Stereoselective crystallization of bile acids

Abstract

Methods for stereoselectively crystallizing 3-keto-5β-cholanic acid (“3-KCA”) and 3-ketoursodeoxycholic acid (“3-KUDCA”) of extremely high purity, salts for carrying out such methods, particularly tert-butylamine and cyclohexylamine salts of 3-KCA, tert-butylamine salts of 3-KUDCA, and crystalline forms of these salts, as well as methods for preparing commercial steroid compounds from these purified materials.

Description

[0001] Invention Field

[0002] The present invention generally relates to methods for purifying 3-keto-5β-cholanic acid (“3-KCA”) and 3-keto-ursodeoxycholic acid (“3-KUDCA”) by crystallization, salts for carrying out these methods, and methods for subsequently preparing commercial steroid compounds from these purified materials. Background of the Invention

[0004] Bile acids and their derivatives have practical value in various medical applications and research projects. They are marketed under the trade name Cholbam. ® The bile acids themselves are approved for the treatment of children and adults with bile acid synthesis disorders caused by a single enzyme deficiency, and for the treatment of peroxidase disorders (e.g., Zellweger syndrome). The effects of 7-ketolithocholic acid on endogenous bile acid synthesis, bile cholesterol saturation, and its potential role as a precursor to chenodeoxycholic acid and ursodeoxycholic acid have been investigated. See Salen et al., Gastroenterology, 1982;83:341-7. Ursodeoxycholic acid (also known as UDCA or ursodiol), marketed under the brand names URSO 250® and URSO Forte® tablets, is approved for the treatment of patients with primary biliary cirrhosis (PBC). Recently, obeticholic acid, marketed under the brand name Ocaliva®, was approved for use in combination with UDCA for the treatment of adults with inadequate response to UDCA or as monotherapy for adults with PBC who cannot tolerate UDCA.

[0005] Despite this significant medical interest in this derivative, its synthesis remains cumbersome and inefficient, and numerous methods have been proposed. Fantin et al., Steroids, November 1993; 58: 524-526, disclose the preparation of 7α-,12α-,12β-hydroxy and 7α-,12α- and 7α-,12β-dihydroxy-3-ketocholanic acids by protecting the 3-keto group to a dimethyl ketal and subsequently reducing the corresponding 7- and 12-oxofunctional groups with sodium borohydride. Galvin reported a method for preparing obeticholic acid by direct alkylation of the C-6 position of 7-ketolithocholic acid (KLCA) in WO2017 / 079062A1. He et al., Steroids, December 2018; 140:173-178, disclosed a synthetic route for the preparation of ursodeoxycholic acid (UDCA) and obeticholic acid (OCA) from inexpensive and readily available cholic acids via a multi-step reaction. Wang et al., Steroids 157 (2020) 108600, also reported a synthetic route for the preparation of ursodeoxycholic acid (UDCA) from commercially available bis(BA) via a multi-step reaction.

[0006] The complexity of synthetic pathways often necessitates the reduction of one or more double bonds in unsaturated intermediates. This is because each steroid compound possesses a unique stereochemistry across multiple chiral centers, typically containing diastereomeric impurities. This is especially true for plant-derived steroids, which often contain 4,5-double bonds and require stereospecific reduction to produce a variety of commercially relevant steroid compounds.

[0007] Currently, there is a need for more efficient methods for the preparation of cholic acid derivatives with high stereoselectivity. In particular, there is a need for more efficient methods for the preparation of 5β-cholic acid and cholanonic acids (including ursodeoxycholic acid and tauroursodeoxycholic acid), as well as starting materials and intermediates. Invention Overview

[0009] The inventors have discovered new crystalline salt forms of 3-KCA and methods for purifying 3-KCA from its 5α isomer using these new forms. Therefore, in one embodiment, the present invention provides a method for preparing 5β-steroid compounds substantially free of 5α isomer impurities, the method comprising selectively crystallizing tert-butylamine (“TBA”) or cyclohexylamine salts of 3-KCA from a solution containing 3-KCA and its 5α isomers. In another embodiment, the present invention provides a tert-butylamine (“TBA”) salt of 3-KCA. In yet another embodiment, the present invention provides a cyclohexylamine salt of 3-KCA.

[0010] The inventors have also discovered novel crystalline salt forms of 3-KUDCA, and methods for purifying 3-KUDCA from its impurities using these novel forms. Therefore, in another embodiment, the present invention provides a method for preparing 3-KUDCA substantially free of its impurities, the method comprising selectively crystallizing tert-butylamine (“TBA”) salts of 3-KUDCA from a solution containing 3-KUDCA and its impurities. In another embodiment, the present invention provides tert-butylamine (“TBA”) salts of 3-KUDCA.

[0011] Further advantages of the invention are set forth in the following description, some of which are obvious from the description or may be learned by practice of the invention. The advantages of the invention will be realized and manifested by means of the elements and combinations particularly pointed out in the appended claims. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and do not constitute a limitation on the claimed invention. Invention Details

[0013] Definition and use of terms

[0014] As used in the specification and claims, the singular forms “an,” “a,” and “the” include plural references unless the context clearly specifies otherwise. For example, the term “specification” refers to one or more specifications for the currently disclosed methods and systems. “Hydrocarbon” includes mixtures of two or more such hydrocarbons, etc. The word “or” or similar terms as used herein mean any member of a particular list, and also include any combination of members of that list.

[0015] As used in this specification and the following claims, the word “comprising” and variations thereof, such as “including” and “containing”, means “including, but not limited to” and are not intended to exclude, for example, other additives, components, integers, or steps. When an element is described as comprising one or more components, steps, or conditions, it should be understood that the element may also be described as “consisting of a component, step, or condition or multiple components, steps, or conditions” or “substantially consisting of a component, step, or condition or multiple components, steps, or conditions.”

[0016] When used herein, the term "about" will compensate for the variability permitted in the pharmaceutical industry and inherent in pharmaceutical products. In one embodiment, the term allows for any variation within 5% of the specification or standard. In another embodiment, the term allows for any variation within 10% of the specification or standard. All numerical values ​​recorded in this application should be interpreted as preceded by the term "about" to provide additional embodiments including 5% and 10% variations.

[0017] When purity is given as a percentage in this document, it will be understood as a weight percentage. Similarly, when yield is expressed as a percentage, it should be understood as the weight of the product actually observed relative to the weight of the product obtained at 100% conversion.

[0018] The term "substantially free" includes "completely free." Therefore, a composition containing one or more impurities that is substantially free of one or more impurities can also be described as having a purity greater than 95%, 98%, 99%, 99.5%, or 99.8%, optionally completely free of one or more impurities relative to them. Similarly, a "substantially pure" compound can be completely pure. Therefore, a substantially pure compound can also be described as having a purity greater than 95%, 98%, 99%, 99.5%, or 99.8%. Purity can be determined relative to one or more specific impurities or all impurities; if no specific impurity is specified, purity is determined relative to all impurities.

[0019] The term "impurity" as used in this document is consistent with that used in the U.S. Food and Drug Administration's Industry Guidance Q3A: Impurities in New Drug Substances (2nd revised edition, June 2008). Impurities can therefore be categorized as follows: organic impurities (process- and drug-related), inorganic impurities, and residual solvents. Organic impurities can arise during the manufacturing process and / or storage of new drug substances. These impurities can be identified or unidentified, volatile or non-volatile, and include raw materials, byproducts, intermediates, degradation products, reagents, ligands, and catalysts. Inorganic impurities may originate from the manufacturing process. These impurities are generally known and identified and include reagents, ligands and catalysts, heavy metals or other residual metals, inorganic salts, and other substances (e.g., filter aids, activated carbon).

[0020] When a method is defined by its constituent steps, it should be understood that a method includes steps that are performed consecutively, simultaneously, or in any order, unless otherwise specified.

[0021] 3-KCA or 3-keto-5β-cholanoic acid has the following chemical structures:

[0022]

[0023] The corresponding 5α stereoisomer of 3-KCA has the following chemical structures:

[0024]

[0025] In various embodiments, 3-KCA or its TBA salt comprises less than 5%, 2%, 1%, 0.5%, or 0.2% of its corresponding 5α stereoisomer. In other embodiments, 3-KCA or its TBA salt is separated from a larger composition or reaction mixture, such that the composition containing 3-KCA or its TBA salt comprises more than 90%, 95%, 98%, 99%, 99.5%, or 99.8% 3-KCA.

[0026] 3-KUDCA or 3-ketoursodeoxycholic acid has the following chemical structure:

[0027]

[0028] In various embodiments, 3-KUDCA or its TBA salt contains less than 5%, 2%, 1%, 0.5%, or 0.2% impurities. In other embodiments, 3-KUDCA or its TBA salt is separated from a larger composition or reaction mixture such that the composition containing 3-KUDCA or its TBA salt contains more than 90%, 95%, 98%, 99%, 99.5%, or 99.8% 3-KUDCA relative to 3-KUDCA and its impurities.

[0029] Ursodeoxycholic acid, 3α,7β-dihydroxy-5β-cholanoic acid, or simply ursodiol or UDCA, has the following chemical structure:

[0030]

[0031] In various embodiments, UDCA comprises less than 5%, 2%, 1%, 0.5%, or 0.2% of its corresponding 5α stereoisomer. In other embodiments, UDCA is isolated from a larger composition or reaction mixture such that the composition containing UDCA comprises greater than 95%, 98%, 99%, or 99.5% UDCA, optionally with a 3α,7β-isomer purity of greater than 95%, 98%, 99%, 99.5%, or 99.8%.

[0032] Taurursodeoxycholic acid, or TUDCA, has the following chemical structure:

[0033]

[0034] In various embodiments, TUDCA comprises less than 5%, 2%, 1%, 0.5%, or 0.2% of its corresponding 5α stereoisomer. In other embodiments, TUDCA is isolated from a larger composition or reaction mixture such that the composition containing TUDCA comprises greater than 95%, 98%, 99%, 99.5%, or 99.8% TUDCA, optionally with a 3α,7β-isomer purity of greater than 95%, 98%, 99%, 99.5%, or 99.8%.

[0035] In any embodiment of the invention, the steroidal compound discussed, whether 3-KCA, 3-KUDCA, UDCA or TUDCA, is preferably plant-derived.

[0036] The term "plant-derived" refers to molecules containing δ-carbon, which are either plant-derived molecules or fossil / animal-plant hybrid derivatives containing predominantly plant-derived carbon. 13 Therefore, plant-derived molecules can be characterized by having more than 50%, 75%, 90%, 95%, 98%, or 99% plant-derived carbon, with the remaining carbon (if any) derived from fossil / animal sources.

[0037] "Plant source" means any source and can be defined as plants, such as trees, shrubs, herbs, grasses, ferns, mosses, flowers, vegetables, and weeds, as well as plant-derived compounds, such as phytosterols and phytosterol derivatives. Plants can be C3 plants, C4 plants, or a combination of both.

[0038] "C3 plants" refers to plants that do not have photosynthetic adaptations that reduce photorespiration. These include rice, wheat, soybeans, most fruits, most vegetables, and all trees.

[0039] "C4 plants" refer to plants in which the light-dependent response and the Calvin cycle are physically separated, with the light-dependent response occurring in mesophyll cells and the Calvin cycle occurring in bundle sheath cells. These include plants such as crabgrass, sugarcane, sorghum, and maize.

[0040] “δ 13 The C value is 13 Isotope measurements of C using the delta notation. 13 The C-value is expressed as a deviation in parts per thousand (‰), for example, a percentage of one thousand relative to the internationally recognized PDB standard (originally referring to belemnites carbonate from the Pee Dee Belemnite Formation in South Carolina, but now more commonly known as Vienna Pee Dee Belemnite (VPDB)). δ 13 The C value is determined using the following formula:

[0041] δ 13 C = ((δ 13 C / δ 12 C) 样品 -(δ 13 C / δ 12 C) PDB ) / ((δ 13 C / δ 12 C) PDB ) 1000

[0042] In any embodiment of the invention, 3-KCA, UDCA, or TUDCA preferably includes δ-corresponding to plant-derived molecules. 13 The C value preferably includes δ values ​​that are less than -15‰, -17.5‰, -20‰, -22.5‰, or -25‰ relative to VPDB. 13C, or in the case of TUDCA, more preferably, includes δ less than -20‰, -22.5‰, or -25‰ relative to VPDB. 13 C.

[0043] discuss

[0044] In a first principal embodiment, the present invention provides a method for preparing a 5β-steroid compound substantially free of 5α-isomer impurities, the method comprising selectively crystallizing tert-butylamine or cyclohexylamine salts of 3-KCA from a solution containing 3-KCA and its 5α-isomer. The method can be carried out in solution using an 80:20 mixture of 3-KCA and its 5α-isomer or any ratio of 3-KCA and its 5α-isomer. Thus, in various embodiments, the solution comprises ≥0.5, 1, 3, 5, 10, or 20% of the 5α-isomer, based on the total weight of the isomer pairs. Furthermore, it should be understood that the method can be performed sequentially two or more times to further purify 3-KCA.

[0045] In other principal embodiments, the present invention provides a method for preparing substantially pure 5β-steroidal compounds, the method comprising selectively crystallizing tert-butylamine salts of 3-KUDCA from a solution containing 3-KUDCA and one or more impurities. The method can be carried out in solution using an 80:20 mixture of 3-KUDCA and impurities or any proportion of 3-KUDCA and its impurities. Thus, in various embodiments, the solution comprises ≥ 0.5, 1, 3, 5, 10, or 20% of 3-KUDCA impurities, based on the total weight of 3-KUDCA and its impurities. Furthermore, it should be understood that the method can be performed sequentially two or more times to further purify 3-KUDCA.

[0046] The method can also be defined based on its stereoselectivity and regioselectivity. Therefore, in any embodiment of the invention, the method will prepare a salt of 3-KCA containing greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8% of 5β-stereochemistry, while excluding 5α-stereochemistry.

[0047] The method can also be defined according to the purity of the final product. Therefore, in any embodiment of the invention, when taking into account 3-KCA or 3-KUDCA and its impurities, the method will prepare 3-KCA or 3-KUDCA salts with a purity greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8%.

[0048] In one embodiment, the method includes selectively crystallizing the tert-butylamine salt of 3-KCA. In various embodiments, the crystalline tert-butylamine salt isomer has a purity greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8%.

[0049] In another embodiment, the method includes selectively crystallizing the cyclohexylamine salt of 3-KCA. In various embodiments, the crystalline cyclohexylamine salt isomer has a purity greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8%.

[0050] In another embodiment, the method includes selectively crystallizing the tert-butylamine salt of 3-KUDCA. In various embodiments, the purity of the crystalline tert-butylamine salt of 3-KUDCA is greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8%, taking into account 3-KUDCA and its impurities.

[0051] Regardless of whether tert-butylamine or cyclohexylamine salts are obtained, the methods for crystallizing 3-KCA salts are similar. In one embodiment, the method includes crystallizing 3-KCA salts from a solution containing a C1-C4 alcohol such as isopropanol, and optionally MTBE (methyl tributyl ether) and / or water.

[0052] In another embodiment, when crystallizing the tert-butylamine salt of 3-KUDCA, the method includes crystallizing the 3-KUDCA salt from a solution containing at least 80 or 90% by volume MTBE, n-butyl acetate, ethyl acetate, or a combination thereof, preferably n-butyl acetate.

[0053] In the preferred method, the temperature is first increased (e.g., 35-50°C). C) 3-KCA and C1-C4 alcohol are mixed to form a solution, which is then mixed directly with the desired amine or with a solution containing the desired amine (e.g., tert-butylamine or cyclohexylamine). 3-KCA and C1-C4 alcohol (e.g., isopropanol) are typically mixed at a ratio of 0.5 g / 10 ml to 10 g / 10 ml, more preferably 1 g / 10 ml to 5 g / 10 ml, or about 2 g / 10 ml.

[0054] In one embodiment, cyclohexylamine or tert-butylamine in a heated solution of MTBE or other acceptable organic solvent (e.g., n-butyl acetate or ethyl acetate) is added to an alcoholic solution of 3-KCA. The volume ratio of the 3-KCA solution to the amine solution is typically about 0.1:1 to about 5:1, or about 0.2:1 to about 1:1, or about 0.5:1. 3-KCA and tert-butylamine or cyclohexylamine are typically combined in a molar ratio of ~1:1. When the 3-KCA solution is mixed with the amine, a solid is typically formed, although at certain concentrations, precipitation requires cooling the mixed solution to, for example, 5-20°C. C or 10-15 C. The precipitate corresponds to the desired crystalline salt.

[0055] UDCA transformation

[0056] In other embodiments, 3-KCA or 3-KUDCA is converted into the desired end product. Thus, in one embodiment, 3-KCA or its cyclohexylamine or tert-butylamine salt comprises a steroidal core, and the method further comprises the following steps sequentially, simultaneously, or in any order: (a) stereoselectively reducing the 3-keto group on the steroidal core to a 3α-hydroxyl group; and (b) stereoselectively hydroxylating the 7-position on the steroidal core to a 7β-hydroxyl group; thereby preparing UDCA or its salt, optionally with a 3α,7β-isomer purity greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8%.

[0057] In another embodiment, 3-KUDCA or its tert-butylamine salt comprises a steroidal core, and the method further comprises stereoselectively reducing the 3-ketone on the steroidal core to a 3α-hydroxyl group; thereby preparing UDCA or its salt, optionally with a purity greater than 90%, 95%, 98%, 99%, 99.5% or 99.8%.

[0058] In another embodiment, 3-KCA or its cyclohexylamine or tert-butylamine salt comprises a steroidal core and further comprises the following steps sequentially, simultaneously, or in any order: (a) stereoselectively reducing the 3-ketone on the steroidal core to a 3α-hydroxyl group in the presence of a 3α-keto reductase; and (b) stereoselectively hydroxylating the 7-position of the steroidal core to a 7β-hydroxyl group in the presence of a 7β-hydroxylase; thereby preparing UDCA or its salt, optionally with a 3α,7β-isomer purity greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8%.

[0059] In another embodiment, 3-KUDCA or its tert-butylamine salt comprises a steroidal core, and further comprises stereoselectively reducing the 3-ketone on the steroidal core to a 3α-hydroxyl group in the presence of a 3α-keto reductase; thereby preparing UDCA or its salt, optionally with a 3α,7β-isomer purity greater than 90%, 95%, 98%, 99%, 99.5% or 99.8%.

[0060] Taurine conjugate

[0061] In the synthesis of TUDCA from UDCA and taurine, UDCA is first converted into an acylating agent, which then reacts with taurine to form TUDCA. UDCA can be converted into a variety of acylating agents suitable for the reactions requiring protection, as commonly taught in Morrison & Boyd, Organic Chemistry, 6th Edition (Benjamin-Cummings Publishing Company) and John Welch, Encyclopedia of Organic Chemistry, Synthesis, Physical Sciences and Technology (3rd Edition), 2003. Commonly used acylating agents suitable for the preparation of amides (such as TUDCA) that can be converted from UDCA include acid anhydrides (including mixed alkoxycarbonyl anhydrides and phosphoric anhydrides), N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, imidazolides, phenyl esters, and acyl halides (also known as acid halides).

[0062] The following approaches can be used to respond to UDCA:

[0063]

[0064] Table 1 lists suitable reagents and exemplary references for converting UDCA into acylating agents, although these reagents are not intended to be limiting, but merely simple examples of a variety of chemical pathways well known to those skilled in the art:

[0065] Table 1

[0066]

[0067] TPP = Triphenylphosphine; NBS = N-bromosuccinimide; Im = Imidazol-1-yl; Et = Ethyl; Me = Methyl; iPr = Isopropyl; Bt = Benzotriazol-1-yl; Su = Succinimide-1-yl; Py = Pyrrolidine-1-yl

[0068] Reference 1 is Montalbetti and Falque, Tetrahedron, 2005, Vol. 61, pp. 10827-10852, and reference 2 is March's Advanced Organic Chemistry, Smith and March, 6th edition, 2007, pp. 1427-1439.

[0069] Therefore, in various sub-implementations, UDCA is converted into an acylating agent, which then reacts with taurine to form TUDCA or a salt thereof. In another embodiment, UDCA is contacted with means for converting 24-carboxylic acid into a derivative that can function as an acylating agent, causing the derivative to react with taurine to form TUDCA or a salt thereof. In these sub-implementations, the method corresponds to the structure of ethyl chloroformate, as specifically described in the examples.

[0070] Enzyme reaction conditions

[0071] In some embodiments, the method further includes (a) enzymatically stereoselectively reducing the 3-keto group on the steroid core (preferably the steroid core of 3-KUDCA) to a 3α-hydroxyl group; and / or (b) enzymatically stereoselectively hydroxylating the 7-position of the steroid core (preferably the steroid core of 3-KCA) to a 7β-hydroxyl group. These enzymatic methods are described in more detail in the examples herein and in WO / 2023 / 081658 (published May 11, 2023) (International Application PCT / US2022 / 079081) (titled "High Purity Non-Animal Derivative TUDCA") and WO / 2023 / 081657 (published May 11, 2023) (International Application PCT / US2022 / 079080) (titled "High Purity Non-Animal Derivative UDCA"). The contents of WO / 2023 / 081658 and WO / 2023 / 081657 are expressly incorporated herein by reference.

[0072] New salts and crystal forms

[0073] When any compound is mentioned herein, whether on its own, in combination with other components, or in a chemical or biological process, it should be understood that the compound can exist or be used in an isolated form. An isolated form means that the compound is preferably present in a solid form and substantially free of any other compound besides the compound described (i.e., <10%, 5%, 3%, or 1% of other compounds).

[0074] In one embodiment, the present invention provides a tert-butylamine salt of 3-KCA, preferably crystalline tert-butylamine 3-KCA. The preferred TBA salts of 3-KCA have a purity greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8%, relative to their 5α stereoisomer (i.e., taking into account the weight of 3-KCA and its 5α stereoisomer).

[0075] The preferred crystal form exhibits 3, 5, 10 or more characteristic peaks reported in the examples and Table A below in its X-ray powder diffraction (XRPD) pattern, preferably denoted as 2-θ, and preferably allowing variability of ±0.2° or 0.1°:

[0076] Table A

[0077]

[0078] In another embodiment, the tert-butylamine salt has a crystalline powder X-ray diffraction pattern comprising at least three characteristic peaks selected from the group denoted by 2θ: 10.404±0.2°, 11.920±0.2°, 14.502±0.2°, 14.925±0.2°, 16.081±0.2°, 16.221±0.2°, 18.103±0.2°, and 20.877±0.2°.

[0079] In another embodiment, the tert-butylamine salt has a crystalline powder X-ray diffraction pattern comprising characteristic peaks denoted by 2θ at 14.502±0.2°, 14.925±0.2°, and 18.103±0.2°.

[0080] In another embodiment, the present invention provides a cyclohexylamine salt of 3-KCA, preferably in its crystalline form. The preferred cyclohexylamine salt of 3-KCA has a purity greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8%, relative to its 5α stereoisomer (i.e., taking into account the weight of 3-KCA and its 5α stereoisomer).

[0081] The preferred crystal form exhibits 3, 5, 10 or more characteristic peaks reported in the examples and Table B below in its X-ray powder diffraction (XRPD) pattern, preferably denoted as 2θ, and preferably allowing variability of ±0.2° or 0.1°:

[0082] Table B

[0083]

[0084] In one embodiment, the cyclohexylamine salt has a crystalline powder X-ray diffraction pattern comprising at least three characteristic peaks selected from the group denoted by 2θ: 9.046±0.2°, 12.907±0.2°, 14.359±0.2°, 15.652±0.2°, 16.170±0.2°, 16.974±0.2°, and 17.508±0.2°.

[0085] In one embodiment, the cyclohexylamine salt has a crystalline powder X-ray diffraction pattern including characteristic peaks denoted by 2θ at 12.907±0.2°, 16.170±0.2°, and 16.974±0.2°.

[0086] In another embodiment, the present invention provides a tert-butylamine salt of 3-KUDCA, preferably crystalline tert-butylamine 3-KUDCA. When taking into account the weight of 3-KUDCA and its impurities, the preferred TBA salts of 3-KUDCA have a purity greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8%.

[0087] The preferred crystal form exhibits 3, 5, 10 or more characteristic peaks reported in the examples and Table C below in its X-ray powder diffraction (XRPD) pattern, preferably denoted as 2θ, and preferably allowing variability of ±0.2° or 0.1°:

[0088] Table C

[0089]

[0090] In another embodiment, the tert-butylamine salt has a crystalline powder X-ray diffraction pattern comprising at least three characteristic peaks selected from the group denoted by 2θ: 11.498±0.2°, 19.002±0.2°, 13.002±0.2°, 20.306±0.2°, 15.496±0.2°, 15.883±0.2°, 12.830±0.2°, and 18.022±0.2°.

[0091] In another embodiment, the tert-butylamine salt has a crystalline powder X-ray diffraction pattern including characteristic peaks denoted by 2θ at 11.498±0.2°, 19.002±0.2°, and 13.002±0.2°.

[0092] The method can also be used to prepare UDCA and TUDCA having the purity described in WO / 2023 / 081658 (published May 11, 2023) (International Application PCT / US2022 / 079081) (titled "High Purity Non-Animal Derivative TUDCA") and WO / 2023 / 081657 (published May 11, 2023) (International Application PCT / US2022 / 079080) (titled "High Purity Non-Animal Derivative UDCA"), according to the methods described in the aforementioned patent disclosures. The disclosures of WO / 2023 / 081658 and WO / 2023 / 081657 are expressly incorporated herein by reference.

[0093] In one embodiment, the present invention provides UDCA or a salt thereof prepared by the method of the present invention. In another embodiment, the present invention provides UDCA or a salt thereof prepared by the method of the present invention, comprising: greater than 95%, 98%, 99%, 99.5% or 99.8% 5β-stereochemistry, optionally with a 3α,7β-isomer purity of greater than 95%, 98%, 99%, 99.5% or 99.8%.

[0094] In one embodiment, the present invention provides TUDCA or a salt thereof prepared by the method of the present invention. In another embodiment, the present invention provides TUDCA or a salt thereof prepared by the method of the present invention, comprising: greater than 95%, 98%, 99%, 99.5% or 99.8% 5β-stereochemistry, optionally with a 3α,7β-isomer purity of greater than 95%, 98%, 99%, 99.5% or 99.8%.

[0095] Source of raw materials

[0096] The raw material sources for 3-KCA, 3-KUDCA, UDCA, and TUDCA can be steroidal compounds, such as cholesterol, stigmasterol, campesterol, and sitosterol, or mixtures thereof, preferably sitosterol. Preferably, the raw material source is a phytosterol, such as sitosterol, stigmasterol, campesterol, and brassosterol, or mixtures thereof. In one embodiment, the phytosterol is primarily derived from soybean or tall oil.

[0097] The origin of carbon atoms can even be determined by measuring δ. 13 The C value is used for further differentiation. For example, US 8,076,156 and “Stable Isotope Ratios as Biomarkers of Diet in Health Studies,” published by DM O’Brien, Annual Reviews (www.annualreviews.org), 2015. The presence of δ-values ​​is due to C 13 This was determined relative to the Pee Dee Belemnite standard, which is based on Cretaceous marine fossils, and this fossil has an unusually high C content. 13 Some biochemical reactions reject it. 13 C, this is why in biological materials 12 The reason for the increase in C concentration. In this way, pure compounds can be used as reference values ​​to distinguish different sources (e.g., plants and animals), as described in application specification 30276 of Guibert et al.'s ThermoScientific: "Detection of the source of squalene and squalane using rapid elemental analysis and δV isotope ratio mass spectrometry".

[0098] Isotope ratios are typically quantified in parts per thousand (‰), a scale known as the delta scale. Specifically, δ... 13 C= (R 样品 / R 标准品 - 1) x 1,000, where R 样品 For the sample 13 C / 12 C isotope ratio, R 标准品The value is 0.0112372, based on the standard Vienna PeeDee Belemnite (VPDB) value. Therefore, 13 One unit of C represents 13 C / 12 The carbon isotope ratio varies by about one unit to the fifth decimal place. Further discussion of this technique can be found, for example, in RN Zare et al., High-precision optical measurements of natural abundance in organic compounds. 13 C / 12 C isotope ratios. 10928–10932, PNAS, July 7, 2009, Vol. 106, No. 27.

[0099] δ 13 The C value can also vary among plants due to different photosynthetic physiology. This can be seen in C3 plants (such as wheat, rice, beans, most fruits and vegetables) exhibiting higher δ values ​​than C4 plants (such as corn, sugarcane, and sorghum). 13 The C-value was observed (“Stable isotope ratios as biomarkers of diet in health studies” by DM O’Brien, Annual Reviews (www.annualreviews.org), 2015).

[0100] In one embodiment, 3-KCA, 3-KUDCA, UDCA, or TUDCA exhibits a δ-reactivity similar to that of 3-KCA, 3-KUDCA, UDCA, or TUDCA obtained from animal sources. 13 δ with different C values 13 C-value. In another embodiment, 3-KCA, 3-KUDCA, UDCA, or TUDCA exhibits a C-value similar to that of 3-KCA, 3-KUDCA, UDCA, and TUDCA derived from mammalian sources. 13 δ with different C values 13 C value.

[0101] The carbon in 3-KCA, 3-KUDCA, UDCA, or TUDCA is preferably derived primarily from plant sources, with only a small (if any) amount of carbon derived from non-plant sources. Therefore, in various preferred embodiments, the carbon in 3-KCA, 3-KUDCA, UDCA, or TUDCA comprises more than 80% plant-derived carbon, with the remainder derived from non-plant sources. More specifically, the carbon on the steroid ring is preferably 100% derived from plant sources, while any attached portions (such as taurine) may be partially or wholly derived from non-plant sources.

[0102] Other implementation plans

[0103] Implementation Scheme 1) is a method for preparing 5β steroidal compounds that are substantially free of 5α isomer impurities, comprising selectively crystallizing tert-butylamine or cyclohexylamine salts of 3-KCA from a solution containing 3-KCA and its 5α isomer.

[0104] Implementation Scheme 2) The method of Implementation Scheme 1, wherein 3-KCA includes δ corresponding to plant-derived molecules. 13 The C value, which optionally includes δ values ​​less than -15‰, -17.5‰, -20‰, -22.5‰, or -25‰ relative to VPDB. 13 C.

[0105] Implementation Scheme 3) The method of Implementation Scheme 1, comprising selectively crystallizing tert-butylamine salt of 3-KCA, optionally with an isomer purity greater than 98%, 99%, 99.5% or 99.8%.

[0106] Implementation Scheme 4) The method of Implementation Scheme 1, comprising selectively crystallizing tert-butylamine salt of 3-KCA from a solution containing C1-C4 alcohol, said solution optionally containing MTBE or water as a co-solvent, optionally with an isomer purity greater than 98%, 99%, 99.5% or 99.8%.

[0107] Implementation Scheme 5) The method of Implementation Scheme 1, comprising selectively crystallizing tert-butylamine salt of 3-KCA from a solution containing isopropanol, said solution optionally containing MTBE or water as a co-solvent, optionally with an isomer purity greater than 98%, 99%, 99.5% or 99.8%.

[0108] Implementation Scheme 6) The method of Implementation Scheme 3, 4 or 5, wherein the tert-butylamine salt comprises a crystalline powder X-ray diffraction pattern comprising at least three characteristic peaks selected from the group denoted by 2θ: 10.404±0.2°, 11.920±0.2°, 14.502±0.2°, 14.925±0.2°, 16.081±0.2°, 16.221±0.2°, 18.103±0.2° and 20.877±0.2°.

[0109] Implementation Scheme 7) The method of Implementation Scheme 1, comprising selectively crystallizing the cyclohexylamine salt of 3-KCA, optionally with an isomer purity greater than 98%, 99%, 99.5% or 99.8%.

[0110] Implementation Scheme 8) The method of Implementation Scheme 1, comprising selectively crystallizing the cyclohexylamine salt of 3-KCA from a solution containing a C1-C4 alcohol, said solution optionally containing MTBE or water as a co-solvent, optionally with an isomer purity greater than 98%, 99%, 99.5% or 99.8%.

[0111] Implementation Scheme 9) The method of Implementation Scheme 1, comprising selectively crystallizing the cyclohexylamine salt of 3-KCA from a solution containing isopropanol, said solution optionally containing MTBE or water as a co-solvent, optionally with an isomer purity greater than 98%, 99%, 99.5% or 99.8%.

[0112] Implementation Scheme 10) The method of Implementation Scheme 7, 8 or 9, wherein the cyclohexylamine salt has a crystalline powder X-ray diffraction pattern comprising at least three characteristic peaks selected from the group denoted by 2θ: 9.046±0.2°, 12.907±0.2°, 14.359±0.2°, 15.652±0.2°, 16.170±0.2°, 16.974±0.2° and 17.508±0.2°.

[0113] Implementation Scheme 11) The method of Implementation Scheme 1, wherein 3-KCA or its cyclohexylamine or tert-butylamine salt comprises a steroid core, further comprising the following steps sequentially, simultaneously or in any order: (a) stereoselectively reducing the 3-keto group on the steroid core to a 3α-hydroxy group; and (b) stereoselectively hydroxylating the 7-position on the steroid core to a 7β-hydroxy group; thereby preparing UDCA or its salt, optionally with a 3α,7β-isomer purity greater than 95%, 98%, 99%, 99.5% or 99.8%.

[0114] Implementation Scheme 12) The method of Implementation Scheme 1, wherein 3-KCA or its cyclohexylamine or tert-butylamine salt comprises a steroid core, further comprising the following steps sequentially, simultaneously or in any order: (a) stereoselectively reducing the 3-ketone on the steroid core to a 3α-hydroxyl group in the presence of a 3α-keto reductase; and (b) stereoselectively hydroxylating the 7-position of the steroid core to a 7β-hydroxyl group in the presence of a 7β-hydroxylase; thereby preparing UDCA or its salt, optionally with a 3α,7β-isomer purity greater than 95%, 98%, 99%, 99.5% or 99.8%.

[0115] Implementation Scheme 13) The method of Implementation Scheme 11 or 12, wherein UDCA comprises 24-carboxylic acid, further comprising the following steps sequentially, simultaneously or in any order: (a) converting the acid into a derivative that can function as an acylating agent; and (b) reacting the derivative with taurine to form TUDCA, optionally with a 3α,7β-isomer purity greater than 95%, 98%, 99%, 99.5% or 99.8%.

[0116] Implementation Scheme 14) The method of Implementation Scheme 11 or 12, wherein UDCA comprises 24-carboxylic acid, further comprising the steps of: (a) contacting the 24-carboxylic acid with a tool for converting the 24-carboxylic acid into a derivative that can function as an acylating agent; and (b) reacting the derivative with taurine to form TUDCA, optionally with a 3α,7β-isomer purity greater than 95%, 98%, 99%, 99.5% or 99.8%.

[0117] Implementation Scheme 15) The method of Implementation Scheme 14, wherein the tool used for conversion is selected from SOCl2, (COCl)2, COCl2, SOBr2, TPP / NBS, Im2CO, EtOCOCl, (CH3)3CCOCl, (MeO)2P(O)Cl, diisopropylcarbodiimide, diisopropylcarbodiimide + HOBt, diisopropylcarbodiimide + HOSu, 2-chloro-1-methylpyridinium iodide, and [(Py)3PBr] + [PF6] - .

[0118] Implementation Scheme 16) The method of Implementation Scheme 1, wherein the solution contains ≥0.5, 1, 3, 5, 10 or 20% of the 5α isomer, based on the total weight of the isomer pairs.

[0119] Implementation scheme 17) tert-butylamine salt of 3-KCA.

[0120] Implementation Scheme 18) The tert-butylamine salt of Implementation Scheme 17 has a crystalline powder X-ray diffraction pattern comprising at least three characteristic peaks selected from the group denoted by 2θ: 10.404±0.2°, 11.920±0.2°, 14.502±0.2°, 14.925±0.2°, 16.081±0.2°, 16.221±0.2°, 18.103±0.2° and 20.877±0.2°.

[0121] Implementation Scheme 19) The tert-butylamine salt of Implementation Scheme 17 has a crystalline powder X-ray diffraction pattern, the pattern including characteristic peaks represented by 2θ at 14.502±0.2°, 14.925±0.2° and 18.103±0.2°.

[0122] Implementation Scheme 20) The tert-butylamine salt of Implementation Scheme 17, including δ-type molecules corresponding to plant-derived molecules. 13 The C value, which optionally includes δ values ​​less than -15‰, -17.5‰, -20‰, -22.5‰, or -25‰ relative to VPDB. 13 C.

[0123] Implementation scheme 21) Cyclohexylamine salt of 3-KCA.

[0124] Implementation Scheme 22) The cyclohexylamine salt of Implementation Scheme 21 has a crystalline powder X-ray diffraction pattern, said pattern including at least three characteristic peaks represented by 2θ selected from the group consisting of: 9.046±0.2°, 12.907±0.2°, 14.359±0.2°, 15.652±0.2°, 16.170±0.2°, 16.974±0.2° and 17.508±0.2°.

[0125] Implementation Scheme 23) The cyclohexylamine salt of Implementation Scheme 21 has a crystalline powder X-ray diffraction pattern, the pattern including characteristic peaks represented by 2θ at 12.907±0.2°, 16.170±0.2° and 16.974±0.2°.

[0126] Implementation Scheme 24) The cyclohexylamine salt of Implementation Scheme 21, including δ-corresponding to plant-derived molecules 13 The C value may optionally include δ13C with a value less than -15‰, -17.5‰, -20‰, -22.5‰, or -25‰ relative to VPDB.

[0127] Implementation Scheme 25) is a method for preparing a 5β steroid compound substantially free of 5α isomer impurities, comprising selectively crystallizing a tert-butylamine salt of 3-KUDCA from a solution containing 3-KUDCA and one or more impurities.

[0128] Implementation Scheme 26) The method of Implementation Scheme 25, wherein 3-KUDCA includes δ corresponding to plant-derived molecules 13 The C value, which optionally includes δ values ​​less than -15‰, -17.5‰, -20‰, -22.5‰, or -25‰ relative to VPDB. 13 C.

[0129] Implementation Scheme 27) The method of Implementation Scheme 25, which includes selectively crystallizing the tert-butylamine salt of 3-KUDCA, optionally with a purity greater than 98%, 99%, 99.5% or 99.8%.

[0130] Implementation Scheme 28) The method of Implementation Scheme 25, comprising selectively crystallizing tert-butylamine salt of 3-KUDCA from a solution comprising MTBE, n-butyl acetate, ethyl acetate or combinations thereof, optionally with a purity greater than 98%, 99%, 99.5% or 99.8%.

[0131] Implementation Scheme 29) The method of Implementation Scheme 27, wherein the tert-butylamine salt comprises a crystalline powder X-ray diffraction pattern comprising at least three characteristic peaks selected from the group consisting of 2θ: 11.498±0.2°, 19.002±0.2°, 13.002±0.2°, 20.306±0.2°, 15.496±0.2°, 15.883±0.2°, 12.830±0.2°, and 18.022±0.2°.

[0132] Implementation Scheme 30) The method of Implementation Scheme 25, wherein 3-KUDCA or its tert-butylamine salt comprises a steroid core, and further comprises stereoselectively reducing the 3-ketone on the steroid core to a 3α-hydroxyl group, thereby preparing UDCA or its salt, optionally with a 3α,7β-isomer purity greater than 95%, 98%, 99%, 99.5% or 99.8%.

[0133] Implementation Scheme 31) The method of Implementation Scheme 25, wherein 3-KUDCA or its tert-butylamine salt comprises a steroid core, and further comprises stereoselectively reducing the 3-ketone on the steroid core to a 3α-hydroxyl group in the presence of a 3α-ketone reductase, thereby preparing UDCA or its salt, optionally with a 3α,7β-isomer purity greater than 95%, 98%, 99%, 99.5% or 99.8%.

[0134] Implementation Scheme 32) The method of Implementation Scheme 30, wherein UDCA comprises 24-carboxylic acid, further comprising the following steps sequentially, simultaneously or in any order: (a) converting the acid into a derivative that can function as an acylating agent; and (b) reacting the derivative with taurine to form TUDCA, optionally with a 3α,7β-isomer purity greater than 95%, 98%, 99%, 99.5% or 99.8%.

[0135] Implementation Scheme 33) The method of Implementation Scheme 30, wherein UDCA comprises 24-carboxylic acid, further comprising the steps of: (a) contacting the 24-carboxylic acid with a tool for converting the 24-carboxylic acid into a derivative that can function as an acylating agent; and (b) reacting the derivative with taurine to form TUDCA, optionally with a 3α,7β-isomer purity greater than 95%, 98%, 99%, 99.5% or 99.8%.

[0136] Implementation Scheme 34) The method of Implementation Scheme 33, wherein the tool used for conversion is selected from SOCl2, (COCl)2, COCl2, SOBr2, TPP / NBS, Im2CO, EtOCOCl, (CH3)3CCOCl, (MeO)2P(O)Cl, diisopropylcarbodiimide, diisopropylcarbodiimide + HOBt, diisopropylcarbodiimide + HOSu, 2-chloro-1-methylpyridinium iodide, and [(Py)3PBr]+ [PF6] - .

[0137] Implementation Scheme 35) The method of Implementation Scheme 25, wherein the solution contains ≥ 0.5, 1, 3, 5, 10 or 20% impurities.

[0138] Implementation scheme 36) Tert-butylamine salt of 3-KUDCA.

[0139] Implementation Scheme 37) The tert-butylamine salt of Implementation Scheme 36 has a crystalline powder X-ray diffraction pattern comprising at least three characteristic peaks selected from the group denoted by 2θ: 11.498±0.2°, 19.002±0.2°, 13.002±0.2°, 20.306±0.2°, 15.496±0.2°, 15.883±0.2°, 12.830±0.2° and 18.022±0.2°.

[0140] Implementation Scheme 38) The tert-butylamine salt of Implementation Scheme 36 has a crystalline powder X-ray diffraction pattern, the pattern including characteristic peaks represented by 2θ at 11.498±0.2°, 19.002±0.2°, and 13.002±0.2°.

[0141] Implementation Scheme 39) The tert-butylamine salt of Implementation Scheme 36, which includes δ corresponding to the plant-derived molecule 13 The C value, which optionally includes δ values ​​less than -15‰, -17.5‰, -20‰, -22.5‰, or -25‰ relative to VPDB. 13 C.

[0142] Example

[0143] In the following embodiments, every effort has been made to ensure the accuracy of numerical values ​​(e.g., quantities, temperatures, etc.), but some errors and deviations should still be considered. The following embodiments are intended to provide a complete disclosure and description, to those skilled in the art, regarding how the methods claimed herein are prepared and evaluated, and are intended purely as examples of the invention and not to limit the scope of the invention as the inventors believe it to be.

[0144] Example 1

[0145] 3-KCA cyclohexylamine salt

[0146] The cyclohexylamine salt of 3-KCA(SH1-75) was prepared according to the following reaction scheme:

[0147]

[0148] Details of the experimental procedure and results are provided below:

[0149] Experimental procedure:

[0150] SH1-75 (2 g) was suspended in 1% aqueous isopropanol (10 ml). The resulting mixture was stirred at 40 °C for 0.5 h to obtain a clear solution. At 40–45 °C, a solution of cyclohexylamine (0.58 g) in MTBE (20 ml) was added to this solution. The reaction mixture was stirred at room temperature for 12 h, then cooled to 5–10 °C. After maintaining this temperature for 1 h, the slurry was filtered, the solid was washed with MTBE (2 ml), and dried under vacuum to give 2 g of cyclohexylamine salt of SH1-75 as a white solid. Yield = 77%.

[0151] The ratio of 5β:5α-isomers was determined by HPLC to be 89.4:10.6.

[0152] purification:

[0153] The solid was transferred to a 50 ml round-bottom flask, diluted with IPA (5 ml), and stirred at room temperature for 1 hour. After 0.5 hours, water (10 ml) was added to the suspension, and the slurry was stirred at room temperature for 2 hours. The mixture was then cooled to 5–10 °C and stirred at the same temperature for 0.5 hours, and the solid was filtered. The wet filter cake was washed with cold water (2 ml) and dried under vacuum to give 1.6 g of SH1-75 cyclohexylamine salt as a yellowish-white solid. Recovery rate = 80%.

[0154] The ratio of 5β:5α-isomers was determined by HPLC to be 93.7:6.3.

[0155] Example 2

[0156] 3-KCA tert-butylamine salt

[0157] The tert-butylamine salt of 3-KCA (SH1-75) was prepared according to the following reaction scheme:

[0158]

[0159] Details of the experimental procedure and results are provided below:

[0160] Experimental procedure:

[0161] SH1-75 (2 g) was suspended in 1% aqueous isopropanol (10 ml). The resulting mixture was stirred at 40 °C for 0.5 h to obtain a clear solution. A solution of tert-butylamine (0.43 g) in MTBE (20 ml) was added to this solution at 40-45 °C. The reaction mixture was stirred at room temperature for 2 h, then cooled to 5-10 °C. After maintaining this temperature for 1 h, the slurry was filtered, the solid was washed with MTBE (2 ml), and dried under vacuum to give 1.8 g of tert-butylamine salt of SH1-75 as a white solid. Yield: 75%.

[0162] The ratio of 5β:5α-isomers was determined by HPLC to be 96.4:3.6.

[0163] purification:

[0164] 1. Suspend 95 g of SH1-75 TBA salt in IPA (238 ml) (2.5 vol.) and stir at room temperature for 1 hour (observe the suspension);

[0165] 2. Add water (475 ml) (5 vol.) to the mixture at room temperature;

[0166] 3. Stir the slurry at room temperature for 2 hours, then stir at 5-10 °C for 1 hour. Filter the solid, wash the wet filter cake with cold water (1 vol.), and vacuum dry to obtain purified SH1-75 TBA salt as a white solid.

[0167] Yield: 66 g (69.5%).

[0168] The ratio of 5β:5α-isomers (w / w%) was determined by HPLC to be 99.70:0.30.

[0169] Example 3

[0170] 3-KUDCA tert-butylamine salt

[0171]

[0172] Experimental procedure:

[0173] 7β-hydroxy-3-ketocholanic acid (3-KUDCA, 14 g) in MTBE (140 ml) was added to a 500 ml multi-necked round-bottom flask equipped with a mechanical stirrer and a dropping funnel. The mixture was stirred at 25–28 °C for 15 minutes to obtain a clear solution. After one hour, a solution of TBA (4.12 ml, 1.1 equiv.) in MTBE (70 ml) was added to this solution. The resulting slurry was stirred at 25–28 °C for 1 hour, followed by stirring at 0–5 °C for another hour. The resulting solid was filtered, and the wet filter cake was washed with MTBE (2 vol.). The product was dried under vacuum at 40–45 °C for 12 hours to obtain 3-KUDCA TBA salt (15 g) as a white solid. Yield: 90%.

[0174] purification:

[0175] 10 g of 3-KUDCA TBA salt was suspended in 50 ml of water at 25-28 °C, and isopropanol (IPA, 10 ml) was added. The resulting suspension was stirred at the same temperature for 2 hours, and then the solid was separated by filtration. The wet solid was washed with IPA (1 ml) and dried under vacuum at 40-45 °C for 12 hours to obtain purified 3-KUDCA TBA salt as a white solid (5 g, 50% recovery). Melting point: 168.4 °C; 1 H NMR (400 MHz, d6-DMSO): δ 3.9-5.8 (bs, 4H), 3.35 (m, 1H), 2.64 (t, J =14Hz, 1H), 2.32(td, J =14, 5Hz, 1H), 2.08 (m, 1H), 1.15-2.00 (m, 22H), 1.11 (s, 9H), 1.01 (m, 1H), 0.96 (s, 3H), 0.89 (d, J =7Hz, 3H), 0.65(s, 3H).

[0176] Example 4

[0177] XRPD analysis of 3-KCA cyclohexylamine salt

[0178] Following the collection conditions in Table 2, the cyclohexylamine salt of 3-KCA in Example 1 was analyzed by X-ray powder diffraction, yielding the results shown in Table 3. Figure 1 The peaks listed in the spectrum.

[0179] Table 2

[0180]

[0181]

[0182] Table 3

[0183]

[0184]

[0185] Example 5

[0186] XRPD analysis of 3-KCA tert-butylamine salt

[0187] Following the acquisition conditions in Table 4, X-ray powder diffraction analysis was performed on the tert-butylamine salt of 3-KCA from Example 2, resulting in the peak list in Table 5. Figure 2 The diffraction pattern.

[0188] Table 4

[0189]

[0190]

[0191] Table 5

[0192]

[0193]

[0194] Example 5

[0195] XRPD analysis of 3-KUDCA tert-butylamine salt

[0196] Following the sampling conditions in Table 6, the 3-KUDCA tert-butylamine salt of Example 3 was analyzed by X-ray powder diffraction, yielding the results shown in Table 7 and... Figure 3 The peaks listed in the spectrum.

[0197] Table 6

[0198]

[0199]

[0200] Table 7

[0201]

[0202]

[0203] Example 7

[0204] Preparation of 3-KCA from tert-butylamine salt

[0205]

[0206] Experimental process

[0207] 500 g of tert-butylamine salt of 3-KCA was suspended in MTBE (3.5 L). 0.5 L of 3N hydrochloric acid was added at 25 °C. After stirring for approximately 30 minutes, the slurry became a clear solution containing two phases. The lower layer was separated and discarded. The upper layer was washed with water (2.5 L) and then concentrated under vacuum to approximately 2 L. The solution was then heated to 40 °C, and 4.0 L of n-heptane was added. After 50 minutes, a suspension was formed. The suspension was slowly cooled to 0–5 °C, held at this temperature for 30 minutes, and then filtered. The wet filter cake was washed with 0.5 L of n-heptane and dried under vacuum to give 3-KCA as a pale yellow solid (315 g, 75% yield).

[0208] The ratio of 5β:5α-isomers (w / w%) was determined by HPLC to be 99.9:0.1.

[0209] The same process can be used to convert the TBA salt of 3-KUDCA to 3-KUDCA.

[0210] Example 8

[0211] UDCA preparation from 3-KCA

[0212]

[0213] 1. The preparation of Pichia pastoris SAND121 was as described in Example 16 of WO 2022 / 115710 A1 and used in Example 19 of the same document.

[0214] 2. 3α-HSDH refers to the 3α-hydroxysteroid dehydrogenase (EC 1.1.1.50) derived from Pseudomonas testosteroni.

[0215] 3. GDH refers to D-glucose dehydrogenase (EC1.1.1.47) derived from Bacillus megaterium.

[0216] 3-KCA hydroxylation

[0217] Pichia pastoris SAND121 (from WO 2022 / 115710) was inoculated into BMG medium supplemented with 100 µg / ml nourseothricin and 100 µg / ml bleomycin, and incubated in Erlenmeyer flasks at 30°C with shaking at 250 RPM for 3 days. Aqueous 5-ALA solution (200 mM) and methanol containing 3-ketolithocholic acid (3-KCA) were added to the culture, and incubation was continued for 1 day. Methanol was added to the culture, and incubation was continued for 1 day. The mixture was acidified to pH 2–3 by adding aqueous hydrochloric acid, and then centrifuged. The solid was treated with acetone, and centrifugation was repeated. The supernatant was concentrated under vacuum to remove most of the acetone, yielding a slurry. The solid was removed by filtration, the product was washed with n-heptane, and dried under vacuum to obtain solid 3-KUDCA.

[0218] Reduce 3-keto in 3-KUDCA

[0219] At room temperature, 3-KUDCA (1 g, 2.56 mmol), dextrose (1.3 g), β-NAD (33 mg), and 250 mM K2HPO4 buffer (70 ml) were added to a 250 ml single-necked round-bottom flask. The mixture was stirred for 0.5 hours to obtain a clear solution. The pH was adjusted to 7.8, and 3α-HSDH (66 mg, derived from *Pseudomonas testis*) was added. Pseudomonas testosteroni) EC1.1.1.50) and GDH (2 mg, from Bacillus megaterium) Bacillus megaterium) (EC 1.1.1.47). The resulting mixture was stirred at room temperature for 20 hours, during which the pH was maintained at 7.8 by periodically adding 1M KOH solution. TLC analysis showed complete conversion of the feedstock.

[0220] The reaction mixture was quenched with 2N hydrochloric acid solution to pH 3-3.5, and the product was then extracted with ethyl acetate (3 × 50 ml) and concentrated under reduced pressure to give UDCA as a white solid (900 mg). 1 H NMR (500 MHz, MeOD): δ 3.44-3.53(m, 2H), 2.30-2.38(m, 1H), 2.18-2.25(m, 1H), 2.03(dt, J =6.5, 2.9Hz, 1H), 1.95-1.79 (m, 5H), 1.56-0.92 (m, 26H), 0.71 (s, 3H).

[0221] Example 9

[0222] TUDCA synthesized from UDCA

[0223] UDCA (5 g) was placed in a 100 mL single-necked round-bottom flask. Acetone (30 mL) was added to obtain a solution. Triethylamine (TEA, 1.7 mL, 0.97 equiv.) was added, and the solution was cooled to 0 °C. Ethyl chloroformate (1.34 g, 0.97 equiv.) was added, and the resulting mixture was stirred at room temperature under nitrogen for 4 hours. The reaction mixture was filtered to remove triethylamine hydrochloride, and the filtrate was added dropwise to an aqueous solution of sodium taurate (prepared by reacting 1.9 g of taurine with 0.6 g of sodium hydroxide in 3.7 mL of water) over 20 minutes. The reaction was continued at room temperature for 1 hour, at which point TLC analysis showed complete conversion.

[0224] Add concentrated hydrochloric acid (1.5 ml) at room temperature to adjust the pH to approximately 1. After stirring for 1 hour, filter the resulting solid (0.9 g). Dilute the filtrate with acetone, stir at room temperature for 36 hours, filter the resulting solid, wash with acetone (10 ml), and dry under vacuum to give TUDCA as a white solid (5.1 g, 80% yield). 1 H NMR (400 MHz, CD3OD): δ 3.62 (t, J =6.8Hz, 2H), 3.52-3.42(m, 2H), 2.97(t, J =6.8Hz, 2H), 2.38-2.28(m, 1H), 2.20-2.10(m, 1H), 2.08-2.00(m, 1H), 1.92-1.75(m, 5H), 1.65-0.96(m, 21H), 0.95(s, 3H), 0.70(s, 3H); 13 CNMR (100MHz, CD3OD): δ 177.97, 72.12, 71.96, 47.45, 56.34, 50.78, 44.79, 44.46, 43.99, 41.52, 40.69, 38.56, 37.96, 37.47, 36.89, 36.07, 35.15, 33.34, 33.18, 30.99, 29.63, 27.91, 23.93, 22.37, 18.96, 12.64; ESI-MS: C 26 H 45 NO6S, m / z499.1 [MH] + .

[0225]

[0226] Various publications have been cited in this application. The disclosures of these publications are incorporated herein by reference in their entirety to more fully describe the state of the art to which this invention pertains. It will be apparent to those skilled in the art that various modifications and variations can be made to this invention without departing from its scope or spirit. Other embodiments of the invention will be apparent to those skilled in the art upon consideration of the specification disclosed herein and the practice of the invention. This specification and embodiments are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the appended claims.

Claims

1. A method for preparing 5β-steroidal compounds substantially free of 5α-isomer impurities, comprising selectively crystallizing tert-butylamine or cyclohexylamine salts of 3-KCA from a solution containing 3-KCA and its 5α-isomer.

2. The method of claim 1, wherein 3-KCA comprises δ corresponding to plant-derived molecules. 13 The C value, which optionally includes δ values ​​less than -15‰, -17.5‰, -20‰, -22.5‰, or -25‰ relative to VPDB. 13 C.

3. The method of claim 1, comprising selectively crystallizing tert-butylamine salt of 3-KCA, optionally with an isomer purity greater than 98%, 99%, 99.5% or 99.8%.

4. The method of claim 1, comprising selectively crystallizing tert-butylamine salt of 3-KCA from a solution containing a C1-C4 alcohol, said solution optionally containing MTBE or water as a co-solvent, optionally with an isomer purity greater than 98%, 99%, 99.5% or 99.8%.

5. The method of claim 1, comprising selectively crystallizing tert-butylamine salt of 3-KCA from a solution containing isopropanol, said solution optionally containing MTBE or water as a co-solvent, optionally with an isomer purity greater than 98%, 99%, 99.5% or 99.8%.

6. The method of claim 3, wherein the tert-butylamine salt comprises a crystalline powder X-ray diffraction pattern, said pattern comprising at least three characteristic peaks selected from the group represented by 2θ: 10.404±0.2°, 11.920±0.2°, 14.502±0.2°, 14.925±0.2°, 16.081±0.2°, 16.221±0.2°, 18.103±0.2° and 20.877±0.2°.

7. The method of claim 1, comprising selectively crystallizing the cyclohexylamine salt of 3-KCA, optionally with an isomer purity greater than 98%, 99%, 99.5% or 99.8%.

8. The method of claim 1, comprising selectively crystallizing the cyclohexylamine salt of 3-KCA from a solution containing a C1-C4 alcohol, said solution optionally containing MTBE or water as a co-solvent, optionally with an isomer purity greater than 98%, 99%, 99.5% or 99.8%.

9. The method of claim 1, comprising selectively crystallizing the cyclohexylamine salt of 3-KCA from a solution containing isopropanol, said solution optionally containing MTBE or water as a co-solvent, optionally with an isomer purity greater than 98%, 99%, 99.5% or 99.8%.

10. The method of claim 7, wherein the cyclohexylamine salt has a crystalline powder X-ray diffraction pattern comprising at least three characteristic peaks selected from the group consisting of 2θ: 9.046±0.2°, 12.907±0.2°, 14.359±0.2°, 15.652±0.2°, 16.170±0.2°, 16.974±0.2°, and 17.508±0.2°.

11. The method of claim 1, wherein 3-KCA or its cyclohexylamine or tert-butylamine salt comprises a steroidal core, further comprising the following steps sequentially, simultaneously, or in any order: (a) Stereoselective reduction of the 3-keto group on the steroid core to a 3α-hydroxy group; and (b) Stereoselectively hydroxylate the 7-position of the steroid core to a 7β-hydroxyl group; UDCA or its salts are thus prepared, optionally with a purity of 3α,7β-isomer greater than 95%, 98%, 99%, 99.5% or 99.8%.

12. The method of claim 1, wherein 3-KCA or its cyclohexylamine or tert-butylamine salt comprises a steroidal core, further comprising the following steps sequentially, simultaneously, or in any order: (a) In the presence of 3α-keto reductase, stereoselectively reducing the 3-keto group on the steroid core to a 3α-hydroxyl group; and (b) In the presence of 7β-hydroxylase, stereoselectively hydroxylate the 7-position of the steroid core to a 7β-hydroxyl group; UDCA or its salts are thus prepared, optionally with a purity of 3α,7β-isomer greater than 95%, 98%, 99%, 99.5% or 99.8%.

13. The method of claim 11, wherein UDCA comprises 24-carboxylic acid, and further comprises the following steps sequentially, simultaneously, or in any order: (a) To convert the acid into a derivative that can function as an acylating agent; and (b) React the derivative with taurine to form TUDCA, optionally with a purity of 3α,7β-isomer greater than 95%, 98%, 99%, 99.5% or 99.8%.

14. The method of claim 11, wherein UDCA comprises 24-carboxylic acid, and further comprises: (a) Contacting 24-carboxylic acid with a tool used to convert 24-carboxylic acid into a derivative that can function as an acylating agent; and (b) React the derivative with taurine to form TUDCA, optionally with a purity of 3α,7β-isomer greater than 95%, 98%, 99%, 99.5% or 99.8%.

15. The method of claim 14, wherein the conversion tool is selected from SOCl2, (COCl)2, COCl2, SOBr2, TPP / NBS, Im2CO, EtOCOCl, (CH3)3CCOCl, (MeO)2P(O)Cl, diisopropylcarbodiimide, diisopropylcarbodiimide + HOBt, diisopropylcarbodiimide + HOSu, 2-chloro-1-methylpyridinium iodide, and [(Py)3PBr] + [PF6] - .

16. The method of claim 1, wherein the solution comprises ≥ 0.5, 1, 3, 5, 10 or 20% of the 5α isomer, based on the total weight of the isomer pairs. 17.3-KCA tert-butylamine salt.

18. The tert-butylamine salt of claim 17, having a crystalline powder X-ray diffraction pattern comprising at least three characteristic peaks selected from the group denoted by 2θ: 10.404±0.2°, 11.920±0.2°, 14.502±0.2°, 14.925±0.2°, 16.081±0.2°, 16.221±0.2°, 18.103±0.2°, and 20.877±0.2°.

19. The tert-butylamine salt of claim 17, having a crystalline powder X-ray diffraction pattern comprising characteristic peaks denoted by 2θ at 14.502±0.2°, 14.925±0.2°, and 18.103±0.2°.

20. The tert-butylamine salt of claim 17, comprising δ corresponding to a plant-derived molecule. 13 The C value, which optionally includes δ values ​​less than -15‰, -17.5‰, -20‰, -22.5‰, or -25‰ relative to VPDB. 13 C. Cyclohexylamine salt of 21,3-KCA.

22. The cyclohexylamine salt of claim 21, having a crystalline powder X-ray diffraction pattern comprising at least three characteristic peaks selected from the group denoted by 2θ: 9.046±0.2°, 12.907±0.2°, 14.359±0.2°, 15.652±0.2°, 16.170±0.2°, 16.974±0.2°, and 17.508±0.2°.

23. The cyclohexylamine salt of claim 21, having a crystalline powder X-ray diffraction pattern comprising characteristic peaks denoted by 2θ at 12.907±0.2°, 16.170±0.2°, and 16.974±0.2°.

24. The cyclohexylamine salt of claim 21, comprising δ corresponding to a plant-derived molecule. 13 The C value, which optionally includes δ values ​​less than -15‰, -17.5‰, -20‰, -22.5‰, or -25‰ relative to VPDB. 13 C.

25. A method for preparing a 5β-steroid compound substantially free of 5α-isomer impurities, comprising selectively crystallizing a tert-butylamine salt of 3-KUDCA from a solution containing 3-KUDCA and one or more impurities.

26. The method of claim 25, wherein 3-KUDCA comprises δ corresponding to plant-derived molecules. 13 The C value, which optionally includes δ values ​​less than -15‰, -17.5‰, -20‰, -22.5‰, or -25‰ relative to VPDB. 13 C.

27. The method of claim 25, comprising selectively crystallizing tert-butylamine salt of 3-KUDCA, optionally with a purity greater than 98%, 99%, 99.5% or 99.8%.

28. The method of claim 25, comprising selectively crystallizing a tert-butylamine salt of 3-KUDCA from a solution comprising MTBE, ethyl n-butyl acetate, or combinations thereof, optionally with a purity greater than 98%, 99%, 99.5%, or 99.8%.

29. The method of claim 27, wherein the tert-butylamine salt comprises a crystalline powder X-ray diffraction pattern, said pattern comprising at least three characteristic peaks selected from the group consisting of 2θ: 11.498±0.2°, 19.002±0.2°, 13.002±0.2°, 20.306±0.2°, 15.496±0.2°, 15.883±0.2°, 12.830±0.2°, and 18.022±0.2°.

30. The method of claim 25, wherein 3-KUDCA or its tert-butylamine salt comprises a steroidal core, and further comprises stereoselectively reducing the 3-ketone on the steroidal core to a 3α-hydroxyl group to prepare UDCA or its salt, optionally with a 3α,7β-isomer purity greater than 95%, 98%, 99%, 99.5% or 99.8%.

31. The method of claim 25, wherein 3-KUDCA or its tert-butylamine salt comprises a steroidal core, and further comprises stereoselectively reducing the 3-ketone on the steroidal core to a 3α-hydroxyl group in the presence of a 3α-keto reductase, thereby preparing UDCA or its salt, optionally with a 3α,7β-isomer purity greater than 95%, 98%, 99%, 99.5% or 99.8%.

32. The method of claim 30, wherein UDCA comprises 24-carboxylic acid, and further comprises the following steps sequentially, simultaneously, or in any order: (a) To convert the acid into a derivative that can function as an acylating agent; and (b) React the derivative with taurine to form TUDCA, optionally with a purity of 3α,7β-isomer greater than 95%, 98%, 99%, 99.5% or 99.8%.

33. The method of claim 30, wherein UDCA comprises 24-carboxylic acid, and further comprises: (a) Contacting 24-carboxylic acid with a tool used to convert 24-carboxylic acid into a derivative that can function as an acylating agent; and (b) React the derivative with taurine to form TUDCA, optionally with a purity of 3α,7β-isomer greater than 95%, 98%, 99%, 99.5% or 99.8%.

34. The method of claim 33, wherein the conversion tool is selected from SOCl2, (COCl)2, COCl2, SOBr2, TPP / NBS, Im2CO, EtOCOCl, (CH3)3CCOCl, (MeO)2P(O)Cl, diisopropylcarbodiimide, diisopropylcarbodiimide + HOBt, diisopropylcarbodiimide + HOSu, 2-chloro-1-methylpyridinium iodide, and [(Py)3PBr] + [PF6] - .

35. The method of claim 25, wherein the solution comprises ≥ 0.5, 1, 3, 5, 10 or 20% impurities. tert-butylamine salt of 36.3-KUDCA.

37. The tert-butylamine salt of claim 36, having a crystalline powder X-ray diffraction pattern comprising at least three characteristic peaks selected from the group denoted by 2θ: 11.498±0.2°, 19.002±0.2°, 13.002±0.2°, 20.306±0.2°, 15.496±0.2°, 15.883±0.2°, 12.830±0.2°, and 18.022±0.2°.

38. The tert-butylamine salt of claim 36, having a crystalline powder X-ray diffraction pattern comprising characteristic peaks denoted by 2θ at 11.498±0.2°, 19.002±0.2°, and 13.002±0.2°.

39. The tert-butylamine salt of claim 36, comprising δ corresponding to a plant-derived molecule. 13 The C value, which optionally includes δ values ​​less than -15‰, -17.5‰, -20‰, -22.5‰, or -25‰ relative to VPDB. 13 C.

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