NZ731924B2

NZ731924B2 - 6-alkyl-7-hydroxy-4-en-3-one steroids as intermediates for the production of steroidal fxr modulators

Info

Publication number: NZ731924B2
Application number: NZ731924A
Authority: NZ
Inventors: Rhys Batchelor; James Boydell; Zofia Komsta; Carl Otter; Laura Wallis; Alexander Weymouthwilson; Wilson Alexander Weymouth
Original assignee: NZP UK Limited
Priority date: 2014-11-19
Filing date: 2015-11-19
Publication date: 2021-03-19

Abstract

The invention relates to compounds of formula (I), wherein R1, R2, Y, R4 and R5 are as defined herein. The compounds are intermediates in the synthesis of synthetic bile acids.

Description

/053516 6—ALKYL—7-HYDROXYENONE STEROIDS AS INTERMEDIATES FOR THE PRODUCTION OF STEROIDAL FXR MODULATORS The t ion relates to compounds which are intermediates in the synthesis of bile acid derivatives with pharmacological activity. In particular, the invention relates to intermediates in the synthesis of obeticholic acid and its analogues. In addition, the invention relates to a method of sizing these intermediates and a method of preparing obeticholic acid and obeticholic acid analogues from the compounds of the invenﬁon.

Bile acids are steroid acids which are found in the bile of mammals and include compounds such as cholic acid, chenodeoxycholic acid, lithocholic acid and deoxycholic acid, all of which are found in humans. Many bile acids are l ligands of the farnesoid X receptor (FXR) which is expressed in the liver and intestine of mammals, including humans.

Bile acids are derivatives of steroids and are ed in the same way. The following shows the general numbering system for steroids and the numbering of the carbon atoms in chenodeoxycholic acid.

General d numbering CDCA numbering Agonists of FXR have been found to be of use in the ent of cholestatic liver disorders including primary biliary cirrhosis and non-alcoholic steatohepatitis (see review by Jonker et al, in Journal of Steroid Biochemistry & Molecular Biology, 2012, 130, 147- 158).

Ursodeoxycholic acid (UDCA), a bile acid originally isolated from the gall bladder of bears, is currently used in the treatment of cholestatic liver disorders, although it s to be inactive at the FXR.

As well as their action at the FXR, bile acids and their derivatives are also modulators of the G protein-coupled receptor TGR5. This is a member of the rhodopsin-like superfamily of G-protein coupled receptors and has an important role in the bile acid signalling network, which ments the role of the FXR.

Because of the importance of FXR and TGR5 agonists in the treatment of tatic liver disorders, efforts have been made to develop new compounds which have agonist activity at these receptors. One particularly active compound is obeticholic acid, which is a potent agonist of both FXR and TGR5. Obeticholic acid is described in WC 02/072598 and EP1568706, both of which describe a process for the ation of obeticholic acid from 7-keto lithocholic acid, which is derived from cholic acid. Further ses for the production of obeticholic acid and its derivatives are described in , US 2009/0062256 and and all of these processes also start from 7-keto lithocholic acid.

It is clear from the number of patent publications directed to processes for the production of obeticholic acid that it is by no means simple to synthesise this nd and indeed the process which is currently used starts from cholic acid, has 12 steps and a low overall yield.

In addition to the inefficiency and high cost of this s, there are also problems with the cost and bility of the starting materials. Cholic acid, the current starting material for the production of obeticholic acid, is a natural bile acid which is usually obtained from the slaughter of cows and other animals. This means that the availability of cholic acid and other bile acids is limited by the number of cattle available for slaughter. Since the incidence of cholestatic liver disease is increasing ide, the demand for synthetic bile acids such as obeticholic acid is also likely to increase and it is doubtful r the supply of naturally derived bile acids will continue to be sufficient to meet demand. 3O Furthermore, the use of a starting material derived from animals means that there is the possibility of the ination of the material with infectious agents such as viruses or prions, which can not only be hazardous to workers but could potentially contaminate the end products if steps are not taken to prevent this.

Although some ts with cholestatic liver disease can be treated with ursodeoxycholic acid, this is also a natural bile acid and faces the same problems of PCT/G82015/053516 limited availability and high cost.

In an attempt to solve the problems associated with the use of bile acids as starting materials, the t inventors have d a process for the synthesis of synthetic bile acid derivatives, such as obeticholic acid, which uses plant sterols as starting materials.

The ors have developed a process for the production of synthetic bile acids which proceeds via novel intermediates and which provides the final product in significantly higher yield than current processes. The s is flexible and can use a y of different starting materials including animal, fungal and plant sterols.

Suitable animal sterols which can be used as starting materials include deoxycholic acid, cholic acid, while fungal sterols include ergosterol.

Plant sterols are widely available at significantly lower cost than bile acids and, indeed, are often waste products of other processes. Suitable plant sterol and plant sterol derivatives which can be used as starting materials e bis-norcholenol (also known as 20-hydroxymethylpregnenone), androstenedione, tenedienedione, dehydroepiandrosterone, stigmasterol, brassicasterol, campesterol and B-sitosterol.

The present ion relates to intermediates in the novel process as well as to ses for preparing the intermediates and processes for converting them to the desired products.

Therefore, in a first aspect of the present invention there is provided a compound of general formula (I): wherein: R1 is 01.4 alkyl optionally substituted with one or more substituents selected from halo, OR6 or NR6R7; PCT/G82015/053516 where each of R6 and R7 is independently selected from H or 01.4 alkyl; R2 is H, halo or OH or a protected OH; Y is a bond or an alkylene, alkenylene or alkynylene linker group having from 1 to 20 carbon atoms and optionally substituted with one or more groups R3; each R3 is independently halo, OR8 or NR8R9; where each of R8 and R9 is independently ed from H or 01.4 alkyl; R4 is C(O)OR”, ”, C(O)NR”R”, OR”, OSi(R”)3, , 802R”, OSOzR”, SOsR”, or OSOsR”; where each R” and Rllis independently: a. hydrogen or b. (31.20 alkyl, C2.2o alkenyl, (32.20 alkynyl, -O-C1.2o alkyl, -O-C2.2o alkenyl or 02-20 alkynyl, any of which is optionally tuted with one or more substituents ed from halo, N02, ON, OR”, SR”, SOzR”, SOsR” or N(R”)2, or a 6- to 14— membered aryl or 5 to 14-membered heteroaryl group, either of which is optionally substituted with 01.5 alkyl, 01.5 haloalkyl, halo, N02, ON, OR”, SR”, SOzR”, 803ng or N(R”)2; or c. a 6- to 14- membered aryl or 5 to 14—membered heteroaryl group either of which is optionally substituted with one or more substituents selected from C1-6 alkyl, 01.5 haloalkyl, halo, N02, ON, OR”, SR”, SOzR”, SOsR” or N(R”)2; d. a polyethylene glycol residue; each R” is independently selected from H, 01.6 alkyl, 01.5 haloalkyl, or a 6- to 14- ed aryl or 5 to 14-membered heteroaryl group either of which is ally substituted with halo, 01.6 alkyl or CH, haloalkyl; each R” is independently a. C1.2o alkyl, C2.2o alkenyl or C2.2o alkynyl optionally substituted with one or more substituents selected from halo, N02, ON, OR”, SR”, SOzR19, SOsR” or 3O N(R”)2, a 6- to 14- membered aryl or 5 to 14-membered heteroaryl group, either of which is optionally substituted with 01.5 alkyl, C1-6 haloalkyl, halo, N02, ON, OR”, 802R”, 803R” or N(R”)2; or b. a 6- to 14- membered aryl or 5 to 14-membered heteroaryl group either of which is optionally substituted with one or more tuents selected from 01.5 alkyl, 01.6 haloalkyl, halo, N02, ON, OR”, SR”, SOzR”, SOgR” or N(R”)2; each R” is independently selected from H, 01.6 alkyl or C16 haloalkyl; R5 is H or OH or a protected OH; or a salt or an isotopic variant thereof.

Compounds of general formula (I) are intermediates in the synthesis of pharmaceutically active compounds such as obeticholic acid and its derivatives.

In the t specification, except where the context es otherwise due to express language or necessary implication, the word “comprises”, or ions such as “comprises” or “comprising” is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

In the present ation the term “01.20n alkyl refers to a straight or branched fully saturated hydrocarbon group having from 1 to 20 carbon atoms. The term encompasses methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl. Other alkyl , for example (3120 alkyl, 01.5 alkyl or 01.3 alkyl are as defined above but contain different numbers of carbon atoms.

The term “01.6 haloalkyl” refers to a straight or branched alkyl group as defined above having from 1 to 6 carbon atoms and substituted with one or more halo atoms, up to perhalo substitution. Examples include trifluoromethyl, chloroethyl and 1,1-difluoroethyl.

The term “C220 alkenyl” refers to a straight or branched arbon group having from 2 to 20 carbon atoms and at least one carbon-carbon double bond. es include ethenyl, propenyl, hexenyl etc.

The term “C220 alkynyl” refers to a ht or branched hydrocarbon group having from 2 to 20 carbon atoms and at least one carbon-carbon triple bond. Examples include ethynyl, propynyl, ynyl etc.

The term “alkylene” refers to a straight or ed fully saturated hydrocarbon chain.

Examples of alkylene groups include -CH2-, 2-, CH(CH3)-CH2-, CH2CH(CH3)-, - CH2CH20H2—, -CH2CH(CH2CH3)- and -CH2CH(CH2CH3)CH2-. 2015/053516 The term “alkenylene” refers to a straight or branched hydrocarbon chain containing at least one carbon-carbon double bond. Examples of alkenylene groups include -CH=CH-, CH3)-, -CH2CH=CH-, -CH=CHCH2-, CH2CH2CH=CH-, CH2CH=C(CH3)- and -CH2CH=C(CH2CH3)-.

The term “alkynylene” refers to a straight or branched hydrocarbon chain containing at least one carbon-carbon triple bond. Examples of alkenylene groups include -CEC-, -CH2CEC-, -CEC-CH2—, CH20H2CEC-, CH2CECCH2- and EC-CH2CHz-.

The terms “aryl” and “aromatic” refer to a cyclic group with aromatic ter having from 6 to 14 ring carbon atoms (unless ise specified) and containing up to three rings. Where an aryl group contains more than one ring, not all rings must be aromatic in character. Examples include phenyl, yl and anthracenyl as well as partially saturated systems such as tetrahydronaphthyl, indanyl and l.

The terms “heteroaryl” and “heteroaromatic” refer to a cyclic group with aromatic character having from 5 to 14 ring atoms (unless otherwise specified), at least one of which is a heteroatom selected from N, O and S, and containing up to three rings., Where a heteroaryl group contains more than one ring, not all rings must be aromatic in character. Examples of heteroaryl groups include pyridine, pyrimidine, indole, benzofuran, benzimidazole and indolene.

The term “halogen” refers to fluorine, chlorine, bromine or iodine and the term ”halo” to fluoro, chloro, bromo or iodo groups.

The term “isotopic variant” refers to isotopically-labelled compounds which are identical to those recited in formula (I) but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature, or in which the proportion of an atom having an atomic mass or mass number found less commonly in nature has been increased (the 3O latter t being referred to as “isotopic enrichment”). es of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, iodine and ne such as 2H (deuterium), 3H, 11C, 130, 14C, 18F, 123i or 125i (e.g. 3H, 11C, 14C, 18F, 123i or 125i), which may be naturally occurring or non-naturally occurring isotopes.

Polyethylene glycol (PEG) is a polyether compound, which in linear form has l formula H-[O CH2—CH2]n-OH. A polyethylene glycol residue is a PEG in which the terminal H is replaced by a bond g it to the der of the molecule.

Branched versions, including hyperbranched and dendritic versions are also plated and are generally known in the art. Typically, a branched polymer has a central branch core moiety and a plurality of linear polymer chains linked to the central branch core. PEG is commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, glycerol oligomers, rythritol and sorbitol. The central branch moiety can also be derived from l amino acids, such as lysine. The branched poly (ethylene glycol) can be represented in general form as R(—PEG-OH)m in which R is derived from a core moiety, such as glycerol, glycerol oligomers, or pentaerythritol, and m represents the number of arms. Multi-armed PEG molecules, such as those described in US 5,932,462; US 5,643,575; US 5,229,490; US 4,289,872; US 2003/0143596; WO 69; and WO 59 may also be used.

The PEG polymers may have an average molecular weight of, for example, 600- 2,000,000 Da, 60,000-2,000,000 Da, 40,000-2,000,000 Da, 0-1,600,000 Da, 800- 000 Da, 600-40,000 Da, 600-20,000 Da, 4,000-16,000 Da, or 8,000-12,000 Da.

The term “protected OH” relates to an OH group protected with any suitable protecting group. For example, the protected OH may be a group R4 as defined above.

Suitable protecting groups include esters such that, for example when R2 and/or R5 is a protected OH, R2 and/or R5 may independently be a group OC(O)R14, where R14 is a group R10 as d above. Silyl ethers are also suitable, and in this case, R2 andlor R5 may independently be a group OSi(R16)3, where each R16 is independently a group R13 as defined above.

Other suitable protecting groups for OH are well known to those of skill in the art (see Wuts, PGM and Greene, TVV (2006) “Greene’s tive Groups in Organic Synthesis”, 3O 41h Edition, John Wiley & Sons, Inc., Hoboken, NJ, USA).

References to a protecting group which is stable in basic conditions mean that the protecting group cannot be removed by ent with a base.

Appropriate salts of the compounds of general formula (I) include basic addition salts such as sodium, potassium, calcium, aluminium, zinc, magnesium and other metal salts as well as choline, diethanolamine, ethanolamine, ethyl diamine, meglumine and other well-known basic addition salts as summarised in Paulekuhn et al., J. Med. Chem. 2007, 50, 6665-6672 and/or known to those d in the art.

In some suitable compounds, the compound of general formula (I) is a compound of general formula (IA): (IA) wherein R1, R2, Y, R4 and R5 are as defined for general formula (I).

In some suitable compounds of general formula (I) and (IA): R1 is 01.4 alkyl optionally tuted with one or more substituents ed from halo, OR6 or NR6R7; where each of R6 and R7 is independently selected from H or 01.4 alkyl; R2 is H, halo or OH; Y is a bond or an ne or alkenylene linker group having from 1 to 6 carbon atoms and optionally substituted with one or more group R3; each R3 is independently halo, OR8 or NR8R9; where each of R8 and R9 is independently selected from H or C1.4 alkyl; R4 is C(O)OR1°, C(O)NR1°R“, °, SOzR‘O, OSOsz, 803R”, or OSO3R1O; or R4 is C(O)OR1°, C(O)NR‘°R“, S(O)R1°, 802R“), or OSOsz; where, in either case, each R10 is hydrogen or 01.5 alkyl or benzyl, either of which may optionally be substituted with one or more halo substituents and R11 is hydrogen or C1-6 alkyl, benzyl, -C1.4 ne-SOsH or -C1.4 alkylene-803(C1.4 alkyl), any of which may optionally be substituted with one or more halo substituents.

In suitable compounds of general formulae (I) and (IA), R1 may be 01.4 alkyl ally substituted with one or more substituents selected from halo, OR6 or NR6R7, where R5 and R7 are each independently H, methyl or ethyl, especially H or methyl.

More suitably, R1 is unsubstituted C14 alkyl.

PCT/G82015/053516 In ularly suitable compounds, R1 is ethyl.

In some compounds of general formula (I) and (IA), Y is a bond. ly in compounds of general ae (I) and (IA), Y is an alkylene or alkenylene linker group having from 1 to 15 carbon atoms, more suitably 1 to 12, 1 to 10 or 1 to 8 carbon atoms and optionally substituted with one or more groups R3 as defined above. lly each R3 is independently halo, OR8 or NR8R9; where each of R8 and R9 is independently selected from H, methyl or ethyl, especially H or methyl.

In some suitable compounds, Y is an unsubstituted alkylene or alkenylene linker having from 1 to 15 carbon atoms, more suitably 1 to 12, 1 to 10 or 1 to 8 carbon atoms.

In some suitable compounds of general formula (I), R2 is H.

In other suitable compounds of general formula (I), R2 is OH.

In still other suitable compounds of general formula (I), R2 is a protected OH group.

When R2 is a protected OH group, it may be a group which is not stable in a basic environment such that treatment with a base converts the protected OH group to OH.

Examples of such groups are well known in the art and include a group OC(O)R14 as defined above in which R14 is a group R10 as defined above for general formula (I).

Particularly suitable R14 groups are as defined for R10 below.

Alternatively, R2 may be a protected OH group which is stable in a basic environment.

Examples of such groups include OSi(R15)3, where each R16 is independently a group R"> as d above.

Particularly suitable R16 groups are as defined for R13 below.

In the compounds of general formulae (I) and (IA) R4 is 1°, OC(O)R1°, C(O)NR1°R11, OR”, 3)3, S(O)R1°, 802R“), OSOsz, 803R”, or OSOsRm.

Suitably, is C(O)OR1°, OR10,803R1°, or OSOst More suitably, R4 is C(O)OR1°, 803R“), or OSOst PCT/G82015/053516 Suitably, each R” and R11 is ndently: a. hydrogen or b. C140 alkyl, C240 alkenyl, C240 alkynyl, -O-C1—1o alkyl, -O-C2—1o alkenyl or -O-C2.1o l, any of which is optionally substituted with one or more substituents as described above;or c. a 6- to 10— membered aryl or 5 to 10-membered heteroaryl group optionally substituted with one or more substituents as described above. d. a polyethylene glycol residue.

More suitably, each R10 and R11 is ndently a. hydrogen or b. C140 alkyl, C240 alkenyl, C240 alkynyl or -O-C140 alkyl optionally substituted with one or more substituents as described above or c. a 6- to 10-membered aryl group optionally substituted with one or more substituents as described above.

Suitably each R” is ndently ed from: a. C140 alkyl, C240 alkenyl or C240 alkynyl optionally substituted with one or more substituents as described above; or b. a 6- to 10- membered aryl or 5 to 10-membered heteroaryl group optionally substituted with one or more substituents as described above.

More suitably, each R” is independently selected from: a. C140 alkyl, C240 l or C240 alkynyl optionally substituted with one or more substituents as described above; or b. a 6- to 10- membered aryl group optionally substituted with one or more tuents as described above. 3O Still more suitably, each R” is independently selected from C140 alkyl or phenyl, either of which is optionally substituted as described above.

Suitable substituents for alkyl, l, alkynyl, alkoxy, alkenyloxy and alkynyloxy R10 and R11 groups and alkyl, alkenyl and alkynyl R” groups include halo, N02, CN, OR”, SR”, SOzR”, 803ng or N(R”)2, or a 6- to 10- ed aryl or 5 to 14-membered heteroaryl group, either of which is optionally substituted with C1.s alkyl, C143 haloalkyl, halo, N02, CN, OR”, SOzR”, SOsR” or N(R19)2; where R” is as defined above.

More suitable substituents for these R10, R11 and R13 groups include halo, OR”, N(R19)2 or a 6- to bered aryl group optionally tuted as described above, more suitably optionally substituted with halo, C14 alkyl, 01.4 haloalkyl, 4 alkyl, -O-C1.4 haloalkyl, -4 alkyl) or -N(Cl.4 alkyl)2; for example fluoro, chloro, methyl, ethyl, trifluoromethyl, methoxy, ethoxy, trifluoromethoxy, amino, methyl amino and dimethylamino.

Suitable substituents for aryl and heteroaryl R10, R11 and R13 groups include 01.6 alkyl, C1. 6 haloalkyl, halo, N02, ON, OR”, SR19 or N(R19)2.

More suitable substituents for these R10, R11 and R13 groups include 01.4 alkyl, C1.4 haloalkyl, halo, OR19 or N(R19)2; in particular, halo, 01.4 alkyl, 01.4 haloalkyl, -O-C1.4 alkyl, - 0-0... haloalkyl, -NH(C1.4 alkyl) or -N(C1.4 alkyl)2.

Specific examples of substituents for aryl and heteroaryl R10, R11 and R13 groups include fluoro, chloro, methyl, ethyl, oromethyl, methoxy, ethoxy, trifluoromethoxy, amino, methyl amino and ylamino.

As set out above, each R19 is independently selected from H, 01.6 alkyl, 01-6 haloalkyl, or a 6- to 14- membered aryl or 5 to 14-membered heteroaryl group either of which is optionally substituted with one or more halo, 01.6 alkyl or 01.6 haloalkyl substituents.

Suitably, R19 is H, 01.6 alkyl, 01.6 haloalkyl, or a 6- to 10- membered aryl or 5 to 10- membered aryl group optionally substituted with one or more halo, (31.4 alkyl or C1. 4 haloalkyl substituents.

More suitably, R19 is H, 01.6 alkyl, 01.6 haloalkyl or phenyl optionally substituted with one or more halo, C1.4 alkyl or 01.4 kyl substituents. 3O Specific examples of R19 include H, methyl, ethyl, oromethyl or phenyl optionally tuted with one or more fluoro, chloro, methyl, ethyl or trifluoromethyl groups.

In some suitable compounds of general formula (I), R5 is H.

In other suitable compounds of general formula (I), R5 is OH.

In still other suitable compounds of general formula (I), R5 is a protected OH group.

PCT/G82015/053516 In still other suitable compounds of general formula (I), R5 is a protected OH group.

When R5 is a protected OH group, it may be a group which is not stable in a basic environment such that treatment with a base converts the protected OH group to OH.

Particularly suitable R14 groups are as d for R10 above.

Alternatively, R5 may be a protected OH group which is stable in a basic environment.

Examples of such groups e OSi(R‘5)3, where each R16 is independently a group R13 as defined above.

Particularly suitable R16 groups are as defined for R13 above.

In some le compounds of general formulae (I) and (IA), independently or in any combination: Y is a bond or an alkylene or alkenylene group having 1 to 3 carbon atoms and is optionally substituted with one or two R3 groups; R4 is C(O)OR1°, SOSRIO, or OSOSRIO, where R10 is as defined above but is more suitably H, 01.6 alkyl or ; and R5 is H or OH.

In some more suitable compounds, independently or in any combination: R1 is ethyl; and/or R2 is H; and/or Y is a bond, -CH2-, -CHzCH2-, -CH=CH- or-CH=C(CHs)-; and/or R4 is C(O)OR1°, where R10 is H, 01.6 alkyl or benzyl; and/or R5 is H.

In some particularly suitable nds of this type, R1 is ethyl and/or R10 is C1-6 alkyl or benzyl.

Particularly suitable compounds of the t ion include (6B, 70c, 22E)ethylhydroxyoxo-4,22—choladienoic acid; (SB, 7a)-6—ethylhydroxy—3-oxochoIenoic acid and 01.6 alkyl and benzyl esters thereof and salts thereof, especially the methyl and ethyl esters.

Compounds of general formula (I) and (IA) may be prepared from compounds of general formula (ll): 0 ., i n R2, R4, R5 and Y are as defined in general formula (I); by selective alkylation with an metallic reagent.

Suitable organometallic reagents include Gilman reagents formed by reaction of an alkyl lithium compound of formula : Rl-Li (XXIV) wherein R1 is as defined for general formula (I); and a copper (I) salt, particularly a copper (I) halide such as copper (I) iodide.

The reaction may be conducted in an organic t such as tetrahydrofuran, other ethers such as diethylether or a mixture f.

Alternatively, the on can be carried out using Grignard reagents RllVng, where R1 is as defined for general formula (I) and X is a halide, for example ethylmagnesium bromide and the reaction is suitably conducted in the presence of a zinc (ll) salt such as zinc chloride and a catalytic amount of a copper (I) or copper(ll) salt or complex, for example copper (I) chloride, copper (ll) de or a copper(l) or copper (ll) acetylacetonate (acac) complex.

The reaction may be carried out in an organic solvent, for example an ether such as THF, 2-methyl THF, methyl ted-butyl ether (tBME), diethyl ether. Surprisingly, the reaction temperature is not particularly significant and while in some cases the on may be d out at reduced temperature, for example at about —25 to 0 °C, it has also been successfully conducted at higher temperatures of up to about 55 °C.

WO 79517 The process for ing a compound of formula (I) from a compound of formula (II) is new and itself forms a part of the invention.

The method is particularly suitable for the preparation of compounds of general formula (I) in which R4 is 1O from compounds of general formula (II) where R4 is also C(O)OR‘°, where R10 is as defined above but is especially H, C1.6 alkyl or benzyl.

Alternatively, compounds of formula (I) can be prepared from other compounds of general formula (I). For example, a compound of general formula (I) in which R4 is C(O)OR10 may be converted to a compound of general a (I) in which R4 is 1°R“, S(O)R1°, 802R”, OSOsz, 803R”, or OSOsRlo.

Compounds of general formula (I) in which R4 is 803R1O may be synthesised from compounds of general formula (I) in which R4 is C(O)OH by the methods taught in W02008/002573, W02010/014836 and W02014/066819.

Thus a compound of formula (I) in which R4 is C(O)OH may be reacted with a C1.s alkanoyl or benzoyl chloride or with a C1.5 alkanoic ide to protect the OH .

The protected compound may then be reacted with a reducing agent such as a hydride, suitably lithium aluminium hydride or sodium borohydride in order to reduce the carboxylic acid group to OH. The alcohol group may be ed by a n, for example e or iodine, using the triphenyl phosphine/imidazole/halogen method described by Classon et al, J. Org. Chem, 1988, 53, 6126-6130. The halogenated compound may then be reacted with sodium sulphite in an alcoholic solvent to give a compound with a 803' Na+ substituent.

A compound of general formula (I) in which R4 is OSOsR1° can be obtained by reacting the alcohol obtained from reducing the protected carboxylic acid as described above with chlorosulfuric acid in the presence of a base such as triethylamine to yield the protected 3O triethylammonium salt. Protecting groups can be removed using base hydrolysis as described above. Reduction of the carboxylic acid followed by reaction of the resultant alcohol with chlorosulfurous acid yields a compound of general formula (I) in which R4 is OSOsz.

Compounds of general formula (I) in which R4 is C(O)NR1°R11 may be prepared from the ylic acid by reaction with an amine of formula H-NR‘OR11 in a suitable solvent with heating. Compounds of general formula (I) in which R4 is C(O)NR1°R11 or O may also be prepared by methods similar to those described by Festa et al, J. Med. Chem, 20933190.1:DCC - 11/26/2020 2014, 57, 495.

Compounds of general a (I) with other R4 groups may be prepared from the above compounds of general formula (I) by methods which are familiar to those of skill in the art.

These methods also form an aspect of the ion.

Compounds of general formula (II) may be prepared from compounds of formula (III): (III) wherein R2, R4, R5 and Y are as defined in l formula (I); by oxidation, for example using bis(monoperoxyphthalate) hexahydrate (MMPP) or 3-Chloroperoxybenzoic acid, (mCPBA).

The reaction using MMPP may be carried out in an organic solvent such as ethyl acetate and if mCPBA is used, the reaction may be carried out in a solvent such as dichloromethane or toluene. Suitably, the reacti on is conducted at or just below the reflux temperature of the solvent.

Compounds of general formula (III) may be prepared from nds of l formula (IV): (IV) wherein R2, R4, R5 and Y are as defined in general formula (I); by reaction with an oxidizing agent such as chloranil.

The reaction may be carried out under acidic conditions, for example in the presence of acetic acid, and in an organic solvent such as toluene. 20933190.1:DCC - 11/26/2020 Some compounds of general formulae (II), (III) and (IV) are known and, for example Uekawa et al in Biosci. Biotechnol. Biochem., 2004, 68, 1332-1337 describe the synthesis of (22E)oxo-4,22-choladienoic acid ethyl ester from stigmasterol followed by its sion to (22E)oxo-4,6,22-cholatrienoic acid ethyl ester, which has the formula: Uekawa et al then go on to describe the conversion of this compound to (6α, 7α, 22E)-6,7- epoxyoxo-4,22-choladienoic acid ethyl ester, a compound of general formula (II) in which R2 and R5 are H, Y is -CH=CH-, and R4 is C(O)OCH2CH3.

Other nds of general formulae (II), (III) and (IV) may be prepared by analogous methods from terols similar to stigmasterol.

Stigmasterol and other phytosterols are plant s and are readily available or may be prepared by known routes.

Compounds of general formula (IV) may also be prepared from compounds of general a (Va): (Va) wherein R2, R4, R5 and Y are as defined in general formula (I); by reaction with m bromide and a base such as lithium carbonate. The reaction may be carried out in a solvent such as N,N-dimethylformamide (DMF) and at a temperature of about 120 °C to 180 °C.

Compounds of general formula (Va) may be obtained by bromination of a compound of l formula (V): PCT/G82015/053516 R5 [I] Y_R4 wherein R2, R4, R5 and Y are as defined in general a (I); using, for example bromine in acetic acid.

Compounds of general formula (V) may be prepared from compounds of general formula (VI): Ho‘“ (VI) wherein R2, R4, R5 and Y are as d in general formula (I); by oxidation, typically with a chromium-based oxidizing agent or with sodium hypochlorite.

Compounds of general formula (VI) in which R4 is C(O)OR1°, where R10 is C1.s alkyl or benzyl may be prepared from compounds of general formula (VI) in which R4 is C(O)OH by esterification, typically by reaction with an appropriate alcohol under acidic conditions.

Compounds of general formula (VI) in which R4 is C(O)OH and R5 is H may be ed from compounds of general formula (VII): 12RO\" (VII) wherein R2 and Y are as d in general formula (I); R4 is C(O)OR1°, where R10 is C1.s alkyl or ; and R12 is a protected OH; by reaction with a reducing agent, typically hydrazine, under basic conditions and in an alcoholic or ic solvent, for example diethylene glycol.

PCT/G82015/053516 Where R12 is a protected OH group which is stable under basic conditions, the reaction may be followed by a reaction to remove the protecting group R12 to leave an OH group.

Protecting groups for OH are discussed above and, for example, R12 may be a group C(O)R‘4, where R14 is as defined above, in particular, 01.5 alkyl or benzyl. Silyl ethers are also suitable, and in this case, R2 and/or R5 may independently be a group Si(R16)3, where R16 is as defined above but is especially 01.6 alkyl or phenyl. Other suitable protecting groups for OH are well known to those of skill in the art (see Wuts, PGM and Greene, TW (2006) “Greene’s tive Groups in Organic Synthesis”, 4th Edition, John Vlﬁley & Sons, Inc., Hoboken, NJ, USA).

Particularly suitable R12 groups e groups which are not stable in the presence of a base since this removes the need for the additional step of removing the protecting group. An example of a group R12 which is not stable in basic ions is a group C(O)R14, where R14 is as defined above, and is ularly 01.6 alkyl or benzyl.

Alternatively, the reaction may be carried out in 2 steps such that the compound of general a (VII) is reacted with a compound of general formula (XXXII): RZO-NH-NHZ (XXXII) n R20 is a leaving group such as toluene sulfonyl or e sulfonyl; to give a compound of l formula (XXXIII): ZORI \N I,“ Y_R4 12R0‘“ (XXXIII) followed by reduction with a suitable reducing agent. Examples of reducing agents which can be used in this reaction include hydrides such as sodium borohydride, sodium cyanoborohydride, lithium aluminum hydride etc.

Compounds of l formula (VII) may be prepared from compounds of general formula (VIII): PCT/G82015/053516 ”Ro‘“ (VIII) wherein R2 and Y are as defined in general formula (I); R4 is C(O)OR1°, where R10 is C145 alkyl or benzyl; and R12 is as defined above, especially -C(O)C1.6 alkyl; by reaction with an oxidizing agent, for example sodium hypochlorite.

The reaction may be carried out under acidic conditions, for example in the presence of acetic acid, and in an organic t such as ethyl acetate.

Compounds of general formula (VIII) may be ed from compounds of general formula (IX): (IX) n R2 and Y are as defined in general formula (I); R4 is C(O)OR1°, where R10 is C1.e alkyl or benzyl; by reaction with an agent suitable to introduce the protecting group R”. For example, when R12 is 4, the compound of general formula (IX) may be reacted with a carboxylic acid anhydride or an acid chloride in the presence of a weak base such as pyridine, suitably catalysed by 4-dimethylaminopyridine (DMAP). The reaction may be conducted in a solvent such as ethyl acetate.

Compounds of general formula (IX) may be prepared by the esterification of compounds of general formula (X): (X) PCT/G82015/053516 wherein R2 and Y are as defined in general formula (I).

The reaction may be carried out by reacting the acid of general formula (X) with a suitable alcohol under acidic conditions.

Compounds of general formula (X) are known. For example, the compound of general formula (X) in which Y is —CH2CH2— and R2 is H is deoxycholic acid, which is readily available from a number of s.

Other bile acids with different values for Y and R2 can be used as ative starting materials.

An alternative route to compounds of general a (IV) is as shown in Scheme 1 in which androstenedione is converted to a compound of general formula (IV) in which R2 and R5 are H; R4 is -C(O)OCH3 and Y is either -CH2CH2— or -CH=CH-.

Scheme 1 O O EtPPh3Br / TsOH tBuOK EtOH THF A o EtO EtO Pelliccari et al, Steroids, 2012, 77, 250 COZMe / MeZAICI Methyl propiolate Dauben and Brookhart J. Am. Chem. Soc, 1981, 103, 237 Pd/BaSO4’ H2 COzMe COZMe and/or 0 O Marker et al, J. Am, Chem. Soc, 1940, 62, 2537 An alternative route to compounds of l formula (III) in which Y is an alkenylene group is by use of an ation on, for example a Horner—Wadsworth-Emmons (HWE) olefination of a nd of l formula (XI): (XI) wherein R2 and R5 are as defined for general formula (I); using a compound of general formula (XII): (I? o 1Oreo—[P OR1O 1ORO (XII) wherein R10 is as defined for general formula (I).

The reaction may be carried out under standard HWE conditions, for example using a base such as sodium hydride.

Compounds of general formula (XII) are y available or may be prepared by methods known to those of skill in the art.

Other olefination reactions, such as Tebbe olefination, Wittig reaction or a Julia- Kocienski olefination, would also give rise to compounds of general formula (III) in which Y is an alkenylene group. These olefination reactions are familiar to a chemist of skill in the art.

Compounds of general formula (XI) may be prepared by reaction of a compound of general formula (XIII) with ozone (XIII) wherein R2 and R5 are as defined for general formula (I) and R15 is C1-6 alkyl.

PCT/G82015/053516 An example of a on of this type is given in US 2,624,748.

Compounds of general formula (XIII) may be prepared by reaction of a nd of general formula (XIV): (XIV) wherein R2 and R5 are as d for general formula (I) and R15 is C1.s alkyl with an acid in a solvent such as methanol. nds of general formula (XIV) may be prepared by oxidation of a compound of general formula (XV): (XV) wherein R2 and R5 are as defined for general formula (I) and R15 is C1.e alkyl using an Oppenauer oxidation.

Examples of the conversion of compounds of general formula (XV) to compounds of general formula (XIII) are taught by Shepherd et al, J. Am. Chem. Soc. 1955, 77, 1212- 1215 and Goldstein, J. Med. Chem. 1996, 39, 5092-5099.

One example of a compound of general formula (XV) is ergosterol, which is a fungal sterol and Scheme 2 below shows the conversion of ergosterol to a compound of general a (III) in which both R2 and R5 are H, Y is CH=CH2 and R4 is C(O)OR1°, where R10 is ethyl.

PCT/G82015/053516 Scheme 2 Goldstein et al Oppenauer HO 0 oxidation J. Med. Chem, 1996, 39, 5092 Ergos erot I Shepherd et al conc. HCI J. Am. Chem. 500., 1955, 77, 1212 MeOH Ozone O O . . Levin and McIntosh, US 2,624,748 olefInatIon 002Et As with the compounds of general formula (I), compounds of general formulae (II) to (X), (Va) and (XXXIII) in which R4 is °, C(O)NR1°R“, S(O)R1°, 803R“), or OSOeR1O may be prepared from the corresponding compounds in which R4 is C(O)OR1O by reaction with an appropriate ts using s well known to those of skill in the art. For example, the methods described in /002573 and W02010/014836 or methods similar to those described by Classon et al, J. Org. Chem, 1988, 53, 6126- 6130 and Festa etal, J. Med. Chem, 2014, 57, 8477—8495.

Compounds of general formula (I) are intermediates in the synthesis of compounds of general a (XVIII): R581 a. Y1-R4 Ho‘“ "OH (XVIII) wherein R1 and R4 are as defined in general formula (I); R2 is H, halo or OH; R53 is H or OH; and Y1 is a bond or an alkylene linker group having from 1 to 20 carbon atoms and optionally substituted with one or more group R3; wherein R3 is as defined for general formula (I).

The compounds of general formula (I) may be converted to compounds of general formula (XVIII) in a 4 step process via intermediates of general formulae (XIX), (XX) and (XXI) as described below.

Therefore, in a further aspect of the invention there is provided a process for the preparation of a compound of general formula (XVIII), the s comprising: i. reducing a compound of general formula (I) using a suitable reducing agent to give a compound of general a (XIX): (XIX) wherein R1, R2, R4 and R5 are as defined in l formula (I); and Y1 is as defined in general formula (XVIII); ii. ing the compound of general formula (XIX) using a suitable ing agent to give a compound of general formula (XX): (XX) wherein R1, R2, R4 and R5 are as defined in general formula (I); and Y1 is as defined in general formula (XVIII); iii. epimerisation of the compound of general formula (XX) to give a compound of general formula (XXI): R5b I" Y1-R4 ; O (XXI) wherein R1 and R4 are as defined in general a (I); Y1 is as defined in general formula ); R2 is H, halo or OH or a protected OH group which is stable under basic conditions; and R5b is H or OH or a protected OH group which is stable under basic conditions; and (iv) reduction of the compound of general formula (XXI) using a suitable reducing agent and, where R2 and/or R5b is a ted OH, l of the protecting group(s), to give a compound of general formula (XVIII) as defined above, wherein removal of the protecting group can take place before or after the reduction; and optionally (v) conversion of a compound of general formula (XVIII) to r compound of general formula (XVIII).

Compounds of general formula (XVIII) are potent agonists of FXR and TGR5 and include obeticholic acid, which is a compound of formula (XVIII) in which R1 is ethyl, R2 and R551 are both H, Y1 is -CHzCH2-, and R4 is .

In the compounds of general formulae (XVIII) to (XXI), more suitable values for R1 and R4 are as defined for general formula (I).

In some compounds of general formulae (XVIII) to (XXI), Y1 is a bond.

In other nds of general formulae (XVIII) to (XXI), Y1 is an alkylene linker group having from 1 to 15 carbon atoms, more suitably 1 to 12, 1 to 10 or 1 to 8 carbon atoms and optionally substituted with one or more groups R3 as defined above. Typically each R3 is independently halo, OR8 or NRSRQ; where each of R8 and R9 is independently selected from H, methyl or ethyl, especially H or methyl.

In some suitable compounds of general formulae (XVIII) to (XXI), Y1 is an tituted alkylene or lene linker having from 1 to 15 carbon atoms, more suitably 1 to 12, 1 RECTIFIEDSHEET (RULE 91) ISA/EP PCT/G82015/053516 to 10 or 1 to 8 carbon atoms.

In step (i) above, the conversion of the compound of general formula (I) to the compound of general formula (XIX) may be d out by hydrogenation, usually catalytic hydrogenation. le catalysts for the catalytic hydrogenation include a palladiumfcarbon, palladium/calcium carbonate, palladium/aluminium oxide, platinum/palladium or Raney nickel catalyst. The reaction may be carried out in an organic solvent, which may be an alcoholic t such as methanol, ethanol or isopropanol; ethyl acetate; pyridine; acetic acid; cyclopentyl methyl ether (CPM E) or N,N- dimethylformamide (DMF). The organic solvent may optionally be mixed with a co- solvent such as e or water and/or a base such as triethylamine may also be added.

The choice of catalyst and solvent affects the ratio of the required product of general formula (XIX): (XIX) to its isomer of general formula (XXX): (XXX) It also affects the rate of sion of the intermediate of formula (XXXI): R5 Y1-R4 0 "’OH to the product.

More ly, a palladium/carbon or palladium/calcium carbonate catalyst is used.

Typically, in the catalyst the palladium is t in an amount of 5-10% by weight with respect to the weight of the matrix (where the matrix is the carbon, calcium carbonate etc).

Solvents which give superior ratios of (XIX): (XXX) include methanol, ethanol and DMF, particularly methanol and DMF.

When methanol is used as the solvent, it may be used alone or in the presence of a base such as triethylamine. Suitably, the amount of triethylamine used is a substoichiometric amount, typically 0.1 to 0.5 equivalents with respect to the amount of ng material of general formula (I).

Methanol in the presence of ylamine gave a ularly high ratio of the required t of general formula (XIX) to isomer of general formula (XXX).

Reactions conducted with methanol as the solvent may be d out at a temperature of about —30 to 25 °C and the temperature has little effect on the ratio of (XIX): (XXX).

When DMF is used as a solvent, it may be mixed with a co-solvent such as acetone, TBME, THF, acetonitrile or acetone/water. Optionally, the solvent contains a base such as triethylamine in a substoichiometric , lly 0.1 to 0.5 equivalents with respect to the amount of starting material of general formula (I).

Reactions conducted using DMF as solvent appear to be more sensitive to temperature than reactions carried out in methanol and the ratio of (XIX) : (XXX) decreases with sing temperature. Suitably, therefore the reaction is conducted at a temperature of 3O —30 to 0 °C, more suitably -20 to —10 °C.

It has been found that the pressure of hydrogen has little effect on the selectivity and therefore the hydrogen pressure is suitably about 1 atmosphere.

Similarly dilution does not appear to have a major impact on the selectivity and therefore the solvent may be used in any convenient amount.

Hydrogenation of a compound of a (I) will also reduce any alkene bonds, if present, in the linker Y.

In step (ii) of the process set out above, the oxidation reaction may be carried out using any suitable method. One suitable method is a Dess-Martin periodinane (1,1,1- triacetoxy—1,1-dihydro-1,2-benziodoxol) oxidation, which may be carried out in a nated solvent such as chloroform or dichloromethane at a temperature of about 15 to 25 °C, suitably at room temperature.

An alternative oxidation method is oxidation using a hypochlorite, for e sodium hypochlorite, under acidic ions, for e provided by acetic acid. The reaction may be carried out in an aqueous solvent and at a temperature of 0 to 15 °C, more usually at about 0 to 10 °C.

Other oxidation methods include a Jones reaction using sodium dichromate or, more usually, chromic trioxide in dilute sulfuric acid. This process is known to be reliable for the clean conversion of bile acid hydroxyl groups to the corresponding keto derivatives (Bortolini et al, J. Org. Chem, 2002, 67, 5802). Alternatively oxidation may be carried out using TEMPO ((2,2,6,6-TetramethyI-piperidiny|)oxy) or a derivative thereof.

The epimerisation reaction of step (iii), suitably comprises treating the compound of general formula (XX) with a base. The compound of general formula (XX) may be dissolved in an alcoholic solvent, optionally mixed with water and contacted with a base, for example sodium or ium hydroxide or a sodium or potassium de, lly an ethoxide.

In the case of compounds of l formula (XX) in which R4 is C(O)OR‘O, where R10 is 01.6 alkyl or benzyl and where a strong base such as sodium or potassium hydroxide is used, the epimerization reaction of step (iii) may be accompanied by hydrolysis to give a 3O compound of general a (XXI) in which R4 is C(O)OH. .

If, in the compound of general a (XX), R2 and/or R5 is a protected OH, for example a group R14, where R14 is as defined above but is especially 01.6 alkyl or benzyl, this will be removed during the epimerisation step to give a compound of general formula (XXI) in which R2 and/or R5b is OH. Other protected OH groups which are stable in basic conditions (for example a group OSi(R15)3 where each R16 is independently as defined above but is especially 01-6 alkyl or phenyl) may be removed before or after step (iv).

In step (iv), the reducing agent is lly a hydride, such as sodium dride which may be used in a solvent such as a e of tetrahydrofuran and water. Typically, this reaction is carried out under basic conditions, for example in the presence of a strong base such as sodium or potassium ide and at a temperature of about 0 to 110°C, more usually 60 to 100 °C. A compound of general formula (XVIII) in which R4 is C(O)OH may be produced by the ion of a compound of general formula (XXI) in which R4 is C(O)OH.

Compounds of general formulae (XVIII) to (XXI) in which R4 is C(O)R1°, 1°R“, S(O)R1°, 802R”, or OSOzR1O may be prepared from the corresponding compounds in which R4 is C(O)OR1O by on with an appropriate ts using methods well known to those of skill in the art.

Compounds of general formulae (XVIII) to (XXI) in which R4 is 803R1O may be synthesised from compounds of general formulae (XVIII) to (XXI) in which R4 is C(O)OH by the methods taught in W02008l002573, W02010/014836 and W02014i066819.

Thus a compound of formula (I) in which R4 is C(O)OH may be reacted with a C1.e alkanoyl or benzoyl chloride or with a C14; ic anhydride to protect the OH groups.

The protected compound may then be reacted with a reducing agent such as a hydride, suitably sodium borohydride in order to reduce the carboxylic acid group to OH. The alcohol group may be replaced by a halogen, for example bromine or iodine, using the triphenyl phosphine/imidazole/halogen method described by Classon et al, J. Org.

Chem, 1988, 53, 6126-6130. The halogenated compound may then be reacted with sodium sulphite in an alcoholic solvent to give a compound with a 803' Na+ substituent. nds of l formulae (XVIII) to (XXI) in which R4 is OSOeR1O can be obtained by reacting the alcohol ed from reducing the protected carboxylic acid with chlorosulfuric acid in the presence of a base such as triethylamine to yield the protected 3O triethylammonium salt. Protecting groups can be removed using base hydrolysis as described above. Reduction of the carboxylic acid followed by reaction of the resultant alcohol with chlorosulfurous acid yields a compound of general formulae (XVIII) to (XXI) in which R4 is 0803?”.

Compounds of general formulae (XVIII) to (XXI) in which R4 is 1°R11 may be prepared from the carboxylic acid by reaction with an amine of formula H-NR‘OR11 in a suitable solvent with heating. Compounds of general formulae (XVIII) to (XXI) in which R4 is 1°R11 or OSOgR1O may also be prepared by methods similar to those described by Festa et al, J. Med. Chem, 2014, 57 (20), 8477—8495. These s also form an aspect of the invention.

A compound of l formula (XVIII) to (XXI) in which R4 is C(O)R1O can be obtained by reduction of a compound in which R4 is C(O)OR1O using one equivalent of diisobutyl aluminium hydride (DIBAL) to obtain an aldehyde in which R4 is C(O)H (see, for example, W02011/014661).

Alternatively, the aldehyde may be prepared by oxidation of a protected compound in which R4 is OH prepared as described above. The oxidation may be Swern oxidation carried out using oxalyl de and dimethyl sulfoxide followed by triethylamine (see, for example Xiang-Dong Zhou et al, Tetrahedron, 2002, 58, 10299). Alternatively, the oxidation may be carried out using an oxidating agent such as pyridinium chlorochromate (PCC) as described by Carnell et a/ (J. Med. Chem, 2007, 50, 2700- 2707).

A compound of general formula (I) in which R4 is C(O)R1O where R10 is other than hydrogen can be obtained by known s, for example by the reaction of the aldehyde in which R4 is C(O)H with a suitable rd reagent, followed by oxidation.

Such methods are well known to those of skill in the art.

The invention will now be described in greater detail with reference to the examples.

In the examples, the following abbreviations were used: AcOH Acetic acid CPME Cyclopentyl methyl ether DMF N,N-dimethylformamide EtOAc Ethyl acetate EtOH Ethanol IPA Isopropyl alcohol MeOH Methanol NEts Triethylamine nBuOAc n—butyl acetate TBME t—butyl methyl ether 2015/053516 THF Tetrahydrofuran TLC Thin layer tography Exam les1to4—S nthesis of 6 5 7d eth lh drox oxo-cho|an-24—oic acid ethyl ester from Stigmasterol.

Example 1 — sis of (22E)oxo-4,6,22-cholatrienoic acid ethyl ester COzEt The starting al, (22E)—3-oxo—4,22-choladienoic acid ethyl ester, was prepared from stigmasterol according to the method described by Uekawa et a/ in Biosci, hnol, Biochem, 2004, 68, 1332-1337. (22E)oxo-4,22-choladienoic acid ethyl ester (1.00 kg, 2.509 mol; 1 eq) was charged to a reaction vessel, ed by AcOH (3 vol, 3.0 L) and toluene (1 vol, 1.0 L) with stirring. Chloranil (0.68 kg, 2.766 mol; 1.1 eq) was then charged and the reaction mixture heated to 100 °C and maintained at this temperature for 1-2 h (IPC by TLC on silica, eluent 3:7 EtOAc : Heptane; Starting Material: Rf 0.50, Product: Rf 0.46; visualise with anisaldehyde . The mixture was then cooled in an ice/water bath to 10 °C and the resulting solid was filtered off. The filter-cake was washed with premixed 3:1 AcOH : Toluene (4 x 0.5 vol) at 5 °C 1r 4 °C and the filtrate concentrated in vacuo at up to 70 °C.

The residue was dissolved in acetone (3 vol), then 3% w/w aq. NaOH (10 vol) was charged dropwise with stirring, maintaining the temperature below 30 °C (exothermic).

The resulting suspension was cooled to 10-15 °C and stirred for 30 mins. The solids were collected by filtration and the filter cake was washed with premixed 1:1 acetone : water (1 x 2 vol then 3 x 1 vol). The filter cake (tan solid) was dried under vacuum at 70- 75 °C, 672 g (68% yield). Characterisation of the compound agrees with the data published in the literature.

Example 2 — (6a., 7a., 22E)-6,7-epoxyoxo-4,22-choladienoic acid ethyl ester 0 i To a solution of (22E)—3—oxo-4,6,22-cho|atrien-24—oic acid ethyl ester (58.0 g, 146.3 mmol) in EtOAc (1.0 L) at reflux was added 80% MMPP (magnesium bis(monoperoxyphthalate) hexahydrate, 197.0 g, ca. 318.6 mmol) in four equal portions at 30 min intervals. The suspension was vigorously stirred at reflux for 5 h and at ambient temperature for a further 16 h. The reaction was then heated to reflux and stirred for an additional 6 h. The mixture was cooled to ca. 50 °C and the solids were filtered and rinsed with hot EtOAc (200 mL). The te was subsequently washed with 20% aq. NaHSOs (100 mL), 1M aq. NaOH (100 mL then 200 mL) and 10% aq. NaCl (250 mL), dried over Na2804, filtered and concentrated in vacuo. The e (yellow solid) was crystallised from m volume of EtOAc at 60 °C to give the e product as off white/pale yellow crystals (25.7 g, 43% yield, prisms). Characterisation of the compound agrees with the data published in the literature.

Example 3 — Synthesis of (613, 7a., 22E)ethylhydroxyoxo-4,22-choladien oic acid ethyl ester 002a Method 1: To a suspension of Cul (1.40 g, 7.35 mmol) in diethyl ether (10 mL), cooled to -78 °C under an argon blanket was charged EtLi (28.8 mL, 14.4 mmol, 0.5 M solution in benzene / cyclohexane). The thick white sion formed was allowed to warm to 0 °C, stirred for 5 mins (forming a dark solution) and cooled to -78 °C. A solution of (60c, 70c, ,7-epoxyoxo-4,22-choladienoic acid ethyl ester (1.00 g, 2.42 mmol) in diethyl ether / THF (24 mL, 3:1) was prepared and charged to the vessel containing the cuprate. THF (1 mL) was used to rinse the vessel that contained the solution of the epoxide and this was also charged to the organocuprate. The reaction mixture was allowed to warm to -4 °C over 30 mins after which time the reaction was complete by PCT/G82015/053516 TLC (silica, 1:1 EtOAc : heptane). After a further 30 mins of stirring at c.a. —4 °C a solution of aq. sat. NH4C| was charged and the mixture was d over 30 mins. The mixture was transferred to a separating funnel and the aqueous phase was removed, along with solid material present at the interface. The organic phase was washed with 5 wt % aq », (2 x 50 mL,.) and water (1 x 50 mL). TBME (50 mL) was used to t the original aqueous phase from the reaction and the combined . The combined organic phases were concentrated and the residue was purified by chromatography using silica (25 g) as the stationary phase (gradient elution with 0-30 % EtOAc in heptane) to give (60, 70c, 22E)ethylhydroxyoxo-4,22-choladienoic acid ethyl ester (0.63 g, 59 °/o). 1H NMR (400 MHz, CDClg): 5 = 8.82 (1H, dd, J = 15.8, 8.9, C22H), 5.75 (1H, s, C4H), .74 (1H, d, J = 15.8, 023H), 4.17 (2H, q, J = 7.1, OCH2CH3), 3.72 (1H, br s, C7H), .25 (5H, m), 2.05-1.98 (2H, m), 1.82-1.10 (23H, m), 0.91 (3H, t, J: 7.4, CH3), 0.77 (3H, s, CH3). 130 NMR (100 MHz, CDCIs): 5 = 199.2, 171.2, 187.1, 154.5, 128.4, 119.0, 71.9, 80.1, 55.3, 54.9, 49.9, 44.3, 42.7, 39.8, 39.1, 38.3, 37.4, 35.8, 34.0, 28.0, 28.3, 23.8, 20.8, 19.7, 19.2, 14.2, 12.8, 12.0; (IR) vmax(cm-1)z 3487, 2939, 2870, 1718, 1851, 1457, 1288, 1229, 1034; HRMS (ESl-TOF) m/z: (M+H)+ calcd for 028H4304 443.3181; found: 443.3158. mp = 59.4 - 82.9 °c Method 2 Zan (32.84 g, 240.9 mmol) was dried under vacuum with slow stirring at 180 °C for 2 h.

The flask was cooled to room temperature under an argon atmosphere and the residue was dissolved in THF (520 mL) and transferred via cannula into a three neck reaction flask equipped with mechanical r and temperature probe. The solution was cooled in an ice bath to 0-3 °C and a 3M solution of EtMgBr in Etzo (80 mL, 240.0 mmol) was added dropwise over 20 mins, maintaining the internal temperature below 10 °C.

Formation of a white precipitate (active zincate species) was observed after addition of ca. 1/3 of the Grignard solution. The mixture was stirred for 1.2 h at 0 °C before a solution of the epoxide (60c, 70c, 22E)—6,7-epoxyoxo-4,22-choladienoic acid ethyl ester (43.0 g, 104.2 mmol) in THF (300 mL) was added dropwise, maintaining the internal temperature below 10 °C. Solid CuCl (1.03 g, 0.104 mmol) was then added in two equal portions with vigorous stirring. After 10 mins the cooling bath was removed and stirring continued at ambient temperature for an additional 1.2 h. The reaction was ed by dropwise on of sat. aq. NH4C| (800 mL) at < 15 °C and stirred for 0.5 h. The mixture was filtered and the solid rinsed with TBME (150 mL). The phases were separated and the aqueous phase extracted with TBME 2><250 mL. The combined c extracts were washed with 10% aq. NaCl (2x200 mL), dried over Na2804, filtered and concentrated in vacuo to give 43.7 g of the crude (68, 70c, 22E)ethylhydroxy oxo-4,22-choladienoic acid ethyl ester as a yellow foam.

Method 3 To a solution of Zan in THF (0.5 M, 8.7 mL, 4.85 mmol, 0.9 eq) was charged ous THF (8.0 mL) and the contents then cooled to —25 °C. A solution of EtMgBr in TBME (1.0 M, 8.7 mL, 8.70 mmol, 1.8 eq) was added over 30 mins and the mixture stirred for 45 mins at —25 °C. Solid CuCl (24 mg, 0.49 mmol, 0.05 eq) was added in one portion and a solution of (601, 7d, 22E)-6,7-epoxyoxo-4,22-choladienoic acid ethyl ester (2.0 g, 4.85 mmol) in THF (8.0 mL) was added dropwise over 30 mins. The ing solid CuCl (24 mg, 0.49 mmol, 0.05 eq) was added half way through the addition of (60, 7d, 22E)—6,7-epoxyoxo-4,22-choladienoic acid ethyl ester. The on was d for 1 h at —25 °C, (TLC 1:1 Heptane2EtOAc, visualised by UV and developed using Ceric um Molybdate stain) and then additional of EtMgBr in TBME (1.0 M, 2.9 mL, 2.91 mmol, 0.6 eq) was added over 10 mins. The reaction was stirred for 0.5 h at —25 °C and then quenched by the addition of sat. aq. NH4C| (5 mL), maintaining the temperature below -5 °C. The inorganic salts were filtered off, rinsed with TBME and the filtrate phases were separated. The aqueous layer extracted with TBME and then the combined 3O organic extracts were washed with sat. aq. NH4C| (3 x 5 mL) and 10% brine (3 >< 6 mL).

The organic phase was concentrated in vacuo at 40 °C to give crude (63, 70c, 22E) ethylhydroxyoxo-4,22-choladienoic acid ethyl ester as a yellow foam (1.91 g).

Method 4 To a solution of Zan in THF (0.5 M, 8.7 mL, 4.85 mmol, 0.9 eq) was charged anhydrous THF ) and the contents then heated to 40 °C. A solution of EtMgBr in TBME (1.0 M, 8.7 mL, 8.70 mmol, 1.8 eq) was added over 30 mins and the mixture stirred for 45 mins at 40 °C. Solid CuCI (24 mg, 0.49 mmol, 0.05 eq) was added in one portion and a solution of (601, 7d, 22E)-6,7-epoxyoxo—4,22-choladienoic acid ethyl ester (2.0 g, 4.85 mmol) in THF (8.0 mL) was added dropwise over 30 mins. The remaining solid CuCI (24 mg, 0.49 mmol, 0.05 eq) was added half way through the addition of (60, 7d, 22E)-6,7-epoxyoxo-4,22-choladienoic acid ethyl ester. The reaction was stirred for 1 h at 40 °C, (TLC 1:1 ezEtOAc, visualised by UV and developed using Ceric Ammonium Molybdate stain) and then quenched by the dropwise addition of sat. aq.

NH4C| (5 mL). The nic salts were filtered off, rinsed with TBME and the filtrate phases were separated. The aqueous layer was extracted with TBME and then the combined organic extracts were washed with sat. aq. NH4C| (3 x 5 mL) and 10% brine (3 x 6 mL). The organic phase was concentrated in vacuo at 40 °C to give crude (68, 70c, 22 E)—6-ethylhydroxyoxo-4,22-choladienoic acid ethyl ester as a yellow foam (2.08 Method 5 To a solution of Zan in THF (0.5 M, 8.7 mL, 4.85 mmol, 0.9 eq) was charged anhydrous THF (8.0 mL) and the contents then cooled to -15 °C. A solution of EtMgBr in THF (1.0 M, 8.7 mL, 8.70 mmol, 1.8 eq) was added over 30 mins and the mixture d for 45 mins at -15 °C. Solid CuCI (24 mg, 0.49 mmol, 0.05 eq) was added in one portion and a solution of (601, 7d, ,7-epoxyoxo—4,22-choladienoic acid ethyl ester (2.0 g, 4.85 mmol) in THF (8.0 mL) was added dropwise over 30 mins. The remaining solid CuCI (24 mg, 0.49 mmol, 0.05 eq) was added half way through the on of (601, )—6,7-epoxyoxo-4,22-choladienoic acid ethyl ester. The reaction stirred for 1 h at -15 °C, (TLC 1:1 HeptanezEtOAc, visualised by UV and developed using Ceric Ammonium Molybdate stain) and then additional EtMgBr in THF (1.0 M, 4.35 mL, 4.36 mmol, 0.9 eq) was added over 15 mins and then ed by the dropwise addition of sat. aq. NH4C| (5 mL). The inorganic salts were filtered off, rinsed with TBME and the filtrate phases were separated. The aqueous phase was extracted with TBME and then 3O the combined organic extracts were washed with sat. aq. NH4C| (3 x 5 mL) and 10% brine (3 x 6 mL). The organic phase was concentrated in vacuo at 40 °C to give crude (68, 70c, 22E)ethylhydroxyoxo-4,22-choladienoic acid ethyl ester as a yellow foam (1.94 g).

Example 4 — Synthesis of (68, 513, 7a.)ethylhydroxyoxo-cholanoic acid ethyl ester COzEt Method 1 To a suspension of 10 wt. % Pd/C (50% wet, 20 mg, 8.6 mol%) in DMF (2 mL) was added a solution of (68, 70c, 22E)ethy|—7-hydroxyoxo-4,22-choladienoic acid ethyl ester (50 mg, 0.11 mmol) in DMF (3 mL) and the reaction mixture was cooled to 0 °C. The flask was evacuated then filled with hydrogen three times with vigorous stirring.

After 3 h the flask was evacuated then filled with argon and the mixture filtered via syringe filter. The mixture was partitioned between TBME (30 mL) and H20 (20 mL). The organic phase was dried 4) and concentrated in vacuo. The crude product (50 mg) was a 14:1 mixture of 58 to 50: isomers sed by 1H NMR) of (6B, 58, 700 ethylhydroxyoxo-cholanoic acid ethyl ester, yield 92%. 1H NMR (700 MHz, CDCIs): 6 = 4.12 (2H, q, J = 7.1, OCH2CH3), 3.71 (1H, br s, C7H), 3.34 (1H, dd, J = 15.5, 13.6, C4H), 2.39-2.32 (2H, m), 2.24-2.20 (1H, m), 2.14-2.09 (2H, m), .91 (4H, m), 1.83-1.79 (2H, m), 1.68-1.63 (2H, m), 1.58 (1H, s), 1.55-1.12 (19H, m), 1.04 (3H, s), 0.95-0.93 (6H, m), 0.88 (1H, J = 7.0), 0.71 (3H, s). 13C NMR (100 MHz, CDCIs): 6 = 213.5, 174.2, 72.1, 60.2, 55.9, 50.2, 49.8, 47.0, 46.7, 42.7, 39.5, 37.7, 36.3, 36.0, 35.7, 35.3, 34.2, 31.3, 31.0, 28.1, 27.7, 24.4, 23.8, 20.8, 18.3, 14.2, 13.9, 11.8. (IR) Vmax(cm'1):3514, 2939, 2870, 1710, 1462, 1377, 1159, 1099, 1032; HRMS (ESI-TOF) m/z: (M-H20+H)+ calcd for C28H4503 429.3369; found: 429.3363.

Method 2 (68, 70c, 22E)ethylhydroxyoxo-4,22-choladienoic acid ethyl ester (20.0 g) was dissolved in DMF (400 mL) and added under argon to solid 10 wt. % Pd/C (50% wet, 10.0 g). The mixture was cooled in an ice-salt bath to approximately -15 °C and the flask was evacuated then filled with hydrogen three times with vigorous ng. The mixture was stirred under an atmosphere of hydrogen for 6 h then the flask was evacuated, filled with argon and filtered through a pad of celite. The catalyst was rinsed with 400 mL of TBME. The filtrate was washed with 10% aq. NaCl (400 mL) and the 2015/053516 aqueous phase extracted with TBME (400 mL). The combined organic phases were washed with 10% aq. NaCl (3 x 200 mL), dried over Na2804, filtered and concentrated in vacuo to give crude (SB, SB, 70c)ethylhydroxyoxo-cholanoic acid ethyl ester (20.0 g, ca. 28:1 5HB:5Ha ratio) as pale yellow oil.

Method 3 % Pd/C was charged to a stainless steel jacketed reaction vessel under an argon atmosphere; DMF was added (20 mL), followed by a solution of crude (66, 70c, 22E) ethylhydroxyoxo-4,22-choladienoic acid ethyl ester from Example 3 ximately 72.6 mmol) in DMF (130 mL). The reaction mixture was cooled to —25 °C (over approximately 40 mins) with vigorous ng (1200 rpm). The reaction vessel was evacuated and charged with hydrogen (10—12 bar) three times. The mixture was stirred for 16 h under an atmosphere of hydrogen (10—12 bar). The vessel was ted, purged with argon and warmed to 20 °C with stirring. TLC of the reaction mixture (1:1 Heptane:EtOAc, developed using Ceric um Molybdate or vanillin dip, Rf values: starting material = 0.42, product = 0.67) indicated complete consumption of the starting material. The sion was diluted with CHSCN (120 mL) and H20 (30 mL) and the suspension filtered via a double GFA filter paper and the filter cake rinsed with CH3CN (60 mL). The mixture was telescoped to the next step without further purification. The mixture contained imately 5% of the 5H-or .

Optimisation The hydrogenation reaction of this example proceeds via the intermediate shown below and produces both the required SHB compound and its 5H0 . A solvent and catalyst screen was carried out to determine reaction conditions which led to the highest yield and the highest ratios of 5H6 isomer to 5H0 isomer.

COZEt COzEt Catalyst, H2 solvent intermediate The solvent screen was performed using 10 wt. % Pd/C catalyst and the reactions were run at room temperature under atmospheric pressure of hydrogen. The reaction run in MeOH in the presence of NEte, was more selective than the one run in neat MeOH, whilst 2015/053516 the addition of 10% of H20 decreased the 55H selectivity. The reaction in DMF ed the best [3201 ratio. The reaction in pyridine gave poor conversion to the required product with mainly starting material and intermediate present in the mixture.

Solvent 5H [3:01 ratio A MeOH 4:1 B MeC»+H20 2:1 C MeOHNEb 7:1 D EtOH 3: 1 E IPA 2:1 F EtOAc 2 : 1 G Pyridine 2 : 1 H AcOH 1:1 I CPME 1:1 J DMF 9 : 1 Reactions in DMF and MeOH were tested at a range of temperatures. For reactions run in DMF temperature has substantial impact on ivity (the selectivity decreases with increasing temperature), while little difference was observed for reactions in MeOH.

Reactions in DMF and MeOH were tested at a range of commercially available 5 and 10 wt. % Pd catalysts, on carbon, calcium carbonate, barium e and aluminium oxide suppon.

The reactions were run in 10 volumes of solvent at -15 °C under atmospheric pressure of hydrogen gas. For reactions run in DMF pressure has lower impact on the selectivity than the temperature. The effect of dilution on the ivity is negligible.

Exam les 5to14—S nthesis of 6 5 7d eth lh drox oxo-cholanoic acid ethyl ester from holic Acid Example 5 — sis of (3d, 56)—3-acetoxyoxo-cholanoic acid methyl ester COZMe A o‘“c To a solution of deoxycholic acid (500 g, 1.27 mol) in MeOH (1.5 L) was charged H2804 (0.68 mL, 12.7 mmol) and the on heated to 64 °C until complete. The reaction was cooled to 55 °C and pyridine (2.06mL, 25.4 mmol) was charged. MeOH (800 mL) was removed by distillation and the reaction cooled to 50 °C. EtOAc (500 mL) was charged and the distillation continued. This co-evaporation was repeated until the MeOH content was <0.5%. The reaction was cooled to 40 °C and EtOAc (1.0 L) was charged followed by Pyridine (134 mL, 1.65 mol) and DMAP (1.1 g, 8.89 mmol). Acetic anhydride (150 mL, 1.58 mmol) was added dropwise and the reaction vessel stirred at 40 °C until complete.

The reaction was cooled to 22 °C and 2M aq. H2804 (1500 mL) added maintaining the temperature below 25 °C. The aqueous phase was removed and the organic phase washed with water (1.2 L), sat. aq. NaHCOs solution (1.2 Lx 2) and water (1.2 L). AcOH (1.0 L) was charged to the c layer, ed by NaBr (6.6 g, 63.5 mmol). Aq. 16.4% NaOCI solution (958 mL, 2.54 mol) was charged dropwise maintaining the reaction temperature below 25 °C. The reaction was d until complete, then cooled to 10 °C and stirred for 90 mins. The resulting solids were collected by filtration, washed with water (3 x 500 mL) and the filter cake dried under vacuum at 40 °C. The solids were crystallised from MeOH (10 vol) to give (301, 5B)—3-acetoxyoxo-cholanoic acid methyl ester as an off white solid (268 g).

Example 6 — Synthesis of (3d, 56)—3-acetoxy-cholanoic acid methyl ester COZMe Aco“' (30, 5B)acetoxy—12-oxo-cholanoic acid methyl ester (268 g, 0.6 mol) was charged to the reaction vessel under argon, followed by AcOH (1.8 L). Tosyl hydrazide (190 g, 1.02 mol) was then added maintaining the reaction temperature at 25 °C. The reaction was stirred until complete and then NaBH4 (113.5 g, 3.00 mol) was charged portion-wise maintaining the temperature below 25 °C. The reaction mixture was stirred until complete and then quenched by the dropwise addition of water (1.34 L) ining the temperature below 25 °C. The reaction mixture was stirred for 30 mins, the resulting solids collected by tion, washed with water (3 x 270 mL) and the solid dried under vacuum at 40 °C. The solids were crystallised from MeOH (3 vol) to give (30, 56)—3- acetoxy-cholan-24—oic acid methyl ester as an off white solid g).

Example 7 — Synthesis of (3d, 53)—3-hydroxy-cholan-24—oic acid (Lithocholic Acid) COZH Ho‘“ To a solution of (3d, 5B)—3-acetoxy-cholanoic acid methyl ester (214.5 g, 0.50 mol) in IPA (536 mL) was charged water (536 mL) and 50% w/w NaOH (99 g, 1.24 mol). The reaction was heated to 50 °C and stirred until complete. 2M H2804 was charged slowly with vigorous stirring until pH 2-3 was obtained and then the reaction cooled to 20 °C.

The resulting solids were collected by filtration, washed with water (3 x 215 mL) and the ant solid dried under vacuum at 40 °C to give (301, 5B)—3-hydroxy-cholanoic acid (176.53 g) Example 8 - Synthesis of (58)—3-oxocholanoic acid ethyl ester COzEt To a solution of (30, 5B)—3-hydroxy—cholanoic acid (10 g, 26.5 mmol) in EtOH (50 mL) was charged H2804 96% (14 uL, 0.27 mmol) and the reaction mixture then heated to reflux for 16 h. Pyridine was then charged, the e stirred for 30 mins and concentrated in vacuo at 40 °C. The e was ved in EtOAc (30 mL) and AcOH (10 mL) and NaBr (136 mg, 1.33 mmol) was then charged. The solution was cooled to 5 °C and NaOCl 9% (27 mL, 39.8 mmol) was charged dropwise maintaining the 20933190.1:DCC - 11/26/2020 temperature below 10 °C. The resulting suspension was warmed to t temperature and stirred for 1 h. The reaction e was cooled to 0 °C for 10 mins, the solids collected by filtration and washed with water (3 x 3 vol). The resultant solid was dried under vacuum at 40 °C to give (5β)oxocholanoic acid ethyl ester (7.83 g). e 9 – Synthesis of (4α, 5β)oxobromo-cholanoic acid ethyl ester To a solution of (5β)oxocholanoic acid ethyl ester (8.0 g, 19.9 mmol) in AcOH (84 mL) was added Br2 in AcOH (16 mL, 21.9 mmol) dropwise over 15 mins. The reaction e was stirred for 10 mins, then diluted with EtOAc (250 mL), washed with water (2 x 200 mL) and concentrated in vacuo at 40 °C. The crude material was ed by column chromatography (30% Heptane: EtOAc) and concentrated in vacuo at 40 °C to give (4α, 5β)oxobromo-cholanoic acid ethyl ester as a pale crystalline solid (7.49g).

Example 10 – Synthesis of 3-oxocholenoic acid ethyl ester To a solution of (4α, 5β)oxobromo-cholanoic acid ethyl ester (4.0 g, 8.33 mmol) in DMF (40 mL) was charged Li2CO3 (4.0 g, 1 mass eq) and LiBr (2.0 g, 0.5 mass eq).

The mixture was heated to 150 °C for 2 h then allowed to cool to ambient temperature and poured onto a mixture of water and ice (200 g, 50 volumes) and AcOH (8 mL). The resulting suspension was stirred for 15 mins, the solids collected by filtration and then purified by column chromatography (30% Heptane: EtOAc) to give 3-oxocholenoic acid ethyl ester as a pale crystalline solid (1.68 g). 20933190.1:DCC - 11/26/2020 Example 11 - Synthesis of 3-oxo-4,6-choladienoic acid ethyl ester. 3-oxocholenoic acid ethyl ester (2.23 g, 5.57 mmol) was charged to a reaction vessel, followed by AcOH (6.7 mL) and toluene (2.23 mL). Chloranil (1.5 g, 6.13 mmol) was charged and the reaction mixture heated to 100 °C for 2 h (IPC by TLC, 3:7 EtOAc: Heptane; visualized with Anisaldehyde stain). The reaction mixture was cooled to 10 °C for 10 mins and the resulting solid removed by filtration. The filter cake was washed with DCM (9 vol) and the resulting filtrate then trated in vacuo at 40 °C. The residue was dissolved in acetone (9 vol) then 3% w/w aq. NaOH (27 vol) was added dropwise maintaining the temperature below 30 °C. The resulting mixture was cooled in an ice bath for 10 mins and the solids collected by filtration. The filter cake was washed with water (2 x 9 vol) and acetone: water 2:1 (4 vol). Purification by column chromatography (0-30% Heptane: EtOAc) gave 3-oxo-4,6-choladienoic acid ethyl ester as a pale lline solid (1.45 g) Example 12 – Synthesis of (6α, 7α)-6,7-epoxyoxocholenoic acid ethyl ester 3-oxo-4,6-choladienoic acid ethyl ester (1.37 g, 4.27 mmol) was charged to a reaction vessel, followed by BHT (23 mg, 0.13 mmol), EtOAc (11mL) and water (3.4 mL) with stirring. The solution was heated to 80 °C and then a solution of mCPBA 70% (1.5 g, 7.51 mmol) in EtOAc (7.5 mL) was added dropwise over 15 mins. The on mixture was d at 70 °C for 2 h (IPC by TLC, 3:7 EtOAc: Heptane; ized with Anisaldehyde stain), cooled to ambient temperature and then washed with 1M aq.NaOH (2 x 20 mL) followed by 10% aq. NaS2O3: 2% NaHCO 3 (3 x 20 mL). The organic phases were dried over Na2SO4 and concentrated in vacuo at 40 °C. The crude solids were crystalized from EtOAc (3 vol) at 60 °C to give an off white solid which was dried under vacuum at 40 °C to give (6α, 7α)-6,7-epoxyoxocholenoic acid ethyl ester (0.90 g). 20933190.1:DCC - 11/26/2020 Example 13 – Synthesis of )ethylhydroxyoxocholenoic acid ethyl ester ZnCl2 (600 mg, 4.25 mmol) was charged to a reaction vessel and dried under vacuum at 180 °C for 1 h. The reaction vessel was cooled to ambient temperature, THF (15 mL) charged and the contents of the reaction vessel cooled to 3 °C. A solution of 3M EtMgBr in Et2O (1.5 mL, 4.25 mmol) was charged to the reaction vessel over 40 mins maintaining the temperature below 5 °C. The reaction mixture was then stirred for 1 h. (6α, 7- epoxyoxocholenoic acid ethyl ester (0.80 g, 1.93 mmol) in THF (6 mL) was charged to the reaction vessel over 40 mins, maintaining the temperature below 5 °C.

CuCl (20 mg, 0.19 mmol) was charged in one portion and the reaction stirred at ambient temperature for 16 h (IPC by TLC, 3:7 EtOAc: Heptane; visualized with Anisaldehyde stain). The reaction mixture was cooled in an ice bath and sat. aq.NH4Cl was added dropwise, maintaining the temperature below 10 °C. The reaction mixture was filtered and the filter cake washed with TBME (12.5 vol). The organic phase of the filtrate was separated and the aqueous phase extracted with TBME (2 x 12.5 vol). The combined organic phases were washed with 5% NaCl (3 x 12.5 vol) and concentrated in vacuo at 40 °C.

Example 14 – Synthesis of (6β, 5β, 7α)ethylhydroxyoxo-cholanoic acid ethyl ester % Pd/C (70 mg) was charged to a reaction vessel under an argon here followed by the crude material from Example 13 in DMF (14.6 mL). The mixture was cooled to -10 °C and the on vessel was ted then filled with hydrogen three times with us stirring. The mixture was stirred under an atmosphere of hydrogen for 24 h while maintaining the temperature at -10 °C (IPC by TLC, eluent 1:1 EtOAc: Heptane; visualized with Anisaldehyde stain) then the flask was evacuated, filled with argon and filtered through a pad of celite and rinsed with DMF (7 mL). 10% Pd/C (70 mg) was recharged to the reaction vessel under an argon atmosphere ed by the DMF reaction mixture. The mixture was cooled to approximately -10 °C and the reaction vessel was evacuated then filled with hydrogen three times with vigorous stirring. The mixture was stirred under an atmosphere of hydrogen for 24 h at -10 °C (IPC by TLC, 1:1 EtOAc: Heptane; visualized with Anisaldehyde stain) then the flask was evacuated, filled with argon and filtered through a pad of celite and washed with TBME (62.5 vol, 50 mL).

The filtrate was washed with 10% aq. NaCl (4 x 25 vol), dried over Na2804, filtered and concentrated in vacuo at 40 °C. Purification by column chromatography (SiOz, 0-30% Heptane: EtOAc) gave (66, 5B, 7d)—6-ethylhydroxyoxo-cholanoic acid ethyl ester (0.17 g). The product was cal to the al obtained from plant origin (SB, 70:, 22E)ethylhydroxyoxo-4,22-choladienoic acid ethyl ester (see Example Examples 15 to 17 — Conversion of (65, 5E, 7d)—6-ethylhydroxyoxo-cholan oic acid ethyl ester to (30;, 5g, 60;, 7a.)ethyl-3,7-dihydroxy-cholanoic acid Example 15 — sis of (6p, 5ﬂ)—3,7-dioxoethyl-cho|anoic acid ethyl ester COZEt Method 1 A solution of Jones’s reagent prepared from Cr03 (1.10 g, 11 mmol) in H2804 (1.4 mL) and made to 5 mL with water was charged dropwise to a solution of (SB, 50, 70c)ethyl- 7—hydroxy—3-oxo-cholan-24—oic acid ethyl ester (0.18 g, 0.40 mmol) in acetone (10 mL) until an orange colour persisted. The reaction mixture was quenched with IPA (1 mL), filtered through a 0.45 pm nylon e filter and the filter was washed with acetone (10 mL). The combined filtrate and wash was concentrated, the residue was ved in EtOAc (20 mL) and washed with water (2 x 10 mL). The s phase was extracted with EtOAc (20 mL), the combined EtOAc phases were concentrated and the residue was dissolved and concentrated from toluene (20 mL) then acetone (20 mL) to give a clear oil containing (SB, 50, 70c)ethylhydroxy-3,7-dioxo-cholanoic acid ethyl ester (185 mg). 1H NMR (700 MHz, : 6 = 4.12 (2H, q, J = 7.1), 2.42 (1H, t, J = 11.4), 2.38-2.17 (6H, m), 2.09-1.74 (9H, m), 1.68-1.11 (17H, m), 0.93 (3H, d, J = 6.5), 0.85 (3H, t, J = 7.4), 0.72 (3H, s). 13c NMR (100 MHz, CDCIs): 5 = 214.5, 211.4, 174.0, 60.1, 57.1, 55.1, 50.3, 48.4, 47.3, 44.9, 43.6, 43.1, 39.2, 35.8, 35.2 (x2), 34.9, 31.3, 30.9, 28.1, 24.6, 23.7, 23.4, 21.7, 18.3, 14.2, 12.6, 12.2. (IR) vmax(cm'1): 2950, 2872, 1709, 1461, 1377, 1304, 1250, 1177, 1097, 1034;HRMS (ESI-TOF) m/z: (M+H)+ calcd for C28H4504 445.3318; found: 445.3312; Method 2 To a solution of (68, 58, 7a)ethylhydroxyoxo-cho|anoic acid ethyl ester (41.0 g crude mass) in ous CH20|2 (600 mL) at 0 °C was added solid DMP (34.0 g, 80.2 mmol) portion-wise over 20 mins (exothermic). The mixture was stirred at 0-5 °C for 2 h, then a further portion of DMP (4.0 g, 9.4 mmol) was added and reaction stirred at 0-5 °C for 1 h. The mixture was filtered through a GFA filter and the solid rinsed with CHzclz (50 mL), the filtrate was stirred usly with 10% aq. 3 and 2% aq. NaHCOs (100 mL) for 20 mins. The phases were separated and the aq. ted with CH2C|2 (2 X 100 mL). The combined organic extracts were washed with 1M NaOH (100 mL). The mixture was diluted with CH2C|2 (300 mL) and phases separated. The c layer was concentrated under reduced pressure and the residue (cloudy brown oil) was dissolved in TBME (600 mL) and washed with 1M NaOH (100 mL) and NaCl (3 X 100 mL). The organic phase was concentrated in vacuo to give a dark yellow runny oil, crude mass 38.1 g. The oil was dissolved in EtOH (400 mL) and stirred with activated charcoal (10 g) at 50 °C, the mixture was then filtered, the charcoal rinsed with EtOH (200 mL) and the filtrate concentrated in vacuo to give (68, 58)—3,7-dioxoethyI-cholanoic acid ethyl ester as a yellow oil (35.9 g).

Method 3 A solution of (68, 58, 70L)ethylhydroxyoxo-cholanoic acid ethyl ester (218 mmol) in DMF (450 ml), CHSCN (540 mL) and H20 (90 mL) was charged into a 2 L vessel and cooled to 9 °C, then AcOH (180 mL) was d, followed by NaBr (4.1 g).

A solution of sodium hypochlorite % w/v, 450 mL) was added dropwise over 1.5 h, maintaining the internal temperature at 5—6 °C, then the mixture was stirred for 5 h at 7 °C. TLC of the reaction e indicated complete consumption of the starting material (IPC by TLC, eluent EtOAc/heptane 3:7, Rf for (68, 58, 7a)ethylhydroxyoxo- cholanoic acid ethyl ester = 0.34; (68, 58)—3,7-dioxoethyl-choIanoic acid ethyl ester = 0.45). A solution of aq. 10% w/v Na2803 (360 mL) was charged dropwise with vigorous stirring, maintaining the internal temperature at 8—10 °C, then H20 (270 mL) was added dropwise and the mixture stirred at 5 °C for 16 h. The solid was filtered and washed with H20 (720 mL). The solid was then dissolved in TBME (1.1 L) and subsequently washed with an aq. NaHCOe, (300 mL) and 10% brine (300 mL). The organic phase was then stirred with activated al (10 g) for 20 mins at 40 °C, d with anhydrous MgSO4 (5 g) and filtered via GFA filter paper, the filter cake was rinsed with TBME (50 mL) and the filtrate concentrated in vacuo to give (63, 5B)—3,7- dioxoethyl-cholanoic acid ethyl ester as light brown oil which fies on ng (82.7 g).

Example 16 — Synthesis of (6a., 5ﬂ)-3,7-dioxoethyl-cholanoic acid COZH O . O Into a 500 mL flask was charged 0.5 vol of 0.5 M NaOH (9 mL) followed by (68, 513)—3,7- dioxoethyl-cholanoic acid ethyl ester from Example 15 (18.00 g, 1 eq) and then IPA (180 mL, 10 vol) (the initial NaOH charge was to avoid the possibility of C3-ketal formation). The mixture was warmed to 60 1r 2 °C and held until a solution was obtained (10-15 mins). The remaining 0.5 M NaOH solution (171 mL, 9.5 vol) was charged over mins and then the reaction was d for a further 3.5 h at 60 1r 2 °C. The IPA was removed under vacuum at 60 °C and then 2M HCI (8 mL) charged to pH 9. EtOAc was charged (90 mL, 5 vol) followed by 2M HCI (54 mL) to pH 1. Vigorous mixing was followed by phase separation. The aqueous phase was back extracted with additional EtOAc (90 mL, 5 vol) and then the combined organic phases were washed with water (54 mL, 3 vol), followed by three portions of 10% aq. NaCl (3 x 54 mL, 3 x 3 vol). The organic phase was treated with ted al (100 mesh powder, 3.37 g, ~0.20 mass eq) for 12 mins and then filtered through GFIB. Concentration at 50 °C in vacuo gave (60c, 5[3)-3,7-dioxoethyl-choIanoic acid as a light yellow foam in quantitative yield.

PCT/G82015/053516 1H NMR (700 MHz, CDCls): 5 = 2.74 (1H, dd, J = 12.8, 5.4), 2.47 (1H, t, J: 12.5), 2.43- 0.90 (32H, m), 0.81 (3H, t, J = 7.4), 0.70 (3H, s). 13c NMR (100 MHz, CDCIs): 5 = 212.1, 210.8, 179.4, 54.9, 52.4, 52.3, 50.0, 48.9, 43.7, 42.7, 38.9, 38.3, 38.7, 38.0, 35.5, 35.2, .9, 30.7, 28.2, 24.8, 22.9, 22.3, 18.8, 18.3, 12.1, 11.8. (IR) vmax(cm-1): 2939, 2873, 1708, 1458, 1382, 1284.8. HRMS (ESl-TOF) m/z: (M+H)+ calcd for Cst41o4 417.3005; found: 417.2997; mp = 71.2-75.9 °c e 17 — Synthesis of (3a., 5]}, 6a., -ethyl-3,7-dihydroxy-cholanoic acid CO2H To a solution of crude (60c, 5B)ethyl-3,7—dioxo-cholan-24—oic acid (21.7 g crude mass) in H20 (260 mL) and 50% NaOH (15.2 mL) at 90 °C was added, dropwise, a solution of NaBH4 (4.4 g, 116.3 mmol) in aq. NaOH (prepared from 25 mL of H20 and 0.8 mL 50% NaOH). The mixture was heated to reflux and stirred for 3 h. The mixture was then cooled to 60 °C and a 2M solution of HCI (200 mL) added dropwise with vigorous stirring. nBuOAc (100 mL) was then charged to the reaction flask and the mixture stirred for a further 20 mins. The phases were separated and the aqueous phase (pH = 1/2) extracted with nBuOAc (100 mL). The combined organic phases were washed with 2M HCI (50 mL) and 10% aq. NaCl (100 mL). The organic solvent was distilled off under d pressure at 70-80 °C. The residue (dense oil) was dissolved in nBuOAc (60 mL) at 70 °C and allowed to gradually cool to room temperature, then stored at 6 °C for 2 h.

The solid was collected via tion, rinsed with cold nBuOAc (20 mL), then dried under vacuum at 70 °C for 5h to give (30, 5B, 601, 7a)ethyl-3,7—dihydroxy-cholanoic acid as a white solid (8.2 g).

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Claims

1. A compound of general formula (I): R1 is C1-4 alkyl optionally substituted with one or more substituents selected from halo, OR6 or NR6R7; 10 where each of R6 and R7 is independently selected from H or C1-4 alkyl; R2 is H, halo or OH or a ted OH; Y is a bond or an alkylene, alkenylene or alkynylene linker group having from 1 to 20 carbon atoms and optionally substituted with one or more groups R3; each R3 is independently halo, OR8 or NR8R9; 15 where each of R8 and R9 is independently selected from H or C1-4 alkyl; R4 is C(O)OR10, OC(O)R10, C(O)NR10R11, OR10, OSi(R13)3, S(O)R10, SO2R10, OSO2R10, SO3R10, or OSO3R10; where each R10 and R11 is independently: 20 a. hydrogen or b. C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl, -O-C1-20 alkyl, -O-C2-20 alkenyl or -OC2-20 alkynyl, any of which is optionally substituted with one or more substituents selected from halo, NO2, CN, OR19, SR19, , SO3R19 or N(R19)2, or a 6- to 14- membered aryl or 5 to 14-membered heteroaryl group, either of which is 25 optionally tuted with C1-6 alkyl, C1-6 haloalkyl, halo, NO2, CN, OR19, SR19, SO2R19, SO3R19 or 2; or c. a 6- to 14- membered aryl or 5 to 14-membered heteroaryl group either of which is optionally substituted with one or more substituents selected from C1-6 alkyl, C1-6 haloalkyl, halo, NO2, CN, OR19, SR19, , SO3R19 or N(R19)2; C:\Interwoven\NRPortbl\DCC\SXD\19271372_1.docx-9/