EP4651902A2 - Asgpr-bindende verbindungen und konjugate - Google Patents

Asgpr-bindende verbindungen und konjugate

Info

Publication number: EP4651902A2
Authority: EP; European Patent Office
Prior art keywords: optionally substituted; certain embodiments; alkyl; alkylene; formula
Prior art date: 2023-01-18
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.): Pending

Application number

EP24705914.0A

Other languages

English (en)

French (fr)

Inventor

Jason G. Lewis

Tao Chen

Darin Hildebrandt

Steven W. RANK

Eric D. Turtle

Shuai ZHENG

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

Lycia Therapeutics Inc

Original Assignee

Lycia Therapeutics Inc

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2023-01-18

Filing date

2024-01-17

Publication date

2025-11-26

2024-01-17 Application filed by Lycia Therapeutics Inc filed Critical Lycia Therapeutics Inc

2025-11-26 Publication of EP4651902A2 publication Critical patent/EP4651902A2/de

Status Pending legal-status Critical Current

Links

Classifications

- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/02—Heterocyclic radicals containing only nitrogen as ring hetero atoms
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/056—Triazole or tetrazole radicals

Definitions

the asialoglycoprotein receptor also known as the Ashwell Morell receptor, is the transmembrane glycoprotein receptor found primarily in hepatocytes which plays an important role in serum glycoprotein homeostasis by mediating the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N-acetylgalactosamine (GalNAc) residues.
ASGPR cycles between endosomes and the cell surface.
the present disclosure provides a class of compounds including a ligand moiety that specifically binds to a cell surface asialoglycoprotein receptor (ASGPR).
ASGPR asialoglycoprotein receptor
the cell surface ASGPR binding compounds can trigger the receptor to internalize into the cell a bound compound.
the ligand moieties of this disclosure can be linked to a variety of moieties of interest without impacting the specific binding to, and function of, the cell surface receptor ASGPR.
conjugates of the ligand moieties linked to a biomolecule such as an antibody
conjugates can harness cellular pathways to remove specific proteins of interest from the cell surface or from the extracellular milieu.
the conjugates described herein may sequester and/or degrade a target molecule of interest in a cell’s lysosome.
compositions comprising such conjugates and methods of using the conjugates to target a polypeptide of interest for sequestration and/or lysosomal degradation, and methods of using the conjugates to treat disorders or disease.
FIG.1 shows a graph of cell fluorescence (MFI) versus antibody conjugate concentration ([Ab]) indicating that various antibody conjugates of exemplary ASGPR binding compounds, and an example M6PR binding compound (520) exhibited comparable robust uptake into HepG2 cells after one hour incubation.
FIG.2A-2D shows graphs of cell fluorescence versus antibody conjugate concentration indicating that various antibody conjugates of exemplary ASGPR binding compounds exhibited robust uptake into HepG2 cells after one hour incubation.
FIG.3 illustrates the fluorescence polarization screening results for example trivalent compounds (1901 (I-171), 1902 (I-172), XB32 and 2101).
FIG.4 illustrates the binding of example monovalent compounds (591, XB20, XB23, XB21, 592 and 593) as a percentage of the activity of reference compound XB149.
FIG.5 illustrates the fluorescence polarization screening results for example monovalent compounds (XB20, XB21, 592, XB23, 591).
FIG.6 shows a graph of cellular uptake of various conjugates of OMA (anti-IgE) with example compounds I-160 to I-163 and I-141 bound to Alexa488 labeled-target IgE in HepG2 cells.
FIG.7 illustrates affinity-dependent clearance of OMA-example compounds (I-160 to I-163) as compared to OMA (reference).
FIG.8 illustrates dose titration OMA-I-163 IgE clearance.
FIG.9 illustrates affinity-dependent clearance of OMA-example compounds (I-160 to I-163) as compared to hIgE (reference).
this disclosure provides classes of compounds including a ligand moiety that specifically binds an ASGPR of a cell of interest.
This disclosure includes compounds of formula (I): X n L Y (I) or a prodrug thereof, or a salt thereof, wherein: X is a moiety that binds to a ASGPR cell surface receptor (e.g., as described herein); n is 1 to 500; L is a linker (e.g., monovalent or multivalent, as described herein, of defined length); and Y is a moiety of interest (e.g., as described herein).
conjugates that comprise a moiety, X, that binds to such an ASGPR internalizing cell surface receptor, for example, for sequestration and/or lysosomal degradation.
this disclosure includes target binding conjugate of formula (I): or a prodrug thereof, or a pharmaceutically acceptable salt thereof, wherein: X is a moiety that binds to an ASGPR cell surface receptor (e.g., as described herein); n is 1 to 500; L is a linker (e.g., monovalent or multivalent, as described herein); m is 1 to 20; Y is a biomolecule that specifically binds an extracellular target molecule.
target binding conjugate is of formula (II’): (II’) or a prodrug thereof, or a salt thereof, wherein: n is 1 to 3; m is 1 to 3; X and Y are each independently as defined herein; each L 1 to L 6 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; and a, b, c, d, and e are each independently 1, 2, 3, 4, or 5. [0020]
the ASGPR binding compounds and conjugates and methods of this disclosure are described in greater detail below. Linkers (L) and moieties of interest (Y) which find use in the ASGPR binding compounds, and the biomolecule conjugates are also described.
ASGPR Ligands [0021] As summarized above, this disclosure provides a class of compounds including a ligand moiety that specifically binds to a cell surface ASGPR.
the ASGPR ligand moieties of this disclosure can be linked to a variety of moieties of interest without impacting the specific binding to, and function of, the cell surface ASGPR.
compounds of this disclosure can utilize the functions of cell surface ASGPRs in a biological system, e.g., for internalization and sequestration of a compound to the lysosome of a cell, and in certain embodiments subsequent lysosomal degradation.
the compounds of this disclosure find use in a variety of applications.
asialoglycoprotein receptor also known as the Ashwell Morell receptor, means the transmembrane glycoprotein receptor found primarily in hepatocytes which plays an important role in serum glycoprotein homeostasis by mediating the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N-acetylgalactosamine (GalNAc) residues.
ASGPR cycles between endosomes and the cell surface.
the ASGPR is Homo sapiens asialoglycoprotein receptor 1 (ASGR1) (see, e.g., NCBI Reference Sequence: NM_001197216).
a compound comprising such ASGPR binding moiety may bind to other receptors, for example, may bind with lower affinity as determined by, e.g., immunoassays or other assays known in the art.
X, or a compound as described herein comprising such X specifically binds to the cell surface ASGPR with an affinity that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the affinity when X or the compound or the conjugate bind to another cell surface receptor.
X or a compound as described herein comprising X specifically binds to ASGPR with an affinity (K d ) 20 mM or less.
affinity (K d ) is 10 mM or less, 1 mM or less, 100 uM or less, 10 uM or less, 1 uM or less, 100 nM or less, 10 nM or less, or 1 nM or less.
the terms “binds,” “binds to,” “specifically binds” or “specifically binds to” in this context are used interchangeably.
the ASGPR binding compounds of this disclosure include a moiety (X) that specifically binds to the cell surface receptor ASGPR.
the ASGPR binding compounds can be monovalent or multivalent (e.g., bivalent or trivalent or of higher valency), where a monovalent compound includes a single ASGPR ligand moiety, and a multivalent compound includes two or more such moieties.
the ASGPR binding moiety X is able to bind to a specific cell surface ASGPR, and direct (or target) the molecule to this receptor.
the ASGPR binding moiety X is capable of binding to the ASGPR and directing (or targeting) a compound or conjugate described herein for internalization and sequestration to the lysosome, and/or subsequent lysosomal degradation.
the ASGPR binding moiety X includes an amino sugar ring derivative of galactose (e.g., N-acetylgalactosamine, and analogs thereof), that is linked via a linking moiety to the 1, 6 or 2-position of the sugar ring.
the linking moiety can be of 1-10 atoms in length, such as 1-6, or 1- 5, 1-4, or 1-3 atoms in length.
the amino sugar ring derivative of galactose is linked via a linking moiety to an oxygen, sulfur, nitrogen or carbon atom at the 1-position of the ring.
the amino sugar ring derivative of galactose is linked via a linking moiety to an oxygen, sulfur, nitrogen or carbon atom the 6-position of the ring. In some embodiments, the amino sugar ring derivative of galactose is linked via a linking moiety to an oxygen, sulfur, nitrogen or carbon atom the 2-position of the ring. In certain embodiments, the amino sugar derivative of galactose is linked via a linking moiety to a heteroaryl group at the 1, 6 or 2 position of the ring. In certain embodiments, the amino sugar derivative of galactose is a bicyclic structure.
the ASGPR binding compounds is monovalent (e.g., in Formula (I), n is 1), such that the ASGPR binding compound includes a single ASGPR ligand moiety (X) that is linked to a moiety of interest (Y) via a linking moiety at the 1, 6, or 2-position of (X).
n is 1, and L comprises a linear linker having a backbone of 20 or more consecutive atoms covalently linking the ASGPR ligand (X) to Y via a linking moiety at any of the 1, 2 or 6-positions of X.
L is 20 to 100 consecutive atoms, such as 25 to 80, 25 to 60, or 25 to 50.
n is 1, and L comprises a backbone of 25 or more consecutive atoms covalently linking the ASGPR ligand (X) to Y.
the ASGPR binding compounds are multivalent (e.g., in Formula (I), n is 2 or more, such that the ASGPR binding compound includes two or more ASGPR ligand binding moieties (X) that are each covalently linked to a moiety of interest (Y) via a branched linker (e.g., L is a branched linker).
the ASGPR binding compound is divalent (e.g., n is 2 in Formula I).
each branch of the branched linker comprises a liner linker of 14 or more consecutive atoms to covalently link a linking moiety of each X to a branching point in the linker.
each branch of the linker includes 14 to 50 consecutive atoms, such as 14 to 40, 14 to 30, or 14 to 20 atoms.
each branch of the linker includes a linear linker of 20 or more consecutive atoms.
the linker comprises a linear linker of 12 or more consecutive atoms to covalently link the branching point of L to a moiety of interest (Y), such as 15 or more, 20 or more, 30 or more, or even more consecutive atoms to covalently link the branching point of L to Y.
Y moiety of interest
the ASGPR ligand moieties e.g., X n -L or (X-L) n of formula (I), and other formulae described herein
the bifunctional molecule specifically bind to ASGPR with an affinity (K d ) of 300 nM or less, such as 100 nM or less, 30 nM or less, 10 nM or less, 3 nM or less, or 1 nM or less.
K d affinity
the terms “binds,” “binds to,” “specifically binds” or “specifically binds to” in this context are used interchangeably.
a compound of formula (I): or a prodrug thereof, or a salt thereof, wherein: n is 1 to 500; m is 1 to 20; Y is a moiety of interest; L is a linker; and X is an asialoglycoprotein receptor (ASGPR) binding moiety of formula (II): wherein: R 1 is selected from –Z 1 –*, –H, –OH, optionally substituted (C 1 -C 6 )alkyl, –OCH 3 ,–OCH 2 CH CH, optionally substituted -S-(C 1 -C 6 )alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, and optionally substituted -S-heteroaryl; R 2 is selected from –Z 1 –*, –NHCOCH 3 , –NHCOCF 3 , –NHCOCH 2 CF 3 ,
Z 1 is a linking moiety selected from -Z 11 -A 1 - and -A 2 -; and -A 1 - and - A 2 - are optionally substituted heterocyclylene; or -A 2 - is optionally substituted isoxazolyl.
R 6 is -OR, optionally substituted (C 1 -C 6 )alkyl, –OC(O)-optionally substituted heteroaryl, -C(O)NH-optionally substituted heteroaryl, -NR xx R yy , optionally substituted aryl, optionally substituted heteroaryl–NHCOR, or –NRCOR, provided the heteroaryl is other than triazole; where each R is independently optionally substituted (C 1 -C 6 )alkyl, optionally substituted aryl, or optionally substituted heteroaryl; R xx and R yy are independently H, optionally substituted (C 1 -C 6 )alkyl, or R xx and R yy can cyclize to form an optionally substituted heterocyclyl.
R 1 is optionally substituted C 2-6 alkyl, optionally substituted -S-(C 1 - C 6 )alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, or optionally substituted -S-heteroaryl.
at least one R 21 is -COR or optionally substituted heteroaryl.
n is 1, 2, or 3; and m is 1-3.
R 6 is -OR, optionally substituted (C 1 -C 6 )alkyl, –OC(O)-optionally substituted heteroaryl, - C(O)NH-optionally substituted heteroaryl, -NR xx R yy , optionally substituted aryl, optionally substituted heteroaryl–NHCOR, or –NRCOR, provided the heteroaryl is other than triazole; where each R is independently optionally substituted (C 1 -C 6 )alkyl, optionally substituted aryl, or optionally substituted heteroaryl; R xx and R yy are independently H, optionally substituted (C 1 -C 6 )alkyl, or R xx and R yy can cyclize to form an optionally substituted heterocyclyl; D) R 1 is optionally substituted C 2-6 alkyl, optionally substituted -S-(C 1 -C 6 )alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substitute
n is 1, 2, or 3; and m is 1-3.
X is of formula (a-II): (a-II).
X is of formula (a-II), R 1 is n-propyl; and R 2 is -Z 1 -*.
Z 1 is a linking moiety selected from -Z 11 -A 1 - and -A 2 -; and -A 1 - and - A 2 - are optionally substituted heterocyclylene.
-L-Y comprises: monocyclic heteroaryl.
-L-Y comprises:
R 6 is -OR, optionally substituted (C 1 -C 6 )alkyl, –OC(O)-optionally substituted heteroaryl, -C(O)NH-optionally substituted heteroaryl, -NR xx R yy , optionally substituted aryl, or optionally substituted heteroaryl, provided the heteroaryl is other than triazole; where R is optionally substituted (C 1 -C 6 )alkyl, optionally substituted aryl, or optionally substituted heteroaryl; R xx and R yy are independently H, optionally substituted (C 1 -C 6 )alkyl, or R xx and R yy can cyclize to form an optionally substituted heterocyclyl.
R 6 is -O-(C 1 -C 6 )alkyl, optionally substituted heterocyclyl, or -O-aryl.
R 1 is optionally substituted C 2-6 alkyl, optionally substituted -S-(C 1 - C 6 )alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-aryl, or optionally substituted -S-heteroaryl
R 1 is optionally substituted C 2-6 alkyl.
at least one R 21 is -COR or optionally substituted heteroaryl.
Y is an antibody or antibody fragment.
n is 1, 2, or 3; and m is 1-3.
the chemoselective ligation group comprises a carboxylic acid or active ester, maleimide, isocyanate or isothiocyanate, alkyl halide, alkyl tosylate, aldehyde, haloacetamide or alpha-leaving group acetamide, 2-sulfonylpyridine, diazirine, sulfonyl halide or vinyl sulfone, hydrazide, hydrazino, hydroxylamino, pyridyl disulfide, (HIPS) hydrazinyl-indolyl group, or (aza-HIPS) hydrazinyl-pyrrolo-pyridinyl group, alkyne or cyclooctyne, azide, or amine.
the chemoselective ligation group comprises a carboxylic acid or active ester, maleimide, iso
A) Z 1 is a linking moiety selected from -Z 11 -A 1 - and -A 2 -; and -A 1 - and -A 2 - are optionally substituted heterocyclylene; or -A 2 - is optionally substituted isoxazolyl;
B) -L-Y comprises: C) R 6 is -OR, optionally substituted (C 1 -C 6 )alkyl, –OC(O)-optionally substituted heteroaryl, - C(O)NH-optionally substituted heteroaryl, -NR xx R yy , optionally substituted aryl, optionally substituted heteroaryl–NHCOR, or –NRCOR, provided the heteroaryl is other than triazole; where each R is independently optionally substituted (C 1 -C 6 )alkyl, optionally substituted aryl, or optionally substituted heteroaryl; R xx and R yyy
the compound of formula (II) is represented by formula (a-II): (a-II).
R 1 is –Z 1 –*, –H, or (C 1 -C 6 )alkyl.
R 2 is –Z 1 –* or –NHCOCH 3 .
R 3 and R 4 are each –H.
R 6 is –OH, –OC(O)R, -NR xx R yy , or aryl; R is (C 1 -C 6 )alkyl; and R xx and R yy cyclize to form an optionally substituted heterocyclyl.
* represents a point of connection of Z 1 to the linker (L).
R 1 is –Z 1 –*.
R 2 is –Z 1 –*.
L comprises of 10 to 60 consecutive linear or branched chain atoms.
L is of formula (IIb’): wherein: each L 1 to L 6 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y.
n is 1-3. In some embodiments of formula (IIb’), n is 1. In some embodiments of formula (IIb’), n is 2. In some embodiments of formula (IIb’), n is 3.
L is of formula (IIb’): wherein: n is 1, 2, or 3; each L 1 to L 6 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y.
a compound of formula (II’): or a prodrug thereof, or a salt thereof, wherein: n is 1 to 3; m is 1 to 20; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; each L 1 to L 6 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; Y is as defined herein; each X is an asialoglycoprotein receptor (ASGPR) binding moiety of formula (a-II): (a-II) wherein: R 1 is selected from –Z 1 –*, –H, –OH, optionally substituted (C 1 -C 6 )alkyl, –OCH 3 , –OCH 2 CH CH, optionally substituted -S-(C 1 -C 6 )alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted -S-
R 1 is selected from –Z 1
each L 1 to L 5 independently comprises one or more linking moieties independently selected from –C 1-20 -alkylene–, –NHC(O)-C 1-6 -alkylene–, –C(O)NH-C 1-6 -alkylene–, –NH- C 1-6 -alkylene–, –NHC(O)NH-C 1-6 -alkylene–, –NHC(S)NH-C 1-6 -alkylene–, –C 1-6 -alkylene–NHC(O)-, – C 1-6 -alkylene–C(O)NH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–NHC(O)NH-, –C 1-6 -alkylene–NHC(S)NH- , -O(CH 2 ) p –, –(OCH 2 CH 2 ) p –, –NHC(O)–, –, –NHC(S)
each R 16 is independently (C 1 -C 6 )alkyl or monocyclic heteroaryl. In some embodiments, each R 16 is independently R”. [0074] In some embodiments, each L 1 to L 5 is independently selected from –C 1-20 -alkylene–, –NHC(O)-C 1-6 -alkylene–, –C(O)NH-C 1-6 -alkylene–, –NH-C 1-6 -alkylene–, –NHC(O)NH-C 1-6 -alkylene–, –NHC(S)NH-C 1-6 -alkylene–, –C 1-6 -alkylene–NHC(O)-, –C 1-6 -alkylene–C(O)NH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–NHC(O)NH-, –C 1-6 -alkylene–NH-,
each L 1 to L 5 is independently selected from –C 1-20 -alkylene–, –NHC(O)-C 1-6 -alkylene–, –C(O)NH-C 1-6 -alkylene–, –NH-C 1-6 -alkylene–, –NHC(O)NH-C 1-6 -alkylene–, –NHC(S)NH-C 1-6 -alkylene–, –C 1-6 -alkylene–NHC(O)-, –C 1-6 -alkylene–C(O)NH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–NHC(O)NH-, –C 1-6 -alkylene–NHC(S)NH-, -O(CH 2 ) p –, –(OCH 2 CH 2 ) p –, –NHC(O)–, –C(O)NH– –NH—NHC(S)NH-
n is 1. [0078] In some embodiments of formula (II’), n is 2. [0079] In some embodiments of formula (II’), n is 3. [0080] In some embodiments, at least one L 1 is –C 1-20 -alkylene– optionally substituted with one to five halo. [0081] In some embodiments, at least one L 1 is -CF 2 CH 2 -. [0082] In some embodiments, at least one L 2 is –(OCH 2 CH 2 ) p –. [0083] In some embodiments, p is 2-3.
At least one L 3 is NHCONH-C 1-6 -alkylene–.
at least one L 4 is –C 1-6 -alkylene–NHCONH-.
at least one L 5 is –(OCH 2 CH 2 ) p –.
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Iia): (Iia) wherein R 2 , R 3 , R 4 , R 6 and Z 1 are as defined herein.
R 6 is selected from –OH, –OC(O)R, and -C(O)NHR; and R 2 is selected from –NHCOCH 3 , –NHCOCF 3 , and –NHCOCH 2 CF 3 .
Z 1 is in a beta configuration, and can be described by formula (Iia-1): (Iia-1).
Z 1 is in an alpha configuration, and can be described by formula (Iia-2) (Iia-2). [0092] In certain embodiments of formula (Iia), (Iia-1) or (Iia-2), Z 1 is -Z 11 -A 1 -, wherein A 1 - is optionally substituted arylene or optionally substituted heteroarylene. In certain embodiments, A 1 is an optionally substituted heteroarylene. In certain embodiments, the heteroarylene is a 5 or 6-membered heteroarylene. In certain embodiments, the heteroarylene is a 5-membered heteroarylene. In certain embodiments, the 5-membered heteroarylene is a triazole.
the triazole is a 1,2,3- triazole moiety.
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (IIIa) or (IIIb): (IIIa) (IIIb) wherein: -Z 11 - is -O-, -S-, -N(R 21 )-, or -C(R 22 ) 2 -, where each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 -C 6 )alkyl, and R 21 is H or optionally substituted (C 1 -C 6 )alkyl; and -A 1 - is arylene, substituted arylene, heteroarylene, or substituted heteroarylene.
Z 11 is -S-.
Z 11 is -C(R 22 ) 2 -.
Z 11 is -CH 2 -.
Z 11 is -C(R 22 ) 2 , where at least one R 22 is H. In certain embodiments, both R 22 are H.
Z 11 is -O-.
Z 11 is -S-.
Z 11 is -N(R 21 ), where R 21 is H or (C 1- C 3 )alkyl.
-A 1 - is triazole.
Z 1 is -C(R 22 ) 2 -triazole-. In certain embodiments, Z 1 is: * * . In certain embodiments, Z 1 is: . [0099] In certain embodiments of formula (Iia), (Iia-1) or (Iia-2), Z 1 is Z 11 . In certain embodiments, Z 11 is -C(R 22 ) 2 . In certain embodiments, at least one R 22 is H. In certain embodiments, both R 22 are H, and Z 11 is -CH 2 -. In certain cases Z 11 is -O-. In certain embodiments, Z 11 is -S-.
Z 11 is -N(R 21 ), where R 21 is H or (C 1 -C 3 )alkyl. [0100] In certain embodiments of formula (Iia), (Iia-1) or (Iia-2), Z 1 is monocyclic 5 or 6-membered heteroaryl or aryl. In certain embodiments, Z 1 is . In certain embodiments, Z 1 .
Z 1 is selected from -O-, -S-, wherein: X 1 is O or S; t is 0 or 1; R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl); and each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
X 1 is O or S
t is 0 or 1
R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl)
each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
Z 1 is optionally substituted (C 1 - C 6 )alkyl.
the alkyl is methyl.
the alkyl is ethyl.
the alkyl is propyl.
the alkyl is butyl.
the alkyl is pentyl.
the alkyl is hexyl.
the ASGPR binding moiety (X) of formula (Iia-1) is selected from one of the following structures:
Z 1 is in a beta configuration and X is of formula (IIIb-2): (IIIb-2) wherein: -A 1 - is arylene, substituted arylene, heteroarylene, or substituted heteroarylene.
a 1 is a triazole.
X is of formula (X A -4).
Z 1 is in a alpha configuration at the 1-position carbon of the galactosamine ring.
Z 1 is S, and each X is of formula (X A -1).
each X is of formula (X A -2). In some embodiments of formula (Iia-1), each X is of formula (X A -3). In some embodiments of formula (Iia-1), each X is of formula (X A -4). In some embodiments of formula (Iia-1), each X is of formula (X A -5). [0107] In certain embodiments, the compound of formula (Iia-2) is selected from one of the following structures: [0108] In some embodiments of formula (Iia-2), each X is of formula (X B -1). [0109] In some embodiments of formula (Iia-2), each X is of formula (X B -2).
each X is of formula (X B -3). [0111] In some embodiments of formula (Iia-2), each X is of formula (X B -4). [0112] In some embodiments of formula (Iia-2), Z 1 is in an alpha configuration and X is of formula (IIIb-1): wherein -A 1 - is arylene, substituted arylene, heteroarylene, or substituted heteroarylene. [0113] In certain embodiments of formula (IIIb-1), A 1 is an optionally substituted heteroarylene. In certain embodiments, the heteroarylene is a 5 or 6-membered heteroarylene. In certain embodiments, the heteroarylene is a 5-membered heteroarylene.
the 5-membered heteroarylene is a triazole. In certain embodiments, the triazole is a 1,2,3-triazole moiety.
the X of formula (IIIb-1) is selected from one of the following structures: [0115] In some embodiments of formula (IIIb-1), each X is of formula (X C -1). [0116] In some embodiments of formula (IIIb-1), each X is of formula (X C -2). [0117] Exemplary ligand moieties that bind ASGPR, and synthons thereof, which can be utilized in the compounds of this disclosure are shown in Tables 1-5. In certain embodiments, the compound of formula (Iia) is a compound shown in Table 1:
Z 1 is in the alpha configuration such that the ASGPR binding moiety X1-X5.1 is derived from formula (Iia-2): 2-linked ASGPR ligand moieties [0119]
the ASGPR binding moiety (X) is linked via the 2-postion of the sugar analog.
the ASGPR binding moiety (X) has a reduced ring carbon at the 1- position relative to a galactosamine derived sugar.
the ASGPR binding moiety (X) of the bifunctional molecules of this disclosure is described by formula (Iib): wherein R 1 , R 3 , R 4 , R 6 , R 11 , and Z 1 are as defined herein.
the ASGPR binding moiety (X) of the compounds of this disclosure are described by formula (Iib’): wherein R 3 -R 4 , R 6 , and Z 1 are as defined herein.
the ASGPR binding moiety (X) of the compounds of this disclosure are described by formula (Iva): wherein R 1 , R 11 , and Z 1 are as defined herein.
Z1 is selected from optionally substituted –(C(R 22 ) 2 ) q -heteroarylene, , wherein q is 0 or 1.
Z 1 is optionally substituted – (C(R 22 ) 2 ) q -triazole wherein q is 0 or 1.
R is H, or C (1-3) -alkyl.
Z 1 is -NR 23 CO-, wherein R 23 is H or C (1-3) -alkyl.
R 23 is H or C (1-3) -alkyl.
Z 1 is selected from optionally substituted –(C(R 22 ) 2 ) q -heteroaryl, , wherein q is 0 or 1.
Z 1 is optionally substituted –(C(R 22 ) 2 ) q -triazole wherein q is 0 or 1.
Z 1 is selected from -O-, - wherein: X 1 is O or S; t is 0 or 1; R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl); and each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
X 1 is O or S
t is 0 or 1
R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl)
each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
the compound of formula of formulae (Iib), (Iib’) or (Iva) is selected from one of the following structures: , wherein R 1A is independently H or (C 1 - 3 )alkyl.
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ivb) or (Ivc): OH OH R 11 R 11 HO O HO O HO R 1 HO R 1 Z 11 A 1 A 2 * * (Ivb) (Ivc), wherein: -Z 11 - is -O-, -S-, -N(R 21 )-, or -C(R 22 ) 2 ; -A 1 - and -A 2 - are optionally substituted arylene or optionally substituted heteroarylene; each R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl; and each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
halogen e.g., F
R 1 is H.
R 2 is –Z 1 –*
R 11 is a group of the formula -CH 2 O- that forms a bridge (i.e., is cyclically linked) to the 1-position carbon atom on the sugar ring.
–Z 1 –* or –Z 1 –L- comprises [0137]
R 11 is H and the compound is of Table 2:
the compound of formula (Iib) is a compound shown in Table 3:
the compound of formula (Iib), the configuration at C1 (i.e., R 1 ) is alpha.
the compound of formula (Iib), the configuration at C1 (i.e., R 1 ) is beta.
the compound of formula (Id’) is a compound shown in Table 4: [0140]
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ivb-1) or (Ivc-1): (Ivb-1) (Ivc-1), wherein R 11 is the bridging moiety that connects the 5-position carbon to the 1-position carbon.
Z 11 is -C(R 22 ) 2 .
at least one R 22 is H.
both R 22 are H.
Z 11 is -O-.
Z 11 is -S-. In certain embodiments, Z 11 is -N(R 21 ), where R 21 is H or (C 1 -C 3 )alkyl. [0142] In certain embodiments of formulae (Ivb), (Ivc), (Ivb-1) or (Ivc-1), -A 1 - and -A 2 - are each independently an optionally substituted heteroarylene.
the heteroarylene is a 5 or 6-membered heteroarylene. In certain embodiments, the heteroarylene is a 5-membered heteroarylene. In certain embodiments, the heteroarylene is a 6-membered heteroarylene.
the A 1 ring is a 5-membered heteroarylene selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, and furan.
the A 1 ring is a 6-membered heteroarylene selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine.
the A 1 ring is triazole.
the A 1 ring is pyridine.
the A 1 ring is pyrimidine.
the A 1 ring is thiadiazole. In certain embodiments, the A 1 ring is a 5 or 6- membered arylene or heteroarylene that is further substituted with one or more substituents. In certain embodiments, the A 1 ring is further substituted with one or more substituents selected from halogen, (C 1- C 6 )alkyl and substituted (C 1- C 6 )alkyl (e.g., CF 3 ).
the A 2 ring is a 5-membered heteroarylene selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, and furan.
the A 2 ring is a 6-membered heteroarylene selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine.
the A 2 ring is triazole.
the A 2 ring is pyridine.
the A 2 ring is pyrimidine.
the A 2 ring is thiadiazole. In certain embodiments, the A 2 ring is a 5 or 6- membered arylene or heteroarylene that is further substituted with one or more substituents. In certain embodiments, the A 2 ring is further substituted with one or more substituents selected from halogen, (C 1- C 6 )alkyl and substituted (C 1- C 6 )alkyl (e.g., CF 3 ).
-Z 11 -A 1 - is a monocyclic 5 or 6- memebered heteroarylene of one of the following structures: [0146] In certain embodiments of formulae (Ivc) or (Ivc-1), -A 2 - is a monocyclic 5 or 6-membered heteroarylene of the following structure: . [0147] It is understood that a variety of substituents can be utilized to connect a particular -Z 11 -A 1 - group to an adjacent linker.
-Z 11 -A 1 - is a monocyclic 5 or 6-membered heteroarylene that is attached to a linking moiety as shown in one of the following structures: [0148] In certain embodiments of formulae (Ivc) or (Ivc-1), -Z 11 -A 1 - is a monocyclic 5 or 6- membered heteroarylene that is attached to a linking moiety as shown in one of the following structures: .
R 1 is H, such that the compound of formula of formulae (Iib), or (Iva)-(Ivc) has no non-hydrogen substituents at the 1-position of the sugar ring.
the compound of formula (Iib) is of any one of formulae (Ivd)-(Ivg): (Ivf), and (Ivg), wherein the A 1 and A 2 rings, R 6 , R 4 , R 3 , R 11 , and R 21 are as defined herein.
the A 1 ring is a 5 or 6-membered arylene or heteroarylene.
the A 1 ring is a 5-membered heteroarylene selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, imidazole, and furan.
the A 1 ring is a 6-membered heteroarylene selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine.
the A 1 ring is triazole.
the A 1 ring is pyridine In certain embodiments the A 1 ring is pyrimidine In certain embodiments, the A 1 ring is thiadiazole. In certain embodiments, the A 1 ring is pyrazine. In certain embodiments, the A 1 ring is a 5 or 6-membered arylene or heteroarylene that is further substituted with one or more substituents. In certain embodiments, the A 1 ring is further substituted with one or more substituents selected from halogen, (C 1- C 6 )alkyl and substituted (C 1- C 6 )alkyl (e.g., CF 3 ).
the A 2 ring is a 5 or 6-membered arylene or heteroarylene.
the A 2 ring is a 5-membered heteroarylene selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, and furan.
the A 2 ring is a 6-membered heteroarylene selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine.
the A 2 ring is triazole.
the A 2 ring is pyridine. In certain embodiments, the A 2 ring is pyrimidine. In certain embodiments, the A 2 ring is thiadiazole. In certain embodiments, the A 2 ring is a 5 or 6-membered arylene or heteroarylene that is further substituted with one or more substituents. In certain embodiments, the A 2 ring is further substituted with one or more substituents selected from halogen, (C 1- C 6 )alkyl and substituted (C 1- C 6 )alkyl (e.g., CF 3 ). [0153] In some embodiments of any one of formulae (Ivd)-(Ivg),the A 1 or A 2 ring is absent.
the A 1 or A 2 ring is phenylene or substituted phenylene.
the A 2 ring is a 5 or 6-membered heteroarylene. In certain cases of formula (Ivd), the A 2 ring is a 5-membered heteroarylene. In certain embodiments of formula (Ivd), the A 2 ring is triazole. In certain embodiments of (Ivd), the A 2 ring is absent.
the A 1 ring is a 5 or 6-membered heteroarylene and R 21 is H.
the A ring is triazole. In certain cases of formula (Ive), the A 1 ring is pyridine. In certain cases of formula (Ive), the A 1 ring is pyrimidine. In certain cases of formula (Ive), the A 1 ring is thiadiazole. In some embodiments of formula (Ive), the A 1 ring is absent and R 21 is H or optionally substituted acyl. In certain embodiments, R 21 is -COCH 3 . In certain embodiments, R 21 is H. [0157] In some embodiments of formula (Ivf), the A 1 ring is a 5 or 6-membered heteroarylene.
the A 1 ring is a 5-membered heteroarylene. In certain embodiments of formula (Ivf), the A 1 ring is triazole. In certain embodiments of (Ivf), the A 1 ring is absent. [0158] In some embodiments of formula (Ivg), the A 2 ring is a 5 or 6-membered heteroarylene. In certain cases of formula (Ivg), the A 2 ring is a 5-membered heteroarylene. In certain embodiments of formula (Ivg), the A 2 ring is triazole. In certain embodiments of (Ivg), the A 2 ring is absent. [0159] In certain embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by any one of formulae (Ivh)-(Ivk):
R 6 , R 4 , R 3 , and R 21 are as defined herein; Y 1 -Y 3 are each independently N or CR 25 ; and R 24 and R 25 are each independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen.
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by any one of formulae (Ivl)-(Ivm): wherein: R 6 , R 4 , R 3 , and R 21 are as defined herein; Y 1 -Y 3 are each independently N or CR 25 ; Y 4 is N or CR 24 ; Y 5 is S, O, or NH; and R 24 and R 25 are each independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen. [0161] In some embodiments of formula (Ivi) at least one of Y 1 to Y 3 is N.
Y 1 to Y 3 are N.
Y 1 and Y 4 are N.
Y 1 and Y 3 are N and Y 2 is CR 25 .
Y 1 and Y 2 are N and Y 3 is CR 25 .
Y 1 and Y 2 are CR 25 and Y 3 is N.
R 6 is H.
R 4 and R 3 are each H. In certain embodiments, at least one of R 4 -R 3 is a promoiety. In certain embodiments, R 4 and R 3 are cyclically linked to form a promoiety (e.g., as described herein).
the compound of formula (Ivi) is of formula (Ivi-1): [0170] wherein R 24 and R 25 are independently selected from H, halogen, (C 1- C 6 )alkyl and substituted (C 1- C 6 )alkyl (e.g., CF 3 ).
R 25 is H.
R 25 is C (1-3) -alkyl, or C (1-3) -fluoroalkyl.
the fluoroalkyl is CF 3 .
R 24 is H.
R 24 is C (1-3) -alkyl, or C (1-3) - fluoroalkyl. In certain embodiments, the fluoroalkyl is CF 3 .
the compound of formula (Ivi-1) is of formula (X D ): [0172]
the compound of formula (Ivk-1) is of formula (X E ): [0173]
the compound of formula (Ivl) is of formula (Ivl-1): wherein: R 6 , R 4 , R 3 , and R 21 are as defined herein; Y 1 -Y 4 are each independently N or CR 25 ; Y 5 is S, O, or NH; and each R 25 is independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen.
each R 25 is H.
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by one of the following structures: .
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by one of the following structures: [0177]
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by one of the following structures: * [0178]
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by one of the following structures: * [0179]
R 1 R 3 , R 4 , and R 11 are H
R 6 is OH: wherein Z 1 is -NH-, -CH 2 -, -S- or -O-.
R 3 , R 4 are H, and R 6 is OH: wherein Z 1 is -NH-, -CH 2 -, -S-, -O-, triazole, 6-linked ASGPR ligand moieties
the ASGPR binding moiety (X) is linked via the 6-postion of the sugar analog.
the ASGPR binding moiety (X) has a reduced ring carbon at the 1- position relative to a galactosamine derived sugar.
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Iic): * wherein R 1 -R 4 and Z 1 are as defined herein.
Z 1 is selected from -O-, -S-, -CONR 21 -, and optionally substituted –(C(R 22 ) 2 ) q - heteroarylene, wherein q is 0 or 1.
Z 1 is -O-.
Z 1 is optionally substituted –(C(R 22 ) 2 ) q -triazole wherein q is 0 or 1.
Z 1 is -Z 11 -A 1 -, wherein -A 1 - is or optionally substituted -A 1 - or optionally substituted arylene. In certain embodiments, -A 1 - is an optionally substituted heteroarylene. In certain embodiments, the heteroarylene is a 5 or 6-membered heteroarylene. In certain embodiments, the heteroarylene is a 5-membered heteroarylene. In certain embodiments, the 5-membered heteroarylene is a triazole. In certain embodiments, the triazole is a 1,2,3-triazole moiety. In certain embodiments, Z 11 is -C(R 22 ) 2 .
At least one R 22 is H. In certain embodiments, both R 22 are H. In certain cases Z 11 is -O-. In certain embodiments, Z 11 is -S-. In certain other cases, Z 11 is -N(R 21 ), where R 21 is H or (C 1 -C 3 )alkyl. In certain embodiments, Z 1 is -C(R 22 ) 2 - * triazole-. In certain embodiments, Z 1 is: . [0186] In certain embodiments of formula (Iic), Z 1 is Z 11 . In certain embodiments, Z 11 is -C(R 22 ) 2 . In certain embodiments, at least one R 22 is H.
both R 22 are H, and Z 11 is -CH 2 -. In certain cases Z 11 is -O-. In certain embodiments, Z 11 is -S-. In certain other cases, Z 11 is -N(R 21 ), where R 21 is H or (C 1- C 3 )alkyl. [0187] In certain embodiments of formula (Iic), Z 1 is monocyclic 5 or 6-membered heteroarylene or arylene.
Z1 is selected from -O-, -S-, -C(R22)2-, -N(R21) - wherein: X 1 is O or S; t is 0 or 1; R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl); and each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
the compound of formula (Iic) is the following structure: .
the compound of formula (Iic) is the following structure: .
R 11 is H and the compound is of Table 5:
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Iid): wherein: R 6 , R 4 , R 3 and Z 1 are as defined herein; Y 6 and Y 5 are each independently selected from -O-, -S-, NR 21 -, and -C(R 22 ) 2 ; R 21 is selected from H, optionally substituted (C 1 -C 6 )alkyl, and -C(O)R 22 ; each R 22 is independently selected from H, halogen and optionally substituted (C 1 -C 6 )alkyl; and ring B is a 5 or 6-membered optionally substituted cyclic group.
Y 5 is connected to the sugar ring via an alpha configuration. In some embodiments of formula (Iid), Y 5 is connected to the sugar ring via a beta configuration.
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Iid’): wherein: R 6 , R 4 , R 3 and Z 1 are as defined herein; Y 5 and Y 6 are each independently selected from -O-, -S-, NR 21 -, and -C(R 22 ) 2 ; R 21 is selected from H, optionally substituted (C 1 -C 6 )alkyl, and -C(O)R 22 ; each R 22 is independently selected from H, halogen and optionally substituted (C 1 -C 6 )alkyl; and ring B is a 5 or 6-membered optionally substituted cyclic group.
Y 5 is O. In certain embodiments, Y 5 is S. In certain embodiments, Y 5 is -NR 21 -. In certain embodiments, Y 5 is -C(R 22 ) 2 and each R 22 is H. [0195] In some embodiments of formula (Iid)-(Iid’) Y 6 is -NR 21 - where R 21 is H. In certain embodiments, Y 6 is -NR 21 - where R 21 is -C(O)R 22 . In certain embodiments, R 22 is methyl.
the B ring is a 5 or 6-membered heterocycle. In certain embodiments, the B ring is a 5-membered heterocycle. In certain embodiments, the B ring is a 6- membered heterocycle.
Z 1 is Z 11 , where Z 11 is selected from -O-, -S-, NR 21 -, and -C(R 22 ) 2 . In certain embodiments, Z 1 is -O-. In certain embodiments, Z 1 is -S-. In certain embodiments, Z 1 is NR 21 where R 21 is H.
Z 1 is -C(R 22 ) 2 where each R 22 is H. [0198] In some embodiments of formula (Iid)-(Iid’) Z 1 is optionally substituted Z 11 -heteroarylene or optionally substituted Z 11 -arylene. In some embodiments, Z 1 is CH 2 -heteroarylene or CH 2 -arylene. In some embodiments of formula (Iid)-(Iid’) Z 1 is optionally substituted amide. In some embodiments of formula (Iid)-(Iid’) Z 1 is optionally substituted sulfonamide.
Z 1 is optionally substituted urea or optionally substituted thiourea.
the compound of formula (Iid)-(Iid’) has one of the following structures: , . [0200]
R 6 is OH.
R 6 is -OC(O)R.
R 6 is -C(O)NHR, where R is an optionally substituted alkyl.
R terminates in an alkenyl or an alkynyl group.
R 6 is optionally substituted triazole.
the triazole is of the following structure: .
R 2 is -NHCOCH 3 .
R 2 is –NHCOCF 3 .
R 2 is –NHCOCH 2 CF 3 .
R 2 is – OH.
R 2 is an optionally substituted triazole.
the triazole in of the following structure: .
R 6 or R 2 when R 6 or R 2 is a substituted triazole, the triazole is a 1,2,3-trizole, and the substituent is at the 4 or 5-position.
the substituent on the triazole moiety includes but is not limited to, an optionally substituted (C 1 - 6 )alkyl, optionally substituted (C 1 - 6 )alkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkaryl, and an optionally substituted alkyheteroaryl. It will be understood that any convenient substituent can be included in the triazole moiety, see, e.g., triazole moieties disclosed in Mamidayala et al, J.
Z 1 , Z 11 , and Z 11 -Ar linking moieties can be considered part of the X group of formula (I).
-Z 1 - can be linked to an - L 1 - moiety (e.g., of the linker as described herein) via a variety of bonds and linking moieties, depending on the method of preparation.
the subject compounds comprise a -Z 1 -L 1 - moiety selected from: wherein each R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl; each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 -C 6 )alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 0 to 6. [0204] In some embodiments, the subject compounds comprise a -Z 1 -L 1 - moiety selected from:
each R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl; each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 -C 6 )alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 1 to 6.
the Z 1 -L 1 - group is , and o is 1 or 2.
the Z 1 -L 1 - group 22 each R is H, and p is 1 or 2.
the Z 1 -L 1 - group is . [0210] In certain embodiments, the Z 1 -L 1 - group are each independently 1-3. [0211] In certain embodiments, the Z 1 -L 1 - group i [0212] In certain embodiments, the Z 1 -L 1 - group i are each independently is 1-3. [0213] In certain embodiments, the Z 1 -L 1 - group is , where x is 0-3. [0214] In certain embodiments, the Z 1 -L 1 - group [0215] In certain embodiments, the Z 1 -L 1 - group is , where R 21 is H, and z is 1-4.
the Z 1 -L 1 - group is , where R 21 is H, and z1 is 1-4. [0217] In certain embodiments, the Z 1 -L 1 - group is , where each R 22 is H, and q is 0-3. In certain embodiments, the Z 1 -L 1 - group is , where each R 22 is H, and q is 1-3. [0218] In certain embodiments, the Z 1 -L 1 - group is , where q is 1-3.
the subject compounds comprise a -Z 1 -L- group selected from: [0220]
the Z 1 -L 1 - group is q O , where q is 1-3. In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. [0221] In certain embodiments, the Z 1 -L 1 - group i . [0222] In certain embodiments, the Z 1 -L 1 - group i . [0223] In certain embodiments, the Z 1 -L 1 - group is . [0224] In certain embodiments, -Z 1 -L 1 - comprises an optionally substituted -NH-heteroarylene-.
the heteroarylene is a triazole. In certain embodiments, the heteroarylene is pyridine. In certain embodiments, the heteroarylene is pyrimidine. In certain embodiments, the heteroarylene is thiadiazole. [0225] In certain embodiments, the -Z 1 -L 1 - comprises a group selected from: wherein each R 21 is independently selected from H, optionally substituted (C 1 -C 6 )alkyl, and optionally substituted acyl; and R 24 and R 25 are each independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen.
the -Z 1 -L 1 - comprises a group selected from: wherein R 24 and R 25 are each independently selected from H, optionally substituted C (1-6) - alkyl, optionally substituted fluoroalkyl, and halogen; and each R 21 is independently selected from H, optionally substituted (C 1 -C 6 )alkyl, and optionally substituted acyl.
R 21 is H.
R 24 is C (1-3) -alkyl, or C (1-3) - fluoroalkyl.
the fluoroalkyl is CF 3 .
R 25 is C (1-3) -alkyl, or C (1-3) -fluoroalkyl.
the fluoroalkyl is CF 3 .
- It is understood that a variety of substituents and chemistries can be utilized to connect a particular X ligand moiety (e.g., as described herein) to an adjacent linker.
a linking moiety of the linker comprises a triazole that derives from a Click chemistry conjugation.
the ASGPR ligand moiety (X) is attached to a linking moiety as shown in one of the following structures: , , [0230]
R 1 R 3 , R 4 , and R 11 are H, and R 6 is OH: wherein Z 1 is triazole, -NH-heteroaryl (e.g., -NH- attached to pyridine, pyrazine, or pyrimidine) , -NH-, - O-, or -CH 2 - , and/or Z 1 is attached to a linking moiety as shown in one of the following structures: [0231]
R 1 R 3 , R 4 , and R 11 are H, and R 6 is OH: wherein Z 1 is attached to a linking moiety as shown in one of the following structures: Additional Exemplary ASGPR Binding Moieties [0232]
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ib): wherein R 1 , R 2 , R 4 , R 5 and Z 1 are as defined herein.
R 1 is selected from –OH, –OC(O)R, and -C(O)NHR; and R 2 is selected from –NHCOCH 3 , –NHCOCF 3 , and – NHCOCH 2 CF 3
Z 1 is in an alpha configuration .
Z 1 is in an alpha configuration and X is of the following formula: .
Z 1 is in an alpha configuration and X is of the following formula: .
Z 1 is in an alpha configuration and X is of the following formula: .
Z 1 is in an alpha configuration and X is of the following formula: .
Z 1 is in an alpha configuration and X is of the following formula: .
Z 1 is in a beta configuration .
Z 1 is in a beta configuration and X is of the following formula: .
Z 1 is in a beta configuration and X is of the following formula: .
Z 1 is in a beta configuration and X is of the following formula: .
Z 1 is in a beta configuration and X is of the following formula: .
Z 1 is in a beta configuration and X is of the following formula: .
Z 1 is in a beta configuration and X is of the following formula: .
Z 1 is Z 11 -Ar , wherein Ar is or optionally substituted heteroaryl or optionally substituted aryl.
Ar is an optionally substituted heteroaryl.
the heteroaryl is a 5 or 6-membered heteroaryl.
the heteroaryl is a 5-membered heteroaryl.
the 5-membered heteroaryl is a triazole.
the triazole is a 1,2,3-triazole moiety.
Z 11 is -C(R 22 ) 2 .
at least one R 22 is H.
both R 22 are H.
Z 11 is -O-.
Z 11 is -S-.
Z 11 is -NR 21 , where R 21 is H or (C 1-3 )alkyl.
Z 1 is -C(R 22 ) 2 -triazole-. In certain * * embodiments, Z 1 is: . In certain embodiments, Z 1 is: . [0250] In certain embodiments of formula (Ib), Z 1 is Z 11 . In certain embodiments, Z 11 is -C(R 22 ) 2 . In certain embodiments, at least one R 22 is H. In certain embodiments, both R 22 are H, and Z 11 is -CH 2 -. In certain cases Z 11 is -O-. In certain embodiments, Z 11 is -S-.
Z 11 is -NR 21 , where R 21 is H or (C1-3)alkyl.
R 21 is H or (C1-3)alkyl.
Z 1 is monocyclic 5 or 6-membered heteroaryl or aryl. In certain embodiments, Z 1 is .
Z 1 is selected from -O-, -S-, -C(R 22 ) 2 -, -NR 21 -, - wherein: X 1 is O or S; t is 0 or 1; R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl); and each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
X 1 is O or S
t is 0 or 1
R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl)
each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
Z 1 is optionally substituted (C 1 -C 6 )alkyl.
the alkyl is methyl.
the alkyl is ethyl.
the alkyl is propyl.
the alkyl is butyl.
the alkyl is pentyl.
the alkyl is hexyl.
the compound of formula (Ib) is selected from one of the following structures: wherein R 5 is independently H or a promoiety.
the compound of formula (Ib) is selected from one of the following structures: wherein R 5 and R 4 independently H or a promoiety, or R 5 and R 4 are cyclically linked to form a promoiety; and n1 is an integer from 1 to 6. [0256] In certain embodiments, the compound of formula (Ib) is selected from one of the following structures: . [0257] In some embodiments, at least one of R 4 -R 5 is of the formula -COCH 3 , -COCH(CH 3 ) 2 or - COC(CH 3 ) 3 . In certain embodiments, at least one of R 4 -R 5 is of the formula -CH 2 OCOC(CH 3 ) 3 .
R 4 -R 5 is of the formula -COC(CH 3 ) 3 or -CH 2 OCOC(CH 3 ) 3 .
R 4 is H and R 5 is selected from -COCH 3 , -COCH(CH 3 ) 2 , -COC(CH 3 ) 3 and - CH 2 OCOC(CH 3 ) 3 .
R 4 is H and R 5 is -COC(CH 3 ) 3 .
R 4 is H and R 5 is -CH 2 OCOC(CH 3 ) 3 .
the compound of formula (Ib) is selected from one of the following structures: wherein n2 is an integer from 1 to 6.
R 5 and R 4 are cyclically linked to form a promoiety.
the compound of formula (Ib) is selected from one of the following structures: wherein n2 is an integer from 1 to 6; and Y 4 is a suitable counterion.
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ic): wherein R 2 -R 5 and Z 1 are as defined herein.
Z 1 is selected from -O-, -S-, -CONR 21 -, and optionally substituted –(C(R 22 ) 2 ) q -heteroaryl, wherein q is 0 or 1.
Z 1 is -O-.
Z 1 is optionally substituted –(C(R 22 ) 2 ) q -triazole wherein q is 0 or 1.
Z 1 is Z 11 -Ar , wherein Ar is or optionally substituted heteroaryl or optionally substituted aryl.
Ar is an optionally substituted heteroaryl.
the heteroaryl is a 5 or 6-membered heteroaryl.
the heteroaryl is a 5-membered heteroaryl.
the 5-membered heteroaryl is a triazole.
the triazole is a 1,2,3-triazole moiety.
Z 11 is -C(R 22 ) 2 .
at least one R 22 is H.
both R 22 are H.
Z 11 is -O-.
Z 11 is -S-.
Z 11 is -NR 21 , where R 21 is H or (C1-3)alkyl.
Z 1 is -C(R 22 ) 2 -triazole-.
Z 1 is: .
Z 1 is Z 11 .
Z 11 is -C(R 22 ) 2 .
at least one R 22 is H.
both R 22 are H, and Z 11 is -CH 2 -.
Z 11 is -O-.
Z 11 is -S-.
Z 11 is -NR 21 , where R 21 is H or (C 1-3 )alkyl.
Z 1 is monocyclic 5 or 6-membered heteroaryl or aryl. In certain embodiments, [0264] In certain embodiments of formula (Ic), Z 1 is selected from -O-, -S-, -C(R 22 ) 2 -, -NR 21 -, - wherein: X 1 is O or S; t is 0 or 1; R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl); and each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
X 1 is O or S
t is 0 or 1
R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl)
the compound of formula (Ic) is the following structure: .
the compound of formula (Ic) is the following structure: .
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Id): wherein R 1 , R 3 -R 5 and Z 1 are as defined herein.
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Id’): [0269] In some embodiments, Z 1 is selected from optionally substituted –(C(R 22 ) 2 ) q -heteroaryl, and [0270] In some embodiments, Z 1 is optionally substituted –(C(R 22 ) 2 ) q -triazole wherein q is 0 or 1. [0271] In some embodiments, Z 1 is . In some embodiments, Z 1 . [0272] In some embodiments, wherein R 23 is H, or C(1-3)-alkyl.
Z 1 is -NR 23 CO-, wherein R 23 is H or C (1-3) -alkyl.
Z 1 is selected from optionally substituted – (C(R 22 ) 2 ) q -heteroaryl, , wherein q is 0 or 1.
Z 1 is optionally substituted –(C(R 22 ) 2 ) q -triazole wherein q is 0 or 1.
R 23 is H, or C(1-3)-alkyl.
Z 1 is -NR 23 CO-, wherein R 23 is H or C (1-3) -alkyl.
Z 1 is monocyclic 5 or 6-membered heteroaryl or aryl.
Z 1 is a monocyclic 5 or 6-memebered heteroaryl of one of the following structures: [0280] In certain embodiments of formula (Id), Z 1 is of one of the following structures: [0281] In certain embodiments of formula (Id), Z 1 is selected from -O-, -S-, -C(R 22 ) 2 -, -NR 21 -, - wherein: X 1 is O or S; t is 0 or 1; R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., C (1-3) -alkyl, such as methyl); and each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl.
X 1 is O or S
t is 0 or 1
R 21 and each R 23 is independently selected from H, and optionally substituted (C 1 -C 6 )al
the compound of formula (Id) is selected from one of the following structures: wherein R 6 is independently H or (C 1 - 3 )alkyl. [0283] In some embodiments of the compound of formula (Id) R 3 is H, such that the compound of formula (Id) has no non-hydrogen substituents at the 1-position of the sugar ring.
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ie’): wherein: R 1 , R 4 , R 5 and R 11 are as defined herein; Z 2 is absent or selected from -O-, -S-, NR 25 -, and -C(R 22 ) 2 , and optionally substituted Z 12 -alkyl; ring A is absent or selected from a 5 or 6-membered optionally substituted aryl and a 5 or 6- membered optionally substituted heteroaryl; Z 3 is a linking moiety selected from Z 12 , optionally substituted alkyl, optionally substituted Z 12 - alkyl, optionally substituted Z 12 -heteroaryl, optionally substituted Z 12 -aryl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amide, optionally substituted sulfonamide, optionally substituted urea, and optionally substituted thiour
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ie’’): [0286] In some embodiments of formula (Ie’), the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (Ie): wherein: R 1 , R 4 , R 5 and R 11 are as defined herein; Z 2 is absent or selected from -O-, -S-, NR 25 -, and -C(R 22 ) 2 , and optionally substituted Z 12 -alkyl; ring A is absent or selected from a 5 or 6-membered optionally substituted aryl and a 5 or 6- membered optionally substituted heteroaryl; Z 3 is a linking moiety selected from Z 12 , optionally substituted alkyl, optionally substituted Z 12 - alkyl, optionally substituted Z 12 -heteroaryl, optionally substituted Z 12 -aryl, optionally
Z 2 is absent.
Z 2 is C(R 22 ) 2 where R 22 is H or optionally substituted (C 1 -C 3 )alkyl.
Z 2 is -CH 2 -.
Z 2 is NR 25 where R 25 is selected from H, optionally substituted (C 1 -C 3 )alkyl and optionally substituted acyl.
Z 2 is -N(COCH 3 )-.
Z 2 is -NH-.
Z 2 is - S-.
Z 2 is O.
the compound of formula (Ie) is of any one of formulae (If)-(Ii): wherein the A ring, R 1 , R 4 , R 5 , R 11 , Z 3 and R 25 are as defined herein.
the A ring is a 5 or 6-membered aryl or heteroaryl.
the A ring is a 5-membered heteroaryl selected from selected from triazole, thiadiazole, thiophene, oxazole, isoxazole, isothiazole, thiazole, oxadiazole, and furan.
the A ring is a 6-membered heteroaryl selected from pyridine, pyrimidine, pyridazine, pyrazine, and triazine. In certain embodiments, the A ring is triazole. In certain embodiments, the A ring is pyridine. In certain embodiments, the A ring is pyrimidine. In certain embodiments, the A ring is thiadiazole. [0290] In some embodiments of any one of formulae (Ie)-(Ii), the A ring is absent. [0291] In some embodiments of any one of formulae (Ie)-(Ii), the A ring is phenyl or substituted phenyl.
the A ring is a 5 or 6-membered heteroaryl. In certain cases of formula (If), the A ring is a 5-membered heteroaryl. In certain embodiments of formula (If), the A ring is triazole. In certain embodiments of (If), the A ring is absent. [0293] In some embodiments of formula (Ig), the A ring is a 5 or 6-membered heteroaryl and R 25 is H. In certain embodiments of formula (Ig), the A ring is triazole. In certain embodiments of formula (Ig), the A ring is pyridine. In certain cases of formula (Ig), the A ring is pyrimidine.
the A ring is thiadiazole. In some embodiments of formula (Ig), the A ring is absent and R 25 is H or optionally substituted acyl. In certain embodiments, R 25 is -COCH 3 . In certain embodiments, R 25 is H. [0294] In some embodiments of formula (Ih), the A ring is a 5 or 6-membered heteroaryl. In certain cases of formula (Ih), the A ring is a 5-membered heteroaryl. In certain embodiments of formula (Ih), the A ring is triazole. In certain embodiments of (Ih), the A ring is absent.
the A ring is a 5 or 6-membered heteroaryl. In certain cases of formula (Ii), the A ring is a 5-membered heteroaryl. In certain embodiments of formula (Ii), the A ring is triazole. In certain embodiments of (Ii), the A ring is absent. [0296] In certain embodiments, the ASGPR binding moiety (X) of the compounds of this disclosure can be described by any one of formulae (Ij)-(Im): (Il), and (Im) wherein: R 1 , R 4 , R 5 , R 11 , Z 3 and R 25 are as defined herein.
Y 1 -Y 3 are each independently N or CR 27 ; and R 24 and R 27 are each independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen.
Z 3 is selected from -O-, -CH 2 O-, - OCH 2 -, optionally substituted -OCH 2 -heteroaryl, optionally substituted -OCH 2 -aryl, optionally substituted -CH 2 O-heteroaryl, and optionally substituted -CH 2 O-aryl.
Z 3 is selected from: [0299] In some embodiments of any one of formulae (Ie)-(Im), Z 3 is selected from -C(R 22 ) 2 -, optionally substituted alkyl, optionally substituted amide, optionally substituted sulfonamide, optionally substituted urea, and optionally substituted thiourea. In some embodiments, Z 3 is -CH 2 -. In some embodiments, Z 3 is -CH 2 CH 2 -. In some embodiments, Z 3 is -CH 2 CH 2 CH 2 -. In some embodiments, Z 3 is -NHSO 2 -(C 1-3 -alkyl).
Z 3 is -N(Ac)-(C 1-3 -alkyl). [0300] In some embodiments of any one of formulae (Ie)-(Im), Z 3 is selected from -S- and -NR 26 -, where R 26 is selected from H and optionally substituted (C 1 -C 3 )alkyl. [0301] In some embodiments of formula (Ik) at least one of Y 1 to Y 3 is N. In certain embodiments, at least two of Y 1 to Y 3 are N. In certain embodiments, Y 1 and Y 3 are N and Y 2 is CR 25 . In certain embodiments, Y 1 and Y 2 are N and Y 3 is CR 25 .
Y 1 and Y 2 are CR 25 and Y 3 is N.
R 25 is H.
R 24 is C (1-3) -alkyl, or C (1-3) -fluoroalkyl.
the fluoroalkyl is CF 3 .
R 1 is OH.
R 4 and R 5 are each H.
at least one of R 4 -R 5 is a promoiety.
R 4 and R 5 are cyclically linked to form a promoiety (e.g., as described herein).
the compound of formula (Ie) is selected from one of the following structures:
the compound of formula (Ie) is selected from one of the following structures: .
the compound of formula (Ie) is: .
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (In’): wherein: R 1 , R 4 , R 5 and Z 1 are as defined herein; Y 1 and Y 2 are each independently selected from -O-, -S-, NR 28 -, and -C(R 22 ) 2 ; R 28 is selected from H, optionally substituted (C 1 -C 6 )alkyl, and -C(O)R 22 ; each R 22 is independently selected from H, halogen and optionally substituted (C 1 -C 6 )alkyl; and ring B is a 5 or 6-membered optionally substituted cyclic group.
Y 1 is connected to the sugar ring via an alpha configuration. In some embodiments of formula (In’), Y 1 is connected to the sugar ring via a beta configuration.
the ASGPR binding moiety (X) of the compounds of this disclosure can be described by formula (In): wherein: R 1 , R 4 , R 5 and Z 1 are as defined herein; Y 1 and Y 2 are each independently selected from -O-, -S-, NR 28 -, and -C(R 22 ) 2 ; R 28 is selected from H, optionally substituted (C 1 -C 6 )alkyl, and -C(O)R 22 ; each R 22 is independently selected from H, halogen and optionally substituted (C 1 -C 6 )alkyl; and ring B is a 5 or 6-membered optionally substituted cyclic group.
Y 1 is O. In certain embodiments, Y 1 is S. In certain embodiments, Y 1 is -NR 28 -. In certain embodiments, Y 1 is -C(R 22 ) 2 and each R 22 is H. [0310] In some embodiments of formula (In)-(In’) Y 2 is -NR 28 - where R 28 is H. In certain embodiments, Y 2 is -NR 28 - where R 28 is -C(O)R 22 . In certain embodiments, R 22 is methyl. [0311] In some embodiments of formula (In)-(In’) the B ring is a 5 or 6-membered heterocycle.
the B ring is a 5-membered heterocycle. In certain embodiments, the B ring is a 6- membered heterocycle. [0312] In some embodiments of formula (In)-(In’) Z 1 is Z 11 , where Z 11 is selected from -O-, -S-, NR 21 -, and -C(R 22 ) 2 . In certain embodiments, Z 1 is -O-. In certain embodiments, Z 1 is -S-. In certain embodiments, Z 1 is NR 21 where R 21 is H. In certain embodiments, Z 1 is -C(R 22 ) 2 where each R 22 is H.
Z 1 is optionally substituted Z 11 -heteroaryl or optionally substituted Z 11 -aryl.
Z 1 is CH 2 -heteroaryl or CH 2 -aryl.
Z 1 is optionally substituted amide.
Z 1 is optionally substituted sulfonamide.
Z 1 is optionally substituted urea or optionally substituted thiourea.
the compound of formula (In)-(In’) has one of the following structures: .
n is 1, and L comprises a linear linker having a backbone of 20 or more consecutive atoms covalently linking X to Y via Z 1 , such as a backbone of 25 or more consecutive atoms, or 30 or more consecutive atoms, and in certain embodiments, up to 100 consecutive atoms.
linker L separates X and Y (or Z 1 ) by a chain of 20 to 50 consecutive atoms.
linker L separates X and Y (or Z 1 ) by a chain of 21 to 50 consecutive atoms, by a chain of 22 to 50 consecutive atoms, by a chain of 23 to 50 consecutive atoms, by a chain of 24 to 50 consecutive atoms, by a chain of 25 to 50 consecutive atoms, by a chain of 26 to 50 consecutive atoms, by a chain of 27 to 50 consecutive atoms, by a chain of 28 to 50 consecutive atoms, or by a chain of 29 to 50 consecutive atoms.
linker L separates X and Y (or Z 1 ) by a chain of 30 to 60 consecutive atoms.
linker L separates X and Y (or Z 1 ) by a chain of 31 to 60 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 32 to 60 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 33 to 60 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 34 to 60 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 35 to 50 consecutive atoms.
linker L separates X and Y (or Z 1 ) by a chain of 36 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 41 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 46 to 50 consecutive atoms. [0316] In certain embodiments of any one of formulae (Ia)-(In), n is 2 or more, and L is a branched linker that covalently links 2 or more X moieties to Y via the linking moiety Z 1 .
n is 2 or more and each branch of L comprises a linear linker of 14 or more consecutive atoms to covalently link via Z 1 each X moiety to a branching point of the linker L, such as 15 or more consecutive atoms, 16 or more consecutive atoms, or 17 or more consecutive atoms, and in certain embodiments, up to 50 consecutive atoms.
each branch of L comprises a linear linker of 14 to 50 consecutive atoms, such as 14 to 45, 14 to 40, 14 to 35 or 14 to 30 consecutive atoms.
each branch of L comprises 14 to 30 consecutive atoms, such as 14 to 29, 14 to 28, 14 to 27, 14 to 26, 14 to 25, 14 to 24, 14 to 23, 14 to 22, 14 to 21, or 14 to 20 consecutive atoms.
L comprises more than 14 consecutive atoms covalently linking each X moiety (via each Z 1 group) to a branching point of the linker.
L comprises 15 consecutive atoms separating each Z 1 group from a branching point of L.
L comprises 16 consecutive atoms separating each Z 1 group from a branching point of L.
L comprises 17 consecutive atoms separating each Z 1 group from a branching point of L.
L comprises 18 consecutive atoms separating each Z 1 group from a branching point of L. In certain embodiments, L comprises 19 consecutive atoms separating each Z 1 group from a branching point of L. In certain embodiments, L comprises 20 consecutive atoms separating each Z 1 group from a branching point of L. In certain other cases, L comprises a liner linker of 20 or more consecutive atoms separating each Z 1 group from a branching point L. [0318] In certain embodiments of any one of formulae (Ia)-(In), n is 2, and L comprises a branched linker having 14 or more consecutive atoms separating each Z 1 group of X from a branching point of L.
n is 3, and L comprises a branched linker having 14 or more consecutive atoms separating each Z 1 group of X from a branching point of L.
the linker L is of the formula (II) (e.g., as described herein).
R 1 is OH. In certain other cases, R 1 is -OC(O)R. In certain embodiments, R 1 is -C(O)NHR, where R is an optionally substituted alkyl.
R terminates in an alkenyl or an alkynyl group.
R 1 is optionally substituted triazole.
the triazole is of the following structure: .
R 2 is -NHCOCH 3 .
R 2 is — NHCOCF 3 .
R 2 is –NHCOCH 2 CF 3 .
R 2 is –OH.
R 2 is an optionally substituted triazole.
the triazole in of the following structure: . [0323] In certain embodiments when R 1 or R 2 is a substituted triazole.
the triazole is a 1,2,3-trizole, and the substituent is at the 4 or 5-position.
the substituent on the triazole moiety includes but is not limited to, an optionally substituted (C 1 - 6 )alkyl, optionally substituted (C 1 - 6 )alkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkaryl, and an optionally substituted alkyheteroaryl. It will be understood that any convenient substituent can be included in the triazole moiety, see, e.g., triazole moieties disclosed in Mamidayala et al, J. Am. Chem. Soc.2012, 134, 1978-1981.
At least one of R 4 -R 5 is a promoiety.
the promoiety is an ester.
at least one of R 4 -R 5 is of the formula -COCH 3 , -COCH(CH 3 ) 2 or -COC(CH 3 ) 3 .
at least one of R 4 -R 5 is of the formula -CH 2 OCOC(CH 3 ) 3
R 4 is a promoiety and R 5 is H.
R 5 is H and R 4 is a promoiety. In certain embodiments, both R 4 and R 5 are both promoieties. In certain embodiments, R 4 and R 5 are cyclically linked to form a promoiety. In certain embodiments, R 4 and R 5 are cyclically linked to form a promoiety of formulae (Io) or (Ip): wherein R 1 -R 3 and Y 4 are as defined herein. [0325] In certain embodiments of any one of formulae (Ia)-(In), both R 4 and R 5 are H. [0326] In certain embodiments of formula (I), n is 2 or 3, and X is selected from one of the wherein R 5 and R 23 are independently H or (C 1 - 3 )alkyl.
n is 1, 2 or 3, and X is selected from one of the following structures: [0328] wherein R 5 and R 4 independently H or a promoiety, or R 5 and R 4 are cyclically linked to form a promoiety; n1 and n2 are each independently an integer from 1 to 6; and Y 4 is a suitable counterion. In some embodiments, Y 4 is sodium. [0329] In certain embodiments of formula (I), n is 1, 2 or 3, and X is selected from one of the following structures: . [0330] In certain embodiments of formula (I), n is 1, 2 or 3, and X is selected from one of the following structures:
n is 1, 2 or 3, and X is selected from one of the following structures: .
n is 1, 2 or 3, and X is the following structure: .
n is 1, 2 or 3, and X is the following structure: .
X is the following structure: .
n is 1 and X is .
-Z 1 - is linked to an -L 1 - moiety (e.g., of the linker of any of formulae (II), (IIa) or (IIb) described herein).
the subject compounds comprise a -Z 1 -L 1 - group selected from: wherein each R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl; each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 -C 6 )alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 1 to 6. [0336] In certain embodiments, the Z 1 -L 1 - group is , and o is 1 or 2.
the Z 1 -L 1 - group each R 22 is H, and p is 1 or 2. [0340] In certain embodiments, the Z 1 -L 1 - group is , where r is 1-3. [0341] In certain embodiments, the Z 1 -L 1 - group are each independently 1-3. [0342] In certain embodiments, the Z 1 -L 1 - group [0343] In certain embodiments, the Z 1 -L 1 - group i are each independently is 1-3. [0344] In certain embodiments, the Z 1 -L 1 - group is , where x is 0-3. [0345] In certain embodiments, the Z 1 -L 1 - group -3.
the Z 1 -L 1 - group i where R 21 is H, and z is 1-4.
the Z 1 -L 1 - group is , where R 21 is H, and z1 is 1-4.
the Z 1 -L 1 - group i is 1-3.
the Z 1 -L 1 - group i where q is 1-3.
the subject compounds comprise a -Z 1 -L- group selected from: [0351] In certain embodiments, the Z 1 -L 1 - group is , where q is 1-3.
q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. [0352] In certain embodiments, the Z 1 -L 1 - group i . [0353] In certain embodiments, the Z 1 -L 1 - group i . [0354] In certain embodiments, the Z 1 -L 1 - group is . [0355] In certain embodiments, -Z 1 -L 1 - comprises an optionally substituted -NH-heteroaryl-. In certain embodiments the heteroaryl is a triazole. In certain embodiments, the heteroaryl is pyridine.
the heteroaryl is pyrimidine In certain cases the heteroaryl is thiadiazole [0356]
the -Z 1 -L 1 - comprises a group selected from: wherein each R 24 is independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen; and each R 25 is independently selected from H, optionally substituted (C 1 -C 6 )alkyl, and optionally substituted acyl.
R 25 is H.
R 24 is C (1-3) -alkyl, or C (1-3) -fluoroalkyl.
the fluoroalkyl is CF 3 .
Exemplary ASGPR Ligands Exemplary moieties that bind ASGPR, and synthons which can be utilized in the preparation of compounds of this disclosure that include the ASGPR ligand of interest are shown in Tables 1-5. [0358] In certain embodiments, the compound of formula (Ib) is a compound shown in Table 1: [0359] In certain embodiments, the compound of formula (Ib) is a compound shown in Table 1A:
the compound of formula (Ib) is a compound shown in Table 1B: [0361] In certain embodiments, the compound of formula (Ic) is a compound shown in Table 2a:
the compound of formula (Id) is a compound shown in Table 3a: [0363] In certain embodiments, the compound of formula (Id) is a compound shown in Table 4:
the compound of formula (Id’) is a compound shown in Table 5: [0365] Additional exemplary moieties that bind ASGPR, and synthons which can be utilized in the preparation of compounds of this disclosure that include the ASGPR ligand of interest are shown in the tables below.
the building blocks described herein can be used to prepare the compounds disclosed herein. As is appreciated by one of skill in the art, reactive functional groups present on the building blocks described herein can be reacted with complimentary functional groups on a linker moiety to bond the ASGPR binding compound X to Y. [0366] For example, compounds of this disclosure can be prepared using the building blocks described herein as exemplified in Scheme 1.
the compounds of this disclosure have various L moieties which may be constructed by coupling X to one or more first portions of the linker L (e.g., an -L 1 - moiety) via Z 1 to provide exemplary ASGPR binding compound X building blocks.
R M1 and R M2 are each independently reactive functional groups for coupling reactions (e.g., alkyne, -N 3 , -C(O)OH, -NH 2 , etc.); and Y’ is Y or a chemoselective a chemoselective ligation group capable of conjugating to an amino acid residue(s) of Y.
R 3 is H such that the ASGPR ligand (X) includes CH 2 at the 1-position, and R 2 is a linking moiety, Z 1 .
Exemplary building blocks that can be used in the preparation of compounds of this disclosure that include ASGPR ligands (X) of interest are shown in Table 7.
the ASGPR ligand (X) building blocks that can be used in the preparation of compounds of this disclosure is a bicyclic structure. Exemplary building blocks that can be used in the preparation of compounds of this disclosure that include ASGPR ligands (X) of interest are shown in Table 8.
Prodrugs [0375] Aspects of this disclosure include prodrugs of any of the ASGPR binding moieties described herein that are incorporated into the compounds and conjugates of this disclosure.
the term “prodrug” refers to an agent which is converted into the drug in vivo by some physiological or chemical process (e.g., a prodrug on being brought to the physiological pH is converted to the desired drug form).
Prodrugs forms of any of the ASGPR binding moieties described herein can be useful because, for example, can lead to particular therapeutic benefits as a consequence of an extension of the half-life of the resulting compound or conjugate in the body or a reduction in the active dose required.
Pro-drugs can also be useful in some situations, as they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The pro-drug may also have improved solubility in pharmacological compositions over the parent drug.
Prodrug derivative of a ASGPR binding moiety generally includes a promoiety substituent at a suitable labile site of the compound. The promoiety refers to the group that is removed by enzymatic or chemical reactions, when a prodrug is converted to the drug in vivo.
the promoiety is a group attached via an ester linkage to a hydroxyl group of the compound or drug.
a prodrug derivative of one or more of the hydroxyl groups of the sugar ring may be incorporated into the compounds.
an ester promoiety can be incorporated at one or more of the hydroxyl groups at the 3 and/or 4 positions of the sugar (e.g., as described herein).
the hydroxyl groups at the 3 and 4 positions of the sugar are cyclically linked to form a promoiety (e.g., as described herein).
Linker Valency [0382]
the ASGPR ligand moieties (X) can be used in a monovalent or multivalent configuration with respect to the binding to ASGPR of the “n” X groups that are displayed on the linker scaffold.
a monovalent configuration includes a single ASGPR ligand moiety (X) per linker of the bifunctional molecule, where it is understood that one or more linkers may be connected to Y.
a multivalent configuration includes two or more such ASGPR ligand moieties per linker (e.g., bivalent or trivalent or of higher valency linker). This disclosure provides particular linker scaffolds and linker valencies that display preferred ASGPR ligand moieties in the bifunctional molecules of this disclosure.
the linked ASGPR ligand moiety (X) of the bifunctional molecule is monovalent (e.g., in Formula (I), n is 1), such that a linker covalently links a single ASGPR ligand moiety (X) via a linking moiety at the 1, 6, or 2-position of the sugar ring analog to a biomolecule (Y).
n is 1, and L comprises a linear linker having a backbone of 20 or more consecutive atoms (e.g., 25 or more) covalently linking the ASGPR ligand X to Y via a linking moiety at any of the 1-, 2- or 6-positions of X.
the linker L includes a backbone of 20 to 100 consecutive atoms linking the ASGPR ligand (X) to Y, such as 25 to 80, 25 to 60, or 25 to 50 consecutive atoms.
the bifunctional molecule is multivalent with respect to X, where in Formula (I), n is 2 or more, such that the conjugate includes two or more ASGPR ligand binding moieties (X) per multivalent linker which connects to Y.
the multivalent linker (L) is a branched linker or a dendrimer linker.
the bifunctional molecule has one or more divalent linkers (e.g., n is 2 in Formula (I)).
each branch of a branched linker includes a linear linker portion covalently connecting each X moiety (via the linking moiety described herein) to a branching point in the branched linker or dendrimer linker.
each branch of the linker includes a linear linker portion having a backbone of 8 or more consecutive atoms, such as 10 or more, 12 or more, 14 or more, 16 or more, 18 or more or 20 or more consecutive atoms between the X ligand moiety and the branching point in the linker.
each branch of the linker includes a linear linker portion having a backbone of 8 to 50 consecutive atoms, such as 10 to 50, 12 to 50, 14 to 50, or 14 to 40, 14 to 30, or 14 to 20 consecutive atoms.
Linkers [0386]
the terms “linker”, “linking moiety” and “linking group” are used interchangeably and refer to a linking moiety that covalently connects two or more moieties, compounds or other biomolecules, such as ligands and proteins of interest.
the linker is divalent and connects two moieties.
the linker is a branched linking group that is trivalent or of a higher multivalency.
the linker that connects the two or more moieties has a linear or branched backbone of 500 atoms or less (such as 400 atoms or less, 300 atoms or less, 200 atoms or less, 100 atoms or less, 80 atoms or less, 60 atoms or less, 50 atoms or less, 40 atoms or less, 30 atoms or less, or even 20 atoms or less) in length, e.g., as measured between the two or more moieties.
500 atoms or less such as 400 atoms or less, 300 atoms or less, 200 atoms or less, 100 atoms or less, 80 atoms or less, 60 atoms or less, 50 atoms or less, 40 atoms or less, 30 atoms or less, or even 20 atoms or less
a linking moiety may be a covalent bond that connects two groups or a linear or branched chain of between 1 and 500 atoms in length, for example of about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 100, 150, 200, 300, 400 or 500 carbon atoms in length, where the linker may be linear, branched, cyclic or a single atom.
one, two, three, four, five or more, ten or more, or even more carbon atoms of a linker backbone may be optionally substituted with heteroatoms, e.g., sulfur, nitrogen or oxygen heteroatom.
linker when the linker includes an ethylene glycol, or longer polyethylene glycol (PEG) linking group, e.g., where every third atom of that segment of the linker backbone is substituted with an oxygen.
PEG polyethylene glycol
the bonds between backbone atoms of a linker may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone.
the linker may include one or more substituent groups, for example an alkyl, aryl or alkenyl group.
a linker may include, without limitations, one or more of the following: oligo(ethylene glycol) (also referred to as PEG), ether, thioether, disulfide, amide, carbonate, carbamate, urea, sulfonamide, thiourea, tertiary amine, alkyl which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1- methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like.
oligo(ethylene glycol) also referred to as PEG
ether also referred to as PEG
thioether disulfide
amide carbonate
carbamate urea
sulfonamide thiourea
tertiary amine alkyl which may be straight or branched, e
the linker backbone may include a cyclic group, for example, an aryl, a heterocycle, a cycloalkyl group or a heterocycle group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone.
a “linker” or linking moiety is derived from a molecule with a reactive terminus, e.g., suitable for conjugation to a protein of interest.
the reactive terminus of the linker precursor includes a chemoselective ligation group capable of conjugating to amino acid residue(s) of a polypeptide.
the chemoselective ligation group conjugates to a cysteine thiol group, or a lysine sidechain amine group of the polypeptide that is accessible.
a variety of conjugation chemistries can be utilized in the conjugtaes of this disclosure (e.g., as described herein).
the chemoselective ligation group is a thiol-reactive group such as maleimide or dibromomaleimide.
the chemoselective ligation group is an amine- reactive group such as an active ester, e.g., perfluorophenyl ester or tetrafluorophenyl ester, or N- hydroxysuccinimidyl ester (NHS) or sulfo-NHS, or as defined herein.
the linker L includes one or more straight or branched-chain carbon moieties and/or polyether (e.g., ethylene glycol) moieties (e.g., repeating units of -CH 2 CH 2 O-), and combinations thereof.
these linkers optionally have amide linkages, urea or thiourea linkages, carbamate linkages, ester linkages, amino linkages, ether linkages, thioether linkages, sulfhydryl linkages, heteroaryl linkages, or other hetero functional linkages.
the linker backbone includes one or more of carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof.
the linker includes one or more of an ether bond, thioether bond, amine bond, amide bond, carbon-carbon bond, carbon-nitrogen bond, carbon-oxygen bond, carbon-sulfur bond, and combinations thereof.
the linker includes a linear structure.
the linker includes a branched structure. In certain embodiments, the linker includes a cyclic structure. In certain embodiments, the linker includes one or more heteroaryl cyclic structures, e.g., a triazole, such as a 1,2,3- traizole.
L is a linker between about 5 ⁇ and about 500 ⁇ . In certain embodiments, L is between about 10 ⁇ and about 400 ⁇ . In certain embodiments, L is between about 10 ⁇ and about 300 ⁇ . In certain embodiments, L is between about 10 ⁇ and about 200 ⁇ . In certain embodiments, L is between about 10 ⁇ and about 100 ⁇ .
linker L separates X (or Z 1 ) and Y by a chain of 10 to 100 consecutive atoms. In certain embodiments, linker L separates X (or Z 1 ) and Y by a chain of 10 to 60 consecutive atoms, by a chain of 12 to 60 consecutive atoms, by a chain of 16 to 50 consecutive atoms, by a chain of 20 to 50 consecutive atoms, by a chain of 30 to 50 consecutive atoms, by a chain of 40 to 50 consecutive atoms. [0391] It is understood that the linker may be considered as connecting directly to a Z 1 group of a ASGPR ligand moiety (X) (e.g., as described herein).
X ASGPR ligand moiety
the linker may be considered as connecting directly to the Z 1 group.
the -Z 1 -L 1 - group e.g., as described herein
the disclosure is meant to include all such configurations of ASGPR ligand moiety (X) and linker (L).
L is a linker of formula (XI): wherein each L 1 and L 3 are independently a linear linking moiety, and L 2 is a branched linking moiety, wherein L 1 to L 3 together provide a linear or branched linker between X and Y; a, b and c are independently 0 or 1; * represents the point of attachment of L 1 to X via Z 1 ; and ** represents the point of conjugation of the linker L to Y; wherein: when n is 1, b is 0 and at least one of a and c is 1; and when n is 2 or 3, a, b and c are each 1.
the linear linker of formula (Xia) has a backbone of 10 or more consecutive atoms covalently linking X to Y via Z 1 , such as a backbone of 12 or more consecutive atoms, 14 or more consecutive atoms, or 16 or more consecutive atoms, and in certain embodiments, up to 100 consecutive atoms.
the linear linker separates X (or Z 1 ) and Y by a chain of 20 to 50 consecutive atoms. In certain embodiments of formula (Xa), the linear linker separates X (or Z 1 ) and Y by a chain of 30 to 60 consecutive atoms.
each L 1 is of formula (XII): wherein: L 10 is a linking moiety, and * represents the point of attachment of L 1 to X via Z 1 ; and L 11 to L 19 are independently absent or a linking moiety, wherein L 10 to L 19 of each L 1 is independently selected from–C 1-6 -alkylene–,-–C 1-12 -alkylene–, – C 1-20 -alkylene–,–NHCO-C 1-6 -alkylene–,
each L 1 is of formula (XII): wherein: L 10 is a linking moiety, and * represents the point of attachment of L 1 to X via Z 1 ; and L 11 to L 19 are independently absent or a linking moiety, wherein L 10 to L 19 of each L 1 is independently selected from –C 1-6 -alkylene–,-CF 2 -, –C 1-12 - alkylene–, –C 1-20 -alkylene–,–NHCO-C 1-6 -alkylene–, –CONH-C 1-6 -alkylene–, –NH-C 1-6 -alkylene–, – NHCONH-C 1-6 -alkylene–, –NHCSNH-C 1-6 -alkylene–, –C 1-6 -alkylene–NHCO-, –C 1-6 -alkylene–CONH-, —C 1-6 -alkylene–CONH-, —C 1-6 -alkylene–CONH-, —C
the linking moiety L 1 includes a linear backbone of 6 to 40 consecutive atoms, such as 10 to 40, 10 to 30, 16 to 30, or 20 to 30 consecutive atoms.
the linking moiety L 1 includes a linear backbone of each L 1 comprises a linear backbone of 6 to 20 consecutive atoms, such as 6 to 16 consecutive atoms, such as 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive atoms.
the linking moiety of formula (XII) includes one or repeating ethylene glycol moieties (e.g., -CH 2 CH 2 O- or -OCH 2 CH 2 -).
the linking moiety of formula (XII) includes 1 to 10 ethylene glycol moieties, such as 1, 2, 3, 4, 5 or 6 ethylene glycol moieties.
the linking moiety of formula (XII) includes one or more triazole (e.g., 1,2,3-triazole) containing linking moieties. It is understood that the triazole may be derived from an azido-alkyne click chemistry and thus have two possible orientations depending on the method of synthesis: [0403]
the triazole containing linking moiety is : wherein w1 and u1 are independently 0 to 12, such as 0, 1, 2, 3, 4, 5 or 6.
L 20 is a branched linking moiety including one or more linking moieties independently selected from amino acid residue (e.g., a residue such as Gly, Ala, beta-Al Glu, Ser, Cys, or a derivative thereof), –NH-CH[(CH 2 ) q ] 2 O– or –NH-C[(CH 2 ) q ] 3 O–, alkylene–, –NHCO-, –CONH–, –NHSO 2 –, –SO 2 NH–, –CO–, –SO 2 –, –O–, –S–, pyrrolidine-2,5-dione, 1,2,3-triazole, –NH–, and –Nme–, –NHC(O)NH–, – NHC(S)
b is 1 and the linking moiety L 2 is selected from one of (L2A)-(L2D): wherein: each Z 2 and Z 3 is independently absent or selected from –NHCO-, –CONH–, –CO–, –O–, –NH–, and –Nme–; x is 1 to 12 (e.g., 1 to 6, or 1 to 3); and y is 0 to 12 (e.g., 1 to 6, or 1 to 3). [0406] In some embodiments of any one of L2A-L2D, Z 2 is –NHCO-. In some embodiments of any one of L2A-L2D, Z 2 is –CONH–.
Z 2 is –CO–. In some embodiments of any one of L2A-L2D, Z 2 is –O–. In some embodiments of any one of L2A-L2D, Z 2 is –NH–. In some embodiments of any one of L2A-L2D, Z 2 is –Nme–. In some embodiments of any one of L2A-L2D, Z 2 is absent. [0407] In some embodiments of any one of L2A-L2D, Z 3 is –NHCO-. In some embodiments of any one of L2A-L2D, Z 3 is –CONH–.
Z 3 is –CO–. In some embodiments of any one of L2A-L2D, Z 3 is –O–. In some embodiments of any one of L2A-L2D, Z 3 is –NH–. In some embodiments of any one of L2A-L2D, Z 3 is –Nme–. In some embodiments of any one of L2A-L2D, Z 3 is absent.
L2A In some embodiments of L2A, Z 2 is –O–, y is 0 and the linking moiety is of the structure L2Ai: [0409] In some embodiments of L2B, Z 2 is –O– or –CO—, and the linking moiety is of the structure L2Bi or L2Bii: [0410] In some embod the linking moiety is of the structure L2Ci, L2Cii, L2Ciii, or L2Civ: [0411] In some embodiments of L2D, Z 2 is absent and the linking moiety is of the structure L2Di: [0412] In some embodiments, of any one of formulae L2A-L2Di, x is 1 to 6.
x is 1 to 3. In certain embodiments, x is 1. In certain embodiments, x is 2. In certain embodiments, x is 3. [0413] In some embodiments of any one of formulae L2A-L2Di, y is 0 to 6. In certain embodiments, y is 0 to 3. In certain embodiments, y is 0. In certain embodiments, y is 1. In certain embodiments, y is 2. In certain embodiments, y is 3.
b is 1 and the linking moiety L 2 is selected from: [0415] In some embodiments of the linker of formula (XI), b is 1 and the linking moiety L 2 is of the formula (XIV): wherein: r is 1 or 2; and when n is 2, r is 1, when n is 3, r is 2. [0416] In some embodiments of the linker of formula (XI), b is 1 and the linking moiety L 2 is of the formula (Xva) or (XVb): wherein: r is 1 or 2; and when n is 2, r is 1, when n is 3, r is 2.
L 2 is of formula (XIIIa) or (XIIIb) and L 2 includes two 2 or more amino acid residues (e.g., 3 or more, or 4 or more amino acid residues, linear or dendrimer).
L 2 includes 4 or more amino acid residues that are branched linking moieties selected from Lys, Orn, Asp, Glu, Ser, and Cys (e.g., where the sidechain, amino and carboxylic acid are each linked to an adjacent moiety).
each L 3 is of the formulae (XVI): wherein: L 30 to L 39 are independently absent or a linking moiety; and Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group of the linker to a compatible group of Y; wherein L 30 to L 39 are each independently selected from –C 1-20 -alkylene–, –NHCO-C 1-6 - alkylene–, –CONH-C 1-6 -alkylene–, –NH C 1-6 -alkylene–, –NHCONH-C 1-6 -alkylene–, – NHCSNH-C 1-6 - alkylene–, –C 1-6 -alkylene–NHCO-, –C 1-6 -alkylene–CONH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–CONH-, –C 1-6 -alkylene–NH-, –C 1-6 -al
the linking moiety of formula (XVI) includes a linear backbone of 6 to 40 consecutive atoms, such as 10 to 40, 10 to 30, or 20 to 30 consecutive atoms.
the linking moiety of formula (XVI) includes repeating ethylene glycol moieties (e.g., -CH 2 CH 2 O- or -OCH 2 CH 2 -).
the linking moiety of formula (XVI) includes 2 to 20 ethylene glycol moieties, such as 2 to 15, 2 to 10, 3 to 20, 3 to 15, 3 to 10, 4 to 15, 5 to 15 or 5 to 10 ethylene glycol moieties.
the linking moiety of formula (XVI) includes 2 or more ethylene glycol moieties, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or even more ethylene glycol moieties.
the linking moiety of formula (XVI) includes one or more triazole linking moieties.
the linker includes one or more 1,2,3-triazole linking moieties.
the one or more 1,2,3-triazoel moieties is selected from one of the following structures: , wherein w1, u1 and q1 are independently 1 to 25 (e.g., 1 to 12, such as 1 to 6).
the linking moiety L 3 includes (C 10 -C 20 -alkylene (e.g., C 12 -alkylene), or –(OCH 2 CH 2 ) p –, where p is 1 to 25, such as 3 to 25, 5 to 24, 7 to 25, 10 to 25, 15 to 25 or 20 to 24.
the linker L is of formula XVII: wherein: a is 0 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2; d is 1 to 6 (e.g., 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); f is 1 to 6 (e.g., 1, 2, or 3); Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group (e.g., as described herein) of a linker precursor to a compatible group of Y.
a is 0 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2; d is 1 to
Z is a residual moiety resulting from the covalent linkage (e.g., via a thioether bond) of a thiol-reactive chemoselective ligation group to one or more cysteine residue(s) of Y.
the thiol-reactive chemoselective ligation group includes maleimide, bromomaleimide, haloacetamide, vinyl sulfone, or thiolactone.
the thiol-reactive group is selected from one of the following structures: wherein: u is 1 to 11 (e.g., 1 to 5); v is 1 to 11 (e.g., 1 to 5); and X is H or Br. [0425] In some embodiments, the thiol-reactive group comprises:
Z is a residual moiety resulting from the covalent linkage (e.g., via an amide bond) of an amine-reactive chemoselective ligation group to one or more lysine residue(s) of Y.
the amine-reactive chemoselective ligation group includes an active ester (e.g., N-hydroxysuccinimidyl (NHS) ester, sulfo-NHS ester, pentafluorophenyl (PFP) ester, tetrafluorophenyl (TFP) ester, or the like).
the linker L includes one of (XVIIIa)-(XVIIIc): wherein: a is 0 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2; d is 1 to 6 (e.g., 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); and f is 1 to 6 (e.g., 1, 2, or 3).
a is 2 to 6, such as 2 to 3. In some embodiments, a is 2. In some embodiments, a is 3. In some embodiments, a is 4. In some embodiments, a is 5. In some embodiments a is 6. [0430] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), b is 1 to 4, such as 1 to 3. In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3.
c is 1 to 4, such as 1 to 3. In some embodiments, c is 1. In some embodiments, c is 2. In some embodiments, c is 3. [0432] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), r is 1. In some embodiments, r is 2. [0433] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), d is 1 to 4, such as 1 to 3. In some embodiments, d is 1. In some embodiments, d is 2. In some embodiments, d is 3.
e is 1 to 5, such as 1 to 3. In some embodiments, e is 1. In some embodiments, e is 2. In some embodiments, e is 3. In some embodiments, e is 4. In some embodiments, e is 5. [0435] In some embodiments of any one of formulae (XVII) or (XVIIIa)-(XVIIIc), f is 1 to 4, such as 1 to 3. In some embodiments, f is 1. In some embodiments, f is 2. In some embodiments, f is 3.
a is 1-4; b is 1-4; c is 1-3; r is 1; d is 1-3; e is 1-6; and f is 1-3.
a is 1-4; b is 1-4; c is 1-3; r is 2; d is 1-3; e is 1-6; and f is 1-3.
a is 2; b is 1; c is 2; r is 1; d is 2; e is 3; and f is 2.
a is 2; b is 1; c is 2; r is 2; d is 2; e is 3; and f is 2.
a is 4; b is 1; c is 2; r is 1; d is 2; e is 3; and f is 2.
a is 4; b is 1; c is 2; r is 2; d is 2; e is 3; and f is 2.
a is 2; b is 2; c is 2; r is 1; d is 2; e is 3; and f is 2.
a is 2; b is 2; c is 2; r is 2; d is 2; e is 3; and f is 2.
a is 0; b is 3; c is 2; r is 2; d is 2; e is 3; and f is 2.
a is 2; b is 4; c is 2; r is 2; d is 2; e is 3; and f is 2.
a is 2; b is 4; c is 2; r is 1; d is 2; e is 3; and f is 2.
the linker L includes L A : (L A ), wherein: Z 4 is selected from -NHC(O)NH-, -NHC(O)-, -C(O)NH-, -O-, -NH-; a is 0 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); d is 1 to 6 (e.g., 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); and f is 1 to 6 (e.g., 1, 2, or 3).
Z 4 is -NHC(O)NH-. In certain embodiments, Z 4 is -NHC(O)-. In certain embodiments, Z 4 is -C(O)NH-. In certain embodiments, Z 4 is -O-. In certain embodiments, Z 4 is -NH-.
a is 1-4; b is 1-4; c is 1-3; d is 1-3; e is 1-6; and f is 1-3. In some embodiments, a is 4; b is 1; c is 2; d is 2; e is 5; and f is 2.
Z 4 is -NHC(O)NH- and a is 1-4; b is 1-4; c is 1-3; r is 1; d is 1-3; e is 1-6; and f is 1-3.
Z 4 is -NHC(O)- and a is 1-4; b is 1-4; c is 1-3; r is 1; d is 1-3; e is 1-6; and f is 1-3.
the linker L includes L B : wherein: a is 0 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2; d is 1 to 6 (e.g., 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); and f is 1 to 6 (e.g., 1, 2, or 3).
a is 1-4; b is 1-4; c is 1-3; r is 1; d is 1-3; e is 1-6; and f is 1-3.
a is 4; b is 1; c is 2; r is 1; d is 2; e is 5; and f is 2.
a is 2; b is 1; c is 2; r is 1; d is 2; e is 3; and f is 2.
a is 4; b is 1; c is 2; r is 1; d is 2; e is 3; and f is 2.
a is 1; b is 2; c is 2; r is 1; d is 2; e is 3; and f is 2.
a is 0; b is 3; c is 2; r is 1; d is 2; e is 3; and f is 2.
L B a is 1-4; b is 1-4; c is 1-3; r is 2; d is 1-3; e is 1-6; and f is 1-3.
a is 2; b is 1; c is 2; r is 2; d is 2; e is 3; and f is 2.
a is 4; b is 1; c is 2; r is 2; d is 2; e is 3; and f is 2.
a is 1; b is 2; c is 2; r is 2; d is 2; e is 3; and f is 2.
a is 0; b is 3; c is 2; r is 2; d is 2; e is 3; and f is 2.
the linker L includes L C : wherein: a is 0 to 12 (e.g., 1 to 6, 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1 to 4, such as 1, 2, or 3); c is 1 to 6 (e.g., 1 to 3, such as 1, 2, or 3); r is 1 or 2; d is 1 to 6 (e.g., 1 to 3, such as 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); and f is 1 to 6 (e.g., 1 to 3, such as 1, 2, or 3).
a is 1-4; b is 1-4; c is 1-3; r is 1; d is 1-3; e is 1-6; and f is 1-3.
a is 2; b is 4; c is 2; r is 1; d is 2; e is 5; and f is 2.
a is 1-4; b is 1-4; c is 1-3; r is 2; d is 1-3; e is 1-6; and f is 1-3.
a is 2; b is 4; c is 2; r is 2; d is 2; e is 5; and f is 2.
-Z 1 - is linked to an -L 1 - moiety (e.g., of the linker as described herein).
the subject compounds comprise a -Z 1 -L 1 - moiety comprising a linking moiety selected from: wherein each R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl; each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 -C 6 )alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 1 to 6.
the Z 1 -L 1 - group is , and o is 1 or 2.
the Z 1 -L 1 - group 22 each R is H, and p is 1 or 2.
the Z 1 -L 1 - group is , where r is 1-3.
the Z 1 -L 1 - group are each independently 1-3.
the Z 1 -L 1 - group i are each independently is 1-3.
the Z -L - group is N N N 1 1 , where x is 0-3.
the Z 1 -L 1 - group R 21 [0468] In certain embodiments, the Z 1 -L 1 - group is , where R 21 is H, and z is 1-4.
R 21 [0469] In certain embodiments, the Z 1 -L 1 - group is , where R 21 is H, and z1 is 1-4.
the Z 1 -L 1 - group is , where each R 22 is H, and q is 1-3.
the Z 1 -L 1 - group i where q is 1-3.
the subject compounds comprise a -Z 1 -L- group comprising a linking moiety selected from: where R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl (e.g., methyl); and each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 -C 6 )alkyl (e.g., methyl).
R 21 is H.
each R 22 is H.
the -Z 1 -L 1 - group is , where q is 1-3. In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. [0474] In certain embodiments, the -Z 1 -L 1 - group is H . [0475] In certain embodiments, -Z 1 -L 1 - includes an optionally substituted -NH-heteroarylene-. In certain embodiments, the heteroarylene is a triazole. In certain embodiments, the heteroarylene is pyridine. In certain embodiments, the heteroarylene is pyrimidine. In certain embodiments, the heteroarylene is thiadiazole.
the -Z 1 -L 1 - includes a group selected from: wherein R 24 and R 25 are each independently selected from H, optionally substituted C (1-6) - alkyl, optionally substituted fluoroalkyl, and halogen; and each R 21 is independently selected from H, optionally substituted (C 1 -C 6 )alkyl, and optionally substituted alkanoyl.
R 21 is H.
R 24 is C (1-3) -alkyl, or C (1-3) -fluoroalkyl.
the fluoroalkyl is CF 3 .
R 25 is C (1-3) -alkyl, or C (1-3) -fluoroalkyl.
the fluoroalkyl is CF 3 .
the linker includes a polypeptide scaffold where some or all of the sidechain groups of the amino acid residues of such a polypeptide scaffold have been modified to attach a X binding moiety (e.g., as described herein). It is understood that X binding moieties (e.g., as described herein) can be conjugated to amino acid residues, such as Asp, Lys, Orn, Glu, and Ser, of a polypeptide containing linker via a convenient conjugation chemistry.
the linker contains a polylysine polypeptide. In some embodiments, the linker contains a polyornithine polypeptide. In some embodiments, the linker contains a polyserine polypeptide. In some embodiments, the linker contains a polyaspartate polypeptide.
the polypeptide backbone of such a linker can be a randomly polymerized polymer having an average length, or a polymer of defined length prepared e.g., in a controlled stepwise fashion. In certain embodiments, the polypeptide linker has a length of 10-100 amino acid residues, such as 20-90, or 20-50 amino acid residues.
the N-terminal or C-terminal of the polypeptide linker is modified to include a linking moiety to an additional X binding moiety (e.g., as described herein).
the N-terminal or C-terminal of the polypeptide linker segment is modified with one or more linking moieties (e.g., as described herein) suitable for attachment to a protein construct (Y) including a polypeptide that specifically binds an autoantibody [0478]
a “linker” or linking moiety is derived from a molecule with two reactive termini, one for conjugation to a moiety of interest (Y), e.g., a biomolecule (e.g., an antibody) and the other for conjugation to a moiety (noted as X) that binds to a ASGPR cell surface receptor.
the polypeptide conjugation reactive terminus of the linker is in some cases a site that is capable of conjugation to the polypeptide through a cysteine thiol or lysine amine group on the polypeptide, and so is can be a thiol-reactive group such as a maleimide or a dibromomaleimide, or as defined herein, or an amine-reactive group such as an active ester (e.g., perfluorophenyl ester or tetrafluorophenyl ester), or as defined herein.
a thiol-reactive group such as a maleimide or a dibromomaleimide, or as defined herein
an amine-reactive group such as an active ester (e.g., perfluorophenyl ester or tetrafluorophenyl ester), or as defined herein.
the linker L comprises one or more straight or branched-chain carbon moieties and/or polyether (e.g., ethylene glycol) moieties (e.g., repeating units of -CH 2 CH 2 O-), and combinations thereof.
these linkers optionally have amide linkages, urea or thiourea linkages, carbamate linkages, ester linkages, amino linkages, ether linkages, thioether linkages, sulfhydryl linkages, heteroaryl linkages, or other hetero functional linkages.
the linker comprises one or more of carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof.
the linker comprises one or more of an ether bond, thioether bond, amine bond, amide bond, carbon-carbon bond, carbon-nitrogen bond, carbon-oxygen bond, carbon-sulfur bond, and combinations thereof.
the linker comprises a linear structure.
the linker comprises a branched structure.
the linker comprises a cyclic structure.
the linker comprises one or more heteroaryl cyclic structures, e.g., a triazole, such as a 1,2,3-traizole.
L is between about 10 ⁇ and about 20 ⁇ in length.
L is between about 15 ⁇ and about 20 ⁇ in length. In certain embodiments, L is about 15 ⁇ in length. In certain embodiments, L is about 16 ⁇ in length. In certain embodiments, L is about 17 ⁇ in length. [0481] In certain embodiments, L is a linker between about 5 ⁇ and about 500 ⁇ . In certain embodiments, L is between about 10 ⁇ and about 400 ⁇ . In certain embodiments, L is between about 10 ⁇ and about 300 ⁇ . In certain embodiments, L is between about 10 ⁇ and about 200 ⁇ . In certain embodiments, L is between about 10 ⁇ and about 100 ⁇ .
L is between about 10 ⁇ and about 20 ⁇ , between about 20 ⁇ and about 30 ⁇ , between about 30 ⁇ and about 40 ⁇ , between about 40 ⁇ and about 50 ⁇ , between about 50 ⁇ and about 60 ⁇ , between about 60 ⁇ and about 70 ⁇ , between about 70 ⁇ and about 80 ⁇ , between about 80 ⁇ and about 90 ⁇ , or between about 90 ⁇ and about 100 ⁇ .
L is a linker between about 5 ⁇ and about 500 ⁇ , which comprises an optionally substituted arylene linked to X, an optionally substituted heteroarylene linked to X, an alkylene group linked to X, or a heteroatom linked to X.
L is a linker between about 10 ⁇ and about 500 ⁇ , which comprises an optionally substituted arylene linked to X, or optionally substituted heteroarylene linked to X, an alkylene group linked to X, or a heteroatom linked to X. In certain embodiments, L is a linker between about 10 ⁇ and about 400 ⁇ , which comprises an optionally substituted arylene linked to X, or optionally substituted heteroarylene linked to X, an alkylene group linked to X, or a heteroatom linked to X.
L is a linker between about 10 ⁇ and about 200 ⁇ , which comprises an optionally substituted arylene linked to X, or optionally substituted heteroarylene linked to X, an alkylene group linked to X, or a heteroatom linked to X.
linker L separates X and Y (or Z 1 ) by a chain of 4 to 500 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 4 to 50 consecutive atoms.
linker L separates X and Y (or Z 1 ) by a chain of 6 to 50 consecutive atoms, by a chain of 11 to 50 consecutive atoms, by a chain of 16 to 50 consecutive atoms, by a chain of 21 to 50 consecutive atoms, by a chain of 26 to 50 consecutive atoms, by a chain of 31 to 50 consecutive atoms, by a chain of 36 to 50 consecutive atoms, by a chain of 41 to 50 consecutive atoms, or by a chain of 46 to 50 consecutive atoms.
linker L separates X and Y (or Z 1 ) by a chain of 6 to 50 consecutive atoms.
linker L separates X and Y (or Z 1 ) by a chain of 11 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 16 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 21 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 26 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 31 to 50 consecutive atoms.
linker L separates X and Y (or Z 1 ) by a chain of 36 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 41 to 50 consecutive atoms. In certain embodiments, linker L separates X and Y (or Z 1 ) by a chain of 46 to 50 consecutive atoms.
linker L separates X and Y (or Z 1 ) by a chain of 4 or 5 consecutive atoms, by a chain of 6 to 10 consecutive atoms, by a chain of 11 to 15 consecutive atoms, by a chain of 16 to 20 consecutive atoms, by a chain of 21 to 25 consecutive atoms, by a chain of 26 to 30 consecutive atoms, by a chain of 31 to 35 consecutive atoms, by a chain of 36 to 40 consecutive atoms, by a chain of 41 to 45 consecutive atoms, or by a chain of 46 to 50 consecutive atoms.
linker L is a chain of 5 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an optionally substituted arylene linked to X, optionally substituted heteroarylene linked to X, optionally substituted alkylene linked to X, or heteroatom linked to X.
linker L is a chain of 7 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an optionally substituted arylene linked to X, optionally substituted heteroarylene linked to X, optionally substituted alkylene linked to X, or heteroatom linked to X.
linker L is a chain of 10 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an optionally substituted arylene linked to X, optionally substituted heteroarylene linked to X, optionally substituted alkylene linked to X, or heteroatom linked to X. In certain embodiments, linker L is a chain of 15 to 400 consecutive atoms separating X and Y (or Z 1 ) and which comprises an optionally substituted arylene linked to X, optionally substituted heteroarylene linked to X, optionally substituted alkylene linked to X, or heteroatom linked to X.
linker L is a chain of 5 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an alkylene, a heteroatom, or optionally substituted heteroarylene linked to X. In certain embodiments, linker L is a chain of 7 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an alkylene, a heteroatom, or optionally substituted heteroarylene linked to X. In certain embodiments, linker L is a chain of 10 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an comprises an alkylene, a heteroatom, or optionally substituted heteroarylene linked to X.
linker L is a chain of 15 to 400 consecutive atoms separating X and Y (or Z 1 ) and which comprises an alkylene, a heteroatom, or optionally substituted heteroarylene linked to X. [0486] In certain embodiments, linker L is a chain of 5 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an optionally substituted triazole linked to X. In certain embodiments, linker L is a chain of 7 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an optionally substituted triazole linked to X.
linker L is a chain of 10 to 500 consecutive atoms separating X and Y (or Z 1 ) and which comprises an optionally substituted triazole linked to X. In certain embodiments, linker L is a chain of 15 to 400 consecutive atoms separating X and Y (or Z 1 ) and which comprises an optionally substituted triazole linked to X. [0487] In certain embodiments, linker L is a chain of 16 to 400 consecutive atoms separating X and Y (or Z) and which comprises an optionally substituted arylene linked to X, optionally substituted heteroarylene linked to X, optionally substituted alkylene linked to X, or a heteroatom linked to X.
the linker may be considered as connecting directly to a Z 1 group of a ASGPR binding moiety (X) (e.g., as described herein). In some embodiments of any of formulae (Ia)- (Ip), the linker may be considered as connecting directly to the Z 1 group.
the -Z 1 -L 1 - group e.g., as described herein
the disclosure is meant to include all such configurations of ASGPR binding moiety (X) and linker (L).
L is a linker of formula (II): wherein L 1 and L 3 are independently a linker, and L 2 is a branched linking moiety, wherein L 1 to L 3 together provide a linear or branched linker between X and Y; a, b and c are independently 0 or 1; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y; wherein: when n is 1, a is 1, and b is 0; when n is >1, a is 1, and b is 1.
L 1 to L 3 each independently comprise one or more linking moieties independently selected from –C 1-20 -alkylene–, –NHCO-C 1-6 -alkylene–, – CONH-C 1-6 -alkylene–, –NH C 1-6 -alkylene–, –NHCONH-C 1-6 -alkylene–, – NHCSNH-C 1-6 -alkylene–, – C 1-6 -alkylene–NHCO-, –C 1-6 -alkylene–CONH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–NHCONH-, –C 1-6 - alkylene–NHCSNH-, -O(CH 2 ) p –, –(OCH 2 CH 2 ) p –, –NHCO—, —CONH–, –NHSO 2 –, –SO 2 NH–
any of L 1 -L 3 comprises repeating ethylene glycol moieties (e.g., -CH 2 CH 2 O- or -OCH 2 CH 2 -).
the linker of formula (II) comprises 1 to 25 ethylene glycol moieties, such as 3 to 25, 5 to 25, 7 to 25, 10 to 25, 15 to 25, 17 to 25, 20 to 25 or 22 to 25 ethylene glycol moieties.
the linker of formulae (II) comprises 3 or more ethylene glycol moieties, such as 5 or more, 7 or more, 10 or more, 15 or more, 20 or more, or even more ethylene glycol moieties.
any of L 1 -L 3 comprises one or more triazole linking moieties.
the linker comprises one or more 1,2,3-triazole linking moieties.
the one or more 1,2,3-triazole moieties is selected from one of the following structures: , wherein w1, u1 and q1 are independently 1 to 25 (e.g., 1 to 12, such as 1 to 6).
n is 1, such that b is 0, and the linker is of the formula (IIa): wherein L 1 and L 3 are independently a linker (e.g., as described herein), wherein L 1 to L 3 together provide a linear linker between X and Y; a is 1; c is 0 or 1; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y.
L 1 and L 3 are independently a linker (e.g., as described herein), wherein L 1 to L 3 together provide a linear linker between X and Y; a is 1; c is 0 or 1; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y.
the linear linker has a backbone of 20 or more consecutive atoms covalently linking X to Y via Z 1 , such as a backbone of 25 or more consecutive atoms, or 30 or more consecutive atoms, and in certain embodiments, up to 100 consecutive atoms.
the linear linker separates X and Y (or Z 1 ) by a chain of 20 to 50 consecutive atoms.
the linear linker separates X and Y (or Z 1 ) by a chain of 21 to 50 consecutive atoms, by a chain of 22 to 50 consecutive atoms, by a chain of 23 to 50 consecutive atoms, by a chain of 24 to 50 consecutive atoms, by a chain of 25 to 50 consecutive atoms, by a chain of 26 to 50 consecutive atoms, by a chain of 27 to 50 consecutive atoms, by a chain of 28 to 50 consecutive atoms, or by a chain of 29 to 50 consecutive atoms.
the linear linker separates X and Y (or Z 1 ) by a chain of 30 to 60 consecutive atoms.
the linear linker separates X and Y (or Z 1 ) by a chain of 31 to 60 consecutive atoms. In certain embodiments, the linear linker separates X and Y (or Z 1 ) by a chain of 32 to 60 consecutive atoms. In certain embodiments, the linear linker separates X and Y (or Z 1 ) by a chain of 33 to 60 consecutive atoms. In certain embodiments, the linear linker separates X and Y (or Z 1 ) by a chain of 34 to 60 consecutive atoms. In certain embodiments, the linear linker L separates X and Y (or Z 1 ) by a chain of 35 to 50 consecutive atoms.
the linear linker L separates X and Y (or Z 1 ) by a chain of 36 to 50 consecutive atoms. In certain embodiments, the linear linker L separates X and Y (or Z 1 ) by a chain of 41 to 50 consecutive atoms. In certain embodiments, the linear linker L separates X and Y (or Z 1 ) by a chain of 46 to 50 consecutive atoms. [0495] In certain other embodiments of formula (II), n is 2 or more, such that L 1 to L 3 together provide a branched linker between X and Y.
n is 2 or more, and L 2 is selected from: wherein each x and y are independently 1 to 10.
L 1 -L 2 comprises a backbone of 14 or more consecutive atoms between X and the branching atom, such as 14 to 50, 14 to 40, 14 to 35 or 14 to 30 consecutive atoms between X and the branching atom.
L 3 comprises a backbone of 10 to 80 consecutive atoms, such as 12 to 70, 12 to 60, or 12 to 50 consecutive atoms.
L comprises of 12 to 70, 12 to 60, 12 to 50, or 10 to 60 consecutive linear or branched chain atoms.
L 3 comprises a linking moiety selected from (C 10 -C 20 -alkylene (e.g., C 12 -alkylene), or –(OCH 2 CH 2 ) p –, where p is 1 to 25, such as 3 to 25, 5 to 24, 7 to 25, 10 to 25, 15 to 25 or 20 to 24.
L is of formula (Iib): wherein each L 1 to L 5 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; a, b, c, d, and e are each independently 0, 1, or 2; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y; wherein: when n is 1, a is 1, and c is 0; and when n is >1, a is 1, and c is 1.
L is of formula (IIb’): wherein: each L 1 to L 6 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y.
L is of formula (IIb’): wherein: each L 1 to L 6 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; a, b, c, d, and e are each independently 1, 2, 3, 4, or 5; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y.
each L 1 to L 5 independently comprises one or more linking moieties independently selected from –C 1-20 -alkylene–, –NHC(O)-C 1-6 -alkylene–, –C(O)NH-C 1-6 -alkylene–, —NH- C 1-6 -alkylene–, –NHC(O)NH-C 1-6 -alkylene–, –NHC(S)NH-C 1-6 -alkylene–, –C 1-6 -alkylene–NHC(O)-, – C 1-6 -alkylene–C(O)NH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–NHC(O)NH-, –C 1-6 -alkylene–NHC(S)NH- , -O(CH 2 ) p –, –(OCH 2 CH 2 ) p –, –NHC(O)–, –, –NHC(S)
each L 1 to L 5 is independently selected from –C 1-20 -alkylene–, – NHC(O)-C 1-6 -alkylene–, –C(O)NH-C 1-6 -alkylene–, –NH-C 1-6 -alkylene–, –NHC(O)NH-C 1-6 -alkylene–, – NHC(S)NH-C 1-6 -alkylene–, –C 1-6 -alkylene–NHC(O)-, –C 1-6 -alkylene–C(O)NH-, –C 1-6 -alkylene–NH-, – C 1-6 -alkylene–NHC(O)NH-, –C 1-6 -alkylene–NHC(S)NH-, -O(CH 2 ) p –, –(OCH 2 CH 2 ) p –, –NHC(O)–, – C(O)NH–, – C(O)NH–,
-(L 1 ) a - comprises an optionally substituted alkyl or ethylene glycol linking moiety.
L 1 comprises an optionally substituted -C 1-6 -alkylene–.
L 1 comprises an ethylene glycol linking moiety.
L 1 is independently selected from: -C 1-6 -alkylene–, –(CH 2 CH 2 O) t –, –-C 1-6 -alkylene-NR 4 CO–, –C 1-6 -alkyleneCONH–,or OCH 2 , wherein t is 1 to 20; and R 4 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl.
L 1 is -C 1-6 -alkylene–, such as -C 1-3 -alkylene–.
L 1 is – (CH 2 CH 2 O) t –, where t is 1 to 20, such as 1 to 15, 1 to 10, 1 to 8, 1 to 6, or 1 to 4.
L 1 is –-C 1-6 -alkylene-NR 4 CO–.
L 1 is –C 1-6 -alkyleneCONH–.
L 1 is or OCH 2 .
one or more L 1 is independently –CH 2 O–; – wherein: R 13 is selected from H, halogen, OH, optionally substituted (C 1 -C 6 )alkyl, optionally substituted (C 1 -C 6 )alkoxy, COOH, NO 2 , CN, NH 2 , -N(R 21 ) 2 , -OCOR 21 , -COOR 21 , -CONHR 21 , and -NHCOR 21 ; each r independently 0 to 20, and any of the L 1 moieties are optionally further substituted.
L 2 is independently selected from: is 1 to 10, u is 0 to 10, w is 1 to 10, and R 4’ is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl.
L 2 is –NR 4’ CO-C 1-6 -alkylene–.
L 2 is – CONR 4’ -C 1-6 -alkylene.
[0510] In certain embodiments, [0511] In certain embodiments, [0512] In certain embodiments, is 1. [0513] In certain embodiments, [0514] In certain embodiments, [0515] In certain embodiments, L 2 is -OCH 2 -.
L 2 is (OCH 2 CH 2 ) q –, and q is 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 certain embodiments, q is 2 to 8, such as 2 to 6 , 4 to 6, or 2 to 4. [0516] In certain embodiments of formula (Iib), L 4 is absent or independently selected from -C 1-6 -alkylene–, –(CH 2 CH 2 O) t –, –-C 1-6 -alkylene-NHCO–, –C 1-6 -alkyleneCONH–,or OCH 2 , wherein t is 1 to 20. In certain embodiments, L 4 is absent.
L 4 is -C 1-6 -alkylene–. In certain embodiments, L 4 is –(CH 2 CH 2 O) t –, where t is 1 to 20, such as 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4 or 1 to 3. In certain embodiments, L 4 is –-C 1-6 -alkylene-NHCO–. In certain embodiments, L 4 is –C 1-6 -alkyleneCONH–. In certain embodiments, L 4 is OCH 2 . [0518] In some embodiments of the subject compounds, n is 1 and L 3 in formula (Iib) is absent.
L 3 of formula (Iib) is a branched linking moiety.
L 3 is a branched linking moiety, e.g., a divalent, or a trivalent linking moiety.
an L 3 linking moiety can be of the one of the following general formula: .
the branched linking moiety can be of higher valency and be described by one of the one of the following general formula: where any two L 3 groups can be directed linked or connected via optional linear linking moieties (e.g., as described herein).
the branched linking moiety can include one, two or more L 3 linking moieties, each being trivalent moieties, which when linked together can provide for multiple branching points for covalent attachment of the ligands and be described by the following general formula: where t is 0 to 500, such as 0 to 100, 0 to 20, or 0 to 10.
the branched linking moiety comprises one or more of: an amino acid residue (e.g., Asp, Lys, Orn, Glu), N-substituted amido (-N(-)C(O)-), tertiary amino, polyol (e.g., O-substituted glycerol), and the like.
an amino acid residue e.g., Asp, Lys, Orn, Glu
N-substituted amido e.g., Asp, Lys, Orn, Glu
N-substituted amido e.g., Asp, Lys, Orn, Glu
N-substituted amido e.g., Asp, Lys, Orn, Glu
N-substituted amido e.g., Asp, Lys, Orn, Glu
N-substituted amido e.g., Asp, Lys, Orn, Glu
each x and y are each independently 1 to 10, such as 1-6, 1-3, e.g., 1 or 2. In certain embodiments, each x is 1, 2 or 3, e.g., 2. [0525]
L 5 is selected from –CH 2 O–; –(CH 2 CH 2 O) t –, wherein: R 13 is selected from H, halogen, OH, optionally substituted (C 1 -C 6 )alkyl, optionally substituted (C 1 -C 6 )alkoxy, COOH, NO 2 , CN, NH 2 , -N(R 21 ) 2 , -OCOR 21 , -COOR 21 , -CONHR 21 , and - NHCOR 21 ; and each r independently 0 to 20, and any of the L 5 moieties are optionally further substituted.
L 5 is –CH 2 O–. In certain embodiments, L 5 is –(CH 2 CH 2 O) t –, where t is 1 to 20, such as 1-15, 1-12, 1-10, 1-8, 1-6, or 1 to 4. In certain embodiments, L 5 is –NR 4 CO–, where R 4 is H, or optionally substituted (C 1 -C 6 )alkyl. In certain embodiments, L 5 is -C 1-6 -alkylene–. [0527] In certain embodiments, L 5 is , where r is 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5.
each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5 and R 13 is H, or optionally substituted (C 1 -C 6 )alkyl.
20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5 and R 13 is H, or optionally substituted (C 1 -C 6 )alkyl.
20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5, and R 13 is H, or optionally substituted (C 1 -C 6 )alkyl.
each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5, and R 13 is H, or optionally substituted (C 1 -C 6 )alkyl.
each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5.
L 5 is , where each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5.
L 5 is , where each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5.
L 5 is , where each r is independently 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5. [0536] In certain embodiments, L 5 is , where r is 0 to 20, such as 0 to 15, 0 to 10, 0 to 8, or 0 to 5.
L 5 comprises one or more of: an amino acid residue (e.g., Asp, Lys, Orn, Glu), an amino acid analogue, N-substituted amido (-N(-)C(O)-), tertiary amino, polyol (e.g., O-substituted glycerol), and the like.
an amino acid residue e.g., Asp, Lys, Orn, Glu
an amino acid analogue e.g., N-substituted amido (-N(-)C(O)-), tertiary amino, polyol (e.g., O-substituted glycerol), and the like.
Analogs of an amino acid include but not limited to, unnatural amino acids, as well as other modifications known in the art.
the amino acid includes L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art
L 1 -L 5 comprises one or more of the following units: R a , where R a is (C 1 -C 6 )alkyl or substituted (C 1 -C 6 )alkyl, e.g., a (C 1 -C 6 )alkyl optionally substituted with amine, a tertiary amine, optionally substituted alkoxy, optionally substituted carboxyl, optionally substituted aryl, or optionally substituted heteroaryl. It is understood that R a can be linked to a M6PR binding moiety.
the linker includes a polypeptide scaffold where some or all of the sidechain groups of the amino acid residues have been modified to attach a ASGPR binding moiety (e.g., as described herein). It is understood that ASGPR binding moieties (e.g., as described herein) can be conjugated to amino acid residues, such as Asp, Lys, Orn, Glu, and Ser, of a polypeptide containing linker via a convenient conjugation chemistry.
the linker contains a polylysine polypeptide.
the linker contains a polyornithine polypeptide.
the linker contains a polyserine polypeptide.
the linker contains a polyaspartate polypeptide.
the polypeptide can be a randomly polymerized polymer having an average length, or a polymer of defined length prepared e.g., in a controlled stepwise fashion.
the polypeptide linker segment has a length of 10-100 amino acid residues, such as 20-90, or 20-50 amino acid residues.
the N-terminal or C-terminal of the polypeptide linker segment is modified to include a linking unit to an additional M6PR binding moiety (e.g., as described herein).
the N-terminal or C-terminal of the polypeptide linker segment is modified with one or more linking units (e.g., as described herein) suitable for attachment to a Y moiety of interest.
a is 1.
at least one of b, c, d, and e is not 0.
b is 1 or 2.
c is 1 or 2.
e is 1 or 2.
b, d and e are independently 1 or 2.
a, b, d, and e are each 1, and c is 0.
the linker comprises 20 to 100 consecutive atoms, such as 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40 or 20 to 30 consecutive atoms.
the linker comprises 25 to 100 consecutive atoms, such as 30 to 100, 35 to 100, 40 to 100, 45 to 100, 50 to 100, 55 to 100, 60 to 100, 65 to 100, 70 to 100, 75 to 100, 80 to 100, 85 to 100, 90 to 100, or 95 to 100 consecutive atoms.
the linker comprises 25 or more consecutive atoms, such as 26 or more, 27 or more, 28 or more, 29 or more or 30 or more consecutive atoms.
the linker comprises 30 or more consecutive atoms, such as 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37, or more, 38 or more, 39 or more, 40 or even more consecutive atoms.
ASGPR binding compounds of this disclosure having a particular configuration with a linker of desired valency and length can specifically bind with high affinity to both the ASGPR and a target simultaneously, and exhibit high uptake activity of a target.
the conjugates of this disclosure can thus provide for sequestering of a target protein in the cell’s lysosome and degrading of the target protein.
conjugates of trivalent ASGPR binding compounds with 14 or more atoms between the ASGPR binding moiety (e.g., Z 1 group) and the branching point of the linker can exhibit superior uptake of cells as compared to conjugates of trivalent ASGPR binding compounds with shorter linkers (e.g., linkers less than 14 atoms) between the ASGPR binding compound (e.g., Z 1 group) and the branching point.
linkers e.g., linkers less than 14 atoms
a conjugate having a 1-triazole moiety and a short linkage (e.g., 6 atoms) from the ASGPR ligand to the branching point of the ligand (I-157, linker length of 6 atoms to branching point) exhibited less uptake activity in HepG2 cells than the conjugate having a 1-triazole moiety and a longer linkage (e.g., 14 atoms) from the ASGPR ligand to the branching point (I-143, length of 14 atoms) (see, e.g., FIG.2A).
multivalent ASGPR binding compounds having a certain linker length range between the ASGPR binding moiety and the linker branching point which provides desirable binding and cellular uptake of a bound target.
conjugates of multivalent ASGPR binding compounds with 12 or more atoms between the branching point of the linker and the Y moiety of interest can exhibit superior uptake of cells as compared to conjugates of multivalent ASGPR binding compounds with shorter linkers (e.g., linkers less than 12 atoms) between the branching point of the linker and the Y moiety of interest.
conjugates of ASGPR binding compounds having more than 12 atoms between the branching point of the linker and Y exhibit comparable uptake activity.
conjugates having longer linkers between the ASGPR linker and Y e.g., conjugates of compounds I- 137, having 81 atoms between the branching point and Y; and I-129, having 33 atoms between the branching point and Y
a reference conjugate e.g., conjugate of compound I-124, having 12 atoms between the branching point and Y
each branch of the linker comprises a linear linker of 14 or more consecutive atoms to covalently link via Z 1 each X moiety to a branching point of the linker.
each branch of the linker comprises a linear linker of 15 or more consecutive atoms to the branching point.
each branch of the linker comprises a linear linker of 16 or more consecutive atoms to the branching point.
each branch of the linker comprises a linear linker of 17 or more consecutive atoms to the branching point.
each branch of the linker comprises a linear linker of 18 or more consecutive atoms to the branching point. In certain embodiments, each branch of the linker comprises a linear linker of 19 or more consecutive atoms to the branching point.
the linker is a branched linker comprising branches covalently linking via Z 1 each X moiety to a branching point of the linker, and a linear linker covalently linking the branching point to Y. In certain embodiments, the linear linker covalently linking the branching point to Y is 12 or more consecutive atoms.
the linear linker covalently linking the branching point to Y is 15 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 20 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 25 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 30 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 40 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 50 or more consecutive atoms.
the linear linker covalently linking the branching point to Y is 60 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 70 or more consecutive atoms. In certain embodiments, the linear linker covalently linking the branching point to Y is 80 or more consecutive atoms.
Exemplary linkers and linking moieties [0548] Exemplary linkers and linking moieties that can be utilized in the preparation of compounds of this disclosure (e.g., that link the ASGPR ligand (X) to the moiety of interest (Y)) are shown in Tables 9-11. [0549] In certain embodiments, the linker is a linear linker or linking moiety as shown in Table 9.
Table 10 includes various linker component synthetic precursors (e.g., linear and branched linker precursors) that can be utilized in the preparation of the subject compounds.
linker component synthetic precursors e.g., linear and branched linker precursors
the linker is a branched linker or linking moiety as shown in Table 11.
Y is a chemoselective ligation group, or a precursor thereof.
a chemoselective ligation group is a group having a reactive functionality or function group capable of conjugation to a compatible group of a second moiety.
chemoselective ligation groups may be one of a pair of groups associated with a conjugation chemistry such as azido-alkyne click chemistry, copper free click chemistry, Staudinger ligation, tetrazine ligation, hydrazine-iso-Pictet-Spengler (HIPS) ligation, cysteine-reactive ligation chemistry (e.g., thiol-maleimide, thiol-haloacetamide or alkyne hydrothiolation), amine-active ester coupling, tyrosine specific conjugation chemistry (e.g., e-Y-CLICK), methionine specific conjugation chemistry (e.g., oxaziridine-based or ReACT chemistry), reductive amination, dialkyl squarate chemistry, etc.
a conjugation chemistry such as azido-alkyne click chemistry, copper free click chemistry, Staudinger ligation, t
Chemoselective ligation groups that may be utilized in linking two moieties, include, but are not limited to, amino (e.g., a N-terminal amino or a lysine sidechain group of a polypeptide), azido, aryl azide, alkynyl (e.g., ethynyl or cyclooctyne or derivative), active ester (e.g., N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester or PFP ester or thioester), haloacetamide (e.g., iodoacetamide or bromoacetamide), chloroacetyl, bromoacetyl, bromomethyl-aryl, chloromethyl-aryl, bromomethyl- heteroaryl, chloromethyl-heteroaryl, hydrazide, maleimide, vinyl sulfone, 2-sulfonyl pyridine,
chemoselective ligation group is capable of spontaneous conjugation to a compatible chemical group when the two groups come into contact under suitable conditions (e.g., copper free Click chemistry conditions). In some instances, the chemoselective ligation group is capable of conjugation to a compatible chemical group when the two groups come into contact in the presence of a catalyst or other reagent (e.g., copper catalyzed Click chemistry conditions).
the chemoselective ligation group is a photoactive ligation group.
a diazirine group can form reactive carbenes, which can insert into C-H, N-H, and O-H bonds of a second moiety.
Y is a precursor of the reactive functionality or function group capable of conjugation to a compatible group of a second moiety.
a carboxylic acid is a precursor of an active ester chemoselective ligation group.
Y is a reactive moiety capable forming a covalent bond to a polypeptide (e.g., with an amino acid sidechain of a polypeptide having a compatible reactive group).
Y is a thio-reactive chemoselective ligation group (e.g., as described in Table 12). In certain embodiments, Y can produce a residual moiety Z resulting from the covalent linkage of a thiol-reactive chemoselective ligation group to one or more cysteine residue(s) of a protein, e.g., Ab.
Y is a Cys-reactive chemoselective ligation group (e.g., a maleimide derivative as described in table 12).
the Cys-reactive chemoselective ligation group includes a maleimide group.
the chemoselective ligation group includes a maleimide group of Table 12, e.g., mal-1 to mal-7.
Y is an amino-reactive chemoselective ligation group (e.g., as described in Table 12).
Y can produce a residual moiety Z resulting from the covalent linkage of an amine-reactive chemoselective ligation group to one or more lysine residue(s) a protein, e.g., Ab.
Y is a Lys-reactive chemoselective ligation group (e.g., an active ester as described in table 12).
the Lys-reactive chemoselective ligation group is a PFP ester.
Exemplary chemoselective ligation groups, and synthetic precursors thereof, which may be adapted for use in the compounds of this disclosure are shown in Table 12.
Table 12 the can represent a point of attachment of Y to a linking moiety or a linked X moiety.
Table 12a shows exemplary residual moieties, wherein the “***” indicates the point of attachment of Y.
This disclosure includes compounds of formula (I) which can include: (1) one or more particular ASGPR ligand (X) (e.g., as described herein, such as ligands X1-X20 of Tables 1-4) or a particular ASGPR ligand (X) (e.g., as described herein), (2) a linker including one or more linking moieties (e.g., as described herein, such as any one or more of the linking moieties of Tables 8 to 10); and (3) a chemoselective ligation group (Y) e.g., as described herein, such as any one of the groups of Table 12).
ASGPR ligand e.g., as described herein, such as ligands X1-X20 of Tables 1-4
a linker including one or more linking moieties e.g., as described herein, such as any one or more of the linking moieties of Tables 8 to 10
Y chemoselective ligation
the chemoselective ligation group can be tailored to provide linkages which confer additional benefits, such as, but not limited to, stability of the conjugate.
the chemoselective ligation group comprises:
Table 13 illustrates various monovalent ligand-linker compounds for use in conjugates of the disclosure.
Tables 14 illustrates various multivalent ligand-linker compounds for use in conjugates of the disclosure.
the compound of formula (I) is an ASGPR binding compound as described in International Application No. WO/2023288033, filed July 14, 2022, and the disclosure of which is herein incorporated by reference in its entirety.
the following Tables illustrate several exemplary ASGPR binding compounds of this disclosure that include a chemoselective ligation group, or a precursor thereof. It is understood that this disclosure includes Y (e.g., as described herein) conjugates of each of the exemplary compounds of Tables 13-23. For example, conjugates where the chemoselective ligation group has been conjugated to a different Y, such as a biomolecule or a small molecule ligand for a target protein.
the chemoselective ligation group of such compounds can be utilized to connect to another Y moiety of interest (e.g., as described below). It is understood that any of these compounds can also be prepared de novo to include an alternative Y moiety of interest (e.g., as described below) rather than the chemoselective ligation group.
such compounds are referred to as a conjugate, e.g., a biomolecule conjugate that specifically binds a target protein.
the present disclosure is meant to encompass stereoisomers of any one of the compounds described herein.
the compound includes an enantiomer of the D- N- acetylgalactosamine (GalNAc), or an analog or derivative of GalNAc.
GalNAc D- N- acetylgalactosamine
Table 19 illustrates exemplary ASGPR binding compounds of this disclosure that include a binding moiety, or a precursor thereof.
Table 20 illustrates exemplary trivalent ASGPR binding intermediate compounds of this disclosure including X groups of formula (Ie).
Table 21 illustrates exemplary monovalent ASGPR binding intermediate compounds of this disclosure that include a promoiety and X groups that are of formula (Ib).
Table 22 illustrates exemplary ASGPR binding intermediate compounds of this disclosure that include X groups that are of formula (In).
Table 23 illustrates exemplary ASGPR binding intermediate compounds.
the present disclosure is meant to encompass stereoisomers of any one of the compounds described herein.
the compound includes an enantiomer of the D-N- acetylgalactosamine (GalNAc), or an analog or derivative of GalNAc.
Conjugates with Moiety of Interest [0580]
the compounds of this disclosure can be referred to as a conjugate, e.g., when the moiety of interest (Y) is a molecule (e.g., as described herein).
conjugates can be prepared by conjugation of a chemoselective ligation group of any one of the compounds described herein with a compatible reactive group of a molecule Y.
the compatible group of the molecule Y can be introduced by modification prior to conjugation, or can be a group present in the molecule.
conjugates can be prepared de novo, e.g., via modification of a Y molecule of interest starting material to introduce a linker, e.g., to which a ligand X can be attached.
the moiety of interest to which the ASGPR binding moiety is linked is a biomolecule.
the moiety of interest is a biomolecule.
the biomolecule is selected from peptide, protein, polynucleotide, polysaccharide, glycan, glycoprotein, lipid, enzyme, antibody, and antibody fragment.
the moiety of interest Y is selected from small molecule, small molecule drug, chemotherapeutic agent, cytotoxic agent, diagnostic agent, dye, fluorophore, and the like.
the moiety of interest is a molecule that specifically binds to a target of interest, i.e., a target-binding moiety.
the conjugates of this disclosure can provide for cellular uptake of the target after it non-covalently binds to the conjugate, and/or degradation.
conjugates of this disclosure having a particular configuration of ASGPR binding moiety of a desired affinity, with a linker of desired valency and length can specifically bind with high affinity to both the ASGPR and the target simultaneously.
the conjugates of this disclosure can thus provide for sequestering of a target protein in the cell’s lysosome and degrading of the target protein.
the moiety of interest is a molecule that does not bind to an extracellular target, but rather is a molecule that is itself desirable to deliver intracellularly.
the moiety of interest is selected from enzymes (e.g., lysosomal enzyme), a nanoparticle, a viral composition (e.g., viral particle), therapeutic protein, therapeutic antibodies and cytotoxic agents.
enzymes e.g., lysosomal enzyme
a nanoparticle e.g., a viral composition
therapeutic protein e.g., therapeutic antibodies and cytotoxic agents.
the moiety of interest is a lysosomal enzyme for delivery to a cell for use in enzyme replacement therapy, such as acid alpha-glucosidase (GAA).
GAA acid alpha-glucosidase
Lysosomal enzymes of interest that may be adapted for use in conjugates of this disclosure include, but are not limited to, acid alpha-glucosidase, acid beta-galactosidase-1, acid sphingomyelinase, alpha-D-mannosidase, alpha- fucosidase, alpha-galactosidase A, alpha-glucosaminide acetyltransferase, alpha-glucosidase, alpha-L- iduronidase, alpha-N-acetylgalactosaminidase, alpha-acetylglucosaminidase, alpha-D-neuraminidase, arylsulfatase A, arylsulfatase B, beta-galactosidase, beta-glucuronidase, beta-mannosidase, cathepsin D, cathepsin K, ceramidase, cysti
aspects of this disclosure include compounds of formula (I) where the moiety of interest Y is a selected from small molecule, dye, fluorophore, monosaccharide, disaccharide, trisaccharide, and biomolecule.
Y is a small molecule that specifically binds to a target molecule, such as a target protein.
Y is a biomolecule.
the biomolecule is selected from protein, polynucleotide, polysaccharide, peptide, glycoprotein, lipid, enzyme, antibody, and antibody fragment.
Y is a biomolecule that specifically binds to a target molecule, such as a target protein.
the compounds of this disclosure can, in certain embodiments, be referred to as a conjugate, e.g., when the moiety of interest (Y) is a molecule such as a biomolecule, where the conjugate can be derived from a conjugation or coupling reaction between a chemoselective ligation group and a compatible group on the biomolecule.
the biomolecule is conjugated via a naturally occurring group of the biomolecule.
the biomolecule is conjugated via a compatible functional group that is introduced into the biomolecule prior to chemoselective conjugation.
the linking moiety between X and Y incorporates the residual group (e.g., Z) that is the product of the chemoselective ligation chemistry.
Z residual group
aspects of this disclosure include compounds of formula (I) where the moiety of interest Y is a moiety that specifically binds to a target molecule, such as a target protein.
the target protein can be the target protein is a membrane bound protein or an extracellular protein.
Y is a biomolecule that specifically binds to a target protein.
the conjugate includes a moiety of interest Y that specifically binds a target protein, and can find use in methods of cell uptake or internalization of the target protein via binding to the cell surface receptor, and eventual degradation of the target protein.
Y is an aptamer that specifically binds to a target molecule, such as a target protein.
Y is a peptide or protein (e.g., peptidic binding motif, protein domain, engineered polypeptide, or glycoprotein) that specifically binds to a target molecule, such as a target protein.
Y is an antibody or antibody fragment that specifically binds to a target molecule, such as a target protein.
Y is a polynucleotide or oligonucleotide that specifically binds to a target molecule, such as a target protein or a target nucleic acid.
one Y biomolecule is conjugated to a single moiety (X) that specifically binds to the cell surface receptor (e.g., ASGPR) via a linker L.
Y can be conjugated to two or more (Xn-L)- groups, wherein each (Xn-L)- group may itself be monovalent or multivalent (e.g., bivalent, trivalent, etc.).
the ratio of linked (Xn-L)- groups to biomolecule can be referred to as 2 or more.
Y is a moiety that specifically binds the target protein and the compound is a conjugate of formula (III’): wherein: n is 1 to 20; m is an average loading of 1 to 80; each X is a moiety that binds to a cell surface ASGPR; each L is a linker; each Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group to a compatible group of Y; and Y is a moiety of interest binds the target protein.
Y is an antibody or an antibody fragment.
Y is an antibody or antibody fragment that specifically binds the target protein and the compound is a conjugate of formula (III): wherein: n is 1 to 20; m is an average loading of 1 to 80; each X is a moiety that binds to a cell surface ASGPR; each L is a linker; each Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group to a compatible group of Ab; and Ab is the antibody or antibody fragment that specifically binds the target protein.
n is 1 to 6.
n is 1, such that the antibody is conjugated to a monovalent ligand and the linker is of the formula (IIa) (e.g., as described herein). In certain cases, n is at least 2, such that the antibody is conjugated to a multivalent ligand. In certain cases, n is 2. In certain cases n is 3. [0595] In certain embodiments of the conjugate of formula (III), Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation moiety of Table 12 (e.g., Table 12a). [0596] In certain embodiments of formula (III) or (III’), each X is independently of formula (Ib) (e.g., as described herein). In certain embodiments, each X is independently selected from a compound of Table 1. In certain embodiments, each X is independently selected from one of the following compounds:
each X is independently selected from one of the following compounds: wherein R 5 and R 4 independently H or a promoiety, or R 5 and R 4 are cyclically linked to form a promoiety; n1 and n2 are each independently an integer from 1 to 6; and Y 4 is a suitable counterion. In some embodiments, Y 4 is sodium.
n is 1 and X is: .
each X is independently of the formula (Ic) (e.g., as described herein).
each X is independently selected from a compound of Table 2 or 2a.
each X is independently of formula (Id) (e.g., as described herein).
each X is independently selected from a compound of Table 3 or 3a.
each X is independently selected from a compound of Table 4.
each X is a compound of Table 5.
each X is independently selected from one of the following compounds: [0602]
L is a linker of formula (II) (e.g., as described herein).
n is 1 to 6. In certain embodiments, n is 1, such that the antibody is conjugated to a monovalent ASGPR ligand and the linker is of the formula (IIa) (e.g., as described herein). In certain embodiments, n is at least 2, such that the antibody is conjugated to a multivalent ASGPR ligand. In certain embodiments, n is 2. In certain cases n is 3. [0604] In certain embodiments of the conjugate of formula (III) or (III’), Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation moiety of Table 9.
Z is a residual moiety resulting from the covalent linkage of a thiol reactive chemoselective ligation group to one or more cysteine residue(s) of Ab.
the thiol-reactive chemoselective ligation group is a maleimide derivative.
Z is a residual moiety resulting from the covalent linkage of an amine-reactive chemoselective ligation group to one or more lysine residue(s) of Ab.
the amine-reactive chemoselective ligation group is an active ester.
the active ester is a PFP ester.
the conjugates with their linker structures described herein have weaker binding affinity to cell surface receptors. Without being bound to any particular mechanism or theory, such weaker binding affinity may be corrected to longer half-life of the conjugates, and may be useful for tuning (e.g., modifying) the pharmacokinetic properties of the conjugates described herein. In certain embodiments, such weaker binding conjugates still have sufficiently robust uptake.
Conjugates of a polypeptide may be made using a variety of bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC- SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo- KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4- vinylsulfone)benzoate).
bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC- SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo- KMUS,
the conjugates described herein may be prepared using any suitable methods as disclosed in the art (see, e.g., Bioconjugate Techniques (Hermanson ed., 2d ed.2008)).
L is bonded through an amide bond to a lysine residue of the polypeptide.
L is bonded through a thioether bond to a cysteine residue of the polypeptide.
L is bonded through an amide bond to a lysine residue of Ab.
L is bonded through a thioether bond to a cysteine residue of Ab. In certain embodiments of the conjugates described herein, L is bonded through two thioether bonds to two cysteine residues of Ab, wherein the two cysteine residues are from an opened cysteine-cysteine disulfide bond in Ab. In certain embodiments, the opened cysteine-cysteine disulfide bond is an interchain disulfide bond. [0610] In certain embodiments of the conjugates described herein, when L is bonded through an amide bond to a lysine residue of a polypeptide (e.g., an antibody), m is an integer from 1 to 80.
m is an integer from 1 to 80.
conjugation to the polypeptide, or the antibody Ab may be via site- specific conjugation. Site-specific conjugation may, for example, result in homogeneous loading and minimization of conjugate subpopulations with potentially altered antigen-binding or pharmacokinetics.
conjugation may comprise engineering of cysteine substitutions at positions on the polypeptide or antibody, e.g., on the heavy and/or light chains of an antibody that provide reactive thiol groups and do not disrupt polypeptide or antibody folding and assembly or alter polypeptide or antigen binding (see, e.g., Junutula et al., J. Immunol. Meth.2008; 332: 41-52; and Junutula et al., Nature Biotechnol.2008; 26: 925-32; see also WO2006/034488 (herein incorporated by reference in its entirety)).
selenocysteine is cotranslationally inserted into a polypeptide or antibody sequence by recoding the stop codon UGA from termination to selenocysteine insertion, allowing site specific covalent conjugation at the nucleophilic selenol group of selenocysteine in the presence of the other natural amino acids (see, e.g., Hofer et al., Proc. Natl. Acad. Sci. USA 2008; 105: 12451-56; and Hofer et al., Biochemistry 2009; 48(50): 12047-57).
Non-limiting techniques that allow for site-specific conjugation to polypeptides or antibodies include engineering of non-natural amino acids, including, e.g., p-acetylphenylalanine (p-acetyl-Phe), p- azidomethyl-N-phenylalanine (p-azidomethyl-Phe), and azidolysine (azido-Lys) at specific linkage sites, and can further include engineering unique functional tags, including, e.g., LPXTG, LLQGA, sialic acid, and GlcNac, for enzyme mediated conjugation. See Jackson, Org. Process Res.
the term “DAR” refers to the average value of “m” or the loading of the conjugate.
the number of “X” moieties (e.g., folate moieties) per each unit of “Xn-L-” or “Xn-” is represented by “n” in formula (III).
the term “valency” or “valencies” refers to the number of “X” moieties per unit (“n”). It will be understood that loading, or DAR, is not necessarily equivalent to the number of “X” moieties per conjugate molecule.
total valency refers to the total number of “X” moieties per conjugate molecule (n x m; total valency).
DAR loading
the conjugates provided herein may include collections of polypeptides, antibodies or antigen binding fragments conjugated with a range of units, e.g., from 1 to 80.
the average number of units per polypeptide or antibody in preparations of the conjugate from conjugation reactions may be characterized by conventional means such as mass spectroscopy.
the quantitative distribution of DAR (loading) in terms of m may also be determined.
the DAR for a conjugate provided herein ranges from 1 to 80. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 70. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 60. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 50. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 40. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 35.
the DAR for a conjugate provided herein ranges from 1 to 30. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 25. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 20. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 18. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 15. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 12. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 10. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 9.
the DAR for a conjugate provided herein ranges from 1 to 8. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 7. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 6. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 5. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 4. In certain embodiments, the DAR for a conjugate provided herein ranges from 1 to 3. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 12. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 10.
the DAR for a conjugate provided herein ranges from 2 to 9. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 8. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 7. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 6. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 5. In certain embodiments, the DAR for a conjugate provided herein ranges from 2 to 4. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 12. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 10.
the DAR for a conjugate provided herein ranges from 3 to 9. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 8. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 7. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 6. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 5. In certain embodiments, the DAR for a conjugate provided herein ranges from 3 to 4.
the DAR for a conjugate provided herein ranges from 1 to about 8; from about 2 to about 6; from about 3 to about 5; from about 3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8; from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3 to about 3.8; or from about 3.3 to about 3.7.
the DAR for a conjugate provided herein is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or more.
the DAR for a conjugate provided herein is about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9. [0617] In some embodiments, the DAR for a conjugate provided herein ranges from 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, or 2 to 13. In some embodiments, the DAR for a conjugate provided herein ranges from 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, or 3 to 13. In some embodiments, the DAR for a conjugate provided herein is about 1. In some embodiments, the DAR for a conjugate provided herein is about 2.
the DAR for a conjugate provided herein is about 3. In some embodiments, the DAR for a conjugate provided herein is about 4. In some embodiments, the DAR for a conjugate provided herein is about 3.8. In some embodiments, the DAR for a conjugate provided herein is about 5. In some embodiments, the DAR for a conjugate provided herein is about 6. In some embodiments, the DAR for a conjugate provided herein is about 7. In some embodiments, the DAR for a conjugate provided herein is about 8. In some embodiments, the DAR for a conjugate provided herein is about 9. In some embodiments, the DAR for a conjugate provided herein is about 10. In some embodiments, the DAR for a conjugate provided herein is about 11.
the DAR for a conjugate provided herein is about 12. In some embodiments, the DAR for a conjugate provided herein is about 13. In some embodiments, the DAR for a conjugate provided herein is about 14. In some embodiments, the DAR for a conjugate provided herein is about 15. In some embodiments, the DAR for a conjugate provided herein is about 16. In some embodiments, the DAR for a conjugate provided herein is about 17. In some embodiments, the DAR for a conjugate provided herein is about 18. In some embodiments, the DAR for a conjugate provided herein is about 19. In some embodiments, the DAR for a conjugate provided herein is about 20.
the DAR for a conjugate provided herein is about 25. In some embodiments, the DAR for a conjugate provided herein is about 30. In some embodiments, the DAR for a conjugate provided herein is about 35. In some embodiments, the DAR for a conjugate provided herein is about 40. In some embodiments, the DAR for a conjugate provided herein is about 50. In some embodiments, the DAR for a conjugate provided herein is about 60. In some embodiments, the DAR for a conjugate provided herein is about 70. In some embodiments, the DAR for a conjugate provided herein is about 80.
a polypeptide may contain, for example, lysine residues that do not react with the compound or linker reagent.
antibodies do not contain many free and reactive cysteine thiol groups which may be linked to a drug unit; indeed most cysteine thiol residues in antibodies exist as disulfide bridges.
an antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups.
DTT dithiothreitol
TCEP tricarbonylethylphosphine
an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.
the compound is conjugated via a lysine residue on the antibody.
the linker unit or a drug unit is conjugated via a cysteine residue on the antibody.
the amino acid that attaches to a unit is in the heavy chain of an antibody.
the amino acid that attaches to a unit is in the light chain of an antibody.
the amino acid that attaches to a unit is in the hinge region of an antibody.
the amino acid that attaches to a unit is in the Fc region of an antibody.
the amino acid that attaches to a unit is in the constant region (e.g., CH1, CH2, or CH3 of a heavy chain, or CH1 of a light chain) of an antibody.
the amino acid that attaches to a unit or a drug unit is in the VH framework regions of an antibody.
the amino acid that attaches to unit is in the VL framework regions of an antibody.
the DAR (loading) of a conjugate may be controlled in different ways, e.g., by: (i) limiting the molar excess of compound or conjugation reagent relative to polypeptide, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reductive conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the polypeptide, such that the number and position of cysteine residues is modified for control of the number and/or position of linker- drug attachments (such as for thiomabs prepared as disclosed in WO2006/034488 (herein incorporated by reference in its entirety)).
m is 1 to 20, such as 2 to 10, 2 to 8, or 2 to 6. In certain embodiments, m is 10 or less.
m is 2 to 8. In certain embodiments, m is 2 to 6. In certain embodiments, m is an average loading of about 4. [0624] It is to be understood that the preparation of the conjugates described herein may result in a mixture of conjugates with a distribution of one or more units attached to a polypeptide, for example, an antibody. Individual conjugate molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, e.g. hydrophobic interaction chromatography, including such methods known in the art. In certain embodiments, a homogeneous conjugate with a single DAR (loading) value may be isolated from the conjugation mixture by electrophoresis or chromatography.
the target-binding moiety can be any moiety that has an affinity for the target of less than 1 ⁇ M, such as 300nM or less, 100nM or less, 30nM or less, 10nM or less, 3nM or less, or 1nM or less, e.g., as measured in an in vitro binding assay.
the target-binding moiety is a biomolecule.
the target-binding moiety is a biomolecule that specifically binds to a target protein.
the biomolecule is selected from peptide, protein, polynucleotide, polysaccharide, glycan, glycoprotein, lipid, enzyme, antibody, and antibody fragment.
the target-binding moiety is a polypeptide (e.g., peptide or protein binding motif, protein domain, engineered polypeptide, or glycoprotein) that specifically binds to a target molecule, such as a target protein.
the target-binding moiety of the bifunctional compound includes a polypeptide that binds to a soluble (e.g., secreted) target protein of interest.
the target-binding is a polypeptide ligand that includes a receptor ligand, or a receptor-binding portion or fragment of the receptor ligand, that binds a target cell surface receptor.
Target-binding polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of naturally occurring amino acids, non-naturally occurring amino acids, and/or amino acid modifications or analogs known in the art. Useful modifications include, e.g., N-terminal acetylation, amidation, methylation, etc.
the target-binding moiety is a polynucleotide that specifically binds to a target molecule, such as a target protein or a target nucleic acid.
a target molecule such as a target protein or a target nucleic acid.
the terms polynucleotide and nucleic acid can be used interchangeably.
the target-binding moiety is a nucleic acid aptamer that specifically binds to a target molecule, such as a target protein.
the target-binding moiety is a glycan.
the target-binding moiety is a glycan epitope for an autoantibody.
the target-binding moiety is an antibody or antibody fragment that specifically binds to a target moiety, such as a target protein.
the ASGPR binding moiety can be site-specifically covalently linked to the antibody or antibody fragment, via an optional linking moiety.
ASGPR binding moiety can be covalently linked to the antibody or antibody fragment via a site-specific cysteine modification on the antibody or antibody fragment (e.g., L443C) and a thiol-reactive chemoselective ligation group.
the bifunctional conjugate of this disclosure includes an antibody (Ab).
Ab is a monoclonal antibody.
Ab is a human antibody.
Ab is a humanized antibody.
Ab is a chimeric antibody.
Ab is a full-length antibody that includes two heavy chains and two light chains.
Ab is an IgG antibody, e.g., is an IgG1, IgG2, IgG3 or IgG4 antibody.
Ab is a single chain antibody.
the target- binding moiety is an antigen-binding fragment of an antibody, e.g., a Fab fragment.
the antibody or antibody fragment specifically binds to a cancer antigen.
the antibody or antibody fragment specifically binds to a hepatocyte antigen.
the antibody or antibody fragment specifically binds to an antigen presented on a macrophage.
the antibody or antibody fragment specifically binds to an intact complement or a fragment thereof.
the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within intact complement or a fragment thereof. [0637] In some embodiments, the antibody or antibody fragment specifically binds to a cell surface receptor. In some embodiments, the antibody or antibody fragment specifically binds to a cell surface receptor ligand. [0638] In some embodiments, the antibody or antibody fragment specifically binds to an epidermal growth factor (EGF) protein, e.g., a human EGF. In some embodiments, the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within an EGF protein.
EGF epidermal growth factor
the antibody or antibody fragment specifically binds to an epidermal growth factor receptor (EGFR) protein, e.g., a human EGFR. In some embodiments, the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within an EGFR protein. In some embodiments, the antibody or antibody fragment comprises the CDRs present in cetuximab. In some embodiments, the antibody or antibody fragment includes the variable light chain and variable heavy chain present in cetuximab. In some embodiments, the antibody is cetuximab. In some embodiments, the antibody or antibody fragment includes the CDRs present in matuzumab.
EGFR epidermal growth factor receptor
the antibody or antibody fragment includes the variable light chain and variable heavy chain present in matuzumab. In some embodiments, the antibody is matuzumab. [0640] In some embodiments, the antibody or antibody fragment specifically binds to vascular endothelial growth factor (VEGF) protein, e.g., human VEGF protein. In some embodiments, the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within a VEGF protein. [0641] In some embodiments, the antibody or antibody fragment specifically binds to a vascular endothelial growth factor receptor (VEGFR) protein, e.g., human VEGFR protein.
VEGF vascular endothelial growth factor
VEGFR vascular endothelial growth factor receptor
the antibody or antibody fragment specifically binds vascular endothelial growth factor receptor 2 (VEGFR2) protein, e.g., a human VEGFR2 protein.
the antibody or antibody fragment specifically binds a vascular endothelial growth factor receptor 3 (VEGFR3) protein, e.g., a human VEGFR3 protein.
the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within a VEGFR protein, a VEGFR2 protein or a VEGFR3 protein.
the antibody or antibody fragment specifically binds to a fibroblast growth factor (FGF), e.g., a human FGF.
FGF fibroblast growth factor
the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within a FGF protein.
the antibody or antibody fragment specifically binds to a fibroblast growth factor receptor (FGFR), e.g., a human FGFR.
FGFR2 fibroblast growth factor receptor 2
FGFR3 fibroblast growth factor receptor 3
the antibody or antibody fragment specifically binds to one or more immunodominant epitope(s) within a FGFR protein, a FGFR2 protein or a FGFR3 protein.
the antibody specifically binds to a receptor tyrosine kinase cMET protein.
the antibody specifically binds to one or more immunodominant epitope(s) within a receptor tyrosine kinase cMET protein.
the antibody specifically binds to a CD47 protein, e.g., a human CD47 protein.
the antibody specifically binds to one or more immunodominant epitope(s) within a CD47 protein. [0646] In some embodiments, the antibody specifically binds to an immune checkpoint inhibitor. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within an immune checkpoint inhibitor. In some embodiments, the antibody specifically binds to a programmed death protein, e.g., a human PD-1. In some embodiments, the antibody specifically binds to one or more immunodominant epitope(s) within PD-1 protein.
the antibody specifically binds to a programmed death ligand-1 (PD- L1) protein, e.g., a human PD-L1. In some embodiments, the antibody specifically binds to one or more immunodominant epitope(s) within PD-L1 protein. [0648] In some embodiments, the antibody binds to TIM3. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within TIM3. [0649] In some embodiments, the antibody specifically binds to a lectin. In some embodiments, the antibody specifically binds to one or more immunodominant epitope(s) within a lectin.
PD- L1 programmed death ligand-1
the antibody binds to SIGLEC. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within SIGLEC. In some embodiments, the antibody binds to a cytokine receptor. In some embodiments, the antibody binds to a one or more immunodominant epitope(s) within cytokine receptor. In some embodiments, the antibody binds to sIL6R. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within sIL6R. In some embodiments, the antibody binds to a cytokine.
the antibody binds to one or more immunodominant epitope(s) within a cytokine. In some embodiments, the antibody binds to MCP-1, TNF (e.g., a TNF- alpha), IL1a, IL1b, IL4, IL5, IL6, IL12/IL23, IL13, IL17 or p40. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within MCP-1, TNF (e.g., a TNF-alpha), IL1a, IL1b, IL4, IL5, IL6, IL12/IL23, IL13, IL17 or p40.
TNF e.g., a TNF-alpha
the antibody binds to a major histocompatibility protein (e.g., a MHC class I or class II molecule). In some embodiments, the antibody binds to one or more immunodominant epitope(s) within a major histocompatibility protein (e.g., a MHC class I or class II molecule). In some embodiments, the antibody binds to beta 2 microglobulin. In some embodiments, the antibody binds to one or more immunodominant epitope(s) within beta 2 microglobulin.
Y is a viral particle, viral capsid, a viral envelope or a viral protein.
the viral composition is a viral particle that comprises a transgene.
the viral protein is a viral capsid protein or a viral envelope protein.
modified viral compositions comprising a viral composition, for example, a virus particle, a virus capsid or a viral protein (e.g., a viral capsid protein or an envelope protein) attached to (e.g., conjugated to, directly or indirectly, for example via an intervening linker sequence) an ASGPR binding moiety that binds to a cell surface receptor.
a modified viral composition comprises a virus particle that comprises a polynucleotide that optionally comprises a transgene, e.g., a transgene useful for therapeutic applications.
the modified viral compositions, e.g., viral conjugates, presented herein may comprise any viral composition described herein e.g., any virus particle, capsid or viral protein, for example capsid protein or envelope protein, or fragment thereof, as described herein.
a viral composition described herein may comprise a virus particle.
the terms “virus particle,” “viral particle,” “virus vector” or “viral vector” are used interchangeably herein.
a “virus particle” refers to a virus capsid and a polynucleotide (DNA or RNA), which may comprise a viral genome, a portion of a viral genome, or a polynucleotide derived from a viral genome (e.g., one or more ITRs), which polynucleotide optionally comprises a transgene.
a virus particle further comprises an envelope (which generally comprises lipid moieties and envelope proteins), surrounding or partially surrounding the capsid.
a viral particle may be referred to as a “recombinant viral particle,” or “recombinant virus particle,” which terms as used herein refer to a virus particle that has been genetically altered, e.g., by the deletion or other mutation of an endogenous viral gene and/or the addition or insertion of a heterologous nucleic acid construct into the polynucleotide of the virus particle.
a recombinant virus particle generally refers to a virus particle comprising a capsid coat or shell (and an optional outer envelope) within which is packaged a polynucleotide sequence that comprises sequences of viral origin and sequences not of viral origin (i.e., a polynucleotide heterologous to the virus).
a viral composition described herein may comprise an “viral capsid,” “empty viral particle,” “empty virus particle,” or “capsid,” or “empty particle” when referred to herein in the context of the virus, which terms as used herein refer to a three-dimensional shell or coat comprising a viral capsid protein, optionally surrounded or partially surrounded by an outer envelope.
the viral composition is a virus particle or a fragment thereof, virus capsid or fragment thereof, a viral protein, for example, a virus capsid protein or fragment thereof or envelope protein, or fragment thereof.
the virus used in a modified viral composition provided herein is adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), rhabdoviruses, murine leukemia virus); herpes simplex virus, coronavirus, reovirus, and the like.
the viral vector, viral particle or viral protein used in the present disclosure is derived from a non- enveloped virus, e.g., an adeno-associated virus (AAV).
lentiviral vectors can be used for CAR-T gene delivery, vaccines, or research tools, e.g., to introduce genes into mature T cells to generate immunity to cancer through the delivery of chimeric antigen receptors (CARs) or cloned T-cell receptors.
CARs chimeric antigen receptors
Naturally occurring AAV forms a virus particle that comprises a three-dimensional capsid coat or shell (a “capsid”) made up of capsid proteins (VP1, VP2 and VP3) and, contained within the capsid, an AAV viral genome.
the modified AAV compositions may comprise any AAV composition described herein, e.g., any AAV particle, capsid or capsid protein, or fragment thereof, as described herein.
AAV capsid protein or “AAV cap protein” refers to a protein encoded by an AAV capsid (cap) gene (e.g., VP1, VP2, and VP3) or a variant or fragment thereof.
the term includes a capsid protein expressed by or derived from an AAV, e.g., a recombinant AAV, such as a chimeric AAV.
the term includes but not limited to a capsid protein derived from any AAV serotype such as AAV1, AAV2, AAV2i8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV rh10, AAV11, AAV12, AAV13, AAV-DJ, AAV3b, AAV LK03, AAV rh74, AAV Anc81, Anc82, Anc83, Anc84, Anc110, Anc113, Anc126, or Anc127, AAV_go.1, AAV hu.37, or AAV rh.8 or a variant thereof.
AAV serotype such as AAV1, AAV2, AAV2i8, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV rh10, AAV11, AAV12, AAV13, AAV-DJ, AAV3b, AAV LK03, AAV
Y is a bridging moiety that specifically binds to a viral composition, for example, a viral particle, viral capsid, viral envelope or viral protein (e.g., a viral capsid protein or envelope protein), wherein the binding is not via a covalent linkage.
a viral particle, viral capsid, viral envelope or viral protein e.g., a viral capsid protein or envelope protein
Any suitable moiety that binds a viral particle, viral capsid, viral envelope or viral protein e.g., a viral capsid protein or envelope protein
a bridging moiety is a polypeptide that specifically binds a viral composition.
the bridging moiety is a polypeptide that binds to a viral composition, e.g., a virus particle, virus capsid, virus envelope, or a viral protein, for example, a viral capsid protein or viral envelope protein.
the bridging composition binds the viral capsid protein or a viral envelope protein, when the viral protein is part of a virus particle.
a bridging moiety is an antibody or antibody fragment (e.g., an antigen binding fragment of an antibody) that specifically binds a viral composition.
a bridging moiety that binds a viral protein may also bind a viral particle, for example, via binding to the viral protein incorporated in a viral particle.
a bridging moiety that binds a viral particle may also bind a viral protein even if the viral protein is not incorporated in a viral particle.
the viral particle can be an AAV virus particle.
the viral protein can be a AAV capsid protein.
the bridging moieties of this disclosure specifically bind to an AAV composition, e.g., an AAV particle, AAV capsid, or AAV viral protein (e.g., an AAV capsid protein, for example, a VP1, VP2 or VP3 protein).
AAV composition e.g., an AAV particle, AAV capsid, or AAV viral protein (e.g., an AAV capsid protein, for example, a VP1, VP2 or VP3 protein).
An antibody or antigen binding fragment that may be utilized in connection with the modified viral compositions provided herein, e.g., in connection with the bridging compositions and bridging moieties presented herein, includes, without limitation, monoclonal antibodies, antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain antibodies, and fragments thereof (e.g., domain antibodies).
the target-binding moiety of the bifunctional compound of this disclosure is a small molecule that specifically binds to a target molecule, such as a target protein.
the bifunctional compound includes a small molecule inhibitor or ligand of a target protein.
a small molecule target-binding moiety can be covalently linked to one or more ASGPR binding moieties via a linker.
the linker can be covalently attached to the small molecule via substitution at any suitable site of the small molecule such that binding to the target protein is substantially retained.
the target-binding moiety is a small molecule inhibitor or antagonist of a target protein (e.g., as described herein).
the target-binding moiety is a small molecule inhibitor or antagonist of VEGF.
the target-binding moiety is a small molecule inhibitor or antagonist of PD-L1.
the target-binding moiety is a small molecule inhibitor or antagonist of EGFR protein, a VEGFR protein, a FGFR2 protein or a FGFR3 protein.
the target-binding moiety is a small molecule inhibitor or antagonist of TNF protein (e.g., TNF-alpha).
TNF-alpha is a soluble cytokine produced by monocytes and macrophages as part of immune and inflammatory processes and is involved in a diverse range of cellular responses including differentiation, proliferation, inflammation, and cell death.
TNF ⁇ is a type II transmembrane protein that can be cleaved and secreted as a soluble form. Both the transmembrane and soluble biologically active forms of TNF ⁇ are homotrimeric complexes that can signal through TNF receptors 1 and 2 (TNF-R1 and TNF-R2).
TNF ⁇ is directly involved in systemic inflammation through the regulation of the intracellular NF- ⁇ B, JNK and p38-MAPK signaling pathways.
the TNF ⁇ binding moiety can be a TNF ⁇ inhibitor, such as a competitive inhibitor of TNF receptor binding or an allosteric inhibitor of TNF signaling.
the compounds of this disclosure can include a potent TNF ⁇ inhibitor, e.g., an inhibitor having sub-micromolar inhibitory activity.
the TNF ⁇ inhibitor is an allosteric inhibitor.
the TNF ⁇ binding moiety is an allosteric desymmetrization TNF ⁇ inhibitor.
An allosteric desymmetrization TNF ⁇ inhibitor refers to a compound that binds to an allosteric site within TNF ⁇ and stabilizes the trimeric unit in a nonsymmetrical conformation that allows the TNF ⁇ trimer to recruit only two out of the three copies of TNF Receptor (TNFR, e.g., TNFR1), leading to an incompetent TNF ⁇ -TNFR signaling complex.
TNFR TNF Receptor
An allosteric desymmetrization TNF ⁇ inhibitor can act via a particular mechanism of action to provide potent inhibitory activity.
the TNF ⁇ inhibitor binding site is a cavity within the TNF ⁇ trimer created via movement of monomer A
the inhibitor stabilizes the TNF ⁇ trimer in an inactive conformation by forming key ⁇ and hydrogen bonding interactions
an allosteric desymmetrization TNF ⁇ inhibitor binds to TNF ⁇ trimer leading to major disruption of one TNFR binding site and minor disruption of a second site, while the third site remains unchanged
the allosteric desymmetrization TNF ⁇ inhibitor modulates TNF-R activity through an allosteric mechanism rather than direct competition with TNFR.
the bifunctional compounds of this disclosure can include a moiety of interest (Y) that specifically binds a target molecule.
the target molecule can be a cell surface molecule or an extracellular molecule.
the target molecule is a cell surface molecule.
cell surface molecule is meant a target molecule associated with a cell membrane, e.g., because the molecule has a domain that inserts into or spans a cell membrane, e.g., a cell membrane- tethering domain or a transmembrane domain.
the cell surface molecule may be any cell surface molecule which is desired for targeted degradation via the endosomal/lysosomal pathway.
the cell surface molecule is a cell surface receptor.
Cell surface receptors of interest include, but are not limited to, stem cell receptors, immune cell receptors, growth factor receptors, cytokine receptors, hormone receptors, receptor tyrosine kinases, a receptor in the epidermal growth factor receptor (EGFR) family (e.g., HER2 (human epidermal growth factor receptor 2), etc.), a receptor in the fibroblast growth factor receptor (FGFR) family, a receptor in the vascular endothelial growth factor receptor (VEGFR) family, a receptor in the platelet derived growth factor receptor (PDGFR) family, a receptor in the rearranged during transfection (RET) receptor family, a receptor in the Eph receptor family, a receptor in the discoidin domain receptor (DDR) family, and a mucin protein (e.g., MUC1 ).
EGFR epidermal growth factor receptor
HER2 human epidermal growth factor receptor 2
FGFR fibroblast growth factor receptor
VEGFR vascular endot
the cell surface molecule is CD71 (transferrin receptor).
the cell surface receptor is an immune cell receptor selected from a T cell receptor, a B cell receptor, a natural killer (NK) cell receptor, a macrophage receptor, a monocyte receptor, a neutrophil receptor, a dendritic cell receptor, a mast cell receptor, a basophil receptor, and an eosinophil receptor.
the moiety of interest (Y) specifically binds a cell surface molecule which mediates its effect not through a specific molecular interaction (and therefore is not susceptible to blocking), but rather through bulk biophysical or aggregate effects.
a non-limiting example of such a cell surface molecule is a mucin.
mucins include, but are not limited to, MUC1 , MUC16, MUC2, MUC5AC, MUC4, CD43, CD45, GPIb, and the like.
cancer cell is meant a cell exhibiting a neoplastic cellular phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, anchorage- independent growth potential, ability to promote tumor growth and/or development in an immunocompromised non-human animal model, and/or any appropriate indicator of cellular transformation.
Cancer cell may be used interchangeably herein with “tumor cell”, “malignant cell” or “cancerous cell”, and encompasses cancer cells of a solid tumor, a semi-solid tumor, a hematological malignancy (e.g., a leukemia cell, a lymphoma cell, a myeloma cell, etc.), a primary tumor, a metastatic tumor, and the like.
the cell surface molecule present on the cancer cell is a tumor-associated antigen or a tumor-specific antigen.
the moiety of interest (Y) specifically binds a cell surface molecule, the cell surface molecule is present on an immune cell.
the cell surface molecule is present on an immune cell selected from a T cell, a B cell, a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a mast cell, a basophil, and an eosinophil.
the cell surface molecule present on the immune cell is an inhibitory immune receptor.
an “inhibitory immune receptor” is a receptor present on an immune cell that negatively regulates an immune response.
Ig superfamily including but not limited to: CD200R, CD300a (IRp60; mouse MAIR-I), CD300f (IREM-1 ), CEACAM1 (CD66a), FcyRIIb, ILT-2 (LIR-1 ; LILRB1 ;
Siglec sialic acid-binding Ig-like lectin
C- type lectins including but not limited to: CLEC4A (DCIR), Ly49Q and MICL. Details regarding inhibitory immune receptors may be found, e.g., in Steevels et al. (2011 ) Eur. J. Immunol.
the cell surface molecule present on the immune cell is a ligand of an inhibitory immune receptor.
the cell surface molecule present on the immune cell is an immune checkpoint molecule.
immune checkpoint molecules to which the moiety of interest (Y) may specifically bind include PD-1, PD-L1, CTLA4, TIM3, LAG3, TIGIT, and a member of the B7 family.
the target molecule is an extracellular molecule.
extracellular molecule is meant a soluble molecule external to the cell membranes of any cells in the vicinity of the soluble molecule.
the extracellular molecule may be any extracellular molecule which is desired for targeted degradation via the endosomal/lysosomal pathway.
the extracellular molecule is a soluble target protein.
the extracellular molecule is a secreted protein that accumulates in disease (e.g., alpha- synuclein), a cholesterol carrier (e.g., ApoB), an infectious disease toxin (e.g., AB toxins, ESAT-6), an infectious particle (e.g., a whole virus, a whole bacterium, etc.), a clotting factor (e.g., Factor IX), the target of any FDA approved antibody that binds to an extracellular molecule (e.g., TNFalpha), any chemokine or cytokine (e.g., mediators of sepsis or chronic inflammation such at IL-1 ), a proteinaceous hormone (e.g., insulin, ACTH, etc.), a proteinaceous mediator of
the target molecule is an extracellular molecule that is an antibody, e.g., an antibody that specifically binds a cell surface molecule or different extracellular molecule.
the antibody is an autoantibody.
the target is a human immunoglobulin A(IgA).
the IgA is a particular antibody that plays a crucial role in the immune function of mucous membranes. In the blood, IgA interacts with an Fc receptor called CD89 expressed on immune effector cells, to initiate inflammatory reactions. Aberrant IgA expression has been implicated in a number of autoimmune and immune-mediated disorders.
the target is a human immunoglobulin G (IgG).
the Fc regions of IgGs include a conserved N- glycosylation site at asparagine 297 in the constant region of the heavy chain.
Various N-glycans can b eattached to this site.
the N-glycan IgG composition has been linked to several autoimmune, infectious and metabolic diseases.
overexpression of IgG4 has been associated with IG4-related diseases.
the target is human immunoglobulin E (IgE).
IgE is a type of immunoglobulin that plays an essential role in type I hypersensitivity, which can manifest into various allergic diseases and conditions.
the extracellular molecule is a ligand for a cell surface receptor.
Cell surface receptor ligands of interest include, but are not limited to, growth factors (e.g., epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and the like), cytokines (e.g., an interleukin, an interferon, a tumor necrosis factor (TNF), a transforming growth factor b (TGF-b), including any particular subtypes of such cytokines), hormones, and the like.
the moiety of interest (Y) specifically binds apolipoprotein E4 (ApoE4).
Pharmaceutical Compositions comprising one or more conjugates disclosed herein and a pharmaceutically acceptable carrier.
the pharmaceutical compositions provided herein contain therapeutically effective amounts of one or more of the conjugates provided herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier.
Pharmaceutical carriers suitable for administration of the conjugates provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
the conjugates described herein can be formulated as the sole pharmaceutically active ingredient in the composition or can be combined with other active ingredients.
the conjugate is formulated into one or more suitable pharmaceutical preparations, such as solutions, suspensions, powders, sustained release formulations or elixirs in sterile solutions or suspensions for parenteral administration, or as transdermal patch preparation and dry powder inhalers.
a conjugate described herein may be mixed with a suitable pharmaceutical carrier.
the concentration of the conjugate in the compositions can, for example, be effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates a condition or disorder described herein or a symptom thereof.
the pharmaceutical compositions provided herein are formulated for single dosage administration.
compositions described herein are provided for administration to a subject, for example, humans or animals (e.g., mammals) in unit dosage forms, such as sterile parenteral (e.g., intravenous) solutions or suspensions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof.
compositions are also provided for administration to humans and animals in unit dosage form, including oral or nasal solutions or suspensions and oil-water emulsions containing suitable quantities of a conjugate or pharmaceutically acceptable derivatives thereof.
the conjugate is, in certain embodiments, formulated and administered in unit-dosage forms or multiple-dosage forms.
Unit-dose forms as used herein refers to physically discrete units suitable for human or animal (e.g., mammal) subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of a conjugate sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes and individually packaged capsules.
Unit-dose forms can be administered in fractions or multiples thereof.
a multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of capsules or bottles. Hence, in specific aspects, multiple dose form is a multiple of unit-doses which are not segregated in packaging.
the conjugates herein are in a liquid pharmaceutical formulation.
Liquid pharmaceutically administrable formulations can, for example, be prepared by dissolving, dispersing, or otherwise mixing a conjugate and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, and the like, to thereby form a solution or suspension.
a pharmaceutical composition provided herein to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, and pH buffering agents and the like.
the injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol.
Other routes of administration may include, enteric administration, intracerebral administration, nasal administration, intraarterial administration, intracardiac administration, intraosseous infusion, intrathecal administration, and intraperitoneal administration.
Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions.
the solutions can be either aqueous or nonaqueous.
suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.
Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.
intravenous or intraarterial infusion of a sterile aqueous solution containing a conjugate described herein is an effective mode of administration.
the pharmaceutical formulations are lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They can also be reconstituted and formulated as solids or gels.
the lyophilized powder is prepared by dissolving a conjugate provided herein, in a suitable solvent.
the lyophilized powder is sterile. Suitable solvents can contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder.
Excipients that can be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent.
a suitable solvent can also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in certain embodiments, about neutral pH.
Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides an example of a formulation.
the resulting solution will be apportioned into vials for lyophilization. Lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature.
the conjugates provided herein can be formulated for local administration or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered.
provided herein are methods of using the conjugates described herein to remove a polypeptide of interest (a target protein) from a cell’s surface. In one aspect, provided herein are methods of using the conjugates described herein to remove a polypeptide of interest (a target protein) from the extracellular milieu. For example, in one embodiment, provided herein are methods of using the conjugates described herein to remove a polypeptide of interest (a target protein) from the surface of a cell by sequestering the target protein in the cell’s lysosome.
provided herein are methods of using the conjugates described herein to remove a polypeptide of interest (a target protein) from the extracellular space (the extracellular milieu) of a cell by sequestering the target protein in the cell’s lysosome.
provided herein are methods of using the conjugates described herein to remove a polypeptide of interest (a target protein) from the extracellular space (the extracellular milieu) of a cell by sequestering the target protein in the cell’s lysosome and degrading the target protein.
Removal of a target protein may refer to reduction, or depletion, of the target protein from the cell surface or from the extracellular space, or the extracellular milieu, that is, a reduction, or depletion, of the amount of the target protein on the cell surface or in the extracellular milieu.
provided herein are methods of using the conjugates described herein to sequester a polypeptide of interest (a target protein) in a cell’s lysosome.
provided herein are methods of using the conjugates described herein to sequester a polypeptide of interest (a target protein) in a cell’s lysosome and to degrade the polypeptide of interest. [0708] In one aspect, provided herein are methods of using the conjugates described herein to degrade a polypeptide of interest (a target protein). [0709] In one aspect, provided herein are methods of depleting a polypeptide of interest (a target protein) described herein by degradation through a cell’s lysosomal pathway.
a polypeptide of interest a target protein described herein by administering to a subject in need thereof an effective amount of a conjugate or pharmaceutically acceptable salt described herein, or a pharmaceutical composition described herein.
the subject is a mammal (e.g., human).
the target protein is a VEGF protein, an EGFR protein, a VEGFR protein, a PD-L1 protein, an FGFR2 protein or an FGFR3 protein.
administering refers to the act of injecting or otherwise physically delivering a substance (e.g., a conjugate or pharmaceutical composition provided herein) to a subject or a patient (e.g., human), such as by mucosal, topical, intradermal, parenteral, intravenous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art.
administering is by intravenous infusion.
a therapeutic e.g., a conjugate or pharmaceutical composition provided herein
administration is by intravenous infusion.
a therapeutic e.g., a conjugate or pharmaceutical composition provided herein
a therapeutic e.g., a conjugate or pharmaceutical composition provided herein
a therapeutic e.g., a conjugate or pharmaceutical composition provided herein
these terms also encompass an amount necessary for the reduction, slowing, or amelioration of the advancement or progression of a given disease, reduction, slowing, or amelioration of the recurrence, development or onset of a given disease, and/or to improve or enhance the prophylactic or therapeutic effect(s) of another therapy or to serve as a bridge to another therapy.
“effective amount” as used herein also refers to the amount of a conjugate described herein to achieve a specified result.
“effective amount” or “therapeutically effective amount” mean that amount of a conjugate or pharmaceutical composition provided herein which, when administered to a human suffering from a cancer, is sufficient to effect treatment for the cancer.
“Treating” or “treatment” of the cancer includes one or more of: (1) limiting/inhibiting growth of the cancer, e.g. limiting its development; (2) reducing/preventing spread of the cancer, e.g. reducing/preventing metastases; (3) relieving the cancer, e.g.
a subject can be a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, goats, rabbits, rats, mice, etc.) or a primate (e.g., monkey and human), for example a human.
a mammal e.g., a human, diagnosed with a disease or disorder provided herein.
the subject is a mammal, e.g., a human, at risk of developing a disease or disorder provided herein.
the subject is human.
therapies can refer to any protocol(s), method(s), compositions, formulations, and/or agent(s) that can be used in the prevention, treatment, management, or amelioration of a disease or disorder or symptom thereof (e.g., a disease or disorder provided herein or one or more symptoms or condition associated therewith).
the terms “therapies” and “therapy” refer to drug therapy, adjuvant therapy, radiation, surgery, biological therapy, supportive therapy, and/or other therapies useful in treatment, management, prevention, or amelioration of a disease or disorder or one or more symptoms thereof.
the term “therapy” refers to a therapy other than a conjugate described herein or pharmaceutical composition thereof.
the disease or disorder is treated by depletion of the target protein by degradation through the lysosomal pathway.
the disease or disorder is treated by depletion of certain proteins, for example, soluble proteins, e.g., secreted proteins, cell surface proteins (for example, cell surface receptor proteins, e.g., tyrosine kinase receptors, soluble cytokine receptors, and immune checkpoint receptors, e.g., EGFR, VEGFR, FGFR, and PD-L1), lectins, complements, lipoproteins, transport proteins, MHC class I and class II molecules, cytokines, chemokines, and/or receptors , or fragments or subunits of any of the foregoing.
the disease or disorder is a cancer.
the cancer is selected from the group consisting of bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, endometrial cancer, hepatocellular carcinoma, kidney cancer, melanoma, myeloid neoplasms, non-small cell lung cancer (NSCLC), Ewing’s sarcoma, and Hodgkin’s Lymphoma.
the cancer is a solid tumor.
the disease or disorder is an inflammatory or autoimmune disease.
the disease or disorder is an inflammatory disease.
the disease or disorder is an autoimmune disease.
the disease or disorder is a viral disease.
the viral disease is hepatitis B. Definitions [0726] It is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. [0727] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of embodiments of the present disclosure.
Proteins may include moieties other than amino acids (e.g., may be glycoproteins, etc.) and/or may be otherwise processed or modified.
a “protein” can be a complete protein chain as produced by a cell (with or without a signal sequence), or can be a protein portion thereof.
a protein can sometimes include more than one protein chain, for example non-covalently or covalently attached, e.g., linked by one or more disulfide bonds or associated by other means.
a polypeptide can occur as a single chain or as two or more associated chains, e.g., may be present as a multimer, e.g., dimer, a trimer.
the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.
polypeptides containing one or more analogs of an amino acid including but not limited to, unnatural amino acids, as well as other modifications known in the art.
Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc.
proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.
proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
antibody and “immunoglobulin” are terms of art and can be used interchangeably herein in their broadest sense and includes certain types of immunoglobulin molecules comprising one or more antigen-binding domains that specifically bind to an antigen or epitope.
an isolated antibody e.g., monoclonal antibody described herein, or an antigen-binding fragment thereof, which specifically binds to a protein of interest, for example, EGFR, is conjugated to one or more lysosomal targeting moieties, for example, via a linker.
an “antigen” is a moiety or molecule that contains an epitope to which an antibody can specifically bind. As such, an antigen is also is specifically bound by an antibody.
the antigen, to which an antibody described herein binds is a protein of interest, for example, EGFR (e.g., human EGFR), or a fragment thereof, or for example, an extracellular domain of EGFR (e.g., human EGFR).
EGFR e.g., human EGFR
An “epitope” is a term known in the art and refers to a localized region of an antigen to which an antibody can specifically bind.
An epitope can be a linear epitope of contiguous amino acids or can comprise amino acids from two or more non-contiguous regions of the antigen.
binds refers to antibody binding to an antigen (e.g., epitope) as such binding is understood by one skilled in the art.
an antigen e.g., epitope
a molecule that specifically binds to an antigen may bind to other polypeptides, generally with lower affinity as determined by, e.g., immunoassays, BiacoreTM, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art.
molecules that specifically bind to an antigen bind to the antigen with an affinity (Kd ) that is at least 2 logs, 2.5 logs, 3 logs, 4 logs lower (higher affinity) than the K d when the molecules bind to another antigen.
Kd affinity
molecules that specifically bind to an antigen do not cross react with other proteins.
EGFR is the protein of interest, molecules that specifically bind to an antigen do not cross react with other non-EGFR proteins.
An antibody specifically includes, but is not limited to, full length antibodies (e.g., intact immunoglobulins), antibody fragments, monoclonal antibodies, polyclonal antibodies,, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain/antibody heavy chain pair, an antibody with two light chain/heavy chain pairs (e.g., identical pairs), intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, bivalent antibodies (including monospecific or bispecific bivalent antibodies), single chain antibodies, or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab’) fragments, F(ab’) 2 fragments, disulfide-linked Fvs (scF
Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class, (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule.
antibodies described herein are IgG antibodies (e.g., human IgG), or a class (e.g., human IgG1, IgG2, IgG3 or IgG4) or subclass thereof.
an antibody is a 4-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs, wherein the amino acid sequences of the H chains are identical and the amino acid sequences of the L chains are identical.
the H and L chains comprise constant regions, for example, human constant regions.
the L chain constant region of such antibodies is a kappa or lambda light chain constant region, for example, a human kappa or lambda light chain constant region.
the H chain constant region of such antibodies comprise a gamma heavy chain constant region, for example, a human gamma heavy chain constant region.
such antibodies comprise IgG constant regions, for example, human IgG constant regions.
the term “constant region” or “constant domain” is a well-known antibody term of art (sometimes referred to as “Fc”), and refers to an antibody portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor.
the terms refer to a portion of an immunoglobulin molecule having a generally more conserved amino acid sequence relative to an immunoglobulin variable domain.
the term “heavy chain” when used in reference to an antibody can refer to any distinct types, e.g., alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) and mu ( ⁇ ), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG 1 , IgG 2 , IgG 3 and IgG 4 .
the term “light chain” when used in reference to an antibody can refer to any distinct types, e.g., kappa ( ⁇ ) of lambda ( ⁇ ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiments, the light chain is a human light chain.
the term “monoclonal antibody” is a well-known term of art that refers to an antibody obtained from a population of homogenous or substantially homogeneous antibodies. The term “monoclonal” is not limited to any particular method for making the antibody. Generally, a population of monoclonal antibodies can be generated by cells, a population of cells, or a cell line.
a “monoclonal antibody,” as used herein, is an antibody produced by a single cell (e.g., hybridoma or host cell producing a recombinant antibody), wherein the antibody specifically binds to an epitope as determined, e.g., by ELISA or other antigen-binding or competitive binding assay known in the art or in the Examples provided herein.
a monoclonal antibody can be a chimeric antibody or a humanized antibody.
a monoclonal antibody is a monovalent antibody or multivalent (e.g., bivalent) antibody.
a monoclonal antibody is a monospecific or multispecific antibody (e.g., bispecific antibody).
variable region refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 100 amino acids in the mature light chain.
Variable regions comprise complementarity determining regions (CDRs) flanked by framework regions (FRs).
CDRs complementarity determining regions
FRs framework regions
the spatial orientation of CDRs and FRs are as follows, in an N-terminal to C-terminal direction: FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4.
the CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen and for the specificity of the antibody for an epitope.
numbering of amino acid positions of antibodies described herein is according to the EU Index, as in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242.
the variable region is a human variable region.
the CDRs of an antibody can be determined according to (i) the Kabat numbering system (Kabat et al. (1971) Ann. NY Acad.
full length antibody “intact antibody” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, and are not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain the Fc region.
Antibody fragments comprise only a portion of an intact antibody, wherein the portion retains at least one, two, three and as many as most or all of the functions normally associated with that portion when present in an intact antibody. In one aspect, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen.
an antibody fragment such as an antibody fragment that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody. Such functions may include FcRn binding, antibody half life modulation, conjugate function and complement binding.
an antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody.
such an antibody fragment may comprise on antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.
Antibody fragments suitable for use in the compounds of this disclosure include, for example, Fv fragments, Fab fragments, F(ab’) 2 fragments, Fab’ fragments, scFv (sFv) fragments, and scFv-Fc fragments.
Polynucleotide or “nucleic acid,” as used interchangeably herein, and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers.
the nucleic acid molecule may be linear or circular.
the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.
a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.
the nucleic acid molecule may be an aptamer.
purified refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance of interest comprises the majority percent of the sample in which it resides.
a substantially purified component comprises 50%, 80%-85%, 90-99%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the sample.
Techniques for purifying polynucleotides, polypeptides and virus particles of interest are well- known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.
treatment “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect, such as reduction of tumor burden.
Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease (as in liver fibrosis that can result in the context of chronic HCV infection); (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease (e.g., reduction in of tumor burden).
the terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to an animal, including, but not limited to, human and non-human primates, including simians and humans; rodents, including rats and mice; bovines; equines; ovines; felines; canines; and the like.
"Mammal” means a member or members of any mammalian species, and includes, by way of example, canines; felines; equines; bovines; ovines; rodentia, etc. and primates, e.g., non-human primates, and humans.
Non-human animal models e.g., mammals, e.g.
a “therapeutically effective amount” or “efficacious amount” means the amount of a compound that, when administered to a mammal or other subject for treating a disease, condition, or disorder, is sufficient to effect such treatment for the disease, condition, or disorder.
the “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.
all alternative isomers are intended to be encompassed within the scope of the claimed subject matter.
the compounds provided herein may be enantiomerically pure, or be stereoisomeric or diastereomeric mixtures.
the compounds provided herein may contain chiral centers. Such chiral centers may be of either the (R) or (S) configurations, or may be a mixture thereof. The chiral centers of the compounds provided herein may undergo epimerization in vivo.
the present disclosure also encompasses all suitable isotopic variants of the compounds according to the present disclosure, whether radioactive or not.
An isotopic variant of a compound according to the present disclosure is understood to mean a compound in which at least one atom within the compound according to the present disclosure has been exchanged for another atom of the same atomic number, but with a different atomic mass than the atomic mass which usually or predominantly occurs in nature.
isotopes which can be incorporated into a compound according to the present disclosure are those of hydrogen, carbon, nitrogen, oxygen, fluorine, chlorine, bromine and iodine, such as 2 H (deuterium), 3 H (tritium), 13 C, 14 C, 15 N, 17 O, 18 O, 18 F, 36 Cl, 82 Br, 123 I, 124 I, 125 I, 129 I and 131 I.
Particular isotopic variants of a compound according to the present disclosure especially those in which one or more radioactive isotopes have been incorporated, may be beneficial, for example, for the examination of the mechanism of action or of the active compound distribution in the body.
Isotopic variants of the compounds according to the present disclosure can be prepared by various, including, for example, the methods described below and in the working examples, by using corresponding isotopic modifications of the particular reagents and/or starting compounds therein.
any of the embodiments described herein are meant to include a salt, a single stereoisomer, a mixture of stereoisomers and/or an isotopic form of the compounds.
a "pharmaceutically acceptable excipient,” “pharmaceutically acceptable diluent,” “pharmaceutically acceptable carrier,” and “pharmaceutically acceptable adjuvant” means an excipient, diluent, carrier, and adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use.
“A pharmaceutically acceptable excipient, diluent, carrier and adjuvant” as used in the specification and claims includes both one and more than one such excipient, diluent, carrier, and adjuvant.
a "pharmaceutical composition” is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human.
a “pharmaceutical composition” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade).
Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, and the like.
pharmaceutically acceptable means being approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and, more particularly in humans.
pharmaceutically acceptable salt refers to those salts which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977).
salts can be prepared in situ during the final isolation and purification of the conjugate compounds, or separately by reacting the free base function or group of a compound with a suitable organic acid.
pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, or salts of an amino group formed with inorganic acids [0761] “Acyl” refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)- , hetero
acyl includes the “acetyl” group CH 3 C(O)- [0762]
alkyl refers to a branched or unbranched saturated hydrocarbon group (i.e., a mono-radical) typically although not necessarily containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like.
alkyl groups herein may contain 1 to about 18 carbon atoms, and such groups may contain 1 to about 12 carbon atoms.
the term "lower alkyl” intends an alkyl group of 1 to 6 carbon atoms.
“Substituted alkyl” refers to alkyl substituted with one or more substituent groups, and this includes instances wherein two hydrogen atoms from the same carbon atom in an alkyl substituent are replaced, such as in a carbonyl group (i.e., a substituted alkyl group may include a -C(O)- moiety).
heteroatom-containing alkyl and “heteroalkyl” refer to an alkyl substituent in which at least one carbon atom is replaced with a heteroatom, as described in further detail infra. If not otherwise indicated, the terms “alkyl” and “lower alkyl” include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkyl or lower alkyl, respectively.
substituted alkyl is meant to include an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain have been optionally replaced with a heteroatom such as -O- , -N-, -S-, -S(O)n- (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) and having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thi
alkenyl refers to a linear, branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, and the like.
alkenyl groups herein may contain 2 to about 18 carbon atoms, and for example may contain 2 to 12 carbon atoms.
lower alkenyl intends an alkenyl group of 2 to 6 carbon atoms.
substituted alkenyl refers to alkenyl substituted with one or more substituent groups
heteroatom-containing alkenyl and “heteroalkenyl” refer to alkenyl in which at least one carbon atom is replaced with a heteroatom.
alkenyl and “lower alkenyl” include linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkenyl and lower alkenyl, respectively.
alkynyl refers to a linear or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, and the like. Generally, although again not necessarily, alkynyl groups herein may contain 2 to about 18 carbon atoms, and such groups may further contain 2 to 12 carbon atoms. The term “lower alkynyl” intends an alkynyl group of 2 to 6 carbon atoms.
substituted alkynyl refers to alkynyl substituted with one or more substituent groups
heteroatom-containing alkynyl and “heteroalkynyl” refer to alkynyl in which at least one carbon atom is replaced with a heteroatom.
alkynyl and “lower alkynyl” include linear, branched, unsubstituted, substituted, and/or heteroatom-containing alkynyl and lower alkynyl, respectively.
alkoxy refers to an alkyl group bound through a single, terminal ether linkage; that is, an "alkoxy” group may be represented as -O-alkyl where alkyl is as defined above.
a "lower alkoxy” group refers to an alkoxy group containing 1 to 6 carbon atoms, and includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc.
Substituents identified as "C1-C6 alkoxy” or “lower alkoxy” herein may, for example, may contain 1 to 3 carbon atoms, and as a further example, such substituents may contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy).
substituted alkoxy refers to the groups substituted alkyl-O-, substituted alkenyl- O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.
aryl refers to an aromatic substituent generally, although not necessarily, containing 5 to 30 carbon atoms and containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety).
Aryl groups may, for example, contain 5 to 20 carbon atoms, and as a further example, aryl groups may contain 5 to 12 carbon atoms.
aryl groups may contain one aromatic ring or two or more fused or linked aromatic rings (i.e., biaryl, aryl-substituted aryl, etc.).
substituted aryl refers to an aryl moiety substituted with one or more substituent groups
heteroatom-containing aryl and “heteroaryl” refer to aryl substituent, in which at least one carbon atom is replaced with a heteroatom, as will be described in further detail infra.
Aryl is intended to include stable cyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated C 3 -C 14 moieties, exemplified but not limited to phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, imidazoyl, pyrimidinyl, and oxazoyl; which may further be substituted with one to five members selected from the group consisting of hydroxy, C 1 -C 8 alkoxy, C 1 -C 8 branched or straight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halogen, trifluoromethyl, cyano, and carboxyl (see e.g.
aryl includes unsubstituted, substituted, and/or heteroatom-containing aromatic substituents.
aralkyl refers to an alkyl group with an aryl substituent
alkaryl refers to an aryl group with an alkyl substituent, wherein “alkyl” and “aryl” are as defined above.
aralkyl and alkaryl groups herein contain 6 to 30 carbon atoms.
Aralkyl and alkaryl groups may, for example, contain 6 to 20 carbon atoms, and as a further example, such groups may contain 6 to 12 carbon atoms.
alkylene refers to a multi-valent (e.g., di-radical alkyl group, tri-radical alkyl group, tetra-radical alkyl group, etc.) . Unless otherwise indicated, such groups include saturated hydrocarbon chains containing from 1 to 24 carbon atoms, which may be substituted or unsubstituted, may contain one or more alicyclic groups, and may be heteroatom-containing. "Lower alkylene” refers to alkylene linkages containing from 1 to 6 carbon atoms.
alkenylene alkynylene
arylene aralkylene
alkarylene refer to di-radical alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively.
the "alkylene” refers to a multi-valent (e.g., di-valent alkyl group, tri-valent alkyl group, tetra-valent alkyl group, etc.).
the terms “alkenylene,” “alkynylene,” “arylene,” “aralkylene,” and “alkarylene” can refer to multi-valent alkenyl, multi-valent alkynyl, multi-valent aryl, multi-valent aralkyl, and multi-valent alkaryl groups, respectively.
amino refers to the group -NRR’ wherein R and R’ are independently hydrogen or nonhydrogen substituents, with nonhydrogen substituents including, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and/or heteroatom-containing variants thereof.
halo and “halogen” are used in the conventional sense to refer to a chloro, bromo, fluoro or iodo substituent.
Carboxyl,” “carboxy” or “carboxylate” refers to –C(O)OH or salts thereof.
Cycloalkyl refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems.
suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like.
Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
substituted cycloalkyl refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy,
heteroatom-containing refers to a molecule, linkage or substituent in which one or more carbon atoms are replaced with an atom other than carbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon, typically nitrogen, oxygen or sulfur.
heteroalkyl refers to an alkyl substituent that is heteroatom-containing
heterocycloalkyl refers to a cycloalkyl substituent that is heteroatom-containing
heterocyclic or “heterocycle” refer to a cyclic substituent that is heteroatom-containing
heteroaryl and “heteroaromatic” respectively refer to “aryl” and “aromatic” substituents that are heteroatom-containing, and the like.
heteroalkyl groups include alkoxyaryl, alkylsulfanyl- substituted alkyl, N-alkylated amino alkyl, and the like.
heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, furyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, etc., and examples of heteroatom-containing alicyclic groups are pyrrolidino, morpholino, piperazino, piperidino, tetrahydrofuranyl, etc. [0779] “Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring.
heteroaryl groups can have a single ring (such as, pyridinyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic, provided that the point of attachment is through an atom of an aromatic ring.
the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N ⁇ O), sulfinyl, or sulfonyl moieties.
heteroaryl substituent can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclo
heterocycle refers to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 15 ring atoms, including 1 to 4 hetero atoms. These ring heteroatoms are selected from nitrogen, sulfur and oxygen, wherein, in fused ring systems, one or more of the rings can be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring.
the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or –SO 2 - moieties.
heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenox
heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,
Hydrocarbyl refers to univalent hydrocarbyl radicals containing 1 to about 30 carbon atoms, including 1 to about 24 carbon atoms, further including 1 to about 18 carbon atoms, and further including about 1 to 12 carbon atoms, including linear, branched, cyclic, saturated and unsaturated species, such as alkyl groups, alkenyl groups, aryl groups, and the like.
a hydrocarbyl may be substituted with one or more substituent groups.
heteroatom-containing hydrocarbyl refers to hydrocarbyl in which at least one carbon atom is replaced with a heteroatom.
hydrocarbyl is to be interpreted as including substituted and/or heteroatom-containing hydrocarbyl moieties.
substituted as in “substituted hydrocarbyl,” “substituted alkyl,” “substituted aryl,” and the like, as alluded to in some of the aforementioned definitions, is meant that in the hydrocarbyl, alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents.
substituents include, without limitation, functional groups, and the hydrocarbyl moieties C 1 -C 24 alkyl (including C 1 -C 18 alkyl, further including C 1 -C 12 alkyl, and further including C 1 -C 6 alkyl), C 2 -C 24 alkenyl (including C 2 -C 18 alkenyl, further including C 2 -C 12 alkenyl, and further including C 2 -C 6 alkenyl), C 2 -C 24 alkynyl (including C 2 -C 18 alkynyl, further including C 2 -C 12 alkynyl, and further including C 2 -C 6 alkynyl), C 5 -C 30 aryl (including C 5 -C 20 aryl, and further including C 5 -C 12 aryl), and C 6 -C 30 aralkyl (including C 6 -C 20 aralkyl, and further including C 6 -C 12 aralkyl).
hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated. Unless otherwise indicated, any of the groups described herein are to be interpreted as including substituted and/or heteroatom-containing moieties, in addition to unsubstituted groups.
“Sulfonyl” refers to the group SO 2 -alkyl, SO 2 -substituted alkyl, SO 2 -alkenyl, SO 2 -substituted alkenyl, SO 2 -cycloalkyl, SO 2 -substituted cylcoalkyl, SO 2 -cycloalkenyl, SO 2 -substituted cylcoalkenyl, SO 2 -aryl, SO 2 -substituted aryl, SO 2 -heteroaryl, SO 2 -substituted heteroaryl, SO 2 -heterocyclic, and SO 2 - substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, wherein al
Sulfonyl includes, by way of example, methyl-SO 2 -, phenyl-SO 2 -, and 4-methylphenyl-SO 2 -.
functional groups chemical groups such as halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2-C24 alkylcarbonato (-O-(CO)-O-
linking or "linker” as in “linking group,” “linker moiety,” etc., is meant a linking moiety that connects two groups via covalent bonds.
the linker may be linear, branched, cyclic or a single atom.
linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and linking moieties containing functional groups including, without limitation: amido (-NH-CO-), ureylene (-NH-CO-NH-), imide (-CO-NH-CO-) , epoxy (-O-), epithio (-S-), epidioxy (-O-O-), carbonyldioxy (-O-CO-O-), alkyldioxy (-O-(CH2)n-O-), epoxyimino (-O-NH-), epimino (-NH-), carbonyl (-CO-), etc.
one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom.
the bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone.
the linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group.
a linker may include, without limitations, poly(ethylene glycol) unit(s) (e.g., -(CH 2 -CH 2 -O)-); ethers, thioethers, amines, alkyls (e.g., (C 1 -C 12 )alkyl) , which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n- pentyl, 1,1-dimethylethyl (t-butyl), and the like.
poly(ethylene glycol) unit(s) e.g., -(CH 2 -CH 2 -O)-
ethers e.g., -(CH 2 -CH 2 -O)-
ethers e.g., -(CH 2 -CH 2 -O)-
ethers e.g.,
the linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone.
a linker may be cleavable or non-cleavable. Any convenient orientation and/or connections of the linkers to the linked groups may be used. [0788] When the term "substituted" appears prior to a list of possible substituted groups, it is intended that the term apply to every member of that group.
substituted alkyl and aryl is to be interpreted as “substituted alkyl and substituted aryl.”
substituted when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.
Each M + may independently be, for example, an alkali ion, such as K + , Na + , Li + ; an ammonium ion, such as + N(R 60 ) 4 ; or an alkaline earth ion, such as [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 , or [Ba 2+ ] 0.5 (“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound as disclosed herein, and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound can serve as the counter ion for such divalent alkali earth ions).
an alkali ion such as K + , Na + , Li +
an ammonium ion such as + N(R 60 ) 4
-NR 80 R 80 is meant to include -NH 2 , -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N- methyl-piperazin-1-yl and N-morpholinyl.
substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R 60 , halo, -O-M + , -OR 70 , -SR 70 , -S – M + , -NR 80 R 80 , trihalomethyl, -CF 3 , -CN, -OCN, -SCN, -NO, -NO 2 , -N 3 , -SO 2 R 70 , -SO 3 – M + , -SO 3 R 70 , -OSO 2 R 70 , -OSO 3 – M + , -OSO 3 R 70 , -PO 3 -2 (M + ) 2 , -P(O)(OR 70 )O – M + , -P(O)(OR 70 ) 2 , -C(O)R 70
substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O-M + , -OR 70 , -SR 70 , -S-M + , -NR 80 R 80 , trihalomethyl, -CF 3 , -CN, -NO, -NO 2 , -S(O) 2 R 70 , -S(O) 2 O-M + , -S(O) 2 OR 70 , -OS(O) 2 R 70 , -OS(O) 2 O-M + , -O S(O) 2 OR 70 , -P(O)(O-) 2 (M + ) 2 , -P(O)(OR 70 )O-M + , -P(O)(OR 70 )(OR 70 ), -C(O
a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.
substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment.
substituent “arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O-C(O)-.
any of the groups disclosed herein which contain one or more substituents it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible.
the subject compounds include all stereochemical isomers arising from the substitution of these compounds.
a substituent may contribute to optical isomerism and/or stereo isomerism of a compound. Salts, solvates, hydrates, and prodrug forms of a compound are also of interest. All such forms are embraced by the present disclosure.
a compound may be a metabolized into a pharmaceutically active derivative.
reference to an atom is meant to include isotopes of that atom.
reference to H is meant to include 1 H, 2 H (i.e., D) and 3 H (i.e., T)
reference to C is meant to include 12 C and all isotopes of carbon (such as 13 C).
the term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, or 3 standard deviations. In certain embodiments, the term “about” or “approximately” means within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.25%, 0.2%, 0.1% or 0.05% of a given value or range.
Embodiment A-1 A cell surface ASGPR binding compound of formula (I): X n L Y ( I) or a prodrug thereof, or a salt thereof, wherein: Y is a moiety of interest; n is 1 to 500; L is a linker; and X is a moiety that binds to a cell surface asialoglycoprotein receptor (ASGPR) of formula (Ia): wherein: R 1 is selected from –OH, –OC(O)R, -C(O)NHR, –Z 1 –*, and optionally substituted triazole, where R is optionally substituted C 1-6 alkyl or optionally substituted aryl; R 2 is selected from–NHCOCH 3 , –NHCOCF 3, –NHCOCH 2 CF 3 , –OH, optionally substituted triazole, and –Z 1 –*; R 3 is selected from –H, –OH, –
Embodiment A-2 The compound of embodiment A-1, wherein each X is independently of formula (Ib): wherein: R 1 is selected from –OH, –OC(O)R, and -C(O)NHR; and R 2 is selected from –NHCOCH 3 , –NHCOCF 3 , and –NHCOCH 2 CF 3 .
Embodiment A-3 The compound of embodiment A-3, wherein Z 1 is selected from -O-, -S-, and -C(R 22 ) 2 -.
Embodiment A-4 The compound of embodiment A-3, wherein Z 1 is Z 11 -Ar , wherein Ar is optionally substituted heteroaryl or optionally substituted aryl.
Embodiment A-5 The compound of embodiment A-4, wherein: Z 11 is O, S, or C(R 22 ) 2 ; and Ar is a monocyclic 5 or 6-membered heteroaryl or aryl.
Embodiment A-6 The compound of embodiment A-5, wherein Z 1 is -C(R 22 ) 2 -triazole-. * [0808] Embodiment A-7: The compound of embodiment A-6, wherein Z 1 is or .
Embodiment A-8 The compound of embodiment A-3, wherein Z 1 is monocyclic 5 or 6- membered heteroaryl or aryl.
Embodiment A-9 The compound of embodiment A-8, wherein .
Embodiment A-10 The compound of embodiment A-2, wherein each X is independently of the formula: wherein R 4 and R 5 are each H.
Embodiment A-11 The compound of embodiment A-10, wherein each X is independently of formula: [0813]
Embodiment A-12 The compound of embodiment A-10, wherein Z 1 is selected from monocyclic 5 or 6-membered heteroaryl, monocyclic 5 or 6-membered aryl and Z 11 -Ar, wherein Ar is optionally substituted heteroaryl or optionally substituted aryl.
Embodiment A-13 The compound of embodiment A-12, wherein each X is [0815]
Embodiment A-14 The compound of embodiment A-2, wherein each X is independently of the formula: wherein R 4 and R 5 are each H.
Embodiment A-19 The compound of embodiment A-18, wherein each X is independently of formula (Ie): wherein: Z 2 is absent or selected from -O-, -S-, NR 25 -, -C(R 22 ) 2 -, and optionally substituted Z 12 -alkyl; ring A is absent or selected from a 5 or 6-membered optionally substituted aryl and a 5 or 6- membered optionally substituted heteroaryl; Z 3 is a linking moiety selected from Z 12 , optionally substituted alkyl, optionally substituted Z 12 - alkyl, optionally substituted Z 12 -heteroaryl, optionally substituted Z 12 -aryl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted amide, optionally substituted sulfonamide, optionally substituted urea, and optionally substituted thiourea; and Z 12 is selected from -CH 2 O-, -O-, -
Embodiment A-20 The compound of embodiment A-17, wherein each X is independently of one of formula (If)-(Ii): [0822]
Embodiment A-21 The compound of embodiment A-20, wherein each X is independently of one of formula (Ij)-(Im) wherein: Y 1 -Y 3 are each independently N or CR 27 ; and R 24 and R 27 are each independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen.
Embodiment A-22 The compound of embodiment A-21, wherein Z 3 is selected from -O-, - CH 2 O-, -OCH 2 -, optionally substituted -OCH 2 -heteroaryl, optionally substituted -OCH 2 -aryl, optionally substituted -CH 2 O-heteroaryl, and optionally substituted -CH 2 O-aryl.
Embodiment A-23 The compound of embodiment A-21 or A-22, wherein X is independently one of the following structures:
Embodiment A-24 The compound of embodiment A-18, wherein X is: .
Embodiment A-26 The compound of embodiment A-25, wherein Z 1 is selected from -O-, - S-, -CONR 21 -, and optionally substituted –(C(R 22 ) 2 ) q -heteroaryl, wherein q is 0 or 1.
Embodiment A-27 The compound of embodiment A-26, wherein Z 1 is -O-.
Embodiment A-28 The compound of embodiment A-36, wherein Z 1 is optionally substituted –(C(R 22 ) 2 ) q -triazole wherein q is 0 or 1.
Embodiment A-29 The compound of embodiment A-28, wherein [0832] Embodiment A-30: The compound of any one of embodiments A-1 to A-20, wherein n is 1, and L comprises a linear linker having a backbone of 20 or more consecutive atoms covalently linking X to Y via Z 1 .
Embodiment A-31 The compound of any one of embodiments A-1 to A-20, wherein n is 2 or more, and L is a branched linker that covalently links 2 or more X moieties to Y via the linking moiety Z 1 .
Embodiment A-32 The compound of any one of embodiment A-1 to A-31, wherein L is of formula (IIb): IIb) wherein each L 1 to L 5 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; a, b, c, d, and e are each independently 0, 1, or 2; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y, wherein when n is >1, d is 1 or 2 and L4 is a branching moiety.
L is of formula (IIb): IIb) wherein each L 1 to L 5 is independently a linking moiety which together provide a linear or branched linker between Z 1 and Y; a, b, c, d, and e are each independently 0, 1, or 2; ** represents the point of attachment to L 1 of X via Z 1 ; and *** represents the point of attachment to Y, wherein when n is
Embodiment A-33 The compound of claim 32, wherein L 1 to L 5 each independently comprise one or more linking moieties independently selected from –C 1-20 -alkylene–, –NHCO-C 1-6 - alkylene–, –CONH-C 1-6 -alkylene–, –NH-C 1-6 -alkylene–, —NHCONH-C 1-6 -alkylene–, – NHCSNH-C 1-6 - alkylene–, –C 1-6 -alkylene–NHCO-, –C 1-6 -alkylene–CONH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene– NHCONH-, –C 1-6 -alkylene–NHCSNH-, -O(CH 2 ) p –, –(OCH 2 CH 2 ) p –, –NHCO—, –CONH–, –NHSO 2 –, – SO
Embodiment A-34 The compound of embodiment A-32 or A-33, wherein L comprises repeating ethylene glycol moieties.
Embodiment A-35 The compound of embodiment A-34, wherein L comprises 1 to 25 ethylene glycol moieties.
Embodiment A-36 The compound of any one of embodiments A-32 to A-35, wherein L comprises one or more 1,2,3-triazole linking moieties.
Embodiment A-37 The compound of any one of embodiments A-32 to A-36, wherein n is 1.
Embodiment A-38 The compound of any one of embodiments A-32 to A-36, wherein n is 2 or more.
Embodiment A-39 The compound of embodiment A-38, wherein L 4 is a branching moiety [0842] wherein each x and y are each independently 1 to 10.
Embodiment A-40 The compound of any one of embodiments A-32 to A-39, wherein L 1 -L 4 comprises a backbone of 14 or more consecutive atoms between X and the branching atom.
Embodiment A-41 The compound of any one of embodiments A-32 to A-40, wherein L 5 comprises a backbone of 10 to 80 consecutive atoms.
Embodiment A-42 The compound of embodiment A-33, wherein L 5 comprises a linking moiety selected from (C 10 -C 20 -alkylene, or –(OCH 2 CH 2 ) p –, where p is 1 to 25.
Embodiment A-43 The compound of any one of embodiments A-32 to A-42, wherein the linker of formula (IIb) comprises a backbone of 20 to 100 consecutive atoms.
Embodiment A-44 The compound of embodiment A-43, wherein the linker of formula (IIb) comprises a backbone of 25 or more consecutive atoms.
Embodiment A-45 The compound of embodiment A-44, wherein the linker of formula (IIb) comprises a backbone of 30 or more consecutive atoms.
Embodiment A-46 The compound of any one of embodiments A-1 to A-45, wherein -Z 1 -L 1 - comprises a group selected from: wherein: each R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl; each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 1 to 6.
Embodiment A-47 The compound of embodiment A-46, wherein -Z 1 -L 1 - comprises a group selected from: wherein q is 1 to 3.
Embodiment A-48 The compound of any one of embodiments A-1 to A-45, wherein -Z 1 -L 1 - comprises an optionally substituted -NH-heteroaryl-.
Embodiment A-49 The compound of embodiment A-48, wherein -Z 1 -L 1 - comprises a group selected from: wherein: each R 24 is independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen; and each R 25 is independently selected from H, optionally substituted (C 1 -C 6 )alkyl, and optionally substituted acyl.
Embodiment A-50 The compound of any one of embodiment A-1 to A-49, wherein Y is selected from small molecule, dye, fluorophore, monosaccharide, polysaccharide, lipid, protein, polynucleotide, enzyme, enzyme substrate, polymer, and chemoselective ligation group or precursor thereof.
Embodiment A-51 The compound of any one of embodiment A-1 to A-49, wherein Y is a moiety that specifically binds an extracellular target protein.
Embodiment A-52 The compound of embodiment A-51, wherein the target protein is a membrane bound protein.
Embodiment A-53 The compound of embodiment A-51, wherein the target protein is a soluble extracellular protein.
Embodiment A-54 The compound of any one of embodiments A-51 to A-53, wherein Y is a target-binding small molecule.
Embodiment A-55 The compound of any one of embodiments A-51 to A-53, wherein Y is a target-binding biomolecule.
Embodiment A-56 The compound of embodiment A-55, wherein the biomolecule is selected from peptide, protein, glycoprotein, polynucleotide, aptamer, and antibody or antibody fragment.
Embodiment A-57 The compound of embodiment A-56, wherein Y is selected from antibody, antibody fragment, chimeric fusion protein, an engineered protein domain, and D-protein binder of target protein.
Embodiment A-58 The compound of embodiment A-55, wherein Y is a protein, n is 1 to 6, and m is 1 to 20.
Embodiment A-59 The compound of any one of embodiment A-51 to A-57, wherein Y is a moiety that specifically binds the target protein and the compound is a conjugate of formula (III’): wherein: n is 1 to 20; m is 1 to 80 (e.g., an average loading or a discrete loading); each X is a moiety that binds to a cell surface ASGPR; each L is a linker; each Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group to a compatible group of Y; and Y is a moiety of interest binds the target protein.
formula (III’) wherein: n is 1 to 20; m is 1 to 80 (e.g., an average loading or a discrete loading); each X is a moiety that binds to a cell surface ASGPR; each L is a linker; each Z is a residual moiety resulting from the covalent linkage
Embodiment A-60 The compound of embodiment A-59, wherein Y is an antibody or an antibody fragment.
Embodiment A-61 The compound of embodiment A-59, wherein Y is selected from chimeric fusion protein, and engineered protein domain.
Embodiment A-62 The compound of any one of embodiments A-59 to A-61, wherein n is 1 to 6.
Embodiment A-63 The compound of any one of embodiments A-59 to A-61, wherein n is 1 to 4.
Embodiment A-64 The compound of any one of embodiments A-59 to A-61, wherein n is 3.
Embodiment A-65 The compound of any one of embodiments A-59 to A-61, wherein n is 2.
Embodiment A-66 The compound of any one of embodiments A-59 to A-61, wherein n is 1.
Embodiment A-67 The compound of any one of embodiments A-59 to A-66, wherein m is 1 to 20.
Embodiment A-68 The compound of any one of embodiments A-59 to A-66, wherein m is 1 to 10.
Embodiment A-69 The compound of any one of embodiments A-59 to A-66, wherein m is 1 to 6.
Embodiment A-70 The compound of any one of embodiments A-59 to A-66, wherein m is 1 to 4.
Embodiment A-71 The compound of any one of embodiments A-59 to A-66, wherein m is 1 to 2.
Embodiment A-72 The compound of any one of embodiments A-59 to A-66, wherein m is 2.
Embodiment A-73 The compound of any one of embodiments A-59 to A-66, wherein m is 1.
Embodiment A-74 The compound of any one of embodiments A-59 to A-73, wherein Z is a residual moiety resulting from the covalent linkage of a thiol-reactive chemoselective ligation group to one or more cysteine residue(s) of Ab.
Embodiment A-75 The compound of embodiment A-74, wherein the thiol-reactive chemoselective ligation group comprises a maleimide.
Embodiment A-76 The compound of any one of embodiments A-59 to A-73, wherein Z is a residual moiety resulting from the covalent linkage of an amine-reactive chemoselective ligation group to one or more lysine residue(s) of Ab.
Embodiment A-77 The compound of embodiment A-76, wherein the amine-reactive chemoselective ligation group comprises a pentafluorophenyl (PFP) active ester.
PFP pentafluorophenyl
Embodiment A-78 A method of internalizing a target protein in a cell comprising a cell surface asialoglycoprotein receptor (ASGPR), the method comprising: contacting a cellular sample comprising the cell and the target protein with an effective amount of a compound according to any one of embodiments A-1 to A-77, wherein the compound specifically binds the target protein and specifically binds the ASGPR to facilitate cellular uptake of the target protein.
ASSGPR cell surface asialoglycoprotein receptor
Embodiment A-79 The method of embodiment A-78, wherein the target protein is a membrane bound protein.
Embodiment A-80 The method of embodiment A-78, wherein the target protein is an extracellular protein.
Embodiment A-81 A method of reducing levels of a target protein in a biological system, the method comprising: contacting the biological system with an effective amount of a compound according to any one of embodiments A-1 to A-77, wherein the compound specifically binds the target protein and specifically binds a ASGPR of cells in the biological system to facilitate cellular uptake and degradation of the target protein.
Embodiment A-82 The method of embodiment A-81, wherein the biological system is a human subject.
Embodiment A-83 The method of any one of embodiments A-81 to A-82, wherein the biological system is an in vitro cellular sample.
Embodiment A-84 The method of any one of embodiments A-81 to A-83, wherein the target protein is a membrane bound protein.
Embodiment A-85 The method of any one of embodiments A-81 to 83, wherein the target protein is an extracellular protein.
Embodiment B-2 The compound of embodiment B-1, wherein each X is independently of formula (IIa): wherein: R 6 is selected from –OH, –OC(O)R, and -C(O)NHR; and R 2 is selected from –NHCOCH 3 , –NHCOCF 3 , and –NHCOCH 2 CF 3 .
Embodiment B-3 The compound of embodiment B-3, wherein Z 1 is selected from -O-, -S-, and -C(R 22 ) 2 -.
Embodiment B-4 The compound of embodiment B-3, wherein Z 1 is Z 11 -Ar , wherein Ar is optionally substituted heteroaryl or optionally substituted aryl.
Embodiment B-5 The compound of embodiment B-4, wherein: Z 11 is O, S, or C(R 22 ) 2 ; and Ar is a monocyclic 5 or 6-membered heteroaryl or aryl.
Embodiment B-6 The compound of embodiment B-5, wherein Z 1 is -C(R 22 ) 2 -triazole-.
Embodiment B-7 The compound of embodiment B-6, wherein Z 1 is .
Embodiment B-8 The compound of embodiment B-3, wherein Z 1 is monocyclic 5 or 6- membered heteroaryl or aryl.
Embodiment B-9 The compound of embodiment B-8, wherein .
Embodiment B-10 The compound of embodiment B-2, wherein each X is independently of the formula: wherein R 3 and R 4 are each H.
Embodiment B-11 The compound of embodiment B-10, wherein each X is independently of formula: [0900]
Embodiment B-12 The compound of embodiment B-10, wherein Z 1 is selected from monocyclic 5 or 6-membered heteroaryl, monocyclic 5 or 6-membered aryl and Z 11 -Ar, wherein Ar is optionally substituted heteroaryl or optionally substituted aryl.
Embodiment B-13 The compound of embodiment B-12, wherein each X is [0902]
Embodiment B-14 The compound of embodiment B-2, wherein each X is independently of the formula: wherein R 3 and R 4 are each H.
Embodiment B-19 The compound of embodiment B-18, wherein each X is independently of formula (IVb) or (IVc): wherein -Z 11 - is -O-, -S-, -N(R 21 )-, or -C(R 22 ) 2 .
Embodiment B-20 The compound of embodiment B-17, wherein each X is independently of formula (IVb-1) or (IVc-1): (IVb-1) (IVc-1) wherein R 11 is the bridging moiety that connects the 5-position carbon to the 1-position carbon.
Embodiment B-21 The compound of embodiment B-19 or B-20, wherein each X is independently of formula ( wherein R 21 and R 22 are independently selected from H, halogen, (C 1- C 6 )alkyl and substituted (C 1- C 6 )alkyl (e.g., CF 3 ).
Embodiment B-22 The compound of embodiment B-21, wherein Z 3 R 21 and R 22 are independently H, or CF 3 .
Embodiment B-23 The compound of embodiment B-21 or B-22, wherein X is independently one of the following structures:
Embodiment B-26 The compound of embodiment B-25, wherein Z 1 is selected from -O-, -S- , -CONR 21 -, and optionally substituted –(C(R 22 ) 2 ) q -heteroaryl, wherein q is 0 or 1.
Embodiment B-27 The compound of embodiment B-26, wherein Z 1 is -O-.
Embodiment B-28 The compound of embodiment B-36, wherein Z 1 is optionally substituted –(C(R 22 ) 2 ) q -triazole wherein q is 0 or 1.
Embodiment B-29 The compound of embodiment B-28, wherein [0918] Embodiment B-30: The compound of any one of embodiments B-1 to B-20, wherein n is 1, and L comprises a linear linker having a backbone of 20 or more consecutive atoms covalently linking X to Y via Z 1 .
Embodiment B-31 The compound of any one of embodiments B-1 to B-20, wherein n is 2 or more, and L is a branched linker that covalently links 2 or more X moieties to Y via the linking moiety Z 1 .
Embodiment B-32 The compound of any one of embodiments B-1 to B-31, wherein L is of formula (XI): wherein each L 1 and L 3 are independently a linear linking moiety, and L 2 is a branched linking moiety, wherein L 1 to L 3 together provide a linear or branched linker between X and Y; a, b and c are independently 0 or 1, wherein: when n is 1, b is 0 and at least one of a and c is 1; and when n is 2 or 3, a, b and c are each 1; * represents the point of connection of L 1 to X via Z 1 ; and ** represents a point of conjugation of the linker L to Y.
L is of formula (XI): wherein each L 1 and L 3 are independently a linear linking moiety, and L 2 is a branched linking moiety, wherein L 1 to L 3 together provide a linear or branched linker between X and Y; a, b
Embodiment B-33 The compound of embodiment B-32, wherein: n is 1; and a is 1, b is 0 and c is 1, whereby L is of formula (XIa): * L1 L3 ** (XIa).
Embodiment B-34 The compound of embodiment B-32, wherein: n is 2; and a is 1, b is 1, and c is 1, whereby L is of formula (XIb): (XIb).
Embodiment B-35 The compound of embodiment B-32, wherein: n is 3; and a is 1, b is 1, and c is 1, whereby L is of formula (XIc): (XIc).
Embodiment B-36 The compound of any one of embodiments B-32 to B-35, wherein each L 1 is of the formula (XII) wherein: L 10 is a linking moiety; and L 11 to L 19 are independently absent or a linking moiety, wherein L 10 to L 19 of each L 1 is each independently selected from –C 1-20 -alkylene–, –NHCO-C 1-6 - alkylene–, –CONH-C 1-6 -alkylene–, –NH-C 1-6 -alkylene–, —NHCONH-C 1-6 -alkylene–, – NHCSNH-C 1-6 - alkylene–, –C 1-6 -alkylene–NHCO-, –C 1-6 -alkylene–CONH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–NHCONH-, –C 1-6 -alkylene–NHCSNH-, -O
Embodiment B-37 The compound of any one of embodiments B-32 to B-36, wherein each L 1 comprises a linear backbone of 6 to 20 consecutive atoms (e.g., 6 to 16 consecutive atoms, such as 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive atoms).
Embodiment B-38 The compound of any one of embodiments B-32, and B-34 to B-37, wherein L 2 is of formula (XIIIa) or (XIIIb): (XIIIa) (XIIIb) wherein: L 20 is a branched linking moiety comprising: a carbon atom or nitrogen atom that is the branching point of the branched linking moiety; and one or more (e.g., 1 to 20, 1 to 10, or 1 to 6) linking moieties independently selected from amino acid residue (e.g., a residue such a s, or a derivative thereof), –NH-CH[(CH 2 ) q ] 2 O– alkylene–, – NHCO-, –CONH–, –NHSO 2 –, –SO 2 NH–, –CO–, –SO 2 –, –O–, –S–, pyrrolidine-2,5-dione, 1,2,3- triazole, –NH–, and
Embodiment B-39 The compound of any one of embodiments B-32, and B-34 to B-38, wherein L 2 comprises a linking moiety selected from: wherein: Z 2 is connected to L 1 , and Z 3 is connected to L 3 ; each Z 2 and Z 3 is independently selected from –NHCO-, –CONH–, –CO–, –O–, –NH–, and – N(CH 3 )–; x is 1 to 12 (e.g., 1 to 6, or 1 to 3); and y is 0 to 12 (e.g., 1 to 6, or 1 to 3).
Embodiment B-40 The compound of embodiment B-39, wherein L 2 comprises a linking moiety selected from: [0929]
Embodiment B-41 The compound of embodiment B-40, wherein L 2 comprises a linking moiety of formula (XIV): wherein: r is 1 or 2; and when n is 2, r is 1, when n is 3, r is 2.
Embodiment B-42 The compound of embodiment B-41, wherein L 2 is of formula (XVa) or (XVb): (XVa) (XVb).
Embodiment B-43 The compound of embodiment B-41 or B-42, wherein L 2 is of formula (XVc) or (XVd): (XVc) (XVc) wherein r is 1 or 2.
Embodiment B-44 The compound of embodiment B-38, wherein L 2 comprises two 2 or more amino acid residues (e.g., 3 or more, or 4 or more amino acid residues, linear or dendrimer).
Embodiment B-45 The compound of embodiment B-38, wherein L 2 comprises 4 or more amino acid residues that are branched linking moieties selected from Lys, Orn, Asp, Glu, Ser, and Cys (e.g., where the sidechain, amino and carboxylic acid are each linked to an adjacent moiety).
Embodiment B-46 The compound of any one of embodiments B-32 to B-45, wherein each L 3 is of the formula (XVI): wherein: L 30 to L 39 are independently absent or a linking moiety; and Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group of the linker to a compatible group of Y; wherein L 30 to L 39 are each independently selected from –C 1-20 -alkylene–, –NHCO-C 1-6 -alkylene–, –CONH-C 1-6 -alkylene–, –NH C 1-6 -alkylene–, –NHCONH-C 1-6 -alkylene–, – NHCSNH-C 1-6 -alkylene–, – C 1-6 -alkylene–NHCO-, –C 1-6 -alkylene–CONH-, –C 1-6 -alkylene–NH-, –C 1-6 -alkylene–CONH
Embodiment B-47 The compound of embodiment B-46, wherein L 3 comprises a linear backbone of 6 to 40 consecutive atoms (e.g., 10 to 30 consecutive atoms, or 20 to 30 consecutive atoms).
Embodiment B-48 The compound of embodiment B-46 or B-47, wherein the linker L has one of the following structures: , , wherein: a is 1 to 12 (e.g., 2 to 6, or 2, or 3); b is 1 to 6 (e.g., 1, 2, or 3); c is 1 to 6 (e.g., 1, 2, or 3); r is 1 or 2; d is 1 to 6 (e.g., 1, 2, or 3); e is b is 1 to 6 (e.g., 1, 2, or 3); f is 1 to 6 (e.g., 1, 2, or 3); g is 1 to 20 (e.g., 1 to 12, or 6 to 20 or 6 to 12); Z is a residual moiety resulting from the
Embodiment B-50 The compound of embodiment B-32, wherein L comprises repeating ethylene glycol moieties.
Embodiment B-51 The compound of embodiment B-32, wherein L comprises 1 to 25 ethylene glycol moieties.
Embodiment B-52 The compound of embodiment B-32, wherein L comprises one or more 1,2,3-triazole linking moieties.
Embodiment B-53 The compound of embodiment B-38, wherein L 2 is a branching moiety selected from: wherein each x and y are each independently 1 to 10.
Embodiment B-54 The compound of any one of embodiments B-32 to B-54, wherein the linker comprises a backbone of 14 or more consecutive atoms between each X and the branching atom.
Embodiment B-55 The compound of any one of embodiments B-32 to B-54, wherein L 3 comprises a backbone of 10 to 80 consecutive atoms.
Embodiment B-56 The compound of embodiment B-55, wherein L 3 comprises a linking moiety selected from (C 10 -C 20 -alkylene, or –(OCH 2 CH 2 ) p –, where p is 1 to 25.
Embodiment B-57 The compound of any one of embodiment B-32 to B-56, wherein the linker of formula (XI) comprises a backbone of 20 to 100 consecutive atoms.
Embodiment B-58 The compound of embodiment B-57, wherein the linker of formula (XI) comprises a backbone of 25 or more consecutive atoms.
Embodiment B-59 The compound of embodiment B-58, wherein the linker of formula (XI) comprises a backbone of 30 or more consecutive atoms.
Embodiment B-60 The compound of any one of embodiments B-1 to B-59, wherein -Z 1 -L 1 - comprises a group selected from: wherein: each R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl; each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl; and o, p, q, r, s, t, u, v, w, x, y, z and z1 are each independently 1 to 6.
each R 21 is independently selected from H, and optionally substituted (C 1 -C 6 )alkyl
each R 22 is independently selected from H, halogen (e.g., F) and optionally substituted (C 1 - C 6 )alkyl
Embodiment B-61 The compound of embodiment B-60, wherein -Z 1 -L 1 - comprises a group selected from: wherein q is 1 to 3.
Embodiment B-62 The compound of any one of embodiments B-1 to B-61, wherein -Z 1 -L 1 - comprises an optionally substituted -NH-heteroaryl-.
Embodiment B-63 The compound of embodiment B-62, wherein -Z 1 -L 1 - comprises a group selected from: wherein: each R 24 is independently selected from H, optionally substituted C (1-6) -alkyl, optionally substituted fluoroalkyl, and halogen; and each R 25 is independently selected from H, optionally substituted (C 1 -C 6 )alkyl, and optionally substituted acyl.
Embodiment B-64 The compound of any one of embodiments B-1 to B-63, wherein Y is selected from small molecule, dye, fluorophore, monosaccharide, polysaccharide, lipid, protein, polynucleotide, enzyme, enzyme substrate, polymer, and chemoselective ligation group or precursor thereof.
Embodiment B-65 The compound of any one of embodiments B-1 to B-64, wherein Y is a moiety that specifically binds an extracellular target protein.
Embodiment B-66 The compound of embodiment B-65, wherein the target protein is a membrane bound protein.
Embodiment B-67 The compound of embodiment B-65, wherein the target protein is a soluble extracellular protein.
Embodiment B-68 The compound of any one of embodiments B-65 to B-67, wherein Y is a target-binding small molecule.
Embodiment B-69 The compound of any one of embodiments B-65 to B-67, wherein Y is a target-binding biomolecule.
Embodiment B-70 The compound of embodiment B-69, wherein the biomolecule is selected from peptide, protein, glycoprotein, polynucleotide, aptamer, and antibody or antibody fragment.
Embodiment B-71 The compound of embodiment B-69, wherein Y is selected from antibody, antibody fragment, chimeric fusion protein, an engineered protein domain, and D-protein binder of target protein.
Embodiment B-72 The compound of embodiment B-69, wherein Y is a protein, n is 1 to 10 (e.g., 1 to 8, 1 to 6, such as 1, 2, 3, 4-5, 5-6, or 6-7), and m is 1 to 20.
Embodiment B-73 The compound of any one of embodiments B-51 to B-57, wherein Y is a moiety that specifically binds the target protein and the compound is a conjugate of formula (III’): wherein: n is 1 to 20; m is 1 to 80 (e.g., an average loading or a discrete loading); each X is a moiety that binds to a cell surface ASGPR; each L is a linker; each Z is a residual moiety resulting from the covalent linkage of a chemoselective ligation group to a compatible group of Y; and Y is a moiety of interest (e.g., a protein, antibody, aptamer, peptide) binds the target protein.
formula (III’) wherein: n is 1 to 20; m is 1 to 80 (e.g., an average loading or a discrete loading); each X is a moiety that binds to a cell surface ASGPR; each L
Embodiment B-74 The compound of embodiment B-59, wherein Y is an antibody or an antibody fragment.
Embodiment B-75 The compound of embodiment B-59, wherein Y is selected from chimeric fusion protein, and engineered protein domain.
Embodiment B-76 The compound of any one of embodiments B-73 to B-75, wherein n is to 10 (e.g., 1 to 8, or 1 to 6).
Embodiment B-77 The compound of any one of embodiments B-73 to B-75, wherein n is 1 to 4.
Embodiment B-78 The compound of any one of embodiments B-73 to B-75, wherein n is 3.
Embodiment B-79 The compound of any one of embodiments B-73 to B-75, wherein n is 2.
Embodiment B-80 The compound of any one of embodiments B-73 to B-75, wherein n is 1.
Embodiment B-81 The compound of any one of embodiments B-73 to B-80, wherein m is 1 to 20.
Embodiment B-82 The compound of any one of embodiments B-73 to B-80, wherein m is 1 to 10.
Embodiment B-83 The compound of any one of embodiments B-73 to B-80, wherein m is 4 to 8 (e.g., 4-6, 4-5, 5-6, or 6-7).
Embodiment B-84 The compound of any one of embodiments B-73 to B-80, wherein m is 1 to 6.
Embodiment B-85 The compound of any one of embodiments B-73 to B-80, wherein m is 1 to 4.
Embodiment B-86 The compound of any one of embodiments B-73 to B-80, wherein m is 1 to 3 (e.g., 1, 2, or 3).
Embodiment B-87 The compound of any one of embodiments B-73 to B-80, wherein m is 2.
Embodiment B-88 The compound of any one of embodiments B-73 to B-80, wherein m is 1.
Embodiment B-89 The compound of any one of embodiments B-73 to B-88, wherein Z is a residual moiety resulting from the covalent linkage of a thiol-reactive chemoselective ligation group to one or more cysteine residue(s) of Ab.
Embodiment B-90 The compound of embodiment B-89, wherein the thiol-reactive chemoselective ligation group comprises a maleimide or phenylene-maleimide.
Embodiment B-91 The compound of any one of embodiments B-73 to B-88, wherein Z is a residual moiety resulting from the covalent linkage of an amine-reactive chemoselective ligation group to one or more lysine residue(s) of Ab.
Embodiment B-92 The compound of embodiment B-91, wherein the amine-reactive chemoselective ligation group comprises a pentafluorophenyl (PFP) active ester.
PFP pentafluorophenyl
Embodiment B-93 A method of internalizing a target protein in a cell comprising a cell surface asialoglycoprotein receptor (ASGPR), the method comprising: contacting a cellular sample comprising the cell and the target protein with an effective amount of a compound according to any one of embodiments B-1 to B-92, wherein the compound specifically binds the target protein and specifically binds the ASGPR to facilitate cellular uptake of the target protein.
Embodiment B-94 The method of embodiment B-93, wherein the target protein is a membrane bound protein.
Embodiment B-95 The method of embodiment B-93, wherein the target protein is an extracellular protein.
Embodiment B-96 A method of reducing levels of a target protein in a biological system, the method comprising: contacting the biological system with an effective amount of a compound according to any one of embodiments B-1 to B-92, wherein the compound specifically binds the target protein and specifically binds a ASGPR of cells in the biological system to facilitate cellular uptake and degradation of the target protein.
Embodiment B-97 The method of embodiment B-96, wherein the biological system is a human subject.
Embodiment B-98 The method of any one of embodiments B-96 to B-97, wherein the biological system is an in vitro cellular sample.
Embodiment B-99 The method of any one of embodiments B-96 to B-98, wherein the target protein is a membrane bound protein.
Embodiment B-100 The method of any one of embodiments B-96 to B-98, wherein the target protein is an extracellular protein.
EXAMPLES [0989] The examples in this section are offered by way of illustration, and not by way of limitation.
Example 1 Preparation of Compounds [0990] The following are illustrative schemes and examples of how the compounds described herein can be prepared and tested. Although the examples can represent only some embodiments, it should be understood that the following examples are illustrative and not limiting. All substituents, unless otherwise specified, are as previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art. The specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare the compounds described herein. Synthesis of compound XC28:
reaction mixture was concentrated, washed with diethyl ether and dried to afford 2-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3- yl)isoindoline-1,3-dione (48-6) as a light purple solid which was used as such for next reaction. Yield: 7.0 g (Crude).
reaction mixture was neutralized with Dowex 50WX8 hydrogen form (200-400 mesh) and filtered through sintered funnel (without celite). The filtrate was concentrated, washed with diethyl ether and dried to afford 2-((3S,4R,5R,6R)-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)isoindoline-1,3-dione (48-8) as an off white solid. Yield: 0.640 g, 83.2 %; ELSD m/z 294.15 [M+1] + .
reaction mixture was cooled, water was added and extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-25 % ethyl acetate in hexane to afford 2- ((3S,4R,5R,6R)-4,5-bis(methoxymethoxy)-6-((methoxymethoxy)methyl)tetrahydro-2H-pyran-3- yl)isoindoline-1,3-dione (48-9) as colourless viscous liquid.
reaction mixture was cooled and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-5 % methanol in dichloromethane to afford (3S,4R,5R,6R)-4,5-bis(methoxymethoxy)-6- ((methoxymethoxy)methyl)tetrahydro-2H-pyran-3-amine (48-10) as colourless viscous liquid. Yield: 0.310 g, 76.99 %; ELSD m/z 296.20 [M+1] + .
reaction mixture was concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-5 % methanol in dichloromethane to afford 2,5-dioxopyrrolidin-1-yl 3,3,3-trifluoropropanoate (2) as colorless viscous liquid. Yield: 0.050 g, 28.57 %; LCMS m/z No ionization; 1 H NMR (400 MHz, CDCl 3 ) ⁇ 3.54-3.47 (m, 2H), 2.87 (s, 4H).
reaction mixture was concentrated to get crude which was purified by prep HPLC (25-33 % acetonitrile in water with 0.1 % trifluoroacetic acid). Fractions containing the desired product were combined and lyophilized to dryness to afford N-((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)-3,3,3- trifluoropropanamide (XC28) as a cream solid.
reaction mixture was concentrated under reduce pressure to get crude which was purified by flash column chromatography on silica gel using 0-50% ethyl acetate in hexane to afford a (S)-3-((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)-5-(methoxymethyl)oxazolidin-2-one (3) as colorless liquid. Yield: 0.2 g; 51.49 %, LCMS m/z 548.0 [M+1] + .
tert-butyl (4-aminobutyl)carbamate (1.1 eq.0.20 g, 1.16 mmol) was added, and stirred the reaction mixture at room temperature for 12 h. After completion (monitored by TLC), water was added, and extracted with dichloromethane.
reaction mixture was filtered on celite pad and washed with methanol. The filtrate was evaporated under reduced pressure to give crude which was again treated with a mixture trifluoroacetic acid and dichloromethane (10 ml, 1:1, v/v) and stirred for 1h at room temperature.
reaction mixture was purified by prep-HPLC (20-30% acetonitrile in water with 0.1% TFA) to give N-(4-aminobutyl)-2- ((3S,4aS,6R,7R,8R,8aR)-7,8-dihydroxy-6-(hydroxymethyl)-2-oxohexahydro-1H,6H-pyrano[2,3- b][1,4]oxazin-3-yl)acetamide (XC25) as colorless semi solid.
reaction mixture was concentrated under vacuum to get crude product which was re-dissolved in ethyl acetate, washed with ice cold water. The organic layer was dried over anhydrous sodium sulfate, concentrated to get crude which was purified by silica gel flash column chromatography using 2-5% methanol in dichloromethane to afford 2-(((3S,4R,5R,6R)-4,5- bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)amino)-N-(2-methoxyethyl)acetamide (3) as green oily liquid.
reaction mixture was quenched by 15 % sodium hydroxide solution, brine solution was added, then extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to give crude N1-((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro- 2H-pyran-3-yl)-N2-(2-methoxyethyl)ethane-1,2-diamine (4) as brown syrup which was used for next reaction without further purification.
reaction mixture was concentrated to give crude residue which was purified by flash column chromatography on silica gel using 50% ethyl acetate in hexane to afford a 1-((3S,4R,5R,6R)-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H- pyran-3-yl)-3-(2-methoxyethyl)imidazolidin-2-one (5) as colorless liquid. Yield: 0.080 g, 69.36 %; LCMS m/z 561.15 [M+1] + .
reaction mixture was concentrated to get crude. which was purified by column chromatography using silica gel (100-200 mesh) and 0-10 % methanol in DCM to afford N-((3aR,4S,7S,8R,8aR)-4-(methoxymethyl)-2,2-dimethylhexahydro-4,7- epoxy[1,3]dioxolo[4,5-d]oxepin-8-yl)-4-(prop-2-yn-1-yloxy)-6-(trifluoromethyl)pyrimidin-2-amine (4) as a white solid. Yield: 0.070 g, 55.07%; LCMS m/z 446.05 [M+1] + .
reaction mixture was purified by prep HPLC (45-55% ACN in H 2 O with 0.1% TFA). Fractions containing desired compound were collected and lyophilized to afford (1S,2R,3R,4R,5S)-1-(methoxymethyl)-4-((4-(prop-2- yn-1-yloxy)-6-(trifluoromethyl)pyrimidin-2-yl)amino)-6,8-dioxabicyclo[3.2.1]octane-2,3-diol (XC11) as an off white solid. Yield: 0.0013 g, 1.37 %; m/z 406.0 [M+1] + .
N-((3aR,4R,7S,7aR)-4-(hydroxymethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran- 7-yl)acetamide (XC17-A, 1.00 eq, 17.0 mg, 0.0693 mmol) was added and after another 10 minutes, 4- methoxyphenol (1.5 eq, 12.9 mg, 0.104 mmol) was added and the reaction was stirred at room temperature for 1 hour then at 50 °C for 18 h.
reaction was purified directly by reversed-phase HPLC (10-50% acetonitrile in water w/0.1% FA) to give N-((3aR,4R,7S,7aR)-4-((4- methoxyphenoxy)methyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)acetamide.
LCMS m/z 351.5 [M+H] + .
reaction mixture was stirred for 16h at room temperature. After completion (monitored by TLC), the reaction mixture was poured into cold 1N HCl, and extracted with dichloromethane. The organic part was then washed with saturated bicarbonate followed by brine, and dried over anhydrous sodium sulfate, filtered, and concentrated to give crude residue which was purified by silica gel flash column chromatography using 0-30% ethyl acetate in hexane to afford 6- ((2R,3S,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-2- yl)hexa-3,5-diyn-1-yl 4-methylbenzenesulfonate (3) as white solid.
reaction mixture was extracted with ethyl acetate, and the collected ethyl acetate was dried over anhydrous sodium sulphate, filtered, and concentrated to give crude (2R,3R,4R,5S,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-oxoethyl)tetrahydro-2H-pyran-3,4-diyl diacetate (4) as colorless syrup. This was immediately used for next step without purification. Yield: 1.4 g (Crude); ELSD-MS m/z 374.19 [M+H] + .
N,N-dibenzyl-2-(2-prop-2- ynoxyethoxy)ethanamine 2 was dissolved in 10 mL toluene then concentrated to dryness and left under high vacuum.
the reaction was filtered then re-cooled in an ice bath before it was treated with 5M aq. sodium hydroxide (350 eq, 214 mL, 1070 mmol) at such a rate as to keep the internal temperature below 24 °C.
5M aq. sodium hydroxide 350 eq, 214 mL, 1070 mmol
the layers were partitioned then the aqueous layer was washed with DCM (80 mL).
the aqueous layer was washed with DCM (50 mL) then the combined organic layer was washed with brine then dried over Na2SO4, filtered, concentrated under reduced pressure and left under high vacuum.
reaction mixture was cooled, water was added, neutralized with solid sodium bicarbonate and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-20 % ethyl acetate in dichloromethane to afford (3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a- tetrahydro-5H-pyrano[3,2-d]thiazole-6,7-diyl diacetate (2) as light yellow viscous liquid.
reaction mixture was concentrated, azeotroped with toluene (2-3 times) and dried to get afford (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6- mercaptotetrahydro-2H-pyran-3,4-diyl diacetate (3) as a light yellow viscous liquid. Yield: 1.6 g (Crude); LCMS m/z 364.10 [M+H].
reaction mixture was concentrated to get crude which was first purified by column chromatography using silica gel (100-200 mesh) and 0-10 % methanol in dichloromethane and then by prep HPLC (10-25 % acetonitrile in water with 0.1 % trifluoroacetic acid).
reaction mixture was concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-5 % methanol in dichloromethane to afford (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((2,2- dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-yl)thio)tetrahydro-2H-pyran-3,4-diyl diacetate (2) as a colorless semi solid.
reaction mixture was neutralized with Dowex 50WX8 hydrogen form (200-400 mesh) and filtered through sintered funnel (without celite). The filtrate was concentrated, washed with diethyl ether and dried to afford tert-butyl (2-(2-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)ethoxy)ethoxy)ethoxy)ethyl)carbamate (3) as an off white solid. Yield: 0.320 g, 88.64 %; LCMS m/z 513.10 [M+H].
reaction mixture stirred at ambient temperature for 30 minutes, at which time 8 mL of 1N aqueous hydrochloric acid was added slowly to achieve approximate final pH of 1-2.
the reaction mixture was concentrated to approximately 1 ⁇ 4 volume and purified by preparatory HPLC, eluting with 1-30% acetonitrile in water with 0.1% trifluoroacetic acid. Fractions containing the desired product were combined and lyophilized to dryness to afford Compound 3 as white solid. Yield: 244 mg (64%); LCMS m/z 423.06 [M+H].
reaction was monitored by ELSD. After completion, reaction mixture was cooled, diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate solution and water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-5 % methanol in dichloromethane to afford (2R,3R,4R,5R,6S)-5-acetamido-2-(acetoxymethyl)-6-((3-methoxy-3-oxopropyl)thio)tetrahydro-2H- pyran-3,4-diyl diacetate (2) as a colorless viscous liquid.
reaction mixture was neutralized with Dowex 50WX8 hydrogen form (200-400 mesh) and filtered through sintered funnel (without celite). The filtrate was concentrated and dried to afford methyl 3-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanoate (3) as an off white solid. Yield: 1.7 g, 59.0 %; LCMS m/z 324.0 [M+H].
reaction mixture was concentrated, methanol was added, neutralized with Dowex 50WX8 hydrogen form (200-400 mesh) and filtered through sintered funnel (without celite). The filtrate was concentrated and dried to afford 3-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanoic acid (4) as an off white sticky solid. Yield: 2.4 g (Crude); LCMS m/z 310.0 [M+H].
reaction mixture was concentrated to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 0-7 % methanol in dichloromethane to afford 3-(((2S,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetoxymethyl)tetrahydro-2H-pyran-2-yl)thio)propanoic acid (XB5) as a colorless viscous liquid.
reaction mixture was quenched with triethyl amine and concentrated under reduced pressure to get crude which was purified by column chromatography using silica gel (100-200 mesh) and 2-5% methanol in dichloromethane to afford (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((3-oxo-1-phenyl-2,7,10-trioxa-4- azatridecan-13-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (3) as colorless viscous liquid.
the mixture was stirred under nitrogen atmosphere at ambient temperature for approximately 15 minutes until completion.
the reaction mixture was diluted with water, which formed a precipitate, then 2 drops of trifluoroacetic acid was added to clear the solution.
the product was isolated from the diluted mixture by preparatory HPLC, eluting with 1-20% acetonitrile in water with 0.1% trifluoroacetic acid.
tert-butyl (2-(2-(2- bromoethoxy)ethoxy)ethyl)carbamate (4a, 0.195 g, 1 eq., 0.625 mmol) was added.
the reaction mixture was stirred at room temperature for 1h. After completion (monitored by TLC), ice-cold water (10 mL) was added to the reaction mixture. Organic part was extracted with ethyl acetate (3 ⁇ 10 mL), combined and dried over anhydrous sodium sulphate.
reaction mixture was cooled down to 0 °C, quenched with the addition of an aqueous sodium thiosulfate solution (1M) and saturated sodium bicarbonate solution. The aqueous layer was extracted with ethyl acetate.
reaction mixture was stirred at room temperature for 1h. Progress of the reaction was monitored by LCMS. After completion of reaction, solvent was evaporated under reduced pressure to obtain crude residue. Crude was purified by RP prep-HPLC (60% acetonitrile in water with 0.1% trifluoroacetic acid). Fractions containing desired product were combined and lyophilized to afford (2R,3R,4R,5S)-5-((4-((2-(2-(2- aminoethoxy)ethoxy)ethoxy)methyl)thiazol-2-yl)amino)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (XB82) as a white sticky solid.
reaction mixture was stirred at room temperature for 12h. After completion the reaction mixture was concentrated under reduced pressure to get a crude which was diluted with water and extracted with ethyl acetate (5 X 120 mL). Organic part was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to afford tert-butyl ((2R,3R,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-propyltetrahydro-2H-pyran-3-yl)carbamate (1a) as off white solid. Yield: 5.0 g, crude; LCMS m/z 306.1[M + H] + .
reaction mixture was neutralized using triethyl amine and concentrated under reduced pressure to afford crude which was purified by silica gel flash column chromatography using 20-50 % ethyl acetate/hexane as eluent to afford tert-butyl ((3aR,4R,6R,7S,7aR)-4-(hydroxymethyl)-2,2-dimethyl-6- propyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-7-yl)carbamate (1) as off white solid.
reaction mixture was stirred at room temperature for 4h. After completion (monitored by TLC), reaction mixture was concentrated under reduce pressure and co-evaporated with dichloromethane three times to obtain crude residue, which was further lyophilized to afford (2R,3R,4R,5R,6R)-5-amino-2-(methoxymethyl)-6- propyltetrahydro-2H-pyran-3,4-diol (3) as light yellow syrup. The crude residue was directly used for next step. Yield: 0.6 g (Crude). LCMS m/z 220.1 [M+H] + .
reaction mixture was concentrated under reduced pressure.
the crude residue was purified by silica gel flash column chromatography using 2-3% methanol in dichloromethane as the eluent to afford tert-butyl (2-(2-((2-(((2R,3R,4R,5R,6R)- 4,5-dihydroxy-6-(methoxymethyl)-2-propyltetrahydro-2H-pyran-3-yl)amino)pyrimidin-4- yl)oxy)ethoxy)ethoxy)ethyl)carbamate (4) as brown gummy liquid. Yield: 0.64 g, 67.1%.
reaction mixture was concentrated under reduced pressure to get crude which was purified by RP prep HPLC (20% acetonitrile in water with 0.1 % acetic acid). Fractions containing desired product were combined and lyophilized to get (2,2-dimethyl- 4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)glycine (3) as a white solid. Yield: 2.5 g, 59.3%.
reaction mixture was concentrated under reduced pressure to afford crude which was again dissolved in tetrahydrofuran (20 mL) and acetic anhydride (2.32 mL, 3 eq., 24.5 mmol) was added at 0 °C and reaction mixture was allowed to stir at room temperature for 36h. After completion, reaction mixture was concentrated to afford crude which was purified by silica gel column chromatography using 2-3% methanol/dichloromethane as eluent to afford 3-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)- 1,2,3-oxadiazol-3-ium-5-olate (4) as yellow liquid.
reaction mixture was quenched with methanol and concentrated under reduced pressure to afford crude which was purified by RP prep-HPLC (40% of acetonitrile in water with 0.1% TFA).
Fractions containing desired product were combined and lyophilized to afford N-((2S,3R,4R,5R,6R)-2-(1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-pyrazol-3-yl)- 4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide (XB84) as colorless foamy solid.
reaction mixture was filtered through syringe filter and washed with methanol. The filtrate was concentrated and dried to obtained crude. Crude was purified by silica gel chromatography using 10% methanol in dichloromethane to afford tert-butyl (2-(2-(3-(2-bromopyrimidin-4- yl)propoxy)ethoxy)ethyl)carbamate (3) as colourless liquid. Yield: 0.11 g, 38.5%; LCMS: m/z 403.9 [M+H].
reaction was monitored by TLC. After completion of reaction, reaction mixture was diluted with ice cold water and extracted with ethyl acetate (3 ⁇ 20 mL). Then, organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford crude tert-butyl (Z)-(2-(2-(2-chloro-2- (hydroxyimino)ethoxy)ethoxy)ethyl)carbamate (4, 1.6 g) which was used for the next step as such.
reaction mixture was concentrated under reduced pressure to afford crude which was purified by silica gel flash column chromatography using 70-80% ethyl acetae- heptane as eluent to afford tert-butyl (2-(2-((5-((2S,3R,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6- ((benzyloxy)methyl)tetrahydro-2H-pyran-2-yl)isoxazol-3-yl)methoxy)ethoxy)ethyl)carbamate (5) as yellowish sticky liquid. Yield: 0.27 g, 65.7%.
reaction mixture was filtered through syringe filter and washed with methanol. The filtrate was concentrated and dried to afford crude tert-butyl (2-(2-(3-(6-(((3S,4R,5R,6R)-4,5-dihydroxy- 6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)pyrazin-2-yl)propoxy)ethoxy)ethyl)carbamate (5) as colour less liquid. Yield: 0.25 g, crude; LCMS: m/z 487.00 [M+H].
reaction mixture was quenched with water (10 mL) and extracted with dichloromethane to obtain crude, which was purified by flash column chromatography (using 10% ethyl acetate/heptane as eluent) to afford tert-butyl(dec-9-yn-1-yloxy)dimethylsilane (2) as colourless liquid.
Methyl iodide (0.566 mL, 1.20 eq., 9.10 mmol) was added and the reaction mixture was allowed to warm to room temperature and stirred for 4h. After completion, the mixture was dried, diluted with water and extracted with ethyl acetate. The combined organic layer was dried over anhydrous sodium sulphate, concentrated under reduced pressure to get crude, which was purified by silica gel flash column chromatography using 20-40% ethyl acetate in heptane as eluent to afford benzyl methyl(2-(2-(prop-2-yn-1- yloxy)ethoxy)ethyl)carbamate (3) as a brown liquid.
reaction mixture was stirred at room temperature under hydrogen for 16 h. Reaction was monitored by ELSD. After completion, reaction mixture was filtered through syringe filter and filtrate was concentrated to get crude which was purified by prep HPLC (14-28 % acetonitrile in water with 0.1 % trifluoroacetic acid) to afford (R)-5-((2-(2-(2 aminoethoxy)ethoxy)ethoxy)methyl)-3- ((3S,4R,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxazolidin-2-one (XB91) as colorless viscous syrup.
reaction mixture was stirred at room temperature for 2h. After completion, the reaction mixture was diluted with water and extracted with dichloromethane. The combined organic layer was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated to get crude which was purified by column chromatography using 10-25% ethyl acetate/heptane as eluent to afford ((3aR,4R,7S,7aR)-7-((tert-butoxycarbonyl)amino)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran- 4-yl)methyl 4-methylbenzenesulfonate (2) as off-white solid.

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