Crystalline and liquid crystalline 25-hydroxy-cholesta-5-ene-3-sodium sulfate and process for the preparation thereof

Abstract

This document describes the crystalline and liquid crystalline forms of sodium 25HC3S. This disclosure includes sodium 25HC3S in forms I, II, III, V, IX, XI, and XIII, and combinations thereof. Pharmaceutical formulations of the said forms or combinations thereof are further disclosed herein, as well as methods for treating or preventing diseases such as hypercholesterolemia, hypertriglyceridemia, and conditions associated with fat accumulation and inflammation (e.g., non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, and atherosclerosis). Methods for preparing 25HC3S are also provided.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 954,279, filed December 27, 2019, the disclosure of which is incorporated herein by reference.

[0003] Introduction

[0004] It has been previously shown that the cholesterol metabolite 5-cholestene-3β-25-diol-3-sulfate (“25HC3S”) reduces lipid biosynthesis and increases cholesterol secretion and degradation, and may be used to treat and prevent hypercholesterolemia, hypertriglyceridemia, and conditions associated with fat accumulation and inflammation (e.g., non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, and atherosclerosis).

[0005] Cholesterol is used by the body to make and repair cell membranes, as well as to synthesize steroid hormones and vitamin D, and is converted into bile acids in the liver. Cholesterol has both exogenous and endogenous sources. The average American consumes about 450 mg of cholesterol per day, with an additional 500 mg to 1,000 mg produced in the liver and other tissues. Another source is the 500 mg to 1,000 mg of bile cholesterol secreted into the intestines daily, of which about 50% is reabsorbed (enterohepatic circulation).

[0006] High serum lipid levels (hypercholesterolemia and hypertriglyceridemia) are associated with the accumulation of cholesterol in the arterial walls and can lead to NAFLD and atherosclerosis. Plaques, characteristic of atherosclerosis, impair blood flow and promote clot formation, and can ultimately lead to death or severe disability through heart attack and / or stroke. Many therapeutic agents for hyperlipidemia have been developed and are widely prescribed by physicians. Unfortunately, only about 35% of patients respond to currently available treatments.

[0007] Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in the United States. This condition is associated with obesity, type II adult-onset diabetes, a sedentary lifestyle, and a high-fat diet. Early stages of NAFLD (i.e., fatty liver) can be reversible with appropriate treatment. However, if left untreated, it can develop into a more difficult-to-treat hepatocellular inflammation (nonalcoholic steatohepatitis, or NASH). Without treatment, NASH can lead to irreversible scarring of liver tissue (fat necrosis) and potentially result in cirrhosis, liver failure, and liver cancer.

[0008] 25HC3S has been disclosed as a pharmaceutically acceptable salt, such as a sodium salt, for example (e.g., U.S. Patent 10,144,759 and Ogawa et al., Steroids, 74, 81-87 (2009)). Crystalline solids are generally more advantageous for processing, storage, and stability than amorphous solids. However, energies may be unfavorable to the easy formation of suitable crystalline solids, and polymorphism may make it impractical to produce stable crystalline solids with specific active pharmaceutical ingredients. In this document, the inventors disclose crystalline sodium 25HC3S, including stable dehydrates, solvates, and hydrates, as well as liquid crystal sodium 25HC3S. Methods for preparing 25HC3S are also provided. Summary of the Invention

[0009] In some aspects of this disclosure, crystalline sodium 25HC3S is provided.

[0010] In some aspects of this disclosure, stable crystalline sodium 25HC3S is provided.

[0011] In other aspects of this disclosure, a hydrate of crystalline sodium 25HC3S is provided.

[0012] In other aspects of this disclosure, a monohydrate of crystalline sodium 25HC3S is provided.

[0013] In other aspects of this disclosure, a dihydrate of crystalline sodium 25HC3S is provided.

[0014] In another aspect of this disclosure, a variable hydrate of crystalline sodium 25HC3S is provided.

[0015] In some aspects of this disclosure, anhydrous crystalline sodium 25HC3S is provided.

[0016] In other aspects of this disclosure, forms I, II, III, IX, XI and XIII of crystalline sodium 25HC3S are provided.

[0017] In another aspect of this disclosure, sodium 25HC3S liquid crystal is provided.

[0018] In some aspects of this disclosure, an intermediate phase of sodium 25HC3S is provided.

[0019] In other aspects of this disclosure, sodium in form V 25HC3S is provided.

[0020] In other aspects of this disclosure, mixtures of two or more of the following forms are provided: sodium form I, sodium form II, sodium form III, sodium form V, sodium form IX, sodium form XI, or sodium form XIII.

[0021] In certain aspects of this disclosure, methods are provided for treating one or more of the following: hypercholesterolemia, hypertriglyceridemia, and conditions associated with fat accumulation and inflammation, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, or atherosclerosis, said methods comprising administering to a patient in need an effective amount of a compound or pharmaceutical composition thereof of crystalline or liquid crystal mesophase 25HC3S sodium.

[0022] In other aspects of this disclosure, pharmaceutical compositions are provided comprising crystalline sodium 25HC3S or liquid crystal sodium 25HC3S or both, and at least one pharmaceutically acceptable excipient.

[0023] In other aspects of this disclosure, pharmaceutical compositions are provided comprising a mixture of two or more of crystalline sodium 25HC3S form I, crystalline sodium 25HC3S form II, crystalline sodium 25HC3S form III, liquid crystal sodium 25HC3S form V, crystalline sodium 25HC3S form IX, crystalline sodium 25HC3S form XI, or crystalline sodium 25HC3S form XIII, and at least one pharmaceutically acceptable excipient.

[0024] This disclosure also includes methods for preparing 25HC3S. In some cases, the method includes contacting 25-hydroxy-(3β)-cholest-5-en-3-ol with a sulfating agent to produce an organic cationic salt of 25-hydroxy-(3β)-cholest-5-en-3-sulfate; and contacting the organic cationic salt of 25-hydroxy-(3β)-cholest-5-en-3-sulfate with at least one metal salt to produce a metal salt of 5-cholest-3β,25-diol 3-sulfate.

[0025] In some cases, the sulfating agent is selected from sulfur trioxide complexes, sulfuric acid compounds, sulfonic acid compounds, and sulfonate compounds. In some cases, the sulfating agent is a sulfur trioxide-pyridine complex. In some cases, the sulfating agent is selected from sulfur trioxide dimethylformamide, sulfur trioxide triethylamine, and sulfur trioxide trimethylamine. In some cases, the sulfating agent is sulfuric acid and acetic anhydride and pyridine. In some cases, the sulfating agent is sulfur trioxide triethylamine and pyridine. In some cases, the sulfating agent is selected from chlorosulfonic acid and pyridine and chlorosulfonic acid and 2,6-dimethylpyridine. In some cases, the sulfating agent is ethyl chlorosulfonate. In some cases, the 25-hydroxy-(3β)-cholest-5-en-3-sulfate organic cationic salt is a pyridinium salt of 25-hydroxy-(3β)-cholest-5-en-3-sulfate. In some cases, the sulfating agent is contacted with an anhydride before contacting 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, the acid anhydride is selected from acetic anhydride, trifluoroacetic anhydride, and trifluoromethanesulfonic anhydride. In some cases, the sulfating agent is contacted with 25-hydroxy-(3β)-cholest-5-en-3-ol in the presence of an organic cationic salt of 25-hydroxy-(3β)-cholest-5-en-3-sulfate. In some cases, the organic cationic salt of 25-hydroxy-(3β)-cholest-5-en-3-sulfate is present as particles (e.g., seed crystals generated in a previous reaction or purification batch).

[0026] In some cases, the sulfating agent is characterized prior to contact with 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, by... 1 The sulfating agent is characterized by ¹H-NMR. In some cases, characterizing the sulfating agent includes determining the extent of degradation of the sulfating agent before contact with 25-hydroxy-(3β)-cholest-5-en-3-ol. In other cases, determining the extent of degradation of the sulfating agent includes determining the amount of impurities in the sulfating agent before contact with 25-hydroxy-(3β)-cholest-5-en-3-ol.

[0027] In some cases, the method includes quenching unreacted sulfation reagents after the formation of an organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate. In some cases, quenching unreacted sulfation reagents includes adding water to the reaction mixture after the formation of the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate. In some cases, quenching unreacted sulfation reagents includes adding water to the reaction mixture, followed by adding at least one base, pyridine, to the reaction mixture. In some cases, the at least one base is selected from trialkylamines such as triethylamine or trimethylamine. In some cases, the at least one base is selected from 2,6-dimethylpyridine or pyridine. In some cases, the base is pyridine. In some cases, unreacted sulfation reagents in the reaction mixture are quenched under slow stirring.

[0028] In some cases, the method includes purifying the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt before contacting it with at least one metal salt. In some cases, the purified 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt has a purity of 70% or greater (such as 80% or greater, 90% or greater, 95% or greater, 98% or greater, or 99% or greater).

[0029] In some cases, purified organic cationic salts of 25-hydroxy-(3β)-cholest-5-en-3-sulfate have one or more sulfation byproducts (e.g., byproducts from the sulfated 25-hydroxy-(3β)-cholest-5-en-3-ol), wherein, relative to the organic cationic salt of 25-hydroxy-(3β)-cholest-5-en-3-sulfate, one or more byproducts are present at 5% w / w or less, 4% w / w or less, 3% w / w or less, 2% w / w or less, or 1%. The disulfation product (i.e., 5-cholestene-3β-25-diol-disulfate) is present in the purified 25-hydroxy-(3β)-cholestene-3-sulfate organic cationic salt composition in amounts of 5% w / w or less, 4% w / w or less, 3% w / w or less, 2% w / w or less, or 1% w / w or less, relative to the 25-hydroxy-(3β)-cholestene-5-ene-3-sulfate organic cationic salt.

[0030] In some cases, the organic cationic salt 25-hydroxy-(3β)-cholest-5-ene-3-sulfate is purified by liquid chromatography. In some cases, the purification of the organic cationic salt 25-hydroxy-(3β)-cholest-5-ene-3-sulfate includes liquid chromatography using a silica gel stationary phase and a mobile phase comprising at least one base. In some cases, said at least one base is pyridine.

[0031] In some cases, one or more fractions collected from liquid chromatography can be combined. In some cases, the combined fractions can be concentrated. In some cases, the combined fractions are concentrated by distillation. In some cases, the combined fractions are concentrated under vacuum. In some cases, the combined fractions are concentrated by distillation under vacuum. In some cases, the combined fractions are contacted with an organic cation salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate. In some cases, the combined fractions are contacted with one or more particles of an organic cation salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate. In some cases, the organic cation salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate (e.g., particles of an organic cation salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate) is contacted with the combined fractions by adding the organic cation salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate during the distillation of the combined fractions. In some cases, the organic cationic salt 25-hydroxy-(3β)-cholest-5-ene-3-sulfate is contacted with the combined fractions by adding the salt after distillation. In other cases, the combined fractions are concentrated and contacted with a composition containing particles of the organic cationic salt 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and at least one solvent.

[0032] In some cases, methods for producing metal salts of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate include contacting an organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate with at least one sodium salt. In some cases, said at least one sodium salt is selected from sodium acetate, sodium iodide, sodium chloride, sodium hydroxide, and sodium methoxide. In some cases, the method includes contacting a pyridinium salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate with sodium iodide to produce sodium 25-hydroxy-(3β)-cholest-5-ene-3-sulfate.

[0033] In some cases, methods for preparing 25-hydroxy-3β-cholestene-5-ene-3-sulfate include contacting 25-hydroxy-(3β)-cholestene-5-ene-3-ol with a sulfur trioxide-pyridine complex to produce 25-hydroxy-(3β)-cholestene-5-ene-3-sulfate pyridinium salt; and contacting 25-hydroxy-(3β)-cholestene-5-ene-3-sulfate pyridinium salt with at least one sodium salt to produce 5-cholestene-3β,25-diol 3-sulfate sodium salt.

[0034] In some cases, methods for preparing 25-hydroxy-3β-cholest-5-en-3-sulfate include contacting (3β)-cholest-5-en-3-ol with a sulfating agent to produce a first (3β)-cholest-5-en-3-sulfate organic cationic salt; contacting the first (3β)-cholest-5-en-3-sulfate organic cationic salt with an organic base to produce a second (3β)-cholest-5-en-3-sulfate organic cationic salt; and oxidizing the second (3β)-cholest-5-en in the presence of at least one surfactant. -3-sulfate organic cationic salt to produce 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt; to produce 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt from 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt by deoxygenation; and to contact 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt with at least one metal salt to produce 5-cholestene-3β,25-diol 3-sulfate metal salt.

[0035] In some cases, the sulfating agent is selected from sulfur trioxide complexes, sulfuric acid compounds, sulfonic acid compounds, and sulfonate / ester compounds. In some cases, the sulfating agent is a sulfur trioxide-pyridine complex. In some cases, the sulfating agent is selected from sulfur trioxide dimethylformamide, sulfur trioxide triethylamine, and sulfur trioxide trimethylamine. In some cases, the sulfating agent is sulfuric acid and acetic anhydride and pyridine. In some cases, the sulfating agent is selected from chlorosulfonic acid and pyridine. In some cases, the sulfating agent is selected from chlorosulfonic acid and 2,6-dimethylpyridine. In some cases, the sulfating agent is selected from ethyl chlorosulfonate. In some cases, the first (3β)-cholest-5-ene-3-sulfate organic cation salt is a (3β)-cholest-5-ene-3-sulfate pyridinium salt.

[0036] In some cases, the organic base contacted with the first (3β)-cholest-5-ene-3-sulfate organic cation salt is selected from hydroxide bases. In some cases, the hydroxide base is selected from tetraethylammonium hydroxide, tetrabutylammonium hydroxide, tetrapropylammonium hydroxide, and tetramethylammonium hydroxide. In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is selected from tetraethylammonium cationic salt, tetrabutylammonium cationic salt, tetrapropylammonium cationic salt, and tetramethylammonium cationic salt.

[0037] In some cases, oxidation of a second (3β)-cholesterol-5-ene-3-sulfate organic cationic salt is performed in the presence of at least one surfactant. In some cases, oxidation of the second (3β)-cholesterol-5-ene-3-sulfate organic cationic salt comprises contacting the second (3β)-cholesterol-5-ene-3-sulfate organic cationic salt with a composition having an oxidizing agent and at least one surfactant. In some cases, oxidation of the second (3β)-cholesterol-5-ene-3-sulfate organic cationic salt to produce a 25-hydroxy-(3β)-cholesterol-(5,6-epoxy)-3-sulfate organic cationic salt comprises contacting the second (3β)-cholesterol-5-ene-3-sulfate organic cationic salt with a composition having potassium persulfate in the presence of at least one surfactant. In some cases, the at least one surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants. Nonionic surfactants can be selected from polyoxyethylene glycol ether surfactants (e.g., polyoxyethylene glycol octylphenol ether), polyoxyethylene glycol alkyl sorbitol esters, alkyl sorbitol esters, block copolymers of polyethylene glycol and polypropylene glycol, and other nonionic surfactants. Anionic surfactants can be selected from surfactants having anionic functional head groups, such as surfactants containing sulfonate / ester, phosphate / ester, sulfate / ester, or carboxylate / ester head groups. For example, anionic surfactants can be selected from alkyl sulfate / esters such as ammonium lauryl sulfate, sodium dioctyl sulfosuccinate, perfluorooctane sulfonate, perfluorononanoate, perfluorooctanoate, linear alkylbenzene sulfonate, alkyl-aryl ether phosphate, sodium lauryl ether sulfate, lignin sulfonate, or sodium stearate, and other anionic surfactants. Cationic surfactants can be selected from surfactants having cationic functional head groups (such as pyridinium or quaternary ammonium head groups). For example, cationic surfactants may be selected from cetyltrimethylammonium bisulfate, tetrabutylammonium bisulfate, cetyltrimethylammonium bromide, tetrabutylammonium bromide, tetrabutylammonium iodide, tetrabutylphosphonium bromide, tetraoctylammonium bromide, tetraoctylammonium iodide, benzyltriethylammonium chloride, benzyltriethylammonium bromide, benzylcetyldimethylammonium chloride, and benzylcetyldimethylammonium bromide. Amphoteric surfactants include cationic and anionic centers, such as sulfobetaine (e.g., 3-[(3-cholanamidopropyl)dimethylammonium]-1-propanesulfonate) or betaine (e.g., cocoamidopropyl betaine). In some cases, the surfactant is Extran laboratory soap, La Parisienne soap, or DL-α-tocopherol methoxy polyethylene glycol succinate (e.g., TPGS-750-M-2).

[0038] In some cases, a second (3β)-cholest-5-ene-3-sulfate organic cationic salt is contacted with an oxidant and at least one ketone in the presence of at least one surfactant. In some cases, the method comprises: contacting an oxidant (e.g., potassium peroxymonosulfate) with at least one ketone in a separate oxidation reaction mixture in the presence of at least one surfactant, and contacting the oxidatively reactive mixture with the second (3β)-cholest-5-ene-3-sulfate organic cationic salt. In some cases, the method comprises oxidizing the second (3β)-cholest-5-ene-3-sulfate organic cationic salt in the presence of water. In some cases, the at least one ketone is selected from tetrahydrothiaran-4-one 1,1-dioxide and haloketones. In some cases, the haloketone is selected from 1,1,1-trifluoro-2-butanone, 4,4-difluorocyclohexanone, 2-2-2-4'-tetrafluoroacetylbenzene, and 1,1,1-trifluoroacetone. In some cases, the at least one ketone is 1,1,1-trifluoro-2-butanone.

[0039] In some cases, oxidation of a second (3β)-cholest-5-ene-3-sulfate organic cation salt involves contacting the second (3β)-cholest-5-ene-3-sulfate organic cation salt with at least one oxidizing agent. In some cases, the at least one oxidizing agent is selected from dioxane. In some cases, the dioxane is produced in situ in a composition having the second (3β)-cholest-5-ene-3-sulfate organic cation salt. In some cases, the dioxane is produced alone (e.g., in a separate reaction vessel such as a flask) and then contacted with a composition having the second (3β)-cholest-5-ene-3-sulfate organic cation salt.

[0040] In some cases, a second (3β)-cholest-5-ene-3-sulfate organic cation salt is oxidized in the presence of at least one base. In some cases, the at least one base is selected from weak bases. In some cases, the at least one base is selected from potassium bicarbonate, sodium bicarbonate, potassium phenolate, sodium citrate buffer, sodium phosphate buffer, potassium formate, and potassium acetate. In some cases, the at least one base is potassium bicarbonate.

[0041] In some cases, the production of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salts from 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salts involves deoxygenation by contacting the 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt with zinc. In some cases, the 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt is contacted with zinc in the presence of at least one halide and at least one acid. In some cases, the at least one halide is selected from iodine and metal halides. In some cases, the metal halide is selected from sodium iodide and lithium iodide. In some cases, the at least one acid is selected from weak acids. In some cases, the at least one acid is selected from acetic acid, hydrochloric acid, citric acid, p-toluenesulfonic acid, formic acid, and methanesulfonic acid. In some cases, the method involves contacting an organic cationic salt of 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate with zinc in the presence of acetic acid to produce an organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate.

[0042] In some cases, methods for producing metal salts of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate include contacting an organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate with at least one sodium salt. In some cases, said at least one sodium salt is selected from sodium acetate, sodium iodide, sodium chloride, sodium hydroxide, and sodium methoxide. In some cases, the method includes contacting a pyridinium salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate with sodium iodide to produce sodium 25-hydroxy-(3β)-cholest-5-ene-3-sulfate.

[0043] Brief description of the attached diagram

[0044] Figure 1 is the XRPD diffraction pattern of crystalline 25HC3S sodium form I.

[0045] Figure 2 is the XRPD diffraction pattern of sodium form II of crystalline 25HC3S.

[0046] Figure 3 is the XRPD diffraction pattern of liquid crystal 25HC3S sodium form V.

[0047] Figure 4 is the XRPD diffraction pattern of V in the sodium form of liquid crystal 25HC3S.

[0048] Figure 5 is the XRPD diffraction pattern of sodium 25HC3S in the form of IX.

[0049] Figure 6 is the XRPD diffraction pattern of XI in the form of crystalline 25HC3S sodium.

[0050] Figure 7 is the XRPD diffraction pattern of XI in the form of crystalline 25HC3S sodium.

[0051] Figure 8 is the XRPD diffraction pattern of crystalline 25HC3S sodium form XIII.

[0052] Figure 9 is the XRPD diffraction pattern of crystalline 25HC3S sodium form XIII.

[0053] Figure 10 shows the index results for crystalline 25HC3S sodium form I.

[0054] Figure 11 is a thermal analysis diagram of crystalline 25HC3S sodium form I.

[0055] Figure 12 shows the variable-temperature XRPD experiment of crystalline 25HC3S sodium form I.

[0056] Figure 13 shows the variable humidity XRPD experiment of crystalline 25HC3S sodium form I and crystalline 25HC3S sodium form XIII.

[0057] Figure 14 is an XRPD diffraction pattern of dehydrated sodium form I from crystalline 25HC3S.

[0058] Figure 15 is an XRPD diffraction pattern (enlarged) of the dehydrated form I of crystalline 25HC3S sodium.

[0059] Figure 16 shows the DVS isotherm for sodium form II of crystalline 25HC3S.

[0060] Figure 17 shows the index results for crystalline 25HC3S sodium form II (99:01 acetonitrile (ACN) / H2O paste, 0.21a). w (3 days).

[0061] Figure 18 shows the index results for crystalline 25HC3S sodium form II (95:05EtOH / H2O paste, 0.30a). w (10 days).

[0062] Figure 19 shows the index results of crystalline 25HC3S sodium form II (acetone paste, 55°C, 1 day).

[0063] Figure 20 shows the variability of XRPD peak positions for the slurry in sodium form II of crystalline 25HC3S indexed in Figures 17-19, ranging from approximately 4°2θ to approximately 11°2θ.

[0064] Figure 21 shows the variability of XRPD peak positions for the slurry in sodium form II of crystalline 25HC3S, indexed in Figures 17-19, ranging from approximately 13°2θ to approximately 21.5°2θ.

[0065] Figure 22 shows the variable humidity XRPD experiment of crystalline 25HC3S sodium form II.

[0066] Figure 23 shows the variability of XRPD peak positions for crystalline 25HC3S sodium form II as observed by variable humidity XRPD, ranging from approximately 7.5°2θ to approximately 10.2°2θ.

[0067] Figure 24 is a TGA thermal analysis diagram of crystalline 25HC3S sodium form II.

[0068] Figure 25 is a DSC thermal analysis diagram of sodium form II of crystalline 25HC3S.

[0069] Figure 26 shows the cyclic DSC experiment of sodium form II of crystalline 25HC3S.

[0070] Figure 27 shows the variable-temperature XRPD experiment of crystalline 25HC3S sodium form II.

[0071] Figure 28 is a TGA thermal analysis diagram of liquid crystal 25HC3S sodium form V.

[0072] Figure 29 is a DSC thermal analysis diagram of liquid crystal 25HC3S sodium form V.

[0073] Figure 30 shows the index results of crystalline 25HC3S sodium form XIII (produced from a mixture of crystalline 25HC3S sodium form I and form XIII exposed to vacuum at 70°C for 2 days).

[0074] Figure 31 shows the XRPD diffraction patterns of crystalline 25HC3S sodium in forms III and IX.

[0075] Figure 32 shows the index results for crystalline 25HC3S sodium form III.

[0076] Figure 33 shows the crystalline 25HC3S sodium form IX in solution. 1 H-NMR spectrum.

[0077] Figure 34 shows sodium 25HC3S in solution. 1 H-NMR spectrum.

[0078] Figure 35 is the XRPD diffraction pattern of sodium form III of crystalline 25HC3S.

[0079] Figure 36 is the XRPD diffraction pattern of crystalline 25HC3S sodium form I.

[0080] Figure 37 is the XRPD diffraction pattern of crystalline 25HC3S sodium form I.

[0081] Figure 38 is the XRPD diffraction pattern of crystalline 25HC3S sodium form I.

[0082] Figure 39 shows the XRPD diffraction patterns of crystalline 25HC3S sodium in forms I and XIII.

[0083] Figure 40 is the XRPD diffraction pattern of crystalline 25HC3S sodium form XIII.

[0084] Figure 41 is the XRPD diffraction pattern of crystalline 25HC3S sodium form XIII.

[0085] Figure 42 is the XRPD diffraction pattern of sodium form II of crystalline 25HC3S.

[0086] Figure 43 is the XRPD diffraction pattern of sodium form II of crystalline 25HC3S.

[0087] Figure 44 is the XRPD diffraction pattern of sodium form II of crystalline 25HC3S.

[0088] Figure 45 is the XRPD diffraction pattern of a mixture of crystalline 25HC3S sodium forms III and IX.

[0089] Figure 46 is the XRPD diffraction pattern of sodium form II of crystalline 25HC3S.

[0090] Figure 47 is the XRPD diffraction pattern of liquid crystalline 25HC3S sodium form V.

[0091] Figure 48 is the XRPD diffraction pattern of liquid crystalline 25HC3S sodium form V.

[0092] Figure 49 is the XRPD diffraction pattern of crystalline 25HC3S sodium form III.

[0093] Figure 50 is the XRPD diffraction pattern of crystalline 25HC3S sodium form III.

[0094] Figure 51 is the XRPD diffraction pattern of sodium 25HC3S in the form of IX.

[0095] Figure 52 is an enlarged XRPD diffraction pattern of crystalline 25HC3S sodium form III.

[0096] Figure 53 is the XRPD diffraction pattern of crystalline 25HC3S sodium form I.

[0097] Figure 54 is the XRPD diffraction pattern of sodium form II of crystalline 25HC3S.

[0098] Figure 55 is the XRPD diffraction pattern of crystalline 25HC3S sodium form I.

[0099] Figure 56 is the XRPD diffraction pattern of liquid crystalline 25HC3S sodium form V.

[0100] Figure 57 These are three different samples of sulfur trioxide pyridine in deuterated acetone. 1 H-NMR spectrum.

[0101] Figure 58A This is a sample of sulfur trioxide-pyridine with 21% impurities in deuterated acetone. 1 Enhancement of the H-NMR spectrum in the region between 8.1 and 9.3 ppm. Figure 58B This is a sample of sulfur trioxide-pyridine with 33% impurities in deuterated acetone. 1 Enhancement of the H-NMR spectrum in the region between 8.1 and 9.3 ppm. Figure 58C This is a sample of sulfur trioxide-pyridine with 36% impurities in deuterated acetone. 1 Enhancement of the H-NMR spectrum in the region between 8.1 and 9.3 ppm.

[0102] Detailed description

[0103] Crystalline and liquid crystalline 25-hydroxy-3β-cholestene-5-ene-3-sulfate (25HC3S)

[0104] Crystalline sodium 25HC3S and liquid crystalline sodium 25HC3S can be readily analyzed by X-ray powder diffraction. The X-ray powder diffraction pattern is an xy plot, where the x-axis is °2θ (diffraction angle) and the y-axis is intensity. The x-axis can also be in the form of d-spacing, which is related to the diffraction angle by Bragg's law, where 2dsinθ = nλ, where d is the d-spacing and λ is the wavelength of the incident X-ray wave. The pattern contains peaks that can be used to characterize crystalline sodium 25HC3S. Unless otherwise specified, peaks are represented by their position on the x-axis, not by their y-axis intensity. It can also occur that, due to sample orientation and the orientation of the sample relative to the instrument, a peak present in one sample on one instrument may not be present in another sample acquired on a different instrument.

[0105] Data from X-ray powder diffraction can be used in a variety of ways to characterize crystalline forms. For example, the entire X-ray powder diffraction pattern output from a diffractometer can be used to characterize sodium 25HC3S. However, smaller subsets of such data may also be, and often are, suitable for characterizing sodium 25HC3S. For example, a set of one or more peaks from such a pattern can be used to characterize crystalline sodium 25HC3S. In this application, as described in Examples 16 and 17, all reported peaks are expressed in °2θ under Cu-Kα radiation. In practice, it is often even possible to characterize such crystalline forms using a single X-ray powder diffraction peak. When crystalline sodium 25HC3S is characterized herein by “one or more peaks” from an X-ray powder diffraction pattern and such peaks are listed, it generally means that any combination of the listed peaks can be used to characterize crystalline sodium 25HC3S. Furthermore, the fact that other peaks are present in the X-ray powder diffraction pattern generally does not negate or otherwise limit the characterization.

[0106] Besides variations in peak intensity, peak positions along the x-axis can also exhibit variability. However, this variability is generally acceptable when reporting peak positions for characterization purposes. Such variability in peak positions along the x-axis can originate from multiple sources (e.g., sample preparation, particle size, water content, solvent content, instrument parameters, data analysis software, and sample orientation). For example, samples of the same crystalline material prepared under different conditions may produce slightly different diffraction patterns, and different X-ray instruments may be operated with different parameters, which can lead to slightly different diffraction patterns from the same crystalline solid.

[0107] Due to this source of variability, the term "about" is typically used to describe X-ray diffraction peaks before the peak value expressed in °2θ. For the purposes of reporting data herein, this value is typically ±0.1°2θ. This generally means that on well-maintained instruments, one can expect a variability of ±0.1°2θ in peak measurements. Unless otherwise stated, X-ray powder diffraction peaks cited herein are generally reported with this variability of ±0.1°2θ, and whenever disclosed herein, regardless of the presence of the term "about," is generally intended to be reported with this variability; however, in some cases, the variability may be as high as ±0.2°2θ or even higher, depending on instrument conditions.

[0108] As described herein, compound 25-hydroxy-3β-cholestene-5-en-3-sulfate (25HC3S) represents [(3S,10R,13R,17R)-17-[(1R)-5-hydroxy-1,5-dimethyl-hexyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecano-1H-cyclopentadien[a]phenanthrene-3-yl]sulfate, compounds of formula I:

[0109]

[0110] This disclosure uses the term "form" to identify different crystalline or liquid crystalline forms of crystalline sodium 25HC3S. Differences in forms can be observed through structural features such as X-ray powder diffraction; properties such as hygroscopicity or thermal behavior; and / or both. The term "Form I" refers to crystalline sodium 25HC3S of form I. Similarly, "Form II" refers to crystalline sodium 25HC3S of form II. Likewise, Form III, Form IX, Form XI, and Form XIII refer to crystalline sodium 25HC3S of forms III, IX, XI, and XIII, respectively. Form V refers to the form of sodium 25HC3S in the liquid crystalline phase.

[0111] The resulting solids were observed using one or more of polarized light microscopy and X-ray powder diffraction. Based on visual examination of the peaks associated with these materials, materials exhibiting unique crystalline X-ray powder diffraction patterns were given names as described in this disclosure.

[0112] Table 1 summarizes some of the experiments performed to obtain sodium 25HC3S in the form reported in this disclosure, namely those related to Examples 1-36.

[0113] In Table 1, cells marked with an asterisk (*) indicate that the identified solid is not one of Form I, Form II, Form III, Form V, Form IX, Form XI, or Form XIII.

[0114] The times, temperatures, and humidity in Table 1 are approximate. The term "B" refers to birefringence, and "NB" refers to non-birefringence when the sample is observed using polarized light microscopy with cross-polarity. Crystalline sodium 25HC3S was screened using solvent-based methods with the various solvents and conditions listed in Table 1. Methods using solvents or solvent mixtures include, for example, cooling the solution, evaporation, antisolvent addition, and suspension (slurry). Variations of these methods can include changes in solvent, solvent mixture, antisolvent, temperature, cooling rate, concentration, addition rate, and mixing order, to name just a few possibilities.

[0115] Table 1-25 Screening of Sodium HC3S

[0116]

[0117]

[0118]

[0119]

[0120] Water activity (α) can be studied through competitive water activity grinding experiments (slurry) in various aqueous solvent mixtures. w The effect of X-ray powder diffraction on the hydration state can be characterized by X-ray powder diffraction. Experiments can be established on various a... w The physically stable form of water activity. Water activity can also be related to relative humidity, because RH% = a w x 100. Therefore, it is possible to directly link the stability of anhydrous / hydrated systems in slurry experiments with their solid-state stability. Slurry techniques that control water activity can provide an accurate method for rapidly predicting the physically stable form in anhydrous / hydrated systems. Results of water activity experiments, conducted at approximately room temperature, are presented in Table 2. Time, temperature, and humidity are approximations. As in Table 1, the term "B" refers to birefringence when observed using a polarized light microscope with cross-polarity.

[0121] Table 2-25 HC3S Sodium Water Activity Pulp

[0122]

[0123]

[0124]

[0125] Several X-ray powder diffraction patterns were indexed. As used herein, “index” generally refers to the process of determining the size and shape of a crystal cell given the peak positions in a diffraction pattern. The term derives from assigning Miller index labels to individual peaks. For example, if all peaks in a pattern are indexed by a single unit cell, this strongly suggests that the sample contains a single crystalline phase. Given an index solution, the unit cell volume can be directly calculated and can be used to determine their solvation state. An index can also be a description of the crystalline form, providing a concise summary of all available peak positions for that phase at a specific thermodynamic state point.

[0126] This document reports several stable crystalline forms of crystalline sodium 25HC3S—forms I, II, IX, XI, and XIII—and one liquid crystal form, form V. In this disclosure, “stable” means that the forms do not readily transform into one another under a given set of conditions. However, metastable forms can transform so readily when exposed to certain conditions. Therefore, a form stable under one set of conditions (e.g., humidity) may be unstable under another set of conditions. Not all forms are indexed herein. Furthermore, form III is a metastable form that can be formed and can be a starting material for form IX. Despite being metastable, form III is sufficiently stable for separation using XRPD.

[0127] In many cases, crystalline sodium 25HC3S is provided, including stable crystalline sodium 25HC3S. Examples of crystalline sodium 25HC3S include anhydrous crystalline sodium 25HC3S, hydrates of crystalline sodium 25HC3S, and solvates of crystalline sodium 25HC3S.

[0128] Hydrates of crystalline sodium 25HC3S include monohydrate, dihydrate, and variable hydrate. Liquid crystal hydrates of sodium 25HC3S are also presented herein.

[0129] In certain cases, the hydrate of crystalline sodium 25HC3S can be characterized by an X-ray powder diffraction pattern containing one or more of the following peaks: (i) peaks less than about 2.8°2θ, such as those between about 2.1°2θ and about 2.6°2θ; (ii) peaks between about 4.3°2θ and about 4.6°2θ; (iii) peaks between about 5.0°2θ and about 5.5°2θ; (iv) peaks between about 8.6°2θ and about 9.1°2θ; and (v) peaks between about 15.0°2θ and about 15.3°2θ. In these and other cases, the hydrate of crystalline sodium 25HC3S can be characterized by an X-ray powder diffraction pattern containing one or more of the following peaks: (i) peaks between about 2.1°2θ and about 2.3°2θ; and (ii) peaks between about 9.9°2θ and about 10.0°2θ.

[0130] In some cases, anhydrous crystalline sodium 25HC3S can be characterized by an X-ray powder diffraction pattern containing one or more of the following peaks: (i) a peak between about 4.5°2θ and about 4.8°2θ, (ii) a peak between about 9.8°2θ and about 9.9°2θ, (iii) a peak between about 14.1°2θ and about 14.3°2θ, and (iv) a peak at about 16.1°2θ.

[0131] In many cases, crystalline sodium 25HC3S in form I is provided in this disclosure. Form I is a dihydrate of crystalline sodium 25HC3S. Form I can be prepared as described in Table 1. For example, sodium 25HC3S is slurried in an acetone / H2O solution, heated, the solution is removed, water is added, and the mixture is left to stand to obtain form I. Form I can also be prepared from methanol. The preparation of form I is further described in Examples 20-23 and in Example 24, it is mixed with form XIII. Table 3 below associates the examples that produce form I with the corresponding XRPD diffraction pattern figures.

[0132] Table 3 - Examples of Form IXRPD / Figures

[0133] Example number Attached Figure 20 36 21 37 22 38 23 55 24 39

[0134] Table 4 shows the peaks found in Figure 55, while the peaks of the other figures in Table 3 are presented on the figures themselves.

[0135] Table 4 - Figure 55 Peak List

[0136]

[0137]

[0138] Form I of crystalline 25HC3S sodium can be characterized by various analytical techniques, including X-ray powder diffraction. An X-ray powder diffraction pattern of form I, or a portion thereof, can be used to identify form I. Form I contains a variety of X-ray powder diffraction peaks, which, individually or together, may aid in the identification of its presence. For example, in many cases, form I can be characterized by an X-ray powder diffraction pattern containing a peak at approximately 2.1°2θ (not all diffraction patterns in this paper show such a low-angle peak). In addition to the peak at approximately 2.1°2θ, X-ray powder diffraction patterns may contain, for example, one or more peaks at approximately 5.4°2θ, approximately 6.5°2θ, approximately 10.8°2θ, and approximately 15.0°2θ.

[0139] In many cases, form I can be characterized by an X-ray powder diffraction pattern containing peaks at approximately 2.1°2θ, approximately 6.5°2θ, and approximately 10.8°2θ. In some cases, the X-ray powder diffraction pattern may further contain one or more peaks at approximately 9.9°2θ, approximately 15.0°2θ, and approximately 15.6°2θ.

[0140] Figure 1 shows an X-ray powder diffraction pattern of a representative sample of Form I, where the x-axis starts at 0°2θ and reaches 40°2θ, while Figure 53 shows the same pattern, but between approximately 3°2θ and approximately 39°2θ. For example, either Figure 1 or Figure 53 can be used to characterize Form I.

[0141] Form I appears to be stable at relative humidity between approximately 38% and approximately 70% RH and between approximately 38% and approximately 70% RH. Therefore, Form I is composed of substances with relative humidity between approximately 0.38 and approximately 0.70a. w and from about 0.38 to about 0.70a w A slurry is produced in an aqueous solvent mixture with a water activity between [value missing]. When exposed to elevated temperatures or humidity conditions close to 0% RH, form I dehydrates to form XIII. Form I is hygroscopic and is believed to be hygroscopic above 0.73a. w Under certain water activity conditions, liquid crystals of a form called form V will form.

[0142] The X-ray powder diffraction pattern of Form I was successfully indexed, indicating that the pattern represents a single crystalline phase (Fig. 10). The indexed result has a monoclinic unit cell containing six 25HC3S sodium molecules. (±5%) of the cell volume leads to The formula unit volume (6 molecules / cell) is (±5%), consistent with the hydrate form. The formula unit volume of form I is approximately larger than that of the anhydrous form (form XIII). The volume difference provides enough space to hold up to 2 mol / mol of water. For reference, one water molecule occupies approximately [missing information - likely a specific volume or area].

[0143] The thermal analysis plot for Form I is shown in Figure 11. The TGA thermal analysis plot provides an 8% weight loss at up to 130 °C, which occurs simultaneously with the extensive dehydration endothermic reaction shown in the DSC thermal analysis plot (Figure 11). Assuming the loss is due to water volatilization, the weight loss corresponds to approximately 2.4 mol / mol of water. The DSC curves in Figure 11 also show endothermic reactions near 168 °C and 182 °C. These events are related to decomposition.

[0144] Dehydration of form I leads to a transformation to form XIII. This transformation was confirmed by variable-temperature X-ray powder diffraction (Fig. 12), which shows form XIII when heated to 170 °C and then cooled to 135 °C (pattern 5 of Fig. 12). In the same experiment, decomposition products identified as trisodium hydrogen disulfate (Na3H(SO4)2) were found at 170 °C. These results are consistent with the thermal analysis plot of form I in Fig. 11.

[0145] Variable humidity X-ray powder diffraction experiments were performed on a mixture consisting primarily of Form I and a small amount of Form XIII, as shown in Figure 13. The laboratory humidity in which the analytical samples were prepared was 15% relative humidity (“RH”). During X-ray powder diffraction analysis, the material was exposed to increasing and then decreasing humidity. The material was identified as Form I under humidity conditions spanning from 25% to 85% RH. However, the material was immediately partially dehydrated back to Form XIII upon exposure to 0% RH. After 20 minutes, it was completely dehydrated to Form XIII at 0% RH. Exposure to 0% RH or 70°C under vacuum dehydrated Form I to Form XIII. Figures 14 and 15 compare the X-ray powder diffraction patterns of the material as Form I dehydrated to Form XIII.

[0146] Further disclosure is of substantially pure form I. As used herein, “substantially pure” generally means the absence of any perceptible amount of the form described herein, except possibly in trace levels of other forms of crystalline sodium 25HC3S, liquid crystalline forms of sodium 25HC3S, or amorphous sodium 25HC3S such as amorphous sodium 25HC3S. Examples of trace levels relative to the total amount of sodium 25HC3S present (based on weight) include totals not exceeding about 10%, 5%, 2%, 1.5%, 1%, 0.5%, 0.25%, 0.1%, or less.

[0147] In many instances of this disclosure, Form II crystalline 25HC3S sodium is provided, including substantially pure Form II crystalline 25HC3S sodium. Form II is a variable hydrate of crystalline 25HC3S sodium. The water content is typically present in a concentration of no more than about 3 moles of water per mole of 25HC3S sodium. In some cases, the molar content of water to 25HC3S sodium is about 1 to 3, including, for example, about 2 to 3. Form II can be prepared as described in Table 1. For example, Form II is obtained by slurrying 25HC3S sodium in acetone and drying it under nitrogen. Form II can also be prepared as described in Examples 27-30. Table 5 below associates examples of producing Form II with corresponding XRPD diffraction pattern figures.

[0148] Table 5 - Examples of Type II XRPD / Figures

[0149] Example number Attached Figure 27 42 28 43 29 44 30 46

[0150] Table 6 shows the peaks found in Figure 46, while the peaks of the other figures in Table 5 are presented on the figures themselves.

[0151] Table 6 - Figure 46 Peak List

[0152]

[0153] Form II can be characterized by various analytical techniques, including by X-ray powder diffraction. An X-ray powder diffraction pattern of Form II, or a portion thereof, can be used to identify Form II. Form II contains a variety of X-ray powder diffraction peaks, which, individually or together, may help identify the presence of Form II. In many cases, Form II can be characterized by an X-ray powder diffraction pattern containing a peak at approximately 2.3°2θ. In addition to the peak at approximately 2.3°2θ, the X-ray powder diffraction pattern may contain, for example, one or more peaks at approximately 4.5°2θ, peaks at approximately 5.0°2θ and approximately 5.1°2θ and between approximately 5.0°2θ and approximately 5.1°2θ, peaks at approximately 5.9°2θ and approximately 6.1°2θ and between approximately 5.9°2θ and approximately 6.1°2θ, and at least one peak at approximately 14.8°2θ and approximately 15.1°2θ and between approximately 14.8°2θ and approximately 15.1°2θ.

[0154] In many cases, form II can be characterized by an X-ray powder diffraction pattern containing peaks at approximately 2.3°2θ and approximately 5.0°2θ. In some cases, the X-ray powder diffraction pattern may contain one or more peaks at approximately 4.5°2θ, approximately 5.9°2θ, approximately 9.1°2θ, and approximately 15.1°2θ.

[0155] Figure 2 is an X-ray powder diffraction pattern for Form II, where the x-axis starts at approximately 0°2θ and extends to approximately 40°2θ, while Figure 54 shows the same pattern, but between approximately 3°2θ and approximately 39°2θ. Either Figure 2 or Figure 54 can be used to characterize Form II.

[0156] Form II is stable under a wide range of temperature and humidity conditions. Form II and essentially pure Form II are described herein. Form II is stable at relative humidity from about 21% to about 30% RH. Therefore, forms with relative humidity of 0.21% and 0.30% are stable. w and in the range of 0.21 to 0.30a w Form II is produced in a slurry of an aqueous solvent mixture with varying water activity. The variable hydrate thus prepared exhibits kinetic stability. Form II has been observed to dehydrate to an anhydrous state without form transformation upon exposure to elevated temperatures or low humidity conditions. In its fully hydrated state, form II appears to contain approximately 3 mol / mol of water (Figure 16 shows the DVS absorption isotherm of form I at 85% RH). Upon prolonged exposure to 75% or higher RH, form II transforms into the liquid crystalline form V of sodium 25HC3S.

[0157] Multiple X-ray powder diffraction patterns of Form II obtained from various experiments have been successfully indexed (Figs. 17, 18, and 19). These indices indicate that the cell volume of Form II varies to accommodate different amounts of water, and therefore Form II is a variable hydrate. The differences in these patterns (attributed to variations in peak positions) suggest that there is a range of observed peaks (Figs. 18 and 19). Figs. 20 and 21 are magnified regions of the X-ray powder diffraction patterns of the slurry. Given that X-ray powder diffraction peak positions are a direct result of cell parameters, an X-ray powder diffraction pattern does not necessarily represent the crystalline form under all conditions due to variable hydration and its influence on lattice parameters. X-ray powder diffraction patterns should be considered as discrete states of the same crystalline phase of Form II. The general trend is that peaks shift to smaller scattering angles as cell volume increases. Anisotropic strain can cause some peaks to shift to higher scattering angles as cell volume increases. Some peak positions are more sensitive to changes in cell volume than others. Peaks sensitive to changes in unit cell volume can be used to determine the unit cell volume of a specific sample. Moving peaks produce X-ray powder diffraction patterns that appear to differ in quality visually, but do not necessarily indicate a change in the crystalline phase.

[0158] Multiple X-ray powder diffraction patterns of Form II were obtained through in-situ variable humidity X-ray powder diffraction experiments generally performed according to Example 18, at ambient temperature and under different RH conditions ranging from 0% to 85% RH. The experiments provide a systematic approach to determine the cell volume change determined by water absorption, where the cell expands / contracts to accommodate water. As expected, no form change occurred; however, peak shifts were observed between patterns due to differences in cell volume (Figs. 22 and 23). At higher humidity, the peaks shifted to smaller scattering angles, suggesting that the cell volume is proportional to the relative humidity and may increase to incorporate water.

[0159] The DVS isotherm provided in Figure 16 shows the change in water content of form II under the same relative humidity conditions, with kinetic form stability confirmed by variable humidity X-ray powder diffraction. Form II increased by approximately 3.5 wt% from 5% to 55% RH (1 mol / mol water), by 7 wt% from 55% to 85% RH (2 mol / mol water), and by an additional 7 wt% from 85% to 95% RH. The instrument timed out above 85% RH, suggesting that further water absorption is possible if stored under these conditions for a longer period. Hysteresis was observed after desorption. The material was recovered and identified as a mixture of form II and form V. Form II remained unchanged by X-ray powder diffraction for up to 4 days under dry nitrogen or for up to 35 days of storage at 0% RH. It converted to form V within 35 days after exposure to 75% RH.

[0160] The largest unit cell volume observed from the indexing results of Form II among the three obtained index solutions is provided in Figure 17. Assuming the orthorhombic unit cell contains 12 sodium 25HC3S molecules, then... The unit volume of the formula will be approximately larger than that of the anhydrous form (form XIII). This corresponds to (±5%) of the unit cell volume. The volume difference provides enough space to accommodate up to 2 mol / mol of water.

[0161] Weight losses of 5% and 6% were observed at temperatures up to 150 °C using TGA (see Figure 24). Assuming the losses are due to water evaporation, these weight losses correspond to approximately 1.5 and 1.8 mol / mol of water, respectively. These losses occurred concurrently with extensive endothermic dehydration observed in DSC (Figure 25). The DSC curves also showed endothermic events near 167 °C and 189 °C. These events are associated with decomposition.

[0162] The cyclic DSC experiments for form II are shown in Figure 26. Extensive desolvation endothermic reaction was observed in the first heating cycle at up to 130 °C (trace 1), consistent with expectations regarding hydrates. However, no event was observed after cooling (trace 2) or after a second heating (trace 3) up to 130 °C. This indicates that no form change occurred after dehydration, and that the material was anhydrous once reheated to 130 °C. The sample then continued to decompose, as confirmed by the endothermic reactions near 167 °C and 188 °C. The kinetic stability of form II after dehydration at elevated temperatures was confirmed by variable-temperature X-ray powder diffraction experiments generally performed according to Example 19 (Figure 27). Form II was still observed by X-ray powder diffraction at 135 °C. Therefore, the results from variable-temperature X-ray powder diffraction and cyclic DSC experiments suggest that form II can be maintained by complete dehydration. In the same variable-temperature X-ray powder diffraction experiment, decomposition products identified as trisodium hydrogen disulfate Na3H(SO4)2 were found at 170 °C.

[0163] In some cases, form XI, including substantially pure form XI, is provided by crystalline sodium 25HC3S. Form XI is a hydrate of crystalline sodium 25HC3S, which can be prepared as described in Table 1. For example, form XI can be prepared by slurrying in diethyl ether and exposing to ambient conditions for 14 days, filtering, and drying under nitrogen. Thus, it can be prepared in a stable form. Furthermore, Example 37 illustrates the preparation of form XI. Form XI can be characterized by various analytical techniques, including by X-ray powder diffraction. An X-ray powder diffraction pattern of form XI, or a portion thereof, can be used to identify form XI. Form XI contains various X-ray powder diffraction peaks, which, individually or together, may aid in the identification of the presence of form XI.

[0164] In many cases, form XI can be characterized by an X-ray powder diffraction pattern containing a peak at approximately 2.6°2θ. Besides the peak at approximately 2.6°2θ, the X-ray powder diffraction pattern may contain, for example, one or more peaks at approximately 3.1°2θ, approximately 3.5°2θ, and approximately 14.5°2θ. The X-ray powder diffraction pattern of form XI is shown in Figures 6 and 7. Figure 6 or Figure 7 can be used to characterize form XI.

[0165] In some cases, this paper provides form V, a readily soluble mesophase of sodium 25HC3S (also referred to as a liquid crystal), including essentially pure form V. The readily soluble mesophase is induced by the presence of a solvent. Furthermore, concentration and temperature are the determining factors for their phase transition. Form V can be prepared as described in Table 1. For example, transparent and viscous gels are prepared from water with a concentration higher than 0.73 Å. wIt is formed from a mixture of aqueous solvents, methanol, and DMSO, with a water activity greater than 0.73a. w Aqueous solvent systems with water activity and methanol form a gel, form V, exhibiting a disordered X-ray powder diffraction pattern. Form V also forms when form I or form II is exposed to a relative humidity of 75% RH or higher. These gels typically do not exhibit birefringence under polarized light microscopy until a shear force is applied, such as sliding between two glass slides. Optical birefringence suggests an ordered phase with at least one dimension of order. Material flow is also observed. Furthermore, form V can be prepared, for example, as described in Examples 34, 35, and 36.

[0166] The mesophase is characterized by its birefringence, which is absent in amorphous solids or isotropic liquids but present in almost all cases of crystalline solids. Furthermore, the material flows like a liquid upon compression rather than fractures and breaks apart like a solid. As a typical mesophase, the X-ray powder diffraction pattern of form V shows few peaks superimposed on a diffuse reflection background (Figure 3).

[0167] In many cases, form V can be characterized by an X-ray powder diffraction pattern containing a peak at approximately 2.2°2θ. In addition to the peak at approximately 2.2°2θ, the X-ray powder diffraction pattern may contain, for example, one or more peaks at approximately 4.4°2θ, approximately 6.6°2θ, and approximately 8.8°2θ.

[0168] In many cases, form V can be characterized by an X-ray powder diffraction pattern containing peaks at approximately 2.2°2θ and approximately 6.6°2θ, as well as a single peak between approximately 4.0°2θ and approximately 6.0°2θ, and between approximately 4.0°2θ and approximately 6.0°2θ. In some cases, the X-ray powder diffraction pattern may further contain one or more peaks at approximately 8.8°2θ, approximately 9.9°2θ, and approximately 14.9°2θ. X-ray powder diffraction patterns for form V are shown in Figures 3 and 4. Either Figure 3 or Figure 4 can be used to characterize form V.

[0169] By TGA, form V exhibited a 7% weight loss up to 147 °C (Fig. 28), which occurred simultaneously with the extensive endothermic dehydration in DSC (Fig. 29). Decomposition-related events were evident above 148 °C.

[0170] The physical stability of form V was demonstrated by X-ray powder diffraction after 25 days of storage under ambient conditions, with no changes observed. Furthermore, the material remained in form V after exposure to 55°C and vacuum for 1 day. Therefore, stable form V is also disclosed herein. Table 7 below associates the examples that produce form V with the corresponding XRPD diffraction patterns.

[0171] Table 7 - Examples of VXRPD Forms / Figures

[0172]

[0173]

[0174] Table 8 shows the peaks found in Figure 56, while the peaks of the other figures in Table 8 are presented on the figures themselves.

[0175] Table 8 - Figure 56 Peak List

[0176]

[0177] In many cases, form XIII crystalline 25HC3S sodium is provided, including substantially pure form XIII crystalline 25HC3S sodium. Form XIII is anhydrous and is observed by dehydration of form I after exposure to 130°C, 70°C, and vacuum, or exposure to humidity conditions close to 0% RH. Under ambient conditions, form XIII rapidly hydrates to form I. Form XIII can also be seen as follows: dissolved in acetonitrile / H2O solution and dried, as shown in Table 1. For example, form XIII can also be prepared as described in Examples 25 and 26.

[0178] The X-ray powder diffraction pattern of form XIII was successfully indexed, indicating that the pattern represents a single crystalline phase (Fig. 30). The indexing result shows a monoclinic unit cell containing six sodium 25HC3S molecules. Therefore, The unit volume of the (±5%) formula is consistent with the anhydrous form and cannot accommodate any additional solvent or water molecules; the cell volume is... (±5%).

[0179] In many cases, form XIII can be characterized by an X-ray powder diffraction pattern containing a peak at approximately 2.3°2θ. In addition to the peak at approximately 2.3°2θ, the X-ray powder diffraction pattern may contain, for example, one or more peaks at approximately 4.6°2θ, approximately 9.3°2θ, approximately 14.3°2θ, and approximately 15.0°2θ (Figures 8 and 9).

[0180] In many cases, form XIII can be characterized by an X-ray powder diffraction pattern containing peaks at approximately 2.3°2θ, approximately 5.4°2θ, approximately 9.3°2θ, and approximately 11.6°2θ. In some cases, the X-ray powder diffraction pattern may further contain one or more peaks at approximately 4.6°2θ and approximately 15.0°2θ. Either Figure 8 or Figure 9 can be used to characterize form XIII.

[0181] Variable humidity X-ray powder diffraction experiments on a mixture consisting primarily of form I and a small amount of form XIII are presented in Figure 13. During the X-ray powder diffraction analysis, the material was exposed to humidity that was first increased and then decreased. These results provide evidence of rapid interconversion between form I and XIII. The mixture of form I and form XIII was clearly visible at 14% RH. Form XIII showed hydration to form I before reaching 25% RH. Conversely, form I showed dehydration to form XIII within minutes of exposure to 0% RH and remained stable under those conditions.

[0182] In many instances of this disclosure, solvates of crystalline 25HC3S sodium are provided, including crystalline solvates of substantially pure 25HC3S sodium.

[0183] In many cases, form III crystalline sodium 25HC3S is provided, including substantially pure form III crystalline sodium 25HC3S. Form III is an ethanol solvate of crystalline sodium 25HC3S. X-ray powder diffraction patterns of form III are shown in Figures 35, 49, and 50. Form III crystallizes only from experiments involving ethanol. Form III is metastable and desolvates to form II under ambient conditions. When exposed to 58°C and vacuum overnight or at a temperature of 130°C, it desolvates to another anhydrous form, form IX (Figure 31). The preparation of form III is further described in Examples 38-39.

[0184] Table 9 shows the peaks found in Figure 50, while the peaks in Figures 35 and 49 are shown on the figures themselves.

[0185] Table 9-Figure 50 Peak List (Form III)

[0186]

[0187] The X-ray powder diffraction pattern of Form III was successfully indexed, indicating that the pattern represents a single crystalline phase, as seen in Figure 32. The indexing result has a triclinic unit cell containing two sodium 25HC3S molecules. Therefore, The volume per unit volume of the formula is consistent with that of the monoethanolate. The volume is approximately larger per unit volume than that of the anhydrous form (form XIII). The volume difference provides sufficient space to accommodate up to 1 mol / mol of ethanol, which further indicates that form III is a monoethanolide.

[0188] In many cases, form III can be characterized by an X-ray powder diffraction pattern containing a peak at approximately 4.9°2θ. Although the peak at approximately 4.9°2θ is not shown in Table 9, a magnified view of the diffraction pattern in Figure 52 shows the presence of the peak at approximately 4.9°2θ. In addition to the peak at approximately 4.9°2θ, the X-ray powder diffraction pattern may contain, for example, one or more peaks at approximately 6.3°2θ, approximately 7.8°2θ, and approximately 9.8°2θ.

[0189] In many cases, form III can be characterized by peaks at approximately 4.9°2θ and approximately 6.3°2θ. In some cases, the X-ray powder diffraction pattern may further contain one or more peaks at approximately 7.8°2θ, approximately 9.8°2θ, approximately 13.3°2θ, and approximately 15.5°2θ. Form III can be characterized by the X-ray powder diffraction pattern shown in Figure 35.

[0190] Regarding form IX, in many cases, form IX can be characterized by an X-ray powder diffraction pattern containing a peak at approximately 4.9°2θ. The X-ray powder diffraction pattern may contain, for example, one or more peaks at approximately 7.9°2θ, approximately 11.2°2θ, approximately 14.1°2θ, approximately 16.1°2θ, and approximately 16.6°2θ.

[0191] In many cases, form IX can be characterized by an X-ray powder diffraction pattern containing peaks at approximately 2.2°2θ, approximately 4.9°2θ, and approximately 7.9°2θ. In some cases, the X-ray diffraction pattern may further contain one or more peaks at approximately 14.1°2θ, approximately 16.1°2θ, and approximately 16.6°2θ. Form IX can be further characterized by Figure 5.

[0192] No residual organic solvents, such as ethanol, were observed. The 1.6% weight loss observed by TGA at up to 145°C is likely due to water evaporation. DSC showed consistent endothermic dehydration, and events above 150°C were associated with decomposition. Form IX was prepared as described in Examples 32 and 33. Figure 5 corresponds to Example 32, and Figure 51 corresponds to Example 33.

[0193] Table 10 shows the peaks found in Figure 51, while the peaks of Figure 5 are presented in this figure.

[0194] Table 10 - Figure 51 Peak List

[0195]

[0196]

[0197] This disclosure also relates to pharmaceutical compositions containing crystalline 25HC3S sodium as disclosed herein. Such pharmaceutical compositions comprise one or more pharmaceutically acceptable excipients and crystalline 25HC3S sodium as described in this disclosure. Such pharmaceutical compositions can be administered orally or formulated for delivery in any effective conventional dose unit, including, for example, immediate, slow, and timed-release oral articles, parenteral, topical, nasal, ocular, optical, sublingual, rectal, vaginal, etc.

[0198] This disclosure further includes mixtures of sodium 25HC3S in forms such as crystalline sodium 25HC3S and each other and / or with readily soluble forms of sodium 25HC3S. For example, mixtures of two or more of crystalline sodium 25HC3S forms I, II, V, IX, XI, or XIII are provided. The amount of each form present in such mixtures ranges from, for example, from about 0.01% to about 99.9% by weight. Other ranges include, by weight, about 0.1% to about 95%, about 0.1% to about 90%, about 0.1% to about 85%, about 0.1% to about 80%, about 0.1% to about 75%, about 0.1% to about 70%, about 0.1% to about 65%, about 0.1% to about 60%, about 0.1% to about 55%, about 0.1% to about 50%, about 0.1% to about 45%, about 0.1% to about 40%, about 0.1% to about 35%, about 0.1% to about 30%, about 0.1% to about 25%, about 0.1% to about 20%, about 0.1% to about 15%, and about 0.1% to about 10%. Other ranges include about 0.1% to about 9% by weight, about 0.1% to about 8%, about 0.1% to about 7%, about 0.1% to about 6%, about 0.1% to about 5%, about 0.1% to about 4%, about 0.1% to about 3%, about 0.1% to about 2%, and about 0.1% to about 1% by weight. Further ranges include about 0.1% to about 0.9% by weight, about 0.1% to about 0.8%, about 0.1% to about 0.7%, about 0.1% to about 0.6%, about 0.1% to about 0.5%, about 0.1% to about 0.4%, about 0.1% to about 0.3%, and about 0.1% to about 0.2% by weight. Other ranges include about 0.01% to about 0.1% by weight. Other ranges include about 0.01% to about 0.09%, about 0.01% to about 0.08%, about 0.01% to about 0.07%, about 0.01% to about 0.06%, about 0.01% to about 0.05%, about 0.01% to about 0.04%, about 0.01% to about 0.03%, and about 0.01% to about 0.02% by weight. Such mixtures may also be present in pharmaceutical compositions comprising one or more pharmaceutically acceptable excipients.

[0199] This disclosure further includes methods and uses for treating human diseases such as hypercholesterolemia, hypertriglyceridemia, and conditions associated with fat accumulation and inflammation (e.g., non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, and atherosclerosis) with effective amounts of the crystalline 25HC3S sodium of this disclosure and / or pharmaceutical compositions containing crystalline 25HC3S sodium.

[0200] Method for preparing 25-hydroxy-cholestene-5-ene-3-sulfate (25HC3S)

[0201] As summarized above, this disclosure also provides methods for preparing 25-hydroxy-cholestene-5-ene-3-sulfates, such as 25-hydroxy-3β-cholestene-5-ene-3-sulfate (25HC3S). Although much of the teaching herein relates to sulfates at the 3β position, the teachings of this disclosure generally also apply to sulfates at the 3α position. The components used in each step of the subject matter methods described herein for preparing 25-hydroxy-3β-cholestene-5-ene-3-sulfate may be purified compositions or crude compositions, as needed. The term "purified" is used in its conventional sense to refer to a composition in which at least some separation or purification process has been performed, such as filtration of the reaction mixture or an aqueous workup. In some cases, purification includes at least one of liquid chromatography, recrystallization, distillation (e.g., azeotropic distillation), and other types of compound purification. For example, compounds described herein can be purified by chromatographic methods such as high-performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC), thin-layer chromatography, rapid column chromatography, and ion-exchange chromatography. Any suitable stationary phase can be used, including normal-phase and reversed-phase phases, as well as ion exchange resins. The mobile phase can be selected from polar and non-polar solvents. In some cases, the mobile phase includes polar solvents. In some cases, the polar solvent is selected from chloroform, dichloromethane, tetrahydrofuran, dichloroethane, acetone, dioxane, ethyl acetate, dimethyl sulfoxide, aniline, diethylamine, nitromethane, acetonitrile, pyridine, isopropanol, ethanol, methanol, ethylene glycol, acetic acid, and water. In some cases, the mobile phase includes non-polar solvents. In some cases, the non-polar solvent is selected from diethyl ether, toluene, benzene, pentane, hexanes, cyclohexane, petroleum ether, and carbon tetrachloride. See, for example, Introduction to Modern Liquid Chromatography, 2nd ed., L.S. Nyder and J.J. Kirkland, John Wiley and Sons, 1979; and Thin Layer Chromatography, E. Stahl, Springer-Verlag, New York, 1969.

[0202] In some cases, the reaction mixture is used as a crude mixture in subsequent steps of the methods described herein, wherein the reaction mixture is not purified or otherwise post-treated. In some cases, the crude mixture comprises a target compound of sufficient purity relative to the crude reaction mixture (in addition to the solvent, when present), such as where the reaction mixture comprises 70% or more of the target compound, such as 75% or more, such as 80% or more, such as 85% or more, such as 90% or more, such as 95% or more, such as 97% or more, such as 99% or more, such as 99.5% or more, such as 99.9% or more, such as 99.99% or more, and includes 99.999% or more, such as by chromatography (e.g., HPLC or SFC), nuclear magnetic resonance spectroscopy (e.g., 1 H NMR or 13The target compound is determined by C NMR or a combination thereof. In some cases, the target compound is present in the reaction mixture at an amount of 30% by weight or greater relative to the crude reaction mixture (in addition to the solvent, when present), such as 40% by weight or greater relative to the crude reaction mixture, such as 50% by weight or greater, such as 60% by weight or greater, such as 70% by weight or greater, such as 75% by weight or greater, such as 80% by weight or greater, such as 85% by weight or greater, such as 90% by weight or greater, such as 95% by weight or greater, such as 97% by weight or greater, such as 99% by weight or greater, such as 99.5% by weight or greater, such as 99.9% by weight or greater, such as 99.99% by weight or greater, and including 99.999% by weight or greater, and the range can be from 5% by weight to 99.999% by weight, such as 30% by weight to 99.99% by weight, 40% by weight to 99.9% by weight, 50% by weight to 99% by weight, 70% by weight to 95% by weight, 75% by weight to 90% by weight, 80% by weight to 99% by weight, or 80% by weight to 95% by weight. In some cases, the target compound is present in the crude reaction mixture at 30 mol% or more (in addition to the solvent, where present), such as 40 mol% or more, such as 50 mol% or more, such as 60 mol% or more, such as 70 mol% or more, such as 75 mol% or more, such as 80 mol% or more, such as 85 mol% or more, such as 90 mol% or more, such as 95 mol% or more, such as 97 mol% or more, such as 99 mol% or more, such as 99.5 mol% or more, such as 99.9 mol% or more, such as 99.99 mol% or more, and includes 99.999 mol% or more, and the range can be from 30 mol% to 99.999 mol%, such as 50 mol% to 99 mol%, 70 mol% to 95 mol%, 75 mol% to 90 mol%, 80 mol% to 99 mol%, or 80 mol% to 95 mol%.

[0203] A method for preparing a 25-hydroxy-3β-cholestear-5-en-3-sulfate metal salt ([(3S,10R,13R,17R)-17-[(1R)-5-hydroxy-1,5-dimethyl-hexyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecylhydro-1H-cyclopentadien[a]phenanthrene-3-yl]sulfate metal salt) according to the present disclosure comprises: contacting 25-hydroxy-(3β)-cholestear-5-en-3-ol with a sulfating agent to produce a 25-hydroxy-(3β)-cholestear-5-en-3-sulfate organic cationic salt; and contacting the 25-hydroxy-(3β)-cholestear-5-en-3-sulfate organic cationic salt with at least one metal salt to produce a 5-cholestear-3β,25-diol 3-sulfate metal salt (Scheme Ia).

[0204] Solution Ia

[0205]

[0206] Solution IA1

[0207]

[0208] 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated by contact with a sulfating agent (Scheme IA1). In some cases, the sulfating agent is selected from sulfur trioxide complexes, sulfuric acid compounds, sulfonic acid compounds, and sulfonate / ester compounds. In some cases, the sulfating agent is selected from sulfur trioxide dimethylformamide, sulfur trioxide triethylamine, and sulfur trioxide trimethylamine. In some cases, the sulfating agent includes sulfuric acid and acetic anhydride and pyridine. In some cases, the sulfating agent includes sulfur trioxide triethylamine and pyridine. In some cases, the sulfating agent is selected from 1) chlorosulfonic acid and pyridine and 2) chlorosulfonic acid and 2,6-dimethylpyridine. In some cases, the sulfating agent is ethyl chlorosulfonate.

[0209] 25-hydroxy-(3β)-cholest-5-en-3-ol can be sulfated at temperatures ranging from -10°C to 50°C, such as from -5°C to 45°C, such as from -4°C to 40°C, such as from -3°C to 35°C, such as from -2°C to 30°C, such as from -1°C to 25°C, and including from 0°C to 20°C. The reaction can be carried out for durations ranging from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, such as from 0.3 hours to 24 hours, such as from 0.4 hours to 21 hours, such as from 0.5 hours to 20 hours, such as from 0.6 hours to 19 hours, such as from 0.7 hours to 18 hours, such as from 0.8 hours to 17 hours, such as from 0.9 hours to 16 hours, and including from 1 hour to 15 hours. The amount of sulfating agent used relative to 25-hydroxy-(3β)-cholest-5-en-3-ol can vary and can be 0.001 equivalents or more, such as 0.01 equivalents or more, such as 0.1 equivalents or more, such as 0.2 equivalents or more, such as 0.3 equivalents or more, such as 0.4 equivalents or more, such as 0.5 equivalents or more, such as 0.6 equivalents or more, such as 0.7 equivalents or more, such as 0.8 equivalents or more, such as 0.9 equivalents or more, such as 1 equivalent or more, such as 1.1 equivalents or more, such as 1.2 equivalents or more, such as 1.3 equivalents or more, such as 1.4 equivalents or more, such as 1.5 equivalents or more, such as 1.6 equivalents or more, such as 1.7 equivalents or more, such as 1.8 equivalents or more, such as 1.9 equivalents or more, such as 2 equivalents or more, such as 3 equivalents or more. More, such as 4 equivalents or more, such as 5 equivalents or more, and including 10 equivalents or more, and relative to 25-hydroxy-(3β)-cholest-5-en-3-ol, the range can be from 0.001 equivalents to 10 equivalents, such as 0.1 equivalents to 10 equivalents, 0.1 equivalents to 8 equivalents, 0.1 equivalents to 5 equivalents, 0.5 equivalents to 10 equivalents, 0.5 equivalents to 8 equivalents, 0.5 equivalents to 5 equivalents, 0.9 Equivalent to 10 equivalents, 0.9 equivalents to 8 equivalents, 0.9 equivalents to 5 equivalents, 1.3 equivalents to 10 equivalents, 1.3 equivalents to 8 equivalents, 1.3 equivalents to 5 equivalents, 1.5 equivalents to 10 equivalents, 1.5 equivalents to 8 equivalents, 1.5 equivalents to 5 equivalents, 2 equivalents to 10 equivalents, 2 equivalents to 8 equivalents, 2 equivalents to 5 equivalents, or 1 equivalent to 2 equivalents, 1 equivalent to 1.5 equivalents, or 1.1-1.2 equivalents.

[0210] In some cases, the method includes sulfating 25-hydroxy-(3β)-cholest-5-en-3-ol in at least one solvent, wherein the 25-hydroxy-(3β)-cholest-5-en-3-sulfate product exhibits low solubility. In some cases, 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated in at least one solvent, wherein the 25-hydroxy-(3β)-cholest-5-en-3-sulfate product exhibits a solubility of 100 mmol / L or less, such as 90 mmol / L or less, such as 80 mmol / L or less, such as 70 mmol / L or less, such as 60 mmol / L or less, such as 50 mmol / L or less, such as 40 mmol / L or less, such as 30 mmol / L or less, such as 20 mmol / L or less, such as 10 mmol / L or less, and includes sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol in at least one solvent, wherein the 25-hydroxy-(3β)-cholest-5-en-3-sulfate product exhibits a solubility of 5 mmol / L or less. In some cases, 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated in at least one solvent, wherein the 25-hydroxy-(3β)-cholest-5-en-3-sulfate product precipitates after formation. In some cases, the at least one solvent is selected from chloroform, dichloromethane, acetone, acetonitrile, toluene, tetrahydrofuran, and methyltetrahydrofuran.

[0211] In some cases, the method involves sulfating 25-hydroxy-(3β)-cholest-5-en-3-ol in a manner sufficient to reduce or eliminate disulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol. In certain circumstances, 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated, and the amount of the disulfate product formed (i.e., 5-cholest-3β-25-diol disulfate, structure IA) is 10% by weight or less of the reaction product formed by contacting 25-hydroxy-(3β)-cholest-5-en-3-ol with a sulfating agent, such as 9% by weight or less, such as 8% by weight or less, such as 7% by weight or less, such as 6% by weight or less, such as 5% by weight or less, such as 4% by weight or less, such as 3% by weight or less, such as 2% by weight or less, such as 1% by weight or less, such as 0.5% by weight or less, such as 0.1% by weight or less, such as 0.01% by weight or less, such as 0.001% by weight or less, and includes cases where 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated. The amount of disulfate product formed is 0.0001% by weight or less, and the range can be from 10% by weight to 0.001% by weight, such as 10% by weight to 0.1% by weight, 10% by weight to 1% by weight, 10% by weight to 2% by weight, 8% by weight to 0.001% by weight, 8% by weight to 0.1% by weight, 8% by weight to 1% by weight, 8% by weight to 2% by weight, 6% by weight to 0.001% by weight, 6% by weight to 0.1% by weight, 6% by weight to 1% by weight, 6% by weight to 2% by weight, 4% by weight to 0.001% by weight, 4% by weight to 0.1% by weight, 4% by weight to 1% by weight, 4% by weight to 2% by weight, 3% by weight to 0.001% by weight, 3% by weight to 0.1% by weight, 3% by weight to 1% by weight, 2% by weight to 0.001% by weight, 2% by weight to 0.1% by weight, or 2% by weight to 1% by weight.

[0212] In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the formed 5-cholest-3β-25-diol-disulfate is 10:1 or more, such as 25:1 or more, such as 50:1 or more, such as 100:1 or more, such as 250:1 or more, such as 500:1 or more, such as 1000:1 or more, such as 2500:1 or more, such as 5000:1 or more, such as 10,000:1 or more, such as 25,000:1 or more, such as 50,000:1 or more, such as 100,000:1 or more, such as 10 6 :1 or more, such as 10 7 :1 or more, such as 10 8:1 or more, including cases where the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the formed 5-cholest-3β-25-diol-disulfate is 10. 9 :1 or more, and the range can be from a weight ratio of 10:1 to 10 9 A weight ratio of 1:1, such as a weight ratio of 10:1 up to 10:1. 6 A weight ratio of 1:1, a weight ratio of 10:1 to 10:1 3 The weight ratio is 1:1, from 10:1 to 100:1, and from 100:1 to 10:1. 9 A weight ratio of 1:1, a weight ratio of 100:1 to 10 6 A weight ratio of 1:1, a weight ratio of 100:1 to 10 3 A weight ratio of 1:1, or 250:1 up to 10. 9 A weight ratio of 1:1, or 250:1 up to 10. 6 A weight ratio of 1:1, or 250:1 up to 10. 3 A weight ratio of 1:1, or 500:1 to 10. 9 A weight ratio of 1:1, or 500:1 to 10. 6 A weight ratio of 1:1, or 500:1 to 10. 3 A weight ratio of 1:1, 10 3 The weight ratio is up to 10:1. 9 A weight ratio of 1:1, 10 3 The weight ratio is up to 10:1. 6 A weight ratio of 1:1, or a weight ratio of 250:1 up to 10. 3 The weight ratio is 1:1.

[0213]

[0214] In some cases, 5-cholesten-3β-25-diol-disulfate, formed upon sulfation of 25-hydroxy-(3β)-cholesten-5-en-3-ol, remains soluble in at least one solvent. In some cases, the 5-cholesten-3β-25-diol-disulfate exhibits high solubility in at least one solvent. In some cases, the 5-cholesten-3β-25-diol-disulfate exhibits solubility of 500 mmol / L or greater, such as 600 mmol / L or greater, such as 700 mmol / L or greater, such as 800 mmol / L or greater, such as 900 mmol / L or greater, and includes solubility of 1 mol / L or more in at least one solvent.

[0215] In some cases, the method further includes separating the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product from the disulfate product (i.e., 5-cholest-3β-25-diol-disulfate). In some cases, the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product from the disulfate product is separated by vacuum filtration. In some cases, the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product from the disulfate product is separated by recrystallization of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product from the disulfate product. In some cases, the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product from the disulfate product is separated by chromatography (e.g., silica gel column chromatography).

[0216] In some cases, 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated in a reaction mixture having a pH ranging from 5.0 to 8.0, such as pH from 5.1 to 7.9, such as pH from 5.2 to 7.8, such as pH from 5.3 to 7.7, such as pH from 5.4 to 7.6, such as pH from 5.5 to 7.5, such as pH from 5.6 to 7.4, such as pH from 5.7 to 7.3, such as pH from 5.8 to 7.2, such as pH from 5.9 to 7.1, and includes 25-hydroxy-(3β)-cholest-5-en-3-ol sulfated in a reaction mixture having a pH ranging from 6.0 to 7.0.

[0217] In some cases, 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated in the presence of an organic cationic salt of 25-hydroxy-(3β)-cholest-5-en-3-sulfate. In some cases, the organic cationic salt of 25-hydroxy-(3β)-cholest-5-en-3-sulfate is present as particles (e.g., seed crystals of the organic cationic salt of 25-hydroxy-(3β)-cholest-5-en-3-sulfate produced in a previous reaction or purification batch). In some cases, sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol in the presence of organic cationic salts of 25-hydroxy-(3β)-cholest-5-en-3-ol (e.g., as particles) is sufficient to reduce the solubility of organic cationic salts of 25-hydroxy-(3β)-cholest-5-en-3-ol generated by the reaction of a sulfating agent with 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, the solubility of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt produced in the reaction mixture is reduced by 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, and includes reducing the solubility of the produced 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt by 99% or more. The size of the organic cationic salt 25-hydroxy-(3β)-cholest-5-en-3-sulfate added to the reaction mixture can vary and can have dimensions (e.g., length, width, or diameter) of 0.01 mm or greater, such as 0.025 mm or greater, such as 0.05 mm or greater, such as 0.075 mm or greater, such as 0.1 mm or greater, such as 0.25 mm or greater, such as 0.5 mm or greater, such as 0.75 mm or greater, such as 1 mm or greater, such as 2 mm or greater, such as 3 mm or greater, such as 4 mm or greater, and including 5 mm or greater. In some cases, the particles of the organic cationic salt 25-hydroxy-(3β)-cholest-5-en-3-ol are added to the reaction mixture immediately after contacting the sulfating agent with 25-hydroxy-(3β)-cholest-5-en-3-ol.In some cases, particles of the organic cationic salt of 25-hydroxy-(3β)-cholest-5-en-3-ol are added to the reaction mixture 1 minute or longer, such as 5 minutes or longer, such as 10 minutes or longer, such as 15 minutes or longer, such as 20 minutes or longer, such as 30 minutes or longer, such as 40 minutes or longer, such as 50 minutes or longer, after contacting the sulfating agent with 25-hydroxy-(3β)-cholest-5-en-3-ol, and including adding particles of the organic cationic salt of 25-hydroxy-(3β)-cholest-5-en-3-ol to the reaction mixture 60 minutes or longer after contacting the sulfating agent with 25-hydroxy-(3β)-cholest-5-en-3-ol.

[0218] In some cases, the sulfated reagent is characterized prior to contact with 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, characterizing the sulfated reagent includes determining the degree of degradation of the sulfated reagent prior to contact with 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, determining the degree of degradation of the sulfated reagent includes determining the amount of impurities in the sulfated reagent prior to contact with 25-hydroxy-(3β)-cholest-5-en-3-ol.

[0219] In some cases, proton nuclear magnetic resonance spectroscopy (NMR spectroscopy) 1 The degradation of the sulfating agent can be determined by H-NMR. The sulfating agent can be sulfated by proton NMR spectroscopy in at least one deuterated solvent. In some cases, the at least one deuterated solvent is deuterated acetone ((CD3)2CO). In some cases, the at least one deuterated solvent is not deuterated benzene (C6D6). In some cases, the at least one deuterated solvent is not deuterated acetonitrile (CD3CN). In some cases, the at least one deuterated solvent is not deuterated chloroform (CD3Cl).

[0220] In some cases, methods for determining the extent of degradation include integrating the chemical shift from 9.2 ppm to 9.3 ppm. 1 One or more peaks in the H-NMR spectrum, and the impurity level of the sulfation reagent calculated based on the integrated peaks. In some cases, methods for determining the extent of degradation include integrating the chemical shift at approximately 9.25 ppm. 1One or more peaks in the H-NMR spectrum, and the impurity level of the sulfating reagent calculated based on the integrated peaks. In some cases, when the impurity level of the sulfating reagent is below a predetermined threshold, such as when the impurity level determined by integrating one or more peaks in the proton NMR spectrum from 9.2 ppm to 9.3 ppm is 25% or lower, such as 24% or lower, such as 23% or lower, such as 22% or lower, such as 21% or lower, such as 20% or lower, such as 19% or lower, such as 18% or lower, such as 17% or lower, such as 16% or lower, such as 15% or lower, such as 14% or lower, such as 13%. Or lower, such as 12% or lower, such as 11% or lower, such as 10% or lower, such as 9% or lower, such as 8% or lower, such as 7% or lower, such as 6% or lower, such as 5% or lower, such as 4% or lower, such as 3% or lower, such as 2% or lower, and including cases where the impurity level determined by one or more peaks in the chemical shift integral proton NMR spectrum from 9.2 ppm to 9.3 ppm is 1% or lower, contacting the sulfation reagent with 25-hydroxy-(3β)-cholest-5-en-3-ol. In certain circumstances, when the impurity level is above a predetermined threshold, such as when the impurity level determined by one or more peaks in the chemical shift integrated proton NMR spectrum from 9.2 ppm to 9.3 ppm is 25% or more, such as 26% or more, such as 27% or more, such as 28% or more, such as 29% or more, such as 30% or more, such as 31% or more, such as 32% or more, such as 33% or more, such as 34% or more, and including cases where the impurity level determined by one or more peaks in the chemical shift integrated proton NMR spectrum from 9.2 ppm to 9.3 ppm is 35% or more, the sulfation reagent is not contacted with 25-hydroxy-(3β)-cholest-5-en-3-ol.

[0221] In some cases, the resulting 25-hydroxy-(3β)-cholest-5-ene-3-sulfate product includes one or more byproducts. In some cases, the byproduct is 5-cholest-3β-25-diol-disulfate. In some cases, the 5-cholest-3β-25-diol-disulfate byproduct is present relative to 25-hydroxy-(3β)-cholest-5-ene-3-sulfate in amounts of 10% by weight or less, such as 9% by weight or less, such as 8% by weight or less, such as 7% by weight or less, such as 6% by weight or less, such as 5% by weight or less, such as 4% by weight or less, such as 3% by weight or less, such as 2% by weight or less, such as 1% by weight or less, such as 0.5% by weight or less, such as 0.1% by weight or less, such as 0.01% by weight. The 5-cholestear-3β-2,5-diol disulfate byproduct is present in the composition produced by sulfated 2,5-hydroxy-(3β)-cholestear-5-en-3-ol in an amount of 0.001% or less, such as 0.001% or less, and may range from 0.1% to 50% by weight, such as 0.5% to 20% by weight or 1% to 12% by weight. In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the formed 5-cholest-3β-25-diol-disulfate byproduct is 10:1 or more, such as 25:1 or more, such as 50:1 or more, such as 100:1 or more, such as 250:1 or more, such as 500:1 or more, such as 1000:1 or more, such as 2500:1 or more, such as 5000:1 or more, such as 10,000:1 or more, such as 25,000:1 or more, such as 50,000:1 or more, such as 100,000:1 or more, such as 10 6 :1 or more, such as 10 7 :1 or more, such as 10 8 :1 or more, including cases where the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the formed 5-cholest-3β-25-diol-disulfate is 10. 9 :1 or more. In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and the formed 5-cholest-3β-25-diol-disulfate ranges from 10:1 to 10:1. 9 :1, such as from 100:1 to 10 8 :1, such as from 1000:1 to 10 7 :1, and includes values ​​from 10000:1 to 10 6 :1.

[0222] This disclosure also includes compositions having 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and 5-cholest-3β-25-diol-disulfate, said 5-cholest-3β-25-diol-disulfate being present in the composition in amounts relative to said 25-hydroxy-(3β)-cholest-5-ene-3-sulfate as follows: 10% by weight or less, such as 9% by weight or less, such as 8% by weight or less, such as 7% by weight or less, such as 6% by weight or less, such as 5% by weight or less, such as 4% by weight or less, such as 3% by weight or less, such as 2% by weight or less, such as 1% by weight or less, such as 0.5% by weight or less, such as 0.1% by weight or less, such as 0.01% by weight or less, such as 0.001% by weight or less, and including 0.00 1 wt% or less, and the range can be from 10 wt% to 0.001 wt%, such as 10 wt% to 0.1 wt%, 10 wt% to 1 wt%, 10 wt% to 2 wt%, 8 wt% to 0.001 wt%, 8 wt% to 0.1 wt%, 8 wt% to 1 wt%, 8 wt% to 2 wt%, 6 wt% to 0.001 wt%, 6 wt% to 0.1 wt%, 6 wt% to 1 wt%, 6 wt% to 2 wt%, 4 wt% to 0.001 wt%, 4 wt% to 0.1 wt%, 4 wt% to 1 wt%, 4 wt% to 2 wt%, 3 wt% to 0.001 wt%, 3 wt% to 0.1 wt%, 3 wt% to 1 wt%, 2 wt% to 0.001 wt%, 2 wt% to 0.1 wt%, or 2 wt% to 1 wt%.

[0223] In some cases, the composition comprises 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and 5-cholest-3β-25-diol-disulfate in a weight ratio of 10:1 or more, such as 25:1 or more, such as 50:1 or more, such as 100:1 or more, such as 250:1 or more, such as 500:1 or more, such as 1000:1 or more, such as 2500:1 or more, such as 5000:1 or more, such as 10,000:1 or more, such as 25,000:1 or more, such as 50,000:1 or more, such as 100,000:1 or more, such as 10 6 :1 or more, such as 10 7 :1 or more, such as 10 8 :1 or more, and including cases where the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to 5-cholest-3β-25-diol-disulfate in the composition is 10. 9 :1 or more. In some cases, the composition includes components from 10:1 to 10 925-hydroxy-(3β)-cholest-5-ene-3-sulfate and 5-cholest-3β-25-diol-disulfate in a weight ratio within the range of 1:1, such as from 100:1 to 10... 8 :1, such as from 1000:1 to 10 7 :1, and includes values ​​from 10000:1 to 10 6 :1.

[0224] In some cases, the byproduct is a sulfated sterol (structure IB).

[0225]

[0226] In some cases, sulfated sterol ([(3S,8S,9S,10R,13R,14S,17R)-17-[(1R)-1,5-dimethylhex-4-enyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecylhydro-1H-cyclopentadieno[a]phenanthrene-3-yl] sulfate) is present in the composition produced by sulfated 25-hydroxy-(3β)-cholest-5-en-3-ol in an amount of 10% by weight or less, such as 9% by weight or less, such as 8% by weight or less, such as 7% by weight or less, such as 6 ...8% by weight or less, such as 8% by weight or less, such as 7% by weight or less, such as 6% by weight or less, such as 8% by weight or less, such as 8% by weight or less, such as 7% by weight or less, such as 6% by weight or less, such as 8% by weight or less, such as 8% by weight or less, such as 8% by weight or less, such as 7% by weight or less, such as 6% by weight or less, such as 8% by weight or less, such as 8% by weight or less, such as 8% by weight or less, such as 7% by weight or less, such as 6% by weight or less, Lower, such as 5% by weight or less, such as 4% by weight or less, such as 3% by weight or less, such as 2% by weight or less, such as 1% by weight or less, such as 0.5% by weight or less, such as 0.1% by weight or less, such as 0.01% by weight or less, such as 0.001% by weight or less, and including cases where the sulfated sterol is present in the composition produced by sulfated 25-hydroxy-(3β)-cholest-5-en-3-ol in an amount of 0.001% by weight or less relative to 25-hydroxy-(3β)-cholest-5-en-3-ol, and the range can be from 0.1% by weight to 10% by weight, such as 0.2% by weight to 5% by weight or 0.3% by weight to 3% by weight. In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the sulfated sterol formed is 10:1 or more, such as 25:1 or more, such as 50:1 or more, such as 100:1 or more, such as 250:1 or more, such as 500:1 or more, such as 1000:1 or more, such as 2500:1 or more, such as 5000:1 or more, such as 10,000:1 or more, such as 25,000:1 or more, such as 50,000:1 or more, such as 100,000:1 or more, such as 10 6 :1 or more, such as 10 7 :1 or more, such as 108 :1 or more, including cases where the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the sulfated sterol formed is 10. 9 :1 or more. In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the sulfated sterol formed ranges from 10:1 to 10:10. 9 :1, such as from 100:1 to 10 8 :1, such as from 1000:1 to 10 7 :1, and includes values ​​from 10000:1 to 10 6 :1.

[0227] This disclosure also includes compositions having 25-hydroxy-(3β)-cholesterol-5-ene-3-sulfate and sulfated sterol, wherein the sulfated sterol is present in the composition in an amount of 10% by weight or less relative to 25-hydroxy-(3β)-cholesterol-5-ene-3-sulfate, such as 9% by weight or less, such as 8% by weight or less, such as 7% by weight or less, such as 6% by weight or less, such as 5% by weight or less, such as 4% by weight or less, such as 3% by weight or less, such as 2% by weight or less, such as 1% by weight or less, such as 0.5% by weight or less, such as 0.1% by weight or less, such as 0.01% by weight or less, such as 0.001% by weight or less, and comprising 0.00% by weight relative to 25-hydroxy-(3β)-cholesterol-5-ene-3-sulfate. 1% w / w or less, and the range can be from 10 wt% to 0.001 wt%, such as 10 wt% to 0.1 wt%, 10 wt% to 1 wt%, 10 wt% to 2 wt%, 8 wt% to 0.001 wt%, 8 wt% to 0.1 wt%, 8 wt% to 1 wt%, 8 wt% to 2 wt%, 6 wt% to 0.001 wt%, 6 wt% to 0.1 wt%, 6 wt% to 1 wt%, 6 wt% to 2 wt%, 4 wt% to 0.001 wt%, 4 wt% to 0.1 wt%, 4 wt% to 1 wt%, 4 wt% to 2 wt%, 3 wt% to 0.001 wt%, 3 wt% to 0.1 wt%, 3 wt% to 1 wt%, 2 wt% to 0.001 wt%, 2 wt% to 0.1 wt%, or 2 wt% to 1 wt%.

[0228] In some cases, the composition comprises 10:1 or more by weight of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and sulfated sterol, such as 25:1 or more, such as 50:1 or more, such as 100:1 or more, such as 250:1 or more, such as 500:1 or more, such as 1000:1 or more, such as 2500:1 or more, such as 5000:1 or more, such as 10,000:1 or more, such as 25,000:1 or more, such as 50,000:1 or more, such as 100,000:1 or more, such as 10 6 :1 or more, such as 10 7 :1 or more, such as 10 8 The composition contains 1 or more, and includes cases where the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to sulfated sterol is 10. 9 :1 or more. In some cases, the composition includes components from 10:1 to 10 9 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and sulfated sterols in a weight ratio ranging from 100:1 to 10:1 8 :1, such as from 1000:1 to 10 7 :1, and includes values ​​from 10000:1 to 10 6 :1.

[0229] In some cases, the byproduct of sulfated 25-hydroxy-(3β)-cholesterol in the 25-hydroxy-(3β)-cholesterol-5-en-3-sulfate composition is a thermal degradation product. In some cases, the byproduct is identified by relative retention time when separating components of the 25-hydroxy-(3β)-cholesterol-5-en-3-sulfate composition by liquid chromatography (e.g., HPLC). In some cases, the byproduct is a sulfated sterol, which has a retention time of about 18.3 minutes when separated by HPLC with a C8 stationary phase running at about 45°C and using a first mobile phase containing a buffer (e.g., an aqueous buffer containing sodium phosphate) and a second mobile phase containing one or more organic solvents (see, for example, Tables 13 and 14 below). In some cases, the first mobile phase is an aqueous buffer. In some cases, the first mobile phase comprises sodium phosphate. In some cases, the second mobile phase is selected from one or more of methoxypropyl acetate, acetonitrile, and methanol. In some cases, the flow rate of the first mobile phase is about 1.0 mL / min. In some cases, the flow rate of the second mobile phase is about 1.0 mL / min or greater. In some cases, 25-hydroxy-(3β)-cholesterol-5-ene-3-sulfate has a retention time of about 7.7 min under the same HPLC conditions. In some cases, when the components of the 25-hydroxy-(3β)-cholesterol-5-ene-3-sulfate composition are separated by HPLC running at about 45 °C with a C8 stationary phase and the components of the composition are separated by a first mobile phase containing a buffer (e.g., an aqueous buffer containing sodium phosphate) and a second mobile phase containing one or more organic solvents (see, for example, Tables 13 and 14 below), the byproduct is a compound with a retention time of about 37.7 min. Although not wishing to be bound by theory, it is believed that the compound with a retention time of about 37.7 min is a sterol. In some cases, the first mobile phase is an aqueous buffer. In some cases, the first mobile phase comprises sodium phosphate. In some cases, the second mobile phase is selected from one or more of methoxypropyl acetate, acetonitrile, and methanol. In some cases, the flow rate of the first mobile phase is about 1.0 mL / min. In some cases, the flow rate of the second mobile phase is about 1.0 mL / min or greater. In some cases, 25-hydroxy-(3β)-cholest-5-ene-3-sulfate has a retention time of about 7.7 min under the same HPLC conditions, resulting in a relative retention time of about 2.4 (=18.3 / 7.7) for sulfated sterols, and a relative retention time of about 4.9 (=37.7 / 7.7) for compounds considered as sterols.

[0230] This disclosure also includes compositions having one or more byproducts of 25-hydroxy-(3β)-cholest-5-en-3-sulfate and sulfated 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, the one or more byproducts are present in the composition in an amount of 10% by weight or less relative to 25-hydroxy-(3β)-cholest-5-ene-3-sulfate, such as 9% by weight or less, such as 8% by weight or less, such as 7% by weight or less, such as 6% by weight or less, such as 5% by weight or less, such as 4% by weight or less, such as 3% by weight or less, such as 2% by weight or less, such as 1% by weight or less, such as 0.5% by weight or less, such as 0.1% by weight or less, such as 0.01% by weight or less, such as 0.001% by weight or less, and include 0.001% by weight or less, and the range can be from 0.1% by weight to 5% by weight, such as 0.2% by weight to 10% by weight or 0.3% by weight to 15% by weight. In some cases, the composition comprises 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and one or more byproducts, said byproducts being present in amounts ranging from 0.0001 wt% to 10 wt% relative to 25-hydroxy-(3β)-cholest-5-ene-3-sulfate, such as from 0.005 wt% to 9.5 wt%, such as from 0.001 wt% to 9.0 wt%, such as from 0.05 wt% to 8.5 wt%, such as from 0.1 wt% to 8.0 wt%, such as from 0.5 wt% to 7.5 wt%, such as from 1 wt% to 7 wt%, such as from 1.5 wt% to 6.5 wt%, and including from 2 wt% to 6 wt%.

[0231] In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to one or more byproducts formed is 10:1 or more, such as 25:1 or more, such as 50:1 or more, such as 100:1 or more, such as 250:1 or more, such as 500:1 or more, such as 1000:1 or more, such as 2500:1 or more, such as 5000:1 or more, such as 10,000:1 or more, such as 25,000:1 or more, such as 50,000:1 or more, such as 100,000:1 or more, such as 10 6 :1 or more, such as 10 7 :1 or more, such as 10 8 :1 or more, including cases where the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to one or more byproducts formed is 10. 9 :1 or more. In some cases, the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to one or more byproducts formed ranges from 10:1 to 10:10.9 :1, such as from 100:1 to 10 8 :1, such as from 1000:1 to 10 7 :1, and includes values ​​from 10000:1 to 10 6 :1.

[0232] In some cases, the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate is a pyridinium salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate (Scheme IA2).

[0233] Solution IA2

[0234]

[0235] In some cases, the sulfating agent is contacted with the acid anhydride before contacting 25-hydroxy-(3β)-cholest-5-en-3-ol. In some cases, the acid anhydride is selected from acetic anhydride, trifluoroacetic anhydride, and trifluoromethanesulfonic anhydride. The amount of acid anhydride relative to 25-hydroxy-(3β)-cholest-5-en-3-ol can vary and can be 0.001 equivalents or more, such as 0.2 equivalents or more, such as 0.3 equivalents or more, such as 0.4 equivalents or more, such as 0.5 equivalents or more, such as 0.6 equivalents or more, such as 0.7 equivalents or more, such as 0.8 equivalents or more, such as 0.9 equivalents or more, such as 1 equivalent or more, such as 1.1 equivalents or more, such as 1.2 equivalents or more, such as 1.3 equivalents or more, such as 1.4 equivalents or more, such as 1.5 equivalents or more, such as 1.6 equivalents or more, such as 1.7 equivalents or more, such as 1.8 equivalents or more, such as 1.9 equivalents or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more. More, including 10 equivalents or more, and the range relative to 25-hydroxy-(3β)-cholest-5-en-3-ol can be from 0.001 equivalents to 10 equivalents, such as 0.1 equivalents to 10 equivalents, 0.1 equivalents to 8 equivalents, 0.1 equivalents to 5 equivalents, 0.5 equivalents to 10 equivalents, 0.5 equivalents to 8 equivalents, 0.5 equivalents to 5 equivalents, 0.9 equivalents to 10 equivalents, 0.9 Equivalent to 8, 0.9 to 5, 1.3 to 10, 1.3 to 8, 1.3 to 5, 1.5 to 10, 1.5 to 8, 1.5 to 5, 2 to 10, 2 to 8, 2 to 5, 0.1 to 1.5, 0.5 to 1.1, or 0.1 to 1.

[0236] In some cases, the method includes quenching (i.e., inactivating) unreacted sulfating agents after the formation of an organic cationic salt of 25-hydroxy-(3β)-cholest-5-en-3-sulfate. In some cases, quenching the sulfating agent includes adding water to the reaction mixture. The amount of water added to the reaction mixture may vary relative to the amount of sulfating agent contacted with 25-hydroxy-(3β)-cholest-5-en-3-ol, and may be 1 equivalent or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, such as 6 equivalents or more, such as 7 equivalents or more, such as 8 equivalents or more, such as 9 equivalents or more, such as 10 equivalents or more, such as 15 equivalents or more, such as 20 equivalents or more, and includes 25 equivalents or more.

[0237] In some cases, quenching the reactivity of unreacted sulfating agents involves adding water to the reaction mixture, followed by the addition of at least one base. In some cases, the at least one base is a trialkylamine, such as trimethylamine or triethylamine. In some cases, the at least one base is 2,6-dimethylpyridine. In some cases, the at least one base is pyridine. Pyridine may be added to the reaction mixture 1 minute or longer after the addition of water, such as 5 minutes or longer, such as 10 minutes or longer, such as 15 minutes or longer, such as 30 minutes or longer, such as 45 minutes or longer, such as 60 minutes or longer, such as 90 minutes or longer, such as 120 minutes or longer, such as 150 minutes or longer, such as 180 minutes or longer, such as 210 minutes or longer, and includes 240 minutes or longer after the addition of water to the reaction mixture. In some cases, pyridine is added to the reaction mixture 60 minutes after the addition of water. The amount of pyridine added to the reaction mixture may vary relative to the amount of sulfating agent and may be 0.001 equivalents or more, such as 0.005 equivalents or more, such as 0.01 equivalents or more, such as 0.05 equivalents or more, such as 0.1 equivalents or more, such as 0.5 equivalents or more, such as 1 equivalent or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, such as 6 equivalents or more, and includes 10 equivalents or more.

[0238] In some cases, unreacted sulfating agents in the reaction mixture are quenched under slow stirring. In some cases, quenching unreacted sulfating agents under slow stirring includes stirring the reaction mixture in a manner sufficient to maintain agglomerates of unreacted sulfating agents in the reaction mixture. In some cases, slow stirring of the reaction mixture is sufficient to cause the agglomerates of unreacted sulfating agents to reduce in size by 10% or less during quenching, such as 9% or less, such as 8% or less, such as 7% or less, such as 6% or less, such as 5% or less, such as 4% or less, such as 3% or less, such as 2% or less, such as 1% or less, and includes cases where the reaction mixture is slowly stirred such that the agglomerates of unreacted sulfating agents reduce in size by 0.1% or less during quenching. In some cases, slow stirring of the reaction mixture is sufficient to keep the agglomerates of unreacted sulfating agents at the bottom of the reaction flask during quenching. In some cases, slow stirring of the reaction mixture is sufficient to result in little or no agglomerates of unreacted sulfating agents present in the stirring vortex of the stirred reaction mixture.

[0239] In some cases, the method includes purifying the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt before contacting it with at least one metal salt. In some cases, the purified 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt has a purity of 97% or greater, such as 98% or greater, such as 99% or greater, such as 99.5% or greater, such as 99.7% or greater, such as 99.9% or greater, and including 99.99% or greater. In some cases, purified organic cationic salts of 25-hydroxy-(3β)-cholest-5-en-3-sulfate have one or more sulfation byproducts (e.g., byproducts from the sulfated 25-hydroxy-(3β)-cholest-5-en-3-ol), wherein one or more byproducts are present relative to the organic cationic salt of 25-hydroxy-(3β)-cholest-5-en-3-sulfate in an amount of 5% w / w or less, such as 4% w / w or less, such as 3% w / w or less, such as 2% w / w or less, such as 1% w / w or less, such as 0.9%. In amounts of w / w or less, such as 0.8% w / w or less, such as 0.7% w / w or less, such as 0.6% w / w or less, such as 0.5% w / w or less, such as 0.4% w / w or less, such as 0.3% w / w or less, such as 0.2% w / w or less, such as 0.1% w / w or less, such as 0.05% w / w or less, such as 0.01% w / w or less, and including amounts of 0.001% w / w or less relative to the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate. In some cases, the disulfation product (i.e., 5-cholestene-3β-25-diol-disulfate) is present in the purified 25-hydroxy-(3β)-cholestene-5-ene-3-sulfate organic cationic salt composition in an amount of 1% w / w or less relative to the 25-hydroxy-(3β)-cholestene-5-ene-3-sulfate organic cationic salt, such as in an amount of 0.9% w / w or less, such as 0.8% w / w or less, such as 0.7% w / w or less. Less, such as 0.6% w / w or less, such as 0.5% w / w or less, such as 0.4% w / w or less, such as 0.3% w / w or less, such as 0.2% w / w or less, such as 0.1% w / w or less, such as 0.05% w / w or less, such as 0.01% w / w or less, and including the presence of an organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate in an amount of 0.001% w / w or less.

[0240] In some cases, 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salts are purified by liquid chromatography. In some cases, purification of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salts involves liquid chromatography using a silica gel stationary phase (e.g., a silica gel plug column, ≥5 mass equivalents). In some cases, 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salts are purified using a silica gel stationary phase and a mobile phase comprising pyridine. In some cases, the mobile phase comprises dichloromethane, methanol, and pyridine. In some cases, the mobile phase comprises a mixture of dichloromethane-methanol (85:15) and pyridine (1%).

[0241] In some cases, one or more fractions collected from the stationary phase can be combined. In some cases, the combined fractions can be concentrated. In some cases, the combined fractions are concentrated by distillation. In some cases, the combined fractions are concentrated under vacuum. In some cases, the combined fractions are concentrated by distillation under vacuum.

[0242] In some cases, contacting the combined fractions with one or more particles of an organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate (e.g., particles from a previously purified sample of the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate is sufficient to precipitate the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate from the combined fractions. In some cases, contacting the particles of the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate with the combined fractions includes adding the particles during the distillation of the combined fractions. In some cases, adding the particles of the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the combined fractions prior to distillation of the combined fractions is performed. In some cases, particles of the organic cation salt 25-hydroxy-(3β)-cholest-5-ene-3-sulfate are added to the combined fractions at the same time as distillation, such as 1 minute or longer after the start of distillation, such as 5 minutes or longer, such as 10 minutes or longer, such as 15 minutes or longer, such as 20 minutes or longer, such as 30 minutes or longer, such as 40 minutes or longer, such as 50 minutes or longer, and including adding particles of the organic cation salt 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the combined fractions at 60 minutes or longer after the start of distillation of the combined fractions. In some cases, fractions distilled and combined under constant pressure may vary such that the pressure changes by 10% or less, such as 9% or less, such as 8% or less, such as 7% or less, such as 6% or less, such as 5% or less, such as 4% or less, such as 3% or less, such as 2% or less, such as 1% or less, and include changes of 0.1% or less. In some cases, the pressure changes during distillation by 10 inHg or less, such as 9 inHg or less, such as 8 inHg or less, such as 7 inHg or less, such as 6 inHg or less, such as 5 inHg or less, such as 4 inHg or less, such as 3 inHg or less, such as 2 inHg or less, such as 1 inHg or less, such as 0.5 inHg or less, such as 0.1 inHg or less, such as 0.05 inHg or less, and include changes of 0.01 inHg or less. In some cases, fractions are combined by distillation under reduced pressure, wherein the pressure is maintained between 15 inHg and 30 inHg, such as from 17.5 inHg to 27.5 inHg, such as from 20 inHg to 25 inHg, such as from 21 inHg to 24 inHg, and including pressures maintained between 22 inHg and 23 inHg.

[0243] In some cases, the combined fractions are concentrated under vacuum, and the concentrated combined fractions are contacted with a composition containing particles of an organic cationic salt of 25-hydroxy-(3β)-cholesterol-5-ene-3-sulfate. In other cases, the concentrated combined fractions are contacted with a composition containing particles of an organic cationic salt of 25-hydroxy-(3β)-cholesterol-5-ene-3-sulfate and at least one solvent. In some cases, the at least one solvent is selected from tetrahydrofurans, such as 2-methyltetrahydrofuran. The concentrated, combined fractions may be contacted with the composition containing particles of the organic cationic salt 25-hydroxy-(3β)-cholesterol-5-ene-3-sulfate for a duration of 0.001 minutes or longer, such as 0.005 minutes or longer, such as 0.01 minutes or longer, such as 0.05 minutes or longer, such as 0.1 minutes or longer, such as 0.5 minutes or longer, such as 1 minute or longer, such as 2 minutes or longer, such as 3 minutes or longer, such as 4 minutes or longer, such as 5 minutes or longer, such as 10 minutes or longer, such as 15 minutes or longer, such as 30 minutes or longer, such as 45 minutes or longer, and including 60 minutes or longer. In some cases, the combined fractions are added dropwise to the composition containing the organic cationic salt 25-hydroxy-(3β)-cholesterol-5-ene-3-sulfate in 2-methyltetrahydrofuran.

[0244] In some cases, the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate is contacted with a metal salt to produce the metal salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate (Scheme IB1).

[0245] Option IB1

[0246]

[0247] In some cases, methods for producing metal salts of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate include contacting the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate with at least one sodium salt. In some cases, said at least one sodium salt is selected from sodium acetate, sodium iodide, sodium chloride, sodium hydroxide, and sodium methoxide. The organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate can be contacted with the metal salt at temperatures ranging from -10°C to 75°C, such as from -5°C to 70°C, such as from -4°C to 65°C, such as from -3°C to 60°C, such as from -2°C to 55°C, such as from -1°C to 50°C, such as from 0°C to 45°C, such as from 5°C to 40°C, and including temperatures from 10°C to 35°C.

[0248] The reaction can last for durations from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, such as from 0.3 hours to 24 hours, such as from 0.4 hours to 21 hours, such as from 0.5 hours to 20 hours, such as from 0.6 hours to 19 hours, such as from 0.7 hours to 18 hours, such as from 0.8 hours to 17 hours, such as from 0.9 hours to 16 hours, and including from 1 hour to 15 hours. The amount of metal salt used relative to the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate can vary and can be 0.0001 equivalents or more, such as 0.001 equivalents or more, such as 0.01 equivalents or more, such as 0.1 equivalents or more, such as 0.2 equivalents or more, such as 0.3 equivalents or more, such as 0.4 equivalents or more, such as 0.5 equivalents or more, such as 0.6 equivalents or more, such as 0.7 equivalents or more, such as 0.8 equivalents or more, such as 0.9 equivalents or more, such as 1 equivalent or more, such as 1.1 equivalents or more, such as 1.2 equivalents or more, such as 1.3 equivalents or more, such as 1.4 equivalents or more, such as 1.5 equivalents or more, such as 1.6 equivalents or more, such as 1.7 equivalents or more, such as 1.8 equivalents or more, such as 1.9 equivalents or more. Equivalents or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, and including 10 equivalents or more, and the range can be from 0.001 equivalents to 10 equivalents, such as 0.1 equivalents to 10 equivalents, 0.1 equivalents to 8 equivalents, 0.1 equivalents to 6 equivalents, 0.1 equivalents to 4 equivalents, 0.1 equivalents to 3 equivalents, 1 equivalent to 10 equivalents, 1 equivalent to 8 equivalents. 1 to 6 equivalent, 1 to 4 equivalent, 1 to 3 equivalent, 1.5 to 10 equivalent, 1.5 to 8 equivalent, 1.5 to 6 equivalent, 1.5 to 4 equivalent, 1.5 to 3 equivalent, 2 to 10 equivalent, 2 to 8 equivalent, 2 to 6 equivalent, 2 to 4 equivalent, or 2 to 3 equivalent, 1 to 100 equivalent, 1 to 5 equivalent, 1 to 2 equivalent.

[0249] In some cases, the method involves contacting pyridinium salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate with sodium iodide to produce sodium salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate (Scheme IB2).

[0250] Option IB2

[0251]

[0252] In some cases, methods for preparing 25-hydroxy-3β-cholestene-5-en-3-sulfate include contacting 25-hydroxy-(3β)-cholestene-5-en-3-ol with a sulfur trioxide-pyridine complex to produce pyridinium salt of 25-hydroxy-(3β)-cholestene-5-en-3-sulfate; and contacting pyridinium salt of 25-hydroxy-(3β)-cholestene-5-en-3-sulfate with a sodium salt to produce sodium salt of 5-cholestene-3β,25-diol 3-sulfate (Scheme Ib).

[0253] Solution Ib

[0254]

[0255] In some cases, methods for preparing 25-hydroxy-3β-cholest-5-en-3-sulfate include contacting (3β)-cholest-5-en-3-ol with a sulfating agent to produce a first (3β)-cholest-5-en-3-sulfate organic cationic salt; contacting the first (3β)-cholest-5-en-3-sulfate organic cationic salt with an organic base to produce a second (3β)-cholest-5-en-3-sulfate organic cationic salt; and oxidizing the second (3β)-cholest-5-en-3-sulfate organic cationic salt in the presence of at least one surfactant. Sulfuric acid organic cation salts are used to produce 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cation salts; 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salts are produced from 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cation salts by deoxygenation; and 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salts are contacted with at least one metal salt to produce 5-cholestene-3β,25-diol 3-sulfate metal salts (Scheme IIa).

[0256] Scheme IIa

[0257]

[0258] In some cases, cholesterol is sulfated with a sulfated reagent (Scheme IIA1). In some cases, the sulfated reagent is selected from sulfur trioxide complexes, sulfuric acid compounds, sulfonic acid compounds, and sulfonate / ester compounds. In some cases, the sulfated reagent is a sulfur trioxide-pyridine complex. In some cases, the sulfated reagent is selected from sulfur trioxide dimethylformamide, sulfur trioxide triethylamine, and sulfur trioxide trimethylamine. In some cases, the sulfated reagent is sulfuric acid and acetic anhydride and pyridine. In some cases, the sulfated reagent is selected from chlorosulfonic acid and pyridine. In some cases, the sulfated reagent is selected from chlorosulfonic acid and 2,6-dimethylpyridine. In some cases, the sulfated reagent is selected from ethyl chlorosulfonate.

[0259] Cholesterol can be sulfated at temperatures ranging from 0°C to 100°C, such as from 5°C to 95°C, such as from 10°C to 90°C, such as from 15°C to 85°C, such as from 20°C to 80°C, such as from 25°C to 75°C, and including from 30°C to 70°C. The reaction can be carried out for durations ranging from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, such as from 0.3 hours to 24 hours, such as from 0.4 hours to 21 hours, such as from 0.5 hours to 20 hours, such as from 0.6 hours to 19 hours, and including from 0.7 hours to 18 hours. The amount of sulfating agent used relative to cholesterol can vary and can be 0.0001 equivalents or more, such as 0.001 equivalents or more, such as 0.01 equivalents or more, such as 0.1 equivalents or more, such as 0.2 equivalents or more, such as 0.3 equivalents or more, such as 0.4 equivalents or more, such as 0.5 equivalents or more, such as 0.6 equivalents or more, such as 0.7 equivalents or more, such as 0.8 equivalents or more, such as 0.9 equivalents or more, such as 1 equivalent or more, such as 1.1 equivalents or more, such as 1.2 equivalents or more, such as 1.3 equivalents or more, such as 1.4 equivalents or more, such as 1.5 equivalents or more, such as 1.6 equivalents or more, such as 1.7 equivalents or more, such as 1.8 equivalents or more, such as 1.9 equivalents or more, such as 2 equivalents or more. More, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, and including 10 equivalents or more, and the range can be from 0.001 equivalents to 10 equivalents, such as 0.1 equivalents to 10 equivalents, 0.1 equivalents to 8 equivalents, 0.1 equivalents to 6 equivalents, 0.1 equivalents to 4 equivalents, 0.1 equivalents to 3 equivalents, 1 equivalent to 10 equivalents, 1 equivalent to 8 equivalents, 1 equivalent to 6 equivalents Equivalents, 1 to 4 equivalents, 1 to 3 equivalents, 1.5 to 10 equivalents, 1.5 to 8 equivalents, 1.5 to 6 equivalents, 1.5 to 4 equivalents, 1.5 to 3 equivalents, 2 to 10 equivalents, 2 to 8 equivalents, 2 to 6 equivalents, 2 to 4 equivalents, 2 to 3 equivalents, 1 to 30 equivalents, 1 to 5 equivalents, or 1 to 2 equivalents.

[0260] Solution IIA1

[0261]

[0262] In some cases, the first (3β)-cholest-5-ene-3-sulfate organic cationic salt is (3β)-cholest-5-ene-3-sulfate pyridinium salt (Scheme IIA2).

[0263] Solution IIA2

[0264]

[0265] In some cases, the first (3β)-cholest-5-ene-3-sulfate organic cation salt (structure IIA) is contacted with an organic base to produce the second (3β)-cholest-5-ene-3-sulfate organic cation salt (structure IIB) (scheme IIB1).

[0266] Solution VB1

[0267]

[0268] In some cases, the organic base contacted with the first (3β)-cholest-5-ene-3-sulfate organic cation salt is selected from hydroxide bases. In some cases, the hydroxide base is selected from tetraethylammonium hydroxide, tetrabutylammonium hydroxide, tetrapropylammonium hydroxide, and tetramethylammonium hydroxide. In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is selected from tetraethylammonium cationic salt, tetrabutylammonium cationic salt, tetrapropylammonium cationic salt, and tetramethylammonium cationic salt. In some cases, the organic base is contacted with the first (3β)-cholest-5-ene-3-sulfate organic cation salt at a temperature ranging from -10°C to 75°C, such as from -5°C to 70°C, such as from -4°C to 65°C, such as from -3°C to 60°C, such as from -2°C to 55°C, such as from -1°C to 50°C, and including from 0°C to 15°C. The reaction can last for durations from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, such as from 0.3 hours to 24 hours, such as from 0.4 hours to 21 hours, such as from 0.5 hours to 20 hours, such as from 0.6 hours to 19 hours, such as from 0.7 hours to 18 hours, such as from 0.8 hours to 17 hours, such as from 0.9 hours to 16 hours, and including from 1 hour to 15 hours.The amount of organic base used relative to the first (3β)-cholesterol-5-ene-3-sulfate organic cation salt can vary and can be 0.0001 equivalents or more, such as 0.001 equivalents or more, such as 0.01 equivalents or more, such as 0.1 equivalents or more, such as 0.2 equivalents or more, such as 0.3 equivalents or more, such as 0.4 equivalents or more, such as 0.5 equivalents or more, such as 0.6 equivalents or more, such as 0.7 equivalents or more, such as 0.8 equivalents or more, such as 0.9 equivalents or more, such as 1 equivalent or more, such as 1.1 equivalents or more, such as 1.2 equivalents or more, such as 1.3 equivalents or more, such as 1.4 equivalents or more, such as 1.5 equivalents or more, such as 1.6 equivalents or more, such as 1.7 equivalents or more, such as 1.8 equivalents or more, such as 1.9 equivalents or more. The amount or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, and including 10 equivalents or more, and the range can be from 0.001 equivalents to 10 equivalents, such as 0.1 equivalents to 10 equivalents, 0.1 equivalents to 8 equivalents, 0.1 equivalents to 6 equivalents, 0.1 equivalents to 4 equivalents, 0.1 equivalents to 3 equivalents, 1 equivalent to 10 equivalents, 1 equivalent to 8 equivalents 1 to 6 equivalent, 1 to 4 equivalent, 1 to 3 equivalent, 1.5 to 10 equivalent, 1.5 to 8 equivalent, 1.5 to 6 equivalent, 1.5 to 4 equivalent, 1.5 to 3 equivalent, 2 to 10 equivalent, 2 to 8 equivalent, 2 to 6 equivalent, 2 to 4 equivalent, 2 to 3 equivalent, 1 to 10 equivalent, 1 to 5 equivalent, or 1 to 2 equivalent.

[0269] In some cases, the method involves contacting a first (3β)-cholest-5-ene-3-sulfate organic cation salt with tetrabutylammonium hydroxide to produce a (3β)-cholest-5-ene-3-sulfate tetrabutylammonium cation salt (structure IIB1) (scheme IIB2).

[0270] Solution VB1

[0271]

[0272] In some cases, a second (3β)-cholest-5-ene-3-sulfate organic cation salt is oxidized to produce 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cation salt (structure IIC) (scheme IIC1).

[0273] Solution IIC1

[0274]

[0275] In some cases, oxidizing a second (3β)-cholest-5-ene-3-sulfate organic cationic salt involves contacting the second (3β)-cholest-5-ene-3-sulfate organic cationic salt with a composition having an oxidizing agent and at least one surfactant.

[0276] In some cases, the at least one surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants. Nonionic surfactants may be selected from polyoxyethylene glycol ethers (e.g., polyoxyethylene glycol octylphenol ether), polyoxyethylene glycol alkyl sorbitol esters, alkyl sorbitol esters, block copolymers of polyethylene glycol and polypropylene glycol, and other nonionic surfactants. Anionic surfactants may be selected from surfactants having anionic functional head groups, such as surfactants containing sulfonate / ester, phosphate / ester, sulfate / ester, or carboxylate / ester head groups. For example, anionic surfactants may be selected from alkyl sulfate / esters such as ammonium lauryl sulfate, sodium dioctyl sulfosuccinate, perfluorooctane sulfonate, perfluorononanoate, perfluorooctanoate, linear alkylbenzene sulfonate, alkyl-aryl ether phosphate, sodium lauryl ether sulfate, lignin sulfonate, or sodium stearate, and other anionic surfactants. Cationic surfactants can be selected from surfactants having cationic functional head groups, such as pyridinium or quaternary ammonium head groups. For example, cationic surfactants can be selected from cetyltrimethylammonium bisulfate, tetrabutylammonium bisulfate, cetyltrimethylammonium bromide, tetrabutylammonium bromide, tetrabutylammonium iodide, tetrabutylphosphonium bromide, tetraoctylammonium bromide, tetraoctylammonium iodide, benzyltriethylammonium chloride, benzyltriethylammonium bromide, benzylcetyldimethylammonium chloride, or benzylcetyldimethylammonium bromide. Amphoteric surfactants include cationic and anionic centers, such as sulfobetaine (e.g., 3-[(3-cholanamidopropyl)dimethylammonium]-1-propanesulfonate) or betaine (e.g., cocoamidopropyl betaine). In some cases, the at least one surfactant is Extran laboratory soap, La Parisienne soap, or DL-α-tocopherol methoxy polyethylene glycol succinate (e.g., TPGS-750-M-2).

[0277] The amount of surfactant used relative to the second (3β)-cholesterol-5-ene-3-sulfate organic cationic salt can vary, wherein in some cases, 0.0001 equivalents or more of surfactant are used, such as 0.001 equivalents or more, such as 0.01 equivalents or more, such as 0.1 equivalents or more, such as 0.2 equivalents or more, such as 0.3 equivalents or more, such as 0.4 equivalents or more, such as 0.5 equivalents or more, such as 0.6 equivalents or more, such as 0.7 equivalents or more, such as 0.8 equivalents or more, such as 0.9 equivalents or more, such as 1 equivalent or more, such as 1.1 equivalents or more, such as 1.2 equivalents or more, such as 1.3 equivalents or more, such as 1.4 equivalents or more, such as 1.5 equivalents or more, such as 1.6 equivalents or more, such as 1.7 equivalents or more, such as 1.8 equivalents or more, such as 1.9 equivalents or more. The amount may be 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, and includes 10 equivalents or more of surfactant, and the range may be from 0.001 equivalents to 10 equivalents, such as 0.1 equivalents to 10 equivalents, 0.1 equivalents to 8 equivalents, 0.1 equivalents to 6 equivalents, 0.1 equivalents to 4 equivalents, 0.1 equivalents to 3 equivalents, 1 equivalent to 10 equivalents, 1 equivalent to 8 equivalents. 1 to 6 equivalent, 1 to 4 equivalent, 1 to 3 equivalent, 1.5 to 10 equivalent, 1.5 to 8 equivalent, 1.5 to 6 equivalent, 1.5 to 4 equivalent, 1.5 to 3 equivalent, 2 to 10 equivalent, 2 to 8 equivalent, 2 to 6 equivalent, 2 to 4 equivalent, 2 to 3 equivalent, 0.1 to 5 equivalent, 0.15 to 1 equivalent, or 0.2 to 0.3 equivalent.

[0278] In some cases, oxidizing a second (3β)-cholest-5-ene-3-sulfate organic cationic salt involves contacting the second (3β)-cholest-5-ene-3-sulfate organic cationic salt with an oxidant and at least one ketone in the presence of at least one surfactant.

[0279] In some cases, the at least one ketone is selected from tetrahydrothiaran-4-one 1,1-dioxide and haloketones. In some cases, the haloketone is selected from 1,1,1-trifluoro-2-butanone, 4,4-difluorocyclohexanone, 2-2-2-4'-tetrafluoroacetylbenzene, and 1,1,1-trifluoroacetone. In some cases, the at least one ketone is 1,1,1-trifluoro-2-butanone. The amount of ketone used relative to the oxidant in the subject reaction can vary and can be 1 equivalent or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, such as 6 equivalents or more, such as 7 equivalents or more, such as 8 equivalents or more, such as 9 equivalents or more, such as 10 equivalents or more, such as 15 equivalents or more, such as 20 equivalents or more, such as 25 equivalents or more, such as 30 equivalents or more, such as 35 equivalents or more, and includes ketones of 50 equivalents or more, and the range can be from 1 equivalent to 50 equivalents. Such as 1 to 35 equivalent, 1 to 25 equivalent, 1 to 15 equivalent, 1 to 10 equivalent, 1 to 8 equivalent, 1 to 5 equivalent, 2 to 50 equivalent, 2 to 35 equivalent, 2 to 25 equivalent, 2 to 15 equivalent, 2 to 10 equivalent, 2 to 8 equivalent, 2 to 5 equivalent, 4 to 50 equivalent, 4 to 35 equivalent, 4 to 25 equivalent, 4 to 15 equivalent, 4 to 10 equivalent, 4 to 8 equivalent, 1 to 50 equivalent, 2 to 25 equivalent, or 5 to 10 equivalent.

[0280] In some cases, the ketone is further purified before use. For example, the ketone may be purified by distillation before use. In other cases, the reactivity of the ketone is tested (e.g., by...). 1 H-NMR testing is used to identify impurities in order to determine whether purification is necessary.

[0281] In some cases, oxidation of a second (3β)-cholest-5-ene-3-sulfate organic cationic salt involves contacting the second (3β)-cholest-5-ene-3-sulfate organic cationic salt with an oxidant and at least one ketone in the presence of at least one surfactant and water. The amount of water present can vary, ranging from 0.0000001% w / v or more of the reaction mixture, such as 0.000001% w / v or more, such as 0.00001% w / v or more, such as 0.0001% w / v or more, such as 0.001% w / v, such as 0.01% w / v or more, such as 0.1% w / v or more, such as 0.05% w / v or more, such as 0.1% w / v or more, such as 0.5% w / v or more, such as 1% w / v or more, such as 5% w / v or more, such as 10% w / v or more, such as 15% w / v or more, and includes 25% w / v or more of the reaction mixture, and the range can be from 0.0000001% w / v to 25% w / v, such as 0.0000001% w / v to 15% w / v, 0.0000001% w / v to 10% w / v. %w / v, 0.0000001%w / v to 5%w / v, 0.0000001%w / v to 1%w / v, 0.001%w / v to 25%w / v, 0.001%w / v to 15%w / v, 0.001%w / v to 10%w / v, 0.001%w / v to 5%w / v, 0.001%w / v to 1%w / v, 0.1%w / v to 25%w / v, 0.1%w / v / v to 15% w / v, 0.1% w / v to 10% w / v, 0.1% w / v to 5% w / v, 0.1% w / v to 1% w / v, 1% w / v to 25% w / v, 1% w / v to 15% w / v, 1% w / v to 10% w / v, 1% w / v to 5% w / v, 0.1% w / v to 50% w / v, 0.1% w / v to 10% w / v, or 0.5% w / v to 1% w / v.

[0282] The second (3β)-cholest-5-ene-3-sulfate organic cation salt can be oxidized at temperatures ranging from -25°C to 50°C, such as from -20°C to 45°C, such as from -15°C to 40°C, such as from -10°C to 35°C, such as from -5°C to 30°C, such as from -1°C to 25°C, and including temperatures from 0°C to 15°C. In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is oxidized at temperatures ranging from 0°C to 5°C. When the reaction mixture includes a certain amount of water, the reaction can be carried out at temperatures ranging from -10°C to 50°C, such as from -5°C to 45°C, such as from 0°C to 40°C, such as from 0°C to 35°C, such as from 0°C to 30°C, such as from 0°C to 25°C, such as from 0°C to 20°C, such as from 0°C to 15°C, and including temperatures from 0°C to 10°C.

[0283] The second (3β)-cholest-5-ene-3-sulfate organic cation salt can be oxidized at pH ranges from 5 to 7.5, such as pH ranges from 5.5 to 7.0, and including pH ranges from 5.5 to 6.5. In some cases, where the reaction mixture contains water (e.g., in a two-phase solvent system), the pH range is from 5.0 to 6.0, such as pH ranges from 5.0 to 5.9, such as pH ranges from 5.0 to 5.8, such as pH ranges from 5.0 to 5.7, such as pH ranges from 5.0 to 5.6, and including pH ranges from 5.0 to 5.5.

[0284] The reaction can last for durations from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, such as from 0.3 hours to 24 hours, such as from 0.4 hours to 21 hours, such as from 0.5 hours to 20 hours, such as from 0.6 hours to 19 hours, such as from 0.7 hours to 18 hours, such as from 0.8 hours to 17 hours, such as from 0.9 hours to 16 hours, and including from 1 hour to 15 hours.

[0285] In some cases, a second (3β)-cholest-5-ene-3-sulfate organic cationic salt is contacted in situ with a composition having potassium persulfate and at least one ketone in the presence of at least one surfactant. In some cases, the method includes contacting potassium persulfate with at least one ketone in the presence of at least one surfactant to form a separate oxidatively reactive mixture, and adding said oxidatively reactive mixture to the second (3β)-cholest-5-ene-3-sulfate organic cationic salt. In these cases, before contacting the oxidatively reactive mixture with the second (3β)-cholest-5-ene-3-sulfate organic cationic salt, the potassium persulfate may be contacted with at least one ketone in the presence of at least one surfactant for a duration of 0.1 minutes or longer, such as 1 minute or longer, such as 2 minutes or longer, such as 3 minutes or longer, such as 5 minutes or longer, and including 10 minutes or longer, and the time range may be from 2 minutes to 180 minutes, such as 3 minutes to 120 minutes, or 4 minutes to 60 minutes. In certain circumstances, potassium peroxymonosulfate can be contacted with at least one ketone in the presence of at least one surfactant to form a separate oxidative reactive mixture, and the oxidative reactive mixture can be immediately contacted with a second (3β)-cholest-5-ene-3-sulfate organic cationic salt. The oxidative reactive mixture can be formed at temperatures ranging from -10°C to 50°C, such as from -5°C to 45°C, such as from -4°C to 40°C, such as from -3°C to 35°C, such as from -2°C to 30°C, such as from -1°C to 25°C, and including temperatures from 0°C to 15°C. Without immediately contacting the oxidizing reactive mixture with a second (3β)-cholest-5-ene-3-sulfate organic cation salt, the oxidizing reactive mixture can be maintained at a temperature ranging from -10°C to 50°C, such as from -5°C to 45°C, such as from -4°C to 40°C, such as from -3°C to 35°C, such as from -2°C to 30°C, such as from -1°C to 25°C, and including from 0°C to 15°C.

[0286] In some cases, the method further includes adding the oxidizing reactive mixture to a second (3β)-cholest-5-ene-3-sulfate organic cation salt. In some cases, the method includes adding the oxidizing reactive mixture dropwise to the second (3β)-cholest-5-ene-3-sulfate organic cation salt. In some cases, the oxidizing reactive mixture is added to the second (3β)-cholest-5-ene-3-sulfate organic cation salt in a metered amount. The metered amount may be added continuously or at predetermined time intervals (e.g., every 30 seconds, 1 minute, 2 minutes, 3 minutes, 5 minutes, or some other interval). In some cases, the oxidizing reactive mixture is added to the second (3β)-cholest-5-ene-3-sulfate organic cation salt by controlled addition, such as with a mechanically or computer-controlled pump, for example, an injection pump. In some cases, the method includes generating the oxidizing reactive mixture and adding a composition containing the second (3β)-cholest-5-ene-3-sulfate organic cation salt to the oxidizing reactive mixture. In some cases, the method involves adding a second (3β)-cholest-5-ene-3-sulfate organic cation salt dropwise to the oxidizing reactive mixture. In other cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is added to the oxidizing reactive mixture in a metered amount. The metered amount can be added continuously or at predetermined time intervals (e.g., every 30 seconds, 1 minute, 2 minutes, 3 minutes, 5 minutes, or some other interval). In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cation salt is added to the oxidizing reactive mixture by controlled addition, such as using a mechanically or computer-controlled pump, for example, an injection pump.

[0287] In some cases, oxidation of a second (3β)-cholest-5-ene-3-sulfate organic cation salt involves contacting the second (3β)-cholest-5-ene-3-sulfate organic cation salt with at least one oxidizing agent. In some cases, said at least one oxidizing agent is selected from dioxane. In some cases, dioxane is produced in situ in a composition having the second (3β)-cholest-5-ene-3-sulfate organic cation salt. In some cases, dioxane is produced alone (e.g., in a separate reaction vessel such as a flask) and added to a composition having the second (3β)-cholest-5-ene-3-sulfate organic cation salt.

[0288] In some cases, a second (3β)-cholest-5-ene-3-sulfate organic cation salt is oxidized in the presence of at least one base. In some cases, the at least one base is selected from weak bases. In some cases, the at least one base is selected from potassium bicarbonate, sodium bicarbonate, potassium phenolate, sodium citrate buffer, sodium phosphate buffer, potassium formate, and potassium acetate. In some cases, the at least one base is potassium bicarbonate. In some cases, at least one base may be added to the reaction mixture over time, such as in metered amounts, wherein the base is added at predetermined time intervals (e.g., every 30 seconds, 1 minute, 2 minutes, 3 minutes, 5 minutes, or some other interval). In some cases, the at least one base may be a composition containing water, wherein the base present in the composition may be 0.0000001% w / v or more, such as 0.000001% w / v or more, such as 0.00001% w / v or more, such as 0.0001% w / v or more, such as 0.001% w / v or more, such as 0.01% w / v or more, such as 0.05% w / v or more, such as 0.1% w / v or more, such as 0.5% w / v or more, such as 1% w / v or more, such as 5% w / v or more, such as 10% w / v or more, such as 15% w / v or more, and includes 25% w / v or more of the composition, and the range may be from 0.0000001% w / v to 25% w / v, such as 0.0000001% w / v to 15% w / v, 0.000000 1% w / v to 10% w / v, 0.0000001% w / v to 5% w / v, 0.0000001% w / v to 1% w / v, 0.001% w / v to 25% w / v, 0.001% w / v to 15% w / v, 0.001% w / v to 10% w / v, 0.001% w / v to 5% w / v, 0.001% w / v to 1% w / v, 0.1% w / v to 25% w / v, 0. 1% w / v to 15% w / v, 0.1% w / v to 10% w / v, 0.1% w / v to 5% w / v, 0.1% w / v to 1% w / v, 1% w / v to 25% w / v, 1% w / v to 15% w / v, 1% w / v to 10% w / v, 1% w / v to 5% w / v, 0.1% w / v to 20% w / v, 0.2% w / v to 15% w / v, or 0.3% w / v to 10% w / v. In some cases, the at least one base may be an aqueous potassium bicarbonate composition.

[0289] In some cases, the second (3β)-cholest-5-ene-3-sulfate organic cationic salt is oxidized as follows: contact with ozone in the presence of cetyltrimethylammonium bisulfate (CTAHS), followed by the addition of trifluorobutanone and potassium bisulfate to form 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt (Scheme IIC2).

[0290] Solution IIC2

[0291]

[0292] In some cases, the method involves in-situ formation of an oxidizing agent with a second (3β)-cholest-5-ene-3-sulfate organic cationic salt, such as contacting potassium persulfate and trifluorobutanone with the second (3β)-cholest-5-ene-3-sulfate organic cationic salt in a reaction mixture in the presence of cetyltrimethylammonium bisulfate (CTAHS). In some cases, in-situ formation of an oxidizing agent with the second (3β)-cholest-5-ene-3-sulfate organic cationic salt includes in-situ formation of dioxane with the second (3β)-cholest-5-ene-3-sulfate organic cationic salt.

[0293] In some cases, the method includes forming dioxane in a separate reaction and adding dioxane to a second (3β)-cholest-5-ene-3-sulfate organic cationic salt. In these cases, potassium peroxymonosulfate may be contacted with trifluorobutanone for 0.1 minutes or longer in the presence of cetyltrimethylammonium bisulfate (CTAHS), followed by contacting the reactive composition with the second (3β)-cholest-5-ene-3-sulfate organic cationic salt for, for, such as, 1 minute or longer, such as, 2 minutes or longer, such as, 3 minutes or longer, such as, 5 minutes or longer, and including 10 minutes or longer, and the time range may be from 0.01 minutes to 120 minutes, such as, 0.1 minutes to 90 minutes or 0.5 minutes to 60 minutes. In certain circumstances, potassium peroxymonosulfate can be contacted with trifluorobutanone in the presence of cetyltrimethylammonium bisulfate (CTAHS) to form an oxidizing reactive composition, which is immediately contacted with a second (3β)-cholest-5-ene-3-sulfate organic cation salt.

[0294] The organic cationic salt 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate was deoxygenated to produce the organic cationic salt 25-hydroxy-(3β)-cholest-5-ene-3-sulfate (Structure IID) (Scheme IID1).

[0295] Solution IID1

[0296]

[0297] In some cases, the production of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salts from 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salts involves deoxygenation by contacting the 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt with zinc. In some cases, the 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt is contacted with zinc in the presence of at least one halide and at least one acid. In some cases, the at least one halide is selected from iodine and metal halides. In some cases, the metal halide is selected from sodium iodide and lithium iodide. In some cases, the at least one acid is selected from weak acids. In some cases, the at least one acid is selected from acetic acid, hydrochloric acid, citric acid, p-toluenesulfonic acid, formic acid, and methanesulfonic acid.

[0298] The amount of reagent used to deoxygenate the organic cationic salt 25-hydroxy-(3β)-cholesterol-(5,6-epoxy)-3-sulfate can vary, wherein in some cases, a reagent of 0.0001 equivalents or more relative to the organic cationic salt 25-hydroxy-(3β)-cholesterol-(5,6-epoxy)-3-sulfate is used, such as 0.001 equivalents or more, such as 0.01 equivalents or more, such as 0.1 equivalents or more, such as 0.2 equivalents or more, such as 0.3 equivalents or more, such as 0.4 equivalents or more, such as 0.5 equivalents or more, such as 0.6 equivalents or more, such as 0.7 equivalents or more, such as 0.8 equivalents or more, such as 0.9 equivalents or more, such as 1 equivalent or more, such as 1.1 equivalents or more, such as 1.2 equivalents or more, such as 1.3 equivalents or more, such as 1.4 equivalents or more, such as 1.5 equivalents or more, such as 1.6 equivalents or more, etc. Such as 1.7 equivalent or more, such as 1.8 equivalent or more, such as 1.9 equivalent or more, such as 2 equivalent or more, such as 3 equivalent or more, such as 4 equivalent or more, such as 5 equivalent or more, and including 10 equivalent or more, and the range can be from 0.001 equivalent to 10 equivalent, such as 0.1 equivalent to 10 equivalent, 0.1 equivalent to 8 equivalent, 0.1 equivalent to 6 equivalent, 0.1 equivalent to 4 equivalent, 0.1 equivalent to 3 equivalent, 1 equivalent Up to 10 equivalent, 1 equivalent to 8 equivalent, 1 equivalent to 6 equivalent, 1 equivalent to 4 equivalent, 1 equivalent to 3 equivalent, 1.5 equivalent to 10 equivalent, 1.5 equivalent to 8 equivalent, 1.5 equivalent to 6 equivalent, 1.5 equivalent to 4 equivalent, 1.5 equivalent to 3 equivalent, 2 equivalent to 10 equivalent, 2 equivalent to 8 equivalent, 2 equivalent to 6 equivalent, 2 equivalent to 4 equivalent, 2 equivalent to 3 equivalent, 1 equivalent to 20 equivalent, 1 equivalent to 10 equivalent, or 4 equivalent to 6 equivalent.

[0299] The 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt can be deoxygenated at temperatures ranging from -10°C to 75°C, such as from -5°C to 70°C, such as from -4°C to 65°C, such as from -3°C to 60°C, such as from -2°C to 55°C, such as from -1°C to 50°C, and including from 0°C to 25°C. The reaction can be carried out for durations ranging from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, such as from 0.3 hours to 24 hours, such as from 0.4 hours to 21 hours, such as from 0.5 hours to 20 hours, such as from 0.6 hours to 19 hours, such as from 0.7 hours to 18 hours, such as from 0.8 hours to 17 hours, such as from 0.9 hours to 16 hours, and including from 1 hour to 15 hours.

[0300] In some cases, the method involves contacting the organic cationic salt of 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate with zinc in the presence of iodine and acetic acid to produce the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate (Scheme IID2).

[0301] Solution IID2

[0302]

[0303] In some cases, the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate (structure IID) is contacted with a metal salt to produce the metal salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate (structure IIE) (Scheme IIE1).

[0304] Scheme IIE1

[0305]

[0306] In some cases, methods for producing metal salts of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate include contacting the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate with at least one sodium salt. In some cases, said at least one sodium salt is selected from sodium acetate, sodium iodide, sodium chloride, sodium hydroxide, and sodium methoxide. The organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate can be contacted with the metal salt at temperatures ranging from -10°C to 75°C, such as from -5°C to 70°C, such as from -4°C to 65°C, such as from -3°C to 60°C, such as from -2°C to 55°C, such as from -1°C to 50°C, such as from 0°C to 45°C, such as from 5°C to 40°C, and including temperatures from 10°C to 35°C.

[0307] The reaction can last for durations from 0.1 hours to 72 hours, such as from 0.2 hours to 48 hours, such as from 0.3 hours to 24 hours, such as from 0.4 hours to 21 hours, such as from 0.5 hours to 20 hours, such as from 0.6 hours to 19 hours, such as from 0.7 hours to 18 hours, such as from 0.8 hours to 17 hours, such as from 0.9 hours to 16 hours, and including from 1 hour to 15 hours. The amount of metal salt used relative to the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate can vary and can be 0.0001 equivalents or more, such as 0.001 equivalents or more, such as 0.01 equivalents or more, such as 0.1 equivalents or more, such as 0.2 equivalents or more, such as 0.3 equivalents or more, such as 0.4 equivalents or more, such as 0.5 equivalents or more, such as 0.6 equivalents or more, such as 0.7 equivalents or more, such as 0.8 equivalents or more, such as 0.9 equivalents or more, such as 1 equivalent or more, such as 1.1 equivalents or more, such as 1.2 equivalents or more, such as 1.3 equivalents or more, such as 1.4 equivalents or more, such as 1.5 equivalents or more, such as 1.6 equivalents or more, such as 1.7 equivalents or more, such as 1.8 equivalents or more, such as 1.9 equivalents or more. Equivalents or more, such as 2 equivalents or more, such as 3 equivalents or more, such as 4 equivalents or more, such as 5 equivalents or more, and including 10 equivalents or more, and the range can be from 0.001 equivalents to 10 equivalents, such as 0.1 equivalents to 10 equivalents, 0.1 equivalents to 8 equivalents, 0.1 equivalents to 6 equivalents, 0.1 equivalents to 4 equivalents, 0.1 equivalents to 3 equivalents, 1 equivalent to 10 equivalents, 1 equivalent to 8 equivalents. 1 to 6 equivalent, 1 to 4 equivalent, 1 to 3 equivalent, 1.5 to 10 equivalent, 1.5 to 8 equivalent, 1.5 to 6 equivalent, 1.5 to 4 equivalent, 1.5 to 3 equivalent, 2 to 10 equivalent, 2 to 8 equivalent, 2 to 6 equivalent, 2 to 4 equivalent, 2 to 3 equivalent, 1 to 20 equivalent, 1 to 10 equivalent, or 1 to 7 equivalent.

[0308] In some cases, the method involves contacting pyridinium salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate with sodium iodide to produce sodium salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate (Scheme IIE2).

[0309] Solution IIE2

[0310]

[0311] Terms and Conditions

[0312] The contents of this disclosure may be further described by one or more of the following non-restrictive provisions.

[0313] Clause 1. Crystalline 25HC3S sodium.

[0314] Clause 1. Crystalline 25HC3S sodium.

[0315] Clause 2. Hydrate of crystalline 25HC3S sodium.

[0316] Clause 3. Monohydrate of crystalline 25HC3S sodium.

[0317] Clause 4. Crystalline sodium 25HC3S dihydrate.

[0318] Clause 5. Variable hydrates of crystalline 25HC3S sodium.

[0319] Clause 6. Crystalline form of sodium 25HC3S, form I.

[0320] Clause 7. A crystalline hydrate of any one of Clauses 2-6, having an X-ray powder diffraction pattern containing a peak at about 2.1°2θ.

[0321] Clause 8. The crystalline hydrate of Clause 1 or 7, further comprising one or more peaks at about 5.4°2θ, about 6.5°2θ, about 10.8°2θ and about 15.0°2θ.

[0322] Clause 9. A crystalline hydrate of any one of Clauses 7-8 and 85-86, wherein the crystalline hydrate has a weight loss of about 8% when heated from ambient temperature to 130°C at a rate of 10°C / min.

[0323] Sodium 25HC3S is a substantially pure crystalline hydrate as specified in any of Clauses 10, 2-9, and 85-86.

[0324] Clause 11. Crystalline hydrates of any one of Clauses 2-10 and 85-86, having approximately The unit cell volume.

[0325] Clause 12. Crystalline hydrates of any of Clauses 2-11 and 85-86, having a monoclinic unit cell.

[0326] Stable crystalline hydrates of any of the clauses 13, 2-12, and 85-86.

[0327] Clause 14. The stable crystalline hydrate of Clause 13, wherein the stable crystalline hydrate is stable at a relative humidity of about 38% to about 70%.

[0328] Clause 15. Form II of crystalline 25HC3S sodium.

[0329] Clause 16. Crystalline hydrates of Clause 2 or 5, wherein the water content is not greater than about 3 moles of water per mole of 25HC3S sodium.

[0330] Clause 17. Crystalline hydrates of Clause 2 or 5, wherein the water content is from about 2 moles of water to about 3 moles of water per mole of 25HC3S sodium.

[0331] Clause 18. Crystalline hydrates of Clause 2 or 5, wherein the water content is from about 1 mole of water to about 3 moles of water / 1 mole of 25HC3S sodium.

[0332] Clause 19. A crystalline hydrate of any of Clauses 15-18, having an X-ray pattern containing a peak at about 2.3°2θ.

[0333] Clause 20. The crystalline hydrate of Clause 2 or 19, further comprising an X-ray pattern containing one or more peaks at about 4.5°2θ, peaks at about 5.0°2θ and about 5.1°2θ and between about 5.0°2θ and about 5.1°2θ, peaks at about 5.9°2θ and about 6.1°2θ and between about 5.9°2θ and about 6.1°2θ, and peaks at about 14.8°2θ and about 15.1°2θ and between about 14.8°2θ and about 15.1°2θ.

[0334] The substantially pure crystalline hydrate of sodium 25HC3S as defined in any of the provisions of Clause 21, Clauses 15-20, and Clauses 87-88.

[0335] Stable crystalline hydrates of any one of Clauses 22, 15-21, and 87-88.

[0336] Clause 23. The stable crystalline hydrate of Clause 22, wherein the stable crystalline hydrate is stable at a relative humidity ranging from about 21% to about 30%.

[0337] Clause 24. Crystalline form of sodium 25HC3S XI.

[0338] Clause 25. A crystalline hydrate of sodium 25HC3S from either Clause 2 or 24, having an X-ray powder diffraction pattern containing a peak at approximately 2.6°2θ.

[0339] Clause 26. A crystalline hydrate of Clause 2 or 25 having an X-ray powder diffraction pattern further comprising one or more peaks at about 3.1°2θ, about 3.5°2θ and about 14.5°2θ.

[0340] Clause 27. The substantially pure crystalline hydrate of sodium 25HC3S as specified in any of Clauses 24-26.

[0341] Clause 28. Stable crystalline hydrates of any of Clauses 24-27.

[0342] Clause 29. Anhydrous crystalline 25HC3S sodium.

[0343] Clause 30. Form XIII crystalline 25HC3S sodium.

[0344] Clause 31. Crystalline sodium 25HC3S of Clauses 1, 29 or 30, having an X-ray powder diffraction pattern containing a peak at approximately 2.3°2θ.

[0345] Clause 32. Crystalline sodium 25HC3S of Clause 1 or 31, having an X-ray powder diffraction pattern containing one or more peaks at about 4.6°2θ, about 9.3°2θ, about 14.3°2θ and about 15.0°2θ.

[0346] Clause 33. Crystalline sodium 25HC3S as described in Clauses 1, 29, or 30, wherein the unit cell has approximately The volume.

[0347] Stable crystalline sodium 25HC3S as specified in Clause 34. Clauses 29-33 and 91-92.

[0348] Clause 35. Crystalline sodium 25HC3S from any of Clauses 29-34 and 91-92, wherein the crystalline sodium 25HC3S is stable at relative humidity in the range of about 0% to about 14% RH.

[0349] Clause 36. Crystalline sodium 25HC3S from any of Clauses 29-35 and 91-92, wherein the crystalline sodium 25HC3S is stable at a temperature of 70°C or higher.

[0350] Clause 37. Crystalline 25HC3S sodium of any of Clauses 29-36 and 91-92, wherein the unit cell is monoclinic.

[0351] Sodium 25HC3S that is substantially pure and crystalline, as specified in any of the provisions of Clause 38, Clauses 30-37, and Clauses 91-92.

[0352] Clause 39. Form IX crystalline 25HC3S sodium.

[0353] Clause 40. Crystalline sodium 25HC3S of Clause 1 or 39, having an X-ray powder diffraction pattern containing a peak at approximately 4.9°2θ.

[0354] Clause 41. The crystalline 25HC3S sodium of Clause 1 or 39 further includes one or more peaks at about 7.9°2θ, about 11.2°2θ, about 14.1°2θ, about 16.1°2θ and about 16.6°2θ.

[0355] Clause 42.25 HC3S Sodium Liquid Crystal.

[0356] Clause 43.25. The intermediate phase of sodium HC3S.

[0357] Clause 44.25 HC3S sodium in form V.

[0358] Clause 45. A liquid crystalline material of Clause 1 or 44 having an X-ray powder diffraction pattern containing a peak at about 2.2°2θ.

[0359] Clause 46. Crystalline material of Clause 1 or 45, further comprising one or more peaks at about 4.4°2θ, about 6.6°2θ and about 8.8°2θ.

[0360] Sodium 25HC3S that is substantially pure and crystalline, as specified in any of the clauses 47, 42-46, and 89-90.

[0361] Stable crystalline sodium 25HC3S as specified in Clause 48. Clauses 42-46 and 89-90.

[0362] Clause 49. Crystalline 25HC3S sodium of Clause 1 or 6, having at least one of the following: an X-ray powder diffraction pattern substantially the same as that in FIG1 and a TGA thermal analysis pattern substantially the same as that shown in FIG11.

[0363] Clause 50. Crystalline 25HC3S sodium of Clause 1 or 6, having at least one of the following: an X-ray powder diffraction pattern substantially the same as that in FIG53 and a TGA thermal analysis pattern substantially the same as that shown in FIG11.

[0364] Clause 51. Crystalline 25HC3S sodium of Clause 1 or 15, having at least one of the following: an X-ray powder diffraction pattern substantially the same as that in FIG2 and a TGA thermal analysis pattern substantially the same as that shown in FIG24.

[0365] Clause 52. Crystalline 25HC3S sodium of Clause 1 or 15, having at least one of the following: an X-ray powder diffraction pattern substantially the same as that in FIG54 and a TGA thermal analysis pattern substantially the same as that shown in FIG24.

[0366] Clause 53. Crystalline 25HC3S sodium of Clause 1 or 44, having at least one of the following: an X-ray powder diffraction pattern substantially the same as that in FIG3 and a TGA thermal analysis pattern substantially the same as that shown in FIG28.

[0367] Clause 54. Crystalline 25HC3S sodium of Clause 1 or 44, having at least one of the following: an X-ray powder diffraction pattern substantially the same as that in FIG4 and a TGA thermal analysis pattern substantially the same as that shown in FIG28.

[0368] Clause 55. Crystalline 25HC3S sodium of Clause 1 or 39, having an X-ray powder diffraction pattern substantially the same as that in Figure 5.

[0369] Clause 56. Crystalline 25HC3S sodium of Clause 1 or 24, having an X-ray powder diffraction pattern substantially the same as that in Figure 6.

[0370] Clause 57. Crystalline 25HC3S sodium of Clause 1 or 24, having an X-ray powder diffraction pattern substantially the same as that in Figure 7.

[0371] Clause 58. Crystalline 25HC3S sodium of Clause 1 or 30, having an X-ray powder diffraction pattern substantially the same as that in Figure 8.

[0372] Clause 59. Crystalline 25HC3S sodium of Clause 1 or 35, having an X-ray powder diffraction pattern substantially the same as that in Figure 9.

[0373] Article 60. A method for treating one or more of hypercholesterolemia, hypertriglyceridemia, and conditions associated with fat accumulation and inflammation, said method comprising administering to a patient in need an effective amount of a compound of sodium 25HC3S from any of Articles 1-59 or 64.

[0374] Clause 61. A pharmaceutical composition comprising sodium 25HC3S of any one of Clauses 1-59, 64, 68-83 and 85-96 and at least one pharmaceutically acceptable excipient.

[0375] Clause 62. A method for treating one or more of hypercholesterolemia, hypertriglyceridemia, and conditions associated with fat accumulation and inflammation, said method comprising administering to a patient in need an effective amount of the pharmaceutical composition of 25HC3S sodium of Clause 61.

[0376] Article 63. The methods of Article 60 or 62, wherein conditions related to fat accumulation and inflammation include non-alcoholic fatty liver disease (NAFLD).

[0377] Clause 64. Stable crystalline 25HC3S sodium.

[0378] Clause 65. The method of Clause 60 for treating non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, and atherosclerosis, said method comprising administering to a patient in need an effective amount of a compound of sodium 25HC3S from any of Clauses 1-59, 64, 68-83, and 85-96.

[0379] Clause 66. Clause 65's method of treating non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic hepatitis, acute kidney injury (AKI), psoriasis, and atherosclerosis, said method comprising administering to a patient in need an effective amount of the pharmaceutical composition of Clause 61's 25HC3S sodium.

[0380] Clause 67. A composition comprising two or more of form I sodium 25HC3S, form II sodium 25HC3S, form III sodium 25HC3S, form V sodium 25HC3S, form IX sodium 25HC3S, form XI sodium 25HC3S, and form XIII sodium 25HC3S.

[0381] Clause 68.25 HC3S sodium in form I has an X-ray powder diffraction pattern that is substantially the same as that in Figure 1.

[0382] Clause 69.25 HC3S sodium in form I has an X-ray powder diffraction pattern that is substantially the same as that in Figure 53.

[0383] Clause 70.25HC3S sodium in form II has an X-ray powder diffraction pattern that is substantially the same as that in Figure 2.

[0384] Clause 71.25 HC3S sodium form II, which has essentially the same X-ray powder diffraction pattern as Figure 54.

[0385] Clause 72.25 HC3S sodium in form XI, which has an X-ray powder diffraction pattern that is substantially the same as that in Figure 6 or Figure 7.

[0386] Clause 73.25 HC3S sodium in form V has an X-ray powder diffraction pattern that is substantially the same as that in Figure 3.

[0387] Clause 74.25 HC3S sodium in form V has an X-ray powder diffraction pattern that is substantially the same as that in Figure 4.

[0388] Clause 75.25 HC3S sodium in form XIII has an X-ray powder diffraction pattern that is substantially the same as that in Figure 8.

[0389] Clause 76.25 HC3S sodium in form XIII has an X-ray powder diffraction pattern that is substantially the same as that in Figure 9.

[0390] Clause 77.25 HC3S sodium in form III, which has essentially the same X-ray powder diffraction pattern as Figure 35.

[0391] Clause 78.25 HC3S sodium in form IX has an X-ray powder diffraction pattern that is substantially the same as that in Figure 5.

[0392] Clause 79. Crystalline form III of sodium 25HC3S.

[0393] Clause 80. Solvates of crystalline 25HC3S sodium.

[0394] Clause 81. Ethanol solvate of crystalline 25HC3S sodium.

[0395] Clause 82. A crystalline solvate of Clause 1 or 79 having an X-ray powder diffraction pattern containing a peak at about 4.9°2θ.

[0396] Clause 83. Crystalline material of Clause 1 or 82, further comprising one or more peaks at about 6.3°2θ, about 7.8°2θ and about 9.8°2θ.

[0397] Clause 84. A pharmaceutical composition comprising two or more and at least one pharmaceutically acceptable excipient selected from form I 25HC3S sodium, form II 25HC3S sodium, form III 25HC3S sodium, form V 25HC3S sodium, form IX 25HC3S sodium, form XI 25HC3S sodium and form XIII 25HC3S sodium.

[0398] Clause 85. Crystalline sodium 25HC3S in form I, having an X-ray powder diffraction pattern containing peaks at about 2.1°2θ, about 6.5°2θ and about 10.8°2θ.

[0399] Clause 86. The crystalline form I of sodium 25HC3S of Clause 85, which further comprises one or more peaks at about 9.9°2θ, about 15.0°2θ and about 15.6°2θ.

[0400] Clause 87. Form II of crystalline 25HC3S sodium, having an X-ray powder diffraction pattern containing peaks at about 2.3°2θ and about 5.0°2θ.

[0401] Clause 88. Clause 87 crystalline 25HC3S sodium form II, which further comprises one or more peaks at about 4.5°2θ, about 5.9°2θ, about 9.1°2θ and about 15.1°2θ.

[0402] Clause 89. Crystalline sodium 25HC3S in form V, having an X-ray powder diffraction pattern comprising peaks at about 2.2°2θ, about 6.6°2θ, and a single peak at about 4.0°2θ and about 6.0°2θ and between about 4.0°2θ and about 6.0°2θ.

[0403] Clause 90. Clause 89 crystalline 25HC3S sodium in the form V, which further comprises one or more peaks at about 8.8°2θ, about 9.9°2θ and about 14.9°2θ.

[0404] Clause 91. Crystalline sodium 25HC3S in form XIII, having an X-ray powder diffraction pattern containing peaks at about 2.3°2θ, about 5.4°2θ, about 9.3°2θ and about 11.6°2θ.

[0405] Clause 92. Clause 91 crystalline 25HC3S sodium in form XIII, further comprising one or more peaks at about 4.6°2θ and about 15.0°2θ.

[0406] Clause 93. Form III of crystalline 25HC3S sodium, having an X-ray powder diffraction pattern containing peaks at approximately 4.9°2θ and approximately 6.3°2θ.

[0407] Clause 94. The crystalline form III of sodium 25HC3S of Clause 93, which further comprises one or more peaks at 7.8°2θ, about 9.8°2θ, about 13.3°2θ and about 15.5°2θ.

[0408] Clause 95. Crystalline sodium 25HC3S in form IX, having an X-ray powder diffraction pattern containing peaks at about 2.2°2θ, about 4.9°2θ and about 7.9°2θ.

[0409] Clause 96. Clause 95 crystalline 25HC3S sodium in form IX, further comprising one or more peaks at about 14.1°2θ, about 16.1°2θ and about 16.6°2θ.

[0410] Clause 97. A pharmaceutical composition comprising crystalline sodium 25HC3S or liquid crystal sodium 25HC3S or both and at least one pharmaceutically acceptable excipient.

[0411] The pharmaceutical compositions of Clause 98 and Clause 97, crystalline sodium 25HC3S or liquid crystal sodium 25HC3S, comprise any one of Clauses 1-59, 64, 68-83 and 85-96.

[0412] The pharmaceutical composition of Clause 99. Clause 97, wherein the crystalline sodium 25HC3S or liquid crystal sodium 25HC3S comprises a mixture of sodium 25HC3S form I and sodium 25HC3S form II.

[0413] Clause 100. A method for producing a metal salt of 5-cholestene-3β,25-diol-3-sulfate, the method comprising: contacting 25-hydroxy-(3β)-cholestene-5-en-3-ol with at least one sulfating agent in at least one solvent to produce an organic cationic salt of 25-hydroxy-(3β)-cholestene-5-en-3-sulfate, wherein the organic cationic salt of 25-hydroxy-(3β)-cholestene-5-en-3-sulfate has low solubility in at least one solvent; and contacting the organic cationic salt of 25-hydroxy-(3β)-cholestene-5-en-3-sulfate with at least one metal salt to produce a metal salt of 5-cholestene-3β,25-diol-3-sulfate.

[0414] Clause 101. The method according to Clause 100, wherein the at least one sulfation agent is a sulfur trioxide complex.

[0415] Clause 102. The method according to Clause 101, wherein the at least one sulfating agent is a sulfur trioxide-pyridine complex.

[0416] Clause 103. The method according to any one of Clauses 100-102, wherein the at least one metal salt is sodium acetate.

[0417] Clause 104. The method according to any one of Clauses 100-102, wherein the at least one metal salt is sodium iodide.

[0418] Clause 105. The method according to any one of Clauses 100-104, wherein at least one sulfating agent is contacted with acetic anhydride prior to contact with 25-hydroxy-(3β)-cholest-5-en-3-ol.

[0419] Clause 106. A method for producing sodium salt of 5-cholestene-3β,25-diol 3-sulfate, the method comprising: contacting 25-hydroxy-(3β)-cholestene-5-en-3-ol with a sulfur trioxide-pyridine complex in at least one solvent to produce pyridinium salt of 25-hydroxy-(3β)-cholestene-5-en-3-sulfate, wherein the pyridinium salt of 25-hydroxy-(3β)-cholestene-5-en-3-sulfate has low solubility in at least one solvent; and contacting the pyridinium salt of 25-hydroxy-(3β)-cholestene-5-en-3-sulfate with a sodium salt to produce sodium salt of 5-cholestene-3β,25-diol 3-sulfate.

[0420] Clause 107. The method according to Clause 106, wherein the sodium salt is sodium acetate.

[0421] Clause 108. The method according to Clause 106, wherein the sodium salt is sodium iodide.

[0422] Clause 109. A method for producing a metal salt of 5-cholestene-3β,25-diol 3-sulfate, said method comprising: contacting (3β)-cholestene-5-en-3-ol with at least one sulfating agent to produce a first (3β)-cholestene-5-en-3-sulfate organic cationic salt; contacting the first (3β)-cholestene-5-en-3-sulfate organic cationic salt with an organic base to produce a second (3β)-cholestene-5-en-3-sulfate organic cationic salt; and oxidizing the second (3β)-cholestene-5-en-3-sulfate organic cationic salt in the presence of at least one surfactant. 5-ene-3-sulfate organic cationic salt to produce 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt; 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt from 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt by deoxygenation; and contacting 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt with at least one metal salt to produce 5-cholestene-3β,25-diol 3-sulfate metal salt.

[0423] Clause 110. The method according to Clause 109, wherein the at least one sulfating agent is a sulfur trioxide complex.

[0424] Clause 111. The method according to Clause 110, wherein the at least one sulfating agent is a sulfur trioxide-pyridine complex.

[0425] Clause 112. The method according to any one of Clauses 109-111, wherein said organic base is tetraethylammonium hydroxide.

[0426] Clause 113. The method according to any one of Clauses 109-112, wherein oxidizing the second (3β)-cholest-5-ene-3-sulfate organic cationic salt comprises contacting the second (3β)-cholest-5-ene-3-sulfate organic cationic salt with a composition comprising an oxidant in the presence of at least one surfactant.

[0427] Clause 114. The method according to Clause 113, wherein the method comprises contacting a second (3β)-cholest-5-ene-3-sulfate organic cationic salt with a composition comprising an oxidant and at least one ketone in the presence of at least one surfactant.

[0428] Clause 115. The method according to Clause 114, wherein the at least one ketone is 1,1,1-trifluoro-2-butanone.

[0429] Clause 116. The method according to any one of Clauses 114-115, wherein the composition comprising an oxidant and at least one ketone further comprises a second (3β)-cholest-5-ene-3-sulfate organic cationic salt and at least one surfactant.

[0430] Clause 117. The method according to any one of Clauses 114-116, wherein oxidizing the second (3β)-cholest-5-ene-3-sulfate organic cationic salt comprises contacting a composition comprising an oxidant and at least one ketone with a second composition comprising the second (3β)-cholest-5-ene-3-sulfate organic cationic salt in the presence of at least one surfactant.

[0431] Clause 118. The method according to any one of Clauses 114-117, wherein the method comprises contacting a second (3β)-cholest-5-ene-3-sulfate organic cation salt with an oxidizing agent in the presence of at least one base.

[0432] Clause 119. The method according to Clause 118, wherein the at least one base is potassium bicarbonate.

[0433] Clause 120. The method according to any one of Clauses 113-119, wherein the oxidant is potassium persulfate.

[0434] Clause 121. The method according to any one of Clauses 109-120, wherein the at least one surfactant is cetyltrimethylammonium hydrogen sulfate.

[0435] Clause 122. The method according to any one of Clauses 109-121, wherein the production of 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt from 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt comprises contacting the 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt with zinc.

[0436] Clause 123. The method according to Clause 122, wherein the method comprises contacting a 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt with zinc in the presence of at least one halide and at least one acid.

[0437] Clause 124. The method according to Clause 123, wherein the at least one acid is acetic acid.

[0438] Clause 125. The method according to any one of Clauses 123-124, wherein the at least one halide is iodine.

[0439] Clause 126. The method according to any one of Clauses 109-125, wherein the at least one metal salt is sodium acetate.

[0440] Clause 127. The method according to any one of Clauses 109-125, wherein the at least one metal salt is sodium iodide.

[0441] Clause 128. A composition comprising: 25-hydroxy-(3β)-cholest-5-ene-3-sulfate; and a sulfated sterol.

[0442] Clause 129. The composition according to Clause 128, wherein the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to sulfated sterol is from 100:1 to 10000:1.

[0443] Clause 130. The composition according to Clause 128, wherein the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to sulfated sterol is from 100:1 to 1000:1.

[0444] Clause 131. A composition comprising: 25-hydroxy-(3β)-cholest-5-ene-3-sulfate; and 5-cholest-3β-25-diol-disulfate.

[0445] Clause 132. The composition according to Clause 131, wherein the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to 5-cholest-3β-25-diol-disulfate is from 100:1 to 10000:1.

[0446] Clause 133. The composition according to Clause 131, wherein the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to 5-cholest-3β-25-diol-disulfate is from 100:1 to 1000:1.

[0447] Clause 134. The composition according to Clause 133, wherein the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to 5-cholest-3β-25-diol-disulfate is from 250:1 to 500:1.

[0448] Clause 135. A method comprising: sulfating 25-hydroxy-(3β)-cholest-5-en-3-ol in at least one solvent to produce 25-hydroxy-(3β)-cholest-5-en-3-sulfate, wherein the 25-hydroxy-(3β)-cholest-5-en-3-sulfate has low solubility in at least one solvent; and precipitating the 25-hydroxy-(3β)-cholest-5-en-3-sulfate in said solvent.

[0449] Clause 136. The method according to Clause 135, wherein 25-hydroxy-(3β)-cholest-5-ene-3-sulfate has a solubility of 100 mmol / L or less in at least one solvent.

[0450] Clause 137. The method according to Clause 136, wherein 25-hydroxy-(3β)-cholest-5-ene-3-sulfate has a solubility of 10 mmol / L or less in at least one solvent.

[0451] Clause 138. The method according to any one of Clauses 135-137, wherein the at least one solvent is selected from chloroform, dichloromethane, acetone, acetonitrile, toluene, tetrahydrofuran, and methyltetrahydrofuran.

[0452] Clause 139. The method according to any one of Clauses 135-138, wherein sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol produces 5-cholest-3β-25-diol disulfate, which remains dissolved in at least one solvent.

[0453] Clause 140. The method according to Clause 139, wherein the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the produced 5-cholest-3β-25-diol-disulfate is from 100:1 to 10000:1.

[0454] Clause 141. The method according to Clause 140, wherein the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the produced 5-cholest-3β-25-diol-disulfate is from 100:1 to 1000:1.

[0455] Clause 142. The method according to any one of Clauses 139-141, wherein 5-cholestyrene-3β-25-diol-disulfate has high solubility in at least one solvent.

[0456] Clause 143. The method according to Clause 142, wherein 5-cholestene-3β-25-diol-disulfate has a solubility of 500 mmol / L or greater in at least one solvent.

[0457] Clause 144. A composition comprising: 25-hydroxy-(3β)-cholest-5-en-3-sulfate; and a byproduct from the sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol, comprising a first compound having a high-performance liquid chromatography (HPLC) retention time of about 18.3 minutes, a second compound having an HPLC retention time of about 37.7 minutes, or any combination thereof, and 25-hydroxy-(3β)-cholest-5-en-3-sulfate having an HPLC retention time of about 7.7 minutes, wherein the first compound, the second compound, and the 25-hydroxy-(3β)-cholest-5-en-3-sulfate are separable by HPLC comprising a C8 stationary phase, said HPLC being operated at about 45°C using a first mobile phase comprising a buffer and a second mobile phase comprising one or more organic solvents.

[0458] Clause 145. The composition according to Clause 144, wherein the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the byproduct is from 100:1 to 10000:1.

[0459] Clause 146. The composition according to Clause 144, wherein the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the byproduct is from 100:1 to 1000:1.

[0460] Clause 147. The composition according to any one of Clauses 144-146, wherein the flow rate of the first mobile phase is about 1.0 mL / min.

[0461] Clause 148. A composition according to any one of Clauses 144-147, wherein the flow rate of the second mobile phase is about 1.0 mL / min.

[0462] Clause 149. A composition according to any one of Clauses 144-148, wherein the first mobile phase comprises sodium phosphate.

[0463] Clause 150. A composition according to any one of Clauses 144-149, wherein the second mobile phase is selected from one or more of methoxypropyl acetate, acetonitrile, and methanol.

[0464] Clause 151. A composition comprising: 25-hydroxy-(3β)-cholest-5-ene-3-sulfate; and a byproduct from the sulfation of 25-hydroxy-(3β)-cholest-5-ene-3-ol, comprising a sulfated sterol, a second compound having an HPLC retention time of about 37.7 minutes, or any combination thereof, and 25-hydroxy-(3β)-cholest-5-ene-3-sulfate having an HPLC retention time of about 7.7 minutes, wherein the sulfated sterol, the second compound, and the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate are separable by HPLC comprising a C8 stationary phase, said HPLC being operated at about 45°C using a first mobile phase comprising a buffer and a second mobile phase comprising one or more organic solvents.

[0465] Clause 152. The composition according to Clause 151, wherein the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the byproduct is from 100:1 to 10000:1.

[0466] Clause 153. The composition according to Clause 151, wherein the weight ratio of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the byproduct is from 100:1 to 1000:1.

[0467] Clause 154. A composition according to any one of Clauses 151-152, wherein the flow rate of the first mobile phase is about 1.0 mL / min.

[0468] Clause 155. A composition according to any one of Clauses 151-154, wherein the flow rate of the second mobile phase is about 1.0 mL / min.

[0469] Clause 156. A composition according to any one of Clauses 151-155, wherein the first mobile phase comprises sodium phosphate.

[0470] Clause 157. A composition according to any one of Clauses 151-156, wherein the second mobile phase is selected from one or more of methoxypropyl acetate, acetonitrile, and methanol.

[0471] Clause 158. A composition according to any one of Clauses 151-157, wherein the second compound is a sterol.

[0472] Clause 159. A method for producing a metal salt of 5-cholestene-3β,25-diol-3-sulfate, the method comprising: contacting 25-hydroxy-(3β)-cholestene-5-en-3-ol with at least one sulfating agent in at least one solvent to produce an organic cationic salt of 25-hydroxy-(3β)-cholestene-5-en-3-sulfate, wherein the organic cationic salt of 25-hydroxy-(3β)-cholestene-5-en-3-sulfate has low solubility in at least one solvent; and contacting the organic cationic salt of 25-hydroxy-(3β)-cholestene-5-en-3-sulfate with at least one metal salt to produce a metal salt of 5-cholestene-3β,25-diol-3-sulfate.

[0473] Clause 160. The method according to Clause 159, wherein the contact with 25-hydroxy-(3β)-cholest-5-en-3-ol is performed before... 1 The sulfation reagent was characterized by H-NMR (proton nuclear magnetic resonance) spectroscopy.

[0474] Clause 161. The method according to Clause 160, wherein the method comprises determining the degree of degradation of the sulfated reagent by determining the amount of impurities present in the sulfated reagent prior to contact with 25-hydroxy-(3β)-cholest-5-en-3-ol.

[0475] Clause 162. The method according to Clause 161, wherein the degree of degradation of the sulfating agent is determined by proton nuclear magnetic resonance spectroscopy as follows: integrating at the chemical shift indicating the presence of impurities in the sulfating agent. 1 One or more peaks in the H-NMR spectrum.

[0476] Clause 163. The method according to Clause 162, wherein the sulfation reagent is subjected to proton nuclear magnetic resonance spectroscopy in at least one deuterated solvent.

[0477] Clause 164. The method according to Clause 163, wherein the at least one deuterated solvent is deuterated acetone.

[0478] Clause 165. The method according to Clause 164, wherein the at least one deuterated solvent is not deuterated benzene, deuterated acetonitrile, or deuterated chloroform.

[0479] Clause 166. The method according to any one of Clauses 162-165, wherein the method comprises calculating the impurity level of the sulfating reagent based on one or more peaks in a chemical shift integrated proton NMR spectrum from 9.2 ppm to 9.3 ppm and the peaks based on the integration.

[0480] Clause 167. The method according to Clause 166, wherein the method includes one or more peaks in a chemical shift integrated proton NMR spectrum of about 9.25 ppm.

[0481] Clause 168. The method according to any one of Clauses 159-167, wherein 25-hydroxy-(3β)-cholest-5-en-3-ol is contacted with the sulfate reagent in the presence of particles of an organic cationic salt of 25-hydroxy-(3β)-cholest-5-en-3-sulfate.

[0482] Clause 169. The method according to Clause 168, wherein the particles of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt have an average particle width of 0.1 mm or greater.

[0483] Clause 170. The method according to any one of Clauses 159-169, wherein said method further comprises quenching unreacted sulfation reagent.

[0484] Clause 171. The method according to Clause 170, wherein quenching unreacted sulfation reagent comprises contacting the reaction mixture with water.

[0485] Clause 172. The method according to Clause 171, wherein one equivalent or more of water is brought into contact with the reaction mixture.

[0486] Clause 173. The method according to any one of Clauses 171-172, wherein quenching unreacted sulfating agents comprises contacting the reaction mixture with water and pyridine.

[0487] Clause 174. The method according to Clause 173, wherein pyridine is contacted with the reaction mixture after a predetermined time period following contact with water.

[0488] Clause 175. The method according to any one of Clauses 170-174, wherein the reactivity of unreacted sulfating agents is quenched under slow stirring.

[0489] Clause 176. The method according to any one of Clauses 159-175, wherein said method further comprises purifying the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate.

[0490] Clause 177. The method according to Clause 176, wherein the organic cation salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate is purified by liquid chromatography.

[0491] Clause 178. The method according to Clause 177, wherein the organic cation salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate is purified by liquid chromatography, said liquid chromatography comprising a silica gel stationary phase and a mobile phase comprising pyridine.

[0492] Clause 179. The method according to Clause 178, wherein the mobile phase comprises dichloromethane, methanol and pyridine.

[0493] Clause 180. The method according to any one of Clauses 178-179, wherein fractions collected from liquid chromatography are combined and concentrated by distillation.

[0494] Clause 181. The method according to any one of Clauses 178-179, wherein fractions collected from liquid chromatography are combined and concentrated under vacuum.

[0495] Clause 182. The method according to any one of Clauses 180-181, wherein the concentrated fraction is contacted with one or more particles of an organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate.

[0496] Clause 183. The method according to Clause 182, wherein particles of the organic cation salt 25-hydroxy-(3β)-cholest-5-ene-3-sulfate are contacted during the distillation of the combined fractions.

[0497] Clause 184. The method according to Clause 182, wherein said method comprises:

[0498] Concentration and merging of fractions under vacuum; and

[0499] The concentrated fraction is contacted with particles of the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and at least one solvent.

[0500] Clause 185. The method according to Clause 184, wherein the at least one solvent is selected from 2-methyltetrahydrofuran, heptane, or a combination thereof.

[0501] Clause 186. The method according to any one of Clauses 184-185, wherein the at least one solvent is 2-methyltetrahydrofuran.

[0502] Clause 187.2 hydrate of crystalline 25HC3S sodium, having an X-ray powder diffraction pattern containing peaks less than about 2.8°2θ.

[0503] Clause 188. The hydrate of crystalline 25HC3S sodium of Clause 187, having a diffraction pattern containing peaks between about 2.1°2θ and about 2.6°2θ.

[0504] Clause 189. The hydrate of crystalline 25HC3S sodium from Clause 188, having a diffraction pattern containing peaks between about 2.1°2θ and about 2.3°2θ.

[0505] The hydrate of crystalline 25HC3S sodium in any of Clauses 190, 2, 187, or 188, having an X-ray powder diffraction pattern containing peaks between about 4.3°2θ and about 4.6°2θ.

[0506] Clause 191. A hydrate of crystalline 25HC3S sodium from any of Clauses 2, 187, 188, 189 or 190, having an X-ray powder diffraction pattern containing peaks between about 5.0°2θ and about 5.5°2θ.

[0507] The hydrate of crystalline 25HC3S sodium in any of Clauses 192, 2, 187-191, having an X-ray diffraction pattern containing peaks between about 8.6°2θ and about 9.1°2θ.

[0508] Hydrate of crystalline 25HC3S sodium from any of Clauses 193, 2, 187-192, having an X-ray powder diffraction pattern containing peaks between about 15.0°2θ and about 15.3°2θ.

[0509] Clause 194. A hydrate of crystalline 25HC3S sodium from any of Clauses 2 or 188, having an X-ray powder diffraction pattern containing peaks between about 9.9°2θ and about 10.0°2θ.

[0510] Clause 195. Anhydrous crystalline sodium 25HC3S of any one of Clauses 29, 30 or 39, having an X-ray powder diffraction pattern containing peaks between about 4.5°2θ and about 4.8°2θ.

[0511] Clause 196. Anhydrous crystalline sodium 25HC3S of any of Clauses 29, 30, 39 or 195, having an X-ray powder diffraction pattern containing peaks between about 9.8°2θ and about 9.9°2θ.

[0512] Clause 197. Anhydrous crystalline sodium 25HC3S of any one of Clauses 29, 30, 39, 195 or 196, having an X-ray powder diffraction pattern containing peaks between about 14.1°2θ and about 14.3°2θ.

[0513] Anhydrous crystalline sodium 25HC3S of any one of Clauses 198, 29, 30, 39, 195, 197 or 197, having an X-ray powder diffraction pattern containing a peak at about 16.1°2θ.

[0514] Experimental Section

[0515] The following embodiments are provided to provide a complete disclosure and description of how to make and use the invention to those skilled in the art. They are not intended to limit the scope of what the inventors consider their invention, nor are they intended to represent that the experiments described below are all or only the experiments performed. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental errors and biases should be taken into account.

[0516] Examples 1-19 relate to samples prepared according to Table 1 and Examples 20, 21, 22, 24, 25, 26, 27, 28, 29, 31, 32, 34, 35, 37, and 38. Examples 23, 30, 33, 36, and 39 were prepared as written; XRPD patterns were collected according to Example 40, and no other analytical data are reported in the disclosure of these examples.

[0517] Example 1 - Analysis of 25HC3S Sodium Used in Screening

[0518] Approximately 10 grams of 25HC3S sodium was used for screening. X-ray powder diffraction analysis identified the material as a physical mixture of hydrates (form I and form II). Solution 1 The H-NMR spectrum is consistent with the chemical structure (see Figure 34 for 25HC3S). 1 (An example of the H-NMR structure). By Karl Fischer titration, the mixture contained 6.1 wt% water. This corresponds to approximately 1.8 mol / mol of water. DVS isotherms indicate that the material is hygroscopic. The mixture increased by approximately 5 wt% in 5–25% RH (1.5 mol / mol water), by 6 wt% in 25–85% RH (1.8 mol / mol water), and by an additional 6 wt% in 85–95% RH. The recovered material was identified by X-ray powder diffraction as predominantly form I, with an additional peak near 8.8° (2θ). This peak is attributed to form V.

[0519] The TGA yielded a 5.4% weight loss up to 128 °C, which coincided with extensive dehydration endothermic events observed in the DSC. The DSC curves also showed endothermic events near 167 °C and 184 °C. These events are associated with decomposition. Hot-stage microscopy correlated with dehydration between 58 °C and 112 °C, followed by decomposition near 163 °C.

[0520] Brief exposure of the mixture to 130°C produces a mixture of forms I, II, and XIII. Furthermore, the peak associated with form II shifts. Exposure to 75% RH for 10 days results in two additional peaks, primarily form I, identified by X-ray powder diffraction, near 4.4°2θ and 8.8°2θ, which are attributed to form V.

[0521] Example 2 - General Screening Method

[0522] Methods using solvents or solvent mixtures include cooling the solution, evaporation, adding antisolvents, and suspension (slurry). Variations in these methods can include changes in the solvent, solvent mixture, antisolvent, temperature, cooling rate, concentration, addition rate, and mixing order, to name just a few possibilities. The resulting solids are observed by polarized light microscopy and / or analyzed by X-ray powder diffraction.

[0523] Specific screening methods are given in Examples 3-7. The methods and results are summarized in, for example, Table 1 and the accompanying drawings of this disclosure.

[0524] Example 3 - Adding Antisolvent

[0525] This allows the solution to come into contact with the antisolvent. Adding these antisolvent additives helps reduce the solubility of the solvent system and induces crystallization.

[0526] Example 4 - Cooling and Slow Cooling

[0527] Prepare solutions in a selected solvent or solvent / antisolvent system. Cool these solutions to below room temperature in a refrigerator for varying durations to attempt to induce nucleation. Record the presence or absence of solids. After observing a sufficient amount of solid for analysis, separate the material. If the amount present is insufficient, further cooling is performed in a refrigerator. Separate the sample for wet analysis or as a dry powder.

[0528] Example 5 - Rapid Evaporation

[0529] Prepare a solution in a selected solvent and stir between additions of aliquots to aid dissolution. Once the mixture is completely dissolved, as determined by visual inspection, filter the solution through a 0.2-μm nylon filter and evaporate it under ambient conditions in an uncapped vial or at ambient temperature under nitrogen. The resulting solids are used for evaluation.

[0530] Example 6 - Slow Evaporation

[0531] Prepare a solution in a selected solvent and stir between additions of aliquots to aid dissolution. Once the mixture is completely dissolved, as determined by visual inspection, filter the solution through a 0.2-μm nylon filter into a sample vial. Cover the vial opening with foil and puncture it three times, then allow it to evaporate under ambient conditions. The resulting solids are used for evaluation.

[0532] Example 7 - Pulp

[0533] A solution is prepared by adding sufficient solid to a given solvent to create an excess of solid. The mixture is then stirred in a sealed vial at ambient temperature or an elevated temperature. After a given period of time, the solid is separated for analysis.

[0534] Example 8 - Differential Scanning Calorimetry (DSC)

[0535] Differential scanning calorimetry (DSC) analysis was performed using a Mettler-Toledo DSC3+ DSC. Temperature calibration was performed using octane, phenyl salicylate, indium, tin, and zinc. The sample was placed in an hermetically sealed or open aluminum DSC pan, and its weight was recorded. A weighing pan configured as the sample pan was placed on the reference side of the unit cell. The sample was analyzed from -30 to 250 °C at a heating rate of 10 °C / min. Although a thermal analysis plot was plotted against the reference temperature (x-axis), the results were reported based on the sample temperature.

[0536] Example 9 - Dynamic Vapor Adsorption / Desorption (DVS)

[0537] Moisture adsorption / desorption data were collected on a VTI SGA-100 Vapor Sorption Analyzer. NaCl and PVP were used as calibration standards. Samples were not dried prior to analysis. Adsorption and desorption data were collected in 10% RH increments from 5% to 95% RH under nitrogen purging. The equilibrium standard used for analysis was a weight change of less than 0.0100% over 5 minutes, with a maximum equilibrium time of 3 hours. No data were corrected for the initial moisture content of the samples.

[0538] Example 10 - Hot Stage Microscopy

[0539] Use installed with SPOT Insight TM Hot-stage microscopy was performed on a Linkam hot stage (FTIR 600) on a Leica DM LP microscope with a color digital camera. Temperature calibration was performed using the USP melting point standard. Samples were placed on coverslips, either clean (neat) or prepared with mineral oil, and a second coverslip was placed on top of the sample. As the stage was heated, each sample was visually observed using a 10x0.22 or 20x0.40 numerical aperture objective lens with orthogonal polarizers and a first-order red compensator. Images were captured using SPOT software (v.4.5.9).

[0540] Example 11 - Karl Fischer

[0541] Carl Fischer (KF) coulometric analysis for water determination was performed using a Mettler Toledo DL39 KF titrator. The NIST traceable water standard (Hydranal Water Standard 1.0) was analyzed to verify the coulometric operation. A blank titration was performed prior to sample analysis. The sample was prepared under ambient conditions, in which a weighed sample was dissolved in approximately 1 mL of Hydranal-Coulomat AD in a pre-dried vial. The entire solution was then passed through the diaphragm into the KF coulometric titrator and mixed for 10 seconds. The sample was then titrated using a generator electrode, which generates iodine via electrochemical oxidation: 2I⁻ → I₂ + 2e⁻. Two replicates were obtained to ensure reproducibility.

[0542] Example 12 - Polarized Light Microscopy

[0543] Polarized light microscopy was performed using a Motic SMZ-168. Each sample was observed using a 10x objective lens with orthogonal polarizers at magnifications from 0.75x to 5.0x.

[0544] Example 13- 1 H NMR spectroscopy

[0545] Obtain the solution from Spectral Data Solutions 1 H NMR spectrum.

[0546] Example 14 - Thermogravimetric Analysis (TGA)

[0547] Thermogravimetric analysis was performed using a Mettler Toledo TGA / DSC3+ analyzer. Temperature calibration was performed using phenyl salicylate, indium, tin, and zinc. Samples were placed in aluminum pans. The open pans were inserted into the TG furnace. The furnace was heated under nitrogen. Each sample was heated from ambient temperature to 350°C at a rate of 10°C / min. Although thermal analysis plots were generated using a reference temperature (x-axis), results were reported based on sample temperature.

[0548] Example 15 - X-ray Powder Diffraction (XRPD)

[0549] XRPD patterns (also known as diffraction patterns) are collected in either transmission or reflection modes.

[0550] Example 16 - Transmission

[0551] X-ray powder diffraction patterns were collected using an incident beam of Cu radiation generated by a long, narrow focusing source, employing a PANalytical X'Pert PRO MPD or PANalytical Empyrean diffractometer. An elliptical graded multilayer mirror was used to focus Cu Kαx-rays through the sample and onto the detector. Prior to analysis, a silicon sample (NIST SRM 640e) was analyzed to verify that the observed Si 111 peak position was consistent with the NIST-certified position. The sample was sandwiched between 3-μm thick films and analyzed within the transmission geometry. Beam stop, short antiscattering extension, and antiscattering knife edge were used to minimize background from air. Soller slits for both the incident and diffracted beams were used to minimize broadening and asymmetry caused by axial divergence. Diffraction patterns were collected using a scanning position-sensitive detector (X'Celerator) at a distance of 240 mm from the sample and Data Collector software v.2.2b or 5.5. Regardless of the instrument used, all images were generated using an instrument labeled X'Pert PROMPD.

[0552] Example 17 - Reflection

[0553] X-ray powder diffraction patterns were collected using a PANalytical X'Pert PRO MPD diffractometer with an incident beam of Cu Kα radiation generated by a long, narrow focusing source and a nickel filter. A symmetrical Bragg-Brentano geometry diffractometer was used. Prior to analysis, a silicon sample (NIST SRM 640e) was analyzed to verify that the observed Si 111 peak position was consistent with the NIST-certified position. The sample was loaded into a bore. An antiscattering slit was used to minimize background caused by air. Soller slits for both the incident and diffracted beams were used to minimize broadening caused by axial divergence. Diffraction patterns were collected using a scanning position-sensitive detector (X'Celerator) at a distance of 240 mm from the sample and Data Collector software v.2.2b.

[0554] Example 18 - Variable Humidity (VH-XRPD)

[0555] X-ray powder diffraction patterns were collected using a PANalytical X'Pert PRO MPD diffractometer with an incident beam of Cu Kα radiation generated by a long, narrow focusing source and a nickel filter. A symmetrical Bragg-Brentano geometry diffractometer was used. Data were collected and analyzed using Data Collector software v.2.2b. Prior to analysis, a silicon sample (NIST SRM640e) was analyzed to verify that the observed Si 111 peak position was consistent with the NIST-certified position. The sample was placed in a nickel-coated copper aperture. An antiscattering slit was used to minimize background from air. Soller slits for both the incident and diffracted beams were used to minimize broadening caused by axial divergence. Diffraction patterns were collected using a scanning position-sensitive detector (X'Celerator) at a distance of 240 mm from the sample and Data Collector software v.2.2b.

[0556] Anton Paar temperature-humidity chambers (THCs) were used for in-situ collection of X-ray powder diffraction patterns as a function of humidity. The temperature of the samples was controlled by a Peltier thermoelectric device located directly beneath the sample holder and monitored by a platinum-100 resistance sensor located within the sample holder. The thermoelectric device was powered and controlled by an Anton Paar TCU 50 interfaced with the Data Collector software.

[0557] Humidity was generated by an RH-200 manufactured by VTI Inc. and carried by a nitrogen gas flow. Humidity and temperature were monitored by a Rotronic HygroClip sensor located next to the sample inside the THC.

[0558] Example 19 - Variable Temperature (VT-XRPD)

[0559] X-ray powder diffraction patterns were collected using a PANalytical X'Pert PRO MPD diffractometer with an incident beam of Cu Kα radiation generated by a long, narrow focusing source and a nickel filter. A symmetrical Bragg-Brentano geometry diffractometer was used. Data were collected and analyzed using Data Collector software v.2.2b. Prior to analysis, a silicon sample (NIST SRM640e) was analyzed to verify that the observed Si 111 peak position was consistent with the NIST-certified position. The sample was placed in a nickel-coated copper aperture. An antiscattering slit was used to minimize background caused by air scattering. Soller slits for both the incident and diffracted beams were used to minimize broadening caused by axial divergence. Diffraction patterns were collected using a scanning position-sensitive detector (X'Celerator) at a distance of 240 mm from the sample.

[0560] Anton Paar TTK 450 was used for in-situ collection of X-ray powder diffraction patterns as a function of temperature. The samples were heated using a resistance heater located directly beneath the sample holder, and the temperature was monitored using a platinum-100 resistance sensor located within the sample holder. The heater was powered and controlled by an Anton Paar TCU 100 interfaced with the Data Collector software.

[0561] Example 20 - Form I - Article 1

[0562] Sodium 25HC3S (164.6 mg) and methanol (2 mL) were heated on a hot plate set at 75 °C until a clear solution was obtained. The solution was filtered through a 0.2 μm nylon filter into 20 mL of dichloromethane. This resulted in the immediate precipitation of a gel, which was harvested by vacuum filtration using water suction. The gel crystallized into form I after separation. Figure 36 is the XRPD diffraction pattern of this product.

[0563] Example 21 - Form I - Article 2

[0564] A slurry was obtained using 64.5 mg of 25HC3S sodium and 1 mL of toluene. The slurry was magnetically stirred at approximately 400 RPM at room temperature for 21 days. Form I was obtained by allowing the suspended solids to settle and decanting excess solution from the solids. Figure 37 is the XRPD diffraction pattern of this product.

[0565] Example 22 - Form I - Article 3

[0566] A slurry was obtained using 41.9 mg of 25HC3S sodium and 12 mL of acetone. The slurry was briefly heated on a hot plate set at 85°C and magnetically stirred at approximately 200 RPM. The slurry was removed from the heat source, and 1 mL of water was added while magnetically stirring at approximately 400 RPM. After almost complete dissolution, a flocculent suspension was formed, which then nucleated into fine dendritic crystals. The slurry was stirred for 4 days. Form I was harvested by vacuum filtration through water suction and dried under nitrogen for approximately 10 minutes. Figure 38 is the XRPD diffraction pattern of this product.

[0567] Example 23 - Form I - Article 4

[0568] Form I was obtained by performing the following dynamic vapor adsorption test:

[0569] Dynamic vapor adsorption (DVS) was measured using an SMS (Surface Measurement System) DVS Intrinsic. The parameters for the DVS test are listed in Table 11.

[0570] Table 11 Parameters of DVS

[0571]

[0572]

[0573] The DVS test results showed that the compound is hygroscopic, and the starting material reversibly absorbs 7.0% water below 30% RH and 8.5% water at 95% RH. Figure 355 is the XRPD diffraction pattern of this product.

[0574] Example 24 - Form I+XIII

[0575] A slurry was prepared using 586.6 mg of 25HC3S sodium and 10 mL of 97:03 v / v acetonitrile / water. The slurry was magnetically stirred at approximately 250 RPM for 4 days at room temperature. The mixture of forms I+XIII was harvested by vacuum filtration through water suction and dried under nitrogen for approximately 30 minutes. Figure 39 shows the XRPD diffraction pattern of this mixture.

[0576] Example 25 - Form XIII - Article 1

[0577] Form XIII was obtained by exposing a mixture of forms I and XIII (obtained from Example 24) to 70°C under vacuum for 2 days. Figure 40 is the XRPD diffraction pattern of this product.

[0578] Example 26 - Form XIII - Article 2

[0579] A mixture of forms I and XIII (obtained from Example 24) separated at 15% RH was successively exposed to 25%, 55%, 75%, 85%, 75%, 25%, and 0% RH. The material was held at each RH condition for a minimum of 1 hour before proceeding to the next condition. Upon exposure to 25% RH, the mixture converted to form I and remained at 55%, 75%, 85%, 75%, and 25% RH. Upon reaching 0% RH, form I converted to a mixture of forms I and XIII. Within 20 minutes of continued exposure to 0% RH, the mixture of forms I and XIII converted to form XIII. Figure 41 is the XRPD diffraction pattern of this article.

[0580] Example 27 - Form II - Article 1

[0581] A slurry was obtained using 75.4 mg of 25HC3S sodium and 20 mL of acetone. The slurry was magnetically stirred at approximately 400 RPM for 14 days at room temperature. Form II was harvested by vacuum filtration through water suction and briefly dried under nitrogen. Figure 42 shows the XRPD diffraction pattern of this product.

[0582] Example 28 - Form II - Article 2

[0583] A slurry was obtained using 66.9 mg of 25HC3S sodium and 19 mL of acetonitrile. The slurry was magnetically stirred at approximately 400 RPM for 14 days at room temperature. Form II was harvested by vacuum filtration through water suction and briefly dried under nitrogen. Figure 43 shows the XRPD diffraction pattern of this product.

[0584] Example 29 - Form II - Article 3

[0585] A slurry was prepared using 75.4 mg of 25HC3S sodium and 8 mL of ethanol. The slurry was filtered through a 0.2 μm nylon filter to obtain a clear solution. Four-mL aliquots of the clear solution were evaporated under ambient conditions until a solid was clearly visible and approximately 0.1 mL of mother liquor remained. Form II was harvested by decantation. Figure 44 shows the XRPD diffraction pattern of this product.

[0586] Example 30 - Form II - Article 4

[0587] Form II was obtained by adding antisolvents (dimethylacetamide (DMA) and methyl isobutyl ketone (MIBK)). The antisolvent method was as follows: a concentrated stock solution of the starting material was prepared in DMA. The solution was stirred and MIBK was rapidly added to induce precipitation. After centrifugation and filtration, the solid for XRPD analysis was separated to produce form II. Figure 46 is the XRPD diffraction pattern of this product.

[0588] Example 31 - Form III + Form IX

[0589] A slurry was prepared using 46.6 mg of 25HC3S sodium and 0.5 mL of ethanol. The slurry was briefly heated on a hot plate set at 75 °C, resulting in solvent absorption. The mixture of forms III and IX was removed from the heat source and dried under nitrogen for approximately 15 minutes. Figure 45 shows the XRPD diffraction pattern of the mixture of forms III and IX.

[0590] Example 32 - Form IX - Article 1

[0591] Form IX was obtained by exposing a mixture of forms III and IX (obtained from Example 31) to 58°C under vacuum for 1 day. Figure 5 is the XRPD diffraction pattern of this product.

[0592] Example 33 - Form IX - Article 2

[0593] Weigh 20.3 mg of sodium 25HC3S into a 3-mL bottle. Place this 3-mL bottle into a 20mL bottle containing 3-4 mL of ethanol and seal the outer bottle. Incubate the system at room temperature for 12 hours, and analyze the separated solids by XRPD. Figure 51 shows the XRPD diffraction pattern of this product.

[0594] Example 34 - Form V - Article 1

[0595] A turbid suspension was obtained using 85.1 mg of 25HC3S sodium and 7 mL of methanol. The suspension was filtered through a 0.2 μm nylon filter to provide a clear solution. Form V was provided by evaporating the solution until it was visually dried under ambient conditions. Figure 47 is the XRPD diffraction pattern of this product.

[0596] Example 35 - Form V - Article 2

[0597] A slurry was prepared using 70.4 mg of 25HC3S sodium and 20 mL of water. The slurry was magnetically stirred at approximately 400 RPM for 14 days. An opaque gel was obtained by vacuum filtration through water suction. The gel was converted to form V after separation. Figure 48 shows the XRPD diffraction pattern of this product.

[0598] Example 36 - Form V - Article 3

[0599] Form V was obtained by adding antisolvents (MeOH solvent and EtOAc antisolvent). The antisolvent method is as follows: a concentrated stock solution of the starting material was prepared in MeOH. The solution was stirred and EtOAC was rapidly added to induce precipitation. After centrifugation and filtration, the solid for XRPD analysis was separated to produce form V. Figure 56 is the XRPD diffraction pattern of this product.

[0600] Example 37 - Form XI

[0601] A slurry was obtained using 97.5 mg of 25HC3S sodium and 20 mL of diethyl ether. The slurry was magnetically stirred at approximately 400 RPM for 14 days. Form XI was harvested by vacuum filtration through water suction and dried under nitrogen. Figures 6 and 7 are XRPD diffraction patterns of this product.

[0602] Example 38 - Form III - Article I

[0603] A turbid suspension was obtained using 97 mg of 25HC3S sodium and 8 mL of ethanol. The suspension was allowed to settle at ambient temperature for approximately one day. The suspension was then centrifuged, and the wet solid was collected by decantation. Figure 49 shows the XRPD diffraction pattern of this product.

[0604] Example 39 - Form III - Article 2

[0605] 1.98 mg of sodium 25HC3S was weighed into a 3 mL glass vial. 3 mL of EtOH was added to the vial at room temperature to form a suspension, and the solution was heated to 50 °C. The solution was filtered through a 0.45 μm PTFE filter at 50 °C, and the filtrate was collected into a clean tubular vial. The solution was cooled to room temperature and then stored at -20 °C. The solid was separated by centrifugation at 14,000 RPM for 5 min, collected, and analyzed by XRPD. Figure 50 shows the XRPD diffraction of this product.

[0606] XRPD analysis of Examples 40-23, 30, 33, 35 and 36.

[0607] Using Panalytical X'Pert 3 Powder XRPD was performed on a Si zero-background support. The 2θ position was calibrated relative to the Panalytical 640Si powder standard. A 4-min method was used for most samples. The dispersion stability of spray-dried samples was analyzed using an 80-min method. Details of the XRPD used in the experiments are listed in Table 12 below.

[0608] Table 12 XRPD Parameters

[0609]

[0610]

[0611] General synthetic procedure for preparing 25-hydroxy-(3β)-cholest-5-ene-3-sulfate

[0612] 25HC3S can be prepared by various methods. This article contains exemplary methods for preparing 25HC3S.

[0613] All temperatures are in degrees Celsius (°C) and are uncorrected. Reagent-grade chemicals and anhydrous solvents were purchased from commercial sources and used without further purification unless otherwise mentioned. Product names were determined using the naming software included in the Biovia Electronic Lab Notebook. Silica gel chromatography was performed on a Teledyne Isco instrument using a pre-packaged, disposable SiO2 stationary column at an eluent flow rate of 15–200 mL / min. Analytical HPLC chromatograms were obtained using an Agilent 1100 series instrument with a DAD detector (190 nm to 300 nm). Mass spectra were recorded at 130 °C using a Waters Micromass ZQ detector. The mass spectrometer was equipped with an electro-ejector ionization (ESI) source operating in cation mode and set to scan between m / z 150–750 at a scan time of 0.3 s. The products and intermediates were analyzed by HPLC / MS as follows: a high pH buffer gradient of 5% to 100% MeCN in H2O (0.03% (NH4)2CO3 / 0.375% NH4OH) was used on a Gemini-NX (5 μM, 2.0 x 30 mm) at 1.8 mL / min for 2.5 min, followed by a run of 3.5 min (B05); and a low pH buffer gradient of 5% to 100% MeCN in H2O (0.1% HCOOH) was used on an EVO C18 (5 μM, 3.0 x 50 mm) at 2.2 mL / min for 2.5 min, followed by a run of 3.5 min (A05). Recordings were performed on a Bruker UltraShield 500 MHz / 54 mm instrument (BZH 43 / 500 / 70B, D221 / 54-3209). 1 1H NMR spectrum. Chemical shifts are reference solvent peaks. 1 In H NMR, the solvent peak appears at 7.26 ppm for CDCl3, at 2.50 ppm for DMSO-d6, and at 3.31 ppm for CD3OD.

[0614] [(3S,10R,13R,17R)-17-[(1R)-5-hydroxy-1,5-dimethyl-hexyl]-10,13-dimethyl-2,3, Synthesis of sodium dodecahydro-1H-cyclopentadien[a]phenanthrene-3-yl]sulfate

[0615]

[0616] A dry 3-necked flask was filled with a pyridine-sulfur trioxide complex (12.45 g, 78 mmol), and the solid was suspended in toluene (1.5 L) and acetic anhydride (7.2 mL, 74.5 mmol). The mixture was stirred at 20 °C for 40 min, and pyridine (60 mL, 745 mmol) was added. The mixture was stirred at 20 °C for 20 min. In a single fraction, (3S,8S,9S,10R,13R,14S,17R)-17-[(1R)-5-hydroxy-1,5-dimethyl-hexyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecylhydro-1H-cyclopentadieno[a]phenanthrene-3-ol (30 g, 74.5 mmol) was added as a solid. The mixture was stirred at 20 °C for 23 h. A sodium acetate aqueous solution (10 wt%, 123 mL, 149 mmol) was added dropwise over 5 min with vigorous stirring. The resulting mixture was stirred at 20 °C for 1 h. The solvent was pumped out of the reactor, and any solids were collected on a glass frit. ACN (700 mL) was added, and the slurry was stirred vigorously for 3 h. The slurry was pumped out of the reactor onto the same glass frit, and the remaining solids in the reactor were resuspended in ACN (700 mL) and stirred for 1 h. The contents of the reactor were then pumped out onto the glass frit. The solids in the glass frit were washed with diethyl ether (750 mL) and then suspended in DMF (800 mL). The mixture was stirred at 20 °C for 1 h. The suspension was filtered, and the filtrate was collected. Diethyl ether (3.2 L) was added to the filtrate with stirring. The resulting solids were collected by vacuum filtration, and the filter cake was washed with diethyl ether (1 L). The solids were dried under reduced pressure to provide the title compound (15 g, 40%) as a solid. 1 H NMR(500MHz,MeOD)δ5.56-5.32(m,1H),4.17(tt,J=11.5,4.8Hz,1H),2.55(dd,J =4.9,2.2Hz,1H),2.47-2.29(m,1H),2.14-2.06(m,2H),2.01(ddd,J=12.4,7.7,5 .1Hz,1H),1.97-1.85(m,2H),1.73-1.22(m,15H),1.20(s,6H),1.19-1.08(m,4H) ,1.07(s,3H),1.04-0.95(m,1H),1.00(d,J=6.5Hz,3H),0.76(s,3H);m / z:ES-[M] - 481.3; LCMS(B05); t R =1.18m.

[0617] [(3S,10R,13R,17R)-17-[(1R)-5-hydroxy-1,5-dimethyl-hexyl]-10,13-dimethyl-2,3, Synthesis of sodium dodecahydro-1H-cyclopentadien[a]phenanthrene-3-yl]sulfate

[0618]

[0619] A sulfur trioxide pyridine complex (4.74 g, 29.8 mmol) was placed in a dry 3-necked flask. The solid was suspended in toluene (500 mL), and acetic anhydride (2.61 mL, 27.67 mmol) was added in a single fraction. The resulting mixture was stirred at 23 °C for 1 h. Pyridine (20 mL, 248.4 mmol) was added, and the mixture was stirred at 23 °C for 5 min. (3S,10R,13R,17R)-17-[(1R)-5-hydroxy-1,5-dimethyl-hexyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecylhydro-1H-cyclopentadieno[a]phenanthrene-3-ol (10 g, 24.83 mmol) was added as a single fraction. The mixture was stirred at 23 °C for 23 h. The reactants were diluted with MeOH (2.01 mL, 49.7 mmol) and stirred at 23 °C for 1 h. The suspension was filtered, and the solid was washed with toluene (2 x 200 mL). The solid was collected and dried under high vacuum to provide a solid. The solid was partially dissolved in ACN (600 mL), and sodium iodide (14.9 g, 99.3 mmol) was added. The mixture was stirred at 23 °C for 10 min, then cooled to 0 °C in an ice bath and stirred for 1.5 h. The suspension was filtered, and the solid was washed with cold ACN (2 x 275 mL) and acetone (2 x 200 mL). The solid was collected and dried under high vacuum to provide the title compound as a solid (7.24 g, 57%). 1 H NMR(500MHz,MeOD)δ5.56-5.32(m,1H),4.17(tt,J=11.5,4.8Hz,1H),2.55(dd,J =4.9,2.2Hz,1H),2.47-2.29(m,1H),2.14-2.06(m,2H),2.01(ddd,J=12.4,7.7,5 .1Hz,1H),1.97-1.85(m,2H),1.73-1.22(m,15H),1.20(s,6H),1.19-1.08(m,4H) ,1.07(s,3H),1.04-0.95(m,1H),1.00(d,J=6.5Hz,3H),0.76(s,3H);m / z:ES-[M] - 481.3; LCMS(B05); t R =1.18m.

[0620] [(3S,10R,13R,17R)-17-[(1R)-5-hydroxy-1,5-dimethyl-hexyl]-10,13-dimethyl-2,3, Synthesis of sodium dodecahydro-1H-cyclopentadien[a]phenanthrene-3-yl]sulfate

[0621]

[0622] The 15L jacketed reactor was heated to 60°C and purified with nitrogen for 1.5 h. The jacket temperature was set to 30°C and 7L of 2-MeTHF was added. (3S,10R,13R,17R)-17-[(1R)-5-hydroxy-1,5-dimethyl-hexyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecylhydro-1H-cyclopentadienyl[a]phenanthrene-3-ol (495 g, 1.23 mol) was added, and the manholes / glassware were rinsed with 6L of 2-MeTHF. The solution was cooled to 25°C, and additional 1L of 2-MeTHF was added, along with a sulfur trioxide pyridine complex (234.8 g, 1.47 mol). The mixture was stirred at 28°C for 24 h. Add 2-MeTHF (2 L), stir the mixture for another 16 h, cool to 20 °C and filter. Wash the solid with 2-MeTHF (3.5 L). Dissolve the solid in a solution of NaOH (118 g, 2.95 mmol) in MeOH (6 L). Stir the mixture at 25 °C for 1 h and then filter on a diatomaceous earth stopper. Concentrate the filtrate to 3.5 L and dilute with ether (8 L). Cool the suspension to 15 °C and filter to provide the title compound as a solid (146.8 g, 24%). Concentrate the filtrate to 1 L and mix again with ether (4 L). Collect the solid by vacuum filtration to provide the title compound as a solid (68.5 g, 11%). Extract the diatomaceous earth with MeOH (2 L), concentrate it to 500 mL and dilute with ether (3 L), and collect the solid by vacuum filtration to provide the title compound as a solid (53.3 g, 8.6%). The fourth batch was separated from the filtrate (11.88 g, 2%). Overall yield: 280.5 g, 45%. 1 H NMR(500MHz,MeOD)δ5.56-5.32(m,1H),4.17(tt,J=11.5,4.8Hz,1H),2.55(dd,J =4.9,2.2Hz,1H),2.47-2.29(m,1H),2.14-2.06(m,2H),2.01(ddd,J=12.4,7.7,5 .1Hz,1H),1.97-1.85(m,2H),1.73-1.22(m,15H),1.20(s,6H),1.19-1.08(m,4H) ,1.07(s,3H),1.04-0.95(m,1H),1.00(d,J=6.5Hz,3H),0.76(s,3H);m / z:ES-[M] - 481.3; LCMS(B05); t R =1.18m.

[0623] [(3S,10R,13R,17R)-17-[(1R)-5-hydroxy-1,5-dimethyl-hexyl]-10,13-dimethyl-2,3, Synthesis of 4,7,8,9,11,12,14,15,16,17-dodecylhydro-1H-cyclopentadien[a]phenanthrene-3-yl]ammonium sulfate

[0624]

[0625] A sulfur trioxide-dimethylformamide complex (42 mg, 0.273 mmol) was added at 0 °C to a stirred solution of (3S,10R,13R,17R)-17-(5-hydroxy-1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecano-1H-cyclopentadieno[a]phenanthrene-3-ol (100 mg, 0.25 mmol) in anhydrous DCM (20 mL). The mixture was stirred at 0 °C for 5 h, and then the reaction mixture was warmed to 20 °C. The mixture was concentrated under reduced pressure to provide a crude solid, which was purified by column chromatography on silica gel (12 g cartridge) eluting with a mixture of DCM and MeOH (0-20%) to provide the impure title compound. m / z:ES-[MH] - 481.

[0626] [(3S,8S,9S,10R,13R,14S,17R)-17-[(1R)-1,5-dimethylhexyl]-10,13-dimethyl-2, 3,4,7,8,9,11,12,14,15,16,17-dodecylhydro-1H-cyclopentadienyl[a]phenanthrene-3-yl]pyridine-1-onium sulfate become

[0627]

[0628] In a dried round-bottom flask, a sulfur trioxide-pyridine complex (4.53 g, 28.5 mmol) was suspended in toluene (240 mL). Acetic anhydride (2.44 mL) was added, followed by pyridine (20.8 mL). The reaction mixture was stirred at 23 °C for 1 h, and cholesterol (10 g, 25.9 mmol) was added as a single fraction. The suspension was stirred at 23 °C for 18 h, filtered through a glass frit, and the solid was washed with toluene (100 mL), followed by washing with hexane (100 mL). The solid was suspended in chloroform (400 mL) and filtered through the same glass frit. The glass frit was washed with chloroform (200 mL) and the filtrate was collected. The filtrate was diluted with hexane to 1.8 L and refrigerated for 1 h. The suspension was filtered; the solid was washed with diethyl ether (100 mL) and dried under high vacuum to provide the title compound as a solid (10.06 g, 71%). 1H NMR(500MHz,MeOD)δ8.89(dd,J=6.6,1.4Hz,2H),8.79-8.61(m,1H),8.27-8.05(m,2H),5. 38(d,J=5.3Hz,1H),4.13(tt,J=11.5,4.7Hz,1H),2.53(ddd,J=13.3,5.0,2.3Hz,1H),2.43 -2.28(m,1H),2.12-2.02(m,2H),2.01-1.94(m,1H),1.94-1.80(m,2H),1.70-0.83(m,20H) ,1.03(s,3H),0.95(d,J=6.6Hz,3H),0.88(dd,J=6.6,1.9Hz,6H),0.72(s,3H); m / z:ES-[M] - 465.3; LCMS(B05); t R =1.40m.

[0629] [(3S,8S,9S,10R,13R,14S,17R)-17-[(1R)-1,5-dimethylhexyl]-10,13-dimethyl-2, 3,4,7,8,9,11,12,14,15,16,17-dodecylhydro-1H-cyclopentadienyl[a]phenanthrene-3-yl]pyridine-1-onium sulfate become

[0630]

[0631] Cholesterol sulfate pyridinium salt was prepared by adding a sulfur trioxide pyridine complex (4.53 g, 28.5 mmol) to a solution of cholesterol (10 g, 25.9 mmol) in 2-MeTHF (250 mL) at 30 °C and stirring the mixture for 16 h. The suspension was then filtered, and the solid was washed with 2-MeTHF (50 mL) to provide the title compound.

[0632] [(3S,8S,9S,10R,13R,14S,17R)-17-[(1R)-1,5-dimethylhexyl]-10,13-dimethyl-2, Synthesis of sodium dodecahydro-1H-cyclopentadien[a]phenanthrene-3-yl]sulfate

[0633]

[0634] Chlorosulfonic acid (0.03 mL, 0.45 mmol) was added to a solution of 2,6-dimethylpyridine (0.08 mL, 0.69 mmol) in acetone (2.5 mL) via a molecular sieve. The solution was stirred at 20 °C for 2 min and then cooled to 0 °C. A solution of cholesterol (100 mg, 0.26 mmol) pre-dried via a molecular sieve in acetone (5 mL) was added dropwise. The mixture was stirred at 0 °C for 2 h and then heated to 20 °C for 16 h. The mixture was filtered and the solid was collected. The solid was then suspended in acetone (10 mL) and an aqueous sodium bicarbonate solution was added until the bubbling subsided. The suspension was filtered and the solid was ground together with MeOH (10 mL) and DCM (10 mL). The solvent was removed under reduced pressure to provide the solid. The solid was ground together with ACN (30 mL), filtered, and the filtrate was lyophilized under low pressure to provide the title compound (7.3 mg, 5.8%) as a solid. 1 H NMR (500MHz, DMSO) δ5.31-5.19(m,1H),4.10(s,1H),3.87-3.78(m,1H),2.42-2.31(m,1H),2.13(dd,J=14.5,7.6Hz,1H ),2.02-1.69(m,5H),1.62-0.95(m,20H),0.94(s,3H),0.89(d,J=6.5Hz,4H),0.84(dd,J=6.6,2.5Hz,7H),0.65(s,3H).

[0635] [(3S,5S,8R,9S,10S,13R,14S,17R)-17-[(1R)-1,5-dimethylhexyl]-10,13-dimethyl- Synthesis of 2,3,4,5,6,7,8,9,11,12,14,15,16,17-Tetradecano-1H-cyclopentadien[a]phenanthrene-3-yl]ammonium sulfate

[0636]

[0637] The sulfur trioxide pyridine complex (300 mg, 1.88 mmol) was added to a solution of cholesterol (300 mg, 0.772 mmol) in pyridine (5.00 mL), and the suspension was stirred at 20 °C for 16 h. The residue was purified by silica gel chromatography (24 g cartridge) with MeOH (5% NH4OH) in DCM, eluting with a mixture of DCM and MeOH (0-30%) to provide the title compound as a solid (314 mg, 84%). 1H NMR(500MHz,DMSO-d6)δ7.08(s,4H),3.97-3.86(m,1H),1.91(dd,J=12.5,3.5Hz,1H),1.86-1.71(m,2H),1.69-1.55(m,3H),1.55-1.41(m ,3H),1.38-1.25(m,5H),1.25-0.90(m,15H),0.88(d,J=6.6Hz,4H),0.84(dd,J=6.6,2.4Hz,7H),0.74(s,3H),0.62(s,3H); m / z: ES[M-NH4] - 467.3; HPLC(BEH Ambicarb / ACN 5-100%)t R = 7.48 min.

[0638] [(3R,8S,9S,10R,13R,14S,17R)-17-[(1R)-1,5-dimethylhexyl]-10,13-dimethyl-2, Synthesis of 3,4,7,8,9,11,12,14,15,16,17-dodecylhydro-1H-cyclopentadien[a]phenanthrene-3-yl]ammonium sulfate

[0639]

[0640] A sulfur trioxide pyridine complex (206 mg, 1.29 mmol) was added to a solution of (3R,8S,9S,10R,13R,14S,17R)-17-[(1R)-1,5-dimethylhexyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecylhydro-1H-cyclopentadieno[a]phenanthrene-3-ol (200 mg, 0.517 mmol) in pyridine (5.00 mL). The suspension was stirred at 20 °C for 16 h and then concentrated under reduced pressure. The residue was purified by silica gel chromatography (24.0 g cartridge) by elution with a mixture of DCM and 5% NH4OH in MeOH (0-30%) to provide the title compound as a solid (160 mg, 64%). 1 H NMR(500MHz,DMSO)δ7.07(s,4H),5.18-5.14(m,1H),4.32-4.27(m,1H),2.40-2.29(m,1H),2.16(dt,J=14.9,2.4Hz,1H),2.01- 1.72(m,4H),1.60-0.96(m,22H),0.94(s,3H),0.90(d,J=6.5Hz,3H),0.84(dd,J=6.6,2.4Hz,6H),0.65(s,3H); m / z: ES[M-NH4] - 465.6; HPLC(BEH AmForm / ACN 5-100%)t R = 2.76 min.

[0641] [(3S,8S,9S,10R,13R,14S,17R)-17-[(1R)-5-hydroxy-1,5-dimethyl-hexyl]-10,13-di Methyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecylhydro-1H-cyclopentadien[a]phenanthrene-3-yl]pyridinium sulfate Synthesis

[0642]

[0643] Acetic anhydride (0.0704 mL, 0.745 mmol) was added to a suspension of sulfur trioxide-pyridine complex (125 mg, 0.782 mmol) in anhydrous toluene (15.0 mL). The suspension was stirred at 20 °C for 40 min, and pyridine (0.600 mL) was added. The suspension was stirred at 20 °C for 20 min. In a separate fraction, (3S,8S,9S,10R,13R,14S,17R)-17-[(1R)-5-hydroxy-1,5-dimethyl-hexyl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecylhydro-1H-cyclopentadieno[a]phenanthrene-3-ol (300 mg, 0.745 mmol) was added as a solid. The suspension was stirred at 20 °C for 20 h. The mixture was filtered over a glass frit to provide the title compound as a solid (329 mg, 92% purity, 72% yield). 1 H NMR (500MHz, DMSO-d6) δ8.99-8.88(m,2H),8.65-8.53(m,1H),8.13-7.97(m,2H),5.30-5.20(m,1H),3.93-3.71(m,1H),2.41-2.32( m / z ES + [M+H] + 481.32; HPLC (DUR B)t R = 1.36 min.

[0644] Synthesis of 3β-25-hydroxycholest-5-ene sulfate (1, as sodium salt)

[0645]

[0646] Preparation of 3β-25-hydroxycholest-5-ene sulfate - Route 1

[0647] 3β,25-dihydroxycholest-5-ene (4.6 g, 0.011 mol) and triethylamine (1.7 mL, 0.023 mol) were suspended in pyridine (57 mL) and heated to 50 °C. A sulfur trioxide trimethylamine complex (3.2 g, 0.023 mol) was added, and the mixture was stirred for 24 hours. Another fraction of the sulfur trioxide trimethylamine complex (0.77 g, 0.006 mol) was added, and the mixture was stirred for another 4 hours. The reaction mixture was distilled to -20% of its initial volume using a jacket at 50 °C. The residue was purified by silica gel chromatography (110 g), eluting with a mixture of ethyl acetate / methanol / triethylamine (90 / 9 / 1 v / v); fractions were analyzed by TLC (4:1 dichloromethane:methanol) using phosphomolybdic acid dye. Fractions containing the 3- and 25-sulfate isomers were combined and evaporated (bath temperature <35 °C). The residue (4.2 g, 0.0072 mol) was pulped in acetonitrile (25 g), treated with 1 N sodium hydroxide (7.2 ml, diluted from 30% sodium hydroxide solution) for 1 hour, and then filtered. The solid was thoroughly washed with acetonitrile (25 g) and dried to constant weight (2.77 g). The solid containing the mixture of 3- and 25-sodium sulfate salts (2.77 g) was ground with ethanol (27.7 g, 10 s) at 50 °C for 1 hour, and then filtered at 5 °C. The separated solid was dried to constant weight (1.2 g). The solid (1.2 g) was suspended in 6:1 acetonitrile / water (10 s) at 30 °C for 30 minutes, and then filtered. Filtration took about 40 minutes. The solid was dried to constant weight (0.86 g) and analyzed.

[0648] Preparation of 3β-25-hydroxycholest-5-ene sulfate - Route 2 - Excess sulfation reagent

[0649] An excess of the sulfur trioxide trimethylamine complex was used to drive the reaction toward the formation of the disulfate. The reactivity of the 3-hydroxy group of 3β,25-dihydroxycholest-5-ene toward sulfation is about 6 times that of the 25-hydroxy group. Providing an excess of the sulfating agent and proceeding the reaction to a high conversion yields a monosulfate with higher positional isomer purity. This result was observed during this synthesis. A solution of 3β,25-dihydroxycholest-5-ene (4.1 g) in pyridine (75 ml) was vacuum distilled to reduce the volume to 50 ml. This was done to remove isopropanol (from recrystallization of the diol) and any present water. Triethylamine (2 equivalents) was added to the reactants at 50 °C for 18 hours, followed by the addition of a total of 1.75 equivalents of the sulfur trioxide trimethylamine complex in fractions (1.0, 0.5, and 0.25 equivalents), and the reaction was allowed to proceed for a total of 43 hours. The reaction mixture was concentrated by vacuum distillation, and the residue was adsorbed onto SiO2 (10 g). Loaded SiO2 was placed on a SiO2 column and eluted with 2-50% methanol / ethyl acetate / 1% triethylamine. Appropriate fractions from the column were combined and evaporated to produce disulfate (3.1 g, 39.7%) and monosulfate (2.6 g, 44.7%). The resulting monosulfate was a 22:1 mixture of 3-sulfate and 25-sulfate. The solid was suspended in acetonitrile (25 g), treated with 1N sodium hydroxide (4.44 mL), and then filtered. A thick gel formed, which was difficult to handle and could not be filtered. The product was suspended in acetonitrile / water. The solvent was removed by rotary evaporation at 40 °C, and the residue was dried in a vacuum oven at 40 °C. The solid was ground with acetone to produce a white solid: 1.27 g, 24.9%. This product showed only 3 β - The product of sulfation, but contaminated by peaks at RRT 8.18 (unknown, 2.0%), RRT 15.17 (diol, 2.2%) and RRT 16.70 (unknown, 1.8%).

[0650] Preparation-scale synthesis

[0651] Synthesis of 3β-25-hydroxycholest-5-ene sulfate (1, as sodium salt)

[0652]

[0653] 3β,25-dihydroxycholest-5-ene (34) (30 g, 74.5 mmol) and dried pyridine (500 mL, Sigma-Aldrich, catalog 270970-1L, lot SHBC6287V) were added in a single batch to a 2 L three-necked round-bottom flask equipped with a top stirrer. A sulfur trioxide-trimethylamine complex (12.2 g, 89.4 mmol, Sigma-Aldrich, catalog 135879-100G, lot MKBH5585V) was added in a single batch. The suspension was stirred overnight at room temperature. The reaction mixture was concentrated, and the residue was purified by column chromatography to yield 25.9 g (59%) of a white solid as a triethylamine salt (HPLC: 98.6% purity). To a suspension of triethylamine salt 34.1 (64 g, 110.1 mmol) in ACN (1 L), 1N NaOH (110 mL, 110.1 mmol, NaOH, Fisher, catalog number 318-3, batch number 034906) was added, and the mixture was stirred at room temperature for 1 h. The solid was filtered, washed with ACN (1 L), and dried under vacuum (P2O5) overnight. Yield: 51.5 g, 93% (HPLC: 98.6% purity).

[0654] After stirring overnight, the reactants formed a gel-like mixture. TLC showed the expected product as the main spot (TLC: 20% MeOH in DCM, R...). f =0.4), starting material (R) f >0.9) and 3β-25-hydroxycholesterol disulfate (R f<0.1) as minor spots. Silica gel (1 kg, Sorbent Technologies, catalog 40930-2.5 kg) was packed to form a 10 cm x 42 cm column. Column equilibration was achieved using 1% triethylamine (Et3N, Fisher, catalog 04885-4, lot 062833) in DCM (2.8 L). The crude residue was dissolved in DCM (200 mL) and Et3N (20 mL) and loaded directly into the column. Triethylamine was used at this stage to avoid decomposition products and disulfates (which form olefins, the latter being very difficult to remove from the product). Initial elution was performed with DCM (1% Et3N) (2 L), followed by 1% MeOH in DCM (1% Et3N) (1 L), 2% MeOH in DCM (1% Et3N) (3 L), and 5% MeOH in DCM (1% Et3N) (1 L). The product was initially eluted in a solution of 2% MeOH in CH2Cl2 (1% Et3N). The collected fraction was concentrated by rotary evaporation below 36 °C (if the temperature was above 45 °C, decomposition of the product was observed in the presence of MeOH). The selected fraction was examined by TLC and NMR. HPLC (Zorbax SB-18, 4.6 x 150 mm, 5 μm, 202 nm, flow rate 0.8 mL / min): Solvent A: MeOH / 5% ACN / 7.4 mM NH4OAc; Solvent B: H2O / 5% ACN / 7.4 mM NH4OAc. Gradient 75% A and 25% B to 100% A. Product: 98.6% purity; 1.4% (starting material 34). HPLC: Durashell C18 (Agela Technologies, 4.6 x 50 mm, 3 μm, Solvent A: MeOH / 5% ACN / 7.4mM NH4OAc; Solvent B: H2O / 5% ACN / 7.4mM NH4OAc. Product: 98.6% purity; 1.4% (starting material 34).

[0655] Large-scale synthesis

[0656] Synthesis of 3β-25-hydroxycholest-5-ene sulfate (1, as sodium salt)

[0657]

[0658] Summary of kilogram-scale preparation

[0659] 3β,25-dihydroxycholest-5-ene (34) (2.6 kg) and pyridine (39.2 kg) were combined and heated to 40 °C with stirring in two 50 L reactors. Sulfur trioxide-trimethylamine (1.1 kg) was added to the mixture and stirred at 40 °C for 6–12 hours until the reaction was complete. The mixture was concentrated to the minimum stirring volume under vacuum distillation and then diluted with dichloromethane and triethylamine.

[0660] The crude reaction mixture in dichloromethane was loaded into a 2.33 ft silica gel-filled container. 3 The fraction was eluted on a stainless steel column (C-105) with dichloromethane (containing 1% methanol and 1% triethylamine). The fraction containing undesirable products was collected in a waste cylinder. The fraction containing the desired products was collected and concentrated in a reactor.

[0661] Acetonitrile, water, and sodium hydroxide were added to a reactor containing the desired product, and the mixture was stirred until the reaction was considered complete. The resulting slurry was cooled to 10–15 °C and filtered to separate compound 1. The cake of separated compound 1 was washed with acetonitrile and then dried under vacuum at 40 °C until constant weight was achieved.

[0662] The solid was filtered, washed with acetonitrile (1 L), and dried under vacuum (P2O5) overnight. Yield: 51.5 g, 93% (HPLC: 98.6% purity).

[0663] discuss

[0664] After 6 hours, HPLC analysis of the reaction mixture showed 44.1% of the remaining starting material. The reaction was considered complete and distilled under vacuum to the minimum stirring volume (step 5.3). Dichloromethane and triethylamine were added to the resulting viscous residue, and the solution was transferred to a clean 5-gallon glass bottle. After incubating the solution overnight, a viscous solid precipitated in the bottle. The solid was filtered off using a benchtop filter. Approximately 1 / 3 of the clear filtrate was loaded onto the top of a C-105 column. The silica gel in the C-105 column was pre-washed with ethyl acetate and methanol and then equilibrated with 1% triethylamine in dichloromethane as eluent.

[0665] Once the crude solution is loaded to the top of the column, eluent is added to maintain a pressure of approximately 10 psi. The eluent is sampled every 10–15 minutes as it exits the column. Pyridine and 3β,25-dihydroxycholest-5-ene were present in the first two samples, but in addition to pyridine and 3β,25-dihydroxycholest-5-ene, the 3β-triethylamine salt and 25-sulfate positional isomers were also detected in the third sample. Due to the minimal separation that occurred, all remaining material was eluted from the column using polar eluents (1% MeOH, 1% NEt3, and 98% DCM). The filtrate was concentrated and combined with the remaining two-thirds of the crude solution from the flask. After distillation, the crude solution was transferred to a clean flask. The eluent exiting the column was analyzed, and the eluent contained 1.7% methanol (…). 1 H NMR area %. The column was equilibrated with eluent (1% triethylamine in dichloromethane) and analyzed with methanol (0.25% methanol). 1 H NMR area (%). During this time, solids begin to form in the bottle. Filter the slurry and collect the filtrate in a clean bottle.

[0666] Approximately one-third of the crude solution was loaded onto a second C-105 column. Eluent was added to the column to maintain a pressure of <5 psi. Analysis of the eluent exiting the column by thin-layer chromatography (TLC) indicated that separation was occurring. Once 3β,25-dihydroxycholest-5-ene was no longer detected by TLC, the separation was completed. 1 ¹H NMR analysis of the eluent ensured separation of the 3β-triethylamine salt from the 25-sulfate positional isomer. The sample was removed from the cylinder containing the eluent; the purity of the 3β-triethylamine salt was 85%, with 15% of the 25-sulfate positional isomer present. 1 ¹H NMR). HPLC weight percentage determination showed that 127 g of the 3β-triethylamine salt / 25-sulfate positional isomer (85% 3β-triethylamine salt) was collected in a cylinder. The purified material was set aside, and the remaining two-thirds of the crude solution was purified by chromatography. The silica gel in the C-105 column was washed with methanol and then equilibrated with 1% triethylamine in dichloromethane (after regeneration, 0.2% methanol in the eluent, via…) 1 H NMR area %.

[0667] pass 1The solid precipitated from the flask was analyzed by ¹H NMR and identified as a quaternary ammonium salt produced from the reaction of dichloromethane with triethylamine (from SO₃NMe₃ reagent) and dichloromethane with triethylamine. The dichloromethane-triethylamine complex was separated by filtration, while the complex was formed in chromatography. Salt formation occurs under ambient conditions and is rapid in some cases under pressure. Equilibrium was established in which the triethylammonium portion of the 3β-triethylamine salt could exchange with the quaternary ammonium salt to produce the quaternary ammonium complex and triethylamine hydrochloride. The equilibrium favored the formation of the quaternary ammonium complex due to the presence of more dichloromethane-triethylamine complex. The triethylamine hydrochloride was separated and characterized.

[0668]

[0669] Of the remaining two-thirds of the crude mixture in the flask, one-third was subjected to chromatography on a fourth column. The pressure was maintained at 0–1 psi throughout the purification process. 3β-triethylamine salt was successfully isolated from the 25-sulfate positional isomer: the purity of 3β-triethylamine salt in the cylinder was 99.79% by HPLC. Approximately 0.050 kg of 34.1 (HPLC wt%) was separated from the column. The silica gel was washed with methanol and regenerated with 1% triethylamine in dichloromethane. The amount of methanol present after regeneration was 0.44% (…). 1 H NMR area %). No separation occurred on this column. No further purification was performed on the material from the fourth column. Cation exchange was performed on the eluent from the third column (approximately 50 g of 3β-triethylamine salt), starting with solvent exchange to acetonitrile. Acetonitrile, water, and 30% sodium hydroxide were added, the mixture was stirred, and then left overnight. After the post-stirring phase, solids were present in the reactor. The mixture was cooled and filtered using a new 8.5” benchtop filter. The filter cake was washed with fresh acetonitrile and dried. 1 The sample was analyzed by ¹H NMR, and a peak consistent with that of the quaternary ammonium salt was observed in the spectrum. The presence of the 25-sulfate positional isomer was confirmed by HPLC.

[0670] The eluent from the second column was concentrated under vacuum and dried to constant weight. 1HNMR analysis of the yellow powder (540 g) showed a ratio of dichloromethane-triethylamine quaternary ammonium salt to monosulfate compound of 3:1. All crude material (540 g) was loaded into a 3 L jacketed reactor. Acetonitrile (1400 g) was added, and the slurry was heated to 50 ± 5 °C and held for 30 min. The slurry was cooled to 26 °C and then filtered. The wet cake was analyzed, and the ratio of the 3β-triethylamine salt / 25-sulfate isomer to the quaternary ammonium salt was approximately 1:1. The purified solid and fresh acetonitrile (1400 g) were returned to the reactor. After 45 min, water (200 g) was added, stirred for 15 min, and then filtered. The granular powder was dried overnight in a vacuum drying oven at 40 °C. The filtrate was concentrated to dryness, and the residue was combined with the dried material and loaded into a 3 L reactor. Acetonitrile (1500 g), 1 N sodium hydroxide (600 g), and 30% sodium hydroxide (40 g) were added sequentially to the reactor. The slurry was stirred for 48 hours and then filtered at ambient temperature. The filter cake was dried to constant weight (173 g) and analyzed by HPLC.

[0671] Purification was performed to separate the 25-sulfate positional isomer from compound 1.

[0672] Several solvents were explored to remove sodium 25-sulfate from compound 1. Dissolving impure compound 1 in a polar solvent followed by the addition of an antisolvent (entries 1 and 2 in Table 13) yielded no solid recovery. Dissolving the material in methanol followed by the addition of acetonitrile (entry 3 in Table 13) resulted in minimal solids. Using 2-propanol (entry 4 in Table 13) and a mixture of methanol and water resulted in a form change, causing the material to become a viscous paste that could not be transferred or filtered. Grinding impure compound 1 with ethanol at 40–50 °C was sufficient to remove most of the 25-sulfate positional isomer (entry 6 in Table 13). A mixture of compound 1 (1 g) and ethanol (10 mL) was heated to reflux, cooled, and filtered. The purified material (55% recovery) was 99.6% pure, with the 25-sulfate and 3β,25-dihydroxycholest-5-ene products reduced to 0.1% and 0.3%, respectively.

[0673] Table 13 - Grinding / Recrystallization of Compound 1

[0674]

[0675] Purification of 3β-triethylamine salt

[0676] The 3β-triethylamine salt was purified to eliminate dichloromethane due to its reactivity with trimethylamine and triethylamine. Purification was achieved using an isocratic solvent system comprising 90% ethyl acetate, 9% methanol, and 1% triethylamine.

[0677] SO 3 NMe3 equivalent optimization

[0678] The amount of SO3NMe3 complex required to completely consume 3β,25-dihydroxycholest-5-ene or to reach the point where disulfide and unreacted starting material byproducts are minimized was determined. A solution of 3β,25-dihydroxycholest-5-ene (0.5 g, 1.0 S) containing triethylamine (0.5 S) in pyridine (18.6 S) was heated to 50 °C. Samples were removed from the reaction every 30 minutes, followed by the addition of the SO3NMe3 complex. After the final addition of the SO3NMe3 complex, the flask was stirred at 50 °C for a total of 24 hours (Table 14). Approximately 1.75 equivalents of the SO3NMe3 complex were sufficient to consume 86.6% of the starting material 3β,25-dihydroxycholest-5-ene (Sample 7, Table 14). Complete consumption of 3β,25-dihydroxycholest-5-ene was achieved after the addition of 2.5 equivalents of the SO3NMe3 complex. As the reaction proceeds, the formation of disulfate competes with the monosulfation of 3β,25-dihydroxycholest-5-ene. The 3β-triethylamine salt is completely converted to disulfate after 24 hours.

[0679] Table 14 - Equivalents of Sulfur Trioxide-Trimethylamine Complex

[0680]

[0681] 100-gram scale synthesis of 3β-25-hydroxycholest-5-ene sulfate (1, as sodium salt)

[0682] A slurry of 3β,25-dihydroxycholest-5-ene (100 g, 1.0 S) and triethylamine (0.5 S) in pyridine (15.6 S) was heated to 50 °C. A SO3NMe3 complex (1.75 equivalents, 0.6 S) was added in one step. The mixture was stirred for 5 hours, and the reaction was then analyzed by HPLC to determine the end of the reaction (Sample 1: 3β-triethylamine salt / 25-sulfate positional isomer (67.1%); 3β,25-dihydroxycholest-5-ene (12.2%); disulfate (20.8%)). The jacket was set to 70 °C, and the reactants were concentrated to less than 20% of their initial volume. The samples were removed, and their stability was analyzed by HPLC (Sample 2: 3β-triethylamine salt / 25-sulfate positional isomer (60.5%); 3β,25-dihydroxycholest-5-ene (10.0%); disulfate (29.5%)). The amount of monosulfate decreased from 67.1% to 60.5% during distillation, while the amount of bisulfate increased by approximately 9%. The amount of 3β,25-dihydroxycholest-5-ene did not decrease significantly during distillation.

[0683] Solids were present in the reactor after a 48-hour stirring phase, and the addition of methanol (0.5S) did not dissolve the solids. The crude material (300g) was purified by silica gel chromatography, eluting with 90% ethyl acetate, 9% methanol, and 1% triethylamine. Silica gel (2.4kg) was slurried in the eluent and packed to form a 5.25” x 28” column. The crude mixture was transferred to the column and purified over 3 days. The eluent was collected in 1L fractions. Fractions 1-7 were free of substances detectable by TLC; fractions 8-11 contained pyridine and 3β,25-dihydroxycholest-5-ene; fractions 12-20 were free of substances detectable by TLC; fractions 21-22 contained unidentified compounds, and fractions 23-59 contained the 3β-triethylamine salt / 25-sulfate positional isomer.

[0684] After column determination by weight percentage analysis, approximately 82 g of the 3β-triethylamine / 25-sulfate positional isomer was isolated (56.5% yield). Following chromatography, the eluent containing the mixture of 3β-triethylamine / 25-sulfate positional isomers was concentrated into a slurry and transferred to a 2 L reactor. The solvent was changed to acetonitrile, the slurry was cooled to 10 °C, and 1 N sodium hydroxide (1.8 s, 1 equivalent, based on 82 g of 3β-triethylamine / 25-sulfate positional isomers) was added over 10 min. The slurry was stirred for 1 h and then filtered. Filtration was very rapid, requiring <5 min. The solid was dried at 40 °C under vacuum to constant weight (70 g, 99% yield for cation exchange). HPLC analysis of the sample (Sample 1, Table 15) showed that the 25-sulfate positional isomer was present at 5.1%. A white powder (70 g) was transferred to a 2 L reactor and pulped at 50 °C with ethanol (700 g) for 1 hour. A change in form was observed by thickening of the pulp mixture after stirring for 30 minutes. The pulp was cooled to 10 °C, stirred for 1 hour, and then filtered at 10 °C. The reactor was rinsed with ethanol (170 g), cooled to 10 °C, and then transferred to a filter as a cake wash. The solid was dried to constant weight (64.6 g, 92.3% recovery) and analyzed by HPLC (Sample 2, Table 15). After milling, the purity of compound 1 increased to 97.4%, but the 25-sulfate isomer was 1.6%. Impure compound 1 (64.6 g, 1.0 S) was pulped in ethanol (581 g, 9 S) at 55 °C for 1.5 hours. The pulp was cooled to 10 °C and then filtered. The reactor and cake were washed with ethanol (84 g) at 10 °C, and the resulting solid was dried under vacuum at 40 °C to constant weight (60.4 g, ethanol present at 5.9%, 87.9% recovery).

[0685] The sample of compound 1 after ethanol grinding was analyzed by HPLC (sample 3, Table 15). The 25-sulfate isomer was purified, but the amount of unknown 1 increased to 0.9%. The purified material (56.8 g) was pulped in acetonitrile (5 s) and water (0.9 s) for 30 min at 30 °C in a 1 L reactor. During this time, the pulp formed stiff peaks, but the paste was easily transferred to a filter using an FMI pump. The reactor and cake were rinsed with fresh acetonitrile (30 g), and the material was dried to constant weight (54.5 g, 90.2% recovery). 1 ¹H NMR analysis showed the absence of ethanol, but the presence of 1.2 wt% water. The purity of the final material was improved to >99% (Sample 4, Table 15). Unknown impurities were present at RRTs of 1.68 and 1.85 at 0.6% and 0.2%, respectively. Taking into account the residual water, the final isolated yield of compound 1 was 43.2% in a 100 g demonstration run.

[0686] Table 15 - Purification of Crude Compound 1

[0687]

[0688] Azeotropic removal of water from 3β,25-dihydroxycholest-5-ene

[0689] A slurry of 3β,25-dihydroxycholest-5-ene (5 g, 1.0 S) and pyridine (15.6 S, 0.016% water, entry 1, Table 16) was heated to 50 °C. A sample of the reaction was removed for water content analysis (0.29%, entry 2, Table 16). The reaction volume was reduced by 50% and sampled for water content analysis (0.042%, entry 3, Table 16). The amount of pyridine collected in the distillate (39 g) was replaced with fresh pyridine in the reactor and sampled again for water analysis (0.027%, entry 4, Table 16). Once the internal temperature reached 50 °C, triethylamine (0.5 S) and SO3NMe3 (0.6 S) were added to the reactor. The thin white slurry became a clear solution within 15 minutes, and the reactants were stirred at 50 °C. Samples were removed at 2 and 3 hours for IPC analysis (entries 1 and 2, Table 17). After 2 hours, only 7.1% of 3β,25-dihydroxycholest-5-ene remained. Azeotropic removal of water before adding SO3NMe3 would increase the consumption of starting materials.

[0690] Table 16 - Moisture Content Analysis

[0691] sample Notes %water 1 pyridine solvent 0.016 2 The reaction solution prior to pyridine distillation 0.285 3 The reaction solution after pyridine distillation 0.042 4 The reaction solution after adding pyridine 0.027

[0692] Table 17 - Reaction termination characteristics determined by HPLC

[0693]

[0694] Ethanol grinding of crude compound 1

[0695]

[0696] Crude compound 1 was suspended in ethanol and heated to 55°C with stirring for 1 hour. The slurry mixture was cooled, filtered, and washed with ethanol. The resulting cake was dried overnight at 50°C. The cake was returned to the reactor and suspended in acetonitrile and water. The mixture was heated to 30°C and stirred for 1 hour. The mixture was then cooled to 15°C, filtered, and washed with acetonitrile and water (90:10). The resulting cake was dried at 50°C for no more than 24 hours until constant weight was achieved. The impurity content in the purified compound 1 was determined by HPLC. (RRT 0.67<0.05%; RRT 0.77<0.05%; RRT 0.79<0.05%; RRT 0.95<0.05%; RRT1.13<0.05%; RRT 1.22<0.05%; RRT 1.31<0.05%; RRT 1.95=0.09%; RRT 2.09<0.05%; RRT 2.67<0.05%; RRT 2.75=0.05%; RRT 3.04<0.05%; RRT 3.23=0.09%; RRT 3.64=0.3%; RRT 5.00<0.05%; total impurities=1.1%.

[0697] The product is derived from the sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol to produce 25-hydroxy-(3β)-cholest-5-en- Identification of 3-sulfate byproducts

[0698] The composition of 25-hydroxy-(3β)-cholest-5-en-3-ol was sulfated in toluene at 23°C with a sulfur trioxide-pyridine complex for 1 hour to produce 25-hydroxy-(3β)-cholest-5-en-3-sulfate. The compounds formed in the reaction mixture during the preparation of the 25-hydroxy-(3β)-cholest-5-en-3-sulfate product were analyzed by high-performance liquid chromatography (HPLC). Tables 13 and 14 provide the HPLC chromatographic conditions. Table 15 lists the retention times of the compounds identified as forming in the reaction mixture when 25-hydroxy-(3β)-cholest-5-en-3-ol was sulfated with the sulfur trioxide-pyridine complex.

[0699] Table 17 - Chromatographic Conditions

[0700]

[0701]

[0702] Table 18 - Chromatographic Conditions - Gradient

[0703] Time (minutes) ％A ％B 0.0 62 38 35 0 100 45 0 100 45.1 62 38 50.0 62 38

[0704] Table 19 - Retention Time

[0705] compound Retention time (min) Pyridine 3.2 25-sulfated cholesterol 6.6 25-Hydroxy-(3β)-Cholester-5-ene-3-sulfate 7.7 Unknown byproduct #1 18.3 25-Hydroxy-(3β)-Cholester-5-en-3-ol 26.5 Unknown byproduct #2 37.7

[0706] Determine the purity of the sulfur trioxide pyridine sulfation reagent

[0707] Proton nuclear magnetic resonance spectroscopy was used to analyze samples of sulfur trioxide pyridine in deuterated solvents. 1 H-NMR). Sulfur pyridine trioxide is a colorless solid that can degrade in the presence of moisture, which can affect the overall yield and reproducibility of sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol. Sulfur pyridine trioxide samples from three batches (AC) were dissolved in deuterated acetone ((CD3)2CO) and proton NMR spectra were recorded using a 500 MHz Bruker spectrometer. Figure 57 ). Figures 58A-58C An enhancement was observed in the NMR spectrum between 8.1 and 9.3 ppm. This was compared with... Figure 58B and 58C Compared to the NMR spectra of batches B and C, Figure 58A The NMR spectrum of batch A showed a smaller peak group at 9.25 ppm. Based on the integrated peak at 9.25 ppm in each spectrum, the impurity level of the sulfation reagent in batch A was calculated to be 21%. Figure 58A The impurity level of the sulfation reagent in batch B was calculated to be 33%. Figure 58B ), and the impurity level of the sulfation reagent in batch C was calculated to be 36% ( Figure 58C ).

[0708] Process parameters for sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol

[0709] Sulfation reactions were investigated to minimize and control the formation of the disulfation product 5-cholestene-3β-25-diol-disulfate.

[0710] The reaction mixture was sulfated with 25-hydroxy-(3β)-cholest-5-en-3-ol particles.

[0711] During the sulfation reaction, the organic cationic salt 25-hydroxy-(3β)-cholest-5-ene-3-sulfate was observed to precipitate as a gel-like solid. Due to its particle size, some of this colloidal material could dissolve in the reaction mixture. To minimize this dissolution effect, it was investigated to add seed crystals of the organic cationic salt 25-hydroxy-(3β)-cholest-5-ene-3-sulfate to the reactants to alter the crystal shape of the product. With the addition of a sulfur trioxide-pyridine complex, the gel-like solid of the organic cationic salt 25-hydroxy-(3β)-cholest-5-ene-3-sulfate generated during the sulfation reaction transformed into an amorphous slurry with a larger particle size. This allowed for control over the solubility of the generated organic cationic salt 25-hydroxy-(3β)-cholest-5-ene-3-sulfate in the reaction mixture. This also led to the minimization of the formation of the disulfation product 5-cholestene-3β-25-diol-disulfate in the reaction mixture.

[0712] Will 25-Hydroxy-(3β)-Cholester-5-en-3-ol Dissolve in 2-methyltetrahydrofuran (30V); and heat to approximately 35-40°C. Cool the solution to approximately 20±5°C and add seed crystals of the organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate. Add the sulfation reagent, sulfur trioxide-pyridine complex, in four portions, spaced 2 hours apart. Add water (2 equivalents) to the slurry and maintain for 1 hour. During this time, reduce stirring to the minimum vortex depth. Add pyridine (2 equivalents) to the 2-methyltetrahydrofuran and maintain the slurry for 12 hours or longer. Collect the crude 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt product by filtration and wash with 2-methyltetrahydrofuran-pyridine (5%). Estimate the presence of the disulfated product 5-cholest-3β-25-diol-disulfate in the crude product to be approximately 2-5%.

[0713] Quenching of unreacted sulfur trioxide-pyridine sulfation reagent

[0714] Excess unreacted sulfur trioxide pyridine sulfation reagent was evaluated using 2 equivalents of water and pyridine to maintain alkaline conditions and avoid hydrolysis of the 25-hydroxy-(3β)-cholesterol-5-ene-3-sulfate organic cationic salt product. In items 1-3 of Table 20, water and pyridine were added simultaneously and held for 1 hour; the product was then separated by vacuum filtration. In item 4 of Table 20, the holding time was extended to simulate time extension. To control the competing reaction between reagent hydrolysis and disulfation, reagent hydrolysis was evaluated by adding water and holding for one hour. This scheme maximized excess hydrolysis. Pyridine was then added to minimize product hydrolysis (item 5, Table 20). As summarized in Table 20, adding water for 1 hour, followed by mixing with pyridine and holding overnight, yielded the highest yield of the 25-hydroxy-(3β)-cholesterol-5-ene-3-sulfate organic cationic salt product and the lowest amount of disulfation product and sterol impurities.

[0715] During the quenching of excess unreacted sulfating reagent, the stirring speed was determined to play a role in the competition between the formation of the disulfation product 5-cholestene-3β-25-diol-disulfate and reagent quenching. At high stirring speeds, the unquenched sulfur trioxide-pyridine complex agglomerates rupture, allowing further reaction with the 25-hydroxy-(3β)-cholestene-3-ene-sulfate organic cationic salt product. At slow stirring speeds, the agglomerated complexes remained at the bottom of the reactor, minimizing this side reaction. The formation of the disulfation product 5-cholestene-3β-25-diol-disulfate was observed in the range of 2–5% under these reaction conditions. The isolated crude 25-hydroxy-(3β)-cholestene-3-ene-sulfate organic cationic salt product was sufficiently stable for further purification.

[0716] Table 20 - Quenching of Unreacted Sulfur Trioxide-Pyridine Sulfation Reagent

[0717]

[0718] *IPC - Process Control

[0719] Liquid chromatography and recrystallization of the organic cationic salt product of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate

[0720] The organic cation salt product of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate was purified using a plug column with a silica gel (≥ stoichiometric) stationary phase and a mobile phase of dichloromethane-methanol (85:15) and pyridine (1%). A column was prepared using silica gel (5 stoichiometric) / DCM-pyridine (1%) in a 1:2 diameter-silica gel ratio. Careful column preparation was performed to avoid disturbing the silica gel top layer. The crude organic cation salt product of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate was dissolved in dichloromethane-methanol (1:1)-pyridine (1%) (2.4V), the solution was packed into the column, and the column was washed with dichloromethane-methanol (15%)-pyridine (1%) (2V). The column was eluted with dichloromethane-methanol (15%)-pyridine (1%) (approximately 75V). Take approximately 10V of sample and monitor it by thin-layer chromatography (mobile phase dichloromethane-methanol 7:3, one drop of pyridine and CAM dye). Combine fractions containing organic cationic salts of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate and exclude fractions containing disulfation products.

[0721] The 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt product was separated and purified from the collected fractions using two different processes:

[0722] Separation and recrystallization process (IP)-A. The product-containing fraction from the stopper column was concentrated using a constant-volume technique. A solution of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt product was added to an initial constant-volume (28V) mixture of 2-methyltetrahydrofuran-heptane (1:2) – seeded with particulates of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt product during simultaneous distillation and addition. The pressure was maintained between 20 and 25 inHg. Under these conditions, the product precipitated immediately and remained as a slurry during distillation. The slurry temperature was adjusted to 20–25 °C and maintained for at least 1 hour. The product was collected by filtration and washed with 2-methyltetrahydrofuran-heptane (1:2), followed by washing with heptane. The collected material was dried under vacuum at 30–35 °C for 24 hours.

[0723] Separation Process (IP)-B. The fraction containing the product from the stopper column is concentrated to approximately 7°C under vacuum. If the solution remains or becomes cloudy or a solid is observed, dichloromethane is added until a clear solution is obtained. This concentrated solution of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt product is added dropwise to a mixture of 2-methyltetrahydrofuran-heptane (1:3) containing the seeds of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt for approximately 1 to 1.5 hours. The product is washed in a vessel with dichloromethane-methanol (1:1) (0.5°C) at 20–25°C for 1 hour. After aging the slurry, the product is collected by filtration and washed with 2-methyltetrahydrofuran-heptane (1:3), followed by washing with heptane. The solid is dried under vacuum at 30–35°C for 24 hours.

[0724] The purity of the 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salt products obtained through each separation and recrystallization process is summarized in Table 21.

[0725] Table 21 - Purity of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cation salt products after separation and recrystallization

[0726]

Claims

1. A method for producing a metal salt of 5-cholestene-3β,25-diol-3-sulfate, the method comprising: 25-hydroxy-(3β)-cholest-5-en-3-ol is contacted with at least one sulfating agent in at least one solvent to produce a precipitate of an organic cationic salt of 25-hydroxy-(3β)-cholest-5-en-3-sulfate, wherein sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol in at least one solvent causes the 25-hydroxy-(3β)-cholest-5-en-3-sulfate product to precipitate after formation; and The organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate is contacted with at least one metal salt to produce the metal salt of 5-cholest-3β,25-diol-3-sulfate.

2. The method according to claim 1, wherein the at least one sulfating agent is selected from sulfur trioxide complexes, sulfuric acid compounds, sulfonic acid compounds, and sulfonate compounds.

3. The method according to claim 1, wherein the at least one sulfating agent is a sulfur trioxide complex.

4. The method according to claim 2, wherein the at least one sulfating agent is a sulfur trioxide-pyridine complex.

5. The method according to any one of claims 1-4, wherein the at least one solvent is selected from chloroform, dichloromethane, acetone, acetonitrile, toluene, tetrahydrofuran, and methyltetrahydrofuran.

6. The method according to any one of claims 1-4, wherein the at least one metal salt is at least one sodium salt.

7. The method according to claim 6, wherein the at least one sodium salt is at least one selected from sodium acetate, sodium iodide, sodium chloride, and sodium methoxide.

8. The method according to claim 5, wherein the at least one metal salt is at least one sodium salt.

9. The method according to claim 8, wherein the at least one sodium salt is at least one selected from sodium acetate, sodium iodide, sodium chloride, and sodium methoxide.

10. The method according to any one of claims 1-4 and 7-9, wherein the at least one metal salt is sodium acetate.

11. The method according to claim 5, wherein the at least one metal salt is sodium acetate.

12. The method according to claim 6, wherein the at least one metal salt is sodium acetate.

13. The method according to any one of claims 1-4 and 7-9, wherein the at least one metal salt is sodium iodide.

14. The method according to claim 5, wherein the at least one metal salt is sodium iodide.

15. The method according to claim 6, wherein the at least one metal salt is sodium iodide.

16. The method according to any one of claims 1-4, 7-9, 11-12 and 14-15, wherein at least one sulfating agent is contacted with acetic anhydride prior to contact with 25-hydroxy-(3β)-cholest-5-en-3-ol.

17. The method of claim 5, wherein at least one sulfating agent is contacted with acetic anhydride prior to contact with 25-hydroxy-(3β)-cholest-5-en-3-ol.

18. The method of claim 6, wherein at least one sulfating agent is contacted with acetic anhydride prior to contact with 25-hydroxy-(3β)-cholest-5-en-3-ol.

19. The method of claim 10, wherein at least one sulfating agent is contacted with acetic anhydride prior to contact with 25-hydroxy-(3β)-cholest-5-en-3-ol.

20. The method of claim 13, wherein at least one sulfating agent is contacted with acetic anhydride prior to contact with 25-hydroxy-(3β)-cholest-5-en-3-ol.

21. A method for producing sodium 5-cholestene-3β,25-diol 3-sulfate, said method comprising: 25-hydroxy-(3β)-cholest-5-en-3-ol is contacted with a sulfur trioxide-pyridine complex in at least one solvent to produce a precipitate of pyridinium salt of 25-hydroxy-(3β)-cholest-5-en-3-sulfate, wherein 25-hydroxy-(3β)-cholest-5-en-3-ol is sulfated in at least one solvent such that pyridinium salt of 25-hydroxy-(3β)-cholest-5-en-3-sulfate precipitates after formation; and 25-hydroxy-(3β)-cholest-5-ene-3-sulfate pyridinium salt was contacted with sodium salt to produce 5-cholest-3β,25-diol 3-sulfate sodium salt.

22. The method according to claim 21, wherein the at least one metal salt is sodium acetate.

23. The method according to claim 21, wherein the at least one metal salt is sodium iodide.

24. A method for producing the metal salt of 5-cholestene-3β,25-diol 3-sulfate, said method comprising: (3β)-cholest-5-en-3-ol is contacted with at least one sulfating agent to produce a first (3β)-cholest-5-en-3-sulfate organic cationic salt; The first (3β)-cholest-5-ene-3-sulfate organic cationic salt is contacted with an organic base to produce the second (3β)-cholest-5-ene-3-sulfate organic cationic salt; Oxidation of a second (3β)-cholest-5-ene-3-sulfate organic cationic salt in the presence of at least one surfactant to produce 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salt; 25-hydroxy-(3β)-cholest-5-ene-3-sulfate organic cationic salts were generated from 25-hydroxy-(3β)-cholest-(5,6-epoxy)-3-sulfate organic cationic salts via deoxygenation; and The organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate is contacted with at least one metal salt to produce the metal salt of 5-cholest-3β,25-diol-3-sulfate.

25. A method comprising: Sulfated 25-hydroxy-(3β)-cholest-5-en-3-ol in at least one solvent to produce a precipitate of 25-hydroxy-(3β)-cholest-5-en-3-sulfate, wherein sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol in at least one solvent causes the 25-hydroxy-(3β)-cholest-5-en-3-sulfate product to precipitate after formation; and 25-hydroxy-(3β)-cholest-5-ene-3-sulfate is precipitated in the solvent.

26. A method for producing a metal salt of 5-cholestene-3β,25-diol-3-sulfate, the method comprising: 25-hydroxy-(3β)-cholest-5-en-3-ol is contacted with at least one sulfating agent in at least one solvent to produce a precipitate of an organic cationic salt of 25-hydroxy-(3β)-cholest-5-en-3-sulfate, wherein sulfation of 25-hydroxy-(3β)-cholest-5-en-3-ol in at least one solvent causes the 25-hydroxy-(3β)-cholest-5-en-3-sulfate product to precipitate after formation; and The organic cationic salt of 25-hydroxy-(3β)-cholest-5-ene-3-sulfate was contacted with sodium hydroxide to produce the metal salt of 5-cholest-3β,25-diol-3-sulfate.

27. The method of claim 26, wherein the at least one sulfating agent is selected from sulfur trioxide complexes, sulfuric acid compounds, sulfonic acid compounds, and sulfonate compounds.

28. The method of claim 26, wherein the at least one sulfating agent is a sulfur trioxide complex.

29. The method of claim 27, wherein the at least one sulfating agent is a sulfur trioxide-pyridine complex.

30. The method according to any one of claims 26-29, wherein the at least one solvent is selected from chloroform, dichloromethane, acetone, acetonitrile, toluene, tetrahydrofuran, and methyltetrahydrofuran.

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