Fluorinated bile acid derivatives

Abstract

The invention relates to compounds of general formula (I): ##STR00001##
wherein R.sup.2a, R.sup.2b, R.sup.3a, R.sup.3b, R.sup.5, Y and R.sup.7 are as defined herein are selective agonists at the FXR receptor and are useful for the treatment or prevention of diseases and conditions including nonalcoholic steatohepatitis (NASH); primary biliary cirrhosis; primary sclerosing cholangitis; biliary atresia; cholestatic liver disease; hepatitis C infection; alcoholic liver disease; fibrosis; and liver damage arising from fibrosis.

Claims

1. A compound of general formula (I): wherein ##STR00131## each of R.sup.2a, R.sup.2b, R.sup.3a and R.sup.3b is independently H or F, and provided that at least one of R.sup.2b and R.sup.3b is F; R.sup.5 is selected from CR.sup.6aR.sup.6bR.sup.8, OR.sup.8, SR.sup.8 and NR.sup.6aR.sup.8; each of R.sup.6a, R.sup.6b and R.sup.8 is independently H or methyl Y is selected from a bond, and a C.sub.1-4 alkylene, or a C.sub.2-4 alkenylene linker group, any of which is optionally substituted with one or more R.sup.10; wherein each R.sup.10 is independently halo or OH; R.sup.7 is selected from C(O)NR.sup.17S(O).sub.2R.sup.15, NR.sup.17C(O)NR.sup.18S(O).sub.2R.sup.15, NR.sup.17C(S)NR.sup.18S(O).sub.2R.sup.15 and NR.sup.17C(NR.sup.20)NR.sup.18S(O).sub.2R.sup.15; R.sup.15 is selected from a 5- to 10-membered aryl ring and a 5- to 10-membered heteroaryl ring, any of which is optionally substituted with one or more substituents selected from C1-6 alkyl, C.sub.1-6 haloalkyl, halo, O(C.sub.1-5 alkyl), and O(C.sub.1-6 haloalkyl); each R.sup.17 and R.sup.18 is independently selected from H and methyl; R.sup.20 is selected from H, methyl and CN; or a salt thereof.

2. The compound according to claim 1, wherein R.sup.3b is F, and each of R.sup.3a, R.sup.2a and R.sup.2b is H.

3. The compound according to claim 1, wherein R.sup.2b is F, and each of R.sup.2a, R.sup.3a and R.sup.3b is H.

4. The compound according to claim 1, wherein: R.sup.3b is F, R.sup.3a is H, one of R.sup.2a and R.sup.2b is F, and the other of R.sup.2a and R.sup.2b is H; or wherein R.sup.3a and R.sup.3b are both F, and R.sup.2a and R.sup.2b are both H.

5. The compound according to claim 1, wherein R.sup.5 is ethyl.

6. The compound according to claim 1, wherein Y is selected from a bond, and a C.sub.1-3 alkylene linker group, which is optionally substituted with one or more OH groups.

7. The compound according to claim 1, wherein R.sup.7 is C(O)NR.sup.17S(O).sub.2R.sup.15, or NR.sup.17C(O)NR.sup.18S(O).sub.2R.sup.15.

8. The compound according to claim 7, wherein, independently or in any combination: each of R.sup.17 and R.sup.18 is H; and/or R.sup.15 is selected from phenyl and a 5- or 6-membered heteroaryl, any of which may be unsubstituted or substituted with one or more substituents selected from C.sub.1-6 alkyl; C.sub.1-6 haloalkyl; halo, O(C.sub.1-6 alkyl), and O(C.sub.1-6 haloalkyl).

9. The compound according to claim 8, wherein R.sup.15 is phenyl, which is unsubstituted or is substituted with a single substituent selected from fluoro, C.sub.1-4 alkyl, C.sub.1-4 fluoroalkyl, O(C.sub.1-4 alkyl) and O(C.sub.1-4 fluoroalkyl).

10. The compound according to claim 1, wherein the compound is selected from: N,N′-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-p-toluene sulfonyl urea (Compound 1); N,N′-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-benzene sulfonyl urea (Compound 2); N,N′-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-4-(tert-butyl) benzene sulfonyl urea (Compound 3); N,N′-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-m-toluene sulfonyl urea (Compound 4); N,N′-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-o-toluene sulfonyl urea (Compound 5); N,N′-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-p-fluorobenzene sulfonyl urea (Compound 6); N,N′-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-m-fluorobenzene sulfonyl urea (Compound 7); N,N′-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-o-fluorobenzene sulfonyl urea (Compound 8); N,N′-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-p-(trifluoromethyl)benzene sulfonyl urea (Compound 9); N,N′-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-m-(trifluoromethyl)benzene sulfonyl urea (Compound 10); N,N′-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-o-(trifluoromethyl)benzene sulfonyl urea (Compound 11); N,N′-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-4-(trifluoromethoxy)benzene sulfonyl urea (Compound 12); N,N′-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-p-methoxybenzene sulfonyl urea (Compound 13); N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-p-trifluoromethoxy benzene sulfonamide (Compound 14); N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-p-fluorobenzene sulfonamide (Compound 15); N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-3-fluorophenyl sulfonamide (Compound 16); N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-2-fluorophenyl sulfonamide (Compound 17); N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-4-trifluoromethylphenyl sulfonamide (Compound 18); N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-3-trifluoromethylphenyl sulfonamide (Compound 19); N-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-2-trifluoromethylphenyl sulfonamide (Compound 20); N,N′-(3α,7α-dihydroxyl-4,4-difluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-benzene sulfonyl urea (Compound 21) N-(3α,7α-dihydroxyl-4,4-difluoro-6α-ethyl-5β-cholan-24-oyl)-benzene sulfonamide (Compound 22); and salts thereof.

11. A method of treating a metabolic syndrome, the method comprising administering an effective amount of the compound according to claim 1 to a patient.

12. A method for the treatment of nonalcoholic steatohepatitis (NASH); primary biliary cirrhosis; primary sclerosing cholangitis; biliary atresia; cholestatic liver disease; hepatitis C infection; alcoholic liver disease; fibrosis; or liver damage arising from fibrosis, the method comprising administering to a patient in need of such treatment an effective amount of a compound according to claim 1.

13. The method according to claim 12 wherein fibrosis is selected from fibrosis of the liver, kidneys and intestines.

14. The method according to claim 13, wherein the liver fibrosis is associated with NASH, alcoholic liver disease or non-alcoholic fatty liver disease, or is associated with an infection, selected from hepatitis B and hepatitis C, or a parasitic liver disease, or is caused by damage induced by a congenital disorder selected from Wilson's disease, Gaucher's disease, glycogen storage disorders, haemochromatosis, Zellweger syndrome, and congenital hepatic fibrosis, or is induced by a drug selected from chlorpromazine, tolbutamide, methotrexate, isoniazid arid methyldopa; and/or fibrosis of the kidneys is associated with a disease selected from diabetic nephropathy, hypertensive nephrosclerosis, glomerulonephritis, interstitial nephritis, glomerulopathy associated with transplant and polycystic kidney disease; and/or intestinal fibrosis is associated with a bowel disorder.

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

16. The pharmaceutical composition according to claim 15, further comprising one or more additional active agents suitable for the treatment of a metabolic syndrome, wherein the metabolic syndrome is selected from nonalcoholic steatohepatitis (NASH); primary biliary cirrhosis; primary sclerosing cholangitis; biliary atresia; cholestatic liver disease; hepatitis C infection; alcoholic liver disease; fibrosis; and liver damage arising from fibrosis.

17. A process for the preparation of a compound according to claim 1, the process comprising: A. for a compound of general formula (I) in which R.sup.7 is NHC(O)N(R.sup.18)S(O).sub.2R.sup.15: reacting a compound of general formula (III): ##STR00132## wherein Y, R.sup.2a, R.sup.2b, R.sup.3a, R.sup.3b and R.sup.5 are as defined in claim 1 and R.sup.40 is a protected OH group; with a sulfonamide of general formula (IV): ##STR00133## wherein R.sup.15 and R.sup.18 are as defined in claim 1 in the presence of a catalyst; to form a compound of general formula (II): ##STR00134## wherein Y, R.sup.2a, R.sup.2b, R.sup.3a, R.sup.3b and R.sup.5 are as defined in claim 1 and R.sup.40 is a protected OH group; and deprotecting the compound of general formula (H); or B. for a compound of general formula (I) in which R.sup.7 is NHC(O)N(R.sup.18)S(O).sub.2R.sup.15: reacting a compound of general formula (XIII): ##STR00135## wherein Y, R.sup.2a, R.sup.2b, R.sup.3a, R.sup.3b and R.sup.5 are as defined in claim 1 and each of R.sup.45 and R.sup.46 is independently a protected OH group; with a sulfonamide of general formula (IV) as defined above in the presence of a catalyst to form a compound of general formula (XII): ##STR00136## wherein Y, R.sup.2a, R.sup.2b, R.sup.3a, R.sup.3b and R.sup.5 are as defined in claim 1 and each of R.sup.45 and R.sup.46 is independently a protected OH group; and deprotecting the compound of general formula (XII); or C. for a compound of general formula (I) in which R.sup.7 is C(O)N(R.sup.17)S(O).sub.2R.sup.15: reacting a compound of general formula (XIII): ##STR00137## wherein Y, R.sup.2a, R.sup.2b, R.sup.3a, R.sup.3b and R.sup.5 are as defined in claim 1 and R.sup.46 is a protected OH group; with a sulfonamide of general formula (XXIV): ##STR00138## wherein R.sup.15 and R.sup.17 are as defined in claim 1; in the presence of a coupling agent and a base; to give a compound of general formula (XXII); ##STR00139## wherein Y, R.sup.2a, R.sup.2b, R.sup.3a, R.sup.3b, R.sup.5, R.sup.15 and R.sup.17 are as defined in claim 1 and R.sup.46 is as defined for general formula (XII); and deprotecting the compound of general formula (XXII).

Description

FIGURES

(1) In the figures, * represents p values<0.05, ** represents p values<0.01 and *** represents p values<0.001.

(2) FIG. 1 shows the change in SHP expression after 24 hours incubation of the human hepatoma cell line Huh7 with control, OCA (at EC.sub.50 and EC.sub.90) and Compound 2 (at EC.sub.50 and EC.sub.90).

(3) FIG. 2 shows the change in OSTα expression after 24 hours incubation of the human hepatoma cell line Huh7 with control, OCA (at EC.sub.50 and EC.sub.90) and Compound 2 (at EC.sub.50 and EC.sub.90).

(4) FIG. 3 shows the change in CYP7A1 expression after 24 hours incubation of the human hepatocellular carcinoma cell line HepG2 with control, OCA (at EC.sub.50 and EC.sub.90) and Compound 2 (at EC.sub.50 and EC.sub.90).

(5) FIG. 4 shows the change in TGFβ1 expression after 24 hours incubation of the human hepatocellular carcinoma cell line HepG2 with control, OCA (at EC.sub.50 and EC.sub.90) and Compound 2 (at EC.sub.50 and EC.sub.90).

(6) FIG. 5 shows the change in SHP expression after 24 hours incubation of the human hepatoma cell line Huh7 with control, OCA (at EC.sub.50 and EC.sub.90) and Compound 14 (at EC.sub.50 and EC.sub.90).

(7) FIG. 6 shows the change in OSTα expression after 24 hours incubation of the human hepatoma cell line Huh7 with control, OCA (at EC.sub.50 and EC.sub.90) and Compound 14 (at EC.sub.50 and EC.sub.90).

(8) FIG. 7 shows the change in CYP7A1 expression after 24 hours incubation of the human hepatocellular carcinoma cell line HepG2 with control, OCA (at EC.sub.50 and EC.sub.90) and Compound 14 (at EC.sub.50 and EC.sub.90).

(9) FIG. 8 shows the change in TGFβ1 expression after 24 hours incubation of the human hepatocellular carcinoma cell line HepG2 with control, OCA (at EC.sub.50 and EC.sub.90) and Compound 14 (at EC.sub.50 and EC.sub.90).

EXAMPLES

(10) In the Examples, the following abbreviations are used. Ac.sub.2O Acetic anhydride DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DMAP Dimethylaminopyridine EDCI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Equiv Equivalents Et.sub.3N Triethylamine EtOAc Ethyl acetate IPA Isopropyl alcohol h Hours HDCA Hyodeoxycholic acid HPLC High performance liquid chromatography LDA Lithium diisopropylamide MeOH Methanol n-BuLi n-Butyl lithium OCA Obeticholic acid PE Petroleum ether PTFE Polytetrafluoroethylene pTSA p-Toluenesulfonic acid RT Room temperature sat Saturated TBAF Tetrabutylammonium fluoride TBDMS-OTf Tert-butyldimethylsilyltrifluoromethane sulfonate TEMPO (2,2,6,6-Tetramethylpiperidin-1-yl)oxidanyl THF Tetrahydrofuran TMS-Cl Trimethylsilylchloride TMS-OTf Trimethylsilyl trifluorotrifluoromethane sulfonate TLC Thin layer chromatography

Example 1—Synthesis of 3α-hydroxyl-4β-fluoro-6α-ethyl-7α-hydroxyl-5β-cholanic Acid Analogues with Sulfonylurea-Substituted Side Chains

A. Methyl 6α-ethyl-3α,7α-dihydroxyl-5β-cholan-24-oate

(11) ##STR00040##

(12) To a solution of OCA (23.5 g, 55.87 mmol) in MeOH (540 mL) at RT was added para-toluenesulfonic acid (1.02 mg, 5.59 mmol, ˜0.1 equiv.) and sonicated at 30° C. for 3 h. Upon completion the reaction mixture was concentrated in vacuo. The residue was dissolved in chloroform (500 mL) and washed with saturated NaHCO.sub.3 (500 mL), H.sub.2O (500 mL) and brine (500 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to yield the title compound as a white solid in quantitative yield. The resulting solid was used without further purification.

(13) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.70 (1H, s), 3.67 (3H, s,), 3.44-3.37 (1H, m), 2.40-2.32 (1H, m), 2.26-2.18 (1H, m), 1.96 (1H, dt, J=12.0, 2.6 Hz), 1.92-1.76 (6H, m), 1.69-1.59 (3H, m), 1.58-1.12 (14H, m), 1.00 (1H, td, 14.2, 3.3 Hz), 0.93 (3H, d, J=6.3 Hz), 0.90 (3H, s), 0.90 (3H, t, J=7.4 Hz), 0.66 (3H, s) ppm.

(14) LRMS (ESI.sup.+) m/z: 452.4 [M+NH.sub.4].sup.+, 100%.

B. Methyl 6α-ethyl-7α-hydroxyl-3-oxo-5β-cholan-24-oate

(15) ##STR00041##

(16) To a stirred solution of methyl 6α-ethyl-3α,7α-dihydroxyl-5β-cholan-24-oate from Step A (9.53 g, 21.9 mmol) in H.sub.2O (22 mL) and tert-butanol (88 mL) at RT was added KBr (5.22 g, 43.9 mmol, ˜2.0 equiv.), KHCO.sub.3 (22.0 g, 219 mmol, ˜10 equiv.) and TEMPO (4.45 g, 28.5 mmol, ˜1.3 equiv.). The reaction mixture was cooled to 0° C. and received NaClO (28 mL, 32.9 mmol, ˜1.5 equiv.) dropwise at a rate of 4 mL per hour over 7 hours. Upon completion the reaction was quenched by the slow addition of 1:1 saturated Na.sub.2S.sub.2O.sub.3 (250 mL) and diluted with EtOAc (200 mL). The organic phase was removed followed by back extraction of the aqueous phase with EtOAc (3×150 mL). Organic phases combined, dried over MgSO.sub.4, filtered and concentrated in vacuo to afford 14.2 g of crude material as an orange oil. The resultant oil was purified via column chromatography (gradient elution of acetone in PE 40-60, 0-20%) to yield the title compound as a white solid (8.48 g, 89%).

(17) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.78 (1H, d, J=2.2 Hz), 3.67 (3H, s), 3.07 (1H, dd, J=15.2, 13.5 Hz), 2.46-2.33 (2H, m), 2.29-1.91 (7H, m), 1.84-1.77 (1H, m). 1.74-1.15 (18H, m), 1.00 (3H, s), 0.94 (3H, d, J=6.5 Hz), 0.91 (3H, t, J=7.4 Hz), 0.70 (3H, s) ppm.

(18) LRMS (ESI.sup.+) m/z: 450.3 [M+NH.sub.4].sup.+, 100%.

C. Methyl 6α-ethyl-4β-fluoro-7α-hydroxyl-3-oxo-5β-cholan-24-oate

(19) ##STR00042##

(20) To a stirred, pre-cooled solution of diisopropylamine (0.78 mL, 5.54 mmol, ˜12 equiv.) in dry THF (6.9 mL) at −78° C. was added n-BuLi in hexanes (1.44 mL, 2.31 mmol, ˜5.0 equiv.) dropwise over 0.25 h under argon. After addition, trimethylsilylchloride (0.29 mL, 2.31 mmol, ˜5.0 equiv.) was added and stirred for 1 h. A solution of methyl 6α-ethyl-7α-hydroxyl-3-oxo-5β-cholan-24-oate from Step B (200 mg, 0.46 mmol) in dry THF (3 mL) and triethylamine (1.16 mL, 8.32 mmol, ˜18 equiv.) were then added. After addition the reaction was gradually allowed to warm to −20° C. and stirred for 2 h. Upon completion the reaction was quenched via the dropwise addition of saturated NaHCO.sub.3 (5 mL) and warmed to RT for 2 h. The organic phase was removed and the aqueous phase back extracted with EtOAc (3×10 mL). Organic phases were combined, washed with brine (30 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to afford 271 mg of crude material as a yellow residue.

(21) To a stirred solution of the resultant crude material in MeCN (13 Ml) was added SELECTFLUOR® and the mixture was stirred for 16 h. Upon completion the reaction mixture was concentrated in vacuo. The residue was dissolved in EtOAC (20 mL) and acidified with 2M HCl (30 mL). The organic phase was removed and the aqueous phase back extracted with EtOAc (3×15 mL). Organic phases were combined, washed with brine (100 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to afford 196 mg of crude material as a green solid. Purification by HPLC using hexane/acetone (90/10) as the eluent yielded an inseparable mix of the title compound and methyl-2β-fluoro-3-oxo-6α-ethyl-7α-hydroxyl-5β-cholan-24-oate as a colourless oil (79 mg, 0.18 mmol, 37% title compound, considering 1% methyl-2β-fluoro-3-oxo-6α-ethyl-7α-hydroxyl-5β-cholan-24-oate contamination by .sup.1H NMR).

(22) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.94 (1H, dd, J=46.5, 10.9 Hz), 3.88 (1H, s), 3.65 (3H, s), 2.49 (1H, td, J=14.6, 5.0 Hz), 2.38-2.09 (4H, m), 2.01-1.30 (18H, m), 1.25-1.14 (3H, m), 1.04 (3H, s), 0.93-0.89 (6H, m), 0.68 (3H, s) ppm.

(23) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): −194.3 (1F, dd, J=46.8, 13.9 Hz) ppm.

(24) LRMS (ESI.sup.+) m/z: 468.4 [M+NH.sub.4].sup.+, 100%.

D. Methyl 6α-ethyl-4β-fluoro-(3α,7α)-dihydroxyl-5β-cholan-24-oate

(25) ##STR00043##

(26) To a stirred solution of methyl 6α-ethyl-4β-fluoro-7α-hydroxyl-3-oxo-5β-cholan-24-oate from Step C (75 mg, 0.17 mmol) in dry THF (6.7 mL) at RT was added NaBH.sub.4 (19 mg, 0.50 mmol, ˜3.0 equiv.) and stirred for 16 h under argon. Upon completion the reaction was quenched via the dropwise addition of H.sub.2O (8 mL) and diluted with EtOAc (10 mL). The organic phase was removed and the aqueous phase back extracted with EtOAc (3×50 mL). Organic phases were combined, washed with H.sub.2O (100 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to afford 77 mg of crude material as a white residue. Purification by HPLC using hexane/acetone (90/10) as the eluent yielded the title compound as a colourless oil (55 mg, 0.12 mmol, 74%).

(27) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.31 (1H, ddd, J=50.0, 10.4, 8.9 Hz), 3.82 (1H, s), 3.67 (3H, s), 3.57-3.50 (1H, m), 2.36 (1H, ddd, J=15.4, 10.1, 5.7 Hz), 2.27-2.18 (1H, m), 1.96-1.92 (2H, m), 1.83-1.07 (23H, m), 0.97 (3H, s), 0.94-0.92 (6H, m), 0.66 (3H, s) ppm.

(28) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−189.0 (1F, d, J=50.3 Hz) ppm.

(29) LRMS (ESI.sup.+) m/z: 470.4 [M+NH.sub.4].sup.+, 100%.

E. 3α, 7α-Dihydroxyl-4β-fluoro-6α-ethyl-5β-cholanic Acid

(30) ##STR00044##

(31) To a stirred solution of methyl 6α-ethyl-4β-fluoro-(3α,7α)-dihydroxyl-5β-cholan-24-oate (58 mg, 0.13 mmol) in MeOH (5 mL) at RT was added NaOH (250 mg, 5% solution) and stirred for 18 h. Upon completion the reaction mixture was concentrated in vacuo and the residue acidified to pH 2 with 1M HCl and diluted with EtOAc (20 mL). The organic phase was removed and the aqueous phase back extracted with EtOAc (3×50 mL). Organic phases were combined, washed with brine (100 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to afford 76 mg of crude material as a yellow oil. Purification by HPLC using hexane/acetone (70/30) as the eluent yielded the title compound as a colourless oil (41 mg, 0.09 mmol, 72%).

(32) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.31 (1H, dt, J=49.9, 9.5 Hz), 3.83 (1H, s), 3.60-3.50 (1H, m), 2.36 (1H, ddd, J=15.5, 10.4, 5.3 Hz), 2.26 (1H, ddd, J=15.8, 9.5, 6.6 Hz), 1.97-1.91 (2H, m), 1.85-1.08 (21H, m), 0.97 (3H, s), 0.95-0.91 (6H, m), 0.67 (3H, s) ppm.

(33) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−188.7 (1F, d, J=48.6 Hz) ppm.

(34) LRMS (ESI.sup.+) m/z: 456.2, [M+NH.sub.4].sup.+, 100%.

(35) Synthesis of Compounds with Sulfonylurea-Substituted Side Chains

(36) The methods below are illustrated for 4β-fluoro derivatives but could also be used for 2β-fluorinated, 4,4-difluorinated or 2,4-difluoroinated compounds.

F. 3α-Acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-5β-cholanic Acid

(37) ##STR00045##

(38) To a stirred solution of 3α, 7α-dihydroxyl-4β-fluoro-6α-ethyl-5β-cholanic acid from Step E (2.08 g, 4.74 mmol) in dry THF (160 mL) at RT under argon was added NaHCO.sub.3 (2.04 g, 23.7 mmol, ˜5.0 equiv.) and Ac.sub.2O (2.29 mL, 23.7 mmol, ˜5.0 equiv.) dropwise over 5 mins. After addition, the reaction mixture was heated at 70° C. for 16 h. Upon completion, the reaction mixture was cooled to RT and quenched by the dropwise addition of H.sub.2O (100 mL), acidified with 1M HCl (20 mL) and diluted with EtOAc (100 mL). The organic phase was removed and the aqueous phase back extracted with EtOAc (3×150 mL). Organic phases were combined, washed with brine (400 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to yield a yellow oil. The resultant oil was purified via column chromatography (gradient elution of MeOH in CH.sub.2Cl.sub.2, 0-3%) to yield the title compound as a white solid (660 mg, 29%).

(39) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.47 (1H, dt, J=49.4, 9.4 Hz), 4.78 (1H, dddd, J=14.1, 11.9, 9.3, 5.0 Hz), 3.84 (1H, s), 2.41 (1H, ddd, J=15.5, 10.2, 5.3 Hz), 2.27 (1H, ddd, J=15.8, 9.7, 6.6 Hz), 2.06 (3H, s), 1.97-1.89 (2H, m), 1.86-1.80 (3H, m), 1.70-1.14 (19H, m), 0.99 (3H, s), 0.94 (3H, d, J=6.2 Hz), 0.92 (3H, t, J=7.1 Hz), 0.67 (3H, s) ppm.

(40) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−188.6 (1F, dt, J=50.3, 12.1 Hz) ppm.

(41) LRMS (ESI.sup.+) m/z: 498.2, [M+NH.sub.4].sup.+, 100%.

G. 3α-Acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-5β-cholan-24-oyl azide

(42) ##STR00046##

(43) To a stirred solution of 3α-acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-5β-cholanic acid from Step F (200 mg, 0.42 mmol) in dry THF (4 mL) at RT was added Et.sub.3N (0.12 mL, 0.83 mmol, ˜2.0 equiv.) dropwise under argon. After addition the reaction mixture was cooled to 0° C. and diphenylphosphoryl azide (0.13 mL, 0.62 mmol, ˜1.5 equiv.) added dropwise. After addition the reaction mixture was stirred for 3 h behind a blast shield. Upon completion the reaction was quenched with brine (5 mL) and diluted with CH.sub.2Cl.sub.2 (5 mL). The organic phase was removed and the aqueous phase back extracted with CH.sub.2Cl.sub.2 (3×5 mL). Organic phases combined, dried over MgSO.sub.4, filtered and concentrated in vacuo at 0° C. to yield a yellow oil. The resulting oil was used without further purification.

(44) .sup.1H NMR—characteristic peaks (400 MHz, CDCl.sub.3): δ 5.47 (1H, ddd, J=49.4, 10.4, 9.2 Hz), 4.82-4.74 (1H, m), 3.83 (1H, s), 2.38 (1H, ddd, J=15.8, 10.0, 5.3 Hz), 2.29-2.23 (1H, m), 2.06 (3H, s), 0.98 (3H, s), 0.920 (3H, d, J=6.5 Hz), 0.919 (3H, t, J=7.2 Hz), 0.67 (3H, s) ppm.

(45) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−186.8 (1F, dt, J=50.3 Hz) ppm.

H. 3α-Acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholan-23-yl isocyanate

(46) ##STR00047##

(47) To a stirred solution of crude oil 3α-acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-5β-cholan-24-oyl azide from Step G (105 mg assumed, 0.42 mmol) in dry toluene (3.1 mL) was heated to 125° C. under argon. After 5 h the reaction was allowed to cool to RT. The resulting solution was used without further purification.

(48) .sup.1H NMR—characteristic peaks (400 MHz, CDCl.sub.3): δ 5.47 (1H, ddd, J=49.4, 10.4, 9.2 Hz), 4.81-4.73 (1H, m), 3.83 (1H, s), 3.35 (1H, ddd, J=12.9, 7.8, 4.5 Hz), 3.30-3.24 (1H, m), 2.05 (3H, s), 0.98 (3H, s), 0.94 (3H, d, J=6.6 Hz), 0.92 (3H, t, J=7.2 Hz), 0.68 (3H, s) ppm.

(49) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−186.5 (1F, dt, J=49.3, 12.7 Hz) ppm.

(50) General Procedure 1 for Conversion of Isocyanate to Sulfonyl Urea.

(51) ##STR00048##

(52) To a stirred crude solution of 3α-acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholan-23-yl isocyanate from Step H in toluene was added sulphonamide (˜1.5 equiv.) and DBU (˜1.5 equiv.) and stirred for a 16 h. Upon completion the reaction was quenched via dropwise addition of 1M HCl (2 mL) and diluted with EtOAc (5 mL). The organic phase was removed and the aqueous phase back extracted with EtOAc (3×5 mL). Organic phases combined, dried over MgSO.sub.4, filtered and concentrated in vacuo. The resultant residue was purified via column chromatography (gradient elution of acetone in PE 40-60, 5-20%) to yield the required sulfonylurea.

N,N′-(3α-Acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholan-23-yl)-p-toluene sulfonyl urea (Intermediate 1)

(53) ##STR00049##

(54) Prepared according to general procedure 1 using 53.4 mg of p-toluenesulfonamide to afford Intermediate 1 as a yellow oil (51.7 mg, 38%).

(55) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.78 (2H, d, J=8.2 Hz), 7.32 (2H, d, J=8.3 Hz), 6.50 (1H, t, J=4.8 Hz), 5.47 (1H, dt, J=49.4, 9.8 Hz), 4.83-4.73 (1H, m), 3.83 (1H, s), 3.33-3.25 (1H, m), 3.19-3.11 (1H, m), 2.44 (3H, s), 2.06 (3H, s), 1.96-1.80 (5H, m), 1.72-1.38 (15H, m), 1.23-1.14 (5H, m), 0.99 (3H, s), 0.93 (3H, d, J=6.6 Hz), 0.92 (3H, t, J=7.5 Hz), 0.65 (3H, s) ppm.

(56) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−186.6 (1F, dt, J=49.0, 12.8 Hz) ppm.

(57) LRMS (ESI.sup.+) m/z: 666.4, [M+NH.sub.4].sup.+, 100%.

N,N′-(3α-Acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholan-23-yl)-benzene sulfonyl urea (Intermediate 2)

(58) ##STR00050##

(59) Prepared according to general procedure 1 using 49.0 mg of benzenesulfonamide to afford Intermediate 2 as a yellow oil (49.9 mg, 38%).

(60) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.90 (2H, d, J=7.6 Hz), 7.63 (1H, t, J=7.0 Hz), 7.50 (2H, t, J=7.7 Hz), 6.60 (1H, s), 5.48 (1H, dt, J=49.2, 9.8 Hz), 4.84-4.74 (1H, m), 3.83 (1H, s), 3.31-3.25 (1H, m), 3.18-3.10 (1H, m), 2.06 (3H, s), 1.95-1.80 (5H, m), 1.69-1.38 (13H, m), 1.29-1.12 (7H, m), 0.99 (3H, s), 0.93 (3H, d, J=6.7 Hz), 0.92 (3H, t, J=7.0 Hz), 0.65 (3H, s) ppm.

(61) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−186.5 (1F, dt, J=48.6, 12.1 Hz) ppm.

(62) LRMS (ESI.sup.+) m/z: 652.3, [M+NH.sub.4].sup.+, 100%.

N,N′-(3α-Acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholan-23-yl)-4-(tert-butyl)benzene sulfonyl urea (Intermediate 3)

(63) ##STR00051##

(64) Prepared according to general procedure 1 using 53.2 mg of 4-(tert-butyl)benzenesulfonamide to afford the Intermediate 3 as a colourless oil (81.6 mg, 71%).

(65) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.81 (2H, d, J=8.7 Hz), 7.54 (2H, d, J=8.7 Hz), 6.56 (1H, s), 5.48 (1H, ddd, J=49.4, 10.4, 9.3 Hz), 4.83-4.73 (1H), 3.84 (1H, s), 3.33-3.29 (1H, m), 3.21-3.14 (1H, m), 2.07 (3H, s), 1.97-1.80 (4H, m), 1.74-1.44 (13H, m), 1.36 (9H, s), 1.29-1.16 (7H, m), 0.99 (3H, s), 0.95 (3H, d, J=6.6 Hz), 0.93 (3H, t, J=7.0 Hz), 0.67 (3H, s) ppm.

(66) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−186.6 (1F, dt, J=49.9, 13.2 Hz) ppm.

(67) LRMS (ESI.sup.+) m/z: 708.4, [M+NH.sub.4].sup.+, 100%.

N,N′-(3α-Acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholan-23-yl)-m-toluene sulfonyl urea (Intermediate 4)

(68) ##STR00052##

(69) Prepared according to general procedure 1 using 42.6 mg of m-toluenesulfonamide to afford Intermediate 4 as a colourless oil (85.7 mg, 80%).

(70) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.71-7.68 (2H, m), 7.45-7.38 (2H, m), 6.54 (1H, s), 5.47 (1H, ddd, J=49.4, 10.3, 9.4 Hz), 4.83-4.73 (1H, m), 3.83 (1H, s), 3.33-3.26 (1H, m), 3.19-3.12 (1H, m), 2.42 (3H, s), 2.06 (3H, s), 1.96-1.80 (4H, m), 1.72-1.11 (21H, m), 0.99 (3H, s), 0.94 (3H, d, J=6.6 Hz), 0.93 (3H, t, J=7.3 Hz), 0.65 (3H, s) ppm.

(71) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−186.6 (1F, dt, J=49.4, 13.4 Hz) ppm.

(72) LRMS (ESI.sup.+) m/z: 666.3, [M+NH.sub.4].sup.+, 100%.

N,N′-(3α-Acetoxy-4β-fluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholan-23-yl)-o-toluene sulfonyl urea (Intermediate 5)

(73) ##STR00053##

(74) Prepared according to general procedure 1 using 42.6 mg of o-toluenesulfonamide to afford Intermediate 5 as a colourless oil (55.4 mg, 51%).

(75) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.94 (1H, dd, J=8.3, 1.0 Hz), 7.51 (1H, td, J=7.6, 1.2 Hz), 7.34 (2H, d, J=7.3 Hz), 6.47 (1H, s), 5.47 (1H, dt, J=49.3, 9.5 Hz), 4.83-4.73 (1H, m), 3.83 (1H, s), 3.28-3.22 (1H, m), 3.15-3.08 (1H, m), 2.65 (3H, s), 2.21-2.17 (1H, m), 2.06 (3H, s), 1.94-1.80 (4H, m), 1.68-1.10 (20H, m), 0.98 (3H, s), 0.92 (3H, d, J=6.7 Hz), 0.90 (3H, t, J=7.3 Hz), 0.63 (3H, s) ppm.

(76) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−186.6 (1F, dt, J=49.9, 12.4 Hz) ppm.

(77) LRMS (ESI.sup.+) m/z: 666.3, [M+NH.sub.4].sup.+, 100%.

General Procedure 2 for Deprotection of 3α-acetate sulfonyl ureas

(78) ##STR00054##

(79) To a flask charged with the protected sulfonyl urea was added a solution of NaOH in MeOH (5% solution, 10 mL) and stirred for 16 h. Upon completion the reaction was acidified to pH 7.0 with 1M HCl and diluted with EtOAc (10 mL). The organic phase was removed and the aqueous phase back extracted with EtOAc (3×10 mL). Organic phases combined, washed with NaHCO.sub.3 solution (50 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo. The resultant residue was purified via column chromatography (gradient elution of MeOH in CH.sub.2Cl.sub.2, 0-5%) to yield the deprotected sulfonyl urea.

N,N′-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-p-toluene sulfonyl urea (Compound 1)

(80) ##STR00055##

(81) Prepared according to general procedure 2 using 49.7 mg of Intermediate 1 to afford Compound 1 as a colourless residue (18.6 mg, 40%).

(82) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.78 (2H, d, J=8.3 Hz), 7.34 (2H, d, J=8.1 Hz), 6.50 (1H, t, J=4.8 Hz), 5.32 (1H, ddd, J=50.0, 10.0, 9.2 Hz), 3.83 (1H, s), 3.59-3.51 (1H, m), 3.33-3.26 (1H, m), 3.21-3.12 (1H, m), 2.46 (3H, s), 1.96-1.06 (26H, m), 0.98 (3H, s), 0.94 (3H, t, J=6.2 Hz), 0.93 (3H, t, J=6.4 Hz), 0.66 (3H, s) ppm.

(83) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−188.8 (1F, dt, J=50.3, 10.4 Hz) ppm.

(84) LRMS (ESI.sup.+) m/z: 624.4, [M+NH.sub.4].sup.+, 100%.

N,N′-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-benzene sulfonyl urea (Compound 2)

(85) ##STR00056##

(86) Prepared according to general procedure 2 using 44.8 mg of Intermediate 2 to afford Compound 2 as a colourless residue (28.5 mg, 64%).

(87) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.91 (2H, d, J=7.5 Hz), 7.65 (1H, t, J=7.3 Hz), 7.54 (2H, t, J=7.8 Hz), 6.51 (1H, s), 5.31 (1H, ddd, J=50.1, 10.3, 9.1 Hz), 3.82 (1H, s), 3.60-3.50 (1H, m), 3.35-3.26 (1H, m), 3.20-3.12 (1H, m), 1.95-1.36 (17H, m), 1.27-1.11 (9H, m), 0.97 (3H, s), 0.94 (3H, d, J=6.2 Hz), 0.93 (3H, t, J=6.7 Hz), 0.65 (3H, s) ppm.

(88) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−189.0 (1F, dt, J=50.3, 12.1 Hz) ppm.

(89) LRMS (ESI.sup.+) m/z: 610.2, [M+NH.sub.4].sup.+, 100%.

N,N′-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-4-(tert-butyl) benzene sulfonyl urea (Compound 3)

(90) ##STR00057##

(91) Prepared according to general procedure 2 using 79.6 mg of Intermediate 3 to afford Compound 3 as a colourless residue (50.7 mg, 65%).

(92) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.82 (2H, d, J=8.6 Hz), 7.53 (2H, t, J=8.4 Hz), 6.54 (1H, s), 5.32 (1H, ddd, J=49.9, 10.3, 9.1 Hz), 3.82 (1H, s), 3.60-3.50 (1H, m), 3.35-3.25 (1H, m), 3.19-3.11 (1H, m), 1.95-1.41 (16H, m), 1.34 (9H, s), 1.28-1.08 (10H, m), 0.97 (3H, s), 0.930 (3H, t, J=6.9 Hz), 0.927 (3H, d, J=6.2 Hz), 0.65 (3H, s) ppm.

(93) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−188.8 (1F, dt, J=50.3, 12.1 Hz) ppm.

(94) LRMS (ESI.sup.+) m/z: 666.4, [M+NH.sub.4].sup.+, 100%.

N,N′-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-m-toluene sulfonyl urea (Compound 4)

(95) ##STR00058##

(96) Prepared according to general procedure 2 using 83.7 mg of Intermediate 4 to afford Compound 4 as a colourless residue (29.0 mg, 37%).

(97) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.71-7.69 (2H, m), 7.45-7.38 (2H, m), 6.52 (1H, s), 5.32 (1H, ddd, J=49.9, 10.4, 8.9 Hz), 3.82 (1H, s), 3.60-3.50 (1H, m), 3.35-3.26 (1H, m), 3.20-3.10 (1H, m), 2.43 (3H, s), 1.95-1.39 (17H, m), 1.28-1.11 (10H, m), 0.97 (3H, s), 0.94 (3H, d, J=6.2 Hz), 0.93 (3H, t, J=6.5 Hz), 0.65 (3H, s) ppm.

(98) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−188.9 (1F, dt, J=48.6, 10.4 Hz) ppm.

(99) LRMS (ESI.sup.+) m/z: 624.3, [M+NH.sub.4].sup.+, 100%.

N,N′-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-o-toluene sulfonyl urea (Compound 5)

(100) ##STR00059##

(101) Prepared according to general procedure 2 using 53.4 mg of Intermediate 5 to afford Compound 5 as a colourless residue (24.2 mg, 48%).

(102) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.94 (1H, d, J=8.1 Hz), 7.52 (1H, td, J=7.6, 1.1 Hz), 7.35 (2H, d, J=7.7 Hz), 6.47 (1H, t, J=4.6 Hz), 5.31 (1H, ddd, J=49.9, 10.4, 8.9 Hz), 3.82 (1H, s), 3.60-3.50 (1H, m), 3.30-3.22 (1H, m), 3.17-3.08 (1H, m), 2.67 (3H, s), 1.94-1.06 (25H, m), 0.97 (3H, s), 0.91 (3H, t, J=7.5 Hz), 0.90 (3H, d, J=6.6 Hz), 0.63 (3H, s) ppm.

(103) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−189.0 (1F, dt, J=48.6, 12.1 Hz) ppm.

(104) LRMS (ESI.sup.+) m/z: 624.3, [M+NH.sub.4].sup.+, 100%.

N,N′-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-p-fluorobenzene sulfonyl urea (Compound 6)

(105) ##STR00060##

(106) This compound was prepared by a method analogous to that described above for Compounds 1-5.

(107) .sup.1H NMR (400 MHz, MeOD): δ 7.93-7. (2H, m), 7.20-7.17 (2H, m), 5.19 (1H, dq, J=49.3, 10.5, 8.9 Hz), 3.65 (1H, s), 3.31 (1H, m), 3.06 (1H, m), 2.95 (1H, m), 1.94-1.06 (21H, m), 0.84-0.76 (9H, m), 0.63 (3H, s) ppm.

(108) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, MeOD): δ−107.29 (1F, m), −186.6 (1F, m) ppm.

N,N′-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-m-fluorobenzene sulfonyl urea (Compound 7)

(109) ##STR00061##

(110) This compound was prepared by a method analogous to that described above for Compounds 1-5.

(111) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.71 (ddd, J=7.8, 1.6, 1.0 Hz, 1H), 7.61 (br dt, J=8.0, 2.1 Hz, 1H), 7.54 (td, J=8.1, 5.3 Hz, 1H), 7.35 (tdd, J=8.3, 2.6, 0.7 Hz, 1H), 6.50 (br t, J=5.1 Hz, 1H), 5.32 (ddd, J=49.9, 10.5, 8.8 Hz, 1H), 3.83 (s, 1H), 3.55 (dddd, J=14.2, 12.0, 8.8, 5.1 Hz, 1H), 3.32 (ddt, J=13.1, 9.5, 5.1 Hz, 1H), 3.18 (dtd, J=13.0, 8.0, 6.1 Hz, 1H), 1.96-1.86 (m, 2H), 1.85-1.74 (m, 2H), 1.71-1.58 (m, 6H), 1.54-1.41 (m, 7H), 1.31-1.08 (m, 9H), 0.98 (s, 3H), 0.95 (d, J=6.5 Hz, 3H), 0.93 (t, J=7.0 Hz, 3H), 0.66 (s, 3H) ppm;

(112) .sup.19F NMR (376 MHz, CDCl.sub.3) δ−108.9 (br s, 1F), −188.9 (br d, J=50.3 Hz, 1F) ppm;

(113) .sup.19F {1H} NMR (376 MHz, CDCl.sub.3) δ−108.9 (s, 1F), −188.9 (s, 1F) ppm;

N,N′-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-o-fluorobenzene sulfonyl urea (Compound 8)

(114) ##STR00062##

(115) This compound was prepared by a method analogous to that described above for Compounds 1-5.

(116) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.91 (ddd, J=7.8, 7.2, 1.7 Hz, 1H), 7.67 (dddd, J=8.3, 7.5, 5.0, 1.7 Hz, 1H), 7.33 (td, J=7.7, 1.1 Hz, 1H), 7.28 (ddd, J=10.0, 8.4, 0.9 Hz, 1H), 6.45 (br t, J=5.1 Hz, 1H), 5.31 (ddd, J=49.9, 10.5, 8.7 Hz, 1H), 3.83 (br s, 1H), 3.55 (dddd, J=13.8, 11.9, 8.9, 5.4 Hz, 1H), 3.29 (ddt, J=12.8, 9.4, 5.3 Hz, 1H), 3.16 (dtd, J=13.5, 7.8, 5.6 Hz, 1H), 1.96-1.74 (m, 4H), 1.71-1.39 (m, 14H), 1.25-1.09 (m, 8H), 0.98 (s, 3H), 0.94 (t, J=7.3 Hz, 3H), 0.93 (d, J=6.6 Hz, 3H), 0.65 (s, 3H) ppm;

(117) .sup.19F NMR (376 MHz, CDCl.sub.3) δ−109.0 (ddd, J=10.4, 6.9, 5.2 Hz, 1F), −189.2 (dt, J=50.3, 11.3 Hz, 1F) ppm;

(118) .sup.19F {1H} NMR (376 MHz, CDCl.sub.3) δ−109.0 (s, 1F), −189.2 (s, 1F) ppm.

N,N′-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-p-(trifluoromethyl)benzene sulfonyl urea (Compound 9)

(119) ##STR00063##

(120) This compound was prepared by a method analogous to that described above for Compounds 1-5.

(121) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.07 (d, J=8.2 Hz, 2H), 7.80 (d, J=8.1 Hz, 2H), 6.46 (br t, J=4.8 Hz, 1H), 5.32 (ddd, J=49.8, 10.4, 8.8 Hz, 1H), 3.82 (s, 1H), 3.55 (dddd, J=14.2, 11.9, 8.8, 5.4 Hz, 1H), 3.28 (ddt, J=13.6, 7.7, 4.9 Hz, 1H), 3.15 (dtd, J=13.5, 7.6, 6.0 Hz, 1H), 1.96-1.75 (m, 4H), 1.72-1.55 (m, 7H), 1.53-1.37 (m, 7H), 1.25-1.06 (m, 8H), 0.97 (s, 3H), 0.933 (d, J=6.1 Hz, 3H), 0.927 (t, J=6.5 Hz, 3H), 0.63 (s, 3H) ppm;

(122) .sup.19F NMR (376 MHz, CDCl.sub.3) δ−63.5 (s, 3F), −188.5 (br d, J=48.6 Hz, 1F) ppm;

(123) .sup.19F {1H} NMR (376 MHz, CDCl.sub.3) δ−63.4 (s, 3F), −188.6 (br s, 1F) ppm.

N,N′-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-m-(trifluoromethyl)benzene sulfonyl urea (Compound 10)

(124) ##STR00064##

(125) This compound was prepared by a method analogous to that described above for Compounds 1-5.

(126) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.17 (s, 1H), 8.11 (d, J=8.0 Hz, 1H), 7.91 (br d, J=7.7 Hz, 1H), 7.71 (t, J=7.9 Hz, 1H), 6.49 (br t, J=4.7 Hz, 1H), 5.32 (ddd, J=49.9, 10.2, 9.1 Hz, 1H), 3.83 (s, 1H), 3.56 (dddd, J=14.1, 11.7, 8.7, 5.1 Hz, 1H), 3.32 (ddt, J=13.5, 9.4, 5.3 Hz, 1H), 3.17 (dtd, J=12.6, 7.8, 6.2 Hz, 1H), 1.96-1.87 (m, 2H), 1.84-1.74 (m, 2H), 1.72-1.58 (m, 6H), 1.53-1.41 (m, 7H), 1.29-1.09 (m, 9H), 0.98 (s, 3H), 0.95 (d, J=6.5 Hz, 3H), 0.93 (t, J=6.9 Hz, 3H), 0.66 (s, 3H) ppm;

(127) .sup.19F NMR (376 MHz, CDCl.sub.3) δ−63.1 (s, 3F), −189.0 (br s, 1F) ppm;

(128) .sup.19F {1H} NMR (376 MHz, CDCl.sub.3) δ−63.1 (s, 3F), −189.0 (br s, 1F) ppm.

N,N′-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-o-(trifluoromethyl)benzene sulfonyl urea (Compound 11)

(129) ##STR00065##

(130) This compound was prepared by a method analogous to that described above for Compounds 1-5.

(131) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.26 (dd, J=6.3, 2.3 Hz, 1H), 7.92 (dd, J=6.6, 2.3 Hz, 1H), 7.77 (m, 2H), 6.35 (br t, J=4.8 Hz, 1H), 5.31 (ddd, J=49.9, 10.4, 8.9 Hz, 1H), 3.82 (s, 1H), 3.56 (dddd, J=14.2, 12.1, 8.7, 5.1 Hz, 1H), 3.29 (ddt, J=13.5, 9.1, 5.1 Hz, 1H), 3.13 (dtd, J=13.5, 7.7, 6.2 Hz, 1H), 1.96-1.72 (m, 5H), 1.70-1.35 (m, 14H), 1.25-1.06 (m, 7H), 0.97 (s, 3H), 0.93 (d, J=7.0 Hz, 3H), 0.92 (t, J=6.5 Hz, 3H), 0.63 (s, 3H) ppm;

(132) .sup.19F NMR (376 MHz, CDCl.sub.3) δ−58.0 (s, 3F), −188.9 (br d, J=48.6 Hz, 1F) ppm;

(133) .sup.19F {1H} NMR (376 MHz, CDCl.sub.3) δ−58.0 (s, 3F), −188.9 (s, 1F) ppm.

N,N′-(3α,7α-Dihydroxyl-6α-ethyl-24-nor-5β-cholan-23-yl)-benzene sulfonyl urea (Comparative Compound A)

(134) ##STR00066##

(135) This compound was prepared by a method analogous to that described above for Compounds 5-9.

(136) .sup.1H NMR (400 MHz, MeOD): δ 7.86-7.81 (2H, m), 7.53 (1H, m), 7.47-7.41 (2H, m), 3.52 (1H, br. s), 3.22 (1H, m), 3.04 (1H, m), 2.93 (1H, m), 1.87-0.83 (25H, m), 0.81-0.76 (9H, m), 0.52 (3H, s) ppm.

Example 2—Alternative Synthesis of Compounds with Sulfonylurea-Substituted Side Chains

(137) The methods below are illustrated for 4β-fluoro derivatives but could also be used for 2β-fluorinated, 4,4-difluorinated or 2,4-difluoroinated compounds.

A. Methyl 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3-oxo-5β-cholan-24-oate

(138) ##STR00067##

(139) To a stirred, pre-cooled solution of diisopropylamine (0.78 mL, 5.54 mmol, ˜12 equiv.) in dry THF (6.9 mL) at −78° C. was added n-BuLi in hexanes (1.44 mL, 2.31 mmol, ˜5.0 equiv.) dropwise over 0.25 h under argon. After addition, trimethylsilylchloride (0.29 mL, 2.31 mmol, ˜5.0 equiv.) was added and stirred for 1 h. A solution of methyl 6α-ethyl-7α-hydroxyl-3-oxo-5β-cholan-24-oate from Example 1, Step B (200 mg, 0.46 mmol) in dry THF (3 mL) and triethylamine (1.16 mL, 8.32 mmol, ˜18 equiv.) were then added. After addition the reaction was gradually allowed to warm to −20° C. and stirred for 2 h. Upon completion the reaction was quenched via the dropwise addition of saturated NaHCO.sub.3 (5 mL) and warmed to RT for 2 h. The organic phase was removed and the aqueous phase back extracted with EtOAc (3×10 mL). Organic phases were combined, washed with brine (30 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to afford 271 mg of crude material as a yellow residue.

(140) To a solution of the crude material (1.16 g, 2.3 mmol) in dry MeCN (55 mL) was charged SELECTFLUOR® (1.23 g, 3.47 mmol). After stirring at RT for 14,5 h the mixture was diluted with ethyl acetate (100 mL) and washed with a mixture of 5% NaHCO.sub.3 (100 mL) and 10% NaCl (50 mL). The aqueous phase was extracted with ethyl acetate (3×100 mL) and the combined organic phases were dried over MgSO.sub.4, filtered and concentrated in vacuo to afford an orange/yellow oil. The crude material was purified by column chromatography (SiO.sub.2, 0-40% EtOAc in heptanes) to afford the title compound as a colourless oil (319.5 mg).

B. Methyl 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-hydroxyl-5β-cholan-24-oate

(141) ##STR00068##

(142) Crude methyl 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3-oxo-5β-cholan-24-oate from Step A (319.5 mg, 0.71 mmol) was dissolved in THF (28 mL) with stirring under argon. NaBH.sub.4 (80.5 mg, 2.13 mmol) was charged and the reaction was stirred at RT for 16.5 h, after which additional NaBH.sub.4 (0.24 g, 6.38 mmol) was charged. The mixture was stirred for an additional 4.5 h then water (20 μL) was charged and the mixture was stirred for ˜60 h. After this time the reaction was quenched by the addition of water (15 mL) and diluted with EtOAc (50 mL). The phases were separated and the aqueous phase was extracted with EtOAc (3×50 mL). The combined extracts were dried over MgSO.sub.4, filtered and concentrated in vacuo to afford a clear syrup (0.34 g). The crude material was purified by column chromatography (SiO.sub.2, 0-40% EtOAc in heptane) to afford the title compound as a clear oil (162.3 mg).

C. Methyl 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-O-tert-butyldimethylsilyl-5β-cholan-24-oate

(143) ##STR00069##

(144) Methyl 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-hydroxyl-5β-cholan-24-oate from Step B (0.48 g, 0.92 mmol) was dissolved in dry DCM (12 mL) and cooled to 0° C. with stirring, under argon. 2,6-lutidine (1.1 mL, 9.17 mmol) was charged followed by the drop-wise addition of TBMDS-OTf (0.32 mL, 1.38 mmol). The reaction was warmed to RT and stirred for 24 h then cooled to 0° C. and quenched by the drop-wise addition of 10% citric acid (5 mL). The phases were separated and the aqueous phase was extracted with DCM (3×5 mL). The combined extracts were washed with 10% citric acid (5 mL), aq. NaHCO.sub.3 (5 mL) and water (5 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to a yellow oil (0.69 g). The crude material was purified by column chromatography (SiO.sub.2, 0-20% EtOAc in heptane) to afford the title compound as a clear oil (0.58 g).

D. 6α-Ethyl-4β-fluoro-7α-trimethylsiloxy-3α-O-tert-butyldimethylsilyl-5β-cholanic Acid

(145) ##STR00070##

(146) Methyl 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-O-tert-butyldimethylsilyl-5β-cholan-24-oate (0.58 g) from Step C was dissolved in IPA (5.8 mL) with stirring. 0.5 M NaOH (5.8 mL) was charged and the reaction was stirred at RT for 15 h. The reaction mixture was concentrated under reduced pressure to ˜half the volume then water (5 mL) was charged and the solution was neutralised by the addition of 2 M H.sub.2SO.sub.4 and diluted with EtOAc (10 mL). The mixture was acidified to pH 1 with 2 M H.sub.2SO.sub.4, the phases were separated and the aqueous phase was extracted with EtOAc (10 mL). The combined extracts were washed with water (5 mL) and brine (5 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to afford a white foam (0.52 g). The crude material was purified by column chromatography (SiO.sub.2, 0-50% acetone in toluene) to afford the title compound as a white solid (0.41 g, 72%).

E. 6α-Ethyl-4β-fluoro-7α-trimethylsiloxy-3α-O-tert-butyldimethylsilyl-5β-cholan-24-oyl azide

(147) ##STR00071##

(148) To a stirred solution of 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-O-tert-butyldimethylsilyl-5β-cholanic acid from Step D (197 mg, 0.32 mmol) in dry THF (3.2 mL) at RT was added Et.sub.3N (0.09 mL, 0.64 mmol, ˜2.0 equiv.) dropwise under argon. After addition the reaction mixture was cooled to 0° C. and diphenylphosphoryl azide (0.1 mL, 0.48 mmol, ˜1.5 equiv.) added dropwise. After addition the reaction mixture was stirred for 2.5 h behind a blast shield. Upon completion the reaction was quenched with brine (3 mL) and extracted with DCM (3×5 mL). The combined organic phases were dried over MgSO.sub.4, filtered and concentrated in vacuo at 0° C. The resulting oil was used without further purification.

General Procedure 3 for Formation of Sulfonylureas

(149) ##STR00072##

(150) A stirred solution of crude 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-O-tert-butyldimethylsilyl-5β-cholan-24-oyl azide from Step E of Example 2 (69 mg) in dry toluene (2.1 mL) was heated to 125° C. under argon. After 5 h the reaction was allowed to cool to RT. The resulting solution was used without further purification. The solution was stirred under argon and the sulfonamide (1.5 equivalents) and DBU (1.5 equivalents) were charged. Upon completion the reaction was quenched via dropwise addition of 1M HCl (1 mL) and diluted with EtOAc (5 mL). The organic phase was removed and the aqueous phase back extracted with EtOAc (3×5 mL). The combined organic phases were washed with brine (3 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo (231.7 mg). The resultant residue was purified via column chromatography to afford the desired sulfonyl urea's as a crude inseparable mixture.

N,N′-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-4-(trifluoromethoxy)benzene sulfonyl urea (Compound 12) and N,N′-(3α,7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-p-methoxybenzene sulfonyl urea (Compound 13)

(151) Compounds 12 and 13 were prepared according to General Procedure 3 above by reaction of the crude isocyanate product with 4-(trifluoromethoxy)benzene sulfonamide and 4-(methoxy)benzene sulfonamide respectively.

(152) In order obtain pure products, the crude Compounds 12 and 13 were converted to protected materials (Intermediates 12 and 13) which were purified and then deprotected to regenerate Compounds 12 and 13. This process is described below.

N,N′-(3α,7α-Di-O-tert-butyldimethylsilyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-4-(trifluoromethoxy)benzene sulfonyl urea (Intermediate 12)

(153) ##STR00073##

(154) N,N′-(3α, 7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-4-(trifluoromethoxy)benzene sulfonyl urea (24.3 mg, 0.036 mmol) was dissolved in dry DCM (1 mL) and cooled to 0° C. with stirring, under argon. 2,6-lutidine (0.04 mL, 0.36 mmol) was charged followed by the drop-wise addition of TBMDS-OTf (0.02 mL, 0.108 mmol). The reaction was warmed to RT and stirred for 1.5 h then cooled to 0° C. and quenched by the drop-wise addition of 10% citric acid (1 mL). The phases were separated and the aqueous phase was extracted with DCM (3×1 mL). The combined extracts were washed with 10% citric acid (1 mL), aq. NaHCO.sub.3 (1 mL) and water (1 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to a yellow oil. The crude material was purified by column chromatography (SiO.sub.2, 0-50% EtOAc in heptane) to afford the title compound as a clear oil (9.4 mg, 33%).

N,N′-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-4-(trifluoromethoxy)benzene sulfonyl urea (Compound 12)

(155) ##STR00074##

(156) N,N′-(3α,7α-di-O-tert-butyldimethylsilyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-4-(trifluoromethoxy)benzene sulfonyl urea (9.4 mg) was dissolved in dry THF (1 mL) with stirring under argon. 1 M TBAF in THF (31 μL, 0.03 mmol) was charged and the reaction was stirred at RT for 6 days. The crude solution was dry loaded onto silica gel and purified by column chromatography (SiO.sub.2, 50-100% EtOAc in heptane) to afford the title compound (1 mg).

N,N′-(3α,7α-Di-O-tert-butyldimethylsilyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-p-methoxybenzene sulfonyl urea (Intermediate 13)

(157) ##STR00075##

(158) N,N′-(3α, 7α-dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-p-methoxybenzene sulfonyl urea (26.1 mg, 0.042 mmol) was dissolved in dry DCM (1 mL) and cooled to 0° C. with stirring, under argon. 2,6-lutidine (0.05 mL, 0.419 mmol) was charged followed by the drop-wise addition of TBMDS-OTf (0.03 mL, 0.126 mmol). The reaction was warmed to RT and stirred for 16 h then cooled to 0° C. and quenched by the drop-wise addition of 10% citric acid (1 mL). The phases were separated and the aqueous phase was extracted with DCM (3×1 mL). The combined extracts were washed with 10% citric acid (1 mL), aq. NaHCO.sub.3 (1 mL) and water (1 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to a yellow oil (28.1 mg). The crude material was purified by column chromatography (SiO.sub.2, 0-80% MeOH in DCM) then re-purified by column chromatography (SiO.sub.2, 0-50% acetone in toluene) to afford the title compound (7.8 mg).

N,N′-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-p-methoxybenzene sulfonyl urea (Compound 13)

(159) ##STR00076##

(160) N,N′-(3α,7α-di-O-tert-butyldimethylsilyl-4β-fluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-p-methoxybenzene sulfonyl urea (7.8 mg) was dissolved in dry THF (1 mL) with stirring under argon. 1 M TBAF in THF (28 μL, 0.03 mmol) was charged and the reaction was stirred at RT for 17 h. The crude solution was dry loaded onto silica gel and purified by column chromatography (SiO.sub.2, 0-80% acetone in toluene) to afford the title compound (3.4 mg, 59.6%).

(161) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.97-7.81 (2H, m), 7.11-7.02 (2H, m), 5.30 (1H, ddd, J=49.6, 10.3, 8.9 Hz), 3.88 (3H, s), 3.76 (1H, br. s), 3.42 (1H, m), 3.15 (1H, m), 3.06 (1H, m), 1.99-0.96 (26H, m), 0.95-0.80 (6H, m), 0.64 (3H, s) ppm.

Example 3—Synthesis of Compounds with Sulfonamide-Substituted Side Chains

(162) The methods below are illustrated for 4β-fluoro derivatives but could also be used for 2β-fluorinated, 4,4-difluorinated or 2,4-difluoroinated compounds. Steps A and B are as for Example 2.

C. 6α-Ethyl-4β-fluoro-7α-trimethylsiloxy-3α-hydroxyl-5β-cholanic Acid

(163) ##STR00077##

(164) Methyl 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-hydroxyl-5β-cholan-24-oate from Step B (162.3 mg) was dissolved in IPA (1.6 mL) with stirring. 0.5 M NaOH (1.6 mL) was charged and the reaction was stirred at RT for 16 h. The reaction mixture was concentrated under reduced pressure to half the volume then water (5 mL) was charged and the solution was neutralised by the addition of 2 M H.sub.2SO.sub.4 and diluted with EtOAc (10 mL). The mixture was acidified to pH 1 with 2 M H.sub.2SO.sub.4, the phases were separated and the aqueous phase was extracted with EtOAc (10 mL). The combined extracts were washed with water (3 mL) and brine (5 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to afford a white foam (151.1 mg). The crude material was purified by column chromatography (SiO.sub.2, 0-80% EtOAc in heptane) to afford the title compound as a clear oil (164.1 mg).

General Procedure 4 for Formation of the Acyl Sulfonamide Side Chain

(165) ##STR00078##

(166) 6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-hydroxyl-5β-cholanic acid (50 mg, 0.11 mmol) was dissolved in dry DCM (2 mL). EDCI (43.7 mg, 0.23 mmol) and DMAP (27.8 mg, 0.23 mmol) followed by the appropriate sulfonamide (3 equivalents). Following an overnight stir at RT water (5 mL) was charged, the phases were separated and the aqueous phase was extracted with DCM (2×5 mL). The combined extracts were washed with 1 M HCl (2 mL) and brine (2 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to afford the crude material as an off white solid.

N-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-p-trifluoromethoxy benzene sulfonamide (Compound 14)

(167) ##STR00079##

(168) Crude N-(6α-ethyl-4β-fluoro-7α-trimethylsiloxy-3α-hydroxyl-5β-cholan-24-oyl)-trifluoromethoxy benzene sulfonamide (81.6 mg), obtained according to General Procedure 4 using trifluoromethoxy benzene sulfonamide was dissolved in dry THF (5 mL) with stirring under argon. 1 M TBAF in THF (0.48 mL, 0.48 mmol) was charged and the reaction was stirred at RT for 17.5 h. The crude solution was dry loaded onto silica gel and purified by column chromatography (SiO.sub.2, 0-100% EtOAc in heptane). Fractions containing the desired product were combined, concentrated in vacuo, dissolved in EtOAc (5 mL) and washed with 2 M HCl (5 mL). The aqueous phase was extracted with EtOAc (2×5 mL) and the combined extracts were dried over MgSO.sub.4, filtered and concentrated in vacuo to a white solid, which was purified by column chromatography (SiO.sub.2, 0-25% acetone in toluene) to afford the title compound as a clear residue (4.9 mg).

(169) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.21-8.10 (2H, m), 7.38-7.36 (2H, dd, J=8.9, 0.8 Hz), 5.31 (1H, ddd, J=49.8, 10.4, 9.0 Hz), 3.82 (1H, br. s), 3.56 (1H, m), 2.31 (1H, ddd, J=15.6, 10.1, 5.0 Hz), 2.17 (1H, m), 1.92-1.07 (23H, m), 0.97 (3H, s), 0.93 (3H, t, J=6.9 Hz), 0.86 (3H, d, J=6.4 Hz), 0.62 (3H, s) ppm.

N-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-p-fluorobenzene sulfonamide (Compound 15)

(170) ##STR00080##

(171) This was prepared by an analogous route to that used for Compound 14 above.

(172) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.11 (m, 2H), 7.23 (m, 2H), 5.31 (ddd, J=49.9, 10.3, 9.1 Hz, 1H), 3.81 (br s, 1H), 3.57 (dddd, J=13.8, 11.6, 8.6, 5.1 Hz, 1H), 2.29 (ddd, J=15.4, 10.0, 5.3 Hz, 1H), 2.17 (ddd, J=15.7, 9.2, 6.4 Hz, 1H), 1.90 (dt, J=12.3, 2.9 Hz, 1H), 1.87-1.57 (m, 9H), 1.53-1.29 (m, 8H), 1.24-1.08 (m, 7H), 0.97 (s, 3H), 0.93 (t, J=7.0 Hz, 3H), 0.86 (d, J=6.4 Hz, 3H), 0.61 (s, 3H) ppm;

(173) .sup.19F NMR (376 MHz, CDCl.sub.3)−103.1 (br s, 1F), −188.8 (br s, 1F) ppm;

(174) .sup.19F {1H} NMR (376 MHz, CDCl.sub.3)−103.1 (br s, 1F), −188.8 (br s, 1F) ppm.

N-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-m-fluorophenyl sulfonamide (Compound 16)

(175) ##STR00081##

(176) This was prepared by an analogous route to that used for Compound 14 above.

(177) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.39 (1H, br s), 7.89 (1H, br d, J=7.8 Hz), 7.78 (1H, br d, J=7.7 Hz), 7.55 (1H, td, J=8.1, 5.4 Hz), 7.36 (1H, td, J=8.3, 1.3 Hz), 5.31 (1H, dt, J=50.0, 9.8 Hz), 3.82 (1H, br s), 3.56 (1H, dddd, J=14.2, 11.0, 8.7, 5.1 Hz), 2.31 (1H, ddd, J=15.0, 9.9, 5.1 Hz), 2.18 (1H, ddd, J=15.3, 9.5, 7.2 Hz), 1.99-1.03 (25H, m), 0.97 (3H, s), 0.94 (3H, t, J=6.6 Hz), 0.87 (3H, d, J=8.1 Hz), 0.62 (3H, s) ppm.

(178) .sup.19F NMR (376 MHz, CDCl.sub.3)−109.52 (1F, br d, J=5.2 Hz), −189.0 (1F, dt, J=50.1, 13.9 Hz) ppm.

(179) .sup.19F {1H} NMR (376 MHz, CDCl.sub.3)−109.52 (1F, s), −189.00 (1F, s) ppm.

N-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-o-fluorophenyl sulfonamide (Compound 17)

(180) ##STR00082##

(181) This was prepared by an analogous route to that used for Compound 14 above.

(182) .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.06 (1H, br s), 8.10 (1H, td, J=7.5, 1.5 Hz), 7.65 (1H, m), 7.35 (1H, t, J=7.6 Hz), 7.22 (1H, t, J=9.2 Hz), 5.31 (1H, dt, J=50.1, 9.5 Hz), 3.81 (1H, br s), 3.57 (1H, dddd, J=14.1, 11.3, 8.6, 5.1 Hz), 2.34 (1H, ddd, J=15.4, 10.0, 5.0 Hz), 2.21 (1H, ddd, J=15.9, 9.3, 6.6 Hz), 2.08-1.02 (25H, m), 0.96 (3H, s), 0.92 (3H, br t, J=6.9 Hz), 0.86 (3H, d, J=6.2 Hz), 0.61 (3H, s) ppm;

(183) .sup.19F NMR (376 MHz, CDCl.sub.3)−110.0 (1F, br s), −189.1 (1F, d, J=46.8 Hz) ppm.

(184) .sup.19F {1H} NMR (376 MHz, CDCl.sub.3)−109.8 (1F, s), −188.9 (1F, s) ppm.

N-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-4-trifluoromethylphenyl sulfonamide (Compound 18)

(185) ##STR00083##

(186) This was prepared by an analogous route to that used for Compound 14 above.

(187) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.22 (2H, d, J=8.3 Hz), 8.00 (1H, br s) 7.83 (2H, d, J=8.4 Hz), 5.30 (1H, dtd, J=50.1, 10.2, 1.6 Hz), 3.81 (1H, br s), 3.56 (1H, dddd, J=13.9, 12.0, 8.6, 5.0 Hz), 2.31 (1H, ddd, J=15.8, 10.3, 5.1 Hz), 2.17 (1H, ddd, J=15.8, 10.0, 6.6 Hz), 1.94-1.05 (25H, m), 0.97 (3H, s), 0.93 (3H, t, J=5.8 Hz), 0.87 (3H, d, J=6.1 Hz), 0.61 (3H, s) ppm.

(188) .sup.19F NMR (376 MHz, CDCl.sub.3)−63.5 (3F, br s), −189.2 (1F, br d, J=48.6 Hz) ppm.

(189) .sup.19F {1H} NMR (376 MHz, CDCl.sub.3)−63.3 (3F, s), −189.0 (1F, s) ppm.

N-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-3-trifluoromethylphenyl sulfonamide (Compound 19)

(190) ##STR00084##

(191) This was prepared by an analogous route to that used for Compound 14 above.

(192) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.32 (2H, m), 8.22 (1H, br s), 7.92 (1H, br d, J=7.8 Hz), 7.72 (1H, t, J=7.9 Hz), 5.30 (1H, dtd, J=49.5, 9.3, 1.2 Hz), 3.81 (1H, br s), 3.54 (1H, m), 2.31 (1H, ddd, J=15.6, 10.1, 5.1 Hz), 2.18 (1H, ddd, J=15.9, 9.2, 6.4 Hz), 1.95-1.04 (25H, m), 0.97 (3H, s), 0.93 (3H, t, J=6.9 Hz), 0.86 (3H, d, J=6.2 Hz), 0.61 (3H, s) ppm.

(193) .sup.19F NMR (376 MHz, CDCl.sub.3)−63.02 (3F, s), −189.11 (1F, br d, J=48.6 Hz) ppm.

(194) .sup.19F {1H} NMR (376 MHz, CDCl.sub.3)−63.02 (3F, s, CF.sub.3), −189.10 (1F, s) ppm.

N-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-2-trifluoromethylphenyl sulfonamide (Compound 20)

(195) ##STR00085##

(196) This was prepared by an analogous route to that used for Compound 14 above.

(197) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.53 (1H, m), 8.09 (1H, br s), 7.90 (1H, m), 7.79 (2H, m), 5.3 (1H, dtd, J=50.0, 9.8, 1.7 Hz), 3.81 (1H, br s), 3.55 (1H, dddd, J=14.2, 11.7, 8.8, 5.0 Hz), 2.31 (1H, ddd, J=15.3, 9.7, 4.8 Hz), 2.17 (1H, m), 1.91 (1H, m), 1.87-1.05 (24H, m), 0.97 (3H, s), 0.93 (3H, t, J=6.9 Hz), 0.86 (3H, d, J=6.2 Hz), 0.61 (3H, s) ppm.

(198) .sup.19F NMR (376 MHz, CDCl.sub.3)−63.02 (3F, s), −189.11 (1F, br d, J=48.6 Hz) ppm.

(199) .sup.19F {1H} NMR (376 MHz, CDCl.sub.3)−63.02 (3F, s), −189.10 (1F, s) ppm.

N-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-cyclopropyl sulfonamide (Comparative Compound B)

(200) ##STR00086##

(201) N-(3α-hydroxyl-4β-fluoro-6α-ethyl-7α-trimethylsiloxy-5β-cholan-24-yl)-cyclopropyl sulfonamide (50 mg) was dissolved in dry THF with stirring under argon. 1 M TBAF in THF (0.3 mL, 0.3 mmol) was charged and the reaction was stirred at RT for 23 h. The reaction was diluted with EtOAc (20 mL) and washed with water (10 mL) and 10% aq. NaCl. The crude solution was dry loaded onto silica gel and purified by column chromatography (SiO.sub.2, 0-50% acetone in toluene) to afford the title compound (5.4 mg).

(202) R.sub.f 0.65 (EtOAc/heptane, 50:50).

(203) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.31 (1H, ddd, J=49.8, 10.7, 8.9 Hz), 3.83 (1H, br. s), 3.55 (1H, m), 2.95 (1H, tt, J=8.1, 4.8 Hz), 2.39 (1H, m), 2.25 (1H, m), 2.01-1.07 (27H, m), 0.98 (3H, s), 0.95 (3H, d, J=6.5 Hz), 0.94 (3H, t, J=7.1 Hz), 0.67 (3H, s) ppm.

N-(3α,7α-Dihydroxyl-4β-fluoro-6α-ethyl-5β-cholan-24-oyl)-methyl sulfonamide (Comparative Compound C)

(204) ##STR00087##

(205) N-(3α-hydroxyl-4β-fluoro-6α-ethyl-7α-trimethylsiloxy-5β-cholan-24-yl)-methyl sulfonamide (50 mg) was dissolved in dry THF with stirring under argon. 1 M TBAF in THF (0.3 mL, 0.3 mmol) was charged and the reaction was stirred at RT for 16 h. The reaction was diluted with EtOAc (5 mL) and washed with brine (3 mL). The crude solution was dry loaded onto silica gel and purified by column chromatography (SiO.sub.2, 0-50% acetone in toluene). Fractions containing the desired product were combined, concentrated under reduced pressure, dissolved in CDCl.sub.3, washed with 2 M HCl and water, filtered through PTFE filter pad and concentrated in vacuo to afford the title compound (7.1 mg).

(206) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.30 (1H, ddd, J=49.8, 10.4, 8.8 Hz), 3.83 (1H, br. s), 3.56 (1H, m), 3.30 (3H, s), 2.39 (1H, ddd, J=15.6, 10.3, 5.1 Hz), 2.25 (1H, m), 2.00-1.08 (23H, m), 0.97 (3H, s), 0.94 (3H, d, J=6.5 Hz), 0.93 (3H, t, J=6.8 Hz), 0.67 (3H, s) ppm.

Example 4—Synthesis of 4,4-difluoro-3α,7α-dihydroxyl-6α-ethyl-5β-cholanic Acid Analogues with Sulfonylurea and Acyl Sulfonamide Side Chains

A. Methyl 6α-ethyl-4,4-difluoro-7α-hydroxyl-3-oxo-5β-cholan-24-oate

(207) ##STR00088##

(208) To a pre-cooled solution of methyl 6α-ethyl-4β-fluoro-7α-hydroxyl-3-oxo-5β-cholan-24-oate (product of Example 1C; 7.30 g, 16.0 mmol) in dry THF (300 mL) at −78° C. was added LDA in hexanes (21.1 mL, 21.1 mmol, ˜1.3 equiv.) dropwise over 0.25 h under argon. After addition, trimethylsilylchloride (2.70 mL, 21.1 mmol, ˜1.3 equiv.) was added as a solution in dry THF (150 mL) and stirred for 1 h. Upon completion, the reaction was quenched via the dropwise addition of saturated NaHCO.sub.3 solution (300 mL) and warmed to RT for 0.25 h. The organic phase was removed and the aqueous phase back extracted with dichloromethane (2×150 mL). Organic phases were combined, washed with brine (300 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to afford the crude material as a yellow residue (3% methyl 6α-ethyl-4β-fluoro-7α-hydroxyl-3-oxo-5β-cholan-24-oate contamination by .sup.19F NMR). The resultant residue was used for the next reaction without further purification.

(209) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz): δ−135.2 (1F, s);

(210) To a stirred solution of the resultant crude material in MeCN (360 mL) was added SELECTFLUOR® (11.4 g, 32.0 mmol, .sup.˜2.0 equiv.) and stirred for 16 h. Upon completion the reaction mixture was concentrated in vacuo. The residue was dissolved in dichloromethane (500 mL) and H.sub.2O (500 mL). The organic phase was removed and the aqueous phase back extracted with dichloromethane (2×250 mL). Organic phases were combined, washed with brine (250 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to afford the crude material as a yellow residue. The resultant residue was used for the next reaction without purification.

(211) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.70 (1H, dq, J=7.7, 3.2 Hz), 3.66 (3H, s), 2.70 (1H, tdd, J=14.2, 5.1, 3.4 Hz), 2.44 (1H, dq, J=15.2, 3.8 Hz), 2.35 (1H, ddd, J=15.5, 10.2, 5.4 Hz, 2.24 (1H, dd, J=9.6, 6.5 Hz), 2.19 (1H, dd, J=10.2, 2.9 Hz), 2.14 (1H, dt, J=17.0, 5.6 Hz), 2.08 (1H, td, J=14.6, 5.8 Hz), 2.00-1.86 (3H, m), 1.84-1.75 (3H, m), 1.73-1.63 (3H, m), 1.58 (1H, dd, J=13.9, 4.8 Hz), 1.54-1.28 (6H, m), 1.25-1.11 (3H, m), 1.10 (3H, s), 0.98 (3H, t, J=7.3 Hz), 0.93 (3H, d, J=6.4 Hz), 0.67 (3H, s) ppm.

(212) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−99.2 (1F, dd, J=263.6, 17.3 Hz), −100.7 (1F, ddd, J=263.6, 29.5, 15.6 Hz) ppm.

(213) LRMS (ESI.sup.+) m/z: 486.6, [M+NH.sub.4].sup.+, 100%.

B. Methyl 4,4-difluoro-(3α,7α)-dihydroxyl-6α-ethyl-5β-cholan-24-oate

(214) ##STR00089##

(215) To a stirred solution of crude methyl 6α-ethyl-4,4-difluoro-7α-hydroxyl-3-oxo-5β-cholan-24-oate from Step A (7.51 g assumed, 16.0 mmol) in dry methanol (500 mL) at RT was added NaBH.sub.4 (3.03 g, 80.1 mmol, ˜5.0 equiv) and stirred for 72 h under argon. Upon completion the reaction was concentrated in vacuo. The residue was dissolved in dichloromethane (500 mL) and H.sub.2O (500 mL). The organic phase was removed and the aqueous phase back extracted with dichloromethane (2×250 mL). Organic phases were combined, washed with brine (250 mL), dried over MgSO.sub.4, filtered over SiO.sub.2 and concentrated in vacuo to afford 7.63 g of crude material as a colourless residue. Purification by flash column chromatography (Biotage SNAP KP-Sil 100 g cartridge) using hexane/acetone (100/0 to 80/20) as the eluent yielded the title compound methyl 4,4-difluoro-(3α,7α)-dihydroxyl-6α-ethyl-5β-cholan-24-oate as a colourless residue (3.09 g, 6.57 mmol, 41% over three steps).

(216) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.76-3.65 (2H, m), 3.67 (3H, s), 2.36 (1H, ddd, J=15.5, 10.3, 5.4 Hz), 2.31-2.19 (2H, m), 2.11 (1H, d, J=5.4 Hz), 2.00-1.92 (3H, m), 1.91-1.30 (16H, m), 1.22-1.10 (4H, m), 1.04 (3H, s), 0.97 (3H, t, J=7.3 Hz), 0.92 (3H, d, J=6.5 Hz), 0.65 (3H, s) ppm.

(217) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−99.3 (1F, d, J=239.3 Hz), −111.4 (1F, dtd, J=239.3, 34.7, 22.5 Hz) ppm.

(218) LRMS (ESI.sup.+) m/z: 488.6, [M+NH.sub.4].sup.+, 100%.

C. 3α, 7α-Dihydroxyl-4,4-difluoro-6α-ethyl-5β-cholanic Acid

(219) ##STR00090##

(220) To a stirred solution of methyl 4,4-difluoro-(3α,7α)-dihydroxyl-6α-ethyl-5β-cholan-24-oate (1.77 g, 3.75 mmol, 1.0 equiv.) from Step B in a solution of 1,4-dioxane (95 mL) and water (35 mL) at RT was added concentrated (37%) hydrochloric acid (11 mL, 9:3:1 ratio). After 1 h at reflux, the reaction mixture was cooled to RT and neutralised with saturated NaHCO.sub.3 solution (50 mL). The organic phase was removed and the aqueous phase back extracted with dichloromethane (3×50 mL). Organic phases were combined, washed with brine (200 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to afford 1.84 g of crude material as a brown oil. Purification by flash column chromatography (Biotage SNAP KP-Sil 50 g cartridge) using hexane/acetone (100/0 to 90/10) as the eluent yielded the title compound 3α, 7α-dihydroxyl-4,4-difluoro-6α-ethyl-5β-cholanic acid as a colourless oil (1.51 g, 3.30 mmol, 88%).

(221) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.78-3.64 (2H, m), 2.39 (1H, ddd, J=15.8, 10.3, 5.3 Hz), 2.25 (1H, ddd, J=15.7, 9.6, 6.4 Hz), 1.98-1.93 (3H, m), 1.86-1.65 (7H, m), 1.60 (1H, d, J=13.0 Hz), 1.49-1.41 (5H, m), 1.40-1.29 (3H, m), 1.25-1.16 (6H, m), 1.04 (3H, s), 0.96 (3H, t, J=7.3 Hz), 0.93 (3H, d, J=6.4 Hz), 0.65 (3H, s) ppm.

(222) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−99.1 (1F, d, J=239.3 Hz), −111.1 (1F, dtd, J=241.0, 38.2, 19.1 Hz) ppm.

(223) LRMS (ESI.sup.+) m/z: 474.6, [M+NH.sub.4].sup.+, 100%.

D. 3α-Acetoxy-4,4-difluoro-6α-ethyl-7α-hydroxyl-5β-cholanic Acid

(224) ##STR00091##

(225) To a stirred solution of 3α, 7α-dihydroxyl-4,4-difluoro-6α-ethyl-5β-cholanic acid (400 mg, 0.88 mmol, 1.0 equiv.) from Step C in dry THF (50 mL) at RT was added sodium hydrogencarbonate (370 mg, 4.38 mmol, ˜5.0 equiv.) and acetic anhydride (0.41 mL, 4.38 mmol, ˜5.0 equiv.). After 16 h at 70° C., the reaction mixture was cooled to RT and quenched by the slow addition of H.sub.2O (50 mL). The organic phase was removed and the aqueous phase back extracted with EtOAc (2×50 mL). Organic phases were combined, washed with saturated NaHCO.sub.3 solution (100 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo to afford 462 mg of crude material as a yellow oil. Purification by flash column chromatography (Biotage SNAP KP-Sil Ultra 25 g cartridge) using hexane/acetone (100/0 to 80/20) as the eluent yielded the title compound 3α-Acetoxy-4,4-difluoro-6α-ethyl-7α-hydroxyl-5β-cholanic acid as a colourless oil (270 mg, 0.54 mmol, 62%).

(226) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.97 (1H, ddd, J=27.5, 10.2, 6.0 Hz), 3.67 (1H, s), 2.54-2.33 (2H, m), 2.30-2.26 (1H, m), 2.24-2.18 (1H, m), 2.13 (3H, s), 2.02-1.79 (6H, m), 1.77-1.62 (3H, m), 1.56-1.42 (6H, m), 1.41-1.21 (5H, m), 1.20-1.13 (3H, m), 1.05 (3H, s), 0.95 (3H, t, J=7.3 Hz), 0.94 (3H, d, J=6.5 Hz), 0.66 (3H, s) ppm.

(227) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−98.2 (1F, d, J=244.5 Hz), −107.1 (1F, ddd, J=242.8, 36.4, 22.5 Hz) ppm.

(228) LRMS (ESI.sup.+) m/z: 516.5, [M+NH.sub.4].sup.+, 100%.

E. 3α-Acetoxy-4,4-difluoro-6α-ethyl-7α-hydroxyl-5β-cholan-24-oyl-azide

(229) ##STR00092##

(230) To a pre-cooled solution of 3α-acetoxy-4,4-difluoro-6α-ethyl-7α-hydroxyl-5β-cholanic acid (240 mg, 0.48 mmol, 1.0 equiv.) from Step D in dry THF (5.3 mL) at 0° C. was added triethylamine (0.14 mL, 0.96 mmol, ˜2.0 equiv.) and diphenylphosphorylazide (0.16 mL, 0.72 mmol, ˜1.5 equiv.). After 3 h the reaction mixture was quenched by the slow addition of brine (10 mL). The organic phase was removed and the aqueous phase back extracted with dichloromethane (3×20 mL). Organic phases were combined, dried over MgSO.sub.4, filtered and concentrated in vacuo at 0° C. to afford the crude material as a pale-yellow oil. The resulting oil was used for the next reaction further purification.

(231) .sup.1H NMR—characteristic peaks (400 MHz, CDCl.sub.3): δ 4.94-4.83 (1H, m), 3.59 (1H, s), 2.30 (1H, ddd, J=15.7, 10.0, 5.4 Hz), 2.04 (3H, s), 0.97 (3H, s), 0.87 (3H, t, J=7.3 Hz), 0.84 (3H, d, J=6.4 Hz), 0.57 (3H, s) ppm.

(232) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−98.2 (1F, d, J=242.8 Hz), −107.2 (1F, dtd, J=242.8, 33.0, 26.0 Hz) ppm.

F. 3α-Acetoxy-4,4-difluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholan-23-yl isocyanate

(233) ##STR00093##

(234) A stirred solution of crude oil 3α-acetoxy-4,4-difluoro-6α-ethyl-7α-hydroxyl-5β-cholan-24-oyl azide from Step E (252 mg assumed, 0.48 mmol) in dry toluene (7.5 mL) was heated to 125° C. under argon. After 4 h the reaction was cooled to RT. The resulting solution was used without further purification.

(235) .sup.1H NMR—characteristic peaks (400 MHz, CDCl.sub.3): δ 4.89 (1H, ddd, J=27.5 10.2, 6.5 Hz), 3.59 (1H, q, J=3.2 Hz), 3.27 (1H, ddd, J=13.1, 7.8, 4.5 Hz), 3.22-3.15 (1H, m), 2.04 (3H, s), 0.97 (3H, s), 0.88 (3H, t, J=7.1 Hz), 0.86 (3H, d, J=6.4 Hz), 0.59 (3H, s) ppm.

(236) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−98.2 (1F, d, J=242.8 Hz), −107.2 (1F, dtd, J=243.2, 37.7, 22.5 Hz) ppm.

G. N,N′-(3α-Acetoxy-4,4-difluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholan-23-yl)-benzene sulfonyl urea

(237) ##STR00094##

(238) Prepared according to general procedure 1 using 113 mg of benzenesulfonamide to afford the title compound N,N′-(3α-acetoxy-4,4-difluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholan-23-yl)-benzene sulfonyl urea as a white residue (235 mg, 0.36 mmol, 75%).

(239) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.90 (2H, dd, J=8.4, 1.1 Hz), 7.63 (1H, tt, J=7.6, 1.1 Hz), 7.49 (2H, t, J=8.1 Hz), 6.53 (1H, s), 4.98 (1H, ddd, J=27.1, 11.3, 4.5 Hz), 3.67 (1H, s), 3.27 (1H, ddd, J=13.5, 8.9, 4.5 Hz), 3.18-3.11 (1H, m), 2.13 (3H, s), 2.02-1.79 (6H, m), 1.75-1.64 (4H, m), 1.62-1.43 (5H, m), 1.42-1.36 (1H, m), 1.28-1.10 (7H, m), 1.05 (3H, s), 0.95 (3H, t, J=7.3 Hz), 0.92 (3H, d, J=6.5 Hz), 0.63 (3H, s) ppm.

(240) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−98.2 (1F, d, J=242.8 Hz), −107.1 (1F, dtd, J=243.2, 37.7, 22.5 Hz) ppm.

(241) LRMS (ESI.sup.+) m/z: 635.8, [M+NH.sub.4].sup.+, 100%.

N,N′-(3α,7α-Dihydroxyl-4,4-difluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-benzene sulfonyl urea (Compound 21)

(242) ##STR00095##

(243) Prepared according to general procedure 2 using 210 mg of N,N-(3α-acetoxy-4,4-difluoro-6α-ethyl-7α-hydroxyl-24-nor-5β-cholan-23-yl)-benzene sulfonyl urea to afford the title compound N,N′-(3α,7α-dihydroxyl-4,4-difluoro-6α-ethyl-24-nor-5β-cholan-23-yl)-benzene sulfonyl urea as a white solid (102 mg, 0.17 mmol, 52%).

(244) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.19 (1H, s), 7.90 (2H, dt, J=7.3, 1.3 Hz), 7.65 (1H, tt, J=7.5, 1.1 Hz), 7.54 (2H, tt, J=7.3, 1.6 Hz), 6.51 (1H, t, J=5.2 Hz), 3.78-3.67 (2H, m), 3.29 (1H, ddt, J=13.9, 10.2, 5.1 Hz), 3.18 (1H, ddd, J=13.6, 8.0, 6.1 Hz), 2.30 (1H, dd, J=31.4, 11.0 Hz), 1.99-1.94 (3H, m), 1.88-1.57 (9H, m), 1.52-1.37 (5H, m), 1.23-1.11 (7H, m), 1.04 (3H, s), 0.97 (3H, t, J=7.3 Hz), 0.93 (3H, d, J=6.5 Hz), 0.64 (3H, s) ppm.

(245) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−99.3 (1F, d, J=239.3 Hz), −111.3 (1F, dtd, J=239.3 Hz) ppm.

(246) LRMS (ESI.sup.+) m/z: 593.6, [M+NH.sub.4].sup.+, 100%.

N-(3α,7α-Dihydroxyl-4,4-difluoro-6α-ethyl-5β-cholan-24-oyl)-benzene sulfonamide (Compound 22)

(247) ##STR00096##

(248) Prepared according to general procedure 4 using 400 mg of 3α, 7α-dihydroxyl-4,4-difluoro-6α-ethyl-5β-cholanic acid from Step C to afford the title compound N-(3α,7α-dihydroxyl-4,4-difluoro-6α-ethyl-5β-cholan-24-oyl)-benzene sulphonamide a white solid (161 mg, 0.27 mmol, 31%).

(249) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.06 (2H, dd, J=7.6, 1.3 Hz), 7.64 (1H, tt, J=7.6, 0.8 Hz), 7.54 (2H, t, J=7.3 Hz), 3.85-3.69 (2H, m), 2.45 (1H, dd, J=32.1, 11.0 Hz), 2.27 (1H, ddd, J=15.3, 10.2, 5.0 Hz), 2.16-2.10 (1H, m), 1.97-1.89 (3H, m), 1.86-1.64 (8H, m), 1.61-1.54 (1H, m), 1.47-1.43 (4H, m), 1.37-1.29 (2H, m), 1.22-1.06 (6H, m), 1.03 (3H, s), 0.97 (3H, t, J=7.3 Hz), 0.84 (3H, d, J=6.2 Hz), 0.59 (3H, s) ppm.

(250) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−99.0 (1F, d, J=239.3 Hz), −111.0 (1F, dtd, J=239.3, 33.0, 26.0 Hz) ppm.

(251) LRMS (ESI.sup.+) m/z: 613.6, [M+NH.sub.4].sup.+, 100%.

Example 5—Synthesis of 6α-ethyl-2α/β,4β-difluoro-(3α,7α)-dihydroxyl-5β-cholanic Acid Analogues with Sulfonylurea and Sulfonamide Side Chains

(252) A. Methyl 6α-ethyl-2α/β,4β-difluoro-7α-hydroxyl-3-oxo-5β-cholan-24-oate

(253) ##STR00097##

(254) To a stirred, pre-cooled solution of 1M LDA in THF/hexanes (1.63 mL, 1.625 mmol) and TMS-Cl (0.21 mL, 1.626 mmol) in dry THF (6 mL) at −78° C. was added a solution of methyl 6α-ethyl-4β-fluoro-7α-hydroxyl-3-oxo-5β-cholan-24-oate (product of Example 1C; 170 mg, 0.325 mmol) in dry THF (2.5 mL) dropwise over 10 mins. After addition the reaction was gradually allowed to warm to RT and stirred for 20 h. Upon completion the reaction mixture was cooled to 0° C. and quenched via dropwise addition of saturated NaHCO.sub.3 (5 mL) and diluted with H.sub.2O (5 mL). The organic phase was removed and the aqueous phase back extracted with EtOAc (3×5 mL). The combined organics were washed with NaHCO.sub.3 (5 mL), H.sub.2O (5 mL) and brine (5 mL). Organic phases were combined, dried over MgSO.sub.4, filtered and concentrated in vacuo to yield a yellow oil. The resultant syrup was used for the next reaction without further purification.

(255) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz): δ−169.9 (1F, s);

(256) To a stirred solution of methyl-3-trimethylsilyl-6α-ethyl-4β-fluoro-7α-hydroxyl-5β-cholan-2-ene-24-oate (assume 0.19 g, 0.325 mmol) in dry MeCN (10 mL) was added SELECTFLUOR® (0.17 g, 0.488 mmol) portion wise and the reaction stirred at RT for 16 h. Upon completion the reaction was diluted with EtOAc (5 mL) and sat. NaHCO.sub.3 (3 mL). Organic phase removed and the aqueous phase back extracted with EtOAc (3×5 mL). Organic phases combined, dried over MgSO.sub.4, filtered and concentrated in vacuo to yield a yellow oil. The procedures described above in Examples 2D and 2E were carried out on the product of Step A to obtain methyl 6α-ethyl-2α/β,4β-difluoro-(3α,7α)-dihydroxyl-5β-cholan-24-oate. This compound can be converted to the equivalent sulfonyl urea or sulfonamide analogues using General Procedures 1 to 4 as described in Examples 1 to 3 above.

Synthesis of 2β-Fluoro Compounds

(257) 2α-Fluoro and 4α-fluoro derivatives of obeticholic acid were prepared as described below.

Example 6—Synthesis of 2β-fluoro-3α,7α-dihydroxyl-6α-ethyl-5β-cholanic Acid Analogues with Sulfonylurea and Sulfonamide Side Chains

A. Methyl-3,7-dioxo-6α-ethyl-5β-cholan-24-oate

(258) ##STR00098##

(259) To a solution of (6α, 5β, 7α)-6-ethyl-7-hydroxy-3,7-dioxo-cholan-24-oic acid prepared as described in WO 2016/079520 (36.0 g, 87.7 mmol, 1.0 equiv.) in methanol (800 mL) at RT was added para-toluenesulfonic acid (1.67 g, 8.78 mmol, ˜0.1 equiv.) and sonicated at 30° C. for 4 hours. The reaction was deemed complete by TLC and the reaction mixture was concentrated in vacuo. The residue was dissolved in chloroform (400 mL) and washed with sat. NaHCO.sub.3 solution (400 mL) and brine (400 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford 37.6 g of crude material as a white solid that was used without further purification (87.3 mmol, 99%).

(260) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.66 (3H, s), 2.74 (1H, m), 2.47 (1H, t, J=11.3 Hz), 2.35 (1H, ddd, J=15.4, 10.0, 5.3 Hz), 2.26-2.14 (6H, m), 2.10-1.77 (6H, m), 1.74-1.35 (7H, m), 1.33 (3H, s), 1.31-1.26 (1H, m), 1.21-0.96 (4H, m), 0.93 (3H, d, J=6.5 Hz), 0.80 (3H, t, J=7.4 Hz), 0.69 (3H, s) ppm.

(261) LRMS (ESI.sup.+) m/z: 448.3 [M+NH.sub.4].sup.+, 100%.

B. Methyl-3β-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-oate and methyl-3α-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-oate

(262) ##STR00099##

(263) To a dry solution of methyl-3,7-dioxo-6α-ethyl-5β-cholan-24-oate of Step A (10.0 g, 23.2 mmol, 1.0 equiv.) in tetrahydrofuran (340 mL) at −78′C. under argon was added L-SELECTRIDE® (35.0 mL; 34.8 mmol, .sup.˜2.5 equiv.) dropwise over 15 minutes. After 10 minutes, the reaction mixture received a solution of hydrogen peroxide (40 mL, 30% v/v) and 2M sodium hydroxide (40 mL) in water (400 mL) at 0° C. After a further 10 minutes, the reaction mixture received 2M hydrochloric acid (130 mL) at RT. The aqueous phase was separated and extracted with ethyl acetate (2×250 mL) and the combined organic fractions were washed with water (500 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford 11.0 g of crude material as a colourless oil. Purification by flash column chromatography (Biotage SNAP KP-Sil 100 g cartridge) using PE 40-60/acetone (90/10 to 80/20) as the eluent yielded an inseparable mixture of compound Methyl-3β-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-oate and methyl-3α-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-oate in a 65:35 ratio as a white residue (7.83 g, 18.1 mmol, 78%). to the mixture was not separated as both compounds lead to the same mixture of alkenes in the next step.

(264) Compound A: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.05 (1H, t, J=2.5 Hz), 3.66 (3H, s), 2.77-2.73 (1H, m), 2.41-2.31 (2H, m), 2.26-2.14 (3H, m), 2.00-1.88 (2H, m), 1.84-1.58 (6H, m), 1.55-1.29 (10H, m), 1.25 (3H, s), 1.15-1.07 (4H, m), 0.92 (3H, d, J=6.5 Hz), 0.81 (3H, t, J=7.4 Hz), 0.66 (3H, s).

(265) LRMS (ESI.sup.+) m/z: 450.3 [M+NH.sub.4].sup.+, 100%.

(266) Compound B: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.66 (3H, s), 3.57-3.48 (1H, m), 2.76-2.67 (1H, m), 2.41-2.32 (2H, m), 2.26-2.14 (3H, m), 2.00-1.88 (2H, m), 1.84-1.59 (6H, m), 1.55-1.29 (10H, m), 1.22 (3H, s), 1.17-1.07 (4H, m), 0.92 (3H, d, J=6.4 Hz), 0.80 (3H, t, J=7.4 Hz), 0.65 (3H, s) ppm.

(267) LRMS (ESI.sup.+) m/z: 450.4 [M+NH.sub.4].sup.+, 100%.

C. Methyl-6α-ethyl-7oxo-5β-chol-2-ene-24-oate and methyl-6α-ethyl-7-oxo-5β-chol-3-ene-24-oate

(268) ##STR00100##

(269) To a solution of methyl-3β-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-oate and methyl-3α-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-oate of Step B (6.31 g, 14.6 mmol, 1.0 equiv.) in dichloromethane (120 mL) at RT was added dimethylaminopyridine (3.56 g, 29.2 mmol, ˜2.0 equiv.). The reaction mixture was cooled to 0° C. and received triflic anhydride (2.57 mL, 15.3 mmol, ˜1.05 equiv.) dropwise over 5 minutes. After 2 hours warming to 12° C. the reaction was deemed complete by TLC and the reaction mixture was quenched with 2M hydrochloric acid (100 mL). The aqueous phase was separated and extracted with dichloromethane (3×100 mL) and the combined organic fractions were washed with brine (200 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford 7.56 g of crude material as an orange oil. Purification by flash column chromatography (Biotage SNAP KP-Sil 100 g cartridge) using PE 40-60/acetone (90/10) as the eluent yielded an inseparable mixture of methyl-6α-ethyl-7oxo-5β-chol-2-ene-24-oate and methyl-6α-ethyl-7-oxo-5β-chol-3-ene-24-oate in a 80:20 ratio as a colourless oil (2.70 g, 6.51 mmol, 45%).

(270) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.63-5.40 (2H, m), 3.66 (3H, s,), 2.74 (1H, dd, J=12.0, 6.6 Hz), 2.34 (2H, tt, J=10.3, 5.1 Hz), 2.27-1.29 (19H, m), 1.27 (2H, s), 1.26 (1H, s), 1.18-0.94 (3H, m), 0.91 (3H, d, J=6.5 Hz), 0.83 (3H, t, J=7.5 Hz), 0.664 (1H, s), 0.657 (2H, s) ppm.

(271) LRMS (ESI.sup.+) m/z: 432.20 [M+NH.sub.4].sup.+, 100%.

D. Methyl-2β,3β-epoxy-6α-ethyl-7-oxo-5β-cholan-24-oate and methyl-3β,4β-epoxy-6α-ethyl-7-oxo-5β-cholan-24-oate

(272) ##STR00101##

(273) To a solution of a 80:20 ratio of methyl-6α-ethyl-7oxo-5β-chol-2-ene-24-oate and methyl-6α-ethyl-7-oxo-5β-chol-3-ene-24-oate of Step C (5.00 g, 12.1 mmol, ˜1.0 equiv.) in dichloromethane (100 mL) at RT was added meta-perchlorobenzoic acid (3.12 g, 18.1 mmol, ˜1.5 equiv.). After 3 hours at RT, the reaction was deemed complete by TLC and the reaction mixture was quenched with sat. Na.sub.2S.sub.2O.sub.3 solution (150 mL). After 10 mins stirring, the aqueous phase was separated and extracted with dichloromethane (3×100 mL) and the combined organic fractions were dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford 5.28 g of crude material as a pale yellow residue. Purification by flash column chromatography (Biotage SNAP KP-Sil 100 g cartridge) using PE 40-60/acetone (95/5 to 90/10) as the eluent yielded an inseparable mixture of methyl-2β,3β-epoxy-6α-ethyl-7-oxo-5β-cholan-24-oate and methyl-3β,4β-epoxy-6α-ethyl-7-oxo-5β-cholan-24-oate as a colourless oil (4.94 g, 11.5 mmol, 95%). Further purification by flash column chromatography (Biotage SNAP KP-Sil 340 g cartridge) using PE 40-60/acetone (95/5 to 90/10) as the eluent yielded compound methyl-2β,3β-epoxy-6α-ethyl-7-oxo-5β-cholan-24-oate as a colourless oil (3.71 g, 8.62 mmol, 72%) and compound methyl-3β,4β-epoxy-6α-ethyl-7-oxo-5β-cholan-24-oate as a colourless oil (1.18 g, 2.74 mmol, 23%).

(274) Compound A: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.66 (3H, s), 3.13 (1H, t, J=2.6 Hz), 3.01 (1H, dd, J=5.5, 4.2 Hz), 2.67 (1H, dd, J=11.5, 6.6 Hz), 2.35 (1H, ddd, J=15.4, 10.2, 5.1 Hz), 2.30-2.18 (3H, m), 2.01-1.65 (7H, m), 1.55-1.20 (10H, m), 1.17 (3H, s), 1.15-0.95 (3H, m), 0.92 (3H, d, J=6.4 Hz), 0.81 (3H, t, J=7.4 Hz), 0.65 (3H, s) ppm.

(275) LRMS (ESI.sup.+) m/z: 448.30 [M+NH.sub.4].sup.+, 100%.

(276) Compound B: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.66 (3H, s), 3.10-3.09 (1H, m), 2.79-2.74 (2H, m), 2.41-2.31 (2H, m), 2.26-2.04 (3H, m), 2.00-1.88 (4H, m), 1.84-1.58 (2H, m), 1.53-1.20 (11H, m), 1.17 (3H, s), 1.14-0.94 (2H, m), 0.91 (3H, d, J=6.3 Hz), 0.90 (3H, t, J=7.5 Hz), 0.66 (3H, s) ppm.

(277) LRMS (ESI.sup.+) m/z: 448.26 [M+NH.sub.4].sup.+, 100%.

E. Methyl-2α-fluoro-3α-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-oate

(278) ##STR00102##

(279) To a dry solution of methyl-2β,3β-epoxy-6α-ethyl-7-oxo-5β-cholan-24-oate of Step D (3.33 g, 7.73 mmol, 1.0 equiv.) in dichloromethane (100 mL) at 0° C. under argon was added HF.pyridine (70%) complex (100 mL, 3.86 mol, ˜500 equiv.) by pouring a freshly opened 100 mL bottle into the cooled reaction flask via a glass funnel whilst under a steady flow of argon. After addition of the reagent, the bottle and funnel were rinsed with dichloromethane (20 mL). After 3 hours at 0° C., the reaction mixture was diluted with dichloromethane (200 mL) and quenched by the slow addition of sat. NaHCO.sub.3 solution (500 mL) and stirred at RT for 1 hour whilst receiving 5.0 g of NaHCO.sub.3 in 100 mg portions. The aqueous phase was then separated and extracted with dichloromethane (3×250 mL) and the combined organic fractions were dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford 3.61 g of crude material as a colourless oil. Purification by flash column chromatography (Biotage SNAP KP-Sil 100 g cartridge) using PE 40-60/acetone (95/5 to 90/150 as the eluent yielded compound methyl-2α-fluoro-3α-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-oate as a white residue (2.39 g, 5.30 mmol, 69%).

(280) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.53 (1H, dq, J=47.0, 2.3 Hz), 4.01 (1H, dq, J=6.6, 2.9 Hz), 3.66 (3H, s), 2.75 (1H, dd, J=13.3, 5.8 Hz), 2.38-2.30 (2H, m), 2.25-2.11 (4H, m), 2.01 (1H, dd, J=12.0, 3.8 Hz), 1.93-1.36 (14H, m), 1.25 (3H, d, J=4.0 Hz), 1.21-1.10 (3H, m), 0.91 (3H, d, J=6.5 Hz), 0.82 (3H, t, J=7.4 Hz), 0.65 (3H, s) ppm

(281) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−184.3 (1F, tt, J=50.3, 8.7 Hz) ppm.

(282) LRMS (ESI.sup.+) m/z: 468.28 [M+NH.sub.4].sup.+, 100%.

F. Methyl-2α-fluoro-3,7-dioxo-6α-ethyl-5β-cholan-24-oate

(283) ##STR00103##

(284) To a solution of methyl-2α-fluoro-3α-hydroxyl-6α-ethyl-7-oxo-5β-cholan-24-oate of Step E (1.00 g, 2.26 mmol, 1.0 equiv.) in dichloromethane (20 mL) at RT was added Dess-Martin periodinane (1.92 g, 4.52 mmol, ˜2.0 equiv.) and H.sub.2O (0.25 mL). After 3 hours at RT, the reaction was deemed complete by TLC and the reaction mixture was quenched with sat. NaHCO.sub.3 solution (25 mL) and filtered over Celite and washed with dichloromethane (90 mL). The aqueous phase was then separated and extracted with dichloromethane (2×50 mL) and the combined organic layers were washed with sat. Na.sub.2S.sub.2O.sub.3 solution (150 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford 1.21 g of crude material as a pale yellow oil. Purification by flash column chromatography (Biotage SNAP KP-Sil 25 g cartridge) using PE 40-60/acetone (95/5 to 90/10) as the eluent yielded compound methyl-2α-fluoro-3,7-dioxo-6α-ethyl-5β-cholan-24-oate as a white residue (568 mg, 1.27 mmol, 56%).

(285) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.69 (1H, ddd, J=50.7, 4.8, 3.4 Hz), 3.68 (3H, s), 2.75 (1H, ddd, J=7.7, 5.0, 4.3 Hz), 2.51-1.94 (12H, m), 1.83-1.40 (9H, m), 1.37 (3H, s), 1.34-1.08 (5H, m), 0.94 (3H, d, J=6.5 Hz), 0.84 (3H, t, J=7.4 Hz), 0.70 (3H, s) ppm.

(286) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−188.2 (1F, ddd, J=51.6, 42.1, 12.1 Hz) ppm.

(287) LRMS (ESI.sup.+) m/z: 466.55 [M+NH.sub.4].sup.+, 100%.

G. 2α-Fluoro-3,7-dioxo-6α-ethyl-5β-cholanic Acid and 2β-fluoro-3,7-dioxo-6α-ethyl-5β-cholanic Acid

(288) ##STR00104##

(289) To a solution of methyl-2α-fluoro-3,7-dioxo-6α-ethyl-5β-cholan-24-oate (product of Step F, 878 mg, 1.95 mmol, 1.0 equiv.) in methanol (20 mL) at RT was added sodium hydroxide (1.0 g). After 19 hours at RT the reaction was deemed complete by TLC and the reaction mixture was acidified to pH 4.0 and concentrated in vacuo. The residue was dissolved in ethyl acetate (50 mL) and washed with 1M hydrochloric acid (50 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford 968 mg of crude material as a colourless oil. Purification by flash column chromatography (Biotage SNAP KP-Sil 25 g cartridge) using dichloromethane/methanol (98/2 to 90/10) as the eluent yielded an inseparable mixture of 2α-fluoro-3,7-dioxo-6α-ethyl-5β-cholanic acid and 2β-fluoro-3,7-dioxo-6α-ethyl-5β-cholanic acid in a 40:60 ratio as a white residue (772 mg, 1.77 mmol, 91%). 2α-fluoro-3,7-dioxo-6α-ethyl-5β-cholanic acid: .sup.1H NMR—characteristic peaks (400 MHz, CDCl.sub.3): δ 4.68 (1H, ddd, J=50.7, 5.1, 3.6 Hz), 2.75 (1H, ddd, J=7.7, 5.0, 4.3 Hz), 1.36 (3H, s), 0.94 (3H, d, J=6.5 Hz), 0.82 (3H, t, J=7.3 Hz), 0.69 (3H, s) ppm.

(290) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−188.2 (1F, ddd, J=51.6, 42.1, 12.1 Hz) ppm.

(291) LRMS (ESI.sup.+) m/z: 452.51 [M+NH.sub.4].sup.+, 100%.

2β-fluoro-3,7-dioxo-6α-ethyl-5β-cholanic Acid

(292) .sup.1H NMR—characteristic peaks (400 MHz, CDCl.sub.3): δ 4.90 (0.6H, ddd, J=48.7, 13.2, 6.1 Hz), 2.75 (1H, ddd, J=7.7, 5.0, 4.3 Hz), 1.39 (2H, s), 0.95 (2H, d, J=6.5 Hz), 0.82 (3H, t, J=7.3 Hz), 0.70 (2H, s) ppm.

(293) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−195.1 (1F, ddd, J=48.6, 10.4, 5.2 Hz) ppm.

(294) LRMS (ESI.sup.+) m/z: 452.51 [M+NH.sub.4].sup.+, 100%.

H. Methyl-2β-fluoro-3,7-dioxo-6α-ethyl-5β-cholan-24-oate

(295) ##STR00105##

(296) To a solution of a 40:60 ratio of 2α-fluoro-3,7-dioxo-6α-ethyl-5β-cholanic acid and 2β-fluoro-3,7-dioxo-6α-ethyl-5β-cholanic acid of Step G (750 mg, 1.72 mmol, ˜1.0 equiv.) in dimethylformamide (17 mL) at RT was added caesium carbonate (840 mg, 2.58 mmol, ˜1.5 equiv). After 20 mins at RT, iodomethane (0.54 mL, 8.59 mmol, ˜5.0 equiv.) was added dropwise. After 19 hours at RT, the reaction was deemed complete by TLC and the reaction mixture was concentrated in vacuo. The residue was dissolved in ethyl acetate (25 mL) and H.sub.2O (20 mL). The aqueous layer was separated and extracted with ethyl acetate (3×25 mL) and the combined organic layers were washed with brine (100 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford a 917 mg of crude material as a pale yellow oil. Purification by flash column chromatography (Biotage SNAP Ultra KP-Sil 25 g cartridge) using PE 40-60/acetone (95/5 to 90/10) as the eluent yielded compound methyl-2β-fluoro-3,7-dioxo-6α-ethyl-5β-cholan-24-oate as a white residue (416 mg, 0.95 mmol, 54%). The corresponding 2α-fluoro derivative was not isolated.

(297) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.69 (1H, ddd, J=48.8, 13.3, 6.4 Hz), 3.67 (3H, s), 2.75 (1H, dd, J=13.1, 5.0 Hz), 2.54-2.44 (2H, m), 2.40-2.32 (2H, m), 2.29-2.13 (3H, m), 2.09 (1H, dt, J=13.0, 3.3 Hz), 2.01-1.91 (2H, m), 1.85-1.65 (5H, m), 1.54-1.42 (2H, m), 1.39 (3H, s), 1.38-1.05 (7H, m), 0.94 (3H, d, J=6.5 Hz), 0.82 (3H, t, J=7.4 Hz), 0.70 (3H, s) ppm.

(298) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−195.1 (1F, ddt, J=48.6, 10.4, 5.2 Hz) ppm.

(299) LRMS (ESI.sup.+) m/z: 466.59 [M+NH.sub.4].sup.+, 100%.

I. Methyl-2β-fluoro-3β,7α-dihydroxyl-6α-ethyl-5β-cholan-24-oate and methyl-2β-fluoro-3α,7α-dihydroxyl-6α-ethyl-5β-cholan-24-oate

(300) ##STR00106##

(301) To a dry solution of methyl-2β-fluoro-3,7-dioxo-6α-ethyl-5β-cholan-24-oate of Step H (390 mg, 0.87 mmol, 1.0 equiv.) in methanol (20 mL) under argon at RT was added sodium borohydride (164 mg, 4.44 mmol, 5.0 equiv.). After 1 hour at RT, the reaction mixture was concentrated in vacuo. The residue was dissolved in dichloromethane (20 mL) and H.sub.2O (20 mL) and the aqueous phase was separated and extracted with dichloromethane (3×20 mL). The combined organic fractions were dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford 446 mg of crude material as a pale yellow oil. Purification by flash column chromatography (Biotage SNAP Ultra KP-Sil 25 g cartridge) using PE 40-60/acetone (95/5 to 90/10) as the eluent yielded compound methyl-2β-fluoro-3β,7α-dihydroxyl-6α-ethyl-5β-cholan-24-oate as a colourless oil (161 mg, 0.36 mmol, 41%) and methyl-2β-fluoro-3α,7α-dihydroxyl-6α-ethyl-5β-cholan-24-oate as a colourless oil (146 mg, 0.32 mmol, 37%).

Methyl-2β-fluoro-3β,7α-dihydroxyl-6α-ethyl-5β-cholan-24-oate

(302) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.63 (1H, dddd, J=47.4, 12.5, 4.4, 3.1 Hz,), 4.15 (1H, q, 3.6 Hz), 3.71 (1H, s), 3.67 (3H, s), 2.36 (1H, ddd, J=15.5, 10.2, 5.3 Hz), 2.33 (1H, ddd, J=16.0, 9.7, 6.6 Hz), 2.12 (1H, td, J=13.2, 2.2 Hz), 2.00 (1H, dt, J=12.5, 3.1 Hz), 1.94-1.78 (5H, m), 1.77-1.54 (6H, m), 1.52-1.25 (8H, m), 1.20-1.12 (4H, m), 1.00 (3H, s), 0.93 (3H, d, J=6.8 Hz), 0.92 (3H, t, J=7.1 Hz), 0.67 (3H, s) ppm.

(303) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−187.1 (1F, dquin, J=46.9, 7.8 Hz) ppm.

(304) LRMS (ESI.sup.+) m/z: 470.64 [M+NH.sub.4].sup.+, 100%.

methyl-2β-fluoro-3α,7α-dihydroxyl-6α-ethyl-5β-cholan-24-oate

(305) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.42 (1H, dddd, J=52.7, 12.5, 8.7, 4.5 Hz), 3.70 (1H, s), 3.67 (3H, s), 3.52 (1H, ddt, 13.8, 12.5, 6.0 Hz), 2.36 (1H, ddd, J=15.4, 10.2, 5.3 Hz), 2.27-2.17 (3H, m), 2.09-1.97 (2H, m), 1.95-1.86 (2H, m), 1.84-1.76 (1H, m), 1.66-1.29 (14H, m), 1.22-1.13 (4H, m), 0.98 (3H, s), 0.93 (3H, d, J=6.5 Hz), 0.92 (3H, t, J=7.2 Hz), 0.67 (3H, s) ppm.

(306) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−188.2 (1F, ddd, J=51.6, 42.1, 12.1 Hz) ppm.

(307) LRMS (ESI.sup.+) m/z: 470.64 [M+NH.sub.4].sup.+, 100%.

J. 2β-Fluoro-3α,7α-dihydroxyl-6α-ethyl-5β-cholanic Acid

(308) ##STR00107##

(309) To a solution of methyl-2β-fluoro-3α,7α-dihydroxyl-6α-ethyl-5β-cholan-24-oate of Step I (119 mg, 0.26 mmol, 1.0 equiv.) in a solution of 1,4-dioxane (9.8 mL) and water (3.6 mL) at RT was added concentrated (37%) hydrochloric acid (1.2 mL, 9:3:1 ratio). After 1 hour at reflux, the reaction was deemed complete by TLC and the reaction mixture was neutralised with sat. NaHCO.sub.3 solution (20 mL). The aqueous phase was extracted with ethyl acetate (3×15 mL) and the combined organic fractions were dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford 141 mg of crude material as a colourless oil. Purification by flash column chromatography (Biotage SNAP Ultra KP-Sil 10 g cartridge) using dichloromethane/methanol (95/5 to 90/10) as the eluent yielded compound 2β-fluoro-3α,7α-dihydroxyl-6α-ethyl-5β-cholanic acid as a white residue (92 mg, 0.21 mmol, 80%).

(310) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.42 (1H, dddd, J=52.6, 12.5, 8.7, 4.3 Hz), 3.69 (1H, s), 3.52 (1H, tdd, 12.0, 8.8, 5.4 Hz), 2.39 (1H, ddd, J=15.5, 10.3, 5.1 Hz), 2.29-2.19 (3H, m), 2.01 (2H, t, J=13.0 Hz), 1.93-1.77 (3H, m), 1.65-1.54 (3H, m), 1.53-1.26 (12H, m), 1.23-1.10 (4H, m), 0.97 (3H, s), 0.94 (3H, d, J=6.5 Hz), 0.91 (3H, t, J=7.1 Hz), 0.66 (3H, s) ppm.

(311) .sup.19F NMR (.sup.1H non-decoupled, 376 MHz, CDCl.sub.3): δ−186.8 (1F, ddq, J=52.9, 13.0, 7.5 Hz,) ppm.

(312) LRMS (ESI.sup.+) m/z: 456.60 [M+NH.sub.4].sup.+, 100%.

(313) This compound can be converted to the equivalent sulfonyl urea or sulfonamide analogues using General Procedures 1 to 4 as described in Examples 1 to 3 above.

BIOLOGICAL EXAMPLES

(314) For Biological Examples 8 and 9 below, all work has been carried out and data has been kindly supplied by Professor Kim Watson and Dannielle Kydd-Sinclair of the University of Reading, UK.

Example 7—Measurement of EC.SUB.50 .and Efficacy at FXR Receptor

(315) The compounds of the invention were assayed for agonist activity at the FXR receptor. Table 1 shows the EC.sub.50 values and efficacy values for example compounds of the present invention compared with the values for comparative example compounds, obeticholic acid and the known FXR agonist GW4064, which has the structure:

(316) ##STR00108##

(317) Efficacy is defined as the maximum point on the dose response curve and the efficacy value for GW4064 in Table 1 has been designated as 100%.

(318) The EC.sub.50 values in Table 1 are normalized against the EC.sub.50 of GW4064, which has been assigned as 25 nM.

(319) Obeticholic acid may be prepared as described in WO 02/072598 or our applications WO 2016/079518, WO 2016/079518, WO 2016/079519 and WO 2016/079520.

(320) FXR EC.sub.50/Efficacy Protocol

(321) Dose-response assays were performed as described in the technical manual of the Human Farnesoid X Receptor (NR1H4, FXR) Reporter Assay System (Indigo Biosciences Human Farnesoid X Receptor (NR1H4, FXR) Reporter Assay System, Technical Manual (version 7.1b), at the World Wide Web (www)indiqobiosciences.com).

(322) FXR reporter cells consisting of an FXR-responsive promoter gene functionally linked to the luciferase gene were defrosted and seeded into a 96-well plate and these cells were immediately dosed with the test compounds at different concentrations (10-0.05 μM) according to the manufacturer's protocol. After 24 h incubation in the presence of the test compound or solvent (DMSO), the cell viability of these treated/untreated reporter cells was measured to eliminate false negative results using the fluorescence-based live cell multiplex (LCM) assay (Indigo Biosciences Live Cell Multiplex Assay, Technical Manual (version 3.1), at the World Wide Web (www)indigobiosciences.com). The fluorescence from the live cells was measured using the plate reader with the filter combination of [485nmEx|535nmEm]. Following this, the induction of luciferase activity, which is the measure of the agonist activity, was quantified by using luminometer (TECAN) according to the manufacturer's protocol.

(323) Positive controls were run in each assay in which the EC.sub.50 values of GW4064 was assigned as 25 nM and GW4064 assigned efficacy of 100%. The efficacy and EC.sub.50 of each test compound was compared to that of GW4064.

(324) The results are set out in Table 1.

(325) TABLE-US-00001 TABLE 1 A- EC.sub.50 Efficacy Compound Ring Side chain (nM) (%) Obeticholic acid No F 09 109 146 GW4064 — —  25 100 Compound 1 4β-F 0  18 193 Compound 2 4β-F  24 195 Compound 3 4β-F  19 151 Compound 4 4β-F  23 148 Compound 5 4β-F  28 166 Compound 6 4β-F  32 187 Compound 7 4β-F  38 131 Compound 8 4β-F  43 159 Compound 9 4β-F  50 158 Compound 11 4β-F 123 104 Compound 12 4β-F 0  27 176 Compound 13 4β-F  48 250 Comparative compound A No F  32 147 Compound 14 4β-F   2.34 219 Compound 15 4β-F  38  98 Compound 16 4β-F  46 143 Compound 17 4β-F  34 121 Compound 18 4β-F  15 148 Compound 19 4β-F  50 136 Comparative compound B 4β-F 158 132 Comparative compound C 4β-F 0 192 130

(326) The compounds of the invention all have FXR agonist activity. All of the compounds apart from Compound 11 have significantly improved EC.sub.50 values compared with obeticholic acid. Furthermore, the efficacy values for all of the compounds of the invention are at least as good as, and in most cases better than, the value for GW4064.

(327) A comparison of the results for Compound 2 with its unfluorinated analogue Comparative Compound A demonstrates that fluorination improves both the EC.sub.50 and the efficacy. A comparison of the results for the aromatic sulfonamide Compounds 14 to 20 with the carbocyclic sulfonamide Comparative Compound B and the methylsulfonamide Comparative Compound C demonstrates the importance of the aromatic substituent on the side chain.

Example 8—Measurement of EC.SUB.50 .and Efficacy at TGR5 Receptor

(328) Compounds 2 and 14, TGR5 (control) and the taurine and glycine conjugates of obeticholic acid were tested for activity at the TGR5 receptor using a HITHUNTER® cAMP assay available from DiscoverX in the agonist mode to monitor the activation of the TGR5 (GPBAR1) receptor through Gi and Gs secondary messenger signalling. Data was normalized to the maximal and minimal response observed in the presence of control ligand (TGR5) and vehicle.

(329) Assay Design: GPCR cAMP Modulation

(330) Cell Handling

(331) 1. cAMP Hunter cell lines were expanded from freezer stocks according to standard procedures.

(332) 2. Cells were seeded in a total volume of 20 μL into white walled, 384-well microplates and incubated at 37° C. for the appropriate time prior to testing.

(333) 3. cAMP modulation was determined using the DiscoverX HITHUNTER® cAMP XS+assay.

(334) Gs Agonist Format

(335) 1. For agonist determination, cells were incubated with sample to induce response.

(336) 2. Media was aspirated from cells and replaced with 15 μL 2:1 HBSS/10 mM Hepes:cAMP XS+ Ab reagent.

(337) 3. Intermediate dilution of sample stocks was performed to generate 4× sample in assay buffer.

(338) 4. 5 μL of 4× sample was added to cells and incubated at 37° C. or room temperature for 30 or 60 minutes. Vehicle concentration was 1%.

(339) Signal Detection

(340) 1. After appropriate compound incubation, assay signal was generated through incubation with 20 μL cAMP XS+ ED/CL lysis cocktail for one hour followed by incubation with 20 μL cAMP XS+ EA reagent for three hours at room temperature.

(341) 2. Microplates were read following signal generation with a PerkinElmer ENVISION™ instrument for chemiluminescent signal detection.

(342) Data Analysis

(343) 1. Compound activity was analyzed using CBIS data analysis suite (ChemInnovation, CA).

(344) 2. For Gs agonist mode assays, percentage activity is calculated using the following formula, where RLU is an abbreviation for relative light units:

(345) $\%\mathrm{Activity}=100\%\times \left(\frac{\mathrm{mean}\mathrm{RLU}\mathrm{of}\mathrm{test}\mathrm{sample}-\mathrm{mean}\mathrm{RLU}\mathrm{of}\mathrm{vehicle}\mathrm{control}}{\mathrm{mean}\mathrm{RLU}\mathrm{of}\mathrm{MAX}\mathrm{control}-\mathrm{mean}\mathrm{RLU}\mathrm{of}\mathrm{vehicle}\mathrm{control}}\right)$

(346) 5. For Gi agonist mode assays, percentage activity is calculated using the following formula:

(347) $\%\mathrm{Activity}=100\%\times \left(\frac{1-\left(\mathrm{mean}\mathrm{RLU}\mathrm{of}\mathrm{test}\mathrm{sample}-\mathrm{mean}\mathrm{RLU}\mathrm{of}\mathrm{MAX}\mathrm{control}\right)}{\mathrm{mean}\mathrm{RLU}\mathrm{of}\mathrm{vehicle}\mathrm{control}-\mathrm{mean}\mathrm{RLU}\mathrm{of}\mathrm{MAX}\mathrm{control}}\right)$
Results

(348) The results are presented in Table 2

(349) TABLE-US-00002 TABLE 2 Compound EC.sub.50 (μM) Max Response TGR5 (control) 0.445 101.26 Compound 2 >100 0 Compound 14 >100 0 Obeticholic acid 0.979 97.70 taurine conjugate Obeticholic acid 1.904 110.9 glycine conjugate

(350) The results demonstrate that, unlike the obeticholic acid conjugates, neither Compound 2 nor Compound 14 has agonist activity at the TGR5 receptor. The compounds are therefore selective FXR agonists.

Example 9—Quantitative Analysis of Ligand-Induced Gene Expression

(351) Compound 2 and obeticholic acid were tested for their effect on the expression of a number of FXR target genes. This example describes cell-based assays and gene expression analysis to observe functional activation of FXR by the compounds of the invention and obeticholic acid at a cellular level.

(352) To assess specific changes in gene expression in response to the test compounds, precise quantification and analysis by quantitative real-time PCR (qPCR) was carried out. Mammalian tissue culture experiments involved seeding Hepatocellular carcinoma, (Huh7) cells, in 6 well plates at a concentration of 1×10.sup.6 cells/well and incubating for 24 hours at 37° C. to allow attachment. Cells were exposed to the respective test compound at either its EC.sub.50 or EC.sub.50, or vehicle (DMSO), for 24 hours.

(353) Compound 2 and OCA were also tested in the human hepatocellular carcinoma cell line, HepG2. HepG2 cells were also incubated with medium containing OCA or Compound 2 at either its EC.sub.50 or EC.sub.50 concentrations for 24 hours.

(354) Testing in Huh7 cells and HepG2 cells was also carried out for Compound 14 in the same way as for Compound 2.

(355) Isolation of Total RNA from Cultured Cells

(356) Total RNA was extracted using the RNAQUEOUS™ Total RNA Isolation kit (Ambion) and all reagents were provided in the kit or prepared according to manufacturer's instructions. For cultured cells, the medium was removed and cells were washed with 1×PBS to remove cellular debris and residual medium. Total RNA was extracted from fresh cells. For 1×10.sup.6 cells, 350 μl lysis buffer was added directly to the well and cells were harvested by scraping with a pipette tip. Lysed cells were combined with an equal volume of 64% ethanol and mixed thoroughly by pipetting. The ethanol-lysate mix was transferred to a column and spun at 12,000×G for 1 minute and flow through discarded. The membrane was washed by adding wash buffer 1 to the column and centrifuging at 12,000×G for 1 minute, before discarding the flow through and repeating this step twice with wash buffer 2. An additional spin with the empty cartridge was included to completely dry the membrane of ethanol. Finally, the total RNA was eluted in 2 sequential aliquots of 50 μl preheated elution buffer (nuclease-free water containing trace amounts of EDTA).

(357) Analysis of RNA Quantity, Purity and Integrity

(358) RNA concentration was quantified by measuring the absorbance at 260 nm, using a Nanodrop Lite spectrophotometer (Thermo Scientific). The purity of RNA was determined by analysing the A.sub.260:280 ratio, where a value of between 1.8 and 2.1 was deemed to be free from protein contamination and acceptable for downstream applications. The integrity of the RNA was determined by running a sample on a denaturing formaldehyde agarose gel. A 1% agarose (Sigma Aldrich), 1×MOPS (Sigma Aldrich), 6.6% formaldehyde (Fisher Scientific) gel was made with the addition of 1×SYBR™ Safe DNA stain (Invitrogen) to visualize the nucleic acids. Prior to loading, equal volumes of formaldehyde loading dye (Ambion) was added to 1 μg RNA and samples were heated at 70° C. for 10 minutes before being immediately snap cooled on ice for 2-3 minutes. The gel was run at 90V for 1 hour 30 minutes and visualised under UV light, using the NuGenius gel doc system (Syngene). The 28S and 18S rRNA bands were scrutinized for sharp, intense bands at approximately 5 kb and 1.9 kb, respectively, where the 28S upper band was expected to be twice the intensity of the 18S lower band for intact RNA. Smearing below the 18S rRNA band was taken to indicate degraded RNA, whilst smearing and/or bands above the 28S rRNA was indicative of DNA contamination. Following analysis, RNA was used immediately for reverse transcription.

(359) Reverse Transcription

(360) Reverse transcription was carried out using ISCRIPT™ Advanced cDNA Synthesis Kit for RT-qPCR (Biorad). To 1 μg DNase-treated RNA, 4 μl of 5×ISCRIPT™ Advanced reaction mix and 1 μl ISCRIPT™ Advanced Reverse Transcriptase was added. Nuclease-free water was added to a final volume of 20 μl and the reaction was incubated at 46° C. for 20 minutes, before inactivation at 95° C. for 1 minute. Newly synthesised cDNA was diluted 10-fold in TE buffer (10 mM Tris pH 8, 1 mM EDTA), aliquoted and stored at −20° C. until use in qPCR experiments.

(361) Qualitative Real-Time PCR Analysis

(362) Reference genes were selected based on data from existing literature. The selected target genes were as set out in Table 3.

(363) TABLE-US-00003 TABLE 3 Selected Target Genes for Qualitative Real-Time PCR Analysis Expected outcome in response to FXR Gene Pathway Activation NR0B2 (SHP) FXR Signalling, BA secretion Upregulated OSTα BA transport across cell membranes Upregulated CYP7A1 BA synthesis Downregulated TGFB1 Fibrosis, tissue remodelling, Downregulated monocyte signalling GAPDH Housekeeping Stable ACTB Housekeeping Stable (BACTIN)
Optimisation of Primers for qPCR

(364) KICQSTART® SYBR® Green Predesigned primers (chosen according to best rank) for the above target genes were purchased from Sigma Life Science. Nuclease-free water was added to the lyophilized primers for a stock concentration of 100 μM. Primers were diluted with nuclease-free water for a working concentration of 10 μM. To test the efficiency, reproducibility and dynamic range of the assay, a ten-fold serial dilution was made; consisting of 5 concentrations of cDNA generated (as outlined above) from Human Reference RNA (Agilent). Following qPCR of these samples, a standard curve was constructed by the threshold cycle (Ct) value (y-axis) versus log cDNA concentration (x-axis). The primer amplification efficiency (E) of one cycle in the exponential phase was determined by the equation E=10.sup.(−1/slope)−1 (Pfaffl, 2001). The accuracy of these qPCR reactions was determined by the R.sup.2 value of the standard curve, with values>0.98 being suitable. The specificity of each primer was determined by melt curve analysis, which was performed at the end of each run, where the production of one peak at one melting temperature indicated the amplification of just one product and, therefore, primers that were highly specific. Amplified products were confirmed by agarose gel electrophoresis (2% Agarose, 1×TAE, run at 100V for 30 minutes) to check amplicon sizes were as expected, and that only one product was seen.

(365) Quantitative PCR

(366) The ready-to-use reaction mastermix, ITAQ™ Universal SYBR® Green Supermix (Biorad) was used for all qPCR reactions. A typical reaction for each gene contained 5 μl 2×ITAQ™ Universal SYBR® Green Supermix, 500 nm forward and 500 nm reverse primers, approximately 15 ng cDNA and nuclease-free water to a final volume of 10 μl. Each target gene, reference gene and no template control were run in triplicate on an Optical MicroAmp 96 well plate (Applied Biosystems). Plates were sealed with an optical adhesive seal (Applied biosystems), briefly placed on a plate shaker to mix the components and centrifuged. Reactions were run using the Applied Biosystem Step One Plus real-time PCR system, using the following cycling conditions; an initial denaturation step at 95° C. for 15 minutes, 40 cycles of amplification, consisting of denaturation step at 94° C. for 15 seconds, combined annealing and extension step at 60° C. for 1 minute, with a single fluorescent measurement. Melting curve analysis was performed straight after each run by increasing the temperature from 60° C. to 95° C. in 0.3° C. increments, and measuring fluorescence dissociation.

(367) Data Analysis

(368) To quantify gene expression, the baseline-corrected C.sub.t value was determined automatically by the qPCR system software (Applied Biosystems). Relative changes in gene expression were determined by the Livak, or ΔΔC.sub.t, method whereby the C.sub.t values of target genes were normalised to C.sub.t value of the reference gene, for both the samples treated with vehicle/untreated samples (control) and the samples treated with compound (test samples). The ΔC.sub.t values for the test samples were then normalised against the ΔC.sub.t values of the control samples. And finally, the expression ratio was calculated using the equation 2.sup.−ΔΔC.sup.t. In total, there were 3 biological replicates and all data are represented as mean±SE. Treatments are compared by one way ANOVA followed by Tukey's and Dunnet's post hoc tests.

(369) Results

(370) Direct target genes, nr0b2 (SHP) and slc51a (OSTα), which are involved in bile acid homeostasis and known to be positively regulated by FXR (Goodwin et al., 2000; Landrier et al., 2006), display significant increases upon Huh7 treatment with Compound 2. SHP expression levels are increased following Compound 2 treatment by between 1.5 (EC.sub.50 concentration) and 2.5 times (EC.sub.50 concentration) (FIG. 1). Ostα mRNA was also considerably upregulated, displaying 11-fold and 18-fold increases in expression, upon treatment with Compound 2, at its EC.sub.50 and EC.sub.90 concentrations respectively (FIG. 2).

(371) FXR activation leads to the suppression of CYP7A1 via both a SHP-mediated, and FGF19-mediated pathway. CYP7A1 is downregulated with increasing concentrations of Compound 2 (FIG. 3).

(372) HepG2 cells display a significant downregulation in TGFβ1 expression levels in response to treatment with Compound 2 at its EC.sub.50 concentration (FIG. 4).

(373) SHP displayed a modest increase whereby treatment with Compound 14 at its EC.sub.90 concentration more than doubled expression levels (FIG. 5). Compound 14 induced highly significant increases in the FXR-target Ostα, with treated cells achieving 20- to 40-times higher mRNA expression levels than vehicle-treated cells (FIG. 6).

(374) At the EC.sub.50 concentration, Compound 14 significantly decreased CYP7A1 expression, reducing it to more than half the levels seen for the vehicle control cells (FIG. 7).

(375) Similarly to Compound 2, Compound 14 induces a decline in TGFβ1 expression with increasing compound concentrations (FIG. 8).

(376) The inventors wish to thank Dannielle Kydd-Sinclair of the University of Reading, who provided much of the background section as well as the methodology and data for the biological examples as mentioned above, and David Evans, Simon Holland and Lawrence Tam of the University of Southampton, who carried out the synthesis of several of the example compounds.

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