Cross-Reference to Related Applications

This patent application is related to Italian Patent Application No. 102022000011705 filed on June 1, 2022, the entire disclosure of which is incorporated herein by reference .

Technical Field

The present invention relates to cholesterol derivatives and to their uses for simultaneously modulating the bile acid receptors, FXR and GPBAR1, and the orphan receptor ROR gamma (RORy) and thus their use in the treatment and/or the prevention of the diseases mediated by the latter.

Background

The strategy of identifying small molecules that can act simultaneously on multiple targets is widely recognized in the identification of new pharmacological approaches to multifactorial pathologies such as chronic inflammatory disorders, including non-alcoholic steatohepatitis, highly prevalent inflammatory liver disease, metabolic syndrome and cancer.

Many intermediates of the cholesterol synthesis or its metabolites act as agonists or inverse agonists of the RORy receptor as well as being able to modulate the activity of the bile acid receptors. The RORy receptor is an orphan receptor, a member of the nuclear receptor family and is involved in a wide variety of physiological processes including development, inflammation, circadian rhythm, immunity and lipid metabolism. RORy is expressed in many tissues, including liver, adipose tissue and muscle tissue.

Like all the nuclear receptors, RORy regulates the transcription of protein-producing genes by binding to specific DNA sequences.

In particular, changes in the cholesterol homoeostasis, which alter the levels of cholesterol or of its metabolites in the cells, may improve or inhibit the transcriptional activity with consequent changes in the physiological processes regulated by the ROR receptors, such as for example the immune response and the regulation of the metabolic pathway. All this can have an impact on different pathologies in which the receptor is involved, such as autoimmune diseases, inflammation, metabolic syndrome, cancer and several neurological disorders.

Among all the possible molecular mechanisms in which the RORy receptor is involved, the most studied and the most interesting one is its involvement in the regulation of the differentiation of the helper 17 (Thl7) T cells, a subtype of CD4 T lymphocytes. These cells are involved in the pathology of autoimmune-based inflammatory disorders and infections .

The main function of Thl7 is the production and the release of a cytokine called interleukin 17A (IL-17A) (Capone and Volpe, Front. Immunol., 2020, doi.org/10 .3389/fimmu.2020.00348). Inverse agonists or antagonists of the RORy receptor are able to suppress Thl7 differentiation and IL-17 production and to protect against tissue inflammation and pathologies associated therewith. In addition, since Thl7 cell differentiation and the same interleukin 17A (IL-17A) itself play a protective role against cancer by activating the CD8+ T cells, RORy is considered as a promising target in immune-based cancer therapy, as a transcriptional factor capable of initiating Thl7 cell differentiation and IL-17 production.

Highly expressed in the liver and the intestine, FXR regulates bile acid homoeostasis and several metabolic pathways, including lipid and glucose metabolism. The FXR agonists have proven to be useful pharmacological tools in metabolic disorders, such as cholestasis, type 2 diabetes, liver fibrosis and the non-alcoholic fatty liver syndrome (NAFLD), atherosclerosis, primary biliary cholangitis and non-alcoholic steatohepatitis (NASH) (Meadows et al., Front. Med., 2020, doi.org/10.3389/fmed.2020.00015). GPBAR1 is instead highly expressed in the liver, the intestine, the muscles, the adipose tissue, the macrophages and the endothelial cells. In the muscle and the brown adipose tissue, GPBAR1 increases the energy expenditure and the oxygen consumption (Watanabe et al. Nature dated 2006, 439, 484). In the entero-endocrine L cells, GPBAR1 activation stimulates glucagon-like peptide (GLP-1) secretion, thereby regulating the blood glucose levels, the gastrointestinal motility, and the appetite (Thomas et al. Cell. Metab. 2009, 10, 167).

GPBAR1 is important in regulating inflammation and immune function. Many innate immune cells express this receptor, such as the monocytes, the macrophages, the NKT cells, the dendritic cells, and mutations of this receptor are associated with an increased risk of developing primitive sclerosing cholangitis and ulcerative colitis.

The need is therefore felt in the art for compounds capable of simultaneously modulating the bile acid receptors, FXR and GPBAR1, and the orphan receptor ROR gamma (RORy) for a new pharmacological approach to multifactorial pathologies .

Summary

The object of the present invention is therefore the identification of new compounds capable of simultaneously modulating the nuclear receptors FXR and RORy and the membrane receptor GPBAR1.

This object is achieved by the present invention in that it relates to compounds of Formula (I) according to claim 1, to their use according to claims 6 and 7 and to a composition thereof according to claim 9. Preferred embodiments are indicated in the dependent claims.

Brief Description of the Drawings

The present invention will now be described in detail with reference to the figures of the accompanying drawings, wherein:

Figure 1 shows the synthetic diagram for the preparation of the compounds of Formula (I);

Figure 2 shows the trend of the (A) body weight percentage and (B) of the colitis activity index (CDAI); (C) Ratio between the colon weight and its length which are measured after sacrifice on day 4 following administration of the compound ROR107 in a first mouse model;

Figure 3 shows the histological analysis by hematoxylin and eosin (H&E) staining of colon sections following administration of the compound ROR107 in a first mouse model;

Figure 4 shows the relative mRNA expression (A) of the cytokines Il-lb, Tnf-a and 11-10, (B) of the macrophage markers Cdllb and Cd38, and (C) of the genes Rory and 11-17 in the colon before and after administration of the compound ROR107 in a first mouse model;

Figure 5 shows the trend of the (A) body weight percentage and B) the corresponding area under the curve; (C) the colitis activity index (CDAI) for the entire duration of the experiment; (D) the number of cells present in the lamina propria of the colon per mg of tissue; (E) the ratio between the weight of the colon and its length measured after sacrifice on day 8 in a second mouse model. The results are the mean ± SEM of 8-12 mice per group. *p< 0.05. The following paragraphs provide the chemical characteristics of the compounds according to the invention and are intended to apply uniformly throughout the description and all claims unless a definition providing a broader definition is expressly stated otherwise.

The term "alkyl" as used herein refers to saturated aliphatic hydrocarbons. This term includes linear (unbranched) chains or branched chains.

Description of Embodiments

Non-limiting examples of alkyl groups according to the invention are, for example, methyl, ethyl, propyl, isopropyl (ipropyl), n-butyl, isobutyl (ibutyl), tert-butyl (t-butyl), n-pentyl, iso-pentyl (hyptyl), n-hexyl and the like.

Unless otherwise indicated, the term "substituted" as used herein means that one or more hydrogen atoms of the aforementioned groups are substituted with another nonhydrogen atom, or a functional group, provided that the normal valencies are maintained and that the substitution results in a stable compound.

Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". The solvates of the compounds of the invention fall within the scope of the invention. The compounds of formula (I) can be easily isolated in association with solvent molecules by crystallisation or evaporation of an appropriate solvent to provide the corresponding solvates.

The compounds of formula (I) can be in crystalline form. In some embodiments, the crystalline forms of the compounds of formula (I) are polymorphic.

The invention in question also includes isotopically- labelled compounds, which are identical to those reported in formula (I), but differ in that one or more atoms are substituted with an atom having an atomic mass or mass number different from the atomic mass or mass number usually present in nature. Examples of isotopes that may be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, and oxygen such as ² H, ³ H, ³¹ C, ¹³ C, ¹⁴ C, The compounds of the present invention containing the aforementioned isotopes and/or other isotopes of other atoms fall within the scope of protection of the present invention. The isotopically-labelled compounds of the present invention, for example those in which radioactive isotopes such as ³ H and ¹⁴ C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated isotopes, i.e. ³ H and carbon-14, i.e. ¹⁴ C, are particularly preferred for their ease of preparation and detectability. The isotopes ⁴¹ C are particularly useful in PET (positron emission tomography) . In addition, the substitution with heavier isotopes such as deuterium, i.e. ² H, may bring some therapeutic advantages resulting from the increased metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, therefore, may be reported in some circumstances. The isotopically-labelled compounds of formula (I) of the present invention can generally be prepared by performing the procedures described in the diagrams and/or in the examples below, substituting a non-isotopically-labelled reagent for a readily available isotopically-labelled reagent.

Certain groups/substituents included in the present invention may be present as isomers. Accordingly, in some embodiments, the compounds of formula (I) may have axial asymmetries and, correspondingly, may exist in the form of optical isomers such as a form (R), a form (S), and the like. The present invention includes within the scope of protection all such isomers, including racemates, enantiomers and mixtures thereof.

In particular, the scope of protection of the present invention includes all stereoisomeric forms, including enantiomers, diastereoisomers and mixtures thereof, including racemates and the general reference to the compounds of formula (I) includes all stereoisomeric forms, unless otherwise indicated.

In general, the compounds of the invention should be considered to exclude those compounds (if any) which are chemically very unstable, either by themselves or in water, to be clearly unsuitable for pharmaceutical use by all routes of administration, regardless of whether it is oral, parenteral or otherwise. Such compounds are known to the skilled chemist.

Finally, the compounds of formula (I) may form salts.

According to a first aspect of the invention, the compounds of formula (I) are provided: ( I ) or its pharmaceutically acceptable salts, solvates or isomers wherein: n is an integer comprised between 2 and 3;

R is selected from the group consisting of

R ₁ is selected from the group consisting of CF ₃ , Co-

₅ alkyl-CH ₂ OH, C _1-5 alkyl-O- C _1-5 alkyl, C _0-5 alkyl-COOH and CN;

10 R2 is selected from the group consisting of H, Ci-salkyl-

CH ₂ OH and C _1-5 alkyl-COOH;

R3 is selected from the group consisting of H, C _0-5 alkyl-

CH ₂ OH and C _0-5 alkyl-COOH, provided that when R is and n=2, Ri is different from meta-CH ₂ OCH ₃ .

In a first embodiment, Ri is selected from the group consisting of CH ₂ OH, COOH, CF ₃ , CN, CH ₂ O-alkyl, (CH ₂ ) ₂ CH ₂ OH, (CH ₂ ) ₂ COOH, (CH ₂ ) ₄ CH ₂ OH, (CH ₂ ) ₄ COOH. In a second embodiment, R ₂ is selected from the group consisting of H, (CH ₂ ) ₃ CH ₂ OH, (CH ₂ ) ₃ COOH, (CH ₂ ) ₄ CH ₂ OH, (CH ₂ ) ₄ COOH.

In a third embodiment, R3 is selected from the group consisting of H, CH ₂ OH, COOH.

In a further embodiment the compounds of formula (I) are selected from the group consisting of:

According to a further embodiment, the compound of formula (I) is selected from the group consisting of:

In a preferred embodiment the compound is ROR107.

A second aspect of the present invention concerns a pharmaceutical composition comprising a compound of Formula

(I) and at least one pharmaceutically acceptable excipient.

A person skilled in the art is aware of a whole variety of such excipient compounds suitable for formulating a pharmaceutical composition.

The compounds of formula (I), together with a conventionally used excipient may be included in pharmaceutical compositions and dosage units thereof and in such form may be used as solids, such as filled tablets or capsules, or liquids such as solutions, suspensions, emulsions, elixirs or capsules filled therewith, all for oral use or in the form of sterile injectable solutions for parenteral administration (including subcutaneous and intravenous use).

Such pharmaceutical compositions and the unit dosage forms thereof may comprise ingredients in conventional percentages, with or without additional compounds or active ingredients, and such unit dosage forms may comprise any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be used.

The pharmaceutical compositions containing a compound of the present invention can be prepared in a manner well known in the pharmaceutical art and comprise at least one active compound. Generally, the compounds of the present invention are administered in a pharmaceutically effective amount. The amount of the compound actually administered will typically be determined by a physician, taking into account relevant circumstances, including the condition to be treated, the route of administration selected, the actual compound administered, the age, the weight and the response of the individual patient, the severity of the patient's symptoms, and the like.

The pharmaceutical compositions of the present invention can be administered by numerous routes including oral, rectal, subcutaneous, intravenous, intramuscular, intranasal and pulmonary routes. The compositions for oral administration can take the form of liquid solutions or suspensions in bulk or in bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate the precise dosing. The expression "unit dosage forms" refers to physically distinct units suitable as unit dosages for human and other mammalian subjects, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect, in association with an acceptable pharmaceutical excipient. The typical unit dosage forms include pre-filled, pre-dosed ampoules or syringes of the liquid compositions or pills, tablets, capsules or similar in the case of solid compositions.

The liquid forms suitable for oral administration may include a suitable aqueous or non-aqueous vehicle with buffering agents, suspending and dispersing agents, dyes, flavours and the like. The solid forms may include, for example, any of the following ingredients, or compounds of similar nature: a binder such as microcrystalline cellulose, tragacanth gum or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate; a flow agent such as colloidal silicon dioxide; a sweetening agent such as sucrose, lactose or saccharin; or a flavouring agent such as peppermint, methyl salicylate or orange flavouring agent.

The injectable compositions are typically based on sterile injectable solution or phosphate buffered solution or other injectable vehicles known in the art.

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

If desired, the tablets may be coated using standard aqueous or non-aqueous techniques. In certain embodiments, such compositions and preparations may contain at least 0.1 percent of active compound. The percentage of active compound in these compositions can be varied, of course, and can suitably be between about 1 percent and about 60 percent of the unit weight. The amount of active compound in such therapeutically useful compositions is such that the therapeutically active dosage will be obtained. The active compound can also be administered intranasally as, for example, liquid drops or sprays.

The tablets, pills, capsules, and the like may also contain a binder such as tragacanth gum, acacia, corn starch, or gelatin; excipients such as calcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, or saccharin. When a dosage unit form is a capsule, it may contain, in addition to the materials of the above type, a liquid medium such as a fatty oil. Various other materials may 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 may contain, in addition to the active ingredient, sucrose as a sweetener, methyl and propyl parabens as preservatives, a dye and a flavouring agent such as cherry or orange flavour. To avoid the breakage during the transit through the upper part of the gastrointestinal tract, the composition is an enteric-coated formulation.

The compositions for pulmonary administration include, but are not limited to, dry powder compositions consisting of powder of a compound of formula (I) and the powder of a suitable vehicle and/or lubricant. The compositions for pulmonary administration may be inhaled from any suitable dry powder inhaler device known to a person skilled in the art.

The administration of the compositions is performed according to a protocol and at a dosage sufficient to reduce inflammation and pain in the subject. 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 may contain from 0.1 to 1000 mg of a compound of formula (I) per each dosage unit for the daily administration.

In some embodiments, the effective amounts for a specific formulation will depend on the severity of the disease, disorder or condition prior to therapy, the health status of the individual and the response to the drug. In some embodiments the dose is in the range from 0.001% by weight to about 60% by weight of the formulation.

When used in combination with one or more of the other active ingredients, the compound of the present invention and the other active ingredient may be used in lower doses than when each is used individually.

As regards the formulations relating to any variety of routes of administration, methods and formulations for drug administration are described in Remington's Pharmaceutical Sciences, 17 ^th Edition, Gennaro et al. Ed., Mack Publishing Co., 1985 and Remington's Pharmaceutical Sciences, Gennaro AR ed. 20 ^th Edition, 2000, Williams & Wilkins PA, USA and Remington: The Science and Practice of Pharmacy, 21 ^st Edition, Lippincott Williams & Wilkins Ed., 2005; and in Loyd V. Allen e Howard C. Ansel, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 10 ^th Edition, Lippincott Williams & Wilkins Ed., 2014.

The components described above for orally administered or injectable compositions are only representative.

The compounds of the present invention may also be administered in sustained release forms or by sustained release drug delivery systems.

A third aspect of the present invention concerns the compounds of formula (I) as described above for use as a medicament .

A compound of Formula (I), as shown above, may be used in the prevention and/or the treatment of a disease mediated by the modulation of FXR, GPBAR1 and RORy receptors.

In particular, the disease is selected from the group consisting of inflammatory disorders, autoimmune diseases, liver diseases, cardiovascular diseases, metabolic disorders, metabolic diseases, infectious diseases, cancer, renal disorders and neurological disorders.

In one embodiment, the autoimmune diseases are selected from the group of rheumatoid arthritis, fibromyalgia, Sjogren's syndrome, scleroderma, Behcet's syndrome, vasculitis and systemic lupus erythematosus, ankylosing spondylitis, multiple sclerosis (MS), uveitis, and psoriasis.

In one embodiment, the liver diseases include primary biliary cirrhosis (PBC), cerebrotendinous xanthomatosis (CTX), primary sclerosing cholangitis (PSC), drug-induced cholestasis, intrahepatic cholestasis of 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 transplantation- associated host disease, living donor transplantation, liver regeneration, congenital liver fibrosis, granulomatous liver disease, intra- or extrahepatic malignancy, Wilson's disease, haemochromatosis, and alpha-l-antitrypsin deficiency.

In one embodiment, the gastrointestinal disorders include inflammatory bowel disease (IBD) (including Crohn's disease, ulcerative colitis and indeterminate 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 tubulointerstitial disease and vascular disorders of the kidney.

In one embodiment, the cardiovascular disease is selected from the group consisting of atherosclerosis, dyslipidaemia, hypercholesterolemia, hypertriglyceridemia, hypertension also known as high blood pressure, inflammatory heart disease including myocarditis and endocarditis, ischemic heart disease, stable angina, unstable angina, myocardial infarction, cerebrovascular disease including ischaemic stroke, pulmonary heart disease including pulmonary hypertension, peripheral artery disease (PAD), also known as peripheral vascular disease (PVD), peripheral artery occlusive disease and peripheral obliterative arteriopathy .

In one embodiment, the metabolic disease is selected from the group consisting of insulin resistance, metabolic syndrome, type I and type II diabetes, hypoglycaemia, and adrenal cortex disorders including adrenal cortex insufficiency.

In one embodiment, the metabolic disorder is selected from the group consisting of obesity and conditions associated with bariatric surgery.

In one embodiment, cancer is selected from the group comprising liver cancer, bile duct cancers, pancreatic cancer, gastric cancer, colorectal cancer, breast cancer, ovarian cancer and pathology associated with resistance to chemotherapy.

In one embodiment, the infectious disease is selected from the group of acquired immunodeficiency syndrome (AIDS) and related disorders, B-virus and C-virus infection.

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

The following abbreviations are used in the accompanying examples: methanol (MeOH), sodium bicarbonate (NaHCO ₃ ), ethyl acetate (EtOAc), dichloromethane (DCM), sodium sulphate (Na2SO4), N,N-dimethylformamide (DMF), hyodeoxycholic acid (HDCA), t- butyldimethylsilyltrifluoromethanesulfonate (TBSOTf), lithium borohydride (LiBH ₄ ), triphenylphosphine (PPhs), diisopropyl azodicarboxylate (DIAD), tetra-n-butylammonium fluoride (TBAF), hydrochloric acid (HC1), triethylamine (TEA), sodium hydroxide (NaOH), tetrahydrofuran (THE), water (H20), deuterated chloroform (CDCI3), deuterated methanol (CD3OD), time (h), room temperature (rt), retention time (t _R ). EXAMPLES

EXAMPLE 1. Synthesis of ROR102-110 and ROR119-120

The synthesis, outlined in Figure 1, envisages the esterification of the hyodeoxycholic acid (HYO) with methanol and p-toluenesulfonic acid under the classic Fisher esterification conditions and the subsequent tosylation to give the 3,6-ditosylate derivative 1. The reaction in the presence of potassium acetate (CH3COOK) to be refluxed in a mixture of DMF:H20 5:1 v/v leads to the C6 elimination and the C3 inversion, giving a mixture of methyl 3-hydroxy-5- cholen-24-oate (2) and its 3-0-acetylated derivative. The subsequent deacetylation with sodium methoxide leads to an efficient and quantitative formation of the compound 2. The reduction of the side chain ester after protection of the C3 alcohol function (2,6-lutidine, t-butyldimethylsilyl- trifluoromethanesulfonate) provides the compound 3.

The alcohol 3 is the key intermediate of the whole synthesis. In fact, this alcohol can be used in different Mitsunobu reactions with different alcohols, to produce, after selective C3 deprotection with TBAF, the compounds ROR101, ROR104, ROR105, ROR106, ROR109, ROR110 and the compound 4.

Finally, the reduction with LiBH ₄ or the basic hydrolysis in the presence of sodium hydroxide of the methyl esters (ROR101, ROR106 and the compound 4) provided the corresponding alcohol and carboxylic acids (alcohols ROR102, ROR107 and R0R119 and acids ROR103, ROR108 and ROR120, respectively) .

General procedures Reaction a). The p-toluene-sulfonic acid (4 eq.) is added at room temperature to a solution of HDCA (5.0 g, 12.7 mmol) in 30 mL of dry MeOH. After Ihr, the reaction is blocked by addition of a saturated aqueous solution of NaHCO ₃ until neutrality and after removal of methanol by evaporation the residue is extracted with EtOAc (3 x 50 mL). The combined organic phases are washed with water and then anhydrified with Na2SO4, and finally concentrated under vacuum to give the methyl ester intermediate in quantitative yield (5.2 g). Reaction b). The ester thus obtained (5.2 g, 12.8 mmol) is solubilized in pyridine and p-toluenesulfonyl chloride (5 eq.) is added to the solution. The reaction is kept under stirring at room temperatures for about 12 hours. When the reaction is complete, the pyridine is removed under vacuum and the reaction is treated with H2O: EtOAc (3 x 50 mL). The combined organic phases are washed with a saturated solution of NaHCO ₃ , with water and then anhydrified with Na2SO4. The evaporation of the solvent under vacuum provides the compound 1 (7.7 g, 84% yield).

Reaction c). A solution of the compound 1 (7.7 g, 10.8 mmol) and of CH3COOK (1 eq), dissolved in water (2 mL) and N,N'- dimethylformamide (10 mL), is refluxed for 3hrs. After this time, the solution is cooled to room temperature and EtOAc and water are added to the solution. The aqueous phase is extracted with EtOAc (3 * 30 mL). The combined organic phases are washed with water, anhydrified (Na2SO4) and the solvent is removed with the rotary evaporator to give the intermediate 3b-acetyl-5-ene (13.2 mmol), which is used directly for the next reaction.

Reaction d). The acetylated intermediate (5.7 g, 13.2 mmol) is treated with a 0.5 M solution of CHsONa (10 eq) in dry methanol and kept under stirring at room temperature for 12 hours. At the end of the reaction, the residue is diluted with water, followed by evaporation of the methanol and treatment with EtOAc (3 x 50 mL). The combined organic phases are extracted once with water, anhydrified with Na2SO4 to give 3.2 g of 2, subsequently purified on a chromatographic column, using 95:5 hexane/EtOAc +0.5% of TEA as eluent mixture. The purification provides 1.5 g of pure compound 2 (3.9 mmol, 36% in two steps).

Reaction e). 2,6-Lutidine (20 eq.) and tertbutyldimethylsilyltrifluoromethanesulfonate (6 eq.) are added to a solution of compound 2 (1.5 g, 3.9 mmol), dissolved in 20 mL of CH2CI2 and placed at 0°C. After 2hrs under stirring at 0°C, an aqueous solution of NaHCO ₄ (IM, 100 mL) is added, the phases are separated and the aqueous phase is extracted with CH2CI2 (3 x 50 mL). The organic phases are washed with IM NaHCO ₄ , water and saturated NaHCO ₃ solution. After evaporation of the solvent under reduced pressure, methyl 3β-tert-butyldimethylsilylose-5-colen-24- oate (2.8 g) was obtained in the form of transparent needles, which is subjected to the next step without any purification. Reaction f). Dry methanol (7 eq.) and LiBH ₄ (2M in THF, 7 eq.) are added to a solution of methyl ester (2.8 g, 5.7 mmol) in dry THF (30 mL) at 0 °C. The resulting mixture is placed under magnetic stirring for 2 hrs at 0°C and then, when the substrate appears finished in TLC, treated by adding a solution of IM NaOH (11.4 mL). The reaction is held Ihr and then the aqueous phase is treated with EtOAc. The combined organic phases are washed with water, anhydrified with Na2SO4 and then concentrated under reduced pressure. The purification on a silica gel column using hexane/EtOAc 9:1 and 0.5% of TEA as eluent mixture provided the compound 3 as white solid (1.5 g, 82% in two steps).

Reaction g). General procedure of the Mitsunobu reaction. DIAD (3.5 eq) is added dropwise to a solution of PPh ₃ (3.5 eq) in dry THF at 0°C. The suspension that forms is placed under stirring for 10 min, then a solution of compound 3 (1.5 g, 3.2 mmol) in dry THF is added to it. After 10 min, a solution of the corresponding phenol or of the methyl 4'- hydroxy- [1,1'-biphenyl]-3-carboxylate in dry THF is added. After a variable time (3hrs-12hrs), water (10 mL) is added and then the reaction mixture is concentrated in the rotary evaporator. The residue is then extracted with EtOAc (3 x 50 mL). The combined organic phases are washed with a solution of 2.5 M KOH and then with water, anhydrified and evaporated on the rotary evaporator to give a yellow oil, which is subject to subsequent deprotection without any purification. Reaction h). The compound is solubilized in dry THF and then a large excess of a solution of tetra-n -butylammonium fluoride (TBAF, IM in dry THF) is added to the solution. When the reaction is complete, the mixture is extracted three times between H2O and EtOAc (3 x 30 mL). Then the combined organic phases are anhydrified, filtered and dried on the rotary evaporator to give a dry residue that will be purified, giving the compounds ROR101, ROR104, ROR105, ROR106, ROR109, ROR110 and 4.

Reaction i). Reduction of the ester with LiBH4. The methyl esters ROR101 (0.0330 mmol), ROR106 (0.055 mmol) and the compound 4 (0.210 mmol) were subjected to reduction with L1BH4, under the same conditions as described in step f, to give the derivatives ROR102, ROR107 and ROR119 as residues, which are then subjected to further chromatographic purification.

Reaction j). General procedure of alkaline hydrolysis. Three aliquots of the methyl esters ROR101 (0.0330 mmol), ROR106 (0.0216 mmol) and 4 (0.210 mmol) are hydrolysed in a basic environment for NaOH (10 eq.) in a MeOH solution: H2O 1:1 v/v (20 mL) under reflux. When the reactions are complete, the resulting solutions are concentrated in vacuo, diluted with water, acidified with 6N HC1 and then extracted with

EtOAc (3 x 50 mL). The combined organic phases are washed with water, anhydrified with Na2SO4 and then evaporated under reduced pressure to give the corresponding carboxylic acids ROR103, ROR108 and ROR120 as crude residues, subsequently subjected to chromatographic purification.

Example 1A. Synthesis of 24-(4-hydroxymethyl)-phenoxy- 5-cholen-3b-ol (ROR102). The residue obtained from the reduction reaction (0.0347 mmol, quantitative yield) is purified by HPLC (Phenomenex Luna C18, 5 pm; 4.6 mm i.d. x 250 mm), using as eluent MeOH/H2O 88:12 (flow 1 mL/min, t _R = 20.0 min) giving an analytical sample of ROR102.

ROR102. C31H46O3; selected values of ³ H NMR (CDCI3, 500 MHz): 7.29 (2H, d, J = 8.3 Hz, H-3',5'), 6.89 (2H, d, J = 8.3 Hz, H-2',6'), 5.37 (1H, br s, H-6), 4.63 (2H, d, J = 5.8 Hz, CH2OH), 3.93 (2H, dd, J = 11.9, 6.3 Hz, H ₂ -24), 3.53 (1H, m, H-3α), 1.02 (3H, s, Me-19), 0.98 (3H, d, J = 6.4 Hz, Me-21), 0.70 (3H, s, Me-18); ¹³ C NMR (CDCI3, 100 MHz) 5 158.8, 140.7, 132.9, 128.6 (2C), 121.7, 114.6 (2C), 71.8, 68.6, 65.1, 56.7, 55.9, 50.1, 42.3, 42.2, 39.7, 37.2, 36.5,

35.5, 32.1, 31.9 (2C), 31.6, 28.2, 25.8, 24.3, 21.0, 19.4,

18.6, 11.9.

Example 2A. Synthesis of 24-(4'-carboxy)-phenoxy-5- cholen-3b-ol (ROR103). The residue obtained from the hydrolysis was purified (0.0434 mmol, quantitative yield) in HPLC (Phenomenex Luna Cl8 column, 5 μm; 4.6 mm i.d. x 250 mm), using an eluent mixture MeOH/H2O 90:10 v/v (flow 1 mL/min, t _R = 19.5 min).

ROR103. C31H44O4; selected values of ³ H NMR (CDCI3, 400 MHz): 8.04 (2H, d, J = 7.9 Hz, H-3',5'), 6.93 (2H, d, J = 7.9 Hz, H-2',6'), 5.36 (1H, d, J = 4.0 Hz, H-6), 4.00 (2H, m, H ₂ -24), 3.53 (1H, m, H-3α), 1.02 (3H, s, Me-19), 0.98 (3H, d, J = 6.4 Hz, Me-21), 0.70 (3H, s, Me-18); ¹³ C NMR (CDCI3, 100 MHz) 5 169.5, 163.5, 140.8, 132.3 (2C), 121.7, 121.2, 114.2 (2C), 71.8, 68.7, 56.8, 55.9, 50.1, 42.4, 42.3, 39.8, 37.3, 36.5, 35.5, 32.0, 31.9 (2C), 31.6, 28.2, 25.7,

24.3, 21.1, 19.4, 18.7, 11.9.

Example 3A. Synthesis of 24-(4'-

(tri luoromethyl)phenoxy)-5-cholen-3b-ol (ROR104). The purification on silica column, using 100% CH2CI2 as eluent provides the compound ROR104 (55%).

ROR104. C31H43F3O2; selected values of ³ H NMR (CDCI3, 400 MHz): 7.54 (2H, d, J = 8.6 Hz, H-3',5'), 6.95 (2H, d, J = 8.6 Hz, H-2',6'), 5.36 (1H, br s, H-6), 3.97 (2H, br s, H ₂ - 24), 3.53 (1H, m, H-3α), 1.02 (3H, s, Me-19), 0.98 (3H, d, J = 6.3 Hz, Me-21), 0.70 (3H, s, Me-18); ¹³ C NMR (CDCI3, 100 MHz) 5161.6, 140.8, 126.8 (2C), 124.5, 122.6, 121.7, 114.2 (2C), 71.7, 68.7, 56.7, 55.9, 50.1, 42.3, 42.2, 39.7, 37.2, 36.5, 35.5, 32.0, 31.9 (2C), 31.6, 28.2, 25.7, 24.2, 21.1,

19.4, 18.6, 11.9. Example 4A. Synthesis of 24-(4'-cyanophenoxy)-5- cholen-3b-ol (ROR105). The purification on silica chromatography column using 100% CH2CI2 as eluent provides ROR105 (1.26 mmol, quantitative yield).

ROR105. C31H43NO2; selected values of ³ H NMR (CDCI3, 400 MHz): 7.57 (2H, d, J = 8.7 Hz, H-3',5'), 6.93 (2H, d, J = 8.7 Hz, H-2',6'), 5.35 (1H, d, J = 4.4 Hz, H-6), 3.97 (2H, dd, J = 9.4, 6.4 Hz, H ₂ -24), 3.52 (1H, m, H-3α), 1.01 (3H, s, Me-19), 0.97 (3H, d, J = 6.5 Hz, Me-21), 0.69 (3H, s, Me-18); ¹³ C NMR (CDCI3, 100 MHz) 5162.4, 140.7, 133.9 (2C), 121.7, 121.6, 115.1 (2C), 103.6, 71.7, 68.8, 56.7, 55.8, 50.0, 42.3, 42.2, 39.7, 37.2, 36.5, 35.4, 31.9, 31.8 (2C), 31.6, 28.2, 25.6, 24.2, 21.0, 19.3, 18.6, 11.9.

Example 5A. Synthesis of 24-(3'-hydroxymethyl)- phenoxy-5-cholen-3b-ol (ROR107). The purification on silica chromatography column using 100% CH2CI2 as eluent provides ROR107 (38%). An analytical sample is obtained in HPLC on a Phenomenex Luna Cl8 column (5 pm; 4.6 mm i.d. x 250 mm), using MeOH/H2O 92:8 v/v as eluent (flow 1 mL/min; t _R = 12.0 min).

ROR107. C31H46O3; selected values of ³ H NMR (CDCI3, 400 MHz): 7.27 (1H, ovl, H-5'), 6.93 (1 H, ovl, H-2'), 6.92 (1H, ovl, H-4'), 6.83 (1H, dd, J = 8.3, 1.9 Hz, H-6'), 5.36 (1H, d, J = 5.0 Hz, H-6), 4.68 (2H, s, CH ₂ OH), 3.94 (2H, m, H ₂ -24), 3.53 (1H, m, H-3α), 1.02 (3H, s, Me-19), 0.98 (3H, d, J = 6.5 Hz, Me-21), 0.70 (3H, s, Me-18); ¹³ C NMR (CDCI3, 100 MHz) 5159.4, 142.4, 140.7, 129.6, 121.7, 118.9, 113.8, 112.9, 71.8, 68.5, 65.4, 56.7, 55.9, 50.1, 42.3, 42.2, 39.8, 37.2, 36.5, 35.5, 32.1, 31.9 (2C), 31.6, 28.2, 25.9, 24.3, 21.0, 19.4, 18.6, 11.9.

Example 6A. Synthesis of 24-(3'-carboxy)-phenoxy-5- cholen-3b-ol (ROR108). The purification on silica chromatography column using CH2CI2: MeOH 9:1 v/v as eluent provides ROR108 with 24.3% yield. An analytical sample is obtained by HPLC separation on Phenomenex Luna C18 column (5 pm; 4.6 mm i.d. x 250 mm), using MeOH/H2O 88:12 v/v as eluent (flow 1 mL/min; t _R =25 min).

ROR108. C31H44O4; selected values of ³ H NMR (CDCI3, 400 MHz): 7.68 (1H, d, J = 7.9 Hz, H-4'), 7.59 (1 H, s, H-2'), 7.37 (1H, t, J = 7.9 Hz, H-5'), 7.14 (1H, dd, J = 7.9, 3.9 Hz, H-6'), 5.36 (1H, br s, H-6), 3.99 (2H, m, H ₂ -24), 3.54 (1H, m, H-3α), 1.02 (3H, s, Me-19), 0.98 (3H, d, J = 6.4

Hz, Me-21), 0.70 (3H, s, Me-18). ¹³ C NMR (CDCI3, 100 MHz) 5

167.4, 159.4, 140.7, 129.9, 122.7, 122.0, 121.1, 120.7,

115.4, 72.1, 69.0, 56.4, 55.9, 50.2, 42.5, 42.2, 40.0, 37.5,

36.6, 35.8, 32.3, 32.1, 31.8, 31.4, 28.5, 26.0, 24.4, 21.2,

19.6, 18.8, 12.1.

Example 7A. Synthesis of 24-(3'-

(trifluoromethyl)phenoxy)-5-cholen-3b-ol (ROR109) . The purification on silica chromatography column using 100% CH2CI2 as eluent provides ROR109 (87.4%).

ROR109. C31H43F3O2; selected values of ³ H NMR (CDCI3, 400 MHz): 7.37 (1H, t, J = 8.2 Hz, H-5'), 7.19 (1H, d, J = 7.5 Hz, H-4'), 7.12 (1 H, s, H-2'), 7.06 (1H, d, J = 8.2 Hz, H- 6'), 5.36 (1H, br s, H-6), 3.96 (2H, m, H ₂ -24), 3.53 (1H, m, H-3α), 1.02 (3H, s, Me-19), 0.98 (3H, d, J = 6.4 Hz, Me- 21), 0.70 (3H, s, Me-18); ¹³ C NMR (CDCI3, 100 MHz) 5 159.2,

140.7, 131.3, 129.8, 124.4, 121.6, 117.9, 117.1, 111.2,

71.7, 68.7, 56.7, 55.9, 50.1, 42.3, 42.2, 39.7, 37.2, 36.4,

35.5, 32.0, 31.9 (2C), 31.6, 28.2, 25.7, 24.2, 21.0, 19.3,

18.6, 11.8.

Example 8A. Synthesis of 24-(3'-cyanophenoxy)-5- cholen-3b-ol (ROR110). The purification on silica chromatography column using 100% CH2CI2 as eluent provides ROR110 with 36% yield.

ROR110. C31H43NO2; selected values of ³ H NMR (CDCI3, 400

MHz): 7.36 (1H, m, H-5'), 7.23 (1H, d, J = 7.5 Hz, H-4'),

7.13 (1 H, s, H-2'), 7.12 (1H, ovl, H-6'), 5.36 (1H, d, J

= 3.8 Hz, H-6), 3.94 (2H, m, H ₂ -24), 3.53 (1H, m, H-3α),

1.02 (3H, s, Me-19), 0.98 (3H, d, J = 6.5 Hz, Me-21), 0.70 (3H, s, Me-18); ¹³ C NMR (CDCI3, 100 MHz) 5 159.2, 140.7, 130.2, 124.2, 121.6, 119.8, 118.8, 117.4, 113.2, 71.8, 68.9,

56.7, 55.9, 50.1, 42.4, 42.3, 39.8, 37.3, 36.5, 35.5, 32.0, 31.9 (2C), 31.6, 28.2, 25.6, 24.2, 21.1, 19.4, 18.6, 11.9.

Example IB. Synthesis of 24-((3''-hydroxymethyl)-[1,1'— biphenyl]-4-yl)oxy-5-cholen-3b-ol (R0R119). The purification on silica chromatography column using hexane: EtOAc 7:3 v/v and 0.5% of TEA, provides ROR119 with 24% yield. An analytical sample is obtained on Nucleodur semipreparative column HPLC (10 pm; 4.6 mm i.d. x 250 mm), using hexane/EtOAc 60:40 v/v as eluent (flow 3 mL/min; tR = 17.5 min).

ROR119. C37H50O3; selected values of ³ H NMR (CDCI3, 400 MHz): 5 7.57 (1H, s, H-2"), 7.53 (2H, d, J = 8.4 Hz, H- 3',5'), 7.50 (1H, d, J = 7.5 Hz, H-4"), 7.42 (1H, t, J = 7.5 Hz, H-6"), 7.31 (1H, d, J = 7.5 Hz, H-5"), 6.97 (2H, d, J = 8.4 Hz, H-2',6'), 5.36 (1H, d, J = 4.0 Hz, H-6), 4.77 (2H, d, J = 4.8 Hz, CH2OH), 3.99 (2H, m, H ₂ -24), 3.54 (1H, m, H-3α), 1.02 (3H, s, Me-19), 0.99 (3H, d, J = 6.6 Hz, Me- 21), 0.71 (3H, s, Me-18); ¹³ C NMR (CDCI3, 100 MHz) 5 158.8,

141.2, 141.1, 140.7, 133.3, 129.0, 128.2 (2C), 126.1, 125.3,

125.2, 121.7, 114.8 (2C), 71.8, 68.6, 65.5, 56.7, 55.9, 53.4, 50.0, 42.3, 42.2, 39.7, 37.2, 36.5, 35.5, 32.0, 31.8, 31.6, 28.2, 25.8, 24.2, 21.0, 19.4, 18.6, 11.9.

Example 2B. Synthesis of 24-((3''-(carboxy)-[1,1'- biphenyl]-4-yl)oxy-5-cholen-3b-ol (ROR120). The purification on silica chromatography column using 100% CH2CI2as eluent provides ROR120 with 64% yield. An analytical sample was obtained on Nucleodur semi-preparative column HPLC (10 pm; 4.6 mm i.d. x 250 mm), using hexane/EtOAc 30:70 as eluent (flow 3 mL/min; tR = 10.0 min).

ROR120. C37H48O4; selected values of (CDCI3, 400 MHz): 8.30 (1H, s, H-2"), 8.04 (1H, d, J = 7.9 Hz, H-4"), 7.81 (1H, d, J = 7.9 Hz, H-6"), 7.57 (2H, d, J = 8.4 Hz, H- 3',5'), 7.53 (1H, t, J = 7.9 Hz, H-5"), 7.00 (2H, d, J = 8.4 Hz, H-2',6'), 5.36 (1H, br s, H-6), 3.99 (2H, m, H ₂ - 24), 3.54 (1H, m, H-3α), 1.02 (3H, s, Me-19), 0.99 (3H, d, J = 6.6 Hz, Me-21), 0.71 (3H, s, Me-18); ¹³ C NMR (CDCI3, 100 MHz) 5 167.7, 159.5, 141.6, 140.7, 133.3, 132.2, 131.7,

129.3, 128.7, 128.5(3C), 122.1, 115.2(20 , 72.1, 68.9,

56.6, 56.2, 50.3, 42.5, 42.2, 39.9, 37.5, 37.1, 35.7, 34.6,

32.3, 32.1, 31.7, 28.5, 26.1, 24.2, 21.2, 19.5, 18.8, 12.1.

The activity data of selected compounds of the invention on the FXR, GPBAR1 and RORy receptors are described in Table 1. In this table, the activities of the compounds are compared with the specific reference compounds and in particular CDCA for FXR, TLCA for GPBAR1. Each compound is tested at a concentration of 10 pM and the activity of the reference compounds is considered to be equal to 100%. Regarding RORy the inverse agonism activity of the compounds was calculated as percentage of inhibition with respect to the untreated cells (100%).

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

For GPBARl-mediated transactivation, the HEK-293T cells were transfected with 200 ng of pGL4.29 (Promega, Madison WI), a reporter vector containing a cAMP response element (CRE) that drives the transcription of the luciferase luc2P reporter gene, with 100 ng of human pCMVSPORT6-GPBARl, and with 100 ng of pGL4.70.

After 24 hrs from transfection, the cells were stimulated for 18 hrs with specific CDCA (lOpM) or TLCA (lOpM) receptor agonists or with the derivatives (lOpM and 50pM).

For the dose-response curves, the cells were stimulated with increasing concentrations of the compounds of interest (0.1-75 pM). After 18 hrs from stimulation, the cell lysates were used to assess the activity of Luciferase and of Renilla by the Dual-Luciferase Reporter assay (E1980, Promega Madison WI). The luminescence was measured using the Glomax 20/20 luminometer (Promega, Madison WI) and the Luciferase activity was normalized with Renilla activity.

The inverse agonism activity assay on RORy was instead performed using the "Human RORyReporter Assay system" kit (IB- IB04001, Indigo Biosciences). This system uses appropriately engineered human cells so as to express high levels of the ROR receptoryconstitutively (Cell Reporter). In particular, the DNA binding domains (DBD) of RORy has been replaced with that of GAL4-DBD yeast.As occurs in vivo, uncharacterized molecular activators present in these cells maintain the receptor in a constant state of activity at high levels. Briefly, 200 pl of Reporter Cells were dispensed into the wells of a 96-well plate. After 4-6 hours of pre-incubation, the growth medium was discarded and 200 pl of treatment medium (containing the compound to be tested) were added to each well. The compounds were tested at concentrations of 1 and 10 pM. In addition, a dose-response curve of a reference inverse agonist, the Ursolic Acid (0.0165-6 pM) was performed and treatment-free control wells (100% activity) were provided. After 24 hrs of incubation, the treatment medium was discarded and 100 pl of Luciferase Detection Reagent were added. The Reporter cells contain the cDNA encoding the enzyme Luciferase firefly (Photinus pyralis) downstream of RORy. The intensity of light emitted by each well was quantified using a plate reader for luminescence and was expressed in Relative Light Units (RLU).

The efficacy of ROR107, GPBAR1 receptor agonist and RORyt antagonist, was determined in two mouse models of colitis mimicking the two main manifestations of IBD (Cronh's disease (CD) and Ulcerative Colitis (UC)). In the first mouse model, colitis was induced in male Balb/c mice through the administration by anal catheter of 1 mg/mouse of trinitrobenzenesulfonic acid (TNBS) dissolved in 50% ethanol. The compound ROR107 was administered daily (from day 0 to day 4) at the concentration of 10, 20 or 30 mg/kg by oral gavage.

The body weight trend and the values of the Colitis Activity Index (CDAI) demonstrate that ROR107 relieves the symptoms of the disease in a dose-dependent manner (Figure 2A and 2B). The 30 mg/kg dose is the most effective one, for example managing to halve the CDAI value on day 4 if compared to the one of the experimental group treated with TNBS alone (Figure 1A). The beneficial effects of the drug treatment are also confirmed by macroscopic and microscopic analysis of the colon (Figure 2C, Figure 3).

The analysis of the gene expression of certain cytokines in the colon shows how the disease induction increases the expression of the pro-inflammatory cytokines Il-lb and Tnf-a while decreasing the expression of the anti-inflammatory cytokine 11-10 (Figure 4A). ROR107 subverts this inflammatory pattern with a dose-dependent effect (Figure 4A), also reducing the expression of CDllb, macrophage markers, and of Cd38, markers specific to the Ml pro-inflammatory macrophage subpopulation (Figure 4B). The analysis of the expression of Rorc, a marker of the Thl7 lymphocytes, shows that the new compound decreases its expression at the colon level indicating a lower influx of these cells into the lamina propria also confirmed by the down-regulation of the expression of the gene encoding 11-17, a cytokine characteristic of Thl7 lymphocytes (Figure 4C).

ROR107 was evaluated in a mouse model of DSS-induced colitis simulating ulcerative colitis. In this model, Gpbarl ^+/+ and Gpbarl -/- mice were used so as to be able to analyse the role of the GPBAR1 receptor in the mechanism of action of the new compound. Colitis was induced by administration of dextran sulfate sodium (DSS) in 3% solution dissolved in drinking water. The compound ROR107 was administered daily (from day 0 to day of sacrifice) at the highest dose equal to 30 mg/kg by oral gavage.

The body weight trend and the area under the graph curve show that even in this second model of colitis ROR107 it relieves the development of the disease (Figure 5A, 5B). The beneficial effect is also partly maintained in Gpbarl -/- mice probably in relation to its antagonistic effect on RORyt (Figure 5A, 5B). The CDAI, which examines the body weight loss, stool consistency and the presence of blood, also indicates that ROR107 is able to alleviate the severity of colitis (Figure 5C). The absence of Gpbarl-/- makes the knock-out mice more susceptible to colitis and also attenuates the beneficial effect of the drug treatment (Figure 5C). In addition, ROR107 statistically reduces the number of immune cells present in the lamina propria of the colon and the ratio between weight and length of the colon which represents a valid indicator of inflammation of the colon (Figure 5D, 5E).