Chemical process for preparing phenylpiperidinyl indole derivatives

Abstract

The present invention relates to a method of synthesizing a compound of formula (I) also referred to as 4-((2S,4S)-(4-ethoxy-1-((5-methoxy-7-methyl-1H-indol-4- yl)methyl)piperidin-2-yl))benzoic acid, or a pharmaceutically acceptable salt thereof, and / or intermediates thereof, their use as pharmaceuticals and pharmaceutical compositions and the use of intermediates for preparing a compound of formula (I), or a pharmaceutically acceptable salt thereof.

Description

[0001] CHEMICAL PROCESS FOR PREPARING PHENYLPIPERIDINYL INDOLE DERIVATIVES

[0002] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003]

[0001] The contents of the electronic sequence listing (PAT059858-PCT-SEC01_Sequence_Listing.xml; Size: 13,408 bytes; and Date of Creation: October 24, 2025) are hereby incorporated by reference in their entirety.

[0004] FIELD OF THE INVENTION

[0005]

[0002] The present invention relates to processes, process steps and intermediates useful in the preparation of phenylpiperidinyl indole derivatives. In particular, the present invention is in the field of organic synthesis and is directed to a method of synthesizing a compound of formula (I), also referred to as 4-((25,45)-(4-ethoxy-l-((5-methoxy-7-methyl-17 / -indol-4-yl)methyl)piperidin-2-yl))benzoic acid, or a pharmaceutically acceptable salt thereof, and / or intermediates thereof, methods for further preparing pharmaceutical compositions of the compound of formula (I), or its intermediates, the use of intermediates for preparing a compound of formula (I) and the intermediates themselves.

[0006] BACKGROUND

[0007]

[0003] The present invention relates to a process for the preparation of phenylpiperidinyl indole derivatives. More particularly, the present invention relates to a process for the preparation of the compound of formula (I)

[0008]

[0009] also referred to as 4-((25,45)-(4-ethoxy-l-((5-methoxy-7-methyl-17 / -indol-4-yl)methyl)piperidin-2-yl))benzoic acid, or a pharmaceutically acceptable salt thereof, which is capable of inhibiting the activation of the alternative pathway of the complement system. The compound is also known as iptacopan or a pharmaceutically acceptable salt thereof. The complement system plays a major role in the innate and adaptive immunity system and comprises a group of proteins that are normally present in an inactive state. These proteins are organized in three activation pathways: the classical, the lectin, and the alternative pathways (Holers, In Clinical Immunology: Principles and practice, ed. R. R. Rich, Mosby Press; 1996, 363-391). Molecules from microorganisms, antibodies or cellular components can activate these pathways resulting in the formation of protease complexes known as the C3-convertase and the C5-convertase. The classical pathway is a calcium / magnesiumdependent cascade, which is normally activated by the formation of antigen-antibody complexes. It can also be activated in an antibody-independent manner by the binding of C-reactive protein complexed to ligand and by many pathogens including gram-negative bacteria. The alternative pathway is a magnesium-dependent cascade, which is activated by deposition and activation of C3 on certain susceptible surfaces (e.g. cell wall polysaccharides of yeast and bacteria, and certain biopolymer materials). The alternative pathway (AP) utilizes C3 fragments (C3b) to opsonize the pathogens hence targeting them for phagocytosis without the need for antibodies. Hyperactivity of the complement system, and in particular in its AP, plays a role in a large number of complement-driven diseases, such as C3 glomerulopathy (C3G), paroxysmal nocturnal hemoglobinuria (PNH) and IgA nephropathy (IgAN). Phenylpiperidinyl indole derivatives, such as compound of formula (I), or a pharmaceutically acceptable salt thereof, play a role in the inhibition of complement factor B, a known critical enzyme for activation of the alternative complement pathway (Lesavre et al., J. Exp. Med. 1978, 148, 1498-1510; Volanakis etal., New Eng. J. Med. 1985, 312, 395-401), which may also be a suitable target for the inhibition of the amplification of the complement pathways. The phenylpiperidinyl indole derivatives, such as compound of formula (I), or a pharmaceutically acceptable salt thereof, and a method for preparing such derivatives, are described in W02015 / 009616. In particular, the compound of formula (I) is described in example 26, of W02015 / 009616. One of the drawbacks of the synthesis was the use of hazardous chemicals (such as sodium hydride, or dimethylacetamide, which represent safety concerns on a larger scale) and the poor enantio- and diastereoselectivity of the steps, leading to unwanted stereoisomers.

[0010]

[0004] Thus, there is a need to provide an alternative reaction route in a process for producing compound of formula (I), or a pharmaceutically acceptable salt thereof, generating less by-products, and easier to handle on a large scale.

[0011] SUMMARY

[0012]

[0005] Chemical processes are usually carried out on a small scale in a research / early development phase, and the scale successively increases in late phase development to finally reach the full size production scale. Upon scaling up a process, topics related to process safety are becoming more and more important, such as health hazards while handling large amount of hazardous and / or toxic chemicals, or environmental hazards.

[0013]

[0006] In an aspect, provided is a method of preparing Compound 5 having a structure of,

[0014]

[0015] (Compound 5),

[0016] wherein R1is Ci-Ce alkyl, such as methyl,

[0017] wherein the method comprises reacting Compound 4 having a structure of

[0018]

[0019] (Compound 4),

[0020] wherein R1is Ci-Ce alkyl, such as methyl;

[0021] with Compound 3 having a structure of

[0022]

[0023] (Compound 3).

[0024]

[0007] In an aspect, provided is a method of preparing a Compound (S)-6,

[0025]

[0026] (Compound (S)-6), wherein R1is Ci-Ce alkyl, such as methyl, wherein the method comprises reacting Compound 5

[0027]

[0028] (Compound 5),

[0029] wherein R1is Ci-Ce alkyl, such as methyl;

[0030] with (i) an enzyme comprising one or more selected from the group consisting of ketoreductase (KRED), alcohol dehydrogenases, and glucose dehydrogenase (GDH); and

[0031] (ii) a co-factor comprising one or more selected from the group consisting of alcohol dehydrogenase, nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD) and pyridoxal monophosphate;

[0032] in an aqueous buffer solution comprising one or more selected from the group consisting of TRIS, HEPES, MOPS, PIPES, borate, glycine, triethanol amine, phosphate, citrate, acetate, and ammonia.

[0033]

[0008] In an aspect, provided is a method of preparing a compound of formula (III),

[0034]

[0035] or a pharmaceutically acceptable salt thereof,

[0036] wherein P3is a protecting group, such as tert-butyloxycarbonyl (Boc), wherein the method comprises the steps of:

[0037] (a) reacting Compound 11

[0038]

[0039] (Compound 11),

[0040] with an alkylating agent, to obtain Compound 12,

[0041]

[0042] (Compound 12);

[0043] (b) reacting Compound 12 to form an indole Compound 14,

[0044]

[0045] (Compound 14);

[0046] (c) reacting Compound 14 with a compound having a formula of (P3)2O, wherein P3is a nitrogen protecting group, such as tert-butyloxycarbonyl (Boc),

[0047] to obtain Compound 9 having a structure of

[0048]

[0049] (Compound 9), or a pharmaceutically acceptable salt thereof, wherein P3is a nitrogen protecting group; and

[0050] (d) reacting Compound 9 with a formylating agent to obtain a compound of formula (III) having a structure of,

[0051]

[0052] pharmaceutically acceptable salt thereof,

[0053] wherein P3is a nitrogen protecting group, such as tert-butyloxy carbonyl (Boc).

[0054]

[0009] In an aspect, the disclosure provides a process for preparing a pharmaceutical composition, comprising a step of formulating the compound of formula (I), or a pharmaceutically acceptable pharmaceutically acceptable salt thereof, prepared by the method of as described herein with a pharmaceutically acceptable excipient.

[0055]

[0010] In an aspect, the disclosure also provides a ketoreductase (KRED) comprising a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid sequence of SEQ ID NO: 1, or a fragment thereof.

[0056] [OH] In an aspect, the disclosure also provides a ketoreductase (KRED) comprising a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid sequence of SEQ ID NO: 3, or a fragment thereof.

[0057]

[0012] In an aspect, the disclosure also provides a ketoreductase (KRED) comprising a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid sequence of SEQ ID NO: 5, or a fragment thereof.

[0058]

[0013] In an aspect, the disclosure also provides a ketoreductase (KRED) comprising a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid sequence of SEQ ID NO: 7, or a fragment thereof.

[0059]

[0014] In an aspect, the disclosure also provides a ketoreductase (KRED) produced from a cell comprising a nucleic acid sequence having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 2.

[0060]

[0015] In an aspect, the disclosure also provides a ketoreductase (KRED) produced from a cell comprising a nucleic acid sequence having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 4.

[0061]

[0016] In an aspect, the disclosure also provides a ketoreductase (KRED) produced from a cell comprising a nucleic acid sequence having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 4.

[0062]

[0017] In an aspect, the disclosure also provides a ketoreductase (KRED) produced from a cell comprising a nucleic acid sequence having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 6.

[0063]

[0018] In an aspect, the disclosure also provides a ketoreductase (KRED) produced from a cell comprising a nucleic acid sequence having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 8.

[0064]

[0019] In an aspect, the disclosure also provides a compound having a structure of

[0065]

[0066] pharmaceutically acceptable salt thereof, wherein R1is Ci-Ce alkyl, such as methyl.

[0067]

[0020] In an aspect, the disclosure also provides a compound having a structure of

[0068]

[0069] pharmaceutically acceptable salt thereof.

[0070]

[0021] In an aspect, the disclosure also provides a compound having a structure of

[0071]

[0072] , or a pharmaceutically acceptable salt thereof.

[0073]

[0022] In an aspect, the disclosure also provides a fumaric acid salt of the compound of the following structure:

[0074]

[0075]

[0023] In an aspect, the disclosure also provides an oxalic acid salt of the compound of the following structure:

[0076]

[0077]

[0024] In an aspect, the disclosure also provides a succinic acid salt of the compound of the following structure:

[0078]

[0079]

[0025] In an aspect, the disclosure also provides a benzoic acid salt of the compound of the following structure:

[0080]

[0081]

[0026] In an aspect, the disclosure also provides a citric acid salt of the compound of the following structure:

[0082]

[0083]

[0027] In an aspect, the disclosure also provides a malonic acid salt of the compound of the following structure:

[0084]

[0085]

[0028] In an aspect, the disclosure also provides a malic acid salt of the compound of the following structure:

[0086]

[0087]

[0029] In an aspect, the disclosure also provides an adipic acid salt of the compound of the following structure:

[0088]

[0089]

[0030] In an aspect, the disclosure also provides a hydrochloric acid salt of the compound of the following structure:

[0090]

[0091]

[0031] In an aspect, the disclosure also provides an acetic acid salt of the compound of the following structure:

[0092]

[0093]

[0032] In an aspect, the disclosure also provides a crystalline form of iptacopan hydrochloride hydrate characterized by having a powder X-ray diffractogram comprising one peak selected from the group consisting of peaks at 28 angles (6.7 ± 0.2)°, (10.0 ± 0.2)°, (13.3 ± 0.2)°, (14.9 ± 0.2)°, and (24.6 ± 0.2)°, e.g., when measured at a temperature in the range of from 20 to 30 °C with Cu-Ka radiation having a wavelength of 0.154 nm (e.g., 1.54190 nm).

[0094]

[0033] In an aspect, the disclosure also provides a crystalline form of iptacopan hydrochloride hydrate characterized by having a powder X-ray diffractogram comprising two peaks selected from the group consisting of peaks at 16 angles (6.7 ± 0.2)°, (10.0 ± 0.2)°, (13.3 ± 0.2)°, (14.9 ± 0.2)°, and (24.6 ± 0.2)°, e.g., when measured at a temperature in the range of from 20 to 30 °C with Cu-Ka radiation having a wavelength of 0.154 nm (e.g., 1.54190 nm).

[0095]

[0034] In an aspect, the disclosure also provides a crystalline form of iptacopan hydrochloride hydrate characterized by having a powder X-ray diffractogram comprising three peaks selected from the group consisting of peaks at 16 angles (6.7 ± 0.2)°, (10.0 ± 0.2)°, (13.3 ± 0.2)°, (14.9 ± 0.2)°, and (24.6 ± 0.2)°, e.g., when measured at a temperature in the range of from 20 to 30 °C with Cu-Ka radiation having a wavelength of 0.154 nm (e.g., 1.54190 nm).

[0096]

[0035] In an aspect, the disclosure also provides a crystalline form of iptacopan hydrochloride hydrate characterized by having a powder X-ray diffractogram comprising four peaks selected from the group consisting of peaks at 16 angles (6.7 ± 0.2)°, (10.0 ± 0.2)°, (13.3 ± 0.2)°, (14.9 ± 0.2)°, and (24.6 ± 0.2)°, e.g., when measured at a temperature in the range of from 20 to 30 °C with Cu-Ka radiation having a wavelength of 0.154 nm (e.g., 1.54190 nm).

[0097]

[0036] In an aspect, the disclosure also provides a crystalline form of iptacopan hydrochloride hydrate characterized by having a powder X-ray diffractogram comprising four peaks selected from the group consisting of peaks at 16 angles (6.7 ± 0.2)°, (10.0 ± 0.2)°, (13.3 ± 0.2)°, (14.9 ± 0.2)°, and (24.6 ± 0.2)°, e.g., when measured at a temperature in the range of from 20 to 30 °C with Cu-Ka radiation having a wavelength of 0.154 nm (e.g., 1.54190 nm).

[0098]

[0037] In some embodiments, the powder X-ray diffractogram further comprises one or more (e.g., one, two, three, four, or five) peaks selected from the group consisting of peaks at 16 angles (7.0 ± 0.2)°, (9.7 ± 0.2)°, (9.8 ± 0.2)°, (11.8 ± 0.2)°, (12.4 ± 0.2)°, (12.6 ± 0.2)°, (15.9 ± 0.2)°, (16.7 ± 0.2)°, (17.1 ± 0.2)°, (17.8 ± 0.2)°, (18.5 ± 0.2)°, (19.1 ± 0.2)°, (19.3 ± 0.2)°, (19.5 ± 0.2)°, (19.6 ± 0.2)°, (20.0 ± 0.2)°, (20.2 ± 0.2)°, (21.8 ± 0.2)°, (22.1 ± 0.2)°, (23.6 ± 0.2)°, (23.8 ± 0.2)°, (24.8 ± 0.2)°, (25.0 ± 0.2)°, (25.4 ± 0.2)°, (26.5 ± 0.2)°, (26.8 ± 0.2)°, (26.9 ± 0.2)°, (27.5 ± 0.2)°, and (29.2 ± 0.2)°, e.g., when measured at a temperature in the range of from 20 to 30 °C with Cu-Ka radiation having a wavelength of 0.154 nm (e.g., 1.54190 nm).

[0099]

[0038] In some embodiments, the powder X-ray diffractogram further comprises one or more (e.g., one, two, three, four, or five) peaks selected from the group consisting of peaks at 20 angles (9.7 ± 0.2)°, (15.9 ± 0.2)°, (17.8 ± 0.2)°, (18.5 ± 0.2)°, (19.5 ± 0.2)°, (19.6 ± 0.2)°, (21.8 ± 0.2)°, (23.6 ± 0.2)°, (26.8 ± 0.2)°, (26.9 ± 0.2)°, (27.5 ± 0.2)°, and (29.2 ± 0.2)°, e.g., when measured at a temperature in the range of from 20 to 30 °C with Cu-Ka radiation having a wavelength of 0.154 nm (e.g., 1.54190 nm).

[0100]

[0039] In an aspect, the disclosure also provides a pharmaceutical composition comprising the crystalline form of iptacopan hydrochloride hydrate disclosed herein.

[0101]

[0040] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0102]

[0041] In some embodiments, the crystalline form or the pharmaceutical composition disclosed herein may be used in a method of treatment or in uses disclosed in WO 2015009616, WO 2019043609, WO 2022013604, WO 2022234541, WO 2022264101, WO 2023137218, WO 2023166487, WO 2024176169, WO 2025046421, and WO 2025172910, the disclosures of which are incorporated here by reference.

[0103]

[0042] In an aspect, the disclosure provides a method of treating a complement mediated disease or disorder, e.g., asthma, arthritis (e.g., rheumatoid arthritis), autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, ischemia-reperfusion injuries, Barraquer-Simons Syndrome, hemodialysis, anca vasculitis, cryoglobulinemia, systemic lupus, lupus erythematosus, psoriasis, multiple sclerosis, transplantation, diseases of the central nervous system such as Alzheimer's disease and other neurodegenerative conditions, atypical hemolytic uremic syndrome (aHUS), glomerulonephritis (including membrane proliferative glomerulonephritis), dense deposit disease, blistering cutaneous diseases (including bullous pemphigoid, pemphigus, and epidermolysis bullosa), ocular cicatrical pemphigoid, MPGN II, PNH, IgAN, C3G, IC-MPGN, LN, gMG, AAV, or aHUS, in a subject, the method comprising administering an effective amount of the crystalline form or pharmaceutical composition to the subject in need thereof.

[0104]

[0043] In an aspect, the disclosure provides use of the crystalline form or pharmaceutical composition in the manufacture of a medicament for the treatment of a complement mediated disease or disorder, e.g., asthma, arthritis (e.g., rheumatoid arthritis), autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, ischemia-reperfusion injuries, Barraquer-Simons Syndrome, hemodialysis, anca vasculitis, cryoglobulinemia, systemic lupus, lupus erythematosus, psoriasis, multiple sclerosis, transplantation, diseases of the central nervous system such as Alzheimer's disease and other neurodegenerative conditions, atypical hemolytic uremic syndrome (aHUS), glomerulonephritis (including membrane proliferative glomerulonephritis), dense deposit disease, blistering cutaneous diseases (including bullous pemphigoid, pemphigus, and epidermolysis bullosa), ocular cicatrical pemphigoid, MPGN II, PNH, IgAN, C3G, IC-MPGN, LN, gMG, AAV, or aHUS.

[0105]

[0044] In an aspect, the disclosure provides the crystalline form or pharmaceutical composition for use in the treatment of a complement mediated disease or disorder, e.g., asthma, arthritis (e.g., rheumatoid arthritis), autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, ischemia-reperfusion injuries, Barraquer- Simons Syndrome, hemodialysis, anca vasculitis, cryoglobulinemia, systemic lupus, lupus erythematosus, psoriasis, multiple sclerosis, transplantation, diseases of the central nervous system such as Alzheimer's disease and other neurodegenerative conditions, atypical hemolytic uremic syndrome (aHUS), glomerulonephritis (including membrane proliferative glomerulonephritis), dense deposit disease, blistering cutaneous diseases (including bullous pemphigoid, pemphigus, and epidermolysis bullosa), ocular cicatrical pemphigoid, MPGN II, PNH, IgAN, C3G, IC-MPGN, LN, gMG, AAV, or aHUS.

[0106]

[0045] Other aspects of the invention are disclosed infra.

[0107] BRIEF DESCRIPTION OF THE DRAWINGS

[0108]

[0046] FIG. 1 is a graph depicting an X-ray powder diffraction (XRPD) spectrum of crystalline form HCof iptacopan hydrochloride hydrate.

[0047] FIG. 2 is a graph depicting a thermogravimetric analysis (TGA) curve of crystalline form HCof iptacopan hydrochloride hydrate.

[0048] FIG. 3 is a graph depicting a differential scanning calorimetry (DSC) curve of crystalline form HCof iptacopan hydrochloride hydrate.

[0109] DETAILED DESCRIPTION

[0110]

[0049] Increasing the amount of reactants and solvents in order to scale up a process to a full size commercial production may be associated with lower yields, or some safety issues while handling large amount of hazardous and / or toxic chemicals.

[0111] In an aspect, the disclosure provides a scalable method that can safely be handled on a larger scale, with reproducible yields, using less hazardous / toxic chemicals. In certain aspects, the synthesis process in the method provides a compound of formula (I), or a salt (e.g., a pharmaceutically acceptable salt) thereof with high enantio- and diastereoselectivity. In certain aspects, the process provides a process using a biocatalyst, e.g., an enzyme or ketoreductase.

[0112] Method of Preparation / Synthesis

[0113]

[0050] In an aspect, the disclosure provides a method of preparing a compound of formula (I), or a salt thereof. In certain aspects, the method includes (i) a process of preparing a compound of formula (II), or a salt (e.g., a pharmaceutically acceptable salt) thereof, (ii) a process of preparing a compound of formula (III), or a salt (e.g., a pharmaceutically acceptable salt) thereof, and (iii) a process of preparing a compound of formula (I), or a salt (e.g., a pharmaceutically acceptable salt) thereof by reacting the compound of formula (II), or a salt (e.g., a pharmaceutically acceptable salt) thereof and the compound of formula (III), or a salt (e.g., a pharmaceutically acceptable salt) thereof. A summary of the overall process is shown in Scheme 1, vide infra.

[0114]

[0115] 9 Compound of Formula (I) Scheme 1

[0116] 1. Synthesis of Compound of Formula (II)

[0117] 1.1 Synthesis of Compound 5

[0118]

[0051] In an aspect, the disclosure provides a process for preparing a compound of formula (II), or a salt thereof, as depicted in Schemes 2 (Method A) and 3 (Method B) below.

[0119]

[0120] Scheme 3

[0121] 1.1.1 Intermediate Compound 3

[0122]

[0052] In an aspect, the disclosure provides a process for preparing Compound 3 as

[0123] shown in Schemes 2 and 3 above, or in Scheme 4 below. The process includes a step of C-acylation, i.e., reacting Compound 1 and Compound 2 in the presence of a coupling reagent to yield Compound 3.

[0124]

[0125] Scheme 4

[0126]

[0053] In certain aspects, excess coupling reagent may be added to Compound 1 and convert an acid (e.g., carboxylic acid) into an acid imidazolide prior to condensation and may enhance reactivity with a neutral magnesium salt of an alkyl malonate to form a β-ketoester.

[0127] In some embodiments, the coupling agent is carbonyldiimidazole (CDI)and can convert a carboxylic acid form of Compound 1 (in THF) into an acid imidazolide (not shown), which then reacts with Compound 2 (i.e. magnesium tert-butyl malonate), to afford the β-ketoester, i.e., Compound 3.

[0128]

[0054] In some embodiments, the reaction may be performed at a temperature ranging from about 5 to about 50 °C, from about 10 to about 40 °C, from about 15 to about 35 °C, or around the room temperature (e.g., from about 15 to about 25 °C). In some embodiments, the reaction is performed around the room temperature, or at about 10 ± 5 °C, about 15 ± 5 °C, or about 20 ± 5 °C.

[0129]

[0055] In some embodiments, the coupling agent (e.g., CDI) may be added to Compound 1 in an amount from about 1 to about 10 eq., from about 1 to about 9 eq., from about 1 to about 8 eq., from about 1 to about 7 eq., from about 1 to about 6 eq., from about 1 to about 5 eq., from about 1 to about 4 eq., from about 1 to about 3 eq., from about 1 to about 2 eq., from about 1 to about 1.5 eq., from about 1 to about 1.4 eq., from about 1 to about 1.3 eq., or from about 1 to about 1.2 eq.. In some embodiments, CDI may be added to Compound 1 at a ratio ranging from about 2 to about 10 eq., from about 2 to about 9 eq., from about 2 to about 8 eq., from about 2 to about 7 eq., from about 2 to about 6 eq., from about 2 to about 5 eq., from about 2 to about 4 eq., from about 2 to about 3 eq., or from about 2 to about 2.5 eq. In some embodiments, CDI may be added to Compound 1 at about 1.1 eq, at about 1.2 eq., at about 1.5 eq., at about 2.0 eq., at about 2.5 eq., at about 3.0 eq., at about 3.5 eq., at about 4.0 eq., at about 4.5 eq., or at about 5.0 eq.

[0130]

[0056] In some embodiments, Compound 2 may be added to a mixture of Compound 1 and CDI at a ratio ranging from about 1 to about 10 eq., from about 1 to about 9 eq., from about 1 to about 8 eq., from about 1 to about 7 eq., from about 1 to about 6 eq., from about 1 to about 5 eq., from about 1 to about 4 eq., from about 1 to about 3 eq., from about 1 to about 2 eq., from about 1 to about 1.5 eq., from about 1 to about 1.4 eq., from about 1 to about 1.3 eq., or from about 1 to about 1.2 eq. In some embodiments, Compound 2 may be added to the mixture of Compound 1 and CDI at a ratio ranging from about 2 to about 10 eq., from about 2 to about 9 eq., from about 2 to about 8 eq., from about 2 to about 7 eq., from about 2 to about 6 eq., from about 2 to about 5 eq., from about 2 to about 4 eq., from about 2 to about 3 eq., or from about 2 to about 2.5 eq. In some embodiments, Compound 2 may be added to the mixture of Compound 1 and CDI at about 1.1 eq, at about 1.2 eq., at about 1.5 eq., at about 2.0 eq., at about 2.5 eq., at about 3.0 eq., at about 3.5 eq., at about 4.0 eq., at about 4.5 eq., or at about 5.0 eq.

[0057] In some embodiments, the reaction mixture including Compound 1, CDI and Compound 2 may be stirred at a temperature range from about 5 to about 50 °C, from about 10 to about 40 °C, from about 15 to about 35 °C, or around the room temperature (e.g., from about 15 to about 25 °C). In some embodiments, the reaction is performed around the room temperature, or at about 10 ± 5 °C, about 15 ± 5 °C, or about 20 ± 5 °C.

[0131]

[0058] In some embodiments, the reaction mixture including Compound 1, CDI and Compound 2 may be stirred for a reaction time of no less than about 1 hour, no less than about 2 hours, no less than about 3 hours, no less than about 4 hours, no less than about 5 hours, no less than about 6 hours, no less than about 7 hours, no less than about 8 hours, no less than about 9 hours, no less than about 10 hours, no less than about 11 hours or no less than about 12 hours.

[0132]

[0059] In some embodiments, the C-acylation reaction may be quenched in an acidic condition. For example, the reaction may be added to a weak acid solution such as citric acid solution. In some embodiments, the resulting product may be separated in the organic layer, e.g., using organic solvent. In some embodiments, the resulting product may be extracted using toluene solution.

[0133] 1.1.2 Synthesis Method A for Compound 5

[0134]

[0060] In an aspect, the disclosure provides a process for preparing Compound 5 as shown in Scheme 5 below. The process includes a step of reacting Compound 3 as prepared above with Compound 4 in acidic condition.

[0135]

[0136] Scheme 5

[0137]

[0061] In Scheme 5, R1is a carboxyl protecting group as described herein.

[0138]

[0062] In some embodiments, R1is C1-C6alkyl, C1-C4alkyl, or C1-C2alkyl. In some embodiments, R1is methyl or ethyl. In some embodiments, R1is methyl.

[0063] In certain aspects, Compound 3 diluted in a toluene solution may be mixed with or added to Compound 4 that contains a carboxyl protecting group (i.e. R1in this disclosure) to afford Compound 5.

[0064] In some embodiments, R1may include a C1-C6-alkyl (e.g., ethyl, methyl, allyl or tert-butyl), or C6-C10-aryl-C1-C6-alkyl (e.g., benzyl), or a silyl group (e.g., SiR11R12R13, wherein R11, R12, and R13are, independently of each other, C1-C7-alkyl, C6-C10-aryl or phenyl-C1-C4-alkyl). In some embodiments, R1is C1-C6alkyl, C1-C4alkyl, or C1-C2alkyl. In some embodiments, R1is methyl or ethyl. In some embodiments, R1is methyl.

[0139]

[0065] In some embodiments, a strong acid may be added to the reaction mixture of Compound 3 and Compound 4 in a toluene solution. In some embodiments, the strong acid may be an acid solution having pKa less than about 2. In some embodiments, the strong acid may be an acid solution having pKa less than about 1.5. In some embodiments, the strong acid may be an acid solution having pKaless than about 1. In some embodiments, the strong acid may be an acid solution having pKaless than about 0.5. In some embodiments, the strong acid may be an acid solution having pKaless than about 0.4. In some embodiments, the strong acid may be an acid solution having pKaless than about 0.3. In some embodiments, the strong acid may be an acid solution having pKaless than about 0.2. In some embodiments, the strong acid may be trifluoroacetic acid (TFA).

[0140]

[0066] In some embodiments, the reaction may be performed at its internal temperature ranging from about 20 to about 80 °C, from about 30 to about 75 °C, from about 45 to about 70 °C, or from about 55 to about 65 °C. In some embodiments, the reaction may be performed at its internal temperature at about 60 ± 5 °C.

[0141]

[0067] In some embodiments, the reaction mixture including Compound 3, Compound 4 and the acid may be stirred for a reaction time of no less than about 1 hour, no less than about 2 hours, no less than about 3 hours, no less than about 4 hours, no than about 5 hours, no less than about 6 hours, no less than about 7 hours, or no less than about less 8 hours. In some embodiments, the reaction mixture including Compound 3, Compound 4 and the acid may be stirred for no less than about 4 hours, no less than about 5 hours, or no less than about 6 hours.

[0142]

[0068] In some embodiments, the reaction mixture may be treated with hydrochloric acid (HC1) in ethyl acetate (EA) solution. In some embodiments, the HC1 / EA treated reaction mixture may be maintained at its internal temperature ranging from about 20 to about 80 °C, from about 30 to about 75 °C, from about 45 to about 70 °C, or from about 55 to about 65 °C. In some embodiments, the reaction mixture added with the HC1 / EA may be stirred for no less than about 4 hours, no less than about 5 hours, or no less than about 6 hours. In some embodiments, the reaction after adding HC1 / EA may be performed at its internal temperature around 60 ± 5 °C. In some embodiments, the reaction mixture treated with HC1 / EA acid may be stirred for a reaction time of no less than about 1 hour, no less than about 2 hours, no less than about 3 hours, or no less than about 4 hours.

[0143]

[0069] In some embodiments, additional HC1 / EA may be added to the reaction mixture. In some embodiments, additionally HC1 / EA treated reaction mixture may be maintained at its internal temperature ranging from about 20 to about 80 °C, from about 30 to about 75 °C, from about 45 to about 70 °C, or from about 55 to about 65 °C. In some embodiments, the reaction mixture added with the HC1 / EA may be stirred for no less than about 4 hours, no less than about 5 hours, or no less than about 6 hours. In some embodiments, the reaction may be performed at its internal temperature around 60 ± 5 °C. In some embodiments, the reaction mixture treated with the additional HC1 / EA may be stirred for a reaction time of no less than about 5 hours, no less than about 6 hours, no less than about 7 hours, no less than about 8 hours, no less than about 9 hours, no less than about 10 hours, no less than about 11 hours, no less than about 12 hours, no less than about 13 hours, no less than about 14 hours, or no less than about 15 hours.

[0144]

[0070] Alternatively, in some embodiments, instead of HC1 / EA solution, the reaction mixture may be treated with hydrochloric acid (HC1) in isopropanol (iPrOH) solution. In some embodiments, the HCl / iPrOH treated reaction mixture may be treated similar to the HC1 / EA treated reaction mixture.

[0145]

[0071] At observation of solid precipitation while stirring, the reaction may be cooled to reach the internal temperature ranging from about 15 to about 40 °C, about 20 to about 30 °C, from about 25 to about 30 °C, or from about 20 to about 25 °C. In some embodiments, the reaction is cooled down to reach its internal temperature at about 25 ± 5 °C. In some embodiments, the cooling down is performed in no less than about 1 hours, no less than about 2 hours, or no less than about 3 hours, while stirring the reaction mixture. In some embodiments, the internal temperature cooled to the internal temperature range of 25 ± 5 °C may be maintained for no less than about 1 hours, no less than about 2 hours, no less than about 3 hours, or no less than about 4 hours. In some embodiments, the solid precipitation of the reaction product, i.e. Compound 5, may be separated (e.g., filtered), purified, washed and / or dried to afford an off-white solid.

[0146] 1.1.3 Synthesis Method B for Compound 5

[0147]

[0072] Alternatively, in an aspect, the disclosure provides a process for preparing Compound 5 as shown in Scheme 6 below. The process includes a step of reacting Compound 3 as prepared above with Compound 4 in a condition.

[0148]

[0149] Scheme 6

[0150]

[0073] In Scheme 6, R1is a carboxyl protecting group as described herein.

[0151]

[0074] In some embodiments, R1is Ci-Ce alkyl, C1-C4 alkyl, or C1-C2 alkyl. In some embodiments, R1is methyl or ethyl. In some embodiments, R1is methyl.

[0152]

[0075] In certain aspects, Compound 3 and Compound 4 are reacted in an organic solvent (e.g., alcohol) together with a weak acid (e.g., acetic acid) and an amine base (e.g., piperidine). In some embodiments, Compound 3 and Compound 4 are mixed at a ratio of about 1: 1 (eq.), of about 1:1.1 (eq.), of about 1:1.2 (eq.), of about 1:1.3 (eq.), of about 1:1.4 (eq.), of about 1:1.5 (eq.), of about 1:1.6 (eq.), of about 1:1.7 (eq.), of about 1:1.8 (eq.), of about 1:1.9 (eq.), or of about 1:2.0 (eq.).

[0153]

[0076] In some embodiments, the weak acid may be added to the reaction mixture of Compound 3 and Compound 4 in an amount of about 1.0 eq., about 1.1 eq., about 1.2 eq., about 1.3 eq., about 1.4 eq., about 1.4 eq., about 1.5 eq., about 1.6 eq., about 1.7 eq., about 1.8 eq., about 1.9 eq., or about 2.0 eq relative to the amount (1 eq.) of Compound 3. In some embodiments, the weak acid may be an acid solution having pKaless than about 10. In some embodiments, the weak acid may be an acid solution having pKaless than about 9. In some embodiments, the weak acid may be an acid solution having pKaless than about 8. In some embodiments, the weak acid may be an acid solution having pKaless than about 7. In some embodiments, the weak acid may be an acid solution having pKaless than about 6. In some embodiments, the weak acid may be an acid solution having pKaless than about 5. In some embodiments, the weak acid may be an acid solution having pKaless than about 4. In some embodiments, the weak acid may be an acid solution having pKaless than about 3. In some embodiments, the weak acid may be acetic acid (AcOH).

[0154]

[0077] In some embodiments, the amine base may be added to the reaction mixture of Compound 3 and Compound 4 in an amount of about 1.0 eq., about 1.1 eq., about 1.2 eq., about 1.3 eq., about 1.4 eq., about 1.4 eq., about 1.5 eq., about 1.6 eq., about 1.7 eq., about 1.8 eq., about 1.9 eq., or about 2.0 eq relative to the amount (1.0 eq.) of Compound 3. In some embodiments, the amine base may be piperidine.

[0155]

[0078] In some embodiments, the amine base may be added to the reaction mixture of Compound 3 and Compound 4 simultaneously with the weak acid (e.g., AcOH). In some embodiments, the amine base may be added to the reaction mixture of Compound 3 and Compound 4 after the weak acid (e.g., AcOH) is added. In some embodiments, the amine base may be added to the reaction mixture of Compound 3 and Compound 4 before the weak acid (e.g., AcOH) is added. In some embodiments, the weak acid may be acetic acid (AcOH).

[0156]

[0079] In some embodiments, the reaction to which AcOH and piperidine are added may be evacuated and backfilled with N2 and the reaction is performed, e.g., by stirring at a reaction temperature in a range from about 20 to about 65 °C, from about 25 to about 60 °C, from about 30 to about 55 °C, or from about 40 to about 50 °C. In some embodiments, the reaction to which AcOH and piperidine are added may be evacuated and backfilled with N2 and the reaction is performed at about 40 ±5 °C, about 45 ±5 °C, or about 50 ±5 °C.

[0157]

[0080] In some embodiments, the process in Scheme 6 may include obtaining Intermediate 5B by quenching the reaction. In some embodiments, Intermediate 5B may be separated (e.g., filtered), purified, washed and / or dried to afford an off-white solid.

[0158]

[0081] In some embodiments, Intermediate 5B is subsequently treated in an organic solvent (e.g., ethyl acetate) and under N2 atmosphere with HC1 / EA (step 2). In some embodiments, Intermediate 5B in the organic solvent and under N2 atmosphere is cooled to a temperature range of from about -10 to about 10 °C, from about -5 to about 5 °C, from about -3 to about 3 °C, or at about 0 °C prior to adding the HC1 / EA. In some embodiments, the HC1 / EA treated reaction mixture may be maintained at its internal temperature ranging from about -10 to about 10 °C, from about -5 to about 5 °C, from about -3 to about 3 °C, or at about 0 °C. In some embodiments, the reaction mixture added with the HC1 / EA may be stirred for no less than about 4 hours, no less than about 5 hours, or no less than about 6 hours.

[0159]

[0082] In some embodiments, the step 2 reaction may be quenched by adding HCl / H₂O and stirring at the reaction temperature in a range from about 20 to about 65 °C, from about 25 to about 60 °C, from about 30 to about 55 °C, or from about 40 to about 50 °C, or at about 45 °C. In some embodiments, the quenching is performed for no less than about 8 hours, no less than about 7 hours, no less than about 6 hours, no less than about 5 hours, no less than about 4 hours, no less than about 3 hours, or no less than about 2 hours.

[0083] In some embodiments, the quenching may be followed by cooling down to the temperature ranging from about 15 °C to about 40 °C, from about 20 to about 30 °C, from about 25 to about 30 °C, or from about 20 to about 25 °C to afford the resulting product, Compound 5. In some embodiments, Compound 5 is obtained as racemic mixture.

[0160]

[0084] In some embodiments, the internal temperature of the cooled reaction may be about 25 ± 5 °C and be maintained for no less than about 1 hours, no less than about 2 hours, no less than about 3 hours, or no less than about 4 hours. In some embodiments, the solid precipitation of the reaction product, Compound 5 is separated (e.g., filtered), purified, washed and / or dried to afford an off-white solid.

[0161]

[0085] In some embodiments, the reaction to convert Intermediate 5B into Compound 5 is cooled down to reach the internal reaction temperature below than about 35 °C, about 30

[0162] °C, about 25 °C, or about 20 °C while stirring the reaction mixture.

[0163] 1.2, Synthesis of Compound of Formula: Compound 5 Compound (SI-6 ->

[0164]

[0165] Compound of Formula (II)

[0166]

[0086] One aspect of the present invention relates to an asymmetric process for preparing a compound of formula (II), or salt thereof, as depicted in Scheme 7 below. In certain

[0167] aspects, the stereocenters in position 2 and in position 4 on the piperidine are obtained in high enantio- and diastereoselectivity.

[0168]

[0169] Scheme 7

[0170] 1.2.1. Synthesis of Compound 6

[0171]

[0087] In an aspect, the disclosure provides a step of treating Compound 5, racemic mixture, under reductive enzymatic conditions to obtain Compound 6 as depicted in Scheme 8 below.

[0172]

[0173] Scheme 8

[0174]

[0088] In Scheme 8, R1is a carboxyl protecting group as described herein.

[0175]

[0089] In some embodiments, R1is Ci-Ce alkyl, C1-C4 alkyl, or C1-C2 alkyl. In some embodiments, R1is methyl or ethyl. In some embodiments, R1is methyl.

[0176]

[0090] In certain aspects, the reductive enzymatic conditions, as disclosed herein, include treating Compound 5, racemic mixture, with an enzyme, a co-factor, in an aqueous buffer solution, optionally in the presence of a surfactant, to provide Compound (S)-C6.

[0177]

[0091] The enzyme used to perform the reaction depicted in Scheme 8 is any enzyme suitable to perform the above transformation. Suitable enzymes for use in the present reaction mixture include one or more selected from ketoreductases (KRED), alcohol dehydrogenases, and glucose dehydrogenase (GDH). In some embodiments, the enzyme is a ketoreductase (KRED). In some embodiments, the reaction can comprise a second enzyme, so-called co-enzyme, for example, glucose dehydrogenase (GDH). In some embodiments, it is appreciated that the ketoreductase (KRED) used in the disclosure may include products that could be purchased from Codexis Inc. (Codex® KRED screening kit), which are described e.g. in WO 2005 / 017135, WO 2008 / 103248, W02009 / 029554, W02009 / 036404, W02016 / 130412, and W02018 / 013710. In some embodiments, it is appreciated that the ketoreductase (KRED) used in the disclosure may include KRED-EW124 that could be purchased from Enzyme Works Inc. China. In some embodiments, the ketoreductase (KRED) may suitably include, but not be limited to, one or more selected from KRED-EW124, KRED-P3-G09, KRED-P1-B02, KRED-P1-C01, KRED-P2-B02, KRED-P2-C02, KRED-P3-B03, KRED-P2-D03, KRED-P2-D11, KRED-P2-D12, KRED-P2-H07, KRED-P3-H12, KRED-101, and KRED-119.

[0178]

[0092] In some embodiments, the ketoreductase (KRED) may have the amino acid sequence of SEQ ID NO: 1 below:

[0179] ( SEQ ID NO: 1 ) MKGFAMLSIGKVGWIEKEKPAPGPFDAIVRPLAVAPCTSDIHTVFGGAGGERHNMILG HEAVGEWEVGSEVKDFKPGDRVWPAITPDWRTSEVQRGYHQHSGGMTAGVKFSNVK D GVFG E F FHV DADMN LAH L P KE I P L EAAVMI P DMMT T G FH GAE L AD I E L GAT VAVL G IGPVGLMAVAGAKLRGAGRIIAVGSRPVCVDAAKYYGATDIVNYKDGPIESQIMNLTE GKGVDAAIIAGGNADIMATAVKIVKPGGTIANVNYFGEGEVLPVPRLEWGCGMAHKTI KGGLCPGGRLRMERLIDLVFYKRVDPSKLVTHVFRGFDNIEKAFMLMKDKPKDLIKPV VILA

[0180]

[0093] In some embodiments, the ketoreductase (KRED) may include a protein having the amino acid sequence of SEQ ID NO: 1, or a fragment thereof. In some embodiments, the ketoreductase (KRED) may include a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid of SEQ ID NO: 1, or a fragment thereof. In some embodiments, the ketoreductase (KRED) may include a protein having a sequence similarity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid of SEQ ID NO: 1, or a fragment thereof.

[0181]

[0094] In some embodiments, the ketoreductase (KRED) may be produced using a nucleic acid sequence of SEQ ID NO: 2 below:

[0182] ( SEQ ID NO: 2 ) ATGAAAGGCTTCGCCATGCTGAGCATCGGCAAAGTGGGTTGGATTGAAAAAGAAAAAC CGGCGCCAGGCCCGTTCGATGCAATTGTGCGCCCTCTGGCAGTAGCGCCGTGTACCAG CGATATTCATACTGTGTTTGGGGGTGCCGGTGGCGAGCGTCACAATATGATTCTGGGC CATGAAGCCGTTGGTGAAGTTGTTGAGGTTGGCAGCGAAGTGAAGGATTTCAAACCGG GCGATCGCGTTGTCGTTCCAGCAATTACCCCGGATTGGCGCACCAGCGAAGTCCAGCG CGGCTACCATCAGCACTCTGGCGGCATGACCGCCGGCGTAAAATTCAGCAATGTAAAG GATGGTGTGTTCGGTGAATTTTTTCACGTTAACGACGCAGACATGAATCTGGCGCACC TGCCGAAAGAAATCCCGCTGGAAGCAGCGGTTATGATTCCGGATATGATGACCACGGG TTTTCACGGCGCAGAGCTGGCGGACATTGAACTGGGCGCTACGGTAGCCGTACTGGGC ATCGGTCCGGTGGGCCTGATGGCAGTTGCAGGCGCTAAGCTGCGCGGCGCAGGTCGTA TTATTGCCGTTGGTTCTCGCCCGGTGTGTGTGGACGCCGCTAAGTATTATGGTGCAAC GGACATTGTCAATTACAAGGACGGCCCAATTGAATCTCAGATCATGAACCTGACGGAA GGTAAAGGCGTTGACGCCGCGATTATCGCTGGCGGCAACGCCGACATCATGGCGACCG CAGTTAAAATCGTCAAGCCAGGTGGTACTATTGCTAACGTCAACTATTTCGGCGAAGG TGAGGTCCTGCCTGTCCCACGTCTGGAATGGGGTTGCGGTATGGCACATAAAACCATT AAAGGTGGCCTGTGCCCAGGCGGCCGTCTGCGTATGGAACGCCTGATCGATCTGGTCT TCTACAAACGCGTGGATCCTAGCAAACTGGTGACTCACGTTTTCCGCGGCTTTGATAA CAT C G AAAAAG CTTTTATGCT GAT G AAAGAT AAAC C GAAAGAT C T GAT T AAAC C G GT T GTCATCCTGGCTTGA

[0183]

[0095] In some embodiments, the KRED may be produced or prepared from a nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the KRED may be produced or prepared in a cell including the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid sequence for producing or preparing the KRED may have sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 2.

[0096] In some embodiments, the ketoreductase (KRED) may have the amino acid sequence of SEQ ID NO: 3 below:

[0184] ( SEQ ID NO: 3 ) MKGFAMLSIGKVGWIEKEKPAPGPFDAIVRPLAVAPCTSDIHTVFEGAGGERHNMILG HEAVGEWEVGSEVKDFKPGDRVWPAITPDWRTSEVQRGYHQHSGGMTAGVKFSNVK D GVFG E F FHV DADMN LAH L P KE I P L EAAVMI P DMMT T G FH GAE L AD I E L GAT VAVL G IGPVGLMAVAGAKLRGAGRIIAVGSRPVCVDAAKYYGATDIVNYKDGPIESQIMNLTE GKGVDAAIIAGGNADIMATAVKIVKPGGTIANVNYFGEGEVLPVPRLEWGCGMAHKTI KGGLCPGGRLRMERLIDLVFYKRVDPSKLVTHVFRGFDNIEKAFMLMKDKPKDLIKPV VILA

[0185]

[0097] In some embodiments, the ketoreductase (KRED) may include a protein having the amino acid sequence of SEQ ID NO: 3, or a fragment thereof. In some embodiments, the ketoreductase (KRED) may include a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid of SEQ ID NO: 3, or a fragment thereof. In some embodiments, the ketoreductase (KRED) may include a protein having a sequence similarity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid of SEQ ID NO: 3, or a fragment thereof.

[0186]

[0098] In some embodiments, the ketoreductase (KRED) may be produced using a nucleic acid sequence of SEQ ID NO: 4 below:

[0187] (SEQ ID NO: 4) ATGAAAGGCTTCGCCATGCTGAGCATCGGCAAAGTGGGTTGGATTGAAAAAGAAAAA CCGGCGCCAGGCCCGTTCGATGCAATTGTGCGCCCTCTGGCAGTAGCGCCGTGTACC AGCGATATTCATACTGTGTTTGAAGGTGCCGGTGGCGAGCGTCACAATATGATTCTG GGCCATGAAGCCGTTGGTGAAGTTGTTGAGGTTGGCAGCGAAGTGAAGGATTTCAAA CCGGGCGATCGCGTTGTCGTTCCAGCAATTACCCCGGATTGGCGCACCAGCGAAGTC CAGCGCGGCTACCATCAGCACTCTGGCGGCATGACCGCCGGCGTAAAATTCAGCAAT GT AAAGG AT GGTGTGTTC GGT GAAT T T T T T C ACGT T AAC GAG GC AG AC AT G AAT CT G GCGCACCTGCCGAAAGAAATCCCGCTGGAAGCAGCGGTTATGATTCCGGATATGATG ACCACGGGTTTTCACGGCGCAGAGCTGGCGGACATTGAACTGGGCGCTACGGTAGCC GTACTGGGCATCGGTCCGGTGGGCCTGATGGCAGTTGCAGGCGCTAAGCTGCGCGGC GCAGGTCGTATTATTGCCGTTGGTTCTCGCCCGGTGTGTGTGGACGCCGCTAAGTAT TATGGTGCAACGGACATTGTCAATTACAAGGACGGCCCAATTGAATCTCAGATCATG AACCTGACGGAAGGTAAAGGCGTTGACGCCGCGATTATCGCTGGCGGCAACGCCGAC ATCATGGCGACCGCAGTTAAAATCGTCAAGCCAGGTGGTACTATTGCTAACGTCAAC TATTTCGGCGAAGGTGAGGTCCTGCCTGTCCCACGTCTGGAATGGGGTTGCGGTATG GCACATAAAACCATTAAAGGTGGCCTGTGCCCAGGCGGCCGTCTGCGTATGGAACGC CTGATCGATCTGGTCTTCTACAAACGCGTGGATCCTAGCAAACTGGTGACTCACGTT TTCCGCGGCTTTGATAACATCGAAAAAGCTTTTATGCTGATGAAAGATAAACCGAAA GATCTGATTAAACCGGTTGTCATCCTGGCTTGA

[0188]

[0099] In some embodiments, the KRED may be produced or prepared from a nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the KRED may be produced or prepared in a cell including the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the nucleic acid sequence for producing or preparing the KRED may have sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 4.

[0189]

[0100] In some embodiments, the ketoreductase (KRED) may have the amino acid sequence of SEQ ID NO: 5 below:

[0190] ( SEQ ID NO: 5 ) MKGFAMLSIGKVGWIEKEKPAPGPFDAIVRPLAVAPCTSDIHTVFEGAWGERHNMILG HEAVGEWEVGSEVKDFKPGDRVWPAITPDWRTSEVQRGYHQHSGGMTAGVKFSNVK D GVFG E F FHV DADMN LAH L P KE I P L EAAVMI P DMMT T G FH GAE L AD I E L GAT VAVL G IGPVGLMAVAGAKLRGAGRIIAVGSRPVCVDAAKYYGATDIVNYKDGPIESQIMNLTE GKGVDAAIIAGGNADIMATAVKIVKPGGTIANVNYFGEGEVLPVPRLEWGCGMAHKTI KGGLCPGGRLRMERLIDLVFYKRVDPSKLVTHVFRGFDNIEKAFMLMKDKPKDLIKPV VILA

[0191]

[0101] In some embodiments, the ketoreductase (KRED) may include a protein having the amino acid sequence of SEQ ID NO: 5, or a fragment thereof. In some embodiments, the ketoreductase (KRED) may include a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid of SEQ ID NO: 5, or a fragment thereof. In some embodiments, the ketoreductase (KRED) may include a protein having a sequence similarity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid of SEQ ID NO: 5, or a fragment thereof.

[0192]

[0102] In some embodiments, the ketoreductase (KRED) may be produced using a nucleic acid sequence of SEQ ID NO: 6 below: (SEQ ID NO: 6) ATGAAAGGCTTCGCCATGCTGAGCATCGGCAAAGTGGGTTGGATTGAAAAAGAAAAA CCGGCGCCAGGCCCGTTCGATGCAATTGTGCGCCCTCTGGCAGTAGCGCCGTGTACC AGCGATATTCATACTGTGTTTGAAGGTGCCTGGGGCGAGCGTCACAATATGATTCTG GGCCATGAAGCCGTTGGTGAAGTTGTTGAGGTTGGCAGCGAAGTGAAGGATTTCAAA CCGGGCGATCGCGTTGTCGTTCCAGCAATTACCCCGGATTGGCGCACCAGCGAAGTC CAGCGCGGCTACCATCAGCACTCTGGCGGCATGACCGCCGGCGTAAAATTCAGCAAT GT AAAGG AT GGTGTGTTC GGT GAAT T T T T T C ACGT T AAC GAG GC AG AC AT G AAT CT G GCGCACCTGCCGAAAGAAATCCCGCTGGAAGCAGCGGTTATGATTCCGGATATGATG ACCACGGGTTTTCACGGCGCAGAGCTGGCGGACATTGAACTGGGCGCTACGGTAGCC GTACTGGGCATCGGTCCGGTGGGCCTGATGGCAGTTGCAGGCGCTAAGCTGCGCGGC GCAGGTCGTATTATTGCCGTTGGTTCTCGCCCGGTGTGTGTGGACGCCGCTAAGTAT TATGGTGCAACGGACATTGTCAATTACAAGGACGGCCCAATTGAATCTCAGATCATG AACCTGACGGAAGGTAAAGGCGTTGACGCCGCGATTATCGCTGGCGGCAACGCCGAC ATCATGGCGACCGCAGTTAAAATCGTCAAGCCAGGTGGTACTATTGCTAACGTCAAC TATTTCGGCGAAGGTGAGGTCCTGCCTGTCCCACGTCTGGAATGGGGTTGCGGTATG GCACATAAAACCATTAAAGGTGGCCTGTGCCCAGGCGGCCGTCTGCGTATGGAACGC CTGATCGATCTGGTCTTCTACAAACGCGTGGATCCTAGCAAACTGGTGACTCACGTT TTCCGCGGCTTTGATAACATCGAAAAAGCTTTTATGCTGATGAAAGATAAACCGAAA GATCTGATTAAACCGGTTGTCATCCTGGCTTGA

[0193]

[0103] In some embodiments, the KRED may be produced or prepared from a nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the KRED may be produced or prepared in a cell including the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the nucleic acid sequence for producing or preparing the KRED may have sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 6.

[0194]

[0104] In some embodiments, the ketoreductase (KRED) may have the amino acid sequence of SEQ ID NO: 7 below:

[0195] ( SEQ ID NO: 7 ) MKGFAMLSIGKVGWIEKEKPAPGPFDAIVRPLAVAPCTSDIHTVFEGAWGERHNMILG HEAVGEWEVGSEVKDFKPGDRVWPAITPDWRTSEVQRGYHQHSGGMTAGVKFGNVK D GVFG E F FHV DADMN LAH L P KE I P L EAAVMI P DMMT T G FH GAE L AD I E L GAT VAVL G IGPVGLMAVAGAKLRGAGRIIAVGSRPVCVDAAKYYGATDIVNYKDGPIESQIMNLTE GKGVDAAIIAGGNADIMATAVKIVKPGGTIANVNYFGSGEVLPVPRLEWGCGMAHKTI KGGLCPGGRLRMERLIDLVFYKRVDPSKLVTHVFRGLDNIEKAFMLMKDKPKDLIKPV VILA

[0196]

[0105] In some embodiments, the ketoreductase (KRED) may include a protein having the amino acid sequence of SEQ ID NO: 7, or a fragment thereof. In some embodiments, the ketoreductase (KRED) may include a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid of SEQ ID NO: 7, or a fragment thereof. In some embodiments, the ketoreductase (KRED) may include a protein having a sequence similarity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid of SEQ ID NO: 7, or a fragment thereof.

[0197]

[0106] In some embodiments, the ketoreductase (KRED) may be produced using a nucleic acid sequence of SEQ ID NO: 8 below:

[0198] (SEQ ID NO: 8) ATGAAAGGCTTCGCCATGCTGAGCATCGGCAAAGTGGGTTGGATTGAAAAAGAA AAACCGGCGCCAGGCCCGTTCGATGCAATTGTGCGCCCTCTGGCAGTAGCGCCGT GTACCAGCGATATTCATACTGTGTTTGAAGGTGCCTGGGGCGAGCGTCACAATAT GATTCTGGGCCATGAAGCCGTTGGTGAAGTTGTTGAGGTTGGCAGCGAAGTGAA GGATTTCAAACCGGGCGATCGCGTTGTCGTTCCAGCAATTACCCCGGATTGGCGC ACCAGCGAAGTCCAGCGCGGCTACCATCAGCACTCTGGCGGCATGACCGCCGGC GTAAAATTCGGTAATGTAAAGGATGGTGTGTTCGGTGAATTTTTTCACGTTAACG ACGCAGACATGAATCTGGCGCACCTGCCGAAAGAAATCCCGCTGGAAGCAGCGG TTATGATCCCAGATATGATGACCACGGGTTTTCACGGCGCAGAGCTGGCGGACAT TGAACTGGGCGCTACGGTAGCCGTACTGGGCATCGGTCCGGTGGGCCTGATGGCA GTTGCAGGCGCTAAGCTGCGCGGCGCAGGTCGTATTATTGCCGTTGGTTCTCGCC CGGTGTGTGTGGACGCCGCTAAGTATTATGGTGCAACGGACATTGTCAATTACAA GGACGGCCCAATTGAATCTCAGATCATGAACCTGACGGAAGGTAAAGGCGTTGA CGCCGCGATTATCGCTGGCGGCAACGCCGACATCATGGCGACCGCAGTTAAAATC GTCAAGCCAGGTGGTACTATTGCTAACGTCAACTATTTCGGCAGCGGTGAGGTCC TGCCTGTCCCACGTCTGGAATGGGGTTGCGGTATGGCACATAAAACCATTAAAGG TGGCCTGTGCCCAGGCGGCCGTCTGCGTATGGAACGCCTGATCGATCTGGTCTTC TACAAACGCGTGGATCCTAGCAAACTGGTGACTCACGTTTTCCGCGGCCTGGATA ACATCGAAAAAGCTTTTATGCTGATGAAAGATAAACCGAAAGATCTGATTAAAC CGGTTGTCATCCTGGCTTGA

[0107] In some embodiments, the KRED may be produced or prepared from a nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the KRED may be produced or prepared in a cell including the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid sequence for producing or preparing the KRED may have sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 8.

[0199]

[0108] In some embodiments, the enzyme (e.g., KRED) is present in the reaction mixture in a concentration suitable to perform the reaction as depicted in Scheme 8 in an amount of about 0.01% to about 100% relative to the amount of Compound 5, racemic mixture. In some embodiments, the enzyme (e.g., KRED) may be present in an amount of about 0.1% to about 75%, about 0.5% to about 50%, about 1% to about 40%, about 2% to about 30%, about 4% to about 25% or about 5% to about 20%, relative to the amount of Compound 5, racemic mixture.

[0200]

[0109] In some embodiments, the reaction as depicted in Scheme 8, further includes a co-factor. The presence and type of the co-factor depends on the enzymatic reaction, which is to be performed. In some embodiments, the co-factor may include one or more selected from the group consisting of nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD), and pyridoxal monophosphate. The co-factor may be used to provide protons, or electrons for the enzymatic reaction. In some embodiments, the co-factor may be present in the reaction mixture in an ionic form, such as, for example, NAD+, NADP+. In some embodiments, the co-factor may be present in the reaction mixture in a protonated form, such as, for example, NAD-H, NADP-H, or NADP-Na. In some embodiments, the reaction is an enzyme-catalyzed reaction. In some embodiments, the reaction mixture may comprise a further enzyme, so called co-enzyme, which regenerates the co-factor. In some embodiments, when NAD, NADP or FAD is used as co-factor, the aqueous reaction mixture may further include a dehydrogenase such as an alcohol dehydrogenase, or a glucose dehydrogenase, and a respective substrate such as an alcohol or glucose. The co-factor may be present in the aqueous reaction mixture in stoichiometric amounts. In some embodiments, the molar amount may be at least as high as the molar amount of Compound 5. In some embodiments, the amount of co-factor is less than the amount of Compound 5, racemic mixture. In some embodiments, the amount of co-factor is in the range from about 0.01% to about 20%, about 0.05% to about 15%, about 0.1% to about 10%, about 0.25% to about 7.5%, or about 0.5% to about 5% relative to the amount of Compound 5, racemic mixture.

[0201] [HO] In some embodiments, in the reaction depicted in Scheme 8, an acid may be used to promote racemization. For example, acids that may be used include, but are not limited to, D-proline, glycine, glutamic acid, aspartic acid, lysine, malonic acid, lactic acid, mandelic acid, and AcOH.

[0202] [Hl] In some embodiments, the reaction is performed in an aqueous buffer solution. The buffer solution should be suitable to keep the pH of the reaction mixture at, or about, a neutral pH. The aqueous reaction mixture may have a pH at which the enzyme is active and stable, and which is suitable for the enzymatic reaction. In some embodiments, the pH value is in the range from about 6.0 to about 8.0. In some embodiments, the pH is from about 6.5 to about 7.5, or about 7.0. In some embodiments, the buffer may be selected from the group consisting of 2-amino-2-(hydroxymethyl)propane- 1,3 -diol (TRIS), 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), 3-(A-morpholino) propanesulfonic acid (MOPS), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), borate, glycine, triethanol amine, phosphate, citrate, acetate and ammonia. In some embodiments, the buffer solution is a phosphate buffered saline (PBS) solution.

[0203]

[0112] In some embodiments, the reaction may be advantageously performed when the enzyme is a ketoreductase (KRED), the co-factor is nicotinamide adenine dinucleotide phosphate (NADP) or NADP-Na, in an aqueous buffer solution, optionally, including a surfactant. In some embodiments, the reaction is performed when the buffer solution is a mixture of a phosphate buffered saline (PBS) solution comprising a TPGS-750-M surfactant. In some embodiments, the reaction is performed at the internal temperature ranging from about 30 °C to about 70 °C, from about 40 °C to about 60 °C, or from about 40 °C to about 50 °C, or at a temperature of about 40 ± 5 °C, about 45 ± 5 °C, or 50 ± 5 °C.

[0204]

[0113] In some embodiments, the reaction under the above described conditions (e.g., using a biocatalyst such as KRED) may provide an environmentally friendly and scalable method of reducing a ketone into an alcohol, as the reaction is performed in aqueous media. Furthermore, because the reaction is diastereoselective, in some embodiments, the desired alcohol may be provided in high yield (e.g., more than about 70%, 80%, 85%, 90%, 95%, or 99%). In some embodiments, the reaction may be efficient to avoid mixtures of diastereoisomers as by-products.

[0205]

[0114] In some embodiments, the reaction may be quenched by deactivating the enzyme (e.g., KRED). In some embodiments, the reaction may be quenched by cooling down the reaction mixture to a temperature ranging from about 0 °C to about 20 °C, or from about 5 °C to about 15 °C, or at a temperature of about 0 ± 5 °C, about 5 ± 5 °C, or 10 ± 5 °C. In some embodiments, the reaction may be quenched by adding an acid, e.g., HC1, and / or by adjusting the pH in a range from about 2.0 to 5.0, from about 2.5 to 4.0, or at about 2.5 ± 1.0, at about 3.0 ± 0.5, or at about 3.0 ± 0.3.

[0206]

[0115] In some embodiments, the resulting product, i.e., Compound 6, is obtained, e.g., as being separated (e.g., filtered), purified, washed and / or dried to afford an off-white solid.

[0207] 1.2,2. Synthesis of compound of formula (II), or a salt thereof

[0208]

[0116] In an aspect, the disclosure provides a step of preparing a compound of formula (II), as depicted in Scheme 9. In some embodiments, the process may include a step of ethylating Compound (S)-6 to obtain the Compound of formula (II).

[0209]

[0210] of Formula (II)

[0211] Scheme 9

[0212]

[0117] In Scheme 9, R1is a carboxy protecting group as described herein.

[0213]

[0118] In some embodiments, R1is Ci-Ce alkyl, C1-C4 alkyl, or C1-C2 alkyl. In some embodiments, R1is methyl or ethyl. In some embodiments, R1is methyl.

[0214]

[0119] In certain aspects, the process may optionally include a step of attaching da protecting group (P2) to Compound (S)-6 as depicted in Scheme 10.

[0215]

[0216] Scheme 10

[0217]

[0120] In Scheme 10, R1is a carboxy protecting group as described herein, and P2is an oxygen protecting group.

[0218]

[0121] In some embodiments, the alcohol group of Compound (S)-(C6) may be protected with an oxygen protecting group P2in the presence of a base and in a solvent, to obtain an intermediate compound of formula (S)-(C6’). In some embodiments, P2may include a silyl group. In some embodiments, P2may be selected from the group consisting of tertbutyldimethylsilyl (TBS), trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), and tert-butyldiphenylsilyl (TBDPS).

[0219]

[0122] In some embodiments, the base may include an amine base. In some embodiments, the base may include one or more selected from triethylamine, pyridine, imidazole, 2,6-lutidine, and dimethylaminopyridine.

[0220]

[0123] In some embodiments, the solvent may include one or more selected from the group consisting of isopropanol. ethanol, dimethylformamide, acetonitrile, tetrahydrofuran, 2-methyl-tetrahydrofuran, dichloromethane (DCM), dichloroethane (DCE), toluene, and heptane.

[0221]

[0124] In certain embodiments, the reaction in the Scheme 10 above may be performed when the alcohol on Compound 6 is protected with an oxygen protecting group P2which is tert-butyldimethylsilyl (TBS), in the presence of imidazole as a base, in a mixture of acetonitrile and isopropanol, to obtain a first intermediate of Compound (S)-6’.

[0222]

[0125] In some embodiments, the oxygen protecting group P2on Compound (S)-6’ may be cleaved first and the resulting alcohol is reacted with an ethylating reagent, in situ, to obtain the Compound of formula (II) as depicted in Scheme 11.

[0223]

[0224] Scheme 11

[0225]

[0126] In Scheme 11, R1is a carboxy protecting group as described herein, and P2is an oxygen protecting group as described herein.

[0226]

[0127] In some embodiments, the ethylating reagents may include one or more selected from ethyl iodide, ethyl bromide, ethyl chloride, ethyl fluoride, diethylsulphate, ethyl triflate (EtOTf), 4-ethylsulfonyltoluene, and 2,4,6-trimethyl-l,3,5-trioxane. In some embodiments, the ethylating reagent may include 2,4,6-trimethyl-l,3,5-trioxane.

[0227]

[0128] In certain aspect, the removal of the oxygen protecting group P2and alkylation of the free alcohol in situ may be carried out with Et3SiH. an ethylating reagent, in the presence of a solvent. In some embodiments, the removal of the oxygen protecting group P2and alkylation of the free alcohol in situ may be carried out with Et3SiH, an ethylating reagent, in the presence of a solvent and a Lewis acid. In some embodiments, the Lewis acid may include one or more selected from TESOTf, TMSBr, BiBr3, TMSOTf, and TBSOTf. In some embodiments, the removing the oxygen protecting group P2may be performed in a solvent that can facilitate the removal of the oxygen protecting group and the alkylation. In some embodiments, the solvent may include one or more selected from the group consisting of dichloromethane, ethyl acetate, 1,4-dioxane, diethyl ether, tetrahydrofuran, methanol and acetonitrile.

[0228]

[0129] In some embodiments, the reaction mixture is performed at the internal temperature ranging from about -10 °C to about 20 °C, from about -5 °C to about 15 °C, or from about 0 °C to about 10 °C, or at a temperature of about -10 ± 10 °C, about -5 ± 10 °C, or 0 ± 5 °C. In some embodiments, the internal temperature may be in a range of about 4 °C to about 5 °C. In some embodiments, the reaction may be stirred for a reaction time of no less than about 1 hour, no less than about 2 hours, no less than about 3 hours, or no less than about 4 hours. In some embodiments, the resulting product, the Compound of formula II, may be obtained, e.g., as being separated (e.g., filtered), purified, washed and / or dried.

[0229]

[0130] In some embodiments, in the reaction in the Scheme 11, the cleavage and replacement of the oxygen protecting group P2on Compound (S)-6 ’ by an ethyl group may be advantageously performed in the presence of TESOTf, E SiH. 2,4,6-trimethyl-l,3,5-trioxane in acetonitrile, at a temperature between 4 °C to 5 °C, to obtain a compound of formula (II), or a salt thereof.

[0230]

[0131] Performing the protection of the alcohol group of Compound 6 in two steps (first protection with P2, second removal of P2 and addition of an ethyl group) under those conditions is particularly advantageous as it provides reactions that are scalable avoiding any hazardous chemicals, such as sodium hydride used in WO2015 / 009616, without impacting the yield of the transformation. Thus, the process produces safely the Compound of formula (II), or a salt thereof.

[0231]

[0132] In certain aspects, the removal of the oxygen protecting group P2and alkylation of the free alcohol may be carried out sequentially, in one pot. In some embodiments, the process includes steps of protecting the alcohol of Compound (S)-6 with an oxygen protecting group P2, to obtain a Compound (S)-6’; and alkylating the protected alcohol of Compound (S)-6’ with an ethyl group, to obtain a compound of formula (II). In some embodiments, the Compound of formula (II) is a fumaric acid salt. In some embodiments, the Compound of formula (II) is an oxalic acid salt. In some embodiments, the Compound of formula (II) is a succinic acid salt. In some embodiments, the Compound of formula (II) is a benzoic acid salt. In some embodiments, the Compound of formula (II) is a citric acid salt. In some embodiments, the Compound of formula (II) is a malonic acid salt. In some embodiments, the Compound of formula (II) is a malic acid salt. In some embodiments, the Compound of formula (II) is an adipic acid salt. In some embodiments, the Compound of formula (II) is a hydrochloric acid salt. In some embodiments, the Compound of formula (II) is an acetic acid salt. In some embodiments, the Compound of formula (II) is a maleic acid salt. In some embodiments, the Compound of formula (II) is a triflate salt.

[0232] 2. Synthesis of Compound of Formula (III)

[0233]

[0133] In an aspect, the disclosure provides a process for preparing a compound of formula (III). The process includes steps of reacting Compound 11 by alkylation to obtain Compound 12, reacting Compound 12 to obtain an indole compound, i.e. Compound 14, adding a nitrogen protecting group to obtain Compound 15, and reacting Compound 15 as depicted in Scheme 12.

[0234]

[0235] Formula (III) Scheme 12

[0236] 2,1, Synthesis of Compound 12

[0237]

[0134] In an aspect, the disclosure provides a process for preparing Compound 12 by reacting Compound 11 with an alkylating reagent (e.g., methyl halide, CH3X where in X is halide (e.g., Cl, Br, or I)), in the presence of a base (e.g., carbonate base or hydroxide), to obtain Compound 12, as depicted in Scheme 13.

[0238]

[0239] Scheme 13

[0240]

[0135] In some embodiments, the methyl halide may be reacted with Compound 11 in an amount of about 1.0 eq., about 1.1 eq., about 1.2 eq., about 1.3 eq., about 1.4 eq., about 1.4 eq., about 1.5 eq., about 1.6 eq., about 1.7 eq., about 1.8 eq., about 1.9 eq., or about 2.0 eq relative to the amount (1 eq.) of Compound 11. In some embodiments, the alkyl halide may include methyl iodide. In some embodiments, the alkyl halide may include methyl bromide. In some embodiments, the alkyl halide may include methyl chloride.

[0136] In some embodiments, the methyl halide may be reacted in situ with Compound 11 and the base. In some embodiments, the base may be an inorganic base, e.g., carbonate base or hydroxide. In some embodiments, the base may include, but not be limited to, Na2CC>3, K2CO3, and CS2CO3. In some embodiments, the base is a carbonate base (e.g., K2CO3). In some embodiments, the base is hydroxide (e.g., KOH).

[0241]

[0137] In some embodiments, the base (e.g., K2CO3 or KOH) may be added to the reaction mixture in an amount of about 1.0 eq., about 1.1 eq., about 1.2 eq., about 1.3 eq., about 1.4 eq., about 1.4 eq., about 1.5 eq., about 1.6 eq., about 1.7 eq., about 1.8 eq., about 1.9 eq., or about 2.0 eq relative to the amount (1 eq.) of Compound 11.

[0242]

[0138] In some embodiments, the reaction is performed at the internal temperature ranging from about 30 °C to about 70 °C, from about 40 °C to about 60 °C, or from about 40 °C to about 50 °C. In some embodiments, the reaction is performed at the internal temperature of about 40 ± 5 °C, about 45 ± 5 °C, or about 50 ± 5 °C.

[0243]

[0139] In some embodiments, the reaction is performed for a reaction time of no less than about 10 hours, no less than about 12 hours, no less than about 14 hours, no less than about 16 hours, no less than about 18 hours, or no less than about 20 hours. In some embodiments, the reaction is quenched by cooling down the reaction mixture to a temperature ranging from about 0 °C to about 30 °C, or from about 20 °C to about 30 °C, or at a temperature of about 25 ± 5 °C. In some embodiments, the resulting product, Compound 12 may be obtained, e.g., as being separated, filtered, washed and dried.

[0244] 2,2, Synthesis of Compound 14

[0245]

[0140] In an aspect, the disclosure provides a process for preparing Compound 13 (i) by reacting Compound 12 with pyrrolidine in the presence of a formylation agent (e.g., DMFDMA) to obtain Compound 13, and (ii) converting Compound 13 into Compound 14, as depicted in Scheme 14.

[0246]

[0247] Scheme 14

[0248]

[0141] In some embodiments, in step 1 of Scheme 14, Compound 12 in DMF may be reacted with a mixture of DMFDMA / pyrrolidine. In some embodiments, the DMFDMA may be added to the reaction mixture in an amount of about 1.0 eq., about 1.5 eq., about 2.0 eq., about 2.5 eq., about 3.0 eq., about 3.5 eq., about 4.0 eq., about 4.5eq., or about 5.0 eq., relative to the amount of Compound 12. In some embodiments, the pyrrolidine may be added to the reaction mixture in an amount of about 1.0 eq., about 1.5 eq., about 2.0 eq., about 2.5 eq., about 3.0 eq., about 3.5 eq., about 4.0 eq., about 4.5eq., or about 5.0 eq., relative to the amount of Compound 12.

[0249]

[0142] In some embodiments, in step 1 of Scheme 14, the reaction may be performed at an internal temperature ranging from about 50 °C to about 100 °C, from about 60 °C to about 90 °C, or from about 70 °C to about 85 °C. In some embodiments, the reaction may be performed at an internal temperature of about 75 ± 5 °C, about 80 ± 5 °C, or about 85 ± 5 °C.

[0250]

[0143] In some embodiments, in step 1 of Scheme 14, the reaction is performed for a reaction time of no less than about 10 hour, no less than about 15 hours, no less than about 20 hours, no less than about 25 hours, or no less than about 30 hours.

[0251]

[0144] In some embodiments, in step 1 of Scheme 14, the reaction may be quenched by cooling down the reaction to a temperature of about from about -10 °C to about 20 °C, from about -5 °C to about 15 °C, or from about 0 °C to about 10 °C. In some embodiments, the reaction may be quenched by cooling down the reaction to a temperature about -10 ± 10 °C, about -5 ± 10 °C, or 0 ± 5 °C. In some embodiments, H2O may be added to the reaction to quench the reaction.

[0252]

[0145] In some embodiments, the resulting product of step 1 in Scheme 14, i.e.

[0253] Compound 13, may be directly used from the reaction mixture, e.g., without separation or drying process.

[0254]

[0146] In some embodiments, in the step 2 of Scheme 14, Compound 13 is reacted with a transition metal catalyst under hydrogen pressure to obtain Compound 14. In some embodiments, the transition metal catalyst may include, not be limited to Pt, Pd, and / or V. In some embodiments, the transition metal catalyst may include Pt and V and Pt and V are supported on a carbon support.

[0255]

[0147] In some embodiments, in the step 2 of Scheme 14, Compound 13 in the presence of the transition metal may be charged with N2 atmosphere, and the reaction atmosphere is purged with hydrogen. In some embodiments, the hydrogen pressure for the reaction may ranges from about 0.1 bar to about 30 bar, from about 0.5 bar to about 20 bar, or from about 1 bar to about 10 bar. In some embodiments, the reaction may be performed at a reaction temperature ranging from about 0 °C to about 40 °C, from about 0 °C to about 25 °C, or from about 0 °C to about 15 °C. In some embodiments, may be performed at the internal temperature of about 20 ± 5 °C, about 15 ± 5 °C, or 10 ± 5 °C. In some embodiments, the reaction in step 2 of Scheme 14 is performed for a reaction time of no less than about 1 hour, no less than about 1.5 hours, or no less than about 2 hours.

[0256]

[0148] Alternatively, in an aspect, the process includes steps of (i) preparing Compound 14 (i) by reacting Compound 12 with an aldehyde (e.g., paraldehyde) in the presence of a base to obtain Compound 16, and (ii) converting Compound 16 into Compound 14, as depicted in Scheme 15.

[0257]

[0258] Scheme 15

[0259]

[0149] In some embodiments, in step 1 of Scheme 15, Compound 12 in a solvent (e.g., DMSO) may be added with paraldehyde, for example, in several portions. In some embodiments, the rection may be performed in the presence of a base. In some embodiments, the reaction may be performed at an internal temperature ranging from about 0 °C to about 30 °C, or from about 20 °C to about 30 °C, or at a temperature of about 25 ± 5 °C.

[0260]

[0150] In some embodiments, the reaction in step 1 of Scheme 15 is performed for a reaction time of no less than about 1 hour, no less than about 1.5 hours, or no less than about 2 hours. In some embodiments, the reaction is quenched by adding a salt solution (e.g., ammonium salt or NH4CI solution). In some embodiments, the resulting product, Compound 16 may be obtained, e.g., as being separated, purified, washed and / or dried.

[0261]

[0151] In some embodiments, in step 2 of Scheme 15, the reaction is performed in the presence of a catalyst. In some embodiments, the catalyst is an organocatalyst suitable for reducing nitroarenes. In some embodiments, the catalyst includes, but not limited to, bis(neopentylglycolato)diboron (B2(neo)2).

[0262]

[0152] In some embodiments, in step 2 of Scheme 15, Compound 16 may be converted into Compound 14 via radiation or photochemical reaction. In some embodiments, Compound 16 may be exposed to light having a wavelength ranging from about 400 to about 700 nm. In some embodiments, Compound 16 may be exposed to light having a wavelength of less than about 200 to about 400 nm. In some embodiments, Compound 16 may be exposed to light having a wavelength in the UV region. In some embodiments, radiation or light exposure may be performed for no less than about 12 hours, no less than about 13 hours, no less than about 14 hours, no less than about 15 hours, no less than about 16 hours, no less than about 17 hours, no less than about 18 hours, no less than about 19 hours, or no less than about 20 hours. In some embodiments, the resulting product, Compound 14 may be obtained, e.g., as being separated, purified, washed and / or dried.

[0263] 2,3, Synthesis of Compound 15

[0264]

[0153] In an aspect, the disclosure provides a process for preparing Compound 15 by protecting the nitrogen in Compound 14.

[0265]

[0266] 1415

[0267] Scheme 16

[0268]

[0154] In Scheme 16, P3is a nitrogen protecting group.

[0269]

[0155] The term "nitrogen protecting group" as used herein refers to any group which is capable of reversibly protecting the nitrogen functionality. In some embodiments, the nitrogen protecting group may include an alkyl (e.g., Ci-Ce-alkyl, Ci-C4-alkyl, or C1-C2- alkyl), (e.g. acetyl, allyl, tert-butyl), which may be substituted or unsubstituted. In some embodiments, the nitrogen protecting group may be C1-C4 alkyl which is mono-, di- or trisubstituted by trialkylsilyl-Ci-C7-alkoxy (e.g., trimethylsilyethoxy), aryl (e.g., phenyl), or an heterocyclic group (e.g., benzyl, cumyl, benzhydryl, pyrrolidinyl, trityl, pyrrolidinylmethyl, 1 -methyl- 1,1 -dimethylbenzyl, (phenyl)methylbenzene). In some embodiments, the aryl or the heterocyclic substituents of the C1-C4 alkyl may be unsubstituted or substituted, for example, by one or more (e.g. two or three) selected from the group consisting of C1-C7- alkyl, hydroxy, Ci-C7-alkoxy, C2-Cs-alkanoyl-oxy, halogen, nitro, cyano, and CF3.

[0270]

[0156] In some embodiments, the nitrogen protecting group may include aryl-Ci-C2- alkoxycarbonyl (e.g., phenyl-Ci-C2-alkoxycarbonyl (e.g., benzyloxycarbonyl (Cbz), benzyloxymethyl (BOM), pivaloyloxymethyl (POM)); Ci-Cio-alkenyloxy carbonyl; Ci-Ce alkylcarbonyl (e.g., acetyl or pivaloyl). In some embodiments, the nitrogen protecting group may include Ce-Cio-arylcarbonyl. In some embodiments, the nitrogen protecting group may include Ci-Ce-alkoxycarbonyl (e.g., tert-butoxycarbonyl (Boc), methylcarbonyl, trichloroethoxycarbonyl (Troc), pivaloyl (Piv), allyloxy carbonyl). In some embodiments, the nitrogen protecting group may include Ce-Cio-aryl-Ci-Ce-alkoxycarbonyl (e.g. 9-fluorenylmethyloxycarbonyl (Fmoc)). In some embodiments, the nitrogen protecting group may include allyl or cinnamyl. In some embodiments, the nitrogen protecting group may include sulfonyl or sulfenyl. In some embodiments, the nitrogen protecting group may include succinimidyl group. In some embodiments, the nitrogen protecting group may include silyl groups (e.g. triarylsilyl, trialkylsilyl, triethylsilyl (TES), trimethylsilylethoxymethyl (SEM), trimethylsilyl (TMS), tri / .sopropylsilyl or tert-butyldimethylsilyl).

[0271]

[0157] In some embodiments, P3may include one or more selected from the group consisting of tert-butyloxycarbonyl (Boc), toluenesulfonyl (Tosyl), and trifluoromethanesulfonyl. In some embodiments, P3may include tert-butyloxycarbonyl (Boc).

[0272]

[0158] In some embodiments, (P3)2O (e.g., (Boc)2O) may be added to Compound 14 in a solvent (e.g., MeCN). In some embodiments, DMAP may be added to Compound 14 in MeCN in an amount of about 0.01 eq., about 0.02 eq., about 0.03 eq., about 0.04 eq., or about 0.05 eq., relative to the amount of Compound 14. In some embodiments, (P3)2O (e.g., (BOC)2O) may be added to Compound 14 in MeCN in an amount of about 1.00 eq., about 1.25 eq., about 1.50 eq., about 1.75 eq., or about 2.0 eq., relative to the amount of Compound 14.

[0273]

[0159] In some embodiments, the reaction may be performed at an internal temperature ranging from about 20 °C to about 60 °C, from about 30 °C to about 50 °C, or from about 40 °C to about 50 °C. In some embodiments, the reaction may be performed at a temperature of about 40 ± 5 °C, 45 ± 5 °C, or 50 ± 5 °C.

[0274]

[0160] In some embodiments, the reaction may be performed for no less than about 10 mins, no less than about 20 mins, no less than about 0.5 hour, no less than about 1 hour, no less than about 2 hours, or no less than about 2 hours.

[0275]

[0161] In some embodiments, H2O may be added to the reaction to quench the reaction.

[0276]

[0162] In some embodiments, in Scheme 16, the reaction may be quenched by cooling down the reaction to a temperature of about from about -10 °C to about 20 °C, from about -5 °C to about 15 °C, or from about 0 °C to about 10 °C. In some embodiments, the reaction may be quenched by cooling down the reaction to a temperature about -10 ± 10 °C, about -5 ± 10 °C, or 0 ± 5 °C.

[0163] In some embodiments, the resulting product, Compound 15 may be obtained, e.g., as being separated, purified, washed and / or dried.

[0277] 2,4, Synthesis of Compound of Formula (III), or a salt thereof

[0278]

[0164] In another embodiment, the present invention provides a process for preparing a compound of formula (III), or a salt thereof. The process includes reacting Compound 15 in the presence of a formylating agent and a catalyst to obtain a compound of formula (III), or a salt (e.g., pharmaceutically acceptable salt) thereof, as depicted below in Scheme 17.

[0279]

[0280] catalyst

[0281] 15 Compound of Formula (III)

[0282] Scheme 17

[0283]

[0165] In Scheme 17, P3is a nitrogen protecting group as described herein. In some embodiments, P3may include one or more selected from the group consisting of tertbutyloxycarbonyl (Boc), toluene sulfonyl (Tosyl), and trifluoromethanesulfonyl. In some embodiments, P3may include tert-butyloxycarbonyl (Boc).

[0284]

[0166] In some embodiments, the formylation reaction may be performed in the presence of a formylating agent. In some embodiments, the formylation may be performed in the presence of a catalyst for electrophilic aromatic substitution. In some embodiments, the catalyst may include a Lewis acid catalyst. In some embodiments, the formylation may be performed in the presence of one or more formylating agents and a Lewis acid catalyst.

[0285]

[0167] In some embodiments, Compound 15 is reacted with a formylating agent that gives a formyl group (-CH=O) to a hydrocarbon (e.g., aryl or phenyl). In some embodiments, the formylating agent may include a formyl group of aldehyde (C-CH=O), formamide (N-CH=O), or formate ester (-O-CH=O). In some embodiments, Compound 15 is reacted with a formylating agent containing formamide (N-CH=O). In some embodiments, the formulating agent may include N-methyl-N-formylaniline.

[0286]

[0168] In some embodiments, the formylating agent may include formamide (e.g., N-methyl-N-formylaniline) and oxalyl dichloride. In some embodiments, the formylating agent may include N-methyl-N-formylaniline and oxalyl dichloride. In some embodiments, the formamide (e.g., N-methyl-N-formylaniline) may be added to the reaction mixture in an amount of about 1.0 eq., about 1.2 eq., about 1.3 eq., about 1.4 eq., about 1.5 eq., about 1.6 eq., about 1.7 eq., about 1.8 eq., about 1.9 eq., or about 2.0 eq., relative to the amount of Compound 15. In some embodiments, the formamide (e.g., N-methyl-N-formylaniline) may be added to the reaction mixture in an amount of about 1.0 eq., about 1.2 eq., about 1.3 eq., about 1.4 eq., about 1.5 eq., about 1.6 eq., about 1.7 eq., about 1.8 eq., about 1.9 eq., or about 2.0eq., relative to the amount of Compound 15. In some embodiments, N-methyl-N-formylaniline may be added to the reaction mixture in an amount of about 1.0 eq., about 1.2 eq., about 1.3 eq., about 1.4 eq., about 1.5 eq., about 1.6 eq., about 1.7 eq., about 1.8 eq., about 1.9 eq., or about 2.0eq., relative to the amount of Compound 15.

[0287]

[0169] In some embodiments, the oxalyl dichloride may be added to the reaction mixture in an amount of about 1.0 eq., about 1.2 eq., about 1.3 eq., about 1.4 eq., about 1.5 eq., about 1.6 eq., about 1.7 eq., about 1.8 eq., about 1.9 eq., or about 2.0eq., relative to the amount of Compound 15.

[0288]

[0170] In some embodiments, the formylating agent may be prepared by admixing formamide (e.g., N-methyl-N-formylaniline) and oxalyl dichloride prior to be added to Compound 15. In some embodiments, the mixture of the formamide (e.g., A-methyl-A-formylaniline) and oxalyl dichloride may be maintained in a temperature of about 10 ± 5 °C, 15± 5 °C, or 20 ± 5 °C. In some embodiments, the mixture of the formamide (e.g., N-methyl-N-formylaniline) and oxalyl dichloride may be stirred for no less than about 5 hours, about 6 hours, about 7 hours, about 8 hours, or about 9 hours.

[0289]

[0171] In some embodiments, the Lewis acid may include one or more elements selected from Al, B, Si, Sn, Ti, Zr, Fe, Co, Zn, Mg, or the like. In some embodiments, the Lewis acid may include one or more selected from AlCl3, FeCl3, MgCl2, TiCl4, BF3, SnCl4, or the like. In some embodiments, the catalyst may include AlCl3. In some embodiments, the catalyst may include FeCl3. In some embodiments, FeCl3 may be added to the reaction mixture in an amount of about 0.8 eq., about 0.9 eq., 1.0 eq., about 1.1 eq., about 1.2 eq., about 1.3 eq., about 1.4 eq., or about 1.5 eq., relative to the amount of Compound 15.

[0290]

[0172] Suitable solvents that can be used for the above formylation reaction are, for example, dimethylformamide (DMF), dimethoxyethane (DME), tetrahydrofuran (THF), 2-methyltetrahydrofuran (Me-THF), dimethyl sulfoxide (DMSO), toluene, acetonitrile or mixtures thereof. In some embodiments, the solvent includes 2-methyltetrahydrofuran (Me-THF). In some embodiments, the solvent includes dimethylformamide (DMF). In some embodiments, the solvent includes THF.

[0291]

[0173] In some embodiments, the formylation reaction may be performed at an internal temperature ranging from about -25 °C to about 10 °C, from about -20 °C to about 0 °C, or from about -15 °C to about 0 °C. In some embodiments, the reaction may be performed at a temperature of about -20 ± 5 °C, -15± 5 °C, or -10 ± 5 °C.

[0292]

[0174] In some embodiments, the formylation reaction may be performed for no less

[0293] than about 10 hours, no less than about 11 hours, no less than about 12 hours, no less than about 13 hours, no less than about 14 hours, no less than about 15 hours, or no less than about 16 hours.

[0294]

[0175] In some embodiments, the resulting product, the Compound of formula (III) may be obtained, e.g., as being separated, recrystallized, purified, washed and / or dried.

[0295] 3. Synthesis of a compound of formula (I)

[0296]

[0176] In an aspect, the disclosure provides a process for preparing a compound of

[0297] formula (I), or a pharmaceutically acceptable salt thereof. The process includes steps of (i) reacting a compound of formula (II), or a salt thereof, with a compound of formula (III), or a salt thereof to obtain Compound 9 and (ii) removing the carboxy protecting group (R1) by hydrolysis to obtain the Compound of formula (I), or a salt (e.g., pharmaceutically acceptable salt) thereof.

[0298]

[0299] Scheme 18

[0300] 3,1, Synthesis of Compound 9

[0301]

[0177] In an aspect, the process includes a step of reacting a compound of formula (II), or a salt thereof, with a compound of formula (III), or a salt thereof, in the presence of a

[0302] catalyst (e.g., iridium (Ir) catalyst) in a solvent to obtain Compound 9 as described in Scheme 19 below.

[0303]

[0304] compound compound

[0305] of formula (III) of formula (II)

[0306]

[0307] Scheme 19

[0308]

[0178] In Scheme 19, R1is a carboxyl protecting group as described herein, and P3is a nitrogen protecting group as described herein.

[0309]

[0179] In some embodiments, the reaction in Scheme 19 may be performed with the catalyst. In some embodiments, the catalyst may include, but not limited to, one or more from the group consisting of [Ru(Triphos)(CO)H2], [Ru(S)-BINAP(p-cymene)Cl]Cl, [Ru(CO)ClH(PPh3)3], [Ru(R)-BINAP (benzene)Cl]Cl, Ir(CO)2acac, Ir(COD)Cl, Ir(CO)3, and IrCl3,xH2O. In some embodiments, the catalyst includes an iridium (Ir) catalyst. In some embodiments, the Ir catalyst includes one or more selected from the group consisting of Ir(CO)2acac, Ir(COD)Cl, Ir(CO)3, and IrCl3,xH2O. In some embodiments, the Ir catalyst includes Ir(CO)2acac. In some embodiments, the Ir catalyst includes IrCl3, xH2O.

[0310]

[0180] In some embodiments, the catalyst (e.g., Ir catalyst) is present in a range from about 0.01 mol% to about 10.0 mol%, from about 0.05 mol% to about 5.0 mol%, or from about 0.1 mol% to about 1.0 mol% based on the total number of moles of the reaction. In some embodiments, the catalyst (e.g., Ir catalyst) is present in a range from about 0.05 mol% to about 1.0 mol%, or from about 0.1 mol% to about 0.5 mol% based on the total number of moles of the reaction.

[0311]

[0181] In some embodiments, the Ir catalyst (e.g., Ir(CO)2acac, or IrCl3,xH2O) is present in a range from about 0.05 mol% to about 1.0 mol%, from about 0.06 mol% to about 1.0 mol%, from about 0.07 mol% to about 1.0 mol%, from about 0.08 mol% to about 1.0 mol%, from about 0.09 mol% to about 1.0 mol%, or from about 0.1 mol% to about 1.0 mol% based on the total number of moles of the reaction. In some embodiments, the Ir catalyst (e.g., Ir(CO)2acac, or IrCl3,xH2O) is present in a range from about 0.05 mol% to about 0.5 mol%, from about 0.06 mol% to about 0.5 mol%, from about 0.07 mol% to about 0.5 mol%, from about 0.08 mol% to about 0.5 mol%, from about 0.09 mol% to about 0.5 mol%, or from about 0.1 mol% to about 0.5 mol% based on the total number of moles of the reaction. In some embodiments, the Ir(CO)2acac catalyst is present at about 0.05 mol%, at about 0.06 mol%, at about 0.07 mol%, at about 0.08 mol%, at about 0.09 mol%, at about 0.1 mol%, at about 0.11 mol%, at about 0.12 mol%, at about 0.13 mol%, at about 0.14 mol%, or at about 0.15 mol% based on the total number of moles of the reaction. In some embodiments, the IrCl3,xH2O catalyst is present at about 0.01 mol%, at about 0.02 mol%, at about 0.03 mol%, at about 0.04 mol%, at about 0.05 mol%, at about 0.06 mol%, at about 0.07 mol%, at about 0.08 mol%, at about 0.09 mol%, or at about 0.1 mol%, based on the total number of moles of the reaction.

[0312]

[0182] In some embodiments, the solvent may include, but not be limited to, one or more selected from methanol, ethanol, isopropanol, ethylene glycol, diethyl carbonate, DMSO, acetonitrile, and tetrahydrofuran. In some embodiments, the solvent may include ethanol.

[0313]

[0183] In some embodiments, the reaction may be performed in the presence of an iridium (Ir) catalyst and under hydrogen (H2) pressure. In some embodiments, the reaction in Scheme 5 may be performed in the presence of an iridium (Ir) catalyst, under hydrogen pressure, in the presence of an additive.

[0314]

[0184] In some embodiments, the reaction may be performed at a pressure of hydrogen (H2) in a range from about 1 bar to about 30 bar, or between about 2.5 bar to about 20 bar. Performing the reaction under those conditions is particularly advantageous as the reaction is highly efficient and the amount of by-product formation is reduced compared to the preparation of Compound 9 described in WO 2015 / 009616 (e.g., Intermediate 4-3, on page 127-128).

[0315]

[0185] In some embodiments, the additive may include a ligand, a base, an acid, or mixtures thereof. In some embodiments, the additive can include, but be not limited to, one or more selected from the group consisting of tetrabutylammonium iodide (TBAI), ((oxydi-2,l-phenylene)bis(diphenylphosphine)) (DPEPhos), triethylamine (EtsN), sodium trifluoromethanesulfonate (NaOTf), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos), l,4-diazabicyclo[2.2.2]octane (DABCO), tris(4-fluorophenyl)phosphine ((4-F-C6H4)3P), acetic acid, N-bromosuccinimide (NBS), and N-chlorosuccinimide (NCS).

[0316]

[0186] In some embodiments, the reaction may be performed when 0.1 mol% of Ir(CO)2acac catalyst is present, in ethanol as solvent, under hydrogen pressure. In some embodiments, the reaction may be performed at a temperature in a range from about room temperature to reflux.

[0187] In some embodiments, the reaction may be performed when 0.05 mol% of IrCh, XH2O catalyst is present, in ethanol as solvent, under hydrogen pressure. In some embodiments, the reaction may be performed at a temperature in a range from about room temperature to reflux.

[0317]

[0188] In some embodiments, the reaction may be performed at a reaction temperature ranging from about 60 °C to about 100 °C, from about 70 °C to about 90 °C, or from about 70 °C to about 80 °C. In some embodiments, may be performed at the internal temperature of about 70 ± 5 °C, about 75 ± 5 °C, or 80 ± 5 °C. In some embodiments, may be performed at the internal temperature of about 75 ± 5 °C.

[0318]

[0189] Alternatively, in an aspect, the disclosure provides a process including a step of obtaining an intermediate compound of formula (Illa). The Compound of formula (Illa) is then reacted with a compound of formula (II), or salt thereof, to obtain Compound 9 as depicted in Scheme 20. In some embodiments, the reactions are performed in the presence of an Iridium catalyst in a solvent, in a hydrogen atmosphere, optionally in the presence of an additive, as described above.

[0319]

[0320] compound compound

[0321] of formula (III) of formula (Illa) 9

[0322] Scheme 20

[0323]

[0190] In Scheme 20, R1is a carboxyl protecting group as described herein, and P3is a nitrogen protecting group as described herein.

[0324]

[0191] In some embodiments, the Compound of formula (Illa) may be obtained under a reducing condition, in In some embodiments, the Compound of formula (Illa) is then reacted with a compound of formula (II), or a salt thereof, in the presence of an Ir catalyst in a solvent, under hydrogen pressure, as described herein. In some embodiments, the Ir catalyst includes one or more selected from the group consisting of Ir(CO)2acac, Ir(COD)Cl, Ir(CO)3, and IrCl3,xH2O. In some embodiments, the Ir catalyst includes Ir(CO)2acac. In some embodiments, the solvent may include, but not be limited to, one or more selected from methanol, ethanol, isopropanol, ethylene glycol, diethyl carbonate, DMSO, acetonitrile, and tetrahydrofuran. In some embodiments, the solvent may include ethanol.

[0325]

[0192] In some embodiments, the Compound of formula (Illa), or a salt thereof, in Scheme 20 is then reacted with a compound of formula (II), or a salt thereof, in the presence of the Ir catalyst in a solvent, under hydrogen pressure, and in the presence of an additive as described herein. In some embodiments, the additive may include a ligand, a base, an acid, or mixtures thereof. In some embodiments, the additive can include, but be not limited to, one or more selected from the group consisting of tetrabutylammonium iodide (TBAI), ((oxydi-2,l-phenylene)bis(diphenylphosphine)) (DPEPhos), triethylamine (EtsN), sodium trifluoromethanesulfonate (NaOTf), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos), 1,4-Diazabicyclo[2.2.2]octane (DABCO), tris(4-fhiorophenyl)phosphine ((4-F-C6H4)3P), acetic acid, N-bromosuccinimide (NBS), and N-chlorosuccinimide (NCS).

[0326] 3,2, Synthesis of Compound of formula (I), or a pharmaceutically acceptable salt thereof

[0327]

[0193] In an aspect, the disclosure provides a process, as depicted in Scheme 21, of converting Compound 9 under hydrolyzing conditions to a compound of formula (I), or a salt (e.g., pharmaceutically acceptable salt) thereof.

[0328]

[0329] p

[0330] Scheme 21

[0331]

[0194] The term “hydrolyzing condition” refers to a reaction condition for the hydrolysis of an ester group (e.g., -CO2R1in Compound 9) to form a carboxylic acid of formula -CO2H. The ester group may be suitably hydrolyzed, for example, under basic conditions (e.g. using an alkali metal base such as NaOH, LiOH or KOH), or under acidic conditions (e.g., using mineral acids, such as HCI, H2SO4, HBr, H3PO4) to provide a carboxylic acid. In addition, the hydrolyzing condition can induce a reaction condition for removing the nitrogen protecting group (e.g., -NP3in Compound 9) from the nitrogen attached thereto.

[0195] In some embodiments, Compound 9, or a salt thereof, is reacted under hydrolyzing conditions to obtain the corresponding carboxylic acid, as depicted in Scheme 21. In some embodiments, the hydrolyzing conditions may be adopted from the description in W02015 / 009616 (example 26, on page 174).

[0332] Embodiments

[0333]

[0196] Certain variants, or alternative processes, to prepare a compound of formula (I), or a pharmaceutically acceptable salt thereof, are described herein below.

[0334]

[0197] In an aspect, provided is a method of preparing a compound of formula (I), or a pharmaceutically acceptable salt thereof, including a process of preparing Compound 9 as described in Section 3.1.

[0335]

[0198] In an aspect, provided is a method of preparing a compound of formula (I), or a pharmaceutically acceptable salt thereof, including steps of:

[0336] (i) preparing a compound of formula (II), as described in Section 1, and

[0337] (ii) preparing a compound of formula (II), as described in Section 2.

[0338]

[0199] In an aspect, provided is a method of preparing a compound of formula (I), or a pharmaceutically acceptable salt thereof, including steps of:

[0339] (i) preparing Compound 5, as described in Section 1.1,

[0340] (ii) preparing Compound 6, as described in Section 1.2.1, and

[0341] (iii) preparing a compound of formula (II), as described in Section 1.2.2.

[0342]

[0200] In an aspect, provided is a method of preparing a compound of formula (I), or a pharmaceutically acceptable salt thereof, including steps of:

[0343] (i) preparing Compound 12, as described in Section 2.1,

[0344] (ii) preparing Compound 14, as described in Section 2.2,

[0345] (iii) preparing Compound 15, as described in Section 2.3, and

[0346] (iv) preparing a compound of formula (III), as described in Section 2.4.

[0347]

[0201] In an aspect, provided is a method of preparing a compound of formula (I), or a pharmaceutically acceptable salt thereof, including steps of:

[0348] (i) preparing Compound 5, as described in Section 1.1,

[0349] (ii) preparing Compound 6, as described in Section 1.2.1,

[0350] (iii) preparing a compound of formula (II), as described in Section 1.2.2,

[0351] (iv) preparing Compound 12, as described in Section 2.1,

[0352] (v) preparing Compound 14, as described in Section 2.2,

[0353] (vi) preparing Compound 15, as described in Section 2.3, and (vii) preparing a compound of formula (III), as described in Section 2.4.

[0354]

[0202] In certain aspects, the method includes the step of preparing Compound 6, as described in Section 1.2.1, by reacting Compound 5 with a KRED.

[0355]

[0203] In an aspect, provided also is a method or process formulating Compound of formula (I), or a pharmaceutically acceptable pharmaceutically acceptable salt thereof, prepared by the method as described herein with a pharmaceutically acceptable excipient.

[0356]

[0204] In an aspect, provided herein a novel ketoreductase (KRED), i.e. a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid sequence of SEQ ID NO: 1, or a fragment thereof.

[0357]

[0205] In an aspect, provided herein a novel ketoreductase (KRED) that may be produced from a cell comprising a nucleic acid sequence having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 2.

[0358] Crystalline Form He

[0359]

[0206] In an aspect, the disclosure provides a crystalline form of iptacopan hydrochloride hydrate characterized by having a powder X-ray diffractogram comprising at least one peak (e.g., one, two, three, four, or five) selected from the group consisting of peaks at 10 angles (6.7 ± 0.2)°, (10.0 ± 0.2)°, (13.3 ± 0.2)°, (14.9 ± 0.2)°, and (24.6 ± 0.2)°, e.g., when measured at a temperature in the range of from 20 to 30 °C with Cu-Ka radiation having a wavelength of 0.154 nm (e.g., 1.54190 nm). Other crystalline forms of iptacopan hydrochloride have been prepared, for example, as disclosed in WO 2021234544.

[0360]

[0207] In some embodiments, the powder X-ray diffractogram further comprises one or more (e.g., one, two, three, four, or five) peaks selected from the group consisting of peaks at 10 angles (7.0 ± 0.2)°, (9.7 ± 0.2)°, (9.8 ± 0.2)°, (11.8 ± 0.2)°, (12.4 ± 0.2)°, (12.6 ± 0.2)°, (15.9 ± 0.2)°, (16.7 ± 0.2)°, (17.1 ± 0.2)°, (17.8 ± 0.2)°, (18.5 ± 0.2)°, (19.1 ± 0.2)°, (19.3 ± 0.2)°, (19.5 ± 0.2)°, (19.6 ± 0.2)°, (20.0 ± 0.2)°, (20.2 ± 0.2)°, (21.8 ± 0.2)°, (22.1 ± 0.2)°, (23.6 ± 0.2)°, (23.8 ± 0.2)°, (24.8 ± 0.2)°, (25.0 ± 0.2)°, (25.4 ± 0.2)°, (26.5 ± 0.2)°, (26.8 ± 0.2)°, (26.9 ± 0.2)°, (27.5 ± 0.2)°, and (29.2 ± 0.2)°, e.g., when measured at a temperature in the range of from 20 to 30 °C with Cu-Ka radiation having a wavelength of 0.154 nm (e.g., 1.54190 nm).

[0208] In a related aspect, the disclosure provides a pharmaceutical composition comprising the crystalline form disclosed herein. The pharmaceutical composition may further include a pharmaceutically acceptable excipient.

[0361]

[0209] In some embodiments, the crystalline form or the pharmaceutical composition disclosed herein may be used in a method of treatment or in uses disclosed in WO 2015009616, WO 2019043609, WO 2022013604, WO 2022234541, WO 2022264101, WO 2023137218, WO 2023166487, WO 2024176169, WO 2025046421, and WO 2025172910, the disclosures of which are incorporated here by reference.

[0362]

[0210] In an aspect, the disclosure provides a method of treating a complement mediated disease or disorder, e.g., asthma, arthritis (e.g., rheumatoid arthritis), autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, ischemia-reperfusion injuries, Barraquer-Simons Syndrome, hemodialysis, anca vasculitis, cryoglobulinemia, systemic lupus, lupus erythematosus, psoriasis, multiple sclerosis, transplantation, diseases of the central nervous system such as Alzheimer's disease and other neurodegenerative conditions, atypical hemolytic uremic syndrome (aHUS), glomerulonephritis (including membrane proliferative glomerulonephritis), dense deposit disease, blistering cutaneous diseases (including bullous pemphigoid, pemphigus, and epidermolysis bullosa), ocular cicatrical pemphigoid, MPGN II, PNH, IgAN, C3G, IC-MPGN, LN, gMG, AAV, or aHUS, in a subject, the method comprising administering an effective amount of the crystalline form or pharmaceutical composition to the subject in need thereof.

[0363]

[0211] In an aspect, the disclosure provides use of the crystalline form or pharmaceutical composition in the manufacture of a medicament for the treatment of a complement mediated disease or disorder, e.g., asthma, arthritis (e.g., rheumatoid arthritis), autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, ischemia-reperfusion injuries, Barraquer-Simons Syndrome, hemodialysis, anca vasculitis, cryoglobulinemia, systemic lupus, lupus erythematosus, psoriasis, multiple sclerosis, transplantation, diseases of the central nervous system such as Alzheimer's disease and other neurodegenerative conditions, atypical hemolytic uremic syndrome (aHUS), glomerulonephritis (including membrane proliferative glomerulonephritis), dense deposit disease, blistering cutaneous diseases (including bullous pemphigoid, pemphigus, and epidermolysis bullosa), ocular cicatrical pemphigoid, MPGN II, PNH, IgAN, C3G, IC-MPGN, LN, gMG, AAV, or aHUS.

[0364]

[0212] In an aspect, the disclosure provides the crystalline form or pharmaceutical composition for use in the treatment of a complement mediated disease or disorder, e.g., asthma, arthritis (e.g., rheumatoid arthritis), autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, ischemia-reperfusion injuries, Barraquer- Simons Syndrome, hemodialysis, anca vasculitis, cryoglobulinemia, systemic lupus, lupus erythematosus, psoriasis, multiple sclerosis, transplantation, diseases of the central nervous system such as Alzheimer's disease and other neurodegenerative conditions, atypical hemolytic uremic syndrome (aHUS), glomerulonephritis (including membrane proliferative glomerulonephritis), dense deposit disease, blistering cutaneous diseases (including bullous pemphigoid, pemphigus, and epidermolysis bullosa), ocular cicatrical pemphigoid, MPGN II, PNH, IgAN, C3G, IC-MPGN, LN, gMG, AAV, or aHUS.

[0365]

[0213] Other embodiments or examples are disclosed infra.

[0366] Definition

[0367]

[0214] The term “catalyst” as used herein refers to a catalytic amount of a chemical agent that enhances the rate of a chemical reaction by lowering the activation energy for the chemical reaction. The catalyst can be a heterogeneous catalyst or a homogenous catalyst. The term “heterogeneous catalyst” refers to a catalyst supported on a carrier, typically although not necessarily a substrate comprised of an inorganic material, for example, a porous material such as carbon, silicon and / or aluminum oxide. The term “homogeneous catalyst” refers to a catalyst that is not supported on a carrier.

[0368]

[0215] The term “one-pot” “or “one-pot process” means that in a series (i.e. in a succession) of reactions, for example two or more successive reactions, each reaction product is provided for the next reaction without isolation and purification. The one-pot processes defined herein encompass not only a series (i.e. a succession) of reactions conducted in a single reaction vessel, but also a series (i.e. a succession) of reactions conducted in a plurality of reaction vessels (e.g., by transferring the reaction mixture from one vessel to other) without isolation and purification. Preferably, the one-pot process is conducted in a single reaction vessel.

[0369]

[0216] The term “ligand” means any compound, achiral or chiral, that can form a complex with a transition metal. The term "chiral" refers to molecules which have the property of non-superimposability on their mirror image partner, while the term "achiral" refers to molecules which are superimposable on their mirror image partner.

[0370]

[0217] The term “amount” herein refers either to the weight of the compounds or to the molar amount of the compounds.

[0371]

[0218] The term “protecting group” may be present and should protect the functional groups concerned against unwanted secondary reactions, such as acylations, etherifications, esterifications, oxidations, solvolysis and similar reactions. It is a characteristic of protecting groups that they lend themselves readily, i.e. without or with very limited undesired secondary reactions, to removal, typically by solvolysis, reduction, photolysis or also by enzyme activity, for example under conditions analogous to physiological conditions, and that they are not present in the end-products. The specialist knows, or can easily establish, which protecting groups are suitable with the reactions mentioned hereinabove and hereinafter. Preferably, if two or more protecting groups are present in one intermediate mentioned, they are chosen so that, if one of the groups needs to be removed, this can be done selectively, e.g. using two or more different protecting groups that are cleavable under different conditions, e.g. one class by mild hydrolysis, the other by hydrolysis under harder conditions, one class by hydrolysis in the presence of an acid, the other by hydrolysis in the presence of a base, or one class by reductive cleavage (e.g. by catalytic hydrogenation), the other by hydrolysis, or the like. Suitable nitrogen protecting groups are conventionally used in peptide chemistry and are described e.g. in the relevant chapters of standard reference works such as J. F. W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press, London and New York 1973; T. W. Greene and P. G. M. Wuts, "Greene's Protective Groups in Organic Synthesis", Fourth Edition, Wiley, New York 2007; in "The Peptides"; Volume 3, Academic Press, London and New York 1981, and in "Methoden der organischen Chemie" (Methods of Organic Chemistry), Houben Weyl, 4th edition, Volume 15 / 1, Georg Thieme Verlag, Stuttgart 1974.

[0372]

[0219] The term "oxygen protecting group" generally comprises any group which is capable of reversibly protecting the oxygen functionality. A hydroxyl protecting group may, for example, be selected from a group comprising (especially consisting of) a silyl protecting group, especially diarylalkyl-silyl, such as diphcnyl- / c / 7-butylsilyl. or more preferably trialkylsilyl, such as / -biity Idi cthyl silyl or trimethylsilyl; an acyl group, e.g. alkanoyl, such as acetyl; benzoyl; alkoxycarbonyl, such as tert-butoxycarbonyl (Boc), or arylalkoxycarbonyl, such as benzyloxy carbonyl; tetrahydropyranyl; unsubstituted or substituted arylalkyl, such as benzyl or -methoxybenzyl, and methoxymethyl. Exemplary hydroxyl protecting groups are acetyl, propionyl, butynyl, pivaloyl, 2-chloroacetyl, benzoyl; carbonate derivatives such as phenoxycarbonyl, t-butoxycarbonyl ethoxycarbonyl, vinyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl and benzyloxycarbonyl; alkyl ether forming groups such as methyl, methoxymethyl, methylthiomethyl, benzyloxymethyl, t-butoxymethyl, 2-methoxyethoxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, tetrahydropyranyl, tetrahydrofuranyl, / -butyl, triphenylmethyl, benzyl, diphenylmethyl, allyl; silyl ether forming groups such as trialkylsilyl, trimethylsilyl, triethylsilyl, / -butyldimcthylsilyl. / -bnty Idiphcny 1 si ly E / .sopropyldialkylsilyl. alkyldiisopropylsilyl, triisopropylsilyl, t- butyldialkyl-silyl; and carbamates such as N-phenylcarbamate or A-imidazoylcarbamate. In particular, a hydroxyl protecting group is a silyl group according to the formula SiR7R8R9, wherein R7, R8and R9are, independently of each other, alkyl or aryl. Examples for R7, R8and R9are methyl, ethyl, isopropyl, t-butyl and phenyl. In particular, R7, R8and R9are ethyl or methyl.

[0373]

[0220] The term "nitrogen protecting group" generally comprise: Ci-Ce-alkyl, preferably Ci-C4-alkyl, more preferably Ci-C2-alkyl, (e.g. acetyl, allyl, tertbutyl) most preferably Cl-alkyl which is mono-, di- or tri-substituted by trialkylsilyl-Ci-C7-alkoxy (e.g. trimethylsilyethoxy), aryl, preferably phenyl, or an heterocyclic group (e.g., benzyl, cumyl, benzhydryl, pyrrolidinyl, trityl, pyrrolidinylmethyl, 1 -methyl- 1,1 -dimethylbenzyl, (phenyl)methylbenzene) wherein the aryl ring or the heterocyclic group is unsubstituted or substituted by one or more, e.g. two or three, residues, e.g. selected from the group consisting of Ci-C?-alkyl, hydroxy, Ci-C?-alkoxy, C2-Cs-alkanoyl-oxy, halogen, nitro, cyano, and CF3; aryl-Ci-C2-alkoxycarbonyl (preferably phenyl-Ci-C2-alkoxycarbonyl (eg. benzyloxycarbonyl (Cbz), benzyloxymethyl (BOM), pivaloyloxymethyl (POM)); Cl-CIO-alkenyloxycarbonyl; Cl-C6alkylcarbonyl (eg. acetyl or pivaloyl); Ce-Cio-arylcarbonyl; Ci-Ce-alkoxycarbonyl (eg. tertbutoxycarbonyl (Boc), methylcarbonyl, trichloroethoxycarbonyl (Troc), pivaloyl (Piv), allyloxy carbonyl); Ce-Cio-aryl-Ci-Ce-alkoxycarbonyl (e.g. 9-fluorenylmethyloxy carbonyl (Fmoc)); allyl or cinnamyl; sulfonyl or sulfenyl; succinimidyl group, silyl groups (e.g. triarylsilyl, trialkylsilyl, triethylsilyl (TES), trimethylsilylethoxymethyl (SEM), trimethylsilyl (TMS), trizsopropylsilyl or tertbutyldimethylsilyl).

[0374]

[0221] As used herein, the term “C1-C12 alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to twelve carbon atoms, and which is attached to the rest of the molecule by a single bond. The term “Ci-Ce alkyl” is to be construed accordingly. Examples of C1-C12 alkyl include, but are not limited to, ethyl, w-propyl. 1 -methylethyl ( / .so-propyl). n-butyl, w-pcntyl 1,1 -dimethylethyl (tert-butyl).

[0375]

[0222] As used herein, the term "Halogen" or “Halo” refers to bromo, chloro, fluoro or iodo.

[0376]

[0223] The term “about”, as used herein, is intended to provide flexibility to a numerical range endpoint, providing that a given value may be “a little above” or “a little below” the endpoint accounting for variations one might see in the measurements taken among different instruments, samples, and sample preparations. The term usually means within 10%, preferably within 5%, and more preferably within 1% of a given value or range.

[0377]

[0224] The term “room temperature” or “ambient temperature” as used herein, unless specified otherwise, means a temperature from 15 to 30 °C, such as from 20 to 30 °C, particularly such as from 20 to 25 °C. The term “internal temperature” as used herein, unless specified otherwise, means the temperature measured inside of the reactor vessel in which the reaction is performed. Such temperature is expressed in degree Celsius. The term “jacket temperature” as used herein, unless specified otherwise, means the temperature measured inside the jacket of the reactor vessel in which the reaction is performed.

[0378]

[0225] The term “stereoisomers” means one of the absolute configurations of a single organic molecule having at least one asymmetric carbon. Also, as used herein, the term refers to any of the various stereo isomeric configurations which may exist for a given compound of the present invention and includes geometric isomers. It is understood that a substituent may be attached at a chiral center of a carbon atom. Therefore, the invention includes enantiomers, diastereomers or racemates of Compound. “Enantiomers” are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1: 1 mixture of a pair of enantiomers is a "racemic” mixture. The term is used to designate a racemic mixture where appropriate.

[0379] "Diastereoisomers” are stereoisomers that have at least two asymmetric atoms, but which are not mirror images of each other. The absolute stereochemistry is specified according to the Cahn- Ingold-Prelog R-S system. When a compound is a pure enantiomer the stereochemistry at each chiral carbon may be specified by either R or. S', Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (dextro- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line. Certain of the compounds described herein contain one or more asymmetric centers or axes and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (. S')-. The present invention is meant to include all such possible isomers, including racemic mixtures, optically pure forms and intermediate mixtures.

[0380]

[0226] In the formulae of the present application the term

[0381]

[0382] " on a C-sp3 indicates the absolute stereochemistry, either ( / ?) or (. S'),

[0383] /

[0384]

[0227] In the formulae of the present application the term " " on a C-sp3 indicates the absolute stereochemistry, either ( / ?) or (. S'),

[0228] The term "resolution" refers to the separation or concentration or depletion of one of the stereoisomers of a molecule.

[0385]

[0229] The term “seed” can be used as a noun to describe one or more crystals of a crystalline compound of same formula as the final compound of the reaction of interest. The term “seed” can also be used as a verb to describe the act of introducing said one or more crystals of a said crystalline compound into an environment (including, but not limited to, for example, a solution, a mixture, a suspension, or a dispersion) thereby resulting in the formation of more crystals of the final compound.

[0386]

[0230] The term “pharmaceutically acceptable salts” or “salt thereof’ refers to salts that can be formed, for example, as acid addition salts, preferably with organic or inorganic acids. For isolation or purification purposes it is also possible to use pharmaceutically unacceptable salts, for example picrates or perchlorates. For therapeutic use, only pharmaceutically acceptable salts or free compounds are employed (where applicable in the form of pharmaceutical preparations), and these are therefore preferred. The salts of the compound of formula (I), and intermediates, as described in the present invention, are preferably pharmaceutically acceptable salts; suitable counter-ions forming pharmaceutically acceptable salts are known in the field. The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0387]

[0231] The term “additive” as used herein refers to a base, an acid, a ligand, or any other chemical species that can enhanced the reactivity of the reaction.

[0388]

[0232] The term “effective amount” of the crystalline form or pharmaceutical composition refers to an amount of the crystalline form or pharmaceutical composition of the present disclosure that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In one non-limiting embodiment, the term “effective amount” refers to the amount of the crystalline form or pharmaceutical composition of the present disclosure that, when administered to a subject, is effective to (1) at least partially alleviating, inhibiting, preventing and / or ameliorating a condition, or a disorder, or a disease or biological process (e.g., tissue regeneration and reproduction) (i) mediated by Factor B, or (ii) associated with Factor B activity, or (iii) characterized by activity (normal or abnormal) of the complement alternative pathway; or (2) reducing or inhibiting the activity of Factor B; or (3) reducing or inhibiting the expression of Factor B; or (4) reducing or inhibiting activation of the complement system and particularly reducing or inhibiting generation of C3a, iC3b, C5a or the membrane attack complex generated by activation of the complement alternative pathway.

[0389]

[0233] As used herein, the terms “treat”, “treatment” and “treating” may also refer to the slowing, reduction or amelioration of the progression or severity of a disorder or disease, e.g., a complement mediated disease or disorder, e.g., asthma, arthritis (e.g., rheumatoid arthritis), autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, ischemiareperfusion injuries, Barraquer-Simons Syndrome, hemodialysis, anca vasculitis, cryoglobulinemia, systemic lupus, lupus erythematosus, psoriasis, multiple sclerosis, transplantation, diseases of the central nervous system such as Alzheimer's disease and other neurodegenerative conditions, atypical hemolytic uremic syndrome (aHUS), glomerulonephritis (including membrane proliferative glomerulonephritis), dense deposit disease, blistering cutaneous diseases (including bullous pemphigoid, pemphigus, and epidermolysis bullosa), ocular cicatrical pemphigoid, MPGN II, PNH, IgAN, C3G, IC-MPGN, LN, gMG, AAV, or aHUS.

[0390]

[0234] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly indicates otherwise.

[0391]

[0235] Similarly, “comprise”, “comprises”, “comprising”, “include”, “includes” and “including” are interchangeable and not intended to be limiting.

[0392] Abbreviations

[0393]

[0394]

[0395]

[0396]

[0397]

[0398] EXAMPLES

[0399]

[0236] The following examples are merely illustrative of the present invention and they should not be considered as limiting the scope of the invention in any way, as these examples, and other equivalents thereof will become apparent to those skilled in the art in the light of the present invention, and the accompanying claims.

[0400] Syntheses

[0401]

[0237] The skilled person will appreciate that the general synthetic routes detailed above show common reactions to transform the starting materials as required. When specific reactions are not provided the skilled person will know that such reactions are well known to those skilled in the art and appropriate conditions considered to be within the skilled person’s common general knowledge. The starting materials are either commercially available compounds or are known compounds and can be prepared from procedures described in the organic chemistry art.

[0402]

[0238] Compounds as described herein, in free form, may be converted into salt form and vice versa, in a conventional manner understood by those skilled in the art. The compounds in free or salt form can be obtained in the form of hydrates or solvates containing a solvent used for crystallization. Compounds described herein can be recovered from reaction mixtures and purified in a conventional manner. Isomers, such as stereoisomers, may be obtained in a conventional manner, e.g. by fractional crystallization or asymmetric synthesis from correspondingly asymmetrically substituted, e.g. optically active, starting materials. The various starting materials, intermediates, and compounds of the preferred embodiments may be isolated and purified, where appropriate, using conventional techniques such as precipitation, filtration, crystallization, evaporation, distillation, and chromatography. Unless otherwise stated. Salts may be prepared from compounds by known salt-forming procedures.

[0403]

[0239] The compounds described herein can be prepared, e.g. using the reactions and techniques described below and in the examples. The reactions may be performed in a solvent appropriate to the reagents and materials employed and suitable for the transformations being effected. It will be understood by those skilled in the art of organic synthesis that the functionality present on the molecule should be consistent with the transformations proposed. This will sometimes require a judgment to modify the order of the synthetic steps or to select one particular process scheme over another in order to obtain a desired compound of the invention.

[0404]

[0240] It would be understood by the skilled person in the art, that the reactions were run on a small scale first in order to access if the starting materials could react in high yields and high purities before to be scalable. The desired compounds obtained during such small scale reaction, that spontaneously crystallized, were used to enhance the latest reactions, using the technique of “seeding”. Here below approximately 1% by weight or less of seeding crystals were added, if needed, to the reaction mixture to generate quicker the spontaneous crystallization of the desired product.

[0405] Measurements methods

[0406]

[0241] Proton-NMR: Measurements were performed on a Bruker 400 MHhz spectrometer. Chemical shifts (δ-values) are reported in ppm downfield and the spectra splitting pattern are designated as singlet (s), doublet (d), triplet (t), quartet (q), quintet (quint), multiplet, unresolved or overlapping signals (m), broad signal (br). Deuterated solvents are given in parentheses.

[0407]

[0242] HPLC: Measurements were performed on an Agilent 1200 HPLC system with high pressure mixing (column: Waters XBridge BEH C18) and an Agilent 1290 UHPLC system (column: Water Acquity BEH C18).

[0408]

[0243] HRMS: Waters ACQUITY UPLC / SYNAPT HDMS QTOF system

[0409]

[0244] LCMS: Waters ACQUITY UPLC / SYNAPT HDMS QTOF system or Agilent 1290 Infinity / MSD LC / MS system Example 1: Preparation of KRED

[0410]

[0245] The engineered ketoreductases were prepared according to the following procedure. The engineered ketoreductase was heterologously expressed in Escherichia coli for production purposes. To start the culture, either a single colony from a plate or a glycerol stock of E. coli containing a plasmid encoding an engineered ketoreductase, was inoculated into 25 mL of LB supplemented with 30 pg / mL CAM and 1% glucose in a 250 ml baffled shake flask. The culture was grown overnight (16-20 hours and OD600>3.8) in an incubator at 37°C, with shaking at 250 rpm. A 1 L shake flask containing 250 mL of TB media with 30 pg / mL CAM, was inoculated with 5 mL of the grown overnight culture. The 250 mL culture was incubated at 30°C, 250 rpm, for 3 - 3.5 hours until OD600reached 0.6–0.8. Expression of the ketoreductase gene was induced by the addition of IPTG to a final concentration of 1 mM, and growth was continued for an additional 18-20 hours. Cells were harvested by transferring the culture into a centrifuge bottle, which was then centrifuged at 7,000 rpm for 5 minutes at 4°C. The supernatant was discarded, and the remaining cell pellet was resuspended in 30 mL of 50mM KPi Buffer pH 8.0, 2 mM MgSO4, 5 pM ZnSO4 and lysed using a LM20 MICROFLUIDIZER® processor system (Microfluidics). Cell debris was removed by centrifugation at 14,000 rpm for 30 minutes at 4°C. The clarified lysate was collected, frozen at -80°C, and then lyophilized, using standard methods known in the art to obtain a dry shake-flask powder comprising crude engineered ketoreductase. To obtain larger quantities of enzyme required for scaled-up reactions, cells were fermented in a bioreactor using standard methods known in the art. In addition to the previously mentioned steps, the downstream processing included a heat purification and a filtration step to further enhance the purity of the resulting enzyme powder.

[0411] Example 2: Synthesis Process Overview

[0412]

[0246] The main synthesis of Compound 10 is described in Scheme A.

[0413]

[0414] Scheme A

[0415]

[0247] The synthesis of Compound 8 is described in Scheme B.

[0416]

[0417] Scheme B Example 3: Synthesis of compound 5

[0418]

[0419] Scheme C

[0420] Synthesis of tert-butyl 5-((tert-butoxycarbonyl)amino)-3-oxopentanoate (3)

[0421]

[0248] Compound 1 (30 g, 1.0 eq) was dissolved in EtOAc (240 ml) and the mixture was stirred for 30 min to get a clear solution. CDI (28.2 g, 1.1 eq) was then charged as solid in several portions while stirring. After charging, the resulting solution was stirred for another 1 h at room temperature. To the reaction mixture was charged compound 2 (72.8 g, 1.2 eq) in several portions. The mixture was stirred at room temperature for no less than 6 h. The reaction was monitored by HPLC. To the reaction mixture was slowly charged with 40% citric acid solution (150 ml) while stirring to quench the reaction. The mixture was stirred for 30 min and the phase was separated. After separation, the organic layer was washed with

[0422] 20% KHCO3 (150 ml) solution and then 2% NaCl solution (90 ml). After washing, the organic layer was first concentrated, then solvent exchanged by toluene (150 ml) to obtain a colorless compound 3 toluene solution. The solvent residue was checked by GC (EA / (EA + toluene) < 2.0%). The 3 / toluene solution was directly used in the next step. (Assay: 25-29%, Assay yield: 94%). LCMS m / z (ESI) [M-l]+ = 286.2.

[0423] Synthesis of methyl 4-(4-oxopiperidin-2-yl)benzoate hydrochloride hydrate (5)

[0424]

[0249] Method A: To a reactor was charged above diluted compound 3 / toluene solution (84.0 g, 20% assay, 1.1 eq) and compound 4 (10.0 g, 1.0 eq) as solid. The mixture was stirred at room temperature, to give a clear solution. TFA (24.3 g, 3.4 eq) was then slowly charged to the mixture. The reaction mixture was warmed up to IT = 60 ± 5 °C and stirred for no less than 4 h. The reaction was monitored by HPLC: compound 3 / (compound 3 + 5A-Int + compound 5) < 3.0%. LCMS (m / z) of 5A-Int: (ESI) [M+l]+ = 334.2. 4 N HC1 / EA solution (30 g) was slowly charged into the above mixture in no less than 2 h. Seed was charged, and the reaction mixture was kept stirring at IT = 60 ± 5 °C for no less than 2 h. 4 N HC1 / EA solution (60 g) was slowly charged into the mixture in no less than 2 h. The reaction mixture was kept stirring at IT = 60 ± 5 °C for no less than 10 h. A lot of solid precipitated out during the above stirring. The reaction was monitored by HPLC: 5A-Int / (5A-Int + 5) < 2.0%. The reaction mixture was slowly cooled down to IT below 25 °C in 2 h and stirred for another 2 h. The suspension was filtered, and the wet cake was washed with acetone (20 g) and further purged under N2 for no less than 1 h to get crude compound 5. Acetone (120 g) and water (10 g) were charged. The resulting suspension was warmed up to IT = 55 ± 5 °C, stirred for 2 h, slowly cooled down to IT = 5 ± 5 °C in no less than 5 h, then stirred for another 5 h at IT = 5 ± 5 °C before fdtration. The wet cake was washed with pre-cooled acetone / water (24 g / 2 g) and then pre-cooled acetone (20 g). After drying at 60 °C under vacuum, 5 was afforded as an off-white solid. (Purity: 98.5%, yield 78%).1H NMR (400 MHz, DMSO) 5 10.50 (s, 1H), 9.95 (s, 1H), 7.80 (d, J= 8.4 Hz, 2H), 7.60 (d, J= 8.4 Hz, 2H), 4.67 (dd, J= 12.9, 3.6 Hz, 1H), 3.64 (s, 3H), 3.40 - 3.34 (m, 1H), 3.23 (dd, J= 12.8, 4.1 Hz, 1H), 2.97 - 2.80 (m, 2H), 2.42 - 2.36 (m, 1H), 2.34 - 2.28 (m, 1H); LCMS m / z (ESI) [M+l]+ = 234.1

[0425]

[0250] Method B: To a 500 mL RBF equipped with condenser was added compound 3 (9.0 g, 1.0 eq.), methyl 4-formylbenzoate (compound 4) (5.65 g, 1.1 eq.), i-PrOH (108.0 mL), then stirred at 25 °C. HO Ac (1.88 g, 1.0 eq.) was charged by one portion, then piperidine (2.66 g, 1.0 eq.) was charged dropwise within 2 min. The mixture was evacuated and backfilled with N2 for 3 times, then warmed up and stirred at 45 °C for 4 hours. The reaction was monitored by HPLC: compound 3 / (3 + 5B-Int) < 5.0%. The mixture was concentrated under vacuum and solvent swapping with EtOAc (90 mL) to obtain a residue mixture (~18 mb, 2 V). EtOAc (180 mL) and 10% Na2SOs in H2O (90 mL) were added at 25 °C to obtain a clear solution. Then 10% NaCl aqueous solution (90 mL) was charged and stirred under 25°C for 30 min. Phase separation and the organic phase with dried with MgSO4 (3.0 g), filtered and t washed with EtOAc (18 mL). The filtrate was concentrated under vacuum, and solvent swapping with n-heptane for 3 times (45 mL each), the mixture was stirred at 25 °C for 1 h, filtered, washed with n-heptane (27 mL) and dried under vacuum for 1 h to obtain crude 5B-Int as an off-white solid, (purity 98.4%, yield 75%). 'H NMR (400 MHz, DMSO) 5 8.04 (d, J= 8.3 Hz, 2H), 7.76 (s, 1H), 7.69 (d, J= 8.3 Hz, 2H), 6.84 (m, 1H), 3.88 (s, 3H), 3.21 (d, J= 5.9 Hz, 2H), 2.99 (t, J= 6.7 Hz, 2H), 1.46 (s, 9H), 1.38 (s, 9H); LCMS m / z (ESI) [M+l]+ = 434.2. To a 100 mL two-neck flask was added 5B-Int (2.85 g, 1.0 eq.), EA (15 mL), and then was evacuated and refilled with N2 for three times. Then it was cooled to 0 °C.

[0426] 4 N HC1 in EA (45 mL) was added and stirred at 0 °C for 3 h. The reaction mixture was transformed to a 250 mL flask and 4 N HC1 in H2O (60 mL) was added and stirred at 45 °C for 4 h. The reaction was monitored by HPLC: 5B-Int / (5 + 5B-Int) < 2.0%. The reaction mixture was slowly cooled down to IT below 25 °C in 2 h and stirred for another 2 h. The suspension was filtered, and the wet cake was washed with acetone (10 g) and further purged under N2 for no less than 1 h to get crude 5. Acetone (60 g) and water (5 g) were charged. The resulting suspension was warmed up to IT = 55 ± 5 °C, stirred for 2 h, slowly cooled down to IT = 5 ± 5 °C in no less than 5 h, then stirred for another 5 h at IT = 5 ± 5 °C before filtration. The wet cake was washed with pre-cooled acetone / water (12 g / 1 g) and then precooled acetone (10 g). After drying at 60 °C under vacuum, 5 was afforded as an off-white solid. (Purity: 98%, yield: 60%). The 1H NMR and MS spectrum are same as method A. Example 4: Synthesis of compound 7

[0427]

[0428] 5 6 7

[0429] Scheme D

[0430] Synthesis of methyl 4-((2S,4S)-4-hvdroxypiperidin-2-yl)benzoate (6)

[0431]

[0251] Preparation of PBS buffer: To a reactor was charged K2HPO4 3H2O (401 g) and KH2PO4 (35 g), followed by 20.3 kg of water. After stirring the mixture at room temperature, to acquire a clear solution, the pH of the PBS buffer was checked to be 7.5 ± 0.3.

[0432]

[0252] To a reactor was charged PBS buffer (504 g) and isopropanol (118 g), followed by L-Proline (4.0 eq, 98.8 g) and 5 (1.0 eq, 57.8 g). After the mixture was stirred at room temperature, to give a clear solution, the pH of the reaction mixture was adjusted to 7.3 ± 0.3 by 5 M KOH aqueous solution. A prepared solution of KRED enzyme (SEQ ID NO: 3; 2.5 g) and NADP sodium salt (2.5 g) in PBS buffer (101 g) was then charged into the reaction mixture. The reaction mixture was warmed up to IT = 40 ± 5 °C and stirred for no less than 7 h. During the reaction, the pH was monitored and controlled to be 7.0 ± 0.3 by 5 M HC1. The reaction was monitored by HPLC. After reaction, the mixture was cooled down to IT = 5 ± 5 °C, and the pH was further adjusted to 3.0 ± 0.3 by 31% HC1 (26 g). The acidic mixture was distilled at JT = 50 ± 5 °C to remove IPA. The solvent residue was monitored by GC. (IPA residue < 5.0%). NaCl (116 g) and MCC (29 g) was charged into the reaction mixture as solid. After stirred at room temperature, for 30 min, the suspension was filtered, and the wet cake was washed with 20% NaCl solution (116 g) twice. The aqueous parts were combined and washed with 2-MeTHF (250 g). To the aqueous phase was then charged 2-MeTHF (400 g). After cooled down to IT = 5 ± 5 °C, the pH of the mixture was adjusted to >9.5 by 50% K2CO3 (290 g). The phase was separated, and the aqueous part was further extracted with 2-MeTHF (250 g). The organic parts were combined, and further solvent exchanged with IP Ac (980 g) to give compound 6 / IPAc solution. The solvent residue was monitored by GC [(2-MeTHF / (2-MeTHF + IP Ac) < 2.0%]. The solution of Compound 6 in IP Ac was passed through an activated carbon filter, and the filter was further washed with IP Ac (98 g). The filtrate (compound 6 / IPAc solution) was directly used to next step (Assay yield 87%). LCMS m / z (ESI) [M+l]+ = 236.1

[0433]

[0253] Other KRED enzymes (SEQ ID NOS: 1, 5, and 7) have also been utilized in this reaction and provided the desired product with similar purity and yield.

[0434] Synthesis of methyl 4-((2S,4S)-4-ethoxypiperidin-2-yl)benzoate trifluoromethanesulfonate

[0435]

[0254] To a reactor was charged compound 6 / IPAc solution (300 g, 10% assay, 1.0 eq). The mixture was distilled under vacuum (JT = 50 °C ) to give a suspension (~110 g). The mixture was refluxed under reduced pressure (JT = 50 °C ) for 2 h to make sure no solid sticks on the reactor wall. The mixture was then gradually cooled down to IT = -10 ± 5 °C in no less than 6 h to give a suspension. TMSOTf (35.4 g, 1.25 eq) was slowly added into the reactor while maintaining IT = -5 ± 10 °C. A clear solution was then formed. Through another adding funnel, Et3SiH (37.5 g, 2.5 eq) was slowly added to the solution while maintaining IT = -5 ± 10 °C. After that, paraldehyde (11.8 g, 0.7 eq) was slowly added through another adding funnel into the reaction mixture while maintaining IT = -5 ± 10 °C. (Note: Seed of 7 (0.03 g) was added when around! of the paraldehyde was charged). The reaction mixture turned from a solution to a suspension, which was kept stirring under IT = -5 ± 5 °C for another 0.5 h before filtered off. The wet cake was washed with pre-cooled IPAc (30 mL) / heptane (30 mL) and dried under 50 °C for 8 h. Compound 7 was afforded as an off-white solid. (Purity: 99%, yield 91%). 1HNMR (400 MHz, DMSO) 58.67 (s, 1H), 8.54 (s, 1H), 7.82 (d, J= 8.4 Hz, 2H), 7.43 (d, J= 8.4 Hz, 2H), 4.25 (t, J= 11.4 Hz, 1H), 3.64 (s, 3H), 3.61 (m, 1H), 3.29 (d, J= 7.0, 1.9 Hz, 2H), 3.02 (m, 2H), 1.96 - 1.70 (m, 3H), 1.69 - 1.54

[0436] (m, 1H), 0.95 (t, J= 7.0 Hz, 3H). LCMS m / z (ESI) [M+l]+ = 264.2

[0437] Example 5: Synthesis of compound 8

[0438]

[0439] Scheme E

[0440] Synthesis of 5-methoxy-l,3-dimethyl-2 -nitrobenzene (12)

[0441]

[0255] To a solution of 3, 5 -dimethyl -4-nitrophenol, compound 11, (10 g, 1.0 eq.) in MeCN (80 mL) was added K2CO3 (12.4 g, 1.5 eq.) and methyl iodide (11.0 g, 1.3 eq.) at 25 °C. The mixture was warmed to 45 °C and stirred for no less than 16 h. The mixture was cooled to 25 °C and filtered off. The organic solution was evaporated under vacuum, diluted with DMF (36 g), evaporated under vacuum to get 40 g 5 -methoxy- 1,3 -dimethyl-2-nitrobenzene, compound 12, in DMF solution which was used in the next step directly. 1H NMR (400 MHz, DMSO-d6): 56.81 (s, 2H), 3.79 (s, 3H), 2.24 (s, 6H). LCMS m / z (ESI) [M+l]+ =182.1.

[0442] Synthesis of (E)-l-(5-methoxy-3-methyl-2-nitrostyryl)pyrrolidine (13)

[0443]

[0256] To DMFDMA (20.7 g, 3 eq.) was added pyrrolidine (12.4 g, 3 eq.) at 25 ± 5 °C

[0444] and the mixture was stirred for 0.5 h. Then DMF solution of compound 12 (52.5 g, 20%

[0445] assay, 1 eq.) was warmed to 80 ± 5 °C (JT < 90 °C) under 400 ± 100 mbar and the DMFDMA / pyrrolidine mixture was added dropwise in 6 h. The mixture was stirred for no less than 20 h. The mixture was cooled to 0 ± 5 °C and H2O (16 g) was added dropwise for 3 h to quench the reaction (Note: exothermic). Then H2O (37 g) was added dropwise for 3 h and hold for 3 h. The mixture was filtered off and the wet cake was washed with MeOH (10 mL) / H2O (10 mL). The wet cake was used in the next step directly without drying. (Yield 80%, purity: 90%). 1H NMR (400 MHz, DMSO-d6): 57.48 (d, J= 12 Hz, 1H), 6.88 (d, J= 4 Hz, 1H), 6.44 (d, J= 4 Hz, 1H), 4.66 (d, J= 12 Hz, 1H), 3.78 (s, 3H), 3.27-3.12 (m, 4H), 2.13 (s, 3H), 1.90-1.79 (m, 4H).

[0446] Synthesis of 5-methoxy-7-methyl-lH-indole (14)

[0447]

[0257] To the hydrogenation kettle was added EtOAc (288 g) and compound 13 wet cake (76 g) at 10 ± 5 °C, and the mixture was stirred until all solid was dissolved. 1% Pt / V / C (1.4 g, 3.5%) was added to the hydrogenation kettle at 10 ± 5 °C. The hydrogenation kettle was purged three times with N2 (3 bar), followed by purging three times with H2 (3 bar). Then the pressure of H2 was adjusted to 1 bar and stirred at 500 rpm at IT = 20 ± 10 °C for 4 h. The mixture was reacted under 6 bar H2 pressure at IT = 20 ± 10 °C for 17 h until almost no H2 consumption. The mixture was filtrated to remove catalyst, and the hydrogenation kettle and residue were washed with EtOAc (36 g) twice. The combined organic solution was washed with a mixed solution of water (240 g), NaHSO₃ (17.2 g) and citric acid monohydrate (100 g) and stirred for 1 h at 25 ± 5 °C. Phase separation and the organic phase was washed with a mixed solution of water (240 g) and citric acid monohydrate (100 g) and stirred for 1 h at 25 ± 5 °C. Phase separation and the organic phase was washed with water (200 g) and stirred for 1 h at 25 ± 5 °C. Phase separation agian to obtain organic phase. Organic phase was then washed with 5% NaHCO3 (200 g) and stirred for 1 h at 25 ± 5 °C and then evaporated under vacuum to get ~68 g residue. The residue was diluted with MeCN (344 g) and then evaporated under vacuum to remove the solvents (distillate around 344 g). The obtained MeCN solution of compound 14 (~68g) was directly used for next step. 1H NMR (400 MHz, CDC13) 58.30 (s, 1H), 7.14 (t, J= 2.8 Hz, 1H), 6.93 (d, J= 2.3 Hz, 1H), 6.65 (d, J= 2.3 Hz, 1H), 6.45 - 6.42 (m, 1H), 3.81 (s, 3H), 2.43 (s, 3H). LCMS m / z (ESI) [M+l]+ = 162.1.

[0448] Synthesis of tert-butyl 5-methoxy-7-methyl-lH-indole-l-carboxylate (15)

[0449]

[0258] To DMAP (0.18 g, 0.03 eq.) was added MeCN solution of compound 14 (24.8 g, 32.3% assay, leq.), and the mixture was stirred at 40 ± 5 °C. BOC2O (13.5 g, 1.25 eq.) in MeCN solution (8 mL) was added drop wise in 2 h, and the mixture was stirred at 40 ± 5 °C for 1 h. To the reaction mixture was added water (1.6 mL) at 40 ± 5 °C, and then 0.2% seeds (0.016g) was added and stirred for 0.5 h to obtain a cloudy suspension. Water (14.4 mL) was added in 2 h and the mixture was stirred for 1 h. The mixture was cooled to 0 ± 5 °C with no less than 4 h, and stirred at 0 ± 5 °C for 1 h, then filtered and washed with cold MeCN / water (16 mL / 8 mL). The solid was dried at 55 °C for no less than 8 h to obtain compound 15 as an off-white solid. 1H NMR (400 MHz, DMSO) 57.34 (m, 1H), 6.72 (m, 1H), 6.48 (m, 1H), 6.35 (m, 1H), 3.52 (s, 3H), 2.27 (s. 3H). 1.35 (s, 9H); LC-MS: m / z[M-tBu+l]+ = 206.1 Synthesis of tert-butyl 4-formyl-5-methoxy-7-methyl-lH-indole-l-carboxylate (8)

[0450]

[0259] To Me-THF (480 ml) was added N-Methyl-N-formylaniline (47 g, 1.5 eq.) at 20±5 °C and the mixture was stirred for 15 min. oxalyl dichloride (47 g, 1.6 eq) was added dropwise to above solution at 20 ± 5 °C, and the mixture was stirred for another 7 h. The mixture was cooled to -15 ± 5 °C, Compound 15 (60 g, 1.0 eq) was added in one portion at - 15 ± 5 °C. The solution of FeC13 (41 g, 1.1 eq.) in Me-THF (240 ml) was added dropwise to above yellow solution (keeping IT< -10 °C ). The reaction mixture was stirred at -15 ± 5 o C for 12 h. 5% Na2SC>4 (480 ml) was added dropwise to reaction mixture (keeping IT < 15 °C ). Phase separation, aqueous layer was discarded, and the organic layer was washed with 5% Na2SC>4 (480 ml), H2O (480 ml) and 5% NaHCCh (180 ml) at 20 ± 5 °C. The organic solution was evaporated under vacuum to ~360 g. The residue was diluted with MeCN (1800 g) and then concentrated to ~600 g. The residual solution was heated to 80 °C and stirred for 0.5 h. The brown solution was cooled to 25 ± 5 °C. Seed (0.12 g) was added, and the mixture was stirred for 0.5 h. H2O (840 g) was added dropwise to above suspension and stirred for another 1 h at 25 ± 5 °C. The mixture was filtered, and the wet cake was washed with a mixed solvent of MeCN (60 mL) and H2O (90 mL). The wet cake was dried under 55 °C for 16 h and compound 8 was obtained as an off-white powder.1H NMR (400 MHz, CDCl3) δ 10.65 (s, 1H), 7.65 (d, J= 3.4 Hz, 1H), 7.49 (d, J= 3.4 Hz, 1H), 6.76(s, 1H), 3.98(s, 3H), 2.70(s, 3H), 1.65(s, 9H). LCMS m / z (ESI) [M+l]+ = 290.1.

[0451] Example 6: Alternative Synthesis of compound 8

[0452]

[0453] Scheme F

[0454] Synthesis of 2-(5-methoxy-3 -methyl -2 -nitrophenyl)ethan-l-ol (16)

[0455]

[0260] To a solution of 5-methoxy-l,3-dimethyl-2 -nitrobenzene 12 (50 g, 1.0 eq.) in DMSO (500 mL) was added t-BuOLi (33.2 g, 1.5 eq.) and the mixture was stirred for 30 minutes at 25 °C. Paraldehyde (10.0 g, 1.2 eq) was added to reaction mixture in several portions. The mixture was stirred at 25 °C for 1.5 h and quenched with saturated NH4C1 aqueous solution. The mixture was extracted with MTBE (150 mL*3) and the combined organic phases were washed 20% NaCl solution (100 mL). The organic phases were washed dried over Na2SC>4, concentrated under vacuum and the residue was purified over silica gel chromatography to get the 2-(5-methoxy-3 -methyl -2 -nitrophenyl)ethan-l-ol, compound 16, as an off white solid (91.5% purity, 68% yield). 1H NMR (400 MHz, DMSO-d6): δ 6.88 – 6.82 (m, 2H), 4.77 (bs, 1H), 3.80 (s, 3H), 2.14 – 2.10 (m, 2H), 2.70 – 2.65 (m, 2H), 2.24 (s, 3H). LCMS m / z (ESI) [M+l]+ = 212.2.

[0456] Synthesis of 5-methoxy-7-methyl-lH-indole (14)

[0457]

[0261] To a flame-dried Schlenk tube with a Teflon-coated magnetic stirring bar were sequentially charged with 2-(5-methoxy-3 -methyl -2 -nitrophenyl)ethan-l-ol 16 (50 mg, 1 eq.), B2nep2 (106.9 mg, 2 eq.), TMP (167.2 mg, 5 eq.), toluene (0.68 mL), and ethylene glycol (75 pL). The mixture was vigorously stirred under the irradiation of a 200 W 405 nm LED at 5 °C for 16 h. The reaction was monitored by HPLC. After completion, the crude mixture was purified on flash column chromatography to afford compound 14 as an off-while solid. (90% purity, 75% yield). 'H NMR (400 MHz, CDCh) 58.30 (s, 1H), 7.14 (t, J= 2.8 Hz, 1H), 6.93 (d, J= 2.3 Hz, 1H), 6.65 (d, J= 2.3 Hz, 1H), 6.45 - 6.42 (m, 1H), 3.81 (s, 3H), 2.43 (s, 3H). LCMS m / z (ESI) [M+l]+ = 162.1. The following steps to convert compound 14 to 8 was same as described above.

[0458] Example 7: Synthesis of Compound 10

[0459]

[0262] The synthesis process of Compound 10 is described in Scheme G

[0460]

[0461] acid = maleic acid, 7a

[0462] Scheme G

[0463]

[0263] Synthesis of compound 9

[0464]

[0465] Scheme H

[0466]

[0264] To the solution of methyl 4-((2S,4S)-4-ethoxypiperidin-2-yl)benzoate trifluoromethanesulfonate 7 (5.0 g, 1.0 eq), tert-butyl 4-formyl-5-methoxy-7-methyl- \H-indole-1 -carboxylate 8 (3.85 g, 1.1 eq) and TEA (3.67 g, 3.0 eq) in Me-THF (50 mL) was added NaBH(OAc)3 (7.69 g, 3.0 eq.) at 20 °C and stirred at 20 °C for no less than 18 h. To the reaction mixture was added 12% aqueous Na2CO3 (37.5 g) at 0 °C. After addition, the

[0467] biphase solution was stirred at 20 °C for 0.5 h followed by stirring at 55 °C for 0.5 h. The aqueous layer was separated. The organic layer was washed with water (20 g) twice followed by concentration. To the residue was added MeCN (40 g) and concentrated to 33 g. The

[0468] above solvent exchange process was further repeated twice. The residue was heated to 80 °C and stirred to get a solution. The solution was cooled to 50 °C in 1 h and stirred at 50 °C for 3 h. The suspension was cooled to 0 °C in 3 h and stirred at 0 °C for 2 h. The suspension was filtered. The wet cake was washed with MeCN (5 g) twice and dried at 50 °C under vacuum for 18 h. Compound 9 was obtained as off-white solid.1H NMR (400 MHz, DMSO-de) 8 ppm 8.13 (d, J= 8.16 Hz, 2H), 7.77 (br d, J=7.84 Hz, 2H), 7.62-7.68 (m, 1H), 6.85 (s, 1H), 6.80 (d, J= 3.76 Hz, 1H), 4.01 (s, 3H), 3.92 (s, 3H), 3.73 (br. s, 1H), 3.55- 3.67 (m, 4H), 3.39 - 3.42 (m, 1H), 2.60 - 2.70 (m, 5H), 1.99 - 2.02(br. d, 1H), 1.82 - 1.90 (m, 2H), 1.74 (s, 9H), 1.64 - 1.70(m, 1H), 1.35 (t, J= 6.97 Hz, 3H).

[0265] Alternatively, compound 9 may be prepared from maleic acid salt 7a as described below. To the solution of methyl 4-((2S,4S)-4-ethoxypiperidin-2-yl)benzoate, maleic acid salt 7a (20.0 g, 1.0 eq), tert-butyl 4-formyl-5-methoxy-7-methyl-1H-indole-1-carboxylate 8 (19.82 g, 1.3 eq) and triethylamine (3.67 g, 3.0 eq) in 2-methyltetrahydrofuran (200 mL) was added NaBH(OAc)3 (23.46 g, 2.1 eq.) at 32 °C in portions and stirred at 32 °C for no less than 13 h. To the reaction mixture was added 12% aqueous Na2CC>3 (157 g) at 5 °C. After addition, the biphase solution was stirred at 20 °C for 0.5 h followed by stirring at 55 °C for 0.5 h. The aqueous layer was separated. The organic layer was washed with water (80 g) twice followed by concentration. To the residue was added MeCN (160 g) and concentrated to 115 g. The above solvent exchange process was further repeated twice. The residue was heated to 80 °C and stirred to get a solution. The solution was cooled to 50 °C in 1 h and stirred at 50 °C for 3 h. The suspension was cooled to 3 °C in 4 h and stirred at 3 °C for 2 h. The suspension was filtered. The wet cake was washed with pre -cooled MeCN (22 g) twice and dried at 50 °C under vacuum for 18 h. Compound 9 was obtained as off-white solid. (99% purity, 92% yield).

[0469]

[0470] Scheme I

[0471]

[0266] To a solution of 4-((2S,4S)-(4-ethoxy-l-((5-methoxy-7-methyl-l H-indol-4-yl)methyl)piperidin- 2-yl))benzoic acid (620 mg, 1.467 mmol) in H2O / CH3CN (10 / 3 mL) was added 5M aq. HC1 (500 pL, 2.500 mmol). The mixture was then lyophilized. The resulting amorphous compound was then suspended in iPrOH (300 mL). The mixture was heated to 70 °C. The mixture turned to a solution after 1.5h. The solution was then cooled to room temperature with stirring for approx. 5h. The resulting solid was collected by filtration. The solid was dried up under high vacuum at 50 °C to afford the title compound as a crystalline solid. 'HNMR (HCI salt, 400 MHz, CD3OD) 5 10.73 (br. s„ 1 H), 8.23 (d, J=8.44 Hz, 2H), 7.74 (d, J=8.44 Hz, 2H), 7.31-7.36 (m, 1 H), 6.77 (s, 1 H), 6.37 (dd, J=1.77, 3.12 Hz, 1 H), 4.33 (d, J=12.72 Hz, 1 H), 4.25 (d, J=12.72 Hz, 1 H), 3.79-3.85 (m, 1 H), 3.76 (s, 3H), 3.51-3.67 (m, 4H), 3.37-3.44 (m, 1 H), 2.51 (s, 3H), 2.21 -2.29 (m, 2H), 1.90-2.15 (m, 2H), 1.31 (t, J=6.97 Hz, 3H).

[0472]

[0267] Compound 10 scale up procedure is as follows. To the suspension of 4-((2. S'.4S')-(4-ethoxy- l-((5-methoxy-7-methyl-lH-indol-4-yl)methyl)piperidin-2-yl))benzoic acid 9 (40.0 g) in 2-methyltetrahydrofuran / MeOH (45.8 g / 26.1 g) was added 32% aqueous NaOH (37.1 g) followed by 22.5 g H2O. The resulting mixture was stirred at 60 °C for no less than 8 h. The mixture was cooled to 23 °C. To the mixture was charged 9.0% aqueous HC1 (176 g) in no less than 1 h, crystal seed (272 mg) and 15% aqueous NaCl (48 g) in no less than 1 h. The resulting suspension was stirred at 23 °C for 5 h followed by charging another portion of 15% aqueous NaCl (432 g) in no less than 4 h. After stirring at 23 °C for another 6 h, the suspension was filtered. The wet cake was washed with 2.0% aqueous NaCl (80 g) twice, water (80 g) twice and dried at 55 °C under vacuum for 10 h. Compound 10 was obtained as off-white to pink to purple solid. (99% purity, 92% yield).

[0473] Example 8: Salts of Compound of Formula (II)

[0474]

[0268] Compound of Formula (II) was subjected to a salt screening study. To a solution of free base of compound of formula (II) in isopropyl acetate (90 mg / mL) was added a solution of an acid in acetone (1.1 equiv., 25%) at 50 °C. The reaction mixture was stirred at the same temperature for 30 min and, if suspension formed, filtered. The resulting wet cake was further washed with isopropyl acetate to obtain the corresponding salt of compound of formula (II). The results of the study are summarized in the table below.

[0475]

[0476]

[0477]

[0269] As shown in the above table, oxalic acid, malonic acid, maleic acid, hydrochloric acid, and acetic acid are particularly suitable salt-forming acids for Compound of Formula (II) salts.

[0478] Example 9: Preparation of Crystalline Form He of the hydrate of a hydrochloric acid salt of the Compound of Formula (I) (iptacopan hydrochloride hydrate)

[0479]

[0270] 50 mg of the hydrochloric acid salt of the compound of Formula (I) from Example 7 were weighed into 1 ml glass vial and 0.1 mL of 2-MeTHF / Methanol (1 / 4, v / v) were added and stirred with a magnetic stirrer to form a clear solution at room temperature (23-25 °C). 0.5 ml of ethyl acetate / water (98 / 2, m / m) were added dropwise, and about 1-2 mg of the compound of Formula (I) seeds were added. After several minutes, a solid precipitate formed. The resulting suspension was filtered after 18 hours, and the recovered solid was dried at 50°C for 4 h in vacuo. The white solid was analyzed by XRPD, DSC and TGA, as well as by NMR (see FIGs. 1, 2, and 3). KF titration indicated that the product is 4.6 wt % water.

[0480]

[0271] X-ray powder diffraction measurement details are summarized in the table below.

[0481] XRPD-method ca. 13 min

[0482] Instrument Bruker D8 Advance

[0483] Detector LYNXEYE (1 D mode) Goniometer radius 280mm

[0484]

[0485]

[0486]

[0273] Among the peaks listed above, peaks at one or more of the following 2θ angles are particularly characteristic of crystalline form He of iptacopan hydrochloride hydrate: (6.7 ± 0.2)°, (10.0 ± 0.2)°, (13.3 ± 0.2)°, (14.9 ± 0.2)°, and (24.6 ± 0.2)°.

Claims

WHAT IS CLAIMED:

1. A method of preparing Compound 5 having a structure of,(Compound 5), wherein R1is Ci-Ce alkyl, such as methyl,wherein the method comprises reacting Compound 4 having a structure of(Compound 4),wherein R1is Ci-Ce alkyl, such as methyl;with Compound 3 having a structure of(Compound 3).

2. The method according to claim 1, further comprising a process of preparing Compound 3 by reacting Compound 1 having a structure of(Compound 1) andCompound 2 having a structure of(Compound 2).

3. The method of claim 2, wherein Compound 1 and Compound 2 are reacted in the presence of carbonyldiimidazole (CDI).

4. The method of claim 3, wherein CDI is added to Compound 1 prior to reacting with Compound 3.

5. The method of claims 1 to 4, wherein Compound 3 and Compound 4 are reacted in the presence of a strong acid.

6. The method of claims 1 to 5, wherein Compound 3 and Compound 4 are reacted in the presence of piperidine.

7. A method of preparing a Compound (S)-6 having a structure of(Compound (S)-6), wherein R1is Ci-Ce alkyl, such as methyl, wherein the method comprises reacting Compound 5 having a structure of(Compound 5),wherein R1is Ci-Ce alkyl, such as methyl;with(i) an enzyme comprising one or more selected from the group consisting of ketoreductase (KRED), alcohol dehydrogenases, and glucose dehydrogenase (GDH); and(ii) a co-factor comprising one or more selected from the group consisting of alcohol dehydrogenase, nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD) and pyridoxal monophosphate;in an aqueous buffer solution comprising one or more selected from the group consisting of TRIS, HEPES, MOPS, PIPES, borate, glycine, triethanol amine, phosphate, citrate, acetate, and ammonia.

8. The method of claim 7, wherein the enzyme is a ketoreductase (KRED).

9. The method of claim 8, wherein the ketoreductase (KRED) is selected from KRED- EW124, KRED-P3-G09, KRED-P1-B02, KRED-P1-C01, KRED-P2-B02, KRED-P2- C02, KRED-P3-B03, KRED-P2-D03, KRED-P2-D11, KRED-P2-D12, KRED-P2-H07, KRED-P3-H12, KRED-101, and KRED-119.

10. The method of claim 8, wherein the KRED comprises a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid sequence of SEQ ID NO: 1, or a fragment thereof.

11. The method of claim 8, wherein the KRED comprises a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid sequence of SEQ ID NO: 3, or a fragment thereof.

12. The method of claim 8, wherein the KRED comprises a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid sequence of SEQ ID NO: 5, or a fragment thereof.

13. The method of claim 8, wherein the KRED comprises a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid sequence of SEQ ID NO: 7, or a fragment thereof.

14. The method of claim 8, wherein the KRED is produced from a cell comprising a nucleic acid sequence having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 2.

15. The method of claim 8, wherein the KRED is produced from a cell comprising a nucleic acid sequence having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 4.

16. The method of claim 8, wherein the KRED is produced from a cell comprising a nucleic acid sequence having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 6.

17. The method of claim 8, wherein the KRED is produced from a cell comprising a nucleic acid sequence having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 8.

18. The method of claims 8 to 17, wherein Compound 5 is reacted with the KRED in the presence of L-proline and / or an acid selected from D-proline, glycine, glutamic acid, aspartic acid, lysine, malonic acid, lactic acid, mandelic acid, and AcOH.

19. The method of claims 8 to 17, wherein Compound 5 is reacted with the KRED in the presence of L-proline.

20. The method of any one of claims 7 to 19, whereinthe method further comprises a step of reacting the Compound (S)-6 having a structure of(Compound (S)-6),wherein R1is Ci-Ce-alkyl, such as methyl;with an ethylating reagent such as 2,4,6-trimethyl-l,3,5-trioxane, to obtain a compound of formula (II) having a structure ofpharmaceutically acceptable salt thereof.

21. The method of any one of claims 7 to 20, wherein the method further comprises a step of:(a) reacting the Compound (S)-6:with a compound comprising an oxygen protecting group P2to obtain a Compound (S)-6’ having a structure of(Compound (S)-6’), wherein R1is Ci-Ce-alkyl, such as methyl, and P2is an oxygen protecting group, such as tert-butyldimethylsilyl (TBS); and(b) reacting the Compound (S)-6’ with an ethylating reagent such as 2,4,6-trimethyl- 1,3,5-trioxane.

22. A method of preparing a compound of formula (III) having a structure ofor a pharmaceutically acceptable salt thereof,wherein P3is a protecting group, such as tert-butyloxycarbonyl (Boc), wherein the method comprises the steps of:(a) reacting Compound 11 having a structure of(Compound 11),with an alkylating agent, to obtain Compound 12 having a structure of(Compound 12);(b) reacting Compound 12 to form an indole Compound 14 having a structure of(Compound 14);(c) reacting Compound 14 with a compound having a formula of (P3)2O, wherein P3is a nitrogen protecting group, such as tert-butyloxycarbonyl (Boc),to obtain Compound 9 having a structure of(Compound 9), or a pharmaceutically acceptable salt thereof, wherein P3is a nitrogen protecting group; and(d) reacting Compound 9 with a formylating agent to obtain a compound of formula (III) having a structure of,pharmaceutically acceptable salt thereof,wherein P3is a nitrogen protecting group, such as tert-butyloxy carbonyl (Boc).

23. The method of claim 22, wherein in the step (b), Compound 12 is reacted with pyrrolidine and a formylation agent to obtain Compound 13 having a structure of(Compound 13).

24. The method of claim 23, wherein Compound 13 is reacted with a transition metal catalyst under hydrogen pressure to obtain Compound 14.

25. The method of any one of claims 22 to 24, wherein Compound 12 is reacted with the pyrrolidine and the methylation agent in the presence of a Lewis acid catalyst.

26. The method of claim 25, wherein the Lewis acid catalyst comprises one or more selected from AlCl3, FeCl3, MgCl2, TiCl4, BF3, and SnCl4.

27. The method of any one of claims 22 to 26, wherein in the step (d), the formylating agent comprises oxalyl dichloride.

28. The method of claim 22 to 27, wherein the formylating agent comprises a formamide, such as N-methyl-N-formylaniline.

29. A method of preparing a compound of formula (I),or a pharmaceutically acceptable salt thereof,the method comprising(i) reacting a compound of formula (II) prepared according to claim 20,pharmaceutically acceptable salt thereof, wherein R1is Ci-Ce alkyl, such as methyl;with a compound of formula (III) prepared according to any one of claims 22 to 28,pharmaceutically acceptable salt thereof, wherein P3is a nitrogen protecting group, such as tert-butyloxycarbonyl (Boc), in the presence of an Iridium catalyst, such as selected from the group consisting of Ir(CO)2acac, Ir(COD)Cl, Ir(CO)3, and IrCl3,xH2O;under hydrogen pressure, such as from 1 to 30 bar;optionally in the presence of an additive comprising one or more selected from the group consisting of TBAI, DPEPhos, Xantphos, DABCO, NaOTf, ((4-F-C6H4)3P), NBS, NCS, triethylamine, and acetic acid to obtain a compound for(ii) reacting Compound 9, under hydrolyzing conditions to obtain a compound of30. A process for preparing a pharmaceutical composition, comprising a step of formulating the compound of formula (I), or a pharmaceutically acceptable pharmaceutically acceptable salt thereof, prepared by the method of any one of claims 1 to 29 with a pharmaceutically acceptable excipient.

31. A ketoreductase (KRED) comprising a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid sequence of SEQ ID NO: 1, or a fragment thereof.

32. A ketoreductase (KRED) comprising a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid sequence of SEQ ID NO: 3, or a fragment thereof.

33. A ketoreductase (KRED) comprising a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid sequence of SEQ ID NO: 5, or a fragment thereof.

34. A ketoreductase (KRED) comprising a protein having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the amino acid sequence of SEQ ID NO: 7, or a fragment thereof.

35. A ketoreductase (KRED) produced from a cell comprising a nucleic acid sequence having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 2.

36. A ketoreductase (KRED) produced from a cell comprising a nucleic acid sequence having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 4.

37. A ketoreductase (KRED) produced from a cell comprising a nucleic acid sequence having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 6.

38. A ketoreductase (KRED) produced from a cell comprising a nucleic acid sequence having a sequence identity of about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, to the nucleotide sequence of SEQ ID NO: 8.

39. A compound having a structure ofpharmaceutically acceptable salt thereof, wherein R1is Ci-Ce alkyl, such as methyl.

40. A compound having a structure ofpharmaceutically acceptable salt thereof.

41. A compound having a structure of, or a pharmaceutically acceptable salt thereof.

42. A fumaric acid salt of the compound of the following structure:

43. An oxalic acid salt of the compound of the following structure:

44. A succinic acid salt of the compound of the following structure:

45. A benzoic acid salt of the compound of the following structure:

46. A citric acid salt of the compound of the following structure:

47. A malonic acid salt of the compound of the following structure:

48. A malic acid salt of the compound of the following structure:

49. An adipic acid salt of the compound of the following structure:

50. A hydrochloric acid salt of the compound of the following structure:

51. An acetic acid salt of the compound of the following structure:

52. A crystalline form of iptacopan hydrochloride hydrate characterized by having a powder X-ray diffractogram comprising one peak selected from the group consisting of peaks at 20 angles (6.7 ± 0.2)°, (10.0 ± 0.2)°, (13.3 ± 0.2)°, (14.9 ± 0.2)°, and (24.6 ± 0.2)°, e.g., when measured at a temperature in the range of from 20 to 30 °C with Cu-Ka radiation having a wavelength of 0.154 nm (e.g., 1.54190 nm).

53. A crystalline form of iptacopan hydrochloride hydrate characterized by having a powder X-ray diffractogram comprising two peaks selected from the group consisting of peaks at 20 angles (6.7 ± 0.2)°, (10.0 ± 0.2)°, (13.3 ± 0.2)°, (14.9 ± 0.2)°, and (24.6 ± 0.2)°, e.g., when measured at a temperature in the range of from 20 to 30 °C with Cu-Ka radiation having a wavelength of 0.154 nm (e.g., 1.54190 nm).

54. A crystalline form of iptacopan hydrochloride hydrate characterized by having a powder X-ray diffractogram comprising three peaks selected from the group consisting of peaks at 20 angles (6.7 ± 0.2)°, (10.0 ± 0.2)°, (13.3 ± 0.2)°, (14.9 ± 0.2)°, and (24.6 ± 0.2)°, e.g., when measured at a temperature in the range of from 20 to 30 °C with Cu-Ka radiation having a wavelength of 0.154 nm (e.g., 1.54190 nm).

55. A crystalline form of iptacopan hydrochloride hydrate characterized by having a powder X-ray diffractogram comprising four peaks selected from the group consisting of peaks at 20 angles (6.7 ± 0.2)°, (10.0 ± 0.2)°, (13.3 ± 0.2)°, (14.9 ± 0.2)°, and (24.6 ± 0.2)°, e.g., when measured at a temperature in the range of from 20 to 30 °C with Cu-Ka radiation having a wavelength of 0.154 nm (e.g., 1.54190 nm).

56. A crystalline form of iptacopan hydrochloride hydrate characterized by having a powder X-ray diffractogram comprising five peaks selected from the group consisting of peaks at28 angles (6.7 ± 0.2)°, (10.0 ± 0.2)°, (13.3 ± 0.2)°, (14.9 ± 0.2)°, and (24.6 ± 0.2)°, e.g., when measured at a temperature in the range of from 20 to 30 °C with Cu-Ka radiation having a wavelength of 0.154 nm (e.g., 1.54190 nm).

57. The crystalline form of any one of claims 52 to 56, wherein the powder X-ray diffractogram further comprises one or more (e.g., one, two, three, four, or five) peaks selected from the group consisting of peaks at 28 angles (7.0 ± 0.2)°, (9.7 ± 0.2)°, (9.8 ± 0.2)°, (11.8 ± 0.2)°, (12.4 ± 0.2)°, (12.6 ± 0.2)°, (15.9 ± 0.2)°, (16.7 ± 0.2)°, (17.1 ± 0.2)°, (17.8 ± 0.2)°, (18.5 ± 0.2)°, (19.1 ± 0.2)°, (19.3 ± 0.2)°, (19.5 ± 0.2)°, (19.6 ± 0.2)°, (20.0 ± 0.2)°, (20.2 ± 0.2)°, (21.8 ± 0.2)°, (22.1 ± 0.2)°, (23.6 ± 0.2)°, (23.8 ± 0.2)°, (24.8 ± 0.2)°, (25.0 ± 0.2)°, (25.4 ± 0.2)°, (26.5 ± 0.2)°, (26.8 ± 0.2)°, (26.9 ± 0.2)°, (27.5 ± 0.2)°, and (29.2 ± 0.2)°, e.g., when measured at a temperature in the range of from 20 to 30 °C with Cu-Ka radiation having a wavelength of 0.154 nm (e.g., 1.54190 nm).

58. The crystalline form of any one of claims 52 to 56, wherein the powder X-ray diffractogram further comprises one or more (e.g., one, two, three, four, or five) peaks selected from the group consisting of peaks at 28 angles (9.7 ± 0.2)°, (15.9 ± 0.2)°, (17.8 ± 0.2)°, (18.5 ± 0.2)°, (19.5 ± 0.2)°, (19.6 ± 0.2)°, (21.8 ± 0.2)°, (23.6 ± 0.2)°, (26.8 ± 0.2)°, (26.9 ± 0.2)°, (27.5 ± 0.2)°, and (29.2 ± 0.2)°, e.g., when measured at a temperature in the range of from 20 to 30 °C with Cu-Ka radiation having a wavelength of 0.154 nm (e.g., 1.54190 nm).

59. A pharmaceutical composition comprising the crystalline form of any one of claims 52 to 58.

60. The pharmaceutical composition of claim 58 further comprising a pharmaceutically acceptable excipient.

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