Stereoselective crystallizations of cholic acids

Abstract

Methods for stereo-selectively crystallizing 3-keto-5β-cholanic acid ("3-KCA") and 3-keto ursodeoxycholic acid ("3-KUDCA") of exceptional purity, salts useful in carrying out such methods, particularly the t-butylamine and cyclohexylamine salts of 3-KCA, the t-butylamine salt of 3-KUDCA, and crystalline forms of such salts, and methods of making commercial steroids from such purified materials.

Description

[0001] STEREOSELECTIVE CRYSTALLIZATIONS OF CHOLIC ACIDS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates generally to methods for purifying 3-keto-5P-cholanic acid (“3-KCA”) and 3 -keto-ursodeoxy cholic acid (“3-KUDCA”) via crystallization, salts useful in carrying out such methods, and methods of subsequently making commercial steroids from such purified materials.

[0004] BACKGROUND OF THE INVENTION

[0005] Cholic acid and its derivatives find utility in numerous medical applications and research initiatives. Cholic acid itself, sold under the brand name Cholbam®, is approved for use as a treatment for children and adults with bile acid synthesis disorders due to single enzyme defects, and for peroxisomal disorders (such as Zellweger syndrome). 7-Ketolithocholic acid has been examined for its effect on endogenous bile acid synthesis, biliary cholesterol saturation, and its possible role as a precursor of chenodeoxycholic acid and ursodeoxycholic acid. See Salen et al., Gastroenterology, 1982;83:341-7. Ursodeoxycholic acid (a / k / a UDCA or ursodiol), sold under the brand name URSO 250® and URSO Forte® tablets, is approved for the treatment of patients with primary biliary cirrhosis (PBC). More recently, obeticholic acid, sold under the brand name Ocaliva®, was approved for the treatment of PBC in combination with UDCA in adults with an inadequate response to UDCA, or as monotherapy in adults unable to tolerate UDCA.

[0006] In spite of this significant medical interest, methods of synthesizing the derivatives remain cumbersome and inefficient, with numerous processes being proposed. Fantin et al., Steroids, 1993 Nov.; 58:524-526, discloses the preparation of 7a-, 12a-, 12p-hydroxy and 7a-, 12a- and 7a-,12P-dihydroxy-3-ketocholanoic acids by protecting the 3-keto group as dimethyl ketal and subsequent reduction with sodium borohydride of the corresponding 7- and 12-oxo functionalities. WO 2017 / 079062 Al by Galvin reports a method of preparing obeticholic acid by direct alkylation at the C-6 position of 7-keto lithocholic acid (KLCA). He et al., Steroids, 2018 Dec;140: 173-178, discloses a synthetic route of producing ursodeoxycholic acid (UDCA) and obeticholic acid (OCA) through multiple reactions from cheap and readily -available cholic acid. Wang et al., Steroids 157 (2020) 108600, similarly report a synthetic route of producing ursodeoxycholic acid (UDCA) through multiple reactions from commercially available bisnoralcohol (BA).

[0007] Complicating the synthetic pathway is the frequent need to reduce one or more double bonds on unsaturated intermediate compounds. Because each steroid has unique stereochemistry at several chiral centers, diastereomeric impurities are often present. This is especially true for plant-derived steroids, which generally have a 4,5-double bond that must be stereo-specifically reduced to produce several commercially relevant steroids.

[0008] What is needed are more efficient processes for making cholic acid derivatives with high stereoselectivity. Particularly needed are more efficient processes for making 5P-cholic and cholanic acids, including ursodeoxycholic acid, tauroursodeoxycholic acid, and starting materials and intermediates therefor.

[0009] SUMMARY OF INVENTION

[0010] The inventors have discovered novel crystalline salt forms of 3-KCA, and methods of using those novel forms to purify 3-KCA from its 5a isomer. Thus, in one embodiment, the invention provides a method of making a 5P steroid substantially free of 5a isomeric impurities comprising selectively crystallizing a t-butylamine (“TBA”) or cyclohexylamine salt of 3-KCA from a solution comprising 3-KCA and its 5a isomer. In another embodiment, the invention provides the t-butylamine (“TBA”) salt of 3-KCA. In another embodiment the invention provides the cyclohexylamine salt of 3-KCA.

[0011] The inventors have further discovered novel crystalline salt forms of 3-KUDCA, and methods of using those novel forms to purify 3-KUDCA from its impurities. Thus, in another embodiment, the invention provides a method of making 3-KUDCA substantially free of its impurities comprising selectively crystallizing a t-butylamine (“TBA”) salt of 3-KUDCA from a solution comprising 3-KUDCA and its impurities. In another embodiment, the invention provides the t-butylamine (“TBA”) salt of 3-KUDCA.

[0012] Additional advantages of the invention are set forth in part in the description that follows, and in part will be obvious from the description or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[0013] DETAILED DESCRIPTION OF THE INVENTION

[0014] Definitions and Use o f Terms

[0015] As used in the specification and claims, the singular forms a, an, and the include plural references unless the context clearly dictates otherwise. For example, the term “a specification” refers to one or more specifications for use in the presently disclosed methods and systems. “A hydrocarbon” includes mixtures of two or more such hydrocarbons, and the like. The word “or” or like terms as used herein means any one member of a particular list and also includes any combination of members of that list.

[0016] As used in this specification and in the claims which follow, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. When an element is described as comprising one or a plurality of components, steps or conditions, it will be understood that the element can also be described as “consisting of’ or “consisting essentially of’ the component, step or condition, or the plurality of components, steps or conditions.

[0017] When used herein the term “about” will compensate for variability allowed for in the pharmaceutical industry and inherent in pharmaceutical products. In one embodiment, the term allows for any variation within 5% of the recited specification or standard. In one embodiment, the term allows for any variation within 10% of the recited specification or standard. All numerical values recited in the application should be interpreted as preceded by the term “about”, thereby providing additional embodiments with 5% variation and 10% variation.

[0018] When purities are given herein based on percentages, they will be understood to be based on weight percent. Likewise, when yields are expressed as percentages, they will be understood to refer to the weight of the resultant product actually observed relative to the weight of the resultant product had there been 100% conversion.

[0019] The term “substantially free” includes completely free. Thus, a composition comprising a compound substantially free of one or more impurities can also be said to have a purity greater than 95%, 98%, 99%, 99.5%, or 99.8%, relative to the one or more impurities, optionally completely free of the one or more impurities. In like manner, a compound which is “substantially pure” can be completely pure. Thus, a compound which is substantially pure can also be said to have a purity greater than 95%, 98%, 99%, 99.5%, or 99.8%. Purity can be judged relative to one or more specified impurities or all impurities and, if no impurities are specified, purity is to be judged relative to all impurities.

[0020] The term “impurity” as used herein is used consistently with the United States Food and Drug Administration’s Guidance for Industry Q3A Impurities in New Drug Substances (June 2008 Revision 2). Impurities can thus be classified into the following categories: organic impurities (process- and drug-related), inorganic impurities, and residual solvents. Organic impurities can arise during the manufacturing process and / or storage of the new drug substance. They can be identified or unidentified, volatile or nonvolatile, and include starting materials, by-products, intermediates, degradation products, reagents, ligands, and catalysts. Inorganic impurities can result from the manufacturing process. They are normally known and identified and include reagents, ligands, and catalysts, heavy metals or other residual metals, inorganic salts, and other materials (e.g., filter aids, charcoal).

[0021] When a method is defined by its constituent steps, it will be understood that the method includes the steps performed consecutively, simultaneously, or in any order, unless specified to the contrary.

[0022] 3-KCA, or 3-keto-5P-cholanic acid, has the following chemical structure:

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

[0024]

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

[0026] 3-KUDCA, or 3-keto ursodeoxycholic acid, has the following chemical structure:

[0027] In various embodiments, the 3-KUDCA or its TBA salt comprises less than 5%, 2%, 1%, 0.5%, or 0.2% impurities. In other embodiments, the 3-KUDCA or its TBA salt is isolated from a larger composition or reaction mixture, such that the composition in which the 3-KUDCA or its TBA salt is present comprises greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8% 3-KUDCA relative to the 3-KUDCA and its impurities.

[0028] Ursodeoxycholic acid, 3a,7p-dihydroxy-5p-cholanic acid, or simply ursodiol or UDCA, has the following chemical structure: In various embodiments, the UDCA comprises less than 5%, 2%, 1%, 0.5%, or 0.2% of its corresponding 5a stereoisomer. In other embodiments, the UDCA is isolated from a larger composition or reaction mixture, such that the composition in which the UDCA is present comprises greater than 95%, 98%, 99%, or 99.5% UDCA, optionally at a 3a,7P-isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.

[0029] Tauroursodeoxy cholic acid, or TUDCA, has the following chemical structure:

[0030] In various embodiments, the TUDCA comprises less than 5%, 2%, 1%, 0.5%, or 0.2% of its corresponding 5a stereoisomer. In other embodiments, the TUDCA is isolated from a larger composition or reaction mixture, such that the composition in which the TUDCA is present comprises greater than 95%, 98%, 99%, 99.5%, or 99.8% TUDCA, optionally at a 3a,7P-isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.

[0031] In any of the embodiments of the current invention, the steroid at issue, whether 3-KCA, 3-KUDCA, UDCA, or TUDCA, is preferably plant derived.

[0032] The term “plant derived” refers to a molecule comprising a 813C value corresponding to a plant derived molecule or a mixed fossil / animal and plant derived molecule, comprising a majority of plant-derived carbons from plant sources. A plant derived molecule can thus be characterized as having greater than 50%, 75%, 90%, 95%, 98%, or 99% plant derived carbons, with the remaining carbons (if any) derived from fossil / animal sources.

[0033] By “plant sources” are meant any source, which may be defined as a plant such as, for example, trees, shrubs, herbs, grasses, fems, mosses, flowers, vegetables, and weeds, as well as compounds derived from plants such as phytosterols, and phytosterol derivatives. The plant can be a C3 plant, a C4 plant, or a combination of both. By “C3 plants” are meant plants that do not have photosynthetic adaptations to reduce photorespiration. This includes plants such as rice, wheat, soybeans, most fruits, most vegetables, and all trees.

[0034] By “C4 plants” are meant plants where the light-dependent reactions and the Calvin cycle are physically separated, and where the light-dependent reactions occur in the mesophyll cells and the Calvin cycle occurs in bundle-sheath cells. This includes plants such as crabgrass, sugarcane, sorghum, and corn.

[0035] “813C value” is an isotopic measurement of the delta notation of13C. 613C values are expressed as a per mil (%o) deviation, e.g., per one thousand, from an internationally accepted PDB standard (originally a carbonate from the Pee Dee Belemnite formation in South Carolina but more commonly today Vienna Pee Dee Belemnite (VPDB)). 513C values are determined using the following formula:

[0036] 813C = ((813C / 812C)sampie - (813C / 812C)PDB) / ((813C / 812C)PDB) * 1000

[0037] In any of the embodiments of this invention, the 3-KCA, UDCA, or TUDCA will preferably comprises a 813C value corresponding to a plant derived molecule, preferably comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 813C relative to VPDB or, in the case of TUDCA, more preferably comprising less than -20%o, -22.5%o, or -25%o 813C relative to VPDB.

[0038] Discussion

[0039] In a first principal embodiment, the invention provides a method of making a 5P steroid substantially free of 5a isomeric impurities comprising selectively crystallizing a t-butylamine or cyclohexylamine salt of 3-KCA from a solution comprising 3-KCA and its 5a isomer. The method can be practiced using an 80:20 mixture of 3-KCA and its 5a isomer, or any ratio of 3-KCA to its 5a isomer in the solution. Thus, in various embodiments, the solution comprises > 0.5, 1, 3, 5, 10, or 20% of the 5a isomer based on the total weight of the isomeric pair. In addition, it will be understood that the method can be practiced two or more times in sequence to further purify the 3-KCA.

[0040] In another principal embodiment, the invention provides a method of making a substantially pure 5p steroid comprising selectively crystallizing a t-butylamine salt of 3-KUDCA from a solution comprising 3-KUDCA and one or more impurities. The method can be practiced using an 80:20 mixture of 3-KUDCA and impurities, or any ratio of 3-KUDCA to its impurities in the solution. 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. In addition, it will be understood that the method can be practiced two or more times in sequence to further purify the 3-KUDCA.

[0041] The process can also be defined based on the stereo- and regio- selectivity of the process. Thus, in any of the embodiments of the current invention, the method will produce salts of 3-KCA having greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8% 5p-stereochemistry, to the exclusion of 5a-stereochemistry.

[0042] The process can also be defined based on purity of the end-product. Thus, in any of the embodiments of the current invention, the method will produce salts of 3-KCA or 3-KUDCA having greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8% purity when considering 3-KCA or 3-KUDCA and its impurities.

[0043] In one embodiment, the method comprises selectively crystallizing the t-butylamine salt of 3-KCA. In various embodiments, the isomeric purity of the crystallized t-butylamine salt is greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8%.

[0044] In another embodiment, the method comprises selectively crystallizing the cyclohexylamine salt of 3-KCA. In various embodiments, the isomeric purity of the crystallized cyclohexylamine salt is greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8%.

[0045] In another embodiment, the method comprises selectively crystallizing the t-butylamine salt of 3-KUDCA. In various embodiments, the purity of the crystallized t-butylamine salt of 3- KUDCA is greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8% when considering 3-KUDCA and its impurities.

[0046] The methods of crystallizing the salt of 3-KCA are similar regardless of whether the t- butylamine or cyclohexylamine salt is obtained. In one embodiment, the method comprises crystallizing the salt of 3-KCA from a solution comprising a C1-C4 alcohol such as isopropanol, and optionally MTBE (methyl tert-butyl ether) and / or water.

[0047] In another embodiment, when the t-butylamine salt of 3-KUDCA is crystallized, the method comprises crystallizing the 3-KUDCA salt from a solution comprising at least 80 or 90 %vol. MTBE, n-butyl acetate, ethyl acetate, or a combination thereof, with n-butyl acetate preferred. In a preferred method, 3-KCA and a C1-C4 alcohol are first mixed at an elevated temperature of, e.g., 35-50 °C, to form a solution, and subsequently mixed either directly with the desired amine, or a solution containing the desired amine e.g., t-butylamine or cyclohexylamine). The 3-KCA and C 1-C4 alcohol e.g., isopropanol) are typically mixed in a ratio of from 0.5g / 10ml to lOg / lOml, more preferably from Ig / lOml to 5g / 10ml, or about 2g / 10ml.

[0048] In one embodiment, the cyclohexylamine or t-butylamine is added to the alcoholic solution of 3-KCA in a heated solution of MTBE or another acceptable organic solvent such as n-butyl acetate or ethyl acetate. The volumetric ratio of the 3-KCA solution to the amine solution will commonly range from about 0.1 :1 to about 5:1, or from about 0.2: 1 to about 1 : 1, or about 0.5: 1. The 3-KCA and t-butylamine or cyclohexylamine will commonly be combined in molar ratios of ~1:1. Upon mixing the solutions of 3-KCA and amine, a solid will typically form, although at some concentrations cooling the combined solutions to, e.g., 5-20 °C or 10-15 °C, will be required for the precipitate to form. The precipitate corresponds to the desired crystalline salt.

[0049] UDCA Conversion

[0050] In other embodiments, the 3-KCA or 3-KUDCA is converted to a desired end-product. Thus, in one embodiment, the 3-KCA, or its cyclohexylamine or t-butylamine salt, comprises a steroidal core, and the method further comprises, sequentially, simultaneously, or in any order: (a) stereo-selectively reducing the 3-ketone on the steroidal core to 3a-hydroxy; and (b) stereo- selectively hydroxylating the 7-position on the steroidal core to 7P-hydroxy; thereby making UDCA or a salt thereof, optionally at a 3a,7P-isomeric purity greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8%.

[0051] In another embodiment, the 3-KUDCA, or its t-butylamine salt, comprises a steroidal core, and the method further comprises stereo-selectively reducing the 3-ketone on the steroidal core to 3a-hydroxy; thereby making UDCA or a salt thereof, optionally at a purity greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8%.

[0052] In another embodiment, the 3-KCA, or its cyclohexylamine or t-butylamine salt, comprises a steroidal core, further comprising, sequentially, simultaneously, or in any order: (a) in the presence of a 3a-ketoreductase, stereo-selectively reducing the 3-ketone on the steroidal core to 3a-hydroxy; and (b) in the presence of a 7p-hydroxylase, stereo-selectively hydroxylating the 7- position on the steroidal core to 7P-hydroxy; thereby making UDCA or a salt thereof, optionally at a 3a,7p-isomeric purity greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8%.

[0053] In another embodiment, the 3-KUDCA, or its t-butylamine salt, comprises a steroidal core, further comprising, in the presence of a 3a-ketoreductase, stereo-selectively reducing the 3-ketone on the steroidal core to 3a-hydroxy; thereby making UDCA or a salt thereof, optionally at a 3a, 7p- isomeric purity greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8%.

[0054] Taurine Conjugation

[0055] In the synthesis of TUDCA from UDCA and taurine, the UDCA is first converted to an acylating agent, which is subsequently reacted with taurine to form TUDCA. The UDCA can be converted to various acylating agents suitable for the claimed reaction, as taught generally by Morrison & Boyd, Organic Chemistry, 6th Edition (Benjamin-Cummings Publishing Company) and John Welch, Organic Chemistry, Synthesis, in Encyclopedia of Physical Science and Technology (Third Edition), 2003. Common acylating agents to which the UDCA can be converted, which are suitable for the production of amides like TUDCA, are acid anhydrides (including alkoxycarbonyl mixed anhydrides and phosphonic anhydrides), N- hydroxysuccinimidyl esters, N-hydroxybenzotriazole esters, imidazolides, phenyl esters, and acyl halides (a / k / a acid halides).

[0056] The reaction with UDCA proceeds according to the following pathway:

[0057] UDCA Acylating Agent TUDCA

[0058] (X = leaving group)

[0059] Table 1 lists suitable reagents and exemplifying references for converting UDCA to an acylating agent, although these reagents are in no way meant to be limiting, but simply exemplary of the numerous chemical pathways well-known to those of skill in the art: Table 1

[0060] TPP = triphenylphosphine; NBS = N-bromosuccinimide; Im = imidazol-l-yl; Et = ethyl; Me = methyl; iPr = isopropyl; Bt = benzotriazol- 1-yl; Su = succinimid-l-yl; Py = pyrrolidin-l-yl where Reference 1 is Montalbetti and Falque, Tetrahedron, 2005, vol. 61, p. 10827-10852, and Reference 2 is March’s Advanced Organic Chemistry, Smith and March, 6thEd., 2007, p. 1427- 1439.

[0061] Thus, in various subembodiments, the UDCA is converted to an acylating agent, which is subsequently reacted with taurine to form TUDCA or a salt thereof. In further embodiments, the UDCA is contacted with means for converting the 24-carboxylic acid to a derivative that can act as an acylating agent, and reacting the derivative with taurine to form TUDCA or a salt thereof. In these subembodiments, the structure corresponding to the means would be ethyl chloroformate, as specifically described in the examples.

[0062] Enzymatic Reaction Conditions

[0063] In some embodiments, the methods further comprise (a) enzymatically stereo-selectively reducing the 3 -ketone on the steroidal core (preferably of 3-KUDCA) to 3a-hydroxy; and / or (b) enzymatically stereo-selectively hydroxylating the 7-position on the steroidal core (preferably of 3-KCA) to 7P-hydroxy. These enzymatic methods are described in greater detail in the examples hereto and in WO / 2023 / 081658 (Published 11 May 2023) (International Application No. PCT / US2022 / 079081) (entitled High Purity Non-Animal Derived TUDCA) and WO / 2023 / 081657 (Published 11 May 2023) (International Application No. PCT / US2022 / 079080) (entitled High Purity Non-Animal Derived UDCA). The disclosures of WO / 2023 / 081658 and WO / 2023 / 081657 are expressly incorporated herein by reference.

[0064] Novel Salts and Crystal Forms

[0065] When any compound is referenced herein, either by itself, in combination with other ingredients, or in a chemical or biological process, it will be understood that the compound can be present in or used as an isolated form. By isolated form it is meant that the compound is preferably present as a solid, and that it is substantially free of any compounds other than the recited compound (i.e. < 10%, 5%, 3%, or 1% other compounds).

[0066] In one embodiment, the invention provides a t-butylamine salt of 3-KCA, preferably crystalline t-butylamine 3-KCA. A preferred TBA salt of 3-KCA has a purity greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8% relative to its 5a stereoisomer (i.e., considering the weight of 3-KCA and its 5a stereoisomer).

[0067] A preferred crystalline form exhibits 3, 5, 10, or more of the characteristic peaks in its X- ray powder diffractogram (XRPD) reported in the Examples and in the following Table A, preferably in terms of 2-theta, and preferably allowing for variability of ± 0.2° or 0.1°:

[0068] Table A In another embodiment, the t-butylamine salt has a crystalline powder x-ray diffraction pattern comprising at least three characteristic peaks, in terms of 2-theta, selected from the group consisting of 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°.

[0069] In another embodiment, the t-butylamine salt has a crystalline powder x-ray diffraction pattern comprising characteristic peaks, in terms of 2-theta, at 14.502 ± 0.2°, 14.925 ± 0.2°, and 18.103 ± 0.2°.

[0070] In another embodiment, the invention provides a cyclohexylamine salt of 3-KCA, preferably in crystalline form. A preferred cyclohexylamine salt of 3-KCA has a purity greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8% relative to its 5a stereoisomer (i.e., considering the weights of 3-KCA and its 5a stereoisomer).

[0071] A preferred crystalline form exhibits 3, 5, 10, or more of the characteristic peaks reported in its X-ray powder diffractogram (XRPD) in the Examples and in the following Table B, preferably in terms of 2-theta, and preferably allowing for variability of ± 0.2° or 0.1°:

[0072] Table B

[0073] In one embodiment, the cyclohexylamine salt has a crystalline powder x-ray diffraction pattern comprising at least three peaks, in terms of 2-theta, 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°.

[0074] In one embodiment, the cyclohexylamine salt has a crystalline powder x-ray diffraction pattern comprising characteristic peaks, in terms of 2-theta, at 12.907 ± 0.2°, 16.170 ± 0.2°, and 16.974 ± 0.2°. In another embodiment, the invention provides a t-butylamine salt of 3-KUDCA, preferably crystalline t-butylamine 3-KUDCA. A preferred TBA salt of 3-KUDCA has a purity greater than 90%, 95%, 98%, 99%, 99.5%, or 99.8% when considering the weights of 3-KUDCA and its impurities.

[0075] A preferred crystalline form exhibits 3, 5, 10, or more of the characteristic peaks in its X- ray powder diffractogram (XRPD) reported in the Examples and in the following Table C, preferably in terms of 2-theta, and preferably allowing for variability of ± 0.2° or 0.1°:

[0076] Table C

[0077] In another embodiment, the t-butylamine salt has a crystalline powder x-ray diffraction pattern comprising at least three characteristic peaks, in terms of 2-theta, selected from the group consisting of 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°. In another embodiment, the t-butylamine salt has a crystalline powder x-ray diffraction pattern comprising characteristic peaks, in terms of 2-theta, at 11.498 ± 0.2°, 19.002 ± 0.2°, 13.002 ± 0.2°.

[0078] The methods can also be used to produce UDCA and TUDCA having the purities described in WO / 2023 / 081658 (Published 11 May 2023) (International Application No. PCT / US2022 / 079081) (entitled High Purity Non-Animal Derived TUDCA) and WO / 2023 / 081657 (Published 11 May 2023) (International Application No. PCT / US2022 / 079080) (entitled High Purity Non-Animal Derived UDCA), and according to the methods described in the foregoing patent publications. The disclosures of WO / 2023 / 081658 and WO / 2023 / 081657 are expressly incorporated herein by reference.

[0079] In one embodiment, the invention provides UDCA or a salt thereof, produced by the methods of the current invention. In another embodiment, the invention provides UDCA or a salt thereof, produced by the methods of the current invention, comprising: greater than 95%, 98%, 99%, 99.5%, or 99.8% 5P-stereochemistry, optionally at a 3a,7P-isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.

[0080] In one embodiment, the invention provides TUDCA, or a salt thereof, produced by the methods of the current invention. In another embodiment, the invention provides TUDCA, or a salt thereof, produced by the methods of the current invention comprising: greater than 95%, 98%, 99%, 99.5%, or 99.8% 5p-stereochemistry, optionally at a 3a,7p-isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.

[0081] Source of Starting Materials

[0082] The source of starting materials for the 3-KCA, 3-KUDCA, UDCA, and TUDCA may be a steroid, such as cholesterol, stigmasterol, campesterol, and sitosterol or mixtures of all of them, preferably sitosterol. Preferably, the source of starting materials will be a plant phytosterol such as sitosterol, stigmasterol, campesterol, and brassicasterol or a mixture thereof. In one embodiment, the phytosterols are mainly of soybean or tall oil origin.

[0083] The origin of the carbon atoms may be even further differentiated by measurement of the 813C value as disclosed e.g., in US 8,076,156 and “Stable Isotope Ratios as biomarkers of diet for health research” by D M. O'Brien, Annual Reviews (www.annualreviews.org), 2015. The 8-value appears as the13C is measured in relation to a standard being Pee Dee Belemnite based on a Cretaceous marine fossil, which had an anomalously high13C. Some biochemical reactions discriminate against13C, which is why the concentration of12C is increased in some biological materials. In this manner, different sources such as plant versus animal may be distinguished using the pure compounds as reference values as described in Application Note 30276 from Thermo Scientific: “Detection of Squalene and Squalane Origin with Flash Elemental Analyzer and Delta V Isotope Ratio Mass Spectrometer” by Guibert et al. (2013).

[0084] Isotope ratios are conveniently quantified in parts per mil (%o) in what is called the 8 notation. Specifically, 613C = (Rsampie / Rstandard - 1) x 1,000 where Rsampie is the13C / 12C isotope ratio of the sample and Rstandard is 0.0112372, which is based on the standard Vienna PeeDee Belemnite (VPDB) value. Thus, 1 unit of13C represents a change of ~1 in the fifth decimal place of the13C / 12C isotope ratio. Further discussion of the technique can be found, for example, in R.N. Zare et al., High-precision optical measurements of13C / 12C isotope ratios in organic compounds at natural abundance. 10928-10932, PNAS luly 7, 2009, vol. 106 no. 27.

[0085] The 813C values may also differ among plants due to their different photosynthetic physiology. This may be observed in C3 plants such as wheat, rice, beans, most fruits, and vegetables which exhibit a higher 813C value than C4 plants such as corn, sugar cane, and sorghum (“Stable Isotope Ratios as biomarkers of diet for health research” by D.M. O'Brien, Annual Reviews (www.annualreviews.org), 2015).

[0086] In one embodiment, the 3-KCA, 3-KUDCA, UDCA, or TUDCA show a 813C value that is different from the 813C value of 3-KCA, 3-KUDCA, UDCA, or TUDCA obtained from animal sources. In a further embodiment, the 3-KCA, 3-KUDCA, UDCA, or TUDCA show a 813C value that is different from the 813C value of 3-KCA, 3-KUDCA, UDCA, and TUDCA obtained from mammal sources.

[0087] The 3-KCA, 3-KUDCA, UDCA, or TUDCA carbons preferably are derived predominantly from plant sources, with only a minor amount (if any) of carbons derived from non-plant sources. Thus, in various preferred embodiments the carbons in the 3-KCA, 3-KUDCA, UDCA, or TUDCA s comprise greater than 80% plant derived carbons, with the remainder derived from non-plant sources. More particularly, the carbons in the steroidal rings are preferably 100% derived from plant sources, while part or all of any appended moieties such as taurine may be derived from nonplant sources.

[0088] ADDITIONAL EMBODIMENTS Embodiment 1) A method of making a 5p steroid substantially free of 5a isomeric impurities comprising selectively crystallizing a t-butylamine or cyclohexylamine salt of 3-KCA from a solution comprising 3-KCA and its 5a isomer.

[0089] Embodiment 2) The method of embodiment 1, wherein the 3-KCA comprises a 813C value corresponding to a plant derived molecule, optionally comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 613C relative to VPDB.

[0090] Embodiment 3) The method of embodiment 1, comprising selectively crystallizing the t- butylamine salt of 3-KCA, optionally at an isomeric purity greater than 98%, 99%, 99.5%, or 99.8%.

[0091] Embodiment 4) The method of embodiment 1, comprising selectively crystallizing the t- butylamine salt of 3-KCA from a solution comprising a C1-C4 alcohol, optionally containing MTBE or water as cosolvents, optionally at an isomeric purity greater than 98%, 99%, 99.5%, or 99.8%.

[0092] Embodiment 5) The method of embodiment 1, comprising selectively crystallizing the t- butylamine salt of 3-KCA from a solution comprising isopropanol, optionally containing MTBE or water as cosolvents, optionally at an isomeric purity greater than 98%, 99%, 99.5%, or 99.8%.

[0093] Embodiment 6) The method of embodiments 3, 4, or 5, wherein the t-butylamine salt comprises a crystalline powder x-ray diffraction pattern comprising at least three characteristic peaks, in terms of 2-theta, selected from the group consisting of 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°.

[0094] Embodiment 7) The method of embodiment 1, comprising selectively crystallizing the cyclohexylamine salt of 3-KCA, optionally at an isomeric purity greater than 98%, 99%, 99.5%, or 99.8%.

[0095] Embodiment 8) The method of embodiment 1, comprising selectively crystallizing the cyclohexylamine salt of 3-KCA from a solution comprising a C1-C4 alcohol, optionally containing MTBE or water as cosolvents, optionally at an isomeric purity greater than 98%, 99%, 99.5%, or 99.8%.

[0096] Embodiment 9) The method of embodiment 1, comprising selectively crystallizing the cyclohexylamine salt of 3-KCA from a solution comprising isopropanol, optionally containing MTBE or water as cosolvents, optionally at an isomeric purity greater than 98%, 99%, 99.5%, or 99.8%. Embodiment 10) The method of embodiments 7, 8, or 9, wherein the cyclohexyl amine salt has a crystalline powder x-ray diffraction pattern comprising at least three peaks, in terms of 2- theta, 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°.

[0097] Embodiment 11) The method of embodiment 1, wherein the 3-KCA, or its cyclohexylamine or t-butylamine salt, comprises a steroidal core, further comprising, sequentially, simultaneously, or in any order: (a) stereo- selectively reducing the 3 -ketone on the steroidal core to 3a-hydroxy; and (b) stereo-selectively hydroxylating the 7-position on the steroidal core to 7P- hydroxy; thereby making UDCA or a salt thereof, optionally at a 3a,7P-isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.

[0098] Embodiment 12) The method of embodiment 1, wherein the 3-KCA, or its cyclohexyl amine or t-butylamine salt, comprises a steroidal core, further comprising, sequentially, simultaneously, or in any order: (a) in the presence of a 3a-ketoreductase, stereo-selectively reducing the 3-ketone on the steroidal core to 3a-hydroxy; and (b) in the presence of a 7P- hydroxylase, stereo-selectively hydroxylating the 7-position on the steroidal core to 7P-hydroxy; thereby making UDCA or a salt thereof, optionally at a 3a,7p-isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.

[0099] Embodiment 13) The method of embodiment 11 or 12, wherein the UDCA comprises a 24-carboxylic acid, further comprising, sequentially, simultaneously, or in any order: (a) converting the acid to a derivative that can act as an acylating agent; and (b) reacting the derivative with taurine to form TUDCA, optionally at a 3a,7p-isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.

[0100] Embodiment 14) The method of embodiment 11 or 12, wherein the UDCA comprises a 24-carboxylic acid, further comprising: (a) contacting the 24-carboxylic acid with means for converting the 24-carboxylic acid to a derivative that can act as an acylating agent; and (b) reacting the derivative with taurine to form TUDCA, optionally at a 3a,7P-isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.

[0101] Embodiment 15) The method of embodiment 14, wherein the means for converting is selected from the group consisting of SOCh, (COCI) , COCh, SOBr , TPP / NBS, Im2C0, EtOCOCl, (CH3)3CCOC1, (MeO)2P(O)Cl, Diisopropylcarbodiimide, Diisopropylcarbodiimide + HOBt, Diisopropylcarbodiimide + HOSu, 2-Chloro-l -methylpyridinium iodide, and [(Py)aPBr]+

[0102] [PF6]-.

[0103] Embodiment 16) The method of embodiment 1, wherein the solution comprises > 0.5, 1, 3, 5, 10, or 20% of the 5a isomer based on the total weight of the isomeric pair.

[0104] Embodiment 17) A t-butylamine salt of 3-KCA.

[0105] Embodiment 18) The t-butylamine salt of embodiment 17 having a crystalline powder x- ray diffraction pattern comprising at least three characteristic peaks, in terms of 2-theta, selected from the group consisting of 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°.

[0106] Embodiment 19) The t-butylamine salt of embodiment 17 having a crystalline powder x- ray diffraction pattern comprising characteristic peaks, in terms of 2-theta, at 14.502 ± 0.2°, 14.925 ± 0.2°, and 18.103 ± 0.2°.

[0107] Embodiment 20) The t-butylamine salt of embodiment 17, comprising a 813C value corresponding to a plant derived molecule, optionally comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 813C relative to VPDB.

[0108] Embodiment 21) A cyclohexylamine salt of 3-KCA.

[0109] Embodiment 22) The cyclohexylamine salt of embodiment 21 having a crystalline powder x-ray diffraction pattern comprising at least three peaks, in terms of 2-theta, 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°.

[0110] Embodiment 23) The cyclohexylamine salt of embodiment 21 having a crystalline powder x-ray diffraction pattern comprising characteristic peaks, in terms of 2-theta, at 12.907 ± 0.2°, 16.170 ± 0.2°, and 16.974 ± 0.2°.

[0111] Embodiment 24) The cyclohexylamine salt of embodiment 21, comprising a 813C value corresponding to a plant derived molecule, optionally comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o SBC relative to VPDB.

[0112] Embodiment 25) A method of making a 50 steroid substantially free of 5a isomeric impurities comprising selectively crystallizing a t-butylamine salt of 3-KUDCA from a solution comprising 3-KUDCA and one or more impurities. Embodiment 26) The method of embodiment 25, wherein the 3-KUDCA comprises a 813C value corresponding to a plant derived molecule, optionally comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 813C relative to VPDB.

[0113] Embodiment 27) The method of embodiment 25, comprising selectively crystallizing the t-butylamine salt of 3-KUDCA, optionally at purity greater than 98%, 99%, 99.5%, or 99.8%.

[0114] Embodiment 28) The method of embodiment 25, comprising selectively crystallizing the t-butylamine salt of 3-KUDCA from a solution comprising MTBE, n-butyl acetate, ethyl acetate, or a combination thereof, optionally at a purity greater than 98%, 99%, 99.5%, or 99.8%.

[0115] Embodiment 29) The method of embodiment 27, wherein the t-butylamine salt comprises a crystalline powder x-ray diffraction pattern comprising at least three characteristic peaks, in terms of 2-theta, selected from the group consisting of 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°.

[0116] Embodiment 30) The method of embodiment 25, wherein the 3-KUDCA, or its t- butylamine salt, comprises a steroidal core, further comprising stereo-selectively reducing the 3- ketone on the steroidal core to 3a-hydroxy, thereby making UDCA or a salt thereof, optionally at a 3a,7p-isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.

[0117] Embodiment 31) The method of embodiment 25, wherein the 3-KUDCA, or its t- butylamine salt, comprises a steroidal core, further comprising, in the presence of a 3a- ketoreductase, stereo-selectively reducing the 3-ketone on the steroidal core to 3a-hydroxy, thereby making UDCA or a salt thereof, optionally at a 3a,7P-isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.

[0118] Embodiment 32) The method of embodiment 30, wherein the UDCA comprises a 24- carboxylic acid, further comprising, sequentially, simultaneously, or in any order: (a) converting the acid to a derivative that can act as an acylating agent; and (b) reacting the derivative with taurine to form TUDCA, optionally at a 3a,7P-isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.

[0119] Embodiment 33) The method of embodiment 30, wherein the UDCA comprises a 24- carboxylic acid, further comprising: (a) contacting the 24-carboxylic acid with means for converting the 24-carboxylic acid to a derivative that can act as an acylating agent; and (b) reacting the derivative with taurine to form TUDCA, optionally at a 3a,7p-isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%. Embodiment 34) The method of embodiment 33, wherein the means for converting is selected from the group consisting of SOCh, (COCI) , COCh, SOBrz, TPP / NBS, Im2C0, EtOCOCl, (ClLQsCCOCl, (Me0)2P(0)Cl, Diisopropylcarbodiimide, Diisopropylcarbodiimide + HOBt, Diisopropylcarbodiimide + HOSu, 2-Chl oro-1 -methylpyridinium iodide, and [(Py)aPBr]+[PF6]-.

[0120] Embodiment 35) The method of embodiment 25, wherein the solution comprises > 0.5, 1, 3, 5, 10, or 20% of impurities.

[0121] Embodiment 36) A t-butylamine salt of 3-KUDCA.

[0122] Embodiment 37) The t-butylamine salt of embodiment 36 having a crystalline powder x- ray diffraction pattern comprising at least three characteristic peaks, in terms of 2-theta, selected from the group consisting of 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°.

[0123] Embodiment 38) The t-butylamine salt of embodiment 36 having a crystalline powder x- ray diffraction pattern comprising characteristic peaks, in terms of 2-theta, at 11.498 ± 0.2°, 19.002 ± 0.2°, 13.002 ± 0.2°.

[0124] Embodiment 39) The t-butylamine salt of embodiment 36, comprising a 813C value corresponding to a plant derived molecule, optionally comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 513C relative to VPDB.

[0125] EXAMPLES

[0126] In the following examples, efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc. , but some errors and deviations should be accounted for. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0127] Example 1

[0128] 3-KCA Cyclohexylamine Salt The cyclohexyl amine salt of 3-KCA (SHI -75) was made according to the following reaction scheme: mw = 374.57 mw = 486.74

[0129] Details of the experimental procedure and results are provided below:

[0130] Experimental procedure:

[0131] SHI-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. To this solution was added a solution of cyclohexylamine (0.58 g) in MTBE (20 ml) at 40-45 °C. The reaction mixture was stirred for 12 h at room temperature and then cooled to 5-10 °C. After being held at this temperature for 1 h, the slurry was filtered, and the solid was washed with MTBE (2 ml) and dried under vacuum to obtain 2 g of the cyclohexylamine salt of SHI-75 as a white solid. Yield = 77%.

[0132] Ratio of 5p:5a-isomer by HPLC = 89.4: 10.6.

[0133] Purification:

[0134] The above solid was transferred to a 50 ml round bottom flask and diluted with IPA (5 ml) and stirred for 1 h at room temperature. To this suspension was added water (10 ml) over a period of 0.5 h, the slurry was stirred for 2 h at RT. The mixture was then cooled to 5-10 °C, stirred at the same temperature for 0.5 h, and the solid was filtered. The wet cake was washed with cold water (2 ml), and dried under vacuum to obtain 1.6 g of SHI-75 cyclohexylamine salt as an off-white solid. Recovery = 80%.

[0135] Ratio of 5P:5a-isomer by HPLC = 93.7:6.3.

[0136] Example 2

[0137] 3-KCA t-Butylamine Salt

[0138] The t-butylamine salt of 3-KCA (SHI -75) was made according to the following reaction scheme: mw = 374.57 mw = 447.70

[0139] Details of the experimental procedure and results are provided below:

[0140] Experimental procedure:

[0141] SHI-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. To this solution was added a solution of t-butylamine (0.43 g) in MTBE (20 ml) at 40-45 °C. The reaction mixture was stirred for 2 h at room temperature and then cooled to 5- 10 °C. After being held at this temperature for 1 h, the slurry was fdtered, and the solid was washed with MTBE (2 ml) and dried under vacuum to obtain 1.8 g of the t- butylamine salt of SHI-75 as a white solid. Yield: 75%.

[0142] Ratio of 5P:5a-isomer by HPLC = 96.4:3.6

[0143] Purification:

[0144] 1. 95 g of SHI-75 TBA salt was suspended in IPA (238 ml) (2.5 vol.), stirred at RT for 1 h (a suspension was observed);

[0145] 2. To this mixture was added water (475 ml) (5 vol.) at RT;

[0146] 3. The slurry was stirred for 2 h at RT followed by 5-10 °C for 1 h and filtered the solid, the wet cake was washed with cold water (1 vol.) and dried under vacuum to obtain purified SHI-75 TBA salt as a white solid.

[0147] Yield: 66 g (69.5%).

[0148] Ratio of 5P:5a-isomer by HPLC (w / w%) = 99.70:0.30.

[0149] Example 3

[0150] 3-KUDCA t-Butylamine Salt

[0151] MW: 390.56

[0152] Experimental procedure:

[0153] To a 500 ml multineck round bottom flask equipped with a mechanical stirrer and addition funnel was charged 7P-hydroxy-3-ketocholanic acid (3-KUDCA, 14 g) in MTBE (140 ml). The mixture was stirred for 15 min at 25-28 °C to obtain a clear solution. To this solution was added a solution of TBA (4.12 ml, 1.1 equiv.) in MTBE (70 ml) over a period of 1 h. The resulting slurry was stirred for 1 h at 25-28 °C followed by another 1 h at 0-5 °C. The resulting solid was filtered, the wet cake was washed using MTBE (2 vol.), and the product was dried under vacuum at 40-45 °C for 12 h to obtain 3-KUDCA TBA salt (15 g) as a white solid. Yield: 90%.

[0154] Purification:

[0155] 3-KUDCA TBA salt (10 g) was suspended in water (50 ml) at 25-28 °C and isopropanol (IP A, 10 ml) was added. The resulting suspension was stirred for 2 h at the same temperature, and then the solid was isolated by filtration. The wet solid and washed with IPA (1 ml) and dried under vacuum at 40-45 °C for 12 h to obtain purified 3-KUDCA TBA salt as a white solid (5 g, 50% recovery). Melting point: 168.4 °C; 'H NMR (400 MHz, d6-DMSO): 8 3.9-5.8 (bs, 4H), 3.35 (m, 1H), 2.64 (t, J = 14 Hz, 1H), 2.32 (td, J = 14, 5 Hz, 1H), 2.08 (m, 1H), 1.15-2.00 (m, 22 H), 1.11 (s, 9H), 1.01 (m, 1H), 0.96 (s, 3H), 0.89 (d, J= 7 Hz, 3H), 0.65 (s, 3H).

[0156] Example 4

[0157] XRPD Analysis of 3-KCA Cyclohexylamine Salt

[0158] The cyclohexylamine salt of 3-KCA of Example 1 was analyzed by x-ray powder diffraction under the acquisition conditions in Table 2, to give the peak listing in Table 3 and pattern in Figure 1.

[0159] Table 2

[0160] Table 3

[0161] Example 5

[0162] XRPD Analysis of 3-KCA t-Butyl amine Salt

[0163] The t-Butylamine salt of 3-KCA of Example 2 was analyzed by x-ray powder diffraction under the acquisition conditions in Table 4, to give the peak listing in Table 5 and pattern in Figure 2.

[0164] Table 4

[0165] Table 5

[0166] Example 5

[0167] XRPD Analysis of 3-KUDCA t-Butylamine Salt

[0168] The t-Butylamine salt of 3-KUDCA of Example 3 was analyzed by x-ray powder diffraction under the acquisition conditions in Table 6, to give the peak listing in Table 7 and pattern in Figure 3.

[0169] Table 6

[0170] Table 7

[0171] Example 7

[0172] Preparation of 3-KCA from 3-KCA t-Butylamine Salt

[0173] Experimental Procedure

[0174] The t-butylamine salt of 3-KCA (500 g) was suspended in MTBE (3.5 L). 3N HC1 (0.5 L) was added at 25 °C. After stirring for ca. 30 min, 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 until a volume of ca. 2 L was reached. It was then heated to 40 °C, and n-heptane (4.0 L) was added over 50 min, resulting in the formation of a suspension. This suspension was cooled slowly to 0-5 °C, and then held at this temperature for 30 min and filtered. The wet cake was washed with n-heptane (0.5 L) and dried under vacuum to give 3-KCA as a pale yellow solid (315 g, 75% yield).

[0175] Ratio of 5P:5a-isomer by HPLC w / w%) = 99.9:0.1.

[0176] The same procedure can be used to convert the TBA salt of 3-KUDCA to 3-KUDCA.

[0177] Example 8

[0178] Preparation of UDCA from 3-KCA

[0179] 1. Pichia pastoris SAND121 is prepared as described in WO 2022 / 115710 A1, example 16, and used according to Example 19 in the same document.

[0180] 2. 3a-HSDH is 3a-Hydroxysteroid Dehydrogenase from Pseudomonas testosteroni (EC 1.1.1.50)

[0181] 3. GDH is D-Glucose Dehydrogenase from Bacillus megaterium (EC 1.1.1.47)

[0182] Hydroxylation of 3-KCA

[0183] Pichia pastoris SAND121 (from WO 2022 / 115710) is used to inoculate BMG medium, supplemented with 100 pg / ml nourseothricin and 100 pg / ml zeocin in an Erlenmeyer flask and incubated at 30°C, shaking at 250 RPM for 3 days. Aqueous 5-ALA solution (200 mM) and methanol containing 3-ketolithocholic acid (3-KCA) are added to the culture, then incubation is continued as before for 1 day. Methanol is added to the culture, then incubation is continued as before for 1 day. The mixture is acidified to pH 2-3 by addition of aq. HC1, and the resulting mixture is centrifuged. The solids are treated with acetone, and centrifugation is repeated. The supernatant is concentrated under vacuum, which after removal of most of the acetone provides a slurry. The solids are removed by filtration, and the product is washed with n-heptane and dried under vacuum to give 3-KUDCA as a solid.

[0184] Reduction of the 3 -ketone in 3-KUDCA

[0185] To a 250 ml single neck round bottom flask were added 3-KUDCA (1 g, 2.56 mmol), dextrose (1.3 g), P-NAD (33 mg) and 250 mM K2HPO4 buffer (70 ml) at room temperature. The mixture was stirred for 0.5 h to get a clear solution. The pH was adjusted to 7.8, and 3a-HSDH (66 mg, from Pseudomonas testosteroni, EC 1.1.1.50) and GDH (2 mg, from Bacillus megaterium, EC 1.1.1.47) were added. The resulting mixture was stirred for 20 h at room temperature, during which, the pH was maintained at 7.8 by the periodic addition of IM KOH solution. TLC analysis showed complete conversion of starting material.

[0186] The reaction mixture was quenched with 2N HC1 solution until the pH reached 3-3.5, and then the product was extracted in EtOAc (3 x 50 ml) and concentrated under reduced pressure to obtain UDCA as a white solid (900 mg). ’H NMR (500 MHz, MeOD): 5 3.44-3.53 (m, 2H), 2.30- 2.38 (m, 1H), 2.18-2.25 (m, 1H), 2.03 (dt, J = 6.5, 2.9 Hz, 1H), 1.95-1.79 (m, 5H), 1.56-0.92 (m, 26H), 0.71 (s, 3H).

[0187] Example 9 Synthesis of TUDCA from UDCA

[0188] UDCA (5 g) was charged to a 100 ml single neck round bottom flask. Acetone (30 ml) was added, resulting in 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 for 4 h at room temperature under N2 atmosphere. The reaction mixture was filtered to remove triethylamine hydrochloride, and the filtrate was added dropwise to an aqueous solution of taurine sodium salt (prepared by reacting 1.9 g taurine with 0.6 g NaOH in 3.7 ml water) at room temperature over a period of 20 minutes. The reaction was continued for another 1 h at room temperature, at which point TLC analysis showed complete conversion.

[0189] Cone. HC1 (1.5 ml), was added at room temperature to adjust the pH to ca. 1 . After stirring for 1 h, the resulting solid was filtered (0.9 g). The filtrate was diluted with acetone, stirred for 36 h at room temperature and the resulting solid was filtered, washed with acetone (10 ml) and dried under vacuum to obtain TUDCA as a white solid (5.1 g, 80% yield). 'H NMR (400 MHz, CD3OD): 8 3.62 (t, J= 6.8 Hz, 2H), 3.52-3.42 (m, 2H), 2.97 (t, J= 6.8 Hz, 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);13C NMR (100 MHz, CD3OD): 8 177.97, 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 for C26H45NO6S m / z 499.1 [M-H]+. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order 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 in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

CLAIMS1) A method of making a 5P steroid substantially free of 5a isomeric impurities comprising selectively crystallizing a t-butylamine or cyclohexylamine salt of 3-KCA from a solution comprising 3-KCA and its 5a isomer.2) The method of claim 1, wherein the 3-KCA comprises a 513C value corresponding to a plant derived molecule, optionally comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 813C relative to VPDB.3) The method of claim 1, comprising selectively crystallizing the t-butylamine salt of 3- KCA, optionally at an isomeric purity greater than 98%, 99%, 99.5%, or 99.8%.4) The method of claim 1, comprising selectively crystallizing the t-butylamine salt of 3-KCA from a solution comprising a C1-C4 alcohol, optionally containing MTBE or water as cosolvents, optionally at an isomeric purity greater than 98%, 99%, 99.5%, or 99.8%.5) The method of claim 1, comprising selectively crystallizing the t-butylamine salt of 3-KCA from a solution comprising isopropanol, optionally containing MTBE or water as cosolvents, optionally at an isomeric purity greater than 98%, 99%, 99.5%, or 99.8%.6) The method of claim 3, wherein the t-butylamine salt comprises a crystalline powder x-ray diffraction pattern comprising at least three characteristic peaks, in terms of 2-theta, selected from the group consisting of 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 at an isomeric purity greater than 98%, 99%, 99.5%, or 99.8%.8) The method of claim 1, comprising selectively crystallizing the cyclohexyl amine salt of 3- KCA from a solution comprising a C1-C4 alcohol, optionally containing MTBE or water as cosolvents, optionally at an isomeric 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 comprising isopropanol, optionally containing MTBE or water as cosolvents, optionally at an isomeric 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 peaks, in terms of 2-theta, 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°.11) The method of claim 1, wherein the 3-KCA, or its cyclohexylamine or t-butylamine salt, comprises a steroidal core, further comprising, sequentially, simultaneously, or in any order:(a) stereo-selectively reducing the 3-ketone on the steroidal core to 3a-hydroxy; and(b) stereo-selectively hydroxylating the 7-position on the steroidal core to 7P- hydroxy; thereby making UDCA or a salt thereof, optionally at a 3a,7 -isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.12) The method of claim 1, wherein the 3-KCA, or its cyclohexylamine or t-butylamine salt, comprises a steroidal core, further comprising, sequentially, simultaneously, or in any order:(a) in the presence of a 3a-ketoreductase, stereo-selectively reducing the 3-ketone on the steroidal core to 3a-hydroxy; and(b) in the presence of a 7P-hydroxylase, stereo-selectively hydroxylating the 7- position on the steroidal core to 7 -hydroxy; thereby making UDCA or a salt thereof, optionally at a 3a,7 -isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.13) The method of claim 11, wherein the UDCA comprises a 24-carboxylic acid, further comprising, sequentially, simultaneously, or in any order:(a) converting the acid to a derivative that can act as an acylating agent; and(b) reacting the derivative with taurine to form TUDCA, optionally at a 3a, 7P- isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.14) The method of claim 11, wherein the UDCA comprises a 24-carboxylic acid, further comprising:(a) contacting the 24-carboxylic acid with means for converting the 24-carboxylic acid to a derivative that can act as an acylating agent; and(b) reacting the derivative with taurine to form TUDCA, optionally at a 3a, 7P- isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.15) The method of claim 14, wherein the means for converting is selected from the group consisting of SOC12, (COC1)2, COC12, SOBr2, TPP / NBS, Im2CO, EtOCOCl,(CHs^CCOCl, (MeO)2P(O)Cl, Diisopropylcarbodiimide, Diisopropylcarbodiimide + HOBt, Diisopropylcarbodiimide + HOSu, 2-Chloro-l -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 5a isomer based on the total weight of the isomeric pair.17) A t-butylamine salt of 3-KCA.18) The t-butylamine salt of claim 17 having a crystalline powder x-ray diffraction pattern comprising at least three characteristic peaks, in terms of 2-theta, selected from the group consisting of 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 t-butylamine salt of claim 17 having a crystalline powder x-ray diffraction pattern comprising characteristic peaks, in terms of 2-theta, at 14.502 ± 0.2°, 14.925 ± 0.2°, and 18.103 ± 0.2°.20) The t-butylamine salt of claim 17, comprising a 813C value corresponding to a plant derived molecule, optionally comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 513C relative to VPDB.21) A cyclohexylamine salt of 3-KCA.22) The cyclohexylamine salt of claim 21 having a crystalline powder x-ray diffraction pattern comprising at least three peaks, in terms of 2-theta, 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°.23) The cyclohexylamine salt of claim 21 having a crystalline powder x-ray diffraction pattern comprising characteristic peaks, in terms of 2-theta, at 12.907 ± 0.2°, 16.170 ± 0.2°, and 16.974 ± 0.2°.24) The cyclohexylamine salt of claim 21, comprising a 813C value corresponding to a plant derived molecule, optionally comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 513C relative to VPDB.25) A method of making a 5p steroid substantially free of 5a isomeric impurities comprising selectively crystallizing a t-butylamine salt of 3-KUDCA from a solution comprising 3- KUDCA and one or more impurities.26) The method of claim 25, wherein the 3-KUDCA comprises a 813C value corresponding toa plant derived molecule, optionally comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 513C relative to VPDB.27) The method of claim 25, comprising selectively crystallizing the t-butylamine salt of 3- KUDCA, optionally at purity greater than 98%, 99%, 99.5%, or 99.8%.28) The method of claim 25, comprising selectively crystallizing the t-butylamine salt of 3- KUDCA from a solution comprising MTBE, n-butyl acetate, ethyl acetate, or a combination thereof, optionally at a purity greater than 98%, 99%, 99.5%, or 99.8%.29) The method of claim 27, wherein the t-butylamine salt comprises a crystalline powder x- ray diffraction pattern comprising at least three characteristic peaks, in terms of 2-theta, selected from the group consisting of 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 the 3-KUDCA, or its t-butylamine salt, comprises a steroidal core, further comprising stereo-selectively reducing the 3 -ketone on the steroidal core to 3a-hydroxy, thereby making UDCA or a salt thereof, optionally at a 3a,7P-isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.31) The method of claim 25, wherein the 3-KUDCA, or its t-butylamine salt, comprises a steroidal core, further comprising, in the presence of a 3 -ketoreductase, stereo-selectively reducing the 3 -ketone on the steroidal core to 3a-hydroxy, thereby making UDCA or a salt thereof, optionally at a 3a,7p-isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.32) The method of claim 30, wherein the UDCA comprises a 24-carboxylic acid, further comprising, sequentially, simultaneously, or in any order:(a) converting the acid to a derivative that can act as an acylating agent; and(b) reacting the derivative with taurine to form TUDCA, optionally at a 3a,7p- isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.33) The method of claim 30, wherein the UDCA comprises a 24-carboxylic acid, further comprising:(a) contacting the 24-carboxylic acid with means for converting the 24-carboxylic acid to a derivative that can act as an acylating agent; and(b) reacting the derivative with taurine to form TUDCA, optionally at a 3 ot^P- isomeric purity greater than 95%, 98%, 99%, 99.5%, or 99.8%.34) The method of claim 33, wherein the means for converting is selected from the group consisting of SOC12, (COC1)2, COCh, SOBr2, TPP / NBS, Im2CO, EtOCOCl, (CHs^CCOCl, (MeO)2P(O)Cl, Diisopropylcarbodiimide, Diisopropylcarbodiimide + HOBt, Diisopropylcarbodiimide + HOSu, 2-Chloro-l-methylpyridinium iodide, and [(Py)3PBr]+[PF6]-.35) The method of claim 25, wherein the solution comprises > 0.5, 1, 3, 5, 10, or 20% of impurities.36) A t-butylamine salt of 3-KUDCA.37) The t-butylamine salt of claim 36 having a crystalline powder x-ray diffraction pattern comprising at least three characteristic peaks, in terms of 2-theta, selected from the group consisting of 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 t-butylamine salt of claim 36 having a crystalline powder x-ray diffraction pattern comprising characteristic peaks, in terms of 2-theta, at 11.498 ± 0.2°, 19.002 ± 0.2°, 13.002 ± 0.2°.39) The t-butylamine salt of claim 36, comprising a 513C value corresponding to a plant derived molecule, optionally comprising less than -15%o, -17.5%o, -20%o, -22.5%o, or -25%o 8I 3C relative to VPDB.

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