EA046251B1 - FXR MODULATING CONNECTIONS (NR1H4) - Google Patents

Description

Cross reference to related applications

This patent application claims priority to U.S. Provisional Patent Application No. 62/792714, filed January 15, 2019, entitled FXR MODULATING COMPOUNDS (NR1H4), the contents of which are incorporated herein in their entirety.

List of sequences

The listing of sequences associated with this application is presented in text format in lieu of paper copy and is thus incorporated herein by reference. The name of the text file containing the sequence list is 1274_SequenceListing. The text file, created on December 17, 2018, is approximately 1 kilobyte in size and is presented electronically using EFS-Web.

Field of technology

The present invention provides compounds that bind to and act as agonists or modulators of the farnesoid X receptor (FXR), and also act as agonists or modulators of FXR. The present invention also relates to the use of said compounds for the treatment and/or prevention of diseases and/or conditions using said compounds.

State of the art

Farnesoid X receptor (FXR), also often referred to as NR1H4 (nuclear receptor subfamily 1, group H, member 4) in the context of the human receptor, is a nuclear hormone receptor. FXR has been associated with a number of biological functions. FXR is primarily expressed in the liver and throughout the gastrointestinal tract, but is also found in the kidneys, adrenal glands, and ovaries. FXR is associated with the control of intracellular gene expression and may be involved in paracrine and endocrine signaling. In the intestine and liver, FXR functions as a regulator of bile acid homeostasis and hepatic lipogenesis. FXR has also been associated with Kupffer cells and liver sinusoidal endothelial cells, where it is thought to have functions related to inflammation, fibrosis, and portal hypertension.

A number of FXR agonists are known and are being studied in connection with a number of physiological conditions, including liver diseases. FXR agonists may be useful in steatosis, lobular inflammation, liver ballooning, and fibrosis.

The action of FXR agonists can lead to different effects in different areas in the body. In the distal small intestine and systemically in organs such as the liver, activation of FXR directly induces the expression and secretion of the hormone FGF19. FGF19 modulates bile acid by down-regulating bile acid synthesis, which may be beneficial in conditions such as liver disease, for example. FXR agonists have also been associated with adverse effects such as pruritus. Such adverse effects and the extent to which they occur may depend on the site of action of the FXR agonists. For example, it has been suggested that pruritus is associated with non-intestinal FXR agonism.

There remains a need for FXR agonists with the required potency, selectivity and reduced harmful effects.

Brief description of the invention

The present invention provides compounds that bind to the NR1H4 receptor (FXR) and act as agonists or modulators of FXR. The present invention also relates to the use of said compounds for the treatment and/or prevention of diseases and/or conditions by binding said nuclear receptor to said compounds.

The present invention provides a compound of formula (I)

F(I), or a pharmaceutically acceptable salt thereof.

In some embodiments, pharmaceutical compositions are provided comprising a compound of formula (I) and a pharmaceutically acceptable excipient.

Some embodiments provide solid forms of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

The present invention also provides methods for treating patients with an FXR-mediated condition, comprising administering a compound of formula (I) to a patient in need thereof.

Description of graphic materials

In fig. 1 shows a graph of plasma FGF19 concentration over time in cynomolgus monkeys following dosing of the compound of Example 1.

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In fig. 2 shows a graph of plasma FGF19 concentration over time in cynomolgus monkeys following dosing of the compound of Comparative Example 1.

In fig. 3 shows a graph of plasma FGF19 concentration over time in cynomolgus monkeys after dosing with the compound of Comparative Example 2.

Fig. 4 is a powder x-ray diffraction pattern of Form I of the mesylate of formula (I).

Fig. 5 is a DSC curve of Form I of the mesylate of formula (I).

Fig. 6 is a TGA curve of Form I of the mesylate of formula (I).

Detailed Description of the Invention

The present invention relates to FXR agonists. The present invention also relates to compositions and methods relating to FXR agonists, as well as the use of such compounds for the treatment and/or prevention of diseases and conditions by binding FXR to these compounds. The present invention also provides compositions and methods for treating and/or preventing liver disease comprising an FXR agonist in combination with one or more additional therapeutic agents.

FXR agonists are expressed in the liver and throughout the gastrointestinal tract, where their action or inaction may play a role in one or more liver diseases such as NASH, PSC and/or liver fibrosis. However, FXR agonists are also found in other areas of the body, in which case their function may vary. FGF19 is the main target gene of FXR in ileal epithelial cells. Physiological activation of ileal FXR by bile acids results in the secretion of FGF19. In the liver, FXR agonism and FGF19 signaling have overlapping and distinct functions. For example, both pathways inhibit bile acid synthesis. FXR agonism in hepatocytes indirectly inhibits many of the same enzymes that are decreased by FGF19.

FXR agonists are suitable for the treatment and prevention of various conditions, including liver disease. Liver disease can include acute or chronic damage to the liver, such as due to infection, injury, abnormal accumulation of normal substances in the blood, or other causes. Although many FXR agonists and related analogues are known, such FXR agonists may have disadvantages, including low efficacy, metabolic problems and/or adverse events.

The present invention discloses FXR agonists and related compositions and methods. The FXR agonists described in the present invention can surprisingly maintain good therapeutic effect while minimizing side effects and metabolic problems.

In some embodiments, the FXR agonists described in the present invention may have desired cellular activity. For example, in some embodiments, high cellular activity may provide greater FXR agonism at lower doses of drug administered compared to compounds having lower cellular activity.

Some embodiments of the present invention provide FXR agonists that can exhibit high levels of FXR agonism in the gastrointestinal tract with reduced systemic FXR agonism. The FXR agonists described in the present invention can cause increased secretion of FGF19 when administered orally, while resulting in a minimal increase in FGF19 when administered intravenously. Reduced systemic FXR agonism may be advantageous, for example, by reducing and/or limiting the potential for certain adverse reactions such as pruritus, or by reducing the risks of potential drug interactions with systemic medications.

In some embodiments, the FXR agonists described herein have good target selectivity. For example, the FXR agonists described in the present invention act agonistically on FXR and do not significantly alter the activity of TGR5 and/or other nuclear hormone receptors associated with FXR. In some embodiments, the FXR agonists described herein preferentially agonize intestinal FXR over hepatic FXR.

Preferably, orally administered FXR agonists described in the present invention can cause a dose-dependent increase in plasma FGF19 levels and a decrease in serum C4 levels, indicating a decrease in bile acid synthesis.

Definitions and general parameters

The following terms and expressions as used herein generally have the meanings set forth below unless the context in which they are used indicates otherwise.

In the present invention, reference to an exemplary value or parameter includes (and describes) implementations that address the specified value or parameter as such. In some embodiments, the term approximately includes the indicated value ± 10%. In other options

- 2 046251 implementation term approximately includes the specified value ± 5%. In some other embodiments, the term approximately includes the indicated value ± 1%. Also, the term about X includes a description of X. In addition, singular forms include plural forms unless the context clearly dictates otherwise. Thus, for example, a reference to a compound includes a plurality of such compounds, and a reference to a study includes a reference to one or more studies and their equivalents known to those skilled in the art.

The invention illustratively described in the present invention can be suitably practiced in the absence of any element or elements, limitation or limitations not specifically disclosed in the present invention. Thus, for example, the terms containing, including, etc. should be read broadly and without limitation. Moreover, the terms and expressions used in the present invention are used as terms of description and not limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the features presented and described, or portions thereof, and implies that various modifications are possible within the scope of scope of the claimed invention.

In some embodiments, the compounds of the present invention may be in the form of a prodrug. The term prodrug is defined in the pharmaceutical field as a biologically inactive derivative of a drug that, when administered to the human body, is converted into the biologically active parent drug by some chemical or enzymatic pathway. Examples of prodrugs include esterified carboxylic acids.

In the human liver, UDP-glucuronosyltransferases act on certain compounds containing amino-, carbamyl, thio-(sulfhydryl) or hydroxyl groups to conjugate uridine diphosphate-a-O-glucuronic acid through glycosidic bonds or to esterify compounds with carboxyl or hydroxyl groups during phase II metabolism. The compounds of the present invention may be glucuronidated, i.e. conjugated with glucuronic acid to produce glucuronides, in particular (B-O)-glucuronides.

One of the stages of bile formation is the conjugation of individual bile acids with an amino acid, in particular glycine or taurine. The compounds of the present invention may be conjugated to glycine or taurine at a substitutable position.

The compounds of the present invention may be in the form of a pharmaceutically acceptable salt. The term pharmaceutically acceptable salts refers to salts derived from pharmaceutically acceptable non-toxic bases or acids, including inorganic bases or acids and organic bases or acids. The compounds of the present invention may be in the form of a pharmaceutically acceptable salt. The term pharmaceutically acceptable salts refers to salts derived from pharmaceutically acceptable non-toxic bases or acids, including inorganic bases or acids and organic bases or acids. If the compounds of the present invention contain one or more acidic or basic groups, the present invention also includes their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically acceptable salts. Thus, the compounds of the present invention which contain acidic groups can be presented with said groups and can be used in accordance with the present invention, for example, as alkali metal salts, alkaline earth metal salts or ammonium salts. More specific examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts, or salts with ammonia or organic amines, such as, for example, ethylamine, ethanolamine, triethanolamine, amino acids, or other bases known to those skilled in the art. Compounds of the present invention containing one or more basic groups, i.e. groups that can be protonated may be present and may be used in accordance with the present invention in the form of their salts formed by addition of inorganic or organic acids. Examples of suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acids, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid , propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid and others acids known to those skilled in the art.

If the compounds of the present invention simultaneously contain acidic and basic groups in the molecule, the present invention, in addition to the salt forms mentioned, also includes internal salts or betaines (zwitterions). The corresponding salts can be prepared by conventional methods known to one skilled in the art, such as, for example, by contacting the corresponding compounds with an organic or inorganic acid, or

- 3 046251 base in a solvent or dispersant, or by anion exchange or cation exchange with other salts.

In some embodiments, a pharmaceutically acceptable salt of a compound of formula (I) includes a zwitterion. For example, a compound of formula (I) can form a zwitterion as follows:

F F

The present invention also includes all salts of the compounds of the present invention which, due to their low physiological compatibility, are not directly suitable for use in pharmaceutical preparations, but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts. Acids and bases suitable for reacting with the parent compound to form pharmaceutically acceptable salts (acid addition salts or base addition salts, respectively) are known to those skilled in the art. Likewise, methods for preparing pharmaceutically acceptable salts from the parent compound (as disclosed) are known to those skilled in the art and are described, for example, in Berge, et al. Journal of Pharmaceutical Science, Jan. 1977, vol. 66, No. 1, and other sources.

In addition, the compounds described in the present invention may be subject to tautomerism. When tautomerism, for example keto-enol tautomerism, of compounds or prodrugs thereof may occur, individual forms, such as, for example, keto and enol forms, as well as mixtures thereof in any ratio, are included within the scope of the present invention. The same applies to stereoisomers, such as, for example, enantiomers, cis/trans isomers, diastereomers, conformational isomers and the like.

The term protecting group refers to a moiety of a compound that masks or alters the properties of a functional group or the properties of the compound as a whole. Chemical protecting groups and deprotection methods are well known in the art. See, for example, Protective Groups in Organic Chemistry, Theodora W. Greene, John Wiley & Sons, Inc., New York, 1991. Protecting groups are commonly used to mask the reactivity of certain functional groups to promote the efficiency of desired chemical reactions, e.g. or breaking chemical bonds in an orderly and planned manner. The term deprotection refers to the removal of a protecting group.

A leaving group includes a molecular fragment that can be removed with a pair of electrons from a covalent bond with the reacting carbon atom during a chemical reaction.

One skilled in the art will recognize that if a list of alternative substituents includes members that, due to valence requirements or other reasons, cannot be used to replace a particular group, the list should be read to the best of one's knowledge, including only those members of the list that are suitable for replacement. specific group.

In addition, the compounds of the present invention may be presented in the form of solvates, such as solvates containing water as the solvate, or pharmaceutically acceptable solvates, such as alcohols, in particular ethanol. The solvate is formed by the interaction of a solvent and a compound.

Some embodiments provide optical isomers, racemates, or other mixtures of isomers of the compounds described herein, or a pharmaceutically acceptable salt or mixture thereof. If necessary, the isomers can be separated using methods known in the art, for example, by liquid chromatography. In these situations, a single enantiomer or diastereomer, i.e. the optically active form can be obtained by asymmetric synthesis or by resolution. The separation can be carried out, for example, by conventional methods such as crystallization in the presence of a separating agent or chromatography, for example using a chiral high performance liquid chromatography (HPLC) column.

A stereoisomer refers to compounds consisting of identical atoms linked by identical bonds, but having different three-dimensional structures that are not interchangeable. The present invention provides various stereoisomers and mixtures thereof and includes enantiomers, which refer to two stereoisomers whose molecules are incompatible mirror images of each other. Diastereomers are stereoisomers that contain at least two asymmetric atoms but are not mirror images of each other.

The compounds described herein and their pharmaceutically acceptable salts may, in some embodiments, contain a center of asymmetricity and, therefore, may have enantiomeric, diastereomeric, and other stereoisomeric forms that can be defined in terms of

- 4 046251 nah absolute stereochemistry, as (R)- or (S)- or as (D)- or (L)- for amino acids. Some embodiments of the invention include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)- or (D)- and (C)-isomers can be prepared using chiral synthons or chiral reagents or can be separated using conventional methods, such as chromatography and fractional crystallization. Traditional methods for preparing/isolating individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or racemate salt or derivative) using, for example, chiral high performance liquid chromatography (HPLC). When the compounds described in the present invention contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise indicated, the compounds are intended to include both E and Z geometric isomers.

Compositions provided by the present invention that contain a compound described herein, or pharmaceutically acceptable salts, an isomer, or a mixture thereof, may include racemic mixtures or mixtures containing an enantiomeric excess of one enantiomer, or individual diastereomers, or diastereomeric mixtures. All such isomeric forms of the specified compounds are expressly included herein in the same manner as if each of the isomeric forms were specifically and separately listed.

Any form or structure provided in the present invention is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds. Isotopically labeled compounds have the structures depicted by the formulas given in the present invention, except that one or more atoms are replaced by an atom having the selected atomic mass or mass number. Examples of isotopes that may be included in the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as, but not limited to, ^2H (deuterium, D), ^3H (tritium), C ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ F, ³¹ P, ³² P, ³⁵ S, ³⁶ Cl and ¹²⁵ I. Various isotopically labeled compounds according to the present invention, for example, compounds in which radioactive isotopes are included, such as ³ H, ¹³ C and ¹⁴ C. Such isotopically labeled compounds may be suitable for use in metabolic studies, reaction kinetics studies, detection and imaging modalities such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including studies distribution of a drug or substrate into tissue, or for radioactive treatment of patients. The isotopically labeled compounds of the present invention and their prodrugs can generally be prepared by following the procedures described in the Schemes or in the Preparation Examples and Methods described below by replacing the non-isotopically labeled reagent with a readily available isotopically labeled reagent.

The present invention also includes deuterated analogues of the compounds described in the present invention, in which from 1 to n hydrogen atoms attached to the carbon atom are replaced by deuterium atoms, where n represents the number of hydrogen atoms in the molecule. Such compounds may exhibit increased resistance to metabolism and therefore may be suitable for increasing the half-life of any compound of formula I when administered to a mammal, such as a human. See, for example, Foster, Deuterium Isotope Effects in Studies of Drug Metabolism, Trends Pharmacol. Sci. 5(12):524-527 (1984). Such compounds are synthesized using methods well known in the art, for example, by using starting materials in which one or more hydrogen atoms are replaced by deuterium.

The deuterium-labeled or deuterium-substituted therapeutic compounds of the present invention may have beneficial DMPK (drug metabolism and pharmacokinetics) distribution, metabolism and excretion (ADME) properties. Substitution with heavier isotopes such as deuterium may provide certain therapeutic benefits due to greater metabolic stability, such as increased in vivo half-life, reduced dosage requirements, and/or improved therapeutic index. The ¹⁸ F labeled compound may be suitable for use in PET or SPECT studies.

The concentration of this heavier isotope, in particular deuterium, can be determined using the isotope enrichment factor. In the compounds of the present invention, any atom not specifically designated as a particular isotope may be any stable isotope of said atom. Unless otherwise indicated, when a position is specifically designated as H or hydrogen, the position is meant to contain hydrogen in its natural isotopic composition. Accordingly, in the compounds of the present invention, any atom specifically designated as deuterium (D) is deuterium.

The present invention further provides pharmaceutical compositions containing a compound of the present invention, or a prodrug thereof, or a pharmaceutically acceptable salt or solvate thereof, as an active ingredient, together with a pharmaceutically acceptable carrier.

Pharmaceutical composition means one or more active ingredients and one or more inert ingredients that constitute the carrier, as well as any product that directly or

- 5 046251 is formed indirectly as a result of the combination, complexation or aggregation of any two or more ingredients, or as a result of the dissociation of one or more ingredients, or as a result of other types of reactions or interactions of one or more ingredients. Accordingly, the pharmaceutical compositions of the present invention may include any composition prepared by mixing at least one compound of the present invention and a pharmaceutically acceptable carrier.

In the present invention, the term pharmaceutically acceptable carrier includes excipients or agents such as solvents, diluents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents and the like, which are not harmful to the described compound or its applications. The use of such carriers and agents for the preparation of compositions of pharmaceutically active substances is well known in the art (see, for example, Remington's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, PA 17th ed. (1985) and Modern Pharmaceutics, Marcel Dekker, Inc., 3rd ed. (ed. G.S. Banker & S. Rhodes)).

The terms therapeutically effective amount and effective amount are used interchangeably and refer to an amount of a compound that is sufficient to provide a treatment, as defined below, when administered to a patient (eg, a human) in need of such treatment in one or more doses. The therapeutically effective amount may vary depending on the patient, the disease being treated, the body weight and/or age of the patient, the severity of the disease, or the route of administration, and will be determined by the qualified prescriber or caregiver.

The term treatment means the administration of a compound or pharmaceutically acceptable salt of formula (I) for the purpose of: (i) delaying the onset of a disease, that is, preventing the development or delaying clinical symptoms of the disease; (ii) slowing down the disease, that is, stopping the development of clinical symptoms; and/or (iii) alleviation of the disease, that is, causing regression of clinical symptoms or their severity.

List of abbreviations and acronyms

Abbreviation Meaning (±)-BINAP (±)-2,2'-Bis-(diphenylphosphino)-1,1'-binaphthalene 2-Me-THF 2-methyltetrahydrofuran ACN or MeCN Acetonitrile drive Water VP Benzyl VOS or Vos otreot-Butyloxycarbonyl BSA Bovine serum albumin

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USSR Balanced salt solution Comput. Calculated DAST (Diethylamino)-sulfur trifluoride DXM Dichloromethane DIBAL-H Diisobutylaluminum hydride DMF A'.L'-dimethylformamide DMSO Dimethyl sulfoxide EA Ethyl acetate EDTA Ethylenediaminetetraacetic acid IER Electrospray ionization Et Ethyl Et ₂ O Diethyl ether EtOAc Ethyl acetate FBS Fetal bovine serum h Watch) HATU 1 -[Bis(dimethylamino)methylene]-\H-1,2,3triazolo[4,5-/>]pyridinium-3-oxide hexafluorophosphate HPLC High performance liquid chromatography ANN Isopropyl alcohol iptg Isopropyl-3-O-1-thiogalactopyranoside LCMS or LC/MS Liquid chromatography/mass spectrometry Me Methyl

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Ministry of Railways Minimum nutrient medium MeOH Methanol MSK Methanesulfonic acid min Minute(s) MS Mass spectrometry m/z Mass to charge ratio NADP Dihydronicotinamide adenine dinucleotide phosphate NML N-methylpyrrolidone NMR Nuclear magnetic resonance spectroscopy n-BuLi //-Butyllithium PE Petroleum ether rpm Revolutions per minute CT Room temperature sat. Saturated TBAF Tetrabutylammonium fluoride TBDMS from/?else-butyldimethylsilyl TBS from/?else-butyldimethylsilyl DARK 2,2,6,6-Tetramethylpiperidine-1-oxyl TFUK Trifluoroacetic acid THF Tetrahydrofuran TMS Trimethylsilyl UHPLC Ultra-high performance liquid chromatography

As used herein, the term FXR agonist refers to any agent capable of binding and activating the farnesoid X receptor (FXR), which may be referred to as the bile acid receptor (BAR) or the NR1H4 receptor (nuclear receptor subfamily 1, group H, member 4). An FXR agonist can act as an agonist or partial agonist for FXR. The agent may be a chemical compound or a biological molecule (eg, a protein or antibody). FXR agonist activity can be measured in several different ways, for example, in an in vitro assay using a cell-free fluorescence resonance energy transfer (FRET) assay as described in Pellicciari, et al. Journal of Medicinal Chemistry, 2002, vol. 15, No. 45:3569-72.

As used herein, an ASK1 inhibitor can be any agent that is capable of inactivating apoptosis signal-regulating kinase 1 (ASK1) protein. The agent may be a chemical compound or a biological molecule (eg, a protein or antibody). ASK1 protein activity can be measured in several different ways. For example, the activity of the ASK1 protein can be determined based on the ability of the ASK1 protein to phosphorylate a substrate protein. Methods for identifying an ASK1 inhibitor are known (see, for example, US Patents 2007/0276050 and 2011/0009410, both of which are incorporated herein by reference in their entirety). Exemplary ASK1 substrate proteins include MAPKK3, MAPKK4, MAPKK6, MAPKK7, or fragments thereof. ASK1 protein activity can also be measured by the level of phosphorylation of the ASK1 protein, for example, the phosphorylation level of the threonine residue in the ASK1 protein corresponding to threonine 838 (T838) of the full-length human ASK1 protein or threonine 845 (T845) of the full-length mouse ASK1 protein. For example, if the ASK1 protein contains the full-length human ASK1 protein sequence, an ASK1 inhibitor can attenuate T838 phosphorylation on the full-length human ASK1 protein sequence. A site-specific antibody to human ASK1 T838 or mouse ASK1 T845 can be used to determine the level of phosphorylation.

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In the present invention, the term ACC inhibitor refers to any agent that is capable of binding and inhibiting acetyl-CoA carboxylase (ACC). ACC inhibitors can act as inhibitors or partial inhibitors of ACC. The agent may be a chemical compound or a biological molecule (eg, a protein or antibody). ACC inhibitor activity can be measured using methods known in the art, such as those described and cited in US Patent No. 8,969,557 and/or US Patent Application Publication No. 20160108061.

In the present invention, a thyroid hormone receptor beta agonist or a THR beta agonist refers to any agent that is capable of binding and activating a thyroid hormone receptor beta, which may be referred to as the NR1A2 receptor (nuclear receptor subfamily 1, group A, member 2). A THR β agonist can act as a THR β agonist or partial agonist. The agent may be a chemical compound or a biological molecule (eg, a protein or antibody). THRβ agonist activity can be measured by known methods.

Connections

The present invention provides a compound of the following formula (I):

F(I) or a pharmaceutically acceptable salt thereof.

In some embodiments, the pharmaceutically acceptable salt is a mesylate salt. For example, a compound of the following formula is proposed:

F

Solid forms

In some embodiments, the present invention provides solid forms of a compound of formula (I) or a pharmaceutically acceptable salt thereof. Solid forms, such as crystalline forms, may provide the advantages of bioavailability and stability, and are suitable for use as an active ingredient in a pharmaceutical composition.

Differences in the crystal structure of a pharmaceutical drug substance or active ingredient may affect dissolution rate (which may affect bioavailability, etc.), processability (e.g., ease of handling, ability to consistently prepare doses of known strength), and stability (e.g., thermal stability, shelf life, etc.) of the pharmaceutical drug or active ingredient. Such variations may affect the preparation or composition of pharmaceutical compositions in various dosage or delivery forms, such as solutions or solid oral dosage form, including tablets and capsules. Compared to other forms, such as non-crystalline or amorphous forms, crystalline forms can provide desired or suitable hygroscopicity, particle size control, dissolution rate, solubility, purity, physical and chemical stability, processability, yield and/or process control.

Some embodiments provide a stable solid form of the compound of formula (I). For example, the mesylate salt of formula (I) can be obtained as a stable crystalline form that is not converted into other polymorphic forms and/or that is formed as the same polymorphic form under different production conditions.

Thus, crystalline forms of a compound of formula (I) may provide advantages such as improvements in: the method of preparation of the compound, the stability or shelf life of the dosage form of the compound, the stability or shelf life of the drug substance of the compound, and/or the bioavailability and/or stability of the compound as an active agent.

In some embodiments, the solid form is Form I of the mesylate of formula (I).

Form I of the mesylate of formula (I) can be characterized by X-ray powder diffraction as a solid form exhibiting a substantially X-ray powder diffraction (XRD) pattern as shown in FIG. 4. Form I of the mesylate of formula (I) may exhibit a differential scanning calorimetry (DSC) thermogram substantially as shown in FIG. 5. Form I of the mesylate of formula (I) may exhibit a thermogravimetric analysis (TGA) thermogram substantially as shown in FIG. 6.

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According to some embodiments of Form I mesylate of formula (I), at least one, at least two, or all of the following statements (a)-(c) apply: (a) Form I mesylate of formula (I) has essentially the following diffraction pattern PRD, as shown in Fig. 4; (b) Form I of the mesylate of formula (I) has essentially the same DSC thermogram as shown in FIG. 5; (c) Form I of the mesylate of formula (I) has essentially the same TGA thermogram as shown in FIG. 6.

In some embodiments, Form I mesylate of formula (I) has at least one, at least two, or at least three of the following properties:

(a) The XRD pattern is essentially the same as shown in FIG. 4, (b) the DSC thermogram is essentially the same as shown in FIG. 5(c) The TGA thermogram is essentially the same as shown in FIG. 6.

In some embodiments, Form I of the mesylate of formula (I) has a PXRD pattern exhibiting at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or at least nine of the highest intensity reflections 20, substantially like the XRD pattern shown in FIG. 4.

In some embodiments, Form I of the mesylate of formula (I) has a PXRD pattern containing 2θ reflections (±0.2° 2θ) at 9.6, 19.3 and 22.6°. In some embodiments, Form I of the mesylate of formula (I) has a PXRD pattern containing 2θ (± 0.2° 2θ) reflections at 9.6, 19.3 and 22.6° and one, two, or three of the 2θ (± 0.2° 2θ) at 3.2, 6.4 and 12.8°. In some embodiments, Form I of the mesylate of formula (I) has a PXRD pattern containing 2θ (± 0.2° 2θ) reflections at 9.6, 19.3 and 22.6° and one, two, or three of the 2θ (± 0.2° 2θ) at 22.1, 25.8 and 29.1°. In some embodiments, Form I of the mesylate of formula (I) has a PXRD pattern containing 2θ reflections (±0.2° 2θ) at 9.6, 19.3, 22.6, 3.2, 6.4, 12.8 , 22.1, 25.8 and 29.1°.

In some embodiments, Form I of the mesylate of formula (I) has a differential scanning calorimetry thermogram containing an endothermic peak with an initial temperature of approximately 221°C.

Pharmaceutical compositions and methods of administration

The present invention further provides pharmaceutical compositions containing at least one compound of the present invention, or a prodrug thereof, or a pharmaceutically acceptable salt or solvate thereof as an active ingredient together with a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention may further contain one or more other compounds as active ingredients, such as a prodrug compound or other nuclear receptor modulators.

The compositions are suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular and intravenous), ocular (ophthalmic), pulmonary (nasal or oral inhalation) or nasal administration, although the most appropriate route in any particular case will depend on the nature and severity of the conditions. to be treated, and the nature of the active ingredient. They may be conveniently presented in unit dosage form and prepared by any of the methods well known in the pharmaceutical field.

In practical use, the compounds of the present invention can be combined as an active ingredient in a uniform mixture with a pharmaceutical carrier in accordance with standard methods for preparing pharmaceuticals. The carrier may take different forms depending on the form of preparation desired for administration, for example, oral or parenteral (including intravenous). When preparing compositions for oral dosage form, any conventional pharmaceutical vehicles may be used, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc., in the case of liquid oral preparations such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants and the like, in the case of solid oral preparations such as, for example, powders, hard and soft capsules and tablets, with solid oral preparations being preferred over liquid preparations.

Because of their ease of administration, tablets and capsules are the most advantageous unit dosage form for oral administration, wherein solid pharmaceutical carriers are used. If desired, tablets can be coated using standard aqueous or non-aqueous methods. Such compositions and preparations must contain at least 0.1 percent active compound. Of course, the percentage of active compound in these compositions may vary and may conveniently range from about 2 to about 60% by weight of the dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage is obtained. Assets

- 10 046251 These compounds can also be administered intranasally, for example in the form of liquid drops or spray.

Tablets, pills, capsules, etc. may also contain a binder such as gum tragacanth, gum acacia, corn starch or gelatin; excipients such as dicalcium phosphate; leavening agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetener such as sucrose, lactose or saccharin. If the unit dosage form is a capsule, in addition to the above types, it may contain a liquid carrier such as a fatty oil.

Various other materials may be present as coatings or to modify the physical form of the unit dosage form. For example, tablets can be coated with shellac, sugar, or both. The syrup or elixir, in addition to the active ingredient, may contain sucrose as a sweetener, methyl and propyl parabens as preservatives, coloring and a flavoring agent such as cherry or orange flavoring.

In some embodiments, the compounds of the present invention may also be used as salts with various countercations to provide an orally available formulation.

The compounds of the present invention can also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerin, liquid polyethylene glycols and their mixtures in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

Pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions for immediate use. In all cases, the form must be sterile and must be fluid enough to be easily syringed. It must be stable under the conditions of production and storage and must be protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyhydric alcohol (eg, glycerin, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof and vegetable oils.

Any suitable route of administration may be used to provide a mammal, particularly a human, with an effective dose of a compound of the present invention. For example, oral, rectal, topical, parenteral, ophthalmic, pulmonary, nasal, and the like may be used. ways. Dosage forms include tablets, lozenges, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like. In some embodiments, the compounds of the present invention are administered orally.

Sets

The present invention also provides kits that include a compound of the present invention or a pharmaceutically acceptable salt, tautomer, stereoisomer, mixture of stereoisomers, prodrug or deuterated analogue thereof, and suitable packaging. In one embodiment, the kit further includes instructions for use. In one aspect, the kit includes a compound of the present invention or a pharmaceutically acceptable salt, tautomer, stereoisomer, mixture of stereoisomers, prodrug or deuterated analogue thereof, and labeling and/or instructions for use of the compounds for the treatment of indications, including diseases or conditions described in the present invention .

The present invention provides articles that contain a compound of the present invention, or a pharmaceutically acceptable salt, tautomer, stereoisomer, mixture of stereoisomers, prodrug or deuterated analogue thereof in a suitable container. The container may be a vial, jar, ampoule, prefilled syringe, or IV bag.

Methods of treatment and application

Treatment is an approach to obtain beneficial or desired results, including clinical results. The beneficial or desired clinical results may include one or more of the following: a) inhibiting a disease or condition (eg, reducing one or more symptoms resulting from a disease or condition and/or reducing the progression of a disease or condition); b) slowing or stopping the development of one or more clinical symptoms associated with a disease or condition (eg, stabilizing the disease or condition, preventing or delaying the worsening or progression of the disease or condition, and/or preventing or delaying the spread (eg, metastasis) of the disease or condition) ; and/or c) relief of the condition, i.e. causing regression of clinical symptoms (for example, improving a disease, providing partial or complete remission of a disease or condition, enhancing the effect of another drug, delaying disease progression, improving quality of life and/or prolonging life).

Prophylaxis or prevention refers to any treatment of a disease or condition that results in the clinical symptoms of the disease or condition not developing. In neko

- 11 046251 In some embodiments, the compounds can be administered to a subject (including a human) who is at risk or has a family history of the disease or condition.

Subject refers to an animal, such as a mammal (including a human), that has been or will be the subject of treatment, observation, or experiment. The methods described in the present invention may be suitable for use in human therapy and/or veterinary applications. In some embodiments, the subject is a mammal. In one embodiment, the subject is a human.

The term therapeutically effective amount or effective amount of a compound described in the present invention, or a pharmaceutically acceptable salt, tautomer, stereoisomer, mixture of stereoisomers, prodrug or deuterated analogue, means an amount sufficient to provide treatment when administered to a subject to provide a therapeutic effect, such as improvement of symptoms or slowing the progression of the disease. For example, a therapeutically effective amount may be an amount sufficient to alleviate a symptom of a disease or condition responsive to FXR agonism. The therapeutically effective amount may vary depending on the subject and the disease or condition being treated, the body weight and age of the subject, the severity of the disease or condition, and the route of administration, which can be readily determined by one skilled in the art.

The present invention also relates to the use of the compounds described in the present invention for the treatment and/or prevention of diseases and/or conditions by binding to said nuclear receptor by said compounds. Furthermore, the present invention relates to the use of said compounds for the production of a medicament for the treatment and/or prevention of diseases and/or conditions by binding said nuclear receptor to said compounds.

In some embodiments, the present invention relates to the use of compounds of formula (I) in the preparation of a medicament for the prevention and/or treatment of chronic intrahepatic or certain forms of extrahepatic cholestatic conditions, liver fibrosis, acute intrahepatic cholestatic conditions, obstructive or chronic inflammatory disorders that occur due to abnormal bile composition, gastrointestinal conditions with reduced absorption of dietary fat and dietary fat-soluble vitamins, inflammatory bowel diseases, disorders of lipid and lipoprotein metabolism, type II diabetes and clinical complications of type I and II diabetes, conditions and diseases that arise as a result of chronic fatty and fibrotic degeneration of organs due to the forced accumulation of lipids and, in particular, triglycerides and subsequent activation of profibrotic pathways, obesity and metabolic syndrome (combined conditions of dyslipidemia, diabetes and abnormally high body mass index), acute myocardial infarction, acute stroke, thrombosis that occurs as an end point of chronic obstructive atherosclerosis, persistent infections with intracellular bacteria or parasitic protozoa, non-malignant hyperproliferative disorders, malignant hyperproliferative disorders, particularly colon adenocarcinoma and hepatocellular carcinoma, hepatic steatosis and related syndromes, liver failure or dysfunction liver as a result of chronic liver disease or surgical resection of the liver, hepatitis B infection, hepatitis C infection and/or cholestatic and fibrotic effects that are associated with alcohol-induced cirrhosis or with viral forms of hepatitis.

The drugs of the present invention can be prepared by conventional methods, including combining a compound of the present invention with a pharmaceutically acceptable carrier.

FXR can modulate both the synthesis of bile acids in the liver and their recycling in the intestine (by regulating bile acid binding proteins). FXR may be involved in the regulation of many different physiological processes that are relevant to the etiology and treatment of various diseases, such as cholesterol gallstones, metabolic disorders such as type II diabetes, dyslipidemia or obesity, chronic inflammatory diseases such as inflammatory bowel diseases, or chronic intrahepatic forms of cholestasis. FXR regulates a complex pattern of response genes in the liver and gastrointestinal tract. Gene products influence various physiological processes. For example, FXR represses the induction of Cyp7A1 by up-regulating the mRNA encoding SHP, another nuclear receptor that is dominantly repressive of LRH-1. Because FXR binds primary bile acids, the end products of this pathway, this can be considered an example of feedback inhibition at the level of gene expression.

FXR ligands induce bile flow and change the composition of bile acids towards a more hydrophilic composition. With the development of the first synthetic FXR ligand GW4064 as a pharmacologically active compound and a semisynthetic artificial bile acid ligand 6-alpha-ethyl-CDCA, it has become possible to analyze the effects of FXR superstimulation with strong

- 12 046251 agonists. Both ligands have been shown to induce bile flow in animals with ligated bile ducts. Additionally, in addition to choleretic effects, hepatoprotective effects may also be observed. These hepatoprotective effects included antifibrotic effects resulting from the repression of tissue inhibitors of matrix metalloproteinases TIMP-1 and 2, the induction of matrix metalloproteinase 2, which degrades collagen deposits, in hepatic stellate cells and the subsequent reduction in levels of alpha-collagen mRNA and transforming growth factor beta (TGF) mRNA. -beta), both of which are profibrotic factors.

In addition, anticholestatic activity has been demonstrated in animal models with ligated bile ducts, as well as in animal models of estrogen-induced cholestasis. Genetically, studies demonstrate that in hereditary forms of cholestasis (progressive familial intrahepatic cholestasis = PFIC, types I-IV), any nuclear localization of FXR is reduced due to a mutation in the FIC1 gene (in PFIC type I, also called Byler's disease) (F. Chen et al. , Gastroenterology 2004, 126, 756; L. Mol. Genet. 2004, 13, 2451) or the levels of the target gene FXR, encoding the phospholipid export pump MDR-3, are reduced (in PFIC type III). There is increasing evidence that FXR binding compounds may demonstrate significant clinical value in a treatment program for chronic cholestatic conditions such as primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC).

FXR agonists can be used to prevent the formation of cholesterol gallstones or to prevent the recurrence of gallstones after surgical removal or shock wave lithotripsy. For example, using the synthetic FXR pharmacological compound GW4064, it can be demonstrated that activation of FXR leads to an improvement in cholesterol saturation index (CSI) and directly to the cessation of gallstone formation in gallstone-susceptible C57L mice, whereas drug treatment does not. has an effect on the formation of gallstones in mice with FXR turned off. Thus, in one embodiment of the present invention, a compound according to formula (I) and pharmaceutical compositions containing said compound are used for the prevention and/or treatment of obstructive or chronic inflammatory disorders that arise due to abnormal bile composition, such as cholelithiasis, also known as cholesterol gallstones.

FXR agonists may be useful in protecting the intestine from neoplastic transformation and from the development of polyps and their progression to adenocarcinoma in the intestine. The absence of FXR leads to a strong increase in the formation of hepatocellular carcinoma (HCC), the most important form of liver cancer. While functional FXR prevents the formation of colon adenocarcinoma and hepatocellular carcinoma, activation of FXR induces liver regeneration after hepatectomy.

The combined hepatoprotective, antitumor, and liver regenerative effects associated with FXR activation can be therapeutically exploited by using FXR agonists to treat severe liver diseases. In one embodiment, the compounds of the present invention and pharmaceutical compositions containing these compounds are used to treat liver diseases such as HCC, stimulate liver regrowth and reduce side effects associated with major liver resection, liver cirrhosis, regardless of etiology, and preventing or treating liver ischemia during liver transplantation or major liver surgery.

In addition, FXR may be a key regulator of serum triglycerides. Activation of FXR by synthetic agonists can result in significant reductions in serum triglyceride levels, primarily as a reduction in VLDL, but also in a reduction in serum total cholesterol. The reduction in serum triglyceride levels is not an isolated effect. Treatment of db/db or ob/ob mice with the synthetic FXR agonist GW4064 results in significant and combined reductions in serum levels of triglycerides, total cholesterol, free fatty acids, and ketone bodies such as 3-OH butyrate. Moreover, activation of FXR is associated with the intracellular insulin signaling pathway in hepatocytes, which leads to decreased glucose production through hepatic gluconeogenesis but is accompanied by an increase in hepatic glycogen. Treatment with FXR has a positive effect on insulin sensitivity as well as glucose tolerance. Weight loss effects were also recently observed in mice overfed with a high lipid diet. This mass loss effect may result from FXR-mediated induction of FGF-19, a fibroblast growth factor known to lead to mass loss and an athletic phenotype. The effects of an FXR agonist on weight loss have been demonstrated.

Accordingly, FXR agonists can be used in a variety of therapeutic ways: FXR binding compounds are believed to be good candidates for the treatment of type II diabetes due to their insulin sensitizing, glycogenogenic and lipid-lowering effects.

- 13 046251

In one embodiment, the compounds of the present invention and pharmaceutical compositions containing these compounds are used for the prevention and/or treatment of type II diabetes, which can be overcome by FXR-mediated up-regulation of systemic insulin sensitivity and intracellular insulin signaling in liver, increased peripheral uptake and metabolization of glucose, increased accumulation of glycogen in the liver, decreased supply of glucose to the serum as a result of gluconeogenesis in the liver.

In another embodiment, the compounds and pharmaceutical compositions are used for the prevention and/or treatment of chronic intrahepatic conditions such as PBC, PSC, progressive familial cholestasis (PFIC), alcohol-induced cirrhosis and related cholestasis, and certain forms of extrahepatic cholestasis or fibrosis. liver.

The present invention also relates to a compound of formula (I) or a pharmaceutical composition containing the same compound for the prevention and/or treatment of gastrointestinal conditions with reduced absorption of dietary fat and dietary fat-soluble vitamins, which can be overcome by increasing the levels of bile acids and phospholipids in the intestines.

In another embodiment, said compound or pharmaceutical composition is used to prevent and/or treat a disease selected from the group consisting of disorders of lipid and lipoprotein metabolism, such as hypercholesterolemia, hypertriglyceridemia and atherosclerosis, as a clinically manifested condition that can be improved by beneficial the effects of FXR in lowering plasma total cholesterol, lowering serum triglyceride levels, increasing hepatic conversion of cholesterol to bile acids, and increasing the clearance and metabolic conversion of VLDL and other lipoproteins in the liver.

In yet another embodiment, the compound or pharmaceutical composition is used for the prevention and/or treatment of diseases in which the combined effects of lipid lowering, anticholestatic and antifibrotic effects of FXR-targeting drugs can be used to treat hepatic steatosis and related syndromes, such as non-alcoholic steatohepatitis (NASH), or to treat the cholestatic and fibrotic effects that are associated with alcohol-induced cirrhosis or viral forms of hepatitis.

In combination with lipid-lowering effects, loss of functional FXR has been shown to result in increased atherosclerosis in ApoE knockout mice. Accordingly, FXR agonists may have clinical value as antiatherosclerotic and cardioprotective drugs. Down-regulation of endothelin-1 in vascular smooth muscle cells may also contribute to these beneficial therapeutic effects.

The present invention also relates to a compound according to formula (I) or a pharmaceutical composition containing the same compound for the prophylactic and post-traumatic treatment of a cardiovascular disorder such as acute myocardial infarction, acute stroke or thrombosis occurring as the end stage of chronic obstructive atherosclerosis.

In addition to controlling the formation of intestinal and colon polyps, FXR appears to be expressed in breast cancer tissues and cell lines, but not in healthy breast tissue, and appears to interact with the estrogen receptor in ER-positive breast cancer cells. Thus, FXR may be a potential target for the treatment of proliferative diseases, especially metastatic cancers that express a small molecule-sensitive form of FXR.

In another embodiment, the compounds and formalceutical compositions are used for the prevention and/or treatment of malignant hyperproliferative disorders, such as various forms of cancer, in particular certain forms of breast, liver or colon cancer, in which intervention through the FXR ligand has a beneficial effect.

FXR may be involved in the control of antibacterial defense in the intestine. FXR agonists may have beneficial effects in the treatment of inflammatory bowel disease (IBD). For example, in forms of IBD that involve the upper (ileal) intestine (eg, Crohn's disease of the ileum), FXR agonists may provide beneficial effects through FXR-mediated control of bacterial growth. In IBD, the intestinal immune system somehow fails to desensitize the adaptive immune response. Bacterial overgrowth can cause a chronic inflammatory response. Therefore, inhibition of bacterial growth through FXR-mediated mechanisms may be a key mechanism for preventing acute inflammatory conditions.

Thus, the present invention also relates to a compound according to formula (I) or a pharmaceutical composition containing the same compound for the prevention and/or treatment of a disease associated with inflammatory bowel disease such as Crohn's disease or ulcerative colitis. It is believed that FXR-mediated restoration of intestinal barrier function and a decrease in non-commensal bacterial load help reduce the impact of bacterial antigens on

- 14 046251 the intestinal immune system and can therefore reduce inflammatory responses.

The present invention also provides a compound or pharmaceutical composition for the prevention and/or treatment of obesity and related disorders such as metabolic syndrome (combined conditions of dyslipidemia, diabetes and abnormally high body mass index), which can be overcome by FXR-mediated reduction in levels serum triglycerides, blood glucose, increased insulin sensitivity, and FXR-mediated weight loss.

In another embodiment, the compounds or pharmaceutical compositions of the present invention are suitable for use in the prevention and/or treatment of clinical complications of type I and type II diabetes. Examples of such complications include diabetic nephropathy, diabetic retinopathy, diabetic neuropathies, or peripheral arterial occlusive disease (PAOD). Other clinical complications of diabetes are also covered by the present invention.

In addition, conditions and diseases that result from chronic fatty and fibrotic degeneration of organs due to the forced accumulation of lipids and in particular triglycerides, and the subsequent activation of profibrotic pathways, can also be prevented and/or treated by administration of the compounds or pharmaceutical composition according to the present invention . Such conditions and diseases include NASH and chronic cholestatic conditions in the liver, glomerulosclerosis and diabetic nephropathy in the kidneys, macular degeneration and diabetic retinopathy in the eyes, and neurodegenerative diseases such as Alzheimer's disease in the brain, or diabetic neuropathies in the peripheral nervous system.

In another embodiment, the compounds or pharmaceutical compositions of the present invention are suitable for the prevention and/or treatment of congenital liver fibrosis.

Dosage

The effective dosage of the active ingredient employed may vary depending on the particular compound employed, the route of administration, the condition being treated, and the severity of the condition being treated. Such dosage can be easily determined by one skilled in the art.

In the treatment or prevention of FXR-mediated conditions for which the compounds of the present invention are indicated, satisfactory results are generally obtained when the compounds of the present invention are administered at a daily dosage of from about 0.1 to about 300 mg per kilogram of animal body weight. In some embodiments, the compounds of the present invention are presented in a single daily dose, or in divided doses for administration two to six times daily, or in a sustained release form. For most large mammals, the total daily dosage is from about 1 to about 1000 mg, or from about 1 to about 50 mg. For a 70 kg adult, the total daily dose is typically from about 7 to about 350 mg. This dosage regimen may be adjusted to ensure optimal therapeutic response. In some embodiments, the total daily dosage is from about 1 to about 900 mg, from about 10 to about 800 mg, from about 20 to about 700 mg, from about 30 to about 600 mg, from about 40 to about 550 mg, from about 50 to about 400 mg, about 50 to about 300 mg, or about 50 to about 200 mg.

The compounds of this application or composition thereof can be administered one, two, three or four times a day using any suitable method described above. In addition, administration or treatment of the compounds can be continued for several days; for example, treatment is typically continued for at least 7 days, 14 days, or 28 days for one treatment cycle. Treatment cycles are well known in cancer chemotherapy and are often interspersed with rest periods of about 1 to 28 days, typically about 7 days or about 14 days between cycles. In other embodiments, treatment cycles may also be continuous.

In a specific embodiment, the methods of the present invention include administering to a subject an initial daily dose of about 1 to 800 mg of a compound described herein and increasing the dose in increments until clinical efficacy is achieved. Increments of approximately 5, 10, 25, 50, or 100 mg may be used to increase the dose. The dosage can be increased daily, every other day, twice a week or once a week.

Combinations

In some embodiments, a compound described herein is administered in combination with one or more additional therapeutic agents to treat or prevent a disease or condition described herein. In some embodiments, the one or more additional therapeutic agents are an ACE inhibitor, an acetyl-CoA carboxylase inhibitor, an adenosine A3 receptor agonist, an adiponectin receptor agonist, an AKT protein kinase inhibitor, an AMP-activated protein kinase (AMPK) inhibitor, an amylin receptor agonist, a receptor antagonist AT-1 angiotensin II, autotaxin inhibitors, bioactive lipid, calcitonin agonist, caspase inhibitor, caspase-3 stimulator, cathepsin inhibitor,

- 15 046251 caveolin 1 inhibitor, CCR2 chemokine antagonist, CCR3 chemokine antagonist, CCR5 chemokine antagonist, chloride channel stimulator, CNR1 inhibitor, cyclin D1 inhibitor, cytochrome P450 7A1 inhibitor, DGAT1/2 inhibitor, dipeptidyl peptidase IV inhibitor, endosialin modulator, eotaxin ligand inhibitor , extracellular matrix protein modulator, farnesoid X receptor agonist, fatty acid synthase inhibitors, FGF1 receptor agonist, fibroblast growth factor ligands (FGF15, FGF-19, FGF-21), galectin-3 inhibitor, glucagon receptor agonist, glucagon-like peptide 1 agonist, G-protein coupled bile acid receptor 1 agonist, Hedgehog (Hh) modulator, hepatitis C virus NS3 protease inhibitor, hepatocyte nuclear factor 4 alpha (HNF4A) modulator, hepatocyte growth factor modulator, HMG-coA reductase inhibitor, IL-10 agonist , IL-17 antagonist, sodium-dependent ileal bile acid cotransporter inhibitor, insulin sensitizer, integrin modulator, interleukin-1 receptor associated kinase 4 (IRAK4) inhibitor, Jak2 tyrosine kinase inhibitor, ketohexokinase inhibitors, klotho beta stimulator, 5-lipoxygenase inhibitor , lipoprotein lipase inhibitor, liver X receptor, LIL gene stimulator, lysophosphatidate-1 receptor antagonist, lysyloxylase homologue 2 inhibitor, matrix metalloproteinase (MMP) inhibitor, MEKK-5 protein kinase inhibitor, membrane copper amine oxidase inhibitor (VAP-1), methionine aminopeptidase inhibitor- 2, methyl-CpG binding protein 2 modulator, microRNA-21(miR-21) inhibitor, mitochondrial uncoupling agent, myelin basic protein stimulator, NACHT LRR PYD 3-domain protein (NLRP3) inhibitor, NAD-dependent sirtuin deacetylase stimulator, inhibitor NADPH oxidase (NOX), nicotinic acid receptor 1 agonist, P2Y13 purinoceptor stimulator, PDE 3 inhibitor, PDE 4 inhibitor, PDE 5 inhibitor, PDGF beta receptor modulator, phospholipase C inhibitor, PPAR alpha agonist, PPAR delta agonist, PPAR gamma agonist, modulator PPAR gamma, protease-activated receptor-2 antagonist, protein kinase modulator, Rho-associated protein kinase inhibitor, sodium-dependent glucose transporter-2 inhibitor, SREBP transcription factor inhibitor, STAT-1 inhibitor, stearoyl-CoA desaturase-1 inhibitor, stimulator of suppressor of cytokine signaling -1, stimulator of suppressor of cytokine signaling-3, transforming growth factor β (TGF-β), transforming growth factor β-activated kinase 1 (TAK1), thyroid hormone receptor beta agonist, TLR-4 antagonist, transglutaminase inhibitor, tyrosine kinase receptor modulator, GPCR modulator, nuclear hormone receptor modulator, WNT modulators or YAP/TAZ modulator.

Non-limiting examples of one or more additional therapeutic agents include ACE inhibitors such as enalapril;

acetyl-CoA carboxylase (ACC) inhibitors such as DRM-01, gemcaben, PF-05175157 and QLT091382;

adenosine receptor agonists such as CF-102, CF-101, CF-502 and CGS21680;

adiponectin receptor agonists such as ADP-355;

amylin/calcitonin receptor agonists such as KBP-042;

AMP-activated protein kinase stimulators such as O-304;

angiotensin II AT-1 receptor antagonists such as irbesartan;

autotaxin inhibitors such as PAT-505, PAT-048, GLPG-1690, X-165, PF-8380 and AM-063; bioactive lipids such as DS-102;

cannabinoid receptor type 1 (CNR1) inhibitors such as namacizumab and GWP-42004; caspase inhibitors such as emricasan;

pan-cathepsin B inhibitors such as VBY-376;

pan-cathepsin inhibitors such as VBY-825;

CCR2/CCR5 chemokine antagonists such as cenicriviroc;

CCR2 chemokine antagonists such as propagermanium;

CCR3 chemokine antagonists such as bertilimumab;

chloride channel stimulants such as cobiprostone;

diglyceride acyltransferase 2 (DGAT2) inhibitors such as IONIS-DGAT2Rx;

dipeptidyl peptidase IV inhibitors such as linagliptin;

eotaxin ligand inhibitors such as bertilimumab;

extracellular matrix protein modulators such as CNX-024;

fatty acid synthase inhibitors such as TVB-2640;

fibroblast growth factor 19 (rhFGF19)/cytochrome P450 (CYP) 7A1 inhibitors such as NGM-282; fibroblast growth factor 21 (FGF-21) ligand such as BMS-986171, BMS-986036;

fibroblast growth factor 21 (FGF-21)/glucagon-like peptide 1 (GLP-1) agonists such as YH-25723;

galectin-3 inhibitors such as GR-MD-02;

glucagon-like peptide 1 (GLP1R) agonists such as AC-3174, liraglutide, semaglutide;

agonists of G-protein coupled bile acid receptor 1 (TGR5), such as RDX-009, INT-777;

- 16 046251 heat shock protein 47 (HSP47) inhibitors, such as ND-L02-s0201;

HMG-CoA reductase inhibitors such as atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin;

IL-10 agonists such as pegilodecakin;

inhibitors of the sodium-dependent ileal bile acid cotransporter such as A4250, volixibate potassium ethanolate hydrate (SHP-262) and GSK2330672;

insulin sensitivity enhancers such as KBP-042, MSDC-0602K, Px-102, RG-125 (AZD4076) and VVP-100X;

ketohexokinase inhibitors such as PF-06835919; a Klotho-beta (KLB)-FGF1c agonist such as NGM-313;

5-lipoxygenase inhibitors such as tipelukast (MN-001);

lipoprotein lipase inhibitors such as CAT-2003;

LRL gene stimulators such as Alipogen tiparvovec;

liver X receptor (LXR) modulators such as PX-L603, PX-L493, BMS-852927, T0901317, GW-3965 and SR-9238;

lysophosphatidate-1 receptor antagonists such as BMT-053011, UD-009, AR-479, ITMN-10534, BMS-986020 and KI-16198;

lysyl oxidase homolog 2 inhibitors such as simtuzumab;

semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP1) inhibitors such as PXS-4728A;

methionine aminopeptidase-2 inhibitors such as ZGN-839;

methyl-CpG binding protein 2 modulators such as mercaptamine;

mitochondrial uncouplers such as 2,4-dinitrophenol;

myelin basic protein stimulants such as olesoxime;

NADPH 1/4 oxidase inhibitors such as GKT-831;

nicotinic acid receptor 1 agonists such as ARI-3037MO; nitazoxinide;

NACHT, LRR and PYD domain containing protein 3 (NLRP3) inhibitors such as KDDF-201406-03 and NBC-6;

nuclear receptor modulators such as DUR-928;

purinoceptor P2Y13 stimulators such as CER-209;

PDE 3/4 inhibitors such as tipelukast (MN-001);

PDE 5 inhibitors such as sildenafil;

PDGF beta receptor modulators such as BOT-191, BOT-509;

PPAR agonists such as elafibranor (GFT-505), MBX-8025, R-enantiomer of deuterated pioglitazone, pioglitazone, DRX-065, saroglitazar and IVA-337;

protease-activated receptor-2 antagonists such as PZ-235;

protein kinase modulators such as CNX-014;

Rho-associated protein kinase (ROCK) inhibitors such as KD-025;

sodium-dependent glucose transporter-2 (SGLT2) inhibitors such as ipragliflozin, remogliflozin etabonate, ertugliflozin, dapagliflozin and sotagliflozin;

SREBP transcription factor inhibitors such as CAT-2003 and MDV-4463;

stearoyl-CoA desaturase-1 inhibitors such as aramchol;

beta thyroid hormone receptor (THR) agonists such as MGL-3196, MGL-3745, VK-2809;

TLR-4 antagonists such as JKB-121;

receptor tyrosine kinase modulators such as CNX-025;

GPCR modulators such as CNX-023; and nuclear hormone receptor modulators such as Px-102.

In some specific embodiments, one or more additional therapeutic agents is selected from A-4250, AC-3174, acetylsalicylic acid, AK-20, AKN-083, Alipogen tiparvovec, aramchol, ARI-3037MO, ASP-8232, atorvastatin, bertilimumab, betaine anhydrous, BAR-704, BI-1467335, BMS-986036, BMS-986171, VMT-053011, VOT-191, VTT-1023, BWD-100, BWL200, CAT-2003, cenicriviroca, CER-209, CF-102 , CGS21680, CNX-014, CNX-023, CNX-024, CNX-025, cobiprostone, colesevelam, dapagliflozin, 16-dehydropregenolone, deuterated R-enantiomer of pioglitazone, 2,4-dinitrophenol, DRX-065, DS-102, DUR -928, EDP-305, elafibranor (GFT-505), emricasan, enalapril, EP-024297, ertugliflozin, evogliptin, EYP-001, F-351, fexaramine, GKT-831, GNF-5120, GR-MD-02, hydrochlorothiazide, icosapent ethyl ester, IMM-124-E, INT-767, IONISDGAT2Rx, INV-33, ipragliflozin, irbesart, propagermanium, IVA-337, JKB-121, KB-GE-001, KBP042, KD-025, M790, M780, M450, metformin, sildenafil, LC-280126, linagliptin, liraglutide, LJN-452, LMB-763, MBX-8025, MDV-4463, mercaptamine, MET-409, MGL-3196, MGL-3745, MSDC0602K, namacizumab, NC-101, ND-L02-s0201, NFX-21, NGM-282, NGM-313, NGM-386, NGM-395, NTX023-1, noursodeoxycholic acid, O-304, obeticholic acid, 25HC3S, olesoxime, PAT- 505, PAT-048, peg-ilodecakin, pioglitazone, pirfenidone, PRI-724, PX20606, Рх-102, PX-L603, PX-L493,

- 17 046251

PXS-4728A, PZ-235, RDX-009, RDX-023, remogliflozin etabonate, repurposed tricaprylin, RG-125 (AZD4076), saroglitazar, semaglutide, simtuzumab, SIPI-7623, solithromycin, sotagliflozin, statins (atorvastatin, fluvastatin, pitavastatin , pravastatin, rosuvastatin, simvastatin), TCM-606F, TERN-101, TEV-45478, tipelukast (MN-001), TLY-012, tropifexor, TRX-318, TVB2640, UD-009, ursodeoxycholic acid, VBY-376, VBY-825, VK-2809, vismodegib, volixibate potassium ethanolate hydrate (SFIP-626), VVP-100X, WAV-301, WNT-974 and ZGN-839.

In some embodiments, the methods and compositions comprise a therapeutically effective amount of an inhibitor of apoptosis signal regulating kinase 1 (ASK1) and a therapeutically effective amount of a farnesoid X receptor (FXR) agonist, wherein the FXR agonist is a compound of formula (I)

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or tautomer thereof.

In some embodiments of the methods and pharmaceutical compositions described herein, the ASK1 inhibitor is a compound of formula (II)

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or tautomer thereof.

ASK1 inhibitors, such as a compound of formula (II), can be synthesized and characterized using methods known to those skilled in the art, such as those described in U.S. documents. 2007/0276050, U.S. 2011/0009410 and U.S. 2013/0197037.

In some embodiments, the methods and compositions comprise a therapeutically effective amount of an acetyl-CoA carboxylase (ACC) inhibitor and a therapeutically effective amount of a farnesoid X receptor (FXR) agonist, wherein the FXR agonist is a compound of formula (I)

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or tautomer thereof.

In some embodiments of the methods and pharmaceutical compositions described in the present invention, the ACC inhibitor is a compound of formula (III)

or a pharmaceutically acceptable salt thereof.

ACC inhibitors, such as a compound of formula (III), can be synthesized and characterized using methods known to those skilled in the art, such as those described in PCT International Application Publication No. WO 2013/071169.

In some embodiments, the methods and compositions comprise a therapeutically effective amount of a thyroid hormone receptor (THR) β agonist in combination with a therapeutically effective amount of a farnesoid X receptor (FXR) agonist, wherein the FXR agonist is a compound of formula (I)

- 18 046251

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or tautomer thereof.

In some embodiments of the methods and pharmaceutical compositions described herein, the THR β agonist is a compound of formula (IV)

or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, or tautomer thereof.

THR β agonists, such as a compound of formula (IV), can be synthesized and characterized using methods known to those skilled in the art.

Examples

The following examples are included to demonstrate specific embodiments in accordance with the present invention. Those skilled in the art will appreciate that the methods presented in the following examples represent methods that function well in the practice of the present invention and, thus, may be considered to constitute specific embodiments of the present invention. However, those skilled in the art will understand that, in the context of the present invention, these examples are illustrative and not exhaustive. In the specific embodiments presented, many changes can be made to achieve the same or similar results without departing from the spirit and scope of the present invention.

The compounds disclosed in the present invention can be prepared in accordance with the methods presented in the following schemes and examples using appropriate materials, and they are further illustrated by the following specific examples. In addition, by using the methods described in the present invention, in combination with the normal skill of one skilled in the art, additional compounds of the present invention claimed in the present invention can be easily prepared. The Examples further illustrate in detail the preparation of the compounds of the present invention. Those skilled in the art will appreciate that known variations of the conditions and processes of the following preparation methods can be used to prepare the compounds. For the synthesis of compounds that are embodiments described in the present invention, inspection of the structure of the compound to be synthesized allows the characteristics of each substituent group to be determined. In some cases, the characteristics of the final product may clearly determine the characteristics of the required starting materials in the context of testing, taking into account the examples given in the present invention. The compounds can be isolated in the form of their pharmaceutically acceptable salts, such as the salts described above. The compounds described in the present invention are generally stable and liberated at room temperature and pressure.

The preparation of the compounds disclosed in the present invention is illustrated below. Unless otherwise specified, variables have the same meaning as described above. The following examples are intended to illustrate specific embodiments in accordance with the present invention. Suitable starting materials, building blocks and reagents used in the synthesis, as described below, are commercially available, for example from Sigma-Aldrich or Acros Organics, or can be prepared using conventional methods described in the literature, for example, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition; John Wiley & Sons or in T. Eicher, S. Hauptmann The Chemistry of Heterocycles; Structures, Reactions, Synthesis and Application, 2nd edition, Wiley-VCH 2003; Fieser et al. Fiesers' Reagents for Organic Synthesis John Wiley & Sons 2000.

Methods

Powder X-ray Diffraction (XRD)

PXR diffraction patterns were recorded on a PANanalytical XPERT-PRO diffractometer under ambient conditions under the following experimental conditions: 45 kV, 40 mA, Κα1=1.5406 A, scanning range from 2 to 40° 2θ, step size 0.0084 or 0.0167° 2θ, measurement time: 5 min.

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Differential scanning calorimetry (DSC)

DSC thermograms were recorded on a TA Instruments Q2000 system equipped with a 50-position automatic sampler. Energy and temperature calibrations were performed using certified indium. Typically, 1-5 mg of each sample in an aluminum tray with pin holes was heated at a rate of 10°C/min from about 25 to about 300°C. Throughout the measurement, a dry nitrogen purge was maintained over the sample at a rate of 50 ml/min. The beginning of the melting endotherm was indicated as the melting point.

Thermogravimetric analysis (TGA)

TGA thermograms were recorded on a TA Instruments Q5000 system equipped with a 25-position automatic sampler. Approximately 1-5 mg of each sample was loaded onto a pre-tared aluminum pan and heated at a rate of 10°C/min from about 25°C to about 350°C. Throughout the measurement, a dry nitrogen purge was maintained over the sample at a rate of 25 ml/min.

General synthesis scheme.

Compounds of formula (Ia) wherein Y is N can be prepared according to the following general synthetic schemes.

In the general synthetic schemes above, A is pyridylene or phenylene, each of which is optionally substituted with one or two groups independently selected from halogen, C _1-4 alkoxy, halogen-C _1-4 alkoxy, C _1-4 alkyl and halogen-C _1-4 alkyl; Q is phenylene or pyridylene, each of which is optionally substituted with one or two substituents independently selected from halogen, methyl, C _1-4 alkoxy, halogen-C _1-4 alkoxy, -CH2F, -CHF2 and -CF3; X is a leaving group, Y is a group such as a halogen, which can be used to form organometallic compounds such as a Grignard reagent, Z is an isoxazole substituted with R ¹ or a pyrazole substituted with a C1-4alkyl or a C3-6cycloalkyl, R ² and R ³ are independently selected from hydrogen, halogen, methoxy, -CF3, -CHF2, -CH2F, -OCH2F, -OCHF2, -OCF3 and methyl; R ⁴ is -CO2R ⁵ or -C(O)NR ⁵ R ⁶ ; R ⁵ represents hydrogen, C 1-6 alkyl or halogen-C _1-6 alkyl; and R ⁶ represents hydrogen or C _1-6 alkyl, wherein said C _1-6 alkyl is optionally substituted with 1-6 substituents independently selected from halogen, -SO3H and -CO2H. PG represents a protecting group, and the remaining variables are as described in the present invention. A compound of formula (C) can be prepared by reacting a compound of formula (A) with a compound of formula (B) in the presence of a base. A compound of formula (D) is prepared from a compound of formula (C) by formation of organometallic compounds such as a Grignard reagent, followed by condensation with suitably protected 3-ketoazetidine. The compound of formula (E) is prepared from the compound of formula (D) under appropriate deprotection conditions. A compound of formula (E) can be combined with a compound of formula (F) in the presence of a base to produce a compound of formula (Ia).

Alternatively, a compound of formula (D) can be prepared by reacting a compound of formula (A) with a compound of formula (G) in the presence of a base.

Suitable compounds of structures (A) and (B) can be prepared in accordance with the methods described in the following examples or using methods known in the art. For example

- 20 046251 measures, X may be a halogen (eg fluorine, bromine, chlorine and/or iodine) and PG may be a tert-butylcarbonyl protecting group (BOC).

Example 1 Preparation of Formula (I)

Stage 1. Preparation of 2,6-dichloro-4-fluorobenzaldehyde oxime.

A suspension of 2,6-dichloro-4-fluorobenzaldehyde (20 g, 100 mmol), hydroxylamine hydrochloride (14 g, 210 mmol) and sodium carbonate (27 g, 260 mmol) in ethanol/water (5:1, 170 ml) was treated sonication for approximately 10 min and then left under stirring overnight at room temperature.

The mixture was diluted with water and ethyl acetate. The aqueous phase was extracted twice with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure to obtain the desired intermediate. LCMS-ESI (m/z): [M+H] ⁺ calc. for C ₇ H ₅ Cl ₂ FNO: 207.97; found: 207.99.

Step 2. Preparation of 2,6-dichloro-4-fluoro-N-hydroxybenzimidoyl chloride

A solution of 2,6-dichloro-4-fluorobenzaldehyde oxime (21 g, 99 mmol) in \,\-dimethylformamide (200 ml) was treated with one portion of N-chlorosuccinimide (1.5 g, 11 mmol). Hydrogen chloride vapor (approximately 40 ml taken from the headspace of the concentrated hydrochloric acid bottle) was bubbled through the mixture. After adding another portion of N-chlorosuccinimide (1.5 g, 90 mmol), two more volumes of hydrogen chloride gas (40 ml x 2) were introduced. Additional N-chlorosuccinimide (12 g, 11 mmol) was added in small portions. When an exotherm of up to 25°C was observed, the mixture was cooled in a water bath at room temperature. The temperature was allowed to drop to approximately 23°C and the remainder of the N-chlorosuccinimide was added in portions. At the end of the addition, LC/MS analysis indicated that the original oxime was retained, so a final portion of N-chlorosuccinimide (1.2 g, 9.0 mmol) was added. The mixture was stirred overnight at room temperature and transferred without treatment to the subsequent synthesis step. LCMS-ESI+ (m/z): [M+H] ⁺ calc. for C ₇ H ₄ Cl ₃ FNO: 241.93; found: 242.20.

Step 3. Preparation of ethyl 5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole-4 carboxylate

A solution of ethylcyclopropyl-3-oxopropanoate (19 g, 120 mmol) in 2-methyltetrahydrofuran at room temperature was treated with triethylamine using a syringe (55 ml, 400 mmol). After 30 minutes of stirring, the reaction mixture containing 2,6-dichloro-4-fluoro-N-hydroxybenzimidoyl chloride (24 g, 99 mmol) was added dropwise through a syringe. At the end of the addition, the mixture was heated at approximately 60°C overnight. The reaction mixture was concentrated under reduced pressure. The residue was diluted with 10% hydrochloric acid and ethyl acetate. The aqueous phase was extracted three times with ethyl acetate. The combined organic extracts were washed once with saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel) to obtain the desired intermediate. LCMS-ESI+ (m/z): [M+H] ⁺ calc. for C ₁₅ H ₁₃ Cl ₂ FNO ₃ : 344.02; found: 344.03.

Step 4. Preparation of (5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)-methanol.

Solution of (ethyl 5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole-4-carboxylate (15 g, 44

- 21046251 mmol) were placed in 2-methyltetrahydrofuran (220 ml) and cooled using a magnetic stirrer in a wet ice/acetone bath to a temperature of about -12 to -10°C. A solution of lithium aluminum hydride (2.0 M in tetrahydrofuran, 53 mmol) was added dropwise. After 30 min of stirring in the bath, the mixture, cooled in an ice water bath, was quenched successively with water (2.0 ml, dropwise very carefully), 15% aqueous sodium hydroxide (2.0 ml) and water (6.0 ml) . The suspension was left stirring at room temperature for 15 minutes before adding anhydrous magnesium sulfate and then stirring for a further hour. The suspension was filtered; the filter cake was washed with ethyl acetate and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel) to obtain the desired intermediate. LCMSIE+ (m/z): [M+H]+ calc. for C13H _n Cl ₂ FNO ₂ : 302.01; found: 302.51.

Step 5. Preparation of 4-(chloromethyl)-5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole ci

F

Thionyl chloride (6.6 ml, 90 mmol) was added to a mixture of (5-cyclopropyl-3-(2,6-dichloro-4fluorophenyl)isoxazol-4-yl)methanol (9.1 g, 30 mmol) in dichloromethane (150 ml ) at room temperature. The mixture was then heated at approximately 45°C for 45 minutes. The mixture was concentrated under reduced pressure. The residue was dissolved in diethyl ether and re-concentrated. This was repeated two more times to obtain the desired intermediate, which was used without further purification. LCMS-ESI+ (m/z): [M+H]+ calc. for C ₁₃ H ₁₀ Cl ₃ FNO: 319.97; found: 320.50.

Step 6. Preparation of 4-((4-bromo-3-chlorophenoxy)methyl)-5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole.

A solution of crude 4-(chloromethyl)-5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole (4.2 g, 13 mmol) in N,N-dimethylformamide (50 ml) was treated with 4-bromo- 3-chlorophenol (2.7 g, 13 mmol), sodium iodide (3.3 g, 22 mmol) and anhydrous potassium carbonate (3.6 g, 26 mmol). The mixture was heated at approximately 60°C for 25 minutes. After cooling, the mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel) to obtain the desired intermediate. LCMS-ESI+ (m/z): [M+H]+ calc. for C ₁₉ H ₁₃ BrCl ₃ FNO ₂ : 489.91; found: 490.10.

Step 7. Preparation of tert-butyl-3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidine -1-carboxylate.

Under argon, a solution of 4-((4-bromo-3-chlorophenoxy)methyl)-5-cyclopropyl-3-(2,6-dichloro-4fluorophenyl)isoxazole (5.1 g, 10 mmol) in tetrahydrofuran (27 ml) treated with isopropylmagnesium chloride/lithium chloride solution (1.3 M, 12 ml, 15 mmol) using a syringe. The resulting mixture was stirred for one hour before adding an additional volume of isopropylmagnesium chloride/lithium chloride solution (1.3 M, 4.0 mL, 5.2 mmol). After 45 minutes of stirring, another portion of isopropylmagnesium chloride/lithium chloride solution (1.3 M, 1.0 mL, 1.3 mmol) was added. After one hour of stirring, tert-butyl 3-oxoazetidine-1-carboxylate (3.8 g, 22 mmol) was added in one portion to the resulting Grignard mixture, which was cooled in a wet ice/acetone bath. After 30 minutes of stirring, an additional portion of tert-butyl 3-oxoazetidine-1-carboxylate (0.80 g, 4.7 mmol) was added. The reaction mixture was quenched with a 10% aqueous solution of citric acid (50 ml). The aqueous phase was extracted three times with ethyl acetate. The combined organic layers were washed once with water and saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel) to obtain the desired intermediate. LCMS-ESI+ (m/z): [M+H]+ calc. for C ₂₇ H ₂₇ Cl ₃ FN ₂ O ₅ : 584.86; found: 483.19.

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Step 8. Preparation of the tosylate salt of 3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4fluorophenyl)isoxazol-4-yl)methoxy)phenyl)azetidin-3-ol.

Para-toluenesulfonic acid monohydrate (3.5 g, 18 mmol) was added to the mixture of tert-butyl-3-(2-chloro4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole-4- yl)methoxy)phenyl)-3-hydroxyazetidine-1-carboxylate (5.3 g, 9.1 mmol) and isopropanol (21 ml). The mixture was heated overnight at approximately 50°C and then cooled in a wet ice/acetone bath while stirring with a magnetic stirrer. The resulting suspension was heated until nearly homogeneous at approximately 50°C. After the mixture had cooled, hexane was added and the resulting suspension was sonicated and then heated to approximately 50°C. The solid was collected by suction filtration, washed with hexane and dried in a vacuum oven at approximately 75°C to obtain the tosylate salt of the desired intermediate. LCMS-ESI+ (m/z): [M+H]+ calc. for C ₂₂ H ₁₉ Cl ₃ FN ₂ O ₃ : 483.04; found: 483.19.

Step 9. Preparation of methyl-6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol4-yl)methoxy)phenyl)-3-hydroxyazetidine- 1-yl)nicotinate.

Mixture of 3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)azetidin-3-ol tosylate (0.47 g , 0.71 mmol), methyl 6-chloronicotinate (0.15 g, 0.90 mmol) and potassium carbonate (0.49 g, 3.6 mmol) in H^dimethylformamide (4 ml) were heated overnight at 80°C. The mixture was partitioned between ethyl acetate and water. The aqueous phase was extracted three times with ethyl acetate. The combined extracts were washed successively with water and saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel) to obtain the desired intermediate. LCMS-ESI+ (m/z): [M+H]+ calc. for C ₂₉ H ₂₄ Cl ₃ FN ₃ O ₅ : 618.07; discovered: 618.41.

Step 10. Preparation of 6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidine- 1-yl)nicotinic acid.

A mixture of methyl 6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidine- 1-yl)isonicotinate (0.32 g, 0.51 mmol) and lithium hydroxide monohydrate (43 mg, 1.0 mmol) in tetrahydrofuran/water (1:1, 10 ml) were stirred at room temperature overnight.

The mixture was concentrated under reduced pressure to remove most of the volatiles. The resulting aqueous mixture was further diluted with water and treated with acetic acid, followed by the addition of a 10% aqueous solution of hydrochloric acid. The resulting precipitate was collected by suction filtration, washed with water and dried in a vacuum oven at a temperature of approximately 50°C to obtain the desired material as the free acid. LCMS-ESI+ (m/z): [M+H]+ calc. for C ₂₈ H ₂₂ Cl ₃ FN ₃ O=: 604.05; discovered: 604.41.

Step 11. Preparation of 6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidine tris salt -1-yl)nicotinic acid.

6-(3 -(2-chloro-4-((5 -cyclopropyl-3 -(2,6-dichloro-4-fluorophenyl)-isoxazol-4-yl)methoxy)phenyl)-3 hydroxyazetidin-1-yl) nicotinic acid (0.29 g, 0.48 mmol) was placed as a suspension in Me

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OH/water (95:5, 2 ml), treated with tromethamine (58 mg, 0.48 mmol), heated at 55°C and concentrated to give the desired acid as the tromethamine salt. ^1H NMR (400 MHz, methanol^ ₄ ) δ 8.65 (dd, J=2.2, 0.8 Hz, 1H), 8.05 (dd, J=8.7, 2.2 Hz, 1H ), 7.38 (d, J=8.3 Hz, 2H), 7.35 (d, J=8.7 Hz, 1H), 6.87 (d, J=2.6 Hz, 1H), 6.78 (dd, J=8.6, 2.6 Hz, 1H), 6.42 (dd, J=8.8, 0.8 Hz, 1H), 4.92 (s, 2H), 4 .57 (dd, J=9.3, 1.1 Hz, 2H), 4.29 (dd, J=9.2, 1.1 Hz, 2H), 3.63 (s, 7H), 2, 32 (tt, J=8.0, 5.5 Hz, 1H), 1.25 -1.11 (m, 4H).

Example 2 Synthesis of Comparative Example 1

6-(3-(2-Chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3hydroxyazetidin-1-yl)-5 -fluoronicotinic acid.

Synthesis of intermediate A.

To a solution of (4-bromo-3-chlorophenoxy)-(tert-butyl)dimethylsilane (60 g, 187 mmol) in THF (500 mL) was added n-BuLi (2.5 M, 75 mL) dropwise at approximately -78 °C in N2 atmosphere. The reaction mixture was stirred at approximately -78°C for 1 hour. A solution of tert-butyl 3-oxoazetidine-1-carboxylate (27 g, 155 mmol) in THF (500 ml) was then added dropwise to the mixture at -78°C C. The reaction mixture was then stirred at approximately 20°C for 3 hours. The reaction mixture was poured into H2O (1 L) and extracted three times with EtOAc (2 L). The combined organic layers were washed with water (1 L), dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel, eluting with 10:1 petroleum ether:EAAc to give 3-(4-((t-butyldimethylsilyl)oxy)-2-chlorophenyl)azetidin-3-ol (intermediate A).

Step 1: tert-butyl 3-(2-chloro-4-hydroxyphenyl)-3-hydroxyazetidine-1-carboxylate

To a solution of tert-butyl 3-(4-((tert-butyldimethylsilyl)oxy)-2-chlorophenyl)-3-hydroxyazetidine-1-carboxylate (intermediate A, 1.27 g, 3.07 mmol) in THF (50, 0 ml) at approximately -10°C, a 1 M solution of TBAF in THF (3.68 ml, 3.68 mmol) was added dropwise. The reaction mixture was stirred for 2 hours and concentrated to give tert-butyl 3-(2-chloro-4-hydroxyphenyl)-3hydroxyazetidine-1-carboxylate, which was used without further purification.

Step 2: 4-(chloromethyl)-5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole

A solution of (5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methanol (45 mg, 2.80 mmol) in DCM (28.0 ml) was cooled to approximately 0°C. Thionyl chloride (1.02 mL, 14.0 mmol) was added and the solution was heated at approximately 45°C for 1 hour. The reaction mixture was concentrated to dryness and used in the next step without purification.

Step 3: tert-butyl-3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4yl)methoxy)phenyl)-3-hydroxyazetidine-1 - carboxylate

A solution of tert-butyl 3-(2-chloro-4-hydroxyphenyl)-3-hydroxyazetidine-1-carboxylate (922 mg, 3.07 mmol) in DMF (28.0 ml) was added to crude 4-(chloromethyl)- 5-cyclopropyl-3-(2,6-dichloro-4fluorophenyl)isoxazole, and then potassium carbonate (773 mg, 5.60 mmol) was added. The mixture was heated at approximately 60°C for 8 hours. The reaction mixture was concentrated, diluted with water and extracted with EtOAc (3 times). The combined organic layers were washed with water and brine, dried over MgSO ₄ , filtered and concentrated. The crude product was purified by silica gel chromatography (DCM/Et ₂ O/MeOH) to give tert-butyl-3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4fluorophenyl)isoxazole- 4-yl)methoxy)phenyl)-3-hydroxyazetidine-1-carboxylate. LCMS-IER+ (m/z):

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[(M+H)-VOC]+calc.: 483.04; found: 483.04.

Step 4: 3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)azetidin-3-ol

To a solution of tert-butyl-3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4yl)methoxy)phenyl)-3-hydroxyazetidine-1-carboxylate (1.52 g, 2.60 mmol) in DCM (130 ml) was added 4 N HCl in 1,4-dioxane (26.0 ml, 104 mmol). The solution was stirred at room temperature for 2.5 hours and concentrated to dryness to give 3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4fluorophenyl)isoxazol-4-yl)methoxy) phenyl)azetidin-3-ol in the form of hydrochloride salt, which was used without further purification. LCMS-ESI+ (m/z): [M+H] ⁺ calc: 483.04; found: 483.03.

Step 5: Methyl 6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidine -1-yl)-5-fluoronicotinate.

Mixture of methyl 6-chloro-5-fluoropyridine (235 mg, 1.24 mmol), 3-(2-chloro-4-((5-cyclopropyl-3-(2,6dichloro-4-fluorophenyl)isoxazole-4- yl)methoxy)phenyl)azetidin-3-ol hydrochloride salt (495 mg, 0.952 mmol) and potassium carbonate (1.05 g, 7.61 mmol) in DMF (30.0 ml) was heated at approximately 60°C for 1 hour. The reaction mixture was concentrated, diluted with water and extracted with EtOAc (3 times). The combined organic layers were washed with brine, dried over MgSO ₄ , filtered and concentrated. The crude mixture was purified by silica gel chromatography (DCM/Et ₂ O/MeOH) to give methyl 6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl) )isoxazol-4yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinate. LCMS-ESI+ (m/z): [M+H]+ calc: 636.07; discovered: 635.96.

Step 6: 6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidine-1 -yl)-5-fluoronicotinic acid (Comparative Example 1).

To a solution of methyl 6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4yl)methoxy)phenyl)-3-hydroxyazetidine-1- yl)-5-fluoronicotinate (364 mg, 0.571 mmol) in THF/water (1:1, 20.0 ml) was added lithium hydroxide monohydrate (41.3 mg, 0.984 mmol). The solution was stirred for 18 hours, concentrated to remove THF and diluted with water (10.0 ml). The pH was adjusted to 3 using 1 N HCl solution. The solids were filtered, washed with water, dissolved in ACN/water and lyophilized to give 6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4fluorophenyl)isoxazol-4- yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinic acid (Comparative Example 1). LCMS-ESI+ (m/z): [M+H]+ calc: 622.05; found: 622.12. ^1H NMR (400 MHz, DMSO-de) δ 12.84 (br. s, 1H), 8.44 (t, J=1.7 Hz, 1H), 7.79-7.63 (m, 3H ), 7.39 (d, J=8.7 Hz, 1H), 6.95 (d, J=2.5 Hz, 1H), 6.77 (dd, J=8.6, 2.6 Hz , 1H), 6.28 (s, 1H), 4.93 (s, 2H), 4.70 (d, J=9.8 Hz, 2H), 4.34 (d, J=9.5 Hz , 2H), 2.50-2.43 (m, 1H), 1.22-1.08 (m, 4H).

Example 3 Activity Test Using FRET

Determination of ligand-mediated cofactor-peptide interactions to quantify ligand binding to the nuclear receptor FXR was performed as follows.

Preparation of human FXR-alpha ligand binding domain (LBD): Human FXR-alpha LSD was expressed in E. coli strain BL21(DE3) as an N-terminal GST-tagged fusion protein. DNA encoding the ligand binding domain of FXR was cloned into the pDEST15 vector (Invitrogen). Expression was controlled by the IPTG-inducible T7 promoter. The amino acid boundaries of the ligand binding domain were amino acids 187-472 from the NM_005123 database (RefSeq). Expression and purification of FXR-LSD: An overnight pre-culture of transformed E. coli strain was diluted 1:20 in LB medium with ampicillin and grown at 30°C to OD ₆₀₀ =0.4-0.6. Gene expression was then induced by adding 0.5 mM IPTG. Cells were incubated for an additional 6 h at 30°C, 180 rpm. Cells were collected by centrifugation (7000xg, 7 min, room temperature). Cells were resuspended in 10 ml of lysis buffer (50 mM glucose, 50 mM Tris pH 7.9, 1 mM EDTA, and 4 mg/ml lysozyme) per liter of original cell culture and left on ice for 30 min. The cells were then sonicated and cell debris was removed by centrifugation (22,000xg, 30 min, 4°C). To 10 ml of supernatant, 0.5 ml of pre-washed glutathione-4B-Sepharose suspension (Qiagen) was added and the resulting suspension was kept under slow rotation for 1 h at 4°C. Glutathione-4B-Sepharose granules were pelleted by centrifugation (2000xg, 15 s, 4°C) and washed twice in wash buffer (25 mM Tris, 50 mM KCl, 4 mM _MgCl2 and 1 M NaCl). The pellet was resuspended in 3 ml of elution buffer per liter of original culture (elution buffer: 20 mM Tris, 60 mM KCl, 5 mM MgCl ₂ and 80 mM glutathione added as powder immediately before use).

The suspension was left to rotate for 15 min at 4°C, the beads were precipitated and eluted again with half the volume of elution buffer compared to the first time. The eluates were pooled and dialyzed overnight in 20 mM Hepes buffer (pH 7.5) containing 60 mM KCl, 5 mM MgCl ₂ as well as 1 mM dithiothreitol and 10% (v/v) glycerol. The protein was analyzed by DNS-PAGE.

The method measures the ability of putative ligands to modulate the interaction between the purified ligand binding domain (LSD) of bacterially expressed FXR and the synthetic

- 25 046251 tic biotinylated peptide based on residues 676-700 of SRC-1 (LCD2, 676-700). The sequence of the peptide used was B-CPSSHSSLTERHKILHRLLQEGSPS-COOH (SEQ ID NO: 1), where the N-terminus is biotinylated (B). The ligand binding domain (LBD) of FXR was expressed as a GST fusion protein in BL-21 cells using the pDEST15 vector. Cells were lysed by sonication and fusion proteins were purified using glutathione-Sepharose (Pharmacia) according to the manufacturer's instructions. Perkin Elmer LANCE technology was used to determine the effect of compounds on the interaction of FXR and peptide. This method is based on binding-dependent energy transfer from a donor to an acceptor of a fluorophore attached to the binding partner of interest. For ease of processing and reduced background, LANCE technology uses common fluorophore tags and time-resolved detection assays to fluorescent compounds in a final volume of 25 μL in a 384-well plate in Tris-based buffer (20 mM TrisHCl pH 7.5; 60 mM KCl , 5 mM MgCl ₂ ; 35 ng/μl BSA) containing 20-60 ng/well of recombinantly expressed FXR-LSD fused to GST, 200-600 nM N-terminal biotinylated peptide representing amino acids 676-700 of SRC1, 200 ng /well streptavidin-xlAPC conjugate (prozyme) and 6-10 ng/well Eu W1024 - anti-GST (Perkin Elmer). The DMSO content in the samples was maintained at 1%. After preparing the assay mixture and diluting the potential FXR modulating ligands, the assay mixture was allowed to equilibrate for 1 h in the dark at room temperature in black 384-well ELISA plates (Greiner). The LANCE signal was detected using a Perkin Elmer VICTOR2VTM multifunction counter. The results were visualized by plotting the ratio of emitted light at 665 and 615 nm. Zero level of FXRpeptide complex formation was observed in the absence of added ligand. Ligands that promote complex formation cause a concentration-dependent, time-resolved increase in the fluorescence signal. Compounds that bind equally well to both monomeric FXR and the FXR-peptide complex are expected to result in no signal change, whereas ligands that bind preferentially to the monomeric receptor will cause a concentration-dependent decrease in the observed signal.

To assess the agonistic potential of the compounds, EC ₅₀ values were determined for the compounds given in the examples and presented in the table below. 1 (FRET EC ₅₀ ). As indicated in table. 1, the compound of Example 1 was evaluated together with Comparative Example 1 and Comparative Example 2 (Example 3 of US Patent Application Publication No. 2017/0355685), the chemical structures of which are shown in the table below.

Example 4 Test in Mammalian One-Hybrid System (M1H)

Determination of ligand-mediated transactivation driven by the Gal4 promoter to quantify ligand binding-mediated activation of FXR was performed as follows.

Part of the cDNA encoding the ligand-binding domain of FXR was cloned into the pCMV-BD vector (Stratagene) as a hybrid with the DNA-binding domain of yeast GAL4 under the control of the CMV promoter. The amino acid boundaries of the ligand-binding domain were amino acids 187-472 from the NM_005123 database (RefSeq). Plasmid pFR-Luc (Stratagene) was used as a reporter plasmid containing a synthetic promoter with five tandem repeats in yeast GAL4-binding sites that drives the expression of the Photinus pyralis (American firefly) luciferase gene as a reporter gene. To increase the accuracy of the experiment, plasmid pRL-CMV (Promega) was cotransfected. pRL-CMV contains a constitutive CMV protomore that controls the expression of Renilla reniformis luciferase. All Gal4 reporter gene assays were performed in HEK293 cells (obtained from DSMZ, Braunschweig, Germany) grown in MPS with L-glutamine and Earle's SCP supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate and 100 units of penicillin/streptavidin per ml at approximately 37°C in a 5% CO2 atmosphere. Media and supplements were obtained from Invitrogen. For the assay, 5 × 10 ⁵ cells per well were seeded in 96-well plates in 100 μl per well of MPS without phenol red and L-glutamine and with Earl's SSR supplemented with PBS (HyClone, South Logan, Utah) treated with 10% charcoal/dextran, 0.1 mM nonessential amino acids, 2 mM glutamine, 1 mM sodium pyruvate, and 100 units penicillin/streptavidin per ml, and incubated at approximately 37°C in a 5% CO2 atmosphere. The next day, cells showed >90% confluence. The medium was removed and cells were transiently transfected using 20 μl per well of OptiMEM polyethyleneimine transfection reagent (OptiMEM, Invitrogen; Polyethyleneimine, Aldrich cat. no. 40827-7), including the three plasmids described above. MPS with the same composition as for cell seeding was added 2-4 hours after adding the transfection mixture. Then, stock solutions of the compounds, previously diluted in MPS, were added (the final carrier concentration did not exceed 0.1%). Cells were incubated for an additional 16 hours before sequentially measuring firefly and renilla luciferase activities in the same cell extract using the Dual-Light-Luciferase-Assay system (Dyer et al., Anal. Biochem. 2000, 282, 158-161). All experiments were performed three times.

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To evaluate the agonistic activity of the compounds given in the examples against FXR, the activity was determined in the M1H assay, and the results are shown below in table. 1 (M1N EC50).

Table 1

Example 5 In vivo pharmacodynamics assessment in cynomolgus monkeys.

The in vivo pharmacodynamics of formula (I) of Example 1, Comparative Example 1 and Comparative Example 2 were determined as follows.

Test sample and its preparation

Doses of oral suspension:

Oral solution doses of Formula (I) (Example 1) were prepared at concentrations of 1 mg/ml in aqueous suspensions of 1% sodium lauryl sulfate (SLS), 1% ethanol and 98% water at pH 2. Oral suspension doses of Comparative Example 1 and comparative example 2 were made at concentrations of 2.5 mg/ml in 1% hydroxypropyl methylcellulose (HPMC), 0.5% Tween 80 and 98.5% water.

Intravenous doses:

Intravenous doses of the compounds of formula (I) (Example 1), Comparative Example 1 and Comparative Example 2 were prepared at concentrations of 0.5 mg/ml in a vehicle containing 5% DMSO, 15% NMP, 60% PEG 300 and water with 1.1 NaOH equivalents.

Animals:

Each dose group consisted of three to six male cynomolgus macaques. At dosing, the animals weighed from 2.4 to 4.4 kg.

Dosage:

Oral test samples were administered to monkeys by gavage at a dose of 2 ml/kg or via nasogastric intubation at a dose of 5 ml/kg to obtain a dose of 5 mg/kg. Intravenous test samples were administered as an approximately 30-minute infusion through an indwelling saphenous or cephalic vein catheter at a dose of approximately 2 mL/kg to produce a 1 mg/kg dose.

Sampling:

Venous blood samples were collected from each animal at predetermined control time points after dosing. Blood samples were collected and transferred into tubes containing the anticoagulant potassium salt (K ₂ )EDTA.

Determination of plasma FGF19 concentrations:

The FGF19 ELISA kit from BioVendor (product number RD191107200R) was used to determine FGF19 concentrations in collected blood samples.

Determination of drug concentration in plasma:

For analysis on an API 5000 LC/MS/MS system, a 50 μL aliquot of each plasma sample was treated with 200 μL of acetonitrile (ACN) containing the internal standard. The above solution was centrifuged at 5000 rpm for 10 min and 50 μl of the supernatant was transferred to a clean 96-well plate, followed by the addition of 200 μl of water. 10 μL aliquots were injected into an API 5000 LC/MS/MS system.

For analysis on an Applied Biosystems API 5500 LC/MS/MS system, a 20 μL aliquot of each plasma sample was treated with 120 μL of acetonitrile (ACN) containing internal standard. The above solution was centrifuged and 100 μl of the supernatant was transferred into a clean 96-well

- 27 046251 plate followed by adding 100 µl of water. Aliquots of 7–10 μL were injected into an API 5500 LC/MS/MS system.

HPLC conditions:

A Zorbax Extend C18 HPLC column (50 x 2.1 mm, 3.5 µm) from Agilent Technologies (product no. 735700-902) (Comparative Example 1 and Comparative Example 2) or a Waters BEN C18 column (50 x 2.1 mm, 1, 7 µm) (example 1). For Zorbax Extend C18 HPLC, mobile phase A contained an aqueous solution of 1% acetonitrile in 10 mM ammonium formate adjusted to pH 3.0 with formic acid, and mobile phase B contained 10% 10 mM ammonium formate in acetonitrile adjusted to pH 4.6 with formic acid. acid. For the Waters BEN C18 column, mobile phase A contained 95% water, 5% acetonitrile, and 0.1% formic acid, and mobile phase B contained 50% methanol, 50% acetonitrile, and 0.1% formic acid. A Thermo Aria multiplexer with two identical Agilent 1200 series bin pumps (P/N G1312A Bin Pump) was used for elution and separation. The elution programs used are given in the following table. 2 for HPLC column Zorbax Extend C18 and in table. 3 for Waters VEN C18 column.

table 2

Time (sec) Stage Consumption (ml/min) Mobile phase A (%) Mobile phase B (%) thirty Loading a sample 0.50 85 15 180 Gradual change in eluent composition 0.50 50 50 90 Gradual change in eluent composition 0.50 99 1 60 Elution 0.50 99 1 120 Recovery equilibrium 0.50 85 15 Table 3 Time (sec) Stage Consumption (ml/min) Mobile phase A (%) Mobile phase B (%) 0 Loading a sample 0.80 45 55 60 Gradual change in eluent composition 0.80 35 65 63 Gradual change in eluent composition 0.80 5 95 78 Gradual change in eluent composition 0.80 5 95 90 Stage 0.80 45 55

An API 5000 triple quadrupole mass spectrometer from AB Sciex, Foster City, CA, was used in multiple reaction monitoring mode to quantify compounds. The applied mass spectrometry parameters are given in the following table. 4.

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Table 4

And he source Spray voltage (V) Gas 1 Gas 2 (prod. (production units) units meas.) meas.) Gas for Impact temperature drying room (manufacturer) chambers (°C) units change) Turbo ion atomizer 5500 70 50 6,550

Oral and IV administration to cynomolgus monkeys

Activation of the farnesoid X receptor in the distal small intestine, as well as systemically in organs such as the liver, directly induces FGF19 expression and secretion. Plasma FGF19 levels were assessed as a pharmacodynamic marker of FXR activation in male cynomolgus monkeys following oral and intravenous dosing of Example 1, Comparative Example 1, or Comparative Example 2. Difference between FGF19 levels after oral (PO) and intravenous (IV) administration. administrations were used to compare the degree of intestinal FXR agonism (via po data analysis) and systemic FXR agonism (via IV data analysis) for each compound administered. Plasma drug and FGF19 levels were measured at various time points over a 24-hour period to obtain pharmacokinetic and pharmacodynamic curves.

In fig. 1 shows a graph of plasma FGF19 concentration over time in cynomolgus monkeys following dosing of the compound of Example 1.

In fig. 2 shows a graph of plasma FGF19 concentration over time in cynomolgus monkeys following dosing of the compound of Comparative Example 1.

In fig. 3 shows a graph of plasma FGF19 concentration over time in cynomolgus monkeys after dosing with the compound of Comparative Example 2.

Pharmacokinetic data are summarized in Table. 5 below.

Table 5

Example Group FGF19 Cmax(pg/ml) FGF19 P1PS ate (pg/ml.h) GS-PPKlast (nM.h) Relationship PPKposl to FG19/GS po/vv Example 1 BB 432 +/- 100 5144+/-2199 3150+/- 1030 1.6 7.8 BY 6260+/-2194 28153 +/- 11539 2250 +/- 1640 12.5 Comparative example 1 BB 2350 +/- 179 22798 +/- 2387 1630 +/- 323 14 1.37 By 3810+/- 195 32085 +/- 4252 1670 +/- 250 19.2 Comparative example 2 BB 1179+/- 226 10757+/- 2551 3140+/- 167 3.4 8.2 By 3186 +/- 856 18193 +/-7134 643 +/-216 28

As reflected in the results, oral administration of Example 1, Comparative Example 1, and Comparative Example 2 caused an increase in plasma FGF19 levels during the dosing interval compared to pre-dose baseline FGF19 levels. This indicates agonism of the intestinal FXR receptor. IV administration of Example 1 did not cause an increase in FGF19, indicating the absence of systemic FXR agonism. In contrast, IV administration of Comparative Examples 1 and 2 increased FGF19 levels compared to pre-dose baseline FGF19 levels, indicating systemic FXR agonism.

These data suggest that ileal exposure to Example 1 and Comparative Example 2 causes activation of FXR in the intestinal epithelium. The data further suggests that systemic exposure to Comparative Example 1 and Comparative Example 2 via IV administration causes greater systemic activation of FXR outside the intestine compared to Example 1.

Example 6 Inhibition of UGT1A1

The compound of formula (I) (Example 1) was tested for inhibition of UGT1A1 and compared with Comparative Example 1. To perform the assay, a sample of the compound was incubated with human UGT1A1 expressed by Supersomes™ (0.25 mg/ml), alamethicin (25 μg/mg) and UDPHA (5 mM) in the presence of the marker substrate estradiol (10 μM) for 30 min at 37°C. A selective UGT1A1 inhibitor, atazanavir, was screened along with test compounds as a positive control. Reactions were completed by quenching an aliquot in two volumes of methanol. Samples were centrifuged at 2500 rpm for 30 min at 4°C, and aliquots of the supernatant were diluted with formic acid in deionized water (final concentration of formic acid 0.1%) with internal standard. General Cyprotex LCMS/MS conditions were used to monitor the formation of estradiol 3-glucuronide. Decreased metabolite formation compared to the control solution

- 29 046251 lem was used to calculate the IC ₅₀ value.

The data showed that the compound of Example 1 demonstrated the most potent inhibition of UGT1A1 compared to the other compounds. Example 1 had an IC ₅₀ of 0.44 μM.

Example 7 Inhibition of CYP2C8, CYP3A4 and CYP2B6

The compound of formula (I) (Example 1) was evaluated for inhibition of CYP2C8, CYP3A4 and CYP2B6 and compared with Comparative Example 1.

CYP2C8

Six sample concentrations of compound (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration 0.25%) were incubated with human liver microsomes (1 mg/ml) and NADPH (1 mM ) in the presence of the marker substrate tolbutamide (120 μM) for 60 min at 37°C. A selective inhibitor of CYP2C9, sulfaphenazole, was screened along with test compounds as a positive control.

CYP3A4

Six concentrations of sample compound (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration 0.26%) were incubated with human liver microsomes (0.1 mg/ml) and NADPH ( 1 mM) in the presence of the marker substrate midazolam (2.5 μM) for 5 min at 37°C. A selective inhibitor of CYP3A4, ketoconazole, was screened along with test compounds as a positive control. Alternatively, six concentrations of sample compound (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration 0.275%) were incubated with human liver microsomes (0.5 mg/ml) and NADPH (1 mM) in the presence of the marker substrate testosterone (50 μM) for 5 min at 37°C. A selective inhibitor of CYP3A4, ketoconazole, was screened along with test compounds as a positive control.

CYP2B6

Six concentrations of the test compound (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration 0.3%) were incubated with human liver microsomes (0.1 mg/ml) and NADPH ( 1 mM) in the presence of the marker substrate bupropion (110 μM) for 5 min at 37°C. A selective inhibitor of CYP2B6, ticlopidine, was screened along with test compounds as a positive control.

For each of the CYP2B6, CYP2C8, and CYP3A4 incubations, reactions were stopped by adding methanol. The samples were then centrifuged and analyzed by LC-MS/MS. Formic acid in deionized water (0.1% final concentration) containing internal standard was added to the final sample prior to analysis. The reduction in metabolite formation compared to the solvent control was used to calculate the IC50.

The data showed that the compound of Example I did not inhibit CYP2B6, while Comparative Example 1 did inhibit CYP2B6.

Example 8 Preparation of Form I mesylate of formula (I)

Form I mesylate of formula (I) was prepared by combining 6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)- 3-hydroxyazetidin-1-yl)nicotinic acid (0.050 g, 0.0827 mmol) with methanesulfonic acid (8 μl, 0.123 mmol) in a vial of isopropyl alcohol (1 ml) to obtain a suspension. The suspension was heated to about 50°C for about 30 minutes, then slowly cooled to room temperature and the suspension was allowed to sit for about 16 hours. The suspension was then filtered and the isolated solids were characterized by PRD as shown in FIG. 4. The solids were then dried in a vacuum oven at approximately 50°C and characterized by PXRD, resulting in the same pattern. The PRD peaks of Form I mesylate of formula (I) included peaks at 3.2, 6.4, 9.6, 12.8, 19.3, 22.1, 22.6, 25.8, 29.1° 2θ. The resulting PRD peaks are presented in the table below.

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Table

No. Pos. [°20] Rel. Int. G%1 1 3.2 55 2 6.4 24 3 8.0 4 4 9.6 78 5 10.6 3 6 12.8 46 7 17.3 4 8 18.2 7 9 19.3 59 10 20.1 2 AND 22.1 23 12 22.6 100 13 23.7 6 14 24.3 10 15 25.8 20 16 26.5 3 17 27.3 5 18 28.1 3 19 29.1 19 20 32.5 12 21 34.4 3 22 35.5 2 23 36.9 9

DSC and TGA analyzes were performed. The DSC thermogram of Form I of the mesylate of formula (I) showed an endotherm at about 221° C. followed by an exotherm as shown in FIG. 5. The TGA thermogram of Form I of the mesylate of formula (I) showed a weight loss of about 0.6% when heated to about 200° C., as shown in FIG. 6.

Form I mesylate of formula (I) was also prepared by taking 6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl) -3-hydroxyazetidin-1-yl)nicotinic acid (3.00 g, 4.96 mmol) as a suspension in MeCN/water (1.5:1 v/v, 15 ml) and treated with aqueous sodium hydroxide (30% wt, 0.56 ml, 5.95 mmol). The resulting solution was then fed over several hours into a second vessel containing a stirred solution of methanesulfonic acid (1.0 mL, 15.9 mmol) in MeCN (15 mL) preheated to about 50°C. The resulting suspension was kept at about 50°C for several hours and then cooled to about 20°C. The suspension was filtered under vacuum and the resulting solids were dried in vacuum at elevated temperature to about 60°C to obtain Form I mesylate of formula (I).

Form I mesylate of formula (I) was also prepared by taking 6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)- 3-Hydroxyazetidin-1-yl)nicotinic acid (0.5 g, 0.83 mmol) suspended in acetone/water (97:3 v/v, 10 ml) and heated to approximately 55°C. Methanesulfonic acid (60 μl, 0.91 mmol) was added to the resulting suspension and the mixture was kept at approximately 55°C for several hours. The suspension was cooled to approximately 20°C and filtered under vacuum. The resulting solids were washed with acetone and dried in vacuo at elevated temperature to about 60°C to obtain Form I mesylate of formula (I).

Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

Thus, it is understood that while the present invention has been specifically presented with preferred embodiments and optional features, modifications, improvements, and changes to the embodiments described in the present invention can be made by those skilled in the art, and that such modifications, improvements, and changes are included in scope of the present invention. The materials, methods and examples presented in the present invention represent preferred embodiments, are given by way of example and do not limit the scope of the present invention.

The present invention has been described broadly and generally in the present invention. Each of the narrower species and subgeneric groups included in the general description of the invention is also part of the present invention. This includes a general description of the present invention with a condition or negative limitation that deletes any member from the class, regardless of whether the material to be deleted was specifically mentioned in the present invention or not.

Moreover, if features or aspects of the present invention are described in terms of Markush groups, those skilled in the art will understand that the present invention is also described in terms of any individual member or subgroup of members of the Markush group.

It will be understood that although the present invention has been described in connection with the embodiments mentioned above

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Claims (8)

The implementations, description and examples above are intended to be illustrative and do not limit the scope of the present invention. Other aspects, advantages and modifications within the scope of the present invention will be apparent to those skilled in the art to which the present invention relates. CLAIM 1. A compound of the following formula (I): F or a pharmaceutically acceptable salt thereof. 2. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof for the treatment of a farnesoid X receptor (FXR) mediated condition. 3. Use of a compound according to claim 2 or a pharmaceutically acceptable salt thereof, characterized in that said FXR-mediated condition is a liver disease. 4. Use of a compound according to any one of claims 2 or 3 or a pharmaceutically acceptable salt thereof, characterized in that said FXR-mediated condition is non-alcoholic steatohepatitis (NASH). 5. Use of a compound according to any one of claims 2, 3 or a pharmaceutically acceptable salt thereof, characterized in that said FXR-mediated condition is primary sclerosing cholangitis (PSC). 6. Use of a compound according to any one of claims 2, 3 or a pharmaceutically acceptable salt thereof, characterized in that said FXR-mediated condition is primary biliary cholangitis (PBC). 7. Use of a compound according to any one of claims 2, 3 or a pharmaceutically acceptable salt thereof, characterized in that said FXR-mediated condition is liver fibrosis. 8. Use of a compound according to claim 2 or 3, characterized in that said FXR-mediated condition is selected from the group consisting of a chronic intrahepatic or extrahepatic cholestatic condition; liver fibrosis; chronic or obstructive inflammatory liver disorder; liver cirrhosis; hepatic steatosis or related syndrome; cholestatic or fibrotic effect associated with alcohol-induced cirrhosis or viral forms of hepatitis; acute or chronic liver failure; liver ischemia after extensive liver resection; chemotherapy-associated steatohepatitis (CHAS); primary biliary cirrhosis (PBC); primary sclerosing cholangitis (PSC); tumor disease of the gastrointestinal tract or liver and inflammatory bowel disease (IBD); lipid metabolism disorders or lipoprotein metabolism disorders; type I diabetes; type II diabetes; clinical complications of type I and type II diabetes, selected from the group consisting of diabetic nephropathy, diabetic neuropathy, diabetic retinopathy and other observed effects of clinically manifested long-term diabetes; non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); obesity; metabolic syndrome, selected from the group consisting of the combined conditions of dyslipidemia, diabetes and abnormally high body mass index; acute myocardial infarction; acute stroke and thrombosis, occurring as the final stage of chronic obstructive atherosclerosis; non-malignant hyperproliferative disorder; a malignant hyperproliferative disorder selected from the group consisting of hepatocellular carcinoma, colon adenoma and polyposis; -