Synthesis of ras inhibitors

Abstract

The present invention relates to Ras inhibitors, intermediates in the synthesis thereto, and methods for preparing the Ras inhibitors and the intermediates.

Description

[0001] SYNTHESIS OF RAS INHIBocNITORS

[0002] BocNACKGROUND

[0003] The vast majority of small molecule drugs act by binding a functionally important pocket on a target protein, thereby modulating the activity of that protein. For example, cholesterol-lowering drugs known as statins bind the enzyme active site of HMG-CoA reductase, thus preventing the enzyme from engaging with its substrates. The fact that many such drug / target interacting pairs are known may have misled some into believing that a small molecule modulator could be discovered for most, if not all, proteins provided a reasonable amount of time, effort, and resources. This is far from the case. Current estimates are that only about 10% of all human proteins are targetable by small molecules. BocNojadzic and BocNuchwald, Curr Top Med Chem 18: 674-699 (2019). The other 90% are currently considered refractory or intractable to above-mentioned small molecule drug discovery. Such targets are commonly referred to as “undruggable.” These undruggable targets include a vast and largely untapped reservoir of medically important human proteins. Thus, there exists a great deal of interest in discovering new molecular modalities capable of modulating the function of such undruggable targets.

[0004] It has been well established in literature that Ras proteins (K-Ras, H-Ras, and N-Ras) play an essential role in various human cancers and are therefore appropriate targets for anticancer therapy. Indeed, mutations in Ras proteins account for approximately 30% of all human cancers in the United States, many of which are fatal. Dysregulation of Ras proteins by activating mutations, overexpression or upstream activation is common in human tumors, and activating mutations in Ras are frequently found in human cancer. For example, activating mutations at codon 12 in Ras proteins function by inhibiting both GTPase-activating protein (GAP)-dependent and intrinsic hydrolysis rates of GTP, significantly skewing the population of Ras mutant proteins to the “on” (GTP-bound) state (Ras(ON)), leading to oncogenic MAPK signaling. Notably, Ras exhibits a picomolar affinity for GTP, enabling Ras to be activated even in the presence of low concentrations of this nucleotide. Mutations at codons 13 (e.g., G13C) and 61 (e.g., Q61 K) of Ras are also responsible for oncogenic activity in some cancers.

[0005] Despite extensive drug discovery efforts against Ras during the last several decades, only two agents targeting the K-Ras G12C mutant have been approved in the U. S. (sotorasib and adagrasib). Additional efforts are needed to uncover additional medicines for cancers driven by the various Ras mutations, and there remains a need for convenient, scalable synthetic methods thereto.

[0006] SUMMARY OF THE INVENTION

[0007] The invention features methods of preparing Compound A, intermediates useful in the synthesis of Compound A, and methods of preparing the intermediates.Compound A, a RAS inhibitor, has the following structure:

[0008]

[0009] Compound A

[0010] In one aspect, the disclosure provides a method of preparing a compound of Formula 1:

[0011]

[0012] Formula 1

[0013] the method including:

[0014] a) reacting a compound of Formula 1 a with an alkylating agent to form a compound of Formula 1b:

[0015]

[0016] Formula 1a Formula 1b

[0017] b) reducing the compound of Formula 1 b to form a compound of Formula 1 c HCI salt:

[0018]

[0019] Formula 1b Formula 1c HCI salt.

[0020] c) converting the compound of Formula 1c HCI salt to form a compound of Formula 1d HCI salt:

[0021] NH HCI

[0022]

[0023] Formula 1c HCI salt Formula 1d HCI salt.an(jd) coupling the compound of Formula 1 d HCI salt with a compound of Formula 1 e to form the compound of Formula 1:

[0024]

[0025] HN— PGaFormula 1d HCI salt Formula 1e

[0026]

[0027] Formula 1

[0028] wherein:

[0029] X is Cl, BocNr, OTf, OMs, OTs, OAc, or OBocNz; and

[0030] PGais a nitrogen protecting group.

[0031] In some embodiments, the reducing (b) includes contacting the compound of Formula 1b with a borohydride reagent.

[0032] In some embodiments, the converting (c) includes contacting the compound of Formula 1c HCI salt with a N-dealkylating agent.

[0033] In some embodiments, the coupling (d) includes contacting the compound of Formula 1d HCI salt with a base.

[0034] In another aspect, the disclosure provides a compound having the structure of Formula 2:

[0035]

[0036] Formula 2

[0037] or a salt thereof, wherein:

[0038] PGa is a nitrogen protecting group; and

[0039]

[0040] In some embodiments, Y is -BocN(OH)2.In yet another aspect, the disclosure provides a method of preparing a compound of Formula 2e:

[0041]

[0042] Formula 2e

[0043] the method including:

[0044] a) coupling a compound of Formula 1 e with a compound of Formula 2a •2HCl to form a compound of Formula 2b:

[0045]

[0046] Formula 1e Formula 2a »2HCI Formula 2b

[0047]

[0048] b) contacting the compound of Formula 2b with a borylation agent to form the compound of Formula 2e:

[0049]

[0050] Formula 2b Formula 2e wherein:

[0051] R1is optionally substituted C1-C6 alkyl;

[0052] PGais a nitrogen protecting group;

[0053] X is Cl, BocNr, OTf, OMs, OTs, OAc, or OBocNz; and

[0054]

[0055] In some embodiments, the borylation agent is bis(pinacolato)diboron, tetrahydroxydiboron, tetramethoxydiboron, tetrahydroxydiboron, or bis(neopentylglycolato)diboron.In some embodiments, the borylating (b) is carried out in the presence of a palladium catalyst.

[0056] In some embodiments,

[0057]

[0058] Y is

[0059] In some embodiments, the method further includes hydrolyzing a compound of Formula 2c to form a compound of Formula 2d:

[0060]

[0061] In some embodiments, the hydrolyzing includes contacting a compound of Formula 2c with a base. In some embodiments, the base is a hydroxide base.

[0062] In another aspect, the disclosure provides a method of preparing Compound 3:

[0063]

[0064] Compound 3

[0065] the method including:

[0066] a) converting Compound 3a to Compound 3b:

[0067]

[0068] Compound 3a Compound 3b.

[0069] b) cyclizing Compound 3b using carbon dioxide to form Compound 3c:

[0070]

[0071] Compound 3b Compound 3c

[0072] c) hydrolyzing Compound 3c to form Compound 3d:

[0073]

[0074] Compound 3c Compound 3d.d) converting Compound 3d to form Compound 3e:

[0075]

[0076] Compound 3d Compound 3e.an(j

[0077] e) cyclizing Compound 3e using formaldehyde to form Compound 3:

[0078]

[0079] Compound 3e Compound 3

[0080] In some embodiments, the converting (a) includes contacting Compound 3a with an ethynyl magnesium halide.

[0081] In some embodiments, the cyclizing (b) is carried out in the presence of a silver catalyst. In some embodiments, the converting (d) is carried out in the presence of an enzyme. In another aspect, the disclosure provides a method of preparing of Compound 4:

[0082]

[0083] Compound 4

[0084] the method including:

[0085] a) converting Compound 3 to Compound 4a:

[0086]

[0087] Compound 3

[0088] b) deprotecting Compound 4a to form Compound 4b • 3HCI:

[0089]

[0090] Compound 4b •3HClc) coupling Compound 4b •3HCl with Compound 4c hydrochloride using a carbonate source to form Compound 4d:

[0091]

[0092] Compound 4b •3HCl Compound 4c HCI Compound 4d

[0093] d) converting Compound 4d to Compound 4:

[0094]

[0095] Compound 4d Compound 4

[0096] In some embodiments, the converting (a) includes contacting Compound 3 with an acyl halide. In some embodiments, the carbonate source of the coupling (c) is bis(trichloromethyl) carbonate. In some embodiments, the converting (d) includes contacting Compound 4d with an iodide salt. In another aspect, the disclosure provides a method of preparing Compound A:

[0097]

[0098] Compound A

[0099] the method including:

[0100] a) coupling Compound 5a with Compound 2 to form Compound 5b:

[0101]

[0102] Compound 5b

[0103]

[0104] Compound 5b Compound 5c

[0105] c) deprotecting Compound 5c to form Compound 5d:

[0106] CF3CF3Compound 5c Compound 5d

[0107]

[0108] d) coupling Compound 5d with Compound 4 to form Compound A:

[0109]

[0110] Compound 5d Compound 4 Compound A In some embodiments, the coupling (a) includes contacting Compound 5a and Compound 2 with a palladium catalyst.

[0111] In some embodiments, the cyclizing (b) includes contacting compound 5b with a carbodiimide coupling reagent.

[0112] In some embodiments, the carbodiimide coupling reagent is EDCI.

[0113] In some embodiments, the coupling (d) includes contacting Compound 5d and Compound 4 with a phosphonium coupling reagent.

[0114] In some embodiments, the phosphonium coupling reagent is PyBocNOP.

[0115] In some embodiments, the method further includes purifying Compound A. In some embodiments, the purifying includes recrystallizing Compound A.Definitions and Chemical Terms

[0116] In this application, unless otherwise clear from context, (i) the term “a” means “one or more”; (ii) the term “or” is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or”; (iii) the terms “comprising” and “including” are understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) where ranges are provided, endpoints are included.

[0117] As used herein, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. In certain embodiments, the term “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated value, unless otherwise stated or otherwise evident from the context (e.g., where such number would exceed 100% of a possible value).

[0118] As used herein, the term “adjacent” in the context of describing adjacent atoms refers to bivalent atoms that are directly connected by a covalent bond.

[0119] A “compound of the present invention” and similar terms as used herein, whether explicitly noted or not, refers to Ras inhibitors described herein (e.g., Compound A) and intermediates in the synthesis thereto, as well as salts (e.g., pharmaceutically acceptable salts), solvates, hydrates, stereoisomers (including atropisomers), and tautomers thereof.

[0120] Those skilled in the art will appreciate that certain compounds described herein can exist in one or more different isomeric (e.g., stereoisomers, geometric isomers, atropisomers, tautomers) or isotopic (e.g., in which one or more atoms has been substituted with a different isotope of the atom, such as hydrogen substituted for deuterium) forms. Unless otherwise indicated or clear from context, a depicted structure can be understood to represent any such isomeric or isotopic form, individually or in combination.

[0121] Compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated.

[0122] Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms.

[0123] In some embodiments, one or more compounds depicted herein may exist in different tautomeric forms. As will be clear from context, unless explicitly excluded, references to such compounds encompass all such tautomeric forms. In some embodiments, tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton. In certain embodiments, a tautomeric form may be a prototropic tautomer, which is an isomeric protonation states having the same empirical formula and total charge as a reference form. Examples of moieties with prototropic tautomeric forms are ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs,amide - imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole. In some embodiments, tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution. In certain embodiments, tautomeric forms result from acetal interconversion.

[0124] Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. Exemplary isotopes that can be incorporated into compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as2H,3H,11C,13C,14C,13N,15N,150,17O,18O,32P,33P,35S,18F,36CI,123l and125l. Isotopically labeled compounds (e.g., those labeled with3H and14C) can be useful in compound or substrate tissue distribution assays. Tritiated (i.e.,3H) and carbon-14 (i.e.,14C) isotopes can be useful for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e.,2H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements). In some embodiments, one or more hydrogen atoms are replaced by2H or3H, or one or more carbon atoms are replaced by13C- or14C-enriched carbon. Positron emitting isotopes such as150,13N,11C, and18F are useful for positron emission tomography (PET) studies to examine substrate receptor occupancy.

[0125] Preparations of isotopically labelled compounds are known to those of skill in the art. For example, isotopically labeled compounds can generally be prepared by following procedures analogous to those disclosed for compounds of the present invention described herein, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0126] As is known in the art, many chemical entities can adopt a variety of different solid forms such as, for example, amorphous forms or crystalline forms (e.g., polymorphs, hydrates, solvate). In some embodiments, compounds of the present invention may be utilized in any such form, including in any solid form. In some embodiments, compounds described or depicted herein may be provided or utilized in hydrate or solvate form.

[0127] At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual subcombination of the members of such groups and ranges. For example, the term “C1-C6 alkyl” is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and Ce alkyl. Furthermore, where a compound includes a plurality of positions at which substituents are disclosed in groups or in ranges, unless otherwise indicated, the present disclosure is intended to cover individual compounds and groups of compounds (e.g., genera and subgenera) containing each and every individual subcombination of members at each position.

[0128] The term “optionally substituted X” (e.g., “optionally substituted alkyl”) is intended to be equivalent to “X, wherein X is optionally substituted” (e.g., “alkyl, wherein said alkyl is optionally substituted”). It is not intended to mean that the feature “X” (e.g., alkyl) perse is optional. As described herein, certain compounds of interest may contain one or more “optionally substituted” moieties. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent, e.g., any of the substituents or groups described herein. Unless otherwise indicated, an “optionally substituted” group may have a suitablesubstituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. For example, in the term “optionally substituted C1-C6 alkyl-C2-Cg heteroaryl,” the alkyl portion, the heteroaryl portion, or both, may be optionally substituted. Combinations of substituents envisioned by the present disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0129] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group may be, independently, deuterium; halogen; -(CH2)o-4R°; -(CH2)o-40R°; -0(CH2)o-4R°;

[0130] -0-(CH2)O-4C(0)OR°; -(CH2)O-4CH(OR°)2; -(CH2)O-4SR°; -(CH2)o-4Ph, which may be substituted with R°; -(CH2)o-40(CH2)o-iPh which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -(CH2)o-40(CH2)o-i-pyridyl which may be substituted with R°; 4-8 membered saturated or unsaturated heterocycloalkyl (e.g., pyridyl); 3-8 membered saturated or unsaturated cycloalkyl (e.g., cyclopropyl, cyclobutyl, or cyclopentyl); -NO2; -CN; -N3; -(CH2)o-4N(R°)2; -(CH2)o-4N(R°)C(0)R°; -N(R°)C(S)R°;

[0131] -(CH2)O-4N(R0)C(0)NR°2; -N(RO)C(S)NR°2; -(CH2)O-4N(R°)C(0)OR°; - N(R°)N(R°)C(O)R°;

[0132] -N(R°)N(RO)C(O)NRO2; -N(R°)N(R°)C(O)OR°; -(CH2)O-4C(0)R°; -C(S)R°; -(CH2)O-4C(0)OR°;

[0133] -(CH2)O-4-C(0)-N(R°)2; -(CH2)O-4-C(0)-N(R°)-S(0)2-R0; -C(NCN)NRO2; -(CH2)O-4C(0)SR°;

[0134] -(CH2)o-4C(0)OsiR°3; -(CH2)o-40C(0)R°; -OC(0)(CH2)o-4SR°; -SC(S)SR°; -(CH2)o-4SC(0)R°;

[0135] -(CH2)O-4C(0)NR°2; -C(S)NRO2; -C(S)SR°; -(CH2)O-40C(0)NR°2; -C(O)N(OR°)R°; -C(O)C(O)R°;

[0136] -C(O)CH2C(O)RO; -C(NOR°)R°; -(CH2)O-4SSR°; -(CH2)O-4S(0)2R°; -(CH2)O-4S(0)2OR°;

[0137] -(CH2)O-40S(0)2R°; -S(O)2NRO2; -(CH2)O-4S(0)R°; -N(RO)S(O)2NR°2; -N(RO)S(O)2R°; -N(OR°)R°;

[0138] -C(NORO)NR°2; -C(NH)NRO2; -P(O)2RO; -P(O)RO2; -P(O)(ORO)2; -OP(O)RO2; -OP(O)(ORO)2;

[0139] -OP(O)(OR°)R°, -SiR°3; -(C1-4 straight or branched alkylene)O-N(R°)2; or-(Ci-4 straight or branched alkylene)C(O)O-N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, -C1-6 aliphatic, -CFhPh, -0(CH2)o-iPh, -CH2-(5-6 membered heteroaryl ring), or a

[0140] 3-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0141] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), may be, independently, halogen,

[0142] -(CH2)O-2R*, -(haloR*), -(CH2)o-2OH, -(CH2)o-2OR*, -(CH2)o-2CH(OR*)2; -O(haloR’), -CN, -N3, -(CH2)O-2C(0)R*, -(CH2)O-2C(0)OH, -(CH2)O-2C(0)OR*, -(CH2)O-2SR*, -(CH2)O-2SH, -(CH2)O-2NH2, -(CH2)O-2NHR*, -(CH2)O-2NR*2, -NO2, -SiR*3, -OsiR*3, -C(O)SR* -(C1-4 straight or branched alkylene)C(O)OR*, or-SSR* wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C1-4 aliphatic, -CFhPh, -0(CH2)o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R' include =O and =S.

[0143] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =O, =S, =NNR*2, =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O)2R*, =NR*, =NOR*, -O(C(R*2))2-3O-, or -S(C(R*2))2-3S-, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR*2)2-3O-, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0144] Suitable substituents on the aliphatic group of R* include halogen, -R*, -(haloR*), -OH, -OR*, -0(haloR*), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or-N02, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently

[0145] C1-4 aliphatic, -CH2Ph, -0(CH2)o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0146] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include -Rt, -NRt2, -C(O)Rt, -C(O)ORt, -C(O)C(O)Rt, -C(O)CH2C(O)Rt, -S(O)2Rt, -S(O)2NRt2, -C(S)NRt2, -C(NH)NRt2, or-N(Rt)S(0)2Rt; wherein each Rt is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted -Oph, or an unsubstituted 3-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of Rt, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0147] Suitable substituents on an aliphatic group of Rt are independently halogen, -R*, -(haloR*), -OH, -OR*, -0(haloR*), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or-N02, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C aliphatic, -CH2Ph, -0(CH2)o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of Rfinclude =O and =S.

[0148] The term “acetyl,” as used herein, refers to the group -C(O)CH3.

[0149] The term “alkoxy,” as used herein, refers to a -O-C1-C20alkyl group, wherein the alkoxy group is attached to the remainder of the compound through an oxygen atom.

[0150] The term “alkyl,” as used herein, refers to a saturated, straight or branched monovalent hydrocarbon group containing from 1 to 20 (e.g., from 1 to 10 or from 1 to 6) carbons. In some embodiments, an alkyl group is unbranched (i.e., is linear); in some embodiments, an alkyl group is branched. Alkyl groups are exemplified by, but not limited to, methyl, ethyl, n- and iso-propyl, n-, sec-, iso- and te / Y-butyl, and neopentyl.

[0151] The term “alkylene,” as used herein, represents a saturated divalent hydrocarbon group derived from a straight or branched chain saturated hydrocarbon by the removal of two hydrogen atoms, and isexemplified by methylene, ethylene, isopropylene, and the like. The term “Cx-Cyalkylene” represents alkylene groups having between x and y carbons. Exemplary values for x are 1, 2, 3, 4, 5, and 6, and exemplary values fory are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 (e.g., Ci-Ce, C1-C10, C2-C20, C2-C6, C2-C10, or C2-C20 alkylene). In some embodiments, the alkylene can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein.

[0152] The term “alkenyl,” as used herein, represents monovalent straight or branched chain groups of, unless otherwise specified, from 2 to 20 carbons (e.g., from 2 to 6 or from 2 to 10 carbons) containing one or more carbon-carbon double bonds and is exemplified by ethenyl, 1 -propenyl, 2-propenyl,

[0153] 2-methyl-1 -propenyl, 1-butenyl, and 2-butenyl. Alkenyls include both cis and trans isomers. The term “alkenylene,” as used herein, represents a divalent straight or branched chain groups of, unless otherwise specified, from 2 to 20 carbons (e.g., from 2 to 6 or from 2 to 10 carbons) containing one or more carbon-carbon double bonds.

[0154] The term “alkynyl,” as used herein, represents monovalent straight or branched chain groups from 2 to 20 carbon atoms (e.g., from 2 to 4, from 2 to 6, or from 2 to 10 carbons) containing a carbon-carbon triple bond and is exemplified by ethynyl, and 1-propynyl.

[0155] The term “amino,” as used herein, represents -N(Ri)2, e.g., -NH2 and -N(CH3)2.

[0156] The term “aminoalkyl,” as used herein, represents an alkyl moiety substituted on one or more carbon atoms with one or more amino moieties.

[0157] The term “aryl,” as used herein, represents a monovalent monocyclic, bicyclic, or multicyclic ring system formed by carbon atoms, wherein the ring attached to the pendant group is aromatic. Examples of aryl groups are phenyl, naphthyl, phenanthrenyl, and anthracenyl. An aryl ring can be attached to its pendant group at any carbon ring atom that results in a stable structure and any of the ring atoms can be optionally substituted unless otherwise specified.

[0158] The term “Co,” as used herein, represents a bond. For example, part of the term -N(C(0)-(Co-Cs alkylene-H)- includes -N(C(C)-(Co alkylene-H)-, which is also represented by -N(C(O)-H)-.

[0159] The terms “carbocyclic” and “carbocyclyl,” as used herein, refer to a monovalent, optionally substituted C3-C12 monocyclic, bicyclic, or tricyclic ring structure, which may be bridged, fused or spirocyclic, in which all the rings are formed by carbon atoms and at least one ring is non-aromatic.

[0160] Carbocyclic structures include cycloalkyl, cycloalkenyl, and cycloalkynyl groups. Examples of carbocyclyl groups are cyclohexyl, cyclohexenyl, cyclooctynyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indenyl, indanyl, decalinyl, and the like. A carbocyclic ring can be attached to its pendant group at any ring atom that results in a stable structure and any of the ring atoms can be optionally substituted unless otherwise specified.

[0161] The term “carbonyl,” as used herein, represents a C(O) group, which can also be represented as C=O.

[0162] The term “carboxyl,” as used herein, means -CO2H, (C=O)(OH), COOH, orC(Q)OH or the unprotonated counterparts.

[0163] The term “cyano,” as used herein, represents a -CN group.

[0164] The term “cycloalkyl,” as used herein, represents a monovalent saturated cyclic hydrocarbon group, which may be bridged, fused or spirocyclic having from three to eight ring carbons, unlessotherwise specified, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cycloheptyl.

[0165] The term “cycloalkenyl,” as used herein, represents a monovalent, non-aromatic, cyclic hydrocarbon group, which may be bridged, fused orspirocyclic having from three to eight ring carbons, unless otherwise specified, and containing one or more carbon-carbon double bonds.

[0166] The term “diastereomer,” as used herein, means stereoisomers that are not mirror images of one another and are non-superimposable on one another.

[0167] The term “enantiomer,” as used herein, means each individual optically active form of a compound of the invention, having an optical purity or enantiomeric excess (as determined by methods standard in the art) of at least 80% (i.e., at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90% and more preferably at least 98%.

[0168] The term “haloacetyl,” as used herein, refers to an acetyl group wherein at least one of the hydrogens has been replaced by a halogen.

[0169] The term “haloalkyl,” as used herein, represents an alkyl moiety substituted on one or more carbon atoms with one or more of the same of different halogen moieties.

[0170] The term “halogen,” as used herein, represents a halogen selected from bromine, chlorine, iodine, or fluorine.

[0171] The term “heteroalkyl,” as used herein, refers to an “alkyl” group, as defined herein, in which at least one carbon atom has been replaced with a heteroatom (e.g., an O, N, or S atom). The heteroatom may appear in the middle or at the end of the radical.

[0172] The term “heteroaryl,” as used herein, represents a monovalent, monocyclic, or polycyclic ring structure that contains at least one fully aromatic ring: i.e., they contain 4n+2 pi electrons within the monocyclic or polycyclic ring system and contains at least one ring heteroatom selected from N, O, or S in that aromatic ring. Exemplary unsubstituted heteroaryl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. The term “heteroaryl” includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heteroaromatic rings is fused to one or more, aryl or carbocyclic rings, e.g., a phenyl ring, ora cyclohexane ring. Examples of heteroaryl groups include, but are not limited to, pyridyl, pyrazolyl, benzooxazolyl, benzoimidazolyl, benzothiazolyl, imidazolyl, thiazolyl, quinolinyl, tetrahydroquinolinyl, and 4-azaindolyl. A heteroaryl ring can be attached to its pendant group at any ring atom that results in a stable structure and any of the ring atoms can be optionally substituted unless otherwise specified. In some embodiments, the heteroaryl is substituted with 1, 2, 3, or 4 substituents groups.

[0173] The term “heterocycloalkyl,” as used herein, represents a monovalent monocyclic, bicyclic, or polycyclic ring system, which may be bridged, fused or spirocyclic, wherein at least one ring is non-aromatic and wherein the non-aromatic ring contains one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. The heterocycloalkyl may have one or more double bonds. Exemplary unsubstituted heterocycloalkyl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. The term “heterocycloalkyl” also represents a heterocyclic compound having a bridged multicyclic structure in which one or more carbons or heteroatoms bridges two non-adjacent members of a monocyclic ring, e.g., a quinuclidinyl group. The term “heterocycloalkyl” includes bicyclic, tricyclic, and tetracyclic groups in which any of the aboveheterocyclic rings is fused to one or more aromatic, carbocyclic, heteroaromatic, or heterocyclic rings, e.g., an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, a pyridine ring, ora pyrrolidine ring. Examples of heterocycloalkyl groups are pyrrolidinyl, piperidinyl, 1,2,3,4-tetrahydroquinolinyl, decahydroquinolinyl, dihydropyrrolopyridine, and decahydronapthyridinyl. A heterocycloalkyl ring can be attached to its pendant group at any ring atom that results in a stable structure and any of the ring atoms can be optionally substituted unless otherwise specified.

[0174] The term “hydroxy,” as used herein, represents a -OH group.

[0175] The term “hydroxyalkyl,” as used herein, represents an alkyl moiety substituted on one or more carbon atoms with one or more -OH moieties.

[0176] The term “isomer,” as used herein, means any tautomer, stereoisomer, atropisomer, enantiomer, or diastereomer of any compound of the invention. It is recognized that the compounds of the invention can have one or more chiral centers or double bonds and, therefore, exist as stereoisomers, such as double-bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). According to the invention, the chemical structures depicted herein, and therefore the compounds of the invention, encompass all the corresponding stereoisomers, that is, both the stereomerically pure form (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereoisomeric mixtures of compounds of the invention can typically be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

[0177] The term “stereoisomer,” as used herein, refers to all possible different isomeric as well as conformational forms which a compound may possess (e.g., a compound of any formula described herein), in particular all possible stereochemically and conformationally isomeric forms, all diastereomers, enantiomers or conformers of the basic molecular structure, including atropisomers. Some compounds of the present invention may exist in different tautomeric forms, all of the latter being included within the scope of the present invention.

[0178] The term “sulfonyl,” as used herein, represents an -S(O)2- group.

[0179] The term “thiocarbonyl,” as used herein, refers to a -C(S)- group.

[0180] As used herein, the term “nitrogen protecting group” refers to a removable chemical moiety on a nitrogen atom in a compound described herein that protects the nitrogen from undesirable side reactions. Non-limiting examples of nitrogen protecting groups include BocNoc (tert-BocNutyloxycarbonyl), Fmoc (fluorenylmethyloxycarbonyl), and CBocNz (benzyl carbamate).

[0181] As used herein, the term “EW-TA-44” refers to an amine transaminase (ATA) enzyme from EnzymeWorks, Inc. The EW-TA-44 enzyme is capable of catalyzing the regio- and stereoselective synthesis of chiral amines, amino acids, and their derivatives from a variety of aliphatic and aromatic ketoacids, aldehydes, ketones, and ketoses.

[0182] Those of ordinary skill in the art, reading the present disclosure, will appreciate that certain compounds described herein may be provided or utilized in any of a variety of forms such as, for example,salt forms, protected forms, pro-drug forms, ester forms, isomeric forms (e.g., optical or structural isomers), isotopic forms, etc. In some embodiments, reference to a particular compound may relate to a specific form of that compound. In some embodiments, reference to a particular compound may relate to that compound in any form. In some embodiments, for example, a preparation of a single stereoisomer of a compound may be considered to be a different form of the compound than a racemic mixture of the compound; a particular salt of a compound may be considered to be a different form from another salt form of the compound; a preparation containing one conformational isomer ((Z) or (E)) of a double bond may be considered to be a different form from one containing the other conformational isomer ((E) or (Z)) of the double bond; a preparation in which one or more atoms is a different isotope than is present in a reference preparation may be considered to be a different form.

[0183] DETAILED DESCRIPTION

[0184] Provided herein are synthetic methods and intermediates for making Ras inhibitors, or salts thereof. The methods and intermediates can be useful for achieving a higher yield, a higher chemical purity, and / or a higher stereoisomeric purity, and a lower cost for the preparation of the Ras inhibitors or the intermediates. Further synthetic details are provided in the Examples.

[0185] The compounds described herein may be prepared using the methods described herein and / or using known organic, inorganic, or enzymatic processes. The synthetic methods may employ the use of commercially available starting materials or starting materials prepared by processes known to those skilled in the art of organic synthesis. These methods include but are not limited to those methods described in the Schemes below and in WO 2021 / 091967, WO 2022 / 235870, and WO 2023 / 060253 the disclosure of each of which is incorporated herein by reference.

[0186] Synthetic Methods

[0187] In one aspect, the disclosure provides a method of preparing a compound of Formula 1:

[0188]

[0189] Formula 1

[0190] the method including:

[0191] a) reacting a compound of Formula 1 a with an alkylating agent to form a compound of Formula 1b:

[0192]

[0193] Formula 1a Formula 1bb) reducing the compound of Formula 1 b to form a compound of Formula 1 c HCI salt:

[0194]

[0195] Formula 1b Formula 1c HCI salt.

[0196] c) converting the compound of Formula 1c HCI salt to form a compound of Formula 1d HCI salt:

[0197]

[0198] Formula 1c HCI salt Formula 1d HCI salt.an(j d) coupling the compound of Formula 1 d HCI salt with a compound of Formula 1 e to form the compound of Formula 1:

[0199] HO / O x NH HCI N— PGaH

[0200]

[0201] HN— PGaFormula 1d HCI salt Formula 1e

[0202]

[0203] X Formula 1 wherein:

[0204] X is Cl, BocNr, OTf, OMs, OTs, OAc, or OBocNz; and

[0205] PGa is a nitrogen protecting group. In some embodiments, PGais BocNoc, Fmoc, or Cbz. In some embodiments, PGais BocNoc. In some embodiments, PGais Fmoc. In some embodiments, PGais CBocNz.

[0206] In some embodiments, the method includes preparing Compound 1:

[0207]

[0208] the method including:

[0209] a) reacting a Compound 1a with an alkylating agent to form Compound 1b:

[0210] BocNr BocNr

[0211] Ph

[0212]

[0213] Compound 1a Compound 1b

[0214] b) reducing the Compound 1 b to form Compound 1 c HCI salt:

[0215]

[0216] Compound 1b Compound 1c HCI salt.c) converting Compound 1c HCI salt to form Compound 1d HCI salt:

[0217] >

[0218]

[0219] Compound 1c HCI salt Compound 1d HCI salt.an(j d) coupling Compound 1d HCI salt with Compound 1e to form Compound 1:

[0220] >

[0221]

[0222] ° ° NHBocNoc Compound 1d HCI salt Compound 1e

[0223]

[0224] Compound 1

[0225] In some embodiments, the alkylating agent of (a) is a benzyl halide. In some embodiments, the benzyl halide is benzyl bromide, benzyl chloride, or benzyl iodide. In some embodiments, the benzyl halide is benzyl bromide. In some embodiments, the reacting (a) is carried out using a 1:1.1 stoichiometric ratio of the compound of Formula 1a to the benzyl halide. In some embodiments, the reacting (a) is carried out at a temperature of at least 50 °C (e.g., 50 °C, 60 °C, or 70 °C). In some embodiments, the reacting (a) is carried out at a temperature from 50 °C to 70 °C. In some embodiments, the reacting (a) is carried out according to the following scheme:

[0226] benzyl halide "sPh

[0227] Ji |j i

[0228]

[0229] acetone

[0230] .. 50 to 70 " C

[0231] Formula 1aW8msgbtFormula 1b

[0232] In some embodiments, the reacting (a) is carried out according to the following scheme:

[0233] BocNr. BocNr,+-- ' benzyl bromide '■'Hz'Ph

[0234] it - - t

[0235] acetone

[0236] Compound 1a

[0237]

[0238] ti.iht Compound 1b

[0239] In some embodiments, the reducing (b) includes contacting the compound of Formula 1b (e.g., Compound 1b) with a borohydride reagent. In some embodiments, the borohydride reagent is sodium triacetoxyborohydride, lithium borohydride, cyanoborohydride, or sodium borohydride. In some embodiments, the borohydride reagent is sodium triacetoxyborohydride. In some embodiments, the reducing (b) is carried out using a 1:6.5 stoichiometric ratio of the compound of Formula 1 b to the borohydride reagent. In some embodiments, the reducing (b) is carried out at a temperature of at least 20 °C (e.g., 20 °C, 25 °C, or 30 °C). In some embodiments, the reducing (b) is carried out at a temperature from 20 °C to 30 °C. In some embodiments, the reducing (b) is carried out according to the following scheme:

[0240] X+„ NaBocNH(OAr:)-< v HCI,

[0241] Yz'Ph - - - - Y ' N - ' ' Ph

[0242]

[0243] IlY DCM / EtOH (10:1) J

[0244] overnighl

[0245] F r o uriwmuuilaa 1b 20 to 30 'C > Formu,la 1.c, HCI sa.lt.In some embodiments, the reducing (b) is carried out according to the following scheme:

[0246] BocNr + HCI

[0247] Y 'N' Th > N '

[0248] DCM / EtOH (10:1)

[0249]

[0250] Compound 1b 25 Compound 1c HCI salt

[0251] In some embodiments, the converting (c) includes contacting the compound of Formula 1c HCI salt (e.g., Compound 1c HCI salt) with an N-dealkylating agent. In some embodiments, the N-dealkylating agent is a chloroethyl chloroformate (e.g., 1-chloroethyl chloroformate or2-chloroethyl chloroformate). In some embodiments, the converting (c) is carried out using a 1:1.5 stoichiometric ratio of the compound of Formula 1c HCI salt to the N-dealkylating agent. In some embodiments, the converting (c) is first carried out at a temperature of 20 °C to 40 °C for 2 hours, then cooled to 0 °C to 5 °C, followed by the addition of the N-dealkylating agent with stirring for 2 hours, and then heated to 0 °C to 5 °C in methanol for 3 hours. In some embodiments, the converting (c) is carried out according to the following scheme:

[0252] q Cl

[0253] X PhX'rpNH HCI

[0254] [[ J 1) DCM.40 °C, 2h

[0255]

[0256] 2) MeOH, 75 °C, 3 h >

[0257] Formula 1c HCI Formula Id HCI

[0258] In some embodiments, the converting (c) is carried out according to the following scheme:

[0259] 0 Cl

[0260] RrHCI,. Rr

[0261] V 'N' " Ph... C...l........... O.....'. ' V NH HCI

[0262] 1) DCM.40QC, 2h

[0263]

[0264] 2) MeOH, 75 °C, 3 h

[0265] Compound 1c HCI Compound Id HCI In some embodiments, the coupling (d) includes contacting the compounds of Formulas 1d and 1e (e.g., Compound 1d HCI and Compound 1e) with a base. In some embodiments, the base is an inorganic base. In some embodiments, the base is a carbonate base. In some embodiments, the base is sodium carbonate, calcium carbonate, or potassium carbonate. In some embodiments, the base is sodium carbonate. In some embodiments, the coupling (d) is carried out using a 1:1.1 stoichiometric ratio of the compound of Formula 1d HCI salt to the compound of Formula 1e. In some embodiments, the coupling (d) is carried out using a 2:1 stoichiometric ratio of the base to the compound of Formula 1d HCI salt. In some embodiments, the coupling (d) is carried out at a temperature of at least 30 °C (e.g., 30 °C, 35 °C, 40 °C, or 50 °C). In some embodiments, the coupling (d) is carried out at a temperature from 30 °C to 50 °C. In some embodiments, the coupling (d) is carried out according to the following scheme:

[0266] „ ~~ HO.. O

[0267] o GH3CN: 10 v

[0268] Y NH HCI O ••••■; f-‘ — PG

[0269] >

[0270] 35-40 ”0 AH

[0271] HN—PGa

[0272]

[0273] Form uta Id Formula 1e For ia 1In some embodiments, the coupling (d) is carried out according to the following scheme:

[0274] Na2CO32.0 eq.

[0275] zp CHjGN W v

[0276] HCI O-T —. H....2... O.....3....5... V..... >.

[0277] L-A 35-40 °C

[0278] NHBocNoC

[0279]

[0280] Compound Id Compound le Compound 1 In another aspect, the disclosure provides a compound having the structure of Formula 2:

[0281]

[0282] Formula 2

[0283] or a salt thereof, wherein:

[0284] PGais a nitrogen protecting group; and

[0285]

[0286] In some embodiments, Y is -BocN(OH)2.

[0287] In some embodiments, the compound of Formula 2 is Compound 2:

[0288] ov irNv°

[0289] OHHNHBocNoc

[0290] HOBocN

[0291]

[0292] OH

[0293] Compound 2

[0294] In yet another aspect, the disclosure provides a method of preparing a compound of Formula 2e:

[0295]

[0296] Formula 2ethe method including:

[0297] a) coupling a compound of Formula 1 e with a compound of Formula 2a •2HCl to form a compound of Formula 2b:

[0298]

[0299] Formula 1e Formula 2a •2HCl Formula 2b

[0300]

[0301] b) contacting the compound of Formula 2b with a borylation agent to form the compound of Formula 2e:

[0302]

[0303] Formula 2b Formula 2e

[0304] wherein:

[0305] R1is optionally substituted C1-C6 alkyl;

[0306] PGais a nitrogen protecting group;

[0307] X is Cl, BocNr, OTf, OMs, OTs, OAc, or OBocNz; and

[0308] Y is

[0309]

[0310] In some embodiments, Y isIn some embodiments, the method includes preparing Compound 2:

[0311]

[0312] Compound 2

[0313] the method including:

[0314] a) coupling Compound 1 with Compound 2a • 2HCl to form Compound 2b:

[0315]

[0316] Compound 1 Compound 2a Compound 2b

[0317]

[0318] b) contacting Compound 2b with a borylation agent to form Compound 2c:

[0319]

[0320] Compound 2b Compound 2c

[0321] In some embodiments, the coupling (a) further includes contacting the compound of Formula 1e (e.g., Compound 1) and the compound of Formula 2a (e.g., Compound 2a • 2HCl) with a carbodiimide coupling reagent and an anti-racemization agent. In some embodiments, the carbodiimide coupling reagent is EDCI. In some embodiments, the anti-racemization agent is HOBocNt Oxyma, HOBocNt, or HOPO. In some embodiments, the anti-racemization agent is HOBocNt. In some embodiments, the coupling (a) is carried out using a 1:1.2 stoichiometric ratio of the compound of Formula 1e to the compound of Formula 2a. In some embodiments, the coupling (a) is carried out using a 1:1.8 stoichiometric ratio of the compound of Formula 1e to the carbodiimide coupling reagent. In some embodiments, the coupling (a) is carried out using a catalytic amount of the anti-racemization agent (e.g., 2 mol%). In some embodiments, the coupling (a) is carried out at a temperature of at least 0 °C (e.g., 0 °C, 5 °C, or 10 °C). In some embodiments, the coupling (a) is carried out at a temperature from 0 °C to 10 °C.In some embodiments, the coupling (a) is carried out according to the following scheme:

[0322] EDCI 1.8 eq.

[0323] HOBocNt 2 mol%

[0324] DCM 10 vol.

[0325] 0-5 °C, 1 h

[0326]

[0327] Formula 1e Formula 2a •2HCl Formula 2b

[0328] In some embodiments, the coupling (a) is carried out according to the following scheme:

[0329] EDCI 1.8 eq.

[0330] HOBocNt 2 mol%

[0331] DCM 10 vol.

[0332] 0-5 °C, 1 h

[0333]

[0334] Compound 1 Compound 2a

[0335] In some embodiments, the borylating (b) is carried out in the presence of a borylation agent. In some embodiments, the borylation agent is bis(pinacolato)diboron, tetrahydroxydiboron, tetramethoxydiboron, tetrahydroxydiboron, or bis(neopentylglycolato)diboron. In some embodiments, the borylation agent is bis(neopentylglycolato)diboron. In some embodiments, the borylating (b) is carried out in the presence of a palladium catalyst. In some embodiments, the palladium catalyst is Pd(dppf)Cl2. In some embodiments, the borylating (b) is carried out in the presence of an acetate salt (e.g., potassium acetate). In some embodiments, the borylating (b) is carried out using a 1:1.5 stoichiometric ratio of the compound of Formula 2b to the borylation agent. In some embodiments, the borylating (b) is carried out using a 1:5 stoichiometric ratio of the compound of Formula 2b to the acetate salt. In some embodiments, the borylating (b) is carried out at a temperature of at least 70 °C (e.g., 70 °C, 80 °C, 85 °C, or 90 °C). In some embodiments, the borylating (b) is carried out at a temperature from 70 °C to 90 °C. In some embodiments, the borylating (b) is carried out according to the following scheme:

[0336] N v --'0

[0337] PG BocNorylation Agent YH

[0338] PGa

[0339] Pd(dppf)Cl₂, AcOK,

[0340] £ ) toluene, 80 ’C, 2 ft

[0341]

[0342] X'

[0343] Formula 2b Formula 2eIn some embodiments, the borylating (b) is carried out according to the following scheme:

[0344] CK N o / -a o--,

[0345] v * r X BocN-BocN'

[0346] OH■'•—a

[0347] f'"^NHBocNoc b— /

[0348] Pd(dppf)Cl₂, AcOK

[0349] f "i totane, BocNO °C, 2 h

[0350]

[0351] Compound 2b Compound 2c

[0352] In some embodiments, the compound of Formula 2e is a compound of Formula 2c:

[0353]

[0354] wherein:

[0355] R1is optionally substituted C1-C6 alkyl; and

[0356] PGais a nitrogen protecting group.

[0357] In some embodiments, the method further includes hydrolyzing a compound of Formula 2c to form a compound of Formula 2d:

[0358]

[0359] In some embodiments, the hydrolyzing further includes contacting the compound of Formula 2c with a base. In some embodiments, the hydroxide base is lithium hydroxide, sodium hydroxide, potassium hydroxide, or calcium hydroxide. In some embodiments, the hydroxide base is lithium hydroxide. In some embodiments, the lithium hydroxide is lithium hydroxide monohydrate. In some embodiments, the hydrolyzing is carried out using a 1:3 stoichiometric ratio of the compound of Formula 2c to the base. In some embodiments, the hydrolyzing is carried out at a temperature of at least 0 °C (e.g., 0 °C, 5 °C, or 10 °C). In some embodiments, the hydrolyzing is carried out at a temperature from 0 °C to 10 °C.In some embodiments, the hydrolyzing is carried out according to the following scheme:

[0360]

[0361] In some embodiments, the hydrolyzing is carried out according to the following scheme:

[0362] LiOH•H2O

[0363]

[0364] In another aspect, the disclosure provides a method of preparing Compound 3:

[0365]

[0366] Compound 3

[0367] the method including:

[0368] a) converting Compound 3a to Compound 3b:

[0369]

[0370] Compound 3a Compound 3b.

[0371] b) cyclizing Compound 3b using carbon dioxide to form Compound 3c:

[0372]

[0373] Compound 3b Compound 3c

[0374] c) hydrolyzing Compound 3c to form Compound 3d:

[0375]

[0376] Compound 3c Compound 3d.d) converting Compound 3d to form Compound 3e:

[0377] XNH2

[0378] (S)

[0379] OHXOH

[0380]

[0381] BocNocN BocNocN

[0382] Compound 3d Compound 3e.an(j

[0383] e) cyclizing Compound 3e using formaldehyde to form Compound 3:

[0384] ' NH2^ NH

[0385] _ I OH

[0386]

[0387] BocNocN

[0388] Compound 3e Compound 3

[0389] In some embodiments, the converting (a) includes contacting Compound 3a with an ethynyl magnesium halide (e.g., ethynyl magnesium bromide or ethynyl magnesium chloride). In some embodiments, the ethynyl magnesium halide is ethynyl magnesium bromide. In some embodiments, the converting (a) is carried out using a 1:1.3 stoichiometric ratio of the compound of Compound 3a to the ethynyl magnesium halide. In some embodiments, the converting (a) is carried out at a temperature of at least 0 °C (e.g., 0 °C, 5 °C, or 10 °C). In some embodiments, the converting (a) is carried out at a temperature from 0 °C to 10 °C.

[0390] In some embodiments, the converting (a) is carried out according to the following scheme:

[0391] , O EthynylMgBocNr in THE (1.3 eq.)

[0392] 1 h

[0393]

[0394] BocNocN BocNocN.

[0395] Compound 3 a Compound 3 b In some embodiments, the cyclizing (b) is carried out in the presence of a silver catalyst. In some embodiments, the silver catalyst is silver iodide. In some embodiments, the cyclizing (b) is carried out in the presence of an acetate salt (e.g., potassium acetate). In some embodiments, the cyclizing (b) is carried out under pressure of at least 0.5 atm carbon dioxide (e.g., 0.5 atm, 1 atm, or 1.5 atm CO2). In some embodiments, the cyclizing (b) is carried out at a temperature of at least 30 °C (e.g., 30 °C, 35 °C, or 40 °C). In some embodiments, the cyclizing (b) is carried out at a temperature from 30 °C to 40 °C. In some embodiments, the cyclizing (b) is carried out according to the following scheme:

[0396] Agl (0.05 eq,), KOAc (0.05 eq.) O

[0397] CO2(1 atm), DMSO (5 V)

[0398] OH35 ''C, 112 h _

[0399] BocNocN

[0400]

[0401] BocNocN Compound 3b Compound 3c

[0402] In some embodiments, the hydrolyzing (c) further includes contacting the Compound 3c with a base. In some embodiments, the hydroxide base is lithium hydroxide, sodium hydroxide, potassium hydroxide, or calcium hydroxide. In some embodiments, the hydroxide base is sodium hydroxide. In some embodiments, the hydrolyzing (c) is carried out using a 1:2 stoichiometric ratio of Compound 3c to the base. In some embodiments, the hydrolyzing (c) is carried out at a temperature of at least 20 °C (e.g., 20°C, 25 °C, or 30 °C). In some embodiments, the hydrolyzing (c) is carried out at a temperature from 20 °C to 30 °C.

[0403] In some embodiments, the hydrolyzing (c) is carried out according to the following scheme:

[0404] o

[0405] Y'K, NaOH {2.0 eq.) in H2O (4 V) (’

[0406] ACN (10 V), 25 °C, 2 h

[0407] BocNocN

[0408]

[0409] J O - - Compound 3c Compound 3d

[0410] In some embodiments, the converting (d) is carried out in the presence of an enzyme. In some embodiments, the enzyme is a transaminase enzyme (e.g., an amine transaminase (ATA) enzyme). In some embodiments, the ATA enzyme is EW-TA-44 (EnzymeWorks, Inc.), EW-ATA-kit (EnzymeWorks, Inc.), or ES-ATA-101ES ~ ATA-166 (SyncoZymes Shanghai Co., Ltd.). In some embodiments, at least 5% (w / w) of enzyme (e.g., 5% (w / w), 8% (w / w), 10% (w / w), 12% (w / w), or 15% (w / w)) is included in the reaction. In some embodiments, 5-15% (w / w) of enzyme is included in the reaction. In some embodiments, the converting (d) further includes contacting Compound 3d with trometamol, isopropylamine (iPrNH2), and pyridoxal phosphate (PLP). In some embodiments, the converting (d) is carried out in a mixture of water and DMSO. In some embodiments, the converting (d) is carried out in a 19:1 mixture of water and DMSO. In some embodiments, the converting (d) is carried out at a temperature of at least 20 °C (e.g., 20 °C, 25 °C, or 30 °C). In some embodiments, the converting (d) is carried out at a temperature from 20 °C to 35 °C.

[0411] In some embodiments, the converting (d) is carried out according to the following scheme:

[0412] (0.1 M Trometamol, 1.0 M iPrNH2

[0413] and 1.0 mM PLP mixture solution)

[0414] EW-TA-44 (10.0 wt.%)

[0415] H2O (19 V), DMSO (1 V)

[0416] BocNocN 25 °C, 2 h

[0417]

[0418] N. J OH

[0419] BocNoc

[0420] Compound 3d Compound 3e

[0421] In some embodiments, the cyclizing (e) is carried out using a 1:40 stoichiometric ratio of Compound 3e to formaldehyde (e.g., formalin or methanal). In some embodiments, the formalin comprises an aqueous solution that is 37% (w / w) formaldehyde. In some embodiments, the cyclizing (e) is carried out at a temperature of at least 20 °C (e.g., 20 °C, 25 °C, or 30 °C). In some embodiments, the cyclizing (e) is carried out at a temperature from 20 °C to 35 °C. In some embodiments, the cyclizing (e) is carried out according to the following scheme:

[0422] NH2methanal (40.0 eq.) NH

[0423] MTBocNE (20 V)

[0424] 25 °C, 19 h

[0425] BocNocN

[0426]

[0427] BocNocN

[0428] Compound 3e Compound 3In yet another aspect, the disclosure provides a method of preparing of Compound 4:

[0429]

[0430] Compound 4

[0431] the method including:

[0432] a) converting Compound 3 to Compound 4a:

[0433]

[0434] Compound 3

[0435] b) deprotecting Compound 4a to form Compound 4b • 3HCI:

[0436]

[0437] Compound 4b •3HCl c) coupling Compound 4b •3HCl with Compound 4c hydrochloride using a carbonate source to form Compound 4d:

[0438]

[0439] Compound 4b •3HCl Compound 4c HCI Compound 4d

[0440] d) converting Compound 4d to Compound 4:

[0441]

[0442] Compound 4d Compound 4 In some embodiments, the converting (a) includes contacting Compound 3 with an acyl halide (e.g., 3-chloropropionyl chloride) and a base. In some embodiments, the base is an inorganic base. In some embodiments, the base is a bicarbonate or carbonate base. In some embodiments, the base is sodium bicarbonate, sodium carbonate, calcium carbonate, or potassium carbonate. In someembodiments, the base is sodium bicarbonate. In some embodiments, the converting (a) is carried out using a 1:1 stoichiometric ratio of Compound 3 to acyl halide (e.g., 3-chloropropionyl chloride). In some embodiments, the converting (a) is carried out using a 1:2 stoichiometric ratio of Compound 3 to base (e.g., sodium bicarbonate). In some embodiments, the converting (a) is carried out at a temperature of at least 0 °C (e.g., 0 °C, 5 °C, or 10 °C). In some embodiments, the converting (a) is carried out at a temperature from 0 °C to 10 °C. In some embodiments, the converting (a) is carried out according to the following scheme:

[0443] 3-Chloropropionyl chloride (1.0 eq.)

[0444] NaHCO3(2.0 eq.)

[0445] MTBocNE (20 V)

[0446] H2O (20 V)

[0447] So7

[0448] BocNoBocNocN O-1O =C. 0.5 h /

[0449]

[0450] BocNocN.

[0451] Compound 3 Compound 4a

[0452] In some embodiments, the deprotecting (b) includes contacting Compound 4a with an acid. In some embodiments, the acid is an inorganic acid. In some embodiments, the acid is hydrochloric acid, sulfuric acid, or nitric acid. In some embodiments, the acid is hydrochloric acid. In some embodiments, the concentration of the acid is 4.0 M (e.g., 4.0 M hydrochloric acid). In some embodiments, the acid is in an organic solvent (e.g., ethyl acetate). In some embodiments, the deprotecting (b) is carried out at a temperature of at least 0 °C (e.g., 0 °C, 5 °C, 10 °C, 15 °C, or 20 °C). In some embodiments, the deprotecting (b) is carried out at a temperature from 0 °C to 20 °C.

[0453] In some embodiments, the deprotecting (b) is carried out according to the following scheme:

[0454] y— Ct Ci

[0455] 4.0 M HCl in ethyl acetate (5 V), ACN (5 V), 15 °C, 1 h •3HCl

[0456] BocN

[0457]

[0458] oBocNocN

[0459] Compound 4a Compound 4b •3HCl

[0460] In some embodiments, the carbonate source of the coupling (c) is bis(trichloromethyl) carbonate (BocNTC). In some embodiments, the coupling (c) further includes contacting Compound 4b • 3HCI with an aromatic base (e.g., pyridine) and a non-nucleophilic base (e.g., N, N-diisopropylethylamine or N-methylmorpholine). In some embodiments, the coupling (c) is carried out using a 1:1 stoichiometric ratio of Compound 4b •3HCl to Compound 4c HCl. In some embodiments, the coupling (c) is carried out using a 1:0.33 stoichiometric ratio of Compound 4b •3HCl to carbonate source (e.g., bis(trichloromethyl) carbonate). In some embodiments, the coupling (c) is carried out using a 1:3 stoichiometric ratio of Compound 4b •3HCl to aromatic base (e.g., pyridine). In some embodiments, the coupling (c) is carried out using a 1:7 stoichiometric ratio of Compound 4b •3HCl to non-nucleophilic base (e.g., N, N-diisopropylethylamine). In some embodiments, the coupling (c) is carried out at a temperature of at least 0 °C (e.g., 0 °C, 5 °C, 10 °C, 15 °C, or 20 °C). In some embodiments, the coupling (c) is carried out at a temperature from 0 °C to 20 °C.In some embodiments, the coupling (c) is carried out according to the following scheme:

[0461] o o | f MeO' rx\

[0462] A.? j Compound 4 HCt (1,0 eq,) ''o' ’'^•MYN'VZ

[0463]

[0464] ” A, 6

[0465] BocN I C CO 33 ea ), pyridine; (30 eq.) •" DlEA(7.0 eq, DCM (10 v)

[0466] Compound 4b •3HCl 0 10 “C, 16 h Compound 4cf

[0467] In some embodiments, the converting (d) includes contacting Compound 4d with an iodide salt (e.g., lithium iodide). In some embodiments, the converting (d) includes contacting Compound 4d with a base. In some embodiments, the base is a non-nucleophilic base (e.g., N, N-diisopropylethylamine, N-methylmorpholine, 1,8-diazabicycloundec-7-ene, or 1,5-diazabicyclo(4.3.0)non-5-ene). In some embodiments, the converting (d) is carried out using a 1:5 stoichiometric ratio of Compound 4d to iodide salt (e.g., lithium iodide). In some embodiments, the converting (d) is carried out using a 1:3 stoichiometric ratio of Compound 4d to non-nucleophilic base (e.g., N, N-diisopropylethylamine). In some embodiments, the converting (d) is carried out at a temperature of at least 50 °C (e.g., 50 °C, 55 °C, 60 °C, 65 °C, or 70 °C). In some embodiments, the converting (d) is carried out at a temperature from 50 °C to 70 °C.

[0468] In some embodiments, the converting (d) is carried out according to the following scheme:

[0469] . ■Cl Q

[0470] A'1)

[0471] LiI (5.0 eq.), DIEA (3.0 eq.)

[0472] 2-Ms THF (10 V) HO'" V

[0473] 60 " C, 40 h 1 A

[0474]

[0475] Compound 4d Compound 4

[0476] In another aspect, the disclosure provides a method of preparing Compound A:

[0477]

[0478] Compound A

[0479] the method including:a) coupling Compound 5a with Compound 2 to form Compound 5b:

[0480]

[0481] Compound 5a Compound 2 Compound 5b

[0482]

[0483] Compound 5b Compound 5c

[0484] c) deprotecting Compound 5c to form Compound 5d:

[0485]

[0486] d) coupling Compound 5d with Compound 4 to form Compound A:

[0487]

[0488] Compound 5d In some embodiments, the coupling (a) includes contacting Compound 5a and Compound 2 with a palladium catalyst. In some embodiments, the palladium catalyst is XPhos Pd G3. In some embodiments, the coupling (a) includes contacting Compound 5a and Compound 2 with a base. In someembodiments, the base is an inorganic base. In some embodiments, the base is a carbonate base. In some embodiments, the base is sodium carbonate, calcium carbonate, or potassium carbonate. In some embodiments, the base is potassium carbonate. In some embodiments, the coupling (a) is carried out using a 1:1.2 stoichiometric ratio of Compound 5a to Compound 2. In some embodiments, the coupling (a) is carried out using a 1:2.5 stoichiometric ratio of Compound 5a to base (e.g., potassium carbonate). In some embodiments, the coupling (a) is carried out at a temperature of at least 20 °C (e.g., 20 °C, 25 °C, or 30 °C). In some embodiments, the coupling (a) is carried out at a temperature from 20 °C to 35 °C. In some embodiments, the coupling (a) is carried out according to the following scheme:

[0489] O NHBocNOC

[0490] EtOH / H2O

[0491]

[0492] on Compound 5a Compound 2 Compound 5b

[0493] In some embodiments, the cyclizing (b) includes contacting compound 5b with a carbodiimide coupling reagent. In some embodiments, the carbodiimide coupling reagent is EDCI. In some embodiments, the cyclizing (b) includes contacting compound 5b with an anti-racemization agent. In some embodiments, the anti-racemization agent is selected from the group consisting of Oxyma, HOBocNt, or HOPO. In some embodiments, the anti-racemization agent is HOBocNt. In some embodiments, the cyclizing (b) includes contacting compound 5b with a base. In some embodiments, the base is a non-nucleophilic base (e.g., N, N-diisopropylethylamine or N-methylmorpholine). In some embodiments, the base is N, N-diisopropylethylamine. In some embodiments, the cyclizing (b) is carried out using a 1:3 stoichiometric ratio of Compound 5b to carbodiimide coupling reagent. In some embodiments, the cyclizing (b) is carried out using a 1:2 stoichiometric ratio of Compound 5b to anti-racemization agent. In some embodiments, the cyclizing (b) is carried out using a 1:2 stoichiometric ratio of Compound 5b to base. In some embodiments, the cyclizing (b) is carried out at a temperature of at least 30 °C (e.g., 30 °C, 35 °C, 38 °C, or 40 °C). In some embodiments, the cyclizing (b) is carried out at a temperature from 30 °C to 40 °C. In some embodiments, the cyclizing (b) is carried out according to the following scheme:

[0494]

[0495] Compound 5b Compound 5c

[0496] In some embodiments, the deprotecting (c) includes contacting compound 5c with an acid. In some embodiments, the acid is an inorganic acid. In some embodiments, the acid is hydrochloric acid, sulfuric acid, or nitric acid. In some embodiments, the acid is hydrochloric acid. In some embodiments, theconcentration of the acid is 4.0 M (e.g., 4.0 M hydrochloric acid). In some embodiments, the acid is in an organic solvent (e.g., 1,4-dioxane). In some embodiments, the deprotecting (c) is carried out at a temperature of at least 20 °C (e.g., 20 °C, 25 °C, 30 °C, or 35 °C). In some embodiments, the deprotecting (c) is carried out at a temperature from 20 °C to 35 °C. In some embodiments, the deprotecting (c) is carried out according to the following scheme:

[0497]

[0498] Compound 5c Compound 5d

[0499] In some embodiments, the coupling (d) includes contacting Compound 5d and Compound 4 with a phosphonium coupling reagent. In some embodiments, the phosphonium coupling reagent is PyBocNOP. In some embodiments, the coupling (d) includes contacting Compound 5d and Compound 4 with an antiracemization agent. In some embodiments, the anti-racemization agent is selected from the group consisting of Oxyma, HOBocNt, or HOPO. In some embodiments, the anti-racemization agent is HOBocNt. In some embodiments, the coupling (d) includes contacting Compound 5d and Compound 4 with a base. In some embodiments, the base is a non-nucleophilic base (e.g., N, N-diisopropylethylamine or N-methylmorpholine). In some embodiments, the coupling (d) is carried out using a 1:1 stoichiometric ratio of Compound 5d to Compound 4. In some embodiments, the coupling (d) is carried out using a 1:1.2 stoichiometric ratio of Compound 5d to phosphonium coupling reagent. In some embodiments, the coupling (d) is carried out using a 1:1 stoichiometric ratio of Compound 5d to anti-racemization agent. In some embodiments, the coupling (d) is carried out using a 1:2 stoichiometric ratio of Compound 5d to base. In some embodiments, the coupling (d) is carried out at a temperature of at least 0 °C (e.g., 0 °C, 5 °C, 10 °C, 15 °C, or 20 °C). In some embodiments, the coupling (d) is carried out at a temperature from 0 °C to 20 °C. In some embodiments, the coupling (d) is carried out according to the following scheme:

[0500] Compound 5d DMSO (5 V) Compound A

[0501]

[0502] 0-20 °C In some embodiments, the method further includes purifying Compound A. In some embodiments, the purifying includes recrystallizing Compound A. In some embodiments, the recrystallizing of Compound A includes the following steps:a) dissolving crude Compound A in ethyl acetate at a temperature of at least 40 °C; b) adding n-heptane to the mixture from step (a) at a temperature of at least 40 °C;

[0503] c) adding a seed of Compound A to the mixture from step (b) at a temperature of at least 25 °C; d) adding n-heptane to the mixture from step (c) to form a solid;

[0504] e) isolating the solid from step (d) and dissolving the solid in a 3:2 mixture of ethyl acetate and n-heptane at a temperature of at least 30 °C to form a solid;

[0505] f) isolating the solid of step (e) and drying the solid under vacuum at a temperature of at least 40 °C;

[0506] g) dissolving the dried solid from step (f) in methyl ethyl ketone at a temperature of at least 40 °C; h) adding methyl tert-butyl ether to the mixture of step (g) at a temperature of at least 40 °C; i) adding a seed of pure Compound A to the mixture of step (h) at a temperature of at least 20 °C; j) adding methyl tert-butyl ether to the mixture of step (i) at a temperature of at least 20 °C to form a solid;

[0507] k) isolating the solid of step (j) and drying the solid under vacuum at a temperature of at least 40 °C;

[0508] l) dissolving the dried solid of step (k) in water at a temperature of at least 30 °C;

[0509] m) isolating the solid of step (I) and drying the solid under vacuum at a temperature of at least 40 °C;

[0510] n) dissolving the dried solid of step (m) in water at a temperature of at least 30 °C; and o) isolating the solid of step (n) and drying the solid under vacuum at a temperature of at least 40 °C.

[0511] EXAMPLES

[0512] The disclosure is further illustrated by the following examples and synthesis examples, which are not to be construed as limiting this disclosure in scope or spirit to the specific procedures herein described. It is to be understood that the examples are provided to illustrate certain embodiments and that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure or scope of the appended claims.

[0513] All variables described in the Examples below have the same definitions as defined in the Summary, above.

[0514] Example 1. Synthetic Procedure for Compound 2 ((S)-1-((S)-3-(5-borono-3,6-dihydropyridin-1(2H)-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylic acid)

[0515] Detailed below is a general synthetic procedure for Compound 2.Synthesis of Compound 2 ((S)-1-((S)-3-(5-borono-3,6-dihydropyridin-1(2H)-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylic acid):

[0516] Step 3 Step 1 Step 2 CnBocNs acetone NaBocNHfOAcfe HCi 6Q ”C ovsmighi bCIWEiOH (W-iY 5} DCM4SeC,2h overnight, ri 2} MeOH, 75 °C, 3 h Compound 1a Compound 1b Compound 1c HCi Compound ki HCil Step 4.0 Step 5

[0517] Step 6 o — >■ I — Lsi -O O— WiBocNoc Compi-unct Its NBocNJCOJ. Compound 2a Pd(dppf)Ct;, AcOK MBocNCN / H2O (3. HOefEDci' toluene. 80 “C. 2 h 46 °C. 2 h DCM, rt, 3 h Compound 1 Compound 2b Compound 2c

[0518] Step 7 L»H. H2O Step 5a CC..!^, Nf.tac V N SOCij, fvteOH OHH’";HO-gXJ Compound 2a -1 Compound 2a OH

[0519]

[0520] Compound 2 Step 1 - Synthesis of Compound 1b (1-benzyl-3-bromopyridin-1-ium)

[0521] Step 1

[0522] BocNr' BocNnBocNr,BocNrY''^N"''''Ph

[0523] °C, overnight (i J

[0524] Compound ta Compound 1b

[0525] Reactor 1 was charged with acetone (280 kg, 10 v) and Compound 1a (36.0 kg, 227.85 mol, 1.0 eq.) into a 1000 L reactor. Reactor 1 was evacuated and backfilled with nitrogen gas (N2) three times. BocNenzyl bromide (42.9 kg, 250.63 mol, 1.1 eq.) was added dropwise to the solution at 20-40 °C within a period of 1 h. The resulting mixture was heated to reflux and maintained at that temperature for 44 h. A sample was taken for IPC analysis (assay by HPLC: 0.48 w% of 1 was detected in the liquid phase). The reaction mixture was cooled to 10-15 °C and stirred for 1 h, and then filtered and washed. The filter cake was washed with acetone (30 kg, 1 v). After drying at 40-45 °C for 14 h under reduced pressure, 70.3 kg of Compound 1b was obtained as a white solid with 99.8% HPLC purity and 101 w% assay by qNMR in a 93.8% corrected yield. The crude product was taken to the next step without further purification.

[0526] Table 1: HPLC Method for Compound 1b

[0527] Instrument: HPLC (e.g., Agilent 1260 series)

[0528] Column: Agilent Poroshell 120 EC-C18 (2.7um, 4.6 x 100mm)

[0529] Mobile Phase: A: 0.1% H3PO4 in Water

[0530] BocN: Methanol

[0531] Gradient: Time (min) BocN (%)

[0532] 0.00 5

[0533]

[0534] 7.00 90

[0535] 12.00 90

[0536] Post time: 3 min

[0537] Flow Rate 1.0 mL / min

[0538] UV Detector Wavelength: 215 nm

[0539] Column Temperature: 30 °C

[0540] Retention Times: Compound 1a = 5.2 min

[0541] Compound 1 b = 4.2 min

[0542]

[0543] MS (ESI+):

[0544] Calculated for C12H11BocNrN (M+H+): 350.13

[0545] Found: 350.10

[0546] 1H NMR of Compound 1b (400 MHz, CD3OD-d4):

[0547] 6 9.48 (d, 1H), 9.08 (dd, 1H), 8.83 (dd, 1H), 8.05 (dd, 1H), 7.54 (m, 2H), 7.48 (m, 3H), 5.87 (s, 2H).

[0548] Step 2 - Synthesis of Compound 1c HCI (1 -benzyl-5-bromo-1,2,3,6-tetrahydropyridine HCI salt)

[0549] Step 2

[0550] NaBocNH(OAc)3< y

[0551]

[0552] DCM / EtOH (10:1)

[0553] Compound 1b overnight, rt Compound 1c HCi A reactor was charged with DCM (530 kg, 20 v), EtOH (16 kg, 1 v) and Compound 1b (20.0 kg, 60.78 mol, 1.0 eq.). The reactor was evacuated and backfilled with nitrogen three times. NaBocNH(OAc)3(83.7 kg, 395.09 mol, 6.5 eq.) was added in 25 portions within a period of 3 h at 0-10 °C. The mixture was warmed to 25-30 °C and maintained for 19 h. A sample was taken for IPC (HPLC purity: 82 A% of 3 and 4 A% of 2). The reaction mixture was cooled to 0-5 °C, and then added into a sat. brine solution (144 kg, 6 v) at 0-10 °C within a period of 1 h. The mixture was stirred at 0-10 °C for 1 h. The resulting mixture was filtered through a pad of diatomite (20 kg, 1 w) and the filter cake was washed with DCM (133 kg, 5 v). The filtrate was separated and water (60 kg, 3 v) was added into the organic phase. Sat. aq. Na2CO3(100 kg, 4.1 v) was added dropwise into the organic phase at 10-20 °C. The organic phase was separated and then washed with sat. aq. Na2CO3(100 kg, 4.1 v). The organic phase was stored at 0-10 °C and combined with the next batch (see below) for purification.

[0554] A reactor was charged with DCM (1325 kg, 20 v), EtOH (40 kg, 1 v) and Compound 1b (50.0 kg, 151.96 mol, 1.0 eq.). The reactor was evacuated and backfilled with nitrogen 3 times. NaBocNH(OAc)3(209.4 kg, 987.72 mol, 6.5 eq.) was added in 25 portions within a period of 3 h at 0-10 °C. The mixture was warmed to 25-30 °C and maintained for 19 h. A sample was taken for IPC (HPLC purity: 84 A% of 3 and 3 A% of 2). The reaction mixture was cooled to 0-5 °C, and then added into a sat. brine solution (360 kg, 6 v) at 0-10 °C within a period of 1 h. The mixture was stirred at 0-10 °C for 1 h. The resulting mixture was filtered through a pad of diatomite (50 kg, 1 w) and washed the filter cake with DCM (331 kg, 5 v). Thefiltrate was separated and water (150 kg, 3 v) was added into the organic phase. Sat. aq. Na2CO3(250 kg, 4.1 v) was added dropwise into the organic phase at 10-20 °C. The organic phase was separated and then washed with sat. aq. Na2CO3(250 kg, 4.1 v). The organic phase was combined with that of the first batch. The resulting mixture was concentrated under reduced pressure to 2 v (140 L) at 30-40 °C under reduced pressure. MTBocNE (518 kg, 10 v) was added to the residue. 10 w / w% of HCI in MTBocNE (anhydrous) (62.1 kg, 170.19 mol, 0.8 eq.) was added dropwise into the solution at 20-30 °C for 1-2 h. The suspension was cooled to 5-10 °C and maintained for 1 h. The mixture was filtered and the filter cake was washed with a 1:10 (v / v) mixture of DCM and MTBocNE (30 kg, 1 v). The wet filter cake was dried at 40-45 °C under reduced pressure (ca. 100 mbar) for 14 h giving 45.5 kg of Compound 1c HCI as a white solid with 93.7% HPLC purity and 93 w% assay by qNMR in a 69% corrected yield.

[0555] Table 2: HPLC Method for Compound 1c HCI

[0556] Reaction monitoring:

[0557] Instrument: HPLC (e.g., Agilent 1100 series)

[0558] Column: Agilent Poroshell 120 EC-C18 (4.6x100 mm, 2.7 pm) Mobile Phase: A: 0.1% H3PO4 in water

[0559] BocN: MeCN

[0560] Gradient: Time (min) %BocN

[0561] 0.00 5

[0562] 7.00 90

[0563] 12.0 90

[0564] Post Time: 3.0 min

[0565] Flow Rate 1.0 mL / min

[0566] UV Detector Wavelength: 210 nm

[0567] Column Temperature: 30 °C

[0568] BocNlank / Diluent MeCN

[0569] Sample Preparation: Transfer 1 drop of reaction solution into a 1.5 mL

[0570] centrifuge tube, add 1 mL diluents and mix well.

[0571] Injection Volume: 1 pL

[0572] Retention Times: Compound 1c HCI = 4.1 min

[0573] Compound 1b = 3.6 min

[0574]

[0575] HPLC purity:

[0576] Instrument: HPLC (e.g., Agilent 1100 series)

[0577] Column: Agilent Poroshell 120 EC-C18 (4.6x100 mm, 2.7 pm) Mobile Phase: A: 0.1% H3PO4 in water

[0578] BocN: MeOH

[0579] Gradient: Time (min) %BocN

[0580] 0.00 5

[0581] 7.00 90

[0582]

[0583] 12.0 90

[0584] Post Time: 3.0 min

[0585] Flow Rate 1.0 mL / min

[0586] UV Detector Wavelength: 215 nm

[0587] Column Temperature: 30 °C

[0588] BocNlank / Diluent MeOH

[0589] Sample Preparation: Transfer s mg of Compound 1b into a 1.5 mL centrifuge tube, add 1 mL diluents and mix well.

[0590] Injection Volume: 1 pL

[0591] Retention Times: Compound 1c HCI = 4.6 min

[0592]

[0593] MS (ESI+):

[0594] Calculated for C12H15BocNrClN (M+H+): 253.16

[0595] Found: 253.20

[0596] 1H NMR of Compound 1c HCI (400 MHz, CD3OD-d4): 67.30 (m, 5H), 6.09 (t, 1H), 3.62 (s, 2H), 3.16 (t, 2H), 2.61 (t, 2H), 2.22 (m, 2H).

[0597] Step 3 - Synthesis of Compound 1d HCI (5-bromo-1,2,3,6-tetrahydropyridine HCI salt)

[0598] Step 3

[0599] q Ci8rcr^'o "^ HCI

[0600] J 1) DCM.40 °C, 2h

[0601]

[0602] 2) MeOH, 75 °C, 3 h

[0603] Compound ic HCI Compound 1d HCI To a reactor was evacuated and backfilled with N2three times. Water (170 kg, 6 v) and EtOAc (180 kg, 7 v) was charged to the reactor. Compound 1c HCI (30.4 kg, 97.96 mol, 1.0 eq., 93 w% assay) was added to the reactor and the mixture was cooled to 0-10 °C. Sat. aq. Na2CO3(270 kg, 8 v) was added dropwise into the reactor at 0-20 °C to adjust the pH to 9-10. The aqueous phases were separated and extracted with EtOAc (127 kg, 5 v). The organic phases were combined and concentrated to 3-4 v at 35-45 °C under reduced pressure. The residue was filtered to remove solids and the filtrate was concentrated to 1 v at 35-45 °C under reduced pressure. DCM (338 kg, 9 v) was added to the concentrate and 4 A molecular sieves (33.8 kg, 1.2 w) was charged to the reactor. The mixture was stirred at 20-30 °C for 2 h. A sample was taken for IPC (water content by KF titration: 100 ppm). The mixture was cooled to 0-5 °C and 1-chloroethyl chloroformate (21 kg, 146.89 mol, 1.5 eq.) was added dropwise into the reactor at 0-5 °C for 1 h. The resulting mixture was agitated for 1 h at 0-5 °C. A sample was taken for IPC (HPLC: 19 A% of intermediate, 61 A% of BocNnCI and 2 A% of Compound 1d HCI). The mixture was filtered to remove the solids. The filtrate was concentrated at 20-40 °C under reduced pressure until no distillate was observed. MeOH (45 kg, 2 v) was added to the concentrate above to give a MeOH solution of the intermediate. The MeOH solution was transferred into an overhead tank connected to a 1000 L reactor. MeOH (157 kg, 7 v) was added to the 1000 L reactor and was heated to reflux. The MeOH solution of theintermediate was added dropwise into reactor at 65-75 °C over 1 h. The resulting mixture was agitated for 30 min at 65-75 °C and was cooled to 20-30 °C. A sample was taken for IPC (HPLC: 62 A% of BocNnCI and 21 A% of Compound 1d HCI were detected). The reaction solution was transferred to a 500 L reactor and concentrated to 1-2 v under reduced pressure at 30-35 °C. MeCN (59 kg, 2.6 v) was added to the reactor and the solution was concentrated to 1-2 v under reduced pressure at 35-45 °C. MeCN (59 kg, 2.6 v) was added to the reactor and the solution was concentrated to 1-2 v under reduced pressure at 35-45 °C. MeCN (29 kg, 1.3 v) was added to the reactor. The temperature was adjusted to 5-10 °C and the suspension was agitated for 1 h at 5-10 °C. The resulting mixture was filtered and the filter cake was rinsed with MeCN (22 kg, 1 v). The wet filter cake was dried under reduced pressure (ca. 100 mbar) at 40-45 °C for 14 h giving 16.3 kg of 4 as a white solid with 99.7% HPLC purity and 98 w% assay by qNMR in an 82% corrected yield.

[0604] Table 3: HPLC Method for Compound 1d HCI

[0605] Reaction monitoring:

[0606] Instrument: HPLC (ARD-S4C1-HPLC-018)

[0607] Column: Agilent Poroshell 120 EC-C18 (4.6x100 mm, 2.7 pm) Mobile Phase: A: 0.1% H3PO4 in water

[0608] BocN: MeCN

[0609] Gradient: Time (min) %BocN

[0610] 0.00 5

[0611] 7.00 90

[0612] 12.0 90

[0613] Post Time: 3.0 min

[0614] Flow Rate 1.0 mL / min

[0615] UV Detector Wavelength: 210 nm

[0616] Column Temperature: 30 °C

[0617] BocNlank / Diluent MeCN

[0618] Sample Preparation: Transfer 1 drop of reaction solution or 20 mg of Compound 1c HCI into a 1.5 mL centrifuge tube, add 1 mL diluents and mix well.

[0619] Injection Volume: 2 pL

[0620] Retention Times: Compound 1c HCI = 4.1 min

[0621] intermediate = 7.5 min

[0622] BocNnCI = 7.2 min

[0623] Compound 1d HCI = 1.8 min

[0624]

[0625] HPLC purity:

[0626] Instrument: HPLC (ARD-S4C1-HPLC-018)

[0627] Column: Waters Atlantis T3 (4.6x150 mm, 3.0 pm)

[0628] Mobile Phase: A: 0.1% H3PO4 in water

[0629]

[0630] BocN: MeCN

[0631] Gradient: Time (min) %BocN

[0632] 0.00 5

[0633] 2.00 5

[0634] 9.00 90

[0635] 15.0 90

[0636] Post Time: 5.0 min

[0637] Flow Rate 1.0 mL / min

[0638] UV Detector Wavelength: 205 nm

[0639] Column Temperature: 30 °C

[0640] BocNlank / Diluent MeCN

[0641] Sample Preparation: Transfer 20 mg of Compound 1d HCI into a 1.5 mL centrifuge tube, add 1 mL diluents and mix well.

[0642] Injection Volume: 2 pL

[0643] Retention Times: Compound 1d HCI = 3.2 min

[0644]

[0645] MS (ESI+):

[0646] Calculated for C5H9BocNrCIN (M+H+): 163.03

[0647] Found: 163.30

[0648] 1H NMR of Compound 1d HCI (400 MHz, CD3OD-d4):

[0649] 66.40 (m, 1 H), 3.93 (d, 2H), 3.33 (m, 2H), 2.50 (m, 2H).

[0650] Step 4 - Synthesis of Compound 1 ((S)-3-(5-bromo-3,6-dihydropyridin-1(2H)-yl)-2-((tert-butoxycarbonyl)amino) propanoic acid)

[0651] Ste 4

[0652] NajCOa 20 ecj.

[0653] ;S r CH3CN 10 v

[0654] ' NH HCI

[0655] 70%

[0656]

[0657] Compound 1d HCI Compound 1e Compound 1

[0658] To a reactor was charged with Compound 1d HCI (9.7 kg, 48.87 mol, 1.0 eq.), MeCN (79 kg, 10 v), and water (35 kg, 3.5 v). Na2CO3(10.4 kg, 97.74 mol, 2.0 eq.) was added to the reactor. The reactor was evacuated and backfilled with nitrogen three times. The mixture was heated to 35-40 °C and tertbuty (S)-(2-oxooxetan-3-yl) carbamate (Compound 1e, 10.98 kg, 53.76 mol, 1.1 eq.) was added in fifteen portions to the reactor at 35-40 °C over a period of 2.5 h. The mixture was stirred at 35-40 °C for 1 h. A sample was taken for IPC (HPLC: 62 A% of Compound 1, 26 A% of intermediate 1 a and 4 A% of intermediate 1b). The reaction solution was cooled to 10-20 °C and filtered through a diatomite (3 kg, 0.3 w) plug. The filter cake was washed with 3:1 (v / v) MeCN / FhO (20 L, 2 v). The resulting mixture was concentrated under reduced pressure at 40-45 °C to 4-5 v. Water (100 kg, 10 v) was added into thereactor. The aqueous phase was extracted with DCM three times (66 kg < 3, 5 vx 3). The aqueous phase was cooled to 0-10 °C and adjusted the pH to 4-5 by adding 2 M formic acid (37.2 kg). The aqueous phase was extracted with DCM three times (133 kg * 2, 10 v * 2; 66 kg * 1, 5 v * 1). The organic phases were combined and concentrated under reduced pressure at 20-40 °C to 3-3.5 v. THF (45 kg, 5 v) was added into the reactor and concentrated under reduced pressure at 30-40 °C to 3-3.5 v. The suspension was cooled to 20-30 °C. THF (18 kg, 2 v) was added into the reactor, n-heptane (83 kg, 12 v) was added into the reactor dropwise over a period of 1 h. The suspension was stirred at 10-20 °C for 12 h. The suspension was cooled to 0-10 °C and filtered. The filter cake was washed with 4:1 (v / v) n-heptane / THF (20 L, 2 v) and then dried at 40-45 °C under reduced pressure over 15 h giving 12.1 kg of Compound 1 as a white solid with 98.7% HPLC purity, 98.9 w% assay by qNMR and 99.2% chiral HPLC purity in a 70% corrected yield.

[0659] Table 4: HPLC Method for step 4

[0660] Instrument: HPLC (e.g., Agilent 1100 series)

[0661] Column: Agilent Poroshell 120 EC-C18 (4.6x100 mm, 2.7 pm) Mobile Phase: A: 0.1% H3PO4 in water

[0662] BocN: MeCN

[0663] Gradient: Time (min) %BocN

[0664] 0.00 5

[0665] 7.00 90

[0666] 12.0 90

[0667] Post Time: 3.0 min

[0668] Flow Rate 1.0 mL / min

[0669] UV Detector Wavelength: 210 nm

[0670] Column Temperature: 30 °C

[0671] BocNlank / Diluent MeCN

[0672] Sample Preparation: Transfer 1 drop of reaction solution or 2 mg of Compound

[0673] 1 into a 1.5 mL centrifuge tube, add 1 mL diluents and mix well.

[0674] Injection Volume: 1 pL

[0675] Retention Times: Compound 1d HCI = 1.8 min

[0676] Compound 1 = 4.2 min

[0677] Intermediate 1 b = 5.5 min

[0678] Intermediate 1a = 5.8 min

[0679]

[0680] MS (ESI+):

[0681] Calculated for C13H21BocNrN2O4(M+H+): 350.23

[0682] Found: 350.20

[0683] 1H NMR of Compound 1 (400 MHz, CD3OD-d4):

[0684] 66.27 (s, 1 H), 4.36 - 4.32 (m, 1 H), 3.75 (s, 2H), 3.24-3.12 (m, 4H), 2.43 (s, 2H), 1.49 (s, 9H).Step 5a - Synthesis of Compound 2a • 2HCl (methyl (S)-hexahydropyridazine-3-carboxylate dihydrochloride salt)

[0685] r Step 5a j ]

[0686] 1 SOCI2, MeOH 1 2HCI

[0687]

[0688] OHBocNOC

[0689] Compound 2a-1 Compound 2a • 2HCl A reactor was charged with Compound 2a-1 (14.5 kg, 43.89 mol, 1.0 eq.) and MeOH (116 kg, 10 v). The reactor was evacuated and backfilled with nitrogen three times. SOCh (10.5 kg, 87.78 mol, 2.0 eq.) was added dropwise into the reactor at 0-30 °C over a 40 min period. The mixture was heated to 30-40 °C and stirred for 42 h. A sample was taken for IPC (HPLC: 97 A% of Compound 2a and 0 A% of Compound 2a-1). The reaction mixture was concentrated to dryness under reduced pressure at 35-40 °C. The residue was dissolved in DCM (30 kg, 1.5 v) and the solution was concentrated to dryness under reduced pressure at 35-40 °C. The residue was dissolved in DCM (38 kg, 2 v) giving 46.4 kg of a DCM solution of Compound 2a with 97.2% HPLC purity and 20 w% assay in a 100% corrected yield.

[0690] Table 5. HPLC method for Compound 2a • 2HCl

[0691] Instrument: HPLC (e.g., Agilent 1100 series)

[0692] Column: Agilent Eclipse Plus C18 (4.6 x 100 mm, 3.5 pm)

[0693] Mobile Phase: A: 10mM (NH₄)₂HPO₄ in water

[0694] BocN: MeCN

[0695] Gradient: Time (min) %BocN

[0696] 0.00 2

[0697] 2.00 2

[0698] 10.0 25

[0699] 15.0 85

[0700] 20.0 85

[0701] Post Time: 5.0 min

[0702] Flow Rate 0.8 mL / min

[0703] UV Detector Wavelength: 210 nm

[0704] Column Temperature: 30 °C

[0705] BocNlank / Diluent MeCN

[0706] Sample Preparation: Transfer 0.1 mL of reaction solution or DCM solution into a 1.5 mL centrifuge tube, add 1 mL diluents and mix well.

[0707] Injection Volume: 1 pL

[0708] Retention Times: Compound 2a-1 = 13.1 min

[0709] Compound 2a = 7.0 min

[0710]

[0711] LCMS (ESI+)

[0712] Calculated for C5H11Cl2N2O2(M+H+): 145.17

[0713] Found: 145.10

[0714] 1H NMR (400 MHz, DMSO-cfe) of Compound 2a«2HCI:

[0715] 6 10.58 (s, 2H), 5.75 (s, 2H), 3.95-3.98 (dd, 1H), 3.68 (s, 3H), 3.10-3.13 (d, 1H), 2.90-2.95 (t, 1 H), 1.89-1.95 (t, 2H), 1.78-1.86 (t, 1 H), 1.57 - 1.65 (q, 1 H).

[0716] Step 5 - Synthesis of Compound 2b (methyl (S)-1-((S)-3-(5-bromo-3,6-dihydropyridin-1(2H)-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate)

[0717] Step 5

[0718] EDCI 1.8 eq HOBocNt 2 mol%

[0719] DCM 10 vol

[0720] 0-5 °C, 1 h

[0721] 78-85%

[0722]

[0723] Compound 1 Compound 2a • 2HCl Compound 2b

[0724] To a reactor was charged the DCM solution of Compound 2a • 2HCl (42.2 kg, contained 8.43 kg of Compound 2a • 2HCl, 38.83 mol, 1.2 equiv.) and DCM (150 kg, 10 v). The mixture was stirred at 10-20 °C for 5 min. The reactor was evacuated and backfilled with nitrogen 3 times. The MTBocNE solution of HCI (3.3 kg, contained 354.5 g of HCI, 9.71 mol, 0.3 eq.) was added into the reactor at 0-5 °C. Compound 1 (11.3 kg, 32.36 mol, 1.0 eq.) was added into the reactor at 0-5 °C. The mixture was stirred at 0-5 °C for 0.5 h. HOBocNt (87.5 g, 0.65 mol, 2 mol%) was added into the reactor at 0-5 °C. EDCI (11.2 kg, 58.25 mol, 1.8 eq.) was added in eight portions into the reactor at 0-5 °C over 50 min. The mixture was stirred at 0-5 °C for 1-2 h. A sample was taken for IPC (HPLC: 88 A% of Compound 2b, 3 A% of Compound 2b-1 and 0 A% of Compound 1). Sat. aq. NaHCO3(13 kg of NaHCO3in 140 kg ofwater) was added into another 1000 L reactor and cooled the solution to -2 to 5 °C. The reaction mixture was added into the 1000 L reactor in a period of 10 min. The phases were separated and the aqueous phase was extracted with DCM (56.5 kg x 1, 5 v x 1). The two DCM solutions were treated separately. The first DCM solution was cooled to 0-10 °C. 2 M formic acid (10.93 kg) was added dropwise such that the pH was adjusted to about 4-5. The phases were separated and the DCM phase was cooled to 0-10 °C. Sat. aq. Na2CO3(12 kg) was added dropwise into the DCM solution such that the pH was about 7-8. The organic phase was washed with water (110 kg, 10 v) and brine (110 L x 2, 10 v x 2) respectively. The DCM phase was concentrated to 2-3 v under reduced pressure at 20-40 °C. Toluene (50 kg, 5 v) was added into the reactor and the solution was concentrated to 2-3 v under reduced pressure at 40-45 °C. The residue was cooled to 15-25 °C giving 27.04 kg of a toluene solution of Compound 2b with 85% HPLC purity, 97.6% chiral HPLC purity and 44.1 w% assay by qNMR (equivalent to 11.9 kg of neat Compound 2b) in a 67% corrected yield. The second isolated portion of DCM solution was concentrated to 2-3 v under reduced pressure 20-40 °C. Toluene (20 kg) was added into the reactor and the solution was concentrated to 1-2 v under reduced pressure at 40- 45 °C. The residue was cooled to 15-25 °C giving 4.46 kg of the toluene solution of Compound 2b with 77% HPLC purity and 53.8 w% assay by qNMR in a 12% corrected yield.Table 6. HPLC method for Compound 2b

[0725] Instrument: HPLC (e.g., Agilent 1100 series)

[0726] Column: Waters Atlantis T3 (4.6 x 150 mm, 3.0 pm)

[0727] Mobile Phase: A: 0.1% H3PO4 in water

[0728] BocN: MeCN

[0729] Gradient: Time (min) %BocN

[0730] 0.00 5

[0731] 10.0 95

[0732] 15.0 95

[0733] Post Time: 3.0 min

[0734] Flow Rate 1.0 mL / min

[0735] UV Detector Wavelength: 210 nm

[0736] Column Temperature: 30 °C

[0737] BocNlank / Diluent MeCN

[0738] Sample Preparation: Transfer 1 drop of reaction solution into a 1.5 mL centrifuge tube, add 1 mL diluents and mix well.

[0739] Injection Volume: 1 pL

[0740] Retention Times: Compound 1 = 5.9 min

[0741] Compound 2b = 6.8 min

[0742] Compound 2b-1 = 7.0 min

[0743]

[0744] LCMS (ESI+):

[0745] Calculated for C19H31BocNrN4O5(M+H+): 476.38

[0746] Found: 476.30

[0747] 1H NMR of Compound 2b (400 MHz, DMSO-cfe):

[0748] 66.51-6.53 (d, 1H), 6.09 (s, 1H), 5.22-5.24 (d, 1H), 5.16-5.19 (m, 1H), 3.98 (s, 1H), 3.69 (s, 3H), 3.58-3.62 (m, 1 H), 3.19 (s, 2H), 2.95 (m, 1H), 2.67 - 2.71 (m, 1 H), 2.48 -2.60 (m, 3H), 2.13 (s, 2H), 1.87 - 1.91 (m, 1 H), 1.72-1.76 (m, 1 H), 1.50 -1.62 (m, 2H),1.38 (s, 9H).Step 6 - Synthesis of Compound 2c (methyl (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(5-(5,5-dimethyl- 1,3,2-dioxaborinan-2-yl)-3,6-dihydropyridin-1(2H)-yl)propanoyl)hexahydropyridazine-3-carboxylate)

[0749] Step 6 O

[0750] -O / "" P. P-\

[0751] TNT X BocN-BocN' K NHBocNoc

[0752] . OHr ^NHBocNoc " _ ''•--Q _ _ b _ _-- /

[0753] xN. Pd(dppf)Ci2, AcOK

[0754] ( 'I toluene, 30 °C, 2 h

[0755]

[0756] Compound 2c

[0757] Compound 2b

[0758] To a reactor was charged Compound 2b (27.7 kg, 25.66 mol, 44.1 w% assay by qNMR (equivalent to 12.2 kg of neat Compound 2b), 1.0 eq.) and toluene (100 kg, 10 v total). The mixture was stirred for 5 min. The reactor was evacuated and backfilled with nitrogen 3 times. BocNis(neopentyl glycolato)diboron (8.7 kg, 38.5 mol, 1.5 eq.) and KOAc (12.6 kg, 128.3 mol, 5.0 eq.) was added into the reactor. The reactor was evacuated and backfilled with nitrogen 3 times. Pd(dppf)Cl2 (564 g, 0.77 mol, 3 mol%) was added into the reactor. The reactor was evacuated and backfilled with nitrogen 6 times. The mixture was heated to 80-85 °C and stirred for 3 h. A sample was taken for IPC (HPLC: 84 A% of Compound 2c and 0 A% of Compound 2b). The reaction mixture was cooled to 10-25 °C. The mixture was filtered and the filter cake was washed with toluene (25 kg x 2, 2 v x 2). The filtrates were extracted with 2M formic acid three times (61 kg x 3, 5 vx 3). The aqueous phases were combined and washed with EtOAc (45 kg, 5 v). The phases were separated and the aqueous phase was cooled to 0-10 °C. The aqueous phase was adjusted pH to 8-9 using sat. aq. Na2CO3(140 kg of water and 36 kg of Na2CO3) at 0-10 °C. The mixture was stirred for 10 min and extracted with MTBocNE three times (50 kg x 3, 5 v x 3). The organic phases were combined and concentrated to 1.5-2 v (15-25 L) under reduced pressure at 40-45 °C. MeOH (50 kg, 5 v) was added into the residue and concentrated to 1.5-2 v (15-25 L) under reduced pressure at 40-45 °C. The residue was cooled to 10-20 °C giving 18.3 kg of a MeOH solution of Compound 2c with 88.6% HPLC purity and 53.0 w% assay by qNMR (equivalent to 9.7 kg of Compound 2c) in an 81% corrected yield.

[0759] Table 7. HPLC method for Compound 2c

[0760] Instrument: HPLC (e.g., Agilent 1100 series)

[0761] Column: Agilent Poroshell 120 EC-C18 (4.6x100 mm, 2.7 pm) Mobile Phase: A: 0.1% H3PO4 in water

[0762] BocN: MeCN

[0763] Gradient: Time (min) %BocN

[0764] 0.00 5

[0765] 7.00 90

[0766] 12.0 90

[0767] Post Time: 3.0 min

[0768] Flow Rate 1.0 mL / min

[0769] UV Detector Wavelength: 210 nm

[0770]

[0771] Column Temperature: 30 °C

[0772] BocNlank / Diluent MeCN

[0773] Sample Preparation: Transfer 1 drop of reaction solution or 10 mg of Compound 2c into a 1.5 mL centrifuge tube, add 1 mL diluents and mix well.

[0774] Injection Volume: 1 pL

[0775] Retention Times: Compound 2b = 4.9 min

[0776] Compound 2c = 4.2 min

[0777]

[0778] LCMS (ESI+):

[0779] Calculated for C24H41BocNN4O7(M+H+): 509.42

[0780] Found: 509.20

[0781] 1H NMR of Compound 2c (400 MHz, DMSO-c / 6)

[0782] 66.34-6.41 (d, 1H), 6.30 (dd, 1H), 5.08-5.13 (m, 2H), 3.96 (s, 1H), 3.60 (s, 2H), 3.53 (s, 4H), 3.09-3.10 (d, 1H), 2.79 - 2.97 (m, 3H), 2.26-2.58 (m, 5H), 2.03 (s, 2H), 1.80-1.82 (d, 1H), 1.66 (d, 1H), 1.44-1.53 (m, 2H), 1.31 (s, 9H), 0.84 (s, 6H).

[0783] Step 7 - Synthesis of Compound 2 ((S)-1-((S)-3-(5-borono-3,6-dihydropyridin-1(2H)-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylic acid)

[0784] Step 7 I " r <1LiOH·H2O

[0785] O

[0786]

[0787] Compound 2c Compound 2

[0788] To a reactor was charged 17.9 kg of Compound 2c (MeOH solution, contained about 9.5 kg of Compound 2c and 1 v MeOH) (18.69 mol, 1.0 eq.) and MeOH (72 kg, 9 v to 10 v). The mixture was stirred for 5 min. The reactor was evacuated and backfilled with nitrogen 3 times. The mixture was cooled to 0-5 °C. LiOH·H2O (2.4 kg, 56.06 mol, 3.0 eq.) in water (68 L, 3.5 v) was added dropwise into the reactor at 0-5 °C over 15 min. The mixture was stirred at 0-5 °C for 2 h. A sample was taken for IPC (HPLC: 90 A% of Compound 2 and 0 A% of Compound 2c). 12 M HCI (4.5 kg) was added into the reaction mixture at 0-5 °C and the pH value was 7-8. The reaction mixture was concentrated under reduced pressure at 40-45 °C until about 55 L of a clear solution remaining. H2O (45 L, 4.5 v) was added into the mixture and the aqueous phase was extracted with MTBocNE seven times (37 kg x 7, 5 v * 7). Solid NaCI (20 kg) was added into the aqueous phase. The aqueous phase was extracted with DCM (20 kg, 1.5 v) and concentrate to dryness under reduced pressure giving 1.2 kg of Compound 2 as a foamy solid with 74 A% HPLC purity which is being stored for possible future use. The aqueous phase was extracted with n-BocNuOH four times (40 kg x 4, 5 v x 4). The n-BocNuOH organic phases were combined and concentrated under reducedpressure (-0.095 Mpa) 45-50 °C (about 90-100 L of suspension remaining). The residue was cooled to 5-15 °C and stirred at 5-15 °C for 1 h. The resulting mixture was filtered and the filter cake was washed twice with n-BocNuOH (1 v x 2). The filtrates were combined and mixed with distilled water (20 kg, 2 v). The mixture was concentrated to about 70-80 L under reduced pressure (-0.095-0.1 Mpa) at 45-50 °C. Distilled water (20 kg, 2 v) was added and the mixture was concentrated to about 50-60 L under reduced pressure (-0.095-0.1 Mpa) at 45-50 °C. Distilled water (20 kg, 2 v) was added and the mixture was concentrated to about 20-30 L under reduced pressure (-0.095-0.1 Mpa) at 45-50 °C. Toluene (36 kg, 4 v) was added and concentrated to about 15 L under reduced pressure (-0.095-0.1 Mpa) at 45-50 °C. The mixture was cooled to 30-40 °C. MTBocNE (7 kg, 1 v) was added and stirred for 5 min giving 23.5 kg of an MTBocNE / n-BocNuOH solution of Compound 2 as a clear solution with 7.2 w% water content by KF titration and 92% HPLC Purity. The MTBocNE / n-BocNuOH solution of Compound 2 was added dropwise into MTBocNE (215 kg, 30 v) over a period of 75 min at 15-25 °C. The resulting suspension was stirred at 10-20 °C for 1 h. The mixture was cooled to 0-5 °C and the suspension was stirred at 0-5 °C for 1 h. The suspension was filtered and the filter cake was washed with MTBocNE (2 v x 2) under N2 protection. The wet filter cake was dried at 20-35 °C under reduced pressure (ca. 100 mbar) to constant weight giving 5.59 kg of Compound 2 with 91.7% HPLC purity, 72.7 w% assay by qNMR, 14.7 w% water content by KF titration as a yellow solid.

[0789] Table 8. HPLC method for Compound 2

[0790] Instrument: HPLC (e.g., Agilent 1100 series)

[0791] Column: Agilent Poroshell 120 EC-C18 (4.6x100 mm, 2.7 pm) Mobile Phase: A: 0.1% H3PO4 in water

[0792] BocN: MeCN

[0793] Gradient: Time (min) %BocN

[0794] 0.00 5

[0795] 10.0 25

[0796] 16.0 90

[0797] 20.0 90

[0798] Post Time: 3.0 min

[0799] Flow Rate 1.0 mL / min

[0800] UV Detector Wavelength: 210 nm

[0801] Column Temperature: 30 °C

[0802] BocNlank / Diluent MeCN

[0803] Sample Preparation: Transfer 1 drop of reaction solution or 2 mg of 5 into a 1.5

[0804] mL centrifuge tube, add 1 mL diluents and mix well.

[0805] Injection Volume: 1 pL

[0806] Retention Times: Compound 2 = 7.5 min

[0807] Compound 2c = 10.6 min

[0808]

[0809] LCMS (ESI-):

[0810] Calculated for C18H31BocNN4O7(M-H+): 425.28

[0811] Found: 425.20

[0812] 1H NMR of Compound 2 (400 MHz, CD3OD)

[0813] 66.34 (s, 1H), 5.37 (s, 1H), 4.28 (s, 1H), 3.55 (s, 2H), 3.33 (s, 1H), 2.45-3.23 (m, 6H), 2.24 (m, 2H), 2.05 (m, 1 H), 1.83 (m, 1 H), 1.59 (m, 2H), 1.42 (s, 9H).

[0814] Example 2. Synthetic Procedure for Compound 4 (N-((S)-3-acryloyl-4-methyl-1-oxa-3,8-diazaspiro[4.5]decane-8-carbonyl)-N-methyl-L-valine)

[0815] Detailed below is a general synthetic procedure for Compound 4.

[0816] Synthesis of Compound 4 (N-((S)-3-acryloyl-4-methyl-1-oxa-3, 8-diazaspiro[4.5]decane-8-carbonyl)-N-methyl-L-valine):

[0817] .

[0818] ^

[0819]

[0820] Step 1 - Synthesis of Compound 3b (tert-butyl 4-ethynyl-4-hydroxypiperidine-1 -carboxylate)

[0821] EthynylMgBocNr in THF (1.3 eq.)

[0822] r - OH

[0823] BocNocN J

[0824]

[0825] 86.2% yield

[0826] Compound 3a. Compound 3b

[0827] Step 1

[0828] To a reactor was charged ethynylmagnesium bromide in THF (52.2 L, 1.3 equiv.) at 25 °C. The solution was cooled and maintained at 0-5 °C. To this mixture was added tert-butyl 4-oxopiperidine-1-carboxylate (Compound 3a, 4.0 kg, 20.08 mol, 1.0 equiv.) portion wise with stirring at 0-5 °C. The resulting solution was maintained at 0°C for 1 hour. A sample was taken and diluted with MeOH to run HPLC analysis (criterion: area% of Compound 3a < 2%, actual result: area% of Compound 3a: 1.2%). To the reaction solution was added 20 wt.% citric acid solutions (20.0 kg) with stirring at 0-5 °C. The solutionwas warmed and maintained at 20-25°C. To this mixture was added n-heptane (17.8 kg). The n-heptane phase was separated and washed with 10 wt.% brine (20.0 kg). The reaction mixture was concentrated under reduced pressure until the residue volume was around 25 L. To the solution was charged n-heptane (13.7 kg). The resultant solution was concentrated under reduced pressure until the residue volume was around 25 L. The solvent swap was repeated for another 2 times. A sample was taken and diluted with NMP to run GC analysis (criterion: area% of THF < 5%, actual result: area% of THF: 4.4%). The resultant solution was stirred for 2 hours at 15-20 °C and filtered. The cake was washed with n-heptane (5.5 kg) and dried to afford Compound 3b as a light yellow solid (3.83 kg, 97.9% a / a purity, 97.7% w / w assay, 16.63 mol, 82.8% yield).

[0829] MS (ESI+) M+1= 152.0

[0830] Molecular formula: C12H19NO3

[0831] 1H NMR of Compound 3b (400 MHz, Chloroform-d) 66.10 (s, 1H), 3.81 (d, J = 4.8 Hz, 2H), 3.28 (ddd, J = 13.2, 9.3, 3.4 Hz, 2H), 1.95 - 1.85 (m, 3H), 1.72 (ddd, J = 13.2, 9.3, 4.0 Hz, 2H), 1.47 (s, 9H).

[0832] Table 9: HPLC Method for step 1.

[0833] In-progress Control from Compound 3a to Compound 3b by HPLC

[0834] Column: Waters Xbridge C18, 4.6x150 mm, 3.5 urn

[0835] A: 0.05% TFA in Water

[0836] Mobile Phase:

[0837] BocN: 0.05% TFA in ACN

[0838] Time (min) A% BocN%

[0839] 0.0 95 5

[0840] Gradient: 8.0 5 95

[0841] 11.0 5 95

[0842] 12.0 95 5

[0843] Flow Rate: 0.8 mL / min

[0844] Post Time 5.0 min

[0845] UV Detector Wavelength: 210 nm

[0846] Column Temperature: 40 °C

[0847] Compound 3a: 7.5 min

[0848] Retention Times:

[0849] Compound 3b: 7.7 min

[0850]

[0851] Step 2 - Synthesis of Compound 3c (tert-butyl 4-methylene-2-oxo-1,3-dioxa-8-azaspiro[4.5] decane-8-carboxylate)

[0852] I ) Agl (0.05 eq.), KOAc (0.05 eq.) <\...o

[0853] CO2(1 atm), DMSO (5 V)

[0854]

[0855] Compound 3bStep 2Compound 3cTo a reactor was charged tert-butyl 4-ethynyl-4-hydroxypiperidine-1 -carboxylate (Compound 3b) (7.3 kg, 32.40 mol, 1.0 equiv.), silver iodide (Agl, 380.4 g, 0.05 equiv.), potassium acetate (KOAc, 150.0 g, 0.05 equiv.), and DMSO (36.5 L 5 V) at 25 °C. The solution was warmed and maintained at 30-35 °C. CO2 was bubbled under surface of mixture for 112 hours at 30-35°C. A sample was taken and diluted with ACN to run HPLC analysis (criterion: area% of Compound 3b < 1%, actual result: area% of Compound 3b: 0.5%). The reaction solution was cooled to 20-25 °C. To the solution was added MTBocNE (30.7 kg) with stirring at 20-25 °C for 2 hours. The solution was filtered to remove insoluble solid and rinsed the solid with MTBocNE (12.3 kg). To the solution was charged H2O (36.5 kg) and MTBocNE (18.4 kg) at 25 °C. The aqueous phase was separated and extracted with MTBocNE (61.3 kg) twice. The combined MTBocNE phase was washed with 10 wt.% brine (73.0 kg) 3 times. The solution was concentrated under reduced pressure to afford tert-butyl 4-methylene-2-oxo-1,3-dioxa-8-azaspiro[4.5] decane-8-carboxylate (Compound 3c) as brown oil.

[0856] Step 3 - Synthesis of Compound 3d (tert-butyl 4-acetyl-4-hydroxypiperidine-1 -carboxylate)

[0857] o

[0858] -0NaOH (2.0 eq.) in H2O (4 V) Y

[0859] ACN (10 V), 25 " C, 2 h f "OH

[0860] BocNocN. J >............................................... BocNocN,.

[0861] 83.4% yield over 2 steps

[0862]

[0863] Compound 3c step 3 Compound 3d

[0864] The solution from the previous step was charged with ACN (45.9 kg) at 25 °C. The solution was charged the solution of NaOH (2.59 kg, 2.0 equiv.) in H2O (29.2 kg) dropwise at 20-25 °C. The resulting solution was maintained at 20-25 °C for 2 hours. A sample was taken and diluted with ACN to run HPLC analysis (criterion: area% of Compound 3c < 1%, actual result: area% of Compound 3c: ND). The solution was concentrated under reduced pressure to remove ACN. The resultant solution was extracted with MTBocNE (46.2 kg) twice and washed with H2O (73.0 kg). The MTBocNE phase was concentrated under reduced pressure to afford light brown oil. The oil was stood for 3 hours to precipitate the solid. The solution was charged the mixture solution of EtOAc (2.97 kg) / n-heptane (22.7 kg) dropwise with stirring at 20-25 °C. The resulting solution was maintained at 20-25°C for 24 hours and filtered. The cake was washed with n-heptane (10.0kg) and dried to afford Compound 3d as off-white solid (6.57 kg, 98.5% a / a purity, 98.6% w / w assay, 26.99 mol, 83.3% yield).

[0865] MS (ESI+) M+1= 170.0

[0866] Chemical formula: C12H21NO4

[0867] 1H NMR of Compound 3d (400 MHz, Chloroform-d) 64.11 - 4.02 (m, 2H), 3.14 (td, J= 13.0, 2.7 Hz, 2H), 2.24 (s, 3H), 1.90 (td, J= 12.9, 4.9 Hz, 2H), 1.48 (s, 9H), 1.42 (dq, J= 13.4, 2.4, 2.0 Hz, 2H).

[0868] Table 10: HPLC Method for steps 2-3 (synthesis of compounds 3c and 3d).

[0869] In-progress Control from Compound 3b to Compound 3d by HPLC

[0870] Column: Waters Xbridge C18, 4.6x150 mm, 3.5 urn

[0871] A: 0.05% TFA in Water

[0872] Mobile Phase:

[0873] BocN: 0.05% TFA in ACN

[0874]

[0875] Time (min) A% BocN%

[0876] 0.0 95 5

[0877] Gradient: 8.0 5 95

[0878] 11.0 5 95

[0879] 12.0 95 5

[0880] Flow Rate: 0.8 mL / min

[0881] Post Time 5.0 min

[0882] UV Detector Wavelength: 210 nm

[0883] Column Temperature: 40 °C

[0884] Compound 3b: 7.7 min

[0885] Retention Times: Compound 3c: 8.8 min

[0886] Compound 3d: 7.4 min

[0887]

[0888] Step 4 - Synthesis of Compound 3e (tert-butyl (S)-4-(1-aminoethyl)-4-hydroxypiperidine-1 -carboxylate)

[0889] (0.1 M TrometamolS 1.0 M IPrNHg

[0890] & 1.0 mM PI.. P mixture solution)

[0891] O EW-TA-44 (tO. O wt.%.)

[0892] H2O (19 V), DMSO (1 V)

[0893] OH 25 °C. 2 h OH

[0894]

[0895] BocNocN BocNocN

[0896] Compound 3d Step 4 Compound 3e

[0897] A reactor was charged with purified water (67.5 kg), trometamol (0.97 kg), and isopropylamine (iPrNH2, 4.75 kg) at 25 °C. The pH of the solution was adjusted to 7.8-8.0 with HCI. To this solution was cooled to 20-25 °C and charged pyridoxal phosphate (PLP, 20 g). The resultant solution was waiting for use. To a reactor was charged tert-butyl 4-acetyl-4-hydroxypiperidine-1 -carboxylate (Compound 3d) (4.0 kg, 16.44 mol, 1.0 equiv.) and DMSO (4.4 kg) at 25 °C. To the solution was charged prepared solution (76 kg) and enzyme (400 g, 10 wt.%, EW-TA-44 (EnzymeWorks, Inc.), EW-ATA-kit (EnzymeWorks, Inc.), or ES-ATA-101ES ~ ATA-166 (SyncoZymes Shanghai Co., Ltd.)). The solution was warmed and maintained at 30 °C for 380 h. Prepared solution was added for maintain the pH at 7.8-8.0. A sample was taken and diluted with ACN to run HPLC analysis (criterion: area% of Compound 3d < 5%, actual result: area% of Compound 3d: 1.7%). The resultant solution was filtered through a pad of celite to remove enzyme and rinsed with mixture solution of DMSO (21.7 kg) and H2O (0.25 kg). The filtrate was concentrated under reduced pressure to afford crude product tert-butyl (S)-4-(1-aminoethyl)-4-hydroxypiperidine-1-carboxylate (Compound 3e) as aqueous solution (71.6 kg, 98.3% a / a purity).

[0898] Steps 5 to 7 - Synthesis of Compound 4b 3 • HCI salt ((S)-3-chloro-1-(4-methyl-1-oxa-3,8-diazaspiro[4.5]decan-3-yl)propan- 1 -one trihydrochloride salt)

[0899] 3-CMoropjopipnyf chiqrsde (I f) eq.) / “Cl fflisshanat <49 M: fct. (26 V) W / 4.0MH(3fflEAI5V; 25 *C. 19 h.. J \ ACW (5 V; 45 *C, 1 Hzf'O )ZBocNod <’0.054 yield oyer 4 steps CompoutMi 3e 8 CompciirKl 3 Step 5 Step 7

[0900]

[0901] Compound 4a ComjimKKi 4b »3HCIA reactor was charged with Compound 3e aqueous solution (35.5 kg) and formalin (methanal, 26.5 kg, 40.0 equiv.) at 25 °C. The solution was warmed to 30°C and stirred for 19 hours. A sample was taken and diluted with ACN to run HPLC analysis (criterion: area% of Compound 3e < 5%, actual result: area% of Compound 3e: 0.0%). The reaction solution was filtered and the filtrate was washed with MTBocNE (16.8 kg). To the aqueous phase was charged NaHCO3(1.33 kg, 2.0 equiv.) and MTBocNE (16.8 kg). The solution was cooled to 0-10 °C. To this solution was added 3-chloropropionyl chloride (1.0 kg, 1.0 equiv.) dropwise at 0-10 °C. The solution was stirred for 0.5 hours at 0-10 °C. A sample was taken and diluted with MeOH to run HPLC analysis (criterion: area% of Compound 3 < 1%, actual result: area% of Compound 3: ND). To the reaction solution was charged MTBocNE (16.8 kg) at 25 °C. The aqueous phase was separated and extracted with MTBocNE (33.6 kg) twice. The combined MTBocNE phase was concentrated under reduced pressure to afford crude product tert-butyl (S)-3-(3-chloropropanoyl)-4-methyl-1-oxa-3,8-diazaspiro[4.5]decane-8-carboxylate (Compound 4a) as faint oil (5.15 kg, 94.8% a / a purity).

[0902] To a reactor was charged Compound 4a and ACN (19.2 kg) at 25 °C. The solution was cooled and maintained at 0-10 °C. To this solution was added 4.0 M HCI in EtOAc (24.5 L) dropwise in 3 hours at 0-10 °C. The solution was warmed to 15 °C and stirred for 1 hour. A sample was taken and diluted with ACN to run HPLC analysis (criterion: area% of Compound 4a < 1%, actual result: area% of Compound 4a: ND). The reaction solution was filtered. The filter cake was washed with EtOAc (4.5 kg) and dried to afford Compound 4b 3 • HCI salt as off-white solid (4.1 kg, 99.6% a / a purity, 68.9% w / w assay based on free, 11.51 mol, 70.0% yield).

[0903] MS (ESI+) M+1= 246.9

[0904] Chemical formula: C11H22Cl4N2O2

[0905] 1H NMR of Compound 4b (400 MHz, DMSO-cfe) 65.09 - 4.85 (m, 2H), 4.07 - 3.85 (m, 1 H), 3.80 (dt, J = 12.9, 6.4 Hz, 2H), 3.13 (dqd, J = 12.2, 7.2, 5.0, 3.4 Hz, 2H), 3.02 - 2.86 (m, 3H), 2.83 - 2.57 (m, 2H), 2.00 - 1.75 (m, 4H), 1.06 (dd, J = 15.9, 6.5 Hz, 3H).

[0906] Table 11: HPLC Method for step 4 and steps 6-7.

[0907] In-progress Control from Compound 3d to Compound 3e and Compound 3 to Compound 4b by HPLC Column: Waters Xbridge C18, 4.6x150 mm, 3.5 urn

[0908] A: 0.05% TFA in Water

[0909] Mobile Phase:

[0910] BocN: 0.05% TFA in ACN

[0911] Time (min) A% BocN%

[0912] 0.0 95 5

[0913] Gradient: 8.0 5 95

[0914] 11.0 5 95

[0915] 12.0 95 5

[0916] Flow Rate: 0.8 mL / min

[0917] Post Time 5.0 min

[0918] UV Detector Wavelength: 210 nm

[0919] Column Temperature: 40 °C

[0920]

[0921] Compound 3d: 7.4 min

[0922] Compound 3: 5.7 min

[0923] Retention Times:

[0924] Compound 4a: 8.0 min

[0925] Compound 4b: 4.8 min

[0926]

[0927] Table 12: HPLC Method for step 5.

[0928] In-progress Control from Compound 3e to Compound 3 by HPLC

[0929] Column: Waters Xbridge C18, 4.6x150 mm, 3.5 urn A: 10 mM NH4OAc in Water

[0930] Mobile Phase:

[0931] BocN: ACN

[0932] Time (min) A% BocN%

[0933] 0.0 65 35

[0934] Gradient: 3.0 65 35

[0935] 10.0 5 95

[0936] 13.0 95 5

[0937] Flow Rate: 1.0 mL / min

[0938] Post Time 5.0 min

[0939] UV Detector Wavelength: 210 nm

[0940] Column Temperature: 40 °C

[0941] Compound 3e: 1.9 min

[0942] Retention Times:

[0943] Compound 3: 4.4 min

[0944]

[0945] Step 8 - Synthesis of Compound 4d (methyl N-((S)-3-(3-chloropropanoyl)-4-methyl-1-oxa-3, 8-diazaspiro[4.5]decane-8-carbonyl)-N-methyl-L-valinate)

[0946] o r~ Cl O! V- / V / "a'TN>

[0947] A ° i rf-tf' ^T'O^ Compound 4c HCI -QN N

[0948]

[0949] BocNTC (0.33 eq.), pyridine {3.0 eq.) •'

[0950] DIEA (7.0 eq,), DCM (10 v)

[0951] Compound 4b •3HCl 0-10 " C,16h Compound 4

[0952] 95.0% yield

[0953] Step8

[0954] To a reactor was charged methyl methyl-L-valinate hydrochloride (Compound 4c HCI) (1.53 kg, 8.42 mol, 1.0 equiv.) and DCM (3.98 kg) at 25 °C. To this solution was added (bis(trichloromethyl) carbonate (BocNTC, 0.83 kg, 0.33 equiv.) in one portion. The solution was cooled and maintained at 0-5 °C. To this mixture was add pyridine (2.0 kg, 3.0 equiv.) dropwise in 2 hours at 0-5 °C. The solution was stirred for 1 hour at 0-5 °C and Compound 4b •3HCl (3000 g, 1.0 eq.) was added. To this solution was added N, N-diisopropylethylamine (DIEA, 2.0 kg, 3.0 equiv.) dropwise in 3 hours and then stirred for 1 hour at 0-5 °C. A sample was taken and diluted with ACN to run HPLC analysis (criterion: area% of Compound 4b < 3%, actual result: area% of Compound 4b: 2.3%).To the reaction solution was charged 10 wt.% citric acid aqueous (30.0 kg) dropwise at 0-5 °C. The solution was stirred for 0.5 hour. The DCM phase was separated and washed with 10 wt.% citric acid aqueous (30.0 kg) and then H2O (30.0 kg). The DCM phase was concentrated under reduced pressure to afford Compound 4d as red oil (3.8 kg, 98.5% a / a purity, 88.5% w / w assay, 8.00 mol, 95.0% yield).

[0955] MS (ESI+) M+1= 418.0

[0956] Chemical formula: C19H32ClN3O5

[0957] 1H NMR of Compound 4d (400 MHz, DMSO-cfe) 6 5.00 (dd, J = 34.1, 3.6 Hz, 1 H), 4.93 - 4.84 (m, 1 H), 3.99 - 3.87 (m, 1 H), 3.86 - 3.77 (m, 3H), 3.34 - 3.25 (m, 2H), 3.03 (dddd, J = 26.4, 12.9, 7.3, 3.2 Hz, 2H), 2.87 (s, 3H), 2.83 - 2.55 (m, 2H), 2.11 (dp, J = 10.1, 6.6 Hz, 1 H), 1.70 - 1.52 (m, 4H), 1.07 (dd, J = 18.3, 6.5 Hz, 3H), 0.89 (dd, J= 13.0, 6.6 Hz, 6H).

[0958] Table 13: HPLC Method for step 8.

[0959] In-progress Control from Compound 4b to Compound 4d by HPLC

[0960] Column: Waters Xbridge C18, 4.6x150 mm, 3.5 urn

[0961] A: 0.05% TFA in Water

[0962] Mobile Phase:

[0963] BocN: 0.05% TFA in ACN

[0964] Time (min) A% BocN%

[0965] 0.0 95 5

[0966] Gradient: 8.0 5 95

[0967] 11.0 5 95

[0968] 12.0 95 5

[0969] Flow Rate: 0.8 mL / min

[0970] Post Time 5.0 min

[0971] UV Detector Wavelength: 210 nm

[0972] Column Temperature: 40 °C

[0973] Compound 4b: 4.8 min

[0974] Retention Times:

[0975] Compound 4d: 8.0 min

[0976]

[0977] Step 9 - Synthesis of Compound 4 (N-((S)-3-acryloyl-4-methyl-1-oxa-3,8-diazaspiro[4.5]decane-8-carbonyl)-N-methyl-L-valine)

[0978] '■(W AW

[0979] 1 I Lil (50 eq } DIEA (30 eq } » 1 1 'K*'

[0980] X o60-40 h_ >. A o

[0981] 72.8% yield

[0982] Step 9

[0983]

[0984] Compound 4d Compound 4

[0985] To a reactor was charged methyl N-((S)-3-(3-chloropropanoyl)-4-methyl-1-oxa-3,8-diazaspiro[4.5]decane-8-carbonyl)-N-methyl-L-valinate (Compound 4d) (2.92 kg, 6.99 mol, 1.0 equiv.) and2-Me THF (28.0 kg) at 25 °C. To the mixture was added DIEA (3.04 kg, 3.0 eq.) dropwise at 20-25°C. The solution was cooled and maintained at 0-5 °C. To this solution was added lithium iodide (Lil, 5.2 kg, 5.0 equiv.) dropwise below 40°C. The solution was stirred for 15 min and then heated to 60°C. A sample was taken and diluted with ACN to run HPLC analysis (criterion: area% of Compound 4d < 1%, actual result: area% of Compound 4d: 0.6%). The reaction solution was filtered to remove the salt. The filtrate was concentrated under reduced pressure until the residue volume was around 15 L. To the solution was charged MTBocNE (12.6 kg) and concentrated under reduced pressure until the residue volume was around 15 L. The solvent swap was repeated for another 2 times. The solution was slurred for 15 hours and filtered. The cake was re-slurred with MTBocNE (12.6 kg) for 5 hours and filtered. Dried the cake to afford Compound 4 as brown solid (5.23 kg, 96.5% a / a purity, 35.6% w / w assay, 5.09 mol, 72.8% yield).

[0986] MS (ESI+) M+1= 368.3

[0987] 1H NMR of Compound 4 (400 MHz, Methanol-*) 66.43 - 6.25 (m, 1H), 5.81 (ddd, J = 14.9, 9.8, 2.4 Hz, 1 H), 5.25 - 5.11 (m, 1 H), 5.07 (q, J = 6.1 Hz, 1 H), 3.97 (dq, J = 7.9, 6.1 Hz, 1 H), 3.88 (td, J = 7.7, 6.2 Hz, 1H), 3.80 - 3.67 (m, 2H), 2.95 (s, 3H), 2.18 (ddd, J = 13.0, 10.5, 6.5 Hz, 1H), 2.09 - 1.99 (m, 1H), 1.99 -1.86 (m, 2H), 1.85 - 1.64 (m, 4H), 1.37 (dd, J = 6.9, 2.7 Hz, 3H), 0.94 (dd, J = 52.8, 6.6 Hz, 6H).

[0988] Table 14: HPLC Method for step 9.

[0989] In-progress Control from Compound 4d to Compound 4 by HPLC

[0990] Column: Waters Xbridge C18, 4.6x150 mm, 3.5 urn

[0991] A: 10 mM NH4OAc in Water

[0992] Mobile Phase:

[0993] BocN: ACN

[0994] Time (min) A% BocN%

[0995] 0.0 95 5

[0996] Gradient: 8.0 5 95

[0997] 11.0 5 95

[0998] 12.0 95 5

[0999] Flow Rate: 0.8 mL / min

[1000] Post Time 5.0 min

[1001] UV Detector Wavelength: 210 nm

[1002] Column Temperature: 40 °C

[1003] Compound 4d: 8.0 min

[1004] Retention Times:

[1005] Compound 4: 5.1 min

[1006]

[1007] Table 15: Purity Determination of Compound 4 by HPLC.

[1008] Column: Ascentis Express C18, 4.6x150mm, 2.7um

[1009] A: 10 mM NH4OAc in Water

[1010] Mobile Phase:

[1011] BocN: ACN

[1012] Gradient: Time (min) A% BocN%

[1013]

[1014] 0.0 90 10

[1015] 10.0 75 25

[1016] 15.0 5 95

[1017] 17.0 5 95

[1018] 18.0 90 10

[1019] Flow Rate: 0.8 mL / min

[1020] Post Time 5.0 min

[1021] UV Detector Wavelength: 210 nm

[1022] Column Temperature: 25 °C

[1023] Retention Times: Compound 4: 8.1 min

[1024]

[1025] Example 3. Synthetic Procedure for Compound 5b (((S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(5-(3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1 -methoxyethyl)pyridin-3-yl)-1 -(2,2,2-trifluoroethyl)-1H-indol-5-yl)-3,6-dihydropyridin-1(2H)-yl)propanoyl)hexahydropyridazine-3-carboxylic acid)

[1026] Detailed below is a general synthetic procedure for Compound 5b.

[1027] Synthesis of Compound 5b (((S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(5-(3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1-(2, 2, 2-trifluoroethyl)-1H-indol-5-yl)-3, 6-dihydropyridin-1(2H)-yl)propanoyl)hexahydropyridazine-3-carboxylic acid):

[1028]

[1029] Compound 5a Compound 2 Compound 5b

[1030] To a reactor was charged EtOH (6.0 L, 10.0V), H2O (1.2 L,2.0V), K2COs (412 g, 2.5equiv.), (S)-3-(5-bromo-2-(2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-3-yl)-2,2-dimethylpropan-1-ol (Compound 5a) (600 g, 1.0 equiv.), and (S)-1-((S)-3-(5-borono-3,6-dihydropyridin-1(2H)-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylic acid (Compound 2) (910 g, 1.2equiv) at 25 °C. N2 gas was bubbled under surface of the mixture for 1 hour at 25°C. To this mixture was added XPhos Pd G3 (20.3g, 0.02 equiv). N2 gas was bubbled under surface of mixture for 0.5 hour at 25°C, after which this mixture was heated and maintained at 80±5°C. The solution was stirred at 80±5°C for 2 hours. A sample was taken and diluted with MeOH to run HPLC analysis (criterion: area% of Compound 5a < 1%). The solution was cooled to 20-25°C, water (4.8 L, 8.0V), and DCM (6.0L, 10V) was charged to the reaction mixture. The 8 batches (600gx8) were combined. The DCM phase was separated and washed with 5% Na2CO3 (twice. (10V x2). To the DCM phase was added H2O (4.2 V), MeOH (2.1V) and aqueous 0.5mol / L HCI to adjust to pH 5. The DCM phase was separated and swapped with 2-Me-THF (2.0Vx5). To this mixture was added H2O (5.0 V),2- Me-THF (10V), and 0.5mol / L HCl.aq. to adjust to pH 2. The aqueous phase was separated and added DCM (5.0 V), MeOH (1.0 V), and 1.0 mol / L NaOH.aq. to adjust to pH 5. The DCM phase was separated and swapped with n-Heptane (2.0Vx5). To this mixture wasadded n-Heptane (5.0 V) and stirred at 15-25°C for not less than 5 hours and filtered. The cake was washed with n-Heptane (1.0 V). The wet cake was dried under vacuum to afford Compound 5b as off-white solid. A sample was taken for GC analysis to check residue MeOH and EtOH (criterion: content of MeOH < 200ppm; content of EtOH < 200ppm, result: content of MeOH =132 ppm; content of EtOH =20 ppm). Compound 5b was used in next step directly (7.48 kg, 93.5% HPLC purity, 84.5% w / w assay, 82.0% yield).

[1031] MS (ESI+) M+1=801.5

[1032] 1H NMR of Compound 5b (400 MHz, DMSO-cfe) 6 8.76 (dd, J = 4.8, 1.7 Hz, 1 H), 7.82 - 7.73 (m, 2H), 7.64 (d, J = 8.6 Hz, 1 H), 7.54 (dd, J = 7.8, 4.7 Hz, 1 H), 7.37 - 7.29 (m, 1 H), 6.71 (s, 1 H), 6.22 (s, 1 H), 5.33 (dq, J = 17.1, 8.8, 8.1 Hz, 2H), 5.16 (d, J = 9.7 Hz, 1 H), 4.48 (dq, J = 16.2, 9.4 Hz, 2H), 4.04 (s, 1H), 3.99 (q, J = 6.2 Hz, 2H), 3.55 (t, J = 8.3 Hz, 1 H), 3.05 (s, 3H), 2.97 (s, 4H), 2.61 (d, J = 14.0 Hz, 1 H), 2.46 (s, 2H), 2.33 (d, J = 14.0 Hz, 1H), 2.08 (s, 2H), 1.93 (d, J = 11.0 Hz, 1H), 1.81 - 1.69 (m, 1H), 1.66 - 1.47 (m, 2H), 1.42 - 1.35 (m, 12H), 0.65 (s, 6H).

[1033] Table 16: HPLC Method for step 10.

[1034] In-progress Control from Compound 5a to Compound 5b by HPLC

[1035] Column: Waters Xbridge C18, 4.6x150 mm, 3.5 urn

[1036] A: 0.05% TFA in Water

[1037] Mobile Phase:

[1038] BocN: 0.05% TFA in ACN

[1039] Time (min) A% BocN%

[1040] 0.0 95 5

[1041] Gradient: 8.0 5 95

[1042] 11.0 5 95

[1043] 12.0 95 5

[1044] Flow Rate: 0.8 mL / min

[1045] Post Time 5.0 min

[1046] UV Detector Wavelength: 210 nm

[1047] Column Temperature: 40 °C

[1048] Compound 2: 6.0 min

[1049] Retention Times: Compound 5b: 7.7 min

[1050] Compound 5a: 9.5 min

[1051]

[1052] Example 4. Synthetic Procedure for Compound 5c (tert-butyl((63S,4S)-12-(2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-11-(2,2,2-trifluoroethyl)-21,22,23,26,61,62,63,64,65,66-decahydro-11H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(5,1)-pyridinacycloundecaphane-4-yl)carbamate)

[1053] Detailed below is a general synthetic procedure for Compound 5c.

[1054] Synthesis of Compound 5c (tert-butyl((63S,4S)-12-(2-((S)-1-methoxyethyl)pyridin-3-yl)-10, 10-dimethyl-5, 7-dioxo- 11-(2, 2, 2-trifluoroethyl)-21, 22, 23, 26, 61, &, 63, 64, 65, & -decahydro- 11H-8-oxa-1(5, 3)-indola-6(1, 3)-pyridazina-2(5,1)-pyridinacycloundecaphane-4-yl)carbamate):

[1055]

[1056] Compound 5b Compound 5c To a reactor was charged DCM (25.0 V), HOBocNT(2.0 eq.), EDCI (3.0 eq.), and DIPEA (2.0 eq.). The solution was warmed and maintained at 35~38°C. To this mixture was added ((S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(5-(3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1 H-indol-5-yl)-3,6-dihydropyridin-1(2H)-yl)propanoyl)hexahydropyridazine-3-carboxylic acid (Compound 5b) (600.0g, dissolved in DCM(5.0V),1.0 equiv.) at 35~38°C in 6 hours. The resulting solution was maintained at 35 ~38°C for 2 hours. A sample was taken and diluted with ACN to run HPLC analysis (criterion: area% of Compound 5b < 1.0%). The reaction mixture was concentrated under reduced pressure to 10 V. To this mixture was added H2O (5.0V) and aq. 3mol / L HCI to adjust to pH 2. The DCM phase was separated, and H2O (5.0V) and 0.5mol / L NaOH.aq. were added to adjust to pH 7. The DCM phase was separated and washed with water (5.0V). The DCM phase was separated and swapped with IPA (2.0Vx3). The suspension was stirred at 20-25°C for no longer than 16 hours and filtered. The cake was washed with IPA (1.0 V). The wet cake was dried under vacuum at 45°C for 24 hours. Compound 5c was obtained as a off-white solid (4.4 kg, 96.7% HPLC purity, 81.7% HPLC assay, 82.7% QNMR assay, residue IPA:13.5% w / w, 61.7% yield).

[1057] MS (ESI+) M+1 =783.5

[1058] 1H NMR of Compound 5c (400 MHz, DMSO-c / e) 6 8.79 (dd, J = 4.8, 1.7 Hz, 1 H), 7.81 (d, J = 7.8 Hz, 1 H), 7.66 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.55 (dd, J = 7.8, 4.7 Hz, 1H), 7.45 (s, 1H), 6.86 (d, J = 8.7 Hz, 1H), 6.33 (s, 1H), 5.44 (d, J = 12.1 Hz, 1H), 5.34 (dt, J = 27.8, 9.0 Hz, 2H), 4.55 (s, 1H), 4.32 (d, J = 13.0 Hz, 1H), 4.08 (d, J = 5.6 Hz, 1H), 3.89 - 3.77 (m, 2H), 3.68 (d, J = 10.6 Hz, 1H), 3.60 (d, J = 11.0 Hz, 1H), 3.17 (d, J = 15.5 Hz, 1H), 3.01 (s, 3H), 2.95 - 2.83 (m, 2H), 2.85 - 2.72 (m, 2H), 2.62 (d, J = 13.2 Hz, 1 H), 2.35 (d, J = 20.2 Hz, 2H), 2.24 (d, J = 17.0 Hz, 1 H), 1.98 (s, 1 H), 1.82 (d, J = 11.8 Hz, 1 H), 1.69 - 1.48 (m, 2H), 1.39 (d, J = 6.1 Hz, 3H), 1.37 (s, 9H), 0.71 (s, 3H), 0.67 (s, 3H).Example 5. Synthetic Procedure for Compound 5d ((63S,4S)-4-amino-12-(2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-11-(2,2,2-trifluoroethyl)-21,22,23,26,61,62,63,64,65,66-decahydro-11H-8-oxa-1 (5, 3)-indola-6(1,3)-pyridazina-2(5,1)-pyridinacycloundecaphane-5, 7-dione) Detailed below is a general synthetic procedure for Compound 5d.

[1059] Synthesis of Compound 5d ((63S,4S)-4-amino-12-(2-((S)-1-methoxyethyl)pyridin-3-yl)-10, 10-dimethyl-11-(2, 2, 2-trifluoroethyl)-21,22, 23, 26,61, 62, 63, 64, 65, 66-decahydro- 11H-8-oxa-1(5, 3)-indola-6( 1,3)-pyridazina-2(5, 1)-pyridinacycloundecaphane-5, 7-dione):

[1060]

[1061] Compound 5c Step 3 Compound 6d

[1062] To a reactor was charged 1,4-dioxane (10.0 V) and tert-butyl((63S,4S)-12-(2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-11-(2,2,2-trifluoroethyl)-21,22,23,26,61,62,63,64,65,66-decahydro-11H-8-oxa-1 (5, 3)-indola-6(1,3)-pyridazina-2(5,1)-pyridinacycloundecaphane-4-yl)carbamate (Compound 5c, 1.9 kg, 1.0 equiv.) at 20~30°C. To this mixture was added HCI in 1,4-dioxane (4M, 10.0V). The reaction mixture was stirred at 20~30 °C for 16 hours. A sample was taken and diluted with ACN and H2O to run HPLC analysis (criterion: area% of Compound 5c < 1%). The reaction mixture was filtered and washed with 1,4-dioxane (5.0Vx2). To a reactor was charged the wet cake (Compound 5d HCI salt), H2O (10.0V), MTBocNE (10.0 V), and 1.0 mol / L NaOH.aq. to adjust to pH 8~9. The MTBocNE phase was separated and washed with water (5.0Vx2). Collected MTBocNE phase and concentrated to dryness. Dried to afford Compound 5d as off-white solid (1.53 kg, 98.1% HPLC purity, 93.7% HPLC assay, 88.3% QNMR assay, residue MTBocNE:45939 ppm; n-Heptane:26333 ppm; 1,4-dioxane:33689 ppm, 98.0% yield corrected by QNMR).

[1063] MS (ESI+) M+1 =683.4

[1064] 1H NMR (400 MHz, DMSO-cfe) 68.78 (dd, J = 4.8, 1.7 Hz, 1 H), 7.80 (d, J = 7.8 Hz, 1 H), 7.65 (d, J = 8.8 Hz, 1H), 7.60 - 7.51 (m, 2H), 7.47 (s, 1H), 6.29 (s, 1H), 5.37 (dq, J = 17.3, 8.6 Hz, 1H), 5.25 (d, J = 12.1 Hz, 1 H), 4.64 - 4.49 (m, 3H), 4.35 (d, J = 12.7 Hz, 1 H), 4.07 (q, J = 6.1 Hz, 1 H), 3.88 - 3.70 (m, 2H), 3.72 - 3.60 (m, 3H), 3.13 (d, J = 15.6 Hz, 1H), 3.02 (s, 3H), 2.81 (tt, J = 14.3, 11.8, 8.6 Hz, 4H), 2.70 - 2.60 (m, 1H), 2.38 (s, 1H), 2.24 (d, J = 17.4 Hz, 1H), 2.00 (d, J = 11.1 Hz, 1 H), 1.89 - 1.77 (m, 1H), 1.71 - 1.47 (m, 2H), 1.39 (d, J = 6.1 Hz, 3H), 0.68 (s, 3H), 0.63 (s, 3H).Table 16: HPLC Method for step 12.

[1065] In-progress Control from Compound 5c to Compound 5d by HPLC

[1066] Column: Waters Xbridge C18, 4.6x150 mm, 3.5 urn

[1067] A: 0.05% TFA in Water

[1068] Mobile Phase:

[1069] BocN: 0.05% TFA in ACN

[1070] Time (min) A% BocN%

[1071] 0.0 70 30

[1072] Gradient: 13.0 5 95

[1073] 15.0 5 95

[1074] 16.0 70 30

[1075] Flow Rate: 0.8 mL / min

[1076] Post Time 5.0 mins

[1077] UV Detector Wavelength: 254 nm

[1078] Column Temperature: 40 °C

[1079] Compound 5d: 6.1 min

[1080] Retention Times:

[1081] Compound 5c: 8.4 min

[1082]

[1083] Example 6. Synthetic Procedure for Compound A ((4S)-3-acryloyl-N-((2S)-1-(((63S,4S)-12-(2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-11 -(2,2,2-trifluoroethyl)- 21,22,23,26,61,62,63,64,65,66-decahydro-11 H-8-oxa-1 (5,3)-indola-6(1,3)-pyridazina-2(5,1 )-pyridinacycloundecaphane-4-yl)amino)-3-methyl-1 -oxobutan-2-yl)-N,4-dimethyl-1 -oxa-3,8-diazaspiro[4.5]decane-8-carboxamide)

[1084] Detailed below is a general synthetic procedure for Compound A.

[1085] Synthesis of Compound A ((4S)-3-acryloyl-N-((2S)-1-(((63S,4S)-12-(2-((S)-1-methoxyethyl)pyridin-3-yl)- 10, 10-dimethyl-5, 7-dioxo-11-(2,2,2-trifluoroethyl)-21,22,23,26, 61, 62, 63, 64, 65, 66-decahydro-11 H-8-oxa- 1 (5,3)-indola-6(1,3)-pyridazina-2(5,1 )-pyridinacycloundecaphane-4-yl)amino)-3-methyl-1 -oxobutan-2-yl)- N, 4-dimethyl-1-oxa-3, 8-diazaspiro[4.5]decane-8-carboxamide):

[1086] CompoiirKi 4 PyBocNOP{1.2eq. J Oxymafi.tteq.) DSEA(2.0eq.) OMACfiSV)

[1087]

[1088] O-XC'^C 1 'it! To a reactorwas charged dimethylacetamide (DMAc, 7.03kg), (63S,4S)-4-amino-12-(2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-11 -(2,2,2-trifluo roethy l)-21,22,23,26,61,62,63,64,65,66-decahydro-11 H-8-oxa-1 (5,3)-indola-6(1,3)-pyridazina-2(5,1)-pyridinacycloundecaphane-5, 7-dione (Compound 5d, 500g, 87.2% w / w assay, 0.64mol, 1.0eq), N-((S)-3-acryloyl-4-methyl-1-oxa-3,8-diazaspiro[4.5]decane-8-carbonyl)-N-methyl-L-valine (Compound 4, 670g, 35% w / w assay, 0.64mol,1. Oeq), and Oxyma (9.1g, 0.64mol, 1. Oeq) at 25°C. The solution was cooled to 0~5°C under N2. To the mixture was charged PyBocNOP (399g, 0.77mol, 1,2eq) at 0~5°C portion-wise. The mixture was stirred at 25°C for 19 hours. A sample was taken and diluted with ACN to run HPLC analysis (result: area% of Compound 5d=0.1%). A sample was taken to check assay of the reaction mixture (5.2%w / w assay, 177.5g Compound A in Reaction solution, in-situ 74.8% yield). The reaction mixture was poured into water (45V) at 25°C, solid precipitated out immediately. The reaction mixture was stirred at 25°C for 1hour, filtered and washed the cake with water to give Compound A as a light-brown wet cake. The aq. mother liquor was extracted with EtOAc (20V) once. A sample of aq. mother liquor was taken for HPLC analysis to check residue Compound A (HPLC showed no Compound A remained in aq. solution after EtOAc extraction). The resultant EtOAc solution was used to dissolve the wet cake of Compound A (solid 1) and washed the EtOAc solution with water (10V) and brine (10V). The EtOAc solution was concentrated under reduced pressure to dryness and afford Compound A as a brown solid (Crude product: 666g, 93.4% a / a purity, 72.5%w / w assay, 70.9% yield)

[1089] MS (ESI+) M+1 =1032.7

[1090] Table 17: HPLC Method for step 13.

[1091] In-progress Control from Compound 5d to Compound A by HPLC

[1092] Column: Waters Xbridge C18, 4.6x150 mm, 3.5 urn

[1093] A: 10mM NH4OAC in Water

[1094] Mobile Phase:

[1095] BocN: ACN

[1096] Time (min) A% BocN%

[1097] 0.0 85 15

[1098] 3.0 60 40

[1099] Gradient: 18.0 5 95

[1100] 21.0 5 95

[1101] 22.0 85 15

[1102] 27.0 85 15

[1103] Flow Rate: 0.8 mL / min

[1104] UV Detector Wavelength: 224 nm and 250 nm

[1105] Column Temperature: 45 °C

[1106] Compound 4: 4.3 min

[1107] Retention Times: Compound 5d: 10.7 min

[1108] Compound A: 13.7 min

[1109]

[1110] Example 7. Recrystallization of (4S)-3-acryloyl-N-((2S)-1-(((63S,4S)-12-(2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-11 -(2,2,2-trifluoroethyl)-21,22,23,26,61,62,63,64,65,66-decahydro-11 H-8-oxa-1 (5,3)-indola-6(1,3)-pyridazina-2(5,1 )-pyridinacycloundecaphane-4-yl)amino)-3-methyl-1 -oxobutan-2-yl)-N,4-dimethyl-1 -oxa-3,8-diazaspiro[4.5]decane-8-carboxamide (Compound A):

[1111] o i 1} EtOAc(2). n-Hep(9.4V}, 40*C. Cooled to 25“C.added ssedfl JHMW) ~"x2} $opwfce<i n-Hep(G.2V} at 25°C trs lOboto and afcred tor 18houts / 3} F-ltsTsscf and charged n'Hep: EtOAc=2:3 (2V) 4} Filtered and dry under vacuum al W'C for iSlsouss 5) MEK (1.5V\ MTBocNE {3V)at Caofod to 28*0. added seed) 6} MTBocNE (7V) dropwtee in Idhouts at20*C 7} Httrsred and charged H2O (lOV) at 38’C

[1112] Recrystallization

[1113]

[1114] Compound A was purified by recrystallization according to the following procedure. To a reactor was Charged EtOAc (1448 mL, 2V) and crude (4S)-3-acryloyl-N-((2S)-1-(((63S,4S)-12-(2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-11 -(2,2,2-trifluoroethyl)- 21,22,23,26,61,62,63,64,65,66-decahydro-11 H-8-oxa-1 (5,3)-indola-6(1,3)-pyridazina-2(5,1)-pyridinacycloundecaphane-4-yl)amino)-3-methyl-1-oxobutan-2-yl)-N,4-dimethyl-1-oxa-3,8-diazaspiro[4.5]decane-8-carboxamide (Compound A, 1000g, 72.4%w / w assay, 0.70mol, 1.0eq.) into reactor at 40°C. (clear solution was obtained). To this mixture was added n-heptane (290 mL, 0.4V) dropwise in 1 hour at 40°C. The mixture was stirred for 30 mins at 40°C; a clear solution was obtained. The mixture was cooled to 25°C and stirred for 15 mins; a clear solution was obtained. The mixture was charged with a seed of Compound A (8.8 g, 82.0%w / w assay, 1% w / w). The mixture was stirred for 30 mins (seed retained). The mixture was stirred at 25 °C for 18 hours, during which solid precipitated out. The suspension was charged with n-heptane (145 mL, 0.2V) dropwise at 25°C in 10 hours and stirred for 18 hours. A sample of cake (94.5% a / a purity) and mother liquor (82.7% a / a purity, Compound A 160.1 mg / mL, 301,4g Compound A dissolved in mother liquor) was taken. The suspension was filtered and afforded Compound A as a light yellow wet solid ~796g. The solid was dissolved in 2:3 n-Hep: EtOAc (1,6L, 2V). The suspension was stirred for 30 mins at 30°C. The suspension was filtered and afforded Compound A as a light yellow wet solid (95.1% a / a purity) with mother liquor (86.9% a / a purity, Compound A 40.3mg / mL, 64.5g Compound A dissolved in mother liquor). The cake was dried under vacuum at 40°C for 19 hours to afford Compound A as a light yellow solid 500.1g (95.5% a / a purity, 80.7% w / w assay, 0.39mol).To a reactor was charged MEK (605mL,1,5V) and Compound A (500.1g, 80.7% w / w assay, 0.39mol, 1. Oeq) at 40°C. The mixture was stirred for 30min at 40°C; a clear solution was obtained. To the mixture was charged MTBocNE (1210mL,3V) dropwise and stirred for 30min at 40°C; a clear solution was obtained. The mixture was cooled to 20°C and stirred for 30 min; a clear solution was obtained. To the mixture was charged seed (5.0g, 96.4% a / a purity, 82.0% w / w assay, 1 % w / w) and stirred for 30 mins at 20°C (seed retained). The suspension was charged with MTBocNE (2825mL, 7V) dropwise in 14 hours at 20°C. The suspension was stirred for 30 hours at 20°C during which solid precipitated out. The suspension was filtered and afforded Compound A as a light yellow wet solid. The cake was dried under vacuum at 40 °C for 19 hours to afford Compound A as a light yellow solid (437.7g, 96.4% a / a purity, 86.8% w / w assay, 0.37mol).

[1115] To a reactor was charged H2O (4.4L, 10V) and Compound A (437.7g 96.4% a / a purity, 86.8% w / w assay, 0.37mol). The suspension was stirred for 19 hours at 30°C. A sample was taken and filtered to give a solid and was dried under vacuum at 40°C for 5 hours (solvent residue: MTBocNE 10300.24ppm, MEK 367.01 ppm, EtOAc 123.11 ppm, n-Hep 269.52ppm). The suspension was filtered and afforded Compound A as an off-white wet solid.

[1116] To a reactor was charged H2O (4.4L, 10V) and the Compound A wet solid. The suspension was stirred for 19h at 30°C. A sample was taken and filtered to give solid and dried under vacuum at 40°C for 5 hours (solvent residue: MTBocNE 7931,97ppm, MEK 206.74ppm, EtOAc 58.65ppm, n-Hep 280.64ppm). The suspension was filtered and afford Compound A as an off-white wet solid. The solid was dried under vacuum at 40°C 19 h and the solid was still wet (~590g, 96.6% a / a purity).

[1117] The Compound A wet solid was dried under vacuum at 40°C for 4 days and afforded purified Compound A as an off-white solid (415g, 96.6% a / a purity, M-2: 0.33% a / a purity, 91.2%, w / w assay, KF=0.78%, 0.37mol, 52.3% yield, solvent residue: MTBocNE 3775 ppm, EtOAc 119ppm, n-Hep 77ppm).

[1118] MS (ESI+) M+1 =1032.7

[1119] 1H NMR of recrystallized Compound A (400 MHz, DMSO-cfe) 68.78 (dd, J = 4.8, 1.7 Hz, 1 H), 7.98 (s, 1 H), 7.80 (d, J = 11 Hz, 1 H), 7.67 (d, J = 8.8 Hz, 1 H), 7.58 (d, J = 8.9 Hz, 1 H), 7.54 (dd, J = 7.7, 4.7 Hz, 2H), 7.45 (s, 1 H), 6.61 (dd, J = 16.7, 10.4 Hz, 1 H), 6.33 - 6.15 (m, 2H), 5.80 - 5.69 (m, 2H), 5.42 (dd, J = 21.9, 10.0 Hz, 2H), 5.11 (dd, J = 38.6, 3.8 Hz, 1H), 5.03 - 4.93 (m, 1 H), 4.61 (s, 1H), 4.28 (d, J = 11.6 Hz, 1H), 4.09 (dq, J = 10.1, 6.2, 5.7 Hz, 2H), 4.00 - 3.74 (m, 4H), 3.66 (d, J = 12.4 Hz, 2H), 3.22 - 2.98 (m, 6H), 2.76 (d, J = 18.3 Hz, 6H), 2.61 (s, 1H), 2.26 (s, 2H), 2.16 - 2.03 (m, 1H), 2.00 (d, J = 11.1 Hz, 1H), 1.82 (d, J = 11.5 Hz, 1 H), 1.65 (tt, J = 36.0, 32.2, 12.8 Hz, 7H), 1.39 (d, J = 6.1 Hz, 3H), 1.13 - 1.07 (m, 3H), 0.87 (d, J = 6.1 Hz, 3H), 0.81 (d, J = 6.6 Hz, 3H), 0.73 (s, 3H), 0.59 (s, 3H).Table 18: Purity Determination of Compound A by HPLC.

[1120] Purity Determination of Compound A by HPLC

[1121] Column: Eclipse plus C18, 3.0x150mm, 3.5um

[1122] A: 10mM NH4OAC in Water

[1123] Mobile Phase:

[1124] BocN: ACN

[1125] Time (min) A% BocN%

[1126] 0.0 85 15

[1127] 3.0 55 45

[1128] 18.0 45 55

[1129] Gradient:

[1130] 27.0 5 95

[1131] 33.0 5 95

[1132] 34.0 85 15

[1133] 39.0 85 15

[1134] Flow Rate: 1.0 mL / min

[1135] UV Detector Wavelength: 250 nm

[1136] Column Temperature: 45 °C

[1137] Retention Times: Compound A: 17.2 min

[1138]

[1139] Other Embodiments

[1140] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features set forth herein.

[1141] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

CLAIMS1. A method of preparing a compound of Formula 1:HO. UDr^NH-PGaFormula 1the method comprising:a) reacting a compound of Formula 1 a with an alkylating agent to form a compound of Formula 1b:Formula 1a Formula 1bb) reducing the compound of Formula 1 b to form a compound of Formula 1 c HCI salt:Formula 1b Formula 1c HCI salt.c) converting the compound of Formula 1c HCI salt to form a compound of Formula 1d HCI salt:X NH HCIFormula 1c HCI salt Formula 1d HCI salt.an(j d) coupling the compound of Formula 1 d HCI salt with a compound of Formula 1 e to form the compound of Formula 1:HN— PGaFormula 1d HCI salt Formula 1eFormula 1 wherein:X is Cl, BocNr, OTf, OMs, OTs, OAc, or OBocNz; andPGais a nitrogen protecting group.

2. The method of claim 1, wherein the reducing (b) comprises contacting the compound of Formula 1 b with a borohydride reagent.

3. The method of claim 1 or 2, wherein the converting (c) comprises contacting the compound of Formula 1c HCI salt with a N-dealkylating agent.

4. The method of any one of claims 1 to 3, wherein the coupling (d) comprises contacting the compound of Formula 1d HCI salt with a base.

5. A compound having the structure of Formula 2:Formula 2or a salt thereof, wherein:PGais a nitrogen protecting group; andY is -BocN(OH)2, -BocN(OCH3)2, -BocN(OC2H5)2,6. The compound of claim 5, wherein Y is -BocN(OH)2.

7. A method of preparing a compound of Formula 2e:Formula 2ethe method comprising:a) coupling a compound of Formula 1 e with a compound of Formula 2a •2HCl to form a compound of Formula 2b:Formula 1e Formula 2a •2HCl Formula 2b; and b) contacting the compound of Formula 2b with a borylation agent to form the compound of Formula 2e:Formula 2b Formula 2e wherein:R1is optionally substituted C1-C6 alkyl;PGais a nitrogen protecting group;X is Cl, BocNr, OTf, OMs, OTs, OAc, or OBocNz; and8. The method of claim 7, wherein the borylation agent is bis(pinacolato)diboron, tetrahydroxydiboron, tetramethoxydiboron, tetrahydroxydiboron, or bis(neopentylglycolato)diboron.

9. The method of claim 7 or 8, wherein the borylating (b) is carried out in the presence of a palladium catalyst.o sBocN-|- 10. The method of claim any one of claims 7 to 9, wherein Y isO?11. The method of claim 10, further comprising hydrolyzing a compound of Formula 2c to form a compound of Formula 2d:

12. The method of claim 11, wherein the hydrolyzing comprises contacting a compound of Formula 2c with a base.

13. The method of claim 12, wherein the base is a hydroxide base.

14. A method of preparing Compound 3:Compound 3the method comprising:a) converting Compound 3a to Compound 3b:Compound 3a Compound 3b.b) cyclizing Compound 3b using carbon dioxide to form Compound 3c:Compound 3b Compound 3cc) hydrolyzing Compound 3c to form Compound 3d:Compound 3c Compound 3d.d) converting Compound 3d to form Compound 3e:Compound 3d Compound 3e.an(je) cyclizing Compound 3e using formaldehyde to form Compound 3:Compound 3e Compound 315. The method of claim 14, wherein the converting (a) comprises contacting Compound 3a with an ethynyl magnesium halide.

16. The method of claim 14 or 15, wherein the cyclizing (b) is carried out in the presence of a silver catalyst.

17. The method of any one of claims 14 to 16, wherein the converting (d) is carried out in the presence of an enzyme.

18. A method of preparing of Compound 4:Compound 4the method comprising:a) converting Compound 3 to Compound 4a:Compound 3b) deprotecting Compound 4a to form Compound 4b • 3HCI:Compound 4b •3HCl c) coupling Compound 4b •3HCl with Compound 4c hydrochloride using a carbonate source to form Compound 4d:Compound 4b »3HCI Compound 4c HCI Compound 4dd) converting Compound 4d to Compound 4:Compound 4d Compound 419. The method of claim 18, wherein the converting (a) comprises contacting Compound 3 with an acyl halide.

20. The method of claim 18 or 19, wherein the carbonate source of the coupling (c) is bis(trichloromethyl) carbonate.

21. The method of any one of claims 18 to 20, wherein the converting (d) comprises contacting Compound 4d with an iodide salt.

22. A method of preparing Compound A:Compound Athe method comprising:a) coupling Compound 5a with Compound 2 to form Compound 5b:Compound 5bCompound 5b Compound 5cc) deprotecting Compound 5c to form Compound 5d:Compound 5cd) coupling Compound 5d with Compound 4 to form Compound A:Compound 5d Compound 423. The method of claim 22, wherein the coupling (a) comprises contacting Compound 5a and Compound 2 with a palladium catalyst.

24. The method of claim 22 or 23, wherein the cyclizing (b) comprises contacting compound 5b with a carbodiimide coupling reagent.

25. The method of claim 24, wherein the carbodiimide coupling reagent is EDCI.

26. The method of any one of claims 22 to 25, wherein the coupling (d) comprises contacting Compound 5d and Compound 4 with a phosphonium coupling reagent.

27. The method of claim 26, wherein the phosphonium coupling reagent is PyBocNOP.

28. The method of any one of claims 22 to 27, wherein the method further comprises purifying Compound A.

29. The method of claim 28, wherein the purifying comprises recrystallizing Compound A.

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