WO2023233346A1 - Dérivés de cholestérol et leurs utilisations - Google Patents

Dérivés de cholestérol et leurs utilisations Download PDF

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Publication number
WO2023233346A1
WO2023233346A1 PCT/IB2023/055628 IB2023055628W WO2023233346A1 WO 2023233346 A1 WO2023233346 A1 WO 2023233346A1 IB 2023055628 W IB2023055628 W IB 2023055628W WO 2023233346 A1 WO2023233346 A1 WO 2023233346A1
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compound
formula
group
cooh
alkyl
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PCT/IB2023/055628
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Angela ZAMPELLA
Stefano Fiorucci
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Precision Bio-Therapeutics S.R.L.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0094—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 containing nitrile radicals, including thiocyanide radicals

Definitions

  • 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.
  • 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.
  • RORy regulates the transcription of protein-producing genes by binding to specific DNA sequences.
  • 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.
  • helper 17 T cells
  • CD4 T lymphocytes a subtype of CD4 T lymphocytes. These cells are involved in the pathology of autoimmune-based inflammatory disorders and infections .
  • Thl7 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).
  • IL-17A interleukin 17A
  • 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.
  • 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.
  • FXR farnesoid X receptor
  • 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).
  • NASH non-alcoholic fatty liver syndrome
  • 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).
  • GLP-1 glucagon-like peptide
  • 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 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.
  • 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;
  • CDAI colitis activity index
  • 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 intestinal 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.
  • alkyl refers to saturated aliphatic hydrocarbons. This term includes linear (unbranched) chains or branched chains.
  • 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.
  • substituted 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.
  • the compounds of formula (I) can be in crystalline form.
  • 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.
  • 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 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) .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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-
  • R2 is selected from the group consisting of H, Ci-salkyl-
  • R3 is selected from the group consisting of H, C 0-5 alkyl-
  • 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.
  • 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.
  • R3 is selected from the group consisting of H, CH 2 OH, COOH.
  • the compound of formula (I) is selected from the group consisting of:
  • the compound is ROR107.
  • a second aspect of the present invention concerns a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the injectable compositions are typically based on sterile injectable solution or phosphate buffered solution or other injectable vehicles known in the art.
  • compositions may be in the form of tablets, pills, capsules, solutions, suspensions, emulsions, powders, suppositories and as sustained release formulations.
  • the tablets may be coated using standard aqueous or non-aqueous techniques.
  • 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.
  • 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.
  • a liquid medium such as a fatty oil.
  • 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.
  • sucrose as a sweetener
  • methyl and propyl parabens as preservatives
  • a dye and a flavouring agent such as cherry or orange flavour.
  • the composition is an enteric-coated formulation.
  • 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.
  • compositions of the present invention are administered according to a protocol and at a dosage sufficient to reduce inflammation and pain in the subject.
  • 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.
  • 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.
  • the compound of the present invention and the other active ingredient may be used in lower doses than when each is used individually.
  • 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.
  • 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.
  • 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.
  • 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.
  • PBC primary biliary cirrhosis
  • CX cerebrotendinous xanthomatos
  • 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.
  • IBD inflammatory bowel disease
  • IBS irritable bowel syndrome
  • the renal disorders include diabetic nephropathy, hypertensive nephropathy, chronic glomerulonephritis including chronic transplant glomerulonephritis, chronic tubulointerstitial disease and vascular disorders of the kidney.
  • 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 .
  • PAD peripheral artery disease
  • PVD peripheral vascular disease
  • peripheral artery occlusive disease peripheral obliterative arteriopathy
  • 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.
  • the metabolic disorder is selected from the group consisting of obesity and conditions associated with bariatric surgery.
  • 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.
  • the infectious disease is selected from the group of acquired immunodeficiency syndrome (AIDS) and related disorders, B-virus and C-virus infection.
  • AIDS acquired immunodeficiency syndrome
  • B-virus B-virus and C-virus infection.
  • the synthesis 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.
  • 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.
  • 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 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).
  • 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
  • Example 1A Synthesis of 24-(4-hydroxymethyl)-phenoxy- 5-cholen-3b-ol (ROR102).
  • Example 2A Synthesis of 24-(4'-carboxy)-phenoxy-5- cholen-3b-ol (ROR103).
  • 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).
  • 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%).
  • 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.
  • 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.
  • Example IB Synthesis of 24-((3''-hydroxymethyl)-[1,1'— biphenyl]-4-yl)oxy-5-cholen-3b-ol (R0R119).
  • 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.
  • the activity data of selected compounds of the invention on the FXR, GPBAR1 and RORy receptors are described in Table 1.
  • 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%.
  • RORy the inverse agonism activity of the compounds was calculated as percentage of inhibition with respect to the untreated cells (100%).
  • 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.
  • IR1 FXR response element
  • 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.
  • CRE cAMP response element
  • the cells were stimulated for 18 hrs with specific CDCA (lOpM) or TLCA (lOpM) receptor agonists or with the derivatives (lOpM and 50pM).
  • 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).
  • Cell Reporter 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.
  • 200 pl of Reporter Cells were dispensed into the wells of a 96-well plate.
  • 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.
  • 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.
  • 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).
  • ROR107 GPBAR1 receptor agonist and RORyt antagonist
  • TNBS trinitrobenzenesulfonic acid
  • 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.
  • ROR107 was evaluated in a mouse model of DSS-induced colitis simulating ulcerative colitis.
  • 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.
  • DSS dextran sulfate sodium
  • 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.

Abstract

La présente invention concerne des composés de formule générale (I) et leurs utilisations pour moduler simultanément les récepteurs d'acides biliaires, FXR et GPBAR1, et le récepteur orphelin ROR gamma (RORγ) et ainsi leur utilisation dans le traitement et/ou la prévention de maladies médiées par ces derniers.
PCT/IB2023/055628 2022-06-01 2023-06-01 Dérivés de cholestérol et leurs utilisations WO2023233346A1 (fr)

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