RU2793266C2 - Isoxazol as fxr receptor agonists - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to new isoxazoles of formula (I) or pharmaceutically acceptable salts thereof, having agonistic activity against the farnesoid FXRreceptor. In this formula, R₁ and R₂ are independently selected from the group consisting of H, halogen and CF ₃, provided that R₁ and R₂ are not H at the same time; R₃ is C₁-C₃ alkyl; n is an integer selected from 1, 2 and 3; R₄ is selected from the group consisting of phenyl unsubstituted or substituted with one R₅ and biphenyl unsubstituted or substituted with one R₅ ; R₅ is selected from the group consisting of COOR₆, CN, hydroxy-C₁-C₃ alkyl, C₁-C₃ alkyl-O-phenyl unsubstituted or substituted with one R₇, and C₁-C₃ alkyl-O-biphenyl , unsubstituted or substituted with one R₇; R₆ is selected from the group consisting of H and C₁-C₃ alkyl, and R₇ is selected from the group consisting of COOR₆, CN and hydroxy-C₁-C₃ alkyl; provided that the compound is not 4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzoic acid. The invention also relates to a pharmaceutical composition containing isoxazole formula (I) and its use as a drug for the prevention and/or treatment of FXR-dependent diseases.

EFFECT: drug for the prevention and/or treatment of FXR-dependent diseases.

9 cl, 1 tbl, 6 ex

I.

Description

Cross-reference to related applications

This patent application claims priority under Italian Patent Application No. 102018000007265 filed July 17, 2018, the entire disclosure of which is incorporated herein by reference.

The field of technology to which the invention belongs

The present invention relates to isoxazole derivatives and their use, in particular in the treatment and/or prevention of FXR-mediated diseases.

Prerequisites for the creation of the invention

The farnesoid X receptor (FXR, also known as BAR (bile acid receptor), NR1H4) is a ligand-dependent transcription factor and belongs to the nuclear hormone receptor superfamily.

Highly expressed in enterohepatic tissues (liver and intestines), FXR regulates bile acid homeostasis, lipoproteins, glucose metabolism, liver regeneration, and cardiovascular disorders (Mangelsdorf, D.J., et al. Cell 1995, 83; 835; Forman, B. M., et al. Cell 1995, 81, 687; Makishima, M., et al. Science 1999, 284, 1362; Parke, D. J., et al. Science 1999, 284, 1365 ; Wang, X., et al. Mol. Cell 1999, 3, 543; Kuipers, F., et al. Rev. Endocr. Metab. Disord. 2004, 5,319; Zhang, et al. Proc. Natl. Acad. Sci (USA 2006, 103, 1006; Matsubara, T., et al. Mol. Cell. Endocrinol. 2013, 368, 17; Claudel, T., et al. Arterioscler. Thromb. Vase. Biol. 2005, 25, 2020) . Specific bile acids bind and activate FXR, the most highly potent being chenodeoxycholic acid (CDCA), which is an endogenous FXR ligand and the primary bile acid found in human bile.

In recent years, there have been reports of various highly active and selective FXR agonists belonging to the classes of steroidal and non-steroidal chemicals such as 6-ECDCA, GW4064 and fexaramine. However, as a result of extended clinical studies, some side effects have been identified in serum lipid deregulation observed in patients with diabetes and hepatic steatosis (Fiorucci C, et al. Expert Opin. Ther. Targets 2014, 18, 1449-59).

In the context of a non-steroidal ligand, GW4064 represents a milestone in the field of isoxazole-type FXR agonists.

Indeed, GW4064 showed limited intestinal absorption (<10%) in rats after oral administration, showing a short terminal phase half-life. In addition, the π-electron system, widely delocalized in a fragment of the stilbene molecule, is responsible for its instability to UV radiation; therefore, GW4064 is potentially toxic to cells. Disclosure of invention

The aim of the present invention is to identify new compounds that act as FXR agonists with improved pharmacokinetic properties compared to GW4064.

This object is achieved by the present invention in relation to selective FXR agonists containing the isoxazole chemical backbone according to paragraph 1, to the pharmaceutical composition according to paragraph 5, to the use according to paragraphs 6 and 7 of the claims. Preferred embodiments of the present invention are set forth in the dependent claims.

Best Mode for Carrying Out the Invention

The following paragraphs contain definitions of various chemical moieties of the compounds of the present invention and are intended to be applied uniformly throughout the specification and in the claims, unless otherwise expressly stated definition contains a broader definition. The term "alkyl", in the context of this document, refers to saturated aliphatic hydrocarbon groups. This term includes normal (non-branched) chains or branched chains.

Non-limiting examples of the alkyl groups of the present invention are, for example, methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, t-butyl, n-pentyl, isopentyl, n-hexyl, and the like.

The term "alkyl", in the context of this document, refers to an alkyl group that is linked to the rest of the compound through an oxygen atom.

The term "halogen", in the context of this document, refers to fluorine, chlorine, bromine and iodine.

Unless otherwise indicated, the term "substituted", in the context of this document, means that at least one hydrogen atom of the above groups is replaced by another non-hydrogen atom or functional group, provided that the normal valency is maintained, and that the substitution results in a stable connection.

The term "pharmaceutically acceptable salts" refers to salts of the following compounds of Formula (I), which retain the desired biological activity and are recognized by regulatory authorities.

As used herein, the term "salt" refers to any salt of a compound of the present invention derived from an inorganic or organic base. Typically, such salts have a physiologically acceptable cation.

In addition, the compounds of Formula (I) can form a salt with a base, and these salts are included in the present invention because they are pharmaceutically acceptable salts.

Compounds of Formula (I) containing acidic protons may be converted into their therapeutically active, non-toxic base addition salt forms, for example metal or amine salts, by treatment with appropriate organic and inorganic bases.

Physiologically or pharmaceutically acceptable salts are particularly suitable for medical use due to their greater aqueous solubility compared to the parent compound.

Pharmaceutically acceptable salts may also be prepared from other salts, including other pharmaceutically acceptable salts of the compounds of Formula (I), using conventional methods.

Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they react or from which they precipitate or crystallize. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". Solvates of the compounds of the present invention are within the scope of the present invention. Compounds of Formula (I) can be easily isolated in combination with solvent molecules by crystallization or evaporation of a suitable solvent to obtain the corresponding solvates.

The compounds of Formula (I) may be in crystalline form. In certain embodiments of the present invention, the crystalline forms of the compounds of Formula (I) are polymorphs.

The subject matter of the present invention also includes isotopically labeled compounds which are identical to those shown in Formula (I) and below, but differ in that at least one atom is replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number commonly found in nature. Examples of isotopes that may be included in the compounds of the present invention and their pharmaceutically acceptable salts include isotopes of hydrogen, carbon, nitrogen, oxygen, such as ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O , ¹⁸ O.

Compounds of the present invention and pharmaceutically acceptable salts of said compounds containing the aforementioned isotopes and/or other isotopes of other atoms are within the scope of the present invention. Isotopically labeled compounds of the present invention, for example, compounds in which radioactive isotopes such as ³ H, ¹⁴ C are incorporated, are suitable for drug and/or substrate tissue distribution assays. Isotopes saturated with tritium, ie ³ H, and carbon-14, ie ¹⁴ C, are particularly preferred because of their ease of preparation and determination. The ¹¹ C isotope is particularly suitable for PET (Positron Emission Tomography). In addition, substitution with heavier isotopes such as deuterium, i.e., ² H, may provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances. The isotopically labeled compounds of Formula (I) of the present invention can generally be prepared by following the procedures outlined in the Schemes and/or the Examples below by substituting a non-isotopically labeled reagent with a readily available isotopically labeled reagent.

Some of the groups/substituents included in the present invention may be present as isomers. Accordingly, in some embodiments, implementation of the present invention, the compounds of Formula (I) may have axial asymmetry and, accordingly, they may exist in the form of optical isomers, such as (R)-form, (S)-form and the like. All such isomers are included within the scope of the present invention, including racemates, enantiomers, and mixtures thereof.

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

In general, the compounds or salts of the present invention are to be interpreted as excluding those compounds (if any) which are so chemically unstable, both on their own and in water, that they are clearly unsuitable for pharmaceutical use by all routes of administration, whether orally, parenterally or otherwise. Such compounds are known to those skilled in the art of chemistry.

According to a first aspect of the present invention, compounds of Formula (I) are provided:

or their pharmaceutically acceptable salts and solvates.

In compounds of Formula (I):

R ₁ and R ₂ are independently selected from the group consisting of H, halogen and CF ₃ , with the proviso that R ₁ and R ₂ are not H at the same time;

R ₃ is selected from the group consisting of C ₁ -C ₃ alkyl and halo-C ₁ -C ₃ alkyl; 11 is an integer selected from 1, 2 and 3;

R ₄ is selected from the group consisting of phenyl unsubstituted or substituted with one R ₅ and biphenyl unsubstituted or substituted with one R ₅ ;

R ₅ is selected from the group consisting of COOR ₆ , CN, hydroxy-C ₁ -C ₃ alkyl, SO ₂ CH ₃ , CF ₃ , C ₁ -C ₃ alkyl-O-phenyl unsubstituted or substituted with one R ₇ , and C ₁ -C ₃ alkyl-O-biphenyl, unsubstituted or substituted with one R ₇ ;

R ₆ is selected from the group consisting of H and C ₁ -C ₃ alkyl, and

R ₇ is selected from the group consisting of COOR ₆ , CN, hydroxy-C ₁ -C ₃ alkyl, SO ₂ CH ₃ and CF ₃ ;

provided that the compound is not 4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzoic acid.

According to one embodiment of the present invention, R4 is selected from the group consisting of

wherein R ₅ is as defined above.

According to one embodiment of the present invention, R ₅ is selected from the group consisting of COOH, COOCH ₃ , CN, -CH ₂ OH, SO ₂ CH ₃ ,

wherein R ₇ is as defined above.

According to one embodiment of the present invention, n is 1.

According to one embodiment of the present invention, R ₁ and R ₂ are independently selected from the group consisting of H, O and CF ₃ and in a preferred embodiment of the present invention, R ₁ and R ₂ are chlorine.

According to one embodiment of the present invention, R ₃ is selected from the group consisting of methyl, ethyl, propyl, isopropyl and CF ₃ .

According to one embodiment of the present invention, R ₇ is selected from the group consisting of COOH, COOCH ₃ , CN, CH ₂ OH, SO ₂ CH ₃ and CF ₃ .

According to one embodiment of the present invention, the compound of Formula (I) is selected from the group consisting of:

The second aspect of the present invention relates to a pharmaceutical composition containing a compound of Formula (I) as disclosed above, comprising 4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzoic acid, and, at least one pharmaceutically acceptable excipient.

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

The compounds of the present invention, together with a commonly used excipient, may be placed in the form of pharmaceutical compositions and unit dosages thereof, and in such form may be used as solids such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs or capsules filled with them, all intended for oral use, or in the form of sterile injectable solutions for parenteral administration (including subcutaneous and intravenous administration).

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

Pharmaceutical compositions containing a compound of the present invention may be prepared in a manner well known in the pharmaceutical art and contain at least one active compound. Typically, the compounds of the present invention are administered in a pharmaceutically effective amount. The amount of compound actually administered will generally be determined by the attending physician, taking into account relevant circumstances, including the disease being treated, the chosen route of administration, the actual compound administered, the age, weight and response of the particular patient, the severity of the patient's symptoms, and the like.

The pharmaceutical compositions of the present invention may be administered by various routes, including oral, rectal, subcutaneous, intravenous, intramuscular, intranasal, and inhalation routes. Compositions for oral administration may take the form of bulk liquid solutions or suspensions, or bulk powders. However, most often the compositions are presented in unit dosage forms, which helps to ensure accurate dosing. The term "unit dosage form" refers to physically discrete units suitable as unit dosages for humans and other mammals, each unit containing a predetermined amount of the active agent calculated to produce the desired therapeutic effect, in combination with a suitable pharmaceutical excipient. Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes containing liquid compositions, or pills, tablets, capsules, or the like when the compositions are in solid form.

Liquid forms suitable for oral administration may include a suitable aqueous or non-aqueous carrier aid with buffers, suspending and dosing agents, coloring agents, flavoring agents, and the like. Solid forms may include, for example, any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth 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 glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavor.

Compositions for injection are typically based on sterile injectable saline or phosphate buffered saline or other injectable vehicles known in the art.

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

If desired, the tablets may be coated using standard aqueous or non-aqueous methods. In some embodiments, implementation of the present invention, such compositions and preparations may contain at least 0.1 percent of the active compound. Of course, the percentage of active compound in these compositions may vary and may well range from about 1 percent to about 60 percent by weight of the unit. The amount of active compound in such therapeutically suitable compositions is such that a therapeutically active dosage will be obtained. The active compounds can also be administered intranasally as, for example, liquid drops or spray.

Tablets, pills, capsules and the like may also contain a binder such as gum tragacanth, gum arabic, corn starch or gelatin; fillers such as calcium phosphate; a disintegrating substance such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetener such as sucrose, lactose or saccharin. When the unit dosage form is a capsule, it may contain, in addition to substances of the above type, a liquid carrier such as a fixed oil. Various other substances may be present as shells or change the physical form of the dosage unit. For example, tablets may be coated with shellac, sugar, or both. A syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl paraben and propyl paraben as preservatives, a coloring agent, and a flavoring agent such as cherry or orange flavor. To prevent splitting during passage through the upper gastrointestinal tract, the composition must be coated with an enteric formulation.

Compositions for administration by inhalation include, but are not limited to, dry powder compositions consisting of a powder of a compound of Formula (I) or a salt thereof and a powder of a suitable carrier and/or lubricant. Compositions for administration by inhalation may be inhaled from any suitable dry powder inhalation device known to those skilled in the art.

The compositions are administered according to the protocol and at a dosage sufficient to reduce inflammation and pain in the patient. In some embodiments, implementation of the present invention, in the pharmaceutical compositions of the present invention, the active substance or active substances are usually included in dosage units. The dosage unit may contain from 0.1 to 1000 mg of the compound of Formula (I) per dosage unit for daily administration.

In some embodiments of the present invention, the amount effective for a particular formulation will depend on the severity of the disease or disorder, prior therapy, the individual's health status, and drug response. In some embodiments, implementation of the present invention, the dose is in the range from 0.001% by weight to about 60% by weight of the composition.

When used in combination with at least one other active ingredient, the compound of the present invention and the other active ingredient can be used at lower doses than when used alone.

With respect to formulations in terms of the variety of routes of administration, methods and formulations for administering drugs are disclosed in Remington's Pharmaceutical Science, 17th edition, Gennaro, et al., ed. Mack Publishing Co., 1985, and Remington's Pharmaceutical Sciences, Gennaro AP, ed., 20th edition, 2000, Williams & Wilkins Pennsylvania, USA, and Remington: The Science and Practice of Pharmacy, 21st edition, ed. Lippincott Williams & Wilkins, 2005; and in Loyd W. Allen and Howard S. Ansel, Ansel Pharmaceutical Dosage Forms and Drug Delivery Systems, 10th edition, ed. Lippincott Williams & Wilkins, 2014.

The components described above for oral or injectable compositions are merely representative.

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

A third aspect of the present invention relates to a compound of Formula (I) as disclosed above, comprising 4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzoic acid, for use as a medical preparation .

A compound of Formula (I) as disclosed above comprising 4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzoic acid may

be used in the prevention and/or treatment of a disease selected from the group consisting of gastrointestinal disorders, liver diseases, cardiovascular disorders, vascular diseases, pulmonary diseases, metabolic pathologies, infectious diseases, cancer, dysfunction kidney, inflammatory diseases, including immune-mediated, and neurological disorders.

In one embodiment of the present invention, immune-mediated inflammatory diseases include autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, scleroderma, also known as systemic sclerosis, spondyloarthritis, vasculitis, sarcoidosis, Mediterranean fever, and other hereditary autoinflammatory diseases, polymyositis and dermatomyositis, Behçet's syndrome.

In one embodiment of the present invention, the infectious diseases are selected from the group of acquired immunodeficiency syndrome (AIDS) and related disorders, infections caused by viruses B and C.

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

In one embodiment of the present invention, the liver diseases include primary biliary cirrhosis (PBC), cerebrotendinal xanthomatosis (CTC), primary sclerosing cholangitis (PSC), drug-induced cholestasis, intrahepatic cholestasis of pregnancy, parenteral nutrition-related cholestasis, bacterial overgrowth, or sepsis. associated cholestasis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver transplantation, congenital liver fibrosis, granulomatous liver disease, intra- or extrahepatic malignancies, Wilson's disease, hemochromatosis and alpha-1 antitrypsin deficiency.

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

In one embodiment of the present invention, renal disorders include diabetic nephropathy, hypertensive nephropathy, chronic glomerulonephritis, including chronic transplant glomerulonephritis, chronic interstitial tubular disease, and renal vascular disease.

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

In one embodiment of the present invention, vascular diseases include pulmonary heart disease such as pulmonary hypertension, peripheral arterial disease (PAD), also known as peripheral vascular disease (PSD), peripheral arterial occlusive disease, and peripheral arteriopathy obliterans.

In one embodiment of the present invention, pulmonary diseases include asthma, cystic fibrosis, obstructive pulmonary disease, interstitial lung disease, including, but not limited to, primary or secondary pulmonary fibrosis.

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

In one embodiment of the present invention, the cancer is selected from the group consisting of liver cancer, various types of bile duct cancer, esophageal cancer, pancreatic cancer, gastric cancer, colon and rectal cancer, breast cancer, ovarian cancer, and disease associated with resistance to chemotherapy.

Compounds of Formula (I), which can be used as a medicinal product and for the prevention and/or treatment of the above diseases, are selected from the group consisting of

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

The following abbreviations are used in the attached examples.

Methylene chloride (CH ₂ Cl ₂ ), hydroxylamine hydrochloride (NH ₂ OH), methyl alcohol (MeOH), potassium carbonate (K ₂ CO ₃ ), sodium sulfate (Na ₂ SO ₄ ), N,N-Diisopropylethylamine (DIPEA), dimethylformamide (DMF), (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), lithium bromide (LiBr), sodium bicarbonate (NaHCO ₃ ), trifluoroacetic acid (TFA), tetrahydrofuran (THF), lithium hydroxide (LiOH), hydrochloric acid (HCl), ethyl acetate (EtOAc), nitrogen (N ₂ ), water (H ₂ O), hour (h), room temperature (rt), retention time (t _R ).

Unless otherwise indicated, ¹ H NMR spectra were recorded on a Varian Inova 400 MHz instrument using CDCl ₃ as solvent, and ¹³ C NMR spectra were recorded on a Varian Inova 100 MHz instrument using CDCl ₃ as solvent. Examples

EXAMPLE 1. OBTAINING BAR2101-2105, BAR2110-2113, BAR2116, BAR2118-2120

Alcohol 1 was prepared by a multi-step procedure involving aldehyde oxime formation, chlorination, β-ketoester mediated isoxazole formation, and diisobutylaluminum hydride mediated ester reduction as previously described (Cipriani, C, et al. Sci. Rep. 2017, 7, 41055) . The key primary alcohol intermediate 1 was then used in numerous Mitsunobu reactions with various phenols (Scheme 1) to produce BAR2101-BAR2103, BAR2110, BAR2113, BAR2116, and BAR2118 in highly efficient chemical yields.

Reduction of LiBH ₄ and alkaline hydrolysis on methyl esters BAR2102 and BAR2110 gave the corresponding alcohols BAR2104 (95% yield) and BAR2112 (quantitative yield), and carboxylic acids BAR2105 (quantitative yield) and BAR2111 (89% yield), respectively.

As a result of the Mitsunobu reaction carried out on alcohol 1 with methyl 4'-hydroxy-[1,1'-biphenyl]-3-carboxylate, limited methyl ester BAR2118 was formed in 68% yield. Reduction and hydrolysis under the same experimental conditions as described above gave the corresponding BAR2120 alcohol and BAR2119 carboxylic acid (83% and 64% chemical yield, respectively).

Scheme 1

a) PPh ₃ , DIAD, THF dry, 0°C; b) LiBH ₄ , MeOH dry, THF dry, 0°C; c) NaOH, MeOH : H ₂ O 1:1 v/v. General procedures

Step a) Mitsunobu reaction. To a solution of PPh ₃ (3.5 eq.) in dry THF at 0°C was added dropwise DIAD (3.5 eq.). The suspension was stirred by shaking for 10 min, then a solution of compound 1 in dry THF was added. After 10 min, a solution of the appropriate phenol with dry THF was added. After 3 hours, overnight, water (10 ml) was added and the reaction mixture was evaporated. Then the residue was extracted with EtOAc (3×50 ml). The combined organic layers were washed with a solution of 2.5 M KOH and water, dried and evaporated to a yellow oil. Purification by silica gel flash chromatography gave BAR2101-2103, BAR2110, BAR2113, BAR2116, BAR2118.

Step b) Recovery of LiBH ₄ . To a solution of BAR2102 or BAR2110 or BAR2118 esters in dry THF (25 ml) at 0°C was added dry methyl alcohol (3.0 eq.) and LiBH ₄ (3.0 eq.). The resulting mixture was stirred with shaking for 4-8 hours at 0°C, then extinguished by adding 1M NaOH (2.0 eq.) and ethyl acetate. The organic phase was washed with water, dried (Na ₂ SO ₄ ) and concentrated to give a crude residue which was purified by HPLC or silica gel column chromatography.

Step c) Alkaline hydrolysis. Another portion of BAR2102 or BAR2110 or BAR2118 was hydrolysed with NaOH (5.0 eq.) in MeOH : H ₂ O 1:1 v/v solution (30 ml). The mixture was stirred for 8 hours under reflux. The resulting solution was then acidified with HCl 6M and extracted with ethyl acetate (3×50 ml). The collected organic phases were washed with brine, dried over anhydrous Na ₂ SO ₄ and evaporated under reduced pressure to give a complex crude residue which was purified by HPLC or flash chromatography.

Example 1A. Preparation of 3-(2,6-dichlorophenyl)-5-isopropyl-4-(phenoxymethyl)isoxazole (BAR2101)

Purification with silica gel (hexane and 0.5% TEA) after step a gave BAR2101 (40%). An analytical sample was prepared by HPLC with Nucleodur 100-5 C18 phase (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (82:18) as eluent (flow rate 1 ml/ min) (t _R = 10 min). BAR2101 C ₁₉ H ₁₇ Cl ₂ NO ₂

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.39 (2H, d, J=7.7 Hz), 7.31 (1H, t, J=7.7 Hz), 7.22 (2H, t, J=7.8 Hz), 6.93 (1H, t, J=7.8 Hz), 6.78 (2H, d, J=7.8 Hz), 4.72 (2H, s), 3 .33 (1H, septet, J=6.9 Hz), 1.41 (6H, d, J=6.9 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz) δ 176.3, 159.1, 158.9, 135.8 (2C), 131.2, 129.4 (2C), 128.0 (3C), 121, 2, 114.7 (2C), 109.4, 59.2, 27.0, 20.8 (2C).

Example 2A. Preparation of methyl 4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzoate (BAR2102)

Purification with silica gel (9:1 hexane/AcOEt and 0.5% TEA) after step a gave BAR2102 (82%). An analytical sample was prepared by HPLC with Nucleodur 100-5 C18 phase (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (82:18) as eluent (flow rate 1 ml/ min) (t _R = 12 min). BAR2102 C ₂₁ H ₁₉ Cl ₂ NO ₄

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.91 (2H, d, J=8.2 Hz), 7.39 (2H, d, J=7.6 Hz), 7.31 (1H, t, J=7.6Hz), 6.78(2H, d, J=8.2Hz), 4.77(2H, s), 3.86(3H, s), 3.32(1H, septet, J=6.7 Hz), 1.42 (6H, d, J=6.7 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.5, 166.7, 161.8, 159.0, 135.7 (2C), 131.5, 131.3 (2C), 128.1 ( 2C), 127.7, 123.0, 114.1 (2C), 108.8, 59.5, 51.9, 27.1, 20.8 (2C).

Example 3A. Preparation of 4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzonitrile (BAR2103)

Purification with silica gel (9:1 hexane/AcOEt and 0.5% TEA) after step a gave BAR2103 (89%). An analytical sample was prepared by HPLC with Nucleodur 100-5 C18 phase (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (82:18) as eluent (flow rate 1 ml/ min) (t _R = 7.4 min). BAR2103 C20H16Cl2N2O2

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.52 (2H, d, J=8.5 Hz), 7.40 (2H, d, J=7.0 Hz), 7.33 (1H, t, J=7.0 Hz), 6.81 (2H, d, J=8.5 Hz), 4.77 (2H, s), 3.31 (1H, septet, J=6.8 Hz) , 1.42 (6H, d, J=6.8 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.61, 161.3, 158.9, 135.7 (2C), 133.9 (2C), 131.4, 128.1 (2C), 127 .5, 118.9, 115.2 (2C), 108.5, 104.5, 59.5, 27.1, 20.8 (2C).

Example 4A. Preparation of 4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)phenyl)methanol (BAR2104)

Purification with silica gel (100% CH ₂ Cl ₂ ) after step b gave BAR2104 (95%). An analytical sample was prepared by HPLC with Nucleodur 100-5 C18 phase (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (70:30) as eluent (flow rate 1 ml/ min) (t _R = 13 min). BAR2104 C ₂₀ H ₁₉ Cl ₂ NO ₃

1H NMR (CDCl ₃ , 400 MHz): δ 7.39 (2H, d, J=7.2 Hz), 7.32 (1H, t, J=7.2 Hz), 7.22 (2H, d , J=7.3 Hz), 6.76(2H, d, J=7.3 Hz), 4.71(2H, s), 4.58(2H, s), 3.32(1H, septet , J=6.6 Hz), 1.41 (6H, d, J=6.6 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.3, 159.1, 157.8, 135.8 (2C), 133.7, 131.2, 128.5 (2C), 128.1 ( 2C), 127.8, 114.8 (2C), 109.4, 64.9, 59.4, 27.1, 20.7 (2C).

Example 5A

Preparation of 4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzoic acid (BAR2105). Purification with silica gel (100% CH ₂ Cl ₂ ) after step c gave BAR2105 (quantitative yield). An analytical sample was prepared by HPLC with Nucleodur 100-5 C18 phase (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (75:25) as eluent (flow rate 1 ml/ min) (t _R = 9.2 min).

BAR2105 C20H17CI2NO4

1H NMR (CDCl ₃ , 400 MHz): δ 7.94 (2H, d, J=6.5 Hz), 7.37 (2H, d, J=7.0 Hz), 7.29 (1H, t , J=7.0 Hz), 6.77 (2H, d, J=6.5 Hz), 4.77 (2H, s), 3.31 (1H, septet, J=6.4 Hz), 1.41 (6H, d, J=6.4 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 177.4, 162.4, 162.3, 158.9, 135.7 (2C), 132.2 (2C), 131.3, 128.0 ( 2C), 127.7, 123.0, 114.2 (2C), 108.8, 59.4, 27.1, 20.8 (2C).

Example 6A. Preparation of 3-(2,6-dichlorophenyl)-5-isopropyl-4-((4-(methylsulfonyl)phenoxy)methyl)isoxazole (BAR2116)

Purification with silica gel (100% CH ₂ Cl ₂ ) after step a gave BAR2116 (67%). An analytical sample was prepared by HPLC with Nucleodur 100-5 C18 phase (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (75:25) as eluent (flow rate 1 ml/ min) (t _R = 13.5 min).

BAR2116 C ₂₀ H ₁₉ Cl ₂ NO ₄ S

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.79 (2H, d, J=8.5 Hz), 7.40 (2H, d, J=7.8 Hz), 7.32 (1H, t, J=7.8 Hz), 6.88 (2H, d, J=8.5 Hz), 4.80 (2H, s), 3.32 (1H, septet, J=6.9 Hz) , 3.0 (3H, s), 1.43 (6H, d, J=6.9 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.6, 162.1, 158.9, 135.7 (2C), 132.8, 131.48, 129.5 (2C), 128.1 ( 2C), 127.5, 114.9 (2C), 108.5, 59.7, 44.2, 27.1, 20.8 (2C).

Example 7A. Preparation of methyl 3-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzoate (BAR2110)

Purification with silica gel (8:2 hexane/AcOEt and 0.5% TEA) after step a gave BAR2110 (57%). An analytical sample was prepared by HPLC with Nucleodur 100-5 C18 phase (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (82:18) as eluent (flow rate 1 ml/ min) (t _R = 16.5 min).

BAR2110 C ₂₁ H ₁₉ Cl ₂ NO ₄

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.60 (1H, d, J=7.8 Hz), 7.43 (1H, s), 7.38 (2H, d, J=7.7 Hz), 7.32-7.25 (2H, m, ovl), 6.94 (1H, d, J=8.3 Hz), 4.77 (2H, s), 3.89 (3H, s ), 3.33 (1H, septet, J=6.9 Hz), 1.42 (6H, d, J=6.9 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.4, 166.7, 159.1, 158.1, 135.7 (2C), 131.4, 131.2, 129.4, 128.1 (2C), 127.8, 122.5, 120.3, 114.6, 109.1, 59.5, 52.1, 27.1, 20.8 (2C).

Example 8A. Preparation of 3-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzonitrile (BAR2113)

Purification with silica gel (9:1 hexane/AcOEt and 0.5% TEA) after step a gave BAR2113 (73%). An analytical sample was prepared by HPLC with Nucleodur 100-5 C18 phase (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (82:18) as eluent (flow rate 1 ml/ min) (t _R = 11.4 min).

BAR2113 C ₂₀ H ₁₆ Cl ₂ N ₂ O ₂

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.40 (1H, s), 7.39 (2H, d, J=7.8 Hz), 7.33 (1H, dd, ovl), 7, 31 (1H, t, ovl), 7.21 (1H, d, J=7.7 Hz), 6.99 (1H, d, J=8.0 Hz), 4.75 (2H, s), 3.31 (1H, septet, J=6.8 Hz), 1.41 (6H, d, J=6.8 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.6, 158.9, 158.1, 135.7 (2C), 131.4, 130.4, 128.1 (2C), 127.6, 125.0, 120.0, 118.4, 117.5, 113.2, 108.6, 59.6, 27.1, 20.8 (2C).

Example 9A. Preparation of 3-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)phenyl)methanol (BAR2112)

Purification after step b by HPLC with Nucleodur 100-5 C18 phase (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (75:25) as eluent (flow rate 1 ml/ min) (t _R = 18 min) gave BAR2112 (quantitative yield).

BAR2112 C ₂₀ H ₁₉ Cl ₂ NO ₃

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.39 (2H, d, J=7.5 Hz), 7.30 (1H, t, J=7.5 Hz), 7.19 (1H, t, J=7.8Hz), 6.90(1H, d, J=7.8Hz), 6.80(1H, s), 6.70(1H, d, J=7.8Hz) , 4.73 (2H, s), 4.61 (2H, d, J=4.1 Hz), 3.33 (1H, septet, J=6.9 Hz), 1.41 (6H, d, J=6.9 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.5, 159.0, 158.5, 142.5, 135.8 (2C), 131.2, 129.5, 128.1 (2C), 127.8, 119.6, 114.1, 112.8, 109.4, 65.0, 59.2, 27.1, 20.8 (2C).

Example 10A. Preparation of 3-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzoic acid (BAR2111)

Purification after HPLC step with Nucleodur 100-5 C18 phase (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (75:25) as eluent (flow rate 1 ml/ min) (t _R = 22.5 min) gave BAR2111 (89%).

BAR2111 C ₂₀ H ₁₇ Cl ₂ NO ₄

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.65 (1H, d, J=7.0 Hz), 7.47 (1H, s), 7.37 (2H, d, J=7.5 Hz), 7.31-7.25 (2H, m, ovl), 6.97 (1H, d, J=7.8 Hz), 4.77 (2H, s), 3.32 (1H, septet , J=7.1 Hz), 1.41 (6H, d, J=7.1 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.41, 159.7, 159.1, 158.2, 135.7 (2C), 131.3, 129.5 (2C), 128.0 ( 2C), 127.6, 123.2, 121.0, 115.2, 109.1, 59.5, 27.1, 20.8 (2C).

Example 11A. Preparation of methyl 4'-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)-[1,1'-biphenyl]-3-carboxylate (BAR2118)

Purification with silica gel (100% CH2CI2) after step a gave BAR2118 (68%). An analytical sample was prepared by HPLC with Nucleodur 100-5 C18 phase (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (90:10) as eluent (flow rate 1 ml/ min) (t _R = 9.9 min).

BAR2118 C ₂₇ H ₂₃ Cl ₂ NO ₄

¹ H NMR (CDCl ₃ , 400 MHz): δ 8.20 (1H, s), 7.98 (1H, d, J=7.7 Hz), 7.71 (1H, d, J=7.7 Hz), 7.49 (2H, d, J=8.5 Hz), 7.48 (1H, t, J=7.7 Hz), 7.42 (2H, d, J=8.0 Hz) , 7.33 (1H, dd, J=7.1, 8.0 Hz), 6.87 (2H, d, J=8.5 Hz), 4.78 (2H, s), 3.95 ( 3H, s), 3.36 (1H, septet, J=7.1 Hz), 1.44 (6H, d, J=7.1 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.4, 167.1, 159.1, 158.1, 140.8, 135.8 (2C), 133.2, 131.2, 131.0 , 130.6, 128.8, 127.8, 127.7, 128.1 (4C), 126.8, 115.1 (2C), 109.3, 59.4, 52.1, 27.1 , 20.8 (2C).

Example 12A. Preparation of (4'-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)-[1,1'-biphenyl]-3-yl)methanol (BAR2120)

Purification with silica gel (9:1 hexane/AcOEt and 0.5% TEA) after step b gave BAR2120 (83%). An analytical sample was prepared by HPLC with Nucleodur 100-5 C18 phase (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (83:17) as eluent (flow rate 1 ml/ min) (t _R = 8.5 min).

BAR2120 C ₂₆ H ₂₃ Cl ₂ NO ₃

¹ H NMR (CD3OD, 400 MHz): δ 7.53 (1H, s), 7.52 (2H, d, J=7.5 Hz), 7.47 (1H, t ovl), 7.46 ( 2H, d, J=8.8 Hz), 7.43 (1H, d, J=7.8 Hz), 7.36 (1H, t, J=7.8 Hz), 7.27 (1H, d, J=7.8 Hz), 6.84(2H, d, J=8.8 Hz), 4.84(2H, s), 4.64(2H, s), 3.43(1H, septet, J=6.7 Hz), 1.42 (6H, d, J=6.7 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.4, 157.8, 154.1, 141.4, 137.4, 135.8 (2C), 134.0, 131.2, 129.0 , 128.1 (4C), 126.0, 125.4, 125.3, 122.8, 115.1 (2C), 109.3, 65.4, 59.4, 27.0, 20.8 (2C).

Example 13A. Preparation of 4'-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)-[1,1'-biphenyl]-3-carboxylic acid (BAR2119)

Purification with silica gel (99:1 CH ₂ Cl ₂ /MeOH) after step c gave BAR2119 (64%). An analytical sample was prepared by HPLC with Nucleodur 100-5 C18 phase (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (83:17) as eluent (flow rate 1 ml/ min) (t _R = 12.5 min).

BAR2119 C ₂₆ H ₂₁ Cl ₂ NO ₄

¹ H NMR (CDCl ₃ , 400 MHz): δ 8.27 (1H, s), 8.04 (1H, d, J=8.0 Hz), 7.76 (1H, d, J=7.7 Hz), 7.51 (2H, d, J=8.2 Hz), 7.50 (1H, t, J=8.0 Hz), 7.42 (2H, d, J=7.6 Hz) , 7.33 (1H, t, J=7.6 Hz), 6.87 (2H, d, J=8.2 Hz), 4.78 (2H, s), 3.36 (1H, septet, J=7.1 Hz), 1.44 (6H, d, J=7.1 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.4, 164.9, 160.6, 158.1, 140.8, 135.8 (2C), 133.1, 132.1, 131.5 , 131.2, 128.8, 128.3, 128.1 (4C), 127.8, 127.7, 115.1 (2C), 109.3, 59.4, 27.1, 20.8 (2C).

EXAMPLE 2 OBTAINING BAR2106-2109, BAR2121-2124, BAR2139, BAR2140 AND BAR2147

BAR2104 prepared according to Scheme 1 was alkylated with six different phenols via the Mitsunobu reaction to give BAR2106, BAR2107, BAR2121, BAR2122, BAR2138 and BAR2144. Reduction of LiBH ₄ and alkaline hydrolysis on methyl esters BAR2106, BAR2122, BAR2138, and BAR2144 resulted in the formation of the corresponding alcohols BAR2108, BAR2124, BAR2140, BAR2146 and acids BAR2109, BAR2123, BAR2139, and BAR2147, respectively (Scheme 2).

Scheme 2

a) PPh ₃ , DIAD, THF dry, 0°C; b) LiBH ₄ , MeOH dry, THF dry, 0°C; c) NaOH, MeOH : H ₂ O 1:1 v/v.

EXAMPLE 2A. Preparation of methyl 4-((4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzyl)oxy)benzoate (BAR2106)

Purification with silica gel (100% CH ₂ Cl ₂ ) after step a gave BAR2106 (63%). Analytical sample BAR2106 was prepared by HPLC with Phenomenex C18 phase (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (85:15) as eluent (flow rate 1 ml/min) (t _R = 10.4 min). BAR2106 C28H25CI2NO5

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.98 (2H, d, J=8.0 Hz), 7.40 (2H, d, J=7.7 Hz), 7.33-7, 26 (3H, m, ovl), 6.96 (2H, d, J=8.0 Hz), 6.79 (2H, d, J=7.7 Hz), 5.00 (2H, s), 4.72(2H, s), 3.88(3H, s), 3.32(1H, septet, J=6.9Hz), 1.41(6H, d, J=6.9Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.4, 166.8, 162.4, 159.1, 158.2, 135.7 (2C), 131.5, 131.2 (2C), 129.2 (2C), 128.8, 128.1 (2C), 127.3, 122.7, 114.9 (2C), 114.4 (2C), 109.3, 69.7, 59, 4, 51.8, 27.1, 20.7 (2C).

EXAMPLE 2B. Preparation of 4-((4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzyl)oxy)benzonitrile (BAR2107)

Purification with silica gel (7:3 hexane/AcOEt and 0.5% TEA) after step a gave BAR2107 (quantitative yield). An analytical sample was prepared by HPLC with Phenomenex C18 5 µm phase (4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (87:13) as eluent (flow rate 1 ml/min) (t _R = 8 min).

BAR2107 C ₂₇ H ₂₂ Cl ₂ N ₂ O ₃

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.58 (2H, d, J=8.5 Hz), 7.40 (2H, d, J=8.0 Hz), 7.32 (1H, t, J=8.4 Hz), 7.27 (2H, d, J=8.4 Hz), 6.98 (2H, d, J=8.5 Hz), 6.80 (2H, d, J=8.0 Hz), 5.00(2H, s), 4.73(2H, s), 3.32(1H, septet, J=6.9 Hz), 1.42(6H, d, J=6.9 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.1, 161.7, 158.8, 158.1, 135.5 (2C), 133.7 (2C), 131.1, 129.0 ( 2C), 128.1, 127.8 (2C), 127.5, 118.9, 115.3 (2C), 114.7 (2C), 109.1, 103.8, 69.7, 59, 2, 26.8, 20.5 (2C),

EXAMPLE 2C. Preparation of (4-((4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzyl)oxy)phenyl)methanol (BAR2108)

Purification after step b by HPLC with Phenomenex C18 phase (5 µm, 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (75:25) as eluent (flow rate 1 ml/min) ( t _R = 24 min) gave BAR2108 (quantitative yield). BAR2108 C27H25CI2NO4

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.39 (2H, d, J=7.9 Hz), 7.31 (1H, ovl), 7.28 (4H, d, ovl), 6, 94 (2H, d, J=8.3 Hz), 6.80 (2H, d, J=8.3 Hz), 4.96 (2H, s), 4.72 (2H, s), 4, 62 (2H, d, J=5.1 Hz), 3.33 (1H, septet, J=6.9 Hz), 1.41 (6H, d, J=6.9 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.5, 159.3, 158.4, 158.2, 135.7 (2C), 133.7, 131.4, 129.8 (2C), 129.2 (2C), 128.8, 128.2 (2C), 128.0, 115.0 (4C), 109.5, 69.8, 64.9, 59.6, 27.3, 20 .9 (2C).

EXAMPLE 2D. Preparation of 4-((4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzyl)oxy)benzoic acid (BAR2109)

Purification with silica gel (7:3 hexane: AcOEt) after step c gave BAR2109 (93%). An analytical sample was prepared by HPLC with Nucleodur VP 100-5 Silica phase (5 µm; 10 mm i.d. × 250 mm) using n-hexane/AcOEt (50:50) as eluent (flow rate 3 ml/min ) (t _R = 15 min).

BAR2109 C ₂₇ H ₂₃ Cl ₂ NO ₅

¹ H NMR (CDCl ₃ , 400 MHz): δ 8.05 (2H, d, J=8.6 Hz), 7.40 (2H, d, J=7.89 Hz), 7.33-7, 29 (3H, m, ovl), 6.99 (2H, d, J=8.6 Hz), 6.81 (2H, d, J=8.4 Hz), 5.03 (2H, s), 4.73 (2H, s), 3.32 (1H, septet, J=6.9 Hz), 1.42 (6H, d, J=6.9 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.4, 163.2, 162.4, 158.9, 158.3, 135.8 (2C), 132.3, 131.2 (2C), 129.2 (2C), 128.7, 128.1 (2C), 127.8, 121.7, 114.9 (2C), 114.5 (2C), 109.3, 69.8, 59, 4, 27.1, 20.8 (2C),

EXAMPLE 2E. Preparation of methyl 3-((4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzyl)oxy)benzoate (BAR2122)

Purification with silica gel (100% CH ₂ Cl ₂ ) after step a gave BAR2122 (61%). An analytical sample was prepared by HPLC with Nucleodur 100-5 C18 phase (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (85:15) as eluent (flow rate 1 ml/ min) (t _R = 18 min).

BAR2122 C ₂₈ H ₂₅ Cl ₂ NO ₅

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.63 (2H, d, J=7.6 Hz), 7.40 (2H, d, J=8.1 Hz), 7.33 (1H, t, J=8.1 Hz), 7.30-7.29(3H, m, ovl), 7.13(1H, d, J=7.9 Hz), 6.79(2H, d, J =8.3 Hz), 5.00 (2H, s), 4.72 (2H, s), 3.91 (3H, s), 3.32 (1H, septet, J=6.9 Hz), 1.41 (6H, d, J=6.9 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.4, 171.1, 169.3, 157.0, 152.9, 135.8 (2C), 131.2, 129.4, 129.2 (4C), 128.1 (2C), 122.2, 120.8, 120.2, 115.0, 114.8 (2C), 109.3, 69.8, 59.4, 52.2, 27.1, 20.7 (2C).

EXAMPLE 2F. Preparation of 3-((4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzyl)oxy)benzonitrile (BAR2121)

Purification with silica gel (100% CH ₂ Cl ₂ ) after step a gave BAR2121 (93%). An analytical sample was prepared by HPLC with Nucleodur 100-5 C18 phase (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (82:18) as eluent (flow rate 1 ml/ min) (t _R = 16 min).

BAR2121 C ₂₇ H ₂₂ Cl ₂ N ₂ O ₃

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.40 (2H, d, J=7.8 Hz), 7.33 (1H, t, J=7.8 Hz), 7.29-7, 23 (4H, m, ovl), 7.16 (2H, ovl), 6.79 (2H, d, J=8.5 Hz), 4.97 (2H, s), 4.72 (2H, s ), 3.32 (1H, septet, J=7.0 Hz), 1.41 (6H, d, J=7.0 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.4, 163.2, 159.1, 158.3, 135.7 (2C), 131.2, 130.4, 129.7, 129.1 (2C), 128.1 (2C), 124.7, 123.9, 120.5, 118.9, 118.7, 114.9, 114.8, 114.6, 109.3, 69.9 , 59.4, 27.1, 20.8 (2C).

EXAMPLE 2G. Preparation of (3-((4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzyl)oxy)phenyl)methanol (BAR2124)

Purification after step b by HPLC with Phenomenex C18 phase 5 µm (4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (80:20) as eluent (flow rate 1 ml/min) (t _R = 10 min) gave BAR2124 (84%).

BAR2124 C ₂₇ H ₂₅ Cl ₂ NO ₄

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.40 (2H, d, J=7.70 Hz), 7.33 (1H, t, J=7.70 Hz), 7.28 (2H, d, J=8.4Hz), 7.27(1H, dd, ovl), 6.98(1H, s), 6.94(1H, d, J=7.5Hz), 6.87( 1H, d, J=8.0 Hz), 6.78 (2H, d, J=8.4 Hz), 4.96 (2H, s), 4.72 (2H, s), 4.66 ( 2H, br s), 3.32 (1H, septet, J=6.9 Hz), 1.41 (6H, d, J=6.9 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.3, 159.1, 158.9, 158.1, 142.5, 135.7 (2C), 131.2, 129.6, 129.0 (3C), 128.0 (2C), 123.2, 119.3, 114.8 (2C), 114.1, 113.2, 109.3, 69.6, 65.2, 59.3, 27.1,

20.8 (2C).

EXAMPLE 2H. Preparation of 3-((4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzyl)oxy)benzoic acid (BAR2123)

Purification with silica gel (8:2 hexane/AcOEt) after step c gave BAR2123 (72%). An analytical sample was prepared by HPLC with Phenomenex C18 5 µm phase (4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (80:20) as eluent (flow rate 1 ml/min) (t _R = 12 min).

BAR2123 C ₂₇ H ₂₃ Cl ₂ NO ₅

¹ H NMR (CD3OD, 400 MHz): δ 7.58 (1H, s), 7.57 (1H, d, J=8.0 Hz), 7.49 (2H, d, J=7.7 Hz ), 7.43 (1H, dd, J=8.0, 8.4 Hz), 7.30 (1H, t, J=7.7 Hz), 7.28 (2H, d, J=8, 6 Hz), 7.10 (1H, d, J=8.4 Hz), 6.77 (2H, d, J=8.6 Hz), 5.00 (2H, s), 4.81 (2H , s), 3.41 (1H, septet, J=7.1 Hz), 1.40 (6H, d, J=7.1 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.3, 169.7, 159.1, 158.6, 158.1, 135.7 (2C), 132.0 (2C), 131.2, 129.5, 129.2, 128.4, 128.0 (2C), 122.7, 120.8, 116.1, 115.3, 114.8 (2C), 109.3, 69.7, 59.3, 27.1, 20.7 (2C).

EXAMPLE 2I. Preparation of 4'-((4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzyl)oxy)-[1,1'-biphenyl]-3-carboxylic acid (BAR2139 )

Purification by HPLC with Nucleodur Sphinx C18 5 µm phase (4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (95:5) and 0.1% TFA as eluent (flow rate 1 ml/ min) gave BAR2139 (t _R = 5 min).

BAR2139 C ₃₃ H ₂₇ Cl ₂ NO ₅

¹ H NMR (CDCl ₃ , 400 MHz): δ 8.27 (1H, br s), 8.02 (1H, d, J=7.7 Hz), 7.78 (1H, d, J=7, 7 Hz), 7.56 (2H, d, J=8.2 Hz), 7.52 (1H, t, J=7.7 Hz), 7.40 (2H, d, J=7.9 Hz ), 7.32 (2H, d, J=8.2 Hz), 7.30 (1H, t, J=7.9 Hz), 6.81 (2H, d, J=8.2 Hz), 5.01 (2H, s), 4.73 (2H, s), 3.33 (1H, septet, J=7.0 Hz), 1.42 (6H, d, J=7.0 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.3, 165.3, 159.1, 158.7, 158.2, 141.1, 135.8 (2C), 132.6, 131.8 , 131.3, 129.5(2C), 129.2(2C), 128.9, 128.4(2C), 128.2(2C), 128.1, 127.8, 115.3(2C ), 114.9 (2C), 109.3, 69.6, 59.6, 27.0, 20.7 (2C).

EXAMPLE 2J. Preparation of (4'-((4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzyl)oxy)-[1,1'-biphenyl]-3-yl)methanol (BAR2140)

Purification by HPLC with Nucleodur Sphinx C18 5 µm phase (4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (85:15) as eluent (flow rate 1 ml/min) gave BAR2140 ( t _R = 12 min).

BAR2140 C ₃₃ H ₂₉ Cl ₂ NO ₄

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.54-7.39 (9H, ovl), 7.27 (2H, d, J=7.8 Hz), 7.01, (2H, d, J=7.9Hz), 6.76(2H, d, J=8.0Hz), 4.98(2H, s), 4.79(2H, s), 4.64(2H, s) , 3.39 (1H, septet, J=7.0 Hz), 1.38 (6H, d, J=7.0 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.4, 159.1, 158.4, 158.2, 141.3, 141.2, 135.8 (2C), 133.7, 131.2 , 129.6, 129.2, 129.0, 128.2 (2C), 128.1 (2C), 127.8 (2C), 126.1, 125.4, 125.3, 115.1 ( 2C), 114.8 (2C), 109.4, 69.6, 65.3, 59.3, 27.0, 20.8 (2C),

EXAMPLE 2K. Preparation of 4'-((4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzyl)oxy)-[1,1'-biphenyl]-4-carboxylic acid (BAR2147 )

Purification by HPLC with Nucleodur Sphinx C18 5 µm phase (4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (90:10) as eluent (flow rate 1 ml/min) (t _R = 8 min).

BAR2147 C ₃₃ H ₂₇ Cl ₂ NO ₅

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.8 (2H, d, J=8.6 Hz), 7.6 (2H, d, J=8.6 Hz), 7.53 (2H, d, J=8.5Hz), 7.40(2H, d, J=7.9Hz), 7.32(3H, ovl), 7.05(2H, d, J=8.5Hz) , 6.81 (2H, d, J=8.2 Hz), 5.01 (2H, s), 4.73 (2H, s), 3.33 (1H, septet, J=7.0 Hz) , 1.42 (6H, d, J=7.0 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.3, 169.3, 159.4, 159.1, 158.2, 145.1, 135.8 (2C), 132.6 (2C), 131.7, 131.3, 129.3, 129.1(2C), 128.4(2C), 128.2(2C), 127.8, 127.1(2C), 115.4(2C) , 114.8 (2C), 109.3, 69.8, 59.4, 27.1, 20.8 (2C),

EXAMPLE 3. OBTAINING BAR2151, BAR2159, BAR2175, BAR2183

Starting with the BAR2112 alcohol and following the procedure described in Example 2 above (Scheme 2), alkylation with four different phenols via Mitsunobu reaction and hydrolysis gave the carboxylic acids BAR2151, BAR2159, BAR2175, BAR2183.

Scheme 3

a) PPh ₃ , DIAD, Phenols-COOCH ₃ , THF dry, 0°C; c) NaOH, MeOH: H ₂ O 1:1 v/v.

EXAMPLE 3A. Preparation of 4'-((3-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzyl)oxy)-[1,1'-biphenyl]-3-carboxylic acid (BAR2151 )

Purification by HPLC with Phenomenex C18 5 µm phase (4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (80:20) as eluent (flow rate 1 ml/min) gave BAR2151 (t _R = 11 minimum).

BAR2151 C ₃₃ H ₂₇ Cl ₂ NO ₅

¹ H NMR (CD3OD, 400 MHz): δ 8.27 (1H, br s), 8.02 (1H, d, J=7.8 Hz), 7.75 (1H, d, J=7.8 Hz), 7.56 (2H, d, J=8.2 Hz), 7.52 (1H, t, J=7.8 Hz), 7.40 (2H, d, J=8.0 Hz) , 7.32 (1H, t, J=8.0 Hz), 7.30 (1H, t, J=7.5 Hz), 7.04 (2H, d, J=8.2 Hz), 7 .00 (1H, d, J=7.5 Hz), 6.85 (1H, s), 6.80 (1H, d, J=7.5 Hz), 5.06 (2H, s), 4 .78 (2H, s), 3.33 (1H, septet, J=7.3 Hz), 1.42 (6H, d, J=7.3 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.4, 165.5, 159.1, 158.7, 158.5, 141.0, 137.7, 135.6 (2C), 132.3 , 131.8, 131.6, 131.4, 129.8, 129.5, 129.1, 128.9, 128.4 (2C), 128.2 (2C), 127.6, 120.4 , 115.1 (2C), 114.5, 113.7, 109.3, 69.8, 59.3, 27.0, 20.7 (2C).

EXAMPLE 3B. Preparation of 4'-((3-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzyl)oxy)-[1,1'-biphenyl]-4-carboxylic acid (BAR2159 )

Purification by HPLC with Nucleodur Sphinx C18 5 µm phase (4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (90:10) as eluent (flow rate 1 ml/min) gave BAR2159 ( t _R = 8 min).

BAR2159 C ₃₃ H ₂₇ Cl ₂ NO ₅

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.90 (2H, d, J=7.7 Hz), 7.70 (2H, d, J=7.7 Hz), 7.56 (2H, d, J=8.2Hz), 7.53(1H, t, J=8.0Hz), 7.40(2H, d, J=7.9Hz), 7.30(1H, t, J=7.9Hz), 7.05(2H, d, J=8.2Hz), 7.02(1H, d, J=8.0Hz), 6.88(1H, s), 6 .76 (1H, d, J=8.0 Hz), 5.03 (2H, s), 4.73 (2H, s), 3.33 (1H, septet, J=7.0 Hz), 1 .42 (6H, d, J=7.0 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.3, 165.3, 159.3, 159.1, 158.2, 141.1, 137.7, 135.8 (2C), 132.6 , 131.5, 131.2, 129.5, 129.2 (2C), 129.1, 128.1 (2C), 128.0 (2C), 123.8 (2C), 115.4 (2C ), 120.0, 114.5, 113.7, 109.3, 69.8, 59.4, 27.0, 20.7 (2C).

EXAMPLE 3C. Preparation of 4-((3-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzyl)oxy)benzoic acid (BAR2175)

Purification by HPLC with Nucleodur Sphinx C18 5 µm phase (4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (80:20) as eluent (flow rate 1 ml/min) gave BAR2175 ( t _R = 13 min).

BAR2175 C ₂₇ H ₂₃ Cl ₂ NO ₅

¹ H NMR (CDCl ₃ , 400 MHz): δ 8.05 (2H, d, J=8.7 Hz), 7.40 (2H, d, J=7.9 Hz), 7.32 (1H, t, J=7.9 Hz), 7.29 (1H, d, J=8.0 Hz), 7.25 (1H, t ovl), 7.0 (2H, d, J=8.7 Hz ), 6.85(1H, s), 6.76(1H, d, J=8.0Hz), 5.07(2H, s), 4.74(2H, s), 3.33(1H , septet, J=7.0 Hz), 1.44 (6H, d, J=7.0 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.4, 163.9, 159.3, 159.0, 158.5, 137.8, 135.8 (2C), 132.3 (2C), 131.5, 131.3, 129.7, 129.5, 128.0 (2C), 120.2, 114.6, 114.4 (2C), 113.6, 109.2, 69.9, 59.3, 27.0, 20.7 (2C).

EXAMPLE 3D. Preparation of 3-((3-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzyl)oxy)benzoic acid (BAR2183)

Purification by HPLC with Nucleodur Sphinx C18 5 µm phase (4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (80:20) as eluent (flow rate 1 ml/min) gave BAR2183 ( t _R = 15 min).

BAR2183 C ₂₇ H ₂₃ Cl ₂ NO ₅

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.72 (1H, d, J=7.5 Hz), 7.67 (1H, br s), 7.41 (1H, ovl), 7.40 (2H, d, ovl), 7.31 (1H, t, ovl), 7.30 (1H, tovl), 7.24 (1H, d, J=8.0 Hz), 7.20 (1H, d, J=8.2 Hz), 6.87 (1H, br s), 6.76 (1H, d, J=8.3 Hz), 5.04 (2H, s), 4.75 (2H , s), 3.34 (1H, septet, J=6.9 Hz), 1.42 (6H, d, J=6.9 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.7, 165.3, 159.0, 158.9, 158.5, 137.7, 135.7 (2C), 131.3, 129.6 , 129.3, 128.9, 128.4, 128.2 (2C), 120.9, 120.5, 116.1, 115.3, 114.7, 113.8, 110.1, 69, 6, 59.3, 27.0, 20.5 (2C).

EXAMPLE 4. OBTAINING BAR2163, BAR2171, BAR2199, BAR2207

Starting with the BAR2120 alcohol and following the procedure described in Examples 2 and 3 above (Scheme 2 or Scheme 3), alkylation with two different phenols and alkaline hydrolysis gave the carboxylic acids BAR2163, BAR2171, BAR2199 and BAR2207 (Scheme 4).

Scheme 4

a) PPh ₃ , DIAD, Phenols-COOCH ₃ , THF dry, 0°C; c) NaOH, MeOH : H ₂ O 1:1 v/v.

EXAMPLE 4A. Preparation of 4-((4'-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)-[1,1'-biphenyl]-3-yl)methoxy)benzoic acid (BAR2163 )

Purification by HPLC with Luna Polar C18 phase 5 µm (4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (85:15) as eluent (flow rate 1 ml/min) gave BAR2163 ( t _R = 13 minimum).

BAR2163 C ₃₃ H ₂₇ Cl ₂ NO ₅

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.60 (2H, d, J=8.8 Hz), 7.55 (1H, br s), 7.51 (1H, d, J=7 .9 Hz), 7.46 (2H, d, J=8.5 Hz), 7.42 (2H, d, J=7.9 Hz), 7.40 (1H, t, ovl), 7, 35 (1H, d, J=7.5 Hz), 7.33 (1H, t, J=7.9 Hz), 7.04 (2H, d, J=8.8 Hz), 6.87 ( 2H, d, J=8.5 Hz), 5.16 (2H, s), 4.77 (2H, s), 3.36 (1H, septet, J=7.0 Hz), 1.44 ( 6H, d, J=7.2 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.4, 165.3, 160.0, 159.1, 157.9, 141.3, 136.2, 135.8 (2C), 134.0 , 133.7, 131.1(3C), 129.2, 128.2(2C), 128.1(2C), 127.8, 126.7, 125.8(2C), 115.6(2C ), 115.0 (2C), 109.3, 70.3, 59.3, 27.1, 20.7 (2C).

EXAMPLE 4B. Preparation of 3-((4'-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)-[1,1'-biphenyl]-3-yl)methoxy)benzoic acid (BAR2171 )

Purification by HPLC with Luna Polar C18 phase 5 µm (4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (85:15) as eluent (flow rate 1 ml/min) gave BAR2171 ( t _R = 20 minimum).

BAR2171 C ₃₃ H ₂₇ Cl ₂ NO ₅

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.51 (1H, br s), 7.51-7.22 (12H, ovl) 6.87 (2H, d, J=8.4 Hz), 5.10 (2H, s), 4.78 (2H, s), 3.36 (1H, septet, J=7.3 Hz), 1.44 (6H, d, J=7.3 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.5, 165.3, 159.1, 158.7, 157.9, 141.2, 136.3, 135.8 (2C), 133.8 , 131.2, 130.4, 129.2, 128.2 (2C), 128.1 (2C), 126.7, 125.8 (2C), 124.8, 120.5, 118.7, 117.8, 115.1(2C), 113.3, 109.4, 70.4, 59.5,27.0, 20.7(2C).

EXAMPLE 4C. Preparation of 3-((4'-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)-[1,1'-biphenyl]-4-yl)methoxy)benzoic acid (BAR2199 )

Purification by HPLC with Luna Polar C18 phase 5 µm (4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (85:15) as eluent (flow rate 1 ml/min) gave BAR2199 ( t _R = 23 minimum).

BAR2199 C ₃₃ H ₂₇ Cl ₂ NO ₅

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.57 (1H, br s), 7.22-7.51 (12H, ovl) 6.87 (2H, d, J=8.1 Hz), 5.16 (2H, s), 5.00 (2H, s), 3.33 (1H, septet, J=7.0 Hz), 1.44 (6H, d, J=7.0 Hz); ¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.5, 169.3, 160.1, 159.3, 158.1, 141.0, 135.8 (2C), 135.6, 133.0 , 131.2(2C), 130.5(2C), 130.1(2C), 129.6, 128.7, 128.2(2C), 128.1(2C), 122.6, 119, 5, 114.9 (2C), 114.4, 109.3, 70.6, 59.8, 27.0, 20.7 (2C).

EXAMPLE 4D. Preparation of 4-((4'-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)-[1,1'-biphenyl]-4-yl)methoxy)benzoic acid (BAR2207 )

Purification by HPLC with Luna Polar C18 phase 5 µm (4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (85:15) as eluent (flow rate 1 ml/min) gave BAR2207 ( t _R = 15 minimum).

BAR2207 C ₃₃ H ₂₇ Cl ₂ NO ₅

¹ H NMR (CDCl ₃ , 400 MHz): δ 8.02 (2H, d, J=8.2 Hz), 7.61 (2H, d, J=8.0 Hz), 7.47 (2H, d, J=7.7 Hz), 7.40 (2H, d, J=7.9 Hz), 7.39 (2H, d, J=7.7 Hz), 7.32 (1H, t, J=7.9Hz), 7.03(2H, d, J=8.0Hz), 6.90(2H, d, J=8.2Hz), 5.20(2H, s), 5 .05 (2H, s), 3.33 (1H, septet, J=7.0 Hz), 1.45 (6H, d, J=7.0 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.3, 165.5, 159.1, 158.7, 158.5, 141.1, 135.8 (2C), 133.0, 132.6 , 131.7, 131.3 (2C), 131.2, 129.2 (2C), 129.1 (2C), 128.9, 128.2 (2C), 128.0 (2C), 115, 4 (2C), 114.9 (2C), 109.3, 70.5, 59.3, 27.0, 20.7 (2C).

EXAMPLE 5 OBTAINING BAR2222-BAR2226, BAR2227 and BAR2228

Following the same procedure as described above for the synthesis of the carboxylic acid BAR2119 (Scheme 1 and Example 12A), and alternately changing the starting material (various aldehydes) or various β-ketoesters to form isoxazole, BAR2222-BAR2226, BAR2227 and BAR2228 were synthesized, respectively. .

Example 5A. Preparation of 4'-((3-(2-chlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)-[1,1'-biphenyl]-3-carboxylic acid (BAR2222)

Purification by HPLC on a Nucleodur Sphinx RP C18 phase column (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (80:20) as eluent (flow rate 1 ml/ min), pure BAR2222 was formed (t _R = 13.1 min).

BAR2222 C ₂₆ H ₂₂ ClNO ₄

¹ H NMR (CDCl ₃ , 400 MHz): δ 8.27 (1H, br s) 8.04 (1H, d, J=7.32 Hz), 7.75 (1H, d, J=7.32 Hz), 7.48-7.51 (5H, m, ovl), 7.42 (1H, t, J=7.41 Hz), 7.35 (1H, t, J=7.41 Hz), 6.86 (2H, d, J=8.05 Hz), 4.84 (2H, s), 3.35 (1H, septet, J=6.93 Hz), 1.44 (6H, d, J = 6.93 Hz).

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.5 (2C), 161.1, 157.6, 140.0, 133.3, 133.1, 131.7, 131.6, 130.9 , 130.8, 129.7, 129.6, 128.2, 128.1(2C), 127.9, 127.8, 126.8, 114.8(2C), 109.2, 59.4 , 26.9, 20.6 (2C).

Example 5B. Preparation of 4'-((3-(2-bromophenyl)-5-isopropylisoxazol-4-yl)methoxy)-[1,1'-biphenyl]-3-carboxylic acid (BAR2223)

Purification by HPLC on a Nucleodur Sphinx RP C18 phase column (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (80:20) as eluent (flow rate 1 ml/ min), pure BAR2223 was formed (t _R = 12.6 min).

BAR2223 C ₂₆ H ₂₂ BrNO ₄

¹ H NMR (CDCl ₃ , 400 MHz): δ 8.26 (1H, br s) 8.04 (1H, d, J=7.85 Hz), 7.77 (1H, d, J=7.85 Hz), 7.64 (1H, d, J=8.20 Hz), 7.53 (1H, t, J=7.85 Hz), 7.51 (1H, t, ovl), 7.51 ( 2H, d, J=8.61Hz), 7.18(1H, t, J=8.20Hz), 6.88(2H, d, J=8.61Hz), 4.79(2H, s), 3.37 (1H, septet, J=7.07 Hz), 1.45 (6H, d, J=7.07 Hz).

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.6, 171.4, 162.6, 157.8, 140.4, 133.6, 132.9, 131.8, 131.1, 130.4, 130.3, 128.5, 128.2, 128.13(2C), 128.1, 127.4, 126.2, 123.0, 115.1(2C), 109.2, 59.6, 27.0, 20.7 (2C).

Example 5C. Preparation of 4'-((5-isopropyl-3-(2-trifluoromethyl)phenyl)isoxazol-4-yl)methoxy)-[1,1'-biphenyl]-3-carboxylic acid (BAR2224)

Purification by HPLC on a Nucleodur Sphinx RP C18 phase column (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (80:20) as eluent (flow rate 1 ml/ min), pure BAR2224 was formed (t _R = 10.8 min).

BAR2224 C ₂₇ H ₂₂ F ₃ NO ₄

¹ H NMR (CDCl ₃ , 400 MHz): δ 8.25 (1H, br s) 8.03 (1H, d, J=7.90 Hz), 7.81 (1H, d, J=7.66 Hz), 7.76 (1H, d, J=7.66 Hz), 7.61 (1H, m, ovl), 7.60 (1H, m, ovl), 7.52-7.51 (4H , ovl), 6.88 (2H, d, J=8.72 Hz), 4.72 (2H, s), 3.33 (1H, septet, J=7.03 Hz), 1.44 (6H , d, J=7.03 Hz).

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.4, 171.0, 161.3, 158.1, 140.9, 133.1, 132.0, 131.8, 131.5, 129, 9, 129.8, 129.7, 128.9, 128.4, 128.3, 128.2 (2C), 126.5, 126.4, 122.2, 115.1 (2C), 109, 4, 59.1, 27.0, 20.8 (2C).

Example 5D. Preparation of 4'-((3-(2-bromo-6-chlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)-[1,1'-biphenyl]-3-carboxylic acid (BAR2225)

Purification by HPLC on a Nucleodur Sphinx RP C18 phase column (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (80:20) as eluent (flow rate 1 ml/ min), pure BAR2225 was formed (t _R = 36.7 min).

BAR2225 C ₂₆ H ₂₁ BrClNO ₄

¹ H NMR (CDCl ₃ , 400 MHz): δ 8.23 (1H, br s) 8.00 (1H, d, J=7.77 Hz), 7.71 (1H, d, J=7.77 Hz), 7.58 (1H, d, J=7.77 Hz), 7.44-7.46 (4H, m, ovl), 7.23 (1H, t, J=8.55 Hz), 6.85 (2H, d, J=8.73 Hz), 4.76 (2H, br s), 3.35 (1H, septet, J=7.03 Hz), 1.43 (6H, d, J=7.03 Hz).

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.4, 171.7, 160.6, 158.2, 140.9, 135.7, 133.0, 131.8, 131.6, 131, 2, 129.7, 128.9, 128.6, 128.4, 128.3(2C), 128.1(2C), 125.0, 115.2(2C), 109.1, 59.4 , 27.0, 20.7 (2C).

Example 5E. Preparation of 4'-((3-(2,6-dibromophenyl)-5-isopropylisoxazol-4-yl)methoxy)-[1,1'-biphenyl]-3-carboxylic acid (BAR2226)

Purification by HPLC on a Nucleodur Sphinx RP C18 phase column (5 µm; 4.6 mm i.d. × 250 mm) using MeOH/H ₂ O (80:20) as eluent (flow rate 1 ml/ min), pure BAR2226 was formed (t _R = 13.3 min).

¹ H NMR (CDCl ₃ , 400 MHz): δ 8.26 (1H, br s) 8.04 (1H, d, J=7.77 Hz), 7.77 (1H, d, J=7.77 Hz), 7.64 (2H, d, J=8.08 Hz), 7.53 (1H, t, J=7.77 Hz), 7.51 (2H, d, J=8.78 Hz) , 7.18 (1H, t, J=8.08 Hz), 6.90 (2H, d, J=8.78 Hz), 4.79 (2H, s), 3.37 (1H, septet, J=7.03 Hz), 1.45 (6H, d, J=7.03 Hz).

¹³ C NMR (CDCl ₃ , 100 MHz): δ 176.5, 171.4, 161.9, 158.1, 140.7, 132.8, 131.9, 131.8, 130.8, 129, 7, 128.7, 128.3, 128.2, 128.2 (2C), 124.9, 124.1, 123.9, 115.0 (2C), 108.9, 59.4, 27, 1, 20.8 (2C).

Example 5F. Preparation of 4'-((3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl)methoxy)-[1,1'-biphenyl]-3-carboxylic acid (BAR2227)

Purification with silica gel after step c gave BAR2227 (98%). An analytical sample was prepared by HPLC with Luna C18 phase (5 µm; 4.6 mm i.d. × 250 mm), buffer B gradient from 40% to 95% in 13 min (buffer A = 95 H ₂ O : 5 CH ₃ CN : 0.1 TFA buffer B = 95 CH ₃ CN : 5 H ₂ O : 0.1 TFA) as eluent (flow rate 1.5 ml/min) (t _R = 13.3 min) .

BAR2227 C ₂₄ H ₁₇ Cl ₂ NO ₄

¹ H NMR (CDCl ₃ , 400 MHz): δ 8.27 (1H, s), 8.04 (1H, d, J=7.7 Hz), 7.77 (1H, d, J=7.7 Hz), 7.51 (2H, d, J=8.2 Hz), 7.53 (1H, t, J=7.7 Hz), 7.43 (2H, d, J=8.0 Hz) , 7.34 (1H, t, J=8.0 Hz), 6.89 (2H, d, J=8.2 Hz), 4.80 (2H, s), 2.59 (3H, s) ;

¹³ C NMR (CDCl ₃ , 100 MHz): δ 168.6, 165.4, 159.0, 158.1, 141.0, 135.8 (2C), 133.1, 131.8 (2C), 131.3, 129.0, 128.5, 128.4, 128.2(2C), 128.1(2C), 127.8, 115.1(2C), 111.2, 59.7, 11 ,6.

5G example. Preparation of 4'-((3-(2,6-dichlorophenyl)-5-propylisoxazol-4-yl)methoxy)-[1,1'-biphenyl]-3-carboxylic acid (BAR2228)

Purification with silica gel after step c gave BAR2228 (quantitative). An analytical sample was prepared by HPLC with Luna C18 phase (5 µm; 4.6 mm i.d. × 250 mm), buffer B gradient from 40% to 95% in 13 min (buffer A = 95 H ₂ O : 5 CH ₃ CN : 0.1 TFA buffer B = 95 CH ₃ CN : 5 H ₂ O : 0.1 TFA) as eluent (flow rate 1.5 ml/min) (t _R = 14.7 min) .

BAR2228 C ₂₆ H ₂₁ Cl ₂ NO ₄

¹ H NMR (CDCl ₃ , 400 MHz): δ 8.26 (1H, s), 8.04 (1H, d, J=7.8 Hz), 7.76 (1H, d, J=7.8 Hz), 7.50 (2H, d, J=8.5 Hz), 7.52 (1H, t, J=7.8 Hz), 7.42 (2H, d, J=8.0 Hz) , 7.34 (1H, t, J=8.0 Hz), 6.88 (2H, d, J=8.5 Hz), 4.78 (2H, s), 2.91 (2H, t, J=7.3 Hz), 1.86 (2H, sextet, J=7.3 Hz), 1.04 (3H, t, J=7.3 Hz);

¹³ C NMR (CDCl ₃ , 100 MHz): δ 172.2, 165.2, 159.0, 158.1, 141.0, 135.8 (2C), 133.0, 131.9, 131.3 (2C), 129.0, 128.5, 128.4, 128.2 (2C), 128.1 (2C), 127.9, 115.1 (2C), 111.0, 59.5, 27 .9, 21.1, 13.6.

EXAMPLE 6. BIOLOGICAL ACTIVITY

The biological activity of selected compounds (Table 1) was tested in vitro using a cellular model transfected with reporter genes at the FXR receptor and compared with the control agonist, chenodeoxycholic acid (CDCA), a primary bile acid that functions as the receptor's endogenous ligand.

HepG2 cells were cultured at 37° C. in E-MEM (Minimum Earl's Salt Medium) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/streptomycin. Transfection experiments were performed using the Fugene HD reagent (Promega) according to the manufacturer's instructions. Cells were seeded in 24-well plates at a density of 5×10 ⁴ cells/well.

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

24 hours after transfection, cells were stimulated with compounds and CDCA as a positive control. To evaluate the EC ₅₀ , dose-response curves were drawn in HepG2 cells transfected as described above and treated with increasing concentrations of compounds (0.1, 0.5, 1, 2.5, 5, 7.5, 10, 12 .5, 25 and 50 µM) (Table 1). After treatment, cells were lysed in 100 μl of lysis buffer (25 mM Tris-phosphate, pH 7.8; 2 mM DTT; 10% glycerol; 1% Triton X-100); 10 μl of cell lysate of each sample was analyzed for luciferase activity using the Dual Luciferase Reporter Assay System (Promega Italia Srl, Milan, Italy) according to the manufacturer's instructions. Luminescence was measured with a Glomax 20/20 luminometer (Promega Italia Srl, Milan, Italy). Luciferase activity (Luciferase Detection Unit, LRL) was normalized with Renilla activity (Renilla Registry Unit, RRR).

FXR activation was also measured in a cell-free assay by alpha screening technology using the Coactivator Recruitment Assay. Anti-GST coated acceptor beads were used to capture the FXR-LBD GST fusion, while the biotinylated SRC-1 peptide was captured by streptavidin donor beads. When illuminated at 680 nm, chemical energy is transferred from the donor to the acceptor beads through the streptavidin-donor/SRC-l-biotin/GSTFXR-LBD/anti-GST acceptor complex and a signal is generated. The assay was performed in white, low-volume, 384-well Optiplates (PerkinElmer) using a final volume of 25 μl containing final concentrations of 10 nM purified GST-labeled FXR-LBD protein, 30 nM biotinylated SRC-1 peptide, 20 mg/ml acceptor beads coated with anti-GST and 10 mg/ml streptavidin donor beads (PerkinElmer). The assay buffer contained 50 mM Tris (pH 7.4), 50 mM potassium chloride (KCl) and 1 mM DTT. The stimulation time for 1 µl of test compound (each at a final concentration of 5 µM) was recorded as 30 minutes at room temperature. The DMSO concentration in each well was maintained at a final concentration of 2%. After addition of the detection mixture (acceptor and donor beads), the plates were incubated in the dark for 3 hours at room temperature and then read in an Envision microplate analyzer (PerkinElmer).

Table 1 shows the efficacy of selected compounds included in Formula (I) in recruiting the SRC-1 coactivator as a percentage of the compound's maximum efficacy relative to CDCA, set at 100%. Results are expressed as the mean of three independent measurements ± standard error. Each ligand was tested at a concentration of 5 μM.

Table 1 presents the potency of the selected compounds included in Formula (I) as EC ₅₀ values (µM) calculated from a transactivation assay based on at least three experiments. Results are expressed as mean ± standard error.

To study the specificity of compounds versus PPARy, HepG2 cells were transiently transfected with 200 ng p(UAS)5XTKLuc reporter vector, 100 ng pGL4.70, and a vector containing the PPARy nuclear receptor ligand binding domain cloned upstream of the GAL4 DNA-binding domain (pSG5 -PPARγLBD-GAL4DBD).

To test the specificity of compounds versus GPBAR1, HEK-293T cells were transiently transfected with the Fugene HD reagent (Promega) using the following vectors: human pCMVSPORT6-GPBAR1, pGL4.29 (Promega), cAMP Response Element (CRE) Reporter Vector ) cloned upstream of the luc2P luciferase reporter gene and pGL4.70.

To investigate compound specificity versus LXRα and LXRβ mediated transactivation, HepG2 cells were transfected with 200 ng p(UAS)5XTKLuc reporter vector, 100 ng vector containing LXRα or LXRβ ligand binding domain cloned upstream of the GAL4 DNA binding domain (i.e. , pSG5-LXRαLBD-GAL4DBD or pSG5-LXRβLBD-GAL4DBD), and 100 ng of pGL4.70 (Promega), a vector encoding the human Renilla gene.

24 hours after transfection, cells were stimulated with specific receptor agonists GW3965 (10 μM), Rosiglitazone (500 nM) and TLCA (10 μM), respectively, or compounds (10 μM).

18 hours after stimulation, the cell lysate was analyzed for luciferase and Renilla activity using the Dual Luciferase Reporter Assay System (E1980, Promega). Luminescence was measured with a Glomax 20/20 luminometer (Promega). Luciferase activity was normalized with Renilla activity.

None of the compounds of Formula (I) showed agonistic/antagonistic activity against LXRs, PPARγ and GPBAR1.

RNA isolation and RT-PCR. HepG2 cells were seeded at a density of 1×10 ⁶ cells/flask in a T25 flask. After overnight incubation, the cells were not fed and then stimulated for 18 hours with 10 μM GW3965 or compounds (0.1, 1 and 5 μM).

Whole RNA was isolated from HepG2 cells or liver tissue using the TPIzol reagent according to the manufacturer's specifications (Invitrogen). One microgram of purified RNA was treated with DNase-I and reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). For real-time PCR, 10 ng of the template was dissolved in 25 µl containing 200 nmol/l of each primer and 12.5 µl of 2x SYBR FAST Universal Premix (Invitrogen). All reactions were run in triplicate and the thermal cycling conditions were as follows: 2 min at 95°C followed by 40 cycles at 95°C for 20 sec and 60°C for 30 sec on a StepOnePlus cycler (Applied Biosystems). Relative mRNA expression was calculated according to the Ct threshold method. The forward and reverse primer sequences were as follows: human GAPDH: gaaggtgaaggtcggagt (SEQ ID: 1) and catgggtggaatcatattggaa (SEQ ID: 2); Human SHP: gctgtctggagtccttctgg (SEQ ID: 3) and ccaatgatagggcgaaagaag (SEQ ID: 4); Mouse GAPDH, ctgagtatgtcgtggagtctac (SEQ ID: 5) and gttggtggtgcaggatgcattg (SEQ ID: 6); mouse SHP acgatcctcttcaacccaga (SEQ ID: 7) and agggctccaagacttcacac (SEQ ID: 8); Mouse FXR: agcttccagggtttcagaca (SEQ ID: 9) and cttccaacaggtctgcatga (SEQ ID: 10); Mouse BSEP: gatgcttcccaagttcaagg (SEQ ID: 11) and taaagaggaaggcgatgagc (SEQ ID: 12).

Animals and protocols. Male C57BL/6N mice were provided by Jackson's Laboratory. Mice were kept under controlled temperature (22°C) and daylight hours (12:12 hour light/dark cycle), mice had unlimited access to standard mouse chow and drinking water, and mice were allowed to acclimatize to these conditions for a period of at least 5 days before inclusion in the experiment. The experimental protocol was approved by the Committee for the Care and Use of Animals of the University of Perugia, as well as the Minister of Health of Italy and the National Institute of Health (Italy), and it complied with the European Guidelines for the Use of Experimental Animals (approval no. 214/2017-PR). The general condition of the animals was monitored daily by a veterinarian in the vivarium. To evaluate in vivo intestinal absorption and successful liver transport, mice were injected with 10 mg/kg of BAR2109 dissolved in methylcellulose daily by gavage (OS) or ip for 3 days. At the end of treatment, the animals were sacrificed and blood and liver were collected for further analysis.

microsomal stability. Male mouse liver microsomes (CD-1) (Sigma-Aldrich) were used. All incubations were performed in duplicate in a shaking water bath at 37°C. Incubation mixtures contained 1 μM compound with 1% DMSO used as an auxiliary transport agent, mouse liver microsome (0.3 mg microsomal protein per ml), 5 mM MgCl ₂ , 1 mM NADP, 5 mM glucose-6 phosphate, 0.4 U/ml ^-1 glucose-6-phosphate dehydrogenase and 50 mM potassium phosphate buffer (pH 7.4) in a final volume of 0.5 ml. Aliquots were removed at 0, 5, 10, 20, 30 and 40 minutes after microsome addition, and the reaction was stopped by adding 200 μl of ice-cold acetonitrile. After 2 hours, the samples were centrifuged for 10 min at 10,000 rpm and the supernatants were transferred into standard calibration cuvettes for LC-MS/MS analysis. Propranolol, known as a drug with high hepatic clearance in rodents, has been used as a quality control compound for microsomal incubations. The linear regression slope of the resulting curve showing the natural logarithm of joint area versus incubation time (-k) was used when converted to in vitro t _1/2 values by t _1/2= -ln(2)/k. In vitro intrinsic clearance (CL _int , expressed in µl/min/mg) was calculated using the following formula: Cl _int = reaction volume (µl)/t _1/2 (min)/liver microsome protein (mg). The percentage of unmodified compound was calculated assuming that the peak area of the compound at time 0 min is 100%.

(мин) 44), BAR2109 (Cl_int 32 мкл/мин/мг,

(мин) 72) и BAR2123 (Cl_int 35 мкл/мин/мг,

(мин) 66), демонстрирующие повышенную метаболическую стабильность в условиях in vitro по отношению к эталонному соединению GW4064 (Clint 56 мкл/мин/мг,

(мин) 41).Preferred examples included in the general formula are BAR2119 ( _Clint 53 µl/min/mg,

(min) 44), BAR2109 (Cl _int 32 µl/min/mg,

(min) 72) and BAR2123 (Cl _int 35 µl/min/mg,

(min) 66), demonstrating increased in vitro metabolic stability relative to reference compound GW4064 (Clint 56 µl/min/mg,

(min) 41).

Preferred examples included in the general formula are BAR2109 and BAR2123, which induce SHP mRNA expression at a concentration of 5 μM by 6, 8 times, and 6.2 times, respectively (reference compound GW4064 was tested at a concentration of 10 μM; induction of SHP mRNA expression = 4, 2 times).

A preferred example included in the general formula is BAR2109, which induces expression of SHP mRNA and BSEP mRNA in the liver when administered orally.

--->

SEQUENCE LIST

<110> BAR PHARMACEUTICALS SOCIETY A LIMIT RESPONSIBILITY

<120> ISOXAZOLE AS FXR RECEPTOR AGONIST

<130> 557-18

<160> 12

<170>BiSSAP 1.3.6

<210> 1

<211> 18

<212> DNA

<213> Faux Sequence

<220>

<223> direct primer

<400> 1

gaaggtgaaggtcggagt 18

<210> 2

<211> 22

<212> DNA

<213> Faux Sequence

<220>

<223> reverse primer

<400> 2

catgggtggaatcatattggaa 22

<210> 3

<211> 20

<212> DNA

<213> Faux Sequence

<220>

<223> Forward primer

<400> 3

gctgtctggagtccttctgg 20

<210> 4

<211> 21

<212> DNA

<213> Faux Sequence

<220>

<223> reverse primer

<400> 4

ccaatgatagggcgaaagaa g 21

<210> 5

<211> 22

<212> DNA

<213> Faux Sequence

<220>

<223> Forward primer

<400> 5

ctgagtatgtcgtggagtctac 22

<210> 6

<211> 22

<212> DNA

<213> Faux Sequence

<220>

<223> reverse primer

<400> 6

gttggtggtgcaggatgcattg 22

<210> 7

<211> 20

<212> DNA

<213> Faux Sequence

<220>

<223> Forward primer

<400> 7

acgatcctcttcaacccaga 20

<210> 8

<211> 20

<212> DNA

<213> Faux Sequence

<220>

<223> reverse primer

<400> 8

agggctccaagacttcacac 20

<210> 9

<211> 20

<212> DNA

<213> Faux Sequence

<220>

<223> direct primer

<400> 9

agcttccagggtttcagaca 20

<210> 10

<211> 20

<212> DNA

<213> Faux Sequence

<220>

<223> reverse primer

<400> 10

cttccaacaggtctgcatga 20

<210> 11

<211> 20

<212> DNA

<213> Faux Sequence

<220>

<223> direct primer

<400> 11

gatgcttcccaagttcaagg 20

<210> 12

<211> 20

<212> DNA

<213> Faux Sequence

<220>

<223> reverse primer

<400> 12

taaaggaaggcgatgagc 20

<---

Claims (50)

1. Compound of formula (I):

or pharmaceutically acceptable salts, characterized in that R ₁ and R ₂ are independently selected from the group consisting of H, halogen and CF ₃ provided that R ₁ and R ₂ are not H at the same time; R ₃ is C ₁ -C ₃ alkyl; n is an integer selected from 1, 2 and 3; R ₄ is selected from the group consisting of phenyl unsubstituted or substituted with one R ₅ and biphenyl unsubstituted or substituted with one R ₅ ; R ₅ is selected from the group consisting of COOR ₆ , CN, hydroxy-C ₁ -C ₃ alkyl, C ₁ -C ₃ alkyl-O-phenyl unsubstituted or substituted with one R ₇ , and C ₁ -C ₃ alkyl-O-biphenyl , unsubstituted or substituted with one R ₇ ; R ₆ is selected from the group consisting of H and C ₁ -C ₃ alkyl, and R ₇ is selected from the group consisting of COOR ₆ , CN and hydroxy-C ₁ -C ₃ alkyl; provided that the compound is not 4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)benzoic acid. 2. The compound according to claim 1, characterized in that R ₄ is selected from the group consisting of

. 3. Connection according to any one of paragraphs. 1 or 2, characterized in that R ₅ is selected from the group consisting of COOH, COOCH ₃ , CN, -CH ₂ OH,

. 4. The connection according to any of the previous paragraphs, characterized in that it is selected from the group including:

5. Pharmaceutical composition for the prevention and/or treatment of FXR-dependent diseases, containing a compound of formula (I):

, characterized in that R ₁ and R ₂ are independently selected from the group consisting of H, halogen and CF ₃ , provided that R ₁ and R ₂ are not H at the same time; R ₃ is C ₁ -C ₃ alkyl; n is an integer selected from 1, 2 and 3; R ₄ is selected from the group consisting of phenyl unsubstituted or substituted with one R ₅ and biphenyl unsubstituted or substituted with one R ₅ ; R ₅ is selected from the group consisting of COOR ₆ , CN, hydroxy-C ₁ -C ₃ alkyl, C ₁ -C ₃ alkyl-O-phenyl unsubstituted or substituted with one R ₇ , and C ₁ -C ₃ alkyl-O-biphenyl , unsubstituted or substituted with one R ₇ ; R ₆ is selected from the group consisting of H and C ₁ -C ₃ alkyl, and R ₇ is selected from the group consisting of COOR ₆ , CN and hydroxy-C ₁ -C ₃ alkyl; or pharmaceutically acceptable salts and at least one pharmaceutically acceptable excipient. 6. The use of a compound of formula (I) as a drug for the prevention and/or treatment of FXR-dependent diseases, wherein the compound of formula (I) is:

, characterized in that R ₁ and R ₂ are independently selected from the group consisting of H, halogen and CF ₃ , provided that R ₁ and R ₂ are not H at the same time; R ₃ is C ₁ -C ₃ alkyl; n is an integer selected from 1, 2 and 3; R ₄ is selected from the group consisting of phenyl unsubstituted or substituted with one R ₅ and biphenyl unsubstituted or substituted with one R ₅ ; R ₅ is selected from the group consisting of COOR ₆ , CN, hydroxy-C ₁ -C ₃ alkyl, C ₁ -C ₃ alkyl-O-phenyl unsubstituted or substituted with one R ₇ , and C ₁ -C ₃ alkyl-O-biphenyl , unsubstituted or substituted with one R ₇ ; R ₆ is selected from the group consisting of H and C ₁ -C ₃ alkyl, and R ₇ is selected from the group consisting of COOR ₆ , CN and hydroxy-C ₁ -C ₃ alkyl; or their pharmaceutically acceptable salts. 7. The use according to claim 6, characterized in that the FXR-dependent disease is selected from the group including disorders of the gastrointestinal tract, liver diseases, disorders of the cardiovascular system, vascular diseases, pulmonary diseases, pathologies of metabolic processes, infectious diseases, cancer, renal dysfunction, inflammatory diseases, including immune-mediated, and neurological disorders. 8. Use according to claim 7, characterized in that said disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, scleroderma, spondylitis, vasculitis, sarcoidosis, Mediterranean fever, polymyositis and dermatomyositis, Behcet's syndrome, acquired immunodeficiency and related disorders, B and C virus infections, Alzheimer's disease and other forms of dementia, Parkinson's disease and other movement disorders, amyotrophic lateral sclerosis and other motor neuron disorders, multiple sclerosis and other demyelinating diseases, myasthenia gravis and muscular dystrophy, primary biliary cirrhosis, cerebrotendinal xanthomatosis, primary sclerosing cholangitis, drug-induced cholestasis, intrahepatic cholestasis of pregnancy, cholestasis associated with parenteral nutrition, cholestasis associated with bacterial proliferation or sepsis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis , liver transplantation, congenital liver fibrosis, granulomatous liver disease, intra- or extrahepatic malignancy, Wilson's disease, hemochromatosis, alpha-1 antitrypsin deficiency, inflammatory bowel disease, Crohn's disease, ulcerative rectocolitis, indeterminate colitis, irritable bowel syndrome, bacterial proliferation , acute and chronic pancreatitis, malabsorption, post-radiation colitis, microscopic colitis, diabetic nephropathy, hypertensive nephropathy, chronic glomerulonephritis, chronic glomerulonephritis after transplantation, chronic tubulointerstitial diseases, vascular diseases of the kidneys, atherosclerosis, arteriosclerosis, dyslipidemia, hypercholesterolemia, hypertriglyceridemia, inflammatory heart disease, myocarditis, endocarditis, ischemic heart disease, stable angina, unstable angina, myocardial infarction, cardiovascular disease, ischemic stroke, pulmonary hypertension, peripheral arterial disease, peripheral arterial occlusive disease, peripheral arteriopathy obliterans, asthma, cystic fibrosis, obstructive pulmonary diseases, interstitial lung diseases, primary or secondary pulmonary fibrosis, insulin resistance, metabolic syndrome, type I and type II diabetes, hypoglycemia, adrenal cortical disease, adrenal insufficiency, obesity, diseases associated with bariatric surgery, liver cancer, various types of bile cancer duct cancer, esophageal cancer, pancreatic cancer, gastric cancer, colon and rectal cancer, breast cancer, ovarian cancer and chemotherapy resistance disease. 9. Application according to any one of paragraphs. 6-8, characterized in that the compound of formula (I) is selected from the group consisting of: