selected from *-C(O)-CH ₂ -CH ₂ -PEG1-**, *-C(O)-CH ₂ -PEG3-**, *-C(O)-CH ₂ -CH ₂ -PEG12**, *-NH-CH2-CH2-PEG1-**, a polyhydroxyalkyl group, and *-C(O)-N(CH ₃ )-CH ₂ -CH ₂ -N(CH ₃ )-C(O)-**, where ** denotes a point of direct or indirect attachment of at least one bridging spacer to the attached group and * denotes a point of direct or indirect attachment of at least one bridging spacer to the peptide group.

In some embodiments, L ₁ is selected from *-C(O)-CH ₂ -CH ₂ -PEG1-**, *-C(O)-CH ₂ -PEG3-**, *-C(O)-CH ₂ -CH ₂ -PEG12**, *-NH-CH2-CH2-PEG1-** and a polyhydroxyalkyl group, where ** denotes a point of direct or indirect attachment of L ₁ to R ¹ and * denotes a point of direct or indirect attachment of L ₁ to Lp.

In some embodiments, m is 1 and L ₂ is -C(O)-N(CH ₃ )-CH ₂ -CH ₂ -N(CH ₃ )-C(O)-.

In some embodiments, the peptide moiety comprises 1-12 amino acid residues. In some embodiments, the peptide moiety (Lp) comprises 1-10 amino acid residues. In some embodiments, the peptide moiety (Lp) comprises 1-8 amino acid residues. In some embodiments, the peptide moiety (Lp) comprises 1-6 amino acid residues. In some embodiments, the peptide moiety comprises 1-4 amino acid residues. In some embodiments, the peptide moiety comprises 1-3 amino acid residues. In some embodiments, the peptide moiety comprises 1-2 amino acid residues. In some cases, the amino acid residues are selected from L-glycine (Gly), L-valine (Val), L-citrulline (Cit), L-cysteine acid (sulfo-Ala), L-lysine (Lys), L-isoleucine (Ile), L-phenylalanine (Phe), L-methionine (Met), L-asparagine (Asn), L-proline (Pro), L-alanine (Ala), L-leucine (Leu), L-tryptophan (Trp), and L-tyrosine (Tyr). For example, the peptide moiety can comprise Val-Cit, Val-Ala, Val-Lys, and/or sulfo-Ala-Val-Ala. In some embodiments, the peptide moiety (Lp) comprises 1 amino acid residue linked to the moiety In some embodiments, the peptide moiety (Lp) comprises a moiety .

In some cases, the peptide moiety comprises a moiety selected from:

In some embodiments, the self-cleaving group comprises para-aminobenzyl carbamate, para-aminobenzyl ammonium, para-amino-(sulfo)benzyl ammonium, para-amino-(sulfo)benzyl carbamate, para-amino-(alkoxy-PEG-alkyl)benzyl carbamate, para-amino-(polyhydroxycarboxytetrahydropyranyl)alkyl benzyl carbamate, or para-amino-(polyhydroxycarboxytetrahydropyranyl)alkyl benzyl ammonium.

In some embodiments, m is 1 and the bridging spacer comprises .

In some embodiments, the linker-drug moiety, -(L-D), is formed from a compound selected from:

,

And

.

In some embodiments, the antibody-drug conjugate comprises a linker-drug moiety, -(L-D), that comprises a formula selected from:

,

,

And

, And

Where is a link to the antibody.

In some embodiments, the antibody-drug conjugate comprises a drug linker moiety, -(L-D), which has the formula (C):

(C),

where: R ¹ is an attached group, L ₁ is a bridging spacer; L _p is a peptide group containing 1-6 amino acids; D is an Mcl-1 inhibitor; G ₁ -L ₂ -A is a self-cleaving spacer; L ₂ is a bond, methylene, neopentylene, or C ₂ -C ₃ alkenylene; A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; L ₃ is a spacer group; and R ² is a hydrophilic group.

In some embodiments, the antibody-drug conjugate comprises a drug linker moiety, -(L-D), having the formula (D):

(D),

where: R ¹ is the attached group; L ₁ is a bridging spacer; Lp is a peptide group containing 1-6 amino acids; A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; L ₃ is a spacer group; and R ² is a hydrophilic group.

In some embodiments, L ₁ comprises: , or *-CH(OH)CH(OH)CH(OH)CH(OH)-**, where each n is an integer from 1 to 12, where * in L ₁ denotes the point of direct attachment to Lp and ** in L ₁ denotes the point of direct attachment to R ¹ .

In some embodiments, L ₁ is and n is an integer from 1 to 12 where * of L ₁ denotes the point of direct attachment to Lp and ** of L ₁ denotes the point of direct attachment to R ¹ .

In some embodiments, L ₁ is and n is 1, where * of L ₁ denotes the point of direct attachment to Lp and ** of L ₁ denotes the point of direct attachment to R ¹ .

In some embodiments, L ₁ is and n is 12, where * of L ₁ denotes the point of direct attachment to Lp and ** of L ₁ denotes the point of direct attachment to R ¹ .

In some embodiments, L ₁ is and n is an integer from 1 to 12, where * of L ₁ denotes a point of direct attachment to Lp and ** of L ₁ denotes a point of direct attachment to R ¹ .

In some embodiments, L ₁ comprises , where * from L ₁ means the point of direct attachment to Lp and ** from L ₁ means the point of direct attachment to R ¹ .

In some embodiments, L ₁ is a bridged spacer comprising:

*-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)O(CH ₂ ) _m SSC(R ³ ) ₂ (CH ₂ ) _m C(=O)NR ³ (CH ₂ ) _m NR ³ C(=O)(CH ₂ ) _m -**; *-C(=O)O(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**; *-C(=O)(CH ₂ ) _m X ₁ (CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n X ₁ (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n X ₁ (CH ₂ ) _n -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n X ₁ (CH ₂ ) _n -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m C(R ³ ) ₂ -** or *-C(=O)(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**, where * from L ₁ denotes the point of direct addition to Lp and ** from L ₁ denotes the point of direct addition to R ¹ , where X ₁ is , or ; and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

In some embodiments, R ² is a hydrophilic group comprising polyethylene glycol, polyalkylene glycol, polyol, polysarcosine, sugar, oligosaccharide, polypeptide, or C ₂ -C ₆ alkyl substituted with 1-3 or groups. In some embodiments, R ² is , , , or , , , where n is an integer from 1 to 6, , or .

In some embodiments, the hydrophilic group comprises a polyethylene glycol of the formula: or , where R is H, -CH ₃ , -CH ₂ CH ₂ NHC(=O)OR _a , -CH ₂ CH ₂ NHC(=O)R _{a ,} or -CH ₂ CH ₂ C(=O)OR _a , R' is OH, -OCH ₃ , -CH ₂ CH ₂ NHC(=O)OR _a , -CH ₂ CH ₂ NHC(=O)R _{a ,} or -OCH ₂ CH ₂ C(=O)OR _a , and each of m and n is an integer from 2 to 25 ( e.g., from 3 to 25).

In some embodiments, the hydrophilic group comprises .

In some embodiments, the hydrophilic group comprises polysarcosine, such as with the following group: , where n is an integer from 3 to 25; and R is H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH.

In some embodiments, L ₃ is a spacer group having the structure ,

Where:

W is -CH ₂ -, -CH ₂ O-, -CH ₂ N(R ^b )C(=O)O-, -NHC(=O)C(R ^b ) ₂ NHC(=O)O-, -NHC(=O)C(R ^b ) ₂ NH-, -NHC(=O)C(R ^b ) ₂ NHC(=O)-, -CH ₂ N(XR ² )C(=O)O-, -C(=O)N(XR ² )-, -CH ₂ N(XR ² )C(=O)-, -C(=O)NR ^b -, -C(=O)NH-, -CH ₂ NR ^b C(=O)-, -CH ₂ NR ^b C(=O)NH-, -CH ₂ NR ^b C(=O)NR ^b -, -NHC(=O)-, -NHC(=O)O-, -NHC(=O)NH-, -OC(=O)NH-, -S(O) ₂ NH-, -NHS(O) ₂ -, -C(=O)-, -C(=O)O- or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl; and

X is a bond, triazolyl, or -CH ₂ -triazolyl-.

In some embodiments, L ₃ is a spacer group having the structure ,

Where:

W is -CH ₂ -, -CH ₂ O-, -CH ₂ N(R ^b )C(=O)O-, -NHC(=O)C(R ^b ) ₂ NHC(=O)O-, -NHC(=O)C(R ^b ) ₂ NH-, -NHC(=O)C(R ^b ) ₂ NHC(=O)-, -CH ₂ N(XR ² )C(=O)O-, -C(=O)N(XR ² )-, -CH ₂ N(XR ² )C(=O)-, -C(=O)NR ^b -, -C(=O)NH-, -CH ₂ NR ^b C(=O)-, -CH ₂ NR ^b C(=O)NH-, -CH ₂ NR ^b C(=O)NR ^b -, -NHC(=O)-, -NHC(=O)O-, -NHC(=O)NH-, -OC(=O)NH-, -S(O) ₂ NH-, -NHS(O) ₂ -, -C(=O)-, -C(=O)O- or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl; and

X is -CH ₂ -triazolyl-C _1-4 alkylene-OC(O)NHS(O) ₂ NH-, -C _4-6 cycloalkylene-OC(O)NHS(O) ₂ NH-, -(CH ₂ CH ₂ O) _n -C(O)NHS(O) ₂ NH-, -(CH ₂ CH ₂ O) _n -C(O)NHS(O) ₂ NH-(CH ₂ CH ₂ O) _n- or -CH ₂ -triazolyl-C _1-4 alkylene-OC(O)NHS(O) ₂ NH-(CH ₂ CH ₂ O) _n- , where each n is independently 1, 2, or 3.

In some embodiments, the attached group is formed by a reaction involving at least one reactive group. In some cases, the attached group is formed by the reaction of a first reactive group attached to a linker and a second reactive group attached to the antibody or an amino acid residue of the antibody.

In some embodiments, at least one of the reactive groups comprises:

thiol,

maleimide,

haloacetamide,

azide,

alkyne,

cyclooctene,

triarylphosphine,

oxanobornadiene,

cyclooctyne,

diaryltetrazine,

monoaryltetrazine,

Norbornen,

aldehyde,

hydroxylamine,

hydrazine,

NH ₂ -NH-C(=O)-,

ketone,

vinyl sulfone,

aziridine,

amino acid residue,

, -ONH ₂ , -NH ₂ , , , -N ₃ , , -SH, -SR ³ , -SSR ⁴ , -S(=O) ₂ (CH=CH ₂ ), -(CH ₂ ) ₂ S(=O) ₂ (CH=CH ₂ ), -NHS(=O) ₂ (CH=CH ₂ ), -NHC(=O)CH ₂ Br, -NHC(=O)CH ₂ I, , -C(O)NHNH ₂ , ,

or ;

Where:

each R ³ is independently selected from H and C ₁ -C ₆ alkyl;

each R ⁴ is 2-pyridyl or 4-pyridyl;

each R ⁵ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl and -OH;

each R ⁶ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl, -NH ₂ , -OCH ₃ , -OCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -CN, -NO ₂ and -OH;

each R ⁷ is independently selected from H, C _1-6 alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C _1-4 alkoxy substituted with -C(=O)OH and C _1-4 alkyl substituted with -C(=O)OH.

In some embodiments, the first reactive group and the second reactive group comprise:

thiol and maleimide,

thiol and haloacetamide,

thiol and vinyl sulfone,

thiol and aziridine,

azide and alkyne,

azide and cyclooctyne,

azide and cyclooctene,

azide and triarylphosphine,

azide and oxanobornadiene,

diaryltetrazine and cyclooctene,

monoaryltetrazine and nonbornene,

aldehyde and hydroxylamine,

aldehyde and hydrazine,

aldehyde and NH ₂ -NH-C(=O)-,

ketone and hydroxylamine,

ketone and hydrazine,

ketone and NH ₂ -NH-C(=O)-,

hydroxylamine and ,

amine and or

CoA or a CoA analogue and a serine residue.

In some embodiments, the attached group comprises a group selected from:

And

disulfide,

Where:

R ³² is H, C _1-4 alkyl, phenyl, pyrimidine, or pyridine;

R ³⁵ is H, C _1-6 alkyl, phenyl, or C _1-4 alkyl substituted with 1-3 -OH groups;

each R ⁷ is independently selected from H, C _1-6 alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C _1-4 alkoxy substituted with -C(=O)OH, and C _1-4 alkyl substituted with -C(=O)OH;

R ³⁷ is independently selected from H, phenyl and pyridine;

q is 0, 1, 2, or 3;

R ⁸ is H or methyl; and

R ⁹ is H, -CH ₃ , or phenyl.

In some embodiments, the peptide moiety (Lp) comprises 1-6 amino acid residues. In some embodiments, the peptide moiety (Lp) comprises 1-4 amino acid residues. In some embodiments, the peptide moiety comprises 1-3 amino acid residues. In some embodiments, the peptide moiety comprises 1-2 amino acid residues. In some embodiments, the amino acid residues are selected from L-glycine (Gly), L-valine (Val), L-citrulline (Cit), L-cysteine acid (sulfo-Ala), L-lysine (Lys), L-isoleucine (Ile), L-phenylalanine (Phe), L-methionine (Met), L-asparagine (Asn), L-proline (Pro), L-alanine (Ala), L-leucine (Leu), L-tryptophan (Trp), and L-tyrosine (Tyr). In some embodiments, the peptide moiety comprises Val-Cit, Phe-Lys, Val-Ala, Val-Lys, Leu-Cit, sulfo-Ala-Val and/or sulfo-Ala-Val-Ala. In some embodiments, Lp is selected from:

In some embodiments, the linker-drug moiety -(L-D) comprises or is formed from a compound of the formula:

, Where:

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment of D; and

D is an inhibitor of Mcl-1.

In some embodiments, the linker-drug moiety -(L-D) comprises or is formed from a compound of the formula:

, Where:

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D; and

D is an inhibitor of Mcl-1.

In some embodiments, the linker-drug moiety -(L-D) comprises or is formed from a compound of the formula:

, Where:

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D; and

D is an inhibitor of Mcl-1.

In some embodiments, the linker-drug moiety -(L-D) comprises or is formed from a compound of the formula:

, Where:

each R is independently selected from H, -CH ₃ and -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D; and

D is an inhibitor of Mcl-1.

In some embodiments, the linker-drug moiety -(L-D) comprises or is formed from a compound of the formula:

, Where:

each R is independently selected from H, -CH ₃ and -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D; and

D is an inhibitor of Mcl-1.

In some embodiments, the linker-drug moiety -(L-D) comprises or is formed from a compound of the formula:

, Where:

Xa is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ - and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D; and

D is an inhibitor of Mcl-1.

In some embodiments, the linker-drug moiety -(L-D) comprises or is formed from a compound of the formula:

, Where:

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D; and

D is an inhibitor of Mcl-1.

In some embodiments, the linker-drug moiety -(L-D) comprises or is formed from a compound of the formula:

, Where:

Xb is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ -, and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D; and

D is an inhibitor of Mcl-1.

In some embodiments, the linker-drug moiety -(L-D) comprises or is formed from a compound of the formula:

, Where:

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D; and

D is an inhibitor of Mcl-1.

In some embodiments, the linker-drug moiety -(L-D) comprises or is formed from a compound of the formula:

, Where:

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D; and

D is an inhibitor of Mcl-1.

In some embodiments, the linker-drug moiety -(L-D) comprises or is formed from a compound of the formula:

, Where:

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and

D is an inhibitor of Mcl-1.

In some embodiments, the linker-drug moiety -(L-D) comprises or is formed from a compound of the formula:

, Where:

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D; and

D is an inhibitor of Mcl-1.

In some embodiments, the linker-drug moiety -(L-D) comprises or is formed from a compound of the formula:

, Where:

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D; and

D is an inhibitor of Mcl-1.

In some embodiments, the linker-drug moiety -(L-D) comprises or is formed from a compound of the formula:

, Where:

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D; and

D is an inhibitor of Mcl-1.

In some embodiments, the linker-drug moiety -(L-D) comprises or is formed from a compound of the formula:

, Where:

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D; and

D is an inhibitor of Mcl-1.

In some embodiments, the linker-drug moiety -(L-D) comprises or is formed from a compound of the formula:

, Where:

each R is independently H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH;

A is a bond, -OC(=O)-*, , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*,

where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D; and

D is an inhibitor of Mcl-1.

In some embodiments, A is a bond.

In some embodiments, R is -CH ₃ .

In some embodiments, the Mcl-1 inhibitor (D) comprises a compound of formula (I):

Where:

Ring D ₀ is a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group,

Ring E ₀ is a furyl, thienyl or pyrrolyl ring,

X ₀₁ , X ₀₃ , X ₀₄ and X ₀₅ are independently of each other a carbon atom or a nitrogen atom,

X ₀₂ is a CR ₀₂₆ group or a nitrogen atom,

means that the ring is aromatic,

Y ₀ is a nitrogen atom or CR ₀₃ group,

Z ₀ is a nitrogen atom or a CR ₀₄ group,

R ₀₁ is a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, a linear or branched (C ₁ -C ₆ )haloalkyl group, a hydroxy group, a hydroxy(C ₁ -C ₆ )alkyl group, a linear or branched (C ₁ -C ₆ )alkoxy group, -S-(C ₁ -C ₆ )alkyl group, a cyano group, a nitro group, -Cy ₀₈ , -(C ₀ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-(C ₁ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-(C ₁ -C ₆ )alkyl-R ₀₁₂ , -C(O)-OR ₀₁₁ , -OC(O)-R ₀₁₁ , -C(O)-NR ₀₁₁ R ₀₁₁ ', -NR ₀₁₁ -C(O)-R ₀₁₁ ', -NR ₀₁₁ -C(O)-OR ₀₁₁ ', -(C ₁ -C ₆ )alkyl-NR ₀₁₁ -C(O)-R ₀₁₁ ', -SO ₂ -NR ₀₁₁ R ₀₁₁ ' or -SO ₂ -(C ₁ -C ₆ )alkyl,

R ₀₂ , R ₀₃ , R ₀₄ and R ₀₅ independently of one another are a hydrogen atom, a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, a linear or branched (C ₁ -C ₆ )haloalkyl, a hydroxy group, a hydroxy(C ₁ -C ₆ )alkyl group, a linear or branched (C ₁ -C ₆ )alkoxy group, -S-(C ₁ -C ₆ )alkyl group, a cyano group, a nitro group, -(C ₀ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-Cy ₀₁ , -(C ₀ -C ₆ )alkyl-Cy ₀₁ , -(C ₂ -C ₆ )alkenyl-Cy ₀₁ , -(C ₂ -C ₆ )alkynyl-Cy ₀₁ , -O-(C ₁ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-(C ₁ -C ₆ )alkyl-R ₀₃₁ , -O-(C ₁ -C ₆ )alkyl-R ₀₁₂ , -C(O)-OR ₀₁₁ , -OC(O)-R ₀₁₁ , -C(O)-NR ₀₁₁ R ₀₁₁ ', -NR ₀₁₁ -C(O)-R ₀₁₁ ', -NR ₀₁₁ -C(O)-OR ₀₁₁ ', -(C ₁ -C ₆ )alkyl-NR ₀₁₁ -C(O)-R ₀₁₁ ', -SO ₂ -NR ₀₁₁ R ₀₁₁ ' or -SO ₂ -(C ₁ -C ₆ )alkyl,

or a pair of (R ₀₁ , R ₀₂ ), (R ₀₂ , R ₀₃ ), (R ₀₃ , R ₀₄ ) or (R ₀₄ , R ₀₅ ) together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containing 5-7 ring members which optionally contains 1-3 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted with 1 or 2 groups selected from halogen, linear or branched (C ₁ -C ₆ )alkyl, (C ₀ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -NR ₀₁₃ R ₀₁₃ ', -(C ₀ -C ₆ )alkyl-Cy ₀₁ or oxo,

R ₀₆ and R ₀₇ independently of one another are a hydrogen atom, a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, a linear or branched (C ₁ -C ₆ )haloalkyl, a hydroxy group, a linear or branched (C ₁ -C ₆ )alkoxy group, -S-(C ₁ -C ₆ )alkyl group, a cyano group, a nitro group, -(C ₀ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-(C ₁ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-Cy ₀₁ , -(C ₀ -C ₆ )alkyl-Cy ₀₁ , -(C ₂ -C ₆ )alkenyl-Cy ₀₁ , -(C ₂ -C ₆ )alkynyl-Cy ₀₁ , -O-(C ₁ -C ₆ )alkyl-R ₀₁₂ , -C(O)-OR ₁₁ , -OC(O)-R ₀₁₁ , -C(O)-NR ₀₁₁ R ₀₁₁ ', -NR ₁₁ -C(O)-R ₀₁₁ ', -NR ₀₁₁ -C(O)-OR ₀₁₁ ', -(C ₁ -C ₆ )alkyl-NR ₀₁₁ -C(O)-R ₀₁₁ ', -SO ₂ -NR ₀₁₁ R ₀₁₁ ' or -SO ₂ -(C ₁ -C ₆ )alkyl,

or a pair (R ₀₆ , R ₀₇ ) fused with two adjacent carbon atoms, together with the carbon atoms to which they are attached, form an aromatic or non-aromatic ring containing 5-7 members in the ring, which optionally contains 1-3 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted with a linear or branched (C ₁ -C ₆ )alkyl group, -NR ₀₁₃ R ₀₁₃ ', -(C ₀ -C ₆ )alkyl-Cy ₀₁ or oxo,

W ₀ is a -CH ₂ - group, -NH - group or an oxygen atom,

R ₀₈ is a hydrogen atom, a linear or branched (C ₁ -C ₈ )alkyl group, a -CHR _0a R _0b group, an aryl group, a heteroaryl group, an aryl(C ₁ -C ₆ )alkyl group or a heteroaryl(C ₁ -C ₆ )alkyl group,

R ₀₉ is a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, -Cy _O2 , -(C ₁ -C ₆ )alkyl-Cy _O2 , -(C ₂ -C ₆ )alkenyl-Cy _O2 , -(C ₂ -C ₆ )alkynyl-Cy _O2 , -Cy _O2 -Cy _O3 , -(C ₂ -C ₆ )alkynyl-O-Cy _O2 , -Cy _O2 -(C ₀ -C ₆ )alkyl-O-(C ₀ -C ₆ )alkyl-Cy _O3 , a halogen atom, a cyano group, -C(O)-R ₀₁₄ or -C(O)-NR ₀₁₄ R ₀₁₄ ',

R ₀₁₀ is a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, an aryl(C ₁ -C ₆ )alkyl group, a (C ₁ -C ₆ )cycloalkylalkyl group, a linear or branched (C ₁ -C ₆ )haloalkyl or -(C ₁ -C ₆ )alkyl-O-Cy ₀₄ ,

or a pair (R ₀₉ , R ₀₁₀ ) condensed with two adjacent carbon atoms, together with the carbon atoms to which they are attached, form an aromatic or non-aromatic ring containing 5-7 members in the ring, which optionally contains 1-3 heteroatoms selected from O, S and N,

R ₀₁₁ and R ₀₁₁ ' independently of one another are a hydrogen atom, an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group or -(C ₀ -C ₆ )alkyl-Cy ₀₁ ,

or a pair (R ₀₁₁ , R ₀₁₁ ') together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5-7 ring members which optionally contains, in addition to the nitrogen atom, 1-3 heteroatoms selected from O, S and N, wherein the nitrogen atom may be substituted by 1 or 2 groups selected from a hydrogen atom and a linear or branched (C ₁ -C ₆ )alkyl group, and wherein one or more carbon atoms of the linear or branched (C ₁ -C ₆ )alkyl group are optionally deuterated,

R ₀₁₂ is -Cy ₀₅ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-O-(C ₀ -C ₆ )alkyl-Cy ₀₆ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-Cy ₀₆ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-NR ₀₁₁ -(C ₀ -C ₆ )alkyl-Cy ₀₆ , -Cy ₀₅ -Cy ₀₆ -O-(C ₀ -C ₆ )alkyl-Cy ₀₇ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-O-(C ₀ -C ₆ )alkyl-Cy ₀₉ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-Cy ₀₉ , -NH-C(O)-NH-R ₀₁₁ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-NR ₀₁₁ -(C ₀ -C ₆ )alkyl-Cy ₀₉ , -C(O)-NR ₀₁₁ R ₀₁₁ ', -NR ₀₁₁ R ₀₁₁ ', -OR ₀₁₁ , -NR ₀₁₁ -C(O)-R ₀₁₁ ', -O-(C ₁ -C ₆ )alkyl-OR ₀₁₁ , -SO ₂ -R ₀₁₁ and -C(O)-OR ₀₁₁ ,

R ₀₁₃ , R ₀₁₃ ', R ₀₁₄ and R ₀₁₄ ' independently of one another are a hydrogen atom or an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group,

R _0a is a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

R _0b is an -OC(O)-OR _0c group, an -OC(O)-NR _0c R _0c ' group, or an -OP(O)(OR _0c ) ₂ group,

R _0c and R _0c ' independently of one another are a hydrogen atom, a linear or branched (C ₁ -C ₈ )alkyl group, a cycloalkyl group, a (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group or a (C ₁ -C ₆ )alkoxycarbonyl(C ₁ -C ₆ )alkyl group,

or a pair (R _0c , R _0c ') together with the nitrogen atom to which they are attached form a non-aromatic ring consisting of 5-7 ring members which may contain, in addition to the nitrogen atom, 1-3 heteroatoms selected from oxygen and nitrogen, wherein the nitrogen is optionally substituted with a linear or branched (C ₁ -C ₆ )alkyl group,

Cy ₀₁ , Cy ₀₂ , Cy ₀₃ , Cy ₀₄ , Cy ₀₅ , Cy ₀₆ , Cy ₀₇ , Cy ₀₈ and Cy ₀₁₀ independently of one another are a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, each of which is optionally substituted,

Cy ₀₉ is ,

or Cy ₀₉ is a heteroaryl group which is substituted by a group selected from -OP(O)(OR ₀₂₀ ) ₂ ; -OP(O)(O ^- M ⁺ ) ₂ ; -(CH ₂ ) _p0 -O-(CHR ₀₁₈ -CHR ₀₁₉ -O) _q0 -R ₀₂₀ ; hydroxy; hydroxy(C ₁ -C ₆ )alkyl; -(CH ₂ ) _r0 -U ₀ -(CH ₂ ) _s0 -heterocycloalkyl; and -U ₀ -(CH ₂ ) _q0 -NR ₀₂₁ R ₀₂₁ ',

R ₀₁₅ is a hydrogen atom; -(CH ₂ ) _p0 -O-(CHR ₀₁₈ -CHR ₀₁₉ -O) _q0 -R ₀₂₀ group; a linear or branched (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group; -U ₀ -(CH ₂ ) _q0 -NR ₀₂₁ R ₀₂₁ 'group; or -(CH ₂ ) _r0 -U ₀ -(CH ₂ ) _s0 -heterocycloalkyl group,

R ₀₁₆ represents a hydrogen atom; a hydroxy group; a hydroxy(C ₁ -C ₆ )alkyl group; -(CH ₂ ) _r0 -U ₀ -(CH ₂ ) _s0 -heterocycloalkyl group; (CH ₂ ) _r0 -U ₀ -V ₀ -OP(O)(OR ₀₂₀ ) ₂ group; -OP(O)(O ^- M ⁺ ) ₂ group; -OS(O) ₂ OR ₀₂₀ group; -S(O) ₂ OR ₀₂₀ group; -(CH ₂ ) _p0 -O-(CHR ₀₁₈ -CHR ₀₁₉ -O) _q0 -R ₀₂₀ group; -(CH ₂ ) _p0 -OC(O)-NR ₀₂₂ R ₀₂₃ group; or -U ₀ -(CH ₂ ) _q0 -NR ₀₂₁ R ₀₂₁ ' group,

R ₀₁₇ is a hydrogen atom; -(CH ₂ ) _p0 -O-(CHR ₀₁₈ -CHR ₀₁₉ -O) _q0 -R ₀₂₀ group; -CH ₂ -P(O)(OR ₀₂₀ ) ₂ group, -OP(O)(OR ₀₂₀ ) ₂ group; -OP(O)(O ^- M ⁺ ) ₂ group; hydroxy group; hydroxy(C ₁ -C ₆ )alkyl group; -(CH ₂ ) _r0 -U ₀ -(CH ₂ ) _s0 -heterocycloalkyl group; -U ₀ -(CH ₂ ) _q0 -NR ₀₂₁ R ₀₂₁ 'group; or aldonic acid,

M ⁺ is a pharmaceutically acceptable monovalent cation,

U ₀ is a bond or oxygen atom,

V ₀ is a -(CH ₂ ) _s0 - group or a -C(O)- group,

R ₀₁₈ is a hydrogen atom or a (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group,

R ₀₁₉ is a hydrogen atom or a hydroxy(C ₁ -C ₆ )alkyl group,

R ₀₂₀ is a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

R ₀₂₁ and R ₀₂₁ ' independently of one another are a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group or a hydroxy(C ₁ -C ₆ )alkyl group,

or a pair (R ₀₂₁ , R ₀₂₁ ') together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5-7 members in the ring, which optionally contains, in addition to the nitrogen atom, 1-3 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted with a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

R ₀₂₂ is a (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group, -(CH ₂ ) _p0 -NR ₀₂₄ R ₀₂₄ ' group or -(CH ₂ ) _p0 -O-(CHR ₀₁₈ -CHR ₀₁₉ -O) _q0 -R ₀₂₀ group,

R ₀₂₃ is a hydrogen atom or a (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group,

or a pair (R ₀₂₂ , R ₀₂₃ ) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5-18 ring members which optionally contains, in addition to the nitrogen atom, 1-5 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted with a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group or a heterocycloalkyl group,

R ₀₂₄ and R ₀₂₄ ' independently of each other are a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

or a pair (R ₀₂₄ , R ₀₂₄ ') together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring consisting of 5-7 ring members which may contain, in addition to the nitrogen atom, 1-3 heteroatoms selected from O, S and N and wherein the resulting ring is optionally substituted with a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

R ₀₂₅ is a hydrogen atom, a hydroxy group or a hydroxy(C ₁ -C ₆ )alkyl group,

R ₀₂₆ is a hydrogen atom, a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group or a cyano group,

R ₀₂₇ is a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

R ₀₂₈ is an -OP(O)(O ^- )(O ^- ) group, -OP(O)(O ^- )(OR ₀₃₀ ) group, -OP(O)(OR ₀₃₀ )(OR ₀₃₀ ') group, -(CH ₂ ) _p0 -O-SO ₂ -O- group, -(CH ₂ ) _p0 -SO ₂ -O- group, -(CH ₂ ) _p0 -O-SO ₂ -OR ₀₃₀ group, -Cy ₀₁₀ , -(CH ₂ ) _p0 -SO ₂ -OR ₀₃₀ group, -OC(O)-R ₀₂₉ group, -OC(O)-OR ₀₂₉ group, or -OC(O)-NR ₀₂₉ R ₀₂₉ 'group;

R ₀₂₉ and R ₀₂₉ ' independently of one another are a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, or a linear or branched amino(C ₁ -C ₆ )alkyl group,

R ₀₃₀ and R ₀₃₀ ' independently of one another are a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group or an aryl(C ₁ -C ₆ )alkyl group,

R0 ₃₁ is or , where the ammonium cation optionally exists as a zwitterionic form or has a monovalent anionic counterion,

n ₀ is an integer equal to 0 or 1,

p ₀ is an integer equal to 0, 1, 2, or 3,

q ₀ is an integer equal to 1, 2, 3, or 4,

r ₀ and s ₀ are independently an integer equal to 0 or 1;

wherein at most one of the R ₀₃ , R ₀₉ or R ₀₁₂ groups, if present, is covalently attached to the linker, and wherein the valence of the atom is not exceeded by one or more substituents bonded thereto,

or an enantiomer, diastereoisomer, atropisomer, deuterated derivative and/or pharmaceutically acceptable salt of any of the above.

In some embodiments, _CyO1 , _CyO2 , _CyO3 , CyO4, _CyO5 , _CyO6 , _CyO7 , _CyO8 , and _CyO10 , independently of one another, are a cycloalkyl group, a heterocycloalkyl group, an aryl group, or a heteroaryl group, each of which is optionally substituted with one or more groups selected from halogen; -( _{C1 - C6} )alkoxy; -( _C1 - _C6 )haloalkyl; -( _C1 - _C6 )haloalkoxy; -( _CH2 ) _p0 -O- _SO2 - _OR030 ; -( _CH2 ) _p0 - _SO2 - _OR030 ; -OP(O)( _OR020 ) ₂ ; -OP(O)(O ^- M ⁺ ) ₂ ; -CH ₂ -P(O)(OR ₀₂₀ ) ₂ ; -(CH ₂ ) _p0 -O-(CHR ₀₁₈ -CHR ₀₁₉ -O) _q0 -R ₀₂₀ ; hydroxy; hydroxy(C ₁ -C ₆ )alkyl; -(CH ₂ ) _r0 -U ₀ -(CH ₂ ) _s0 -heterocycloalkyl; or -U ₀ -(CH ₂ ) _q0 -NR ₀₂₁ R ₀₂₁ '.

In some embodiments, D comprises a compound of formula (II):

Where:

Z ₀ is a nitrogen atom or a CR ₀₄ group,

R ₀₁ is a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, a linear or branched (C ₁ -C ₆ )haloalkyl group, a hydroxy group, a linear or branched (C ₁ -C ₆ )alkoxy group, -S-(C ₁ -C ₆ )alkyl group, a cyano group, -Cy _08, -NR ₀₁₁ R ₀₁₁ ',

R ₀₂ , R ₀₃ and R ₀₄ independently of one another are a hydrogen atom, a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, a linear or branched (C ₁ -C ₆ )haloalkyl, a hydroxy group, a linear or branched (C ₁ -C ₆ )alkoxy group, -S-(C ₁ -C ₆ )alkyl group, a cyano group, a nitro group, -(C ₀ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-Cy ₀₁ , -(C ₀ -C ₆ )alkyl-Cy ₀₁ , -(C ₂ -C ₆ )alkenyl-Cy ₀₁ , -(C ₂ -C ₆ )alkynyl-Cy ₀₁ , -O-(C ₁ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-(C ₁ -C ₆ )alkyl-R ₀₃₁ , -C(O)-OR ₀₁₁ , -OC(O)-R ₀₁₁ , -C(O)-NR ₀₁₁ R ₀₁₁ ', -NR ₀₁₁ -C(O)-R ₀₁₁ ', -NR ₀₁₁ -C(O)-OR ₀₁₁ ', -(C ₁ -C ₆ )alkyl-NR ₀₁₁ -C(O)-R ₀₁₁ ', -SO ₂ -NR ₀₁₁ R ₀₁₁ ' or -SO ₂ -(C ₁ -C ₆ )alkyl,

or a pair of (R ₀₂ , R ₀₃ ) or (R ₀₃ , R ₀₄ ) together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containing 5-7 ring members which optionally contains 1-3 heteroatoms selected from O, S and N, wherein the ring is optionally substituted with a group selected from linear or branched (C ₁ -C ₆ )alkyl, -NR ₀₁₃ R ₀₁₃ ', -(C ₀ -C ₆ )alkyl-Cy ₀₁ and oxo,

R ₀₆ and R ₀₇ independently of one another are a hydrogen atom, a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, a linear or branched (C ₁ -C ₆ )haloalkyl, a hydroxy group, a linear or branched (C ₁ -C ₆ )alkoxy group, -S-(C ₁ -C ₆ )alkyl group, a cyano group, a nitro group, -(C ₀ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-Cy ₀₁ , -(C ₀ -C ₆ )alkyl-Cy ₀₁ , -(C ₂ -C ₆ )alkenyl-Cy ₀₁ , -(C ₂ -C ₆ )alkynyl-Cy ₀₁ , -O-(C ₁ -C ₆ )alkyl-R ₀₁₂ , -C(O)-OR ₀₁₁ , -OC(O)-R ₀₁₁ , -C(O)-NR ₀₁₁ R ₀₁₁ ', -NR ₀₁₁ -C(O)-R ₀₁₁ ', -NR ₀₁₁ -C(O)-OR ₀₁₁ ', -(C ₁ -C ₆ )alkyl-NR ₀₁₁ -C(O)-R ₀₁₁ ', -SO ₂ -NR ₀₁₁ R ₀₁₁ ' or -SO ₂ -(C ₁ -C ₆ )alkyl,

or a pair of (R ₀₆ , R ₀₇ ) fused with two adjacent carbon atoms, together with the carbon atoms to which they are attached, form an aromatic or non-aromatic ring containing 5-7 members in the ring, which optionally contains 1-3 heteroatoms selected from O, S and N, and wherein the resulting ring is optionally substituted with a group selected from a linear or branched (C ₁ -C ₆ )alkyl group, -NR ₀₁₃ R ₀₁₃ ', -(C ₀ -C ₆ )alkyl-Cy ₀₁ and oxo,

R ₀₈ is a hydrogen atom, a linear or branched (C ₁ -C ₈ )alkyl group, an aryl group, a heteroaryl group, an aryl-(C ₁ -C ₆ )alkyl group or a heteroaryl(C ₁ -C ₆ )alkyl group,

R ₀₉ is a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, -Cy _O2 , -(C ₁ -C ₆ )alkyl-Cy _O2 , -(C ₂ -C ₆ )alkenyl-Cy _O2 , -(C ₂ -C ₆ )alkynyl-Cy _O2 , -Cy _O2 -Cy _O3 , -(C ₂ -C ₆ )alkynyl-O-Cy _O2 , -Cy _O2 -(C ₀ -C ₆ )alkyl-O-(C ₀ -C ₆ )alkyl-Cy _O3 , a halogen atom, a cyano group, -C(O)-R ₀₁₄ , -C(O)-NR ₀₁₄ R ₀₁₄ ',

R ₀₁₁ and R ₀₁₁ ' independently of one another are a hydrogen atom, an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group or -(C ₀ -C ₆ )alkyl-Cy ₀₁ ,

or a pair (R ₀₁₁ , R ₀₁₁ ') together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5-7 ring members which optionally contains, in addition to the nitrogen atom, 1-3 heteroatoms selected from O, S and N, wherein the N atom is optionally substituted with a linear or branched (C ₁ -C ₆ )alkyl group and wherein one or more carbon atoms of the linear or branched (C ₁ -C ₆ )alkyl group are optionally deuterated,

R ₀₁₂ is -Cy ₀₅ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-Cy ₀₆ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-O-(C ₀ -C ₆ )alkyl-Cy ₀₆ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-NR ₀₁₁ -(C ₀ -C ₆ )alkyl-Cy ₀₆ , -Cy ₀₅ -Cy ₀₆ -O-(C ₀ -C ₆ )alkyl-Cy ₀₇ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-Cy ₀₉ , -NH-C(O)-NH-R ₀₁₁ , -C(O)-NR ₀₁₁ R ₀₁₁ ', -NR ₀₁₁ R ₀₁₁ ', -OR ₀₁₁ , -NR ₀₁₁ -C(O)-R ₀₁₁ ', -O-(C ₁ -C ₆ )alkyl-OR ₀₁₁ , -SO ₂ -R ₀₁₁ or -C(O)-OR ₀₁₁ ,

R ₀₁₃ , R ₀₁₃ ', R ₀₁₄ and R ₀₁₄ ' independently of one another are a hydrogen atom or an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group,

Cy ₀₁ , Cy ₀₂ , Cy ₀₃ , Cy ₀₅ , Cy ₀₆ , Cy ₀₇ and Cy ₀₈ independently of one another are an optionally substituted cycloalkyl group, an optionally substituted heterocycloalkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group,

Cy ₀₉ is , where R ₀₁₅ , R ₀₁₆ and R ₀₁₇ are as defined for formula (I),

R ₀₃₁ is , where R ₀₂₇ and R ₀₂₈ are as defined for formula (I), wherein at most one of the R ₀₃ , R ₀₉ or R ₀₁₂ groups, if present, is covalently attached to the linker,

or an enantiomer, diastereoisomer, atropisomer, deuterated derivative and/or pharmaceutically acceptable salt of any of the above.

In some embodiments, D comprises a compound of formula (III):

Where:

R ₀₁ is a linear or branched (C ₁ -C ₆ )alkyl group,

R ₀₃ is -O-(C ₁ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ' or ,

where R ₀₁₁ and R ₀₁₁ ' independently of one another are a hydrogen atom, an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group or -(C ₀ -C ₆ )alkyl-Cy ₀₁ ;

or a pair (R ₀₁₁ , R ₀₁₁ ') together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5-7 members in the ring, which optionally contains, in addition to the nitrogen atom, 1-3 heteroatoms selected from O, S and N, wherein the N atom may be substituted by 1 or 2 groups selected from a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

and where R ₀₂₇ is a hydrogen atom and R ₀₂₈ is a -(CH ₂ ) _p0 -O-SO ₂ -O ^- group or a -(CH ₂ ) _p0 -SO ₂ -OR ₀₃₀ group;

R ₀₉ is a linear or branched (C ₂ -C ₆ )alkynyl group or -Cy ₀₂ ,

R ₀₁₂ is -Cy ₀₅ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-Cy ₀₆ or -Cy ₀₅ -(C ₀ -C ₆ )alkyl-Cy ₀₉ ,

Cy ₀₁ , Cy ₀₂ , Cy ₀₅ and Cy ₀₆ independently of one another are a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, each of which is optionally substituted,

Cy₀₉ is ,

R ₀₁₅ , R ₀₁₆ and R ₀₁₇ are as defined for formula (I),

where at most one of the R ₀₃ , R ₀₉ or R ₀₁₂ groups, if present, is covalently attached to the linker,

or an enantiomer, diastereoisomer, atropisomer, deuterated derivative and/or pharmaceutically acceptable salt of any of the above.

In some embodiments, Cy _O1 , Cy _O2 , Cy _O5 , Cy _O6 , independently of one another, are a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, each of which is optionally substituted with one or more groups selected from halogen; -(C ₁ -C ₆ )alkoxy; -(C ₁ -C ₆ )haloalkyl; -(C ₁ -C ₆ )haloalkoxy; -(CH ₂ ) _p0 -O-SO ₂ -OR ₀₃₀ ; -(CH ₂ ) _p0 -SO ₂ -OR ₀₃₀ ; -OP(O)(OR ₀₂₀ ) ₂ ; -OP(O)(O ^- M ⁺ ) ₂ ; -CH ₂ -P(O)(OR ₀₂₀ ) ₂ ; -(CH ₂ ) _p0 -O-(CHR ₀₁₈ -CHR ₀₁₉ -O) _q0 -R ₀₂₀ ; hydroxy; hydroxy(C ₁ -C ₆ )alkyl; -(CH ₂ ) _r0 -U ₀ -(CH ₂ ) _s0 -heterocycloalkyl; or -U ₀ -(CH ₂ ) _q0 -NR ₀₂₁ R ₀₂₁ '.

In some embodiments, R ₀₁ is methyl or ethyl.

In some embodiments, R ₀₃ is -O-CH ₂ -CH ₂ -NR ₀₁₁ R ₀₁₁ ' in which R ₀₁₁ and R ₀₁₁ ' form, together with the nitrogen atom bearing them, a piperazinyl group that may be substituted with a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group.

In some embodiments, R ₀₃ comprises the formula: , where R ₀₂₇ is a hydrogen atom and R ₀₂₈ is a -(CH ₂ ) _p0 -O-SO ₂ -OR ₀₃₀ group, p ₀ is an integer equal to 0, 1, 2 or 3; and where R ₀₃₀ is a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group or an aryl(C ₁ -C ₆ )alkyl group.

In some embodiments, R ₀₃ comprises the formula: ,

Where is a connection with the linker.

In some embodiments, Cy ₀₁ , Cy ₀₂ , Cy ₀₃ , Cy ₀₄ , Cy ₀₅ , Cy ₀₆ , Cy ₀₇ , Cy ₀₈ and Cy ₀₁₀ , independently of one another, are an optionally substituted cycloalkyl group, an optionally substituted heterocycloalkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group, wherein the optional substituents are selected from optionally substituted linear or branched (C ₁ -C ₆ )alkyl, optionally substituted linear or branched (C ₂ -C ₆ )alkenyl group, optionally substituted linear or branched (C ₂ -C ₆ )alkynyl group, optionally substituted linear or branched (C ₁ -C ₆ )alkoxy, optionally substituted (C ₁ -C ₆ )alkyl-S-, hydroxy, oxo (or N-oxide, where applicable), nitro, cyano, -C(O)-OR ₀ ', -OC(O)-R ₀ ', -C(O)-NR ₀ 'R ₀ '', -NR ₀ 'R ₀ '', -(C=NR ₀ ')-OR ₀ '', linear or branched (C ₁ -C ₆ ) haloalkyl, trifluoromethoxy or halogen, where R ₀ ' and R ₀ '' each independently is a hydrogen atom or an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group and where one or more of the carbon atoms of the linear or branched (C ₁ -C ₆ )alkyl group is optionally deuterated.

In some embodiments, R ₀₉ is a Cy ₀₂ group, preferably an aryl group, more preferably a phenyl group. In some embodiments, Cy ₀₂ is an optionally substituted aryl group.

In some embodiments, Cy ₀₅ comprises a heteroaryl group selected from a pyrazolyl group and a pyrimidinyl group.

In some embodiments, Cy ₀₅ is a pyrimidinyl group.

In some embodiments, Cy ₀₅ is a pyrimidinyl group and Cy ₀₆ is a phenyl group.

In some embodiments, linker (L) is attached to D via a covalent bond from L to R ₀₃ of formula (I), (II), or (III). In some embodiments, linker (L) is attached to D via a covalent bond from L to R ₀₉ of formula (I), (II), or (III).

In some embodiments, D comprises:

or , or

an enantiomer, a diastereoisomer, an atropisomer, a deuterated derivative and/or a pharmaceutically acceptable salt of any of the above.

In some embodiments, -(LD) is formed from a compound selected from Table A or an enantiomer, diastereoisomer, atropisomer, deuterated derivative, and/or pharmaceutically acceptable salt ^thereof . For compounds in Table A, depending on their electronic charge, these compounds may contain one pharmaceutically acceptable monovalent anionic counterion _M1- ^. In some embodiments, the monovalent anionic counterion _M1- may be selected from biromide, chloride, iodide, acetate, trifluoroacetate, benzoate, mesylate, tosylate, triflate, formate, or the like. In some embodiments, the monovalent anionic counterion _M1- is ^{trifluoroacetate} or formate.

Table A. Typical linker-drug groups

In some embodiments, the antibody-drug conjugate has a formula a according to any of the structures shown in Table 1a.

Table 1a. ADC structures

L86-P1 =anti-CD74 antibody or its antigen-binding fragment
The ADCs shown above can also be represented by the following formula:
Ab-(LD) _p ,
Where is an anti-CD74 antibody or antigen-binding fragment thereof covalently linked to the payload linker (L/P) depicted above; p is an integer of 1-16. In some embodiments, p is an integer of 1-8. In some embodiments, p is an integer of 1-5. In some embodiments, p is an integer of 2-4. In some embodiments, p is 2. In some embodiments, p is 4. In some embodiments, p is determined by liquid chromatography-mass spectrometry (LC-MS).

In some embodiments, the antibody-drug conjugate has a formula according to any of the structures depicted in Table 1b.

Table 1b. ADC Structures ADC structure Target L/P Name CD33 L23-C3 CD33 L24-C1 CD33 L13-C4 CD33 L14-C3 CD33 L15-C5 CD33 L16-C3 CD33 L17-C3 CD33 L18-C3 CD33 L19-C3 CD33 L20-C6 CD33 L24-C6 CD33 L24-C7 HER2 L24-C1 HER2 L24-C1 HER2 L24-C6 HER2 L24-C7 BCMA L21-C1 BCMA L9-C1 BCMA L9-C8 BCMA L22-C1 BCMA L9-C10 BCMA L9-C9 BCMA L9-C11 BCMA L9-C12 BCMA L9-C13 CD33 L29-C3 BCMA
DANAPA L27-P1 BCMA
DANAPA L28-P1 BCMA L26-P1 BCMA L9-P14 BCMA L9-P15 BCMA L25-P1 BCMA L9-P16 BCMA L9-P17 CD46 L9-P1 The ADCs shown above can also be represented by the following formula:
Ab-(LD) _p ,
Where is an anti-CD74 antibody or antigen-binding fragment thereof covalently linked to the payload linker (L/P) depicted above; p is an integer of 1-16. In some embodiments, p is an integer of 1-8. In some embodiments, p is an integer of 1-5. In some embodiments, p is an integer of 2-4. In some embodiments, p is 2. In some embodiments, p is 4. In some embodiments, p is determined by liquid chromatography-mass spectrometry (LC-MS).

As used herein, "L/P" refers to a linker-payload, linker-drug, or linker-compound disclosed herein, and the terms "L#-P#" and "L#-C#" are used interchangeably to refer to a particular linker-drug disclosed herein, while the codes "P#" and "C#" are used interchangeably to refer to a particular compound, unless otherwise specified. For example, both "L1-C1" and "L1-P1" refer to the same linker-payload structure described herein, while "C1" and "P1" denote the same compound disclosed herein, including an enantiomer, diastereoisomer, atropisomer, deuterated derivative, and/or pharmaceutically acceptable salt of any of the above.

In some embodiments, the antibody or antigen-binding fragment binds to a target antigen on a cancer cell. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2, Nectin4, TROP2, LIV1, CD46, or GPNMB. In some embodiments, the target antigen is 4-1BB, 5AC, 5T4, alpha-fetoprotein, angiopoietin 2, ASLG659, TCLI, BMPRIB, Brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (mimicking), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR I), CD22 (B cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B cell differentiation antigen CD72), CD79a, CD80, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), Epicialin, ERBB3, ETBR (endothelin receptor type B), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extra domain-B, burn receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, receptor kinase human scattering factor, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily translocation receptor 2-associated), Lewis-Y antigen, LY64 (RP105), MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen, PSCA (prostate stem cell antigen precursor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-associated gene I, TAG-72, TEMI, Tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential-acting cation channel superfamily transient receptor M member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or Vimentin. In some embodiments, the target antigen is BCMA, CD33, PCAD, HER2, CD38, CD46, CD48, or CD79b. In some embodiments, the target antigen is BCMA, CD33, CD48, PCAD, or HER2. In some embodiments, the target antigen is CD38 or CD48.

In some embodiments, the antibody or antigen-binding fragment is an anti-BCMA antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 15 (HCDR1), SEQ ID NO: 16 (HCDR2), and SEQ ID NO: 17 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 18 (LCDR1), SEQ ID NO: 19 (LCDR2), and SEQ ID NO: 20 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the antibody or antigen-binding fragment comprises an IgG1 heavy chain constant domain or a modified IgG1 heavy chain constant domain. In some embodiments, the IgG1 heavy chain constant domain comprises a cysteine residue (C) at position 152 and position 375. In some embodiments, the IgG1 heavy chain constant domain comprises a cysteine residue (C) at position 156 and position 379. In some embodiments, the antibody or antigen-binding fragment comprises an Ig kappa light chain constant domain.

In some embodiments, the antibody or antigen-binding fragment is an anti-CD33 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:21 (HCDR1), SEQ ID NO:22 (HCDR2), and SEQ ID NO:23 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:24 (LCDR1), SEQ ID NO:25 (LCDR2), and SEQ ID NO:26 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4. In some embodiments, the antibody or antigen-binding fragment comprises an IgG1 heavy chain constant domain or a modified IgG1 heavy chain constant domain. In some embodiments, the IgG1 heavy chain constant domain comprises a glutamine residue (Q) at position 297. In some embodiments, the antibody or antigen-binding fragment comprises an Ig kappa light chain constant domain.

In some embodiments, the antibody or antigen-binding fragment is an anti-PCAD antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:33 (HCDR1), SEQ ID NO:34 (HCDR2), and SEQ ID NO:35 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:36 (LCDR1), SEQ ID NO:37 (LCDR2), and SEQ ID NO:38 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8.

In some embodiments, the antibody or antigen-binding fragment is an anti-HER2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:39 (HCDR1), SEQ ID NO:40 (HCDR2), and SEQ ID NO:41 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:42 (LCDR1), SEQ ID NO:43 (LCDR2), and SEQ ID NO:44 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the antibody or antigen-binding fragment comprises an IgG1 heavy chain constant domain or a modified IgG1 heavy chain constant domain. In some embodiments, the IgG1 heavy chain constant domain comprises a glutamine residue (Q) at position 297. In some embodiments, the IgG1 heavy chain constant domain comprises a serine residue (S) at position 297. In some embodiments, the antibody or antigen-binding fragment comprises an Ig kappa light chain constant domain.

In some embodiments, the antibody or antigen-binding fragment is an anti-CD38 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is an anti-CD46 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is an anti-CD48 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is an anti-CD79b antibody or antigen-binding fragment.

The present document, in some embodiments, also provides compositions comprising multiple copies of an antibody-drug conjugate (e.g., any of the exemplary antibody-drug conjugates described herein). In some embodiments, the average p of the antibody-drug conjugate in the composition is from about 2 to about 4.

Also provided herein, in some embodiments, are pharmaceutical compositions comprising an antibody-drug conjugate (e.g., any of the exemplary antibody-drug conjugates described herein) or composition (e.g., any of the exemplary compositions described herein) and a pharmaceutically acceptable carrier.

Additionally, in some embodiments, the present disclosure provides therapeutic uses of the disclosed compounds and ADC compositions, such as in the treatment of cancer. In some embodiments, the present disclosure provides methods for treating cancer (e.g., a cancer that expresses an antigen targeted by an antibody or antigen-binding fragment of an ADC, such as BCMA, CD33, PCAD, or HER2). In some embodiments, the present disclosure provides methods for reducing or slowing the growth of a cancer cell population in a subject. In some embodiments, the present disclosure provides methods for determining whether a subject suffering from cancer or suspected of having cancer will respond to treatment with a compound or ADC composition disclosed herein.

An exemplary embodiment is a method of treating a subject having or suspected of having cancer, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions described herein). In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2, Nectin4, TROP2, LIV1, CD46, or GPNMB. In some embodiments, the target antigen is 4-1BB, 5AC, 5T4, alpha-fetoprotein, angiopoietin 2, ASLG659, TCLI, BMPRIB, Brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (mimicking), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR I), CD22 (B cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B cell differentiation antigen CD72), CD79a, CD80, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), Epicialin, ERBB3, ETBR (endothelin receptor type B), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extra domain-B, burn receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, receptor kinase human scattering factor, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily translocation receptor 2-associated), Lewis-Y antigen, LY64 (RP105), MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen, PSCA (prostate stem cell antigen precursor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-associated gene I, TAG-72, TEMI, Tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential-acting cation channel superfamily transient receptor M member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or Vimentin. In some embodiments, the target antigen is BCMA, CD33, PCAD, HER2, CD38, CD46, CD48, or CD79b. In some embodiments, the target antigen is BCMA, CD33, CD48, PCAD, or HER2. In some embodiments, the target antigen is CD38 or CD48. In some embodiments, the cancer is a tumor or a hematological cancer. In some embodiments, the cancer is breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer is lymphoma or gastric cancer.

Another exemplary embodiment is a method for reducing or inhibiting tumor growth in a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions described herein). In some embodiments, the tumor expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2, Nectin4, TROP2, LIV1, CD46, or GPNMB. In some embodiments, the target antigen is 4-1BB, 5AC, 5T4, alpha-fetoprotein, angiopoietin 2, ASLG659, TCLI, BMPRIB, Brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (mimicking), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR I), CD22 (B cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B cell differentiation antigen CD72), CD79a, CD80, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), Epicialin, ERBB3, ETBR (endothelin receptor type B), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extra domain-B, burn receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, receptor kinase human scattering factor, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily translocation receptor 2-associated), Lewis-Y antigen, LY64 (RP105), MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen, PSCA (prostate stem cell antigen precursor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-associated gene I, TAG-72, TEMI, Tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential-acting cation channel superfamily transient receptor M member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or Vimentin. In some embodiments, the target antigen is BCMA, CD33, PCAD, HER2, CD38, CD46, CD48, or CD79b. In some embodiments, the target antigen is BCMA, CD33, CD48, PCAD, or HER2. In some embodiments, the target antigen is CD38 or CD48. In some embodiments, the tumor is breast cancer, gastric cancer, bladder cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer, hepatocellular carcinoma, melanoma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the tumor is gastric cancer. In some embodiments, administration of the antibody-drug conjugate, composition, or pharmaceutical composition reduces or inhibits tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%.

Another exemplary embodiment is a method for reducing or slowing the growth of a population of cancer cells in a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions described herein). In some embodiments, the population of cancer cells expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2, Nectin4, TROP2, LIV1, CD46, or GPNMB. In some embodiments, the target antigen is 4-1BB, 5AC, 5T4, alpha-fetoprotein, angiopoietin 2, ASLG659, TCLI, BMPRIB, Brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (mimicking), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR I), CD22 (B cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B cell differentiation antigen CD72), CD79a, CD80, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), Epicialin, ERBB3, ETBR (endothelin receptor type B), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extra domain-B, burn receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, receptor kinase human scattering factor, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily translocation receptor 2-associated), Lewis-Y antigen, LY64 (RP105), MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen, PSCA (prostate stem cell antigen precursor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-associated gene I, TAG-72, TEMI, Tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential-acting cation channel superfamily transient receptor M member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or Vimentin. In some embodiments, the target antigen is BCMA, CD33, PCAD, HER2, CD38, CD46, CD48, or CD79b. In some embodiments, the target antigen is BCMA, CD33, CD48, PCAD, or HER2. In some embodiments, the target antigen is CD38 or CD48. In some embodiments, the population of cancer cells is derived from a tumor or hematological cancer. In some embodiments, the population of cancer cells is derived from breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer cell population is derived from lymphoma or gastric cancer. In some embodiments, administration of the antibody-drug conjugate, composition, or pharmaceutical composition reduces the cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, administration of the antibody-drug conjugate, composition, or pharmaceutical composition slows the growth of a cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%.

Another exemplary embodiment is an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions described herein) for use in treating a subject suffering from or suspected of having cancer. In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2, Nectin4, TROP2, LIV1, CD46, or GPNMB. In some embodiments, the target antigen is 4-1BB, 5AC, 5T4, alpha-fetoprotein, angiopoietin 2, ASLG659, TCLI, BMPRIB, Brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (mimicking), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR I), CD22 (B cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B cell differentiation antigen CD72), CD79a, CD80, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), Epicialin, ERBB3, ETBR (endothelin receptor type B), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extra domain-B, burn receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, receptor kinase human scattering factor, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily translocation receptor 2-associated), Lewis-Y antigen, LY64 (RP105), MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen, PSCA (prostate stem cell antigen precursor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-associated gene I, TAG-72, TEMI, Tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential-acting cation channel superfamily transient receptor M member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or Vimentin. In some embodiments, the target antigen is BCMA, CD33, PCAD, HER2, CD38, CD46, CD48, or CD79b. In some embodiments, the target antigen is BCMA, CD33, CD48, PCAD, or HER2. In some embodiments, the target antigen is CD38 or CD48. In some embodiments, the cancer is a tumor or a hematological cancer. In some embodiments, the cancer is breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer is lymphoma or gastric cancer.

Another exemplary embodiment is the use of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions described herein) in the treatment of a subject suffering from or suspected of having cancer. In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2, Nectin4, TROP2, LIV1, CD46, or GPNMB. In some embodiments, the target antigen is 4-1BB, 5AC, 5T4, alpha-fetoprotein, angiopoietin 2, ASLG659, TCLI, BMPRIB, Brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (mimicking), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR I), CD22 (B cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B cell differentiation antigen CD72), CD79a, CD80, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), Epicialin, ERBB3, ETBR (endothelin receptor type B), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extra domain-B, burn receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, receptor kinase human scattering factor, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily translocation receptor 2-associated), Lewis-Y antigen, LY64 (RP105), MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen, PSCA (prostate stem cell antigen precursor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-associated gene I, TAG-72, TEMI, Tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential-acting cation channel superfamily transient receptor M member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or Vimentin. In some embodiments, the target antigen is BCMA, CD33, PCAD, HER2, CD38, CD46, CD48, or CD79b. In some embodiments, the target antigen is BCMA, CD33, CD48, PCAD, or HER2. In some embodiments, the target antigen is CD38 or CD48. In some embodiments, the cancer is a tumor or a hematological cancer. In some embodiments, the cancer is breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer is lymphoma or gastric cancer.

Another exemplary embodiment is the use of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions described herein) in a method for the manufacture of a medicament for the treatment of a subject suffering from or suspected of having cancer. In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2, Nectin4, TROP2, LIV1, CD46, or GPNMB. In some embodiments, the target antigen is 4-1BB, 5AC, 5T4, alpha-fetoprotein, angiopoietin 2, ASLG659, TCLI, BMPRIB, Brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (mimicking), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR I), CD22 (B cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B cell differentiation antigen CD72), CD79a, CD80, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), Epicialin, ERBB3, ETBR (endothelin receptor type B), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extra domain-B, burn receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, receptor kinase human scattering factor, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily translocation receptor 2-associated), Lewis-Y antigen, LY64 (RP105), MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen, PSCA (prostate stem cell antigen precursor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-associated gene I, TAG-72, TEMI, Tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential-acting cation channel superfamily transient receptor M member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or Vimentin. In some embodiments, the target antigen is BCMA, CD33, PCAD, HER2, CD38, CD46, CD48, or CD79b. In some embodiments, the target antigen is BCMA, CD33, CD48, PCAD, or HER2. In some embodiments, the target antigen is CD38 or CD48. In some embodiments, the cancer is a tumor or a hematological cancer. In some embodiments, the cancer is breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer is lymphoma or gastric cancer.

Another exemplary embodiment is a method for determining whether a subject suffering from or suspected of having cancer will respond to treatment with an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions described herein) by providing a biological sample from the subject; contacting the sample with an antibody-drug conjugate; and detecting binding of the antibody-drug conjugate to cancer cells in the sample. In some embodiments, the cancer cells in the sample express a target antigen. In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2, Nectin4, TROP2, LIV1, CD46, or GPNMB. In some embodiments, the target antigen is 4-1BB, 5AC, 5T4, alpha-fetoprotein, angiopoietin 2, ASLG659, TCLI, BMPRIB, Brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (mimicking), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR I), CD22 (B cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B cell differentiation antigen CD72), CD79a, CD80, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), Epicialin, ERBB3, ETBR (endothelin receptor type B), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extra domain-B, burn receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, receptor kinase human scattering factor, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily translocation receptor 2-associated), Lewis-Y antigen, LY64 (RP105), MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen, PSCA (prostate stem cell antigen precursor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-associated gene I, TAG-72, TEMI, Tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential-acting cation channel superfamily transient receptor M member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or Vimentin. In some embodiments, the target antigen is BCMA, CD33, PCAD, HER2, CD38, CD46, CD48, or CD79b. In some embodiments, the target antigen is BCMA, CD33, CD48, PCAD, or HER2. In some embodiments, the target antigen is CD38 or CD48. In some embodiments, the cancer is a tumor or hematological cancer. In some embodiments, the cancer is a tumor or hematological cancer. In some embodiments, the cancer is breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer is lymphoma or gastric cancer. In some embodiments, the sample is a tissue biopsy sample, a blood sample, or a bone marrow sample.

Methods for preparing the disclosed ADC compounds and compositions are also described. A typical embodiment is a method for producing an antibody-drug conjugate by reacting an antibody or antigen-binding fragment with a cleavable linker attached to an Mcl-1 inhibitor under conditions permitting conjugation.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 shows an example of site-specific conjugation of antibodies using bacterial transglutaminase (BTG).

Figure 2A shows the dose-response curve of free Mcl-1 payload (P1), CD38-targeting ADC, Mcl-1, and non-isotype-targeting ADC on A4-Fuk cells. Figure 2B shows the dose-response curve of free Mcl-1 payload (P1), CD38-targeting ADC, Mcl-1, and non-isotype-targeting ADC on KMS021 BM cells.

Figure 3A shows the dose-response curve of free Mcl-1 (P1), CD48-targeting ADC, Mcl-1, and non-isotype-targeting ADC payload on NCI-H929 cells. Figure 3B shows the dose-response curve of free Mcl-1 (P1), CD48-targeting ADC, Mcl-1, and non-isotype-targeting ADC payload on OPM-2 cells. Figure 3C shows the dose-response curve of free Mcl-1 (P1), CD48-targeting ADC, Mcl-1, and non-CD48-targeting ADC payload on AMO1 cells.

Figure 4 shows the dose-response curve of the payload of free Mcl-1 (P1), CD79b-targeting Mcl-1 ADC, and non-targeting isotype ADC on RS4 cells; 11.

Figure 5A shows the dose-response curve of free Mcl-1 (P1) payload and HER2-targeting Mcl-1 ADC on HCC1954 cells. Figure 5B shows the dose-response curve of free Mcl-1 (P1) payload and HER2-targeting Mcl-1 ADC on HCC2218 cells.

Figure 6A shows the dose-response curve for the free Mcl-1 payload (P1), CD33-targeting Mcl-1 ADC, and non-targeting isotype ADC on AMO1-CD33 clone D2 cells. Figure 6B shows the dose-response curve for the free Mcl-1 payload (P1), CD33-targeting Mcl-1 ADC, and non-targeting isotype ADC on MOLM-13 cells.

Figure 7 shows the dose-response curve for the free Mcl-1 (P1) payload, BCMA-targeting Mcl-1 ADC, and non-isotype-targeting ADC on NCI-H929 cells.

Figures 8A and 8B show the results of the MTT cell viability assay. All tested anti-CD33 ADCs and their corresponding payloads caused a dose-dependent decrease in the viability of AMO1-CD33 clone D2 cells; however, no significant effect was observed with the corresponding naked antibody.

Figures 9A and 9B show the results of the CTG cell viability assay. All tested anti-HER2 ADCs and their corresponding payloads caused a dose-dependent reduction in HCC1954 cell viability, while no significant effect was observed with the corresponding naked antibody.

Figures 10A, 10B, 10C, and 10D show the results of the MTT cell viability assay. All tested anti-BCMA ADCs and their corresponding payloads caused a dose-dependent reduction in NCI-H929 cell viability; however, no significant effect was observed with the corresponding naked antibody.

Figure 11 shows tumor volume ( ^mm3 ) of NCI-H929-inoculated female SCID mice after treatment with isotype control IgG1-Linker-Payload with silent Fc, Fc-silenced anti-BCMA_CysmAb, or Fc-silenced anti-BCMA_CysmAb_L7-P1 (10 and/or 30 mg/kg as a single IV dose, n=6).

Figure 12 shows the proportion (%) of body weight loss in NCI-H929-inoculated female SCID mice treated with isotype control IgG1-Linker-Payload Fc-silenced, anti-BCMA_CysmAb Fc-silenced, or anti-BCMA_CysmAb Fc-silenced Fc_L7-P1 (10 and/or 30 mg/kg administered intravenously once, n=6).

Fig. 13A shows the results of a CTG cell viability assay testing anti-CD48 ADC in NCI-H929 cells. Fig. 13B shows the results of a CTG cell viability assay testing anti-CD48 ADC in KHM1B cells. Fig. 13C shows the results of a CTG cell viability assay testing anti-CD48 ADC in KMS21BM cells.

Figure 14 shows tumor volume ( ^mm3 ) of H929-grafted female SCID mice as a function of time after randomization (days) following treatment with Fc-silenced anti-BCMA_CysmAb or various anti-BCMA_CysmAb Fc ADCs (20 mg/kg administered intravenously once, n=6).

Figure 15 shows the % body weight loss of H929-inoculated female SCID mice over time after randomization (days) following treatment with Fc-silenced anti-BCMA_CysmAb or various Fc-silenced anti-BCMA_CysmAb ADCs (20 mg/kg administered intravenously once, n=6).

Figure 16 shows tumor volume ( ^mm3 ) of H929-transplanted female SCID mice as a function of time after randomization (days) following treatment with Fc-silenced IgG1-Linker-Payload, Fc-silenced anti-CD48_CysmAb, or Fc-silenced anti-CD48_CysmAb L5-P1 (30 mg/kg as a single intravenous injection, n=8).

Figure 17 shows % body weight loss in H929-inoculated female SCID mice over time after randomization (days) following treatment with Fc-silenced IgG1-Linker-Payload, Fc-silenced anti-CD48_CysmAb, or Fc-silenced anti-CD48_CysmAb L5-P1 (30 mg/kg, administered intravenously once, n=8).

Figure 18 shows the results of the CTG cell viability assay. Anti-CD46-Mcl-1 ADC Ab C - L9-C1 and the corresponding payload C1 caused a dose-dependent decrease in KMS21BM cell viability, while no significant effect was observed with the corresponding naked antibody.

Figures 19A and 19B show the results of the MTT cell viability assay. All ADCs caused a dose-dependent reduction in the viability of clone D2 AMO1-CD33 cells when administered alone. Interestingly, ADC activity was significantly improved when combined with the BCL2 inhibitor compound A1, whereas no significant effect was observed after treatment of these cells with the corresponding naked antibody alone or in combination with 1 μM compound A1.

Figures 20A and 20B show the results of the MTT cell viability assay. All anti-BCMA-Mcl-1 ADCs caused a dose-dependent reduction in AMO1 cell viability. Interestingly, ADC activity was significantly enhanced in combination with the BCL2 inhibitor compound A1, whereas no significant effect was observed after treatment of these cells with the corresponding naked antibody alone or in combination with 1 μM compound A1.

Figures 21A and 21B show the results of the MTT cell viability assay. All anti-BCMA-Mcl-1 ADCs caused a dose-dependent decrease in H929 cell viability. Interestingly, ADC activity was significantly improved in combination with the BCL2 inhibitor compound A1, whereas no significant effect was observed after treatment of these cells with the corresponding naked antibody alone or in combination with 1 μM compound A1.

Fig. 22 shows the inhibitory activity of the CD48-MCL-1 antibody-drug conjugate CD48-L7-P1, IgG1 isotype ADC-L7-P1 and MCL-1 free payload P1 against KMS-21BM, KMS-20, KMS-27 and NCI-H929.

Fig. 23A and 23B show the inhibitory activities of the CD48 MCL-1 antibody-drug conjugate CD48-L7-P1, IgG1 isotype ADC-L7-P1, and MCL-1 free payload P1 in combination with venetoclax against KMS-21BM, KMS-20, KMS-27, and NCI-H929.

DETAILED DESCRIPTION OF ILLUSTRATIVE IMPLEMENTATION OPTIONS

The compositions and methods described may be more readily understood by reference to the following detailed description taken together with the accompanying figures which form part of this description.

As used herein, descriptions refer to compositions and methods of using compositions. If a description describes or claims a feature or embodiment associated with a composition, such feature or embodiment applies equally to methods of using the composition. Similarly, if a description describes or claims a feature or embodiment associated with a method of using a composition, such feature or embodiment applies equally to the composition.

When a range of values is specified, it includes embodiments utilizing any particular value within the range. Furthermore, references to values specified in ranges include each value within the range. All ranges include their endpoints and may be combined. When values are specified as approximations using the preceding "about," it is to be understood that a particular value constitutes another embodiment. Reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise. The use of "or" will mean "and/or" unless the specific context of its use dictates otherwise. All references cited herein are incorporated by reference for all purposes. If a reference and a specification conflict, the specification shall control.

Unless the context of the description indicates otherwise, such as in the absence of symbols indicating a specific linkage point(s), when a structure or fragment of a structure is drawn, it may be used alone or attached to other components of the ADC, and this may occur in any orientation, such as with the antibody attached at any suitable attachment point to a chemical moiety such as a drug-linker. However, where indicated, the ADC components are attached in the orientation shown in a given formula. For example, if formula (1) is described as Ab-(LD) _p , and the "-(LD)" group is described as , then the developed structure of formula (1) is She is not. .

It should be appreciated that certain features of the described compositions and methods, which for clarity are described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the described compositions and methods, which for brevity are described in the context of a single embodiment, may also be presented separately or in any sub-combination.

In the context of the present application, an antibody-drug conjugate can be identified using a naming convention in the general format "antigen-target/antibody-linker-payload." For example, if an antibody-drug conjugate is called "target X-L0-P0," such a conjugate will comprise an antibody that binds target X, a linker designated as L0, and a payload designated as P0. Alternatively, if an antibody-drug conjugate is called "anti-target X-L0-P0," such a conjugate may comprise an antibody that binds target X, a linker designated as L0, and a payload designated as P0. In another embodiment, if the antibody-drug conjugate is called "AbX-L0-P0," such a conjugate may comprise an antibody designated AbX, a linker designated L0, and a payload designated P0. A control antibody-drug conjugate comprising a non-specific, isotype control antibody may be called "isotype control IgG1-L0-P0" or "IgG1-L0-P0."

Any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds. Isotopically labeled compounds have the structures depicted by the formulas given herein, except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Isotopes that may be included in the compounds of the present invention include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, such as ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ F, and ³⁶ Cl. Accordingly, it should be understood that the present disclosure includes compounds that include one or more of any of the above-mentioned isotopes, including, for example, radioactive isotopes such as ³ H and ¹⁴ C, or those in which non-radioactive isotopes such as ² H and ¹³ C are present. Such isotopically labeled compounds are useful in metabolism studies (with ¹⁴ C), reaction kinetics studies (e.g., with ² H or ³ H), in detection or imaging methods such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including analysis of drug or substrate distribution in tissues, or in radioactive treatment of patients. In particular, ¹⁸ F or a labeled compound may be particularly desirable for PET or SPECT studies. Isotopically labeled compounds can generally be prepared by conventional methods known to those skilled in the art, for example, using suitable isotopically labeled reagents in place of a previously used unlabeled reagent.

Definitions

The description and claims use various terms related to aspects of the description. Such terms should be given their ordinary meaning in the art unless otherwise specified. Other specifically defined terms should be interpreted in accordance with the definitions provided herein.

As used herein, the singular forms "a," "an," "having," "having," as in "having the chemical formula," "including," and "comprising" are to be construed as open terms (i.e., meaning "including, but not limited to") unless otherwise specified. Furthermore, whenever "comprising" or another open term is used in an embodiment, it is to be understood that the same embodiment may be more narrowly claimed using the intermediate term "consisting essentially of" or the closed term "consisting of."

The term "about" or "approximately," when used in the context of numerical values and ranges, refers to values or ranges that are approximately or close to the stated values or ranges, so that the embodiment can be performed as intended, as is apparent to one skilled in the art from the instructions provided herein. In some embodiments, "about" means plus or minus 20%, 15%, 10%, 5%, 1%, 0.5%, or 0.1% of the numerical value. In one embodiment, the term "about" refers to a range of values that are 10% greater than or less than the stated value. In another embodiment, the term "about" refers to a range of values that are 5% greater than or less than the stated value. In another embodiment, the term "about" refers to a range of values that are 1% greater than or less than the stated value.

The terms "antibody-drug conjugate,""antibodyconjugate,""conjugate,""immunoconjugate," and "ADC" are used interchangeably and refer to one or more therapeutic compounds (e.g., an Mcl-1 inhibitor) that are linked to one or more antibodies or antigen-binding fragments. In some embodiments, an ADC is defined by the general formula: Ab-(LD) _p (Formula 1), where Ab = antibody or antigen-binding fragment, L = linker group, D = drug moiety (e.g., Mcl-1 inhibitor drug moiety), and p = number of drug moieties per antibody or antigen-binding fragment. In ADCs containing an Mcl-1 inhibitor drug moiety, " p " refers to the number of Mcl-1 inhibitor compounds linked to the antibody or antigen-binding fragment.

The term "antibody" is used in its broadest sense to refer to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or combinations thereof, through at least one antigen-recognition site in the variable region of the immunoglobulin molecule. An antibody may be polyclonal or monoclonal, multi- or single-chain, or intact immunoglobulin and may be obtained from natural sources or from recombinant sources. An "intact" antibody is a glycoprotein that typically contains at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated VL) and a light chain constant region. The light chain constant region consists of a single CL domain. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with antigen. The constant regions of antibodies can mediate the binding of immunoglobulin to tissues or host factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The antibody may be a monoclonal antibody, a human antibody, a humanized antibody, a camelid antibody, or a chimeric antibody. Antibodies may be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. An antibody may be an intact antibody or an antigen-binding fragment thereof.

The term "antibody fragment," "antigen-binding fragment," or "functional antibody fragment," as used herein, refers to at least one portion of an antibody that retains the ability to specifically interact with (e.g., by binding, sterically hindering, stabilizing/destabilizing, spatially distributing) an epitope of an antigen (e.g., BCMA, CD33, PCAD, or HER2). Antigen-binding fragments may also retain the ability to be internalized into an antigen-expressing cell. In some embodiments, antigen-binding fragments also retain immune effector activity. The terms "antibody," "antibody fragment," "antigen-binding fragment," and the like are intended to encompass the use of binding domains from antibodies in the context of larger macromolecules, such as ADCs. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), an Fd fragment consisting of VH and CH1 domains, linear antibodies, single-domain antibodies such as sdAb (VL or VH), camelid VHH domains, multispecific antibodies formed from antibody fragments such as a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region, and isolated CDR or other epitope-binding fragments of the antibody. The antigen-binding moiety can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, bispecific or multispecific antibody constructs, ADCs, v-NARs, and bis-scFv (see, e.g., Holliger and Hudson (2005) Nat Biotechnol. 23(9):1126–36). Antigen-binding moieties can also be grafted to polypeptide-based scaffolds such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies). The term "scFv" refers to a fusion protein comprising at least one antigen-binding fragment comprising a light chain variable region and at least one antigen-binding fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are linked continuously, for example, via a synthetic linker, for example, a short flexible polypeptide linker, and capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, the scFv may have the VL and VH variable regions in any order, for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL. Antigen-binding fragments are prepared using conventional techniques known to those skilled in the art, and the binding fragments are screened for utility (e.g., binding affinity, internalization) in the same manner as intact antibodies. Antigen-binding fragments, for example, can be prepared by cleavage of the intact protein, such as with a protease or chemical cleavage.

The term "complementarity-determining region" or "CDR," as used herein, refers to the amino acid sequences in the variable regions of an antibody that confer antigen specificity and binding affinity. For example, in general, there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991) "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (Kabat numbering scheme); Al-Lazikani et al. (1997) J Mol Biol. 273(4):927-48 (the "Chothia" numbering scheme); ImMunoGenTics (IMGT) numbering (Lefranc (2001) Nucleic Acids Res. 29(1):207-9; Lefranc et al. (2003) Dev Comp Immunol. 27(1):55-77) (the "IMGT" numbering scheme); or a combination thereof. In the combined Kabat and Chothia numbering scheme for a given CDR region (e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, or LC CDR3), in some embodiments, the CDRs correspond to amino acid residues that are defined as part of the Kabat CDRs together with amino acid residues that are defined as part of the Chothia CDRs. In this context, CDRs defined according to the Chothia numbering scheme are also sometimes referred to as "hypervariable loops".

In some embodiments, according to Kabat, the amino acid residues of the CDRs in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1) (e.g., insert(s) after position 35), 50-65 (HCDR2), and 95-102 (HCDR3); and the amino acid residues of the CDRs in the light chain variable domain (VL) are numbered 24-34 (LCDR1) (e.g., insert(s) after position 27), 50-56 (LCDR2), and 89-97 (LCDR3). In some embodiments, according to Chothia, the amino acid residues of the CDRs in VH are numbered 26-32 (HCDR1) (e.g., insert(s) after position 31), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in the VL are numbered 26-32 (LCDR1) (e.g., insert(s) after position 30), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of Kabat and Chothia, in some embodiments, the CDRs comprise or consist of, for example, amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. In some embodiments, according to IMGT, the amino acid residues of the CDRs in VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the amino acid residues of the CDRs in VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). In some embodiments, according to IMGT, the CDR regions of the antibody can be determined using the IMGT/DomainGap Align program.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically comprise multiple antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as it is obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present disclosure can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be produced by recombinant DNA techniques (see, e.g., U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described by Clackson et al. (1991) Nature 352:624-8 and Marks et al. (1991) J Mol Biol. 222:581-97, for example. The term also includes preparations of antibody molecules with single-molecule composition. The composition of monoclonal antibodies exhibits a single binding specificity and affinity for a particular epitope.

The monoclonal antibodies described herein may be non-human, human, or humanized. The term specifically includes "chimeric" antibodies in which a portion of the heavy and/or light chain is identical to or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remaining portion of the chain(s) is identical to or homologous to the corresponding sequences in antibodies derived from another species or belonging to a different class or subclass of antibodies, as well as fragments of such antibodies, provided that they specifically bind to the target antigen and/or exhibit the desired biological activity.

As used herein, the term "human antibody" refers to an antibody produced by a human or to an antibody having the amino acid sequence of an antibody produced by a human. The term includes antibodies having variable regions in which both the framework region and the CDR regions are derived from sequences of human origin. Furthermore, if the antibody comprises a constant region, the constant region is also derived from such human sequences, for example, human germline sequences or mutated versions of human germline sequences, or antibodies containing consensus framework sequences derived from analysis of human framework region sequences, for example, as described in Knappik et al. ((2000) J Mol Biol. 296(1):57-86). The structures and arrangement of immunoglobulin variable domains, such as CDRs, can be determined using well-known numbering schemes, such as the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia and/or the ImMunoGenTics (IMGT) numbering scheme. Human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or somatic mutation in vivo, or conservative substitution to improve stability or production). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term "recombinant human antibody" as used herein refers to a human antibody that is produced, expressed, created, or isolated by recombinant methods, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes, or a hybridoma derived therefrom, antibodies isolated from a host cell transformed to express a human antibody, such as from a transfectoma, antibodies isolated from a recombinant, combinatorial library of human antibodies, and antibodies produced, expressed, created, or isolated by any other methods that involve splicing all or part of the human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, however, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, somatic mutagenesis in vivo) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist in the human germline antibody repertoire in vivo.

As used herein, the term "chimeric antibody" refers to antibodies in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. In some cases, the variable regions of both the heavy and light chains correspond to the variable regions of antibodies derived from one species, with the desired specificity, affinity, and potency, while the constant regions are homologous to antibodies derived from another species (e.g., humans) to minimize the immune response in the latter species.

As used herein, the term "humanized antibody" refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies, as well as human antibodies. Such antibodies are a type of chimeric antibody that contains a minimal sequence derived from a non-human immunoglobulin. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to loops of a non-human immunoglobulin, and all or substantially all of the framework regions (FRs) are portions of a human immunoglobulin sequence. A humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. A humanized antibody can be further modified by substituting residues in either the Fv framework region and/or the substituted non-human residues to refine and optimize the specificity, affinity, and/or activity of the antibody.

The term "Fc region," as used herein, refers to a polypeptide comprising CH3, CH2, and at least a portion of the hinge region of an antibody constant domain. Optionally, the Fc region may include the CH4 domain present in some classes of antibodies. The Fc region may include the entire hinge region of the antibody constant domain. In some embodiments, an antibody or antigen-binding fragment comprises an Fc region and a CH1 region of an antibody. In some embodiments, an antibody or antigen-binding fragment comprises an Fc region and a CH3 region of an antibody. In some embodiments, an antibody or antigen-binding fragment comprises an Fc region, a CH1 region, and a kappa/lambda region from an antibody constant domain. In some embodiments, an antibody or antigen-binding fragment comprises a constant region, such as a heavy chain constant region and/or a light chain constant region. In some embodiments, such a constant region is modified compared to a wild-type constant region. That is, a polypeptide may contain alterations or modifications of one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and/or the light chain constant domain (CL). Examples of modifications include additions, deletions, or substitutions of one or more amino acids in one or more domains. Such alterations may be included to optimize effector function, half-life, etc.

The term "internalization" as used herein in relation to an antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment that is capable of passing through the lipid bilayer membrane of a cell into an internal compartment (i.e., "internalizing") upon binding to the cell, preferably into a destructive compartment in the cell. For example, an internalizing anti-HER2 antibody is an antibody that is capable of entering a cell upon binding to HER2 on the cell membrane. In some embodiments, the antibody or antigen-binding fragment used in the ADCs described herein targets a cell surface antigen (e.g., BCMA, CD33, PCAD, or HER2) and is an internalizing antibody or internalizing antigen-binding fragment (i.e., the ADC passes through the cell membrane upon binding the antigen). In some embodiments, the internalizing antibody or antigen-binding fragment binds to a receptor on the cell surface. An internalizing antibody or internalizing antigen-binding fragment that targets a receptor on the cell membrane can induce receptor-mediated endocytosis. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment is introduced into the cell via receptor-mediated endocytosis.

The term "non-internalizing," as used herein in relation to an antibody or antigen-binding fragment, refers to an antibody or antigen-binding fragment that remains on the cell surface after binding to the cell. In some embodiments, the antibody or antigen-binding fragment used in the ADCs described herein targets a cell surface antigen and is a non-internalizing antibody or non-internalizing antigen-binding fragment (i.e., the ADC remains on the cell surface and is not transported across the cell membrane after binding the antigen). In some embodiments, the non-internalizing antibody or antigen-binding fragment binds a non-internalizing receptor or other cell surface antigen. Examples of non-internalizing cell surface antigens include, but are not limited to, CA125 and CEA, and antibodies that bind to non-internalizing antigen targets are also known in the art (see, e.g., Bast et al. (1981) J Clin Invest. 68(5):1331-7; Scholler and Urban (2007) Biomark Med. 1(4):513-23; and Boudousq et al. (2013) PLoS One 8(7):e69613).

The term "B-cell maturation antigen" or "BCMA" as used herein refers to any native form of human BCMA (also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17)). The term encompasses full-length human BCMA (e.g., UniProt reference sequence: Q02223; SEQ ID NO: 72) as well as any form of human BCMA that may result from cellular processing. The term also encompasses functional variants or fragments of human BCMA, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human BCMA (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer only to the wild-type protein). BCMA may be isolated from humans or may be produced recombinantly or synthetically.

The term "anti-BCMA antibody" or "antibody that binds to BCMA" as used herein refers to any form of antibody or antigen-binding fragment thereof that binds, e.g., specifically binds, to BCMA. The term encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antigen-binding fragments if they bind, e.g., specifically bind, to BCMA. WO 2012/163805 discloses and is incorporated by reference exemplary BCMA-binding sequences, including exemplary anti-BCMA antibody sequences. In some embodiments, the anti-BCMA antibody used in the ADCs described herein is an internalizing antibody or an internalizing antigen-binding fragment. J6M0 (WO 2012/163805) is an exemplary anti-BCMA antibody.

The term "myeloid cell surface antigen CD33" or "CD33" as used herein refers to any native form of human CD33 (also known as sialic acid-binding Ig-like lectin 3 (SIGLEC3)). The term includes full-length human CD33 (e.g., UniProt reference sequence: P20138; SEQ ID NO: 73), as well as any form of human CD33 that may result from cellular processing. The term also includes functional variants or fragments of human CD33, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human CD33 (i.e., variants and fragments are included unless the context indicates that the term is used to refer only to the wild-type protein). CD33 may be isolated from humans or may be produced recombinantly or synthetically.

The term "anti-CD33 antibody" or "antibody that binds to CD33" as used herein refers to any form of antibody or antigen-binding fragment thereof that binds, for example, specifically binds, to CD33. The term encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antigen-binding fragments if they bind, for example, specifically bind, to CD33. US 2013/0078241 discloses and is incorporated by reference herein exemplary CD33-binding sequences, including exemplary anti-CD33 antibody sequences. In some embodiments, the anti-CD33 antibody used in the ADCs described herein is an internalizing antibody or an internalizing antigen-binding fragment. MuMy9-6ch (US 2013/0078241) is an exemplary anti-CD33 antibody.

The term "P-cadherin" or "PCAD" as used herein refers to any native form of human PCAD (also known as type 1 cadherin 3 or CDH3). The term encompasses full-length human PCAD (e.g., UniProt reference sequence: P22223; SEQ ID NO: 74), as well as any form of human PCAD that may result from cellular processing. The term also encompasses functional variants or fragments of human PCAD, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human PCAD (i.e., variants and fragments are included unless the context indicates that the term is used to refer only to the wild-type protein). PCAD may be isolated from humans or may be produced recombinantly or synthetically.

The term "anti-PCAD antibody" or "antibody that binds to PCAD" as used herein refers to any form of antibody or antigen-binding fragment thereof that binds, for example, specifically binds, to PCAD. The term encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antigen-binding fragments if they bind, for example, specifically bind, to PCAD. WO 2016/203432 discloses, and is incorporated by reference herein, exemplary PCAD-binding sequences, including exemplary anti-PCAD antibody sequences. In some embodiments, the anti-PCAD antibody used in the ADCs described herein is an internalizing antibody or an internalizing antigen-binding fragment. NOV169N31Q (WO 2016/203432) is an exemplary anti-PCAD antibody.

As used herein, the term "human epidermal growth factor receptor 2," "HER2," or "HER2/NEU" refers to any native form of human HER2. The term includes full-length human HER2 (e.g., UniProt reference sequence: P04626; SEQ ID NO: 75), as well as any form of human HER2 that may result from cellular processing. The term also includes functional variants or fragments of human HER2, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human HER2 (i.e., variants and fragments are included unless the context indicates that the term is used to refer only to the wild-type protein). HER2 may be isolated from humans or may be produced recombinantly or synthetically.

The term "anti-HER2 antibody" or "antibody that binds to HER2" as used herein refers to any form of antibody or antigen-binding fragment thereof that binds, for example, specifically binds, to HER2. The term encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antigen-binding fragments if they bind, for example, specifically bind, to HER2. U.S. Patent Nos. 5,821,337 and 6,870,034 disclose, and are incorporated herein by reference, exemplary HER2-binding sequences, including exemplary anti-HER2 antibody sequences. In some embodiments, the anti-HER2 antibody used in the ADCs described herein is an internalizing antibody or an internalizing antigen-binding fragment. Trastuzumab (U.S. Patent Nos. 5,821,337 and 6,870,034; see also Molina et al. (2001) Cancer Res. 61(12):4744-9) is a typical anti-HER2 antibody.

The term "cluster of differentiation 38" or "CD38" as used herein refers to any native form of human CD38 (also known as ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase). The term includes full-length human CD38 (e.g., UniProt reference sequence: P28907; SEQ ID NO: 76), as well as any form of human CD38 that may result from cellular processing. The term also encompasses functional variants or fragments of human CD38, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human CD38 (i.e., variants and fragments are included unless the context indicates that the term is used to refer only to the wild-type protein). CD38 may be isolated from humans or may be produced recombinantly or synthetically.

The term "cluster of differentiation 48" or "CD48" as used herein refers to any native form of human CD48 (also known as B-lymphocyte activation marker 1 (BLAST-1) or signaling lymphocyte activation molecule 2 (SLAMF2)). The term includes full-length human CD48 (e.g., UniProt reference sequence: P09326; SEQ ID NO: 77), as well as any form of human CD48 that may result from cellular processing. The term also includes functional variants or fragments of human CD48, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human CD48 (i.e., variants and fragments are included unless the context indicates that the term is used to refer only to the wild-type protein). CD48 may be isolated from humans or may be produced recombinantly or synthetically.

The term "cluster of differentiation 79b" or "CD79b" as used herein refers to any native form of human CD79b (also known as the beta chain of B-cell antigen receptor complex-associated protein). The term includes full-length human CD79b (e.g., UniProt reference sequence: P40259; SEQ ID NO: 78), as well as any form of human CD79b that may result from cellular processing. The term also encompasses functional variants or fragments of human CD79b, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human CD79b (i.e., variants and fragments are included unless the context indicates that the term is used to refer only to the wild-type protein). CD79b may be isolated from humans or may be produced recombinantly or synthetically.

As used herein, the term "binding specificity" refers to the ability of an individual antibody or antigen-binding fragment to preferentially react with one antigenic determinant over another antigenic determinant. The degree of specificity indicates the extent to which an antibody or fragment preferentially binds to one antigenic determinant over another antigenic determinant. Furthermore, as used herein, the terms "specific,""specificallybinds," and "binds specifically" refer to the binding reaction between an antibody or antigen-binding fragment (e.g., an anti-HER2 antibody) and a target antigen (e.g., HER2) in a heterogeneous population of proteins and other biologics. Antibodies can be tested for binding specificity by comparing binding to a relevant antigen with binding to an irrelevant antigen or mixture of antigens under a given set of conditions. If an antibody binds to a relevant antigen with at least 2, 5, 7, 10, or more times greater affinity than to an irrelevant antigen or mixture of antigens, it is considered specific. A "specific antibody" or "target-specific antibody" is an antibody that binds only to the target antigen (e.g., BCMA, CD33, PCAD, or HER2) but does not bind (or shows minimal binding) to other antigens. In some embodiments, an antibody or antigen-binding fragment that specifically binds a target antigen (e.g., BCMA, CD33, PCAD, or HER2) has a K _D of less than 1 x 10 ^-6 M, less than 1 x 10 ^-7 M, less than 1 x 10 ^-8 M, less than 1 x 10 ^-9 M, less than 1 x 10 ^-10 M, less than 1 x 10 ^-11 M, less than 1 x 10 ^-12 M, or less than 1 x 10 ^-13 M. In some embodiments, the K _D is from 1 to 500 pM. In some embodiments, the K _D is from 500 pM to 1 μM, from 1 μM to 100 nM, or from 100 mM to 10 nM.

As used herein, the term "affinity" refers to the strength of interaction between an antibody and an antigen at individual antigenic sites. Without being bound by theory, within each antigen-binding site, the variable region of the antibody's "arm" interacts with the antigen at multiple sites through weak non-covalent forces; the more interactions, the stronger the affinity. Antibody binding affinity is the sum of the attractive and repulsive forces acting between the antigenic determinant and the antigen-binding site.

The term "k _on " or "k _a " refers to the rate constant of antibody-antigen association to form an antibody/antigen complex. The rate can be determined using standard assays such as surface plasmon resonance, biolayer inferometry, or ELISA.

The term "k _off " or "k _d " refers to the rate constant for the dissociation of an antibody from the antibody/antigen complex. The rate can be determined using standard assays such as surface plasmon resonance, biolayer inferometry, or ELISA.

The term "K _D " refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. K _D is calculated as k _a /k _d . The rate can be determined using standard assays such as surface plasmon resonance, biolayer inferometry, or ELISA.

The term "epitope" refers to the portion of an antigen that can be recognized and specifically bound by an antibody (or antigen-binding fragment). Epitope determinants typically consist of chemically active surface groups of molecules, such as amino acids or the side chains of carbohydrates or sugars, and may have specific three-dimensional structural characteristics as well as specific charge characteristics. When the antigen is a polypeptide, epitopes can be formed from contiguous amino acids or non-contiguous amino acids superimposed by tertiary folding of the polypeptide. An epitope can be "linear" or "conformational." Conformational and linear epitopes differ in that binding to the former, but not to the latter, is lost in the presence of denaturing solvents. An epitope associated with an antibody (or antigen-binding fragment) can be identified using any epitope mapping method known in the art, including X-ray crystallography to identify the epitope by direct visualization of the antigen-antibody complex, as well as monitoring the binding of the antibody to fragments or mutated variants of the antigen, or monitoring the solvent accessibility of different parts of the antibody and antigen. Examples of strategies used for antibody epitope mapping include, but are not limited to, array-based oligopeptide scanning, limited proteolysis, site-directed mutagenesis, high-throughput mutagenesis mapping, hydrogen-deuterium exchange, and mass spectrometry (see, e.g., Gershoni et al. (2007) BioDrugs 21:145-56; and Hager-Braun and Tomer (2005) Expert Rev Proteomics 2:745-56).

Competitive binding and epitope binding can also be used to identify antibodies with identical or overlapping epitopes. Competitive binding can be assessed using a cross-blocking assay, such as the one described in "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory, Harlow and Lane ( ^1st edition 1988, ^2nd edition 2014). In some embodiments, competitive binding is identified when a test antibody or binding protein reduces the binding of a reference antibody or binding protein to a target antigen, such as BCMA, CD33, PCAD, or HER2 (e.g., a binding protein comprising CDRs and/or variable domains selected from the domains identified in Tables 3-5), by at least about 50% in a cross-blocking assay (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or more, or any other percentage in between), and/or vice versa. In some embodiments, competitive binding may occur due to common or similar (e.g., partially overlapping) epitopes, or due to steric hindrance when antibodies or binding proteins bind to adjacent epitopes (see, e.g., Tzartos, Methods in Molecular Biology (Morris, ed. (1998) vol. 66, pp. 55-66)). In some embodiments, competitive binding can be used to sort groups of binding proteins that have similar epitopes. For example, binding proteins that compete for binding can be "pooled" as a group of binding proteins that have overlapping or adjacent epitopes, while those that do not compete are placed in a separate group of binding proteins that do not have overlapping or adjacent epitopes.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms encompass amino acid polymers comprising two or more amino acids linked to each other by peptide bonds, amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The terms include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins, among others. The terms also include naturally occurring peptides, recombinant peptides, synthetic peptides, or combinations thereof. Unless otherwise specified, a particular polypeptide sequence also implicitly includes conservatively modified variants thereof.

A "recombinant" protein refers to a protein (e.g., an antibody) produced using recombinant techniques, such as through the expression of recombinant nucleic acid.

An "isolated" protein refers to a protein that is not accompanied by at least some of the material with which it is normally associated in its natural state. For example, a naturally occurring polynucleotide or polypeptide present in a living organism is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the living organism is isolated. This definition includes the production of an antibody in a variety of organisms and/or host cells known in the art.

An "isolated antibody," as used herein, is an antibody that has been identified and separated from one or more (e.g., a majority) of the components (by mass) of its original medium, such as components of a hybridoma cell culture or other cell culture used to produce it. In some embodiments, the separation is performed in a manner that sufficiently removes components that might otherwise affect the suitability of the antibody for desired applications (e.g., therapeutic use). Methods for producing isolated antibodies are known in the art and include, but are not limited to, protein A chromatography, anion exchange chromatography, cation exchange chromatography, virus retention filtration, and ultrafiltration.

As used herein, the term "variant" refers to a nucleic acid sequence or amino acid sequence that differs from a reference nucleic acid sequence or amino acid sequence, respectively, but retains one or more biological properties of the reference sequence. A variant may comprise one or more amino acid substitutions, deletions, and/or insertions (or corresponding codon substitutions, deletions, and/or insertions) relative to the reference sequence. Changes in a nucleic acid variant may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid sequence, or may result in amino acid substitutions, additions, deletions, fusions, and/or truncations. In some embodiments, a nucleic acid variant described herein encodes an amino acid sequence identical to the sequence encoded by an unmodified nucleic acid, or encodes a modified amino acid sequence that retains one or more functional properties of the unmodified amino acid sequence. Changes in the sequence of peptide variants are typically limited or conservative, such that the sequences of the unmodified peptide and the variant are generally very similar and identical in many regions. In some embodiments, the peptide variant retains one or more functional properties of the unmodified peptide sequence. The variant and unmodified peptide may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination.

A nucleic acid or peptide variant may be a naturally occurring variant or a variant not known to occur naturally. Nucleic acid and peptide variants can be produced by mutagenesis, direct synthesis, or other methods known in the art. A variant does not necessarily require physical manipulation of the reference sequence. If the sequence contains a nucleic acid or amino acid different from the reference sequence, it is considered a "variant," regardless of how it was synthesized. In some embodiments, a variant has high sequence identity (i.e., 60% or higher nucleic acid or amino acid sequence identity) compared to the reference sequence. In some embodiments, a peptide variant includes polypeptides containing amino acid substitutions, deletions, and/or insertions, so long as the polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity to a reference sequence or to a corresponding segment (e.g., a functional fragment) of a reference sequence, such as those variants that also retain one or more functions of the reference sequence. In some embodiments, a nucleic acid variant comprises polynucleotides containing amino acid substitutions, deletions, and/or insertions, provided that the polynucleotide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% nucleic acid sequence identity to a reference sequence or to a corresponding segment (e.g., a functional fragment) of a reference sequence.

The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. For nucleic acid sequences, conservatively modified variants refer to those nucleic acids that encode identical or substantially identical amino acid sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at each position where alanine is specified by a codon, that codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variation. Each nucleic acid sequence herein that encodes a polypeptide also describes all possible silent variations of the nucleic acid. One skilled in the art will understand that every codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to form a functionally identical molecule. Accordingly, every silent variation of a nucleic acid encoding a polypeptide is implicitly present in each described sequence. For polypeptide sequences, conservatively modified variants include individual substitutions, deletions, or additions to the polypeptide sequence that result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitutions that provide functionally similar amino acids are well known in the art.

The term "conservative sequence modifications" as used herein refers to amino acid modifications that do not significantly affect or alter the binding characteristics of, for example, an antibody or antigen-binding fragment comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into an antibody or antigen-binding fragment using standard methods known in the art, such as, for example, site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in some embodiments, one or more amino acid residues in an antibody can be replaced with other amino acid residues from the same side chain family, and the altered antibody can be tested using the functional assays described herein.

As used herein, the term "homologous" or "identity" refers to the identity of a subunit sequence between two polymer molecules, such as between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; for example, if a position in each of two DNA molecules is occupied by an adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions. For example, if half (e.g., five positions in a ten-subunit polymer) of the positions in two sequences are matching or homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matching or homologous, the two sequences are 90% homologous.

The "sequence identity" fraction can be determined by comparing two optimally aligned sequences within a comparison window, where the amino acid sequence fragment within the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The fraction can be calculated by determining the number of positions at which an identical amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity fraction. The result is the sequence identity fraction of the subject sequence relative to the query sequence. The sequence identity fraction between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that must be introduced for optimal alignment of the two sequences. Typically, the amino acid identity or homology between the proteins described herein and variants thereof, including variants of target antigens (such as BCMA, CD33, PCAD, or HER2) and variants of antibody variable domains (including individual CDR variants), is at least 80% of the sequences depicted herein, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, nearly 100%, or 100% identity or homology.

Comparison of sequences and determination of the proportion of identity between two sequences can be performed using a mathematical algorithm. In some embodiments, the proportion of identity between two amino acid sequences is determined using the Needleman and Wunsch algorithm ((1970) J Mol Biol. 48:444-53), which is included in the GAP program in the GCG software package, using either the Blossum 62 matrix or the PAM250 matrix with a gap mass of 16, 14, 12, 10, 8, 6, or 4 and a length mass of 1, 2, 3, 4, 5, or 6. In some embodiments, the proportion of identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix and a gap mass of 40, 50, 60, 70, or 80 and a length mass of 1, 2, 3, 4, 5, or 6. A typical set of parameters is the Blossum 62 scoring matrix with a gap penalty 12, a gap extension penalty of 4, and a gap frameshift penalty of 5. The proportion of identity between two amino acid or nucleotide sequences can also be determined using the Meyers and Miller algorithm ((1989) CABIOS 4:11–17), which has been incorporated into the ALIGN program (version 2.0) using the PAM120 mass residue table, a gap extension penalty of 12, and a gap frameshift penalty of 4.

The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, an extract obtained from biological materials, or a combination of two or more of these. The term "therapeutic agent" or "drug" refers to an agent capable of modulating a biological process and/or possessing biological activity. Mcl-1 inhibitors and ADCs containing them, as described herein, are exemplary therapeutic agents.

The term "chemotherapeutic agent" or "anticancer agent" is used herein to refer to all agents that are effective in the treatment of cancer (regardless of the mechanism of action). Inhibition of metastasis or angiogenesis is often a property of a chemotherapeutic agent. Chemotherapeutic agents include antibodies, biomolecules, and small molecules, and encompass Mcl-1 inhibitors and ADCs containing them, as described herein. A chemotherapeutic agent can be a cytotoxic or cytostatic agent. The term "cytostatic agent" refers to an agent that inhibits or suppresses cell growth and/or cell proliferation. The term "cytotoxic agent" refers to a substance that causes cell death, primarily by interfering with the expression activity and/or function of the cell.

The term "myeloid cell leukemia 1" or "Mcl-1" as used herein refers to any native form of human Mcl-1, an anti-apoptotic member of the Bcl-2 protein family. The term encompasses full-length human Mcl-1 (e.g., UniProt reference sequence: Q07820; SEQ ID NO: 63), as well as any form of human Mcl-1 that may result from cellular processing. The term also encompasses functional variants or fragments of human Mcl-1, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human Mcl-1 (i.e., variants and fragments are included unless the context indicates that the term is used only to refer to the wild-type protein). Mcl-1 may be isolated from humans or may be produced recombinantly or synthetically.

The term "inhibit," "inhibition," or "inhibiting" as used herein means a reduction in a biological activity or process by a measurable amount and may include, but does not require, complete prevention or inhibition. In some embodiments, "inhibition" means a reduction in the expression and/or activity of Mcl-1 and/or one or more upstream modulators or their downstream targets.

The term "Mcl-1 inhibitor" as used herein refers to an agent capable of reducing the expression and/or activity of Mcl-1 and/or one or more upstream modulators or downstream targets thereof. Exemplary Mcl-1 modulators (including exemplary Mcl-1 inhibitors) are described in WO 2015/097123; WO 2016/207216; WO 2016/207217; WO 2016/207225; WO 2016/207226; WO 2017/125224; WO 2019/035899, WO 2019/035911, WO 2019/035914, WO 2019/035927, US 2019/0055264, WO 2016/033486, WO 2017/147410, WO 2018/183418 and WO 2017/182625, each of which is incorporated herein by reference as exemplary Mcl-1 modulators, including exemplary Mcl-1 inhibitors, that can be included as drug components in the disclosed ADCs. For example, exemplary Mcl-1 inhibitors that can be included as drug moieties in the disclosed ADCs have the formula:

where each variable is defined as in WO2019/035911; WO 2019/035899; WO 2019/035914; or WO 2019/035927. Specific examples include, for example,

(P15), wherein each compound as a drug payload can be conjugated to an antibody or a linker via the N-methyl nitrogen atom in the piperazinyl moiety of the compound. As used herein, the terms "derivative" and "analog" when referring to an Mcl-1 inhibitor or the like mean any such compound that retains substantially the same, similar, or enhanced biological function or activity compared to the parent compound, but has an altered chemical or biological structure.

As used herein, "Mcl-1 inhibitor drug moiety," "Mcl-1 inhibitor," and the like refer to a component of an ADC or composition that provides a structure of an Mcl-1 inhibitor compound or a compound modified to attach to an ADC that retains substantially the same, similar, or enhanced biological function or activity as compared to the parent compound. In some embodiments, the Mcl-1 inhibitor drug moiety is component (D) in an ADC of formula (1).

As used herein, the term "cancer" refers to the presence of cells exhibiting characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and/or certain morphological features. Cancer cells can often be in the form of a tumor or mass, but such cells can exist separately within a subject or circulate in the bloodstream as independent cells, such as leukemia or lymphoma cells. The term "cancer" includes all types of cancer and cancer metastases, including hematologic malignancies, solid tumors, sarcomas, carcinomas, and other types of solid and non-solid tumors. Hematologic cancers can include B-cell malignancies, blood cancers (leukemias), plasma cell cancers (myelomas, such as multiple myeloma), or lymph node cancers (lymphomas). Common B-cell malignancies include chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma. Leukemias may include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), acute monocytic leukemia (AMoL), etc. Lymphomas may include Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc. Other hematological cancers may include myelodysplasia syndrome (MDS). Solid tumors may include carcinomas such as adenocarcinoma, for example, breast cancer, pancreatic cancer, prostate cancer, colon or colorectal cancer, lung cancer, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, cholangiocarcinoma, glioma, melanoma, etc. In some embodiments, the cancer is breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, cancer ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer is lymphoma or gastric cancer.

As used herein, the term "tumor" refers to any tissue mass that results from excessive growth or proliferation of cells, benign or malignant, including precancerous lesions. In some embodiments, the tumor is breast cancer, gastric cancer, bladder cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer, hepatocellular carcinoma, melanoma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the tumor is gastric cancer.

The terms "tumor cell" and "cancer cell" may be used interchangeably herein and refer to individual cells or the entire cell population derived from a tumor or cancer, including both non-tumorigenic cells and cancer stem cells. The terms "tumor cell" and "cancer cell" will be modified by the term "non-tumorigenic" if they refer exclusively to cells that lack the capacity to renew and differentiate, to distinguish these cells from cancer stem cells.

The term "target negative," "target antigen negative," or "antigen negative" as used herein refers to the absence of target antigen expression by a cell or tissue. The term "target positive," "target antigen positive," or "antigen positive" refers to the presence of target antigen expression. For example, a cell or cell line that does not express a target antigen can be described as target negative, while a cell or cell line that does express a target antigen can be described as target positive.

The terms "subject" and "patient" are used interchangeably herein to refer to any human or non-human animal in need of treatment. Non-human animals include all vertebrates (e.g., mammals and non-mammals), such as any mammal. Non-limiting examples of mammals include humans, chimpanzees, great apes, monkeys, cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, and guinea pigs. Non-limiting examples of non-mammals include birds and fish. In some embodiments, the subject is a human.

The term "subject in need of treatment" as used herein refers to a subject who would benefit from treatment (e.g., treatment with one or more exemplary ADC compounds described herein) in a biological, medical, or quality of life sense.

As used herein, the term "treat," "treatment," or "treatment" refers to any improvement in any consequence of a disease, disorder, or condition, such as prolonged survival, reduced morbidity, and/or reduced side effects from an alternative therapeutic method. In some embodiments, treating comprises delaying or alleviating the disease, disorder, or condition (i.e., slowing, stopping, or reducing the progression of the disease or at least one of its clinical symptoms). In some embodiments, treating comprises delaying, alleviating, or improving at least one physical parameter of the disease, disorder, or condition, including those that may not be perceptible to the patient. In some embodiments, treating comprises modulating the disease, disorder, or condition either physically (e.g., stabilizing a significant symptom), physiologically (e.g., stabilizing a physical parameter), or both. In some embodiments, treating comprises administering a disclosed compound or ADC composition to a subject, such as a patient, to obtain the treatment benefits listed herein. Treatment may include treating, curing, alleviating, delaying, preventing, alleviating, modifying, curing, ameliorating, ameliorating, improving, or influencing a disease, disorder, or condition (e.g., cancer), the symptoms of a disease, disorder, or condition (e.g., cancer), or a predisposition to a disease, disorder, or condition (e.g., cancer). In some embodiments, in addition to treating a subject suffering from a disease, disorder, or condition, the composition described herein may also be administered prophylactically to prevent or reduce the likelihood of developing the disease, disorder, or condition.

As used herein, the term "prevent," "prevention," or "prophylaxis" of a disease, disorder, or condition refers to the prophylactic treatment of a disease, disorder, or condition; or to the delay of the onset or progression of a disease, disorder, or condition.

As used herein, the term "pharmaceutical composition" refers to a formulation of the composition, such as a compound or ADC composition, in addition to at least one other (and optionally more than one other) component suitable for administration to a subject, such as a pharmaceutically acceptable carrier, stabilizer, diluent, dispersing agent, suspending agent, thickening agent, and/or excipient. The pharmaceutical compositions provided herein are in a form that allows administration and subsequently provides the desired biological activity of the active ingredient(s) and/or to achieve a therapeutic effect. The pharmaceutical compositions provided herein preferably do not contain additional components that are unacceptably toxic to the subject to which the preparation is to be administered.

As used herein, the terms "pharmaceutically acceptable carrier" and "physiologically acceptable carrier," which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to the subject and does not interfere with the biological activity and properties of the administered ADC compound or composition and/or any additional therapeutic agent in the composition. Pharmaceutically acceptable carriers may enhance or stabilize the composition or may be used to facilitate formulation. Pharmaceutically acceptable carriers may include solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrating agents, lubricants, sweeteners, flavoring agents, coloring agents, and the like and combinations thereof known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except where any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated. The carrier may be selected to minimize adverse side effects in the subject and/or to minimize degradation of the active ingredient(s). Any of these formulations may also include an adjuvant.

As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of the active ingredient. Formulations for parenteral administration may, for example, contain excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, or hydrogenated naphthalenes. Other typical excipients include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, ethylene-vinyl acetate copolymer particles, and surfactants, including, for example, polysorbate 20.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt that does not abolish the biological activity and properties of the compounds of the invention and does not cause significant irritation to the subject to which it is administered. Examples of such salts include, but are not limited to: (a) acid addition salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like; and salts formed with organic acids, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, polygalacturonic acid and the like; and (b) salts formed from elemental anions such as chlorine, bromine and iodine. See, for example, Haynes et al., “Commentary: Occurrence of Pharmaceutically Acceptable Anions and Cations in the Cambridge Structural Database,” J. Pharmaceutical Sciences, vol. 94, no. 10 (2005), and Berge et al., “Pharmaceutical Salts,” J. Pharmaceutical Sciences, vol. 66, no. 1 (1977), which are incorporated herein by reference.

In some embodiments, depending on their electronic charge, the antibody-drug conjugates (ADCs), linkers, payloads, and linker-payloads described herein may comprise a monovalent anionic counterion _M1- ^. Any suitable anionic counterion can be used. In some embodiments, the monovalent anionic counterion is a pharmaceutically acceptable monovalent anionic counterion. In some embodiments, the monovalent anionic counterion _M1- can be selected from bromide, chloride, ^iodide , acetate, trifluoroacetate, benzoate, mesylate, tosylate, triflate, formate, or the like. In some embodiments, _the monovalent anionic counterion ^M1- is trifluoroacetate or formate.

As used herein, the term "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a compound described herein, such as an ADC compound or composition described herein, to achieve the desired therapeutic result (i.e., inhibition of enzyme or protein activity, improvement of symptoms, alleviation of symptoms or conditions, delay in disease progression, reduction in tumor size, inhibition of tumor growth, prevention of metastasis). In some embodiments, the therapeutically effective amount does not cause unwanted side effects. In some embodiments, the therapeutically effective amount causes or causes side effects, but only those that are acceptable to the treating physician given the patient's condition. In some embodiments, the therapeutically effective amount is effective for the measurable destruction, reduction, and/or inhibition of the growth or spread of cancer cells, the size or number of tumors, and/or another measure of the level, stage, progression, and/or severity of cancer. This term also applies to a dose that will cause a specific response in target cells, such as a reduction, slowing, or inhibition of cell growth. A therapeutically effective amount can be determined by initially administering a low dose and then gradually increasing this dose until the desired effect is achieved. A therapeutically effective amount can also vary depending on the intended use (in vitro or in vivo) or the subject and the disease state being treated, such as the subject's body weight and age, the severity of the disease state, the route of administration, and the like, which can be easily determined by one of skill in the art. The specific amount can vary depending on, for example, a particular pharmaceutical composition, the subject and their age, as well as the existing health condition or risk to the health condition, the dosing regimen to be followed, the severity of the disease, whether administered in combination with other agents, the time of administration, the tissue into which they are administered, and the physical delivery system in which they are carried. In the case of cancer, a therapeutically effective amount of an ADC can reduce the number of cancer cells, reduce the size of a tumor, inhibit (e.g., slow or stop) tumor metastasis, inhibit (e.g., slow or stop) tumor growth, and/or alleviate one or more symptoms.

As used herein, the term "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a compound described herein, such as an ADC compound or composition described herein, that is effective at dosages and for periods of time necessary to achieve the desired prophylactic result. Typically, since the prophylactic dose is used in subjects before or at an earlier stage of a disease, the prophylactically effective amount will be less than the therapeutically effective amount. In some embodiments, the prophylactically effective amount can prevent the onset of disease symptoms, including cancer-related symptoms.

The term " p " or "drug load" or "drug:antibody ratio" or "drug-to-antibody ratio" or "DAR" refers to the number of drug moieties per antibody or antigen-binding fragment, i.e., the drug load or the number of -LD groups per antibody or antigen-binding fragment (Ab) in an ADC of Formula (1). In ADCs containing an Mcl-1 inhibitor drug moiety, " p " refers to the number of Mcl-1 inhibitor compounds linked to the antibody or antigen-binding fragment. For example, if two Mcl-1 inhibitor compounds are linked to the antibody or antigen-binding fragment, p = 2. In compositions containing multiple copies of an ADC of Formula (1), the "average p " refers to the average number of -LD groups per antibody or antigen-binding fragment, and is also referred to as the "average drug load."

Antibody-drug conjugates

Antibody-drug conjugate (ADC) compounds disclosed herein include compounds with anticancer activity. In particular, the ADC compounds comprise an antibody or antigen-binding fragment conjugated (i.e., covalently attached by a linker) to a drug moiety (e.g., an Mcl-1 inhibitor), wherein the drug moiety, when not conjugated to the antibody or antigen-binding fragment, has a cytotoxic or cytostatic effect. In some embodiments, the drug moiety, when not conjugated to the antibody or antigen-binding fragment, is capable of reducing the expression and/or activity of Mcl-1 and/or one or more upstream modulators or their downstream targets. Without being bound by theory, by targeting the expression and/or activity of Mcl-1 in some embodiments, the ADCs described herein may be potent anticancer agents. Furthermore, without being limited by theory, by conjugating a drug moiety to an antibody that binds an antigen associated with expression in a tumor cell or cancer, an ADC may provide improved activity, better cytotoxic specificity, and/or reduced off-target killing compared to the drug when administered alone.

In some embodiments, therefore, the ADC components are selected to (i) preserve one or more therapeutic properties exhibited by the antibody and drug moieties upon isolation, (ii) maintain the specific binding properties of the antibody or antigen-binding fragment; (iii) optimize drug payload and drug-to-antibody ratio; (iv) provide delivery, e.g., intracellular delivery, of the drug moiety through stable attachment to the antibody or antigen-binding fragment; (v) maintain the stability of the ADC as an intact conjugate until transport or delivery to the target site; (vi) minimize aggregation of the ADC before or after administration; (vii) exhibit a therapeutic effect, e.g., a cytotoxic effect, of the drug moiety upon cleavage or other release mechanism in the cellular environment; (viii) exhibit in vivo anti-cancer therapeutic efficacy comparable to or superior to that of the antibody and drug moieties upon isolation; (ix) minimize off-target killing by the drug moiety; and/or (x) exhibiting desired pharmacokinetic and pharmacodynamic properties, formability, and toxicological/immunological profiles. Each of these properties may yield an improved ADC for therapeutic use (Ab et al. (2015) Mol Cancer Ther. 14:1605-13).

The ADC compounds of the present invention can selectively deliver an effective dose of a cytotoxic or cytostatic agent to cancer cells or tumor tissue. In some embodiments, the cytotoxic and/or cytostatic activity of the ADC depends on the expression of the target antigen in the cell. In some embodiments, the disclosed ADCs are particularly effective in killing cancer cells expressing the target antigen, with minimal off-target killing. In some embodiments, the disclosed ADCs do not exhibit cytotoxic and/or cytostatic effects on cancer cells that do not express the target antigen.

Examples of cancers expressing BCMA include, but are not limited to, multiple myeloma (Cho et al. (2018) Front Immunol. 9:1821).

Examples of cancers expressing CD33 include, but are not limited to, colorectal cancer, pancreatic cancer, lymphoma, and leukemia (e.g., acute myeloid leukemia) (Human Protein Atlas; Walter (2014) Expert Opin Ther Targets 18(7):715-8).

Examples of cancers expressing PCAD include, but are not limited to, breast cancer, gastric cancer, endometrial cancer, ovarian cancer, pancreatic cancer, bladder cancer, prostate cancer, and melanoma (Vieira and Paredes (2015) Mol Cancer 14:178).

Examples of cancers that express HER2 include, but are not limited to, breast cancer, gastric cancer, bladder cancer, urothelial cell carcinoma, esophageal cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., endometrial serous carcinoma), salivary duct cancer, cervical cancer, endometrial cancer, and ovarian cancer (English et al. (2013) Mol Diagn Ther. 17:85-99).

In some aspects, provided herein are ADC compounds comprising an antibody or antigen-binding fragment thereof (Ab), an Mcl-1 inhibitor drug moiety (D), and a linker group (L) that covalently attaches the Ab to D. In some embodiments, provided is an antibody or antigen-binding fragment that is capable of binding to a tumor-associated antigen (e.g., BCMA, CD33, PCAD, or HER2), for example, with high specificity and high affinity. In some embodiments, the antibody or antigen-binding fragment is internalized into a target cell upon binding, for example, in a degradation compartment within the cell. In some embodiments, the ADC is internalized upon binding to a target cell, undergoes degradation, and releases the Mcl-1 inhibitor drug moiety to kill cancer cells. The Mcl-1 inhibitor drug moiety may be released from the antibody and/or the ADC linker moiety by enzymatic action, hydrolysis, oxidation, or any other mechanism.

A typical ADC has formula (1):

Ab-(LD) p (1)

where Ab=antibody or antigen-binding fragment, L=linker group, D=Mcl-1 inhibitor drug moiety, and p =number of Mcl-1 inhibitor drug moieties per antibody or antigen-binding fragment.

Antibodies

An antibody or antigen-binding fragment (Ab) of formula (1) includes any antibody or antigen-binding fragment that specifically binds to a target antigen on a cell. In some embodiments, an antibody or antigen-binding fragment (Ab) of formula (1) includes within its scope any antibody or antigen-binding fragment that specifically binds to a target antigen on a cancer cell. An antibody or antigen-binding fragment thereof can bind to a target antigen with a dissociation constant (K _D ) of ≤1 mM, ≤100 nM, or ≤10 nM, or any amount in between, as determined, for example, by a BIAcore® assay. In some embodiments, the K _D is from 1 pM to 500 pM. In some embodiments, the K _D is from 500 pM to 1 μM, from 1 μM to 100 nM, or from 100 mM to 10 nM.

In some embodiments, the antibody or antigen-binding fragment is a four-chain antibody (also referred to as an immunoglobulin, full-length, or intact antibody) comprising two heavy chains and two light chains. In some embodiments, the antibody or antigen-binding fragment is an antigen-binding fragment of an immunoglobulin. In some embodiments, the antibody or antigen-binding fragment is an antigen-binding fragment of an immunoglobulin that retains the ability to bind a target cancer antigen and/or provide at least one immunoglobulin function.

In some embodiments, the antibody or antigen-binding fragment is an internalizing antibody or an internalizing antigen-binding fragment thereof. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment thereof binds to a cancer target antigen expressed on the cell surface and enters the cell after binding. In some embodiments, the Mcl-1 inhibitor drug moiety of the ADC is released from the antibody or antigen-binding fragment of the ADC after the ADC enters and is present in a cell expressing the cancer target antigen (i.e., after the ADC has been internalized), for example, by cleavage, degradation of the antibody or antigen-binding fragment, or any other suitable release mechanism.

In some embodiments, antibodies contain mutations that mediate reduced or absent antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In some embodiments, these mutations are known as Fc silencing mutations, Fc silenced mutations, or Fc silenced mutations. In some embodiments, amino acid residues L234 and L235 of the IgG1 constant region are replaced with A234 and A235 (also known as "LALA"). In some embodiments, amino acid residue N297 of the IgG1 constant region is replaced with A297 (also known as "N297A"). In some embodiments, amino acid residues D265 and P329 of the IgG1 constant region are replaced with A265 and A329 (also known as "DAPA"). Other Fc silencing mutations of the antibody can also be used. In some embodiments, Fc silencing mutations are used in combination, such as D265A, N297A, and P329A (also known as “DANAPA”).

The amino acid sequences of exemplary antibodies of the present invention, in addition to exemplary target antigens, are presented in Tables C, D, and E.

Table 2. Antibodies presented in the examples Antibody target Antibody code Reference mAb BCMA BCMA or Ab B J6M0 CD33 CD33ch or Ab G MuMy9-6ch CD33 CD33 gemtuzumab PCAD PCAD NOV169N31Q HER2/NEU HER2 or Ab T trastuzumab CD38 CD38 daratumumab CD46 CD46 or Ab C Anti-CD46 CD48 CD48 SGN-CD48A CD79b CD79b polatuzumab

Table 3. Amino acid sequences of the variable regions of mAb mAb IgG chain SEQ ID NO Amino acid sequence J6M0 VH 1 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSS J6M0 VL 2 DIQMTQSPSSLSSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIK MuMy9-6ch VH 3 QVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFKGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGAGTTVTVSS MuMy9-6ch VL 4 NIMLTQSPSSLAVSAGEKVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLIISSVQSEDLAIYYCHQYLSSRTFGGGTKLEIK gemtuzumab VH 5 EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSS gemtuzumab VL 6 DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVK NOV169N31Q VH 7 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSQSAAWNWIRQSPSRGLEWLGRIYYRSKWYNDYALSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGEGYGREGFAIWGQGTLVTVSS NOV169N31Q VL 8 DIQMTQSPSSLSASVGDRVTITCRASQTISNTLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLSWFTFGQGTKVEIK trastuzumab VH 9 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS trastuzumab VL 10 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT daratumumab VH 11 EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS daratumumab VL 12 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEI SGN-48A VH 13 QVQLVQSGSELKKPGASVKVSCKASGYTFTDFGMNWVRQAPGQGLEWMGWINTFTGEPSYGNVFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARRHGNGNVFDSWGQGTLVTVSS SGN-48A VL 14 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSNIHWYQQKPDQSPKLLIKYTSESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSNSWPLTFGGGTKVEIK polatuzumab VH 80 EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYWIEWVRQAPGKGLEWIGEILPGGGDTNYNEIFKGRATFSADTSKNTAYLQMNSLRAEDTAVYYCTRRVPIRLDYWGQGTLVTVSS polatuzumab VL 81 DIQLTQSPSSLSSASVGDRVTITCKASQSVDYEGDSFLNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPLTFGQGTKVEIK Anti-CD46 VH 90 QVQLVQSGGGVVQPGRSLRLACAASGLTVNNYAMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGGGYFDLWGRGTLVTVSS Anti-CD46 VL 91 QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNNNRP SGVPDRFSGSKSGTSASLAI TGLQAEDEA DYYCSSYTSGTWLFG GGTKLTVL

Table 4. Amino acid sequences of mAb CDRs mAb IgG chain SEQ ID NO Amino acid sequence J6M0 HCDR1 15 GGTFSNYWMH J6M0 HCDR2 16 ATYRGHSDTYYNQKFKG J6M0 HCDR3 17 GAIYNGYDVLDN J6M0 LCDR1 18 SASQDISNYLN J6M0 LCDR2 19 YTSNLHS J6M0 LCDR3 20 QQYRKLPWT MuMy9-6ch HCDR1 21 GYTFTSYYIH MuMy9-6ch HCDR2 22 VIYPGNDDISYNQKFKG MuMy9-6ch HCDR3 23 EVRLRYFDV MuMy9-6ch LCDR1 24 KSSQSVFFSSSQKNYLA MuMy9-6ch LCDR2 25 WASTRES MuMy9-6ch LCDR3 26 HQYLSSRT gemtuzumab HCDR1 27 GYTITDSNIH gemtuzumab HCDR2 28 YIYPYNGGTDYNQKFKN gemtuzumab HCDR3 29 GNPWLAY gemtuzumab LCDR1 30 RASESLDNYGIRFLT gemtuzumab LCDR2 31 AASNQGS gemtuzumab LCDR3 32 QQTKEVPWS NOV169N31Q HCDR1 33 TCAISGDSVSSQSAAWN NOV169N31Q HCDR2 34 RIYYRSKWYNDYALSVKS NOV169N31Q HCDR3 35 GEGYGREGFAI NOV169N31Q LCDR1 36 RASQTISNTLA NOV169N31Q LCDR2 37 AASNLQS NOV169N31Q LCDR3 38 QQYLSWFT trastuzumab HCDR1 39 GFNIKDTYIH trastuzumab HCDR2 40 RIYPTNGYTRYADSVKG trastuzumab HCDR3 41 WGGDGFYAMDV trastuzumab LCDR1 42 RASQDVNTAVAW trastuzumab LCDR2 43 SASFLES trastuzumab LCDR3 44 QQHYTTPPT daratumumab HCDR1 45 GFTFNSFAMS daratumumab HCDR2 46 AISGSGGGTYYADSVKG daratumumab HCDR3 47 DKILWFGEPVFDY daratumumab LCDR1 48 RASQSVSSYLA daratumumab LCDR2 49 DASNRAT daratumumab LCDR3 50 QQRSNWPPT SGN-48A HCDR1 51 GYTFTDFGMN SGN-48A HCDR2 52 WINTFTGEPSYGNVFKG SGN-48A HCDR3 53 RHGNGNVFDS SGN-48A LCDR1 54 RASQSIGSNIH SGN-48A LCDR2 55 YTSESIS SGN-48A LCDR3 56 QQSNSWPLT polatuzumab HCDR1 82 GYTFSSYWIE polatuzumab HCDR2 83 EILPGGGDTNYNEIFKG polatuzumab HCDR3 84 RVPIRLDY polatuzumab LCDR1 85 ITCKASQSVDYEGDSFLN polatuzumab LCDR2 86 AASNLES polatuzumab LCDR3 87 QQSNEDPLT

Table 5. Amino acid sequences of full-length Ig mAb chains mAb IgG chain SEQ ID NO Amino acid sequence J6M0 Heavy chain 57 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK J6M0 Light chain 58 DIQMTQSPSSLSSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC MuMy9-6ch Heavy chain 59 QVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFKGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGAGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK MuMy9-6ch Light chain 60 NIMLTQSPSSLAVSAGEKVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLIISSVQSEDLAIYYCHQYLSSRTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC gemtuzumab Heavy chain 61 EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK gemtuzumab Light chain 62 DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVE VKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC NOV169N31Q Heavy chain 63 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSQSAAWNWIRQSPSRGLEWLGRIYYRSKWYNDYALSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGEGYGREGFAIW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK NOV169N31Q Light chain 64 DIQMTQSPSSLSASVGDRVTITCRASQTISNTLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLSWFTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC trastuzumab Heavy chain 65 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK trastuzumab Light chain 66 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC daratumumab Heavy chain 67 EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPCPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK daratumumab Light chain 68 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SGN-48A Heavy chain 69 QVQLVQSGSELKKPGASVKVSCKASGYTFTDFGMNWVRQAPGQGLEWMGWINTFTGEPSYGNVFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARRHGNGNVFDSWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SGN-48A Light chain 70 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSNIHWYQQKPDQSPKLLIKYTSESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSNSWPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Polatuzumab Heavy chain 88 EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYWIEWVRQAPGKGLEWIGEILPGGGDTNYNEIFKGRATFSADTSKNTAYLQMNSLRAEDTAVYYCTRRVPIRLDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPCDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Polatuzumab Light chain 89 DIQLTQSPSSSLSASVGDRVTITCKASQSVDYEGDSFLNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPLTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Table 6. Typical amino acid sequences of Mcl-1 and target antigen Mcl-1/antigen SEQ ID NO Amino acid sequence Mcl-1 71 MFGLKRNAVIGLNLYCGGAGLGAGSGGATRPGGRLLATEKEASARREIGGGEAGAVIGGSAGASPPSTLTPDSRRVARPPPIGAEVPDVTATPARLLFFAPTRRAAPLEEMEAPAADAIMSPEEELDGYEPEPLGKRPAVLPLLELVGESGNNTSTDGSLPSTPPPAEEEEDELY RQSLEIISRYLREQATGAKDTKPMGRSGATSRKALETLRRVGDGVQRNHETAFQGMLRKLDIKNEDDVKSLSRVMIHVFSDGVTNWGRIVTLISFGAFVAKHLKTINQESCIEPLAESITDVLVRTKRDWLVKQRGWDGFVEFFHVEDLEGGIRNVLLAFAGVAGVGAGLAYLIR BCMA 72 MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR CD33 73 MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSA APTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ PCAD 74 MGLPRGPLASLLLLQVCWLQCAASEPCRAVFREAEVTLEAGGAEQEPGQALGKVFMGCPGQEPALFSTDNDDFTVRNGETVQERRSLKERNPLKIFPSKRILR RHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKETGWLLLNKPLDREEIAKYELFGHAVSENGASVEDPMNISIIVTD QNDHKPKFTQDTFRGSVLEGVLPGTSVMQVTATDEDDAIYTYNGVVAYSIHSQEPKDPHDLMFTIHRSTGTISVISSGLDREKVPEYTLTIQATDMDGDGSTT TAVAVVEILDANDNAPMFDPQKYEAHVPENAVGHEVQRLTVTDLDAPNSPAWRATYLIMGGDDGDHFTITTHPESNQGILTTRKGLDFEAKNQHTLYVEVTNEA PFVLKLPTSTATIVVHVEDVNEAPVFVPPSKVVEVQEGIPTGEPVCVYTAEDPDKENQKISYRILRDPAGWLAMDPDSGQVTAVGTLDREDEQFVRNNIYEVM VLAMDNGSPPTTGTGTLLLTLIDVNDHGPVPEPRQITICNQSPVRQVLNITDKDLSPHTSPFQAQLTDDSDIYWTAEVNEEGDTVVLSLKKFLKQDTYDVHLSL SDHGNKEQLTVIRATVCDCHGHVETCPGPWKGGFILPVLGAVLALLFLLLVLLLLVRKKRKIKEPLLLPEDDTRDNVFYYGEEGGGEEDQDYDITQLHRGLEAR PEVVLRNDVAPTIIPTPMYRPRPANPDEIGNFIENLKAANTDPTAPPYDTLLVFDYEGSGSDAASLSSLTSSASDQDQDYDYLNEWGSRFKKLADMYGGGEDD HER2/NEU 75 MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQ RNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTL VCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHH NTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCP INCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSR LLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERL PQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGL QSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV CD38 76 MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLL GYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI CD48 77 MCSRGWDSCLALELLLLPLSLLVTSIQGHLVHMTVVSGSNVTLNISESLPENYKQLTWFYTFDQKIVEWDSRKSKYFESKFKGRVRLDPQSGALYISKVQKEDNSTYIMRVLKKTGNEQEW KIKLQVLDPVPKPVIKIEKIEDMDDNCYLKLSCVIPGESVNYTWYGDKRPFPKELQNSVLETTLMPHNYSRCYTCQVSNSVSSKNGTVCLSPPCTLARSFGVEWIASWLVVTVPTILGLLLT CD79b 78 MARLALSPVPSHWMVALLLLLSAEPVPAARSEDRYRNPKGSACSRIWQSPRFIARKRGFTVKMHCYMNSASGNVSWLWKQEMDENPQQLKLEKGRMEESQNESLATLTIQGIRF EDNGIYFCQQKCNNTSEVYQGCGTELRVMGFSTLAQLKQRNTLKDGIIMIQTLLIILFIIVPIFLLLDKDDSKAGMEEDHTYEGLDIDQTATYEDIVTLRTGEVKWSVGEHPGQE

In some embodiments, an antibody or antigen-binding fragment of an ADC described herein may comprise any set of heavy and light chain variable domains listed in the tables above, or a set of six CDRs from any set of heavy and light chain variable domains listed in the tables above. In some embodiments, an antibody or antigen-binding fragment of an ADC described herein may comprise amino acid sequences that are conservatively modified and/or homologous to the sequences listed in the tables above, so long as the ADC retains the ability to bind to its target cancer antigen (e.g., with a K _D of less than 1 x 10 ^-8 M) and retains one or more functional properties of the ADC described herein (e.g., the ability to be internalized, to bind to a target antigen, such as an antigen expressed on a tumor or other cancer cell, etc.).

In some embodiments, the antibody or antigen-binding fragment of the ADC described herein further comprises human heavy and light chain constant domains or fragments thereof. For example, the antibody or antigen-binding fragment of the disclosed ADCs may comprise a human IgG heavy chain constant domain (such as IgG1) and a human kappa or lambda chain constant domain. In some embodiments, the antibody or antigen-binding fragment of the disclosed ADCs comprises a human immunoglobulin G subtype 1 (IgG1) heavy chain constant domain with a human Ig kappa light chain constant domain.

In some embodiments, the target cancer antigen for the ADC is BCMA.

In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 15, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 16, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 17; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 18, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 19, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 20.

In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO: 1 and the light chain variable region amino acid sequence of SEQ ID NO: 2, or sequences that are at least 95% identical to the sequences described. In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2.

In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the anti-BCMA antibody comprises a human IgG1 heavy chain constant domain or a modified IgG1 constant domain. In some embodiments, the IgG1 heavy chain constant domain comprises a cysteine residue (C) at amino acid positions corresponding to 152 and 375 in the wild-type (unmodified) IgG1 heavy chain constant domain, numbered according to the EU numbering system. In some embodiments, the IgG1 heavy chain constant domain comprises a cysteine residue (C) at amino acid positions corresponding to 156 and 379 in the wild-type (unmodified) IgG1 heavy chain constant domain. In some embodiments, the anti-BCMA antibody comprises a human Ig kappa light chain constant domain or a modified Ig kappa light chain constant domain.

In some embodiments, the anti-BCMA antibody comprises the heavy chain amino acid sequence of SEQ ID NO:57 or a sequence that is at least 95% identical to SEQ ID NO:57, and the light chain amino acid sequence of SEQ ID NO:58 or a sequence that is at least 95% identical to SEQ ID NO:58. In some embodiments, the anti-BCMA antibody comprises the heavy chain amino acid sequence of SEQ ID NO:57 and the light chain amino acid sequence of SEQ ID NO:58, or sequences that are at least 95% identical to the sequences described. In some embodiments, the anti-BCMA antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 57 and a light chain sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 58. In some embodiments, the anti-BCMA antibody is J6M0 (WO 2012/163805), or an antigen-binding fragment thereof.

In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof comprises three CDRs of the heavy chain and three CDRs of the light chain of J6M0, or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions, or substitutions of HCDR1 (SEQ ID NO:15), HCDR2 (SEQ ID NO:16), HCDR3 (SEQ ID NO:17); LCDR1 (SEQ ID NO:18), LCDR2 (SEQ ID NO:19), and LCDR3 (SEQ ID NO:20).

In some embodiments, the cancer antigen target for the ADC is CD33.

In some embodiments, the anti-CD33 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 21, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 22, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 23; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 24, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 25, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 26.

In some embodiments, the anti-CD33 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4. In some embodiments, the anti-CD33 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO:3 and the light chain variable region amino acid sequence of SEQ ID NO:4, or sequences that are at least 95% identical to the sequences described. In some embodiments, the anti-CD33 antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:4.

In some embodiments, the anti-CD33 antibody or antigen-binding fragment thereof is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the anti-CD33 antibody comprises a human IgG1 heavy chain constant domain or a modified IgG1 constant domain. In some embodiments, the IgG1 heavy chain constant domain comprises a glutamine residue (Q) at the amino acid position corresponding to 297 in the wild-type (unmodified) IgG1 heavy chain constant domain. In some embodiments, the anti-CD33 antibody comprises a human Ig kappa light chain constant domain or a modified Ig kappa light chain constant domain.

In some embodiments, the anti-CD33 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:59 or a sequence that is at least 95% identical to SEQ ID NO:59, and the light chain amino acid sequence of SEQ ID NO:60 or a sequence that is at least 95% identical to SEQ ID NO:60. In some embodiments, the anti-CD33 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:59 and the light chain amino acid sequence of SEQ ID NO:60, or sequences that are at least 95% identical to the sequences described. In some embodiments, the anti-CD33 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 59 and a light chain sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 60. In some embodiments, the anti-CD33 antibody is MuMy9-6ch (US 2013/0078241), or an antigen-binding fragment thereof.

In some embodiments, the anti-CD33 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs of MuMy9-6ch, or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions, or substitutions of HCDR1 (SEQ ID NO:21), HCDR2 (SEQ ID NO:22), HCDR3 (SEQ ID NO:23); LCDR1 (SEQ ID NO:24), LCDR2 (SEQ ID NO:25), and LCDR3 (SEQ ID NO:26).

In some embodiments, the anti-CD33 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 27, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 28, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 29; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 30, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 31, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 32.

In some embodiments, the anti-CD33 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the anti-CD33 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO:5 and the light chain variable region amino acid sequence of SEQ ID NO:6, or sequences that are at least 95% identical to the sequences described. In some embodiments, the anti-CD33 antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:5 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:6.

In some embodiments, the anti-CD33 antibody or antigen-binding fragment thereof is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the anti-CD33 antibody comprises a human IgG1 heavy chain constant domain or a modified IgG1 constant domain. In some embodiments, the IgG1 heavy chain constant domain comprises a cysteine residue (C) at amino acid positions corresponding to 152 and 375 in the wild-type (unmodified) IgG1 heavy chain constant domain, numbered according to the EU numbering system.

In some embodiments, the anti-CD33 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:61 or a sequence that is at least 95% identical to SEQ ID NO:61, and the light chain amino acid sequence of SEQ ID NO:62 or a sequence that is at least 95% identical to SEQ ID NO:62. In some embodiments, the anti-CD33 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:61 and the light chain amino acid sequence of SEQ ID NO:62, or sequences that are at least 95% identical to the sequences described. In some embodiments, the anti-CD33 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 61 and a light chain sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 62. In some embodiments, the anti-CD33 antibody is gemtuzumab, or an antigen-binding fragment thereof.

In some embodiments, the anti-CD33 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs of gemtuzumab, or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions, or substitutions of HCDR1 (SEQ ID NO:27), HCDR2 (SEQ ID NO:28), HCDR3 (SEQ ID NO:29); LCDR1 (SEQ ID NO:30), LCDR2 (SEQ ID NO:31), and LCDR3 (SEQ ID NO:32).

In some embodiments, the cancer antigen target for the ADC is PCAD.

In some embodiments, the anti-PCAD antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 33, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 34, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 35; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 36, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 37, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 38.

In some embodiments, the anti-PCAD antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the anti-PCAD antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO:7 and the light chain variable region amino acid sequence of SEQ ID NO:8, or sequences that are at least 95% identical to the sequences described. In some embodiments, the anti-PCAD antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8.

In some embodiments, the anti-PCAD antibody or antigen-binding fragment thereof is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the anti-PCAD antibody comprises a human IgG1 heavy chain constant domain or a modified IgG1 constant domain. In some embodiments, the IgG1 heavy chain constant domain comprises a cysteine residue (C) at amino acid positions corresponding to 152 and 375 in the wild-type (unmodified) IgG1 heavy chain constant domain, numbered according to the EU numbering system.

In some embodiments, the anti-PCAD antibody comprises the heavy chain amino acid sequence of SEQ ID NO:63 or a sequence that is at least 95% identical to SEQ ID NO:63, and the light chain amino acid sequence of SEQ ID NO:64 or a sequence that is at least 95% identical to SEQ ID NO:64. In some embodiments, the anti-PCAD antibody comprises the heavy chain amino acid sequence of SEQ ID NO:63 and the light chain amino acid sequence of SEQ ID NO:64, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-PCAD antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 63 and a light chain sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 64. In some embodiments, the anti-PCAD antibody is NOV169N31Q (WO 2016/203432), or an antigen-binding fragment thereof.

In some embodiments, the anti-PCAD antibody or antigen-binding fragment thereof comprises three CDRs of the heavy chain and three CDRs of the light chain of NOV169N31Q, or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions, or substitutions of HCDR1 (SEQ ID NO:33), HCDR2 (SEQ ID NO:34), HCDR3 (SEQ ID NO:35); LCDR1 (SEQ ID NO:36), LCDR2 (SEQ ID NO:37), and LCDR3 (SEQ ID NO:38).

In some embodiments, the cancer antigen targeted by the ADC is HER2.

In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 39, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 40, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 41; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 42, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 43, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 44.

In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO:9 and the light chain variable region amino acid sequence of SEQ ID NO:10, or sequences that are at least 95% identical to the sequences described. In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10.

In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the anti-HER2 antibody comprises a human IgG1 heavy chain constant domain or a modified IgG1 constant domain. In some embodiments, the IgG1 heavy chain constant domain comprises a glutamine residue (Q) at the amino acid position corresponding to 297 in the wild-type (unmodified) IgG1 heavy chain constant domain. In some embodiments, the IgG1 heavy chain constant domain comprises a serine residue (S) at the amino acid position corresponding to 297 in the wild-type (unmodified) IgG1 heavy chain constant domain. In some embodiments, the IgG1 heavy chain constant domain comprises a cysteine residue (C) at amino acid positions corresponding to 152 and 375 in the wild-type (unmodified) IgG1 heavy chain constant domain, numbered according to the EU numbering system. In some embodiments, the anti-HER2 antibody comprises a human Ig kappa light chain constant domain or a modified Ig kappa light chain constant domain.

In some embodiments, the anti-HER2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:65 or a sequence that is at least 95% identical to SEQ ID NO:65, and the light chain amino acid sequence of SEQ ID NO:66 or a sequence that is at least 95% identical to SEQ ID NO:66. In some embodiments, the anti-HER2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:65 and the light chain amino acid sequence of SEQ ID NO:66, or sequences that are at least 95% identical to the sequences described. In some embodiments, the anti-HER2 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:65 and a light chain sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:66. In some embodiments, the anti-HER2 antibody is trastuzumab (U.S. Pat. Nos. 5,821,337 and 6,870,034; see also Molina et al. (2001) Cancer Res. 61(12):4744-9), or an antigen-binding fragment thereof.

In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs of trastuzumab, or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions, or substitutions of HCDR1 (SEQ ID NO:39), HCDR2 (SEQ ID NO:40), HCDR3 (SEQ ID NO:41); LCDR1 (SEQ ID NO:42), LCDR2 (SEQ ID NO:43), and LCDR3 (SEQ ID NO:44).

In some embodiments, the cancer antigen target for the ADC is CD38.

In some embodiments, the anti-CD38 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 45, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 46, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 47; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 48, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 49, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 50.

In some embodiments, the anti-CD38 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CD38 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO: 11 and the light chain variable region amino acid sequence of SEQ ID NO: 12, or sequences that are at least 95% identical to the sequences described. In some embodiments, the anti-CD38 antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12.

In some embodiments, the anti-CD38 antibody or antigen-binding fragment thereof is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the anti-CD38 antibody comprises a human IgG1 heavy chain constant domain or a modified IgG1 constant domain. In some embodiments, the IgG1 heavy chain constant domain comprises a cysteine residue (C) at amino acid positions corresponding to 152 and 375 in the wild-type (unmodified) IgG1 heavy chain constant domain, numbered according to the EU numbering system.

In some embodiments, the anti-CD38 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:67 or a sequence that is at least 95% identical to SEQ ID NO:67, and the light chain amino acid sequence of SEQ ID NO:68 or a sequence that is at least 95% identical to SEQ ID NO:68. In some embodiments, the anti-CD33 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:67 and the light chain amino acid sequence of SEQ ID NO:68, or sequences that are at least 95% identical to the sequences described. In some embodiments, the anti-CD38 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 67 and a light chain sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 68. In some embodiments, the anti-CD38 antibody is daratumumab, or an antigen-binding fragment thereof.

In some embodiments, the anti-CD38 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs of gemtuzumab, or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions, or substitutions of HCDR1 (SEQ ID NO:45), HCDR2 (SEQ ID NO:46), HCDR3 (SEQ ID NO:47); LCDR1 (SEQ ID NO:48), LCDR2 (SEQ ID NO:49), and LCDR3 (SEQ ID NO:50).

In some embodiments, the cancer antigen target for the ADC is CD46.

In some embodiments, the anti-CD46 antibody or antigen-binding fragment is as described in WO2018/089807, incorporated herein by reference. In some embodiments, the anti-CD46 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:90 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:91. In some embodiments, the anti-CD46 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO:90 and the light chain variable region amino acid sequence of SEQ ID NO:91, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD46 antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:90 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:91.

In some embodiments, the cancer antigen target for the ADC is CD48.

In some embodiments, the anti-CD48 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 51, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 52, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 53; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 54, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 55, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 56.

In some embodiments, the anti-CD48 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-CD48 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO: 13 and the light chain variable region amino acid sequence of SEQ ID NO: 14, or sequences that are at least 95% identical to the sequences described. In some embodiments, the anti-CD48 antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14.

In some embodiments, the anti-CD48 antibody or antigen-binding fragment thereof is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the anti-CD48 antibody comprises a human IgG1 heavy chain constant domain or a modified IgG1 constant domain. In some embodiments, the IgG1 heavy chain constant domain comprises a cysteine residue (C) at amino acid positions corresponding to 152 and 375 in the wild-type (unmodified) IgG1 heavy chain constant domain, numbered according to the EU numbering system.

In some embodiments, the anti-CD48 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:69 or a sequence that is at least 95% identical to SEQ ID NO:69, and the light chain amino acid sequence of SEQ ID NO:70 or a sequence that is at least 95% identical to SEQ ID NO:70. In some embodiments, the anti-CD48 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:69 and the light chain amino acid sequence of SEQ ID NO:70, or sequences that are at least 95% identical to the sequences described. In some embodiments, the anti-CD48 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 69 and a light chain sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 70. In some embodiments, the anti-CD48 antibody is SGN-48A, or an antigen-binding fragment thereof.

In some embodiments, the anti-CD48 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs of gemtuzumab, or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions, or substitutions of HCDR1 (SEQ ID NO:51), HCDR2 (SEQ ID NO:52), HCDR3 (SEQ ID NO:53); LCDR1 (SEQ ID NO:54), LCDR2 (SEQ ID NO:55), and LCDR3 (SEQ ID NO:56).

In some embodiments, the cancer antigen target for the ADC is CD79B.

In some embodiments, the anti-CD48 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 82, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 83, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 84; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 85, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 86, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 87.

In some embodiments, the anti-CD79B antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:80 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:81. In some embodiments, the anti-CD79B antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO:80 and the light chain variable region amino acid sequence of SEQ ID NO:81, or sequences that are at least 95% identical to the sequences described. In some embodiments, the anti-CD79B antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 80 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 81.

In some embodiments, the anti-CD79B antibody or antigen-binding fragment thereof is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the anti-CD79B antibody comprises a human IgG1 heavy chain constant domain or a modified IgG1 constant domain. In some embodiments, the IgG1 heavy chain constant domain comprises a cysteine residue (C) at amino acid positions corresponding to 152 and 375 in the wild-type (unmodified) IgG1 heavy chain constant domain, numbered according to the EU numbering system.

In some embodiments, the anti-CD79B antibody comprises the heavy chain amino acid sequence of SEQ ID NO:88 or a sequence that is at least 95% identical to SEQ ID NO:88, and the light chain amino acid sequence of SEQ ID NO:89 or a sequence that is at least 95% identical to SEQ ID NO:89. In some embodiments, the anti-CD33 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:88 and the light chain amino acid sequence of SEQ ID NO:89, or sequences that are at least 95% identical to the sequences described. In some embodiments, the anti-CD79B antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 88 and a light chain sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 89. In some embodiments, the anti-CD79B antibody is polatizumab, or an antigen-binding fragment thereof.

In some embodiments, the anti-CD79B antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs of gemtuzumab, or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions, or substitutions of HCDR1 (SEQ ID NO:82), HCDR2 (SEQ ID NO:83), HCDR3 (SEQ ID NO:84); LCDR1 (SEQ ID NO:85), LCDR2 (SEQ ID NO:86), and LCDR3 (SEQ ID NO:87).

Residues in two or more polypeptides are said to "match" if the residues occupy similar positions within the polypeptide structures. Similar positions in two or more polypeptides can be determined by aligning the polypeptide sequences based on amino acid sequence or structural similarity. Those skilled in the art understand that it may be necessary to introduce gaps in any sequence to obtain a satisfactory alignment.

In some embodiments, amino acid substitutions are single residues. Insertions will typically be on the order of about 1 to about 20 amino acid residues, although significantly larger insertions may be tolerated as long as biological function (e.g., binding to a target antigen) is maintained. Deletions typically range from 1 to 20 amino acid residues, although in some cases, deletions may be much larger. Substitutions, deletions, insertions, or any combination thereof can be used to produce the final derivative or variant. Typically, these changes are made to a few amino acids to minimize alteration of the molecule, particularly the immunogenicity and specificity of the antigen-binding protein. However, larger changes may be tolerated under certain circumstances. Conservative substitutions can be made according to the following map, presented in Table 1.

Table 7 Initial balance Typical replacements Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

In some embodiments where antibody variant sequences are used in ADCs, the variants typically exhibit the same qualitative biological activity and will elicit the same immune response, although variants can also be selected to modify the characteristics of the antigen-binding proteins, if desired. Alternatively, a variant can be engineered to alter the biological activity of the antigen-binding protein. For example, glycosylation sites can be altered or removed.

Various antibodies can be used with the ADCs used herein to target cancer cells. As demonstrated below, useful linkers in the ADCs described herein are unexpectedly effective with various tumor antigen-targeting antibodies. Suitable antigens expressed on cancer cells but not on healthy cells, or expressed on cancer cells at higher levels than on healthy cells, are known in the art, as are antibodies directed against them. Additional antibodies against these target antigens can be prepared by those skilled in the art. These antibodies can be used with the linkers and Mcl-1 inhibitor payloads described herein. In some embodiments, the antibody or antigen-binding fragment targets BCMA, and the BCMA-targeting antibody or antigen-binding fragment is J6M0. In some embodiments, the antibody or antigen-binding fragment targets CD33, and in some embodiments, the CD33-targeting antibody or antigen-binding fragment is MuMy9-6ch. In some embodiments, the antibody or antigen-binding fragment targets PCAD, and in some embodiments, the PCAD-targeting antibody or antigen-binding fragment is NOV169N31Q. In some embodiments, the antibody or antigen-binding fragment targets HER2, and in some embodiments, the HER2-targeting antibody or antigen-binding fragment is trastuzumab. In some embodiments, while the described Mcl-1 inhibitor linkers and payloads are unexpectedly effective with several different tumor-targeting antibodies, BCMA-targeted antibodies such as J6M0, CD33-targeted antibodies such as MuMy9-6ch, PCAD-targeted antibodies such as NOV169N31Q, and HER2-targeted antibodies such as trastuzumab provide particularly improved drug-to-antibody ratio, aggregation level, stability (i.e., in vitro and in vivo stability), tumor targeting (i.e., cytotoxicity, efficacy), minimal off-target killing, and/or treatment efficacy. Increased treatment efficacy can be measured in vitro or in vivo and can include a decrease in tumor growth rate and/or a reduction in tumor volume.

In some embodiments, alternative antibodies to the same targets or antibodies to different antigen targets are used that provide at least some of the beneficial functional properties described above (e.g., improved stability, improved tumor targeting, improved treatment efficacy, etc.). In some embodiments, some or all of these beneficial functional properties are observed when the described linkers and Mcl-1 inhibitor payloads are conjugated to alternative BCMA, CD33, CD46, CD48, PCAD, or HER2 targeting antibodies or antigen-binding fragments. In some other embodiments, some or all of these beneficial functional properties are observed when the described linkers and Mcl-1 inhibitor payloads are conjugated to a BCMA targeting antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment targets BCMA. In some embodiments, the BCMA targeting antibody or antigen-binding fragment is J6M0. In other embodiments, some or all of these favorable functional properties are observed when the described linkers and Mcl-1 inhibitor payloads are conjugated to a CD33 targeting antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment targets CD33. In some embodiments, the CD33-targeting antibody or antigen-binding fragment is MuMy9-6ch. In other embodiments, some or all of these favorable functional properties are observed when the described linkers and Mcl-1 inhibitor payloads are conjugated to a PCAD targeting antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment targets PCAD. In some embodiments, the PCAD targeting antibody or antigen-binding fragment is NOV169N31Q. In other embodiments, some or all of these beneficial functional properties are observed when opsin linkers and Mcl-1 inhibitor payloads are conjugated to a HER2-targeted antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment targets HER2. In some embodiments, the HER2-targeted antibody or antigen-binding fragment is trastuzumab.

Linkers

In some embodiments, the linker in the ADC is sufficiently extracellularly stable to be therapeutically effective. In some embodiments, the linker is stable outside of cells such that the ADC remains intact when present in extracellular conditions (e.g., prior to transport or delivery into a cell). The term "intact" as used in the context of an ADC means that the antibody or antigen-binding fragment remains attached to the drug moiety (e.g., an Mcl-1 inhibitor).

As used herein, the term "stable" in the context of a linker or an ADC comprising a linker means that no more than 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% of the linkers (or any percentage therebetween) in a sample of the ADC are cleaved (or, in the case of the entire ADC, otherwise remain intact) when the ADC is present under extracellular conditions. In some embodiments, the linkers and/or ADCs described herein are stable compared to alternative linkers and/or ADCs with alternative linkers and/or an Mcl-1 inhibitor payload. In some embodiments, the ADCs described herein can remain intact for more than about 48 hours, more than 60 hours, more than about 72 hours, more than about 84 hours, or more than about 96 hours.

[0038] One can determine whether a linker is stable extracellularly, for example, by incorporating the ADC into plasma for a predetermined period of time (e.g., 2, 4, 6, 8, 16, 24, 48, or 72 hours) and then quantifying the amount of free drug moieties present in the plasma. Stability can allow the ADC time to localize to target cancer cells and prevent premature release of the drug moiety, which can reduce the therapeutic index of the ADC by indiscriminately damaging both normal and cancerous tissues. In some embodiments, the linker is stable outside the target cell and releases the drug moiety from the ADC once inside the cell so that the drug can bind to its target. Thus, an effective linker will: (i) maintain the specific binding properties of the antibody or antigen-binding fragment; (ii) provide delivery, such as intracellular delivery, of the drug moiety through stable attachment to an antibody or antigen-binding fragment; (iii) remain stable and intact until the ADC is transported or delivered to the target site; and (iv) provide a therapeutic effect, such as a cytotoxic effect, of the drug moiety upon cleavage or an alternative release mechanism.

Linkers can affect the physicochemical properties of ADCs. Since many cytotoxic agents are hydrophobic in nature, linking them to an antibody via an additional hydrophobic group can lead to aggregation. ADC aggregates are insoluble and often limit the achievable drug load on the antibody, which can negatively impact ADC efficacy. Protein aggregates of biologics are also typically associated with increased immunogenicity. As demonstrated below, the linkers described herein result in ADCs with low levels of aggregation and desirable drug loads.

A linker can be "cleavable" or "non-cleavable" (Ducry and Stump (2010) Bioconjugate Chem. 21:5-13). Cleavable linkers are designed to release the drug moiety (e.g., an Mcl-1 inhibitor) upon exposure to certain environmental factors, such as internalization into the target cell, whereas non-cleavable linkers typically rely on degradation of the antibody or antigen-binding moiety itself.

As used herein, the term "alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation. The term "C ₁ -C ₆ alkyl," as used herein, refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to six carbon atoms and attached to the remainder of the molecule by a single bond. Non-limiting examples of "C ₁ -C ₆ alkyl" groups include methyl (C ₁ alkyl), ethyl (C ₂ alkyl), 1-methylethyl (C ₃ alkyl), n-propyl (C ₃ alkyl), isopropyl (C ₃ alkyl), n-butyl (C ₄ alkyl), isobutyl (C ₄ alkyl), sec-butyl (C ₄ alkyl), tert-butyl (C ₄ alkyl), n-pentyl (C ₅ alkyl), isopentyl (C ₅ alkyl), neopentyl (C ₅ alkyl), and hexyl (C ₆ alkyl).

As used herein, the term "alkenyl" refers to a group of radicals with a straight or branched hydrocarbon chain consisting solely of carbon and hydrogen atoms, containing at least one double bond. The term " _C2 - _C6 alkenyl" as used herein refers to a group of radicals with a straight or branched hydrocarbon chain consisting solely of carbon and hydrogen atoms, containing at least one double bond, having from two to six carbon atoms, which is attached to the remainder of the molecule by a single bond. Non-limiting examples of " _C2 - _C6 alkenyl" groups include ethenyl ( _C2 alkenyl), prop-1-enyl ( _C3 alkenyl), but-1-enyl ( _C4 alkenyl), pent-1-enyl ( _C5 alkenyl), pent-4-enyl ( _C5 alkenyl), penta-1,4-dienyl ( _C5 alkenyl), hexa-1-enyl ( _C6 alkenyl), hexa-2-enyl ( _C6 alkenyl), hexa-3-enyl ( _C6 alkenyl), hexa-1,4-dienyl ( _C6 alkenyl), hexa-1,5-dienyl ( _C6 alkenyl), and hexa-2,4-dienyl ( _C6 alkenyl). The term " _C2 - _C3 alkenyl" as used herein refers to a group of radicals with a straight or branched hydrocarbon chain consisting solely of carbon and hydrogen atoms, containing at least one double bond, having from two to three carbon atoms that are attached to the remainder of the molecule by a single bond. Non-limiting examples of " _C2 - _C3 alkenyl" groups include ethenyl ( _C2 alkenyl) and prop-1-enyl ( _C3 alkenyl).

As used herein, the term "alkylene" refers to a divalent, straight- or branched-chain hydrocarbon radical consisting solely of carbon and hydrogen atoms and containing no unsaturation. The term "C ₁ -C ₆ alkylene" as used herein refers to a divalent, straight- or branched-chain hydrocarbon radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, and having from one to six carbon atoms. Non-limiting examples of "C ₁ -C ₆ alkylene" groups include methylene (C ₁ alkylene), ethylene (C ₂ alkylene), 1-methylethylene (C ₃ alkylene), n-propylene (C ₃ alkylene), isopropylene (C ₃ alkylene), n-butylene (C ₄ alkylene), isobutylene (C ₄ alkylene), sec-butylene (C ₄ alkylene), tert-butylene (C ₄ alkylene), n-pentylene (C ₅ alkylene), isopentylene (C ₅ alkylene), neopentylene (C ₅ alkylene), and hexylene (C ₆ alkylene).

As used herein, the term "alkenylene" refers to a divalent straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms and containing at least one double bond. The term " _C2 - _C6 alkenylene" as used herein refers to a group of divalent straight or branched hydrocarbon chain radicals consisting solely of carbon and hydrogen atoms, containing at least one double bond and having from two to six carbon atoms. Non-limiting examples of " _C2 - _C6 alkenylene" groups include ethenylene ( _C2 alkenylene), prop-1-enylene ( _C3 alkenylene), but-1-enylene ( _C4 alkenylene), pent-1-enylene ( _C5 alkenylene), pent-4-enylene ( _C5 alkenylene), penta-1,4-dienylene ( _C5 alkenylene), hexa-1-enylene ( _C6 alkenylene), hexa-2-enylene ( _C6 alkenylene), hexa-3-enylene ( _C6 alkenylene), hexa-1,4-dienylene ( _C6 alkenylene), hexa-1,5-dienylene ( _C6 alkenylene) and hexa-2,4-dienylene ( _C6 alkenylene). The term " _C2 - _C6 alkenylene" as used herein refers to a group of radicals with a divalent straight or branched hydrocarbon chain consisting solely of carbon and hydrogen atoms, containing at least one double bond and having from two to three carbon atoms. Non-limiting examples of " _C2 - _C3 alkenylene" groups include ethenylene ( _C2 alkenylene) and prop-1-enylene ( _C3 alkenylene).

As used herein, the term "cycloalkyl" or " _C3 - _C8 cycloalkyl" refers to a saturated, monocyclic, fused bicyclic, fused tricyclic, or bridged polycyclic ring system. Non-limiting examples of fused bicyclic or bridged polycyclic ring systems include bicyclo[1.1.1]pentane, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, bicyclo[3.2.1]octane, bicyclo[2.2.2]octane, and adamantanyl. Non-limiting examples of a monocyclic _C3 - _C8 cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups.

As used herein, the term "haloalkyl" refers to a straight or branched alkyl chain substituted with one or more halogen groups in place of hydrogen atoms along the hydrocarbon chain. Examples of halogen groups suitable for substitution in a haloalkyl group include fluorine, bromine, chlorine, and iodine. A haloalkyl group may include substitution with multiple halogen groups in place of hydrogen atoms in the alkyl chain, where said halogen groups may be attached to the same carbon atom or to a different carbon atom in the alkyl chain.

As used herein, the alkyl, alkenyl, alkynyl, alkoxy, amino, aryl, heteroaryl, cycloalkyl and heterocycloalkyl groups may be optionally substituted with 1 to 4 groups selected from optionally linear or branched (C ₁ -C ₆ )alkyl, optionally substituted linear or branched (C ₂ -C ₆ )alkenyl group, optionally substituted linear or branched (C ₂ -C ₆ )alkynyl group, optionally substituted linear or branched (C ₁ -C ₆ )alkoxy, optionally substituted (C ₁ -C ₆ )alkyl-S-, hydroxy, oxo (or N-oxide, if possible), nitro, cyano, -C(O)-OR ₀ ', -OC(O)-R ₀ ', -C(O)-NR ₀ 'R ₀ '', -NR ₀ 'R ₀ '', -(C=NR ₀ ')-OR ₀ '', linear or branched (C ₁ -C ₆ ) haloalkyl, trifluoromethoxy or halogen, where R ₀ ' and R ₀ '' each independently is a hydrogen atom or an optionally substituted linear or branched (C ₁ -C ₆ ) alkyl group, and where one or more of the carbon atoms of the linear or branched (C ₁ -C ₆ ) alkyl group is optionally deuterated.

_The term "polyoxyethylene,""polyethyleneglycol," or "PEG" as used herein refers to a straight chain, branched chain, or star-shaped configuration consisting of ( _OCH2CH2 ) groups. In some embodiments, the polyethylene or _PEG group is -( _OCH2CH2 ) _t *-, where t is 1-40 or 4-40, and where "-" indicates the end directed toward the self-cleaving spacer, and "*-" denotes the point of attachment to the end of the R' group, where R' is OH, _{OCH3 ,} or _OCH2CH2C (=O)OH. In other embodiments, the polyethylene or _{PEG group is -(CH2CH2O ) t} *-, where t is 1-40 or 4-40 and where "-" indicates the end directed toward the self-cleaving spacer and "*-" denotes the point of attachment to the end of the R" group, where R" is H, _{CH3 ,} or _CH2CH2C (=O)OH. For example, the term "PEG12" as used herein means that t is 12.

As used herein, the term "polyalkylene glycol" refers to a straight chain, branched chain, or star-shaped configuration consisting of (O( _CH2 ) _m ) _n groups. In some embodiments, the polyethylene or PEG group is -(O( _CH2 ) _m ) _t *-, where m is 1-10, t is 1-40 or 4-40, and where "-" indicates the end directed toward the self-cleaving spacer and "*-" denotes the point of attachment to the end of the R' group, where R' is OH, _OCH3 , _{or OCH2CH2C} (=O)OH. In other embodiments, the polyethylene or PEG group is -(( _CH2 ) _mO ) _t *-, where m is 1-10, t is 1-40 or 4-40, and where "-" indicates the end directed toward the self-cleaving spacer and "*-" denotes the point of attachment to the end of the R'' group, where R'' is -H, _{CH3 ,} or _CH2CH2C (=O)OH.

The term "reactive group," as used herein, means a functional group capable of forming a covalent bond with a functional group of an antibody, antibody fragment, or another reactive group attached to an antibody or antibody fragment. Non-limiting examples of such functional groups include the reactive groups from Table 8 presented herein.

The term "attached group" or "coupling group," as used herein, refers to a divalent moiety that links a bridging spacer to an antibody or fragment thereof. An attached group or coupling group is a divalent moiety formed by a reaction between a reactive group and a functional group on an antibody or fragment thereof. Non-limiting examples of such divalent moieties include the divalent chemical moieties listed in Table 8 and Table 9 provided herein.

The term "bridged spacer," as used herein, refers to one or more linker components that are covalently linked together to form a divalent moiety that links a divalent peptide spacer to a reactive group, links a divalent peptide spacer to a linking group, or links an attached group to at least one cleavable group. In some embodiments, the "bridged spacer" comprises a carboxyl group attached to the N-terminus of the divalent peptide spacer via an amide bond.

The term "spacer group" as used herein refers to one or more linker components that are covalently linked together to form a moiety that links a self-cleaving spacer to a hydrophilic group.

The term "bivalent peptide spacer" as used herein refers to a bivalent linker comprising one or more amino acid residues covalently linked together to form a moiety that links a bridging spacer to a self-cleaving spacer. The one or more amino acid residues may be amino acid residues selected from alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), norvaline (Nva), norleucine (Nle), selenocysteine (Sec), pyrrolysine (Pyl), homoserine, homocysteine, and desmethylpyrrolysine.

In some embodiments, a "divalent peptide spacer" is a combination of 2 to 4 amino acid residues, wherein each residue is independently selected from an amino acid residue selected from alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), norvaline (Nva), norleucine (Nle), selenocysteine (Sec), pyrrolysine (Pyl), Homoserine, homocysteine, and desmethylpyrrolysine, such as -ValCit*; -CitVal*; -AlaAla*; -AlaCit*; -CitAla*; -AsnCit*; -CitAsn*; -CitCit*; -ValGlu*; -GluVal*; -SerCit*; -CitSer*; -LysCit*; -CitLys*; -AspCit*; -CitAsp*; -AlaVal*; -ValAla*; -PheAla*; -AlaPhe*; -PheLys*; -LysPhe*; -ValLys*; -LysVal*; -AlaLys*; -LysAla*; -PheCit*; -CitPhe*; -LeuCit*; -CitLeu*; -IleCit*; -CitIle*; -PheArg*; -ArgPhe*; -CitTrp*; -TrpCit*; -PhePheLys*; -LysPhePhe*; -DPhePheLys*; -DLysPhePhe*; -GlyPheLys*; -LysPheGly*; -GlyPheLeuGly- [SEQ ID NO:160]; -GlyLeuPheGly- [SEQ ID NO:161]; -AlaLeuAlaLeu- [SEQ ID NO:162], -GlyGlyGly*; -GlyGlyGlyGly- [SEQ ID NO:163]; -GlyPheValGly- [SEQ ID NO:164]; and -GlyValPheGly- [SEQ ID NO:165], where “-” denotes the point of attachment to the bridging spacer and “*” denotes the point of attachment to the self-cleaving spacer.

The term "linker component" as used herein refers to a chemical group that is part of a linker. Examples of linker components include: an alkylene group: -( _CH2 ) _n- , which may be linear or branched (where in this case _n is _1-18 ); an alkenylene group; an alkynylene group; an alkenyl group; an _alkynyl group; an ethylene glycol unit: _-OCH2CH2- or _-CH2CH2O- ; a polyethylene glycol unit: ( _-CH2CH2O- ) _x (where x in this case is 2-20); -O-; -S-; carbonyl: -C(=O); ester: C(=O)-O or OC(=O); carbonate: -OC(=O)O-; amine: -NH-; tertiary amine; amide: -C(=O)-NH-, -NH-C(=O)- or -C(=O)N(C _1-6 alkyl); carbamate: -OC(=O)NH- or -NHC(=O)O; urea: -NHC(=O)NH; sulfonamide: -S(O) ₂ NH- or -NHS(O) ₂ ; ether: -CH ₂ O- or -OCH ₂ -; alkylene substituted with one or more groups independently selected from carboxy, sulfonate, hydroxyl, amine, amino acid, saccharide, phosphate and phosphonate); alkenylene substituted with one or more groups independently selected from carboxy, sulfonate, hydroxyl, amine, amino acid, saccharide, phosphate and phosphonate); alkynylene substituted with one or more groups independently selected from carboxy, sulfonate, hydroxyl, amine, amino acid, saccharide, phosphate and phosphonate); _C1 - _C10 alkylene in which one or more methylene groups is substituted with one or more -S-, -NH- or -O- groups; ring systems having two available points of attachment, such as divalent rings selected from phenyl (including 1,2-, 1,3- and 1,4-disubstituted phenyls), _C5 - _C6 heteroaryl, _C3 - _C8 cycloalkyl (including 1,1-disubstituted cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl and 1,4-disubstituted cyclohexyl) and _C4 - _C8 heterocycloalkyl; an amino acid residue selected from alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), norvaline (Nva), norleucine (Nle), selenocysteine (Sec), pyrrolysine (Pyl), homoserine, homocysteine, and desmethylpyrrolysine; a combination of 2 or more amino acid residues, wherein each residue is independently selected from an amino acid residue selected from alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), norvaline (Nva), norleucine (Nle), selenocysteine (Sec), pyrrolysine (Pyl), homoserine, homocysteine, and desmethylpyrrolysine, such as Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; Val-Ala; Phe-Lys; Lys-Phe; Val-Lys; Lys-Val; Ala-Lys; Lys-Ala; Phe-Cit; Cit-Phe; Leu-Cit; Cit-Leu; Ile-Cit; Cit-Ile; Phe-Arg; Arg-Phe; Cit-Trp; and Trp-Cit; and a self-cleaving spacer, wherein the self-cleaving spacer comprises one or more protecting (helper) groups that are susceptible to acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase-induced cleavage, phosphodiesterase-induced cleavage, phosphatase-induced cleavage, protease-induced cleavage, lipase-induced cleavage, or disulfide bond cleavage.

Non-limiting examples of such self-cleaving spacers include:

And , Where

PG is a protective (helper) group;

X _a is O, NH or S;

X _b is O, NH, NCH ₃ or S;

X _c is O or NH;

Y _a is CH ₂ , CH ₂ O or CH ₂ NH;

Y _b is CH ₂ , O or NH;

Y _c is a bond, CH ₂ , O or NH, and

LG is a leaving group such as the drug group (D) of the linker-drug group of the invention.

Additional non-limiting examples of such self-cleaving spacers are described in Angew. Chem. Int. Ed. 2015, 54, 7492–7509.

Additionally, the linker component can be a chemical group that readily forms upon reaction between two reactive groups. Non-limiting examples of such chemical groups are given in Table 8.

Table 8 Reactive group 1 (RG1) Reactive group 2 (RG2) Chemical group thiol thiol -S-S- thiol maleimide thiol haloacetamide azide alkyne or azide triarylphosphine azide cyclooctyne or azide oxanobornadiene triarylphosphine azide oxanobornadiene azide alkyne azide or cyclooctyne azide or cyclooctene diaryltetrazine or diaryltetrazine cyclooctene or monoaryltetrazine Norbornen Norbornen monoaryltetrazine aldehyde hydroxylamine aldehyde hydrazine aldehyde NH ₂ -NH-C(=O)- ketone hydroxylamine ketone hydrazine ketone NH ₂ -NH-C(=O)- hydroxylamine aldehyde hydroxylamine ketone hydrazine aldehyde hydrazine ketone NH ₂ -NH-C(=O)- aldehyde NH ₂ -NH-C(=O)- ketone haloacetamide thiol maleimide thiol vinyl sulfone thiol thiol vinyl sulfone aziridine thiol or thiol aziridine or hydroxylamine hydroxylamine -NH ₂ , amide -NH ₂ , amide CoA or CoA analogue Serine residue pyridyldithiol thiol disulfide

wherein: R ³² in Table 8 is H, C _1-4 alkyl, phenyl, pyrimidine, or pyridine; R ³⁵ in Table 8 is H, C _1-6 alkyl, phenyl, or C _1-4 alkyl substituted with 1-3 -OH groups; each R ⁷ in Table 8 is independently selected from H, C _1-6 alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C _1-4 alkoxy substituted with -C(=O)OH, and C _1-4 alkyl substituted with -C(=O)OH; R ³⁷ in Table 8 is independently selected from H, phenyl, and pyridine; q in Table 8 is 0, 1, 2, or 3; R ⁸ and R ¹³ in Table 8 are H or methyl; and R ⁹ and R ¹⁴ in Table 2 are H, -CH ₃ or phenyl; R in Table 8 is H or any replaceable substituent; and R ⁵⁰ in Table 8 is H.

Additionally, the linker component may be a group listed in Table 9 below.

Table 9.

each R ⁷ is independently selected from H, C _1-6 alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C _1-4 alkoxy substituted with -C(=O)OH, and C _1-4 alkyl substituted with -C(=O)OH;

each R ¹² is independently selected from H and C ₁ -C ₆ alkyl

R ⁸ is H or methyl;

R ⁹ is H, -CH ₃ or phenyl;

each R ²⁵ is independently selected from H or C _1-4 alkyl;

each R ¹⁸ is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl that is substituted with azido, and C ₁ -C ₆ alkyl that is substituted with 1-5 hydroxyl;

q is 0, 1, 2, or 3;

l is 1, 2, 3, 4, 5, or 6;

R ²⁶ is ,

R ³² is independently selected from H, C _1-4 alkyl, phenyl, pyrimidine and pyridine;

R ³³ is independently selected from ,

R ³⁴ is independently selected from H, C _1-4 alkyl and C _1-6 haloalkyl, and

R ^aa is the side chain of an amino acid.

In this document, when a partial structure of a compound is illustrated, a wavy line ( ) indicates the point of attachment of the partial structure to the rest of the molecule.

The terms "self-cleaving spacer" and "self-cleaving group" as used herein refer to a moiety comprising one or more helper groups (TG) that are activated by acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase-induced cleavage, phosphodiesterase-induced cleavage, phosphatase-induced cleavage, protease-induced cleavage, lipase-induced cleavage, or disulfide bond cleavage, and upon activation, the protecting group is removed, generating a cascade of disassembly reactions leading to the transient sequential release of the leaving group. Such a cascade of reactions may include, but is not limited to, 1,4-, 1,6-, or 1,8-elimination reactions.

Non-limiting examples of a self-cleaving spacer or group include:

And

, where such groups may be optionally substituted, and

Where:

TG is a helper group;

X _a is O, NH or S;

X _b is O, NH, NCH ₃ or S;

X _c is O or NH;

Y _a is CH ₂ , CH ₂ O or CH ₂ NH;

Y _b is CH ₂ , O or NH;

Y _c is a bond, CH ₂ , O or NH, and

LG is a leaving group such as the drug group (D) of the linker-drug group of the invention.

Additional non-limiting examples of self-cleaving spacers are described in Angew. Chem. Int. Ed. 2015, 54, 7492–7509.

In a particular embodiment, the self-cleaving spacer is a group having the structure , where Lp is an enzymatically cleavable divalent peptide spacer, and A, D, L ₃ and R ² are as defined herein.

In a preferred embodiment, the self-cleaving spacer is a group having the structure , where Lp is an enzymatically cleavable divalent peptide spacer and D, L ₃ and R ² are as defined herein. In some embodiments, D is an MCl1 inhibitor comprising a quaternized tertiary amine.

In other preferred embodiments, the self-cleaving spacer is a group having the structure , where Lp is an enzymatically cleavable divalent peptide spacer and D, L ₃ and R ² are as defined herein.

The term "hydrophilic group" as used herein refers to a moiety that has hydrophilic properties that increase the water solubility of the drug group (D) when the drug group (D) is attached to a linker group of the invention. Examples of such hydrophilic groups include, but are not limited to, polyethylene glycols, polyalkylene glycols, sugars, oligosaccharides, C ₂ -C ₆ alkyl polypeptides substituted with 1-3 in groups.

Drug groups

In some embodiments, an intermediate that is a precursor to a linker group reacts with a moiety of a drug (e.g., an Mcl-1 inhibitor) under appropriate conditions. In some embodiments, reactive groups are used on the drug and/or the intermediate or linker. The reaction product between the drug and the intermediate, or the derivatized drug (drug plus linker), is subsequently reacted with an antibody or antigen-binding fragment under conditions that facilitate conjugation of the drug and the intermediate or the derivatized drug and the antibody or antigen-binding fragment. Alternatively, the intermediate or linker may first react with the antibody or antigen-binding fragment or the derivatized antibody or antigen-binding fragment and then react with the drug or derivatized drug.

A number of different reactions are available for covalently attaching a drug moiety and/or a linker group to an antibody or antigen-binding fragment. This is often achieved by reacting one or more amino acid residues of the antibody or antigen-binding fragment, including the amine groups of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl groups of cysteine, and various aromatic amino acid groups. For example, non-specific covalent attachment can be accomplished using a carbodiimide reaction to couple the carboxy (or amino) group on the drug moiety to an amino (or carboxy) group on the antibody or antigen-binding fragment. Furthermore, bifunctional agents such as dialdehydes or imidoesters can also be used to couple the amino group of the drug moiety to the amino group of the antibody or antigen-binding fragment. A Schiff base reaction is also available for coupling drugs (e.g., an Mcl-1 inhibitor) to coupling agents. This method involves oxidizing the periodate of the drug, which contains glycol or hydroxy groups, to form an aldehyde, which is then reacted with the coupling agent. The coupling occurs through the formation of a Schiff base with the amino groups of the coupling agent. Isothiocyanates can also be used as coupling agents for covalently coupling drugs to coupling agents. Other methods are known to those skilled in the art and are within the scope of the present disclosure. Examples of drug moieties that can be prepared and coupled to an antibody or antigen-binding fragment using various chemistries known in the art include Mcl-1 inhibitors, such as the Mcl-1 inhibitors described and exemplified herein.

Suitable drug groups may include a compound of formulas (I), (II), (III) or an enantiomer, diastereoisomer, atropisomer, deuterated derivative, and/or addition salt with a pharmaceutically acceptable acid or base thereof. Furthermore, the drug group may include any of the Mcl-1 inhibitor (D) compounds described herein.

As used herein, "atropisomers" are stereoisomers arising from hindered rotation about a single bond, where the energy difference due to steric strain or other factors creates a barrier to rotation high enough to allow the isolation of individual conformers (Bringmann et al. Angew. Chem. Int. Ed. 2005, 44, 5384-5427). For example, for a compound of formula (II) of the invention, the atropisomers may be as follows:

.

For example, a preferred atropisomer might be ( 5S _a ), also called (5 aS ).

The group of the medicinal product according to the invention may be one of the compounds described in International Patent Application Publications Nos. WO 2015/097123; WO 2016/207216; WO 2016/207217; WO 2016/207225; WO 2016/207226; WO 2017/125224; WO 2019/035899; WO 2019/035911; WO 2019/035914; WO 2019/035927; WO 2016/033486; WO 2017/147410; WO 2018/183418; and WO 2017/182625 and US Patent Application Publication No. 2019/0055264, each of which is incorporated herein by reference in its entirety.

In some embodiments, the drug group as described may comprise a compound of formula (I):

where ring D ₀ is a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group,

ring E ₀ is a furyl, thienyl or pyrrolyl ring,

X ₀₁ , X ₀₃ , X ₀₄ and X ₀₅ are independently of each other a carbon atom or a nitrogen atom,

X ₀₂ is a CR ₀₂₆ group or a nitrogen atom,

means that the ring is aromatic,

Y ₀ is a nitrogen atom or CR ₀₃ group,

Z ₀ is a nitrogen atom or a CR ₀₄ group,

R ₀₁ is a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, a linear or branched (C ₁ -C ₆ )haloalkyl group, a hydroxy group, a hydroxy(C ₁ -C ₆ )alkyl group, a linear or branched (C ₁ -C ₆ )alkoxy group, -S-(C ₁ -C ₆ )alkyl group, a cyano group, a nitro group, -Cy _08, -(C ₀ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-(C ₁ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-(C ₁ -C ₆ )alkyl-R ₀₁₂ , -C(O)-OR ₀₁₁ , -OC(O)-R ₀₁₁ , -C(O)-NR ₀₁₁ R ₀₁₁ ', -NR ₀₁₁ -C(O)-R ₀₁₁ ', -NR ₀₁₁ -C(O)-OR ₀₁₁ ', -(C ₁ -C ₆ )alkyl-NR ₀₁₁ -C(O)-R ₀₁₁ ', -SO ₂ -NR ₀₁₁ R ₀₁₁ ' or -SO ₂ -(C ₁ -C ₆ )alkyl,

R ₀₂ , R ₀₃ , R ₀₄ and R ₀₅ independently of one another are a hydrogen atom, a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, a linear or branched (C ₁ -C ₆ )haloalkyl, a hydroxy group, a hydroxy(C ₁ -C ₆ )alkyl group, a linear or branched (C ₁ -C ₆ )alkoxy group, -S-(C ₁ -C ₆ )alkyl group, a cyano group, a nitro group, -(C ₀ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-Cy ₀₁ , -(C ₀ -C ₆ )alkyl-Cy ₀₁ , -(C ₂ -C ₆ )alkenyl-Cy ₀₁ , -(C ₂ -C ₆ )alkynyl-Cy ₀₁ , -O-(C ₁ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-(C ₁ -C ₆ )alkyl-R ₀₃₁ , -O-(C ₁ -C ₆ )alkyl-R ₀₁₂ , -C(O)-OR ₀₁₁ , -OC(O)-R ₀₁₁ , -C(O)-NR ₀₁₁ R ₀₁₁ ', -NR ₀₁₁ -C(O)-R ₀₁₁ ', -NR ₀₁₁ -C(O)-OR ₀₁₁ ', -(C ₁ -C ₆ )alkyl-NR ₀₁₁ -C(O)-R ₀₁₁ ', -SO ₂ -NR ₀₁₁ R ₀₁₁ ' or -SO ₂ -(C ₁ -C ₆ )alkyl,

or a pair of (R ₀₁ , R ₀₂ ), (R ₀₂ , R ₀₃ ), (R ₀₃ , R ₀₄ ) or (R ₀₄ , R ₀₅ ) together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containing 5-7 ring members which optionally contains 1-3 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted with 1 or 2 groups selected from halogen, linear or branched (C ₁ -C ₆ )alkyl, (C ₀ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -NR ₀₁₃ R ₀₁₃ ', -(C ₀ -C ₆ )alkyl-Cy ₀₁ or oxo,

R ₀₆ and R ₀₇ independently of one another are a hydrogen atom, a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, a linear or branched (C ₁ -C ₆ )haloalkyl, a hydroxy group, a linear or branched (C ₁ -C ₆ )alkoxy group, -S-(C ₁ -C ₆ )alkyl group, a cyano group, a nitro group, -(C ₀ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-(C ₁ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-Cy ₀₁ , -(C ₀ -C ₆ )alkyl-Cy ₀₁ , -(C ₂ -C ₆ )alkenyl-Cy ₀₁ , -(C ₂ -C ₆ )alkynyl-Cy ₀₁ , -O-(C ₁ -C ₆ )alkyl-R ₀₁₂ , -C(O)-OR ₀₁₁ , -OC(O)-R ₀₁₁ , -C(O)-NR ₀₁₁ R ₀₁₁ ', -NR ₀₁₁ -C(O)-R ₀₁₁ ', -NR ₀₁₁ -C(O)-OR ₀₁₁ ', -(C ₁ -C ₆ )alkyl-NR ₀₁₁ -C(O)-R ₀₁₁ ', -SO ₂ -NR ₀₁₁ R ₀₁₁ ' or -SO ₂ -(C ₁ -C ₆ )alkyl,

or a pair (R ₀₆ , R ₀₇ ) fused with two adjacent carbon atoms, together with the carbon atoms to which they are attached, form an aromatic or non-aromatic ring containing 5-7 members in the ring, which optionally contains 1-3 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted with a linear or branched (C ₁ -C ₆ )alkyl group, -NR ₀₁₃ R ₀₁₃ ', -(C ₀ -C ₆ )alkyl-Cy ₀₁ or oxo,

W ₀ is a -CH ₂ - group, -NH- group or an oxygen atom,

R ₀₈ is a hydrogen atom, a linear or branched (C ₁ -C ₈ )alkyl group, a -CHR _0a R _0b group, an aryl group, a heteroaryl group, an aryl(C ₁ -C ₆ )alkyl group or a heteroaryl(C ₁ -C ₆ )alkyl group,

R ₀₉ is a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, -Cy _O2 , -(C ₁ -C ₆ )alkyl-Cy _O2 , -(C ₂ -C ₆ )alkenyl-Cy _O2 , -(C ₂ -C ₆ )alkynyl-Cy _O2 , -Cy _O2 -Cy _O3 , -(C ₂ -C ₆ )alkynyl-O-Cy _O2 , -Cy _O2 -(C ₀ -C ₆ )alkyl-O-(C ₀ -C ₆ )alkyl-Cy _O3 , a halogen atom, a cyano group, -C(O)-R ₀₁₄ or -C(O)-NR ₀₁₄ R ₀₁₄ ',

R ₀₁₀ is a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, an aryl(C ₁ -C ₆ )alkyl group, a (C ₁ -C ₆ )cycloalkylalkyl group, a linear or branched (C ₁ -C ₆ )haloalkyl or -(C ₁ -C ₆ )alkyl-O-Cy ₀₄ ,

or a pair (R ₀₉ , R ₀₁₀ ) condensed with two adjacent carbon atoms, together with the carbon atoms to which they are attached, form an aromatic or non-aromatic ring containing 5-7 members in the ring, which optionally contains 1-3 heteroatoms selected from O, S and N,

R ₀₁₁ and R ₀₁₁ ' independently of one another are a hydrogen atom, an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group or -(C ₀ -C ₆ )alkyl-Cy ₀₁ ,

or a pair (R ₀₁₁ , R ₀₁₁ ') together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5-7 ring members, which optionally contains, in addition to the nitrogen atom, 1-3 heteroatoms selected from O, S and N, wherein the N atom may be substituted by 1 or 2 groups selected from a linear or branched (C ₁ -C ₆ )alkyl group, and wherein one or more carbon atoms of the linear or branched (C ₁ -C ₆ )alkyl group are optionally deuterated,

R ₀₁₂ is -Cy ₀₅ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-O-(C ₀ -C ₆ )alkyl-Cy ₀₆ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-Cy ₀₆ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-NR ₀₁₁ -(C ₀ -C ₆ )alkyl-Cy ₀₆ , -Cy ₀₅ -Cy ₀₆ -O-(C ₀ -C ₆ )alkyl-Cy ₀₇ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-O-(C ₀ -C ₆ )alkyl-Cy ₀₉ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-Cy ₀₉ , -NH-C(O)-NH-R ₀₁₁ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-NR ₀₁₁ -(C ₀ -C ₆ )alkyl-Cy ₀₉ , -C(O)-NR ₀₁₁ R ₀₁₁ ', -NR ₀₁₁ R ₀₁₁ ', -OR ₀₁₁ , -NR ₀₁₁ -C(O)-R ₀₁₁ ', -O-(C ₁ -C ₆ )alkyl-OR ₀₁₁ , -SO ₂ -R ₀₁₁ , -C(O)-OR ₀₁₁ ,

R ₀₁₃ , R ₀₁₃ ', R ₀₁₄ and R ₀₁₄ ' independently of one another are a hydrogen atom or an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group,

R _0a is a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

R _0b is an -OC(O)-OR _0c group, an -OC(O)-NR _0c R _0c ' group, or an -OP(O)(OR _0c ) ₂ group,

R _0c and R _0c ' independently of one another are a hydrogen atom, a linear or branched (C ₁ -C ₈ )alkyl group, a cycloalkyl group, a (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group or a (C ₁ -C ₆ )alkoxycarbonyl(C ₁ -C ₆ )alkyl group,

or a pair (R _0c , R _0c ') together with the nitrogen atom to which they are attached form a non-aromatic ring consisting of 5-7 ring members which may contain, in addition to the nitrogen atom, 1-3 heteroatoms selected from oxygen and nitrogen, wherein the nitrogen is optionally substituted with a linear or branched (C ₁ -C ₆ )alkyl group,

Cy ₀₁ , Cy ₀₂ , Cy ₀₃ , Cy ₀₄ , Cy ₀₅ , Cy ₀₆ , Cy ₀₇ , Cy ₀₈ and Cy ₀₁₀ independently of each other are an optionally substituted cycloalkyl group, an optionally substituted heterocycloalkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group,

Cy ₀₉ is ,

or Cy ₀₉ is a heteroaryl group which is substituted by a group selected from -OP(O)(OR ₀₂₀ ) ₂ ; -OP(O)(O ^- M ⁺ ) ₂ ; -(CH ₂ ) _p0 -O-(CHR ₀₁₈ -CHR ₀₁₉ -O) _q0 -R ₀₂₀ ; hydroxy; hydroxy(C ₁ -C ₆ )alkyl; -(CH ₂ ) _r0 -U ₀ -(CH ₂ ) _s0 -heterocycloalkyl; and -U ₀ -(CH ₂ ) _q0 -NR ₀₂₁ R ₀₂₁ ',

R ₀₁₅ is a hydrogen atom; -(CH ₂ ) _p0 -O-(CHR ₀₁₈ -CHR ₀₁₉ -O) _q0 -R ₀₂₀ group; a linear or branched (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group; -U ₀ -(CH ₂ ) _q0 -NR ₀₂₁ R ₀₂₁ 'group; or -(CH ₂ ) _r0 -U ₀ -(CH ₂ ) _s0 -heterocycloalkyl group,

R ₀₁₆ represents a hydrogen atom; a hydroxy group; a hydroxy(C ₁ -C ₆ )alkyl group; -(CH ₂ ) _r0 -U ₀ -(CH ₂ ) _s0 -heterocycloalkyl group; (CH ₂ ) _r0 -U ₀ -V ₀ -OP(O)(OR ₀₂₀ ) ₂ group; -OP(O)(O ^- M ⁺ ) ₂ group; -OS(O) ₂ OR ₀₂₀ group; -S(O) ₂ OR ₀₂₀ group; -(CH ₂ ) _p0 -O-(CHR ₀₁₈ -CHR ₀₁₉ -O) _q0 -R ₀₂₀ group; -(CH ₂ ) _p0 -OC(O)-NR ₀₂₂ R ₀₂₃ group; or -U ₀ -(CH ₂ ) _q0 -NR ₀₂₁ R ₀₂₁ ' group,

R ₀₁₇ is a hydrogen atom; -(CH ₂ ) _p0 -O-(CHR ₀₁₈ -CHR ₀₁₉ -O) _q0 -R ₀₂₀ group; -CH ₂ -P(O)(OR ₀₂₀ ) ₂ group, -OP(O)(OR ₀₂₀ ) ₂ group; -OP(O)(O ^- M ⁺ ) ₂ group; hydroxy group; hydroxy(C ₁ -C ₆ )alkyl group; -(CH ₂ ) _r0 -U ₀ -(CH ₂ ) _s0 -heterocycloalkyl group; -U ₀ -(CH ₂ ) _q0 -NR ₀₂₁ R ₀₂₁ 'group; or aldonic acid,

M ⁺ is a pharmaceutically acceptable monovalent cation,

U ₀ is a bond or oxygen atom,

V ₀ is a -(CH ₂ ) _s0 - group or a -C(O)- group,

R ₀₁₈ is a hydrogen atom or a (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group,

R ₀₁₉ is a hydrogen atom or a hydroxy(C ₁ -C ₆ )alkyl group,

R ₀₂₀ is a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

R ₀₂₁ and R ₀₂₁ ' independently of one another are a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group or a hydroxy(C ₁ -C ₆ )alkyl group,

or a pair (R ₀₂₁ , R ₀₂₁ ') together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5-7 members in the ring, which optionally contains, in addition to the nitrogen atom, 1-3 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted with a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

R ₀₂₂ is a (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group, -(CH ₂ ) _p0 -NR ₀₂₄ R ₀₂₄ ' group or -(CH ₂ ) _p0 -O-(CHR ₀₁₈ -CHR ₀₁₉ -O) _q0 -R ₀₂₀ group,

R ₀₂₃ is a hydrogen atom or a (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group,

or a pair (R ₀₂₂ , R ₀₂₃ ) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5-18 ring members which optionally contains, in addition to the nitrogen atom, 1-5 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted with a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group or a heterocycloalkyl group,

R ₀₂₄ and R ₀₂₄ ' independently of each other are a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

or a pair (R ₀₂₄ , R ₀₂₄ ') together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring consisting of 5-7 ring members which may contain, in addition to the nitrogen atom, 1-3 heteroatoms selected from O, S and N and wherein the resulting ring is optionally substituted with a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

R ₀₂₅ is a hydrogen atom, a hydroxy group or a hydroxy(C ₁ -C ₆ )alkyl group,

R ₀₂₆ is a hydrogen atom, a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group or a cyano group,

R ₀₂₇ is a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

R ₀₂₈ is an -OP(O)(O ^- )(O ^- ) group, -OP(O)(O ^- )(OR ₀₃₀ ) group, -OP(O)(OR ₀₃₀ )(OR ₀₃₀ ') group, -(CH ₂ ) _p0 -O-SO ₂ - group, -(CH ₂ ) _p0 -SO ₂ -O ^- group, -(CH ₂ ) _p0 -O-SO ₂ -OR ₀₃₀ group, -Cy ₀₁₀ , -(CH ₂ ) _p0 -SO ₂ -OR ₀₃₀ group, -OC(O)-R ₀₂₉ group, -OC(O)-OR ₀₂₉ group, or -OC(O)-NR ₀₂₉ R ₀₂₉ 'group;

R ₀₂₉ and R ₀₂₉ ' independently of one another are a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, or a linear or branched amino(C ₁ -C ₆ )alkyl group,

R ₀₃₀ and R ₀₃₀ ' independently of one another are a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group or an aryl(C ₁ -C ₆ )alkyl group,

R ₀₃₁ is or

, where the ammonium ion does not necessarily exist in the zwitterionic form or have a monovalent anionic counterion,

n ₀ is an integer equal to 0 or 1,

p ₀ is an integer equal to 0, 1, 2, or 3,

q ₀ is an integer equal to 1, 2, 3, or 4,

r ₀ and s ₀ are independently an integer equal to 0 or 1;

wherein at most one of the R ₀₃ , R ₀₉ or R ₀₁₂ groups, if present, is covalently attached to the linker, and

where the valence of an atom is not exceeded by one or more substituents bound to it,

or an enantiomer, diastereoisomer, atropisomer, deuterated derivative and/or pharmaceutically acceptable salt of any of the above.

In some embodiments, the drug group as described may comprise a compound of formula (II):

Where:

Z ₀ is a nitrogen atom or a CR ₀₄ group,

R ₀₁ is a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, a linear or branched (C ₁ -C ₆ )haloalkyl group, a hydroxy group, a linear or branched (C ₁ -C ₆ )alkoxy group, -S-(C ₁ -C ₆ )alkyl group, a cyano group, -Cy _08, -NR ₀₁₁ R ₀₁₁ ',

R ₀₂ , R ₀₃ and R ₀₄ independently of one another are a hydrogen atom, a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, a linear or branched (C ₁ -C ₆ )haloalkyl, a hydroxy group, a linear or branched (C ₁ -C ₆ )alkoxy group, -S-(C ₁ -C ₆ )alkyl group, a cyano group, a nitro group, -(C ₀ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-Cy ₀₁ , -(C ₀ -C ₆ )alkyl-Cy ₀₁ , -(C ₂ -C ₆ )alkenyl-Cy ₀₁ , -(C ₂ -C ₆ )alkynyl-Cy ₀₁ , -O-(C ₁ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-(C ₁ -C ₆ )alkyl-R ₀₃₁ , -C(O)-OR ₀₁₁ , -OC(O)-R ₀₁₁ , -C(O)-NR ₀₁₁ R ₀₁₁ ', -NR ₀₁₁ -C(O)-R ₀₁₁ ', -NR ₀₁₁ -C(O)-OR ₀₁₁ ', -(C ₁ -C ₆ )alkyl-NR ₀₁₁ -C(O)-R ₀₁₁ ', -SO ₂ -NR ₀₁₁ R ₀₁₁ ' or -SO ₂ -(C ₁ -C ₆ )alkyl,

or a pair of (R ₀₂ , R ₀₃ ) or (R ₀₃ , R ₀₄ ) together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containing 5-7 ring members which optionally contains 1-3 heteroatoms selected from O, S and N, wherein the ring is optionally substituted with a group selected from linear or branched (C ₁ -C ₆ )alkyl, -NR ₀₁₃ R ₀₁₃ ', -(C ₀ -C ₆ )alkyl-Cy ₀₁ and oxo,

R ₀₆ and R ₀₇ independently of one another are a hydrogen atom, a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, a linear or branched (C ₁ -C ₆ )haloalkyl, a hydroxy group, a linear or branched (C ₁ -C ₆ )alkoxy group, -S-(C ₁ -C ₆ )alkyl group, a cyano group, a nitro group, -(C ₀ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', -O-Cy ₀₁ , -(C ₀ -C ₆ )alkyl-Cy ₀₁ , -(C ₂ -C ₆ )alkenyl-Cy ₀₁ , -(C ₂ -C ₆ )alkynyl-Cy ₀₁ , -O-(C ₁ -C ₆ )alkyl-R ₀₁₂ , -C(O)-OR ₀₁₁ , -OC(O)-R ₀₁₁ , -C(O)-NR ₀₁₁ R ₀₁₁ ', -NR ₀₁₁ -C(O)-R ₀₁₁ ', -NR ₀₁₁ -C(O)-OR ₀₁₁ ', -(C ₁ -C ₆ )alkyl-NR ₀₁₁ -C(O)-R ₀₁₁ ', -SO ₂ -NR ₀₁₁ R ₀₁₁ ' or -SO ₂ -(C ₁ -C ₆ )alkyl,

or a pair of (R ₀₆ , R ₀₇ ) fused with two adjacent carbon atoms, together with the carbon atoms to which they are attached, form an aromatic or non-aromatic ring containing 5-7 members in the ring, which optionally contains 1-3 heteroatoms selected from O, S and N, and wherein the resulting ring is optionally substituted with a group selected from a linear or branched (C ₁ -C ₆ )alkyl group, -NR ₀₁₃ R ₀₁₃ ', -(C ₀ -C ₆ )alkyl-Cy ₀₁ and oxo,

R ₀₈ is a hydrogen atom, a linear or branched (C ₁ -C ₈ )alkyl group, an aryl group, a heteroaryl group, an aryl(C ₁ -C ₆ )alkyl group or a heteroaryl(C ₁ -C ₆ )alkyl group,

R ₀₉ is a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, -Cy _O2 , -(C ₁ -C ₆ )alkyl-Cy _O2 , -(C ₂ -C ₆ )alkenyl-Cy _O2 , -(C ₂ -C ₆ )alkynyl-Cy _O2 , -Cy _O2 -Cy _O3 , -(C ₂ -C ₆ )alkynyl-O-Cy _O2 , -Cy _O2 -(C ₀ -C ₆ )alkyl-O-(C ₀ -C ₆ )alkyl-Cy _O3 , a halogen atom, a cyano group, -C(O)-R ₀₁₄ , -C(O)-NR ₀₁₄ R ₀₁₄ ',

R ₀₁₁ and R ₀₁₁ ' independently of one another are a hydrogen atom, an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group or -(C ₀ -C ₆ )alkyl-Cy ₀₁ ,

or a pair (R ₀₁₁ , R ₀₁₁ ') together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5-7 ring members, which optionally contains, in addition to the nitrogen atom, 1-3 heteroatoms selected from O, S and N, wherein the N atom is optionally substituted with a linear or branched (C ₁ -C ₆ )alkyl group, and wherein one or more carbon atoms of the linear or branched (C ₁ -C ₆ )alkyl group are optionally deuterated,

R ₀₁₂ is -Cy ₀₅ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-Cy ₀₆ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-O-(C ₀ -C ₆ )alkyl-Cy ₀₆ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-NR ₀₁₁ -(C ₀ -C ₆ )alkyl-Cy ₀₆ , -Cy ₀₅ -Cy ₀₆ -O-(C ₀ -C ₆ )alkyl-Cy ₀₇ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-Cy ₀₉ , -NH-C(O)-NH-R ₀₁₁ , -C(O)-NR ₀₁₁ R ₀₁₁ ', -NR ₀₁₁ R ₀₁₁ ', -OR ₀₁₁ , -NR ₀₁₁ -C(O)-R ₀₁₁ ', -O-(C ₁ -C ₆ )alkyl-OR ₀₁₁ , -SO ₂ -R ₀₁₁ or -C(O)-OR ₀₁₁ ,

R ₀₁₃ , R ₀₁₃ ', R ₀₁₄ and R ₀₁₄ ' independently of one another are a hydrogen atom or an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group,

Cy ₀₁ , Cy ₀₂ , Cy ₀₃ , Cy ₀₅ , Cy ₀₆ , Cy ₀₇ and Cy ₀₈ independently of one another are an optionally substituted cycloalkyl group, an optionally substituted heterocycloalkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group,

Cy ₀₉ is , where R ₀₁₅ , R ₀₁₆ and R ₀₁₇ are as defined for formula (I),

R ₀₃₁ is , where R ₀₂₇ and R ₀₂₈ are as defined for formula (I),

where at most one of the R ₀₃ , R ₀₉ or R ₀₁₂ groups, if present, is covalently attached to the linker,

or an enantiomer, diastereoisomer, atropisomer, deuterated derivative and/or pharmaceutically acceptable salt of any of the above.

In some embodiments, the drug group as described may comprise a compound of formula (III):

Where:

R ₀₁ is a linear or branched (C ₁ -C ₆ )alkyl group,

R ₀₃ is -O-(C ₁ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ' or

,

where R ₀₁₁ and R ₀₁₁ ' independently of one another are a hydrogen atom, an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group or -(C ₀ -C ₆ )alkyl-Cy ₀₁ ;

or a pair (R ₀₁₁ , R ₀₁₁ ') together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5-7 members in the ring, which optionally contains, in addition to the nitrogen atom, 1-3 heteroatoms selected from O, S and N, where the N atom may be substituted with 1 or 2 groups selected from a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

and where R ₀₂₇ is a hydrogen atom and R ₀₂₈ is a -(CH ₂ ) _p0 -O-SO ₂ -O- group or a -(CH ₂ ) _p0 -SO ₂ -OR ₀₃₀ group;

R ₀₉ is a linear or branched (C ₂ -C ₆ )alkynyl group or -Cy ₀₂ ,

R ₀₁₂ is -Cy ₀₅ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-Cy ₀₆ or -Cy ₀₅ -(C ₀ -C ₆ )alkyl-Cy ₀₉ ,

Cy ₀₁ , Cy ₀₂ , Cy ₀₅ and Cy ₀₆ independently of one another are a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, each of which is optionally substituted,

Cy ₀₉ is ;

p0, R ₀₁₅ , R ₀₁₆ and R ₀₁₇ are as defined for formula (I),

where at most one of the R ₀₃ , R ₀₉ or R ₀₁₂ groups, if present, is covalently attached to the linker,

or an enantiomer, diastereoisomer, atropisomer, deuterated derivative and/or pharmaceutically acceptable salt of any of the above.

In some embodiments, Cy ₀₁ , Cy ₀₂ , Cy ₀₃ , Cy ₀₄ , Cy ₀₅ , Cy ₀₆ , Cy ₀₇ , Cy ₀₈ and Cy ₀₁₀ , independently of one another, are an optionally substituted cycloalkyl group, an optionally substituted heterocycloalkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group, wherein the optional substituents are selected from optionally substituted linear or branched (C ₁ -C ₆ )alkyl, optionally substituted linear or branched (C ₂ -C ₆ )alkenyl group, optionally substituted linear or branched (C ₂ -C ₆ )alkynyl group, optionally substituted linear or branched (C ₁ -C ₆ )alkoxy, optionally substituted (C ₁ -C ₆ )alkyl-S-, hydroxy, oxo (or N-oxide, where possible), nitro, cyano, -C(O)-OR ₀ ', -OC(O)-R ₀ ', -C(O)-NR ₀ 'R ₀ '', -NR ₀ 'R ₀ '', -(C=NR ₀ ')-OR ₀ '', linear or branched (C ₁ -C ₆ )haloalkyl, trifluoromethoxy or halogen, where R ₀ ' and R ₀ '' each independently is a hydrogen atom or an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group, and where one or more of the carbon atoms of the linear or branched (C ₁ -C ₆ )alkyl group is optionally deuterated.

In some embodiments, the drug group (D) comprises:

or an enantiomer, diastereoisomer, atropisomer, deuterated derivative and/or pharmaceutically acceptable salt of any of the above.

Additionally, the drug group description may contain one of the following:

,

or .

In some embodiments, the linker-drug (or “linker-payload”) moiety -(L-D) may comprise compounds from Table A or an enantiomer, diastereoisomer, atropisomer, deuterated derivative, and/or pharmaceutically acceptable salt of any of the above.

Drug load

The drug load is represented by p and is also referred to herein as the drug-to-antibody ratio (DAR). The drug load can vary from 1 to 16 parts of drug per antigen-binding fragment. In some embodiments, p is an integer from 1 to 16. In some embodiments, p is an integer from 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is an integer from 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments, p is an integer from 1 to 16. In some embodiments, p is an integer from 1 to 8. In some embodiments, p is an integer from 1 to 5. In some embodiments, p is an integer from 2 to 4. In some embodiments, p is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, p is 2. In some embodiments, p is 4.

The drug load may be limited by the number of attachment sites of the antibody or antigen-binding fragment. In some embodiments, the linker group (L) of the ADC is attached to the antibody or antigen-binding fragment via a chemically active group on one or more amino acid residues on the antibody or antigen-binding fragment. For example, the linker can be attached to the antibody or antigen-binding fragment via a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., to the N- or C-terminus, to the epsilon amino group of one or more lysine residues, to the free carboxylic acid group of one or more glutamic acid or aspartic acid residues, or to the sulfhydryl group of one or more cysteine residues). The site to which the linker is attached may be a natural residue in the amino acid sequence of the antibody or antigen-binding fragment, or it may be introduced into the antibody or antigen-binding fragment, for example, by recombinant DNA technology (e.g., by introducing a cysteine residue into the amino acid sequence) or by protein biochemistry (e.g., by reduction, pH adjustment, or hydrolysis).

In some embodiments, the number of drug moieties that can be conjugated to an antibody or antigen-binding fragment is limited by the number of free cysteine residues. For example, if the attachment is a cysteine thiol group, the antibody may have only one or more cysteine thiol groups or may have only one or more sufficiently reactive thiol groups through which a linker can be attached. Antibodies generally do not contain a large number of free and reactive cysteine thiol groups that can be linked to a drug moiety. Indeed, most cysteine thiol groups in antibodies are involved in either interchain or intrachain disulfide bonds. Therefore, conjugation to cysteines in some embodiments may require at least partial reduction of the antibody. Excessive linker-toxin binding to an antibody can destabilize the antibody by reducing cysteine residues capable of forming disulfide bonds. Therefore, the optimal drug:antibody ratio should enhance the potency of the ADC (by increasing the number of attached drug moieties per antibody) without destabilizing the antibody or antigen-binding fragment. In some embodiments, the optimal ratio may be 2, 4, 6, or 8. In some embodiments, the optimal ratio may be 2 or 4.

In some embodiments, the antibody or antigen-binding fragment is exposed to reducing conditions prior to conjugation to generate one or more free cysteine residues. The antibody, in some embodiments, can be reduced with a reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) under partial or complete reducing conditions to generate reactive cysteine thiol groups. Unpaired cysteines can be formed by partial reduction with limited molar equivalents of TCEP, which can reduce the interchain disulfide bonds that link the light chain and heavy chain (one pair per H-L pair) and the two heavy chains in the hinge region (two pairs per H-H pair in the case of human IgG1), while leaving intrachain disulfide bonds intact (Stefano et al. (2013) Methods Mol Biol. 1045:145-71). In embodiments of the invention, disulfide bonds in antibodies are reduced electrochemically, for example, by using a working electrode that applies alternating reducing and oxidizing voltages. This approach can enable online coupling of disulfide reduction to an analytical device (e.g., an electrochemical detection device, an NMR spectrometer, or a mass spectrometer) or a chemical separation device (e.g., a liquid chromatograph (e.g., HPLC) or an electrophoresis device (see, for example, US 2014/0069822)). In some embodiments, the antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups on amino acid residues, such as cysteine.

The drug loading of an ADC can be controlled in a variety of ways, such as by: (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody; (ii) limiting the time or temperature of the conjugation reaction; (iii) partial or limiting reducing conditions for modification of the cysteine thiol; and/or (iv) recombinantly engineering the amino acid sequence of the antibody such that the number and position of cysteine residues is varied to control the number and/or position of linker-drug attachments.

In some embodiments, free cysteine residues are introduced into the amino acid sequence of an antibody or antigen-binding fragment. For example, cysteine engineered antibodies can be produced by replacing one or more amino acids of the parent antibody with the amino acid cysteine. Any form of antibody can be engineered in this manner, i.e., mutated. For example, a fragment of the parent Fab antibody can be engineered to form a cysteine-engineered Fab called a "ThioFab." Similarly, the parent monoclonal antibody can be engineered to form a "ThioMab." A single-site mutation results in one engineered cysteine residue in a ThioFab, whereas a single-site mutation results in two engineered cysteine residues in a ThioMab due to the dimeric nature of the IgG antibody. DNA encoding an amino acid sequence variant of a parent polypeptide can be prepared by a variety of methods known in the art (see, for example, the methods described in WO 2006/034488). These methods include, but are not limited to, site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared DNA encoding the polypeptide. Recombinant antibody variants can also be constructed by restriction fragment manipulation or by overlap-extension PCR with synthetic oligonucleotides. ADCs of formula (1) include, but are not limited to, antibodies that contain 1, 2, 3, or 4 engineered cysteine amino acids (Lyon et al. (2012) Methods Enzymol. 502:123-38). In some embodiments, one or more free cysteine residues are already present in the antibody or antigen-binding fragment without the use of engineering, in which case the existing free cysteine residues can be used to conjugate the antibody or antigen-binding fragment to a drug moiety.

If more than one nucleophilic group reacts with a drug-linker intermediate or a linker moiety reagent followed by a drug-moiety reagent in a reaction mixture containing multiple copies of an antibody or antigen-binding fragment and a linker moiety, then the resulting product may be a mixture of ADC compounds with a distribution of one or more drug moieties attached to each copy of the antibody or antigen-binding fragment in the mixture. In some embodiments, the drug load in the ADC mixture resulting from the conjugation reaction ranges from 1 to 16 drug moieties attached to the antibody or antigen-binding fragment. The average number of drug moieties per antibody or antigen-binding fragment (i.e., the average drug load or average p ) can be calculated by any conventional method known in the art, for example, using mass spectrometry (e.g., liquid chromatography-mass spectrometry (LC-MS)) and/or high performance liquid chromatography (e.g., HILC-HPLC). In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is determined using liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is from about 1.5 to about 3.5, from about 2.5 to about 4.5, from about 3.5 to about 5.5, from about 4.5 to about 6.5, from about 5.5 to about 7.5, from about 6.5 to about 8.5, or from about 7.5 to about 9.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is from about 2 to about 4, from about 3 to about 5, from about 4 to about 6, from about 5 to about 7, from about 6 to about 8, from about 7 to about 9, from about 2 to about 8, or from about 4 to about 8.

In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 2. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is 2.

In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 4. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, or about 4.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is 4.

In some embodiments, the term "about" when used in relation to the average number of drug moieties per antibody or antigen-binding fragment means plus or minus 20%, 15%, 10%, 5%, or 1%. In one embodiment, the term "about" refers to a range of values that are 10% greater than or less than the stated value. In another embodiment, the term "about" refers to a range of values that are 5% greater than or less than the stated value. In another embodiment, the term "about" refers to a range of values that are 1% greater than or less than the stated value.

Individual ADC compounds or "species" can be identified in the mixture using mass spectroscopy and separated, for example, by UPLC or HPLC, such as hydrophobic interaction chromatography (HIC-HPLC). In some embodiments, a homogeneous or nearly homogeneous ADC product with a single loading value can be isolated from the conjugation mixture, for example, by electrophoresis or chromatography.

In some embodiments, a higher drug load (e.g., p >16) may cause aggregation, insolubility, toxicity, or loss of cellular permeability of some antibody-drug conjugates. Increased drug loads may also negatively impact the pharmacokinetics (e.g., clearance) of some ADCs. In some embodiments, a lower drug load (e.g., p <2) may reduce the efficacy of certain ADCs against cells expressing the target. In some embodiments, the drug load for ADCs disclosed herein is from about 2 to about 16, from about 2 to about 10, from about 2 to about 8; from about 2 to about 6; from about 2 to about 5; from about 3 to about 5; from about 2 to about 4; or from about 4 to about 8.

In some embodiments, an average loading of about 2 is achieved, for example, by using a partial reduction in intrachain disulfides on the antibody or antigen-binding fragment, provides beneficial properties. In some embodiments, an average loading of about 4 or about 6 or about 8 is achieved, for example, by using a partial reduction in intrachain disulfides on the antibody or antigen-binding fragment, provides beneficial properties. In some embodiments, a drug loading and/or an average loading of less than about 2 may result in an unacceptably high level of unconjugated antibody species that may compete with the ADC for binding to the CD74 target antigen and/or provide a decrease in the effectiveness of the treatment. In some embodiments, a drug loading and/or an average drug loading of greater than about 16 may result in an unacceptably high level of product heterogeneity and/or ADC aggregation. An average drug loading of greater than 16 may also affect the stability of the ADC due to the loss of one or more chemical bonds necessary for stabilizing the antibody or antigen-binding fragment.

The present disclosure includes methods for producing the disclosed ADCs. Briefly, the ADCs comprise an antibody or antigen-binding fragment as the antibody or antigen-binding fragment, a drug moiety (e.g., an Mcl-1 inhibitor), and a linker that connects the drug moiety and the antibody or antigen-binding fragment. In some embodiments, the ADCs can be prepared using a linker having reactive functional groups for covalent attachment to the drug moiety and the antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is functionalized to provide a functional group that interacts with the linker or drug-linker intermediate. For example, in some embodiments, a cysteine thiol of the antibody or antigen-binding fragment can form a bond with a reactive functional group of the linker or drug-linker intermediate to form an ADC. In some embodiments, the antibody or antigen-binding fragment is produced with bacterial transglutaminase (BTG), reactive glutamines specifically functionalized with an amine-containing cyclooctyne BCN ( N -[( 1R , 8S , 9s )-bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl]-1,8-diamino-3,6-dioxaoctane) group. In some embodiments, site-specific conjugation of a linker or drug-linker intermediate to the BCN group of the antibody or antigen-binding fragment occurs, for example, as described and illustrated herein. The creation of ADCs can be carried out by methods known to one of skill in the art.

In some embodiments, an ADC is produced by contacting an antibody or antigen-binding fragment with a linker and a drug moiety (e.g., an Mcl-1 inhibitor) sequentially, such that the antibody or antigen-binding fragment is first covalently linked to the linker, and then a preformed antibody-linker intermediate is reacted with the drug moiety. The antibody-linker intermediate may or may not undergo a purification step before contact with the drug moiety. In other embodiments, an ADC is produced by contacting an antibody or antigen-binding fragment with a linker-drug compound previously formed by reacting the linker with the drug moiety. The preformed linker-drug compound may or may not undergo a purification step before contact with the antibody or antigen-binding fragment. In other embodiments, the antibody or antigen-binding fragment is contacted with a linker and a drug moiety in a single reaction mixture, allowing for the simultaneous formation of covalent bonds between the antibody or antigen-binding fragment and the linker, and between the linker and the drug moiety. This method for producing an ADC may include a reaction in which the antibody or antigen-binding fragment is contacted with the antibody or antigen-binding fragment before adding the linker to the reaction mixture, and vice versa. In some embodiments, the ADC is produced by reacting the antibody or antigen-binding fragment with a linker attached to a drug moiety, such as an Mcl-1 inhibitor, under conditions that allow conjugation.

ADCs obtained according to the methods described above can be subjected to a purification step. The purification step can include any biochemical methods known in the art for purifying proteins, or any combination thereof. These include, but are not limited to, tangential flow filtration (TFF), affinity chromatography, ion exchange chromatography, charge-based or isoelectric point chromatography, mixed-mode chromatography such as CHT (ceramic hydroxyapatite), hydrophobic interaction chromatography, size-exclusion chromatography, dialysis, filtration, selective precipitation, or any combination thereof.

Therapeutic uses and compositions

Described herein are methods for using the compositions described herein, for example, the ADC compounds and compositions described herein, in treating a subject for a disorder, for example, cancer. The compositions, for example, ADCs, can be administered alone or in combination with at least one additional inactive and/or active agent, for example, at least one additional therapeutic agent, and can be administered in any pharmaceutically acceptable composition, dosage, and dosing regimen. The effectiveness of the treatment can be assessed for toxicity, as well as efficacy indicators, and adjusted accordingly. Efficacy measures include, but are not limited to, a cytostatic and/or cytotoxic effect observed in vitro or in vivo, a reduction in tumor volume, inhibition of tumor growth, and/or prolonged survival.

Methods for determining whether an ADC has a cytostatic and/or cytotoxic effect on a cell are known. For example, the cytotoxic or cytostatic activity of an ADC can be measured, for example, by treating mammalian cells expressing the ADC's target antigen CD74 in cell culture medium; culturing the cells for a period of from about 6 hours to about 6 days; and measuring cell viability (e.g., using the CellTiter-Glo® (CTG) test or the MTT cell viability assay). In vitro cellular assays can also be used to measure the viability (proliferation), cytotoxicity, and apoptosis induction (caspase activation) of an ADC.

To determine cytotoxicity, necrosis or apoptosis (programmed cell death) can be measured. Necrosis is typically accompanied by increased cell membrane permeability, cell swelling, and membrane rupture. Apoptosis can be quantified, for example, by measuring DNA fragmentation. Commercial photometric assays for quantifying DNA fragmentation in vitro are available. Examples of such assays, including TUNEL (which detects the incorporation of labeled nucleotides into fragmented DNA) and ELISA-based assays, are described in Biochemica (1999) 2:34-7 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in the cell. For example, as with necrosis, loss of cell membrane integrity can be determined by measuring the uptake of certain dyes (e.g., a fluorescent dye such as acridine orange or ethidium bromide). A method for measuring the number of apoptotic cells was described by Duke and Cohen, Current Protocols in Immunology (Coligan et al., eds. (1992) pp. 3.17.1-3.17.16). Cells can also be labeled with a DNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide), and the cells are observed for chromatin condensation and clumping at the edge of the inflammatory site along the inner nuclear membrane. In some embodiments, apoptosis can also be determined by screening for caspase activity. In some embodiments, the Caspase-Glo® assay can be used to measure caspase-3 and caspase-7 activity. In some embodiments, the assay provides a luminogenic caspase-3/7 substrate in a reagent optimized for caspase activity, luciferase activity, and cell lysis. In some embodiments, addition of the Caspase-Glo® 3/7 reagent in an add-mix-measure format can result in cell lysis, followed by cleavage of the substrate by the caspase and the creation of a luminescent signal, such as a "glow-type" signal, produced by the luciferase. In some embodiments, the luminescence can be proportional to the amount of caspase activity present and can serve as an indicator of apoptosis. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane blebbing, and a decrease in cell size. Detection of any of these effects on cancer cells indicates that the ADC is useful in cancer treatment.

Cell viability can be measured, for example, by determining the cell uptake of a dye such as neutral red, trypan blue, crystal violet, or ALAMAR™ blue (see, e.g., Page et al. (1993) Intl J Oncology 3:473-6). In such an assay, cells are incubated in a medium containing the dye, the cells are washed, and the remaining dye, reflecting the cell uptake of the dye, is measured spectrophotometrically.

Cell viability can also be measured, for example, by quantifying ATP, an indicator of metabolically active cells. In some embodiments, the in vitro potency and/or cell viability of the resulting ADCs or Mcl-1 inhibitor compounds can be assessed using the CellTiter-Glo® Cell Viability Assay (CTG), as described in the examples provided herein. In this assay, in some embodiments, a single reagent (CellTiter-Glo® reagent) is added directly to cells cultured in serum-supplemented medium. The addition of the reagent results in cell lysis and the generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture.

Cell viability can also be measured, for example, by measuring the reduction of tetrazolium salts. In some embodiments, the in vitro potency and/or cell viability of the resulting ADCs or Mcl-1 inhibitor compounds can be assessed using the MTT cell viability assay, as described in the examples provided herein. In this assay, in some embodiments, yellow tetrazolium MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) is reduced by metabolically active cells, in part by dehydrogenase enzymes, to form reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan can then be solubilized and quantified spectrophotometrically.

In some aspects, the present disclosure relates to a method for killing, inhibiting, or modulating the growth of a cancer cell or tissue by disrupting the expression and/or activity of Mcl-1 and/or one or more upstream modulators or their downstream targets. The method can be used with any subject in whom disrupting the expression and/or activity of Mcl-1 provides a therapeutic effect. Subjects who may benefit from disrupting the expression and/or activity of Mcl-1 include, but are not limited to, those who have or are at risk of developing cancer, such as a tumor or hematological cancer. In some embodiments, the cancer is breast cancer, multiple myeloma, mantle cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer is lymphoma or gastric cancer.

In some embodiments, the disclosed ADCs can be administered to any cell or tissue that expresses BCMA, such as a tumor cell or tissue expressing BCMA. An exemplary embodiment includes a method of killing a cancer cell or tissue expressing BCMA. The method can be used with any cell or tissue expressing BCMA, such as a cancer cell or metastatic lesion. Non-limiting examples of BCMA-expressing cancer include multiple myeloma (Cho et al. (2018) Front Immunol. 9:1821). Non-limiting examples of BCMA-expressing cells include NCI-H929 multiple myeloma cells and cells containing a recombinant nucleic acid encoding BCMA, or a portion thereof.

In some embodiments, the disclosed ADCs can be administered to any cell or tissue that expresses CD33, such as a tumor cell or tissue expressing CD33. An exemplary embodiment includes a method of killing CD33-expressing cancer cells or tissues. The method can be used with any cell or tissue expressing CD33, such as a cancer cell or metastatic lesion. Non-limiting examples of cancers expressing CD33 include colorectal cancer, pancreatic cancer, lymphoma, and leukemia (e.g., acute myeloid leukemia) (Human Protein Atlas; Walter (2014) Expert Opin Ther Targets 18(7):715-8). Non-limiting examples of cells expressing CD33 include MOLM-13 leukemia cells and cells containing a recombinant nucleic acid encoding CD33 or a portion thereof.

In some embodiments, the disclosed ADCs can be administered to any cell or tissue that expresses PCAD, such as cancer cells or tissues expressing PCAD. An exemplary embodiment includes a method of killing a cancer cell or tissue expressing PCAD. The method can be used with any cell or tissue expressing PCAD, such as a cancer cell or metastatic lesion. Non-limiting examples of cancers expressing PCAD include breast cancer, gastric cancer, endometrial cancer, ovarian cancer, pancreatic cancer, bladder cancer, prostate cancer, and melanoma (Vieira and Paredes (2015) Mol Cancer 14:178).

In some embodiments, the disclosed ADCs can be administered to any cell or tissue expressing HER2, such as HER2-expressing cancer cells or tissues. An exemplary embodiment includes a method of killing a cancer cell or tissue expressing HER2. The method can be used with any cell or tissue expressing HER2, such as a cancer cell or metastatic lesion. Non-limiting examples of malignant tumors expressing HER2 include breast cancer, gastric cancer, bladder cancer, urothelial cell carcinoma, esophageal cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., endometrial serous carcinoma of the uterus), salivary duct carcinoma, cervical cancer, endometrial cancer, and ovarian cancer (English et al. (2013) Mol Diagn Ther. 17:85-99). Non-limiting examples of HER2-expressing cells include HCC1954 and HCC2218 breast cancer cells, as well as cells containing recombinant nucleic acid encoding HER2 or a portion thereof.

Exemplary methods include the steps of contacting a cell with an ADC, as described herein, in an effective amount, i.e., an amount sufficient to kill the cell. The method can be used on cells in culture, such as in vitro, in vivo, ex vivo, or in situ. For example, cells that express HER2 (e.g., cells collected from a tumor or metastatic lesion biopsy; cells from an established cancer cell line; or recombinant cells) can be cultured in vitro in a culture medium, and the contacting step can be affected by adding the ADC to the culture medium. The method results in the killing of HER2-expressing cells, including, in particular, HER2-expressing cancer cells. Alternatively, the ADC can be administered to the subject by any suitable route (e.g., intravenously, subcutaneously, or by direct contact with tumor tissue) to exert an effect in vivo. This approach can be applied to antibodies targeting other cell surface antigens (e.g., BCMA, CD33, PCAD).

The in vivo effect of the described ADC therapeutic composition can be assessed in a suitable animal model. For example, xenogenic cancer models can be used, in which cancer explants or passaged xenograft tissues are administered to immunocompromised animals, such as nude or SCID mice (Klein et al. (1997) Nature Med. 3:402-8). Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression, or metastasis, among other things.

In vivo assays that assess the promotion of tumor death by mechanisms such as apoptosis can also be used. In some embodiments, xenografts from tumor-bearing mice treated with a therapeutic composition can be examined for apoptotic foci and compared with untreated control mice bearing the xenograft. The extent to which apoptotic foci are detected in the tumors of treated mice indicates the therapeutic efficacy of the composition.

The present document also provides methods for treating a disorder, such as cancer. The compositions described herein, such as the ADCs described herein, can be administered to a non-human mammal or a human for therapeutic purposes. The therapeutic methods include administering to a subject having cancer or suspected of having cancer a therapeutically effective amount of a composition comprising an Mcl-1 inhibitor, such as an ADC, wherein the inhibitor is linked to a targeting antibody that binds to an antigen (1) expressed on a cancer cell (2) accessible for binding, and/or (3) localized or preferentially expressed on the surface of a cancer cell compared to a non-cancerous cell.

An exemplary embodiment is a method of treating a subject having or suspected of having cancer, comprising administering to the subject a therapeutically effective amount of a composition described herein, e.g., an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions described herein). In some embodiments, the cancer expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2, Nectin4, TROP2, LIV1, CD46, or GPNMB. In some embodiments, the target antigen is 4-1BB, 5AC, 5T4, alpha-fetoprotein, angiopoietin 2, ASLG659, TCLI, BMPRIB, Brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (mimicking), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR I), CD22 (B cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B cell differentiation antigen CD72), CD79a, CD80, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), Epicialin, ERBB3, ETBR (endothelin receptor type B), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extra domain-B, burn receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, receptor kinase human scattering factor, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily translocation receptor 2-associated), Lewis-Y antigen, LY64 (RP105), MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen, PSCA (prostate stem cell antigen precursor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-associated gene I, TAG-72, TEMI, Tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential-acting cation channel superfamily transient receptor M member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or Vimentin. In some embodiments, the target antigen is BCMA, CD33, PCAD, HER2, CD38, CD46, CD48, or CD79b. In some embodiments, the target antigen is BCMA, CD33, CD48, PCAD, or HER2. In some embodiments, the target antigen is CD38 or CD48. In some embodiments, the cancer is a tumor or a hematological cancer. In some embodiments, the cancer is breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer is lymphoma or gastric cancer.

Another exemplary embodiment is a method for delivering an Mcl-1 inhibitor to a cell expressing BCMA, comprising conjugating the Mcl-1 inhibitor to an antibody that immunospecifically binds to an epitope of BCMA and exposing the cell to the ADC. Typical cancer cells that express BCMA, for which the ADCs of the present invention are indicated, include multiple myeloma cells.

Another exemplary embodiment is a method of delivering an Mcl-1 inhibitor to a cell expressing CD33, comprising conjugating the Mcl-1 inhibitor to an antibody that immunospecifically binds to an epitope of CD33 and exposing the cell to an ADC. Typical cancer cells expressing CD33 for which the ADCs of the present invention are indicated include leukemia cells.

Another exemplary embodiment is a method for delivering an Mcl-1 inhibitor to a cell expressing PCAD, comprising conjugating the Mcl-1 inhibitor to an antibody that immunospecifically binds to an epitope of PCAD and exposing the cell to an ADC. Exemplary cancer cells expressing PCAD, for which the ADCs of the present disclosure are indicated, include breast cancer and gastric cancer cells.

Another exemplary embodiment is a method for delivering an Mcl-1 inhibitor to a cell expressing HER2, comprising conjugating the Mcl-1 inhibitor to an antibody that immunospecifically binds to an epitope of HER2 and exposing the cell to an ADC. Typical cancer cells expressing HER2, for which ADCs are specified according to the present disclosure, include breast cancer cells.

In some aspects, the present disclosure further provides methods for reducing or inhibiting tumor growth (e.g., a BCMA-expressing tumor, a CD33-expressing tumor, a PCAD-expressing tumor, a HER2-expressing tumor) comprising administering a therapeutically effective amount of an ADC or a composition comprising an ADC. In some embodiments, the treatment is sufficient to reduce or suppress tumor growth in a patient, reduce the number or size of metastatic lesions, reduce tumor burden, reduce primary tumor burden, reduce invasiveness, prolong survival time, and/or maintain or improve quality of life. In some embodiments, the tumor is resistant or refractory to treatment with an antibody or antigen-binding fragment of an ADC (e.g., an anti-BCMA antibody, an anti-CD33 antibody, an anti-PCAD antibody, an anti-HER2 antibody) when administered alone, and/or the tumor is resistant or refractory to treatment with an Mcl-1 inhibitor drug group when administered alone.

An exemplary embodiment is a method of reducing or inhibiting tumor growth in a subject, comprising administering to the subject a therapeutically effective amount of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions described herein). In some embodiments, the tumor expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2, Nectin4, TROP2, LIV1, CD46, or GPNMB. In some embodiments, the target antigen is 4-1BB, 5AC, 5T4, alpha-fetoprotein, angiopoietin 2, ASLG659, TCLI, BMPRIB, Brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (mimicking), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR I), CD22 (B cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B cell differentiation antigen CD72), CD79a, CD80, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), Epicialin, ERBB3, ETBR (endothelin receptor type B), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extra domain-B, burn receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, receptor kinase human scattering factor, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily translocation receptor 2-associated), Lewis-Y antigen, LY64 (RP105), MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen, PSCA (prostate stem cell antigen precursor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-associated gene I, TAG-72, TEMI, Tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential-acting cation channel superfamily transient receptor M member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or Vimentin. In some embodiments, the target antigen is BCMA, CD33, PCAD, HER2, CD38, CD46, CD48, or CD79b. In some embodiments, the target antigen is BCMA, CD33, CD48, PCAD, or HER2. In some embodiments, the target antigen is CD38 or CD48. In some embodiments, the tumor is breast cancer, gastric cancer, bladder cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer, hepatocellular carcinoma, melanoma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the tumor is gastric cancer. In some embodiments, administration of the ADC, composition, or pharmaceutical composition reduces or inhibits tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, compared to untreated growth.

Another exemplary embodiment is a method for delaying or slowing tumor growth in a subject, comprising administering to the subject a therapeutically effective amount of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein). In some embodiments, the tumor expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2, Nectin4, TROP2, LIV1, CD46, or GPNMB. In some embodiments, the target antigen is 4-1BB, 5AC, 5T4, alpha-fetoprotein, angiopoietin 2, ASLG659, TCLI, BMPRIB, Brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (mimicking), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR I), CD22 (B cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B cell differentiation antigen CD72), CD79a, CD80, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), Epicialin, ERBB3, ETBR (endothelin receptor type B), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extra domain-B, burn receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, receptor kinase human scattering factor, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily translocation receptor 2-associated), Lewis-Y antigen, LY64 (RP105), MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen, PSCA (prostate stem cell antigen precursor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-associated gene I, TAG-72, TEMI, Tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential-acting cation channel superfamily transient receptor M member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or Vimentin. In some embodiments, the target antigen is BCMA, CD33, PCAD, HER2, CD38, CD46, CD48, or CD79b. In some embodiments, the target antigen is BCMA, CD33, CD48, PCAD, or HER2. In some embodiments, the target antigen is CD38 or CD48. In some embodiments, the tumor is breast cancer, gastric cancer, bladder cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer, hepatocellular carcinoma, melanoma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the tumor is gastric cancer. In some embodiments, administration of the ADC, composition, or pharmaceutical composition delays or slows tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, compared to untreated growth.

In some aspects, the present disclosure further provides methods for reducing or slowing the growth of a population of cancer cells (e.g., a population of cancer cells expressing BCMA, a population of cancer cells expressing CD33, a population of cancer cells expressing PCAD, a population of cancer cells expressing HER2), comprising administering a therapeutically effective amount of an ADC or a composition comprising an ADC.

An exemplary embodiment is a method for reducing or slowing the growth of a cancer cell population in a subject, comprising administering to the subject a therapeutically effective amount of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions described herein). In some embodiments, the cancer cell population expresses a target antigen. In some embodiments, the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2, Nectin4, TROP2, LIV1, CD46, or GPNMB. In some embodiments, the target antigen is 4-1BB, 5AC, 5T4, alpha-fetoprotein, angiopoietin 2, ASLG659, TCLI, BMPRIB, Brevican BCAN, BEHAB, C242 antigen, C5, CA-125, CA-125 (mimicking), CA-IX (carbonic anhydrase 9), CCR4, CD140a, CD152, CD19, CD20, CD200, CD21 (C3DR I), CD22 (B cell receptor CD22-B isoform), CD221, CD23 (gE receptor), CD28, CD30 (TNFRSF8), CD37, CD4, CD40, CD44 v6, CD51, CD52, CD70, CD72 (Lyb-2, B cell differentiation antigen CD72), CD79a, CD80, CEA, CEA-related antigen, ch4D5, CLDN18.2, CRIPTO (CR, CRI, CRGF, TDGF1), CTLA-4, CXCR5, DLL4, DR5, E16 (LATI, SLC7A5), EGFL7, EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5), Epicialin, ERBB3, ETBR (endothelin receptor type B), FCRHI (Fc receptor-like protein I), FcRH2 (IFGP4, IRTA4, SPAPI, SPAP IB, SPAP IC), fibronectin extra domain-B, burn receptor, GD2, GD3 ganglioside, GEDA, HER1, HER2/neu, HER3, HGF, HLA-DOB, HLA-DR, receptor kinase human scattering factor, IGF-I receptor, IL-13, IL20R (ZCYTOR7), IL-6, ILGF2, ILFRIR, integrin u, IRTA2 (immunoglobulin superfamily translocation receptor 2-associated), Lewis-Y antigen, LY64 (RP105), MCP-I, MDP (DPEPI), MPF, MSLN, SMR, mesothelin, megakaryocyte, PD-I, PDCDI, PDGF-R u, prostate-specific membrane antigen, PSCA (prostate stem cell antigen precursor), PSCA hlg, RANKL, RON, SDCI, Sema Sb, STEAP I, STEAP2, PCANAP I, STAMP I, STEAP2, STMP, prostate cancer-associated gene I, TAG-72, TEMI, Tenascin C, TENB2, (TMEFF2, tomoregulin, TPEF, HPPI, TR), TGF-IJ, TRAIL-E2, TRAIL-R1, TRAIL-R2, T17M4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential-acting cation channel superfamily transient receptor M member 4), TWEAK-R, TYRP I (glycoprotein 75), VEGF, VEGF-A, EGFR-I, VEGFR-2, or Vimentin. In some embodiments, the target antigen is BCMA, CD33, PCAD, HER2, CD38, CD46, CD48, or CD79b. In some embodiments, the target antigen is BCMA, CD33, CD48, PCAD, or HER2. In some embodiments, the target antigen is CD38 or CD48. In some embodiments, the population of cancer cells is derived from a tumor or hematological cancer. In some embodiments, the population of cancer cells is derived from breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer cell population is derived from lymphoma or gastric cancer. In some embodiments, administration of the ADC, composition, or pharmaceutical composition reduces the cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% compared to the untreated population. In some embodiments, administration of the ADC, composition, or pharmaceutical composition slows the growth of a cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, compared to untreated proliferation.

The present document also provides methods for determining whether a subject having or suspected of having cancer will respond to treatment with the described ADCs and compositions. An exemplary embodiment is a method for determining whether a subject having or suspected of having cancer will respond to treatment with an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions described herein) by providing a biological sample from the subject; contacting the sample with an ADC; and detecting binding of the ADC to cancer cells in the sample. In some embodiments, the sample is a tissue biopsy sample, a blood sample, or a bone marrow sample. In some embodiments, the method comprises providing a biological sample from the subject; contacting the sample with an ADC; and detecting one or more markers of cancer cell death in the sample (e.g., increased expression of one or more apoptotic markers, decreased growth of a cancer cell population in culture, etc.).

Furthermore, therapeutic uses of the described ADCs and compositions are provided herein. An exemplary embodiment is an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions described herein) for use in treating a subject having or suspected of having a cancer (e.g., a BCMA-expressing cancer, a CD33-expressing cancer, a PCAD-expressing cancer, a HER2-expressing cancer). Another exemplary embodiment is the use of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions described herein) in treating a subject having or suspected of having a cancer (e.g., a BCMA-expressing cancer, a CD33-expressing cancer, a PCAD-expressing cancer, a HER2-expressing cancer). Another exemplary embodiment is the use of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions described herein) in a method for the manufacture of a medicament for the treatment of a subject suffering from or suspected of having a cancer (e.g., a BCMA-expressing cancer, a CD33-expressing cancer, a PCAD-expressing cancer, a HER2-expressing cancer). Methods for identifying subjects suffering from a cancer that expresses a target antigen (e.g., BCMA, CD33, PCAD, HER2) are known in the art and can be used to identify patients suitable for treatment with the described compound or ADC composition.

Furthermore, the ADCs of the present invention can be administered to a non-human mammal expressing an antigen to which the ADC is capable of binding, for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models can be useful for assessing the therapeutic efficacy of the described ADCs (e.g., testing dosages and administration time courses).

Therapeutic compositions used in the practice of the above methods can be included in pharmaceutical compositions containing a pharmaceutically acceptable carrier suitable for the desired delivery route. A typical embodiment is a pharmaceutical composition comprising an ADC as described herein and a pharmaceutically acceptable carrier, for example, one suitable for the selected route of administration, for example, intravenous administration. The pharmaceutical composition may also contain one or more additional inactive and/or therapeutic agents that are suitable for the treatment or prevention of, for example, cancer (e.g., a standard treatment, etc.). The pharmaceutical composition may also contain one or more carrier, excipient, and/or stabilizer components, and the like. Methods for formulating such pharmaceutical compositions and suitable formulations are known in the art (see, for example, "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA).

Suitable carriers include any material that, when combined with the therapeutic composition, maintains the antitumor function of the therapeutic composition and does not typically react with the patient's immune system. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of the following: water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, mesylate, and the like, as well as combinations thereof. In many cases, isotonic agents are included in the formulation, for example, sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride. Pharmaceutically acceptable carriers may additionally contain minor amounts of excipients such as wetting or emulsifying agents, preservatives, or buffers that enhance the shelf life or potency of the ADC.

The pharmaceutical composition of the present invention can be administered by a variety of routes known in the art. The route and/or method of administration can vary depending on the desired results. In some embodiments, the therapeutic composition is solubilized and administered by any route capable of delivering the therapeutic composition to the site of cancer. Potentially effective routes of administration include, but are not limited to, parenteral (e.g., intravenous, subcutaneous), intraperitoneal, intramuscular, intratumoral, intradermal, intraorgan, orthotopic, and the like. In some embodiments, administration is intravenous, subcutaneous, intraperitoneal, or intramuscular. The pharmaceutically acceptable carrier should be suitable for the route of administration, for example, intravenous or subcutaneous administration (e.g., by injection or infusion). Depending on the route of administration, the active compound(s), i.e., the ADC and/or any additional therapeutic agent, may be coated with a material to protect the compound(s) from acids and other environmental conditions that may inactivate the compound(s). Administration may be systemic or topical.

The therapeutic compositions described herein can be sterile and stable under the conditions of manufacture and storage, and can be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injection and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The form depends on the intended route of administration and therapeutic use. In some embodiments, the described ADCs can be included in a pharmaceutical composition suitable for parenteral administration. The injection solution can consist of either a liquid or lyophilized dosage form in a flint or amber vial, ampoule, pre-filled syringe, or other known delivery or storage device. In some embodiments, one or more ADCs or pharmaceutical compositions are supplied as a dry, sterilized, lyophilized powder or anhydrous concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to a suitable concentration for administration to a subject.

Typically, a therapeutically effective amount or an effective amount of the described composition, for example, the described ADC, is used in the pharmaceutical compositions described herein. A composition, for example, containing an ADC, can be formulated into a pharmaceutically acceptable dosage form using conventional methods known in the art. Dosages and administration protocols for cancer treatment using the aforementioned methods will vary depending on the method and the target cancer and will generally depend on a number of other factors considered in the art.

Dosage regimens for the compositions described herein, for example, those containing an ADC alone or in combination with one additional inactive and/or active therapeutic agent, can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus of one or both agents can be administered at one time, several divided doses can be administered over a predetermined period of time, or the dose of one or both agents can be proportionally increased or decreased as indicated by the exigency of the therapeutic situation. In some embodiments, treatment involves a single bolus or repeated administration of the ADC preparation by a suitable route of administration. In some embodiments, the ADC is administered to the patient daily, weekly, monthly, or at any time in between. For any particular subject, specific dosing regimens can be adjusted over time in accordance with individual needs and the professional judgment of the treating physician. Compositions for parenteral administration can be formulated as a unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier.

Dosage values for compositions containing an ADC and/or any additional therapeutic agent(s) can be selected based on the unique characteristics of the active compound(s) and the specific therapeutic effect to be achieved. A physician or veterinarian can begin administering doses of the ADC used in the pharmaceutical composition at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. In general, effective doses of the compositions of the present invention for the treatment of cancer can vary depending on many different factors, including the route of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. The selected dosage level may also depend on a variety of pharmacokinetic factors, including the activity of the specific compositions or their ester, salt, or amide; the route of administration; the time of administration; the rate of elimination of the specific compound; the duration of treatment; other drugs, compounds, and/or materials used in combination with the specific compositions; the age, gender, body weight, condition, general health, and previous medical history of the patient being treated; and similar factors. Treatment dosages may be titrated to optimize safety and efficacy.

The toxicity and therapeutic efficacy of the compounds described herein can be determined using standard pharmaceutical procedures in cell culture or animal models. For example, LD50, ED50, EC50, and IC50 can be determined, and the dose ratio between toxic and therapeutic effects (LD50/ED50) can be calculated as a therapeutic index. Data obtained from in vitro and in vivo assays can be used to estimate or determine a dose range for use in humans. For example, the compositions and methods described herein can initially be evaluated in xenogeneic cancer models (e.g., the NCI-H929 mouse model of multiple myeloma).

In some embodiments, the ADC or ADC-containing composition is administered once. In other embodiments, the ADC or ADC-containing composition is administered multiple times. The intervals between single doses can be, for example, daily, weekly, monthly, or annually. The intervals can also be irregular, based on measuring the levels of the administered agent (e.g., ADC) in the patient's blood, in order to maintain a relatively constant plasma concentration of the agent. The dosage and frequency of administration of the ADC or ADC-containing composition can also vary depending on whether the treatment is prophylactic or therapeutic. For prophylactic purposes, a relatively small dose can be administered at relatively infrequent intervals over an extended period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively higher dosage at relatively shorter intervals is sometimes required until the progression of the disease is reduced or stopped and, possibly, until the patient shows a partial or complete improvement in one or more symptoms of the disease. After this, the patient may be prescribed a lower dose, for example, a prophylactic regimen.

The aforementioned therapeutic approaches may be combined with any of a variety of additional surgical, chemotherapeutic, or radiation therapy regimens. In some embodiments, the ADCs or compositions described herein are co-formulated and/or co-administered with one or more additional therapeutic agents, such as one or more chemotherapeutic agents or one or more standard agents for the specific condition being treated.

Also provided are kits for use in the therapeutic and/or diagnostic applications described herein. Such kits may include a carrier, package, or container that is divided into compartments for receiving one or more containers, such as vials, tubes, and the like, where each container contains one of the individual elements used in the method described herein. A label may be present on or with the container(s) indicating that the ADC or composition in the kit is used for a specific therapy or non-therapeutic application, such as prognostic, prophylactic, diagnostic, or laboratory use. The label may also indicate directions for in vivo or in vitro use, such as those described herein. Directions and other information may also be included in package inserts or label(s) that are in or on the kit. The label may be on or associated with the container. The label may be on the container when the letters, numbers, or other characters that form the label are molded or etched on the container itself. A label may be associated with the container when present in a container or carrier that also supports the container, such as a package insert. The label may indicate that the ADC or composition in the kit is used to diagnose or treat a condition, such as cancer, as described herein.

In some embodiments, the kit comprises an ADC or a composition comprising an ADC. In some embodiments, the kit further comprises one or more additional components, including, but not limited to: instructions for use; other reagents, such as a therapeutic agent (e.g., a standard of care agent); devices, containers, or other materials for preparing the ADC for administration; pharmaceutically acceptable carriers; and devices, containers, or other materials for administering the ADC to a subject. The instructions for use may include guidance on therapeutic use, including suggested dosages and/or routes of administration, such as for a patient with or suspected of having cancer. In some embodiments, the kit comprises an ADC and instructions for using the ADC to treat, prevent, and/or diagnose cancer.

COMBINATION THERAPIES

In some embodiments, the present disclosure provides methods of treatment in which the antibody-drug conjugates described herein are administered in combination with one or more additional therapeutic agents. Examples of combination partners are described herein.

In some embodiments, the combination described herein comprises a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is selected from PDR001 (Novartis), Nivolumab (Bristol-Myers Squibb), Pembrolizumab (Merck & Co), Pidilizumab (CureTech), MEDI0680 (Medimmune), REGN2810 (Regeneron), TSR-042 (Tesaro), PF-06801591 (Pfizer), BGB-A317 (Beigene), BGB-108 (Beigene), INCSHR1210 (Incyte), or AMP-224 (Amplimmune). In some embodiments, the PD-1 inhibitor is PDR001. PDR001 is also known as spartalizumab.

In some embodiments, the combination described herein comprises a LAG-3 inhibitor. In some embodiments, the LAG-3 inhibitor is selected from LAG525 (Novartis), BMS-986016 (Bristol-Myers Squibb), or TSR-033 (Tesaro).

In some embodiments, the combination described herein comprises a TIM-3 inhibitor. In some embodiments, the TIM-3 inhibitor is MBG453 (Novartis), TSR-022 (Tesaro), LY-3321367 (Eli Lilly), Sym23 (Symphogen), BGB-A425 (Beigene), INCAGN-2390 (Agenus), BMS-986258 (BMS), RO-7121661 (Roche), or LY-3415244 (Eli Lilly).

In some embodiments, the combination described herein comprises a PDL1 inhibitor. In one embodiment, the PDL1 inhibitor is selected from FAZ053 (Novartis), atezolizumab (Genentech), durvalumab (Astra Zeneca), or avelumab (Pfizer).

In some embodiments, the combination described herein comprises a GITR agonist. In some embodiments, the GITR agonist is selected from GWN323 (NVS), BMS-986156, MK-4166 or MK-1248 (Merck), TRX518 (Leap Therapeutics), INCAGN1876 (Incyte/Agenus), AMG 228 (Amgen), or INBRX-110 (Inhibrx).

In some embodiments, the combination described herein comprises an IAP inhibitor. In some embodiments, the IAP inhibitor comprises LCL161 or a compound described in International Publication No. WO 2008/016893.

In one embodiment, the combination comprises an mTOR inhibitor, such as RAD001 (also known as everolimus).

In one embodiment, the combination comprises an HDAC inhibitor, such as LBH589. LBH589 is also known as panobinostat.

In one embodiment, the combination comprises an IL-17 inhibitor, such as CJM112.

In some embodiments, the combination described herein comprises an estrogen receptor (ER) antagonist. In some embodiments, the estrogen receptor antagonist is used in combination with a PD-1 inhibitor, a CDK4/6 inhibitor, or both. In some embodiments, the combination is used to treat ER-positive (ER+) cancer or breast cancer ( e.g., ER+ breast cancer).

In some embodiments, the estrogen receptor antagonist is a selective estrogen receptor modulator (SERD). SERDs are estrogen receptor antagonists that bind to the receptor and result in,For example, To degradation or suppression of the receptor (Boer K.et al., (2017)Therapeutic Advances in Medical Oncology9(7): 465-479). ER is a hormone-activated transcription factor that is important for,For example, growth, development and physiology of the human reproductive system. ER is activated,For example, the hormone estrogen (17beta estradiol). ER expression and signaling are involved in cancer (For example,breast cancer),For example, ER positive (ER+) breast cancer. In some embodiments, the SERD is selected from LSZ102, fulvestrant, brilanestrant, or elacestrant.

In some embodiments, the SERD comprises a compound described in International Publication No. WO 2014/130310, which is incorporated herein by reference in its entirety.

In some embodiments, the SERD comprises LSZ102. LSZ102 has the chemical name: (E)-3-(4-((2-(2-(1,1-difluoroethyl)-4-fluorophenyl)-6-hydroxybenzo[b]thiophen-3-yl)oxy)phenyl)acrylic acid. In some embodiments, the SERD comprises fulvestrant (CAS Registry Number: 129453-61-8) or a compound described in International Publication No. WO 2001/051056, which is incorporated herein by reference in its entirety. In some embodiments, the SERD comprises elacestrant (CAS Registry Number: 722533-56-4) or a compound described in U.S. Patent No. 7,612,114, which is incorporated herein by reference in its entirety. Elacetrant is also known as RAD1901, ER-306323, or (6R)-6-{2-[ethyl({4-[2-(ethylamino)ethyl]phenyl}methyl)amino]-4-methoxyphenyl}-5,6,7,8-tetrahydronaphthalen-2-ol. Elacetrant is an orally bioavailable, nonsteroidal, combined selective estrogen receptor modulator (SERM) and SERD. Elacetrant is also described in, e.g. , Garner F et al. (2015) Anticancer Drugs 26(9):948-56. In some embodiments, the SERD is brilanestrant (CAS Registry Number: 1365888-06-7) or a compound described in International Publication No. WO 2015/136017, which is incorporated by reference in its entirety.

In some embodiments, the SERD is selected from RU 58668, GW7604, AZD9496, bazedoxifene, pipendoxifene, arzoxifene, OP-1074, or acolbifene, such as described in McDonell et al. (2015) Journal of Medicinal Chemistry 58(12) 4883-4887.

Other exemplary estrogen receptor antagonists are described, for example, in WO 2011/156518, WO 2011/159769, WO 2012/037410, WO 2012/037411, and US 2012/0071535, all of which are incorporated herein by reference in their entireties.

In some embodiments, the combination described herein comprises a cyclin-dependent kinase 4 or 6 (CDK4/6) inhibitor. In some embodiments, the CDK4/6 inhibitor is used in combination with a PD-1 inhibitor, an estrogen receptor (ER) antagonist, or both. In some embodiments, the combination is used to treat ER-positive (ER+) cancer or breast cancer ( e.g., ER+ breast cancer). In some embodiments, the CDK4/6 inhibitor is selected from ribociclib, abemaciclib (Eli Lilly), or palbociclib.

In some embodiments, the CDK4/6 inhibitor comprises ribociclib (CAS Registry Number: 1211441-98-3) or a compound described in U.S. Patent Nos. 8,415,355 and 8,685,980, which are incorporated by reference in their entireties.

In some embodiments, the CDK4/6 inhibitor comprises a compound described in International Publication No. WO 2010/020675 and U.S. Patent Nos. 8,415,355 and 8,685,980, which are incorporated by reference in their entireties.

In some embodiments, the CDK4/6 inhibitor comprises ribociclib (CAS Registry Number: 1211441-98-3). Ribociclib is also known as LEE011, KISQALI®, or 7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide.

In some embodiments, the CDK4/6 inhibitor comprises abemaciclib (CAS Registry Number: 1231929-97-7). Abemaciclib is also known as LY835219 or N-[5-[(4-ethyl-1-piperazinyl)methyl]-2-pyridinyl]-5-fluoro-4-[4-fluoro-2-methyl-1-(1-methylethyl)-1H-benzimidazol-6-yl]-2-pyrimidinamine. Abemaciclib is a CDK inhibitor selective for CDK4 and CDK6 and is described, for example , in Torres-Guzman R et al. (2017) Oncotarget 10.18632/oncotarget.17778.

In some embodiments, the CDK4/6 inhibitor comprises palbociclib (CAS Registry Number: 571190-30-2). Palbociclib is also known as PD-0332991, IBRANCE®, or 6-acetyl-8-cyclopentyl-5-methyl-2-{[5-(1-piperazinyl)-2-pyridinyl]amino}pyrido[2,3-d]pyrimidin-7(8H)-one. Palbociclib inhibits CDK4 with an IC50 of 11 nM and inhibits CDK6 with an IC50 of 16 nM and is described, for example , in Finn et al. (2009) Breast Cancer Research 11(5):R77.

In some embodiments, the combination described herein comprises a chemokine (C-X-C motif) receptor 2 (CXCR2) inhibitor. In some embodiments, the CXCR2 inhibitor is selected from 6-chloro-3-((3,4-dioxo-2-(pentan-3-ylamino)cyclobut-1-en-1-yl)amino)-2-hydroxy-N-methoxy-N-methylbenzenesulfonamide, danirixin, reparixin, or navarixin.

In some embodiments, the CSF-1/1R binding agent is selected from a macrophage colony-stimulating factor (M-CSF) inhibitor, such as a monoclonal antibody or Fab to M-CSF ( e.g. , MCS110), a CSF-1R tyrosine kinase inhibitor ( e.g., 4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide or BLZ945), a receptor tyrosine kinase (RTK) inhibitor ( e.g., pexidartinib), or an antibody targeting CSF-1R ( e.g. , emactuzumab or FPA008). In some embodiments, the CSF-1/1R inhibitor is BLZ945. In some embodiments, the CSF-1/1R binding agent is MCS110. In other embodiments, the CSF-1/1R binding agent is pexidartinib.

In some embodiments, the combination described herein comprises a c-MET inhibitor. C-MET, a receptor tyrosine kinase overexpressed or mutated in many types of tumor cells, plays a key role in tumor cell proliferation, survival, invasion, metastasis, and tumor angiogenesis. Inhibition of c-MET can induce cell death in tumor cells overexpressing the c-MET protein or expressing constitutively activated c-MET protein. In some embodiments, the c-MET inhibitor is selected from capmatinib (INC280), JNJ-3887605, AMG 337, LY2801653, MSC2156119J, crizotinib, tivantinib, or golvatinib.

In some embodiments, the combination described herein comprises a transforming growth factor beta (also known as TGF-β, TGFβ, TGFb, or TGF-beta, used interchangeably herein) inhibitor. In some embodiments, the TGF-β inhibitor is selected from fresolimumab or XOMA 089.

In some embodiments, the combination described herein comprises an adenosine A2a (A2aR) receptor antagonist ( e.g., an A2aR pathway inhibitor, e.g., an adenosine inhibitor, e.g., an A2aR or CD-73 inhibitor). In some embodiments, the A2aR antagonist is used in combination with a PD-1 inhibitor and one or more ( e.g., two, three, four, five, or all) CXCR2 inhibitors, a CSF-1/1R binding agent, a LAG-3 inhibitor, a GITR agonist, a c-MET inhibitor, or an IDO inhibitor. In some embodiments, the combination is used to treat pancreatic cancer, colorectal cancer, gastric cancer, or melanoma ( e.g., refractory melanoma). In some embodiments, the A2aR antagonist is selected from PBF509 (NIR178) (Palobiofarma/Novartis), CPI444/V81444 (Corvus/Genentech), AZD4635/HTL-1071 (AstraZeneca/Heptares), Vipadenant (Redox/Juno), GBV-2034 (Globavir), AB928 (Arcus Biosciences), theophylline, istradefylline (Kyowa Hakko Kogyo), Tozadenant/SYN-115 (Acorda), KW-6356 (Kyowa Hakko Kogyo), ST-4206 (Leadiant Biosciences/SC or Preladenden Merck/Schering). Without wishing to be bound by theory, it is believed that in some embodiments, inhibition of A2aR results in upregulation of IL-1b.

In some embodiments, the combination described herein comprises an indoleamine 2,3-dioxygenase (IDO) and/or tryptophan 2,3-dioxygenase (TDO) inhibitor. In some embodiments, the IDO inhibitor is used in combination with a PD-1 inhibitor and one or more ( e.g., two, three, four, or all) TGF-β inhibitors, an A2aR antagonist, a CSF-1/1R binding agent, a c-MET inhibitor, or a GITR agonist. In some embodiments, the combination is used to treat pancreatic cancer, colorectal cancer, gastric cancer, or melanoma ( e.g., refractory melanoma). In some embodiments, the IDO inhibitor is selected from (4E)-4-[(3-chloro-4-fluoroanilino)-nitrosomethylidene]-1,2,5-oxadiazol-3-amine (also known as epacadostat or INCB24360), indoximod (NLG8189), (1-methyl-D-tryptophan), α-cyclohexyl-5H-imidazo[5,1-a]isoindole-5-ethanol (also known as NLG919), indoximod, BMS-986205 (formerly F001287).

In some embodiments, the combination described herein comprises a galectin inhibitor, such as galectin-1 or galectin-3. In some embodiments, the combination comprises a galectin-1 inhibitor and a galectin-3 inhibitor. In some embodiments, the combination comprises a bispecific inhibitor ( e.g., a bispecific antibody molecule) that targets both galectin-1 and galectin-3. In some embodiments, the galectin inhibitor is used in combination with one or more therapeutic agents described herein. In some embodiments, the galectin inhibitor is selected from the anti-galectin antibody molecule GR-MD-02 (Galectin Therapeutics), Galectin-3C (Mandal Med), Anginex, or OTX-008 (OncoEthix, Merck).

In some embodiments, the combination described herein comprises a MEK inhibitor. In some embodiments, the MEK inhibitor is selected from Trametinib, Selumetinib, AS703026, BIX 02189, BIX 02188, CI-1040, PD0325901, PD98059, U0126, XL-518, G-38963, or G02443714. In some embodiments, the MEK inhibitor is Trametinib.

In one embodiment, the combination described herein includes an interleukin-1 beta (IL-1β) inhibitor. In some embodiments, the IL-1β inhibitor is selected from canakinumab, gevokizumab, anakinra, or rilonacept.

In some embodiments, the combination described herein comprises an IL-15/IL-15Ra complex. In some embodiments, the IL-15/IL-15Ra complex is selected from NIZ985 (Novartis), ATL-803 (Altor), or CYP0150 (Cytune).

In some embodiments, the combination described herein comprises a mouse dual microprotein homolog (MDM2) inhibitor. The human MDM2 homolog is also known as HDM2. In some embodiments, the MDM2 inhibitor described herein is also known as an HDM2 inhibitor. In some embodiments, the MDM2 inhibitor is selected from HDM201 or CGM097.

In one embodiment, the MDM2 inhibitor comprises (S)-1-(4-chlorophenyl)-7-isopropoxy-6-methoxy-2-(4-(methyl(((1r,4S)-4-(4-methyl-3-oxopiperazin-1-yl)cyclohexyl)methyl)amino)phenyl)-1,2-dihydroisoquinolin-3(4H)-one (also known as CGM097) or a compound described in PCT Publication No. WO 2011/076786 for the treatment of a disorder, such as a disorder described herein). In one embodiment, the therapeutic agent described herein is used in combination with CGM097.

In some embodiments, the combination described herein comprises a hypomethylating agent (HMA). In some embodiments, the HMA is selected from decitabine or azacitidine.

In some embodiments, the combination described herein comprises an inhibitor that acts on any of the Bcl2 family of pro-survival proteins. In some embodiments, the combination described herein comprises a Bcl-2 inhibitor. In some embodiments, the Bcl-2 inhibitor is venetoclax:

(venetoclax).

In one embodiment, the Bcl-2 inhibitor is selected from the compounds described in WO 2013/110890 and WO 2015/011400. In some embodiments, the Bcl-2 inhibitor comprises navitoclax (ABT-263), ABT-737, BP1002, SPC2996, APG-1252, obatoclax mesylate (GX15-070MS), PNT2258, Zn-d5, BGB-11417, or oblimersen (G3139). In some embodiments, the Bcl-2 inhibitor is N-(4-hydroxyphenyl)-3-[6-[(3S)-3-(morpholinomethyl)-3,4-dihydro-1H-isoquinoline-2-carblonyl]-1,3-benzodioxol-5-yl]-N-phenyl-5,6,7,8-tetrahydroindolizine-1-carboxamide, Compound A1:

compound A1).

In some embodiments, the Bcl-2 inhibitor is (S)-5-(5-chloro-2-(3-(morpholinomethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)phenyl)-N-(5-cyano-1,2-dimethyl-1H-pyrrol-3-yl)-N-(4-hydroxyphenyl)-1,2-dimethyl-1H-pyrrole-3-carboxamide), Compound A2:

(connection A2).

In one embodiment, the antibody-drug conjugates or combinations described herein are suitable for the in vivo treatment of cancer. For example, the combination can be used to suppress the growth of cancer tumors. The combination can also be used in combination with one or more of: a standard treatment ( e.g., for cancer or infectious diseases), a vaccine ( e.g., a therapeutic cancer vaccine), cell therapy, radiation therapy, surgery, or any other therapeutic agent or treatment for a disorder described herein. For example, to achieve antigen-specific immunity enhancement, the combination can be administered together with the antigen of interest. The combination described herein can be administered in any order or simultaneously.

ADDITIONAL IMPLEMENTATION OPTIONS

The description provides the following additional embodiments for linker-drug moieties, antibody-drug conjugates, linker moieties, and conjugation methods.

Linker-drug group

In some embodiments, a linker-drug moiety of the invention may be a compound having the structure (A') or a pharmaceutically acceptable salt thereof:

formula (A')

Where:

R ¹ is a reactive group;

L ₁ is a bridging spacer;

Lp is a divalent peptide spacer;

GL ₂ -A is a self-cleaving spacer;

R ² is a hydrophilic group;

L ₂ is a bond, methylene, neopentylene, or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D;

L ₃ is a spacer group; and

D is a drug group that is capable of inhibiting the activity of the MCl-1 protein when isolated from, for example, antibody-drug conjugates or immunoconjugates described herein.

Certain aspects and examples of the linker-drug moiety of the invention are presented in the following list of enumerated embodiments. It should be understood that the features identified in each embodiment may be combined with other identified features to produce additional embodiments of the present invention.

Embodiment 1. A compound of formula (A') or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is a reactive group;

L ₁ is a bridging spacer;

Lp is a divalent peptide containing two to four amino acid residues;

GL ₂ -A is a self-cleaving spacer;

R ² is a hydrophilic group;

L ₂ is a bond, methylene, neopentylene, or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D;

L ₃ is a spacer group; and

D is a drug group as defined herein, such as an MCl-1 inhibitor.

Embodiment 2. A compound of formula (A') or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is a reactive group;

L ₁ is a bridging spacer;

Lp is a divalent peptide containing two to four amino acid residues;

group selected from:

, where * from means the point of attachment to D (for example, to the N or O group of a drug), *** of means the point of attachment to Lp;

R ² is a hydrophilic group;

L ₂ is a bond, methylene, neopentylene, or a C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D;

L ₃ is a spacer group; and

D is a group of drug as defined herein, such as an MCl-1 inhibitor.

Embodiment 3. A compound of formula (A') or a pharmaceutically acceptable salt thereof having a structure of formula (B'):

Formula (B')

Where:

R ¹ is a reactive group;

L ₁ is a bridging spacer;

Lp is a divalent peptide containing two to four amino acid residues;

R ² is a hydrophilic group;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D;

L ₃ is a spacer group; and

D is a drug group as defined herein and containing N or O, where D is connected to A via a direct bond from A to the N or O of the drug group.

Embodiment 4. A compound of formula (A') or any one of embodiments 1-3, or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is , -ONH ₂ , -NH ₂ , , , -N ₃ , , -SH, -SR ³ , -SSR ⁴ , -S(=O) ₂ (CH=CH ₂ ), -(CH ₂ ) ₂ S(=O) ₂ (CH=CH ₂ ), -NHS(=O) ₂ (CH=CH ₂ ), -NHC(=O)CH ₂ Br, -NHC(=O)CH ₂ I, , -C(O)NHNH ₂ , ,

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)(CH ₂ ) _m -**;

*-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)O(CH ₂ ) _m SSC(R ³ ) ₂ (CH ₂ ) _m C(=O)NR ³ (CH ₂ ) _m NR ³ C(=O)(CH ₂ ) _m -**;

*-C(=O)O(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**;

*-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**;

*-C(=O)(CH ₂ ) _m X ₁ (CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n X ₁ (CH ₂ ) _n -**;

*-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n -**;

*-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n X ₁ (CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n X ₁ (CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n C(=O)NH(CH ₂ ) _m -**;

*-C(=O)(CH ₂ ) _m C(R ³ ) ₂ -**; or

*-C(=O)(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**,

where * of L ₁ denotes the point of attachment to Lp, and ** of L ₁ denotes the point of attachment to R ¹ ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

each R ³ is independently selected from H and C ₁ -C ₆ alkyl;

R ⁴ is 2-pyridyl or 4-pyridyl;

each R ⁵ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl and -OH;

each R ⁶ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl, -NH ₂ , -OCH ₃ , -OCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -CN, -NO ₂ and -OH;

each R ⁷ is independently selected from H, C _1-6 alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C _1-4 alkoxy substituted with -C(=O)OH, and C _1-4 alkyl substituted with -C(=O)OH;

X ₁ is or ;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer containing an amino acid residue selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan and tyrosine;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)C(R ^b ) ₂ NHC(=O)O-**, -NHC(=O)C(R ^b ) ₂ NH-**, -NHC(=O)C(R ^b ) ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W, and the * of X denotes the point of attachment to R ² ; and

* from L ₃ means the point of attachment to R ² ;

And

D is a drug group as defined herein and containing N or O, where D is connected to A via a direct bond from A to the N or O of the drug group.

Embodiment 5. A compound of formula (A') or any one of embodiments 1-4, or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is , -ONH ₂ , or ;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * of L ₁ denotes the point of attachment to Lp, and ** of L ₁ denotes the point of attachment to R ¹ ;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer selected from (ValCit), (PheLys), (ValAla), (ValLys) and (LeuCit), where * of Lp denotes the point of attachment to L ₁ , and ** of Lp denotes the point of attachment to the -NH- group of G;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -NHC(=O)CH ₂ NH-**, -NHC(=O)CH ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W, and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D; and

D is a drug group as defined herein and containing N or O, where D is connected to A via a direct bond from A to the N or O of the drug group.

Embodiment 6. A compound of formula (A') or any one of embodiments 1-5, or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is ;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * of L ₁ denotes the point of attachment to Lp and ** of L ₁ denotes the point of attachment to R ¹ ;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer selected from (ValCit), where * of Lp denotes the point of attachment to L ₁ and ** of Lp denotes the point of attachment to the -NH- group G;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W, and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D;

And

D is a drug group as defined herein and containing N or O, where D is connected to A via a direct bond from A to the N or O of the drug group.

Embodiments 7. A compound of formula (A') or any one of embodiments 1-6, or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is ;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * of L ₁ denotes the point of attachment to Lp, and ** of L ₁ denotes the point of attachment to R ¹ ;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer selected from (ValCit), where * of Lp denotes the point of attachment to L ₁ , and ** of Lp denotes the point of attachment to the -NH- group G;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-** or -NHC(=O)NH-**, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

A is a bond or -OC(=O)*, where * denotes the point of attachment to D;

And

D is a drug group as defined herein and containing N or O, where D is connected to A via a direct bond from A to the N or O of the drug group.

Embodiment 8. A compound of formula (A') or any one of embodiments 1-7, or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is ;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * of L ₁ denotes the point of attachment to Lp, and ** of L ₁ denotes the point of attachment to R ¹ ;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer selected from (ValCit), where * of Lp denotes the point of attachment to L ₁ , and ** of Lp denotes the point of attachment to the -NH- group G;

L ₃ is a spacer group having the structure ,

Where

W is _-CH2O -**, _-CH2N ( ^Rb )C(=O)O-**, -NHC(=O) _CH2NHC (=O)O-**, _-CH2N ( ^XR2 )C(=O)O-** or -C(=O)N( ^XR2 )-**, where each ^Rb is independently selected from H, _C1 - _C6 alkyl or _C3 - _C8 cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

A is a bond or -OC(=O)*, where * denotes the point of attachment to D;

And

D is a drug group as defined herein and containing N or O, where D is connected to A via a direct bond from A to the N or O of the drug group.

Embodiment 9. A compound of formula (A') or any one of embodiments 1-8, or a pharmaceutically acceptable salt thereof, wherein R ¹ is a reactive group selected from Table 8.

Embodiment 10. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is , -ONH ₂ , -NH ₂ , , , , -N ₃ , , -SH, -SR ³ , -SSR ⁴ , -S(=O) ₂ (CH=CH ₂ ), -(CH ₂ ) ₂ S(=O) ₂ (CH=CH ₂ ), -NHS(=O) ₂ (CH=CH ₂ ), -NHC(=O)CH ₂ Br, -NHC(=O)CH ₂ I, , -C(O)NHNH ₂ , , ,

Embodiment 11. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is , -ONH ₂ , -NH ₂ , , , , -N ₃ , , -SH, -SR ³ , -SSR ⁴ , -S(=O) ₂ (CH=CH ₂ ), -(CH ₂ ) ₂ S(=O) ₂ (CH=CH ₂ ), -NHS(=O) ₂ (CH=CH ₂ ), -NHC(=O)CH ₂ Br, -NHC(=O)CH ₂ I, , -C(O)NHNH ₂ , , , or .

Embodiment 12. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is , -ONH ₂ , , , , .

Embodiment 13. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is , -ONH ₂ , or .

Embodiment 14. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, wherein R ¹ is .

Embodiment 15. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, wherein R ¹ is -ONH ₂ .

Embodiment 16. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, wherein: R ¹ is or .

Embodiment 17. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is or .

Embodiment 18. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, having the structure:

, Where

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

Embodiment 19. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, having the structure:

, Where

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

Embodiment 20. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, having the structure:

, Where

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

Embodiment 21. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, having the structure:

, Where

each R is independently selected from H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

Embodiment 22. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, having the structure:

, Where

each R is independently selected from H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

Embodiment 23. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, having the structure:

, Where

Xa is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ - and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH.

Embodiment 24. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, having the structure:

, Where

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

Embodiment 25. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, having the structure:

, Where

Xb is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ -, and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH.

Embodiment 26. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, having the structure:

.

Embodiment 27. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, having the structure:

.

Embodiment 28. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, having the structure:

.

Embodiment 29. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, having the structure:

.

Embodiment 30. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, having the structure:

.

Embodiment 31. A compound of formula (A') or any one of embodiments 1-9, or a pharmaceutically acceptable salt thereof, having the structure of the compound in Table A.

Embodiment 32. A linker-drug group linker of formula (A') having a structure of formula (C'),

formula (C')

Where

L ₁ is a bridging spacer;

Lp is a divalent peptide spacer;

GL ₂ -A is a self-cleaving spacer;

R ² is a hydrophilic group;

L ₂ is a bond, methylene, neopentylene, or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D,

And

L ₃ is a spacer group.

Embodiment 33. The linker of embodiment 32, wherein:

L ₁ is a bridging spacer;

Lp is a divalent peptide containing two to four amino acid residues;

GL ₂ -A is a self-cleaving spacer;

R ² is a hydrophilic group;

L ₂ is a bond, methylene, neopentylene, or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D,

And

L ₃ is a spacer group.

Embodiment 34. The linker of embodiment 32 or 33, wherein:

L ₁ is a bridging spacer;

Lp is a divalent peptide containing two to four amino acid residues;

group selected from:

, where * from means the point of attachment to D (for example, to the N or O group of a drug), *** of means the point of attachment to Lp;

R ² is a hydrophilic group;

L ₂ is a bond, methylene, neopentylene, or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D,

And

L ₃ is a spacer group.

Embodiment 35. The linker of any one of embodiments 32-34, wherein:

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)(CH ₂ ) _m -**; *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)O(CH ₂ ) _m SSC(R ³ ) ₂ (CH ₂ ) _m C(=O)NR ³ (CH ₂ ) _m NR ³ C(=O)(CH ₂ ) _m -**;

*-C(=O)O(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**;

*-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**; *-C(=O)(CH ₂ ) _m X ₁ (CH ₂ ) _m -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n X ₁ (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n -**;

*-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n X ₁ (CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n X ₁ (CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n C(=O)NH(CH ₂ ) _m -**;

*-C(=O)(CH ₂ ) _m C(R ³ ) ₂ -** or *-C(=O)(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**, where * of L ₁ denotes the point of attachment to Lp;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

each R ³ is independently selected from H and C ₁ -C ₆ alkyl;

X ₁ is or ;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer containing an amino acid residue selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan and tyrosine;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)C(R ^b ) ₂ NHC(=O)O-**, -NHC(=O)C(R ^b ) ₂ NH-**, -NHC(=O)C(R ^b ) ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W, and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² .

Embodiment 36. The linker of any one of embodiments 32-35, wherein:

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * of L ₁ denotes the point of attachment to Lp;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer selected from (ValCit), (PheLys), (ValAla), (ValLys) and (LeuCit), where * of Lp denotes the point of attachment to L ₁ ;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -NHC(=O)CH ₂ NH-**, -NHC(=O)CH ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W, and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

And

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D.

Embodiment 37. The linker of any one of embodiments 32-36, wherein:

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * of L ₁ denotes the point of attachment to Lp;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer selected from (ValCit), where * of Lp denotes the point of attachment to L ₁ , and ** of Lp denotes the point of attachment to the -NH- group G;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH2N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W, and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

And

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D.

Embodiment 38. The linker of any one of embodiments 32-37, wherein:

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * of L ₁ denotes the point of attachment to Lp;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer selected from (ValCit), where * from Lp denotes the point of attachment to L ₁ ;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH2N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-** or -NHC(=O)NH-**, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W, and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

And

A is a bond or -OC(=O)*, where * denotes the point of attachment to D.

Embodiment 39. The linker of any one of embodiments 32-38, wherein:

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * of L ₁ denotes the point of attachment to Lp;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer selected from (ValCit), where * from Lp denotes the point of attachment to L ₁ ;

L ₃ is a spacer group having the structure ,

Where

W is _-CH2O -**, -CH2N(Rb)C(=O)O-**, -NHC(=O) _CH2NHC (=O)O-**, _-CH2N ( ^XR2 )C(=O)O-** or -C(=O)N( ^XR2 )-**, where each ^Rb is independently selected from H, _C1 - _C6 alkyl or _C3 - _C8 cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W, and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

And

A is a bond or -OC(=O)*, where * denotes the point of attachment to D.

Embodiment 40. A linker of formula (C') having a structure having a structure of formula (D'),

formula (D')

Where

L ₁ is a bridging spacer;

Lp is a divalent peptide spacer;

R ² is a hydrophilic group;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D,

And

L ₃ is a spacer group.

Embodiment 41. The linker of embodiment 40, wherein:

L ₁ is a bridging spacer;

Lp is a divalent peptide containing two to four amino acid residues;

R ² is a hydrophilic group;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D,

And

L ₃ is a spacer group.

Embodiment 42. The linker of embodiment 40 or 41, wherein:

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)(CH ₂ ) _m -**; *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)O(CH ₂ ) _m SSC(R ³ ) ₂ (CH ₂ ) _m C(=O)NR ³ (CH ₂ ) _m NR ³ C(=O)(CH ₂ ) _m -**;

*-C(=O)O(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**;

*-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**; *-C(=O)(CH ₂ ) _m X ₁ (CH ₂ ) _m -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n X ₁ (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n -**;

*-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n X ₁ (CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n X ₁ (CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m C(R ³ ) ₂ -** or

*-C(=O)(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**, where * from L ₁ denotes the point of attachment to Lp;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

each R ³ is independently selected from H and C ₁ -C ₆ alkyl;

X ₁ is or ;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer containing an amino acid residue selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan and tyrosine;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH2N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W, and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² .

Embodiment 43. The linker of any one of embodiments 40-42, wherein:

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * of L ₁ denotes the point of attachment to Lp;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer selected from (ValCit), (PheLys), (ValAla), (ValLys) and (LeuCit), where * of Lp denotes the point of attachment to L ₁ , and ** of Lp denotes the point of attachment to the -NH- group G;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH2N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

And

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D.

Embodiment 44. The linker of any one of embodiments 40-43, wherein:

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * of L ₁ denotes the point of attachment to Lp;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer selected from (ValCit), where * of Lp denotes the point of attachment to L ₁ , and ** of Lp denotes the point of attachment to the -NH- group G;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W, and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

And

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D.

Embodiment 45. The linker of any one of embodiments 40-44, wherein:

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * of L ₁ denotes the point of attachment to Lp;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer selected from (ValCit), where * of Lp denotes the point of attachment to L ₁ , and ** of Lp denotes the point of attachment to the -NH- group G;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-** or -NHC(=O)NH-**, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W, and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

And

A is a bond or -OC(=O)*, where * denotes the point of attachment to D.

Embodiment 46. The linker of any one of embodiments 40-45, wherein:

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * of L ₁ denotes the point of attachment to Lp;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer selected from (ValCit), where * of Lp denotes the point of attachment to L ₁ , and ** of Lp denotes the point of attachment to the -NH- group G;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-** or -C(=O)N(XR ² )-**, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W, and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

And

A is a bond or -OC(=O)*, where * denotes the point of attachment to D.

Embodiment 47. The linker of any one of embodiments 32-46, having the structure:

, Where

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

Embodiment 48. The linker of any one of embodiments 32-46, having the structure:

, Where

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

Embodiment 49. The linker of any one of embodiments 32-46, having the structure:

, Where

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

Embodiment 50. The linker of any one of embodiments 32-46, having the structure:

, Where

each R is independently selected from H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

Embodiment 51. The linker of any one of embodiments 32-46, having the structure:

, Where

each R is independently selected from H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

Embodiment 52. The linker of any one of embodiments 32-46, having the structure:

, Where

Xa is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ - and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH.

Embodiment 53. The linker of any one of embodiments 32-46, having the structure:

, Where

R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH.

Embodiment 54. The linker of any one of embodiments 32-46, having the structure:

, Where

Xb is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ - and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH.

Embodiment 55. The linker of any one of embodiments 32-46, having the structure:

.

Embodiment 56. The linker of any one of embodiments 32-46, having the structure:

.

Embodiment 57. The linker of any one of embodiments 32-46, having the structure:

.

Embodiment 58. The linker of any one of embodiments 32-46, having the structure:

Embodiment 59. The linker of any one of embodiments 32-46, having the structure:

.

For illustrative purposes, the general reaction schemes depicted herein represent potential synthetic routes to the compounds of the present invention, as well as key intermediates. For a more detailed description of individual reaction steps, see the Examples section below. Although specific starting materials and reagents are depicted in the schemes and discussed below, other starting materials and reagents can be readily substituted to provide a variety of derivatives and/or reaction conditions. Furthermore, many of the compounds prepared by the methods described below can be further modified in light of this description using conventional chemistry well known to those skilled in the art.

As an example, the general synthesis of compounds of formula (B') is shown below in Scheme 1.

Scheme 1

Antibody-drug conjugates according to the invention

The present invention provides antibody-drug conjugates, also referred to herein as immunoconjugates, that comprise linkers that contain one or more hydrophilic groups.

The antibody-drug conjugates of the invention have a structure of formula (E'):

Formula (E')

Where:

Ab is an antibody or its fragment;

R ¹⁰⁰ is a combining group;

L ₁ is a bridging spacer;

Lp is a divalent peptide spacer;

GL ₂ -A is a self-cleaving spacer;

R ² is a hydrophilic group;

L ₂ is a bond, methylene, neopentylene, or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D;

L ₃ is a spacer group;

D is a drug group as defined herein, such as an MCl-1 inhibitor, and may contain N or O, where D may be connected to A by a direct linkage from A to the N or O of the drug group,

And

y is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Certain aspects and examples of the antibody-drug conjugate of the invention are presented in the following list of enumerated embodiments. It should be understood that the features identified in each embodiment may be combined with other identified features to obtain additional embodiments of the present invention.

Embodiment 60. Immunoconjugate of formula (E') where:

Ab is an antibody or its fragment;

R ¹⁰⁰ is a combining group;

L ₁ is a bridging spacer;

Lp is a divalent peptide containing two to four amino acid residues;

GL ₂ -A is a self-cleaving spacer;

R ² is a hydrophilic group;

L ₂ is a bond, methylene, neopentylene, or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D;

L ₃ is a spacer group;

D is a drug group as defined herein where D is connected to A via a direct linkage from A to D (e.g., an N or O drug group),

And

y is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 61. An immunoconjugate of formula (E') or according to embodiment 60, wherein:

Ab is an antibody or its fragment;

R ¹⁰⁰ is a combining group;

L ₁ is a bridging spacer;

Lp is a divalent peptide containing two to four amino acid residues;

group selected from:

, where * from means the point of attachment to D (for example, to the N or O group of a drug), *** of means the point of attachment to Lp;

R ² is a hydrophilic group;

L ₂ is a bond, methylene, neopentylene, or C ₂ -C ₃ alkenylene;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D;

L ₃ is a spacer group;

D is a drug group as defined herein and containing N or O, where D is connected to A via a direct bond from A to the N or O of the drug group,

And

y is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 62. An immunoconjugate of formula (E') or any of embodiments 60-61, having a structure of formula (F'),

formula (F')

Where:

Ab is an antibody or its fragment;

R ¹⁰⁰ is a combining group;

L ₁ is a bridging spacer;

Lp is a divalent peptide containing two to four amino acid residues;

R ² is a hydrophilic group;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D;

L ₃ is a spacer group;

D is a drug group as defined herein and containing N or O, where D is connected to A via a direct bond from A to the N or O of the drug group,

And

y is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 63. An immunoconjugate of formula (D') or any of embodiments 60-62, wherein:

Ab is an antibody or its fragment;

R ¹⁰⁰ is , , -S-, -C(=O)-, -ON=***, -NHC(=O)CH ₂ -***, -S(=O) ₂ CH ₂ CH ₂ -***, -(CH ₂ ) ₂ S(=O) ₂ CH ₂ CH ₂ -***, -NHS(=O) ₂ CH ₂ CH _2- ***, -NHC(=O)CH ₂ CH ₂ -***, -CH ₂ NHCH ₂ CH ₂ -***, -NHCH ₂ CH ₂ -***, , , ,

or , where *** of R ¹⁰⁰ denotes the point of attachment to Ab;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)(CH ₂ ) _m -**; *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)O(CH ₂ ) _m SSC(R ³ ) ₂ (CH ₂ ) _m C(=O)NR ³ (CH ₂ ) _m NR ³ C(=O)(CH ₂ ) _m -**;

*-C(=O)O(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**;

*-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**; *-C(=O)(CH ₂ ) _m X ₁ (CH ₂ ) _m -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n X ₁ (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n -**;

*-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n X ₁ (CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n X ₁ (CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m C(R ³ ) ₂ -** or *-C(=O)(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**, where * of L ₁ denotes the point of attachment to Lp and ** of L ₁ denotes the point of attachment to R ¹⁰⁰ ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

each R ³ is independently selected from H and C ₁ -C ₆ alkyl;

R ⁴ is 2-pyridyl or 4-pyridyl;

each R ⁵ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl and -OH;

each R ⁶ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl, -NH ₂ , -OCH ₃ , -OCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -CN, -NO ₂ and -OH;

each R ⁷ is independently selected from H, C _1-6 alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C _1-4 alkoxy substituted with -C(=O)OH, and C _1-4 alkyl substituted with -C(=O)OH;

X ₁ is or ;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer containing an amino acid residue selected from valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan and tyrosine;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(Rb)C(=O)O-**, -NHC(=O)C(R ^b ) ₂ NHC(=O)O-**, -NHC(=O)C(R ^b ) ₂ NH-**, -NHC(=O)C(R ^b ) ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W, and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

D is a drug group as defined herein and containing N or O, where D is connected to A via a direct bond from A to the N or O of the drug group,

And

y is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 64. An immunoconjugate of formula (D') or any of embodiments 60-63, wherein:

Ab is an antibody or its fragment;

R ¹⁰⁰ is , or , where *** of R ¹⁰⁰ denotes the point of attachment to Ab;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * of L ₁ denotes the point of attachment to Lp, and ** of L ₁ denotes the point of attachment to R ¹⁰⁰ ;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer selected from (ValCit), (PheLys), (ValAla), (ValLys) and (LeuCit), where * of Lp denotes the point of attachment to L ₁ , and ** of Lp denotes the point of attachment to the -NH- group G;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -NHC(=O)CH ₂ NH-**, -NHC(=O)CH ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W, and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D;

D is a drug group as defined herein and containing N or O, where D is connected to A via a direct bond from A to the N or O of the drug group,

And

y is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 65. An immunoconjugate of formula (E') or any of embodiments 60-64, wherein:

Ab is an antibody or its fragment;

R ¹⁰⁰ is , where *** of R ¹⁰⁰ denotes the point of attachment to Ab;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * of L ₁ denotes the point of attachment to Lp, and ** of L ₁ denotes the point of attachment to R ¹⁰⁰ ;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer selected from (ValCit), where * of Lp denotes the point of attachment to L ₁ , and ** of Lp denotes the point of attachment to the -NH- group G;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl, and * of A denotes the point of attachment to D;

D is a drug group as defined herein and containing N or O, where D is connected to A via a direct bond from A to the N or O of the drug group,

And

y is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 66. An immunoconjugate of formula (E') or any of embodiments 60-65, wherein:

Ab is an antibody or its fragment;

R ¹⁰⁰ is , where *** of R ¹⁰⁰ denotes the point of attachment to Ab;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * of L ₁ denotes the point of attachment to Lp and ** of L ₁ denotes the point of attachment to R ¹⁰⁰ ;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer selected from (ValCit), where * of Lp denotes the point of attachment to L ₁ , and ** of Lp denotes the point of attachment to the -NH- group G;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-** or -NHC(=O)NH-**, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is a bond, triazolylmo or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W, and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

A is a bond or -OC(=O)*, where * denotes the point of attachment to D;

D is a drug group as defined herein and containing N or O, where D is connected to A via a direct bond from A to the N or O of the drug group,

And

y is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 67. An immunoconjugate of formula (E') or any of embodiments 60-66, wherein:

Ab is an antibody or its fragment;

R ¹⁰⁰ is , where *** of R ¹⁰⁰ denotes the point of attachment to Ab;

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -, where * of L ₁ denotes the point of attachment to Lp, and ** of L ₁ denotes the point of attachment to R ¹⁰⁰ ;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

each t is independently selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30;

Lp is a divalent peptide spacer selected from (ValCit), where * of Lp denotes the point of attachment to L ₁ , and ** of Lp denotes the point of attachment to the -NH- group G;

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(Rb)C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-** or -C(=O)N(XR ² )-**, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl, and where the ** of W denotes the point of attachment to X;

X is ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W, and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² ;

R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, sugar, oligosaccharide, polypeptide or C ₂ -C ₆ alkyl substituted with 1-3 in groups;

A is a bond or -OC(=O)*, where * denotes the point of attachment to D;

D is a drug group as defined herein and containing N or O, where D is connected to A via a direct bond from A to the N or O of the drug group,

And

y is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 68. An immunoconjugate of formula (E') or any of embodiments 60-63, wherein

R ¹⁰⁰ is , , -S-, -C(=O)-, -ON=***, -NHC(=O)CH ₂ -***, -S(=O) ₂ CH ₂ CH ₂ -***, -(CH ₂ ) ₂ S(=O) ₂ CH ₂ CH ₂ -***, -NHS(=O) ₂ CH ₂ CH _2- ***, -NHC(=O)CH ₂ CH ₂ -***, -CH ₂ NHCH ₂ CH ₂ -***, -NHCH ₂ CH ₂ -***, ,

or , where *** of R ¹⁰⁰ denotes the point of attachment to Ab.

Embodiment 69. An immunoconjugate of formula (E') or any of embodiments 60-63, wherein

R ¹⁰⁰ is , or , where *** of R ¹⁰⁰ denotes the point of attachment to Ab.

Embodiment 70. An immunoconjugate of formula (E') or any of embodiments 60-63, wherein

R ¹⁰⁰ is , or , where *** of R ¹⁰⁰ denotes the point of attachment to Ab.

Embodiment 71. An immunoconjugate of formula (E') or any of embodiments 60-70 having the structure:

, Where

R is H, _-CH3 , or _-CH2CH2C (=O ₎ OH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

Embodiment 72. An immunoconjugate of formula (E') or any of embodiments 60-70 having the structure:

, Where

R is H, _-CH3 , or _-CH2CH2C (=O ₎ OH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

Embodiment 73. An immunoconjugate of formula (E') or any of embodiments 60-70 having the structure:

, Where

R is H, _-CH3 , or _-CH2CH2C (=O ₎ OH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

Embodiment 74. An immunoconjugate of formula (E') or any of embodiments 60-70 having the structure:

, Where

each R is independently selected from H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 75. An immunoconjugate of formula (E') or any of embodiments 60-70 having the structure:

, Where

each R is independently selected from H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 76. An immunoconjugate of formula (E') or any of embodiments 60-70 having the structure:

, Where

Xa is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ - and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

Embodiment 77. An immunoconjugate of formula (E') or any of embodiments 60-70 having the structure:

, Where

R is H, _-CH3 , or _-CH2CH2C (=O ₎ OH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

Embodiment 78. An immunoconjugate of formula (E') or any of embodiments 60-70 having the structure:

, Where

Xb is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ - and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

Embodiment 79. An immunoconjugate of formula (E') or any of embodiments 60-70 having the structure:

, where y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

Embodiment 80. An immunoconjugate of formula (E') or any of embodiments 60-70 having the structure:

, where y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

Embodiment 81. An immunoconjugate of formula (E') or any of embodiments 60-70 having the structure:

, where y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

Embodiment 82. An immunoconjugate of formula (E') or any of embodiments 60-70 having the structure:

, where y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

Embodiment 83. An immunoconjugate of formula (E') or any of embodiments 60-70 having the structure:

, where y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

Certain aspects and examples of linker-drug moieties, linkers, and antibody-drug conjugates of the invention are presented in the following list of additional enumerated embodiments. It should be understood that the features identified in each embodiment may be combined with other identified features to yield additional embodiments of the present invention.

Embodiment 84. A compound of formula (A') or any of embodiments 1-2, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-39, and an immunoconjugate of formula (E') or any of embodiments 60-61, wherein:

G is , where * of G denotes the attachment point to L ₂ and ** of G denotes the attachment point to L ₃ and *** of G denotes the attachment point to Lp.

Embodiment 85. A compound of formula (A') or any of embodiments 1-2, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-39, and an immunoconjugate of formula (E') or any of embodiments 60-61, wherein:

G is , where * of G denotes the attachment point to L ₂ and ** of G denotes the attachment point to L ₃ and *** of G denotes the attachment point to Lp.

Embodiment 86. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70, wherein:

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)(CH ₂ ) _m -**; *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**;

*-C(=O)O(CH ₂ ) _m SSC(R ³ ) ₂ (CH ₂ ) _m C(=O)NR ³ (CH ₂ ) _m NR ³ C(=O)(CH ₂ ) _m -**;

*-C(=O)O(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**;

*-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**; *-C(=O)(CH ₂ ) _m X ₁ (CH ₂ ) _m -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n X ₁ (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n -**;

*-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n X ₁ (CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n X ₁ (CH ₂ ) _n -**;

*-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m C(R ³ ) ₂ -** or *-C(=O)(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**, where * of L ₁ denotes the point of attachment to Lp and ** of L ₁ denotes the point of attachment to R ¹ if present or ** of L ₁ denotes the point of attachment to R ¹⁰⁰ if present.

Embodiment 87. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70, wherein:

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**; *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m C(R ³ ) ₂ -** or *-C(=O)(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**, where * of L ₁ denotes the point of attachment to Lp and ** of L ₁ denotes the point of attachment to R ¹ if present or ** of L ₁ denotes the point of attachment to R ¹⁰⁰ if present.

Embodiment 88. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70, wherein:

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**; or *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**, where * of L ₁ denotes the point of attachment to Lp and ** of L ₁ denotes the point of attachment to R ¹ if present or ** of L ₁ denotes the point of attachment to R ¹⁰⁰ if present.

Embodiment 89. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70, wherein:

L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -** or *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**, where * of L ₁ denotes the point of attachment to Lp and ** of L ₁ denotes the point of attachment to R ¹ if present or ** of L ₁ denotes the point of attachment to R ¹⁰⁰ if present.

Embodiment 90. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70, wherein L ₁ is *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**, wherein * of L ₁ denotes the point of attachment to Lp and ** of L ₁ denotes the point of attachment to R ¹ if present or ** of L ₁ denotes the point of attachment to R ¹⁰⁰ if present.

Embodiment 91. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70, wherein L ₁ is *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**, wherein * of L ₁ denotes the point of attachment to Lp and ** of L ₁ denotes the point of attachment to R ¹ if present or ** of L ₁ denotes the point of attachment to R ¹⁰⁰ if present.

Embodiment 92. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70, wherein L ₁ is *-C(=O)(CH ₂ ) _m -**, wherein * of L ₁ denotes the point of attachment to Lp and ** of L ₁ denotes the point of attachment to R ¹ if present or ** of L ₁ denotes the point of attachment to R ¹⁰⁰ if present.

Embodiment 93. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70, wherein L ₁ is *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**, wherein * of L ₁ denotes the point of attachment to Lp and ** of L ₁ denotes the point of attachment to R ¹ if present or ** of L ₁ denotes the point of attachment to R ¹⁰⁰ if present.

Embodiment 94. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-93, wherein Lp is an enzymatically cleavable divalent peptide spacer.

Embodiment 95. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-94, wherein Lp is a divalent peptide spacer comprising an amino acid residue selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine.

Embodiment 96. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-95, wherein Lp is a divalent peptide comprising two to four amino acid residues.

Embodiment 97. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-96, wherein Lp is a divalent peptide comprising two to four amino acid residues, wherein each is independently selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, methionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine.

Embodiment 98. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-97, wherein:

Lp is a divalent peptide spacer selected from (ValCit), (PheLys), (ValAla), (ValLys) and (LeuCit), where * of Lp denotes the point of attachment to L ₁ and ** of Lp denotes the point of attachment to the -NH- group of formula (B') or ** of Lp denotes the point of attachment to G of formula (A').

Embodiment 99. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-98, wherein:

Lp is (ValCit), where * of Lp denotes the point of attachment to L ₁ and ** of Lp denotes the point of attachment to the -NH- group of formula (B') or ** of Lp denotes the point of attachment to G of formula (A').

Embodiment 100. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-98, wherein:

Lp is (PheLys), where * of Lp denotes the point of attachment to L ₁ and ** of Lp denotes the point of attachment to the -NH- group of formula (B') or ** of Lp denotes the point of attachment to G of formula (A').

Embodiment 101. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-98, wherein:

Lp is (ValAla), where * of Lp denotes the point of attachment to L ₁ and ** of Lp denotes the point of attachment to the -NH- group of formula (B') or ** of Lp denotes the point of attachment to G of formula (A').

Embodiment 102. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-98, wherein:

Lp is (ValLys), where * of Lp denotes the point of attachment to L ₁ and ** of Lp denotes the point of attachment to the -NH- group of formula (B') or ** of Lp denotes the point of attachment to G of formula (A').

Embodiment 103. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-98, wherein:

Lp is (LeuCit), where * of Lp denotes the point of attachment to L ₁ and ** of Lp denotes the point of attachment to the -NH- group of formula (B') or ** of Lp denotes the point of attachment to G of formula (A').

Embodiment 104. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-103, wherein L ₂ is a bond, methylene, or C ₂ -C ₃ alkenylene.

Embodiment 105. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-104, wherein L ₂ is a bond or methylene.

Embodiment 106. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-105, wherein L ₂ is a bond.

Embodiment 107. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-105, wherein L ₂ is methylene.

Embodiment 108. A compound of formula (A') or any of embodiments 1-30, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-83 or any of embodiments 84-107, wherein:

A is a bond, -OC(=O)-, -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)- or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl or a C ₃ -C ₈ cycloalkyl.

Embodiment 109. A compound of formula (A') or any of embodiments 1-30, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-83 or any of embodiments 84-107, wherein A is a bond or -OC(=O).

Embodiment 110. A compound of formula (A') or any of embodiments 1-30, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-83 or any of embodiments 84-109, wherein A is a bond.

Embodiment 111. A compound of formula (A') or any of embodiments 1-30, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-83 or any of embodiments 84-109, wherein A is -OC(=O).

Embodiment 112. A compound of formula (A') or any of embodiments 1-30, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-83 or any of embodiments 84-107, wherein:

A is or .

Embodiment 113. A compound of formula (A') or any of embodiments 1-30, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-83 or any of embodiments 84-107, wherein:

A is -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)- or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl or a C ₃ -C ₈ cycloalkyl.

Embodiment 114. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-113, wherein:

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)C(R ^b ) ₂ NHC(=O)O-**, -NHC(=O)C(R ^b ) ₂ NH-**, -NHC(=O)C(R ^b ) ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² .

Embodiment 115. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-114, wherein:

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -NHC(=O)CH ₂ NH-**, -NHC(=O)CH ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl and where the ** of W denotes the point of attachment to X;

X is a bond;

And

* from L ₃ means the point of attachment to R ² .

Embodiment 116. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-115, wherein:

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -NHC(=O)CH ₂ NH-**, -NHC(=O)CH ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl and where the ** of W denotes the point of attachment to X;

X is triazolyl, where the *** of X denotes the point of attachment to W and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² .

Embodiment 117. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-115, wherein:

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -NHC(=O)CH ₂ NH-**, -NHC(=O)CH ₂ NHC(=O)-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, -CH ₂ N(XR ² )C(=O)-**, -C(=O)NR ^b -**, -C(=O)NH-**, -CH ₂ NR ^b C(=O)-**, -CH ₂ NR ^b C(=O)NH-**, -CH ₂ NR ^b C(=O)NR ^b -**, -NHC(=O)-**, -NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O) ₂ NH-**, -NHS(O) ₂ -**, -C(=O)-, -C(=O)O-** or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl and where the ** of W denotes the point of attachment to X;

X is ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² .

Embodiment 118. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-115, wherein:

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl and where the ** of W denotes the point of attachment to X;

X is a bond, triazolyl, or ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² .

Embodiment 119. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-115, wherein:

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl and where the ** of W denotes the point of attachment to X;

X is a bond;

And

* from L ₃ means the point of attachment to R ² .

Embodiment 120. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-115, wherein:

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl and where the ** of W denotes the point of attachment to X;

X is triazolyl, where the *** of X denotes the point of attachment to W and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² .

Embodiment 121. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-115, wherein:

L ₃ is a spacer group having the structure ,

Where

W is -CH ₂ O-**, -CH ₂ N(R ^b )C(=O)O-**, -NHC(=O)CH ₂ NHC(=O)O-**, -CH ₂ N(XR ² )C(=O)O-**, -C(=O)N(XR ² )-**, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl or C ₃ -C ₈ cycloalkyl and where the ** of W denotes the point of attachment to X;

X is ***-CH ₂ -triazolyl-*, where the *** of X denotes the point of attachment to W and the * of X denotes the point of attachment to R ² ;

And

* from L ₃ means the point of attachment to R ² .

Embodiment 122. A compound of formula (A') or any of Embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of Embodiments 32-46, and an immunoconjugate of formula (E') or any of Embodiments 60-70 or any of Embodiments 84-121, wherein R ² is a hydrophilic group selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide, or C ₂ -C ₆ alkyl substituted with 1-3 in groups.

Embodiment 123. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-122, wherein R ² is a sugar.

Embodiment 124. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-122, wherein R ² is an oligosaccharide.

Embodiment 125. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-122, wherein R ² is a polypeptide.

Embodiment 126. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-122, wherein R ² is a polyalkylene glycol.

Embodiment 127. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-122, wherein R ² is a polyalkylene glycol having the structure -(O(CH ₂ ) _m ) _t R', where R' is OH, OCH ₃ , or OCH ₂ CH ₂ C(=O)OH, m is 1-10, and t is 4-40.

Embodiment 128. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-122, wherein R ² is a polyalkylene glycol having the structure -((CH ₂ ) _m O) _t R''-, wherein R'' is H, CH ₃ , or CH ₂ CH ₂ C(=O)OH, m is 1-10, and t is 4-40.

Embodiment 129. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-122, wherein R ² is polyethyleneglycol.

Embodiment 130. A compound of formula (A') or any of Embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of Embodiments 32-46, and an immunoconjugate of formula (E') or any of Embodiments 60-70 or any of Embodiments 84-122, wherein R ² is a polyethyleneglycol having the structure -(OCH ₂ CH ₂ ) _t R', wherein R' is OH, OCH ₃ , or OCH ₂ CH ₂ C(=O)OH and t is 4-40,

Embodiment 131. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-122, wherein R ² is polyethyleneglycol having the structure -(CH ₂ CH ₂ O) _t R''-, wherein R'' is H, CH ₃ , or CH ₂ CH ₂ C(=O)OH and t is 4-40.

Embodiment 132. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-122, wherein:

R ² is , or , where * from R ² denotes the point of attachment to X or L ₃ .

Embodiment 133. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-122, wherein:

R ² is or , where * from R ² denotes the point of attachment to X or L ₃ .

Embodiment 134. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-122, wherein:

R ² is or , where * from R ² denotes the point of attachment to X or L ₃ .

Embodiment 135. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-122, wherein:

R ² is , where * from R ² denotes the point of attachment to X or L ₃ .

Embodiment 136. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-135, wherein:

X ₁ is or .

Embodiment 137. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-135, wherein:

X ₁ is or .

Embodiment 138. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-137, wherein:

each m is independently selected from 1, 2, 3, 4, and 5.

Embodiment 139. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-137, wherein:

each m is independently selected from 1, 2, and 3.

Embodiment 140. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-139, wherein:

each n is independently selected from 1, 2, 3, 4, and 5.

Embodiment 141. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-139, wherein:

each n is independently chosen from 1, 2, and 3.

Embodiment 142. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-141, wherein:

each t is independently selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

Embodiment 143. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-141, wherein:

each t is independently selected from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

Embodiment 144. A compound of formula (A') or any of embodiments 1-17, or a pharmaceutically acceptable salt thereof, a linker of formula (C') or any of embodiments 32-46, and an immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-141, wherein:

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18.

Embodiment 145. An immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-144, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

Embodiment 146. An immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-144, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

Embodiment 147. An immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-144, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Embodiment 148. An immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-144, wherein y is 1, 2, 3, 4, 5, 6, 7, or 8.

Embodiment 149. An immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-144, wherein y is 1, 2, 3, 4, 5, or 6.

Embodiment 150. An immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-144, wherein y is 1, 2, 3, or 4.

Embodiment 151. An immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-144, wherein y is 1 or 2.

Embodiment 152. An immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-144, wherein y is 2.

Embodiment 153. An immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-144, wherein y is 4.

Embodiment 154. An immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-144, wherein y is 6.

Embodiment 155. An immunoconjugate of formula (E') or any of embodiments 60-70 or any of embodiments 84-144, wherein y is 8.

Embodiment 156. A compound of formula (A') or any of embodiments 1-30, or a pharmaceutically acceptable salt thereof, an immunoconjugate of formula (E') or any of embodiments 60-70, or any of embodiments 84-155, wherein D is an MCl-1 inhibitor when isolated from immunoconjugates.

Other linker groups

Other examples of linker groups that are suitable for preparing the MCl-1 inhibitor ADCs or immunoconjugates described herein include those described in international application publications such as WO2018200812, WO2017214456, WO2017214458, WO2017214462, WO2017214233, WO2017214282, WO2017214301, WO2017214322, WO2017214335, WO2017214339, WO2016094509, WO2016094517 and WO2016094505, the contents of each of which are incorporated by reference in their entirety.

For example, the MCl-1 inhibitor immunoconjugates described herein may have a linker-payload structure (“-L-D”) selected from:

,

Where:

Lc is a linker component, and each Lc is independently selected from a linker component described herein;

x is an integer chosen from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20;

y is an integer chosen from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20;

p is an integer chosen from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

D is an MCl-1 inhibitor described herein;

and each cleavage element ( _CE ) is independently selected from a self-cleaving spacer and a group that is susceptible to cleavage selected from acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase-induced cleavage, phosphodiesterase-induced cleavage, phosphatase-induced cleavage, protease-induced cleavage, lipase-induced cleavage, or disulfide bond cleavage.

In some embodiments, L has a structure selected from the following, or L comprises a structural component selected from the following: ,

And .

In some embodiments, Lc is a linker component and each Lc is independently selected from

,

And

.

In some embodiments, linker L comprises a linker component that is selected from:

-**C(=O)O(CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m -; -**C(=O)O(CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m O(CH ₂ ) _m -;

-**C(=O)O(CH ₂ ) _m NR ¹¹ C(=O)X _1a X _2a C(=O)(CH ₂ ) _m -;

-**C(=O)OC(R ¹² ) ₂ (CH ₂ ) _m NR ¹¹ C(=O)X _1a X _2a C(=O)(CH ₂ ) _m -;

-**C(=O)O(CH ₂ ) _m NR ¹¹ C(=O)X _1a X _2a C(=O)(CH ₂ ) _m O(CH ₂ ) _m -;

-**C(=O)O(CH ₂ ) _m NR ¹¹ C(=O)X _1a X _2a C(=O)(CH ₂ ) _m O(CH ₂ ) _m C(=O)-;

-**C(=O)O(CH ₂ ) _m NR ¹¹ C(=O)X ₄ C(=O)NR ¹¹ (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m O(CH ₂ ) _m -;

-**C(=O)O(CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)(CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m -;

-**C(=O)X ₄ C(=O)NR ¹¹ (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m O(CH ₂ ) _m -;

-**C(=O)(CH ₂ ) _m NR ¹¹ C(=O)X _1a X _2a C(=O)(CH ₂ ) _m -;

-**C(=O)O(CH ₂ ) _m X ₆ C(=O)X _1a X _2a C(=O)(CH ₂ ) _m -;

-**C(=O)(CH ₂ ) _m NR ¹¹ C(=O)((CH ₂ ) _m O) _n (CH ₂ ) _m -

-**C(=O)O(CH ₂ ) _m X ₆ C(=O)(CH ₂ ) _m -; -**C(=O)O(CH ₂ ) _m X ₆ C(=O)(CH ₂ ) _m O(CH ₂ ) _m -;

-**C(=O)O(CH ₂ ) _m X ₆ C(=O)X _1a X _2a C(=O)(CH ₂ ) _m -;

-**C(=O)O(CH ₂ ) _m X ₆ C(=O)X _1a X _2a C(=O)(CH ₂ ) _m O(CH ₂ ) _m -;

-**C(=O)O(CH ₂ ) _m X ₆ C(=O)X _1a X _2a C(=O)(CH ₂ ) _m O(CH ₂ ) _m C(=O)-;

-**C(=O)O(CH ₂ ) _m X ₆ C(=O)X ₄ C(=O)NR ¹¹ (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m O(CH ₂ ) _m -;

-**C(=O)X ₄ C(=O)X ₆ (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m O(CH ₂ ) _m -;

-**C(=O)(CH ₂ ) _m X ₆ C(=O)X _1a X _2a C(=O)(CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)(CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)(CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)(CH ₂ ) _m X ₃ (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)((CH ₂ ) _m O) _n (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m X ₃ (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O))X ₅ C(=O)((CH ₂ ) _m O) _n (CH ₂ ) _m X ₃ (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)(CH ₂ ) _m NR ¹¹ C(=O)((CH ₂ ) _m O) _n (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)(CH ₂ ) _m NR ¹¹ C(=O)((CH ₂ ) _m O) _n (CH ₂ ) _m X ₃ (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ (CH ₂ ) _m X ₃ (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ ((CH ₂ ) _m O) _n (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ ((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ ((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m X ₃ (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ ((CH ₂ ) _m O) _n (CH ₂ ) _m X ₃ (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ (CH ₂ ) _m NR ¹¹ ((CH ₂ ) _m O) _n (CH ₂ ) _m -; -**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)(CH ₂ ) _m NR ¹¹ ((CH ₂ ) _m O) _n (CH ₂ ) _m X ₃ (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ (CH ₂ ) _m -; -**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ C(=O)((CH ₂ ) _m O) _n (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)X ₅ (CH ₂ ) _m X ₃ (CH ₂ ) _m -; -**C(=O)O(CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m -; -**C(=O)O(CH ₂ ) _m NR ¹¹ (CH ₂ ) _m -;

-**C(=O)O(CH ₂ ) _m NR ¹¹ (CH ₂ ) _m C(=O)X _2a X _1a C(=O)-;

-**C(=O)O(CH ₂ ) _m X ₃ (CH ₂ ) _m -; -**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m X ₃ (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m -; -**C(=O)O(CH ₂ ) _m NR ¹¹ C(=O(CH ₂ ) _m X ₃ (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m X ₃ (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n X ₃ (CH ₂ ) _m -; -**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m X ₃ (CH ₂ ) _m -;

-**C(=O)O((CH ₂ ) _m O) _n (CH ₂ ) _m C(=O)NR ¹¹ (CH ₂ ) _m -; -**C(=O)O(CH ₂ ) _m C(R ¹² ) ₂ -;

-**C(=O)OCH ₂ ) _m C(R ¹² ) ₂ SS(CH ₂ ) _m NR ¹¹ C(=O)(CH ₂ ) _m -, and

-**C(=O)O(CH ₂ ) _m C(=O)NR ¹¹ (CH ₂ ) _m -, where: ** denotes the point of attachment to the drug group (D) and the other end may be connected to R ¹⁰⁰ , i.e., the coupling group, as described herein;

Where:

X _1a is or , where * denotes the point of attachment to X _2a ;

X _2a is selected from ,

And

; where * denotes the point of attachment to X _1a ;

X ₃ is or ;

X ₄ is -O(CH ₂ ) _n SSC(R ¹² ) ₂ (CH ₂ ) _n - or -(CH ₂ ) _n C(R ¹² ) ₂ SS(CH ₂ ) _n O-;

X ₅ is or , where ** denotes orientation in the direction of the drug group;

X ₆ is or , where ** denotes orientation in the direction of the drug group;

each R ¹¹ is independently selected from H and C ₁ -C ₆ alkyl;

each R ¹² is independently selected from H and C ₁ -C ₆ alkyl;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18.

Conjugation methods

The present invention provides various methods for conjugating linker-drug moieties of the invention to antibodies or antibody fragments to produce antibody-drug conjugates that comprise a linker having one or more hydrophilic groups.

The general reaction scheme for the formation of an antibody-drug conjugate of formula (E') is shown in Scheme 2 below:

Scheme 2

where: RG ₂ is a reactive group that reacts with a compatible R ¹ group to form the corresponding R ¹⁰⁰ group (such groups are shown in Table 2 and Table 3). D, R ¹ , L ₁ , Lp, Ab, y and R ¹⁰⁰ are as defined herein.

Scheme 3 further illustrates this general approach to preparing antibody-drug conjugates of formula (E'), wherein the antibody contains reactive groups (RG ₂ ) that react with an R ¹ group (as defined herein) to covalently attach a linker-drug group to the antibody via an R ¹⁰⁰ group (as defined herein). For illustrative purposes only, Scheme 3 shows an antibody having four RG ₂ groups.

Scheme 3

.

In one aspect, linker-drug moieties are conjugated to antibodies via modified cysteine residues in the antibodies (see, for example, WO2014/124316). Scheme 4 illustrates this approach to preparing antibody-drug conjugates of formula (E') wherein a free thiol group is created from engineered cysteine residues in the antibody reacting with an R ¹ group (where R ¹ is maleimide) to covalently attach the linker-drug moiety to the antibody via an R ¹⁰⁰ group (where R ¹⁰⁰ is a succinimide ring). For illustrative purposes only, Scheme 4 shows an antibody having four free thiol groups.

Scheme 4

In another aspect, linker-drug moieties are conjugated to antibodies via lysine residues in the antibodies. Scheme 5 illustrates this approach to preparing antibody-drug conjugates of formula (E') wherein the free amine group of the lysine residues in the antibody reacts with the ^R1 group (where ^R1 is an NHS ester, pentafluorophenyl, or tetrafluorophenyl) to covalently attach the linker-drug moiety to the antibody via the ^R100 group (where ^R100 is an amide). For illustrative purposes only, Scheme 5 shows an antibody having four amine groups.

In another aspect, linker-drug moieties are conjugated to antibodies via the formation of an oxime bridge on naturally occurring disulfide bridges of the antibody. The oxime bridge is created by first forming a ketone bridge by reducing an interchain disulfide bridge of the antibody and re-bridge formation using 1,3-dihaloacetone (e.g., 1,3-dichloroacetone). Subsequent reaction with a linker-drug moiety containing hydroxylamine creates an oxime bond (oxime bridge), which attaches the linker-drug moiety to the antibody (see, e.g., WO2014/083505). Scheme 6 illustrates an approach to preparing antibody-drug conjugates of formula (E').

Scheme 6

.

The general reaction scheme for the preparation of antibody-drug conjugates of formula (F') is shown in Scheme 7 below:

Scheme 7

where: RG ₂ is a reactive group that reacts with a compatible R ¹ group to produce the corresponding R ¹⁰⁰ group (such groups are shown in Table 2 and Table 3). D, R ¹ , L ₁ , Lp, Ab, y and R ¹⁰⁰ are as defined herein.

Scheme 8 further illustrates this general approach to preparing antibody-drug conjugates of formula (F'), wherein the antibody contains reactive groups (RG ₂ ) that react with an R ¹ group (as defined herein) to covalently attach a linker-drug group to the antibody via an R ¹⁰⁰ group (as defined herein). For illustrative purposes only, Scheme 8 shows an antibody having four RG ₂ groups.

Scheme 8

.

In one aspect, linker-drug moieties are conjugated to antibodies via modified cysteine residues in the antibodies (see, for example, WO2014/124316). Scheme 9 illustrates this approach to preparing antibody-drug conjugates of formula (F') wherein a free thiol group is created from engineered cysteine residues in the antibody reacting with an R ¹ group (where R ¹ is maleimide) to covalently attach the linker-drug moiety to the antibody via an R ¹⁰⁰ group (where R ¹⁰⁰ is a succinimide ring). For illustrative purposes only, Scheme 9 shows an antibody having four free thiol groups.

Scheme 9

In another aspect, linker-drug moieties are conjugated to antibodies via lysine residues in the antibodies. Scheme 10 illustrates this approach to preparing antibody-drug conjugates of formula (F') wherein the free amine group of the lysine residues in the antibody reacts with the ^R1 group (where ^R1 is an NHS ester, pentafluorophenyl, or tetrafluorophenyl) to covalently attach the linker-drug moiety to the antibody via the ^R100 group (where ^R100 is an amide). For illustrative purposes only, Scheme 10 shows an antibody having four amine groups.

Scheme 10

In another aspect, linker-drug moieties are conjugated to antibodies via the formation of an oxime bridge on naturally occurring disulfide bridges of the antibody. The oxime bridge is created by first forming a ketone bridge by reducing an interchain disulfide bridge of the antibody and re-bridge it using 1,3-dihaloacetone (e.g., 1,3-dichloroacetone). Subsequent reaction with a linker-drug moiety containing hydroxylamine creates an oxime bond (oxime bridge), which attaches the linker-drug moiety to the antibody (see, e.g., WO2014/083505). Scheme 11 illustrates an approach to preparing antibody-drug conjugates of formula (F').

Scheme 11

.

Protocols are also provided for certain aspects of the analytical methodology for evaluating the antibody conjugates of the invention. Such analytical methodology and results can demonstrate that the conjugates have favorable properties, such as properties that facilitate their manufacture, make them easier to administer to patients, are more effective, and/or are potentially safer for patients. One example is molecular size determination using size-exclusion chromatography (SEC), where the amount of a desired antibody species in a sample is determined relative to the amount of high-molecular-weight contaminants (e.g., dimer, multimer, or aggregated antibody) or low-molecular-weight contaminants (e.g., antibody fragments, degradation products, or individual antibody chains present in the sample). In general, higher amounts of monomer and lower amounts of, for example, aggregated antibody are desirable due to the impact of, for example, aggregates on other properties of the antibody sample, such as, but not limited to, clearance rate, immunogenicity, and toxicity. Another example is the determination of hydrophobicity by hydrophobic interaction chromatography (HIC), where the hydrophobicity of a sample is assessed relative to a set of standard antibodies with known properties. In general, a low hydrophobicity is desirable due to the influence of hydrophobicity on other properties of the antibody sample, such as, but not limited to, aggregation, aggregation over time, adhesion to surfaces, hepatotoxicity, clearance rate, and pharmacokinetic effects. Damle, N.K., Nat Biotechnol. 2008; 26(8):884–885; Singh, S.K., Pharm Res. 2015; 32(11):3541–71. When measured by hydrophobic interaction chromatography, higher hydrophobicity index values (i.e., faster elution from the HIC column) reflect lower hydrophobicity of the conjugates. As shown in the examples below, most of the antibody conjugates tested exhibited a hydrophobicity index greater than 0.8. In some embodiments, antibody conjugates are provided that have a hydrophobicity index of 0.8 or greater, as determined by hydrophobic interaction chromatography.

EXAMPLES

The following examples provide illustrative embodiments of the description. One of ordinary skill in the art will recognize numerous modifications and variations that can be made without changing the spirit or scope of the description. Such modifications and variations are intended to be within the scope of the description. The examples provided are not intended to limit the description in any way.

Example 1. Synthesis and characterization of linkers, linker payloads and their precursors.

Generic linkers, payload linkers, and their precursors are synthesized using the generic methods described in this example.

Abbreviations:

CuI: copper(I) iodide

DCC: dicyclohexylcarbodiimide

DCM: dichloromethane

DEA: N-ethylethanamine

DIPEA: N,N-diisopropylethylamine

DMF: dimethylformamide

DMSO: dimethyl sulfoxide

EEDQ: ethyl 2-ethoxy-2H-quinoline-1-carboxylate

Fmoc-Cit-OH: (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-ureidopentanoic acid

GBTU: hexafluorophosphate (2-( 1H- benzotriazol-1-yl)-1,1,3,3-tetramethyluronium

HOAt: 1-Hydroxy-7-azabenzotriazole

MgSO ₄ ; magnesium sulfate

NH ₄ Cl: ammonium chloride

NMP: N-methylpyrrolidone

Pd(PPh ₃ ) ₂ Cl ₂ ; dichloro-tri(triphenylphosphine)palladium

PBr ₃ : tribromophosphane

Pt/C 10%: platinum on carbon 10%

SOCl ₂ : thionyl chloride

THF: tetrahydrofuran

TBAI: Tetrabutylammonium iodide

TFA: trifluoroacetic acid

Materials, methods and general techniques:

All reagents, obtained from commercial sources, were used without further purification. Anhydrous solvents were obtained from commercial sources and used without further drying. Flash chromatography was performed on a CombiFlash Rf (Teledyne ISCO) with prepacked silica gel cartridges (Macherey-Nagel Chromabond Flash). Thin-layer chromatography was performed on 5×10 cm plates coated with Merck Type 60 F254 silica gel. Microwave heating was performed on a CEM Discover® instrument.

¹ H NMR measurements were performed on a Bruker Avance 400 MHz or Avance Neo 500 MHz spectrometer using DMSO -d ₆ or CDCl ₃ as solvent. ¹ H NMR data are reported as chemical shift values in parts per million (ppm) using the residual solvent peak (2.50 ppm for DMSO- d ₆ and 7.26 ppm for CDCl ₃ ) as an internal standard. Splitting patterns are denoted as: s (singlet), d (doublet), t (triplet), qv (quartet), quint (quintet), m (multiplet), bs (broad singlet), bt (broad triplet), dd (doublet of doublets), td (triplet of doublets), dt (doublet of triplets), ddd (doublet of doublets of doublets). IR measurements were performed on a Bruker Tensor 27 equipped with an ATR Golden Gate (SPECAC) unit. HRMS measurements were performed on an LTQ OrbiTrap Velos Pro mass spectrometer (ThermoFisher Scientific). Samples were dissolved in _CH3CN / _H2O (2/1: v/v) in the concentration range from approximately 0.01 to 0.05 mg/mL and injected into the source with a 2 µL injection at a flow rate of 0.1 mL/min. ESI ionization parameters were capillary ion transfer, 3.5 kV, and 350°C. All spectra were acquired in positive ionization mode with a resolving power of 30,000 or 60,000 using a fixed mass.

HRMS measurements were performed on an LTQ OrbiTrap Velos Pro mass spectrometer (ThermoFisher Scientific GmbH, Bremen, Germany). Samples were dissolved in _CH3CN / _H2O (2/1: v/v) in the concentration range of approximately 0.01 to 0.05 mg/mL and injected into the source with a 2 μL injection at a flow rate of 0.1 mL/min. ESI ionization parameters were 3.5 kV and 350°C capillary ion transfer. All spectra were acquired in positive ionization mode with a resolving power of 30,000 or 60,000 using a fixed mass.

UPLC®-MS:

UPLC®-MS data are obtained using an instrument with the following parameters (Table 10):

Table 10. UPLC ® -MS parameters

Preparative HPLC:

Preparative HPLC (“prep-HPLC”) data are collected using an instrument with the following parameters (Table 11):

Table 11. Prep-HPLC parameters

Three methods of prep-HPLC are tried:

TFA method: solvent: A water + 0.05% TFA, B acetonitrile + 0.05% TFA, gradient from 5 to 100% B over 15-30 CV

NH ₄ HCO ₃ method: solvent: A water + 0.02 M NH ₄ HCO ₃ , B acetonitrile/water 80/20 + 0.02 M NH ₄ HCO ₃ , gradient from 5 to 100% B over 15-30 CV

Neutral method: solvent: A water, B acetonitrile, gradient from 5 to 100% B over 15-30 CV

All fractions containing the pure compound are combined and freeze-dried to obtain the compound as an amorphous powder.

Preparative SLC purification:

Preparative chiral SLC is performed on a PIC solution Prep200 system. The sample is dissolved in ethanol at a concentration of 150 mg/mL. The mobile phase is maintained isocratically at 40% ethanol/ _CO2 . The instrument is equipped with a Chiralpak IA column and a 3 mL loop. The ABPR (automatic back pressure regulator) is set to 100 bar.

Obtaining L23-P3:

(2R)-2-[(5 Sa )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-(2-azidoethoxy)acetyl]amino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

Step 1: (2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-N-[4-(hydroxymethyl)phenyl]-5-ureido-pentanamide

To a solution of 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetic acid (purchased from Broadpharm, 1.4 g, 6 mmol) in THF (20 mL) were added 1-hydroxypyrrolidine-2,5-dione (690 mg, 6 mmol) and N,N'-dicyclohexylcarbodiimide (1.2 g, 6 mmol). The reaction mixture was stirred at room temperature overnight. The precipitate was filtered off and the filtrate was concentrated to give (2,5-dioxopyrrolidin-1-yl) 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetate (1.9 g, 6 mmol), used immediately without further purification.

To a solution of (2,5-dioxopyrrolidin-1-yl) 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetate (1.6 g, 4.85 mmol) in DMF (15 mL) was added (2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]-N-[4-(hydroxymethyl)phenyl]-5-ureidopentanamide (1.96 g, 5.17 mmol). The mixture was stirred at room temperature for 2 h and concentrated. The residue was diluted in water (20 mL) and acetonitrile (5 mL) and stirred at room temperature overnight. The mixture was purified by C18 reverse phase chromatography using the neutral method to give (2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methylbutanoyl]amino]-N-[4-(hydroxymethyl)phenyl]-5-ureidopentanamide (1.07 g, 1.8 mmol). ^1H NMR (400 MHz, DMSO-d6): δ 9.95 (s, 1H), 8.3 (d, 1H), 7.55 (d, 2H), 7.46 (d, 1H), 7.22 (d, 2H), 5.98 (t, 1H), 5.4 (s, 1H), 5.08 (t, 1H), 4.43 (d, 2H), 4.4 (q, 1H), 4.33 (dd, 1H), 3.95 (s, 2H), 3.6 (m, 10H), 3.38 (t, 2H), 3 (m, 2H), 2 (m, 1H), 1.7/1.6 (2m, 2H), 1.5-1.3 (m, 2H), 0.89/0.82 (2d, 6H).

Step 2: [4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl (4-nitrophenyl)carbonate

To a solution of (2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methylbutanoyl]amino]-N-[4-(hydroxymethyl)phenyl]-5-ureidopentanamide (100 mg, 0.168 mmol) in DMF (30 mL) were added DIPEA (32 μL, 0.179 mmol) and bis(4-nitrophenyl)carbonate (100 mg, 0.329 mmol). The mixture was stirred at room temperature for 4 h and concentrated to dryness. The residue was purified by silica gel chromatography (gradient methanol in dichloromethane) to give 4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl (4-nitrophenyl)carbonate (65 mg, 0.088 mmol), ¹ H NMR (400 MHz, DMSO-d6): δ 9.95 (s, 1H), 8.3 (d, 1H), 7.55 (d, 2H), 7.46 (d, 1H), 7.22 (d, 2H), 5.98 (t, 1H), 5.4 (s, 1H), 5.08 (t, 1H), 4.43 (d, 2H), 4.4 (q, 1H), 4.33 (dd, 1H), 3.95 (s, 2H), 3.6 (m, 10H), 3.38 (t, 2H), 3 (m, 2H), 3.02-2.95 (m, 2H), 2 (m, 1H), 1.7 (m, 1H), 1.6 (m, 1H), 0.89 (d, 3H), 0.82 (d, 3H).

Stage 3: (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid L23-P3

To a solution of ((2R)-2-[(5 S _a )-5-[3-chloro-2-methyl-4-(2-piperazin-1-ylethoxy)phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid P3 (147 mg, 0.17 mmol) in DMF (16 ml) were added sequentially DIPEA (85 μl, 0.51 mmol), 4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl (4-Nitrophenyl)carbonate (136 mg, 0.179 mmol), 2,6-lutidine (99 µL, 0.85 mmol), and HOAt (7 mg, 0.05 mmol). The mixture was stirred at room temperature overnight and purified by reverse-phase C18 prep-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the NH ₄ HCO ₃ method to afford L23-P3 (110 mg, 0.074 mmol). ¹ H NMR (400 MHz, DMSO-d6): δ 10.05 (s, 1H), 8.87 (d, 1H), 8.59 (s, 1H), 8.32 (d, 1H), 7.67 (br s, 1H), 7.59 (d, 2H), 7.52 (dd, 1H), 7.45 (td, 1H), 7.44 (d, 1H), 7.36 (dl, 1H), 7.29 (m, 2H), 7.27 (d, 2H), 7.2 (t, 2H), 7.19 (d, 1H), 7.14 (d, 1H), 7.13 (t, 1H), 7.03 (t, 1H), 6.99 (d, 1H), 6.71 (t, 1H), 6.24 (dl, 1H), 5.99 (t, 1H), 5.48 (dd, 1H), 5.41 (shs, 1H), 5.23 (m, 2H), 4.97 (s, 2H), 4.39 (m, 1H), 4.32 (dd, 1H), 4.21 (m, 2H), 3.95 (m, 2H), 3.75 (s, 3H), 3.65-3.50 (m, 10H), 3.34 (m, 2H), 3.02/2.95 (m, 2H), 2.73 (t, 2H), 2.49/2.3 (m, 2H), 2.45 (m, 4H), 2.3 (m, 4H), 2 (m, 1H), 1.82 (s, 3H), 1.7/1.59 (m, 2H), 1.44/1.37 (m, 2H), 0.87 (d, 3H), 0.82 (d, 3H). ¹³ C NMR (100 MHz, DMSO-d6): δ 158.3, 152.9, 131.6, 131.6, 131.3, 131.3, 131, 129, 128.8, 121, 120.8, 119.5, 116.4, 116.1, 112.8, 112.4, 111.2, 74.5, 70.1, 69.3, 67.7, 66.4, 57, 56.7, 56.2, 53.7, 53.2, 50.4, 43.6, 39, 32.8, 31.6, 29.6, 27.3, 19.3, 17.7. IR wavelength (cm ^-1 ): 3500-2500, 2106, 1656. HR-IER+: m/z [M+H]+=1479.5422/1479.5405 (measured/theoretical).

Obtaining L24-P1:

(2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid

Step 1: (2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-N-[4-(bromomethyl)phenyl]-5-ureido-pentanamide

To a solution of (2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-N-[4-(hydroxymethyl)phenyl]-5-ureidopentanamide (330 mg, 0.55 mmol; prepared according to step 1 of the synthesis of L23-P3) in THF (10 mL) was added dropwise at 0 °C a solution of 1 M phosphorus tribromide in dichloromethane (1 mL, 1 mmol). The mixture was stirred at 0 °C for 1 h and finely ground NaHCO ₃ (100 mg) was added. After 10 min of stirring, the reaction mixture was diluted with ethyl acetate and filtered. The organic layer was dried over magnesium sulfate and concentrated. The residue of (2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methylbutanoyl]amino]-N-[4-(bromomethyl)phenyl]-5-ureidopentanamide (283 mg, 0.43 mmol) was used without further purification. HR-ESI+: m/z [M+H]+=595.3200/595.3198 (measured/theoretical).

Stage 2: ((2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid L24-P1

To a solution of ethyl (2R)-2-[(5S _a )-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate dihydrochloride ( P1 ) (345 mg, 0.355 mmol) in DMF (1 ml) was added (2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-N-[4-(bromomethyl)phenyl]-5-ureidopentanamide (233 mg, 0.355 mmol) and DIPEA (50 μL, 0.304 mmol). The mixture was stirred at room temperature overnight. A solution of lithium hydroxide monohydrate (15 mg, 3.55 mmol) in water (0.5 mL) was added, and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was purified by reverse-phase C18 prep-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the TFA method to afford L24-P1 (80 mg, 0.054 mmol). ^1H NMR (400 MHz, DMSO-d6): δ 13.2 (m, 1H), 10.25 (m, 1H), 8.88 (d, 1H), 8.6 (s, 1H), 8.36 (d, 1H), 7.72 (d, 2H), 7.63 (d, 1H), 7.52 (dd, 1H), 7.46 (t, 1H), 7.44 (m, 1H), 7.43 (m, 2H), 7.37 (d, 1H), 7.3 (dd, 2H), 7.21 (t, 2H), 7.2 (d, 1H), 7.15 (d, 1H), 7.15 (t, 1H), 7.03 (t, 1H), 7 (t, 1H), 6.72 (t, 1H), 6.22 (d, 1H), 6 (t, 1H), 5.52 (m, 2H), 5.49 (dd, 1H), 5.25 (dd, 2H), 4.5 (shs, 2H), 4.39 (m, 1H), 4.32 (m, 1H), 4.25 (m, 2H), 3.95 (shs, 2H), 3.76 (s, 3H), 3.4/3.24 (m, 4H), 3.35 (m, 2H), 3.28/2.51 (m, 2H), 3.04/2.83 (m, 4H), 3.02/2.96 (m, 2H), 2.92 (m, 2H), 2.87 (s, 3H), 1.99 (m, 1H), 1.83 (s, 3H), 1.69/1.61 (m, 2H), 1.46/1.38 (m, 2H), 0.88/0.82 (m, 6H). ¹³ C NMR (125 MHz, DMSO-d6): δ 134.2, 131.4, 131.3, 131.3, 131.2, 130.7, 128.7, 120.9, 120.5, 119.2, 116.3, 115.8, 112.7, 112.3, 111, 74, 70.2, 69.6, 67.8, 58.9, 56.9, 56.1, 55.4, 54, 50.5, 46.6, 44.9, 39, 32.7, 31.6, 29.8, 27.5, 19.7/18.4, 18. IR wavelength (cm ^-1 ): 3700-2200, 3000-2000, 2109, 1662, 1250-1050. HR-IER+: m/z [M+Na]+=1473.5656/1473.5628 (measured/theoretical).

Obtaining L13-C4:

(2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-[4-(phosphonomethyl)phenyl]pyrimidin-4-yl]methoxy]phenyl]propanoic acid

Step 1: Synthesis of (2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-N-[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]-3-methyl-butanamide

To a solution of (2S)-2-amino-N-[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxoethyl]-3-methylbutanamide (0.9 g, 3.07 mmol; prepared according to step 3 of the L18-C3 synthesis) and 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (purchased from Broadpharm, 2 g, 3.07 mmol) in DMF (20 mL) were added sequentially DIPEA (1 mL, 6.13 mmol), 3-(ethyliminomethyleneamino)propyl dimethyl ammonium chloride (EtOH) and 2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (purchased from Broadpharm, 2 g, 3.07 mmol). chloride (EDC) (0.65 g, 3.37 mmol) and [dimethylamino(triazolo[4,5-b]pyridin-3-yloxy)methylene]-dimethyl-ammonium hexafluorophosphate (GATU) (1.28 g, 3.37 mmol). The mixture was stirred at room temperature overnight and purified _by C18 prep reverse phase HPLC by direct loading of the reaction mixture onto an Xbridge column and using the _NH4HCO3 method to give (2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-N-[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]-3-methyl-butanamide (1.64 g, 1.81 mmol). ^1H NMR (400 MHz, DMSO-d6): δ 9.82 (m, 1H), 8.14 (d, 1H), 7.87 (d, 1H), 7.54 (d, 2H), 7.23 (d, 2H), 5.08 (t, 1H), 4.43 (d, 2H), 4.39 (m, 1H), 4.2 (m, 1H), 3.65-3.44 (m, 48H), 3.39 (t, 2H), 2.50-2.30 (m, 2H), 1.97 (m, 1H), 1.31 (d, 3H), 0.87/0.84 (m, 6H). IR wavelength (cm ^-1 ): 3600-3200, 3287, 2106, 1668, 1630, 1100. HR-IER+: m/z [M+H]+=919.5265/919.5234 (measured/theoretical).

Stage 2: [4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate

To a solution of (2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-N-[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]-3-methyl-butanamide (210 mg, 0.228 mmol) in a mixture of THF and dichloromethane (5 and 2.5 ml, respectively) were added pyridine (30 μl, 0.479 mmol) and 4-nitrophenyl chloroformate (97 mg, 0.479 mmol) sequentially. The reaction mixture was stirred at room temperature for 3 h and further portions of 4-nitrophenyl chloroformate were added. (40 mg, 0.197 mmol) and pyridine (30 μl, 0.479 mmol). The reaction mixture was stirred at 0°C for 55 h and evaporated to dryness. The residue was purified by silica gel chromatography (gradient of MeOH in dichloromethane) to give [4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (118 mg, 0.110 mmol). ¹H NMR (400 MHz, DMSO-d6): δ 10.00 (s, 1H), 8.31 (d, 2H), 8.19 (d, 1H), 7.88 (d, 1H), 7.64 (d, 2H), 7.58 (d, 2H), 7.41 (d, 2H), 5.25 (s, 2H), 4.39 (m, 1H), 4.21 (m, 1H), 3.63-3.47 (m, 48H), 3.39 (t, 2H), 2.50-2.35 (m, 2H), 1.98 (m, 1H), 1.31 (d, 3H), 0.89/0.85 (m, 6H). IR wavelength (cm^-1): 3278, 2108, 1763, 1633, 1526, 1525, 1350, 1215, 1110.

Stage 3: (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-[4-(phosphonomethyl)phenyl]pyrimidin-4-yl]methoxy]phenyl]propanoic acid ( L13-C4 )

To a solution of [4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (52 mg, 47.6 μmol) in DMF (5 mL) was added sequentially (2R)-2-[(5S _a )-5-[3-chloro-2-methyl-4-(2-piperazin-1-ylethoxy)phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-[4-(phosphonomethyl)phenyl]pyrimidin-4-yl]methoxy]phenyl]propanoic acid C4 (36.7 mg, 39.7 μmol) and DIPEA (26 μL, 108 μmol). The reaction mixture was stirred at room temperature for 1 h and purified by C18 reverse-phase prep-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the NH ₄ HCO ₃ method to afford L13-C4 (36 mg, 19 μmol). ^1H NMR (400 MHz, DMSO-d6): δ 10.1 (brs, 1H), 8.81 (brs, 1H), 8.55 (m, 1H), 8.32 (brs, 1H), 8.19 (d, 2H), 8.02 (brs, 1H), 7.66 (m, 1H), 7.58 (d, 2H), 7.37 (d, 1H), 7.29 (dd, 2H), 7.28 (d, 2H), 7.25 (d, 2H), 7.19 (t, 2H), 7.17 (d, 1H), 7.08 (t, 1H), 6.96 (d, 1H), 6.68 (t, 1H), 6.21 (d, 1H), 5.5 (m, 1H), 5.22 (m, 2H), 4.96 (s, 2H), 4.4 (m, 1H), 4.2 (dd, 1H), 4.18 (m, 2H), 3.62/3.41 (m, 24H), 3.5 (m, 4H), 3.38 (m, 2H), 3.28 (m, 4H), 2.87 (m, 2H), 2.7 (m, 2H), 2.48/2.36 (m, 2H), 2.41 (m, 4H), 1.99 (m, 1H), 1.79 (s, 3H), 1.3 (d, 3H), 0.87/0.83 (m, 6H). ¹³ C NMR (100 MHz, DMSO-d6): δ 130.7, 130.7, 130.6, 130.3, 129, 128.4, 127.4, 121, 119.6, 116.3, 116.1, 112.1, 70.2/67.3, 69.5, 67.5, 66.4, 58.2, 56.4, 53.2, 50.3, 49.6, 43.8, 36.3, 31, 19, 18.5, 17.8. ¹⁹ F NMR (376 MHz, DMSO-d6): δ -112.4. ³¹ P NMR (200 MHz, DMSO-d6): δ 17.8. IR wavelength (cm ^-1 ): 3290, 2102, 1698, 1651, 1237, 1094, 833, 756. HR-ESI+: m/z [M+H]+=1867.7129/1867.7154 (measured/theoretical).

Obtaining L19-C3:

(2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

Step 1: [4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate

To a suspension of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxoethyl]carbamoyl]-2-methylpropyl]carbamate (1 g, 1.66 mmol) in a THF/dichloromethane mixture (100 and 30 ml, respectively) were added pyridine (269 μl, 3.32 mmol) and 4-nitrophenyl chloroformate (670 mg, 3.30 mmol) sequentially. The reaction mixture was stirred at room temperature overnight and another portion of 4-nitrophenyl chloroformate (335 mg, 1.66 mmol) was added. The reaction mixture was stirred at room temperature for 3 h, concentrated and the residue was purified by silica gel chromatography (gradient with ethyl acetate in heptane) to give [4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (658 mg, 0.97 mmol). ^1H NMR (400 MHz, DMSO-d6): δ 10.07 (m, 1H), 8.31 (d, 2H), 8.19 (d, 1H), 7.89 (d, 2H), 7.74 (t, 2H), 7.64 (d, 2H), 7.57 (d, 2H), 7.41 (m, 2H), 7.41 (d, 2H), 7.4 (m, 1H), 7.32 (t, 2H), 5.24 (s, 2H), 4.43 (m, 1H), 4.36-4.19 (m, 3H), 3.92 (dd, 1H), 2 (m, 1H), 1.32 (d, 3H), 0.9/0.87 (m, 6 H). IR wavelength (cm ^-1 ): 3350-3200, 1760, 1690, 1670, 1630, 1523, 1290.

Stage 2: (2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]pipe razin-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propane acid

To a solution of (2R)-2-[ (5S _a )- 5-[3-chloro-2-methyl-4-(2-piperazin-1-ylethoxy)phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid C3 (100 mg, 0.116 mmol) in DMF (1 ml) were added sequentially [4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (87 mg, 0.128 mmol) and DIPEA (38 μl, 0.232 mmol). The reaction mixture was stirred at room temperature overnight and concentrated. The residue was taken up in water and filtered to give (2R)-2-[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (110 mg, 0.078 mmol), used without further purification in the next step. ^1H NMR (400 MHz, DMSO-d6): δ 10.05 (bs, 1H), 8.88 (d, 1H), 8.57 (s, 1H), 8.23 (d, 1H), 7.88 (d, 2H), 7.75 (m, 1H), 7.74 (2d, 2H), 7.58 (d, 2H), 7.53 (dd, 1H), 7.45 (m, 1H), 7.45 (d, 1H), 7.41 (m, 1H), 7.4 (m, 2H), 7.31 (m, 2H), 7.29 (m, 2H), 7.26 (d, 2H), 7.2 (t, 2H), 7.18 (m, 1H), 7.14 (d, 1H), 7.11 (t, 1H), 7.03 (t, 1H), 6.98 (d, 1H), 6.69 (t, 1H), 6.2 (d, 1H), 5.46 (d, 1H), 5.22 (m, 2H), 4.97 (s, 2H), 4.42 (t, 1H), 4.26 (m, 2H), 4.21 (m, 1H), 4.2 (m, 2H), 3.91 (m, 1H), 3.75 (s, 3H), 3.35/2.45 (m, 2H), 3.29 (m, 4H), 2.73 (t, 2H), 2.44 (m, 4H), 1.99 (m, 1H), 1.8 (s, 3H), 1.29 (d, 3H), 0.88/0.85 (m, 6H). ¹³ C NMR (100 MHz, DMSO-d6): δ 158.3, 152.7, 131.6, 131.4, 131.3, 131.1, 131.1, 128.9, 128.5, 128, 127.6, 125.8, 120.9, 120.5, 120.5, 119.4, 116.4, 116, 112.7, 112.2, 111.1, 69.4, 67.8, 66.5, 66.1, 60.7, 56.8, 56.1, 53.2, 49.6, 47.1, 43.8, 33.3, 30.9, 19.7, 18.9, 18.1.

Stage 3: (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chl or-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propane acid

To a solution of (2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (176 mg, 0.125 mmol) in DMF (3 ml) piperidine (300 μl, 1.25 mmol) was added dropwise at 0°C. The reaction mixture was stirred at room temperature for 1 h and concentrated. The residue was purified by C18 prep reverse phase HPLC by direct loading of the reaction mixture onto an Xbridge column and using the NH ₄ HCO ₃ method to afford (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (130 mg, 0.11 mmol). ^1H NMR (400 MHz, DMSO-d6): δ 10.2 (s, 1H), 8.9 (d, 1H), 8.6 (dl, 1H), 8.55 (s, 1H), 7.85 (d, 1H), 7.6 (d, 2H), 7.55 (dd, 1H), 7.45 (m, 2H), 7.25 (d, 2H), 7.25 (m, 4H), 7.2 (m, 3H), 7.15 (d, 1H), 7.1 (t, 1H), 7.05 (t, 1H), 6.95 (d, 1H), 6.65 (t, 1H), 6.15 (d, 1H), 5.4 (dd, 1H), 5.2 (m, 2H), 4.95 (s, 2H), 4.45 (m, 1H), 4.2 (m, 2H), 3.75 (s, 3H), 3.4/2.35 (m, 2H), 3.3 (m, 5H), 2.6 (t, 2H), 2.4 (m, 4H), 2 (m, 3H), 1.8 (s, 3H), 1.3 (d, 3H), 0.9/0.85 (m, 6H). IR wavelength (cm ^-1 ): 3600-2500, 1678.

Stage 4: (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid L19-C3

To a solution of 2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (50 mg, 0.042 mmol) in DMF (0.3 ml) was added sequentially DIPEA (14 μl, 0.085 mmol), [Dimethylamino-(2,5-dioxopyrrolidin-1-yl)oxy-methylene]-dimethyl-ammonium tetrafluoroborate (14 mg, 0.046 mmol) and a solution of 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetic acid (28 mg, 0.12 mmol) in DMF (0.5 mL) were added. The reaction mixture was stirred at room temperature for 2 h and purified by C18 reverse-phase prep-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the NH ₄ HCO ₃ method to afford L19-C3 (22 mg, 0.016 mmol). ^1H NMR (400 MHz, DMSO-d6): δ 10.02 (s, 1H), 8.88 (d, 1H), 8.4 (d, 1H), 7.72 (bs, 1H), 7.58 (s, 1H), 7.58 (d, 2H), 7.53 (d, 1H), 7.45 (d, 1H), 7.45 (t, 1H), 7.38 (d, 1H), 7.29 (dd, 2H), 7.27 (d, 2H), 7.2 (t, 2H), 7.18 (d, 1H), 7.14 (d, 1H), 7.11 (t, 1H), 7.03 (t, 1H), 6.98 (d, 1H), 6.7 (t, 1H), 6.21 (d, 1H), 5.46 (dd, 1H), 5.23 (m, 2H), 4.97 (s, 2H), 4.4 (m, 1H), 4.29 (dd, 1H), 4.22 (m, 2H), 3.94 (s, 2H), 3.75 (s, 3H), 3.65-3.53 (m, 10H), 3.35 (m, 2H), 3.3 (m, 4H), 3.3/2.5 (m, 2H), 2.73 (t, 2H), 2.44 (m, 4H), 2 (m, 1H), 1.81 (s, 3H), 1.3 (d, 3H), 0.88/0.82 (m, 6H). ¹³ C NMR (100 MHz, DMSO-d6): δ 158, 152.7, 131.4, 131.4, 131.3, 131.1, 131.1, 128.9, 128.6, 120.9, 120.7, 119.5, 116.2, 112.5, 112.1, 111.1, 70.4, 70.4, 69.7, 67.5, 66.2, 56.8, 56.7, 56.1, 53.3, 50.4, 49.5, 43.8, 31.7, 19.5, 0.82, 18.3, 18.2. ¹⁹ F NMR (376 MHz, DMSO-d6): δ -112.3. IR wavelength (cm ^-1 ): 3294, 2104, 1697, 1663, 1288, 1238, 1120, 1076, 1051, 1020, 833,755. HR-ESI+: m/z [M+H]+=1395.5083/1395.5070 (measured/theoretical).

Obtaining L15-C5:

(2R)-3-[2-[[2-[3-[[[2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]ethoxy-hydroxy-phosphoryl]oxy-hydroxy-phosphoryl]oxymethyl]phenyl]pyrimidin-4-yl]methoxy]phenyl]-2-[(5S _a )-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-propanoic acid

Stage 1: (2R)-2-[(5S _a )-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-[3-(phosphonooxymethyl)phenyl]pyrimidin-4-yl]methoxy]phenyl]propanoic acid; bis 2,2,2-trifluoroacetic acid

To a solution of ethyl (2R)-2-[(5S _a )-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-[3-(hydroxymethyl)phenyl]pyrimidin-4-yl]methoxy]phenyl]propanoate (110 mg, 0.123 mmol; prepared according to WO 2016/207216) in THF (0.5 ml) was added diphosphoryl chloride (51 μl, 0.368 mmol) dropwise at -40°C under argon. The reaction mixture was stirred at -40°C for 30 min. Another portion of diphosphoryl chloride (10 μL, 0.074 mmol) was added at -40°C, and the reaction mixture was stirred at -40°C for 20 min, quenched by adding aqueous saturated potassium carbonate solution (0.1 mL), and warmed to room temperature. The pH was adjusted to 10 with potassium carbonate (powder), and the reaction mixture was stirred for 20 min at room temperature. The reaction mixture was acidified to pH 2 by slow addition of aqueous 2 M HCl solution at 0°C and extracted with dichloromethane (4 times). The combined organic layers were concentrated, diluted with dioxane (3 mL), and a solution of lithium hydroxide monohydrate (17 mg, 0.403 mmol) in water (0.3 mL) was added. The reaction mixture was stirred at room temperature for 4 days, neutralized with aqueous 4 M HCl (0.4 mL, 0.4 mmol) and evaporated. The residue was purified by reverse phase C18 prep-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the TFA method to afford (2R)-2-[(5S _a )-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-[3-(phosphonooxymethyl)phenyl]pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetic acid as the 2TFA salt (41 mg, 43 μmol). MS (ESI) m/z [M+2H]/2+=487.5.

Step 2: 2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]ethyl dihydrogen phosphate

To a solution of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (200 mg, 0.311 mmol) in dichloromethane (2 ml) were added 1-hydroxypyrrolidine-2,5-dione (79 mg, 0.684 mmol), 3-(ethyliminomethyleneamino)propyl-dimethyl-ammonium chloride (107 mg, 0.56 mmol). The reaction mixture was stirred at room temperature overnight, diluted with dichloromethane, separated with saturated aqueous NaHCO ₃ solution and extracted with dichloromethane. The combined organic layers were washed with brine, dried over magnesium sulfate, and concentrated to approximately 1 mL. The residue was diluted with DMF (1 mL), 2-aminoethyl dihydrogen phosphate (30 mg, 0.214 mmol) was added, and the reaction mixture was stirred at 80°C overnight, diluted with dichloromethane, washed with water. The aqueous layer was separated and freeze-dried to give 2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]ethyl dihydrogen phosphate (165 mg, 0.2 mmol). ^1H NMR (400 MHz, DMSO-d6): δ 3.45-3.65 (m, 53H), 3.26-3.39 (m, 2H), 3.12 (m, 2H), 2.27 (t, 2H). HR-IER+: m/z [M+H]+=767.3697/767.3686 (measured/theoretical).

Step 3: (2R)-3-[2-[[2-[3-[[[2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]ethoxy-hydroxy-phosphoryl]oxy-hydroxy-phosphoryl]oxymethyl]phenyl]pyrimidin-4-yl]methoxy]phenyl]-2-[(5S _a )-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-propanoic acid ( L15-C5 )

To a solution of 2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]ethyl dihydrogen phosphate (49 mg, 0.064 mmol) in DMF (0.2 mL) were successively added di(imidazol-1-yl)methanone (11 mg, 0.066 mmol), triethylamine (17 μL, 0.066 mmol) and 4Å molecular sieves (50 mg). The reaction mixture was stirred at room temperature for 2 h. The solid was removed by filtration and the filtrate was treated with zinc chloride (23 mg, 0.172 mmol) and (2R)-2-[(5S _a )-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-[3-(phosphonooxymethyl)phenyl]pyrimidin-4-yl]methoxy]phenyl]propanoic acid; bis 2,2,2-trifluoroacetic acid (41 mg, 0.043 mmol). The mixture was heated to 50°C overnight. The reaction mixture was purified by C18 prep _- RP-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the _NH4HCO3 method to afford L15-C5 (11 mg, 6 μmol). HR-ESI+: m/z [M+H]+=1703.5962/1703.5959 (measured/theoretical).

Obtaining L17-C3:

(2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2S,3R,4S,5R)-6-azido-2,3,4,5-tetrahydroxy-hexyl]amino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetic acid

To a solution of (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (230 mg, 0.194 mmol; prepared according to step 5 of preparation L19-C3) and 6-deoxy-6-azido-D-galactose (120 mg, 0.584 mmol; prepared according to Ekholm et al., ChemMedChem 2016, 11, 2501-2505) in DMSO/water 80/20 mixture containing 1% of DIPEA (20 mL) was added sodium cyanoborohydride (24 mg, 0.389 mmol) at room temperature. The reaction mixture was heated at 65°C for 48 h. Then, another portion of sodium cyanoborohydride (24 mg, 0.389 mmol) and 6-deoxy-6-azido-D-galactose (120 mg, 0.584 mmol) were added at room temperature. The reaction mixture was heated at 65°C for another 48 h and purified by C18 prep-RP-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the TFA method to afford L17-C3 (38 mg, 28 μmol). ^1H NMR (400 MHz, DMSO-d6): δ 13.2 (brs, 1H), 10.2 (s, 1H), 8.88 (d, 1H), 8.85 (d, 1H), 8.62 (s, 1H), 8.53 (brs, 1H), 7.63 (d, 1H), 7.59 (d, 2H), 7.52 (d, 1H), 7.45 (t, 1H), 7.42 (d, 1H), 7.33 (dd, 2H), 7.33 (d, 2H), 7.27 (d, 1H), 7.27 (d, 1H), 7.21 (t, 2H), 7.15 (t, 1H), 7.04 (t, 1H), 7.01 (d, 1H), 6.73 (t, 1H), 6.21 (d, 1H), 5.51 (d, 1H), 5.28/5.22 (m, 2H), 5.04 (shs, 2H), 4.52 (m, 1H), 4.49 (m, 2H), 4.12 (m, 1H), 3.89 (m, 1H), 3.78 (m, 1H), 3.76 (s, 3H), 3.63 (m, 6H), 3.42/3.21 (m, 2H), 3.38 (m, 1H), 3.37 (m, 1H), 3.28/2.52 (m, 2H), 3.22 (m, 4H), 2.96 (m, 2H), 2.21 (m, 1H), 1.86 (s, 3H), 1.36 (d, 3H), 1.03/0.94 (m, 6H). ¹³ C NMR (125 MHz, DMSO-d6): δ 157.8, 152.5, 131.4, 131.3, 131.3, 130.6, 129.1, 129, 128.8, 120.8, 120.6, 119.4, 116.2, 116.1, 112.3, 111.3, 111.3, 74.2, 71.3, 70.4, 69.5, 69.2, 67.1, 65.6, 64.5, 64.5, 56.2, 54.8, 54.2, 51.9, 50.3, 49.9, 32.7, 29.4, 19.3, 18.9, 18. ¹⁹ F NMR (470 MHz, DMSO-d6): δ -74.4, -112.1. IR wavelength (cm ^-1 ): 2200-3500, 2104, 1669, 1181, 1132, 798, 758, 720. HR-ESI+: m/z [M+H]+=1369.4918/1369.4913 (measured/theoretical).

Obtaining L24-P7:

(2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-3-chloro-2-ethyl-phenyl]-6-prop-1-ynyl-thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid

To a solution of (2S)-2-[[(2S)-2-[[2-(2-azidoethoxy)acetyl]amino]-3-methyl-butanoyl]amino]-N-[4-(bromomethyl)phenyl]-5-ureido-pentanamide (72 mg, 0.109 mmol) in THF (5 ml) were added sequentially (2R)-2-[(5S _a )-5-[3-chloro-2-ethyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-prop-1-ynyl-thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid P7 (30 mg, 0.036 mmol) and DIPEA (19 μl, 0.108 mmol). The reaction mixture was stirred overnight at room temperature and purified by C18 prep-RP-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the TFA method to afford L24-P7 (25 mg, 18 μmol). ^1H NMR (400 MHz, DMSO-d6): δ 10.25 (s, 1H), 8.85 (d, 1H), 8.62 (s, 1H), 8.35 (d, 1H), 7.72 (d, 2H), 7.6 (d, 1H), 7.5 (d, 1H), 7.45 (t, 1H), 7.43 (d, 2H), 7.4 (d, 1H), 7.22 (d, 1H), 7.17 (m, 1H), 7.15 (m, 1H), 7.13 (d, 1H), 7.02 (t, 1H), 7 (d, 1H), 6.78 (t, 1H), 6.3 (d, 1H), 5.98 (shs, 1H), 5.5 (dd, 1H), 5.4 (ws, 1H), 5.28/5.2 (m, 2H), 4.5 (ws, 2H), 4.38 (m, 1H), 4.3 (dd, 1H), 4.25 (m, 2H), 3.94 (ws, 2H), 3.74 (s, 3H), 3.70/3.50 (m, 10H), 3.50 (m, 8H), 3.35 (t, 2H), 3.22/2.5 (m, 2H), 3.0 (m, 2H), 2.95 (t, 2H), 2.9 (shs, 3H), 2.55/2.4 (m, 2H), 2.0 (s, 3H), 1.98 (m, 1H), 1.70/1.30 (m, 4H), 0.88/0.82 (m, 6H), 0.72 (t, 3H). IR wavelength (cm ^-1 ): 3321, 2111, 1660, 1188, 1124, 798,756,719. HR-ESI+: m/z [M+H-CF3COOH]+=1409.59077/1409.5903 (measured/theoretical).

Obtaining L24-P6:

(2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-[2-(hydroxymethyl)phenyl]pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid

To a solution of (2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methylbutanoyl]amino]-N-[4-(bromomethyl)phenyl]-5-ureidopentanamide (55.3 mg, 84 μmol) in DMF (1 mL) was added sequentially ethyl (2R)-2-[(5S _a )-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-[2-(hydroxymethyl)phenyl]pyrimidin-4-yl]methoxy]phenyl]propanoate (53.2 mg, 59 μmol; synthesized according to EP 2 886 545) and DIPEA (44 μL, 0.252 mmol). The reaction mixture was stirred at room temperature for 1 h and concentrated under reduced pressure. The residue was diluted with dioxane (1 mL) and a solution of lithium hydroxide monohydrate (14 mg, 0.0334 mmol) in water (0.3 mL) was added. The reaction mixture was stirred at room temperature overnight, neutralized by adding aqueous 1 M HCl (0.33 mL, 0.33 mmol), and concentrated under reduced pressure. The crude product was purified by reverse-phase C18 prep-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the TFA method to afford L24-P6 (47 mg, 32 μmol). ^1H NMR (400 MHz, DMSO-d6): δ 10.27 (s, 1H), 8.94 (d, 1H), 8.61 (s, 1H), 8.38 (d, 1H), 7.93 (d, 1H), 7.73 (d, 2H), 7.68 (t, 1H), 7.66 (d, 1H), 7.5 (t, 1H), 7.45 (d, 1H), 7.43 (d, 2H), 7.38 (d, 1H), 7.37 (m, 1H), 7.3 (dd, 2H), 7.21 (d, 1H), 7.2 (t, 2H), 7.16 (t, 1H), 7.02 (d, 1H), 6.72 (t, 1H), 6.21 (d, 1H), 6.01 (m, 1H), 5.5 (d, 1H), 5.4 (m, 1H), 5.3 (m, 2H), 4.8 (s, 2H), 4.39 (m, 1H), 4.32 (dd, 1H), 4.25 (m, 2H), 3.95 (s, 2H), 3.57 (m, 16H), 3.42/3.26 (m, 2H), 3.36 (m, 2H), 3.29/2.51 (m, 2H), 3.11/2.92 (m, 8H), 2.98 (m, 2H), 2.97 (m, 2H), 1.99 (m, 1H), 1.83 (s, 3H), 1.68/1.62 (m, 2H), 1.45/1.39 (m, 2H), 0.88/0.82 (m, 6H). ¹³ C NMR (100 MHz, DMSO-d6): δ 158.2, 152.1, 134.2, 131.4, 131.3, 130.9, 130.8, 130.2, 128.7, 128.1, 127, 120.8, 119.3, 116.3, 115.7, 112.2, 111, 74, 70.5, 70.1, 69.5, 67.7, 62.3, 58.8, 57.2, 55.5, 54.1, 50.5, 46.6, 38.9, 32.5, 31.5, 29.6, 27.6, 19.6, 18.6, 18.3. ¹⁹ F NMR (376 MHz, DMSO-d6): δ -74.6, -112.5. IR wavelength (cm ^-1 ): 3303, 2104, 1730, 1662, 1182, 1124, 833,796,761. HR-ESI+: m/z [M+2H]/2+=726.2957/726.2941 (measured/theoretical).

Obtaining L20-C6:

(2R)-3-[2-[[2-[2-[[2-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]ethyl-methyl-carbamoyl]oxymethyl]phenyl]pyrimidin-4-yl]methoxy]phenyl]-2-[(5S _a )-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-propanoic acid

Step 1: Ethyl (2R)-3-[2-[[2-[2-[[2-[tert-butoxycarbonyl(methyl)amino]ethyl-methyl-carbamoyl]oxymethyl]phenyl]pyrimidin-4-yl]methoxy]phenyl]-2-[(5S _a )-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxypropanoate

To the solution (ethyl (2R)-2-[(5S_a)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-[2-(hydroxymethyl)phenyl]pyrimidin-4-yl]methoxy]phenyl]propanoate (50 mg, 55 μmol; synthesized according to EP 2 886 545) in dichloromethane (0.5 ml) was successively added 4-nitrophenyl chloroformate (19 mg, 94 μmol) and DIPEA (69 μl, 0.5 mmol). The reaction mixture was stirred at room temperature for 1 h and tert-butyl N-methyl-N-[2-(methylamino)ethyl]carbamate was added. (54 mg, 0.287 mmol). The mixture was stirred at room temperature overnight and concentrated under reduced pressure. The residue was purified by silica gel chromatography (gradient of methanol in dichloromethane) to give ethyl (2R)-3-[2-[[2-[2-[[2-[tert-butoxycarbonyl(methyl)amino]ethyl-methyl-carbamoyl]oxymethyl]phenyl]pyrimidin-4-yl]methoxy]phenyl]-2-[(5S_a)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxypropanoate (30 mg, 27 μmol).¹H NMR (500 MHz, DMSO-d6): δ 9.00 (d, 1H), 8.58 (s, 1H), 7.98 (m, 1H), 7.61 (d, 1H), 7.51 (t, 1H), 7.48 (d, 1H), 7.45 (t, 1H), 7.31 (dd, 2H), 7.31 (d, 1H), 7.22 (t, 2H), 7.18 (t, 1H), 7.17 (d, 1H), 7.02 (d, 1H), 6.76 (t, 1H), 6.32 (d, 1H), 5.52 (dd, 1H), 5.47 (shs, 2H), 5.26 (m, 2H), 4.2 (m, 2H), 4.07 (m, 2H), 3.24/3.17 (2m, 4H), 3.17/2.6 (2m, 2H), 2.77/2.64 (m, 6H), 2.7 (m, 2H), 2.49/2.28 (m, 8H), 2.12 (shs, 3H), 1.87 (s, 3H), 1.3 (3s, 9H), 1.07 (t, 3H). ¹³C NMR (125 MHz, DMSO-d6): δ 158.2, 152.4, 131, 130.1, 130.1, 129, 128.3, 128.2, 121.5, 121.4, 120.9, 116.3, 115.8, 112, 111.1, 74.1, 69.2, 68.1, 65.6, 61.2, 56.8, 55.2, 53.1, 46.5, 45.9, 34.5, 32.4, 28.3, 17.4, 14.9.1⁹F NMR (470 MHz, DMSO-d6): δ -112.2. IR wavelength (cm^-1): 1750, 1693, 1221/1160/1120, 834/756.

Step 2: 2R)-3-[2-[[2-[2-[[2-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]ethyl-methyl-carbamoyl]oxymethyl]phenyl]pyrimidin-4-yl]methoxy]phenyl]-2-[(5Sa)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-propanoic acid L20-C6

To a solution of ethyl (2R)-3-[2-[[2-[2-[[2-[tert-butoxycarbonyl(methyl)amino]ethyl-methyl-carbamoyl]oxymethyl]phenyl]pyrimidin-4-yl]methoxy]phenyl]-2-[(5S_a)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxypropanoate (25 mg, 22 μmol) in dichloromethane (0.5 mL) was added at 0°C with trifluoroacetic acid (35 μL, 447 mmol). The reaction mixture was stirred at room temperature for 6 h and concentrated under reduced pressure. The residue was diluted with DMF (0.5 mL) and [4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methylbutanoyl]amino]-5-ureidopentanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (20 mg, 22 μmol; prepared according to step 3 of preparation of L23-P3) and DIPEA (78 μL, 0.447 mmol) were added sequentially. The reaction mixture was stirred at room temperature overnight, concentrated under reduced pressure, diluted with dioxane (0.5 mL), and treated with a solution of lithium hydroxide monohydrate (3.7 mg, 89 μmol) in water (0.3 mL). The reaction mixture was stirred at room temperature overnight, neutralized at 0°C by dropwise addition of an aqueous 1M HCl solution to pH 7, and concentrated under reduced pressure.

The crude product was purified by C18 prep-reversed-phase HPLC by direct loading of the reaction mixture onto an Xbridge column and using the NH ₄ HCO ₃ method to afford L20-C6 (13 mg, 8 µmol). ^1H NMR (500 MHz, DMSO-d6): δ 8.88 (m, 1H), 8.54 (s, 1H), 7.97 (d, 1H), 7.77 (m, 1H), 7.6 (d, 2H), 7.5 (m, 1H), 7.47 (m, 1H), 7.46 (m, 1H), 7.41 (d, 1H), 7.29 (dd, 2H), 7.21 (t, 2H), 7.19 (d, 1H), 7.18 (m, 2H), 7.12 (t, 1H), 6.97 (d, 1H), 6.7 (t, 1H), 6.19 (d, 1H), 5.49 (d, 1H), 5.45 (m, 4H), 5.23 (m, 2H), 4.89 (m, 2H), 4.4 (m, 1H), 4.32 (dd, 1H), 4.22 (m, 2H), 3.94 (s, 2H), 3.56 (m, 10H), 3.39/2.44 (m, 2H), 3.34 (t, 2H), 3.28 (m, 4H), 2.99 (m, 2H), 2.75/2.7 (m, 6H), 2.73 (m, 2H), 2.5/2.37 (m, 8H), 2.18 (s, 3H), 2.04 (m, 1H), 1.81 (s, 3H), 1.74/1.62 (m, 2H), 1.46/1.38 (m, 2H), 0.86/0.8 (m, 6H). ¹³ C NMR (125 MHz, DMSO-d6): δ 158.3, 152.9, 131.5, 131.4, 131.3, 131, 130, 128.3, 128.3, 128, 127.7, 120.8, 119.3, 116.2, 115.6, 112.1, 111.1, 75.3, 70.5, 70.2, 69.2, 67.6, 66.6, 65.4, 57.2, 56.7, 55.1/52.9, 54, 50.5, 46.5, 45.1, 39.1, 34.4, 31.5, 29.6, 27.4, 19.9, 18.2, 18. ¹⁹ F NMR (470 MHz, DMSO-d6): δ -112.5. IR wavelength (cm ^-1 ): 3323, 2106, 1691, 1660, 1220, 1120, 1051, 759. HR-ESI+: m/z [M+H]+=1609.6517/1609.6500 (measured/theoretical).

Obtaining L22-C1:

(2R)-2-[(5Sa)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-yl-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid;2,2,2-trifluoroacetate;2,2,2-trifluoroacetic acid

Stage 1: (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; bis-2,2,2-trifluoroacetic acid

To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate (200 mg, 0.388 mmol) in DMF (20 mL) were added triphenylphosphine (152 mg, 0.581 mmol) and N-bromosuccinimide (103 mg, 0.581 mmol) sequentially. The reaction mixture was stirred at room temperature overnight and (2R)-2-[ (5S _a )- 5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid C1 (302 mg, 345 mmol) and DIPEA (120 μL, 0.691 mmol) were added. The reaction mixture was stirred at room temperature for 2 s and diethylamine (49 μL, 486 mmol) was added. The reaction mixture was stirred at room temperature for 24 h, concentrated under reduced pressure and purified by C18 prep reverse phase HPLC by direct loading of the reaction mixture onto an Xbridge column and using the TFA method to give (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; bis-2,2,2-trifluoroacetic acid (253 mg, 0.220 mmol). ^1H NMR (400 MHz, DMSO-d6): δ 10.4 (s, 1H), 8.89 (d, 1H), 8.75 (d, 1H), 8.61 (s, 1H), 8.08 (big, 3H), 7.72 (d, 2H), 7.63 (d, 1H), 7.52 (d, 1H), 7.46 (t, 1H), 7.45 (d, 2H), 7.39 (d, 1H), 7.31 (dd, 2H), 7.21 (d, 1H), 7.21 (t, 2H), 7.15 (d, 1H), 7.15 (t, 1H), 7.04 (t, 1H), 7.01 (d, 1H), 6.72 (t, 1H), 6.22 (d, 1H), 5.5 (dd, 1H), 5.25 (m, 2H), 4.53 (m, 2H), 4.52 (m, 1H), 4.28 (m, 2H), 3.76 (s, 3H), 3.62 (m, 1H), 3.43/3.29 (m, 4H), 3.28/2.5 (m, 2H), 3.13/2.94 (m, 4H), 3.01 (m, 2H), 2.9 (br s, 3H), 2.07 (m, 1H), 1.84 (d, 3H), 1.36 (d, 3H), 0.95 (d, 6H). ¹³ C NMR (125 MHz, DMSO-d6): δ 253, 158.2, 134.3, 131.5, 131.4, 131.4, 131.3, 131, 128.9, 121.1, 120.6, 119.5, 116.3, 115.9, 113, 112.3, 111.1, 74.1, 69.8, 67.5, 58.7, 57.9, 56.5, 55.4, 49.8, 46.5, 45.2, 32.9, 30.4, 18.6, 18.4, 18.3. ¹⁹ F NMR (470 MHz, DMSO-d6): δ -74, -112.6.

Stage 2: (2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; bis-2,2,2-trifluoroacetic acid L22-C1

To a solution of (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; To bis-2,2,2-trifluoroacetic acid (150 mg, 0.130 mmol) in DMF (0.4 mL) was added (2,5-dioxopyrrolidin-1-yl) 3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoate (60 mg, 194 mmol). The reaction mixture was stirred at room temperature for 3 h, concentrated under reduced pressure, and purified by C18 reverse-phase prep-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the TFA method to afford L22-C1 (67 mg, 37 μmol). ^1H NMR (400 MHz, DMSO-d6): δ 10.14 (s, 1H), 8.88 (d, 1H), 8.61 (s, 1H), 8.22 (d, 1H), 7.84 (d, 1H), 7.73 (d, 2H), 7.63 (d, 1H), 7.52 (dd, 1H), 7.45 (d, 1H), 7.44 (d, 2H), 7.38 (d, 1H), 7.31 (dd, 2H), 7.21 (d, 1H), 7.21 (t, 2H), 7.15 (t, 1H), 7.14 (d, 1H), 7.02 (t, 1H), 7.01 (d, 1H), 7 (s, 2H), 6.71 (t, 1H), 6.21 (d, 1H), 5.5 (dd, 1H), 5.25 (m, 2H), 4.53 (shs, 2H), 4.38 (m, 1H), 4.25 (m, 2H), 4.19 (dd, 1H), 3.76 (s, 3H), 3.58 (m, 2H), 3.54 (t, 2H), 3.48 (m, 2H), 3.43/3.3 (m, 4H), 3.28/2.51 (m, 2H), 3.16/2.98 (m, 4H), 3.04 (m, 2H), 2.91 (shs, 3H), 2.43/2.33 (m, 2H), 1.93 (m, 1H), 1.84 (s, 3H), 1.31 (d, 3H), 0.87/0.82 (m, 6H). ¹³ C NMR (100 MHz, DMSO-d6): δ 158, 152.8, 135.2, 134, 131.4, 131.3, 131.3, 131.2, 131, 128.9, 120.8, 120.6, 119.3, 116.3, 115.8, 112.4, 112.3, 111.1, 74.2, 69.6, 67.4, 67.4, 67.1, 67, 58.4, 57.9, 56.2, 55.2, 49.7, 46.5, 45.1, 37.1, 36.3, 32.7, 30.9, 19.6, 18.5, 18.2, 18.2. ¹⁹ F NMR (376 MHz, DMSO-d6): δ -74.6, -112.2. IR wavelength (cm ^-1 ): 2000-3500, 1760/1705, 1733, 1668, 1180/1128, 829/798/758/720/696. HR-ESI+: m/z [M+H]+=1345.4944/1345.4954 (measured/theoretical)

Obtaining L9-C9:

3-[4-[[2-[(2R)-2-carboxy-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-yl-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-ethyl]phenoxy]methyl]pyrimidin-2-yl]benzenesulfonate; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid

Step 1: (2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-N-[4-(hydroxymethyl)phenyl]-5-ureido-pentanamide

To a solution of 3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoic acid (855 mg, 4.01 mmol) in THF (42 ml) were added N,N'-dicyclohexylmethanediimine (1.05 g, 5.08 mmol) and 1-hydroxypyrrolidine-2,5-dione (510 mg, 4.43 mmol). The reaction mixture was stirred at room temperature for 20 h. The precipitate was removed by filtration and the filtrate was added to a solution of (2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-N-[4-(hydroxymethyl)phenyl]-5-ureido-pentanamide (1.27 g, 3.35 mmol) in DMF (42 mL). The reaction mixture was stirred at room temperature for 20 h, diluted with diethyl ether (250 mL). The solid was isolated by filtration to give (2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methylbutanoyl]amino]-N-[4-(hydroxymethyl)phenyl]-5-ureidopentanamide (1.81 g, 3.15 mmol) as a white solid.¹H NMR (400 MHz, DMSO-d6): δ 9.87 (s, 1H), 8.05 (d, 1H), 7.82 (d, 1H), 7.53 (d, 2H), 7.21 (d, 2H), 7.00 (s, 2H), 5.95 (t, 1H), 5.39 (s, 2H), 5.07 (t, 1H), 4.41 (d, 2H), 4.34-4.40 (m, 1H), 4.18-4.22 (m, 1H), 3.42-3.65 (m, 4H), 2.88-3.02 (m, 2H), 2.73 (s, 2H), 2.28-2.45 (m, 2H), 1.91-1.99 (m, 1H), 1.53-1.75 (m, 2H), 1.30-1.147 (m, 2H), 0.85 (d, 3H), 0.81 (d, 3H). ¹³C NMR (125 MHz, DMSO-d6): δ 171.05, 170.83, 170.32, 170.09, 158.82, 137.49, 137.37, 134.50, 126.88, 118.81, 66.66, 66.53, 62.57, 57.49, 53.06, 36.74, 35.76, 30.51, 29.31, 26.79, 25.20, 19.16, 18.07. MS (ESI) m/z [M+H]+=575.2.

Step 2: (2S)-N-[4-(bromomethyl)phenyl]-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanamide

To a solution of (2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methylbutanoyl]amino]-N-[4-(hydroxymethyl)phenyl]-5-ureidopentanamide (37.2 mg, 65 μmol) in THF (1 mL) was added phosphorus tribromide (45 μL, 97 mmol) dropwise at 0°C under argon. The reaction mixture was stirred at 0°C for 1 h and at room temperature for 2 h. The progress of the reaction mixture was monitored by UPLC-MS: an aliquot was treated with a large excess of morpholine in acetonitrile, followed by the formation of the corresponding morpholine addition product. The reaction mixture was diluted with THF (3 mL), quenched by adding 2 drops of saturated NaHCO ₃ solution, stirred for 5 min at room temperature, dried over magnesium sulfate, and filtered. The residue containing crude (2S)-N-[4-(bromomethyl)phenyl]-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methylbutanoyl]amino]-5-ureidopentanamide (45 mg, 65 μmol theoretical) was used directly in the next step. MS (ESI) m/z [M+H]+ = 662.62 (morpholine addition product)

Step 3: 3-[4-[[2-[(2R)-2-carboxy-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-yl-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-ethyl]phenoxy]methyl]pyrimidin-2-yl]benzenesulfonate L9-C9

To a solution of (2R)-2-{[( 5S _a )-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(3-sulfophenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid C9 (15 mg, 16 mmol) in DMF (0.8 mL) was added a solution of (2S)-N-[4-(bromomethyl)phenyl]-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanamide (45 mg crude, 65 μmol theoretical from step 2) in THF (1 mL) and DIPEA (14 μL, 81 μmol). The reaction mixture was stirred at room temperature and heated at 50°C for 2 h. The crude product was purified by C18 prep-reversed-phase HPLC by direct loading of the reaction mixture onto an Xbridge column and using the TFA method to afford L9-C9 (5.2 mg, 3.5 μmol). HR-ESI+: m/z [M+H]+=1481.4917/1481.4896 (measured/theoretical).

Getting L9-C13:

(2R)-2-[6-(3-amino-4,5-difluorophenyl)-(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid;2,2,2-trifluoroacetate;2,2,2-trifluoroacetic acid

The procedure is the same as in the method for synthesizing L9-C9 , replacing C9 used in step 3 with (2R)-2-{[( 5S _a )-6-(3-amino-4,5-difluorophenyl)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid C13 and using the TFA method for purification. ^1H NMR (400 MHz, DMSO-d6): δ 10.2 (s), 8.9 (d, 1H), 8.6 (s, 1H), 8.12 (d), 7.8 (d), 7.7 (d, 2H), 7.6 (d, 1H), 7.5 (d, 1H), 7.45 (d, 2H), 7.42 (m, 1H), 7.32 (d, 1H), 7.2 (d, 1H), 7.18 (m, 1H), 7.18 (m, 1H), 7.02 (d, 1H), 7 (s, 2H), 7 (m, 1H), 6.75 (t, 1H), 6.65 (d, 1H), 6.25 (d, 1H), 6.15 (dd, 1H), 5.98 (m, 1H), 5.48 (dd, 1H), 5.4 (ws, 1H), 5.24 (dd, 2H), 4.51 (ws, 2H), 4.38 (m, 1H), 4.28 (m, 2H), 4.22 (m, 1H), 3.80-3.40 (m, 8H), 3.75 (s, 3H), 3.26 (m, 4H), 3.1 (m, 2H), 2.98 (m, 4H), 2.9 (shs, 3H), 2.9/2.5 (2m, 2H), 2.43/2.3 (2m, 2H), 1.92 (m, 1H), 1.88 (s, 3H), 1.70-1.30 (m, 4H), 0.82 (2d, 6H). ¹⁹ F NMR (470 MHz, DMSO-d6): δ -74.3, -139.3, -160.4. HR-ESI+: m/z [M+H]+=1464.5482/1464.5449 (measured/theoretical).

Obtaining L14-C3:

(2S,3S,4R,5R,6S)-6-[2-[(5S _a )-5-[[(2S)-2-[[(2S)-2-[[2-(2-azidoethoxy)acetyl]amino]-3-methyl-butanoyl]amino]propanoyl]amino]-2-[[4-[2-[4-[4-[(1R)-1-carboxy-2-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]ethoxy]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy]ethyl]piperazine-1-carbonyl]oxymethyl]phenyl]ethyl]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid

Stage 1: 2-iodo-4-nitrobenzoic acid

To a solution of 2-amino-4-nitrobenzoic acid (10.0 g, 54.90 mmol) in acetonitrile (280 mL) was added p-toluenesulfonic acid monohydrate (32.0 g, 168.2 mmol). The mixture was stirred at room temperature for 15 min, then a solution of sodium nitrite (8.00 g, 115.9 mmol) and potassium iodide (24.0 g, 144.6 mmol) in water (140 mL) were added dropwise over 15 min. The reaction mixture was stirred for 19 h. After completion of the reaction, the mixture was quenched with sodium thiosulfate (13.02 g, 82.36 mmol) and acidified with an aqueous solution of 3 M hydrochloride (25 mL). The aqueous layer was extracted with ethyl acetate (2×250 mL) and the combined organic layers were washed with aqueous 1 M HCl solution (100 mL), dried over sodium sulfate, filtered and concentrated to dryness. The resulting residue was taken up in dichloromethane (1 L) and washed with aqueous 1 M HCl solution (100 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to dryness to give 2-iodo-4-nitrobenzoic acid (15.0 g, 51.2 mmol) as an orange powder. ¹ H NMR (400 MHz, DMSO-d6): δ 13.8 (br s, 1H), 8.64 (s, 1H), 8.27 (d, 1H), 7.86 (d, 1H).

Step 2: (2-iodo-4-nitro-phenyl)methanol

To a solution of 2-iodo-4-nitrobenzoic acid (5.0 g, 17.06 mmol) in THF (70 mL) was added a solution of 1 M borane in THF (85 mL, 85.0 mmol). The reaction mixture was stirred at 65°C for 4 h. After completion of the reaction, the reaction mixture was cooled to room temperature and quenched by adding methanol (200 mL). The mixture was stirred at room temperature for 30 min, then concentrated to dryness. The crude product was purified by silica gel chromatography (gradient with ethyl acetate in cyclohexane) to give (2-iodo-4-nitrophenyl)methanol (3.38 g, 12.11 mmol) as a yellow solid. ^1H NMR (400 MHz, DMSO-d6): δ 8.54 (d, 1H), 8.29 (dd, 1H), 7.70 (d, 1H), 5.82 (t, 1H), 4.47 (d, 2H).

Step 3: (4-amino-2-iodo-phenyl)methanol

To a solution of (2-iodo-4-nitrophenyl)methanol (3.70 g, 13.26 mmol) in ethanol (100 mL) and water (25 mL) were added successively iron (3.70 g, 66.25 mmol) and ammonium chloride (800 mg, 14.96 mmol). The reaction mixture was stirred for 3 h at 80°C. After completion of the reaction, the reaction mixture was filtered over Celite®, washed with ethanol and concentrated to dryness. The resulting residue was taken up in ethyl acetate (100 mL) and washed with saturated sodium hydrogen carbonate solution (100 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to dryness to give (4-amino-2-iodophenyl)methanol (2.48 g, 9.95 mmol) as a yellow oil. ^1H NMR (400 MHz, DMSO-d6): δ 7.02-7.10 (m, 2H), 6.57 (d, 1H), 5.16 (s, 2H), 4.97 (t, 1H), 4.28 (d, 2H).

Step 4: 4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodoaniline

To a solution of (4-amino-2-iodophenyl)methanol (3.51 g, 13.37 mmol) in dichloromethane (150 mL) was added imidazole (0.95 g, 13.95 mmol). The mixture was cooled to 0°C, then a solution of tert-butyl chlorodimethylsilane (2.40 mL, 13.85 mmol) in dichloromethane (150 mL) was added dropwise over 15 min. The ice bath was removed and the reaction mixture was stirred at room temperature for 16 h. After completion of the reaction, the reaction mixture was quenched with methanol (20 mL) and concentrated to dryness. The crude product was purified by silica gel chromatography (gradient with ethyl acetate in cyclohexane) to give 4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodoaniline (3.64 g, 10.03 mmol, 75%) as a yellow oil. ¹ H NMR (400 MHz, DMSO-d6): δ 7.05 (s, 1H), 7.03 (d, 1H), 6.55 (d, 1H), 5.24 (s, 2H), 4.46 (s, 2H), 0.88 (s, 9H), 0.06 (s, 6H).

Step 5: (2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoic acid

To a solution of (2S)-2-aminopropanoic acid (3.22 g, 36.09 mmol) in water (90 mL) were added sequentially sodium carbonate (7.29 g, 68.74 mmol) and a solution of (2,5-dioxopyrrolidin-1-yl) (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoate (15.0 g, 34.37 mmol) in dimethoxyethane (90 mL). The reaction mixture was stirred for 16 h at room temperature. After completion of the reaction, the mixture was acidified to pH 1 with an aqueous 1 M hydrochloride solution, then the aqueous layer was extracted with ethyl acetate (3×500 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to dryness to give a crude mixture, which was triturated with diethyl ether (50 mL) to give (2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoic acid (11.25 g, 27.41 mmol) as a white solid. ¹ H NMR (400 MHz, DMSO-d6) δ 12.48 (s, 1H), 8.21 (d, 1H), 7.89 (d, 2H), 7.72-7.79 (m, 2H), 7.28-7.46 (m, 5H), 4.15-4.32 (m, 4H), 3.90 (t, 1H), 1.90-2.02 (m, 1H), 1.28 (d, 3H), 0.86-0.90 (m, 6H).

Step 6: 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-2-[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodoanilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate

To a solution of (2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]amino]propanoic acid (1.50 g, 3.65 mmol) in dichloromethane (18 mL) and methanol (18 mL) were added sequentially 4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodoaniline (1.33 g, 3.65 mmol) and ethyl 2-ethoxy-2H-quinoline-1-carboxylate (1.36 g, 5.48 mmol). The colorless suspension was stirred for 16 h at room temperature. After concentration to dryness, the crude product was purified by silica gel chromatography (gradient ethyl acetate in cyclohexane) and then C18 chromatography (gradient methanol in water) to give 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-2-[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodoanilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate (1.18 g, 1.56 mmol) as a white solid. ^1H NMR (400 MHz, DMSO-d6): δ 10.05 (s, 1H), 8.16-8.24 (m, 2H), 7.88 (d, 2H), 7.71-7.77 (m, 2H), 7.55 (d, 1H), 7.37-7.48 (m, 3H), 7.27-7.37 (m, 3H), 4.56 (s, 2H), 4.38 (t, 1H), 4.18-4.33 (m, 3H), 3.91 (t, 1H), 2.08-2.20 (m, 1H), 1.30 (d, 3H), 0.83-0.95 (m, 15H), 0.06 (s, 6H).

Step 7: (3R,4S,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-one

A suspension of (3R,4S,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-ol (30.0 g, 55.49 mmol) in DMSO (120 mL) was stirred for 30 min at room temperature (until complete solubilization), then acetic anhydride (90 mL) was added dropwise at room temperature over 15 min. The beige solution was stirred for 16 h, then cooled to 0°C and 1 M aqueous hydrochloride solution (100 mL) was slowly added. The reaction mixture was stirred for 20 min at room temperature, then acetic acid was evaporated. The resulting residue was diluted with water (200 mL) and ethyl acetate (200 mL). The aqueous layer was extracted with ethyl acetate (2 x 200 mL) and the combined organic layers were washed with water (2 x 500 mL), saturated sodium hydrogen carbonate solution (2 x 500 mL), then dried over sodium sulfate, filtered and concentrated to dryness to give a crude mixture. The crude product was purified by silica gel chromatography (gradient with ethyl acetate in cyclohexane) to give (3R,4S,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-one (25.05 g, 46.51 mmol) as a colorless oil. ¹ H NMR (400 MHz, DMSO-d6): δ 7.19-7.39 (m, 20H), 4.85 (d, 1H), 4.57-4.72 (m, 5H), 4.46-4.56 (m, 3H), 4.36 (d, 1H), 3.98-4.05 (m, 1H), 3.84-3.92 (m, 1H), 3.65-3.76 (m, 2H).

Step 8: (3R,4S,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)-2-(2-trimethylsilylethynyl)tetrahydropyran-2-ol

To a solution of trimethylsilylacetylene (24 mL, 168.6 mmol) in THF (325 mL) was added a solution of 2.5 M butyl lithium in hexane (59.41 mL, 148.5 mmol) over 20 min at -78°C. The colorless solution was stirred for 45 min at -78°C and then for 45 min at 0°C. The reaction mixture was cooled to -78°C, and a solution of (3R,4S,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-one (25.0 g, 46.41 mmol) in THF (325 mL) was added dropwise over 45 min. The reaction mixture was stirred for 4 h at this temperature, then quenched with water (200 mL). The aqueous layer was extracted with ethyl acetate (2×200 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to dryness to give (3R,4S,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)-2-(2-trimethylsilylethynyl)tetrahydropyran-2-ol (29.56 g, 46.41 mmol) as a beige oil containing two diastereomers in a 4/6 ratio. ¹ H NMR (400 MHz, DMSO-d6): δ 7.13-7.43 (m, 20H), 4.87-4.99 (m, 1H), 4.65-4.83 (m, 4H), 3.43-3.57 (m, 3H), 3.70-3.85 (m, 2H), 3.55-3.68 (m, 3H), 3.43-3.53 (m, 2H), 0.11-0.22 (m, 9H).

Step 9: Trimethyl-[2-[(2S,3S,4R,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-yl]ethynyl]silane

To a solution of (3R,4S,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)-2-(2-trimethylsilylethynyl)tetrahydropyran-2-ol (29.56 g, 46.42 mmol) in acetonitrile (83 mL) and dichloromethane (193 mL) was added a solution of triethylsilane (44.98 mL, 278.5 mmol) in an acetonitrile/dichloromethane mixture (37 mL/18 mL) over 20 min at -15°C, then a solution of boron trifluoride diethyl etherate (23.53 mL, 185.7 mmol) in acetonitrile (37 mL) over 30 min at -15°C. The colorless solution was stirred for 5 h at the same temperature, then diluted with water (500 mL). The aqueous layer was extracted with ethyl acetate (2×500 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to dryness to give trimethyl [2-[(2S,3S,4R,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-yl]ethynyl]silane (28.82 g, 46.41 mmol) as a brown oil. ¹ H NMR (400 MHz, DMSO-d6): δ 7.10-7.44 (m, 20H), 4.93 (d, 1H), 4.67-4.86 (m, 4H), 4.43-4.57 (m, 3H), 4.16-4.28 (m, 1H), 3.42-3.68 (m, 6H), 0.15 (s, 9H).

Step 10: (2R,3R,4R,5S,6S)-3,4,5-tribenzyloxy-2-(benzyloxymethyl)-6-ethynyl-tetrahydropyran

To a solution of trimethyl-[2-[(2S,3S,4R,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-yl]ethynyl]silane (28.80 g, 46.39 mmol) in methanol (1.12 L) and dichloromethane (240 mL) was added aqueous sodium hydroxide solution 1 M (80 mL). The beige solution was stirred for 1 h at room temperature, then acidified to pH = 1 with aqueous hydrochloride solution 1 M and diluted with water (500 mL). Methanol was evaporated, and then the aqueous layer was extracted with ethyl acetate (2 × 1 L). The combined organic layers were dried over sodium sulfate, filtered and concentrated to dryness. The crude product was purified by silica gel chromatography (gradient with ethyl acetate in cyclohexane) to give (2R,3R,4R,5S,6S)-3,4,5-tribenzyloxy-2-(benzyloxymethyl)-6-ethynyl-tetrahydropyran (20.00 g, 36.45 mmol) as a colorless oil. ¹ H NMR (400 MHz, DMSO-d6): δ 3.42-3.67 (m, 7H), 4.17 (d, 1H), 4.44-4.56 (m, 3H), 4.67-4.86 (m, 4H), 4.90 (d, 1H), 7.15-7.40 (m, 20H).

Step 11: (2S,3R,4R,5S,6R)-2-ethynyl-6-(hydroxymethyl)tetrahydropyran-3,4,5-triol

To a solution of (2R,3R,4R,5S,6S)-3,4,5-tribenzyloxy-2-(benzyloxymethyl)-6-ethynyltetrahydropyran (20.00 g, 36.45 mmol) in ethanethiol (400 mL) was added boron trifluoride diethyl etherate (147.8 mL, 1166 mmol) dropwise at room temperature over 5 min. The beige solution was stirred for 16 h at room temperature, then cooled to 0°C and equipped with a gas trap containing a saturated aqueous solution of sodium hypochlorite. A saturated aqueous solution of sodium hydrogen carbonate (500 mL) was added dropwise at 0°C over 1 h (formation of carbon dioxide). After concentration to dryness, the crude product was purified by silica gel chromatography (gradient methanol in dichloromethane) to give (2S,3R,4R,5S,6R)-2-ethynyl-6-(hydroxymethyl)tetrahydropyran-3,4,5-triol (4.05 g, 21.52 mmol) as a white solid. ^1H NMR (400 MHz, DMSO-d6): δ 5.28 (d, 1H), 4.99 (d, 1H), 4.91 (d, 1H), 4.52 (t, 1H), 3.77 (d, 1H), 3.60-3.69 (m, 1H), 3.35-3.43 (m, 1H), 3.32 (s, 1H), 2.97-3.13 (m, 4H).

Step 12: Methyl (2S,3S,4R,5R,6S)-6-ethynyl-3,4,5-trihydroxy-tetrahydropyran-2-carboxylate

To a solution of (2S,3R,4R,5S,6R)-2-ethynyl-6-(hydroxymethyl)tetrahydropyran-3,4,5-triol (4.05 g, 21.52 mmol) in saturated aqueous sodium hydrogen carbonate (81 mL) and THF (81 mL) was added (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (168 mg, 1.08 mmol). The yellow suspension was cooled to 0°C and 1,3-dibromo-5,5-dimethylimidazolidine-2,4-dione (12.31 g, 43.04 mmol) was added portionwise over 30 min. The reaction mixture was stirred for 4 h at 0°C, then quenched by the addition of methanol (40 mL). After stirring for 30 min at this temperature, a saturated aqueous solution of potassium carbonate (10 ml) and dichloromethane (100 ml) are added. The organic layer is extracted with water (2 x 200 ml), then the combined aqueous layers are acidified to pH = 1 with an aqueous solution of 3 M hydrochloride and concentrated to dryness. The resulting residue is taken up in methanol (100 ml) and an aqueous solution of 3 M hydrochloride (20 ml). The mixture is concentrated to dryness and coevaporated several times with methanol (4 x 100 ml). The crude product was purified by silica gel chromatography (gradient methanol in dichloromethane, cerium developer) to give methyl (2S,3S,4R,5R,6S)-6-ethynyl-3,4,5-trihydroxy-tetrahydropyran-2-carboxylate (3.00 g, 13.88 mmol) as a beige solid. ^1H NMR (400 MHz, DMSO-d6): δ 5.46 (d, 1H), 5.32 (d, 1H), 5.18 (d, 1H), 3.93-4.00 (m, 1H), 3.75 (dd, 1H), 3.65 (s, 3H), 3.40-3.44 (m, 1H), 3.31 (s, 1H), 3.09-3.19 (m, 2H).

Step 13: Methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-ethynyl tetrahydropyran-2-carboxylate

To a solution of methyl (2S,3S,4R,5R,6S)-6-ethynyl-3,4,5-trihydroxytetrahydropyran-2-carboxylate (3.00 g, 13.88 mmol) in DMF (37.5 mL) and pyridine (12.5 mL) was added N,N-dimethylpyridin-4-amine (84.8 mg, 0.693 mmol). The reaction mixture was cooled to 0°C, then acetic anhydride (20.0 mL, 213 mmol) was added dropwise over 5 min. The colorless solution was stirred for 3 h at room temperature, then diluted with aqueous 1 M hydrochloride solution (200 mL). The aqueous layer was extracted with ethyl acetate (2×200 mL). The combined organic layers were washed with 1 M aqueous hydrochloride solution (2×200 mL), then with saturated aqueous potassium carbonate solution (200 mL), then dried over sodium sulfate, filtered, and concentrated to dryness to give the crude mixture. The crude product was purified by silica gel chromatography (gradient with ethyl acetate in cyclohexane, cerium developer) to give methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-ethynyl tetrahydropyran-2-carboxylate (4.60 g, 13.44 mmol) as a white solid. ¹ H NMR (400 MHz, DMSO-d6): δ 5.33 (t, 1H), 4.93-5.01 (m, 2H), 4.70 (d, 1H), 4.44 (d, 1H), 3.67 (s, 1H), 3.64 (s, 3H), 2.02 (s, 3H), 1.94-2.01 (m, 6H).

Step 14: Methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]ethynyl]tetrahydropyran-2-carboxylate

To a solution of methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-ethynyl-tetrahydropyran-2-carboxylate (496 mg, 1.45 mmol) in DMF (7.3 ml) were successively added 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-2-[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodoanilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate (730 mg, 0.966 mmol), DIPEA (738 μl, 4.47 mmol), copper iodide (18.4 mg, 96.6 mmol) and dichloro-bis-(triphenylphosphine)palladium(II) (67.8 mg, 96.6 mmol). The yellow solution was washed with argon and then stirred for 16 h at room temperature. After dilution with water (100 mL), the aqueous layer was extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with water (2 x 200 mL) and saturated aqueous ammonium chloride solution (2 x 200 mL), then dried over sodium sulfate, filtered, and concentrated to dryness. The crude product is purified by silica gel chromatography (gradient with ethyl acetate in cyclohexane) to give methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]ethynyl]tetrahydropyran-2-carboxylate (782 mg, 0.806 mmol) as a yellow solid.¹H NMR (400 MHz, DMSO-d6): δ 10.09 (s, 1H). 8.20 (d, 1H), 7.89 (d, 2H), 7.70-7.78 (m, 3H), 7.55 (d, 1H), 7.32-7.46 (m, 4H), 7.27-7.32 (m, 2H), 5.41 (t, 1H), 4.96-5.14 (m, 3H), 4.67 (s, 2H), 4.51 (d, 1H), 4.36-4.44 (m, 1H), 4.16-4.32 (m, 3H), 3.88-3.95 (m, 1H), 3.64 (s, 3H), 1.94-2.07 (m, 10H), 1.30 (d, 3H), 0.84-0.93 (m, 15H), 0.08 (s, 6H).

Step 15: Methyl (3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]ethyl]tetrahydropyran-2-carboxylate

Solution of methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]ethynyl]tetrahydropyran-2-carboxylate (750 mg, 0.773 mmol) in THF (15 mL) was washed with argon. Dry 5% platinum on carbon (75 mg, 50% w/w) was added. The reaction mixture was washed successively with argon, H₂and stirred for 16 h at room temperature under H atmosphere₂(P atm.). The reaction mixture is filtered through a pad of Celite®, washed with THF, then concentrated to dryness. The entire sequence of actions (add dry 5% platinum on carbon (75 mg, 50% w/w)), stirring for 16 h at room temperature under H₂(1 bar) and filtration through a pad of Celite®) was repeated 4 more times. The crude product was purified by silica gel chromatography (gradient with ethyl acetate in cyclohexane) to give methyl (3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]ethyl]tetrahydropyran-2-carboxylate (470 mg, 0.483 mmol) as a white solid.¹H NMR (400 MHz, DMSO-d6): δ 9.90 (s, 1H), 8.16 (d, 1H), 7.89 (d, 2H), 7.70-7.78 (m, 2H), 7.37-7.49 (m, 4H), 7.27-7.32 (m, 3H), 7.23 (d, 1H), 5.29 (t, 1H), 4.95 (t, 1H), 4.78 (t, 1H), 4.60 (s, 2H), 4.34-4.44 (m, 2H), 4.16-4.32 (m, 3H), 3.88-3.95 (m, 1H), 3.72-3.79 (m, 1H), 3.64 (s, 3H), 2.69-2.78 (m, 1H), 2.50-2.60 (m, 1H), 1.92-2.03 (m, 10H), 1.55-1.75 (m, 2H), 1.30 (d, 3H), 0.84-0.93 (m, 15H), 0.05 (s, 6H).

Step 16: Methyl (3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]-2-(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate

To a solution of methyl (3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]ethyl]tetrahydropyran-2-carboxylate (470 mg, 0.483 mmol) in THF (540 μL) and water (540 μL) was added acetic acid (1.6 mL, 28.28 mmol). The colorless solution was stirred for 16 h at room temperature and then diluted with water (100 mL). The aqueous layer was extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with water (2 x 200 mL) and saturated aqueous sodium hydrogen carbonate solution (200 mL), then dried over sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by silica gel chromatography (gradient with ethyl acetate in cyclohexane) to give methyl (3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]-2-(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate (354 mg, 0.412 mmol) as a white solid.¹H NMR (400 MHz, DMSO-d6): δ 9.87 (s, 1H), 8.16 (d, 1H), 7.89 (d, 2H), 7.70-7.78 (m, 2H), 7.37-7.50 (m, 4H), 7.27-7.37 (m, 3H), 7.25 (d, 1H), 5.29 (t, 1H), 4.91-4.98 (m, 2H), 4.78 (t, 1H), 4.34-4.44 (m, 4H), 4.16-4.32 (m, 3H), 3.88-3.95 (m, 1H), 3.72-3.79 (m, 1H), 3.64 (s, 3H), 2.64-2.73 (m, 1H), 2.50-2.60 (m, 1H), 1.92-2.03 (m, 10H), 1.69-1.79 (m, 1H), 1.52-1.65 (m, 1H), 1.30 (d, 3H), 0.84-0.93 (m, 6H).

Step 17: Methyl (3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]phenyl]ethyl]tetrahydropyran-2-carboxylate

To a solution of methyl (3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]-2-(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate (310 mg, 0.361 mmol) in THF (7.75 mL) were added pyridine (146 μL, 1.80 mmol) and 4-nitrophenyl chloroformate successively (182 mg, 0.901 mmol). The white suspension was stirred for 16 h at room temperature, then concentrated to dryness to give the crude mixture. The crude product was purified by silica gel chromatography (gradient with ethyl acetate in dichloromethane) to give methyl (3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]phenyl]ethyl]tetrahydropyran-2-carboxylate (257 mg, 0.251 mmol) as a white solid.¹H NMR (400 MHz, DMSO-d6): δ 10.04 (s, 1H), 8.31 (d, 2H), 8.20 (d, 1H), 7.89 (d, 2H), 7.66-7.78 (m, 2H), 7.56 (d, 2H), 7.28-7.52 (m, 8H), 5.31 (t, 1H), 5.25 (s, 2H), 4.96 (t, 1H), 4.79 (t, 1H), 4.40 (d, 2H), 4.16-4.32 (m, 3H), 3.88-3.95 (m, 1H), 3.74-3.83 (m, 1H), 3.61 (s, 3H), 2.74-2.84 (m, 1H), 2.60-2.71 (m, 1H), 1.90-2.03 (m, 10H), 1.72-1.83 (m, 1H), 1.58-1.71 (m, 1H), 1.30 (d, 3H), 0.82-0.94 (m, 6H). LC-MS: MS (ESI) m/z [M+Na]+=1047.6.

Stage 18: (2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]-2-[2-[(2SR,3SR,4RS,5SR,6SR)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2-yl]ethyl]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To a solution of (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-(2-piperazin-1-ylethoxy)phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid(C3)(118 mg, 0.121 mmol) in dimethylformamide (3.0 ml) was successively added a solution of methyl (3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]phenyl]ethyl]tetrahydropyran-2-carboxylate (130 mg, 0.127 mmol) in dimethylformamide (3.0 mL) and DIPEA (60 μL, 0.363 mmol). The reaction mixture was stirred at room temperature for 2 h. (2R)-2-[(5S_a)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]-2-[2-[(2SR,3SR,4RS,5SR,6SR)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2-yl]ethyl]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid is obtained as a solution in dimethylformamide and used similarly in the next step. UPLC-MS: MS (ESI) m/z [M+H]+=1745.6+1747.6.

Stage 19: (2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]-2-[2-[(2SR,3SR,4RS,5SR,6SR)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2-yl]ethyl]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To the solution 2SR,3SR,4RS,5RS,6SR)-6-[2-[(5S_a)-5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino]-2-[[4-[2-[4-[4-[(1R)-1-carboxy-2-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]ethoxy]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy]ethyl]piperazine-1-carbonyl]oxymethyl]phenyl]ethyl]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid (0.121 mmol) in DMF (3.0 mL) from step 18 was sequentially added methanol (2 mL) and lithium hydroxide monohydrate (64.0 mg, 1.52 mmol) in water (2 mL). The reaction mixture was stirred at room temperature for 1 h. The crude product was purified by reverse-phase C18 prep-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the NH method.₄HCO₃to obtain (2R)-2-[(5S_a)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]-2-[2-[(2SR,3SR,4RS,5SR,6SR)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2-yl]ethyl]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (124 mg, 0.0895 mmol) in the form of a white powder. UPLC-MS: MS (ESI) m/z [M+H]+=1384.3+1386.3.

Step 20: (2,3,4,5,6-pentafluorophenyl) 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetic acid 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetate

A solution of 2,3,4,5,6-pentafluorophenol is added to a solution of 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetic acid (75 mg, 0.342 mmol) in a THF solution (500 µl) (75.5 mg, 0.410 mmol) in THF (500 μL) and a solution of N,N'-dicyclohexylmctanediimine (84.7 mg, 0.410 mmol) in THF (500 μL) were added. The reaction mixture was stirred for 15 h at room temperature, and the progress of the reaction mixture was monitored by UPLC-MS. (2,3,4,5,6-pentafluorophenyl) 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetate was obtained as a THF solution by simple filtration of the suspension on a small disposable frit. This solution was used without further purification in the next step. UPLC-MS: MS (ESI) m/z [M-N2+H]+=372.3.

Stage 21: (2SR,3SR,4RS,5RS,6SR)-6-[2-[(5S _a )-5-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-2-[[4-[2-[4-[4-[(1R)-1-carboxy-2-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]ethoxy]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy]ethyl]piperazine-1-carbonyl]oxymethyl]phenyl]ethyl]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid L14-C3

To the solution (2R)-2-[(5S_a)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]-2-[2-[(2SR,3SR,4RS,5SR,6SR)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2-yl]ethyl]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (118 mg, 0.085 mmol) in DMF (500 μL) were added sequentially a solution of (2,3,4,5,6-pentafluorophenyl) 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetate (0.342 mmol) in THF from step 20 and DIPEA (42.2 μL, 0.256 mmol). The reaction mixture was stirred for 1 h at room temperature, and the reaction progress was monitored by UPLC-MS. The crude product was purified by C18 prep-HPLC with reversed phase by direct loading of the reaction mixture onto an Xbridge column and using the NH method.₄HCO₃with receiptL14-C3in the form of a white powder. UPLC-MS: MS (ESI) m/z [M+H]+=1599.0+1601.2. IR wavelength (cm)^-1): 3263, 2105, 1652, 1600, 1284/1240/1089, 756. ¹H NMR(400 MHz, DMSO-d6): δ 9.98 (s), 8.85 (d, 1H), 8.52 (s, 1H), 8.38 (d, 1H), 7.93 (d, 1H), 7.56 (d, 1H), 7.5 (t, 1H), 7.49 (d, 1H), 7.47 (d, 1H), 7.44 (d, 1H), 7.42 (s, 1H), 7.3 (dd, 2H), 7.22 (d, 1H), 7.2 (t, 2H), 7.19 (t, 1H), 7.13 (d, 1H), 7.08 (t, 1H), 7.02 (t, 1H), 6.95 (d, 1H), 6.65 (t, 1H), 6.11 (d, 1H), 5.43 (d, 1H), 5.27/5.2 (m, 2H), 4.93 (shs, 2H), 4.38 (m, 1H), 4.35/4.2 (2m, 2H), 4.3 (m, 1H), 3.94 (s, 2H), 3.75 (s, 3H), 3.58 (m, 10H), 3.57 (m, 1H), 3.51/2.29 (2dd, 2H), 3.35 (m, 2H), 3.25 (m, 4H), 3.2 (m, 1H), 3.2 (m, 1H), 3.06 (m, 1H), 2.96 (m, 1H), 2.75 (m, 2H), 2.72/2.5 (m, 2H), 2.41 (m, 4H), 2 (m, 1H), 1.99/1.6 (m, 2H), 1.8 (s, 3H), 1.3 (d, 3H), 0.88/0.82 (2d, 6H). ¹³C NMR (100 MHz, DMSO-d6): δ 158.2, 152.7, 131.9, 131.4, 131.4, 131.3, 131.1, 131, 127.8, 120.6, 120.5, 120.1, 116.8, 116.3, 116, 112.6, 112, 111.6, 79.6, 79.6, 78.5, 76.8, 74.2, 73.1, 70.3, 70.3, 69.3, 66.3, 65.1, 56.8, 56.3, 56.1, 52.6, 50.3, 49.4, 43.8, 34.2, 33.5, 31.7, 28, 19.6/18.4, 18.2, 18. ¹⁹F NMR (376 MHz, DMSO-d6): δ -112.5.HR-IER+: m/z [M+H]+=1599.5724 (1599.5704) (measured/theoretical)

Obtaining L18-C3:

(2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[2-(2-azidoethoxy)acetyl]amino]-3-sulfo-propanoyl]amino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

Step 1: 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate

To a solution of (2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]amino]propanoic acid (6.0 g, 14.6 mmol; prepared according to step 5 of preparation L14-C3 ) in dichloromethane (70 mL) and methanol (30 mL) were added sequentially (4-aminophenyl)methanol (2.16 g, 17.5 mmol) and ethyl 2-ethoxy-2H-quinoline-1-carboxylate (5.42 g, 21.93 mmol). The red solution was stirred at room temperature for 16 h (precipitation occurred after a few minutes). After completion of the reaction, the reaction mixture was diluted with diethyl ether (70 mL). The resulting precipitate was filtered and dried to give 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate (5.16 g, 10.01 mmol) as a beige solid. ¹ H NMR (400 MHz, DMSO-d6): δ 9.91 (s, 1H), 8.15 (d, 1H), 7.89 (d, 2H), 7.70-7.78 (m, 2H), 7.53 (d, 2H), 7.38-7.46 (m, 3H), 7.29-7.35 (m, 2H), 7.23 (d, 2H), 5.08 (t, 1H), 4.37-4.50 (m, 3H), 4.16-4.34 (m, 3H), 3.91 (t, 1H), 1.92-2.02 (m, 1H), 1.30 (d, 3H), 0.83-0.91 (m, 6H).

Step 2: (2S)-2-amino-N-[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]-3-methyl-butanamide

To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxoethyl]carbamoyl]-2-methylpropyl]carbamate (5.16 g, 10.01 mmol) in DMF (120 mL) was added piperidine (52 mL, 525 mmol). The reaction mixture was stirred for 2 h at room temperature, then the piperidine was evaporated and the resulting solution was diluted with water (500 mL). The resulting solid was filtered off and the filtrate was washed twice with diethyl ether (2 x 500 mL). The aqueous layer was concentrated to dryness to give the crude reaction mixture. The crude product was purified by silica gel chromatography (gradient methanol (containing 7M ammonia) in dichloromethane) to give (2S)-2-amino-N-[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]-3-methyl-butanamide (2.02 g, 6.89 mmol) as a beige solid. ^1H NMR (400 MHz, DMSO-d6): δ 10.0 (s, 1H), 8.17 (s, 1H), 7.53 (d, 2H), 7.23 (d, 2H), 5.12 (t, 1H), 4.39-4.52 (m, 3H), 2.96-3.02 (m, 1H), 1.86-1.97 (m, 1H), 1.70 (shs, 2H), 1.29 (d, 3H), 0.88 (d, 3H), 0.78 (d, 3H).

Step 3: [(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-oxo-3-sodiumoxypropyl]sulfonyloxysodium

To a solution of [(2R)-2-amino-3-oxo-3-sodiumoxypropyl]sulfonyloxysodium monohydrate (3.00 g, 12.98 mmol) in water (127 mL) was added sodium carbonate (4.13 g, 38.94 mmol). A solution of 9H-fluoren-9-ylmethyl carbonochloridate (3.69 g, 14.28 mmol) in dioxane (127 mL) was added dropwise over 15 min at room temperature. The mixture was stirred at this temperature for 4 h. After completion of the reaction, the mixture was neutralized to pH = 7 with an aqueous solution of 1 M HCl, diluted with a saturated aqueous solution of sodium hydrogen carbonate (50 mL) and concentrated to dryness. The crude product was purified by C18 reverse phase chromatography using a neutral method to give [(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-oxo-3-sodiumoxypropyl]sulfonyloxysodium (4.4 g, 10.11 mmol) as a white solid. ¹ H NMR (400 MHz, DMSO-d6): δ 7.88 (d, 2H), 7.70 (d, 2H), 7.39-7.44 (m, 2H), 7.29-7.36 (m, 2H), 6.71 (s, 1H), 3.84-4.25 (m, 4H), 2.73-2.91 (m, 2H).

Step 4: [(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-[[(1S)-1-[[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-3-oxo-propyl]sulfonyloxysodium

To a solution of (2S)-2-amino-N-[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxoethyl]-3-methylbutanamide (1.19 g, 4.04 mmol) in DMF (395 mL) were added [(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-oxo-3-sodiumoxypropyl]sulfonyloxysodium (4.40 g, 10.11 mmol), DIPEA (6.01 mL, 36.38 mmol), and HBTU (3.83 g, 10.11 mmol) sequentially. The white suspension was stirred for 22 h at room temperature and then cooled to 0°C. Dilution with water (1.5 L), saturated sodium carbonate solution (20 mL), and solid sodium chloride gave a white emulsion which was filtered and the filtrate concentrated to dryness to give the crude mixture. The crude product was purified by reverse-phase C18 chromatography (methanol in water gradient) to give [(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-[[(1S)-1-[[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxoethyl]carbamoyl]-2-methyl-propyl]amino]-3-oxo-propyl]sulfonyloxysodium (936 mg, 1.36 mmol) as a beige solid. ¹ H NMR (400 MHz, DMSO-d6): δ 9.39 (s, 1H). 8.25-8.31 (m, 1H), 8.11-8.17 (m, 1H), 7.89 (d, 2H), 7.70 (d, 2H), 7.64 (d, 2H), 7.50-7.55 (m, 1H), 7.38-7.46 (m, 2H), 7.29-7.35 (m, 2H), 7.20 (d, 2H), 5.07 (s, 1H), 4.51 (s, 1H), 4.42 (s, 2H), 4.19-4.33 (m, 4H), 4.01 (s, 1H), 2.90-3.10 (m, 2H), 2.08-2.20 (m, 1H), 1.31 (d, 3H), 0.8-0.93 (m, 6H).

Stage 5: (2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-[[(1S)-2-methyl-1-[[(1S)-1-methyl-2-[4-[(4-nitrophenoxy)carbonyloxymethyl]anilino]-2-oxo-ethyl]carbamoyl]propyl]amino]-3-oxo-propane-1-sulfonate

To a suspension of [(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-[[(1S)-1-[[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-3-oxo-propyl]sulfonyloxysodium (600 mg, 0.87 mmol) in THF (24 ml) add DIPEA (432 μl, 2.61 mmol), then 4-nitrophenyl chloroformate (439 mg, 2.17 mmol). The mixture was stirred at room temperature for 4 h. Additional 4-nitrophenyl chloroformate (439 mg, 2.17 mmol) was added and the reaction mixture was stirred at room temperature for another 16 h. Additional 4-nitrophenyl chloroformate (439 mg, 2.17 mmol) was added. After 5 h of stirring at room temperature, the mixture was concentrated to dryness and purified by silica gel chromatography (gradient with ethyl acetate in cyclohexane) and then by C18 reverse phase chromatography using the neutral method to give (2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-[[(1S)-2-methyl-1-[[(1S)-1-methyl-2-[4-[(4-nitrophenoxy)carbonyloxymethyl]anilino]-2-oxo-ethyl]carbamoyl]propyl]amino]-3-oxo-propane-1-sulfonate (303 mg, 0.32 mmol) as a white solid. ¹ H NMR (400 MHz, DMSO-d6): δ 9.52 (s, 1H), 8.25-8.37 (m, 3H), 8.06-8.24 (m, 4H), 7.89 (d, 2H), 7.76 (d, 2H), 7.70 (d, 2H), 7.49-7.61 (m, 3H), 7.35-7.45 (m, 4H), 7.26-7.35 (m, 2H), 5.23 (s, 2H), 4.48 (s, 1H), 4.20-4.33 (m, 4H), 4.01 (s, 1H), 3.57-3.66 (m, 2H), 3.10-3.18 (m, 2H), 2.90-3.10 (m, 2H), 2.08-2.20 (m, 1H), 1.33 (d, 3H), 1.21-1.26 (m, 15H), 0.86-0.92 (m, 6H). UPLC-MS: MS (ESI) m/z [MH]-: 830.5.

Stage 6: (2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)butanoyl]amino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxy arbonyl]piperazin-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propane acid

To a solution of (2R)-2-[(5S _a )-5-[3-chloro-2-methyl-4-(2-piperazin-1-ylethoxy)phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-Trifluoroacetic acid ( C3 ) (128 mg, 0.149 mmol) in DMF (1.5 mL) was successively added with a solution of (2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-[[(1S)-2-methyl-1-[[(1S)-1-methyl-2-[4-[(4-nitrophenoxy)carbonyloxymethyl]anilino]-2-oxo-ethyl]carbamoyl]propyl]amino]-3-oxo-propane-1-sulfonate (150 mg, 0.156 mmol) in DMF (1.5 mL) and DIPEA (77 μL, 0.468 mmol). The reaction mixture was stirred for 2 h at room temperature and the progress of the reaction mixture was monitored by UPLC-MS. (2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)butanoyl]amino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid is obtained as a solution in dimethylformamide, which is used similarly in the next step. UPLC-MS: MS (ESI) m/z [M+H]+=1553.2+1555.3.

Stage 7: (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2S)-2-aminobutanoyl]amino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl] ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propane acid

To a solution of (2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)butanoyl]amino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (0.156 mmol) in dimethylformamide (3 ml) obtained in the previous step, piperidine (30.6 μl, 0.312 mmol). The reaction mixture was stirred at room temperature for 15 h, and the progress of the reaction mixture was monitored by UPLC-MS. The crude product was purified by C18 prep-reverse phase HPLC by direct loading of the reaction mixture onto an Xbridge column and using the NH ₄ HCO ₃ method to afford (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2S)-2-aminobutanoyl]amino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (148 mg = 0.111 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+H]+=1331.4+1333.5.

Stage 8: (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]butanoyl]amino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetic acid L18-C3

To the solution(2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2S)-2-aminobutanoyl]amino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (148 mg, 0.111 mmol) in DMF (1.5 ml) was successively added to a solution of (2,3,4,5,6-pentafluorophenyl) 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetate (0.596 mmol; obtained according to step 20 of preparationL14-C3) in THF (1 ml) and DIPEA (74 µl, 0.447 mmol). The reaction mixture was stirred for 1 h at room temperature, and the reaction progress was monitored by UPLC-MS. The crude product was purified by C18 prep-HPLC with reversed phase by direct loading of the reaction mixture onto an Xbridge column and using the NH method.₄HCO₃with receiptL18-C3(60 mg, 0.0389 mmol) as a white powder. IR wavelength (cm^-1): 3288, 2101, 1659, 1237,1039, 833,755.¹H NMR(400 MHz, DMSO-d6): δ (m, 10H), 9.42 (s, 1H), 8.88 (d, 1H), 8.58 (s, 1H), 8.32 (d, 1H), 8.18 (d, 1H), 8.12 (d, 1H), 7.71 (m, 1H), 7.7 (d, 2H), 7.54 (dd, 1H), 7.46 (d, 1H), 7.39 (d, 1H), 7.29 (dd, 2H), 7.25 (d, 2H), 7.21 (t, 2H), 7.18 (d, 1H), 7.15 (d, 1H), 7.13 (t, 1H), 7.04 (t, 1H), 6.99 (d, 1H), 6.71 (t, 1H), 6.22 (d, 1H), 5.47 (m, 1H), 5.23 (AB, 2H), 4.98 (s, 2H), 4.71 (sq, 1H), 4.3 (m, 1H), 4.24/4.19 (2m, 2H), 3.97 (dd, 1H), 3.92 (m, 2H), 3.76 (s, 3H), 3.37 (t, 2H), 3.31 (m, 4H), 3.12/2.97 (2dd, 2H), 2.74 (t, 2H), 2.45 (m, 4H), 2.15 (m, 1H), 1.81 (s, 3H), 1.33 (d, 3H), 0.91 (2d, 6H). ¹³C NMR (100 MHz, DMSO-d6): δ 157.9, 152.3, 131.3, 131.2, 131.1, 131, 130.7, 128.9, 128.6, 120.7, 120.4, 119.6, 116.4, 112.7, 112, 111.3, 70.6, 70.3, 69.4, 67.5, 66.3, 59.7, 56.7, 56.1, 53.4, 52.5, 50.8, 50.4, 49.9, 44, 29.9, 19.6, 17.8, 17.6. ¹⁹F NMR (376 MHz, DMSO-d6): δ ppm. 112.3. HR-IER+: m/z [M+H]+=1546.503 (1546.5009) (measured/theoretical).

Obtaining L16-C3:

(2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-6-amino-2-[[(2S)-2-[[2-(2-azidoethoxy)acetyl]amino]-3-methyl-butanoyl]amino]hexanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

Step 1: (2S)-6-(tert-butoxycarbonylamino)-2-[[2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]hexanoic acid

To a solution of (2S)-2-amino-6-(tert-butoxycarbonylamino)hexanoic acid (2.96 g, 12 mmol) and sodium hydrogen carbonate (1.01 g, 12 mmol) in water (30 mL) was added a solution of (2,5-dioxopyrrolidin-1-yl) 2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoate (5.0 g, 11.5 mmol) in dimethoxyethane (30 mL), THF (15 mL) was added to improve solubility. The reaction mixture was stirred at room temperature for 16 h. Aqueous hydrochloric acid 1 M (15 mL) was added, and the aqueous layer was extracted with ethyl acetate (3×75 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to dryness to afford the crude compound. Trituration with dichloromethane/pentane with sonication afforded (2S)-6-(tert-butoxycarbonylamino)-2-[[2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]hexanoic acid (4.9 g, 8.63 mmol) as a white solid. ^1H NMR (400 MHz, DMSO-d6): δ 12.48 (s, 1H), 7.89 (d, 2H), 7.74 (t, 2H), 7.28-7.44 (m, 6H), 6.73 (s, 1H), 4.10-4.33 (m, 5H), 3.9 (t, 1H), 2.82-2.90 (m, 2H), 1.52-1.73 (m, 2H), 1.34 (s, 9H), 1.22-1.31 (m, 4H), 0.83-0.91 (m, 6H).

Step 2: 9H-fluoren-9-ylmethyl N-[1-[[(1S)-5-(tert-butoxycarbonylamino)-1-[[4-(hydroxymethyl)phenyl]carbamoyl]pentyl]carbamoyl]-2-methyl-propyl]carbamate

To a solution of (2S)-6-(tert-butoxycarbonylamino)-2-[[2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]hexanoic acid (1.5 g, 2.64 mmol) in dichloromethane (19 mL) and methanol (9.5 mL) was added (4-aminophenyl)methanol (651.0 mg, 5.28 mmol) in methanol (1.5 mL). Ethyl 2-ethoxy-2H-quinoline-1-carboxylate (1.31 g, 5.28 mmol) was then added. The reaction mixture was stirred at room temperature for 16 h, then concentrated to dryness. The crude product was purified by silica gel chromatography (gradient methanol in dichloromethane) to give 9H-fluoren-9-ylmethyl N-[1-[[(1S)-5-(tert-butoxycarbonylamino)-1-[[4-(hydroxymethyl)phenyl]carbamoyl]pentyl]carbamoyl]-2-methylpropyl]carbamate (544 mg, 0.80 mmol) as a pale red solid. ^1H NMR (400 MHz, DMSO-d6): δ 9.93 (s, 1H), 8.01 (d, 1H), 7.89 (d, 2H), 7.74 (t, 2H), 7.52 (d, 2H), 7.37-7.45 (m, 3H), 7.32 (t, 2H), 7.22 (d, 2H), 6.71 (s, 1H), 5.08 (shs, 1H), 4.43 (d, 2H), 4.21-4.40 (m, 4H), 3.92 (t, 1H), 2.83-2.91 (m, 2H), 1.94-2.01 (m, 1H), 1.55-1.74 (m, 2H), 1.21-1.42 (m, 4H), 1.33 (s, 9H), 0.87 (t, 6H).

Step 3: [4-[[(2S)-6-(tert-butoxycarbonylamino)-2-[[2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]hexanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate

To a solution of 9H-fluoren-9-ylmethyl N-[1-[[(1S)-5-(tert-butoxycarbonylamino)-1-[[4-(hydroxymethyl)phenyl]carbamoyl]pentyl]carbamoyl]-2-methylpropyl]carbamate (600.0 mg, 0.892 mmol) in THF (19 mL) was added pyridine (361 μL, 4.46 mmol) followed by 4-nitrophenyl chloroformate (448 mg, 2.22 mmol). The mixture was stirred at room temperature for 16 h, then concentrated to dryness. The crude product was purified by silica gel chromatography (gradient with ethyl acetate in cyclohexane) to give [4-[[(2S)-6-(tert-butoxycarbonylamino)-2-[[2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]hexanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (524 mg; 0.62 mmol; 70%) as a pale pink solid. ^1H NMR (400 MHz, DMSO-d6): δ 10.13 (s, 1H), 8.31 (d, 2H), 8.1 (d, 1H), 7.89 (d, 2H), 7.74 (t, 2H), 7.63 (d, 2H), 7.57 (d, 2H), 7.28-7.45 (m, 7H), 6.72 (s, 1H), 5.24 (s, 2H), 4.35-4.42 (m, 1H), 4.27-4.33 (m, 1H), 4.22 (s, 2H), 3.92 (t, 1H), 2.83-2.91 (m, 2H), 1.96-2.00 (m, 1H), 1.58-1.73 (m, 2H), 1.20-1.30 (m, 4H), 1.33 (s, 9H), 0.86 (t, 6H). ¹³ C NMR (100 MHz, DMSO-d6): δ 171.22, 170.67, 156.1, 155.5, 155.27, 151.92, 145.15, 143.87, 143.75, 140.68, 139.34, 129.43, 129.31, 127.6, 127.03, 125.38, 125.32, 122.58, 120.07, 119.11, 77.28, 70.23, 65.67, 60.11, 54.89, 53.43, 46.67, 31.69, 30.39, 29.22, 28.23, 22.74, 19.19, 18.26. LC-MS: MS (ESI) m/z [M+Na]+=837.4.

Stage 4: (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-6-(tert-butoxycarbonylamino)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]hexanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To a solution of (2R)-2-[(5S _a )-5-[3-chloro-2-methyl-4-(2-piperazin-1-ylethoxy)phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid C3 (166.3 mg, 0.170 mmol) in DMF (1.5 ml) was successively added a solution of [4-[[(2S)-6-(tert-butoxycarbonylamino)-2-[[2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]hexanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (150 mg, 0.179 mmol) in DMF (1.5 mL) and DIPEA (85 μL, 0.510 mmol). The reaction mixture was stirred for 1 h at room temperature, and the progress of the reaction mixture was monitored by UPLC-MS. The crude product was purified by C18 reverse-phase prep-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the NH ₄ HCO ₃ method to afford (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-6-(tert-butoxycarbonylamino)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]hexanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (134 mg, 0.0859 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+H]+=1559.1+1561.3, [M+Na]+: 1581.0+1583.2. IR Wavelength (cm ^-1 ): 3309, 1698, 1238, 1162, 757, 744. ¹ H NMR (400 MHz, DMSO-d6): δ 10.05 (s, 1H), 8.87 (d, 1H), 8.6 (m, 1H), 8.06 (d, 1H), 7.88 (d, 2H), 7.74 (2d, 2H), 7.64 (m, 1H), 7.57 (d, 2H), 7.52 (dd, 1H), 7.44 (t, 1H), 7.43 (d, 1H), 7.4 (t, 2H), 7.35 (d, 1H), 7.3 (t, 2H), 7.3 (dd, 2H), 7.26 (d, 2H), 7.2 (t, 2H), 7.18 (d, 1H), 7.14 (d, 1H), 7.12 (t, 1H), 7.03 (t, 1H), 6.99 (d, 1H), 6.72 (t, 1H), 6.71 (m, 1H), 6.24 (d, 1H), 5.49 (dd, 1H), 5.23 (m, 2H), 4.97 (s, 2H), 4.38 (m, 1H), 4.29/4.23 (m, 2H), 4.22 (m, 1H), 4.2 (m, 2H), 3.92 (dd, 1H), 3.74 (s, 3H), 3.29 (m, 4H), 3.29/2.5 (2dd, 2H), 2.87 (m, 2H), 2.74 (t, 2H), 2.45 (m, 4H), 1.99 (m, 1H), 1.82 (s, 3H), 1.68/1.6 (2m, 2H), 1.36/1.28 (2m, 4H), 1.32 (s, 9H), 0.86 (2d, 6H). ¹³ C NMR (100 MHz, DMSO-d6): δ 158, 131.4, 131.2, 131.2, 131, 130.8, 128.9, 128.5, 127.9, 127.5, 125.6, 120.8, 120.5, 120.4, 119.3, 116, 115.9, 112.4, 112.2, 111.2, 74.1, 69.2, 67.9, 66.7, 66.2, 60.6, 56.6, 56.2, 53.8, 53, 47.1, 43.7, 40, 32.6, 32.2, 30.6, 29.8/23.2, 28.5, 18.8, 18.1. ¹⁹ F NMR (376 MHz, DMSO-d6): δ -112.

Stage 5: (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-6-(tert-butoxycarbonylamino)hexanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To the solution (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-6-(tert-butoxycarbonylamino)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]hexanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (134 mg, 0.0859 mmol) in dimethylformamide (3 ml) was added piperidine (17 μl, 0.172 mmol). The reaction mixture was stirred at room temperature for 18 h, and the reaction progress was monitored by UPLC-MS. The crude product was purified by C18 prep-HPLC with reversed phase by direct loading of the reaction mixture onto an Xbridge column and using the NH method.₄HCO₃to obtain (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-6-(tert-butoxycarbonylamino)hexanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (88 mg, 0.0658 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+H]+=1337.4+1339.4, [M+Na]+=1359.4+1361.4. IR wavelength (cm^-1): 3307, 1683, 1290, 1238, 1162, 835, 754.¹H NMR (400 MHz, DMSO-d6) δ ppm. 10.23 (s, 1H), 8.88 (d, 1H), 8.53 (m, 1H), 8.47 (w, 1H), 7.86 (d, 1H), 7.58 (d, 2H), 7.54 (d, 1H), 7.48 (d, 1H), 7.45 (t, 1H), 7.27 (dd, 2H), 7.25 (d, 2H), 7.19 (t, 2H), 7.18 (d, 1H), 7.14 (d, 1H), 7.08 (t, 1H), 7.03 (t, 1H), 6.96 (t, 1H), 6.72 (t, 1H), 6.67 (t, 1H), 6.14 (d, 1H), 5.42 (d, 1H), 5.21 (m, 2H), 4.97 (s, 2H), 4.4 (m, 1H), 4.21 (m, 2H), 3.75 (s, 3H), 3.42/2.35 (m, 2H), 3.29 (m, 4H), 3.24 (m, 1H), 2.87 (q, 2H), 2.72 (t, 2H), 2.43 (m, 4H), 1.99 (m, 1H), 1.78 (s, 3H), 1.7/1.61 (2m, 2H), 1.36 (m, 2H), 1.34 (s, 9H), 1.26 (m, 2H), 0.89/0.82 (2d, 6H). ¹³C NMR (100 MHz, DMSO-d6): δ ppm 158.4, 131.3, 131.2, 131.1, 131, 128.4, 128, 120.8, 120.6, 120.4, 119.7, 116.1, 115.9, 112.7, 111.7, 111.2, 76.2, 69.2, 67.4, 66.3, 59.2, 56.6, 56.3, 53.6, 53.1, 43.8, 40.1, 33.2, 32.4, 31.3, 29.3, 28.8, 22.9, 19.7/17.5, 17.9.¹⁹F NMR (376 MHz, DMSO-d6): δ ppm. -112.4.

Stage 6: (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-6-(tert-butoxycarbonylamino)hexanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To a solution of (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-6-(tert-butoxycarbonylamino)hexanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (82 mg, 0.0613 mmol) in DMF (500 μl) was successively added a solution of (2,3,4,5,6-pentafluorophenyl) 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetate (0.245 mmol; prepared according to step 20 of preparation L14-C3 ) in THF and DIPEA (30.4 μL, 0.184 mmol). The reaction mixture was stirred for 1 h at room temperature, and the progress of the reaction mixture was monitored by UPLC-MS. The crude product was purified by C18 reverse-phase prep-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the NH ₄ HCO ₃ method to afford (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-6-(tert-butoxycarbonylamino)hexanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (60 mg, 0.0386 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+H]+=1552.2+1554.2, [M+Na]+=1574.1+1576.3.

Stage 7: (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-6-amino-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]hexanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid L16-C3

To the solution (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-6-(tert-butoxycarbonylamino)hexanoyl]amino]phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (23 mg, 0.0148 mmol) in dichloromethane (3 ml) was added 2,2,2-trifluoroacetic acid (400 µl, 4.57 mmol). The reaction mixture was stirred for 2 h at room temperature, and the progress of the reaction mixture was monitored by UPLC-MS. The crude product was purified by C18 prep-HPLC with reversed phase by direct loading of the reaction mixture onto an Xbridge column and using the NH method.₄HCO₃with receiptL16-C3(5 mg, 0.00344 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+H]+=1552.4+1554.5, [M+Na]+=1574.4+1576.4. IR wavelength (cm^-1): 3250, 2250-3500, 2102, 1660, 1288, 1238, 1121, 833, 755.¹H NMR(400 MHz, DMSO-d6): δ ppm. 10.24 (s, 1H), 8.85 (d, 1H), 8.49 (s, 1H), 8.49 (d, 1H), 7.98 (d, 1H), 7.6 (d, 2H), 7.55 (d, 1H), 7.51 (d, 1H), 7.5 (d, 1H), 7.46 (t, 1H), 7.26 (d, 2H), 7.25 (dd, 2H), 7.18 (t, 2H), 7.17 (d, 1H), 7.14 (d, 1H), 7.05 (t, 1H), 7.02 (t, 1H), 6.89 (d, 1H), 6.6 (t, 1H), 6.05 (d, 1H), 5.32 (d, 1H), 5.21/5.15 (m, 2H), 5.02/4.96 (m, 2H), 4.36 (sq, 1H), 4.31 (dd, 1H), 4.2 (m, 2H), 3.94 (s, 2H), 3.77 (s, 3H), 3.58 (m, 10 H), 3.46/2.28 (d+t, 2H), 3.34 (t, 2H), 3.29 (m, 4 H), 2.8 (m, 2H), 2.67 (t, 2H), 2.43 (m, 4 H), 2.01 (m, 1H), 1.75 (s, 3 H), 1.69/1.6 (2m, 2H), 1.51 (m, 2H), 1.31 (m, 2H), 0.86/0.8 (2d, 6 H). ¹³C NMR (100 MHz, DMSO-d6): δ ppm. 157.9, 153.7, 131.4, 131.4, 131.3, 131.1, 130.9, 129.3, 127.6, 120.9, 120.4, 119.8, 116.2, 116.1, 112.6, 111.8, 111.8, 78.1, 70.5, 70.4, 69.3, 66.6, 66.6, 56.9, 56.5, 56.3, 54, 52.3, 50.4, 44, 39, 33.8, 32.2, 31.8, 28.2, 23.1, 19.7/18.1, 18.3.¹⁹F NMR (376 MHz, DMSO-d6): δ ppm -112.6. HR-IER+: m/z [M+H]+=1452.5661 (1452.5648) (measured/theoretical).

Obtaining L21-C1:

(2S,3S,4R,5R,6S)-6-[2-[2-[[4-[2-[4-[4-[(1R)-1-carboxy-2-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]ethoxy]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy]ethyl]-1-methyl-pipe razin-1-yl]methyl]-5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]ethyl]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid; 2,2,2-trifluoroacetate

Step 1: tert-butyl-[(2-iodo-4-nitro-phenyl)methoxy]-dimethyl-silane

To a solution of (2-iodo-4-nitrophenyl)methanol (172 g, 61.64 mmol; prepared according to step 2 of the preparation of L14-C3 ) in dichloromethane (300 mL) was added imidazole (5.04 g, 73.97 mmol). The mixture was cooled to 0 °C, then a solution of tert-butyl chlorodimethylsilane (11.15 g, 73.97 mmol) in dichloromethane (300 mL) was added dropwise over 15 min. The ice bath was removed and the reaction mixture was stirred at room temperature for 16 h. After completion of the reaction, the reaction mixture was quenched with methanol (20 mL) and concentrated to dryness. The crude product was purified by silica gel chromatography (gradient with ethyl acetate in cyclohexane) to give tert-butyl-[(2-iodo-4-nitro-phenyl)methoxy]-dimethyl-silane (19.65 g, 49.96 mmol) as a white solid. ¹ H NMR (400 MHz, DMSO-d6): δ 8.57 (s, 1H), 8.31 (d, 1H), 7.66 (d, 1H), 4.67 (s, 2H), 0.92 (s, 9H), 0.14 (s, 6H).

Step 2: Methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-nitro-phenyl]ethynyl]tetrahydropyran-2-carboxylate

To a solution of tert-butyl [(2-iodo-4-nitro-phenyl)methoxy]-dimethylsilane (3.0 g, 7.63 mmol) in DMF (55 mL) were added sequentially methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-ethynyl tetrahydropyran-2-carboxylate (3.39 g, 9.92 mmol; prepared according to step 13 of preparation L14-C3 ), DIPEA (5.80 mL, 35.09 mmol), copper iodide (145 mg, 0.763 mmol), and dichlorobis-(triphenylphosphine)palladium(II) (535 mg, 0.763 mmol). The yellow solution was washed with argon and stirred for 16 h at room temperature. After dilution with water (300 mL), the aqueous layer was extracted with ethyl acetate (2 x 300 mL). The combined organic layers were washed with water (2 x 300 mL) then dried over sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by silica gel chromatography (gradient with ethyl acetate in cyclohexane) to give methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-nitrophenyl]ethynyl]tetrahydropyran-2-carboxylate (4.01 g, 6.60 mmol) as a beige solid. ^1H NMR (400 MHz, DMSO-d6): δ 8.32 (dd, 1H), 8.19 (d, 1H), 7.75 (d, 1H), 5.45 (t, 1H), 5.16 (t, 1H), 5.02-5.07 (m, 2H), 4.82 (s, 2H), 4.55 (d, 1H), 3.65 (s, 3H), 1.98-2.07 (m, 9H), 0.92 (m, 9H), 0.14 (s, 6H).

Step 3: Methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-(hydroxymethyl)-5-nitro-phenyl]ethynyl]tetrahydropyran-2-carboxylate

To a solution of methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-nitro-phenyl]ethynyl]tetrahydropyran-2-carboxylate (4.01 g, 6.60 mmol) in THF (48 mL) and water (48 mL) was added acetic acid (193 mL, 3.36 mol). The colorless solution was stirred for 2 days at room temperature then diluted with water (300 mL). The aqueous layer was extracted with dichloromethane (2 x 300 mL). The combined organic layers were washed with water (2 x 300 mL) and saturated aqueous sodium hydrogen carbonate solution (400 mL), then dried over sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by silica gel chromatography (gradient with ethyl acetate in cyclohexane) to give methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-(hydroxymethyl)-5-nitro-phenyl]ethynyl]tetrahydropyran-2-carboxylate (2.67 g, 5.41 mmol) as a white solid. ^1H NMR (400 MHz, DMSO-d6): δ 8.29 (dd, 1H), 8.15 (d, 1H), 7.79 (d, 1H), 5.68 (t, 1H), 5.45 (t, 1H), 5.16 (t, 1H), 5.02-5.07 (m, 2H), 4.62 (d, 2H), 4.55 (d, 1H), 3.65 (s, 3H), 1.98-2.07 (m, 9H).

Step 4: Methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-amino-2-(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate

A solution of methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-(hydroxymethyl)-5-nitro-phenyl]ethynyl]tetrahydropyran-2-carboxylate (2.67 g, 5.41 mmol) in THF (59 mL) was washed with argon. Dry 5% platinum on carbon (1.34 g, 50% w/w) was added. The reaction mixture was washed successively with argon, H ₂ and stirred for 2 days at room temperature under H ₂ (P atm). The reaction mixture was filtered through a pad of Celite®, washed with ethyl acetate/methanol 9/1 solution (500 mL), then concentrated to dryness. The entire sequence (addition of dry 5% platinum on carbon (1.34 g, 50% w/w), stirring for 16 h at room temperature under H ₂ (P atm), and filtration through a pad of Celite®) was repeated to achieve complete conversion. The crude product was purified by silica gel chromatography (gradient with ethyl acetate in cyclohexane) to give methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-amino-2-(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate (1.12 g, 2.40 mmol) as a white solid. ¹ H NMR (400 MHz, DMSO-d6): δ 6.93 (d, 1H). 6.67-6.33 (m, 2H), 5.30 (t, 1H), 4.96 (t, 1H), 4.88 (s, 2H), 4.81 (t, 1H), 4.61 (t, 1H), 4.39 (d, 1H), 4.29-4.24 (m, 2H), 3.78-3.72 (m, 1H), 3.65 (s, 3H), 2.65-2.54 (m, 2H), 2.07-1.98 (m, 9H), 1.79-1.68 (m, 1H), 1.63-1.52 (m, 1H).

Step 5: Methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-(tert-butoxycarbonylamino)-5-ureidopentanoyl]amino]-2-(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate

To a solution of methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-amino-2-(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate (1.00 g, 2.14 mmol) in DMF (21 mL) were added sequentially (2S)-2-(tert-butoxycarbonylamino)-5-ureidopentanoic acid (589 mg, 2.14 mmol), DIPEA (707 μL, 4.28 mmol) and HBTU (1.22 g, 3.21 mmol). The reaction mixture was stirred for 72 h at room temperature. After completion of the reaction, the mixture was diluted with water (100 mL) and concentrated to dryness. The crude product was purified by silica gel chromatography (gradient methanol in dichloromethane) to give methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-(tert-butoxycarbonylamino)-5-ureidopentanoyl]amino]-2-(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate (1.05 g, 1.45 mmol) as a beige solid. ^1H NMR (400 MHz, DMSO-d6): δ 9.82 (s, 1H), 7.35-7.42 (m, 2H), 7.24 (d, 1H), 6.95 (d, 1H), 5.94 (t, 1H), 5.37 (s, 2H), 5.30 (t, 1H), 4.91-4.99 (m, 2H), 4.79 (t, 1H), 4.36-4.42 (m, 3H), 4.01-4.08 (m, 1H), 3.76 (t, 1H), 3.65 (s, 3H), 2.95-3.04 (m, 2H), 2.54-2.65 (m, 2H), 1.98-2.07 (m, 9H), 1.68-1.79 (m, 1H), 1.49-1.63 (m, 3H), 1.30-1.42 (m, 11H).

Step 6: Methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]-2-(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate

To a solution of methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-(tert-butoxycarbonylamino)-5-ureidopentanoyl]amino]-2-(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate (950 mg, 1.31 mmol) in dichloromethane (7.5 ml) was added trifluoroacetic acid (1.9 ml, 25.6 mmol) at 0°C. The reaction mixture was stirred at room temperature for 3 h. After completion of the reaction, the reaction mixture was concentrated to dryness and co-evaporated with toluene (2×50 ml) to give the crude compound.

To this crude solution in DMF (13 mL) were added sequentially (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoic acid (467 mg, 1.38 mmol), DIPEA (867 μL, 5.24 mmol), and HBTU (845 mg, 2.23 mmol). The reaction mixture was stirred for 16 h at room temperature. After completion of the reaction, saturated aqueous hydrogen carbonate solution (20 mL) was added. The mixture was stirred at room temperature for 1 h, diluted with water (100 mL), and concentrated to dryness. The crude product was purified by silica gel chromatography (methanol in dichloromethane gradient) and then by C18 reverse phase chromatography using the neutral method to give methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]-2-(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate (680 mg, 0.720 mmol) as a white solid. LC-MS: MS (ESI) m/z [M+H]+=946.3. ¹ H NMR (400 MHz, DMSO-d6): δ 9.90 (s, 1H). 8.07 (d, 2H), 7.89 (d, 2H), 7.74 (t, 2H), 7.44-7.38 (m, 3H), 7.36-7.28 (m, 3H), 7.24 (d, 1H), 5.94 (t, 1H), 5.37 (s, 2H), 5.30 (t, 1H), 4.99-4.92 (m, 2H), 4.79 (t, 1H), 4.42-4.36 (m, 4H), 4.32-4.19 (m, 3H), 3.94-3.90 (m, 1H), 3.76 (t, 1H), 3.65 (s, 3H), 2.99-2.94 (m, 2H), 2.65-2.54 (m, 2H), 2.07-1.98 (m, 10H), 1.70-1.55 (m, 4H), 1.46-1.36 (m, 2H), 0.89-0.84 (m, 6H). ¹³ C NMR (100 MHz, DMSO-d6): δ 171.19, 170.33, 169.58, 169.45, 169.27, 167.77, 158.81, 156.12, 143.89, 143.76, 140.69, 139.48, 137.54, 134.88, 128.44, 127.62, 127.06, 125.35, 120.08, 119.42, 116.65, 75.78, 74.61, 72.65, 71.20, 69.49, 65.68, 60.49, 60.10, 53.14, 52.40, 46.68, 32.32, 30.43, 29.54, 27.19, 26.77, 20.39, 20.34, 20.24, 19.22, 18.25.

Step 7: Methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-(bromomethyl)-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]ethyl]tetrahydropyran-2-carboxylate

To a solution of methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]-2-(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate (154 mg, 0.163 mmol) in THF (8.2 ml) were added triphenylphosphine (85.4 mg, 0.326 mmol) and 1-bromopyrrolidine-2,5-dione (58.0 mg, 0.326 mmol). The reaction mixture was stirred for 2 h at room temperature. The reaction mixture's progress is monitored by UPLC-MS: an aliquot is treated with a large excess of MeOH, followed by the formation of the corresponding methyl ester. The expected bromide derivative is stable under UPLC-MS conditions. After 5 h, triphenylphosphine (85.4 mg, 0.326 mmol) and 1-bromopyrrolidine-2,5-dione (58.0 mg, 0.326 mmol) are added, and the reaction mixture is stirred for 15 h at room temperature. The obtained crude methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-(bromomethyl)-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]phenyl]ethyl]tetrahydropyran-2-carboxylate was used similarly in the next step. UPLC-MS: MS (ESI) m/z [M+Ome-Br+H]+=960.7.

Stage 8: (2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]-2-[2-[(2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2-yl]ethyl]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To the solution Methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-(bromomethyl)-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]ethyl]tetrahydropyran-2-carboxylate (0.167 mmol) in DMF from the previous step (step 7) is sequentially added (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-(2-piperazin-1-ylethoxy)phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid(C1)(143 mg, 0.163 mmol) and DIPEA (114 μL, 0.652 mmol). The reaction mixture was stirred for 15 h at room temperature, and the progress of the reaction mixture was monitored by UPLC-MS (an aliquot was treated with a large excess of MeOH). The crude product was purified by C18 reverse-phase prep-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the TFA method to give (2R)-2-[(5Sa)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]-2-[2-[(2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2-yl]ethyl]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (21.3 mg, 0.0111 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+H]+=1802.9+1804.9. IR wavelength (cm^-1): 1755, 1672, 1226,1201,1130.¹H NMR (400 MHz, DMSO-d6) δ ppm. 13.3 (shs, 1H), 10.2 (s, 1H), 8.88 (d, 1H), 8.61 (s, 1H), 8.14 (d, 1H), 7.88 (d, 2H), 7.73 (dd, 2H), 7.65 (d, 1H), 7.63 (d, 1H), 7.62 (m, 1H), 7.54 (shs, 1H), 7.51 (dd, 1H), 7.45 (t, 1H), 7.4 (t, 2H), 7.38 (m, 1H), 7.32 (t, 2H), 7.3 (dd, 2H), 7.2 (d, 1H), 7.2 (t, 2H), 7.15 (t, 1H), 7.15 (d, 1H), 7.03 (t, 1H), 7.01 (dd, 1H), 6.72 (t, 1H), 6.22 (d, 1H), 6 (ws, 1H), 5.51 (dd, 1H), 5.34 (t, 1H), 5.3 (ws, 2H), 5.27/5.21 (m, 2H), 4.98 (t, 1H), 4.85 (t, 1H), 4.57/4.49 (m, 2H), 4.39 (m, 1H), 4.35 (d, 1H), 4.27 (m, 2H), 4.26 (m, 2H), 4.23 (m, 1H), 3.93 (t, 1H), 3.76 (s, 3H), 3.71 (m, 1H), 3.64 (s, 3H), 3.4 (m, 4H), 3.29/2.51 (2dd, 2H), 3.13/2.94 (2m, 4H), 3 (m, 2H), 2.98 (m, 2H), 2.93 (shs, 3H), 2.81 (m, 2H), 1.99/1.95 (3s, 9H), 1.98 (m, 1H), 1.84 (s, 3H), 1.77/1.59 (2m, 2H), 1.64 (2m, 2H), 1.41 (2m, 2H), 0.88/0.85 (2d, 6H). ¹³C NMR (100 MHz, DMSO-d6): δ ppm. 158.1, 152.9, 135.6, 131.5, 131.4, 131.3, 131.2, 131, 128.9, 128.1, 127.5, 125.7, 120.9, 120.6, 120.4, 120.3, 117, 116, 116, 112.8, 112.2, 111.2, 75.6, 74.9, 73.8, 72.9, 71.4, 69.6, 69.4, 67.5, 66.1, 60.4, 58.3, 56, 55.4, 53.9, 52.8, 47.2, 46.2, 44.3, 39, 32.8, 32.8, 31, 29.6, 27.4, 27.2, 21.1, 19.5/18.7, 18.1.¹⁹F NMR (376 MHz, DMSO-d6) δ ppm. -74, -112.

Stage 9: (2S,3S,4R,5R,6S)-6-[2-[(5S _a )-5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]-2-[[4-[2-[4-[4-[(1R)-1-carboxy-2-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]ethoxy]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy]ethyl]-1-methyl-piperazin-1-yl-1-yl]methyl]phenyl]ethyl]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid

To the solution (2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]-2-[2-[(2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2-yl]ethyl]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (21.3 mg, 0.0111 mmol) in methanol (6.0 mL) was added a solution of lithium hydroxide monohydrate (4.66 mg µL, 0.111 mmol) in water (4 mL). The reaction mixture was stirred at room temperature for 60 h, and the progress of the reaction mixture was monitored by UPLC-MS. The crude product was purified by C18 reverse-phase prep-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the TFA method to afford (2S,3S,4R,5R,6S)-6-[2-[(5S _a )-5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]-2-[[4-[2-[4-[4-[(1R)-1-carboxy-2-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]ethoxy]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy]ethyl]-1-methyl-piperazin-1-yl-1-yl]methyl]phenyl]ethyl]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid (47.8 mg, 0.029 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+H]+=1440.6+1442.6.

Step 10: (2S,3S,4R,5R,6S)-6-[2-[2-[[4-[2-[4-[4-[(1R)-1-carboxy-2-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]ethoxy]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy]ethyl]-1-methyl-pipe razin-1-yl]methyl]-5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]ethyl]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid; 2,2,2-trifluoroacetate L21-C1

To a solution of (2S,3S,4R,5R,6S)-6-[2-[(5S _a )-5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]-2-[[4-[2-[4-[4-[(1R)-1-carboxy-2-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]ethoxy]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy]ethyl]-1-methyl-piperazin-1-ium-1-yl]methyl]phenyl]ethyl]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid (47.1 mg, 0.0282 mmol) in DMF (1.5 mL) were added successively a solution of (2,5-dioxopyrrolidin-1-yl) 3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoate (purchased from Broadpharm, 13.1 mg, 0.0423 mmol) in DMF (500 µL) and DIPEA (17.2 µL, 0.0988 mmol).

The reaction mixture was stirred for 1 h at room temperature, and the progress of the reaction mixture was monitored by UPLC-MS. The crude product was purified by C18 prep-RP-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the TFA method to afford L21-C1 (65 mg, 0.0310 mmol) as a white powder. HR-ESI+: m/z [M+H]+ = 1635.6093 (1635.6068) (measured/theoretical).

Getting L9-C1:

(2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid

Step 1: 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-1-[[4-(bromomethyl)phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]carbamate

A solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-1-[[4-(bromomethyl)phenyl]carbamoyl]-4-ureidobutyl]carbamoyl]-2-methylpropyl]carbamate (150 mg, 0.249 mmol) in THF (3.8 mL) was cooled to 0 °C. At 0 °C, tribromophosphamide (1 M in dichloromethane) (374 μL, 0.249 mmol) was added dropwise. The reaction mixture was stirred for 5 min at 0 °C and 1 h at room temperature. The progress of the reaction mixture was monitored by UPLC-MS (an aliquot was treated with a large excess of MeOH). The reaction mixture was diluted with ethyl acetate (3 mL) and washed with an aqueous saturated solution of sodium hydrogen carbonate (1×6 mL). The organic layer was dried over magnesium sulfate and filtered. DMF (10 mL) was added and ethyl acetate and THF were evaporated. The resulting solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-1-[[4-(bromomethyl)phenyl]carbamoyl]-4-ureidobutyl]carbamoyl]-2-methylpropyl]carbamate was used as is in the next step. UPLC-MS: MS (ESI) m/z [M+Na]+=686.5+688.6.

Stage 2: (2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-yl-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-1-[[4-(bromomethyl)phenyl]carbamoyl]-4-ureidobutyl]carbamoyl]-2-methylpropyl]carbamate (0.249 mmol) in DMF from the previous step (step 1) were added sequentially DMF (10 mL), (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-(2-piperazin-1-ylethoxy)phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (C1) (218 mg, 0.249 mmol) and DIPEA (130 µl, 0.748 mmol). The reaction mixture was stirred for 15 h at room temperature, and the progress of the reaction mixture was monitored by UPLC-MS (an aliquot was treated with a large excess of MeOH). The resulting solution of (2R)-2-[(5Sa)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid in DMF was used as is in the next step. UPLC-MS: MS (ESI) m/z [M+Na]+=1458.7+1460.7.

Stage 3: (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; bis 2,2,2-trifluoroacetic acid

To a solution of (2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (0.249 mmol) in dimethylformamide (3 mL) obtained in the previous step (step 2) was added piperidine (49.3 μL, 0.499 mmol). The reaction mixture was stirred at room temperature for 5 h, and the progress of the reaction mixture was monitored by UPLC-MS. The crude product was purified by C18 prep reverse phase HPLC by direct loading of the reaction mixture onto an Xbridge column and using the TFA method to give (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; bis 2,2,2-trifluoroacetic acid (31.2 mg = 0.0213 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+Na]+ = 1236.7+1238.7.

Stage 4: (2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid L9-C1

To a solution of (2R)-2-[(5Sa)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (31.2 mg, 0.0213 mmol) in DMF (1.5 ml) was successively added a solution of (2,5-dioxopyrrolidin-1-yl) 3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoate (23.8 mg, 0.0768 mmol) in DMF (500 μL) and DIPEA (31.2 μL, 0.179 mmol). The reaction mixture was stirred for 15 h at room temperature, and the progress of the reaction mixture was monitored by UPLC-MS. The crude product was purified by C18 prep-HPLC with reversed phase by direct loading of the reaction mixture onto an Xbridge column and using the TFA method to obtainL9-C1(6 mg, 0.00303 mmol) as a white powder. HR-IER+: m/z [M]+=1431.5437 (1431.5433) (measured/theoretical).

Getting L9-C8:

(2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(3-sulfooxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid

Step 1: (2R)-2-[(5Sa)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(3-sulfooxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-1-[[4-(bromomethyl)phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]carbamate (0.0230 mmol; obtained according to step 1 of preparationL9-C1) in DMF (3 ml) are successively added DMF (5 ml), ammonium; [3-[4-[[2-[(2R)-2-carboxy-2-[(5Sa)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-ethyl]phenoxy]methyl]pyrimidin-2-yl]phenyl] sulfate(C8)(22 mg, 0.0230 mmol) and DIPEA (12 μL, 0.069 mmol). The reaction mixture was stirred for 15 h at room temperature, and the progress of the reaction mixture was monitored by UPLC-MS (an aliquot was treated with a large excess of MeOH). The resulting solution of (2R)-2-[(5Sa)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid in DMF was used in the next step. UPLC-MS: MS (ESI) m/z [M-SO₃H]+=1444.8+1446.7.

Stage 2: (2R)-2-[(5S _a )-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(3-sulfooxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; bis 2,2,2-trifluoroacetic acid

To a solution of (2R)-2-[(5Sa)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(3-sulfooxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (0.0230 mmol) in dimethylformamide (3 ml) obtained in the previous step (step 1), piperidine (9 μl, 0.0920 mmol). The reaction mixture was stirred at room temperature for 5 h, and the progress of the reaction mixture was monitored by UPLC-MS. The crude product was purified by C18 prep reverse phase HPLC by direct loading of the reaction mixture onto an Xbridge column and using the TFA method to afford [3-[4-[[2-[(2R)-2-[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-2-carboxyethyl]phenoxy]methyl]pyrimidin-2-yl]phenyl] sulfate; bis 2,2,2-trifluoroacetic acid (10.0 mg = 0.00633 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M-SO ₃ ]+ = 1224.12.

Stage 3: (2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(3-sulfooxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid

To the solution (2R)-2-[(5S_a)_-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(3-sulfooxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (10 mg, 0.00653 mmol) in DMF (1 ml) was successively added to a solution of (2,5-dioxopyrrolidin-1-yl) 3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoate (3.1 mg, 0.00980 mmol) in DMF (500 µl) and DIPEA (4 µl, 0.0229 mmol). The reaction mixture was stirred for 15 h at room temperature, and the progress of the reaction mixture was monitored by UPLC-MS. The crude product was purified by C18 prep-HPLC with reversed phase by direct loading of the reaction mixture onto an Xbridge column and using the TFA method to obtainL9-C8(6 mg, 0.00401 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+Na]+=1519.5+1521.2, [M+H-SO3]+ 1417.7+1419.6. HR-ESI+: m/z [M+H]+=1497.486 (1497.4845) (measured/theoretical).

Getting L9-C10:

(2R)-2-[(5S _a )5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-[4-fluoro-3-(2,2,2-trifluoroethoxy)phenyl]thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid

The procedure is the same as the synthesis method of L9-C9 , replacing C9 used in step 3 with (2R)-2-[((5Sa)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-[4-fluoro-3-(2,2,2-trifluoroethoxy)phenyl]thieno[2,3-d]pyrimidin-4-yl)oxy]-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid C10 and using the TFA method for purification. HR-ESI+: m/z [M+H]+=1529.543/1529.5413 (measured/theoretical).

Getting L9-C11:

(2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-yl-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(4-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid.

The procedure is the same as the synthesis method of L9-C9 , replacing C9 used in step 3 with (2R)-2-{[(5Sa)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(4-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid C11 and using the TFA method for purification. HR-ESI+: m/z [M+H]+=1431.5442/14.31.5433 (measured/theoretical).

Getting L9-C12:

(2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2,2,2-trifluoroethyl)pyrazol-3-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid

The procedure is the same as the synthesis method of L9-C9 , replacing C9 used in step 3 with (2R)-2-{[(5Sa)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl]methoxy}phenyl)propanoic acid C12 using the TFA method for purification. HR-ESI+: m/z [M+H]+ = 1395.5048/1395;5045 (measured/theoretical).

Obtaining L9-C14:

(2R)-2-[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-yl-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(3-hydroxy-2-methoxy-phenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate

Step 1: (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxy-3-sulfooxy-phenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

500 mg ethyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[(2-chloropyrimidin-4-yl)methoxy]phenyl]propanoate (0.60 mmol, WO2016/207216 Preparation 1 ) and 202 mg (3-hydroxy-2-methoxy-phenyl)boronic acid (1.20 mmol) were dissolved in 9 ml of 1,4-dioxane, then 42 mg Pd( _PPh3 ) _{2Cl2 (} 0.06 mmol), 588 mg _Cs2CO3 (1.80 mmol) and 9 ml of water were _added and the mixture was stirred under _N2 at 70°C until complete conversion. Then it is diluted with water, neutralized _with 2 M aqueous HCl solution and extracted with DCM. The combined organic phase is dried over _Na2SO4 , filtered and the filtrate is concentrated under reduced pressure. The crude ester is purified by flash chromatography using heptane, EtOAc and 0.7 M _NH3 solution in MeOH as eluents to give a mixture of diastereoisomers. Then it is dissolved in 23.6 mL pyridine, 0.97 mL _SO3 ×pyrimidine (5.98 mmol) is added and the mixture is stirred at 70°C until complete conversion. Then it is concentrated under reduced pressure and dissolved in 2 mL dioxane, then 200 mg KOH (3.57 mmol) and 1 mL water is added. The mixture is stirred at rt until complete hydrolysis. It was then neutralized with 2 M aqueous HCl and directly injected onto a prep-RP-HPLC using 25 mM aqueous NH ₄ HCO ₃ and MeCN as eluents. The later eluted diastereomer was collected as the title product. ^1H NMR (500 MHz, DMSO- _d6 ) δ: 8.92 (d, 1H), 8.63 (s, 1H), 7.68 (dd, 1H), 7.63 (d, 1H), 7.34 (d, 1H), 7.30 (dd, 2H), 7.29 (d, 1H), 7.20 (t, 2H), 7.16 (t, 1H), 7.15 (d, 1H), 7.10 (t, 1H), 7.02 (d, 1H), 6.73 (t, 1H), 6.38 (d, 1H), 5.50 (dd, 1H), 5.29/5.23 (d+d, 2H), 4.21/4.16 (m+m, 2H), 3.84 (s, 3H), 3.25/2.55 (dd+dd, 2H), 3.18-2.75 (m, 10H), 2.65 (br s, 3H), 1.82 (s, 3H). HRMS calculated for C ₄₇ H ₄₄ N ₆ O ₁₀ S ₂ ClF: 970.2233; found 971.2297 (M+H).

Step 2: (2R)-2-[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(3-hydroxy-2-methoxy-phenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate L9-C14

To a solution of (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxy-3-sulfooxy-phenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (20.0 mg; 0.0206 mmol) in DMF (309 μl) was successively added (2S)-N-[4-(chloromethyl)phenyl]-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanamide (17.5 mg; 0.0206 mmol), DIPEA (10.8 μL; 0.0618 mmol) and TBAI (1 mg; 0.0027 mmol). The reaction mixture was stirred at 70°C for 18 h. The crude product was purified by C18 reversed-phase prep-HPLC by direct loading of the reaction mixture onto an X-Bridge column and using the TFA method to afford L9-C14 (10.5 mg; 0.00725 mmol) as a white powder. HR-ESI+: m/z [M+H]+=1448.5437/1448.5466 [measured/theoretical].

Obtaining L9-P15:

(11R,20R)-23,26-dichloro-20-[[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-yl-1-yl]methyl]-3-(4- fluorophenyl)-14-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]-24,25-dimethyl-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20,2,2,12,5,113,17,09,28]octacosa-1(24),2,5(28),6,8,13,15,17(27),22,25-decaene-11-carboxylic acid; 2,2,2-trifluoroacetate

To a solution of (11R,20R)-23,26-dichloro-3-(4-fluorophenyl)-14-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]-24,25-dimethyl-20-[(4-methylpiperazin-1-yl)methyl]-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20,2,2,12,5,113,17,09,28]octacose-1(25),2,5(28),6,8,13,15,17(27),22(26),23-decaene-11-carboxylic acid P15 (obtained according to WO 2019/035914; 10.0 mg; 0.0105 mmol) in DMF (630 μl), (2S)-N-[4-(bromomethyl)phenyl]-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methylbutanoyl]amino]-5-ureidopentanamide (10.0 mg, 0.0158 mmol), DIPEA (5.5 μL, 0.0315 mmol) and TBAI (0.5 mg, 0.0010 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 0.5 h. The crude product was purified by C18 reverse phase prep-HPLC by direct loading of the reaction mixture onto an X-Bridge column and using the TFA method to afford L9-P15 (11.9 mg, 0.00733 mmol) as a white powder. HR-IER+: m/z [M-CF ₃ CO ₂ ]+=1507.5183/1507.5155

Obtaining L9-P16:

(11R,20R)-23,26-dichloro-14-[[2-[4-[[(2S)-1,4-dioxan-2-yl]methoxymethyl]-4-fluoro-cyclohexyl]pyrimidin-4-yl]methoxy]-20-[[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pe [antanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]methyl]-3-(4-fluorophenyl)-24,25-dimethyl-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20,2,2,12,5,113,17,09,28]octacosa-1(25),2,5(28),6,8,13,15,17(27),22(26),23-decaene-11-carboxylic acid; 2,2,2-trifluoroacetate

To a solution of (11R,20R)-23,26-dichloro-14-[[2-[4-[[(2S)-1,4-dioxan-2-yl]methoxymethyl]-4-fluoro-cyclohexyl]pyrimidin-4-yl]methoxy]-3-(4-fluorophenyl)-24,25-dimethyl-20-[(4-methylpiperazin-1-yl)methyl]-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20,2,2,12,5,113,17,09,28]octacose-1(24),2,5(28),6,8,13,15,17(27),22,25-decaene-11-carboxylic acid P16 (obtained according to WO 2019/035911; 14.7 mg; 0.0137 mmol) in DMF (1 mL) were sequentially added (2S)-N-[4-(bromomethyl)phenyl]-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methylbutanoyl]amino]-5-ureidopentanamide (13.1 mg; 0.0205 mmol) and DIPEA (7.1 μL; 0.0410 mmol). The reaction mixture was stirred at room temperature for 2 h. The crude product was purified by direct loading of the reaction mixture on an X-Bridge column using the TFA method to afford L9-P16 (7.9 mg; 0.00420 mmol) as a white powder. IR (cm ^-1 ): 3327, 1768/1706, 1666, 1199/1118, 831/798. HR-ESI+: m/z [M-CF ₃ CO ₂ ]+=1631.6071/1631.6054 [measured/theoretical]

Obtaining L9-P17:

(11R,20R)-23,26-dichloro-14-[[2-[4-[[(2S)-1,4-dioxan-2-yl]methoxy]cyclohexyl]pyrimidin-4-yl]methoxy]-20-[[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentane oil]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]methyl]-3-(4-fluorophenyl)-24,25-dimethyl-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20,2,2,12,5,113,17,09,28]octacosa-1(24),2,5(28),6,8,13,15,17(27),22,25-decaene-11-carboxylic acid; 2,2,2-trifluoroacetate

To a solution of (11R,20R)-23,26-dichloro-14-[[2-[4-[[(2S)-1,4-dioxan-2-yl]methoxy]cyclohexyl]pyrimidin-4-yl]methoxy]-3-(4-fluorophenyl)-24,25-dimethyl-20-[(4-methylpiperazin-1-yl)methyl]-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20,2,2,12,5,113,17,09,28]octacose-1(24),2,5(28),6,8,13,15,17(27),22,25-decaene-11-carboxylic acid P17 (obtained according to WO 2019/035911; 14.5 mg; 0.0139 mmol) in DMF (1 mL), (2S)-N-[4-(bromomethyl)phenyl]-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methylbutanoyl]amino]-5-ureidopentanamide (13.3 mg, 0.0208 mmol) and DIPEA (7.3 μL, 0.0417 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 8 h. The crude product was purified by direct loading of the reaction mixture on an X-Bridge column and using the TFA method to afford L9-P17 (7.0 mg, 0.00437 mmol) as a white powder. IR (cm ^-1 ): 3700-2400, 1771/1738/1705, 1665, 1194/1128. HR-IER+: m/z [M-CF ₃ CO ₂ ]+=1599.6013/1599.5992. HR-IER+: m/z [M-CF ₃ CO ₂ +H]2+=800.3049/800.3035 [measured/theoretical]

Obtaining L25-P1:

2-[[4-[2-[4-[4-[(1R)-1-carboxy-2-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]ethoxy]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy]ethyl]-1-methyl-piperazin-1-yl-1-yl]methyl]-5-[[(2S)-2-[[1-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethylcarbamoyl]cyclobutanecarbonyl]amino]-5-ureidopentanoyl]amino]benzenesulfonate; 2,2,2-trifluoroacetic acid

Step 1: tert-butyl 1-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethylcarbamoyl] cyclobutanecarboxylate

To a solution of 1-tert-butoxycarbonylcyclobutanecarboxylic acid (58.6 mg; 0.293 mmol) in DCM (5.85 mL), 1-[2-(2-aminoethoxy)ethyl]pyrrole-2,5-dione (53.9 mg; 0.293 mmol), EDC (84.2 mg; 0.439 mmol), HOBt (59.3 mg; 0.439 mmol), and DIPEA (204 μL; 1.17 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 18 h. The progress of the reaction mixture was monitored by UPLC-MS. The reaction mixture was concentrated to dryness and solubilized in DMF (1 mL) and the solution was purified on an X-Bridge C ₁₈ column by direct loading of the reaction mixture onto the column and using the TFA method to give the title compound (57.3 mg; 0.156 mmol). IR (cm ^-1 ): 3390, 1697/1666. ¹ H NMR (400 MHz, DMSO-d6) δ ppm 7.5 (t, 1H), 7.02 (s, 2H), 3.55/3.5 (2t, 4H), 3.38 (t, 2H), 3.17 (q, 2H), 2.33 (m, 4H), 1.77 (m, 2H), 1.38 (s, 9H). UPLC-MS: MS(ESI): m/z [M+Na]+=389.26 [M+H-tBu]+=311.22

Step 2: 1-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethylcarbamoyl]cyclobutanecarboxylic acid

To a solution of tert-butyl 1-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethylcarbamoyl]cyclobutanecarboxylate (7 mg, 0.0191 mmol) in DCM (0.175 mL) was added TFA (51.2 μL, 0.668 mmol). The reaction mixture was stirred at room temperature for 3.5 h, then concentrated to dryness to give the title compound (5.8 mg, 0.0187 mmol) as a colorless oil. The crude product was used in the next step. UPLC-MS: MS(ESI): m/z [M+H]+=311.35, [M+Na]+=333.37

Step 3: (2,3,4,5,6-pentafluorophenyl) 1-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethylcarbamoyl]cyclobutanecarboxylate

To a solution of 1-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethylcarbamoyl]cyclobutanecarboxylic acid (18.2 mg; 0.0587 mmol) in THF (3 mL), 2,3,4,5,6-pentafluorophenol (13.0 mg; 0.0704 mmol) and DCC (14.5 mg; 0.0704 mmol) were added successively. The reaction mixture was stirred at room temperature for 15 h and the progress of the reaction mixture was monitored by UPLC-MS. The reaction mixture was suspended, the precipitate was filtered and washed with THF (1 mL) to obtain a solution of (2,5-dioxopyrrolidin-1-yl) 1-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethylcarbamoyl]cyclobutanecarboxylate in THF. The crude product solution was used in step 9. UPLC-MS: MS(ESI): m/z [M+H]+=477.28, [M+Na]+=499.23

Step 4: Methyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate

To a solution of (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid P1 (5.0 g, 5.712 mmol) in DCM (25 mL) and methanol (25 mL), a solution of diazomethyl(trimethyl)silane (2 M in Et ₂ O) (5.712 mL, 11.42 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 2 h, and the progress of the reaction mixture was monitored by UPLC-MS. Upon completion, the reaction mixture was quenched by slow addition of acetic acid until the color turned yellow to red and concentrated to dryness to give the crude mixture. The crude product was purified by silica gel chromatography (methanol in DCM gradient) to give the title compound (4.52 g, 5.082 mmol). UPLC-MS: MS(ESI): m/z [M+H]+=889.27+891.6, [M+Na]+=911.31, [M+2H]2+= 445.59. IR (cm ^-1 ): 1753, 1238/1053. ¹ H NMR (400 MHz, DMSO-d6) δ ppm. 8.6 (s, 1H), 8.45 (d, 1H), 7.6 (d, 1H), 7.52 (dd, 1H), 7.45 (td, 1H), 7.3 (m, 3H), 7.25-7.1 (m, 5H), 7.02 (t+d, 2H), 6.78 (t, 1H), 6.31 (dd, 1H), 5.52 (dd, 1H), 5.25 (AB, 2H), 4.2 (m, 2H), 3.78/3.65 (2s, 6H), 3.2/2.58 (2dd, 2H), 2.71 (t, 2H), 2.5/2.3 (2m, 8H), 2.12 (s, 3H), 1.88 (c, 3H).

Step 5: 5-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-ureidopentanoyl]amino]-2-(hydroxymethyl)benzenesulfonic acid

To a solution of Fmoc-Cit-OH (2.224 g, 5.596 mmol) in DCM (22.2 mL) and methanol (22.2 mL) were added sodium 5-amino-2-(hydroxymethyl)benzenesulfonate (1.89 mg, 8.395 mmol) and EEDQ (2.768 g, 11.19 mL) sequentially. The reaction mixture was stirred at room temperature for 25 h, then concentrated to dryness. The crude product was purified by silica gel chromatography (methanol in DCM gradient) to give the title compound (2.81 g, 4.823 mmol) as a white powder. MK (cm ^-1 ): 3700-3000, 1660 (large), 1180. ¹ H NMR (400 MHz, DMSO-d6) δ ppm. 10.02 (s, 1H), 7.88 (m, 3H), 7.76 (2t, 2H), 7.7 (dd, 1H), 7.61 (d, 1H), 5.99 (t, 1H), 5.38 (m, 2H), 5.03 (t, 1H), 4.72 (d, 2H), 4.3-4.2 (m, 3H), 4.15 (m, 1H), 3.06-2.90 (m, 2H), 1.75-1.30 (m, 4H). UPLC-MS: MS(ESI): m/z [M+H]+=583.42, [M+Na]+=565.31.

Step 6: 2-(chloromethyl)-5-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-ureidopentanoyl]amino]benzenesulfonic acid

To a solution of 5-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-ureidopentanoyl]amino]-2-(hydroxymethyl)benzenesulfonic acid (543.6 mg, 0.933 mmol) in NMP (5 mL) a solution of SOCl ₂ (68.1 μL, 0.933 mmol) in NMP (200 μL) was added at room temperature. The reaction mixture was stirred at room temperature for 15 min, and the progress of the reaction mixture was monitored by UPLC-MS. To achieve complete conversion, SOCl ₂ (68 μL) was added 7 more times. Excess _SOCl2 was evaporated under vacuum and the residue was purified by direct loading of the reaction mixture on an Oasis column using the TFA method to afford the title compound (362 mg, 0.512 mmol) as a white solid. UPLC-MS: MS(ESI): m/z [M+H]+=601.19+603.23 [M+Na]+=622.93

Step 7: 2-[[4-[2-[2-chloro-4-[6-(4-fluorophenyl)-4-[(1R)-2-methoxy-1-[[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]methyl]-2-oxo-ethoxy]thieno[2,3-d]pyrimidin-5-yl]-3-methyl-phenoxy]ethyl]-1-methyl-piperazin-1-yl-1-yl]methyl]-5-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-ureido-pentanoyl]amino]benzenesulfonate

To a solution of 2-(chloromethyl)-5-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-ureidopentanoyl]amino]benzenesulfonic acid (195.6 mg, 0.277 mmol) from step 6 in NMP solution (10 mL) were added sequentially methyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (123 mg, 0.138 mmol) from step 4, DIPEA (385 μL, 2.213 mmol) and TBAI (10 mg, 0.027 mmol). The reaction mixture was stirred at 70°C for 12 h, and the progress of the reaction mixture was monitored by UPLC-MS. The crude product in NMP solution was used directly in the next step. UPLC-MS: MS(ESI): m/z [M+H]+=1231.12+1233.45, [M+2H]2+=616.34+617.37

Step 8: 5-[[(2S)-2-amino-5-ureidopentanoyl]amino]-2-[[4-[2-[4-[4-[(1R)-1-carboxy-2-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]ethoxy]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy]ethyl]-1-methyl-piperazin-1-yl-1-yl]methyl]benzenesulfonate; 2,2,2-trifluoroacetic acid

To the previous solution of 2-[[4-[2-[2-chloro-4-[6-(4-fluorophenyl)-4-[(1R)-2-methoxy-1-[[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]methyl]-2-oxo-ethoxy]thieno[2,3-d]pyrimidin-5-yl]-3-methyl-phenoxy]ethyl]-1-methyl-piperazin-1-yl-1-yl]methyl]-5-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-ureidopentanoyl]amino]benzenesulfonate in NMP, a solution of lithium hydroxide monohydrate (82.2 mg; 1.106 mmol) in water (4 ml) was added. The reaction mixture was stirred at room temperature for 1.5 h, and the progress of the reaction mixture was monitored by UPLC-MS. The crude product solution was purified by directly loading the reaction mixture onto an X-Bridge column using the TFA method to afford the title compound (45.6 mg, 0.0374 mmol) as a white powder. UPLC-MS: MS(ESI): m/z [M+H]+=1217.46, [M+Na]+=1241.16, [M+2H]2+=609.61

Step 9: 2-[[4-[2-[4-[4-[(1R)-1-carboxy-2-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]ethoxy]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy]ethyl]-1-methyl-piperazin-1-yl-1-yl]methyl]-5-[[(2S)-2-[[1-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethylcarbamoyl]cyclobutanecarbonyl]amino]-5-ureidopentanoyl]amino]benzenesulfonate L25-P1

To a solution of 5-[[(2S)-2-amino-5-ureidopentanoyl]amino]-2-[[4-[2-[4-[4-[(1R)-1-carboxy-2-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]ethoxy]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy]ethyl]-1-methyl-piperazin-1-ium-1-yl]methyl]benzenesulfonate (22.6 mg; 0.0186 mmol) in DMF (1.4 ml), a THF solution of (2,3,4,5,6-pentafluorophenyl) 1-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethylcarbamoyl]cyclobutanecarboxylate (from step 3) (26.8 mg, 0.0562 mmol) and DIPEA (12.9 µL, 0.0742 mmol). The reaction mixture was stirred at room temperature for 2 h. The crude product solution was purified by direct loading of the reaction mixture on an X-Bridge column and using the TFA method to afford L25-P1 (7.5 mg, 0.0050 mmol) as a white powder. IR (cm ^-1 ): 3321, 1705/1624, 1666, 1581, 1180/1124, 833/798/756/719/696. ¹ H NMR (400/500 MHz, DMSO-d6) δ ppm. 10.4 (s), 8.88 (d, 1H), 8.61 (s, 1H), 8.13 (df, 1H), 7.92 (dd, 1H), 7.78 (d), 7.74 (t), 7.63 (d, 1H), 7.52 (dd, 1H), 7.47 (d, 1H), 7.46 (t, 1H), 7.38 (d, 1H), 7.3 (dd, 2H), 7.23 (d, 1H), 7.21 (t, 2H), 7.16 (t, 1H), 7.14 (d, 1H), 7.03 (t, 1H), 7.01 (d, 1H), 7 (s, 2H), 6.73 (t, 1H), 6.22 (d, 1H), 5.99 (m), 5.55 (sl), 5.5 (dd, 1H), 5.25 (AB, 2H), 5.1 (sl, 2H), 4.37 (m, 1H), 4.33 (m, 2H), 3.76 (s, 3H), 3.7 (m, 10H), 3.55 (m, 2H), 3.5 (m, 2H), 3.42 (m, 2H), 3.28/2.52 (2dd, 2H), 3.21 (m, 2H), 3.04 (w, 3H), 2.97 (m, 2H), 2.4 (m, 4H), 1.85 (w, 3H), 1.74/1.62 (2m, 2H), 1.73 (m, 2H), 1.43/1.35 (2m, 2H). ¹³ C NMR (400/500 MHz, DMSO-d6) δ ppm. 157.5, 152.8, 135.4, 134.9, 131.5, 131.4, 131.4, 131.2, 131.1, 128.7, 121, 120.6, 119.2, 119.2, 116.3, 116, 112.8, 112.2, 111, 74, 69.5, 68.9, 67.4, 66.6, 56.2, 55.3/46.5, 54.1, 45.7, 39.4, 39.2, 37.2, 32.9, 29.7, 29.7, 27.3, 18, 16. ¹⁹ F NMR (400/500 MHz, DMSO-d6) δ ppm. -74.6, -112.2. HR-IER+: m/z [M+H]+=1509.4867/1509.4851 [measured/theoretical]

Obtaining L26-P1:

(2R)-2-[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]propyl]phenyl]methyl]-4-methyl-piperazin-4-yl-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate; 2,2,2-trifluoroacetate

Stage 1: 3-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prop-1-yne

To a solution of 2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol (1.95 g, 6.50 mmol) in THF (25.0 mL) was added sodium hydride (260.0 mg, 6.57 mmol) at 0°C. After 5 min, a solution of 3-bromoprop-1-yne in toluene (1.42 mL, 13.14 mmol) was added. The reaction mixture was stirred at 0°C for 1 h and 2 days at room temperature, and the progress of the reaction mixture was monitored by UPLC-MS. The reaction mixture was then filtered and the filtrate was concentrated to dryness and purified by silica gel chromatography (DCM/methanol gradient) to afford the title compound (1.74 g, 4.12 mmol) as a colorless oil. ¹ H NMR (CDCl ₃ ): 2.43 (t, 1H, J = 2.4 Hz), 3.37 (s, 3H), 3.53-3.55 (m, 2H), 3.64-3.70 (m, 30H), 4.20 (d, 2H, J = 2.4 Hz).

Step 2: 9H-fluoren-9-ylmethyl N-[(1S)-1-[[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodo-phenyl]carbamoyl]-4-ureido-butyl]carbamate

To a solution of [[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodoaniline (10.0 g, 27.52 mmol) in methanol (70 mL) and DCM (140 mL), Fmoc-Cit-OH (12.0 g, 30.28 mmol) and EEDQ (8.17 g, 33.03 mmol) were added successively. The reaction mixture was stirred for 14 h at room temperature. After completion of the reaction, the resulting residue was purified by column chromatography on silica gel using DCM/methanol (100/0-88/12) as eluent to give the title compound (17.09 g, 21.97 mmol) as a white solid. ^1H NMR (DMSO): δ 0.09 (s, 6H), 0.91 (s, 9H), 1.38-1.48 (m, 2H), 1.59-1.68 (m, 2H), 2.93-3.05 (m, 2H), 4.06-4.15 (m, 2H), 4.20-4.29 (m, 3H), 4.56 (s, 2H), 5.41 (s, 2H), 5.98 (t, 1H, J =5.5 Hz), 7.30-7.43 (m, 5H), 7.55 (dd, 1H, J =8.8, 2.1 Hz), 7.69 (d, 1H, J =7.8 Hz), 7.74 (dd, 1H, J =7.2, 3.4 Hz), 7.89 (d, 1H, J =7.5 Hz), 8.25 (d, 1H, J =1.5 Hz), 10.12 (s, 1H).

Step 3: (2S)-2-amino-N-[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodophenyl]-5-ureidopentanamide

To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodophenyl]carbamoyl]-4-ureidobutyl]carbamate (17.08 g, 23.00 mmol) in THF (120 mL) was added 2M dimethylamine in THF (44.5 mL, 89.00 mmol). The reaction mixture was stirred for 15 h at room temperature. After concentration to dryness, the resulting residue was purified by column chromatography on C ₁₈ using water/acetonitrile (98/02-0/100) as eluent to give the title compound (5.47 g, 10.50 mmol) as a white solid. ^1H NMR (DMSO): δ 0.0 (s, 6H), 0.81 (s, 9H), 1.27-1.38 (m, 3H), 1.47-1.53 (m, 1H), 2.83-2.89 (m, 2H), 3.16-3.19 (m, 1H), 4.46 (s, 2H), 5.26 (s, 2H), 5.82 (t, 1H, J =5.6 Hz), 7.24 (d, 1H, J =8.5 Hz), 7.50 (dd, 1H, J =8.3, 2.0 Hz), 8.17 (d, 1H, J =2.0 Hz).

Step 4: 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-1-[[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodo-phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]carbamate

To a solution of (2S)-2-amino-N-[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodophenyl]-5-ureidopentanamide (3.00 g, 5.76 mmol) in 2-methyltetrahydrofuran (240 mL), Fmoc-Val-Osu (8.65 g, 8.65 mmol) and DIPEA (1.90 mL, 11.53 mmol) were added successively. The reaction mixture was stirred for 15 h at room temperature. The reaction mixture was filtered through a sintered funnel, and the recovered solid was washed with 2-methyltetrahydrofuran (2×250 mL), then dried under high vacuum to give the title compound (3.57 g, 4.24 mmol) as a white solid. ^1H NMR (DMSO): δ 0.10 (s, 6H), 0.83-0.95 (m, 15H), 1.27-1.52 (m, 2H), 1.52-1.75 (m, 2H), 1.93-2.07 (m, 1H), 2.88-3.09 (m, 2H), 3.93 (t, 1H, J =8.0 Hz), 4.17-4.49 (m, 4H), 4.56 (s, 2H), 5.40 (s, 2H), 5.96 (t, 1H, J =5.6 Hz), 7.27-7.37 (m, 3H), 7.37-7.48 (m, 3H), 7.54 (d, 1H, J =8.0 Hz), 7.74 (t, 2H, J =7.2 Hz), 7.88 (d, 2H, J =7.6 Hz), 8.13 (d, 1H, J =7.6 Hz), 8.22 (s, 1H), 10.11 (s, 1H).

Step 5: 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-1-[[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-[3-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prop-1-ynyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]carbamate

To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-1-[[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodophenyl]carbamoyl]-4-ureidobutyl]carbamoyl]-2-methylpropyl]carbamate (1.23 g; 1.46 mmol) in dimethylformamide (15.40 ml), 3-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prop-1-yne (930.0 mg; 2.20 mmol) and DIPEA (2.47 ml; 14.92 mmol) were added successively. After 3 argon flushes, Pd( _PPh3 ) _2Cl2 ( ₂₂₀ mg, 0.307 mmol) and CuI (68.0 mg, 0.36 mmol) were added and the reaction mixture was flushed with argon 3 times. The reaction mixture was stirred for 3 h at room temperature and the progress of the reaction mixture was monitored by UPLC-MS. The mixture was diluted with isopropyl acetate (200 mL) and washed with brine (3×150 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated to dryness. The crude product was purified by C18 reverse-phase prep-HPLC by direct loading of the reaction mixture onto an X-Bridge column using the neutral method to afford the title compound (790.0 mg, 0.70 mmol) as a pale yellow gum. ^1H NMR (DMSO): δ 0.08 (s, 6H), 0.85-0.90 (m, 15H), 1.36-1.45 (m, 2H), 1.58-1.71 (m, 2H), 1.97-2.00 (m, 1H), 2.93-3.03 (m, 2H), 3.23 (s, 3H), 3.40-3.43 (m, 2H), 3.49-3.52 (m, 25H), 3.56-3.58 (m, 2H), 3.63-3.66 (m, 2H), 3.93 (dd, 1H, J = 8.9, 6.9 Hz), 4.23-4.32 (m, 3H), 4.37-4.43 (m, 3H), 4.75 (s, 2H), 5.39 (s, 2H), 5.97 (t, 1H, J =6.1 Hz), 7.30-7.43 (m, 6H), 7.51-7.54 (m, 1H), 7.72-7.78 (m, 3H), 7.88 (d, 2H J =7.5 Hz), 8.12 (d, 2H, J =7.4 Hz), 10.10 (s, 1H).

Step 6: 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-1-[[4-(hydroxymethyl)-3-[3-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prop-1-ynyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]carbamate

To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-1-[[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-[3-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prop-1-ynyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]carbamate (452 mg; 0.40 mmol) in tetrahydrofuran (0.60 mL) and water (0.90 mL), acetic acid (4.17 mL; 72.78 mmol) was added. The reaction mixture was stirred for 22 h at room temperature, and the progress of the reaction mixture was monitored by UPLC-MS. After concentration to dryness, the crude product was purified by C18 prep-reversed-phase HPLC by direct loading of the reaction mixture onto an X-Bridge column using the neutral method to afford the title compound (327 mg, 0.32 mmol) as a white gum. ¹ H NMR (DMSO): δ 0.87 (dd, 6H, J = 11.7, 6.8 Hz), 1.36-1.45 (m, 2H), 1.58-1.71 (m, 2H), 1.97-2.00 (m, 1H), 2.93-3.02 (m, 2H), 3.23 (s, 3H), 3.31 (s, 5H), 3.40-3.43 (m, 2H), 3.48-3.53 (m, 21H), 3.54-3.64 (m, 6H), 3.91-3.95 (m, 1H), 4.23-4.42 (m, 4H), 4.56 (d, 2H, J =5.5 Hz), 5.19 (t, 1H, J =5.6 Hz), 5.39 (s, 2H), 5.96 (t, 1H, J =5.8 Hz), 7.30-7.34 (m, 2H), 7.39-7.43 (m, 4H), 7.50-7.52 (m, 1H), 7.72-7.76 (s, 3H), 7.88 (d, 1H J =7.5 Hz), 8.12 (d, 2H, J =7.4 Hz), 10.06 (s, 1H).

Step 7: 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-1-[[4-(hydroxymethyl)-3-[3-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]carbamate

To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-1-[[4-(hydroxymethyl)-3-[3-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prop-1-ynyl]phenyl]carbamoyl]-4-ureidobutyl]carbamoyl]-2-methylpropyl]carbamate (327.0 mg; 0.32 mmol) in THF (3.7 ml), acetic acid (0.37 ml) was added. After 3 argon purges, Pt/C 5% (195 mg) was added, and after 3 more argon purges, the reaction mixture was placed under a hydrogen atmosphere and stirred for 18 h at room temperature, and the reaction progress was monitored by UPLC-MS. The mixture was filtered through PTFE, and the filtrate was concentrated to dryness, then triturated with dichloromethane/pentane (1/4 mixture, 50 mL). The precipitate was isolated by filtration to yield, after drying, the title compound (130 mg; 0.13 mmol) as a white solid. ^1H NMR (DMSO): δ 0.85-0.89 (m, 6H), 1.23-1.46 (m, 2H), 1.56-1.76 (m, 4H), 1.97-2.02 (m, 1H), 2.56-2.60 (m, 2H), 2.91-3.04 (m, 2H), 3.23 (s, 3H), 3.38-3.43 (m, 4H), 3.48-3.54 (m, 30H), 3.93 (dd, 1H, J =8.9, 6.9 Hz), 4.21-4.31 (m, 3H), 4.38-4.41 (m, 1H), 4.45 (d, 2H, J =5.3 Hz), 4.94 (t, 1H, J =5.3 Hz), 5.37 (s, 2H), 5.95 (t, 1H, J =5.8 Hz), 7.25 (d, 1H, J =8.3 Hz), 7.30-7.34 (m, 2H), 7.39-7.43 (s, 5H), 7.72-7.76 (m, 2H), 7.88 (d, 1H J =7.5 Hz), 8.06 (d, 2H, J =7.6 Hz), 9.88 (s, 1H). UPLC-MS: MS (ESI) m/z [M+H]+=1026.52

Step 8: 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-1-[[4-(bromomethyl)-3-[3-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propyl]phenyl]carbamoyl]-4-ureidobutyl]carbamoyl]-2-methylpropyl]carbamate

To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-1-[[4-(hydroxymethyl)-3-[3-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propyl]phenyl]carbamoyl]-4-ureidobutyl]carbamoyl]-2-methylpropyl]carbamate (60 mg; 0.0584 mmol) in THF (6.6 ml), PBr ₃ (1 M solution in THF) (0.0877 ml; 0.0877 mmol) was added dropwise at 0°C. The solution was then stirred for 3 hours at room temperature. The reaction progress was monitored by UPLC-MS after the addition of an aliquot of morpholine to react with the expected bromine compound. The reaction mixture was treated with an aqueous saturated solution of NH ₄ Cl (50 µL). After 5 min, the mixture was dried over MgSO ₄ , filtered, and washed with THF (2 mL) to obtain the title bromine compound as a THF solution, which was used crude in the next step. UPLC-MS analysis was performed after the addition of methanol and morpholine.

Step 9: (2R)-2-[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]-2-[3-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]propyl]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid;2,2,2-trifluoroacetate

To a THF solution of 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-1-[[4-(bromomethyl)-3-[3-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]carbamate from the previous step (0.0584 mmol) was successively added DMF (1.5 ml), (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid P1 (46.1 mg, 0.0527 mmol) and DIPEA (0.173 mL, 0.995 mmol). The reaction mixture was stirred for 20 h at room temperature and the progress of the reaction mixture was monitored by UPLC-MS. The crude mixture containing the expected title compound and the deprotected Fmoc compound (expected in step 10) was used in the next deprotection step. UPLC-MS: MS (ESI) m/z [M-Fmoc+H+H]+=1660.99

Step 10: (2R)-2-[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propyl]phenyl]methyl]-4-methyl-piperazin-4-yl-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid

Piperidine (11.6 μL, 0.117 mmol) was added to the crude mixture from the previous step in DMF. The reaction mixture was stirred at room temperature for 15 h, and the progress of the reaction mixture was monitored by UPLC-MS. After completion of the reaction, the crude product was purified by C18 prep-reversed-phase HPLC by directly loading the reaction mixture onto an X-Bridge column using the TFA method to afford the title compound (29.2 mg, 0.0155 mmol) as a white powder. IR: 3600-2300, 1672, 1602, 1541+1516. HR-ESI+: m/z [M-CF ₃ COO]+ = 1660.7574 (1660.7575 theoretical)

Step 11: (2R)-2-[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methylbutanoyl]amino]-5-ureidopentanoyl]amino]-2-[3-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]propyl]phenyl]methyl]-4-methyl-piperazin-4-yl-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate;2,2,2-trifluoroacetate L26-P1

To a solution of (2R)-2-[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propyl]phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid (42.5 mg; 0.0225 mmol) in DMF (1.28 mL) was sequentially added with a solution of (2,5-dioxopyrrolidin-1-yl) 3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoate (Brodpharm 21854) (7.71 mg; 0.0247 mmol) and DIPEA (19.6 μL; 0.112 mmol). The reaction mixture was stirred at room temperature for 15 h, and the progress of the reaction mixture was monitored by UPLC-MS. The crude product was purified by C18 prep-reversed-phase HPLC by direct loading of the reaction mixture onto an X-Bridge column using the TFA method to afford L26-P1 (28 mg; 0.0151 mmol) as a white powder. HR-ESI+: m/z [M-CF ₃ COO]+=1855.8105 (1855.8106 theoretical)

Obtaining L27-P1:

(2R)-2-[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-3-methyl-5-ureido-pentanoyl]amino]-2-sulfo-phenyl]methyl]-4-methyl-piperazin-4-yl-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

Step 1: 2-(chloromethyl)-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]benzenesulfonate

5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]-2-(hydroxymethyl)benzenesulfonic acid (300 mg; 0.4263 mmol) was dissolved in anhydrous NMP (6 ml) at room temperature. In parallel, a solution of SOCl ₂ (206 μl) in NMP (6 ml) was prepared. A solution of 900 μl of SOCl ₂ was added to the reaction mixture 6 times over 75 min. After the last addition, the reaction mixture was stirred at room temperature for 15 min. The crude product was purified by direct loading of the reaction mixture onto an Oasis column using the TFA method to afford the title compound (138 mg, 0.1971 mmol) as a white powder. ¹ H NMR (400 MHz, DMSO-d6) δ ppm. 10.15+8.1+7.42+6.0 (s+2d+m, 4H), 7.9 (m, HH), 7.75 (m, 3H), 7.42+7.31 (2m, 5H), 5.23 (s, 2H), 4.4 (m, 1H), 4.3-4.2 (m, 3H), 3.95 (dd, 1H), 3.0 (m, 2H), 2.0 (m, 1H), 1.7+1.6 (2m, 2H), 1.48+1.37 (2m, 2H), 0.88 (2d, 6H). HR-ESI+: m/z [M+H]+=700.2199/700.2202 [measured/theoretical]

Step 2: 5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]-2-[[4-[2-[2-chloro-4-[6-(4-fluorophenyl)-4-[(1R)-2-methoxy-1-[[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]methyl]-2-oxo-ethoxy]thieno[2,3-d]pyrimidin-5-yl]-3-methyl-phenoxy]ethyl]-1-methyl-piperazin-1-yl-1-yl]methyl]benzenesulfonic acid

To a solution of 2-(chloromethyl)-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]benzenesulfonate (82.4 mg; 0.1177 mmol) in anhydrous NMP (2.5 ml), DIEA (94 μl; 0.540 mmol) was added at room temperature, then methyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (60 mg; 0.067 mmol) and TBAI (12.4 mg; 0.034 mmol). The reaction mixture was stirred at 80°C for 4 h and overnight at room temperature. Then, 2-(chloromethyl)-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]benzenesulfonate (14 mg; 0.017 mmol) was added again, followed by TBAI (17 μL; 0.0337 mmol) and the reaction mixture was stirred at 80°C for 4 h and then overnight at room temperature. The Fmoc deprotection step was performed by adding DEA (53 μL; 0.515 mmol) to the reaction mixture and stirring at room temperature overnight. Purification was accomplished by direct injection of the mixture onto an Oasis column _eluted with a gradient of solution A: _H2O / _CH3CN / _NH4HCO3 (1960 mL/400 mL/3.16 g) to solution B: _CH3CN / _H2O / _NH4HCO3 ( ₁₆₀₀ mL/400 mL/3.16 g) to afford the title compound (17 mg; 0.009 mmol). UPLC-MS: MS (ESI) m/z [M]+=1329

Step 3: (2R)-2-[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]-2-sulfo-phenyl]methyl]-4-methyl-piperazin-4-yl-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To a mixture of 5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]-2-[[4-[2-[2-chloro-4-[6-(4-fluorophenyl)-4-[(1R)-2-methoxy-1-[[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]methyl]-2-oxo-ethoxy]thieno[2,3-d]pyrimidin-5-yl]-3-methyl-phenoxy]ethyl]-1-methyl-piperazin-1-ium-1-yl]methyl]benzenesulfonic acid (18 mg; 0.014 mmol) in dioxane/water (1 ml/1 ml) was added LiOH ^. H ₂ O (2.3 mg; 0.054 mmol) was added and the reaction mixture was stirred at room temperature for 4 h. The solution was adjusted to pH 6-7 with 1N HCl and the dioxane was evaporated under reduced pressure. Purification was accomplished by direct injection of the mixture onto an Oasis column eluted with a gradient of solution A: H ₂ O/CH ₃ CN/NH ₄ HCO ₃ (1960 mL/40 mL/3.16 g) to solution B: CH ₃ CN/H ₂ O/NH ₄ HCO ₃ (1600 mL/400 mL/3.16 g) to afford the title compound (11 mg; 0.008 mmol).

Step 4: (2R)-2-[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-3-methyl-5-ureido-pentanoyl]amino]-2-sulfo-phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid L27-P1

To a solution of (2R)-2-[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]-2-sulfo-phenyl]methyl]-4-methyl-piperazin-4-yl-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (10.5 mg; 0.007 mmol) in DMF (0.4 ml) was added (2,5-dioxopyrrolidin-1-yl) 3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoate (5.7 mg; 0.018 mmol) was added and the solution was stirred at room temperature for 4 h. The solution was purified on an X-Bridge C ₁₈ column by direct loading of the reaction mixture onto the column and using the TFA method to afford L27-P1 (10 mg; 0.006 mmol). HR-ESI+: [M+H]+ 1511.5018/1511.5002 [measured/theoretical]

Obtaining L28-P1:

(2R)-2-[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]-2-sulfo-phenyl]methyl]-4-methyl-piperazin-4-yl-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate

Step 1: 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-2-[4-(chloromethyl)-3-methyl-anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate

To a solution of 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]-2-(hydroxymethyl)benzenesulfonate (504.1 mg, 0.816 mmol) in NMP (5 mL), a solution of _SOCl2 (60 μL, 0.816 mmol) in NMP (500 μL) was added 6 times over 75 min. The reaction mixture was stirred at room temperature for 15 min. The crude product was purified by direct loading of the reaction mixture on an Oasis column using the TFA method to afford (337 mg) as a mixture of 70% of the title compound (384 mmol) and 30% of the starting material (170 mmol) as a white powder. IR (cm ^-1 ): 3600-2400, 1688+1648, 1599, 1518, 1022. UPLC-MS: MS (ESI) m/z [M+H]+=614.17+616.18 (Cl)

Step 2: (2R)-2-[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino]-2-methyl-phenyl]methyl]-4-methyl-piperazin-4-yl-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate

To a solution of methyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (152 mg; 0.171 mmol) in NMP (4.5 ml), 9H-fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-2-[4-(chloromethyl)-3-methyl-anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate (150 mg; 0.171 mmol), DIPEA (238 μl; 1.37 mmol) and TBAI (76 mg; 0.205 mmol). The reaction mixture was stirred at 80°C for 28 h. The reaction mixture was cooled to room temperature. Then a solution of LiOH.H ₂ O (13.7 mg, 0.342 mmol) in water (500 μL) was added. The reaction mixture was stirred at room temperature for 48 h. The crude product was purified by reversed-phase C18 prep-HPLC by direct loading of the reaction mixture onto an X-Bridge column and using the TFA method to afford the title compound (40 mg; 0.0325 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M]+=1230.61+1232.61 (Cl)

Step 3: (2R)-2-[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]-2-sulfo-phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate L28-P1

To a solution of (2R)-2-[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino]-2-methyl-phenyl]methyl]-4-methyl-piperazin-4-ium-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate (6.0 mg, 0.0049 mmol) in DMF solution (180 μL) was sequentially added with (2,5-dioxopyrrolidin-1-yl) 3-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethylcarbamoyl]oxetane-3-carboxylate (2.3 mg, 0.0073 mmol) and DIPEA (3.0 μL, 0.017 mmol). The reaction mixture was stirred at room temperature for 1.5 h and monitored by UPLC-MS. The crude product was purified by direct loading of the reaction mixture onto an X-Bridge column using the TFA method to afford L28-P1 (2.9 mg, 0.0020 mmol) as a white powder. HR-IER+: m/z [M+H]+=1425.4534/ 1425.4527 [measured/theoretical]

Obtaining L29-C3:

(2R)-2-[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-(2-azidoethoxy)acetyl]amino]-3-methyl-butanoyl]amino]propanoyl]amino]-2-sulfo-phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

Step 1: Sodium; 5-[[(2S)-2-(tert-butoxycarbonylamino)propanoyl]amino]-2-(hydroxymethyl)benzenesulfonate

To a solution of Boc-L-Ala-OH (588 mg, 3.11 mmol) in DMF (38.6 ml), GATU (1.77 g, 4.67 mmol), sodium 5-amino-2-(hydroxymethyl)benzenesulfonate (771 mg, 3.42 mmol) and DIPEA (1.29 ml, 7.78 mmol) were added successively. The reaction mixture was stirred for 16 h at room temperature, then concentrated to dryness and co-evaporated with water to give the crude reaction mixture. The resulting residue was purified by column chromatography on silica gel using ethyl acetate/methanol 95:5-80:20 as eluent to give the title compound (1.17 g, 2.95 mmol) as a white solid. ¹ H NMR (DMSO): δ 1.24 (s, 9H), 1.38 (m, 3H), 4.05-1.44 (m, 1H), 4.73 (d, 2H, J =4.8 Hz), 5.04 (t, 1H, J =5.6 Hz); 6.97-7.02 (m, 1H), 7.33 (d, 1H, J =8 Hz), 7.65-7.70 (m, 1H), 7.83 (s, 1H), 9.91 (s, 1H).

Step 2: 5-[[(2S)-2-aminopropanoyl]amino]-2-(hydroxymethyl)benzenesulfonate, hydrochloride

Sodium 5-[[(2S)-2-(tert-butoxycarbonylamino)propanoyl]amino]-2-(hydroxymethyl)benzenesulfonate (1.17 g, 2.95 mmol, 1 eq) was suspended in a solution of 4N HCl in dioxane (10 mL). The mixture was stirred at room temperature for 2 h and then concentrated to dryness to give the crude mixture (982 mg, 2.95 mmol) as a white solid. ^1H NMR (DMSO): δ 1.45 (d, 3H, J =5.6 Hz), 3.91-4.0 (m, 1H), 4.76 (s, 2H), 7.41 (d, 1H, J =7.6 Hz), 7.66 (d, 1H, J =7.6 Hz), 7.85 (s, 1H), 8.17 (s, 2H), 10.44 (s, 1H).

Step 3: 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]-2-(hydroxymethyl)benzenesulfonate

To a solution of 5-[[(2S)-2-aminopropanoyl]amino]-2-(hydroxymethyl)benzenesulfonate hydrochloride (981 mg, 2.95 mmol) in DMF (34.5 mL) were added Fmoc-L-Val-Osu (1.29 g, 2.95 mmol, 1 eq.) and DIPEA (975 µL, 5.9 mmol). The mixture was stirred overnight at room temperature, then concentrated to dryness and co-evaporated with water to give a crude mixture. The resulting residue was purified by column chromatography on silica gel using ethyl acetate/methanol 95:5-80:20 as eluent to give the title compound (1.28 g, 2.072 mmol) as a colorless oil. ^1H NMR (DMSO): δ 0.80-0.92 (m, 6H), 1.30 (d, 3H, J =6.4 Hz), 2.02-2.10 (m, 1H), 4.17-4.31 (m, 3H), 4.37-4.44 (m, 1H), 4.73 (d, 2H, J =5.6 Hz), 5.04 (t, 1H, J =6.4 Hz), 7.28-7.36 (m, 3H), 7.37-7.47 (m, 3H), 7.66 (d, 1H, J =8.4 Hz), 7.71-7.77 (m, 2H), 7.83-7.85 (m, 1H), 7.88 (d, 2H, J =7.6 Hz), 8.14 (d, 1H, J =6.4 Hz), 9.99 (s, 1H).

Step 4: 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]benzenesulfonic acid

To a suspension of 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]-2-(hydroxymethyl)benzenesulfonate (1.28 g, 2.07 mmol) in THF (65 mL) was added pyridine (875 µL, 10.8 mmol) followed by 4-nitrophenyl chloroformate (1.09 g, 5.41 mmol). The mixture was stirred overnight at room temperature. Additional 4-nitrophenyl chloroformate (1.09 g, 5.41 mmol, 2.5 eq) was then added. After 5 hours of stirring at room temperature, the mixture was concentrated to dryness, then purified by column chromatography on C ₁₈ using water/acetonitrile 90/10-0/100 as eluent for 30 minutes. Acetonitrile was removed from the combined tubes, and the residue was lyophilized to afford the title compound (650 mg; 0.83 mmol) as a white solid. ^1H NMR (DMSO): δ 0.88 (m, 6H), 1.31 (d, 3H, J =4.8 Hz), 1.97-2.03 (m, 1H), 3.92 (t, 1H, J =6.8 Hz), 4.23 (s, 2H), 4.24-4.34 (m, 1H), 4.42 (t, 1H J =5.6 Hz), 5.69 (s, 2H), 7.30-7.48 (m, 6H), 7.62 (d, 2H, J =8 Hz), 7.72-7.76 (m, 3H), 7.89 (d, 2H, J =6.4 Hz), 7.94 (s, 1H), 8.18 (d, 1H, J= 5.6 Hz), 8.33 (d, 2H, J =7.6 Hz), 10.11 (s, 1H). ¹³ C NMR (DMSO): δ 18.01, 18.26, 19.21, 30.4, 46.66, 49.05, 59.91, 65.67, 67.82, 117.7, 119.1, 120.06, 122.66, 125.37, 126.33, 127.05, 127.62, 128.0, 138.06, 140.67, 143.77, 143.86, 145.1, 146.23, 151.96, 155.47, 156.12, 171.0, 171.15. LCMS (2-100 ACN/ _H2O +0.05% TFA): 90.41% Tr=12.7 min. Positive mode 578.41 determined. Negative mode 759.17 determined.

Step 5: (2S)-2-[[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]-2-sulfo-phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]methyl]-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To a suspension of (2S)-2-[[5-[3-chloro-2-methyl-4-(2-piperazin-1-ylethoxy)phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]methyl]-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetic acid (178.4 mg; 0.183 mmol) in DMF (1.5 ml), 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]benzenesulfonic acid (150 mg; 0.192 mmol) and DIPEA (91 μl; 0.549 mmol) were added successively. The mixture was stirred overnight at room temperature for 15 minutes. The crude product was purified by direct loading of the reaction mixture onto an X-Bridge column using the NH ₄ HCO ₃ method to afford the title compound (176 mg, 0.118 mmol) as a white solid. UPLC-MS: MS (ESI) m/z [M+H]+=1482.15+1484.56(Cl)

Step 6: (2S)-2-[[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino]-2-sulfo-phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]methyl]-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To a solution of (2S)-2-[[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]-2-sulfo-phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]methyl]-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (176 mg; 0.118 mmol) in DMF (1.0 mL), piperidine (24.17 μL; 0.237 mmol) was added. The reaction mixture was stirred at room temperature for 18 h. The crude product was purified by direct loading of the reaction mixture onto an X-Bridge column using the NH ₄ HCO ₃ method to afford the title compound (102 mg, 0.0809 mmol) as a white powder. IR (cm ^-1 ): 3620-2680, 1683, 1235. UPLC-MS: MS (ESI) m/z [M+H]+=1260.37+1262.37(Cl). ¹ H NMR (400 MHz, DMSO-d6) δ ppm. 10.20 (m, NH), 8.90 (m, 2H), 8.90 (m, 1H), 8.60 (m, NH), 8.60 (s, 1H), 7.90 (m, 1H), 7.78 (m, 1H), 7.70 (d, 1H), 7.55 (d, 1H), 7.48 (t, 1H), 7.45 (d, 1H), 7.3/7.2 (m, 4H), 7.20 (m, 1H), 7.20 (d, 1H), 7.15 (t, 1H), 7.15 (d, 1H), 7.05 (t, 1H), 7.00 (d, 1H), 6.7 (t, 1H), 6.2 (d, 1H), 5.48 (s, 2H), 5.50 (m, 1H), 5.23 (s, 2H), 4.50 (m, 1H), 4.25 (m, 2H), 3.75 (s, 3H), 3.4 (m, 4H), 3.40 (m, 1H), 3.35 (m, 1H) 2.80 (m, 2H), 2.5 (m, 4H), 2.50 (m, 1H), 2.05 (m, 1H), 1.80 (s, 3H), 1.30 (d, 3H), 0.90 (dd, 6H)

Step 7: (2,3,4,5,6-pentafluorophenyl) 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetate

To a solution of 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetic acid (74 mg; 0.317 mmol) in THF (500 μl) were added sequentially 2,3,4,5,6-pentafluorophenol (70 mg; 0.380 mmol) in THF solution (500 μl) and DCC (78.5 mg; 0.380 mmol) in THF solution (500 μl). The reaction mixture was stirred at room temperature for 18 h. The crude suspension was filtered through a cotton pad in a Pasteur pipette, and the solution was used without further processing in step 8.

Step 8: (2R)-2-[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-(2-azidoethoxy)acetyl]amino]-3-methyl-butanoyl]amino]propanoyl]amino]-2-sulfo-phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid L29-C3

To a solution of (2S)-2-[[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino]-2-sulfo-phenyl]methoxycarbonyl]piperazin-1-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]methyl]-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (100 mg; 0.0793 mmol) in DMF (500 μl) was successively added a solution of (2,3,4,5,6-pentafluorophenyl) 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetate in THF from step 7 (0.317 mmol) and DIPEA (39.3 µL, 0.238 mmol) were added. The reaction mixture was stirred at room temperature for 15 min. The crude product was purified by direct loading of the reaction mixture onto an X-Bridge column using the NH ₄ HCO ₃ method to afford L29-C3 (43 mg, 0.0291 mmol) as a white powder. IR (cm ^-1 ): 3257, 2102, 1663, 1236/1082, 834/756. ¹ H NMR (400 MHz, DMSO-d6) δ ppm. 10.03 (s, NH), 8.87 (d, 1H), 8.59 (sNC, 1H), 8.35 (d, NH), 7.89 (dph, 1H), 7.69 (dd, 1H), 7.66 (m, 1H), 7.53 (d, 1H), 7.45 (t, 1H), 7.36 (d, 1H), 7.29 (dd, 2H), 7.21 (d, 1H), 7.20 (t, 2H), 7.19 (d, 1H), 7.14 (d, 1H), 7.13 (m, 1H), 7.09 (m, NH), 7.03 (t, 1H), 7.00 (d, 1H), 6.72 (t, 1H), 6.24 (d, 1H), 5.48 (dNC, 1H), 5.44 (s, 2H), 5.23(AB, 2H), 4.40 (t, 1H), 4.30 (dd, 1H), 4.24 (m, 2H), 3.94 (s, 2H), 3.75 (s, 3H), 3.58 (m, 10H), 3.38 (m, 4H), 3.36 (t, 2H), 3.30 (NC, 1H), 2.75 (t, 2H), 2.50 (m, 4H), 2.50 (m, 1H), 2.00 (m, 1H), 1.51 (s, 3H), 1.30 (d, 3H), 0.88/0.82 (2d, 6H). ¹³ C NMR (100/126 MHz, DMSO-d6) δ ppm. 158.2, 131.6, 131.1, 130.9, 130.9, 130.9, 130.9, 128.3, 126.7, 120.7, 120.4, 119.3, 117.9, 116.2, 112.5, 112.1, 110.8, 70.2, 70.2; 69.6, 67.7, 64.0, 56.7, 56.5, 55.8, 53.2, 50.4, 49.2, 43.8, 31.7, 19.7, 18.7, 18.4, 17.8. ¹⁹ F NMR (376/470 MHz, DMSO-d6) δ ppm -112.0. HR-ESI+: m/z [M+H]+=1475.4643/1475.4638; [2M+H+Ca]3+=996.6289/996.6273; [M+2H]2+=738.2378/738.2355; [M+H+Na]2+=749.2255/749.2265.

Example 2. Synthesis and characterization of additional linkers, payload linkers, and their precursors.

Generic linkers, payload linkers, and their precursors are synthesized using the generic methods described in this example.

Synthesis of 2-(bromomethyl)-4-nitrobenzoic acid

To a stirred solution of 2-methyl-4-nitrobenzoic acid (300 g, 1.5371 mol) in CCl ₄ (3000 mL) were added NBS (300.93 g, 1.6908 mol) and AIBN (37.86 g, 0.2305 mol) at room temperature. The reaction mixture was stirred at 80°C for 16 h. The reaction mixture was monitored by TLC analysis. The reaction mixture was diluted with saturated NaHCO ₃ solution (2 L) and extracted with ethyl acetate (2×2 L). The combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel using 2-3% ethyl acetate in petroleum ether as eluent to afford 2-(bromomethyl)-4-nitrobenzoic acid. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.35 (d, J=2.0 Hz, 1H), 8.20 (q, J=8.8, 2.4 Hz, 1H), 8.12 (d, J=8.8 Hz, 1H), 4.97 (s, 2H), 4.00 (s, 3H).

Synthesis of 4-nitro-2-((prop-2-yn-1-yloxy)methyl)benzoic acid

To a mixture of 2-(bromomethyl)-4-nitrobenzoic acid (250 g, 0.9122 mol) in ACN (5000 mL) were added prop-2-yn-1-ol (255.68 g, 265.50 mL, 4.5609 mol, d = 0.963 g/mL) and Cs ₂ CO ₃ (743.03 g, 2.2805 mol) at room temperature. The resulting mixture was heated to 80 °C for 16 h. The reaction mixture was filtered through a pad of celite, washed with ethyl acetate (2 L). The filtrate was concentrated under reduced pressure. The resulting crude compound was added to a saturated NaHCO ₃ solution (1 L) and the aqueous layer was acidified to pH 2 using 2N HCl (2 L). After filtration and vacuum drying, 4-nitro-2-((prop-2-yn-1-yloxy)methyl)benzoic acid is obtained. ¹ H NMR (400 MHz, DMSO): δ 13.61 (br s, 1H), 8.37 (d, J=2.4 Hz, 1H), 8.23 (dd, J=2.4, 8.4 Hz, 1H), 8.10 (d, J=8.8 Hz, 1H), 4.95 (s, 2H), 4.37 (d, J=2.4 Hz, 2H), 3.52 (t, J=2.4 Hz, 1H)

Synthesis of methyl 4-nitro-2-((prop-2-yn-1-yloxy)methyl)benzoate

To a stirred solution of 4-nitro-2-((prop-2-yn-1-yloxy)methyl)benzoic acid (130 g, 0.5527 mol) in MeOH (1300 mL) was slowly added SOCl ₂ (526.08 g, 320.78 mL, 4.4219 mol, d = 1.64 g/mL) at 0 °C. The reaction was stirred at 70 °C for 4 h. The reaction solvent was evaporated under reduced pressure. The resulting residue was dissolved in ethyl acetate (1000 mL) and washed with sat. NaHCO ₃ (600 mL), water (500 mL), and brine (500 mL). The separated organic layer was dried over sodium sulfate, filtered and evaporated under reduced pressure to give methyl 4-nitro-2-((prop-2-yn-1-yloxy)methyl)benzoate. ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.56 (t, J=0.8 Hz, 1H), 8.18-8.09 (m, 2H), 5.03 (s, 2H), 4.35 (d, J=2.4 Hz, 2H), 3.96 (s, 3H), 2.49 (t, J=2.4 Hz, 1H).

Synthesis of methyl 4-amino-2-((prop-2-yn-1-yloxy)methyl)benzoate

To a solution of methyl 4-nitro-2-((prop-2-yn-1-yloxy)methyl)benzoate (110 g, 0.4414 mol) in a mixture of EtOH (1100 mL) and H ₂ O (550 mL) were added Fe powder (197.21 g, 3.5310 mol) and NH ₄ Cl (188.88 g, 3.5310 mol) at room temperature. The resulting mixture was heated at 80°C for 16 h. The reaction mixture was cooled to room temperature and filtered through celite® and washed with ethyl acetate (2 L). The filtrate was concentrated under reduced pressure to half the volume. Ethyl acetate (1.5 L) was added to the residue and the two layers were separated, and the aqueous layer was extracted with ethyl acetate (2 L). The combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield the crude product. Purification of _SiO2 by column chromatography (15-20% ethyl acetate in petroleum ether) afforded methyl 4-amino-2-((prop-2-yn-1-yloxy)methyl)benzoate. ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.67 (d, J=8.8 Hz, 1H), 6.78 (t, J=1.6 Hz, 1H), 6.48 (q, J=8.4, 2.4 Hz, 1H), 4.79 (s, 2H), 4.25 (d, J=2.4 Hz, 2H), 3.70 (d, J=4.0 Hz, 3H), 3.42 (t, J=2.4 Hz, 1H).

Synthesis of (4-amino-2-((prop-2-yn-1-yloxy)methyl)phenyl)methanol

To a stirred solution of THF (1000 mL) was added LiAlH4 (1 M in THF) (21.23 g, 798.2 mmol, 798.2 mL) slowly at 0 °C. A solution of methyl 4-amino-2-((prop-2-yn-1-yloxy)methyl)benzoate (70 g, 319.3 mmol) in THF (800 mL) was added slowly at 0 °C. The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was cooled to 0 °C, then water (22 mL) was added very slowly, and then 20% NaOH (22 mL) and water (66 mL) were added. The reaction mixture was stirred at 0 °C for 30 min. Anhydrous sodium sulfate was added to absorb excess water. The mixture was filtered through celite®. The filter cake was washed with ethyl acetate (1000 mL) and 10% MeOH/DCM (500 mL). The filtrate was concentrated under reduced pressure. The resulting crude compound was purified by _SiO2 column chromatography (35-40% ethyl acetate in petroleum ether as eluent) to afford (4-amino-2-((prop-2-yn-1-yloxy)methyl)phenyl)methanol. ¹ H NMR (400 MHz, CDCl ₃ ): δ 6.98 (d, J=8.0 Hz, 1H), 6.56 (d, J=2.4 Hz, 1H), 6.43 (dd, J=2.4, 8.0 Hz, 1H), 4.98 (s, 2H), 4.64 (t, J=5.2 Hz, 1H), 4.47 (s, 2H), 4.34 (d, J=5.6 Hz, 2H), 4.15 (d, J=2.4 Hz, 2H), 3.46 (t, J=2.4 Hz, 1H).

Synthesis of (9H-fluoren-9-yl)methyl (S)-(1-((4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate

To a solution of (4-amino-2-((prop-2-yn-1-yloxy)methyl)phenyl)methanol (1.92 g, 10.04 mmol, 1.0 eq.), (9H-fluoren-9-yl)methyl (S)-(1-amino-1-oxo-5-ureidopentan-2-yl)carbamate (3.99 g, 10.04 add N,N-diisopropylethylamine (2.62 mL, 15.06 mmol, 1.5 equiv). After stirring at ambient temperature for 1 s, the mixture was poured into water (200 mL). The resulting solids were filtered, washed with water, and dried under vacuum to give (9H-fluoren-9-yl)methyl (S)-(1-((4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate. LCMS: MH+=571.5; Rt=0.93 min (2 min acidic method).

Synthesis of (S)-2-amino-N-(4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)-5-ureidopentanamide

To (9H-fluoren-9-yl)methyl (S)-(1-((4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)carbamate (6.08 g, 10.65 mmol, 1.0 equiv) was added dimethylamine (2 M in THF, 21.31 mL, 42.62 mmol, 4 equiv). After stirring at ambient temperature for 1.5 h, the supernatant solution was decanted from the resulting gum-like residue. The residue was triturated with ether (3 x 50 mL), and the resulting solids were filtered, washed with ether, and dried under vacuum. (S)-2-amino-N-(4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)-5-ureidopentanamide is obtained. LCMS: MH+ 349.3; Rt=0.42 min (2 min acidic method).

Synthesis of tert-butyl ((S)-1-(((S)-1-((4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

To a solution of (S)-2-amino-N-(4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)-5-ureidopentanamide (3.50 g, 10.04 mmol, 1.0 equiv), (tert-butoxycarbonyl)-L-valine (2.62 g, 12.05 mmol, 1.2 equiv) and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (4.58 g, 12.05 mmol, 1.2 equiv) in DMF (10 mL) was added N,N-diisopropylethylamine (3.50 mL, 20.08 mmol, 2.0 equiv). After stirring at ambient temperature for 2 h, the mixture was poured into water (200 mL), and the resulting suspension was extracted with EtOAc (3×100 mL). The combined organic layers were dried over sodium sulfate and concentrated in vacuo. After purification with ISCO SiO ₂ chromatography (0-20% methanol/dichloromethane), tert-butyl ((S)-1-(((S)-1-((4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate was obtained. ¹ H NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H), 7.96 (d, J=7.7 Hz, 1H), 7.55 (dq, J=4.9, 2.2 Hz, 2H, aryl), 7.32 (d, J=8.9 Hz, 1H, aryl), 6.76 (d, J=8.9 Hz, 1H), 5.95 (t, J=5.8 Hz, 1H), 5.38 (s, 2H), 5.01 (t, J=5.5 Hz, 1H), 4.54 (s, 2H), 4.45 (dd, J=25.2, 5.3 Hz, 3H), 4.20 (d, J=2.4 Hz, 2H), 3.83 (dd, J=8.9, 6.7 Hz, 1H), 3.49 (t, J=2.4 Hz, 1H), 2.97 (dg, J=26.0, 6.5 Hz, 2H), 1.96 (g, J=6.6 Hz, 1H), 1.74-1.50 (m, 2H), 1.39 (m, 11H), 0.84 (dd, J=16.2, 6.7 Hz, 6H). LCMS: Mna+ 570.5; Rt=0.79 min (2 min sour method).

Synthesis of tert-butyl ((S)-1-(((S)-1-((4-(chloromethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

To a solution of tert-butyl ((S)-1-(((S)-1-((4-(hydroxymethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (2.00 grams, 3.65 mmol, 1.0 equiv.) in acetonitrile (13.3 mL) at 0 ^o C was added thionyl chloride (0.53 mL, 7.30 mmol, 2.0 equiv.). After stirring in an ice bath for one hour, the solution was diluted with water (40 mL) and the resulting white precipitate was collected by filtration, air-dried and dried under high vacuum to give tert-butyl ((S)-1-(((S)-1-((4-(chloromethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate. LCMS: Mna+ 588.5; Rt=2.17 min (5 min acidic method).

Synthesis of 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzoic acid

To a solution of 6-nitroisobenzofuran-1(3H)-one (90 g, 502.43 mmol, 1.00 equiv.) in MeOH (1000 mL) and KOH (28.19 g, 502.43 mmol, 1.00 equiv.) in H ₂ O (150 mL) was added. The brown mixture was stirred at 25°C for 1.5 h. The brown mixture was concentrated under reduced pressure to give a residue and dissolved in DCM (2000 mL). TBDPSCl (296.91 g, 1.08 mol, 277.49 ml, 2.15 equiv.) and imidazole (171.03 g, 2.51 mol, 5.00 equiv.) were added to the mixture, and stirred at 25°C for 12 h. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=1/0, 1/1), and 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzoic acid was obtained as a white solid. ¹ H NMR (400 MHz, METHANOL-d4) δ ppm. 1.13 (s, 9 H) 5.26 (s, 2 H) 7.34-7.48 (m, 6 H) 7.68 (bd, J=8 Hz, 4 H) 8.24 (bd, J=8 Hz, 1 H) 8.46 (bd, J=8 Hz, 1 H) 8.74 (s, 1 H).

Synthesis of (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)methanol

To a mixture of 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzoic acid (41 g, 94.14 mmol, 1 eq.) in THF (205 mL) was added BH ₃ . THF (1 M, 470.68 mL, 5 eq.). The yellow mixture was stirred at 60 °C for 2 h. MeOH (400 mL) was added to the mixture and concentrated under reduced pressure to give a residue. Then H ₂ O (200 mL) and DCM (300 mL) were added, extracted with DCM (3 x 200 mL), washed with brine (300 mL), dried over anhydrous MgSO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=1/0, 1/1). (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)methanol was obtained as a white solid. ¹ H NMR (400 MHz, METHANOL-d4) δ ppm 1.10 (s, 9 H) 4.58 (s, 2 H) 4.89 (s, 2 H) 7.32-7.51 (m, 6 H) 7.68 (dd, J=8, 1.38 Hz, 4 H) 7.76 (d, J=8 Hz, 1 H) 8.15 (dd, J=8 2.26 Hz, 1 H) 8.30 (d, J=2 Hz, 1 H).

Synthesis of 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzaldehyde

To a solution of (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)methanol (34 g, 80.65 mmol, 1 eq.) in DCM (450 mL) was added MnO ₂ (56.09 g, 645.22 mmol, 8 eq.). The black mixture was stirred at 25 °C for 36 h. MeOH (400 mL) was added to the mixture and concentrated under reduced pressure to give a residue. Then H ₂ O (200 mL) and DCM (300 mL) were added, extracted with DCM (3 × 200 mL), washed with saturated brine (300 mL), dried over anhydrous MgSO ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (CH ₂ Cl ₂ = 100%). 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzaldehyde was obtained as a white solid. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.14 (s, 9 H) 5.26 (s, 2 H) 7.34-7.53 (m, 6 H) 7.60-7.73 (m, 4 H) 8.13 (d, J=8 Hz, 1 H) 8.48 (dd, J=8, 2.51 Hz, 1 H) 8.67 (d, J=2 Hz, 1 H) 10.16 (s, 1 H).

Synthesis of N-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)prop-2-yn-1-amine

To a solution of 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzaldehyde (12.6 g, 30.03 mmol, 1 equiv.) in DCM (130 mL) were added prop-2-yn-1-amine (4.14 g, 75.08 mmol, 4.81 mL, 2.5 equiv.) and MgSO ₄ (36.15 g, 300.33 mmol, 10 equiv.), then the suspension was stirred at 25 °C for 24 h. A little of the reaction solution was taken and treated with NaBH ₄ , TLC showed the formation of one new spot. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. (E)-N-[[2-[[tert-butyl(diphenyl)silyl]oxymethyl]-5-nitro-phenyl]methyl]prop-2-yn-1-imine was obtained as a yellow solid. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm. 1.11 (s, 9 H) 2.48 (t, J=2.38 Hz, 1 H) 4.52 (t, J=2.13 Hz, 2 H) 5.09 (s, 2 H) 7.35-7.49 (m, 6 H) 7.63-7.72 (m, 4 H) 7.79 (d, J=8.53 Hz, 1 H) 8.25 (dd, J=8.53, 2.51 Hz, 1 H) 8.68 (d, J=2.26 Hz, 1 H) 8.84 (t, J=1.88 Hz, 1 H).

(E)-N-[[2-[[tert-butyl(diphenyl)silyl]oxymethyl]-5-nitro-phenyl]methyl]prop-2-yn-1-imine (12 g, 26.28 mmol, 1 eq) was dissolved in MeOH (100 mL) and THF (50 mL), then NaBH ₄ (1.49 g, 39.42 mmol, 1.5 eq) was added and the yellow mixture was stirred at -20 °C for 2 h. LCMS showed the determination of the desired compound. The reaction mixture was quenched by the addition of MeOH (200 mL) at -20 °C and then concentrated under reduced pressure to give a residue. The residue was dissolved in EtOAc (500 mL), washed with brine (150 mL), dried over _{anhydrous Na2SO4} , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash chromatography on silica gel (eluent 0-10% ethyl acetate/petroleum ether gradient). N-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)prop-2-yn-1-amine was obtained as a pale yellow oil. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm. 1.12 (s, 9 H) 2.13 (t, J=2.38 Hz, 1 H) 3.33 (d, J=2.51 Hz, 2 H) 3.80 (s, 2 H) 4.93 (s, 2 H) 7.36-7.49 (m, 6 H) 7.69 (dd, J=7.91, 1.38 Hz, 4 H) 7.77 (d, J=8.53 Hz, 1 H) 8.16 (dd, J=8.41, 2.38 Hz, 1 H) 8.24 (d, J=2.26 Hz, 1 H).

Synthesis of (9H-fluoren-9-yl)methyl (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(prop-2-yn-1-yl)carbamate

To a solution of N-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)prop-2-yn-1-amine (9 g, 19.62 mmol, 1 eq.) and FMOC-OSU (7.28 g, 21.59 mmol, 1.1 eq.) in dioxane (90 mL) was added sat. _NaHCO3 (90 mL) and the white suspension was stirred at 20 °C for 12 h. The reaction mixture was diluted with _H2O (150 mL) and extracted with EtOAc (150 mL x 2). The combined organic layers were washed with brine (200 mL), dried over _{anhydrous Na2SO4} , filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash chromatography on silica gel (eluent 0-30% ethyl acetate/petroleum ether). (9H-fluoren-9-yl)methyl (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(prop-2-yn-1-yl)carbamate (7.7 g, 11.08 mmol, 56.48% yield, 98% purity) was obtained as a white solid. ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.12 (s, 9 H) 2.17 (bd, J=14.31 Hz, 1 H) 3.87-4.97 (m, 9 H) 6.98-8.28 (m, 21 H).

Synthesis of (9H-fluoren-9-yl)methyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate

To an ice-cooled solution of (9H-fluoren-9-yl)methyl (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(prop-2-yn-1-yl)carbamate (5.0 g, 7.34 mmol, 1.0 equiv.) in 10% AcOH/CH ₂ Cl ₂ (100 mL) was added Zn (7.20 g, 110 mmol, 15 equiv.). The ice bath was removed and the resulting mixture was stirred for 2 h, during which time it was filtered through a pad of celite®. The volatiles were removed in vacuo and the residue was dissolved in EtOAc, washed with _NaHCO3 (sat.), NaCl (sat.), dried over _MgSO4 , filtered, concentrated and subjected to ISCO _SiO2 chromatography (0-75% EtOAc/Heptane) to afford (9H-fluoren-9-yl)methyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate. LCMS: MH+=651.6; Rt=3.77 min (5 min acidic method).

Synthesis of (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate

To (9H-fluoren-9-yl)methyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate (2.99 g, 4.59 mmol, 1.0 eq.) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanoic acid (1.72 g, 4.59 mmol, 1.0 eq.) in CH ₂ Cl ₂ (40 mL) was added ethyl 2-ethoxyquinoline-1(2H)-carboxylate (2.27 g, 9.18 mmol, 2.0 eq.). After stirring for 10 min, MeOH (1 mL) was added and the solution became homogeneous. The reaction mixture was stirred for 16 h, the volatiles were removed in vacuo and purified by ISCO SiO ₂ chromatography (0-15% MeOH/CH ₂ Cl ₂ ) to give (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate. LCMS: MH+=1008.8; Rt=3.77 min (5 min acidic method).

Synthesis of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate

To (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate (1.60 g, 1.588 mmol, 1.0 eq) was added 2M dimethylamine in MeOH (30 mL, 60 mmol, 37 eq) and THF (10 mL). After standing for 3 h, the volatiles were removed in vacuo and the residue was triturated with Et ₂ O to remove FMOC deprotection byproducts. To the resulting solid were added _CH2Cl2 ( ₁₆ mL) and pyridine (4 mL), and propargyl chloroformate (155 μL, 1.588 mmol, 1.0 equiv) was added to the heterogeneous solution. After stirring for 30 min, additional propargyl chloroformate (155 μL, 1.588 mmol, 1.0 equiv) was added. After stirring for another 20 min, MeOH (1 mL) was added to quench the remaining chloroformate, and the volatiles were removed under vacuum. Purification of ISCO SiO ₂ by chromatography (0-15% MeOH/CH ₂ Cl ₂ ) afforded prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate. LCMS: MH+=867.8; Rt=3.40 min (5 min acidic method).

Synthesis of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(prop-2-yn-1-yl)carbamate

To a solution of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-1-yl)carbamate (984 mg, 1.135 mmol, 1.0 equiv.) in THF (7.5 mL) was added 1.0 M tetrabutylammonium fluoride in THF (2.27 mL, 2.27 mmol, 2.0 equiv.). After standing for 6 h, the volatiles were removed in vacuo and the residue was purified by ISCO SiO ₂ chromatography (0-40% MeOH/CH ₂ Cl ₂ ) to give prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(prop-2-yn-1-yl)carbamate. LCMS: MH+=629.6; Rt=1.74 min (5 min acidic method).

Synthesis of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(prop-2-yn-1-yl)carbamate

To prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(prop-2-yn-1-yl)carbamate (205 mg, 0.326 mmol, 1.0 equiv) in _CH2Cl2 (10 mL) was added pyridine (158 μL, 1.96 mmol, 5 equiv). The heterogeneous mixture was cooled in a 0 ^o C ice bath and thionyl chloride (71 μL, 0.98 mmol, 3 equiv) _was added. After stirring in an ice bath for 3 h, the reaction mixture was purified directly by ISCO SiO ₂ chromatography (0-30% MeOH/CH ₂ Cl ₂ ) to afford prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(prop-2-yn-1-yl)carbamate. LCMS: MH+=647.6; Rt=2.54 min (5 min acidic method).

Synthesis of 2-(hydroxymethyl)-N-methyl-5-nitrobenzamide

To a stirred suspension of 6-nitroisobenzofuran-1(3H)-one (500 g, 2.79 mol) in MeOH (1500 mL) was added MeNH ₂ (3.00 kg, 29.94 mol, 600 mL, 31.0% purity) at 25°C and stirred for 1 h. The solid was filtered and washed with water twice (600 mL) and dried under high vacuum to give a residue. The product 2-(hydroxymethyl)-N-methyl-5-nitrobenzamide was obtained as a white solid. LCMS: Rt = 0.537 min, MS m/z = 193.2. ¹ H NMR: 400 MHz DMSO δ 8.57 (bd, J=4.4 Hz, 1H), 8.31 (dd, J=2.4, 8.6 Hz, 1H), 8.21 (d, J=2.4 Hz, 1H), 7.86 (d, J=8.8 Hz, 1H), 5.54 (t, J=5.6 Hz, 1H), 4.72 (d, J=5.5 Hz, 2H), 2.78 (d, J=4.4 Hz, 3H).

Synthesis of (2-((methylamino)methyl)-4-nitrophenyl)methanol

To a solution of 2-(hydroxymethyl)-N-methyl-5-nitrobenzamide (560 g, 2.66 mol) in THF (5000 mL) was cooled to 0 °C, then BH ₃ -Me ₂ S (506 g, 6.66 mol) (2.0 M in THF) was added dropwise over 60 min and heated to 70 °C for 5 h. LCMS showed consumption of the starting material. Upon completion, 4 M HCl (1200 mL) in methanol was added to the reaction mixture at 0 °C and heated at 65 °C for 8 h. The reaction mixture was cooled to 0 °C, the solid was filtered and concentrated under reduced pressure. The product (2-((methylamino)methyl)-4-nitrophenyl)methanol (520 g) was obtained as a white solid. LCMS: Rt=0.742 min, MS m/z=197.1 [M+H]+. ¹ H NMR: 400 MHz DMSO δ 9.25 (br s, 2H), 8.37 (d, J=2.4 Hz, 1H), 8.14 (dd, J=2.4, 8.5 Hz, 1H), 7.63 (d, J=8.4 Hz, 1H), 5.72 (br s, 1H), 4.65 (s, 2H), 4.15 (br s, 2H), 2.55-2.45 (m, 3H)

Synthesis of 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethanamine

To a solution of (2-((methylamino)methyl)-4-nitrophenyl)methanol (520 g, 2.65 mol) and imidazole (721 g, 10.6 mol) in DCM (2600 mL) cooled to 0°C was added TBDPS-CL (1.09 kg, 3.98 mol, 1.02 L) dropwise and stirred for 2 h. The mixture was poured into ice water (1000 mL) and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered and evaporated in vacuo to give the crude product. The crude product was purified by chromatography on silica gel eluting with ethyl acetate:petroleum ether (10/1 to 1) to give a residue. The product 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethanamine was obtained as a yellow liquid. LCMS: product: Rt=0.910 min, MS m/z=435.2 [M+H]+. ¹ H NMR: 400 MHz _CDCl3 δ 8.23 (d, J=2.4 Hz, 1H), 8.15 (dd, J=2.4, 8.4 Hz, 1H), 7.76 (d, J=8.4 Hz, 1H), 7.71-7.66 (m, 4H), 7.50-7.37 (m, 6H), 4.88 (s, 2H), 3.65 (s, 2H), 2.39 (s, 3H), 1.12 (s, 9H)

Synthesis of (9H-fluoren-9-yl)methyl (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate

To a solution of 1-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)-N-methylmethanamine (400 g, 920.3 mmol) in THF (4000 mL) were added FMOC-OSU (341.5 g, 1.01 mol) and Et ₃ N (186.2 g, 1.84 mol, 256.2 mL). The mixture was stirred at 25 °C for 1 h. The mixture was poured into water (1600 mL) and extracted with ethyl acetate (1000 mL x 2). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered and evaporated under vacuum to give the crude product. The crude product was purified by silica gel chromatography eluting with petroleum ether:ethyl acetate (1/0 to 1/1) to give (9H-fluoren-9-yl)methyl (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate as a white solid. LCMS: Rt=0.931 min, MS m/z=657.2 [M+H]+. ¹ H NMR: EW16000-26-P1A, 400 MHz CDCl ₃ δ 8.21-7.96 (m, 1H), 7.87-7.68 (m, 3H), 7.68-7.62 (m, 4H), 7.62-7.47 (m, 2H), 7.47-7.28 (m, 9H), 7.26-7.05 (m, 2H), 4.81 (br s, 1H), 4.62-4.37 (m, 4H), 4.31-4.19 (m, 1H), 4.08-3.95 (m, 1H), 2.87 (br d, J=5.2 Hz, 3H), 1.12 (s, 9H).

Synthesis of (9H-fluoren-9-yl)methyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate

A solution of (9H-fluoren-9-yl)methyl (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate (3.0 g, 4.57 mmol, 1.0 equiv.) in MeOH (90 mL) and EtOAc (30 mL) was degassed and flushed with a balloon of N ₂ through a three-way stopcock. After repeating degassing/flushing with N ₂ 2x, 10% Pd/C deGussa type (0.486 g, 0.457 mmol, 0.1 equiv.) was added. The resulting mixture was degassed and flushed with a balloon of H ₂ through a three-way stopcock. After repeating degassing/flushing with H ₂ 2x, the reaction was stirred under H ₂ balloon pressure for 4 h. The reaction mixture was degassed and flushed with N ₂ , filtered through a pad of Celite, further eluting with MeOH. After removing the volatiles in vacuo and pumping under high vacuum, (9H-fluoren-9-yl)methyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate was obtained. LCMS: MH+ = 627.7; Rt = 1.59 min (2 min acidic method).

Synthesis of (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate

To (9H-fluoren-9-yl)methyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (2.86 g, 4.56 mmol, 1.0 equiv) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanoic acid (1.71 g, 4.56 mmol, 1.0 equiv) in 2:1 CH ₂ Cl ₂ /MeOH (60 mL) was added ethyl 2-ethoxyquinoline-1(2H)-carboxylate (2.256 g, 9.12 mmol, 2.0 equiv). The homogeneous solution was stirred for 16 h, at which time additional (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanoic acid (0.340 g, 0.2 equiv) and ethyl 2-ethoxyquinoline-1(2H)-carboxylate (0.452 g, 0.4 equiv) were added to complete the reaction. After stirring for an additional 5 h, the volatiles were removed in vacuo and purified by ISCO SiO ₂ chromatography (0-5% MeOH/CH ₂ Cl ₂ ) to afford (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate. LCMS: MH+=984.1; Rt=1.54 min (2 min acidic method).

Synthesis of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate

To (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (2.05 g, 2.085 mmol, 1.0 eq.) in THF (10 mL) was added 2.0 M dimethyl amine in MeOH (10.42 mL, 20.85 mmol, 10 eq.). After stirring for 16 h, volatiles were removed in vacuo. The residue was dissolved in _CH2Cl2 ( ₂₀ mL) and DIEA (0.533 mL, 4.17 mmol, 2 eq.) and propargyl chloroformate (0.264 mL, 2.71 mmol, 1.3 eq.) was added. After stirring at rt for 16 h, the reaction mixture was diluted _{with CH2Cl2} (20 mL), washed with _NaHCO3 (sat.), NaCl (sat.), dried over _MgSO4 , filtered, concentrated and purified _by ISCO _SiO2 chromatography (0-15% MeOH/ _CH2Cl2 ) to give prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate. LCMS: MH+=843.8; Rt=1.35 min (2 min acidic method).

Synthesis of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate

To a 0 ^o C solution of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (1.6 g, 1.90 mmol, 1.0 equiv.) in THF (10.0 mL) was added 1.0 M tetrabutylammonium fluoride in THF (3.80 mL, 3.80 mmol, 2.0 equiv.). After warming to room temperature and stirring for 16 h, the volatiles were removed in vacuo, the residue was dissolved in EtOAc, washed with NaHCO ₃ (sat.), NaCl (sat.), dried over MgSO ₄ , filtered, concentrated, and the residue was purified by ISCO SiO ₂ chromatography (0-30% MeOH/CH ₂ Cl ₂ ) to give prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate. LCMS: MH+=605.7; Rt=0.81 min (2 min acidic method).

Synthesis of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(methyl)carbamate

To prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (350 mg, 0.579 mmol, 1.0 equiv) in _CH2Cl2 (10 mL) was added pyridine (0.278 mL, 3.47 mmol, 6 equiv). The heterogeneous mixture was cooled in _a 0 ^o C ice bath and thionyl chloride (0.126 mL, 1.73 mmol, 3 equiv) was added. After stirring in an ice bath for 3 h, the reaction mixture was purified by ISCO SiO ₂ chromatography (0-30% MeOH/CH ₂ Cl ₂ ) to give prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(prop-2-yn-1-yl)carbamate. LCMS: MH+=623.7; Rt=2.19 min (5 min acidic method).

Synthesis of (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate

To (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (2.6 g, 2.64 mmol, 1.0 eq.) dissolved in THF (20 mL) were added acetic acid (0.757 mL, 13.22 mmol, 5.0 eq.) and 1.0 M TBAF in THF (2.91 mL, 2.91 mmol, 1.1 eq.). The solution was stirred for 72 h, during which time the volatiles were removed in vacuo. Purification of ISCO SiO ₂ by chromatography (0-30% MeOH/CH ₂ Cl ₂ ) afforded (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate. LCMS: MH+=745.5; Rt=1.07 min (2 min acidic method).

Synthesis of (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(methyl)carbamate

To (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (200 mg, 0.269 mmol, 1.0 eq.) in _{CH2Cl2 (} 10 mL) was added pyridine (0.130 mL, 1.61 mmol, 6 eq.). The heterogeneous mixture was cooled in a 0 ^o C ice bath and thionyl chloride (0.059 mL, 0.806 mmol, 3 eq.) was added. After rapid stirring in an ice bath, the reaction mixture was stirred with warming to room temperature for 2 h. The reaction mixture was purified by ISCO SiO ₂ chromatography (0-30% MeOH/CH ₂ Cl ₂ ) to afford (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(methyl)carbamate. LCMS: MH+=763.2; Rt=1.18 min (2 min acidic method).

GENERAL METHOD 1

Synthesis 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-in-1-yl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)-4-(2-(4-(4-( (R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

To (R)-2-((5-(3-chloro-2-methyl-4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)propanoic acid hydrochloride (73.8 mg, 0.81 mmol, 1.0 equiv.) and prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(prop-2-yn-1-yl)carbamate (78 mg, 0.122 mmol, 1.5 equiv.) was dissolved in DMF (0.5 mL), DIEA (70 μL, 0.405 mmol, 5.0 equiv.) was added, followed by tetrabutylammonium iodide (25.4 mg, 0.069 mmol, 0.85 equiv.). After stirring for 5 h, the reaction mixture was diluted with DMSO (3 mL) and purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, 0.1% NH₄OH modifier). After lyophilization, 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium is obtained. LCMS: M+=1486.3; Rt=2.70 min (5 min alkaline method).

GENERAL METHOD 2

Synthesis of 1-(2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)am ino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidine-4- yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

To 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (24 mg, 0.016 mmol, 1.0 equiv.) and 25-azido-2,5,8,11,14,17,20,23-octaoxapentacosane (26.5 mg, 0.065 mmol, 4 equiv) was added with t-BuOH (1 mL). The mixture was degassed under room vacuum and purged with a balloon of N₂through a 3-way stopcock. Degassing/purging is repeated 3 times. 16 mg/mL aqueous sodium ascorbate solution (297 µL, 0.024 mmol, 1.5 equiv) is added and the solution is degassed and purged with N₂three times. 4 mg/mL aqueous copper sulfate solution (298 µL, 0.0048 mmol, 0.3 equiv) is added and the solution is degassed and purged with N₂three times. After stirring under N₂for 3 h, the reaction mixture was diluted with DMSO (3 ml) and purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 1-(2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)methyl)-4-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-1- ... (ino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. MSVR M+=2307.0730, Rt=2.69 min (5 min acidic method).

GENERAL METHOD 3

Synthesis of 1-(2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1- (yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (L1-P1)

K 1-(2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)am (ino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (19 mg, 0.0082 mmol, 1.0 equiv) was added 25% TFA/CH₂Cl₂(2 ml). After standing for 45 min, the volatiles are removed in vacuo, CH₂Cl₂and the volatiles were removed in vacuo and pumped down. The residue was dissolved in DMF (1 mL) and DIEA (22 μL, 0.124 mmol, 15 equiv) and 2,5-dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (5.1 mg, 0.016 mmol, 2 equiv) were added. After standing for 18 h, the solution was diluted with DMSO (3 mL) and purified by RP-ISCO gold chromatography. After lyophilization, 1-(2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1- (yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium(L1-P1). MSVR: M+=2399.0797, Rt=2.43 min (5 min acid run). ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 0.83 (dd,J=13.82, 6.72 Hz, 6 H) 1.30-1.52 (m, 2 H) 1.55-1.76 (m, 2 H) 1.83 (s, 3 H) 1.88-2.08 (m, 1 H) 2.28-2.46 (m, 7 H) 2.73-2.84 (m, 4 H) 2.84-3.08 (m, 8 H) 3.15-3.27 (m, 3 H) 3.43-3.66 (m, 68 H) 3.73-3.83 (m, 7 H) 4.16-4.30 (m, 3 H) 4.32-4.43 (m, 2 H) 4.47 (shs, 6 H) 4.60 (shs. 3 H) 5.16-5.30 (m, 3 H) 5.40 (shs, 2 H) 5.44-5.52 (m, 1 H) 5.99 (pieces, J=5.07 Hz, 1 H) 6.21 (d,J=6.48 Hz, 1 H) 6.71 (t,J=7.40 Hz, 1 H) 6.97-7.04 (m, 2 H), 7.00 (s, 2 H) 7.12-7.23 (m, 5 H) 7.27-7.54 (m, 8 H) 7.63 (d,J=5.14 Hz, 1 H) 7.78-7.94 (m, 3 H) 7.99 (shs, 1 H) 8.05-8.23 (m, 2 H) 8.60 (s, 1 H) 8.88 (d, J=5.13 Hz, 1 H) 10.24 (shs, 1 H).

Synthesis of 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((((1-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)((1-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl) xakosyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidine -4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 2 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (30 mg, 0.020 mmol, 1.0 eq.) and 1-azido-3,6,9,12,15,18,21,24-octaoxahexacosane-27-oic acid (28.3 mg, 0.061 mmol, 3 equiv) gave 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((((1-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)((1-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)((1-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexa (xacosyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+=2420.0867, Rt=2.57 min (5 min acidic method).

EXAMPLE 2: Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((((1-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-1H-1,2,3-triazol-4-yl)methoxy) carbonyl)((1-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium (L10-P1)

Following GENERAL PROCEDURE 3 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((((1-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)((1-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl) (xacosyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (30 mg, 0.012 mmol, 1.0 equiv.) was obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((((1-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-1H-1,2,3-triazol-4-yl)methoxy) carbonyl)((1-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium (L10-P1) . HRMS: M+=2515.0879, Rt=2.43 min (5 min acidic method). ¹ H NMR (400 MHz, DMSO -d ₆ ) δ ppm. 0.83 (dd, J =13.82, 6.72 Hz, 6 H) 1.30-1.52 (m, 2 H) 1.55-1.76 (m, 2 H) 1.83 (s, 3 H) 1.88-2.08 (m, 1 H) 2.28-2.46 (m, 7 H) 2.73-2.84 (m, 4 H) 2.84-3.08 (m, 8 H) 3.15-3.27 (m, 3 H) 3.43-3.66 (m, 68 H) 3.73-3.83 (m, 7 H) 4.16-4.30 (m, 3 H) 4.32-4.43 (m, 2 H) 4.47 (shs, 6 H) 4.60 (shs, 3 H) 5.16-5.30 (m, 3 H) 5.40 (shs, 2 H) 5.44-5.52 (m, 1 H) 5.99 (piece, J = 5.07 Hz, 1 H) 6.21 (d, J = 6.48 Hz, 1 H) 6.71 (t, J = 7.40 Hz, 1 H) 6.97-7.04 (m, 2 H), 7.00 (s, 2H) 7.12-7.23 (m, 5 H) 7.27-7.54 (m, 8 H) 7.63 (d, J = 5.14 Hz, 1 H) 7.78-7.94 (m, 3 H) 7.99 (shs, 1 H) 8.05-8.23 (m, 2 H) 8.60 (s, 1 H) 8.88 (d, J =5.13 Hz, 1 H) 10.24 (shs, 1 H).

Synthesis 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)- 1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 1 with (R)-2-((5-(3-chloro-2-methyl-4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)propanoic acid hydrochloride (300 mg, 0.329 mmol, 1.0 eq.) and prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(methyl)carbamate (246 mg, 0.395 mmol, 1.2 equiv.) yield 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+=1461.5800, Rt=2.53 min (5 min acidic method).

Synthesis 1-(2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl )(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopent anamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)e toxic)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 2 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (20 mg, 0.14 mmol, 1.0 eq.) and 25-azido-2,5,8,11,14,17,20,23-octaoxapentacosane (16.8 mg, 0.041 mmol, 3.0 equiv.) is obtained 1-(2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. MSHR: M+=1872.8359, Rt=2.56 min (5 min acidic method).

Synthesis of 1-(2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutane ido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (L4-P1)

Following the GENERAL METHOD 3 s 1-(2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl )(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopenta Namido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)eth oxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-iem (16.9 mg, 0.009 mmol, 1.0 equiv.), 1-(2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutane ido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (L4-P1) . HRMS: M+= 1967.8375, Rt=2.46 min (5 min acidic method).

Synthesis 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((((1-( 26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)(methyl)a mino)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl) ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 2 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (12 mg, 0.0082 mmol, 1.0 eq.) and 1-azido-3,6,9,12,15,18,21,24-octaoxahexacosan-27-oic acid (11.5 mg, 0.025 mmol, 3.0 equiv.) is obtained 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((((1-(2 6-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)(methyl)am ino)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)e toxic)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. MSHR: M+=1928.8459, Rt=2.52 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((((1-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahec sacosyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-di hydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium (L3-P1)

Following the general 3 s technique 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((((1-(2 6-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)(methyl)am ino)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)e toxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-iem (12 mg, 0.006 mmol, 1.00 equiv) yields 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((((1-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahec sacosyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium (L3-P1) . HRMS: M+=2024.8516, Rt=2.42 min (5 min acidic method).

Synthesis of 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

To 4-methoxybenzyl (R)-2-((5-(3-chloro-2-methyl-4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)propanoate (160 mg, 0.161 mmol, 1.0 eq.) and (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(methyl)carbamate (153 mg, 0.201 mmol, 1.25 equiv.) dissolved in DMF (2 mL), DIEA (0.056 mL, 0.321 mmol, 2.0 equiv.) was added, followed by tetrabutylammonium iodide (65.3 mg, 0.177 mmol, 1.1 equiv.). After standing for 16 h, 2.0 dimethylamine in THF (0.804 mL, 1.67 mmol, 10 equiv.) was added. After standing for 2 h, the volatiles were removed in vacuo, DMSO (6 mL) was added, and the solution was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium is obtained. MSHR: M+=1499.3700; Rt=2.59 min (5 min acidic method).

Synthesis of 1-(4-((R)-2-((R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2-methyl-3-oxo-4,7,10,13,16,19,22,25,28-nonaoxa-2-azanonacosyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

To 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (40 mg, 0.027 mmol, 1.0 equiv.) and 2,5-dioxopyrrolidin-1-yl (2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl) carbonate (30.8 mg, 0.059 mmol, 2.2 equiv.) dissolved in DMF (1.5 mL) was added DIEA (0.009 mL, 0.053 mmol, 2.0 equiv.). After standing for 1 h, DMSO (3 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 1-(4-((R)-2-((R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2-methyl-3-oxo-4,7,10,13,16,19,22,25,28-nonaoxa-2-azanonacosyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium is obtained. MSHR: M+=1909.3800; Rt=2.92 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((R)-2-((R)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(2-methyl-3-oxo-4,7,10,13,16,19,22,25,28-nonaoxa-2-azanonacosyl)benzyl)-1-methylpiperazin-1-ium (L2-P1)

Following the GENERAL METHOD 3 s 1-(4-((R)-2-((R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2-methyl-3-oxo-4,7,10,13,16,19,22,25,28-nonaoxa-2-azanonacosyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (25.3 mg, 0.013 mmol, 1.0 equiv.), obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((R)-2-((R)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(2-methyl-3-oxo-4,7,10,13,16,19,22,25,28-nonaoxa-2-azanonacosyl)benzyl)-1-methylpiperazin-1-ium (L2-P1) . HRMS: M+= 1884.7900, Rt=2.50 min (5 min acidic method).

Synthesis 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(80-carboxy-2-methyl -3-oxo-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78-pentacosaoxa-2-azaoctacontyl) benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

To 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (150 mg, 0.093 mmol, 1.0 equiv.) and 79-((2,5-dioxopyrrolidin-1-yl)oxy)-79-oxo-4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-pentacosaoxanone heptacontanoic acid (134 mg, 0.102 mmol, 1.1 equiv) was dissolved in DMF (2 mL) and DIEA (0.081 mL, 0.464 mmol, 5.0 equiv) was added. After standing for 18 h, DMSO (6 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(80-carboxy-2-methyl-3-oxo-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78-pentacosaoxa-2-azaoctacontyl) is obtained benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. MSHR: M+=2700.8701; Rt=2.83 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(80-carboxy-2-methyl-3-oxo-6,9,12,15,18,21,24,27,30,33,36 ,39,42,45,48,51,54,57,60,63,66,69,72,75,78-pentacosaoxa-2-azaoctacontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5 -dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium (L11-P1)

Following GENERAL PROCEDURE 3 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(80-carboxy-2-methyl-3-oxo-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78-pentacosaoxa-2-azaoctaconyl)b (enzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium, is obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(80-carboxy-2-methyl-3-oxo-6,9,12,15,18,21,24,27,30,33,36 ,39,42,45,48,51,54,57,60,63,66,69,72,75,78-pentacosaoxa-2-azaoctacontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium ( L11-P1) . HRMS: M+=2674.8201, Rt=2.44 min (5 min acidic method).

Synthesis 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-met oxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 1 with (R)-2-((5-(3-chloro-2-methyl-4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)propanoic acid (50 mg, 0.057 mmol, 1.0 eq.) and tert-butyl ((S)-1-(((S)-1-((4-(chloromethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (34.1 mg, 0.069 mmol, 1.2 eq.), 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium is obtained. LCMS: M+=1337.2, Rt=1.11 min (2 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium or (2R)-2-[(5S _a )-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-yl-1-yl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (L9-P1)

Following GENERAL PROCEDURE 3 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (55 mg, 0.041 mmol, 1.0 equiv.), 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium ( L9-P1) . HRMS: M+=1431.5400, Rt=2.50 min (5 min acidic method).

Synthesis of 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 1 with 4-methoxybenzyl (R)-2-((5-(3-chloro-2-methyl-4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)propanoate (85 mg, 0.085 mmol, 1.0 eq.) and tert-butyl ((S)-1-(((S)-1-((4-(chloromethyl)-3-((prop-2-yn-1-yloxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (58 mg, 0.102 mmol, 1.2 equiv) yield 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. MSHR: M+=1524.6200, Rt=2.95 min (5 min acidic method).

Synthesis 1-(2-(((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl l)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benz yl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 2 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (20 mg, 0.014 mmol, 1.0 eq.) and 25-azido-2,5,8,11,14,17,20,23-octaoxapentacosane (5.8 mg, 0.014 mmol, 1 equiv.) is obtained 1-(2-(((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. LCMS: [(M+)+H+]+2/2=908.5, Rt=1.15 min (2 min acidic method).

Synthesis of 1-(2-(((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureide (opentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (L8-P1)

Following GENERAL PROCEDURE 3 with 1-(2-(((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (20 mg, 0.010 mmol, 1.0 equiv.) yields 1-(2-(((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureide (opentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (L8-P1) . HRMS: M+= 1908.8097, Rt=2.37 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 3 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (123.1 mg, 0.075 mmol, 1.0 equiv.), 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium. HRMS: M+= 1499.5601, Rt=2.50 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((1-(26-carboxy-3,6,9,12,15,18,21,24-o ktaoxahexacosyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium (L7-P1)

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium (40 mg, 0.027 mmol, 1.0 equiv.) and 1-azido-3,6,9,12,15,18,21,24-octaoxahexacosane-27-oic acid (37.4 mg, 0.080 mmol, 3.0 equiv.) gave 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((1-(26-carboxy-3,6,9,12,15,18,21,24-o (((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium (L7-P1) . HRMS: M+=1965.5601, Rt=2.35 min (5 min acidic method).

Synthesis 1-(2-(((1-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatrigeptacontan-73-yl)- 1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamide o)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-and l)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (L5-P1)

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium (20.4 mg, 0.014 mmol, 1.0 equiv.) and 73-azido-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatriheptacontane (28.1 mg, 0.025 mmol, 1.5 equiv.) are obtained 1-(2-(((1-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatrigeptacontan-73-yl)- 1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamide (o)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (L5-P1) . HRMS: M+=2613.2100, Rt=2.44 min (5 min acidic method).

Synthesis of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate

A solution of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (249 mg, 0.412 mmol) and 4-nitrophenyl (4-nitrosophenyl) carbonate (356 mg, 1.24 mmol, 3.0 equiv.) in DMF (2 mL) was stirred until homogeneous and kept for 16 h. The solution was diluted with DMSO (6 mL) and purified RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, without modifier). After lyophilization, prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate is obtained. LC/MS MH+=770.7, Rt=2.45 min (5 min acidic method).

Synthesis of prop-2-yn-1-yl (5-((R)-2-((R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((methyl(2-(methylamino)ethyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate

To a solution of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (100 mg, 0.130 mmol) in DMF (1 mL) was added N,N'-dimethylethylenediamine (22.90 mg, 0.260 mmol), then DIPEA (0.113 mL, 0.650 mmol) was added at room temperature. The resulting solution was stirred at room temperature overnight. The reaction mixture was diluted with DMSO, purified RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, 0.1% NH4OH modifier). After lyophilization, prop-2-yn-1-yl (5-((R)-2-((R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((methyl(2-(methylamino)ethyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate is obtained. LCMS: MH+=719.9, Rt=0.73 min (2 min acidic method).

Synthesis of (R)-4-(2-(2-chloro-4-(6-(4-fluoro-3-hydroxyphenyl)-4-((1-methoxy-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium

To a solution of (R)-4-(2-(4-(4-(1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluoro-3-hydroxyphenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium or (2R)-2-[(5S _a )-5-[3-chloro-2-methyl-4-[2-[4-methyl-4-(3-sulfopropyl)piperazin-4-yl-1-yl]ethoxy]phenyl]-6-(4-fluoro-3-hydroxy-phenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (100 mg, 0.099 mmol) in MeOH (1.5 ml) were added a few drops of H₂SO₄(conc.). After stirring overnight, MeOH was removed in vacuo. The residue was dissolved in DMSO and purified RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, (R)-4-(2-(2-chloro-4-(6-(4-fluoro-3-hydroxyphenyl)-4-((1-methoxy-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium is obtained. MSVR M+=1027.2900, Rt=2.31 min (5 min acidic method).

Synthesis 4-(2-(4-(6-(3-(((2-((((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentane mido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl) carbamoyl)oxy)-4-fluorophenyl)-4-(((R)-1-methoxy-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium

To a solution of (R)-4-(2-(2-chloro-4-(6-(4-fluoro-3-hydroxyphenyl)-4-((1-methoxy-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium (50 mg, 0.049 mmol, 1.0 equiv.) in CH₂Cl₂(1 ml) at 0^oTo C was added TEA (34 µL, 0.243 mmol, 5.0 equiv), followed by 4-nitrophenyl chloroformate (10.8 mg, 0.054 mmol, 1.1 equiv). After stirring for 15 min, a solution of prop-2-yn-1-yl (5-((R)-2-((R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((methyl(2-(methylamino)ethyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate (66.3 mg, 0.092 mmol, 2.0 equiv) in DMF (1 mL), followed by DIEA (40 µL, 0.231 mmol, 5.0 equiv). After stirring for 2 h, the volatiles were removed in vacuo, the solution was diluted with DMSO (3 ml) and purified RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 4-(2-(4-(6-(3-(((2-(((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl) is obtained carbamoyl)oxy)-4-fluorophenyl)-4-(((R)-1-methoxy-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium. MSVR M+=1771.6700, Rt=2.57 min (5 min acidic method).

Synthesis 4-(2-(4-(6-(3-(((2-((((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentane amido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl )carbamoyl)oxy)-4-fluorophenyl)-4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl) ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium

To the solution 4-(2-(4-(6-(3-(((2-((((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamide o)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamo yl)oxy)-4-fluorophenyl)-4-(((R)-1-methoxy-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropane -2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium (23 mg, 0.013 mmol, 1.0 equiv.) in THF (1 mL) was added 2N LiOH (0.032 mL, 0.065 mmol, 5 equiv.). After stirring for 2 h, the solution was neutralized with AcOH and purified RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 4-(2-(4-(6-(3-(((2-(((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-4-fluorophenyl)-4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium is obtained. MSVR M+=1757.6200, Rt=2.46 min (5 min acidic method).

Synthesis 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(3-(((2-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-in-1-yloxy)carbonyl)amino)methyl)benzyl)oxy)carbonyl)(methyl) amino)ethyl)(methyl)carbamoyl)oxy)-4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium

Following the GENERAL PROCEDURE 3 m 4-(2-(4-(6-(3-(((2-(((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl) carbamoyl)oxy)-4-fluorophenyl)-4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium (17 mg, 0.0097 mmol, 1.0 equiv.) was obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(3-(((2-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl) (carbamoyl)oxy)-4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium. HRMS: M+= 1852.5200, Rt=2.29 min (5 min acidic method).

Synthesis of 4-(2-(4-(6-(3-(((2-((((2-((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl) (oxy)-4-fluorophenyl)-4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium (L12-P2)

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(3-(((2-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)oxy) (methyl)amino)ethyl)(methyl)carbamoyl)oxy)-4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium (10 mg, 0.0054 mmol, 1.0 equiv.) and 25-azido-2,5,8,11,14,17,20,23-octaoxapentacosane (4.4 mg, 0.011 mmol, 2 equiv.) were obtained 4-(2-(4-(6-(3-(((2-((((2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentane (mido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-4-fluorophenyl)-4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium (L12-P2 ). HRMS: M+=2261.8601, Rt=2.24 min (5 min acidic method).

Synthesis 4-(2-(4-(6-(3-((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)oxy)-4-fluorophenyl )-4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium

GENERAL PROCEDURE 1 was followed using (R)-4-(2-(2-chloro-4-(6-(4-fluoro-3-hydroxyphenyl)-4-((1-methoxy-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium (40 mg, 0.039 mmol, 1.0 equiv.) and prop-2-yn-1-yl (5-((R)-2-((R)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(methyl)carbamate (36.4 mg, 0.058 mmol, 1.5 equiv), with the modification that after alkylation, 2N LiOH (0.097 mL, 0.195 mmol, 5.0 equiv) was added and stirred for 2 h before neutralization and purification by RP-HPLC. After lyophilization, 4-(2-(4-(6-(3-((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)oxy)-4-fluorophenyl)-4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium is obtained. MSHR: M+=1599.5856, Rt=1.34 min (2 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(3-((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)oxy)-4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium

Following GENERAL PROCEDURE 3 with 4-(2-(4-(6-(3-((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)oxy)-4-fluorophenyl)-4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium (46 mg, 0.029 mmol, 1.0 eq.), 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(3-((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)oxy)-4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium are obtained. MSHR: M+= 1694.5699, Rt=2.55 min (5 min acidic method).

Synthesis of 4-(2-(4-(6-(3-((2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutane (mido)-5-ureidopentanamido)benzyl)oxy)-4-fluorophenyl)-4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium (L4-P2)

Following the GENERAL METHOD 2 s 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(3-((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-1-yloxy)carbonyl)amino)methyl)benzyl)oxy)-4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium (44 mg, 0.026 mmol, 1.0 equiv) and 25-azido-2,5,8,11,14,17,20,23-octaoxapentacosane (21.2 mg, 0.052 mmol, 2 equiv) yield 4-(2-(4-(6-(3-((2-((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutane (mido)-5-ureidopentanamido)benzyl)oxy)-4-fluorophenyl)-4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-1-ium (L4-P2) . HRMS: M+=2103.8000, Rt=2.47 min (5 min acidic method).

GENERAL METHOD 4:

Synthesis 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(39-methyl -38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-aazatetracontan-40-yl)benzyl)-4-(2-(2-chloro-4 -(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

To 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (60 mg, 0.040 mmol, 1.0 equiv.) and 2,5-dioxopyrrolidin-1-yl 2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane-38-oate (35.7 mg, 0.052 mmol, 1.3 equiv.) dissolved in DMF (1 mL) was added DIPEA (0.035 mL, 0.200 mmol, 5.0 equiv.). After standing for 18 h, DMSO (2 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H2O, 0.1% TFA modifier). After lyophilization, 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(39-methyl -38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-aazatetracontan-40-yl)benzyl)-4-(2-(2-chloro-4 -(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. MSVR: [M+Na]+=2092.9399; Rt=2.88 min (5 min sour method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(39-methyl-38-ok co-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-aazatetracontan-40-yl)benzyl)-1-methylpiperazin-1-ium (L32-P1)

Following GENERAL PROCEDURE 3 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(39-methyl-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-aazatetracontan-40-yl)benzyl)-4-(2-(2-chloro-4 -(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (43.4 mg, 0.021 mmol, 1 eq.) was obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5- (dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(39-methyl-38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-aazatetracontan-40-yl)benzyl)-1-methylpiperazin-1-ium. HRMS: [M+Na]+= 2066.8799; Rt=2.44 min (5 min acidic method).

Synthesis 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(51-methyl-50- oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecaoxa-51-azadopentacontan-52-yl)benzyl)-4-(2-( 2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 4 with 2,5-dioxopyrrolidin-1-yl 2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecaoxapentacontan-50-oate (44.8 mg, 0.021 mmol, 1 eq.), 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(51-methyl-50- oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecaoxa-51-azadopentacontan-52-yl)benzyl)-4-(2-( 2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+= 2246.0400; Rt=2.88 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(51-methyl-50-oxo-2,5,8,11, 14,17,20,23,26,29,32,35,38,41,44,47-hexadecaoxa-51-azadopentacontan-52-yl)benzyl)-1-methylpiperazin-1-ium (L31-P1)

Following GENERAL PROCEDURE 3 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(51-methyl-50-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecaoxa-51-azadopentacontan-52-yl)benzyl)-4-(2-( 2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (47.5 mg, 0.021 mmol, 1 eq.) was obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(51-methyl-50-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecaoxa-51-azadopentacontan-52-yl)benzyl)-1-methylpiperazin-1-ium. HRMS: M+= 2221.0000; Rt=2.45 min (5 min acidic method).

Synthesis of 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(75-methyl-74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azahexaheptacontan-76-yl) benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 4 with 2,5-dioxopyrrolidin-1-yl 2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxatetraheptacontan-74-oate (52.6 mg, 0.043 mmol, 1.3 eq.), 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(75-methyl-74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azahexaheptacontan-76-yl)b ((2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+= 2598.2500; Rt=2.88 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-i l)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(75-methyl-74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azahexaheptacontan-76-yl)benzyl)-1-methylpiperazin-1-ium (L30-P1)

Following GENERAL PROCEDURE 3 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(75-methyl-74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azahexaheptacontan-76-yl)b (4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (38.8 mg, 0.014 mmol, 1 equiv.) was obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2 ,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl ()ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(75-methyl-74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azahexaheptacontan-76-yl)benzyl)-1-methylpiperazin-1-ium. HRMS: M+= 2573.2000; Rt=2.47 min (5 min acidic method).

Synthesis 1-(2-(((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-N-methyl-5-oxopentanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 4 with 5-(tert-butyl) 1-(2,5-dioxopyrrolidin-1-yl) (((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate (39.0 mg, 0.075 mmol, 1.1 equiv) gave 1-(2-(((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-N-methyl-5-oxopentanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2 -chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+= 1906.8101; Rt=3.03 min (5 min acidic method).

Synthesis of 1-(2-(((S)-2-amino-5-(tert-butoxy)-N-methyl-5-oxopentanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

TO 11-(2-(((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-N-methyl-5-oxopentanamido)me til)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-( 2-Chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (38.8 mg, 0.026 mmol, 1.0 equiv.) dissolved in DMSO (2 mL) was added dimethylamine (0.192 mL, 0.384 mmol, 20 equiv.) After standing for 4 h, DMSO (2 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 1-(2-(((S)-2-amino-5-(tert-butoxy)-N-methyl-5-oxopentanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium is obtained. MSHR: M+=1684.4000; Rt=2.64 min (5 min acidic method).

GENERAL METHOD 5

Synthesis of 1-(2-(((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-N-methyl-3-sulfopropanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

To (((9H-fluoren-9-yl)methoxy)carbonyl)(sulfo)-D-alanine (55.2 mg, 0.141 mmol, 1.3 eq.) and 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate (V) (41.3 mg, 0.109 mmol, 1.0 eq.) dissolved in DMF (2 ml), add DIPEA (0.024 ml, 0.138 mmol, 8.0 eq.). After standing for 10 min, 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (200 mg, 0.109 mmol, 1.0 equiv.) was added. After standing for 2.5 h, DMSO (4 ml) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H ₂ O, 0.1% TFA modifier). After lyophilization, 1-(2-(((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-N-methyl-3-sulfopropanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium is obtained. MSHR: M+=1872.7000; Rt=3.09 min (5 min acidic method).

Synthesis of 1-(2-(((R)-2-amino-N-methyl-3-sulfopropanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

To 1-(2-(((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-N-methyl-3-sulfopropanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (173 mg, To the mixture (0.087 mmol, 1.0 equiv.) dissolved in THF (2 mL), dimethylamine (0.870 mL, 1.740 mmol, 20 equiv.) was added. After standing for 5 h, all volatiles were removed.in a vacuumThe solid is triturated with diethyl ether. DMSO (2 mL) is added and the solution is purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 1-(2-(((R)-2-amino-N-methyl-3-sulfopropanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium is obtained. MSHR: M+=1650.5800; Rt=2.71 min (5 min acidic method).

Synthesis of 1-(2-(((R)-2-amino-N-methyl-3-sulfopropanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (L70-P1)

Following GENERAL PROCEDURE 3 with 1-(2-(((S)-2-amino-5-(tert-butoxy)-N-methyl-5-oxopentanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (145 mg, 0.088 mmol, 1 equiv.) yield 1-(2-(((R)-2-amino-N-methyl-3-sulfopropanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. MSHR: M+= 1625.5601; Rt=2.32 min (5 min acidic method).

GENERAL METHOD 6

Synthesis of 1-(2-(((S)-5-(tert-butoxy)-N-methyl-5-oxo-2-(2-sulfoacetamido)pentanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

To 2-sulphonacetic acid (4.83 mg, 0.035 mmol, 2.0 eq.) and 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate (V) (9.85 mg, 0.026 mmol, 1.5 eq.), dissolved in DMF (1 ml), add DIPEA (0.024 ml, 0.138 mmol, 8.0 eq.). After standing for 10 min, 1-(2-(((S)-2-amino-5-(tert-butoxy)-N-methyl-5-oxopentanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (29.1 mg, 0.017 mmol, 1.0). After standing for 45 min, DMSO (2 ml) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 1-(2-(((S)-5-(tert-butoxy)-N-methyl-5-oxo-2-(2-sulfoacetamido)pentanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium is obtained. MSHR: M+=1806.7000; Rt=3.10 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((S)-4-carboxy-N-methyl-2-(2-sulfoacetamido)butanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium (L71-P1)

Following GENERAL PROCEDURE 3 with 1-(2-(((S)-5-(tert-butoxy)-N-methyl-5-oxo-2-(2-sulfoacetamido)pentanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (23.7 mg, 0.011 mmol, 1 equiv.) are obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((S)-4-carboxy-N-methyl-2-(2-sulfoacetamido)butanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium. HRMS: M+= 1725.5900; Rt=2.39 min (5 min acidic method).

Synthesis 1-(2-((S)-40-(3-(tert-butoxy)-3-oxopropyl)-42-methyl-38,41-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-3 9,42-diazatritetracontan-43-yl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanam ido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 4 with 1-(2-(((S)-2-amino-5-(tert-butoxy)-N-methyl-5-oxopentanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (30 mg, 0.018 mmol, 1.0 equiv) and Mal-PEG12-NHS ester (18.31 mg, 0.027 mmol, 1.5 equiv) gave 1-(2-((S)-40-(3-(tert-butoxy)-3-oxopropyl)-42-methyl-38,41-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,42-diazatritetracontan-43-yl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanes (do)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+= 2255.0400; Rt=2.97 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-((S)-40-(2-carboxyethyl)-42-methyl-38,41-dioxo-2,5,8,1 1,14,17,20,23,26,29,32,35-dodecaoxa-39,42-diazatritetracontan-43-yl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-d ihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium (L72-P1)

Following GENERAL PROCEDURE 3 with 1-(2-((S)-40-(3-(tert-butoxy)-3-oxopropyl)-42-methyl-38,41-dioxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,42-diazatritetracontan-43-yl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanes (do)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (20.1 mg, 8.91 μmmol, 1 equiv.) was obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-((S)-40-(2-carboxyethyl)-42-methyl-38,41-dioxo-2,5,8,1 1,14,17,20,23,26,29,32,35-dodecaoxa-39,42-diazatritetracontan-43-yl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium. HRMS: [M]+= 2173.9199; Rt=2.40 min (5 min acidic method).

Synthesis of 1-(2-((2-(bis(2-(tert-butoxy)-2-oxoethyl)amino)-N-methylacetamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 5 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (17.0 mg, 0.011 mmol, 1.0 equiv.) and bis(2-(tert-butoxy)-2-oxoethyl)glycine (10.97 mg, 0.017 mmol, 1.5 equiv.) is obtained 1-(2-((2-(bis(2-(tert-butoxy)-2-oxoethyl)amino)-N-methylacetamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+=1784.8000; Rt=3.31 min (5 min acidic method).

Synthesis of 1-(2-((2-(bis(carboxymethyl)amino)-N-methylacetamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (L67-P1)

Following GENERAL PROCEDURE 3 with 1-(2-((2-(bis(2-(tert-butoxy)-2-oxoethyl)amino)-N-methylacetamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (9.2 mg, 5.12 μmmol, 1 equiv.) yields 1-(2-((2-(bis(carboxymethyl)amino)-N-methylacetamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. MSHR: M+= 1647.6100; Rt=2.28 min (5 min acidic method).

General Methodology 7

Synthesis of 1-(2-(((S)-3-amino-4-(tert-butoxy)-N-methyl-4-oxobutanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

The reagents are used as a stock solution in DMF. DIPEA (22.68 μL, 0.130 mmol, 8.0 equiv) is added to (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanoic acid (8.04 mg, 161 μL, 0.020 mmol, 1.2 equiv) and 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate (V) (6.81 mg, 681 μL, 0.018 mmol, 1.1 equiv). After standing for 10 min, 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (30 mg, 353 μl, 0.016 mmol, 1.0 equiv) was added. After standing for 45 min, dimethyl amine (163 μL, 0.326 mmol) was added. After standing for 16 h, DMSO (2 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 1-(2-(((S)-3-amino-4-(tert-butoxy)-N-methyl-4-oxobutanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium is obtained. MSHR: M+=1670.2300; Rt=2.69 min (5 min acidic method).

Synthesis of 1-(2-(((S)-4-(tert-butoxy)-N-methyl-4-oxo-3-(2-sulfoacetamido)butanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 6 with 1-(2-(((S)-3-amino-4-(tert-butoxy)-N-methyl-4-oxobutanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (20.6 mg, 10.23 μmol, 1 eq.) are obtained 1-(2-(((S)-4-(tert-butoxy)-N-methyl-4-oxo-3-(2-sulfoacetamido)butanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. MSHR: M+= 1792.6899; Rt=3.17 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((S)-3-carboxy-N-methyl-3-(2-sulfoacetamido)propanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium (L79-P1)

Following GENERAL PROCEDURE 3 with 1-(2-(((S)-4-(tert-butoxy)-N-methyl-4-oxo-3-(2-sulfoacetamido)butanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (25.3 mg, 0.012 mmol, 1 equiv.) are obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((S)-3-carboxy-N-methyl-3-(2-sulfoacetamido)propanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium. HRMS: M+= 1711.5699; Rt=2.48 min (5 min acidic method).

Synthesis of 4-benzyl 1-(tert-butyl) ((3-(tert-butoxy)-3-oxopropoxy)carbonyl)-L-aspartate

To tert-butyl 3-hydroxypropanoate (111 mg, 0.760 mmol, 1 eq.) and bis(4-nitrophenyl) carbonate (289 mg, 0.950 mmol, 1.2 eq.) dissolved in DMF (2 mL) was added DIPEA (0.221 mL, 1.267 mmol, 2.0 eq.). After standing for 1 h, 4-benzyl 1-(tert-butyl) L-aspartate (200 mg, 0.633 mmol) was added. After standing for 16 h, DMSO (4 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 4-benzyl 1-(tert-butyl) ((3-(tert-butoxy)-3-oxopropoxy)carbonyl)-L-aspartate is obtained. MSVR: [M+H]+=452.4; Rt=2.68 min (5 min sour method).

Synthesis of ((S)-4-(tert-butoxy)-3-(((3-(tert-butoxy)-3-oxopropoxy)carbonyl)amino)-4-oxobutanoic acid

To 4-benzyl 1-(tert-butyl) ((3-(tert-butoxy)-3-oxopropoxy)carbonyl)-L-aspartate (52.7 mg, 0.117 mmol) dissolved in MeOH (2 mL) was added palladium hydroxide (8.20 mg, 0.012 mmol, 0.1 equiv). The reaction atmosphere was changed to hydrogen. After stirring for 16 h, the reaction mixture was filtered through a pad of celite. The filtrate was removed in vacuo to give 4-benzyl 1-(tert-butyl) ((3-(tert-butoxy)-3-oxopropoxy)carbonyl)-L-aspartate.

Synthesis 1-(2-((S)-5-(tert-butoxycarbonyl)-2,13,13-trimethyl-3,7,11-trioxo-8,12-dioxa-2,6-diazatetradecyl)-4 -((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chl or-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 7 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (20 mg, 235 µl, 0.011 mmol, 1 eq.) and 4-benzyl 1-(tert-butyl) ((3-(tert-butoxy)-3-oxopropoxy)carbonyl)-L-aspartate (7.84 mg, 204 µl, 0.022 mmol, 2 equiv) gave 1-(2-((S)-5-(tert-butoxycarbonyl)-2,13,13-trimethyl-3,7,11-trioxo-8,12-dioxa-2,6-diazatetradecyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro p-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+= 1842.8000; Rt=3.23 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((S)-3-carboxy-3-(((2-carboxyethoxy)carbonyl)amino)-N-methylpropanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium (L102-P1)

Following GENERAL PROCEDURE 3 with 1-(2-((S)-5-(tert-butoxycarbonyl)-2,13,13-trimethyl-3,7,11-trioxo-8,12-dioxa-2,6-diazatetradecyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro p-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (17.6 mg, 0.008 mmol, 1 equiv.) was obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((S)-3-carboxy-3-(((2-carboxyethoxy)carbonyl)amino)-N-methylpropanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium. MSHR: M+= 1705.6200; Rt=2.41 min (5 min acidic method).

Synthesis of tert-butyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((4-nitrophenoxy)carbonyl)-L-serinate

To tert-butyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-serinate (300 mg, 0.782 mmol, 1 equiv.) and bis(4-nitrophenyl) carbonate (357 mg, 1.174 mmol, 1.5 equiv.) dissolved in DMF (2 mL) was added DIPEA (0.136 mL, 0.782 mmol, 1.0 equiv.). After standing for 16 h, DMSO (4 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, tert-butyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((4-nitrophenoxy)carbonyl)-L-serinate is obtained. MSHR: M+= 566.4; Rt=3.01 min (5 min acidic method).

Synthesis of 1-(2-(((((S)-2-amino-3-(tert-butoxy)-3-oxopropoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

To tert-butyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((4-nitrophenoxy)carbonyl)-L-serinate (7.14 mg, 143 µl, 0.013 mmol, 1.2 equiv.) and 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (20 mg, 235 μl, 0.011 mmol, 1.0 eq.) was added DIPEA (15.1 μl, 0.087 mmol, 8.0 equiv). After standing for 16 h, dimethyl amine (109 µl, 0.217 mmol, 20 equiv) was added. The reaction mixture was stirred at RT for 1 h. DMSO (4 ml) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 1-(2-(((((S)-2-amino-3-(tert-butoxy)-3-oxopropoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium is obtained. MSHR: M+= 1686.7200; Rt=2.71 min (5 min acidic method).

Synthesis of 1-(2-(((((S)-3-(tert-butoxy)-3-oxo-2-(2-sulfoacetamido)propoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 6 with 1-(2-(((((S)-2-amino-3-(tert-butoxy)-3-oxopropoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (22.9 mg, 0.011 mmol, 1 equiv.) are obtained 1-(2-(((((S)-3-(tert-butoxy)-3-oxo-2-(2-sulfoacetamido)propoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. MSHR: M+= 1808.6899; Rt=3.18 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((((S)-2-carboxy-2-(2-sulfoacetamido)ethoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium (L77-P1)

Following GENERAL PROCEDURE 3 with 1-(2-(((((S)-2-amino-3-(tert-butoxy)-3-oxopropoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (15.3 mg, 7.11 μmol, 1 equiv.) are obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((((S)-2-carboxy-2-(2-sulfoacetamido)ethoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium. MSHR: M+= 1727.5800; Rt=2.47 min (5 min acidic method).

Synthesis of 4-benzyl 1-(tert-butyl) (4-(diethoxyphosphoryl)butanoyl)-L-aspartate

To 4-benzyl 1-(tert-butyl) L-aspartate (200 mg, 0.633 mmol, 1.0 equiv.) and 4-(diethoxyphosphoryl)butanoic acid (213 mg, 0.950 mmol, 1.5 equiv.) dissolved in DMF (2 ml) were added dicyclohexylmctanediimine (157 mg, 0.760 mmol, 1.2 equiv.), 1H-[1,2,3]triazolo[4,5-b]pyridin-1-ol hydrate (146 mg, 0.950 mmol, 1.5 equiv.) and DIPEA (0.110 ml, 0.633 mmol, 1.0 equiv.). After standing for 16 h, DMSO (4 ml) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 4-benzyl 1-(tert-butyl) (4-(diethoxyphosphoryl)butanoyl)-L-aspartate is obtained. MSVR: [M+H]+=486.4; Rt=2.15 min (5 min sour method).

Synthesis of (S)-4-(tert-butoxy)-3-(4-(diethoxyphosphoryl)butanamido)-4-oxobutanoic acid

To 4-benzyl 1-(tert-butyl) (4-(diethoxyphosphoryl)butanoyl)-L-aspartate (YUBI5-040-EXP082-001 (100 mg, 0.206 mmol, 1.0 equiv.)) dissolved in MeOH (2 mL) was added palladium hydroxide (14.46 mg, 0.021 mmol, 0.1 equiv.). The reaction atmosphere was exchanged for hydrogen. After stirring for 16 h, the reaction mixture was filtered through a pad of celite. The filtrate was discarded.in a vacuumto obtain (S)-4-(tert-butoxy)-3-(4-(diethoxyphosphoryl)butanamido)-4-oxobutanoic acid. MSHR: [M+H]+= 396.3; Rt=1.35 min (5 min sour method).

Synthesis of 1-(2-(((S)-4-(tert-butoxy)-3-(4-(diethoxyphosphoryl)butanamido)-N-methyl-4-oxobutanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 7 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (30 mg, 353 µl, 0.016 mmol, 1 eq.) and (S)-4-(tert-butoxy)-3-(4-(diethoxyphosphoryl)butanamido)-4-oxobutanoic acid (12.87 mg, 161 µl, 0.033 mmol, 2 eq.) was obtained 1-(2-(((S)-4-(tert-butoxy)-3-(4-(diethoxyphosphoryl)butanamido)-N-methyl-4-oxobutanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. MS: M/2+= 940.3; Rt=2.60 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((S)-3-carboxy-3-(4-(diethoxyphosphoryl)butanamido)-N-methylpropanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium (L103-P1)

Following the GENERAL METHOD 3 s 1-(2-(((S)-4-(tert-butoxy)-3-(4-(diethoxyphosphoryl)butanamido)-N-methyl-4-oxobutanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (20 mg, 0.009 mmol, 1 equiv.), obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((S)-3-carboxy-3-(4-(diethoxyphosphoryl)butanamido)-N-methylpropanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium. MSHR: M+= 1795.6700; Rt=2.44 min (5 min acidic method).

Synthesis of 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-2,6-diamino-N-methylhexanamido)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 7 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (20 mg, 0.011 mmol, 1 eq.) and (S)-2,5-bis((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pentanoic acid (7.51 mg, 0.013 mmol, 1.2 eq.) was obtained 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-2,6-diamino-N-methylhexanamido)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+= 1627.4000; Rt=2.48 min (5 min acidic method).

Synthesis of 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-N-methyl-2,6-bis(2-sulfoacetamido)hexanamido)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 6 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-2,6-diamino-N-methylhexanamido)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (9.7 mg, 0.0049 mmol, 1 equiv.), 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-N-methyl-2,6-bis(2-sulfoacetamido)hexanamido)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium is obtained. HRMS: M+= 1871.6700; Rt=3.02 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-N-methyl-2,6-bis(2-sulfoacetamido)hexanamido)methyl)benzyl)-1-methylpiperazin-1-ium (L78-P1)

Following GENERAL PROCEDURE 3 with -(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-N-methyl-2,6-bis(2-sulfoacetamido)hexanamido)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (15 mg, 7.55 μmol, 1 equiv.), 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-N-methyl-2,6-bis(2-sulfoacetamido)hexanamido)methyl)benzyl)-1-methylpiperazin-1-ium is obtained. MSHR: M+= 1846.6000; Rt=2.49 min (5 min acidic method).

Synthesis of 4-benzyl 1-(tert-butyl) ((2S,3S,4S,5R,6S)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2-carbonyl)-L-aspartate

To (2S,3S,4S,5R,6S)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2-carboxylic acid (344 mg, 0.950 mmol) and 4-benzyl 1-(tert-butyl) L-aspartate (300 mg, 0.950 mmol) dissolved in DMF (3.2 mL) were added DIPEA (0.165 mL, 0.950 mmol, 1.0 eq.), 1H-[1,2,3]triazolo[4,5-b]pyridin-1-ol hydrate (154 mg, 0.997 mmol, 1.05 eq.) and 3-(((ethylimino)methylene)amino)-N, N-dimethylpropan-1-amine hydrochloride (191 mg, 0.997 mmol, 1.05 equiv.). After standing for 16 h, DMSO (4 ml) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 4-benzyl 1-(tert-butyl) ((2S,3S,4S,5R,6S)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2-carbonyl)-L-aspartate is obtained. MSHR: M+=641.5; Rt=2.55 min (5 min acidic method).

Synthesis of (S)-4-(tert-butoxy)-4-oxo-3-((2S,3S,4S,5R,6S)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2-carboxamido)butanoic acid

Palladium hydroxide (11.26 mg, 0.016 mmol, 0.1 equiv) was added to 4-benzyl 1-(tert-butyl) ((2S,3S,4S,5R,6S)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2-carbonyl)-L-aspartate (100 mg, 0.160 mmol, 1.0 equiv) dissolved in MeOH (2 mL). The reaction atmosphere was exchanged for hydrogen. After stirring for 16 h, the reaction mixture was filtered through a pad of celite. The filtrate is removed in vacuo to give (S)-4-(tert-butoxy)-4-oxo-3-((2S,3S,4S,5R,6S)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2-carboxamido)butanoic acid. HRMS: [M-H]-= 532.3, Rt=1.71 min (5 min acidic method).

Synthesis of 1-(2-(((S)-4-(tert-butoxy)-N-methyl-4-oxo-3-((2R,3R,4R,5S,6R)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2-carboxamido)butanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido) benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 7 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (30 mg, 0.016 mmol, 1.0 eq.) and (S)-4-(tert-butoxy)-4-oxo-3-((2S,3S,4S,5R,6S)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2-carboxamido)butanoic acid (12.2 mg, 968 µl, 0.023 mmol, 1.4 equiv.) was obtained 1-(2-(((S)-4-(tert-butoxy)-N-methyl-4-oxo-3-((2R,3R,4R,5S,6R)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2-carboxamido)butanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido) benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+=2014.7800; Rt=3.21 min (5 min acidic method).

Synthesis of 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-3-carboxy-N-methyl-3-((2R,3R,4R,5S,6R)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2-carboxamido)propanamido)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

K 1-(2-(((S)-4-(tert-butoxy)-N-methyl-4-oxo-3-((2R,3R,4R,5S,6R)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2-carboxamido)butanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido) benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (22.7 mg, 0.009 mmol, 1.0 eq.) dissolved in DCM (32 mL) was added TFA (0.67 mL). After stirring for 45 min, the solvent was removed in vacuo to give 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-3-carboxy-N-methyl-3-((2R,3R,4R,5S,6R)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2-carboxamido)propanamido)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+=1738.6200; Rt=2.30 min (5 min acidic method).

Synthesis of 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-3-carboxy-N-methyl-3-((2R,3R,4R,5S,6S)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxamido)propanamido)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

K 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-3-carboxy-N-methyl-3-((2R,3R,4R,5S,6R)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2-carboxamido)propanamido)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (22.7 mg, 0.009 mmol, 1.0 equiv.) dissolved in THF (1 mL) and MeOH (1 mL), lithium hydroxide (5.04 mg, 0.120 mmol, 10 equiv.) was added. After stirring for 2 h, the solvent was removedin a vacuum. Water (1 ml), TFA (0.2 ml), MeCN (1 ml) and DMSO (4 ml) were added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-3-carboxy-N-methyl-3-((2R,3R,4R,5S,6S)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxamido)propanamido)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium is obtained. MSHR: M+=1570.5900; Rt=2.02 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((S)-3-carboxy-N-methyl-3-((2R,3R,4R,5S,6S)-3,4,5,6 -tetrahydroxytetrahydro-2H-pyran-2-carboxamido)propanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium (L68-P1)

K 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-3-carboxy-N-methyl-3-((2R,3R,4R,5S,6S)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxamido)propanamido)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (10.8 mg, 0.0056 mmol, 1.0 equiv) and 2,5-dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (5.25 mg, 0.017 mmol, 3.5 equiv) dissolved in DMF (1 mL) was added DIPEA (7.86 µL, 0.045 mmol, 8 equiv). After standing for 1.5 h, DMSO (2 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((S)-3-carboxy-N-methyl-3-((2R,3R,4R,5S,6S)-3,4,5,6 -tetrahydroxytetrahydro-2H-pyran-2-carboxamido)propanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium. MSHR: M+=1765.6500; Rt=2.31 min (5 min acidic method).

Synthesis of 4-benzyl 1-(tert-butyl) ((((3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)-L-aspartate

To ((3R,4S,5S,6S)-2-hydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (254 mg, 0.760 mmol, 1.2 equiv.) and bis(4-nitrophenyl) carbonate (289 mg, 0.950 mmol, 1.5 equiv.) dissolved in DMF (2 mL) was added DIPEA (0.221 mL, 1.267 mmol, 2.0 equiv.). After standing for 1 h, 4-benzyl 1-(tert-butyl) L-aspartate (200 mg, 0.633 mmol, 1.0 equiv.) was added. After standing for 16 h, DMSO (6 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 4-benzyl 1-(tert-butyl) ((((3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)-L-aspartate is obtained. MSVR: [M-H]-= 638.4; Rt=2.61 min (5 min sour method).

Synthesis of (3S)-4-(tert-butoxy)-4-oxo-3-(((((3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)butanoic acid

To 4-benzyl 1-(tert-butyl) ((((3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)-L-aspartate (50 mg, 0.078 mmol, 1.0 equiv.) dissolved in MeOH (2 mL) was added palladium hydroxide (5.49 mg, 7.82 μmol, 0.1 equiv.). The reaction atmosphere was exchanged with hydrogen. After stirring for 16 h, the reaction mixture was filtered through a pad of celite. The filtrate was removed in vacuo to give (3S)-4-(tert-butoxy)-4-oxo-3-(((((3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)butanoic acid. HRMS: [M-H]-: 548.4, Rt=1.79 min (5 min acidic method).

Synthesis of 1-(2-(((3S)-4-(tert-butoxy)-N-methyl-4-oxo-3-(((((3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)butanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-urea dopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 7 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (30 mg, 0.016 mmol, 1.0 eq.) and (3S)-4-(tert-butoxy)-4-oxo-3-(((((3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)butanoic acid (12.5 mg, 0.250 ml, 0.023 mmol, 1.4 equiv.) was obtained 1-(2-(((3S)-4-(tert-butoxy)-N-methyl-4-oxo-3-(((((3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)butanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureide (opentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+=2030.7900; Rt=3.19 min (5 min acidic method).

Synthesis of 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((3S)-3-carboxy-N-methyl-3-(((((3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)propanamido)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

K 1-(2-(((3S)-4-(tert-butoxy)-N-methyl-4-oxo-3-(((((3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)butanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-urea (dopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (25.8 mg, 10.86 μmol, 1.0 equiv.) dissolved in DCM (2 mL) was added TFA (0.67 mL). After stirring for 2 h, the solvent was removed in vacuo to give 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((3S)-3-carboxy-N-methyl-3-(((((3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)propanamido)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. MSHR: M+=1754.6200; Rt=2.31 min (5 min acidic method).

Synthesis 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((3S)-3-carboxy-3-(((((3R,4S, 5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)-N-methylpropanamide o)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)e toxic)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

K 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((3S)-3-carboxy-N-methyl-3-(((((3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)propanamido)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (26 mg, 0.012 mmol, 1.0 equiv.) dissolved in THF (1 mL) and MeOH (1 mL) was added lithium hydroxide (5.20 mg, 0.124 mmol, 10 equiv.). After stirring for 2 h, the solvent was removed.in a vacuum. Water (1 ml), TFA (0.2 ml), MeCN (1 ml) and DMSO (4 ml) were added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((3S)-3-carboxy-3-(((((3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)-N-methylpropanamido)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium is obtained. MSHR: M+=1614.5800; Rt=2.04 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((3S)-3-carboxy-3-((((3R,4S,5S,6S)-6-carboxy-3,4,5-trihydro roxytetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)-N-methylpropanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium (L69-P1)

To 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((3S)-3-carboxy-3-(((((3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)-N-methylpropanamido)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (10 mg, 5.11 μmol, 1.0 equiv) and 2,5-dioxopyrrolidin-1-yl 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanoate (4.75 mg, 0.015 mmol, 3.5 equiv) dissolved in DMF (1 mL) was added DIPEA (7.12 μL, 0.041 mmol, 8 equiv). After standing for 1.5 h, DMSO (2 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((3S)-3-carboxy-3-((((3R,4S,5S,6S)-6-carboxy-3,4,5-trihyd roxytetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)-N-methylpropanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium. MSHR: M+=1809.6300; Rt=2.32 min (5 min acidic method).

Synthesis of 1-(2-((3-(2-(2-aminoethoxy)ethoxy)-N-methylpropanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

A mixture of 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (45 mg, 0.026 mmol), 1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecane-13-oic acid (12 mg, 0.030 mmol), HBTU (12 mg, 0.032 mmol) and DIPEA (0.023 mL, 0.13 mmol) in DMF (1 mL) were stirred at RT for 30 min. Me ₂ NH (2 M in THF, 0.065 mL, 0.13 mmol) was added and the mixture was stirred at RT for 1 h. Additional Me ₂ NH (2 M in THF, 0.1 mL, 0.2 mmol) was added. The mixture was continued to stir at RT for 1 h, diluted with DMSO (3 ml) and the solution was purified by RP-HPLC ISCO gold chromatography (MeCN/H ₂ O, 0.1% TFA modifier). After lyophilization, 1-(2-((3-(2-(2-aminoethoxy)ethoxy)-N-methylpropanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium is obtained. MSHR: M+= 1658.7200, Rt=2.57 min (5 min acidic method).

Synthesis of 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2-methyl-3,13-dioxo-15-phosphono-6,9-dioxa-2,12-diazapentadecyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

A mixture of 3-phosphopropionic acid (11 mg, 0.071 mmol), HBTU (27 mg, 0.071 mmol) and DIPEA (0.060 ml, 0.34 mmol) in DMF (0.5 ml) was stirred at RT for 10 min. This mixture was added to a solution of 1-(2-((3-(2-(2-aminoethoxy)ethoxy)-N-methylpropanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (40 mg, 0.021 mmol) and DIPEA (0.010 mL, 0.057 mmol) in DMF (0.5 mL). The mixture was stirred at RT for 2 days. The mixture was diluted with DMSO (3 mL) and the solution was purified by RP-HPLC ISCO gold chromatography (MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2-methyl-3,13-dioxo-15-phosphono-6,9-dioxa-2,12-diazapentadecyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium is obtained. HRMS: M+= 1794.7100, Rt=2.78 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(2-methyl-3,13-dioxo-15-phosphono-6,9-dioxa-2,12-diazapentadecyl)benzyl)-1-methylpiperazin-1-ium (L41-P1)

FollowingGENERAL METHOD 3(except that the product after the first stage with TFA/CH₂Cl₂purified by RP-HPLC ISCO gold chromatography [MeCN/H₂O, 0.1% NH₄OH modifier]) with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2-methyl-3,13-dioxo-15-phosphono-6,9-dioxa-2,12-diazapentadecyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium, is obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(2-methyl-3,13-dioxo-15-phosphono-6,9-dioxa-2,12-diazapentadecyl)benzyl)-1-methylpiperazin-1-ium. HRMS: M+= 1769.4500, Rt=2.33 min (5 min acidic method).

Synthesis 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2,5,8,11,14,17,20,23,26,29,32,3 5,38,44,44-pentadecamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42-tetradecaoxo-43-oxa-2,5,8,11,14,17,20,23,26,29,32,35,38-t (ridecaazapentatetracontyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

To a stirred solution of 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (43.6 mg, 0.025 mmol, 1.0 equiv.), 3,6,9,12,15,18,21,24,27,30,33,36,42,42-tetradecamethyl-4,7,10,13,16,19,22,25,28,31,34,37,40-tridecaoxo-41-oxa-3,6,9,12,15,18,21,24,27,30,33,36-dodecaazatritetracontanoic acid (25.9 mg, 0.025 mmol, 1.0 equiv.) and GATU (10.5 mg, 0.028 mmol, 1.1 equiv.) in DMF (0.25 mL) were added DIPEA (22 μL, 0.126 mmol, 5.0 equiv.). The resulting solution was stirred at ambient temperature for 1 hour. The reaction mixture was diluted with 1 ml DMSO and purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H ₂ O, 0.1% TFA modifier). After lyophilization, 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2,5,8,11,14,17,20,23,26,29,32,35 ,38,44,44-pentadecamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42-tetradecaoxo-43-oxa-2,5,8,11,14,17,20,23,26,29,32,35,38-tr idecaazapentatetracontyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS M+ 2508.1499 Rt=2.78 min (5 min acidic method).

Synthesis of 1-(2-(41-carboxy-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39-tridecaoxo-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaazagenetetracontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethox i)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimide in-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (L35-P1)

Following GENERAL PROCEDURE 3 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2,5,8,11,14,17,20,23,26,29,32,35,38,44,44-pentadecamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42-tetradecaoxo-43-oxa-2,5,8,11,14,17,20,23,26,29,32,35,38-tr idecaazapentatetracontyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (50.4 mg, 0.019 mmol, 1.0 equiv.) was obtained 1-(2-(41-carboxy-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39-tridecaoxo-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaazahentetracontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethox (i)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+ 2427.0400, rt=2.30 min (5 min acidic method).

Synthesis 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,4 7,50,53,56,62,62-genicosamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,4 2,45,48,51,54,57,60-icosaoxo-61-oxa-2,5,8,11,14,17,20,23,26,29,32, 35,38,41,44,47,50,53,56-nonadecaazatrihexacontyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

To a stirred solution of 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (31.9 mg, 0.018 mmol, 1.0 equiv.), 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,60,60-icosamine-4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58-nonadecaoxo-59-oxa-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54-octadecaazagenehexacontanoic acid (26.8 mg, 0.018 mmol, 1.0 equiv.) and GATU (7.7 mg, 0.020 mmol, 1.1 equiv.) DIPEA (16 μL, 0.092 mmol, 5.0 equiv.) was added to DMF (0.25 mL). The resulting solution was stirred at ambient temperature for 1 h. The reaction mixture was diluted with 1 mL DMSO and purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H ₂ O, 0.1% TFA modifier). After lyophilization, 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47 ,50,53,56,62,62-genicosamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42 ,45,48,51,54,57,60-icosaoxo-61-oxa-2,5,8,11,14,17,20,23,26,29,32,3 5,38,41,44,47,50,53,56-nonadecaazatrihexacontyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS M+ 2934.3601 Rt=2.73 min (5 min acidic method).

Synthesis of 1-(2-(59-carboxy-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56-nonadecamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57-nonadecaoxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56-nonadecaazanonepentacontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5 -dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (L36-P1)

Following GENERAL PROCEDURE 3 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,62,62-henycosamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60-icosaoxo-61-oxa-2,5,8,11,14,17,20,23,26,29,32,3 5,38,41,44,47,50,53,56-nonadecaazatrihexacontyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (45.5 mg, 0.015 mmol, 1.0 equiv.) was obtained 1-(2-(59-carboxy-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53 ,56-nonadecamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54 ,57-nonadecaoxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56-nonadecaazanonepentacontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5 -dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+ 2853.2766, rt=2.20 min (5 min acidic method).

Synthesis of 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,6 2,65,68,71,74,80,80-heptacosamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,4 5,48,51,54,57,60,63,66,69,72,75,78-hexacosaoxo-79-oxa-2,5,8,11,14,17,20,2 3,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74-pentacosaazagenoctacontyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)ox i)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

To a stirred solution of 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (27.8 mg, 0.016 mmol, 1.0 equiv.), 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,78,78-hexacosamethyl-4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55, 58,61,64,67,70,73,76-pentacosaoxo-77-oxa-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaazanone heptacontanoic acid (30.2 mg, 0.016 mmol, 1.0 equiv) and GATU (6.7 mg, 0.018 mmol, 1.1 equiv) in DMF (0.25 mL) were added DIPEA (14 μL, 0.080 mmol, 5.0 equiv). The resulting solution was stirred at ambient temperature for 1 h. The reaction mixture was diluted with 1 ml DMSO and purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H ₂ O, 0.1% TFA modifier). After lyophilization, 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,6) was obtained 2,65,68,71,74,80,80-heptacosamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,4 5,48,51,54,57,60,63,66,69,72,75,78-hexacosaoxo-79-oxa-2,5,8,11,14,17,20,2 3,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74-pentacosaazagenoctacontyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS M+ 3360.5798 Rt=2.68 min (5 min acidic method).

Synthesis 1-(2-(77-carboxy-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59, 62,65,68,71,74-pentacosamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48, 51,54,57,60,63,66,69,72,75-pentacosaoxo-2,5,8,11,14,17,20,23,26,29,32,35,3 8,41,44,47,50,53,56,59,62,65,68,71,74-pentacoseazaheptaheptacontyl)-4-((S)-2 -((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (L37-P1)

Following GENERAL PROCEDURE 3 with 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,6 2,65,68,71,74,80,80-heptacosamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,4 5,48,51,54,57,60,63,66,69,72,75,78-hexacosaoxo-79-oxa-2,5,8,11,14,17,20,2 3,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74-pentacosaazagenoctacontyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (41.3 mg, 0.012 mmol, 1.0 equiv.) was obtained 1-(2-(77-carboxy-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59, 62,65,68,71,74-pentacosamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48, 51,54,57,60,63,66,69,72,75-pentacosaoxo-2,5,8,11,14,17,20,23,26,29,32,35,3 8,41,44,47,50,53,56,59,62,65,68,71,74-pentacoseazaheptaheptacontyl)-4-((S)-2 -((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+ 3279.4678, rt=2.21 min (5 min acidic method).

Synthesis of Tert-butyl 1-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)-3,6,9,12,15,18-hexaoxagenicosane-21-oate

To 2-azidoethan-1-ol (105 mg, 1.21 mmol) in CH ₂ Cl ₂ (15 mL) was added sulfur isocyanatide chloride (0.105 mL, 1.21 mmol) at 0 °C. The mixture was stirred at 0 °C for 30 min, then TEA (0.336 mL, 2.41 mmol) and tert-butyl 1-amino-3,6,9,12,15,18-hexaoxagenicosan-21-oate (518 mg, 1.27 mmol) in CH ₂ Cl ₂ (1 mL) were added. The mixture was stirred at 0 °C for 1 h and rt for 2 h, then quenched with sat. NH ₄ Cl and 1 N HCl (2.4 mL). The aqueous layer was extracted with CH ₂ Cl ₂ (5X). The organic layers were dried over _{anhydrous Na2SO4} , filtered _, and concentrated by rotary evaporation to give a clear oil. Purification by flash chromatography (0-15% MeOH in _CH2Cl2 , ELSD determination) gave tert-butyl 1-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)-3,6,9,12,15,18-hexaoxagenicosan-21-oate as a clear oil (474 mg, 0.788 mmol): LCMS: M+NH4+=619.5, Rt=0.94 min (acidic, 2 min). ¹ H NMR (400 MHz, DMSO-d6) δ 11.33 (s, 1H), 7.76 (s, 1H), 4.28-4.20 (m, 2H), 3.60 (td, J=5.6, 5.0, 2.9 Hz, 4H), 3.55-3.45 (m, 22H), 3.16-3.04 (m, 2H), 2.46-2.38 (m, 2H), 1.41 (s, 9H).

Synthesis of 1-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)-3,6,9,12,15,18-hexaoxagenicosan-21-oic acid

To tert-butyl 1-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino) _{-3,6,9,12,15,18} -hexaoxagenicosan-21-oate (145 mg, 0.241 mmol) in _CH2Cl2 (1 mL) at 0°C was added TFA (1 mL, 12.98 mmol). The mixture was stirred at RT for 1.45 h, then concentrated by rotary evaporation at 25°C on a water bath and dried under high vacuum for 30 min. The residue was azeotropically dried with anhydrous. toluene (3 x 2 mL) and dried under vacuum overnight to give 1-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)-3,6,9,12,15,18-hexaoxagenicosan-21-oic acid as a clear oil (147 mg, 89% w/w of theoretical yield): LCMS: M+=546.3, 0.65 min (acidic, 2 min, ELSD); ^1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 7.77-7.71 (m, 1H), 4.25-4.19 (m, 2H), 3.63-3.55 (m, 4H), 3.54-3.45 (m, 22H), 3.07 (sq, J=6.0 Hz, 2H), 2.47-2.40 (m, 2H).

Synthesis 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2- methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium trifluoroacetate

To 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium trifluoroacetate (321 mg, 0.210 mmol) at 0°C was added 25% TFA in CH ₂ Cl ₂ (12.3 mL, 40.0 mmol). The reaction mixture was raised to RT, stirred for 1 h, then concentrated under high vacuum at RT on a water bath. The crude product was diluted with DMSO (3 mL) and purified by RP-HPLC ISCO gold chromatography (150 g, 10-80% MeCN/H ₂ O, 0.1% TFA modifier). After lyophilization, 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium trifluoroacetate was obtained as a white powder (224 mg, 0.158 mmol): HRMS: M+=1304.5100, Rt=2.15 min (5 min acidic)

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium trifluoroacetate

Following GENERAL PROCEDURE 3 with 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium trifluoroacetate (164 mg, 0.115 mmol) and Mal-Peg1-NHS ester (72 mg, 0.23 mmol), 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium trifluoroacetate was obtained as a white powder (170 mg, 0.105 mmol), HRMS: M+ =1499.5699, Rt=2.45 min (5 min sour); ¹ H NMR (400 MHz, DMSO-d6) δ 10.16 (s, 1H), 8.81 (d, J=5.1 Hz, 1H), 8.54 (s, 1H), 8.07 (d, J=7.2 Hz, 1H), 7.75 (d, J=8.5 Hz, 1H), 7.69 (d, J=6.9 Hz, 2H), 7.56 (d, J=5.1 Hz, 1H), 7.45 (dd, J=7.5, 1.8 Hz, 1H), 7.42-7.29 (m, 3H), 7.24 (dd, J=8.9, 5.4 Hz, 2H), 7.18-7.05 (m, 5H), 6.99-6.91 (m, 4H), 6.65 (t, J=7.4 Hz, 1H), 6.15 (d, J=7.0 Hz, 1H), 5.91 (s, 1H), 5.43 (dd, J=9.8, 3.5 Hz, 1H), 5.23-5.12 (m, 2H), 4.54 (d, J=11.2 Hz, 4H), 4.33-4.09 (m, 7H), 3.69 (s, 3H), 3.42-2.78 (m, 32H, overlay with DMSO), 2.39-2.20 (m, 2H), 1.91-1.81 (m, 1H), 1.77 (s, 3H), 1.46 (dd, J=93.8, 31.3 Hz, 3H), 0.76 (dd, J=13.8, 6.7 Hz, 6H); 19F NMR (376 MHz, DMSO-d6): -112.18 ppm.

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((1-(2-(((N-(20-carboxy-3,6,9,12,15,18-hexaoxaicosyl l)sulfamoyl)carbamoyl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium trifluoroacetate (L59-P1)

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium trifluoroacetate (32 mg, 0.021 mmol) and 1-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)-3,6,9,12,15,18-hexaoxagenicosan-21-oic acid (26.2 mg, 0.043 mmol), 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((1-(2-(((N-(20-carboxy-3,6,9,12,15,18-hexaoxaicosin (l)sulfamoyl)carbamoyl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium trifluoroacetate is obtained as a white powder: HRMS: M+ = 2044.7700, Rt = 2.36 min (5 min acidic).

Synthesis of 3-(2-(2-(2-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)ethoxy)ethoxy)ethoxy)propanoic acid

To 2-azidoethan-1-ol (105 mg, 1.21 mmol) in CH ₂ Cl ₂ (15 mL) was added sulfur isocyanatide chloride (0.105 mL, 1.21 mmol) at 0 °C. The mixture was stirred at 0 °C for 30 min. TEA (0.336 mL, 2.41 mmol) and tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoate (401 mg, >80% technical grade, 1.447 mmol) in CH ₂ Cl ₂ (1 mL) were added at 0 °C. After stirring at 0 °C for 1 h, then at rt for 1 s, the mixture was quenched with sat. NH ₄ Cl and 1 N HCl (2.4 mL). The aqueous layer was extracted _{with CH2Cl2} (5X). The organic layers were dried over anh. _Na2SO4 , _{filtered ,} and concentrated by rotary evaporation to give a clear oil. After purification by flash chromatography (0-15% MeOH in _CH2Cl2 , ELSD determination), tert-butyl 3-(2-(2-(2-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)ethoxy)ethoxy)ethoxy)propanoate was obtained as a thick clear oil (356 mg, 0.910 mmol): LCMS: MS+NH4+=487.4, Rt=0.90 min (acidic, 2 min, ELSD); ¹ H NMR (400 MHz, DMSO-d6) δ 11.31 (s, 1H), 7.78-7.70 (m, 1H), 4.26-4.19 (m, 2H), 3.58 (td, J=5.6, 5.0, 3.7 Hz, 4H), 3.53-3.39 (m, 10H), 3.10-3.03 (m, 2H), 2.44-2.39 (m, 2H), 1.40 (s, 9H).

To tert-butyl 3-(2-(2-(2-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)ethoxy)ethoxy)ethoxy)propanoate (162 mg, 0.345 mmol) in CH ₂ Cl ₂ (1 mL) at 0°C was added TFA (1 mL, 13.0 mmol). After stirring at RT for 2 h, the mixture was concentrated by rotary evaporation on a water bath at 25°C. The residue was dried under high vacuum for 30 min, azeotropically distilled with anhydrous toluene (3X 2 mL) and in vacuo overnight to give 3-(2-(2-(2-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)ethoxy)ethoxy)ethoxy)propanoic acid as a thick oil (139 mg, 0.335 mmol): LCMS: MS+NH4+=431.4, Rt=0.62 min (acidic, 2 min, ELSD): ¹ H NMR (400 MHz, DMSO-d6) δ 11.32 (d, J=15.4 Hz, 1H), 7.78-7.69 (m, 1H), 4.27-4.15 (m, 2H), 3.67-3.54 (m, 8H), 3.49-3.42 (m, 4H), 3.07 (sq, J=6.0 Hz, 2H), 2.47-2.39 (m, 4H).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((1-(2-(((N-(2-(2-(2-(2-(2-carboxyethoxy)ethoxy)ethoxy)eth (yl)sulfamoyl)carbamoyl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium trifluoroacetate (L60-P1)

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium trifluoroacetate (15 mg, 10 μmol) and 3-(2-(2-(2-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)ethoxy)ethoxy)ethoxy)propanoic acid (12 mg, 0.029 mmol), 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((1-(2-(((N-(2-(2-(2-(2-carboxyethoxy)ethoxy)ethoxy)eth (yl)sulfamoyl)carbamoyl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium trifluoroacetate was obtained as a white powder: HRMS: MS+=1912.7000, Rt=2.38 min (5 min acidic).

Synthesis of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate

A mixture of tert-butyl (2-(2-hydroxyethoxy)ethyl)carbamate ( ₁₅₃₅ mg, 7.48 mmol), _Ag2CO3 ( ₈₂₄₈ mg, 14.96 mmol), and a piece of crystalline iodine in _CH2Cl2 (6 mL) was stirred with powdered 4A molecular sieves (1400 mg) for 15 min. To the mixture was added ( 2R,3R,4S,5R,6R )-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate (2050 mg, 4.99 mmol) _{in CH2Cl2 (} 6.00 mL), also stirred with powdered 4A molecular sieves (1400 mg) for 15 min before addition. The resulting mixture was covered with aluminum foil and stirred at RT for 60 h and then filtered through Celite washing with EtOAc. The filtrate was concentrated to give a clear oil, which was purified _by flash chromatography (0-10% MeOH in _CH2Cl2 , ELSD determination) to give, after concentration of appropriate fractions, a thick oil (640 mg) as a 47/53% mixture of the desired ( 2R,3R,4S,5R,6R )-2-(acetoxymethyl)-6-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate and ( 2S,3aR,5R,6R,7S,7aR )-5-(acetoxymethyl)-2-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethoxy)-2-methyltetrahydro-5H-[1,3]dioxolo[4,5-b]pyran-6,7-diyl-diacetate: LCMS: MS+=536.4, Rt=0.96 min (2 min, acid, ELSD); ¹ H NMR (400 MHz, DMSO-d6) δ 6.77 (s, 2H), 5.78 (s, 2H), 5.76 (s, 1H), 5.28 (t, J=9.5 Hz, 1H), 5.04 (t, J=3.1 Hz, 1H), 4.91 (t, J=9.7 Hz, 1H), 4.87-4.74 (m, 3H), 4.38 (ddd, J=5.2, 3.1, 0.9 Hz, 1H), 4.24-4.10 (m, 3H), 4.07-3.96 (m, 2H), 3.92 (dt, J=8.8, 4.1 Hz, 1H), 3.80 (dt, J=11.2, 4.4 Hz, 1H), 3.68-3.60 (m, 1H), 3.57-3.45 (m, 7H), 3.38 (td, J=6.2, 1.5 Hz, 7H), 3.07 (dt, J=6.1, 3.0 Hz, 4H), 2.08 (s, 3H), 2.06 (s, 3H), 2.04 (s, 6H), 2.01 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.65 (s, 3H), 1.39 (s, 19H).

Synthesis of (2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(2-(2-aminoethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate

To a 47/53% mixture of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate and (2S,3aR,5R,6R,7S,7aR)-5-(acetoxymethyl)-2-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethoxy)-2-methyltetrahydro-5H-[1,3]dioxolo[4,5-b]pyran-6,7-diyl diacetate (622 mg, 1.161 mmol) in CH ₂ Cl ₂ (16 mL) at 0°C was added TFA (4.0 mL, 52 mmol). The reaction mixture was raised to rt and stirred for 1 h. The mixture was concentrated and the residue was dried in vacuo for 60 min to give a light yellow oil. The crude product was diluted with DMSO (4 mL) and purified by RP-HPLC ISCO gold chromatography (5-60% MeCN/H ₂ O, 0.1% TFA modifier, ELSD determination). After lyophilization of the appropriate fractions, (2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(2-(2-aminoethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate, as the TFA salt, was obtained as a white powder (195 mg, 0.355 mmol): LCMS: MS+ = 436.4, Rt = 0.58 min (acidic, 2 min); ¹ H NMR (400 MHz, DMSO-d6) δ 7.72 (s, 3H), 5.27 (t, J=9.4 Hz, 1H), 4.92 (t, J=9.6 Hz, 1H), 4.86-4.75 (m, 2H), 4.22-4.15 (m, 1H), 4.07-3.94 (m, 3H), 3.89-3.53 (m, 14H, mixed with DMSO), 3.03-2.91 (m, 2H), 2.03 (s, 3H), 2.01 (s, 3H), 1.99 (s, 3H), 1.94 (s, 3H).

Synthesis of 2-azidoethyl (N-(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)sulfamoyl)carbamate

To 2-azidoethan-1-ol (16 mg, 0.18 mmol) in CH ₂ Cl ₂ (2.5 mL) was added sulfur isocyanatide chloride (0.016 mL, 0.184 mmol) at 0 °C. The mixture was stirred at 0 °C for 1 h, then TEA (0.128 mL, 0.919 mmol) and ( 2R,3R,4S,5R,6R )-2-(acetoxymethyl)-6-(2-(2-aminoethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate, as the TFA salt (116 mg, 0.211 mmol) in CH ₂ Cl ₂ (1.5 mL) were added. After stirring at 0 °C for 1 h, then at rt for 1 h, the mixture was quenched with sat. NH ₄ Cl and 1 N HCl (0.919 mL, 0.919 mmol). The aqueous layer was extracted with CH ₂ Cl ₂ (5X). The organic layer was washed over anh. Na ₂ SO ₄ , filtered and concentrated by rotary evaporation to give (2R,3R,4S,5R,6R)-2- (acetoxymethyl)-6-(2-(2-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate as a clear oil (121 mg): LCMS: MS+=628, Rt=0.85 min (acidic, 2 min, ELSD); ¹ H NMR (400 MHz, DMSO-d6) δ 11.32 (s, 1H), 7.79-7.71 (m, 1H), 5.26 (t, J=9.5 Hz, 1H), 4.90 (t, J=9.7 Hz, 1H), 4.85-4.73 (m, 2H), 4.25-4.14 (m, 3H), 4.06-3.94 (m, 2H), 3.79 (ddd, J=11.3, 5.3, 3.8 Hz, 1H), 3.68-3.40 (m, 8H), 3.16-3.00 (m, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H), 1.94 (s, 3H).

The product obtained above was dissolved in dioxane (3 mL) and cooled at 0°C. LiOH.H ₂ O (0.5 M in water, 2.94 mL, 1.47 mmol) was added. The resulting clear solution was stirred at RT for 1 h and then quenched at 0°C with HCl (5N, 0.147 mL, 0.735 mmol). The mixture was concentrated by rotary evaporation at 20°C on a water bath to remove most of the dioxane. The residual solution (ca. 3 mL) was purified by prep-HPLC (Sunfire 5 μm 30×50 mm column, 2-12% acetonitrile with 0.1% FA in water. Flow rate: 75 mL/min., MS 459.3, 476.3 determination) to give, after removal of the solvent of the appropriate fractions by lyophilization, 2-azidoethyl (N-(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)sulfamoyl)carbamate as a semi-solid (64 mg, 0.14 mmol): LCMS: M+NH4+=477.3, MS-=458 (acidic, 2 min, ELSD); ¹ H NMR (400 MHz, DMSO-d6) δ 11.32 (s, 1H), 7.71 (s, 1H), 4.96 (d, J=4.9 Hz, 1H), 4.89 (dd, J=13.7, 4.8 Hz, 2H), 4.47 (t, J=5.9 Hz, 1H), 4.22 (t, J=5.0 Hz, 2H), 4.15 (d, J=7.8 Hz, 1H), 3.90-3.81 (m, 1H), 3.67 (ddd, J=12.0, 5.7, 2.0 Hz, 1H), 3.62-3.39 (m, 8H), 3.19-2.90 (m, 6H).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-meth (ylbutanamido)-5-ureidopentanamido)-2-(((1-(2-(((N-(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)sulfamoyl)carbamoyl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzyl)-1-methylpiperazin-1-ium trifluoroacetate (L63-P1)

FollowingGENERAL METHOD 2with 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium trifluoroacetate (36 mg, 0.022 mmol) and 2-azidoethyl (N-(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)sulfamoyl)carbamate (22 mg, 0.048 mmol), 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-meth (ylbutanamido)-5-ureidopentanamido)-2-(((1-(2-(((N-(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)sulfamoyl)carbamoyl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzyl)-1-methylpiperazin-1-ium trifluoroacetate obtained in the form of a white powder: MSHR: MS+=1958.6899, Rt=2.31 min (5 min acidic);¹H NMR (400 MHz, DMSO-d6) δ 11.17 (s, 1H), 10.16 (s, 1H), 8.81 (d, J=5.2 Hz, 1H), 8.54 (s, 1H), 8.07 (d, J=13.5 Hz, 2H), 7.74 (d, J=8.6 Hz, 1H), 7.68 (kv, J=4.3, 2.7 Hz, 3H), 7.56 (d, J=5.1 Hz, 1H), 7.45 (dd, J=7.6, 1.8 Hz, 1H), 7.42-7.28 (m, 3H), 7.23 (ddd, J=8.5, 5.4, 2.6 Hz, 2H), 7.17-7.04 (m, 5H), 6.99-6.91 (m, 4H), 6.65 (t, J=7.4 Hz, 1H), 6.14 (dd, J=7.6, 1.8 Hz, 1H), 5.93 (s, 1H), 5.43 (dd, J=9.8, 3.6 Hz, 1H), 5.23-5.11 (m, 2H), 4.59 (dd, J=11.3, 7.4 Hz, 8H), 4.42-4.06 (m, 12H), 3.53-3.17 (m, 27H, DMSO overlap), 3.09-2.80 (m, 17H, DMSO overlap), 2.39-2.20 (m, 3H), 1.86 (h, J=6.9 Hz, 1H), 1.77 (s, 3H), 1.68-1.21 (m, 5H), 0.76 (dd, J=13.8, 6.8 Hz, 6H); ¹⁹F NMR (376 MHz, DMSO-d6): -112.18 ppm.

Synthesis of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-((2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate

A mixture of tert-butyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (1046 mg, 4.20 mmol), Ag ₂ CO ₃ (4627 mg, 8.39 mmol), and a piece of crystalline iodine in CH ₂ Cl ₂ (3 mL) was stirred with powdered 4A molecular sieves (700 mg) for 15 min. To the mixture was added (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate (1150 mg, 2.80 mmol) in CH ₂ Cl ₂ (3.00 mL) (also stirred with powdered 4A molecular sieves (700 mg) for 15 min). The resulting mixture was covered with aluminum foil and stirred at RT for 60 h, then filtered through Celite, rinsing with EtOAc. The filtrate was concentrated to yield a clear oil. Purification by flash chromatography (20-100% EtOAc in heptane, ELSD determination) gave a 28/72% mixture of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-((2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate and ( 2S,3aR,5R,6R,7S,7aR )-5-(acetoxymethyl)-2-((2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)oxy)-2-methyltetrahydro-5H-[1,3]dioxolo[4,5-b]pyran-6,7-diyl diacetate as a thick clear oil (307 mg, 0.529 mmol): LCMS: MS+=580.4, Rt=0.96 min (acids, 2 min, ELSD only); ¹ H NMR (400 MHz, DMSO-d6) δ 6.74 (s, 3H), 5.76 (s, 8H), 5.26 (t, J=9.5 Hz, 1H), 5.02 (t, J=3.1 Hz, 3H), 4.90 (t, J=9.7 Hz, 1H), 4.84-4.72 (m, 5H), 4.37 (dd, J=5.2, 3.1, 0.9 Hz, 3H), 4.18 (dd, J=12.2, 5.0 Hz, 1H), 4.11 (d, J=4.5 Hz, 5H), 4.08-3.94 (m, 4H), 3.91 (dt, J=8.6, 4.1 Hz, 3H), 3.85-3.75 (m, 1H), 3.66-3.58 (m, 1H), 3.58-3.51 (m, 7H), 3.42-3.31 (m, 14H), 3.06 (kv, J=5.9 Hz, 8H), 2.07 (s, 7H), 2.05 (s, 7H), 2.02 (s, 10H), 1.99 (d, J=1.0 Hz, 6H), 1.99 (s, 3H), 1.94 (s, 3H), 1.63 (s, 7H), 1.38 (s, 36H), 1.18 (t, J=7.1 Hz, 4H).

Synthesis of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate

To a mixture of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-((2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate and ( 2S,3aR,5R,6R,7S,7aR )-5-(acetoxymethyl)-2-((2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)oxy)-2-methyltetrahydro-5H-[1,3]dioxolo[4,5-b]pyran-6,7-diyl diacetate (323 mg, 0.557 mmol) in CH ₂ Cl ₂ (8 mL) at 0°C was added TFA (1.9 ml, 25 mmol). After stirring at RT for 45 min, the mixture was concentrated and the residue was dried under vacuum for 60 min to give a light yellow oil. Purification by flash chromatography (0-20% MeOH in _CH2Cl2 , with 0.2% _NH4OH modifier in MeOH, ELSD determination) gave (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(2-(2-aminoethoxy)ethoxy)ethoxy ₎ tetrahydro-2H-pyran-3,4,5-triyl triacetate as a clear oil (82 mg, 0.171 mmol). LCMS: MS+=480.4, Rt=0.61 min (acidic, 2 min, ELSD); ¹ H NMR (400 MHz, DMSO-d6) δ 7.76 (s, 2H), 5.28 (t, J=9.4 Hz, 1H), 4.93 (t, J=9.7 Hz, 1H), 4.86-4.75 (m, 2H), 4.20 (dd, J=12.2, 5.0 Hz, 1H), 4.08-3.95 (m, 2H), 3.88-3.79 (m, 1H), 3.67-3.53 (m, 11H, DMSO overlay), 3.00 (kv, J=5.5 Hz, 2H), 2.04 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.96 (s, 3H).

Synthesis of 2-azidoethyl (N-(2-(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)sulfamoyl)carbamate

To 2-azidoethan-1-ol (12.5 mg, 0.144 mmol) in CH ₂ Cl ₂ (2 mL) was added sulfur isocyanatide chloride (0.012 mL, 0.14 mmol) at 0 °C. The mixture was stirred at 0 °C for 45 min, then TEA (0.040 mL, 0.29 mmol) and (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (77 mg, 0.16 mmol) in CH ₂ Cl ₂ (1 mL) were added. After stirring at 0 °C for 1 h, then at RT for 1 h, the mixture was quenched with sat. NH ₄ Cl and 1 N HCl (0.29 mL). The aqueous layer was extracted with CH ₂ Cl ₂ (5X). The organic layers were dried over anh. Na ₂ SO ₄ , filtered and concentrated by rotary evaporation to give (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(2-(2-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate as a clear oil (75 mg): LCMS: 0.97 min, MS+=672.4, 96, Rt -0.87 min (acidic, 2 min, ELSD).

LiOH.H ₂ O (0.5 M in water, 3.45 mL, 1.72 mmol) was added to the product obtained above in dioxane (4 mL) at 0°C. The resulting clear solution was stirred at RT for 1 h and then concentrated by rotary evaporation from 20°C on a water bath. The residue was purified by prep-HPLC (Sunfire 5 μm 30×50 mm column, 2-12% acetonitrile with 0.1% FA in water. Flow rate: 75 mL/min, MS 503.5, 520.3 determination) to give, after lyophilization, 2-azidoethyl (N-(2-(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)sulfamoyl)carbamate as a clear thin film (22 mg, 0.044 mmol): LCMS: MS+=504.3, Rt=0.52 min (acidic, 2 min, ELSD); ¹ H NMR (400 MHz, DMSO-d6) δ 11.31 (s, 1H), 7.74 (s, 1H), 4.96 (d, J=4.9 Hz, 1H), 4.89 (dd, J=12.5, 4.8 Hz, 2H), 4.47 (t, J=5.9 Hz, 1H), 4.22 (t, J=5.0 Hz, 2H), 4.15 (d, J=7.8 Hz, 1H), 3.93-3.82 (m, 1H), 3.67 (dd, J=11.2, 5.8 Hz, 1H), 3.62-3.40 (m, 12H), 3.17-2.90 (m, 6H).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbut anamido)-5-ureidopentanamido)-2-(((1-(2-(((N-(2-(2-(2-((2-((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)sulfamoyl)carbamoyl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzyl)-1-methylpiperazin-1-ium trifluoroacetate (L62-P1)

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium trifluoroacetate (32 mg, 0.020 mmol) and 2-azidoethyl (N-(2-(2-(2-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2=yl)oxy)ethoxy)ethoxy)ethyl)sulfamoyl)carbamate (21 mg, 0.042 mmol), 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutyl anamido)-5-ureidopentanamido)-2-(((1-(2-(((N-(2-(2-(2-((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)sulfamoyl)carbamoyl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzyl)-1-methylpiperazine-1-ium trifluoroacetate was obtained as a white powder: HRMS: MS+=2002.7100, Rt=2.37 min (5 min acidic).

Synthesis of Di-tert-butyl 3,3 ' -((3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoyl)azandiyl)dipropionate

To a mixture of di-tert-butyl 3,3'-azanediyl dipropionate (403 mg, 1.47 mmol), 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid ( ₅₀₅ mg, 1.62 mmol) and DIPEA (0.309 mL, 1.77 mmol) in _CH2Cl2 (3 mL) was added N- (dimethylaminopropyl) -N′ -ethylcarbodiimide hydrochloride HCl (367 mg, 1.92 mmol). After stirring at RT for 2 h, the mixture was quenched with sat. _NH4Cl and extracted _{with CH2Cl2} (3X). The combined organic phase was washed with brine, dried over anh. _Na2SO4 , _filtered and concentrated. The resulting crude product was purified by flash chromatography (0-50% EtOAc in heptane) to give di-tert-butyl 3,3'-((3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoyl)azanediyl)dipropionate as a white foam (290 mg, 0.511 mmol): LCMS: MS+=567.5, Rt=1.32 min (acid, 2 min); PMR: ¹ H NMR (400 MHz, DMSO-d6) δ 7.94-7.88 (m, 2H), 7.70 (d, J=7.5 Hz, 2H), 7.47-7.40 (m, 2H), 7.40-7.31 (m, 2H), 7.22 (t, J=5.7 Hz, 1H), 4.31 (d, J=6.8 Hz, 2H), 4.26-4.18 (m, 1H), 3.56-3.03 (m, 8H), 2.50-2.38 (m, 4H), 1.41 (s, 9H), 1.40 (s, 9H).

Synthesis of di-tert-butyl 3,3 ' -((3-aminopropanoyl)azanediyl)dipropionate, 1-(9H-fluoren-9-yl)-N,N-dimethylmethanamine

To di-tert-butyl 3,3'-((3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoyl)azanediyl)dipropionate (288 mg, 0.508 mmol) in CH ₂ Cl ₂ (3 mL) was added dimethylamine (2 N in THF, 1 mL, 2 mmol). The mixture was stirred at RT for 1 h. More dimethylamine (2 N in THF, 1 mL, 2 mmol) was added. After stirring for an additional 4 h, the mixture was concentrated and the residue was purified by flash chromatography (0-25% MeOH in _CH2Cl2 , ₃₇ min, 0.2% _NH4OH modifier in MeOH, ELSD determination) to give di-tert-butyl 3,3'-((3-aminopropanoyl)azanediyl)dipropionate, 1-(9H-fluoren-9-yl)-N,N-dimethylmethanamine as a light brown oil (62 mg, 0.472 mmol): LCMS: MS+=345.4, Rt=0.76 min (acidc, 2 min, ELSD); ¹ H NMR (400 MHz, DMSO -d 6) δ 4.05 (s, 2H), 3.51 (t, J= 7.2 Hz, 2H), 3.43 (t, J= 7.3 Hz, 2H), 3.31 (s, 2H), 2.79 (t, J= 6.4 Hz, 2H), 2.55-2.45 (m, 17H, overlap with DMSO), 2.41 (t, J= 7.3 Hz, 2H), 1.41 (s, 9H), 1.40 (s, 9H).

Synthesis of 3,3 ' -((3-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)propanoyl)azanediyl)dipropionic acid

To 2-azidoethan-1-ol (16 mg, 0.18 mmol) in CH₂Cl₂(2.5 mL) was added sulfur isocyanate chloride (0.016 mL, 0.18 mmol) at 0°C. The mixture was stirred at 0°C for 30 min, then TEA (0.051 mL, 0.37 mmol) and di-tert-butyl 3,3'-((3-aminopropanoyl)azanediyl)dipropionate (73 mg, 0.21 mmol) in CH₂Cl₂(1 ml). After stirring at 0°C for 1 h and then at rt for 1 h, the mixture was quenched with sat. NH₄Cl and 1 N HCl (0.37 mL). The aqueous layer was extracted with CH₂Cl₂(5X). The organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated by rotary evaporation. The resulting residue was purified by flash chromatography (0-10% MeOH in CH₂Cl₂, ELSD and UV214 determination) to obtain di-tert-butyl 3,3'-((3-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)propanoyl)azanediyl) dipropionate as a thick clear oil (70 mg, 0.13 mmol): LCMS MS+= 537.4, Rt=1.08 min (acidic, 2 min, ELSD);¹H NMR (400 MHz, DMSO-d6) δ 11.39 (s, 1H), 7.65-7.59 (m, 1H), 4.28-4.23 (m, 2H), 3.63-3.59 (m, 2H), 3.54-3.47 (m, 2H), 3.47-3.38 (m, 2H), 3.18-3.09 (m, 2H), 2.61-2.54 (m, 4H), 2.43 (dd, J=8.5, 6.0 Hz, 2H), 1.43 (s, 9H), 1.42 (s, 9H).

To the above product in _CH2Cl2 ( ₂ mL) at 0°C was added TFA (2 mL). After stirring at RT for 1.5 h, the mixture was concentrated by rotary evaporation at 25°C on a water bath. The residue was dried under high vacuum for 30 min, then azeotropically distilled with anhydrous toluene (3X 3 mL) and then dried under high vacuum overnight to give 3,3'-((3-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)propanoyl)azanediyl)dipropionic acid as a white solid (72 mg, 77% w/w of theoretical yield. It was used directly in the next step): LCMS MS+=425.3 , Rt=0.52 min (acidic, 2 min, ELSD); ¹ H NMR (400 MHz, DMSO-d6) δ 12.28 (s, 1H), 11.35 (s, 1H), 7.58 (t, J=5.8 Hz, 1H), 4.26-4.20 (m, 2H), 3.61-3.55 (m, 2H), 3.50 (t, J=7.4 Hz, 2H), 3.42 (t, J=7.4 Hz, 2H), 3.12 (kv, J=6.8 Hz, 2H), 2.56 (dd, J=15.1, 7.4 Hz, 4H), 2.43 (t, J=7.4 Hz, 2H), 2.08 (s, 1H).

Synthesis of 1-(2-(((1-(2-(((N-(3-(bis(2-carboxyethyl)amino)-3-oxopropyl)sulfamoyl)carbamoyl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylb ((2-(4-(4-((2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium trifluoroacetate (L61-P1)

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium trifluoroacetate (20 mg, 0.012 mmol) and 3,3'-((3-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)propanoyl)azanediyl)dipropionic acid (15 mg, 0.027 mmol), 1-(2-(((1-(2-(((N-(3-(bis(2-carboxyethyl)amino)-3-oxopropyl)sulfamoyl)carbamoyl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylb ((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazine-1-ium trifluoroacetate is obtained as a white powder: HRMS: MS+=1923.6500 Rt=2.34 min (5 min acidic); ¹ H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 10.16 (s, 1H), 8.81 (d, J=5.1 Hz, 1H), 8.54 (s, 1H), 8.08 (d, J=13.7 Hz, 2H), 7.77-7.50 (m, 5H), 7.47-7.20 (m, 6H), 7.18-7.04 (m, 5H), 6.99-6.90 (m, 4H), 6.65 (t, J=7.4 Hz, 1H), 6.14 (dd, J=7.6, 1.7 Hz, 1H), 5.92 (s, 1H), 5.43 (dd, J=9.8, 3.5 Hz, 1H), 5.24-5.11 (m, 3H), 4.65-4.51 (m, 9H), 4.45-3.81 (m, 52H, DMSO overlay), 3.68 (s, 3H), 3.54-2.77 (m, 31H), 2.39-2.20 (m, 5H), 1.86 (kv, J=6.8 Hz, 1H), 1.77 (s, 3H), 1.69-1.00 (m, 6H), 0.76 (dd, J=13.9, 6.8 Hz, 6H); 19F NMR (376 MHz, DMSO-d6): -112.16 ppm.

Synthesis 1-(2-(((1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triaz ol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl) -4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 2 with 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (87.5 mg, 0.063 mmol, 1.0 equiv.) and mPEG12-Azide (73.3 mg, 0.125 mmol, 2 equiv.), 1-(2-(((1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. MSHR: M+= 1889.8544, Rt=2.19 min (5 min acidic method).

Synthesis 1-(2-(((1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl)methox i)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamide (o)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (L104-P1)

Following the GENERAL METHOD 3 s 1-(2-(((1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (22 mg, 0.011 mmol, 1.0 equiv.), 1-(2-(((1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido )-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+= 2084.9099, Rt=2.45 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 3 with 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (155 mg, 0.119 mmol, 1.0 eq.) gave 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium. HRMS: M+= 1499.5699, Rt=2.39 min (5 min acidic method).

Synthesis of 1-(2-(((1-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecaoxanone-tetracontan-49-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutyl ((tanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (L34-P1)

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium (50 mg, 0.033 mmol, 1.0 equiv.) and m-PEG16-azide (from Broadpharm BP-23558) (50.8 mg, 0.067 mmol, 2 equiv.) gave 1-(2-(((1-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecaoxanone-tetracontan-49-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutyl ((4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+=2261.0196, Rt=2.28 min (5 min acidic method).

Synthesis of 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((1-((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2 -yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl )methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 2 with 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (87.5 mg, 0.063 mmol, 1.0 equiv.) and 1-azido-1-deoxy-beta-D-lactopyranoside (22.99 mg, 0.063 mmol, 1 equiv.) yields 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((1-((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2 -yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+= 1671.6400, Rt=1.95 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((1-((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy -6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium (L46-P1)

Following GENERAL PROCEDURE 3 with 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((1-((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2 -yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (35 mg, 0.020 mmol, 1.0 equiv.) was obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((1-((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy- 6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazin-1-ium. HRMS: M+= 1866.6899, Rt=2.28 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((6S,9S,12S)-9-isopropyl-2,2-dimethyl-4,7,10-trioxo-6-(prop-2-yn-1-yl)-12-(3-ureidopropyl)-3-oxa-5,8,11-triazatridecane-13-amido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium

To a mixture of Boc-propargyl-Gly-OH (40 mg, 0.188 mmol, 1 equiv) and GATU (71.3 mg, 0.188 mmol, 1 equiv) in DMF (0.5 mL) was added DIPEA (65.5 μL, 0.375 mmol, 2 equiv). The mixture was stirred at RT for 30 min. Then a solution of 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (245 mg, 0.188 mmol, 1 eq.) in DMF (1 ml) was added to the reaction mixture. The reaction mixture was stirred at RT for 30 min. The crude mixture was separated on a C18 column (100 cartridge, MeCN/water with 0.1% formic acid, 0-100% over 15CV) to give 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((6S,9S,12S)-9-isopropyl-2,2-dimethyl-4,7,10-trioxo-6-(prop-2-yn-1-yl)-12-(3-ureidopropyl)-3-oxa-5,8,11-triazatridecane-13-amido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium. MSHR: M+= 1499.6000, Rt= 2.91 min (5 min acidic method).

Synthesis of 1-(4-((S)-2-((S)-2-((S)-2-aminopent-4-ynamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

At 0 ^o C in an ice-water bath, 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(4-((6S,9S,12S)-9-isopropyl-2,2-dimethyl-4,7,10-trioxo-6-(prop-2-yn-1-yl)-12-(3-ureidopropyl)-3-oxa-5,8,11-triazatridecane-13-amido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-1-methylpiperazin-1-ium (56 mg, 0.037 mmol) was added TFA (25% in DCM) 2 ml, then the reaction mixture was raised to RT and stirred for 1 h. The crude mixture was concentrated under high vacuum. The mixture was then dissolved in MeOH and purified on a C-18 column (50 g cartridge, MeCN/water with 0.1% formic acid 0-100% over 16 CV) to give 1-(4-((S)-2-((S)-2-((S)-2-aminopent-4-ynamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+= 1399.5400 , Rt= 2.17 min (5 min acidic method).

Synthesis of 1-(4-((S)-2-((S)-2-((S)-2-amino-3-(1-((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(((1-((2S,3S,4S,5R,6S)-3,4-dihydroxy-6-(hydroxymethyl)-5- (((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

Following GENERAL PROCEDURE 2 with 1-(4-((S)-2-((S)-2-((S)-2-aminopent-4-ynamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (44 mg, 0.031 mmol, 1.0 eq.) and 1-azido-1-deoxy-beta-D-lactopyranoside (69.2 mg, 0.188 mmol, 6 equiv.) is obtained 1-(4-((S)-2-((S)-2-((S)-2-amino-3-(1-((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(((1-((2S,3S,4S,5R,6S)-3,4-dihydroxy-6-(hydroxymethyl)-5- (((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+= 2133.7800 , Rt= 1.95 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((1-((2S,3S,4S,5R,6S)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl )-4-((2S,5S,8S)-8-((1-((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methyl)-15-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-4,7,10-trioxo-2-(3-ureidopropyl)-13-oxa-3,6,9-triazapentadecanamido)benzyl)-1-methylpiperazin-1-ium (L47-P1)

Following GENERAL PROCEDURE 3 with 1-(4-((S)-2-((S)-2-((S)-2-amino-3-(1-((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(((1-((2S,3S,4S,5R,6S)-3,4-dihydroxy-6-(hydroxymethyl)-5- (((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)benzyl)-4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (19 mg, 0.009 mmol, 1.0 equiv.) was obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(((1-((2S,3S,4S,5R,6S)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl )-4-((2S,5S,8S)-8-((1-((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-1H-1,2,3-triazol-4-yl)methyl)-15-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-4,7,10-trioxo-2-(3-ureidopropyl)-13-oxa-3,6,9-triazapentadecanamido)benzyl)-1-methylpiperazin-1-ium. MSHR: M+= 2328.8301, Rt=2.15 min (5 min acidic method).

1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(78-carboxy-2-methyl-3-oxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4-diazaoctaheptacone thyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium

A mixture of 1-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontane-75-oic acid (50 mg, 0.044 mmol), bis(4-nitrophenyl) carbonate (13 mg, 0.043 mmol) and DIPEA (20 μl, 0.12 mmol) in THF (2 ml) was stirred at rt for 2 h. The mixture was concentrated by bubbling nitrogen gas through it. The resulting solid residue was taken up in DMF (1 ml). 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium (50 mg, 0.029 mmol) and DIPEA (100 μl, 0.573 mmol) were added. The mixture was stirred at RT for 5 min. The mixture was diluted with DMSO (2 ml) and the solution was purified by RP-HPLC ISCO gold chromatography (MeCN/H₂O, 0.1% TFA modifier). After lyophilization, 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(78-carboxy-2-methyl-3-oxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4-diazaoctaheptaconte (yl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium. HRMS: M+= 2671.2700, Rt=2.88 min (5 min acidic method).

4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(78-carboxy-2-methyl-3-oxo-7,10,13,16,19,22,25,28,31,34,37,4 0,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4-diazaoctaheptacontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2 ,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazine-1-ium (L42-P1)

FollowingGENERAL METHOD 3c 1-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(78-carboxy-2-methyl-3-oxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4-diazaoctaheptaconte yl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1-methylpiperazin-1-ium, is obtained 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1-(2-(78-carboxy-2-methyl-3-oxo-7,10,13,16,19,22,25,28,31,34,37,4 0,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4-diazaoctaheptacontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2 ,5-dihydro-1H-pyrrol-1-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-1-methylpiperazine-1-ium (L42-P1). MSVR: M+= 2646.7700, Rt=2.38 min (5 min acidic method).

The following compounds are prepared using procedures similar to those described above.

The synthetic methods for preparing polyethylene glycols in L43-P1, L44-P1 and L45-P1 are described below.

Synthesis of 2-oxo-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75-tetracozaoxa-3-azaoctaheptacontane-78-oic acid

To a stirred solution of 1-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontane-75-oic acid (100 mg, 0.087 mmol, 1.0 equiv) and DIPEA (24.8 mg, 34 μL, 0.192 mmol, 2.2 equiv) in dichloromethane (0.5 mL) was added acetic anhydride (8.9 mg, 8.25 μL, 1.0 equiv). The resulting mixture was stirred at ambient temperature for 1.5 h. The solvent was removed under reduced pressure. The resulting residue was taken up in DMSO (1 mL) and purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H ₂ O, 0.1% TFA modifier). After lyophilization, 2-oxo-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75-tetracozaoxa-3-azaoctaheptacontane-78-oic acid (62.3 mg, 0.052 mmol, 60% yield) was obtained. LC/MS [MH]- 1187.3 Rt = 0.75 min (2 min acidic method). ¹ H NMR (400 MHz, DMSO-d6) δ 7.86 (s, 1H), 3.60 (t, J=6.4 Hz, 3H), 3.50 (d, J=4.9 Hz, 91H), 3.40 (t, J=5.9 Hz, 2H), 3.18 (q, J=5.8 Hz, 2H), 2.44 (t, J=6.4 Hz, 2H), 1.80 (s, 3H).

Synthesis of 4-oxo-3,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77-pentacosaoxa-5-azaoctacontane-80-oic acid

To a stirred solution of 1-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontane-75-oic acid (100 mg, 0.087 mmol, 1.0 equiv) and DIPEA (24.8 mg, 34 μL, 0.192 mmol, 2.2 equiv) in dichloromethane (0.5 mL) was added ethyl chloroformate (9.5 mg, 8.34 μL, 1.0 equiv). The resulting mixture was stirred at ambient temperature for 1.5 h. The solvent was removed under reduced pressure. The resulting residue was taken up in DMSO (1 mL) and purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H ₂ O, 0.1% TFA modifier). After lyophilization, 4-oxo-3,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77-pentacosaoxa-5-azaoctacontane-80-oic acid (75 mg, 0.062 mmol, 71% yield) was obtained. LC/MS [MH]- 1217.3 Rt=0.81 min (2 min acidic method). ¹ H NMR (400 MHz, DMSO-d6) δ 7.03 (s, 1H), 3.97 (q, J=7.1 Hz, 2H), 3.60 (t, J=6.4 Hz, 2H), 3.50 (d, J=5.0 Hz, 92H), 3.40 (t, J=6.1 Hz, 2H), 3.11 (kv, J=5.9 Hz, 2H), 2.45 (kv, J=6.5 Hz, 2H), 1.15 (t, J=7.1 Hz, 3H,).

Synthesis of 4-oxo-2,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77-pentacosaoxa-5-azaoctacontane-80-oic acid

To a stirred solution of 1-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontane-75-oic acid (100 mg, 0.087 mmol, 1.0 eq.) and DIPEA (24.8 mg, 34 μL, 0.192 mmol, 2.2 eq.) in dichloromethane (0.5 mL) was added methoxyacetyl chloride (11.36 mg, 9.57 μL, 1.2 eq.). The resulting mixture was stirred at ambient temperature for 1.5 h. The solvent was removed under reduced pressure. The resulting residue was taken up in DMSO (1 mL) and purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H ₂ O, 0.1% TFA modifier). After lyophilization, 4-oxo-2,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77-pentacosaoxa-5-azaoctacontane-80-oic acid (69 mg, 0.057 mmol, 65% yield) was obtained. LC/MS [MH]- 1217.4 Rt = 0.75 min (2 min acidic method). ¹ H NMR (400 MHz, DMSO-d6) δ 7.68 (s, 1H), 3.79 (s, 2H), 3.60 (t, J=6.4 Hz, 2H), 3.51 (s, 92H), 3.43 (t, J=6.0 Hz, 2H), 3.30 (s, 3H), 3.26 (kv, J=6.0 Hz, 2H), 2.44 (t, J=6.4 Hz, 2H).

Example 3. Synthesis and characterization of Mcl-1 payloads

Typical payloads are synthesized using the typical methods described in this example.

Obtaining C1:

( 2R )-2-{[( 5S _a )-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3 -d ]pyrimidin-4-yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid

C1 was prepared according to Example 30 in WO 2015/097123.

Obtaining C2:

(2 R )-2-[(5 S _a )-5-[3-chloro-2-methyl-4-[2-[4-methyl-4-(3-sulfopropyl)piperazin-4-yl-1-yl]ethoxy]phenyl]-6-(4-fluoro-3-hydroxyphenyl)thieno[2,3- d ]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

Step A: 5-bromo-4-chloro-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidine

4.49 g of 5-bromo-4-chloro-6-iodo-thieno[2,3-d]pyrimidine (11.96 mmol; prepared according to WO 2015/097123, preparation 1a) and 4.31 g of (4-fluoro-3-tetrahydropyran-2-yloxyphenyl)boronic acid (17.94 mmol) were dissolved in 60 mL of THF, then 134 mg of Pd(Oac) ₂ (0.60 mmol), 508 mg of tBuXPhos (1.20 mmol), 11.69 g of Cs ₂ CO ₃ (35.88 mmol) and 60 mL of water were added and the mixture was stirred at 70 °C under N ₂ until the conversion ceased. Then it was diluted with water, neutralized with 2 M aqueous HCl solution and extracted with DCM. The combined organic layer was dried over _Na2SO4 , filtered _, and the filtrate was concentrated under reduced pressure. The crude product was purified by flash chromatography using heptane and EtOAc as eluents to give 5-bromo-4-chloro-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidine. ¹ H NMR (500 MHz, DMSO-d ₆ ) δ: 9.02 (s, 1H), 7.64 (dd, J = 7.9, 2.1 Hz, 1H), 7.47 (dd, J = 11.0, 8.5 Hz, 1H), 7.36 (m, 1H), 5.63 (m, 1H), 3.81 (m, 1H), 3.61 (m, 1H), 1.94-1.78 (m, 3H), 1.69-1.50 (m, 3H). ¹³ C NMR (125 MHz, DMSO-d ₆ ) δ: 166.6, 153.9, 153.1, 152.7, 144.3, 139.2, 127.7, 126.6, 124.2, 119.9, 117.1, 100.7, 97.2, 61.6, 29.5, 24.5, 18.2. HRMS calculated for C ₁₇ H ₁₃ N ₂ O ₂ SBrClF: 441.9554; found 442.9624 (M+H).

Step B: Ethyl (2R)-2-[5-bromo-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate

3.09 g of 5-bromo-4-chloro-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidine (6.97 mmol), 3.28 g of ethyl (2R)-2-hydroxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (8.02 mmol; prepared according to WO 2015/097123, preparation 3bs) were dissolved in 70 ml of tert- butanol, then 6.82 g of Cs ₂ CO ₃ (20.9 mmol) were added and the mixture was stirred at 70 °C under N ₂ until the conversion ceased. Then it was diluted with water, neutralized with 2 M aqueous HCl solution and extracted with DCM. The combined organic layer was _dried over _Na2SO4 , filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by flash chromatography using heptane and EtOAc as eluents to give ethyl (2R)-2-[5-bromo-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin- ₄ -yl] _methoxy ] _phenyl ]propanoate as a mixture of diastereoisomers. HRMS calculated for _{C40H36N4O7SBrF} : 814.1472; found 815.1539 (M+H).

Step C: Ethyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate

3.65 g of ethyl (2R)-2-[5-bromo-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (4.47 mmol) and 2.12 g of 1-[2-[2-chloro-3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]ethyl]-4-methyl-piperazine (5.36 mmol; prepared according to WO 2015/097123, preparation 5b) were dissolved in 22 ml of dioxane, then 315 mg of PdCl ₂ ×AtaPhos (0.45 mmol) were added. 4.37 g _{of Cs2CO3 (} 13.41 mmol) and 22 mL of water were added, and the mixture was stirred at 70°C under _N2 until complete conversion. It was then diluted with water, neutralized with 2 M aqueous HCl, and extracted with EtOAc. The combined organic layer was dried over _Na2SO4 , filtered _, and the filtrate was concentrated under reduced pressure. The crude product was purified by flash chromatography using EtOAc and MeOH followed by DCM and MeOH as eluents to give ethyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluoro-3-tetrahydropyran-2-yloxyphenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate as a mixture of two pairs of diastereoisomers. HRMS calculated for C ₅₄ H ₅₆ N ₆ O ₈ SClF: 1002.3553; found 1003.3614 and 1003.3622 (M+H).

Step D: (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

3.47 g of ethyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (3.46 mmol) were dissolved in 35 ml of dioxane, then 1.45 g of LiOH×H ₂ O (34.6 mmol) and 35 ml of water were added. The mixture was stirred at room temperature until complete hydrolysis. Then it was diluted with water, acidified to pH 4 with 2 M aqueous HCl solution and extracted with DCM. The combined organic phase was dried over Na ₂ SO ₄ , filtered, and the filtrate was concentrated under reduced pressure. The atropisomers were purified and separated by preparative reverse-phase chromatography using 25 mM aqueous NH ₄ HCO ₃ and MeCN as eluents. The last eluted pair of atropisomers was isolated as (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid. HRMS calculated for C ₅₂ H ₅₂ N ₆ O ₈ SClF: 974.3240; found 975.3303 (M+H).

Step E: (4-methoxyphenyl)methyl (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate

2.39 g of (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (2.45 mmol), 1.13 g of DTBAD (4.91 mmol) and 1.29 g of _PPh3 (4.91 mmol) were dissolved in 49 ml of toluene, then 0.61 ml of PMB-OH (4.91 mmol) was added and the reaction mixture was stirred at 50°C until complete conversion. The mixture was then diluted with DCM and then concentrated under reduced pressure and then purified by flash chromatography using heptane and EtOAc as eluents to give (4-methoxyphenyl)methyl (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate as a mixture of diastereoisomers. HRMS calculated for C ₆₀ H ₆₀ N ₆ O ₉ SClF: 1094.3815; found 1095.3880 (M+H).

Step F: (2 R )-2-[(5 S _a )-5-[3-chloro-2-methyl-4-[2-[4-methyl-4-(3-sulfopropyl)piperazin-4-yl-1-yl]ethoxy]phenyl]-6-(4-fluoro-3-hydroxyphenyl)thieno[2,3- d ]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (C2)

600 mg of (4-methoxyphenyl)methyl (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (0.548 mmol) were dissolved in 11 ml of MeCN, then 0.48 ml of oxathiolane 2,2-dioxide (5.48 mmol) was added and the mixture was stirred under N ₂ at 60°C until complete conversion. It is then concentrated under reduced pressure, dissolved in 8 mL of DCM, 2.2 mL of TFA is added, and the mixture is stirred at room temperature until complete cleavage of THP and PMB. It is then concentrated (the heating bath is removed). It is dissolved in 10 mL of THF and concentrated again under reduced pressure at 30°C. The crude product is purified by preparative reverse-phase chromatography using 5 mM aqueous NH ₄ HCO ₃ and MeCN as eluents to afford C2 . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 13.19 (brs, 1H), 10.16 (brs, 1H), 8.89 (d, J =5.2 Hz, 1H), 8.58 (s, 1H), 7.68 (brs, 1H), 7.52 (dd, J =7.5, 1.8 Hz, 1H), 7.46 (m, 1H), 7.33 (d, J =8.3 Hz, 1H), 7.22-7.09 (m, 4H), 7.06-7.00 (m, 2H), 6.86 (dd, J =8.3, 2.0 Hz, 1H), 6.74 (t, J =7.4 Hz, 1H), 6.66 (m, 1H), 6.23 (d, J =6.7 Hz, 1H), 5.46 (dd, J =9.8, 3.3 Hz, 1H), 5.27 (d, J =15.2 Hz, 1H), 5.22 (d, J =15.2 Hz, 1H), 4.23 (m, 2H), 3.76 (s, 3H), 3.46 (m, 2H), 3.41-3.23 (m, 5H), 2.97 (s, 3H), 2.94-2.77 (m, 6H), 2.48 (m, 1H), 2.45 (t, J =7.0 Hz, 2H), 2.00-1.90 (m, 2H), 1.86 (s, 3H). ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ: 170.8, 166.2, 165.9, 164.7, 157.7, 157.2, 155.4, 153.6, 152.7, 152.3, 149.9, 145.1, 137.0, 135.9, 131.0, 130.8, 130.3, 129.2, 128.34, 128.32, 128.2, 128.0, 122.0, 120.5, 120.1, 118.8, 118.2, 116.6, 115.6, 112.2, 111.9, 110.6, 73.3, 69.0, 67.3, 59.2, 59.1, 55.71, 55.68, 47.6, 46.1, 31.8, 18.1, 17.6. HRMS calculated for C ₅₀ H ₅₀ N ₆ O ₁₀ S ₂ ClF: 1012.2703; found 1013.2775 (M+H).

Obtaining C3:

(2R)-2-{[(5Sa)-5-{3-chloro-2-methyl-4-[2-(piperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid

C3 is prepared according to Example 744 in WO 2015/097123.

Obtaining C4:

(2R)-2-{[(5Sa)-5-{3-chloro-2-methyl-4-[2-(piperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid

Step 1: Ethyl (2R)-2-[5-bromo-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[(2-chloropyrimidin-4-yl)methoxy]phenyl]propanoate

To a solution of ethyl (2R)-3-[2-[(2-chloropyrimidin-4-yl)methoxy]phenyl]-2-hydroxypropanoate (25 g, 74.2 mmol) in THF (38 mL) were added sequentially 5-bromo-6-(4-fluorophenyl)-4-iodothieno[2,3-d]pyrimidine (23 g, 67.5 mmol) and cesium carbonate (67 g, 203 mmol). The reaction mixture was heated at reflux overnight and the volatiles were evaporated. The residue was diluted with ethyl acetate and water (500 and 400 mL, respectively), and the solution was filtered. The organic layer was separated, washed with brine, dried over magnesium sulfate, and concentrated in vacuo. The residue was purified by silica gel chromatography (gradient with ethyl acetate in petroleum ether) to give ethyl (2R)-2-[5-bromo-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[(2-chloropyrimidin-4-yl)methoxy]phenyl]propanoate as a slightly orange solid. ¹ H NMR (400 MHz, DMSO-d6): δ 8.83 (d, 1H), 8.65 (s, 1H), 7.75 (m, 2H), 7.71 (d, 1H), 7.48 (d, 1H), 7.45 (m, 2H), 7.25 (t, 1H), 7.06 (d, 1H), 6.95 (t, 1H), 5.75 (dd, 1H), 5.28 (2*d, 2H), 4.18 (q, 2H), 3.6/3.3 (2*dd, 2H), 1.12 (t, 3H). IR wavelength (cm ^-1 ): 1749.

Step 2: (4-bromo-2-chloro-3-methyl-phenoxy)-triisopropyl-silane

To a solution of 4-bromo-2-chloro-3-methylphenol (100 g, 482 mmol) in dichloromethane (1.5 L) was added imidazole (82 g, 1.2 mol) and chloro(triisopropyl)silane was added dropwise over 1 h. (102 mL, 482 mmol). The reaction mixture was stirred at room temperature for 1 h and water (500 mL) was added. The organic layers were washed with saturated brine (200 mL), dried over magnesium sulfate, and concentrated. The residue was used without further purification.¹H NMR (400 MHz, CDCl₃): δ 7.48 (s, 1H), 7.2 (dd, 1H), 6.7 (d, 1H), 1.3 (m, 3H), 1.1 (2s, 18H).

Step 3: tert-butyl-[2-chloro-3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]-dimethylsilane

To a solution of (4-bromo-2-chloro-3-methyl-phenoxy)-triisopropyl-silane (27.2 g, 71.9 mmol) in THF (350 mL) at -78°C under argon was added dropwise over 30 min a solution of 1.6 M n-butyl lithium in THF (49.5 mL, 79.9 mmol). The reaction mixture was stirred at -78°C for 2 h and a solution of 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (16.1 g, 86.4 mmol) in THF (50 mL) was added dropwise over 30 min. After stirring for 2 h at -78°C, the reaction mixture was quenched by slow addition of water (20 mL) and warmed to room temperature, diluted with water (200 mL) and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over magnesium sulfate and concentrated in vacuo. The residue was used without further purification. ¹ H NMR (400 MHz, DMSO-d6): δ 7.5 (d, 1H), 6.82 (d, 1H), 2.52 (s, 3H), 1.32 (m, 3H), 1.3 (s, 12H), 1.08 (s, 18H).

Step 4: 2-chloro-3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol

To a solution of tert-butyl [2-chloro-3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]-dimethylsilane (25.4 g, 59.8 mmol) in THF (750 mL) was added dropwise at room temperature a solution of 1 M tetrabutylammonium fluoride in THF (90 mL, 90 mmol). The reaction mixture was stirred for 2 h, concentrated, diluted with ethyl acetate, partitioned with water and extracted with ethyl acetate. The combined organic layers were washed with saturated brine, dried over magnesium sulfate and concentrated in vacuo. The residue was purified by silica gel chromatography (gradient of ethanol in dichloromethane) to give 2-chloro-3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol. ¹ H NMR (400 MHz, DMSO-d6): δ 10.4 (m, 1H), 7.4 (d, 1H), 6.8 (d, 1H), 2.5 (s, 3H), 1.3 (s, 12H). IR wavelength (cm ^-1 ): 3580-3185, 1591, 857, 827.

Step 5: Ethyl (2R)-2-{[(5S _a )-5-(3-chloro-4-hydroxy-2-methylphenyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-{2-[(2-chloropyrimidin-4-yl)methoxy]phenyl}propanoate

To a solution of ethyl (2R)-2-[5-bromo-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[(2-chloropyrimidin-4-yl)methoxy]phenyl]propanoate (43.8 g, 61.2 mmol) and 2-chloro-3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (19.7 g, 73.5 mmol) in THF/H ₂ O 1/1 (800 mL) was added cesium carbonate (40 g, 122 mmol). The reaction mixture was degassed by bubbling argon through the solution for 20 min and bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II) (4.35 g, 6.1 mmol) was added. The reaction mixture was heated at 80°C under argon overnight. The reaction mixture was diluted with water, partitioned with ethyl acetate, and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over magnesium sulfate, and concentrated in vacuo. The residue was purified by silica gel chromatography (gradient methanol in dichloromethane) to give ethyl (2R)-2-[5-(3-chloro-4-hydroxy-2-methyl-phenyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[(2-chloropyrimidin-4-yl)methoxy]phenyl]propanoate as an 85/15 mixture of diastereoisomers (aS/aR or S _a /R _a ). Optically pure aS (or S _a ) was obtained by preparative SLC purification.

Step 6: tert-butyl 4-(2-{2-chloro-4-[4-{[(2R)-3-{2-[(2-chloropyrimidin-4-yl)methoxy]phenyl}-1-ethoxy-1-oxopropan-2-yl]oxy}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-3-methylphenoxy}ethyl)piperazine-1-carboxylate

Diisopropyl azodicarboxylate (2.33 g, 10 mmol) was added to a solution of triphenylphosphine (2.66 g, 10 mmol) in THF at room temperature under argon. After stirring for 15 min, a solution of tert-butyl 4-(2-hydroxyethyl)piperazine-1-carboxylate (2.33 g, 10 mmol) in THF (8 ml) was added. The reaction mixture was stirred at room temperature for 1 h, then a solution of (2R)-2-[(5 S _a )-5-(3-chloro-4-hydroxy-2-methyl-phenyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[(2-chloropyrimidin-4-yl)methoxy]phenyl]propanoic acid (3.57 g, 5 mmol) in THF (8 mL) was added dropwise. The reaction mixture was stirred at room temperature for 96 h and concentrated. The residue is purified by silica gel chromatography (gradient methanol (containing 7M ammonia) in dichloromethane) to give tert-butyl 4-(2-{2-chloro-4-[4-{[(2R)-3-{2-[(2-chloropyrimidin-4-yl)methoxy]phenyl}-1-ethoxy-1-oxopropan-2-yl]oxy}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-3-methylphenoxy}ethyl)piperazine-1-carboxylate, ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.95 (d, 1H), 8.58 (s, 1H), 8.32 (d, 2H), 7.58 (d, 1H), 7.41 (dd, 2H), 7.32 (d, 1H), 7.29 (dd, 2H), 7.22 (t, 2H), 7.21 (d, 1H), 7.19 (t, 1H), 7.05 (d, 1H), 6.75 (t, 1H), 6.31 (dd, 1H), 5.53 (dd, 1H), 5.29 (dd, 2H), 4.2 (m, 2H), 4.05 (q, 2H), 3.97 (m, 4H), 3.3 (m, 2H), 3.2 (t, 4H), 3.19/2.59 (m, 2H), 2.72 (t, 2H), 2.4 (t, 4H), 1.87 (s, 3H), 1.37 (s, 9H), 1.18 (t, 6H), 1.05 (t, 3H). ¹³ C NMR (125 MHz, CDCl ₃ ): δ 158, 152, 131, 131, 130, 130, 128, 127, 120.5, 116, 116, 112, 110, 73, 68.5, 67, 62, 61, 56, 52, 43, 32, 32, 28, 17, 16, 14.

Step 7: tert-Butyl 4-(2-{2-chloro-4-[4-{[(2R)-3-{2-[(2-{4-[(diethoxyphosphoryl)methyl]phenyl}pyrimidin-4-yl)methoxy]phenyl}-1-ethoxy-1-oxopropan-2-yl]oxy}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-3-methylphenoxy}ethyl)piperazine-1-carboxylate

To a solution of tert-butyl 4-(2-{2-chloro-4-[4-{[(2R)-3-{2-[(2-chloropyrimidin-4-yl)methoxy]phenyl}-1-ethoxy-1-oxopropan-2-yl]oxy}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-3-methylphenoxy}ethyl)piperazine-1-carboxylate (337 mg, 0.367 mmol) and [4-(diethoxyphosphorylmethyl)phenyl]boronic acid (200 mg, 0.735 mmol) in dioxane (2.5 ml), water (2.5 ml) and cesium carbonate (241 mg, 0.735 mmol) were added. The reaction mixture was degassed by bubbling argon through the solution for 30 min, bis(triphenylphosphine)palladium(II) dichloride (2.5 mg, 3.6 μmol) was added, and the reaction mixture was heated under microwave irradiation in a sealed vessel at 90°C for 3 h. The reaction mixture was diluted with ethyl acetate and water. The aqueous layer was extracted with ethyl acetate, and the combined organic layers were washed with brine, dried over magnesium sulfate, and concentrated in vacuo. The residue is purified by silica gel chromatography (gradient methanol in dichloromethane) to give tert-butyl 4-(2-{2-chloro-4-[4-{[(2R)-3-{2-[(2-{4-[(diethoxyphosphoryl)methyl]phenyl}pyrimidin-4-yl)methoxy]phenyl}-1-ethoxy-1-oxopropan-2-yl]oxy}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-3-methylphenoxy}ethyl)piperazine-1-carboxylate, ¹ H NMR (500 MHz, DMSO-d6): δ 8.95 (d, 1H), 8.58 (s, 1H), 8.32 (d, 2H), 7.58 (d, 1H), 7.41 (dd, 2H), 7.32 (d, 1H), 7.29 (dd, 2H), 7.22 (t, 2H), 7.21 (d, 1H), 7.19 (t, 1H), 7.05 (d, 1H), 6.75 (t, 1H), 6.31 (dd, 1H), 5.53 (dd, 1H), 5.29 (2*d, 2H), 4.2 (m, 2H), 4.05 (q, 2H), 3.97 (m, 4H), 3.3 (m, 2H), 3.2 (t, 4H), 3.19/2.59 (2*dd, 2H), 2.72 (t, 2H), 2.4 (t, 4H), 1.87 (s, 3H), 1.37 (s, 9H), 1.18 (t, 6H), 1.05 (t, 3H). ¹³ C NMR (125 MHz, DMSO-d6) δ 158, 152, 131, 131, 130, 130, 128, 127, 120.5, 116, 116, 112, 110, 73, 68.5, 67, 62, 61, 56, 52, 43, 32, 32, 28, 17, 16, 14

Step 8: Synthesis of (2R)-2-[(5S _a )-5-[3-chloro-2-methyl-4-(2-piperazin-1-ylethoxy)phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-[4-(phosphonomethyl)phenyl]pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To a solution of tert-butyl 4-(2-{2-chloro-4-[4-{[(2R)-3-{2-[(2-{4-[(diethoxyphosphoryl)methyl]phenyl}pyrimidin-4-yl)methoxy]phenyl}-1-ethoxy-1-oxopropan-2-yl]oxy}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-3-methylphenoxy}ethyl)piperazine-1-carboxylate (540 mg, 0.486 mmol) in dichloromethane (5 mL) was added bromotrimethylsilane (186 μL, 1.46 mmol). The reaction mixture was heated at reflux overnight. Another portion of bromotrimethylsilane was added at room temperature (186 μL, 1.46 mmol), and the reaction mixture was heated at reflux for 20 h and concentrated to dryness. The residue was taken up in methanol, stirred at room temperature for 3 h, and concentrated to give a brown viscous oil, which was diluted with dioxane (4 mL) and water (4 mL). Lithium hydroxide monohydrate (100 mg, 24 mmol) was added portionwise, and the reaction mixture was stirred at room temperature for 1 h, heated at 45°C for 3 h, and concentrated. The residue was diluted with water (5 mL), acidified to pH 2 by dropwise addition of an aqueous solution of 2 M HCl. The precipitate was filtered, washed with THF and purified by C18 prep-RP-HPLC by direct loading of the reaction mixture onto an Xbridge column and using the NH ₄ HCO ₃ method to afford C4 . ¹ H NMR (500 MHz, DMSO-d6): δ 8.88 (bd, 1 H), 8.25 (d, 2 H), 7.75 (t, 1 H), 7.59 (s, 1 H), 7.52 (d, 1 H), 7.35 (d, 2 H), 7.23 (dd, 2 H), 7.18 (d, 1 H), 7.15 (t, 2 H), 7.11 (t, 1 H), 7.02 (d, 1 H), 6.82 (d, 1 H), 6.64 (m, 1 H), 5.51 (d, 1 H), 5.28/5.07 (m, 2 H), 3.82/3.55 (2m, 2 H), 3.35/2.55 (br s, 2 H), 2.81 (d, 2 H), 2.55 (m, 4 H), 2.4/2.27 (2m, 2 H), 2.21 (m, 4 H), 1.65 (br s, 3 H). ¹³ C NMR (125 MHz, DMSO-d6): δ 131.5, 130.2, 129.7, 127.4, 127.2, 120.3, 115.9, 115.3, 111.9, 110.3, 75.1, 69.2, 67.3, 56.4, 49.9, 42.4, 40, 38.9, 18.1. ³¹ P NMR (200 MHz, DMSO-d6): δ 15 HR-ESI+: m/z [M+H]+=925.2356/925.2346 (measured/theoretical)

Obtaining C5:

(2R)-2-{[(5Sa)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(4-hydroxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid

C5 is prepared according to Example 3 in WO 2016/207216.

Obtaining C6:

( 2R )-2-{[( 5S _a )-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-[2-({2-[2-(hydroxymethyl)phenyl]pyrimidin-4-yl}methoxy)phenyl]propanoic acid

C6 is prepared according to Example 728 in WO 2015/097123.

Getting C7:

(2R)-2-[(5S _a )-5-[3-chloro-2-ethyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-prop-1-ynyl-thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

Step A: Ethyl (2R)-2-(5-iodo-6-prop-1-ynyl-thieno[2,3-d]pyrimidin-4-yl)oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate

5.0 g of 4-chloro-5-iodo-6-prop-1-ynyl-thieno[2,3-d]pyrimidine (15.0 mmol; prepared according to WO 2015/097123, preparation 2f) and 6.10 g of ethyl (2R)-2-hydroxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (15.0 mmol; prepared according to WO 2015/097123, preparation 3bs) were dissolved in 150 mL of tert- butanol, then 14.7 g of Cs ₂ CO ₃ (45.0 mmol) were added and the mixture was stirred under N ₂ at 50 °C until the conversion ceased. Then water and brine were added and the mixture was extracted with EtOAc. The combined organic layer was _dried over _Na2SO4 , filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by flash chromatography using heptane and EtOAc as eluents to give ethyl (2R)-2-(5-iodo-6-prop-1-ynyl-thieno[2,3-d]pyrimidin-4-yl)oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 8.89 (d, J = 5.1 Hz, 1H), 8.59 (s, 1H), 7.62 (d, J = 5.2 Hz, 1H), 7.55 (dd, J = 7.5, 1.6 Hz, 1H), 7.51 (dd, J = 7.5, 1.6 Hz, 1H), 7.43 (m, 1H), 7.26 (m, 1H), 7.11 (m, 2H), 7.02 (td, J = 7.5, 0.9 Hz, 1H), 6.94 (td, J = 7.4, 0.8 Hz, 1H), 5.79 (dd, J = 9.1, 4.6 Hz, 1H), 5.31 (d, J =14.9 Hz, 1H), 5.26 (d, J =14.9 Hz, 1H), 4.13 (m, 2H), 3.76 (s, 3H), 3.60 (dd, J =13.8, 4.5 Hz, 1H), 3.33 (m, 1H), 2.21 (s, 3H), 1.10 (t, J =7.1 Hz, 3H). ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ: 169.4, 166.5, 165.7, 164.8, 161.3, 157.7, 155.8, 153.6, 132.2, 131.0, 130.8, 128.3, 124.0, 120.9, 120.1, 115.5, 112.2, 112.0, 110.5, 98.9, 79.5, 74.4, 74.3, 69.1, 61.1, 55.7, 13.9, 4.6.

Step B: 2-chloro-3-ethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol

33.7 g of [2-chloro-3-ethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]-triisopropylsilane (76.9 mmol; prepared according to WO 2015/097123, preparation 5e) were dissolved in 600 mL of THF and cooled to 0°C, then 92.3 mL of TBAF (92.3 mmol, 1 M solution in THF) were added dropwise and the mixture was stirred until complete conversion. It was then diluted with brine, acidified with citric acid, and then extracted with DCM. The combined organic layer was dried over Na ₂ SO ₄ , filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by flash chromatography using heptane and EtOAc as eluents to give 2-chloro-3-ethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol. ¹ H NMR (400 MHz, CDCl ₃ ) δ: 7.63 (d, J = 8.2 Hz, 1H), 6.86 (d, J = 8.2 Hz, 1H), 5.87 (s, 1H), 3.09 (q, J = 7.44 Hz, 2H), 1.33 (s, 12H), 1.15 (t, J = 7.44 Hz, 3H).

Step C: Ethyl (2R)-2-[5-(3-chloro-2-ethyl-4-hydroxy-phenyl)-6-prop-1-ynyl-thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate

353 mg of ethyl (2R)-2-(5-iodo-6-prop-1-ynyl-thieno[2,3-d]pyrimidin-4-yl)oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (0.50 mmol) and 282 mg of 2-chloro-3-ethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (0.55 mmol) were dissolved in 2 ml of dioxane, then 35 mg of PdCl2 _× AtaPhos (0.05 mmol), 326 mg _{of Cs2CO3} (1.00 mmol) and 1 ml of water were added and the mixture was stirred in a microwave reactor at 100°C under _N2 atmosphere for 20 minutes. It is then diluted with brine, acidified to pH 5 with 1 _M aqueous HCl, and extracted with DCM. The combined organic layer is dried over _Na2SO4 , filtered, and the filtrate is concentrated under reduced pressure. The crude product is purified by flash chromatography using heptane and EtOAc as eluents. It was then further purified by preparative reverse phase chromatography using 5 mM aqueous _NH4HCO3 and _MeCN as eluents to give ethyl (2R)-2-[5-(3-chloro-2-ethyl-4-hydroxy-phenyl)-6-prop-1-ynyl-thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate as a 2:1 mixture of atropisomers. ¹ H NMR (500 MHz, DMSO-d ₆ ) δ: 10.35/10.29 (s, 1H), 8.93 (d, J =5.1 Hz, 1H), 8.59/8.57 (s, 1H), 7.63/7.60 (d, J =5.1 Hz, 1H), 7.54-6.93 (m, 8H), 6.84/6.74 (t, J =7.5 Hz, 1H), 6.43/6.18 (dd, J =7.5, 1.4 Hz, 1H), 5.51/5.40 (m, 1H), 5.30-5.16 (m, 2H), 4.17-3.99 (m, 2H), 3.76/3.75 (s, 3H), 3.34-3.14 (m, 1H), 2.93-2.35 (m, 3H), 2.02/1.98 (s, 3H), 1.08/1.04 (t, J =7.0 Hz, 3H), 0.94/0.76 (t, J =7.5 Hz, 3H).

Step D: (2R)-2-[(5Sa)-5-[3-chloro-2-ethyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-prop-1-ynyl-thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid, C7

322 mg of ethyl (2R)-2-[5-(3-chloro-2-ethyl-4-hydroxyphenyl)-6-prop-1-ynyl-thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (0.44 mmol), 190 mg of 2-(4-methylpiperazin-1-yl)ethanol (1.32 mmol) and 346 mg of _PPh3 (1.32 mmol) were dissolved in 10 ml of dry troluene, then 304 mg of DTBAD (1.32 mmol) were added and the mixture was stirred at 50°C under _N2 atmosphere until the conversion was complete. The mixture was then concentrated under reduced pressure and the residue was purified by flash chromatography using heptane, EtOAc and MeOH as eluents, then further purified by preparative reverse-phase chromatography using 5 mM aqueous NH ₄ HCO ₃ and MeCN as eluents to afford the intermediate ester. It was dissolved in 2 mL of dioxane, then 84 mg LiOH×H ₂ O (2.00 mmol) and 1 mL of water were added. The mixture was stirred at 50°C until complete hydrolysis. It was then diluted with brine, neutralized with 2 M aqueous HCl and extracted with DCM. The combined organic phase was dried over Na ₂ SO ₄ , filtered and the filtrate was concentrated under reduced pressure. Atropisomers are purified and separated by preparative reverse-phase chromatography using 5 mM aqueous NH ₄ HCO ₃ and MeCN as eluents. The later eluted atropisomer is isolated as C7 . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 8.88 (d, J = 5.2 Hz, 1H), 8.60 (s, 1H), 7.76 (d, J = 5.0 Hz, 1H), 7.54 (dd, J = 7.6, 1.8 Hz, 1H), 7.46 (m, 1H), 7.26 (d, J = 8.5 Hz, 1H), 7.20-7.13 (m, 3H), 7.04 (td, J = 7.5, 0.9 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.78 (t, J = 7.5 Hz, 1H), 6.32 (dd, J = 7.4, 1.5 Hz, 1H), 5.48 (dd, J =9.7, 2.9 Hz, 1H), 5.27 (d, J =15.0 Hz, 1H), 5.19 (d, J =15.0 Hz, 1H), 4.23 (m, 2H), 3.76 (s, 3H), 3.31 (m, 1H), 2.75 (m, 2H), 2.47 (m, 1H), 2.64-2.36 (m, 10H), 2.22 (s, 3H), 2.01 (s, 3H), 0.74 (t, J =7.5 Hz, 3H). ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ: 165.95, 165.89, 164.7, 157.8, 157.2, 155.3, 154.0, 141.6, 135.6, 131.0, 130.8, 130.1, 128.4, 128.0, 127.2, 121.4, 120.1, 117.8, 112.2, 111.7, 97.2, 74.9, 68.9, 67.1, 56.1, 55.7, 54.0, 32.7, 24.4, 13.1, 4.4. HRMS calculated for C ₄₅ H ₄₅ N ₆ O ₆ SCl: 832.2810; found 833.2878 (M+H).

Obtaining C8:

(2R)-2-[(5S _a )-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(3-sulfooxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

210 mg of ethyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(3-hydroxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (0.24 mmol; prepared according to WO 2016/207216, preparation 2) were dissolved in 9.3 ml of pyridine, then 0.38 ml of SO ₃ ×pyridine (2.36 mmol) was added and the mixture was stirred at 70°C until complete conversion. The mixture was then concentrated under reduced pressure and the residue was dissolved in 2 ml of dioxane, then 200 mg of KOH (3.56 mmol) and 1 ml of water were added. The mixture was stirred at 70°C until the Et ester was completely hydrolyzed. It was then neutralized with 5 M aqueous HCl and purified by preparative reverse-phase chromatography (direct injection) using 25 mM aqueous NH ₄ HCO ₃ and MeCN as eluents to afford C8 . ¹ H NMR (500 MHz, DMSO-d ₆ ) δ: 8.93 (d, J =5.1 Hz, 1H), 8.63 (s, 1H), 8.27 (m, 1H), 8.13 (m, 1H), 7.61 (d, J =5.1 Hz, 1H), 7.43 (t, J =7.8 Hz, 1H), 7.33 (d, J =7.8 Hz, 1H), 7.32-7.27 (m, 3H), 7.23-7.13 (m, 4H), 7.05 (d, J =7.8 Hz, 1H), 6.73 (t, J =7.5 Hz, 1H), 6.31 (dd, J =7.5, 1.3 Hz, 1H), 5.51 (dd, J =9.8, 3.5 Hz, 1H), 5.34 (d, J =15.2 Hz, 1H), 5.27 (d, J =15.2 Hz, 1H), 4.24-4.08 (m, 2H), 3.26 (dd, J =14.3, 3.4 Hz, 1H), 3.10-2.52 (m, 14H), 1.82 (s, 3H). ¹³ C NMR (125 MHz, DMSO-d ₆ ) δ: 170.8, 166.5, 166.4, 162.8, 162.7, 158.3, 155.3, 154.0, 153.6, 152.9, 137.8, 136.8, 135.9, 131.12, 131.05, 130.4, 130.3, 129.1, 128.5, 128.2, 127.8, 124.4, 123.5, 122.7, 121.9, 120.5, 120.2, 118.8, 116.0, 115.9, 112.0, 110.5, 73.5, 69.1, 67.2, 55.4, 43.1, 31.8, 17.5. HRMS calculated for C ₄₆ H ₄₂ N ₆ O ₉ S ₂ ClF: 940.2127; found 941.2191 (M+H).

Obtaining C9:

2 R )-2-{[5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3- d ]pyrimidin-4-yl]oxy}-3-(2-{[2-(3-sulfophenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid

750 mg ethyl (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[(2-methylsulfanylpyrimidin-4-yl)methoxy]phenyl]propanoate (0.89 mmol; prepared according to WO 2015/097123, preparation 10a) were dissolved in 9 ml THF, then 726 mg [3-(2,2-dimethylpropoxysulfonyl)phenyl]boronic acid (2.67 mmol), 62 mg Pd(PPh ₃ ) ₄ (0.05 mmol) and 509 mg thiophene-2-carbonyloxycopper (2.67 mmol) were added and The mixture was stirred at 75°C until complete conversion. It was then concentrated under reduced pressure and purified by flash chromatography using heptane, EtOAc, and 0.7 M NH ₃ in MeOH as eluents. It was then dissolved in 20 mL of 1,1,1,3,3,3-hexafluoropropan-2-ol, 4.5 mL of TFA was added, and the mixture was stirred at 80°C until complete hydrolysis of the sulfone ester. The mixture was concentrated under reduced pressure and then dissolved in 5 mL of dioxane, followed by the addition of 210 mg LiOH×H ₂ O (5.00 mmol) and 2 mL of water. The mixture was stirred at room temperature until complete hydrolysis. It was then diluted with brine, neutralized with 2 M aqueous HCl, and extracted with DCM. The combined organic phase was dried over _Na2SO4 , filtered _, and the filtrate was concentrated under reduced pressure. The resulting atropisomers were purified and separated by preparative reverse-phase chromatography using 25 mM aqueous _NH4HCO3 and _MeCN as eluents, then further purified using 0.1% aqueous TFA and MeCN as eluents to afford C9 . ¹ H NMR (500 MHz, DMSO-d ₆ ) δ: 13.19 (br s, 1H), 9.48 (br s, 1H), 8.94 (d, J = 5.1 Hz, 1H), 8.74 (t, J = 1.6 Hz, 1H), 8.65 (s, 1H), 8.37 (dt, J = 7.8, 2.9 Hz, 1H), 7.78 (dt, J = 7.6, 1.5 Hz, 1H), 7.60 (d, J = 5.1 Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.29 (m, 3H), 7.22-7.14 (m, 3H), 7.13-7.05 (m, 2H), 6.74 (t, J =7.5 Hz, 1H), 6.38 (d, J =7.6 Hz, 1H), 5.53 (dd, J =9.6, 3.6 Hz, 1H), 5.37 (d, J =15.3 Hz, 1H), 5.31 (d, J =15.3 Hz, 1H), 4.2 (m, 2H), 3.50-2.88 (m, 11H), 2.76 (s, 3H), 2.61 (dd, J =14.2, 9.7 Hz, 1H), 1.79 (s, 3H). ¹³ C NMR (125 MHz, DMSO-d ₆ ) δ: 170.8, 166.5, 163.0, 162.7, 161.1, 158.4, 155.4, 153.3, 152.9, 148.6, 136.6, 136.0, 131.1, 130.1, 129.0, 128.5, 128.3, 128.1, 127.9, 125.3, 124.4, 121.9, 120.5, 118.8, 116.2, 116.0, 115.9, 112.1, 110.5, 73.2, 69.1, 66.5, 55.0, 51.7, 49.7, 42.1, 31.5, 17.5. HRMS calculated for C ₄₆ H ₄₂ N ₆ O ₈ S ₂ ClF: 924.2178; found 925.2274 (M+H).

Getting C10:

(2R)-2-[(5S _a )-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-[4-fluoro-3-(2,2,2-trifluoroethoxy)phenyl]thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

250 mg of ethyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-iodo-thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (0.27 mmol; prepared according to WO 2015/097123, preparation 30) and 79 mg of [4-fluoro-3-(2,2,2-trifluoroethoxy)phenyl]boronic acid (0.40 mmol) were dissolved in 1 ml of THF, then 3.0 mg of PdOAc ₂ (0.013 mmol), 5.7 mg of tBuXPhos (0.013 mmol), 174 mg of Cs ₂ CO ₃ were added (0.53 mmol) and 0.27 mL of water and the mixture was stirred at 70 °C under N ₂ for 2 h. Then it was diluted with brine and extracted with 2-MeTHF. The combined organic layer was dried over Na ₂ SO ₄ , filtered and the filtrate was concentrated under reduced pressure. The crude ether was purified by flash chromatography using heptane, EtOAc and 0.7 M NH ₃ solution in MeOH as eluents. Then it was dissolved in 5.3 mL of dioxane, followed by the addition of 64 mg LiOH×H ₂ O (1.52 mmol) and 1.3 mL of water. The mixture was stirred at room temperature until complete hydrolysis. Then it was diluted with brine, neutralized with 2 M aqueous HCl and extracted with DCM. The combined organic phase was dried over _Na2SO4 , filtered _, and the filtrate was concentrated under reduced pressure. The atropisomers were purified and separated by preparative reverse-phase chromatography using 25 mM aqueous _NH4HCO3 and _MeCN as eluents. The atropisomer eluted later was isolated as C10 . ¹ H NMR (500 MHz, DMSO-d ₆ ) δ: 8.91 (d, J =5.2 Hz, 1H), 8.57 (s, 1H), 7.82 (d, J =5.1 Hz, 1H), 7.53 (dd, J =7.5, 1.7 Hz, 1H), 7.45 (m, 2H), 7.27 (dd, J =11.0, 8.6 Hz, 1H), 7.22 (d, J =8.6 Hz, 1H), 7.16-7.09 (m, 3H), 7.03 (t, J =7.5 Hz, 1H), 6.98 (d, J =8.3 Hz, 1H), 6.92 (m, 1H), 6.69 (t, J =7.4 Hz, 1H), 6.16 (d, J =7.2 Hz, 1H), 5.47 (dd, J =10.3, 2.6 Hz, 1H), 5.25 (d, J =15.1 Hz, 1H), 5.19 (d, J =15.1 Hz, 1H), 4.75-4.53 (m, 2H), 4.21 (t, J =5.5 Hz, 2H), 3.75 (s, 3H), 3.41 (d, J =12.0 Hz, 1H), 2.77-2.30 (m, 12H), 2.24 (s, 3H), 1.81 (s, 3H). ¹³ C NMR (125 MHz, DMSO-d ₆ ) δ: 171.3, 166.2, 166.0, 164.6, 163.5, 157.9, 157.2, 155.3, 153.7, 153.1, 152.1, 150.5, 144.6, 135.8, 131.0, ,130.8, 130.7, 129.6, 129.1, 128.4, 128.1, 127.8, 125.4, 123.7, 122.0, 120.4, 120.1, 118.8, 117.0, 116.7, 115.6, 112.2, 111.7, 110.5, 74.8, 68.9, 67.2, 65.6, 56.0, 55.7, 53.8, 52.0, 44.6, 32.7, 17.5. HRMS calculated for C ₄₉ H ₄₅ N ₆ O ₇ SClF ₄ : 972.2695; found 973.2761 (M+H).

Getting C11:

( 2R)-2-{[(5Sa)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(4-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid

C11 is prepared according to Example 107 in WO 2015/097123.

Obtaining C12

( 2R )-2-{[( 5S _a )-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3 -d ]pyrimidin-4-yl]oxy}-3-(2-{[1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl]methoxy}phenyl)propanoic acid

C12 is prepared according to Example 77 in WO 2015/097123.

Obtaining C13

(2R)-2-[(5S _a )-6-(3-amino-4,5-difluorophenyl)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

250 mg of ethyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-iodo-thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (0.27 mmol; prepared according to WO 2015/097123, preparation 30) and 102 mg of 2,3-difluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.41 mmol) were dissolved in 1 ml of THF, then 3.0 mg of PdOAc ₂ (0.013 mmol), 5.7 mg of tBuXPhos (0.013 mmol), 174 mg _Cs2CO3 (0.53 mmol ₎ and 0.27 mL water were added and the mixture was stirred at 70°C under _N2 for 2 h. Then it was diluted with brine and extracted with ₂ -MeTHF. The combined organic layer was dried over _Na2SO4 , filtered and the filtrate was concentrated under reduced pressure. The crude ester was purified by flash chromatography using heptane, EtOAc and 0.7 M _NH3 in MeOH as eluents. Then it was dissolved in 0.7 mL dioxane, followed by the addition of 60 mg LiOH× _H2O (1.43 mmol) and 0.18 mL water. The mixture was stirred at room temperature until complete hydrolysis. Then it was diluted with brine, neutralized with 2 M aqueous HCl and extracted with DCM. The combined organic phase was dried over _Na2SO4 , filtered _, and the filtrate was concentrated under reduced pressure. The atropisomers were purified and separated by preparative reverse-phase chromatography using 25 mM aqueous _NH4HCO3 and _MeCN as eluents. The atropisomer eluted later was isolated as C13 . ¹ H NMR (500 MHz, DMSO-d ₆ ) δ: 8.89 (d, J =5.2 Hz, 1H), 8.56 (s, 1H), 7.76 (d, J =5.0 Hz, 1H), 7.53 (dd, J =7.6, 1.8 Hz, 1H), 7.45 (m, 1H), 7.36 (d, J =8.6 Hz, 1H), 7.20 (d, J =8.7 Hz, 1H), 7.13 (m, 2H), 7.03 (td, J =7.5, 1.0 Hz, 1H), 6.99 (d, J =8.1 Hz, 1H), 6.71 (t, J =7.3 Hz, 1H), 6.62 (m, 1H), 6.21 (d, J =7.5, 1.3 Hz, 1H), 6.12 (m, 1H), 5.73 (s, 2H), 5.46 (dd, J =10.1, 3.1 Hz, 1H), 5.25 (d, J =15.1 Hz, 1H), 5.19 (d, J =15.2 Hz, 1H), 4.22 (m, 2H), 3.75 (s, 3H), 3.35 (m, 1H), 2.73 (m, 2H), 2.65-2.35 (m, 9H), 2.22 (s, 3H), 1.85 (s, 3H). ¹³ C NMR (125 MHz, DMSO-d ₆ ) δ: 171.1, 166.0, 165.3, 163.3, 157.8, 157.2, 155.3, 153.7, 152.9, 149.0, 138.9, 137.1, 135.8, 131.0, 130.8, 130.4, 128.7, 128.4, 128.1, 127.8, 125.2, 122.0, 120.4, 120.1, 118.9, 115.6, 112.2, 111.9, 110.6, 103.2, 74.5, 68.9, 67.1, 56.0, 55.7, 53.9, 52.1, 44.7, 32.6, 17.6. HRMS calculated for C ₄₇ H ₄₄ N ₇ O ₆ SClF ₂ : 907.2730; found 908.2803 (M+H).

Obtaining C14

(2 R )-2-{[(5Sa)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3- d ]pyrimidin-4-yl]oxy}-3-(2-{[2-(3-hydroxy-2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid

210 mg of ethyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[(2-chloropyrimidin-4-yl)methoxy]phenyl]propanoate (0.25 mmol, WO2016/207216 Preparation 1 ) and 84 mg of (3-hydroxy-2-methoxy-phenyl)boronic acid (0.50 mmol) were dissolved in 3.8 ml of 1,4-dioxane, then 18 mg of Pd( _PPh3 ) _2Cl2 ( _0.025 mmol), 240 mg _{of Cs2CO3} (0.75 mmol) and 3.8 ml of water were added and the mixture was stirred under _N2 atmosphere at 70°C until complete conversion. It was then diluted with water, neutralized with 2 M aqueous HCl and extracted with DCM. The combined organic phase was dried over Na ₂ SO ₄ , filtered and the filtrate was concentrated under reduced pressure. The crude ester was purified by flash chromatography using heptane, EtOAc and 0.7 M NH ₃ in MeOH as eluents to give a mixture of diastereoisomers. It was dissolved in 2 mL dioxane, then 245 mg LiOH×H ₂ O (5.85 mmol) and 1 mL water were added. The mixture was stirred at rt until complete hydrolysis. It was then neutralized with 2 M aqueous HCl and directly injected onto a prep-RP-HPLC using 0.1% aqueous TFA and MeCN as eluents. The later eluted diastereoisomer is isolated as C14 . ^1H NMR (500 MHz, DMSO- _d6 ) δ: 9.53 (bs, 1H), 8.91 (d, 1H), 8.56 (s, 1H), 7.79 (d, 1H), 7.42 (d, 1H), 7.26 (m, 2H), 7.19 (d, 1H), 7.18 (m, 2H), 7.12 (t, 1H), 7.06 (dd, 1H), 6.98 (m, 1H), 6.98 (m, 1H), 6.97 (d, 1H), 6.68 (t, 1H), 6.16 (d, 1H), 5.47 (m, 1H), 5.27/5.20 (d+d, 2H), 4.26/4.19 (m+m, 2H), 3.76 (s, 3H), 3.38/2.42 (dd+dd, 2H), 2.74 (m, 2H), 2.55 (b, 4H), 2.47 (b, 4H), 2.25 (s, 3H), 1.80 (s, 3 H). HRMS calculated for C ₄₇ H ₄₄ N ₆ O ₇ SClF: 890.2665; found 891.2721 (M+H).

Obtaining a P15

(11R,20R)-23,26-dichloro-3-(4-fluorophenyl)-14-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]-24,25-dimethyl-20-[(4-methylpiperazin-1-yl)methyl]-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20,2,2,12,5,113,17,09,28]octacosa-1(25),2,5(28),6,8,13,15,17(27),22(26),23-decaene-11-carboxylic acid

P15 is obtained according to Example 116 in WO 2019/035914.

Obtaining P16

(11R,20R)-23,26-dichloro-14-[[2-[4-[[(2S)-1,4-dioxan-2-yl]methoxymethyl]-4-fluoro-cyclohexyl]pyrimidin-4-yl]methoxy]-3-(4-fluorophenyl)-24,25-dimethyl-20-[(4-methylpiperazin-1-yl)methyl]-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20,2,2,12,5,113,17,09,28]octacosa-1(24),2,5(28),6,8,13,15,17(27),22,25-decaene-11-carboxylic acid

P16 is prepared according to Example 28 in WO 2019/035911.

Obtaining P17

(11R,20R)-23,26-dichloro-14-[[2-[4-[[(2S)-1,4-dioxan-2-yl]methoxy]cyclohexyl]pyrimidin-4-yl]methoxy]-3-(4-fluorophenyl)-24,25-dimethyl-20-[(4-methylpiperazin-1-yl)methyl]-10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20,2,2,12,5,113,17,09,28]octacosa-1(24),2,5(28),6,8,13,15,17(27),22,25-decaene-11-carboxylic acid

P17 is prepared according to Example 44 in WO 2019/035911.

Example 4: Synthesis and characterization of an ADC inhibitor of Mcl-1

Exemplary antibody-drug conjugates (ADCs) are synthesized using the exemplary methods described in this example. The antibodies used to prepare the exemplary ADCs are described in Table 12. The term "CysmAb" refers to cysteine mutations in the antibody heavy chain that are used to conjugate the linker-payload to the antibody via maleimide groups. In some embodiments, the CysmAb mutations contain E152C and S375C (numbered according to the EU system). The mutations may also be numbered E156C and S379C, which correspond to the E152C and S375C mutations according to the EU system.

Table 12. Antibodies used for the synthesis of typical ADCs Antibody designation Antibody Mutation SEQ ID NO
(VL, VH) Allotype Ab G MuMy9-6 ch
(anti-CD33) N297Q SEQ ID NOs
4.3 G1m3 Ab T trastuzumab
(anti-HER2) N297S SEQ ID NOs
10.9 G1m3 Ab T trastuzumab
(anti-HER2) N297Q SEQ ID NOs
10.9 G1m3 BCMA or Ab B J6M0
(anti-BCMA) E156C, S379C (or E152C S375C (EU) (CysmAb)) SEQ ID NOs
2.1 G1m3 BCMA with silent Fc or Ab B with silent Fc J6M0
(anti-BCMA) E156C, S379C (or E152C S375C(EU) (CysmAb)) with silent Fc SEQ ID NOs
2.1 G1m3 PCAD NOV169N31Q E152C S375C EU (CysmAb) SEQ ID NOs 8, 7 G1m3 Ab C Anti-CD46 E156C S379C (or E152C S375C (EU)) SEQ ID Nos. 91, 90 G1m3

BTG: The antibody (Ab G and Ab T) is endowed with glutamines capable of reacting with bacterial transglutaminase (BTG), specifically functionalized with an amine containing cyclooctyne BCN (N-[(1R,8S,9s)-Bicynlo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl]-1,8-diamino-3,6-dioxaoctane, also known as BCN-POE3-NH2, CAS number: 1263166-93-3). Site-specific conjugation of the antibody to the BCN moiety using bacterial transglutaminase is accomplished using the methods described in Innate Pharma 2013 (presented at the ADC Summit, San Francisco, California, Oct. 15, 2013), WO 2017/059160, and WO 2016/144608, all of which are incorporated herein by reference for such methods. These modifications allowed the described azide-containing precursors to be conjugated using Method A and Method B, as described below. See also FIG. 1 .

Redox: The antibody (anti-CD33, anti-BCMA, anti-PCAD) is incubated with RMP Protein A (GE) resin at a ratio of 10 mg Ab per 1 mL of resin in PBS for 15 minutes with mixing in a disposable column of appropriate size. Cysteine HCl is added to a final concentration of 20 mM and incubated with mixing for 30 minutes at room temperature to allow reactive cysteines to deblock. The resin is quickly washed with 50 column volumes of PBS on a vacuum manifold. The resin is then resuspended in an equal volume of PBS containing 250 nM _CuCl2 . Reformation of interchain disulfides of the antibody is monitored at time points. At each time point, 25 μl of the resin suspension is removed, 1 μl of 20 mM MC-valcit-MMAE is added, and the tube is vortexed several times. The resin is centrifuged, the supernatant is removed, and then eluted with 50 μl of antibody elution buffer (Thermo Fisher Scientific). The resin is pelleted, and the supernatant is analyzed by separated phase chromatography using an Agilent PLRP-S 4000A 5 μm, 4.6 × 50 mm column (buffer A contains water, 0.1% TFA, buffer B contains acetonitrile, 0.1% TFA, the column is maintained at 80°C, flow rate 1.5 ml/min).

After determining that the antibody has reformed its interchain disulfide bonds, the resin is washed with 10 column volumes of PBS, resuspended in an equal volume of PBS, and allowed to drain to allow the resin to settle freely. Reoxidized antibodies are eluted from the Protein A resin with antibody elution buffer and neutralized with 1/10 volume of 1 M Tris, pH 9.0. The antibody buffer is then exchanged for PBS using PD-10 columns. The protein concentration is adjusted to 5 mg/mL and aliquoted for single-use conjugations (see Method C1-C18 below).

Method A (DAR2) : Propylene glycol (PG, 1.5 mL, 20% of the binding volume) was added to an antibody (Ab) solution (3 mg/mL, 3.3 mL). The mixture was vortexed for 30 seconds, followed by the addition of a 4-fold molar excess of the payload linker (5 mM, 533.8 µL in PG/DMA). The reaction mixture was stirred at room temperature overnight at 64 rpm. The solution was centrifuged (14,000 G at 4°C) for 20 minutes and then loaded onto a HiLoad 26/600 Superdex 200 pg chromatography column (GE Healthcare, 28989336) SEC. ADC was eluted with 20% PG in PBS (Sigma, P3813, 10PAK) followed by two dialysis cycles (16 and 4 hours) in PBS 1x pH 7.4 (Sigma, P3813, 10PAK). ADC was concentrated using Vivaspin 20, 50KD, PES (Sartorius Stedim, VS2031), sterile filtered through a 0.2 μm sterile PES filter, 25 mm (Whatmann, G896-2502), and stored at 4°C.

Alternatively, propylene glycol (PG, 673 μL, 20% binding volume) is added to the antibody (Ab) solution (1.8 mg/mL, 2.8 mL, 5 mg). The mixture is vortexed for 30 seconds, followed by the addition of a 4-fold molar excess of the payload linker (5 mM, 27 μL in PG/DMA). The reaction mixture is stirred at room temperature overnight at 64 rpm. The solution is centrifuged (14,000 G at 4°C) for 20 minutes and then loaded onto a HiLoad 26/600 Superdex 200 pg chromatography column (GE Healthcare, 28989336) SEC. ADC was eluted with 20% PG in PBS (Sigma, P3813, 10PAK) followed by two dialysis cycles (16 and 4 hours) in PBS 1x pH 7.4 (Sigma, P3813, 10PAK). ADC was concentrated using Vivaspin 20, 50KD, PES (Sartorius Stedim, VS2031), sterile filtered through a 0.2 μm sterile PES filter, 25 mm (Whatmann, G896-2502), and stored at 4°C.

Method B (DAR4) : Propylene glycol (PG, 1.5 mL, 20% of the binding volume) was added to the Ab solution (3 mg/mL, 3.3 mL). The mixture was vortexed for 30 sec, followed by the addition of a 10-fold molar excess of the linker-warhead payload (5 mM, 533.8 µL in PG/DMA). The reaction mixture was stirred at room temperature overnight at 64 rpm. The solution was then incubated with a 10-fold molar excess of DBCO-containing Tentagel resin (0.1-0.2 mmol/g, Iris Biotech, CS-0477.0500) for 6 h. The solution was then centrifuged (14,000 G at 4°C) for 20 minutes and then loaded onto a HiLoad 26/600 Superdex 200 pg chromatography column (GE Healthcare, 28989336) SEC. ADC was eluted with 20% PG in PBS (Sigma, P3813, 10PAK) followed by two dialysis cycles (16 and 4 hours) in PBS 1X pH 7.4 (Sigma, P3813, 10PAK). ADC was concentrated using Vivaspin 20, 50KD, PES (Sartorius Stedim, VS2031), sterile filtered through a 0.2 μm sterile PES filter, 25 mm (Whatmann, G896-2502) and stored at 4°C.

Alternatively, propylene glycol (PG, 266 μL, 20% of the binding volume) is added to the Ab solution (4.5 mg/mL, 1.4 mL, 6.3 mg). The mixture is vortexed for 30 sec, followed by the addition of a 10-fold molar excess of the payload linker (5 mM, 84 μL in PG/DMA). The reaction mixture is stirred at room temperature overnight at 64 rpm. This solution is incubated with a 10-fold molar excess of DBCO-containing Tentagel resin (0.1-0.2 mmol/g, Iris Biotech, CS-0477.0500) for 6 h. The solution was then centrifuged (14,000 G at 4°C) for 20 min and loaded onto a HiLoad 26/600 Superdex 200 pg chromatography column (GE Healthcare, 28989336) SEC. ADC was eluted with 20% PG in PBS (Sigma Life Science, P3813, 10PAK) followed by two dialysis cycles (16 and 4 h) in PBS 1x pH 7.4 (Sigma Life Science, P3813, 10PAK). The conjugate was concentrated using Vivaspin 20, 50KD, PES (Sartorius Stedim, VS2031), sterile filtered through a 0.2 μm sterile PES filter, 25 mm (Whatmann, G896-2502) and stored at 4°C.

Method C: The antibody was bound to rmp Protein A resin (GE Healthcare) at a ratio of 10 mg Ab per 1 ml of resin in PBS for 30 min by mixing in a Biorad-sized disposable column. To deblock reactive cysteines, cysteine hydrochloride monohydrate was added to a final concentration of 20 mM. The mixture was stirred for 30 min at room temperature, washing the resin five times with 50 column volumes of PBS on a vacuum manifold. The resin was then resuspended in an equal volume of PBS containing 250 nM _CuCl2 and incubated for 1 hour and 30 min. The reoxidized antibody was washed five times with 50 column volumes of PBS on a vacuum manifold and resuspended in an equal volume of PBS. A 10-fold molar excess of 20 mM linker-payload solution and an equal volume of DMSO are added to the mixture. The mixture is incubated at room temperature for 2 hours. As a conjugation control, 20 μl of the resin suspension is centrifuged, the supernatant is removed, and then eluted with 40 μl of antibody elution buffer (Thermo Fisher Scientific). The supernatant is analyzed by reverse-phase chromatography using an Agilent PLRP-S 4000A 5 μm, 4.6×50 mm column (buffer A is water, 0.1% TFA, buffer B is acetonitrile, 0.1% TFA, the column is maintained at 80°C, flow rate is 1.5 ml/min). After removing excess payload linker by washing the resin 5 times with 50 column volumes of PBS on a vacuum manifold, ADCs were eluted from protein A with antibody elution buffer and neutralized with 1/10 volume of 1 M Tris pH 9.0. All example ADCs (except Ab C - L9-C1, Ab B - L9-P15, Ab B - L26-P1, Ab B - L9-P16, Ab B - L9-P17) were purified on a HiLoad 26/600 Superdex 200 pg SEC chromatography column (GE Healthcare, 28989336), eluting with 20% PG in PBS (Sigma Life Science, P3813, 10PAK). ADC was buffer exchanged by dialyzing into PBS 1X pH 7.4 (Sigma Life Science, P3813, 10PAK), concentrated using Vivaspin 20, 50KD, PES (Sartorius Stedim, VS2031), sterile filtered through a 0.2 µm sterile PES filter, 25 mm (Whatmann, G896-2502) and stored at 4°C. For ADC Ab C - L9-C1, Ab B - L9-P15, Ab B - L26-P1, Ab B - L9-P16 and Ab B - L9-P17, they were subjected to buffer exchange only via dialysing into PBS 1X pH 7.4 (Sigma Life Science, P3813, 10PAK), concentrated using Vivaspin 20, 50KD, PES (Sartorius Stedim, VS2031), sterile filtered through a 0.2 μm sterile PES filter, 25 mm (Whatmann, G896-2502) and stored at 4°C.

Alternatively, the antibody is incubated with RMP Protein A (GE) resin at a ratio of 10 mg Ab to 1 mL resin in PBS for 15 min with mixing in a disposable column of appropriate size. Cysteine HCl is added to a final concentration of 20 mM and incubated with mixing for 30 min at room temperature to allow reactive cysteines to deblock. The resin is quickly washed with 50 column volumes of PBS on a vacuum manifold. The resin is then resuspended in an equal volume of PBS containing 250 nM CuCl ₂ . Reformation of antibody interchain disulfides is monitored at time points. At each time point, 25 μL of the resin suspension is removed, 1 μL of 20 mM MC-valcit-MMAE is added, and the tube is tapped several times. The resin is centrifuged, the supernatant is removed, and then eluted with 50 µl of antibody elution buffer (Thermo). The resin is pelleted, and the supernatant is analyzed by reverse-phase chromatography using an Agilent PLRP-S 4000A 5 µm, 4.6 x 50 mm column (buffer A: water, 0.1% TFA, buffer B: acetonitrile, 0.1% TFA, column maintained at 80°C, flow rate: 1.5 ml/min). After the antibody has been determined to have reformed its interchain disulfide bonds, the resin is washed with 10 column volumes of PBS and resuspended in an equal volume of PBS. Eight to 12 equivalents of the linker-payload in DMSO are added to the reaction to a final concentration of 10% DMSO and then incubated at room temperature for 3 hours. The resin is then washed with 50 column volumes of PBS. The ADC is eluted from the protein A resin using antibody elution buffer. The ADC buffer is then replaced with PBS or another suitable buffer, and preparative size exclusion chromatography (S200 Increase; GE) is performed to remove aggregates if required. The following analyses are performed: analytical SEC to determine the monomer fraction, mass spectroscopy to determine DAR, LAL test to determine the endotoxin load, and protein concentration is determined by A280 using the excitation factor and the molecular weight of the antibody.

Method C1 - (CD33-L1-P1) : L1-P1 (2.53 µl of a 20 mM solution in DMSO, 0.051 µmol, 7.5 equiv) was added to a solution of CD33 antibody (1.0 mg, 154 µl of a 6.5 mg/ml solution in 1X PBS buffer). The resulting mixture was shaken at 400 rpm at ambient temperature for 1 h and then purified by ultracentrifugation (4 ml Amicon 10 kDa cut-off membrane filter, dilute the sample to a total volume of 4 ml with PBS buffer, then centrifugate for 10 min at 7500 x g, repeat 6 times). After dilution to 5.0 mg/ml , CD33-L1-P1 (0.842 mg, 0.0053 μmol, 79%) was obtained. The following analyses were performed: analytical size-exclusion chromatography (SEC) to determine the monomer fraction, mass spectroscopy (MS) to determine DAR, and protein concentration determined by A280 using the excitation factor and the molecular weight of the antibody. HRMS data (protein method) indicated a mass of 157884 with a DAR of 3.9, based on a comparison of the MS peak intensities of the DAR3 and DAR4 species. SEC indicated aggregation </=1%, based on a comparison of the high molecular weight absorption peak area at 210 and 280 nm with that of the monomeric ADC.

Method C2 - (PCAD-L1-P1): FollowingMethod C1with PCAD antibody (2.0 antibody, 357 µl of 5.6 antibody/ml solution, 0.013 µmol, 1.0 equiv.) andL1-P1(5.04 μl of 20 mM solution in DMSO, 0.101 μmol, 7.5 equiv.) is obtainedPCAD-L1-P1(1.73 antibody, 0.0109 μmol, 81%). HRMS data (protein method) showed a mass of 158268, with a DAR of 3.9. SEC showed </=1% aggregation.

Method C3 - (PCAD-L10-P1): FollowingMethod C1with PCAD antibody (2.0 antibody, 357 µl of 5.6 antibody/ml solution, 0.013 µmol, 1.0 equiv.) andL10-P1(5.04 μl of 20 mM solution in DMSO, 0.101 μmol, 7.5 equiv.) is obtainedPCAD-L10-P1(1.97 antibody, 0.012 μmol, 92%). Protein-based HRMS data showed a mass of 158,731, with a DAR of 3.9. SEC showed 1% aggregation.

Method C4 - (CD33-L10-P1): FollowingMethod C1with CD33 antibody (1.1 antibody, 169 μl of 6.5 antibody/ml solution, 0.0074 μmol, 1.0 equiv.) andL10-P1(2.53 μl of 20 mM solution in DMSO, 0.051 μmol, 6.8 equiv.) is obtainedCD33-L10-P1(1.08 antibody, 0.0068 µmol, 92%). Protein-based HRMS data showed a mass of 158,346, with a DAR of 3.9. SEC showed 1% aggregation.

Method C5 - (PCAD-L4-P1): FollowingMethod C1with PCAD antibody (2.0 antibody, 357 µl of 5.6 antibody/ml solution, 0.013 µmol, 1.0 equiv.) andL4-P1(5.38 µl of 20 mM solution in DMSO, 0.108 µmol, 8.0 equiv.) is obtainedPCAD-L4-P1(2.1 antibody, 0.013 μmol, 99%). HRMS data (protein method) showed a mass of 156533, with a DAR of 4.2. SEC showed 1% aggregation.

Method C6 - (PCAD-L3-P1) : Following Method C1 with PCAD antibody (1.0 antibody, 179 µL of a 5.6 antibody/mL solution, 0.0067 µmol, 1.0 equiv.) and L3-P1 (2.69 µL of a 20 mM solution in DMSO, 0.054 µmol, 8.0 equiv.), PCAD-L3-P1 (1.05 antibody, 0.0067 µmol, 99%) was obtained. HRMS data (protein method) showed a mass of 156,763, with a DAR of 3.9. SEC showed 1% aggregation.

Method C7 - (CD33-L2-P1): FollowingMethod C1with CD33 antibody (2.0 antibody, 351 μl of 5.7 antibody/ml solution, 0.013 μmol, 1.0 equiv.) andL2-P1(5.4 μl of 20 mM solution in DMSO, 0.108 μmol, 8.0 equiv.) is obtainedCD33-L2-P1(2.1 antibody, 0.013 μmol, 99%). Protein-based HRMS data showed a mass of 155821, with a DAR of 3.9. SEC showed 1% aggregation.

Method C8 - (PCAD-L9-P1): FollowingMethod C1with PCAD antibody (2.0 antibody, 357 µl of 5.6 antibody/ml solution, 0.013 µmol, 1.0 equiv.) andL9-P1(6.73 μl of 20 mM solution in DMSO, 0.13 μmol, 10.0 equiv.) is obtainedPCAD-L9-P1(1.90 antibody, 0.012 μmol, 91%). HRMS data (protein method) showed a mass of 154392, with a DAR of 3.9. SEC showed </= 1% aggregation.

Method C9 - (CD33-L9-P1): FollowingMethod C1with CD33 antibody (1.0 antibody, 154 μl of 6.5 antibody/ml solution, 0.0067 μmol, 1.0 equiv.) andL9-P1(3.37 µl of 20 mM solution in DMSO, 0.067 µmol, 10.0 equiv.) is obtainedCD33-L9-P1(0.84 antibody, 0.0055 μmol, 81%). HRMS data (protein method) showed a mass of 154009, with a DAR of 3.9. SEC showed 1% aggregation.

Method C10 - (CD33-L8-P1): FollowingMethod C1with CD33 antibody (1.0 antibody, 154 μl of 6.5 antibody/ml solution, 0.0067 μmol, 1.0 equiv.) andL8-P1(3.43 µl of 30 antibody/ml solution in DMSO, 0.054 µmol, 8.0 equiv.) is obtainedCD33-L8-P1(0.91 antibody, 0.0058 μmol, 87%). HRMS data (protein method) showed a mass of 1555921, with a DAR of 3.9. SEC showed 1% aggregation.

Method C11 - (PCAD-L8-P1): FollowingMethod C1with PCAD antibody (2.5 antibody, 472 µl of 5.6 antibody/ml solution, 0.017 µmol, 1.0 equiv.) andL8-P1(10.7 µl of 30 antibody/ml solution in DMSO, 0.17 µmol, 10.0 equiv.) is obtainedPCAD-L8-P1(1.57 antibody, 0.010 μmol, 60%). HRMS data (protein method) showed a mass of 156307, with a DAR of 3.9. SEC showed </= 1% aggregation.

Method C12 - (CD33-L7-P1): FollowingMethod C1with CD33 antibody (1.0 antibody, 154 μl of 6.5 antibody/ml solution, 0.0067 μmol, 1.0 equiv.) andL7-P1(3.54 µl of 30 antibody/ml solution in DMSO, 0.054 µmol, 8.0 equiv.) is obtainedCD33-L7-P1(0.56 antibody, 0.0036 μmol, 53%). HRMS data (protein method) showed a mass of 156151, with a DAR of 3.9. SEC showed 1% aggregation.

Method C13 - (PCAD-L7-P1): FollowingMethod C1with PCAD antibody (2.5 antibody, 472 µl of 5.6 antibody/ml solution, 0.017 µmol, 1.0 equiv.) andL7-P1(11.03 µl of 30 antibody/ml solution in DMSO, 0.17 µmol, 10.0 equiv.) is obtainedPCAD-L7-P1(2.60 antibody, 0.017 μmol, 99%). HRMS data (protein method) showed a mass of 156539, with a DAR of 3.9. SEC showed </= 1% aggregation.

Method C14 - (BCMA-L5-P1): FollowingMethod C1with BCMA antibody (2.0 antibody, 250 µl of 8.0 antibody/ml solution, 0.013 µmol, 1.0 equiv.) andL5-P1(5.38 µl of 20 mM solution in DMSO, 0.108 µmol, 8 equiv.) is obtainedBCMA-L5-P1(2.1 antibody, 0.013 μmol, 99%). HRMS data (protein method) showed a mass of 159197 with a DAR of 3.9. SEC showed 1% aggregation.

Method C15 - (CD33-L12-P2): FollowingMethod C1with CD33 antibody (2.0 antibody, 345 µl of 5.8 antibody/ml solution, 0.013 µmol, 1.0 equiv.) andL12-P2(4.05 μl of 20 mM solution in DMSO, 0.081 μmol, 6.0 equiv.) is obtainedCD33-L12-P2(2.1 antibody, 0.013 μmol, 99%). Protein-based HRMS data showed a mass of 157,331, with a DAR of 3.9. SEC showed 1% aggregation.

Method C16 - (BCMA-L12-P2): FollowingMethod C1with BCMA antibody (2.0 antibody, 250 µl of 8.0 antibody/ml solution, 0.013 µmol, 1.0 equiv.) andL12-P2(3.03 μl of 20 mM solution in DMSO, 0.061 μmol, 4.5 equiv.) is obtainedBCMA-L12-P2(2.1 antibody, 0.013 μmol, 99%). Protein-based HRMS data showed a mass of 157,792, with a DAR of 3.8. SEC showed 1% aggregation.

Method C17 - (CD33-L4-P2): FollowingMethod C1with CD33 antibody (2.0 antibody, 345 µl of 5.8 antibody/ml solution, 0.013 µmol, 1.0 equiv.) andL4-P2(3.03 μl of 20 mM solution in DMSO, 0.061 μmol, 4.5 equiv.) is obtainedCD33-L4-P2(2.1 antibody, 0.013 μmol, 99%). HRMS data (protein method) showed a mass of 156,700, with a DAR of 3.9. SEC showed 1% aggregation.

Method C18 - (BCMA-L4-P2): FollowingMethod C1with BCMA antibody (2.0 antibody, 250 µl of 8.0 antibody/ml solution, 0.013 µmol, 1.0 equiv.) andL4-P2(3.03 μl of 20 mM solution in DMSO, 0.061 μmol, 4.5 equiv.) is obtainedBCMA-L4-P2(2.1 antibody, 0.013 μmol, 99%). HRMS data (protein method) showed a mass of 157164, with a DAR of 3.9. SEC showed 1% aggregation.

Other ADCs described in the examples, such as CD38 Mcl1 inhibitor ADC, CD48 Mcl1 inhibitor ADC, and CD79b Mcl1 inhibitor ADC (e.g., described in Tables 24-26), are synthesized by methods similar to those described above.

Analytical methods

Liquid Chromatography-Mass Spectrometry (LC/MS) I

General methodology The drug-to-antibody ratio (DAR) of representative ADCs is determined by liquid chromatography-mass spectrometry (LC/MS) according to one of the following methods (e.g., LC-I, LCII, and LC-III). For all LC methods, mobile phase A is MS-grade purified water (Biosolve, Dieuze, France, 00232141B1BS), mobile phase B is MS-grade acetonitrile (Biosolve, Dieuze, France, 0001204101BS), and mobile phase D is MS-grade purified water supplemented with 1% formic acid (FA) (Honeywell/Fluka, Bucharest, Romania, 56302). Mobile phase D is fixed at 10% to maintain a mobile phase composition of 0.1% FA, and the column temperature is set at 80°C. The general MS method is optimized for all synthesized ADCs except one ADC where native MS is used to determine the average DAR (see MS-I and Table 22 below).

LC-I : ADC is loaded onto a MassPREP Micro desalting column (2.1 x 5.0 mm, Waters, Saint-Quentin-en-Yvelines, France, 186004032). For starting material mass analysis, a desalting step is performed for 0.5 min with 5% mobile phase B at a flow rate of 0.5 mL/min. An elution step is performed with a gradient from 0.51 min at 5% B to 2.0 min at 90% B at a flow rate of 0.2 mL/min. Two washing steps are set from 2.1 min to 2.7 min and from 2.8 min to 3.4 min at 5% B to 90% B at a flow rate of 0.5 mL/min. Finally, a conditioning step is used at 3.5 min for 0.5 min at 5% B (0.5 mL/min).

For ADC analysis under reduced conditions, a desalting step is performed for 0.5 min at 5% B at a flow rate of 0.2 mL/min. This is followed by an elution step with a gradient from 0.51 min at 10% B to 7.61 min at 80% B at a flow rate of 0.2 mL/min. Two wash steps are set from 8.1 min to 8.6 min and from 8.7 min to 9.2 min from 5% B to 90% B (0.5 mL/min). Finally, a conditioning step is performed at 9.3 min for 0.5 min at 5% B at a flow rate of 0.5 mL/min.

LC-II : ADC is loaded onto a MabPac RP column (2.1 x 100 mm, 4 μm, Thermo Fisher Scientific, Rockford, IL, 088647). For analysis under both initial and reducing conditions, a desalting step is performed for 1.4 min at 20% B at a flow rate of 0.4 mL/min. Then, an elution step is performed with a gradient from 1.5 min at 20% B to 11.5 min at 70% B at a flow rate of 0.3 mL/min. A wash step is set from 11.75 min to 13.75 min at 90% B at a flow rate of 0.5 mL/min. Finally, a conditioning step is used at 14.0 min for 1.0 min at 20% B at a flow rate of 0.4 mL/min.

LC-III : ADC was loaded onto Bioresolve RP mAb Polyphenyl, 450A, 2.7 μm, 2.1 x 150 mm (Waters, Saint-Quentin-en-Yvelines, France, 186008946). For analysis under both initial and reducing conditions, a desalting step was performed for 1.4 min at 20% B with a flow rate of 0.4 mL/min. An elution step was performed with a gradient from 1.5 min at 20% B to 11.5 min at 70% B with a flow rate of 0.3 mL/min. A wash step was set from 11.75 min to 13.75 min at 90% B with a flow rate of 0.5 mL/min. Finally, a conditioning step was used at 14.0 min for 1.0 min at 20% B with a flow rate of 0.4 mL/min.

MS-I The ADC buffer is exchanged for an MS-compatible buffer and injected directly into a hybrid Q-TOF MS instrument (Synapt G2-S HDMS, Waters, Manchester, UK) equipped with an automated chip-based nanoESI source (Triversa Nanomate, Advion Biosciences, Ithaca, NY, USA) operating in positive ionization mode. Instrumental MS parameters are adjusted to preserve the native 3D structure in the gas phase and ensure efficient desolvation and ion transfer.

LC-MS analysis was performed using a Waters UPLC H-Class Bio chromatograph system coupled with an ESI Xevo G2 XS Q-TOF mass spectrometer (Waters, Manchester, UK). ADC was analyzed either intact (without pretreatment) or after reduction with 5 mM (final concentration) dithiothreitol DTT (Thermo Scientific, Rockford, IL, 20291). The treated ADC was then analyzed using any of the above-mentioned LC-I, LC-II , or LC-III (Table 22). Electrospray ionization time-of-flight mass spectra of the analytes were acquired using MassLynx™ data acquisition software (Waters, Manchester, UK). For the single native MS experiment, ADC was analyzed using the MS-I method to determine the average DAR. The resulting intensity versus m/z spectra are then deconvoluted using the maximum entropy (MaxEnt) method in MassLynx™ software to determine the mass of each intact antibody species or each recovered antibody fragment, depending on the processing used. Finally, the DAR is determined from the deconvoluted spectra or UV chromatogram by summing the integrated MS (total ion current) or UV (280 nm) peak areas of the unconjugated and conjugated species (mAb or associated fragment), weighted by multiplying each area by the amount of attached drug. To determine the DAR from the UV chromatogram, the relative area fraction of each species is multiplied by the amount of attached drug. The summed weighted areas are divided by the sum of the total areas, and the results yield the final average DAR for the total ADC.

Liquid chromatography-mass spectrometry (LC/MS) II

LC/MS data were obtained using an instrument with the following parameters (Table 7):

The methods used to obtain LC/MS data are as follows (Tables 14-16):

High-resolution mass spectrometry

The MSVR data are collected using a tool with the following parameters (Table 17):

The methods used to obtain HRMS data for linkers/payloads and synthetic intermediates are as follows (Tables 18 and 19):

The method used to generate the HRMS data for ADCs is as follows (Table 20):

Size exclusion chromatography (SEC) I

Size exclusion chromatography (SEC) is used to determine the quality of ADC and the aggregation fraction (%) after purification. The analysis is performed on a Superdex 200 Increase 5/150 GL analytical column (GE Healthcare, 28990945) under isocratic conditions with 100% PBS pH 7.4 (Sigma, P3813, 10PAK), flow rate of 0.45 ml/min for 12 minutes. The % aggregated fraction of the ADC sample is quantified based on the absorbance peak area at 280 nm. The calculation is based on the ratio of the high-molecular weight eluent at 280 nm to the sum of the absorbance peak area at the same wavelength of the high-molecular weight and monomeric eluents, multiplied by 100%.

Size exclusion chromatography (SEC) II

SEC data are obtained using an instrument with the following parameters (Table 21) and a run time of 12 minutes:

Results

Some of the conjugations are performed on antibodies in the 2-20 mg range. All ADCs presented in the examples are characterized by analytical size exclusion chromatography Superdex 200 Increase 5/150 GL (GE Healthcare, 28990945) to determine the proportion of monomers and LC-MS to determine the DAR. The characteristics of typical ADCs are listed in Table 22 (coupling method and LC/MS, aggregation state and DAR). Average DAR values are determined using the above-mentioned LC/MS methods (LC/MS I), and the proportion of aggregation is determined using the above-mentioned SEC methods (SEC I).

Table 22. Analytical characterization of ADCs and conjugation methodology ADS Conjugation method LC-MS method DAR % aggr. (according to SEC I) Ab G - L23-C3 B LC-I 4 1.3 Ab G - L24-C1 B LC-I 3.6 1 Ab G - L13-C4 B MS-I 3.8 1 Ab G - L14-C3 B LC-II 4 0.9 Ab G - L15-C5 B LC-I 3.7 0.8 Ab G - L16-C3 B LC-I 4 0.5 Ab G - L17-C3 B LC-I 4 0.6 Ab G - L18-C3 B LC-II 4 1 Ab G - L19-C3 B LC-II 4 0.9 Ab G - L20-C6 B LC-III 4 1.7 Ab G - L24-C6 B LC-III 4 0.5 Ab G - L24-C7 B LC-III 4 1 Ab T - L24-C1 A LC-I 2 0.5 Ab T - L24-C1 B LC-I 4 0.5 Ab T - L24-C6 B LC-III 4 1.8 Ab T - L24-C7 B LC-III 4 0.9 Ab B - L21-C1 C LC-III 3.7 1.7 Ab B - L9-C1 C LC-III 3.4 0.9 Ab B - L9-C8 C LC-III 3.6 2 Ab B with silent Fc - L22-C1 C LC-III 4.1 0.5 Ab B - L9-C10 C LC-III 3.4 0.2 Ab B - L9-C9 C LC-III 4.2 0.9 Ab B - L9-C11 C LC-III 3.3 0.2 Ab B - L9-C12 C LC-III 3.3 0.2 Ab B - L9-C13 C LC-III 3.3 <1 Ab G - L29-C3 B LC-II 4 0.2 Ab B with silent Fc - L27-P1 C LC-III 4.1 0.2 Ab B with silent Fc - L28-P1 C LC-III 4.0 1.1 Ab B - L26-P1 C LC-III 3.5 0.4 Ab B - L9-C14 C LC-III 5.3 0.2 Ab C - L9-C1 C LC-III 3.1 4.4 Ab B - L9-P15 C LC-III 3.4 1 Ab B - L25-P1 C LC-III 3.7 3.1 Ab B - L9-P16 C LC-III 3.4 0.2 Ab B - L9-P17 C LC-III 3.5 0.2

Example 5: In Vitro Assessment I

Inhibition of Mcl-1 by fluorescence polarization (FP)

The relative binding affinity of each compound is determined using fluorescence polarization (FP). The method utilizes a fluorescein-labeled ligand (Fluorescein-βAla-Ahx-A-REIGAQLRRMADDLNAQY-OH; MW 2765) that binds to the Mcl-1 protein (UniProt reference sequence: Q07820; SEQ ID NO: 48), resulting in an increase in the measured anisotropy in millipolarization units (mP) using a reader. Adding a compound that competitively binds to the same site as the ligand increases the fraction of unbound ligand in the system, as indicated by a decrease in mP units.

An 11-point serial dilution of each compound in DMSO was prepared and 2 μL was transferred to a low-binding, flat-bottomed 384-well plate (final DMSO concentration 5%). 38 μL of buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], 150 mM NaCl, 0.05% Tween 20, pH 7.4) containing fluorescein-labeled ligand (final concentration 1 nM) and Mcl-1 protein (final concentration 5 nM) were then added.

Assay plates are incubated for ~2 hours at room temperature before FP measurement on a Biomek Synergy2 reader (Exc. 528 nm, Evaporation 640 nm, Threshold 510 nm) and mP units are calculated. Binding of increasing doses of the test compound is expressed as a proportion of the mP reduction compared to a window established between the “5% DMSO only” and “100% inhibition” controls. Eleven-point dose-response curves are constructed with XL-Fit software using a 4-parameter logistic model (sigmoidal dose-response model) to determine inhibitory concentrations that produce a 50% reduction in mP (IC ₅₀ ). The results are presented in Table 17 below. The results indicate that the test compounds inhibit the interaction between the Mcl-1 protein and the fluorescein-labeled peptide.

In vitro cytotoxicity

Cytotoxicity studies were performed on the NCI-H929 multiple myeloma cell line. Cells were distributed into microplates and exposed to test compounds for 48 hours. Cell viability was then determined using the Microculture Tetrazolium Assay (Carmichael et al. (1987) Cancer Res. 47:936-42).

The results are expressed as IC ₅₀ (the concentration of the compound that inhibits cell viability by 50%) and are presented in Table 23 below. The results demonstrate that the test compounds are cytotoxic to NCI-H929 cells.

Table 23. IC ₅₀ Mcl-1 inhibition and cytotoxicity in NCI-H929 cells Payload IC ₅₀ (M) Mcl-1 FP IC ₅₀ (M) MTT H929 Payload IC ₅₀ (M) Mcl-1 FP IC ₅₀ (M) MTT H929 C1 2.8E-09 3.0E-09 C8 3.0E-09 5.74E-07 C2 2.6E-09 8.51E-06 C9 2.5E-09 2.27E-06 C3 9.48E-10 4.2E-08 C10 3.4E-09 1.35E-08 C4 2.6E-09 3.21E-06 C11 5.3E-09 7.0E-09 C5 4.05E-09 4.08E-09 C12 5.9E-09 4.2E-08 C6 1.18E-09 3.0E-09 C13 2.2E-09 3.76E-09 C7 2.6E-09 1.88E-09 C14 8.4E-10 2.61E-09

Example 6: In Vitro Assessment II

Mcl-1 ADC is being tested on ten endogenous cancer cell lines and one isogenic cell line overexpressing the targets of interest. Six targets are being evaluated: HER2, BCMA, CD38, CD48, CD79b, and CD33.

Cell lines

- HER2 : HCC1954 (ATCC No. CRL-2338 cultured in RPMI-1640+10% FBS) and HCC2218 (ATCC No. CRL-2343 cultured in RPMI-1640+10% FBS).

- BCMA : NCI-H929 (ATCC No. CRL-9068 cultured in RPMI-1640+10% FBS+0.05 mM 2-mercaptoethanol).

- CD38 : A4-Fuk (JCRB No. JCRB0097 cultured in RPMI-1640+10% FBS) and KMS-21BM (JCRB No. JCRB1185 cultured in RPMI-1640+10% FBS).

- CD48 : NCI-H929 (ATCC #CRL-9068 cultured in RPMI-1640+10% FBS+0.05 mM 2-mercaptoethanol), OPM-2 (DSMZ #ACC-50 cultured in RPMI-1640+10% FBS) and AMO1 (DSMZ #ACC-538 cultured in RPMI-1640+20% FBS).

- CD79b :RS4;11 (ATCC No. CRL-1873 cultured in RPMI-1640+10% FBS).

- CD33 : MOLM-13 (DSMZ #ACC-554 cultured in RPMI-1640+10% FBS) and an isogenic cell line, AMO1-CD33 clone D2. The AMO1 cell line was transfected to generate a stable AMO1 cell line expressing the exogenous protein of interest, CD33, AMO1-CD33 clone D2 (cultured in RPMI-1640+20% FBS).

Inhibition of cell proliferation and survival

The ability of Mcl-1 ADC to inhibit cell proliferation and cell viability was assessed using the Promega CellTiter-Glo® Proliferation Assay. Cell lines were cultured in a medium optimal for their growth at 5% _CO2 , 37°C in a tissue culture incubator. Before plating for the proliferation assay, cells were split at least 2 days prior to the assay to ensure optimal growth density. On the day of plating, adherent cells were removed from the tissue culture cell using 0.25% trypsin. Cell viability and cell density were determined using a cell counter (Vi-Cell XR Cell Viability Analyzer, Beckman Coulter). Cells with viability greater than 85% were seeded in 384-well, white, clear-bottomed plates treated with TC (Corning cat. #3765). Cells are seeded at a density of 1,000 cells per well in 45 µl of standard growth medium. The plates are incubated at 5% _CO2 , 37°C overnight in a tissue culture incubator. The following day, Mcl-1-free payload (P1), targeted Mcl-1 ADC, and non-targeted isotype ADC are prepared at 10X in standard growth medium. The prepared drugs are then added to the cells, yielding final concentrations of 0.0005-500 nM and a final volume of 50 µl per well. Each drug concentration is tested in quadruple amounts. Plates are incubated at 5% _CO2 , 37°C for 5 days in a tissue culture incubator, after which cell viability is assessed by the addition of 25 µl of CellTiter Glo® (Promega, cat. #G7573), a reagent that lyses cells and measures total adenosine triphosphate (ATP). Plates are incubated at room temperature for 10 minutes to stabilize luminescent signals before reading using a luminescence plate reader (EnVision Multilabel Plate Reader, PerkinElmer). To assess the effect of drug treatment, luminescence counts from wells containing untreated cells (100% viability) are used to normalize treated samples. A variable slope model is used to fit a nonlinear regression curve to the data in GraphPad PRISM version 7.02. The IC ₅₀ and Amax values are extrapolated from the obtained curves.

Result

Dose-response curves for representative cancer cell lines expressing the six targets of interest are shown in FIG. 2A - FIG. 7 . Treatment concentrations required to inhibit 50% of cell growth or viability (IC50) were calculated with the representative IC50 values for the test cell lines summarized in Tables 24-29.

Representative cancer cell lines were shown to be sensitive to the Mcl-1 (P1) payload, with IC50 values ranging from 0.038 to 5.6 nM activity, with the exception of HER2-positive cancer cell lines, in which IC50 values ranged from 10 to 60 nM. The CD38-targeted Mcl-1 ADC CD38-L7-P1, tested in A4-Fuk and KMS-21BM cells, demonstrated enhanced in vitro potency compared to the non-targeted isotype-matched control ADC IgG-L7-P1. However, maximal cell growth inhibition reached 50% ( FIG. 2A and FIG. 2B ). Despite its sensitivity to the Mcl-1 payload, P1, the CD48-targeting Mcl-1 ADC, CD48-L7-P1, did not induce inhibition of cell proliferation and was similar to the non-targeting isotype-matched control ADC, IgG-L7-P1, in this experiment ( FIG. 3A - FIG. 3C ). A similar trend was observed for the CD79b-targeting Mcl-1 ADC, CD79b-L7-P1 ( FIG. 4 ). The HER2-targeting Mcl-1 ADC, Her2-L9-P1, showed cytotoxic activity against HCC2218 cells similar to that of the Mcl-1 payload (P1), but was inactive against HCC1954 cells ( FIG. 5A and FIG. 5B ). Cell proliferation of clone D2 of the isogenic AMO1-CD33 cell line is inhibited by CD33-L2-P1, CD33-L3-P1, and CD33-L7-P1 with IC50s ranging from 0.089 to 0.476 nM. Little activity is observed with the matched non-targeted Mcl-1 ADC, IgG-L7-P1. CD33-L12-P2 and CD33-L4-P2 are inactive on clone D2 of the isogenic AMO1-CD33 cell line. Only ADC CD33-L7-P1 is tested on MOLM-13 cells, and its activity is moderate despite the low background activity of the matched non-targeted Mcl-1 ADC, IgG-L7-P1 ( FIG. 6A and FIG. 6B ). BCMA-targeted ADCs using different linkers (L1, L3-L5, L7-L10) exhibit similar cytotoxic activity, inhibiting NCI-H929 cell proliferation. An isotype-matched non-targeted control, IgG-L7-P1, exhibits some activity against the cell line at the highest concentration tested, 500 nM. BCMA-L12-P2 and BCMA-L4-P2 ADCs are inactive in the NCI-H929 cell line ( FIG. 7 ).

These studies demonstrate that Mcl-1 ADCs are capable of inhibiting cell proliferation in various cancer cell lines expressing BCMA, CD33, and, to a lesser extent, CD38. Mcl-1 ADCs exhibit mild cytotoxic activity compared to isotype-matched non-targeted controls in cancer cell lines expressing CD38, CD79b, and HER2.

Table 24. CD38 Mcl-1 ADC CD38 A4FUK KMS21BM L/P or connection no. IC50 (nM) Amax IC50 (nM) Amax L7-P1 0.494 81.08 ** ** P1 ** ** ** **

** tested, but the curve did not converge

Table 25. CD48 Mcl-1 ADC CD48 H929 OPM2 AMO1 L/P or connection no. IC50 (nM) Amax IC50 (nM) Amax IC50 (nM) Amax L7-P1 3,708 ~186.6 ** ** 0.696 79.31 P1 0.394 25.79 0.136 22.97 0.038 19.97

** tested, but the curve did not converge

Table 26. CD79b Mcl-1 ADC CD79b RS;411 L/P or connection no. IC50 (nM) Amax L7-P1 1114,000 194.40 L9-P1 0.150 88.91 L10-P1 0.150 87.70 P1 0.299 132.40

** tested, but the curve did not converge

Table 27. HER2 Mcl-1 ADC HER2 HCC1954 HCC2218 L/P or connection no. IC50 (nM) Amax IC50 (nM) Amax L9-P1 ** ** ** ** P1 63.52 64.80 14.08 100.40

** tested, but the curve did not converge

Table 28. CD33 Mcl-1 ADC CD33 MOLM13 AMO1-CD33 L/P or connection no. IC50 (nM) Amax IC50 (nM) Amax L1-P1 >0.701 100.61 L2-P1 >150 -- 0.476 84.11 L3-P1 0.089 108.90 L6-P1 >0.644 103.75 L7-P1 >150 -- 0.368 69.23 L8-P1 >0.733 101.22 L10-P1 >0.757 99.65 L12-P2 ** ** L4-P2 ** ** P1 2,440 128.80 1,530 103.00

** tested, but the curve did not converge

Table 29. BCMA Mcl-1 ADC BCMA H929 L/P or connection no. IC50 (nM) Amax L1-P1 4,103 101.90 L3-P1 4,264 103.70 L4-P1 2,456 105.20 L5-P1 6,270 100.00 L7-P1 3,054 107.60 L8-P1 2,134 116.10 L9-P1 2,989 93.79 L10-P1 10,100 93.65 L12-P2 ** ** L4-P2 ** ** P1 5,608 89.16

** tested, but the curve did not converge

Example 7. In vitro evaluation of anti-CD33 ADC (site-specific Gln conjugation)

AMO1-CD33 Construction of an engineered cell line (clone D2)

AMO1 is a human multiple myeloma cell line that does not express CD33. To generate an AMO1 clone expressing hCD33, AMO1 cells were transduced with lentiviral particles (GE Healthcare - Dharmacon) encoding hCD33 (clone ID: PLOHS 100004356) at a multiplicity of infection (MOI) of 8 in the presence of 8 μg/ml polybrene. Cells were sorted based on GFP reporter gene expression, and the presence of hCD33 on the cell surface was assessed by flow cytometry. AMO1-CD33 cells were then sorted as single cells to isolate clones with different levels of hCD33 expression. Clone AMO1-CD33 D2 was selected based on its high hCD33 expression measured by flow cytometry.

Cell viability using the MTT assay

The MTT colorimetric assay is based on the mitochondrial reduction of tetrazolium salts by living cells. The number of viable cells is proportional to the production of formazan salts, which can be measured spectrophotometrically at 540 nm.

AMO1-CD33 clone D2 cells were cultured in RPMI 1640 medium supplemented with 20% heat-inactivated fetal bovine serum, penicillin (100 IU/ml), streptomycin (100 μg/ml), and L-glutamine (2 mM). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO ₂ . Cells were seeded in 96-well microwell plates (150 μl per well) and exposed to ADC for 48 h (3.16-fold serial dilution; 8 concentrations each, triplicate). After the incubation time, 15 μl of MTT solution (5 mg/ml) were added per well, and the cells were incubated for another 4 h. Next, 100 µl of 10% sodium dodecyl sulfate (SDS)/10 mM HCl is added to the well, and the plate is incubated overnight before measuring the absorbance at 540 nm. IC50 values are calculated using a standard four-parameter curve fit. For this assay, the IC50 is defined as the compound concentration at which the MTT signal is reduced to 50% of that measured for the control. Each experiment is performed at least twice with reproducible results.

Results : As shown in FIGS. 8A and 8B , all tested anti-CD33 ADCs and their corresponding payloads caused a dose-dependent decrease in the viability of AMO1-CD33 clone D2 cells in the MTT assay, while no significant effect was observed with the corresponding naked antibody.

Example 8. In vitro evaluation of anti-HER2 ADC (site-specific Gln conjugation)

Cell viability using CTG assay

HCC1954 cells were cultured in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 IU/mL), streptomycin (100 μg/mL), and L-glutamine (2 mM). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO ₂ . Cells were seeded in 96-well microwell plates and exposed to ADC for 120 h (5-fold serial dilutions; 9 concentrations each, in triplicate). The effect of ADC on cell viability was assessed after 5 days of incubation at 37°C/5% CO ₂ by quantifying cellular ATP levels using the CellTiterGlo® reagent at 75 μL of reagent per well. All conditions were tested in triplicate. Luminescence is quantified using a multifunctional plate reader. IC50 values are calculated using a standard four-parameter curve fit. For this assay, the IC50 is defined as the compound concentration at which the CTG signal is reduced to 50% of that measured for the control. Each experiment is performed at least twice, ensuring reproducible results.

Results : As shown in FIGS. 9A and 9B , all tested anti-HER2 ADCs and their corresponding payloads caused a dose-dependent reduction in HCC1954 cell viability in the CTG assay; whereas no significant effect was observed with the corresponding naked antibody.

Example 9. In vitro evaluation of the effect of anti-BCMA ADC (site-specific Cys conjugation) on the viability of NCI-H929 (multiple myeloma) cells using the MTT assay.

The MTT colorimetric assay is based on the mitochondrial reduction of tetrazolium salts by living cells. The number of viable cells is proportional to the production of formazan salts, which can be measured spectrophotometrically at 540 nm.

NCI-H929 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 IU/mL), streptomycin (100 μg/mL), and L-glutamine (2 mM). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO ₂ . Cells were seeded in 96-well microwell plates (150 μL per well) and exposed to ADC for 48 h (3.16-fold serial dilution; 8 concentrations each, triplicate). After the incubation time, 15 μL of MTT solution (5 mg/mL) were added per well, and the cells were incubated for another 4 h. Next, 100 µl of 10% sodium dodecyl sulfate (SDS)/10 mM HCl is added to the well, and the plate is incubated overnight before measuring the absorbance at 540 nm. IC50 values are calculated using a standard four-parameter curve fit. For this assay, the IC50 is defined as the compound concentration at which the MTT signal is reduced to 50% of that measured for the control. Each experiment is performed at least twice to ensure reproducibility of results.

Results : As shown in FIGS. 10A, 10B, 10C , and 10D , all tested anti-BCMA-Mcl1i ADCs and their corresponding payloads caused a dose-dependent reduction in NCI-H929 cell viability in the MTT assay, while no significant effect was observed with the corresponding naked antibody.

Example 10: In vitro assessment - anti-CD48ADC.

NCI-H929, KHM1B, and KMS21BM cells were cultured in RPMI supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 IU/mL), streptomycin (100 μg/mL), and L-glutamine (2 mM). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO ₂ . Cells were seeded in 96-well microwell plates and exposed to ADC for 72 h (3.16-fold serial dilution; 8 concentrations each, in triplicate). The effect of ADC on cell viability was assessed after 3 days of incubation at 37°C/5% CO ₂ by quantifying cellular ATP levels using the CellTiterGlo® reagent at 75 μL of reagent per well. All conditions are tested in triplicate. Luminescence is quantified using a multi-plate reader. IC50 values are calculated using a standard four-parameter curve fit. For this assay, IC50 is defined as the compound concentration at which the CTG signal is reduced to 50% of that measured for the control. Each experiment is performed at least twice with reproducible results.

Results : The effect of the tested anti-CD48 ADC on cell viability in the CTG assay is shown in FIG. 13A (NCI-H929), FIG. 13B (KHM1B), and FIG. 13C (KMS21BM). It is shown that the tested anti-CD48 ADC causes a clear decrease in the viability of NCI-H929, KHM1B, and KMS21BM cells in the CTG assay.

Example 11. In Vivo Assessment I

Generic BCMA-targeted ADCs formulated in phosphate-buffered saline (PBS) are evaluated for in vivo therapeutic efficacy in the NCI-H929 murine multiple myeloma model following intravenous (IV) administration.

Materials and methods.

The following compounds are used in this example (ADCs are synthesized using the methods described in Example 4): IgG1-Linker-Payload with Silenced Fc, Anti-BCMA_CysmAb with Silenced Fc Ab, Anti-BCMA_CysmAb with Silenced Fc_L7-P1.

NCI-H929 cells (ATCC #CRL-9068) were cultured in RPMI supplemented with 10% FBS. Cells were resuspended in 100% Matrigel (BD Biosciences), and 0.1 mL of Matrigel containing 5 × 10 ⁶ cells was inoculated subcutaneously into the right flank of female SCID mice (Charles River). When tumors reached the appropriate volume, mice were randomly assigned to six animals per group using Easy stat software. IgG1-Linker-Payload with Silenced Fc, Anti-BCMA_CysmAb with Silenced Fc, and Anti-BCMA_CysmAb with Silenced Fc_L7-P1 (10 mg/kg or 30 mg/kg) were administered once intravenously in PBS. Body weight was monitored three times a week, and tumor size was measured using electronic calipers. Tumor volume is estimated by measuring the minimum and maximum tumor diameters using the formula: (minimum diameter) ² (maximum diameter)/2. On the final day, in at least half of the animals from all groups still present in the study (d17), tumor growth inhibition is calculated using the formula:

where DTV (delta tumor volume) at Dx is calculated as TV at Dx - TV under random distribution.

The response is assessed as follows: CR (complete response) if the tumor size is ≤25 ^mm3 for at least three measurements, PR (partial response) if the tumor size is between 25 ^mm3 and 114 ^mm3 (half the initial size) for at least three measurements. Mice are sacrificed at the first measurement in which the tumor volume exceeds 2000 ^mm3 or at the first sign of deterioration in the animal's health. All experiments are carried out in accordance with French regulations in force in 2018. SCID mice are maintained in accordance with established regulations.

Results

The efficacy of anti-BCMA_CysmAb with silenced Fc_L7-P1 in NCI-H929 xenografts is illustrated in FIG. 11 . Treatment begins 11 days after tumor cell inoculation (mean size 229.2 mm ³ ). IgG1-Linker-Payload with silenced Fc (non-targeted ADC FS), anti-BCMA_CysmAb with silenced Fc (naked antibody FS), and anti-BCMA_CysmAb with silenced Fc_L7-P1 (BCMA-targeted ADC FS) are administered as a single IV dose of 10 and/or 30 mg/kg. At day 17, the tumor growth inhibition (%TGI) induced by BCMA-targeted ADC FS at a dose of 30 mg/kg was greater than that of naked antibody FS (TGI = 99.2% vs. 34.7%, respectively, p < 0.0001) and the corresponding non-targeted ADC FS (TGI = 37.7%, p < 0.0001), as shown in FIG. 11 and Table 30. At a dose of 10 mg/kg, naked antibody FS does not show antitumor activity, while BCMA-targeted ADC FS induced a TGI of 75.6% (p = 0.0038). Complete regression (CR) was achieved exclusively in animals treated with BCMA-targeted ADC FS. Clinically significant body weight loss or other clinical signs were not observed as a result of treatment ( FIG. 12 ).

Table 30: Tumor growth inhibition Treatment Dose/Regimen %TGI (d17) %CR %PR IgG1-Dinker-Payload with Silenced Fc 30MK QD, BB 37.7 0 0 anti-BCMA_CysmAb With silent Fc 10MK QD, BB 0.5 0 0 anti-BCMA_CysmAb With silent Fc 30MK QD, BB 34.7 0 0 anti-BCMA_CysmAb with silent Fc_L7-P1 10MK QD, BB 75.6* 50 0 anti-BCMA_CysmAb with silent Fc_L7-P1 30MK QD, BB 99.2* 100 0

* p-value <= 0.05 compared with control group.

Example 12. In Vivo Assessment II

In vivo therapeutic efficacy of three BCMA-targeted ADCs was assessed in the H929 murine multiple myeloma model following intravenous (IV) administration.

Materials and methods.

The following compounds are used in this example (ADCs were synthesized using the methods described in Example 4): Fc-silenced anti-BCMA_CysmAb Ab, Fc-silenced anti-BCMA_CysmAb Ab, Fc-silenced anti-BCMA_CysmAb Ab B with silent Fc - L27-P1 and anti-BCMA_CysmAb with silent Fc _L5-P1.

H929 cells obtained from ATCC were cultured in RPMI supplemented with 10% FBS. Cells were resuspended in 100% matrigel (BD Biosciences) and 0.1 ml containing 5×10⁶cells were inoculated subcutaneously into the right flank of female SCID mice obtained from Charles River. When tumors reached the appropriate volume, mice were randomly assigned to six animals per group using Easy stat software. Anti-BCMA_CysmAb with silent Fc, anti-BCMA_CysmAb with silent Fc_L11-P1/L5-P1,Ab B with silent Fc -L27-P1(20 mg/kg) was administered intravenously in 20 mM histidine/NaCl. The mice's body weight was monitored three times a week, and tumor size was measured using electronic calipers. Tumor volume was estimated by measuring the minimum and maximum tumor diameters using the formula: (minimum diameter)²(maximum diameter)/2. On the last day, in at least half of the animals from all groups still present in the study (d14), tumor growth inhibition is calculated using the formula:

where DTV (delta tumor volume) at Dx is calculated as TV at Dx - TV under random distribution.

The response is assessed as follows: CR (complete response) if the tumor size is ≤25 ^mm3 for at least three measurements, PR (partial response) if the tumor size is between 25 ^mm3 and 90.6 ^mm3 (half the initial size) for at least three measurements. Mice are sacrificed at the first measurement in which the tumor volume exceeds 2000 ^mm3 or at the first sign of deterioration in the animal's health. All experiments are carried out in accordance with French regulations in force in 2018 after approval by the Servier Research Institute (IdRS) Ethical Committee. SCID mice are maintained in accordance with established regulations.

Results

The efficacy of Fc-silenced anti-BCMA_CysmAb ADCs containing different linkers in H929 xenografts is illustrated in FIG. 14 . Treatment begins 11 days after tumor cell inoculation (mean cell size 181.3 mm ³ ). Fc-silenced anti-BCMA_CysmAb (naked antibody FS) and different Fc-silenced anti-BCMA_CysmAb ADCs (BCMA-targeted ADC FS) are administered intravenously at a single dose of 20 mg/kg. At day 14, the 3 BCMA-targeted FS ADCs were found to cause significant tumor growth inhibition (%TGI) compared to the naked FS antibody (TGI= 84.3%, 96.5%, 108.6% vs. -1.7%, respectively, p<0.01), as shown in FIG. 14 and Table 31. Complete regression (CR) was achieved exclusively in animals treated with the BCMA-targeted FS ADCs. No clinically significant weight loss or other clinical signs were observed as a result of treatment ( FIG. 15 ).

Table 31 : Inhibition of H929 tumor growth by treatment with Fc-silenced anti-BCMA_CysmAb or different Fc-silenced anti-BCMA_CysmAb ADCs (20 mg/kg, administered as a single IV dose) Treatment Dose/Regimen %TGI (d14) %CR %PR anti-BCMA_CysmAb With silent Fc 20MK QD, BB -1.7 0 0 anti-BCMA_CysmAb With silent Fc_L11-P1 20MK QD, BB 108.6** 33.3 0 Ab B With silent Fc - L27-P1 20MK QD, BB 96.5*** 33.3 0 anti-BCMA_CysmAb With silent Fc_L5-P1 20MK QD, BB 84.3** 16.7 0

*p<=0.05, **p<=0.01, ***p<=0.001 compared with the control group

Example 13. In Vivo Assessment III

In vivo therapeutic efficacy of CD48-targeted ADCs formulated in phosphate-buffered saline (PBS) was evaluated in the H929 multiple myeloma model following intravenous (IV) administration.

Materials and methods.

The following compounds are used in this example: IgG1-Linker-Payload with Silenced Fc, Anti-CD-48_CysmAb with Silenced Fc, Anti-CD-48_CysmAb with Silenced Fc_L5-P1. The anti-CD48 antibody used in the examples herein is humanized MEM102 (hMEM102), as described in U.S. Patent Publication No. 20180092984, which is engineered with CysmAb mutations to enhance conjugation to the linker-payload. The ADC is synthesized by the methods described in Example 4.

H929 cells obtained from ATCC were cultured in RPMI supplemented with 10% FBS. Cells were resuspended in 100% matrigel (BD Biosciences), and 0.1 mL containing 5 × 10 ⁶ cells was inoculated subcutaneously into the right flank of female SCID mice provided by Charles River. When tumors reached the appropriate volume, mice were randomly assigned to eight animals per group using Easy stat software. IgG1-Linker-Payload with silent Fc, anti-CD-48_CysmAb with silent Fc, and anti-CD-48_CysmAb with silent Fc_L5 -P1 (30 mg/kg) were administered once intravenously in PBS. Body weight was monitored three times a week, and tumor size was measured using electronic calipers. Tumor volume is estimated by measuring the minimum and maximum tumor diameters using the formula: (minimum diameter) ² (maximum diameter)/2. On the final day, in at least half of the animals from all groups still present in the study (d16), tumor growth inhibition is calculated using the formula:

where DTV (delta tumor volume) at Dx is calculated as TV at Dx - TV under random distribution.

Mice are sacrificed at the first measurement of tumor volume exceeding 2000 ^mm³ or at the first sign of deterioration in animal health. All experiments are conducted in accordance with French regulations in effect as of 2018, following approval by the Servier Research Institute (IdRS) Ethics Committee. SCID mice are maintained in accordance with established guidelines.

Results

The efficacy of Fc-silenced anti-CD-48_CysmAb_L5 -P1 on H929 xenografts is illustrated in FIG. 16 . Treatment was started 10 days after tumor cell inoculation (mean size 133.1 mm ³ ). Silenced Fc IgG1 Linker Payload (non-targeted ADC FS), Fc-silenced anti-CD-48_CysmAb (naked antibody FS), and Fc-silenced anti-CD-48_CysmAb_L5 -P1 (CD-48-targeted ADC FS) were administered intravenously at a single dose of 30 mg/kg. At day 16, tumor growth inhibition (%TGI) induced by CD-48-targeted ADC FS at 30 mg/kg was greater than that of naked antibody FS (TGI= 98.8% vs. -22.9%, respectively, p<0.001) and the corresponding non-targeted ADC FS (TGI= -50.4%), as shown in FIG. 16 and Table 32. No clinically significant weight loss or other clinical signs were observed as a result of treatment ( FIG. 17 ).

Table 32. Inhibition of H929 tumor growth by treatment with IgG1-Linker-Payload with silent Fc, anti-CD-48_CysmAb with silent Fc, anti-CD-48_CysmAb with silent Fc_L5-P1 (30 mg/kg, administered single IV) Treatment Dose/Regimen %TGI (d16) IgG1-Linker-Payload with Silenced Fc 30MK QD, BB -50.4 anti-CD-48_CysmAb with silent Fc 30MK QD, BB -22.9 anti-CD-48_CysmAb with silent Fc_ L5-P1 30MK QD, BB 98.2***

***p<=0.001 compared to the control group.

Example 14. In vitro evaluation of the effect of anti-CD46-Mcl-1 ADC (Ab C-L9-C1) and corresponding payload on the viability of KMS21BM (multiple myeloma) cells using the CTG assay.

KMS21BM cells were cultured in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 IU/ml), streptomycin (100 μg/ml), and L-glutamine (2 mM) at 37°C in a humidified atmosphere containing 5% _CO2 . KMS21BM cells were seeded in 96-well microwell plates and exposed to ADC for 72 h (3.16-fold serial dilution; 9 concentrations each, triplicate). The effect of ADC, naked antibody, or the corresponding C1 payload on cell viability was assessed after 3 days of incubation at 37°C/5% _CO2 by quantifying cellular ATP levels using CellTiterGlo at 75 μl reagent per well. All conditions were tested in triplicate. Luminescence was quantified on a multi-plate reader. The _IC50 (Table 33) was calculated using a standard four-parameter curve fit. The _IC50 was defined as the compound concentration at which the CTG signal was reduced to 50% of that measured for the control. Each experiment was performed at least twice with reproducible results. As shown in FIG. 18 , the anti-CD46-Mcl-1 ADC ( Ab C-L9-C1 ) and the corresponding payload C1 caused a dose-dependent decrease in KMS21BM cell viability in the CTG assay, while no significant effect was observed with the corresponding naked antibody.

Table 33. IC ₅₀ results KMS21BM cell line IC ₅₀ (nM) Naked Ab C >300 Ab C - L9-C1 87.82 Payload C1 2.31

Example 15. In vitro assessment of the effect of anti-CD33-Mcl-1 ADC in combination with the BCL2 inhibitor compound A1 on the viability of AMO-1-CD33 clone D2 cells using the MTT assay.

AMO1-CD33 clone D2 cells were cultured in RPMI 1640 medium supplemented with 20% heat-inactivated fetal bovine serum, penicillin (100 IU/ml), streptomycin (100 μg/ml), and L-glutamine (2 mM). Cells were cultured at 37°C in a humidified atmosphere containing 5% _CO2 . Cells were seeded in 96-well microwell plates (150 μl/well) and treated with ADC, naked antibody, or the corresponding payloads for 48 h (3.16-fold serial dilutions; 8 concentrations each, triplicate) in the absence or presence of 1 μM BCL2 inhibitor compound A1. After the incubation time, 15 µl of MTT solution (5 mg/ml) is added to the well, and the cells are incubated for another 4 hours. Then, 100 µl of 10% sodium dodecyl sulfate (SDS)/10 mM HCl is added to the well, and the plate is incubated overnight before measuring the absorbance at 540 nm. The _IC50 is calculated using a standard four-parameter curve fit. The _IC50 is defined as the compound concentration at which the MTT signal is reduced to 50% of that measured for the control. Each experiment is performed at least twice with reproducible results.

As shown in FIGS. 19A and 19B , all ADCs caused a dose-dependent reduction in the viability of AMO1-CD33 clone D2 cells when administered alone. Interestingly, ADC activity was significantly improved in combination with the BCL2 inhibitor compound A1, whereas no significant effect was observed after treatment of these cells with the corresponding naked antibody alone or in combination with 1 μM compound A1.

Example 16. In vitro evaluation of the effect of anti-BCMA-Mcl-1 ADC as a single agent and in combination with the BCL2 inhibitor compound A1 on the viability of AMO1 cells using the MTT assay.

The MTT colorimetric assay is based on the mitochondrial reduction of tetrazolium salts by living cells. The number of viable cells is proportional to the production of formazan salts, which can be measured spectrophotometrically at 540 nm.

AMO1 cells were cultured in RPMI 1640 medium supplemented with 20% heat-inactivated fetal bovine serum, penicillin (100 IU/ml), streptomycin (100 μg/ml), and L-glutamine (2 mM). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO ₂ . Cells were seeded in 96-well microwell plates (150 μl per well) and exposed to ADC or the corresponding loads for 48 h (3.16-fold serial dilutions; 8 concentrations each, three times) in the absence or presence of 1 μM compound A1. At the end of the incubation time, 15 μl of MTT solution (5 mg/ml) were added per well, and the cells were incubated for another 4 h. Then, 100 μL of 10% sodium dodecyl sulfate (SDS)/10 mM HCl is added per well, and the plate is incubated overnight before measuring the absorbance at 540 nm. _IC50 values are calculated using a standard four-parameter curve fit. For this assay, the _IC50 is defined as the concentration of compound at which the MTT signal is reduced to 50% of that measured for the control. Each experiment is performed at least twice with reproducible results. As shown in FIGS. 20A and 20B , all anti-BCMA-Mcl-1 ADCs tested cause a dose-dependent decrease in AMO1 cell viability in the MTT assay. Interestingly, ADC activity was significantly improved in combination with the BCL2 inhibitor compound A1, while no significant effect was observed with the corresponding naked antibody as a single agent or in combination with 1 μM compound A1.

Example 17. In vitro evaluation of the effect of anti-BCMA-Mcl-1 ADC as a single agent and in combination with the BCL2 inhibitor Compound A1 on the viability of NCI-H929 cells using the MTT assay.

The MTT colorimetric assay is based on the mitochondrial reduction of tetrazolium salts by living cells. The number of viable cells is proportional to the production of formazan salts, which can be measured spectrophotometrically at 540 nm.

NCI-H929 cells were cultured in RPMI 1640 medium supplemented with 20% heat-inactivated fetal bovine serum, penicillin (100 IU/mL), streptomycin (100 μg/mL), and L-glutamine (2 mM). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO ₂ . Cells were seeded in 96-well microwell plates (150 μL per well) and exposed to ADC or the corresponding loads for 48 h (3.16-fold serial dilutions; 8 concentrations each, in triplicate) in the absence or presence of 1 μM compound A1. At the end of the incubation time, 15 μL of MTT solution (5 mg/mL) were added per well, and the cells were incubated for another 4 h. Then, 100 μL of 10% sodium dodecyl sulfate (SDS)/10 mM HCl is added per well, and the plate is incubated overnight before measuring the absorbance at 540 nm. _IC50 values are calculated using a standard four-parameter curve fit. For this assay, the _IC50 is defined as the compound concentration at which the MTT signal is reduced to 50% of that measured for the control. Each experiment is performed at least twice with reproducible results. As shown in FIGS. 21A and 21B , all anti-BCMA-Mcl-1 ADCs tested cause a dose-dependent decrease in the viability of NCI-H929 cells in the MTT assay. Interestingly, ADC activity was significantly improved in combination with the BCL2 inhibitor compound A1, while no significant effect was observed with the corresponding naked antibody as a single agent or in combination with 1 μM compound A1.

Example 18 In vitro assessment - anti-CD48ADC.

Cell lines

A CD48-MCL-1 drug conjugate, CD48-L7-P1, an IgG1-L7-P1 isotype ADC, and an MCL-1-free P1 payload are tested in four endogenous cancer cell lines. KMS-21BM (JCRB #JCRB1185, cultured in RPMI-1640+10% FBS), KMS-20 (JCRB #JCRB1196, cultured in RPMI-1640+10% FBS), KMS-27 (JCRB #JCRB1188, cultured in RPMI-1640+10% FBS), and NCI-H929 (ATCC #CRL-9068, cultured in RPMI-1640+10% FBS+0.05 mM 2-mercaptoethanol).

Inhibition of cell proliferation and survival

The ability of the CD48-MCL-1 conjugate and the drug CD48-L7-P1, the IgG1-L7-P1 ADC, and the MCL-1-free P1 payload to inhibit cell proliferation and survival was assessed using the Promega CellTiter-Glo® Proliferation Assay.

Cell lines are cultured in a medium optimal for their growth at 5% _CO2 , 37°C in a tissue culture incubator. Before plating for proliferation analysis, cells are split at least 2 days before the assay to ensure optimal growth density. On the day of plating, adherent cells are removed from the tissue culture core using 0.25% trypsin. Cell viability and cell density are determined using a cell counter (Vi-Cell XR Cell Viability Analyzer, Beckman Coulter). Cells with viability greater than 85% are seeded into 384-well, white, clear-bottomed plates treated with TC (Corning cat. #3765). Cells are seeded at a density of 1,000 cells per well in 45 µl of standard growth medium. Plates are incubated at 5% _CO2 , 37°C overnight in a tissue culture incubator. The following day, MCL-1-free payload (P1), MCL-1-targeted ADC, and non-targeted isotype ADC are prepared at 10X in standard growth medium. The prepared drugs are then added to cells, giving final concentrations of 0.0005–500 nM and a final volume of 50 µL per well. Each drug concentration is tested in quadruple amounts. Plates are incubated at 5% _CO2 , 37°C for 5 days in a tissue culture incubator, after which cell viability is assessed by the addition of 25 µL of CellTiter Glo® (Promega, cat. #G7573), a reagent that lyses cells and measures total adenosine triphosphate (ATP). The plates are incubated at room temperature for 10 minutes to stabilize the luminescent signals before reading them using a luminescence plate reader (EnVision Multilabel Plate Reader, PerkinElmer). To assess the effect of drug treatment, luminescence counts from wells containing untreated cells (100% viability) are used to normalize the treated samples. A variable slope model is used to fit a nonlinear regression curve to the data in GraphPad PRISM version 7.02. IC ₅₀ and Amax values are extrapolated from the obtained curves.

Result

Dose-response curves of representative cancer cell lines expressing the CD48 target of interest are shown in FIG. 22 (square = CD48-L7-P1, circle = IgG-L7-P1, triangle = MCL1-free P1 payload). Treatment concentrations required to inhibit 50% of cell growth or survival ( _IC50 ) were calculated using the representative IC50 values for the test cell lines provided in Table 34.

Table 34 : CD48 MCL1 ADC CD48-L7-P1 IgG-L7-P1 P1 IC50 (nM) Amax IC50 (nM) Amax IC50 (nM) Amax KMS-21BM 0.257 112.2 >200 -- 0.941 85.27 KMS-20 >200 -- >200 -- 4,946 94.34 KMS-27 >200 -- >200 -- 6,976 88.98 NCI-H929 0.298 81.17 >200 -- 2,039 109.1

Example 19. In vitro evaluation - the effect of anti-CD48 ADC as a single agent and in combination with venetoclax on the viability of various cancer cell lines.

Cell lines

CD48 MCL-1 antibody drug conjugates are tested against four endogenous cancer cell lines: KMS-21BM (JCRB #JCRB1185, cultured in RPMI-1640+10% FBS), KMS-20 (JCRB #JCRB1196, cultured in RPMI-1640+10% FBS), KMS-27 (JCRB #JCRB1188, cultured in RPMI-1640+10% FBS), and NCI-H929 (ATCC #CRL-9068, cultured in RPMI-1640+10% FBS+0.05 mM 2-mercaptoethanol).

Inhibition of cell proliferation and survival

The ability of the CD48-MCL-1 drug conjugate to inhibit cell proliferation and survival was assessed using the Promega CellTiter-Glo® Proliferation Assay.

Cell lines are cultured in a medium optimal for their growth at 5% _CO2 , 37°C in a tissue culture incubator. Before plating for proliferation analysis, cells are split at least 2 days before the assay to ensure optimal growth density. On the day of plating, adherent cells are removed from the tissue culture core using 0.25% trypsin. Cell viability and cell density are determined using a cell counter (Vi-Cell XR Cell Viability Analyzer, Beckman Coulter). Cells with viability greater than 85% are seeded into 384-well, white, clear-bottomed plates treated with TC (Corning cat. #3765). Cells are seeded at a density of 1,000 cells per well in 45 µl of standard growth medium. Plates are incubated at 5% _CO2 , 37°C overnight in a tissue culture incubator. The following day, MCL-1-free payload (P1), MCL-1-targeted ADC, and non-targeted isotype ADC are prepared at 10X in standard growth medium. The prepared drugs are then added to cells, giving final concentrations of 0.0005–500 nM and a final volume of 50 µL per well. Each drug concentration is tested in quadruple amounts. Plates are incubated at 5% _CO2 , 37°C for 5 days in a tissue culture incubator, after which cell viability is assessed by the addition of 25 µL of CellTiter Glo® (Promega, cat. #G7573), a reagent that lyses cells and measures total adenosine triphosphate (ATP). The plates are incubated at room temperature for 10 minutes to stabilize the luminescent signals before reading them using a luminescence plate reader (EnVision Multilabel Plate Reader, PerkinElmer). To assess the effect of drug treatment, luminescence counts from wells containing untreated cells (100% viability) are used to normalize the treated samples. A variable slope model is used to fit a nonlinear regression curve to the data in GraphPad PRISM version 7.02. IC ₅₀ and Amax values are extrapolated from the obtained curves.

Result

Dose-response curves of representative cancer cell lines expressing the CD48 target of interest are shown in FIGS. 23 and 23B . Treatment concentrations required to inhibit 50% of cell growth or survival ( _IC50 ) were calculated using the representative IC50 values for the test cell lines provided in Tables 35-37.

Table 35: P1+Venetoclax P1 P1+5 nM Venetoclax P1+50 nM Venetoclax IC50 (nM) Amax IC50 (nM) Amax IC50 (nM) Amax KMS-21BM 5,732 94.25 5,573 100.8 5,229 99.41 KMS-27 153.1 -- 20.05 110.5 11.99 114.7 KMS-20 34.71 65.65 30.55 75.53 36.16 82.77 NCI-H929 10.73 90.09 8,686 91.53 8.29 103.4

Table 36: CD48-L7-P1+ Venetoclax CD48-L7-P1 CD48-L7-P1+ 5 nM Venetoclax CD48-L7-P1+50 nM Venetoclax IC50 (nM) Amax IC50 (nM) Amax IC50 (nM) Amax KMS-21BM 0.3649 90.67 0.4164 90.69 0.301 84.86 KMS-20 10.6 39.56 >25 -- >25 -- KMS-27 >25 -- >25 -- >25 -- NCI-H929 0.2259 92.5 0.2279 82.63 0.2317 81.49

Table 37: IgG1- L7-P1+ Venetoclax

IgG1-L7-P1 IgG1-L7-P1+5 nM Venetoclax IgG1-L7-P1+50 nM Venetoclax IC50 (nM) Amax IC50 (nM) Amax IC50 (nM) Amax KMS-21BM >200 -- >100 -- 149.9 116.1 KMS-20 >200 -- >200 -- >200 -- KMS-27 >200 -- >200 -- >100 -- NCI-H929 138.4 70.94 >100 -- 135.3 81.87

Example 20. Evaluation of ADC activity in vitro on a panel of cancer cell lines.

In this example, the following compounds are used: CD33_CysmAb-L7-P1, CLL1_CysmAb-L7-P1, FLT3_CysmAb-L7-P1, cKIT_CysmAb-L7-P1, CD123_CysmAb-L7-P1, BCMA_CysMab-L7-P1, BCMA_CysMab-L7-P1, BCMA_Cys_CysmAb-L7-P1, CD38_CysmAb-L7-P1, CD48_CysmAb-L7-P1, SLAMF7_CysmAb-L7-P1, IgG1_CysmAb-L7-P1 isotype, and free payload P1. The anti-CD33 antibody used in this example is gemtuzumab. The anti-CLL1 antibody used in this example is m6E7, as described in WO2016/205200. The anti-cKIT antibody used in this example is described in WO2014/150937. The CD123 antibody used in this example is CSL362, also known as talacotuzumab. The anti-BCMA antibody used in this example is J6M0, also known as belantamab. The anti-CD138 antibody used in this example is indatuximab. The anti-CD38 antibody used in this example is daratumumab. The anti-CD48 antibody used in this example is humanized MEM102 (hMEM102), described in U.S. Patent Publication No. 20180092984. The anti-SLAMF7 antibody used in this example is azintuxizumab. Each of these antibodies was engineered with CysmAb mutations to facilitate linker conjugation, as described above. The ADCs were synthesized using the methods described in Example 4.

Antibody-drug conjugates are tested against cancer cell lines obtained from the American Type Culture Collection (ATCC) or from cell lines derived from patient-derived xenograft models. Cells are cultured in a growth medium at 5% _CO2 and 37°C in a tissue culture incubator. Before seeding for the proliferation assay, cells are split at least 2 days prior to analysis to ensure optimal growth density. On the day of seeding, cells are recovered from tissue culture flasks using 0.25% trypsin. Cell viability and cell density are determined using a cell counter (Vi-Cell XR Cell Viability Analyzer, Beckman Coulter). Cells with a viability greater than 85% are seeded into 384-well plates with a white, transparent bottom (Greiner cat. no. 781098) at a density of 1000 cells per well in 50 µl of standard growth medium. The plates are incubated at 37°C overnight in a tissue culture incubator.

ADCs are prepared in standard phosphate-buffered saline to the desired concentrations. A 10-dilution series is prepared for each ADC. The prepared drugs are then added to the cells, resulting in final concentrations of 0.000005–300 nM. An acoustic transduction device (Echo555, Beckman Coulter) is used to add the ADCs to the cells. Each treatment is tested in triplicate on assay plates. The plates are incubated at 37°C overnight or for 5 days in a tissue culture incubator. The ability of the ADCs to inhibit proliferation and cell viability is assessed using the Promega CellTiter-Glo® Proliferation Assay. The plates are incubated at room temperature for 20 minutes to stabilize the luminescent signals before being read using a multimodal plate reader (Pherastar, BMG). Luminescence counts of untreated cells are performed the day after plating (measurements on day 0) and after 5 days of treatment (measurements on day 5). Untreated cell measurements on day 5 are compared with measurements on day 0. Assays with at least one cell doubling during the incubation period are considered valid. To assess the effect of drug treatment, the amount of luminescence in wells containing untreated cells (100% viability) is used to normalize for treated samples. Treatment concentrations required to inhibit 50% of cell growth or viability (GI50) are calculated using a four-parameter logistic regression equation (Tables 38 and 39).

Table 38. In vitro activity of ADCs against different AML cell lines CD33 CLL-1 FLT3 CKIT CD123 Isotype IgG P1 cell line GI50 (μM) Amax GI50 (μM) Amax GI50 (μM) Amax GI50 (μM) Amax GI50 (μM) Amax GI50 (μM) Amax GI50 (μM) Amax 21435hx >0.3 80 0.154 45 0.001 34 >0.3 63 >0.3 90 >0.3 91 0.012 -91 5339hx >0.3 116 >0.3 103 >0.3 69 >0.3 53 >0.3 99 >0.3 96 0.005 -83 5340hx 0.063 12 0.162 15 0.019 -24 0.0004 -54 0.140 -6 >0.3 73 0.001 -92 5979hx 0.141 11 0.042 0 0.014 -12 0.015 0 0.041 31 0.2 35 0.001 -83 aml193 >0.3 106 >0.3 107 >0.3 110 >0.3 111 >0.3 108 >0.3 99 0.006 -87 cmk >0.3 107 >0.3 101 >0.3 104 >0.3 100 >0.3 100 >0.3 95 >10 100 eol1 0.129 -3 >0.3 137 0.197 -2 0.203 9 >0.3 115 0.2 31 0.001 -91 f36p >0.3 107 >0.3 99 >0.3 107 >0.3 100 >0.3 110 >0.3 97 >10 51 hel >0.3 119 >0.3 108 >0.3 111 >0.3 99 >0.3 108 >0.3 84 6,086 4 hel9217 >0.3 104 >0.3 99 >0.3 102 >0.3 97 >0.3 103 >0.3 87 5,283 -1 hl60 >0.3 110 >0.3 106 >0.3 115 >0.3 112 >0.3 103 >0.3 113 0.087 -71 kasumi1 >0.3 107 >0.3 115 >0.3 94 >0.3 107 >0.3 140 >0.3 108 0.037 -87 kasumi6 >0.3 82 >0.3 128 >0.3 73 >0.3 112 >0.3 110 >0.3 93 0.004 -96 kg1 >0.3 120 >0.3 100 >0.3 101 >0.3 98 >0.3 104 >0.3 97 0.861 -69 ko52 >0.3 114 >0.3 114 >0.3 115 >0.3 118 >0.3 101 >0.3 110 0.500 -75 m07e >0.3 112 >0.3 101 >0.3 100 >0.3 104 >0.3 100 >0.3 93 0.094 -74 molm13 >0.3 75 >0.3 51 >0.3 65 >0.3 62 >0.3 95 >0.3 73 0.002 -54 molm16 >0.3 107 >0.3 102 >0.3 103 >0.3 99 >0.3 106 >0.3 86 0.045 -76 monomac1 >0.3 51 0.007 18 0.006 22 0.042 30 >0.3 51 >0.3 55 0.001 -66 monomac6 >0.3 69 0.008 12 0.002 28 0.237 47 >0.3 61 >0.3 93 0.001 -43 mv411 >0.3 59 0.097 30 0.046 30 >0.3 95 >0.3 80 >0.3 54 0.001 -72 nb4 >0.3 110 >0.3 106 >0.3 106 >0.3 105 >0.3 102 >0.3 89 0.003 -62 nomo1 >0.3 114 >0.3 102 0.292 49 >0.3 98 >0.3 86 >0.3 75 0.001 -80 ociaml2 >0.3 109 >0.3 103 >0.3 100 >0.3 103 >0.3 100 >0.3 87 0.127 -50 ociaml3 >0.3 110 >0.3 112 >0.3 112 >0.3 111 >0.3 106 >0.3 108 0.138 1 ociaml5 >0.3 115 >0.3 105 >0.3 107 >0.3 103 >0.3 100 >0.3 98 0.265 4 ocim1 >0.3 112 >0.3 110 >0.3 113 >0.3 106 >0.3 103 >0.3 105 >10 86 p31fuj >0.3 120 >0.3 107 >0.3 105 >0.3 106 >0.3 93 >0.3 95 0.116 -73 pl21 >0.3 109 >0.3 109 >0.3 115 >0.3 107 >0.3 103 >0.3 100 0.350 -55 sigm5 >0.3 109 >0.3 104 >0.3 105 >0.3 106 >0.3 100 >0.3 100 0.118 -21 skm1 >0.3 107 >0.3 107 >0.3 107 >0.3 106 >0.3 105 >0.3 100 0.119 -49 tf1 >0.3 97 >0.3 100 >0.3 99 >0.3 99 >0.3 113 >0.3 66 0.239 10 thp1 >0.3 110 >0.3 111 >0.3 71 >0.3 103 >0.3 110 >0.3 102 0.028 -72

Table 39: In vitro activity of ADCs against different MM cell lines BCMA CD138 CD38 CD48 SLAMF7 Isotype IgG P1 Cell Line GI50 (μM) Amax GI50 (μM) Amax GI50 (μM) Amax GI50 (μM) Amax GI50 (μM) Amax GI50 (μM) Amax GI50 (μM) Amax amo1 >0.3 52 0.001 -1 >0.3 72 >0.3 55 >0.3 88 >0.3 112 0.005 -20 ejm >0.3 117 >0.3 121 >0.3 113 >0.3 115 >0.3 124 >0.3 116 0.061 -93 h929 0.003 -71 0.002 29 >0.3 87 0.0014 17 >0.3 83 >0.3 118 0.001 -74 karpas620 >0.3 104 >0.3 110 >0.3 106 >0.3 108 >0.3 115 >0.3 112 0.205 -43 ke97 >0.3 129 >0.3 119 >0.3 123 >0.3 66 >0.3 126 >0.3 117 7,913 21 khm1b >0.3 98 0.000389 -16 0.000015 38 0.000566 11 0.008368 25 >0.3 117 0.002 -84 kmm1 >0.3 96 >0.3 101 >0.3 96 >0.3 96 >0.3 107 >0.3 107 0.154 -67 kms11 >0.3 94 >0.3 109 >0.3 94 >0.3 97 >0.3 104 >0.3 102 0.098 -62 kms12bm >0.3 102 >0.3 144 >0.3 127 >0.3 130 >0.3 142 >0.3 125 0.011 -79 kms20 >0.3 112 >0.3 75 >0.3 111 >0.3 105 >0.3 117 >0.3 112 0.008 -88 kms21bm 0.008547 5 0.000632 -42 0.012261 -28 0.000544 7 0.028214 4 >0.3 126 0.001 -76 kms26 >0.3 118 >0.3 111 >0.3 116 >0.3 109 >0.3 126 >0.3 126 0.060 -49 kms27 >0.3 104 >0.3 111 >0.3 104 >0.3 103 >0.3 107 >0.3 103 0.001 -89 kms28bm >0.3 105 >0.3 123 >0.3 115 >0.3 112 >0.3 115 >0.3 121 0.006 -42 kms34 >0.3 117 >0.3 137 >0.3 122 >0.3 133 >0.3 134 >0.3 130 0.498 -64 L363 >0.3 71 >0.3 94 >0.3 88 >0.3 83 >0.3 86 >0.3 124 0.003 -47 lp1 >0.3 106 >0.3 131 >0.3 115 >0.3 122 >0.3 128 >0.3 126 0.007 -58 molp8 0.253701 46 0.005143 20 0.029991 11 >0.3 61 >0.3 67 >0.3 67 0.001 -74 opm2 >0.3 110 >0.3 58 >0.3 117 >0.3 107 >0.3 118 >0.3 119 0.001 -77 rpmi8226 >0.3 122 >0.3 84 >0.3 153 >0.3 147 >0.3 141 >0.3 127 0.002 -91 skmm2 >0.3 112 >0.3 117 >0.3 115 >0.3 118 >0.3 113 >0.3 104 >10 112 u266b1 >0.3 107 >0.3 121 >0.3 114 >0.3 115 >0.3 120 >0.3 106 0.283 -24

--->

SEQUENCE LIST

<110> NOVARTIS AG

LES LABORATOIRES SERVIER

<120> MCL-1 INHIBITOR ANTIBODY-DRUG CONJUGATES AND

METHODS OF THEIR APPLICATION

<130> 132043-00120

<140> PCT/US2020/033615

<141> 2020-05-19

<150> 62/850,098

<151> 2019-05-20

<160> 97

<170> PatentIn version 3.5

<210> 1

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 1

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ala Thr Tyr Arg Gly His Ser Asp Thr Tyr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ala Ile Tyr Asn Gly Tyr Asp Val Leu Asp Asn Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 2

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 2

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Arg Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 3

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 3

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Val

35 40 45

Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 4

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 4

Asn Ile Met Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Phe Phe Ser

20 25 30

Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ile

65 70 75 80

Ile Ser Ser Val Gln Ser Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln

85 90 95

Tyr Leu Ser Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 5

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 5

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Ile Thr Asp Ser

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Lys Asn Arg Ala Thr Leu Thr Val Asp Asn Pro Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Tyr Cys

85 90 95

Val Asn Gly Asn Pro Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 6

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 6

Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Leu Asp Asn Tyr

20 25 30

Gly Ile Arg Phe Leu Thr Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Met Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Lys

85 90 95

Glu Val Pro Trp Ser Phe Gly Gln Gly Thr Lys Val Glu Val Lys

100 105 110

<210> 7

<211> 123

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 7

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Gln

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Ile Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Leu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Gly Glu Gly Tyr Gly Arg Glu Gly Phe Ala Ile

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 8

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 8

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Asn Thr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Ser Trp Phe Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 9

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 10

<211> 109

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr

100 105

<210> 11

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 11

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 12

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 12

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105

<210> 13

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 13

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Phe Thr Gly Glu Pro Ser Tyr Gly Asn Val Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Gly Asn Gly Asn Val Phe Asp Ser Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 14

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 14

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Asn

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Thr Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 15

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 15

Gly Gly Thr Phe Ser Asn Tyr Trp Met His

1 5 10

<210> 16

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 16

Ala Thr Tyr Arg Gly His Ser Asp Thr Tyr Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 17

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 17

Gly Ala Ile Tyr Asn Gly Tyr Asp Val Leu Asp Asn

1 5 10

<210> 18

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 18

Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 19

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 19

Tyr Thr Ser Asn Leu His Ser

1 5

<210> 20

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 20

Gln Gln Tyr Arg Lys Leu Pro Trp Thr

1 5

<210> 21

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 21

Gly Tyr Thr Phe Thr Ser Tyr Tyr Ile His

1 5 10

<210> 22

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 22

Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 23

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 23

Glu Val Arg Leu Arg Tyr Phe Asp Val

1 5

<210> 24

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 24

Lys Ser Ser Gln Ser Val Phe Phe Ser Ser Ser Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 25

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 25

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 26

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 26

His Gln Tyr Leu Ser Ser Arg Thr

1 5

<210> 27

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 27

Gly Tyr Thr Ile Thr Asp Ser Asn Ile His

1 5 10

<210> 28

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 28

Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asn

<210> 29

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 29

Gly Asn Pro Trp Leu Ala Tyr

1 5

<210> 30

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 30

Arg Ala Ser Glu Ser Leu Asp Asn Tyr Gly Ile Arg Phe Leu Thr

1 5 10 15

<210> 31

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 31

Ala Ala Ser Asn Gln Gly Ser

1 5

<210> 32

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 32

Gln Gln Thr Lys Glu Val Pro Trp Ser

1 5

<210> 33

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 33

Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Gln Ser Ala Ala Trp

1 5 10 15

Asn

<210> 34

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 34

Arg Ile Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Leu Ser Val

1 5 10 15

Lys Ser

<210> 35

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 35

Gly Glu Gly Tyr Gly Arg Glu Gly Phe Ala Ile

1 5 10

<210> 36

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 36

Arg Ala Ser Gln Thr Ile Ser Asn Thr Leu Ala

1 5 10

<210> 37

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 37

Ala Ala Ser Asn Leu Gln Ser

1 5

<210> 38

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 38

Gln Gln Tyr Leu Ser Trp Phe Thr

1 5

<210> 39

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 39

Gly Phe Asn Ile Lys Asp Thr Tyr Ile His

1 5 10

<210> 40

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 40

Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 41

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 41

Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Val

1 5 10

<210> 42

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 42

Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp

1 5 10

<210> 43

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 43

Ser Ala Ser Phe Leu Glu Ser

1 5

<210> 44

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 44

Gln Gln His Tyr Thr Thr Pro Pro Thr

1 5

<210> 45

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 45

Gly Phe Thr Phe Asn Ser Phe Ala Met Ser

1 5 10

<210> 46

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 46

Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 47

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 47

Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr

1 5 10

<210> 48

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 48

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210> 49

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 49

Asp Ala Ser Asn Arg Ala Thr

1 5

<210> 50

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 50

Gln Gln Arg Ser Asn Trp Pro Pro Thr

1 5

<210> 51

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 51

Gly Tyr Thr Phe Thr Asp Phe Gly Met Asn

1 5 10

<210> 52

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 52

Trp Ile Asn Thr Phe Thr Gly Glu Pro Ser Tyr Gly Asn Val Phe Lys

1 5 10 15

Gly

<210> 53

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 53

Arg His Gly Asn Gly Asn Val Phe Asp Ser

1 5 10

<210> 54

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 54

Arg Ala Ser Gln Ser Ile Gly Ser Asn Ile His

1 5 10

<210> 55

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 55

Tyr Thr Ser Glu Ser Ile Ser

1 5

<210> 56

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 56

Gln Gln Ser Asn Ser Trp Pro Leu Thr

1 5

<210> 57

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 57

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ala Thr Tyr Arg Gly His Ser Asp Thr Tyr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ala Ile Tyr Asn Gly Tyr Asp Val Leu Asp Asn Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Cys Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Cys Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 58

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 58

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Arg Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 59

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 59

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Ile His Trp Ile Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Val

35 40 45

Gly Val Ile Tyr Pro Gly Asn Asp Asp Ile Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Val Arg Leu Arg Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Cys Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Cys Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 60

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 60

Asn Ile Met Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Phe Phe Ser

20 25 30

Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Ile Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ile

65 70 75 80

Ile Ser Ser Val Gln Ser Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln

85 90 95

Tyr Leu Ser Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 61

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 61

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Ile Thr Asp Ser

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Lys Asn Arg Ala Thr Leu Thr Val Asp Asn Pro Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Tyr Cys

85 90 95

Val Asn Gly Asn Pro Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Cys Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Cys Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 62

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 62

Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Leu Asp Asn Tyr

20 25 30

Gly Ile Arg Phe Leu Thr Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Met Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Lys

85 90 95

Glu Val Pro Trp Ser Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 63

<211> 453

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 63

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Gln

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Ile Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Leu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Gly Glu Gly Tyr Gly Arg Glu Gly Phe Ala Ile

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Cys Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Cys Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 64

<211> 213

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 64

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Asn Thr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Ser Trp Phe Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 65

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 65

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 66

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 66

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 67

<211> 452

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 67

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Cys Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Cys Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 68

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 68

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 69

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 69

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Phe Thr Gly Glu Pro Ser Tyr Gly Asn Val Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Gly Asn Gly Asn Val Phe Asp Ser Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Cys Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Cys Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 70

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 70

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Asn

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Thr Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 71

<211> 350

<212> PROTEIN

<213> Homo sapiens

<400> 71

Met Phe Gly Leu Lys Arg Asn Ala Val Ile Gly Leu Asn Leu Tyr Cys

1 5 10 15

Gly Gly Ala Gly Leu Gly Ala Gly Ser Gly Gly Ala Thr Arg Pro Gly

20 25 30

Gly Arg Leu Leu Ala Thr Glu Lys Glu Ala Ser Ala Arg Arg Glu Ile

35 40 45

Gly Gly Gly Glu Ala Gly Ala Val Ile Gly Gly Ser Ala Gly Ala Ser

50 55 60

Pro Pro Ser Thr Leu Thr Pro Asp Ser Arg Arg Val Ala Arg Pro Pro

65 70 75 80

Pro Ile Gly Ala Glu Val Pro Asp Val Thr Ala Thr Pro Ala Arg Leu

85 90 95

Leu Phe Phe Ala Pro Thr Arg Arg Ala Ala Pro Leu Glu Glu Met Glu

100 105 110

Ala Pro Ala Ala Asp Ala Ile Met Ser Pro Glu Glu Glu Leu Asp Gly

115 120 125

Tyr Glu Pro Glu Pro Leu Gly Lys Arg Pro Ala Val Leu Pro Leu Leu

130 135 140

Glu Leu Val Gly Glu Ser Gly Asn Asn Thr Ser Thr Asp Gly Ser Leu

145 150 155 160

Pro Ser Thr Pro Pro Pro Ala Glu Glu Glu Glu Asp Glu Leu Tyr Arg

165 170 175

Gln Ser Leu Glu Ile Ile Ser Arg Tyr Leu Arg Glu Gln Ala Thr Gly

180 185 190

Ala Lys Asp Thr Lys Pro Met Gly Arg Ser Gly Ala Thr Ser Arg Lys

195 200 205

Ala Leu Glu Thr Leu Arg Arg Val Gly Asp Gly Val Gln Arg Asn His

210 215 220

Glu Thr Ala Phe Gln Gly Met Leu Arg Lys Leu Asp Ile Lys Asn Glu

225 230 235 240

Asp Asp Val Lys Ser Leu Ser Arg Val Met Ile His Val Phe Ser Asp

245 250 255

Gly Val Thr Asn Trp Gly Arg Ile Val Thr Leu Ile Ser Phe Gly Ala

260 265 270

Phe Val Ala Lys His Leu Lys Thr Ile Asn Gln Glu Ser Cys Ile Glu

275 280 285

Pro Leu Ala Glu Ser Ile Thr Asp Val Leu Val Arg Thr Lys Arg Asp

290 295 300

Trp Leu Val Lys Gln Arg Gly Trp Asp Gly Phe Val Glu Phe Phe His

305 310 315 320

Val Glu Asp Leu Glu Gly Gly Ile Arg Asn Val Leu Leu Ala Phe Ala

325 330 335

Gly Val Ala Gly Val Gly Ala Gly Leu Ala Tyr Leu Ile Arg

340 345 350

<210> 72

<211> 184

<212> PROTEIN

<213> Homo sapiens

<400> 72

Met Leu Gln Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser

1 5 10 15

Leu Leu His Ala Cys Ile Pro Cys Gln Leu Arg Cys Ser Ser Asn Thr

20 25 30

Pro Pro Leu Thr Cys Gln Arg Tyr Cys Asn Ala Ser Val Thr Asn Ser

35 40 45

Val Lys Gly Thr Asn Ala Ile Leu Trp Thr Cys Leu Gly Leu Ser Leu

50 55 60

Ile Ile Ser Leu Ala Val Phe Val Leu Met Phe Leu Leu Arg Lys Ile

65 70 75 80

Asn Ser Glu Pro Leu Lys Asp Glu Phe Lys Asn Thr Gly Ser Gly Leu

85 90 95

Leu Gly Met Ala Asn Ile Asp Leu Glu Lys Ser Arg Thr Gly Asp Glu

100 105 110

Ile Ile Leu Pro Arg Gly Leu Glu Tyr Thr Val Glu Glu Cys Thr Cys

115 120 125

Glu Asp Cys Ile Lys Ser Lys Pro Lys Val Asp Ser Asp His Cys Phe

130 135 140

Pro Leu Pro Ala Met Glu Glu Gly Ala Thr Ile Leu Val Thr Thr Lys

145 150 155 160

Thr Asn Asp Tyr Cys Lys Ser Leu Pro Ala Ala Leu Ser Ala Thr Glu

165 170 175

Ile Glu Lys Ser Ile Ser Ala Arg

180

<210> 73

<211> 364

<212> PROTEIN

<213> Homo sapiens

<400> 73

Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala

1 5 10 15

Met Asp Pro Asn Phe Trp Leu Gln Val Gln Glu Ser Val Thr Val Gln

20 25 30

Glu Gly Leu Cys Val Leu Val Pro Cys Thr Phe Phe His Pro Ile Pro

35 40 45

Tyr Tyr Asp Lys Asn Ser Pro Val His Gly Tyr Trp Phe Arg Glu Gly

50 55 60

Ala Ile Ile Ser Arg Asp Ser Pro Val Ala Thr Asn Lys Leu Asp Gln

65 70 75 80

Glu Val Gln Glu Glu Thr Gln Gly Arg Phe Arg Leu Leu Gly Asp Pro

85 90 95

Ser Arg Asn Asn Cys Ser Leu Ser Ile Val Asp Ala Arg Arg Arg Asp

100 105 110

Asn Gly Ser Tyr Phe Phe Arg Met Glu Arg Gly Ser Thr Lys Tyr Ser

115 120 125

Tyr Lys Ser Pro Gln Leu Ser Val His Val Thr Asp Leu Thr His Arg

130 135 140

Pro Lys Ile Leu Ile Pro Gly Thr Leu Glu Pro Gly His Ser Lys Asn

145 150 155 160

Leu Thr Cys Ser Val Ser Trp Ala Cys Glu Gln Gly Thr Pro Pro Ile

165 170 175

Phe Ser Trp Leu Ser Ala Ala Pro Thr Ser Leu Gly Pro Arg Thr Thr

180 185 190

His Ser Ser Val Leu Ile Ile Thr Pro Arg Pro Gln Asp His Gly Thr

195 200 205

Asn Leu Thr Cys Gln Val Lys Phe Ala Gly Ala Gly Val Thr Thr Glu

210 215 220

Arg Thr Ile Gln Leu Asn Val Thr Tyr Val Pro Gln Asn Pro Thr Thr

225 230 235 240

Gly Ile Phe Pro Gly Asp Gly Ser Gly Lys Gln Glu Thr Arg Ala Gly

245 250 255

Val Val His Gly Ala Ile Gly Gly Ala Gly Val Thr Ala Leu Leu Ala

260 265 270

Leu Cys Leu Cys Leu Ile Phe Phe Ile Val Lys Thr His Arg Arg Lys

275 280 285

Ala Ala Arg Thr Ala Val Gly Arg Asn Asp Thr His Pro Thr Thr Gly

290 295 300

Ser Ala Ser Pro Lys His Gln Lys Lys Ser Lys Leu His Gly Pro Thr

305 310 315 320

Glu Thr Ser Ser Cys Ser Gly Ala Ala Pro Thr Val Glu Met Asp Glu

325 330 335

Glu Leu His Tyr Ala Ser Leu Asn Phe His Gly Met Asn Pro Ser Lys

340 345 350

Asp Thr Ser Thr Glu Tyr Ser Glu Val Arg Thr Gln

355 360

<210> 74

<211> 829

<212> PROTEIN

<213> Homo sapiens

<400> 74

Met Gly Leu Pro Arg Gly Pro Leu Ala Ser Leu Leu Leu Leu Gln Val

1 5 10 15

Cys Trp Leu Gln Cys Ala Ala Ser Glu Pro Cys Arg Ala Val Phe Arg

20 25 30

Glu Ala Glu Val Thr Leu Glu Ala Gly Gly Ala Glu Gln Glu Pro Gly

35 40 45

Gln Ala Leu Gly Lys Val Phe Met Gly Cys Pro Gly Gln Glu Pro Ala

50 55 60

Leu Phe Ser Thr Asp Asn Asp Asp Phe Thr Val Arg Asn Gly Glu Thr

65 70 75 80

Val Gln Glu Arg Arg Ser Leu Lys Glu Arg Asn Pro Leu Lys Ile Phe

85 90 95

Pro Ser Lys Arg Ile Leu Arg Arg His Lys Arg Asp Trp Val Val Ala

100 105 110

Pro Ile Ser Val Pro Glu Asn Gly Lys Gly Pro Phe Pro Gln Arg Leu

115 120 125

Asn Gln Leu Lys Ser Asn Lys Asp Arg Asp Thr Lys Ile Phe Tyr Ser

130 135 140

Ile Thr Gly Pro Gly Ala Asp Ser Pro Pro Glu Gly Val Phe Ala Val

145 150 155 160

Glu Lys Glu Thr Gly Trp Leu Leu Leu Asn Lys Pro Leu Asp Arg Glu

165 170 175

Glu Ile Ala Lys Tyr Glu Leu Phe Gly His Ala Val Ser Glu Asn Gly

180 185 190

Ala Ser Val Glu Asp Pro Met Asn Ile Ser Ile Ile Val Thr Asp Gln

195 200 205

Asn Asp His Lys Pro Lys Phe Thr Gln Asp Thr Phe Arg Gly Ser Val

210 215 220

Leu Glu Gly Val Leu Pro Gly Thr Ser Val Met Gln Val Thr Ala Thr

225 230 235 240

Asp Glu Asp Asp Ala Ile Tyr Thr Tyr Asn Gly Val Val Ala Tyr Ser

245 250 255

Ile His Ser Gln Glu Pro Lys Asp Pro His Asp Leu Met Phe Thr Ile

260 265 270

His Arg Ser Thr Gly Thr Ile Ser Val Ile Ser Ser Gly Leu Asp Arg

275 280 285

Glu Lys Val Pro Glu Tyr Thr Leu Thr Ile Gln Ala Thr Asp Met Asp

290 295 300

Gly Asp Gly Ser Thr Thr Thr Ala Val Ala Val Val Glu Ile Leu Asp

305 310 315 320

Ala Asn Asp Asn Ala Pro Met Phe Asp Pro Gln Lys Tyr Glu Ala His

325 330 335

Val Pro Glu Asn Ala Val Gly His Glu Val Gln Arg Leu Thr Val Thr

340 345 350

Asp Leu Asp Ala Pro Asn Ser Pro Ala Trp Arg Ala Thr Tyr Leu Ile

355 360 365

Met Gly Gly Asp Asp Gly Asp His Phe Thr Ile Thr Thr His Pro Glu

370 375 380

Ser Asn Gln Gly Ile Leu Thr Thr Arg Lys Gly Leu Asp Phe Glu Ala

385 390 395 400

Lys Asn Gln His Thr Leu Tyr Val Glu Val Thr Asn Glu Ala Pro Phe

405 410 415

Val Leu Lys Leu Pro Thr Ser Thr Ala Thr Ile Val Val His Val Glu

420 425 430

Asp Val Asn Glu Ala Pro Val Phe Val Pro Pro Ser Lys Val Val Glu

435 440 445

Val Gln Glu Gly Ile Pro Thr Gly Glu Pro Val Cys Val Tyr Thr Ala

450 455 460

Glu Asp Pro Asp Lys Glu Asn Gln Lys Ile Ser Tyr Arg Ile Leu Arg

465 470 475 480

Asp Pro Ala Gly Trp Leu Ala Met Asp Pro Asp Ser Gly Gln Val Thr

485 490 495

Ala Val Gly Thr Leu Asp Arg Glu Asp Glu Gln Phe Val Arg Asn Asn

500 505 510

Ile Tyr Glu Val Met Val Leu Ala Met Asp Asn Gly Ser Pro Pro Thr

515 520 525

Thr Gly Thr Gly Thr Leu Leu Leu Thr Leu Ile Asp Val Asn Asp His

530 535 540

Gly Pro Val Pro Glu Pro Arg Gln Ile Thr Ile Cys Asn Gln Ser Pro

545 550 555 560

Val Arg Gln Val Leu Asn Ile Thr Asp Lys Asp Leu Ser Pro His Thr

565 570 575

Ser Pro Phe Gln Ala Gln Leu Thr Asp Asp Ser Asp Ile Tyr Trp Thr

580 585 590

Ala Glu Val Asn Glu Glu Gly Asp Thr Val Val Leu Ser Leu Lys Lys

595 600 605

Phe Leu Lys Gln Asp Thr Tyr Asp Val His Leu Ser Leu Ser Asp His

610 615 620

Gly Asn Lys Glu Gln Leu Thr Val Ile Arg Ala Thr Val Cys Asp Cys

625 630 635 640

His Gly His Val Glu Thr Cys Pro Gly Pro Trp Lys Gly Gly Phe Ile

645 650 655

Leu Pro Val Leu Gly Ala Val Leu Ala Leu Leu Phe Leu Leu Leu Val

660 665 670

Leu Leu Leu Leu Val Arg Lys Lys Arg Lys Ile Lys Glu Pro Leu Leu

675 680 685

Leu Pro Glu Asp Asp Thr Arg Asp Asn Val Phe Tyr Tyr Gly Glu Glu

690 695 700

Gly Gly Gly Glu Glu Asp Gln Asp Tyr Asp Ile Thr Gln Leu His Arg

705 710 715 720

Gly Leu Glu Ala Arg Pro Glu Val Val Leu Arg Asn Asp Val Ala Pro

725 730 735

Thr Ile Ile Pro Thr Pro Met Tyr Arg Pro Arg Pro Ala Asn Pro Asp

740 745 750

Glu Ile Gly Asn Phe Ile Ile Glu Asn Leu Lys Ala Ala Asn Thr Asp

755 760 765

Pro Thr Ala Pro Pro Tyr Asp Thr Leu Leu Val Phe Asp Tyr Glu Gly

770 775 780

Ser Gly Ser Asp Ala Ala Ser Leu Ser Ser Leu Thr Ser Ser Ala Ser

785 790 795 800

Asp Gln Asp Gln Asp Tyr Asp Tyr Leu Asn Glu Trp Gly Ser Arg Phe

805 810 815

Lys Lys Leu Ala Asp Met Tyr Gly Gly Gly Glu Asp Asp

820 825

<210> 75

<211> 1255

<212> PROTEIN

<213> Homo sapiens

<400> 75

Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu

1 5 10 15

Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys

20 25 30

Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His

35 40 45

Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr

50 55 60

Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val

65 70 75 80

Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu

85 90 95

Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr

100 105 110

Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro

115 120 125

Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser

130 135 140

Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln

145 150 155 160

Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn

165 170 175

Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys

180 185 190

His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser

195 200 205

Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys

210 215 220

Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys

225 230 235 240

Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu

245 250 255

His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val

260 265 270

Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg

275 280 285

Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu

290 295 300

Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln

305 310 315 320

Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys

325 330 335

Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu

340 345 350

Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys

355 360 365

Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp

370 375 380

Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe

385 390 395 400

Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro

405 410 415

Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg

420 425 430

Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu

435 440 445

Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly

450 455 460

Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val

465 470 475 480

Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr

485 490 495

Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His

500 505 510

Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys

515 520 525

Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys

530 535 540

Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys

545 550 555 560

Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys

565 570 575

Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp

580 585 590

Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu

595 600 605

Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln

610 615 620

Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys

625 630 635 640

Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser

645 650 655

Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly

660 665 670

Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg

675 680 685

Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly

690 695 700

Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu

705 710 715 720

Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys

725 730 735

Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile

740 745 750

Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu

755 760 765

Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg

770 775 780

Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu

785 790 795 800

Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg

805 810 815

Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly

820 825 830

Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala

835 840 845

Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe

850 855 860

Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp

865 870 875 880

Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg

885 890 895

Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val

900 905 910

Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala

915 920 925

Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro

930 935 940

Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met

945 950 955 960

Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe

965 970 975

Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu

980 985 990

Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu

995 1000 1005

Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr

1010 1015 1020

Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly

1025 1030 1035

Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg

1040 1045 1050

Ser Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu

1055 1060 1065

Glu Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser

1070 1075 1080

Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu

1085 1090 1095

Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser

1100 1105 1110

Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val

1115 1120 1125

Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln Pro

1130 1135 1140

Asp Val Arg Pro Gln Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro

1145 1150 1155

Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu

1160 1165 1170

Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly

1175 1180 1185

Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala

1190 1195 1200

Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp

1205 1210 1215

Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro

1220 1225 1230

Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr

1235 1240 1245

Leu Gly Leu Asp Val Pro Val

1250 1255

<210> 76

<211> 300

<212> PROTEIN

<213> Homo sapiens

<400> 76

Met Ala Asn Cys Glu Phe Ser Pro Val Ser Gly Asp Lys Pro Cys Cys

1 5 10 15

Arg Leu Ser Arg Arg Ala Gln Leu Cys Leu Gly Val Ser Ile Leu Val

20 25 30

Leu Ile Leu Val Val Val Leu Ala Val Val Val Pro Arg Trp Arg Gln

35 40 45

Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg Phe Pro Glu Thr Val Leu

50 55 60

Ala Arg Cys Val Lys Tyr Thr Glu Ile His Pro Glu Met Arg His Val

65 70 75 80

Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala Phe Ile Ser Lys

85 90 95

His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro Leu Met Lys Leu

100 105 110

Gly Thr Gln Thr Val Pro Cys Asn Lys Ile Leu Leu Trp Ser Arg Ile

115 120 125

Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln Arg Asp Met Phe Thr

130 135 140

Leu Glu Asp Thr Leu Leu Gly Tyr Leu Ala Asp Asp Leu Thr Trp Cys

145 150 155 160

Gly Glu Phe Asn Thr Ser Lys Ile Asn Tyr Gln Ser Cys Pro Asp Trp

165 170 175

Arg Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe Trp Lys Thr Val

180 185 190

Ser Arg Arg Phe Ala Glu Ala Ala Cys Asp Val Val His Val Met Leu

195 200 205

Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser Thr Phe Gly Ser

210 215 220

Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln Thr Leu Glu Ala

225 230 235 240

Trp Val Ile His Gly Gly Arg Glu Asp Ser Arg Asp Leu Cys Gln Asp

245 250 255

Pro Thr Ile Lys Glu Leu Glu Ser Ile Ile Ser Lys Arg Asn Ile Gln

260 265 270

Phe Ser Cys Lys Asn Ile Tyr Arg Pro Asp Lys Phe Leu Gln Cys Val

275 280 285

Lys Asn Pro Glu Asp Ser Ser Cys Thr Ser Glu Ile

290 295 300

<210> 77

<211> 243

<212> PROTEIN

<213> Homo sapiens

<400> 77

Met Cys Ser Arg Gly Trp Asp Ser Cys Leu Ala Leu Glu Leu Leu Leu

1 5 10 15

Leu Pro Leu Ser Leu Leu Val Thr Ser Ile Gln Gly His Leu Val His

20 25 30

Met Thr Val Val Ser Gly Ser Asn Val Thr Leu Asn Ile Ser Glu Ser

35 40 45

Leu Pro Glu Asn Tyr Lys Gln Leu Thr Trp Phe Tyr Thr Phe Asp Gln

50 55 60

Lys Ile Val Glu Trp Asp Ser Arg Lys Ser Lys Tyr Phe Glu Ser Lys

65 70 75 80

Phe Lys Gly Arg Val Arg Leu Asp Pro Gln Ser Gly Ala Leu Tyr Ile

85 90 95

Ser Lys Val Gln Lys Glu Asp Asn Ser Thr Tyr Ile Met Arg Val Leu

100 105 110

Lys Lys Thr Gly Asn Glu Gln Glu Trp Lys Ile Lys Leu Gln Val Leu

115 120 125

Asp Pro Val Pro Lys Pro Val Ile Lys Ile Glu Lys Ile Glu Asp Met

130 135 140

Asp Asp Asn Cys Tyr Leu Lys Leu Ser Cys Val Ile Pro Gly Glu Ser

145 150 155 160

Val Asn Tyr Thr Trp Tyr Gly Asp Lys Arg Pro Phe Pro Lys Glu Leu

165 170 175

Gln Asn Ser Val Leu Glu Thr Thr Leu Met Pro His Asn Tyr Ser Arg

180 185 190

Cys Tyr Thr Cys Gln Val Ser Asn Ser Val Ser Ser Lys Asn Gly Thr

195 200 205

Val Cys Leu Ser Pro Pro Cys Thr Leu Ala Arg Ser Phe Gly Val Glu

210 215 220

Trp Ile Ala Ser Trp Leu Val Val Thr Val Pro Thr Ile Leu Gly Leu

225 230 235 240

Leu Leu Thr

<210> 78

<211> 229

<212> PROTEIN

<213> Homo sapiens

<400> 78

Met Ala Arg Leu Ala Leu Ser Pro Val Pro Ser His Trp Met Val Ala

1 5 10 15

Leu Leu Leu Leu Leu Ser Ala Glu Pro Val Pro Ala Ala Arg Ser Glu

20 25 30

Asp Arg Tyr Arg Asn Pro Lys Gly Ser Ala Cys Ser Arg Ile Trp Gln

35 40 45

Ser Pro Arg Phe Ile Ala Arg Lys Arg Gly Phe Thr Val Lys Met His

50 55 60

Cys Tyr Met Asn Ser Ala Ser Gly Asn Val Ser Trp Leu Trp Lys Gln

65 70 75 80

Glu Met Asp Glu Asn Pro Gln Gln Leu Lys Leu Glu Lys Gly Arg Met

85 90 95

Glu Glu Ser Gln Asn Glu Ser Leu Ala Thr Leu Thr Ile Gln Gly Ile

100 105 110

Arg Phe Glu Asp Asn Gly Ile Tyr Phe Cys Gln Gln Lys Cys Asn Asn

115 120 125

Thr Ser Glu Val Tyr Gln Gly Cys Gly Thr Glu Leu Arg Val Met Gly

130 135 140

Phe Ser Thr Leu Ala Gln Leu Lys Gln Arg Asn Thr Leu Lys Asp Gly

145 150 155 160

Ile Ile Met Ile Gln Thr Leu Leu Ile Ile Leu Phe Ile Ile Val Pro

165 170 175

Ile Phe Leu Leu Leu Asp Lys Asp Asp Ser Lys Ala Gly Met Glu Glu

180 185 190

Asp His Thr Tyr Glu Gly Leu Asp Ile Asp Gln Thr Ala Thr Tyr Glu

195 200 205

Asp Ile Val Thr Leu Arg Thr Gly Glu Val Lys Trp Ser Val Gly Glu

210 215 220

His Pro Gly Gln Glu

225

<210> 79

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 79

Gly Phe Leu Gly

1

<210> 80

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 80

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Gly Gly Asp Thr Asn Tyr Asn Glu Ile Phe

50 55 60

Lys Gly Arg Ala Thr Phe Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Arg Val Pro Ile Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 81

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 81

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Asp Tyr Glu

20 25 30

Gly Asp Ser Phe Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 82

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 82

Gly Tyr Thr Phe Ser Ser Tyr Trp Ile Glu

1 5 10

<210> 83

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 83

Glu Ile Leu Pro Gly Gly Gly Asp Thr Asn Tyr Asn Glu Ile Phe Lys

1 5 10 15

Gly

<210> 84

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 84

Arg Val Pro Ile Arg Leu Asp Tyr

1 5

<210> 85

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 85

Ile Thr Cys Lys Ala Ser Gln Ser Val Asp Tyr Glu Gly Asp Ser Phe

1 5 10 15

Leu Asn

<210> 86

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 86

Ala Ala Ser Asn Leu Glu Ser

1 5

<210> 87

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 87

Gln Gln Ser Asn Glu Asp Pro Leu Thr

1 5

<210> 88

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 88

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Gly Gly Asp Thr Asn Tyr Asn Glu Ile Phe

50 55 60

Lys Gly Arg Ala Thr Phe Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Arg Val Pro Ile Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Cys Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Cys Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 89

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 89

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Asp Tyr Glu

20 25 30

Gly Asp Ser Phe Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 90

<211> 116

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 90

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Leu Thr Val Asn Asn Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Gly Gly Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 91

<211> 109

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 91

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Gly

85 90 95

Thr Trp Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 92

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 92

Gly Leu Phe Gly

1

<210> 93

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 93

Ala Leu Ala Leu

1

<210> 94

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 94

Gly Gly Gly Gly

1

<210> 95

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 95

Gly Phe Val Gly

1

<210> 96

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 96

Gly Val Phe Gly

1

<210> 97

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<220>

<221> MOD_RES

<222> (1)..(1)

<223> Beta-Ala

<220>

<221> MOD_RES

<222> (2)..(2)

<223> Ahh

<400> 97

Xaa Xaa Ala Arg Glu Ile Gly Ala Gln Leu Arg Arg Met Ala Asp Asp

1 5 10 15

Leu Asn Ala Gln Tyr

20

<---

Claims (563)

1. An antibody-drug conjugate of formula (1) Ab-(LD) _p (1), where Ab is an antibody or its antigen-binding fragment; D is an inhibitor of Mcl-1; L is a linker that covalently attaches Ab to D; and p is an integer between 1 and 16, where D is a compound of formula (III) or its enantiomer, diastereoisomer, atropisomer and/or pharmaceutically acceptable salt, where R ₀₁ is a linear or branched (C ₁ -C ₆ )alkyl group, R ₀₃ is -O-(C ₁ -C ₆ )alkyl-NR ₀₁₁ R ₀₁₁ ', or , where R ₀₁₁ and R ₀₁₁ ' independently of one another are a hydrogen atom, an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group or -(C ₀ -C ₆ )alkyl-Cy ₀₁ ; or a pair (R ₀₁₁ , R ₀₁₁ ') together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5-7 members in the ring, which optionally contains, in addition to the nitrogen atom, 1-3 heteroatoms selected from O, S and N, wherein the N atom may be substituted by 1 or 2 groups selected from a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group, and where R ₀₂₇ is a hydrogen atom and R ₀₂₈ is a -(CH ₂ ) _p0 -O-SO ₂ -O- group or a -(CH ₂ ) _p0 -SO ₂ -OR _{030 -} group; R ₀₉ is a linear or branched (C ₂ -C ₆ )alkynyl group or -Cy ₀₂ , R ₀₁₂ is -Cy ₀₅ , -Cy ₀₅ -(C ₀ -C ₆ )alkyl-Cy ₀₆ or -Cy ₀₅ -(C ₀ -C ₆ )alkyl-Cy ₀₉ , Cy ₀₁ , Cy ₀₂ , Cy ₀₅ and Cy ₀₆ independently of one another are a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, each of which is optionally substituted, Cy ₀₉ is , R ₀₁₅ is a hydrogen atom; -(CH ₂ ) _p0 -O-(CHR ₀₁₈ -CHR ₀₁₉ -O) _q0 -R ₀₂₀ group; a linear or branched (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group; -U ₀ -(CH ₂ ) _q0 -NR ₀₂₁ R ₀₂₁ 'group; or -(CH ₂ ) _r0 -U ₀ -(CH ₂ ) _s0 -heterocycloalkyl group, R ₀₁₆ represents a hydrogen atom; a hydroxy group; a hydroxy(C ₁ -C ₆ )alkyl group; -(CH ₂ ) _r0 -U ₀ -(CH ₂ ) _s0 -heterocycloalkyl group; (CH ₂ ) _r0 -U ₀ -V ₀ -OP(O)(OR ₀₂₀ ) ₂ -group; -OP(O)(O ^- M ⁺ ) ₂ -group; -OS(O) ₂ OR ₀₂₀ -group; -S(O) ₂ OR ₀₂₀ -group; -(CH ₂ ) _p0 -O-(CHR ₀₁₈ -CHR ₀₁₉ -O) _q0 -R ₀₂₀ -group; -(CH ₂ ) _p0 -OC(O)-NR ₀₂₂ R ₀₂₃ -group; or -U ₀ -(CH ₂ ) _q0 -NR ₀₂₁ R ₀₂₁ '-group, R ₀₁₇ is a hydrogen atom; -(CH ₂ ) _p0 -O-(CHR ₀₁₈ -CHR ₀₁₉ -O) _q0 -R ₀₂₀ group; -CH ₂ -P(O)(OR ₀₂₀ ) ₂ group, -OP(O)(OR ₀₂₀ ) ₂ group; -OP(O)(O ^- M ⁺ ) ₂ group; hydroxy group; hydroxy(C ₁ -C ₆ )alkyl group; -(CH ₂ ) _r0 -U ₀ -(CH ₂ ) _s0 -heterocycloalkyl group; -U ₀ -(CH ₂ ) _q0 -NR ₀₂₁ R ₀₂₁ 'group; or aldonic acid, M ⁺ is a pharmaceutically acceptable monovalent cation, U ₀ is a bond or oxygen atom, V ₀ is a -(CH ₂ ) _s0 group or a -C(O) group, R ₀₁₈ is a hydrogen atom or a (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group, R ₀₁₉ is a hydrogen atom or a hydroxy(C ₁ -C ₆ )alkyl group, R ₀₂₀ is a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group, R ₀₂₁ and R ₀₂₁ ' independently of one another are a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group or a hydroxy(C ₁ -C ₆ )alkyl group, or a pair (R ₀₂₁ , R ₀₂₁ ') together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5-7 members in the ring, R ₀₂₂ is a (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group, -(CH ₂ ) _p0 -NR ₀₂₄ R ₀₂₄ ' group, or -(CH ₂ ) _p0 -O-(CHR ₀₁₈ -CHR ₀₁₉ -O) _q0 -R ₂₀ group, R ₀₂₃ is a hydrogen atom or a (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group, or a pair (R ₀₂₂ , R ₀₂₃ ) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5-18 ring members which optionally contains, in addition to the nitrogen atom, 1-5 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted with a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group or a heterocycloalkyl group, R ₀₂₄ and R ₀₂₄ ' independently of each other are a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group, or a pair (R ₀₂₄ , R ₀₂₄ ') together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring consisting of 5-7 ring members which may contain, in addition to the nitrogen atom, 1-3 heteroatoms selected from O, S and N, and wherein the resulting ring is optionally substituted with a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group, R ₀₃₀ is a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, or an aryl(C ₁ -C ₆ )alkyl group; p ₀ is an integer equal to 0, 1, 2, or 3, q ₀ is an integer equal to 1, 2, 3, or 4, r ₀ and s ₀ are independently an integer equal to 0 or 1; and where one of the R ₀₃ , R ₀₉ or R ₀₁₂ groups, if present, is covalently attached to the linker. 2. The antibody-drug conjugate of claim 1, wherein p is an integer from 1 to 6 or from 2 to 4; or p is 2 or 4; or p is determined by liquid chromatography-mass spectrometry (LC-MS). 3. An antibody-drug conjugate according to claim 1 or 2, characterized in that L contains: attached group; at least one bridging spacer group; and at least one cleavable group, optionally at least one cleavable group containing a pyrophosphate group and/or a self-cleavable group. 4. The antibody-drug conjugate according to claim 3, characterized in that -(L-D) has the formula (A) (A), where R ¹ is the attached group; L ₁ is a bridging spacer group; E is a split group. 5. The antibody-drug conjugate of claim 3 or 4, wherein the cleavable group comprises a pyrophosphate group or the cleavable group comprises . 6. An antibody-drug conjugate according to claim 3 or 4, characterized in that the bridging spacer group contains: (i) a polyoxyethylene (PEG) group; (ii) a PEG group selected from PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14 and PEG15; (iii) -CO-CH ₂ -CH ₂ -PEG12- group; (iv) a butanoyl, pentanoyl, hexanoyl, heptanoyl or octanoyl group; or (v) hexanoyl group. 7. The antibody-drug conjugate according to claim 6, characterized in that (i) the attached group is formed from at least one reactive group selected from a maleimide group, a thiol group, a cyclooctyne group and an azido group; optionally wherein: a) the maleimide group has the structure ; b) the azido group has the structure -N=N ⁺ =N ^- ; c) the cyclooctyne group has the structure or , Where is a linkage to an antibody or antigen-binding fragment; or d) the cyclooctyne group has the structure , Where is a linkage to an antibody or antigen-binding fragment; or (ii) the attached group has a formula containing or , Where is a linkage to an antibody or antigen-binding fragment. 8. The antibody-drug conjugate of claim 7, wherein the antibody or antigen-binding fragment is connected to the linker (L) by an attached group selected from And , Where is a linkage to an antibody or antigen-binding fragment and where is a bond with a bridging spacer group. 9. The antibody-drug conjugate of claim 8, wherein the bridging spacer group is -CO-CH ₂ -CH ₂ -PEG12-. 10. The antibody-drug conjugate of claim 8 or 9, wherein the bridging spacer group is linked to a cleavable group; optionally, the cleavable group is -pyrophosphate-CH ₂ -CH ₂ -NH ₂ -. 11. The antibody-drug conjugate of any one of claims 8-10, wherein the cleavable group is linked to the Mcl-1 inhibitor (D) or the cleavable group is linked to the Mcl-1 inhibitor (D) group via a phenylpyrimidinyl group. 12. An antibody-drug conjugate according to any one of paragraphs 1-3, characterized in that the linker comprises: affiliated group, at least one bridging spacer group, peptide group and at least one cleavable group. 13. The antibody-drug conjugate of claim 12, wherein -(L-D) has the formula (B) (B), where R ¹ is the attached group; L ₁ is a bridging spacer; Lp is a peptide group containing 1-6 amino acid residues, or Lp contains a group ; E is a split group; L ₂ is a bridging spacer; m is 0 or 1; and D is an inhibitor of Mcl-1. 14. The antibody-drug conjugate according to claim 12 or 13, characterized in that (i) the attached group is formed from at least one reactive group selected from a maleimide group, a thiol group, a cyclooctyne group and an azido group; optionally wherein: a) the maleimide group has the structure ; b) the azido group has the structure -N=N ⁺ =N ^- ; c) the cyclooctyne group has the structure or , Where is a linkage to an antibody or antigen-binding fragment; or d) the cyclooctyne group has the structure , Where is a linkage to an antibody or antigen-binding fragment; or (ii) the attached group has a formula containing or , Where is a linkage to an antibody or antigen-binding fragment. 15. An antibody-drug conjugate according to any one of paragraphs 12-14, characterized in that: (i) at least one bridging spacer comprises a PEG group, optionally the PEG group is selected from PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14 and PEG15; or (ii) at least one bridging spacer is selected from *-C(O) _-CH2 - _CH2- PEG1-**, *-C(O) _-CH2 -PEG3-**, *-C(O)-CH2- _{CH2 -} PEG12**, *-NH-CH2-CH2-PEG1-**, a polyhydroxyalkyl group and *-C(O)-N( _CH3 ) _-CH2 - _CH2 -N( _CH3 )-C(O)-**, where ** denotes the point of direct or indirect attachment of at least one bridging spacer to the attached group and * denotes the point of direct or indirect attachment of at least one bridging spacer to the peptide group. 16. The antibody-drug conjugate of any one of claims 12 to 15, wherein L ₁ is selected from *-C(O)-CH ₂ -CH ₂ -PEG1-**, *-C(O)-CH ₂ -PEG3-**, *-C(O)-CH ₂ -CH ₂ -PEG12**, *-NH-CH2-CH2-PEG1-** and a polyhydroxyalkyl group, where ** denotes the point of direct or indirect attachment of L ₁ to R ¹ and * denotes the point of direct or indirect attachment of L ₁ to Lp. 17. The antibody-drug conjugate of any one of claims 12-16, wherein m is 1 and L ₂ is -C(O)-N(CH ₃ )-CH ₂ -CH ₂ -N(CH ₃ )-C(O)-. 18. An antibody-drug conjugate according to any one of paragraphs 12-17, characterized in that (i) the peptide moiety comprises 1-6, 1-4, 1-3 or 1-2 amino acid residues, optionally the amino acid residues are selected from L-glycine (Gly), L-valine (Val), L-citrulline (Cit), L-cysteine acid (sulfo-Ala), L-lysine (Lys), L-isoleucine (Ile), L-phenylalanine (Phe), L-methionine (Met), L-asparagine (Asn), L-proline (Pro), L-alanine (Ala), L-leucine (Leu), L-tryptophan (Trp) and L-tyrosine (Tyr); (ii) the peptide group comprises Val-Cit, Val-Ala, Val-Lys and/or sulfo-Ala-Val-Ala; (iii) the peptide group is selected from: 19. The antibody-drug conjugate of any one of claims 12 to 18, wherein (i) the cleaving group comprises pyrophosphate and/or a self-cleaving group; (ii) the cleaving group comprises a self-cleaving group; or (iii) the cleaving group comprises a self-cleaving group comprising para-aminobenzyl carbamate, para-aminobenzyl ammonium, para-amino-(sulfo)benzyl ammonium, para-amino-(sulfo)benzyl carbamate, para-amino-(alkoxy-PEG alkyl)benzyl carbamate, para-amino-(polyhydroxycarboxytetrahydropyranyl)alkyl benzyl carbamate, or para-amino-(polyhydroxycarboxytetrahydropyranyl)alkyl benzyl ammonium. 20. The antibody-drug conjugate of any one of claims 13-19, wherein m is 0 or 1 or m is 1 and the bridging spacer comprises . 21. An antibody-drug conjugate according to any one of paragraphs 13-20, characterized in that -(L-D) is formed from a compound selected from: , And . 22. An antibody-drug conjugate according to any one of paragraphs 13-21, characterized in that -(L-D) comprises a formula selected from: , , And , Where is a linkage to an antibody or antigen-binding fragment. 23. The antibody-drug conjugate of claim 1 or 2, wherein -(L-D) has the formula (C) (C), where R ¹ is the attached group; L ₁ is a bridging spacer; Lp is a peptide group containing 1-6 amino acids; D is an inhibitor of Mcl-1; G ₁ -L ₂ -A is a self-cleaving spacer; L ₂ is a bond, methylene, neopentylene, or C ₂ -C ₃ alkenylene; A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; L ₃ is a spacer group; and R ₂ is a hydrophilic group. 24. The antibody-drug conjugate of claim 23 or a pharmaceutically acceptable salt thereof, wherein -(L-D) has the formula (D) (D) where R ¹ is the attached group; L ₁ is a bridging spacer; Lp is a peptide group containing 1-6 amino acids; A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; L ₃ is a spacer group; and R ₂ is a hydrophilic group. 25. The antibody-drug conjugate of claim 23 or 24, characterized in that: (1) L ₁ contains or *-CH(OH)CH(OH)CH(OH)CH(OH)-**, where each n is an integer from 1 to 12, where * of L ₁ denotes a point of direct attachment to Lp and ** of L ₁ denotes a point of direct attachment to R ¹ ; (2) L ₁ is and n is an integer from 1 to 12 or n is 1 or n is 12, where * of L ₁ denotes a point of direct attachment to Lp and ** of L ₁ denotes a point of direct attachment to R ¹ ; (3) L ¹ is and n is an integer from 1 to 12, where * of L ₁ denotes the point of direct attachment to Lp and ** of L ₁ denotes the point of direct attachment to R ¹ ; (4) L ₁ contains , where * from L ₁ means the point of direct attachment to Lp and ** from L ₁ means the point of direct attachment to R ¹ ; (5) L ₁ is a bridging spacer containing: *-C(=O)(CH ₂ ) _m O(CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m -**; *-C(=O)NH((CH ₂ ) _m O) _t (CH ₂ ) _n -**; *-C(=O)O(CH ₂ ) _m SSC(R ³ ) ₂ (CH ₂ ) _m C(=O)NR ³ (CH ₂ ) _m NR ³ C(=O)(CH ₂ ) _m -**; *-C(=O)O(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m NH(CH ₂ ) _n C(=O)-**; *-C(=O)(CH ₂ ) _m X ₁ (CH ₂ ) _m -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n X ₁ (CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n -**; *-C(=O)(CH ₂ ) _m NHC(=O)(CH ₂ ) _n X ₁ (CH ₂ ) _n -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n NHC(=O)(CH ₂ ) _n X ₁ (CH ₂ ) _n -**; *-C(=O)((CH ₂ ) _m O) _t (CH ₂ ) _n C(=O)NH(CH ₂ ) _m -**; *-C(=O)(CH ₂ ) _m C(R ³ ) ₂ -**; or *-C(=O)(CH ₂ ) _m C(=O)NH(CH ₂ ) _m -**, where * from L ₁ means the point of direct attachment to Lp and ** from L ₁ means the point of direct attachment to R ¹ ; X ₁ is or ; And each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30; and each R ³ is independently selected from H and C ₁ -C ₆ alkyl. 26. The antibody-drug conjugate of any one of claims 23-25, wherein R ² is a hydrophilic group comprising polyethylene glycol, polyalkylene glycol, polyol, polysarcosine, sugar, oligosaccharide, polypeptide, or C ₂ -C ₆ alkyl substituted with 1-3 or in groups. 27. The antibody-drug conjugate of any one of paragraphs 23-26, characterized in that R ² is where n is an integer from 1 to 6, 28. The antibody-drug conjugate of claim 23 or 24, wherein the hydrophilic group comprises: (i) polysarcosine, for example with the following group: , where n is an integer between 3 and 25; and R is H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH; or (ii) polyethylene glycol of the formula or , where R is H, -CH ₃ , CH ₂ CH ₂ NHC(=O)OR _a , -CH ₂ CH ₂ NHC(=O)R _a or -CH ₂ CH ₂ C(=O)OR _a , R' is OH, -OCH ₃ , -CH ₂ CH ₂ NHC(=O)OR _a , -CH ₂ CH ₂ NHC(=O)R _a or -OCH ₂ CH ₂ C(=O)OR _a , where R _a is H or C _1-4 alkyl optionally substituted with either OH or C _1-4 alkoxyl, and each of m and n is independently an integer between 2 and 25. 29. The antibody-drug conjugate according to any one of paragraphs 23-27, characterized in that the hydrophilic group contains . 30. An antibody-drug conjugate according to any one of paragraphs 23-29, characterized in that: (i) L ₃ is a spacer group having the structure , where W is -CH ₂ -, -CH ₂ O-, -CH ₂ N(R ^b )C(=O)O-, -NHC(=O)C(R ^b ) ₂ NHC(=O)O-, -NHC(=O)C(R ^b ) ₂ NH-, -NHC(=O)C(R ^b ) ₂ NHC(=O)-, -CH ₂ N(XR ² )C(=O)O-, -C(=O)N(XR ² )-, -CH ₂ N(XR ² )C(=O)-, -C(=O)NR ^b -, -C(=O)NH-, -CH ₂ NR ^b C(=O)-, -CH ₂ NR ^b C(=O)NH-, -CH ₂ NR ^b C(=O)NR ^b -, -NHC(=O)-, -NHC(=O)O-, -NHC(=O)NH-, -OC(=O)NH-, -S(O) ₂ NH-, -NHS(O) ₂ -, -C(=O)-, -C(=O)O- or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl; and X is a bond, triazolyl, or -CH ₂ -triazolyl-; or (ii) L ₃ is a spacer group having the structure , where W is -CH ₂ -, -CH ₂ O-, -CH ₂ N(R ^b )C(=O)O-, -NHC(=O)C(R ^b ) ₂ NHC(=O)O-, -NHC(=O)C(R ^b ) ₂ NH-, -NHC(=O)C(R ^b ) ₂ NHC(=O)-, -CH ₂ N(XR ² )C(=O)O-, -C(=O)N(XR ² )-, -CH ₂ N(XR ² )C(=O)-, -C(=O)NR ^b -, -C(=O)NH-, -CH ₂ NR ^b C(=O)-, -CH ₂ NR ^b C(=O)NH-, -CH ₂ NR ^b C(=O)NR ^b -, -NHC(=O)-, -NHC(=O)O-, -NHC(=O)NH-, -OC(=O)NH-, -S(O) ₂ NH-, -NHS(O) ₂ -, -C(=O)-, -C(=O)O- or -NH-, where each R ^b is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl; and X is -CH ₂ -triazolyl-C _1-4 alkylene-OC(O)NHS(O) ₂ NH-, -C _4-6 cycloalkylene-OC(O)NHS(O) ₂ NH-, -(CH ₂ CH ₂ O) _n -C(O)NHS(O) ₂ NH-, -(CH ₂ CH ₂ O) _n -C(O)NHS(O) ₂ NH-(CH ₂ CH ₂ O) _n- or -CH ₂ -triazolyl-C _1-4 alkylene-OC(O)NHS(O) ₂ NH-(CH ₂ CH ₂ O) _n- , where each n is independently 1, 2, or 3. 31. An antibody-drug conjugate according to any one of claims 3-30, characterized in that the attached group is formed by a reaction containing at least one reactive group. 32. An antibody-drug conjugate according to any one of paragraphs 3-31, characterized in that the attached group is formed by the interaction of: the first reactive group that is attached to the linker, and a second reactive group that is attached to the antibody or antigen-binding fragment or is an amino acid residue of the antibody or antigen-binding fragment, where optionally, (i) at least one of the reactive groups contains: thiol, maleimide, haloacetamide, azide, alkyne, cyclooctene, triarylphosphine, oxanobornadiene, cyclooctyne, diaryltetrazine, monoaryltetrazine, Norbornen, aldehyde, hydroxylamine, hydrazine, NH ₂ -NH-C(=O)-, ketone, vinyl sulfone, aziridine, amino acid residue, , -ONH ₂ , -NH ₂ , , , -N ₃ , , -SH, -SR ³ , -SSR ⁴ , -S(=O) ₂ (CH=CH ₂ ), -(CH ₂ ) ₂ S(=O) ₂ (CH=CH ₂ ), -NHS(=O) ₂ (CH=CH ₂ ), -NHC(=O)CH ₂ Br, -NHC(=O)CH ₂ I, , -C(O)NHNH ₂ , , ; wherein each R ³ is independently selected from H and C ₁ -C ₆ alkyl; each R ⁴ is 2-pyridyl or 4-pyridyl; each R ⁵ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl and -OH; each R ⁶ is independently selected from H, C ₁ -C ₆ alkyl, F, Cl, -NH ₂ , -OCH ₃ , -OCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -CN, -NO ₂ and -OH; each R ⁷ is independently selected from H, C _1-6 alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C _1-4 alkoxy substituted with -C(=O)OH _, and C _1-4 alkyl substituted with -C(=O)OH; (ii) the first reactive group and the second reactive group comprise: thiol and maleimide, thiol and haloacetamide, thiol and vinyl sulfone, thiol and aziridine, azide and alkyne, azide and cyclooctyne, azide and cyclooctene, azide and triarylphosphine, azide and oxanobornadiene, diaryltetrazine and cyclooctene, monoaryltetrazine and norbornene, aldehyde and hydroxylamine, aldehyde and hydrazine, aldehyde and NH ₂ -NH-C(=O)-, ketone and a hydroxylamine, ketone and hydrazine, ketone and NH ₂ -NH-C(=O)-, hydroxylamine and , amine and or or CoA or a CoA analogue and a serine residue. 33. The antibody-drug conjugate of any one of claims 3-32, wherein the attached group comprises a group selected from: amide; ; And disulfide, where R ³² is H, C _1-4 alkyl, phenyl, pyrimidine or pyridine; R ³⁵ is H, C _1-6 alkyl, phenyl, or C _1-4 alkyl substituted with 1-3 -OH groups; each R ⁷ is independently selected from H, C _1-6 alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C _1-4 alkoxy substituted with -C(=O)OH, and C _1-4 alkyl substituted with -C(=O)OH; R ³⁷ is independently selected from H, phenyl and pyridine; q is 0, 1, 2 or 3; R ⁸ is H or methyl; and R ⁹ is H, -CH ₃ , or phenyl. 34. The antibody-drug conjugate of any one of claims 23 to 33, wherein the peptide moiety comprises 1-4, or 1-3, or 1-2 amino acid residues, optionally the amino acid residues are selected from L-glycine (Gly), L-valine (Val), L-citrulline (Cit), L-cysteine acid (sulfo-Ala), L-lysine (Lys), L-isoleucine (Ile), L-phenylalanine (Phe), L-methionine (Met), L-asparagine (Asn), L-proline (Pro), L-alanine (Ala), L-leucine (Leu), L-tryptophan (Trp) and L-tyrosine (Tyr). 35. The antibody-drug conjugate of any one of claims 23-33, wherein the peptide group comprises Val-Cit, Phe-Lys, Val-Ala, Val-Lys, Leu-Cit, sulfo-Ala-Val and/or sulfo-Ala-Val-Ala. 36. The antibody-drug conjugate according to any one of paragraphs 23-35, characterized in that Lp is selected from: 37. An antibody-drug conjugate according to any one of paragraphs 23-36, characterized in that -(L-D) comprises or is formed from a compound of the formula: (1) , where R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH; A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and D is an Mcl-1 inhibitor as defined in paragraph 1; (2) , where R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH; A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and D is an Mcl-1 inhibitor as defined in paragraph 1; (3) , where R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH; A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and D is an Mcl-1 inhibitor as defined in paragraph 1; (4) , where each R is independently selected from H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH; A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and D is an Mcl-1 inhibitor as defined in paragraph 1; (5) , where each R is independently selected from H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH; A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and D is an Mcl-1 inhibitor as defined in paragraph 1; (6) , where Xa is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ - and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH; A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and D is an Mcl-1 inhibitor as defined in paragraph 1; (7) , where R is H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH; A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and D is an Mcl-1 inhibitor as defined in paragraph 1; (8) , where Xb is -CH ₂ -, -OCH ₂ -, -NHCH ₂ -, or -NRCH ₂ - and each R is independently H, -CH ₃ , or -CH ₂ CH ₂ C(=O)OH; A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and D is an Mcl-1 inhibitor as defined in paragraph 1; (9) , where A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and D is an Mcl-1 inhibitor as defined in paragraph 1; (10) , where A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and D is an Mcl-1 inhibitor as defined in paragraph 1; (11) , where A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and D is an Mcl-1 inhibitor as defined in paragraph 1; (12) , where A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and D is an Mcl-1 inhibitor as defined in paragraph 1; (13) , where A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and D is an Mcl-1 inhibitor as defined in paragraph 1; (14) , where A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and D is an Mcl-1 inhibitor as defined in paragraph 1; (15) , where A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and D is an Mcl-1 inhibitor as defined in paragraph 1; or (16) , where each R is independently H, -CH ₃ or -CH ₂ CH ₂ C(=O)OH; A is a bond, -OC(=O)-*, , , -OC(=O)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )C(=O)-* or -OC(=O)N(CH ₃ )C(R ^a ) ₂ C(R ^a ) ₂ N(CH ₃ )C(=O)-*, where each R ^a is independently selected from H, C ₁ -C ₆ alkyl and C ₃ -C ₈ cycloalkyl and * of A denotes the point of attachment to D; and D is an Mcl-1 inhibitor as defined in paragraph 1. 38. The antibody-drug conjugate of any one of claims 23-37, wherein A is a bond and/or R is -CH ₃ . 39. An antibody-drug conjugate according to any one of claims 1-38, wherein R ₀₁ is methyl or ethyl. 40. The antibody-drug conjugate of any one of claims 1 to 39, wherein R ₀₃ is -O-CH ₂ -CH ₂ -NR ₀₁₁ R ₀₁₁ ', wherein R ₀₁₁ and R ₀₁₁ ', together with the nitrogen atom to which they are attached, form a piperazinyl group, which may be substituted with a group selected from a hydrogen atom and a linear or branched (C ₁ -C ₆ )alkyl group. 41. An antibody-drug conjugate according to any one of paragraphs 1-39, characterized in that R ₀₃ contains the formula , where R ₀₂₇ is a hydrogen atom and R ₀₂₈ is a -(CH ₂ ) _p0 -SO ₂ -OR ₀₃₀ group. 42. The antibody-drug conjugate according to claim 39, characterized in that R ₀₃ has the formula , Where is a connection with the linker. 43. The antibody-drug conjugate of any one of claims 1-42, wherein R ₀₉ is Cy ₀₂ . 44. The antibody-drug conjugate of claim 43, wherein Cy ₀₂ is an optionally substituted aryl group. 45. An antibody-drug conjugate according to any one of claims 1-44, wherein Cy ₀₅ contains a heteroaryl group selected from a pyrazolyl group and a pyrimidinyl group. 46. An antibody-drug conjugate according to any one of claims 1-45, wherein Cy ₀₅ is a pyrimidinyl group. 47. The antibody-drug conjugate of any one of claims 1 to 46, wherein L is attached to D by a covalent bond from L to R ₀₃ ; or L is attached to D by a covalent bond from L to R ₀₉ . 48. An antibody-drug conjugate according to any one of paragraphs 1-47, characterized in that: (1) D contains: , or its enantiomer, diastereoisomer, atropisomer and/or pharmaceutically acceptable salt; (2) D contains: , or its enantiomer, diastereoisomer, atropisomer and/or pharmaceutically acceptable salt; (3) D contains: , or its enantiomer, diastereoisomer, atropisomer and/or pharmaceutically acceptable salt; (4) D contains: , or its enantiomer, diastereoisomer, atropisomer and/or pharmaceutically acceptable salt; (5) D contains: , or its enantiomer, diastereoisomer, atropisomer and/or pharmaceutically acceptable salt; (6) D contains: , or its enantiomer, diastereoisomer, atropisomer and/or pharmaceutically acceptable salt; (7) D contains: , or its enantiomer, diastereoisomer, atropisomer and/or pharmaceutically acceptable salt; (8) D contains: , or its enantiomer, diastereoisomer, atropisomer and/or pharmaceutically acceptable salt; (9) D contains: , or its enantiomer, diastereoisomer, atropisomer and/or pharmaceutically acceptable salt; (10) D contains: , or its enantiomer, diastereoisomer, atropisomer and/or pharmaceutically acceptable salt; (11) D contains: , or its enantiomer, diastereoisomer, atropisomer and/or pharmaceutically acceptable salt; (12) D contains: , or its enantiomer, diastereoisomer, atropisomer and/or pharmaceutically acceptable salt; or (13) D contains: , or its enantiomer, diastereoisomer, atropisomer and/or pharmaceutically acceptable salt; (14) D contains: , or its enantiomer, diastereoisomer, atropisomer and/or pharmaceutically acceptable salt. 49. Antibody-drug conjugate of formula (1) Ab-(LD) _p (1), where Ab is an antibody or its antigen-binding fragment, p is an integer between 1 and 16; and -(L-D) is formed from a compound selected from the group consisting of: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , And , or a pharmaceutically acceptable salt thereof. 50. An antibody-drug conjugate according to any one of claims 1-49, wherein the antibody or antigen-binding fragment binds to a target antigen on a cancer cell. 51. The antibody-drug conjugate according to claim 50, characterized in that: (i) the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2, Nectin4, TROP2, LIV1, CD46, or GPNMB; (ii) the target antigen is BCMA, CD33, PCAD, HER2, CD38, CD46, CD48, or CD79b; or (iii) the target antigen is BCMA, CD33, CD48, PCAD or HER2. 52. An antibody-drug conjugate according to any one of claims 1-51, wherein the antibody or antigen-binding fragment is an anti-BCMA antibody or antigen-binding fragment. 53. The antibody-drug conjugate of claim 52, wherein the antibody or antigen-binding fragment comprises: (a) three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 15 (HCDR1), SEQ ID NO: 16 (HCDR2) and SEQ ID NO: 17 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 18 (LCDR1), SEQ ID NO: 19 (LCDR2), and SEQ ID NO: 20 (LCDR3); or (b) the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. 54. The antibody-drug conjugate according to paragraph 52 or 53, characterized in that: (a) the antibody or antigen-binding fragment comprises an IgG1 heavy chain constant domain or a modified IgG1 heavy chain constant domain, optionally the IgG1 heavy chain constant domain comprises a cysteine residue (C) at position 152 and position 375 or the IgG1 heavy chain constant domain comprises a cysteine residue (C) at position 156 and position 379; and/or (b) the antibody or antigen-binding fragment comprises a constant domain of the kappa light chain of Ig. 55. The antibody-drug conjugate of any one of claims 1-54, wherein the antibody or antigen-binding fragment is an anti-CD33 antibody or antigen-binding fragment. 56. The antibody-drug conjugate according to claim 55, characterized in that: (a) the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 21 (HCDR1), SEQ ID NO: 22 (HCDR2), and SEQ ID NO: 23 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 24 (LCDR1), SEQ ID NO: 25 (LCDR2), and SEQ ID NO: 26 (LCDR3); and/or (b) the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4. 57. The antibody-drug conjugate of claim 55 or 56, characterized in that: (a) the antibody or antigen-binding fragment comprises an IgG1 heavy chain constant domain or a modified IgG1 heavy chain constant domain, optionally the IgG1 heavy chain constant domain comprises glutamine (Q) at position 297; and/or (b) the antibody or antigen-binding fragment comprises a constant domain of the kappa light chain of Ig. 58. The antibody-drug conjugate of any one of claims 1-51, wherein the antibody or antigen-binding fragment is an anti-PCAD antibody or antigen-binding fragment. 59. The antibody-drug conjugate according to claim 58, characterized in that: (a) the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 33 (HCDR1), SEQ ID NO: 34 (HCDR2), and SEQ ID NO: 35 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 36 (LCDR1), SEQ ID NO: 37 (LCDR2), and SEQ ID NO: 38 (LCDR3); and/or (b) the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. 60. The antibody-drug conjugate of any one of claims 1-51, wherein the antibody or antigen-binding fragment is an anti-HER2 antibody or antigen-binding fragment. 61. The antibody-drug conjugate according to claim 60, characterized in that: (a) the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 39 (HCDR1), SEQ ID NO: 40 (HCDR2), and SEQ ID NO: 41 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 42 (LCDR1), SEQ ID NO: 43 (LCDR2), and SEQ ID NO: 44 (LCDR3); and/or (b) the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10. 62. An antibody-drug conjugate according to claim 60 or 61, characterized in that: (a) the antibody or antigen-binding fragment comprises an IgG1 heavy chain constant domain or a modified IgG1 heavy chain constant domain, optionally the IgG1 heavy chain constant domain comprises glutamine (Q) at position 297 or the IgG1 heavy chain constant domain comprises serine (S) at position 297; and/or (b) the antibody or antigen-binding fragment comprises a constant domain of the kappa light chain of Ig. 63. The antibody-drug conjugate of any one of claims 1-51, wherein the antibody or antigen-binding fragment is an anti-CD38 antibody or antigen-binding fragment; an anti-CD46 antibody or antigen-binding fragment; an anti-CD48 antibody or antigen-binding fragment; or an anti-CD79b antibody or antigen-binding fragment. 64. A composition for treating cancer comprising a therapeutically effective amount of multiple copies of an antibody-drug conjugate according to any one of claims 1-63, wherein the average p of the antibody-drug conjugate in the composition is from about 2 to about 16, such as from about 2 to about 8, such as from about 2 to about 4, wherein the average p refers to the average number of -LD groups per antibody or antigen-binding fragment thereof. 65. A pharmaceutical composition for treating cancer, comprising a therapeutically effective amount of an antibody-drug conjugate according to any one of claims 1-63 or a composition according to claim 64 and a pharmaceutically acceptable carrier. 66. A method of treating a subject having or suspected of having cancer, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate according to any one of claims 1-63, a composition according to claim 64, or a pharmaceutical composition according to claim 65. 67. The method according to claim 66, characterized in that the cancer expresses a target antigen. 68. The method according to claim 66 or 67, characterized in that the cancer is a tumor or a hematological cancer, preferably the cancer is breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer or spleen cancer. 69. A method for reducing or inhibiting tumor growth in a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate according to any one of claims 1-63, a composition according to claim 64, or a pharmaceutical composition according to claim 65. 70. The method according to claim 69, characterized in that the tumor expresses a target antigen. 71. The method according to claim 69 or 70, wherein the tumor is breast cancer, stomach cancer, bladder cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer, hepatocellular cancer, melanoma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, or spleen cancer. 72. The method of any one of paragraphs 69-71, wherein administering the antibody-drug conjugate, composition, or pharmaceutical composition reduces or inhibits tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. 73. A method for reducing or slowing the growth of a cancer cell population in a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate according to any one of claims 1-63, a composition according to claim 64, or a pharmaceutical composition according to claim 65. 74. The method according to claim 73, characterized in that the population of cancer cells expresses a target antigen. 75. The method according to claim 73 or 74, characterized in that the population of cancer cells originates from a tumor or hematological cancer, preferably the population of cancer cells originates from breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer or spleen cancer. 76. The method of any one of claims 73-75, wherein administering the antibody-drug conjugate, composition, or pharmaceutical composition reduces the cancer cell population or slows the growth of the cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. 77. A method for determining whether a subject having or suspected of having cancer will respond to treatment with the antibody-drug conjugate of any one of claims 1-63, the composition of claim 64, or the pharmaceutical composition of claim 65, comprising providing a biological sample from the subject; contacting the sample with the antibody-drug conjugate; and detecting binding of the antibody-drug conjugate of any one of claims 1-63 to cancer cells in the sample. 78. The method according to claim 77, characterized in that the cancer cells in the sample express the target antigen. 79. The method according to claim 77 or 78, characterized in that the cancer expresses the target antigen. 80. The method according to any one of claims 77-79, characterized in that the cancer is a tumor or a hematological cancer, preferably the cancer is breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer or spleen cancer. 81. The method according to any one of paragraphs 77-80, characterized in that the sample is a tissue biopsy sample, a blood sample, or a bone marrow sample. 82. The method of any one of claims 66-81, wherein (i) the target antigen is BCMA, CD33, HER2, CD38, CD48, CD79b, PCAD, CD74, CD138, SLAMF7, CD123, CLL1, FLT3, CD7, CKIT, CD56, DLL3, DLK1, B7-H3, EGFR, CD71, EPCAM, FOLR1, ENPP3, MET, AXL, SLC34A2, Nectin4, TROP2, LIV1, CD46, or GPNMB; (ii) the target antigen is BCMA, CD33, PCAD, HER2, CD38, CD46, CD48, or CD79b; or (iii) BCMA, CD33, CD48, PCAD, or HER2. 83. A method for producing an antibody-drug conjugate according to any one of claims 1-63, comprising reacting an antibody or antigen-binding fragment with a cleavable linker attached to an MCL1 inhibitor under conditions allowing conjugation. 84. The method according to any one of paragraphs 66-82, further comprising administering to a subject in need thereof at least one additional therapeutic agent, preferably characterized in that the one additional therapeutic agent is a Bcl-2 inhibitor, more preferably the one additional therapeutic agent is venetoclax; compound A1; or connection A2. 85. A compound having one of the following structures: ; ; ; ; or , or a pharmaceutically acceptable salt thereof. 86. The compound according to claim 85, characterized in that the compounds are used as an intermediate product of the synthesis for obtaining an antibody-drug conjugate of formula (1) according to claim 1. 87. Compounds according to claim 85 or their addition salt with a pharmaceutically acceptable acid or base for use as pro-apoptotic agents.