BR112016028085B1 - CHOLANE DERIVATIVES FOR USE IN THE TREATMENT AND/OR PREVENTION OF DISEASES MEDIATED BY FXR AND TGR5/GPBAR1 - Google Patents

Abstract

CHOLANE DERIVATIVES FOR USE IN THE TREATMENT AND/OR PREVENTION OF DISEASES MEDIATED BY FXR AND TGR5/GPBAR1. The present invention relates to compounds having cholane scaffolds of formula (I), said compounds for use in the treatment and/or prevention of diseases mediated by FXR and TGR5/GPBAR1. The present invention relates to compounds having cholane scaffolds of formula (I), said compounds for use in the treatment and/or prevention of diseases mediated by FXR and TGR5/GPBAR1.

Description

FIELD OF INVENTION

[001] The present invention relates to compounds having cholane scaffolds of formula (I) for use in the treatment and/or prevention of diseases mediated by FXR and TGR5/GPBAR1.

STATE OF THE TECHNIQUE

[002] Bile acids (BAs) are signaling molecules that interact with two types of specific cellular receptors, intracellular nuclear receptors and cell surface receptors. The nuclear receptors include a farnesoid

[003] Highly expressed in enterohepatic tissues (liver and intestine), FXR regulates bile acid homeostasis, metabolic pathways also including lipid and glucose homeostasis (Zhang et al. Proc. Natl. Acad. Sci. USA 2006 , 103, 1006). Additionally, FXR agonists provide anti-inflammatory, antifibrotic and anticancer effects (Renga et al. FASEB J. 2012, 26, 3021-3031).

[004] A bile acid cell surface receptor (GPBAR1, M-BAR1, GP-BAR1, TGR5) belongs to the rhodopsin-type superfamily of G protein-coupled receptors (Takeda et al. FEBS Lett. 2002, 520, 97; Kawamata et al. J. Biol. Chem. 2003, 278, 9435).

[005] A ligand binding to TGR5/GPBAR1 results in an increase in intracellular cAMP levels with a consequent activation of a signaling cascade. GPBAR1 is highly expressed in the liver and intestine, but also in muscles, brain, adipose tissue, macrophages and endothelial cells. In muscle and brown adipose tissue, TGR5/GPBAR1 increases energy expenditure and oxygen consumption (Watanabe et al. Nature 2006, 439, 484) in entero-endocrine L cells. Activation of TGR5/GPBAR1 stimulates the secretion of glucagon-like peptide (GLP)-1, an incretin that improves the release of glucose, thus regulating blood glucose levels, gastrointestinal motility and appetite (Thomas, et al. Cell. Metab. 2009 , 10, 167).

[006] Chemically, BAs are truncated cholesterol side chain derivatives. Its molecular repertoire is generated, firstly, in the liver with the production of primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). A microbiotransformation in the intestine generates secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA). In the human body, bile acids are conjugated to glycine and taurine. Activity toward the two BA receptors is structure dependent with CDCA being the most potent endogenous FXR activator, and LCA and TLCA the strongest natural TGR5/GPBAR1 agonists.

[007] Cholestatic pruritus has been cited as a severe side effect associated with the use of FXR agonists in PBC, and a recent study indicated TGR5/GPBAR1 as the molecular target involved in the development of this side effect (Alemi et al. J. Clin. Invest. 2013, 123, 15131530).

[008] Patent document WO2013192097 describes 6-alpha-ethyl-chenodeoxycholic acid (6-ECDCA), a potent and selective transmitted X-ray beam agonist with anticholestatic effect.

[009] Patent document WO2008002573 describes bile acid derivatives as FXR ligands for preventing or treating FXR-mediated diseases or conditions.

[010] Patent document WO2010014836 and Sato H. (J Med Chem. 2008, 51, 4849) describe TGR5 modulators.

[011] D’Amore C. et al. (J. Med. Chem. 2014, 57, 937) describes the design, synthesis, and biological evaluation of GP-BAR1/FXR dual agonists. D’Amore et al. describes the compounds BAR502 and BAR504 as synthesis intermediates.

[012] Iguchi Y. et al. (J Lipid Res. 2010, 51, 1432) describes that bile alcohols function as TGR5 ligands.

[013] The compound BAR107 is exposed as a synthesis intermediate by Kihira K. et al. (Steroids 1992, 57(4), 193 198).

[014] Swaan P. W. et al. (J. Comp.-Aid. Mol. Des. 1997, 11, 581-588) in a molecular modeling of the tested intestinal bile acid vehicle ursocholate (there, compound 15, here, BARn406) among a set of acid conjugates biliary. BARn406 was found to have an undetectable ability to inhibit taurocholic acid transport in CaCo-2 cells.

[015] Burns et al. (Steroids 2011, 76(3), 291-300) describes the synthesis and olfactory activity of sulfated bile acid-5 derivatives in sea lamprey (Petromyzon marinus). There, exposed compound 9 (here, compound BAR407) did not elicit an olfactory response.

[016] The objective of the present invention is the identification of new compounds containing the chemical framework of cholane and which modulate FXR and/or TGR5/GPBAR1.

SUMMARY OF THE INVENTION

[017] The subject of the present invention is a compound of formula (I) wherein R1 is OH or H; R2 is Et, =CH-CH3; R3 is OH; n is 0, 1, or 3; R is CH2OH, COOH, CH2OSO3H or CN; provided that when R2 is Et or =CH-CH3 and R3 is OH: if n is 0 or 1 then R will be CH2OH or CN when R1 is alpha-OH or R will be COOH, CH2OH or CH2OSO3H when R1 is beta-OH or H ; if n is 3 then R1 and R are as defined above; including pharmacologically acceptable inorganic and organic salts, amino acid solvates or conjugates thereof; excluding a compound in which

[018] The compounds as described above have been shown to be modulators of FXR or/and TGR5/GPBAR1 and, therefore, are useful for the treatment of diseases mediated by FXR and TGR5/GPBAR1.

[019] Therefore, for one aspect, the present invention relates to a compound for use as a medicine, said compound of formula (I) wherein R1 is OH or H; R2 is Et, =CH-CH3; R3 is OH; n is 0, 1, or 3; R is CH2OH, COOH, CH2OSO3H or CN provided that when R2 is Et or =CH-CH3 and R3 is OH: if n is 0 or 1 then R will be CH2OH or CN when R1 is alpha-OH or R will be COOH, CH2OH, CH2OSO3H or COOH when R1 is beta-OH or H; if n is 3 then R1 and R are as defined above; when R2 is H: if R1 is alpha-OH and R3 is beta-OH then R will be CH2OH or CH2OSO3H when n is 0 or R will be CH2OH or COOH when n is 3; if R1 is H, n is 1 and R3 is alpha-OH then R will be CH2OSO3H; if R1 and R3 are H then R will be CH2OSO3H or COOH when n is 0 or R will be CH2OSO3H when n is 1; including pharmacologically acceptable inorganic and organic salts, amino acid solvates or conjugates thereof.

[020] For a further aspect, the present invention relates to a compound of formula (I) as described above, including the compounds BAR107, BAR504 and BAR502, for use in the prevention and/or treatment of gastrointestinal disorders, liver diseases , cardiovascular diseases, atherosclerosis, metabolic diseases, infectious diseases, cancer, renal disorders, inflammatory disorders and neurological disorders (such as a stroke), said compounds of formula (I) as described above.

[021] The present invention also relates to a process for preparing a compound, as described above.

DETAILED DESCRIPTION OF THE INVENTION

[022] According to the invention, those compounds are preferred in which R2 is Et or =CH-CH3 and R3 is OH; more preferably, those compounds wherein n is 0 or 1, R is CH2OH or CN and R1 is alpha-OH or n is 0 or 1, R is COOH, CH2OH or CH2OSO3H and R1 is beta-OH or H.

[023] When R2 is Et or =CH-CH3 and R3 is OH, a compound selected from the group consisting of

[024] According to the invention, those compounds are also preferred in which R2 is Et or =CH-CH3, R3 is OH, n is 3 and R1 and R are as defined above; More preferred are those compounds in which R2 is Et, R3 is alpha-OH, n is 3 and R1 is alpha-OH and R is as defined above.

[025] A compound selected from the group consisting of

[026] Among these, a compound selected from the group consisting of

[027] Particularly preferred is a compound according to the invention, which is selected from the group consisting of:

[028] The compounds according to the invention, including compounds BAR107, BAR504, and BAR502, have been shown to be highly selective modulators of FXR or TGR5/GPBAR1 or dual modulators of FXR and TGR5/GPBAR1, and are therefore useful as medicines in particular for use in the prevention and/or treatment of gastrointestinal disorders, liver diseases, cardiovascular diseases, atherosclerosis, metabolic diseases, metabolic disorders, infectious diseases, cancer, kidney disorders, inflammatory disorders and neurological disorders, such as a stroke.

[029] In certain embodiments, the liver disease is selected from the group consisting of chronic liver diseases, including primary biliary cirrhosis (PBC), cerebrotendinous xanthomatosis (CTX), primary sclerosing cholangitis (PSC), drug-induced cholestasis, cholestasis intrahepatic pregnancy, cholestasis associated with parenteral nutrition, cholestasis associated with bacterial overgrowth and sepsis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH ), liver transplant-associated graft-versus-host disease, living donor transplantation, liver regeneration, congenital hepatic fibrosis, granulomatous liver disease, intra- or extrahepatic malignancy, Wilson's disease, hemochromatosis, and alpha 1 deficiency - antitrypsin.

[030] In certain embodiments, the gastrointestinal disease is selected from the group consisting of inflammatory stomach disease (IBD) (including Crohn's disease, ulcerative colitis and indeterminate colitis), irritable stomach syndrome (IBS), bacterial overgrowth, pancreatitis acute and chronic, malabsorption, post-radiation colitis and microscopic colitis.

[031] In certain embodiments, kidney disease is selected from the group consisting of diabetic nephropathy, hypertensive nephropathy, chronic glomerular disease, including chronic glomerulonephritis and chronic transplant glomerulopathy, chronic tubulointerstitial disorders and vascular disorders of the kidney.

[032] In certain embodiments, cardiovascular disease is selected from the group consisting of atherosclerosis, dyslipidemia, hypercholesterolemia, hypertriglyceridemia, hypertension also known as arterial hypertension, inflammatory heart disease including myocarditis and endocarditis, stable angina due to ischemic heart disease, unstable angina, myocardial infarction, cerebrovascular disease including ischemic stroke, pulmonary heart disease including pulmonary hypertension, peripheral artery disease (PAD), also known as peripheral vascular disease (PVD), peripheral artery occlusive disease, and peripheral obliterating arteriopathy.

[033] In certain embodiments, the metabolic disease is selected from the group consisting of insulin resistance, metabolic syndrome, Type I and Type II diabetes, hypoglycemia, adrenal cortex disorders including adrenal cortex insufficiency.

[034] In certain embodiments, a metabolic disorder is selected from the group consisting of obesity and conditions associated with bariatric surgery.

[035] In certain embodiments, a cancer is selected from the group of liver cancer, bile duct cancers, pancreatic cancer, gastric cancer, colorectal cancer, breast cancer, ovarian cancer and conditions associated with chemotherapy resistance.

[036] In certain embodiments, an infectious disorder is selected from the group of human immunodeficiency-associated disease (AIDS) and related disorders, virus B and virus C infection.

[037] In certain embodiments, an inflammatory disorder is selected from the group of rheumatoid arthritis, fibromyalgia, Syogren's syndrome, scleroderma, Behcet's syndrome, vasculitis and systemic lupus erythematosus.

[038] Data on the activity of certain compounds of the invention on FXR and TGR5/GPBAR1 are described in the following table. In this table, the activities for the compounds of the invention on FXR and GPBAR1 were compared with those of reference compounds: that is, CDCA for FXR and TLCA for TGR5/GPBAR1. Each compound was tested at a concentration of 10 microM and a transactivation activity of CDCA over FXR and TLCA in CRE (i.e., TGR5/GPBAR1) was considered equal to 100%. Table 1

[039] For one aspect, the present invention relates to compounds of formula (I), wherein the compounds are dual FXR and TGR5/GPBAR1 agonists. An example selected from this group is BAR502. Surprisingly, BAR502 does not induce itching when administered to animals rendered cholestatic by the administration of ANIT or Estrogen. In cholestatic syndromes, the accumulation of bile acids in the body is thought to cause itching. Recently, TGR5/GPBAR1 was shown to mediate itch caused by DCA and LCA administration (Alemi et al. J. Clin. Invest. 2013, 123, 1513-1530). In clinical trials, administration to patients suffering from primary biliary cirrhosis (PBC) of obeticholic acid resulted in severe itching in approximately 80% of patients. A specific and surprising advantage of BAR502 is that this agent does not induce itching when administered to animals rendered cholestatic by administration of a-naphthyl isothiocyanate (ANIT) or 17a-ethinyl estradiol (two validated models of cholestasis). In this experimental setting, administration of BAR502 increases survival, attenuates serum alkaline phosphatase levels, and robustly modulates liver expression of canonical FXR target genes including OSTa, BSEP, SHP, and MDR1, without the induction of pruritus. In the 17a-ethinyl estradiol model, BAR502 attenuates cholestasis and reformats the bile acid pool without inducing itch, demonstrating that in non-obstructive cholestasis models, BAR502 attenuates liver damage without causing itch.

[040] In one aspect, the present invention relates to compounds of formula (I) wherein the compounds are highly selective FXR agonists, with no effects on GPBAR1 when administered alone, but effective in inhibiting GPBAR1 activation caused by TLCA (10 µM), thus behaving as GPBAR1 antagonists. In another aspect, the present invention relates to compounds of formula (I) wherein the compounds are highly selective GPBAR1 agonists with no effects on FXR. In a further aspect, the present invention relates to compounds of formula (I) wherein the compounds are highly selective antagonists of GPBAR1 without effects on FXR.

[041] The present invention also relates to processes for preparing a compound of formula (I) as described above.

[042] For one aspect, the present invention relates to a process for preparing a compound of formula (I) as described above, wherein R is CH2OH, said process comprising contacting a compound of corresponding formula (I) in that R is COOMe with LiBH4.

[043] For one aspect, the present invention relates to a process for preparing a compound of formula (I) as described above wherein R is COOH, said process comprising subjecting a compound of corresponding formula (I) where R is COOMe for alkaline hydrolysis; preferably with 5% NaOH in MeOH/H2O.

[044] For one aspect, the present invention relates to a process for preparing a compound of formula (I) as described above wherein R is CH2OSO3H and salts thereof, said process comprising contacting a compound of corresponding formula ( II) wherein n and R2 are as described above and R4 and R5, if different from H, are OP, where P is an alcohol protection function, with a trialkylamine-sulfur trioxide to obtain a compound of formula (III) R2, R4 and R5 are as described above.

[045] Then, from a compound of formula (III) as described above, the corresponding compound of formula (I) can be obtained by deprotecting the hydroxyl functions at C3 and C7.

[046] For one aspect, the present invention relates to a process for preparing a compound of formula (I) as described above, wherein n= 3 said process comprising subjecting a compound of corresponding formula (IV) where m=n-2=1, R4 and R5, if other than H, are OP, where P is an alcohol protection function, and R2 is as described above (preferably R2 is H or alpha Et), for one-pot Swern oxidation/Wittig C2 homologation to obtain a protected methyl ester of formula (V) where m=1, R2 is as described above (preferably R2 is H or alpha-Et), R4 and R5, if other than H, are OP, where P is an alcohol protection function.

[047] Swern oxidation is a chemical reaction whereby a primary or secondary alcohol is oxidized to an aldehyde or a ketone using oxalyl chloride, dimethyl sulfoxide (DMSO) and an organic base, such as triethylamine.

[048] The Wittig reaction, or Wittig olefination, is a chemical reaction of an aldehyde or a ketone with triphenyl phosphonium ylide (often called a Wittig reagent) to provide an alkene or triphenylphosphine oxide. The triphenylphosphonium ylide is preferably methyl(triphenylphosphoranylidene)acetate.

[049] Preferably, the above process may further comprise subjecting a compound of formula (V) as described above to catalytic hydrogenation, thus ensuring a compound of formula (VI) where n=m+2=3, R2 is as described above, R4 and R5, if other than H, are OP, where P is an alcohol protection function.

[050] Preferably OP is a silyl ether, more preferably, t-butyldimethylsilyl ether. Therefore, deprotection is preferably carried out by acid hydrolysis, preferably by treatment with HCl.

[051] For one aspect, the present invention relates to a process for preparing a compound of formula (I) as described above wherein n=0, said process comprising contacting a compound of formula (XII) with HCOOH and HClO4 and subsequently contacting the resulting compound with TFA, trifluoroacetic anhydride and NaNO2 to obtain a compound of formula (VII)

[052] Simple and well-known chemical transformations can then lead from a compound of formula (VII) to a compound of formula (I) as described above, so that the CN-group can be hydrolyzed to COOH, as well such as the OCHO group can be hydrolyzed to a hydroxyl group or the =O group can be reduced to a hydroxyl group.

[053] For one aspect, the present invention relates to a process for preparing a compound of formula (I) as described above in which R2 is =CH-CH3 or Et and R3 is not H, said process comprising the subjecting a compound of formula (VIII) to an aldol condensation thereby contacting a compound of formula (VIII) where n = 0.1, P is an alcohol protection function, preferably OAc, with alkyl lithium, such as nBuLi, and, subsequently, with acetaldehyde, preferably also in the presence of BF3(OEt)2, to obtain a compound of formula (IX) where n and P are as described above.

[054] An aldol condensation is an aldol addition reaction, which could involve the nucleophilic addition of a ketone enolate to an aldehyde, in which, once formed, the aldol product loses a water molecule to form an a,ß-saturated carbonyl compound.

[055] By subjecting a compound of formula (IX) to catalytic hydrogenation, preferably with H2 in the presence of Pd(OH)2/C, a compound of formula (X) can be obtained

[056] For one aspect, the present invention relates to a process for preparing a compound of formula (I) in which R2 is alpha-Et, said process comprising contacting a compound of formula (XIV) where n is 0 or 1, R8 is beta-OH, OAc or H;

[057] with MeONa/MeOH to obtain epimerization of the C6 stereocenter, thus obtaining a compound of formula (XI) where n is 0 or 1, R8 is as described above. In the case where R8 is OAc, treatment with MeONa/MeOH simultaneously ensures the hydrolysis of the C3 acetoxy group, thus obtaining a compound of formula (XI) in which R8 is OH.

[058] A carbonyl reduction at C7 can be obtained by contacting a compound of formula (X) with NaBH4 or Ca(BH4)2 to obtain a mixture of beta-OH (up to 70% in the case of compounds BAR501 and BARn501) and alpha-OH at C7. A subsequent treatment with LiBH4 reduces, if present, the methyl ester function on a side chain to -CH2OH and the OAc protecting group at C3 to OH.

[059] A carbonyl reduction at C7 can be obtained by contacting a compound of formula (XI) or a corresponding compound having COOH in the side chain, with LiBH4 obtaining almost exclusively alpha-OH at C7. Simultaneously, LiBH4 treatment reduces, if present, the methyl ester function on a side chain to -CH2OH and the OAc protecting group on C3 to OH. Subjecting a compound of formula (IX) to a reduction of NaBH4 followed by a treatment with LiBH4 produced reduction at C7 and the side chain with a simultaneous deprotection, in particular deacetylation, at C3, to obtain a compound of formula (I) where R1 is alpha-OH, R2 is =CH-CH3, R3 is beta-OH, n=0.1 and R is CH2OH.

[060] Subjecting the above compound of formula (I) in which R1 is alpha-OH, R2 is =CH-CH3, R3 is beta-OH, n=0.1 and R is CH2OH to a catalytic hydrogenation, preferably with H2 and Pd(OH)2/C, a compound of formula (I) can be obtained in which R1 is alpha-OH, R2 is alpha-Et, R3 is beta-OH, n=0.1 and R is CH2OH

[061] For one aspect, the present invention relates to a process for preparing a compound of formula (I) in which R1 is beta-OH, said process comprising starting from a compound of formula (XIII) wherein R2 is Et or H, preferably Et, and there is inversion of the C3 hydroxy configuration by a treatment with tosyl chloride in the presence of a base then followed by a treatment with CH3COOK.

[062] For one aspect, the present invention relates to a process for preparing a compound of formula (I) in which R1 is H, said process comprising subjecting a compound of formula (XIII) (XIII) as described above to a tosylation and elimination at a C-3 hydroxyl group followed by a double bond reduction.

[063] Tosylation is preferably carried out with TsCl and pyridine.

[064] An elimination is preferably carried out with LiBr and Li2CO3 in DMF at a reflux temperature.

[065] A double bond reduction is preferably carried out by catalytic hydrogenation, preferably with H2 and Pd(OH)2/C.

[066] The present invention could be better understood in light of the examples and experimental section below.

EXPERIMENTAL SECTION Chemical EXAMPLE 1. Preparation of compounds of formula (I) in which R2=H EXAMPLE 1A. Synthesis of bis-homoursodeoxycholane derivatives

[067] A four-step reaction sequence in 1, including protection of alcoholic functions at C3 and C7, a side-chain methyl ester reduction, and a subsequent one-pot Swern oxidation/Wittig C2 homologation provided the protected methyl ester of ?24,25- bis-homoUDCA. A side chain double bond hydrogenation and an alcohol function deprotection provided the bis-homoUDCA methyl ester 4, which was used as a starting material in the preparation of BAR305 and its corresponding alcohol, BAR304, through a treatment with LiOH and LiBH4. , respectively. a) 2,6-lutidine, t-butyldimethylsilyl trifluoromethanesulfonate, CH2Cl2, 0 °C; b) LiBH4, dry MeOH, THF, 0 °C, quantitative production in two steps; c) DMSO, oxalyl chloride, dry TEA, CH2Cl2, -78 °C then methyl(triphenylphosphoranylidene)acetate, 76%; d) H2, Pd(OH)2/C Degussa type, THF/MeOH 1:1, quantitative production; e) HCl 37%, MeOH, quantitative production; f) 5% NaOH in MeOH/H2O 1:1 v/v, 60%; g) LiBH4, dry MeOH, THF, 0 °C, 77%.

Step a, b) Preparation of 3a, 7ß-di(tert-butyldimethylsilyloxy)-5ß-cholan-24-ol (2)

[068] Compound 1 (1.2 g, 3 mmol) was protected in both alcoholic functions following the same synthetic procedure described in J. Med. Chem. 2014, 57, 937 to obtain 1.9 g of methyl 3 a, 7 ß -di(tert-butyldimethylsilyloxy)-5Q-cholan-24-oate(quantitative production) in the form of colorless needles, which was subjected to the next step without any purification.

[069] Methanol (850 µL, 21 mmol) and LiBH4 (10.5 mL, 2M in THF, 21 mmol) were added to a solution of methyl ester (1.9 g, 3 mmol) in dry THF (30 mL) at 0 °C following the same synthetic procedure described in J. Med. Chem. 2014, 57, 937. A silica gel purification (hexane/ethyl acetate 99:1 and 0.5% TEA) provided 2 as a white solid (1.8 g, quantitative yield).

Step c) One-pot preparation of methyl 3a, 7ß-di(tert-butyldimethylsilyloxy)-25, 26-bishomo-5ß-col-24-en-26-oate (3).

[070] DMSO (2.1 mL, 30 mmol) was added dropwise for 15 min to a solution of oxalyl chloride (7.5 mL, 15 mmol) in dry dichloromethane (30 mL) at -78 °C under an argon atmosphere. After 30 min, a solution of 2 (1.8 g, 3 mmol) in dry CH2Cl2 was added through a cannula and the mixture was stirred at -78 °C for 30 min. Et3N (2.5 mL, 18 mmol) was added dropwise. After 1 h methyl(triphenylphosphoranylidene)acetate (2.0 g, 6 mmol) was added and the mixture was allowed to warm to room temperature. A saturated NaCl solution was added and the aqueous phase was extracted with diethyl ether (3x100 mL). The combined organic phases were washed with water, dried (Na2SO4) and concentrated. Purification by silica gel (hexane - ethyl acetate 95:5 and 0.5% TEA) provided compound 3 as a colorless oil (1.5 g, 76%). Step d) Preparation of methyl 3a, 7ß-di(tert-butyldimethylsilyloxy)-25, 26-bishomo-5ß-cholan-26-oate. A solution of compound 3 (1.5 g, 2.3 mmol) in dry THF/dry MeOH (25 mL/25 mL, v/v) was hydrogenated in the presence of 5 wt% Pd(OH)2 on activated carbon of Degussa type (20 mg) following the same synthetic procedure described in J. Med. Chem. 2014, 57, 937 providing methyl 3a, 7ß-di(tert-butyldimethylsilyloxy)-25, 26-bishomo-5ß-cholan-26-oate (1.5 g, quantitative production) which was subjected to step e) without purification.

Step e) Preparation of methyl 3a, 7ß-dihydroxy-25, 26-bishomo-5ß-cholan-26-oate (4)

[071] Methyl 3 a, 7 ß -di(tert-butyldimethylsilyloxy)-25, 26-bishomo-5 ß -cholan-26-oate (1.5 g) was dissolved in methanol (70 mL). HCl (2 mL, 37% v/v) was added to the solution following the same synthetic procedure described in J. Med. Chem. 2014, 57, 937 providing 4 as a colorless amorphous solid (1.0 g, quantitative production). Step f) Preparation of 3a, 7ß-dihydroxy-25, 26-bishomo-5e-cholan-26-oic acid (BAR305). A portion of compound 4 (430 mg, 1 mmol) was hydrolyzed with NaOH (400 mg, 10 mmol) in a 1:1 v/v MeOH:H2O solution (20 mL) for 4 h at reflux. An analytical sample was purified by HPLC on a Nucleodur 100-5 C18 (5 µm; 4.6 mm i.d. x 250 mm) with MeOH/H2O (95:5) as an eluent (flow rate 1 mL/min) (tR=5 min ). BAR305: C26H44O4

[072] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.47 (2H, m, H-3 and H-7), 2.27 (2H, t, J = 7 .2 Hz, H2-25), 0.96 (3H, s, H3-19), 0.94 (3H, d, J = 6.5 Hz, H3-21), 0.70 (3H, s, H3-18).

[073] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d178.2, 72.1, 71.9, 57.6, 56.7, 44.8, 44.5, 44 ,0, 41.6, 40.7, 38.6, 38.0, 36.9, 36.8, 36.1, 35.3, 35.2, 30.9, 29.8, 27.9 , 26.7, 26.6, 23.9, 22.4, 19.3, 12.7. Step g) 25, 26-bishomo-5ß-colan-3a, 7ß, 26-triol (BAR304). Compound 4 (500 mg, 1.2 mmol) was reduced under the same operating conditions described in step b). Purification by silica gel (CH2Cl2/methanol 9:1) provided BAR304 as a colorless oil (375 mg, 77%). An analytical sample was purified by HPLC on a Nucleodur 100-5 C18 (5 µm; 4.6 mm i.d. x 250 mm) with MeOH/H2O (85:15) as an eluent (flow rate 1 mL/min) (tR=9 min ). BAR 304: C26H46O3

[074] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.53 (2H, t, J = 6.5 Hz, H2-26), 3.48 (2H, m, H-3 and H-7), 0.95 (3H, s, H3-19), 0.93 (3H, d, J = 6.5 Hz, H3-21), 0.70 (3H, s, H3-18).

[075] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d72.1, 71.9, 63.0, 57.5, 56.7, 44.7, 44.4, 44 ,0, 41.6, 40.7, 38.5, 37.9, 37.2, 37.0, 36.1, 35.2, 33.7, 30.9, 29.8, 27.9 , 27.4, 27.1, 23.9, 22.4, 19.4, 12.7.

EXAMPLE 1B. Synthesis of 3a,7ß-dihydroxy-24-nor-5ß-colan-23-yl-23-sodium sulfate (BAR106) and 3a,7ß-dihydroxy-24-nor-5ß-colan-23-ol (BAR107)

[076] BAR106 was prepared starting from UDCA by a reaction sequence comprising performance on the hydroxyl groups, a degradation of Beckmann carbon one at C24 and a transformation of the C23 hydroxyl group into the corresponding methyl ester intermediate. A protection on the hydroxyl groups at C-3 and C-7 as silyl esters, a reduction to C23 methyl ester, a sulfation to a C23 primary alcohol function and finally a deprotection of crude BAR106 supplied as ammonium salt. Purification on Amberlite and then by HPLC provided a title BAR106 as a sodium salt. a) HCOOH, HClO4, 96%; b) TFA, trifluoroacetic anhydride, NaNO2, 96%; c) KOH 30% in MeOH/H2O 1:1 v/v, 97%; d) p-TsOH, dry MeOH, 98%; e) 2,6-lutidine, t-butyldimethylsilyl trifluoromethanesulfonate, CH2Cl2, 0 °C, 88%; f) LiBH4, dry MeOH, THF, 0 °C, quantitative production; g) Et3N.SO3, DMF, 95 °C; h) HCl 37%, MeOH, then Amberlite CG-120, MeOH, 86% in two steps; i) LiBH4, dry MeOH, THF, 0 °C, quantitative production.

Steps a, d) Preparation of methyl 3a,7ß-dihydroxy-24-nor-5ß-cholan-23-oate (6).

[077] Ursodeoxycholic acid (2.0 g, 5.1 mmol) was transformed into methyl 3a,7ß-dihydroxy-24-nor-5ß-cholan-23-oate (6, 1.6 g, 87%) followed by if the same synthetic procedure described in J. Med. Chem. 2014, 57, 937. Step e) Preparation of methyl 3a, 7ß-di(tert-butyldimethylsilyloxy)-5ß-cholan-24-oate

[078] Compound 6 (1.2 g, 3.0 mmol) was protected in hydroxyl groups under the same operating condition described in example 1A step a). Purification by flash chromatography on silica gel using hexane/ethyl acetate 9:1 and 0.5% triethylamine as an eluent provided a protected methyl ester (1.6 g, 88%). Stepf) Preparation of 3a, 7ß-di(tert-butyldimethylsilyl0xy)-5 ß -colan-24-ol (7)

[079] A side chain methyl ester (818 mg, 1.3 mmol) was reduced under the same operating condition described in example 1A step b). Purification by flash chromatography on silica gel using hexane/ethyl acetate 98:2 and 0.5% triethylamine as an eluent, afforded 7 (770 mg, quantitative yield).

Steps g, h) Preparation of 3a,7ß-dihydroxy-24-nor-5ß-cholan-23-yl-23-sodium sulfate (BAR106)

[080] The trilethylamine-sulfur trioxide complex (2.0 g, 11 mmol) was added to a solution of 7 (660 mg, 1.1 mmol) in dry DMF (25 mL) following the same synthetic procedure described in J. Med. Chem. 2014, 57, 937. HPLC on a Nucleodur 100-5 C18 (5 µm; 10 mm i.d. x 250 mm) with MeOH/H2O (65:35) as an eluent (flow rate 3 mL/min), provided 442 mg ( 86% by two steps) of BAR106 (tR=8.4 min).

BAR 106: C23H39NaO6S

[081] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 4.04 (2H, m, H2-23), 3.48 (2H, m, H-3 and H-7 ), 1.00 (3H, d, J = 6.5 Hz, H3-21), 0.97 (3H, s, H3-19), 0.72 (3H, s, H3-18).

Step i) Preparation of 3a, 7ß-dihydroxy-24-nor-5ß-colan-23-ol (BAR107)

[082] Compound 6 was transformed into BAR107 in the same operating condition described in step f. BAR107: C23H40O3

[083] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d3.60 (1H, m, H-7), 3.51 (1H, m, H-3), 3.50 (2H, m, H2-23), 0.97 (3H, d, ovl, H3-21), 0.96 (3H, s, H3-19), 0.72 (3H, s, H3-18) .

[084] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 72.1, 71.9, 60.8, 57.5, 57.1, 44.8, 44.5, 44.0, 41.6, 40.7, 39.9, 38.6, 38.0, 36.1, 35.2, 34.1, 31.0, 29.8, 27.9, 23, 9, 22.4, 19.5, 12.6;

Example 1C. Synthesis of 7a-hydroxy-5ß-cholan-24-yl-24-sodium sulfate (BAR402)

[085] A tosylation and elimination at a C-3 hydroxyl group in methyl ester 8 followed by a reduction of a subsequent treatment with LiBH4 DMF, reflux, c) H2, Pd(OH)2, THF/MeOH 1:1, temperature subsequent with LiBH4 and a regioselective sulfation at a C-24 primary hydroxyl group afforded BAR402. a) p-TsCl, pyridine, quantitative production; b) LiBr, Li2CO3, DMF, reflux, c) H2, Pd(OH)2, THF/MeOH 1:1, room temperature, quantitative production; d) LiBH4, dry MeOH, THF, 0 °C, 79%; e) Et3N.SO3, DMF, 95 °C. Steps ac) Preparation of methyl 7-keto-5β-cholan-24-oate (9)

[086] To a solution of 8 (965 mg, 2.5 mmol) in dry pyridine (100 mL), tosyl chloride (4.7 g, 25.0 mmol) was added, and the mixture was stirred at room temperature for 4 hours. It was poured into cold water (150 mL) and extracted with CH2Cl2 (3 x 150 mL). The combined organic layer was washed with a saturated solution of NaHCO3 (150 mL), and water (150 mL), and then dried over anhydrous MgSO4 and evaporated in vacuo to provide 1.4 g of methyl 3a-tosyloxy-7-keto- 5ß-colan-24-oate (quantitative production). Lithium bromide (434 mg, 5.0 mmol) and lithium carbonate (370 mg, 5.0 mmol) were added to a solution of 3 a -tosyloxy-7-keto-5 ß -cholan-24-oate (1 .4 g, 2.5 mmol) in dry DMF (30 mL), and the mixture was refluxed for 2 h. After cooling to room temperature, the mixture was slowly poured into a 10% HCl solution (20 mL) and extracted with CH2Cl2 (3 x 50 mL). The combined organic layer was successively washed with water, saturated with a solution of NaHCO3 and water, and then dried over anhydrous MgSO4 and evaporated to dryness to provide 965 mg of residue oils (quantitative production), which was subjected to the next step without any purification.

[087] A hydrogenation in Pd(OH)2 in the same operating condition described in example 1A, step d provided 975 mg of 9 (quantitative production), which was submitted to the next step without any purification.

Step d) Preparation of 5ß-colan-7a,24-diol

[088] A treatment with LiBH4 in compound 9 in the same operating condition described in example 1A step b and purification by silica gel (ethyl acetate-hexane, 85:15) provided 5ß-cholan-7a,24-diol as a white solid (714 mg, 79%). Step e) Preparation of 7a-hydroxy-5ß-cholan-24-yl-24-sodium sulfate (BAR402). A C24 sulfation was carried out under the same operating conditions described as in example 1B step g) to provide crude BAR402 as an ammonium salt. RP18/HPLC on a Nucleodur 100-5 C18 (5 µm; 10 mm i.d. x 250 mm) with MeOH/H2O (90:10) as an eluent (flow rate 3 mL/min) afforded BAR402 (tR= 6.6 min ) as a sodium salt. BAR402: C24H41NaO5S

[089] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent d 3.96 (2H, t, J = 6.6 Hz, H2-24), 3.78 (1H, br s, H- 7), 0.96 (3H, d, J = 6.5 Hz, H3-21), 0.92 (3H, s, H3-19), 0.69 (3H, s, H3-18);

[090] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 69.7, 69.4, 57.7, 51.6, 45.0, 43.8, 41.2, 41.0, 39.0, 37.1, 37.0 36.3, 34.2, 33.3, 31.7, 29.5, 29.0, 27.3, 24.8, 24.3 , 22.7, 21.9, 19.2, 12.3.

EXAMPLE 2. Preparation of compounds of formula (I) in which R2=Et or =CH-CH3 EXAMPLE 2A. Synthesis of 6ß-ethyl-3a,7-dihydroxy-5ß-colan-24-ol (BAR501)

[091] A methyl ester formation and acetylation at a C-3 hydroxyl group in 7-KLCA provided an intermediate 10 in a two-step production of 84%. An aldol addition to a silylic enol ether intermediate generated 11 which was hydrogenated at the exocyclic double bond (H2 in Pd(OH)2) yielding 12 in an 80% yield over three steps. A treatment with NaBH4 in methanol followed by a reduction with LiBH4 in the crude reaction product provided a mixture whose HPLC purification (88% MeOH:H2O) provided pure BAR501 in a yield of 79% with respect to its C7 epimer, BAR504- 6b. a) p-TsOH, dry MeOH; b) acetic anhydride, pyridine, 84% production by two stages; c) DIPA, n-BuLi, TMSCl, dry TEA, dry THF -78 °C; d) acetaldehyde, BF (OEt), CHCl, -60 °C, 80% in two steps; e) H, Pd(OH)2, THF/MeOH 1:1, quantitative production; f) NaBH, MeOH; g) LiBH, dry MeOH, THF, 0 °C, 79% in two steps.

[092] Steps a-d). Preparation of methyl 3a-acetoxy-6-ethylidene-7-keto-5ß-cholan-24-oate (11)

[093] To a solution of 7-ketolithocholic acid (5 g, 12.8 mmol), dissolved in 100 mL of dry methanol, p-toluenesulfonic acid (11 g, 64.1 mmol) was added. The solution was allowed to stand at room temperature for 2 h. The mixture was cooled abruptly by adding a saturated NaHCO3 solution. After evaporation of methanol, the residue was extracted with EtOAc (3x150 mL). The combined extract was washed with brine, dried with Na2SO4, and evaporated to provide the methyl ester as an amorphous solid (5.13 g, quantitative yield).

[094] In the solution of the methyl ester (5.13 g, 12.7 mmol) in dry pyridine (100 mL), an excess of acetic anhydride (8.4 mL, 89 mmol) was added. When the reaction was complete, the pyridine was concentrated under vacuum. The residue was poured into cold water (100 mL) and extracted with AcOEt (3x150 mL). The combined organic phases were dried (Na2SO4) and concentrated to give a residue which was further purified by flash chromatography on silica gel using 8:2 hexane/ethyl acetate and 0.5% triethylamine as an eluent (4.8 g of 10 as a white solid, 84% two-step production).

[095] To a solution of diisopropylamine (23 mL, 0.16 mol) in dry THF (50 mL) was added dropwise a solution of n-butyllithium (60 mL, 2.5 M in hexane, 0.15 mol ) at -78 °C. After 30 min, trimethylchlorosilane (27.1 mL, 0.21 mol) was added. After an additional 30 min, a solution of compound 10 (4.8 g, 10.7 mmol) in dry THF (70 mL) was added. The reaction was stirred at -78 °C for an additional 45 min and then triethylamine (54 mL, 0.38 mol) was added. After 1 h, the reaction mixture was allowed to warm to -20 °C, treated with a saturated aqueous NaHCO3 solution (100 mL) and brought to room temperature in 2 h. The aqueous phase was extracted with ethyl acetate (3x50 mL). The combined organic phases were then washed with saturated NaHCO3 solution, water and brine. After drying over anhydrous Na2SO4, the residue was evaporated under vacuum to provide 6 g of yellow residue, which was diluted in dry CH2Cl2 (50 mL) and cooled to -78 °C. Into this stirred solution, acetaldehyde (3 mL, 53 mmol) and BFa^OEt2 (13.5 mL, 0.107 mol) were added dropwise. The reaction mixture was stirred for 2 h at −60 °C and allowed to warm to room temperature. The mixture was quenched with a saturated aqueous solution of NaHCO3 and extracted with CH2Cl2. The combined organic phases were washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuo.

[096] Purification by silica gel (hexane - ethyl acetate 9:1 and 0.5% TEA) provided compound 11 (4.1 g, 80%). An NMR analysis demonstrated a diastomeric E/Z ratio >95%. The E configuration in the exocyclic double bond was established by a Ha-26 (d 1.67)/H-5 (d 2.62) dipolar coupling in a Noesy spectrum (400 MHz, mixing time 400 ms).

(E )-3a-acetoxy-6-ethylidene-7-keto-5ß-cholan-24-oate (11) : C29H44O5

[097] 1H NMR was recorded on Varian Inova 400 MHz, using CDCl3 as solvent: d 6.16 (1H, q, J = 7.0 Hz, H-25), 4.74 (1H, m, H-3), 3.64 (3H, s, COOCH3), 2.62 (1H, dd, J = 13.0, 3.6 Hz, H-5), 1.98 (3H, s, COCH3) , 1.67 (3H, d, J = 7.0 Hz, H3-26), 1.00 (3H, s, H3-19), 0.92 (3H, d, J = 6.0 Hz, H3 -21), 0.67 (3H, s, H3-18).

[098] 13C NMR was recorded on Varian Inova 100 MHz, using CDCl3 as solvent: d 204.5, 174.6, 170.7, 143.1, 130.2, 72.5, 54.5, 51.4, 50.7, 48.6, 45.2, 43.5, 39.1, 38.9, 35.1, 34.9, 34.1, 33.4, 31.0, 30, 9, 28.4, 25.9 (2C), 22.8, 21.4, 21.2, 18.4, 12.7, 12.2.

Steps e) Preparation of methyl 3a-acetoxy-6 ß-ethyl-7-keto-5 ß -cholan-24-oate (12).

[099] A solution of 11 (4.0 g, 8.5 mmol) in dry THF/dry MeOH (100 mL, 1:1 v/v) was hydrogenated in the presence of 20% by weight Pd(OH)2 in activated charcoal (100 mg) type Degussa. The mixture was transferred to a standard PARR apparatus and washed with nitrogen and then hydrogen several times. The apparatus was shaken under 50 psi (344.74 kPa) of H2. The reaction was stirred at room temperature for 8 h. The catalyst was filtered through Celite, and the recovered filtrate was concentrated in vacuo to provide 12 (4.0 g, quantitative yield).

Methyl 3a-acet0xy-6ß-ethyl-7-keto-5ß-colan-24-oate (12) : C29H46O5

[100] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d4.65 (1H, m, H—3), 3.66 (3H, s, COOCH3), 2.56 (1H , t, J = 11.5 Hz, H-8), 2.35 (1H, m, H-23a), 2.22 (1H, m, H-23b), 1.99 (3H, s, COCH3 ), 1.22 (3H, s, H3- 19), 0.92 (3H, d, J = 6.3 Hz, H3-21), 0.83 (3H, t, J = 7.2 Hz, H3-26), 0.67 (3H, s, H3-18).

[101] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 214.7, 174.3, 170.2, 72.6, 61.7, 54.8, 51.3, 49.0, 48.5, 45.3, 42.7, 42.3 (2C), 38.6, 35.4, 35.1, 35.0, 31.0, 30.8, 28.0 (2C), 26.4, 25.7, 24.7, 21.3, 21.1, 18.2, 12.9, 11.9.

[102] The ß configuration of the ethyl group at C-6 was determined by dipolar couplings H3-26 (d 0.83)/ H3-I9 (d 1.22) and H-8 (d 2.56)/H- 25 (d 1.83) in a Noesy spectrum (400 MHz, mixing time 400 ms).

Steps f, g) Preparation of 6ß-ethyl-3a-dihydroxy-5ß-cholan-24-ol (BAR501).

[103] To a methanol solution of compound 12 (1.18 g, 2.5 mmol), a large excess of NaBH4 was added at 0 °C. The mixture was left at room temperature for 2 h and then water and MeOH were added dropwise over a period of 15 min at 0 °C with effervescence being observed. After evaporation of the solvents, the residue was diluted with water and extracted with AcOEt (3x50 mL). The combined extract was washed with brine, dried with Na2SO4, and evaporated to provide 1.3 g of a crude residue which was subjected to the next step without further purification. The crude residue was treated with LiBH4 (2 M in THF) under the same operating conditions described in example 1A step b). Purification by HPLC on a Nucleodur 100-5 C18 (5 µm; 10 mm i.d. x 250 mm) with MeOH/H2O (88:12) as an eluent (flow rate 3 mL/min), provided 802 mg of BAR501 (79% , tR= 11 min).

[104] Alternatively, step f was carried out with Ca(BH4)2, produced in situ.

[105] To a solution of compound 12 (500 mg, 1.05 mmol) and absolute ethanol (4 mL) at 0 °C, CaCl2 (466 mg, 4.2 mmol) was added. To the same solution was added a solution of NaBH4 (159 mg, 4.2 mmol) in absolute ethanol (4 mL). After 4 h at −5 °C, MeOH was added dropwise. Then after evaporation of the solvents, the residue was diluted with water and extracted with AcOEt (3x50 mL). The combined extract was washed with brine, dried with Na2SO4, and evaporated to provide 500 mg of a crude residue which was subjected to step g without further purification.

BAR501: C26H46O3

[106] 1H NMR was recorded on Varian Inova 700 MHz, using CD3OD as solvent: d3.74 (1H, dd, J = 10.3, 6.0 Hz, H- 7), 3.51 (1H , ovl, H-3), 3.49 (2H, ovl, H2-24), 1.00 (3H, s, H3-19), 0.97 (3H, d, J = 6.5 Hz, H3 -21), 0.96 (3H, t, J = 7.6 Hz, H3-26), 0.72 (3H, s, H3-18).

[107] 13C NMR was recorded on Varian Inova 175 MHz, using CD3OD as solvent0: d75.3 71, 9, 63, 6, 57.5, 56.5, 51, 6, 45.7, 44, 9, 42.1, 41.5, 40.4, 40.3, 37.1, 35.8, 32.4, 30.7, 30.3, 29.7, 29.6, 28.3, 26.2, 23.4, 22.1, 19.4, 14.8, 12.7.

Example 2B. Preparation of 6ß-ethyl-3a7a-dihydroxy-5ß-cholan-24-ol (BAR504-6b)

[108] BAR504-6b was prepared as described in Example 2A (tR= 20.4 min).

BAR504-6b: C26H46O3

[109] 1H NMR was recorded on Varian Inova 700 MHz, using CD3OD as solvent: d 3.60 (1H, s, H-7), 3.51 (2H, m, H2- 24), 3, 35 (1H, ovl, H-3), 2.30 (1H, q, J = 13.5 Hz, H-4a), 0.97 (3H, d, J = 6.8 Hz, H3-21) , 0.95 (3H, t, J = 7.3 Hz, H3-26), 0.94 (3H, s, H3-19), 0.70 (3H, s, H3-18).

[110] 13C NMR was recorded on Varian Inova 175 MHz, using CD3OD as solvent: d 71.9, 71.8, 62.7, 56.8, 51.7, 50.5, 46.7, 42.5, 41.4, 40.1, 36.6, 36.4, 36.2, 36.0, 33.2, 32.4, 30.1, 29.5, 28.8, 28, 5, 25.3, 23.9, 20.7, 18.4, 13.7, 11.4.

EXAMPLE 2C. Synthesis of 6a-ethyl-3a7a-dihydroxy-24-nor-5ß-cholan-23-ol (BAR502), 6ß-ethyl-3a7ß-dihydroxy-24-nor-5ß-cholan-23-ol (BARn501) and 6ß- ethyl-3a7a-dihydroxy-24-nor-5ß-cholan-23-ol (BARn504-6b)

[111] 7-KLCA (1 g, 2.56 mmol) was subjected to Beckmann degradation at C24 and methylation at C-23 providing 13 in 66% production. An acetylation at C-3 and alkylation provided 14 which was hydrogenated providing 15. A treatment with MeONa/MeOH provided a hydrolysis at C-3 and epimerization at C-6. A simultaneous reduction in a methyl ester function at C-23 and a C-7 carbonyl group provided BAR502 in 89% production. Intermediate 15 (250 mg, 0.54 mmol) was also used as a starting material in the preparation of BARn501 and BARn504-6b. a) HCOOH, HClO4; b) TFA, trifluoroacetic anhydride, NaNO2; c) 30% KOH in MeOH/H2O 1:1 v/v, 88% in three steps; d) p- TsOH, dry MeOH; e) acetic anhydride, pyridine; f) DIPA, n-BuLi, TMSCl, dry TEA, dry THF -78 °C; g) acetaldehyde, BF (OEt), CHCl, -60 °C, 60% in four steps; h) H2, Pd(OH)2, THF/MeOH 1:1, quantitative production; i) MeONa, MeOH; j) LiBH4, MeOH, dry THF, 0 °C, 70% in two steps; k) NaBH4, dry MeOH, 0 °C; l) LiBH4, MeOH, dry THF, 0 °C, 77% in two steps.

Steps a-d) Preparation of methyl 7-keto-24-nor-LCA (13)

[112] Compound 13 (660 mg, 1.69 mmol, 66% by four steps) was prepared from 7-KLCA in the same operating condition described in example 1B, steps a-d). Steps e-h) Preparation of methyl 3a-acetoxy-6ß-ethyl-7-keto-24-nor-5ß-cholan-23-oate (15). Compound 13 (660 mg, 1.69 mmol) was subjected to the same operating condition described in example 2A, steps b-d to obtain 603 mg of 14 (78% by three steps). An NMR analysis demonstrated a diastomeric E/Z ratio >95%. The E configuration in the exocyclic double bond was established by a H3-25 (d1.67)/H-5 (d 2.61) dipolar coupling in a Noesy spectrum (400 MHz, mixing time 400 ms).

(E) -3a-acet0xy-6-ethylidene-7-keto-24-nor-5ß-cholan-23-oate (14): C28H42O5

[113] 1H NMR was recorded on Varian Inova 400 MHz, using CDCl3 as solvent: d 6.17 (1H, q, J = 7.2 Hz, H-24), 4.75 (1H, m, H-3), 3.64 (3H, s, COOCH3), 2.61 (1H, dd, J = 13.1, 4.0 Hz, H-5), 1.98 (3H, s, COCH3) , 1.67 (3H, d, J = 7.2 Hz, H3-25), 1.00 (3H, s, H3-19), 0.97 (3H, d, J = 6.8 Hz, H3 -21), 0.67 (3H, s, H3-18).

[114] 13C NMR was recorded on Varian Inova 100 MHz, using CDCl3 as solvent: d 204.5, 174.2, 170.5, 143.0, 130.6, 72.5, 54.7, 51.4, 50.7, 48.6, 45.3, 43.7, 41.5, 39.1, 38.8, 34.6, 34.2, 33.6, 33.4, 28, 5, 25.9 (2C), 22.8, 21.3 (2C), 19.7, 12.7, 12.1.

[115] A hydrogenation in Pd(OH)2 under the same operating condition described in example 2A, step e, provided 600 mg of 15 (quantitative production).

[116] The ß configuration of the ethyl group at C-6 was determined by dipolar couplings H3-25 (d 0.83)/ H3-19 (d 1.22) in a Noesy spectrum (400 MHz, mixing time 400 ms). 3a-acet0xi-6ß-ethyl-7-keto-24-nor-5ß-cholan-23-oate (15): C28H44O5

[117] 1H NMR was recorded on Varian Inova 400 MHz, using CDCI3 as solvent: d 4.65 (1H, m, H-3), 3.67 (3H, s, COOCH3), 2.60 ( 1H, t, J = 11.2 Hz, H-8), 2.43 (1H, dd, J = 14.2, 2.6 Hz, H-22a), 1.98 (3H, s, COCH3) , 1.88 (1H, m ovl, H-6), 1.22 (3H, s, H3-19), 0.98 (3H, d, J = 6.4 Hz, H3-21), 0, 83 (3H, t, J = 7.0 Hz, H3-25), 0.70 (3H, s, H3-18).

[118] 13C NMR was recorded on Varian Inova 100 MHz, using CDCl3 as solvent: d 215.3, 174.0, 170.5, 72.8, 61.9, 55.0, 51.4, 49.2, 48.7, 45.5, 42.9, 42.6, 41.4, 38.7 (2C), 35.6, 35.3, 34.9, 28.3 (2C), 26.5, 25.9, 24.8, 21.4, 21.3, 19.6, 13.0, 12.1. Steps i, j) Preparation of 6a-ethyl-3a7a-dihydroxy-24-nor-5ß-cholan-23-ol (BAR502)

[119] To a solution of compound 15 (450 mg, 1.0 mmol) and dry methanol (4 mL), MeONa (20 mL, 0.5 M in MeOH, 10 mmol) was added. After 24 h, H2O was added dropwise. Then after evaporation of the solvents, the residue was diluted with water and extracted with AcOEt (3x50 mL). The combined extract was washed with water, dried with Na2SO4, and evaporated to provide 16 which was subjected to step g without further purification.

Methyl 6a-ethyl-3a-hydroxy-7-keto-24-nor-5ß-cholan-23-oate (16): C26H42O4

[120] 1H NMR was recorded on Varian Inova 400 MHz, using CDCl3 as solvent: d 3.64 (3H, s, COOCH3), 3.45 (1H, m, H-3), 2.83 ( 1H, q, J = 7.3 Hz, H-6), 2.51 (1H, t, J = 11.2 Hz, H-8), 2.45 (1H, dd, J = 14.5, 3.2 Hz, H-22a), 1.26 (3H, s, H3-19), 0.98 (3H, d, J = 6.6 Hz, H3-21), 0.81 (3H, t , J = 7.0 Hz, H3-25), 0.73 (3H, s, H3-18).

[121] 13C NMR was recorded on Varian Inova 100 MHz, using CDCI3 as solvent: d 214.9, 175.4, 71.6, 56.2, 53.2, 52.0, 51.9, 51.0, 50.5, 45.2, 43.8, 42.2, 40.2, 36.7, 35.3, 34.8, 32.5, 30.5, 29.4, 25, 6, 24.0, 22.9, 20.1, 20.0, 12.6, 12.4.

[122] Compound 16 was subjected to reduction with LiBH4 under the same operating condition described in example 1A, step g. A silica gel chromatography eluting with hexane/EtOAc 6:4 provided BAR502 (274 mg, 70% by two steps). An analytical sample was obtained by HPLC on a Nucleodur 100-5 C18 (5 µm; 4.6 mm i.d. x 250 mm) with MeOH/H2O (88:12) as an eluent (flow rate 1 mL/min, tR=10.8 min).

BAR502: C25H44O3

[123] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.65 (1H, s, H-7), 3.61 (1H, m, H- 23a), 3, 53 (1H, m, H-23b) 3.31 (1H, m, H-3), 0.97 (3H, d, J = 6.6 Hz, H3-21), 0.92 (3H, s , H3-19), 0.91 (3H, t, J = 7.0 Hz, H3-25), 0.71 (3H, s, H3-18).

[124] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 73.2, 71.1, 60.7, 57.7, 51.4, 46.9, 43.8, 42.9, 41.3, 40.9, 39.8, 36.7, 36.5, 34.6, 34.5, 34.2, 31.2, 29.4, 24.5, 23, 7, 23.4, 21.8, 19.3, 12.1, 11.9. Steps k, l). Preparation of 6ß-ethyl-3a7ß-dihydroxy-24-nor-5ß-cholan-23-ol (BARn501) and 6ß-ethyl-3a7a-dihydroxy-24-nor-5ß-cholan-23-ol (BARn504-6b). Compound 15 (100 mg, 0.22 mmol) was subjected to the same operating condition described in example 2A, steps f-g. Purification by HPLC on a Nucleodur 100-5 C18 (5 µm; 10 mm i.d. x 250 mm) with MeOH/H2O (86:14) as an eluent (flow rate 3 mL/min), provided 47 mg of BARn501 (54% , tR= 11 min) and 20 mg of BARn504-6b (23%, tR= 15 min). BARn501: C25H44O3

[125] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.73 (1H, dd, J = 10.5, 5.5 Hz, H- 7), 3.61 ( 1H, m, H-23a), 3.51 (1H, m, ovl, H-23b), 3.51 (1H, m, ovl, H-3), 0.98 (3H, d, ovl, H3 -21), 0.97 (3H, s, H3-19), 0.96 (3H, t, ovl, H3-25), 0.70 (3H, s, H3-18).

[126] The 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 75.2, 71.8, 60.8, 57.5, 56.6, 51.5, 45.5, 44.8, 42.0, 41.4, 40.7, 40.3, 39.9, 36.9, 36.0, 34.2, 30.5, 29.6, 28.3, 26, 2, 23.4, 22.0, 19.4, 14.7, 12.9. BARn504-6b: C25H44O3

[127] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d3.63 (1H, m, H-23a), 3.60 (1H, m, H-7), 3.55 (1H, m, H-23b), 3.37 (1H, m, H-3), 2.30 (1H, q, J = 12.5 Hz, H-4a), 0.97 (3H, d , J = 6.6 Hz, H3-21), 0.95 (3H, s, H3-19), 0.95 (3H, t, J = 7.0 Hz, H3-25), 0.72 ( 3H, s, H3-18).

[128] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 72.8, 72.7, 60.8, 57.9, 52.7, 51.4, 47.5, 43.7, 42.3, 41.0, 39.9, 37.5, 37.3, 36.7, 34.2, 33.3, 31.0, 29.6, 29.4, 26, 2, 24.8, 21.6, 19.3, 14.5, 12.1.

2D EXAMPLE. Synthesis of 6-ethylidene-3a,7ß-dihydroxy-5ß-cholan-24-ol (BAR503), 6a-ethyl-3a7ß-dihydroxy-5ß-cholan-24-ol (BAR501-6a), 6-ethylidene-3a, 7ß-dihydroxy-24-nor-5ß-cholan- 23-ol (BARn503) and 6a-ethyl-3a7ß-dihydroxy-24-nor-5ß-cholan- 23-ol (BARn501-6a)

[129] Intermediate 11 was subjected to a reduction with NaBH4 followed by a treatment with LiBH4. Alternatively, a treatment with LiAlH4 continued in a direct manner, providing a concomitant reduction in C-24 and C-7. BAR503 was also used as a starting material for BAR501-6a by hydrogenation on a Pd(OH)2 catalyst. The same synthetic protocol was performed on intermediate 14 producing the corresponding derivatives 23, BARn503 and BARn501-6a. a) NaBH4, MeOH; b) LiBH4, dry MeOH, THF, 0 °C, 85% in two steps; c) H2, Pd(OH)2, THF:MeOH 1:1 v/v. Steps a, b). Preparation of 6-ethylidene-3a, 7ß-dihydroxy-5ß-cholan-24-ol (BAR503) and 6-ethylidene-3a,7ß-dihydroxy-24-nor-5ß-cholan-23-ol (BARn503).

[130] Compound 11 (1 g, 2.11 mmol) was subjected to the same operating condition described in example 2A, steps f, g. Purification by HPLC on a Nucleodur 100-5 C18 (5 µm; 10 mm i.d. x 250 mm) with MeOH/H2O (88:12) as an eluent (flow rate 3 mL/min), provided 727 mg of BAR503 (85% in two stages, tR= 9.2 min). Alternatively, a LiAlH4 treatment in 11 provided BAR503.

BAR503: C26H44O3

[131] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 5.66 (1H, q, J = 6.9 Hz, H-25), 3.90 (1H, d, J = 9.8 Hz, H-7), 3.55 (1H, m, H-3), 3.50 (2H, m, H2-24), 2.50 (1H, dd, J = 4, 0, 13.1 Hz, H-5), 1.62 (3H, d, J = 6.9 Hz, H3-26), 0.97 (3H, d, J = 6.8 Hz, H3-21 ), 0.81 (3H, s, H3-19), 0.70 (3H, s, H3-18).

[132] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d142.7, 114.5, 73.4, 71.1, 63.6, 57.1, 56.1, 45 ,2, 44.9, 44.2, 40.7, 40.2, 36.3, 36.2, 35.9, 34.7, 32.4, 30.2, 29.5, 28.8 , 27.4, 22.6, 21.5, 18.5, 11.8, 11.7.

[133] The same synthetic protocol was performed on intermediate 14. A purification on a Nucleodur 100-5 C18 (5 µm; 10 mm i.d. x 250 mm) with MeOH/H2O (86:14) as an eluent (flow rate 3 mL /min), provided BARn503 (tR= 8 min).

BARn503: C25H42O3

[134] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 5.66 (1H, q, J = 6.8 Hz, H-24), 3.92 (1H, d, J = 9.9 Hz, H-7), 3.60 (1H, m, H-23a), 3.56 (1H, m, H-3), 3.55 (1H, m, H-23b) , 2.52 (1H, dd, J = 3.7, 13.2 Hz, H-5), 1.63 (3H, d, J = 6.8 Hz, H3-25), 0.98 (3H , d, J = 6.5 Hz, H3-21), 0.95 (3H, s, H3-19), 0.71 (3H, s, H3-18).

[135] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 143.7, 115.4, 74.1, 71.8, 60.8, 58.0, 57.1, 46.1, 45.9, 45.1, 41.6, 41.1, 39.9, 37.0, 36.4, 35.8, 34.1, 30.9, 29.8, 28, 1, 23.5, 22.5, 19.5, 12.7, 12.6.

Step c). Preparation of 6a-ethyl-3adihydroxy-5ß-cholan-24-ol (BAR501-6a) and 6a-ethyl-3a7ß-dihydroxy-24-nor-5ß-cholan-23-ol (BARn501-6a)

[136] BAR503 (350 mg, 0.86 mmol) was subjected to the same operating condition described in example 2A step and, obtaining BAR501-6a in quantitative production. BAR501-6a: C26H46O3

[137] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.50 (2H, t, J = 6.8 Hz, H3-24), 3.44 (1H, m, H-3), 3.07 (1H, t, J = 9.8 Hz, H-7), 0.96 (3H, d, J = 6.8 Hz, H3-21), 0.95 (3H , s, H3-19), 0.86 (3H, t, J = 7.4 Hz, H3-26), 0.71 (3H, s, H3-18).

[138] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 76.5, 72.3, 63.6, 57.9, 57.3, 46.3, 45.0, 44.8, 41.8, 41.0, 39.9, 37.0, 36.4, 35.5, 33.3, 31.3, 31.0, 30.3, 29.8, 27, 8, 24.3, 22.5, 22.0, 19.3, 12.8, 11.8.

[139] BARn503 was subjected to the same operating condition described in example 2A step and, obtaining BARn501-6a in quantitative production.

BARn501-6a: C25H44O3

[140] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.62 (1H, m, H-23a), 3.54 (1H, m, H-23b), 3, 45 (1H, m, H-3), 3.08 (1H, t, J = 9.8 Hz, H-7), 0.97 (3H, d, J = 6.5 Hz, H3-21) , 0.95 (3H, s, H3-19), 0.86 (3H, t, J = 7.4 Hz, H3-25), 0.73 (3H, s, H3-18).

[141] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 76.4, 72.5, 60.8, 57.9, 57.2, 46.2, 45.1, 44.7, 41.8, 41.2, 40.0, 39.8, 36.5, 35.6, 34.2, 31.2, 30.9, 29.9, 27.9, 24, 1, 22.7, 22.0, 19.5, 12.7, 11.7.

EXAMPLE 2E Synthesis of 6a-ethyl-3a,7a-dihydroxy-24-nor-5ß-cholan-23-nitrile (BAR506)

[142] 7-KLCA was transformed into nitrile 17 following the same synthetic procedure described in Example 1B steps ab. An alkylation followed by a double bond reduction and epimerization at C-6 in the same operating condition described in example 2A step cd and example 2C step i, respectively provided 18. A treatment with LiBH4 as in example 2C step j provided the desired hydroxyl group 7Q in BAR506. HCOOH, HClO4; b) TFA, trifluoroacetic anhydride, NaNO2; c) DIPA, n-BuLi, TMSCl, dry TEA, dry THF -78 °C; d) acetaldehyde, BF (OEt), CHCl, -60 °C; e) H, Pd(OH)2, THF/MeOH 1:1; f) MeONa, MeOH; g) LiBH4, MeOH, dry THF, 0 °C

BAR506: C25H41NO2

[143] 1H NMR was recorded on Varian Inova 700 MHz, using CD3OD as solvent: d 3.66 (1H, br s, H-7), 3.31 (1H, ovl, H-3), 2 .46 (1H, dd, J = 3.8, 16.9 Hz, H-22a), 2.34 (1H, dd, J = 7.4, 16.9 Hz, H-22b), 1.16 (3H, d, J = 6.5 Hz, H3- 21), 0.91 (3H, t, J = 7.5 Hz, H3-25), 0.92 (3H, s, H3-19), 0.73 (3H, s, H3-18).

[144] 13C NMR was recorded on Varian Inova 175 MHz, using CD3OD as solvent: d 120.3, 72.9, 70.9, 56.1, 51.5, 46.7, 43.4, 42.9, 41.4, 40.2, 36.5, 36.2, 34.3 (2C), 34.2, 30.7, 29.2, 24.9, 24.4, 23.4 , 23.3, 21.9, 18.5, 12.1, 11.6. EXAMPLE 2F. Synthesis of 6a-ethyl-7a-hydroxy-5ß-cholan-24-ol (BAR701), 6a-ethyl-7a-hydroxy-5ß-cholan-24-yl 24-sodium sulfate (BAR701solf), 6e-ethyl-7e -hydroxy-5e-cholan-24-ol (BAR702), 6a-ethyl-7a-hydroxy-5e-cholan-24-ol (BAR703), 6a-ethyl-7a-hydroxy-5e-cholan-24-oic acid ( BAR704), sodium taurine sulfate 6a-ethyl-7a-hydroxy-5e-cholan-24-oil (BART704), 6ß-ethyl-7 a -hydroxy-5e-cholan-24-ol (BAR705) and acid 6 a - ethyl-7 ß -hydroxy-5 ß -colan-24-oic (BAR711)

[145] Compound 12 was treated with MeONa in methanol to obtain deacetylation at C-3 and inversion at C-6. Tosylation, elimination, and hydrogenation of the double bond in ring A provided 20. A hydrolysis to a methyl ester function followed by a LiBH4 treatment provided BAR704 at a high chemical yield. Intermediate 20 was also used as a starting material for BAR701. A C-24 sulfation of a small aliquot of BAR701 provided BAR701solf. Pd(OH)2, THF/MeOH 1:1, room temperature, 88% by two steps; e) NaOH, MeOH:H2O 1:1 v/v, 82%; f) LiBH4, dry MeOH, THF, 0 °C, 83%; g) DMT-MM, Et3N, taurine, dry DMF; h) LiBH4, dry MeOH, THF, 0 °C, 77%; i) Et3N.SO3, DMF, 95 °C.

Steps a-f) Preparation of 6-ethyl-7-hydroxy-5-cholan-24-oic acid (BAR704).

[146] Compound 12 (500 mg, 1.05 mmol) was treated with MeONa (2.1 mL, 0.5 M in MeOH, 1.05 mmol) in MeOH (5 mL) overnight under the same condition operative part of Example 2C step i. A tosylation on the crude reaction product under the same operating condition as in Example 1C, step a, provided 19 (620 mg, quantitative production by two steps). Intermediate 19 (500 mg, 0.85 mmol) was subjected to the same operating condition as in Example 1C, steps b, c, to obtain 312 mg of 20 (88% by two steps). Compound 20 (200 mg, 0.48 mmol) was hydrolyzed with NaOH (96 mg, 2.4 mmol) in a 1:1 v/v MeOH:H2O solution (10 mL) under the same operating condition as in Example 1A step f. A crude carboxylic acid intermediate (190 mg, 0.47 mmol) was treated with LiBH4 (1.65 mL, 2 M in THF, 3.3 mmol) and MeOH (133 µL, 3.3 mmol) in dry THF ( 5 mL). Purification by silica gel (CH2Cl2-MeOH 99:1) provided 157 mg of BAR704 (83%). In the same embodiments, a treatment with LiBH4 after alkaline hydrolysis produced small amounts (around 10%) of 6ü-ethyl-7ü-hydroxy-5D-cholan-24-oic acid (BAR711) which was isolated by purification in a Nucleodur 100-5 C18 (5 µm; 10 mm i.d. x 250 mm) with MeOH/H2O (88:12) as an eluent (flow rate 3 mL/min, tR= 16 min).

BAR704: C26H44O3

[147] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d3.65 (1H, br s, H-7), 2.34 (1H, m, H-23a), 2, 20 (1H, m, H-23b), 0.96 (3H, d, J = 6.3 Hz, H3- 21), 0.92 (3H, s, H3-19), 0.89 (3H, t, J = 7.4 Hz, H3-26), 0.70 (3H, s, H3-18).

[148] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 178.0, 71.6, 57.4, 51.7, 48.7, 43.8, [1] 3 , 41.5, 41.1, 39.3, 37.4, 36.8, 34.6, 32.5 (2C), 29.3, [2] 8, 25.1, 24.6 (2C ), 23.5, 22.5, 22.0, 18.8, 12.2, 12.1. BAR711: C26H44O3

[149] 1H NMR was recorded on Varian Inova 500 MHz, using CD3OD as solvent: d 3.08 (1H, t, J = 9.6 Hz, H-7), 2.32 (1H, m, H-23a), 2.20 (1H, m, H-23b), 0.96 (3H, d, J = 6.2 Hz, H3-21), 0.95 (3H, s, H3-19) , 0.85 (3H, t, J = 7.0 Hz, H3-26), 0.70 (3H, s, H3-18).

Step g) Preparation of 6a-ethyl-7a-hydroxy-5ß-cholan-24-oil sodium taurine sulfate (BART704)

[150] An aliquot of BAR704 (10 mg, 0.024 mmol) in dry DMF (5 mL) was treated with DMT-MM (20.5 mg, 0.07 mmol) and triethylamine (83 µL, 0.6 mmol) and the mixture was stirred at room temperature for 10 min. Then, to the mixture was added taurine (18 mg, 0.14 mmol). After 3 h, the reaction mixture was concentrated under vacuum and dissolved in water (5 mL). A purification at C18 on a silica gel column and then HPLC on a Nucleodur 100-5 C18 (5 µm; 10 mm i.d. x 250 mm) with MeOH/H2O (83:17) as an eluent (flow rate 3 mL/min) , provided 4.5 mg BART704 (tR = 10 min). BART704: C28H48NNaO5S

[151] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.65 (1H, br s, H-7), 3.58 (2H, t, J = 7.0 Hz , CH2-N), 2.96 (2H, t, J = 9.6 Hz, CH2-S), 2.25 (1H, m, H-23a), 2.10 (1H, m, H-23b ), 0.97 (3H, d, J = 6.4 Hz, H3-21), 0.92 (3H, s, H3-19), 0.89 (3H, t, J = 7.1 Hz, H3- 26), 0.70 (3H, s, H3-18).

Step h) Preparation of 6a-ethyl-7a-hydroxy-5ß-cholan-24-ol (BAR701)

[152] Compound 20 (100 mg, 0.24 mmol) was treated in the same operating condition as Example 2C step j. Purification by HPLC on a Nucleodur 100-5 C18 (5 µm; 10 mm i.d. x 250 mm) with MeOH/H2O (92:8) as an eluent (flow rate 3 mL/min), provided 64 mg of BAR701 (tR= 31 min) and a small amount of 6a-ethyl-7ß-hydroxy-5ß-cholan-24-ol (BAR703) (8 mg, tR = 24.8 min).

BAR701: C26H46O2

[153] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d3.65 (1H, br s, H-7), 3.51 (2H, m, H2-24), 0, 97 (3H, d, J = 6.3 Hz, H3-21), 0.92 (3H, s, H3-19), 0.89 (3H, t, J = 7.3 Hz, H3-26) , 0.71 (3H, s, H3-18).

[154] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 71.6, 63.6, 57.6, 51.8, 48.7, 43.7, 43.3, 41, 5, 41.1, 39.3, 37.5, 37.0, 34.6, 33.2, 30.3, 29.4, 43.4, 25.1, 24.6 (2C), 23.5 , 22.5, 22.0, 19.2, 12.3, 12.1.

BAR703 C26H46O2

[155] 1H NMR was recorded on Varian Inova 500 MHz, using CD3OD as solvent: d 3.51 (2H, m, H2-24), 3.07 (1H, t, J = 10.0 Hz, H-7), 0.96 (3H, d, J = 6.6 Hz, H3-21), 0.84 (3H, t, J = 7.0 Hz, H3-26), 0.95 (3H , s, H3-19), 0.71 (3H, s, H3-18).

[156] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 76, 3, 63, 6, 57.9, 56, 8, 46, 3, 45, 3, 45.0, 44.7, 41.7, 41.1, 38.8, 37.0, 36.3, 33.3, 30.3, 28.1, 27.9 (2C), 25.0 (2C), 22.0, 21.9 (2C), 19.4, 12.7, 11.6. Step i) Preparation of 6a-ethyl-7a-hydroxy-5ß-cholan-24-yl 24-sodium sulfate (BAR701solf)

[157] A C-24 sulfation on a small aliquot of BAR701 was carried out under the same operating conditions described as in example 1B step g) to provide crude BAR701solf as an ammonium salt. RP18/HPLC on a Nucleodur 100-5 C18 (5 µm; 10 mm i.d. x 250 mm) with MeOH/H2O (82:18) as an eluent (flow rate 3 mL/min) afforded BAR701solf (tR = 14.2 min ) as a sodium salt.

BAR701solf: C26H45 NaO5S

[158] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.96 (2H, t, J = 6.3 Hz, H2-24), 3.64 (1H, br s, H -7), 0.96 (3H, d, J = 6.6 Hz, H3-21), 0.91 (3H, s, H3-19), 0.88 (3H, t, J = 7.4 Hz, H3-26), 0.69 (3H, s, H3-18). Preparation of 6ß-ethyl-7ß-hydroxy-5ß-cholan-24-ol (BAR702) and 6ß-ethyl-7a-hydroxy-5ß-cholan-24-ol (BAR705)

[159] Compound 12 (500 mg, 1.05 mmol) was treated with MeONa (2.1 mL, 0.5 M in MeOH, 1.05 mmol) in MeOH (10 mL) in the same operating condition as Example 2C , step i, except for the reaction time (2 h). A tosylation of the crude reaction product under the same operating condition as in Example 1C, step a, provided 21 (620 mg, quantitative production by two steps). Intermediate 21 (600 mg, 1.02 mmol) was subjected to the same operating condition as in Example 1C, steps b, c, to obtain 400 mg of 22 (94%). Compound 22 (350 mg, 0.84 mmol) was reduced with NaBH4/LiBH4 under the same operating condition as in Example 2A steps f, g. Purification by HPLC on a Nucleodur 100-5 C18 (5 µm; 10 mm id x 250 mm) with MeOH/H2O (92:8) as an eluent (flow rate 3 mL/min), provided 180 mg of BAR702 (tR= 25 min) and 75.4 mg of BAR705 (tR= 13 min). Alternatively, the step was carried out with Ca(BH4)2, produced in situ. a) MeONa, MeOH; b) p-TsCl, pyridine, quantitative production in two steps; c) LiBr, Li2CO3, DMF, reflux; d) H2, Pd(OH)2, THF/MeOH 1:1, room temperature, 94% in two steps; e) NaBH4, MeOH; f) LiBH4, dry MeOH, THF, 0 °C, 78% in two steps.

BAR702: C26H46O2

[160] 1H NMR was recorded on Varian Inova 700 MHz, using CD3OD as solvent: d 3.67 (1H, dd, J = 8.7, 4.7 Hz, H- 7), 3.51 ( 2H, m, H2-24), 0.98 (3H, s, H3-19), 0.97 (3H, d, J = 6.6 Hz, H3-21), 0.96 (3H, t, J = 7.4 Hz, H3-26), 0.71 (3H, s, H3-18).

[161] 13C NMR was recorded on Varian Inova 175 MHz, using CD3OD as solvent: d 75.5, 63.8, 57.6, 56.5, 44.2, 43.7, 42.8, 41.0, 40.9, 40.8, 38.2 (2C), 36.9, 34.4, 32.8, 29.7, 43.5, 27.0, 26.1, 24.7, 22 ,2, 22.0 (2C), 19.2, 13.9, 12.3.

BAR705: C26H46O2

[162] 1H NMR was recorded on Varian Inova 700 MHz, using CD3OD as solvent: d3.59 (1H, br s, H-7), 3.51 (2H, m, H2-24), 2, 23 (1H, dq, J = 13.9, 4.0 Hz, H-4a), 0.97 (3H, d, J = 6.6, H3-21), 0.95 (3H, t, J = 7.1 Hz, H3-26), 0.94 (3H, s, H3-19), 0.70 (3H, s, H3-18).

[163] 13C NMR was recorded on Varian Inova 175 MHz, using CD3OD as solvent: d 73.1, 63.2, 57.3, 52.8, 51.4, 49.4, 43.8, 41.3, 39.7, 37.4 (2C), 37.2, 34.2, 32.6 (2C), 29.9, 29.5, 28.9, 28.3, 27.3, 24.5, 21.7, 21.2, 18.8, 14.3, 12.3. EXAMPLE 2G. Synthesis of 6a-ethyl-3ß,7a-dihydroxy-5ß-cholan-24-oic acid (BAR710), 6a-ethyl-3ß,7a-dihydroxy-5ß-cholan-24-oil sodium taurine sulfate (BART710), 6a -ethyl-3ß,7a- dihydroxy-5-cholan-24-ol (BAR706), sodium 6ß-ethyl-3ß,7ß- dihydroxy-5ß-cholan-24-yl 24- sulfate (BAR706solf), 6ß-ethyl- 3ß,7a-dihydroxy-5ß-cholan-24-ol (BAR707), 6ß-ethyl-3ß,7ß-dihydroxy-5ß-cholan-24-ol (BAR708) and 6ß-ethyl-3ß,7a- dihydroxy-5ß- cholan-24-ol (BAR709) and 6a-ethyl-3ß,7ß-dihydroxy-5ß-cholan-24-oic acid (BAR712).

[164] In a convergent protocol inversion at C-3 in derivative 19 followed by a treatment with MeONa/MeOH provided 23 which was used as starting material in the synthesis of BAR706, BAR706solf, BAR710 and BART710. An inversion at C-3 followed by a reduction at C-7 and C-24 produced BAR708 and BAR709. CH3COOK, DMF:H2O 5:1 v/v; b) NaOMe, MeOH, 74% in two steps; c) NaOH, MeOH:H2O 1:1 v/v; d) LiBH4, dry MeOH, THF, 0 °C, 65% in two steps; e) DMT-MM, Et3N, taurine, dry DMF; f) LiBH4, dry MeOH, THF, 0 °C, 58%; g) Et3N.SO3, DMF, 95 °C; h) CH3COOK, DMF:H2O 5:1; i) NaBH4, MeOH; j) LiBH4, dry MeOH, THF, 0 °C, 74% for three steps.

Steps a-d) Preparation of 6a-ethyl-3ß,7a-dihydroxy-5ß-cholan-24-oic acid (BAR710)

[165] A solution of 21 (600 mg, 1.0 mmol) and CH3COOK (98 mg, 1.0 mmol) dissolved in water (2 mL) and N,N'-dimethylformamide (DMF, 10 mL) was placed in reflux for 2 h. The solution was cooled to room temperature and then ethyl acetate and water were added. The separated aqueous phase was extracted with ethyl acetate (3 x 30 mL). The combined organic phases were washed with water, dried (Na2SO4) and evaporated to dryness to provide 600 mg of mixture. Purification by silica gel (hexane - ethyl acetate 8:2 and 0.5% TEA) provided 350 mg of oily oil. A C-6 inversion at the same operating condition as described in Example 2C step i, provided 23 (320 mg, 74% by two steps) which was subjected to a hydrolysis followed by a treatment with LiBH4 as described in Example 2F, steps e, f. Purification by HPLC on a Nucleodur 100-5 C18 (5 µm; 10 mm i.d. x 250 mm) with MeOH/H2O (88:12) as an eluent (flow rate 3 mL/min), provided 208 mg of BAR710 (65% , tR= 11 min). Alternatively, an inversion at C-3 in 21 followed by alkaline hydrolysis and then LiBH4 treatment provided BAR710 in a direct manner.

[166] In the same embodiments, a treatment with LiBH4 after alkaline hydrolysis produced small amounts (around 10%) of 6a-ethyl-3e,7e-dihydroxy-5e-cholan-24-oic acid (BAR712) which was isolated by HPLC purification on a Nucleodur 100-5 C18 (5 µm; 10 mm i.d. x 250 mm) with MeOH/H2O (88:12) as an eluent (flow rate 3 mL/min, tR= 8 min)

BAR710: C26H44O4

[167] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.97 (1H, br s, H-3), 3.67 (1H, br s, H-7), 2.33 (1H, m, H-23a), 2.21 (1H, m, H-23b), 0.96 (3H, d, J = 6.5 Hz, H3-21), 0.94 ( 3H, s, H3-19), 0.91 (3H, t, J = 7.6 Hz, H3-26), 0.70 (3H, s, H3-18).

[168] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 178.3, 71.3, 67.5, 57.4, 51.7, 43.8, 42.8, 41.5, 41.2, 41.0, 37.0, 36.7, 33.8, 32.4, 32.0, 31.1 (2C), 29.3, 28.3, 24.6 , 24.2, 23.3, 22.2, 18.8, 12.3, 12.2.

BAR712: C26H44O4

[169] 1H NMR was recorded on Varian Inova 500 MHz, using CD3OD as solvent: d 4.01 (1H, br s, H-3), 3.06 (1H, t, J = 9.7 Hz , H-7), 2.32 (1H, m, H-23a), 2.19 (1H, m, H- 23b), 0.97 (3H, s, H3-19), 0.96 (3H , d, ovl, H3-21), 0.87 (3H, t, J = 7.7 Hz, H3-26), 0.71 (3H, s, H3-18).

Step e) Preparation of 6a-ethyl-3ß,7a-dihydroxy-5ß-cholan-24-oil sodium taurine sulfate (BART710)

[170] An aliquot of BAR710 (10 mg) was treated in the same operating condition as in Example 2F step g.

BART710: C28H48NNaO6S

[171] 1H NMR was recorded on Varian Inova 500 MHz, using CD3OD as solvent: d 3.97 (1H, br s, H-3), 3.67 (1H, br s, H-7), 3.59 (2H, t, J = 6.8 Hz, CH2-N), 2.96 (2H, t, J = 6.8 Hz, CH2-S), 0.97 (3H, d, J = 6.4 Hz, H3-21), 0.95 (3H, s, H3-19), 0.91 (3H, t, J = 7.1 Hz, H3-26), 0.70 (3H, s , H3- 18).

Steps f) Preparation of 6a-ethyl-3ß,7a-dihydroxy-5ß-cholan-24-ol (BAR706) and 6a-ethyl-3ß,7ß-dihydroxy-5ß-cholan-24-ol (BAR707)

[172] Intermediate 23 (500 mg, 1.16 mmol) was treated with LIBH4 (4 mL, 8.1 mmol) and MeOH (327 ÜL, 8.1 mmol) in dry THF (10 mL) as described in Example 2C, step j. Purification by HPLC on a Nucleodur 100-5 C18 (5 µm; 10 mm i.d. x 250 mm) with MeOH/H2O (88:12) as an eluent (flow rate 3 mL/min), provided BAR706 (250 mg, tR= 12.6 min) and a small amount of 6a-ethyl-3ß,7ß-dihydroxy-5ß-cholan-24-ol (BAR707) (23 mg, tR = 8.2 min). BAR706: C26H46O3

[173] 1H NMR was recorded on Varian Inova 500 MHz, using CD3OD as solvent: d 3.97 (1H, br s, H-3) , 3.66 (1H, br s, H-7), 3.51 (2H, m, H2-24), 0.96 (3H, d, J = 6.6 Hz, H3-21), 0.94 (3H, s, H3-19), 0.91 ( 3H, t, J = 7.5 Hz, H3-26), 0.70 (3H, s, H3-18).

[174] 13C NMR was recorded on Varian Inova 125 MHz, using CD3OD as solvent: d 71.4, 67.4, 63.6, 57.6, 51.7, 43.7, 42.8, 41.5, 41.2, 41.1, 37.1 (2C), 33.8, 33.2, 31.3 (2C), 30.3, 29.4, 28.3, 24.6, 24.2, 23.3, 22.3, 19.2, 12.7, 12.1.

BAR707: C26H46O3

[175] 1H NMR was recorded on Varian Inova 500 MHz, using CD3OD as solvent: d 4.01 (1H, br s, H-3), 3.51 (2H, m, H2-24), 3 .05 (1H, t, J = 9.7 Hz, H-7), 0.97 (3H, s, H3- 19), 0.96 (3H, d, J = 6.4 Hz, H3-21 ), 0.88 (3H, t, J = 7.6 Hz, H3-26), 0.72 (3H, s, H3-18).

[176] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 76.3, 67.1, 63.6, 57.8, 56.8, 45.0, 44.8, 44.5, 41.7, 40.3, 39.1, 37.0, 35.9, 33.0, 31.2, 30.2, 29.8, 28.4, 28.0, 27, 9, 24.8, 22.9, 21.8, 19.3, 12.8, 11.6.

Step g) Preparation of 6a-ethyl-3ß,7a-dihydroxy-5ß-cholan-24-yl 24-sodium sulfate (BAR706solf)

[177] A C-24 sulfation on a small aliquot of BAR706 was carried out under the same operating conditions described as in example 1B step g.

BAR706solf: C26H45NaO6S

[178] 1H NMR was recorded on Varian Inova 500 MHz, using CD3OD as solvent: d 3.97 (1H, br s ovl, H-3), 3.96 (2H, t ovl, H2-24) , 3.65 (1H, br s, H-7), 0.96 (3H, d, J = 6.6 Hz, H3-21), 0.94 (3H, s, H3-19), 0, 90 (3H, t, J = 7.5 Hz, H3-26), 0.70 (3H, s, H3-18).

Steps h, i, j) Preparation of 6ß-ethyl-3ß,7ß-dihydroxy-5ß-cholan-24-ol (BAR708) and 6ß-ethyl-3ß,7a-dihydroxy-5ß-cholan-24-ol (BAR709) .

[179] Compound 19 was treated in the same operating condition as step a. A reduction with 100 mg NaBH4/LiBH4 (0.23 mmol) under the same operating conditions as in Example 2A, steps f, g, provided a mixture whose HPLC purification (88% MeOH:H2O) provided pure 6ß-ethyl-3ß7ß- dihydroxy-5ß-cholan-24-ol (BAR708) (48.3 mg, tR= 11 min) and 6e-ethyl-3e7a-dihydroxy-5e-cholan-24-ol (BAR709) (20.7 mg, tR= 13 min). Alternatively, step i was performed with Ca(BH4)2, produced in situ.

BAR708: C26H46O3

[180] 1H NMR was recorded on Varian Inova 700 MHz, using CD3OD as solvent: d 3.59 (1H, br s, H-3), 3.57 (1H, dd, J = 12.6, 2.3 Hz, H-7), 3.51 (2H, m, H2-24), 0.98 (3H, s, H3-19), 0.96 (3H, ovl, H3-21), 0 .96 (3H, t, ovl, H3-26), 0.70 (3H, s, H3-18).

[181] 13C NMR was recorded on Varian Inova 175 MHz, using CD3OD as solvent: dQ 75.2, 68.3, 63.6, 58.3, 57.1, 45.7 (2C), 44 ,2, 41.8 (2C), 41.2, 40.0, 37.0, 35.9, 33.3, 31.1, 30.3, 29.4 (2C), 26.6 (2C ), 23.2 (2C), 19.3, 13.0, 12.3.

BAR709: C26H46O3

[182] 1H NMR was recorded on Varian Inova 700 MHz, using CD3OD as solvent: ô 3.91 (1H, br s, H-3), 3.60 (1H, br s, H-7), 3.51 (2H, m, H2-24), 2.45 (1H, t, J = 13.3 Hz, H- 4a), 0.97 (3H, s, H3-19), 0.97 ( 3H, ovl, H3-21), 0.95 (3H, t, J = 7.4 Hz, H3-26), 0.71 (3H, s, H3-18).

[183] 13C NMR was recorded on Varian Inova 175 MHz, using CD3OD as solvent: ô 72.8, 67.4, 63.4, 57.2, 51.3, 51.2, 43.2, 41.6, 40.5, 37.3, 37.1 (2C), 36.9, 34.0, 33.3, 32.1, 30.3, 29.3, 28.9, 28.6 , 26.3, 24.9, 22.0, 19.3, 13.8, 12.1. EXAMPLE 2H. Synthesis of 6a-ethyl-7a-hydroxy-24-nor-5ß-cholan-23-oic acid (BARn704), sodium taurine sulfate 6a-ethyl-7a-hydroxy-24-nor-5ß-cholan-23-oil ( BARTn704), 6a-ethyl-7a-hydroxy-24-nor-5ß-cholan-23-ol (BARn701) and 6a-ethyl-7a-hydroxy-24-nor-5ß-cholan-23-yl 23- sodium sulfate (BARn701solf)

[184] BARn704, BARTn704, BARn701 and BARn701solf were prepared starting from 15 and following the same synthetic protocol described for their C24 homologues (Example 2F, steps ai). a) MeONa, MeOH; b) p-TsCl, pyridine, 73% in two steps; c)LiBr, Li2CO3, DMF, reflux, d) H2, room temperature, quantitative production in two stages; e) NaOH, MeOH:H2O 1:1 v/v, 80%; f) LiBH4, dry MeOH, THF, 0 °C, 92%; g) DMT-MM, Et3N, taurine, dry DMF; h) LiBH4, dry MeOH, THF, 0 °C, 70%; i) Et3N.SO3, DMF, 95 °C.

BARn704: C25H42O3

[185] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.64 (1H, br s, H-7), 2.41 (1H, dd, J = 11.0, 2.6 Hz, H-22a), 1.00 (3H, d, J = 6.0 Hz, H3- 21), 0.90 (3H, s, H3-19), 0.87 (3H, t , J = 7.4 Hz, H3-25), 0.71 (3H, s, H3-18).

[186] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 178.9, 71.6, 57.5, 51.7, 48.6, 43.8, 43.4, 43.3, 41.5, 40.9, 39.1, 37.4, 35.2, 34.6, 29.4, 28.8, 25.0, 24.6 (2C), 23.5 , 22.4, 22.0, 20.1, 12.2, 12.1.

BARTn704: C27H46NNaO5S

[187] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.64 (1H, br s, H-7), 3.59 (2H, t, J = 6.8 Hz , CH2-N), 2.96 (2H, t, J = 6.8 Hz, CH2-S), 2.40 (1H, dd, J = 11.0, 2.8 Hz, H-22a), 1.00 (3H, d, J = 6.0 Hz, H3-21), 0.89 (3H, s, H3-19), 0.86 (3H, t, J = 7.4 Hz, H3- 25), 0.70 (3H, s, H3-18).

BARn701: C25H44O2

[188] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.66 (1H, br s, H-7), 3.61 (1H, m, H-23a), 3 .54 (1H, m, H-23b), 0.96 (3H, d, J = 6.7 Hz, H3- 21), 0.91 (3H, s, H3-19), 0.89 (3H , t, J = 7.3 Hz, H3-25), 0.70 (3H, s, H3-18).

[189] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 71.5, 60.8, 57.8, 51.7, 48.6, 43.8, 43.3, 41.5, 41.1, 39.9, 39.2, 37.4, 34.5, 34.2, 29.4, 28.8, 25.0, 24.6 (2C), 23.5 , 22.5, 22.0, 19.4, 12.2, 12.1.

BARn701solf: C25H43NaO5S

[190] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 4.02 (2H, m, H2-23), 3.65 (1H, br s, H-7), 0 .97 (3H, d, J = 6.7 Hz, H3-21), 0.91 (3H, s, H3- 19), 0.88 (3H, t, J = 7.5 Hz, H3-25 ), 0.70 (3H, s, H3-18). EXAMPLE 2I. Synthesis of 6a-ethyl-3ß,7a-dihydroxy-24-nor-5ß-cholan-23-0ic acid (BARn710), sodium taurine sulfate 6a-ethyl-3ß,7a-dihydroxy-24-nor-5ß-cholan- 23-oil (BARTn710), 6a-ethyl-3ß,7a-dihydroxy-24-nor-5ß-cholan-23-ol (BARn706), and 6a-ethyl-3ß,7a-dihydroxy-24-nor-5ß- sulfate sodium cholan-23-yl 23- (BARn706solf) a) CH3COOK, DMF:H2O 5:1 v/v; b) NaOH, MeOH:H2O 1:1 v/v; c) LiBH4, dry MeOH, THF, 0 °C, 58% in two steps; d) DMT-MM, Et3N, taurine, dry DMF; e) LiBH4, dry MeOH, THF, 0 °C, 57%; f) Et3N.SO3, DMF, 95 °C.

[191] BARn710, BARTn710, BARn706 and BARn706solf were prepared starting from 24 and following the same synthetic protocol described for their C24 homologues (Example 2G, steps c-g).

BARn710: C25H42O4

[192] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.97 (1H, br s, H-3), 3.66 (1H, br s, H-7), 2.42 (1H, dd, J = 11.3, 3.3 Hz, H-22a), 1.02 (3H, d, J = 6.0 Hz, H3-21), 0.94 (3H, s, H3-19), 0.91 (3H, t, J = 7.3 Hz, H3-25), 0.73 (3H, s, H3-18).

[193] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d 177.7, 71.3, 67.4, 57.4, 51.8, 43.8 (2C), 42 .8, 41.5, 41.2, 41.0, 37.0, 35.1, 33.8, 31.3, 31.2, 29.4, 28.3, 24.6, 24.2 , 23.3, 22.2, 20.0, 12.2, 12.1.

BARTn710: C27H46NNaO6S

[194] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.97 (1H, br s, H-3), 3.66 (1H, br s, H-7), 3.59 (2H, t, J = 6.8 Hz, CH2-N), 2.96 (2H, t, J = 6.8 Hz, CH2-S), 2.42 (1H, dd, J = 11.3, 3.3 Hz, H-22a), 1.00 (3H, d, J = 6.3 Hz, H3-21), 0.94 (3H, s, H3-19), 0.90 (3H, t, J = 7.0 Hz, H3-25), 0.70 (3H, s, H3-18).

BARn706: C25H44O3

[195] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.97 (1H, br s, H-3), 3.67 (1H, br s, H-7), 3.61 (1H, m, H-23a), 3.55 (1H, m, H-23b), 0.97 (3H, d, J = 6.6 Hz, H3-21), 0.95 ( 3H, s, H3-19), 0.91 (3H, t, J = 7.4 Hz, H3-25), 0.71 (3H, s, H3-18).

[196] 13C NMR was recorded on Varian Inova 100 MHz, using CD3OD as solvent: d71.4, 67.4, 60.8, 57.9, 51.8, 43.8, 42.8, 41 .5, 41.2, 41.1, 39.9, 37.0, 34.2, 33.8, 31.3, 31.2, 29.4, 28.3, 24.6, 24.2 , 23.3, 22.3, 19.4, 12.2, 12.1. BARn706solf: C25H43NaO6S

[197] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 4.05 (2H, m, H2-23), 3.97 (1H, br s, H-3), 3 .66 (1H, br s, H-7), 1.00 (3H, d, J = 6.0 Hz, H3- 21), 0.94 (3H, s, H3-19), 0.91 ( 3H, t, J = 6.9 Hz, H3-25), 0.71 (3H, s, H3-18). EXAMPLE 2J. Preparation of 6a-ethyl-3a, 7a-dihydroxy-25, 26-bishomo-5ß-cholan-26-0ic acid (BAR802), 6a-ethyl-3a, 7a-dihydroxy-25, 26-bishomo-5ß-cholan- 26-ol (BAR803), and 6a-ethyl-3a, 7a-dihydroxy-25, 26-bishomo-5ß-cholan-26-yl-26-sodium sulfate (BAR804) BAR804 a) 2,6-lutidine, t-butyldimethylsilyl trifluoromethanesulfonate, CH2Cl2, 0 °C; b) LiBH4, dry MeOH, THF, 0 °C, 68% in two steps; c) DMSO, oxalyl chloride, dry TEA, CH2Cl2, -78 °C then methyl(triphenylphosphoranylidene)acetate, 79%; d) H2, Pd(OH)2/C Degussa type, THF/MeOH 1:1, quantitative production; e) HCl 37%, MeOH, 87%; f) 5% NaOH in MeOH/H2O 1:1 v/v, 89%; g) LiBH4, dry MeOH, THF, 0 °C, 78%; h) Et3N.SO3, DMF, 95 °C, 25 %.

[198] BAR802-804 were prepared following the same synthetic protocols as in example 1A, steps a-g. Sulfation on a small aliquot of BAR803 under the same operating conditions as in Example 1B step g provided BAR804.

BAR802: C28H48O4

[199] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.66 (1H, br s, H-7), 3.31 (1H, m ovl, H-3), 2.24 (2H, t, J = 7.3 Hz, H2-25), 0.95 (3H, d, J = 6.4 Hz, H3-21), 0.92 (3H, s, H3- 19), 0.91 (3H, t, J = 6.9 Hz, H3-28), 0.70 (3H, s, H3-18).

[200] 13C NMR was recorded on Varian Inova 175 MHz, using CD3OD as solvent: d 186.7, 73.3, 71.3, 57.7, 51.7, 47.0, 43.7, 43.1, 41.6, 41.1, 37.1, 36.9, 36.8, 36.6, 34.5, 34.4 (2C), 31.3, 29.4, 27.0 (2C), 24.6, 23.8, 23.5, 22.0, 19.2, 12.2, 12.0.

BAR803: C28H50O3

[201] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.64 (1H, br s, H-7), 3.53 (2H, t, J = 6.6 Hz , H2-26), 3.30 (1H, m ovl, H-3), 0.94 (3H, d, J = 6.7 Hz, H3-21), 0.91 (3H, s, H3- 19), 0.90 (3H, t, J = 7.0 Hz, H3-28), 0.68 (3H, s, H3-18).

[202] 13C NMR was recorded on Varian Inova 175 MHz, using CD3OD as solvent: d 73.3, 71.3, 63.1, 57.7, 51.7, 47.0, 43.7, 43.1, 41.6, 41.1, 37.1, 36.8 (2C), 36.6, 34.5, 34.4, 33.7, 31.3, 29.5, 27.4 , 27.0, 24.6, 23.8, 23.5, 22.0, 19.3, 12.3, 12.0.

BAR804: C28H49NaO6S

[203] 1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: d 3.99 (2H, t, J = 6.6 Hz, H2-26), 3.65 (1H, br s , H-7), 3.31 (1H, m ovl, H-3), 0.94 (3H, d, J = 6.2 Hz, H3-21), 0.91 (3H, s, H3- 19), 0.90 (3H, t, J = 7.0 Hz, H3-28), 0.69 (3H, s, H3-18).

[204] Biological Activities. Activity of selected compounds was tested in vitro using a whole cell model transfected with reporter genes to establish selectivity of compounds shown in Table 1 toward FXR and TGR5/GPBAR1 compared to chenodeoxycholic acid (CDCA) and TLCA. . CDCA is a primary bile acid that functions as an endogenous ligand for FXR, while TLCA is a physiological ligand for TGR5/GPBAR1. In this assay, HepG2 cells (a liver-derived cell line) were cultured at 37°C in Earl's salts minimum essential medium containing 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% % penicillin/streptomycin. HEK-293T cells were cultured at 37°C in D-MEM containing 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/streptomycin. Transfection experiments were performed using Fugene HD according to manufacturing specifications. Cells were plated in a 24-well plate at 5 x 10 cells/well. For FXR-mediated transactivation, HepG2 cells were transfected with 100 ng of pSG5-FXR, 100 ng of pSG5-RXR, 100 ng of pGL4.70 a vector encoding the human Renilla gene 250 ng of the p(hsp27) reporter vector -TK-LUC containing the FXR response element IR1 cloned from the heat shock protein 27 (hsp27) promoter.

[205] For GPBAR1-mediated transactivation, HEK-293T cells were transfected with 200 ng of pGL4.29, a reporter vector containing a cAMP response element (CRE) that drives transcription of the luciferase reporter gene luc2P, with 100 ng of pCMVSPORT6-human GPBAR1, and with 100 ng of pGL4.70 a vector encoding the human Renilla gene. In control experiments, HEK-293T cells were transfected only with pGL4.29 and pGL4.70 vectors to exclude any possibility that the compounds could activate the CRE in a GPBAR1-independent manner. At 24 h posttransfection, cells were stimulated for 18 h with 10 μM TLCA as a control agent or putative GPBAR1 agonists as the same concentration. After treatments, cells were lysed in 100 µL of a lysis buffer (25 mM Tris-phosphate, pH 7.8; 2 mM DTT; 10% glycerol; 1% Triton X100), and 20 DL of cell lysate were assayed. for luciferase activity using the luciferase assay system. Luminescence was measured using the Glomax 20/20 luminometer. Luciferase activities were normalized against Renilla activities. Antagonism against FXR of GPBAR1/TGR5 was measured as a percentage of activity in a transactivation assay following TLCA activity as an example of agonism.

[206] Animals and protocols. Mice lacking GPBAR1 (GPBAR1- B6=GPBAR12/2 mice, generated directly on C57BL/6NCrl background), and C57BL/6NCrl congenic littermates were housed under controlled temperatures (22 °C) and photoperiods (12:12 hours of light/dark cycle), left with unrestricted access to standard rat chow and tap water and allowed to acclimate to these conditions for at least 5 days before inclusion in an experiment. Scratch Test. Male GPBAR1-/- mice and their congenic littermates (8-12 weeks old) were used for their studies. The fur at the base of the neck was removed, and the mice were placed in individual cylinders on a glass shelf. A circle approximately 0.5 cm in diameter was drawn on the neck and test agents injected into this area. Rats were acclimatized to the experimental room, restraint apparatus, and investigators for 2 2-h periods on 2 successive days before the experiments. Scratching behavior was quantified by 2 observers unaware of the agents or genotypes tested. A scratch was defined as raising the hindlimb to the injection site and then placing the paw on the floor, regardless of the number of strokes. If counts differed by more than 5 scratches over a 30-min period, both observers re-evaluated the records. Results were expressed as the number of scratching events during 30 or 60 min of observation. The test agents were: DCA (25 µg), TLCA (25 µg), UDCA (25 µg), and BAR502 (25 µg), or with betulinic acid (50 µg), oleanolic acid (50 µg). LCA and DCA were dissolved in DMSO and the other agents in 0.9% NaCl (10 µL). In another experimental scenario, GPBAR1-/- mice and their congenic littermates were administered alpha-naphthylisothiocyanate (ANIT) (25 mg/kg, per os) dissolved in olive oil or olive oil alone (control mice) or with the combination of ANIT plus BAR502 (15 mg/kg once daily, per os) for 10 days. On day 5, spontaneous scratching was assessed for 60 min and after a subcutaneous injection of 25 µg DCA. Serum levels of total bilirubin, aspartate aminotransferase (AST), and alkaline phosphatase were measured by a routine clinical chemistry test on a Hitachi 717 automatic analyzer. For the estrogen model, wild-type C57BL6 mice were administered 10 mg/ kg i.p. with 17a-Ethinylestradiol (17aE2) dissolved in PEG or PEG alone (control rats) or the combination of 17aE2 and BAR502 (15 mg/kg daily, per os) for 8 days. At the end of the study, spontaneous scratching and scratching induced by s.c. injection of 25 µg DCA were recorded. Gallbladder weight and serum bilirubin and alkaline phosphatase levels were also measured. For all studies, animals were visually assessed at least twice daily from Monday through Friday and once daily on the weekend by investigators and daily trained animal facility personnel. Animals were weighed daily and sacrificed at the indicated time points or when their clinical condition became critical, as assessed by a body weight reduction greater than 25% of baseline body weight at 7 days. Furthermore, animals were sacrificed when, on daily assessment, they demonstrated an inability to climb or move. Rats were euthanized by an overdose of pentobarbital sodium (>100 mg/kg i.p.).

Claims (17)

1. Compound of formula (I) characterized by the fact that R1 is OH or H; R2 is Et, =CH-CH3; R3 is OH; n is 0, 1, or 3; R is CH2OH, COOH, CH2OSO3H or CN; provided that when R2 is Et or =CH-CH3 and R3 is OH: if n is 0 or 1 then R will be CH2OH or CN when R1 is alpha-OH or R will be COOH, CH2OH or CH2OSO3H when R1 is beta-OH or H ; if n is 3 then R1 and R are as defined above; including pharmaceutically acceptable inorganic and organic salts, amino acid solvates or conjugates thereof; excluding a compound in which 2. Compound according to claim 1, characterized by the fact that n is 0 or 1, R is CH2OH or CN and R1 is alpha-OH or n is 0 or 1, R is COOH, CH2OH or CH2OSO3H and R1 is beta-OH or H. 3. Compound according to claim 1, characterized by the fact that R2 is Et, n is 3, R1 is alpha-OH and R3 is alpha-OH. 4. Use of a compound to prepare a medicine, said compound of formula (I) (I) characterized by the fact that R1 is OH or H; R2 is Et, =CH-CH3; R3 is OH; n is 0, 1, or 3; R is CH2OH, COOH, CH2OSO3H or CN; provided that when R2 is Et or =CH-CH3 and R3 is OH: if n is 0 or 1 then R will be CH2OH or CN when R1 is alpha-OH or R will be CH2OH, CH2OSO3H or COOH when R1 is beta-OH or H ; if n is 3 then R1 and R are as defined above; including pharmacologically acceptable inorganic and organic salts, solvates or amino acid conjugates thereof. 5. Use according to claim 4, characterized in that said compound of formula (I) is as defined in any one of claims 2 to 5. 6. Use according to claim 3, characterized in that said compound of formula (I) is selected from the group consisting of 7. Use of compound for preparation of a medicine, such as an FXR and/or TGR5/GPBAR1 modulator, for the prevention and/or treatment of gastrointestinal disorders, liver diseases, cardiovascular diseases, atherosclerosis, metabolic diseases, infectious diseases, cancer , renal disorders, inflammatory disorders and neurological disorders, said compound of formula (I) characterized in that it is as defined in any one of claims 4 to 6. 8. Pharmaceutical composition, characterized by the fact that it comprises a compound of formula (I) and at least one other pharmaceutical ingredient; said compound of formula (I) as defined in any one of claims 4 to 6. 9. Process for preparing a compound of formula (I), according to any one of claims 1 to 4 or as defined in any one of claims 6 to 8, characterized in that R2 is =CH-CH3 or Et and R3 is not H, said process comprising subjecting a compound of formula (VIII) to an aldol condensation thereby contacting a compound of formula (VIII) where n is 0 or 1, P is a hydroxyl protecting group, with alkyl lithium, such as nBuLi, and, subsequently, with acetaldehyde, preferably in the presence also of BF3(OEt)2, to obtain a compound of formula (IX) where n and P are as described above. 10. Process, according to claim 9, characterized by the fact that the compound of formula (IX) is subjected to catalytic hydrogenation, to obtain a compound of formula (X) 11. Process according to claim 10, characterized in that the compound of formula (X) is subjected to a carbonyl reduction at C7 by contacting the compound of formula (X) with NaBH4 or Ca(BH4)2 for the obtaining a mixture of alpha-OH and beta-OH in C7; and subsequently subjected to a LiBH4 treatment, which completely reduces the methyl ester function in the para-CH2OH side chain. 12. Process for preparing a compound of formula (I), according to any one of claims 1 to 4 or as defined in any one of claims 6 to 8, characterized by the fact that R2 is alpha-Et, said process comprising contact with MeONa/MeOH of a compound of formula (XIV) where n is 0 or 1, R8 is beta-OH, OAc or H to obtain epimerization of the C6 stereocenter, thus obtaining a compound of formula (XI) wherein, in the case where R8 is OAc, treatment with MeONa/MeOH simultaneously ensures the hydrolysis of C3 acetoxy group. 13. Process according to claim 12, characterized in that a C7 carbonyl reduction is obtained by contacting a compound of formula (XI) or a corresponding compound having COOH in the side chain, with LiBH4 obtaining almost exclusively alpha- OH at C7; wherein LiBH4 treatment simultaneously reduces, if present, the methyl ester function in the side chain to -CH2OH and ceases to protect OAc if present at C3. 14. Process for preparing a compound of formula (I), according to any one of claims 1 to 2 and 4 to 5 or as defined in any one of claims 6 to 8, characterized by the fact that n = 3, said process comprising subjecting a compound of corresponding formula (IV) where m=n-2=1, R4 and R5, if other than H, are OP, where P is an alcohol protection function, and R2 is H or Et, one-pot Swern oxidation/homologation of Wittig C2 to obtain a protected methyl ester of formula (V) wherein m=1, R2 is as defined in claim 1, R4 and R5, if other than H, are OP, wherein P is an alcohol protection function. 15. Process for preparing a compound of formula (I), according to any one of claims 1 to 3 or 5 or according to any one of claims 6 to 8, characterized by the fact that n=0, said process comprising contact a compound of formula (XII) with HCOOH and HClO4 and subsequently contacting the resulting compound with TFA, trifluoroacetic anhydride and NaNO2 to obtain a compound of formula (VII) 16. Process for preparing a compound of formula (I), according to any one of claims 1 to 5 or according to any one of claims 6 to 8, characterized in that R1 is beta-OH, said process comprising start from a compound having a formula (XIII) where R2 is Et, n is 0 or 1 and invert the C3 hydroxyl configuration by a treatment with tosyl chloride in the presence of a base then followed by a treatment with CH3COOK. 17. Process for preparing a compound of formula (I), according to any one of claims 1 to 5 or according to any one of claims 6 to 8, characterized in that R1 is H, said process comprising subjecting of a compound of formula (XIII) where n is 0 or 1, R2 Et or H, to a tosylation and elimination at a C-3 hydroxyl group followed by a double bond reduction via a catalytic hydrogenation.