Prodrugs of betahistine and their use in therapy

Abstract

Herein is disclosed prodrugs of betahistine such as a compound has Formula (I): or a pharmaceutically acceptable salt thereof, wherein A is -C(=O)-R1, and R1 is a C1-C30 linear or branched organic group, optionally including one or more heteroatoms selected from O, N, S, and P. Also provided are methods of using the compound to treat various neurological and psychiatric conditions.

Description

[0001] NOVEL PRODRUGS OF BETAHISTINE

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to U.S. Provisional Application Serial No. 63 / 687,954, filed on August 28, 2024, the content of which is incorporated herein by reference in its entirety.

[0004] FIELD OF THE INVENHON

[0005] The present invention relates to novel prodrugs of betahistine. The present invention further relates to pharmaceutical compositions of said derivatives and methods of treating various neurological disorders.

[0006] BACKGROUND OF THE INVENTION

[0007] Obsessive-compulsive disorder (OCD) is a chronic and highly debilitating neuropsychiatric disorder characterized by compulsions and / or obsessions. An obsession is a thought, feeling, or image that is of an intrusive, distressing, or of repetitive nature. Compulsions (also sometimes called rituals) are repetitive behaviors that an individual feels compelled to perform in response to their obsessions, and can often be irrational or clearly excessive, difficult to control, and stereotyped in nature.

[0008] Betahistine is employed in the therapeutic management of vertigo and Meniere's disease. It has been postulated that betahistine may also be useful to treat some neurological and neuropsychiatric conditions, including OCD. Betahistine is a partial Hl receptor agonist and a H3 receptor inverse agonist / antagonist which is thought to function by enhancing blood flow within the stapedial artery in the inner ear, consequently reducing vestibular pressure and mitigating symptoms such as vertigo and tinnitus. However, the clinical use of betahistine is not without limitations. Its relatively short half-life necessitates multiple daily doses, and it may cause gastrointestinal side effects in certain patients. Thus, there remains a need in derivatives of betahistine with improved pharmacological properties and bioavailability.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention is directed to derivatives of betahistine, which are prodrug molecules with improved pharmacological properties.

[0011] 1

[0012] 20300-WO-PCT / / 103241-016PCT In an embodiment, provided is a compound having Formula I:

[0013] Formula I, or a pharmaceutically acceptable salt thereof, wherein A is -C(C=O)R1, and R1is a C1-C30 linear or branched organic group, optionally including one or more heteroatoms selected from O, N, S, and P.

[0014] Also provided is a compound having Formula II:

[0015] Formula II, or a pharmaceutically acceptable salt thereof, wherein:

[0016] (i) n=0, and B is

[0017] (a) -CH(NH2)-CH2-S-S-CH2-CH(NH2)- wherein one of both amino groups can be optionally protected an acetyl (Ac), tert-butyloxycarbonyl (Boc), or benzyloxycarbonyl (Cbz) group, or

[0018] (b) -O-(Ci-C6alkylene)-O- or (c) -O-(Ci-C6alkylene)-O-C(=O)-O-(Ci-C6alkylene)-O-, or

[0019] Also provided is a compound having Formula III: or a pharmaceutically acceptable salt thereof, wherein

[0020] 2

[0021] 20300-WO-PCT / / 103241-016PCT R90is H, phenyl, or C1-C3 alkyl.

[0022] Also provided is a c compound having Formula IV: Formula IV, or a pharmaceutically acceptable salt thereof, wherein

[0023] R is:

[0024] (i) -C(R62a)(R62b)-R91, wherein R62aand R62bare independently H or C1-C3 alkyl, R91is an unsaturated cyclic carbonate ester, optionally substituted with C1-C3 alkyl;

[0025] (ii) -C(R63a)(R63b)-NR92-C(=O)-phenyl, wherein the phenyl is substituted with -O- CR64aR64b-O-C(=O)-R93, wherein R63a, R63b, R64a, and R64bare independently H or C1-C3 alkyl, and R93is Ci-C& alkyl; or

[0026] (iii) OH or Ci-Ce alkoxy.

[0027] In another embodiment, provided is a method of treating an obsessive-compulsive related disorder (OCRD), including administering to a subject in need of such treatment, a therapeutically effective amount of a compound according to one or more of Formula I, Formula II, Formula III, or Formula IV, or a pharmaceutically acceptable salt thereof.

[0028] In another embodiment, provided is a method of treating a tic disorder, including but not limited to Tourette’s Syndrome, wherein the method includes administering to a subject in need of such treatment, a therapeutically effective amount of a compound according to one or more of Formula I, Formula II, or Formula III, Formula IV, or a pharmaceutically acceptable salt thereof.

[0029] In another embodiment, provided is a method of treating autism spectrum disorder including administering to a subject in need of such treatment an effective amount of a compound according to one or more of Formula I, Formula II, Formula III, Formula IV, or a pharmaceutically acceptable salt thereof.

[0030] In another embodiment, provided is a method of treating a glutamate excitotoxicity related disorder including administering to a subject in need of such treatment an effective amount of a compound according to one or more of Formula I, Formula II, Formula III, Formula IV, or a pharmaceutically acceptable salt thereof.

[0031] 3

[0032] 20300-WO-PCT / / 103241-016PCT DETAILED DESCRIPTION OF THE INVENTION

[0033] The following detailed description is provided to aid those skilled in the art in practicing the present invention. Exemplary embodiments will hereinafter be described in detail. However, these embodiments are only exemplary, and the present disclosure is not limited thereto but rather is defined by the scope of the appended claims. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present disclosure. Accordingly, the embodiments are merely described below, by referring to structures and schemes, to explain aspects of the present description.

[0034] Terminology

[0035] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. The term "or" means "and / or." Expressions such as "at least one of," when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

[0036] It is understood that the terms "comprises" and / or "comprising," or "includes" and / or "including" when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description is for describing particular embodiments only and is not intended to be limiting. It will be further understood that the terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0038] As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application. In instances where a term is not specifically defined herein, that term

[0039] 4

[0040] 20300-WO-PCT / / 103241-016PCT is given an art-recognized meaning by those of ordinary skill applying that term in context to its use in describing the present invention.

[0041] The articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article unless the context clearly indicates otherwise. By way of example, "an element" means one element or more than one element.

[0042] “Alkyl” includes both branched and straight chain saturated aliphatic hydrocarbon groups, having the specified number of carbon atoms. For example, Ci-C& alkyl means that the straight or branched chain may include from 1 to 6 carbon atoms. If an R group can be “Ci-Ce alkyl,” the same R group can also optionally be C1-C5 alkyl, C1-C4 alkyl, or C1-C3 alkyl. As would be apparent from the context, in some cases “alkyl” is also intended to encompass internal linking groups which bridge two substituents. Such internal alkyl linking groups may be alternatively referred to as alkylene groups, which are bivalent saturated aliphatic radicals such as in the context of linking groups. An example of an alkylene group is methylene, i.e., -CH2-. Alkyl groups may generally be substituted at one or more positions to replace a hydrogen atom with a different atom or constituent atom of another group.

[0043] An alkoxy group indicates "alkyl-O-", wherein the alkyl group may be the same as described above. Non-limiting examples of the alkoxy group may include a methoxy group, an ethoxy group, a propoxy group, a 2-propoxy group, a butoxy group, a t-butoxy group, a pentyloxy group, and a hexyloxy group.

[0044] “Cycloalkyl” means a monovalent group having one or more saturated rings in which all ring members are carbon. For example, “C4-C7 cycloalkyl” means one or more rings having between 4 to 7 carbon atoms total (by way of non-limiting example, cyclobutyl having 4 carbon atoms, cyclohexyl having 6 carbon atoms, norbornane having 7 carbon atoms as a bridged ring system, and spiro[2.3]hexane having 6 carbon atoms as a spiro system). Cycloalkyl groups may generally be substituted at one or more positions to replace a hydrogen atom with a different atom or constituent atom of another group.

[0045] “Heterocycloalkyl” refers to one or more rings including at least one heteroatom independently selected from S, N, or O. For example, C4-C7 heterocycloalkyl means one or more rings having between 4 and 7 atoms total, where at least one atom is a heteroatom other than C, such as S, N, O, or P. In some embodiments, a heterocycloalkyl may contain one, two, three, or

[0046] 5

[0047] 20300-WO-PCT / / 103241-016PCT four heteroatoms. Heterocycloalkyl groups may generally be substituted at one or more positions to replace a hydrogen atom or lone electron pair with a different atom or constituent atom of another group.

[0048] “Cyclic carbonate ester” refers to a cyclic molecule that has a carbonate group linked to two carbon atoms within the ring structure. “Unsaturated cyclic carbonate ester” is a cyclic carbonate ester having a double bond within the ring.

[0049] Natural amino acids are known to one of ordinary skill in the art and may include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, threonine, tryptophan, tyrosine, and valine.

[0050] As used herein, when specific definition is not otherwise provided, the term "substituted" refers to a group substituted with deuterium, a halogen (-F, -Cl, -Br, -I), a hydroxy group (-OH), an amino group (-NH2), a carboxyl group (-CO2H), a substituted or unsubstituted C1-C10 amine group, a nitro group (-NO2), a C1-C10 alkyl group, a C3-C10 cycloalkyl group, a C6-C12 aryl group, a C1-C10 alkoxy group, a C1-C10 trifluoroalkyl group such as a trifluoromethyl group (-CF3) and the like, or a cyano group (-CN) instead of at least one hydrogen of a substituting group or compound.

[0051] Certain compounds are described herein using a general formula that includes variables, e.g., RCR5. Unless otherwise specified, each variable within such a formula is defined independently of other variables.

[0052] If stereochemistry is not indicated, a name or structural representation includes any stereoisomer or any mixture of stereoisomers. Also encompassed are all isotopic variants having natural or enriched isotopic content such as deuterated compounds or other isotopically enriched forms.

[0053] The prodrug compounds described herein may contain one or more chiral centers, and may therefore include optical isomers, i.e. enantiomers, and diastereomers. The compounds may be obtained as racemic mixtures or enantiomerically pure R and S stereoisomers and pharmaceutically acceptable salts thereof. Cis and trans isomers of alkenes and imines or other double-bonded species are also contemplated. Certain stereoisomers may be obtained by standard techniques known in the art such as asymmetric synthesis, diastereomeric recrystallization, kinetic

[0054] 6

[0055] 20300-WO-PCT / / 103241-016PCT resolution, chromatographic or other separation techniques, etc. Chromatographic techniques may include column chromatography, thin-layer chromatography, and high-performance liquid chromatography (HPLC), and may incorporate chiral columns which generally have a chiral stationary phase. Chromatographic and other separation techniques are well-known in the art and standard procedures may be followed to obtain enantiomerically pure or diastereomerically pure compounds.

[0056] Unless otherwise indicated, any reference to a compound herein by structure, name, or any other means includes pharmaceutically acceptable salts; alternate solid forms, such as polymorphs, solvates, and hydrates, etc.

[0057] "Pharmaceutically acceptable salt" includes derivatives of the disclosed compounds wherein the parent compound is modified by making salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines. For example, acid salts can include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid and the like; as well as those derived from organic acids such as acetic acid, trifluoroacetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxylmaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, mesylic acid, esylic acid, besylic acid, sulfanilic acid, 2- acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethane disulfonic acid, oxalic acid, isethionic acid, H00C-(CH2)n-C00H where n is 0-4, and the like. Lists of additional suitable salts may be found, e.g., in G. Steffen Paulekuhn, et al., Journal of Medicinal Chemistry 2007, 50, 6665 and Handbook of Pharmaceutically Acceptable Salts: Properties, Selection and Use, P. Heinrich Stahl and Camille G. Wermuth Editors. Wiley-VCH, 2002.

[0058] Solvates are compounds formed by solvation, which is the combination of solvent molecules with molecules or ions of the solute. Solvation is an interaction of a solute with the solvent, which leads to stabilization of the solute species in the solution. One may also refer to the solvated state, whereby an ion in a solution is complexed by solvent molecules. The difference in the physical properties of different solvates and polymorphic forms thereof results from different orientation and intermolecular interactions of adjacent molecules in the solid. Polymorphic forms

[0059] 7

[0060] 20300-WO-PCT / / 103241-016PCT of compounds or solvates can be distinguished by X-ray diffraction and by other methods such as, infrared spectroscopy or Raman spectroscopy.

[0061] The term "hydrate" refers to a compound that is associated with water. Typically, the number of water molecules contained in a hydrate of a compound is in a definite ratio to the number of compound molecules in the hydrate. Therefore, a hydrate of a compound may be represented, for example, by the general formula R xI O, wherein R is the compound, and x is a number greater than 0. A given compound may form more than one type of hydrate, including, e.g., monohydrates (x is 1), lower hydrates (x is a number greater than 0 and smaller than 1, e.g., hemihydrates (R O.5H2O) ), and polyhydrates (x is a number greater than 1, e.g., dihydrates (R 2 H2O) and hexahydrates (R 6H2O)).

[0062] The term "polymorph" refers to a crystalline form of a compound (or a salt, hydrate, or solvate thereof). All polymorphs have the same elemental composition. Different crystalline forms usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate. Various polymorphs of a compound can be prepared by crystallization under different conditions.

[0063] A “patient’' or a '‘subject” is a human or non-human animal. The term “non-human animal” includes, for example, vertebrates such as non-human primates, horses, livestock such as sheep and cows, pets such as dogs and cats, and rodents such as mice, rats and guinea pigs. In some embodiments, the subject is a human.

[0064] “Treatment” or “treating” means alleviating, inhibiting, ameliorating, relieving, reducing, and / or slowing a condition or a disease in a subject.

[0065] ’The term "therapeutically effective amount" is the amount sufficient to effect treatment, as defined herein, when administered to a subject in need of such treatment.

[0066] BETAHISTINE PRODRUGS

[0067] In an embodiment, the betahistine prodrug is a compound having the following Formula I, or a pharmaceutically acceptable salt thereof:

[0068] 8

[0069] 20300-WO-PCT / / 103241-016PCT

[0070] Formula I, wherein A is -C^C^-R1, and R1is a C1-C30 or C3-C30 linear or branched organic group, optionally including one or more heteroatoms selected from O, N, S, and P. Preferably, R1includes at least one of O, N, S, or P or at least two of O, N, S, or P. In an embodiment, R1is a C1-C30 linear or branched organic group including N and O.

[0071] In another embodiment of Formula I,

[0072] A may be -C(=O)-O-CR10aR10b-O-Rn, wherein

[0073] R10aand R10bmay independently be H or Ci-C& alkyl, and

[0074] R11may be an ester bond-linked natural amino acid, or an ester bond-linked dipeptide of two natural amino acids, wherein an amino group of the natural amino acid is optionally protected with an acetyl (Ac), tert-butyloxycarbonyl (Boc), or benzyloxycarbonyl (Cbz) group, (e.g., Compounds 28-30, 57, 77, 78, 81). The compounds can be synthesized in accordance with Scheme 3.

[0075] In another embodiment of Formula I,

[0076] A may be -C(=O)-O-CR10aR10b-O-Rn,

[0077] R10aand R10bmay independently be H or Ci-C& alkyl, and

[0078] R11may be an ester bond-linked natural amino acid, wherein an amino group of the natural amino acid is substituted with D, and

[0079] D can be -C(=O)-CR43aR43b-NR44aR44b, wherein R43a, R43b, and R44amay independently be H or Ci-Ce alkyl, and R44bis H, an acetyl (Ac), tert-butyloxy carbonyl (Boc), or benzyloxycarbonyl (Cbz) group, (e.g., Compounds 31, 32, 54-56) The compounds can be prepared in accordance with Scheme 3.

[0080] Alternatively, D can be -C(=O)-CR38aR38b-O-C(=O)-R39, wherein R38a, R38b, and R39may independently be H or Ci-Ce alkyl, (e.g., Compounds 33-40 and 42-53) In an embodiment, the natural amino acid may be proline. The compounds can be prepared in accordance with Scheme 3.

[0081] 9

[0082] 20300-WO-PCT / / 103241-016PCT Alternatively, D may be -C(=O)-CR40aR40b-O-R41, wherein R40aand R40bmay independently be H or Ci-Ce alkyl, and R41is an ester bond-linked natural amino acid, (e.g., Compound 41) The compounds can be prepared in accordance with Scheme 3.

[0083] Alternatively, D may be -C(=O)-O-CR45aR45b-R46, wherein R45aand R45bmay independently be H or Ci-Ce alkyl, and R46is an unsaturated cyclic carbonate ester optionally substituted with one or more Ci-Ce alkyl or C1-C3 alkyl. Examples of the unsaturated cyclic O

[0084] ■ carbonate ester can include 2-oxo-l,3-dioxol groupr7° , wherein R70may be H, Ci-Ce alkyl or C1-C3 alkyl, (e.g., Compound 94) The compounds can be prepared in accordance with Scheme 27.

[0085] In another embodiment of Formula I,

[0086] A may be -C(=O)-O-CR10aR1(,b-O-(C=O)-Rn, wherein:

[0087] R10aand R10bmay independently be H or C1-C3 alkyl, and

[0088] R11may be a peptide bond-linked natural amino acid, a peptide bond-linked dipeptide of two natural amino acids (same or different), or a peptide bond-linked tripeptide of three natural amino acids (same or different), wherein a carboxylic acid group of the natural amino acid is optionally protected as a C1-C4 alkyl ester or a benzyl ester, (e.g., Compounds 22-27, 63, 64, 73, 76)

[0089] Alternatively, R11may be a peptide bond-linked dipeptide of two amino acids, and R11may be -NR35-CR36aR36b-C(=O)-NR37aR37b. R35, R36a, R36b, and R37amay be H or Ci-C6alkyl, and R37bmay be -R95-C(=O)-OR96, wherein R95may be C1-6 alkylene, and R96may be H or Ci-Ce alkyl, (e.g., Compound 25) The compounds can be prepared in accordance with Schemes 7-10.

[0090] Alternatively, R11may be -NR35-CR36aR36b-C(=O)-OR37, wherein R35, R36a, R36b, and R37may independently be H or Ci-Ce alkyl, (e.g., Compounds 62, 63) The compounds can be prepared in accordance with Scheme 7, for example.

[0091] In another embodiment of Formula I,

[0092] A may be -C(=O)-O-CR10aR1(,b-O-(C=O)-Rn, wherein

[0093] R10aand R10bmay independently be H or Ci-C& alkyl, and

[0094] R11may be H or Ci-Ce alkyl, for example isopropyl or / -butyl. (e.g., Compounds 13-16,

[0095] 10

[0096] 20300-WO-PCT / / 103241-016PCT 96) Examples of methods to prepare the compounds can be found in Scheme 2 or 28.

[0097] Alternatively, R11may be -R20-R21, wherein R20may be Ci-Cr> alkylene, and R21may be

[0098] Cs-Cio heterocycloalkyl with S and N ring atoms. An example (e.g., Compound 99) The compounds can be synthesized in accordance with Scheme 3.

[0099] Alternatively, R11may be benzyl or phenyl, wherein said benzyl and phenyl can independently be substituted with -CR22aR22b-O-C(=O)-R23or -OP(=O)(OR24a)(OR24b), wherein R22aand R22bcan independently be H or Ci-C& alkyl, R23may be Ci-Ce alkyl or phenyl, and R24aand R24bcan independently be H or Ci-Ce alkyl, or C1-C3 alkyl, or methyl, (e.g., Compounds 70, 92, 112) The compounds can be prepared in accordance with Schemes 3 or 25.

[0100] In another embodiment of Formula I,

[0101] A may be -C(=O)-O-Rn, wherein

[0102] R11may be Ci-C& alkyl, (e.g., Compounds 17-18) The compounds can be prepared in accordance with Scheme 4.

[0103] Alternatively, R11may be -(Ci-Ce alkylene)n-R25wherein R25can be a cyclic carbonate ester, or a cyclic acetyl, said cyclic carbonate ester and cyclic acetyl optionally substituted with Ci-C6alkyl, C1-C3 alkyl or methyl, and n is 0 or 1. (e.g., Compounds 19, 88 and 102) Examples

[0104] 0A

[0105] H°oi °i of R25includer7°, wherein R70is H, Ci-Ce alkyl or C1-C3 alkyl such as methyl, ’~i“vor O^O

[0106] "AT'W. (e.g., Compounds 19, 88, 102) The compounds may be prepared in accordance with Scheme 5 or 23.

[0107] Alternatively, R11may be -(Ci-Ce alkylene)-R26wherein R26can be a monosaccharide. The monosaccharide of R26can have the chemical formula G>H 12O6. The monosaccharide of R26can have a chemical structure selected from:

[0108] 11

[0109] 20300-WO-PCT / / 103241-016PCT

[0110]

[0111] (e.g., Compound 87) The compounds can be prepared in accordance with Scheme 22.

[0112] Alternatively, R11may be Ci-C& alkyl, C5-C6 cycloalkyl such as cyclopentyl, or C4-C6 heterocycloalkyl such as furanyl, wherein each of Ci-Ce alkyl, C5-C6 cycloalkyl and C4-C6 heterocycloalkyl can be substituted with one or more OH, for example one OH, two OH, or three OH groups, (e.g., Compounds 89, 90, 103, 104, 105) The compounds can be prepared in accordance with Scheme 4.

[0113] Alternatively, R11may be -R70-(OR27)n, wherein R70is a Ci-C& linking group such as an alkylene, n is 1 or 2, and R27is an ester-bonded linked natural amino acid, (e.g., Compound 93) The compounds can be prepared in accordance with Scheme 26.

[0114] Alternatively, R11may be -C-R28aR28b-C(R29a)(R29b)(R30), wherein R28a, R28band R29acan independently be H or Ci-Ce alkyl, R29bis -NR100, wherein R100may be H or an acetyl (Ac), tertbutyloxycarbonyl (Boc), or benzyloxycarbonyl (Cbz) group, and R30may be -C(=O)OR30a, wherein R30amay be H or Ci-Ce alkyl, (e.g., Compounds 106 and 107) The compounds can be prepared in accordance with Scheme 4.

[0115] In an embodiment of Formula I,

[0116] A may be a peptide bond-linked natural amino acid, a peptide bond-linked dipeptide of two natural amino acids (same or different), a peptide bond-linked tripeptide of three natural amino acids (same or different), or a peptide bond-linked cystine, (e.g., Compounds 1-12, 20, 83, 84, 97, 98) Optionally, an amino group of the natural amino acid is protected with an acetyl (Ac), tertbutyloxycarbonyl (Boc), or benzyloxycarbonyl (Cbz) group, (e.g., Compounds 20, 84) The compounds can be synthesized in accordance with Scheme 1 or 20. As illustrated in these schemes, peptide bond-linked means that a carboxyl group and an amine group are connected by a peptide bond (amide).

[0117] In another embodiment of Formula I,

[0118] A may be -C(=O)-CR10aR10b-NRnR12, wherein

[0119] 12

[0120] 20300-WO-PCT / / 103241-016PCT R10aand R10bmay independently be H or Ci-C& alkyl (e.g., CH3),

[0121] R11may be H,

[0122] R12may be -C(=O)-CR13aR13b-NR14aR14b,

[0123] R13aand R13bmay be H,

[0124] R14amay be H, and

[0125] R14bmay be H or Ci-Ce alkyl, (e.g., Compounds 9-11 and other dipeptide analogs) These compounds can be synthesis in accordance with Scheme 1.

[0126] In another embodiment of Formula I,

[0127] A may be -C(=O)-CR10aR10b-NRnR12, wherein

[0128] Ri°a, R10b, and R11may independently be H or Ci-Ce alkyl (e.g., CH3),

[0129] R12may be -C(=O)-CR13aR13b-NR14aR14b,

[0130] R13a, R13b, andR14amay independently be H or Ci-Ce alkyl (e.g., CH3),

[0131] R14bmay be -C(=O)-CR15aR15b-NR16aR16b,

[0132] R15aand R15bmay independently be H or Ci-C& alkyl (e.g., CH3),

[0133] R16amay be H, and

[0134] R16bmay be H or Ci-Ce alkyl. (Compound 12 and other tripeptide analogs) These compounds can be prepared in accordance with Scheme 1.

[0135] In another embodiment of Formula I,

[0136] A can be -C(=O)-R30a-R30b, wherein R30amay be Ci-Ce alkylene, and R30bmay be a monosaccharide, (e.g., Compound 86) The monosaccharide can be the chemical formula CeHnOe and can have a structure as described herein. The compounds can be prepared in accordance with Scheme 21.

[0137] In another embodiment of Formula I,

[0138] A may be -C(=O)-R32-R33, wherein R32may be Ci-C& alkylene, and R33may be phenyl substituted with -O(C=O)R34, and optionally one or more Ci-Ce alkyl, and R34may be Ci-Ce alkyl, (e.g., Compound 101) The compounds can be prepared in accordance with Scheme 29.

[0139] In another embodiment of Formula I,

[0140] A may be -C(=O)-O-CR10aR10b-O-(C=O)Rn, wherein

[0141] R10aand R10bmay independently be H or Ci-C& alkyl (e.g., C1-C3 alkyl), and

[0142] R11may be -(CR12aR12b)n-NR13R14, wherein

[0143] 13

[0144] 20300-WO-PCT / / 103241-016PCT n may be 0 or 1 ,

[0145] R12aand R12bmay independently be H or Ci-Ce alkyl (e.g., CH3),

[0146] R13may be H or Ci-Ce alkyl (e.g., C1-C3 alkyl),

[0147] R14may be -C(=O)-CR15aR15b-NR16aR16b, an acetoxyacetyl group, or -C(=O)-CR47aR47b- O-R80,

[0148] R15aand R15bmay be H,

[0149] R16amay be H, an acetyl (Ac), tert-butyloxycarbonyl (Boc), or benzyloxycarbonyl (Cbz) group,

[0150] R16bmay be H or Ci-Ce alkyl, and

[0151] R47aandR47bmay independently be H or Ci-C& alkyl (e.g., C1-C3 alkyl), and

[0152] R80may be an ester-bonded linked natural amino acid, (e.g., Compounds 31, 32, 58-62) The compounds can be prepared in accordance with Scheme 3 or Scheme 11 .

[0153] In another embodiment of Formula I,

[0154] A may be -C(=O)-O-CR10aR10b-ORn, wherein

[0155] R10aand R10bmay independently be H or Ci-C& alkyl (e.g., C1-C3 alkyl), and

[0156] R11may be -P(=O)(OR12)(OR13), wherein

[0157] R12and R13may independently be H or Ci-C& alkyl such as C1-C3 alkyl . (e.g., Compounds 67-69) The compounds can be prepared in accordance with Scheme 14 or 15.

[0158] In another embodiment, the betahistine prodrug is a compound having Formula II:

[0159] Formula II, or a pharmaceutically acceptable salt thereof, wherein

[0160] (i) n=0, and B can be

[0161] (a) -CH(NH2)-CH2-S-S-CH2-CH(NH2)- wherein one or both amino groups can be optionally protected as an acetyl (Ac), tert-butyloxycarbonyl (Boc), or benzyloxycarbonyl (Cbz) group, or

[0162] 14

[0163] 20300-WO-PCT / / 103241-016PCT (b) -O-(Ci-C6alkylene)-O- or (c) -O-(Ci-C6alkylene)-O-C(=O)-O-(Ci-C6alkylene)-O-, or

[0164] — O — C2-6 alkyl — O —

[0165] O

[0166] (ii) n=l, and B can be I . (e.g., Compounds 21, 85, 91, 108, 109, 110,

[0167] 111) In an embodiment, n is 0 and B is -O-(Ci-C6 alkylene)-O-, for example, n can be 0 and B can be -O-(Ci-C4 alkylene)-O-.

[0168] In another embodiment the betahistine derivative may be the following compound: , or a pharmaceutically acceptable salt thereof, wherein R10aand R10bmay independently be H or Ci-Ce alkyl (e.g., C1-C3 alkyl).

[0169] In another embodiment, the betahistine prodrug is a compound having Formula III or a pharmaceutically acceptable salt thereof, wherein R90may be H, phenyl, Ci-C& alkyl, (e.g., Compound 66) The compounds can be synthesized in accordance with Scheme 13.

[0170] In another embodiment, the betahistine prodrug is a compound having Formula IV: Formula IV, or a pharmaceutically acceptable salt thereof.

[0171] In an embodiment, R can be -P(=O(OR60a)(OR60b), wherein R60aand R60bare independently H or phenyl, (e.g., Compound 65)

[0172] In another embodiment, R is -C(R62a)(R62b)-R91, wherein R62aand R62bmay independently be H or Ci-C& alkyl, and R91may be an unsaturated cyclic carbonate ester such as l,3-dioxol-2- one, optionally substituted with Ci-Ce alkyl.

[0173] In another embodiment, R can be -C(R63a)(R63b)-NR92-C(=O)-phenyl, wherein the phenyl can be substituted with -OCR64aR64bO-C(=O)-R93, wherein R63a, R63b, R64a, and R64bare

[0174] 15

[0175] 20300-WO-PCT / / 103241-016PCT independently H or Ci-Ce alkyl, and R93is Ci-Ce alkyl, (e.g., Compounds 80, 82) The compounds can be synthesized in accordance with Scheme 12, 18, or 19.

[0176] In another embodiment, R can be OH or Ci-Ce alkoxy (e.g., Compounds 71, 72).

[0177] In an embodiment, the betahistine prodrug is a compound selected from the group consisting of the following compounds, or a pharmaceutically acceptable salt thereof:

[0178] 16

[0179] 20300-WO-PCT / / 103241-016PCT

[0180]

[0181] 17

[0182] 20300-WO-PCT / / 103241-016PCT

[0183]

[0184] 18

[0185] 20300-WO-PCT / / 103241-016PCT

[0186]

[0187] 19

[0188] 20300-WO-PCT / / 103241-016PCT

[0189]

[0190] 20

[0191] 20300-WO-PCT / / 103241-016PCT

[0192]

[0193] The betahistine prodrug can also be the following compounds, or a pharmaceutically acceptable salt thereof:

[0194] 21

[0195] 20300-WO-PCT / / 103241-016PCT

[0196]

[0197] 20300-WO-PCT / / 103241-016PCT

[0198]

[0199] 20300-WO-PCT / / 103241-016PCT

[0200]

[0201] 20300-WO-PCT / / 103241-016PCT

[0202]

[0203] 20300-WO-PCT / / 103241-016PCT

[0204] Examples of the pharmaceutically acceptable salts of the compound of Formula (I),

[0205] Formula (II), Formula (III), or Formula (IV) include:

[0206] 26

[0207] 20300-WO-PCT / / 103241-016PCT

[0208] There is an inadvertent typo in Formula (I) of U.S. Provisional Application No.

[0209] 63 / 687,954 filed August 28, 2024, which is the priority document to the present application. In Formula (I), R1should be CR10aR10bNR11R12, not (C=O)CR1(,aR10bNRnR12. In light of the disclosure of the priority document, this inadvertent typo would be obvious to a person of ordinary skill in the art.

[0210] SYNTHETIC SCHEMES

[0211] 20300-WO-PCT / / 103241-016PCT The schemes below show general methodologies for the synthesis of the compounds of the present invention. It should be appreciated that variations of these schemes are contemplated to produce the various compounds described herein. The starting materials useful for making the compounds of the present invention are readily commercially available or can be prepared by those skilled in the art.

[0212] Scheme 1

[0213] Amide and diamide compounds described herein can be prepared according to scheme 1.

[0214] In step 1, betahistine is coupled to a nitrogen protected amino acid (N-PG-aa) with a typical amino acid coupling reagent, for example, BOP or HATU, in the presence of an appropriate base, for example DIPEA or TEA. Step 2 represents the removal of the protecting group (PG) under appropriate conditions (for example TFA to remove Boc or piperidine to remove Fmoc) to produce mono-amide amine analogs. Although not depicted in scheme 1, the amino acid could be a N- protected proline. Additionally, the Ri substituent on the N-PG-aa may need to be protected. For example, the amine of the Ri of lysine can be protected with a Boc group and the alcohol of the

[0215] Ri of N-Boc serine can be protected with a t-butyl group. To produce diamides, appropriately protected amino acids are coupled to the product of step 2 with conventional methods. In step 3, the protecting groups are cleaved under appropriate conditions.

[0216] Scheme 2

[0217] 28

[0218] 20300-WO-PCT / / 103241-016PCT

[0219] Compounds described herein can be synthesized according to scheme 2. Step 1 consists of coupling a chloroalkyl carbonochloridate with 4-nitrophenol. In step 2, the product of step 1 is reacted with a carboxylic acid, optionally in the presence of a reagent such as silver oxide, diisopropylethylamine (DIPEA) or potassium carbonate. In step 3, betahistine reacts with the product of step 2 via nucleophilic substitution to displace the nitrophenol.

[0220] Scheme 3

[0221] Compounds described herein can be synthesized according to scheme 3. Step 1 consists of reacting betahistine with a chloroalkyl carbonochloridate. Step 2 consists of coupling a chloroalkyl carbamate with a carboxylic acid in the presence of a reagent such as silver oxide, diisopropylethylamine (DIPEA) or potassium carbonate. If the R2 group contains functionality that contains a protecting group, it can be removed by conventional methods. For example, a trityl protected thiol can be cleaved with TFA and TIPS or a Boc protected amine can be removed with TFA or HC1.

[0222] Scheme 4

[0223] Compounds described herein can be synthesized according to scheme 4. This consists of reacting betahistine with an alkyl carbonochloridate to form a carbamate. The carbonochloridates can be purchased or prepared from an alkyl alcohol through conventional methods, for example by treatment with phosgene. In some examples, substituents on the R-group would require a protecting group. If this is the case, the product of the reaction above would be treated with

[0224] 29

[0225] 20300-WO-PCT / / 103241-016PCT appropriate conditions to remove the protecting group. For example, a silyl group would be removed with TBAF or a tert-butyl ester would be treated with trifluoroacetic acid to form a carboxylic acid. Additional functionalization on the subsequent alcohol or carboxylic acid can be envisaged. For example, an alcohol can be converted to a carbonochloridate with a reagent such as phosgene and subsequently treated with an amine such as betahistine to form a bis-carbamate analog.

[0226] Scheme 5

[0227] Compounds described herein can be synthesized according to scheme 5. Step 1 consists of reacting an alcohol with 4-nitrophenyl carbonochloridate. Step 2 consists of coupling an alkyl 4-nitrophenyl carbonate with betahistine to form a carbamate.

[0228] Scheme 6

[0229] Compounds described herein can be synthesized according to scheme 6. Chloroalkyl methyl(2-(pyridin-2-yl)ethyl)carbamate can be reacted with carbon dioxide and a primary or secondary amine under basic conditions to provide a carbamoyloxy carbamate.

[0230] Scheme 7

[0231] Compounds described herein can be synthesized according to scheme 7. In step 1, a chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate is reacted with carbon dioxide and an aminoalkanoate under basic conditions to provide a carbamoyloxycarbamate. In step 2, the ester is converted to a carboxylic acid. This can be accomplished under hydrogenation conditions from a benzyl ester, under basic conditions (for example with LiOH / water) from an alkyl ester or under acidic conditions from a tertbutyl ester. Additionally, removal of a protecting group on the R2position may be performed.

[0232] Scheme 8

[0233] 30

[0234] 20300-WO-PCT / / 103241-016PCT

[0235] Compounds described herein can be synthesized according to scheme 8. In step 1, a chloroalkyl methyl(2-(pyridin-2-yl)ethyl)carbamate is reacted with carbon dioxide, a base such as cesium carbonate and a carboxylate protected peptide to provide a carbamoyloxycarbamate. In step 2, the ester is converted to a carboxylic acid. This can be accomplished under hydrogenation conditions from a benzyl ester, under basic conditions (for example with LiOH / water) from an alkyl ester or under acidic conditions from a tert-butyl ester.

[0236] Scheme 9

[0237] Compounds described herein can be synthesized according to scheme 9. Step 1 consists of coupling carboxylic acid (prepared as described in scheme 7) with the nitrogen of a benzyl protected amino acid (for example Val-OBn or Sarocine-OBn). Step 2 consists of removal of the benzyl group with hydrogen and a catalyst such as Pd / C.

[0238] Scheme 10

[0239] Compounds described herein can be synthesized according to scheme 10. In step 1, chloroalkyl methyl(2-(pyridin-2-yl)ethyl)carbamate is reacted with carbon dioxide and proline benzyl ester under basic conditions to provide a carbamoyloxycarbamate. In step 2, under hydrogenation conditions, the ester is converted to the carboxylic acid.

[0240] Scheme 11

[0241] 31

[0242] 20300-WO-PCT / / 103241-016PCT Compounds described herein can be synthesized according to scheme 11. Step 1 consists of a coupling of an amino ester to a chloroalkylchloroformate. In step 2, the resulting chloromethoxycarbonyl is reacted with betahistine in the presence of carbon dioxide and a base such as cesium carbonate.

[0243] Scheme 12

[0244] The reaction in scheme 12 consists of reacting betahistine with diphenyl phosphorochloridate to provide diphenyl methyl(2-(pyridin-2-yl)ethyl)phosphoramidate.

[0245] Scheme 13

[0246] The reaction in scheme 13 consists of reacting betahistine with phenyl phosphorodichloridate to provide methyl((methyl[2-(pyridin-2- yl)ethyl]amino)(phenoxy)phosphoryl) [2-(pyridin-2-yl)ethyl]amine.

[0247] Scheme 14

[0248] Compounds described herein can be synthesized according to scheme 14. Steps 1 consists of treating a methyl-(2-pyridin-2-yl-ethyl)-carbamic acid chloroalkyl ester with phosphoric acid dibenzyl ester in the presence of a base such as potassium carbonate. Step 2 consists of removing the benzyl group under a hydrogen atmosphere in the presence of a catalyst such as with palladium to provide a phosphoric acid.

[0249] Scheme 15

[0250] Compounds described herein can be synthesized according to scheme 15. Methyl-(2- pyridin-2-yl-ethyl)-carbamic acid chloroalkyl ester is reacted with dialkyl hydrogen phosphate in

[0251] 32

[0252] 20300-WO-PCT / / 103241-016PCT the presence of a base such as potassium carbonate to provide a dialkoxyphosphoryloxymethyl carbamate.

[0253] Scheme 16 Compounds described herein can be synthesized according to scheme 16. In step 1, the secondary amine of betahistine is protected with a Boc group. In step 2, the pyridine nitrogen is oxidized with a reagent such as MCPBA. In step 3 the Boc protecting group is removed with acid. Scheme 17 Compounds described herein can be synthesized according to scheme 17. In this example,

[0254] 2-vinylpyridine is reacted with hydroxyl or methyl hydroxyl methyl amine to provide the N-0 analogs of betahistine.

[0255] Scheme 18 Compounds described herein can be synthesized according to scheme 17. Betahistine is coupled with 4-(chloromethyl)-5-methyl-l ,3-dioxol-2-one in the presence of a base such as triethyl amine.

[0256] Scheme 19

[0257] 33

[0258] 20300-WO-PCT / / 103241-016PCT Compounds described herein can be synthesized according to scheme 19. Step 1 consists of a reaction of 2-hydroxybenzamide with iodoalkyl alkylate to provide an intermediate, which in step 2 is reacted with betahistine and formaldehyde to form the aminal linker.

[0259] Scheme 20

[0260] Compounds described herein can be synthesized according to scheme 20. Betahistine is coupled with an acyl cysteine dimer in the presence of appropriate coupling conditions to form the mono- or di-betahistine analog.

[0261] Scheme 21

[0262] Compounds described herein can be synthesized according to scheme 21. In step 1, betahistine is reacted with a hydroxyl substituted alkyl acid or corresponding metal salt in the presence of conventional amino acid coupling conditions such as BOP and DIPEA. Step 2 consists of reacting the alcohol with an appropriately protected alpha-bromo sugar analog such as ((2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate to provide an ether. Step 3 consists of removal of the protecting groups to provide the sugar analog.

[0263] Scheme 22

[0264] 34

[0265] 20300-WO-PCT / / 103241-016PCT

[0266] Compounds described herein can be synthesized according to scheme 22. In step 1 betahistine is reacted with a carbonochloridate such as 4-nitrophenyl carbonochloridate. Step 2 consists of forming a carbamate linkage by a reaction with a dihydroxyl substituted alkyl. In step 3 this product is treated with an appropriately protected alpha-bromo sugar analog such as ((2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate to provide an ether. Step 3 consists of removal of protecting groups.

[0267] Scheme 23

[0268] Compounds described herein can be synthesized according to scheme 23. In step 1 2,2- dimethyl-l,3-dioxan-5-yl carbonochloridate is prepared by treatment of 2,2-dimethyl-l,3-dioxan- 5-ol with triphosgene. In step 2 this product is reacted with betahistine to provide the corresponding carbamate. Removal of the ketal in the presence of acid such as para-toluene sulfonic acid provides l,3-dihydroxypropan-2-yl methyl(2-(pyridin-2-yl)ethyl)carbamate which can be treated with triphosgene to afford 2-oxo-l,3-dioxan-5-yl methyl(2-(pyridin-2-yl)ethyl)carbamate as shown in step 4 of scheme 23.

[0269] Compounds described herein can be synthesized according to scheme 24. In step 1 a diol such as ethane- 1,2-diol is treated with triphosgene to afford a carbonochloridate. In step 2 the

[0270] 35

[0271] 20300-WO-PCT / / 103241-016PCT carbonochloridate is reacted with betahistine to form a carbamate. In step 3 the alcohol is treated with 1,1 -carbonyldiimidazole to provide the carbonate dimer.

[0272] Compounds described herein can be synthesized according to scheme 25. Step 1 consists of reacting 2-(4-((bis(benzyloxy)phosphoryl)oxy)phenyl)acetic acid with chloromethyl methyl(2- (pyridin-2-yl)ethyl)carbamate in the presence of silver oxide to form an intermediate which, in step 2, is subjected to hydrogen atmosphere in the presence of an appropriate catalyst such as Pd-

[0273] C to remove benzyl protecting groups to form the corresponding phosphoric acid.

[0274] Scheme 26

[0275] Compounds described herein can be synthesized according to scheme 26. In step 1 a nitrogen protected amino acid is coupled with l,3-dihydroxypropan-2-yl methyl(2-(pyridin-2- yl)ethyl)carbamate in the presence of a coupling reagent such as DCC and a base such as DMAP to provide a diester. In step 2 the protecting group (PG) is cleaved with reagents such as hydrochloric acid to remove Boc groups or piperidine to remove Fmoc groups.

[0276] Scheme 27

[0277] Compounds described herein can be synthesized according to scheme 27. Step 1 entails the reaction of 4-(hydroxymethyl)-5-methyl-l,3-dioxol-2-one 4-(hydroxymethyl)-5-methyl-l,3- dioxol-2-one with 4-nitrophenyl carbonochloridate in the presence of a base such as pyridine to provide (5-methyl-2-oxo-l,3-dioxol-4-yl)methyl (4-nitrophenyl) carbonate. In step 2 this compound is reacted with an amino ester in the presence of a base such as triethylamine to afford a carbamate. In step 3 the ester is converted to a carboxylic acid. This can be accomplished under hydrogenation conditions from a benzyl ester, under basic conditions (for example with

[0278] 36

[0279] 20300-WO-PCT / / 103241-016PCT LiOH / water) from an alkyl ester or under acidic conditions from a tert-butyl ester. Step 4 consists of the treatment of the carboxylic acid with a chloroalkyl methyl(2-(pyridin-2- yl)ethyl)carbamate.

[0280] Scheme 28

[0281] Compounds described herein can be synthesized according to scheme 28. Betahistine is reacted with a nitrophenoxy)carbonyl)oxy)alkyl alkylate to displace the 4-nitrophenoxyl to provide the betahistine analog.

[0282] Scheme 29

[0283] Compounds described herein can be synthesized according to scheme 29. Step 1 consists of reacting 2-(4-((tert-butyldimethylsilyl)oxy)-2-methylbutan-2-yl)-3,5-dimethylphenol with a carboxylic acid in the presence of a coupling conditions, for example with DCC and DMAP, to provide the corresponding ester. Step 2 consists of removing the silyl group to form the alcohol by treatment with an acid such as acetic acid or with a fluorine source such as TBAF. In step 3 the alcohol is oxidized to the carboxylic acid. This transformation can be accomplished via two steps, by initially forming the aldehyde by treatment of the primary alcohol with an oxidizing reagent such as PCC followed by further conversion of the aldehyde to the carboxylic acid under oxidative conditions such as with NaCICh / EbCh. Alternatively, the alcohol can be directly converted to the carboxylic acid with a reagent such as KMNO4. In step 4 the carboxylic acid is reacted with betahistine under conventional coupling conditions including BOP / DIPEA.

[0284] Scheme 30

[0285] 37

[0286] 20300-WO-PCT / / 103241-016PCT

[0287]

[0288] A method to prepare a trimer of betahistine is described in scheme 30. In step 1, (2,2- dimethyl-l,3-dioxolan-4-yl)methyl carbonochloridate is reacted with betahistine in the presence of base such as DIPEA to form a carbamate. Step 2 consists of removal of the ketal with an acid such as / v / ra-toluenesulfonic acid to form the diol, which is then selectively protected on the less sterically hindered primary alcohol with a protecting group (PG) such a / c / 7-butyldimethylsilyl group. In step 3, the secondary alcohol is functionalized as a carbonochloridate with a reagent such as phosgene and subsequently reacted with betahistine to form the carbamate. The alcohol protecting group is then removed by conventional methods, for example a silyloxyl group can be removed with acid or TBAF, to expose the alcohol. In step 3, the alcohol is converted into the carbonochloridate with triphosgene and then reacted with betahistine to form the carbamate.

[0289] METHODS OF TREATMENT

[0290] Further aspects of the disclosure are methods for treating an obsessive-compulsive related disorder (OCRD) in a subject in need thereof by administration of a prodrug compound according to Formulae I-IV, or a pharmaceutically acceptable salt thereof. The OCRD can include obsessive- compulsive disorder (OCD), hoarding disorder, trichotillomania (hair-pulling disorder), excoriation (skin-picking) disorder, body dysmorphic disorder, substance / medication-induced obsessive-compulsive and related disorder, obsessive-compulsive disorder due to another medical condition.

[0291] Also provided for are methods for treating a glutamate excitotoxicity related disorder in a subject in need thereof by administration of a compound according to Formulae I-IV, or a pharmaceutically acceptable salt thereof. The glutamate excitotoxicity related disorder can include

[0292] 38

[0293] 20300-WO-PCT / / 103241-016PCT amyotrophic lateral sclerosis (ALS), Parkinson’s disease, traumatic brain injury, concussion, postconcussion syndrome, cerebral infarction, stroke, multiple sclerosis, Huntington’s disease, schizophrenia, seizure, epilepsy, epileptic syndrome, status epilepticus, refractory focal epilepsy, autoimmune encephalitis, infectious encephalitis, viral encephalitis, acute disseminated encephalomyelitis, and mitochondrial encephalitis.

[0294] Also provided for are methods for treating autism spectrum disorder in a subject in need thereof by administration of a compound according to Formulae I-IV, or a pharmaceutically acceptable salt thereof.

[0295] Also provided for are methods for treating a tic disorder in a subject in need thereof by administration of a compound according to Formulae I-IV, or a pharmaceutically acceptable salt thereof. The tic disorder can include Tourette’s Syndrome, Tourette’s Disorder, chronic motor tic disorder, chronic vocal tic disorder, transient tic disorder, and substance-induced tic disorder.

[0296] Also provided for are methods for treating Tourette’s Syndrome in a subject in need thereof by administration of a compound according to Formulae I-IV, or a pharmaceutically acceptable salt thereof.

[0297] Methods of treatment can include providing certain dosage amounts of the betahistine prodrug compound to a patient. Dosage levels of each compound of from about 0.1 mg to about 250 mg per kilogram of body weight per day are useful in the treatment of the above-indicated disease or conditions. In an embodiment, the compound or a pharmaceutically acceptable salt thereof can be administered such that the total weekly dose is from 5 to 2000 mg. Frequency of dosage may also vary depending on the compound used and the particular disease treated. For example, the compound or a pharmaceutically acceptable salt thereof can be administered once per day, twice per day, three times per day, or more than three times per day. However, for treatment of most diseases and disorders, a dosage regimen of 4 times daily or less can be used and in certain embodiments a dosage regimen of 1 or 2 times daily is used. In certain embodiments 5 mg to 1000 mg, or 25 mg to 400 mg of a betahistine prodrug compound is provided daily to a patient.

[0298] The betahistine prodrug compound as described herein may be the only active agent administered (monotherapy) or may be combined with one or more other therapeutically active compounds (combination, adjunct, or augmentation therapy). The other therapeutically active

[0299] 39

[0300] 20300-WO-PCT / / 103241-016PCT compound can include a monoamine oxidase inhibitor (MAO-I), a serotonin / norepinephrine dual reuptake inhibitor (SNRI), an antidepressant, a glutamate-modulating agent, a benzothiazole or benzothiazole derivative, a serotonin reuptake inhibitor (SRI), a skeletal muscle relaxant, an antipsychotic agent, an antidepressant, an anticholinergic, an anticonvulsant, a benzodiazepine or benzodiazepine derivative, and any combination thereof.

[0301] The standard routes of administration described by the FDA are contemplated herein as shown in Table 1 below (FDA Routes of Administration; retrieved from the official FDA website)

[0302] Table 1 : FDA Routes of Administration

[0303] 40

[0304] 20300-WO-PCT / / 103241-016PCT

[0305] 41

[0306] 20300-WO-PCT / / 103241-016PCT

[0307] 42

[0308] 20300-WO-PCT / / 103241-016PCT

[0309] In some embodiments, the compounds of Formulae I-IV or a pharmaceutically acceptable salt thereof may be administered orally, intravenously, sublingually, buccally, transdermally, or intranasally.

[0310] The compounds or a pharmaceutically acceptable salt thereof can be administered in combination with a neuromodulation technique selected from the group consisting of transcranial direct current stimulation (tDCS), transcranial magnetic stimulation (TMS), deep brain stimulation (DBS), and a combination thereof.

[0311] The betahistine prodrug compounds disclosed herein can be administered as the neat chemicals but are preferably administered as a pharmaceutical composition comprising the compound and one or more pharmaceutically acceptable carriers, excipients, or diluents. Examples of such carriers are well known to those skilled in the art and can be prepared in accordance with acceptable pharmaceutical procedures, such as, for example, those described in Remington's Pharmaceutical Sciences, 17th edition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is incorporated by reference herein for all purposes. As used herein, “pharmaceutically acceptable” refers to a substance that is

[0312] 43

[0313] 20300-WO-PCT / / 103241-016PCT acceptable for use in pharmaceutical applications from a toxicological perspective and does not adversely interact with the active ingredient. Accordingly, pharmaceutically acceptable carriers are those that are compatible with the other ingredients in the formulation and are biologically acceptable. Supplementary active ingredients can also be incorporated into the pharmaceutical compositions.

[0314] The formulator will understand that excipients are used primarily to serve in delivering a safe, stable, and functional pharmaceutical, serving not only as part of the overall vehicle for delivery but also as a means for achieving effective absorption by the recipient of the active ingredient. An excipient may fill a role as simple and direct as being an inert filler, or an excipient may be part of a pH stabilizing system or coating.

[0315] Compounds of the present disclosure can be administered orally or parenterally, neat or in combination with conventional pharmaceutical carriers. Applicable solid carriers can include one or more substances which can also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents, or encapsulating materials. Oral formulations containing a compound disclosed herein can comprise any conventionally used oral form, including tablets, capsules, buccal forms, troches, lozenges and oral liquids, suspensions or solutions. In powders, the carrier can be a finely divided solid, which is an admixture with a finely divided compound. In tablets, a compound disclosed herein can be mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets can contain up to 99% of the compound(s).

[0316] Capsules can contain mixtures of one or more compound(s) disclosed herein with inert filler(s) and / or diluent(s) such as pharmaceutically acceptable starches (e.g., corn, potato or tapioca starch), sugars, artificial sweetening agents, powdered celluloses (e.g., crystalline and microcrystalline celluloses), flours, gelatins, gums, and the like.

[0317] Useful tablet formulations can be made by conventional compression, wet granulation or dry granulation methods and utilize pharmaceutically acceptable diluents, binding agents, lubricants, disintegrants, surface modifying agents (including surfactants), suspending or stabilizing agents, including, but not limited to, magnesium stearate, stearic acid, sodium lauryl sulfate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, microcrystalline cellulose, sodium carboxymethyl cellulose, carboxymethylcellulose calcium,

[0318] 44

[0319] 20300-WO-PCT / / 103241-016PCT polyvinylpyrrolidine, alginic acid, acacia gum, xanthan gum, sodium citrate, complex silicates, calcium carbonate, glycine, sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, low melting waxes, ion exchange resins, benzyl alcohol, eucalyptol, gelatin, limonene, mannitol, menthol, menthone, menthyl acetate, sucralose, and vanillin. Surface modifying agents include nonionic and anionic surface modifying agents. Representative examples of surface modifying agents include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum silicate, and triethanolamine. Oral formulations herein can utilize standard delay or time-release formulations to alter the absorption of the compound(s). The oral formulation can also consist of administering a compound disclosed herein in water or fruit juice, containing appropriate solubilizers or emulsifiers as needed.

[0320] Liquid carriers can be used in preparing solutions for oral or parenteral administration (such as intravenous, intramuscular, or other injections), including suspensions, emulsions, syrups, elixirs, and additionally for inhaled delivery. A compound of the present teachings can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, or a mixture of both, or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers, and osmo-regulators. Examples of liquid carriers for oral and parenteral administration include, but are not limited to, water (particularly containing additives as described herein, e.g., cellulose derivatives such as a sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and poly hydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration, the carrier can be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are used in sterile liquid form compositions for parenteral administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellants.

[0321] Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intraperitoneal or subcutaneous injection. Sterile injectable solutions can also be administered intravenously. Compositions for oral administration can be in either liquid or solid form.

[0322] 45

[0323] 20300-WO-PCT / / 103241-016PCT Preferably the pharmaceutical composition is in unit dosage form, for example, as tablets, capsules, powders, solutions, suspensions, emulsions, granules, or suppositories. In such form, the pharmaceutical composition can be further sub-divided to contain appropriate quantities of the compound. The unit dosage forms can be packaged compositions, for example, packeted powders, vials, ampoules, prefilled syringes or sachets containing liquids. Alternatively, the unit dosage form can be a capsule or tablet itself, or it can be the appropriate number of any such compositions in package form. Such doses can be administered in any manner useful in directing the compound(s) to the recipient's bloodstream, including orally, via implants, parenterally (including intravenous, intraperitoneal and subcutaneous injections), rectally, vaginally, and transdermally.

[0324] In some cases it may be desirable to administer a compound directly to the airways of the patient, using devices such as, but not limited to, metered dose inhalers, breath- operated inhalers, multidose dry-powder inhalers, pumps, squeeze-actuated nebulized spray dispensers, aerosol dispensers, and aerosol nebulizers. For administration by intranasal or intrabronchial inhalation, the compounds of the present teachings can be formulated into a liquid composition, a solid composition, or an aerosol composition. The liquid composition can include, by way of illustration, one or more compounds of the present teachings dissolved, partially dissolved, or suspended in one or more pharmaceutically acceptable solvents and can be administered by, for example, a pump or a squeeze-actuated nebulized spray dispenser. The solvents can be, for example, isotonic saline or bacteriostatic water. The solid composition can be, by way of illustration, a powder preparation including one or more compounds of the present teachings intermixed with lactose or other inert powders that are acceptable for intrabronchial use, and can be administered by, for example, an aerosol dispenser or a device that breaks or punctures a capsule encasing the solid composition and delivers the solid composition for inhalation. The aerosol composition can include, by way of illustration, one or more compounds of the present teachings, propellants, surfactants, and co-solvents, and can be administered by, for example, a metered device. The propellants can be a chlorofluorocarbon (CFC), a hydrofluoroalkane (HF A), or other propellants that are physiologically and environmentally acceptable.

[0325] Compounds described herein can be administered parenterally or intraperitoneally. Solutions or suspensions of these compounds or pharmaceutically acceptable salts thereof can be prepared in water suitably mixed with a surfactant such as hydroxylpropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Under

[0326] 46

[0327] 20300-WO-PCT / / 103241-016PCT ordinary conditions of storage and use, these preparations typically contain a preservative to inhibit the growth of microorganisms.

[0328] The pharmaceutical forms suitable for injection can include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In some embodiments, the form can be sterile and its viscosity permits it to flow through a syringe. The form preferably is stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0329] The invention is further illustrated by the following non-limiting examples.

[0330] EXAMPLES

[0331] LCMS METHODS

[0332] 47

[0333] 20300-WO-PCT / / 103241-016PCT

[0334] HPLC Methods

[0335] Method A: Column:X-Bridge C8 (50 X 4.6 mm); 3.5pm; Mobile phase A: 10 mM ammonium acetate in water; Mobile phase B: ACN; Flow: 1.0 mL / min. Method B: Column: X-Bridge C8 (50 X 4.6 mm); 3.5 pm; Mobile phase A: 0.1% Formic acid in water; Mobile phase B: ACN; Flow: 1.0 mL / min.

[0336] Method C: Column: XBridge C8 (50 X 4.6 mm); 3.5 pm; Flow Rate: 1.0 mL / min; Mobile Phase

[0337] A: 0.1% TFA in water; Mobile Phase B: ACN

[0338] Method D: Column: YMC Triart Hilic (250 X 4.6 mm); 5 pm; Flow Rate: 0.8 mL / min; Mobile Phase A : 0.1% TFA in water; Mobile Phase B: ACN

[0339] Method E: Column: X-Bridge C8(50 X 4.6 m); 3.5 pm; Mobile phase A: l 0 mM Ammonium Bicarbonate in water; Mobile phase B: ACN; Flow: 1.0 mL / min.

[0340] 48

[0341] 20300-WO-PCT / / 103241-016PCT Method F: Column: Acclaim Polar C18 (250 X 4.6 mm), 5.0 m; Mobile phase A: lOmM Ammonium Bicarbonate in water; Mobile phase B: ACN; Flow: 0.8 mL / min.

[0342] Pren-HPLC Methods

[0343] Method A: Mobile phase AHO mM NH4HCO3 in water, B: ACN; flow rate: 12 mL / min; column: X-Bridge Cl 8 (250 x 19 mm, 5 pm).

[0344] Method B: Mobile phase AHO mM NH4OAC in MQ water, Mobile phase B: ACN; flow rate: 12 mL / min; column: X-Bridge Cl 8 (250 x 19 mm, 5 pm).

[0345] Method C: Mobile phase A: 0.1% Formic acid in MQ water: Mobile phase B: ACN; flow rate: 12 mL / min; column: X-select Cl 8 (250 x 19 mm, 5 pm).

[0346] Method D: Mobile phase A: 0.1% TFA in MQ water: Mobile phase B: ACN; flow rate: 12 mL / min; column: Sunfire Cl 8 (250 x 19 mm, 5 pm).

[0347] Method E: Mobile phase A: 10 mM NH4HCO3 in water, Mobile phase B: ACN; flow rate: 12 mL / min; column: YMC Actus triart C18 (19*250 mm. 5 pm).

[0348] SFC Method

[0349] Method A: Instrument: PIC 100; Column: YMC EP-2(250*30) mm, 5pm

[0350] Mobile Phase: CO2: 0.5% IP Am in IPA (70:30)%, Total Flow: 70 mL / min

[0351] Back pressure: 100 bar, Wavelength: 254 nm, Cycle time: 5.0 min

[0352] SYNTHESIS OF SELECTED COMPOUNDS

[0353] Cmpd 1 : (S)-2,6-Diamino-N-methyl-N-(2-(pyridin-2-yl)ethyl)hexanamide

[0354] Step 1: di-tert-Butyl (6-(methyl(2-(pyridin-2-yl)ethyl)amino)-6-oxohexane-l,5-diyl)(S)- dicarbamate

[0355] To a stirred solution of / V-methyl-2-(pyridin-2-yl)ethan- l -amine dihydrochloride (181 mg, 0.866 mmol) in DMF (2 mL) at 25 °C was added / V, / V-diisopropylethylamine (373 mg, 2.89 mmol). After 5 min, ( I / 7-benzo [d] [ 1 ,2, 3 Jtriazol- 1 -yl)oxy)tris(dimethy lamino)phosphonium hexafluorophosphate (V) (BOP) (255 mg, 0.577 mmol) and A^A^-bis^ert-butoxycarbonyl)- / ,-

[0356] 49

[0357] 20300-WO-PCT / / 103241-016PCT lysine (200 mg, 0.577 mmol) were added to the reaction mixture and stirring was continued for 16 h. The reaction mixture was quenched with ice cooled water (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layer was washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure to afford crude material (420 mg). The crude material was purified by chromatography (230-400 mesh silica, 25 g snap, 4% MeOH in DCM) to afford di-tert-butyl (6-(methyl(2-(pyridin-2-yl)ethyl)amino)-6-oxohexane-l,5-diyl)(S)-dicarbamate as colorless oil (250 mg, 93%). LCMS (ES+): m / z = 465.3 [M+H]+.

[0358] Step 2: (S)-2,6-Diamino-N-methyl-N-(2-(pyridin-2-yl)ethyl)hexanamide (Cmpd 1)

[0359] To a stirred solution of di-tert-butyl (6-(methyl(2-(pyridin-2-yl)ethyl)amino)-6-oxohexane-l,5- diyl)(S)-dicarbamate (200 mg, 0.430 mmol) in 1,4-dioxane (2 mL) at 25 °C was added HC1 in dioxane (4M, 1.076 ml, 4.30 mmol) and the reaction mixture was stirred for 2 h. Reaction progress was monitored by LCMS and TLC. The reaction mixture was concentrated under vacuum to afford crude material which was purified by prep-HPLC (Method E) to afford (S)-2,6-diamino-N-methyl- N-(2-(pyridin-2-yl)ethyl)hexanamide (Cmpd 1) as a yellow viscous liquid (32.3 mg, 28%). LCMS (ES+): m / z = 265.2 [M+H]+.

[0360] Cmpd 2: (5)-2-Amino- / V-methyl-N-(2-(pyridin-2-yl)ethyl)propanamide

[0361] Step 1: / er / - Butyl (5)-(l-(methyl(2-(pyridin-2-yl)ethyl)amino)-l-oxopropan-2-yl)carbamate To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (332 mg, 1.59 mmol) in DMF (2 mL) at 25 °C was added / V, / V-diisopropylethylamine (0.923 ml, 5.29 mmol). After 5 min of stirring at same temperature, ( l / 7-benzo[< / ][ l ,2,3]triazol- l - yl)oxy)tris(dimethylamino)phosphonium hexafluorophosphate (V) (468 mg, 1.06 mmol) and (tert- butoxy carbonyl )- / ,-alanine (200 mg, 1.06 mmol) were added and stirring was continued for 16 h at 25 °C. Reaction progress was monitored by UPLC and TLC. The reaction mixture was quenched with ice cooled water (5 mL) and extracted with EtOAc (3 x 10 mL). The combined organic phases were washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure to afford crude material (550 mg). The crude material was purified by silica gel

[0362] 50

[0363] 20300-WO-PCT / / 103241-016PCT chromatography (230-400 mesh, 25 g snap, 4% MeOH in DCM) to afford tert-butyl fS -( l - (methyl(2-(pyridin-2-yl)ethyl)amino)-l-oxopropan-2-yl)carbamate as a pale-yellow liquid (310 mg, 95%). LCMS (ES+): m / z = 308.2 [M+H]+.

[0364] Step 2: (xS')-2-Ainin()- V-inethyl- V-(2-(pyridin-2-yl)ethyl)propanainide (Cmpd 2)

[0365] To a stirred solution of tert-butyl (5)-(l-(methyl(2-(pyridin-2-yl)ethyl)amino)-l-oxopropan-2- yl)carbamate (100 mg, 0.325 mmol) in 1,4-dioxane (1 mL) at 25 °C was added HC1 in dioxane (4 M, 0.81 mL, 3.3 mmol) and reaction mixture was stirred for 2 h at 25 °C. Reaction progress was monitored by LCMS and TLC. The reaction mixture was concentrated under vacuum to afford crude product which was purified by prep-HPLC (Method E) to afford (S)-2-amino-A-methyl-7V- (2-(pyridin-2-yl)ethyl)propanamide (Cmpd 2) as a colorless oil (13.5 mg, 19.9%). LCMS (ES+): m / z = 208.2 [M+H]+.

[0366] Cmpd 3: (5)-2-Amino- / V,3-dimethyl- / V-(2-(pyridin-2-yl)ethyl)butanamide hydrochloride

[0367] Step 1: tert-Butyl (5)-(3-methyl- 1-( met hyl(2-(py rid in-2-yl )ethyl )amino)- l-oxobutan-2- yl)carbamate

[0368] To a stirred solution of A-methyl-2-(pyridin-2-yl)ethan- l -amine dihydrochloride (300 mg, 1.44 mmol) in DMF (5 mL) at 25 °C was added A,A-diisopropylethylamine (1.25 mL, 7.17 mmol). After 5 min of stirring at same temperature, (tert-butoxycarbonyl)-L- valine (468 mg, 2.15 mmol) followed by HATU (1.09 g, 2.87 mmol) were added to the reaction mixture and stirring was continued for 16 h at 25 °C. Reaction progression was monitored by UPLC and TLC. The reaction mixture was quenched with ice cooled water (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layer was washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude material was purified by prep-HPLC (Method B) to afford tert-butyl (5)-(3-methyl-l-(methyl(2-(pyridin-2-yl)ethyl)amino)-l-oxobutan-2- yl)carbamate as an off-white gummy solid (367 mg, 76%). LCMS (ES+): m / z = 336.3 [M+H]+.

[0369] 51

[0370] 20300-WO-PCT / / 103241-016PCT Step 2: ( xS')-2- inino- V.3-d imet hyl-V-( 2-( py rid in-2-yl (ethyl (but ana inide hydrochloride (Cmpd 3)

[0371] To a stirred solution of tert-butyl (5)-(3 -methyl- l-(methyl(2-(pyridin-2-yl)ethyl)amino)-l- oxobutan-2-yl)carbamate (360 mg, 1.07 mmol) in 1,4-di oxane (5 mL) at 25 °C was added HC1 in dioxane (4M, 0.805 mL, 3.22 mmol) and reaction mixture was stirred for 2 h. Reaction progression was monitored by LCMS and TLC. The reaction mixture was concentrated under vacuum and the resulting material was lyophilized to afford (5)-2-amino- / V,3-dimethyl- / V-(2- (pyridin-2-yl)ethyl)butanamide hydrochloride (Cmpd 3) as a pale yellow gummy solid (135 mg, 46.2%). LCMS (ES+): m / z = 236.2 [M+H]+.

[0372] Cmpd 4: (2S,3S)-2-Amino-N,3-dimethyl-N-(2-(pyridin-2-yl)ethyl)pentanamide

[0373] Step 1: tert- Butyl ((25,35)-3-methyl-l-(methyl(2-(pyridin-2-yl)ethyl)amino)-l-oxopentan-2- yl)carbamate

[0374] To a stirred solution of 7V-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (271 mg, 1.297 mmol) in DMF (2 mL) at 25 °C was added / V, / V-diisopropylethylamine (0.755 mL, 4.32 mmol). After 5 min, ((77 / -benzo[<7][l,2,3]triazol-l-yl)oxy)tris(dimethylamino)phosphonium hexafluorophosphate (V) (BOP) (382 mg, 0.865 mmol) and (tert-butoxycarbonyl)-L-isoleucine (200 mg, 0.865 mmol) were added to the reaction mixture and stirring was continued for 16 h. Reaction progression was monitored by UPLC and TLC. The reaction mixture was quenched with ice cooled water (5 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layer was washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure to afford crude material (590 mg). The crude material was purified by column chromatography (230- 400 mesh silica, 25 g snap, 4% MeOH in DCM) to afford tert- butyl ((25, 3S)-3 -methyl- 1- (methyl(2-(pyridin-2-yl)ethyl)amino)-l-oxopentan-2-yl)carbamate as a colorless oil (320 mg, 98%). LCMS (ES+): m / z = 350.2 [M+H]+.

[0375] Step 2: (2S,3S)-2-Amino-N,3-dimethyl-N-(2-(pyridin-2-yl)ethyl)pentanamide (Cmpd 4)

[0376] 52

[0377] 20300-WO-PCT / / 103241-016PCT To a stirred solution of tert-butyl ((2S,3S)-3-methyl-l-(methyl(2-(pyridin-2-yl)ethyl)amino)-l- oxopentan-2-yl)carbamate (200 mg, 0.572 mmol) in 1,4-dioxane (2 mb) at 25 °C was added HC1 in dioxane (4M, 1.43 mb, 5.72 mmol) and reaction mixture was stirred for 2 h. Reaction progression was monitored by LCMS and TLC. The reaction mixture was concentrated under vacuum, co-evaporated with di chloromethane (2 x 10 mL), washed with hexane (2 x 10 mb), and dried under vacuum to afford crude material (161 mg). Crude material was purified by prep-HPLC (Method E) to afford (2S,3S)-2-amino-N,3-dimethyl-N-(2-(pyridin-2-yl)ethyl)pentanamide (Cmpd 4) as a pale yellow viscous liquid (67.9 mg, 47.4%). LCMS (ES+): m / z = 250.2 [M+H]+

[0378] Cmpd 5: (S)-2-Amino-N-methyl-3-phenyl-N-(2-(pyridin-2-yl)ethyl)propanamide hydrochloride

[0379] Step 1: tert-Butyl (S)-(l-(methyl(2-(pyridin-2-yl)ethyl)amino)-l-oxo-3-phenylpropan-2- yl)carbamate

[0380] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (177 mg, 0.848 mmol) in DMF (3 mL) was added N,N-Diisopropylethylamine (292 mg, 2.262 mmol) at 25 °C. After 5 min of stirring at same temperature, (tert-butoxycarbonyl)-L-phenylalanine (150 mg, 0.565 mmol) followed by BOP (250 mg, 0.565 mmol) were added to the reaction mixture and stirring was continued for 16 h at 25 °C. The reaction was monitored by UPLC-MS. The mixture was diluted with EtOAc (10 mL) and water (10 mL), the organic layer was separated and washed with brine (10 mL), dried over Na2SO4 and concentrated, to afford crude material (400 mg). The crude material was purified by Isolera-Biotage (silica gel 100-200 mesh, eluted with 3% MeOH in DCM), to afford tert-butyl (S)-(l-(methyl(2-(pyridin-2-yl)ethyl)amino)-l-oxo-3-phenylpropan-2- yl)carbamate as a pale brown oil (150 mg, 68.5%). LCMS (ES+): m / z = 384.3 [M+H]+.

[0381] Step 2: (S)-2-Amino-N-methyl-3-phenyl-N-(2-(pyridin-2-yl)ethyl)propanamide hydrochloride (Cmpd 5)

[0382] To a stirred solution of tert-butyl (S)-(l-(methyl(2-(pyridin-2-yl)ethyl)amino)-l-oxo-3- phenylpropan-2-yl) carbamate (150 mg, 0.391 mmol) in 1,4-dioxane (2 mL) was added 4M HCI in

[0383] 53

[0384] 20300-WO-PCT / / 103241-016PCT dioxane (1 mL, 32.9 mmol) and the reaction mixture was stirred at 25 °C for 3h. The reaction was monitored by UPLC-MS. The mixture was concentrated and co-evaporated with DCM (2 x 10 mL), washed with hexane (2 x 5 mL), and dried under vacuum to get 120 mg of crude material which was purified by C-l 8 reverse phase column chromatography (ammonium bicarbonate buffer in H2O-CH3CN mixture, 0-70%) to afford (S)-2-amino-N-methyl-3-phenyl-N-(2-(pyridin-2- yl)ethyl)propanamide hydrochloride) (Cmpd 5) as a brown oil (25 mg, 99 %). LCMS (ES+): m / z = 284.2 [M+H]+.

[0385] Cmpd 6: (S)-N-Methyl-N-(2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide

[0386] Step 1: tert-Butyl (S)-2-(methyl(2-(pyridin-2-yl)ethyl)carbamoyl)pyrrolidine-l-carboxylate To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (219 mg, 1.05 mmol) in DMF (1 mL) was added N,N-diisopropylethylamine (0.607 mL, 3.48 mmol) at 25 °C. After 5 min, (tert-butoxycarbonyl)-L-proline (150 mg, 0.697 mmol) followed by BOP (370 mg, 0.836 mmol) were added to the reaction mixture and stirring was continued for 16 h. The reaction was monitored by UPLC and TLC. The reaction mixture was quenched with ice cooled water (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layer was washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude material was purified by Isolera-Biotage chromatography (silica gel 230-400 mesh, eluted with 3% MeOH in DCM) to afford tert-butyl (S)-2-(methyl(2-(pyridin-2-yl)ethyl)carbamoyl)pyrrolidine-l- carboxylate as a light brown gum (190 mg, 81%). LCMS (ES+): m / z = 334.2 [M+H]+.

[0387] Step 2: (S)-N-Methyl-N-(2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide (Cmpd 6)

[0388] To a stirred solution of tert-butyl (S)-2-(methyl(2-(pyridin-2-yl)ethyl)carbamoyl)pyrrolidine-l- carboxylate (185 mg, 0.555 mmol) in 1,4-dioxane (2 mL), was added 4M HC1 in dioxane (0.169 mL, 5.55 mmol) at 25 °C and reaction mixture was stirred for 3 h at 25 °C. The reaction was monitored by LCMS and TLC. The reaction mixture was concentrated under vacuum, coevaporated with DCM (2 x 10 mL), washed with hexane (2 x 5 mL), and dried under vacuum to provide crude material (130 mg). The crude material was purified by prep-HPLC (Method B) to

[0389] 54

[0390] 20300-WO-PCT / / 103241-016PCT afford (S)-N-methyl-N-(2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide (Cmpd 6) as a pale yellow oil (40 mg, 30.7%). LCMS (ES+): m / z = 234.2 [M+H]+.

[0391] Cmpd 7 : (S)-2-Amino-3-hydroxy-N-methyl-N-(2-(pyridin-2-yl)ethyl)propanamide

[0392] Step 1: tert-Butyl (S)-(3-(tert-butoxy)-l-(methyl(2-(pyridin-2-yl)ethyl)amino)-l-oxopropan- 2-yl)carbamate

[0393] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (360 mg, 1.72 mmol) in DMF (5 mL) at 25 °C was added N,N-diisopropylethylamine (1.00 mL, 5.74 mmol). After 5 min, ((lH-benzo[d][l,2,3]triazol-l-yl)oxy)tris(dimethylamino)phosphonium hexafluorophosphate (V) (609 mg, 1.38 mmol) followed by N-(tert- butoxy carbonyl)-O-(tert- butyl)-L-serine (300 mg, 1.15 mmol) were added to the reaction mixture and stirring was continued for 16 h. The reaction was monitored by UPLC and TLC. The reaction mixture was quenched with ice cooled water (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layer was washed with brine (10 mL), dried over ISfeSC and concentrated under reduced pressure. The crude material was purified by column chromatography (230-400 mesh silica, 25g snap, 0-100 % EtOAc in petroleum ether) to afford tert-butyl (S)-(3-(tert-butoxy)-l-(methyl(2-(pyridin-2- yl)ethyl)amino)-l-oxopropan-2-yl)carbamate as a gummy solid (410 mg, 94%). LCMS (ES+): m / z = 380.2 [M+H]+.

[0394] Step 2: (S)-2-Amino-3-hydroxy-N-methyl-N-(2-(pyridin-2-yl)ethyl)propanamide (Cmpd 7)

[0395] To a stirred solution of tert-butyl (S)-(3-(tert-butoxy)-l-(methyl(2-(pyridin-2-yl)ethyl)amino)-l- oxopropan-2-yl)carbamate (400 mg, 1.05 mmol) in DCM (10 mL) was added TFA (0.161 mL, 2.11 mmol) at 0 °C and then the reaction mixture was stirred at 25 °C for 16 h. The reaction was monitored by TLC and UPLC-MS. The reaction mixture was concentrated, then purified by prep- HPLC (Method A) to afford (S)-2-amino-3-hydroxy-N-methyl-N-(2-(pyridin-2- yl)ethyl)propanamide (Cmpd 7) as an off-white viscous oil (12 mg, 5%). LCMS (ES+): m / z = 224.1 [M+H]+.

[0396] Cmpd 8: (S)-3-Amino-4-(methyl(2-(pyridin-2-yl)ethyl)amino)-4-oxobutanoic acid

[0397] 55

[0398] 20300-WO-PCT / / 103241-016PCT

[0399] Step 1: tert-Butyl (S)-3-((tert-butoxycarbonyl)amino)-4-(methyl(2-(pyridin-2- yl)ethyl)amino)-4-oxobutanoate

[0400] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (217 mg, 1.04 mmol) in DMF (2 mL) at 25 °C was added N,N-diisopropylethylamine (447 mg, 3.46 mmol). After 5 min, (( 1 H-benzo [d] [1,2, 3 Jtriazol- 1 -yl)oxy)tris(dimethy lamino)phosphonium hexafluorophosphate(V) (306 mg, 0.691 mmol) and (S)-4-(tert-butoxy)-2-((tert- butoxycarbonyl)amino)-4-oxobutanoic acid (200 mg, 0.691 mmol) were added to the reaction mixture and stirring was continued for 16 h. The reaction was monitored by UPLC and TLC. The reaction mixture was quenched with ice cooled water (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layer was washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure to afford crude material (450 mg). The crude material was purified by column chromatography (silica gel 230-400 mesh, 25g snap, 4% MeOH in DCM) to afford tert-butyl (S)-3-((tert-butoxycarbonyl)amino)-4-(methyl(2-(pyridin-2-yl)ethyl)amino)-4- oxobutanoate as a colorless oil (210 mg, 49.2%). LCMS (ES+): m / z = 408.3 [M+H]+.

[0401] Step 2: (S)-3-amino-4-(methyl(2-(pyridin-2-yl)ethyl)amino)-4-oxobutanoic acid (Cmpd 8):

[0402] To a stirred solution of tert-butyl (S)-3-((tert-butoxycarbonyl)amino)-4-(methyl(2-(pyridin-2- yl)ethyl)amino)-4-oxobutanoate (200 mg, 0.491 mmol) in DCM (2 mL), was added TFA (0.189 mL, 2.45 mmol) at 0 °C and the reaction mixture was stirred for 2 h at 25 °C. Upon completion of reaction, monitored by UPLC and TLC, the mixture was concentrated under vacuum to afford crude product. The crude product was purified by prep-HPLC (Method E) to afford (S)-3-amino- 4-(methyl(2-(pyridin-2-yl)ethyl)amino)-4-oxobutanoic acid (Cmpd 8) (40.2 mg, 32.4%) as a white solid. LCMS (ES+): m / z = 252.1 [M+H]+.

[0403] Cmpd 9: 2-(2-Aminoacetamido)-N-methyl-N-(2-(pyridin-2-yl)ethyl)acetamide

[0404] 56

[0405] 20300-WO-PCT / / 103241-016PCT Step 1: tert-Butyl (2-((2-(methyl(2-(pyridin-2-yl)ethyl)amino)-2-oxoethyl)amino)-2- oxoethyl)carbamate

[0406] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (500 mg, 2.39 mmol) in DMF (8 mL) at 25 °C was added N,N-diisopropylethylamine (2.08 mL, 12.0 mmol). After 5 min, (tert-butoxycarbonyl)glycylglycine (833 mg, 3.59 mmol) followed by HATU (182 mg, 4.78 mmol) were added to the reaction mixture and stirring was continued for 16 h. The reaction was monitored by UPLC and TLC. The reaction mixture was concentrated and purified by C-18 reverse phase column chromatography (ammonium bicarbonate buffer in H2O-CH3CN mixture, 0-70%) to afford tert-butyl (2-((2-(methyl(2-(pyridin-2-yl)ethyl)amino)-2- oxoethyl)amino)-2-oxoethyl)carbamate as an off-white gummy liquid (724 mg, 72.4%). LCMS (ES+): m / z = 351.2 [M+H]+.

[0407] Step 2: 2-(2-aminoacetamido)-N-methyl-N-(2-(pyridin-2-yl)ethyl)acetamide (Cmpd 9)

[0408] To a stirred solution of tert-butyl (2-((2-(methyl(2-(pyridin-2-yl)ethyl)amino)-2-oxoethyl)amino)- 2-oxoethyl)carbamate (720 mg, 2.06 mmol) in 1,4-dioxane (5 mL) at 25 °C was added 4M HC1 solution in dioxane (2.57 mL, 10.3 mmol) and the reaction mixture was stirred for 2 h. Reaction progression was monitored by LCMS and TLC. The reaction mixture was concentrated under vacuum to afford crude material (680 mg) which was purified by SFC Achiral Method A to afford 2-(2-aminoacetamido)-N-methyl-N-(2-(pyridin-2-yl)ethyl)acetamide (Cmpd 9) (105 mg, 0.406 mmol, 19.8%) as a brown viscous oil. LCMS (ES+): m / z = 251.3 [M+H]+.

[0409] Cmpd 10: (S)-2-(2-Aminoacetamido)-N-methyl-N-(2-(pyridin-2-yl)ethyl)propanamide

[0410] Step 1: tert-Butyl (S)-(l-(methyl(2-(pyridin-2-yl)ethyl)amino)-l-oxopropan-2-yl)carbamate

[0411] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (497 mg, 2.38 mmol) in DMF (3 mL) at 25 °C was added N,N-diisopropylethylamine (1.41 mL, 7.93 mmol).

[0412] 57

[0413] 20300-WO-PCT / / 103241-016PCT After 5 min, ((lH-benzo[d][l,2,3]triazol-l-yl)oxy)tris(dimethylamino)phosphonium hexafluorophosphate(V) (701 mg, 1.59 mmol) and tert-butyl (S)-(l-(methyl(2-(pyridin-2- yl)ethyl)amino)-l-oxopropan-2-yl)carbamate (475 mg, 1.07 mmol) were added to the reaction mixture and stirring was continued for 16 h. The reaction was monitored by UPLC and TLC. The reaction mixture was quenched with ice cooled water (5 mL) and extracted with EtOAc (3 x 10 mb). The combined organic layer was washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure. The resulting residue was purified by column chromatography (silica-gel, 230-400, 0 - 20% MeOH in DCM). The pure fractions were evaporated and dried under vacuum to afford tert-butyl (S)-(l-(methyl(2-(pyridin-2- yl)ethyl)amino)-l-oxopropan-2-yl)carbamate as yellow liquid (475 mg, 67%). LCMS (ES+): m / z = 308.2 [M+H]+.

[0414] Step 2: (S)-2-Amino-N-methyl-N-(2-(pyridin-2-yl)ethyl)propenamide hydrochloride

[0415] To a stirred solution of tert-butyl (S)-(l-(methyl(2-(pyridin-2-yl)ethyl)amino)-l-oxopropan-2- yl)carbamate (157 mg, 0.511 mmol) in 1 ,4-dioxane (2 mL) at 25 °C was added 4M HC1 in dioxane (1.28 mL, 5.11 mmol) and reaction mixture was stirred for 2 h at 25 °C. The reaction was monitored by LCMS and TLC. The reaction mixture was concentrated under vacuum to afford crude (S)-2-amino-N-methyl-N-(2-(pyridin-2-yl)ethyl)propenamide hydrochloride as yellow solid (140 mg, 99%). LCMS (ES+): m / z = 208.2 [M+H]+.

[0416] Step 3: tert-Butyl (S)-(2-((l-(methyl(2-(pyridin-2-yl)ethyl)amino)-l-oxopropan-2-yl)amino)- 2-oxoethyl)carbamate

[0417] To a stirred solution of (tert-butoxycarbonyl)glycine (85 mg, 0.485 mmol) in DMF (2 mL) were added benzotriazole- l-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (322 mg, 0.728 mmol) and N,N-diisopropylethylamine (0.423 mL, 2.43 mmol) at 0 °C and the mixture was stirred for 5 min. (S)-2-amino-N-methyl-N-(2-(pyridin-2-yl)ethyl)propenamide (201 mg, 0.728 mmol) was added to the reaction mixture and it was stirred at 25 °C for 16 h. The reaction was monitored by TLC and UPLC. The reaction mixture was quenched with ice cold water (5 mL) and extracted with EtOAc (3 X 10 mL). The combined organic layer was washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure to afford crude material (200 mg). The crude product was purified by flash column chromatography (silica-gel, 230-400, 0 - 20% MeOH in DCM) to afford tert-butyl (S)-(2-((l-(methyl(2-(pyridin-2-yl)ethyl)amino)-l-

[0418] 58

[0419] 20300-WO-PCT / / 103241-016PCT oxopropan-2-yl)amino)-2-oxoethyl)carbamate as a yellow liquid (135 mg, 42%). LCMS (ES+): m / z = 365.2 [M+H]+.

[0420] Step 4: (S)-2-(2-Aminoacetamido)-N-methyl-N-(2-(pyridin-2-yl)ethyl)propanamide (Cmpd 10)

[0421] To a stirred solution of tert-butyl (S)-(2-((l-(methyl(2-(pyridin-2-yl)ethyl)amino)-l-oxopropan-2- yl)amino)-2-oxoethyl)carbamate (135 mg, 0.370 mmol) in 1,4-dioxane (1.3 mb) at 25 °C was added HCI in dioxane (4M, 1.1 mL, 4.3 mmol) and reaction mixture was stirred for 2 h at 25 °C. The reaction was monitored by LCMS and TLC. The reaction mixture was concentrated under vacuum. The resulting residue was purified by prep-HPLC (Method A) to afford (S)-2-(2- aminoacetamido)-N-methyl-N-(2-(pyridin-2-yl)ethyl)propanamide (Cmpd 10) as yellow gum (10 mg, 10%). LCMS (ES+): m / z = 265.2 [M+H]+.

[0422] Cmpd 11: 2-(2-(tert-Butylamino)acetamido)-N-methyl-N-(2-(pyridin-2-yl)ethyl)acetamide

[0423] Step 1: tert-Butyl (2-(methyl(2-(pyridin-2-yl)ethyl)amino)-2-oxoethyl)carbamate

[0424] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (358 mg, 1.71 mmol) in DMF (7 mL), N,N-disopropylethylamine (0.997 mL, 5.71 mmol) was added at 25 °C. After 5 min, (benzotriazol- l-yloxytris(dimethylamino)phosphonium hexafluorophosphate) (BOP) (505 mg, 1.14 mmol) and (tert-butoxycarbonyl)glycine (200 mg, 1.14 mmol) were added to the reaction mixture and stirring was continued for 16 h. Completion of reaction was monitored by UPLC and TLC. The reaction mixture was quenched with ice cooled water (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layer was washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure to afford crude (450 mg). The crude material was purified by silica gel 230-400, 25 g snap, 4% MeOH in DCM) to afford pure tert-

[0425] 59

[0426] 20300-WO-PCT / / 103241-016PCT butyl (2-(methyl(2-(pyridin-2-yl)ethyl)amino)-2-oxoethyl)carbamate as a yellow viscous liquid (210 mg, 50.8%). LCMS (ES+): m / z = 294.1 [M+H]+.

[0427] Step 2: 2-Amino-N-methyl-N-(2-(pyridin-2-yl)ethyl)acetamide hydrochloride

[0428] To a stirred solution of tert-butyl (2-(methyl(2-(pyridin-2-yl)ethyl)amino)-2-oxoethyl)carbamate (200 mg, 0.682 mmol) in 1,4-dioxane (2 mL) was added 4 M HC1 in dioxane (0.25 mL, 6.82 mmol) at 25 °C and reaction mixture was further stirred for 2 h at 25 °C. The reaction was monitored by LCMS and TLC. The reaction mixture was concentrated under high vacuum to afford crude 2-amino-N-methyl-N-(2-(pyridin-2-yl)ethyl)acetamide hydrochloride (165 mg, 98%) as a pale yellow liquid. LCMS (ES+): m / z = 194.1 [M+H]+.

[0429] Step 3: tert-Butyl tert-butyl(2-((2-(methyl(2-(pyridin-2-yl)ethyl)amino)-2-oxoethyl)amino)- 2-oxoethyl)carbamate

[0430] To a stirred solution of 2-amino-N-methyl-N-(2-(pyridin-2-yl)ethyl)acetamide (170 mg, 0.880 mmol) in N,N-dimethylformamide (5 mL) was added N,N-diisopropylethylamine (0.768 mL, 4.40 mmol) at 25 °C. After 5 min, (benzotriazol- l-yloxytris(dimethylamino)phosphonium hexafluorophosphate) (BOP) (506 mg, 1.144 mmol) and N-(tert- butoxy carbonyl)-N-(tert- butyl)glycine (305 mg, 1.32 mmol) were added to the reaction mixture and stirring was continued for 16 h at 25 °C. The reaction was monitored by UPLC and TLC. The reaction mixture was quenched with ice cooled water (15 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layer was washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure to afford crude (420 mg). The crude material was purified by silica gel chromatography (230-400 mesh, 25 g snap, 4% MeOH in DCM) to afford tert-butyl tert-butyl(2-((2-(methyl(2-(pyridin-2-yl)ethyl)amino)-2-oxoethyl)amino)-2- oxoethyl)carbamate (200 mg, 54.8% yield) as colorless oil. LCMS (ES+): m / z = 407.3 [M+H]+.

[0431] Step 4: 2-(2-(tert-Butylamino)acetamido)-N-methyl-N-(2-(pyridin-2-yl)ethyl)acetamide (Cmpd 11)

[0432] To a stirred solution of tert-butyl tert-butyl(2-((2-(methyl(2-(pyridin-2-yl)ethyl)amino)-2- oxoethyl)amino)-2-oxoethyl)carbamate (200 mg, 0.492 mmol) in 1,4-dioxane (2 mL) at 25 °C was added 4(M) HC1 in dioxane (1.230 ml, 4.92 mmol) and reaction mixture was further stirred for 2 h at 25 °C. The reaction was monitored by LCMS and TLC. The reaction mixture was concentrated under vacuum to afford crude product which was purified by prep-HPLC method-E to afford 2-

[0433] 60

[0434] 20300-WO-PCT / / 103241-016PCT (2-(tert-butylamino)acetamido)-N-methyl-N-(2-(pyridin-2-yl)ethyl)acetamide as a pale yellow sticky solid (57.2 mg, 37.8%). LCMS (ES+): m / z = 307.3 [M+H]+.

[0435] Cmpd 12: 2-(2-(2-Aminoacetamido)acetamido)-N-methyl-N-(2-(pyridin-2- yl)ethyl)acetamide

[0436] Step 1: tert-Butyl (2-((2-((2-(methyl(2-(pyridin-2-yl)ethyl)amino)-2-oxoethyl)amino)-2- oxoethyl)amino)-2-oxoethyl)carbamate

[0437] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (200 mg, 0.956 mmol) in DMF (3 mL) N,N-diisopropylethylamine (618 mg, 4.78 mmol) was added at 25 °C. After 5 min of stirring, (tert-butoxycarbonyl)glycylglycylglycine (415 mg, 1.43 mmol) followed by HATU (727 mg, 1.91 mmol) were added and the reaction mixture was stirred at 25 °C for 3 h. The reaction was monitored by TLC and UPLC-MS. The reaction mass was purified by C-18 reverse phase column chromatography using ammonium bicarbonate buffer in H2O-CH3CN mixture (0- 70%) to afford tert-butyl (2-((2-((2-(methyl(2-(pyridin-2-yl)ethyl)amino)-2-oxoethyl)amino)-2- oxoethyl)amino)-2-oxoethyl)carbamate as a colorless viscous oil (240 mg, 57.9% yield). LCMS (ES+): m / z = 408.2 [M+H]+.

[0438] Step 2: 2-(2-(2-Aminoacetamido)acetamido)-N-methyl-N-(2-(pyridin-2-yl)ethyl)acetamide (Cmpd 12)

[0439] To a stirred solution of tert-butyl (2-((2-((2-(methyl(2-(pyridin-2-yl)ethyl)amino)-2- oxoethyl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (240 mg, 0.589 mmol) in 1,4-dioxane (5 mL) was added 4M HCI in dioxane (0.294 mL, 1.18 mmol) at 25 °C. Then the reaction mixture was stirred at 25 °C for 16 h. The reaction was monitored by TLC and LC-MS. The reaction mixture was concentrated to afford crude. Crude was purified by prep-HPLC Method-A to isolate pure fractions which were lyophilized to afford 2-(2-(2-aminoacetamido)acetamido)-N-methyl-N- (2-(pyridin-2-yl)ethyl)acetamide) as a colorless liquid (41 mg, 22%). LCMS (ES+): m / z = 308.1 [M+H]+.

[0440] Cmpd 13: l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl isobutyrate

[0441] 61

[0442] 20300-WO-PCT / / 103241-016PCT

[0443] Step-1: 1-Chloroethyl (4-nitrophenyl) carbonate

[0444] To a solution of 4-nitrophenol (1.50 g, 10.8 mmol) in DCM (30 mL) at 0 °C was added pyridine (0.87 mL, 10.8 mmol) followed by dropwise addition of 1 -chloroethyl carbonochloridate (1.70 g, 11.9 mmol). The resulting reaction mixture was then stirred at 0 °C for 30 min followed by stirring at 15 °C for 2.5 h. The progress of the reaction was monitored by TLC analysis. After completion of the reaction, the mixture was concentrated under reduced pressure to get crude compound which was partitioned between EtO Ac- water, and extracted with EtOAc (3 x 30 mL). The combined EtOAc extracts were washed with brine, dried over Na2SC>4, filtered, and concentrated under reduced pressure to afford 1 -chloroethyl (4-nitrophenyl) carbonate (2.60 g, 10.53 mmol, 98% yield) as a white solid. The crude compound was used in the next step without further purification.

[0445] Step-2: l-((4-Nitrophenoxy)carbonyl)oxy)ethyl isobutyrate

[0446] To a solution of 1-chloroethyl (4-nitrophenyl) carbonate (500 mg, 2.04 mmol) in isobutyric acid (3.70 mL, 40.7 mmol), Ag2O (472 mg, 2.04 mmol) was added, and the mixture was stirred at 95 °C for 2 h. The progress of the reaction was monitored by TLC analysis. The mixture was diluted with EtOAc (20 mL), filtered through a Celite® pad, and the filtrate was basified with with 10 mM aqueous NH4HCO3 solution (10 mL) and extracted with EtOAc (2 x 50 mL). The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to afford l-((4- nitrophenoxy)carbonyl)oxy)ethyl isobutyrate as a yellow liquid (470 mg, 78%). 'H-NMR (400 MHz, CDCh): 8 8.28-8.31 (m, 2H), 7.44-7.40 (m, 2H), 2.67-2.60 (m, 1H), 1.64 (d, J = 5.60 Hz, 3H), 1.36 (d, J= 7.20 Hz, 1H), 1.23 (dd, J= 0.80, 7.00 Hz, 6H).

[0447] Step 3: l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl isobutyrate (Cmpd 13)

[0448] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (0.27 g, 1.29 mmol) in THE (5 mL) at 25 °C was added triethylamine (0.9 mL, 6.5 mmol) and the mixture was stirred for 15 min. l-(((4-Nitrophenoxy)carbonyl)oxy)ethyl isobutyrate (0.461 g, 1.55 mmol) was added and the reaction mixture was stirred at room temperature for 1 h. The crude was diluted with EtOAc (10 mL) and washed with water (10 mL) and brine (10 mL), then dried over Na2SO4 and

[0449] 62

[0450] 20300-WO-PCT / / 103241-016PCT concentrated, to afford crude material (400 mg). The crude material was purified by Isolera- Biotage (silica gel 100-200 mesh, eluted with 3% MeOH in DCM) to afford l-((methyl(2-(pyridin- 2-yl)ethyl)carbamoyl)oxy)ethyl isobutyrate as a brown oil (100 mg, 26%). LCMS (ES+): m / z = 295.1 [M+H]+.

[0451] Cmpd 14: 2-Methyl-l-((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)propyl isobutyrate

[0452] Step 1: l-Chloro-2-methylpropyl (4-nitrophenyl) carbonate

[0453] To a stirred solution of 4-nitrophenol (2.0 g, 14 mmol) in DCM (8 mL) was added pyridine (1.2 mb, 14.4 mmol) and the mixture was stirred for 10 min. 1 -Chloro-2-methylpropyl carbonochloridate (2.7 mL, 18.7 mmol) was dissolved in DCM (2 mL) and added to the reaction mixture dropwise at 0 °C. The reaction mixture was stirred at 25 °C for 16 h. The reaction was monitored by TLC. The reaction mixture was quenched with water (10 mL) and extracted with DCM (2 x 10 mL). Combined organic layers were washed with 10% aqueous NaHCO, solution (10 mL), dried over Na2SO4 and concentrated under reduced pressure to afford crude material as a yellow liquid (4.18 g). The crude product was purified by flash column chromatography (silica- gel, 230-400, eluting with EtOAc - petroleum ether, 0 - 20%). The pure fractions were evaporated and dried under high vacuum to afford 1 -chloro-2-methylpropyl (4-nitrophenyl) carbonate as a pale-yellow solid (3.3 g, 83% yield). ‘H-NMR (400 MHz, DMSO-d6): 88.35 (d, J= 9.20 Hz, 2H), 7.64 (d, J= 9.20 Hz, 2H), 6.44 (d, J= 4.00 Hz, 1H), 2.34-2.29 (m, 1H), 1.05 (d, J= 20.00 Hz, 6H).

[0454] Step 2: 2-Methyl-l-(((4-nitrophenoxy)carbonyl)oxy)propyl isobutyrate

[0455] To a stirred solution of 1 -chloro-2-methylpropyl (4-nitrophenyl) carbonate (1.00 g, 3.65 mmol) in isobutyric acid (3.39 mL, 36.5 mmol) was added silver oxide (0.847 g, 3.65 mmol) at 25 °C. The reaction was stirred at 95 °C for 2 h. The reaction was monitored by TLC. The reaction mixture was filtered through Celite®- and the filtrate was basified with with 10 mmol aqueous NH4HCO3 solution (10 mL) and extracted with EtOAc (2 x 10 mL). The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to afford crude (800 mg). The crude product was purified by flash column chromatography (silica-gel, 230-400, 0 - 20% EtOAc in petroleum

[0456] 63

[0457] 20300-WO-PCT / / 103241-016PCT ether eluent) to afford 2-methyl-l-(((4-nitrophenoxy)carbonyl)oxy)propyl isobutyrate as a yellow liquid (173 mg, 14.4%). ‘H-NMR (400 MHz, DMSO-d6): 8 8.34 (dd, J= 3.20, 9.40 Hz, 2H), 7.58 (dd, = 2.80, 9.40 Hz, 2H), 6.53 (d, J= 6.80 Hz, 1H), 2.70-2.61 (m, 1H), 2.17-2.10 (m, 1H), 1.15- 1.07 (m, 6H), 8 1.00 (d, J= 2.40 Hz, 3H), 0.98 (d, J= 2.40 Hz, 3H).

[0458] Step 3: 2-Methyl-l-((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)propyl isobutyrate (Cmpd 14)

[0459] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (93 mg, 0.45 mmol) in THF (1 mL) was added triethylamine (0.124 mL, 0.889 mmol) at 0 °C and the mixture was stirred for 5 min. 2-Methyl-l-(((4-nitrophenoxy)carbonyl)oxy)propyl isobutyrate (174 mg, 0.534 mmol) was added and the reaction mixture was stirred at 0 °C for 5 min. The reaction was monitored by TLC and LCMS. The reaction mixture was quenched with water (5 mL) and extracted with EtOAc (2 x 5 mL). The combined organic layer was dried over Na2SC>4 and concentrated under reduced pressure to afford crude material (130 mg). The crude product was purified by flash column chromatography (silica-gel, 230-400, 0 - 70% EtOAc in petroleum ether as eluent) to afford 2-methyl-l-((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)propyl isobutyrate as pale yellow liquid (9.1 mg, 6.1%). LCMS (ES+): m / z = 323.1 [M+H]+.

[0460] Cmpd 15: l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl pivalate

[0461] Step-1: 1-Chloroethyl (4-nitrophenyl) carbonate

[0462] To a solution of 4-nitrophenol (1.50 g, 10.8 mmol) in DCM (30 mL) at 0 °C was added pyridine (0.87 mL, 10.8 mmol) followed by dropwise addition of 1 -chloroethyl carbonochloridate (1.70 g, 11.9 mmol). The resulting reaction mixture was then stirred at 0 °C for 30 min followed by stirring at 15 °C for 2.5 h. The progress of the reaction was monitored by TLC analysis. After completion of reaction, the reaction mixture was concentrated under reduced pressure to get crude compound which was partitioned between EtOAc- water and extracted with EtOAc (3 x 30 mL). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure

[0463] 20300-WO-PCT / / 103241-016PCT to get crude 1 -chloroethyl (4-nitrophenyl) carbonate (2.60 g, 10.5 mmol, 98% yield) as a white solid. The crude compound was used in the next step without further purification.

[0464] Step 2: l-(((4-Nitrophenoxy)carbonyl)oxy)ethyl pivalate

[0465] To a stirred solution of 1 -chloroethyl (4-nitrophenyl) carbonate (1.56 g, 6.35 mmol) in pivalic acid (14.3 mL, 127 mmol) was added Ag2O (1.47 g, 6.35 mmol) at 25 °C. The reaction was stirred at 95 °C for 2 h. The reaction was monitored by TLC. The reaction mixture was filtered through Celite® and the filtrate was basified with with 10 mM aqueous NH4HCO3 solution (10 mL) and extracted with EtOAc (2 x 50 mL). The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to afford l-(((4-nitrophenoxy)carbonyl)oxy)ethyl pivalate as a yellow solid (1.46 g, 20%). The compound was used without further purification. ’H-NMR (400 MHz, CDCh): 8 8.33 (dd, J= 9.20 Hz, 2H), 7.45 (dd, J= 9.20 Hz, 2H), 6.53 (q, J= 6.00 Hz, 1H), 1.95 (d, J = 6.00 Hz, 3H), 1.26 (t, J = 10.00 Hz, 9H).

[0466] Step 3: l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl pivalate (Cmpd 15):

[0467] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (250 mg, 1.196 mmol) in THF (5 mL) was added triethylamine (0.500 mL, 3.59 mmol) at 0 °C and the mixture was stirred for 5 min. l-(((4-Nitrophenoxy)carbonyl)oxy)ethyl pivalate (558 mg, 1.79 mmol) was added to the reaction mixture and it was stirred at 25 °C for 3 h. The reaction was monitored by TLC and LCMS. The reaction mixture was quenched with water (5 mL) and extracted with EtOAc (2 x 5 mL). The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to afford crude material which was purified by prep-HPLC (Method E) to afford 1- ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl pivalate as a brown viscous oil (193 mg, 52%). LCMS (ES+): m / z = 309.2 [M+H]+.

[0468] Cmpd 16: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl isobutyrate

[0469] Step 1: Chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate

[0470] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (1.00 g, 4.78 mmol) in DCM (7 mL) was added triethylamine (1.30 mL, 9.56 mmol) at 25 °C and the mixture

[0471] 65

[0472] 20300-WO-PCT / / 103241-016PCT was stirred for 10 min. Chloromethyl carbonochloridate (0.51 mL, 5.74 mmol) was dissolved in DCM (3 mL) and added to the reaction mixture dropwise at 0 °C. The reaction mixture was stirred at 25 °C for 16 h. Reaction progress was monitored by TLC. The reaction mixture was quenched with water (10 mL) and extracted with DCM (2 x 10 mL). Combined organic layer was washed with 10% aqueous solution of NaHCO, (10 mL), dried over Na2SC>4 and concentrated to afford crude as yellow liquid. The crude product was purified by flash column chromatography (silica- gel, 230-400, eluted with 0 - 20% MeOH in DCM) to afford chloromethyl methyl(2-(pyridin-2- yl)ethyl)carbamate as an orange gum (100 mg, 3%). LCMS (ES+): m / z = 229.1 [M+H]+.

[0473] Step 2: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl isobutyrate (Cmpd 16)

[0474] To a stirred solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (80 mg, 0.35 mmol) in isobutyric acid (649 pL, 7.00 mmol) was added Ag2O (81 mg, 0.35 mmol) and the reaction was stirred at 95 °C for 2 h. The reaction was monitored by UPLC. The reaction mixture was diluted with DCM (10 mL) and filtered through Celite®. The filtrate was basified with 10% solution of NaHCO, (10 mL) and extracted with DCM (2 x 5 mL). The combined organic layers were concentrated to afford crude product. The crude product was purified by prep-HPLC Method E to afford ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl isobutyrate as a yellow gum (8 mg, 7.8%). LCMS (ES+): m / z = 281.1 [M+H]+.

[0475] Cmpd 17: Ethyl methyl(2-(pyridin-2-yl)ethyl)carbamate 0 °C-RT, 2h Cmpd 17

[0476] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (100 mg, 0.478 mmol) in DCM (2 mL) at 0 °C was added triethylamine (0.326 mL, 2.39 mmol) and the mixture was stirred for 15 min. Ethyl chloroformate (62.3 mg, 0.574 mmol) was added to the reaction mixture and it was stirred at 25 °C for 1 h. The reaction was monitored by TLC. The reaction mixture was quenched with water (5 mL) and extracted with DCM (2 x 5 mL). The combined organic layer was dried over Na2SC>4 and evaporated under reduced pressure to afford crude. The crude product was purified by flash column chromatography (silica-gel, 230-400, eluted with 0 -

[0477] 66

[0478] 20300-WQ-PCT / / 103241-016PCT 70% EtOAc in petroleum ether) to afford ethyl methyl(2-(pyridin-2-yl)ethyl)carbamate as a colorless viscous oil (35 mg, 34.6%). LCMS (ES+): m / z = 209.1 [M+H]+.

[0479] Cmpd 18: Methyl methyl(2-(pyridin-2-yl)ethyl)carbamate

[0480] Cmpd 18

[0481] 49.2%

[0482] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (250 mg, 1.20 mmol) in in DCM (2 mL) at 0 °C was added triethylamine (0.834 mL, 5.98 mmol) and the mixture was stirred for 15 min. Methyl chloroformate (113 mg, 1.20 mmol) was added to the reaction mixture and it was stirred at 25 °C for 1 h. The reaction was monitored by TLC. The reaction mixture was quenched with water (5 mL) and extracted with DCM (2 x 5 mL). The combined organic layer was dried over Na2SO4 and evaporated under reduced pressure to afford crude material which was purified by flash column chromatography (silica-gel, 230-400, eluted with 0 - 100% EtOAc in petroleum ether) to afford methyl methyl(2-(pyridin-2-yl)ethyl)carbamate as white viscous liquid (115 mg, 49%). LCMS (ES+): m / z = 195.1 [M+H]+.

[0483] Cmpd 19: (5-Methyl-2-oxo-l,3-dioxol-4-yl)methyl methyl(2-(pyridin-2-yl)ethyl)carbamate

[0484] Step 1: (5-Methyl- 2-oxo- l,3-dioxol-4-yl)methyl (4-nitrophenyl) carbonate

[0485] 4-(Hydroxymethyl)-5-methyl-l,3-dioxol-2-one (1.5 g, 11.5 mmol) was dissolved in CHCh (21 mL) and the mixture was cooled to 0 °C. Pyridine (1.0 mL, 12 mmol) was added dropwise to the solution and the reaction mixture was stirred at 0 °C for 5 min. Then 4-nitrophenyl carbonochloridate (2.51 g, 12.5 mmol) was added, and the resulting reaction mixture was stirred at 25 °C for 16 h. The progress of the reaction was monitored by TLC. After the completion of reaction, saturated aqueous NaHCO, was added (25 mL), the mixture was extracted with DCM (3

[0486] 67

[0487] 20300-WO-PCT / / 103241-016PCT x 30 mL), then washed with brine (25 mL) and the organic layer was separated, dried over Na2SC>4 and concentrated under reduced pressure to get crude compound. The crude residue was purified by flash chromatography (silica gel mesh 230-400, eluent: 90-100 % EtOAc in petroleum ether followed by 10% MeOH-DCM) to afford (5-methyl-2-oxo-l,3-dioxol-4-yl)methyl (4-nitrophenyl) carbonate as an off-white solid (2.4 g, 70%). 'H-NMR (400 MHz, DMSO-t / e): 8 8.33 (d, J= 12.4 Hz, 2H), 7.59 (d, J= 12.4 Hz, 2H), 5.19 (s, 2H), 2.19 (s, 3H).

[0488] Step 2: (5-Methyl- 2-oxo- l,3-dioxol-4-yl)methyl methyl(2-(pyridin-2-yl)ethyl)carbamate (Cmpd 19)

[0489] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (150 mg, 0.717 mmol) in CH3CN (2 mL) was added triethylamine (0.501 mL, 3.59 mmol) and the mixture was stirred for 15 min. (5-Methyl-2-oxo-l,3-dioxol-4-yl)methyl (4-nitrophenyl) carbonate (254 mg, 0.861 mmol) was added and the reaction mixture stirred was at 25 °C for 1 h. Completion of the reaction was monitored by TLC and LCMS. The reaction mixture was evaporated to afford a yellow material. The crude product was purified by reverse phase Cl 8 column chromatography using water: ACN (0 - 70%) as eluent. The pure fraction was lyophilized, to afford a colorless viscous oil. The product was further purified by flash column chromatography (silica-gel, 230- 400, eluted with 0 - 70% EtOAc in petroleum ether) to afford (5-methyl-2-oxo-l,3-dioxol-4- yl)methyl methyl(2-(pyridin-2-yl)ethyl)carbamate as white viscous oil (21.31 mg, 8.76%). LCMS (ES+): m / z = 293.1 [M+H]+.

[0490] Cmpd 20: 2-Acetamido-N-methyl-4-(methylthio)-N-(2-(pyridin-2-yl)ethyl)butanamide

[0491] Step 1: Acetylmethionine

[0492] To a stirred solution of DL-methionine (0.500 g, 3.35 mmol) in THE (5 mL) was added NaOH (2 M aqueous solution, 6.5 ml, 13 mmol). The mixture was cooled to 0 °C and then a solution of acetyl chloride (0.359 ml, 5.03 mmol) in THF (2.5 mL) was added slowly to the solution at 0° C. The reaction mixture was stirred at 25 °C for 3 h. and then the solution was acidified with

[0493] 68

[0494] 20300-WO-PCT / / 103241-016PCT concentrated HC1 to pH=2 and extracted with EtOAc (3 x 10 mL). The combined organic layer was washed with water (10 mL) and brine (10 mL), dried over Na2SC>4 and concentrated under reduced pressure to afford crude acetylmethionine as an off-white solid (180 mg, 26%). LCMS (ES+): m / z = 192.1 [M+H]+.

[0495] Step 2: 2-Acetamido-N-methyl-4-(methylthio)-N-(2-(pyridin-2-yl)ethyl)butanamide (Cmpd 20)

[0496] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (236 mg, 1.13 mmol) in DME (5 mL) were added N,N-diisopropylethylamine (0.656 ml, 3.76 mmol) and acetylmethionine (180 mg, 0.941 mmol) followed by ((lH-benzo[d][l,2,3]triazol-l- yl)oxy)tris(dimethylamino)phosphonium hexafluorophosphate (V) (BOP) (458 mg, 1.04 mmol). The mixture was stirred for 1 h at 25 °C. The reaction was monitored by UPLC and TLC. The reaction mixture was quenched with ice cooled water (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layer was washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure to afford crude material (300 mg). The crude material was purified by Prep-HPLC Method B to afford 2-acetamido-N-methyl-4-(methylthio)-N-(2- (pyridin-2-yl)ethyl)butanamide as brown oil (100 mg, 33%). LCMS (ES+): m / z = 310.2 [M+H]+.

[0497] Cmpd 21: Methylene bis(methyl(2-(pyridin-2-yl)ethyl)carbamate)

[0498] A mixture of A-methyl-2-(pyridin-2-yl)ethan-l -amine (1.4 g, 10 mmol) and CS2CO3 (1.4 g, 4.3 mmol) in DMF (30 mL) with a balloon attachment was treated with dry ice to produce a CO2 atmosphere. The mixture stirred for 2 h then chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate hydrochloride salt (0.32 g, 1.4 mmol) was added and the mixture stirred for 18 h. The reaction mixture was diluted with brine (100 mL) and water (100 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic layers were dried (Mg2SC>4) and evaporated. The crude product was purified by chromatography on silica gel eluted with a gradient of methanol in dichloromethane (0-20%) to leave the pure product as a light green oil (78 mg, 15%). LCMS (ES+): m / z = 373.2 [M+H]+.

[0499] Cmpd 22: ((((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)-L-valine

[0500] 69

[0501] 20300-WO-PCT / / 103241-016PCT

[0502] Step 1: Benzyl ((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)-L- val inate

[0503] A mixture of valine benzyl ester hydrochloride (1.0 g, 4.1 mmol) and CS2CO3 (3.9 g, 12 mmol) in DMF (10 mL) was fitted with a balloon and adaptor. The mixture was treated with dry ice to form a CO2 atmosphere and stirred for 2 h. To the reaction mixture was added chloromethyl methyl(2- (pyridin-2-yl)ethyl)carbamate hydrochloride salt (136 mg, 0.51 mmol) and stirring continued for 18 h. The mixture was diluted with water (150 mL) and extracted with ethyl acetate (2 X 100 mL). The combined organic layers were dried (Mg2SC>4) and evaporated. The crude product was purified by reversed phase HPLC (Sunfire column, 5-95% water / acetonitrile with 0.1% TFA) and the product fractions were combined and treated with saturated sodium bicarbonate solution. The solution was extracted with ethyl acetate (2 X 100 mL), combined, dried (Mg2SC>4) and evaporated to leave product as a thick oil (54 mg, 24%). LCMS (ES+): m / z = 444 [M+H]+.

[0504] Step 2: ((((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)-L-valine (Cmpd 22)

[0505] The benzyl ester (50 mg, 0.11 mmol) was hydrogenated at 1 atmosphere over 10% Pd / C (55% water, 20 mg) in methanol (3 mL) for 2 h. The catalyst was filtered and the filtrate evaporated to leave the product as a thick gum (38 mg, 98%). LCMS (ES+): m / z = 354.2 [M+H]+.

[0506] Cmpd 23: ((((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy) methoxy)carbonyl)-L-alanyl-L- valine

[0507] Step 1: Benzyl ((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)-L- alanyl-L-val inate

[0508] A mixture of alanine valine benzyl ester hydrochloride (1.4 g, 4.4 mmol) and CS2CO3 (3.2 g, 10 mmol) in DMF (20 mL) was fitted with a balloon and adaptor. The mixture was treated with dry

[0509] 70

[0510] 20300-WO-PCT / / 103241-016PCT ice to form a CO2 atmosphere and stirred for 2 h. To the reaction mixture was added chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate hydrochloride salt (147 mg, 0.55 mmol) and stirring continued for 72 h. The mixture was diluted with water (150 mL) and extracted with ethyl acetate (2 X 100 mL). The combined organic layers were dried (Mg2SO4) and evaporated. The crude product was purified by chromatography on silica gel using a gradient of methanol in dichloromethane (0-20%). The product was further purified by chromatography on silica gel using a gradient of ethyl acetate in hexanes (50-100%) to leave product as a thick oil (60 mg, 21%). LCMS (ES+): m / z = 567.4 [M+H]+.

[0511] Step 2: ((((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)-L-alanyl-L- valine (Cmpd 23)

[0512] The benzyl ester (52 mg, 0.10 mmol) was hydrogenated at 1 atmosphere over 10% Pd / C (55% water, 20 mg) in methanol (2 mL) for 2 h. The catalyst was filtered and the filtrate evaporated to afford ((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)-L-alanyl-L-valine (Cmpd 23) as a thick gum (39 mg, 92%). LCMS (ES+): m / z = 425.2 [M+H]+.

[0513] Cmpd 24: ((((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl) glycyl-L- valine and Cmpd 73: ((((Methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)glycine

[0514] Stepl : Benzyl ((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)glycinate

[0515] A mixture of glycine benzyl ester hydrochloride (1.6 g, 8.0 mmol) and CS2CO3 (3.2 g, 10 mmol) in DMF (30 mL) was fitted with a balloon and adaptor. The mixture was treated with dry ice to form a CO2 atmosphere and stirred for 2 h. To the reaction mixture was added chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate hydrochloride salt (250 mg, 1.1 mmol) and stirring continued for 18 h. The mixture was diluted with water (150 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic layers were dried (Mg2SC>4) and evaporated. The crude

[0516] 71

[0517] 20300-WO-PCT / / 103241-016PCT product was purified by reversed phase HPLC (Sunfire column, 5-95% water / acetonitrile with 0.1% TFA) and the product fractions were combined and treated with saturated sodium bicarbonate solution. The solution was extracted with ethyl acetate (2 x 100 mL), combined, dried (Mg2SO4) and evaporated to leave product as a thick oil (57 mg, 13%).

[0518] Step 2: ((((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)glycine (cmpd 73)

[0519] Benzyl ((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)glycinate (57 mg, 0.14 mmol) was hydrogenated at 1 atmosphere over 10% Pd / C (55% water, 20 mg) in methanol (2 mL) for 2 h. The catalyst was filtered and the filtrate evaporated to leave the product as a thick gum (42 mg, 96%). 'H NMR (400 MHz, Methanol-^) 8 = 8.56 (d, 7=4.9 Hz, 1H), 7.89 (t, 7=7.7 Hz, 1H), 7.45 (d, 7=7.9 Hz, 1H), 7.39 (dd, 7=5.5, 7.0 Hz, 1H), 5.74 (d, 7=16.9 Hz, 2H), 3.92 (s, 2H), 3.72 (t, 7=7.2 Hz, 2H), 3.18 - 3.06 (m, 2H), 2.98 - 2.87 (m, 3H). LCMS (ES+): m / z = 312 [M+H]+.

[0520] Step 3: Benzyl ((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)glycyl-L- val inate

[0521] ((((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)glycine (42 mg, 0.14 mmol), valine benzyl ester hydrochloride (34 mg, 0.14 mmol) and DIPEA (54 mg, 0.42 mmol, 75 mL) were dissolved in DMF (1.5 mL) and treated with HATU (53 mg, 0.14 mmol). The reaction mixture stirred for 90 min and was diluted with ethyl acetate (50 mL). The solution was washed with water (2 x 25 mL), 1 M NaOH (2 x 25 mL) and brine (25 mL). The organic phase was dried (MgSO-i) and evaporated. The crude product was purified by chromatography on silica gel eluted with a gradient of ethyl acetate in hexanes (50-100%) to leave a thick gum (40 mg, 57%). 'H NMR (400 MHz, MethanoL74) 8 = 8.55 (d, 7=4.9 Hz, 1H), 7.90 - 7.80 (m, 1H), 7.51 - 7.40 (m, 7H), 7.40 - 7.32 (m, 1H), 5.77 (q, 7=6.2 Hz, 1H), 5.73 - 5.67 (m, 1H), 5.33 - 5.16 (m, 2H), 4.51 - 4.43 (m, 1H), 4.02 - 3.84 (m, 2H), 3.72 (t, 7=7.2 Hz, 2H), 3.10 (q, 7=7.3 Hz, 2H), 2.92 (d, 7=2.8 Hz, 4H), 2.31 - 2.15 (m, 1H), 1.07 - 0.92 (m, 6H).

[0522] Step 4: ((((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)glycyl-L-valine (Cmpd 24)

[0523] Benzyl ((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)glycyl-L-valinate (40 mg, 0.080 mmol) was hydrogenated at 1 atmosphere over 10% Pd / C (55% water, 10 mg) in

[0524] 72

[0525] 20300-WO-PCT / / 103241-016PCT methanol (1.5 mL) for 2 h. The catalyst was filtered and the filtrate evaporated to provide ((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)glycyl-L-valine (Cmpd 24) as a thick oil (31 mg, 95%). LCMS (ES+): m / z = 411.27 [M+H]+.

[0526] Cmpd 25: N-Methyl-N-(((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl) glycyl)glycine

[0527] Step 2 Cmpd 25

[0528] Step 1: Benzyl 7V-methyl-7V-(((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy) methoxy)carbonyl)glycyl)glycinate

[0529] ((((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)glycine (22 mg, 0.070 mmol) prepared from the above procedure, sarcosine benzyl ester hydrochloride (17 mg, 0.080 mmol) and DIPEA (27 mg, 0.21 mmol, 38 mL) were dissolved in DMF (1 mL) and treated with HATU (27 mg, 71 pmol). The reaction mixture stirred for 18 h and was purified directly by reversed phase HPLC (Cl 8, 5-95% water / acetonitrile with 0.1% TFA). The product fractions were combined and treated with saturated sodium bicarbonate solution and the product was extracted with ethyl acetate (2 X 50 mL), combined, dried (Mg2SOi) and evaporated to leave product as a thick oil (17 mg, 51%). 'H NMR (400 MHz, Methanol-^) 8 = 8.33 (d, 7=2.6 Hz, 1H), 7.70 - 7.57 (m, 1H), 7.32 - 7.18 (m, 5H), 7.17 - 7.10 (m, 1H), 5.63 - 5.42 (m, 2H), 5.13 - 4.99 (m, 2H), 3.96 (s, 4H), 3.51 (t, 7=7.0 Hz, 2H), 2.98 (m, 3H), 2.93 - 2.79 (m, 3H), 2.71 (m, 3H). LCMS (ES+): m / z = 473 [M+H]+.

[0530] Step 2: N-Methyl-N-(((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl) glycyl)glycine (Cmpd 25):

[0531] Benzyl A-methyl-A-(((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy) methoxy)carbonyl) glycyl)glycinate (17 mg, 0.036 mmol) was hydrogenated at 1 atmosphere over 10% Pd / C (55% water, 7 mg) in methanol (1.5 mL) for 2 h. The catalyst was filtered and the filtrate evaporated to provide A-methyl-A-(((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy) methoxy) carbonyl)glycyl)glycine (Cmpd 25) as a thick oil (14 mg, 100%). LCMS (ES+): m / z = 383 [M+H]+.

[0532] 73

[0533] 20300-WO-PCT / / 103241-016PCT Cmpd 26: ((((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy) methoxy)carbonyl)-L-proline

[0534] Step 1: 2-Benzyl l-(((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl) (S)-pyrrolidine- 1,2-dicarboxylate

[0535] A mixture of proline benzyl ester hydrochloride (2.0 g, 8.3 mmol) and CS2CO3 (3.9 g, 12 mmol) in DMF (30 mL) was fitted with a balloon and adaptor. The mixture was treated with dry ice to form a CO2 atmosphere and stirred for 2 h. To the reaction mixture was added chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate hydrochloride salt (265 mg, 1.0 mmol) and stirring continued for 18 h. The mixture was diluted with water (150 mL) and extracted with ethyl acetate (2 X 100 mL). The combined organic extracts were washed with brine (50 mL), dried (Mg2SC>4) and evaporated. The residue was purified by flash chromatography (silica gel) eluting with a linear gradient of 50-100% ethyl acetate in hexanes and further purified by flash chromatography (silica gel) eluting with a linear gradient of 0-15% methanol in dichloromethane to provide 2-benzyl 1- (((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl) (S)-pyrrolidine-l,2-di carboxylate as a thick oil (100 mg, 23%). LCMS (ES+): m / z = 442 [M+H]+.

[0536] Step 2: ((((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)-L-proline (Cmpd 26)

[0537] 2-Benzyl 1 -(((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl) (S)-pyrrolidine- 1 ,2- dicarboxylate (100 mg, 0.23 mmol) was hydrogenated at 1 atmosphere over 10% Pd / C (55% water, 25 mg) in methanol (3 mL) for 2 h. The catalyst was filtered and the filtrate evaporated to leave the product as a thick oil (83 mg, 100%). LCMS (ES+): m / z = 352.28 [M+H]+.

[0538] Cmpd 27: ((l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethoxy)carbonyl)-L-proline

[0539] 74

[0540] 20300-WO-PCT / / 103241-016PCT Step 1: 2-Benzyl l-(l-((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl) (2S)-pyrrolidine- 1,2-dicarboxylate

[0541] A mixture of proline benzyl ester hydrochloride (1.2 g, 5.0 mmol) and CS2CO3 (2.4 g, 7.5 mmol) in DMF (20 mL) was fitted with a balloon and adapter. The mixture was treated with dry ice to form a CO2 atmosphere and stirred for 2 h. To the reaction mixture was added 1 -chloroethyl methyl(2-(pyridin-2-yl)ethyl)carbamate hydrochloride salt (173 mg, 0.62 mmol) and stirring continued for 18 h at 50 °C. After cooling to room temperature, the mixture was diluted with water (150 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic extracts were washed with brine (50 mL), dried (Mg2SO4) and concentrated. The crude product was purified by flash chromatography (silica gel) eluting with a linear gradient of 0-15% methanol in dichloromethane and further purified by reversed phase HPLC (Cl 8, eluting with 5-95% water / acetonitrile with 0.1% TFA). Product fractions were combined, treated with saturated sodium bicarbonate solution and extracted with ethyl acetate (2 X 100 mL). The combined organic phases were washed with brine, dried (MgSCU) and concentrated to provide 2-benzyl 1-(1- ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl) (2S)-pyrrolidine-l,2-di carboxy late as a thick oil (55 mg, 19%). LCMS (ES+): m / z = 456 [M+H]+.

[0542] Step 2: ((l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethoxy)carbonyl)-L-proline (Cmpd 27)

[0543] 2-Benzyl 1 -(1 -((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl) (2S)-pyrrolidine- 1 ,2- dicarboxylate (55 mg, 0.12 mmol) was hydrogenated at 1 atmosphere over 10% Pd / C (55% water, 16 mg) in methanol (3 mL) for 2 h. The catalyst was filtered and the filtrate concentrated to provide ((l-((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethoxy)carbonyl)-L-proline (Cmpd 27) as a thick oil (44 mg, 100%). LCMS (ES+): m / z = 366.25 [M+H]+.

[0544] Cmpd 28: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl acetyl-L-cysteinate

[0545] Step 1: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl V-acetyLS- trityl cysteinate

[0546] 75

[0547] 20300-WO-PCT / / 103241-016PCT A mixture of A-acetyl-S-trityl-L-cysteine (203 mg, 0.5 mmol), chloromethyl methyl(2-(pyridin-2- yl)ethyl)carbamate (115 mg, 0.5 mmol) and A,A-diisopropylethylamine (0.17 ml, 1.0 mmol) in dichloromethane (5 mL) was stirred under room temperature for 18 h. The reaction mixture was diluted with water (5 mL) and extracted with dichloromethane (2 x 5 mL). The combined organic extracts were dried (Mg2SC>4) and evaporated. The residue was purified by flash chromatography (silica gel) eluting with a linear gradient of 0-10% methanol in dichloromethane to provide ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl A-acetyl-5-tritylcysteinate as a colorless oil (100 mg, 34%). LCMS (ES+): m / z = 598.36 [M+H]+.

[0548] Step 2: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl acetyl-L-cysteinate (Cmpd 28)

[0549] A mixture of ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl A-acetyl-S-tritylcysteinate 24 (100 mg, 0.17 mmol), trifluoroacetic acid (38 mg, 0.33 mmol), triisopropylsilane (52 mg, 0.33 mmol) and dichloromethane (2 mL) was stirred at room temperature for 2 h. The reaction mixture was diluted with water (5 mL) and extracted with dichloromethane (2 x 5 mL). The combined organic extracts were dried (Mg2SC>4) and evaporated. The residue was purified by flash chromatography on silica gel eluted with a gradient of methanol in dichloromethane (0-20%) to provide ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl acetyl-L-cysteinate (Cmpd 28) as a colorless oil (39 mg, 65%). LCMS (ES+): m / z = 356.17 [M+H]+.

[0550] Cmpd 29: 1-Benzyl 2-(((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl) (S)- pyrrolidine-l,2-dicarboxylate

[0551] To a suspension of K2CO3 (0.484 g, 3.50 mmol) in anhydrous DMF in a 40 mL vial at room temperature under N2 was added A-benzyloxycarbonyl-L-proline (0.218 g, 0.875 mmol). The mixture was stirred for 15 minutes before adding methyl-(2-pyridin-2-yl-ethyl)-carbamic acid chloromethyl ester (0.40 g, 1.75 mmol) and it was stirred at room temperature for 64 h. The reaction mixture was partitioned between EtOAc (60 mL) and water (15 mL) and separated. The aqueous layer was re-extracted with EtOAc (60 mL). The organic layers were combined and

[0552] 76

[0553] 20300-WO-PCT / / 103241-016PCT washed with water (3 x 10 mL), then brine (10 mL), then dried with Na2SC>4, filtered, and concentrated. The crude product was purified on silica gel (0 to 100% 25% iPrOH in EtOAc / hexanes over 15 minutes) to provide 1 -benzyl 2-(((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methyl) (S)-pyrrolidine-l,2-dicarboxylate (Cmpd 29) as a clear yellow oil (0.39 g, 100%). LCMS (ES+): m / z = 442.1 [M+H]+.

[0554] Cmpd 30: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((benzyloxy)carbonyl)-L- val inate

[0555] Cmpd 30

[0556] To a suspension of K2CO3 (0.484 g, 3.50 mmol) in anhydrous DMF in a 40 mL vial at room temperature under N2 was added A-carbobenzyloxy-L-valine (0.22 g, 0.88 mmol). The mixture was stirred for 15 minutes before adding methyl-(2-pyridin-2-yl-ethyl)-carbamic acid chloromethyl ester (0.40 g, 1.75 mmol) and stirring at room temperature for 16 h. The reaction mixture was partitioned between EtOAc (60 mL) and water (15 mL) and separated. The aqueous layer was re-extracted with EtOAc (60 mL). The organic layers were combined and washed with water (3 x 10 mL), then brine (10 mL), then dried with Na2SO4, filtered, and concentrated. The residue was purified on silica gel (0 to 100% (25% iPrOH in EtOAc) / hexanes over 20 minutes) to provide ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((benzyloxy)carbonyl)-L- valinate as a clear, colorless oil (0.19 g, 50%). LCMS (ES+): m / z = 444 [M+H]+.

[0557] Cmpd 31: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl N-((benzyloxy)carbonyl)-N- methylglycylglycinate

[0558] Step 1: [2-(Benzyloxycarbonyl-methyl-amino)-acetylamino]-acetic acid ethyl ester

[0559] 77

[0560] 20300-WO-PCT / / 103241-016PCT To a solution of Cbz-Sar-OH (1.18 g, 4.30 mmol) and DIPEA (2.50 mL, 1.85 g, 14.3 mmol) in anhydrous di chloromethane in a small flask at room temperature under N2 was added HATU (1.77 g, 4.66 mmol). The mixture was stirred for 15 minutes before adding glycine ethyl ester hydrochloride (0.50 g, 3.58 mmol) and stirring for 40 h. The reaction mixture was partitioned between EtOAc (60 mL) and sat. NaHCO, solution (15 mL) and separated. The aqueous layer was re-extracted with EtOAc (60 mL). The combined organic extracts were washed with water (15 mL), then brine (10 mL), then dried with Na2SO4, filtered, and concentrated. The crude product was purified on silica gel (0 to 100% ethyl acetate - hexanes) to provide crude [2- (benzyloxycarbonyl-methyl-amino)-acetylamino] -acetic acid ethyl ester as a clear, colorless oil (1.25 g). LCMS (ES+): m / z = 309 [M+H]+.

[0561] Step 2: [2-(Benzyloxycarbonyl-methyl-amino)-acetylamino]-acetic acid

[0562] To a solution of [2-(benzyloxycarbonyl-methyl-amino)-acetylamino]-acetic acid ethyl in a mixture 1:1 ( / v) THF / MeOH (18 mL) at room temperature under N2 was added a 1 M solution of LiOH in water (3.94 mL, 3.94 mmol) and the mixture was stirred for 16 h. The reaction mixture was concentrated to dryness, then stored in the refrigerator over the weekend. The residue was suspended in H2O (20 mL) and washed with Et2O (60 mL), and the organic layer was discarded. The aqueous layer was acidified to pH 1 - 2 by adding 10% aqueous KHSO4 solution and then extracted with DCM (3 x 40 mL). The organic layers were combined and washed with brine (15 mL), then dried with Na2SO4, filtered, and concentrated to yield the desired product as a clear, colorless oil, which eventually became a white solid (0.91 g). The material was used in the next step without further purification. LCMS (ES+): m / z = 281 [M+H]+.

[0563] Step 3: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl N-((benzyloxy)carbonyl)-N- methylglycylglycinate (Cmpd 31)

[0564] To a suspension of K2CO3 (0.453 g, 3.28 mmol) in anhydrous DMF (4 mL) in a 40 mL vial at room temperature under N2 was added [2-(benzyloxycarbonyl-methyl-amino)-acetylamino] -acetic acid (0.23 g). The mixture was stirred for 15 minutes before adding methyl-(2-pyridin-2-yl-ethyl)- carbamic acid chloromethyl ester (0.300 g, 1.31 mmol) and stirring for 16 h. The reaction mixture was partitioned between EtOAc (90 mL) and sat. NH4CI solution (15 mL) and separated. The aqueous layer was re-extracted with EtOAc (30 mL). The organic layers were combined and washed with water (2 x 10 mL), then sat. NaHCO-, solution (15 mL), and brine (15 mL), then dried with Na2SO4, filtered, and concentrated. The residue was purified on silica gel (0 to 100% 25% in

[0565] 78

[0566] 20300-WO-PCT / / 103241-016PCT 75% EtOAc) / hexanes over 20 minutes) to yield ((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methyl N-((benzyloxy)carbonyl)-N-methylglycylglycinate (Cmpd 31) as a clear, yellow oil (0.10 g, 26%). LCMS (ES+): m / z = 473 [M+H]+.

[0567] Cmpd 32: l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl N-((benzyloxy)carbonyl)-

[0568] N-methylglycylglycinate

[0569] Cmpd 32

[0570] A mixture of K2CO3 (0.428 g, 3.10 mmol), [2-(benzyloxycarbonyl-methyl-amino)-acetylamino]- acetic acid (0.217 g, 0.775 mmol), and methyl-(2-pyridin-2-yl-ethyl)-carbamic acid chloromethyl ester (0.300 g, 1.24 mmol) in anhydrous DMF (5 mL) in a 40 mL vial at room temperature under N2 was stirred for 16 h. The reaction was heated at 40 °C for 4 h, then at 50 °C overnight, then 60 °C for a further overnight. The reaction mixture was partitioned between EtOAc (90 mL) and sat. NH4CI solution (15 mL) and separated. The aqueous layer was re-extracted with EtOAc (30 mL). The organic layers were combined and washed with water (2 x 10 mL), then sat. NaHCO-, solution (15 mL), and brine (15 mL), then dried with Na2SO4, filtered, and concentrated. The residue was purified on silica gel (0 to 100% [25% iPrOH in EtOAc] / hexanes over 20 minutes) to provide 1- ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl N-((benzyloxy)carbonyl)-N- methylglycylglycinate (Cmpd 32) as a clear, yellow oil (29 mg, 8%). LCMS (ES+): m / z = 487 [M+H]+.

[0571] Cmpd 33: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2-acetoxyacetyl)-D- prolinate, 2,2,2-trifluoroacetate salt

[0572] Step 1: Benzyl (2R)-l-[2-(acetyloxy)acetyl]pyrrolidine-2-carboxylate

[0573] 79

[0574] 20300-WO-PCT / / 103241-016PCT To a stirred solution of (R)-benzyl pyrrolidine-2-carboxylate hydrochloride (0.26 g, 1.1 mmol) in DMF (3 mL) at 25 °C was added A, V-diisopropylethylamine (0.52 mL, 3.0 mmol). After 5 min of stirring at same temperature, 2-acetoxyacetic acid (0.13 g, 1.1 mmol) was added followed by HATU (0.45 g, 1.19 mmol) and the reaction mixture was stirred at 25 °C for 3 h. The reaction mixture was taken up into ethyl acetate and washed with water then brine. The organic phase was dried over Na2SO4 and the solvent was removed in vacuo. The residue was purified by flash chromatograph eluting with a linear gradient of 0-2% MeOH / methylene chloride to afford benzyl (2R)-l-[2-(acetyloxy)acetyl]pyrrolidine-2-carboxylate (200 mg, 61 %). LCMS (ES+): m / z = 306.1 [M+H]+.

[0575] Step 2: (2R)-l-[2-(Acetyloxy)acetyl]pyrrolidine-2-carboxylic acid

[0576] To a mixture of benzyl (2R)-l-[2-(acetyloxy)acetyl]pyrrolidine-2-carboxylate (200 mg, 0.66 mmol) in EtOH (4 mL) was added 10% Pd / C (50 mg). The reaction was stirred under a hydrogen at 1 atm for 16 h. The mixture was then filtered through Celite® and the solvent was removed in vacuo to provide (2R)-l-[2-(acetyloxy)acetyl]pyrrolidine-2-carboxylic acid (120 mg, 84.5%. LCMS (ES+): m / z =216.0 [M+H]+.

[0577] Step 3: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2-acetoxyacetyl)-D-prolinate, 2,2,2-trifluoroacetate salt (Cmpd 33)

[0578] To a stirred solution of chloromethyl methyl2-(pyridin-2-yl)ethylcarbamate hydrochloride (93 mg, 0.35 mmol) in DMF (2 mL) was added cesium carbonate (171 mg, 0.52 mmol) and (2R)-l-[2- (acetyloxy)acetyl]pyrrolidine-2-carboxylic acid (75 mg, 0.35 mmol). The reaction mixture was stirred at 50 °C for 4 h. The mixture was filtered and the filtrate was purified by C-l 8 reverse phase column chromatography eluting with a linear gradient of 5-95% H2O / CH3CN (containing 0.1% TFA) to provide ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2-acetoxyacetyl)-D- prolinate, 2,2,2-trifluoroacetate salt (Cmpd 33) as a colorless viscous oil (62 mg, 34%). LCMS (ES+): m / z = 408.02 [M+H]+.

[0579] Cmpd 34: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ( 2-acet oxy acet y !)- / )- prolinate

[0580] 80

[0581] 20300-WO-PCT / / 103241-016PCT Step 1: Benzyl (2-acetoxyacetyl)-D-prolinate

[0582] To a stirred solution of 2-acetoxyacetic acid (425 mg, 3.6 mmol) in N, / V-dimethylformamide (40 mL) was added benzyl D-prolinate (1.04 g, 4.32 mmol), l-[bis(dimethylamino)methylene]-177- l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (2.05 g, 5.4 mmol) and triethylamine (1.26 mL, 9.00 mmol) at 25 °C and the reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were dried over Na2SO4 and the solvent was removed in vacuo. The residue was purified by flash column chromatography (silica-gel, 230-400) eluting with a linear gradient of 0 - 50% ethyl acetate in hexane to afford benzyl (2-acetoxyacetyl)- / )-prolinate as a colorless viscous oil (420 mg, 38%). LCMS (ES+): m / z = 306.45 [M+H]+.

[0583] Step 2: (2-Acetoxyacetyl)-D-proline

[0584] To a stirred solution of benzyl (2-acetoxyacetyl)- / )-prolinate (420 mg, 1.37 mmol) in methanol (20 mL) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times. The reaction was stirred at 25 °C under hydrogen atmosphere (balloon) for 18 h, filtered through Celite® and concentrated under reduced pressure. The crude product was carried to the next step without further purification. LCMS (ES+): m / z = 216.25 [M+H]+.

[0585] Step3: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2-acetoxyacetyl)-D-prolinate (Cmpd 34)

[0586] To a stirred solution of 1 -chloroethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (121 mg) in N,N- dimethylformamide (5 mL) was added (2-acetoxyacetyl)- / )-proline (107 mg, 0.5 mmol) and cesium carbonate (325 mg, 1 mmol) at 25 °C and the reaction mixture was stirred at 50 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over Na2SO4 and the solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Cl 8, 10-60% water / acetonitrile with 0.1% TFA) to afford ((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methyl (2-acetoxyacetyl)-D-prolinate (Cmpd 34) as a colorless viscous oil (10 mg, 5% yield). LCMS (ES+): m / z = 422.0 [M+H]+.

[0587] 81

[0588] 20300-WO-PCT / / 103241-016PCT Cmpd 35: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2-acetoxyacetyl)-£- prolinate

[0589] Step 1: Benzyl (2-acetoxyacetyl)-Z-prolinate

[0590] To a stirred solution of 2-acetoxyacetic acid (425 mg, 3.6 mmol) in N, / V-dimethylformamide (40 mL) was added benzyl L-prolinate (1.04 g, 4.32 mmol), l-[bis(dimethylamino)methylene]-lH- l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (2.05 g, 5.4 mmol) and triethylamine (1.3 mL, 9.0 mmol) at 25 °C and the mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were dried over Na2SC>4 and the solvent was removed in vacuo. The residue was purified by flash column chromatography (silica- gel, 230-400) eluting with 0 - 50% ethyl acetate in hexane to afford benzyl (2-acetoxyacetyl)- / .- prolinate as a colorless viscous oil (420 mg, 38%). LCMS (ES+): m / z = 306.47 [M+H]+.

[0591] Step 2: (2-Acetoxyacetyl)-L-proline

[0592] To a stirred solution of benzyl (2-acetoxyacetyl)- / .-prol inate (420 mg, 1.37 mmol) in methanol (20 mL) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times. The reaction was stirred at 25 °C under hydrogen atmosphere (balloon) for 18 h, filtered through Celite® and concentrated under reduced pressure. The crude product was carried to the next step without further purification. LCMS (ES+): m / z = 216.25 [M+H]+.

[0593] Step 3: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2-acetoxyacetyl)-£-prolinate (Cmpd 35)

[0594] To a stirred solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (146 mg) in N,N- dimethylformamide (5 mL) was added (2-acetoxyacetyl)-L-proline (108 mg, 0.5 mmol) and cesium carbonate (285 mg, 0.75 mmol) at 25 °C and the mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over Na2SC>4, and the

[0595] 82

[0596] 20300-WO-PCT / / 103241-016PCT solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Cl 8, 5-60% water / acetonitrile with 0.1% TFA) to afford ((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methyl (2-acetoxyacetyl)-L-prolinate (Cmpd 35) as a colorless viscous oil (50 mg, 25%). LCMS (ES+): m / z = 408.07 [M+H]+.

[0597] Cmpd 36: l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl (2-acetoxyacetyl)-L- prolinate

[0598] To a stirred solution of 1 -chloroethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (121 mg, 0.5 mmol) in A,A-dimethylformamide (5 mL) was added (2-acetoxyacetyl)- / .-proline (107 mg, 0.5 mmol) and cesium carbonate (325 mg, 1 mmol) at 25 °C. The reaction mixture was stirred at 50 °C for 18 h. Completion of reaction was monitored by LCMS and TLC. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over Na2SO4, and the solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Cl 8, 10-60% water / acetonitrile with 0.1% TFA) to afford l-((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2-acetoxyacetyl)- / .- prolinate (Cmpd 36) as a colorless viscous oil (40 mg, 19%). LCMS (ES+): m / z = 422.00 [M+H]+.

[0599] Cmpd 37: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2-(propionyloxy)acetyl)- Z-prolinate

[0600] Step 1: Benzyl (2-(propionyloxy)acetyl)-Z-prolinate

[0601] To a stirred solution of 2-(propionyloxy)acetic acid (626 mg, 3.6 mmol) in N,N- dimethylformamide (40 mL) was added benzyl L-prolinate (1.04 g, 4.32 mmol), 1- [bis(dimethylamino)methy lene] -177-1,2,3 -triazolo [4, 5 -b]pyridinium 3 -oxide hexafluorophosphate (2.05 g, 5.4 mmol) and triethylamine (1.3 mL, 9 mmol) at 25 °C and the reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated

[0602] 83

[0603] 20300-WO-PCT / / 103241-016PCT sodium bicarbonate solution (50 mL) and extracted with ethyl acetate (3 x 50 mb). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash column chromatography (silica-gel, 230-400) eluting with a linear gradient of 0 - 50% ethyl acetate in hexane to afford benzyl (2-(propionyloxy)acetyl)-L-prolinate as a colorless viscous oil (540 mg, 40%).

[0604] Step 2: (2-(Propionyloxy)acetyl)-£-proline

[0605] To a stirred solution of benzyl (2-(propionyloxy)acetyl)-L-prolinate (540 mg, 1.49 mmol) in methanol (40 mL) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times. The reaction was stirred at 25 °C under hydrogen atmosphere (balloon) for 18 h, filtered through Celite® and concentrated under reduced pressure to provide (2-(propionyloxy)acetyl)-L-proline (364 mg, 90%). LCMS (ES+): m / z = 230.36 [M+H]+.

[0606] Step 3: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2-(propionyloxy)acetyl)-£- prolinate (Cmpd 37)

[0607] To a stirred solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (146 mg, 0.6 mmol) in A, / V-dimethylformamide (5 mL) was added (2-(propionyloxy)acetyl)-L-proline (108 mg, 0.5 mmol) and cesium carbonate (285 mg, 0.75 mmol) at 25 °C and the mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over Na2SO4 and the solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Cl 8, 5-60% water / acetonitrile with 0.1% TFA) to afford ((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methyl (2-(propionyloxy)acetyl)-L-prolinate (Cmpd 37) as a colorless viscous oil (90 mg, 42% yield). LCMS (ES+): m / z = 422.0 [M+H]+.

[0608] Cmpd 38: (((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2-(propionyloxy)acetyl)- D-prolinate

[0609] 84

[0610] 20300-WO-PCT / / 103241-016PCT

[0611] Step 1: 2-(Benzyloxy)-2-oxoethyl propionate

[0612] To a stirred solution of benzyl 2-hydroxyacetate (665 mg, 4 mmol) in dichloromethane (40 mL) was added acetic anhydride (0.7 mL, 6 mmol), tri ethylamine (1.1 mL, 8 mmol) and 4- dimethylaminopyridine (5 mg, 0.04 mmol) at 25 °C and the mixture was stirred at 25 °C for 18 h. The reaction mixture was quenched with water (50 mL) and extracted with methylene chloride (3 x 50 mL). The combined organic extracts were dried over ISfeSC and the solvent was removed in vacuo. The residue was purified by flash column chromatography (silica-gel, 230-400) eluting with 0 - 10% ethyl acetate in hexane to afford 2-(benzyloxy)-2-oxoethyl propionate as a colorless viscous oil (800 mg, 90 %). LCMS (ES+): m / z = 223.35 [M+H]+.

[0613] Step 2: 2-(Propionyloxy)acetic acid

[0614] To a stirred solution of 2-(benzyloxy)-2-oxoethyl propionate (800 mg, 3.9 mmol) in methanol (40 mL) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times. The reaction was stirred at 25 °C under hydrogen atmosphere (balloon) for 18 h, filtered through Celite® and concentrated under reduced pressure to provide 2-(propionyloxy)acetic acid. (425 mg, 89%). LCMS (ES+): m / z = 133.21 [M+H]+.

[0615] Step 3: Benzyl (2-(propionyloxy)acetyl)-Z)-prolinate

[0616] To a stirred solution of 2-(propionyloxy)acetic acid (626 mg, 3.6 mmol) in V, V- dimethylformamide (40 mL) was added benzyl D-prolinate (1.04 g, 4.32 mmol), 1- [bis(dimethylamino)methy lene] - H- 1 ,2, 3 -triazolo [4, 5 -b]pyridinium 3 -oxide hexafluorophosphate (2.05 g, 5.4 mmol) and triethylamine (1.26 mL, 9 mmol) at 25 °C and the mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were dried over Na2SO4 and the solvent was removed in vacuo. The residue was purified

[0617] 85

[0618] 20300-WO-PCT / / 103241-016PCT by flash column chromatography (silica-gel, 230-400) eluting with 0 - 50% ethyl acetate in hexane to afford benzyl (2-(propionyloxy)acetyl)-D-prolinate as a colorless viscous oil (540 mg, 40%).

[0619] Step 4: (2-( Propionyloxy (acetyl)- / )- proline

[0620] To a stirred solution of benzyl (2-(propionyloxy)acetyl)-D-prolinate (540 mg, 1.49 mmol) in methanol (40 mb) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times. The reaction was stirred at 25 °C under hydrogen atmosphere (balloon) for 18 h, filtered through Celite® and concentrated under reduced pressure to provide (2-(propionyloxy)acetyl)-D-proline. (364 mg, 90%). LCMS (ES+): m / z = 230.36 [M+H]+.

[0621] Step 5: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2-(propionyloxy)acetyl)-Z>- prolinate (Cmpd 38)

[0622] To a stirred solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (146 mg, 0.6 mmol) in A,A-dimethylformamide (5 mL) was added (2-(propionyloxy)acetyl)- / )-proline (115mg, 0.5 mmol) and cesium carbonate (285 mg, 0.75 mmol) at 25 °C and the mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Cl 8, 5-60% water / acetonitrile with 0.1% TLA) to afford ((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methyl (2-(propionyloxy)acetyl)-D-prolinate (Cmpd 38) as a colorless viscous oil (90 mg, 43%). LCMS (ES+): m / z = 422.00 [M+H]+.

[0623] Cmpd 39: l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl (2-(propionyloxy)acetyl)- / Lprolinate

[0624] To a stirred solution of 1 -chloroethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (121 mg, 0.5 mmol) in A,A-dimethylformamide (10 mL) was added (2-(propionyloxy)acetyl)-D-proline (115 mg, 0.5 mmol) and cesium carbonate (325 mg, 1 mmol) at 25 °C and the mixture was stirred at 50 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and

[0625] 86

[0626] 20300-WO-PCT / / 103241-016PCT extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over Na2SC>4 and the solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Cl 8, 5-60% water / acetonitrile with 0.1% TFA) to afford 1 -((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)ethyl (2-(propionyloxy)acetyl)-D-prolinate (Cmpd 39) as a colorless viscous oil (40 mg, 18% yield). LCMS (ES+): m / z = 436.53 [M+H]+.

[0627] Cmpd 40: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((R)-2-acetoxypropanoyl)-

[0628] D-prolinate, 2,2,2-trifluoroacetate salt

[0629] Step 1: Benzyl (2R)-l-[(2R)-2-(acetyloxy)propanoyl]pyrrolidine-2-carboxylate

[0630] To a stirred solution of (R)-benzyl pyrrolidine-2-carboxylate hydrochloride (0.24 g, 1.0 mmol) in DMF (4 mL) at 25 °C was added / V, / V-diisopropylethylamine (0.52 mL, 3.0 mmol). After 5 min of stirring at same temperature, (R)-2-acetoxypropanoic acid (0.15 g, 1.1 mmol) was added followed by HATU (0.42 g, 1.1 mmol) and the reaction mixture was stirred at 25 °C for 3 h. The reaction mixture was taken up into ethyl acetate and washed with water then brine. The organic phase was dried over Na2SO4 and the solvent was removed in vacuo. The residue was purified by flash chromatograph eluting with a linear gradient of 0-2% MeOH / DCM to afford benzyl (2R)-1-[(2R)- 2-(acetyloxy)propanoyl]pyrrolidine-2-carboxylate (330 mg, quantitative yield). LCMS (ES+): m / z = 320.0 [M+H]+.

[0631] Step 2: (2R)-l-[(2R)-2-(Acetyloxy)propanoyl]pyrrolidine-2-carboxylic acid

[0632] Benzyl (2R)-l-[(2R)-2-(acetyloxy)propanoyl]pyrrolidine-2-carboxylate (0.33 g, 1.0 mmol) was taken up into EtOH (4 mL) and 10% Pd / C (50 mg) was added. The reaction was stirred under H2 at 1 atm overnight. The reaction was then filtered through Celite® and the solvent was removed in vacuo to provide (2R)-l-[(2R)-2-(acetyloxy)propanoyl]pyrrolidine-2-carboxylic acid (130 mg, 57 %. LCMS (ES+): m / z =230.0 [M+H]+.

[0633] 87

[0634] 20300-WO-PCT / / 103241-016PCT Step 3: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((R)-2-acetoxypropanoyl)-D- prolinate, 2,2,2-trifluoroacetate salt (Cmpd 40)

[0635] To a stirred solution of chloromethyl methyl2-(pyridin-2-yl)ethylcarbamate hydrochloride (75 mg, 0.28 mmol) in DMF (2 mL) was added cesium carbonate (137 mg, 0.42 mmol) and (2R)-1-[(2R)- 2-(acetyloxy)propanoyl]pyrrolidine-2-carboxylic acid (65 mg, 0.28 mmol) and the reaction mixture was stirred at 50 °C for 4 h. The mixture was filtered and the filtrate was purified by C-18 reverse phase column chromatography eluting with a linear gradient of 5-95% H2O / CH3CN (containing 0.1% TFA) to provide ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((R)-2- acetoxypropanoyl)-D-prolinate, 2,2,2-trifluoroacetate salt (Cmpd 40) as a colorless viscous oil (90 mg, 60% yield). LCMS (ES+): m / z = 422.36 [M+H]+.

[0636] Cmpd 41: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2-((L-valyl)oxy)acetyl)-D- prolinate, 2,2,2-trifluoroacetate salt

[0637] Step 1: / er / - Butyl (5)-l-((((benzyloxy)carbonyl)methoxy)carbonyl)-2- methylpropylcarbamate

[0638] To a stirred mixture of benzyl 2-hydroxyacetate (1.50 g, 9.03 mmol), Boc-valine (1.88 g, 9.93 mmol), and 4-(dimethylamino)pyridine (55 mg, 0.45 mmol) in dichloromethane (15 mL) was added l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.08 g, 10.84 mmol). After stirring for 16 h, the mixture was diluted with dichloromethane (20 mL) and washed with 0.5 M HCI (20 mL) followed by saturated sodium bicarbonate (20 mL). The organic phase was dried over Na2SC>4 and the solvent was removed in vacuo. The residue was purified by flash chromatography (40 g silica, eluting with a linear gradient of 0 to 30% EtOAc / hexanes) to provide tert-butyl (S)-l-((((benzyloxy)carbonyl)methoxy)carbonyl)-2-methylpropylcarbamate (2.33 g, 71%) as a clear oil. 'H NMR (400 MHz, Chloroform-7) 5 = 7.47 - 7.29 (m, 5H), 5.20 (s, 2H), 5.00 (d, 7=9.2 Hz, 1H), 4.92 - 4.50 (m, 2H), 4.34 (dd, 7=4.3, 9.1 Hz, 1H), 2.35 - 2.17 (m, 1H), 1.51 - 1.40 (m, 9H), 1.01 (d, 7=6.8 Hz, 3H), 0.93 (d, 7=6.8 Hz, 3H). LCMS (ES+): m / z = 266.7 [M+H-

[0639] 88

[0640] 20300-WO-PCT / / 103241-016PCT Boc]+.

[0641] Step 2: 2-(((tert-Butoxycarbonyl)-Z-valyl)oxy)acetic acid

[0642] A solution of tert-butyl (S)-l-((((benzyloxy)carbonyl)methoxy)carbonyl)-2-methylpropyl carbamate (2.02 g, 6.02 mmol) in ethyl acetate (60 mL) was prepared under nitrogen atmosphere and Pd / C (10%, 200 mg) was added. A vacuum was applied, followed by a hydrogen balloon. This was repeated three times, and the mixture was stirred under a hydrogen atmosphere (balloon) for 16 h. The reaction mixture was vacuum filtered through Celite® and the filtrate was concentrated in vacuo to a clear oil which was dried under high vacuum to provide 2-(((tert- butoxycarbonyl)-Z-valyl)oxy)acetic acid (1.72 g, quantitative yield).JH NMR (400 MHz, Chloroform- ) 8: 5.03 (d, 7=9.2 Hz, 1H), 4.88 - 4.61 (m, 2H), 4.28 (dd, 7=5.1, 8.6 Hz, 1H), 2.35 - 2.15 (m, 1H), 1.46 (s, 9H), 1.09 - 0.88 (m, 6H). LCMS (ES+): m / z = 176.4 [M-Boc]+.

[0643] Step 3: Benzyl (2-(((tert-butoxycarbonyl)-Z-valyl)oxy)acetyl)-Z)-prolinate

[0644] To a mixture of (R)-benzyl pyrrolidine-2-carboxylate hydrochloride salt (0.58 g, 2.41 mmol) and 2-(((tert-butoxycarbonyl)-L-valyl)oxy)acetic acid (0.73 g, 2.65 mmol) in DMF (5.5 mL) at 0 °C was added HATU (1.10 g, 2.89 mmol) followed by A,A-diisopropylethylamine (1.26 mL, 7.23 mmol). The mixture was allowed to warm to room temperature and stirred for 100 min. The mixture was diluted with EtOAc (25 mL) and water (20 mL). The layers were separated, and the aqueous phase was extracted with EtOAc (10 mL). The combined organic extracts were washed with 0.3 M HC1 (20 mL) and brine (20 mL), dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash chromatography (40 g silica, eluting with a linear gradient of 15 to 85% EtOAc / Hex) to provide benzyl (2-((( / / 7-butoxycarbonyl)-L- valyl)oxy)acetyl)-D-prolinate (0.99 g, 89%) as a clear oil. 'H NMR (400 MHz, Chloroform-7) 8 = 7.46 - 7.30 (m, 5H), 5.26 - 5.11 (m, 2H), 5.05 (d, 7=8.9 Hz, 1H), 4.84 - 4.69 (m, 2H), 4.67 - 4.43 (m, 1H), 4.42 - 4.26 (m, 1H), 3.73 - 3.41 (m, 2H), 2.41 - 1.82 (m, 5H), 1.52 - 1.37 (m, 9H), 1.08 - 0.88 (m, 6H). LCMS (ES+): m / z = 464.1 [M+H]+.

[0645] Step 4: (2-(((tert-Butoxycarbonyl)-L-valyl)oxy)acetyl)-Z)-proline

[0646] A solution of benzyl (2-((( / r7 -butoxycarbonyl)-L-valyl)oxy)acetyl)- / )-prol inate (0.84 g, 1.92 mmol) in ethyl acetate (60 mL) was prepared under nitrogen atmosphere and Pd / C (10%, 90 mg) was added. A vacuum was applied, followed by a hydrogen balloon. This was repeated three times, and the mixture was stirred under a hydrogen atmosphere (balloon) overnight. The reaction

[0647] 89

[0648] 20300-WO-PCT / / 103241-016PCT mixture was vacuum filtered through Celite®, and the filtrate was concentrated in vacuo to a clear oil which was dried under high vacuum to provide (2-(((terZ-butoxycarbonyl)-L-valyl)oxy)acetyl)- D-proline (0.68 g, quantitative yield). LCMS (ES+): m / z = 396.0 [M+Na]+.

[0649] Step 5: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2-(((ter / -butoxycarbonyl)-Z- valyl)oxy)acetyl)-Z)-prolinate

[0650] Cesium carbonate (0.24 g, 0.722 mmol) was added to a solution of (2-((( / / 7-butoxycarbonyl)-A- valyl)oxy)acetyl)-D-proline (0.16 g, 0.44 mmol) and chloromethyl methyl(2-(pyridin-2- yl)ethyl)carbamate ethylcarbamate hydrochloride salt (0.12 g, 0.44 mmol) in DMF (2.2 mL). The mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with water (4 mL) and EtOAc (6 mL) and stirred for a further 15 minutes. The layers were separated, and the aqueous phase was extracted with EtOAc (5 mL). The combined organic extracts were washed with water (6 mL), and brine (6 mL), dried over Na2SO4, and the solvent was removed in vacuo. The residue was purified by flash chromatography (25 g silica, eluting with a linear gradient of 0.75% to 5% MeOH / DCM) to provide ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2- ((( / r 7-butoxycarbonyl)-A-valyl)oxy)acetyl)- / )-prolinate (0.191 g, 77%) as a clear oil.JH NMR (400 MHz, Chloroform- ) 8= 8.54 (d, 7=4.8 Hz, 1H), 7.62 (t, 7=7.6 Hz, 1H), 7.26 - 7.11 (m, 2H), 5.89 - 5.65 (m, 2H), 5.18 - 4.94 (m, 1H), 4.87 - 4.65 (m, 2H), 4.63 - 4.22 (m, 2H), 3.74 - 3.41 (m, 4H), 3.13 - 2.94 (m, 2H), 2.94 - 2.79 (m, 3H), 2.40 - 2.15 (m, 2H), 2.14 - 1.80 (m, 3H), 1.52 - 1.38 (m, 9H), 1.08 - 0.87 (m, 6H). LCMS (ES+): m / z = 565.3 [M+H]+.

[0651] Step 6: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2-((L-valyl)oxy)acetyl)-D- prolinate, 2,2,2-trifluoroacetate salt (Cmpd 41)

[0652] To a solution of ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2-((( / c / 7-butoxycarbonyl)- L-valyl)oxy)acetyl)-D-prolinate (119 mg, 0.21 mmol) in methylene chloride (4 mL) was added a solution of 1 : 1 (y / v) TEADCM (6 mL) slowly. After stirring for 35 minutes at room temperature, toluene (3 mL) was added to the reaction mixture, the volatiles were removed in vacuo, and the resulting clear residue was dried under high vacuum. The resulting clear oil was taken up in a minimum amount of 9: 1 (v / v) water / CFLCN, frozen in a dry ice / acetone bath, and lyophilized to provide ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (2-((L-valyl)oxy)acetyl)-D- prolinate, 2,2,2-trifluoroacetate salt (Cmpd 41) (121 mg, 83%) as a clear solid.

[0653] Cmpd 42: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl

[0654] 90

[0655] 20300-WO-PCT / / 103241-016PCT ((7?)-2-acetoxy-3-methylbutanoyl)-£-prolinate

[0656] Step 1: Benzyl ( / ?)-2-hydroxy-3-niethyl but anoate

[0657] To a stirred solution of ( / ?)-2-hydroxy-3 -methylbutanoic acid (1.18 g, 10 mmol) in acetonitrile (100 mL) was added benzyl bromide (1.2 mL, 10 mmol) and potassium carbonate (2.07 g, 15 mmol) at 25 °C and the reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography (silica-gel, 230-400) eluting with a linear gradient of 0 - 40% ethyl acetate in hexane to afford benzyl (7?)-2-hydroxy-3-methylbutanoate as a colorless viscous oil (1.50 g, 72%). LCMS (ES+): m / z = 209.19 [M+H]+. 'H NMR (400 MHz, Chloroform- ) 57.47 - 7.28 (m, 5H), 5.32 - 5.12 (m, 2H), 4.09 (m, 1H), 2.09 (m, 1H), 1.01 (d, J= 6.9 Hz, 3H), 0.85 (m, 3H).

[0658] Step 2: Benzyl (R)-2-acetoxy-3-methylbutanoate

[0659] To a stirred solution of benzyl ( / ?)-2-hydroxy-3-methylbutanoate (0.82 g, 4.0 mmol) in dichloromethane (40 mL) was added acetic anhydride (0.57 mL, 6.0 mmol), triethyl amine (1.1 mL, 8.0 mmol) and 4-dimethylaminopyridine (5 mg, 0.04 mmol) at 25 °C and the reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was quenched with water (50 mL) and extracted with methylene chloride (3 x 50 mL). The combined organic extracts were dried over Na2SC>4 and the solvent was removed in vacuo. The residue was purified by flash column chromatography (silica-gel, 230-400) eluting with a linear gradient of 0 - 10% ethyl acetate in hexane to afford benzyl ( / ?)-2-acetoxy-3-methylbutanoate as a colorless viscous oil (1.00 g, 99% yield). LCMS (ES+): m / z = 251.35 [M+H]+. 'H NMR (400 MHz, Chloroform- ) 8 7.45 - 7.29 (m, 5H), 5.18 (m, 2H), 4.88 (d, J= 4.6 Hz, 1H), 2.35 - 2.19 (m, 1H), 2.14 (m, 3H), 1.02 - 0.85 (m, 6H).

[0660] Step 3: (7?)-2-acetoxy-3-methylbutanoic acid

[0661] 91

[0662] 20300-WO-PCT / / 103241-016PCT To a stirred solution of benzyl ( / ?)-2-acetoxy-3-methylbutanoate (1.0 g, 4.0 mmol) in methanol (40 mb) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times. The reaction was stirred at 25 °C under hydrogen atmosphere (balloon) for 18 h. The mixture filtered through Celite® and concentrated under reduced pressure to provide ( / ?)-2-acetoxy-3 -methylbutanoic acid (580 mg, 90% yield). LCMS (ES+): m / z = 161.41 [M+H]+.1H NMR (400 MHz, Chloroform- ) 8 5.01 - 4.77 (m, 1H), 2.28 (m, 1H), 2.15 (m, 3H), 1.01 (m, 6H).

[0663] Step 4: Benzyl ((R)-2-acetoxy-3-methylbutanoyl)-D-prolinate

[0664] To a stirred solution of ( / ?)-2-acetoxy-3 -methylbutanoic acid (580 mg, 3.6 mmol) in V,2V- dimethylformamide (40 mL) was added benzyl L-prolinate (1.04 g, 4.32 mmol), 1- [bis(dimethylamino)methy lene] - 1 H- 1 ,2, 3 -triazolo [4, 5 -b]pyridinium 3 -oxide hexafluorophosphate (2.05 g, 5.4 mmol) and triethylamine (1.26 mL, 9 mmol) at 25 °C and the reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were dried over Na2SO4 and the solvent was removed in vacuo. The residue was purified by flash column chromatography (silica-gel, 230-400) eluting with a linear gradient of 0 - 50% ethyl acetate in hexane to afford benzyl (( / )-2-acetoxy-3-methylbutanoyl)- / .-prolinate as a colorless viscous oil (600 mg, 46 %). LCMS (ES+): m / z = 348.41 [M+H]+. 'H NMR (400 MHz, Chloroform- ) 87.39 - 7.29 (m, 5H), 5.25 - 5.07 (m, 2H), 4.95 (d, J= 7.7 Hz, 1H), 3.98 (m, 1H), 3.60 - 3.46 (m, 1H), 2.30 - 2.07 (m, 4H), 2.03 (m, 3H), 1.94 - 1.86 (m, 2H), 1.10 - 0.87 (m, 6H).

[0665] Step 5: ((7?)-2-Acetoxy-3-methylbutanoyl)-£-proline

[0666] To a stirred solution of benzyl (( / ?)-2-acetoxy-3-methylbutanoyl)- / .-prol inate (600 mg, 1.72 mmol) in methanol (40 mL) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times. The reaction was stirred at 25 °C under hydrogen atmosphere (balloon) for 18 h. The reaction mixture was filtered through Celite® and concentrated under reduced pressure. The crude product was carried to the next step without further purification. LCMS (ES+): m / z = 258.53 [M+H]+.

[0667] Step 6: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((7?)-2-acetoxy-3- methylbutanoyl)-£-prolinate (Cmpd 42)

[0668] 92

[0669] 20300-WO-PCT / / 103241-016PCT To a stirred solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (43 mg, 0.19 mmol) in A,A-dimethylformamide (5 mL) was added (( / ?)-2-acetoxy-3-methylbutanoyl)- / .-proline (50 mg, 0.19 mmol) and cesium carbonate (124 mg, 0.38 mmol) at 25 °C and the reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over ISfeSCU and the solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Sunfire column, 5-60% water / acetonitrile with 0.1% TFA) to afford ((methyl(2- (pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (( / ?)-2-acetoxy-3-methylbutanoyl)- / .-prolinate (Cmpd 42) as a colorless viscous oil (60 mg, 34% yield). LCMS (ES+): m / z = 450.11 [M+H]+.

[0670] Cmpd 43: l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl ((7?)-2-acetoxy-3- methylbutanoyl)-£-prolinate

[0671] To a stirred solution of 1 -chloroethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (95 mg, 0.39 mmol) in N, / V-di methyl formamide (5 mL) was added (( / )-2-acetoxy-3 -methyl butanoyl )- / .-proline (100 mg, 0.39 mmol) and cesium carbonate (254 mg, 0.78 mmol) at 25 °C and the reaction mixture was stirred at 50 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over ISfeSCU and the solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Sunfire column, 5-60% water / acetonitrile with 0.1% TFA) to afford 1 -((methyl(2- (pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl (( / ?)-2-acetoxy-3-methylbutanoyl)- / .-prolinate (Cmpd 43) as a colorless viscous oil (50 mg, 28% yield). LCMS (ES+): m / z = 464.08 [M+H]+.

[0672] 93

[0673] 20300-WO-PCT / / 103241-016PCT Cmpd 44: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((N)-2-acetoxy-3- methylbutanoyl)-Z)-prolinate

[0674] Step 1: Benzyl (N)-2-hydroxy-3-methylbutanoate

[0675] To a stirred solution of (S)-2-hydroxy-3 -methylbutanoic acid (1.18 g, 10 mmol) in acetonitrile (100 mL) was added benzyl bromide (1.2 mL, 10 mmol) and potassium carbonate (2.07 g, 15 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 18 h. The solvent was removed in vacuo and the residue was purified by flash chromatography (silica-gel, 230-400) eluting with a linear gradient of 0-40% ethyl acetate / hexanes to afford benzyl (S)-2-hydroxy-3-methylbutanoate as a colorless viscous oil (1.30 g, 63% yield). LCMS (ES+): m / z = 209.2 [M+H]+. NMR (400 MHz, Chloroform- ) 8 7.47 - 7.28 (m, 5H), 5.32 - 5.12 (m, 2H), 4.09 (dd, J= 6.2, 3.4 Hz, 1H), 2.09 (m, 1H), 1.01 (d, J= 6.9 Hz, 3H), 0.83 (d, J= 6.8 Hz, 3H).

[0676] Step 2: Benzyl (N)-2-acetoxy-3-methylbutanoate

[0677] To a stirred solution of benzyl (S)-2-hydroxy-3-methylbutanoate (816 mg, 4.0 mmol) in dichloromethane (40 mL) was added acetic anhydride (0.57 mL, 6 mmol), triethylamine (1.1 mL, 8.0 mmol) and 4-dimethylaminopyridine (5 mg, 0.04 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was quenched with water (50 mL) and extracted with methylene chloride (3 x 50 mL). The combined organic extracts were dried over Na2SO4and the solvent was removed in vacuo. The residue was purified by flash chromatography (silica-gel, 230-400) eluting with a linear gradient of 0-10% ethyl acetate in hexanes to afford benzyl (S)-2- acetoxy-3-methylbutanoate as a colorless viscous oil (1.0 g, 99 %). LCMS (ES+): m / z = 251.35 [M+H]+. 'H NMR (400 MHz, Chloroform- ) 87.45 - 7.29 (m, 5H), 5.18 (m, 2H), 4.88 (d, J= 4.5 Hz, 1H), 2.35 - 2.19 (m, 1H), 2.14 (m, 3H), 1.02 - 0.85 (m, 6H).

[0678] Step 3: (N)-2-Acetoxy-3-methylbutanoic acid

[0679] 94

[0680] 20300-WO-PCT / / 103241-016PCT To a stirred solution of benzyl (8)-2-acetoxy-3-methylbutanoate (1.0 g, 4 mmol) in methanol (40 mb) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times. The mixture was then stirred at 25 °C under a hydrogen atmosphere for 18 h. The reaction mixture filtered through Celite® and concentrated under reduced pressure to provide (S)-2-acetoxy-3 -methylbutanoic acid (627 mg, 98%). LCMS (ES+): m / z = 161.41 [M+H]+. Tl NMR (400 MHz, Chloroform- ) 8 5.01 - 4.77 (m, 1H), 2.27 (m, 1H), 2.15 (m, 3H), 1.02 (m, 6H).

[0681] Step 4: Benzyl ((N)-2-acetoxy-3-methylbutanoyl)-Z)-prolinate

[0682] To a stirred solution of (5)-2-acetoxy-3 -methylbutanoic acid (580 mg, 3.6 mmol) in V.7V- dimethylformamide (40 mL) was added benzyl D-prol inate (1.04 g, 4.32 mmol) and 1- [bis(dimethylamino)methy lene] -177-1,2,3 -triazolo [4, 5 -b]pyridinium 3 -oxide hexafluorophosphate (2.05 g, 5.4 mmol) and triethylamine (1.26 mL, 9 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash chromatography (silica-gel, 230-400) eluting with a linear gradient of 0-50% ethyl acetate in hexanes to afford benzyl (fS')-2-acetoxy-3-methylbutanoyl)- / )-prol inate as a colorless viscous oil (600 mg, 46% yield). LCMS (ES+): m / z = 348.05 [M+H]+. 'H NMR (400 MHz, Chloroform - ) 87.39 - 7.29 (m, 5H), 5.25 - 5.07 (m, 2H), 4.95 (m, 1H), 3.98 (m, 1H), 3.60 - 3.46 (m, 1H), 2.30 - 2.07 (m, 4H), 2.03 (m, 3H), 1.94 - 1.86 (m, 2H), 1.10 - 0.87 (m, 6H).

[0683] Step 5: ((N)-2-Acetoxy-3-methylbutanoyl)-Z)-proline

[0684] To a stirred solution of benzyl(fS')-2-acetoxy-3 -methyl butanoyl )- / )-prol inate (600 mg, 1.72 mmol) in methanol (40 mL) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times and the mixture was then stirred at 25 °C under a hydrogen atmosphere for 18 h. The reaction mixture filtered through Celite® and concentrated under reduced pressure. The crude product was carried to the next step without further purification. LCMS (ES+): m / z = 258.53 [M+H]+.

[0685] Step 6: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((N)-2-acetoxy-3- methylbutanoyl)-Z)-prolinate (Cmpd 44)

[0686] 95

[0687] 20300-WO-PCT / / 103241-016PCT To a stirred solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (43 mg, 0.19 mmol) in A, / V-dimethylformamide (5 mL) was added (fS -2-acetoxy-3 -methyl butanoyl )- / )-proline (50 mg, 0.19 mmol) and cesium carbonate (124 mg, 0.38 mmol) at 25 °C and the reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over ISfeSCU and the solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Cl 8, 5-60% water / acetonitrile with 0.1% TFA) to afford ((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methyl (fS')-2-acetoxy-3-methylbutanoyl)- / )-prolinate (Cmpd 44) as a colorless viscous oil (20 mg, 23% yield). LCMS (ES+): m / z = 450.11 [M+H]+.

[0688] Cmpd 45: l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl ((5)-2-acetoxy-3- methylbutanoyl)-D-prolinate

[0689] To a stirred solution of 1 -chloroethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (95 mg, 0.39 mmol) in A,A-dimethylformamide (5 mL) was added (fS')-2-acetoxy-3-methylbutanoyl)- / )-proline (100 mg, 0.39 mmol) and cesium carbonate (254 mg, 0.78 mmol) at 25 °C and the mixture was stirred at 50 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Sunfire column, 5-60% water / acetonitrile with 0.1% TFA) to afford l-((methyl(2-(pyridin- 2-yl)ethyl)carbamoyl)oxy)ethyl (fS')-2-acetoxy-3-methylbutanoyl)- / )-prolinate (Cmpd 45) as a colorless viscous oil (50 mg, 28% yield). LCMS (ES+): m / z = 464.08 [M+H]+.

[0690] Cmpd 46: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ( ( / ?)-3-met hy l-2- (propionyloxy)butanoyl)-£-prolinate

[0691] 96

[0692] 20300-WO-PCT / / 103241-016PCT

[0693] Step 1: Benzyl ( / ?)-3-inethyl-2-( propionyloxy (but anoate

[0694] To a stirred solution of benzyl ( / ?)-2-hydroxy-3-methylbutanoate (816 mg, 4.0 mmol) in methylene chloride (40 mL) was added propionic anhydride (0.77 mb, 6 mmol), triethylamine (1.1 mL, 8 mmol) and 4- dimethylaminopyridine (5 mg, 0.04 mmol) at 25 °C and the reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was quenched with water (50 mL) and extracted with methylene chloride (3 x 50 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash column chromatography (silica-gel, 230-400) eluting with a linear gradient of 0 - 10% ethyl acetate in hexane to afford benzyl (7?)-3-methyl-2-(propionyloxy)butanoate as a colorless viscous oil (1057 mg, 99%). LCMS (ES+): m / z = 265.37 [M+H]+. 'H NMR (400 MHz, Chloroform- ) 57.47 - 7.30 (m, 5H), 5.32 - 5.05 (m, 2H), 4.89 (d, J= 4.5 Hz, 1H), 2.43 (m, 2H), 2.33 - 2.20 (m, 1H), 1.16 (t, J= 7.5 Hz, 3H), 1.04 - 0.81 (m, 6H).

[0695] Step 2: (7?)-3-Methyl-2-(propionyloxy (butanoic acid

[0696] To a stirred solution of benzyl (R)-3-methyl-2-(propionyloxy)butanoate (1.06 g, 4.0 mmol) in methanol (40 mL) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times and the reaction was stirred at 25 °C under hydrogen atmosphere (balloon) for 18. The reaction mixture filtered through celite and concentrated under reduced pressure to provide ( / ?)-3-methyl-2-(propionyloxy (butanoic acid (663 mg, 95%). LCMS (ES+): m / z = 175.45 [M+H]+. 'H NMR (400 MHz, Chloroform- ) 84.88 (m, 1H), 2.44 (m, 2H), 2.39 - 2.13 (m, 1H), 1.19 (m, 3H), 1.16 - 0.84 (m, 6H).

[0697] Step 3: Benzyl ((7?)-3-methyl-2-(propionyloxy)butanoyl)-£-prolinate

[0698] To a stirred solution of (R)-3-methyl-2-(propionyloxy)butanoic acid (626 mg, 3.6 mmol) in N,N- dimethylformamide (40 mL) was added benzyl E-prolinate (1.04 g, 4.32 mmol), 1- [bis(dimethylamino)methy lene] - 1 H- 1 ,2, 3 -triazolo [4, 5 -b]pyridinium 3 -oxide

[0699] 97

[0700] 20300-WO-PCT / / 103241-016PCT hexafluorophosphate (2.05 g, 5.4 mmol) and triethylamine (1.26 mL, 9 mmol) at 25 °C and the reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash column chromatography (silica-gel, 230-400) eluting with a linear gradient of 0 - 10% ethyl acetate in hexane to afford benzyl (( / ?)-3-methyl-2- (propionyloxy)butanoyl)-L-prolinate as a colorless viscous oil (540 mg, 40%). LCMS (ES+): m / z = 362.28 [M+H]+. Tl NMR (400 MHz, Chloroform- ) 5 7.21 - 7.02 (m, 5H), 5.01 - 4.88 (m, 2H), 4.78 (m, 1H), 4.29 (m, 1H), 3.88 (m, 1H), 3.49 - 3.28 (m, 2H), 2.29 - 2.12 (m, 2H), 2.03 (m, 1H), 1.98 - 1.84 (m, 1H), 1.80 (m, 1H), 1.77 - 1.63 (m, 1H), 0.97 - 0.88 (m, 3H), 0.88 - 0.54 (m, 6H).

[0701] Step 4: ((7?)-3-Methyl-2-(propionyloxy)butanoyl)-£-proline

[0702] To a stirred solution of benzyl ((7?)-3-methyl-2-(propionyloxy)butanoyl)-L-prolinate (540 mg, 1.49 mmol) in methanol (40 mL) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times. The reaction mixture was stirred at 25 °C under hydrogen atmosphere (balloon) for 18 h, filtered through Celite® and concentrated under reduced pressure to provide (( / )-3 -methyl -2-(propionyl oxy (butanoyl )- / .- proline (364 mg, 90 % yield). LCMS (ES+): m / z = 272.65 [M+H]+.

[0703] Step 5: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((7?)-3-methyl-2-

[0704] (propionyloxy)butanoyl)-£-prolinate (Cmpd 46)

[0705] To a stirred solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (228 mg, 1 mmol) in A, A-dimethylformamide (10 mL) was added (( / ?)-3-methyl-2-(propionyloxy)butanoyl)- / .- proline (271 mg, 1 mmol), and cesium carbonate (652 mg, 2 mmol) at 25 °C and the mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Sunfire column, 5-60% water / acetonitrile with 0.1% TFA) to afford ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (( / ?)-3-methyl-2-(propionyloxy)butanoyl)- L-prolinate (Cmpd 46) as a colorless viscous oil (20 mg, 4%). LCMS (ES+): m / z = 464.08 [M+H]+.

[0706] 98

[0707] 20300-WO-PCT / / 103241-016PCT Cmpd 47: l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl ((R)-3-methyl-2-

[0708] (propionyloxy)butanoyl)-L-prolinate

[0709] To a stirred solution of 1 -chloroethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (242 mg, 1.0 mmol) in A,A-dimethylformamide (10 mL) was added (( / ?)-3-methyl-2-(propionyloxy)butanoyl)-L- proline (271 mg, 1 mmol), and cesium carbonate (652 mg, 2.0 mmol) at 25 °C and the mixture was stirred at 50 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Cl 8, 5-60% water / acetonitrile with 0.1% TFA) to afford l-((methyl(2- (pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl ((R)-3-methyl-2-(propionyloxy)butanoyl)-L-prolinate (Cmpd 47) as a colorless viscous oil (90 mg, 18% yield). LCMS (ES+): m / z = 478.18 [M+H]+.

[0710] Cmpd 48: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((5)-3-methyl-2-

[0711] (propionyloxy )butanoyl)- / )-prolinate

[0712] Step 1: Benzyl fS')-3-inethyl-2-( propionyloxy (butanoate

[0713] To a stirred solution of benzyl (5)-2-hydroxy-3-methylbutanoate (816 mg, 4.0 mmol) in dichloromethane (40 mL) was added propionic anhydride (0.77 mL, 6.0 mmol), tri ethyl amine (1.1 mL, 8.0 mmol) and 4-dimethylaminopyridine (5 mg, 0.04 mmol) at 25 °C and the reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was quenched with water (50 mL) and extracted with methylene chloride (3 x 50 mL). The combined organic extracts were dried over sodium

[0714] 99

[0715] 20300-WO-PCT / / 103241-016PCT sulfate and the solvent was removed in vacuo. The residue was purified by flash chromatography (silica-gel, 230-400) eluting with a linear gradient of 0-10% ethyl acetate in hexanes to afford benzyl (5)-3-methyl-2-(propionyloxy)butanoate as a colorless viscous oil (1.04 g, 98% yield). LCMS (ES+): m / z = 265.37 [M+H]+. 'H NMR (400 MHz, Chloroform- ) 8 7.47 - 7.30 (m, 5H), 5.32 - 5.05 (m, 2H), 4.89 (d, J = 4.5 Hz, 1H), 2.43 (m, 2H), 2.33 - 2.20 (m, 1H), 1.16 (m, 3H), 1.04 - 0.81 (m, 6H).

[0716] Step 2: (N)-3-Methyl-2-(propionyloxy)butanoic acid

[0717] To a stirred solution of benzyl (S)-3-methyl-2-(propionyloxy)butanoate (1.04 g, 3.9 mmol) in methanol (40 mb) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times. The reaction was stirred at 25 °C under hydrogen atmosphere for 18 h. The reaction mixture filtered through Celite® and concentrated under reduced pressure to afford (S)-3-methyl-2-(propionyloxy)butanoic acid (651 mg, 96%). LCMS (ES+): m / z = 175.45 [M+H]+. 'H NMR (400 MHz, Chloroform- ) 8 4.88 (m, 1H), 2.44 (m, 2H), 2.39 - 2.13 (m, 1H), 1.19 (m, 3H), 1.16 - 0.84 (m, 6H).

[0718] Step 3: Benzyl ((N)-3-methyl-2-(propionyloxy)butanoyl)-Z)-prolinate

[0719] To a stirred solution of (5)-3-methyl-2-(propionyloxy)butanoic acid (0.63 g, 3.6 mmol) in N,N- dimethylformamide (40 mL) was added benzyl D-prolinate (1.04 g, 4.32 mmol), 1- [bis(dimethylamino)methy lene] -177-1,2,3 -triazolo [4, 5 -b]pyridinium 3 -oxide hexafluorophosphate (2.05 g, 5.4 mmol) and triethylamine (1.26 mL, 9 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash chromatography (silica-gel, 230-400) eluting with a linear gradient of 0-50% ethyl acetate in hexanes to afford benzyl (CS')-3 -methyl-2-(propionyloxy (butanoyl )- / )-prol inate as a colorless viscous oil (540 mg, 40%). LCMS (ES+): m / z = 362.23 [M+H]+. 'H NMR (400 MHz, Chloroform-<7) 8 7.21 - 7.02 (m, 5H), 5.01 - 4.88 (m, 2H), 4.78 (m, 1H), 4.29 (m, 1H), 3.88 (m, 1H), 3.49 - 3.28 (m, 2H), 2.29 - 2.12 (m, 2H), 2.03 (m, 1H), 1.98 - 1.84 (m, 1H), 1.80 (m, 1H), 1.77 - 1.63 (m, 1H), 0.97 - 0.88 (m, 3H), 0.88 - 0.54 (m, 6H).

[0720] Step 4: ((N)-3-Methyl-2-(propionyloxy)butanoyl)-Z)-proline

[0721] 100

[0722] 20300-WO-PCT / / 103241-016PCT To a stirred solution of benzyl ((5)-3-methyl-2-(propionyloxy)butanoyl)-D-prolinate (540 mg, 1.49 mmol) in methanol (40 mL) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times. The reaction was stirred at 25 °C under hydrogen atmosphere for 18 h. The reaction mixture was filtered through Celite® and concentrated under reduced pressure to provide ((5 -3 -methyl -2-(propionyl oxy (butanoyl )- / )- proline (364 mg, 90%). LCMS (ES+): m / z = 272.65 [M+H]+.

[0723] Step 5: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((5)-3-methyl-2-

[0724] (propionyloxy)butanoyl)-Z)-prolinate (Cmpd 48)

[0725] To a stirred solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (52 mg, 0.19 mmol) in A,A-dimethylformamide (5 mL) was added ((6 -3 -methyl -2-(propionyl oxy (butanoyl )- / )- proline (50 mg, 0.19 mmol), and cesium carbonate (124 mg, 0.38 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over sodium sulfate and the residue was purified by reversed phase HPLC (Cl 8, eluting with a linear gradient of 5-60% water / acetonitrile with 0.1% TLA) to afford ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((S)-3-methyl-2-(propionyloxy)butanoyl)- D-prolinate trifluoroacetate salt (Cmpd 48) as a colorless viscous oil (10 mg, 11%). LCMS (ES+): m / z = 464.08 [M+H]+.

[0726] Cmpd 49: l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl ((A)-3-methyl-2-

[0727] (propionyloxy )butanoyl)- / )-prolinate l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl ((A)-3-inethyl-2-( propionyloxy) butanoyl)-Z)-prolinate (Cmpd 49)

[0728] To a stirred solution of 1 -chloroethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (95 mg, 0.39 mmol) in A, A-dimethylformamide (5 mL) was added ((A)-3-methyl-2-(propionyloxy)butanoyl)- / )-proline (106 mg, 0.39 mmol), and cesium carbonate (254 mg, 0.78 mmol) at 25 °C and the reaction mixture was stirred at 50 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were

[0729] 101

[0730] 20300-WO-PCT / / 103241-016PCT dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Sunfire column, 5-60% water / acetonitrile with 0.1% TFA) to afford 1- ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl ((S)-3-methyl-2-(propionyloxy)butanoyl)-D- prolinate (Cmpd 49) as a colorless viscous oil (60 mg, 32% yield). LCMS (ES+): m / z = 478.18 [M+H]+.

[0731] Cmpd 50: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((7?)-2-(butyryloxy)-3- methylbutanoyl)-Z-prolinate

[0732] Step 1: Benzyl (7?)-2-(butyryloxy)-3-methylbutanoate

[0733] To a stirred solution of benzyl ( / ?)-2-hydroxy-3-methylbutanoate (0.82 g, 4.0 mmol) in dichloromethane (40 mb) was added butyric anhydride (0.98 mL, 6.0 mmol), tri ethylamine (1.1 mb, 8.0 mmol) and 4-dimethylaminopyridine (5 mg, 0.04 mmol) at 25 °C and the mixture was stirred at 25 °C for 18 h. The reaction mixture was quenched with water (50 mL) and extracted with methylene chloride (3 x 50 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash column chromatography (silica-gel, 230-400) eluting with a linear gradient of 0 - 10% ethyl acetate in hexane to afford benzyl (7?)-2-(butyryloxy)-3-methylbutanoate as a colorless viscous oil (1100 mg, 99% yield). LCMS (ES+): m / z = 279.5 [M+H]+. 'H NMR (400 MHz, Chloroform- ) 8 7.44 - 7.30 (m, 5H), 5.27 - 5.09 (m, 2H), 4.88 (m, 1H), 2.38 (m, 2H), 2.24 (m, 1H), 1.68 (m, 2H), 1.14 - 0.85 (m, 9H).

[0734] Step 2: (R)-2-( Butyryloxy)-3-methylbutanoic acid

[0735] To a stirred solution of benzyl ( / ?)-2-(butyryloxy)-3-methylbutanoate (1.1 g, 4 mmol) in methanol (40 mL) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times. The reaction was stirred at 25 °C under hydrogen atmosphere (balloon) for 18 h, filtered through Celite® and concentrated under reduced pressure

[0736] 102

[0737] 20300-WO-PCT / / 103241-016PCT to provide (7?)-2-(Butyryloxy)-3-methylbutanoic acid. (0.72 g, 95%). LCMS (ES+): m / z = 189.51 [M+H]+. 'H NMR (400 MHz, Chloroform- ) 8 4.99 - 4.70 (m, 1H), 2.39 (m, 2H), 2.28 (m, 1H), 1.69 (m, 2H), 1.23 - 0.82 (m, 9H).

[0738] Step 3: Benzyl ((7?)-2-(butyryloxy)-3-methylbutanoyl)-£-prolinate

[0739] To a stirred solution of (7?)-2-(butyryloxy)-3 -methylbutanoic acid (0.72 g, 3.8 mmol) in V, V- dimethylformamide (40 mL) was added benzyl L-prol inate (1.04 g, 4.3 mmol), 1- [bis(dimethylamino)methy lene] - 1 H- 1 ,2, 3 -triazolo [4, 5 -b]pyridinium 3 -oxide hexafluorophosphate (2.05 g, 5.4 mmol) and triethylamine (1.26 mL, 9 mmol) at 25 °C and the mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (50 mL) and extracted with ethyl acetate (3 x 50 mL). The residue was purified by flash column chromatography (silica-gel, 230-400) eluting with a linear gradient of 0 - 50% ethyl acetate in hexane to afford benzyl (( / ?>-2-(butyryloxy)-3-methylbutanoyl)- / .-prolinate as a colorless viscous oil (600 mg, 42%). LCMS (ES+): m / z = 376.41 [M+H]+.

[0740] Step 4: ((7?)-2-(Butyryloxy)-3-methylbutanoyl)-L-proline

[0741] To a stirred solution of benzyl ((7?)-2-(butyryloxy)-3-methylbutanoyl)-L-prolinate (600 mg, 1.49 mmol) in methanol (40 mL) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times. The reaction was stirred at 25 °C under hydrogen atmosphere (balloon) for 18 h. Completion of the reaction was monitored by LCMS and TLC. The reaction mixture was filtered through Celite® and concentrated under reduced pressure. The crude product was carried to the next step without further purification. LCMS (ES+): m / z = 286.52 [M+H]+.

[0742] Step 5: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((7?)-2-(butyryloxy)-3- methylbutanoyl)-£-prolinate (Cmpd 50)

[0743] To a stirred solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (228 mg, 1.0 mmol) in A,A-dimethylformamide (10 mL) was added ((7?)-2-(butyryloxy)-3-methylbutanoyl)-L-proline (285 mg, 1.0 mmol) and cesium carbonate (652 mg, 2 mmol) at 25 °C and the reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by reversed-phase HPLC (Cl 8, 5-60% water / acetonitrile with 0.1% TEA) to afford ((methyl(2-

[0744] 103

[0745] 20300-WO-PCT / / 103241-016PCT (pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (( / ?)-3-methyl-2-(propionyloxy)butanoyl)- / .-prolinate (Cmpd 50) as a colorless viscous oil (50 mg, 10 %). LCMS (ES+): m / z = 478.12 [M+H]+.

[0746] Cmpd 51: l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl ((7?)-2-(butyryloxy)-3- methylbutanoyl)-Z-prolinate

[0747] To a stirred solution of 1 -chloroethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (242 mg, 1.0 mmol) in A,A-dimethylformamide (10 mL) was added (( / ?)-2-(butyryloxy)-3-methylbutanoyl)- / .-proline (285 mg, 1 mmol), and cesium carbonate (652 mg, 2.0 mmol) at 25 °C and the reaction mixture was stirred at 50 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by reversed-phase HPLC (Cl 8, 5-60% water / acetonitrile with 0.1% TLA) to afford l-((methyl(2- (pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl (( / )-2-(butyryloxy)-3-methylbutanoyl)- / .-prolinate (Cmpd 51) as a colorless viscous oil (50 mg, 10 %). LCMS (ES+): m / z = 492.15 [M+H]+.

[0748] Cmpd 52: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((5)-2-(butyryloxy)-3- methylbutanoyl)-Z)-prolinate

[0749] Step 1: Benzyl (5)-2-(butyryloxy)-3-methylbutanoate

[0750] To a stirred solution of benzyl (S)-2-hydroxy-3-methylbutanoate (816 mg, 4 mmol) in dichloromethane (40 mL) was added butyric anhydride (0.98 mL, 6 mmol), tri ethyl amine (1.1 mL, 8 mmol), and 4-dimethylaminopyridine (5 mg, 0.04 mmol) at 25 °C. The reaction mixture

[0751] 104

[0752] 20300-WO-PCT / / 103241-016PCT was stirred at 25 °C for 18 h. The reaction mixture was quenched with water (50 mL) and extracted with methylene chloride (3 x 50 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash column chromatography (silica-gel, 230-400) eluting with a linear gradient of 0 - 10% ethyl acetate in hexane to afford benzyl (5)-2-(butyryloxy)-3-methylbutanoate as a colorless viscous oil (1.10 g, 99 %). LCMS (ES+): m / z = 279.47 [M+H]+. 'H NMR (400 MHz, Chloroform- ) 57.44 - 7.30 (m, 5H), 5.27 - 5.09 (m, 2H), 4.89 (d, J = 4.5 Hz, 1H), 2.38 (m, 2H), 2.24 (m, 1H), 1.68 (h, J = 7.4 Hz, 2H), 1.14 - 0.84 (m, 9H).

[0753] Step 2: (N)-2-(Butyryloxy)-3-methylbutanoic acid

[0754] To a stirred solution of benzyl (5)-2-(butyryloxy)-3-methylbutanoate (1.10 g, 4.0 mmol) in methanol (40 mL) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times. The reaction was stirred at 25 °C under hydrogen atmosphere (balloon) for 18 h. The reaction mixture filtered through Celite® and concentrated under reduced pressure to provide (5)-2-(butyryloxy)-3-methylbutanoic acid (715 mg, 95 % yield). LCMS (ES+): m / z = 189.51 [M+H]+. 'H NMR (400 MHz, Chloroform- ) 84.99

[0755] - 4.75 (m, 1H), 2.39 (m, 2H), 2.26 (m, 1H), 1.69 (h, J= 7.4 Hz, 2H), 1.23 - 0.80 (m, 9H).

[0756] Step 3: Benzyl ((N)-2-(butyryloxy)-3-methylbutanoyl)-D-prolinate

[0757] To a stirred solution of (8)-2-(butyryloxy)-3 -methylbutanoic acid (715 mg, 3.8 mmol) in N,N- dimethylformamide (40 mL) was added benzyl D-prolinate (1.04 g, 4.32 mmol), 1- [bis(dimethylamino)methy lene] -177-1,2,3 -triazolo [4, 5 -b]pyridinium 3 -oxide hexafluorophosphate (2.05 g, 5.4 mmol) and triethylamine (1.26 mL, 9 mmol) at 25 °C and the mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash column chromatography (silica-gel, 230-400) eluting with a linear gradient of 0

[0758] - 50% ethyl acetate in hexane to afford benzyl ((S)-2-(butyryloxy)-3-methylbutanoyl)-D-prolinate as a colorless viscous oil (600 mg, 42 %). LCMS (ES+): m / z = 376.31 [M+H]+.

[0759] Step 4: ((N)-2-(Butyryloxy)-3-methylbutanoyl)-D-proline

[0760] To a stirred solution of benzyl ((S)-2-(butyryloxy)-3-methylbutanoyl)-D-prolinate (600 mg, 1.49 mmol) in methanol (40 mL) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The

[0761] 105

[0762] 20300-WO-PCT / / 103241-016PCT reaction vessel was evacuated and purged with hydrogen three times. The reaction was stirred at 25 °C under hydrogen atmosphere (balloon) for 18 h. The reaction mixture filtered through Celite® and concentrated under reduced pressure to provide ( fS')-2-(butyryloxy)-3 -methyl butanoyl )- / )- proline (364 mg, 90 % yield). LCMS (ES+): m / z = 286.52 [M+H]+.

[0763] Step 5: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ( ( )-2-( butyryl oxy )-3- methylbutanoyl)-Z)-prolinate (Cmpd 52)

[0764] To a stirred solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (54 mg, 0.19 mmol) in A, A-dimethylformamide (5 mb) was added (fS')-2-(butyryloxy)-3-methylbutanoyl)- / )-proline (50 mg, 0.19 mmol) and cesium carbonate (124 mg, 0.38 mmol) at 25 °C and the mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mb) and extracted with ethyl acetate (3 x 5 mb). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Sunfire column, 5-60% water / acetonitrile with 0.1% TFA) to afford ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((5)-3-methyl-2-(propionyloxy)butanoyl)- D-prolinate (Cmpd 52) as a colorless viscous oil (15 mg, 17 % yield). LCMS (ES+): m / z = 478.03 [M+H]+.

[0765] Cmpd 53: l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl ( ( )-2-( butyryl oxy )-3- methylbutanoyl)-Z)-prolinate

[0766] To a stirred solution of 1 -chloroethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (100 mg, 0.39 mmol) in A,A-dimethylformamide (5 mb) was added ( (A)-2-(butyryloxy)-3 -methyl butanoyl )- / )- proline (111 mg, 0.39 mmol) and cesium carbonate (254 mg, 0.78 mmol) at 25 °C and the reaction mixture was stirred at 50 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Sunfire column, 5-60% water / acetonitrile with 0.1% TFA) to afford 1 -((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl ((5)-2-(butyryloxy)-3-

[0767] 106

[0768] 20300-WO-PCT / / 103241-016PCT methylbutanoyl)- / )-prolinate (Cmpd 53) as a colorless viscous oil (60 mg, 31 %). LCMS (ES+): m z = 492.15 [M+H]+

[0769] Cmpds 54 and 55: l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl N- ((benzyloxy)carbonyl)-N-methylglycyl-L-valinate (Cmpd 54) and ((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methyl N-((benzyloxy)carbonyl)-N-methylglycyl-L-valinate (Cmpd 55)

[0770] Step 1: Methyl (2S)-2-(2- { [(benzyloxy)carbonyl] (methyl)amino} acetamido)-3- methylbutanoate

[0771] To a mixture of 2-(methyl-(phenylmethoxycarbonyl)amino)acetic acid (0.80 g, 3.58 mmol) and A, A-diisopropylethylamine (2.08 mL, 1.54 g, 11.9 mmol) in anhydrous methylene chloride (14.9 mb) at room temperature under a nitrogen atmosphere was added HATU (1.47 g, 3.88 mmol) and the mixture was stirred for 10 minutes. L- Valine methyl ester hydrochloride (0.50 g, 2.98 mmol) was added, and the resulting mixture was stirred at room temperature for 4 days. The reaction mixture was diluted with ethyl acetate (90 mL) and washed with saturated ammonium chloride solution (15 mL). The aqueous phase was extracted with ethyl acetate (30 mL) and the combined organic extracts were washed with water (2 x 15 mL), saturated NaHCO, solution (15 mL), and brine (15 mL), then dried over sodium sulfate, filtered, and the solvent removed in vacuo. The residue was purified by flash chromatography (silica gel) eluting with a linear gradient of 0-75% ethyl acetate / hexanes over 25 minutes to provide methyl (2S)-2-(2- ([(benzyloxy)carbonyl](methyl)amino)acetamido)-3-methylbutanoate as a colorless oil (0.65 g, 65%). 'H NMR (400 MHz, Chloroform- ) 8 = 7.35 (br s, 5H), 6.65 - 6.15 (m, 1H), 5.18 (br s, 2H), 4.54 (dd, J=4.5, 8.8 Hz, 1H), 4.08 - 3.99 (m, 1H), 3.96 - 3.87 (m, 1H), 3.72 (s, 3H), 3.04 (s, 3H), 2.14 (br s, 1H), 0.94 - 0.80 (m, 6H). LCMS (ES+): m / z = 337 [M+H]+.

[0772] Step 2: Lithium (2S)-2-(2-{[(benzyloxy)carbonyl](methyl)amino}acetamido)-3-methyl butanoate

[0773] 107

[0774] 20300-WO-PCT / / 103241-016PCT To a solution of methyl (2S)-2-(2-{[(benzyloxy)carbonyl](methyl)amino}acetamido)-3- methylbutanoate (0.65 g, 1.92 mmol) in 1: 1 (y / v) THF / MeOH (9.6 mL) was added a 1 M solution of lithium hydroxide in water (1.92 mL, 1.92 mmol). The mixture was stirred at room temperature for 16 h. A further aliquot of a 1 M solution of lithium hydroxide in water (0.192 mL, 0.192 mmol) was added and the mixture was stirred for an additional 48 h. The solvent was removed in vacuo and the residue was dried under high vacuum to provide lithium (2S)-2-(2- {[(benzyloxy)carbonyl](methyl)amino}acetamido)-3-methyl butanoate as a white solid (0.47 g, 75 %). 'H NMR (400 MHz, DMSO- ,) 8 = 7.38 - 7.30 (m, J=19.9 Hz, 5H), 5.10 - 4.99 (m, 2H), 3.99 - 3.85 (m, 2H), 3.79 (dd, J=4.3, 7.8 Hz, 1H), 2.87 (d, J=11.3 Hz, 3H), 2.11 - 1.93 (m, 1H), 0.88 - 0.69 (m, 6H). LCMS (ES+): m / z = 345 [M+Na]+.

[0775] Step 3: ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl N-((benzyloxy)carbonyl)-N- methylglycyl-L-valinate (Cmpd 55)

[0776] To a suspension of sodium bicarbonate (0.48 g, 3.50 mmol) and lithium 2-[2-(benzyloxycarbonyl- methyl-amino)-acetylamino]-3-methyl-butyrate (0.23 g, 0.701 mmol) in anhydrous DMF (3.5 mL) at room temperature under a nitrogen atmosphere was added chloromethyl A-methyl-A-[2- (pyridin-2-yl)ethyl] carbamate (0.24 g, 1.05 mmol) and the mixture was stirred at room temperature for 66 h. The mixture was partitioned between ethyl acetate (60 mL) and saturated NaHCO, solution (15 mL) and the aqueous layer was extracted with ethyl acetate (60 mL). The combined organic extracts were washed with water (3 x 10 mL), then brine (15 mL), then dried over sodium sulfate, filtered, and the solvent was removed in vacuo. The residue was purified by flash chromatography (silica gel), eluting with a linear gradient of 0 to 100% [1:3 (v / v) iPrOH / EtOAc] / hexanes over 20 minutes to provide ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl N- ((benzyloxy)carbonyl)-N-methylglycyl-L-valinate (Cmpd 55) as a colorless oil (0.12 g, 33 %). LCMS (ES+): m / z = 515 [M+H]+.

[0777] Step 3: l-((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl N-((benzyloxy)carbonyl)-N- methylglycyl-L-valinate (Cmpd 54)

[0778] A mixture of 1 -chloroethyl A-methyl- V-[2-(pyridin-2-yl)ethyl] carbamate (0.25 g, 1.05 mmol), lithium 2-[2-(benzyloxycarbonyl-methyl-amino)-acetylamino]-3-methyl-butyrate (0.23 g, 0.70 mmol) and NaHCO, (0.484 g, 3.50 mmol) in anhydrous DMF (3.5 mL) was stirred at room temperature for 16 h. The reaction mixture was then heated at 50 °C for a further 16 h and at room temperature for a further 5 days. The mixture was partitioned between ethyl acetate (60 mL) and

[0779] 108

[0780] 20300-WO-PCT / / 103241-016PCT saturated NaHCO, solution (15 mL) and the aqueous phase was extracted with ethyl acetate (60 mL). The combined organic extracts were washed with water (3 x 10 mL), then brine (10 mL), then dried over sodium sulfate, filtered, and the solvent was removed in vacuo. The residue was purified by flash chromatography (silica gel) eluting with a linear gradient of 0 to 100% [1 :3 ( / v) iPrOH / EtOAc] / hexanes over 16 minutes to provide 1 -((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)ethyl N-((benzyloxy)carbonyl)-N-methylglycyl-L-valinate, (Cmpd 54) as a clear yellow oil (0.019 g, 5.2%). LCMS (ES+): m / z = 529 [M+H]+.

[0781] Cmpd 56: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl N-((benzyloxy)carbonyl)-

[0782] N-methylglycyl-L-phenylalaninate

[0783] Step 1: Methyl (2S)-2-(2-{[(benzyloxy)carbonyl](methyl)amino}acetamido)-3- phenylpropanoate

[0784] To a mixture of 2-(methyl-(phenylmethoxycarbonyl)amino)acetic acid (0.62 g, 2.78 mmol) and N, / V-diisopropylethylamine (1.62 mL, 1.20 g, 9.27 mmol) in anhydrous methylene chloride (12 mL) at room temperature under a nitrogen atmosphere was added HATU (1.15 g, 3.01 mmol) and the mixture was stirred for 10 minutes. L-Phenylalanine methyl ester hydrochloride (0.500 g, 2.32 mmol) was added, and the resulting mixture was stirred at room temperature for 4 days. The reaction mixture was diluted with ethyl acetate (70 mL) and washed with saturated ammonium chloride solution (15 mL). The aqueous phase was extracted with ethyl acetate (60 mL) and the combined organic extracts were washed with water (2 x 15 mL), saturated NaHCO, solution (15 mL), and brine (15 mL), then dried over sodium sulfate, filtered, and the solvent was removed in vacuo. The residue was purified by flash chromatography (silica gel) eluting with a linear gradient of 0-75% ethyl acetate / hexanes over 25 min to provide methyl (2S)-2-(2- {[(benzyloxy)carbonyl](methyl)amino}acetamido)-3-phenylpropanoate as a colorless oil (0.87 g, 97.3%). 'H NMR (400 MHz, Chloroform- ) 8 = 7.41 - 7.18 (m, 8H), 7.13 - 6.94 (m, 2H), 6.55 - 6.11 (m, 1H), 5.14 (br. s., 2H), 4.86 (q, J=6.1 Hz, 1H), 3.91 (s, 2H), 3.72 (s, 3H), 3.21 - 2.99 (m, 2H), 2.89 (s, 3H). LCMS (ES+): m / z = 385 [M+H]+.

[0785] 109

[0786] 20300-WQ-PCT / / 103241-016PCT Step 2: Lithium (2S)-2-(2-{[(benzyloxy)carbonyl](methyl)amino}acetamido)-3- phenylpropanoate

[0787] To a solution of methyl (2S)-2-(2-{[(benzyloxy)carbonyl](methyl)amino}acetamido)-3- phenylpropanoate (0.87 g, 2.26 mmol) in 1: 1 (y / v) THF / MeOH (11.2 mL) was added a 1 M solution of lithium hydroxide in water (2.37 mL, 2.37 mmol). The mixture was stirred at room temperature for 16 h. A further aliquot of a 1 M solution of lithium hydroxide in water (0.23 mL, 0.23 mmol) was added and the mixture was stirred for an additional 4 h. The solvent was removed in vacuo and the residue was dried under high vacuum to provide lithium (2S)-2-(2- {[(benzyloxy)carbonyl](methyl)amino}acetamido)-3-phenylpropanoate as a white solid (0.80 g, 95%). 'H NMR (400 MHz, DMSO-d6) 8 = 7.38 - 7.29 (m, 6H), 7.17 - 7.06 (m, 5H), 5.04 (d, J=16.8 Hz, 2H), 4.02 - 3.94 (m, 1H), 3.88 - 3.74 (m, 2H), 3.10 - 3.01 (m, 1H), 2.92 (dd, J=5.5, 12.9 Hz, 1H), 2.74 (d, 7=4.3 Hz, 3H). LCMS: M+H+= 371

[0788] Step 3: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl N-((benzyloxy)carbonyl)-N- methylglycyl-L-phenylalaninate (Cmpd 56)

[0789] A mixture of chloromethyl A-methyl-7V-[2-(pyridin-2-yl)ethyl]carbamate (0.273 g, 1.20 mmol), lithium 2-[2-(benzyloxycarbonyl-methyl-amino)-acetylamino]-3-methyl-butyrate (0.300 g, 0.797 mmol) and NaHCO, (0.27 g, 3.19 mmol) in anhydrous DMF (8.0 mL) was stirred at room temperature for 16 h. The mixture was partitioned between ethyl acetate (60 mL) and saturated NaHCO, solution (15 mL) and the aqueous phase was extracted with ethyl acetate (60 mL). The combined organic extracts were washed with water (3 x 10 mL), then brine (10 mL), then dried over sodium sulfate, filtered, and the solvent was removed in vacuo. The residue was purified by flash chromatography (silica gel) eluting with a linear gradient of 0 to 100% [1:3 (y / v) iPrOH / EtOAc] / hexanes over 15 minutes to provide ((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methyl N-((benzyloxy)carbonyl)-N-methylglycyl-L-phenylalaninate (Cmpd 56) as a colorless oil (0.29 g, 66%). LCMS (ES+): m / z = 563 [M+H]+.

[0790] Cmpd 57: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((benzyloxy)carbonyl)-£- alaninate

[0791] 110

[0792] 20300-WO-PCT / / 103241-016PCT

[0793] To a stirred solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (114 mg, 0.5 mmol) in A(A^-dimethylformamide (5 mL) was added ((benzyloxy)carbonyl)-L-alanine (112 mg, 0.5 mmol) and / V-diisopropylethylamine (0.17 mL, 1 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash chromatography (silica-gel, 230-400) eluting with a linear gradient of 0-10% methanol in dichloromethane to afford ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl ((benzyloxy)carbonyl)-L-alaninate (Cmpd 57) as a colorless viscous oil (30 mg, 15 %). LCMS (ES+): m / z = 416.2 [M+H]+

[0794] Cmpd 58: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl V-(2-acetoxyacetyl)-V- methylglycinate

[0795] Step 1: Benzyl V-(2-acetoxyacetyl)- V-inethylglycinate

[0796] To a stirred solution of 2-acetoxyacetic acid (1.18 g, 10 mmol) in A(A^-dimethylformamide (40 mL) was added benzyl methylglycinate hydrochloride (2.15 g, 10 mmol), 1- [bis(dimethylamino)methy lene] - H- 1 ,2, 3 -triazolo [4, 5 -b]pyridinium 3 -oxide hexafluorophosphate (4.80 g, 12.6 mmol) and tri ethylamine (2.83 mL, 20 mmol) at 25 °C and the mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash column chromatography (silica-gel, 230-400) eluting with a linear gradient of 0 - 50% ethyl acetate in hexane. The pure fractions were evaporated and dried under vacuum to

[0797] 111

[0798] 20300-WO-PCT / / 103241-016PCT afford benzyl / V-(2-acetoxyacetyl)- / V-methylglycinate as a colorless viscous oil (1.2 g, 43 % yield). LCMS (ES+): m / z = 280.58 [M+H]+.

[0799] Step 2: V-(2-acetoxyacetyl)- V-methyl«lycine

[0800] To a stirred solution of benzyl / V-(2-acetoxyacetyl)- / V-methylglycinate (279 mg, 1 mmol) in methanol (10 mb) was added palladium on carbon (10 mg, 0.01 mmol) at 25 °C. The reaction vessel was evacuated and purged with hydrogen three times. The reaction was stirred at 25 °C under hydrogen atmosphere (balloon) for 18 h, filtered through Celite® and concentrated under reduced pressure. The crude product was carried to the next step without further purification. LCMS (ES+): m / z = 190.26 [M+H]+.

[0801] Step 3: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl V-( 2-acet oxy acetyl )-V- methylglycinate (Cmpd 58)

[0802] To a stirred solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (114 mg, 0.5 mmol) in / V-dimethylformamide (10 mL) was added V-(2-acetoxyacetyl)- V-methylglycine (95 mg, 0.5 mmol) and cesium carbonate (325 mg, 1 mmol) at 25 °C and the reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Cl 8, 5-60% water / acetonitrile with 0.1% TFA) to afford ((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methyl / V-(2-acetoxyacetyl)- / V-methylglycinate (Cmpd 58) as a colorless viscous oil (10 mg, 5 % yield). LCMS (ES+): m / z = 382.95 [M+H]+.

[0803] Cmpd 59: l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl V-( 2-acet oxy acetyl )-V- methylglycinate

[0804] To a stirred solution of 1 -chloroethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (121 mg, 0.5 mmol) in A, / V-dimethylformamide (5 mL) was added V-(2-acetoxyacetyl)- V-methylgly cine (95 mg, 0.5 mmol) and cesium carbonate (325 mg, 1 mmol) at 25 °C and the reaction mixture was stirred at 50 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over

[0805] 112

[0806] 20300-WO-PCT / / 103241-016PCT sodium sulfate and the solvent was removed in vacuo. The residue was purified by reversed phase HPLC (Cl 8, 10-60% water / acetonitrile with 0.1% TFA) to afford 1 -((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)ethyl A-(2-acetoxyacetyl)- V-methylglycinate (Cmpd 59) as a colorless viscous oil (15 mg, 8 %). LCMS (ES+): m / z = 396.16 [M+H]+.

[0807] Cmpd 60: [Methyl((2-[((methyl[2-(pyridin-2-yl)ethyl]carbamoyl)oxy)methoxy]-2- oxoethyl))carbamoyl] methyl (2S)-2-amino-3-methylbutanoate

[0808] Step 1: 2-((2-(Benzyloxy)-2-oxoethyl)(methyl)amino)-2-oxoethyl (tert-butoxycarbonyl)-Z- val inate

[0809] To a mixture of benzyl 2-(methylamino)acetate hydrochloride (0.56 g, 2.58 mmol) and 2-(((tert- butoxycarbonyl)-Z-valyl)oxy)acetic acid (0.78 g, 2.83 mmol) in DMF (15 mL) at 0 °C was added HATU (1.18 g, 3.10 mmol) followed by A,A-diisopropylethylamine (1.35 mL, 7.74 mmol). The mixture was warmed to room temperature and stirred for 3 h. The mixture was diluted with EtOAc (15 mL) and water (10 mL). The layers were separated, and the aqueous phase was extracted with EtOAc (10 mL). The combined organic extracts were washed with 0.3 M HC1 (10 mL) and brine (10 mL), dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash chromatography (40 g silica, eluting with a linear gradient of 15 to 85% EtOAc / Hex) to provide 2-((2-(benzyloxy)-2-oxoethyl)(methyl)amino)-2-oxoethyl (tert-butoxycarbonyl)-Z- valinate (0.91 g, 81%) as a clear oil. NMR (400 MHz, Chloroform-<7) 8 = 7.49 - 7.30 (m, 5H), 5.28 - 5.11 (m, 2H), 5.11 - 4.54 (m, 3H), 4.43 - 3.94 (m, 3H), 3.12 - 2.91 (m, 3H), 2.42 - 2.15 (m, 1H), 1.46 (s, 9H), 1.09 - 0.91 (m, 6H). LCMS (ES+): m / z = 459.1 [M+Na]+.

[0810] Step 2: V-(2-( (( / c / 7- Bu t oxy car bony l)- / .-valyl)oxy )acetyl )- V-inethyl«lycine

[0811] A solution of 2-((2-(benzyloxy)-2-oxoethyl)(methyl)amino)-2-oxoethyl ( / c / 7-butoxycarbonyl)-A- valinate (0.910 g, 1.92 mmol) in ethyl acetate (60 mL) was prepared under nitrogen atmosphere.

[0812] 113

[0813] 20300-WO-PCT / / 103241-016PCT Pd / C (10%, 90 mg) was added. A vacuum was applied, followed by a hydrogen balloon. This was repeated three times, and the mixture was stirred under a hydrogen atmosphere (balloon) for 16 h. The reaction mixture was vacuum filtered through Celite®, and the filtrate was concentrated in vacuo to a clear oil which was dried under high vacuum to provide A-(2-((( / c / 7-butoxycarbony I )- A-valyl)oxy)acetyl)- / V-methylglycine (0.75 g, quantitative yield).JH NMR (400 MHz, Chloroform-d) 8 = 8.64 - 7.55 (bs, 1H), 5.22 - 4.61 (m, 3H), 4.41 - 3.93 (m, 3H), 3.16 - 2.89 (m, 3H), 2.46 - 2.15 (m, 1H), 1.56 - 1.36 (m, 9H), 1.10 - 0.83 (m, 6H). LCMS (ES+): m / z = 369.8 [M+Na]+.

[0814] Step 3: 3,10-Dimethyl-4,8,ll-trioxo-l-(pyridin-2-yl)-5,7-dioxa-3,10-diazadodecan-12-yl (tert- butoxycarbonyl)-Z-val inate

[0815] Cesium carbonate (0.22 g, 0.67 mmol) was added to a solution of A-(2-((( / / 7-butoxycarbonyl)-A- valyl)oxy)acetyl)-A-methylglycine (0.15 g, 0.42 mmol) and chloromethyl methyl(2-(pyridin-2- yl)ethyl)carbamate ethylcarbamate hydrochloride salt (0.11 g, 0.42 mmol) in DMF (1.5 mb) and the mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with water (4 mL) and EtOAc (6 mL). The layers were separated, and the aqueous phase was extracted with EtOAc (5 mL). The combined organic extracts were washed with water (6 mL), and brine (6 mL), dried over sodium sulfate, and the solvent was removed in vacuo. The residue was purified by flash chromatography (25 g silica, eluting with a linear gradient of 0.75% to 5% MeOH / DCM) to provide 3,10-dimethyl-4,8,l l-trioxo-l-(pyridin-2-yl)-5,7-dioxa-3,10-diazadodecan-12-yl (tert- butoxycarbonyl)-L-valinate (0.13 g, 58%) as a clear oil. 'H NMR (400 MHz, Chloroform-<7) 8 = 8.54 (d, 7=5.0 Hz, 1H), 7.71 - 7.53 (m, 1H), 7.25 - 7.07 (m, 2H), 5.91 - 5.66 (m, 2H), 5.14 - 4.54 (m, 3H), 4.40 - 4.02 (m, 3H), 3.77 - 3.57 (m, 2H), 3.19 - 2.94 (m, 5H), 2.91 - 2.75 (m, 3H), 2.28 (br s, 1H), 1.54 - 1.37 (m, 9H), 1.10 - 0.82 (m, 6H). LCMS (ES+): m / z = 539.3 [M+H]+.

[0816] Step 4: 3,10-Dimethyl-4,8,ll-trioxo-l-(pyridin-2-yl)-5,7-dioxa-3,10-diazadodecan-12-yl L- valinate bis(2,2,2-trifluoroacetate) (Cmpd 60)

[0817] A solution was prepared of 3,10-dimethyl-4,8,l l-trioxo-l-(pyridin-2-yl)-5,7-dioxa-3,10- diazadodecan- 12-yl ( / / 7-butoxycarbonyl)-A-val inate (67 mg, 0.124 mmol) in DCM (4 mL). A solution of 1 :1 ( / v) TFA / DCM (6 mL) was added slowly. After stirring for 35 minutes at room temperature, toluene (3 mL) was added to the reaction mixture, the volatiles were removed in vacuo, and the resulting clear residue was dried under high vacuum. The resulting clear oil was taken up in a minimum amount of 9: 1 water / MeCN, frozen in a dry ice / acetone bath, and

[0818] 114

[0819] 20300-WO-PCT / / 103241-016PCT lyophilized to provide the 3,10-dimethyl-4,8,l l-trioxo-l-(pyridin-2-yl)-5,7-dioxa-3,10- diazadodecan- 12-yl L-valinate bis(2,2,2-trifluoroacetate) (Cmpd 60) (76 mg, 92%) as a colorless solid. 'H NMR (400 MHz, Methanol-^) 8 = 8.86 - 8.70 (m, 1H), 8.50 (dq, J=1.6, 7.9 Hz, 1H), 8.03 - 7.85 (m, 2H), 5.79 - 5.48 (m, 2H), 5.23 - 4.95 (m, 2H), 4.33 - 4.10 (m, 2H), 4.10 - 3.98 (m, 1H), 3.75 (t, J=6.5 Hz, 2H), 3.29 - 3.22 (m, 1H), 3.15 - 3.05 (m, 3H), 3.03 - 2.90 (m, 4H), 2.46 - 2.26 (m, 1H), 1.20 - 1.03 (m, 6H). LCMS (ES+): m / z = 439.9 [M+H]+.

[0820] Cmpd 61: Ethyl 3,9-dimethyl-4,8-dioxo-ll-(pyridin-2-yl)-5,7-dioxa-3,9-diazaundecanoate

[0821] Step 1: (Chloromethoxy carbonyl-methyl-amino)-acetic acid ethyl ester

[0822] To a stirred solution of methylamino-acetic acid ethyl ester (1.0 g, 6.5 mmol) and N,N- diisopropylethylamine (1.80 mL, 10.33 mmol) in anhydrous dichloromethane (15 mL) was slowly added chloromethylchloroformate (0.6 mL, 6.75 mmol) and the resulting mixture was stirred at 25 °C for 16 h. Water was added and the mixture was extracted with dichloromethane (3x 35 mL). The combined organic extracts were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to afford a light brown oil which was used without further purification in the next step.

[0823] Step 2: (Methyl-([methyl-(2-pyridin-2-yl-ethyl)-carbamoyloxy]-methoxycarbonyl)-amino)- acetic acid ethyl ester (Cmpd 61)

[0824] A stirred mixture of methyl-(2-pyridin-2-yl-ethyl)-amine (0.6 mL, 4.3 mmol) and cesium carbonate (2.9 g, 9.00 mmol) in anhydrous DMF (15 mL) was flushed with nitrogen and two one- inch pieces of solid carbon dioxide were added. The vessel was fitted with a balloon containing a two-inch piece of solid carbon dioxide and the resulting mixture was stirred at 25 °C for 4 h. A solution of (chloromethoxycarbonyl-methyl-amino)-acetic acid ethyl ester in anhydrous DMF (5 mL) was slowly added to the reaction mixture and stirring continued, maintaining carbon dioxide atmosphere at 25 °C for 16 h. The mixture was concentrated under reduced pressure, partitioned between ethyl acetate and water and extracted with ethyl acetate (3 x 40 mL). The combined organic extracts were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel) eluting with a

[0825] 115

[0826] 20300-WO-PCT / / 103241-016PCT linear gradient of 0-15% methanol with 5% ammonium hydroxide / dichloromethane to afford ethyl 3,9-dimethyl-4,8-dioxo-l l-(pyridin-2-yl)-5,7-dioxa-3,9-diazaundecanoate (Cmpd 61) as a clear liquid (41 mg, 2.7 %) LCMS (ES+): m / z = 354.95 [M+H]+.

[0827] Cmpd 62: Methyl ((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl) oxy)methoxy)carbonyl)-L- alaninate and Cmpd 63: ((((Methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)-Z-alanine

[0828] Step 1: Methyl ((((methyl (2-(pyridin-2-yl)ethyl)carbamoyl)oxy)meth oxy)carbonyl)-L- alaninate (Cmpd 62)

[0829] To a stirred solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (228 mg, 1.0 mmol) in / / , / / -di methylformamide (5 mL) was added methyl A -al an inate (103 mg, 1.0 mmol) and cesium carbonate (650 mg, 2 mmol) at 25 °C. The reaction mixture was stirred under a carbon dioxide atmosphere at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash chromatography (silica-gel, 230-400) eluting with a linear gradient of 0-10% methanol in dichloromethane to afford methyl ((((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)-Z-alaninate (Cmpd 62) as a colorless viscous oil (30 mg, 9 %). LCMS (ES+): m / z = 340.17 [M+H]+.

[0830] Step 2: ((((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)-Z-alanine (Cmpd 63)

[0831] To a stirred solution of methyl((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy) methoxy) carbonyl)-Z-alaninate (5 mg, 0.015 mmol) in methanol (1 mL) was added a 1 M solution of lithium hydroxide in water (0.015 mL, 0.015 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 18 h. The reaction was quenched with saturated ammonium chloride solution (10 mL) and extracted with methylene chloride (3 x 5 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash

[0832] 116

[0833] 20300-WO-PCT / / 103241-016PCT chromatography (silica-gel, 230-400) eluting with a linear gradient of 0-10% methanol in dichloromethane to afford ((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)-Z- alanine (Cmpd 63) as a colorless viscous oil (5 mg, 94 %). LCMS (ES+): m / z = 325.96 [M+H]+.

[0834] Cmpd 64: tert-Butyl ((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl) oxy)methoxy)carbonyl)-Z- histidinate

[0835] Step 1: tert- Butyl 7Va-((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)-7Vt- trityl-Z-histidinate

[0836] To a stirred solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (228 mg, 1.0 mmol) in A,A-dimethylformamide (5 mb) was added tert- butyl TV-trity l-A-histidinate (453 mg, 1.0 mmol), cesium carbonate (650 mg, 2.0 mmol) and potassium iodide (332 mg, 2.0 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 18 h. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash chromatography (silica-gel, 230-400) eluting with a linear gradient of 0-10% methanol in dichloromethane to afford tert-butyl Aa-((((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)-M-trityl-Z-histidinate as a colorless viscous oil (33 mg, 5 % yield). LCMS (ES+): m / z = 690.37 [M+H]+. 'H NMR (400 MHz, Methanol-^) 8 8.77 (m, 1H), 8.71 (m, 1H), 8.50 (m, 1H), 7.92 (m, 2H), 7.47 - 7.31 (m, 9H), 7.31 - 7.07 (m, 7H), 5.64 - 5.32 (m, 2H), 4.52 - 4.32 (m, 1H), 3.70 (m, 2H), 3.37 - 3.15 (m, 4H), 3.15 - 3.01 (m, 1H), 2.92 (m, 3H), 1.58 - 1.25 (m, 9H).

[0837] Step 2: tert-Butyl((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)-Z- histidinate (Cmpd 64)

[0838] To a stirred solution of tert-butyl / V'-((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl) oxy)methoxy)carbonyl)-M-trityl-Z-histidinate (33 mg, 0.05 mmol) in methylene chloride (0.5 mL) was added trifluoroacetic acid (0.02 mL, 0.24 mmol) at 25 °C. The reaction mixture was stirred at 25 °C for 1 hour. The solvent was removed in vacuo and the residue was purified by reversed phase HPLC (Cl 8, 5-50% water / acetonitrile with 0.1% TFA) to afford tert-butyl ((((methyl(2-

[0839] 117

[0840] 20300-WO-PCT / / 103241-016PCT (pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)carbonyl)-L-histidinate trifluoroacetate salt (Cmpd 64) as a colorless viscous oil (10 mg, 47 % yield). LCMS (ES+): m / z = 391.94 [M+H-tBu]+.

[0841] Cmpd 65: Diphenyl methyl(2-(pyridin-2-yl)ethyl)phosphoramidate

[0842] Cmpd 65

[0843] To a stirred solution of V-methyl-2-(pyridin-2-yl)ethan-l -amine (100 mg, 0.74 mmol) in anhydrous THF (5 mL) at 0 °C was added diphenyl phosphorochloridate (200 mg, 0.74 mmol) over a period of approximately 3 min and the mixture was stirred at 0 °C for 2 h. The resulting suspension was centrifuged and the solvent decanted. The solids were stirred with diethyl ether, centrifuged and the solvent decanted. The combined organic solutions were evaporated in vacuo and the residue was purified by reverse phase chromatography (Cl 8) eluting with a linear gradient of 5-95% CH3CN / H2O (containing 0.05% TFA) to provide diphenyl methyl(2-(pyridin-2- yl)ethyl)phosphoramidate (Cmpd 65) as a thick oil (109 mg, 31 %). LCMS (ES+): m / z = 369.18 [M+H]+.

[0844] Cmpd 66: Methyl((methyl[2-(pyridin-2-yl)ethyl]amino)(phenoxy)phosphoryl)[2-(pyridin-2- yl)ethyl] amine bis trifluoroacetic acid salt

[0845] To a stirred solution of phenyl phosphorodichloridate (156 mg, 0.74 mmol) and N,N- diisopropylethylamine diisopropylethylamine (194 mg, 269 pL, 1.5 mmol) in anhydrous THF (5 mL) at 0 °C was added / V-methyl-2-(pyridin-2-yl)ethan- l -amine (100 mg, 0.74 mmol) over a period of approximately 3 min and the mixture was stirred at 0 °C for 2 h. Water (0.5 mL) was added and the mixture was stirred at 0 °C for a further 30 min. The solvent was removed in vacuo and the residue was purified by reverse phase chromatography to provide methyl((methyl[2- (pyridin-2-yl)ethyl]amino)(phenoxy)phosphoryl) [2-(pyridin-2-yl)ethyl]amine bis trifluoroacetic acid salt (Cmpd 66) (26 mg, 24 % yield). LCMS (ES+): m / z = 411.27 [M+H]+.

[0846] Cmpd 67: Methyl-(2-pyridin-2-yl-ethyl)-carbamic acid phosphonooxymethyl ester

[0847] 118

[0848] 20300-WO-PCT / / 103241-016PCT

[0849] Step 1: Methyl-(2-pyridin-2-yl-ethyl)-carbamic acid benzyloxy-hydroxy- phosphoryloxymethyl ester

[0850] To a stirred solution of methyl-(2-pyridin-2-yl-ethyl)-carbamic acid chloromethyl ester (200 mg, 0.87 mmol) in DMF (2 mL) was added phosphoric acid dibenzyl ester (292 mg, 1.05 mmol) followed by potassium carbonate (240 mg, 1.74 mmol). The reaction mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated under reduced pressure and partitioned between dichloromethane and water. The aqueous phase was extracted with dichloromethane (3 x 15 mL) and the combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel) eluting with a linear gradient of 0-15% methanol with 5% ammonium hydroxide / dichloromethane to afford methyl-(2-pyridin-2-yl-ethyl)-carbamic acid benzyloxy-hydroxy- phosphoryloxymethyl ester as colorless oil (130 mg, 39 %). LCMS (ES+): m / z = 381.17 [M+H]+.

[0851] Step 2: Methyl-(2-pyridin-2-yl-ethyl)-carbamic acid phosphonooxymethyl ester (Cmpd 67) A reaction vessel containing a stirred solution of methyl-(2-pyridin-2-yl-ethyl)-carbamic acid benzyloxy-hydroxy-phosphoryloxymethyl ester (130 mg, 0.34 mmol) in ethanol (10 mL) at 25 °C was evacuated and flushed with nitrogen three times, then palladium on carbon 55% wet (52 mg) was added. The mixture was then stirred under 1 atm hydrogen and at 25 °C for 16 h. The reaction mixture was filtered through a plug of Celite® and the filtrate was concentrated under reduced pressure. The residue was purified by semi-prep HPLC (Cl 8) eluting with a linear gradient of 0-65% CH3CN-H2O with 0.1% TFA to provide methyl-(2-pyridin-2-yl-ethyl)- carbamic acid phosphonooxymethyl ester (Cmpd 67) as a colorless liquid (75 mg, 76%).

[0852] Cmpd 68: ((Diethoxyphosphoryl)oxy)methyl methyl(2-(pyridin-2-yl)ethyl)carbamate

[0853] Cmpd 68

[0854] To a solution of chloromethyl methyl-2-(pyridin-2-yl)ethylcarbamate (150 mg, 0.66 mmol) in anhydrous DMF (1.5 mL) was added potassium carbonate (182 mg, 1.32 mmol) and diethyl

[0855] 119

[0856] 20300-WO-PCT / / 103241-016PCT hydrogen phosphate (122 mg, 1.32 mmol). The reaction vessel was flushed with nitrogen and stirred for 18 h at room temperature. The solvent was removed in vacuo and the residue was partitioned between ethyl acetate and brine. The layers were separated, the organic phase was dried over sodium sulfate, and the solvent was removed in vacuo. The residue was purified by flash chromatography (silica gel) eluting with a linear gradient of 0-10% (methanol with 5% ammoniumhydroxide] / dichloromethane) to provide ((diethoxyphosphoryl)oxy)methyl methyl(2- (pyridin-2-yl)ethyl)carbamate (Cmpd 68) as a white foam (41 mg, 18%). LCMS (ES+): m / z = 347.95 [M+H]+.

[0857] Cmpd 69: Methyl-(2-pyridin-2-yl-ethyl)-carbamic acid l-(diethoxy-phosphoryloxy)-ethyl ester phosphoric acid

[0858] To a stirred solution of phosphoric acid diethyl ester (0.55 g, 3.6 mmol) in DMF (7.5 mL) was added methyl-(2-pyridin-2-yl-ethyl)-carbamic acid 1 -chloro-ethyl ester (0.68 g, 3.0 mmol) and potassium carbonate (0.83 g, 6.0 mmol). The resulting mixture was stirred at 25 °C for 16 h. The solvent was removed under reduced pressure and the residue was purified by semi-prep HPLC

[0859] (Cl 8) eluting with a linear gradient of 0-65% ACN-H2O with 0.1% TFA to provide (+ / -) methyl-

[0860] (2-pyridin-2-yl-ethyl)-carbamic acid l-(diethoxy-phosphoryloxy)-ethyl ester (Cmpd 69) as a pale yellow liquid (37 mg, 2.85%). LCMS (ES+): m / z = 361.10 [M+H]+.

[0861] Cmpd 70: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl 2-

[0862] ((benzoyloxy)methyl)benzoate

[0863] To a solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate hydrochloride salt (50 mg, 0.19 mmol) and 2-((benzoyloxy)methyl)benzoic acid (58 mg, 0.23 mmol) in DMF (5 mL) was added cesium carbonate (196 mg, 0.60 mmol) and the mixture was stirred at room temperature for 2 h. The mixture was diluted with water and extracted with ethyl acetate (2x). The combined

[0864] 120

[0865] 20300-WO-PCT / / 103241-016PCT organic extracts were washed with saturated brine, dried over magnesium sulfate and the solvent was removed in vacuo. The residue was purified by flash chromatography (silica gel, eluting with a linear gradient of 35-100% ethyl acetate / hexanes) to provide ((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methyl 2-((benzoyloxy)methyl)benzoate (Cmpd 70) (72 mg) as an oil. LCMS (ES+): m / z = 499.0 [M+H]+.

[0866] Cmpd 71: / V-Methyl- / V-(2-(pyridin-2-yl)ethyl)hydroxylamine

[0867] Cmpd 71

[0868] 2-Vinylpyridine (0.32 mL, 3 mmol) andN-methylhydroxylamine (334 mg, 4.0 mmol) were mixed and stirred at 25 °C for 30 minutes. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash column chromatography (silica-gel, 230-400) eluting with a linear gradient of 0 - 50% ethyl acetate in hexane to afford / V-methyl- / V-(2-(pyridin-2-yl)ethyl)hydroxylamine (Cmpd 71) as a brown oil (250 mg, 55 %). LCMS (ES+): m / z = 153.48 [M+H]+.

[0869] Cmpd 72: / V / >-Dimethyl- / V-(2-(pyridin-2-yl)ethyl)hydroxylamine

[0870] Cmpd 72

[0871] 2-Vinylpyridine (0.32 mL, 3 mmol) and N, O-dimethylhydroxylamine hydrochloride (0.39 g, 4.0 mmol) were mixed and stirred at 25 °C for 30 min. The reaction mixture was diluted with saturated sodium bicarbonate solution (10 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash column chromatography (silica-gel, 230-400) eluting with a linear gradient of 0 - 50% ethyl acetate to afford A(O-dimethyl-A^-(2-(pyridin-2-yl)ethyl)hydroxylamine (Cmpd 72) as brown oil (220 mg, 44 % yield). LCMS (ES+): m / z = 167.48 [M+H]+.

[0872] Cmpd 76: ((l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl) oxy)ethoxy)carbonyl)glycine

[0873] 121

[0874] 20300-WO-PCT / / 103241-016PCT

[0875] ((l-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethoxy)carbonyl)-glycine (Cmpd 76) was synthesized in a similar manner to (Cmpd 73) from 1 -chloroethyl methyl(2-(pyridin-2- yl)ethyl)carbamate hydrochloride and glycine benzyl ester hydrochloride. LCMS (ES+): m / z = 326.2 [M+H]+.

[0876] Cmpd 77: ((((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl) oxy) methoxy)carbonyl)-L-histidine

[0877] Step 1: ((((Methyl (2-(pyridin-2-yl)ethyl)carbamoyl) oxy) met boxy )carbonyl )-L- V-Boc- (trityl)histidine

[0878] To a mixture of / V-Boc His(Trt)OH (3.0 g, 6.0 mmol) and cesium carbonate (2.3 g, 7.0 mmol) in DMF (20 mL) was added methyl-(2-pyridin-2-yl-ethyl)-carbamic acid chloromethyl ester (0.2 g, 0.75 mmol) and the mixture was stirred at room temperature for 18 h. The mixture was diluted with water and extracted with ethyl acetate (x2). The combined organic extracts were washed with brine, dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash chromatography (Silica, eluting with a linear gradient of 50-100% ethyl acetate / hexanes) to provide ((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl) oxy) methoxy)carbonyl)-L- V-Boc- (trityl)histidine (0.29 g, 56%) as a viscous oil.

[0879] Step 2: ((((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl) oxy) methoxy)carbonyl)-L- histidine (Cmpd 77)

[0880] To a solution of ((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl) oxy) methoxy)carbonyl)-L- V-Boc- (trityl)histidine (100 mg, 0.14 mmol) in methylene chloride (2 mL) was added trifluoroacetic acid (2 mL) and triethyl silane (93 pL, 0.58 mmol) and the mixture was stirred at room temperature for 2 h. The solvent was evaporated, and the residue was partitioned between methylene chloride and water. The layers were separated, and the aqueous phase was washed with methylene chloride and hexanes and then lyophilized to provide ((((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)

[0881] 122

[0882] 20300-WO-PCT / / 103241-016PCT methoxy)carbonyl)-L- histidine trifluoroacetate (Cmpd 77) (100 mg, 100%). LCMS (ES+): m / z = 348.2 [M+H]+.

[0883] Cmpd 78: ((((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl) oxy)ethoxy)carbonyl)-L-histidine

[0884] ((((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl) oxy)ethoxy)carbonyl)-L-histidine Cmpd 78 was synthesized in a similar manner to Cmpd 77 from 1 -chloroethyl methyl(2-(pyridin-2- yl)ethyl)carbamate hydrochloride and A-Boc His(Trt)OH. LCMS (ES+): m / z = 362.8 [M+H]+.

[0885] Cmpd 80: 4-Methyl-5-((methyl(2-(pyridin-2-yl)ethyl)amino)methyl)-l,3-dioxol-2-one

[0886] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine hydrochloride (0.20 g, 1.2 mmol) in DCM (10 ml) was added tri ethylamine (0.807 mL, 5.79 mmol). After 5 min, 4-(chloromethyl)- 5-methyl-l,3-dioxol-2-one (0.258 g, 1.74 mmol) was added. The mixture was stirred at room temperature for 16 h, then concentrated under reduced pressure. The resulting residue was dissolved in EtOAc (10 mL), washed with water and brine, then dried over sodium sulfate and concentrated under reduced pressure. The crude was purified by Isolera-Biotage using silica gel 230-400 mesh and eluted with 3% MeOH in DCM, to afford 4-methyl-5-((methyl(2-(pyridin-2- yl)ethyl)amino)methyl)-l,3-dioxol-2-one (100 mg, 33% yield) as a light brown gum. LCMS (Method C): m / z = 249.1 (M++ H), Rt. 0.29 mm, 99.82% (Max). HPLC-Method E; RT: 3.73 mm.

[0887] Cmpd 81: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl L-leucinate hydrochloride

[0888] Step 1: Chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate

[0889] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (750 mg, 3.59 mmol) in DCM (8 mL) at 25 °C was added DIPEA (1391 mg, 10.76 mmol). After 5 min,

[0890] 123

[0891] 20300-WO-PCT / / 103241-016PCT chloromethyl carbonochloridate (601 mg, 4.66 mmol) was added to the reaction mixture at 0 °C and reaction was stirred for 2 h at 25 °C. The reaction was monitored by UPLC. The mixture was quenched with ice cooled water (10 mL) and extracted with DCM (3 X 10 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, and concentrated under reduced pressure to afford crude chloromethyl methyl(2-(pyridin-2- yl)ethyl)carbamate (800 mg, 3.11 mmol, 87% yield) as a pale yellow liquid. LCMS (Method A): m / z = 229.1 (M++ H), Rt.= 1.43 mm, 89.76 % (Max).

[0892] Step 2: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (tert-butoxycarbonyl)-L- leucinate

[0893] To a vial containing chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (297 mg, 1.30 mmol) and (tert-butoxycarbonyl)-L-leucine (250 mg, 1.08 mmol) were added monosilver(I) oxide (250 mg, 1.08 mmol) and DMF (0.25 mL) at 25 °C. The reaction mixture was stirred for 3 h at 90 °C under dark conditions. The reaction was monitored by UPLC. The reaction mixture was diluted with DCM (15 mL) and then filtered through Celite®. The filtrate was concentrated under reduced pressure to afford crude as a dark brown liquid. The crude was purified by 230-400 mesh silica using 25 g snap, eluting with 80% ethyl acetate in pet-ether to afford ((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methyl (tert-butoxycarbonyl)-L-leucinate (180 mg, 0.417 mmol, 38.5% yield) as a pale yellow viscous liquid. LCMS (Method A): m / z = 424.2 (M++ H), Rt.= 1.91 min, 98.08 % (Max).

[0894] Step 3: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl L-leucinate hydrochloride (Cmpd 81)

[0895] To a stirred solution of ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (tertbutoxy carbonyl)-L-leucinate (50 mg, 0.118 mmol)) in 1,4-dioxane (0.1 mL) was added HC1 (0.295 mL, 1.181 mmol) at 25 °C. The reaction mixture was stirred for 2 h at 25 °C. The reaction was monitored by UPLC. The reaction mixture was concentrated under reduced pressure. The product was triturated several times with n-hexane to afford (methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methyl L-leucinate hydrochloride (27.30 mg, 0.072 mmol, 61.0% yield) as a pale yellow sticky solid. LCMS (Method B): m / z = 324.2 (M++ H), Rt. = 2.03 min, 96.16 % (Max). HPLC (Method E); RT: 4.28 mm.

[0896] 124

[0897] 20300-WO-PCT / / 103241-016PCT Cmpd 82: (2-(((Methyl(2-(pyridin-2-yl)ethyl)amino)methyl)carbamoyl)phenoxy)methyl pivalate

[0898] Step 1: (2-Carbamoylphenoxy)methyl pivalate

[0899] To a stirred solution of 2-hydroxybenzamide (100 mg, 0.729 mmol) in acetone (1 mL) was added iodomethyl pivalate (177 mg, 0.729 mmol) at room temperature. Then the reaction was stirred for 16 h. The reaction was monitored by UPLC. The reaction mixture was filtered and the filtrate was concentrated. The crude was diluted with DCM and water (10 mL each) and the layers were separated. The organic layer was washed with brine (10 mL), dried over sodium sulfate and concentrated to afford (2-carbamoylphenoxy)methyl pivalate (140 mg, 0.525 mmol, 72.1% yield) as a white solid. LCMS (Method A): m / z = 252.1 (M++ H), Rt. 0.89 mm, 94.4% (Max).1!! NMR (300 MHz, DMSO-d6): 8 7.80 - 7.69 (m, 1H), 7.57 (bs, 2H), 7.56 - 7.45 (m, 1H), 7.32 - 7.21 (m, 1H), 7.20 - 7.11 (m, 1H), 5.90 - 5.77 (m, 2H), 1.12 - 1.03 (m, 10H).

[0900] Step 2: (2-(((Methyl(2-(pyridin-2-yl)ethyl)amino)methyl)carbamoyl)phenoxy)methyl pivalate (Cmpd 82)

[0901] To a stirred solution of (2-carbamoylphenoxy)methyl pivalate (100 mg, 0.398 mmol) in MeOH (5 mL) was added 37% formaldehyde (48.4 mg, 0.597 mmol), followed by N-methyl-2-(pyridin-2- yl)ethan-l -amine dihydrochloride (83 mg, 0.40 mmol) at room temperature and the mixture was stirred for 16 h. The reaction was monitored by TLC. The reaction mixture was concentrated and purified by Prep-HPLC (method-B) to get (2-(((methyl(2-(pyridin-2- yl)ethyl)amino)methyl)carbamoyl)phenoxy)methyl pivalate (20 mg, 0.048 mmol, 12.1% yield) as a white viscous oil. LCMS (Method A): m / z = 400.3 (M++ H), Rt. 0.65 min, 94.7% (Max). HPLC- Method E; RT: 3.35 min, 96.3%

[0902] Cmpd 83: (R)-2-Acetamido-3-mercapto-N-methyl-N-(2-(pyridin-2-yl)ethyl)propanamide

[0903] 125

[0904] 20300-WO-PCT / / 103241-016PCT

[0905] Step 1: N-Acetyl-S-trityl-L-cysteine

[0906] To a stirred solution of acetyl-L-cysteine (500 mg, 3.06 mmol) in DMF (5 mL) was added (chloromethanetriyl)tribenzene (854 mg, 3.06 mmol) at 25 °C and the reaction mixture was stirred at 25 °C for 16 h. The reaction was monitored by LC-MS. The reaction mass was taken to the next step as such. LCMS (Method C): m / z = 404.0 (M+- H), Rt. 1.124 min, 77.42% (Max).

[0907] Step 2: (7?)-2-Acetamido-N-methyl-N-(2-(pyridin-2-yl)ethyl)-3-(tritylthio)propanamide

[0908] To N-acetyl-S-trityl-L-cysteine (1.2 g, 2.3 mmol) was added N-methyl-2-(pyridin-2-yl)ethan-l- amine dihydrochloride (0.572 g, 2.73 mmol) and DIPEA (1.59 mL, 9.11 mmol) followed by EDC (0.874 g, 4.56 mmol) and HOBt (0.698 g, 4.56 mmol) at 0 °C. Then the reaction mixture was stirred at 25 °C for 16 h. The reaction was monitored by LC-MS. The reaction mass was concentrated under reduced pressure to afford crude. The crude was purified by prep-HPLC (Method E). The pure fraction was lyophilized to afford (R)-2-acetamido-N-methyl-N-(2-(pyridin- 2-yl)ethyl)-3-(tritylthio)propanamide (584 mg, 1.11 mmol, 48.8% yield) as an off-white viscous oil. LCMS (Method A): m / z =

[0909] 524.3 (M++H), Rt. 2.036 mm, 99.72% (Max).

[0910] Step 3: (R)-2-Acetamido-3-mercapto-N-methyl-N-(2-(pyridin-2-yl)ethyl)propenamide (Cmpd 83)

[0911] To a stirred solution of (R)-2-acetamido-N-methyl-N-(2-(pyridin-2-yl)ethyl)-3- (tritylthio)propanamide (570 mg, 1.09 mmol) in DCM (2 mL) was added triisopropylsilane (0.446 mL, 2.18 mmol) followed by TFA (0.167 mL, 2.18 mmol) at 0 °C. Then the reaction mixture was stirred at 25 °C for 4 h. The reaction was monitored by LC-MS. The reaction mass was concentrated under reduced pressure to afford crude. The crude was purified by reverse phase chromatography (Method E). The compound was re-purified by prep-HPLC (Method-G). The pure fraction was lyophilized to afford (R)-2-acetamido-3-mercapto-N-methyl-N-(2-(pyridin-2- yl)ethyl)propanamide (50 mg, 0.172 mmol, 15.8% yield) as an off-white viscous oil.

[0912] LCMS (Method A): m / z = 282.1 (M+H), Rt. 0.998 mm, 93.16% (Max). HPLC (Method A): RT: 3.238 min.

[0913] 126

[0914] 20300-WO-PCT / / 103241-016PCT Cmpd 84: S-(((R)-2- Acetamido- 3-(methyl(2-(pyridin-2-yl)ethyl)amino)-3-oxopropyl)thio)-

[0915] N-acetyl-D-cysteine

[0916] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (300 mg, 1.43 mmol) in DMF (5 mL) was added DIPEA (0.832 mL, 4.78 mmol). Then S-(((R)-2-acetamido-2- carboxy ethyl)thio)-N-acetyl-D-cysteine (310 mg, 0.956 mmol) was added followed by DMTMM (529 mg, 1.91 mmol) at 25 °C. The reaction mixture was stirred at 80 °C for 16 h, then concentrated under reduced pressure. The resulting residue was purified by prep-HPLC (Method-F). The pure fractions were lyophilized to afford S-(((R)-2-acetamido-3-(methyl(2-(pyridin-2-yl)ethyl)amino)- 3-oxopropyl)thio)-N-acetyl-D-cysteine (60 mg, 0.13 mmol, 13.9 % yield) as a white solid. LCMS (Method A): m / z = 441.1 (M++ H), Rt. 0.777 mm, 95.59% (Max). HPLC-(Method A): RT: 2.603 min.

[0917] Cmpd 85: Dimer of (5)-2-acetamido-3-mercapto-N-methyl-N-(2-(pyridin-2- yl)ethyl)propanamide

[0918] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (161 mg, 7.71 mmol) in DMF (8 mL), DIPEA (1.34 mL, 7.71 mmol) was added. Then S-(((R)-2-acetamido-2- carboxyethyl)thio)-N-acetyl-D-cysteine (500 mg, 1.54 mmol) was added followed by DMTMM (171 mg, 6.17 mmol) at 25 °C. The reaction mixture was stirred at 80 °C for 16 h. The reaction was monitored by LCMS. To the reaction mass, water was added, and the mixture was washed with ethyl acetate (30 mL). The aqueous layer was concentrated under reduced pressure. The crude material was purified by prep-HPLC (Method-H). The pure fraction was lyophilized to afford dimer of (S)-2-acetamido-3-mercapto-N-methyl-N-(2-(pyridin-2-yl)ethyl)propanamide (160 mg,

[0919] 127

[0920] 20300-WO-PCT / / 103241-016PCT 0.285 mmol, 18.48 % yield) as an off-white gummy solid. LCMS (Method A): m / z = 561.2 (M++H), Rt. 1.173 mm, 98.85% (Max). HPLC (Method A): RT: 3.474 mm.

[0921] Cmpd 86: N-Methyl-N-(2-(pyridin-2-yl)ethyl)-4-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-

[0922] (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)butanamide

[0923] Step 1: Lithium 4-hydroxybutanoate

[0924] To a stirred solution of dihydrofuran-2(3H)-one (500 mg, 5.81 mmol) in methanol (5 mL) and water (2.0 mL) was added LiOH (278 mg, 11.6 mmol) at 25 °C and the mixture was stirred at 25 °C for 16 h. The reaction was monitored by LC-MS. The reaction mass was concentrated under reduced pressure to afford crude lithium 4-hydroxybutanoate (1.2 g, crude) as a white solid. Purification was not performed.

[0925] LCMS (Method C): m / z = 103.2 (M+-H), Rt. 0.153 mm, 99.88% (Max). ‘H-NMR (400 MHz, DMSO-d6): 8 6.32 (s, 1H), 3.42-3.36 (m, 2H), 2.02 (t, J= 6.60 Hz, 2H), 1.54 (t, J = 6.00 Hz, 2H).

[0926] Step 2: 4-Hydroxy-N-methyl-N-(2-(pyridin-2-yl)ethyl)butanamide

[0927] To a stirred solution ofN-methyl-2-(pyridin-2-yl)ethan-l-amine dihydrochloride (1.4 g, 6.7 mmol) in DMF (10 mL) was added lithium 4-hydroxybutanoate (1.10 g, 10.0 mmol). Then DIPEA (5.83 m , 33.5 mmol) followed by BOP (5.92 g, 13.4 mmol) were added at 25 °C and the reaction mixture was stirred at 25 °C for 16 h. The reaction was monitored by LC-MS. Water was added, and the mixture was extracted with 10% methanol in DCM (30 mL x 5). The combined organic layers were concentrated under reduced pressure. The crude material was purified by flash column chromatography (230-400 nm silica mesh, 0-100% ethyl acetate in pet-ether gradient and 10% methanol in DCM). The pure fraction was concentrated under reduced pressure to afford 4- hydroxy-N-methyl-N-(2-(pyridin-2-yl)ethyl)butanamide (3.5 g, 6.46 mmol, 96% yield) as an off- white gummy liquid. LCMS (Method A): m / z = 223.2 (M+-H), Rt. 0.953 min, 41.34% (Max).

[0928] 128

[0929] 20300-WO-PCT / / 103241-016PCT Step 3: (2R,3R,4S,5R,6R)-2-(Acetoxymethyl)-6-(4-(methyl(2-(pyridin-2-yl)ethyl)amino)-4- oxobutoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0930] To a stirred solution of ((2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran- 3,4,5-triyl triacetate (1.00 g, 2.43 mmol) in anhydrous diethyl ether (15 mL) was added 4-hydroxy- N-methyl-N-(2-(pyridin-2-yl)ethyl)butanamide (1.98 g, 3.65 mmol). Then silver carbonate (2.01 g, 7.30 mmol) in presence of molecular sieves (4A) was added and the reaction mixture was stirred at 25 °C for 16 h. The reaction was monitored by LC-MS. The mixture was filtered and concentrated under reduced pressure to afford crude. The crude was purified by flash column 25 g (230-400 nm silica, 0-100% ethyl acetate in pet-ether gradient). The pure fraction was concentrated to afford (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(4-(methyl(2-(pyridin-2- yl)ethyl)amino)-4-oxobutoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (260 mg, 0.249 mmol, 10.25% yield) as an off white gummy solid. LCMS (Method A): m / z = 553.2 (M++H), Rt. 1.516 min, 53.37% (Max)

[0931] Step 4: N-Methyl-N-(2-(pyridin-2-yl)ethyl)-4-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-

[0932] (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)butanamide (Cmpd 86)

[0933] To a stirred solution of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(4-(methyl(2-(pyridin-2- yl)ethyl)amino)-4-oxobutoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (260 mg, 0.471 mmol) in DCM (5 mL) was added sodium methoxide in 25% methanol (0.215 mL, 0.941 mmol) at 0 °C. Then the reaction mixture was stirred at 25 °C for 5 h. The reaction was monitored by LC-MS. The reaction mass was quenched with water and extracted with 10% methanol in DCM (30 mL*5). The collected organic layer was dried with sodium sulfate, filtered and concentrated under reduced pressure to afford crude. The crude was purified by prep-HPLC (Method-F). The pure fraction was lyophilized to afford N-methyl-N-(2-(pyridin-2-yl)ethyl)-4-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy- 6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)butanamide (20 mg, 0.051 mmol, 11% yield) as an off white viscous oil. LCMS (Method C): m / z = 385.2 (M++H), Rt. 0.812 min, 99.89% (Max). HPLC (Method A): RT: 2.775 mm.

[0934] Cmpd 87: 3-(((2R,3R,4S,5S,6R)-3,4,5-Trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran- 2-yl)oxy)propyl methyl(2-(pyridin-2-yl)ethyl)carbamate

[0935] 129

[0936] 20300-WO-PCT / / 103241-016PCT

[0937] Step 1: 4-Nitrophenyl methyl(2-(pyridin-2-yl)ethyl)carbamate

[0938] To a stirred solution of 4-nitrophenyl carbonochloridate (1.00 g, 4.96 mmol) in DCM (10 mL) was added N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (1.04 g, 4.96 mmol) followed by TEA (2.1 mL, 15 mmol) at 25 °C. Then the reaction mass was stirred at 25 °C for 16 h. The reaction was monitored by LCMS. The reaction mass was washed with water and extracted with DCM (50 mL*3). The collected organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to afford crude. The crude was purified by column chromatography using 25 g snap (230-400 nm silica mesh, 0-100% ethyl acetate in pet-ether gradient). The pure fraction was concentrated under reduced pressure to afford 4-nitrophenyl methyl(2-(pyridin-2-yl)ethyl)carbamate (900 mg, 2.77 mmol, 55.9% yield) as a brown liquid. LCMS (Method C): m / z = 302.1 (M++H), Rt. 0.919 mm, 92.96% (Max). 'H-NMR (300 MHz, DMSO-d6): 8 8.53-8.49 (m, 1H), 8.29-8.22 (m, 2H), 7.70-7.76 (m, 1H), 7.39-7.33 (m, 2H), 7.20- 7.27 (m, 2H), 3.82-3.64 (m, 2H), 3.03 (s, 3H), 2.92 (s, 2H).

[0939] Step 2: 3-Hydroxypropyl methyl(2-(pyridin-2-yl)ethyl)carbamate

[0940] To a stirred solution of 4-nitrophenyl methyl(2-(pyridin-2-yl)ethyl)carbamate (900 mg, 2.99 mmol) in acetonitrile (10 mL) was added propane- 1,3 -diol (0.322 mL, 4.48 mmol) followed by CS2CO3 (2.92 g, 8.96 mmol) at 25 °C. Then the reaction mass was stirred at 80 °C for 5 h. The reaction was monitored by LCMS. The reaction mass was filtered and concentrated under reduced pressure to afford crude. The crude was purified by 25 g snap (230-400 nm silica mesh, 0-100% ethyl acetate in pet-ether gradient and 0-10% methanol in DCM). The pure fraction was concentrated under reduced pressure to afford 3-hydroxypropyl methyl(2-(pyridin-2- yl)ethyl)carbamate (555 mg, 1.83 mmol, 61.4% yield) as a yellow liquid. LCMS (Method C): m / z = 239.1 (M++H), Rt. 0.576 mm, 78.71% (Max).

[0941] 130

[0942] 20300-WO-PCT / / 103241-016PCT Step 3: (2R,3R,4S,5R,6R)-2-(Acetoxymethyl)-6-(3-((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)propoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0943] To a stirred solution of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5- triyl triacetate (104 mg, 2.52 mmol)) in anhydrous THF (10 mL) was added 3-hydroxypropyl methyl(2-(pyridin-2-yl)ethyl)carbamate (500 mg, 2.10 mmol). Then silver carbonate (1736 mg, 6.29 mmol) in presence of molecular sieves (4 A) was added and the reaction mixture was stirred at 80 °C for 16 h. The reaction was monitored by LC-MS. The reaction mass was filtered and concentrated under reduced pressure to afford crude (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(3- ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)propoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (180 mg, 0.109 mmol, 5.21% yield) as an off-white solid. (Method C): m / z = 569.1 (M++H), Rt. 0.926 mm, 34.50% (Max).

[0944] Step 4: 3-(((2R,3R,4S,5S,6R)-3,4,5-Trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)propyl methyl(2-(pyridin-2-yl)ethyl)carbamate (Cmpd 87)

[0945] To a stirred solution of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(3-((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)propoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (180 mg, 0.317 mmol) in DCM (5 mL) was added sodium methoxide in 25% methanol (0.145 mL, 0.633 mmol) at 0 °C. Then the reaction mixture was stirred at 25 °C for 2 h. The reaction was monitored by TLC. The reaction mass was quenched with water and concentrated under reduced pressure to afford crude. The crude was purified by prep-HPLC (Method A). The pure fraction was concentrated below 40 °C and lyophilized to afford 3-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl methyl(2-(pyridin-2-yl)ethyl)carbamate (76.0 mg, 0.189 mmol, 59.7% yield) as an off-white liquid. LCMS (Method C): m / z = 401.7 (M++H), Rt. 1.966 mm, 99.90% (Max). HPLC (Method A): RT: 2.856 mm.

[0946] Cmpd 88: 2-Oxo-l,3-dioxan-5-yl methyl(2-(pyridin-2-yl)ethyl)carbamate and Cmpd 89: l,3-dihydroxypropan-2-yl methyl(2-(pyridin-2-yl)ethyl)carbamate

[0947] Step 1: 2,2-Dimethyl-l,3-dioxan-5-yl carbonochloridate

[0948] To a stirred solution of triphosgene (1.35 g, 4.54 mmol) in THF (10 mL), charcoal (0.018 g, 1.5 mmol) was added at 25 °C. After 5 min 2,2-dimethyl-l,3-dioxan-5-ol (1.00 g, 7.57 mmol)

[0949] 131

[0950] 20300-WO-PCT / / 103241-016PCT dissolved in THF was added dropwise to the reaction mixture at 0 °C and the reaction mixture was stirred for 3 h at 25 °C. The reaction was monitored by TLC. After 3 h the reaction mixture was filtered through the Celite®. The solution was used in the next step.

[0951] Step 2 : 2,2-Dimethyl-l,3-dioxan-5-yl methyl(2-(pyridin-2-yl)ethyl)carbamate

[0952] To a stirred solution of 2,2-dimethyl-l,3-dioxan-5-yl carbonochloridate (1.20 g, 6.17 mmol) obtained from previous step, DIPEA (4.31 mL, 24.7 mmol) was added at 0 °C. N-Methyl-2- (pyridin-2-yl)ethan-l -amine dihydrochloride (1.55 g, 7.40 mmol) was added to solution at 25 °C and the reaction mixture was stirred for 16 h at 25 °C. The reaction mixture was concentrated under reduced pressure. The resulting crude was purified by chromatography (230-400 mesh silica using 50 g snap, 80% EtOAc in pet-ether) to afford 2,2-dimethyl-l,3-dioxan-5-yl methyl(2- (pyridin-2-yl)ethyl)carbamate (610 mg, 1.78 mmol, 28.9% yield) as a pale-yellow liquid. LCMS (Method F): m / z = 295.2 (M++ H), Rt.= 0.746 mm, 86.53 % (Max).

[0953] Step 3: l,3-Dihydroxypropan-2-yl methyl(2-(pyridin-2-yl)ethyl)carbamate (Cmpd 89)

[0954] To a stirred solution of 2,2-dimethyl-l,3-dioxan-5-yl methyl(2-(pyridin-2-yl)ethyl)carbamate (450 mg, 1.53 mmol) in MeOH (5 mL), 4-methylbenzenesulfonic acid hydrate (233 mg, 1.22 mmol) was added at 0 °C and the mixture was stirred for 16 h at 25 °C. The reaction was monitored by UPLC. The reaction mixture was concentrated under reduced pressure and crude was purified by reverse phase chromatography (C-18, 100 g column, eluted with 45% acetonitrile and ammonium bicarbonate in water) to afford l,3-dihydroxypropan-2-yl methyl(2-(pyridin-2-yl)ethyl)carbamate (120 mg, 0.434 mmol, 28.4% yield) as a pale yellow viscous liquid. LCMS (Method B): m / z = 255.1 mm (M++ H), Rt. = 1.12 mm, 92.84% (Max).

[0955] Step 4: 2-Oxo-l,3-dioxan-5-yl methyl(2-(pyridin-2-yl)ethyl)carbamate (Cmpd 88)

[0956] To a stirred solution of triphosgene (28.0 mg, 0.094 mmol) in THF (1 mL), charcoal (0.378 mg, 0.031 mmol) was added at 25 °C. After 5 min l,3-dihydroxypropan-2-yl methyl(2-(pyridin-2- yl)ethyl)carbamate (40 mg, 0.16 mmol) dissolved in THF was added dropwise at 0 °C and the reaction mixture was stirred for 3 h at 25 °C. The reaction was monitored by UPLC. After 3 h the reaction mixture was filtered through the Celite® and the filtrate was concentrated under reduced pressure. The resulting residue was purified by prep-HPLC (Method A) to afford 2-oxo-l,3- dioxan-5-yl methyl(2-(pyridin-2-yl)ethyl)carbamate (18 mg, 0.062 mmol, 39.4% yield) as a

[0957] 132

[0958] 20300-WO-PCT / / 103241-016PCT yellow liquid. LCMS (Method B): m / z = 281.1 (M++ H), Rt. = 1.55 min, 97.06% (Max). HPLC

[0959] (Method E): Rt: 3.51 mm.

[0960] Cmpd 90 and Cmpd 91: 2-Hydroxy ethyl carbonochloridate and

[0961] (carbonylbis(oxy))bis(ethane-2,l-diyl) bis(methyl(2-(pyridin-2-yl)ethyl)carbamate)

[0962] Step 1: 2-Hydroxy ethyl carbonochloridate

[0963] To a stirred solution of ethane- 1,2-diol (100 mg, 1.61 mmol) in ACN (1 mL) were added triphosgene (478 mg, 1.61 mmol) and DIPEA (0.281 mL, 1.61 mmol) dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 1 h. Completion of reaction was confirmed by TLC. The reaction mixture was evaporated to dryness to afford crude 2-hydroxy ethyl carbonochloridate (540 mg) as a yellow solid.

[0964] Step 2: 2-Hydroxyethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (Cmpd 90)

[0965] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (500 mg, 2.39 mmol) in ACN (1 mL) were added TEA (1.30 mL, 9.56 mmol) and 2-hydroxyethyl carbonochloridate (447 mg, 3.59 mmol) at room temperature. The reaction mixture was stirred at room temperature for 36 h. Completion of reaction was confirmed by UPLC. The reaction mixture was quenched with water (5 mL) and extracted with DCM (2 x 5 mL). The combined organic extracts were dried over sodium sulfate and evaporated. The crude product was purified by flash column chromatography (silica-gel, 230-400) using MeOH - DCM (0 - 20%) as eluent. The pure fractions were evaporated and dried under high vacuum to afford crude (250 mg). The crude product (100 mg) was purified by prep-HPLC (Method A). The pure fractions were lyophilized to afford 2-hydroxyethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (31 mg, 0.135 mmol, 5.7% yield) as pale yellow viscous oil. LCMS (Method A): m / z = 225.1 (M++ H), Rt. 1.266 mm, 98.69% (Max). HPLC (Method E); Rt: 2.953 mm.

[0966] Step 3 (Carbonylbis(oxy))bis(ethane-2,l-diyl) bis(methyl(2-(pyridin-2-yl)ethyl)carbamate) (Cmpd 91)

[0967] To a stirred solution of 2-hydroxyethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (300 mg, 1.34 mmol) in THE (3 mL) were added 1 , 1 -carbonyldiimidazole (1.09 g, 6.69 mmol) and DIPEA (1.17

[0968] 133

[0969] 20300-WQ-PCT / / 103241-016PCT mL, 6.69 mmol) dropwise at room temperature. The reaction mixture was stirred at 80 °C for 32 h. Completion of reaction was confirmed by LCMS. The reaction mixture was quenched with water (10 mL) and extracted with ethyl acetate (2 x 10 mL). The combined organic extracts were dried over sodium sulfate and evaporated. The crude product was purified by flash column chromatography (silica-gel, 230-400) using MeOH - DCM (0 - 10%) as eluent. The pure fractions were evaporated and dried under high vacuum to afford crude. The crude product was purified by prep-HPLC (Method A). The prep fractions were lyophilized to afford (carbonylbis(oxy))bis(ethane-2,l-diyl) bis(methyl(2-(pyridin-2-yl)ethyl)carbamate) (9 mg, 0.018 mmol, 1.361% yield) as a pale yellow viscous oil. LCMS (Method B): m / z = 475.2 (M++ H), Rt. 2.032 mm, 94.31% (Max). HPLC (Method E); RT: 4.470 mm.

[0970] Cmpd 92: 4-(2-(((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)-2-oxoethyl)phenyl hydrogen phosphate

[0971] Step 1: Methyl 2-(4-((bis(benzyloxy)phosphoryl)oxy)phenyl)acetate

[0972] To a stirred solution of methyl 2-(4-hydroxyphenyl)acetate (3.00 g, 18.1 mmol) in dichloromethane (30 mL) were added dibenzyl diisopropylphosphoramidite (12.5 g, 36.1 mmol) and tetrazole (2.82 g, 36.1 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 3 h. Hydrogen peroxide (4.42 g, 39.0 mmol) dissolved in acetonitrile (2 mL) was added to the reaction mixture at 0 °C and it was stirred for 1 h at 0 °C. Completion of reaction was confirmed by UPLC. The reaction mixture was quenched with a saturated solution of sodium metabisulphite (3 mL) and extracted with dichloromethane (2 x 15 mL). The combined organic extracts were dried over sodium sulfate and evaporated. The crude product was purified by flash column chromatography (silica-gel, 230-400) using ethyl acetate - petroleum ether (0 - 30%) as an eluent. The pure fractions were evaporated and dried under vacuum to afford methyl 2-(4- ((bis(benzyloxy)phosphoryl)oxy)phenyl)acetate (3.00 g, 6.75 mmol, 37.4% yield) as a pale yellow liquid. LCMS (Method C): m / z = 427.1 (M++ H), Rt. 1.180 mm, 96.44% (Max).

[0973] Step 2: 2-(4-((bis(Benzyloxy)phosphoryl)oxy)phenyl)acetic acid

[0974] 134

[0975] 20300-WO-PCT / / 103241-016PCT To a stirred solution of methyl 2-(4-((bis(benzyloxy)phosphoryl)oxy)phenyl)acetate (1.0 g, 2.3 mmol) in THF (3.5 mL) was added a solution of lithium hydroxide (0.112 g, 4.69 mmol) in water (3.5 mL) dropwise at room temperature. After 10 min completion of reaction was confirmed by UPLC. The reaction mixture was evaporated to dryness. The crude product was dissolved in water (20 mL) and washed with 10% isopropyl alcohol in chloroform (4 x 10 mL). The aqueous layer was acidified with 10% aqueous solution of citric acid (pH = 6.5) and extracted with 10% isopropyl alcohol in chloroform (4 x 10 mL). The combined organic layers were dried over sodium sulfate and evaporated to afford 2-(4-((bis(benzyloxy)phosphoryl)oxy)phenyl)acetic acid (740 mg, 1.60 mmol, 68.1% yield) as a transparent liquid. LCMS (Method C): m / z = 413.0 (M++ H), Rt. 1.082 min, 89.08% (Max).

[0976] Step 3: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl 2-(4- ((bis(benzyloxy)phosphoryl)oxy)phenyl)acetate

[0977] To a stirred solution of 2-(4-((bis(benzyloxy)phosphoryl)oxy)phenyl)acetic acid (300 mg, 0.727 mmol) in toluene (3 mL) were added chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (333 mg, 1.46 mmol) and silver oxide (169 mg, 0.727 mmol) at room temperature. The reaction mixture was stirred at 65 °C for 2 h. Completion of reaction was monitored by LCMS. The reaction mixture was filtered through Celite® and washed with dichloromethane (2 x 4 mL). The filtrate was diluted with dichloromethane (3 mL), washed with water (5 mL), dried over anhydrous sodium sulfate and evaporated. The crude product was purified by reverse phase Cl 8 column chromatography using water - acetonitrile (0 - 55%) as an eluent. The pure fractions were lyophilized to afford ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl 2-(4-

[0978] ((bis(benzyloxy)phosphoryl)oxy)phenyl)acetate (220 mg, 0.349 mmol, 48.0% yield) as a paleyellow liquid.

[0979] LCMS (Method F): m / z = 605.2 (M++ H), Rt. 1.188 mm, 96.66% (Max).

[0980] Step 4: Ammonium 4-(2-(((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methoxy)-2- oxoethyl)phenyl hydrogen phosphate (Cmpd 92)

[0981] To a stirred solution of ((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl 2-(4- ((bis(benzyloxy)phosphoryl)oxy)phenyl)acetate (120 mg, 0.198 mmol) in methanol (1 mL) was added Pd-C (52.8 mg, 0.050 mmol). The reaction mixture was stirred under 1 kg of hydrogen atmosphere at room temperature for 2 h. Completion of reaction was monitored by LCMS. The reaction mixture was filtered through Celite® and washed with methanol (2 x 10 mL). The filtrate

[0982] 135

[0983] 20300-WO-PCT / / 103241-016PCT was evaporated. The crude product was purified by prep-HPLC (Method A). The pure fractions were lyophilized to afford ammonium 4-(2-(((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methoxy)-2-oxoethyl)phenyl hydrogen phosphate (38 mg, 0.082 mmol, 41.4% yield) as an off white solid. LCMS (Method B): m / z = 425.1 (M++ H), Rt. 0.968 min, 95.54% (Max). HPLC (Method E); Rt: 2.420 mm.

[0984] Cmpd 93: 2-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)propane-l,3-diyl (2S,2'S)-bis(2- amino-3-methylbutanoate) dihydrochloride

[0985] Step 1: 2,2-Dimethyl-l,3-dioxan-5-yl carbonochloridate

[0986] To a stirred solution of triphosgene (1.35 g, 4.54 mmol) in THF (10 mL), charcoal (0.018 g, 1.5 mmol) was added at 25 °C. After 5 min a solution of 2,2-dimethyl-l,3-dioxan-5-ol (1.0 g, 7.8 mmol) in THF was added dropwise to the reaction mixture at 0 °C and reaction was stirred for 3 h at 25 °C. The reaction was monitored by TLC. After 3 h the reaction mixture was filtered through the Celite® and the filtrate was used for the next step.

[0987] Step 2: 2,2-Dimethyl-l,3-dioxan-5-yl methyl(2-(pyridin-2-yl)ethyl)carbamate

[0988] To a stirred solution of 2,2-dimethyl-l,3-dioxan-5-yl carbonochloridate (1.0 g, 5.1 mmol) in THF (as obtained from Step 1 ) at 0 ° C was added DIPEA (1.99 g, 15.4 mmol) . The mixture was warmed to 25 °C and then N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (1.08 g, 5.14 mmol) and DIPEA (2 eq) were added. The reaction was monitored by UPLC. After 16 h, the mixture was concentrated under reduced pressure, then diluted with DCM (20 mL), filtered and concentrated under reduced pressure to afford 2,2-dimethyl-l,3-dioxan-5-yl methyl(2-(pyridin-2- yl)ethyl)carbamate (1.2 g, 2.85 mmol, 55% yield) as a yellow liquid. LCMS (Method F): m / z = 295.1 (M++ H), Rt.= 0.75 mm, 84.18 % (Max).

[0989] Step 3: l,3-Dihydroxypropan-2-yl methyl(2-(pyridin-2-yl)ethyl)carbamate

[0990] 136

[0991] 20300-WO-PCT / / 103241-016PCT To a stirred solution of 2,2-dimethyl-l,3-dioxan-5-yl methyl(2-(pyridin-2-yl)ethyl)carbamate (1.00 g, 3.40 mmol) in MeOH (10 mL), 4-methylbenzenesulfonic acid hydrate (0.517 g, 2.72 mmol) was added at 25 °C. The reaction was monitored by UPLC. After 16 h the reaction mixture was concentrated under reduced pressure and the residue was purified by reverse phase chromatography using C-l 8 100 g column, product was eluted at 45% acetonitrile and ammonium bicarbonate in water to afford pure l,3-dihydroxypropan-2-yl methyl(2-(pyridin-2- yl)ethyl)carbamate (810 mg, 3.09 mmol, 91% yield) as a colorless gummy liquid. LCMS (Method B): m / z = 255.1 mm (M++ H), Rt. = 1.19 mm, 97.55% (Max).

[0992] Step 4: 2-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)propane-l,3-diyl (2S,2'S)-bis(2- ((tert-butoxycarbonyl)amino)-3-methylbutanoate)

[0993] To a stirred solution of l,3-dihydroxypropan-2-yl methyl(2-(pyridin-2-yl)ethyl)carbamate (400 mg, 1.57 mmol) in THF (5 mL) and traces of DMF (to solubilize), DCC (714 mg, 3.46 mmol) and DMAP (423 mg, 3.46 mmol) were added at 25 °C. (tert-Butoxycarbonyl)-L-valine (718 mg, 3.30 mmol) was added and the mixture was stirred for 24 h at 25 °C. The reaction was monitored by UPLC. The reaction mixture was quenched with ice cooled water (7 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, and concentrated under reduced pressure to afford crude (370 mg). The crude was purified by 230-400 mesh silica using 25 g snap, product was eluted at 40% ethyl acetate in pet-ether to afford pure 2-((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)propane- 1,3 -diyl (2S,2'S)-bis(2-((tert-butoxycarbonyl)amino)-3-methylbutanoate) (250 mg, 0.379 mmol, 24.1% yield) as a yellow liquid. LCMS (Method F): m / z = 653.3 min (M++ H), Rt. = 1.29 min, 99.66 % (Max).

[0994] Step 5: 2-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)propane-l,3-diyl (2S,2'S)-bis(2- amino-3-methylbutanoate) dihydrochloride (Cmpd 93)

[0995] To a stirred solution of 2-((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)propane- 1,3 -diyl (2S,2'S)-bis(2-((tert-butoxycarbonyl)amino)-3-methylbutanoate) (100 mg, 0.153 mmol) in 1,4- dioxane (1 mL) hydrochloric acid in dioxane (0.383 mL, 1.53 mmol) was added at 25 °C. The reaction was monitored by UPLC. After 2 h, the reaction mixture was concentrated under reduced pressure, then concentrated two times after addition of DCM (5 mL) and triturated two times with hexane (5 mL) to afford pure 2-((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)propane- 1,3 -diyl (2S,2'S)-bis(2-amino-3-methylbutanoate) dihydrochloride (46 mg, 0.085 mmol, 56% yield) as a

[0996] 137

[0997] 20300-WO-PCT / / 103241-016PCT colorless gummy liquid. LCMS (Method B): m / z: 453.3 (M++ H), Rt. = 1.98 min, 98.57 % (Max). HPLC (Method E): Rt = 4.19 mm.

[0998] Cmpd 94: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (((5-methyl-2-oxo-l,3- dioxol-4-yl)methoxy)carbonyl)-L-leucinate

[0999] Step 1: (5-Methyl- 2-oxo- l,3-dioxol-4-yl)methyl (4-nitrophenyl) carbonate

[1000] To a stirred solution of 4-(hydroxymethyl)-5-methyl-l,3-dioxol-2-one (2.00 g, 15.4 mmol) in CHCh (20 mL) at 0 °C, pyridine (1.34 g, 16.9 mmol) was added. After 5 min the reaction mixture was cooled to 0 °C and 4-nitrophenyl carbonochloridate (3.41 g, 16.9 mmol) was added. The reaction was monitored by UPLC. After stirring for 16 h at 25 °C the mixture was quenched with ice cooled water (20 mL) and extracted with DCM (3 x 20 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, and concentrated under reduced pressure to afford crude (2.3 g). The crude was purified by 230-400 mesh silica using 50 g snap, product was eluted at 20% ethyl acetate in pet-ether to afford pure (5-methyl-2-oxo-l,3-dioxol-4- yl)methyl (4-nitrophenyl) carbonate (910 mg, 2.96 mmol, 19.3% yield) as a yellow solid. LCMS (Method C): m / z = 146.2 (M++ H), Rt.= 0.96 mm, 96.02% (Max).

[1001] Step 2: tert-Butyl (((5-methyl-2-oxo-l,3-dioxol-4-yl)methoxy)carbonyl)-L-leucinate

[1002] To a stirred solution of tert-butyl L-leucinate hydrochloride (300 mg, 1.34 mmol) in acetonitrile (4 mL) at 25 °C, cesium carbonate (1.31 g, 4.02 mmol) was added. After 5 min (5-methyl-2-oxo- l,3-dioxol-4-yl)methyl (4-nitrophenyl) carbonate (475 mg, 1.61 mmol) was added and reaction mixture was stirred for 16 h at 25 °C. The reaction was monitored by UPLC. The reaction mixture was quenched with ice cooled water (10 mL) and then extracted with ethyl acetate (3 x 15 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, and concentrated under reduced pressure to afford crude (120 mg). The crude was purified by prep-HPLC (Method C) to afford tert-butyl (((5-methyl-2-oxo-l,3-dioxol-4- yl)methoxy)carbonyl)-L-leucinate (120 mg, 0.346 mmol, 25.8% yield) as a yellow solid. LCMS (Method C): m / z = 132.3 (M++ H), Rt.= 1.18 mm, 99.88% (Max).

[1003] Step 3: (((5-Methyl-2-oxo-l,3-dioxol-4-yl)methoxy)carbonyl)-L-leucine

[1004] 138

[1005] 20300-WO-PCT / / 103241-016PCT To a stirred solution of tert- butyl (((5-methyl-2-oxo-l,3-dioxol-4-yl)methoxy)carbonyl)-L- leucinate (130 mg, 0.379 mmol) in DCM (1.5 mL), TFA (0.146 mb, 1.89 mmol) was added at 0 °C and the mixture was stirred for 2 h at 25 °C. The reaction was monitored by UPLC. The reaction mixture was concentrated under reduced pressure to afford (((5-methyl-2-oxo-l,3-dioxol-4- yl)methoxy)carbonyl)-L-leucine (115 mg, 0.364 mmol, 96% yield) as a yellow solid. LCMS (Method C): m / z = 244.1 min (M++ H), Rt. = 0.80 min, 91.49 % (Max).

[1006] Step 4: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl (((5-methyl-2-oxo-l,3-dioxol- 4-yl)methoxy)carbonyl)-L-leucinate (Cmpd 94)

[1007] To a stirred solution of (((5-methyl-2-oxo-l,3-dioxol-4-yl)methoxy)carbonyl)-L-leucine (100 mg, 0.348 mmol) in toluene (1 mL), chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (96 mg, 0.42 mmol) and silver(I) oxide (81 mg, 0.35 mmol) were added at 25 °C and reaction mixture was stirred for 2 h at 65 °C under dark condition. The reaction was monitored by UPLC. The reaction mixture was diluted with DCM and filtered through Celite®. The filtrate was concentrated under reduced pressure. The crude was purified by 230-400 mesh silica using 12+M column, product was eluted at 80% ethyl acetate in pet-ether to afford ((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)methyl (((5-methyl-2-oxo-l,3-dioxol-4-yl)methoxy)carbonyl)-L- leucinate (8.0 mg, 0.017 mmol, 4.8% yield) as a colorless gummy liquid. Product purification was strictly carried out under normal phase chromatography. LCMS (Method B): m / z: 480.2 (M++ H), Rt. = 2.67 mm, 97.07 % (Max). HPLC (Method E): Rt: 5.44 mm.

[1008] Cmpd 95: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl pivalate

[1009] Cmpd 95

[1010] To a stirred solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (150 mg, 0.656 mmol) in pivalic acid (1.34 g, 13.1 mmol) was added silver oxide (152 mg, 0.656 mmol) at room temperature. The reaction mixture was stirred at 95 °C for 2 h. Completion of reaction was confirmed by LCMS. The reaction mixture was diluted with dichloromethane and filtered through Celite®. The filtrate was basified with 10% solution of ammonium bicarbonate and extracted with ethyl acetate (2*5 mL). The organic layers were concentrated. The crude product was purified by prep-HPLC (Method A). The prep fractions were lyophilized to afford ((methyl(2-(pyridin-2-

[1011] 139

[1012] 20300-WO-PCT / / 103241-016PCT yl)ethyl)carbamoyl)oxy)methyl pivalate (15 mg, 0.050 mmol, 7.7% yield) as a yellow a viscous liquid. LCMS (Method A): m / z = 295.1 (M++ H), Rt. 1.688 mm, 98.57% (Max). HPLC-Method E; Rt: 5.174 min.

[1013] Cmpd 96: 2-Methyl-l-((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)propyl pivalate

[1014] To a stirred solution of N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (500 mg, 2.39 mmol) in THF (10 mL) was added 2-methyl-l-(((4-nitrophenoxy)carbonyl)oxy)propyl pivalate (1.52 g, 2.87 mmol) followed by TEA (1.00 mL, 7.17 mmol) at 25 °C. Then the reaction mixture was stirred at 25 °C for 3 h. The reaction was monitored by LC-MS. To the mixture water was added and it was extracted with ethyl acetate (50 mL * 4). The collected organic layer was concentrated under reduced pressure to afford crude. Crude was purified by prep-HPLC (Method A). The pure fraction was lyophilized to afford desired compound with impurities. Then the compound was purified by column chromatography using 10 g (230-400 nm silica mesh) 0-100% ethyl acetate in pet-ether gradient. The pure fraction was concentrated and lyophilized to afford 2- methyl-l-((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)propyl pivalate (170 mg, 0.500 mmol, 20.9% yield) as an off-white viscous oil. LCMS (Method B): m / z = 337.2 (M++H), Rt. 3.220 min, 98.39% (Max). HPLC-(Method A): Rt: 6.309 mm.

[1015] Cmpd 97: (S)-l-(L-Valyl)-N-methyl-N-(2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide hydrochloride

[1016] Step 1: tert-Butyl ((S)-3-methyl-l-((S)-2-(methyl(2-(pyridin-2- yl)ethyl)carbamoyl)pyrrolidin-l-yl)-l-oxobutan-2-yl)carbamate

[1017] To a stirred solution of (tert-butoxycarbonyl)-L-valyl-L-proline (330 mg, 1.05 mmol) in DMF (1 m ) was added N-methyl-2-(pyridin-2-yl)ethan-l -amine (143 mg, 1.05 mmol) and DIPEA (0.917 mL, 5.25 mmol) followed by BOP (511 mg, 1.16 mmol). The reaction mixture was

[1018] 140

[1019] 20300-WO-PCT / / 103241-016PCT stirred at room temperature for 16 h. Completion of reaction was monitored by UPLC. The reaction mixture was concentrated to get crude gummy oil, then purified by prep-HPLC (Method B) to get tert-butyl ((S)-3-methyl-l-((S)-2-(methyl(2-(pyridin-2-yl)ethyl)carbamoyl)pyrrolidin-l- yl)-l-oxobutan-2-yl)carbamate (200 mg, 0.458 mmol, 43.6% yield) as a pale-yellow gum. LCMS (Method C): m / z = 433.1 (M++ H), Rt. 0.635 mm, 99.2% (Max). HPLC-Method E; Rt: 4.79 mm, 99.9%.

[1020] Step 2: (S)-l-(L-Valyl)-N-methyl-N-(2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide hydrochloride

[1021] To a stirred solution of tert-butyl ((S)-3-methyl-l-((S)-2-(methyl(2-(pyridin-2- yl)ethyl)carbamoyl)pyrrolidin-l-yl)-l-oxobutan-2-yl)carbamate (50 mg, 0.12 mmol) in dioxane (2 mL) was added HC1 (4 M in dioxane) (0.058 mL, 0.23 mmol) at 0 °C. Then the mixture was stirred at room temperature for 2 h. The mixture was concentrated, co-evaporated with DCM (2 x 5 mL) and dried to afford a pale brown gum. The compound was lyophilized, to get (S)-l-(L-valyl)-N- methyl-N-(2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide hydrochloride (25 mg, 0.067 mmol, 58.0% yield) as an off-white sticky solid. LCMS (Method C): m / z = 333.3 (M++ H), Rt. 0.17 min, 99.9% (Max). HPLC-Method E; Rt: 2.79 mm, 98.05%

[1022] Cmpd 98: (S)-3-Amino-4-((S)-2-(methyl(2-(pyridin-2-yl)ethyl)carbamoyl)pyrrolidin-l-yl)- 4-oxobutanoic acid

[1023] Step 1: ((S)-4-(tert-Butoxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutanoyl)-L-proline

[1024] To a stirred solution of (S)-4-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutanoic acid (0.50 g, 1.73 mmol) in DMF (5 mL) at 25 °C, DIPEA (0.906 mL, 5.18 mmol) and BOP (0.764 g, 1.73 mmol) were added. After 5 min L-proline (0.239 g, 2.07 mmol) was added and reaction mixture was stirred for 16 h at 25 °C. The reaction was monitored by UPLC. The reaction mixture was quenched with ice cooled water (10 mL) and extracted with ethyl acetate (3 X 10 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, and concentrated under reduced pressure to afford crude (1.5 g). The crude was purified by 230-

[1025] 141

[1026] 20300-WO-PCT / / 103241-016PCT 400 mesh silica using 25 g snap, product was eluted at 7% methanol in DCM to afford (S)-4-(tert- butoxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutanoyl)-L-proline (0.154 g, 0.397 mmol, 23% yield) as a yellow liquid. LCMS (Method C): m / z = 387.1 (M++ H), Rt.= 0.91 min, 23.31% (Max).

[1027] Step 2 : tert-Butyl (S)-3-((tert-butoxycarbonyl)amino)-4-((S)-2-(methyl(2-(pyridin-2- yl)ethyl)carbamoyl)pyrrolidin-l-yl)-4-oxobutanoate

[1028] To a stirred solution of (S)-4-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutanoyl)-L- proline (300 mg, 0.776 mmol) in DMF (4 mL), DIPEA (301 mg, 2.33 mmol) was added at 25 °C. After 5 min BOP (343 mg, 0.776 mmol) and N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (195 mg, 0.932 mmol) were added and the reaction mixture was stirred for 16 h at 25 °C. The reaction was monitored by UPLC. The reaction mixture was quenched with ice cooled water (15 mL) and extracted with ethyl acetate (3 x 15 mL). The combined organic phases were washed with brine (10 mL), dried over sodium sulfate, and concentrated under reduced pressure to afford crude (630 mg). The crude was purified by 230-400 mesh silica using 25 g snap, product was eluted at 20% ethyl acetate in pet-ether to afford tert-butyl (S)-3-((tert- butoxycarbonyl)amino)-4-((S)-2-(methyl(2-(pyridin-2-yl)ethyl)carbamoyl)pyrrolidin-l-yl)-4- oxobutanoate (90 mg, 0.169 mmol, 21.8% yield) as a yellow liquid. LCMS (Method L): m / z = 505.2 (M++ H), Rt.= 1.00 mm, 95.64% (Max).

[1029] Step 3: (S)-3-Amino-4-((S)-2-(methyl(2-(pyridin-2-yl)ethyl)carbamoyl)pyrrolidin-l-yl)-4- oxobutanoic acid

[1030] To a stirred solution of tert-butyl (S)-3-((tert-butoxycarbonyl)amino)-4-((S)-2-(methyl(2-(pyridin-

[1031] 2-yl)ethyl)carbamoyl)pyrrolidin-l-yl)-4-oxobutanoate (65 mg, 0.13 mmol) in DCM (1 mL) at 0 °C, TLA (0.049 mL, 0.644 mmol) was added and reaction mixture was stirred for 2 h at 25 °C. The reaction was monitored by UPLC. The reaction mixture was concentrated under reduced pressure to afford crude (52 mg). The crude was purified by prep-HPLC (Method E) to afford (S)-

[1032] 3-amino-4-((S)-2-(methyl(2-(pyridin-2-yl)ethyl)carbamoyl)pyrrolidin-l-yl)-4-oxobutanoic acid (21.57 mg, 0.062 mmol, 48.0% yield) as a white solid. LCMS (Method B): m / z = 349.2 min (M++ H), Rt. = 0.89 mm, 97.46 % (Max). HPLC (Method E): Rt: 2.33 mm.

[1033] Cmpd 99: ((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl 5-((3aS,4S,6aR)-2- oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoate

[1034] 142

[1035] 20300-WO-PCT / / 103241-016PCT

[1036] To a stirred solution of chloromethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (1.00 g, 4.37 mmol) and 5-((3aS,4S,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoic acid (1.07 g, 4.37 mmol) in DMF (2 mL) was added silver oxide (1.01 g, 4.37 mmol) and the reaction mixture was stirred at 100 °C for 2 h. Completion of reaction was monitored by UPLC and TLC. The reaction mixture was filtered through Celite® and filtrate was quenched with sodium bicarbonate solution (50 mL) and extracted with DCM (3 x 100 mL). The organic layer was dried over sodium sulfate and concentrated under high vacuum to afford crude product which was purified through reverse phase column to yield (methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)methyl 5-((3aS,4S,6aR)-2- oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanoate (247 mg, 0.561 mmol, 12.8% yield) as a yellowish solid. LCMS (Method B): m / z = 437.1 (M++ H), Rt = 1.973 mm, 98.76%. HPLC (Method B): Rt = 2.885 mm, 98.23%

[1037] Cmpd 101: 3,5-Dimethyl-2-(2-methyl-4-(methyl(2-(pyridin-2-yl)ethyl)amino)-4-oxobutan-2- yl)phenyl isobutyrate

[1038] Step 1: 2-(4-Hydroxy-2-methylbutan-2-yl)-3,5-dimethylphenol

[1039] To a stirred solution of 4,4,5,7-tetramethylchroman-2-one (2.00 g, 9.79 mmol) in THF (5 mL) was added a solution of LAH (12.2 mL, 24.5 mmol, 2M in THF) at 0 °C. Then the reaction mixture was stirred at 25 °C for 2 h. The reaction was monitored by TLC. The reaction mixture was quenched with ethyl acetate and saturated aqueous sodium sulfate and then filtered through Celite® and extracted with ethyl acetate (30 mL*4). The organic layer was dried with sodium sulfate, filtered and concentrated under reduced pressure to afford crude. The crude was recrystallized with

[1040] 143

[1041] 20300-WO-PCT / / 103241-016PCT DCM to afford 2-(4-hydroxy-2-methylbutan-2-yl)-3,5-dimethylphenol (2.04 g, 9.70 mmol, 99% yield) as a white solid. LCMS (Method B): m / z = 207.1 (M- H)', Rt. 2.607 min, 99.05 % (Max)

[1042] Step 2: 2-(4-((tert-Butyldimethylsilyl)oxy)-2-methylbutan-2-yl)-3,5-dimethylphenol

[1043] To a stirred solution of 2-(4-hydroxy-2-methylbutan-2-yl)-3,5-dimethylphenol (2.00 g, 9.60 mmol) in DCM (20 mb) was added DMAP (1.88 g, 15.4 mmol) followed by TBDMS-C1 (1.74 g, 11.5 mmol) in DCM at 0 °C. Then the reaction mixture was stirred at 25 °C for 2 h. The reaction was monitored by TLC. The reaction was quenched with water and then extracted with DCM (50 mL*3). The collected organic layer was washed with sodium bicarbonate solution, dried over sodium sulfate, filtered, and concentrated under reduced pressure to afford crude. Crude was washed with pet-ether and filtered, then dried under vacuum to afford 2-(4-((tert- butyldimethylsilyl)oxy)-2-methylbutan-2-yl)-3, 5 -dimethylphenol (2.1 g, 6.5 mmol, 67.6% yield) as a white solid. 'H NMR (400 MHz, DMSO-d6) 8 = 9.09 - 8.86 (m, 1H), 6.48 - 6.09 (m, 2H), 3.44 - 3.36 (m, 2H), 2.37 (s, 3H), 2.12 - 2.05 (m, 5H), 1.46 (s, 6H), 0.87 - 0.77 (m, 9H), -0.06 (s, 6H)

[1044] Step 3: 2-(4-((tert-Butyldimethylsilyl)oxy)-2-methylbutan-2-yl)-3,5-dimethylphenyl isobutyrate

[1045] To a stirred solution of 2-(4-((tert-butyldimethylsilyl)oxy)-2-methylbutan-2-yl)-3,5- dimethylphenol (700 mg, 2.17 mmol) in DCM (15 mL) was added isobutyric acid (287 mg, 3.26 mmol) followed by DMAP (26.5 mg, 0.217 mmol) at 25 °C. Then DCC (896 mg, 4.34 mmol) was added. Then the reaction mixture was stirred at 25 °C for 16 h. The reaction was monitored by TLC. The reaction was quenched with water followed by sodium bicarbonate solution, then extracted with DCM (50 mL*3). The collected organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to afford a brown liquid. The crude was purified by column chromatography with 25 g snap (230-400 nm silica mesh) eluting with 0-50% ethyl acetate in pet-ether gradient. The pure fraction was concentrated under reduced pressure to afford 2-(4-((tert-butyldimethylsilyl)oxy)-2-methylbutan-2-yl)-3,5-dimethylphenyl isobutyrate (560 mg, 1.42 mmol, 65.7% yield) as a yellow viscous liquid. 'H NMR (400 MHz, DMSO-de) 8 = 6.82 (d, J= 1.5 Hz, 1H), 6.53 (d, J = 1.4 Hz, 1H), 3.46 - 3.38 (m, 2H), 2.80 - 2.70 (m, 1H), 2.47 (s, 3H), 2.18 (s, 3H), 1.41 (s, 6H), 1.23 (d, J= l.Q Hz, 7H), 0.81 (s, 9H), -0.06 (s, 6H)

[1046] Step 4: 2-(4-Hydroxy-2-methylbutan-2-yl)-3,5-dimethylphenyl isobutyrate

[1047] 144

[1048] 20300-WO-PCT / / 103241-016PCT To a stirred solution of 2-(4-((tert-butyldimethylsilyl)oxy)-2-methylbutan-2-yl)-3,5- dimethylphenyl isobutyrate (550 mg, 1.40 mmol) in THF (5.0 mL) and water (5.0 mb) was added AcOH (15.0 mL, 1.40 mmol) at 0 °C and the reaction mixture was stirred at 25 °C for 16 h. The reaction was monitored by LCMS and TLC. The reaction mixture was concentrated under reduced pressure and quenched with water, then extracted with ethyl acetate (25 mL *2). The collected organic layer was washed with brine (15 mL*3). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to afford crude product 2-(4-hydroxy-2-methylbutan-2- yl)-3,5-dimethylphenyl isobutyrate (360 mg, 1.28 mmol, 91% yield) as a brown gum. NMR (300 MHz, DMSO-d6) 8 = 6.90 - 6.76 (m, 1H), 6.51 (d, J= 2.0 Hz, 1H), 4.29 - 4.15 (m, 1H), 3.27 - 3.14 (m, 2H), 2.77 (quin, J = 7.0 Hz, 1H), 2.48 (s, 2H), 2.18 (s, 3H), 2.01 - 1.87 (m, 2H), 1.40 (s, 6H), 1.25 (s, 3H), 1.22 (s, 3H).

[1049] Step 5: 3,5-Dimethyl-2-(2-methyl-4-oxobutan-2-yl)phenyl isobutyrate (SYN2400561-047)

[1050] To a stirred solution of 2-(4-hydroxy-2-methylbutan-2-yl)-3,5-dimethylphenyl isobutyrate (350 mg, 1.26 mmol) in DCM (6 mL) was added PCC (542 mg, 2.51 mmol) at 25 °C. Then the reaction mass was stirred at 25 °C for 16 h. The progress of the reaction was monitored by TLC and 2,4 DNP stain. The reaction mixture was filtered through a Celite® bed. The filtrate was concentrated under reduced pressure and used in the next reaction without further purification.1H NMR (400 MHz, DMSO-d6) 8 = 9.51 - 9.44 (m, 1H), 8.88 - 8.85 (m, 1H), 8.47 - 8.40 (m, 1H), 7.97 - 7.92 (m, 1H), 6.87 - 6.82 (m, 1H), 6.59 - 6.51 (m, 1H), 2.84 - 2.77 (m, 3H), 2.21 - 2.16 (m, 3H), 1.50 - 1.46 (m, 6H), 1.26 - 1.23 (m, 6H).

[1051] Step 6: 3-(2-(Isobutyryloxy)-4,6-dimethylphenyl)-3-methylbutanoic acid

[1052] To a stirred solution of 3,5-dimethyl-2-(2-methyl-4-oxobutan-2-yl)phenyl isobutyrate (330 mg, 1.19 mmol) in water (2.0 mL) and ACN (6.0 mL) was added sodium chlorite (162 mg, 1.79 mmol), sodium dihydrogen phosphate (43.0 mg, 0.36 mmol) and hydrogen peroxide (0.131 mL, 1.67 mmol) at 0 °C. Then the reaction mixture was stirred at 25 °C for 16 h. The progress of the reaction was monitored by TLC. The reaction mixture was concentrated under reduced pressure. Then 1 N HC1 was added till the pH was brought to 3-4. Then the mixture was extracted several times with ethyl acetate. The combined organics were dried over sodium sulfate, filtered, and concentrated under reduced pressure to desired product. LCMS (Method B): m / z = 293.1 (M++ H), Rt. 1.965 min, 43.19 % (Max).

[1053] 145

[1054] 20300-WO-PCT / / 103241-016PCT Step 7 : 3,5-Dimethyl-2-(2-methyl-4-(methyl(2-(pyridin-2-yl)ethyl)amino)-4-oxobutan-2- yl)phenyl isobutyrate (Cmpd 101)

[1055] To a stirred solution of 3-(2-(isobutyryloxy)-4,6-dimethylphenyl)-3-methylbutanoic acid (320 mg, 1.09 mmol) in DMF (8 mL) was added N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (229 mg, 1.09 mmol) followed by BOP (726 mg, 1.64 mmol) and DIPEA (4.43 mL, 3.28 mmol) at 25 °C. Then the reaction mixture was stirred at 25 °C for 16 h. Completion of reaction was monitored by LCMS. The reaction was quenched with water and extracted with ethyl acetate (15 mL *4). The collected organic layer was washed with brine (10 mL*3). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to afford crude product as a brown liquid. The crude product was purified by prep-HPLC by method A. The prep fractions were lyophilized to afford 3,5-dimethyl-2-(2-methyl-4-(methyl(2-(pyridin-2-yl)ethyl)amino)-4- oxobutan-2-yl)phenyl isobutyrate (45.3 mg, 0.110 mmol, 10.0% yield) as a brown gum. LCMS (Method B): m / z = 411.3 (M++ H), Rt. 3.164 mm, 99.61 % (Max). HPLC (Method A): Rt: 6.202 mm.

[1056] Cmpd 102: 2,2-Dimethyl-l,3-dioxan-5-yl methyl(2-(pyridin-2-yl)ethyl)carbamate

[1057] Cmpd 102

[1058] Step 1: 2,2-Dimethyl-l,3-dioxan-5-yl carbonochloridate

[1059] To a stirred solution of 2,2-dimethyl-l,3-dioxan-5-ol (50 mg, 0.38 mmol) in THF (5 mL) was added charcoal (4.54 mg, 0.378 mmol) followed by triphosgene (67.4 mg, 0.227 mmol) at 25 °C. Then the reaction mixture was stirred at 25 °C for 3 h. The reaction mass was filtered through Celite®, and filtrate was concentrated and used as such in the next step.

[1060] Step 2: 2,2-Dimethyl-l,3-dioxan-5-yl methyl(2-(pyridin-2-yl)ethyl)carbamate

[1061] To a stirred solution of 2,2-dimethyl-l,3-dioxan-5-yl (4-nitrophenyl) carbonate (100 mg, 0.336 mmol) in THF (10 mL) was added N-methyl-2-(pyridin-2-yl)ethan-l -amine (50.4 mg, 0.370 mmol) followed by DIPEA (0.176 mL, 1.01 mmol) at 25 °C. Then the reaction mixture was stirred at 25 °C for 16 h. The reaction was monitored by LCMS. To the reaction mixture, water was added

[1062] 146

[1063] 20300-WO-PCT / / 103241-016PCT and it was extracted with DCM (10 mL*3). The collected organic layer was dried with sodium sulfate, filtered and concentrated.

[1064] The crude was purified by prep-HPLC (Method B). LCMS (Method B): m / z = Not ionised, Rt. 1.94 min, 93.0% (Max). HPLC (Method E); Rt: 4.12 mm, 96.75%

[1065] Cmpd 103: l-Hydroxypropan-2-yl methyl(2-(pyridin-2-yl)ethyl)carbamate

[1066] Cmpd 103

[1067] Step 1: l-((tert-Butyldimethylsilyl)oxy)propan-2-yl carbonochloridate:

[1068] To a stirred solution of l-((tert-butyldimethylsilyl)oxy)propan-2-ol (250 mg, 1.31 mmol) in THF (5.0 mL) was added activated charcoal (1.6 mg, 0.13 mmol) followed by triphosgene (234 mg, 0.788 mmol). Then the reaction mass was stirred at 25 °C for 3 h. The reaction was monitored by TLC. The reaction mass was filtered and taken to the next step as such.

[1069] Step 2: l-((tert-Butyldimethylsilyl)oxy)propan-2-yl methyl(2-(pyridin-2-yl)ethyl)carbamate To a solution of l-((tert-butyldimethylsilyl)oxy)propan-2-yl carbonochloridate (260 mg, 1.03 mmol) in THF (10 mL) was added N-methyl-2-(pyridin-2-yl)ethan-l-amine dihydrochloride (215 mg, 1.03 mmol) followed by DIPEA (399 mg, 3.09 mmol) at 25 °C. Then the reaction mixture was stirred at 25 °C for 16 h. The progress of the reaction was monitored by LCMS. After completion of reaction, the reaction was quenched with water and extracted with ethyl acetate (50 mL*4). The collected organic layer was washed with brine (30 mL*3). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to afford crude l-((tert- butyldimethylsilyl)oxy)propan-2-yl methyl(2-(pyridin-2-yl)ethyl)carbamate (320 mg, 0.866 mmol, 84 % yield) as a brown liquid. LCMS (Method C): m / z = 353.2 (M++ H), Rt. 0.953 min, 95.39 % (Max).

[1070] Step 3: l-Hydroxypropan-2-yl methyl(2-(pyridin-2-yl)ethyl)carbamate (Cmpd 103)

[1071] To a solution of l-((tert-butyldimethylsilyl)oxy)propan-2-yl methyl(2-(pyridin-2- yl)ethyl)carbamate (300 mg, 0.851 mmol) in MeOH (5.0 mL) was added pTSA (162 mg, 0.851 mmol) at 0 °C. Then the reaction mixture was stirred at 25 °C for 2 h. The reaction was monitored by LCMS. The reaction mass was concentrated under reduced pressure, quenched with water, and extracted with ethyl acetate (25 mL *4). The collected organic layer was washed with brine (15

[1072] 147

[1073] 20300-WO-PCT / / 103241-016PCT mL*3). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to afford crude l-hydroxypropan-2-yl methyl(2-(pyridin-2-yl)ethyl)carbamate as a brown gummy liquid. The crude product was purified by prep-HPLC (Method A). The pure fractions were lyophilized to afford 1 -hydroxypropan-2-yl methyl(2-(pyridin-2-yl)ethyl)carbamate (22 mg, 0.092 mmol, 11 % yield) as a pale-yellow gum. LCMS (Method B): m / z = 239.1 (M++ H), Rt. 1.462 min, 99.92 % (Max). HPLC (Method E): Rt: 3.323 mm.

[1074] Cmpd 104: (lS,2R)-2-Hydroxycyclopentyl methyl(2-(pyridin-2-yl)ethyl)carbamate

[1075] TBS-CI, Im,

[1076] Step 1: (lR,2S)-2-((tert-Butyldimethylsilyl)oxy)cyclopentan-l-ol

[1077] To a stirred solution of (lR,2S)-cyclopentane-l,2-diol (500 mg, 4.90 mmol) in THF (5 mL) was added imidazole (1.00 g, 14.7 mmol), DMAP (60 mg, 4.9 mmol) and TBSC1 (738 mg, 4.90 mmol).

[1078] The reaction mixture was stirred at room temperature for 2 h. Completion of reaction was monitored by TLC. The reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate and evaporated to afford crude product. This crude product was purified by flash column chromatography (silica-gel 230-400 mesh) using ethyl acetate\hexane (0 -20%) as an eluent. The pure fractions were evaporated and dried under vacuum to afford (lR,2S)-2-((tert- butyldimethylsilyl)oxy)cyclopentan-l-ol (390 mg, 1.80 mmol, 36.8% yield) as a colorless liquid. ‘H-NMR (400 MHz, DMSO-d6): 8 = 4.48 - 4.42 (m, 1H), 4.20 - 4.12 (m, 1H), 4.03 - 3.97 (m, 1H), 3.84 - 3.73 (m, 2H), 3.50 - 3.41 (m, 2H), 0.93 - 0.85 (m, 9H), 0.11 - 0.06 (m, 6H).

[1079] Step 2: (lR,2S)-2-((tert-Butyldimethylsilyl)oxy)cyclopentyl carbonochloridate

[1080] To a stirred solution of triphosgene (466 mg, 1.57 mmol) and charcoal (3.77 mg, 0.314 mmol) in

[1081] THF (5 mL) was added (lR,2S)-2-((tert-butyldimethylsilyl)oxy)cyclopentan-l-ol (340 mg, 1.57

[1082] 148

[1083] 20300-WO-PCT / / 103241-016PCT mmol) at 0 °C. The reaction was stirred at 25 °C for 3 h. Completion of reaction was monitored by TLC. The reaction mixture was filtered through Celite® and the filtrate was concentrated under reduced pressure to afford (lR,2S)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl carbonochloridate (340 mg, 1.22 mmol, 78% yield) as a brown liquid. This crude was directly taken to the next step without further purification.

[1084] Step 3: (lS,2R)-2-((tert-Butyldimethylsilyl)oxy)cyclopentyl methyl(2-(pyridin-2- yl)ethyl)carbamate

[1085] To a stirred solution of (lR,2S)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl carbonochloridate (350 mg, 1.26 mmol) and N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (262 mg, 1.26 mmol) in THF (5 mL) was added DIPEA (1.07 mL, 6.28 mmol) at 0 °C and the reaction mixture was stirred for 16 h at 25 °C. Completion of reaction was monitored by UPLC and TLC. The reaction mixture was quenched with sodium bicarbonate solution at 0 °C and extracted with DCM (3 x 20 mL). The organic layer was washed with brine (25 mL) and water (20 mL), dried over sodium sulfate and concentrated under vacuum to afford crude product. This crude product was purified by flash column chromatography (silica-gel 230-400 mesh) using ethyl acetate\hexane (0 -20%) as an eluent. The pure fractions were evaporated and dried under vacuum to afford (1 S,2R)- 2-((tert-butyldimethylsilyl)oxy)cyclopentyl methyl(2-(pyridin-2-yl)ethyl)carbamate (450 mg, 1.12 mmol, 89% yield) as a brown liquid. LCMS (Method C): m / z = 379.1 (M++ H), Rt = 1.056 mm, 94.50%.

[1086] Step 4: (lS,2R)-2-Hydroxycyclopentyl methyl(2-(pyridin-2-yl)ethyl)carbamate (Cmpd 104)

[1087] To a stirred solution of (lS,2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl methyl(2-(pyridin-2- yl)ethyl)carbamate (450 mg, 1.19 mmol) in MeOH (5 mL) was added p-toluenesulfonic acid monohydrate (226 mg, 1.19 mmol). The reaction mixture was stirred at room temperature for 1 h. Completion of reaction was monitored by UPLC and TLC. The reaction mixture was concentrated under reduced pressure and then diluted with water and extracted with DCM. The organic layer was dried over sodium sulfate and concentrated under vacuum to afford crude product. The crude product was purified by reverse phase chromatography (Method E). The pure fractions were lyophilized to afford (1 S,2R)-2-hydroxy cyclopentyl methyl(2-(pyridin-2-yl)ethyl)carbamate (37.2 mg, 0.141 mmol, 11.8 % yield) as a brown gum. LCMS (Method B): m / z = 265.2 (M++ H), Rt = 2.777 mm, 99.07%. HPLC (Method A): Rt = 3.692 mm, 99.81%.

[1088] 149

[1089] 20300-WO-PCT / / 103241-016PCT Cmpd 105: (3S,4R)-4-Hydroxytetrahydrofuran-3-yl methyl(2-(pyridin-2- yl)ethyl)carbamate

[1090] TBS-CI, Im,

[1091] Cmpd 105

[1092] Step 1 : (3R,4S)-4-((tert-Butyldimethylsilyl)oxy)tetrahydrofuran-3-ol

[1093] To a stirred solution of ((3R,4S)-tetrahydrofuran-3,4-diol (1.00 g, 9.61 mmol) in DMF (10 mL) was added imidazole (1.96 g, 28.8 mmol), DMAP (0.117 g, 0.961 mmol) and TBS-CI (1.45 g, 9.61 mmol). The reaction mixture was stirred at room temperature for 2 h. Completion of reaction was monitored by TLC. The reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate and evaporated to afford crude product. This crude product was purified by flash column chromatography (silica-gel 230-400 mesh) using ethyl acetate\hexane (0 -20%) as an eluent. The pure fractions were evaporated and dried under high vacuum to afford (3R,4S)-4-((tert- butyldimethylsilyl)oxy)tetrahydrofuran-3-ol (850 mg, 3.89 mmol, 40.5% yield) as a colorless liquid. ‘H-NMR (400 MHz, DMSO-d6): 8 = 4.46 - 4.37 (m, 1H), 4.19 - 4.12 (m, 1H), 4.03 - 3.96 (m, 1H), 3.84 - 3.73 (m, 2H), 3.50 - 3.42 (m, 2H), 0.90 - 0.85 (m, 9H), 0.10 - 0.04 (m, 6H).

[1094] Step 2: (3R,4S)-4-((tert-Butyldimethylsilyl)oxy)tetrahydrofuran-3-yl carbonochloridate

[1095] To a stirred solution triphosgene (1.15 g, 3.89 mmol) and charcoal (9.34 mg, 0.778 mmol) in THF (5 mL) was added (3R,4S)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-ol (850 mg, 3.89 mmol) at 0 °C. The reaction was further stirred at 25 °C for 3 h. Completion of reaction was monitored by TLC. The reaction mixture was filtered through celite and the filtrate was directly taken to the next step without concentration.

[1096] 150

[1097] 20300-WO-PCT / / 103241-016PCT Step 3: (3S,4R)-4-((tert-Butyldimethylsilyl)oxy)tetrahydrofuran-3-yl methyl(2-(pyridin-2- yl)ethyl)carbamate

[1098] To the filtrate that was generated in Step-2 containing (3R,4S)-4-((tert- butyldimethylsilyl)oxy)tetrahydrofuran-3-yl carbonochloridate in THF was added DIPEA (3.4 m , 19.5 mmol) and N-methyl-2-(pyridin-2-yl)ethan-l -amine hydrochloride (673 mg, 3.9 mmol) at 0 °C and the reaction mixture was stirred for 16 h at 25 °C. Completion of reaction was monitored by UPLC and TLC. The reaction mixture was quenched with sodium bicarbonate solution at 0 °C and extracted with DCM. The organic layer was washed with brine and water, dried over sodium sulfate and concentrated under vacuum to afford crude product. This crude product was purified by flash column chromatography (silica-gel 230-400 mesh) using ethyl acetate\hexane (0-20%) as an eluent. The pure fractions were evaporated and dried under vacuum to afford (3S,4R)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl methyl(2- (pyridin-2-yl)ethyl)carbamate (200 mg, 0.433 mmol, 14.3%) as a brown liquid. LCMS (Method C): m / z = 381.3 (M++ H), Rt = 0.847 mm, 82.46%.

[1099] Step 4: (3S,4R)-4-Hydroxytetrahydrofuran-3-yl methyl(2-(pyridin-2-yl)ethyl)carbamate (Cmpd 105)

[1100] To a stirred solution of (3S,4R)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl methyl(2- (pyridin-2-yl)ethyl)carbamate (100 mg, 0.263 mmol) in THF (1 m ) was added a solution of TBAF (0.263 mb, 0.263 mmol, IM in THF). The reaction mixture was stirred at room temperature for 1 h. Completion of reaction was monitored by UPEC and TEC. The reaction mixture was concentrated under reduced pressure and then diluted with water and extracted with DCM. The organic layer was dried over sodium sulfate and concentrated under vacuum to afford crude product. The crude product was purified by prep-HPEC (Method E). The pure fractions were lyophilized to afford (3S,4R)-4-hydroxytetrahydrofuran-3-yl methyl(2-(pyridin-2- yl)ethyl)carbamate (4.3 mg, 0.015 mmol, 5.68% yield) as a yellowish gum. LCMS (Method A): m / z = 267.1 (M++ H), Rt = 1.328 mm, 92.78%. HPLC (Method A): Rt = 3.108 mm, 92.44%.

[1101] Cmpd 106: O-(Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)-L-serine

[1102] Step 1: tert-Butyl N-(tert-butoxycarbonyl)-O-(chlorocarbonyl)-L-serinate

[1103] 151

[1104] 20300-WO-PCT / / 103241-016PCT To a stirred solution of triphosgene (341 mg, 1.15 mmol) in THF (5 mL) at 25 °C was added charcoal (4.60 mg, 0.383 mmol). After 5 min the mixture was cooled to 0 °C and tert-butyl (tert- butoxycarbonyl)-L-serinate (500 mg, 1.91 mmol) dissolved in THF was added dropwise. The reaction mixture was warmed to 25 °C and stirred for 3 h. According to TLC a new non-polar spot was observed. The mixture was filtered through Celite® and used directly in the next step.

[1105] Step 2: tert-Butyl N-(tert-butoxycarbonyl)-O-(methyl(2-(pyridin-2-yl)ethyl)carbamoyl)-L- serinate

[1106] To a stirred solution of tert-butyl N-(tert-butoxycarbonyl)-O-(chlorocarbonyl)-L-serinate (1.20 g, 3.71 mmol) in THF (15 mL) at 0 °C was added DIPEA (2.59 mL, 14.8 mmol). The mixture was warmed to 25 °C and then N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (0.930 g, 4.45 mmol) and DIPEA (1 eq) were added. The reaction was monitored by UPLC. After 16 h the mixture was concentrated under reduced pressure. The crude was purified by 230-400 mesh silica using 50 g snap, product was eluted at 50% ethyl acetate in pet-ether to afford tert-butyl N-(tert- butoxycarbonyl)-O-(methyl(2-(pyridin-2-yl)ethyl)carbamoyl)-L-serinate (410 mg, 0.852 mmol, 23.0% yield) as a pale yellow liquid. LCMS (Method F): m / z = 424.2 (M++ H), Rt.= 1.10 min, 88.59% (Max).

[1107] Step 3: O-(Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)-L-serine (Cmpd 106)

[1108] To a stirred solution of tert-butyl N-(tert-butoxycarbonyl)-O-(methyl(2-(pyridin-2- yl)ethyl)carbamoyl)-L-serinate (100 mg, 0.236 mmol) in DCM (1 mL), TFA (0.182 mL, 2.36 mmol) was added at 0 °C and mixture was stirred for 5 h at 25 °C. The reaction was monitored by UPLC. The mixture was concentrated under reduced pressure. The crude was purified by prep- HPLC (Method B) to afford pure O-(methyl(2-(pyridin-2-yl)ethyl)carbamoyl)-L-serine (29 mg, 0.11 mmol, 46% yield) as a white solid. LCMS (Method B): m / z = 268.1 (M++ H), Rt. = 0.79 min, 99.14% (Max). HPLC (Method F): Rt: 5.93 mm.

[1109] Cmpd 107: O-(Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)-L-threonine

[1110] Step 1: Methyl N-(tert-butoxycarbonyl)-O-(chlorocarbonyl)-L-threoninate

[1111] 152

[1112] 20300-WO-PCT / / 103241-016PCT To a stirred solution of triphosgene (0.763 g, 2.57 mmol) in THF (1 mL) at 25 °C charcoal (10.3 mg, 0.857 mmol) was added. After 5 min the mixture was cooled to 0 °C and methyl (tert- butoxycarbonyl)-L-threoninate (1.00 g, 4.29 mmol) dissolved in THF was added dropwise. The mixture was warmed to 25 °C and stirred for 3 h. According to TLC a new non-polar spot was observed. The mixture was filtered through the Celite® and the filtrate was used as is in the next step.

[1113] Step 2 : Methyl N-(tert-butoxycarbonyl)-O-(methyl(2-(pyridin-2-yl)ethyl)carbamoyl)-L- threoninate

[1114] To a stirred solution of methyl N-(tert-butoxycarbonyl)-O-(chlorocarbonyl)-L-threoninate (1.0 g, 3.4 mmol) in THF at 0 °C, DIPEA (0.897 mL, 5.14 mmol) was added. The mixture was warmed to 25 °C and then N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (0.778 g, 3.72 mmol) and DIPEA (2 eq) were added. The reaction was monitored by UPLC. After 16 h the reaction mixture was concentrated under reduced pressure and diluted with DCM (20 mL). The mixture was filtered, and filtrate was concentrated under reduced pressure to afford crude methyl N-(tert- butoxycarbonyl)-O-(methyl(2-(pyridin-2-yl)ethyl)carbamoyl)-L-threoninate (1.31 g, 2.98 mmol, 88% yield) as a yellow liquid. LCMS (Method F): m / z = 396.2 (M++ H), Rt.= 0.96 min, 90.38% (Max).

[1115] Step 3: O-(Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)-L-threonine (Cmpd 107)

[1116] To a stirred solution of methyl N-(tert-butoxycarbonyl)-O-(methyl(2-(pyridin-2- yl)ethyl)carbamoyl)-L-threoninate (750 mg, 1.89 mmol) in water (4 mL) and THF (4 mL), lithium hydroxide hydrate (80 mg, 1.9 mmol) was added. The reaction mixture was stirred for 2 h at 25 °C, then concentrated under reduced pressure and treated with 1.5N HC1 to remove Boc group. The crude was purified by prep-HPLC (Method C) to afford pure O-(methyl(2-(pyridin-2- yl)ethyl)carbamoyl)-L-threonine (99.5 mg, 0.347 mmol, 18.3% yield). LCMS (Method B): m / z = 282.1 (M++ H), Rt. = 0.88 mm, 99.68% (Max). HPLC (Method A): Rt: 3.86 mm.

[1117] Cmpd 108: Propan e-l,2-diyl bis(methyl(2-(pyridin-2-yl)ethyl)carbamate)

[1118] Step 1: 2-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)propyl carbonochloridate

[1119] 153

[1120] 20300-WO-PCT / / 103241-016PCT To a stirred solution of 1 -hydroxypropan-2-yl methyl(2-(pyridin-2-yl)ethyl)carbamate (90.0 mg, 0.378 mmol, Cmpd 103) in THF (2.0 mL) was added activated charcoal (0.45 mg, 0.038 mmol) followed by triphosgene (67.2 mg, 0.227 mmol). Then the reaction mixture was stirred at 25 °C for 2 h. The progress of the reaction was monitored by TLC. The reaction mixture was filtered and taken to the next step as such.

[1121] Step 2: Propane-1, 2-diyl bis(methyl(2-(pyridin-2-yl)ethyl)carbamate) (Cmpd 108)

[1122] To a stirred solution of 2-((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)propyl carbonochloridate (110 mg, 0.366 mmol) and N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (76 mg, 0.37 mmol) in THF (3.0 mL) was added DIPEA (0.191 mL, 1.10 mmol) at 0 °C. Then the mixture was stirred at room temperature for 6 h. The reaction was monitored by LCMS. The reaction mixture was concentrated under reduced pressure, quenched with H2O and extracted with ethyl acetate (25 mL *4). The collected organic layer was washed with brine (15 mL*3). The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by prep-HPLC (method A). The pure fractions were lyophilized to afford propane- 1,2- diyl bis(methyl(2-(pyridin-2-yl)ethyl)carbamate) (90.0 mg, 0.225 mmol, 61.4% yield) as a paleyellow gum. LCMS (Method B): m / z = 401.2 (M++ H), Rt. 2.047 mm, 99.89 % (Max) HPLC (Method E): RT: 4.370 mm

[1123] Cmpd 109: Ethan e-1, 2-diyl bis(methyl(2-(pyridin-2-yl)ethyl)carbamate

[1124] Step 1: 2-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl carbonochloridate

[1125] To a stirred solution of triphosgene (192 mg, 0.647 mmol) and charcoal (31.1 mg, 2.59 mmol) in THF (5.0 mL) was added 2-hydroxyethyl methyl(2-(pyridin-2-yl)ethyl)carbamate (290 mg, 1.29 mmol) at 0 °C. The mixture was stirred at 25 °C for 3 h. Completion of reaction was monitored by TLC. The reaction mixture was filtered to afford 2-((methyl(2-(pyridin-2- yl)ethyl)carbamoyl)oxy)ethyl carbonochloridate (310 mg, 1.081 mmol, 84% yield) as a white transparent liquid.

[1126] Step 2: Ethane-1, 2-diyl bis(methyl(2-(pyridin-2-yl)ethyl)carbamate (Cmpd 109)

[1127] 154

[1128] 20300-WO-PCT / / 103241-016PCT To a stirred solution of 2-((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)ethyl carbonochloridate (290 mg, 1.01 mmol) in THF (5.0 mb) was added N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (212 mg, 1.01 mmol) followed by DIPEA (392 mg, 3.03 mmol) at 0 °C. Then the reaction mixture was stirred at room temperature for 16 h. Completion of reaction was monitored by TLC and LCMS. The reaction mixture was quenched with water and extracted with ethyl acetate (50 mL *4). The collected organic layer was washed with brine (30 mL *3). The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by prep HPLC (method A). The pure fractions were lyophilized to afford ethane- 1,2- diyl bis(methyl(2-(pyridin-2-yl)ethyl)carbamate) (30.8 mg, 0.079 mmol, 7.79% yield) as a paleyellow gummy. LCMS (Method D): m / z = 387.1 (M++ H), Rt. 1.609 min, 99.11 % (Max). HPLC- (Method E): Rt: 4.094 min.

[1129] Cmpd 110: Propan e-l,3-diyl bis(methyl(2-(pyridin-2-yl)ethyl)carbamate)

[1130] Step 1: 3-Hydroxypropyl carbonochloridate

[1131] To a stirred solution of triphosgene (0.975 g, 3.29 mmol) and charcoal (16 mg, 1.3 mmol) in THF (10 mL) at 0°C was added propane- 1,3 -diol (500 mg, 6.57 mmol). The mixture was stirred at 25 °C for 3 h. Completion of reaction was monitored by TLC. The reaction mixture was filtered through Celite®, and the filtrate was used in the next step.

[1132] Step 2: 3-Hydroxypropyl methyl(2-(pyridin-2-yl)ethyl)carbamate

[1133] To a stirred solution of 3 -hydroxypropyl carbonochloridate (500 mg, 3.61 mmol) in THF (10 mL), was added DIPEA (3.15 mL, 15.0 mmol) at 0 °C and the mixture was stirred for 15 min. Then N- methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (755 mg, 3.61 mmol) was added and reaction mixture was stirred for 16 h at 25 °C. Completion of reaction was monitored by UPLC and TLC. The reaction mixture was diluted with water and extracted with DCM. The organic layer

[1134] 20300-WO-PCT / / 103241-016PCT was dried over sodium sulfate and concentrated under vacuum to afford crude product as 3- hydroxypropyl methyl(2-(pyridin-2-yl)ethyl)carbamate (450 mg, 1.70 mmol, 47.1% yield). LCMS (Method C): m / z = 239.2 (M++ H), Rt = 0.207 mm, 30.41%.

[1135] Step 3: 3-((Methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)propyl carbonochloridate

[1136] To a stirred solution of triphosgene (0.280 g, 0.944 mmol) and charcoal (4.54 mg, 0.378 mmol) in THF (10 mL) was added 3-hydroxypropyl methyl(2-(pyridin-2-yl)ethyl)carbamate(450 mg, 1.89 mmol) at 0°C. The reaction was stirred at 25 °C for 3 h. Completion of reaction was monitored by TLC. The reaction mixture was filtered through Celite®, and the filtrate was taken directly to the next step.

[1137] Step 4: Propane-1, 3-diyl bis(methyl(2-(pyridin-2-yl)ethyl)carbamate) (Cmpd 110)

[1138] To a stirred solution of 3-((methyl(2-(pyridin-2-yl)ethyl)carbamoyl)oxy)propyl carbonochloridate (450 mg, 1.50 mmol) in THF (10 mL), was added DIPEA (1.31 mL, 7.48 mmol) at 0 °C and the mixture was stirred for 15 min. Then N-methyl-2-(pyridin-2-yl)ethan-l -amine dihydrochloride (0.313 mg, 1.496 mmol) was added and reaction mixture was stirred for 16 h at 25 °C. Completion of reaction was monitored by UPLC and TLC. The reaction mixture was diluted with water and extracted with DCM. The organic layer was dried over sodium sulfate and concentrated under vacuum to afford crude product. The crude product was purified by prep-HPLC (Method-B) to afford propane- 1,3 -diyl bis(methyl(2-(pyridin-2-yl)ethyl)carbamate) (43.2 mg, 0.107 mmol, 7.13% yield) as a brownish gum. LCMS (Method B): m / z = 401.2 (M++ H), Rt = 1.975 min, 99.32%. HPLC (Method E): Rt = 4.243 mm, 98.88%.

[1139] Cmpd 111: Propan e-1, 2, 3-triyl tris(methyl(2-(pyridin-2-yl)ethyl)carbamate)

[1140] Step 1: (2,2-Dimethyl-l,3-dioxolan-4-yl)methyl carbonochloridate

[1141] To a stirred solution of triphosgene (2.69 g, 9.08 mmol) in THF (20 mL), charcoal (0.036 g, 3.03 mmol) was added. After 5 min the mixture was cooled to 0 °C and (2,2-dimethyl-l,3-dioxolan-4-

[1142] 156

[1143] 20300-WO-PCT / / 103241-016PCT yl)methanol (2.00 g, 15.1 mmol) dissolved in THF was added dropwise and then the mixture was stirred for 3 h at 25 °C. According to TLC a new non-polar spot was observed. After 3 h the reaction mixture was filtered through the Celite®. The filtrate was taken to the next step as such.

[1144] Step 2: (2,2-Dimethyl-l,3-dioxolan-4-yl)methyl methyl(2-(pyridin-2-yl)ethyl)carbamate

[1145] To a stirred solution of (2,2-dimethyl-l,3-dioxolan-4-yl)methyl carbonochloridate (2.00 g, 10.3 mmol) in THF (from previous step), DIPEA (5.38 mL, 30.8 mmol) was added at 0 °C. N-methyl- 2-(pyridin-2-yl)ethan-l -amine dihydrochloride (2.36 g, 11.3 mmol) was added at 25 °C and the mixture was stirred for 16 h at 25 °C. The reaction was monitored by UPLC. The reaction mixture was concentrated under reduced pressure. The residue was diluted with DCM and filtered. Filtrate was concentrated under reduced pressure. The crude was purified by 230-400 mesh silica using 100 g snap, product was eluted at 95% ethyl acetate in pet-ether to afford (2,2-dimethyl-l,3- dioxolan-4-yl)methyl methyl(2-(pyridin-2-yl)ethyl)carbamate (1.53 g, 5.04 mmol, 49.1% yield) as a yellow liquid. LCMS (Method F): m / z = 295.2 (M++ H), Rt.= 0.75 min, 97.91% (Max).

[1146] Step 3: 2,3-Dihydroxypropyl methyl(2-(pyridin-2-yl)ethyl)carbamate

[1147] To a stirred solution of (2,2-dimethyl-l,3-dioxolan-4-yl)methyl methyl(2-(pyridin-2- yl)ethyl)carbamate (1.90 g, 6.45 mmol) in MeOH (20 mL), 4-methylbenzenesulfonic acid hydrate (0.859 g, 4.52 mmol) was added. The reaction was monitored by UPLC. After stirring for 3 h at 25 °C the reaction mixture was concentrated under reduced pressure and the resulting crude product was purified by reverse phase chromatography using a C-18 100 g column, eluting with 45% acetonitrile in 10 mmol ammonium bicarbonate to afford 2, 3 -dihydroxypropyl methyl(2- (pyridin-2-yl)ethyl)carbamate (1.20 g, 4.58 mmol, 70.9% yield) as a pale-yellow viscous liquid. LCMS (Method F): m / z = 255.1 (M++ H), Rt = 0.47 mm, 97.85% (Max).

[1148] Step 4: 3-((tert-Butyldimethylsilyl)oxy)-2-hydroxypropyl methyl(2-(pyridin-2- yl)ethyl)carbamate

[1149] To a stirred solution of 2,3-dihydroxypropyl methyl(2-(pyridin-2-yl)ethyl)carbamate (860 mg, 3.38 mmol) in DCM (10 mL) at 25 °C, imidazole (345 mg, 5.07 mmol) and DMAP (83 mg, 0.68 mmol) were added. After 5 min, the mixture was cooled to 0 °C and tert-butylchlorodimethylsilane (561 mg, 3.72 mmol) was added. The re...

Claims

1. CLAIMSWHAT IS CLAIMED IS:

1. A compound having Formula I:I, or a pharmaceutically acceptable salt thereof, wherein:A is — C(=O)-R1, whereinR1is a C1-C30 linear or branched organic group, optionally including one or more heteroatoms selected from O, N, S, and P.

2. The compounds according to Claim 1, whereinA is -C(=O)-O-CR10aR1(,b-ORn, whereinR10aand R10bare independently H or Ci-Ce alkyl, andR11is an ester bond-linked natural amino acid, or an ester bond-linked dipeptide of two natural amino acids, wherein an amino group of the natural amino acid is optionally protected with an acetyl (Ac), tert-butyloxycarbonyl (Boc), or benzyloxycarbonyl (Cbz) group.

3. The compounds according to Claim 1, whereinA is -C(=O)-O-CR10aR10b-O-Rn, whereinR10aand R10bare independently H or Ci-C& alkyl, andR11is an ester bond-linked natural amino acid, wherein an amino group of the natural amino acid is substituted with D, wherein D is(i) -C(=O)-CR43aR43b-NR44aR44b, wherein R43a, R43b, and R44aare independently H or Ci-Ce alkyl, and R44bis H, an acetyl (Ac), tert-butyloxycarbonyl (Boc), or benzyloxycarbonyl (Cbz) group,(h) -C(=O)-CR38aR38b-O-C(=O)-R39, wherein R38a, R38b, and R39are independently H or Ci-Ce alkyl,19720300-WO-PCT / / 103241-016PCT(iii) -C(=O)-CR40aR40b-O-R41, wherein R40aand R40bare independently H or Ci- G> alkyl, and R41is an ester bond-linked natural amino acid, or(iv) -C(=O)-O-CR45aR45b-R46, wherein R45aand R45bare H or Ci-C6alkyl, and R46is an unsaturated cyclic carbonate ester, optionally substituted with C1-C3 alkyl.

4. The compounds according to Claim 1, whereinA is -C(=O)-O-CR10aR1(,b-O-C(=O)-Rn, whereinR10aand R10bare independently H or Ci-C& alkyl, andR11is(i) a peptide bond-linked natural amino acid, a peptide bond-linked dipeptide of two natural amino acids, or a peptide bond-linked tripeptide of three natural amino acids, wherein a carboxylic acid group of the natural amino acid is optionally protected as a Ci- C4 alkyl ester or a benzyl ester,(ii) -NR35-CR36aR36b-C(=O)-NR37aR37b, wherein R35, R36a, R36b, and R37aare independently H or Ci-Ce alkyl, and R37bis -R95-C(=O)-OR96, wherein R95is C1-6 alkylene, and R96is H or Ci-Ce alkyl, or(hi) -NR35-CR36aR36b-C(=O)-OR37, wherein R35, R36a, R36b, and R37are independently H or Ci-Ce alkyl.

5. The compounds according to Claim 1, whereinA is -C(=O)-O-CR10aR1(,b-O-C(=O)-Rn, whereinR10aand R10bare independently H or Ci-C& alkyl,R11is H, Ci-Ce alkyl, -R20-R21, a substituted benzyl, or a substituted phenyl,R20is Ci-Ce alkylene,R21is Cs-Cio heterocycloalkyl with S and N ring atoms, wherein said substituted benzyl and substituted phenyl are independently substituted with -CR22aR22b-O-C(=O)-R23, or -OP(=O)(OR24a)(OR24b), and wherein R22a- and R22bare independently H or Ci-Ce alkyl,R23is Ci-Ce alkyl or phenyl, andR24aand R24bare independently H or Ci-Ce alkyl.19820300-WO-PCT / / 103241-016PCT6. The compounds according to Claim 1, whereinA is -C(=O)-O-Rn, whereinR11is:(i) Ci-C6alkyl,(ii) -(Ci-C6alkylene)n-R25, wherein R25is a cyclic carbonate ester, or a cyclic acetyl, said cyclic carbonate ester and said cyclic acetyl each optionally substituted with one or more Ci-Ce alkyl, and n is 0 or 1,(iii) -Ci-Ce alkylene-R26wherein R26is a monosaccharide,(iv) Ci-C6alkyl, C5-C6 cycloalkyl, or C4-C6 heterocycloalkyl, each of which is substituted with one or more OH,(v) -R70-(OR27)n, wherein R70is a Ci-Ce alkyl linking group, n is 1 or 2, and R27is an ester-bond linked natural amino acid, or(vi) -CR28aR28b-C(R29a)(R29b)(R30), wherein R28a, R28band R29aare independently H or Ci-Ce alkyl, R29bis -NR100, wherein R100is H or an acetyl (Ac), tertbutyloxycarbonyl (Boc), or benzyloxycarbonyl (Cbz) group, and R30is -C(=O)OR30a, wherein R30ais H or Ci-Ce alkyl.

7. The compound according to Claim 1, wherein A is a peptide bond-linked natural amino acid, a peptide bond-linked dipeptide of two natural amino acids, a peptide bond-linked tripeptide of three natural amino acids, or a peptide bond-linked cystine, wherein an amino group of the natural amino acid is optionally protected with an acetyl (Ac), tert-butyloxy carbonyl (Boc), or benzyloxycarbonyl (Cbz) group.

8. The compounds according to Claim 1, whereinA is -C(=O)-CR10aR1(,b-NRnR12, whereinR10aand R10bare independently H or Ci-C& alkyl,R11is H,R12is -C(=O)-CR13aR13b-NR14aR14b,R13aand R13bare H,R14ais H, andR14bis H or Ci-C6alkyl.19920300-WO-PCT / / 103241-016PCT9. The compounds according to Claim 1, whereinA is -C(=O)-CR10aR1(,b-NRnR12, whereinRi°a, R10b, and R11are independently H or Ci-Cr> alkyl,R12is -C(=O)-CR13aR13b-NR14aR14b,R13a- R13b, andR14aare independently H or Ci-Ce alkyl,R14bis -C(=O)-CR15aR15b-NR16aR16b,R15aand R15bare independently H or Ci-C& alkyl,R16ais H, andR16bis H or Ci-C6alkyl.

10. The compound according to Claim 1 , wherein A is -C(=O)-R30a-R30b, and wherein R30ais Ci-Ce alkylene, and R30bis a monosaccharide.

11. The compound according to Claim 1, wherein A is -C(=O)-R32-R33, wherein R32is Ci-C6alkylene, and R33is phenyl substituted with -O(C=O)-R34and optionally one or more Ci-C6alkyl, and R34is Ci-C6alkyl.

12. The compounds according to Claim 1, whereinA is -C(=O)O-CR10aR1(,b-O(C=O)-Rn, whereinR10aand R10bare independently H or Ci-C& alkyl, andR11is -(CR12aR12b)n-NR13R14, wherein n is 0 or 1 ,Ri23, R12b, and R13are independently H or Ci-Ce alkyl,R14is -C(=O)-CR15aR15b-NR16aR16b, an acetoxyacetyl group, or -C(=O)-CR47aR47b.O.R80R15aand R15bare H,R16ais H, an acetyl (Ac), tert-butyloxycarbonyl (Boc), or benzyloxycarbonyl (Cbz) group,R16bis H or Ci-C6alkyl,R47aandR47bmay be independently H or C1-C3 alkyl, and20020300-WO-PCT / / 103241-016PCTR80may be an ester-bonded linked natural amino acid.

13. The compounds according to Claim 1, whereinA is -C(=O)-O-CR10aR1(,b-O-Rn, whereinR10aand R10bare independently H or Ci-C& alkyl, andR11is -P(=O)(OR12)(OR13), whereinR12is R13may independently be H or Ci-Ce alkyl.

14. A compound having Formula IIFormula II, or a pharmaceutically acceptable salt thereof, wherein(i) n=0, and B is(a) -CH(NH2)-CH2-S-S-CH2-CH(NH2)- wherein one or both amino groups can be optionally protected as an acetyl (Ac), tert-butyloxycarbonyl (Boc), or benzyloxycarbonyl (Cbz) group, or(b) -O-(Ci-C6alkylene)-O- or (c) -O-(Ci-C6alkylene)-O-C(=O)-O-(Ci-C6alkylene)-O-, or15. A compound having Formula IIIFormula III,20120300-WO-PCT / / 103241-016PCTor a pharmaceutically acceptable salt thereof, whereinR90is H, phenyl, or Ci-Ce alkyl.

16. A compound having Formula IVFormula IV, or a pharmaceutically acceptable salt thereof, whereinR is:(i) -C(R62a)(R62b)-R91, wherein R62aand R62bare independently H or Ci-Ce alkyl, R91is an unsaturated cyclic carbonate ester, optionally substituted with Ci-C& alkyl;(ii) -C(R63a)(R63b)-NR92-C(=O)-phenyl, wherein the phenyl is substituted with -O- CR64aR64b-O-C(=O)-R93, wherein R63a, R63b, R64a, and R64bare independently H or Ci-Ce alkyl, and R93is Ci-C& alkyl; or(iii) R is OH or Ci-Ce alkoxy.

17. A compound selected from the following compounds or a pharmaceutically acceptable salt thereof:20220300-WO-PCT / / 103241-016PCT20300-WO-PCT / / 103241-016PCT20300-WO-PCT / / 103241-016PCT20520300-WO-PCT / / 103241-016PCT20300-WO-PCT / / 103^41 -016PCT20300-WO-PCT / / 103241-016PCT20300-WO-PCT / / 103241-016PCT20300-WO-PCT / / 103241-016PCT20300-WO-PCT / / 103241-016PCT18. A method of treating an obsessive-compulsive related disorder (OCRD), comprising administering to a subject in need of such treatment, a therapeutically effective amount of the compound according to any one of Claims 1-17 or a pharmaceutically acceptable salt thereof.

19. The method according to Claim 18, wherein the OCR!) is selected from the group consisting of obsessive-compulsive disorder (OCD), hoarding disorder, trichotillomania (hairpulling disorder), excoriation (skin-picking) disorder, body dysmorphic disorder, substance / medication-induced obsessive-compulsive and related disorder, obsessive-compulsive disorder due to another medical condition.

20. The method according to Claim 18, wherein the OCRD is obsessive compulsive disorder (OCD).21120300-WO-PCT / / 103241-016PCT21. A method of treating Tourette’s Syndrome, comprising administering to a subject in need of such treatment, a therapeutically effective amount of the compound according to any one of Claims 1-17 or a pharmaceutically acceptable salt thereof.

22. A method of treating a glutamate exci tot oxi city related disorder, comprising administering to a subject in need of such treatment an effective amount of the compound according to any one of Claims 1-17 or a pharmaceutically acceptable salt thereof.

23. The method according to Claim 22, wherein the glutamate excitotoxicity related disorder is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Parkinson’s disease, traumatic brain injury, concussion, post-concussion syndrome, cerebral infarction, stroke, multiple sclerosis, Huntington’s disease, schizophrenia, seizure, epilepsy, epileptic syndrome, status epilepticus, refractory focal epilepsy, autoimmune encephalitis, infectious encephalitis, viral encephalitis, acute disseminated encephalomyelitis, and mitochondrial encephalitis.

24. A method of treating autism spectrum disorder, comprising administering to a subject in need of such treatment an effective amount of the compound according to any one of Claims 1-17 or a pharmaceutically acceptable salt thereof.

25. A method of treating a tic disorder, comprising administering to a subject in need of such treatment, a therapeutically effective amount of the compound according to any one of Claims 1-17 or a pharmaceutically acceptable salt thereof.

26. The method according to Claim 25, wherein the tic disorder is selected from the group consisting of Tourette’s Syndrome, Tourette’s Disorder, chronic motor tic disorder, chronic vocal tic disorder, transient tic disorder, and substance-induced tic disorder.

27. The method according to any of Claims 18-26, wherein the compound or a pharmaceutically acceptable salt thereof is administered in combination with one or more other therapeutically active compounds.21220300-WQ-PCT / / 103241-016PCT28. The method according to Claim 27, wherein the other therapeutically active compound is selected from the group consisting of a monoamine oxidase inhibitor (MAO-I), a serotomn / norepinephrine dual reuptake inhibitor (SNR1), an antidepressant, a glutamate- modulating agent, a benzothiazole or benzothiazole derivative, a serotonin reuptake inhibitor (SRI), a skeletal muscle relaxant, an antipsychotic agent, an antidepressant, an anticholinergic, an anticonvulsant, a benzodiazepine or benzodiazepine derivative, and any combination thereof.

29. The method according to any of Claims 18-28, wherein the compound or a pharmaceutically acceptable salt thereof is administered orally, intravenously, sublingually, buccally, transdernially, or intranasally.

30. The method according to Claim 29, wherein the compound or a pharmaceutically acceptable salt thereof is administered orally.

31. The method according to any of Claims 18-30, wherein the compound or a pharmaceutically acceptable salt thereof is administered in combination with a neuromodulation technique selected from the group consisting of transcranial direct current stimulation (tDCS), transcranial magnetic stimulation(TMS), deep brain stimulation (DBS), and a combination thereof.

32. The method according to any of Claims 18-30, wherein the compound or a pharmaceutically acceptable salt thereof is administered once per day, twice per day, three times per day, or more than three times per day.

33. The method according to any of Claims 18-32, wherein said the compound or a pharmaceutically acceptable salt thereof is administered such that the total weekly dose is from 5 mg to 2000 mg.21320300-WO-PCT / / 103241-016PCT

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