WO2025049912A1

WO2025049912A1 - Methods of manufacturing highly stereochemically pure inhibitors of z-at polymerization

Info

Publication number: WO2025049912A1
Application number: PCT/US2024/044680
Authority: WO
Inventors: Pauline BOURBON; Colm Campbell; Claire O'BRIEN; Heather Ann WENZEL; Warren PADEN; Gerard Phillip ROBERTSON; John George BOWEN
Original assignee: Biomarin Pharmaceutical Inc
Current assignee: Biomarin Pharmaceutical Inc
Priority date: 2023-08-31
Filing date: 2024-08-30
Publication date: 2025-03-06
Anticipated expiration: 2026-02-28
Also published as: AR133704A1

Abstract

The present disclosure provides methods for synthesizing compounds of Formula (I-A) such as compounds wherein R₁ is F, R₂ is propyl, n is 2, one R is F, and one R is Cl in high stereochemical purity without the need for chiral separations. Compositions having such compounds with high stereochemical purity are also provided. The compounds and compositions may be used in the treatment of diseases mediated by Z-α1-trypsin polymerization.

Description

COMPOUNDS AND COMPOSITIONS OF HIGHLY STEREOCHEMICALLY PURE INHIBITORS OF Z-AT POLYMERIZATION AND METHODS OF MANUFACTURING THE SAME 1. CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/535,850, filed August 31, 2023, the disclosure of which is incorporated by reference herein in its entirety. 2. FIELD [0002] The present disclosure relates generally to methods of making compounds useful in treating diseases, disorders, and conditions associated with alpha-1-antitrypsin. 3. BACKGROUND [0003] Alpha-1-antitrypsin or α1antitrypsin (A1AT, sometimes referred to as AAT) is a protease inhibitor belonging to the serpin superfamily. It is a protein made in hepatocytes (as well as in other cells) and secreted into the blood where it functions to limit enzymatic activity of key proteases, in particular, neutrophil elastase. In its absence, the activity of key proteases including neutrophil elastase is unchecked, resulting in excessive breakdown of elastin and connective tissues. [0004] Alpha-1-antitrypsin or α1antitrypsin deficiency (A1ATD, also referred to as AATD or AATLD) is an autosomal co-dominant genetic disorder usually caused by mutations in the SERPINA1 gene. Most severe cases of A1ATD are caused by homozygosity for the mutant Z allele (protease inhibitor [Pi]ZZ), in which a single amino acid substitution (E342K) produces a thermodynamically unstable protein. This unstable protein readily forms an unstable intermediate which in turn accepts the reactive center loop (RCL) of other A1AT proteins, forming large intracellular polymers. As a result, the mutated Z form of the A1AT protein (Z A1AT) is poorly secreted and a substantial reduction in the median plasma concentration of A1AT is observed. The organ most commonly affected by accumulation of Z-A1AT is the liver, where polymer accumulation can lead to liver fibrosis and other forms of liver damage. Polymers of Z-A1AT are also found in other tissues including blood, lungs, and skin. Polymers have been shown to be pro-inflammatory and may contribute to pathology in tissues where they are found, particularly the lung and the skin. 1 NAI-1541064841v1 [0005] There are currently no available pharmacological therapies approved for A1AT- associated liver disease; current treatments focus on supportive measures. In severe cases damage to the liver is so great that a liver transplant is necessary. [0006] WO 2019/243841 A1, which is incorporated herein by reference in its entirety, describes hydroxy-carboxamides that can reduce Z-A1AT polymerization; however, it fails to describe compositions of such hydroxy-carboxamides that have high stereochemical purity or methods of producing such compounds in a highly stereochemically pure form. 4. SUMMARY [0007] In one aspect, the present disclosure provides a method of making a compound of Formula (I-A): ,

wherein: R1 is selected from the group consisting of F, Cl, Br, I, and C1-6 alkyl; R2 is C1-6 alkyl; each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C_1-6 alkyl; and n is an integer from 0-5; wherein the method comprises: contacting a compound of Formula (II-A) with a compound of Formula (III), optionally in the presence of a base, to make a compound of Formula (IV-A): 2 NAI-1541064841v1 R_n MX_p ₂

, wherein: Pg is a protecting group; n is 0, 1, 2, 3, 4, or 5; X is Cl, Br, or I; and M is an alkali metal and p is 0; or M is an alkaline earth metal and p is 1; and converting the compound of Formula (IV-A) to a compound of Formula (I-A) , wherein the

(I-A) comprises contacting the compound of Formula (IV-A) with a reducing agent. In one embodiment, the reducing agent is a stereospecific or a stereoselective reducing agent. In one embodiment, the stereoselective or stereospecific reducing agent is (S,S)-Ts-Deneb, (S,S)-Ms- Deneb, or aluminum isopropoxide. In a particular embodiment, the stereoselective or stereospecific reducing agent is aluminum isopropoxide. [0008] In one embodiment, the C1-6 alkyl is selected from the group consisting of CH3, CH₂CH₃, CH₂CH₂CH₃, and CH(CH₃)₂. In one embodiment, n is 2. In one embodiment, n is not 0 and each R is independently selected from the group consisting of F, Cl, and Br. In one embodiment, the protecting group Pg is selected from the group consisting of 9- fluorenylmethyloxycarbonyl (Fmoc), p-methoxybenzyl carbonyl (Moz or MeOZ), tert- 3 NAI-1541064841v1 butyloxycarbonyl (Boc), carboxybenzyl (Cbz), acetyl (Ac), benzoyl (Bz), benzyl (Bn), carbamate, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts), trichloroethyl chloroformate (Troc), and a sulfonamide. In one embodiment, the protecting group Pg is tert-butyloxycarbonyl (Boc). In one embodiment, the MXp moiety in Formula (III) is MgCl. [0009] In one embodiment, the base is selected from the group consisting of a Grignard reagent, a lithium aluminum alkyl hydride, an alkyllithium compound, and an aryllithium compound. [0010] In one embodiment, the compound of Formula (II-A) is contacted with a non- nucleophilic base and the reaction proceeds as shown below, generating an intermediate of Formula (IIc-A): R_n MX_p R₂ .

of Formula (II-A) to the compound of Formula (IIc-A). When employed, the non-nucleophilic base is a base that deprotonates the compound of Formula (II-A) to form the intermediate compound of Formula (IIc-A) without significant nucleophilic attack on an electrophilic site of Compound (II-A), such as the carbonyl carbon. However, minor amounts of one or more side-reactions, even nucleophilic side-reactions, are acceptable. In one embodiment, the non-nucleophilic base is selected from the group consisting of a Grignard reagent, a lithium aluminum alkyl hydride, an alkyllithium compound, and an aryllithium compound. [0012] In one embodiment, the converting of the compound of Formula (IV-A) to the compound of Formula (I-A) comprises: contacting the compound of Formula (IV-A) with a reducing agent to make a compound of Formula (V-A): 4 NAI-1541064841v1 . [0013] In pecific reducing agent. In one embodiment, the stereoselective or stereospecific reducing agent is (S,S) -Ts- Deneb, (S,S)-Ms-Deneb, or aluminum isopropoxide. In a particular embodiment, the stereoselective or stereospecific reducing agent is aluminum isopropoxide. [0014] In one embodiment, the converting of the compound of Formula (IV-A) to the compound of Formula (I-A) comprises or further comprises: contacting the compound of Formula (V-A) with a deprotecting agent to make a compound of Formula (VI-A): . [0015] In

salt. [0016] In one embodiment, the converting of the compound of Formula (VI-A) to the compound of Formula (I-A) comprises or further comprises: contacting the compound of Formula (VI-A) or salt thereof with a compound of Formula (VII) to make the compound of Formula (I-A): . NAI-

[0017] In one embodiment, the compound of Formula (II-A) is made by a method comprising converting a compound of Formula (IIa-A) to an activated intermediate: .

[0018] In one embodiment, the converting of the compound of Formula (IIa-A) to an activated intermediate comprises contacting a compound of Formula (IIa-A) with a coupling agent to make the activated intermediate. In one embodiment, the method further comprises contacting the activated intermediate with N,O-dimethylhydroxylamine to make a compound of Formula (II-A): .

contacting a compound of Formula (VIIa) with a catalyst to make a compound of Formula (VIIb): contacting

to make the compound of Formula (VII): 6 NAI-1541064841v1 . [0020] In one e y a method comprising: contacting a compound of Formula (VIIa) with tert-butyldimethylsilyl chloride to make a compound of Formula (VIII): ;

chloride and n- butyl lithium to make an intermediate of Formula (VIIIa); ;

intermediate of Formula (VIIIb); 7 NAI-1541064841v1 nd ke a compound of Formula (VII):

. [0021]

(VIIa) is contacted with tert- butyldimethylsilyl chloride to make a compound of Formula (VIII) in the presence of a base. In one embodiment, the base is sodium hydride (NaH), lithium hydride (LiH), an alkyl lithium base (e.g., methyllithium, ethyllithium, isopropyllithium, n-butyllithium, sec-butyllithium, tert- butyllithium), or a borohydride salt (e.g., NaBH4 or KBH4). In one embodiment, the base is NaH. [0022] In some embodiments, the carbon dioxide is pressurized carbon dioxide. [0023] In some embodiments, the bis-silyl deprotecting agent comprises an acid, such as phosphoric acid. [0024] In another aspect, the present disclosure provides a method of making N-[(1R)-1-[(S)- (2-chloro-3-fluorophenyl)hydroxymethyl]butyl]-7-fluoro-2,3-dihydro-2-oxo-1H-indole-4- carboxamide (Compound 1-A): 8 NAI-1541064841v1 , the method comprising: contacting tert-butyl (R)-(1-(methoxy(methyl)amino)-1-oxopentan-2-yl)carbamate (2-A) with a compound of Formula 3, optionally in the presence of a base, to make tert-butyl (R)-(1-(2- chloro-3-fluorophenyl)-1-oxopentan-2-yl)carbamate (Compound 4-A): ,

X is Cl, Br, or I; and M is an alkali metal and p is 0; or M is an alkaline earth metal and p is 1; and converting Compound 4-A to Compound 1-A:

4- A with a reducing agent. 9 NAI-1541064841v1 [0025] In one embodiment, Compound 2-A is contacted with Formula 3 and optionally, a base, in the presence of 2-methyltetrahydrofuran (2-MeTHF). [0026] In one embodiment, the reducing agent is a stereospecific or stereoselective reducing agent. In one embodiment, the stereoselective or stereospecific reducing agent is (S,S) -Ts- Deneb, (S,S)-Ms-Deneb, or aluminum isopropoxide. In a particular embodiment, the stereoselective or stereospecific reducing agent is aluminum isopropoxide. [0027] In one embodiment, M is an alkaline earth metal and X is chloride. In one embodiment, M is magnesium. In one embodiment, the MXp moiety of the compound of Formula 3 is MgCl. [0028] In one embodiment, Compound 2-A is contacted with a non-nucleophilic base and the reaction proceeds as shown below, generating an intermediate of Compound 2-A: .

2- A to form intermediate Compound 2c-A without significant nucleophilic attack on an electrophilic site of Compound 2-A, such as the carbonyl carbon. However, minor amounts of one or more side-reactions, even nucleophilic side-reactions, are acceptable. In one embodiment, the non-nucleophilic base is selected from the group consisting of a Grignard reagent, a lithium aluminum alkyl hydride, an alkyllithium compound, and an aryllithium compound. [0030] In one embodiment, Compound 2-A is made by a method comprising: contacting (R)-2-((tert-butoxycarbonyl)amino)pentanoic acid (Compound 2a-A) with a coupling agent to make an activated intermediate; and contacting the activated intermediate with N,O-dimethylhydroxylamine to make tert- butyl (R)-(1-(methoxy(methyl)amino)-1-oxopentan-2-yl)carbamate (Compound 2-A): 10 NAI-1541064841v1 . [00 embodiment, the coupling agent is CDI and Compound 2a-A is contacted with CDI at a temperature of about 0 °C. In one embodiment, (R)-2-((tert-butoxycarbonyl)amino)pentanoic acid is contacted with CDI in the presence of 2-MeTHF. [0032] In one embodiment, Compound 2a-A is obtained by desalting a salt of Compound 2a- A, such as a dicyclohexylammonium (DCHA) salt, followed by distillation. Desalting can occur in the presence of acid, such as hydrochloric acid or sulfuric acid; the acid and concentration of the acid that is selected depends on the identity of the salt of Compound 2a-A. The free base Compound 2a-A, as shown in the reaction scheme herein, is then contacted with the coupling agent. [0033] In one embodiment, the contacting of the activated intermediate with N,O- dimethylhydroxylamine is in the presence of N,N-dimethylformamide (DMF) and 2-MeTHF. [0034] In one embodiment, the method of making the compound of Formula (3) comprises contacting 1-bromo-2-chloro-3-fluorobenzene with isopropyl magnesium chloride. In one embodiment, the contacting is carried out in tetrahydrofuran (THF) or 2-MeTHF. [0035] In one embodiment, the converting of Compound 4-A to Compound 1-A comprises: contacting Compound 4-A with a reducing agent to make tert-butyl ((1S,2R)-1-(2-chloro-3- fluorophenyl)-1-hydroxypentan-2-yl)carbamate (Compound 5-A): .

embodiment, Compound 5-A is contacted with the reducing agent in the presence of isopropyl 11 NAI-1541064841v1 alcohol and toluene. In one embodiment, Compound 5-A is contacted with the reducing agent at an elevated temperature, for example, a temperature of about 50 °C. [0037] In one embodiment, the converting of Compound 4-A to Compound 1-A comprises: contacting Compound 4-A with a stereoselective or stereospecific reducing agent. In one embodiment, the stereoselective or stereospecific reducing agent is (S,S) -Ts-Deneb, (S,S)-Ms- Deneb, or aluminum isopropoxide. In a particular embodiment, the stereoselective or stereospecific reducing agent is aluminum isopropoxide. [0038] In one embodiment, the converting of Compound 4-A to Compound 1-A comprises or further comprises: contacting Compound 5-A with a deprotecting agent to make (1S,2R)-2-amino-1-(2- chloro-3-fluorophenyl)pentan-1-ol hydrochloride (Compound 6-A): .

or further comprises: contacting Compound 6-A with 7-fluoro-2-oxoindoline-4-carboxylic acid (Compound 7) to make Compound 1-A: .

12 NAI-1541064841v1 contacting 4-bromo-7-fluoroindolin-2-one (Compound 7a) with a suitable catalyst to make methyl 7-fluoro-2-oxoindoline-4-carboxylate (Compound 7b): 7: . [0041]

the presence of carbon monoxide gas and a base. In one embodiment, the catalyst is a palladium catalyst. In one embodiment, the base is selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, triethylamine, N-diisopropylethylamine, pyridine, 4-dimethylpyridine, potassium acetate, sodium methoxide, potassium tert-butoxide, and any combination thereof. In one embodiment, the hydrolyzing agent is selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium methoxide, potassium tert-butoxide, or any combination thereof. [0042] In one embodiment, Compound 7 is made by a method comprising: contacting 4-bromo-7-fluoroindolin-2-one (Compound 7a) with tert-butyldimethylsilyl chloride to make 4-bromo-1-(tert-butyldimethylsilyl)-2-((tert-butyldimethylsilyl)oxy)-7-fluoro- 1H-indole (Compound 8): 13 NAI-1541064841v1 F F ; contacting lithium to make

intermediate 8a: ;

7:

.

NAI-1541064841v1

[0043] In another aspect, the present disclosure provides a compound of N-[(1R)-1-[(S)-(2- chloro-3-fluorophenyl)hydroxymethyl]butyl]-7-fluoro-2,3-dihydro-2-oxo-1H-indole-4- carboxamide (Compound 1-A): ,

which is made by any method as disclosed herein. [0044] In another aspect, the present disclosure provides a composition comprising Compound 1-A, wherein the stereochemical purity of the compound in the composition is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or even at least about 99.8%. In one embodiment, the composition further comprises a pharmaceutically acceptable excipient. [0045] In another aspect, the present disclosure provides a composition comprising N-[(1R)- 1-[(S)-(2-chloro-3-fluorophenyl)hydroxymethyl]butyl]-7-fluoro-2,3-dihydro-2-oxo-1H-indole-4- carboxamide (Compound 1-A): ,

wherein the stereochemical purity of (Compound 1-A) is 90% or greater, optionally 95% or greater, further optionally 98.5% or greater, yet further optionally 99% or greater, still further optionally 99.5% or greater, and yet still further optionally 99.8% or greater. [0046] In one embodiment, the composition comprises no more than 10% of a combined amount of Compounds 1-B, 1-C, and 1-D: 15 NAI-1541064841v1 [0047] In one embodiment, the composition comprises no more than 5% of a combined amount of Compounds 1-B, 1-C, and 1-D, optionally no more than 1.5% of a combined amount of Compounds 1-B, 1-C, and 1-D, further optionally no more than 1% of a combined amount of Compounds 1-B, 1-C, and 1-D, further optionally no more than 0.5% of a combined amount of Compounds 1-B, 1-C, and 1-D, still further optionally no more than 0.2% of a combined amount of Compounds 1-B, 1-C, and 1-D. In one embodiment, the composition further comprises a pharmaceutically acceptable excipient. [0048] In one embodiment, the composition consists essentially of N-[(1R)-1-[(S)-(2-chloro- 3-fluorophenyl)hydroxymethyl]butyl]-7-fluoro-2,3-dihydro-2-oxo-1H-indole-4-carboxamide (Compound 1-A): 16 NAI-1541064841v1 . [0049] In one embodiment, the stereochemical purity of (Compound 1-A) is about 90% or greater, optionally about 95% or greater, optionally about 98.5% or greater, further optionally about 99% or greater, further optionally about 99.5% or greater, still further optionally about 99.8% or greater. [0050] In another aspect, the present disclosure provides a pharmaceutical composition comprising a composition as disclosed herein and at least one pharmaceutically acceptable excipient. 5. BRIEF DESCRIPTION OF THE DRAWINGS [0051] FIG.1 and FIG.2 provide a dose response curve of Compounds 1-A, 1-B, 1-C, and 1-D with 1000 nM zA1AT and 300 nM mA1AT. [0052] FIG.3 and FIG.4 provide a thermostability dose response curve of Compounds 1-A, 1-B, 1-C, and 1-D with 1000 nM zA1AT and 300 nM mA1AT. [0053] FIG.5 provides data related to calreticulin levels in PiZ mice after treatment with Compound 1-A. [0054] FIG.6 provides data related to prolyl 4-hydroxylase beta polypeptide levels in PiZ mice after treatment with Compound 1-A. [0055] FIG.7 provides data related to heat shock protein 5 levels in PiZ mice after treatment with Compound 1-A. [0056] FIG.8 provides data related to Factor VII coagulation factor levels in PiZ mice after treatment with Compound 1-A. [0057] FIG.9 provides data related to C5 complement levels in PiZ mice after treatment with Compound 1-A. [0058] FIG.10 provides data related to thyroxine-binding globulin levels in PiZ mice after treatment with Compound 1-A. 17 NAI-1541064841v1 6. DETAILED DESCRIPTION [0059] In view of the foregoing, a problem to be solved relates to the provision of one or more pharmaceutical compounds, preferably one or more inhibitors of Z-A1AT polymerization, in a highly stereochemically pure form, for example, as a composition having a single active stereoisomer of a compound that inhibits Z-A1AT polymerization that is present as no less than 90%, preferably no less than 95%, more preferably no less than 98.5%, even more preferably no less than 99%, yet more preferably no less than 99.5%, and still more preferably no less than 99.75%, and most preferably no less than 99.9%, by weight, of the weight of all stereoisomers of the compound. In a more specific variation of this problem, the inhibitor of Z-A1AT polymerization is N-[(1R)-1-[(S)-(2-chloro-3-fluorophenyl)hydroxymethyl]butyl]-7-fluoro-2,3- dihydro-2-oxo-1H-indole-4-carboxamide. [0060] Another problem to be solved is provision of a method of synthesis of a highly stereochemically pure form of a compound of Formula (I) , wherein:

R1 is selected from the group consisting of F, Cl, Br, I, and C1-6 alkyl; R₂ is C_1-6 alkyl; each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C_1-6 alkyl; and n is an integer from 0-5. [0061] In particular cases, the foregoing problem can relate to a method of synthesis of compounds of Formula (I) having (1S, 2R) stereochemistry, and most particularly methods that provide N-[(1R)-1-[(S)-(2-chloro-3-fluorophenyl)hydroxymethyl]butyl]-7-fluoro-2,3-dihydro-2- oxo-1H-indole-4-carboxamide (Formula (I)) in high stereochemical purity; however, in other cases it may relate to synthesis of highly stereochemically pure forms of other isomers. A more 18 NAI-1541064841v1 specific version of this problem is to provide a method that is practical and economical to implement on an industrial scale, such as an asymmetric synthesis, and most particularly a method that does not require the use of chiral separation techniques to provide a composition having a single stereoisomer in sufficiently high stereochemical purity. In some cases, the stereochemical purity can be sufficiently high when, in the composition the desired stereoisomer (which is typically the 1S,2R stereoisomer) is present in sufficiently high amount compared to the undesired stereoisomers that the undesired stereoisomers do not have any meaningful biological effect on a subject to whom the composition is delivered. In other cases, the stereochemical purity can be sufficiently high when it is acceptable to a regulatory authority such as the US FDA. [0062] Aspects of solutions to one, some, or all of the foregoing problems are provided herein. In brief, one aspect, the present disclosure provides a method of synthesizing a compound of Formula (I) , wherein:

R1 is selected from the group consisting of F, Cl, Br, I, and C1-6 alkyl; R₂ is C_1-6 alkyl; each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C_1-6 alkyl; and n is an integer from 0-5. [0063] In another aspect, the present disclosure provides a method of making a compound of Formula (I-A) 19 NAI-1541064841v1 ^OH R₁ ,

wherein: R1 is selected from the group consisting of F, Cl, Br, I, and C1-6 alkyl; R2 is C1-6 alkyl; each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C_1-6 alkyl; and n is an integer from 0-5, wherein the method produces the compound at high stereochemical purity without any need for chiral separation. For example, using the methods disclosed herein, a compound of Formula (I- A) may be produced at a stereochemical purity of 90% by weight or greater, 95% by weight or greater, 99% by weight or greater, 99.5% by weight or greater, 99.8% by weight or greater, or even 99.9% by weight or greater, relative to the total weight the compound of Formula (I-A) and all of its stereoisomers. 6.1. Definitions [0064] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art of the present disclosure. As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. [0065] In some embodiments, chemical structures are disclosed with a corresponding chemical name. In case of conflict, the chemical structure controls the meaning, rather than the name. [0066] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context otherwise, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural. 20 NAI-1541064841v1 [0067] As used herein and unless otherwise specified, “stereoisomers” refer to compounds that have the same chemical formula and whose atoms have the same connectivity, but which have different orientations in space. In some cases, stereoisomers can be isolated from mixtures by methods known to those skilled in the art. For analytical purposes, chiral high pressure liquid chromatography (HPLC) can be used, though this method may not provide enough material for industrial or commercial use. In some cases, chiral salts can be separated and crystallized; however, this requires asymmetric conditions that are often impractical for use on a commercial or industrial scale. In particular methods of this disclosure, stereoisomers can be prepared by asymmetric syntheses that provides the desired stereoisomer in a form that is substantially free of other, undesired stereoisomers. Particular compounds as described herein are present as individual stereoisomers that are substantially free of other stereoisomers, or alternatively, as mixtures of various stereoisomers. [0068] The term “alkyl” refers to monovalent aliphatic hydrocarbyl groups that lack a double or triple bonds between carbon atoms, and that preferably have from one to twelve carbon atoms, such as one to 10, one to 6, one to 5, one to 4, or one to three carbon atoms. This term includes, by way of example, linear, branched, and cyclic hydrocarbyl groups such as methyl (-CH₃), ethyl (-CH₂CH₃), n-propyl (-CH₂CH₂CH₃), isopropyl (CH(CH₃)₂), cyclopropyl (-CH)₃, n-butyl (-CH2CH2CH2CH3), isobutyl (-CH2CH(CH3)2), sec-butyl (CH(CH3)(CH3CH2)), t-butyl (-C(CH₃)₃), n-pentyl (-CH₂CH₂CH₂CH₂CH₃), and neopentyl (CH₂C(CH₃)₃). [0069] The term “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and/or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, rats, mice, and/or dogs; and/or birds, including commercially relevant birds such as chickens, ducks, geese, quails, and/or turkeys. In certain embodiments, the subject is a human. As used herein and unless otherwise specified, a human subject to which administration of a therapeutic agent (e.g., a compound as described herein) is contemplated in order to treat, prevent, or manage a disease, disorder, or condition, or symptoms thereof, is also called a “patient.” [0070] As used herein and unless otherwise specified, the terms “treatment” and “treating” refer to therapeutic or palliative measures. Beneficial or desired clinical results include, but are 21 NAI-1541064841v1 not limited to, alleviation, in whole or in part, of symptoms associated with a disease, disorder, or condition, diminishment of the extent of disease, stabilization (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state (e.g., one or more symptoms of the disease), and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. In one embodiment, “treatment” comprises administration of a therapeutic agent (e.g., a compound as described herein) after manifestation of the unwanted condition (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof). In some particular embodiments, “treatment” can mean reducing rate at which A1AT polymers accumulate in the liver, or preferably halting the accumulation of A1AT polymers in the liver, or more preferably reducing the amount of A1AT polymers in the liver (“amount” in this context is usually measured as a concentration but might also refer to weight.) In some particular embodiments, “treatment” can mean increasing the secretion of A1AT from the liver, usually as measured by an increase in the concentration of A1AT outside of the liver. [0071] An “effective amount,” as used herein, refers to an amount that is sufficient to achieve a desired biological effect. A “therapeutically effective amount,” as used herein, refers to an amount that is sufficient to achieve a desired therapeutic effect. In some particular embodiments, “effective amount” can mean an amount effective for reducing rate at which A1AT polymers accumulate in the liver, or preferably effective for halting the accumulation of A1AT polymers in the liver, or more preferably effective for reducing the amount of A1AT polymers in the liver (“amount” in this context is usually measured as a concentration but might also refer to weight.) In some particular embodiments, “effective amount” can mean an amount effective for increasing the secretion of A1AT from the liver, usually as measured by an increase in the concentration of A1AT outside of the liver. [0072] As used herein, and unless otherwise specified, the terms “about” and “approximately,” when used in connection with doses, amounts, or weight percent of ingredients of a composition or a dosage form, mean a dose, amount, or weight percent that is recognized by one of ordinary skill in the art to provide a pharmacological effect equivalent to that obtained from the specified dose, amount, or weight percent. In certain embodiments, the terms “about” and “approximately,” when used in this context, contemplate a dose, amount, or weight percent 22 NAI-1541064841v1 within 30%, within 20%, within 15%, within 10%, or within 5%, of the specified dose, amount, or weight percent. [0073] As used herein, and unless otherwise specified, the terms “about” when used in connection with a temperature is a temperature that is recognized by one of ordinary skill in the art to result in an equivalent outcome, e.g., result in the same product. In certain embodiments, the terms “about” and “approximately,” when used in this context, contemplate a temperature that is ± 5 °C, ± 4 °C, ± 3 °C, ± 2 °C, ± 1 °C, or ± 0.5 °C from the specified temperature. [0074] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. [0075] Any of the compounds disclosed herein may be provided and/or utilized in the form of a salt. One of skill in the art will readily recognize the variety of salts that may be employed within the context of this methods disclosed herein. In one embodiment, a salt may be a “pharmaceutically acceptable salt”, which refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. [0076] As used herein, and unless otherwise specified, the term “stereochemical purity” and its cognates refers to the weight percent of one stereoisomer of a compound, typically a specified stereoisomer of the compound, in relation to the total amount of all stereoisomers of that compound. When the compound is in a composition that also contains other components (i.e., components other than stereoisomers of the compound at issue), the “stereochemical purity” does not consider the amount of the compound relative to other components. For example, if a composition contains a hypothetical desired stereoisomer of Compound 1 having a stereochemical purity of at least 90%, then at least 90% of the weight of all stereoisomers of Compound 1 in the composition is the desired stereoisomer, regardless of how much Compound 1 is in the composition. The detection of desired stereoisomer versus all or other stereoisomers can be accomplished by any method apparent to a person of ordinary skill in the art, including, but not limited to, chiral high pressure liquid chromatography (HPLC) and NMR spectroscopy. 23 NAI-1541064841v1 [0077] As used herein, it will be understood that a specific percentage of stereochemical purity of a desired stereoisomer (e.g., 90%, 95%, 98.5%, 99%, 99.5%, or 99.8%) refers to the specific percentage by weight of that stereoisomer based on the weight of all stereoisomers of the same compound. Thus, for example, a stereochemical purity of 90%, 95%, 98.5%, 99%, 99.5%, or 99.8% also means 90% by weight, 95% by weight, 98.5% by weight, 99% by weight, 99.5% by weight, or 99.8% by weight, respectively. [0078] As used herein, the term “stereochemically pure” with respect to Compound 1-A means that a composition comprising Compound 1-A contains no detectable amount of any other stereoisomer of Compound 1. [0079] In certain embodiments, the compositions provided herein comprise stereochemically pure Compound 1-A. In certain embodiments, the compositions provided herein comprise Compound 1-A, wherein the stereochemical purity of Compound 1-A is 99.8% or greater. In certain embodiments, the compositions provided herein comprise Compound 1-A, wherein the no other stereoisomer of Compound 1 is present in a detectable amount. [0080] The disclosure can be understood more fully by reference to the following detailed description and illustrative examples, which are intended to exemplify non-limiting embodiments. 6.2. Methods of Synthesizing Compounds of Formula (I) [0081] In one aspect, the methods of synthesizing a compound of Formula (I) , wherein:

R1 is selected from the group consisting of F, Cl, Br, I, and C1-6 alkyl; R₂ is C_1-6 alkyl; each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C1-6 alkyl; and 24 NAI-1541064841v1 n is an integer from 0-5, wherein the method comprises: contacting a compound of Formula (II) with a compound of Formula (III), optionally in the presence of a base, to make a compound of Formula (IV) as shown in SCHEME I: ,

wherein: Pg is a protecting group; n is 0, 1, 2, 3, 4, or 5; X is Cl, Br, or I; and M is an alkali metal and p is 0; or M is an alkaline earth metal and p is 1; and converting the compound of Formula (IV) to a compound of Formula (I) as shown in SCHEME II: .

[0082] With reference to the compound of Formula (I), in one embodiment, the C1-6 alkyl of R₂ is selected from the group consisting of CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₂)₂ (CH)₃, and CH(CH3)2. 25 NAI-1541064841v1 [0083] With reference to the compound of Formula (I), in one embodiment, n is 0-5. In one embodiment, n is 0, 1, or 2. In one embodiment, n is 1. In one embodiment, n is 2. In one embodiment, n is not 0. [0084] With reference to the compound of Formula (I), in one embodiment, and each R is independently selected from the group consisting of F, Cl, Br, and I. In one embodiment, each R is independently selected from the group consisting of F, Cl, and Br. [0085] With reference to the compound of Formula (I), in one embodiment, n is not 0 and each R is independently selected from the group consisting of F, Cl, and Br. [0086] With reference to the compounds of Formulas (II), (IIC), and (IV), in one embodiment, the protecting group Pg is selected from the group consisting of 9- fluorenylmethyloxycarbonyl (Fmoc), p-methyoxybenzyl carbonyl (Moz or MeOZ), tert- butyloxycarbonyl (Boc), carboxybenzyl (Cbz), acetyl (Ac), benzoyl (Bz), benzyl (Bn), carbamate, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts), trichloroethyl chloroformate (Troc), allyloxycarbonyl, trityl, 1-(4,4-dimethyl-2,6- dioxocyclohexy-1-ylidene)-3-methylbutyl, 4-methyltrityl (Mtt), 2,4 dinitrobenzenesulfonyl (dNBS), and a sulfonamide. In one embodiment, the protecting group Pg is tert- butyloxycarbonyl (Boc). [0087] In one embodiment, M of the MXp moiety of Formula (III) is an alkali metal. In one embodiment, M of the MX_p moiety of Formula (III) is magnesium. In one embodiment, the MX_p moiety of Formula (III) is MgCl or MgBr. In another embodiment, M of the MX_p moiety is lithium. [0088] In one embodiment, the compound of Formula (III) is made by contacting 1-bromo- 2-chloro-3-fluorobenzene with isopropyl magnesium chloride in 2-MeTHF. [0089] In one embodiment, the base is selected from the group consisting of a Grignard reagent, a lithium aluminum alkyl hydride, an alkyllithium compound, and an aryllithium compound. [0090] In one embodiment, the compound of Formula (II) is contacted with a non- nucleophilic base and the reaction proceeds as shown below in SCHEME I', generating an intermediate of Formula (IIc): 26 NAI-1541064841v1 , wherein: R₂ is C_1-6 alkyl; each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C_1-6 alkyl; n is an integer from 0-5; Pg is a protecting group; X is Cl, Br, or I; and M is an alkali metal and p is 0; or M is an alkaline earth metal and p is 1. [0091] When employed, the non-nucleophilic base is a base that deprotonates the compound of Formula (II) to form the intermediate compound of Formula (IIc) without significant nucleophilic attack on an electrophilic site of the compound of Formula (II), such as the carbonyl carbon. However, minor amounts of one or more side-reactions, even nucleophilic side- reactions, are acceptable. In one embodiment when it is employed, the non-nucleophilic base is selected from the group consisting of a Grignard reagent, a lithium aluminum alkyl hydride, an alkyllithium compound, and an aryllithium compound. [0092] In one embodiment, a non-nucleophilic base is not employed. [0093] In one embodiment, the compound of Formula (IV) may be made via a Suzuki coupling reaction between the compound of Formula (II) and a compound of Formula (IIIa) in the presence of a catalyst, for example, a palladium catalyst, as shown in SCHEME Ia below, 27 NAI-1541064841v1 , SCHEME Ia wherein: R2 is C1-6 alkyl; each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C_1-6 alkyl; n is an integer from 0-5; Pg is a protecting group; and Y1 and Y2 are each independently OH or OR3, wherein R3 is C1-6 alkyl. [0094] In one embodiment, the converting of the compound of Formula (IV) to the compound of Formula (I) comprises contacting the compound of Formula (IV) with a reducing agent to make a compound of Formula (V) as shown in SCHEME III: .

wherein: R2 is C1-6 alkyl; each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C1-6 alkyl; n is an integer from 0-5; and Pg is a protecting group. 28 NAI-1541064841v1 [0095] In one embodiment, the converting of the compound of Formula (IV) to the compound of Formula (I) comprises or further comprises contacting the compound of Formula (V) with a deprotecting agent to make a compound of Formula (VI) or a salt thereof, as shown in SCHEME IV: .

[0096] one to the compound of Formula (I) comprises or further comprises contacting the compound of Formula (VI), or salt thereof, with a compound of Formula (VII) to make the compound of Formula (I) as shown in SCHEME V: ,

wherein: R₁ is selected from the group consisting of F, Cl, Br, I, and C_1-6 alkyl; R2 is C1-6 alkyl; each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C_1-6 alkyl; and n is an integer from 0-5. 29 NAI-1541064841v1 [0097] In one embodiment, the converting of the compound of Formula (IV) to the compound of Formula (I) comprises: contacting the compound of Formula (IV) with a reducing agent to make a compound of Formula (V): ;

a agent to a compound of Formula (VI) or salt thereof:

contacting the compound of Formula (VI) or a salt thereof with a compound of Formula (VII) to make the compound of Formula (I): .

[0098] In one embodiment, the compound of Formula (IV) is contacted with a reducing agent selected from the group consisting of NaBH3CN, NaBH3(OAc), Ce(BH4)3, Ca(BH4)3, 30 NAI-1541064841v1 Al(BH4)3, LiBH4, LiBHEt3, sodium isopropoxide, L-selectride, K-selectride, lithium aluminum hydride, and sodium borohydride to make a compound of Formula (V). In one embodiment, the reducing agent is sodium borohydride. In one embodiment, the compound of Formula (IV) is contacted with aluminum isopropoxide to make a compound of Formula (V). In one embodiment, the contacting is at an elevated temperature, such as at a temperature of about 50 °C. In one embodiment, the compound of Formula (IV) is contacted with aluminum isopropoxide to make a compound of Formula (V), wherein the contacting is in the presence of isopropyl alcohol and toluene. [0099] In one embodiment, the compound of Formula (V) is contacted with a deprotecting agent to make a compound of Formula (VI) or a salt thereof. In one embodiment, the deprotecting agent is hydrochloric acid. In one embodiment, the deprotecting agent is trifluoroacetic acid. [0100] In one embodiment, the compound of Formula (VI) or salt thereof is contacted with the compound of Formula (VII) in the presence of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC), 4-dimethylaminopyridine (DMAP), and DMF. [0101] In one embodiment, the compound of Formula (VI) or salt thereof is contacted with the compound of Formula (VII) in the presence of O-(benzotriazol-1-yl)-N,N,N′,N′- tetramethyluronium tetrafluoroborate (TBTU), N,N-diisopropylethylamine (DIPEA), and acetonitrile. [0102] In one embodiment, the compound of Formula (II) may be prepared by an initial step of converting a compound of Formula (IIa): to an activated intermediate.

[0103] As used herein “activated intermediate” is an intermediate state that is formed during the conversion of reactants into products. As used herein, “activating agent” and “coupling agent” are used interchangeably. In one embodiment, a compound of Formula (IIa) is contacted with an activating agent or a coupling agent to make the activated intermediate. In this 31 NAI-1541064841v1 embodiment, “activating agent” and “coupling agent” refer to a reagent that generates an activated intermediate that, when reacted with N,O-dimethylhydroxylamine, couples the N,O- dimethylhydroxylamine thereto resulting in a compound of Formula (II): . [0104] The coupling agent is not one of skill in the art will recognize

that a wide variety of coupling agents may a compound of Formula (II) from a compound of Formula (IIa). In one embodiment, the coupling agent is selected from the group consisting of EDC/hydroxybenzotriazole (HOBt), EDC/DMAP, DIPEA/(benzotriazol-1- yloxytris(dimethylamino)phosphonium hexafluorophosphate) (BOP), 4-methylmorpholine (NMM)/isobutyl chloroformate (IBCF), N,N- dicyclohexylcarbodiimide (DCC)/DIPEA/DMAP, PCl₃ phosphorus trichloride, and CDI. In one embodiment, the coupling agent is CDI, and the activated intermediate is a compound of Formula (II'): . [0105] In one embodiment, a

is obtained by desalting a salt of Formula (IIa), such as a DCHA salt, followed by distillation. Desalting can occur in the presence of acid, such as hydrochloric acid or sulfuric acid; the acid and concentration of the acid that is selected depends on the identity of the salt of Formula (IIa). The compound of Formula (IIa), in its desalted form as shown in SCHEME VI below, is then contacted with the coupling agent. [0106] In one embodiment, the activated intermediate may then be contacted with N,O- dimethylhydroxylamine to make the compound of Formula (II) as shown in SCHEME VI: 32 NAI-1541064841v1 . [0107] In one embodiment, the activated intermediate is contacted with N,O- dimethylhydroxylamine in the presence of 2-MeTHF, DMF, THF, DCM, cyclopentyl methyl ether (CPME), ethyl tetrabutyl ether (ETBE), n-butyl-pyrrolidone (NBP), dimethyl sulfoxide (DMSO), t-butanol, acetonitrile, or a mixture of any of the foregoing, to make the compound of Formula (II). In one embodiment, the activated intermediate is contacted with N,O- dimethylhydroxylamine in the presence of 2-MeTHF to make the compound of Formula (II). In one embodiment, the activated intermediate is contacted with N,O-dimethylhydroxylamine in the presence of 2-MeTHF and DMF to make the compound of Formula (II). [0108] In one embodiment, the compound of Formula (VII) is made by a method comprising: contacting a compound of Formula (VIIa) with a catalyst to make a compound of Formula (VIIb) as shown in SCHEME VII:

contacting the compound of Formula (VIIb) with a hydrolyzing agent to make the compound of Formula (VII) as shown in SCHEME VIII: 33 NAI-1541064841v1 . [0109] In one embodiment, the hydrolyzing agent is selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium methoxide, potassium tert-butoxide, or any combination thereof. [0110] In another embodiment, the compound of Formula (VII) is made by a method comprising: contacting a compound of Formula (VIIa) with tert-butyldimethylsilyl chloride to make a compound of Formula (VIII) as shown in SCHEME IX below: ;

contacting the compound of Formula (VIII) with isopropyl magnesium chloride and n- butyl lithium to make an intermediate of Formula (VIIIa) as shown in SCHEME X below: ; NAI-

SCHEME X contacting the intermediate of Formula (VIIIa) with carbon dioxide to make intermediate of Formula (VIIIb) as shown in SCHEME XI below:

contacting the intermediate of Formula (VIIIb) with a deprotecting agent to make a compound of Formula (VII) as shown in SCHEME XII below: .

[0111] In some embodiments, the compound of Formula (VIIa) is contacted with tert- butyldimethylsilyl chloride to make a compound of Formula (VIII) in the presence of a base. In one embodiment, the base comprises sodium hydride (NaH), lithium hydride (LiH), an alkyl lithium base (e.g., methyllithium, ethyllithium, isopropyllithium, n-butyllithium, sec- butyllithium, tert-butyllithium), or a borohydride salt (e.g., NaBH₄ or KBH₄). In one embodiment, the base comprises NaH. [0112] In some embodiments, the carbon dioxide is pressurized carbon dioxide. [0113] In some embodiments, the bis-silyl deprotecting agent comprises an acid (e.g., ammonium chloride, aqueous phosphoric acid, acidic aqueous THF, or acidic methanol) or a 35 NAI-1541064841v1 fluoride (e.g., ammonium fluoride, tetrabutylammonium fluoride, tetrabutylammonium chloride, potassium fluoride, or aqueous hydrofluoric acid. In one embodiment, the bis-silyl deprotecting agent comprises aqueous phosphoric acid. 6.3. Methods of Synthesizing Compound 1 [0114] In one embodiment, the compound of Formula (I) is N-(1-(2-chloro-3-fluorophenyl)- 1-hydroxypentan-2-yl)-7-fluoro-2-oxoindoline-4-carboxamide (Compound 1): . [0115] Therefore, a method of making N-

(1-(2-chloro-3-fluorophenyl)-1-hydroxypentan-2-yl)-7-fluoro-2-oxoindoline-4-carboxamide (Compound 1), wherein the method comprises: contacting tert-butyl (1-(methoxy(methyl)amino)-1-oxopentan-2-yl)carbamate (Compound 2) with a compound of Formula 3, optionally in the presence of a base, to make tert- butyl-(1-(2-chloro-3-fluorophenyl)-1-oxopentan-2-yl)carbamate (Compound 4) as shown in SCHEME 1: Cl ,

wherein: X is Cl, Br, or I; and M is an alkali metal and p is 0; or 36 NAI-1541064841v1 M is an alkaline earth metal and p is 1; and converting Compound 4 to Compound 1, as shown in SCHEME 2: .

[0116] one an alkaline earth metal. In one embodiment, M of the MXp moiety of the compound of Formula 3 is magnesium. In one embodiment, the MXp moiety of the compound of Formula 3 is MgCl. [0117] In one embodiment, a base is not employed when contacting tert-butyl (1- (methoxy(methyl)amino)-1-oxopentan-2-yl)carbamate (Compound 2) with a compound of Formula 3. In one embodiment, when a base is employed, the base is selected from the group consisting of a Grignard reagent, a lithium aluminum alkyl hydride, an alkyllithium compound, and an aryllithium compound. [0118] In another embodiment, Compound 4 may be made via a Suzuki coupling reaction between Compound 2 and a compound of Formula 3a in the presence of a catalyst, for example, a palladium catalyst, as shown in SCHEME 1a below, ,

wherein Y₁ and Y₂ are each independently OH or OR₃, wherein R₃ is C_1-6 alkyl. [0119] In one embodiment, the compound of Formula 3 is made by contacting 1-bromo-2- chloro-3-fluorobenzene with isopropyl magnesium chloride in 2-MeTHF. 37 NAI-1541064841v1 [0120] In one embodiment, Compound 2 is contacted with a non-nucleophilic base and the reaction proceeds as shown below in SCHEME 1', generating an intermediate of Compound 2c: .

[0121] When employed, the non-nucleophilic base is typically a base that deprotonates Compound 2 to form intermediate Compound 2c without significant nucleophilic attack on an electrophilic site of Compound 2, such as the carbonyl carbon. However, minor amounts of one or more side-reactions, even nucleophilic side-reactions, are acceptable. In one embodiment, the non-nucleophilic base is selected from the group consisting of a Grignard reagent, a lithium aluminum alkyl hydride, an alkyllithium compound, and an aryllithium compound. [0122] In one embodiment, the converting of Compound 4 to Compound 1 comprises contacting Compound 4 with a reducing agent to make tert-butyl-1-(2-chloro-3-fluorophenyl)-1- hydroxypentan-2-yl)carbamate (Compound 5), as shown in SCHEME 3: .

[0123] In one embodiment, the converting of Compound 4 to Compound 1 comprises or further comprises: contacting Compound 5 with a deprotecting agent to make 2-amino-1-(2-chloro-3- fluorophenyl)pentan-1-ol hydrochloride (Compound 6), as shown in SCHEME 4: 38 NAI-1541064841v1 . [0124] In one embodiment, the deprotecting agent is hydrochloric acid and Compound 6 is produced as a hydrochloride salt. [0125] In one embodiment, the converting of Compound 4 to Compound 1 comprises or further comprises: contacting Compound 6 with 7-fluoro-2-oxoindoline-4-carboxylic acid (Compound 7) to make Compound 1, as shown in SCHEME 5: .

[0126] In one embodiment, the converting of Compound 4 to Compound 1 comprises: contacting Compound 4 with a reducing agent to make tert-butyl-1-(2-chloro-3- fluorophenyl)-1-hydroxypentan-2-yl)carbamate (Compound 5): ; NAI-

contacting Compound 5 with a deprotecting agent to make 2-amino-1-(2-chloro-3- fluorophenyl)pentan-1-ol (Compound 6) or a salt thereof:

or a acid (Compound 7) to make Compound 1: F H N F O .

[0127] In one embodiment, the deprotecting agent is an acid, and particularly hydrochloric acid. In one embodiment when hydrochloric acid is used, Compound 6 is produced as a hydrochloride salt. In embodiments where other acids are used, Compound 6 can be produced as another salt or even as a free base. [0128] In one embodiment, Compound 4 is contacted with a reducing agent to make Compound 5, wherein the reducing agent is aluminum isopropoxide. In one embodiment, Compound 4 is contacted with a reducing agent to make Compound 5, wherein the contacting is at a temperature of about 50 °C. In one embodiment, Compound 4 is contacted with a reducing agent to make Compound 5, wherein the contacting is in the presence of isopropyl alcohol and toluene. 40 NAI-1541064841v1 [0129] In one embodiment, Compound 5 is contacted with a deprotecting agent to make Compound 6, wherein the deprotecting agent is hydrochloric acid and Compound 6 is made as a hydrochloride salt. [0130] In one embodiment, Compound 6 (or salt thereof) is contacted with 7-fluoro-2- oxoindoline-4-carboxylic acid (Compound 7) in the presence of EDC, DMAP and an organic solvent. In one embodiment, the organic solvent is selected from the group consisting of DMF, DMSO, dimethyl carbonate (DMC), THF, dichloromethane (DCM), or a mixture of any of the foregoing. In one embodiment, Compound 6 (or salt thereof) is contacted with 7-fluoro-2- oxoindoline-4-carboxylic acid (Compound 7) in the presence of EDC, DMAP, and DMF. [0131] In one embodiment, Compound 6 is contacted with 7-fluoro-2-oxoindoline-4- carboxylic acid (Compound 7) in the presence of TBTU and a base. In one embodiment, the base is selected from the group consisting of DIPEA, trimethylamine (TMA), diethylamine (DEA), triethylamine (TEA), and dimethylethanolamine (DMAE). In one embodiment, Compound 6 is contacted with 7-fluoro-2-oxoindoline-4-carboxylic acid (Compound 7) in the presence of TBTU and DIPEA. In one embodiment, Compound 6 is contacted with 7-fluoro-2-oxoindoline-4- carboxylic acid (Compound 7) in the presence of TBTU, DIPEA, and acetonitrile. [0132] Compound 7 may be prepared by methods known in the art, for example, as disclosed herein and in Chinese Patent Publication No. CN113801053 A, which is incorporated by reference in its entirety. [0133] In one embodiment, Compound 2 may be prepared an initial step of converting 2- ((tert-butoxycarbonyl)amino)pentanoic acid (Compound 2a): to an activated intermediate.

[0134] In one embodiment, Compound 2a is contacted with a coupling agent or activating agent to make the activated intermediate. As used in this embodiment, “activating agent” and “coupling agent” are used interchangeably and refer to a reagent that generates an activated intermediate that, when reacted with N,O-dimethylhydroxylamine, couples the N,O- dimethylhydroxylamine thereto resulting in Compound 2: 41 NAI-1541064841v1 . [0135] The coupling agent is not d and one of skill in the art will recognize that a wide variety of coupling agents may be used to generate Compound 2 from Compound 2a. In one embodiment, the coupling agent is selected from the group consisting of EDC/HOBt, EDC/DMAP, DIPEA/BOP, NMM/ICBF, DCC/DIPEA/DMAP, PCl₃ (phosphorus trichloride), and CDI. In one embodiment, the coupling agent is CDI, and the activated intermediate is Compound 2': . [0136] In one embodiment, by desalting a salt of Compound 2a,

such as a DCHA salt, followed by distillation. Desalting can occur in the presence of acid, such as hydrochloric acid or sulfuric acid; the acid that is selected depends on the identity of the salt of Compound 2a. The free base of Compound 2a, as shown in SCHEME 6 below, is then contacted with the coupling agent. [0137] In one embodiment, the activated intermediate may then be contacted with N,O- dimethylhydroxylamine to make Compound 2, as shown in SCHEME 6: .

[0138] In one embodiment, Compound 2 is prepared by a method comprising: 42 NAI-1541064841v1 contacting 2-((tert-butoxycarbonyl)amino)pentanoic acid (Compound 2a) with a coupling agent to make an activated intermediate; and contacting the activated intermediate with N,O-dimethylhydroxylamine to make tert- butyl-(1-(methoxy(methyl)amino)-1-oxopentan-2-yl)carbamate (2). [0139] In one embodiment, the activated intermediate is contacted with N,O- dimethylhydroxylamine in the presence of 2-MeTHF, DMF, THF, DCM, CPME, ETBE, NBP, DMSO, t-butanol, acetonitrile, or a mixture of any of the foregoing, to make Compound 2. In one embodiment, the activated intermediate is contacted with N,O-dimethylhydroxylamine in the presence of 2-MeTHF to make Compound 2. In one embodiment, the activated intermediate is contacted with N,O-dimethylhydroxylamine in the presence of 2-MeTHF and DMF to make Compound 2. [0140] Compound 7 may be made by a method as described herein (e.g., according to SCHEMES VII and VIII and in the Examples) and by process described elsewhere, e.g., in Chinese Patent Publication No. CN113801053 A, which is incorporated by reference in its entirety. [0141] In one embodiment, Compound 7 is made by a method comprising: contacting 4-bromo-7-fluoroindolin-2-one (Compound 7a) with a suitable catalyst to make methyl 7-fluoro-2-oxoindoline-4-carboxylate (Compound 7b) as shown in SCHEME 7 below:

contacting Compound 7b with a hydrolyzing agent to make Compound 7 as shown in SCHEME 8 below: 43 NAI-1541064841v1 . [0142] In one embodiment, the hydrolyzing agent is selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium methoxide, potassium tert-butoxide, or any combination thereof. [0143] In one embodiment, Compound 7 is made by a method comprising: contacting 4-bromo-7-fluoroindolin-2-one (Compound 7a) with tert-butyldimethylsilyl chloride to make 4-bromo-1-(tert-butyldimethylsilyl)-2-((tert-butyldimethylsilyl)oxy)-7-fluoro- 1H-indole (Compound 8) as shown in SCHEME 9 below: ;

contacting Compound 8 with isopropyl magnesium chloride and n-butyl lithium to make Intermediate 8a as shown in SCHEME 10 below: ; NAI-

contacting Intermediate 8a with a carbon dioxide to make Intermediate 8b as shown in SCHEME 11 below:

a agent to 7 as shown in SCHEME 12 below:

.

SCHEME 12

[0144] In some embodiments, Compound 7a is contacted with tert-butyldimethylsilyl chloride to make Compound 8 in the presence of a base. In one embodiment, the base comprises sodium hydride (NaH), lithium hydride (LiH), an alkyl lithium base (e.g., methyllithium, ethyllithium, isopropyllithium, n-butyllithium, sec-butyllithium, tert-butyllithium), or a borohydride salt (e.g., NaBH4 or KBH4). In one embodiment, the base comprises NaH. [0145] In some embodiments, the carbon dioxide is pressurized carbon dioxide. [0146] In some embodiments, the bis-silyl deprotecting agent comprises an acid (e.g., ammonium chloride, aqueous phosphoric acid, acidic aqueous THF, or acidic methanol) or a fluoride (e.g., ammonium fluoride, tetrabutylammonium fluoride, tetrabutylammonium chloride, potassium fluoride, or aqueous hydrofluoric acid). In one embodiment, the bis-silyl deprotecting agent comprises aqueous phosphoric acid. 45 NAI-1541064841v1 6.4. Methods of Making Stereoisomers of Formula (I) [0147] In one embodiment, the compound of Formula (I) is a compound of Formula (I-A), (I-B), (I-C), or (I-D), as shown below: R O ₁ R_n ^H _H

wherein: R₁ is selected from the group consisting of F, Cl, Br, I, and C_1-6 alkyl; R2 is C1-6 alkyl; 46 NAI-1541064841v1 each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C1-6 alkyl; and n is an integer from 0-5. [0148] With reference to the compounds of Formula (I-A), (I-B), (I-C), and (I-D), in one embodiment, R1 is F, R2 is propyl, n is 2, one R is F, and one R is Cl. [0149] In one embodiment, the compound of Formula (I) is a compound of Formula (I-A'), (I-B'), (I-C'), or (I-D'), as shown below:

wherein: 47 NAI-1541064841v1 R1 is selected from the group consisting of F, Cl, Br, I, and C1-6 alkyl; R2 is C1-6 alkyl; and each of R' and R'' is independently selected from the group consisting of F, Cl, Br, I, CN, and C1-6 alkyl. [0150] With reference to the compounds of Formula (I-A'), (I-B'), (I-C'), and (I-D'), in one embodiment, R₁ is F, R₂ is propyl, one of R' and R'' is F, and one of R' and R'' is Cl. [0151] In one embodiment, the compound of Formula (I) is Compound 1-A (N-[(1R)-1-[(S)- (2-chloro-3-fluorophenyl)hydroxymethyl]butyl]-7-fluoro-2,3-dihydro-2-oxo-1H-indole-4- carboxamide), Compound 1-B (N-[(1R)-1-[(R)-(2-chloro-3-fluorophenyl)hydroxymethyl]butyl]- 7-fluoro-2,3-dihydro-2-oxo-1H-indole-4-carboxamide), Compound 1-C (N-[(1S)-1-[(S)-(2- chloro-3-fluorophenyl)hydroxymethyl]butyl]-7-fluoro-2,3-dihydro-2-oxo-1H-indole-4- carboxamide), or Compound 1-D (N-[(1S)-1-[(R)-(2-chloro-3- fluorophenyl)hydroxymethyl]butyl]-7-fluoro-2,3-dihydro-2-oxo-1H-indole-4-carboxamide), shown below: 48 NAI-1541064841v1

[0152] Each of the

, , , , or Compounds 1-A, 1- B, 1-C, and 1-D may be prepared using the methods disclosed herein. In some embodiments, one 49 NAI-1541064841v1 or more intermediates generated during the preparation are subjected to a chiral separation to improve stereochemical purity. [0153] For example, in one embodiment, the compounds of Formula (I-A) and (I-B) may be prepared by the following reactions (SCHEMES VI-A, I-A and III-A) to yield compounds of Formula (V-A) and (V-B):

SCHEME VI-A

[0154] The compound Formula of (V-A) and (V-B) may be deprotected and subjected to chiral separation (in any order) and each deprotected separated stereoisomer reacted separately 50 NAI-1541064841v1 with a compound of Formula (VII) to make the compounds of Formula (I-A) and (I-B) as shown in SCHEME V-A and V-B below:

[0155] In some embodiments, the compounds of Formula (V-A) and (V-B) are deprotected with hydrochloric acid or trifluoracetic acid. In some embodiments, the compounds of Formulas (VI-A) and (VI-B) are produced as salts, e.g., hydrochloride salts. [0156] In some embodiments, the compound of Formula (IV-A) may be made via a Suzuki coupling reaction between the compound of Formula (II-A) and a compound of Formula (IIIa) in the presence of a catalyst, for example, a palladium catalyst, as shown in SCHEME I-Aa below, 51 NAI-1541064841v1 , SCHEME I-Aa wherein: R2 is C1-6 alkyl; each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C1-6 alkyl; n is an integer from 0-5; Pg is a protecting group; and Y₁ and Y₂ are each independently OH or OR₃, wherein R₃ is C_1-6 alkyl. [0157] Compounds of Formula (I-C) and (I-D) may be prepared by the following reactions (SCHEMES VI-C, I-C, and III-C) to yield compounds of Formula (V-C) and (V-D):

SCHEME VI-C 52 NAI-1541064841v1

SCHEME III-C

[0158] In one embodiment, the compound of Formula (IV-C) may be made via a Suzuki coupling reaction between a compound of Formula (II-C) and a compound of Formula (IIIa) in the presence of a catalyst, for example, a palladium catalyst, as shown in SCHEME I-Ca below, ,

wherein: R₂ is C_1-6 alkyl; 53 NAI-1541064841v1 each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C1-6 alkyl; n is an integer from 0-5; Pg is a protecting group; and Y1 and Y2 are each independently OH or OR3, wherein R3 is C1-6 alkyl. [0159] The compound Formula of (V-C) and (V-D) may be subjected to chiral separation and each separated stereoisomer deprotected, then reacted separately with a compound of Formula (VII) to make the compounds of Formula (I-C) and (I-D) as shown in SCHEME V-C and V-D below: R₁ NH

[0160] Within the context of the aforementioned schemes and the compounds depicted therein, R1 is selected from the group consisting of F, Cl, Br, I, and C1-6 alkyl; R₂ is C_1-6 alkyl; 54 NAI-1541064841v1 each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C1-6 alkyl; and n is an integer from 0-5. [0161] In another aspect, the present disclosure provides a method of making a compound of Formula (I-A) via a method that produces a high yield of the compound with high stereochemical purity without the use of any chiral separation, as was described in other embodiments herein. [0162] In one embodiment, Formula (I-A) may be made with high stereochemical purity via a method comprising: contacting a compound of Formula (II-A) with a compound of Formula (III), optionally in the presence of a base, to make a compound of Formula (IV-A):

, SCHEME I-A wherein: Pg is a protecting group; n is 0, 1, 2, 3, 4, or 5; X is Cl, Br, or I; and M is an alkali metal and p is 0; or M is an alkaline earth metal and p is 1; and converting the compound of Formula (IV-A) to a compound of Formula (I-A) 55 NAI-1541064841v1 R O^H ₁ R R O _n _{n H}

, SCHEME II-A wherein the converting of the compound of Formula (IV-A) to the compound of Formula (I-A) comprises contacting the compound of Formula (IV-A) with at least one reducing agent that is a stereospecific reducing agent or a stereoselective reducing agent. In one embodiment, the stereoselective or stereospecific reducing agent is a sterically hindered reducing agent. In one embodiment, the stereoselective or stereospecific reducing agent is (S,S)-Ts-Deneb, (S,S)-Ms- Deneb, or aluminum isopropoxide. In a particular embodiment, the stereoselective or stereospecific reducing agent is aluminum isopropoxide. [0163] In one embodiment, R2 is C1-6 alkyl selected from the group consisting of CH3, CH₂CH₃, CH₂CH₂CH₃, and CH(CH₃)₂. [0164] In one embodiment, n is 0, 1, or 2. In one embodiment, n is 1. In one embodiment, n is 2. In one embodiment, n is not 0. [0165] In one embodiment, each R is independently selected from the group consisting of F, Cl, Br, and I. In one embodiment, each R is independently selected from the group consisting of F, Cl, and Br. [0166] In one embodiment, n is not 0 and each R is independently selected from the group consisting of F, Cl, and Br. [0167] In one embodiment, n is 2 and each R is independently selected from the group consisting of F, Cl, and Br. [0168] In one embodiment, n is 2 and each R is independently F or Cl. [0169] In one embodiment, the protecting group Pg is selected from the group consisting of 9-fluorenylmethyloxycarbonyl (Fmoc), p-methyoxybenzyl carbonyl (Moz or MeOZ), tert- butyloxycarbonyl (Boc), carboxybenzyl (Cbz), acetyl (Ac), benzoyl (Bz), benzyl (Bn), carbamate, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), 56 NAI-1541064841v1 tosyl (Ts), trichloroethyl chloroformate (Troc), and a sulfonamide. In one embodiment, the protecting group Pg is tert-butyloxycarbonyl (Boc). [0170] In one embodiment, M of the MX_p moiety in Formula (III) is an alkaline earth metal. In one embodiment, M of the MXp moiety in Formula (III) is magnesium. In one embodiment, the MXp moiety in Formula (III) is MgCl. [0171] In one embodiment, the compound of Formula (III) is made by contacting 1-bromo- 2-chloro-3-fluorobenzene with isopropyl magnesium chloride in 2-MeTHF. [0172] In one embodiment, Formula (II-A) is contacted with a non-nucleophilic base and the reaction proceeds as shown below in SCHEME I-A', generating an intermediate of Formula (IIc-A): .

[0173] The non-nucleophilic base is typically a base that deprotonates the compound of Formula (II-A) to form the intermediate of Formula (II-A) without significant nucleophilic attack on an electrophilic site of the compound of Formula (II-A), such as the carbonyl carbon. However, minor amounts of one or more side-reactions, even nucleophilic side-reactions, are acceptable. In one embodiment, the non-nucleophilic base is selected from the group consisting of a Grignard reagent, a lithium aluminum alkyl hydride, an alkyllithium compound, and an aryllithium compound. In another embodiment, the non-nucleophilic base is not employed. [0174] In another embodiment, the compound of Formula (IV-A) may be made via a Suzuki coupling reaction between the compound of Formula (II-A) and a compound of Formula (IIIa) in the presence of a catalyst, for example, a palladium catalyst, as shown in SCHEME I-Aa below, 57 NAI-1541064841v1 , wherein: R2 is C1-6 alkyl; each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C_1-6 alkyl; n is an integer from 0-5; Pg is a protecting group; and Y₁ and Y₂ are each independently OH or OR₃, wherein R₃ is C_1-6 alkyl. [0175] In one embodiment, the converting of the compound of Formula (IV-A) to the compound of Formula (I-A) comprises contacting the compound of Formula (IV-A) with a stereospecific reducing agent or a stereoselective or reducing agent to make a compound of Formula (V-A) as shown below in SCHEME III':

.

SCHEME III' [0176] In one embodiment, the converting of the compound of Formula (IV-A) to the compound of Formula (I-A) comprises or further comprises contacting the compound of Formula (V-A) with a deprotecting agent to make a compound of Formula (VI-A) as shown below in SCHEME IV-A: 58 NAI-1541064841v1 . SCHEME IV-A [0177] In one embodiment, Formula (VI-A) is produced as a salt, e.g., hydrochloride salt. [0178] In one embodiment, the converting of the compound of Formula (IV-A) to the compound of Formula (I-A) comprises or further comprises contacting the compound of Formula (VI-A), or salt thereof, with a compound of Formula (VII) to make the compound of Formula (I-A) as shown below in SCHEME V-A: .

[0179] In one embodiment, the converting of the compound of Formula (IV) to the compound of Formula (I-A) comprises: contacting the compound of Formula (IV-A) with a stereoselective or stereospecific reducing agent to make a compound of Formula (V-A) as shown in SCHEME III-A': ; NAI-

contacting the compound of Formula (V-A) with a deprotecting agent to make a compound of Formula (VI-A) or salt thereof as shown in SCHEME IV-A:

a to make the compound of Formula (I-A) as shown in SCHEME V-A: .

[0180] In one embodiment, the stereospecific/stereoselective reducing agent of SCHEME III-A' is (S,S)-Ts-Deneb, (S,S)-Ms-Deneb, or aluminum isopropoxide. In one embodiment, the stereospecific/stereoselective reducing agent is aluminum isopropoxide. In one embodiment, the compound of Formula (IV-A) is contacted with aluminum isopropoxide to make a compound of Formula (V-A). In one embodiment, the compound of Formula (IV-A) is contacted with aluminum isopropoxide to make a compound of Formula (V-A) at a temperature of about 50 °C. In one embodiment, the compound of Formula (IV-A) is contacted with aluminum isopropoxide to make a compound of Formula (V-A), wherein the contacting is in the presence of isopropyl alcohol and toluene. [0181] In one embodiment, the compound of Formula (V-A) is contacted with a deprotecting agent to make a compound of Formula (VI-A), wherein the deprotecting agent is hydrochloric acid. In one embodiment Formula (VI-A) is produced as a salt, e.g., hydrochloride salt. 60 NAI-1541064841v1 [0182] In one embodiment, the compound of Formula (VI-A), or salt thereof, is contacted with the compound of Formula (VII) in the presence of EDC, DMAP and an organic solvent. In one embodiment, the organic solvent is selected from the group consisting of DMF, DMSO, DMC, THF, DCM, and a mixture of any of the foregoing. In one embodiment, Compound 6 (or salt thereof) is contacted with 7-fluoro-2-oxoindoline-4-carboxylic acid (Compound 7) in the presence of EDC, DMAP, and DMF. In one embodiment, the compound of Formula (VI-A), or salt thereof, is contacted with the compound of Formula (VII) in the presence of EDC, DMAP, and DMF. [0183] In one embodiment, the compound of Formula (VI-A), or salt thereof, is contacted with the compound of Formula (VII) in the presence of TBTU and a base. In one embodiment, the base is selected from the group consisting of DIPEA, TMA, DEA, TEA, and DMAE. In one embodiment, the compound of Formula (VI-A), or salt thereof, is contacted with the compound of Formula (VII) in the presence of TBTU, DIPEA, and acetonitrile. [0184] In one embodiment, the compound of Formula (II-A) may be prepared by an initial step of converting a compound of Formula (IIa-A):

to an activated intermediate. [0185] In one embodiment, a compound of Formula (IIa-A) is contacted with a coupling agent to make the activated intermediate. In this embodiment, “coupling agent” refers to a reagent that generates an activated intermediate that, when reacted with N,O- dimethylhydroxylamine, couples the N,O-dimethylhydroxylamine thereto resulting in a compound of Formula (II-A): .

61 NAI-1541064841v1 [0186] The coupling agent is not particularly limited and one of skill in the art will recognize that a wide variety of coupling agents may be used to generate a compound of Formula (II-A) from a compound of Formula (IIa-A). In one embodiment, the coupling agent is selected from the group consisting of EDC/HOBt, EDC/DMAP, DIPEA/BOP, NMM/IBCF, DCC/DIPEA/DMAP, PCl3 phosphorus trichloride, and CDI. In one embodiment, the coupling agent is CDI, and the activated intermediate is a compound of Formula (II-A'): .

[0187] In one embodiment, a compound of Formula (IIa-A) is contacted with a coupling agent to make the activated intermediate. In one embodiment, the compound of Formula (IIa-A) is contacted with a coupling agent in the presence of 2-MeTHF. In one embodiment, the compound of Formula (IIa-A) is contacted with a coupling agent at a temperature of about 0 °C. [0188] In one embodiment, the activated intermediate may then be contacted with N,O- dimethylhydroxylamine to make the compound of Formula (II-A) as shown in SCHEME VI-A below:

. SCHEME VI-A [0189] In one embodiment, the activated intermediate is contacted with N,O- dimethylhydroxylamine in the presence of 2-MeTHF, DMF, THF, DCM, CPME, ETBE, NBP, DMSO, t-butanol, acetonitrile, or a mixture of any of the foregoing, to make the compound of Formula (II-A). In one embodiment, the activated intermediate is contacted with N,O- dimethylhydroxylamine in the presence of 2-MeTHF to make the compound of Formula (II-A). 62 NAI-1541064841v1 In one embodiment, the activated intermediate is contacted with N,O-dimethylhydroxylamine in the presence of 2-MeTHF and DMF to make the compound of Formula (II-A). [0190] A compound of Formula (VII) may be prepared by methods known in the art, for example, as disclosed in Chinese Patent Publication No. CN113801053 A, which is incorporated by reference in its entirety. In one embodiment, the compound of Formula (VII) is made by a method comprising: contacting a compound of Formula (VIIa) with a catalyst to make a compound of Formula (VIIb) as shown in SCHEME VII below:

contacting the compound of Formula (VIIb) with a hydrolyzing agent to make the compound of Formula (VII) as shown in SCHEME VIII below: .

[0191] In one embodiment, the hydrolyzing agent is selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium methoxide, potassium tert-butoxide, or any combination thereof. [0192] In another embodiment, the compound of Formula (VII) is made by a method comprising: contacting a compound of Formula (VIIa) with tert-butyldimethylsilyl chloride to make a compound of Formula (VIII) as shown in SCHEME IX below: 63 NAI-1541064841v1 ; contacting the compound of Formula (VIII) with isopropyl magnesium chloride and n- butyl lithium to make an intermediate of Formula (VIIIa) as shown in SCHEME X below:

; SCHEME X contacting the intermediate of Formula (VIIIa) with carbon dioxide to make intermediate of Formula (VIIIb) as shown in SCHEME XI below:

contacting the intermediate of Formula (VIIIb) with a deprotecting agent to make a compound of Formula (VII) as shown in SCHEME XII below: 64 NAI-1541064841v1

. [0193] In some embodiments, the compound of Formula (VIIa) is contacted with tert- butyldimethylsilyl chloride to make a compound of Formula (VIII) in the presence of a base. In one embodiment, the base comprises sodium hydride (NaH), lithium hydride (LiH), an alkyl lithium base (e.g., methyllithium, ethyllithium, isopropyllithium, n-butyllithium, sec- butyllithium, tert-butyllithium), or a borohydride salt (e.g., NaBH4 or KBH4). In one embodiment, the base comprises NaH. [0194] In some embodiments, the carbon dioxide is pressurized carbon dioxide. [0195] In some embodiments, the bis-silyl deprotecting agent comprises an acid (e.g., ammonium chloride, aqueous phosphoric acid, acidic aqueous THF, or acidic methanol) or a fluoride (e.g., ammonium fluoride, tetrabutylammonium fluoride, tetrabutylammonium chloride, potassium fluoride, or aqueous hydrofluoric acid. In one embodiment, the bis-silyl deprotecting agent comprises aqueous phosphoric acid. [0196] In another aspect, the present disclosure provides a compound of Formula (I-A) , wherein:

R1 is selected from the group consisting of F, Cl, Br, I, and C1-6 alkyl; 65 NAI-1541064841v1 R2 is C1-6 alkyl; each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C1-6 alkyl; and n is an integer from 0-5, wherein the compound of Formula (I-A) is prepared by any of the methods disclosed herein and wherein the method produces the compound at high stereochemical purity without any need for chiral separation. For example, using the methods disclosed herein, a composition comprising a compound of Formula (I-A) may be produced, wherein the composition exhibits a stereochemical purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at last about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.8%, and wherein the composition is produced without subjecting the compounds of Formulas (IV-A), (V-A), (VI-A) or (I-A) to a chiral separation. 6.5. Methods of Synthesizing Stereoisomers of Compound 1 [0197] With reference to compounds of Formulas (I-A), (I-B), (I-C), and (I-D), in one embodiment, R₁ is F, R₂ is propyl, n is 2, one R is F, and one R is Cl. As such, the compounds of Formulas (I-A), (I-B), (I-C), and (I-D) are Compounds 1-A, 1-B, 1-C, and 1-D. Therefore, in another aspect, the present disclosure provides a method of making Compounds 1-A, 1-B, 1-C, and 1-D. [0198] In one embodiment, Compounds 1-A and 1-B may be prepared by the reaction described in SCHEMES 6-A,1-A, and 3-A to yield Compounds 5-A and 5-B:

SCHEME 6-A 66 NAI-1541064841v1

. [0199] Compound 5-A and 5-B may be subjected to chiral separation and each separated stereoisomer reacted separately with a deprotecting agent to make Compounds 6-A and 6-B as shown in SCHEME 4-A and 4-B below: . NAI-

SCHEME 4-B [0200] In one embodiment, the deprotecting agent is hydrochloric acid and Compounds 6-A and 6-B are produced as a hydrochloride salt. [0201] Each of Compounds 6-A and 6-B may then be reacted with Compound 7 to make Compounds 1-A and 1-B as shown in SCHEME 5-A and SCHEME 5-B below: .

[0202] Compounds 1-C and 1-D may be prepared first by preparing Compound 5-C and 5-D by the reactions shown in SCHEMES 6-C, 1-C, and 3-C:

. [0203] Compound 5-C and 5-D may then be subjected to chiral separation and each separated stereoisomer reacted separately with a deprotecting agent to make Compounds 6-C and 6-D, or salts thereof, as shown in SCHEME 4-C and 4-D below:

69 NAI-1541064841v1 Cl OH Cl OH deprotecting agent F F .

[0204] In one embodiment, the deprotecting agent is hydrochloric acid and Compounds 6-C and 6-D are produced as a hydrochloride salt. [0205] Each of Compounds 6-C and 6-D (or salts thereof) may then be reacted with Compound 7 to make Compounds 1-C and 1-D as shown in SCHEME 5-C and SCHEME 5-D below: . NAI-

[0206] In one embodiment, Compound 4-C may be made via a Suzuki coupling reaction between Compound 2-C and a compound of Formula 3a in the presence of a catalyst, for example, a palladium catalyst, as shown in SCHEME 1-Ca below,

, SCHEME 1-Ca wherein Y₁ and Y₂ are each independently OH or OR₃, wherein R₃ is C_1-6 alkyl. [0207] In another aspect, the present disclosure provides a method of making Compound 1-A via a method that produces a high yield of the compound with high stereochemical purity without the use of any chiral separation, as was described in other embodiments herein. [0208] In one embodiment, Compound 1-A with high stereochemical purity may be made via a method comprising: contacting tert-butyl (R)-(1-(methoxy(methyl)amino)-1-oxopentan-2-yl)carbamate (Compound 2-A) with a compound of Formula 3, optionally in the presence of a base, to make tert-butyl (R)-(1-(2-chloro-3-fluorophenyl)-1-oxopentan-2-yl)carbamate (Compound 4-A) as shown in SCHEME 1-A below:

, SCHEME 1-A wherein: X is Cl, Br, or I; and 71 NAI-1541064841v1 M is an alkali metal and p is 0; or M is an alkaline earth metal and p is 1; and converting Compound 4-A to Compound 1-A as shown in SCHEME 2-A below:

SCHEME 2-A wherein the converting of Compound 4-A to Compound 1-A according to SCHEME 2-A comprises contacting Compound 4-A with a stereospecific reducing agent or a stereoselective reducing agent. In one embodiment, the stereospecific/stereoselective reducing agent is (S,S)-Ts- Deneb, (S,S)-Ms-Deneb, or aluminum isopropoxide. In one embodiment, the stereospecific/stereoselective reducing agent is aluminum isopropoxide. [0209] In one embodiment, M of the MXp moiety of the compound of Formula 3 is an alkaline earth metal. In one embodiment, M of the MX_p moiety of the compound of Formula 3 is magnesium. In one embodiment, the MXp moiety of the compound of Formula 3 is MgCl. In one embodiment, the non-nucleophilic base is selected from the group consisting of a Grignard reagent, a lithium aluminum alkyl hydride, an alkyllithium compound, and an aryllithium compound. [0210] In one embodiment, the compound of Formula 3 is made by contacting 1-bromo-2- chloro-3-fluorobenzene with isopropyl magnesium chloride in 2-MeTHF. [0211] In one embodiment, Compound 2-A is contacted with a non-nucleophilic base and the reaction proceeds as shown below, generating an intermediate of Compound 2c-A as shown in SCHEME 1-A' below: 72 NAI-1541064841v1 . [0212] In another embodiment, Compound 4-A may be made via a Suzuki coupling reaction between Compound 2-A and a compound of Formula 3a in the presence of a catalyst, for example, a palladium catalyst, as shown in SCHEME 1-Aa below, ,

wherein Y₁ and Y₂ are each independently OH or OR₃, wherein R₃ is C_1-6 alkyl. [0213] In one embodiment, the converting of Compound 4-A to Compound 1-A comprises contacting Compound 4-A with a stereospecific reducing agent or a stereoselective reducing agent to make tert-butyl ((1S,2R)-1-(2-chloro-3-fluorophenyl)-1-hydroxypentan-2-yl)carbamate (Compound 5-A) as shown in SCHEME 3-A' below:

. NAI-1541064841v1

[0214] In one embodiment, the converting of Compound 4-A to Compound 1-A comprises or further comprises: contacting Compound 5-A with a deprotecting agent to make (1S,2R)-2-amino-1-(2- chloro-3-fluorophenyl)pentan-1-ol, or a salt thereof (Compound 6-A) as shown in SCHEME 4- A below: .

[0215] In one embodiment, the deprotecting agent is hydrochloric acid and a hydrochloride salt of Compound 6-A is formed. [0216] In one embodiment, the converting of Compound 4-A to Compound 1-A comprises or further comprises: contacting Compound 6-A (or a salt thereof) with 7-fluoro-2-oxoindoline-4-carboxylic acid (Compound 7) to make Compound 1-A as shown in SCHEME 5-A below: .

[0217] In one embodiment, the converting of Compound 4-A to Compound 1-A comprises: contacting Compound 4-A with a stereospecific reducing agent or a stereoselective reducing agent to make tert-butyl ((1S,2R-1-(2-chloro-3-fluorophenyl)-1-hydroxypentan-2- yl)carbamate (Compound 5-A) as shown in SCHEME 3-A' below: 74 NAI-1541064841v1 ; contacting Compound 5-A with a deprotecting agent to make (1S,2R)-2-amino-1-(2- chloro-3-fluorophenyl)pentan-1-ol (Compound 6-A), or a salt thereof as shown in SCHEME 4- A below:

contacting Compound 6-A with 7-fluoro-2-oxoindoline-4-carboxylic acid (Compound 7) to make Compound 1-A as shown in SCHEME 5-A below: .

[0218] In one embodiment, Compound 4-A is contacted with a stereospecific reducing agent or a stereoselective reducing agent to make Compound 5-A. In one embodiment, the stereospecific/stereoselective reducing agent is (S,S) -Ts-Deneb, (S,S)-Ms-Deneb, or aluminum isopropoxide. In one embodiment, the stereospecific/stereoselective reducing agent is aluminum isopropoxide. In one embodiment, Compound 4-A is contacted with aluminum isopropoxide to 75 NAI-1541064841v1 make Compound 5-A, wherein the contacting is at a temperature of about 50 °C. In one embodiment, Compound 4-A is contacted with aluminum isopropoxide to make Compound 5-A, wherein the contacting is in the presence of isopropyl alcohol and toluene. [0219] In one embodiment, Compound 5-A is contacted with a deprotecting agent to make Compound 6-A, wherein the deprotecting agent is hydrochloric acid. [0220] In one embodiment, Compound 6-A (or salt thereof) is contacted with 7-fluoro-2- oxoindoline-4-carboxylic acid (Compound 7) in the presence of EDC, DMAP and an organic solvent. In one embodiment, the organic solvent is selected from the group consisting of DMF, DMSO, DMC, THF, DCM, and a mixture of any of the foregoing. In one embodiment, Compound 6-A (or salt thereof) is contacted with 7-fluoro-2-oxoindoline-4-carboxylic acid (Compound 7) in the presence of EDC, DMAP, and DMF. [0221] In one embodiment, Compound 6-A (or salt thereof) is contacted with 7-fluoro-2- oxoindoline-4-carboxylic acid (Compound 7) in the presence of TBTU and a base. In one embodiment, the base is selected from the group consisting of DIPEA, TMA, DEA, TEA, and DMAE. In one embodiment, Compound 6-A (or salt thereof) is contacted with 7-fluoro-2- oxoindoline-4-carboxylic acid (Compound 7) in the presence of TBTU and DIPEA. In one embodiment, Compound 6-A (or salt thereof) is contacted with 7-fluoro-2-oxoindoline-4- carboxylic acid (Compound 7) in the presence of TBTU, DIPEA, and acetonitrile. [0222] In one embodiment, Compound 2-A may be prepared an initial step of converting (R)-2-((tert-butoxycarbonyl)amino)pentanoic acid (Compound 2a-A):

to an activated intermediate. [0223] In one embodiment, Compound 2a-A is contacted with a coupling agent to make the activated intermediate. In this embodiment, “coupling agent” refers to a reagent that generates an activated intermediate that, when reacted with N,O-dimethylhydroxylamine, couples the N,O- dimethylhydroxylamine thereto resulting in Compound 2-A: 76 NAI-1541064841v1 . [0224] The coupling agent is not particularly limited and one of skill in the art will recognize that a wide variety of coupling agents may be used to generate Compound 2-A from Compound 2a-A. In one embodiment, the coupling agent is CDI, and the activated intermediate is Compound 2-A': .

[0225] In one embodiment, Compound 2a-A is contacted with a coupling agent to make the activated intermediate. In one embodiment, Compound 2a-A is contacted with a coupling agent in the presence of 2-MeTHF. In one embodiment, Compound 2a-A is contacted with a coupling agent at a temperature of about 0 °C. [0226] In one embodiment, the activated intermediate may then be contacted with N,O- dimethylhydroxylamine to make Compound 2-A as shown in SCHEME 6-A below:

. SCHEME 6-A [0227] In one embodiment, Compound 2-A is prepared by a method comprising: contacting (R)-2-((tert-butoxycarbonyl)amino)pentanoic acid (Compound 2a-A) with a coupling agent to make an activated intermediate; and 77 NAI-1541064841v1 contacting the activated intermediate with N,O-dimethylhydroxylamine to make tert- butyl (R)-(1-(methoxy(methyl)amino)-1-oxopentan-2-yl)carbamate (Compound 2-A) as shown in SCHEME 6-A below:

. SCHEME 6-A [0228] In one embodiment, the activated intermediate is contacted with N,O- dimethylhydroxylamine in the presence of 2-MeTHF, DMF, THF, DCM, CPME, ETBE, NBP, DMSO, t-butanol, acetonitrile, or a mixture of any of the foregoing, to make Compound 2-A. In one embodiment, the activated intermediate is contacted with N,O-dimethylhydroxylamine in the presence of 2-MeTHF to make Compound 2-A. In one embodiment, the activated intermediate is contacted with N,O-dimethylhydroxylamine in the presence of 2-MeTHF and DMF to make Compound 2-A. [0229] In one embodiment, Compound 7 is made by a method comprising: contacting 4-bromo-7-fluoroindolin-2-one (Compound 7a) with a suitable catalyst to make methyl 7-fluoro-2-oxoindoline-4-carboxylate (Compound 7b) as shown in SCHEME 7 below:

contacting Compound 7b with a hydrolyzing agent to make Compound 7 as shown in SCHEME 8 below: 78 NAI-1541064841v1 . [0230] In one embodiment, the hydrolyzing agent is selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium methoxide, potassium tert-butoxide, or any combination thereof. [0231] In one embodiment, Compound 7 is made by a method comprising: contacting 4-bromo-7-fluoroindolin-2-one (Compound 7a) with tert-butyldimethylsilyl chloride to make 4-bromo-1-(tert-butyldimethylsilyl)-2-((tert-butyldimethylsilyl)oxy)-7-fluoro- 1H-indole (Compound 8) as shown in SCHEME 9 below: ;

a agent to 7 as shown in SCHEME 12 below:

.

SCHEME 12

[0232] In some embodiments, Compound 7a is contacted with tert-butyldimethylsilyl chloride to make Compound 8 in the presence of a base. In one embodiment, the base comprises sodium hydride (NaH), lithium hydride (LiH), an alkyl lithium base (e.g., methyllithium, ethyllithium, isopropyllithium, n-butyllithium, sec-butyllithium, tert-butyllithium), or a borohydride salt (e.g., NaBH4 or KBH4). In one embodiment, the base comprises NaH. [0233] In some embodiments, the carbon dioxide is pressurized carbon dioxide. [0234] In some embodiments, the bis-silyl deprotecting agent comprises an acid (e.g., ammonium chloride, aqueous phosphoric acid, acidic aqueous THF, or acidic methanol) or a fluoride (e.g., ammonium fluoride, tetrabutylammonium fluoride, tetrabutylammonium chloride, potassium fluoride, or aqueous hydrofluoric acid). In one embodiment, the bis-silyl deprotecting agent comprises aqueous phosphoric acid. 80 NAI-1541064841v1 [0235] In one embodiment, the aforementioned method of producing Compound 1-A at high stereochemical purity is achieved without subjecting the compound or any precursors thereto to any chiral separation. For example, using the methods disclosed herein, a composition comprising Compound 1-A may be produced, wherein the composition exhibits a stereochemical purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at last about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.8%, and wherein the composition is produced without subjecting compounds 4-A, 5-A, 6-A, or 1-A to a chiral separation. In one embodiment, a composition comprising Compound 1-A may be produced, wherein the composition consists essentially of Compound 1-A, wherein the stereochemical purity of (Compound 1-A) is about 90% or greater, optionally about 95% or greater, optionally about 98.5% or greater, further optionally about 99% or greater, further optionally about 99.5% or greater, still further optionally about 99.8% or greater. In one embodiment, a composition comprising Compound 1-A may be produced, wherein the composition comprises stereochemically pure Compound 1-A. In certain embodiments, the stereochemical purity of the Compound 1-A in the composition is 99.8% or greater. 6.6. Pharmaceutical Compositions [0236] In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound of Formula (I-A), (I-B), (I-C), or (I-D), such as Compound 1-A, 1-B, 1- C, or 1-D, synthesized as disclosed in any of Sections 6.2, 6.3, 6.4, and 6.5 herein, and a pharmaceutically acceptable excipient. [0237] In another aspect, the present disclosure provides a pharmaceutical composition comprising a composition of a compound of Formula (I-A), such as Compound 1-A, and a pharmaceutically acceptable excipient, wherein the composition of the compound of Formula (I- A) has a stereochemical purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at last about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.8%, wherein the composition is produced without subjecting the product of contacting the compound of Formula (VI-A), or salt thereof, with the compound of Formula (VII) to a chiral separation. [0238] In another aspect, the present disclosure provides a pharmaceutical composition comprising a composition of Compound 1-A and a pharmaceutically acceptable excipient, 81 NAI-1541064841v1 wherein the composition of Compound 1-A has a stereochemical purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at last about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.8%, and wherein the composition is produced without subjecting the product of contacting Compound 6-A with 7-fluoro-2-oxoindoline-4-carboxylic acid (Compound 7) to a chiral separation. In one embodiment, the present disclosure provides a pharmaceutical composition comprising a composition of Compound 1-A and a pharmaceutically acceptable excipient, wherein the composition consists essentially of Compound 1-A, wherein the stereochemical purity of Compound 1-A is about 90% or greater, optionally about 95% or greater, optionally about 98.5% or greater, further optionally about 99% or greater, further optionally about 99.5% or greater, still further optionally about 99.8% or greater. [0239] In another aspect, the present disclosure provides a pharmaceutical composition comprising a composition of Compound 1-A and a pharmaceutically acceptable excipient, wherein the composition is stereochemically pure with respect to Compound 1-A. In certain embodiments, the stereochemical purity of the composition is 99.8% or greater with respect to Compound 1-A. [0240] In some embodiments, higher stereochemical purity, for example, 98% or higher (e.g., 99.8% or greater), of a pharmaceutical composition comprising Compound 1-A is associated with increased tolerability, for example, minimal nausea and/or emesis, in a subject to which the pharmaceutical composition has been administered. In certain embodiments, higher stereochemical purity, for example, 98% or higher (e.g., 99.8% or greater), of a pharmaceutical composition comprising Compound 1-A is associated with no nausea in a subject to which the pharmaceutical composition has been administered. In certain embodiments, higher stereochemical purity, for example, 98% or higher (e.g., 99.8% or greater), of a pharmaceutical composition comprising Compound 1-A is associated with no emesis in a subject to which the pharmaceutical composition has been administered. In certain embodiments, higher stereochemical purity, for example, 98% or higher (e.g., 99.8% or greater), of a pharmaceutical composition comprising Compound 1-A is associated with no nausea or emesis in a subject to which the pharmaceutical composition has been administered. [0241] In one embodiment, the present disclosure provides a pharmaceutical composition comprising a composition or a compound of Formula (I-A), such as Compound 1-A and one or 82 NAI-1541064841v1 more pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical preparations may be for use in treating or preventing a disease, disorder, or condition as described herein or for use in the manufacture of a medicament for treating or preventing a disease, disorder, or condition as disclosed herein. [0242] Pharmaceutical compositions may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, inhaled, intranasal, topical (including buccal, sublingual, or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the pharmaceutically acceptable excipient(s). [0243] Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions. [0244] For instance, for oral administration in the form of a tablet or capsule, an active ingredient (e.g., Compound 1 or a stereoisomer thereof) can be combined with an oral, non-toxic pharmaceutically acceptable inert excipient such as ethanol, glycerol, water, and the like. Flavoring, preservative, dispersing and coloring agent can also be present. [0245] Pharmaceutical compositions may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient, vehicle or carrier. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules, or the like in the case of solid compositions. [0246] In one embodiment, a unit dosage composition contains a daily dose or sub-dose, or an appropriate fraction thereof, of an active ingredient. Such unit doses may therefore be administered once or more than once a day. Such pharmaceutical compositions may be prepared by any of the methods well known in the pharmacy art. 83 NAI-1541064841v1 [0247] It should be understood that in addition to the ingredients particularly mentioned above, the compositions may include other agents conventional in the art having regard to the type of formulation in question. 6.7. Therapeutic Methods [0248] In another aspect, the present disclosure a method of treating a subject in need thereof with a compound (e.g., Compound 1 or any of its stereoisomers, e.g., 1-A) synthesized as disclosed in any of Sections 6.2, 6.3, 6.4, and 6.5 herein. [0249] Such a compound may be administered in a pharmaceutical composition as described in Section 6.6, in an effective amount, which will be readily determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient’s symptoms, and the like. [0250] In one embodiment, the present disclosure provides a method of treating a disease, condition, or disorder related to the polymerization of alpha-1-antitrypsin in a subject in need thereof comprising administering to the subject a therapeutically effective amount of Compound 1 or any of its stereoisomers, e.g., 1-A. In one embodiment, the present disclosure provides use of Compound 1 or any of its stereoisomers, e.g., 1-A, in the manufacture of a medicament for use treating a disease, condition, or disorder related to the polymerization of alpha-1-antitrypsin in a subject in need thereof. In one embodiment, the disease or disorder related to the polymerization of alpha-1 antitrypsin is with a Z-AT mutation of alpha-1 antitrypsin. In one embodiment, the disease, disorder, or condition related to the polymerization of alpha-1-antitrypsin is liver disease. [0251] For the treatment of liver disease, the compounds or pharmaceutical compositions of the invention may be administered with other therapeutic agents useful in treating these diseases. Therefore, in another aspect, the present disclosure provides a method of treating a disease, disorder, or condition related to the polymerization of alpha-1-antitrypsin (e.g., liver disease), comprising administering a therapeutically effective amount of Compound 1 or any of its stereoisomers, e.g., 1-A, together with a second therapeutically active agent. In one embodiment, the present disclosure provides use of Compound 1 or any of its stereoisomers in the manufacture of a medicament for use treating a disease, condition, or disorder related to the polymerization of alpha-1-antitrypsin (e.g., liver disease) in a subject in need thereof, wherein 84 NAI-1541064841v1 the medicament is intended to be administered to the subject together with a second therapeutic agent effective to treat liver disease. 7. EXAMPLES Example 1: Synthesis of Compound 1-A (N-[(1R)-1-[(S)-(2-chloro-3- fluorophenyl)hydroxymethyl]butyl]-7-fluoro-2,3-dihydro-2-oxo-1H-indole-4-carboxamide) Step 1: Preparation of tert-butyl (R)-(1-(methoxy(methyl)amino)-1-oxopentan-2- yl)carbamate (Compound 2-A) Part A [0252] (R)-2-((tert-butoxycarbonyl)amino)pentanoic acid dicyclohexylamine (1 eq), was added to a reactor followed by 2-MeTHF (10 vol) and agitated. The slurry was cooled to -10 °C to -5 °C after which 10% aqueous sulfuric acid (5 vol) was added slowly over a period of 15 minutes maintaining the temperature at or below 20 °C. The mixture was then warmed to room temperature and stirred for at least 30 minutes. The mixture was allowed to settle, and the aqueous (bottom) layer was discharged. The remaining organic layer was washed with 10% aqueous sulfuric acid, a mixture of water and brine, and brine. Azeodistillation followed by filtration provided a solution of (R)-2-((tert-butoxycarbonyl)amino)pentanoic acid in 2-MeTHF. Part B [0253]

(1.2 eq), and 2-MeTHF (4 vol) were combined and agitated. TEA (1.2 eq) was slowly added over a period of 30 minutes at a temperature of 30 °C or less. The reaction mixture was stirred at room temperature for at least one hour then cooled to -2 °C to 2 °C. 85 NAI-1541064841v1 Part C [0254]

under nitrogen atmosphere. The slurry was cooled to -2 to 2 °C after which the (R)-2-((tert- butoxycarbonyl)amino)pentanoic acid solution in 2-MeTHF was added slowly while maintaining a temperature of no more than 2 °C, and the reaction mixture was stirred for at least 1 hour. [0255] The reaction mixture was then slowly added to the Part B reactor while maintaining the temperature of the reaction mixture at or below 2 °C. The reaction mixture was then warmed to room temperature and stirred for at least 3 hours. The reaction mixture was then slowly quenched with HCl (1N) at a temperature of 25 °C or less, followed by washing with HCl (1 N), with potassium phosphate dibasic (5% aqueous), with water, and with brine. The product was azeotropically distilled, cooled to room temperature, and filtered to yield a solution of tert-butyl (R)-(1-(methoxy(methyl)amino)-1-oxopentan-2-yl)carbamate (2-A) in 2-MeTHF. Step 2: Preparation of tert-butyl (R)-(1-(2-chloro-3-fluorophenyl)-1-oxopentan-2- yl)carbamate (Compound 4-A) Part A

[0256] A solution of isopropylmagnesium chloride (2 M in THF, 1.87 eq) was added to 1- bromo-2-chloro-3-fluorobenzene (1.7 eq) in 2-MeTHF (3.8 vol) maintaining the temperature of the reaction mixture at or below 5 °C. The reaction mixture was stirred for at least 2.5 hours. Part B

(methoxy(methyl)amino)-1-oxopentan-2-yl)carbamate (Compound 2-A) and the reaction mixture was cooled to -10 to -5 °C. A solution of isopropylmagnesium chloride (2 M in THF, 1.0 eq) was then added over a period of 1 hour, maintaining the temperature at or below 5 °C. Part C

maintaining a temperature at or below 15 °C. The reaction mixture was then warmed to 46 °C to 48 °C over a period of 2 hours then stirred at that temperature for 7 hours. The reaction mixture was cooled to -5 °C – 0 °C, then slowly quenched with acetic acid (6 eq) at room temperature, followed by washing with acetic acid, with water and brine, with aqueous potassium phosphate dibasic (15%), with water and brine, and with brine. The reaction mixture was then distilled to change solvents to 2-propanol. Following cooling to -5 °C to 0 °C over 2 hours, water (8 vol) was charged to crystallize the product, which was then isolated by filtration and dried to yield tert-butyl (R)-(1-(2-chloro-3-fluorophenyl)-1-oxopentan-2-yl)carbamate (Compound 4-A). 87 NAI-1541064841v1 Step 1-3: Preparation of Compound 6-A

- isopropoxide (0.2 eq) at room temperature. The reaction mixture was heated to 50 °C over 2 hours and agitated at the same temperature for at least 10 hours. The reaction mixture was then cooled to room temperature and quenched with HCl (1N), followed by washing with HCl (1 N), with potassium phosphate dibasic (5% aqueous), with water, and with brine. [0260] The reaction mixture was then distilled to switch solvents to toluene, filtered, and cyclopentyl methyl ether (CMPE, 4 vol) was added. HCl (3 M) in CPME (7 eq) was then added slowly over a period of 1 hour while maintaining the temperature below 30 °C. The mixture was then stirred at room temperature for at least 24 hours. (1S,2R)-2-amino-1-(2-chloro-3- fluorophenyl)pentan-1-ol hydrochloride (Compound 6-A) was isolated by filtration, and dried. Step 1-4: Preparation of Compound 1-A

[0261] 7-Fluoro-2-oxoindoline-4-carboxylic acid (Compound 7, 1.2 eq), DMAP (2.5 eq) and EDC (1.5 eq) were charged to a pre-cooled reactor at 0 °C under nitrogen. DMF (5.5 vol) was added followed by (1S,2R)-2-amino-1-(2-chloro-3-fluorophenyl)pentan-1-ol hydrochloride (Compound 6-A). The reaction mixture was warmed to room temperature and stirred for at least 4 hours. Ethyl acetate (8 vol) was added, and the reaction mixture cooled to 13 – 17 °C.1 N HCl 88 NAI-1541064841v1 (5 vol) was then charged to the reactor and the reaction mixture was agitated. The reaction mixture was then filtered, and the filtrate allowed to settle, after which the lower aqueous phase was removed from the upper organic phase. The organic phase was then washed with 1 N HCl and brine, potassium phosphate dibasic (5%) and brine, and then with water and brine, followed by brine. The reaction mixture was then distilled with ethyl acetate. The temperature was adjusted to 50-55 °C, and SilaMet S thiol resin (10 wt. %) was added and the resulting mixture stirred at the same temperature for at least 3 hours. The reaction mixture was then filtered, and the solvent was switched to ethanol. The reaction mixture was cooled to 15-20 °C over at least 1.5 h and stirred. Heptane (27 vol) was then added to obtain a slurry. Compound 1-A was isolated by filtration, washed with 4:1 heptane/ethanol, dried, and analyzed by chiral HPLC, which revealed no detectable amount of Compound 1-B, 1-C, or 1-D. Due to instrument error of ± 0.2%, the stereochemical purity of the obtained compound was determined to be 99.8% or greater. [0262] First Exemplary Synthesis of Compound 7: 4-bromo-7-fluoroindoline-2,3-diketone (50 g, 205 mmol) and hydrazine hydrate (15.4 g (250 mmol, 80%) was added to ethylene glycol (250 mL) in a 500 mL three-necked flask and the reaction mixture was stirred for 30 minutes maintaining the temperature at 55-65 ℃. The reaction was allowed to occur for 2-6 hours. The temperature was lowered to room temperature, and filtration was carried out to obtain 60 g of a crude product. In a separate three-neck flask, ethylene glycol (500 mL) and sodium acetate (1.7 g, 20.5 mmol) were added to the crude product (60 g) and the reaction temperature was raised to 145 ℃ after which the contents of the flask were concentrated under reduced pressure to a third of the original volume, cooled to 0-10 ℃, filtered, leached with 100 mL of water, and dried in vacuum to obtain 40 g of 4-bromo-7-fluoroindolin-2-one. Methanol (300 mL), 4-bromo- 7-fluoroindolin-2-one (30 g), TEA (27 mL), and (dppf)PdCl2 (21.55 g) were charged to a 500 mL pressure reactor, which was purged with CO three times, maintaining the pressure of CO in the reaction kettle at 0.5 mPa and the temperature at 115-125 ℃. The reaction was carried out for 10 h after which the reaction mixture was cooled to room temperature and concentrated. DMF and DCM were added, and the mixture was stirred for 30 minutes then filtered to obtain 23 g of 4-(but-1-en-2-yl)-7-fluoroindolin-2-one. Methanol (100 mL) and 4-(but-1-en-2-yl)-7- fluoroindolin-2-one (20 g, 95.7 mmol) were charged to a 250 mL three-neck flask and sodium hydroxide (7.7 g, 191 mmol) and water (100 mL) were added. The reaction mixture was stirred 89 NAI-1541064841v1 for 30 minutes at a temperature of 40 to 60 ℃ for 5 hours. The reaction mixture was then concentrated, and the pH adjusted to 1-2 with 1 M hydrochloric acid. The mixture was filtering to obtain 17.7 g of Compound 7. [0263] Second Exemplary Synthesis of Compound 7:

to a mixture of sodium hydride in mineral oil (60% w/w, 2.76 eq) and THF (3.0 vols) at 5 ± 5 °C in a first vessel. The mixture was stirred for 10 mins at 5 ± 5 °C and a solution of tert- butyldimethylsilyl chloride (TBDMS-Cl, 2.56 eq) in THF (2.5 vols) was charged at 5 ± 5 °C. The mixture was warmed to 20 to 25 °C and stirred for 2 hours until reaction completion. The reaction mixture was cooled to -10 °C. A biphasic mixture of 1 M potassium phosphate buffer (10.0 vols) and n-heptane (12.0 vols) was cooled to 15 ± 5 °C in a second vessel. The contents of the first vessel were added to the second vessel over approximately 1 hour, during which time off-gassing was controlled by the rate of addition and a temperature of 15 ± 5 °C was maintained. The residue in the first vessel was rinsed into the second vessel with THF (1.0 vols). The biphasic mixture was warmed to 25 ± 5 °C and the two phases were separated. The organic layer was washed with water (10.0 vols) at 25 ± 5 °C and distilled in vacuo to 8 volumes at approximately 50 °C. n-Heptane (3.0 vols) was added, and the mixture was distilled in vacuo to 8 volumes at approximately 50 °C. The mixture was cooled to 25 ± 5 °C and analyzed for THF content by GC-HS. Basic aluminum oxide (0.1% w/w) was added, followed by rinsing with n- heptane (1.0 vols) and the mixture was stirred at 25 ± 5 °C for about 1 hour. The slurry was filtered into a third vessel, followed by a line rinse with n-heptane (2.0 vols). The following procedure was repeated three times: isopropanol (9.0 vols) was added, and the solution was distilled to 7 to 8 volumes at NMT 60 °C. The mixture was heated at 75 to 80 °C until dissolution was confirmed. The solution was cooled to 17 ± 3 °C at 0.2 °C/min and held at this temperature for approximately 22 hours. The solid was filtered off, washed with isopropanol (4.0 90 NAI-1541064841v1 vols) at 17 ± 3 °C, and dried in vacuo at 50 ± 5 °C. This resulted in 4-bromo-1-(tert- butyldimethylsilyl)-2-((tert-butyldimethylsilyl)oxy)-7-fluoro-1H-indole as Compound 8:

THF (6.8 vols) was cooled to 4°C in a vessel. Isopropyl magnesium chloride in THF (2 M, 0.47 eq) was added to the vessel over 8 minutes, followed by the addition of n-butyl lithium in hexanes (2.5 M, 0.93 eq) over 10 minutes at 0-5 °C. The mixture was stirred for 15 minutes. Gaseous carbon dioxide (1.5 eq) was added by sparging, with the gas addition rate adjusted as needed to maintain the reaction temperature ≤10 °C, after which time the mixture was stirred at 0 °C for 70 minutes. The mixture was diluted with methanol (1.4 vol) then 85% w/w H₃PO₄ (2.5 eq) was added. The mixture was warmed to 25°C over 3 hours and stirred for at least 16 hours. Water (6 vols) was added and the mixture was agitated for 30 minutes. The pH of the mixture was adjusted to pH ≤ 2 by addition of 85% w/w aqueous H₃PO₄ and stirred for another 30 minutes. The solids were isolated by filtration, washed with water (2 x 1.4 vols) THF:MeOH (85:15 v/v, 1.4 vol), then MeOH (1.4 vol) and dried at 50 °C under vacuum to give Compound 7. 91 NAI-1541064841v1 Example 2: Synthesis of N-[(1S)-1-[(S)-(2-chloro-3-fluorophenyl)hydroxymethyl]butyl]-7- fluoro-2,3-dihydro-2-oxo-1H-indole-4-carboxamide (Compound 1-C) and (N-[(1S)-1-[(R)- (2-chloro-3-fluorophenyl)hydroxymethyl]butyl]-7-fluoro-2,3-dihydro-2-oxo-1H-indole-4- carboxamide) (Compound 1-D) Step 2-1: Preparation of tert-butyl (S)-(1-(methoxy(methyl)amino)-1-oxopentan-2- yl)carbamate Part A [0266] (S)-2-((tert-butoxycarbonyl)amino)pentanoic acid dicyclohexylamine (1 eq) was added to a reactor followed by 2-MeTHF (10 vol) and agitated. The slurry was cooled to -10 °C to -5 °C after which 10% aqueous sulfuric acid (5 vol) was added slowly over a period of 15 minutes maintaining the temperature at or below 20 °C. The mixture was then warmed to room temperature and stirred for at least 30 minutes. The mixture was allowed to settle, and the aqueous (bottom) layer was discharged. The remaining organic layer was washed sequentially with 10% aqueous sulfuric acid, a mixture of water and brine, and brine. Azeodistillation with 2- MeTHF followed by filtration provided a solution of (S)-2-((tert- butoxycarbonyl)amino)pentanoic acid in 2-MeTHF. Part B [0267]

(1.2 eq), and 2-MeTHF (4 vol) were combined and agitated. TEA (1.2 eq) was slowly added over a period of 30 minutes at a temperature of 30 °C or less. The reaction mixture was stirred at room temperature for at least one hour then cooled to -2 °C to 2 °C. Part C NAI-

[026 itated under nitrogen atmosphere. The slurry was cooled to -2 to 2 °C after which the (S)-2-((tert- butoxycarbonyl)amino)pentanoic acid solution in 2-MeTHF was added slowly while maintaining a temperature of no more than 2 °C and the reaction mixture was stirred for at least 1 hour. [0269] The reaction mixture was slowly added to the Part B reactor while maintaining the temperature of the reaction mixture at or below 2 °C. The reaction mixture was warmed to room temperature and stirred for at least 3 hours. The reaction mixture was washed at a temperature of 25 °C or less with HCl (1N), with potassium phosphate dibasic (5% aqueous), with water, and with brine. The product was azeotropically distilled, cooled to room temperature, and filtered to yield a solution of tert-butyl (S)-(1-(methoxy(methyl)amino)-1-oxopentan-2-yl)carbamate in 2- MeTHF. Step 2-2: Preparation of tert-butyl (S)-(1-(2-chloro-3-fluorophenyl)-1-oxopentan-2- yl)carbamate Part A [0270]

to 1- bromo-2-chloro-3-fluorobenzene (1.7 eq) in 2-MeTHF (3.8 vol) maintaining the temperature of the reaction mixture at or below 5 °C. The reaction mixture was stirred for at least 2.5 hours. 93 NAI-1541064841v1 Part B -1-

oxopentan- was to - to -5 °C. A solution of isopropylmagnesium chloride (2 M in THF, 1.0 eq) was then added over a period of 1 hour, maintaining the temperature at or below 5 °C. Part C

[0272] The product of Step 2-2, Part A was slowly added to the reactor from Step 2-2, Part B while maintaining a temperature at or below 15 °C. The reaction mixture was warmed to 46 °C to 48 °C over a period of 2 hours then stirred at that temperature for 7 hours, before quenching with aqueous acetic acid. The reaction mixture was washed sequentially with acetic acid, water and brine, potassium phosphate dibasic, and brine. The product was concentrated in vacuo and dried to obtain tert-butyl (S)-(1-(2-chloro-3-fluorophenyl)-1-oxopentan-2-yl)carbamate as an oil with a purity of 68.5%. Step 2-3: Preparation of (1S,2S)-2-amino-1-(2-chloro-3-fluorophenyl)pentan-1-ol hydrochloride and (1R,2S)-2-amino-1-(2-chloro-3-fluorophenyl)pentan-1-ol hydrochloride NAI-

[0273] Tert-butyl (S)-(1-(2-chloro-3-fluorophenyl)-1-oxopentan-2-yl)carbamate was dissolved in methanol (10.5 volumes). The mixture was heated to 55 °C to 60 °C and sodium borohydride (2 eq) was added in portions over 30 minutes while monitoring gas evolution. The mixture was heated and maintained at a temperature of 55 °C to 60 °C for 1.5 hours. The reaction mixture was then cooled to room temperature and quenched with aqueous HCl (1 M). The mixture was extracted with EtOAc (x3), and the combined organic layer washed with water and brine, dried over MgSO₄, and concentrated to an oil under reduced pressure. [0274] The product was dissolved in cyclopentyl methyl ether (CMPE, 4 vols) in toluene. HCl in CPME (7 eq) was added slowly over a period of 1 hour while maintaining the temperature below 30 °C. The mixture was stirred at room temperature for at least 24 hours before the solvent was removed in vacuo to yield (1S,2S)-2-amino-1-(2-chloro-3- fluorophenyl)pentan-1-ol hydrochloride and (1R,2S)-2-amino-1-(2-chloro-3- fluorophenyl)pentan-1-ol hydrochloride, which were separated by repeated flash column chromatography using a BIOTAGE^® silica gel column with MTBE/hexane gradient from 5% to 35% MTBE as the eluent to provide the isolated compounds. Step 2-4: Preparation of Compound 1-C and Compound 1-D [0275]

cooled reactors at 0 °C under nitrogen containing 7-fluoro-2-oxoindoline-4-carboxylic acid (1.2 eq), DMAP (2.5 eq) and EDC (1.5 eq). DMF (5.5 vol) was added to each reactor. Each reaction mixture was further treated as follows: 95 NAI-1541064841v1 [0276] Each reaction mixture was warmed to room temperature and stirred for at least 4 hours. Ethyl acetate (8 vol) was added, and the reaction mixture cooled to 13 – 17 °C.1 N HCl (5 vol) was charged to the reactor and the reaction mixture was agitated. The reaction mixture was filtered, and the filtrate allowed to settle, after which the lower aqueous phase was removed from the upper organic phase. The organic phase was then washed with 1 N HCl and brine, potassium phosphate dibasic (5%) and brine, water and brine, and brine. The reaction mixture was then distilled with ethyl acetate. The temperature was adjusted to 50-55 °C, and SilaMet S thiol resin (10 wt. %) was added and the resulting mixture stirred at the same temperature for at least 3 hours. The reaction mixture was then filtered, the solvent was switched to ethanol. The reaction mixture was cooled to 15-20 °C over at least 1.5 h and stirred. Heptane (27 vol) was then added to obtain a slurry. Compound 1-C and Compound 1-D were each isolated by filtration, washed with 4:1 heptane/ethanol and dried. Example 3: Synthesis of (N-[(1R)-1-[(R)-(2-chloro-3-fluorophenyl)hydroxymethyl]butyl]-7- fluoro-2,3-dihydro-2-oxo-1H-indole-4-carboxamide) (Compound 1-B) Step 3-1: Preparation of tert-butyl (R)-(1-(methoxy(methyl)amino)-1-oxopentan-2- yl)carbamate Part A [0277] (R)-2-((tert-butoxycarbonyl)amino)pentanoic acid dicyclohexylamine (1 eq) was added to a reactor followed by 2-MeTHF (10 vol) and agitated. The slurry was cooled to -10 °C to -5 °C after which 10% aqueous sulfuric acid (5 vol) was added slowly over a period of 15 minutes maintaining the temperature at or below 20 °C. The mixture was warmed to room temperature and stirred for at least 30 minutes. The mixture was allowed to settle, and the aqueous (bottom) layer was discharged. The remaining organic layer was washed sequentially with 10% aqueous sulfuric acid, a mixture of water and brine, and brine. Azeodistillation followed by filtration provided a solution of tert-butyl (R)-(1-(methoxy(methyl)amino)-1- oxopentan-2-yl)carbamate in 2-MeTHF. 96 NAI-1541064841v1 Part B [0278] In (1.2

eq), and 2-MeTHF (4 vol) were combined and agitated. TEA (1.2 eq) was slowly added over a period of 30 minutes at a temperature of 30 °C or less. The reaction mixture was stirred at room temperature for at least one hour then cooled to -2 °C to 2 °C. Part C

[0279] a separate reactor, was and agitated under nitrogen atmosphere. The slurry was cooled to -2 to 2 °C after which the (R)-2-((tert- butoxycarbonyl)amino)pentanoic acid solution in 2-MeTHF was added slowly while maintaining a temperature of no more than 2 °C and the reaction mixture was stirred for at least 1 hour. [0280] The reaction mixture was then slowly added to the Part B reactor while maintaining the temperature of the reaction mixture at or below 2 °C. The reaction mixture was warmed to room temperature and stirred for at least 3 hours. The reaction mixture was washed at a temperature of 25 °C or less with HCl (1N), with potassium phosphate dibasic (5% aqueous), with water, and with brine. The product was azeotropically distilled, cooled to room temperature, 97 NAI-1541064841v1 and filtered to yield a solution of tert-butyl (R)-(1-(methoxy(methyl)amino)-1-oxopentan-2- yl)carbamate in 2-MeTHF. Step 3-2: Preparation of tert-butyl (R)-(1-(2-chloro-3-fluorophenyl)-1-oxopentan-2- yl)carbamate Part A [0281] A was added to 1-

bromo-2-chloro-3-fluorobenzene (1.7 eq) in 2-MeTHF (3.8 vol) maintaining the temperature of the reaction mixture at or below 5 °C. The reaction mixture was stirred for at least 2.5 hours. Part B [0282]

-1- oxopentan-2-yl)carbamate from Step 3-1 and the reaction mixture was cooled to -10 to -5 °C. A solution of isopropylmagnesium chloride (2 M in THF, 1.0 eq) was then added over a period of 1 hour, maintaining the temperature at or below 5 °C. Part C [0283]

3-2, Part B while maintaining a temperature at or below 15 °C. The reaction mixture was then warmed to 46 °C to 48 °C over a period of 2 hours then stirred at that temperature for 7 hours. The reaction mixture was then slowly quenched with acetic acid (6 eq) at room temperature, followed by washing with acetic acid, with water and brine, with aqueous potassium phosphate dibasic 98 NAI-1541064841v1 (15%), with water and brine, and with brine. The reaction mixture was then distilled to change solvents to 2-propanol. Following cooling to -5 °C to 0 °C over 2 hours, water (8 vol) was charged to crystallize the product, which was then isolated by filtration and dried to yield tert- butyl (R)-(1-(2-chloro-3-fluorophenyl)-1-oxopentan-2-yl)carbamate as an oil. Step 3-3: Preparation of (1R,2R)-2-amino-1-(2-chloro-3-fluorophenyl)pentan-1-ol hydrochloride

dissolved in methanol (10.5 volumes). The mixture was heated to 55 °C to 60 °C and sodium borohydride (2 eq) was added in portions over 30 minutes while monitoring gas evolution. The mixture was heated and maintained at a temperature of 55 °C to 60 °C for 1.5 hours. [0285] The reaction mixture was cooled to room temperature and washed with HCl (1 N), with aqueous potassium phosphate, dibasic (5%), water, and with brine. The (R,R) stereoisomer was purified by removing the solvent under vacuum and subjected to repeated flash column chromatography using a BIOTAGE^® silica gel column with MTBE/hexane gradient from 5% to 35% MTBE as the eluent then purified a second time by flash chromatography using a BIOTAGE silica gel column with a gradient of DCM/diethyl ether from 11% to 66% as the eluent. [0286] The (R,R) stereoisomer was reacted with cyclopentyl methyl ether (CMPE, 4 vol) in toluene. HCl in CPME (7 eq) was added slowly over a period of 1 hour while maintaining the temperature below 30 °C. The mixture was stirred at room temperature for at least 24 hours before the solvent was removed in vacuo to yield (1R,2R)-2-amino-1-(2-chloro-3- fluorophenyl)pentan-1-ol chloride. Step 4: Preparation of Compound 1-B 99 NAI-1541064841v1 [0287] n charged to a pre-cooled reactor at 0 °C under nitrogen containing 7-fluoro-2-oxoindoline-4-carboxylic acid (1.2 eq), DMAP (2.5 eq) and EDC (1.5 eq). DMF (5.5 vol) was then added. The reaction mixture was warmed to room temperature and stirred for at least 4 hours. Ethyl acetate (8 vol) was added, and the reaction mixture cooled to 13 – 17 °C.1 N HCl (5 vol) was then charged to the reactor and the reaction mixture was agitated. The reaction mixture was filtered, and the filtrate allowed to settle, after which the lower aqueous phase was removed from the upper organic phase. The organic phase was washed with 1 N HCl and brine, potassium phosphate dibasic (5%) and brine, water and brine, and brine. The reaction mixture was distilled with ethyl acetate. The temperature was adjusted to 50-55 °C, and SilaMet S thiol resin (10 wt. %) was added and the resulting mixture stirred at the same temperature for at least 3 hours. The reaction mixture was filtered, the solvent was switched to ethanol. The reaction mixture was cooled to 15- 20 °C over at least 1.5 h and stirred. Heptane (27 vol) was added to obtain a slurry. Compound 1- B was then isolated by filtration, washed with 4:1 heptane/ethanol and dried. Example 4: A1AT Activity Assay [0288] The assay protocol for Northwest Life Science Specialties (NWLSS) was followed with the following modifications or substitutions when needed. 1. The excess assay buffer in the NWLSS kit can be substituted with tris-buffered saline with 0.1% TWEEN^® 20 (TBST) that has been pH adjusted to pH 8.0. 2. N-succinyl-Ala-Ala-Ala-p-nitroanilide (NSAN) reagent from the NWLSS kit can be substituted with NSAN (Millipore Sigma S4760). NSAN stock reagent was made by resuspending powder to 6 mM (10X) in NWLSS assay buffer and then making a 1:10 dilution in NWLSS assay buffer for a working solution at 600 µM the day of use.6 mM NSAN reagent was stored at -80 °C. 100 NAI-1541064841v1 3. Elastase calibrator and elastase reagent in the NWLSS kit can be substituted with porcine elastase. Stock solution of elastase can be made by resuspending porcine elastase to ~120 µM in 50 mM sodium acetate, 200 mM NaCl, pH 5.5 buffer. This low pH buffer prevents elastase self-cleavage which can occur at NWLSS Assay Buffer pH 8.5. Stock solution can be aliquoted, and flash frozen for long term storage at -80 °C. Working elastase was made by diluting stock solution to 600 nM in NWLSS assay buffer for elastase calibrator and then making a 1:2 dilution of elastase calibrator in NWLSS assay buffer for elastase reagent (300 nM). [0289] Compounds were weighed out and resuspended in the desired vehicle. Compounds were then serially diluted as needed in vehicle in a PCR plate. Compound dilution series were then diluted in NWLSS assay buffer, in non-binding plates, until the final concentration needed for testing was achieved. Most dilutions aimed to start the series at a final concentration of 20 µM. A1AT was diluted to desired concentration in a 15 mL conical tube with NWLSS assay buffer.110 µL of A1AT was added to appropriate wells of a PCR plate.10 µL of 12X diluted compound was added to 110 µL A1AT in PCR plate and mixed 5 times. Samples were incubated for 1 hour at room temperature in the dark. [0290] For animal studies, purified A1AT concentrations were calculated from A1AT ELISA. A1AT samples were diluted to 0.054 mg/mL (1200 nM) or 0.027 (600 nM) starting concentrations (based on limited concentration) in appropriate matrix buffer (mixed elution buffer and wash buffer in appropriate ratio).1200 nM A1AT samples were diluted 1:4 in assay buffer and 600 nM A1AT samples were diluted 1:2 in assay buffer before loading in plates. [0291] An elastase calibrator standard curve was prepared by making 2-fold serial dilutions in NWLSS assay buffer. In a clear bottom 96-well assay plate, elastase calibrator standard or elastase reagent was added at 50 µL/well. NWLSS assay buffer was added to standard wells at 50 µL/well and A1AT samples and controls to appropriate elastase reagent wells at 50µL/well. The plates were mixed in a horizontal plate shaker at 400 RPM for 1 minute then incubated for 45 minutes at room temperature in the dark.600 µM NSAN to each well at 50 µL/well. The plates were then mixed in a horizontal plate shaker at 400 RPM for 1 minute then incubated for 15 minutes at room temperature in the dark. Glacial acetic acid was added to each well at 10 µL/well to stop the reaction. Plates were mixed in a horizontal plate shaker at 400 RPM for 1 minute then read on a SpectraMax i3 plate reader at 410 nm. Elastase inhibition and A1AT 101 NAI-1541064841v1 inhibition were calculated per the equations below, wherein X is elastase activity the raw optical density data obtained from the spectrophotometric analysis: Elastase % Inhibition = 100 * [1–(X–average of “no elastase”)/(average of “elastase alone”– average of “no elastase”)] A1AT % Inhibition = 100 – Elastase % Inhibition [0292] Data analysis was performed using SoftmaxPro and GraphPad Prism. Non-linear regression analysis to determine IC₅₀/EC₅₀ for each compound was performed with GraphPad Prism and is reported in Tables 1 and 2 below: Table 1 IC₅₀ (nM) H A1AT f 1 A 1 B 1 C 1 D

EC₅₀ (nM)

(R,R), 1-C (S,S) and 1-D (R,S) with 1000 nM zA1AT and 300 nM mA1AT. Example 5: Thermal Shift Analyses [0294] To determine the binding and thermostability provided by Compound 1 to mA1AT and zA1AT, thermal shift assays were performed. The thermal shift assay was carried out using the same samples used in the activity assay of Example 4 with protein concentrations ranging from 300 nM to 1 µM (1000 mM). Generally, 1-2 µM gave a more reliable and robust signal in this assay. [0295] Briefly, Prometheus NT.48 capillaries (no significant difference was seen using either standard or high sensitivity capillaries) were placed in each well of the 96-well PCR plate one row at a time. After opening the Prometheus NT.48 drawer and removing the magnetic holder, the plate was then turned sideways at almost 90° angle to promote capillary action to fill liquid to 102 NAI-1541064841v1 the top of the capillaries. Each capillary was then loaded one at a time into the slots on the Prometheus NT.48 stage. The magnetic holder was then gently placed over the loaded capillaries and the drawer was then closed. PR. ThermControl v2.1.6 software was used to start data collection. During the discovery scan, excitation ranging from 20-40% performed best as per Nanotemper peak excitation guidelines. Standard melting scan was performed with a temperature ramp of 1.0 °C/min from 20 °C to 95 °C. Default 350 nm/330 nm ratios and the first derivative of these graphs were used to detect inflection points. Inflection points represent the melting temperature of the domains of protein. Sometimes manual selection of inflection points was needed for heterogenous slopes. Non-linear regression analysis to determine EC₅₀ for each compound was performed with GraphPad Prism. [0296] For the ligand binding assay, 10 mM of each stereoisomer of N-(1-(2-chloro-3- fluorophenyl)-1-hydroxypentan-2-yl)-7-fluoro-2-oxoindoline-4-carboxamide in DMSO was subjected to a SPECTRUMSCREEN binding assay (Eurofins Scientific, Luxembourg). IC₅₀ values were determined by a non-linear, least squares regression analysis using MATHIQ (ID Business Solutions Ltd., UK). Only ligands against which at least one stereoisomer showed 50% or greater inhibition of stimulation are shown in these results. The specific conditions for each of ligand is shown in Table 3 below. Table 3: Ligand Conditions L_{igand Source Vehicle} ^Incubation T_{ime/Temp Buffer} A 1 A A

103 NAI-1541064841v1 [0297] Results for the ligand binding analysis for each tested compound is shown below in Table 4. Table 4: Results of Ligand Binding Analysis Ligand Compound 1-A Compound 1-B Compound 1-C Compound 1-D Adenosine A3 ^{81 81 83 90}

A (S,R), 1-B (R,R), 1-C (S,S) and 1-D (R,S) with 1000 nM zA1AT and 300 nM mA1AT. Example 6: Beagle Dog Study [0299] Three batches of N-[(1R)-1-[(S]-(2-chloro-3-fluorophenyl)hydroxymethyl]butyl]-7- fluoror-2,3-dihydro-2-oxo-1H-indonel-4-caboxamide were used during the study. Batch 1 had a total purity of 96% and a stereochemical purity of 94.7%. Batch 2 had a total purity of 98% and a stereochemical purity of 99.8%. Batch 3 had a total purity of 97.8% and a stereochemical purity of 99.9%. In this context, the stereochemical purity relates to the amount of the Compound 1-A. [0300] All doses were administered by oral gavage in a vehicle of 50% propylene glycol, 30% Labrasol, 20% Transcutol HP, and 0.5 mg/mL HPMC. On days where two doses were administered, the second daily dose of each day was administered 12 hours +/- 30 minutes after the start of the first daily dose. Two groups of animals, each group having one male and one female Beagle dog were used for this study. [0301] In the study, the first day of dosing is denoted as day 1, and subsequent days numbered accordingly regardless of whether an animal was dosed on that day. [0302] Beginning on day 1, Group 1 animals were subjected to twice daily dosing twice a day with Batch 1 at 15 mg/kg/dose (30 mg/kg/day) at a dose volume of 5 mL/kg. This dosing resulted in frequent emesis in both animals. A dose holiday was initiated after the first dose on day 3. Dosing was resumed on day 7 with the dose volume decreased to 1 mL/kg but at the same 104 NAI-1541064841v1 dose level of 15 mg/kg/dose (30 mg/kg/day). Animals exhibit no adverse effects on day 7, but frequent emesis resumed on days 8 and 9. [0303] On day 12, Group 2 animals were given two doses of vehicle only (no drug) at a dose volume of 1 mL/kg followed by one dose of vehicle only (no drug) at a dose volume of 1 mL/kg the morning of day 13. No emesis was noted. [0304] On day 22, Group 2 animals were administered two doses of Batch 2 at a dose level of 15 mg/kg/dose (30 mg/kg/day) at a dose volume of 1 mL/kg. No emesis was noted. [0305] On days 43-45 Group 2 animals were administered two doses of Batch 3 per day at a dose level of 15 mg/kg/dose (30 mg/kg/day) at a dose volume of 1 mL/kg. No emesis was noted. Example 7: Treating Fibrosis with Compound 1-A [0306] Female PiZ mice or WT littermates (JAX stock #035411, Jackson Laboratories, Sacramento, California, USA) were assigned to untreated baseline control groups or treatment groups at approximately 12 weeks of age. Animals were dosed twice daily orally (gavage) with 0 (vehicle), 10, 30, 50 or 100 mg/kg/dose BID (two times per day) with Compound 1-A (0, 20, 60, 100, or 200 mg/kg/day). Compared to WT controls, hepatocytes of baseline control PiZ mice and vehicle-treated PiZ mice contained cytoplasmic eosinophilic globules, staining strongly magenta with PAS-D stain. After 30 days, there was progression of disease in vehicle-treated animals compared to baseline controls. [0307] Microscopic evaluation of livers from control animals showed randomly distributed, scattered foci of mixed inflammation (minimal to mild), typically centered on degenerate hepatocytes. Treatment with Compound 1-A not only prevented formation of AAT polymers in hepatocytes but was also associated with reduced severity of inflammation, which was sustained during a 6-day recovery period, demonstrating stabilizing Z-AAT and subsequent prevention of polymer formation reduces pro-necro-inflammatory pathways in the liver. [0308] In particular, proteomic analyses of plasma from Compound 1-A-treated PiZ mice showed significant decreases in proteins associated with ER stress and protein folding pathways such as calreticulin, heat shock protein 5, and prolyl 4-hydroxylase beta polypeptide in mice treated with Compound 1-A given 50 or 100 mg/kg/dose BID (100 or 200 mg/kg/day) compared with vehicle-treated PiZ mice, as shown in Table 5 and in FIGs.5, 6, and 7. 105 NAI-1541064841v1 Table 5: Effect of Compound 1-A on Certain Biomarkers PiZ Mice: PiZ Mice: PiZ Mice: PiZ Mice: Biomarker 200 mg/kg/day 100 mg/kg/day Baseline Control Vehicle 9 6 4 ed

in WT mice. Asterisks indicate significant differences compared with PiZ mice treated with vehicle (analyzed by one-way ANOVA followed by Fisher’s least significant difference [LSD] procedure) as follows: ** = p<0.01; *** = p < 0.001, **** = p < 0.0001. Error bars show standard deviation. The reduction in ER stress and UPR markers in Compound 1-A-treated mice suggests that diminishing the accumulation of Z-AAT in the liver reduces pathways that may lead to hepatocyte cell death. [0310] Further, as shown in Table 6 and FIGs.8, 9, and 10, treatment with Compound 1-A at 50 or 100 mg/kg/dose BID for 30 days normalized levels of thyroxine-binding globulin (hormone transport protein), Factor VII (coagulation factor), and C5 Complement (complement system protein), which suggests that the biosynthetic function of the liver was effectively restored to WT levels. Table 6: Effect of Compound 1-A on Certain Biomarkers PiZ Mice: PiZ Mice: PiZ Mice: PiZ Mice: 5 6 6

detected in WT mice. Asterisks indicate significant differences compared with PiZ mice treated with vehicle (analyzed by one-way ANOVA followed by Fisher’s least significant difference [LSD] procedure) as follows: * = p < 0.05; ** = p < 0.01; *** = p < 0.001, **** = p < 0.0001. 106 NAI-1541064841v1 Error bars show standard deviation. Together, these results demonstrate treatment with Compound 1-A reduces necro-inflammatory pathways, suggesting its pharmacologic action of stabilizing monomers of human AAT may allow for prevention and potentially reversal of fibrosis in the liver. Normalization of liver function as demonstrated by restoration of biosynthetic pathways further supports this conclusion. * * * * * [0312] Throughout this application, various publications, patents, patent applications and other documents have been referenced. The disclosures of these publications, patents, patent applications and other documents in their entireties are hereby incorporated by reference in this application for all purposes, including in order to more fully describe the state of the art to which this the subject matter disclosed herein pertains. Although the disclosed subject matter has been described with reference to the examples provided above, it should be understood that various modifications could be made without departing from the spirit of the disclosed subject matter. Many variations will become apparent to those skilled in the art upon review of this specification. 107 NAI-1541064841v1

Claims

CLAIMS What is claimed is: 1. A method of making a compound of Formula (I-A): , wherein:

R1 is selected from group C1-6 alkyl; R2 is C1-6 alkyl; each R is independently selected from the group consisting of F, Cl, Br, I, CN, and C_1-6 alkyl; and n is an integer from 0-5; the method comprising: contacting a compound of Formula (II-A) and a compound of Formula (III), optionally in the presence of a base, to make a compound of Formula (IV-A):

, wherein: Pg is a protecting group; and M is an alkali metal, p is 0, and X is Cl, Br, or I; or M is alkaline earth metal, p is 1, and X is Cl, Br, or I; and 108 NAI-1541064841v1 converting the compound of Formula (IV-A) to a compound of Formula (I-A) , wherein the

Formula (I-A) comprises contacting the compound of Formula (IV-A) with a stereoselective or stereospecific reducing agent. 2. The method of claim 1, wherein the converting of the compound of Formula (IV- A) to the compound of Formula (I-A) comprises: contacting the compound of Formula (IV-A) with the stereoselective or stereospecific reducing agent to make a compound of Formula (V-A):

. 3. The method of claims 1 or claim 2, wherein the converting of the compound of Formula (IV-A) to the compound of Formula (I-A) comprises or further comprises: contacting the compound of Formula (V-A) with a deprotecting agent to make a compound of Formula (VI-A) or a salt of Formula (VI-A):

. 4. The method of any one of claims 1-3, wherein the converting of the compound of Formula (IV-A) to the compound of Formula (I-A) comprises or further comprises: 109 NAI-1541064841v1 contacting the compound of Formula (VI-A), or salt thereof, with a compound of Formula (VII) to make the compound of Formula (I-A): .

5. any one (II-A) is made by a method comprising converting a compound of Formula (IIa-A) to an activated intermediate: .

6. The method of any one of claims 2-5, wherein the converting of the compound of Formula (II-A) to an activated intermediate comprises contacting a compound of Formula (IIa- A) with a coupling agent to make an activated intermediate. 7. The method of claim 5 or claim 6, which further comprises contacting the activated intermediate with N,O-dimethylhydroxylamine to make a compound of Formula (II- A): . NAI-

8. The method of any one of claims 1-7, wherein the C1-6 alkyl is selected from the group consisting of CH3, CH2CH3, CH2CH2CH3, and CH(CH3)2. 9. The method of any one of claims 1-8, wherein n is 2. 10. The method of any one of claims 1-9, wherein n is not 0 and each R is independently selected from the group consisting of F, Cl, and Br. 11. The method of any one of claims 1-10, wherein the protecting group Pg is selected from the group consisting of 9-fluorenylmethyloxycarbonyl (Fmoc), p-methyoxybenzyl carbonyl (Moz or MeOZ), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), acetyl (Ac), benzoyl (Bz), benzyl (Bn), carbamate, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts), trichloroethyl chloroformate (Troc), and a sulfonamide. 12. The method of claim 11, wherein the protecting group Pg is tert-butyloxycarbonyl (Boc). 13. The method of any one of claims 1-12, wherein the MX_p moiety in Formula (III) is MgCl. 14. The method of any one of claims 1-13, wherein the compound of Formula (VII) is made by a method comprising: contacting a compound of Formula (VIIa) with a catalyst to make a compound of Formula (VIIb): contacting

to make the compound of Formula (VII): 111 NAI-1541064841v1

. 15. A method of making a compound of Formula (VII):

, wherein R1 is selected from the group consisting of F, Cl, Br, I, and C1-6 alkyl, and wherein the method comprises: contacting a compound of Formula (VIIa) with tert-butyldimethylsilyl chloride to make a compound of Formula (VIII): ;

chloride and n- butyl lithium to make an intermediate of Formula (VIIIa) 112 NAI-1541064841v1

; intermediate of Formula (VIIIb):

a compound of Formula (VII):

.

16. The method of claim 15, wherein Formula (VIII) is contacted with isopropyl magnesium chloride and n-butyl lithium in the presence of a base, optionally wherein the base comprises sodium hydride. 113 NAI-1541064841v1

17. The method of claim 15 or claim 16, wherein the carbon dioxide is pressurized carbon dioxide. 18. The method of any one of claims 15-17, wherein the bis-silyl deprotecting agent comprises an acid, optionally wherein the bis-silyl deprotecting agent comprises phosphoric acid. 19. The method of any one of claims 1-13, wherein the compound of Formula (VII) is made by the method of any one of claim 15-18. 20. The method of any one of claims 6-14, wherein the coupling agent is carboyldiimidazole (CDI). 21. The method of any one of claims 6-20, wherein the activated intermediate is a compound of Formula (IIa-A'): .

22. A method of making N-[(1R)-1-[(S)-(2-chloro-3- fluorophenyl)hydroxymethyl]butyl]-7-fluoro-2,3-dihydro-2-oxo-1H-indole-4-carboxamide (Compound 1-A): ,

the method comprising: contacting tert-butyl (R)-(1-(methoxy(methyl)amino)-1-oxopentan-2-yl)carbamate (Compound 2-A) with a of Formula 3, optionally in the presence of a base, to make tert-butyl (R)-(1-(2-chloro-3-fluorophenyl)-1-oxopentan-2-yl)carbamate (Compound 4-A): 114 NAI-1541064841v1 , wherein: M is an alkali metal, p is 0, and X is Cl, Br, or I; or M is an alkaline earth metal, p is 1, and X is Cl, Br, or I; and converting Compound 4-A to Compound 1-A: , wherein

A with a stereoselective or stereospecific reducing agent. 23. The method of claim 22, wherein the converting of Compound 4-A to Compound 1-A comprises: contacting 4-A with a stereoselective or stereospecific reducing agent to make tert-butyl ((1S,2S)-1-(2-chloro-3-fluorophenyl)-1-hydroxypentan-2-yl)carbamate (Compound 5):

.

24. The method of claim 22 or claim 23, wherein the converting of Compound 4-A to Compound 1-A comprises or further comprises: 115 NAI-1541064841v1 contacting Compound 5-A with a deprotecting agent to make (1S,2R)-2-amino-1-(2- chloro-3-fluorophenyl)pentan-1-ol hydrochloride (Compound 6-A): .

4- A to Compound 1-A comprises or further comprises contacting Compound 6-A with 7-fluoro-2- oxoindoline-4-carboxylic acid (Compound 7) to make Compound 1-A: .

26. The method of claim 22, which further comprises: contacting (R)-2-((tert-butoxycarbonyl)amino)pentanoic acid (Compound 2a-A) with a coupling agent to make an activated intermediate; and contacting the activated intermediate with N,O-dimethylhydroxylamine to make tert- butyl (R)-(1-(methoxy(methyl)amino)-1-oxopentan-2-yl)carbamate (Compound 2-A): .

27. The method of claim 26, wherein the coupling agent is carboyldiimidazole (CDI). 28. The method of claim 27, wherein the activated intermediate is Compound 2-A': .

29. The method of any one of claims 1-28, wherein the base is present and is selected from the group consisting of a Grignard reagent, a lithium aluminum alkyl hydride, an alkyllithium compound, and an aryllithium compound. 30. The method of any one of claims 1-29, wherein M is an alkaline earth metal and X is chloride. 31. The method of any one of claims 1-30, wherein M is magnesium. 32. The method of any one of claims 23-31, wherein the MXp moiety of the compound of Formula 3 is MgCl. 33. The method of claim 32, wherein the making of compound of Formula 3 comprises contacting 1-bromo-2-chloro-3-fluorobenzene with isopropyl magnesium chloride in 2-methyltetrahydrofuran (2-MeTHF). 34. The method of any one of claims 22-33, wherein Compound 7 is made by a method comprising: contacting 4-bromo-7-fluoroindolin-2-one (Compound 7a) with a suitable catalyst to make methyl 7-fluoro-2-oxoindoline-4-carboxylate (Compound 7b): NAI-1541064841v1

contacting Compound 7b with a hydrolyzing agent to make Compound 7: . 35.

the catalyst in the presence of carbon monoxide gas and a base. 36. The method of claim 34 or claim 35, wherein the catalyst is a palladium catalyst. 37. The method of claim 35 or 36, wherein the base is selected from the group consisting of sodium hydroxide, lithium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, triethylamine, N- diisopropylethylamine, pyridine, 4-dimethylpyridine, potassium acetate, sodium methoxide, potassium tert-butoxide, and any combination thereof. 38. The method of any one of claims 34-37, wherein the hydrolyzing agent is selected from the group consisting of sodium hydroxide, lithium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium methoxide, potassium tert-butoxide, or any combination thereof. 39. The method of claims 3-21 or 24-38, wherein the deprotecting agent is hydrochloric acid. 40. A method of making Compound 7:

118 NAI-1541064841v1 wherein the method comprises: contacting 4-bromo-7-fluoroindolin-2-one (Compound 7a) with tert-butyldimethylsilyl chloride to make 4-bromo-1-(tert-butyldimethylsilyl)-2-((tert-butyldimethylsilyl)oxy)-7-fluoro- 1H-indole (Compound 8): ; contacting

lithium to make Intermediate 8a: ;

7: 119 NAI-1541064841v1

. 41. The method of claim 40, wherein Compound 7 is contacted with isopropyl magnesium chloride and n-butyl lithium in the presence of a base, optionally wherein the base comprises sodium hydride. 42. The method of claim 40 or claim 41, wherein the carbon dioxide is pressurized carbon dioxide. 43. The method of any one of claims 40-42, wherein the bis-silyl deprotecting agent comprises an acid, optionally wherein the bis-silyl deprotecting agent comprises phosphoric acid. 44. The method of any one of claims 22-33, wherein Compound 7 is made the method of any one of claims 40-43. 45. The method of any one of claims 25-44, wherein Compound 6-A is contacted with Compound 7 in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 4- (dimethylamino)pyridine (DMAP), and N,N-dimethylformamide (DMF). 46. The method of any one of claims 24-44, wherein Compound 6-A is contacted with Compound 7 in the presence of O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU). 47. The method of any one of claims 1-40, wherein the stereoselective or stereospecific reducing agent is (S,S)-Ts-Deneb, (S,S)-Ms-Deneb, or aluminum isopropoxide. 48. The method of any one of claims 1-47, wherein the stereoselective or stereospecific reducing agent is aluminum isopropoxide. 120 NAI-1541064841v1

49. The method of any one of claims 24-48, wherein Compound 5-A is contacted with the stereoselective or stereospecific reducing agent in the presence of isopropyl alcohol and toluene. 50. The method of any one of claims 24-49, wherein Compound 5-A is contacted with the stereoselective or stereospecific reducing agent at a temperature of about 50 °C. 51. The method of any one of claims 26-50, wherein Compound 2-A is contacted with the compound of Formula 3 in the presence of 2-MeTHF. 52. The method of any one of claims 26-51, wherein the coupling agent is CDI and (R)-2-((tert-butoxycarbonyl)amino)pentanoic acid is contacted with CDI at a temperature of about 0 °C. 53. The method of claim 52, wherein (R)-2-((tert-butoxycarbonyl)amino)pentanoic acid is contacted with CDI in the presence of 2-MeTHF. 54. The method of any one of claims 5-21 or claims 26-53, wherein the contacting of the activated intermediate with N,O-dimethylhydroxylamine is in the presence of DMF and 2- MeTHF. 55. A compound which is N-[(1R)-1-[(S)-(2-chloro-3- fluorophenyl)hydroxymethyl]butyl]-7-fluoro-2,3-dihydro-2-oxo-1H-indole-4-carboxamide (Compound 1-A): F Cl ^OH ,

made by the method of any one of claims 1-54. 56. A composition comprising the compound of claim 55, wherein the stereochemical purity of the compound in the composition is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at 121 NAI-1541064841v1 least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.8%. 57. A pharmaceutical composition comprising the composition of claim 56 and a pharmaceutically acceptable excipient. 58. A composition comprising N-[(1R)-1-[(S)-(2-chloro-3- fluorophenyl)hydroxymethyl]butyl]-7-fluoro-2,3-dihydro-2-oxo-1H-indole-4-carboxamide (Compound 1-A): ,

wherein the stereochemical purity of the composition with regards to Compound 1-A is about 90% or greater, optionally about 95% or greater, optionally about 98.5% or greater, further optionally about 99% or greater, further optionally about 99.5% or greater, still further optionally about 99.8% or greater. 59. The composition of claim 58, wherein the composition comprises no more than 10% of a combined amount of Compounds 1-B, 1-C, and 1-D:

122 NAI-1541064841v1 . 60. The composition of claim 58 or claim 59, wherein the composition comprises no more than 5% of a combined amount of Compounds 1-B, 1-C, and 1-D, optionally no more than 1.5% of a combined amount of Compounds 1-B, 1-C, and 1-D, further optionally no more than 1% of a combined amount of Compounds 1-B, 1-C, and 1-D, further optionally no more than 0.5% of a combined amount of Compounds 1-B, 1-C, and 1-D, still further optionally no more than 0.2% of a combined amount of Compounds 1-B, 1-C, and 1-D. 61. A pharmaceutical composition comprising the composition of any one of claims 58-60, and at least one pharmaceutically acceptable excipient. 62. A composition consisting essentially of N-[(1R)-1-[(S)-(2-chloro-3- fluorophenyl)hydroxymethyl]butyl]-7-fluoro-2,3-dihydro-2-oxo-1H-indole-4-carboxamide (Compound 1-A): F Cl ^OH . NAI-1541064841v1

63. The composition of claim 62, wherein the stereochemical purity of Compound 1- A is about 90% or greater, optionally about 95% or greater, optionally about 98.5% or greater, further optionally about 99% or greater, further optionally about 99.5% or greater, still further optionally about 99.8% or greater. 64. A pharmaceutical composition comprising the composition of claim 62 or claim 63 and at least one pharmaceutically acceptable excipient. 124 NAI-1541064841v1