Process for preparing (2s,4s,4's,6s)-2-methyl-6-(1-methyl-1h-1,2,3-triazol-4-YL)-2',-(trieluoromethyl)-4',5'-dihydrospiro[piperidine-4,7'-thieno [2,3-c] pyran] -4'-ol

Abstract

The disclosure provides processes of preparing (2S,4S,4'S,6S)-2-methyl-6-(1-methyl-1H-1,2,3-triazol-4-yl)-2'-(trifluoromethyl)-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-4'-ol (Compound (I)), stereoisomers of Compound (I), deuterated derivatives of Compound (I), and deuterated derivatives of stereoisomers of Compound (I).

Description

PROCESS FOR PREPARING (2S,4S,4’S,6S)-2-METHYL-6-(l-METHYL-lH- l,2,3-TRIAZOL-4-YL)-2,-(TRIFLUOROMETHYL)-4,,5,- DIHYDROSPIRO [PIPERIDINE-4,7’-THIENO [2,3-C] PYRAN] -4’-OL

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 537,769, filed on September 11, 2023, and U.S. Provisional Application No. 63 / 539,056, filed on September 18, 2023, the contents of both of which are incorporated by reference in their entirety.

[0002] This disclosure provides processes for the synthesis of compounds that can inhibit apolipoprotein LI (APOL1). Such compounds have been shown to be useful in treating APOL1 -mediated diseases, e.g, pancreatic cancer, focal segmental glomerulosclerosis (FSGS), and / or non-diabetic kidney disease (NDKD).

[0003] FSGS is a rare kidney disease with an estimated global incidence of 0.2 to 1.1 / 100, 000 / y ear. FSGS is a disease of the podocyte (glomerular visceral epithelial cells) responsible for proteinuria and progressive decline in kidney function. NDKD is a kidney disease involving damage to the podocyte or glomerular vascular bed that is not attributable to diabetes. NDKD is a disease characterized by hypertension and progressive decline in kidney function. Human genetics support a causal role for the G1 and G2APOL1 variants in inducing kidney disease. Individuals with 2APOL1 alleles are at increased risk of developing end-stage kidney disease (ESKD), including primary (idiopathic) FSGS, human immunodeficiency virus (HlV)-associated FSGS, NDKD, arterionephrosclerosis, lupus nephritis, microalbuminuria, and chronic kidney disease. See, P. Dummer etal., Semin Nephrol. 2015, 35(3): 222-236.

[0004] FSGS and NDKD can be divided into different subgroups based on the underlying etiology. One homogeneous subgroup of FSGS is characterized by the presence of independent common sequence variants in the apolipoprotein LI (APOL1) gene termed G1 and G2, which are referred to as the “APOL1 risk alleles.” G1 encodes a correlated pair of non-synonymous amino acid changes (S342G and I384M), G2 encodes a 2 amino acid deletion (N388del:Y389del) near the C terminus of the protein, and GO is the ancestral (low risk) allele. A distinct phenotype of NDKD is found in patients with APOL1 genetic risk variants as well. In both APOL1 -mediated FSGS and NDKD, higher levels of proteinuria and a more accelerated loss of kidney function occur in patients with two risk alleles compared to patients with the same disease who have no or just 1AP0L1 genetic risk variant. Alternatively, in AMKD, higher levels of proteinuria and accelerated loss of kidney function can also occur in patients with one risk allele. See, G. Va| gel2019, 47(8): 1209-1217.

[0005] APOL1 is a 44 kDa protein that is only expressed in humans, gorillas, and baboons. The APOL1 gene is expressed in multiple organs in humans, including the liver and kidney. APOL1 is produced mainly by the liver and contains a signal peptide that allows for secretion into the bloodstream, where it circulates bound to a subset of high- density lipoproteins. APOL1 is responsible for protection against the invasive parasite, Trypanosoma brucei brucei (T. b. brucei). APOL1 is endocytosed by T. b. brucei and transported to lysosomes, where it inserts into the lysosomal membrane and forms pores that lead to parasite swelling and death.

[0006] While the ability to lyse T. b. brucei is shared by all 3 APOL1 variants (GO, Gl, and G2), APOL1 Gl and G2 variants confer additional protection against parasite species that have evolved a serum resistant associated protein (SRA) which inhibits APOL1 GO; APOL1 Gl and G2 variants confer additional protection against trypanosoma species that cause sleeping sickness. Gl and G2 variants evade inhibition by SRA; Gl confers additional protection against T. b. gambiense (which causes West African sleeping sickness) while G2 confers additional protection against T. b. rhodesiense (which causes East African sleeping sickness).

[0007] In the kidney, APOL1 is expressed in podocytes, endothelial cells (including glomerular endothelial cells), and some tubular cells. Podocyte-specific expression of APOL1 Gl or G2 (but not GO) in transgenic mice induces structural and functional changes, including albuminuria, decreased kidney function, podocyte abnormalities, and glomerulosclerosis. Consistent with these data, Gl and G2 variants of APOL1 play a causative role in inducing FSGS and accelerating its progression in humans. Individuals with APOL1 risk alleles (z.e., homozygous or compound heterozygous for the APOL1 Gl or APOL1 G2 alleles) have increased risk of developing FSGS and they are at risk for rapid decline in kidney function if they develop FSGS. Thus, inhibition of APOL1 could have a positive impact in individuals who harbor APOL1 risk alleles.

[0008] Although normal plasma concentrations of APOL1 are relatively high and can vary at least 20-fold in humans, circulating APOL1 is not causally associated with kidney disease. However, APOL1 in the kidney is thought to be responsible for the development of kidney diseases, including FSGS and NDKD. Under certaincircumstances, AP0L1 protein synthesis can be increased by approximately 200-fold by pro-inflammatory cytokines such as interferons or tumor necrosis factor-a. In addition, several studies have shown that APOL1 protein can form pH-gated Na+ / K+pores in the cell membrane, resulting in a net efflux of intracellular K+, ultimately resulting in activation of local and systemic inflammatory responses, cell swelling, and death.

[0009] The risk of ESKD is substantially higher in people of recent sub-Saharan African ancestry as compared to those of European ancestry. In the United States, ESKD is responsible for nearly as many lost years of life in women as from breast cancer and more lost years of life in men than from colorectal cancer.

[0010] FSGS and NDKD are caused by damage to podocytes, which are part of the glomerular filtration barrier, resulting in proteinuria. Patients with proteinuria are at a higher risk of developing end-stage kidney disease (ESKD) and developing proteinuria- related complications, such as infections or thromboembolic events. There is no standardized treatment regimen nor approved drugs for FSGS or NDKD. Currently, FSGS and NDKD are managed with symptomatic treatment (including blood pressure control using blockers of the renin angiotensin system), and patients with FSGS and heavy proteinuria may be offered high dose steroids. Current therapeutic options for NDKD are anchored on blood pressure control and blockade of the renin angiotensin system.

[0011] Corticosteroids, alone or in combination with other immunosuppressants, induce remission in a minority of patients (e.g, remission of proteinuria in a minority of patients) and are associated with numerous side effects. However, remission is frequently indurable even in patients initially responsive to corticosteroid and / or immunosuppressant treatment. As a result, patients, in particular individuals of recent sub-Saharan African ancestry with two APOL1 risk alleles, experience rapid disease progression leading to end-stage renal disease (ESRD). Illustratively, in view of evidence that APOL1 plays a causative role in inducing and accelerating the progression of kidney disease, inhibition of APOL1 should have a positive impact on patients with APOL1 mediated kidney disease, particularly those who carry two APOL1 risk alleles (z.e., are homozygous or compound heterozygous for the G1 or G2 alleles).

[0012] Additionally, APOL1 is an aberrantly expressed gene in multiple cancers (Lin et al., Cell Death and Disease 2021, 12(8):760). Recently, APOL1 was found to be abnormally elevated in human pancreatic cancer tissues compared with adjacent tissuesand was associated with poor prognosis in pancreatic cancer patients. In in vivo and in vitro experiments, knockdown of APOL1 significantly inhibited cancer cell proliferation and promoted the apoptosis of pancreatic cancer cells.

[0013] One promising APOL1 inhibitor is Compound I:Compound I which has been disclosed in WO 2022 / 047031 and WO 2023 / 028237, along with methods of use and methods of synthesizing Compound I. Both WO 2022 / 047031 and WO 2023 / 028237 are incorporated herein by reference in their entirety and particularly for their synthetic processes of producing Compound I.

[0014] Treatment of APOL1 -mediated diseases with inhibitors such as Compound I require efficient processes for synthesis. Especially when larger quantities of a compound are to be produced, factors relating to safety, efficiency, and simplicity of conducting reactions exhibit increased importance. Although WO 2022 / 047031 and WO 2023 / 028237 disclose useful methods for accessing APOL1 inhibitors such as Compound I, there is an unmet need for improved processes that provide a safer, more scalable, and more efficient way of accessing these compounds.

[0015] Thus, one aspect of the present disclosure provides improved processes for preparing Compound I, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing, as well as intermediates for use in the synthesis of Compound I, its deuterated derivatives, and pharmaceutically acceptable salts. These novel processes address shortcomings of prior synthesis methods and exhibit unexpected advantages that previous methods lacked. In some embodiments, these advancements may include but are not limited to a reduction in reaction times, increased overall yield of the desired compounds, higher material throughput, simplified reaction setup, and no need for hazardous and / or costly reagents.Brief Description of the Drawings

[0016] FIG. 1 depicts an exemplary setup for the synthesis of Compound S4 from Compound S3 in a flow system: [1] A vessel containing a solution of Compound S3 inan appropriate solvent, such as acetonitrile; [2] a vessel containing a solution of an appropriate acid, such as benzenesulfonic acid, in an appropriate solvent, such as acetonitrile; [3] a vessel containing a solution of a bromination reagent, such as NBS, in an appropriate solvent, such as acetonitrile; [4] a light source, such as an LED light, of appropriate wavelength, such as 450 nm, irradiates the reaction, such as a coil, [5] through with the reaction mixture flows; [6] a vessel containing a solution of the quencher, such as 1,3-dimethoxybenzene, in an appropriate solvent, such as acetonitrile; [7] a back pressure regulator; [8] a vessel to collect the mixture containing the desired product (Compound S4); [9] a pump to deliver reactants and reagents;

[0010] tubes or lines through which the reactants, reagents, and reaction mixture flow; and

[0011] mixer to achieve efficient mixing of the reaction components.Detailed DescriptionDefinitions

[0017] The term “APOL1,” as used herein, means apolipoprotein LI protein, and the term "APOI.I ” means apolipoprotein LI gene.

[0018] The term “APOL1 mediated disease” refers to a disease or condition associated with aberrant APOL1 (e.g, certain APOL 1 genetic variants; elevated levels of APOL1). In some embodiments, an APOL1 mediated disease is an APOL1 mediated kidney disease. In some embodiments, an APOL1 mediated disease is associated with patients having two APOL1 risk alleles, e.g, patients who are homozygous or compound heterozygous for the G1 or G2 alleles. In some embodiments, an APOL1 mediated disease is associated with patients having oneAPOLl risk allele.

[0019] The term “APOL1 mediated kidney disease” refers to a disease or condition that impairs kidney function and can be attributed to APOL1. In some embodiments, APOL1 mediated kidney disease is associated with patients having two APOL1 risk alleles, e.g, patients who are homozygous or compound heterozygous for the G1 or G2 alleles. In some embodiments, the APOL1 mediated kidney disease is chosen from ESKD, NDKD, FSGS, HIV-associated nephropathy, arterionephrosclerosis, lupus nephritis, microalbuminuria, and chronic kidney disease. In some embodiments, the APOL1 mediated kidney disease is chronic kidney disease or proteinuria.

[0020] The term “FSGS,” as used herein, means focal segmental glomerulosclerosis, which is a disease of the podocyte (glomerular visceral epithelial cells) responsible for proteinuria and progressive decline in kidney function. In some embodiments, FSGS isassociated with two APOL1 risk alleles.

[0021] The term “NDKD,” as used herein, means non-diabetic kidney disease, which is characterized by severe hypertension and progressive decline in kidney function. In some embodiments, NDKD is associated with two APOL1 risk alleles.

[0022] The terms “ESKD” and “ESRD” are used interchangeably herein to refer to end stage kidney disease or end stage renal disease. ESKD / ESRD is the last stage of kidney disease, i.e., kidney failure, and means that the kidneys have stopped working well enough for the patient to survive without dialysis or a kidney transplant. In some embodiments, ESKD / ESRD is associated with two APOL1 risk alleles.

[0023] As used herein, the term “ambient conditions” means room temperature, open air, and uncontrolled humidity conditions. The terms “room temperature” and “ambient temperature” mean 15 °C to 30 °C.

[0024] Methods of adding (a process generally referred to as “protecting”) and removing (process generally referred to as “deprotecting”) protecting groups are well-known in the art and available, for example, in P. J. Kocienski, Protecting Groups, 3rdedition (Thieme, 2005), and in Greene and Wuts, Protective Groups in Organic Synthesis, 4th edition (John Wiley & Sons, New York, 2007), both of which are hereby incorporated by reference in their entirety.

[0025] As used herein, the term “stereoisomer” refers to both enantiomers and diastereomers.

[0026] It will be appreciated that certain compounds of this invention may exist as separate stereoisomers or enantiomers and / or mixtures of those stereoisomers or enantiomers. As used in the chemical structures disclosed herein, a “wedge” (-^) or “hash” (••'''') bond to a stereogenic atom indicates a chiral center of known absolute stereochemistry (i.e., one stereoisomer). As used in the chemical structures disclosed herein, a “wavy” bond (^ ) to a stereogenic atom indicates a chiral center of unknown absolute stereochemistry (i.e., one stereoisomer). As used in the chemical structures disclosed herein, a “wavy” bond f ^ ) to a double-bonded carbon indicates a mixture of E!Z isomers. As used in the chemical structures disclosed herein, a(“straight”) bond to a stereogenic atom indicates where there is a mixture (e.g., a racemate or enrichment). As used herein, two(“straight”) bonds to a double-bonded carbon indicates that the double bond possesses the E!Z stereochemistry as drawn. As used in the chemicalAstructures disclosed herein, a ' (a “wavy” line perpendicular to a “straight” bond to group “A”) indicates that group “A” is a substituent whose point of attachment is at the end of the bond that terminates at the “wavy” line. As used herein, a stereogenic atom that is notated with an (R) or (S) indicates the stereochemical designation of the stereogenic atom under the Cahn-Ingold-Prelog convention.

[0027] The term “compound,” when referring to a compound of this disclosure, refers to a collection of molecules having an identical chemical structure unless otherwise indicated as a collection of stereoisomers (for example, a collection of racemates, a collection of cis / trans stereoisomers, or a collection of (E) and (Z) stereoisomers), except that there may be isotopic variation among the constituent atoms of the molecules. Thus, it will be clear to those of skill in the art that a compound represented by a particular chemical structure containing indicated deuterium atoms will also contain lesser amounts of isotopologues having hydrogen atoms at one or more of the designated deuterium positions in that structure. The relative amount of such isotopologues in a compound of this disclosure will depend upon a number of factors, including the isotopic purity of reagents used to make the compound and the efficiency of incorporation of isotopes in the various synthesis steps used to prepare the compound. However, as set forth above, the relative amount of such isotopologues in total will be less than 49.9% of the compound. In other embodiments, the relative amount of such isotopologues in total will be less than 47.5%, less than 40%, less than 32.5%, less than 25%, less than 17.5%, less than 10%, less than 5%, less than 3%, less than 1%, or less than 0.5% of the compound.

[0028] In some embodiments, the disclosure also is directed to processes for preparing isotope-labelled compounds of the herein disclosed compounds, wherein the formula and variables of such compounds are each independently as described herein, provided that one or more atoms therein have been replaced by an atom or atoms having an atomic mass or mass number which differs from the atomic mass or mass number of the atom which usually occurs naturally (isotope-labelled). Examples of isotopes which are commercially available and suitable for the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, for example2H,3H,13C,14C,15N,18O,170,31P,32P,35S,18F, and36C1, respectively.

[0029] In the compounds of this disclosure, any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom. Unless otherwisestated, when a position is designated specifically as “H” or “hydrogen,” the position is understood to have hydrogen at its natural abundance isotopic composition.

[0030] “Selected from” and “chosen from” are used interchangeably herein.

[0031] As used herein, the term “solvent” refers to any liquid in which the product is at least partially soluble (solubility of product >1 g / L). Non-limiting examples of suitable solvents that may be used in methods of this disclosure include dichloromethane (CH2CI2), chlorobenzene, fluorobenzene, benzonitrile, sulfolane, acetic acid (AcOH), water, acetonitrile (MeCN), ethyl acetate (EtOAc), isopropyl acetate (IP Ac), isopropyl alcohol (IP A), 2-methyl tetrahydrofuran (2-Me THF), methyl- / c 7-butyl ether (MTBE), and 1,4-di oxane.

[0032] As used herein, the term “batch process” refers to a process that is executed in fixed reactors and is composed of discrete operations, each of fixed duration and separated by fixed hold times.

[0033] As used herein, the term “flow process” refers to a process in which a product is made continuously. Operations may be integrated into the process in a continuous manner, avoiding the need for discete steps separated by fixed hold times.

[0034] As used herein, the term “halogen” or “halo” means F, Cl, Br, or I.

[0035] As used herein, the term “transition metal complex” may comprise a central atom or ion and a surrounding array of bound molecules or ions, which are known as ligands or complexing agents. Transition metal complexes may include organotransition metal complexes (such as ferricyanide (III) and its reduced ferrocyanide (II) counterpart), or complexes where non-metal atoms other than carbon, such as heteroatoms (e.g, N, S, O, and P), are datively bonded to the transition metal center. Non-limiting examples of transition metal complexes include rhodium complexes (e.g, (pentamethylcyclopentadi enyl)rhodium(III) dichloride dimer), ruthenium complexes (e.g, ruthenium hexamine complexes), and iron complexes.

[0036] As used herein, a “rhodium complex” may comprise a central rhodium atom or ion and one or more ligands. The ligand(s) of the rhodium complex is / are not particularly limited as long as the rhodium complex functions as a catalyst. Non-limiting examples of ligands, which may be optionally substituted, include bipyridyl, phenanthroline, imidazole, thiolene, thiolate, thioether, sulfide, porphyrins, pyrrole, pyrrazole, thiazole, diazole, triazole, picolinate, carboxylate, oxo, and quinone. Non-limiting examples of monodentate ligands, which may be optionally substituted, include hydrates / hydroxo,aminates, acetates, thiolates, halides, thiocyanates, and cyanides. Non-limiting examples of multidentate ligands, which may be optionally substituted, include aminoacetates, N- tosylated l,2-diphenyl-l,2-ethylenediamines, ethylenediaminetetraacetic acid, hydroxy ethylethylenediamine triacetic acid, nitrilotriacetic acid, [3-alaninediacetic acid, methyleneglycine diacetic acid, iminodisuccinate, glutamate N,N'-bisdiacetic acid, ethylenediamine disuccinic acid, diethylenetriaminepentaacetic acid, polyethers, poly carboxylates, phosphonates, and polyamines. A rhodium compound may be (pentamethylcyclopentadi enyl)rhodium(III) dichloride dimer.

[0037] As used herein, the term “rhodium catalyst” refers to the active rhodium complex that promotes (or catalyzes) the desired chemical transformation. An example of a desired chemical transformation includes but is not limited to a stereoselective transfer hydrogenation.

[0038] As used herein, the term "biocatalyst" may include natural, semi-synthetic, or metabolically engineered catalytic substances that are isolated from biological sources and synthetic catalytic molecules that mimic biological pathways. Non-limiting examples of a biocatalysts include ketoreductases (KRED) (also known as carbonyl reductases (CRED)), which are a class of proteins / enzymes that can catalyze the reduction of ketones to alcohols. Examples of KRED and CRED include but are not limited to CRED-33 and CRED-A231 (proprietary products distributed by Almac Sciences), and GDH-102 (a proprietary product distributed by Almac Sciences).

[0039] Non-limiting examples of reducing agents that may be used in this disclosure include, for example NADP.

[0040] Non-limiting examples of hydrogen donor agents that may be used in this disclosure include, for example, formic acid, sodium formate, hydrogen (H2), glycerol, methanol, ethanol, isopropanol, and dihydroanthracene.

[0041] Non-limiting examples of amine bases that may be used in this disclosure include, for example, primary amines, secondary amines, tertiary amines, aromatic amines, heterocyclic amines, and amidine bases, such as triethylamine, diisopropylethylamine, and dicyclohexylamine.

[0042] Non-limiting examples of carbonate bases that may be used in this disclosure include, for example, monobasic and dibasic carbonates such as sodium bicarbonate (NaHCCE), and potassium carbonate (K2CO3).

[0043] Non-limiting examples of hydroxide bases that may be used in this disclosureinclude, for example, lithium hydroxide (LiOH), sodium hydroxide (NaOH), and potassium hydroxide (KOH).

[0044] Non-limiting examples of phosphate bases that may be used in this disclosure include, for example, monobasic, dibasic, and tribasic phosphates, such as tribasic potassium phosphate (K3PO4) and dibasic potassium phosphate (K2HPO4).

[0045] Non-limiting examples of acetate bases that may be used in this disclosure include, for example, lithium acetate, sodium acetate, potassium acetate, and caesium acetate.

[0046] Non-limiting examples of acids that may be used in this disclosure include, for example, trifluoroacetic acid (TFA), hydrochloric acid (HC1), methanesulfonic acid (MsOH), phosphoric acid (H3PO4), and sulfuric acid (H2SO4).

[0047] Non-limiting examples of organic acids that may be used in this disclosure include, for example, formic acid, acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, and malonic acid.

[0048] Non-limiting examples of inorganic acids that may be used in this disclosure include, for example, hydrochloric acid (HC1), nitric acid (HNO3), phosphoric acid (H3PO4), and sulfuric acid (H2SO4).

[0049] Non-limiting examples of a carboxylic acid that may be used in this disclosure are formic acid, acetic acid, and trichloroacetic acid.

[0050] Non-limiting examples of sulfonic acids that may be used in this disclosure include, for example, p-toluenesulfonic acid, benzenesulfonic acid (BSA), triflic acid (TfOH), and methanesulfonic acid (MsOH).

[0051] Non-limiting examples of brominating agents that may be used in this disclosure include, for example, JV-bromosuccinimide, JV-bromophthalimide, JV-bromosaccharin, N- bromoacetamide, bromine (Bn), Br2-l,4-di oxane complex, l,3-dibromo-5,5- dimethylhydantoin, monosodiumbromoisocyanurate, and dibromoisocyanuric acid.

[0052] Non-limiting examples of V-oxide reagents that may be used in this disclosure include, for example, 2-methylpyridine V-oxide, 2,6-dimethylpyridine V-oxide, 2- ethylpyridine V-oxide, isoquinoline V-oxide, 4-methylmorpholine V-oxide, 4- methylpyridine JV-oxide, trimethylamine V-oxide, pyridine N-oxide. 3 -methylpyridine N- oxide, and 4-methoxypyridine V-oxide.

[0053] As used herein, a “light source” is a lamp, LED, or other device capable of emitting light including that of a requisite wavelength to induce a desired photochemicalreaction. Examples of suitable light sources include, for example, a 450 nm light emitting LED, a 420 nm light emitting LED, and a 365 nm light emitting LED.

[0054] As used herein, a “quench solution” is a solution containing an agent that, when exposed to the reaction solution, will react with and consume a component of the reaction solution, e.g, residual reagent or reaction by-product. In the case of the bromination reaction, the quench solution is used to e.g, consume excess brominating agent. Non-limiting examples of quench solutions include, for example, electron rich aromatic compounds, such as 1,3-dimethoxybenzene in a suitable solvent.

[0055] As used herein, the terms “about” and “approximately,” when used in connection with amounts, volumes, reaction times, reaction temperatures, etc. mean an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In some embodiments, the terms “about” and “approximately” mean within 1, 2, 3, or 4 standard deviations. In certain embodiments, the terms “about” and “approximately” mean within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or range. In preferred embodiments, “about” and “approximately” mean within 10 % of a given value or range. In even more preferred embodiments, “about” and “approximately” mean within 5 % of a given value or range. As used herein, the symbolappearing immediately before a numerical value has the same meaning as the terms “about” and “approximately.”Synthesis of Compound I

[0056] The disclosure features processes for preparing Compound I.

[0057] In some embodiments, Compound I is prepared according to Scheme 1:

[0058] In some embodiments, Compound I is prepared according to Scheme 2:

[0059] In some embodiments, Compound S3 is prepared by reacting Compound SI:with Compound S2:to produce Compound S3.

[0060] In some embodiments, reacting Compound SI with Compound S2 is performed in the presence of an acid.

[0061] In some embodiments, reacting Compound SI with Compound S2 is performed in the presence of a Bronsted acid.

[0062] In some embodiments, reacting Compound SI with Compound S2 is performed in the presence of a sulfonic acid.

[0063] In some embodiments, the sulfonic acid is methanesulfonic acid (MsOH).

[0064] In some embodiments, reacting Compound SI with Compound S2 is performed in dichloromethane.

[0065] In some embodiments, reacting Compound SI with Compound S2 is performed at about 39 °C.

[0066] In some embodiments, reacting Compound SI with Compound S2 is performed at about 45 °C.

[0067] In some embodiments, converting Compound S3 to Compound S5-MsOHcomprises:(i) converting Compound S3 to Compound S4:(ii) converting Compound S4 to Compound S5:(iii) converting Compound S5 to Compound S5-MsOH:

[0068] In some embodiments, converting Compound S3 to Compound S4 is performed in a batch process.

[0069] In some embodiments, converting Compound S3 to Compound S4 is performed in a flow process.

[0070] In some embodiments, converting Compound S3 to Compound S4 is performed in the presence of an acid and a brominating reagent.

[0071] In some embodiments, converting Compound S3 to Compound S4 is performed in the presence of an acid and a brominating reagent, wherein Compound S3, the acid, and the brominating reagent are each dissolved in an appropriate solvent in separate vessels. In some embodiments, Compound S3, the acid, and the brominating reagent are combined through a feed line.

[0072] In some embodiments, converting Compound S3 to Compound S4 is performed in the presence of a sulfonic acid and a brominating reagent.

[0073] In some embodiments, converting Compound S3 to Compound S4 is performed in the presence of (1) one or more of mineral acid, sulfonic acid, camphor sulfonic acid, benzenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, sulfuric acid,phosphoric acid, hydrochloric acid, triflic acid, and trifluoroacetic acid and (2) one or more of N-bromosuccinimide, N-bromophthalimide, N-bromosaccharin, N- bromoacetamide, bromine, Br2-l,4-di oxane complex, l,3-dibromo-5,5- dimethylhydantoin, monosodiumbromoisocyanurate, and dibromoisocyanuric acid.

[0074] In some embodiments, converting Compound S3 to Compound S4 is performed in the presence of (1) one or more of mineral acid, sulfonic acid, camphor sulfonic acid, benzenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, sulfuric acid, phosphoric acid, hydrochloric acid, triflic acid, and trifluoroacetic acid and (2) one or more of N-bromosuccinimide, N-bromophthalimide, and l,3-dibromo-5,5- dimethylhydantoin.

[0075] In some embodiments, converting Compound S3 to Compound S4 is performed in the presence of benzenesulfonic acid and N-bromosuccinimide.

[0076] In some embodiments, converting Compound S3 to Compound S4 is performed by quenching the reaction with a quench solution.

[0077] In some embodiments, converting Compound S3 to Compound S4 is performed by quenching the reaction with a solution of an electron-rich aromatic compound in a suitable solvent.

[0078] In some embodiments, converting Compound S3 to Compound S4 is performed by quenching the reaction with a solution of 1,3 -dimethoxy benzene in acetonitrile.

[0079] In some embodiments, converting Compound S3 to Compound S4 is performed by quenching the reaction with a solution of 1,3-dimethoxy benzene and 2,6-lutidine in acetonitrile.

[0080] In some embodiments, converting Compound S3 to Compound S4 is performed in the presence of irradiation.

[0081] In some embodiments, converting Compound S3 to Compound S4 is performed in the presence of irradiation with light. In some embodiments, the irradiation with light is directed at a coil through which a mixture of Compound S3, the acid, and the brominating reagent flow.

[0082] In some embodiments, converting Compound S3 to Compound S4 is performed in the presence of 365 nm wavelength LED irradiation. In some embodiments, converting Compound S3 to Compound S4 is performed in the presence of 420 nm wavelength LED irradiation. In some embodiments, converting Compound S3 to Compound S4 is performed in the presence of 450 nm wavelength LED irradiation. Insome embodiments, the 450 nm blue light emitting LED irradiates a coil through which Compound S3, the acid, and the brominating reagent flow. In some embodiments, converting Compound S3 to Compound S4 is performed in the presence of 400 nm to 450 nm wavelength LED irradiation.

[0083] In some embodiments, converting Compound S3 to Compound S4 is performed in a suitable solvent.

[0084] In some embodiments, converting Compound S3 to Compound S4 is performed in a polar solvent.

[0085] In some embodiments, converting Compound S3 to Compound S4 is performed in a protic polar solvent. In some embodiments, converting Compound S3 to Compound S4 is performed in an aprotic polar solvent.

[0086] In some embodiments, converting Compound S3 to Compound S4 is performed in acetonitrile, di chloromethane, chlorobenzene, fluorobenzene, benzonitrile, 1,4- dioxane, sulfolane, acetic acid, or a combination of any of the foregoing.

[0087] In some embodiments, converting Compound S3 to Compound S4 is performed in acetonitrile.

[0088] In some embodiments, converting Compound S3 to Compound S4 is performed at a temperature between 5 °C and 25 °C. In some embodiments, converting Compound S3 to Compound S4 is performed at 25 °C.

[0089] In some embodiments, converting Compound S4 to Compound S5 is performed in the presence of a DMSO and a base. In some embodiments, converting Compound S4 to Compound S5 is performed in the presence of a DMSO and an amine base. In some embodiments, converting Compound S4 to Compound S5 is performed in the presence of a DMSO and triethylamine.

[0090] In some embodiments, converting Compound S4 to Compound S5 is performed in the presence of an N-oxide reagent and a base.

[0091] In some embodiments, converting Compound S4 to Compound S5 is performed in the presence of an N-oxide reagent and an amine base.

[0092] In some embodiments, converting Compound S4 to Compound S5 is performed in the presence of an N-oxide reagent and a carbonate base.

[0093] In some embodiments, converting Compound S4 to Compound S5 is performed in the presence of an N-oxide reagent and a phosphate base.

[0094] In some embodiments, converting Compound S4 to Compound S5 is performedin the presence of an N-oxide reagent and an acetate base.

[0095] In some embodiments, converting Compound S4 to Compound S5 is performed in the presence of (1) one or more of 4-chloropyridine N-oxide, 2-methyl-4-nitropyridine N-oxide, 2-chloro-4-nitropyridine N-oxide, 2-chloropyridine N-oxide, and 2- cyanopyridine N-oxide, 2-methylpyridine N-oxide (2 -picoline N-oxide), 2,6- dimethylpyridine N-oxide, 2-ethylpyridine N-oxide, isoquinoline N-oxide, 4- methylmorpholine N-oxide, 4-methylpyridine N-oxide, trimethylamine N-oxide, pyridine N-oxide, 3 -methylpyridine N-oxide, and 4-methoxypyridine N-oxide and (2) one or more of triethylamine, diisopropylethylamine, dicyclohexylamine, sodium bicarbonate, potassium carbonate, potassium acetate, potassium phosphate tribasic, and potassium phosphate dibasic.

[0096] In some embodiments, converting Compound S4 to Compound S5 is performed in the presence of 2-picoline N-oxide and triethylamine.

[0097] In some embodiments, converting Compound S4 to Compound S5 is performed in a suitable solvent.

[0098] In some embodiments, converting Compound S3 to Compound S4 is performed in a polar solvent.

[0099] In some embodiments, converting Compound S4 to Compound S5 is performed in an aprotic polar solvent.

[0100] In some embodiments, converting Compound S4 to Compound S5 is performed in acetonitrile, dichloromethane, chlorobenzene, or a mixture of any of the foregoing.

[0101] In some embodiments, converting Compound S4 to Compound S5 is performed in acetonitrile.

[0102] In some embodiments, converting Compound S4 to Compound S5 is performed at about 60 °C.

[0103] In some embodiments, Compound S5 is purified by washing with one or more of an aqueous solution of N-acetyl-L-cysteine and ammonium persulfate. In some embodiments, Compound S5 is purified by washing with an aqueous solution of ammonium persulfate and NaHSOs. In some embodiments, Compound S5 is purified by washing with an aqueous solution of ammonium persulfate and sodium metabisulfite. In some embodiments, Compound S5 is purified by washing with an aqueous solution of ammonium persulfate and KHSO3. In some embodiments, Compound S5 is purified by washing with an aqueous solution of ammonium persulfate and thiomalic acid. In someembodiments, Compound S5 is purified by washing with air and an aqueous solution of NaHSOs. In some embodiments, Compound S5 is purified by washing with an aqueous solution of ammonium persulfate and thiosalicyclic acid.

[0104] In some embodiments, converting Compound S5 to Compound S5-MsOH is performed in the presence of methanesulfonic acid.

[0105] In some embodiments, converting Compound S5 to Compound S5-MsOH is performed in a suitable solvent.

[0106] In some embodiments, converting Compound S5 to Compound S5-MsOH is performed in a polar solvent.

[0107] In some embodiments, converting Compound S5 to Compound S5-MsOH is performed in a protic polar solvent.

[0108] In some embodiments, converting Compound S5 to Compound S5-MsOH is performed in isopropanol.

[0109] In some embodiments, converting Compound S5 to Compound S5-MsOH is performed at 75 °C.

[0110] In some embodiments, Compound I is prepared by converting CompoundS5 MsOH:to Compound I.

[0111] In some embodiments, converting Compound S5-MsOH to Compound I is performed in the presence of a transition metal complex, one or more ligands, a hydrogen donor reagent, and a base.

[0112] In some embodiments, converting Compound S5-MsOH to Compound I is performed in the presence of a Rh-based or Ru-based hydrogenation catalyst, a hydrogen donor agent, one or more ligands, and an amine base.

[0113] In some embodiments, converting Compound S5-MsOH to Compound I is performed in the presence of pentamethylcyclopentadi enyl)rhodium(III) dichloride dimer, (17?,27?)-(-)-JV-(4-toluenesulfonyl)-l,2-diphenylethylenediamine, formic acid, and triethylamine.

[0114] In some embodiments, converting Compound S5-MsOH to Compound I is performed in the presence of pentamethylcyclopentadi enyl)rhodium(III) dichloride dimer, ( I / ?.2 / ?)-(-)- -(4-toluenesulfonyl)- 1 ,2-di phenylethylenediamine. sodium formate, triethylamine, water, and acetonitrile.

[0115] In some embodiments, converting Compound S5-MsOH to Compound Compound I is performed in the presence of 0.25 mol% pentamethylcyclo- pentadienyl)rhodium(III) dichloride dimer. In some embodiments, converting Compound S5-MsOH to Compound I is performed in the presence of 0.03 mol% pentamethylcyclopentadienyl)rhodium(III) dichloride dimer. In some embodiments, converting Compound S5-MsOH to Compound I is performed in the presence of 0.05 mol% pentamethylcyclopentadienyl)rhodium(III) dichloride dimer.

[0116] In some embodiments, converting Compound S5-MsOH to Compound I is performed in the presence of 0.6 mol% (17?,27?)-(-)- V-(4-toluenesulfonyl)-l,2- diphenylethylenediamine. In some embodiments, converting Compound S5-MsOH to Compound I is performed in the presence of 0.072 mol% (17?,27?)-(-)- V-(4- toluenesulfonyl)-l,2-di phenylethylenediamine. In some embodiments, converting Compound S5-MsOH to Compound I is performed in the presence of 0.12 mol% (17?,27?)-(-)-JV-(4-toluenesulfonyl)-l,2-diphenylethylenediamine.

[0117] In some embodiments, converting Compound S5-MsOH to Compound I is performed in a suitable solvent.

[0118] In some embodiments, converting Compound S5 to Compound S5-MsOH is performed in a polar solvent.

[0119] In some embodiments, converting Compound S5 to Compound S5-MsOH is performed in an aprotic polar solvent.

[0120] In some embodiments, converting Compound S5-MsOH to Compound I is performed in acetonitrile, dichloromethane, ethyl acetate, 2-MeTHF, or a mixture of any of the foregoing.

[0121] In some embodiments, converting Compound S5-MsOH to Compound I is performed in acetonitrile.

[0122] In some embodiments, converting Compound S5-MsOH to Compound I is performed a 0 °C to 5 °C. In some embodiments, converting Compound S5-MsOH to Compound I is performed at 5 °C.

[0123] In some embodiments, Compound I is prepared by converting Compound S5:to Compound I.

[0124] In some embodiments, converting Compound S5 to Compound I is performed in the presence of a transition metal complex, one or more ligands, a hydrogen donor reagent, and a base.

[0125] In some embodiments, converting Compound S5 to Compound I is performed in the presence of a Rh-based or Ru-based hydrogenation catalyst, a hydrogen donor agent, one or more ligands, and an amine base.

[0126] In some embodiments, converting Compound S5 to Compound I is performed in the presence of pentamethylcyclopentadienyl)rhodium(III) dichloride dimer, (\R.2R)-(-)- #-(4-toluenesulfonyl)- l .2-di phenyl ethylenedi amine. formic acid, and tri ethylamine.

[0127] In some embodiments, converting Compound S5 to Compound I is performed in the presence of pentamethylcyclopentadienyl)rhodium(III) dichloride dimer, (\R.2R)-(-)- JV-(4-toluenesulfonyl)- 1, 2-di phenyl ethylenediamine, sodium formate, tri ethylamine, water, and acetonitrile.

[0128] In some embodiments, converting Compound S5 to Compound I is performed in the presence of a suitable amount of pentamethylcyclopentadi enyl)rhodium(III) dichloride dimer.

[0129] In some embodiments, converting Compound S5 to Compound I is performed in the presence of 0.05 mol% pentamethylcyclopentadienyl)rhodium(III) dichloride dimer.

[0130] In certain embodiments, converting Compound S5 to Compound I is performed in the presence of 0.03 mol% pentamethylcyclopentadienyl)rhodium(III) di chloride dimer. In certain embodiments, converting Compound S5 to Compound I is performed in the presence of 0.05 mol% pentamethylcyclopentadienyl)rhodium(III) di chloride dimer.

[0131] In some embodiments, converting Compound S5 to Compound I is performed in the presence of a suitable amount of (17?,27?)-(-)- V-(4-toluenesulfonyl)-l,2- diphenylethylenediamine.

[0132] In some embodiments, converting Compound S5 to Compound I is performed inthe presence of 0.12 mol% (17?,27?)-(-)-JV-(4-toluenesulfonyl)-l,2- diphenylethylenediamine.

[0133] In certain embodiments, converting Compound S5 to Compound I is performed in the presence of 0.072 mol% (17?,27?)-(-)-JV-(4-toluenesulfonyl)-l,2- diphenylethylenediamine. In certain embodiments, converting Compound S5 to Compound I is performed in the presence of 0.012 mol% (17?,27?)-(-)- V-(4- toluenesulfonyl)-l,2-diphenylethylenediamine.

[0134] In some embodiments, converting Compound S5 to Compound I is performed in a suitable solvent.

[0135] In some embodiments, converting Compound S5 to Compound S5 is performed in a polar solvent.

[0136] In some embodiments, converting Compound S5 to Compound S5 is performed in an aprotic polar solvent.

[0137] In some embodiments, converting Compound S5 to Compound I is performed in acetonitrile, dichloromethane, ethyl acetate, 2-MeTHF, or a mixture of any of the foregoing.

[0138] In some embodiments, converting Compound S5 to Compound I is performed in acetonitrile.

[0139] In some embodiments, converting Compound S5 to Compound I is performed at 5 °C. In some embodiments, converting Compound S5 to Compound I is performed at 0 °C to 5 °C.

[0140] In some embodiments, Compound I is crystallized from a suitable solvent.

[0141] In some embodiments, Compound I is crystallized from acetonitrile. In some embodiments, crystallization from acetonitrile affords a Compound I acetonitrile-solvate.

[0142] In some embodiments, Compound I is purified through crystallization from a Ci- Ce alkyl alcohol and water. In some embodiments, Compound I is purified through crystallization from ethanol and water. In some embodiments, Compound I is purified through crystallization from methanol and water. In some embodiments, crystallization from alcohol and water affords a hydrate form of Compound I.

[0143] In some embodiments, Compound I is prepared using a compound selected from

[0144] There are advantages to forming Compound I according to Scheme 1 and the embodiments described above. These advantages are even more pronounced when Compound I is manufactured on a larger scale. One or more of the present transformations are considered preferrable over the synthesis routes disclosed in PCT / US2021 / 047754 and PCT / US2022 / 041534.

[0145] The conversion of Compound S3 to S5-MsOH through the presently provided sequence exhibits advantages over previous approaches, wherein one or more steps within the sequence are preferrable over the previous ones.

[0146] Some of the previous synthesis routes involved a step that required oxygen as oxidant, which is hazardous and may limit options for manufacturing. In some embodiments, the present approach employs easy-to-handle bromination reagents and sulfonic acids and does not require a catalyst for the benzylic oxidation. In some embodiments, the target product is afforded in higher yield and with increased efficiency compared to some of the previous photocatalytic oxidation reactions.

[0147] Some of the previous synthesis routes required separate protection / deprotection steps that added to the overall step count and led to reduced overall yield and efficiency. In some embodiments, the present reactions do not require separate protection / deprotection steps, thus enabling a more efficient synthesis of the target product.

[0148] Some of the previous synthesis routes involved a complex flow system design, which made reaction setup more difficult, more expensive, and limited manufacturing options. In some embodiments, the present approach uses simpler equipment design, which facilitates reaction setup and translates to a more robust process.

[0149] Some of the previous synthesis routes involve bromination reactions that requiredthe presence of potentially thermally hazardous and difficult-to-handle radical initiator reagents (e.g, AIBN). In some embodiments, the present bromination step does not require such reagents. In some embodiments, the present bromination proceeds significantly faster and leads to higher yield than the previous photocatalytic oxidation, thus enabling higher material throughput and efficiency.

[0150] In some embodiments, the conversion of Compound S5-MsOH to Compound I exhibits advantages over previous approaches. For example, no protecting group is required to enable reduction, so no separate deprotection step is required to furnish the free piperidine amine after reduction of the carbonyl group and the reaction is carried out on protecting group-free substrate. In some embodiments, the reaction conditions for the carbonyl group reduction step do not involve sub-zero temperatures. In some embodiments, rhodium is purged sufficiently well in the routine isolation step that no separate removal step is required, whereas some of the previous routes required a separate step for removal of residual transition metal impurities from the product.Alternative Synthesis of Compound I

[0151] In some embodiments, Compound I is prepared according to Scheme 3:

[0152] In some embodiments, Compound I is prepared according to Scheme 4:

[0153] In some embodiments, Compound I is prepared by converting CompoundS5 MsOH:to Compound I.

[0154] In some embodiments, Compound I is prepared by converting Compound S5:Compound I.

[0155] In some embodiments, converting Compound S5 or S5-MsOH to Compound I is performed at a pH<7.

[0156] In some embodiments, converting Compound S5 or S5-MsOH to Compound I is performed at a pH of ~5.16.

[0157] In some embodiments, converting Compound S5 or S5-MsOH to Compound I is performed in the presence of a ketoreductase and a suitable reducing agent.

[0158] In some embodiments, converting Compound S5 or S5-MsOH to Compound I is performed in the presence of CRED-33, GDH-102, and NADP.

[0159] In some embodiments, converting Compound S5 or S5-MsOH to Compound I is performed in the presence of CRED-A231, GDH-102, and NADP.

[0160] In some embodiments, converting Compound S5 or S5-MsOH to Compound I is performed in a suitable solvent.

[0161] In some embodiments, converting Compound S5 or S5-MsOH to Compound I is performed in a polar solvent.

[0162] In some embodiments, converting Compound S5 or S5-MsOH to Compound I is performed in an aprotic polar solvent.

[0163] In some embodiments, converting Compound S5 or S5-MsOH to Compound I is performed in isopropylacetate, ethyl acetate, or a mixture of the foregoing.

[0164] In some embodiments, converting Compound S5 or S5-MsOH to Compound I is performed in isopropyl acetate. In some embodiments, converting Compound S5 orS5-MsOH to Compound I is performed in ethyl acetate.

[0165] In some embodiments, converting Compound S5 or S5-MsOH to Compound I is performed at about 30 °C.

[0166] Some of the previous synthesis routes required separate protection / deprotection steps that added to the overall step count and led to reduced overall yield and efficiency. The present route does not exhibit these drawbacks, thus enabling a more efficient synthesis of the target product.

[0167] In some embodiments, the conversion of Compound S5 or S5-MsOH to Compound I under the above-recited conditions exhibits advantages over previous approaches. In some embodiments, the present reaction does not require cooling of the reaction mixture and is amenable to producing Compound I on an increased scale without reduction in reaction efficiency or yield. In some embodiments, no separate deprotecting step is required to furnish the free piperidine amine after reduction of the carbonyl group. In some embodiments, the approach does not require a transition metal catalyst such as a rhodium catalyst, which, when used, can sometimes necessitate subsequent removal of toxic residual metal from the target product.Alternative Synthesis of Compound S4

[0168] In some embodiments, Compound S5-MsOH is prepared according to Scheme 5:

[0169] In some embodiments, the conversion of Compound S3 to Compound S4 entails mixing all starting materials and reagents into a single stock solution prior to irradiation.

[0170] In some embodiments, the conversion of Compound S3 to Compound S4 entails preparation of separate starting material and reagent solutions.

[0171] In some embodiments, a separate solution comprising Compound S3 is prepared. In some embodiments, a separate solution comprising benzenesulfonic acid is prepared. In some embodiments, a separate solution comprising NBS is prepared. In some embodiments, a separate solution comprising 1,3-dimethoxybenzene is prepared. In some embodiments, each of the solutions is prepared with acetonitrile as the solvent. In some embodiments, each of the solutions is protected from light and held at 20 °C toimprove stability of one or more of Compound S3, benzenesulfonic acid, NBS, and 1,3- dimethoxybenzene.

[0172] In some embodiments, the solutions each containing one of Compound S3, benzenesulfonic acid, NBS, and 1,3 -dimethoxy benzene are combined according to Fig. 1

[0173] In some embodiments, the solution containing Compound S3 is combined with the solution containing benzenesulfonic acid in a feed line. In some embodiments, the combined solution of Compound S3 and benzenesulfonic acid is further combined with the solution containing NBS. In some embodiments, the combined solution containing Compound S3, benzenesulfonic acid, and NBS is subjected to photoreaction in a 10 mL coil by irradiation with 450 nm light, and a mean residence time of 18 sec. In some embodiments, after the photoreaction, the solution containing Compound S3, benzenesulfonic acid, NBS, and Compound S4 is subjected to quenching by combination with a solution containing 1,3 -dimethoxy benzene. In some embodiments, the solution containing Compound S3, benzenesulfonic acid, NBS, Compound S4, and 1,3- dimethoxybenzene is collected in a collection vessel.

[0174] In some embodiments, the conversion of Compound S3 to Compound S4 by mixing of each of the starting materials and reagents in feed lines as described above reduces the decomposition of one or more of Compound S3, benzenesulfonic acid, NBS, and 1,3-dimethoxybenzene. In some embodiments, the mixing of each of the starting materials and reagents provided by separate feed lines as described above leads to improved stability of Compound S3 and NBS.Purification of Compound S5-MsOH

[0175] In some embodiments, Compound S5-MsOH is used as obtained through the reaction described herein without additional purification. In some embodiments, Compound S5-MsOH further purified prior to the next reaction step. In some embodiments, it is preferrable to subject Compound S5-MsOH to an additional purification step to improve downstream product purity.

[0176] In some embodiments, the photobromination oxidation sequence (Compound S3 —> Compound S4 —> Compound S5) results in impurities that are difficult to remove and may affect downstream processing. By implanting an aqueous wash with a combination of APS (ammonium persulfate) and NAC (N-acetylcysteine) (or one of the combinations provided in paragraph

[0103] above) during the work up, the presence of these impuritiescan be significantly reduced. Without being bound by theory, it is believed that APS and NAC (or one of the combinations provided in paragraph

[0103] above) selectively react with the by-products and result in compounds more amenable to removal by various purification methods as compared with the unreacted impurities.

[0177] In some embodiments, 0.05 equiv of APS is added. In some embodiments, 0.1 equiv of APS is added. In some embodiments, the treatment with 0.05 equiv of APS results in <0.1 area% of the undesired byproduct in the isolated Compound S5-MsOH.Crystallization of Compound I

[0178] In some embodiments, Compound I can be further purified by crystallization. In some embodiments, Compound I is crystallized from a mixture of water and methanol. In some embodiments, Compound I is crystallized from a mixture of water and ethanol. In some embodiments, the crystallization from methanol / water is preferrable over the crystallization from ethanol / water for impurity purging.Examples

[0179] In order that the disclosure described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this disclosure in any manner.

[0180] Methods of preparation, along with the structure of and physicochemical data, for Compound I are reported in International Application No. PCT / US2021 / 047754, filed on August 26, 2021, and in International Application No. PCT / US2022 / 041534, filed on August 25, 2022, the contents of which are incorporated herein by reference.

[0181] The compounds of the disclosure may be made according to standard chemical practices or as described herein.

[0182] One of ordinary skill in the art will appreciate that the methods described herein are generally applicable to other stereoisomers of the compounds described herein.

[0183] Should the name of a compound conflict with the structure of the compound anywhere in the present application, the structure supersedes the name and is intended to be controlling.

[0184] Throughout the following synthetic schemes and in the descriptions for preparing compounds, the following abbreviations are used:AbbreviationsACN or MeCN acetonitrileAcOH acetic acidAP area percentAPS ammonium persulfateBSA benzenesulfonic acidBPR back pressure regulatorCRED carbonyl reductaseDCM dichloromethaneDIPEA AJV-diisopropylethylamineDMAP A-dimethylaminopyridineDMB 1,3 -dimethoxy benzeneDMF dimethylformamideDMSO dimethyl sulfoxideDSC differential scanning calorimetry e.r. enantiomeric ratioEtOAc ethyl acetateEtOH ethanolGC gas chromatographyGDH glucose dehydrogenase h hoursHPLC high-performance liquid chromatographyIPA isopropyl alcohol / -PrOAc isopropyl acetateKRED ketoreductaseLCMS liquid chromatography mass spectrometryLDA lithium diisopropyl amideLED light-emitting diodeMeOH methanol min minutesMsOH methanesulfonic acidMTBE methyl tert-butyl etherNAC N-acetyl-L-cysteineNADP nicotinamide adenine dinucleotide phosphate NaHCCE sodium bicarbonateNaOH sodium hydroxideNBS N-bromosuccinimideNMP A-methyl pyrrolidinePTSA -toluenesul Tonic acid monohydrateRPM revolutions per minute sec secondsTEA triethylamineTFA trifluoroacetic acidTGA thermogravimetric analysisTHF tetrahydrofuranTLC thin layer chromatography(7?. / ?)-TsDPEN ( / . / )-N-( / Mol uenes ul Tony I)- 1.2-di phenyl ethylenedi amineExample 1: Synthesis of Compound I from S5-MsOHStep 1. Synthesis of (2S,4S,6S)-2-methyl-6-(l-methyl-lH-l,2,3-triazol-4-yl)-2'-( trifluoromethyl) -4 ',5'-dihydrospiro[piperidine-4, 7'-thieno[2, 3-c ]pyran ] (S3)

[0185] A solution of Compound SI (70 g, 0.360 mol, 1.0 equiv) and S2 (74.2 g, 0.378 mol, 1.05 equiv) in DCM (210 mL, 3 vol) was cooled to 5 °C. Methanesulfonic acid (210.6 mL, 3.24 mol, 9.0 equiv) was charged to the reactor while maintaining an internal temperature <30 °C. The resulting reaction mixture was heated to 39 °C. After 18 h, HPLC analysis indicated >99% conversion to Compound S3. The reaction mixture was cooled to 30 °C, charged with DCM (280 mL, 4 vol), and further cooled to 0 °C. The pH was adjusted to pH 10 with 4 M sodium hydroxide (830 mL). The organic layer was separated, and the aqueous phase was back extracted with DCM (350 mL, 5 vol). Thecombined organics were washed with water (350 mL, 5 vol) and concentrated at reduced pressure to 3.5 total volumes. The mixture was charged with MTBE (350 mL, 5 vol) and concentrated under reduced pressure to 3.5 total volumes. This put / take cycle was repeated three additional times and the resulting 3.5 vol mixture was diluted with MTBE (455 mL, 6.5 vol) to provide a 10 vol mixture. The slurry was heated to 50 °C, stirred for 5 h, then charged with n-heptane (700 mL, 10 vol) over 2 h. The resulting suspension was cooled to 20 °C over 5 h and stirred for 18 h. The suspension was filtered, washed with 1 :2 MTBE / n-heptane (2 x 140 mL, 2 x 2 vol) and dried under vacuum while flushing with nitrogen at 50 °C for 18 h to give 103 g of Compound S3 (77% yield). Step 2. Synthesis of (2S,4S,6S)-4'-bromo-2-methyl-6-(l-methyl-lH-l,2,3-triazol-4-yl)-2'- ( trifluoromethyl) -4 'f '-dihydrospiro [piperidine-4, 7'-thieno[2, 3-c ]pyran ] (S4)

[0186] To an appropriately sized amber flask was added S3 (5 g, 1.0 equiv) followed by acetonitrile (75 mL, 15 vol). Benzenesulfonic acid (4.25 g, 2.0 equiv) was added followed by N-bromosuccinimide (1.31 g, 2.63 equiv). The mixture was agitated until solids were fully dissolved. The Vapourtec flow system (E-Series equipped with UV-150 photochemical reactor) was prepared by adjusting according to the parameters described in Table 1.Table 1. Settings and Parameters for Vapourtec Flow System

[0187] Acetonitrile was pumped though the reactor to condition the lines. Feed lines were then switched from solvent to reaction solution. Post reaction stream was collected at the outlet. The combined solution of S4 was charged to a suitably sized vessel and cooled to 0-5 °C. In a separate container, a quench solution of 1,3-dimethoxybenzene (93mg, 0.05 eq) and 2,6-lutidine (72 mg, 0.05 eq) in acetonitrile (1 mL, 0.1 vol) was prepared and mixed to ensure full dissolution of the components. The quench solution was then added to the product solution while keeping the internal temperature below 15 °C. After holding at 0-5 °C for 15 min the solution was warmed to 20 °C and held at that temperature for 30 min. This quenched solution of S4 was used as-is in Step 3. Step 3. Synthesis of (2S,4S,6S)-2-methyl-6-(l-methyl-lH-l,2,3-triazol-4-yl)-2'- (trifluoromethyl)spiro[piperidine-4, 7'-thieno[2,3-c]pyran]-4'(5'H)-one (S5)

[0188] The quenched solution of S4 from Step 2 was treated with a solution of 2-picoline N-oxide (1.5 eq, 2.198 g, 20.14 mmol) in acetonitrile (0.2 vol, 1 mL). After stirring for 5 min at 20 °C under nitrogen, triethylamine (4.0 eq, 7.49 mL, 53.70 mmol) was added over 10 min. The resulting reaction solution was heated to 60 °C and stirred overnight. The reaction solution was cooled to 20 °C, diluted with water (3 vol, 15 mL), and concentrated to 5 total volumes. The resulting solution was diluted with 0.5 M aqueous sodium carbonate solution (5 vol, 25 mL) and isopropyl acetate (5 vol, 25 mL). The phases were mixed, settled, and separated. The aqueous phase was back-extracted with isopropyl acetate (5 vol, 25 mL). The organic phases were combined and concentrated to 5 total volumes. Isopropanol (10 vol, 50 mL) was added, and the solution was concentrated to 5 total volumes. Isopropanol (5 vol, 25 mL) was added, and the solution was again concentrated to 4 total volumes.

[0189] The 4-volume solution of S5 in isopropanol was heated to 75 °C and methanesulfonic acid (1.0 eq, 0.87 mL, 13.43 mmol) was added at that temperature over 2 h during which time a slurry formed. The slurry was stirred an additional 2 h at 75 °C then cooled to 20 °C over 4 h and stirred at that temperature for 17 h. The slurry was filtered under vacuum and washed with isopropanol (3 x 1 vol, 3 x 5 mL). The solids were rinsed once more with isopropanol (1 vol, 5 mL) and dried further under vacuum. S5 MsOH was isolated in 63% yield.Step 4. Synthesis of (2S,4S,4'S,6S)-2-methyl-6-(l-methyl-lH-l,2,3-triazol-4-yl)-2'- ( trifluoromethyl) -4 'f '-dihydrospiro [piperidine-4, 7'-thieno[2, 3-c ]pyran ]-4 '-ol (Compound I)

[0190] A flask was charged with (pentamethylcyclopentadienyl)rhodium(III) dichloride dimer (19.2 mg, 0.0311 mmol, 0.0025 equiv), and (17?,27?)-(-)-A-(4-toluenesulfonyl)-l,2- diphenylethylenediamine (27.3 mg, 0.0746 mmol, 0.0060 equiv). The head space was flushed with nitrogen for 5 min. Acetonitrile (7.2 mL, 0.24 vol) was added and themixture was agitated for 1 h at 22 °C (“catalyst solution”). To a separate flask was charged acetonitrile (16.8 mL. 0.56 vol) and triethylamine (10.4 mL, 7.55 g, 74.6 mmol,1.2 equiv). The solution of triethylamine was cooled to 5 °C, charged with formic acid (2.58 mL, 3.15 g, 68.4 mmol, 1.1 equiv) over 5 min, then warmed to 22 °C. To a separate, jacketed reactor, was added S5-MsOH (30.0 g, 62.2 mmol, 1 equiv, containing S5-MsOH (24.0 g)) followed by acetonitrile (96 mL, 3.2 vol) and the mixture was stirred at 20 °C. The resulting thick suspension was treated with triethylamine (8.66 mL, 6.29 g,62.2 mmol, 1.0 equiv) then cooled to 5 °C over 30 min. The prepared catalyst solution was charged to the reactor over 10 min. After 10 min of incubation, the pre-mixed triethylamine / formic acid solution was charged to the reactor over 3 h. The resulting thin suspension was stirred at 5 °C for additional 19 h. Once the reaction reached >99.8% conversion by HPLC analysis, 1 M sodium hydroxide (75 mL, 1.2 equiv, 2.5 vol) was added over 10 min at 5 °C. The solution was warmed to 20 °C over 30 min and charged with methyl tert-butyl ether (190 mL, 6.4 vol). The mixture was agitated for 0.5 h at20 °C. Stirring was stopped and the phases were allowed to settle for 0.5 h. The aqueous layer was removed, and the organic phase was charged with water (75 mL, 2.5 vol). The mixture was agitated for 0.5 h at 20 °C. Stirring was stopped and phases were allowed to settle for 0.5 h. The aqueous layer was removed, and the organic phase was concentrated under reduced pressure at 40 °C to 2.4 total volumes. Acetonitrile (120 mL, 4 vol) was added, and the mixture was again concentrated at 40 °C to 2.4 total volumes. This put / take cycle was repeated four additional times. The resulting solution was heated to 58 °C and held at that temperature for 30 min. The solution was cooled to 52 °C over 15 min. Seeds crystals (60 mg, 0.25 wt%) were charged and the resulting thin suspension was held at 52 °C for 1 h, cooled to 20 °C over 3 h and held at 20 °C for 11 h. The slurry was filtered, and the solids were washed with acetonitrile (4 x 12 mL, 2 vol). The solids were dried under vacuum for 30 min to give Compound I (19.23 g) in 73.5% yield and 99.7% AUC purity as off-white solid.

[0191] An optional crystallization was applied by adding the isolated Compound I (16.0 g) to a 100 mL reactor followed by acetonitrile (40 mL). The suspension was heated to 61 °C and held at that temperature for 30 min. The solution was cooled to 55 °C over 15 min. Seed crystals (80 mg, 0.5 wt% Compound I) were charged and the thin suspension was held at 55 °C for 1 h, cooled to 20 °C over 5 h and held at 20 °C for 13 h. The slurry was filtered, and the solid was washed with acetonitrile (2 x 8 mL, 1 vol). The solid wasdried under vacuum for 30 min to give Compound I (14.6 g) in 90.5% yield and >99.9% AUC purity as off-white solid.

[0192] Seed Preparation Procedure for (2S,4S,4'S,6S)-2-methyl-6-(l-methyl-lH-l,2,3- triazol-4-yl)-2'-(trifluoromethyl)-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]- 4'-ol (Compound I)

[0193] Compound S5-MsOH (1.00 g, 2.02 mmol) was placed in a 50 mL round bottom flask with a magnetic stir bar and diluted with acetonitrile (3.4 mL, 46 mmol) to give a slurry. The flask was placed in an ice bath under nitrogen. Triethylamine (0.2822 mL, 2.025 mmol) was added, creating a nearly clear solution, which was stirred for 10 min. A prepared stock solution of the catalyst and ligand was added to deliver the pentamethylcyclopentadienylrhodium(III)chloride dimer (0.0006258 g, 0.001012 mmol) and (R,R)-TsDPEN (0.0008905 g, 0.002430 mmol) in acetonitrile (0.24 mL). The reaction solution was stirred for several minutes. A prepared stock solution of formic acid (0.1025 g, 2.227 mmol) and triethylamine (0.2459 g, 2.430 mmol) in acetonitrile (0.56 mL) was added over 3 h. After stirring for 16 h, the reaction was deemed complete. The reaction mixture was cooled on an ice bath and charged with 1 M NaOH (2.4 vol) and MTBE (6.4 vol). After mixing for several minutes, the phases were split and separated. The organic phase was further washed with water (2.4 vol). The aqueous phase was removed, and the organic phase was concentrated under reduced pressure to 2.4 volumes. Acetonitrile (4 vol) was charged, and the solution was concentrated under reduced pressure to 2.4 volumes. This put / take cycle was repeated three more times to give a 2.4 volume solution. A stir bar was added, inducing crystallization, and the resulting slurry was heated to 58 °C. After holding for 1 h at 58 °C, the slurry was cooled to 20 °C over 3 h and stirred at that temperature overnight. The slurry was filtered under vacuum and the resulting solid was dried to provide Compound I in approximately Tl% yield.Example 2: Synthesis of Compound I from S5

[0194] S5 (1.00 g) in acetonitrile (4 vol) was treated with pre-mixed (lR,2R)-(-)-N-(4- toluenesulfonyl)-l,2-diphenylethylenediamine (0.0012 equiv) and pentamethyl cyclopentadienylrhodium(III) chloride dimer (0.0005 equiv) in acetonitrile (0.3 vol) at 20 °C. The reaction was cooled 5 °C and treated with a pre-mixed solution of triethylamine (1.2 equiv) and formic acid (1.1 equiv) in acetonitrile (anhydrous, 0.7 vol) over 3 h. Compound I seeds (5 mg) were added to form a suspension. The suspension was held at 5 °C for 18 h to give Compound I (>99.9% conversion). The suspension was heated to 65 °C to give a solution and concentrated to 3.5 volumes. Acetonitrile (3 vol) was added, and the solution was concentrated under reduced pressure to 3.5 volumes. The solution was cooled to 55 °C and Compound I seeds (5 mg) were added. The reaction was held at 55 °C for 1 h, cooled to 20 °C over 3 h and held at 20 °C for 18 h. The resulting solid was filtered, rinsed with acetonitrile (2 x 0.5 vol) and dried to give 0.94 g of Compound I (84% yield).Example 3: Alternative Synthesis of Compound I from S5-MsOHSynthesis of (2S, 4S,4'S, 6S) -2-methyl-6-( 1 -methyl- 1H-1, 2, 3-triazol-4-yl)-2'- (trifluoromethyl)-4',5'-dihydrospiro[piperidine-4, 7'-thieno[2,3-c]pyran]-4'-ol (Compound I)

[0195] 0.1 M, pH 7 potassium phosphate buffer (240 mL) was added to the reactor and stirred at 500 rpm (overhead stirring with a pitched blade impeller) followed by the addition of glucose monohydrate (15.37 g, 1.2 eq). A clear colorless solution was obtained. A temperature probe was inserted, and the reactor contents were warmed (target 30 °C). S5-MsOH (31.22 g) was added to the reactor, dissolving rapidly to give an orange solution. Isopropylacetate (250 mL) was added and calibrated pH probe was inserted and attached to a pH-stat. Once the reactor contents settled at 30 °C, the pH was adjusted to 7.0 by addition of 20% K2CO3 solution via pH-stat (total added = ~33 mL). CRED-33 (250 mg, 1% w / w), GDH-102 (125 mg, 0.5% w / w) and NADP (125 mg, 0.5%) were dissolved in 0.1 M, pH 7 potassium phosphate buffer (7.5 mL) in a COC vial and added to the reactor to initiate the reaction. The COC vial was then rinsed withadditional 0.1 M, pH 7 potassium phosphate buffer (2.5 mL) and added to the reactor. The reaction was stirred at 500 rpm and maintained at pH 7.0 by pH-stat controlled addition of 20% K2CO3 solution. The reaction was sampled periodically for HPLC analysis, diluted to an appropriate concentration with acetonitrile, centrifuged to sediment precipitated solids, and the supernatant analyzed. After 70 min, the reaction had reached 99.7% conversion.

[0196] A volume of 20% K2CO3 solution was added during the reaction = ~20 mL. EtOAc (250 mL) was added to the completed reaction mixture and stirring was continued (at a reduced speed of 350 rpm) for 8 min then allowed to settle for 25 min. This gave a lower clear pale yellow aqueous layer and an upper orange emulsion. After removal of the aqueous layer, the organic / emulsion layer was stirred again for 30 sec then allowed to settle overnight. The aqueous phase was removed, and the rag layer was kept with the organic phase. The combined aqueous phase was added back to the reactor along with EtOAc (250 mL) and stirred at 350 rpm for 15 min then allowed to settle. This separated into a lower pale yellow aqueous layer and an upper very pale-yellow organic layer with some emulsion present. The layers were separated, and any emulsion was kept with the organic layer. After a third extraction of the aqueous layer with EtOAc (250 mL), the upper organic layer was colorless with minimal emulsion present. The layers were separated. All organic / emulsion layers were combined in the reactor along with 20% brine (250 mL) and stirred at 600 rpm for 5 min, then allowed to settle. This gave a lower colorless aqueous layer and an upper orange organic layer with some rag present at the interface. The brine layer was removed, and the organic and rag layer was filtered through a pad of Celite (~1 cm depth, pre-wetted with EtOAc). This removed all remaining rag / emulsion. A small amount of separated aqueous was removed from the filtrate via pipette, then the product solution was dried (MgSO4), filtered and concentrated in vacuo to afford Compound I as a solid (24.27 g) in 90% yield.Example 4: Alternative Synthesis I of Compound I from S5Synthesis of (2S, 4S,4'S, 6S) -2-methyl-6-( 1 -methyl- 1H-1, 2, 3-triazol-4-yl)-2'- (trifluoromethyl)-4',5'-dihydrospiro[piperidine-4, 7'-thieno[2,3-c]pyran]-4'-ol (Compound I)

[0197] Glucose monohydrate (769 mg, 1.2 eq) was measured into a 50 mL 3-neck round bottom flask. 0.1 M, pH 7 potassium phosphate buffer buffer (10 mL) was added, and the resulting mixture was stirred in an oil bath at 30 °C to provide a homogeneous solution. A calibrated pH probe was introduced and connected to a pH-stat. Compound S5 (1.25 g) was dissolved in isopropyl acetate (12.5 mL) and added. CRED-33 (12.5 mg, 1% w / w), NADP (6.25 mg, 0.5% w / w) and glucose dehydrogenase, GDH-102 (6.25 mg, 0.5% w / w) were dissolved in 0.1 M, pH 7 potassium phosphate buffer (2.5 mL) and added to initiate the reaction. The mixture was stirred. The pH was maintained at 7 by pH stat controlled addition of 20% K2CO3 solution. After 1 h, HPLC analysis indicated the reaction had achieved >99% conversion. EtOAc (25 mL) was added to the mixture. After stirring for several minutes, the mixture was filtered through a pad of celite. The reaction flask was rinsed with EtOAc (25 mL), which was then used to rinse the celite pad. The layers were separated, and the aqueous phase was extracted twice more with EtOAc (25 mL x 2). The combined organics were dried over MgSO4, filtered, and concentrated in vacuo to afford Compound I as a white solid in 96% yield.Example 5: Alternative Synthesis II of Compound I from S5-MsOHSynthesis of (2S, 4S,4'S, 6S) -2-methyl-6-( 1 -methyl- 1H-1, 2, 3-triazol-4-yl)-2'- (trifluoromethyl)-4',5'-dihydrospiro[piperidine-4, 7'-thieno[2,3-c]pyran]-4'-ol (Compound I)

[0198] Compound S5-MsOH (96.4% qNMR potency, containing ~0.6 wt% isopropanol, 3.00 g, 5.99 mmol) and D-(+)-glucose monohydrate (1.54 g, 7.79 mmol, 1.3 eq) were placed in a 250 mL three-neck round bottom flask fitted with a mechanical stirrer. To the solids was added 1 M potassium phosphate buffer (30 mL, 10 volumes) under stirring to give a hazy mixture. (1 M potassium phosphate buffer was prepared by dissolvingpotassium phosphate dibasic (K2HPO4, 10.79 g, 61.9 mmol) and potassium phosphate monobasic (KH2PO4, 5.23 g, 38.4 mmol) in water and diluting to 100 mL).

[0199] Ethyl acetate (24 mL, 8 volumes) was added to give a clear, biphasic solution. Once a clear solution formed, an aqueous solution of K2CO3 (20% in water, 2.1 mL, 0.7 volumes) was poured into the reaction solution and the biphasic solution was stirred for 30 min to 60 min at 30 °C with a nitrogen sweep of the reaction headspace.

[0200] While the biphasic mixture was stirring, CRED-33 (0.0386 g), GDH-102 (0.0201 g), and NADP+ (beta-nicotinamide adenine dinucleotide phosphate sodium salt (95%, 0.0190 g, 0.026 mmol) were combined in a vial, diluted with 1 M potassium phosphate buffer (1 mL, 0.33 volumes), agitated to give a more homogeneous mixture, and then added to the stirring biphasic reaction solution at 30 °C. 1 M potassium phosphate buffer (0.8 mL, 0.27 volumes) and (0.6 mL, 0.2 volumes) (0.8 volumes of buffer total to transfer the enzyme reagents to the reaction) was used to rinse and transfer the reagent solution to the reaction. The resultant reaction mixture was stirred at 30 °C for 1 h to 3 h, until <1 area percent (AP) Compound S5 remained.

[0201] The temperature of the reaction mixture was maintained at 30 °C, and an aqueous solution of K2CO3 solution (20% in water, 9 mL, 3 volumes) was added to adjust the pH of the aqueous phase to pH 8.5 to 9. Additional K2CO3 solution may be added as needed to achieve the target pH value. The biphasic solution was stirred for 15 min to 30 min and then allowed to settle for 15 min, and the lower aqueous phase was removed. The 30 °C organic solution / emulsion / residual aqueous phase was filtered through a prewetted Celite frit (Celite 545, 3 g, 100 wt% of Compound S5-MsOH starting material) with EtOAc under reduced pressure. The reaction flask was rinsed at 30 °C with a mixture of EtOAc (24 mL, 8 volumes) and water (1.5 mL, 0.5 volumes) under stirring to remove the gel-like residue and then filtered through the Celite frit under reduced pressure.

[0202] The filtrate was concentrated to a ~5 volume solution (~15 mL solution total) at 30 °C. Methanol (21 mL, 7 volumes) was added, and the mixture was concentrated to a ~5 volume solution (~15 mL). Methanol (21 mL, 7 volumes) was added again, and the mixture was concentrated to a ~5 volume solution (~15 mL). The ~5 volume methanol mixture was diluted with water (3 mL, 1 volume) and heated to 50 °C to form a clear solution. Water (4.5 mL, 1.5 volumes) was added over 1 h, and the solution was seeded with 0.5-1 wt% Compound LH2O seeds (obtained from crystallization from methanoland water or ethanol and water as described above) and held for 1 h resulting in a suspension. Water (10.5 mL, 3.5 volumes) was added over 1 h. The slurry was cooled to ambient temperature over 3 h and stirred overnight. The slurry was filtered, and the flask and solids were rinsed with a methanol / water mixture (3 mL total, 1 volume; 1 mL methanol : 2 mL water). The solids were dried at ambient temperature to provide Compound I as a white solid in 85-88% yield (potency corrected) and >99.8% AUC HPLC purity.Example 6: Alternative Synthesis III of Compound I from S5-MsOHSynthesis of (2S, 4S,4'S, 6S) -2-methyl-6-( 1 -methyl- 1H-1, 2, 3-triazol-4-yl)-2'- (trifluoromethyl)-4',5'-dihydrospiro[piperidine-4, 7'-thieno[2,3-c]pyran]-4'-ol (Compound I)

[0203] Compound S5-MsOH (94.5% qNMR potency, containing ~0.8 wt% isopropanol, 1.00 g, 1.96 mmol) and D-(+)-glucose monohydrate (0.505 g, 2.55 mmol, 1.3 eq) were placed in a 50 mL round bottom flask fitted with a magnetic stir bar. To the solids was added 1 M potassium phosphate buffer (10 mL, 10 volumes) under stirring to provide a cloudy mixture. Ethyl acetate (8 mL, 8 volumes) was added to give a clear, biphasic solution. (Note: 1 M potassium phosphate buffer was prepared by dissolving potassium phosphate dibasic (K2HPO4, 10.79 g, 61.9 mmol) and potassium phosphate monobasic (KH2PO4, 5.23 g, 38.4 mmol) in water and diluting to 100 mL).

[0204] Once a clear solution was formed, an aqueous solution of K2CO3 solution (20% in water, 0.7 mL, 0.7 volumes) was poured into the reaction solution and the biphasic solution was stirred for 30 min to 60 min at 30 °C with a nitrogen sweep of the reaction headspace.

[0205] While the biphasic mixture was stirring, CRED-33 (0.0127 g), GDH-102 (0.0062 g) and NADP+ (beta-nicotinamide adenine dinucleotide phosphate sodium salt, 95%, R595, 0.0062 g, 0.0084 mmol) were combined in vial, diluted with 1 M potassium phosphate buffer (0.3 mL, 0.3 volumes), agitated to give a more homogeneous mixture, and then added to the vigorously stirring biphasic reaction solution at 30 °C. Additional 1M potassium phosphate buffer (0.3 mL, 0.3 volumes) and (0.2 mL, 0.2 volumes) (0.8 volumes of buffer total to transfer the enzyme reagents to the reaction) was used to rinse and transfer the reagent mixture to the reaction. The resultant reaction mixture was stirred at 30 °C for 1 h to 3 h, until <1 AP Compound S5 remained.

[0206] The reaction was maintained at 30 °C and an aqueous solution of K2CO3 (20% in water, 3 mL, 3 volumes) was added to adjust the pH of the aqueous phase to pH 8.5 to 9. Additional K2CO3 solution may be added as needed to achieve the desired pH. The biphasic solution was stirred for 15 min to 30 min, and then allowed to settle for 1 h. The lower, clear aqueous phase was removed, and the 30 °C organic solution / emulsion / residual aqueous phase was filtered through a pre-wetted Celite frit (Celite 545, 1 g, 100 wt% vs Compound S5-MsOH starting material). The reaction flask was rinsed at 30 °C with a mixture of EtOAc (8 mL, 8 volumes) and water (0.5 mL, 0.5 volumes), and the resulting rinsate was passed through the Celite frit under reduced pressure.

[0207] The filtrate was concentrated to a -5 volume solution (~5 mL) at 30 °C. Ethanol (7 mL, 7 volumes) was added, and the resultant solution was concentrated to a -3 volume mixture (~3 mL) with solids present. The -3 volume ethanol mixture was heated to 50 °C and water (5 mL, 5 volumes) was added over 1 h to give a clear solution. The resultant solution was seeded with 0.5-1 wt% Compound LH2O seeds and held at that temperature for 1 h to give a slurry. Additional water (8 mL, 8 volumes; 13 volumes water total for a 1 :6.5 ratio of ethanol to water) was added over 1 h. The resultant slurry was cooled to ambient temperature over 3 h and stirred at ambient for 15 h to 16 h. The slurry was filtered under reduced pressure, and the flask and solids were rinsed with a 1:6 mixture of ethanol: water (1 mL, 1 volume). The solids were dried at ambient temperature under vacuum filtration to provide Compound I as a white solid in -84% yield (potency corrected) and 99.7 area% HPLC purity.Example 7: Alternative Synthesis of Compound S4Synthesis of (2S, 4S, 6S)-4 '-bromo-2-methyl-6-( 1 -methyl- 1H-1, 2, 3-triazol-4-yl)-2 '-( trifluoromethyl) -4 ',5'-dihydrospiro[piperidine-4, 7'-thieno[2, 3-c ]pyran ] (S4)

[0208] A Vapourtec E-Series flow system equipped with an UV-150 photochemical reactor and two additional pumps as shown in Fig. 1 was set up according to the parameters described in Table 2. Light source, temperature settings and flow rates were checked and confirmed, and, if necessary, a pump recalibration was performed.Table 2. Settings and Parameters for Vapourtec Flow System

[0209] Acetonitrile was pumped through the reactor to condition the lines. To an appropriately sized container was charged Compound S3 (500 g, 1 equiv) followed by acetonitrile (4 L, 8 vol), and the mixture was agitated until full dissolution was achieved. The so-obtained solution (“Solution 1”) was held at 20 °C and protected from light until further use. To an appropriately sized container was charged benzenesulfonic acid (425.9 g, 1.9 eq (corrected for purity)) followed by acetonitrile (0.5 L, 1 vol), and the mixture was agitated until full dissolution was achieved. The so-obtained solution (“Solution 2”) was held at 20 °C and protected from light until further use. To an appropriately sized container was charged N-bromosuccinimide (263.6 g, 1.1 eq) followed by acetonitrile (2 L, 4 vol), and the mixture was agitated until full dissolution was achieved. The so-obtained solution (“Solution 3”) was held at 20 °C and protected from light until further use. To an appropriately sized container was charged 1, 3- dimethoxybenzene (9.3 g, 0.05 eq) followed by acetonitrile (0.1 L, 0.2 vol) and the mixture was agitated until full dissolution was achieved. The so-obtained solution (“Solution 4”) was held at 20 °C and protected from light until further use. The flow system was equipped with Solutions 1-4 as indicated in Fig. 1. Then, the feed lines were switched from solvent to reaction solutions, and the solutions were collected at the outlet in fractions. The fractions were analyzed by HPLC and a conversion of >95% wasconfirmed for each fraction. The fractions were combined in an appropriately sized vessel. The so-obtained quenched solution of Compound S4 was used as-is in Step 3, and the Vapourtec system was flushed with acetonitrile, followed by water, and then 2- propanol for storage until further use.Example 8: Alternative Synthesis of Compound S5-MsOHStep 1. Synthesis of (2S,4S,6S)-4'-bromo-2-methyl-6-(l-methyl-lH-l,2,3-triazol-4-yl)-2'- ( trifluoromethyl) -4 'f '-dihydrospiro [piperidine-4, 7'-thieno[2, 3-c ]pyran ] (S4)

[0210] Five bromination reactions were performed, and the resulting solutions combined for the subsequent reaction. For each bromination reaction, Compound S3 (1.00 g, 2.68 mmol) and benzenesulfonic acid (2 equiv, 0.85 g, 5.37 mmol) were combined in a 100 mL round bottom flask equipped with a magnetic stir bar and diluted with acetonitrile (14 volumes, 14.0 mL). N-bromosuccinimide (1.1 equiv, 0.526 g, 2.95 mmol) was added in one bolus as a solid. Acetonitrile (1 volume, 1 mL) was used to rinse the NBS in the flask. The reaction was placed under nitrogen, the blue LED lights were turned on, and the reaction solution was stirred vigorously for 1 h. Thereafter, the reaction was quenched through addition of a solution of 1,3 -dimethoxy benzene (0.1 eq, 0.037 g, 0.27 mmol) in acetonitrile (0.25 volumes, 0.25 mL) while maintaining the internal temperature at <25 °C. The resultant solution of Compound S4 was stirred at ambient temperature for 1 h.Step 2. Synthesis of (2S,4S,6S)-2-methyl-6-(l-methyl-lH-l,2,3-triazol-4-yl)-2'- (trifluoromethyl)spiro[piperidine-4, 7'-thieno[2,3-c]pyran]-4'(5'H)-one (S5)

[0211] The five 1 g quenched bromination reaction solutions containing Compound S4 were combined in a 3-neck, 250 mL round bottom flask fitted with a reflux condenserand internal temperature probe and placed under nitrogen. To the quenched bromination reaction was added a pre-mixed solution of 2-methylpyridine V-oxide (1.5 eq, 2.2 g, 20.1 mmol) in acetonitrile (0.5 volumes, 2.5 mL) via syringe over 1-2 min. Tri ethylamine (4.0 eq, 7.5 mL, 53.7 mmol) was then added to the reaction via syringe over 1-2 min. The resultant reaction solution was heated to 60 °C, stirred for 16 h at this temperature, and then cooled to ambient temperature. To the ambient temperature reaction solution, water (3 volumes, 15 mL) was added, and the resultant solution was concentrated to 5 to 6 volumes (30-36 mL). Isopropyl acetate (5 volumes, 25 mL) and 0.5 M sodium carbonate solution (5 volumes, 25 mL) were added, and the phases were mixed, settled, and the aqueous phase removed.

[0212] The aqueous phase was extracted with isopropyl acetate (5 volumes, 25 mL) and the two isopropyl acetate phases were combined and concentrated to a ~6 volume solution. The ~6 volume isopropyl acetate solution containing Compound S5 was transferred to a three-neck 250 mL round bottom flask fitted with a magnetic stirrer, internal temperature probe, and nitrogen inlet / outlet. A fresh solution of N-acetyl-L- cysteine (1.0 eq, 2.19 g, 13.4 mmol) in water (4 volumes, 20.0 mL, 1110 mmol) was prepared separately and stirred at ambient temperature until full dissolution was achieved, and then added to the reaction solution via syringe over <5 min. A fresh solution of ammonium persulfate (0.05 eq, 0.153 g, 0.67 mmol) in water (0.25 volumes, 1.25 mL, 69.4 mmol) was separately prepared at ambient temperature and stirred until full dissolution was achieved. The resultant solution was added to the reaction over <5 min via syringe. Water (0.25 volumes, 1.25 mL, 69.4 mmol) was used as a rinse to add all the APS solution to the reaction. The resultant biphasic reaction solution was stirred vigorously at ambient temperature. If required, the reaction may be re-dosed after 3-6 h with another freshly pre-mixed solution of ammonium persulfate (0.05 eq, 0.153 g, 0.67 mmol) in water (0.25 volumes, 1.25 mL, 69.4 mmol) and water (0.25 volumes, 1.25 mL, 69.4 mmol) to rinse. The reaction was allowed to stir overnight at ambient temperature.

[0213] To the reaction solution stirring at ambient temperature was added 1 M sodium carbonate solution (4 volumes, 20 mL) over 30 min. The phases were mixed, settled, and the aqueous phase was removed. To the remaining organic phase was added water (3 volumes, 15 mL) and isopropanol (2 volumes, 10 mL). The phases were mixed, settled, and the lower aqueous phase removed. The resultant phase containing CompoundS5, isopropanol, and iPrOAc was concentrated to a ~5-6 volume solution. The solvent was swapped to isopropanol by three separate additions of isopropanol (7 volumes, 35 mL) followed by concentration to a ~5 volume solution after each one. A ~5 volume (~25 mL) solution in isopropanol was targeted at the end.

[0214] The ~5 volume solution of Compound S5 in isopropanol was placed in a 3-neck 250 mL round bottom flask fitted with a mechanical stirrer, reflux condenser, and internal temperature probe and placed under nitrogen. The solution was heated to 70 °C and held at that temperature for ~30 minutes until a clear solution formed.Methanesulfonic acid (1.0 eq, 0.87 mL, 13.4 mmol) was added over 30 min, forming a slurry. Upon complete addition, the slurry was held at 70 °C for 1 h. If no slurry had formed, the solution would have been seeded at this point and held for an additional 1 h at that temperature. The slurry was then cooled to ambient temperature over 5 h and stirred overnight (—15 hours) at ambient temperature. The slurry was filtered under reduced pressure through a coarse glass frit. Isopropanol (4 x 5 mL) was used to rinse the flask and solids. The material was dried under vacuum filtration and subsequently placed in a 50 °C vacuum oven overnight, which provided 4.4 g of Compound S5-MsOH as an off-white solid and with a potency adjusted yield of 61%.

[0215] All publications and patents referred to in this disclosure are incorporated herein by reference in their entirety to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference in their entirety. Should the meaning of the terms in any of the patents or publications incorporated by reference conflict with the means of the terms used in this disclosure, the meaning of the terms defined in this disclosure is intended to be controlling.

[0216] The foregoing discussion discloses and describes merely exemplary embodiments of this disclosure. One skilled in the art will readily recognize, from such discussion and from the accompanying claims, that various changes, modifications, and variations can be made therein without departing from the spirit and scope of this disclosure as defined in the following claims.

Claims

CLAIMS1. A method for preparing Compound I:comprising:(a) Converting Compound S4:into Compound S5:(b) converting Compound S5 into Compound I.A method for preparing Compound I:comprising:(a) converting Compound S4:into Compound S5:(b) converting Compound S5 into Compound S5-MsOH:(c) converting Compound S5-MsOH into Compound I.A method for preparing Compound I:comprising:(a) converting Compound S3:into Compound S4:(b) converting Compound S4 into Compound S5:(c) converting Compound S5 into Compound I.A method for preparing Compound I:comprising:(a) converting Compound S3:into Compound S4:(b) converting Compound S4 into Compound S5:(c) converting Compound S4 into Compound S5-MsOH:(d) converting Compound S5-MsOH into Compound I.

5. The method of claims 3 or 4, wherein Compound S3:is obtained through reaction of Compound SI:with Compound S2:

6. The method of claims 1 or 3, wherein converting Compound S5 into Compound I is performed in the presence of at least one catalyst, a hydrogen donor agent, and optionally one or more of a base and a ligand.

7. The method of claims 2 or 4, wherein converting Compound S5-MsOH into Compound I is performed in the presence of at least one catalyst, a hydrogen donor agent, and optionally one or more of a base and a ligand.

8. A method for preparing Compound I:comprising converting Compound S5:into Compound I, wherein converting Compound S5 into Compound I is performed in the presence of at least one catalyst, a hydrogen donor agent, and optionally one or more of a base and a ligand.

9. A method for preparing Compound I:comprising converting Compound S5-MsOH:into Compound I, wherein converting Compound S5-MsOH into Compound I is performed in the presence of at least one catalyst, a hydrogen donor agent, and optionally one or more of a base and a ligand.

10. The method of any one of claims 6-9, wherein the at least one catalyst is selected from a rhodium catalyst, CRED-33, CRED-A231, and GDH-102.

11. The method of any one of claims 6-9, wherein the at least one catalyst is a rhodium catalyst.

12. The method of claim 10 or 11, wherein the rhodium catalyst is (pentamethylcyclopentadi enyl)rhodium(III) dichloride dimer.

13. The method of any one of claims 6-9, wherein the hydrogen donor agent is selected from formic acid, sodium format, and NADP.

14. The method of any one of claims 6-9, wherein the base is tri ethylamine.

15. The method of any one of claims 6-9, wherein the ligand is (R,R)-N-(p- toluenesulfonyl)-l,2-diphenylethylenediamine ((R,R)-TsDPEN).

16. The method of any one of claims 6-9, wherein the at least one catalyst is pentamethylcyclopentadienyl)rhodium(III) dichloride dimer, the hydrogen donor agent is formic acid, the base is tri ethylamine, and the ligand is (R,R)-N-(p-toluenesulfonyl)-l,2- diphenylethylenediamine ((R,R)-TsDPEN).

17. The method of any one of claims 6-9, wherein converting Compound S5 into Compound I is performed at a pH value of 4-6.

18. The method of any one of claims 6-9, wherein converting Compound S5-MsOH into Compound I is performed at a pH value of 4-6.

19. The method of any one of claims 6-18, wherein Compound I is purified through crystallization from a Ci-Ce alkyl alcohol and water.

20. The method of claim 19, wherein the Ci-Ce alkyl alcohol is selected from ethanol and methanol.

21. The method of claim 19, wherein the Ci-Ce alkyl alcohol is methanol.

22. A method for preparing Compound S5-MsOH:comprising converting Compound S5:into Compound S5-MsOH.

23. The method of claims 2, 4, or 22, wherein converting Compound S5 intoCompound S5-MsOH is performed in the presence of methanesulfonic acid.

24. The method of claim 23, wherein the methanesulfonic acid is added to Compound S5 over 2 h.

25. A method for preparing Compound S5:comprising converting Compound S4:into Compound S5.

26. The method of claims 1-4 or 25, wherein converting Compound S4 into Compound S5 is performed in the presence of an oxidant and a base.

27. The method of claim 26, wherein the oxidant is an N-oxide reagent.

28. The method of claim 27, wherein the N-oxide reagent is selected from 4- chloropyridine N-oxide, 2-methyl-4-nitropyridine N-oxide, 2-chloro-4-nitropyridine N- oxide, 2-chloropyridine N-oxide, and 2-cyanopyridine N-oxide, 2-methylpyridine N- oxide, 2,6-dimethylpyridine N-oxide, 2-ethylpyridine N-oxide, isoquinoline N-oxide, 4- methylmorpholine N-oxide, 4-methylpyridine N-oxide, trimethylamine N-oxide, pyridine N-oxide, 3 -methylpyridine N-oxide, and 4-methoxypyridine N-oxide.

29. The method of claims 27 or 28, wherein the A-oxide reagent is picoline A-oxide.

30. The method of claim 25, wherein the base is selected from amine bases, carbonate bases, and phosphate bases.

31. The method of claim 30, wherein the amine base is selected from triethylamine, diisopropylethylamine, and dicyclohexylamine.

32. The method of claim 30, wherein the carbonate base is selected from sodium bicarbonate and potassium carbonate.

33. The method of claim 30, wherein the acetate base is potassium acetate.

34. The method of claim 30, wherein the phosphate base is tribasic potassium phosphate or dibasic potassium phosphate.

35. The method of any one of claims 26-31, wherein the base is triethylamine.

36. The method of any one of claims 26-35, wherein the oxidant is picoline V-oxide and the base is triethylamine.

37. The method of any one of claims 25-36, wherein Compound S5 is purified by washing with one or more of an aqueous solution of N-acetyl-L-cysteine and ammonium persulfate.

38. A method for preparing Compound S4:comprising converting Compound S3:into Compound S4.

39. The method of any one of claims 3, 4, or 38, wherein converting Compound S3 into Compound S4 is performed in a batch process.

40. The method of any one of claims 3, 4, or 38, wherein converting Compound S3 into Compound S4 is performed in a flow process.

41. The method of any one of claims 3, 4, or 38-40, wherein converting Compound S3 into Compound S4 is performed in the presence of a light source.

42. The method of claim 37, wherein the light source is a 450 nm blue light emitting LED.

43. The method of any one of claims 3, 4, or 38-42, wherein converting Compound S3 into Compound S4 is performed in the presence of an acid and a brominating reagent.

44. The method of claim 43, wherein Compound S3, the acid, and the brominating reagent are each dissolved in an appropriate solvent in a separate vessel.

45. The method of claim 44, wherein Compound S3, the acid, and the brominating reagent are combined through a feed line.

46. The method of claim 45, wherein the 450 nm blue light emitting LED irradiates a coil through which Compound S3, the acid, and the brominating reagent flow.

47. The method of any one of claims 43-46, wherein the acid is selected from sulfonic acids, phosphoric acid, hydrochloric acid, triflic acid, and trifluoroacetic acid.

48. The method of claim 47, wherein the sulfonic acid is selected from benzenesulfonic acid, methanesulfonic acid, and -toluenesulfonic acid.

49. The method of any one of claims 43-46, wherein the brominating reagent is selected from JV-bromosuccinimide, N -bromophthalimide, N -bromosaccharin, N - bromoacetamide, bromine, Br2-l,4-di oxane complex, l,3-dibromo-5,5- dimethylhydantoin, monosodiumbromoisocyanurate, and dibromoisocyanuric acid.

50. The method of claim 43, wherein the acid is benzenesulfonic acid and the brominating agent is N -bromosuccinimide.

51. The method of any one of claims 3, 4, or 38-50, wherein converting Compound S3 into Compound S4 comprises quenching the reaction with a quench solution.

52. The method of claim 51, wherein the quench solution comprises 1,3- dimethoxybenzene.

53. The method of claims 51 or 52, wherein the quench solution is treated with a solution of 2-picoline V-oxide and heated to 60 °C.

54. A compound of Formula S4:tautomers thereof, deuterated derivatives of those compounds and tautomers, and pharmaceutically acceptable salts of any of the foregoing.

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