WO2024259310A2 - Composés antagonistes de pcsk9 perméables passifs - Google Patents

Composés antagonistes de pcsk9 perméables passifs Download PDF

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Publication number
WO2024259310A2
WO2024259310A2 PCT/US2024/034113 US2024034113W WO2024259310A2 WO 2024259310 A2 WO2024259310 A2 WO 2024259310A2 US 2024034113 W US2024034113 W US 2024034113W WO 2024259310 A2 WO2024259310 A2 WO 2024259310A2
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mmol
added
int
synthesis
afford
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WO2024259310A3 (fr
Inventor
Abbas M. Walji
Harold B. Wood
Yuhua Huang
Thomas Joseph Tucker
Yi-Heng Chen
Mark W. Embrey
Fa-Xiang Ding
Jennifer HANISAK
Charles Lee Jayne
Chengwei WU
Jennifer Marie JOHNSTON
Shawn P. Walsh
Zhongxiang Sun
Danila Branca
Stefania Colarusso
Anilkumar G. Nair
Michael Man-Chu Lo
Peter Orth
Sookhee Nicole Ha
Liangqin Guo
Ling Tong
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Merck Sharp and Dohme LLC
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Merck Sharp and Dohme LLC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains four or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains four or more hetero rings

Definitions

  • PCSK9 Proprotein convertase subtilisin-kexin type 9
  • NARC-1 neural apoptosis-regulated convertase 1
  • PCSK9 belongs to the mammalian proprotein convertase family of serine proteases and contains an N-terminal signal sequence, a prodomain, a catalytic domain, and a C-terminal domain. Seidah et al., 2012 Nat. Rev. Drug Discov.11:367-383.
  • PCSK9 is transcriptionally regulated by sterol regulatory element-binding proteins (“SREBP”), as found with other genes involved in cholesterol metabolism (Maxwell et al., 2003 J. Lipid Res.44:2109-2119) and as found with other genes implicated in lipoprotein metabolism (Dubuc et al., 2004 Arterioscler.
  • SREBP sterol regulatory element-binding proteins
  • PCSK9 promoters possess two conserved sites involved in cholesterol regulation, a sterol regulatory element and an Sp1 site. [0005] In the endoplasmic reticulum, PCSK9 catalyzes autocleavage between residues Gln-152 and Ser-153 (Naureckiene et al., 2003 Arch. Biochem. Biophys.420:55-67; Seidah et al., 2003 Proc. Natl. Acad. Sci. U.S.A.100:928-933). The prodomain remains tightly associated with the catalytic domain during subsequent trafficking through the trans-Golgi network.
  • mice lacking PCSK9 or in which PCSK9 mRNA has been lowered by antisense oligonucleotide inhibitors have higher levels of hepatic LDLR protein and a greater ability to clear circulating LDL-C (Rashid et al., 2005 PNAS 102:5374-5379; and Graham et al., 2007 J. Lipid Res.48(4):763-767). Additionally, lowering PCSK9 levels in cultured human hepatocytes by siRNA was found to result in higher LDLR protein levels and an increased ability to take up LDL-C (Benjannet et al., 2004 J. Biol.
  • PCSK9 The expression or upregulation of PCSK9 is associated with increased plasma levels of LDL cholesterol, and the corresponding inhibition or lack of expression of PCSK9 is associated with reduced LDL cholesterol plasma levels. Decreased levels of LDL cholesterol associated with sequence variations in PCSK9 have been found to confer protection against coronary heart disease (Cohen, 2006 N. Engl. J. Med.354:1264-1272). [0009] Thus, identification of compounds and/or agents that antagonize PCSK9's LDL regulating property. However, in general, because PCSK9 circulates in blood and has modest binding affinity to cell surface LDL receptors, attempts to utilize this mechanism in treatment of diseases related to high serum LDL levels have largely been focused on the use of large biomolecules, for example, antibodies.
  • the compounds of the invention have a structure in accordance with the structural Formula (I): (I), or a pharmaceutically acceptable salt thereof, wherein ring A, B, R 1 , R 2 , R 3 , R 4 and R 5 are as defined below.
  • the invention provides pharmaceutical compositions comprising at least one compound of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. Such compositions according to the invention may optionally further include one or more additional therapeutic agents as described herein.
  • the invention provides a method for treating hypercholesterolemia. The method comprises administering to a patient in need thereof a therapeutically effective amount of one of the above compositions.
  • the representation contemplates all stereochemical and spatial orientations of the structural features.
  • particular asymmetric carbon centers are structurally represented using conventional “Solid Wedge” and “Hash Wedge” bonding representation. Absolute configuration has not been determined for the example compounds but has been assigned by analogy to specific example compounds of known stereochemical configurations (determined by X-ray crystallography) prepared using the same or analogous reaction conditions and starting reagents and isolated under the same chromatographic conditions.
  • the separation of a mixture of stereoisomers can be carried out at an intermediate step during the synthesis of a compound of Formula I or it can be done on a final racemic product.
  • absolute stereochemistry is determined by X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing a stereogenic center of known configuration.
  • the invention includes all such isomers, as well as salts, solvates (including hydrates) and solvated salts of such racemates, enantiomers, diastereomers, and mixtures thereof.
  • the invention also embraces isotopically-labeled compounds which are structurally identical to those recited herein, but for the fact that a statistically significant percentage of one or more atoms in that form of the compound are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number of the most abundant isotope usually found in nature, thus altering the naturally occurring abundance of that isotope present in a compound of the invention.
  • the invention includes all suitable isotopic variations of the compounds of Formula I.
  • isotopes that can be preferentially incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, iodine, fluorine and chlorine, for example, but not limited to 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I, and 125 I. It will be appreciated that other isotopes may be incorporated by known means also.
  • certain isotopically labeled compounds of the invention are recognized as being particularly useful in compound and/or substrate tissue distribution assays using a variety of known techniques.
  • compounds of the invention contemplate isotopic substitution with different isotopic forms of hydrogen (H), including protium ( 1 H) and deuterium ( 2 H or D).
  • H isotopic forms of hydrogen
  • protium 1 H
  • deuterium 2 H or D
  • Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples.
  • Isotopically enriched compounds within Formula I can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Schemes and Examples herein using appropriate isotopically enriched reagents and/or intermediates.
  • a wavy line terminates a conventional bond (as opposed to connecting two atoms within a structure) it indicates a point of bonding to a structure, e.g.: [0021] indicates that a secondary-butyl moiety is bonded via the methylene group through the bond terminating with the wavy line.
  • a dash is employed to indicate the point of bonding to the indicated substrate, e.g.: —CH2—C(O)—CH2Cl indicates the acetyl chloride moiety is bonded via the methylene portion of the moiety.
  • L linker (L) is – CH2-CF2-
  • a and B are portions of the compound flanking the linker, respectively, on the left and on the right
  • the definition of L includes only A–CH2-CF2- B, not A–CF2-CH2-B.
  • any variable e.g., n, R 5 , etc.
  • its definition on each occurrence is independent of its definition at every other occurrence unless otherwise specified at the point of definition.
  • a “stable” compound is a compound which can be prepared and isolated and whose structure and properties remain or can be caused to remain essentially unchanged for a period of time sufficient to allow use of the compound for the purposes described herein (e.g., therapeutic administration to a subject).
  • the compounds of the invention are limited to stable compounds embraced by Formula I.
  • any variable or moiety is expressed in the form of a range, e.g. (—CH2—)1-4, both of the extremes of the specified range are included (i.e., 1 and 4 in the example) as well as all of the whole number values in between (i.e., 2 and 3 in the example).
  • m is an integer between 0 and 4
  • the range is inclusive of the numbers 0 and 4 and includes 0, 1, 2, 3, and 4.
  • halogen includes fluorine, chlorine, bromine and iodine unless specified otherwise at the point of use.
  • subjects refers to an animal, preferably a mammal, and in particular a human or a non-human animal including livestock animals and domestic animals including, but not limited to, cattle, horses, sheep, swine, goats, rabbits, cats, dogs, and other mammals in need of treatment.
  • the subject is preferably a human.
  • administration and variants thereof (e.g., “administering” a compound) in reference to a compound of Formula I means providing the compound, or a pharmaceutically acceptable salt thereof, to a subject in need of treatment.
  • the invention provides a compound of Formula I I, or a pharmaceutically acceptable salt thereof, wherein X is H, F, Cl, or Br; R 1 is selected from the group consisting of -NR 6 CO(C1-C4)alkyl, pyrimidinyl-CONR 5 -, R 3 is -O(C1-C4)alkyl, optionally substituted with one to six F; R 4 is H or -(C1-C4)alkyl; R 5 , at each occurrence, is independently selected from H, -(C1-C4)alkyl, and a bond; R 6 , at each occurrence, is independently selected from H and -(C1-C4)alkyl; is a linker selected from the group consisting of
  • linker selected from the group consisting of
  • the invention provides the compound of any of embodiments 1 or 2, wherein the linker B is selected from the group consisting of:
  • the invention provides the compound of the embodiment 1, wherein A is B is
  • the invention provides the compound of embodiment 4, wherein A is and B is selected from the group consisting of [0034]
  • the invention provides the compound of embodiment 5, wherein at least one of the two R 5 groups is -(C 1 -C 4 )alkyl.
  • the invention provides the compound of embodiment 5, wherein both R 5 groups are -(C1-C4)alkyl.
  • the invention provides the compound of any one of embodiments 6 or 7, wherein at least one occurrence of R 5 is -CH3.
  • the invention provides the compound of any one of embodiments 6 or 7, wherein each occurrence of R 5 and R 6 is -CH 3 , and R 7 is H.
  • the invention provides the compound of embodiment 1, wherein A is selected from the group consisting of B is selected from the group consisting of [0039]
  • the invention provides the compound of embodiment 10, wherein B is selected from the group consisting of
  • the invention provides the compound of any one of embodiments 1- 11, wherein X is F.
  • the invention provides a compound of embodiment 1 selected from the group consisting of:
  • the invention provides a composition comprising at least one compound of any of embodiments 1 to 13, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.
  • the invention provides a method of treating hypercholesterolemia, comprising administering to a patient in need thereof a therapeutically effective amount of a composition of embodiment 14.
  • the invention provides a compound having the structure pharmaceutically acceptable salt thereof.
  • the invention provides a composition comprising the compound of embodiment 16 and at least one pharmaceutically acceptable excipient.
  • the invention provides a method of treating hypercholesterolemia, comprising administering to a patient in need thereof a therapeutically effective amount of a composition of embodiment 17.
  • the invention provides a compound having the structure
  • the invention provides a composition comprising the compound of embodiment 19 and at least one pharmaceutically acceptable excipient.
  • the invention provides a method of treating hypercholesterolemia, comprising administering to a patient in need thereof a therapeutically effective amount of a composition of embodiment 20.
  • the invention provides a compound having the structure pharmaceutically acceptable salt thereof.
  • the invention provides a composition comprising the compound of embodiment 22 and at least one pharmaceutically acceptable excipient.
  • the invention provides a method of treating hypercholesterolemia, comprising administering to a patient in need thereof a therapeutically effective amount of a composition of embodiment 23.
  • the invention provides a compound having the structure
  • the invention provides a composition comprising the compound of the embodiment 25 and at least one pharmaceutically acceptable excipient.
  • the invention provides a method of treating hypercholesterolemia, comprising administering to a patient in need thereof a therapeutically effective amount of a composition of embodiment 26.
  • the invention provides a compound having the structure pharmaceutically acceptable salt thereof.
  • the invention provides a composition comprising the compound of embodiment 28 and at least one pharmaceutically acceptable excipient.
  • the invention provides a method of treating hypercholesterolemia, comprising administering to a patient in need thereof a therapeutically effective amount of a composition of embodiment 29.
  • salts of compounds of the invention includes any of the following: acidic salts formed with inorganic and/or organic acids, basic salts formed with inorganic and/or organic bases, zwitterionic, and quaternary ammonium complexes.
  • Salts of compounds of the invention may be formed by methods known to those of ordinary skill in the art, for example, by reacting a compound of the invention with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in aqueous medium followed by lyophilization.
  • Compounds of the invention contain tri-coordinate nitrogen atoms, for example, primary, secondary or tertiary amino moieties, wherein, as is known, the lone pair of electrons residing on the nitrogen atom may be protonated with an appropriate acid or alkylated with an appropriate reagent, for example, alkyl bromide, under the appropriate reaction conditions to provide tetracoordinate charged nitrogen stabilized by an anion generated in the process, for example, a halogen ion or conjugate base. Accordingly, compounds of the invention may be prepared in the form of a free-base or isolated in the form of a quaternary complex or a salt complex.
  • salts of the inventive compounds whether acidic salts formed with inorganic and/or organic acids, basic salts formed with inorganic and/or organic bases, salts formed which include zwitterionic character, for example, where a compound contains both a basic moiety, for example, but not limited to, a nitrogen atom, for example, an amine, pyridine or imidazole, and an acidic moiety, for example, but not limited to a carboxylic acid, and quaternary ammonium complexes are included in the scope of the inventive compounds described herein.
  • structural representation of compounds of the invention also include all other forms of such compounds discussed above.
  • one aspect of the invention is the provision of compounds of the invention in the form of a pharmaceutically acceptable salt, zwitterionic complex or quaternary ammonium complex.
  • the compounds of the invention may form such complexes, including where a tetracoordinate nitrogen can be quaternerized or protonated and the charged nitrogen form stabilized by an associated anion.
  • pharmaceutically acceptable salt refers to a salt (including a quaternary ammonium complex and an inner salt such as a zwitterion complex) which possesses effectiveness similar to or greater than a free-base form of the compound and which is not biologically or otherwise undesirable (e.g., is neither toxic nor otherwise deleterious to the recipient thereof).
  • the invention contemplates both freebase forms of the compounds of the invention and all available salts, including salts which are generally recognized as safe for use in preparing pharmaceutical formulations and those that may be formed presently within the ordinary skill in the art and are later classified as being “generally recognized as safe” for use in the preparation of pharmaceutical formulations, termed herein as “pharmaceutically acceptable salts.”
  • freebase compounds may be prepared by controlling the conditions of isolation of the compound during synthesis or by neutralization and ion exchange from salt forms of compounds of the invention.
  • Examples of pharmaceutically acceptable acid salts include, but are not limited to, acetates, including trifluoroacetate salts, adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates, methanesulfonates, methyl sulfates, 2-naphthalenesulfonates, nicotinates, nitrates, o
  • Examples of pharmaceutically acceptable basic salts include, but are not limited to, ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, aluminum salts, zinc salts, salts with organic bases (for example, organic amines) such as benzathines, diethylamine, dicyclohexylamines, hydrabamines (formed with N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines, N-methyl-D-glucamides, t-butyl amines, piperazine, phenylcyclohexyl-amine, choline, tromethamine, and salts with amino acids such as arginine, lysine and the like.
  • organic bases for example, organic amines
  • organic bases for example, organic amines
  • Basic nitrogen- containing groups may be converted to an ammonium ion or quaternized with agents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g. decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g. benzyl and phenethyl bromides), and others.
  • lower alkyl halides e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialkyl sulfates e.g., dimethyl, diethyl, dibutyl, and
  • salts of compounds include, but are not limited to, fluoride, chloride, bromide and iodide.
  • salts of compounds are intended to be pharmaceutically acceptable salts within the scope of the invention.
  • purified in purified form or “in isolated and purified form” for a compound refers to the physical state of said compound after being isolated from a synthetic process or natural source or combination thereof.
  • purified in purified form or “in isolated and purified form” for a compound refers to the physical state of said compound after being obtained from a purification process or processes described herein or well known to the skilled artisan, and in sufficient purity to be characterized by standard analytical techniques described herein or well known to the skilled artisan.
  • Compounds of the invention include any form of the compound including in situ in a reaction mixture as well as in isolated and purified form obtained by routine techniques. Also included are polymorphic forms of the compounds of the invention and solvates and prodrugs thereof.
  • Certain compounds of the invention may exist in different tautomeric forms, for example, but are not limited to, ketone/enol tautomeric forms, imine-enamine tautomeric forms, and for example heteroaromatic forms such as the following moieties:
  • ketone/enol tautomeric forms for example, but are not limited to, imine-enamine tautomeric forms, and for example heteroaromatic forms such as the following moieties:
  • presenting a structural representation of any tautomeric form of a compound which exhibits tautomerism is meant to include all such tautomeric forms of the compound. Accordingly, where compounds of the invention, their salts, and solvates and prodrugs thereof, exist in different tautomeric forms or in equilibrium among such forms, all such forms of the compound are embraced by, and included within the scope of the invention.
  • the invention provides pharmaceutical compositions comprising one or more compounds of the invention.
  • pharmaceutical composition comprises at least one pharmaceutically active compound and at least one excipient, and is intended to encompass both the combination of the specified ingredients in the specified amounts, and any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
  • an excipient is any constituent which adapts the composition to a particular route of administration or aids the processing of a composition into a dosage form without itself exerting an active pharmaceutical effect.
  • compositions comprise more than one excipient depending upon the route of administration and the characteristics of the active being administered.
  • excipients which impart to the composition properties which make it easier to handle or process include, but are not limited to, lubricants or pressing aids in powdered medicaments intended to be tableted, and emulsion stabilizers in compositions in which the active is present in the form of an emulsion.
  • excipients which adapt a composition to a desired route of administration are, for example, but not limited to, for oral administration, absorption enhancers promoting absorption from the gastrointestinal tract, for transdermal or transmucosal administration, penetration enhancers, for example, those employed in adhesive skin “patch” or compositions for buccal administration.
  • excipients are collectively termed herein “a carrier.”
  • formulations may comprise up to about 95 percent active ingredient and the balance carrier, although formulations with different ratios may be prepared.
  • acceptable pharmaceutical compositions contain a suitable concentration of the active that an effective amount of the PCSK9 antagonist can be provided in an individual dosage form of acceptable volume based upon the route of administration such that it can provide a therapeutic serum level of the active for an acceptable period of time in a subject to whom the composition is administered and the composition will retain biological activity during storage within an acceptable temperature range for an acceptable period of time.
  • composition refers both to a bulk composition, that is, formulated material that has not yet been formed into individual dosage units for administration, and the composition contained within individual dosage units. While compositions of the invention may be employed in bulk form, it will be appreciated that for most applications compositions will be incorporated into a dosage form providing individual units suitable for administration to a patient, each dosage form comprising an amount of the selected composition which contains an effective amount of said one or more compounds of Formula I.
  • suitable dosage forms include, but are not limited to, dosage forms adapted for: (i) oral administration, e.g., a liquid, gel, powder, solid or semi-solid pharmaceutical composition which is loaded into a capsule or pressed into a tablet and may comprise additionally one or more coatings which modify its release properties, for example, coatings which impart delayed release or formulations which have extended release properties; (ii) a dosage form adapted for administration through tissues of the oral cavity, for example, a rapidly dissolving tablet, a lozenge, a solution, a gel, a sachet or a needle array suitable for providing intramucosal administration; (iii) a dosage form adapted for administration via the mucosa of the nasal or upper respiratory cavity, for example a solution, suspension or emulsion formulation for dispersion in the nose or airway; (iv) a dosage form adapted for transdermal administration, for example, a patch, cream or gel; (v) a dosage form adapted for intradermal administration, for example,
  • a dosage form adapted for intramuscular administration for example, an injectable solution or suspension, and which may be adapted to form a depot having extended release properties
  • a dosage form adapted for drip intravenous administration for example, a solution or suspension, for example, as an IV solution or a concentrate to be injected into a saline IV bag
  • IV drip intravenous administration
  • a dosage form adapted for subcutaneous administration including administration over an extended time period by implanting a rod or other device which diffuses the compound into the surround tissue and thereby provides a continuous serum therapeutic level
  • a dosage form adapted for delivery via rectal or vaginal mucosa for example, a suppository.
  • compositions can be solid, semi-solid or liquid.
  • Solid, semi-solid and liquid form preparations can be adapted to a variety of modes of administration, examples of which include, but are not limited to, powders, dispersible granules, mini-tablets, beads, which can be used, for example, for tableting, encapsulation, or direct administration.
  • liquid form preparations include, but are not limited to, solutions, suspensions and emulsions which for example, but not exclusively, can be employed in the preparation of formulations intended for ingestion, inhalation or intravenous administration (IV), for example, but not limited to, administration via drip IV or infusion pump, intramuscular injection (IM), for example, of a bolus which is released over an extended duration, direct IV injection, or adapted to subcutaneous routes of administration.
  • IV intravenous administration
  • IM intramuscular injection
  • Other routes of administration that are contemplated include intranasal administration, or for administration to some other mucosal membrane.
  • Formulations prepared for administration to various mucosal membranes may also include additional components adapting them for such administration, for example, viscosity modifiers.
  • compositions suitable for use in a solid oral dosage form are preferable routes of administration for a compound of the invention or a salt thereof
  • a composition of the invention may be formulated for administration via other routes mentioned above.
  • aerosol preparations for example, suitable for administration via inhalation or via nasal mucosa
  • solid form preparations which are intended to be converted, shortly before use, to a suspension or a solution, for example, for oral or parenteral administration.
  • transdermal compositions can also take the form of creams, lotions, aerosols and/or emulsions and can be provided in a unit dosage form which includes a transdermal patch of any known in the art, for example, a patch which incorporates either a matrix comprising the pharmaceutically active compound or a reservoir which comprises a solid or liquid form of the pharmaceutically active compound.
  • compositions may be formulated by any number of strategies known in the art, see, e.g., McGoff and Scher, 2000 Solution Formulation of Proteins/Peptides: McNally, E. J., ed. Protein Formulation and Delivery.
  • the invention provides methods of employing PCSK9-specific antagonist compounds described herein for antagonizing PCSK9 function; said methods of which are further described below.
  • antagonizing refers to providing to the affected tissue(s) a substance which opposes the action of, inhibits, counteracts, neutralizes, or curtails one or more functions of PCSK9 in the affected tissues.
  • Inhibition or antagonism of one or more of PCSK9-associated functional properties can be readily determined according to methodologies known to the art (see, e.g., Barak & Webb, 1981 J. Cell Biol.90:595-604; Stephan & Yurachek, 1993 J. Lipid Res. 34:325330; and McNamara et al., 2006 Clinica Chimica Acta 369:158-167) as well as those described herein.
  • PCSK9-specific antagonist in accordance with the invention antagonizes PCSK9 functioning to the point that there is a decrease of at least 10%, of the measured parameter including but not limited to the activities disclosed herein, and more preferably, a decrease of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95% of the measured parameter.
  • the invention provides a method for antagonizing the activity of PCSK9, which comprises contacting a cell, population of cells or tissue sample capable of being affected by PCSK9 (i.e., which expresses and/or comprises LDL receptors) with a PCSK9-specific antagonist disclosed herein under conditions that allow said antagonist to bind to PCSK9 when present and inhibit PCSK9's inhibition of cellular LDL uptake.
  • a cell is a human cell.
  • the present invention provides a method for antagonizing the activity of PCSK9 in a subject, which comprises administering to the subject a therapeutically effective amount of a PCSK9-specific antagonist of the present invention.
  • the methods for antagonizing PCSK9 function are for the treatment, as defined herein, of a PCSK9- associated disease, disorder, or condition or, alternatively, for providing therapy in a disease, disorder or condition that could benefit from the effects of a PCSK9 antagonist.
  • the invention thus, contemplates the use of PCSK9-specific antagonists described herein in various methods of treatment where antagonizing PCSK9 function is desirable.
  • the term “method of treatment” relates to a course of action resulting in a change in at least one symptom of a disease state which can be prophylactic or therapeutic in nature.
  • the invention relates to a method of treatment for a condition associated with and/or attributed to PCSK9 activity, or a condition where the functioning of PCSK9 is contraindicated for a particular subject, the method comprising administering to the subject a therapeutically effective amount of a PCSK9-antagonist compound of Formula I, or pharmaceutically acceptable salt thereof.
  • the condition is atherosclerosis, hypercholesterolemia, coronary heart disease, metabolic syndrome, acute coronary syndrome or related cardiovascular disease or a cardiometabolic conditions.
  • the condition is a disease state or condition in which PCSK9 activity is contraindicated.
  • Methods of treatment in accordance with the invention comprise administering to an individual a therapeutically (or prophylactically) effective amount of a PCSK9-specific antagonist of the invention.
  • a therapeutically (or prophylactically) effective amount of a PCSK9-specific antagonist of the invention refers to the amount necessary at the intended dosage to achieve the desired therapeutic and/or prophylactic effect for the period desired.
  • the desired effect may be, for example, the alleviation, amelioration, reduction, or cessation of at least one symptom associated with the treated condition.
  • a PCSK9 antagonist compound of the invention is administered in the form of a pharmaceutical composition as described herein.
  • Dosing of antagonist therapeutics is well within the realm of the skilled artisan, see, e.g., Lederman et al., 1991 Int. J.
  • the subject may need, or desire, treatment for an existing disease or medical condition.
  • the subject “in need” of treatment of an existing condition encompasses both a determination of need by a medical professional as well as the desire of the subject for such treatment.
  • “administration” and its variants are each understood to include provision of the compound or its salt and the other agents contemporaneously or simultaneously or over a course of separate administrations over a period of time.
  • the agents of a combination are administered at the same time, they can be administered together in a single composition, or they can be administered separately.
  • a “combination” of active agents can be a single composition containing all the active agents or multiple compositions each containing one or more of the active agents.
  • a combination can be either a single composition comprising both agents or two separate compositions each comprising one of the agents.
  • compositions and combinations of the invention are suitably administered in effective amounts.
  • effective amount means the amount of active compound sufficient to antagonize PCSK9 and thereby elicit the response being sought (i.e., induce a therapeutic response in the treatment or management of conditions associated with or impacted by PCSK9 function, including, but not limited to atherosclerosis, hypercholesterolemia, coronary heart disease, metabolic syndrome, acute coronary syndrome and related cardiovascular disease, and cardiometabolic conditions in an animal or human).
  • the actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage regimen for a particular situation is within the skill in the art, for example, as described in the standard literature, for example, as described in the “Physicians' Desk Reference” (PDR), e.g., 1996 edition (Medical Economics Company, Montvale, N.J.07645-1742, USA), the Physician's Desk Reference, 56th Edition, 2002 (published by Medical Economics company, Inc.
  • PDR Physical Economics Company, Montvale, N.J.07645-1742, USA
  • the PCSK9-specific antagonist may be administered to an individual by any route of administration appreciated in the art, including but not limited to oral administration, administration by injection (specific embodiments of which include intravenous, subcutaneous, intraperitoneal, or intramuscular injection), or administration by inhalation, intranasal, or topical administration, either alone or in combination with other agents designed to assist in the treatment of the individual.
  • the PCSK9-specific antagonist may also be administered by injection devices, injector pens, needleless devices, and subcutaneous patch delivery systems.
  • the route of administration should be determined based on several considerations appreciated by the skilled artisan including, but not limited to, the desired physiochemical characteristics of the treatment.
  • One or more additional pharmacologically active agents may be administered in combination with a compound of Formula I.
  • An additional active agent (or agents) is intended to mean a pharmaceutically active agent (or agents) that is active in the body, including pro-drugs that convert to pharmaceutically active form after administration, which are different from the compound of Formula I, and includes free acid, free base and pharmaceutically acceptable salts of said additional active agents.
  • any suitable additional active agent or agents including but not limited to anti-hypertensive agents, anti-atherosclerotic agents such as a lipid modifying compound, anti-diabetic agents and/or anti-obesity agents may be used in any combination with the compound of Formula I in a single dosage formulation (a fixed dose drug combination), or may be administered to the subject in one or more separate dosage formulations which allows for concurrent or sequential administration of the active agents (co-administration of the separate active agents).
  • angiotensin converting enzyme inhibitors e.g., alacepril, benazepril, captopril, ceronapril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, imidapril, lisinopril, moveltipril, perindopril, quinapril, ramipril, spirapril, temocapril, and trandolapril); angiotensin II receptor antagonists (e.g., losartan i.e., COZAAR®, valsartan, candesartan, olmesartan, telmesartan and any of these drugs used in combination with hydrochlorothiazide such as HYZAAR®); neutral endopeptidase inhibitors (e.g., thiorphan and phosphoram
  • sildenafil, tadalfil and vardenafil vasodilators; calcium channel blockers (e.g., amlodipine, nifedipine, verastrial, diltiazem, gallopamil, niludipine, nimodipins, nicardipine); potassium channel activators (e.g., nicorandil, pinacidil, cromakalim, minoxidil, aprilkalim, loprazolam); diuretics (e.g., hydrochlorothiazide); sympatholitics; beta-adrenergic blocking drugs (e.g., propranolol, atenolol, bisoprolol, carvedilol, metoprolol, or metoprolol tartate); alpha adrenergic blocking drugs (e.g., doxazocin, prazocin or alpha methyldopa); central alpha ad
  • lipid lowering agents e.g., HMG-CoA reductase inhibitors such as simvastatin and lovastatin which are marketed as ZOCOR® and MEVACOR® in lactone pro-drug form and function as inhibitors after administration; and pharmaceutically acceptable salts of dihydroxy open ring acid HMG-CoA reductase inhibitors such as atorvastatin (particularly the calcium salt sold in LIPITOR®), rosuvastatin (particularly the calcium salt sold in CRESTOR®), pravastatin (particularly the sodium salt sold in PRAVACHOL®), fluvastatin (particularly the sodium salt sold in LESCOL®), crivastatin, and pitavastatin; a cholesterol absorption inhibitor such as ezetimibe (ZETIA®) and ezetimibe in combination with any other lipid lowering agents such as the HMG-CoA reductase inhibitors noted above and particularly with simvastatin (VYTORIN®) or with atorva
  • CETP inhibitors e.g., anacetrapib, torcetrapib, and evacetrapib
  • inhibitors of fructose 1,6-bisphosphatase e.g., such as those disclosed in U.S. Pat.
  • the compounds of the invention can be prepared readily according to the schemes, descriptions, and specific examples provided below, or modifications thereof, using readily available starting materials, reagents, and conventional synthetic procedures. In these reactions, it is also possible to make use of variants which are themselves known to those of ordinary skill in this art but are not mentioned in detail.
  • the general procedures for making the compounds of the invention can be readily understood and appreciated by one skilled in the art from viewing these schemes and descriptions.
  • MS refers to Mass spectrum associated with peaks obtained by UPLC-MS as described below.
  • Total ion current (TIC) and Dad UV chromatographic traces together with MS and UV spectra associated with peaks were obtained using a UPLC/MS Waters Acquity TM system VbfZaaVU hZeY KFB:(F;8 VieV_UVU v UVeVTe ⁇ c T ⁇ fa]VU e ⁇ R MReVcd CZTc ⁇ Rdd OG TM Mass spectrometer operating in positive or negative electron spray ionization mode [LC/MS-ES(+or-)].
  • Chromatography details include Column A: Waters BEH C18 Column (130 ⁇ , 1.7 ⁇ m; column size: 2.1 * 100 mm or 2.1 * 50 mm; column temperature 45°C). Column B: Waters BEH C4 Column (300 ⁇ , 1.7 ⁇ m; column size: 2.1 * 100 mm or 2.1 * 50 mm, column temperature 45°C).
  • Mobile phase 1 (A) 0.1% HCOOH in HPLC water and (B) 0.1% HCOOH in HPLC acetonitrile; or mobile phase 2: (A) 0.1% TFA in HPLC water and (B) 0.1% TFA in HPLC acetonitrile; Z_[VTeZ ⁇ _ g ⁇ ]f ⁇ V5 , lB6 W] ⁇ h cReV5 +)/ ⁇ B* ⁇ Z_6 KL hRgV]V_XeY v 7 -,/ _ ⁇ 6 CRdd cR_XV5 ⁇ I$&%5 100-800 amu or 100-1000 amu or 100-1800 amu or 400-1800 amu; ES (-)100-800 amu.
  • UV detection range 400-1200 nm.
  • the usage of this methodology is indicated by UPLC-MS in the analytic characterization of the described compounds.
  • Confirmation of identity and purity assessment of final compounds were performed by UPLC-MS using a reverse phase Waters Acquity TM UPLC-MS system. Column: Waters BEH C18 Column (130 ⁇ , 1.7 ⁇ m, column size 2.1 * 100 mm, column temperature 45°C).
  • Step C Synthesis of intermediate A-3
  • Intermediate A-2 137.5 mg, 0.785 mmol was dissolved in DMF (7845 ⁇ l) and (S)-1- (((9H-fluoren-9-yl)methoxy)carbonyl)-2-methylpyrrolidine-2-carboxylic acid (414 mg, 1.177 mmol), HATU (418 mg, 1.098 mmol), and DIPEA (410 ⁇ l, 2.354 mmol) were added. After stirring at room temp for 1 hour, the mixture was diluted with EtOAc, washed three times with water, then brine, dried over magnesium sulfate and concentrated.
  • Step E Synthesis of intermediate A-5 [0101]
  • Intermediate A-4 (176 mg, 0.614 mmol) was dissolved in DMF (6145 ⁇ l) and (S)-2- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-methoxyphenyl)propanoic acid (385 mg, 0.922 mmol), HATU (327 mg, 0.860 mmol), and DIPEA (322 ⁇ l, 1.843 mmol) were added in 1 mL DMF. After stirring at room temp for 1 hour, the mixture was diluted with EtOAc, washed three times with water, then brine, dried over magnesium sulfate, and concentrated.
  • Step G Synthesis of intermediate A-7
  • Intermediate A-6 (294.2 mg, 0.635 mmol) was dissolved in DMF (6346 ⁇ l)) and ((2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methoxybutanoic acid (338 mg, 0.952 mmol)), HATU (338 mg, 0.888 mmol), and DIPEA (332 ⁇ l, 1.904 mmol) were added in 1 mL DMF. After stirring at room temp 1.5 hours, the mixture was diluted with EtOAc, washed three times with water, then brine, dried over magnesium sulfate, and concentrated.
  • Step B Synthesis of intermediate F-2
  • Step C Synthesis of intermediate F-3
  • Intermediate F-2 (680 mg, 1.048 mmol) was dissolved in DCM (10.5 mL) and TFA (1.211 mL, 15.72 mmol) was added. The reaction was stirred at room temperature for 1.5 h. The reaction solution was diluted in 15 mL DCM and poured directly onto a 10 g Agilent Bond Elut SCX column. The column was flushed with MeOH to remove excess TFA, then eluted with 7N NH 3 /MeOH solution to afford F-3.
  • LC/MS: [M+H] + 549.36.
  • Step D Synthesis of intermediate F-4
  • Intermediate F-3 (453 mg, 0.826 mmol) was dissolved in DMF (8257 ⁇ l) and DIPEA (361 ⁇ l, 2.064 mmol) and acetic anhydride (93 ⁇ l, 0.991 mmol) were added. After stirring at room temp for 1.5 hours, the reaction solution was diluted with EtOAc, washed with water four times, then washed with brine, dried over magnesium sulfate and concentrated to afford F-4.
  • LC/MS: [M+H] + 591.4.
  • Step E Synthesis of intermediate F-5 [0117]
  • Intermediate F-4 (0.3391 g, 0.574 mmol) was dissolved in DMF (7 mL) and cesium carbonate (0.636 g, 1.952 mmol) was added followed by allyl bromide (0.176 ml, 2.038 mmol). The reaction was stirred at room temperature for 18 h. Water was added, and precipitate was filtered to afford F-5.
  • LC/MS: [M+H] + 631.39.
  • Step F - Synthesis of intermediate F [0118]
  • Intermediate F-5 (0.291 g, 0.461 mmol) was dissolved in THF (8.8 mL), MeOH (2.2 mL), and water (4.4 mL), and LiOH (0.055 g, 2.307 mmol) was then added. After stirring at room temperature for 7 h, the reaction was concentrated to ⁇ 1/2 vol and acidified with 1N HCl to pH 4-5. EtOAc was added and the layers were separated. The aqueous layer was extracted with EtOAc (3x15 mL). The combined organic layer was washed with brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo.
  • Step B Synthesis of intermediate G-2 [0120]
  • Intermediate G-1 (3.39 g, 11.67 mmol) was dissolved in DCM (60 ml) and DIPEA (2.039 ml, 11.67 mmol) was added followed by dropwise addition of CBz-Cl (1.633 ml, 11.44 mmol) at 0 °C. After stirring for 30 min, the reaction mixture was quenched at 0 °C with saturated aqueous sodium bicarbonate and extracted with DCM. The organic fractions were combined and washed with brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo.
  • Step C Synthesis of intermediate G-3
  • Intermediate G-2 (4.30 g, 10.13 mmol) was dissolved in DCM (25 ml) at room temperature and HCl 4N in dioxane (10.13 ml, 40.5 mmol) was added. After stirring for 3 h, the reaction was concentrated. To the residue, DCM, toluene, and acetonitrile was added, and the mixture was concentrated again. The mixture was dissolved in 30% acetonitrile and lyophilized to afford G-3.
  • LC/MS: [M+H] + 325.28.
  • Step D Synthesis of intermediate G-4 [0122]
  • Intermediate G-3 (238 mg, 0.678 mmol) and benzyl allyl(4-(2- aminoethyl)benzyl)carbamate 0395898-0161 (200 mg, 0.616 mmol) were dissolved in DMF (3082 ⁇ l), and HATU (258 mg, 0.678 mmol) was added, followed by DIPEA (323 ⁇ l, 1.849 mmol). After stirring at room temp for 2 hours, the mixture was diluted with EtOAc, washed thrice with water, then washed with brine, dried over magnesium sulfate, and concentrated.
  • Step F Synthesis of intermediate G-6 [0124]
  • Intermediate G-5 (176.4 mg, 0.405 mmol) and (S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-(4-methoxyphenyl)propanoic acid (178 mg, 0.425 mmol) were dissolved in DMF (2025 ⁇ l) and HATU (159 mg, 0.417 mmol) and DIPEA (177 ⁇ l, 1.012 mmol) were then added. After stirring at room temp for 1.5 hours, the reaction mixture was diluted with EtOAc and washed with water three times, then brine, then dried over magnesium sulfate and concentrated.
  • Step H Synthesis of intermediate G-8 [0126]
  • Intermediate G-7 (213 mg, 0.348 mmol) was dissolved in DMF (1738 ⁇ l), and then (2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methoxybutanoic acid (130 mg, 0.365 mmol), HATU (139 mg, 0.365 mmol), and DIPEA (182 ⁇ l, 1.043 mmol) were added. After stirring at room temp for 1.5 hours, the reaction mixture was diluted with EtOAc, washed thrice with water, then washed with brine, dried over magnesium sulfate, and concentrated.
  • Step D Synthesis of intermediate H-4 [0131]
  • Intermediate H-3 (1g, 1.687 mmol) was dissolved in DMF (5.62 ml) and cesium carbonate (1.869 g, 5.74 mmol) was added, followed by 3-bromoprop-1-ene (0.511 ml, 5.91 mmol). After stirring overnight at room temp, the reaction mixture was diluted with EtOAc, washed thrice with water, then washed with brine, dried over magnesium sulfate, and concentrated to obtain H-4.
  • LC/MS: [M+H] + 633.36.
  • Step C- Synthesis of intermediate J-3 [0137]
  • Intermediate J-2 (0.634 g, 3.87 mmol) and I (1.167 g, 3.52 mmol) was dissolved in DMF (10 ml), and HATU (1.406 g, 3.70 mmol) and DIPEA (1.846 ml, 10.57 mmol) were added.
  • the aqueous phase was partitioned between saturated sodium bicarbonate (100 mL) and DCM (100 mL). Next, the aqueous phase was further extracted with DCM (2x100 mL).
  • Step E- Synthesis of intermediate J-5 [0139] Intermediate J-4 (0.70 g, 1.858 mmol) was dissolved in DCM (30 ml) at 0 °C, and DIPEA (1.298 ml, 7.43 mmol) and 4-nitrobenzenesulfonyl chloride (0.453 g, 2.043 mmol) were added. After stirring at 0 °C for 0.5 h, the solution was partitioned between DCM (200 mL) and water. Next, the aqueous phase was further extracted with DCM (2x100 mL).
  • Step C Synthesis of intermediate M-3
  • Intermediate L (388 mg, 1.071 mmol) and M-2 (235 mg, 1.071 mmol) and HATU (489 mg, 1.285 mmol) were dissolved in DMF (10 ml) at 0°C and DIPEA (0.746 ml, 4.28 mmol) was then added. The mixture was allowed to warm up to rt and stirred for 4h. The reaction was quenched with water and extracted with EtOAc. The combined organic phase was washed with water (x2), dried over magnesium sulfate, and concentrated.
  • Step E Synthesis of intermediate M-5 [0151] DIPEA (698 ⁇ l, 4.01 mmol) was added dropwise to a stirred mixture of intermediate K (321 mg, 1.002 mmol) and M-4 (465mg, 1.002 mmol) and HATU (457 mg, 1.203 mmol) in DMF (13 mL). After stirring at 0°C, the reaction was then warmed up to room temperature for 1hr. The mixture was partitioned between EtOAc and water. The organic phase was separated, washed with water (X2), dried (MgSO4) and the volatiles removed under reduced pressure.
  • Step B Synthesis of intermediate N-2 [0155]
  • Intermediate N-1 (0.76 g, 2.115 mmol) was dissolved in DCM (3 ml) and TFA (3 ml, 38.9 mmol) was then added. After stirring at RT for 40 min, the reaction mixture was concentrated and the residue was dissolved in DCM/toluene (1:1, 10 mL) and treated with HCl (2.64 ml, 10.57 mmol). The resulting solution was concentrated to afford N-2.
  • LC/MS: [M+H] + 260.1.
  • Step C Synthesis of intermediate N-3 [0156] To the solution of intermediate N-2 (0.625 g, 2.115 mmol) and N-(tert-butoxycarbonyl)- O-methyl-L-threonine (0.518 g, 2.221 mmol) in DMF (8 ml) was added HATU (0.844 g, 2.221 mmol) and DIPEA (2.216 ml, 12.69 mmol). After stirring at RT for 1h, the reaction mixture was partitioned between EtOAc (200 mL) and brine (100 mL). The organic phase was further washed with brine (2x100 mL), dried over sodium sulfate, concentrated.
  • Step D Synthesis of intermediate N [0157]
  • Intermediate N-3 (0.780 g, 1.644 mmol) was dissolved in DCM (3 ml) and TFA (4 ml, 51.9 mmol) was then added. After stirring at RT for 30min, the reaction mixture was concentrated and the residue was dissolved in ACN/water (3:1, 20 mL) and treated with HCl (3.29 ml, 3.29 mmol). The resulting solution was lyophilized to afford N.
  • LC/MS: [M+H] + 375.3.
  • Step B Synthesis of intermediate O-2
  • Intermediate O-1 25 mg, 0.034 mmol
  • tert- butyl 4-(methylamino)piperidine-1-carboxylate 14.73 mg, 0.069 mmol
  • DIPEA 30.0 ⁇ l, 0.172 mmol
  • the reaction mixture was diluted with EtOAc, washed with water three times, then brine, dried over magnesium sulfate and concentrated to afford O-2.
  • LC/MS: [M+H] + 861.66.
  • Step C Synthesis of intermediate O [0160]
  • Intermediate O-2 (70 mg, 0.081 mmol) was dissolved in THF (1548 ⁇ l), MeOH (774 ⁇ l), and Water (387 ⁇ l) and LiOH (9.73 mg, 0.406 mmol) was then added. After stirring at room temp for 2 hours, the reaction mixture was evaporated under vacuum. Next it was acidified with 1N HCl to pH ⁇ 3 and extracted 3 times with EtOAc. washed with brine, dried over magnesium sulfate and concentrated to afford O.
  • LC/MS: [M+H] + 847.56.
  • Step B Synthesis of intermediate P-2
  • Intermediate P-1 (316 mg, 0.986 mmol) and allyl bromide (0.341 ml, 3.95 mmol)) were dissolved in dry DMF (3 ml)) and sodium hydride (71.0 mg, 1.775 mmol) was then added. After stirring at room temp overnight, it was added ⁇ 30 mL water, extracted 3 times with EtOAc, washed with water twice, then brine, dried over magnesium sulfate and concentrated to afford P- 2.
  • LC/MS: [M+H] + 361.25.
  • Step C Synthesis of intermediate P [0163]
  • Intermediate P-2 (372 mg, 1.032 mmol) was dissolved in DCM (10 mL) and TFA (1193 ⁇ l, 15.48 mmol) was then added. After stirring at room temp for 1 hour, the reaction mixture was concentrated and purified via flash column chromatography on a 12g silica gold column using 0- 50-100% [3:1 EtOAc:EtOH] in hexane gradient to afford P.
  • LC/MS: [M+H] + 261.28.
  • ALLYL BROMIDE (2.058 ml, 23.78 mmol) was then added, and the reaction was stirred at room temperature for 20 minutes, followed by addition of 1mL more allyl bromide. After 15 minutes, 1mL more allyl bromide was added and stirred for 20more minutes. After cooled in ice bath, it was quenched slowly with water to quench. 1N aq NaOH (20mL) was added after 20 minutes, followed by additional 1N aq HCl ( ⁇ 25 ml) after 30 minutes to adjust pH to 4-5. The mixture was concentrated, redissolved in water and EtOAc and then acidified with 1N aq HCl to pH 2-3, then was diluted with brine and extracted 3X EtOAc.
  • Step B Synthesis of Intermediate W-2 [0174] Intermediate W-1 (5.87 g, 21.62 mmol) was dissolved in DCM (25 ml) and TFA (25 ml, 324 mmol) was then added. After stirring at room temperature for 20 minutes, the reaction was concentrated in vacuo and then concentrated in vacuo 2X from toluene to afford W-2, which was used crude in the next step. LCMS: 172.1 (M+1) + .
  • Intermediate W-2 (6.17 g, 21.62 mmol) and SODIUM CARBONATE (4.58 g, 43.2 mmol) were dissolved in water (130 mL) and acetone (130 mL) and Fmoc-OSu (7.29 g, 21.62 mmol) was then added.
  • the resultant suspension was diluted with water (40 mL) and acetone (40 mL). After stirring overnight at room temperature, the reaction was acidified reaction to pH 2-3 with 1N aq HCl ( ⁇ 60mL) and concentrated in vacuo, then diluted with water and brine and extracted with DCM (3X). The combined organic phase was dried over Na2SO4, filtered, concentrated.
  • Step B – Synthesis of Intermediate X-2 [0177] In flame dried flask under an atmosphere of nitrogen, intermediate X-1 (1330 mg, 3.95 mmol) was dissolved in anhydrous DMF (30 ml) and cooled in ice bath. Next sodium hydride (125 mg, 4.94 mmol) was added portion wise. After stirring 20 minutes at 0 o C, 3-bromoprop-1- yne (80% in xylene) (0.529 ml, 4.74 mmol) was added dropwise. After stirring at 0 o C for an hour, lithium hydroxide (1M aq) (6 ml, 6.00 mmol) was added dropwise.
  • Step D Synthesis of Intermediate X [0179] Intermediate X-3 (1173 mg, 3.95 mmol) and SODIUM CARBONATE (838 mg, 7.91 mmol) were dissolved in acetone (30 mL) and water (60 mL) and a solution of FMOC-OSU (1334 mg, 3.95 mmol) in 30 mL acetone was then added. After stirring at room temperature for an hour, 1N aq HCl ( ⁇ 11 mL) was added dropwise to adjust pH ⁇ 3.
  • Step B Synthesis of Intermediate Int 3 [0183] Intermediate Int 3a (425 mg, 0.617 mmol) was dissolved in THF (10 ml) and LiOH (1M, aq) (1.234 ml, 1.234 mmol) was then added, followed by methanol (5 ml). After stirring at RT for 45 mins, the reaction was quenched with 1.5 mL 1N aq. HCl, diluted with brine, and extracted with EtOAc (3X). The combined organic phase was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo.
  • Step B Synthesis of Intermediate Int 5 [0186] The above filtered solid Int 5a was added DCM (13 ml), DIEA (0.848 ml, 4.85 mmol), and ACETIC ANHYDRIDE (0.343 ml, 3.64 mmol). After stirring at RT for an hour, aq.1N HCl was added to adjusted pH to ⁇ 2. The mixture was diluted with water and extracted with DCM (3x).
  • Step B Synthesis of Intermediate Int 6b [0188] Intermediate Int 6a (170 mg, 0.271 mmol) was dissolved in MeOH (10 ml) and bubbled with nitrogen gas. Next 10% Pd-C (30 mg, 0.282 mmol) was added, and the flask was evacuated with H2 and back filled three times. After stirring at RT for 3 hours, the reaction was filtered through syringe filter and concentrated in vacuo.
  • Step D Synthesis of intermediate Y [0196] Intermediate Y-3 (320 mg, 0.509 mmol) was dissolved in THF (2121 ⁇ l) and MeOH (2121 ⁇ l) and Water (2121 ⁇ l) and LITHIUM HYDROXIDE MONOHYDRATE (85 mg, 2.036 mmol) were then added at room temperature.
  • Step B Synthesis of intermediate Z-2 [0198] Intermediate Z-2 (500 mg, 1.290 mmol) was dissolved in DCM (2mL) and 2,2,2- trifluoroacetic acid (2 ml, 1.290 mmol) was then added under N2. The solution was concentrated and dried over high vacuum overnight.
  • N-ethyl-N-isopropylpropan-2-amine (0.899 ml, 5.16 mmol) was added dropwise to the mixture the above TFA salt and(S)-2-((tert-butoxycarbonyl)amino)-3-(4- methoxyphenyl)propanoic acid (381 mg, 1.290 mmol) and HATU (589 mg, 1.548 mmol) in DMF (12.90 ml). After stirring at room temperature for overnight, the mixture was partitioned between EtOAc and water. The organic phase was separated, washed with water (X2), dried (MgSO4) and concentrated under reduced pressure.
  • Step C Synthesis of intermediate Z-3 [0200]
  • Intermediate Z-2 (488 mg, 0.864 mmol) was dissolved in DCM (1mL) and 4N hydrogen chloride (1ml, 4.00 mmol) was added under N2. After stirring at room temperature for 4 hours, the solution was concentrated and dried over high vacuum to dryness.
  • N-ethyl-N-isopropylpropan-2-amine (0.542 ml, 3.11 mmol) was added dropwise to the mixture the above HCl salt and(2S,3R)-2-((tert-butoxycarbonyl)amino)-3-methoxybutanoic acid (202 mg, 0.864 mmol) and HATU (361 mg, 0.951 mmol) in DMF (4.32 ml) at 0°C and the mixture was allowed to warm up to room temperature and stirred for overnight. The mixture was partitioned between EtOAc and water. The organic phase was separated, washed with water (X2), dried (MgSO4) and the volatiles removed under reduced pressure.
  • Step E Synthesis of intermediate Z [0202]
  • Intermediate Z (426 mg, 0.627 mmol) was dissolved in DCM (2.5ml) and 2,2,2- trifluoroacetic acid (71.5 mg, 0.627 mmol) was then added under N2. After stirring for 1h, the solution was concentrated to dried over high vacuum to dryness. The residue was added ether and allowed to sit for 10min, solid precipitate out. The solvent was decanted and the solid was dried to afford intermediate Z as a solid.
  • Step B Synthesis of intermediate Int 7b [0204] Intermediate Int 7a (0.330 g, 1.019 mmol) was dissolved in DCM (4 ml) and HCl (4N HCl in dioxane) (2.038 ml, 8.15 mmol) was then added. The resulting solution was stirred at RT for 1h. The solvent was concentrated to afford Int 7b.
  • Step D Synthesis of intermediate Int 7
  • Intermediate Int 7c (0.224 g, 0.51 mmol) was dissolved in DCM (3 ml) and HCl (4M in dioxane) (0.638 ml, 2.55 mmol) was then added. After stirring at room temperature for 1hour, the solvent was concentrated to afford intermediate Int 7. LC/MS 339.27 (M+1) + .
  • Preparation of Intermediate Int 8 [0207] Intermediate F-4 (0.49 g, 0.830 mmol) was dissolved in THF (6 ml) and MeOH (2ml) and lithium hydroxide (2.489 ml, 2.489 mmol) was added dropwise.
  • Step B Synthesis of intermediate Int 10b
  • DIPEA 0.177 ml, 1.015 mmol
  • HATU 79 mg, 0.207 mmol
  • the reaction mixture was purified by preparative HPLC reverse phase (SunFire C-18, 19x150mm), and eluted with Acetonitrile/Water + 0.05%TFA (10-100% Acetonitrile in water) to afford Int 10b.
  • Step C Synthesis of intermediate Int 10
  • Intermediate Int 10b (90 mg, 0.190 mmol) was dissolved in THF (1.8ml), MeOH (1.2 ml) and Water (0.6 ml) and LiOH (1M in H2O) (0.570 ml, 0.570 mmol) was added. After stirring at room temperature for 2 hours, the solvent was concentrated under reduced pressure and the residue was purified by preparative HPLC reverse phase (SunFire C-18, 19x150mm), and eluted with Acetonitrile/Water + 0.05%TFA (10-100% Acetonitrile in water) to yield product Int 10.
  • Step B Synthesis of Int 12b (2S,3S)-1-benzyl 2-methyl 3-hydroxypyrrolidine-1,2-dicarboxylate [0219] To the solution of Int 12a (7.48 g, 28.2 mmol) in MeOH (80 ml) was added TMS- Diazomethane (70.5 ml, 141 mmol) dropwise. After stirring at rt for 10min, the reaction was quenched by addition of acetic acid (ca.400 ⁇ L) dropwise.
  • Step C Synthesis of Int 12c [0220] To solution of Int 12b (2.06 g, 7.38 mmol) in DCM (100 mL) was bubbled with N2 for 30min, then RHODIUM(II) ACETATE DIMER (0.326 g, 0.738 mmol) was added.
  • Step B Synthesis of Int 13 [0222] Int 13a (1.795 g, 4.77 mmol) was dissolved in Acetone (100 ml) and Water (100 ml) at 0 °C and sodium carbonate (1.011 g, 9.54 mmol) and FMOC-OSU (1.770 g, 5.25 mmol) were then added. After stirring at RT for 2 hours, the volatile was removed on rotary evaporator, the aqueous phase was acidified to pH 3, the precipitate was extracted with EtOAc (3x100 mL) and the combined organic phase was dried over Na2SO4, concentrated.
  • Step C Synthesis of Int 14c [0225] Int 14b (404 mg, 1.134 mmol) was dissolved in CH2Cl2 (20 ml) and DIEA (1.188 ml, 6.80 mmol) and acetic anhydride (0.160 ml, 1.700 mmol) were then added. After stirring at rt for 2h, the reaction solution was concentrated, and the residue was purified on reverse phase MPLC (130g) using 5-70%acetonitrile (0.05%TFA) to afford Int 14c as an oil. LC/MS 399.3 (M+1) + .
  • Step D Synthesis of Int 14d [0226] To the solution of Int 14d (438 mg, 1.099 mmol) in MeOH (20 ml) was added Pd/C (117 mg, 0.110 mmol), and BOC-Anhydride (0.383 ml, 1.649 mmol). The resulting mixture was hydrogenated at rt for 2h, then filtered through celite under N2. Next the filtrate was concentrated, and the residue was purified on silica gel column (80g) using 10- 100%EtOAc/hexane as and eluted solvents to afford Int 14d as an oil. LC/MS 365.2 (M+1) + .
  • Step E Synthesis of Int 14 [0227] To the solution of Int 14d (0.33 g, 0.906 mmol) in THF (6 ml), MeOH (2 ml), and Water (2 ml) at 0 °C was added LiOH (3.62 ml, 3.62 mmol) dropwise. After stirring at 0 °C for 2h, the volatile was then evaporated on rotary evaporator and the aqueous phase was acidified to pH 4, then extracted with DCM (3x50 mL). The combined organic phase was dried over Na2SO4, concentrated to afford Int 14 as an oil. LC/MS 351.2. (M+1) + .
  • Step B Synthesis of Int 15 [0229] Int 15a (2.08 g, 6.77 mmol) was dissolved in CH2Cl2 (30 ml) and methanamine (40% in water) (0.791 ml, 10.15 mmol) was then added.
  • Step B Synthesis of Int 16b [0231] Int 16a (2.099 g, 7.74 mmol) was dissolved in CH2Cl2 (20 ml) and (9H-fluoren-9- yl)methanol (1.822 g, 9.28 mmol), DIC (1.446 ml, 9.28 mmol), and DMAP (0.095 g, 0.774 mmol) were added.
  • Step C Synthesis of Int 16c [0232] Int 16b (3.99 g, 7.63 mmol) was dissolved in CH2Cl2 (10 ml) and TFA (11.76 ml, 153 mmol) was then added.
  • Step D Synthesis of Int 16d [0233] To the solution of Int 16c (3.54 g, 7.64 mmol) in Acetone (110 ml) and Water (100 ml) was added FMOC-OSU (2.71 g, 8.02 mmol). The resulting mixture was stirred at rt for 2h. Next the volatile was evaporated on rotary evaporator.
  • Step E Synthesis of Int 16 [0234] To the solution of Int 16d (4.23 g, 7.40 mmol) in Dioxane (80 ml) and Water (40 ml) was added sodium periodate (6.33 g, 29.6 mmol) and osmium tetroxide (2.351 g, 0.370 mmol).
  • Step B Synthesis of Int 17b [0236] To the solution of Int 17a (980 mg, 1.114 mmol) in Acetonitrile (20 ml) was added PIPERIDINE (0.551 ml, 5.57 mmol). The resulting solution was stirred at rt for 1.5h. Next the mixture was filtered, and the filtrate was concentrated.
  • Step C Synthesis of Int 17c [0237] To the solution of Int 17b (160 mg, 0.330 mmol) in DMF (2 ml) was added HATU (132 mg, 0.347 mmol) and DIEA (0.115 ml, 0.660 mmol).
  • Step B Synthesis of Int 18 [0239] To the solution of Int 18 (297 mg, 0.431 mmol) in THF (6 ml), MeOH (2 ml), and Water (2 ml) at 0 °C was added LiOH (1.725 ml, 1.725 mmol) dropwise. The resulting solution was stirred at 0 °C for 5h.
  • Step B Synthesis of Int 20b [0242] To the solution of Int 20a (0.67 g, 1.854 mmol) in THF (10 ml) at 0 °C was added 9- BBN (0.5M in THF) (11.12 ml, 5.56 mmol) dropwise. The resulting solution was stirred at rt for 1h.
  • Step C Synthesis of Int 20c [0243] To the solution of Int 20b (358 mg, 0.943 mmol) and Int 19 (328 mg, 1.132 mmol) in THF (10 ml) at 0 °C was added TRIPHENYLPHOSPHINE (371 mg, 1.415 mmol) and DIAD (0.275 mL, 1.415 mmol). The resulting solution was stirred from 0 °C to rt for 2h.
  • Step D Synthesis of Int 20 [0244] To the solution of Int 20c (155 mg, 0.259 mmol) in MeOH (10 ml) was added 10%Pd/C (27.6 mg, 0.026 mmol).
  • Step B Synthesis of intermediate Int 22
  • UPLC-MS: [M+H] + 529.2.
  • Step C Synthesis of intermediate Int 23 [0252]
  • Int 23b (1 g, 1.836 mmol) and p-toluensulfonic acid monohydrate (3.49 g, 18.36 mmol) were dissolved in water (4.67 ml) and acetone (14 ml). The mixture was heated at 80 °C for 1h under microwave irradiation. Then the mixture was extracted with Et 2 O. The phases were separated and the organic washed with NaHCO 3 two times. Then the aqueous phase was basified with NaHCO 3 and extracted with EtOAc.
  • Step B - Synthesis of intermediate Int 28b [0258] Intermediate Int 28a (2.50 g, 4.43 mmol) was dissolved in DCM (2 mL) was NBS (0.79 g, 4.43 mmol) was added at room temperature. The reaction solution was refluxed at 50 °C for 66 h.
  • Step C Synthesis of intermediate Int 28c
  • allyltributylstannane 201 mg, 0.617 mmol
  • Pd(PPh 3 ) 4 35.7 mg, 0.309 mmol
  • the reaction solution was stirred at 80 °C for 4 h.
  • the resulting solution was allowed to warm to 25 °C and saturated aqueous NaCl (20 mL) was added to the solution.
  • the solution was extracted with EA (4 x 50 mL) and the organic layer was dried over anhydrous Na 2 SO 4 and filtered. The filtrate was concentrated under reduced pressure.
  • Step D Synthesis of intermediate Int 28d
  • LiBr (0.93 g, 10.73 mmol
  • the reaction solution was stirred at 60 °C for 16 h.
  • the resulting solution was concentrated under reduced pressure.
  • the residue was purified by a silica gel column chromatography, and eluted with gradient 0 - 30% PE in EA. The fractions containing the desired product were combined and concentrated under reduced pressure to afford Int 28d as a solid.
  • Step E Synthesis of intermediate Int 28 [0261]
  • Intermediate Int 28d (2.20 g, 3.46 mmol) was dissolved in THF (90 mL) and LiOH (69 mL, 6.92 mmol, 0.1 N in water) was then added at 0 °C.
  • the reaction solution was stirred for 4 h at room temperature.
  • the resulting solution was adjusted pH to 4 ⁇ 5 with HCl (1M).
  • the solution was extracted with EA (3 x 100 mL) and the combined organic layer was washed with brine (3 x 100 mL), dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure.
  • Step D Synthesis of intermediate Int 29d
  • Intermediate Int 29c was dissolved in THF (5 ml) and aq 1M lithium hydroxide (4.23 ml, 4.23 mmol) was added and stirred at RT overnight.
  • DCM was added and the reaction solution was acidified by 1N HCl.
  • the mixture was extracted with DCM for 2 times.
  • the organic layers were combined and separated, washed with brine, separated, dried over MgSO4, filtered, and concentrated to afford Int 29d as a solid, which was use crude in the next step.
  • Step B Synthesis of Intermediate Int 32 [0268] To a solution of Int 32-a (10.0 g, 64.4 mmol) in 1,4-dioxane (150 mL) and water (150 mL) was added Fmoc-OSu (65.2 g, 193 mmol). The reaction solution was stirred for 35 h at room temperature. The pH value of the solution was adjusted to 5 with aqueous HCl (4 M). The solution was extracted with EA (2 x 200 mL). The combined organic layers were washed with brine (2 x 200 mL) and concentrated under reduced pressure.
  • Step B Synthesis of intermediate Int 30b
  • methyl 4- oxobutanoate 0.15 g, 2.72 mmol
  • TEA 1.42 mL, 10.2 mmol
  • MgSO 4 2.04 g, 17.0 mmol
  • Step C - Synthesis of intermediate Int 30c [0271] To a stirred solution of intermediate Int 30b (500 mg, 1.53 mmol) in toluene (1 mL) and water (100 ⁇ L) were added allylboronic pinacol ester (309 mg, 1.84 mmol), cataCXium A Pd G2 (102 mg, 0.153 mmol) and Cs2CO3 (1.80 g, 5.52 mmol) at room temperature. The reaction solution was stirred at 80 °C for 4 h. The resulting solution was concentrated under reduced pressure and purified by a silica gel column chromatography and eluted with gradient 0% - 50% EA in PE.
  • Step D Synthesis of intermediate Int 30
  • Step 2 N-((12S,13S,9S,12S,E)-9-acetamido-12-((1-((E)-4-((1-(tert-butoxycarbonyl)piperidin-4- yl)oxy)but-2-en-1-yl)-5-fluoro-1H-indol-3-yl)methyl)-10,13-dioxo-2-oxa-11-aza-1(3,1)- pyrrolidina-7(1,3)-benzenacyclotridecaphan-4-ene-12-carbonyl)-O-methyl-L-threonyl-L-serine [0276] LiOH ⁇ H2O (45 mg, 1.1 mmol) was added to a solution of Int 31a (370 mg, 0.35 mmol) in 1:1:1 THF:MeOH:H 2 O (3.5 mL).
  • Step 3 N-((12S,13S,9S,12S,E)-9-acetamido-12-((5-fluoro-1-((E)-4-(piperidin-4-yloxy)but-2-en- 1-yl)-1H-indol-3-yl)methyl)-10,13-dioxo-2-oxa-11-aza-1(3,1)-pyrrolidina-7(1,3)- benzenacyclotridecaphan-4-ene-12-carbonyl)-O-methyl-L-threonyl-L-serine [0277] HCl (4 M in dioxane, 0.89 mL, 3.5 mmol) was added to a solution of Int 31b (370 mg, 0.35 mmol) in DCM (1.8 mL).
  • Step 5 N-((3aS,6E,15S,18S,20Z,27E,36R,39S,41aS)-22-fluoro-36-(iodomethyl)-39-((R)-1- methoxyethyl)-16,35,38,41,45-pentaoxo- 3,3a,5,8,14,15,16,17,18,19,26,29,32,33,35,36,37,38,39,40,41,41a-docosahydro-2H,31H-31,34- ethano-1,18-methano-9,13:20,25-di(metheno)benzo[u]pyrrolo[3,2- b][1,15]dioxa[5,8,11,20,26]pentaazacyclooctatriacontin-15-yl)acetamide [0279] Iodine (33 mg, 0.13 mmol) was added to a mixture of Int 31d (
  • reaction block containing the vials was sealed, placed on a tumble stirrer, and heated to 40 °C for 18 h.
  • the reaction mixtures were allowed to cool to 23 °C, quenched with 1% AcOH in DMSO, filtered.
  • the resulting crude mixtures were purified by reverse-phase high-performance liquid chromatography.
  • Step B Synthesis of Int 33b [0283] To a solution of Int 33a (10.0 g, 29.4 mmol) in THF (150 mL) was added LiOH (294 mL, 58.8 mmol, 0.2 N in water) at 0 °C. The reaction solution was stirred at 25 °C for 2 h.
  • Step C Synthesis of Int 33 [0284] To a solution of Int 33b (1.30 g, 3.99 mmol) in dry THF (30 mL) was added NaH (0.479 g, 12.0 mmol, 60% in mineral oil) at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 10 min. Iodomethane (4.53 g, 31.9 mmol) was added to the mixture in dropwise. The reaction solution was stirred at 25 °C for 16 h. The reaction was quenched with water (50 mL). The pH value of the reaction solution was adjusted to 3 with aqueous HCl (0.1 M). The solution was extracted with DCM (3 x 200 mL).
  • Step B Synthesis of Int 34b [0286] To a stirred solution of intermediate Int 34a (6.38 g, 28.1 mmol) in THF (20 mL) was added HCl in dioxane (4.0 M, 60 mL) at room temperature. The reaction mixture was stirred at 25 °C for 3 h. The solvent was concentrated under reduced pressure. The residue was dissolved in THF (100 mL) and Water (100 mL). Sodium hydrogen carbonate (9.43 g, 112 mmol) and benzyl carbonochloridate (7.18 g, 42.1 mmol) were added sequentially at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at 25 °C for 12 h.
  • Step C Synthesis of Int 34c [0287] To a solution of intermediate Int 34b (1.0 g, 3.83 mmol) and 3-bromoprop-1-ene (3.33 mL, 38.3 mmol) in DCM (25 mL) was added (1,3-dimesitylimidazolidin-2-ylidene)(5-(N,N- dimethylsulfamoyl)-2-isopropoxybenzylidene)ruthenium(VI) chloride (0.28 g, 0.38 mmol) under nitrogen atmosphere at room temperature. The mixture was refluxed for 5 h then the solvent was evaporated under reduced pressure.
  • Step D Synthesis of Int 34d [0288] To a solution of (S)-tert-butyl 2-(hydroxymethyl)-2-methylpyrrolidine-1-carboxylate (1.86 g, 8.64 mmol) in THF (15 mL) was added sodium hydride (311 mg, 7.77 mmol) under nitrogen atmosphere at 0 o C. The solution was stirred for 15 min, then intermediate Int 34c (1.53 g, 4.32 mmol) in THF (15 mL) and tetrabutylammonium bromide (278 mg, 0.86 mmol) were added to the solution. The mixture was stirred at room temperature for 12 h and monitored periodically by LCMS.
  • Step E Synthesis of Int 34 [0289] To a stirred solution of Int 34d (1.46 g, 2.99 mmol) in THF (20 mL) was added 10% Palladium on activated carbon (354 mg, 2.99 mmol) under nitrogen atmosphere. The reaction mixture was degassed with hydrogen for three times and stirred at room temperature for 12 h under hydrogen. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure to afford Int 34 as an oil. The crude product was used in the next step without further purification. LCMS (ESI) calculated for C19H36N2O4 [M + H] + : 357.3, found 357.3.
  • Example 1 Synthesis of Example compound 10 (Ex-10) Step A - Synthesis of intermediate Ex-10a
  • Intermediate A 63 mg, 0.109 mmol
  • intermediate F 67.1 mg, 0.109 mmol
  • HATU 43.5 mg, 0.114 mmol
  • DIPEA 47.4 ⁇ l, 0.272 mmol
  • Example 2 Synthesis of Example compound 11 (Ex-11) Step A - Synthesis of intermediate Ex-11a
  • Intermediate H (93 mg, 0.151 mmol) and intermediate G (100 mg, 0.137 mmol) were dissolved in DMF (1374 ⁇ l) and HATU (57.5 mg, 0.151 mmol) was then added, followed by DIPEA (71.8 ⁇ l, 0.412 mmol). After stirring at room temp for 1 hour, the reaction mixture was diluted with EtOAc, washed with water three times, brine, dried over magnesium sulfate and concentrated. The residue was purified via flash column chromatography on a 12g silica gold column, and eluted with 10% MeOH in DCM to afford Ex-11a.
  • Example 3 Synthesis of Example compound 22 (Ex-22) Step A - Synthesis of intermediate Ex-22a
  • Intermediate L 37 mg, 0.052 mmol
  • intermediate J (27.5 mg, 0.054 mmol) were dissolved in dry DMF (1397 ⁇ l) and cooled to 0 °C.
  • HATU (20.64 mg, 0.054 mmol
  • DIPEA 45.0 ⁇ l, 0.258 mmol
  • the mixture was loaded directly onto HPLC using a SunFire prep C18 OTD column, 19 x 100 mm, 10-100% [ACN with 0.05% TFA] in [water w/0.05% TFA] gradient to afford Ex-22a.
  • Step B Synthesis of intermediate Ex-25b
  • Intermediate Ex-25a (6 mg, 4.99 ⁇ mol) was dissolved in THF (285 ⁇ l)), MeOH (142 ⁇ l), and Water (71.2 ⁇ l) and LiOH (0.597 mg, 0.025 mmol) was then added. After stirring at room temp for 2 hours, it was quenched with 1N HCl, extracted three times with EtOAc, dried over magnesium sulfate and concentrated to afford Ex-25b.
  • LC/MS: [M+H] + 1189.45.
  • Step D Synthesis of Example Ex-25 [0304]
  • AOP (4.27 mg, 9.64 ⁇ mol) and DIPEA (7.86 ⁇ l, 0.045 mmol) were dissolved in dry DCM (161 ⁇ l) and intermediate Ex-25c (7 mg, 6.43 ⁇ mol) in DMF (6603 ⁇ l) was added dropwise. After stirring at room temp overnight, it was diluted with EtOAc, washed with water three times, brine, dried over magnesium sulfate and concentrated. The residue was purified via HPLC using a SunFire prep C18 OTD column, 19 x 100 mm, 10-100% [ACN with 0.05% TFA] in [water w/0.05% TFA] gradient to afford Ex-25.
  • Example 6 Synthesis of Example compound 31 (Ex-31) Step A - Synthesis of intermediate Ex-31 [0309]
  • Example Ex-20 (9.3 mg, 8.94 ⁇ mol) was dissolved in MeOH (2980 ⁇ l) and Pd-C (0.952 mg, 8.94 ⁇ mol) was then added. After the flask was evacuated and backfilled with Hydrogen (1 atm via balloon) three times, the reaction was stirred at room temp for 2 hours and then filtered through celite. The residue was dissolved in ACN/Water and dried via lyophilization to afford Ex-31.
  • LC/MS: [M+H] + 1044.59.
  • Example 7 Synthesis of Example compound 33 (Ex-33) Step A - Synthesis of intermediate Ex-33a [0310]
  • Intermediate S (22.8 mg, 0.030 mmol) was dissolved in dry DMF (297 ⁇ l) and cooled to 0 °C.
  • Intermediate P (20.48 mg, 0.044 mmol), HATU (16.91 mg, 0.044 mmol), and DIPEA (25.9 ⁇ l, 0.148 mmol) were then added and the reaction was stirred at 0 °C for 30 min. Next the mixture was diluted with EtOAc, washed with water three times, brine, dried over magnesium sulfate and concentrated.
  • Step D Synthesis of Example Ex-34/Ex-35 [0317]
  • Intermediate Ex-34c (30 mg, 0.027 mmol) was dissolved in dry DMF (2720 ⁇ l) and cooled to 0 °C.
  • HATU (10.36 mg, 0.027 mmol)
  • DIPEA (28.5 ⁇ l, 0.163 mmol) were then added. After stirring for 1 hour, the reaction was diluted with EtOAc, washed with water three times, brine, dried over magnesium sulfate and concentrated.
  • Example 9 Synthesis of Example compound 38 (Ex-38) Step A - Synthesis of intermediate Ex-38a [0318]
  • Intermediate F-4 100 mg, 0.169 mmol
  • cesium carbonate 132 mg, 0.406 mmol
  • 2-(bromomethyl)-5,8-dioxaspiro[3.4]octane 70.1 mg, 0.339 mmol
  • the reaction was diluted with EtOAc, washed with water three times, brine, dried over magnesium sulfate and concentrated.
  • Step F Synthesis of intermediate Ex-38f
  • Intermediate Ex-38e (26.6 mg, 0.022 mmol) was dissolved in MeOH (748 ⁇ l) and formaldehyde (16.71 ⁇ l, 0.224 mmol) and acetic acid (6.42 ⁇ l, 0.112 mmol) were then added.
  • sodium cyanoborohydride (11.28 mg, 0.180 mmol) was added, followed by magnesium sulfate. After stirring at room temp for 2 hours, the reaction was quenched with sat aq. sodium bicarbonate solution. evaporated volatiles, extracted with EtOAc three times, washed with brine, dried over magnesium sulfate and concentrated to afford Ex-38f.
  • Step H Synthesis of intermediate Ex-38h
  • Intermediate Ex-38h (18.6 mg, 0.017 mmol) was dissolved in dry DMF (3726 ⁇ l) and cooled to 0 °C.
  • Example 10 Synthesis of Example compound 39 (Ex-39) Step A - Synthesis of intermediate Ex-39a
  • Intermediate S-4 110 mg, 0.152 mmol
  • intermediate U 69.4 mg, 0.304 mmol
  • DIPEA 133 ⁇ l, 0.760 mmol
  • the reaction was diluted with EtOAc, washed with water 3 times, brine, dried over magnesium sulfate and concentrated.
  • the residue was purified via flash column chromatography on a 4g silica gold column using 30-70% [3:1 EtOAc:EtOH] in hexane gradient to afford Ex-39a.
  • Step D Synthesis of intermediate Ex-39d
  • Intermediate Ex-39c 17.7 mg, 0.021 mmol
  • intermediate N 11.54 mg, 0.031 mmol
  • HATU 11.72 mg, 0.031 mmol
  • DIPEA 17.90 ⁇ l, 0.103 mmol
  • Step G Synthesis of Example Ex-39 [0334]
  • Intermediate Ex-39f (3.8 mg, 3.44 ⁇ mol) was dissolved in dry DMF (749 ⁇ l) and DCM (6739 ⁇ l) and cooled to 0 °C.
  • HATU (1.310 mg, 3.44 ⁇ mol) was added, followed by DIPEA (3.01 ⁇ l, 0.017 mmol).
  • DIPEA 3.01 ⁇ l, 0.017 mmol
  • Step A – Synthesis of Intermediate Ex-01a The peptide was synthesized using Fmoc/t-Bu chemistry on cysteamine 4-methoxytrityl resin with a CEM Liberty automated microwave peptide synthesizer. The peptide sequence was synthesized on a 0.125 mmol scale, using single-couplings of 4 equivalents of Fmoc protected amino acids as a 0.2M DMF solution along with 3.6 eq of HATU as a 0.45 M DMF solution and 8 eq of 2M DIEA in NMP, and heated at 75C for 300 seconds. Fmoc deprotections were performed using 20%(V/V) piperidine in DMF at 90C for 90 seconds.
  • Fmoc-AAs were coupled to the resin in this order: (S)-1-(((9H-fluoren-9- yl)methoxy)carbonyl)-2-methylpyrrolidine-2-carboxylic acid, (S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-(4-methoxyphenyl)propanoic acid, N-(((9H-fluoren-9- yl)methoxy)carbonyl)-O-(tert-butyl)-L-threonine, (2S,3S)-1-(((9H-fluoren-9- yl)methoxy)carbonyl)-3-(allyloxy)pyrrolidine-2-carboxylic acid (Intermediate W), (S)-2-((((9H- fluoren-9-yl)methoxy)carbonyl)amino)-3-(5-fluoro-1-(proparoyl
  • Intermediate Ex-01a (68.2 mg, 0.063 mmol) was dissolved in a degassed solution of acetonitrile (62 ml) and 20mM aq. NH 4 HCO 3 (28 ml).
  • Intermediate Ex-01b (77 mg, 0.063 mmol) was dissolved in degassed t-BuOH (50 ml) and water (25 ml) under an atmosphere of nitrogen gas and put into a preheated oil bath at 50 o C to raise to target temperature.
  • tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (7.5 mg, 0.014 mmol) and sodium ascorbate (22 mg, 0.111 mmol) were then added. After all solid was dissolved, tetrakis(acetonitrile)copper(I) hexafluorophosphate (5.3 mg, 0.014 mmol) was added.
  • Step D Synthesis of Example Ex-01 andEx-02 [0347]
  • Intermediate Ex-01c 37 mg, 0.030 mmol was dissolved in degassed acetic acid (2 ml) and DCM (40 ml) with continued bubbling of nitrogen and 1,3-Bis(2,4,6-trimethylphenyl)-4,5- dihydroimidazol-2-ylidene[2-(i-propoxy)-5-(N,N-dimethylaminosulfonyl)phenyl] methyleneruthenium(II) dichloride (Zhan Catalyst-1B) (16.4 mg, 0.023 mmol) was then added. The reaction was sealed and put in preheated oil bath at 50 o C for 100 minutes.
  • Intermediate Ex-07a (693 mg, 2.55 mmol) was dissolved in DMF (17 ml) and DIEA (1.784 ml, 10.22 mmol) was added, followed by methylamine (2M THF) (2.55 ml, 5.11 mmol) and then HATU (1020 mg, 2.68 mmol).
  • 2M THF 2.55 ml, 5.11 mmol
  • HATU 1020 mg, 2.68 mmol
  • 1N aq. HCl was added to neutralize the mixture, followed by dilution with water and extraction with DCM (3X). The combined organic phase was dried over Na2SO4, filtered, and concentrated in vacuo.
  • Step F Synthesis of Intermediate Ex07f [0355] Intermediate Int 3 (44.6 mg, 0.066 mmol) and Ex-07e (39 mg, 0.066 mmol) were dissolved in DMF (2 ml) and DIEA (29 ⁇ L, 0.166 mmol), and then AOP (CAS: 156311-85-2) (30.7 mg, 0.069 mmol) was added, followed by 20 ⁇ L DIEA to adjust pH to 9-10.
  • AOP CAS: 156311-85-2
  • Example 14 Synthesis of Example compound 08 (Ex-08) [0359]
  • Example Ex-07 (isomeric mixture) (2.8 mg, 2.67 ⁇ mol) was dissolved in ethanol (4 ml) and nitrogen gas was bubbled through the solution.
  • Example 15 Synthesis of Example compound 14 (Ex-14) Step A – Synthesis of Intermediate Ex-14a [0360] (S)-2-amino-3-(5-fluoro-1H-indol-3-yl)propanoic acid (3000 mg, 13.50 mmol) and BOC-Anhydride (3.29 ml, 14.18 mmol) were dissolved in DMA (30 ml) and Methanol (10 ml) and triethylamine (2.070 ml, 14.85 mmol) was then added. After stirring reaction at RT for an hour, the mixture was cooled in ice bath and a solution of conc.
  • Intermediate Ex-14f (112 mg, 0.168 mmol) and (S)-2-((tert-butoxycarbonyl)amino)-3- (4-methoxyphenyl)propanoic acid (64.5 mg, 0.218 mmol) were dissolved in DCM (5 ml) and DIEA (0.088 ml, 0.504 mmol) was then added, followed by AOP (CAS: 156311-85-2) (89 mg, 0.202 mmol). Next DIEA (44uL) was added to adjust pH to >8.
  • Intermediate Ex-32b (2000 mg, 4.75 mmol) was dissolved in anhydrous DMF (25 ml) under an atmosphere of nitrogen gas and IODOMETHANE (4 ml, 64.0 mmol) and SILVER OXIDE (5600 mg, 24.17 mmol) were then added.
  • IODOMETHANE (4 ml, 64.0 mmol)
  • SILVER OXIDE 5600 mg, 24.17 mmol
  • Step K Synthesis of Intermediate Ex-32k
  • Intermediate Ex-32j (0.013 mmol) was dissolved in DMF (2 ml) and DIEA (13.70 ⁇ l, 0.078 mmol), intermediate V (8.38 mg, 0.020 mmol) and HATU (6.46 mg, 0.017 mmol) were then added. After stirring at RT for 2 hours, the reaction was added DIETHYLAMINE (200 ⁇ l, 1.914 mmol) and stirred at RT for 10 mins and then concentrated in vacuo. The residue was redissolved in DMF (1 mL) and DIEA (25 ⁇ l, 0.14 mmol) and Ac2O (12.33 ⁇ l, 0.131 mmol) were added.
  • Example 17 Synthesis of Example compound 03 (Ex-03) Step A - Synthesis of Intermediate Ex-03a (tert-butyl 3-(azidomethyl)phenethylcarbamate) [0391] To a solution of tert-butyl 3-(hydroxymethyl)phenethylcarbamate (2 g, 7.96 mmol) in THF 40 ml, 2-azido-1,3-dimethyl-1H-imidazol-3-ium hexafluorophosphate(V) (3.15 g, 11.14 mmol), and DBU (2.79 g, 11.14 mmol) were added. After stirring at room temperature for 10 min, the reaction was quenched with sat.
  • Trityl resin was first loaded with 2 eq of (S)-1-(((9H-fluoren-9- yl)methoxy)carbonyl)-2-methylpyrrolidine-2-carboxylic acid and 4 eq DIEA. It was heated at 50oC in microwave for 10 min for 2 times. Then, the synthesis was continued by standard SPPS using Fmoc/t-Bu chemistry on a CEM Liberty Blue automated peptide synthesizer (CEM Corp.).
  • Reaction conditions were as follows: Deprotection Conditions: 20% piperidine (v/v) in DMF (2 X 2 min at 75 °C); Residue Coupling Conditions: 5 eq (relative to resin) of activated amino acid (5 mL of a 0.2 M amino acid stock solution in DMF) was delivered to the resin, followed by 5 eq of HATU activator (2 mL of a 0.45 M solution in DMF), and 10 eq of DIEA (1 mL of a 2M solution in NMP) and allowed to react for 5 min at 75 °C.
  • Fmoc-AAs were coupled to the resin in this order: (S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4- methoxyphenyl)propanoic acid (Y01), (2S,3R)-2-((((9H-fluoren-9-yl)methoxy) carbonyl) amino)-3-(tert-butoxy)butanoic acid (Thr), (2S,3S)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-3- (allyloxy)pyrrolidine-2-carboxylic acid (P42), S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-(5-fluoro-1-(-(prop-2-yn-1-yl)-1H-indol-3-yl)propanoic acid (W18), (S)-2-((((
  • Example 18 Synthesis of Example compound 04 (Ex-04) Synthesis of peptidyl resin EX-04a [0398] Peptidyl resin Ex-04a was synthesized by standard SPPS using Fmoc/t-Bu chemistry. The first residue Fmoc-(2S,3R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-3-(allyloxy)pyrrolidine- 2-carboxylic acid was coupled manually to Fmoc-MBHA-ProTide resin (0.19 mmol/g, CEM), using 2 eq of AA:2 eq HATU: 4 eq of DIEA, room temp. stirred for 2 hours, two times.
  • Example 19 Synthesis of Example compound 05 (Ex-05) Synthesis of Intermediate Ex-05a [0402] The peptidyl resin Ex-05a was synthesized using Fmoc-protected amino acids on a solid-phase 2- chlorotrityl chloride resin (0.16 mmol, 0.6 mmol/g, CreoSalus).
  • Trityl resin was first loaded with 2 eq of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4- methoxyphenyl)propanoic acid using and 4 eq DIEA and was heated at 50oC microwave for10 min for 2 times. Then, the loaded resin was synthesized by standard SPPS using Fmoc/t-Bu chemistry on a CEM Liberty Blue automated peptide synthesizer (CEM Corp.).
  • Reaction conditions were as follows: Deprotection Conditions: 20% piperidine (v/v) in DMF (2 X 2 min at 75 °C); Residue Coupling Conditions: 6 eq (relative to resin) of activated amino acid (5 mL of a 0.2 M amino acid stock solution in DMF) was delivered to the resin, followed by 6 eq of HATU activator (2 mL of a 0.45 M solution in DMF), and 12 eq of DIEA (1 mL of a 2M solution in NMP) and allowed to react for 5 min at 75 °C.
  • Fmoc-AAs were coupled to the resin in this order: (2S,3S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methoxybutanoic acid, (2S,3S)-1- (((9H-fluoren-9-yl)methoxy)carbonyl)-3-(prop-2-yn-1-yloxy)pyrrolidine-2-carboxylic acid, (S)- 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(1-allyl-5-fluoro-1H-indol-3-yl)propanoic acid, (S)-(9H-fluoren-9-yl)methyl (1-(3-(azidomethyl)phenyl)-3-oxopropan-2-yl)carbamate, and acetic anhydride for capping.
  • Example Ex-05 (16 mg, 0.015 mmol) was synthesized with Zhan catalyst (6.53 mg, 9.06 ⁇ mol) by following general RCM protocol to afford Ex-05 (7.8 mg). LC/MS 1031.3 (M+1) + .
  • Example 20 Synthesis of Example compound 6 (Ex-06) Synthesis of Intermediate Ex-06a [0407]
  • the peptidyl resin Ex-06a was synthesized by standard SPPS using Fmoc/t-Bu chemistry on a CEM Liberty Blue automated peptide synthesizer (CEM Corp.) on Ethyl indole AM resin (0.1 mmol, Novabiochem, 0.73 mmol/g).
  • Reaction conditions were as follows: Deprotection Conditions: 20% piperidine (v/v) in DMF (2 X 2 min at 75 °C); Residue Coupling Conditions: 5 eq (relative to resin) of activated amino acid (5 mL of a 0.2 M amino acid stock solution in DMF) was delivered to the resin, followed by 4.5 eq of HATU activator (1 mL of a 0.45 M solution in DMF), and 10 eq of DIEA (0.5 mL of a 2M solution in NMP) and allowed to react for 2 min at 90 °C.
  • Fmoc-AAs were coupled to the resin in this order: (S)-2-((((9H-fluoren- 9-yl)methoxy)carbonyl)amino)-3-(4-methoxyphenyl)propanoic acid, (2S,3S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-methoxybutanoic acid , (2S,3S)-1-(((9H-fluoren-9- yl)methoxy)carbonyl)-3-(prop-2-yn-1-yloxy)pyrrolidine-2-carboxylic acid , (S)-2-((((9H-fluoren- 9-yl)methoxy)carbonyl)amino)-3-(1-allyl-5-fluoro-1H-indol-3-yl)propanoic acid , (S)-(9H- fluoren-9-yl)methyl (1
  • Example Ex-06 [0410] Intermediate Ex-06b (16 mg, 0.014 mmol) was converted to Ex-06 (12 mg) by following general RCM protocol. LC/MS 1102.3 (M+1) + .
  • Example 21 Synthesis of Example compound 9 (Ex-09) Step A - Synthesis of intermediate Ex-09a [0411] N-ethyl-N-isopropylpropan-2-amine (0.052 ml, 0.296 mmol) was added dropwise to intermediate Y (50.5 mg, 0.082 mmol), intermediate Z (57 mg, 0.082 mmol), and HATU (37.5 mg, 0.099 mmol) in DMF (0.5 ml) at 0°C and the mixture was allowed to warm up to room temperature and stirred for 1h.
  • Step B Synthesis of Example Ex-09 [0412] Copper(II) sulfate (74.6 mg, 0.468 mmol) in Water (1 ml) was added dropwise to a mixture of intermediate Ex-09a and sodium (R)-5-((S)-1,2-dihydroxyethyl)-4-hydroxy-2-oxo- 2,5-dihydrofuran-3-olate (185 mg, 0.935 mmol) in tBuOH (20 ml) and Water (10 ml) and the resulting reaction mixture was stirred at room temperature for 2h. The tBuOH was removed under reduced pressure and sat. aq. ammonium chloride and EtOAc were added. The aqueous phase was separated and further extracted with EtOAc. The combined organic phase was washed with brine, dried (MgSO4) and concentrated under reduced pressure. The residue was purified by high throughput purification to afford Ex-09 as a solid.
  • Example 22 Synthesis of Example compound 24 (Ex-24) Step A - Synthesis of intermediate Ex-24a [0413] Tert-butyl 4-(azidomethyl)piperidine-1-carboxylate (363.4 mg, 1.512 mmol) was dissolved in DCM (5 mL) and TFA (2 mL, 26.0 mmol) was then added. After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure to afford Ex-24a as an oil. LC/MS 140.94 (M+1) + .
  • Step B Synthesis of intermediate Ex-24b
  • Boc-N-methyl-O-methyl-L-tyrosine dicyclohexylammonium salt (741 mg, 1.511 mmol) and HATU (574 mg, 1.511 mmol) were dissolved in DMF (8 mL) in a methanol/ice bath and Ex- 24a (384 mg, 1.511 mmol) and Hunig's base (0.264 mL, 1.511 mmol) were then added. The reaction was allowed to warm to ambient overnight.
  • Step F Synthesis of intermediate Ex-24f [0418]
  • Intermediate Y (94 mg, 0.153 mmol) and HATU (116 mg, 0.306 mmol) were dissolved in DMF (3 mL) in a methanol/ice bath and intermediate Ex-24e and Hunig's Base (0.080 mL, 0.459 mmol) were then added. After stirring at room temperature overnight, the reaction crude was purified by reverse phase chromatography (C18, 43 g cartridge), and eluted with an acetonitrile/water/0.1% v/v formic acid mixture (0% to 100 %) to afford Ex-24f. LC/MS 1065.65 (M+Na) + .
  • Example 23 Synthesis of Example compound 17 (Ex-17) Step A - Synthesis of intermediate Ex-17a [0420] To the solution of intermediate Int 7 (80 mg, 0.213 mmol) and Int 8(123 mg, 0.213 mmol) in DMF (2ml) at room temperature was added HATU (85 mg, 0.224 mmol) and DIPEA (0.149 ml, 0.854 mmol).
  • Step B Synthesis of intermediate Ex-17b
  • Intermediate Ex-17a (170 mg, 0.190 mmol) and cesium carbonate (185 mg, 0.569 mmol) were dissolved in DMF (2 ml) and tert-butyl acrylate (0.110 ml, 0.758 mmol) was then added. After stirring at 50°C for 2 hours, the reaction mixture was diluted with EtOAc and washed with water, the organic phase was dried over Na2SO4, concentrated and the residue was purified by flash column chromatography on silica gel (ISCO 40 g column, and eluted with MeOH/DCM(0-10% MeOH in DCM) to afford intermediate Ex-17b. LC/MS 1025.43 (M+1) + .
  • Step C Synthesis of intermediate Ex-17c
  • Intermediate Ex-17b 160 mg, 0.156 mmol was dissolved in DCM (1.5 ml) and TFA (0.481 ml, 6.24 mmol) was then added. After stirring for 2 hours, the reaction solution was concentrated under reduced pressure and the residue was purified by preparative HPLC reverse phase (SunFire C-18, 19x150mm), and eluted with Acetonitrile/Water + 0.05%TFA (10-100% Acetonitrile in water) to yield product Ex-17c.
  • Step D Synthesis of intermediate Ex-17d
  • HATU 14.42 mg, 0.038 mmol
  • DIPEA 0.025 ml, 0.144 mmol
  • Step F Synthesis of intermediate Ex-17f [0425]
  • Intermediate Ex-17e (36.0 mg, 0.033 mmol) was dissolved in THF (0.4ml) and LiOH (0.330 ml, 0.330 mmol) was then added. After stirring at room temperature for 2 hours, the solvent was removed under reduced pressure and the residue was purified by preparative HPLC reverse phase (SunFire C-18, 19x150mm), and eluted with Acetonitrile/Water + 0.05%TFA (10- 100% Acetonitrile in water) to yield Ex-17f.
  • Step G Synthesis of Example Ex-17 [0426] To the solution of intermediate Ex-17f (20 mg, 0.018 mmol) in DMF (2.5 ml) and DCM (30 ml) was added HATU (7.03 mg, 0.018 mmol) and DIPEA (0.015 ml, 0.088 mmol). The resulting solution was stirred at RT for 2 hours. The solvent was removed under reduced pressure and the residue was purified by preparative HPLC reverse phase (SunFire C-18, 19x150mm), and eluted with Acetonitrile/Water + 0.05%TFA (20-60% Acetonitrile in water) to afford Ex-17. LC/MS 1021.37 (M+1) + .
  • Example 24 Synthesis of Example compound 21 (Ex-21) Step A - Synthesis of intermediate Ex-21a [0427] To the solution of Int 10 (11.48 mg, 0.025 mmol) and Int 11 (17 mg, 0.024 mmol) in DMF (0.4 ml) was added DIPEA (0.021 ml, 0.119 mmol) and HATU (9.50 mg, 0.025 mmol) at room temperature. After stirring for 1 hour, the crude mixture was purified by preparative HPLC reverse phase (SunFire C-18, 19x150mm), and eluted with Acetonitrile/Water + 0.05%TFA (10- 100% Acetonitrile in water) to afford intermediate Ex-21a. LC/MS 1058.37 (M+1) + .
  • Step B Synthesis of intermediate Ex-21b
  • Ex-21a 15 mg, 0.014 mmol
  • DCM 0.2ml
  • imidazole 3.86 mg, 0.057 mmol
  • triphenylphosphine 13.87 mg, 0.053 mmol
  • iodine 11.51 mg, 0.045 mmol
  • the mixture was directly loaded on silica gel column (ISCO 24 g column and eluted with MeOH/DCM (0-10 % MeOH in DCM) to afford intermediate Ex-21b as a solid.
  • Step D - Synthesis of Example Ex-21 [0430] To a vial was added intermediate Ex-21c (5.7 mg, 4.41 ⁇ mol), nickel (II) chloride ethylene glycol dimethyl ether complex (1.936 mg, 8.81 ⁇ mol) and pyridine-2,6- bis(carboximidamide) dihydrochloride (2.080 mg, 8.81 ⁇ mol), then followed by DMA (176 ⁇ l) and ZINC (1.267 mg, 0.019 mmol) in glovebox. The reaction solution was stirred at 45°C for 20h in the glovebox.
  • Example 25 Synthesis of Example compound 13 (Ex-13) Step A: Synthesis of intermediate Ex-13a [0431] To the solution of Intermediate C (1.297 g, 3.58 mmol) and Int 12 (1.021 g, 3.94 mmol) in DMF (30 ml) was added HATU (1.497 g, 3.94 mmol) and DIEA (1.251 ml, 7.16 mmol. After stirring at rt for 1h, the mixture was partitioned between EtOAc (200 mL) and brine (100 mL).
  • Ex-13b (TFA salt) as a powder.
  • HCl (17.16 ml, 1.716 mmol) dropwise was added to the solution of Ex-13b (0.53 g, 0.858 mmol) in Acetonitrile (100 ml) and Water (50 ml) at 0 °C.
  • HCl 17.16 ml, 1.716 mmol
  • Step D Synthesis of Ex-13d [0434]
  • Intermediate Ex-13c (0.64 g, 0.766 mmol) was dissolved in CH2Cl2 (6 ml) and TFA (6 ml, 78 mmol) was then added. After stirring at rt for 1h, it was concentrated and the residue was dissolved in DCM (10 mL) and toluene (6 mL), then concentrated again. The resulting residue was redissolved in DCM (10 mL) and treated with HCl (0.957 ml, 3.83 mmol), then concentrated to afford Ex-13d (hydrochloride salt) as an oil.
  • Step E Synthesis of Ex-13e [0435] To the solution of Ex-13d (549 mg, 0.766 mmol) in DMF (25 ml) and CH2Cl2 (500 ml) was added HATU (350 mg, 0.919 mmol), followed by addition of DIEA (0.803 ml, 4.60 mmol) dropwise. After stirring at rt for 1h, the reaction solution was concentrated and the residue was purified on reverse phase MPLC (C18, 275 g column), using 0-60%acetonitrile (0.05%TFA) as and eluted solvents to afford Ex-13e as a powder. LC/MS 662.2 (M+1) + .
  • Step F Synthesis of Ex-13f [0436] To the solution of Ex-13e (383 mg, 0.579 mmol) in THF (6 ml), Water (2 ml), and MeOH (2 ml) at 0 °C was added LiOH (1.736 ml, 1.736 mmol) dropwise. After stirring at 0 °C for 4h, then rt for 1h, the volatile was evaporated, and the aqueous phase was acidified to pH 3, extracted with 30%Isopropanol/DCM (3x100 mL). The combined organic phase was dried over Na2SO4, concentrated and the residue was lyophilized from acetonitrile/water (2:1, 30 mL) to Ex-13f as a powder.
  • Step H Synthesis of Example Ex-13 [0438]
  • the solution of Ex-13g (84 mg, 0.077 mmol) in CH2Cl2 (140 ml) was bubbled with N2 for 30min, then was added ZHAN CATALYST-1B (28.4 mg, 0.039 mmol).
  • the resulting solution was further bubbled with N2 for 30min, then heated at 50 °C for 5h. It was then concentrated, and the residue was purified on Gilson using 5-75%acetonitrile (0.05%TFA), and the product was repurified on silica gel column (40 g) using 0-10%MeOH/DCM as and eluted solvents to Ex-13as a solid.
  • Example 26 Synthesis of Example compound (Ex-19) Step A: Synthesis of Ex-19 [0439] To the solution of Int 17 (trifluoroacetic acid 1:1) (275 mg, 0.259 mmol) and Intermediate J (138 mg, 0.272 mmol) in DMF (4 ml) at 0 °C was added HATU (104 mg, 0.272 mmol) and DIEA (0.272 ml, 1.556 mmol). After stirring at 0 °C for 45 min, the reaction solution was partitioned between EtOAc (200 mL) and brine (100 mL) and then washed with sat. Na2CO3 (50 mL).
  • Step E Synthesis of Ex-19e [0443] To the solution of Ex-19e (190 mg, 0.170 mmol) in CH2Cl2 (4 ml) was added HCl (4N in dioxane) (1.278 ml, 5.11 mmol). After stirring at rt for 2h, the reaction solution was concentrated and the residue was dissolved in acetonitrile/water (10 mL, 2:1) to afford Ex-19e as a solid. LC/MS 1015.9 (M+1) + .
  • Step F Synthesis of Ex-19 [0444] To the solution of Ex-19e (48.8 mg, 0.045 mmol) in DMF (1 ml) was added ACETIC ANHYDRIDE (8.46 ⁇ l, 0.090 mmol) and DIEA (0.047 ml, 0.269 mmol). After stirring at rt for 30min, the reaction solution was then purified on Gilson (C18 column) using 25-43%acetonitrile (0.05%TFA) as and eluted solvents to afford Ex-19 as a powder. LC/MS 1057.8 (M+1) + .
  • Step A Synthesis of Ex-20a [0445] To the solution of Int 18 (520 mg, 0.771 mmol) and Intermediate N (380 mg, 0.925 mmol) in DMF (12 ml) at 0 °C was added 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3- tetramethylisouronium hexafluorophosphate (322 mg, 0.848 mmol) and DIEA (0.942 ml, 5.39 mmol).
  • Step B Synthesis of Ex-20b [0446] To the solution of Ex-20a (883 mg, 0.856 mmol) in THF (21 ml), MeOH (7.00 ml), and Water (7 ml) at 0 °C was added LiOH (3.43 ml, 3.43 mmol). After stirring at 0 °C for 2h, the volatile was evaporated, and the aqueous phase was acidified to pH 4. The mixture was extracted with DCM (3x100 mL). The combined organic phase was dried over Na2SO4, concentrated to afford Ex-20b as a solid. LC/MS 1017.8 (M+1) + .
  • Step C Synthesis of Ex-20c [0447] To the solution of Intermediate J-2 (38.6 mg, 0.236 mmol) and Ex-20b (200 mg, 0.197 mmol) in DMF (4 ml) at 0 °C was added HATU (90 mg, 0.236 mmol) and DIEA (0.275 ml, 1.573 mmol). After stirring at 0 °C for 30min, it was purified on reverse phase MPLC (C18, 86 g) using 5-100%acetonitrile (0.05%TFA) as and eluted solvents to afford Ex-20c as a powder. LC/MS 1126.9 (M+1) + .
  • Step D Synthesis of Ex-20d [0448]
  • the solution of Ex-20c (180 mg, 0.160 mmol) in CH2Cl2 (400 ml) was bubbled with N2 for 1h, then was added ZHAN CATALYST-1B (58.6 mg, 0.080 mmol).
  • the resulting solution was further bubbled with N2 for 30min, then heated at 50 °C for 3h. It was then quenched by addition of vinyl ethyl ether (1 mL). After stirring at rt open to air for 10min, it was concentrated, and the residue was purified on silica gel column using 0-10%MeOH/DCM as and eluted solvents to afford Ex-20d as a solid.
  • Step F Synthesis of Ex-20 and Ex-54 [0450] To the solution of Ex-20e (29 mg, 0.028 mmol) in DMF (1 ml) was added DIEA (0.029 ml, 0.168 mmol) and acetic anhydride (5.29 ⁇ l, 0.056 mmol), the resulting solution was stirred at rt for 30min, then purified on reverse phase Gilson C18 using 35-64% acetonitrile(0.05%TFA)/water(0.05%TFA) as and eluted solvents to give Ex-20 as a powder LC/MS 1040.7 (M+1) + ; and slow eluant Ex-54. LC/MS 1040.8 (M+1) + .
  • Step A Synthesis of Ex-28a [0451] To the solution of Int 21 and Intermediate F-4 (100 mg, 0.169 mmol) in DMF (1 ml) was added Cs2CO3 (165 mg, 0.508 mmol). After stirring at rt overnight, the mixture was partitioned between EtOAc (150 mL) and water (100 mL). The organic phase was further washed with water (2x100 mL), dried over Na2SO4, concentrated and the residue was purified on silica gel column using 0-10%MeOH/DCM as and eluted solvents to afford Ex-28a as an oil. LC/MS 844.8 (M+1) + .
  • Step B Synthesis of Ex-28b [0452] To the solution of Ex-28a (136 mg, 0.161 mmol) in THF (3 ml), MeOH (1 ml), and Water (1 ml) at 0 °C was added LiOH (0.500 ml, 0.500 mmol) dropwise. After stirring at 0 °C for 3h, the volatile was evaporated and the mixture was acidified to pH 3-4, extracted with DCM (3x70 mL). The combined organic phase was dried over Na2SO4, concentrated to afford Ex-28b as an oil. LC/MS 830.7 (M+1) + .
  • Step C Synthesis of Ex-28c [0453] To the solution of Ex-28b (60.6 mg, 0.073 mmol) Intermediate N (30 mg, 0.073 mmol) in DMF (2 ml) was added HATU (28.3 mg, 0.074 mmol) and DIEA (0.077 ml, 0.438 mmol). After stirring at rt for 45min, the mixture was partitioned between EtOAc (150 mL) and brine (100 mL).
  • Example 29 Synthesis of Example compound 45 (Ex-45) Step A: Synthesis of Ex-45a [0457] To the solution of intermediate Ex-20b in DMF (6 ml) at 0 °C was added HATU (101 mg, 0.265 mmol) and DIEA (0.232 ml, 1.327 mmol). After stirring at 0 °C for 60min, the mixture was partitioned between EtOAc (250 mL) and brine (200 mL). Next the organic phase was washed with brine (2x200 mL), dried overNa2SO4, concentrated and the residue was purified on silica gel column (80g) using 0-10%MeOH/DCM as and eluted solvents to afford Ex- 45a as an oil.
  • Example 30 Synthesis of Example compound 47 (Ex-47) Synthesis of Ex-47 [0461] To the solution of dimethylglycine hydrochloride (7.72 mg, 0.055 mmol) in DMF (0.5 ml) was added HATU (21.03 mg, 0.055 mmol) and DIEA (0.039 ml, 0.221 mmol).
  • Example 31 Synthesis of Example compound 48 (Ex-48) Synthesis of Ex-48 [0462] Intermediate Ex-45c (8 mg, 7.63 ⁇ mol) was dissolved in DMF (1 ml) and DIEA (8.00 ⁇ l, 0.046 mmol) and ACETIC ANHYDRIDE (1.440 ⁇ l, 0.015 mmol) were then added. After stirring at rt for 30min, the mixture was purified on reverse phase Gilson using 10-90% acetonitrile (0.05%TFA)/water (0.05%TFA) as and eluted solvents to afford Ex-48 as a powder. LC/MS 1054.7 (M+1) + .
  • Example 32 Synthesis of Example compound 49 (Ex-49) Synthesis of Ex-49 [0463] To the solution of 2-methoxyacetic acid (11.00 mg, 0.122 mmol) in DMF (1 ml) was added HATU (46.4 mg, 0.122 mmol) and DIEA (0.043 ml, 0.244 mmol). After stirring at rt for 30min, 0.2 mL of the above solution was added to a solution of Ex-45c (16 mg, 0.015 mmol) and DIEA (8.00 ⁇ l, 0.046 mmol) in DMF (0.5 ml).
  • Example 33 Synthesis of Example compound 46 (Ex-46) Synthesis of Ex-46 [0464] To the solution of Ex-47 (3.7 mg, 3.05 ⁇ mol) in MeOH (4 ml) was added Pd/C (1.300 mg, 1.222 ⁇ mol). The resulting mixture was hydrogenated at rt via H2 Balloon for 2h. then the mixture was filtered through celite.
  • Example 34 Synthesis of Example compounds 50 and 51 (Ex-50 and Ex-51) Synthesis of Ex-50 and Ex-51 [0465] To the solution of Ex-20e (33.8 mg, 0.033 mmol) and dimethylglycine hydrochloride (5.47 mg, 0.039 mmol) in DMF (1.5 ml) was added HATU (14.91 mg, 0.039 mmol) and DIEA (0.046 ml, 0.261 mmol).
  • Example 35 Synthesis of Example compound 53 (Ex-53) Synthesis of Ex-53 [0466] To the solution of Ex-50 (68 mg, 0.057 mmol) in MeOH (20 ml) was added Pd/C (19.04 mg, 0.036 mmol).
  • Example 36 Synthesis of Example compounds 27 and 52 (Ex-27 and Ex-52) Step A: Synthesis of Ex-27a [0467] To the solution of intermediate Int 20 (75 mg, 0.147 mmol) and Intermediate J (82 mg, 0.162 mmol) in DMF (4 ml) was added HATU (61.7 mg, 0.162 mmol) and DIEA (0.129 ml, 0.737 mmol). After stirring at rt for 1h, the solution was partitioned between EtOAc (150 mL) and brine (100 mL).
  • Step D Synthesis of Ex-27d [0470] To the solution of Ex-27c (173 mg, 0.144 mmol) in THF (6 ml), Water (2 ml) and MeOH (2 ml) at 0 °C was added LiOH (0.575 ml, 0.575 mmol) dropwise. After stirring at 0 °C for 2h, the volatile was evaporated, and the aqueous phase was acidified to pH 3-4 with 1 N HCl. The mixture was extracted with DCM (3x80 mL). The combined organic phase was concentrated to afford Ex-27d as an oil. LC/MS 1190.9 (M+1) + .
  • Step E Synthesis of Ex-27e [0471]
  • Intermediate Ex-27d (0.150 g, 0.126 mmol) was dissolved in CH2Cl2 (2 ml) and TFA (2 ml, 26.0 mmol) was then added. After stirring at rt for 45min, the mixture was concentrated and the residue was dissolved in acetonitrile/water (3:1, 30 mL), and treated with HCl (0.630 ml, 0.630 mmol). The resulting mixture was lyophilized to afford Ex-27e as a powder.
  • Step F Synthesis of Ex-27f [0472] To the solution of Ex-27e (142 mg, 0.126 mmol) in DMF (10 ml) and CH2Cl2 (200.00 ml) at 0 °C was added HATU (47.9 mg, 0.126 mmol) and DIEA (0.132 ml, 0.756 mmol). After stirring at 0 °C for 2.5h, the volatile was evaporated, and the residue was then partitioned between EtOAc (200 mL) and brine (100 mL).
  • Example 37 Synthesis of Example compound 41 (Ex-41) (HCl salt) Step A - Synthesis of intermediate Ex-41a
  • a solution of Int 22 (214 mg, 0.379 mmol) and Int 24 (275 mg, 0.379 mmol) in DMF (3 ml) was treated with HATU (144 mg, 0.379 mmol) and DIPEA (0.199 mL, 1.138 mmol). After stirring for 2h, the reaction mixture was diluted with EtOAc and washed with 0.1N HCl and then with saturated aqueous solution of NaHCO3 and brine.
  • Step E Synthesis of intermediate Ex-41e
  • Intermediate Int-41d (107 mg, 0.089 mmol) was dissolved in DCM (2 ml) and treated with acetyl chloride (6.96 ⁇ l, 0.098 mmol) and DIPEA (0.062 ml, 0.356 mmol). After stirring for 16 hours, the solvents were removed under reduced pressure to afford the crude product which was purified by reverse phase flash chromatography (120 g C18, elution water 0.1%TFA/CH 3 CN 0.1%TFA from 100:0 to 50:50 in 10CV, then to 50:50 in 6CV) to give after lyophilization Ex-41e as beige solid.
  • Step G Synthesis of intermediate Ex-41g
  • Ex-41f (81 mg, 0.071 mmol) dissolved in THF/H 2 O 1.1 (2 ml) and treated with LiOH (1.135 mg, 0.047 mmol) for 2h. Then the solvents were removed under reduced pressure to give Ex-41g.
  • UPLC-MS: [M+H] + 1199.8.
  • Step H Synthesis of intermediate Ex-41h [0481] Intermediate Ex-41g was dissolved in THF (1 ml) and treated with 4M HCl in dioxane (0.019 ml, 0.075 mmol). After stirring for 5 h, the solvents were removed under reduced pressure to give Ex-41h.
  • Example 41 Synthesis of Example compound 36 (Ex-36) (HCl salt) Step A - Synthesis of intermediate Ex-36a
  • Intermediate Int 23b (216 mg, 0.396 mmol), Int 22 (235 mg, 0.417 mmol) and HATU (151 mg, 0.396 mmol) were dissolved in DMF (4 ml) and DIPEA (0.218 ml, 1.250 mmol) was added. After stirring for 5 min, the reaction was quenched with AcOH and concentrated to dryness. The crude product was dissolved with EtOAc and washed with 0.1 M HCl, NaHCO3 saturated aqueous solution and brine.
  • Step D Synthesis of intermediate Ex-36d
  • Intermediate Ex-36c (128 mg, 0.121 mmol) was dissolved in dry DCE (128 ml) under N2.
  • GrubbsII (20.48 mg, 0.024 mmol) was added and the reaction was stirred at 60 °C under N2 for 45 min.
  • SiliaMetS DMT resin (212 mg, 0.121 mmol) was added and stirred at room temperature for 1h, filtered and evaporated to afford Ex-36d.
  • UPLC-MS: [M+H] + 1033.3.
  • Step F Synthesis of intermediate Ex-36f
  • Intermediate Ex-36e (83 mg, 0.084 mmol) and tert-butyl 2,7-diazaspiro[3.5]nonane-7- carboxylate (26.6 mg, 0.117 mmol) were dissolved in dry MeOH (2 ml). AcOH was added to adjust pH to 5. After stirring for 20 min, sodium cyanoborohydride (7.38 mg, 0.117 mmol) was added and stirred at room temperature overnight. Then the reaction mixture was concentrated to dryness. The residue was dissolved with EtOAc and washed with NaHCO3 saturated aqueous solution and brine.
  • Step C Synthesis of intermediate Ex-12c
  • N-ethyl-N-isopropylpropan-2-amine (0.799 ml, 4.59 mmol) was added to the mixture of (S)-2-((tert-butoxycarbonyl)amino)-3-(4-methoxyphenyl)propanoic acid (339 mg, 1.147 mmol) and intermediate Ex-12b (236mg, 1.147 mmol) and 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)- 1,1,3,3-tetramethylisouronium hexafluorophosphate(V) (524 mg, 1.377 mmol) in DCM (9 ml)/DMF (1 ml) at 0°C.
  • Step D Synthesis of intermediate Ex-12d
  • Intermediate Ex-12c (128 mg, 0.296 mmol) was dissolved in DCM (10.00 ml) and then added 4N HCl in dioxane (0.490 ml, 1.958 mmol). After stirring under N2 for 1h, the reaction was concentrated to afford an HCl salt solid.
  • N-ethyl-N-isopropylpropan-2-amine (0.532 ml, 3.05 mmol) was added dropwise to the above HCl salt (341 mg, 0.764 mmol) and(2S,3R)-2-((tert- butoxycarbonyl)amino)-3-methoxybutanoic acid (178 mg, 0.764 mmol)and HATU (348 mg, 0.916 mmol) in DMF (1 ml) / DCM (4 ml) at 0°C and allowed to warm up to room temperature and stirred for overnight. The mixture was partitioned between EtOAc and water.
  • N-ethyl-N-isopropylpropan-2-amine (0.362 ml, 2.081 mmol) was added to a mixture of Int 28 (241 mg, 0.520 mmol) and the above formed HCl salt of Ex-12e and HATU (237 mg, 0.624 mmol) in DCM (1.000 ml)/ DMF (1ml) at 0°C and allowed to warm up to rt and stirred for 4h. The reaction was quenched with water and extracted with EtOAc. The mixture was washed with water (x2). The org layer was separated and dried over MgSO4, filtered, and concentrated.
  • Step G Synthesis of intermediate Ex-12g
  • Intermediate Ex-12f 400 mg, 0.392 mmol in anhydrous CH2Cl2 (100ml) was added dropwise to a solution of Zhan catalyst (115 mg, 0.157 mmol) in anhydrous DCM (800 ml) in 90 min and the resulting reaction mixture was stirred at room temperature under an atmosphere of nitrogen for a period of 3h then was heated at 40°C for 3h. After stirring at rt overnight, more Zhan catalyst (0.4eq) was added and heated at 45°C for 3 hr then was stirred at RT overnight.
  • Step J Synthesis of intermediate Ex-12j [0513] N-ethyl-N-isopropylpropan-2-amine (0.036 ml, 0.207 mmol) was added to the mixture of Ex-12i (43 mg, 0.052 mmol) and (S)-3-(3-allylphenyl)-2-((tert- butoxycarbonyl)amino)propanoic acid (15.80 mg, 0.052 mmol) and 2-(3H-[1,2,3]triazolo[4,5- b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate(V) (23.61 mg, 0.062 mmol) in DCM (1 ml)/DMF (1ml) and stirred at rt overnight.
  • Step K Synthesis of intermediate Ex-12k
  • Intermediate Ex-12j (57 mg, 0.051 mmol) was dissolved in anhydrous CH2Cl2 (10ml) and to a solution of Zhan catalyst(1,3-dimesitylimidazolidin-2-ylidene)(5-(N,N- dimethylsulfamoyl)-2-isopropoxybenzylidene)ruthenium(VI) chloride (14 mg, 0.019 mmol) in anhydrous DCM (80 ml) was added dropwise in 30minx3 and the resulting reaction mixture was stirred at room temperature under an atmosphere of nitrogen for a period of 3h.
  • Zhan catalyst(1,3-dimesitylimidazolidin-2-ylidene)(5-(N,N- dimethylsulfamoyl)-2-isopropoxybenzylidene)ruthenium(VI) chloride 14 mg, 0.019 mmol
  • anhydrous DCM 80 ml
  • Step M Synthesis of Example Ex-12 [0516] To a flask containing intermediate Ex-12l (20 mg, 0.018 mmol) in DCM (2 ml) was added N-ethyl-N-isopropylpropan-2-amine (0.013 ml, 0.072 mmol) followed by N- acetoxysuccinimide (4.27 mg, 0.027 mmol). The reaction was stirred at room temperature for overnight. EtOAc and water were added to the reaction. The aqueous layer was extracted with EtOAc. The combined organic layer was washed with Brine, dried over Na 2 SO 4 , and concentrated in vacuo to give a solid. The crude was sent to HTP for purification to afford Example Ex-12 as a solid.
  • Example 44 Synthesis of Example compound 29 (EX-29) Step A - Synthesis of intermediate Ex-29 [0517] N-ethyl-N-isopropylpropan-2-amine (2.64 ml, 15.17 mmol) was added to the mixture of 2-allylpyrrolidine hydrochloride (560 mg, 3.79 mmol) and BOC-METYR(ME)-OH DCHA (2047mg, 4.17 mmol) and 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3- tetramethylisouronium hexafluorophosphate(V) (1731 mg, 4.55 mmol) in DMF (1 ml) / DCM (10 ml) at 0°C.
  • N-ethyl-N-isopropylpropan-2-amine (1.071 ml, 6.15 mmol) was added to the mixture of TFA salt of N-(tert-butoxycarbonyl)-O-methyl-L-threonine (395 mg, 1.692 mmol) and the TFA salt of Ex-29a (619 mg, 1.538 mmol) and 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)- 1,1,3,3-tetramethylisouronium hexafluorophosphate(V) (643 mg, 1.692 mmol) in DMF (1 ml) / DCM (10 ml) at 0°C.
  • N-ethyl-N-isopropylpropan-2-amine (0.995 ml, 5.71 mmol) was added to a mixture of the above obtained TFA salt of Ex-29b and (2S,3S)-1-(tert-butoxycarbonyl)-3- hydroxypyrrolidine-2-carboxylic acid (396 mg, 1.713 mmol) and 2-(3H-[1,2,3]triazolo[4,5- b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate(V) (651 mg, 1.713 mmol) in DCM (10 ml)/DMF (1 ml) at 0°C.
  • Step E Synthesis of intermediate Ex-29e
  • Intermediate Ex-29d 350 mg, 0.394 mmol
  • anhydrous CH2Cl2 25ml
  • Zhan catalyst(1,3-dimesitylimidazolidin-2-ylidene)(5-(N,N- dimethylsulfamoyl)-2-isopropoxybenzylidene)ruthenium(VI) chloride 116 mg, 0.157 mmol
  • anhydrous DCM 800 ml
  • 30minx3 anhydrous DCM
  • Example 45 Synthesis of Example compound 18 (Ex-18) Step A - Synthesis of intermediate Ex-18a [0526] To the solution of intermediate Ex-12b (1.105 g, 3.90 mmol) and N-BOC-N-METHYL- 4-METHOXY-L-PHENYLALANINE (1.568 g, 5.07 mmol) in DMF (10 ml) was added HATU (1.928 g, 5.07 mmol) and DIEA (4.09 ml, 23.40 mmol). After stirring at rt overnight, the solution was partitioned between EtOAc (200 mL) and brine (100 mL).
  • Step C Synthesis of intermediate Ex-18c
  • N-ethyl-N-isopropylpropan-2-amine (0.680 ml, 3.91 mmol) was added to a mixture of intermediate Ex-18b and (2S,3S)-1-(tert-butoxycarbonyl)-3-hydroxypyrrolidine-2-carboxylic acid (271 mg, 1.172 mmol) and 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3- tetramethylisouronium hexafluorophosphate(V) (446 mg, 1.172 mmol) in DCM (10 ml)/DMF (1 ml) at 0°C and allowed to warm up to rt and stirred for 4h.
  • N-ethyl-N-isopropylpropan-2-amine (0.251 ml, 1.440 mmol) was added to the mixture of above HCl salt and intermediate Int 29 and 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3- tetramethylisouronium hexafluorophosphate(V) (164 mg, 0.432 mmol) in DCM (1 ml)/DMF (1ml) and stirred at rt overnight. The mixture was partitioned between EtOAc and water. The organic phase was separated, washed with water (X2), dried (MgSO4) and the volatiles removed under reduced pressure.
  • Intermediate Ex-18d (194 mg, 0.188 mmol) in anhydrous CH2Cl2 (100ml) was added dropwise to a solution of Zhan catalyst(1,3-dimesitylimidazolidin-2-ylidene)(5-(N,N- dimethylsulfamoyl)-2-isopropoxybenzylidene)ruthenium(VI) chloride (138 mg, 0.188 mmol) in anhydrous DCM (400 ml) over 30minx3 and the resulting reaction mixture was stirred at room temperature under an atmosphere of nitrogen for a period of 3h.
  • Zhan catalyst(1,3-dimesitylimidazolidin-2-ylidene)(5-(N,N- dimethylsulfamoyl)-2-isopropoxybenzylidene)ruthenium(VI) chloride 138 mg, 0.188 mmol
  • anhydrous DCM 400 ml
  • Step H – Synthesis of intermediate Ex-18h [0534] The crude intermediate Ex-18g above and Int 5 were dissolved in DMF (657 ⁇ l) with HATU (30.0 mg, 0.079 mmol). The reaction was evacuated and purged with N2. DIEA (41.3 ⁇ l, 0.236 mmol) was added and the reaction was stirred at ambient temperature for 90 minutes. The reaction was diluted with ethyl acetate and washed with 10% aq LiCl solution. The organics were dried with sodium sulfate, filtered, and concentrated.
  • Example 46 Synthesis of Example compound 23 (Ex-23) Step A – Synthesis of intermediate Ex-23a [0536] Intermediate Ex-18g (30mg, 0.031 mmol) and Int 30 were dissolved in DMF (44.7 ⁇ l) with HATU (14.27 mg, 0.038 mmol). The reaction was evacuated and purged with N2.
  • Intermediate Ex-23a (10 mg, 8.97 ⁇ mol) in anhydrous CH2Cl2 (1.5 ml) was added dropwise to a solution of Zhan catalyst(1,3-dimesitylimidazolidin-2-ylidene)(5-(N,N- dimethylsulfamoyl)-2-isopropoxybenzylidene)ruthenium(VI) chloride (2.63 mg, 3.59 ⁇ mol) in anhydrous DCM (13.500 ml) over 15 min and the resulting reaction mixture was stirred at room temperature under an atmosphere of nitrogen for 6h. The reaction mixture was filtered through celite and then removed solvent under reduced pressure.
  • Example 47 Synthesis of Example compounds 15 and 16 (Ex-15 and Ex-16) Step A: Synthesis of Ex-15a [0538] To a stirred solution of Int 33 (200 mg, 0.62 mmol) in DCM (2 mL) was added (E)-tert- butyl N,N'-diisopropylcarbamimidate (619 mg, 3.09 mmol) at room temperature. The reaction solution was stirred at 50 °C for 3 h. The resulting solution was quenched with water (30 mL) and extracted with EA (3 x 60 mL). The combined organic layer was washed with brine (3 x 50 mL), dried over anhydrous Na 2 SO 4, and filtered.
  • E tert- butyl N,N'-diisopropylcarbamimidate
  • Step B Synthesis of Ex-15b [0539] To a solution of Ex-15a (150 mg, 0.39 mmol) in THF (1.5 mL) was added HCl (4 M) in 1,4-dioxane (1.5 mL). The reaction solution was stirred for 4 h at room temperature. The resulting solution was concentrated under reduced pressure to afford Ex-15b hydrochloride salt as an oil. LCMS 280.3 –M - HCl + H] + .
  • Step C Synthesis of Ex-15c [0540] To a stirred solution of (S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoic acid (76.0 mg, 0.350 mmol) in DMF (2 mL) were added HATU (160 mg, 0.420 mmol), Ex-15b (110 mg, 0.35 mmol) and DIEA (0.24 mL, 1.40 mmol) at 0 °C under nitrogen atmosphere. The reaction solution was stirred at room temperature for 4 h. The resulting solution was quenched with water (50 mL), extracted with EA (3 x 50 mL).
  • Step F Synthesis of Ex-15f [0543] To a solution of Intermediate F-4 (50.0 mg, 0.085 mmol) in DMF (1 mL) was added NaH (3.56 mg, 0.089 mmol, 60% in mineral oil) at -10 °C. The reaction solution was stirred at - 10 °C for 0.5 h. Then intermediate Ex-15e (45.6 mg, 0.093 mmol) was added to the solution at - 10 °C. The reaction solution was stirred at -10 °C for 2 h.
  • Step G Synthesis of Ex-15g [0544] To a stirred solution of Ex-15f (30.0 mg, 0.03 mmol) in THF (1 mL) and water (0.1 mL) was added LiOH (2.16 mg, 0.09 mmol) at 0 °C. The reaction solution was stirred at room temperature for 16 h. The pH value of the solution was adjusted to 5 with 1 N HCl and the resulting solution was lyophilized to afford Ex-15g as a solid.
  • Step H Synthesis of Ex-15h [0545] To a stirred solution of Ex-15g (400 mg, 0.410 mmol) in DMF (5 mL) were added HATU (170 mg, 0.450 mmol), (S)-tert-butyl 2-((S)-2-amino-N,3-dimethylbutanamido)-3-(4- methoxy-3-methylphenyl)propanoate hydrochloride (185 mg, 0.450 mmol) and DIEA (0.28 mL, 1.62 mmol) at 0 °C under nitrogen atmosphere. The reaction solution was stirred at room temperature for 2 h.
  • Step I Synthesis of Ex-15i [0546] To a stirred solution of Ex-15h (200 mg, 0.150 mmol) in DCM (3 mL) was added TFA (3 mL) at room temperature. The reaction solution was stirred at room temperature for 2 h. The solution was concentrated under reduced pressure and co-evaporated with toluene and DCM. The solution was dissolved in DCM (2 mL) and treated with HCl (4 N) in dioxane (0.3 mL). The solution was concentrated under reduced pressure. The residue was re-dissolved in ACN (2 mL) and water (5 mL), and 1 N HCl (0.2 mL) added at 0 °C, and lyophilized to afford Ex-15i as a solid.
  • Step K Synthesis of Ex-15 and Ex-16 [0548]
  • Example 48 Synthesis of Example compound 44 (Ex-44) (HCl salt) [0549]
  • Compound Ex-44 was prepared from Ex-27and Int 22 by applying the synthetic procedure used for Ex-41. [0550] The crude cyclic peptide was purified by preparative reversed-phase high performance liquid chromatography (RP-HPLC) [Luna C18 (Phenomenex) (column size 30 * 250 mm, 100 ⁇ , 5 ⁇ m).
  • Example 49 Synthesis of Example compound 30 (Ex-30) Step A-Synthesis of Ex-30a [0551] To a stirred solution of Intermediate F-4 (200 mg, 0.339 mmol) in DMF (4 mL) were added Cs2CO3 (331 mg, 1.016 mmol) and tert-butyl (3-bromopropyl)(methyl)carbamate (256 mg, 1.016 mmol) at 25 °C under nitrogen atmosphere. The reaction solution was stirred at 50 °C for 2 h. The reaction was quenched with water (60 mL).
  • Step D Synthesis of Ex-30d
  • Pd-C 192 mg, 0.180 mmol, 10%
  • the mixture was degassed with hydrogen for 3 times at room temperature for 1 h.
  • the solid was filtered out and the filtrate was concentrated under reduced pressure to afford 120 mg of Ex-30d as a solid.
  • Example 50 Synthesis of Example compounds 55A, 55B, and 56
  • the structures of compounds 55A, 55B, and 56 are as follows. These compounds were synthesized using the following reaction scheme. Step 1: Synthesis of Int-55-1 [0561] To a 40- ml vial with a magnetic stir bar, tert-butyl 4-(hydroxymethyl)piperidine-1- carboxylate (Aldrich) (500 mg, 2.322 mmol) and SODIUM HYDRIDE (60% in oil) (139 mg, 3.48 mmol) was charged dry THF (11.6 ml) under N 2 atmosphere. The mixture was stirred at room temperature for about 5 min or until bubbling ceased.
  • Aldrich 500 mg, 2.322 mmol
  • SODIUM HYDRIDE 50% in oil
  • ALLYL BROMIDE (322 ⁇ l, 3.72 mmol) was added. The mixture was stirred at room temperature for about 1h. LCMS check showed desired product, but the reaction was not complete. The mixture was left stirring at room temperature overnight. LCMS check showed desired product as the major product. [0562] The resulting reaction mixture was quenched with addition of MeOH ( ⁇ 5 ml), and concentrated in vacuo. The residue was purified by normal phase chromatography (Isco system using 40 g ISCO RediSep silica gold column and eluted with 0-100% EtOAc/hexane).
  • Step 2 Synthesis of Int-55-2 [0563] To a stirred solution of tert-butyl 4-((allyloxy)methyl)piperidine-1-carboxylate (Int-1, 520 mg, 2.036 mmol) in CH2Cl2 (17 ml) was added TFA (18.8 ml, 244 mmol). The mixture was stirred at room temperature for about 65 min. LCMS check showed reaction completed. The mixture was concentrated.
  • Step 3 Synthesis of Int-55-3 [0564] To a stirred solution of (S)-2-((2S,3R)-2-((tert-butoxycarbonyl)amino)-3-methoxy-N- methylbutanamido)-3-(4-methoxyphenyl)propanoic acid (155 mg, 0.365 mmol) and HATU (Aldrich) (146 mg, 0.383 mmol) in DMF (3651 ⁇ l) were added DIEA (Aldrich) (255 ⁇ l, 1.461 mmol) and 4-((allyloxy)methyl)piperidine HCl (Int-55-2, 77 mg, 0.402 mmol) at 0 o C.
  • DIEA Aldrich
  • reaction mixture was then concentrated and purified by normal phase chromatography using 40 g Isco silica gel gold column and 0-100% EtOAc-EtOH (3:1)/hexane as eluent to give the desired product methyl (12S,13S,9S,12S)-12-((1-allyl-5-fluoro- 1H-indol-3-yl)methyl)-9-((tert-butoxycarbonyl)amino)-4,10,13-trioxo-2-oxa-5,11-diaza-1(3,1)- pyrrolidina-7(1,3)-benzenacyclotridecaphane-12-carboxylate (Int-55-5, 1861 mg) a solid.
  • Example 51 Synthesis of Example compounds 57, 58, and 59 The structures of Example Compounds 57, 58, and 59 are: These compounds were synthesized as described below.
  • Example compound 57 [0585] To a solution of compound Int-57-10 (30 mg, 0.029 mmol) and DIPEA (0.020 ml, 0.114 mmol) in DMF (2ml) and CH 2 Cl 2 (40ml) at r.t. was added HATU (13.02 mg, 0.034 mmol). The resulting solution was stirred at r.t. for 1h. LCMS showed that the desired mass was present.
  • reaction mixture was purified by preparative HPLC reverse phase (SunFire C-18, 19x150mm), and eluted with Acetonitrile/Water + 0.05%TFA (5-80% Acetonitrile in water) to yield tert-butyl ((R)-1- ((R)-3-hydroxypyrrolidin-1-yl)-3-(4-methoxyphenyl)-1-oxopropan-2-yl)carbamate as a solid (exact mass of 364.20).
  • a solution containing 40 nM biotinylated PCSK9 + 10 nM Lance ULight Streptavidin is made in 50 mM HEPES pH 7.4, 0.15 M NaCl, 5 mM CaCl2, 0.01% BSA, and 0.01% Surfactant P20.
  • a separate solution containing 40 nM rhLDLR-6 ⁇ His+10 nM Eu-W1024 anti-6 ⁇ His is made in the same buffer system.
  • An Echo is used to transfer 0.750 ⁇ l of compound to an assay plate followed by the addition of 15 ⁇ l of PCSK9+Ulight and 15 ⁇ l of LDLR+Eu.
  • the final assay volume is 30.750 ⁇ l containing 20 nM PCSK9, 5 nM Ulight, 20 nM LDLR, and 5 nM Eu.
  • the reaction is incubated at room temperature for at least two hours prior to fluorescence measurements using an Envision Multilabel Reader.1050 values are determined by fitting data to a sigmoidal dose- response curve using nonlinear regression.
  • Counts (B-counts) of the europium-labeled LDLR are followed to observe if compounds are adversely affecting LDLR. A fall off of the B-counts is likely indicates a false positive of inhibition.
  • AF AlexaFluor647
  • a separate solution containing 40 nM of the AlexaFluor tagged cyclic peptide is made in the same buffer system.
  • An Echo is used to transfer 0.750 ⁇ l of compound to an assay plate followed by the addition of 15 ⁇ l of PCSK9+Stept-Eu and 15 ⁇ l of AF peptide.
  • the final assay volume is 30.750 ⁇ l containing 0.5 nM PCSK9, 1.25 nM Strep-Eu, and 20 nM AF cyclic peptide.
  • the reaction is incubated at room temperature for at least two hours prior to fluorescence measurements using an Envision Multilabel Reader.
  • IC 50 values are determined by fitting data to a sigmoidal dose- response curve using nonlinear regression. Ki is then calculated from the IC 50 and the K D of AF cyclic peptide.
  • the peptide was synthesized on a 0.250 mmol scale on CEM Liberty Blue, Microwave synthesizer using Fmoc/tBu chemistry on PS Rink-Amide MBHA resin, 0.32 mmol g n, .
  • the assembly was performed using single couplings using 4 eq of Fmoc protected amino acid 0.2M in DMF, 4 eq of 0.5M HATU in DMF, 4 eq of 2M DIPEA (double coupling for Tyr). Fmoc deprotection cycles were performed using 20% (V/V) piperidine in DMF.
  • the sequence of Fmoc protected amino acids and building blocks used are: 1.
  • Step B Synthesis of Intermediate Compound Int-B: As Described for Reagent B [0610] Purified by RP-HPLC (Waters Deltapak C4, double cartridge, 40 ⁇ 100 mm, 15 ⁇ m, 300 ⁇ ; 15% to 35% ACN/water+0.1% TFA modifier over 20 min). Collected fractions lyophilized to afford 35 mg of Intermediate Compound Int-B. LCMS Anal.
  • a solution containing 1 nM biotinylated PCSK9 + 2.5 nM Lance Streptavidin Europium (Strep-Eu) is made in 50 mM HEPES pH 7.4, 0.15 M NaCl, 5 mM CaCl2, 0.01% BSA, and 0.01% Surfactant P20.
  • a separate solution containing 1920 nM of the AlexaFluor tagged cyclic peptide is made in the same buffer system.
  • An Echo is used to transfer 0.075 ⁇ l of compound plus 0.675 ⁇ l of DMSO to each well of an assay plate followed by the addition of 15 ⁇ l of PCSK9+Stept-Eu and 15 ⁇ l of AF peptide.
  • the final assay volume is 30.750 ⁇ l containing 0.5 nM PCSK9, 1.25 nM Strep-Eu, and 960 nM AF cyclic peptide.
  • the reaction is incubated at room temperature for at least two hours prior to fluorescence measurements using an Envision Multilabel Reader.
  • IC 50 values are determined by fitting data to a sigmoidal dose-response curve using nonlinear regression. Ki is then calculated from the IC 50 and the K D of AF cyclic peptide.
  • Reagent B was prepared by the following procedure.
  • the peptide was synthesized on a 0.250 mmol scale on CEM Liberty Blue, Microwave synthesizer using Fmoc/tBu chemistry on PS Rink-Amide MBHA resin, 0.32 mmol g n, .
  • the assembly was performed using single-couplings using 4 eq of Fmoc protected amino acid 0.2M in DMF, 4 eq of 1M Oxyme in DMF, 4 eq of 0.5M N,N-diisopropylcarbodiimide (DIC) (double coupling for Y01).
  • Fmoc deprotection cycles were performed using 20% (V/V) piperidine in DMF.
  • the peptide was cleaved from solid support using 50 ml of TFA solution (v/v) (91% TFA, 5% H2O, 4% TIPS) for approximately 1.5 hours, at room temperature. The resin was filtered, washed with TFA and solution concentrated to dryness and lyophilized. Lyophilization afforded Intermediate Compound Int. A (300 mg), which was used as crude in the next step. LCMS anal. calculated. C63H79F2N15O13S2: 1356.53, found: 1356.9 (M+1) + .
  • Step B Synthesis of Intermediate Compound Int-B [0617] Crude Int-A (0.22 mmol) was redissolved in 24 ml of DMF.6 ml of 1M aqueous solution of sodium bicarbonate was added to raise the pH to 7. Then 0.26 mmol of 1,3- bis(bromomethyl)benzene (0.1 M in DMF) was added dropwise.
  • Step C Synthesis of Compound Reagent B [0618] Intermediate Compound Int-B (15 mg) was dissolved in 0.2 ml of dry DMSO. Then 15 mg of ALEXAFLUOR 647NHS Ester (A37566, Life technology) dissolved in 1.5 ml of dry DMSO was added.20 ⁇ L of dry DIPEA was added.
  • PAMPA Parallel Artificial Membrane Passive Permeability
  • MS analysis was performed on a Waters I-Class Acquity Ultra High-Pressure Liquid Chromatography system (UHPLC) coupled with a Waters Xevo TQ-S triple quadrupole mass spectrometer.
  • the gradient was held at 98% A for 0.1 minutes then ramped linearly to 90% mobile phase B over 0.9 minutes, held for 0.5 minutes before returning to the initial conditions.
  • a Waters HSS T32.1x50 mm 1.8 ⁇ m particle size column was used and held at 50 oC. Parent to product transitions monitored m/z 776.2 to m/z 678.4 and m/z 523.9 to m/z 784.5 for MK-0616 and the angiotensin II internal standard, respectively.
  • a MK-0616 standard curve (1.6nM- 5 uM) and was prepared by dilution of 2mM DMSO stock into donor buffer using a Hewlett Packard D300 system. Initial, acceptor, donor and standard curve plates were matrix matched and a 4x volume of internal standard solution added (250ng/mL Angiotensin II in acetonitrile).
  • MK-0616 concentration data was imported into BioAssay (Perkin Elmer) for calculation of P eff and percentage recovery.
  • HRUZ ⁇ RTeZgZej was determined by liquid scintillation counting in a MicroBeta Wallac Trilux scintillation counter.

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Abstract

Des composés de formule (I), ou un sel pharmaceutiquement acceptable de ceux-ci (I), où A, B, R1, R2, R3, R4 et<sp />R5 sont tels que définis dans la description. Ces composés sont des antagonistes de PCSK9 et ont une perméabilité passive (ou intrinsèque) élevée de telle sorte qu'ils peuvent être utilisés sans agent d'amélioration de la perméabilité. L'invention concerne également des compositions pharmaceutiques comprenant les composés de formule I ou leurs sels, et des méthodes de traitement de maladies cardiovasculaires et d'états liés à l'activité du PCSK9, par exemple l'athérosclérose, l'hypercholestérolémie, la coronaropathie, le syndrome métabolique, le syndrome coronarien aigu ou les maladies cardiovasculaires et cardiométaboliques associées.
PCT/US2024/034113 2023-06-15 2024-06-14 Composés antagonistes de pcsk9 perméables passifs Pending WO2024259310A2 (fr)

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JOP20190150A1 (ar) * 2018-06-21 2019-12-21 Merck Sharp & Dohme مركبات مناهضة لـ pcsk9
ES3015132T3 (en) * 2018-06-21 2025-04-29 Ucb Holdings Inc Cyclic polypeptides for pcsk9 inhibition
US11306125B2 (en) * 2018-06-21 2022-04-19 Merck Sharp & Dohme Corp. PCSK9 antagonists bicyclo-compounds

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