TECHNICAL FIELD

The present invention relates to hyodeoxycholic acid derivatives and their uses, in particular in the treatment and/or prevention of FXR, LXR and TGR5/GPBAR1 mediated diseases.

BACKGROUND ART

Bile acids (BAs) are ligands interacting with at least four types of receptors belonging to the nuclear receptor (NR) super- family: farnesoid X receptor (FXR), identified as the sensor of endogenous bile acids (Makishima et al Science 1999, 284, 1362. Parchi et al Science 1999, 284, 1365), costitutive androstane receptor (CAR, identified in Saini et al. Mol . Pharmacol. 2004, 65, 292-300), pregnane X receptor (PXR, identified in Staudinger et al. Proc.Natl. Acad. Sci. U.S.A. 2001, 98, 3369), liver X receptor (LXR, identified in Song et al. Steroids 2000, 65, 423) and vitamin D receptor (VDR, identified in Makishimaet al. Science 2002, 296, 1313) .

In addition, the secondary bile acids activate G-protein- coupled receptors (GPCR) , including GPBAR1 (also known as M-BAR, TGR5, or BG37) (Takeda et al . FEBS Lett. 2002, 520, 97; Kawamata et al J. Biol. Chem., 2003, 278, 9435) .

Highly expressed in entero-hepatic tissues (liver and intestine) , FXR regulates bile acid homeostasis and some metabolic pathways such as lipid and glucose metabolism. FXR agonists also show anti-inflammatory, anti-fibrotic and anti-cancer effects (Renga et al. FASEB J. 2012, 26, 3021-3031).

The LXRs act as sensors of oxysterols and regulate genes involved in lipid and cholesterol metabolism. The physiological impact of the LXRs is associated with the communication interface of lipid metabolism and inflammation. Therefore, LXRs have been identified as promising pharmacological targets for indications such as hypercholesterolemia, atherosclerosis and cardiovascular diseases (Colin et al. J. Med. Chem. 2014, 57, 7182) .

In the brain, LXRs regulate the expression of the genes linked to the transport of cholesterol, and show anti-inflammatory effects in microglia. Deletion of LXRa or LXRβ in a transgenic mouse model of Alzheimer's disease led to a marked increase in amyloid-β deposition and an Alzheimer's disease-like pathology. Several studies have shown that synthetic LXR agonists have therapeutic effects in mouse models of Alzheimer's disease (Hong et al. Nat. Rev. Drug. Discov. 2014, 6, 433) .

TGR5/GPBAR1 ligands increase the intracellular concentrations of cAMP with consequent activation of a signalling cascade. GPBAR1 is highly expressed in the liver and in the intestine, but also in the muscles, in the adipose tissue, in the macrophages and in the endothelial cells. In muscles and brown adipose tissue, GPBAR1 increases energy expenditure and oxygen consumption (Watanabe et al. Nature del 2006, 439, 484) . In the entero-endocrine L cells, GPBAR1 activation stimulates the secretion of glucagon-like peptide (GLP- 1), regulating glucose blood levels, gastrointestinal motility and appetite (Thomas et al. Cell. Metab. 2009, 10, 167) .

GPBAR1 appears relevant also for the pathogenesis of atherosclerosis. In a mouse model of atherosclerosis, GPBAR1 activation reduces the size of atheroma plaques by decreasing the intra-plaque inflammation, the macrophage count within the plaque, and the macrophage activity (Pols et al. Cell Metabolism 2011, 14, 747) . GPBAR1 is also expressed in astrocytes and neurons and in both cell types, the receptor is sensitive to the neurosteroids , leading to a rapid increase in the intracellular concentrations of cAMP and calcium. Even more interesting, activation of this receptor may reduce neuro-inflammation and improve neurological deficits during hepatic encephalopathy (McMillin et al. J. Neurochem. 2015, 135,

R=OH R1 =H CDCA HDCA

R=OH R1 =OH CA

R=H R1 =OH DCA

R=H R1 =H LCA

In chemical terms, bile acids are cholesterol derivatives provided with a truncated lateral chain.

They are generated firstly in the liver with the production of primary bile acids (cholic acid (CA) and chenodeoxycholic acid (CDCA) ) .

Microbiological transformation 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 .

DISCLOSURE OF INVENTION

The object of the present invention is the identification of new compounds deriving from the hyodeoxycholic acid (HDCA) able to modulate FXR, LXR and TGR5/GPBAR1.

Said object is achieved by the present invention, relative to hyodeoxycholic acid derivatives according to claim 1 and to the use thereof according to claims 8 and 9.

BEST MODE FOR CARRYING OUT THE INVENTION

Surprisingly, the inventors have found that, although the hyodeoxycholic acid is not able to effectively modulate the activity of these receptors, slight modifications in its lateral chain result in compounds able to transactivate them in a selective or also dual manner .

In a first embodiment of the invention, compounds are provided having formula (I) :

wherein :

Ri is selected from the group consisting of:

a) -(CH2)n ^~ R2, wherein n=l-6 and R2 is selected from the group consisting of H, isopropyl, phenyl, and wherein R3 and R are simultaneously H or CH3, or alternatively R3 is H and R4 is selected from the group consisting of CH2OH and COOH;

b) - ( CH2 ) _n -R2 wherein n=4-6 e R2 is selected from the group consisting of CH2OH and COOH;

c) - ( CH2 ) _m -CH=C (R5R6) wherein m is 1-4, R5 and R6 are simultaneously H or CH3 , or alternatively R5 is H and R6 is phenyl; with the proviso that the compound having formula (I)

is not:

BAR 1 145 CAS: 53241 -60-4

In one embodiment, compounds are provided having formula (IA) :

wherein n=l-6 and R2 is selected from the group consisting isopropyl, phenyl, and wherein R3 and R are simultaneously H or CH3, or alternatively R3 is H and R is selected from the group consisting of CH2OH and COOH;

with the proviso that the compound having formula (IA) is not:

BAR1107 BAR1125 CAS: 53241-60-4

Preferably, R2 is wherein R3 and R4 are simultaneously H or CH3 , or alternatively R3 is H and R4 is selected from the group consisting of CH2OH and COOH.

In an alternative embodiment of the compounds of formula (IA), n can vary between 4 and 6 and R2 is selected from the group consisting of CH ₂ 0H and COOH.

In a further embodiment, compounds are provided having formula

(IB) :

wherein m is 1-4, R5 and R6 are simultaneously H or CH3 or alternatively R5 is H and R6 is phenyl;

with the proviso that the compound having formula (I) is not:

BAR1 103 BAR1 1 ₄ 5

In particular, the compounds of the invention are selected from the group consisting of:

BAR1130 BAR1128 R1159

BAR1106

from the group consisting of:

According to a second embodiment of the invention, a pharmaceutical composition is provided comprising the compounds of formula I, as described previously.

The composition of the invention can be formulated in a pharmaceutical form which further contains at least one pharmacologically acceptable excipient, for example a carrier, a stabilizer or a diluent.

A person skilled in the art is familiar with a wide variety of said carriers, diluents or excipient compounds suitable for formulating a pharmaceutical composition.

The active ingredients of the composition of the invention, together with an adjuvant, a carrier, a diluent or an excipient used conventionally, can take the form of pharmaceutical compositions and unit doses thereof, and in said form can be used as solids, tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs or capsules filled with the same, all for oral use, or in the form of injectable sterile solutions for parenteral administration (including subcutaneous and intravenous use) . Said pharmaceutical compositions and the unit dosage forms thereof can comprise ingredients in conventional proportions, with or without additional compounds or active ingredients, and said unit dosage forms can contain any suitable effective quantity of the active ingredient commensurate with the daily dosage range to be used.

The pharmaceutical compositions can be prepared in a manner well known in pharmaceutical technique and comprise at least one active compound. Generally, the compounds of this invention are administered in a pharmaceutically effective quantity. The quantity of the compound actually administered will be typically determined by a doctor, in the light of the relevant circumstances, including the condition to be treated, the administration route chosen, the effective compound administered, the age, weight and response of the individual patient, the gravity of the patient's symptoms and similar .

The compositions of the present invention can be administered via various routes, including oral, rectal, cutaneous or subcutaneous, intravenous, intramuscular, intranasal and pulmonary routes. The compositions for oral administration can take the form of bulk liquid solutions or suspensions or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term "unit dosage forms" refers to physically discrete units suitable as unit doses for humans and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, combined with a suitable pharmaceutical excipient. The typical unit dosage forms include pre-measured vials or syringes, pre-filled with the liquid compositions, or pills, tablets, capsules or similar in the case of solid compositions.

The liquid forms suitable for oral administration can include a suitable aqueous or non-aqueous carrier with buffers, suspension and dispersion agents, colourings, flavourings and similar. The solid forms can include, for example, any of the following ingredients, or compounds of similar nature: a ligand like microcrystalline cellulose, tragacanth or gelatin; an excipient like starch or lactose, a disintegrating agent like alginic acid, Primogel or maize starch; a lubricant like magnesium stearate; a slip agent like colloidal silicon dioxide; a sweetening agent like sucrose or saccharin; or a flavouring agent like peppermint, methyl salicylate or an orange flavouring.

Injectable compositions are typically based on injectable sterile saline solution or saline solution buffered with phosphate or other injectable carriers known in the art.

The pharmaceutical compositions can be in the form of tablets, pills, capsules, solutions, suspensions, emulsions, powders, suppositories and as sustained release formulations.

If desired, the tablets can be coated by means of standard aqueous or non-aqueous techniques. In some embodiments, said compositions and preparations can contain at least 0.1 percent of active compound. The percentage of active compound in these compositions can, naturally, be varied and can be expediently between approximately 1 percent and approximately 60 percent of the weight of the unit. The quantity of active compound in said therapeutically useful compositions is such that a therapeutically active dosage will be obtained. The active compounds can also be administered intranasall , for example liquid drops or spray.

The tablets, pills, capsules and similar can also contain a ligand like tragacanth, acacia, maize starch or gelatin; excipients like dicalcium phosphate; a disintegrating agent like maize starch, potato starch, alginic acid; a lubricant like magnesium stearate; and a sweetening agent like sucrose, lactose or saccharin. When a unit dosage form is a capsule, it can contain, in addition to the materials of the above type, a liquid carrier like a fatty oil. Various other materials can be present as coatings or to modify the physical form of the dosage unit. For example, the tablets can be coated with shellac, sugar or both. A syrup or elixir can contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl- propylparabens as preservatives, a colouring agent and a flavouring agent like cherry or orange flavouring. To avoid breakage during transit through the upper portion of the gastrointestinal tract, the composition can be a gastro-protected formulation.

The compositions for pulmonary administration include, without limitations, dry powder compositions of the active ingredients, and the powder of a suitable carrier and/or lubricant. The compositions for pulmonary administration can be inhaled by any suitable dry powder inhaler known to a person skilled in the art.

The compositions are administered according to a protocol and at a dosage sufficient to reduce the inflammation and pain in the patient. In some embodiments, in the pharmaceutical compositions of the present invention the active ingredient or the active ingredients are generally formulated in dosage units . The dosage unit can contain 0.1 to 1000 mg of an active ingredient per dosage unit for daily administration .

In some embodiments, the effective guantities for a specific formulation will depend on the gravity of the disease, disorder or condition, the previous treatment, the state of health of the individual and response to the drug. In some embodiments, the dose is in the range from 0.001% by weight to approximately 60% by weight of the formulation.

A third embodiment of the invention refers to the use of the compounds having formula (I) including

BAR1145 532 ₄ 1 -60-4

as a medicament .

Particularly preferred compounds for use as a medicament are: 14

More in particular, the compounds for use as a medicament are:

In particular, the medicament is suitable for modulating the activity of the FXR, LXR and TGR5/GPBAR1 receptors in a single or dual manner, more in particular the medicament is suitable for use in the prevention and/or in treatment of a disorder selected from the group consisting of gastrointestinal disorders, liver diseases, cardiovascular and vascular diseases, pulmonary and metabolic diseases, infectious diseases, cancer, renal disorders, inflammatory disorders including immune-mediated disorders, and neurological disorders .

In one embodiment, the immune-mediated inflammatory disorders include autoimmune disorders such as systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome , scleroderma also known as systemic sclerosis, spondylarthritis , vasculitis, sarcoidosis, Mediterranean fever, and other hereditary autoinflammatory diseases, polymyositis and dermatomyositis, Behcet's syndrome. In one embodiment, the infectious diseases are selected from the group of Acquired Immuno-Deficiency Syndrome (AIDS) and related disorders, virus B and virus C infections.

In one embodiment, neurological disorders include Alzheimer' s disease and other forms of dementia, Parkinson and other movement disorders, amyotrophic lateral sclerosis and other motor neuron disorders, multiple sclerosis and other demyelinating diseases, ischemic stroke, myasthenia and muscular dystrophy.

In one embodiment, the liver disorders include primary biliary cirrhosis (PBC) , cerebrotendinous xanthomatosis (CTX) , primary sclerosing cholangitis (PSC) , drug induced cholestasis, intrahepatic cholestasis of pregnancy, parenteral nutrition associated cholestasis, bacterial overgrowth or sepsis associated cholestasis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD) , nonalcoholic steatohepatitis (NASH) , liver transplant, congenital hepatic fibrosis, granulomatous liver disease, intra- or extrahepatic malignancy, Wilson's disease, hemochromatosis, and alpha 1- antitrypsin deficiency.

In one embodiment, the gastrointestinal disorders include inflammatory bowel disease (IBD) (including Crohn's disease, ulcerative colitis and undetermined colitis), irritable bowel syndrome (IBS), bacterial overgrowth, acute and chronic pancreatitis, malabsorption, post-radiation colitis, and microscopic colitis .

In one embodiment, the renal disorders include diabetic nephropathy, hypertensive nephropathy, chronic glomerulonephritis including chronic transplant glomerulonephritis, chronic tubule interstitial diseases and vascular disorders of the kidney.

In one embodiment, the cardiovascular diseases include atherosclerosis, arteriosclerosis, dyslipidemia, hypercholesterolemia, hypertriglyceridemia, hypertension also known as arterial hypertension, inflammatory heart diseases including myocarditis and endocarditis, ischemic heart disease, stable angina, unstable angina, myocardial infarction, cerebrovascular diseases including ischemic stroke.

In one embodiment, the vascular diseases include pulmonary heart disease such as pulmonary hypertension, peripheral artery disease (PAD) , also known as peripheral vascular disease (PVD) peripheral artery occlusive disease, and peripheral obliterative arteriopathy .

In one embodiment, pulmonary disorders include asthma, cystic fibrosis, obstructive respiratory diseases, interstitial lung disease including, but not limited to, primary or secondary pulmonary fibrosis .

In one embodiment, the metabolic disease is selected from the group of diseases comprising insulin resistance, metabolic syndrome, Type I and Type II diabetes, hypoglycaemia, disorders of the adrenal cortex including adrenal cortex insufficiency. Metabolic diseases also include obesity and conditions associated with bariatric surgery .

In one embodiment, cancer is selected from the group comprising liver cancer, bile duct cancers, esophageal cancer, pancreatic cancer, gastric cancer, colon-rectal cancer, breast cancer, ovarian cancer and the condition associated with chemotherapy resistance. According to a further aspect of the invention, compounds of formula IB are provided for use as selective LXRa agonists. In particular, said compounds of formula IB are selected from the group consisting of BAR1103, BAR1106, BAR1107, BAR1124 and BAR1125.

According to a further aspect of the invention, compounds of formula IA are provided for use as selective GPBAR1 agonists. In particular, said compounds of formula IA are selected from the group consisting ofBAR1115, BAR1126, BAR1131 and BAR1147.

According to a further aspect of the invention, compounds of formula IA or IB are provided for use as dual LXRa/GPBARl agonists. Particularly preferred is BAR1130.

Further characteristics of the present invention will result from the following description of some merely illustrative and non- limiting examples.

The following abbreviations are used in the attached examples.

Methylene chloride (CH2CI2) , methanol (MeOH) , p-toluenesulfonic acid (p-TsOH) , lithium borohydride (L1BH4) , tetrahydrofuran (THF) , dimethylsulfoxide (DMSO) , triethylamine (TEA) , sodium bicarbonate (NaHC03) , ethyl acetate (EtOAc) , sodium sulphate (Na2S0.j) , sodium hydrogen sulphate (NaHSCM) , sodium hydroxide (NaOH) , triethylamine (Et3 ) , sodium chloride (NaCl) , diethyl ether (Et20) , n-butyl lithium (n-BuLi), hydrochloric acid (HC1) , meta-chloroperbenzoic acid (MCPBA) , chloroform (CHCI3) , hydrogen (¾) , palladium hydroxide (Pd(0H)2), water (H2O) , pyridine (Pyr) , acetic anhydride (AC2O) , copper acetate (Cu (OAc) 2) , lead acetate (Pb (OAc) 4) , sodium methoxide (CH30Na) , diatomaceous earth (Celite) , formic acid (HCOOH) , perchloric acid (HCIO4) , sodium nitrite (NaNC ) , potassium hydroxide (KOH) , hour (h) , room temperature (rt), retention time (tr _< ) .

When not otherwise indicated, ¹ H NMR was recorded on Varian Inova 400 MHz, using CDCI3 as solvent, and ¹³ C NMR was recorded on Varian Inova 100 MHz, using CDCI3 as solvent.

Examples

Preparation of aldehyde 4

A four-step reaction sequence on HDCA, including preparation of lateral chain methyl ester, protection of alcoholic functions at C- 3 and C-6, reduction of the lateral chain methyl ester and subsequent Swern oxidation gave the protected aldehyde (4), as starting material for the preparation of BAR1103-BAR1107 , BAR1115, BA 1124-1126, and

a) p-TsOH, anhydrous MeOH, quantitative yield; b) 2 , 6-lut idine, t- butyldimethylsilyltrifluoromethanesulfonate, CH2CI2, 0°C, quantitative yield; c) LiBH ₄ , anhydrous MeOH, THF, 0°C, 56%; d) DMSO, oxalyl chloride, anhydrous TEA, CH2CI2, -78°C, quantitative yield. Steps a-d) Preparation of aldehyde 4

To a solution of HDCA (6 g, 15.3 mmol) in 50 mL of anhydrous methanol, p-toluenesulfonic acid (10 g, 58.1 mmol) was added at rt . After lh, the mixture was quenched by addition of a saturated solution of NaHC03 until neutrality and, after evaporation, the residue was extracted with EtOAc (3x100 mL) . The combined extracts were washed with a saline solution, dried on a2S04, and evaporated to give the methyl ester 1 in quantitative yield. 2,6-Lutidine (17.4 mL, 150 mmol) and tert- butyldimethylsilyltrifluoromethanesulfonate (10.3 mL, 45 mmol) were added at 0°C to a solution of 1 (6.1 g, 15 mmol) in 50 mL of CH2CI2. After 2h stirring at 0°C, the reaction was quenched by addition of aqueous NaHS04 (1M, 100 mL) . The layers were separated and the aqueous phase was extracted with CH2CI2 (3x100 mL) . The combined organic phases were washed with NaHSC , water, saturated aqueous NaHC03, saline solution and evaporated in vacuo to give protected methyl ester 2 in quantitative yield. To a solution of 2 (9.5 g, 15 mmol) in anhydrous THF (50 mL) and anhydrous methanol (1.82 mL, 45 mmol), LiBEU (22.5 mL, 2M in THF, 45 mmol) was added at 0°C. After stirring for 2h at 0°C, the mixture was quenched by addition of NaOH 1M and then EtOAc. The organic phase was washed with water, dried ( a2S0 ) and concentrated. Purification on silica gel (hexane/EtOAc 9:1 and 0.5% TEA) gave alcohol 3 in 56% yield (5 g, 8.3 mmol) . DMSO (6.6 mL, 92 mmol) was added dropwise for 15 min to a solution of oxalyl chloride (23.0 mL, 46 mmol) in anhydrous CH2CI2 (50 mL) at -78°C under argon atmosphere. After 30 min, a solution of alcohol 3 (4 g, 6.6 mmol) in anhydrous CH2CI2 was added via cannula and the mixture was stirred at -78°C for 30 min. Et3 (13.8 mL, 99 mmol) was added dropwise followed by saturated solution of NaCl . The aqueous phase was extracted with Et20 (3x100 mL) . The combined organic phase was washed with water, dried (Na2S04) and concentrated to give aldehyde 4 (4 g) as a colourless oil in quantitative yield.

EXAMPLE 1A. Synthesis of BAR1103, BAR1107, BAR1115 and BAR1128

Wittig olefination on aldehyde 4 with isopropyl triphenylphosphonium iodide gave the protected intermediate 5. Removal of the protective groups at C3 and C6 gave BAR1103, which was used as starting material both for the hydrogenation (BAR1107) and for the epoxidation (BAR1115) . Wittig olefination with isobutyl triphenylphosphonium iodide followed by deprotection and hydrogenat ion on lateral chain dou

a) n-BuLi, isopropyl triphenylphosphonium iodide, anhydrous THF, r.t., 84%; b) HC1 37%, MeOH, quantitative yield; c) MCPBA, anhydrous CHC1 ₃ , 87%; d) H ₂ , Pd(0H) ₂ , anhydrous TH : MeOH 1:1, quantitative yield; e) n-BuLi, isobutyl triphenylphosphonium iodide, anhydrous THF, r.t.; f) HC1 37%, MeOH; g) H ₂ , Pd(0H) ₂ , anhydrous THF : MeOH 1:1. Steps a,b) . Preparation of BAR1103

To a solution of isopropyl triphenylphosphonium iodide (5.4 g, 12.5 mmol) in THF (2 mL) , n-BuLi (5 mL, 12.5 mmol) was added dropwise at rt until the solution turned a red colour. After 30 min, a solution of aldehyde 4 (1.5 g, 2.5 mmol) in THF (5 mL) was added. After lh, the mixture was quenched by addition of a saturated aqueous solution of NaHC03 (50 mL) and extracted with EtOAc (3x50 mL) . The organic phase was dried (Na ₂ S04) and concentrated. Purification on silica gel (hexane) gave 5 in 84% yield.

To a solution of 5 (1.3 g, 2.1 mmol) in MeOH, 1 mL of HC1 37% v/v was added. After lh, silver carbonate was added, the reaction mixture was centrifuged, and the supernatant was concentrated in vacuo to give BAR1103 as a colourless amorphous solid (850 mg, quantitative yield) . An analytic sample was purified by HPLC on a Nucleodur 100-5 C18 (5 m; 10 mm i.d. x 250 mm) with MeOH/H ₂ 0 (96:4) as eluent (flow rate 3 mL/min, tR= 12 min) .

δ _Η 5.10 (1H, t, J=6.7 Hz, H-24), 4.08(1H, dt, J=11.9, 4.6 Hz, Η-6β), 3.64(1H, m, Η-3β), 1.68 (3H, s, Me-26), 1.60 (3H, s, Me-27), 0.92 (3H, d, J=7.0 Hz, Me-21), 0.91(3H, s, Me-19), 0.65(3H, s, H3-I8); 5c 130.9, 125.3, 71.6, 68.2, 56.2 (x2C) , 48.4, 42.9, 40.0, 39.9, 36.4, 36.2, 35.6, 35.3, 35.0, 34.9, 30.3, 29.3, 28.4, 25.7, 24.7, 24.3, 23.3, 20.9, 18.4, 17.7, 12.1.

Step c) . Preparation of BAR1115

To a solution of BAR1103 (500 mg, 1.24 mmol) in anhydrous CHCI3, meta-chloroperbenzoic acid (643 mg, 3.73 mmol) was added at rt . After over-night stirring, the mixture was extracted with an aqueous solution of Na ₂ S0 ₃ (5%) and CH2CI2 (3x50 mL) to give BAR1115 (450 mg, 87%)) . An analytic sample was purified by HPLC on a Phenomenex Luna C18 (5 μπι; 4.6 mm i.d. x 250 mm) with MeOH/H ₂ 0 (85:15) as eluent (flow rate 1 mL/min, tR = 15.0 min) .

The configuration at C24 as S was determined by comparison with the ¹³ C-NMR data reported in literature for the compound 24 (S) , 25- epoxycholesterol ( J. Org. Chem. 1998, 63, 9919) .

BAR1115: C2 H ₄₆ 0 ₃

δ _Η .06(1Η, dt, J=12.0, 4.6 Hz, Η-6β), 3.62 (1H, m, Η-3β), 2.66 (1H, t, J=6.0 Hz, H-24), 0.91 (3H, d, J=7.0 Hz, Me-21), 0.89 (3H, s, Me- 19), 0.65 (3H, s, Me-18) . 5c 71.6, 68.1, 64.7, 58.4, 56.1 (x2C) , 48.5, 42.9, 40.0, 39.8, 35.7, 35.6, 35.2, 35.0, 34.9, 32.5, 30.5, 29.3, 28.5, 24.4, 25.7, 24.9, 23.4, 20.9, 18.6 (x2C) , 11.9.

Step d) . Preparation of BAR1107

A solution of BAR1103 (350 mg, 0.87 mmol) in anhydrous

THF/anhydrous MeOH (5 mL/5 mL, v/v) was hydrogenated in the presence of Pd(0H)2 5% wt on activated carbon. After 12h, the catalyst was filtered through Celite, and the recovered filtrate was concentrated under vacuum to give a colourless amorphous solid (350 mg, quantitative yield) . An analytic sample was purified by HPLC on a Nucleodur 100-5 C18 (5 urn; 10 mm i.d. x 250 mm) with MeOH/H ₂ 0 (95:5) as eluent (flow rate 3 mL/min, tR= 8 min) .

BAR1107: C27H48O2

δ _Η 4.06 (1H, dt, J= 11.9, 4.6 Hz, Η-6β), 3.62 (1H, m, Η-3β), 0.91 (3H, s, Me-19), 0.90 (3H, d, J= 7.0 Hz, Me-21), 0.87 (3H, d, J= 6.6 Hz, Me-26), 0.86 (3H, d, J= 6.6 Hz, Me-27), 0.64 (3H, s, Me-18) . 5c 71.6, 68.2, 56.3, 56.2, 48.4, 42.9, 40.0, 39.9, 39.4, 36.4, 36.0, 35.9, 35.6, 35.5, 34.9, 30.4, 29.3, 28.4, 28.2, 22.7 (x2C) , 24.4, 23.9, 23.3, 20.9, 18.8, 12.1.

Steps e-g) . Preparation of BAR1128

BAR1128 was prepared from aldehyde 4 (1 g, 1.65 mmol), in the same operative conditions described in steps a, b) and d) , example 1A using isobutyl triphenylphosphonium iodide in step a. HPLC purification on Nucleodur 100-5 C18 (5 μπι; 4.6 mm i.d. x 250 mm) with MeOH/H ₂ 0 (96:4) as eluent (flow 1 mL/min) gave BAR1128 (t _R =16 min) .

BAR1128: C28H50O2 ¹ E NMR was recorded on Varian Inova 400 MHz, in CD3OD as solvent: 6H 4.01 (1H, dt, J=11.9, 4.5 Hz, Η-6β), 3.50 (1H, m, Η-3β), 0.87 (6H, d, J=6.7 Hz, Me-27 and Me-28), 0.93 (3H, d, J=6.6 Hz, Me-21), 0.92 (3H, s, Me-19), 0.68 (3H, s, Me-18) .

5 _C 71.6, 68.1, 56.2 (x2C) , 48.4, 42.8, 39.9, 39.8, 39.1, 36.0, 35.9, 35.7, 35.5, 35.1, 34.8, 30.3, 29.1, 28.2, 28.0, 27.9, 26.3, 24.2, 23.7, 23.6, 23.5, 20.7, 18.6, 12.0.

EXAMPLE IB. Synthesis of BAR1124, BAR1125, and BAR1126

Wittig olefination on aldehyde 4 with methyl triphenylphosphonium iodide gave the protected intermediate 6. Removal of the protecting groups at C3 and C6 gave BAR1124, which was used as starting material in the double bond hydrogenation reaction and in the epoxidation to give BAR1125 and BAR1126 respectivel .

BAR1126

a) i-BuLi, triphenylphosphonium iodide, anhydrous THF, rt, 60%; b) HC1 37%, MeOH, quantitative yield; c) H ₂ , Pd(OH) ₂ , anhydrous THF : MeOH 1:1, 70%; d) MCPBA, anhydrous CHCI3, quantitative yield.

Steps a, b) . Synthesis of BAR1124 BAR1124 (370 mg, 0.99 mmol) was prepared from aldehyde 4 (1 g, 1.65 mmol, 60% yield) in the same operative conditions described in steps a) and b) example 1A using methyl triphenylphosphonium iodide in step a. An analytic sample was purified by HPLC on a Nucleodur 100-5 C18 (5 μπι; 10 mm i.d. x 250 mm) with MeOH/H ₂ 0 (96 : 4) as eluent (flow 3 mL/min, t _R =ll min) .

BAR1124: C ₂₅ H ₄₂ 0 ₂

δ _Η 5.80 (1H, m, H-24), 4.99 (1H, d, J=17.1 Hz, H-25), 4.91 (1H, d, J=9.8 Hz, H-25), 4.05 (1H, dt, J=12.0, 4.5 Hz, Η-6β), 3.62 (1H, m, Η-3β) , 0.91 (3H, s, Me-19), 0.92 (3H, d, J=7.0 Hz, Me-21), 0.64 (3H, s, Me-18) .

5c 139.6, 113.9, 71.6, 68.1, 56.2, 56.1, 48.4, 42.8, 39.9, 39.8, 36.0, 35.5, 35.3, 35.2, 35.1, 34.8, 30.5, 30.2, 29.2, 28.2, 24.2, 23.5, 20.7, 18.4, 12.0.

Step c) . Synthesis of BAR1125

Reduction on BAR1124 (125 mg, 0.33 mmol) in the same operative conditions described in step d) example 1A followed by purification by HPLC on Nucleodur 100-5 C18 (5 μπι; 4.6 mm i.d. x 250 mm) with MeOH/H ₂ 0 (90 : 10) as eluent (flow 1 mL/min) gave 87 mg of BAR1125 (t _R =18.6 min, 70%) .

BAR1125: C ₂₅ H ₄₄ 0 ₂

¹ H NMR was recorded on Varian Inova 400 MHz, in CD3OD as solvent: 5H 3.99 (1H, dt, J=12.2, 4.6 Hz, Η-6β), 3.50 (1H, m, Η-3β), 0.92 (3H, s, Me-19), 0.93 (3H, d, J=6.8 Hz, Me-21), 0.90 (t, J=6.8 Hz, Me-25), 0.68 (3H, s, Me-18) .

5c 71.6, 68.1, 56.2, 56.1, 48.4, 42.8, 39.9, 39.8, 35.9, 35.7, 35.5, 35.2, 35.0, 34.8, 30.2, 29.2, 28.3, 28.2, 24.2, 23.5, 23.1, 20.7, 18.6, 14.2, 12.0.

Step d) . Synthesis of BAR1126

Epoxidation on BAR1124 (125 mg, 0.33 mmol) in the same operative conditions described in step c) example 1A gave BA 1126 as a mixture of the two diastereoisomers at C24 on the lateral chain in quantitative yield.

BAR1126: C25H42O3

δ _Η .07 (1H, dt, J=11.9, 4.4 Hz, Η-6β), 3.63 (1H, m, Η-3β), 2.88 (1H, m, H-24), 2.75(1H, m, H-25) , 2.47 (1H, m, H-25) , 0.93 (3H, d, J=6.7 Hz, Me-21), 0.91 (3H, s, Me-19), 0.64 (3H, s, Me-18) .

5c 71.6, 68.1, 52.9, 52.7, 56.0 (x2C) , 48.5, 42.9, 39.9, 39.8, 36.0, 35.5 (x2C) , 35.1, 34.9, 32.0, 30.3, 29.3, 29.1, 28.2, 24.2, 23.5,

20.7, 18.7, 12.1.

EXAMPLE 1C. Synthesis of BAR1104, BAR1105 and BAR1106.

Wittig olefination on aldehyde 4 with benzyl triphenylphosphonium iodide and deprotection of the hydroxyls led to the formation of 7 as a mixture of the two diastereoisomers at the lateral chain double bond. HPLC purification gave BAR1104 and BAR1105. Hydrogenation on the mixture gave BAR1106.

a) Ώ-BuLi, benzyl triphenylphosphonium iodide, anhydrous THF; b) HC1 37%, MeOH, 67% over two steps; c) H2 , Pd(OH) 2 degussa type, anhydrous THF : MeOH 1:1, quantitative yield.

Steps a, b) . Synthesis of BAR1104 and BAR1105

Compound 7 (125 mg, 0.28 mmol, 68% over two steps) was prepared as a mixture of diastereoisomers at the lateral chain double bond, from aldehyde 4 (250 mg, 0.41 mmol) in the same operative conditions described in steps a) and b) example 1A using benzyl triphenylphosphonium iodide in step a) . An aliquot was purified by HPLC on Nucleodur 100-5 C18 (5 μπι ; 10 mm i.d. x 250 mm) with MeOH/H ₂ 0 (96:4) as eluent (flow 3 mL/rain) to give BAR1104 (tR=13.6 min) and BAR1105 (t _R =16.2min) .

BAR1104: C31H46O2

δ _Η 7.33-7.21 (5H, Ph) , 6.39 (1H, d, J=11.7 Hz, H-25) , 5.64 (1H, dt, J=11.7, 7.3 Hz, H-24), 4.05 (1H, dt, J=12.0, 4.5 Hz, Η-6β), 3.62 (1H, m, Η-3β) , 2.38 (1H, m, H-23) , 2.22 (1H, m, H-23) , 0.91 (3H, s, Me-19), 0.89 (3H, d, J=6.8 Hz, Me-21), 0.64 (3H, s, Me-18) .

5c 137.8, 133.6, 128.7, 128.6, 128.2, 126.4, 71.6, 68.2, 56.2 (x2C) , 48.4, 42.9, 40.0, 39.9, 36.4, 36.2, 35.6, 35.5, 35.1 (x2C) , 30.4, 29.3, 28.4, 25.5, 24.4, 23.3, 20.9, 18.5, 12.1.

BAR1105: C31H46O2

δ _Η 7.33-7.18 (5H, Ph) , 6.38 (1H, d, J=15.7 Hz, H-25), 6.20 (1H, dt, J=15.7, 6.8 Hz, H-24), 4.05 (1H, dt, J=12.1, 4.5 Hz, Η-6β), 3.62 (1H, m, Η-3β) , 2.28 (1H, m, H-23), 2.10 (1H, m, H-23), 0.96 (3H, d, J=6.4 Hz, Me-21), 0.91 (3H, s, Me-19), 0.65 (3H, s, Me-18) .

5c 138.0, 131.5, 129.3, 128.4, 126.8, 125.9, 71.6, 68.2, 56.2 (x2C) , 48.4, 42.9, 40.0, 39.9, 36.4, 35.8, 35.6, 35.5, 35.2, 35.1, 30.4, 29.8, 29.3, 28.4, 24.4, 23.4, 20.9, 18.6, 12.1.

Step c) . Synthesis of BAR1106

Hydrogenation on 7 (40 mg, 0.09 mmol) in the same operative conditions described in step d) example 1A gave BAR1106 in quantitative yield. An analytic sample was purified by HPLC on Nucleodur 100-5 C18 (5 μπι; 4.6 mm i.d. x 250 mm) with MeOH/H ₂ 0 (96 : 4) as eluent (flow 1 mL/min, t _R = .6 min) .

BAR1106: C31H48O2

δ _Η 7.27-7.16 (5H, Ph) , 4.05 (1H, dt , J=11.9, 4.5 Hz, Η-6β) , 3.62 (1H, m, Η-3β) , 2.60 (2H, m, H-25) , 0.90 (3H, s, Me-19), 0.89 (3H, d, J=6.8 Hz, Me-21), 0.64 (3H, s, Me-18) .

5c 143.0, 128.4, 128.3, 125.6, 71.6, 68.2, 56.2 (x2C) , 48.4, 42.9, 40.0, 39.9, 36.3, 36.1, 35.7, 35.6, 35.5, 35.2 (x2C) , 32.0, 30.4, 29.3, 28.4, 26.0, 24.4, 23.4, 20.9, 18.6, 12.1.

EXAMPLE 2. Synthesis of BAR1129-BAR1131 , and BAR1145-BAR1147

Beckmann degradation of one carbon at C24 on HDCA, as reported in J. Med. Chem. 2014, 57, 937-954, followed by transformation of the nitrile group at C23 in the corresponding methyl ester gave the intermediate 9 as starting material in the synthesis of BAR1129— BAR1131 and BAR1145-BAR1147.

a) HCOOH, HC10 ₄ ; b) TFA, trifluoroacet ic anhydride, NaN0 ₂ , quantitative yield over two steps; c) KOH 30%, ethylene glycol; d) p-TSOH, anhydrous MeOH, quantitative yield over two steps; e) 2,6- lutidine, t-butyldimethylsilyltrifluoromethanesulfonate, CH2CI2, 0°C; f) L1BH4, anhydrous MeOH in anhydrous THF, quantitative yield over two steps; g) DMSO, oxalyl chloride, anhydrous TEA, CH2CI2, — 78°C, 94% over three steps; h) n-BuLi, isopropyl triphenylphosphonium iodide, anhydrous THF; i) HC1 37%, MeOH, 80% over two steps; j) H ₂ , Pd(OH) ₂ /C degussa type, THF/MeOH 1:1, 90%; k) MCPBA, anhydrous CHCI3, 80%; 1) n-BuLi, methyl triphenylphosphonium iodide, anhydrous THF, rt ; m) HC1 37%, MeOH, 95% over two steps; n) H ₂ , Pd(0H) ₂ , anhydrous THF : MeOH 1:1, 88%; o) MCPBA, anhydrous CHCI3, 96% .

Steps a, b) . Preparation of 8

Intermediate 8 was prepared from HDCA (2 g, 5 mmol) in quantitative yield as described in J. Med. Chem. 2014, 57, 937-954. Steps c-f) . Preparation of 10

The raw compound 8 (2 g, 5 mmol) was refluxed in ethylene glycol (50 mL) and KOH (30%) for 24h. H2O was added and the resulting solution was neutralized with HC1 6N. The residue was extracted with dichloromethane (3x50 mL) . The combined organic layers were washed with a saline solution, dried and concentrated to give the corresponding de-formylated carboxylic acid.

Said compound was transformed into the methyl ester 9 (1.9 g, quantitative yield) in the same operative conditions described in step a) of preparation of the aldehyde 4.

The compound 9 (1.9 g, 4.8 mmol) was transformed into the corresponding protected methyl ester (2.85 g, 4.6 mmol, quantitative yield) in the same operative conditions described in step b) of preparation of the aldehyde 4.

To a solution of the above intermediate (2.85 g, 4.6 mmol) in anhydrous THF (50 mL) and anhydrous MeOH (1.3 mL, 32 mmol), L1BH4 (16 mL, 2M in THF, 32 mmol) was added at 0°C. Treatment as described in step c) of preparation of the aldehyde 4 gave alcohol 10 in quantitative yield (2.66 g, 4.5 mmol) .

Step g) . Preparation of aldehyde 11.

Aldehyde 11 (1 g, 1.7 mmol, 94%) was prepared from 10 (1 g, 1.8 mmol) in the same operative conditions described in step d) for the preparation of aldehyde 4.

Steps h) , i) . Preparation of BAR1129

Wittig olefination on aldehyde 11 (500 mg, 0.85 mmol) was performed with the same synthesis protocol described in step a) , example 1A. Treatment of intermediate 12 in the same operative conditions described in step b) , example 1A, gave BAR1129 (264 mg, 0.7 mmol, 80% over two steps) . An analytic sample was purified by HPLC on Nucleodur 100-5 C18 (5 μιη; 10 mm i.d. x 250 mm) with MeOH/H ₂ 0 (96:4) as eluent (flow 3 mL/min, t _R =16 min) .

BAR1129: C26H ₄₄ 0 ₂

¾ NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: δ _Η 5.12 (1H, brt, J=6.8 Hz, H-23) , 4.01(1H, dt, J=11.9, 4.6 Hz, H- 6β) , 3.50 (1H, m, Η-3β), 1.70 (3H, s, Me-26), 1.59 (3H, s, Me-25), 0.93 (3H, s, Me-19), 0.90(3H, d, J=6.7 Hz, Me-21), 0.69 (3H, s, Me- 18) .

¹³ C NMR was recorded on Varian Inova 100 MHz, using CD30D as solvent: 5c 132.8, 124.2, 72.4, 68.6, 57.6 (x2C) , 49.9, 44.1, 41.3 (x2C) ,

38.2, 37.0, 36.8, 36.2, 35.6, 35.5, 31.1, 29.9, 29.5, 26.1, 25.5, 24.1, 21.9, 19.2, 18.0, 12.4.

Step j) . Preparation of BAR1130

BAR1129 (100 mg, 0.3 mmol) was hydrogenated in the same operative conditions described in step d) , example 1A. HPLC purification on Nucleodur 100-5 C18, (5 μπι; 10 mm i.d. x 250 mm) with MeOH/H ₂ 0 (96:4) as eluent (flow 3 mL/min) gave BAR1130 (t _R =18.4 min) in 90% yield (90 mg, 0.2 mmol) .

BAR1130: C26H46O2

¹ H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: δ _Η 4.01 (1H, dt, J=12.1, 4.6 Hz, Η-6β), 3.51 (1H, m, Η-3β), 0.94 (3H, s, Me-19), 0.93(3H, d, J=6.5Hz, Me-21), 0.89 (3H, d, J=6.0 Hz, Me- 25), 0.87(3H, d, J=6.0 Hz, Me-26), 0.68 (3H, s, Me-18) .

5c 71.6, 68.1, 56.2 (x2C) , 48.4, 42.8, 39.9, 39.8, 35.9, 35.8, 35.5,

35.3, 35.1, 34.8, 33.5, 30.3, 29.2, 28.4, 28.2, 24.2, 23.5, 23.0, 22.4, 20.7, 18.7, 12.0.

Step k) . Preparation of BAR1131

Epoxidation on BAR1129 (100 mg, 0.3 mmol) in the same operative conditions described in step c) , example 1A, gave BAR1131 (97 mg, 0.2 mmol, 80%) . HPLC purification on Nucleodur 100-5 C18, (5 μπι; 10 mm i.d. x 250 mm) with MeOH/H ₂ 0 (90:10) as eluent (flow 3 mL/min) gave pure BAR1131 (tR=10.4 min) . The configuration at C23 as S was determined by comparison with the ¹³ C-NMR data reported in literature for the compound 24 (S) , 25-epoxycholesterol (J ^" . Org. Chem. 1998, 63, 9919) .

BAR1131: C26H44O3

¹ H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: δ _Η 4.01 (1H, dt, J=12.0, 4.6 Hz, Η-6β), 3.50 (1H, m, Η-3β), 2.80 (1H, dd, J=6.5, 4.6 Hz, H-23), 1.29(3H, s, Me-25), 1.25(3H, s, Me-26), 1.07 (3H, d, J=6.4 Hz, Me-21), 0.93 (3H, s, Me-19), 0.72 (3H, s, Me- 18) .

5c 71.6, 68.1, 63.4, 58.7, 56.2, 56.1, 48.4, 42.9, 39.9, 39.8, 36.0,

35.5, 35.4, 35.3, 35.1, 34.8, 30.3, 29.2, 28.4, 24.2, 23.5, 24.8, 20.7, 19.2, 18.9, 11.9.

Steps 1), m) . Preparation of BAR1145

Wittig olefination on aldehyde 11 (500 mg, 0.85 mmol) followed by acidic hydrolysis with the same synthesis protocol described in steps a) and b) , example IB, gave BAR1145 in 95% yield over two steps (290 mg, 0.8 mmol) . An analytic sample was purified by HPLC on Nucleodur 100-5 C18 (5 μπι; 10 mm i.d. x 250 mm) with MeOH/H ₂ 0 (96 : 4) as eluent (flow 3 mL/min, tR=ll min) .

BAR1145 : C24H40O2 δ _Η 5.77 (1H, m, H-23), 5.01 (1H, ovl, H-24), 4.99 (1H, ovl, H-24), 4.07 (1H, dt, J=12.1, 4.6 Hz, Η-6β), 3.63 (1H, m, Η-3β), 0.92 (3H, d, J=6.6 Hz,Me-21), 0.91 (3H, s, Me-19), 0.66 (3H, s, Me-18) .

5c 137.3, 115.7, 71.6, 68.1, 56.1, 55.8, 48.4, 42.8, 40.5, 39.9, 39.8, 35.9, 35.8, 35.5, 35.1, 34.8, 30.3, 29.2, 28.2, 24.2, 23.5, 20.7, 18.6, 12.0.

Step n) . Preparation of BAR1146

BAR1145 (100 mg, 0.3 mmol) reduction in the same operative conditions described in step c) , example IB, followed by HPLC purification on Nucleodur 100-5 C18 (5 μπι; 4.6 mm i.d. x 250 mm) with MeOH/H ₂ 0 (96:4) as eluent (flow 1 mL/min) , gave 88 mg (0.2 mmol, 88%) of BAR1146 (t _R =8 min) .

BAR1146: C24 H 42 O2

δ _Η 4.06 (1H, dt, J=12.1, 4.6 Hz, Η-6β), 3.62 (1H, m, Η-3β), 0.91 (3H, s, Me-19), 0.89 (3H, d, J=7.0 Hz, Me-21), 0.86 (3H, t, J=7.6 Hz, Me- 23) , 0.64 (3H, s, Me-18) .

5c 71.6, 68.2, 56.0, 55.9, 48.4, 42.7, 39.9, 39.8, 38.2, 36.4, 35.5, 35.4, 35.1, 34.8, 30.2, 29.3, 28.2, 24.3, 23.5, 20.8, 19.2, 18.6, 12.0, 14.5.

Step o) . Preparation of BAR1147

Epoxidation on BAR11 5 (100 mg, 0.3 mmol) in the same operative conditions described in step d) , example IB, gave BAR1147 (108 mg, 0.3 mmol, 96%) . HPLC purification on Nucleodur 100-5 C18 (5 μπι; 4.6 mm i.d. x 250 mm) with MeOH/H ₂ 0 (80:20) as eluent (flow 1 mL/min,

BAR1147: C24 H 40 O3

δ _Η 4.07 (1H, dt, J=12.0, 4.4 Hz, Η-6β), 3.64 (1H, m, Η-3β), 2.95 (1H, m, H-23), 2.80 (1H, m, H-24), 2.48 (1H, m, H-24), 1.04 (3H, d, J=6.7 Hz, Me-21), 0.92 (3H, s, Me-19), 0.68 (3H, s, Me-18) .

5c 71.6, 68.0, 53.2, 56.3, 56.1, 50.7, 48.4, 43.0, 39.8, 39.7, 38.5, 36.0, 35.5, 35.1, 34.9, 33.6, 30.2, 29.2, 28.2, 24.2, 23.4, 20.7, 19.2, 12.1.

EXAMPLE 3. Synthesis of BAR1132 and BAR1133

One pot Swern oxidation/Wittig C2 homologation on alcohol 3 performed as described in J. Med. Chem. 2014, 57, 7687-7701 generated the intermediate 13, the protected methyl ester of A ²⁴ ' ²⁵ -bis- homoEOCA . Lateral chain double bond hydrogenation and alcoholic function deprotection gave bis- omoHDCA methyl ester 14, which was used as starting material in the synthesis of BAR1132 and BAR1133.

a) DMSO, oxalyl chloride, anhydrous TEA, CH2CI2, -78°C then methyl (triphenylphosphoranylidene ) acetate ; b) ¾, Pd(OH)2/C degussa type, THF/EtOH 1:1; c) HC1 37%, MeOH, 56% yield over three steps; d) NaOH 5% in MeOH/H ₂ 0 1:1 v/v, 76%; e) LiBH ₄ , anhydrous MeOH, THF, 0°C, 57%.

Steps a-c) . Preparation of the methyl ester 14

Swern oxidation and ittig olefination on alcohol 3 (1 g, 1.6 mmol) followed by deprotection in acid condition and reduction of Δ ²⁴ _' ²⁵ double bond as detailed in J. Med. Chem.2014, 57, 7687-7701 provided 400 mg of ester 13 (56% yield) .

Steps d) , e) . Preparation of BAR1132 and BAR1133

Methyl ester hydrolysis or alternatively reduction by treatment with LIBH ₄ provided BAR1132 (147 mg, 0.35 mmol, 76%) and BAR1133 (106 mg, 0.26 mmol, 57%), respectively.

An analytic sample of BAR1132 was purified on Nucleodur 100-5 C18 (5 μπι; 10 mm i.d. x 250 mm) with MeOH/H ₂ 0 (88:12) as eluent (flow 3 mL/min, tR = 11.8 min) . An analytic sample of BAR1133 was purified on Nucleodur 100-5 C18 (5 urn; 4.6 mm i.d. x 250 mm) with MeOH/H ₂ 0 (80:20) as eluent (flow 1 mL/min, tR=22.6 min) .

BAR1132: C26H44O4

¹ H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: δ _Η 4.01 (1H, dt, J=12.0, 4.5 Hz, Η-6β), 3.51 (1H, m, Η-3β), 0.94 (3H, d, J=6.6 Hz, Me-21), 0.93 (3H, s, Me-19), 0.69 (3H, s, Me-18) .

1 ³ C NMR was recorded on Varian Inova 400 MHz, using CDsODas solvent: 5 _C 177.8, 72.4, 68.7, 57.7, 57.6, 49.9, 44.1, 41.4, 41.3, 37.1, 36.9, 36.8, 36.7, 36.2, 35.6, 34.1, 31.1, 30.0, 29.3, 26.7, 26.6, 25.3,

24.0, 21.9, 19.1, 12.4.

BAR1133: C26H46O3

1H NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: δ _Η 4.01 (1H, dt, J=12.0, 4.6 Hz, Η-6β), 3.54 (2H, t, J=6.6 Hz, H ₂ - 25), 3.51 (1H, m, Η-3β), 0.94 (3H, d, J=6.6 Hz, Me-21), 0.93 (3H, s, Me-19), 0.69 (3H, s, Me-18) .

¹³ C NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: 5c 72.4, 68.6, 63.0, 57.7, 57.6, 50.0, 44.0, 41.4, 41.3, 37.1, 36.9, 36.8 (x2C) , 36.2, 35.6, 33.7, 31.1, 29.9, 29.3, 27.4, 27.0, 25.3,

24.1, 21.9, 19.2, 12.5. EXAMPLE 4. Synthesis of BAR1167a and BAR1167b

The allylic alcohol 15 was obtained from compound 13, the protected methyl ester of Δ ²⁴ _' ²⁵ -bis- omoHDCA . Enantioselective Sharpless epoxidation on compound 15 followed by deprotection and HPLC purification gave BAR1167a and BAR1167b.

a) DIBAL-H, anhydrous THF, -78°C, 55% yield; b) D- (-) -DIPT, Ti(0iPr) ₄ , TBHP, MS, -20°C, 90%; c) L- ( +) -DIPT, Ti (OiPr) ₄ , TBHP, MS, -20°C, 85%; d) TBAF, anhydrous THF, quantitative yield.

Step a) Preparation of compound 15. To a solution of compound 13 (1.0 g, 1.5 mmol) in anhydrous THF at -78°C, 2.0 mL of DIBAL-H (1.5 M in THF, 3.0 mmol) were added dropwise. After 3h (TLC monitoring), the reaction was quenched for 2h with saturated potassium sodium tartrate tetrahydrate (25 mL) and the mixture was extracted with CH2CI2 (50 mL x 3) . Organic layer was dried over a2S04, concentrated in vacuo and purified on Si02 chromatography (hexane/EtOAc, 99:1) affording pure compound 15 (526 mg, 55%) .

Step b, d) Preparation of BAR1167a

To a 50 mL round bottom flask under argon were added molecular sieves (4 A, 1.2 mg) in anhydrous CH ₂ C1 ₂ (5 mL) . Then Ti (Oi-Pr) ₄ (58 μL, 0.2 mmol) and D- (-) -DIPT (50 μΐ,, 0.24 mmol) were added at -20°C. The solution was allowed to stir for 5 min, then compound 15 (0.25 g, 0.39 mmol) and TBHP (1.4 mL of a 5.5 M solution in decane, 8 mmol) were added. After 5h, a solution of tartaric acid (36 mg, 0.24 mmol) and FeS04 (133 mg, 0.48 mmol) in 20 mL of water were added and the mixture was stirred at 0°C. After 10 min, the cooling bath was removed and after separation of the aqueous layer, the organic layer was washed once with water, dried over Na2S04 and concentrated. The resulting oil was diluted with Et2<D (50 mL) and cooled in an ice bath, and then NaOH (2 mL of 30% solution in brine) was added; the mixture was stirred at 0°C for 1 h, and then the ether phase was washed with brine, dried over Na2S0 and concentrated. S1O2 chromatography (hexane/EtOAc, 95:5) afforded 230 mg of pure compound (90%) . The deprotection of TBS group with TBAF afforded the corresponding alcohol. An analytic sample of BAR1167a was purified on Phenomenex 100-5 C18 (5 μπι; 4.6 mm i.d. x 250 mm) with MeOH/H ₂ 0

(80:20) as eluent (flow 1 mL/min, tR = 8 min) .

BAR1167a: C26H44O4

1E NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: δ _Η 4.07 (1H, dt, J =12.0, 4.5 Hz, Η-6β), 3.92 (1H, m, H-25), 3.59 (2H, m, Η-3β, H-24), 2.93 (2H, m, H ₂ -26), 0.94 (3H, d, ovl, Me-21), 0.91 (3H, s, Me-19), 0.64 (3H, s, Me-18).

¹³ C NMR was recorded on Varian Inova 400 MHz, using CDCI3 as solvent: 5c.71.6, 68.0, 61.7, 58.5, 56.1, 56.0 (2C) , 48.4, 42.9, 39.9, 39.8, 35.9, 35.5, 35.4, 35.0, 34.8, 31.7, 30.3, 29.1, 28.2, 28.1, 24.1,

23.4, 20.8, 18.5, 12.2.

Step c, d) Preparation of BAR1167b

Sharpless epoxidation using L-( + )-DIPT (instead of D- (-) -DIPT) and TBAF deprotection on compound 15 (250 mg, 0.4 mmol) in the same operative conditions described in step b) , example 4, gave BAR1167b (142 mg, 0.34 mmol, 85%) . HPLC purification was performed on Phenomenex 100-5 C18 (5 im; 4.6 mm i.d. x 250 mm) with MeOH/H ₂ 0

(80:20) as eluent (flow 1 mL/min, tR = 12 min)

BAR1167b: C26H44O4

¹ E NMR was recorded on Varian Inova 400 MHz, using CD3OD as solvent: δ _Η 4.07 (1H, dt, J =12.0, 4.5 Hz, Η-6β), 3.91 (1H, m, H-25) , 3.59

(2H, m, Η-3β, H-24) , 2.93 (2H, m, H ₂ -26) , 0.92 (3H, d, ovl, Me-21) ,

0.91 (3H, s, Me-19) , 0.65 (3H, s, Me-18) .

5 _C 71.6, 68.0, 61.7, 58.3, 56.4, 56.1, 56.0, 48.4, 42.9, 39.9, 39.8,

35.9, 35.4, 35.3, 35.0, 34.8, 32.1, 30.3, 29.1, 28.3, 28.1, 24.1,

23.4, 20.8, 18.4, 12.1.

EXAMPLE 5. Biological activity

The biological activity of the selected compounds (Table 1) was tested in vitro using a cell model transfected with reporter genes, on the receptors FXR, TGR5/GPBAR1 and LXRa in comparison with the control agonists: chenodeoxycholic acid (CDCA) , t aurolithocholic acid (TLCA) and GW3965.

CDCA is a primary bile acid that functions as an endogenous ligand of the receptor FXR; GW3965 (CAS: 405911-17-3) is a synthetic non steroidal compound, agonist of the receptor LXR, with activity both on the receptor LXR and on the receptor LXR ; TLCA is a physiological ligand of the receptor TGR5/GPBAR1. HepG2 cells were cultured at 37°C in E-MEM medium (Earl's salt Minimum Essential Medium) with the addition of 10% fetal bovine serum (FBS), 1% L- glutamine, and 1% penicillin/streptomycin. The HEK-293T cells were cultured at 37°C in D-MEM medium (Dulbecco's Minimum Essential Medium), with the addition of 10% fetal bovine serum (FBS), 1% L- glutamine, and 1% penicillin/streptomycin. The transfection experiments were performed using the reagent Fugene HD (Promega) according to the manufacturer's instructions. The cells were plated in 24-well plates at 5 10 ⁴ cells/well.

For the FXR mediated transactivation, HepG2 cells were transfected with 100 ng of the vector pSG5-FXR, 100 ng of the vector pSG5-RXR, 100 ng of the vector pGL4.70 Renilla, a plasmid encoding the human Renilla gene, and 200 ng of the reporter vector p(hsp27)- TK-LUC containing the FXR responsive-element IR1 cloned from the promoter of heat shock protein 27 (hsp27) .

For the GPBAR1 mediated transactivation, HEK-293T cells were transfected with 200 ng of the plasmid pGL4.29 (Promega), a reporter vector containing the cAMP response element (CRE) cloned upstream of the luciferase reporter gene luc2P, 100 ng of the vector pCMVSport6-human GPBAR1, and 100 ng of the vector pGL4.70 Renilla, a plasmid encoding the human Renilla gene. In control experiments, HEK-293T cells were transfected only with vectors pGL4.29 (Promega) and pGL4.70 Renilla, to exclude any possibility that compounds could activate the CRE independently of the GPBARl.

For the LXRa mediated transactivation, HepG2 cells were transfected with 200 ng reporter vector p (UAS) 5XTKLuc, 100 ng of a vector containing the ligand binding domain (LBD) of LXRa cloned upstream of the GAL4-DNA binding domain (i.e. pSG5-LXRaLBD-GAL4DBD) and 100 of the vector pGL4.70 Renilla, a plasmid encoding for the Renilla human gene.

For the LXR mediated transactivation, HepG2 cells were transfected with 200 ng reporter vector p (UAS) 5XTKLuc, 100 ng of a vector containing the ligand binding domain (LBD) of LXR3 cloned upstream of the GAL4-DNA binding domain (i.e. pSG5-LXR3LBD-GAL4DBD) and 100 of the vector pGL4.70 Renilla, a plasmid encoding for the Renilla human gene.

For the PXR mediated transactivation, HepG2 cells were transfected with 100 ng pSG5-PXR, 100 ng pSG5-RXR, 100 ng pGL4.70 Renilla, a plasmid encoding for the Renilla human gene, and 200 ng of the reporter vector containing the PXR target gene promoter CYP3A4 , cloned upstream of the luciferase gene (pCYP3A4 promoter- TKLuc) .

At 24 h post-transfection, cells were stimulated for 18 h with 10 μΜ CDCA, TLCA, GW3965 or Rifaximin (10 μΜ) , used as control agents, or with the compounds to be tested, at the same concentration. After treatments, cells were lysed in 100 iL of lysis buffer (25 mM Tris-phosphate, pH 7.8; 2 mM DTT; 10% glycerol; 1% Triton X-100) ; 10 L of the cellular lysate of each sample were assayed for luciferase activity using Dual Luciferase Reporter Assay System (Promega Italia S.r.l., Milan, Italy) according to the manufacturer's instructions. Luminescence was measured using Glomax 20/20 luminometer (Promega Italia S.r.l., Milan, Italy) . The luciferase activity (Luciferase Recording Unit, RLU) was normalized using the activity of the Renilla (Renilla Recording Unit, RRU) .

Table 1 reports the activity of selected compounds included in formula I towards FXR, LXRa and TGR5 /GPBAR1. Table 1 reports the efficacy of the selected compounds compared to that of the reference compounds, CDCA for FXR, taurolithocholic acid (TLCA) for TGR5/GPBAR1 and GW3965 (CAS: 405911-17-3) for LXRa, for which the transactivation activity was considered equal to 100% and with respect to the HDCA starting bile acid. Each compound was tested at the concentration of 10 μΜ.

Table 1

BAR1115 77% 295% 27% 171% 5% 15%

BAR1124 25% 98% 43% 276% 5% 15%

BAR1125 27% 104% 54% 348% 4% 12%

BAR1126 92% 260% 21% 134% 6% 19%

BAR1129 53% 202% 36% 232% 7% 22%

BAR1130 55% 212% 109% 703% 4% 13%

BAR1131 102% 369% 40% 259% 13% 42%

BAR1132 75% 287% 6% 35% 64% 205%

BAR1133 83% 318% 7% 47% 60% 134%

BAR1145 69% 264% 6 39% 5% 15%

BAR11 6 54% 206% 24% 153% 5% 15%

BAR1147 102% 369% 11% 72% 7% 21%

In some aspects, the invention is related to compounds belonging to formula IB, which are selective LXRa agonists. Examples are BAR1103, BAR1106, BAR1107, BAR1124 and BAR1125 that demonstrated a significant increase in efficacy when compared to HDCA. BAR1124 and BAR1125 showed EC50 values on LXRa of 2.7 μΜ e 5.1 μΜ, respectively.

In some aspects, the invention is related to compounds belonging to formula IA, which are selective GPBAR1 agonists. Examples are the epoxides BAR1115, BAR1126, BAR1131 and BAR1147. Selected examples in the above group are BAR1131 and BAR1147, with an efficacy of 102% with respect to TLCA and an efficacy of 332% with respect to HDCA.

In some aspects, the invention is related to compounds belonging to formula IA and IB, which are dual LXRa/GPBARl agonists. A selected example is BAR1130 with an efficacy comparable to the potent synthetic agonist G 3965 and with an efficacy of 703% with respect to HDCA on LXRa and with an efficacy of 55% with respect to TLCA and of 201% with respect to HDCA on GPBARl. Furthermore, BAR1130 showed EC ₅ o values of 4.9 μΜ e 3.2 μΜ on GPBARl and LXRa respectively.

The dual activity of BAR1130 in modulating GPBARl and LXRa is of particular interest. The agonistic activity on GPBARl could reduce the risk of hepatic steatosis associated with the activation of LXRa in the liver.

None of the compounds belonging to formula I showed activity on the isoform β of LXR and on PXR.