CN112805279A - Isoxazole derivative and preparation method and application thereof - Google Patents

Abstract

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

Description

Isoxazole derivative and preparation method and application thereof Technical Field

The present invention relates generally to compounds useful for the treatment of diseases or disorders mediated by Farnesoid X Receptor (FXR), and more particularly to FXR agonist compounds, as well as 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 use.

Background

Farnesoid X receptors (FXR, NR1H4) are expressed in The liver, The entire gastrointestinal tract including The esophagus, stomach, duodenum, small intestine, colon, kidney and adrenal gland (Kuipers, f. et al, The Farnesoid X Receptor (FXR) as a Modulator of double Acid Metabolism, rev. endogrine metal. disorders, 2004, 5: 319-. FXR is a member of transcription factors known to be activated as ligands for nuclear receptors. Bile acids such as chenodeoxycholic acid (CDCA) or its taurine or glycine amide conjugates are endogenous ligands of FXR. FXR is activated upon binding of bile acid to FXR, and expression of various genes, including those involved in bile acid, cholesterol, triglyceride, lipoprotein homeostasis in liver and circulatory system, is controlled by heterodimer complexes with Retinoid X Receptors (RXR) (Kalaany, N.Y.; Mangelsdorf, D.J.; LXRS and FXR: the yin and yang of cholesterol and fat metabolism, Annu.Rev. Physiol., 2006, 68, 159. 191; Calif., A.C.; Tontonoz, P.; transcription of metabolism by the nuclear-activated receptors LXR and FXR, Nat.Rev. mol. biol., cell, 13, 213. 224). FXR also appears to be involved in paracrine and endocrine signaling by up-regulating Fibroblast growth factor 15 (rodents) or Fibroblast growth factor 19 (monkeys, humans) (t.inagaki et al, fibre growth factor 15 functions as an enterohepatic signal to regulated biological acid homeostasis. cell method, 2005, 2(4), 217-225).

Bile acids are amphiphilic molecules that form micelles and emulsify lipids in the diet. If the concentration of bile acid is too high, cytotoxicity may occur, and thus there is a mechanism for strictly controlling the concentration of bile acid physiologically. FXR plays a key role in controlling Bile acids to maintain a stable state in vivo (Makishima, M.; nucleic acids as Targets for Drug Development: Regulation of Cholesterol and Bile Acid Metabolism by nucleic acids Receptors, J.Pharmacol. Sci., 2005, 97: 177-183).

In addition, FXR has also been shown to regulate complex biological processes beyond metabolism, such as liver regeneration or integrity of the intestinal barrier. FXR also controls the intestinal and hepatic immune systems and has some anti-inflammatory effect (Modica, s.; Gadaleta, r.m.; moschatta, a.; differentiation the nuclear double acid receptor FXR paramigim, nuclear.receptor.signal., 2010, 8, e 005).

Obeticholic Acid (Obeticholic Acid, 6-Et CDCA) is an FXR receptor agonist with greater activity than the endogenous ligand CDCA and has been shown to have significant improvement in insulin sensitivity in phase IIa clinical studies of non-alcoholic fatty liver disease (NAFLD) and other metabolic beneficial effects (Mudaliar, S., Henry, R.R.; Sanyal, A.J., Et al, effectiveness and safety of the fatty X receptor agonist in tissue with type 2 reagents and non-alcoholic fatty liver disease, Gastroenterology, 574, 3, 145, 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 have improved liver function impairment (Nevens, f., Andreone, p., Mazzella, g., etc., The first primary bipolar renal (PBC) phase 3 tertiary in two decades-an international study of The FXR aginst inflammatory acid in PBC polypeptides, j.hepatol., 2014, 60, S525-S526).

WO2012087519 discloses a method for the treatment of a condition mediated by FXR with an agonist or partial agonist of FXR. However, FXR agonist compounds disclosed in the prior art still have deficiencies in pharmacodynamic or pharmacokinetic properties.

Summary of The Invention

The present invention relates generally to compounds of general 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

R is selected from hydrogen, halogen and C_1-6Alkyl and C_1-6A haloalkyl group.

Another aspect of the invention is a pharmaceutical composition comprising a compound of the 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, 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 condition mediated by FXR.

The invention also includes a method of preventing or treating a disease or disorder mediated by FXR, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the general formula (I) or a stereoisomer, tautomer, polymorph, solvate (such as 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 condition mediated by FXR, comprising:

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

b) optionally a second container comprising at least one further therapeutic agent as a second therapeutic agent, or a pharmaceutical composition comprising said further therapeutic agent as a second pharmaceutical composition; and

c) optionally, packaging instructions.

The invention also includes a compound of the general formula (I) or a stereoisomer, a tautomer, a polymorph, a solvate (such as a hydrate), a pharmaceutically acceptable salt, an ester, a metabolite, an N-oxide, a chemically protected form or a prodrug thereof, or a pharmaceutical composition thereof, for use in the prevention or treatment of a disease or disorder mediated by FXR.

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

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

The compound of the general formula (I) has excellent in vivo or in vitro pharmacodynamic or pharmacokinetic properties, shows good FXR activation activity and activation effect and excellent stability of liver microsomes, and thus has good pharmaceutical activity and metabolic advantages.

Brief description of the drawings

FIG. 1 shows the effect of administering compound C3 in a mouse fatty liver model, where a denotes p.ltoreq.0.05 compared to control; b represents p ≦ 0.05 compared to the model group.

Detailed Description

Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and molecular formulae. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that the invention is not limited to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the invention 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 invention. The present invention is in no way limited to the methods and materials described. In the event that one or more of the cited documents, patents, and similar materials differ or contradict this application (including but not limited to defined terms, usage of terms, described techniques, etc.), this application controls.

Definition of

Unless defined otherwise below, all technical and scientific terms used herein are intended to have the same meaning as 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 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 substituents. In some embodiments, the alkyl group has 1 to 6 carbon atoms (C)_1-6). In other embodiments, the alkyl group has 1 to 4 carbon atoms (C)_1-4) Especially 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 group, 2-methyl-2-butyl group, 3-methyl-1-butyl group, 2-methyl-1-butyl group, 1-hexyl group, 2-hexyl group, 3-hexyl group, 2-methyl-2-pentyl group (-C (CH).₃) ₂CH ₂CH ₂CH 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 a bond of a substituent is shown through a bond connecting two atoms in a ring, then such substituent may be bonded to any ring atom in the substitutable ring.

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

The term "stereoisomers" refers to compounds having the same chemical composition but differing in the spatial arrangement of atoms or groups.

"diastereomer" refers to a stereoisomer that has two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. 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 nonsuperimposable mirror images of each other.

The stereochemical definitions and rules used herein generally follow the general definitions of S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic 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 present 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 description of 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 prefixes d and l or (+) and (-) are used to indicate the sign of a compound rotating plane polarized light, where (-) or l means that the compound is left-handed. Compounds with the prefix (+) or d are 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. A50: 50 mixture of enantiomers is referred to as a racemic mixture or racemate, which may occur without stereoselectivity or stereospecificity in the chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers, with no optical activity. In one aspect, stereoisomers of the invention may exist in predominant forms, 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.

Where the process for preparing the compounds of the invention produces 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, for example by formation of diastereomeric pairs by salt formation with an optically active acid such as (-) -di-p-toluoyl-d-tartaric acid and/or (+) -di-p-toluoyl-l-tartaric acid, followed by fractional crystallization and regeneration of the free base. The compounds can also be resolved by the formation of diastereomeric esters or amides, followed by chromatographic purification and removal of the chiral auxiliary. Alternatively, the compounds can be resolved using a chiral HPLC column.

The term "tautomer" or "tautomeric form" refers to energetically different structural isomers that can interconvert through a low energy barrier. For example, proton tautomers (also referred to as proton transfer tautomers) include interconversions by proton transfer, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions through recombination of some of the bonding 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 ratio.

It will be appreciated that certain compounds of the invention may be present in free form for use in therapy or, where appropriate, in the form of a pharmaceutically acceptable derivative 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, which upon administration to a subject in need thereof are capable of providing, directly or indirectly, a compound of the present invention or a metabolite or residue 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 (such as hydrates), pharmaceutically acceptable salts, esters, metabolites, N-oxides of said compound of formula (I) as well as 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, isonicotinates, salicylates, acid citrates, oleates, tannins, pantothenate, gluconates, glucuronates, saccharinates, and pamoate (i.e., 1' -methylene-bis (2-hydroxy-3-naphthoate)) and the like. A pharmaceutically acceptable salt may include the inclusion of another molecule of a counterion. The counter ion may be any organic or inorganic ion that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. The plurality of charged atoms may be part of a pharmaceutically acceptable salt and may have a plurality of counter ions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counter ions.

If the compound of the invention is a base, the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, by treating the free base with an inorganic acid or with an organic acid such as salicylic acid, pyranoside acids such as glucuronic or galacturonic acid, alpha-hydroxy acid amino acids, aromatic acids, and the like.

If the compound of the invention is an acid, the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example, by treating the free acid with an inorganic or organic base such as an amine (primary, secondary or tertiary), an 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 comprising 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 hydrolysable esters, which can be hydrolysed under physiological conditions to release the compound of formula (I) of the invention in free acid or alcohol form. The compounds of the general formula (I) according to the invention may themselves also be esters.

The compounds of the invention may be present in the form of solvates, such as hydrates, wherein the compounds of the invention comprise as structural element of the crystal lattice of the compound a polar solvent, such as in particular water, methanol or ethanol. The amount of polar solvent, particularly water, may be present in stoichiometric or non-stoichiometric proportions.

A "metabolite" is a product produced by the in vivo metabolism of a particular compound or salt thereof. Metabolites of compounds can be identified using conventional techniques known in the art and their activity can be determined using assays such as those described herein. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound. 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, since the available lone pair is required for oxidation of the nitrogen to the oxide; one skilled in the art will recognize nitrogen-containing heterocycles that are 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 oxidation of heterocycles and tertiary amines with peroxy acids such as peracetic and m-chloroperbenzoic acid (m-CPBA), hydrogen peroxide, alkyl hydroperoxides such as t-butyl hydroperoxide, sodium perborate, and dioxiranes (dioxiranes) such as dimethyldioxirane. 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-; s.v.ley, ed., Pergamon Press; M.Tisler and B.Stanovnik, Comprehensive Heterocyclic Chemistry, vol.3, pp 18-20.

In 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 concerned, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved by conventional protecting Groups, for example, as described in Protective Groups in Organic Chemistry, ed.j.f.w.mcomie, Plenum Press, 1973; and T.W.Greene & P.G.M.Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which are incorporated herein by reference. The protecting group 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 treatment methods 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 is converted in vivo to the above-described compounds upon administration to a subject. Conventional methods for selecting and preparing suitable Prodrug derivatives are described, for example, in "Design of produgs", ed.h. bundgaard, Elsevier, 1985.

Any formula or structure shown herein, including compounds of formula (I), is also intended to represent unlabeled forms and isotopically labeled forms of the compounds. Isotopically labeled compounds have the structure shown 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 contained in compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as, but not limited to²H (deuterium, D),³H (tritium),¹¹C、 ¹³C、 ¹⁴C、 ¹⁵N、 ¹⁸F、 ³¹P、 ³²P、 ³⁵S、 ³⁶Cl and¹²⁵I. various isotopically-labeled compounds of the invention, for example, those comprising³H、 ¹³C and¹⁴those of the radioactive isotopes of C. Such isotopically labeled compounds are useful in metabolic studies, reaction kinetic studies, 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.¹⁸The F-labeled compounds are useful for PET or SPECT studies. Isotopically labeled compounds of the present invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and the preparative procedures described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent. Furthermore, with heavier isotopes, in particular deuterium (i.e. deuterium)²H 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 indexAnd then. It is to be understood that deuterium is considered as a substituent in the compounds of formula (I) in this case. The concentration of such heavier isotopes, particularly deuterium, can be defined by the isotopic enrichment factor. In the compounds of the present invention, any atom not specifically designated as a particular isotope is intended to represent any stable isotope of that atom. Unless otherwise indicated, when a position is explicitly designated as "H" or "hydrogen," it is understood that the position has hydrogen in its natural abundance isotopic composition. Thus, in the compounds of the present invention, any atom explicitly designated as 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 product which results, directly or indirectly, from a combination of compounds of formula (I) of the present invention.

Compound (I)

In some embodiments, the present invention provides 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,

wherein:

x is CH or N; and is

R is selected from hydrogen, halogen and C_1-6Alkyl and C_1-6A haloalkyl group.

In some embodiments, the present invention provides a compound, or a stereoisomer, tautomer, polymorph, solvate (such as hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, chemically protected form or prodrug thereof, wherein R is selected from the group consisting of hydrogen, fluoro, methyl and trifluoromethyl.

In some embodiments, the present invention provides a compound or a stereoisomer, tautomer, polymorph, solvate (such as 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 present 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 X is N; and R is hydrogen.

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

the compounds of the general formula (I) according to the invention may contain asymmetric or chiral centers and may thus exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers thereof, as well as mixtures thereof, e.g., 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, the cis and trans forms as well as mixtures thereof are encompassed within the scope of the present invention. In the structures shown herein, all stereoisomers are considered and included as compounds of the present invention if the stereochemistry of any particular chiral atom is not specified. If stereochemistry is indicated by a solid or dashed wedge representing a particular 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 contemplated to be 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

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

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

Pharmaceutically acceptable carriers that may be employed in the pharmaceutical compositions of the present 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. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. 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 optionally contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents. 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 systemically and/or locally. For this purpose, they may be administered by a suitable route, for example by injection, intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular or transdermal administration; or by oral, buccal, nasal, transmucosal, topical, in the form of ophthalmic preparations 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 uses

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

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

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

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

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

inflammatory bowel disease, dyslipidemia, atherosclerosis, diabetes and related diseases; lipid and lipoprotein disorders; type II diabetes and clinical complications of 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 fibrotic conditions due to forced lipid, particularly triglyceride, accumulation and subsequent activation of the profibrotic pathway, such as non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH); obesity or metabolic syndrome (dyslipidaemia, diabetes and co-morbidities with abnormally high body mass index);

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

As used herein, unless otherwise indicated, the term "treating" or "treatment" means reversing, alleviating, inhibiting the progression of the indicated disease or disorder or one or more symptoms of such disease or disorder.

As used herein, "individual" includes a human or non-human animal. Exemplary human individuals include human individuals (referred to as patients) having a disease (e.g., a disease described herein) or normal individuals. "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, livestock 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 above-mentioned therapeutic efficacy when administered.

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 dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is noted that dosage values may vary with the type and severity of the condition being alleviated, and may include single or multiple doses. It is further understood that for any particular individual, the specific dosage regimen will be adjusted over time according to the individual need and the professional judgment of the person administering the composition or supervising the administration of the composition.

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

The amount or amount of a compound of the invention in the pharmaceutical composition may be from about 0.01mg to about 1000mg, suitably 0.1-500mg, preferably 0.5-300mg etc.

Combination therapy

The compounds of general 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 general formula (I) are combined with other therapeutic agents, e.g., having anti-hyperproliferative efficacy, in the pharmaceutical compositions or dosage regimens as a combination therapy. The other therapeutic agent may be, for example, a chemotherapeutic agent. The pharmaceutical composition or other therapeutic agent of the dosing regimen preferably has complementary activities to the compound of formula (I) such that they do not adversely affect each other. Such compounds are suitably present in combination in an amount effective for the intended purpose.

The combination therapy may be administered on a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations. 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 preferably there is a period of time during which both (or all) active agents exert their biological activity simultaneously.

Suitable dosages for any of the above-mentioned concurrently administered agents are those currently used and may be reduced due to the combined (synergistic) action of the newly identified drug with other therapeutic agents or treatments.

Combination therapy may provide "synergy" and prove "synergistic," i.e., the effect achieved when the active ingredients are used together is greater than the sum of the effects produced when the compounds are used separately. When the active ingredient: (1) when co-formulated and administered or delivered simultaneously in a combined unit dose formulation; (2) when delivered alternately or in parallel as separate formulations; or (3) by some other scheme, a synergistic effect may 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. Generally in alternation therapy, an effective dose of each active ingredient is administered sequentially, i.e. consecutively, whereas in combination therapy, effective doses of two or more active ingredients are administered together.

In a particular embodiment of treatment, a compound of 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 may be combined with other therapeutic agents, such as those described herein, and also with surgical treatment and radiotherapy. Thus, the combination therapy of the present invention comprises the 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, chemically protected 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 to achieve the desired combined therapeutic effect.

Metabolites of compounds of formula (I)

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

Metabolites are typically produced by preparing radioisotopes of the invention (e.g.¹⁴C or³H) Labeled compounds are identified by parenterally administering them to an animal such as rat, mouse, guinea pig, monkey, or human at a detectable dose (e.g., greater than about 0.5mg/kg), metabolizing for a sufficient time (typically about 30 seconds to 30 hours), and isolating their conversion products from urine, blood, or other biological samples. These products are easy to isolate because they are labelled (others are isolated by using antibodies capable of binding to epitopes remaining in the metabolite). Metabolite structure is determined in conventional manner, e.g. by MS, LC/MS or NMR analysis. Analysis of the metabolites was performed in the same manner as in conventional drug metabolism studies well known to those skilled in the art. Metabolites, as long as they are not found in vivo, can be used in diagnostic assays to therapeutically administer the compounds of the present invention.

Medicine box

In other embodiments of the invention, "kits" are provided that contain materials useful for treating the above-described diseases or conditions. The kit comprises a container containing a compound of general formula (I), a stereoisomer, a tautomer, a polymorph, a solvate (e.g. a hydrate), a pharmaceutically acceptable salt, an ester, a metabolite, an 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 typically contained in commercial packages of therapeutic products that contain information regarding indications, usage, dosages, administration, contraindications and/or warnings associated with the use of the therapeutic products. 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 to treat a condition, 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 for use in treating a selected condition, such as cancer. In addition, the label or package insert can 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 can also indicate that the composition can 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 condition mediated by FXR, or a pharmaceutical composition comprising the other therapeutic agent, as a second pharmaceutical composition. Thus, in some embodiments, the kit can 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 an additional therapeutic agent, the kit may further comprise instructions for administering the first and second pharmaceutical compositions simultaneously, sequentially or separately 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 commercial and user, including other buffers, diluents, fillers, 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 kits preferably comprise a plurality of unit doses. Such kits may include cards with the dosage positioned for their intended use. One example of such a kit is a "blister pack". Blister packaging is well known in the packaging industry and is widely used for packaging pharmaceutical unit dosage forms. If desired, a memory aid may be provided in the form of a number, letter or other indicia or calendar insert, for example, specifying the day of administration in a treatment schedule.

Process for the preparation of compounds

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

wherein:

Hal ¹、Hal ²and Hal³Each independently of the other, identical or different halogen, for example F, Cl, Br or I, preferably Cl or Br;

PG ¹is an amino protecting group, preferably tert-butyloxycarbonyl (Boc);

PG ²as a carboxyl protecting group, preferred isIs C_1-6Alkyl, more preferably methyl;

y is a boronic acid or boronic ester group, preferably-B (OH)₂Or

The remaining groups are as defined above;

the reaction conditions for each step were 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, 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 alkoxide or carbonate of an alkali metal) and/or a catalyst. The catalyst may be a catalyst system comprising a crown ether, which may be selected from 15-crown-5 and 18-crown-6; the carbonate of the 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 and potassium ethoxide. Preferably, the alkali metal alkoxide and the catalyst are sodium tert-butoxide and/or potassium tert-butoxide in combination with 15-crown-5 and/or 18-crown-6, preferably sodium tert-butoxide in combination with 15-crown-5 or potassium tert-butoxide in combination 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: removal of PG from Compound IN-3¹To 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, ethyl chloride, dichloroethane, trichloroethane), N-dimethylformamide, N-dimethylacetamide, and any combination thereof, preferably dichloromethane. The reaction can 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., a carboxylic acid 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.

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

IN some embodiments, compound IN-4 is subjected to a substitution reaction with compound IN-a to give 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 ℃, e.g. 30-140 ℃, preferably 25 ℃, 50 ℃, 100 ℃ or 130 ℃, preferably 80 ℃. The substitution reaction is preferably carried out for a suitable time, for example 2 to 24 hours, 2 to 18 hours or 2 to 12 hours, for example 5, 8 or 10 hours.

IN other embodiments, compound IN-4 is coupled with 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, palladium triphenylphosphine, 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, 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 to 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-5 is subjected to a metal-catalyzed coupling reaction with the compound IN-b to give the compound IN-6. The metal-catalyzed coupling reaction is carried out by conventional methods. For example: the compound IN-5 and the 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 to react at a temperature of 50 ℃ to 120 ℃ (preferably 80 ℃ or 90 ℃) for 8 to 24 hours (preferably 8 hours or 12 hours), optionally under nitrogen protection. The organic solvent is N, N-dimethylformamide, tetrahydrofuran, 1, 4-dioxane, toluene or DME and the like. The palladium catalyst is tris (dibenzylideneacetone) dipalladium, [1, 1 '-bis (diphenylphosphino) ferrocene ] dichloropalladium, triphenylphosphine palladium and palladium acetate, preferably [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium or tris (dibenzylideneacetone) dipalladium and the like. The base is preferably an inorganic base such as potassium carbonate, cesium carbonate, sodium bicarbonate or potassium bicarbonate, and the like.

Step E: removal of PG from Compound IN-6²A 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.), N-methylpyrrolidone, N-dimethylformamide, N-dimethylacetamide, 1, 4-dioxane, dimethylsulfoxide 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 to 5 hours or 6 to 15 hours, for example 2, 3 or 4 hours or overnight.

IN other embodiments, the present 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 selection of a particular compound or condition will depend on the particular synthetic procedure to be performed and the identity of the molecule or molecules to be transformed, but 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 carried out under anhydrous or inert atmosphere, etc.) can 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 of preparing compounds of formula (I). One 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 can be substituted to provide various derivatives and/or reaction conditions. In addition, many of the example compounds produced by the methods described herein may be further modified using conventional chemistry well known to those skilled in the art, with reference to this disclosure.

In preparing the compounds of formula (I), it may be desirable to protect the remote functional groups (e.g., carboxyl or amino groups) of the intermediates. The need for such protection may vary with the nature of the distal functional group and the conditions of the preparation method. The necessity of such protection is readily determined by the person skilled in the art. For a summary 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 provided to limit the scope of the invention.

The structure of the compound is determined by nuclear magnetic resonance¹H-NMR) or Mass Spectrometry (MS).¹The H-NMR shifts (δ) are given in parts per million (ppm). Chemical shift is at 10^-6(ppm) is given as a unit.

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

Preparation of high performance liquid phase a shimadzu preparative high performance liquid chromatograph was used.

Thin layer chromatography silica gel plate (TLC) an aluminum plate (20X 20cm) from Merck was used, 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 a developing system of: 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 solvent needs to be adjusted, or triethylamine and the like need to be further added, so as to realize separation and purification of the product.

Use of microwave reaction

Initiator + (400W, RT-300 ℃) microwave reactor.

The column chromatography generally uses 200-300 mesh silica gel as a carrier. The system of eluents comprises: the volume ratio of the solvent is adjusted according to different polarities of the compounds, and a small amount of triethylamine can be added for adjustment.

Unless otherwise specified, the reaction temperatures of the examples are room temperature (20 ℃ C. to 30 ℃ C.).

The reagents used in the present invention are commercially available from Acros Organics, Aldrich Chemical Company, Shanghai Teber Chemical science and technology, Inc., and the like.

In the conventional syntheses, as well as in the examples and intermediate preparations, the abbreviations have the following meanings.

Abbreviations	Means of	Abbreviations	Means of
TLC	Thin layer chromatography	LC-MS	Liquid chromatogram-mass spectrum combined instrument
DMF	N, N-dimethylformamide	DME	Ethylene glycol dimethyl ether

Preparation example of intermediate:

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 is as follows: (E) preparation of (E) -2- (trifluoromethoxy) benzaldoxime (T1-b)

2- (trifluoromethoxy) benzaldehyde (T1-a) (150g, 788.98mmol) was dissolved in a mixed solvent of ethanol (1000mL) and water (1000mL), hydroxylamine hydrochloride (65.79g, 946.77mmol) was added with mechanical stirring to precipitate a white solid, and a 1M aqueous solution of sodium hydroxide (789mL, 788.98mmol) was further added. The reaction mixture was reacted at 25 ℃ for 2 hours, 1M hydrochloric acid (2000mL) was added to the mixture to adjust the system pH to about 5, followed by suction filtration and drying at 50 ℃ overnight to obtain compound (T1-b) (150g, yield: 92%).

The second step is that: preparation of (Z) -N-hydroxy-2- (trifluoromethoxy) iminebenzyl chloride (T1-c)

Compound (T1-b) (150g, 731.23mmol) was dissolved in DMF (1000mL), and N-chlorosuccinimide (117.17g, 877.48mmol) was added under mechanical stirring at 0 ℃ to react at 0 ℃ for 1 hour. Water (2000mL) was added to the reaction, followed by extraction with ethyl acetate (1000 mL. times.3). The organic layer was dried over anhydrous sodium sulfate (500g), filtered and concentrated to obtain compound (T1-c) (160g, yield: 91%).

The third step: preparation of methyl 5-cyclopropyl-3- (2- (trifluoromethoxy) phenyl) isoxazole-4-carboxylate (T1-d)

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

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

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

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

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

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

The 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) (85g, 167.15mmol) was dissolved in dichloromethane (500mL) and dioxane hydrochloride solution (4M, 500mL) was added. The reaction was carried out at room temperature overnight. The reaction mixture was spin-dried to give compound (T1-h) (60g, 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)

The compound (T1-h) (7g, 15.73mmol) was dissolved in DMF (50mL), followed by addition of N, N-diisopropylethylamine (10.17g, 78.67mmol) and 2, 4-dibromothiazole (4.59g, 18.88mmol) and reaction at 100 ℃ for 12 hours. Water (300mL) was added to the reaction solution, and the mixture was dried over ethyl acetate (1000mL) and anhydrous sodium sulfate (20g), filtered, and the organic phase was concentrated and then purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 10/1-6/1) to obtain compound (T1) (2.8g, 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 is as follows: 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) (1g, 1.75mmol) was dissolved in 1, 4-dioxane (20mL) at room temperature, followed by addition of methyl 4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzoate (532.50mg, 1.93mmol), [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (143.16mg, 195.66. mu. mol) and potassium carbonate (483.85mg, 3.50mmol), and displacement with nitrogen 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 (100mL × 3), the obtained organic phase was dried over anhydrous sodium sulfate (20g), filtered and concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate ═ 10/1-6/1) to obtain the title compound (1.12g) of this step.

MS m/z(ESI)：626.1[M+H] ⁺。

The second step is that: 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) (390mg, 625.31. mu. mol) was dissolved in tetrahydrofuran (5mL), and an aqueous solution (2mL) of sodium hydroxide (25.01mg, 625.31. mu. mol) was added to conduct a reaction at 25 ℃ for 4 hours. The reaction solution was adjusted to pH 4 with 1M dilute hydrochloric acid, extracted with ethyl acetate (100 mL. times.3), 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 obtain the title compound (150 mg).

MS m/z(ESI)：612.1[M+H] ⁺。

¹H-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 is as follows: 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) (500mg, 876.55mmol) was dissolved in 1, 4-dioxane (20mL) at room temperature, followed by addition of methyl 3-methyl-4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzoate (266.25mg, 964.20 μmol), [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (71.58mg, 97.83 μmol) and potassium carbonate (241.93mg, 1.75mmol), and nitrogen substitution 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 (100mL × 3), the obtained organic phase was dried over anhydrous sodium sulfate (20g) and concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 10/1-6/1) to obtain the title compound (400mg) of this step.

MS m/z(ESI)：640.2[M+H] ⁺。

The second step is that: 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) (300mg, 468.98. mu. mol) was dissolved in tetrahydrofuran (5mL), and an aqueous solution (2mL) of sodium hydroxide (18.76mg, 468.98. mu. mol) was added to conduct a reaction at 25 ℃ for 4 hours. The reaction solution was adjusted to pH 4 with 1M dilute hydrochloric acid, extracted with ethyl acetate (100 mL. times.3), 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 to 6: 1) to obtain the title compound (280 mg).

MS m/z(ESI)：612.1[M+H] ⁺。

¹H-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 is as follows: 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) (650mg, 1.14mmol) was dissolved in 1, 4-dioxane (20mL) at room temperature, followed by addition of methyl 3-fluoro-4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzoate (248.13mg, 1.25mmol), [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (93.06mg, 127.18. mu. mol) and potassium carbonate (314.50mg, 2.28mmol), and displacement with nitrogen 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 (100mL × 3), the obtained organic phase was dried over anhydrous sodium sulfate (20g), and concentrated by filtration, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate ═ 10/1) to obtain the title compound (350mg) of this step.

MS m/z(ESI)：644.1[M+H] ⁺。

The second step is that: 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) (390mg, 625.31. mu. mol) was dissolved in tetrahydrofuran (5mL), and an aqueous solution (2mL) of sodium hydroxide (25.01mg, 625.31. mu. mol) was added to conduct a reaction at 25 ℃ for 4 hours. The reaction solution was adjusted to pH 4 with 1M dilute hydrochloric acid, extracted with ethyl acetate (100mL × 3), the organic phase was dried over anhydrous sodium sulfate and concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate ═ 10/1-6/1) to obtain the title compound (1.01 g).

MS m/z(ESI)：620.1[M+H] ⁺。

¹H-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 is as follows: 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) (400mg, 701.24. mu. mol) was dissolved in 1, 4-dioxane (20mL) at room temperature, followed by addition of methyl 2-fluoro-4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzoate (152.70mg, 771.36. mu. mol), [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (57.27mg, 78.26. mu. mol) and potassium carbonate (193.54mg, 1.40mmol), and nitrogen substitution 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 (100mL × 3), the obtained organic phase was dried over anhydrous sodium sulfate (20g), and concentrated by filtration, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate ═ 10/1) to obtain the title compound (200mg) of this step.

MS m/z(ESI)：630.1[M+H] ⁺。

The second step is that: 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) (200.00mg, 310.73. mu. mol) was dissolved in tetrahydrofuran (5mL), and an aqueous solution (2mL) of sodium hydroxide (12.43mg, 310.73. mu. mol) was added to conduct a reaction at 25 ℃ for 4 hours. The reaction solution was adjusted to pH 4 with 1M dilute hydrochloric acid, extracted with ethyl acetate (100 mL. times.3), the organic phase was dried over anhydrous sodium sulfate and concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate: 10/1-6/1) to obtain the title compound (170 mg).

MS m/z(ESI)：620.1[M+H] ⁺。

¹H-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 is as follows: 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) (0.4g, 701.24. mu. mol) was dissolved in 1, 4-dioxane (20mL) at room temperature, followed by addition of methyl 4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2- (trifluoromethyl) benzoate (277.78mg, 841.48. mu. mol), [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (57.27mg, 70.12. mu. mol) and potassium carbonate (193.83mg, 1.40mmol), and displacement with nitrogen 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 (100mL × 3), the obtained organic phase was dried over anhydrous sodium sulfate (20g), filtered and concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate ═ 15/1-8/1) to obtain the title compound (0.36g) of this step.

MS m/z(ESI)：694.1[M+H] ⁺。

The second step is that: 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) (360.00mg, 518.99. mu. mol) was dissolved in tetrahydrofuran (5mL), and an aqueous solution (2mL) of sodium hydroxide (20.71mg, 518.99. mu. mol) was added to conduct a reaction at 25 ℃ for 4 hours. The reaction solution was adjusted to pH 4 with 1M dilute hydrochloric acid, extracted with ethyl acetate (100 mL. times.3), the organic phase was dried over anhydrous sodium sulfate and concentrated, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate. 10/1-6/1) to obtain the title compound (45 mg).

MS m/z(ESI)：680.1[M+H] ⁺。

¹H-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 is as follows: 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) (50mg, 87.65. mu. mol in 1, 4-dioxane (20mL) was dissolved at room temperature, subsequently, methyl 5- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) picolinate (27.67mg, 105.19. mu. mol), [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (7.16mg, 8.77. mu. mol) and potassium carbonate (12.11mg, 87.65. mu. mol) were added, the reaction was carried out 2 to 3 times by nitrogen substitution, 80 ℃ was carried out for 8 hours, the reaction solution was suction-filtered with celite, the filtrate was washed with ethyl acetate (100 mL. times.3), the resulting organic phase was dried over anhydrous sodium sulfate (20g) and then concentrated by filtration, and the resulting residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 10/1-6/1) to give the title compound (20mg) of this step.

MS m/z(ESI)：626.1[M+H] ⁺。

The second step is that: 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) (200mg, 316.61. mu. mol) was dissolved in tetrahydrofuran (5mL), and an aqueous solution (2mL) of sodium hydroxide (12.66mg, 316.61. mu. mol) was added to conduct a reaction at 25 ℃ for 4 hours. The reaction solution was adjusted to pH 4 with 1M dilute hydrochloric acid, extracted with ethyl acetate (100 mL. times.3), 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 obtain the title compound (25 mg).

MS m/z(ESI)：618.1[M+H] ⁺。

¹H-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 assay

Experimental example 1: bile acid receptor FXR coactivator binding assay

1. Test method

Invitrogen LanthaScreen was used^TMThe TR-FRET Farnesoid X Receptor activator Assay kit measures the FXR activation effect of a compound.

And (3) incubating the receptor and the compounds to be detected with different concentrations at room temperature, adding the fluorescence-labeled co-activation factor short peptide and the terbium-labeled antibody, reacting at room temperature, and detecting the FRET signal. Calculating the activation activity EC of the compound to be detected on FXR by using a four-parameter fitting equation with receptor-free proteome as a blank₅₀And the maximum activation effect value E_max：

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

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

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

relative activation effect (%) < Emax/Emax'). times.100%

Where Emax represents the maximum activation effect value of the tested compounds of the present invention and Emax' represents the maximum activation effect value of CDCA, both calculated by the formulas shown above.

2. Test results

TABLE 1 EC against FXR for the Compounds tested according to the invention₅₀

Compound numbering	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 of 4.43. mu.M relative to chenodeoxycholic acid (CDCA)₅₀The compounds tested had lower EC₅₀(0.002-0.133. mu.M), indicating that the compound of the present invention has better FXR activating activity.

TABLE 2 relative activating Effect of Compounds tested according to the invention on FXR

Compound numbering	Relative activation effect
CDCA	100％
C3	154％
C4	137％
C5	196％
C6	177％

The data in table 2 show that the maximum activation effect values of the tested compounds of the invention are higher than that of chenodeoxycholic acid (CDCA), indicating that the compounds of the invention have very good maximum activation effect on FXR.

EC of comprehensive Table 1 and Table 2₅₀Values and relative activation effect data show that the compounds of the invention have a better activating effect on FXR.

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 ensure that the plasmid highly expresses the FXR and the human BSEP luciferase reporter gene. Transfected cells were digested, resuspended, counted, and then plated in multi-well plates. mu.L of each test compound was added to a multiwell plate to give 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 to give a final concentration of 0.5%. After the test compound and the cells are incubated for 18h, a Brigh-GloTM detection reagent is added, a chemiluminescence unit value (RLU) is detected by using a multifunctional full-automatic enzyme standard instrument, and the relative signal percentage (%) of each test compound concentration is calculated by taking the signal value of a blank hole (containing no test compound) as 100%. Fitting the tested compounds EC with SigmaPlut 10 software using a four parameter model ₅₀And maximum agonistic effect Emax (percent relative signal).

2. Test results

The results of the measurement are shown in Table 3 below.

TABLE 3

Compound numbering	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％

Data in Table 3 show thatIn vitro cell assay₅₀The value is between 0.014 mu M and 0.058 mu M, and the Emax value is more than 250 percent. The compounds of the invention were shown to have good FXR activation activity in an in vitro cell assay.

Experimental example 3: liver microsome stability test

1. The experimental method comprises the following steps:

the test compound (50. mu.L) was mixed with each genus liver microsome (100. mu.L), and after preincubation at 37 ℃ for 5 minutes, NADPH (50. mu.L) was added and incubated for 0, 30, and 60 minutes at incubation concentrations of 1. mu.M, 1mM, and 0.5mg/mL for the test compound, NADPH, and liver microsome enzymes, respectively. Glacial acetonitrile (200. mu.L) was added to stop the reaction, an appropriate volume of internal standard (rivaroxaban) was added, the supernatant was vortexed, centrifuged and examined.

The detection method comprises the following steps:

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

The half-life period and the liver clearance of the drug are calculated by measuring the sample concentration of different incubation times and plotting Ln (drug residual amount%) to the incubation time to obtain a rate constant, and the metabolic stability of the drug in the liver microsome is evaluated by the half-life period and the liver clearance value of the drug.

2. The experimental results are as follows:

TABLE 4

TABLE 5

And (4) conclusion: as can be seen from the experimental data in tables 4 and 5, the compound of the present invention has the advantages of slow clearance in liver microsomes, good stability and good pharmacokinetic properties.

EXAMPLE 4 rat Pharmacokinetic (PK) study

The compounds of the invention were administered to male SD rats by Intravenous (IV) and intragastric (PO) administration, respectively, and pharmacokinetic profiles were examined. IV and PO doses were 1mg/kg and 5mg/kg respectively, IV vehicle system was 5% DMSO: 5% Solutol: 90% saline, PO administration vehicle system 0.5% MC (methyl cellulose). Blood was collected at various time points after IV and PO administration, anticoagulated with K2-EDTA, centrifuged to obtain plasma samples, stored at-80 ℃. Plasma samples were processed for precipitated protein and analyzed by LC-MS/MS.

Pharmacokinetic parameters were calculated using the WinNonlin 6.3 software using a non-compartmental model, and 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 present invention administered IV at a dose of 1mg/kg have excellent drug exposure in rats.

TABLE 7 pharmacokinetic parameters of PO dosed Compounds in rats

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

Combining tables 6 and 7, the 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_lastValue, C_maxThe value and bioavailability have better pharmacokinetic property in rats.

Experimental example 5 efficacy test of mouse fatty liver model

Test method

Male C57 mice (purchased from Witongliwa) aged 8-10 weeks were used, and the animals were randomly divided into 5 groups by body weight before molding, namely a normal group (group 1), a model group (group 2), and a C3 compound administration group (30 mg/kg). The normal group was given normal feed, the model group and the group given compound C3 were fed MCD feed (methionine choline deficient feed, Research Diets). At the same time of molding, the group of C3 compounds was administered by single gavage daily for 28 consecutive days. The normal group and the model group were given 0.5% MC (methyl cellulose). See table 8 for details.

TABLE 8 grouping and dosing Table

Detecting the index

The drug effect index is as follows: animals were dissected after the administration on day 28, liver HE was stained, and pathological scoring was performed (standard for scoring, referred to "Chinese guidelines for diagnosis and treatment of non-alcoholic fatty liver disease").

Pharmacokinetic indexes are as follows: at 0.5h, 1h, 4h after the 28 th day dosing, blood, liver and small intestine tissue samples were taken from the animals and drug concentrations were measured using LC-MS/MS.

Test results

The result of the drug effect is as follows: as can be seen in FIG. 1, the fatty liver score in the model group was significantly higher than that in the normal group, indicating that the mouse fatty liver model was successfully obtained. In addition, the fatty score of the C3 compound 30mg/kg dose group was significantly different from that of the model group.

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

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

Sample(s)	Blood plasma	Liver disease	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 data in the table above, compound C3 has a distribution in both the liver and intestine, and the exposure of the liver and intestine is higher than that of the plasma, and the exposure of the liver is higher.

Conclusion of the experiment

Compound C3 was able to inhibit MCD feed-induced liver steatosis and was significantly improved relative to the model group. Compound C3 was more highly distributed in the liver than plasma, indicating that it was effectively enriched at the target site.

Various modifications of the invention 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, including all patents, patent applications, journal articles, books, and any other publications, cited in this application is hereby incorporated by reference in its entirety.

Claims (9)

1. 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,

   

   wherein:

   x is CH or N; and is

   R is selected from hydrogen, halogen and C_1-6Alkyl and C_1-6A haloalkyl group.
2. The compound of claim 1, or a stereoisomer, tautomer, polymorph, solvate (such as hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, chemically protected form thereof, or prodrug thereof, wherein R is selected from the group consisting of hydrogen, fluoro, methyl, and trifluoromethyl.
3. A compound according to claim 1 or 2, or a stereoisomer, tautomer, polymorph, solvate (such as hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, chemically protected form or prodrug thereof, wherein said compound is selected from the group consisting of:

   
4. a pharmaceutical composition comprising at least one compound of any one of claims 1-3 or a stereoisomer, tautomer, polymorph, solvate (such as hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, chemically protected form or prodrug thereof, and one or more pharmaceutically acceptable carriers.
5. The pharmaceutical composition of claim 4, in a form selected from the group consisting of: tablets, capsules, troches, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, and syrups.
6. A kit, comprising:

   a) a first container comprising as a first therapeutic agent at least one compound of any one of claims 1-3 or a stereoisomer, tautomer, polymorph, solvate (such as hydrate), pharmaceutically acceptable salt, ester, metabolite, N-oxide, chemically protected form or prodrug thereof, or a pharmaceutical composition of claim 4 or 5 as a first pharmaceutical composition;

   b) optionally a second container comprising at least one further therapeutic agent as a second therapeutic agent, or a pharmaceutical composition comprising said further therapeutic agent as a second pharmaceutical composition; and

   c) optionally, packaging instructions.
7. Use of a compound according to any one of claims 1-3, or a stereoisomer, a tautomer, a polymorph, a solvate (such as a hydrate), a pharmaceutically acceptable salt, an ester, a metabolite, an N-oxide, a chemically protected form or a prodrug thereof, or a pharmaceutical composition according to claim 4 or 5, for the preparation of a medicament for the prophylaxis or treatment of a disease or condition mediated by farnesoid X receptors.
8. A process for the preparation of a compound of general formula (I) according to any one of claims 1 to 3, comprising the steps of:

   

   wherein:

   Hal ¹、Hal ²and Hal³Each independently of the other, identical or different halogen, for example F, Cl, Br or I, preferably Cl or Br;

   PG ¹is an amino protecting group, preferably tert-butyloxycarbonyl (Boc);

   PG ²is a carboxyl protecting group, preferably C_1-6Alkyl, more preferably methyl;

   y is a boronic acid or boronic ester group, preferably-B (OH)₂Or

   

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

   the reaction conditions for each step were as follows:

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

   and B: removal of PG from Compound IN-3¹To give compound IN-4;

   and 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 from Compound IN-6²To give a compound of general formula (I).
9. A compound of the general formula (IN-6) or a pharmaceutically acceptable salt thereof,

   

   wherein each group is as defined in claim 1, 2 or 8;

   the compound is preferably selected from:

   

   

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