CN112805279B - Isoxazole derivative, preparation method and application thereof - Google Patents

Abstract

Relates to an isoxazole derivative, a preparation method and application thereof. In particular, farnesoid X Receptor (FXR) agonist compounds and stereoisomers, tautomers, polymorphs, solvates (e.g., hydrates), pharmaceutically acceptable salts, esters, metabolites, N-oxides and chemically protected forms and prodrugs thereof are provided. Also provided are methods of preparing the compounds, intermediates, pharmaceutical compositions and kits comprising the compounds, and their use for treating FXR mediated diseases or conditions.

Description

Isoxazole derivative, preparation method and application thereof

Technical Field

The present invention relates generally to compounds useful in the treatment of diseases or conditions mediated by the Farnesoid X Receptor (FXR), and more particularly to FXR agonist compounds, and stereoisomers, tautomers, polymorphs, solvates (e.g., hydrates), pharmaceutically acceptable salts, esters, metabolites, N-oxides, and chemically protected forms and prodrugs thereof. The invention also relates to processes for the preparation of said compounds, intermediates, pharmaceutical compositions and kits comprising said compounds and their therapeutic uses.

Background

Farnesoid X receptors (FXR, NR1H 4) are expressed in the liver, the entire gastrointestinal tract including esophagus, stomach, duodenum, small intestine, colon, kidneys and adrenal glands (Kupers, F. Et al, the Farnesoid X Receptor (FXR) as Modulator of Bile Acid Metabolism, rev. Endocrine Metab. Disorders,2004, 5:319-326). FXR is a member of the transcription factors known to be activated as ligands for nuclear receptors. Bile acids such as chenodeoxycholic acid (CDCA) or taurine or glycine amide conjugates thereof are endogenous ligands for FXR. FXR is activated upon binding of bile acids to FXR, and expression of various genes is controlled by heterodimer complexes with Retinol X Receptor (RXR), including gene expression involved in bile acid, cholesterol, triglycerides, lipoprotein homeostasis in the liver and circulatory system (kalan, n.y.; mangelsdorf, d.j.; LXRS and FXR: the yin and yang of cholesterol and fat metabolism, annu. Rev. Physiol.,2006, 68, 159-191; calkin, a.c.; tonthoz, p.; transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR, nat.Rev.Mol.Cell biol.; 2012, 13, 213-224). FXR also appears to be involved in paracrine and endocrine signaling by upregulation of either fibroblast growth factor 15 (rodents) or fibroblast growth factor 19 (monkeys, humans) (t.inagaki et al Fibroblast growth factor 15functions asan enterohepatic signal to regulate bile acid homeostasis.Cell Metab, 2005,2 (4), 217-225).

Bile acids are amphiphilic molecules that form micelles and emulsify lipids in the diet. Cytotoxicity is also generated if the concentration of bile acid is too high, so there is a mechanism of strictly controlling the concentration of bile acid physiologically. FXR plays a key role in controlling bile acids to remain stable in vivo (Makishima, M.; nuclear Receptors as Targets for Drug Development: regulation of Cholesterol and Bile Acid Metabolism byNuclear Receptors, J.Pharmacol. Sci.; 2005, 97:177-183).

Furthermore, FXR has also been demonstrated to regulate complex biological processes beyond metabolism, such as liver regeneration or intestinal barrier integrity. FXR also has control over the immune system of the intestine and liver, and has a certain anti-inflammatory effect (Modica, S.; gadaleta, R.M.; moschetta, A.; deciphering the nuclear bile acid receptor FXR paradigm, nucl. Recept. Signal.; 2010,8, e 005).

Obeticholic Acid (6-Et CDCA) is an FXR receptor agonist that is more active than the endogenous ligand CDCA, and shows significant improvement in insulin sensitivity and other metabolic beneficial effects in phase IIa clinical studies of non-alcoholic fatty liver disease (NAFLD) (Mudaliar, S.; henry, r.r.; sanyal, a.j., et al, efficacy and safety of the farnesoid X receptor agonist Obeticholic Acid in patientts with type 2diabetes and nonalcoholic fatty liver disease,Gastroenterology,2013, 145, 574-582). Phase IIb studies of obeticholic acid showed that 72 weeks of treatment was also beneficial for histopathological improvement of non-alcoholic hepatitis (NASH). In phase III studies of Primary Biliary Cirrhosis (PBC), patients were improved for liver function damage (Nevens, F., andreone, P., mazzella, G., et al, the first Primary Biliary Cirrhosis (PBC) phase 3trial in two decades-an international study of the FXR agonist obeticholic acid in PBC patients, J.hepatol.,2014, 60, S525-S526).

WO2012087519 discloses a method for treating FXR mediated disorders by agonists or partial agonists of FXR. However, FXR agonist compounds disclosed in the prior art still have drawbacks in terms of pharmacodynamic or pharmacokinetic properties.

Summary of The Invention

The present invention relates generally to compounds of formula (I) or stereoisomers, tautomers, polymorphs, solvates (e.g. hydrates), pharmaceutically acceptable salts, esters, metabolites, N-oxides, chemically protected forms or prodrugs thereof,

wherein:

x is CH or N; and is also provided with

R is selected from hydrogen, halogen and C _1-6 Alkyl and C _1-6 A haloalkyl group.

Another aspect of the invention is a pharmaceutical composition comprising a compound of formula (I) or a stereoisomer, tautomer, polymorph, solvate (e.g. hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, chemically protected form or prodrug thereof, and one or more pharmaceutically acceptable carriers. The pharmaceutical composition may further comprise one or more other therapeutic agents suitable for preventing or treating a disease or disorder mediated by FXR.

The invention also includes a method of preventing or treating a disease or condition mediated by FXR, comprising administering to a subject in need thereof a therapeutically effective amount of the compound of formula (I) or a stereoisomer, tautomer, polymorph, solvate (e.g., hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, chemically protected form or prodrug thereof, or the pharmaceutical composition.

The invention also includes a kit for preventing or treating a disease or disorder mediated by FXR comprising:

a) A first container comprising as a first therapeutic agent at least one compound of said general formula (I) or a stereoisomer, tautomer, polymorph, solvate (e.g. hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, chemically protected form or prodrug thereof, or said pharmaceutical composition as a first pharmaceutical composition;

b) An optional second container comprising at least one other therapeutic agent as a second therapeutic agent, or a pharmaceutical composition comprising the other therapeutic agent as a second pharmaceutical composition; and

c) Optionally package insert.

The invention also includes the compounds of formula (I) or stereoisomers, tautomers, polymorphs, solvates (e.g. hydrates), pharmaceutically acceptable salts, esters, metabolites, N-oxides, chemically protected forms or prodrugs thereof or the pharmaceutical compositions thereof for use in the prevention or treatment of diseases or conditions mediated by FXR.

The invention also includes the use of a compound of formula (I) or a stereoisomer, tautomer, polymorph, solvate (e.g. hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, chemically protected form or prodrug thereof, or of the pharmaceutical composition, for the manufacture of a medicament for the prophylaxis or treatment of a disease or condition mediated by FXR.

The application also includes processes for preparing the compounds of the application and corresponding intermediates.

The compounds of general formula (I) according to the application have excellent in vivo or in vitro pharmacodynamic or pharmacokinetic properties, show good FXR activation activity and activation and excellent liver microsomal stability, and thus have good pharmaceutical activity and metabolic advantages.

Brief description of the drawings

FIG. 1 shows the effect of compound C3 administration in a mouse fatty liver model, wherein a represents p.ltoreq.0.05 compared to the control group; b represents p.ltoreq.0.05 compared with the model group.

Detailed Description

Reference will now be made in detail to certain embodiments of the application, examples of which are illustrated in the accompanying structures and formulas. While the application will be described in conjunction with the enumerated embodiments, it will be understood that the application is not limited to those embodiments. On the contrary, the application is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the application as defined by the appended claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the present application. The present application is in no way limited to the methods and materials described. In the event of one or more of the cited documents, patents and similar materials being different or contradictory to the present application (including but not limited to defined terms, term usage, described techniques, etc.), the present application controls.

Definition of the definition

Unless defined otherwise hereinafter, all technical and scientific terms used herein are intended to be identical to what is commonly understood by one of ordinary skill in the art.

The terms "comprising," "including," "having," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

The term "alkyl" as used herein means a compound having 1 to 6 carbon atoms (C _1-6 ) Wherein the alkyl group may be optionally substituted with one or more (e.g., 1, 2, 3 or 4) suitable substituentsAnd (3) group substitution. In some embodiments, the alkyl groups have 1 to 6 carbon atoms (C _1-6 ). In other embodiments, the alkyl groups have 1 to 4 carbon atoms (C _1-4 ) In particular 1 to 3 carbon atoms (C _1-3 ) Or 1 to 2 carbon atoms (C _1-2 ). Examples of alkyl groups include, but are not limited to: methyl (Me), ethyl (Et), 1-propyl (n-Pr), 2-propyl (i-Pr or isopropyl), 1-butyl (n-Bu or n-butyl), 2-methyl-1-propyl (i-Bu or isobutyl), 2-butyl (s-Bu or sec-butyl), 2-methyl-2-propyl (t-Bu or tert-butyl), 1-pentyl (n-pentyl), 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl (-C (CH) ₃ ) ₂ CH ₂ CH ₂ CH ₃ ) 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2, 3-dimethyl-2-butyl, 3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like.

The term "halo" or "halogen" as used herein includes F, cl, br or I. "halo" includes, but is not limited to, mono-, di-or tri-substituted, and the halogen atoms used for substitution may be the same or different. The term "haloalkyl" as used herein includes fluoroalkyl, chloroalkyl, bromoalkyl, or iodoalkyl.

When the bond of a substituent is shown as a bond through the ring connecting two atoms, then such substituent may be bonded to any ring-forming atom in the substitutable ring.

The term "chiral" refers to molecules that have non-superimposability of a mirror image pair, while the term "achiral" refers to molecules that may be superimposed on their mirror image pair.

The term "stereoisomers" refers to compounds having the same chemical composition but different spatial arrangements of atoms or groups.

"diastereoisomers" refers to stereoisomers which have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. Mixtures of diastereomers can be separated by high resolution analytical methods such as electrophoresis and chromatography.

"enantiomer" refers to two stereoisomers of a compound that are non-superimposable mirror images of each other.

The stereochemical definitions and rules used herein generally follow S.P. Parker, ed., mcGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, new York; and Eliel, e. And Wilen, s., "Stereochemistry ofOrganic Compounds", john Wiley & Sons, inc., new York,1994. The compounds of the invention may contain asymmetric or chiral centers and thus exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the invention, including but not limited to diastereomers, enantiomers and atropisomers thereof, as well as mixtures thereof, such as racemic mixtures, are intended to form part of the present invention. Many organic compounds exist in optically active form, i.e., they have the ability to rotate the plane of plane polarized light. In the expression optically active compounds, the prefixes D and L, or R and S, are used to denote the absolute configuration of the chiral center of the molecule. The prefix d and 1 or (+) and (-) is used to indicate the sign of the compound to rotate plane polarized light, where (-) or 1 means that the compound is left-handed. The compound with the prefix (+) or d is dextrorotatory. For a particular chemical structure, these stereoisomers are identical, except that they are mirror images of each other. Particular stereoisomers may also be referred to as enantiomers, and mixtures of such isomers are often referred to as enantiomeric mixtures. Enantiomer 50: the 50 mixture is referred to as a racemic mixture or racemate, which may occur without stereoselectivity or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers, without optical activity. In one aspect, stereoisomers of the invention may exist in a predominant form, for example, greater than 50% ee (enantiomeric excess), greater than 80% ee, greater than 90% ee, greater than 95% ee, or greater than 99% ee.

In the case where the process for preparing the compounds of the invention yields a mixture of stereoisomers, these isomers may be separated by conventional techniques such as preparative chromatography. The compounds may be prepared in racemic form, or individual enantiomers may be prepared by enantioselective synthesis or by resolution. For example, the compounds may be resolved into their enantiomeric components by standard techniques, such as by salt formation with optically active acids such as (-) -di-p-toluoyl-d-tartaric acid and/or (+) -di-p-toluoyl-1-tartaric acid, followed by fractional crystallization and regeneration of the free base. The compounds can also be resolved by formation of diastereomeric esters or amides, followed by chromatographic purification and removal of the chiral auxiliary. Alternatively, the compounds may be resolved using a chiral HPLC column.

The term "tautomer" or "tautomeric form" refers to energy-different structural isomers that can be converted to each other by a low energy barrier. For example, proton tautomers (also known as proton transfer tautomers) include interconversions by proton transfer, such as keto-enol and imine-enamine isomerisation. Valence tautomers include interconversions by recombination of some of the bond-forming electrons.

The present invention encompasses all possible crystalline forms or polymorphs of the compound of formula (I), which may be a single polymorph or a mixture of more than one polymorph in any proportion.

It will be appreciated that certain compounds of the invention may exist in free form for use in therapy or, where appropriate, in the form of pharmaceutically acceptable derivatives thereof. According to the present invention, pharmaceutically acceptable derivatives include, but are not limited to: pharmaceutically acceptable salts, esters, solvates, metabolites, N-oxides, and chemically protected forms and prodrugs thereof, can provide, directly or indirectly, a compound of the invention or a metabolite or residue thereof upon administration to an individual in need thereof.

Thus, when reference is made herein to "a compound of formula (I)," a compound of the invention "or" a compound of formula (I) of the invention ", it is also intended to encompass solvates (e.g. hydrates), pharmaceutically acceptable salts, esters, metabolites, N-oxides of said compound of formula (I) and chemically protected forms and prodrugs thereof.

The term "pharmaceutically acceptable salt" as used herein refers to pharmaceutically acceptable organic or inorganic salts of the compounds of the present invention. Exemplary salts include, but are not limited to, isonicotinate, salicylate, acid citrate, oleate, tannate, pantothenate, gluconate, glucuronate, gluconate, pamoate (i.e., 1' -methylene-bis (2-hydroxy-3-naphthoate)), and the like. The pharmaceutically acceptable salt may include inclusion of another molecule of the counter ion. The counter ion may be any organic or inorganic ion that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. The plurality of charged atoms may have a plurality of counter ions where the plurality is part of a pharmaceutically acceptable salt. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counter ions.

If the compounds of the present invention are bases, the desired pharmaceutically acceptable salts can be prepared by any suitable method available in the art, for example, treating the free base with an inorganic acid or with an organic acid such as salicylic acid, pyranoside acids, e.g., glucuronic or galacturonic acid, alpha-hydroxy acid amino acids, aromatic acids, and the like.

If the compounds of the present invention are acids, the desired pharmaceutically acceptable salts can be prepared by any suitable method, for example, treating the free acid with an inorganic or organic base such as an amine (primary, secondary or tertiary), alkali metal hydroxide or alkaline earth metal hydroxide, and the like. Illustrative examples of suitable salts include, but are not limited to, organic salts derived from amino acids and cyclic amines, and inorganic salts.

The term "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other components of the formulation and/or the mammal being treated therewith.

The term "ester" as used herein means an ester derived from a compound of formula (I), including physiologically hydrolyzable esters, which can be hydrolyzed under physiological conditions to release the compound of formula (I) of the present invention in the form of a free acid or alcohol. The compounds of the general formula (I) according to the invention may also be esters per se.

The compounds of the invention may exist in the form of solvates (e.g. hydrates) wherein the compounds of the invention comprise a polar solvent as a structural element of the compound lattice, in particular, for example, water, methanol or ethanol. The polar solvent, in particular water, may be present in stoichiometric or non-stoichiometric amounts.

A "metabolite" is a product of a particular compound or salt thereof produced by metabolism in vivo. Metabolites of compounds may be identified using conventional techniques known in the art and their activity may be determined using assays such as those described herein. Such products may result from, for example, oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the compound being administered. Accordingly, the present invention includes metabolites of the compounds of the present invention, including compounds produced by a method comprising contacting a compound of formula (I) of the present invention with a mammal for a period of time sufficient to produce a metabolite thereof.

Those skilled in the art will appreciate that not all nitrogen-containing heterocycles are capable of forming N-oxides, as nitrogen requires available lone pairs to oxidize to oxides; those skilled in the art will recognize nitrogen-containing heterocycles capable of forming N-oxides. Those skilled in the art will also recognize that tertiary amines are capable of forming N-oxides. Synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art and include oxidizing heterocycles and tertiary amines with peroxyacids such as peracetic acid and m-chloroperoxybenzoic acid (m-CPBA), hydrogen peroxide, alkyl hydroperoxides such as t-butyl hydroperoxide, sodium perborate, and dioxiranes (dioxiranes) such as dimethyl dioxirane. These methods for preparing N-oxides have been widely described and reviewed in the literature, see for example: t.l. Gilchrist, comprehensive Organic Synthesis, vol.7, pp 748-750; ley, ed., pergamon Press; tisler and B.Stanovnik, comprehensive Heterocyclic Chemistry, vol.3, pp 18-20.

During any process for preparing the compounds of the present invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules of interest, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved by conventional protecting groups, for example, in Protective Groups in Organic Chemistry, ed.J.F.W.McOmie, plenum Press,1973; and those described in T.W.Greene & P.G.M.Wuts, protective Groups in Organic Synthesis, john Wiley & Sons,1991, which are incorporated herein by reference. The protecting groups may be removed at a suitable subsequent stage using methods known in the art.

The present invention further includes within its scope prodrugs of the compounds of the present invention. Typically such prodrugs will be functional derivatives of the compounds which are readily convertible in vivo into the desired therapeutically active compound. Thus, in these instances, the term "administering" as used in the methods of treatment of the present invention shall include treating various diseases or conditions with a prodrug form of one or more of the claimed compounds, but which converts to the above-described compounds in vivo upon administration to a subject. Conventional methods for selecting and preparing suitable prodrug derivatives are described, for example, in "Design of Prodmg", ed.H. Bundgaard, elsevier, 1985.

Any formula or structure shown herein, including compounds of formula (I), is also intended to represent unlabeled and isotopically-labeled forms of the compounds. Isotopically-labeled compounds have structures represented by the formulae given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be included in compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as, but not limited to ² H (deuterium, D), ³ H (tritium), ¹¹ C、 ¹³ C、 ¹⁴ C、 ¹⁵ N、 ¹⁸ F、 ³¹ p、 ³² P、 ³⁵ S、 ³⁶ Cl and Cl ¹²⁵ I. Various isotopically-labeled compounds of the present invention, for example, comprise, for example ³ H、 ¹³ C and C ¹⁴ Those of the radioisotope of C. Such isotope labelsThe compounds described can be used in metabolic studies, in reaction kinetics studies, in detection or imaging techniques, such as Positron Emission Tomography (PET) or single photon emission tomography (SPECT), including drug or substrate tissue distribution assays, or in the radiation treatment of patients. Deuterium labeled or substituted therapeutic compounds of the present invention may have improved DMPK (drug metabolism and pharmacokinetics) properties with respect to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements. ¹⁸ F-labeled compounds can be used in PET or SPECT studies. Isotopically-labeled compounds of the present invention and prodrugs thereof can generally be prepared by carrying out the methods disclosed in the schemes or examples and by preparing methods described below by substituting a readily available isotopically-labeled reagent for a non-isotopically-labeled reagent. Furthermore, the use of heavier isotopes, in particular deuterium (i.e ² H or D) substitution may provide certain therapeutic advantages due to greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements or improved therapeutic index. It is to be understood that deuterium in this case is considered a substituent in the compound of formula (I). The concentration of such heavier isotopes, particularly deuterium, can be defined by the isotopic enrichment coefficient. In the compounds of the present invention, any atom not explicitly specified as a particular isotope is intended to represent any stable isotope of that atom. Unless otherwise indicated, when a position is explicitly indicated as "H" or "hydrogen," it is understood that the position has hydrogen of its natural abundance isotopic composition. Thus, in the compounds of the present invention, any atom explicitly labeled deuterium (D) is intended to represent deuterium.

The term "pharmaceutical composition" as used herein includes products comprising a therapeutically effective amount of a compound of formula (I) of the present invention, as well as any products resulting directly or indirectly from the combination of compounds of formula (I) of the present invention.

Compounds of formula (I)

In some embodiments, the invention provides compounds of formula (I) or stereoisomers, tautomers, polymorphs, solvates (e.g. hydrates), pharmaceutically acceptable salts, esters, metabolites, N-oxides, chemically protected forms or prodrugs thereof,

wherein:

x is CH or N; and is also provided with

R is selected from hydrogen, halogen and C _1-6 Alkyl and C _1-6 A haloalkyl group.

In some embodiments, the invention provides compounds or stereoisomers, tautomers, polymorphs, solvates (e.g., hydrates), pharmaceutically acceptable salts, esters, metabolites, N-oxides, chemically protected forms or prodrugs thereof, wherein R is selected from the group consisting of hydrogen, fluoro, methyl and trifluoromethyl.

In some embodiments, the invention provides a compound or a stereoisomer, tautomer, polymorph, solvate (e.g., hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, chemically protected form or prodrug thereof, wherein X is CH; and R is selected from hydrogen, fluoro, methyl and trifluoromethyl.

In some embodiments, the invention provides a compound or a stereoisomer, tautomer, polymorph, solvate (e.g., hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, chemically protected form or prodrug thereof, wherein X is N; and R is hydrogen.

In some embodiments, the invention provides a compound or a stereoisomer, tautomer, polymorph, solvate (e.g., hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, chemically protected form or prodrug thereof, wherein the compound is selected from the group consisting of:

the compounds of the general formula (I) according to the invention may contain asymmetric centers or chiral centers and thus may exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the invention, including but not limited to diastereomers, enantiomers and atropisomers thereof, as well as mixtures thereof, such as racemic mixtures, are intended to form part of the present invention.

Furthermore, the present invention encompasses all diastereomers, including cis-trans (geometric) isomers and conformational isomers. For example, if the compounds of formula (I) contain double bonds or fused rings, both cis and trans forms and mixtures thereof are contemplated as being within the scope of the present invention. In the structures shown herein, all stereoisomers are considered and encompassed as compounds of the invention unless stereochemistry of any particular chiral atom is indicated. If stereochemistry is indicated with a solid wedge line or a dashed line representing a specific configuration, the stereoisomer is so indicated and defined.

The compounds of the present invention may exist in unsolvated forms as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and the present invention is intended to encompass both solvated and unsolvated forms.

The compounds of the invention may also exist in different tautomeric forms and all such forms are intended to be encompassed within the scope of the invention.

It is also to be understood that combinations of any two or more of the embodiments are also included within the scope of the present invention.

Pharmaceutical composition

In another aspect, the present invention provides a pharmaceutical composition comprising at least one compound of formula (I) of the present invention as described above or a stereoisomer, tautomer, polymorph, solvate (e.g. hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, chemically protected form or prodrug thereof, and one or more pharmaceutically acceptable carriers. In some embodiments, the pharmaceutical composition may further comprise one or more additional therapeutic agents, e.g., additional therapeutic agents suitable for preventing or treating a disease or disorder mediated by FXR.

By "pharmaceutically acceptable carrier" is meant a diluent, adjuvant, excipient or vehicle with which the active ingredient is administered, and which is suitable for contacting the tissues of humans and/or other animals within the scope of sound medical judgment without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the invention include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, water is an exemplary carrier. Physiological saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, in particular for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents, as desired. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1990).

The pharmaceutical compositions of the present invention may act systematically and/or locally. For this purpose, they may be administered by a suitable route, for example by injection, intravenously, intra-arterially, subcutaneously, intraperitoneally, intramuscularly or transdermally; or by oral, buccal, nasal, transmucosal, topical, in the form of an ophthalmic formulation or by inhalation.

For these routes of administration, the pharmaceutical compositions of the present invention may be administered in suitable dosage forms.

Such dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.

Therapeutic use

Another aspect of the invention provides therapeutic uses of the compounds and pharmaceutical compositions.

Thus, in some embodiments, the invention relates to a method of preventing or treating a disease or condition mediated by FXR, comprising administering to a subject in need thereof a therapeutically effective amount of at least one compound of formula (I) of the invention or a stereoisomer, tautomer, polymorph, solvate (e.g. hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, chemoprotective form or prodrug thereof, or administering a pharmaceutical composition of the invention.

In other embodiments, the invention relates to the use of at least one compound of the general formula (I) according to the invention or a stereoisomer, tautomer, polymorph, solvate (e.g. hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, chemically protected form or prodrug thereof, or a pharmaceutical composition according to the invention, for the preparation of a medicament for the prophylaxis or treatment of a disease or condition mediated by FXR.

Such FXR mediated diseases or conditions include, but are not limited to:

chronic intrahepatic or some form of extrahepatic cholestatic disorder; liver fibrosis; obstructive or chronic inflammatory disorders of the liver; cirrhosis of the liver; fatty liver and complications thereof; cholestatic and fibrotic effects associated with alcohol-induced cirrhosis or with viral infectious forms of hepatitis; liver failure or liver ischemia after segmental hepatectomy; chemotherapy-associated steatohepatitis (CASH); acute liver failure;

inflammatory bowel disease, dyslipidemia, atherosclerosis, diabetes and related diseases; lipid and lipoprotein disorders; clinical complications of type II diabetes and type I and type II diabetes, including diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, and other observed effects of clinically significant long-term diabetes; conditions and diseases caused by chronic fatty and fibrous changes due to forced accumulation of lipids, particularly triglycerides, followed by activation of pro-fibrotic pathways, such as non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH); obesity or metabolic syndrome (dyslipidemia, diabetes and a combination of conditions with abnormally high body mass index);

Acute myocardial infarction, acute stroke, or thrombosis occurring as an endpoint of chronic obstructive atherosclerosis; non-malignant hyperproliferative disorders and malignant hyperproliferative disorders, in particular hepatocellular carcinoma, colon adenoma and polyposis, colon adenoma, breast cancer, pancreatic cancer, butcher's esophageal cancer and other forms of neoplastic diseases of the gastrointestinal tract and liver.

The term "treating" as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progression of the indicated disease or condition or one or more symptoms of such disease or condition.

As used herein, "individual" includes human or non-human animals. Exemplary human individuals include human individuals (referred to as patients) or normal individuals suffering from a disease (e.g., a disease described herein). "non-human animals" in the context of the present invention include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, domestic animals and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

The term "therapeutically effective amount" as used herein refers to the amount of a compound that will achieve the therapeutic efficacy described above after administration.

The dosing regimen may be adjusted to provide the most desirable response. For example, a single bolus may be administered, several divided doses may be administered over time, or the doses may be proportionally reduced or increased as indicated by the urgent need for a therapeutic situation. It is noted that the dosage value may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is further understood that for any particular individual, the particular dosage regimen will be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions.

The amount of the compound of the invention administered will depend on the severity of the individual, disorder or condition being treated, the rate of administration, the disposition of the compound and the discretion of the prescribing physician. Generally, an effective dose is about 0.0001 to about 50mg, for example about 0.01 to about 10 mg/kg/day per kg body weight per day (single or divided administration). For a 70kg human, this amounts to about 0.007 mg/day to about 3500 mg/day, for example about 0.7 mg/day to about 700 mg/day. In some cases, dosage levels not higher than the lower limit of the aforementioned range may be sufficient, while in other cases larger doses may still be employed without causing any adverse side effects, provided that the larger dose is first divided into several smaller doses for administration throughout the day.

The compounds of the present invention may be present in the pharmaceutical composition in an amount of about 0.01mg to about 1000mg, suitably 0.1-500mg, preferably 0.5-300mg, etc.

Combination therapy

The compounds of formula (I) may be used alone or in combination with one or more other therapeutic agents suitable for the prevention or treatment of diseases or conditions mediated by FXR. In some embodiments, the compounds of formula (I) are combined with other therapeutic agents, e.g., having anti-hyperproliferative efficacy, in the pharmaceutical composition or as a dosing regimen for combination therapy. The other therapeutic agent may be, for example, a chemotherapeutic agent. The pharmaceutical compositions or other therapeutic agents of the dosing regimen preferably have complementary activity to the compounds of formula (I) so that they do not adversely affect each other. Such compounds are suitably present in combination in amounts effective for the intended purpose.

The combination therapy may be administered simultaneously or sequentially on a regimen of administration. When administered sequentially, the combination may be administered in two or more administrations. The combined administration includes simultaneous administration using separate pharmaceutical compositions or a single pharmaceutical composition comprising a compound of formula (I) and the other therapeutic agent, as well as sequential administration in any order, wherein there is preferably a period of time in which both (or all) active agents exert their biological activity simultaneously.

Suitable dosages of any of the above co-administered agents are those currently in use and may be reduced due to the combined (synergistic) effect of the newly identified drug with other therapeutic agents or treatments.

Combination therapy may provide "synergistic effects" and prove "synergistic", i.e., the effect achieved by the active ingredients when taken together is greater than the sum of the effects produced by the compounds when taken separately. When the active ingredient: (1) When co-formulated and co-administered or delivered in a combined unit dose formulation; (2) when delivered alternately or in parallel as separate formulations; or (3) through some other scheme, a synergistic effect can be achieved. When delivered in alternation therapy, a synergistic effect may be achieved when the compounds are administered or delivered sequentially, e.g., by separate injections in separate syringes, by separate pills or capsules, or by separate infusions. Typically, in alternating therapy, an effective dose of each active ingredient is administered sequentially, i.e., continuously, while in combination therapy, an effective dose of two or more active ingredients is administered together.

In a particular embodiment of the treatment, the compounds of formula (I) or stereoisomers, tautomers, polymorphs, solvates (e.g. hydrates), pharmaceutically acceptable salts, esters, metabolites, N-oxides, chemically protected forms or prodrugs thereof may be combined with other therapeutic agents such as those described herein, as well as with surgical treatment and radiation therapy. Thus, the combination therapy of the invention comprises administration of at least one compound of formula (I) or a stereoisomer, tautomer, polymorph, solvate (e.g. hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, chemoprotective form or prodrug thereof, and the use of at least one other therapeutic method. The amounts of the compound of formula (I) and the other therapeutic agent, and the relative timing of administration, are selected in order to achieve the desired combined therapeutic effect.

Metabolites of compounds of the general formula (I)

In vivo metabolites of the compounds of formula (I) described herein are also within the scope of the invention. Such products may result from, for example, oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the compound being administered. Accordingly, the present invention includes metabolites of compounds of formula (I), including compounds made by a process of contacting a compound of the present invention with a mammal for a time sufficient to produce the metabolites thereof.

Metabolites are typically produced by preparing a radioisotope of the present invention (e.g ¹⁴ C or ³ H) The labeled compound is parenterally administered to an animal such as a rat, mouse, guinea pig, monkey, or human in a detectable dose (e.g., greater than about 0.5 mg/kg), metabolized for a period of time sufficient (typically about 30 seconds to 30 hours), and then its conversion products isolated from urine, blood, or other biological samples for identification. These products are easy to isolate because they are labeled (others are isolated by using antibodies that bind to epitopes remaining in the metabolite). The metabolite structures are determined in a conventional manner, for example by MS, LC/MS or NMR analysis. Analysis of metabolites was performed in the same manner as conventional drug metabolism studies well known to those skilled in the art. Metabolites may be used in diagnostic assays to administer the compounds of the invention therapeutically, provided they are not found in vivo.

Medicine box

In other embodiments of the present invention, a "kit" is provided that comprises materials for treating the above-described diseases or disorders. The kit comprises a container comprising a compound of formula (I), a stereoisomer, tautomer, polymorph, solvate (e.g., hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, or a chemically protected form or prodrug thereof, as a first therapeutic agent, or a pharmaceutical composition of the invention as a first pharmaceutical composition. In some embodiments, the kit may further comprise a label or package insert on or with the container. The term "package insert" refers to instructions that are typically included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings associated with the use of the therapeutic product. Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. The container may be made of various materials such as glass and plastic. The container may contain a compound of formula (I) or a formulation thereof effective for treating a disorder, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used to treat a selected disorder, such as cancer. In addition, the label or package insert may indicate that the patient to be treated is a patient suffering from a disease or condition such as cirrhosis, a hyperproliferative disorder, atherosclerosis, type I diabetes, and the like, and may indicate that the composition may be used to treat other conditions. In other embodiments, the kit further comprises a second container comprising as a second therapeutic agent at least one other therapeutic agent suitable for preventing or treating a disease or disorder mediated by FXR, or a pharmaceutical composition comprising said other therapeutic agent as a second pharmaceutical composition. Thus, in some embodiments, the kit may include instructions for administering the first therapeutic agent or first pharmaceutical composition and the second therapeutic agent or second pharmaceutical composition, if present. For example, if the kit comprises a first composition comprising a compound of formula (I) and a second pharmaceutical composition comprising the other therapeutic agent, the kit may further comprise instructions for simultaneous, sequential or separate administration of the first and second pharmaceutical compositions to a subject in need thereof. Alternatively or additionally, the kit may further comprise a third container comprising a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution and dextrose solution. The kit may also include other materials desirable to the business and user, including other buffers, diluents, fillers, injection needles, and syringes.

In other embodiments, the kit is suitable for delivering a solid oral form of a compound of formula (I), such as a tablet or capsule. Such a kit preferably comprises a plurality of unit doses. Such kits may include cards with doses positioned for their intended use. One example of such a kit is a "blister pack". Blister packages are well known in the packaging industry and are widely used for packaging pharmaceutical unit dosage forms. If desired, a memory aid may be provided in the form of, for example, numbers, letters or other indicia or calendar inserts specifying the days on which the medication may be administered in the treatment schedule.

Process for the preparation of compounds

In some embodiments, the present invention provides a process for preparing a compound of formula (I) of the present invention, comprising the steps of:

wherein:

Hal ¹ 、Hal ² and Hal ³ Each independently is the same or different halogen, e.g., F, cl, br or I, preferably C1 or Br;

PG ¹ an amino protecting group, preferably t-butoxycarbonyl (Boc);

PG ² is a carboxyl protecting group, preferably C _1-6 Alkyl, more preferably methyl;

y is a boric acid or borate group, preferably-B (OH) ₂ Or alternatively

The remaining groups are as defined above;

the reaction conditions of each step are as follows:

Step A: reacting compound IN-1 with compound IN-2 to give compound IN-3;

the reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of linear or cyclic ethers (e.g., tetrahydrofuran or diethyl ether, etc.), N-methylpyrrolidone, N-dimethylformamide, N-dimethylacetamide, 1, 4-dioxane, dimethylsulfoxide, and any combination thereof, preferably tetrahydrofuran or N, N-dimethylformamide. The reaction is preferably carried out in the presence of a suitable base (e.g. an alkali metal alkoxide or carbonate) and/or a catalyst. The catalyst may be a catalyst system comprising a crown ether, which crown ether may be selected from 15-crown ether-5 and 18-crown ether-6; the carbonate of an alkali metal is, for example, potassium carbonate or cesium carbonate; the alkali metal alkoxide may be selected from sodium tert-butoxide, potassium tert-butoxide, sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide. Preferably, the alkali metal alkoxide and catalyst is a combination of sodium t-butoxide and/or potassium t-butoxide with 15-crown-5 and/or 18-crown-6, preferably a combination of sodium t-butoxide with 15-crown-5 or a combination of potassium t-butoxide with 18-crown-6. The reaction is preferably carried out at a suitable temperature. The temperature is preferably room temperature (20-30 ℃) or 50-100 ℃ (e.g. 50-80 ℃). The reaction is preferably carried out for a suitable time, for example 1 to 24 hours, for example 5 to 15 hours.

And (B) step (B): removal of PG IN Compound IN-3 ¹ A group to give compound IN-4;

the reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of halogenated hydrocarbons (e.g., dichloromethane, chloroform, chloroethane, dichloroethane, trichloroethane), N-dimethylformamide, N-dimethylacetamide, and any combination thereof, with dichloromethane being preferred. The reaction may be carried out under acidic conditions, for example in a solution of 1, 4-dioxane in hydrogen chloride; or in the presence of a suitable organic acid (e.g., carboxylic or halogenated acid, including but not limited to formic acid, fluoroacetic acid, difluoroacetic acid, trifluoroacetic acid, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, and combinations thereof, preferably trifluoroacetic acid). The reaction is preferably carried out at a suitable temperature. The temperature is preferably room temperature (20-30 ℃). The reaction is preferably carried out for a suitable time, for example 1 to 5 hours or 6 to 15 hours, for example 2 hours, 4 hours or overnight.

Step C: reacting compound IN-4 with compound IN-a to give compound IN-5;

IN some embodiments, compound IN-4 is substitution reacted with compound IN-a to yield compound IN-5. The substitution reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of N, N-dimethylformamide, N-dimethylacetamide, tetrahydrofuran, N-methylpyrrolidone, dimethylsulfoxide, and any combination thereof, preferably N, N-dimethylformamide or N, N-dimethylacetamide. The substitution reaction is preferably carried out in the presence of a suitable base. Preferably, the base is an organic base (e.g., an organic amine such as triethylamine, N-diisopropylethylamine, N-methylmorpholine or pyridine, preferably triethylamine or N, N-diisopropylethylamine) or an inorganic base (e.g., an alkali metal salt, preferably potassium carbonate). The substitution reaction is preferably carried out at a suitable temperature. The temperature may be 20-150 ℃, for example 30-140 ℃, preferably 25 ℃, 50 ℃, 100 ℃ or 130 ℃, preferably 80 ℃. The substitution reaction is preferably carried out for a suitable time, for example from 2 to 24 hours, from 2 to 18 hours or from 2 to 12 hours, for example 5, 8 or 10 hours.

IN other embodiments, compound IN-4 is coupled to compound IN-a to provide compound IN-5. The coupling reaction is preferably carried out in the presence of a metal catalyst and a base. Preferably, the metal catalyst is a palladium metal catalyst such as tris (dibenzylideneacetone) dipalladium, [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium, triphenylphosphine palladium, palladium acetate, preferably tris (dibenzylideneacetone) dipalladium. The base is an inorganic base such as potassium carbonate, cesium carbonate, sodium bicarbonate, potassium bicarbonate, preferably cesium carbonate. Preferably, the coupling reaction is carried out in the presence of an organophosphorus compound derived from biphenyl, said organophosphorus compound being selected from BINAP, ruPhos and XPhos, preferably BINAP. Preferably, the coupling reaction is carried out in a suitable organic solvent, which may be selected from benzene, toluene and xylene, for example toluene. Preferably, the coupling reaction is carried out under a suitable protective atmosphere (e.g., nitrogen atmosphere). Preferably, the coupling reaction is carried out at a suitable temperature, which may be 70-100 ℃, preferably 80 ℃. Preferably, the coupling reaction is carried out for a suitable time, for example 1-3 hours, for example 2 hours.

Step D: reacting compound IN-5 with compound IN-b to give compound IN-6;

preferably, the compound IN-6 is obtained by a metal catalyzed coupling reaction of the compound IN-5 with the compound IN-b. The metal catalytic coupling reaction is carried out by a conventional method. For example: compound IN-5 and compound IN-b are dissolved IN a solvent (e.g., water, an organic solvent, or a mixed solvent of an organic solvent and water), and a palladium catalyst and a base are added, and reacted at a temperature of 50 ℃ to 120 ℃ (preferably 80 ℃ or 90 ℃) for 8 to 24 hours (preferably 8 hours or 12 hours), optionally under a nitrogen atmosphere. The organic solvent is N, N-dimethylformamide, tetrahydrofuran, 1, 4-dioxane, toluene, DME or the like. The palladium catalyst is tris (dibenzylideneacetone) dipalladium, [1,1 '-bis (diphenylphosphino) ferrocene ] palladium dichloride, triphenylphosphine palladium, palladium acetate, preferably [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride or tris (dibenzylideneacetone) dipalladium and the like. The base is preferably an inorganic base such as potassium carbonate, cesium carbonate, sodium bicarbonate, potassium bicarbonate, or the like.

Step E: removal of PG IN Compound IN-6 ² A group to give a compound of general formula (I);

The reaction is preferably carried out in a suitable organic solvent (which may be selected from the group consisting of linear or cyclic ethers (e.g. tetrahydrofuran or diethyl ether, etc.), M-methylpyrrolidone, N-dimethylformamide, N-dimethylacetamide, 1, 4-dioxane, dimethyl sulfoxide, and any combination thereof, preferably tetrahydrofuran). The reaction is preferably carried out in the presence of an alcohol or water and a base. The alcohol may be, for example, methanol or ethanol. The base may be selected from alkali metal hydroxides, which may be selected from lithium hydroxide, sodium hydroxide and potassium hydroxide. The reaction is preferably carried out at a suitable temperature. The temperature may be from room temperature to 80 ℃, for example 25 ℃ or 40-60 ℃. The reaction is preferably carried out for a suitable time, for example 2-5 hours or 6-15 hours, for example 2, 3 or 4 hours or overnight.

IN other embodiments, the invention provides a compound of formula (IN-6) or a pharmaceutically acceptable salt thereof,

wherein each group is as defined above;

the compound is preferably selected from:

the term "suitable" as used herein means that the choice of a particular compound or condition will depend on the particular synthetic procedure to be performed and the nature of the molecule or molecules to be converted, but such choice is within the ability of one skilled in the art. All process/method steps described herein are performed under conditions sufficient to provide the indicated products. Those skilled in the art will appreciate that all reaction conditions (including, for example, reaction solvent, reaction time, reaction temperature, and whether the reaction should be performed in an anhydrous or inert atmosphere, etc.) may be varied to optimize the yield of the desired product and that such variations are within the ability of those skilled in the art.

The examples provide exemplary methods for preparing compounds of formula (I). Those skilled in the art will appreciate that other synthetic routes may be used to synthesize the compounds of formula (I). Although specific starting materials and reagents are described and discussed in the examples, other starting materials and reagents may be substituted to provide various derivatives and/or reaction conditions. In addition, many of the example compounds prepared by the methods described herein may be further modified using conventional chemistry well known to those skilled in the art with reference to the present disclosure.

In preparing compounds of formula (I), it may be desirable to protect the distal functional group (e.g., carboxyl or amino) of the intermediate. The need for such protection may vary with the nature of the distal functional group and the conditions of the preparation process. The necessity of such protection is readily determined by one skilled in the art. For an overview of protecting groups and their use, see t.w. greene, protective Groups in Organic Synthesis, john Wiley & sons.new york.1991.

Examples

The invention is further described below in connection with examples, which are not intended to limit the scope of the invention.

The structure of the compound is that nuclear magnetic resonance is adopted ¹ H-NMR) or Mass Spectrometry (MS). ¹ The H-NMR shift (. Delta.) is given in parts per million (ppm). Chemical shift is 10 ^-6 (ppm) is given as a unit.

MS was determined using an Agilent (ESI) mass spectrometer.

The preparation of the high performance liquid phase uses Shimadzu to prepare a high performance liquid chromatograph.

Thin layer chromatography silica gel plate (TLC) was carried out using an aluminum plate (20X 20 cm) produced by Merck, and GF 254 (0.4-0.5 mm) was used for separation and purification by thin layer chromatography.

The reaction was monitored by Thin Layer Chromatography (TLC) or LC-MS using the following developing reagent system: dichloromethane and methanol systems, n-hexane and ethyl acetate systems or petroleum ether and ethyl acetate systems. According to different polarities of the compounds, the volume ratio of the solvents is required to be regulated, or triethylamine and the like are required to be further added, so that the separation and purification of the products are realized.

Microwave reaction is usedInitiator+ (400W, RT-300 ℃) microwave reactor.

Column chromatography generally uses 200-300 mesh silica gel as a carrier. The system of the eluent comprises: the volume ratio of the solvent in the methylene dichloride and methanol systems and the normal hexane and ethyl acetate systems is adjusted according to the polarity of the compounds, and can be adjusted by adding a small amount of triethylamine.

The reaction temperature of the examples was room temperature (20 ℃ C. To 30 ℃ C.) unless otherwise indicated.

The reagents used in the present invention are available from Acros Organics, aldrich Chemical Company, shanghai Tebert chemical technologies, inc.

In the conventional synthesis and examples and intermediate preparations, the abbreviations have the following meanings.

Abbreviations (abbreviations)	Meaning of	Abbreviations (abbreviations)	Meaning of
				TLC	Thin layer chromatography	LC-MS	Liquid chromatograph-mass spectrometer
DMF	N, N-dimethylformamide	DME	Ethylene glycol dimethyl ether

Intermediate preparation example:

intermediate preparation example 1: preparation of 4- ((((1R, 3r, 5S) -8- (4-bromothiazol-2-yl) -8-azabicyclo [3.2.1] oct-3-yl) oxy) methyl) -5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazole (T1)

The first step: (E) Preparation of 2- (trifluoromethoxy) benzaldehyde oxime (T1-b)

2- (trifluoromethoxy) benzaldehyde (T1-a) (150 g,788.98 mmol) was dissolved in a mixed solvent of ethanol (1000 mL) and water (1000 mL), hydroxylamine hydrochloride (65.79 g,946.77 mmol) was added under mechanical stirring, a white solid was precipitated, and 1M aqueous sodium hydroxide solution (789 mL,788.98 mmol) was further added. The reaction mixture was subjected to suction filtration as it was with 1M hydrochloric acid (2000 mL) added to the mixture to adjust the pH of the system to about 5, followed by drying at 50℃overnight, to give compound (T1-b) (150 g, yield: 92%).

And a second step of: preparation of (Z) -N-hydroxy-2- (trifluoromethoxy) iminobenzyl chloride (T1-c)

Compound (T1-b) (150 g,731.23 mmol) was dissolved in DMF (1000 mL) and N-chlorosuccinimide (117.17 g,877.48 mmol) was added with mechanical stirring at 0deg.C and reacted at 0deg.C for 1 hour. Water (2000 mL) was added to the reaction, followed by extraction with ethyl acetate (1000 mL. Times.3). The organic layer was dried over anhydrous sodium sulfate (500 g), and the compound (T1-c) was obtained after filtration and concentration (160 g, yield: 91%).

And a third step of: preparation of 5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazole-4-carboxylic acid methyl ester (T1-d)

Methyl 3-cyclopropyl-3-oxopropionate (189.87 g,1.34 mol) was added to compound (T1-c) (160 g,667.84 mmol), and the reaction was stirred at-5℃and triethylamine (500 mL) was added dropwise and reacted overnight at-5 ℃. The reaction solution was poured into water (20L), and mechanically stirred for 30 minutes, and a solid was found, which was suction-filtered to give a yellow solid, which was dried overnight at 50℃to give compound (T1-d) (120 g, yield: 55%).

Fourth step: preparation of (5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methanol (T1-e)

Compound (T1-d) (120 g,366.69 mmol) was dissolved in toluene, and the reaction was stirred at-10℃and 2M diisobutylaluminum hydride (550 mL,1.10 mol) was added dropwise thereto and reacted overnight at room temperature. The reaction solution was poured into methanol (1000 mL) to which ice cubes were added, water (3000 mL) was added under mechanical stirring, and suction filtration was performed to obtain a yellow solid, the filtrate was extracted with ethyl acetate (2000 mL. Times.3), dried over anhydrous sodium sulfate (500 g), filtered, and the organic phase was dried by spinning to obtain compound (T1-e) (100 g, yield: 91%).

Fifth step: preparation of 4- (chloromethyl) -5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazole (T1-f)

Benzotriazole (59.65 g,500.74 mmol) was dissolved in dichloromethane, and the reaction mixture was stirred at-5℃to drop thionyl chloride (59.65 g,501.39 mmol), stirred at room temperature for half an hour, then a dichloromethane solution (500 mL) of compound (T1-e) (100 g,334.17 mmol) was added and reacted at room temperature for 6 hours. The reaction solution was suction-filtered, and the filtrate was dried by spin-drying to give compound (T1-f) (106 g, yield: 94%).

Sixth step: preparation of (1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester (T1-g)

Compound (T1-f) (74.77 g,328.94 mmol) was dissolved in tetrahydrofuran (500 mL) followed by the addition of 18-crown-6 (118.56 g,448.55 mmol). The reaction was stirred at 0deg.C, potassium tert-butoxide (50.33 g,448.55 mmol) was added, followed by stirring at room temperature, tert-butyl (1R, 3r, 5S) -3-hydroxy-8-azabicyclo [3.2.1] octane-8-carboxylate (95 g,299.03 mmol) was added and the reaction was carried out overnight at room temperature. The reaction mixture was dried by spinning, ethyl acetate (1500 mL) and water (1500 mL) were added to the residue, and the organic phase was washed twice with saturated brine (1500 mL), and after concentrating the organic phase, the organic phase was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10/1-5/1) to give compound (T1-g) (85 g, yield: 56%).

Seventh step: preparation of 4- (((1R, 3r, 5S) -8-azabicyclo [3.2.1] oct-3-yloxy) methyl) -5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazole (T1-h)

Compound (T1-g) (85 g,167.15 mmol) was dissolved in dichloromethane (500 mL) followed by the addition of dioxane hydrochloride solution (4M, 500 mL). The reaction was carried out at room temperature overnight. The reaction mixture was dried by spin-drying to give Compound (T1-h) (60 g, yield: 81%).

Eighth step: preparation of 4- ((((1R, 3r, 5S) -8- (4-bromothiazol-2-yl) -8-azabicyclo [3.2.1] oct-3-yl) oxy) methyl) -5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazole (T1)

Compound (T1-h) (7 g,15.73 mmol) was dissolved in DMF (50 mL) and then N, N-diisopropylethylamine (10.17 g,78.67 mmol) and 2, 4-dibromothiazole (4.59 g,18.88 mmol) were added and reacted at 100℃for 12 hours. The reaction mixture was added with water (300 mL), dried over ethyl acetate (1000 mL), anhydrous sodium sulfate (20 g), filtered, and the organic phase was concentrated and purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10/1-6/1) to give compound (T1) (2.8 g, yield: 31%).

Example 1: preparation of 4- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl) thiazol-4-yl) benzoic acid (C1)

The first step: preparation of methyl 4- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl) thiazol-4-yl) benzoate (C1-a)

Compound (T1) (1 g,1.75 mmol) was dissolved in 1, 4-dioxane (20 mL) and then methyl 4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzoate (532.50 mg,1.93mmo 1), [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (143.16 mg, 195.66. Mu. Mol) and potassium carbonate (483.85 mg,3.50 mmol) were added and nitrogen was displaced 2-3 times. The reaction was carried out at 80℃for 8 hours. The reaction solution was suction-filtered through celite, the filtrate was washed with ethyl acetate (100 ml×3), the obtained organic phase was dried over anhydrous sodium sulfate (20 g), and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10/1-6/1) to give the title compound (1.12 g) of the present step.

MS m/z(ESI)：626.1[M+H] ⁺ 。

And a second step of: preparation of 4- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl) thiazol-4-yl) benzoic acid (C1)

Compound (C1-a) (390 mg, 625.31. Mu. Mol) was dissolved in tetrahydrofuran (5 mL), and an aqueous solution (2 mL) of sodium hydroxide (25.01 mg, 625.31. Mu. Mol) was added thereto to react at 25℃for 4 hours. The reaction solution was adjusted to pH 4 with 1M dilute hydrochloric acid, extracted with ethyl acetate (100 ml×3), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10/1-6/1) to give the title compound (150 mg).

MS m/z(ESI)：612.1[M+H] ⁺ 。

¹ H-NMR(400MHz，DMSO-d ₆ )δ：7.96(s，1H)，7.68(dd，J＝18.6，7.7Hz，1H)，7.58(dd，J＝13.9，6.8Hz，1H)，7.45(s，1H)，4.36(s，1H)，4.15(s，1H)，2.36(s，1H)，2.04(d，J＝15.2Hz，1H)，1.82(s，1H)，1.70(d，J＝14.9Hz，1H)，1.39-1.26(m，1H)，1.13(d，J＝22.2Hz，1H)。

Example 2: preparation of 4- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl) thiazol-4-yl) -3-methylbenzoic acid (C2)

The first step: preparation of methyl 4- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl) thiazol-4-yl) -3-methylbenzoate (C2-a)

Compound (T1) (500 mg,876.55 mmol) was dissolved in 1, 4-dioxane (20 mL) and methyl 3-methyl-4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzoate (266.25 mg, 964.20. Mu. Mol), [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (71.58 mg, 97.83. Mu. Mol) and potassium carbonate (241.93 mg,1.75 mmol) were then added and nitrogen was replaced 2-3 times. The reaction was carried out at 80℃for 8 hours. The reaction solution was filtered with celite, the filtrate was washed with ethyl acetate (100 ml×3), the obtained organic phase was dried over anhydrous sodium sulfate (20 g) and concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10/1-6/1) to give the title compound (400 mg) of the present step.

MS m/z(ESI)：640.2[M+H] ⁺ 。

And a second step of: preparation of 4- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl) thiazol-4-yl) -3-methylbenzoic acid (C2)

Compound (C2-a) (300 mg, 468.98. Mu. Mo 1) was dissolved in tetrahydrofuran (5 mL), and an aqueous solution (2 mL) of sodium hydroxide (18.76 mg, 468.98. Mu. Mol) was added thereto to react at 25℃for 4 hours. The reaction solution was adjusted to pH 4 with 1M diluted hydrochloric acid, extracted with ethyl acetate (100 ml×3), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=10:1-6:1) to give the title compound (280 mg).

MS m/z(ESI)：612.1[M+H] ⁺ 。

¹ H-NMR(400MHz，DMSO-d ₆ )δ：7.84(s，1H)，7.80(d，J＝8.1Hz，1H)，7.74-7.68(m，1H)，7.66(d，J＝6.2Hz，1H)，7.58(dd，J＝13.7，6.4Hz，1H)，7.06(s，1H)，4.35(s，1H)，4.11(s，1H)，2.51(d，J＝15.3Hz，4H)，2.41-2.32(m，1H)，2.04(d，J＝14.7Hz，1H)，1.94(s，1H)，1.82(s，2H)，1.69(d，J＝14.6Hz，1H)，1.19-1.14(m，2H)，1.10(m，2H).

Example 3: preparation of 4- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl ] thiazol-4-yl) -3-fluorobenzoic acid (C3)

The first step: preparation of methyl 4- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl) thiazol-4-yl) -3-fluorobenzoate (C3-a)

Compound (T1) (650 mg,1.14 mmol) was dissolved in 1, 4-dioxane (20 mL) and then methyl 3-fluoro-4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzoate (248.13 mg,1.25 mmol), [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (93.06 mg, 127.18. Mu. Mol) and potassium carbonate (314.50 mg,2.28 mmol) were added and nitrogen was replaced 2-3 times. The reaction was carried out at 80℃for 8 hours. The reaction solution was suction-filtered through celite, the filtrate was washed with ethyl acetate (100 ml×3), the obtained organic phase was dried over anhydrous sodium sulfate (20 g), and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10/1) to give the title compound (350 mg) of the present step.

MS m/z(ESI)：644.1[M+H] ⁺ 。

And a second step of: preparation of 4- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl ] thiazol-4-yl) -3-fluorobenzoic acid (C3)

Compound (C3-a) (390 mg, 625.31. Mu. Mo 1) was dissolved in tetrahydrofuran (5 mL), and an aqueous solution (2 mL) of sodium hydroxide (25.01 mg, 625.31. Mu. Mol) was added thereto to react at 25℃for 4 hours. The reaction solution was adjusted to pH 4 with 1M dilute hydrochloric acid, extracted with ethyl acetate (100 ml×3), and the organic phase was concentrated by drying over anhydrous sodium sulfate, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10/1-6/1) to give the title compound (1.01 g).

MS m/z(ESI)：620.1[M+H] ⁺ 。

¹ H-NMR(400MHz，DMSO-d ₆ )δ：7.71(t，J＝7.8Hz，1H)，7.66(d，J＝7.4Hz，1H)，7.63-7.54(m，2H)，7.50(d，J＝11.7Hz，1H)，7.33(s，1H)，4.35(s，1H)，4.12(s，1H)，2.39-2.33(m，1H)，2.03(d，J＝14.5Hz，1H)，1.80(s，2H)，1.69(d，J＝14.7Hz，1H)，1.33-1.24(m，1H)，1.16(m，1H)，1.10(m，1H)。

Example 4: preparation of 4- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl) thiazol-4-yl) -2-fluorobenzoic acid (C4)

The first step: preparation of methyl 4- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl) thiazol-4-yl) -2-fluorobenzoate (C4-a)

Compound (T1) (400 mg, 701.24. Mu. Mol) was dissolved in 1, 4-dioxane (20 mL) and then methyl 2-fluoro-4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzoate (152.70 mg, 771.36. Mu. Mol), [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (57.27 mg, 78.26. Mu. Mol) and potassium carbonate (193.54 mg,1.40 mmol) were added and nitrogen was replaced 2-3 times. The reaction was carried out at 80℃for 8 hours. The reaction solution was filtered with celite, the filtrate was washed with ethyl acetate (100 ml×3), the obtained organic phase was dried over anhydrous sodium sulfate (20 g), and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10/1) to give the title compound (200 mg) of the present step.

MS m/z(ESI)：630.1[M+H] ⁺ 。

And a second step of: preparation of 4- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl) thiazol-4-yl) -2-fluorobenzoic acid (C4)

Compound (C4-a) (200.00 mg, 310.73. Mu. Mol) was dissolved in tetrahydrofuran (5 mL), and an aqueous solution (2 mL) of sodium hydroxide (12.43 mg, 310.73. Mu. Mol) was added thereto to react at 25℃for 4 hours. The reaction solution was adjusted to pH 4 with 1M dilute hydrochloric acid, extracted with ethyl acetate (100 ml×3), and the organic phase was concentrated by drying over anhydrous sodium sulfate, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10/1-6/1) to give the title compound (170 mg).

MS m/z(ESI)：620.1[M+H] ⁺ 。

¹ H-NMR(400MHz，DMSO-d ₆ )δ：7.85(d，J＝8.1Hz，1H)，7.72(dd，J＝17.5，10.3Hz，1H)，7.66(d，J＝7.3Hz，1H)，7.58(dd，J＝13.5，6.3Hz，1H)，7.37(d，J＝1.9Hz，1H)，4.36(s，1H)，4.15(s，1H)，2.37(d，J＝4.9Hz，1H)，2.03(d，J＝14.7Hz，1H)，1.82(s，2H)，1.70(d，J＝14.6Hz，1H)，1.33-1.24(m，1H)，1.15(dd，J＝11.0，6.1Hz，1H)，1.10(d，J＝3.0Hz，1H)。

Example 5: preparation of 4- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl) thiazol-4-yl) -2- (trifluoromethyl) benzoic acid (C5)

The first step: preparation of methyl 4- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl) thiazol-4-yl) -2- (trifluoromethyl) benzoate (C5-a)

Compound (T1) (0.4 g, 701.24. Mu. Mol) was dissolved in 1, 4-dioxane (20 mL) and then methyl 4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2- (trifluoromethyl) benzoate (277.78 mg, 841.48. Mu. Mol), [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (57.27 mg, 70.12. Mu. Mol) and potassium carbonate (193.83 mg,1.40 mmol) were added and nitrogen was replaced 2-3 times. The reaction was carried out at 80℃for 8 hours. The reaction solution was suction-filtered through celite, the filtrate was washed with ethyl acetate (100 ml×3), the obtained organic phase was dried over anhydrous sodium sulfate (20 g), and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=15/1-8/1) to give the title compound (0.36 g) of the present step.

MS m/z(ESI)：694.1[M+H] ⁺ 。

And a second step of: preparation of 4- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl) thiazol-4-yl) -2- (trifluoromethyl) benzoic acid (C5)

Compound (C5-a) (360.00 mg, 518.99. Mu. Mol) was dissolved in tetrahydrofuran (5 mL), and an aqueous solution (2 mL) of sodium hydroxide (20.71 mg, 518.99. Mu. Mol) was added thereto to react at 25℃for 4 hours. The reaction solution was adjusted to pH 4 with 1M dilute hydrochloric acid, extracted with ethyl acetate (100 ml×3), and the organic phase was concentrated by drying over anhydrous sodium sulfate, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10/1-6/1) to give the title compound (45 mg).

MS m/z(ESI)：680.1[M+H] ⁺ 。

¹ H-NMR(400MHz，DMSO-d ₆ )δ：8.21(s，1H)，8.16(d，J＝7.3Hz，1H)，7.83(d，J＝7.2Hz，1H)，7.64(dd，J＝18.9，11.7Hz，1H)，7.57(d，J＝12.8Hz，1H)，4.33(s，1H)，4.12(s，1H)，2.33(s，1H)，2.01(d，J＝12.4Hz，1H)，1.79(s，1H)，1.68(m，1H)，1.19-1.01(m，1H)。

Example 6: preparation of 5- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl) thiazol-4-yl) picolinic acid (C6)

The first step: preparation of methyl 5- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl) thiazol-4-yl) picolinate (C6-a)

Compound (T1) (50 mg, 87.65. Mu. Mol) was dissolved in 1, 4-dioxane (20 mL), followed by addition of methyl 5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) picolinate (27.67 mg, 105.19. Mu. Mol), [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (7.16 mg, 8.77. Mu. Mol) and potassium carbonate (12.11 mg, 87.65. Mu. Mol), nitrogen substitution was performed for 2-3 times.80℃for 8 hours.

MS m/z(ESI)：626.1[M+H] ⁺ 。

And a second step of: preparation of 5- (2- ((1R, 3r, 5S) -3- ((5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methoxy) -8-azabicyclo [3.2.1] oct-8-yl) thiazol-4-yl) picolinic acid (C6)

Compound (C6-a) (200 mg, 316.61. Mu. Mol) was dissolved in tetrahydrofuran (5 mL), and an aqueous solution (2 mL) of sodium hydroxide (12.66 mg, 316.61. Mu. Mol) was added thereto to react at 25℃for 4 hours. The reaction solution was adjusted to pH 4 with 1M dilute hydrochloric acid, extracted with ethyl acetate (100 ml×3), and the organic phase was dried over anhydrous sodium sulfate and concentrated by filtration, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10/1-6/1) to give the title compound (25 mg).

MS m/z(ESI)：618.1[M+H] ⁺ 。

¹ H-NMR(400MHz，DMSO-d ₆ )δ：8.88(s，1H)，8.20(dd，J＝8.1，2.1Hz，1H)，7.93(d，J＝8.2Hz，1H)，7.71-7.61(m，1H)，7.55(t，J＝7.5Hz，1H)，7.44(s，1H)，4.32(s，1H)，4.11(s，1H)，3.52(s，1H)，2.33(dd，J＝10.8，5.9Hz，1H)，2.01(d，J＝13.7Hz，1H)，1.79(s，2H)，1.67(d，J＝14.5Hz，1H)，1.13(m，1H)，1.09-1.03(m，1H)。

Biological assays

Experimental example 1: bile acid receptor FXR cofactor binding assay

1. Test method

Invitrogen LanthaScreen is used ^TM The TR-FRET Famesoid X Receptor Coactivator Assay kit measures the activation of FXR by a compound.

After incubating the receptor with the compound to be tested with different concentrations at room temperature, adding fluorescent-labeled coactivator short peptide and terbium-labeled antibody, and detecting FRET signal after reacting at room temperature. Calculating activation activity EC of a test compound on FXR by using a four-parameter fitting equation and taking a receptor-free protein group as a blank ₅₀ Maximum activation effect value E _max ：

y＝Emin+(Emax-Emin)/(1+(x/EC ₅₀ )^(-Hillslope))

Wherein y is FRET binding signal, emax and Emin are upper and lower asymptote estimates, respectively, of the fitted curve, x is the logarithmic concentration of the compound, and Hillslope is the slope of the curve.

In addition, with chenodeoxycholic acid (i.e., CDCA) as a positive control, the relative activating effect of the tested compounds of the present invention was calculated by the following formula:

relative activation effect (%) = (Emax/Emax') ×100%

Wherein Emax represents the maximum activation effect value of the compounds tested according to the invention and Emax' represents the maximum activation effect value of CDCA, both calculated by the formula shown above.

2. Test results

TABLE 1 EC of tested compounds of the invention on FXR ₅₀

Numbering of compounds	EC 50 (μM)
		CDCA	4.43
C3	0.133
		C4	0.022
C5	0.002
		C6	0.031

The data in Table 1 show an EC value of 4.43. Mu.M relative to chenodeoxycholic acid (CDCA) ₅₀ The compounds tested have lower EC ₅₀ (0.002-0.133. Mu.M) indicating that the compounds of the present invention have better activating activity for FXR.

TABLE 2 relative activating effect of the tested compounds of the invention on FXR

Numbering of compounds	Relative activation effect
		CDCA	100％
C3	154％
		C4	137％
C5	196％
		C6	177％

The data in Table 2 show that the compounds tested in accordance with the present invention have a higher maximum activation effect than chenodeoxycholic acid (CDCA), indicating that the compounds of the present invention have a good maximum activation effect on FXR.

EC of table 1 and table 2 are combined ₅₀ The values and relative activation effect data show that the compounds of the invention have a better activation effect on FXR.

Experimental example 2 luciferase reporter Gene detection experiment

1. Test method

Human embryonic kidney cells HEK293 were cultured in DMEM medium containing 10% fbs. And co-transfecting the plasmid to enable the plasmid to express FXR and a human BSEP luciferase reporter gene. Transfected cells were digested, resuspended, counted, and then plated into multiwell plates. 10. Mu.L of the test compounds were added to the well plates at respective final concentrations of 64. Mu.M, 16. Mu.M, 4. Mu.M, 1. Mu.M, 0.25. Mu.M, 0.0625. Mu.M, 0.0156. Mu.M, 0.0039. Mu.M, 0.000975. Mu.M, 0.000244. Mu.M, 0. Mu.M, and DMSO at a final concentration of 0.5%. After the test compound is incubated with the cells for 18 hours, brigh-GloTM detection reagent is added, a multifunctional full-automatic enzyme-labeled instrument is used for detecting chemiluminescence unit value (RLU), and the relative signal percentage (%) under the concentration of each test compound is calculated by taking the signal value of a blank hole (without the test compound) as 100%. Tested chemicals were fitted using a four parameter model with SigmaPlat 10 softwareCompound EC ₅₀ And a maximum agonism Emax (relative signal percentage).

2. Test results

The measurement results are shown in Table 3 below.

TABLE 3 Table 3

Numbering of compounds	EC 50 (μM)	Emax
			C1	0.058±0.023	252％
C2	0.033±0.018	320％
			C3	0.037±0.019	343％
C4	0.021±0.006	322％
			C5	0.014±0.005	301％

The data in Table 3 shows that the compounds tested were measured in vitroCentering EC ₅₀ The value is between 0.014 mu M and 0.058 mu M, and the Emax value is more than 250%. The compounds of the invention are shown to have good FXR activation activity in vitro cell assays.

Experimental example 3: liver microsome stability experiment

1. The experimental method comprises the following steps:

the test compound (50. Mu.L) was mixed with each species of liver microsomes (100. Mu.L), and after pre-incubation at 37℃for 5 minutes, NADPH (50. Mu.L) was added thereto, and incubation was performed for 0, 30, and 60 minutes, with incubation concentrations of the test compound, NADPH, and liver microsome enzyme being 1. Mu.M, 1mM, and 0.5mg/mL, respectively. The reaction was quenched by addition of glacial acetonitrile (200 μl), and then an appropriate volume of internal standard (rivaroxaban) was added, the supernatant was vortexed and centrifuged for detection.

The detection method comprises the following steps:

LC-MS/MS, mass spectrum API 5500, liquid phase is Shimadzu LC-30AD system. The chromatographic column is Hypersil GOLD C18, particle size of 1.9 μm, 50×2.1mm; the mobile phase A is water+0.1% formic acid, and the mobile phase B is acetonitrile; the flow rate was 0.55mL/min and the column temperature was 40 ℃. The ion source is adopted as ESI source positive ion mode, and the scanning mode is Multiple Reaction Monitoring (MRM).

The half life and liver clearance of the drug were calculated by measuring the sample concentrations at different incubation times and plotting "Ln (drug residual%)" against "incubation time" to obtain a rate constant, and the metabolic stability of the drug in the liver microsomes was evaluated by the half life and liver clearance values of the drug.

2. Experimental results:

TABLE 4 Table 4

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TABLE 5

Conclusion: from the experimental data in tables 4 and 5, it can be seen that the compounds of the present invention have slower clearance in liver microsomes, better stability and good pharmacokinetic properties.

Experimental example 4 rat Pharmacokinetic (PK) study

The pharmacokinetic profile was examined by administering the compounds of the invention to male SD rats Intravenously (IV) and intragastrically (PO), respectively. The IV and PO doses were 1mg/kg and 5mg/kg, respectively, with the IV dosing vehicle system 5% DMSO:5% solutol:90% physiological saline and 0.5% MC (methylcellulose) as PO administration vehicle system. Blood is collected at different time points after IV and PO administration, anticoagulated by K2-EDTA, and plasma samples are obtained after centrifugation and stored at-80 ℃. Plasma samples were subjected to LC-MS/MS analysis after treatment with precipitated proteins.

Pharmacokinetic parameters were calculated using the non-compartmental model using WinNonlin 6.3 software, the results are shown in tables 6 and 7.

Table 6. Pharmacokinetic parameters of iv administered compounds in rats

The data in Table 6 show that compounds C3, C5 of the invention, administered at a dose IV of 1mg/kg, have excellent drug exposure in rats.

TABLE 7 pharmacokinetic parameters of PO-administered Compounds in rats

The data in Table 7 show that compounds C3, C5 of the present invention, administered at a dose of PO of 5mg/kg, have excellent drug exposure and bioavailability in rats.

Taken together, tables 6 and 7, compounds C3, C5 of the present invention have excellent plasma drug exposure and oral bioavailability in rats by IV and PO administration.

Other compounds of the invention also have better AUC _last Value, C _max The value and bioavailability have better pharmacokinetic properties in rats.

Experimental example 5 mice fatty liver model efficacy test

Test method

Male C57 mice (purchased from Vetong Liwa) 8-10 weeks old were used, and animals were randomly divided into 5 groups according to body weight before molding, which were normal group (group 1), model group (group 2), and C3 compound administration group (30 mg/kg), respectively. The normal group was given normal feed, the model group and the C3 compound-fed group were fed MCD feed (methionine choline deficient feed, research Diets). At the same time of molding, the C3 compound group was administered by single gastric lavage daily for 28 days. The normal and model groups were given 0.5% mc (methylcellulose). Details are shown in Table 8.

TABLE 8 grouping and dosing schedules

Detection index

Drug efficacy index: animals were dissected after day 28 dosing, liver HE staining was performed for pathology scoring (scoring criteria reference "guidelines for diagnosis and treatment of chinese non-alcoholic fatty liver disease").

Pharmacokinetic index: 0.5h, 1h, 4h after day 28 dosing, blood, liver and small intestine tissue samples were taken from animals and drug concentrations were measured using LC-MS/MS.

Test results

Efficacy results: as can be seen from fig. 1, the model group fatty transformation score was significantly higher than that of the normal group, indicating successful acquisition of the mouse fatty liver model. Furthermore, the 30mg/kg dose of the C3 compound group showed a significant difference in the fat change score compared to the model group.

Pharmacokinetic results: pharmacokinetic data obtained in the experiment are shown in table 9.

TABLE 9 PK data (30 mg/kg) from C3 in vivo efficacy test

Sample of	Plasma of blood	Liver	Small intestine
				Time point/concentration	ng/ml	ng/g	ng/g
0.25h	373±242	3263±1429	1115±488
				1h	1353±204	12700±4260	4940±57
4h	2212±462	13040±4218	6105±2471

As can be seen from the above table, compound C3 was distributed throughout the liver and intestine with higher liver and intestine exposure than plasma.

Conclusion of the test

Compound C3 was able to inhibit MCD feed-induced liver steatosis with significant improvement over the model group. Compound C3 was distributed higher in the liver than in plasma, indicating that it was effectively enriched at the target site.

Various modifications of the application, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this disclosure (including all patents, patent applications, journal articles, books, and any other publications) is hereby incorporated by reference in its entirety.

Claims (15)

1. A compound of formula (I) or a stereoisomer, tautomer, pharmaceutically acceptable salt thereof,

wherein:

x is CH or N; and is also provided with

R is selected from hydrogen, halogen and C _1-6 Alkyl and C _1-6 A haloalkyl group.

2. The compound of claim 1, or a stereoisomer, tautomer, pharmaceutically acceptable salt thereof, wherein R is selected from the group consisting of hydrogen, fluoro, methyl, and trifluoromethyl.

3. A pharmaceutical composition comprising at least one compound according to any one of claims 1-2, or a stereoisomer, tautomer, pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

4. A pharmaceutical composition comprising at least one compound or a stereoisomer, tautomer, pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers selected from the group consisting of water, peanut oil, soybean oil, mineral oil, sesame oil, physiological saline, aqueous glycerol, starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, ethanol, optionally with minor amounts of wetting agents, emulsifying agents or pH buffering agents;

The compound is selected from the group consisting of:

5. the pharmaceutical composition of claim 3 or 4, which is in a form selected from the group consisting of: tablets, capsules, troches, hard candies, powders, sprays, creams, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs and syrups.

6. A kit, comprising:

a) A first container comprising at least one compound of any one of claims 1-2, or a stereoisomer, tautomer, pharmaceutically acceptable salt thereof, as a first therapeutic agent, or a pharmaceutical composition of any one of claims 3-5, as a first pharmaceutical composition;

b) An optional second container comprising at least one other therapeutic agent as a second therapeutic agent, or a pharmaceutical composition comprising the other therapeutic agent as a second pharmaceutical composition; and

c) Optionally package insert.

7. Use of a compound according to any one of claims 1-2, or a stereoisomer, tautomer, pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 3-5, for the manufacture of a medicament for the prevention or treatment of a disease or condition mediated by farnesoid X receptors.

8. A process for preparing a compound of general formula (I) according to any one of claims 1 to 2, which comprisesThe method comprises the following steps:

wherein:

Hal ¹ 、Hal ² and Hal ³ Each independently is the same or different halogen;

PG ¹ is an amino protecting group;

PG ² is a carboxyl protecting group;

y is boric acid or a borate group;

the remaining groups are as defined in claim 1 or 2;

the reaction conditions of each step are as follows:

step A: reacting compound IN-1 with compound IN-2 to give compound IN-3;

and (B) step (B): removal of PG IN Compound IN-3 ¹ A group to give compound IN-4;

step C: reacting compound IN-4 with compound IN-a to give compound IN-5;

step D: reacting compound IN-5 with compound IN-b to give compound IN-6; and

step E: removal of PG IN Compound IN-6 ² A group to give a compound of the general formula (I).

9. The method of claim 8, wherein Hal ¹ 、Hal ² And Hal ³ Each independently F, cl, br or I.

10. The method of claim 8, wherein Hal ¹ 、Hal ² And Hal ³ Each independently is Cl or Br.

11. The method of claim 8, wherein PG ¹ Is tert-butyloxycarbonyl.

12. The method of claim 8, wherein PG ² Is C _1-6 An alkyl group.

13. The method of claim 8, wherein PG ² Is methyl.

14. The method of claim 8, wherein Y is-B (OH) ₂ Or alternatively

15. A compound of the general formula (IN-6) or a pharmaceutically acceptable salt thereof,

wherein each group is as defined in any one of claims 8 or 13.