FLUORINATED BILE ACIDS

Abstract

Compounds of general formula (1):

##STR00001##

wherein R.sup.1, R.sup.2 and R.sup.3 are as defined herein;
are of use in the treatment and prevention of neurodegenerative disorders including Alzheimer's disease and Parkinson's disease.

Claims

1. A compound of general formula (I): ##STR00095## wherein one of R.sup.1 and R.sup.2 is F, and the other of R.sup.1 and R.sup.2 is H or F; Y is selected from a bond, and a C.sub.1-20 alkylene, C.sub.2-20 alkenylene or C.sub.2-20 alkynylene linker group; R.sup.3 is selected from C(O)OR.sup.12, C(O)NR.sup.12R.sup.13, S(O).sub.2R.sup.12, OS(O).sub.2R.sup.12, S(O).sub.2OR.sup.12, OS(O).sub.2OR.sup.12, S(O).sub.2NR.sup.12R.sup.13, C(O)NR.sup.12S(O).sub.2R.sup.13, NHC(O)NR.sup.12S(O).sub.2R.sup.13, OP(O)(OR.sup.12).sub.2, C(O)NR.sup.12[CH(R.sup.15)].sub.nR.sup.16 and C(O)NR.sup.12C(O)CH.sub.2NR.sup.12[CH(R.sup.15)].sub.nR.sup.16; each R.sup.12 is independently selected from H and C.sub.1-6 alkyl optionally substituted by one or more substituents selected from halo, OR.sup.10, NR.sup.10R.sup.11, R.sup.16 and aryl; each R.sup.10 and R.sup.11 is independently selected from H and C.sub.1-6 alkyl; R.sup.13 is H, C.sub.1-6 alkyl optionally substituted by one or more substituents selected from halo and aryl; or a 3- to 8-membered carbocyclic ring or heterocyclic ring, wherein said carbocyclic or heterocyclic ring is optionally substituted with one or more substituents selected from ═O and R.sup.16; or a phenyl or 5- or 6-membered heteroaryl ring, wherein said phenyl or heteroaryl ring is optionally substituted with a substituent R.sup.16; or when R.sup.3 is C(O)NR.sup.12R.sup.13 or S(O).sub.2NR.sup.12R.sup.13, R.sup.12 and R.sup.13 together with the nitrogen atom to which they are attached form a 3- to 8-membered heterocyclic ring which optionally contains one or more further hetero atoms selected from N, O and S; and is optionally substituted with one or more substituents selected from CH.sub.2C(O)OH, C(O)OH, C.sub.1-6 alkyl, C(O)OC.sub.1-6 alkyl, S(O).sub.2OH, ═O and ═N—OH; and is optionally fused to a phenyl group unsubstituted or substituted with one or more substituents selected from halo and nitro; n is 1, 2 or 3; each R.sup.15 is independently selected from H and C.sub.1-6 alkyl optionally substituted by one or more substituents selected from halo, phenyl and 5- or 6-membered heteroaryl; a 3- to 8-membered cycloalkyl group; or a group R.sup.14, where R.sup.14 is a side chain of an amino acid; or when n is 2 or 3, two R.sup.15 groups together with the carbon atoms to which they are attached, and optionally an intervening carbon atom where present, can combine to form —(CH.sub.2).sub.p— such that the group [CH(R.sup.15)]n is a 3- to 8 membered carbocyclic ring; p is 1, 2, 3, 4, 5 or 6; R.sup.16 is selected from C(O)OH, S(O).sub.2OH, S(O).sub.2(C.sub.1-6 alkyl), OS(O).sub.2OH and P(O)(OH).sub.2; or a pharmaceutically acceptable salt or isotopic variant thereof.

2. (canceled)

3. A compound according to claim 1 which is a compound of general formula (IA), (IB), (IC) or (ID): ##STR00096## wherein R.sup.1, R.sup.2, Y and R.sup.3 are as defined above for general formula (I).

4. A compound according to claim 3 which is a compound of general formula (IA).

5. A compound according to claim 3 which is a compound of general formula (IB).

6. A compound according to claim 1, wherein both R.sup.1 and R.sup.2 are F.

7. (canceled)

8. A compound according to claim 1, wherein Y is —CH.sub.2CH.sub.2—.

9. (canceled)

10. A compound according to claim 1, wherein R.sup.12 is selected from H methyl, and ethyl, and is optionally substituted with R.sup.16 or N(R.sup.10)(R.sup.11); or R.sup.3 is selected from C(O)NR.sup.12S(O).sub.2R.sup.13, NHC(O)NR.sup.12S(O).sub.2R.sup.13, C(O)NR.sup.12[CH(R.sup.15)].sub.nR.sup.16, and C(O)NR.sup.12C(O)CH.sub.2NR.sup.12[CH(R.sup.15)].sub.nR.sup.16; and R.sup.12 is H or methyl; and/or R.sup.13 is a 5- or 6-membered carbocyclic ring or heterocyclic ring optionally substituted with R.sup.16 or ═O; or R.sup.13 is phenyl optionally substituted with R.sup.16; or R.sup.3 is selected from C(O)NR.sup.12R.sup.13 and S(O).sub.2NR.sup.12R.sup.13 and R.sup.12 and R.sup.13 together with the nitrogen atom to which they are attached form a 5- or 6-membered heterocyclic ring optionally substituted with one or more substituents selected from R.sup.16 and ═O and optionally comprising one or more further heteroatoms selected from O, N and S; and/or R.sup.16 is selected from C(O)OH, S(O).sub.2OH, S(O).sub.2(C.sub.1-6 alkyl), and OS(O).sub.2OH; or R.sup.16 is selected from C(O)OH, S(O).sub.2OH, OS(O).sub.2OH and P(O)(OH).sub.2, and the compound is in the form of a pharmaceutically acceptable salt.

11-12. (canceled)

13. A compound according to claim 1 wherein R.sup.3 is C(O)NR.sup.12[CH(R.sup.15)].sub.nR.sup.16 or a pharmaceutically acceptable salt thereof, wherein R.sup.12 is H, methyl or methyl substituted with R.sup.16; where R.sup.16 is C(O)OH, S(O).sub.2OH, S(O).sub.2(C.sub.1-6 alkyl) or OS(O).sub.2OH.

14-15. (canceled)

16. A compound according to claim 1, wherein: R.sup.3 is C(O)NR.sup.12CH(R.sup.14)C(O)OH, wherein R.sup.12 is H or methyl and R.sup.14 is H; or R.sup.3 is C(O)NR.sup.12CH(R.sup.15)CH(R.sup.15)S(O).sub.2OH, wherein R.sup.12 is H or methyl and both R.sup.15 moieties are H.

17. (canceled)

18. A compound according to claim 1, wherein R.sup.3 is C(O)NR.sup.12R.sup.13 or a pharmaceutically acceptable salt thereof.

19-20. (canceled)

21. A compound according to claim 1 selected from: 2β-fluorochenodeoxycholic acid (Compound 1); 2β-fluoro-3β,7α-dihydroxy-5β-cholanic acid (Compound 2); 2α-fluoro-3β,7α-dihydroxy-5β-cholanic acid (Compound 3); 2α-fluoro-3β,7β-dihydroxy-5β-cholanic acid (Compound 4); 2α-fluoro-3α,7α-dihydroxy-5β-cholanic acid (Compound 5); 2α-fluoro-3α,7β-dihydroxy-5β-cholanic acid (Compound 6); 2,2-difluoro-3β,7β-dihydroxy-5β-cholanic acid (Compound 7); 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (Compound 8); 2,2-difluoro-3α,7β-dihydroxy-5β-cholanic acid (Compound 9); N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-ethylsulfonic acid (Compound 10); N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-propanoic acid (Compound 11); N-(methyl),N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-acetic acid (Compound 12); N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-trans-2-cyclohexane carboxylic acid (Compound 13); 1-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-piperidine-3-carboxylic acid (Compound 14); 3-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-4-thiazolidine-carboxylic acid (Compound 15); N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-morpholine (Compound 16); N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-methylcarboxylic acid (Compound 17) N-(carboxymethyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-2-amino acetic acid (Compound 18); N-(methyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide) ethylsulfonic acid (Compound 19); 3-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl) amino-propanesulfonic acid (Compound 20); N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide) methanesulfonic acid (Compound 21); N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-2-aminoethyl sulfuric acid (Compound 22); O-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-2-hydroxy ethyl sulfonic acid (Compound 23); N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl) aniline-2-sulfonic acid (Compound 24); N-(cyclohexyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-3-amino-propanesulfonic acid (Compound 25); N-(cyclohexyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-2-amino-ethanesulfonic acid (Compound 26); N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl) 2-aminoethyl methyl sulfone (Compound 27); N-(ethyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-3-amino-tetrahydrothiophene dioxide (Compound 28); N-(2-(diisopropylamino)ethyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-3-amino-tetrahydrothiophene dioxide (Compound 29); N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-thiomorpholine-dioxide (Compound 30); N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl) 1,1-dioxidotetrahydro-2H-thiopyran-3-ylamine (Compound 31); and pharmaceutically acceptable salts thereof.

22-23. (canceled)

24. A method for the treatment of a neurodegenerative disorder, the method comprising administering to a patient in need of such treatment an effective amount of a compound according claim 1.

25. (canceled)

26. A method according to claim 24, wherein the neurodegenerative disorder is Parkinson's disease, and the compound is selected from the compounds of general formula (IA) and (ID); ##STR00097## wherein one of R.sup.1 and R.sup.2 is F, and the other of R.sup.1 and R.sup.2 is H or F; Y is selected from a bond, and a C.sub.1-20 alkylene, C.sub.2-20 alkenylene or C.sub.2-20 alkynylene linker group; R.sup.3 is selected from C(O)OR.sup.12, C(O)NR.sup.12R.sup.13, S(O).sub.2R.sup.12, OS(O).sub.2R.sup.12, S(O).sub.2OR.sup.12, OS(O).sub.2OR.sup.12, S(O).sub.2NR.sup.12R.sup.13, C(O)NR.sup.12S(O).sub.2R.sup.13, NHC(O)NR.sup.12S(O).sub.2R.sup.13, OP(O)(OR.sup.12).sub.2, C(O)NR.sup.12[CH(R.sup.15)].sub.nR.sup.16 and C(O)NR.sup.12C(O)CH.sub.2NR.sup.12[CH(R.sup.15)].sub.nR.sup.16; each R.sup.12 is independently selected from H and C.sub.1-6 alkyl optionally substituted by one or more substituents selected from halo, OR.sup.10, NR.sup.10R.sup.11, R.sup.16 and aryl; each R.sup.10 and R.sup.11 is independently selected from H and C.sub.1-6 alkyl; R.sup.13 is H, C.sub.1-6 alkyl optionally substituted by one or more substituents selected from halo and aryl; or a 3- to 8-membered carbocyclic ring or heterocyclic ring, wherein said carbocyclic or heterocyclic ring is optionally substituted with one or more substituents selected from ═O and R.sup.16; or a phenyl or 5- or 6-membered heteroaryl ring, wherein said phenyl or heteroaryl ring is optionally substituted with a substituent R.sup.16; or when R.sup.3 is C(O)NR.sup.12R.sup.13 or S(O).sub.2NR.sup.12R.sup.13, R.sup.12 and R.sup.13 together with the nitrogen atom to which they are attached form a 3- to 8-membered heterocyclic ring which optionally contains one or more further hetero atoms selected from N, O and S; and is optionally substituted with one or more substituents selected from CH.sub.2C(O)OH, C(O)OH, C.sub.1-6 alkyl, C(O)OC.sub.1-6 alkyl, S(O).sub.2OH, ═O and ═N—OH; and is optionally fused to a phenyl group unsubstituted or substituted with one or more substituents selected from halo and nitro; n is 1, 2 or 3; each R.sup.15 is independently selected from H and C.sub.1-6 alkyl optionally substituted by one or more substituents selected from halo, phenyl and 5- or 6-membered heteroaryl; a 3- to 8-membered cycloalkyl group; or a group R.sup.14, where R.sup.14 is a side chain of an amino acid; or when n is 2 or 3, two R.sup.15 groups together with the carbon atoms to which they are attached, and optionally an intervening carbon atom where present, can combine to form —(CH.sub.2).sub.p— such that the group [CH(R.sup.15)]n is a 3- to 8 membered carbocyclic ring; p is 1, 2, 3, 4, 5 or 6; and R.sup.16 is selected from C(O)OH, S(O).sub.2OH, S(O).sub.2(C.sub.1-6 alkyl), OS(O).sub.2OH and P(O)(OH).sub.2.

27. A method according to claim 26, wherein the compound is selected from: 2,2-difluoro-3β,7β-dihydroxy-5β-cholanic acid (Compound 7); difluoro-3α,7β-dihydroxy-5β-cholanic acid (Compound 9) N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-ethylsulfonic acid (Compound 10); N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-methylcarboxylic acid (Compound 17); and N-(methyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide) ethylsulfonic acid (Compound 19).

28. A eompound for use, a use or a method according to claim 24, wherein the neurodegenerative disorder is Alzheimer's disease, and wherein the compound is 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (Compound 8).

29. (canceled)

30. A pharmaceutical composition comprising a compound according to claim 1, and a pharmaceutically acceptable excipient or carrier.

31. A process for the preparation of a compound according to claim 1, comprising: A. for a compound of general formula (IB) or (IC) as defined in claim 3 in which R.sup.1 is F and R.sup.3 is C(O)OR.sup.12a: wherein R.sup.12a is C.sub.1-6 alkyl optionally substituted by one or more halo or aryl groups: treating with an acid a compound of general formula (II): ##STR00098## wherein Y and R.sup.3 are as defined in claim 1; R.sup.12a is C.sub.1-6 alkyl optionally substituted by one or more halo or aryl groups; and R.sup.21 is an OH protecting group which is acid labile; B. for a compound of general formula (IA) or (IC) as defined in claim 3 in which R.sup.2 is F and R.sup.3 is C(O)OR.sup.12a, wherein R.sup.12a is as defined for general formula (II): reducing a compound of general formula (XIIa): ##STR00099## wherein Y is as defined in claim 1 and R.sup.12a is as defined for general formula (II); C. for a compound of general formula (IB) or (ID) as defined in claim 3 in which R.sup.2 is F and R.sup.3 is C(O)OR.sup.12a, wherein R.sup.12a is as defined for general formula (II): reducing a compound of general formula (XIIb): ##STR00100## wherein Y is as defined in claim 1 and R.sup.12a is as defined for general formula (II); D. for a compound of general formula (IA) as defined in claim 3 in which both R.sup.1 and R.sup.2 are F and R.sup.3 is C(O)OR.sup.12a, wherein R.sup.12a is as defined for general formula (II): reacting with an acid a compound of general formula (XXI): ##STR00101## wherein Y is as defined in claim 1 and R.sup.12a and R.sup.21 are as defined for general formula (II); E. for a compound of general formula (IB) or (ID) as defined in claim 3 in which both R.sup.1 and R.sup.2 are F and R.sup.3 is C(O)OR.sup.12a, wherein R.sup.12a is as defined for general formula (II): reducing a compound of general formula (XXII): ##STR00102## wherein Y is as in claim 1 and R.sup.12a and R.sup.21 are as defined for general formula (II); F. for a compound of general formula (I) in which R.sup.3 is C(O)OH: hydrolysing a compound of general formula (I) in which R.sup.3 is C(O)R.sup.12a, wherein R.sup.12a is as defined above for general formula (II); G. for a compound of general formula (I) in which R.sup.3 is C(O)NR.sup.12R.sup.13: reacting a compound of general formula (I) in which R.sup.3 is C(O)OH with an amine of general formula:
H—NR.sup.12R.sup.13 wherein R.sup.12 and R.sup.13 are as defined in claim 1; in the presence of a coupling reagent and an amine; H. for a compound of general formula (I) in which R.sup.3 is C(O)NR.sup.12[CH(R.sup.15)].sub.nR.sup.16: reacting a compound of general formula (I) in which R.sup.3 is C(O)OH with a compound of general formula (XL):
HNR.sup.12[CH(R.sup.15)].sub.nR.sup.16  (XL) wherein R.sup.12, R.sup.15, n and R.sup.16 are as defined in claim 1; in the presence of a coupling agent and an amine; I. for a compound of general formula (I) in which R.sup.3 is C(O)NR.sup.12CH(R.sup.14)C(O)OH: reacting a compound of general formula (I) in which R.sup.3 is C(O)OH by reaction with an amino acid of general formula (XLI): ##STR00103## wherein R.sup.12 and R.sup.14 are as defined in claim 1; in the presence of a coupling agent and an amine; J. for a compound of general formula (I) in which R.sup.3 is C(O)NR.sup.12CH(R.sup.15)CH(R.sup.15)S(O).sub.2OH: reacting a compound of general formula (I) in which R.sup.3 is C(O)OH by reaction with a compound of general formula (XLII): ##STR00104## wherein R.sup.12 and R.sup.15 are as defined in claim 1; in the presence of a coupling agent and an amine; K. for a compound of general formula (I) in which R.sup.3 is C(O)NR.sup.12S(O).sub.2R.sup.13: reacting a compound of general formula (I) in which R.sup.3 is C(O)OH with a compound of formula:
NHR.sup.12S(O).sub.2R.sup.13 wherein R.sup.12 and R.sup.13 are as defined in claim 1, in the presence of a coupling reagent and an amine; L. for a compound of general formula (I) in which R.sup.3 is NHC(O)NR.sup.12S(O).sub.2R.sup.13: reacting a compound of general formula (I) in which R.sup.3 is C(O)OH as follows: ##STR00105## wherein R.sup.1, R.sup.2, R.sup.12 and R.sup.13 are as defined in claim 1; M. for a compound of general formula (I) in which R.sup.3 is S(O).sub.2OR.sup.12: reacting a compound of general formula (I) in which R.sup.3 is C(O)OH with a C.sub.1-6 alkanoyl or benzoyl chloride or with a C.sub.1-6 alkanoic anhydride to give a protected intermediate; and converting the carboxylic acid group of the protected intermediate to OH by reduction with a hydride reducing agent to give a reduced intermediate; and halogenating the reduced intermediate to give a halogenated intermediate in which the OH group is replaced with a halogen; and reacting the halogenated intermediate with sodium sulphite in an alcoholic solvent; N. for a compound of general formula (I) in which R.sup.3 is OS(O).sub.2R.sup.12: reacting a compound of general formula (I) in which R.sup.3 is C(O)OR.sup.12 with a C.sub.1-6 alkanoyl or benzoyl chloride or with a C.sub.1-6 alkanoic anhydride to protect any OH groups; and converting the C(O)OR.sup.12 of the protected intermediate to OH by reduction with a hydride reducing agent to give a reduced intermediate; and reacting the reduced intermediate with chlorosulfonic acid in the presence of a base to give a protected product; and base hydrolysis of the protected product to remove the protecting groups; or O. for a compound of general formula (I) in which R.sup.3 is S(O).sub.2R.sup.12: reacting the reduced intermediate of (M) or (N) above with Lawesson's reagent followed by oxidation of the resultant product.

32. A compound according to claim 1 which is 2,2-difluoro-3β,7β-dihydroxy-5β-cholanic acid (Compound 7), or a pharmaceutically acceptable salt thereof.

33. A compound according to claim 1 which is 2,2-difluoro-3α,7β-dihydroxy-5β-cholanic acid (Compound 9), or a pharmaceutically acceptable salt thereof.

34. A compound according to claim 1 which is N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-ethylsulfonic acid (Compound 10), or a pharmaceutically acceptable salt thereof.

35. A compound according to claim 1 which is N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-methylcarboxylic acid (Compound 17), or a pharmaceutically acceptable salt thereof.

36. A compound according to claim 1 which is N-(methyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide) ethylsulfonic acid (Compound 19), or a pharmaceutically acceptable salt thereof.

37. A compound according to claim 1 which is 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (Compound 8), or a pharmaceutically acceptable salt thereof.

Description

[0233] The invention will now be described in greater detail with reference to the following examples and to the figures in in which:

[0234] FIG. 1 (FIGS. 1A, 1B and 1C) shows data from fibroblast cell lines from controls and from 6 sporadic Parkinson's disease (sPD) patients. Controls are compared with untreated sPD, fibroblasts, sPD fibroblasts incubated with UDCA and sPD fibroblasts incubated with Compound 7; FIG. 1A: basal oxygen consumption; FIG. 1B: maximal respiratory rate; FIG. 1C: ATP linked respiration.

[0235] FIG. 2 shows the extracellular acidification rate in fibroblast cell lines from controls and from 6 sporadic Parkinson's disease (sPD) patients. Control fibroblasts are compared with untreated sPD, fibroblasts, sPD fibroblasts incubated with UDCA and sPD fibroblasts incubated with Compound 7.

[0236] FIG. 3 shows mitochondrial respiratory chain complex I activity in homogenate from a MTPT mouse which was either untreated or treated with Compound 7 at a dose of 4 mg/kg or 12 mg/kg

[0237] FIG. 4 shows a comparison of mitochondrial respiratory chain complex I activity in left striatum mouse brain homogenate from a mouse which is treated with vehicle, with vehicle+12 mg Compound 7, with MPTP or with MTPT plus 1 mg, 4 mg or 12 mg Compound 7.

ABBREVIATIONS

[0238] AcOH Acetic acid [0239] Boc t-butyloxycarbonyl [0240] tBuOH t-butanol [0241] BzOH Benzoic acid [0242] Calcd Calculated [0243] mCPBA Meta-chloroperbenzoic acid [0244] d Days [0245] DAST Diethylaminosulfur trifluoride [0246] DCM Dichloromethane [0247] DEAD Diethyl azodicarboxylate [0248] DIPA di-isopropyl alcohol [0249] DIPEA N,N-diisopropylethylamine [0250] DMF N,N-dimethylformamide [0251] EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide [0252] Equiv Equivalents [0253] EtOAc Ethyl acetate [0254] EtOH Ethanol [0255] Et.sub.3N Triethylamine [0256] h Hours [0257] HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate [0258] HOBt Hydroxybenzotriazole [0259] HPLC High performance liquid chromatography [0260] MeOH Methanol [0261] Mol Moles [0262] MOM Methoxymethyl [0263] NaOMe Sodium methoxide [0264] PE Petroleum ether [0265] PPh3 triphenylphosphine [0266] RM Reaction mixture [0267] RT Room temperature [0268] sat Saturated [0269] SM Starting material [0270] Tf.sub.2O Trifluromethanesulfonic anhydride (triflic anhydride) [0271] THF Tetrahydrofuran [0272] TLC Thin layer chromatograpy [0273] UDCA Ursodeoxycholic acid

[0274] General Procedures

[0275] General Procedure A for 24-Carboxylic Acid Protection as Methyl Ester

[0276] The method of Pelliciari was used (ACS Med. Chem. Lett. 2012, 3, 273-277). Free bile acid (25.0 g, 64 mmol, 1 equiv) was dissolved in HPLC grade MeOH (20 volumes) before adding p-toluene sulfonic acid (0.1 equiv) and sonicating at 30° C. for 2 h. Once deemed complete by TLC analysis the solvent was removed in vacuo, before dissolving the residue in EtOAc (16 volumes), washing the organics with sat. NaHCO.sub.3 (×2), water and brine. The organic phase was then dried (Na.sub.2SO.sub.4) and concentrated to yield the methyl ester.

[0277] General Procedure B for 3-Keto/7-Keto Reduction Using NaBH.sub.4/CeCl.sub.3

[0278] Using the conditions of Černý (Steroids 2012, 77, 1233-1241). To a solution of the ketone (1 equiv) and CeCl.sub.3 (1.2 equiv) in MeOH (˜50 volumes) and EtOAc (2 mL) was added NaBH.sub.4 (1.1 equiv) over the course of 5 min. The solution was stirred for 30 min, at which point further NaBH.sub.4 (1 equiv) was added and stirred for a further 30 min to drive the reaction towards completion. Reaction quenched with ice cold 2M HCl and the aqueous washed with EtOAc (×2). The combined organic were washed with sat NaHCO.sub.3 and water, dried (Na.sub.2SO.sub.4) and concentrated then purified by column chromatography to afford both the α-OH and β-OH epimers.

[0279] General Procedure C for Saponification of Methyl Ester Using LiOH in MeOH

[0280] To a solution of the methyl ester (43 mg, 0.11 mmol, 1 equiv) in MeOH (˜70 volumes) was added 2M LiOH (10 equiv) and the solution allowed to stir until complete at RT. Solvent removed in vacuo and the crude residue acidified with 2M HCl, before extracting with EtOAc (×2). Combined organics washed with water and brine, dried (Na.sub.2SO.sub.4) and concentrated to yield the free acid.

[0281] General Procedure D for Saponification of Methyl Ester Using LiOH in THF

[0282] The methyl ester (1.0 equiv.) was dissolved in THF (˜20 volumes) and added LiOH (2M in H.sub.2O, 10 equiv.). After stirring at room temperature until complete, the reaction mixture was reduced in vacuo, the resulting residue was acidified with 2M HCl and the aqueous phase was extracted with EtOAc (×2). The combined organic phases were then washed with water and brine before drying over Na.sub.2SO.sub.4 and reducing in vacuo to afford the free acid.

[0283] General Procedure E for Methanolysis of Acetate/Benzoate

[0284] The acetate/benzoate protected bile acid (3.1 g, 6.12 mmol, 1 equiv) was dissolved in dry MeOH (˜10 volumes) before the addition of 25% NaOMe in MeOH (˜6 volumes) and the RM stirred at RT. On completion the reaction was acidified to pH 4-5 with 2M HCl and diluted with H.sub.2O. The aqueous phase was extracted with DCM (×2), combined organics washed with NaHCO.sub.3, dried (Na.sub.2SO.sub.4) and concentrated to afford the deprotected material. Used without further purification.

[0285] General Procedure F for Saponification of Ester Using NaOH

[0286] To a round-bottom flask were added the ester (1.0 eq), NaOH (12.95 eq) and MeOH (HPLC grade, 8.35 mL/mol). The reaction mixture was stirred at room temperature overnight. Upon completion indicated by TLC analysis, the solvent was removed under reduced pressure. The residue was diluted with water, acidified with aqueous HCl and extracted with EtOAc (×3). The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated in vacuo to afford the desired compound (purification by flash chromatography if needed).

[0287] General Procedure G for Reduction of Ketone Derivatives Using NaBH.sub.4

[0288] To a round bottom flask were added the ketone (1.0 eq), sodium borohydride (2.4 eq) and anhydrous THF (˜40 vol). The reaction mixture was stirred at room temperature overnight, quenched with H.sub.2O and extracted with ethyl acetate (×3). The combined organic layer was washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude was purified by flash chromatography, and by HPLC if needed.

[0289] General Procedure K for Protection of Secondary Alcohol as a MOM Ether

[0290] To a solution of starting secondary alcohol (1 equiv) in dry DCM (˜17 volumes) was added DIPEA (3 equiv) and MOM-CI (5 equiv) at 0° C. The reaction mixture was warmed to room temperature before allowing to stir overnight. Once complete, the reaction mixture was quenched with water (˜3.4 mL/mmol) and methanol (˜3.4 mL/mmol) before separating the layers and extracting the aqueous with EtOAc (×4) and washing the combined organics with brine (×2). The organic phase was then dried (Na.sub.2SO.sub.4) and concentrated in vacuo to yield the crude material. The crude was purified by flash chromatography (HPLC if needed) to afford the desired compound.

[0291] General Procedure L for Cleavage of MOM-Group Using HCl

[0292] The MOM protected material (1.5 g, 1.76 mmol, 1 equiv) was dissolved in MeOH (˜30 volumes) and 2M HCl (˜5.7 mL/mmol), then the mixture was warmed to 70° C. for 5 hr. Reaction mixture was cooled, and concentrated in vacuo, azeotroping to complete dryness (MeOH×3, CHCl.sub.3×1) to yield the desired material.

[0293] General Procedure M for Secondary Alcohol Oxidation Using Dess-Martin Periodinane

[0294] To a solution of starting secondary alcohol (1.0 equiv.) in dichloromethane at 0° C. was added Dess-Martin periodinane (˜1.2 equiv.) portion-wise over 10 mins. After 18 hours warming to RT, the reaction was deemed complete by TLC and the reaction mixture was quenched by the addition of sat. Na.sub.2S.sub.2O.sub.3 solution and sat. NaHCO.sub.3 solution. The aqueous phase was separated and extracted with dichloromethane (×3) and the combined organic fractions were washed with sat. NaHCO.sub.3 solution, water and brine, dried over MgSO.sub.4, filtered and concentrated in vacuo to afford the crude desired product. The crude was purified by flash chromatography (HPLC if needed) to afford the desired compound.

[0295] General Procedure N for Hydrogenation/Hydrogenolysis Using Catalyst

[0296] To a round-bottom flask was added benzyl protected bile acid (1.0 eq), catalyst [Pd/C or PtO.sub.2] (10 mol %) and solvent [MeOH, EtOH, etc]. The reaction mixture was degassed with hydrogen gas and then stirred under H.sub.2 at atmospheric pressure or high pressure for 16-72 hours. The catalyst was filtered through Celite and the filtrate was concentrated and purified by flash chromatography.

[0297] General Procedure O for Preparation of Silyl Enol Ether from Ketone Derivatives

[0298] To a round-bottom flask were added DIPA (12.6 eq) and THF (1.25 mL/mmol). The solution was cooled to −78° C., then n-butyllithium (2.5 M in hexanes, 12 eq) was added dropwise and stirred at −78° C. for 30 min. TMSCI (10 eq) was added and stirred for 20 min. A solution of the ketone derivative (1 eq) in THF (6.5 mL/mmol) was then added dropwise in 10 min and stirred at this temperature for 45 min, followed by the addition of triethylamine (18 eq) and stirred for 1 h. The reaction mixture was warmed to −20° C., quenched with saturated NaHCO.sub.3 solution and warmed to room temperature in 2 h. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (×3). The combined organic layer was washed with saturated NaHCO.sub.3 solution, water, brine, dried over Na.sub.2SO.sub.4, filtered and concentrated to afford the desired sily enol ether intermediate which was used for further reaction without any purification.

[0299] General Procedure P for Eletrophilic Fluorination of Silyl Enol Ether Using Selectfluor in DMF

[0300] To a round bottom flask were the silyl enol ether derivative (1.0 eq), DMF (2 mL/mmol) and a solution of Selectfluor (1.5 eq) in DMF (3 mL/mmol) at 0° C. The reaction mixture was stirred at room temperature overnight, quenched with H.sub.2O and extracted with ethyl acetate (×4). The combined organic layer was washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude was purified by flash chromatography, and by HPLC if needed.

[0301] General Procedure Q for the Formation of Conjugates

[0302] Fluorinated bile acid (1 equiv.) was dissolved in dry DMF (12 vol) with stirring under argon. HATU (1 equiv.), DIPEA (3.0 equiv.) and amino acid (1.1 equiv.) added and the reaction stirred at RT for 16 h. Upon completion, the reaction mixture was dry loaded directly onto silica and purified via C18 chromatography (gradient elution of MeOH in H.sub.2O, 0-100%) to yield the conjugate as either a DIPEA salt or free acid. DIPEA salts were then treated with a sodium ion exchange column to yield the desired sodium salt of the compounds as residues.

Example 1—Synthesis of 2β-Fluoro Compounds

A. Methyl 3α,7α-dihydroxy-5β-cholanoate (1A.1)

[0303] ##STR00031##

[0304] The method of Pellicari was used (ACS Med. Chem. Lett. 2012, 3, 273-277). CDCA (25.0 g, 64 mmol, 1 equiv) was dissolved in HPLC grade MeOH (500 mL) before adding p-toluene sulfonic acid (1.21 g, 6.4 mmol, 0.1 equiv) and sonicating at 30° C. for 2 h. Once deemed complete by TLC analysis the solvent was removed in vacuo, before dissolving the residue in EtOAc (400 mL), washing the organics with sat. NaHCO.sub.3 (2×150 mL), water (250 mL) and brine (250 mL). The organic phase was then dried (Na.sub.2SO.sub.4) and concentrated to yield target compound as a white/pale yellow solid (26.0 g, quantitative). (General procedure A).

[0305] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.84 (1H, q, J=2.4 Hz), 3.66 (3H, s), 3.44 (1H, tt, J=10.9, 4.5 Hz), 2.34 (1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.28-2.15 (2H, m), 2.12-0.97 (26H, m), 0.93 (3H, d, J=6.2 Hz), 0.90 (3H, s), 0.65 (3H, s) ppm.

[0306] LRMS (ESI.sup.+) m/z: 429.1 [M+Na].sup.+.

B. Methyl 7α-hydroxy-3-oxo-5β-cholanoate (1B.1)

[0307] ##STR00032##

[0308] To a solution of methyl 3α,7α-dihydroxy-5β-cholanoate (10.0 g, 24.6 mmol, 1 equiv) in water (25 mL) and t-butanol (100 mL) was added KBr (5.9 g, 49.0 mmol, 2 equiv), KHCO.sub.3 (24.6 g, 246 mmol, 10 equiv) and TEMPO (5.0 g, 32.0 mmol, 1.3 equiv). The solution was cooled to 0° C. before adding ˜11% NaClO solution (54.2 mL, 73.2 mmol, 3.0 equiv) portion wise over the course of 6 h. The reaction was quenched with slow addition of sodium thiosulfate solution (300 mL, 1.2 M, 350 mmol). The aqueous was extracted with EtOAc (2×300 mL), which were combined and washed with brine (300 mL) and water (300 mL) before drying (Na.sub.2SO.sub.4) and removing the solvent in vacuo. The resulting bright red thick oily crude (15 g) was purified using flash chromatography (PE/EtOAc: 80:20->65:35) to yield a white solid (6.5 g, 16.0 mmol, 66%).

[0309] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.93 (1H, br s), 3.67 (3H, s), 3.40 (1H, t, J=14.4 Hz), 2.46-1.10 (30H, m), 1.01 (3H, s), 0.95 (3H, d, J=6.6 Hz), 0.71 (3H, s) ppm.

[0310] LRMS (ESI.sup.+) m/z: 422.1 [M+NH.sub.4].sup.+, 427.1 [M+Na].sup.+.

C. Methyl 7α-methoxymethoxyl-3-oxo-5β-cholanoate (1C.1)

[0311] ##STR00033##

[0312] To a solution of methyl 7α-hydroxy-3-oxo-5β-cholanoate (3.0 g, 7.41 mmol, 1 equiv) in dry DCM (50 mL) was added DIPEA (3.83 mL, 22.2 mmol, 3 equiv) and MOM-CI (2.82 mL, 37.1 mmol, 5 equiv) at 0° C. The reaction mixture was warmed to room temperature before allowing to stir overnight. Once complete, the reaction mixture was quenched with water (25 mL) and methanol (25 mL) before separating the layers and extracting the aqueous with EtOAc (4×75 mL) and washing the combined organics with brine (2×150 mL). The organic phase was then dried (Na.sub.2SO.sub.4) and concentrated in vacuo to yield 3.8 g of crude material which was purified by flash chromatography (PE/EtOAc: 75:25) yielding a white solid (3.10 g, 6.9 mmol, 93%).

[0313] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.68 (1H, d, J=6.8 Hz), 4.55 (1H, d, J=6.8 Hz), 3.72-3.62 (4H, m), 3.43-3.28 (4H, m), 2.49-1.05 (27H, m), 1.03 (3H, s), 0.94 (3H, d, J=6.4 Hz), 0.69 (3H, s) ppm.

[0314] LRMS (ESI.sup.+) m/z: 449.3 [M+H].sup.+, 471.1 [M+Na].sup.+.

D. Methyl 7α-methoxymethoxyl-3-trimethylsilyloxy-5β-chol-2-eneoate (1D.1) and methyl 7α-methoxymethoxyl-3-trimethylsilyloxy-5β-chol-3-eneoate (ID.2)

[0315] ##STR00034##

[0316] Following method of Barlow et al (Eur. J. Med. Chem. 2011, 46, 1545-1554). To a solution of methyl 7α-methoxymethoxyl-3-oxo-5β-cholanoate (1.0 g, 2.23 mmol, 1 equiv) in dry DCM (20 mL) at 0° C. was added Et.sub.3N (0.62 mL, 4.46 mmol, 2 equiv) and trimethylsilyl triflate (0.44 mL, 2.45 mmol, 1.1 equiv). The reaction mixture was allowed to stir for 1 hr before diluting with further DCM (150 mL) and quenching with sat. NaHCO.sub.3 (100 mL). The layers were separated and the aqueous was extracted with further DCM (3×100 mL), which were combined and washed with brine (150 mL), dried (Na.sub.2SO.sub.4) and concentrated to yield a colourless oil (1.2 g) which contained methyl 7α-methoxymethoxyl-3-trimethylsilyloxy-5β-chol-2-eneoate (IA.1) and methyl 7α-methoxymethoxyl-3-trimethylsilyloxy-5β-chol-3-eneoate (1A.2) in a roughly 1:1 ratio. This crude material was used in subsequent steps without further purification.

[0317] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.80-4.44 (3H, m), 3.67 (3H, s), 3.66-3.56 (1H, m), 3.40 (1.5H, s), 3.36 (1.5H, s), 2.51-1.08 (31H, m), 0.99-0.95 (3H, m), 0.93 (3H, d, J=6.2 Hz), 0.66 (1.5H, br. s), 0.65 (1.5H, br. s), 0.21-0.15 (9H, m) ppm.

E. Methyl 2β-fluoro-7α-methoxymethoxyl-3-oxo-5β-cholanoate (1E.1) and methyl 4β-fluoro-7α-methoxymethoxyl-3-oxo-5β-cholanoate (1E.2)

[0318] ##STR00035##

[0319] Following the method of Fujimoto et al (Bioorg. Med. Chem. Lett. 2011, 21, 6409-6413). To a solution of 7α-methoxymethoxyl-3-trimethylsilyloxy-5β-chol-2-eneoate and methyl 7α-methoxymethoxyl-3-trimethylsilyloxy-5β-chol-3-eneoate (1.10 g, 2.2 mmol, 1 equiv) in dry acetonitrile was added Selectfluor@ (1.20 g, 3.3 mmol, 1.5 equiv), allowing the reaction mixture to stir at room temperature for 4 h. The solvent was removed in vacuo before diluting with EtOAc (100 mL) and water (100 mL). The layers were separated before extracting the aqueous with further EtOAc (2×100 mL). The combined organics were then washed with brine (150 mL), dried (Na.sub.2SO.sub.4) and concentrated to yield 1.05 g of a pale yellow solid crude. The crude material was purified using flash chromatography (PE/EtOAc: 80:20) to yield methyl 2β-fluoro-7α-methoxymethoxyl-3-oxo-5β-cholanoate as a white solid (377 mg, 0.81 mmol, 36% over two steps) and methyl 4β-fluoro-7α-methoxymethoxyl-3-oxo-5β-cholanoate as a white solid (321 mg, 0.69 mmol, 31% over two steps).

[0320] 1E.1: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.05 (1H, ddd, J=49.4, 13.5, 5.6 Hz), 4.67 (1H, d, J=6.8 Hz), 4.54 (1H, d, J=6.8 Hz), 3.67 (4H, s), 3.48 (1H, t, J=13.9 Hz), 3.37 (3H, s), 2.52 (1H, dt, J=12.7, 6.1 Hz), 2.42-1.12 (27H, m), 1.08 (3H, s), 0.95 (3H, d, J=6.6 Hz), 0.69 (3H, s) ppm.

[0321] .sup.19F NMR (CDCl.sub.3, 376 MHz): 5-195.19 (ddt, J=49.2, 9.6, 6.2 Hz) ppm.

[0322] LRMS (ESI.sup.+) m/z: 484.2 [M+NH.sub.4].sup.+, 489.1 [M+Na].sup.+.

[0323] 1E.2: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.78 (1H, dd, J=46.8, 11.7 Hz), 4.77 (1H, d, J=6.8 Hz), 4.59 (1H, d, J=6.8 Hz), 3.78 (1H, q, J=2.7 Hz), 3.67 (3H, s), 3.40 (3H, s), 2.52 (1H, td, J=14.4, 4.9 Hz), 2.42-1.11 (24H, m), 1.07 (3H, s), 0.94 (3H, d, J=6.4 Hz), 0.70 (3H, s) ppm.

[0324] .sup.19F NMR (CDCl.sub.3, 376 MHz): 5-200.67 (ddd, J=46.8, 12.1, 6.9 Hz) ppm.

[0325] LRMS (ESI.sup.+): m/z 484.2 (M+NH.sub.4).sup.+, 489.1 (M+Na).sup.+.

F. Methyl 2β-fluoro-3α-hydroxy-7α-methoxymethoxyl-5β-cholanoate (1F.1) and methyl 2β-fluoro-3p-hydroxy-7α-methoxymethoxyl-5β-cholanoate (1F.2)

[0326] ##STR00036##

[0327] To a solution of methyl 2β-fluoro-7α-methoxymethoxyl-3-oxo-5β-cholanoate (260 mg, 0.56 mmol, 1 equiv) in anhydrous tetrahydrofuran (20 mL) was added sodium borohydride (64 mg, 1.70 mmol, 3 equiv) and the reaction mixture allowed to stir overnight at room temperature. Once deemed compete by TLC analysis the reaction was diluted with EtOAc (150 mL) and quenched with water (100 mL), separating the layers before extracting the aqueous with further EtOAc (2×100 mL). The combined organics were then washed with water (150 mL) and brine (150 mL) before drying (Na.sub.2SO.sub.4) and concentrating in vacuo to yield 306 mg of a pale crude oil. The crude was purified by flash chromatography (PE/EtOAc: 65:35) to yield methyl 2β-fluoro-3α-hydroxy-7α-methoxymethoxyl-5β-cholanoate as a white solid (144 mg, 0.307 mmol, 55%) and methyl 2β-fluoro-3p-hydroxy-7α-methoxymethoxyl-5β-cholanoate as a colourless gum (88 mg, 0.19 mmol, 34%).

[0328] 1F.1: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.68 (1H, d, J=6.8 Hz), 4.54 (1H, d, J=7.1 Hz), 4.40 (1H, dddd, J=52.3, 12.0, 8.6, 4.4 Hz), 3.66 (3H, s), 3.62-3.47 (3H, m), 3.37 (3H, s), 2.48-1.01 (27H, m), 0.99 (3H, s), 0.92 (3H, d, J=6.4 Hz), 0.64 (3H, s) ppm.

[0329] .sup.19F NMR (CDCl.sub.3, 376 MHz): 5-187.47 (ddq, J=52.3, 12.7, 7.1 Hz) ppm.

[0330] LRMS (ESI.sup.+) m/z: 486.1 [M+NH.sub.4].sup.+, 491.2 [M+Na].sup.+.

[0331] 1F.2: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.72-4.51 (3H, m), 4.20-4.09 (1H, m), 3.66 (3H, s), 3.59 (1H, d, J=2.4 Hz), 3.38 (3H, s), 2.45 (1H, ddd, J=15.2, 12.4, 2.2 Hz), 2.35 (1H, ddd, J=15.0, 10.3, 5.1 Hz), 2.22 (1H, ddd, J=15.6, 9.5, 6.5 Hz), 2.06-1.05 (25H, m), 1.02 (3H, s), 0.93 (3H, d, J=6.6 Hz), 0.65 (3H, s) ppm.

[0332] .sup.19F NMR (CDCl.sub.3, 376 MHz): 5-187.31 (dquin, J=47.2, 7.5 Hz) ppm.

[0333] LRMS (ESI.sup.+) m/z: 486.0 [M+NH.sub.4].sup.+, 491.1 [M+Na].sup.+.

G. 2β-fluorochenodeoxycholic acid (Compound 1)

[0334] ##STR00037##

[0335] Using general procedure L, followed by general procedure C, methyl 2β-fluoro-3α-hydroxy-7α-methoxymethoxyl-5β-cholanoate (1F.1; 118 mg, 0.25 mmol, 1 equiv) was deprotected to yield 2β-fluorochenodeoxycholic acid (Compound 1) as a pale yellow solid (72 mg, 0.18 mmol, 70%).

[0336] Compound 1: .sup.1H NMR (400 MHz, CD.sub.3OD): δ 4.32 (1H, dddd, J=52.5, 12.5, 8.6, 4.0 Hz), 3.78 (1H, q, J=2.3 Hz), 3.44 (1H, tdd, J=12.0, 8.7, 5.0 Hz), 2.43 (1H, q, J=13.2 Hz), 2.33 (1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.25-2.11 (2H, m), 2.04 (1H, dt, J=12.4, 2.9 Hz), 1.99-1.04 (22H, m), 1.00 (3H, s), 0.97 (3H, d, J=6.5 Hz), 0.70 (3H, s) ppm.

[0337] .sup.19F NMR (376 MHz, CD.sub.3OD): 5-186.77 (ddq, J=52.2, 11.9, 7.8 Hz) ppm.

[0338] LRMS (ESI.sup.+) m/z: 393.1 [M+H−H.sub.2O].sup.+, 821.2 [2M+H].sup.+, 843.3 [2M+Na].sup.+.

H. 2β-fluoro-3β,7α-dihydroxy-5β-cholanic acid (Compound 2)

[0339] ##STR00038##

[0340] Using general procedure L, followed by general procedure C, methyl 2β-fluoro-3β-hydroxy-7α-methoxymethoxyl-5β-cholanoate (1F.2; 25 mg, 0.053 mmol, 1 equiv) was deprotected to yield 2β-fluoro-3β,7α-dihydroxy-5β-cholanic acid (Compound 2) as a gummy solid (21 mg, 0.05 mmol, 96%).

[0341] Compound 2: .sup.1H NMR (400 MHz, Acetone-D.sub.6): δ 10.42 (1H, br. s), 4.58 (1H, dddd, J=47.7, 12.1, 4.4, 2.8 Hz), 4.12-4.00 (1H, m), 3.80 (1H, q, J=2.4 Hz), 3.59 (1H, br. s), 3.29 (1H, br. s), 2.61 (1H, ddd, J=15.3, 12.8, 2.3 Hz), 2.34 (1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.21 (1H, ddd, J=15.5, 9.6, 6.4 Hz), 2.03-1.03 (31H, m), 1.01 (3H, s), 0.97 (3H, d, J=6.5 Hz), 0.70 (3H, s) ppm;

[0342] .sup.19F NMR (376 MHz, Acetone-D.sub.6): 5-186.90 (dquin, J=47.6, 7.6 Hz) ppm.

[0343] LRMS (ESI.sup.+) m/z: 393.4 [M+H−H.sub.2O].sup.+, 373.4 [M+H−H.sub.2O—HF].sup.+, 355.5 [M+H−2H.sub.2O—HF].sup.+.

Example 2—Synthesis of 2α-Fluoro Compounds

A. Methyl 7-oxo-5β-chol-2-eneoate (2A.1) and methyl 7-oxo-5β-chol-3-eneoate (2A.2)

[0344] ##STR00039##

[0345] Methyl 3α-hydroxyl-7-oxo-5β-cholanoate (60 g, 148 mmol, 1.0 equiv; synthesised from 7-ketolithocholic acid using procedure A) and DMAP (30 g, 122 mmol, 2.0 equiv) were dissolved in DCM (500 mL) and cooled to 0° C. on ice. Triflic anhydride (26.1 mL, 156 mmol, 1.05 equiv) was then added over the course of 15 mins. The reaction was stirred at 0° C. for 2 hours, although there was no reaction progress. Reaction was then slowly warmed to 10-12° C. and progress monitored via TLC. Deemed complete after 2 h, RM quenched with 2M HCl (500 mL) and stirred at RT for 10 mins. Layers separated and aqueous extracted with brine (500 mL), dried (Na.sub.2SO.sub.4) and concentrated to yield 78 g of a brown gummy solid. Crude purified via flash chromatography (Petrol ether/EtOAc 95/5.fwdarw.90:10) to yield a mixture of alkenes methyl 7-oxo-5β-chol-2-eneoate and methyl 7-oxo-5β-chol-3-eneoate as a colourless gum (37.5 g, 97 mmol, 66%).

[0346] 2A.1 and 2A.2: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.67-5.30 (2H, m), 3.65 (3H, s), 2.89-2.79 (1H, m), 2.58-2.48 (1H, m), 2.44-1.26 (21H, m), 1.23 (2H, s), 1.21 (1H, s), 0.91-0.88 (3H, m), 0.65 (3H, m) ppm.

[0347] LRMS (ESI.sup.+) m/z: 387.2 [M+H].sup.+, 404.2 [M+NH.sub.4].sup.+.

B. Methyl 2β,3β-epoxy-7-oxo-5β-cholanoate (2B.1) and methyl 3β,4β-epoxy-7-oxo-5β-cholanoate (2B.2)

[0348] ##STR00040##

[0349] The mixture of alkenes methyl 7-oxo-5β-chol-2-eneoate and methyl 7-oxo-5β-chol-3-eneoate (20 g, 51.8 mmol, 1 equiv) was dissolved in DCM (200 mL) at room temperature, before the addition of mCPBA (19.1 g, 77.7 mmol, 1.5 equiv). The reaction was deemed complete after 1.5 h, with the mixture changing from a solution to a suspension over the course of the reaction. The reaction was quenched with sat. aq. Na.sub.2S.sub.2O.sub.3 (150 mL) and allowed to stir for 30 mins. Further DCM (200 mL) and H.sub.2O (150 mL) added to aid solvation. Layers separated and aqueous extracted with further DCM (200 mL), then the combined organics were washed with sat. aq. NaHCO.sub.3 (200 mL) and dried (Na.sub.2SO.sub.4) and concentrated to yield 20.5 g of a pale yellow, gummy solid. Crude purified via flash chromatography (Petrol ether/EtOAc: 92.5:7.5->92:8->80:10->88:12->80:20) to yield the pure Δ3β,4β-epoxide (2.00 g) along with 80% pure Δ2β,3β-epoxide (1.85 g) and a significant amount of mixed fractions (8.5 g). The mixed fractions were re-purified (Petrol ether/EtOAc: 93:7->92:8->91:9->80:10->88:12->85:15->80:20) to yield the pure Δ3β,4β-epoxide (0.8 g) along with 80% pure Δ3β,4β-epoxide (2.15 g) and 60% pure Δ2,3-epoxide (1.30 g). Overall, methyl 2β,3β-epoxy-7-oxo-5β-cholanoate was isolated as a white crystalline solid (˜2.3 g, 5.8 mmol, 11%), along with methyl 3β,4β-epoxy-7-oxo-5β-cholanoate as a white solid (˜4.5 g, 11.3 mmol, 22%)

[0350] 2B.1: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.63 (3H, s), 3.13-3.09 (1H, m), 2.98 (1H, dd, J=5.3, 4.4 Hz), 2.78 (1H, dd, J=12.3, 4.3 Hz), 2.39-2.11 (5H, m), 1.91 (6H, m), 1.61-1.17 (10H, m), 1.13 (3H, m), 1.09-0.92 (2H, m), 0.89 (3H, d, J=6.5 Hz), 0.63 (3H, s) ppm.sup.−

[0351] LRMS (ESI.sup.+) m/z: 403.1 [M+H].sup.+, 425.2 [M+Na].sup.+, 403.1 [M+H-MeCN].sup.+.

[0352] 2B.2: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.64 (3H, s), 3.16-3.13 (1H, m), 2.89 (1H, dd, J=12.6, 7.0 Hz,), 2.82 (1H, d, J=3.8 Hz), 2.42-1.17 (24H, m), 1.13 (3H, s), 0.89 (3H, d, J=6.5 Hz), 0.64 (3H, s) ppm.sup.−

[0353] LRMS (ESI.sup.+) m/z: 403.2 [M+H].sup.+, 425.2 [M+Na].sup.+.

C. Methyl 2α-fluoro-3β-hydroxy-7-oxo-5β-cholanoate (2C.1)

[0354] ##STR00041##

[0355] To a solution of methyl 2β,3β-epoxy-7-oxo-5β-cholanoate (830 mg, 2.06 mmol, 1 equiv) in dry DCM (25 mL) was cooled to 0° C., before adding 70% HF.pyridine (830 μL) and allowing to warm to RT. Deemed complete after 2 d, reaction cooled to 0° C. again and carefully quenched with drop-wise addition of saturated NaHCO.sub.3 (20 mL). Layers separated and aqueous extracted with further DCM (20 mL); combined organics washed with 2M HCl and brine (30 mL each), dried (Na.sub.2SO.sub.4) and concentrated to 840 mg of a white foamy solid. Crude purified via flash chromatography (PE/EtOAc: 70:30) to yield methyl 2α-fluoro-3p-hydroxy-7-oxo-5β-cholanoate as a gummy solid (700 mg, 1.66 mmol, 80%).

[0356] .sup.1H NMR (400 MHz, CDCl.sub.3): 5.53 (1H, dq, J=47.0, 2.6 Hz), 4.04-3.96 (1H, m), 3.65 (3H, s), 2.87 (1H, dd, J=12.7, 6.1 Hz), 2.42-1.25 (25H, m), 1.22 (3H, s), 1.20-1.00 (3H, m), 0.90 (3H, d, J=6.4 Hz), 0.64 (3H, s) ppm.

[0357] .sup.19F NMR (CDCl.sub.3, 376 MHz): 5-184.60 (tt, J=48.6, 8.7 Hz) ppm.

[0358] LRMS (ESI.sup.+) m/z: 423.1 [M+H].sup.+, 445.1 [M+Na].sup.+, 845.5 [2M+H].sup.+.

D. Methyl 2α-fluoro-3α-benzoyloxy-7-oxo-5β-cholanoate (12D.1)

[0359] ##STR00042##

[0360] To a solution of methyl 2α-fluoro-3p-hydroxy-7-oxo-5β-cholanoate (1.05 g, 2.5 mmol, 1 equiv), PPh.sub.3 (980 mg, 3.7 mmol, 1.5 equiv) and benzoic acid (450 mg, 3.7 mmol, 1.5 equiv) in dry THF (25 mL) was added DEAD (650 μL, 3.7 mmol, 1.5 equiv). The solution was allowed to stir at 30° C. over the weekend, at which point crude .sup.19F NMR indicated roughly 40% conversion to desired benzoate. Further PPh.sub.3, BzOH and DEAD (1.5 equiv each) was added and reaction allowed to stir O/N, at which point conversion was =60%. Further PPh.sub.3, benzoic acid and DEAD (0.5 equiv each) added, stirred overnight and 80% conversion reached. More PPh.sub.3, benzoic acid and DEAD (0.5 equiv each) added and stirred O/N once more, although no further progress noted. Solvent removed in vacuo and crude bright yellow material separated via flash chromatography (PE/EtOAc: 98:2->95:5->85:15->70:30->0:100) to yield 285 mg of methyl 2α-fluoro-3α-benzoyloxy-7-oxo-5β-cholanoate (=90% pure) along with 1.28 g of additional mixed fractions.

[0361] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.04 (2H, dd, J=7.8, 1.2 Hz), 7.56 (1H, tt, J=7.6, 1.2 Hz), 7.44 (2H, t, J=7.8 Hz), 5.12-4.79 (2H, m), 3.67 (3H, s), 2.92 (1H, dd, J=12.6, 5.9 Hz), 2.53-1.29 (21H, m), 1.26 (3H, s), 1.24-1.04 (4H, m), 0.93 (3H, d, J=6.4 Hz), 0.67 (3H, s) ppm.

[0362] .sup.19F NMR (CDCl.sub.3, 376 MHz): 5-199.45 (tdd, J=49.9, 28.6, 8.7 Hz) ppm.

[0363] LRMS (ESI.sup.+) m/z: 527.2 [M+H].sup.+, 544.1 [M+NH.sub.4].sup.+, 549.1 [M+Na].sup.+.

E. Methyl 2α-fluoro-3α-hydroxy-7-oxo-5β-cholanoate (2E.1)

[0364] ##STR00043##

[0365] Using the method of Zhao et al (Eur. J. Org. Chem., 2005, 2005, 4414-4427). A mixture of methyl 2α-fluoro-3α-benzoyloxy-7-oxo-5β-cholanoate (400 mg, 0.76 mmol, 1 equiv) and potassium carbonate (20 mg, 0.15 mmol, 0.2 equiv) were suspended in dry MeOH (20 mL) and allowed to stir for 16 h at RT. After 16 h reaction mixture had formed a colourless solution, and was deemed complete by TLC analysis. Solvent removed in vacuo and crude residue taken up between EtOAc/H.sub.2O (5 mL each) and aqueous extracted with further EtOAc (2×5 mL). Combined organics dried (Na.sub.2SO.sub.4) and concentrated to yield 320 mg of a pale gum. Crude purified via flash chromatography (PE/acetone: 70:30) to yield methyl 2α-fluoro-3α-hydroxy-7-oxo-5β-cholanoate (275 mg, 0.65 mmol, 86%) as a gummy solid.

[0366] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.71 (1H, d, J=52.0 Hz), 3.62 (3H, s), 3.59-3.45 (1H, m), 2.84 (1H, dd, J=12.5, 6.0 Hz), 2.43 (1H, d, J=8.3 Hz), 2.39-2.26 (3H, m), 2.24-2.08 (2H, m), 2.01-1.20 (18H, m), 1.18 (3H, s), 1.14-1.02 (3H, m), 0.88 (3H, d, J=6.5 Hz), 0.61 (3H, s) ppm.

[0367] .sup.19F NMR (CDCl.sub.3, 376 MHz): 5-202.32 (tdd, J=51.2, 29.5, 8.7 Hz) ppm.

[0368] LRMS (ESI.sup.+) m/z: 423.4 [M+H].sup.+.

F. Methyl 2α-fluoro-3β,7α-dihydroxy-5β-cholanoate (2F.1) and methyl 2α-fluoro-3β,7β-dihydroxy-5β-cholanoate (2F.7)

[0369] ##STR00044##

[0370] Using general procedure B, methyl 2α-fluoro-3p-hydroxy-7-oxo-5β-cholanoate (300 mg, 0.71 mmol, 1 equiv) was reduced. Crude material purified via flash chromatography (PE/EtOAc: 65:35.fwdarw.55:45) to yield methyl 2α-fluoro-3β,7α-dihydroxy-5β-cholanoate (140 mg, 0.33 mmol, 46%) and methyl 2α-fluoro-3β,7β-dihydroxy-5β-cholanoate (107 mg, 0.25 mmol, 35%).

[0371] 2F.1: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.55 (1H, dq, J=47.4, 2.8 Hz), 4.00 (1H, dq, J=7.2, 3.4 Hz), 3.86 (1H, q, J=2.6 Hz), 3.66 (3H, s), 2.72 (1H, tt, J=14.3, 2.4 Hz), 2.35 (1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.22 (1H, ddd, J=15.7, 9.4, 6.5 Hz), 2.11-1.06 (27H, m), 0.97 (3H, s), 0.92 (3H, d, J=6.5 Hz), 0.66 (3H, s) ppm.

[0372] .sup.19F NMR (376 MHz, CDCl.sub.3): 5-184.70 (tt, J=48.6, 8.7 Hz) ppm;

[0373] LRMS (ESI.sup.+) m/z: 447.3 [M+Na].sup.+.

[0374] 2F.2: .sup.1H NMR (400 MHz, CDCl.sub.3): 54.50 (1H, dq, J=47.3, 2.8 Hz), 3.95 (1H, dq, J=7.2, 3.4 Hz), 3.63 (3H, s), 3.55 (1H, td, J=9.7, 5.1 Hz), 2.32 (1H, ddd, J=15.4, 10.2, 5.0 Hz), 2.19 (1H, ddd, J=15.6, 9.4, 6.5 Hz), 2.11-1.02 (27H, m), 0.96 (3H, s), 0.90 (3H, d, J=6.4 Hz), 0.65 (3H, s) ppm.

[0375] .sup.19F NMR (376 MHz, CDCl.sub.3): 5-184.43 (tt, J=47.7, 8.7 Hz) ppm.

[0376] LRMS (ESI.sup.+) m/z: 447.2 [M+Na].sup.+.

G. Methyl 2α-fluoro-3α,7α-dihydroxy-5β-cholanoate (2G.1) and methyl 2α-fluoro-3α,7β-dihydroxy-5β-cholanoate (2G.2)

[0377] ##STR00045##

[0378] Using general procedure B, methyl 2α-fluoro-3α-hydroxy-7-oxo-5β-cholanoate (270 mg, PGP 0.64 equiv, 1 equiv) was reduced. Crude purified via flash chromatography (PE/acetone: 75:25) to yield 33 mg of pure 7α-OH analogue along with 140 mg of a mixture of both 7α-OH and 7B—OH epimers. The mixture was re-purified via flash chromatography (PE/EtOAc 60:40.fwdarw.50:50) to yield further pure methyl 2α-fluoro-3α,7α-dihydroxy-5β-cholanoate (total—74 mg, 0.17 mmol, 27%) and pure methyl 2α-fluoro-3α,7β-dihydroxy-5β-cholanoate (45 mg, 0.11 mmol, 17%), both as gummy solids.

[0379] 2G.1: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.67 (1H, d, J=52.1 Hz), 3.78 (1H, q, J=2.4 Hz), 3.59 (3H, s), 3.37 (1H, dddd, J=28.5, 12.0, 4.4, 2.5 Hz), 2.46 (1H, q, J=13.0 Hz), 2.37-2.23 (2H, m), 2.20-2.10 (1H, m), 2.04-0.88 (27H, m), 0.88-0.83 (6H, m), 0.59 (3H, s) ppm.

[0380] .sup.19F NMR (CDCl.sub.3, 376 MHz): 5-202.71 (tdd, J=52.0, 27.7, 8.7 Hz) ppm.

[0381] LRMS (ESI.sup.+) m/z: 407.4 [M+H−H.sub.2O].sup.+, 387.3 [M+H−H.sub.2O—HF].sup.+. 2G.2: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.74 (1H, d, J=52.1 Hz), 3.66 (3H, s), 3.63-3.35 (2H, m), 2.43-2.29 (2H, m), 2.28-2.14 (1H, m), 2.08-1.00 (29H, m), 0.96 (3H, s), 0.92 (3H, d, J=6.2 Hz), 0.67 (3H, s) ppm.

[0382] .sup.19F NMR (CDCl.sub.3, 376 MHz): 5-202.49 (tdd, J=52.0, 29.5, 8.7 Hz) ppm.

[0383] LRMS (ESI.sup.+) m/z 407.4 [M+H−H.sub.2O].sup.+, 387.3 [M+H−H.sub.2O—HF].sup.+.

H. 2α-fluoro-3β,7α-dihydroxy-5β-cholanic acid (Compound 3)

[0384] ##STR00046##

[0385] Using general procedure C, methyl 2α-fluoro-3β,7α-dihydroxy-5β-cholanoate (105 mg, 0.25 mmol, 1 equiv) was hydrolysed to yield 2α-fluoro-3β,7α-dihydroxy-5β-cholanic acid (Compound 3) (96 mg, 0.23 mmol, 94%) as a pale solid.

[0386] .sup.1H NMR (400 MHz, acetone-D.sub.6): δ 10.51 (1H, br. s), 4.58 (1H, dq, J=48.0, 2.7 Hz), 3.96 (1H, dq, J=7.2, 3.4 Hz), 3.91 (1H, q, J=2.8 Hz), 2.89 (1H, tt, J=14.2, 2.6 Hz), 2.43 (1H, ddd, J=15.5, 10.7, 5.3 Hz), 2.30 (1H, ddd, J=15.0, 9.4, 6.7 Hz), 2.20-2.06 (4H, m), 2.01-1.13 (21H, m), 1.11-0.97 (6H, m), 0.78 (3H, s) ppm.

[0387] .sup.19F NMR (376 MHz, acetone-D.sub.6): 5-184.37 (tt, J=49.4, 8.7 Hz) ppm.

[0388] LRMS (ESI.sup.−) m/z: 409.1 [M−H].sup.−, 819.5 [M−H].sup.−.

I. 2α-fluoro-3β,7β-dihydroxy-5β-cholanic acid (Compound 4)

[0389] ##STR00047##

[0390] Using general procedure C, methyl 2α-fluoro-3β,7β-dihydroxy-5β-cholanoate (90 mg, 0.21 mmol, 1 equiv) was hydrolysed to yield 2α-fluoro-3β,7β-dihydroxy-5β-cholanic acid (Compound 4) (80 mg, 0.19 mmol, 93%) as a colourless solid.

[0391] .sup.1H NMR (400 MHz, acetone-D.sub.6): δ 10.43 (br. s), 4.58 (1H, dq, J=48.0, 3.1 Hz), 3.99 (1H, dq, J=7.1, 3.3 Hz), 3.57 (1H, tdd, J=10.2, 5.0, 1.0 Hz), 2.42 (1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.29 (1H, ddd, J=15.8, 9.2, 6.8 Hz), 2.22-2.06 (4H, m), 2.04-1.14 (23H, m), 1.07 (3H, s), 1.05 (3H, d, J=6.6 Hz), 0.79 (3H, s) ppm.

[0392] .sup.19F NMR (376 MHz, acetone-D.sub.6): 5-184.29 (tt, J=49.4, 8.7 Hz) ppm.

[0393] LRMS (ESI.sup.−) m/z: 409.1 [M−H].sup.−, 819.5 [2M−H].sup.−.

J. 2α-fluoro-3α,7α-dihydroxy-5β-cholanic acid (Compound 5)

[0394] ##STR00048##

[0395] Using general procedure C, methyl 2α-fluoro-3α,7α-dihydroxy-5β-cholanoate (74 mg, 0.17 mmol, 1 equiv) was hydrolysed to yield 2α-fluoro-3α,7α-dihydroxy-5β-cholanic acid (Compound 5) (65 mg, 0.16 mmol, 93%) as a colourless solid.

[0396] .sup.1H NMR (400 MHz, acetone-D.sub.6): δ 4.65 (dq, J=52.3, 1.7 Hz), 3.81 (1H, q, J=2.8 Hz), 3.40 (1H, dddd, J=29.7, 12.0, 3.9, 2.1 Hz), 2.69 (1H, q, J=12.6 Hz), 2.39-2.16 (3H, m), 2.02-1.01 (25H, m), 0.98-0.91 (6H, m), 0.69 (3H, s) ppm.

[0397] .sup.19F NMR (376 MHz, acetone-D.sub.6): 5-200.79 (tdd, J=51.2, 29.5, 8.7 Hz) ppm.

[0398] LRMS (ESI.sup.−) m/z: 841.4 [2M+H].sup.+, 393.4 [M+H−H.sub.2O].sup.+, 375.4 [M+H−H.sub.2O—HF].sup.+, 373.4 [M+H−2H.sub.2O].sup.+.

K. 2α-fluoro-3α,7β-dihydroxy-5β-cholanic acid (Compound 6)

[0399] ##STR00049##

[0400] Using general procedure C, methyl 2α-fluoro-3α,7β-dihydroxy-5β-cholanoate (44 mg, 0.10 mmol, 1 equiv) was hydrolysed to yield 2α-fluoro-3α,7β-dihydroxy-5β-cholanic acid (Compound 6) (40 mg, 0.97 mmol, 97%) as a colourless solid.

[0401] .sup.1H NMR (400 MHz, acetone-D.sub.6): δ 4.67 (1H, dq, J=52.1, 1.7 Hz), 3.61-3.42 (2H, m), 2.39-2.15 (3H, m), 2.02-1.06 (29H, m), 0.99-0.93 (6H, m), 0.70 (3H, s) ppm.

[0402] .sup.19F NMR (376 MHz, acetone-D.sub.6): 6-200.58 (tdd, J=50.7, 30.3, 6.9 Hz) ppm.

[0403] LRMS (ESI.sup.−) m/z: 393.4 [M+H−H.sub.2O].sup.+, 375.4 [M+H−H.sub.2O—HF].sup.+, 373.4 [M+H−2H.sub.2O].sup.+.

Example 3—Synthesis of 2,2-difluorinated Analogues

A. Methyl 3α,7β-dihydroxy-5β-cholanoate (3A.1)

[0404] ##STR00050##

[0405] Using general procedure A, UDCA (100 g, 250 mmol, 1 equiv) was protected to yield methyl 3α,7β-dihydroxy-5β-cholanoate as a white solid (103 g, 250 mmol, quantitative).

[0406] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.66 (3H, s), 3.73-3.63 (2H, m), 3.58 (2H, td, J=10.4, 5.3 Hz), 2.35 (1H, ddd, J=15.3, 10.1, 4.8 Hz), 2.21 (1H, ddd, J=15.6, 9.6, 6.4 Hz), 1.99 (1H, dt, J=12.3, 2.8 Hz), 1.95-0.97 (26H, m), 0.94 (3H, s), 0.92 (3H, d, =6.4 Hz), 0.67 (3H, s) ppm.

[0407] LRMS (ESI.sup.+) m/z: 389.5 [M+H−H.sub.2O].sup.+, 371.5 [M+H−2H.sub.2O].sup.+.

[0408] Data consistent with literature (except m.p.); see J. Ren, Y. Wang, J. Wang, J. Lin, K. Wei, R. Huang, Steroids 2013, 78, 53-58.

B. Methyl 3α-acetoxy-7β-hydroxy-5β-cholanoate (3B.1)

[0409] ##STR00051##

[0410] Methyl 3α,7β-dihydroxy-5β-cholanoate (30.0 g, 73.8 mmol, 1 equiv), acetic anhydride (35 mL, 369 mmol, 1 equiv) and NaHCO.sub.3 (37.2 g, 443 mmol, 6 equiv) were taken up in THF (600 mL) and the reaction mixture was warmed to 85° C. overnight. Reaction mixture was cooled, filtered and the supernatant concentrated in vacuo to yield a crude residue. This was taken up in EtOAc and brine (300 mL each), the layers were then separated and the aqueous extracted with further EtOAc (2×200 mL). The combined organics were dried (Na.sub.2SO.sub.4) and concentrated to yield 37 g of clear gum/liquid. The crude was purified via flash chromatography (pet ether/EtOAc: 85:15->80:20->70:30) to yield methyl 3α-acetoxy-7p-hydroxy-5β-cholanoate as a gummy solid (25.3 g, 56.4 mmol, 76%).

[0411] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.64 (1H, tt, J=10.5, 5.5 Hz), 3.64 (3H, s), 3.55 (1H, ddd, J=11.5, 8.7, 5.1 Hz), 2.33 (1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.20 (1H, ddd, J=15.6, 9.6, 6.4 Hz), 2.00 (3H, s), 1.97-0.98 (24H, m), 0.93 (3H, s), 0.90 (3H, d, J=6.4 Hz), 0.65 (3H, s) ppm.

[0412] LRMS (ESI.sup.+) m/z: 471.5 [M+Na].sup.+, 371.4 [M+H−H.sub.2O—HOAc].sup.+.

C. Methyl 3α-acetoxy-7β-methoxymethoxyl-5β-cholanoate (3C.1)

[0413] ##STR00052##

[0414] Using general procedure K, methyl 3α-acetoxy-7p-hydroxy-5β-cholanoate (72 g, 160.5 mmol) was protected as a MOM ether. Crude purified via flash chromatography (pet ether/EtOAc: 85:15->80:20->70:30->60:40) to yield methyl 3α-acetoxy-7β-methoxymethoxyl-5β-cholanoate as a gummy solid (62 g, 126 mmol, 79%).

[0415] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.70-4.61 (1H, m), 4.60 (2H, s), 3.64 (3H, s), 3.39-3.25 (4H, m), 2.33 (ddd, J=15.5, 11.0, 5.0 Hz), 2.19 (1H, ddd, J=15.5, 9.6, 6.4 Hz), 2.00 (3H, s), 1.90-0.98 (25H, m), 0.94 (3H, s), 0.90 (3H, d, J=6.4 Hz), 0.65 (3H, s) ppm.

[0416] LRMS (ESI.sup.+) m/z: 515.5 [M+Na].sup.+, 371.5 [M+H−HOCH.sub.2OCH.sub.3—HOAc].sup.+.

D. Methyl 3α-hydroxy-7β-methoxymethoxyl-5β-cholanoate (3D.1)

[0417] ##STR00053##

[0418] Using general procedure E, methyl 3α-acetoxy-7β-methoxymethoxyl-5β-cholanoate (82 g, 166 mmol, 1 equiv) was hydrolysed to yield methyl 3α-hydroxy-7β-methoxymethoxyl-5β-cholanoate as a pale yellow gum (75 g, 166 mmol, quantitative yield).

[0419] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.61 (2H, s), 3.65 (3H, s), 3.56 (1H, tt, J=10.5, 5.0 Hz), 3.41-3.25 (4H, m), 2.34 (1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.20 (1H, ddd, J=15.5, 9.6, 6.4 Hz), 2.02-1.90 (1H, m), 1.89-1.72 (6H, m), 1.70-0.97 (19H, m), 0.94 (3H, s), 0.90 (3H, d, J=6.4 Hz), 0.66 (3H, s) ppm.

[0420] LRMS (ESI.sup.+) m/z: 371.5 [M+H−HOCH.sub.2OCH.sub.3—H.sub.2O].sup.+.

E. Methyl 7β-methoxymethoxyl-5β-chol-2-enoate (3E.1) and methyl 7β-methoxymethoxyl-5β-chol-3-enoate (3E.2)

[0421] ##STR00054##

[0422] Methyl 3α-hydroxy-7β-methoxymethoxyl-5β-cholanoate (75 g, 166 mmol, 1 equiv) was dissolved in DCM (650 mL) and cooled to 5° C. on ic, before the addition of lutidine (58 mL<500 mmol, 3 equiv) and Tf.sub.2O (31 mL, 183 mmol, 1.1 equiv). Reaction mixture warmed to 8-10° C. for 1 h however reaction incomplete, further lutidine (25 mL) and Tf.sub.2O (15 mL), and RM further warmed to 12-14° C. for a further 1.5 h. Reaction deemed complete by TLC analysis. Reaction mixture dry loaded onto silica, and purified via flash chromatography (pet ether/EtOAc: 98:2.fwdarw.97:3->95:5) to yield an inseparable mixture of methyl 7β-methoxymethoxyl-5β-chol-2-enoate and methyl 7β-methoxymethoxyl-5β-chol-3-enoate as a pale yellow gum (64.1 g, 148 mmol, 89%).

[0423] 3E.1/3E.2: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.74-5.34 (2H, m), 4.68-4.62 (2H, m), 3.66 (3H, s), 3.37 (3H, s), 3.13 (1H, td, J=10.2, 5.0 Hz), 2.32 (1H, ddd, J=15.5, 10.3, 5.0 Hz), 2.26-2.16 (1H, m), 2.15-1.00 (27H, m), 0.98 (2H, s), 0.92 (2H, d, J=6.4 Hz), 0.86 (2H, d, J=6.6 Hz), 0.68 (3H s) ppm.

F. Methyl 2α-acetoxy-3β-hydroxy-7β-methoxymethoxyl-5β-cholanoate (3F.1) and methyl 3β,4β-epoxy-7β-methoxymethoxyl-5β-cholanoate (3F.2)

[0424] ##STR00055##

[0425] A mixture of methyl 7β-methoxymethoxyl-5β-chol-2-enoate and methyl 7β-methoxymethoxyl-5β-chol-3-enoate (63.0 g, 146 mmol, 1 equiv), along with mCPBA (54.0 g, 1.5 equiv) was dissolved in DCM and stirred for 1 h at RT. RM quenched with sat. aq. Na.sub.2S.sub.2O.sub.3 (250 mL) and stirred for 20 min at RT. Layers separated and aqueous extracted with DCM (300 mL). Combined organics washed with sat. aq. NaHCO.sub.3 (300 mL), dried (Na.sub.2SO.sub.4) and concentrated, to yield 72 g of a pale yellow gum containing an inseparable mixture of Δ2β,3β- and Δ3β,4β-epoxides (assume quantitative yield). This mixture was then dissolved in AcOH (600 mL), and warmed to 50° C. for 16 hr. The reaction mixture was concentrated in vacuo, then azeotroped (EtOAc×3, DCM×1), before the crude was purified via flash chromatography (pet ether/EA:85:15.fwdarw.80:20.fwdarw.70:30.fwdarw.60:40.fwdarw.50:50) to yield methyl 2α-acetoxy-3p-hydroxy-7β-methoxymethoxyl-5β-cholanoate as a gummy solid (11.1 g, 21.9 mmol, 15%—2 steps) and methyl 3β,4β-epoxy-7β-methoxymethoxyl-5β-cholanoate as a gummy solid (40.5 g, 90 mmol, 62%—2 steps).

[0426] 3F.1: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.74 (1H, q, J=3.9 Hz), 4.62 (2H, s), 3.79 (1H, q, J=3.7 Hz), 3.64 (3H, s), 3.36-3.31 (4H, m), 2.33 (1JH, ddd, J=15.5, 11.0, 5.0 Hz), 2.20 (1H, ddd, J=15.5, 9.6, 6.4 Hz), 2.03 (3H, s), 2.01-0.99 (29H, m), 0.98 (3H, s), 0.90 (3H, d, J=6.4 Hz), 0.65 (3H, s) ppm.

[0427] LRMS (ESI.sup.+) m/z: 531.6 [M+Na].sup.+, 387.4 [M+H−HOCH.sub.2OCH.sub.3—HOCH.sub.2OCH.sub.3].sup.+.

[0428] 3F.2: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.64 (2H, s), 3.64 (3H, s), 3.35 (3H, s), 3.19 (1H, br. s), 3.10 (1H, td, J=10.8, 4.5 Hz), 2.88 (1H, d, J=3.7 Hz), 2.32 (1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.19 (1H, ddd, J=15.5, 9.6, 6.4 Hz), 2.12-2.04 (1H, m), 2.03-0.95 (25H, m), 0.90 (3H, d, J=6.4 Hz), 0.87 (3H, s), 0.65 (3H, s) ppm.

[0429] LRMS (ESI.sup.+) m/z: 417.4 [M, partial -MOM cleavage].sup.+, 387.4 [M+H−HOCH.sub.2OCH.sub.3].sup.+.

G. Methyl 2α-acetoxy-3β,7β-dimethoxymethoxyl-5β-cholanoate (3G.1)

[0430] ##STR00056##

[0431] Using general procedure K, methyl 2α-acetoxy-3p-hydroxy-7β-methoxymethoxyl-5β-cholanoate (11.0 g, 21.6 mmol, 1 equiv) was protected as the MOM derivative to yield methyl 2α-acetoxy-3β,7β-dimethoxymethoxyl-5β-cholanoate as a pale yellow oil/gum (13.0 g, quantitative).

[0432] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.83 (1H, q, J=3.1 Hz), 4.62 (2H, s), 4.62 (2H, s), 3.67 (1H, q, J=2.9 Hz), 3.63 (3H, s), 3.33 (3H, s), 3.33 (3H, s), 3.32-3.30 (1H, m), 2.32 (1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.19 (1H, ddd, J=15.6, 9.4, 6.5 Hz), 2.01 (3H, s), 1.99-1.25 (25H, m), 0.95 (3H, s), 0.89 (3H, d, J=6.4 Hz), 0.65 (3H, s) ppm.

[0433] LRMS (ESI.sup.+) m/z: 575.6 [M+Na].sup.+, 491.6 [M−HOCH.sub.2OCH.sub.3].sup.+, 429.5 [M−2HOCH.sub.2OCH.sub.3].sup.+.

H. Methyl 2α-hydroxy-3β,7β-dimethoxymethoxyl-5β-cholanoate (3H.1)

[0434] ##STR00057##

[0435] Using general procedure E, methyl 2α-acetoxy-3β,7β-dimethoxymethoxyl-5β-cholanoate (13.0 g, 21.6 mmol, 1 equiv) was methanolysed to yield methyl 2α-hydroxy-3β,7β-dimethoxymethoxyl-5β-cholanoate as a pale yellow gum (9.5 g, 18.3 mmol, 85%).

[0436] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.65 (1H, d, J=6.8 Hz), 4.62 (1H, d, J=6.8 Hz), 4.60 (1H, d, J=6.8 Hz), 4.57 (1H, d, J=6.8 Hz), 3.65-3.59 (4H, m), 3.43-3.36 (1H, m), 3.34 (3H, s), 3.33-3.31 (1H, m), 3.31 (3H, s), 2.96 (1H, br. s), 2.29 (1H, ddd, J=15.6, 10.5, 5.0 Hz), 2.16 (1H, ddd, J=15.6, 9.4, 6.5 Hz), 1.97-1.23 (21H, m), 1.16-0.96 (4H, m), 0.92 (3H, s), 0.86 (3H, d, J=6.4 Hz), 0.61 (3H, s) ppm.

[0437] LRMS (ESI.sup.+) m/z: 533.7 [M+Na].sup.+, 399.5 [M−HOCH.sub.2OCH.sub.3—H.sub.2O—OMe].sup.+, 387.4 [M+H−2HOCH.sub.2OCH.sub.3].

I. Methyl 2-oxo-3β,7β-dimethoxymethoxyl-5β-cholanoate (3I.1)

[0438] ##STR00058##

[0439] Using general procedure M, methyl 2α-hydroxy-3β,7β-dimethoxymethoxyl-5β-cholanoate (9.2 g, 18.0 mmol 1 equiv) was oxidised, then purified via flash chromatography (pet ether/EtOAc: 80:20->70:30->65:35) to yield methyl 2-oxo-3β,7β-dimethoxymethoxyl-5β-cholanoate as a pale gummy solid (8.5 g, 16.7 mmol, 93%).

[0440] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.62 (2H, d, J=7.0 Hz), 4.58 (2H, d, J=7.0 Hz), 3.76 (1H, t, J=2.6 Hz), 3.63 (3H, s), 3.34 (3H, s), 3.32 (3H, s), 3.31-3.23 (1H, m), 2.64 (1H, d, J=12.8 Hz), 2.38-2.03 (5H, m), 2.01-1.25 (14H, m), 1.21-1.10 (2H, m), 1.09 (3H, s), 0.87 (3H, d, J=6.4 Hz), 0.63 (3H, s) ppm.

[0441] LRMS (ESI.sup.+) m/z: 531.6 [M+Na].sup.+, 477.6 [M-OMe].sup.+, 415.5 [M−HOCH.sub.2OCH.sub.3—OMe].sup.+.

J. Methyl 2,2-difluoro-3β,7β-dimethoxymethoxyl-5β-cholanoate (3J.1) and methyl 2-fluoro-3β,7β-dimethoxymethoxyl-5β-chol-1-enoate (3J.2)

[0442] ##STR00059##

[0443] Methyl 2-oxo-3β,7β-dimethoxymethoxyl-5β-cholanoate (8.0 g, 15.7 mmol, 1 equiv) was dissolved in DCM (40 mL) before the addition of DAST (1004 mL, 78.6 mmol, 5 equiv) and the reaction mixture stirred at RT for 5 hr. Mixture was then diluted with DCM (100 mL) before adding dropwise to an ice-cold sat. aq. solution of NaHCO.sub.3 (150 mL), then stirred for 20 mins. Layers were separated then aqueous was extracted with DCM (100 mL), combined organics were then dried (Na.sub.2SO.sub.4) and concentrated to yield 7.5 g of a pale brown gum/oil. Crude purified via flash chromatography (pet ether/EtOAc: 90:10.fwdarw.85:15.fwdarw.80:20) to yield methyl 2,2-difluoro-3β,7β-dimethoxymethoxyl-5β-cholanoate (1.75 g, 3.3 mmol, 21%) along with methyl 2-fluoro-3β,7β-dimethoxymethoxyl-5β-chol-1-enoate (970 mg, 1.9 mmol, 12%) both as gummy solids.

[0444] 3J.1: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.72 (1H, d, J=6.6 Hz), 4.66 (1H, d, J=6.6 Hz), 4.63 (2H, s), 3.81 (1H, br. s), 3.66 (3H, s), 3.38 (3H, s), 3.36 (3H, s), 3.31-3.21 (1H, s), 2.34 (1H, ddd, J=15.6, 10.5, 5.0 Hz), 2.21 (1H, ddd, J=15.6, 9.4, 6.5 Hz), 2.12 (1H, br. s), 2.05-1.08 (25H, m), 1.04 (3H, s), 0.92 (3H, d, J=6.4 Hz), 0.67 (3H, s) ppm.

[0445] .sup.19F NMR (376 MHz, CDCl.sub.3): δ −99.89 (d, J=259.0 Hz), −102.89 (ddt, J=250.6, 40.7, 5.0 Hz) ppm

[0446] LRMS (ESI.sup.+) m/z: 553.5 [M+Na].sup.+, 437.5 [M−HOCH.sub.2OCH.sub.3—OCH.sub.3].sup.+.

[0447] 3J.2: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.39 (1H, d, J=17.7 Hz), 4.71 (1H, d, J=6.8 Hz), 4.69 (1H, d, J=6.8 Hz), 4.64 (2H, s), 4.12-4.08 (1H, m), 3.66 (3H, s), 3.40 (3H, s), 3.36 (3H, s), 3.27-3.17 (1H, m), 2.34 (1H, ddd, J=15.6, 10.5, 5.0 Hz), 2.21 (1H, ddd, J=15.6, 9.4, 6.5 Hz), 2.12-1.14 (24H, m), 1.12 (3H, s), 1.10-0.96 (2H, m), 0.91 (3H, d, J=6.4 Hz), 0.68 (3H, s) ppm.

[0448] .sup.19F NMR (376 MHz, CDCl.sub.3): 5-115.57 (dt, J=17.0, 8.5 Hz) ppm.

[0449] LRMS (ESI.sup.+) m/z: 448.6[M+H−HOCH.sub.2OCH.sub.3].sup.+, 387.4 [M+H−2HOCH.sub.2OCH.sub.3].sup.+.

K. Methyl 2,2-difluoro-3β,7β-dihydroxy-5β-cholanoate (3K.1)

[0450] ##STR00060##

[0451] Following general procedure L, methyl 2,2-difluoro-3β,7β-dimethoxymethoxyl-5β-cholanoate (1.5 g, 1.76 mmol, 1 equiv) deprotected to yield methyl 2,2-difluoro-3β,7β-dihydroxy-5β-cholanoate as a gummy solid (1.3 g, quantitative yield).

[0452] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.89 (1H, t, J=5.7 Hz), 3.67 (3H, s), 3.54 (1H, ddd, J=11.6, 9.2, 5.1 Hz), 2.36 (1H, ddd, J=15.6, 10.5, 5.0 Hz), 2.22 (1H, ddd, J=15.6, 9.4, 6.5 Hz), 2.17-1.07 (27H, m), 1.05 (3H, s), 0.93 (3H, d, J=6.4 Hz), 0.69 (3H, s) ppm.

[0453] .sup.19F NMR (376 MHz, CDCl.sub.3): 5-100.05 (d, J=251.4 Hz), −105.16 (ddt, J=252.1, 40.5, 7.2 Hz) ppm.

[0454] LRMS (ESI.sup.+) m/z: 425.5 [M+H−H.sub.2O].sup.+, 405.5 [M+H−H.sub.2O—HF].sup.+.

L. Methyl 2,2-difluoro-3,7-dioxo-5β-cholanoate (3L.1)+hydrate (3L.2)

[0455] ##STR00061##

[0456] Using general procedure M, methyl 2,2-difluoro-3β,7β-dihydroxy-5β-cholanoate (1.0 g, 2.26 mmol, 1 equiv) was oxidised to yield a mixture of methyl 2,2-difluoro-3,7-dioxo-5β-cholanoate+hydrate (900 mg, 2.05 mmol, 91%—combined yield).

[0457] 3L.1 .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.67 (3H, s), 2.93 (1H, ddd, J=13.2, 5.5, 0.8 Hz), 2.77-2.63 (2H, m), 2.52-1.38 (23H, m), 1.37 (3H, s), 1.35-0.96 (6H, m), 0.94 (3H, d, J=6.5 Hz), 0.70 (3H, s) ppm.

[0458] .sup.19F NMR (376 MHz, CDCl.sub.3): 5-104.37 (ddd, J=263.6, 39.9, 12.1 Hz), −111.15 (dq, J=263.6, 5.0 Hz) ppm.

[0459] LRMS (ESI.sup.+) m/z: 439.5 [M+H].sup.+.

[0460] 3L.2 LRMS (ESI.sup.+) m/z: 457.5 [M+H].sup.+.

M. Methyl 2,2-difluoro-3α,7α-dihydroxy-5β-cholanoate (3M.1)+methyl 2,2-difluoro-3α,7β-dihydroxy-5β-cholanoate (3M.2)

[0461] ##STR00062##

[0462] Using general procedure B, a mixture of methyl 2,2-difluoro-3,7-dioxo-5β-cholanoate and the hydrate (750 mg, 1.71 mmol, 1 equiv) were reduced. Crude was purified via flash chromatography (petrol ether/EtOAc: 70:30->60:40->50:50) to yield methyl 2,2-difluoro-3α,7α-dihydroxy-5β-cholanoate (300 mg, 0.68 mmol, 40%) and methyl 2,2-difluoro-3α,7β-dihydroxy-5β-cholanoate (26 mg, 0.06 mmol, 4%).

[0463] 3M.1: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.86 (1H, q, J=2.7 Hz), 3.67 (3H, s), 3.65-3.56 (1H, m), 2.54 (1H, q, J=13.2 Hz), 2.47-2.31 (2H, m), 2.28-2.17 (1H, m), 2.05-1.04 (19H, m), 1.00 (3H, s), 0.94 (3H, d, J=6.5 Hz), 0.67 (3H, s) ppm.

[0464] .sup.19F NMR (376 MHz, CDCl.sub.3): 5-101.58 (dquin, J=235.8, 5.2 Hz), −119.95 (dddd, J=235.8, 39.9, 20.8, 10.4 Hz) ppm.

[0465] LRMS (ESI.sup.+) m/z: 425.5 [M+H−H.sub.2O].sup.+.

[0466] 3M.2: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.73 (1H, ddt, J=19.7, 10.9, 5.4 Hz), 3.67 (3H, s), 3.57 (1H, ddd, J=11.3, 9.5, 5.1 Hz), 2.43-2.30 (2H, m), 2.22 (2H, ddd, J=15.7, 9.4, 6.6 Hz), 2.04-1.05 (31H, m), 1.02 (3H, s), 0.93 (3H, d, J=6.4 Hz), 0.68 (3H, s) ppm.

[0467] .sup.19F NMR (376 MHz, CDCl.sub.3): 5-101.31 (dquin, J=237.6, 5.2 Hz), −119.06 (dddd, J=237.6, 38.2, 19.0, 10.2 Hz) ppm.

[0468] LRMS (ESI.sup.+) m/z: 425.5 [M+H−H.sub.2O].sup.+.

N. 2,2-difluoro-3β,7β-dihydroxy-5β-cholanic acid (Compound 7)

[0469] ##STR00063##

[0470] Using general procedure C, methyl 2,2-difluoro-3β,7β-dihydroxy-5β-cholanoate (60 mg, 0.14 mmol, 1 equiv) was hydrolysed to yield 2,2-difluoro-3β,7β-dihydroxy-5β-cholanic acid (Compound 7) as a pale solid (50 mg, 0.12 mmol, 83%).

[0471] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.89 (1H, br. s), 3.55 (1H, ddd, J=11.4, 9.2, 5.2 Hz), 2.39 (1H, ddd, J=15.6, 10.5, 5.0 Hz), 2.26 (1H, ddd, J=15.8, 9.4, 6.5 Hz), 2.18-1.07 (26H, m), 1.05 (3H, s), 0.94 (3H, d, J=6.5 Hz), 0.69 (3H, s) ppm.

[0472] .sup.19F NMR (376 MHz, CDCl.sub.3): 5-100.00 (d, J=253.2 Hz), −105.12 (ddt, J=251.4, 41.6, 8.0 Hz) ppm.

[0473] LRMS (ESI.sup.+) m/z: 411.5 [M+H−H.sub.2O].sup.+, 391.5 [M+H−H.sub.2O—HF].sup.+.

O. 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (Compound 8)

[0474] ##STR00064##

[0475] Using general procedure C, methyl 2,2-difluoro-3α,7α-dihydroxy-5β-cholanoate (75 mg, 0.17 mmol, 1 equiv) was hydrolysed to yield 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (Compound 8) as a white solid (70 mg, 0.16 mmol, 96%).

[0476] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 33.86 (1H, q, J=2.0 Hz), 3.62 (1H, ddt, J=19.7, 10.9, 5.4 Hz), 2.54 (1H, q, J=13.0 Hz), 2.44-2.35 (2H, m), 2.25 (1H, ddd, J=15.9, 9.6, 6.5 Hz), 2.02-1.02 (25H, m), 0.99 (3H, s), 0.94 (3H, d, J=6.4 Hz), 0.67 (3H, s) ppm.

[0477] .sup.19F NMR (376 MHz, CDCl.sub.3): δ −119.67 (d, J=235.8 Hz), −119.67 (dddd, J=235.8, 38.2, 19.1, 10.4 Hz) ppm.

[0478] LRMS (ESI.sup.+) m/z: 411.5 [M+H−H.sub.2O].sup.+, 393.4 [M+H−2H.sub.2O].sup.+.

P 2,2-difluoro-3,7-dioxo-5β-cholanic acid (Comparative Compound E)+hydrate (3P.1)

[0479] ##STR00065##

[0480] Using general procedure C, methyl 2,2-difluoro-3,7-dioxo-5β-cholanoate and the hydrate (60 mg, 0.14 mmol, 1 equiv) were hydrolysed to yield 2,2-difluoro-3,7-dioxo-5β-cholanic acid (Compound E) and the hydrate as a white solid, as a mixture of ketone/hydrate/acetal adducts (55 mg, 0.13 mmol, 93%).

[0481] .sup.1H NMR (400 MHz, CD3CN): δ 2.93 (1H, dd, J=13.1, 5.9 Hz), 2.90-2.84 (1H, m), 2.70-1.97 (9H, m), 1.93-0.93 (19H, m), 0.91 (2H, d, J=6.4 Hz), 0.89 (1H, d, J=6.4 Hz), 0.67 (2H, s), 0.64 (1H, s) ppm.

[0482] .sup.19F NMR (376 MHz, CD3CN): 6-103.96 (1F, ddd, J=261.6, 38.1, 15.0 Hz), −108.59 (0.2F, ddd, J=246.2, 39.0, 11.3 Hz), −110.91 (1F, dq, J=263.6, 5.2 Hz), −113.71-112.69 (0.1F, dq, J=246.2, 5.2 Hz), −116.15 (0.1F, dq, J=246.2, 5.2 Hz), −117.09 (0.2F, dq, J=246.2, 5.2 Hz) ppm.

[0483] LRMS (ESI.sup.+) m/z: Ketone: 425.5 [M+H].sup.+; Hydrate: 443.5 [M+H].sup.+; Hemi-acetal: 457.7 [M+H].sup.+, 439.5 [M+H−H.sub.2O].sup.+; Acetal: 443.5 [M+H].sup.+, 439.5 [M+H-MeOH].sup.+.

Q. 2,2-difluoro-3α,7β-dihydroxy-5β-cholanic acid (Compound 9)

[0484] ##STR00066##

[0485] Using general procedure C, methyl 2,2-difluoro-3α,7β-dihydroxy-5β-cholanoate (25 mg, 0.06 mmol, 1 equiv) was hydrolysed to yield 2,2-difluoro-3α,7β-dihydroxy-5β-cholanic acid (Compound 9) as a gummy solid (20 mg, 0.05 mmol, 78%).

[0486] .sup.1H NMR (400 MHz, CD30D): 53.69 (1H, ddt, J=21.0, 11.0, 5.0 Hz), 3.44 (1H, ddd, J=11.5, 9.8, 5.0 Hz), 2.38-2.26 (2H, m), 2.25-2.14 (2H, m), 2.08-1.06 (32H, m), 1.03 (3H, s), 0.96 (3H, d, J=6.5 Hz), 0.72 (3H, s) ppm.

[0487] .sup.19F NMR (376 MHz, CD30D): 5-101.64 (dquin, J=239.3, 5.0 Hz), −120.18 (dddd, J=239.3, 38.2, 20.8, 10.4 Hz) ppm.

[0488] LRMS (ESI.sup.+) m/z: 411.4 [M+H−H.sub.2O].sup.+, 393.3 [M+H−2H.sub.2O].sup.+.

Example 4—Synthesis of Conjugates of Compound 9

A. Sodium N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-ethylsulfonic acid (Compound 10)

[0489] ##STR00067##

[0490] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (7.4 mg, 0.017 mmol) was conjugated to yield sodium N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-ethylsulfonic acid as a clear residue (7.4 mg, 77%).

[0491] .sup.1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.61-3.56 (2H, m), 3.40 (1H, ddd, J=14.6, 9.7, 5.2 Hz), 2.95 (2H, app. t., J=6.9 Hz), 2.24 (1H, ddd, J=15.0, 10.6, 5.2 Hz), 2.12-1.07 (23H, m), 1.05 (3H, s), 0.97 (3H, d, J=6.5 Hz), 0.71 (3H, s) ppm.

[0492] .sup.19F NMR (376 MHz, MeOD) 5-101.00 (d, J=251.4 Hz), −105.55 (ddt, J=250.3, 40.3, 7.8 Hz) ppm.

[0493] LRMS (ESI.sup.−): [M-Na].sup.− Calcd. 534.2706; found 534.2709.

B. Sodium N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-propanoic acid (Compound 11)

[0494] ##STR00068##

[0495] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield sodium N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-propanoic acid as a clear residue (17.66 mg, 73%).

[0496] .sup.1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.45-3.35 (3H, m), 2.40-2.30 (2H, m), 2.23 (1H, m), 2.12-1.06 (23H, m), 1.05 (3H, s), 0.96 (3H, d, J=6.4 Hz), 0.71 (3H, s) ppm.

[0497] .sup.19F NMR (376 MHz, MeOD) 5-100.62 (d, J=250.4 Hz), −105.55 (ddt, J=250.4, 40.8, 7.6 Hz) ppm.

[0498] HRMS (ESI.sup.+): [M+Na].sup.+ Calcd. 522.3002; found 522.3007.

C. N-(methyl),N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-acetic acid (Compound 12)

[0499] ##STR00069##

[0500] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (25.36 mg, 0.059 mmol) was conjugated to yield N-(methyl),N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-acetic acid as a clear residue (13.3 mg, 45%).

[0501] .sup.1H NMR (400 MHz, MeOD) δ 3.96 (2H, br. d, J=18.1 Hz), 3.78 (1H, br. s), 3.40 (1H, m), 3.08 (1.5H, s), 2.94 (1.5H, s), 2.48 (1H, m), 2.41-1.06 (23H, m), 1.05 (1.5H, s), 1.05 (1.5H, s), 1.00 (1.5H, d, J=6.5 Hz), 0.96 (1.5H, d, J=6.5 Hz), 0.73 (1.5H, s), 0.71 (1.5H, s) ppm;

[0502] .sup.19F NMR (376 MHz, MeOD) 5-100.62 (d, J=250.7 Hz), −105.55 (ddt, J=250.0, 40.2, 7.2 Hz) ppm.

[0503] HRMS (ESI.sup.+): [M+Na].sup.+ Calcd. 522.3002; found 522.2998.

D. Sodium N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-trans-2-cyclohexane carboxylic acid (Compound 13)

[0504] ##STR00070##

[0505] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield Sodium N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-trans-2-cyclohexane carboxylic acid as a clear residue (18.18 mg, 68%).

[0506] .sup.1H NMR (400 MHz, MeOD) δ 3.89-3.72 (2H, m), 3.40 (1H, m), 2.28-1.06 (33H, m), 1.05 (3H, s), 0.96 (3H, d, J=6.5 Hz), 0.70 (3H, s) ppm.

[0507] .sup.19F {.sup.1H} NMR (376 MHz, MeOD) 5-100.61 (d, J=249.4 Hz), −105.55 (d, J=250.2, 4 Hz) ppm.

[0508] HRMS (ESI.sup.+): [M+H].sup.+ Calcd. 554.3652; found 554.3656.

E. Sodium 1-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-piperidine-3-carboxylate (Compound 14)

[0509] ##STR00071##

[0510] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield sodium 1-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-piperidine-3-carboxylate as a residue (21.88 mg, 83%).

[0511] .sup.1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.41 (1H, ddd, J=14.8, 9.9, 5.2 Hz), 2.57-1.07 (33H, m), 1.05 (3H, s), 0.99 (3H, d, J=6.5 Hz), 0.72 (3H, s) ppm.

[0512] .sup.19F NMR (376 MHz, MeOD) 5-100.63 (d, J=250.3 Hz), −105.55 (ddt, J=249.8, 40.9, 7.8 Hz) ppm.

[0513] HRMS (ESI.sup.+): [M+H].sup.+ Calcd. 540.3495; found 540.3507.

F. Sodium 3-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-4-thiazolidine-carboxylate (Compound 15)

[0514] ##STR00072##

[0515] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield sodium 3-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-4-thiazolidine-carboxylate as a residue (21.41 mg, 81%).

[0516] .sup.1H NMR (400 MHz, MeOD) δ 4.90-4.78 (0.4H, m), 4.77 (0.6H, d, J=9.8 Hz), 4.71 (0.4H, d, J=8.4 Hz), 4.60 (0.4H, d, J=8.4 Hz), 4.59 (0.6H, m), 4.48 (0.6H, d, J=9.8 Hz), 3.78 (1H, br. s), 3.46-3.15 (3H, m), 2.58-1.07 (24H, m), 1.04 (3H, br. s), 0.99 (1.2H, d, J=6.5 Hz), 0.96 (1.8H, d, J=6.5 Hz), 0.73 (1.2H, s), 0.71 (1.8H, s) ppm.

[0517] .sup.19F NMR (376 MHz, MeOD) 5-100.63 (d, J=249.1 Hz), −105.52 (ddt, J=250.1, 41.4, 7.8 Hz) ppm.

[0518] HRMS (ESI.sup.+): [M+H].sup.+ Calcd. 544.2903; found 544.2904.

G. N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-morpholine (Compound 16)

[0519] ##STR00073##

[0520] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-morpholine as a clear residue (9.83 mg, 65%).

[0521] .sup.1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.72-3.59 (4H, m), 3.59-3.50 (4H, m), 3.40 (1H, ddd, J=14.8, 9.7, 5.2 Hz), 2.42 (1H, m), 2.30 (1H, m), 2.13-1.06 (22H, m), 1.05 (3H, s), 0.99 (3H, d, J=6.5 Hz), 0.72 (3H, s) ppm.

[0522] .sup.19F NMR (376 MHz, MeOD) 5-101.64 (d, J=250.3 Hz), −105.55 (ddt, J=249.6, 40.6, 7.8 Hz) ppm

[0523] HRMS (ESI.sup.+): [M+H].sup.+ Calcd. 498.3389; found 498.3394.

H. Sodium N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-methylcarboxylic acid (Compound 17)

[0524] ##STR00074##

[0525] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield sodium N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-methylcarboxylic acid as a clear residue (18.12 mg, 77%).

[0526] .sup.1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.75 (2H, s), 3.40 (1H, ddd, J=14.8, 9.7, 5.2 Hz), 2.30 (1H, m), 2.13-1.06 (23H, m), 1.05 (3H, s), 0.98 (3H, d, J=6.4 Hz), 0.72 (3H, s) ppm.

[0527] .sup.19F NMR (376 MHz, MeOD) 5-101.61 (d, J=249.7 Hz), −105.55 (ddt, J=249.6, 40.6, 7.8 Hz) ppm.

[0528] HRMS (ESI.sup.+): [M+H].sup.+ Calcd. 486.3026; found 486.3029.

I. Disodium N-(carboxymethyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-2-amino acetate (Compound 18)

[0529] ##STR00075##

[0530] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield disodium N-(carboxymethyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-2-amino acetate as a clear residue (14.81 mg, 59%).

[0531] .sup.1H NMR (400 MHz, MeOD) δ 4.00 (4H, d, J=19.4 Hz), 3.78 (1H, br. s), 3.40 (1H, ddd, J=14.8, 9.7, 5.2 Hz), 2.42 (1H, m), 2.23 (1H, m), 2.14-1.07 (22H, m), 1.05 (3H, s), 0.96 (3H, d, J=6.4 Hz), 0.71 (3H, s) ppm.

[0532] .sup.19F NMR (376 MHz, MeOD) 5-100.65 (d, J=250.7 Hz), −105.55 (ddt, J=249.6, 40.6, 7.8 Hz) ppm.

[0533] HRMS (ESI.sup.+): [M+H].sup.+ Calcd. 544.3080; found 544.3081.

J. Sodium N-(methyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide) ethylsulfonic acid (Compound 19)

[0534] ##STR00076##

[0535] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield sodium N-(methyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide)-ethylsulfonic acid as a clear residue (9.49 mg, 36%).

[0536] .sup.1H NMR (400 MHz, MeOD) δ 3.84-3.68 (3H, m), 3.40-3.36 (1H, m), 3.11 (1.5H, s) 3.08-2.99 (2H, m), 2.92 (1.5H, m), 2.57-1.07 (23H, m), 1.05 (3H, s), 0.99 (1.5H, d, J=6.5 Hz), 0.98 (1.5H, d, J=6.5 Hz), 0.72 (3H, s) ppm.

[0537] .sup.19F NMR (376 MHz, MeOD) 5-100.64 (d, J=249.4 Hz), −105.54 (ddt, J=250.3, 40.3, 7.8 Hz) ppm.

[0538] HRMS (ESI.sup.+): [M+Na].sup.+ Calcd. 572.2828; found 572.2830.

K. Sodium 3-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl) amino-propanesulfonate (Compound 20)

[0539] ##STR00077##

[0540] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (25.0 mg, 0.058 mmol) was conjugated to yield sodium 3-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl) amino-propanesulfonate as a clear residue (25.44 mg, 76%).

[0541] .sup.1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.40 (1H, ddd, J=14.6, 9.7, 5.2 Hz), 3.33-3.25 (2H, m), 2.86-2.78 (2H, m), 2.24 (1H, m), 2.17-1.07 (25H, m), 1.05 (3H, s), 0.97 (3H, d, J=6.5 Hz), 0.72 (3H, s) ppm.

[0542] .sup.19F NMR (376 MHz, MeOD) 5-100.59 (d, J=250.1 Hz), −105.55 (ddt, J=249.4, 40.8, 7.5 Hz) ppm.

[0543] HRMS (ESI.sup.+): [M+Na].sup.+ Calcd. 594.2647; found 594.2648.

L Sodium N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide) methanesulfonic acid (Compound 21)

[0544] ##STR00078##

[0545] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (25.0 mg, 0.058 mmol) was conjugated to yield sodium N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-amide) methanesulfonic acid as a clear residue (27.63 mg, 87%).

[0546] .sup.1H NMR (400 MHz, MeOD) δ 4.32 (1H, d, J=17.8 Hz), 4.28 (1H, d, J=17.8 Hz), 3.78 (1H, br. s), 3.41 (1H, ddd, J=14.6, 9.8, 5.1 Hz), 2.33 (1H, m), 2.25-1.07 (23H, m), 1.05 (3H, s), 0.98 (3H, d, J=6.4 Hz), 0.72 (3H, s) ppm.

[0547] .sup.19F NMR (376 MHz, MeOD) 5-100.61 (d, J=250.8 Hz), −105.55 (ddt, J=249.4, 40.8, 7.5 Hz) ppm.

[0548] HRMS (ESI.sup.+): [M−2H+D+2Na].sup.+ Calcd. 567.2397; found 567.2387.

M. Sodium N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-2-aminoethyl sulfate (Compound 22)

[0549] ##STR00079##

[0550] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (30.0 mg, 0.070 mmol) was conjugated to yield sodium N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-H NMR (400 MHz, MeOD) δ 4.04 (2H, app. t, J=5.5 Hz), 3.78 (1H, br. s), 3.44 (2H, app. t, J=5.5 Hz), 3.41 (1H, m), 2.26 (1H, ddd, J=15.5, 10.5, 5.2 Hz), 2.18-1.06 (23H, m), 1.05 (3H, s), 0.98 (3H, d, J=6.5 Hz), 0.71 (3H, s) ppm.

[0551] .sup.19F NMR (376 MHz, MeOD) 5-100.59 (d, J=250.0 Hz), −105.48 (ddt, J=249.4, 40.8, 7.5 Hz) ppm.

[0552] HRMS (ESI.sup.+): [M+Na].sup.+ Calcd. 574.2621; found 574.2620.

N. Sodium O-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-2-hydroxy ethyl sulfonic acid (Compound 23)

[0553] ##STR00080##

[0554] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield sodium 0-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-2-hydroxy ethyl sulfonic acid as a clear residue (15.06 mg, 58%).

[0555] .sup.1H NMR (400 MHz, MeOD) δ 4.42 (2H, app. t, J=7.0 Hz), 3.78 (1H, br. s), 3.41 (1H, ddd, J=14.8, 9.8, 5.0 Hz), 3.12 (2H, app. t, J=7.2 Hz), 2.39 (1H, m), 2.26 (1H, m), 2.14-1.06 (22H, m), 1.05 (3H, s), 0.95 (3H, d, J=6.7 Hz), 0.71 (3H, s) ppm.

[0556] .sup.19F NMR (376 MHz, MeOD) 5-100.65 (d, J=250.5 Hz), −105.48 (ddt, J=249.8, 40.9, 7.6 Hz) ppm.

[0557] HRMS (ESI.sup.+): [M+Na].sup.+ Calcd. 581.2331; found 581.2332.

O. Sodium N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl) aniline-2-sulfonic acid (Compound 24)

[0558] ##STR00081##

[0559] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield sodium N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl) aniline-2-sulfonic acid as a white residue (24.04 mg, 85%).

[0560] .sup.1H NMR (400 MHz, MeOD) δ 8.30 (1H, d, J=8.2 Hz), 7.86 (1H, dd, J=7.9, 1.5 Hz), 7.40 (1H, m), 7.12 (1H, m), 3.79 (1H, br. s), 3.41 (1H, ddd, J=14.8, 9.8, 5.0 Hz), 2.50 (1H, m), 2.34 (1H, m), 2.15-1.08 (22H, m), 1.05 (3H, s), 1.02 (3H, d, J=6.3 Hz), 0.73 (3H, s) ppm.

[0561] .sup.19F NMR (376 MHz, MeOD) 5-100.62 (d, J=251.2 Hz), −105.49 (ddt, J=249.3, 40.6, 7.2 Hz) ppm.

[0562] HRMS (ESI.sup.+): [M−H+2Na].sup.+ Calcd. 628.2491; found 628.2494.

P. Sodium N-(cyclohexyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-3-amino-propanesulfonate (Compound 25)

[0563] ##STR00082##

[0564] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield sodium N-(cyclohexyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-3-amino-propanesulfonate as a clear residue (27.97 mg, 92%).

[0565] .sup.1H NMR (400 MHz, MeOD) δ 4.17 (0.4H, m), 3.78 (1H, br. s), 3.62 (0.6H, m), 3.45-3.33 (3H, m), 2.87-2.75 (2H, m), 2.41 (1H, m), 2.30 (1H, m), 2.14-1.07 (34H, m), 1.05 (3H, br. s), 1.02-0.97 (3H, m), 0.73 (1.8H, s), 0.72 (1.2H, s) ppm.

[0566] .sup.19F NMR (376 MHz, MeOD) 5-100.60 (d, J=249.8 Hz), −105.48 (ddt, J=250.1, 41.0, 7.6 Hz) ppm.

[0567] HRMS (ESI.sup.+): [M+Na].sup.+ Calcd. 654.3610; found 654.3606.

Q. Sodium N-(cyclohexyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-2-amino-ethanesulfonate (Compound 26)

[0568] ##STR00083##

[0569] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield sodium N-(cyclohexyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-2-amino-ethanesulfonate as a clear residue (28.41 mg, 95%).

[0570] .sup.1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.73-3.56 (3H, m), 3.40 (1H, ddd, J=14.7, 9.7, 5.0 Hz), 3.09-2.96 (2H, m), 2.44 (1H, m), 2.31 (1H, m), 2.15-1.07 (32H, m), 1.05 (3H, br. s), 1.02-0.97 (3H, m), 0.73 (1.8H, s), 0.72 (1.2H, s) ppm.

[0571] .sup.19F NMR (376 MHz, MeOD) 5-100.57 (d, J=251.0 Hz), −105.49 (ddt, J=250.3, 40.9, 7.0 Hz) ppm.

[0572] HRMS (ESI.sup.+): [M−H+2Na].sup.+ Calcd. 662.3273; found 662.3285.

R. N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl) 2-aminoethyl methyl sulfone (Compound 27)

[0573] ##STR00084##

[0574] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl) 2-aminoethyl methyl sulfone as a clear residue (22.49 mg, 90%).

[0575] .sup.1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.62 (2H, app. t, J=6.7 Hz), 3.40 (1H, ddd, J=14.7, 9.6, 4.9 Hz), 3.29 (2H, app. t, J=6.5 Hz), 2.99 (3H, s), 2.26 (1H, ddd, J=15.3, 10.4, 5.3 Hz), 2.17-1.06 (23H, m), 1.05 (3H, s), 0.97 (3H, d, J=6.4 Hz), 0.71 (3H, s) ppm. .sup.19F NMR (376 MHz, MeOD) 5-100.60 (d, J=249.7 Hz), −105.49 (ddt, J=249.9, 41.2, 7.1 Hz) ppm.

[0576] HRMS (ESI.sup.+): [M+Na].sup.+ Calcd. 556.2879; found 556.2873.

S. N-(ethyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-3-amino-tetrahydrothiophene dioxide (Compound 28)

[0577] ##STR00085##

[0578] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield N-(ethyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-3-amino-tetrahydrothiophene dioxide as a clear residue (20.71 mg, 77%).

[0579] .sup.1H NMR (400 MHz, MeOD) δ 4.56 (1H, m), 3.78 (1H, br. s), 3.51 (4H, m), 3.33-3.21 (2H, m), 3.08 (1H, m), 2.63-1.06 (29H, m), 1.05 (3H, s), 0.99 (3H, d, J=6.5 Hz), 0.72 (3H, s) ppm.

[0580] .sup.19F NMR (376 MHz, MeOD) 5-100.59 (d, J=250.4 Hz), −105.48 (ddt, J=250.4, 41.5, 7.4 Hz) ppm.

[0581] HRMS (ESI.sup.+): [M+H].sup.+ Calcd. 574.3372; found 574.3370.

T. N-(2-(diisopropylamino)ethyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-3-amino-tetrahydrothiophene dioxide (Compound 29)

[0582] ##STR00086##

[0583] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield N-(2-(diisopropylamino)ethyl)-N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-3-amino-tetrahydrothiophene dioxide as a residue (23.90 mg, 76%).

[0584] .sup.1H NMR (400 MHz, MeOD) δ 3.78 (1H, br. s), 3.50-0.88 (m), 0.72 (3H, s) ppm.

[0585] .sup.19F NMR (376 MHz, MeOD) 5-100.56 (d, J=250.5 Hz), −105.49 (m) ppm.

[0586] HRMS (ESI.sup.+): [M+H].sup.+ Calcd. 673.4420; found 673.4430.

U. N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-thiomorpholine-dioxide (Compound 30)

[0587] ##STR00087##

[0588] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl)-thiomorpholine-dioxide as a clear residue (22.2 mg, 87%).

[0589] .sup.1H NMR (400 MHz, MeOD) δ 4.09-3.93 (4H, m), 3.78 (1H, br. s), 3.40 (1H, ddd, J=14.6, 9.6, 5.1 Hz), 3.23-3.04 (4H, m), 2.52 (1H, m), 2.38 (1H, m), 2.14-1.07 (22H, m), 1.05 (3H, s), 0.99 (3H, d, J=6.6 Hz), 0.73 (3H, s) ppm.

[0590] .sup.19F NMR (376 MHz, MeOD) 5-100.63 (d, J=249.3 Hz), −105.54 (ddt, J=250.6, 41.6, 7.5 Hz) ppm.

[0591] HRMS (ESI.sup.+): [M+H].sup.+ Calcd. 546.3059; found 546.3065.

V. N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl) 1,1-dioxidotetrahydro-2H-thiopyran-3-ylamine (Compound 31)

[0592] ##STR00088##

[0593] Using general procedure Q, 2,2-difluoro-3α,7α-dihydroxy-5β-cholanic acid (20.0 mg, 0.047 mmol) was conjugated to yield N-(2,2-difluoro-3β,7β-dihydroxy-5β-cholan-24-oyl) 1,1-dioxidotetrahydro-2H-thiopyran-3-ylamine as a residue (22.44 mg, 88%).

[0594] .sup.1H NMR (400 MHz, MeOD) δ 4.26 (1H, tt, J=11.1, 3.6 Hz), 3.78 (1H, br. s), 3.40 (1H, ddd, J=14.5, 10.0, 5.1 Hz), 3.27 (1H, m), 3.08-2.91 (3H, m), 2.30-1.06 (26H, m), 1.05 (3H, s), 0.97 (3H, d, J=6.5 Hz), 0.71 (3H, s) ppm.

[0595] .sup.19F NMR (376 MHz, MeOD) 5-100.63 (d, J=250.3 Hz), −105.49 (ddt, J=250.7, 41.5, 7.9 Hz) ppm.

[0596] HRMS (ESI.sup.+): [M+H].sup.+ Calcd. 560.3216; found 560.3217.

Comparative Example 5—Synthesis of 2-fluoroalkene Compounds

A. Methyl 2-fluoro-3β,7β-dihydroxy-5β-chol-1-enoate (4A.1)

[0597] ##STR00089##

[0598] Using general procedure L, methyl 2-fluoro-3β,7β-dimethoxymethoxyl-5β-chol-1-enoate from Example 4J (900 mg, 1.76 mmol, 1 equiv) was deprotected to yield methyl 2-fluoro-3β,7β-dihydroxy-5β-chol-1-enoate as a white gummy solid (750 mg, quantitative yield).

[0599] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.34 (1H, d, J=17.6 Hz), 4.20 (1H, ddd, J=7.7, 4.7, 1.3 Hz), 3.67 (3H, s), 3.50 (1H, ddd, J=11.2, 9.6, 4.8 Hz), 2.35 (1H, ddd, J=15.6, 10.5, 5.0 Hz), 2.22 (1H, ddd, J=15.6, 9.4, 6.5 Hz), 2.12 (1H, td, J=14.0, 5.3 Hz), 2.04-1.21 (23H, m), 1.12 (3H, d, J=0.7 Hz), 0.92 (3H, d, J=6.5 Hz), 0.70 (3H, s) ppm.

[0600] .sup.19F NMR (376 MHz, CDCl.sub.3): 5-117.65 (dt, J=17.0, 8.5 Hz) ppm.

[0601] LRMS (ESI.sup.+) m/z: 405.5 [M+H−H.sub.2O].sup.+, 387.5 [M+H−2H.sub.2O].sup.+.

B. Methyl 2-fluoro-3,7-di-oxo-5β-chol-1-enoate (4B.1)

[0602] ##STR00090##

[0603] Using general procedure M, methyl 2-fluoro-3β,7β-dihydroxy-5β-chol-1-enoate (600 mg, 1.42 mmol, 1 equiv) was oxidised, then purified via flash chromatography (Petrol ether/EtOAc: 80:20->70:30->60/40) to yield methyl 2-fluoro-3,7-di-oxo-5β-chol-1-enoate as a gummy solid (400 mg, 0.96 mmol, 67%).

[0604] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 6.34 (1H, d, J=14.7 Hz), 3.66 (3H, s), 2.89 (1H, dd, J=12.8, 5.9 Hz), 2.61 (1H, dtd, J=13.5, 5.8, 2.0 Hz), 2.55-2.11 (7H, m), 2.07 (1H, dd, J=13.2, 2.0 Hz), 2.04-1.71 (5H, m), 1.51 (3H, s), 1.49-0.94 (8H, m), 0.92 3H, (d, J=6.4 Hz), 0.70 (3H, s) ppm.

[0605] .sup.19F NMR (376 MHz, CDCl.sub.3): δ −131.67 (dd, J=15.6, 3.5 Hz), ppm.

[0606] LRMS (ESI.sup.+) m/z: 419.5 [M+H].sup.+, 460.5 [M+H+MeCN].sup.+.

C. Methyl 2-fluoro-3α,7α-dihydroxy-5β-chol-1-enoate (4C.1)

[0607] ##STR00091##

[0608] Using general procedure B, methyl 2-fluoro-3β,7β-dihydroxy-5β-chol-1-enoate (400 mg, 0.96 mmol, 1 equiv) was reduced, then purified via flash chromatography (Petrol ether/EtOAc: 70:30->60:40->50:50) to yield methyl 2-fluoro-3α,7α-dihydroxy-5β-chol-1-enoate as a gummy solid (99 mg, 0.22 mmol, 25%).

[0609] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.25 (1H, d, J=18.2 Hz), 4.35 (1H, t, J=7.9 Hz), 3.85 (1H, q, J=1.7 Hz), 3.67 (3H, s), 3.65 (1H, d, J=1.8 Hz), 2.53-2.32 (2H, m), 2.29-2.17 (1H, m), 2.04-1.07 (29H, m), 1.04 (3H, d, J=0.9 Hz), 0.93 (3H, d, J=6.4 Hz), 0.68 (3H, s) ppm.

[0610] .sup.19F NMR (376 MHz, CDCl.sub.3): δ −125.36 (dd, J=19.1, 6.9 Hz) ppm.

[0611] LRMS (ESI.sup.+) m/z: 405.5 [M+H−H.sub.2O].sup.+, 387.5 [M+H−2H.sub.2O].sup.+.

D 2-fluoro-3β,7β-dihydroxy-5β-chol-1-enic acid (Comparative Compound F)

[0612] ##STR00092##

[0613] Using general procedure C, methyl 2-fluoro-3β,7β-dihydroxy-5β-chol-1-enoate from step A (60 mg, 0.14 mmol, 1 equiv) was hydrolysed to yield 2-fluoro-3β,7β-dihydroxy-5β-chol-1-enic acid (Compound F) as a white solid (40 mg, 0.10 mmol, 70%).

[0614] .sup.1H NMR (400 MHz, CD.sub.3OD): δ 5.32 (1H, d, J=17.7 Hz), 4.10 (1H, ddd, J=8.0, 4.5, 1.0 Hz), 3.38 (1H, td, J=10.5, 4.8 Hz), 2.40-1.15 (29H, m), 1.13 (3H, s), 0.95 (3H, d, J=6.5 Hz), 0.73 (3H, s) ppm.

[0615] .sup.19F NMR (376 MHz, CD.sub.3OD): 6-117.71 (dt, J=15.6, 7.6 Hz) ppm.

[0616] LRMS (ESI.sup.+) m/z: 391.5 [M+H−H.sub.2O].sup.+, 373.5 [M+H−2H.sub.2O].sup.+.

E. 2-fluoro-3,7-dioxo-5β-chol-1-enic acid (Comparative Compound G)

[0617] ##STR00093##

[0618] Using general procedure C, methyl 2-fluoro-3,7-di-oxo-5β-chol-1-enoate from step B (50 mg, 0.12 mmol, 1 equiv) was hydrolysed to yield 2-fluoro-3,7-dioxo-5β-chol-1-enic acid (Compound G) as a white solid (45 mg, 0.11 mmol, 93%).

[0619] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 6.34 (1H, d, J=14.7 Hz), 2.89 (1H, dd, J=13.0, 6.1 Hz), 2.62 (1H, dtd, J=13.5, 5.8, 2.0 Hz), 2.55-1.73 (13H, m), 1.51 (3H, s), 1.49-0.95 (9H, m), 0.93 (3H, d, J=6.4 Hz), 0.70 (3H, s) ppm.

[0620] .sup.19F NMR (376 MHz, CDCl.sub.3): δ −131.63 (dd, J=13.9, 3.5 Hz) ppm.

[0621] LRMS (ESI.sup.+) m/z: 405.4 [M+H].sup.+, 446.5 [M+H+MeCN].sup.+.

F. 2-fluoro-3α,7α-dihydroxy-5β-chol-1-enic acid (Comparative Compound H)

[0622] ##STR00094##

[0623] Using general procedure C, methyl 2-fluoro-3α,7α-dihydroxy-5β-chol-1-enoate from step C (50 mg, 0.11 mmol, 1 equiv) was hydrolysed to yield 2-fluoro-3α,7α-dihydroxy-5β-chol-1-enic acid (Compound H) as a pale solid (45 mg, 0.11 mmol, 97%).

[0624] .sup.1H NMR (400 MHz, Acetone-D.sub.6): δ 5.16 (1H, d, J=18.6 Hz), 4.23 (1H, ddd, J=9.0, 7.0, 2.5 Hz), 4.07 (1H, br. s), 3.82 (1H, q, J=2.7 Hz), 3.32 (1H, br. s), 2.54 (1H, td, J=13.7, 10.0 Hz), 2.34 (1H, ddd, J=15.5, 11.0, 5.0 Hz), 2.21 (1H, ddd, J=15.6, 9.4, 6.5 Hz), 2.02-1.06 (25H, m), 1.05 (3H, d, J=1.0 Hz), 0.96 (3H, d, J=6.6 Hz), 0.71 (3H, s) ppm.

[0625] .sup.19F NMR (376 MHz, Acetone-D.sub.6): 5-123.32 (dd, J=19.1, 6.9 Hz) ppm.

[0626] LRMS (ESI.sup.+) m/z: 373.5 [M+H−2H.sub.2O].sup.+.

BIOLOGICAL EXAMPLES

Abbreviations

[0627]

TABLE-US-00001 BDNF Brain-derived neurotrophic factor Cy3 Cyanine 3 DAPI 4′,6-diamidino-2-phenylindole DAT Anti-dopamine transporter antibody dcAMP Dibutyryl cyclic adenosine monophosphate DMEM Dulbecco Modified Eagle Medium EDTA Ethylene diamine tetra-acetic acid FGFb Basic fibroblast growth factor FGF8 Fibroblast growth factor 8 GDNF Glial cell line-derived neurotrophic factor HBSS Hanks' balanced salt solution HEPES 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid iNPC Induced neural progentitor cell MEM Minimum essential medium MMP Mitochondrial membrane potential MPTP 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine NEEA Non-essential amino Acid cell culture supplement PBS Phosphate buffered saline PD Parkinson's disease (sPD is sporadic Parkinson's disease) TGF-b3 Transforming growth factor geta-3 TMRM Tetramethylrhodamine methyl ester perchlorate Tuj Anti-beta-tubulin III antibody

Example 6

A. Culture of Primary Fibroblasts, Generation and Culture of iNPC's

[0628] Fibroblasts were cultured in DMEM (Invitrogen) and routinely subcultured every 3-5 days using trypsin to dissociate them. Induced neural progenitor cells (iNPC's) were generated as previously described (Meyer et al, “Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS” Proc Natl Acad Sci USA 2014). iNPC's were maintained in DMEM/Ham F12 (Invitrogen); N2, B27 supplements (Invitrogen) and FGFb (Peprotech) in fibronectin (Millipore) coated tissue culture dishes and routinely subcultured every 2-3 days using accutase to detach them.

B. Dopaminergic Neuron Differentiation of iNPC's

[0629] Briefly, iNPCs are plated in a 6-well plate and cultured for 2 days in DMEM/F-12 medium with Glutamax™ supplemented with 1% NEAA, 2% B27 (Gibco) and 2.5 μM of DAPT. On day 3, DAPT is removed and the medium is supplemented with 1 μM smoothened agonist (SAG) and FGF8 (75 ng/ml) for additional 10 days. Neurons are replated at this stage. Subsequently SAG and FGF8 are withdrawn and replaced with BDNF (30 ng/ml), GDNF (30 ng/ml), TGF-b3 (2 mM) and dcAMP (2 mM, Sigma) for 15 days.

C. Immunofluorescence Staining and ELISA

[0630] Cells are plated into 96 well plates and fixed using 4% paraformaldehyde for 30 minutes. After PBS washes cells are permeabilised using 0.1% Triton™ X-100 for 10 minutes and blocked using 5% goat serum for 1 hour. Cells are incubated with primary antibodies tyrosine hydroxylase (St John's Laboratory); DAT (Abcam); Tuj (Millipore); Tom20 (BD Biosciences); activated caspase 3 (Cell Signaling); alpha synuclein (Cell Signaling); phosphorylated alpha synuclein (Millipore) at 4° C. for 16 hours. Cells are washed using PBS-Tween® and incubated with Alexa Fluor™-conjugated secondary antibodies 488 and 568 (Invitrogen) and Hoescht (Sigma) 1 μM prior to imaging. Imaging was performed using the Opera Phenix™ high content imaging system (Perkin Elmer).

[0631] Dopamine ELISA is performed using Dopamine research ELISA kit (Labor Diagnostika Nord GmbH&Co. KG) as per manufacturers instructions. Dopamine release is obtained incubating the cells at 37° C. using three different conditions at the same time per line. Medium is removed in all wells then the first well is incubated with HBSS with Ca.sup.2+ and Mg.sup.2+ (Gibco by Life Technologies) for 30 minutes, the second well is incubated in HBSS with Ca.sup.2+ and Mg.sup.2+ for 15 minutes and then 56 mM KCl (Fisher chemical) is added for another 15 minutes and the third well is incubated with HBSS without Ca.sup.2+ and Mg.sup.2+ (Gibco by Life Technologies) but with 2 mM EDTA for 15 min and then 56 mM KCl is added for another 15 minutes. Straight away media is collected in an eppendorf and cells are harvested using Accutase®, centrifuge at 400 g for 4 min and resuspended in 10 μl of PBS. EDTA 1 mM and Sodium Metabisulfite (Sigma) 4 mM are added to both the media and pellet to preserve the dopamine.

[0632] MMP Protocol

[0633] Fibroblasts were cultured and plated into a Griener black 384 pClear® plate at 10000 cells per well in 50 μl of media volume. The plates are left overnight in an incubator to allow the fibroblasts to adhere to the plate surface. The following morning the Glucose based medium is replaced with 25 μl of Galactose based media. The plates were then dosed with the compounds using an ECHO® 550 liquid handling system. The wells were dosed to provide an 8-point concentration range of 0.06 nM-300 nM of compound. After dosing the wells are topped up with a further 25 μl of Galactose based medium and then left in an incubator for 24 hours. After 24 hours, the medium is removed from the wells and replaced with 25 ul phenol free Minimal essential medium with 100 nM TMRM (Sigma) and 10 μM Hoechst Stain (Sigma). The plate is returned to the incubator for another hour after which the stain medium is removed and replaced with 25 ul Phenol free MEM. The plate is then imaged using an IN Cell high content microscope (GE Healthcare) with 10 fields of view per well in 2 channels, Cy3 excitation 542 nm, emission 604-64 nm; and the DAPI excitation 350 nm, emission 450-55 nm at 37° C. with CO.sub.2. After imaging the plate is disposed of and the images are Data mined using the INCell developer Toolkit (GE Healthcare).

[0634] ATP Protocol

[0635] The ATP protocol is generally as described in Mortiboys et al, “Mitochondrial function and morphology are impaired in parkin-mutant fibroblasts”, Ann Neurol. 2008 November; 64(5):555-65. Briefly, fibroblasts were cultured as and plated into white 384 well plates with 5000 cells per well in 50 μl of media volume. The plates are left overnight in an incubator to allow the fibroblasts to adhere to the plate surface. The following morning the Glucose based medium is replaced with 25 μl of Galactose based medium. The compounds are added to the plates using a ECHO 550 liquid handling system. The wells were dosed to provide an 8-point concentration range of 0.06 nM-300 nM of compound. After dosing the wells are topped up with a further 25 μl of Galactose based medium and then left in an incubator for 24 hours. Following this incubation the medium is removed from the plate and the wells are washed twice with sterile PBS. The wells are filled with 25 μl of Sterile PBS followed by 12.5 μl of Lysis solution from the ATPlite™ Luminescence ATP detection assay system (Perkin Elmer), including 16 cell free wells to use as blank controls. The plate is then placed on a rotary shaker for 5 mins at 700 rpm. Following the shaking 12.5 μl of ATP substrate solution (Perkin Elmer) is added to each well and a further 5 min of shaking. The plate is then placed in darkness for 10 minutes prior to reading. Using a PHERAStar® plate reader, luminescence intensity is recorded. Following the ATP assay the plates are immediately assayed for DNA content in a CyQUANT® assay.

[0636] Immediately following The ATP assay DNA content is assessed with the CyQUANT® NF Cell Proliferation Assay Kit (ThermoFisher). CyQUANT® buffer is prepared immediately before the assay and is comprised of 1 ul CyQUANT® dye per ml×1 HBSS solution. 12.5 ul of CyQUANT® buffer is added to each well. Plate left in incubator for 1 hour then read on a PHERAStar® Plate reader with excitation at 497 nm and emission at 520 nm. ATP Quantification for each well is determined using the following formula:

[00001]$\mathrm{ATP}\mathrm{score}=\frac{\mathrm{ATPlite}\mathrm{blank}\mathrm{corrected}\mathrm{value}}{\mathrm{CyQUANT}\mathrm{blank}\mathrm{corrected}\mathrm{value}}$

[0637] Data analysis for primary screen assays.

[0638] After the assays had been repeated in triplicate per line and compound the data was then inputted into Graph pad Prism 7 software suite where a dose response curve is generated using the default “[Agonist] vs response (three parameters)” equation.

[00002]$y=\mathrm{Bottom}+x\times \frac{\mathrm{Top}-\mathrm{Bottom}}{\mathrm{EC}50+x}$

[0639] From this EC.sub.50 values, lowest response and maximal response were taken and used to calculate the Geometric mean between the 5 different lines assessed.

[0640] Results derived from compounds that showed an Ambiguous result from the “[Agonist] vs response (three parameters)” equation were excluded from the Geometric mean calculations due to the high skew that was introduced by their inclusion.

[0641] Seahorse Assay

[0642] Fibroblasts are plated into Seahorse 24 well plates with 50,000 cells per well. Cells are left to attach for 2 days. Media is changed to Seahorse DMEM media with glucose and sodium pyruvate and left to equilibrate at 37 degrees normal air CO.sub.2 for 1 hour. The plate is entered into the Seahorse machine and run on a program of mix (2 minutes), wait (3 minutes) and measure (3 minutes). After three measurements of basal respiration and ECAR; 0.5 μM oligomycin is injected after which another three measurements are taken; then 0.5 μM of FCCP is injected and three measurements taken and finally 1 μM rotenone is injected and three measurements taken. After all measurements are complete, cells are stained with 10 μM Hoescht and imaged using the InCell to count the number of cells per well for normalisation. This classical ritochondrial stress test experimental protocol allows us to calculate the basal mitochondrial respiration, ATP linked respiration (the amount which is coupled to ATP generation), the maximal and spare respiratory capacity, the non-mitochondrial respiration rate and the extracellular acidification rate which is a proxy measure of glycosolysis.

[0643] Complex I Assay

[0644] Ex vivo mice brain was homogenated in a buffer of 250 mM sucrose, 20 mM HEPES, 3 mM EDTA, pH 7.5 at 4° C. Homogenisation was carried out using a Dounce homogenizer, for cortex samples, and by repetitive passage through a 0.5 mm syringe for isolated striatum. Samples were then incubated with 30 μl of detergent from the AbCam colorimetric Complex I assay kit on ice for 20 minutes. Samples are then centrifuged at 13,000 rpm for 30 mins. Triplicate samples per condition were blocked using the kit blocking buffer on the AbCam colorimetric Complex I assay kit plate for 3 hours. Samples are then washed using the kit wash buffer 3 times before the addition of the kit assay buffer containing NADH and colorimetric dye. The assay plate is read on a plate reader in a kinetic assay programme reading 450 nm in a 30 second interval for 50 minutes.

D. Mitochondrial Function and Morphology Measurements in iNeurons

[0645] Cells are plated in 96 well plates; for live imaging cells are incubated for one hour at 37 degree with 80 nM tetramethlyrhodamine (TMRM), 1 μM LysoTracker® Green (Invitrogen) and Hoechst Stain solution (Sigma) at 1 μM before imaging using Opera Phenix™. Cellular ATP measurements are undertaken using ATPlite kit (Perkin Elmer) as per manufacturer's instructions. Mitochondrial reactive oxygen species generation was assessed using mitochondrial NpFR2 (probe; a kind gift from Dr Liz New, University of Sydney, Australia) at 20 μM and Hoechst stain solution at 1 μM for 30 mins at 37° C., then the dyes are removed and cells images using Opera Phenix™. Images generated from the live imaging experiments were analysed using Harmony® (Perkin Elmer software). We developed protocols in order to segment nucleus, cell boundary and processes, mitochondria, lysosomes, autophagosomes. We only analysed the z projection images collected from the z stacks.

[0646] Results

[0647] Fibroblasts

[0648] The mitochondrial membrane potential was measured in fibroblasts from 6 (Table 1) or 3 (Table 2) patients with sporadic Parkinson's disease when treated with Compounds of the invention or Comparator compounds. The results are shown in Tables 1 and 2, where “Bottom”=max response with lowest dose of compound (0.06 nM) and “top”=max response with highest dose of compound (300 nM).

TABLE-US-00002 TABLE 1 Mitochondrial Membrane Potential data from 6 sporadic PD patient fibroblasts Compound E F G H 2 7 9 Bottom (% of vehicle treated) 104.9 110 103.4 99.83 107.3 100.4 109.8 Top (% of vehicle treated) 111.8 108.1 123.3 109.1 112 114 112.4 EC.sub.50 (nM) 23.29 0.6738 282.1 4.581 20.82 4.066 2.186

TABLE-US-00003 TABLE 2 Mitochondrial Membrane Potential data from 3 sporadic PD patient fibroblasts Compound 14 17 19 20 22 27 30 31 Bottom (% of vehicle treated) 94.3 88.9 95.9 99.8 99.3 102.6 98.4 99.6 Top (% of vehicle treated) 166.8 152.3 143.1 147.1 145.8 158.9 155.6 151.1 EC.sub.50 (nM) 18.9 127.6 104.4 17.7 45.4 171.1 21.01 5.5 NB: The sPD patients have a mean reduction in MMP compared to controls of 18%; therefore increase of MMP from the vehicle treated sPD patient level of 118% would restore MMP to control levels.

[0649] Cellular ATP levels were measured in fibroblasts from 6 (Table 3) or 3 (Table 4) patients with sporadic Parkinson's disease when treated with Compounds of the invention or Comparator compounds. The results are shown in Tables 3 and 4, where “Bottom”=max response with lowest dose of compound (0.06 nM) and “top”=max response with highest dose of compound (300 nM).

TABLE-US-00004 TABLE 3 Cellular ATP levels data from 6 sporadic PD patient fibroblasts Compound E F G H 2 7 9 Bottom (% of vehicle treated) 81.59 99.91 83.3 87.64 79.9 112 99.04 Top (% of vehicle treated) 135.5 111.9 92.72 120.8 112.6 171.8 102.9 EC.sub.50 (nM) 18.79 1.912 58.35 0.1247 1.078 0.4293 21.32

TABLE-US-00005 TABLE 4 Cellular ATP levels data from 3 sporadic PD patient fibroblasts Compound 14 17 19 20 22 27 30 31 Bottom (% of vehicle treated) 107 100.7 98.8 102.4 95.9 101.4 96.7 108.1 Top (% of vehicle treated) 126.9 132.2 122.3 121.7 115.9 113.8 115.3 133 EC.sub.50 (nM) 1.3 17.2 8.9 34.7 6.2 16.6 4.2 13.3 NB: sPD fibroblasts have an average reduction of 24% of cellular ATP levels as compared to controls. Therefore a % of vehicle treated sPD fibroblasts of 124% is an increase to control ATP levels.

[0650] The MMP and ATP assays described above along with a toxicity measure comprise the primary screen of the Compounds in primary patient fibroblasts. When considering which compound is most active in the primary screens all information is taken into account including EC50 values indicating potency and % maximal responses for both assays; based upon the combined activity expert biologists take decisions for each compound.

[0651] Oxygen Consumption data obtained from the seahorse assay for 6 sporadic PD patient fibroblast lines and 6 controls are shown in FIGS. 1A, 1B and 1C, from which it can be seen that sPD fibroblasts show a significant reduction of basal mitochondrial respiration of 42% (***p<0.005), maximal respiration of 48% (*p<0.05) and ATP linked respiration of 18% (*p<0.05). This is not improved by treatment with UDCA however treatment with Compound 7 significantly improves all three mitochondrial parameters. Note both compounds are dosed at EC90 concentrations which is 100 nM for UDCA, 50 nM for Compound 7. Therefore it is clear Compound 7 provides a greater increase in mitochondrial function as measured by oxygen consumption and it provides this affect at a lower concentration.

[0652] Extracellular Acidification Rate in 6 sporadic fibroblasts and 6 controls. sPD fibroblasts have a significant reduction in ECAR (a proxy measure of glycolysis) by 44% as compared to controls (***p<0.005). As shown in FIG. 2, this is not altered by treatment with UDCA but is significantly improved by treatment with Compound 7.

[0653] The above data shows the mitochondrial protective effects of the compounds in primary fibroblasts from sPD patients however the cell type which is primarily affected in PD is the dopaminergic neuron. The data below shows the results obtained from dopaminergic neurons derived from three sPD patients; these cultures are approximately 96% dopaminergic neurons and currently this methodology is the only protocol to generate such a pure dopaminergic culture from patient cells (method developed by Mortiboys, University of Sheffield); therefore this is the patient derived model which represents most closely the neurons affected in PD.

[0654] Table 5 below shows the results for mitochondrial function and neuronal morphology measurements in iNeurons from sPD patients vs controls when untreated or when treated with either UDCA or Compound 7.

TABLE-US-00006 TABLE 5 iNeurons % of level in iNeurons from Controls untreated sPD patients sPD patients sPD treated with treated with Parameter patients UDCA Compound 7 ATP normalisation 50 82 101 (% of controls) ATP EC.sub.50 (nM) 5 0.8 MMP normalisation 52 73 100 (% of controls) MMP EC.sub.50 (nM) 7.3 0.52 ROS (Normalisation) 115 110 103 (% controls) Neuronal morphology 48 64 78 (elongation; % controls) Activated caspase 3 162 143 100 levels Dopamine level 0 No effect ~5% increase in dopamine release

[0655] The data in Table 5 show very clearly that Compound 7 provides a protective effect on both mitochondrial parameters in sPD derived dopaminergic neurons in addition to improving neuronal morphology and reducing apoptosis levels (as measured by activated caspase 3 levels). Apoptosis is a major mechanism of cell death of the dopaminergic neurons in culture and of dopaminergic neurons in patients with PD.

[0656] In vivo mouse data with Compound 7.

[0657] FIG. 3 shows that mitochondrial respiratory chain complex I activity is significantly increased in wild type mouse whole brain homogenate after dosing with Compound 7 4 mg/kg and 12 mg/kg in a dose dependent fashion. With a 4 mg/kg dose, the increase is approximately 380% and with 12 mg/kg dose, there is an increase in Complex I activity of approximately 800% compared to untreated controls (*p<0.05).

[0658] FIG. 4 shows results for Complex 1 activity in left striatum mouse brain homogenate of mice dosed with: [0659] Vehicle [0660] Vehicle+12 mg Compound 7 [0661] MPTP [0662] MPTP+1 mg Compound 7 [0663] MPTP+4 mg Compound 7 [0664] MPTP+12 mg Compound 7

[0665] Treatment with Compound 7 alone causes an increase in complex I activity of approximately 30% over untreated controls (*p<0.05). Treatment with MPTP causes a reduction in complex I activity of 50% compared with untreated controls (**p<0.01) but treatment concurrently with 1 mg Compound 7 and MPTP prevents the MPTP induced loss of complex I activity and retains complex I at normal levels (**p<0.01 as compared to MPTP treatment alone), with increased doses of Compound 7 concurrently with MPTP appears to prevent any loss of complex I activity by MPTP.

[0666] Activity of Compounds in Alzheimer's Disease Patient Fibroblasts

[0667] Fibroblasts from both sAD and familial AD (PSEN1 mutants) were tested using the same primary screening assay for total cellular ATP levels. sAD and PSEN1 patient fibroblasts have a reduction of 21% as compared to controls. Data shown in the Table 6 below is the mean increase in ATP levels after 24 hour treatment with compounds at 100 nM concentration. As cells have an average decrease in ATP levels of 21%, an increase by 21% restores to control levels, anything over 21% is increasing beyond control levels.

TABLE-US-00007 TABLE 6 Compound UDCA 2 7 8 % increase over 12% 65% 24% 65% vehicle treated

[0668] The data clearly show that treatment with compounds 2, 7 and 8 have a more beneficial restoration of cellular ATP levels than UDCA treatment. Furthermore compounds 2 and 8 are particularly effective increasing ATP levels dramatically.