EA043973B1 - FXR MODULATING CONNECTIONS (NR1H4) - Google Patents

Description

Cross reference to related applications

This application claims priority to U.S. Provisional Application No. 62/349,490, filed June 13, 2016, the contents of which are incorporated herein by reference 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 list of sequences is 1165_PF_ST25.txt. The text file, created on May 16, 2017, is approximately 550 bytes in size and is presented electronically using EFS-Web.

Field of technology

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 compounds for the treatment and/or prevention of diseases and/or conditions by binding to said nuclear receptor by said compounds.

State of the art

Multicellular organisms depend on complex mechanisms for transmitting information between cells and body compartments. The information transmitted can be very complex and can lead to changes in genetic programs involved in cell differentiation, proliferation or reproduction. The signals, or hormones, are often small molecules such as peptides, fatty acids, or cholesterol derivatives.

Many of these signals exert their influence by essentially changing the transcription of certain genes. One well-studied group of proteins that mediate cellular responses to various signals is the family of transcription factors known as nuclear receptors, hereinafter referred to as NRs. Members of this group include receptors for steroid hormones, vitamin D, ecdysone, cis- and trans-retinoic acid, thyroid hormone, bile acids, cholesterol derivatives, fatty acids (and other peroxisomal proliferators), as well as so-called orphan receptors, proteins, which are structurally similar to other members of this group, but for which no ligands are known. Orphan receptors may indicate unknown signaling pathways in the cell or may be nuclear receptors that function without ligand activation. Transcriptional activation by some of these orphan receptors can occur in the absence of an exogenous ligand and/or through signal transduction pathways originating from the cell surface.

Overall, three functional domains have been identified in NR. The amino-terminal domain is believed to have some regulatory function. This is followed by a DNA binding domain (hereinafter referred to as the DBD), which typically contains two zinc finger elements and recognizes a specific hormonal response element (hereinafter referred to as the HRE) in the promoters of responsive genes. Specific amino acid residues in the DBD have been shown to confer DNA sequence binding specificity. The ligand binding domain (hereinafter referred to as LBD) is located in the carboxy-terminal region of known NRs.

In the absence of the hormone, the LBD appears to prevent the DBD from interacting with its HRE. Hormone binding likely results in a conformational change in the NR and thus eliminates this obstruction. NR without LBD constitutively activates transcription, but at a low level.

Coactivators or transcription activators are believed to be the link between sequence-specific transcription factors, the basic transcription machinery, and also influence the chromatin structure of the target cell. Several proteins, such as SRC-1, ACTR, and Grip1, interact with NR in a ligand-enhanced manner.

Nuclear receptor modulators, such as steroid hormones, influence the growth and function of specific cells by binding to intracellular receptors and forming nuclear receptor-ligand complexes. Nuclear receptor-hormone complexes then interact with HREs at the control region of specific genes and alter specific gene expression.

Farnesoid X receptor alpha (hereinafter also commonly referred to herein as NR1H4 in the context of the human receptor) is a prototypic type 2 nuclear receptor that activates genes upon binding to the promoter region of target genes in a heterodimeric manner with the retinoid X receptor. The corresponding physiological ligands of NR1H4 are bile acids. The most potent ligand is chenodeoxycholic acid (CDCA), which regulates the expression of several genes involved in bile acid homeostasis. Farnesol and derivatives, collectively called farnesoids, were originally described to activate the rat ortholog at high concentrations, but they do not activate the human or mouse receptor. FXR is expressed in the liver, throughout the gastrointestinal tract, including the esophagus, stomach, duodenum, small intestine, colon, ovary, adrenal gland, and kidney. Besides controlling inside

- 1 043973 cellular gene expression FXR also appears to be involved in paracrine and endocrine signaling through up-regulation of the expression of the cytokine, fibroblast growth factor 15 (rodents) or 19 (monkeys, humans).

Although many FXR agonists are known, there is a need to provide improved FXR agonists.

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 compounds for the treatment and/or prevention of diseases and/or conditions by binding to said nuclear receptor by said compounds.

The present invention provides compounds according to formula (I) z ^0H (I) wherein 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-Smalkoxy, -CH2F, -CHF2 and -CF3,

Y represents N or CH;

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;

Z is an isoxazole substituted with R ¹ or a pyrazole substituted with R ¹ ;

R ¹ represents C ₁ . ₄ -alkyl or C ₃ . ₆ -cycloalkyl, where the specified C ₁ . _{The 4} -alkyl is optionally substituted with 1-3 substituents independently selected from fluorine, hydroxyl, C ₁ . ₃ -alkoxy and fluoro-C ₁ . ₃ -alkoxy, and said 6-cycloalkyl is optionally substituted with 1-3 substituents independently selected from fluorine, hydroxyl, C ₁ . ₃ -alkyl, fluoro-C ₁ . ₃ -alkyl, C ₁ . ₃ -alkoxy and fluoro-C ₁ . ₃ -alkoxy;

R ² and R ³ are independently selected from hydrogen, halogen, methoxy, -CF ₃ , -CHF2, -CH2F, -OCH2F, -OCHF2, -OCF3 and methyl;

R ⁴ is -CO2R ⁵ or -C(O)NR5R 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, where the specified C ₁ . _{The 6} -alkyl is optionally substituted with 1-6 substituents independently selected from halogen, -SO3H and -CO2H;

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

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

Also provided herein are methods of treating patients with an FXR-mediated condition, comprising administering a compound of formula (I) to a patient in need thereof.

Description of the figures

Fig. 1 - plasma content of the substance in example 3 and comparison example 2 depending on the levels of FGF19 in plasma in cynomolgus macaques;

fig. 2 - FGF19 levels obtained in cynomolgus monkeys with increasing oral doses of Example 3 and Comparative Example 2.

Detailed Description of the Invention

Definitions.

The following description presents exemplary embodiments of the present technology. However, it should be recognized that the above description is not intended to limit the scope of the present invention and is intended to describe exemplary embodiments.

The following words, phrases and symbols, as used herein, generally have the meanings set forth below, unless the context in which they are used indicates otherwise.

The invention illustrated herein may be suitably practiced in the absence of any element or elements, limitation or restrictions not specifically disclosed herein. Thus, for example, the terms containing, including, etc. should be read broadly and without limitation. In addition, terms and expressions used herein are used as terms of description and not limitation, and

- 2 043973 the use of such terms and expressions does not imply the exclusion of any equivalents of the presented and described distinctive features or fragments thereof and implies that various modifications are possible within the scope of the claimed invention.

A prime (-) that is not between two letters or symbols is used to indicate where a substituent is attached. For example, -C(O)NH2 is attached via a carbon atom. A prime at the beginning or end of a chemical group is used for convenience; chemical groups may be depicted with or without one or more strokes without losing their normal meaning. A wavy line drawn through the structure indicates where the group attaches. Unless chemically or structurally required, directionality is not indicated or implied by the order in which a chemical group is written or named.

The prefix C _uv indicates that the following group contains from and to v carbon atoms. For example, Ci-6 alkyl indicates that the alkyl group contains from 1 to 6 carbon atoms.

As used herein, 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 embodiments, the term approximately includes the indicated value of ±5%. In some other embodiments, the term approximately includes the indicated value of ±1%. Also, the term about X includes a description of X. In addition, singular forms include plural forms unless the context clearly indicates 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.

In the context of the present invention, alkyl refers to a saturated hydrocarbon chain, which may be straight or branched. In the context of the present invention, C ₁ . ₆ -alkyl refers to a saturated alkyl chain containing from 1 to 6 carbon atoms, which may be straight or branched. Examples thereof include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl and n-hexyl.

The term haloalkyl means that one or more hydrogen atoms in the alkyl chain have been replaced by a halogen. A non-limiting example is CF ₃ .

A cycloalkyl group denotes a saturated or partially unsaturated mono-, bi- or spirocyclic hydrocarbon ring system.

Alkoxy refers to -O-alkyl, where alkyl is as defined herein. Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, n-hexoxy and 1,2-dimethylbutoxy.

Halogen or halogen- refers to the F, Cl, Br or I atom. Hydroxyl or hydroxy refers to -OH.

Haloalkoxy refers to an alkoxy group, as defined herein, where one or more hydrogen atoms in the alkyl chain are replaced by a halogen.

Fluoroalkyl refers to an alkyl group, as defined herein, where one or more hydrogen atoms in the alkyl chain are replaced by a fluorine atom.

Fluoroalkoxy refers to an alkoxy group, as defined herein, where one or more hydrogen atoms in the alkyl chain are replaced by a fluorine atom.

The terms optional or optional mean that the event or circumstance described below may or may not occur, and that the description includes cases in which the specified event or circumstance occurs and cases in which it does not occur. In addition, the term optionally substituted refers to any one or more hydrogen atoms at the specified atom or group, which may or may not be replaced by a moiety other than hydrogen.

In addition, the compounds of the present invention may be subject to tautomerism. When tautomerism, for example keto-enol tautomerism, may occur, of the compounds of the present invention or their prodrugs, 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, conformers, 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.

- 3 043973

One skilled in the art will appreciate that if a list of alternative substituents includes members that, due to valency 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 list members that are suitable to replace a particular group. groups.

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-α-D-glucuronic acid through glycosidic bonds or to esterify compounds with carboxyl or hydroxyl groups in the process 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. 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 applicable 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, in the form of 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 or amino acids. Compounds of the present invention which contain 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 addition salts with 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 one 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 them with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts. 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.

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

Some embodiments provide optical isomers, racemates, or other mixtures of the compounds described herein, or pharmaceutically acceptable salts or mixtures thereof. If desired, the isomers can be separated using methods known in the art, for example, liquid chromatography. In these situations, a single enantiomer or diastereomer, i.e. optically active form, can be obtained by asymmetric synthesis or by

- 4 043973 divisions. 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 different 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 contain a center of assymmetry and, therefore, may have enantiomeric, diastereomeric and other stereoisomeric forms, which can be defined in terms of absolute stereochemistry as (R) or (S)- or as (D)- or (L)- for amino acids. The present invention is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)- or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents or can be separated using conventional methods, such as chromatography and fractional crystallization. Conventional 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 herein 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 herein 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 herein 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 herein, 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) , ⁿ 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 ^3H , ^13C and ^14C . Such isotopically labeled compounds may be suitable for use in metabolic studies, reaction kinetics studies, detection and imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT) , including studies of drug or substrate distribution in 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 of formula (I) in which 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):524527 (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.

Labeled or deuterium-substituted therapeutic compounds of the present invention may have improved 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 such a heavier isotope, in particular deuterium, can be determined using

- 5 043973 cabbage soup isotope enrichment coefficient. 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 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 substance.

Pharmaceutical composition means one or more active ingredients and one or more inert ingredients that constitute the carrier substance, as well as any product that is formed directly or indirectly by the combination, complexation or aggregation of any two or more ingredients, or by the dissociation of one or more 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 include any composition prepared by mixing at least one compound of the present invention and a pharmaceutically acceptable carrier substance.

List of abbreviations and acronyms

Abbreviation Meaning (±)-BINAP (±)-2,2'-Bis(diphenylphosphino)-1,1'-binaphthalene 2-Me-THF 2-Methyltetrahydrofuran ACN or MeCN Acetonitrile water Water VP Benzyl VOS or Vos tert-Butyloxycarbonyl BSA Bovine serum albumin USSR Balanced salt solution cal cd Calculated DAST (Diethylamino)sulfur trifluoride DXM Dichloromethane DIBAL-H Diisobutylaluminum hydride DMF Dimethylformamide DMSO Dimethyl sulfoxide EA Ethyl acetate EDTA Ethylenediaminetetraacetic acid IER Electrospray ionization Et Ethyl E1gO Diethyl ether EtOAc Ethyl acetate FBS Fetal bovine serum h Watch) HATU 1 - [Bis(dimethylamino)methylene]-177-1,2,3-triazolo[4,5 b}pyridinium-3-oxide hexafluorophosphate HPLC High performance liquid chromatography ANN Isopropyl alcohol iptg Isopropyl-3-O-1-thiogalactopyranoside

- 6 043973

LCMS or LC/MS Liquid chromatography/mass spectrometry Me Methyl MEM Minimum nutrient medium MeOH Methanol min Minute(s) MS Mass spectrometry m/z Mass to charge ratio NADP Dihydronicotinamide adenine dinucleotide phosphate NMP N-methylpyrrolidone NMR Nuclear magnetic resonance spectroscopy H-BuLi n-Butyllithium rpm Revolutions per minute PE Petroleum ether CT Room temperature us. Saturated TBAF Tetrabutylammonium fluoride TBDMS tert -B util di methyl si l i l TBS tert -B util di methyl si l i l TEMPO 2,2,6,6-T ethramethylpiperidine-1-oxyl TFC Trifluoroacetic acid THF T etragide rofuran TMS Trimethylsilyl UHPLC Ultra-high performance liquid chromatography

Connections.

The present invention provides compounds according to formula (I)

F (I) wherein Q is phenylene or pyridylene, each of which is optionally substituted with one or two substituents independently selected from halogen, methyl, C1. ₄ -alkoxy, halogen-C1. ₄ alkoxy, -CH2F, -CHF2 and -CF3,

Y represents N or CH;

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;

Z is an isoxazole substituted with R ¹ or a pyrazole substituted with R ¹ ;

R ¹ is C _1-4 -αalkyl or C _{3_6} -cycloalkyl, wherein said C _{1_4} -alkyl is optionally substituted with 1-3 substituents independently _{selected from} fluorine, hydroxyl, C _1-3 -αalkoxy and fluoro-C ₁ . ₃ -alkoxy, and said C ₃ . _{The 6} -cycloalkyl is optionally substituted with 1-3 substituents independently selected from fluorine, hydroxyl, C ₁ . ₃ -alkyl, fluoro-C ₁ . ₃ -alkyl, C ₁ . ₃ -alkoxy and fluoro-C ₁ . ₃ -alkoxy;

R2 and ^R3 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 ;

- 7 043973

R ⁵ represents hydrogen, C1-6-alkyl or halogen- _C1-6 -alkyl; And

R6 is hydrogen or _{C1_6 -} alkyl, wherein said _{C1_6} - _alkyl is optionally substituted with 1-6 substituents independently selected from halogen, _-SO3H and -CO2H;

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

In one embodiment, compounds of formula (Ia) are provided:

(1a) wherein Q is phenylene or pyridylene, each of which is optionally substituted with one or two substituents independently selected from halogen, methyl, _{C1_4 -} alkoxy, halogen-C1. ₄ alkoxy, -CH2F, -CHF2 and -CF3,

Y represents N or CH;

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;

R ¹ is C ₁ _ ₄ -alkyl or C ₃ _ ₆ -cycloalkyl, wherein said C ₁ _ ₄ -alkyl is optionally substituted with 1-3 substituents independently selected from fluorine, hydroxyl, C ₁ _ ₃ -alkoxy and fluoro-C _{1_3} -alkoxy, and said _{C3_6 -} cycloalkyl is optionally substituted with 1-3 substituents independently _selected from fluorine, hydroxyl, _C1 . ₃ -alkyl, fluoro-C ₁ . ₃ -alkyl, C ₁ . ₃ -alkoxy and fluorine-C ₁ . ₃ -alkoxy;

R ² and R ³ are independently selected from hydrogen, halogen, methoxy, -CF ₃ , -CHF ₂ , -CH ₂ F, -OCH ₂ F, -OCHF ₂ , -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;

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;

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

In one embodiment, compounds of formula (Ia) are provided:

where Q is phenylene, optionally substituted with one or two halogens;

Y represents N or CH;

_{A is pyridylene, optionally substituted with one or two groups independently selected from halogen and C 1_4 -alkoxy} ;

_R ¹ represents C _{1_4} -alkyl or C _{3_6} -cycloalkyl _;

R ² and R ³ are independently selected from hydrogen and halogen;

R ⁴ is -CO2R ⁵ or -C(O)NR ⁵ R ⁶ ;

R ⁵ represents hydrogen; And

R ⁶ is C ₁ _ ₂ alkyl, optionally substituted with -CO2H or -SO ₃ H;

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

In one embodiment, Q is phenylene or pyridylene, each of which is optionally substituted with one or two substituents independently selected from halogen, methyl, -CHF2 and _-CF3 . In some embodiments, Q is phenylene, optionally substituted with one or two substituents independently selected from halogen, methyl, and -CF ₃ . In some embodiments, Q is pyridylene, optionally substituted with one or two substituents independently selected from halogen, methyl, and -CF3.

In one embodiment, Q is phenylene, optionally substituted with odo

- 8 043973 neem or two halogens. In some embodiments, Q is pyridylene, optionally substituted with one or two halogens. In some embodiments, Q is phenylene, optionally substituted with one or two chlorine atoms. In some embodiments, Q is pyridylene, optionally substituted with one or two chlorine atoms.

In one embodiment, Q is phenylene substituted with one chlorine atom. In some embodiments, Q is pyridylene substituted with one chlorine atom.

In one embodiment, R ¹ is C1-4 alkyl. In some embodiments, R ¹ is C3-6-cycloalkyl. In some embodiments, R ¹ is cyclopropyl or methyl. In some embodiments, R ¹ is cyclopropyl.

In one embodiment, both R ² and R ³ are non-hydrogen atoms. In some embodiments, R ² and R ³ are independently selected from hydrogen, halogen, methoxy, -OCHF2, -OCF3 and methyl. In some embodiments, R ² and R ³ are independently selected from halogen, methoxy, -OCHF2, -OCF3 and methyl.

In one embodiment, R ² and R ³ are halogen. In some embodiments, R ² and R ³ are chlorine.

In one embodiment, one of R ² and R ³ is halogen and the other is hydrogen. In one embodiment, one of R ² and R ³ is chlorine and the other is hydrogen. In some embodiments, one of R ² and R ³ is fluorine and the other is hydrogen.

In one embodiment, Y is N. In some embodiments, Y is CH.

In one embodiment, A is pyridylene, optionally substituted with one or two halogens. In some embodiments, A is pyridylene, optionally substituted with one or two C _1-4 alkoxy.

In one embodiment, A is pyridylene substituted with one fluorine atom. In some embodiments, A is pyridylene substituted with one methoxy. In one embodiment, A is unsubstituted pyridylene.

In one embodiment, A is phenylene, optionally substituted with one or two halogens. In one embodiment, A is phenylene, optionally substituted with one or two C _1-4 alkoxy.

In one embodiment, A is phenylene substituted with one fluorine atom. In one embodiment, A is phenylene substituted with one methoxy. In one embodiment, A is unsubstituted phenylene.

In one embodiment, R ⁴ is -CO2R ⁵ and R ⁵ is hydrogen. In one embodiment, R ⁴ is -CO2R ⁵ and R ⁵ is C _1-6 alkyl or halo-C _1-6 alkyl.

In one embodiment, R ⁴ is -C(O)NR ⁵ R ⁶ , R ⁵ is C1-6 alkyl or halo-C1-6 alkyl, and R ⁶ is C1-2 alkyl, wherein said C _{1 -2} -alkyl is substituted with -SO3H or _-CO2H .

In one embodiment, R ⁴ is -C(O)NR ⁵ R ⁶ , R ⁵ is hydrogen, and R ⁶ is C _1-2 alkyl, wherein said C _1-2 alkyl is substituted with -SO3H or - CO ₂ N.

In one embodiment, R4-A is

wherein the pyridylene 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.

In one embodiment, R4-A is

F

In one embodiment, R4-A is

- 9 043973

In one embodiment, R4-A is

In one embodiment, a connection selected from the group consisting of

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

In one embodiment, the present invention provides a compound having the following formula:

- 10 043973 or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention provides a compound having the following formula:

F

The chemical names of each of these compounds are presented below in the table. 1.

Table 1

Example Structure IUPAC name 1 F 2-(3-(2-chloro-4-((5cyclopropyl-3 -(2,4difluorophenyl)isoxazol-4yl)methoxy)phenyl)-3 hydroxyazetidin-1 yl)isonicotinic acid 2 but-<1 ^Cl ClxJ^.CI Ψ F 2-(3-(2-chloro-4-((5cyclopropyl-3-(2,6-dichloro4-fluorophenyl)isoxazol-4yl)methoxy)phenyl)-3 hydroxyazetidin-1yl)isonicotinic acid 3 /H Υθ BUT \=N ^v )= ^{7 Cl} ΟΙ^Χ,ΟΙ Ψ F 6-(3-(2-chloro-4-((5cyclopropyl-3-(2,6-dichloro4-fluorophenyl)isoxazol-4yl)methoxy)phenyl)-3 hydroxyazetidin-1 -yl)-5 fluoronicotinic acid 4 HO^^N ^Cl CI^X^CI V F 6-(3-(2-chloro-4-((3-(2,6dichloro-4-fluorophenyl)-5methylisoxazol-4yl)methoxy)phenyl)-3 hydroxyazetidin-1 -yl)-5 fluoronicotinic acid 5 F ° ^Н /ГХ^°\ЧЧ HO “γΥ' HU2U Η J F 6-(3-(2-chloro-4-((4cyclopropyl-1-(2,6-dichloro4-fluorophenyl)-1H-pyrazol-5yl)methoxy)phenyl)-3 hydroxyazetidin-1 -yl)-5 fluoronicotinic acid 6 \-o ΟΗ/Γ γ°Ύ ь 4ΥΎ/ / ck/Lxi < J ci if η N^x 1 J ^N OMe F 5-((1 S,3 S)-3-(2-chloro-4-((5cycloπpoπyl-3-(2,6-dichloro4-fluorophenyl)isoxazol-4yl)methoxy)phenyl)-3 hydroxycyclobutyl)-6methoxynicotinic acid

- 11 043973

7 o O' ^Z ^)=\ V _o ) e ·: O □: 2-(6-(3 -(2-chloro-4-((5cyclopropyl-3 -(2,6-dichloro4-fluorophenyl)isoxazol-4yl)methoxy)phenyl)-3 hydroxyazetidin-1 -yl)-5 fluoronicotinamido) ethane-1 sulfonic acid 8 O o ° OTO t (6-(3 -(2-chloro-4-((5 cyclopropyl-3 -(2,6-dichloro4-fluorophenyl)isoxazol-4yl)methoxy)phenyl)-3 hydroxyazetidin-1 -yl)-5 fluoronicotinoyl)glycine

Pharmaceutical compositions and methods of administration.

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

The pharmaceutical composition of the present invention may further contain one or more other compounds as active ingredients, such as a prodrug 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 substance in accordance with standard methods for preparing pharmaceuticals. The carrier substance may take different forms depending on the form of preparation desired for administration, for example, oral or parenteral (including intravenous). In the preparation of 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 carrier substances 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, where 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 percent to about 60 percent by weight of the dosage form. The amount of active compound in such therapeutically useful compositions is such that it provides an effective dosage. The active 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 substance 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 may contain sugar in addition to the active ingredient

- 12 043973 rose as a sweetener, methyl and propyl parabens as preservatives, coloring and flavoring such as cherry or orange flavoring.

Since the compounds of the present invention are generally carboxylic acids or the like anionic isosteres, and since the salt forms of ionic compounds can significantly affect bioavailability, the compounds of the present invention can also be used as salts with various countercations to provide an orally available formulation. Such pharmaceutically acceptable cations may be, among others, mono or bivalent ions such as ammonium, alkali metal, sodium or potassium or alkaline earth metal cations, magnesium or calcium, certain pharmaceutically acceptable amines such as tris(hydroxymethyl)aminomethane, ethylenediamine, diethylamine, piperazine and others, or some cationic amino acids such as lysine or arginine.

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 administration. 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 substance 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 herein document.

The invention provides articles that contain a compound described herein, 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 or cure is an approach to obtain beneficial or desired results, including clinical results. 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) alleviation of the condition, i.e. causing regression of clinical symptoms (eg, improving disease status, 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 survival).

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 some embodiments, the compounds may be administered to a subject (including a human) who is at risk or has a family history of a 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 this document

- 13 043973 those 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 herein, or a pharmaceutically acceptable salt, tautomer, stereoisomer, mixture of stereoisomers, prodrug or deuterated analogue thereof, 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 sensitive to inhibition of Cot activity. 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 said compounds for the treatment and/or prevention of diseases and/or conditions by binding said nuclear receptor to said compounds. In addition, 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.

Also provided herein are methods of treating a patient with an FXR-mediated condition, comprising administering a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In some embodiments, a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, is provided for use in treating an FXR-mediated condition.

In some embodiments, a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing a compound of formula (I) or a pharmaceutically acceptable salt thereof is provided for the preparation of a medicament for treating an FXR-mediated condition.

In some embodiments, the FXR-mediated condition is a chronic intrahepatic or some form of extrahepatic cholestatic condition;

liver fibrosis;

obstructive inflammatory liver disorder;

chronic inflammatory liver disorder;

cirrhosis of the liver;

hepatic steatosis or related syndrome;

cholestatic or fibrotic effects that are associated with alcohol-induced cirrhosis or viral forms of hepatitis;

liver failure or liver ischemia after extensive liver resection;

chemotherapy-associated steatohepatitis (CASH);

acute liver failure; or inflammatory bowel disease.

In some embodiments, the FXR-mediated condition is a disorder of lipid and lipoprotein metabolism;

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; or thrombosis, which occurs as the final stage of chronic obstructive atherosclerosis.

In some embodiments, the FXR-mediated condition is a non-malignant hyperproliferative disorder; and a malignant hyperproliferative disorder selected from the group consisting of hepatocellular carcinoma, colon adenoma and polyposis;

- 14 043973 colon adenocarcinoma;

mammary cancer;

pancreatic adenocarcinoma;

Barrett's esophagus; or other forms of tumor diseases of the gastrointestinal tract and liver.

In some embodiments, the FXR-mediated condition is non-alcoholic steatohepatitis (NASH).

In some embodiments, the present invention relates to the use of compounds in accordance with formula (I) for 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, which arise due to abnormal bile composition, gastrointestinal conditions with reduced absorption of dietary fat and fat-soluble vitamins from food, inflammatory bowel diseases, disorders of lipid and lipoprotein metabolism, type II diabetes and clinical complications of type I and type II diabetes , conditions and diseases that arise as a result of chronic fatty and fibrous 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 the end stage of chronic obstructive atherosclerosis, persistent infections with intracellular bacteria or parasitic protozoa, non-malignant hyperproliferative disorders, malignant hyperproliferative disorders, in particular adenocarcinoma of the colon and hepatocellular carcinoma, hepatic steatosis or related syndromes, liver failure or liver dysfunction resulting from chronic liver disease or surgical resection of the liver, hepatitis B infection, hepatitis C infection and/or cholestatic or fibrotic effects that are associated with alcohol-induced cirrhosis or viral forms of hepatitis.

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

FXR is believed to be a nuclear bile acid receptor. Thus, it modulates both the synthesis of bile acids in the liver and their recycling in the intestine (by regulating bile acid binding proteins). In addition, in addition to bile acid physiology, FXR appears to 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 disease or chronic intrahepatic forms of cholestasis, and many other diseases.

FXR regulates a complex pattern of response genes in the liver and gastrointestinal tract. Gene products influence various physiological processes. The first regulatory network that was analyzed in the functional analysis of FXR was the regulation of bile acid synthesis. While LXRs induce the key cholesterol-to-bile acid conversion enzyme, Cyp7A1, through induction of the regulatory nuclear receptor LRH-1, FXR represses the induction of Cyp7A1 through up-regulation of the mRNA encoding SHP, another nuclear receptor that is dominantly repressive over LRH -1. Because FXR binds the end products of this pathway, primary bile acids such as cholic acid (CA) or CDCA, this can be considered an example of feedback inhibition at the level of gene expression. In parallel with the repression of bile acid synthesis by SHP, FXR induces a number of so-called ABC transporters (for the ATP-binding cassette), which are responsible for the export of toxic bile acids from the cytosol of hepatocytes into the canaliculi, the small branches of the bile ducts in which bile is produced. This hepatoprotective function of FXR first became apparent in an analysis of FXR knockout mice, which demonstrated under- or overexpression of several ABC transporters in the liver. Further detailed analysis revealed that the main bile salt export pump, BSEP or ABCB11 (as well as the key enzyme that mediates lipid transfer from lipoproteins to phospholipids, PLTP) and two key tubular membrane transporters for phospholipids, MRP-2 (ABCC4) and MDR -3 (ABCB4), are direct targets for ligand-directed transcriptional activation by FXR.

The fact that FXR appears to be a major metabolite sensor and regulator for bile acid synthesis, export, and recycling implies the use of FXR ligands to induce bile flow and alter bile acid composition toward a more hydrophilic composition. With the development of the first synthetic FXR ligand GW4064 as a pharmacological agent

- 15 043973 active compound and semi-synthetic artificial bile acid ligand 6-alpha-ethylCDCA, it became possible to analyze the effect of FXR superstimulation with strong 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. This hepatoprotective effect was further narrowed to an antifibrotic effect, which results from the repression of the tissue inhibitors of matrix metalloproteinases, TIMP1 and 2, the induction of matrix metalloproteinase 2, which cleaves collagen deposits, in hepatic stellate cells and the subsequent decrease in the levels of alpha-collagen mRNA and transforming growth factor beta mRNA. (TRF-beta), which are both profibrotic factors, under the action of FXR agonists. 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. Alvarez et al., Hum. Mol. Genet. 2004, 13, 2451) or levels of the target gene FXR, encoding the phospholipid export pump MDR-3, are reduced (in PFIC type III). Taken together, they form a growing body of evidence that FXR binding compounds will demonstrate significant clinical value in a treatment program for chronic cholestatic conditions such as primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC).

The profound effect that FXR activation has on the metabolism and excretion of bile acids has implications not only for cholestatic syndromes, but even more directly for therapy against gallstone formation. Cholesterol gallstones are formed due to the low solubility of cholesterol, which is actively pumped out of the liver cells into the cavities of the tubules. The relative percentages of the three main components, bile acids, phospholipids and free cholesterol, determine the formation of mixed micelles and, therefore, the apparent solubility of free cholesterol in bile. FXR polymorphism, according to quantitative trait loci mapping, is one of the factors contributing to cholelithiasis. Using the synthetic FXR pharmacologically active 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 has no effect on gallstone formation. stones in FXR knockout mice.

These results identify FXR as a good target for the development of small molecule agonists that can be used to prevent the formation of cholesterol gallstones or to prevent the recurrence of gallstones after surgical removal or shock wave lithotripsy.

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.

In addition to its strong hepatoprotective and choleretic as well as antifibrotic effects that FXR exhibits upon small molecule-stimulated activation in the liver, FXR appears to play a role in protecting the intestine from neoplastic transformation and from the development of polyps and their progression to adenocarcinoma in the intestine. Similar to the situation in the intestine, the absence of FXR leads to a strong increase in the formation of hepatocellular carcinoma (HCC), the most prominent 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.

Since the discovery of the first synthetic FXR agonist and its administration to rodents, it has become apparent that FXR is a key regulator of serum triglycerides. Over the past six years, a wealth of evidence has been published showing that activation of FXR by synthetic agonists leads to

- 16 043973 significant reduction in serum triglyceride levels, mainly in the form of a reduction in VLDL, but also in a reduction in total serum cholesterol.

But 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 results in decreased glucose production through hepatic gluconeogenesis but an accompanying 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 FGF19, 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.

Taken together, these pharmacological effects of 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.

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 absorption 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 fat-soluble vitamins from food, which can be overcome by increased levels of bile acids and phospholipids in the intestine.

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.

It has also been shown that, in combination with lipid-lowering effects, loss of functional FXR leads to increased atherosclerosis in ApoE knockout mice. Therefore, 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, which occurs 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.

This allows us to consider FXR also as a potential target for the treatment of proliferative diseases, especially metastatic forms of cancer that express sensitive

- 17 043973 to small molecules form FXR.

In another embodiment, the compounds and pharmaceutical compositions are used for the prevention and/or treatment of malignant hyperproliferative disorders, such as various forms of cancer, particularly certain forms of breast, liver, or colon cancer, which benefit from intervention by an FXR ligand.

Finally, FXR also appears to be involved in the control of antibacterial defense in the intestine, although the exact mechanism is not presented. However, based on the published data, it can be concluded that treatment with FXR agonists may have a beneficial effect in the treatment of inflammatory bowel diseases (IBD), in particular those forms in which the upper (ile) part of the intestine is affected (for example, Crohn's disease ileum) because it appears to be the site of FXR-mediated control of bacterial growth. In IBD, the desensitization of the adaptive immune response is somehow disrupted in the intestinal immune system. 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 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 reduction of non-commensal bacterial load help reduce the exposure of the intestinal immune system to bacterial antigens and may 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, and 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 subsequent activation of profibrotic pathways can also be prevented and/or treated by administration of the compounds or pharmaceutical composition of 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.

Dosage.

The effective dosage of the active ingredient used may vary depending on the particular compound used, 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 milligram to about 1000 milligrams per kilogram of body weight of the animal. 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 ranges from about 1 milligram to about 1000 milligrams, or from about 1 milligram to about 50 milligrams. For a 70 kg adult, the total daily dose is typically from about 7 milligrams to about 350 milligrams. This dosage regimen may be adjusted to ensure optimal therapeutic response. In some embodiments, the total daily dosage is from about 1 milligram to about 900 milligrams, from about 10 milligrams to about 800 milligrams, from about 20 milligrams to about 700 milligrams, from about 30 milligrams to about 600 milligrams, from about 40 milligrams to about 550 milligrams, or about 50 milligrams to about 400 milligrams.

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, the introduction

- 18 043973 or treatment with compounds can be continued for several days; for example, treatment is typically continued for at least 7, 14, or 28 days per 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 provided herein 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.

Combination therapies.

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 A3 adenosine receptor agonist, an adiponectin receptor agonist, an AKT protein kinase inhibitor, an AMP-activated protein kinase (AMPK) inhibitor, an amylin receptor agonist , angiotensin II AT-1 receptor antagonist, autotaxin inhibitors, bioactive lipid, calcitonin agonist, caspase inhibitor, caspase-3 stimulator, cathepsin inhibitor, caveolin 1 inhibitor, CCR2 chemokine antagonist, CCR3 chemokine antagonist, CCR5 chemokine antagonist, chloride channel stimulator, inhibitor CNR1, 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, growth factor ligands fibroblasts (FGF15, FGF19, FGF21), 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, nuclear factor modulator hepatocyte 4-alpha (HNF4A), hepatocyte growth factor modulator, HMG-CoA reductase inhibitor, IL-10 agonist, IL17 antagonist, ileal sodium-dependent bile acid cotransporter inhibitor, insulin sensitivity enhancer, integrin modulator, inhibitor of associated interleukin-1 receptor kinase 4 (IRAK4), Jak2 tyrosine kinase inhibitor, Kloto-beta stimulator, 5-lipoxygenase inhibitor, lipoprotein lipase inhibitor, liver X receptor, LPL gene stimulator, lysophosphatidate-1 receptor antagonist, lysyl oxidase homolog 2 inhibitor, matrix metalloproteinase (MMP) inhibitor , MEKK-5 protein kinase inhibitor, membrane copper amine oxidase (VAP-1) inhibitor, methionine aminopeptidase-2 inhibitor, methyl-CpG binding protein 2 modulator, microRNA-21 (miR-21) inhibitor, mitochondrial uncoupling protein, core protein stimulator myelin, NACHT, LRR and PYD domain-containing protein 3 (NLRP3) inhibitor, NAD-dependent deacetylase sirtuin stimulator, NADPH oxidase (NOX) inhibitor, nicotinic acid receptor 1 agonist, purinoceptor P2Y13 stimulator, PDE 3 inhibitor, PDE 4 inhibitor PDE 5, PDGF receptor beta modulator, phospholipase C inhibitor, PPAR alpha agonist, PPARdelta agonist, PPAR gamma agonist, PPAR gamma modulator, protease-activated receptor-2 antagonist, protein kinase modulator, Rho-associated protein kinase inhibitor, sodium glucose transporter inhibitor -2, SREBP transcription factor inhibitor, STAT-1 inhibitor, stearoyl-CoA desaturase inhibitor-1, cytokine signal suppressor stimulator-1, cytokine signal suppressor stimulator-3, transforming growth factor-β (TGF-β), transforming growth factor-activated factor-β kinase 1 (TAK1), thyroid hormone receptor beta agonist, TLR-4 antagonist, transglutaminase inhibitor, receptor tyrosine kinase 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 0-304;

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;

- 19 043973 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;

farnesoid X receptor (FXR) agonists such as AGN-242266, AKN-083, EDP-305, GNF5120, LJN-452, LMB-763, obeticholic acid, Px-102, Px-103, M790, M780, M450, M480, РХ20606, EYP-001 and INT-2228;

farnesoid X receptor (FXR)/G protein-coupled bile acid receptor 1 (TGR5) agonists such as INT-767;

fatty acid synthase inhibitors such as TVB-2640;

fibroblast growth factor 19 (rhFGF19)/cytochrome P450 (CYP) 7A1 inhibitors such as NGM282;

fibroblast growth factor 21 (FGF21) ligand such as BMS-986171, BMS-986036;

fibroblast growth factor 21 (FGF21)/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, INT777;

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 A-4250, 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;

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;

LPL gene stimulators such as alipogene 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 homologue 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 uncoupling proteins such as 2,4-dinitrophenol;

myelin basic protein stimulants such as olesoxime;

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

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

NACHT, LRR and PYD domain containing protein 3 (NLRP3) inhibitors such as KDDF-20140603 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;

- 20 043973 sodium glucose transporter 2 (SGLT2) inhibitors such as ipragliflozin, remogliflozin etabonate, ertugliflozin, dapagliflozin and sotagliflozin;

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

stearoyl-CoA desaturase-1 inhibitors such as aramchol;

thyroid hormone receptor beta 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 modulator 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, Alipogen tiparvovec, aramchol, ARI-3037MO, ASP-8232, bertilimumab, betaine anhydrous, BI-1467335, BMS-986036, BMS986171, VMT-053011, VOT-191, VTT-1023, SAT-2003, tsenikriviroka, CER-209, CF-102, CGS21680, CNX-014, CNX-023, CNX-024, CNX-025, cobiprostone, colesevelam, dapagliflozin, R-enantiomer of deuterated pioglitazone, 2,4-dinitrophenol, DRX-065, DS-102, DUR-928, EDP-305, elafibranor (GFT-505), emricasan, enalapril, ertugliflozin, evogliptin, F -351, GKT-831, GNF-5120, GRMD-02, hydrochlorothiazide, eicosapentaenoic acid ethyl ester, IMM-124-E, INT-767, IONISDGAT2Rx, ipragliflozin, irbesartan, propagermanium, IVA-337, JKB-121, KB- GE-001, KBP-042, KD-025, M790, M780, M450, metformin, sildenafil, LC-280126, linagliptin, liraglutide, LJN452, LMB-763, MBX-8025, MDV-4463, mercaptamine, MGL-3196, MGL-3745, MSDC-0602K, namacizumab, NC-101, ND-L02-S0201, NGM-282, NGM-313, NGM-386, NGM-395, nor-ursodeoxycholic acid, O-304, obeticholic acid, 25HC3S, Olezoxime, PAT-505, PAT-048, pegilodecakin, pioglitazone, pirfenidone, PRI-724, PX20606, Px-102, PX-L603, PX-E493, PXS-4728A, PZ-235, RDX-009, remogliflozin etabonate, RG -125 (AZD4076), saroglitazar, semaglutide, simtuzumab, solithromycin, sotagliflozin, statins (atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin), TCM-606F, TEV-45478, tipelukast (MN-001), TLY-012, TRX-318, TVB-2640, UD-009, ursodeoxycholic acid, VBY-376, VBY-825, VK-2809, vismodegib, volixibate potassium ethanolate hydrate (SHP-626), VVP-100X, WAV-301, WNT-974 and ZGN-839.

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 methods for its practice. However, those skilled in the art will appreciate that, in the context of the present invention, many changes can be made to the specific embodiments presented to achieve the same or similar result without departing from the spirit and scope of the present invention.

The compounds of the present invention can be prepared in accordance with the methods presented in the following schemes and examples using appropriate materials, and are further illustrated by the following specific examples. In addition, by using the methods described herein in combination with the normal skill of one skilled in the art, additional compounds of the present invention as claimed herein can be readily prepared. However, the compounds illustrated in the examples should not be construed as constituting the only example of the compounds covered by the present invention. The example further illustrates 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. The characteristics of the final product will generally clearly define the characteristics of the required starting materials through simple testing, taking into account the examples given in this document. The compounds of the present invention are generally isolated in the form of their pharmaceutically acceptable salts, such as the salts described above. In general, the compounds described herein are generally stable and liberated at room temperature and pressure.

The amino-free bases corresponding to the isolated salts can be prepared by neutralization with a suitable base, such as aqueous solutions of sodium hydrogen carbonate, sodium carbonate, sodium hydroxide and potassium hydroxide, and extraction of the liberated amino-free base into an organic solvent followed by evaporation. The amino-free base thus isolated can be further converted into another pharmaceutically acceptable salt by dissolution in an organic solvent followed by addition of an appropriate acid and subsequent evaporation, precipitation or crystallization. acids,

- 21 043973 carboxyl-free corresponding to the isolated salts can be prepared by neutralization with a suitable acid, such as aqueous solutions of hydrochloric acid, sodium hydrogen sulfate, sodium dihydrogen phosphate, and extraction of the liberated carboxyl-free acid into an organic solvent followed by evaporation. The carboxyl-free acid thus isolated can be further converted into another pharmaceutically acceptable salt by dissolution in an organic solvent followed by addition of an appropriate base and subsequent evaporation, precipitation or crystallization.

The preparation of the compounds of the present invention is illustrated below. Unless otherwise indicated on the diagrams, the variables have the same meaning as described above. The following examples are intended to illustrate specific embodiments of 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 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.

General synthesis scheme.

Compounds of formula (I) where Y is N can be prepared according to the following general synthetic scheme:

In the general synthesis scheme presented above, X represents a leaving group, PG represents a protecting group, and the remaining variables are as described herein. 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 to produce a compound of formula (C). The compound of formula (D) is prepared from the compound of formula (C) under appropriate deprotection conditions. A compound of formula (D) can be combined with a compound of formula (E) in the presence of a base to produce a compound of formula (I).

Suitable compounds of structures (A) and (B) can be prepared in accordance with the specific methods described in the following examples, or using methods known in the art. In some embodiments, X is halogen. In some embodiments, the PG is a BOC.

General synthesis 1

Step 1: 2-(3-hydroxyazetidin-1-yl)isonicotinonitrile (1a).

- 22 043973

Potassium carbonate (4.6 g, 33 mmol) was added to 2-chloro-4-pyridinecarbonitrile (2.0 g, 14.4 mmol) and 3-hydroxyazetidin hydrochloride (1.7 g, 16 mmol) in NMP (12 ml) at room temperature and the mixture was heated to 80°C for 2 hours in a sealed tube. The mixture was cooled to room temperature, treated with H ₂ O and extracted with EtOAc. The organic layers were washed with brine, dried with Na ₂ SO ₄ and concentrated. Purification by chromatography (on a 24 g ISCO silica gel column) using a 1:1 hexane/EtOAc - 100% EtOAc gradient provided 2-(3-hydroxyazetidin-1yl)isonicotinonitrile (1a).

Step 2: 2-(3-oxoazetidin-1-yl)isonicotinonitrile (1b).

N-methylmorpholine (1.9 g, 16 mmol) and then tetrapropylammonium perruthenate (190 mg, 0.5 mmol) were added to 2-(3-hydroxyazetidin-1-yl)isonicotinonitrile (1.9 g, 10.7 mmol ) in CH ₂ Cl ₂ (200 ml) using molecular sieves (1 g, activated powder, 4 A) at room temperature. After 20 minutes of vigorous stirring, the mixture was filtered through a pad of celite and concentrated. Purification by chromatography (on a 24 g ISCO silica gel column) using a 100% hexane to 1:3 hexane/EtOAc gradient provided 2-(3-oxoazetidin-1-yl)isonicotinonitrile (1b).

Preparation 1c: (4-bromo-3-chlorophenoxy)(tert-butyl)dimethylsilane (1c).

Imidazole (164 g, 2.41 mol) was added to a solution of 4-bromo-3-chlorophenol (250 g, 1.21 mol) and TBSCl (272 g, 1.81 mol) in DMF (2.0 L). The reaction mixture was then stirred at 30°C for 12 hours. The reaction mixture was immersed in H ₂ O (3 L) and extracted twice with EtOAc (2 L). The combined organic layers were washed with H ₂ O (1 L) and brine (1 L), dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. Purification by silica gel chromatography eluting with petroleum ether gave (4-bromo-3-chlorophenoxy)(tert-butyl)dimethylsilane (1c).

Step 3: 2-(3-(4-((tert-butyldimethylsilyl)oxy)-2-chlorophenyl)-3-hydroxyazetidin-1-yl)isonicotinonitrile (1d).

Isopropylmagnesium chloride-lithium chloride complex (1.3 ml, 1.7 mmol, 1.5 M solution in THF) was added dropwise to (4-bromo-3-chlorophenoxy)(tert-butyl)dimethylsilane (1c, 370 mg, 1 .15 mmol) in THF (0.9 ml) at room temperature. After 3 hours, the reaction mixture was cooled to 0°C and treated with one portion of 2-(3-oxoazetidin-1-yl)isonicotinonitrile (199 mg, 1.15 mmol) as a solid. After 1 hour, the reaction was quenched with H ₂ O and EtOAc. The organic layer was washed with brine, dried with Na ₂ SO ₄ and concentrated. Purification by chromatography (on a 4 g ISCO silica gel column) using a 100% hexane to 1:3 hexane/EtOAc gradient gave 2-(3-(4-((tert-butyldimethylsilyl)oxy)-2-chlorophenyl)-3- Hydroxyazetidin-1-yl)isonicotinonitrile (1d).

Step 4: 2-(3-(2-chloro-4-hydroxyphenyl)-3-hydroxyazetidin-1-yl)isonicotinonitrile (1e).

To a solution of 2-(3-(4-((tert-butyldimethylsilyl)oxy)-2-chlorophenyl)-3-hydroxyazetidin-1-yl)isonicotinonitrile (1d) (180 mg, 0.43 mmol) in 2-Me- THF (4 ml) was added to a 1M solution of TBAF in THF (0.6 ml, 0.59 mmol) at room temperature. After 30 min, the reaction was quenched with water, and the mixture was extracted with EtOAc. The organic phase was washed with brine (10 ml), dried with Na ₂ SO ₄ and concentrated to give 2-(3-(2-chloro-4-hydroxyphenyl)-3-hydroxyazetidin-1-yl)isonicotinonitrile (1e), which was used without additional cleaning.

General synthesis 2

Step 1: 2,6-dichloro-4-fluorobenzaldehyde oxime (2b).

Suspension of 2,6-dichloro-4-fluorobenzaldehyde (6.0 g, 31.2 mmol), NH2OH-HC1 (4.3 g, 62.4 mmol), Na ₂ CO ₃ (8.3 g, 78.7 mmol) in ethanol-water (50 ml, 5:1) was stirred at room temperature for 3 hours. The reaction mixture was condensed under vacuum and the residue was treated with water and then extracted with ethyl acetate. The ethyl acetate layer was washed with brine, dried over Na ₂ SO ₄ and concentrated to give 2,6-dichloro-4-fluorobenzaldehyde oxime (2b).

Step 2: 2,6-dichloro-4-fluoro-N-hydroxybenzimidoyl chloride (2c).

To a solution of 2,6-dichloro-4-fluorobenzaldehyde oxime (2b, 5.5 g, 26.7 mmol) in DMF (10 ml) add

- 23 043973 N-chlorosuccinimide (4.3 g, 32.0 mmol). The reaction mixture was stirred at RT for 1 hour. The reaction was quenched with H2O and the mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to give 2,6-dichloro-4fluoro-N-hydroxybenzimidole chloride (2c), which was used in the next step without further purification.

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

Et ₃ N (10.8 g, 107.2 mmol) was added to a solution of 3-cyclopropyl-3-oxopropionic acid ethyl ester (5.0 g, 32.0 mmol) in 30 ml THF. The reaction mixture was stirred at RT in for 30 minutes, and then the reaction mixture from the previous step (2,6-dichloro-4fluoro-N-hydroxybenzimidoyl chloride (2c)) was added dropwise. The resulting mixture was stirred for 2 hours at RT. The solvent was removed and the residue was partitioned between 100 ml water and 50 ml EtOAc. The organic layer was washed with brine, dried, filtered, concentrated and purified on a silica gel column (PE/EA=10/1) to obtain ethyl 5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole-4carboxylate (2d).

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

LiAlH4 ( 11.1 ml, 11.1 mmol, 1M solution in hexane) at 0°C. The reaction mixture was stirred for 30 minutes. 1.0 ml of water was added, and then 2.0 g of 10% NaOH solution, 3.0 ml of water were added. The mixture was filtered and concentrated. The crude product was purified by a silica gel column (PE/EA=2/1) to give (5-cyclopropyl-3-(2,6-dichloro-4fluorophenyl)isoxazol-4-yl)methanol (2e). LCMS (PER): m/z 302.0 (M+1)+. ¹ I-NMR (500 MHz, CDCl ₃ ): δ 7.22-7.20 (d, J=8.5 Hz, 2H), 4.42-4.41 (d, J=6.0 Hz, 2H), 2.19-2.16 (m, 1H), 1.41-1.39 (m, 1H), 1.29-1.26 (m, 2H), 1.16-1.13 ( m, 2H).

General synthesis 3

3c 3d

Step 1: methyl 5-fluoro-6-(3-hydroxyazetidin-1-yl)nicotinate (3a).

A mixture of azetidin-3-ol hydrochloride (2.8 g, 26 mmol), methyl 6-bromo-5-fluoronicotinate (5.0 g, 21 mmol) and potassium carbonate (7.4 g, 53 mmol) in DMF ( 100 ml) was heated at 65°C for 19 hours. The mixture was purified by flash chromatography (silica gel) to obtain the desired product. LCMS-ESI+ (m/z): [M+H]+ calculated for C ₁₀ H ₁₂ FN ₂ O ₃ : 227.1; set: 227.0.

Step 2: methyl 5-fluoro-6-(3-oxoazetidin-1-yl)nicotinate (3b).

A solution of methyl 5-fluoro-6-(3-hydroxyazetidin-1-yl)nicotinate (4.7 g, 21 mmol) in dichloromethane (270 ml) was treated with Dess-Martin periodinane (9.7 g, 23 mmol). After stirring for 6 hours at room temperature, an additional portion of Dess-Martin periodinane (1.5 g) was added and the mixture was left to stir overnight at room temperature. After stirring overnight, the mixture was treated with an aqueous sodium thiosulfate solution and a saturated aqueous sodium hydrogencarbonate solution. The aqueous phase was extracted three times with dichloromethane. The combined extracts were dried over anhydrous magnesium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified twice by flash chromatography (silica gel) to obtain the target material. LCMS-ESI+ (m/z): [M+H ₂ O+H]+ calculated for C ₁₀ H ₁₂ FN ₂ O ₄ : 243.1; set: 243.0.

Step 3: Methyl 6-(3-(4-((tert-butyldimethylsilyl)oxy)-2-chlorophenyl)-3-hydroxyazetidin-1-yl)5-fluoronicotinate (3 s).

A solution of (4-bromo-3-chlorophenoxy)(tert-butyl)dimethylsilane (4.5 g, 14 mmol) in 2-methyltetrahydrofuran (14 ml) was treated with isopropylmagnesium chloride/lithium chloride solution (Aldrich, 1.3 M, 11 ml , 15 mmol) by adding drops through a syringe. The resulting mixture was stirred for approximately one hour and then cooled in an ice water bath. Methyl 5-fluoro6-(3-oxoazetidin-1-yl)nicotinate (2.0 g, 8.9 mmol) was added in portions over 2 hours. The mixture was kept overnight at room temperature. The reaction was quenched with a 10% aqueous solution of citric acid. The aqueous phase was extracted three times with ethyl acetate. The combined organic layers were washed once with saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure to give the crude title product, which was used without further purification. LCMS-ESI+ (m/z): [M+H] ⁺ calculated for C ₂₂ H ₂₉ ClFN2O ₄ Si: 467.2; installed: 467.1.

Step 4: Methyl 6-(3-(2-chloro-4-hydroxyphenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinate (3d).

Crude methyl 6-(3-(4-((tert-butyldimethylsilyl)oxy)-2-chlorophenyl)-3-hydroxyazetidin-1yl)-5-fluoronicotinate (ca. 10 mmol) was dissolved in tetrahydrofuran (70 ml) and treated with the solution tetra-n-butylammonium fluoride (Aldrich, 1.0 M solution in THF, 18 ml, 18 mmol). The mixture was kept at room temperature until reaction was complete as determined by LC/MS and then purified by flash chromatography (silica gel) to yield intermediate 3d. LCMS-ESI+ (m/z): [M+H] ⁺ calculated for C ₁₆ H ₁₅ ClFN ₂ O ₄ : 353.1; set: 353.0.

Example 1: 5-((4-bromo-3-chlorophenoxy)methyl)-4-cyclopropyl-1-(2,6-dichlorophenyl)-1H-pyrazole.

Step 1: 2,4-difluorobenzaldehyde oxime

The title compound was prepared according to the method described in General Synthesis 2, Step 1, starting with 2,4-difluorobenzaldehyde (10 g, 70 mmol).

Step 2: 2,4-difluoro-N-hydroxybenzimidoyl chloride ^H R CI

F

The title compound was prepared according to the method described in General Synthesis 2, Step 2, starting with 2,4-difluorobenzaldehyde oxime (9 g, 57 mmol).

Step 3: Ethyl 5-cyclopropyl-3-(2,4-difluorophenyl)isoxazole-4-carboxylate

T\F ⁰

W

F

The title compound was prepared according to the method described in General Synthesis 2, Step 3, starting with 2,4-difluoro-N-hydroxybenzimidoyl chloride (11 g, 57 mmol).

Step 4: (5-cyclopropyl-3-(2,4-difluorophenyl)isoxazol-4-yl)methanol ^y-o

Λ__F O

F

The title compound was prepared according to the method described in General Synthesis 2, Step 4, starting with ethyl 5-cyclopropyl-3-(2,4-difluorophenyl)isoxazole-4-carboxylate (2.2 g, 8 mmol).

Step 5: 4-(chloromethyl)-5-cyclopropyl-3-(2,4-difluorophenyl)isoxazole

Thionyl _chloride ( ₁₆₄ µl, 2 .3 mmol) at 0°C. The mixture was heated to a temperature

- 25 043973 reverse condensation for 15 minutes and cooled to room temperature. The mixture was concentrated in vacuo. More CH2Cl2 (5 ml) was added and the mixture was concentrated again. This process was repeated a third time to remove excess thionyl chloride. The crude residue was used in the next step without further purification.

Step 6: 2-(3 -(2-chloro-4-((5-cyclopropyl-3 -(2,4-difluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3 hydroxyazetidin-1 -yl)isonicotinonitrile

4-(Chloromethyl)-5-cyclopropyl-3-(2,4-difluorophenyl)isoxazole (113 mg, 0.45 mmol), 2-(3-(2-chloro-4hydroxyphenyl)-3-hydroxyazetidin-1-yl )isonicotinonitrile (intermediate 1e) (149 mg, 0.5 mmol) and K ₂ CO ₃ (124 mg, 0.9 mmol) were combined in anhydrous DMF (2.3 ml) at room temperature. The mixture was heated to 65°C under a nitrogen atmosphere. After 2 h, the solution was cooled to room temperature, the reaction was quenched with H2O, and the mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. Purification by chromatography: ISCO (12 g silica gel column) using a gradient of 100% CH ₂ Cl ₂ 3:1 CH ₂ Cl ₂ /premixed solution 60:35:5 CH ₂ Cl ₂ :Et ₂ O:MeOH gave title compound.

Step 7: 2-(3-(2-chloro-4-((5-cyclopropyl-3-(2,4-difluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3hydroxyazetidin-1-yl)isonicotinic acid ( example 1)

A 10M aqueous sodium hydroxide solution (0.67 ml) was added to 2-(3-(2-chloro-4-((5-cyclopropyl-3(2,4-difluorophenyl)isoxazol-4-yl)methoxy)phenyl)- 3-hydroxyazetidin-1-yl)isonicotinonitrile (210 mg, 0.39 mmol) in ethanol (2 ml) and H2O (2 ml) at room temperature and the mixture was heated at 60°C for 90 min in a sealed tube. The mixture was cooled to room temperature and the pH was adjusted to approximately 5 using a 1M HCl solution, causing a precipitate to precipitate from the solution. The solution was filtered and the solid was washed with Et ₂ O and dried in vacuo to give 2-(3-(2-chloro-4-((5cyclopropyl-3-(2,4-difluorophenyl)isoxazol-4-yl)methoxy)phenyl) -3-hydroxyazetidin-1yl)isonicotinic acid (example 1). ¹ H-NMR (300 MHz, DMSO-d6) ¹ H-NMR (300 MHz, DMSO-d6) δ 13.41 (s, 1H), 8.19 (dd, J=5.2, 0.8 Hz , 1H), 7.59 (td, J=8.5, 6.5 Hz, 1H), 7.49 -7.34 (m, 2H), 7.28 - 7.15 (m, 1H), 7.05 - 6.96 (m, 2H), 6.88 - 6.74 (m, 2H), 6.20 (s, 1H), 5.00 (s, 2H), 4.47 (d, J=9.3 Hz, 2H), 4.18 (d, J=9.2 Hz, 2H), 2.40 (tt, J=8.3, 5.3 Hz, 1H), 1.20 - 1.00 (m, 4H). MS (ESI) (m/z) 554.0 (M+H).

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

- 26 043973

Preparation of intermediate A.

To a solution of (4-bromo-3-chlorophenoxy)(tert-butyl)dimethylsilane (1c, 60 g, 187 mmol) in THF (500 ml) n-BuLi (2.5 M, 75 ml) was added dropwise at -78 °C in N2 atmosphere. The reaction mixture was stirred at -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. The reaction mixture was then stirred at 20°C for 3 hours. The reaction mixture was immersed in 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-((tert-butyldimethylsilyl)oxy)-2chlorophenyl)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 -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)-3-hydroxyazetidine-1-carboxylate, which was used without further purification.

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

(5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methanol solution (2e); 845 mg, 2.80 mmol) in DCM (28.0 ml) was cooled to 0°C. Thionyl chloride (1.02 mL, 14.0 mmol) was added and the solution was heated at 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-4-yl)methoxy)phenyl)-3-hydroxyazetidin- 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 60°C for 8 hours. The reaction mixture was concentrated, diluted with water and extracted with EtOAc (3x). 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-4-fluorophenyl) isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidine-1-carboxylate. LCMS-ESI+ (m/z): [(M+H)BOC] ⁺ calculated: 483.04; installed: 483.04.

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

- 27 043973 nil)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 ambient 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 the hydrochloride salt, which was used without further purification. LCMS-ESI+ (m/z): [M+H] ⁺ calculated: 483.04; set: 483.03.

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

Mixture of methyl 2-bromopyridine-4-carboxylate (0.466 g, 2.16 mmol), 3-(2-chloro-4-((5-cyclopropyl-3(2,6-dichloro-4-fluorophenyl)isoxazole-4 -yl)methoxy)phenyl)azetidin-3-ol as hydrochloride salt (1.02 g, 1.96 mmol), cesium carbonate (2.56 g, 7.85 mmol), (±)-BINAP (0.244 g , 0.392 mmol), palladium acetate trimer (88.0 mg, 0.131 mmol) and 1,4-dioxane (40.0 ml) were heated at 85°C for 18 hours. The reaction mixture was cooled to room temperature, filtered through celite and purified by silica gel chromatography (acetone/hexane) to give methyl 2-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro4-fluorophenyl)isoxazol-4-yl)methoxy )phenyl)-3-hydroxyazetidin-1-yl)isonicotinate. LCMS-ESI+ (m/z): [M+H] ⁺ calculated: 618.08; set: 618.20.

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

To a solution of 2-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidine-1- yl)isonicotinate (617 mg, 0.997 mmol) in THF/water (1:1, 10 ml) was added lithium hydroxide monohydrate (83.6 mg, 1.99 mmol). The solution was stirred for 90 minutes, concentrated to remove THF and diluted with water. While stirring, acetic acid (0.23 mL, 3.99 mmol) was added causing solids to precipitate. The solids were filtered, washed with water, IPA and ether and dried under vacuum to give 2-(3-(2-chloro4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl )methoxy)phenyl)-3-hydroxyazetidin-1yl)isonicotinic acid (example 2). LCMS-ESI+ (m/z): [M+H] ⁺ calculated: 604.06; set: 604.15. ¹ H-NMR (300 MHz, DMSO-d ₆ ) δ 13.47 (br s, 1H), 8.18 (dd, J=5.3, 0.8 Hz, 1H), 7.69 (d , J=8.5 Hz, 2H), 7.37 (d, J=8.7 Hz, 1H), 7.02 (dd, J=5.3, 1.4 Hz, 1h), 6.93 (d, J=2.6 Hz, 1H), 6.86 (width s, 1h), 6.75 (dd, J=8.6, 2.6 Hz, 1H), 6.20 (s, 1H), 4.91 (s, 2H), 4.49 (d, J=9.3 Hz, 2H), 4.19 (d, J=9.3 Hz, 2H), 2.46 - 2, 37 (m, 1H), 1.23 - 1.04 (m, 4H).

Example 3: 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

Cl

Intermediate A

Stage 1

F F

Example 3

- 28 043973

Steps 1-4 were the same as described in the synthesis of Example 2.

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 in the form of hydrochloride salt (495 mg, 0.952 mmol) and potassium carbonate (1.05 g, 7.61 mmol) in DMF (30.0 ml) was heated at 60 ° C in for 1 hour. The reaction mixture was concentrated, diluted with water and extracted with EtOAc (3x). 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-4-yl)methoxy)phenyl)-3hydroxyazetidin-1-yl)-5-fluoronicotinate. LCMS-ESI+ (t/z): [M+H] ⁺ calculated: 636.07; set: 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 (example 3).

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 1N. 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 (example 3). LCMS-ESI+ (t/z): [M+H] ⁺ calculated: 622.05; set: 622.12. ¹ H-NMR (400 MHz, DMSO-d ₆ ) 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).

Intermediate 4: (3-(2,6-dichloro-4-fluorophenyl)-5-methylisoxazol-4-yl)methanol

According to general synthesis 2, starting with 2,6-dichloro-4-fluorobenzaldehyde in step 1, and using acetoacetic acid ethyl ester in step 3, (3-(2,6-dichloro-4fluorophenyl)-5-methylisoxazol- 4-yl)methanol (intermediate 4). LCMS-ESI+ (m/z): [M+H] ⁺ calculated: 276.00; set: 276.05.

Example 4: Preparation of 6-(3-(2-chloro-4-((3-(2,6-dichloro-4-fluorophenyl)-5-methylisoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidine-1 -yl)-5-fluoronicotinic acid

Following the general method described for Example 3, using intermediate 4, 6-(3-(2-chloro-4-((3-(2,6-dichloro-4-fluorophenyl)-5-methylisoxazole-4 -yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinic acid. LCMS-ESI+ (m/z): [M+H] ⁺ calculated: 596.04; set: 596.12. ¹ H-NMR (400 MHz, DMSO-de) δ 12.82 (br s, 1H), 8.44 (t, J=1.6 Hz, 1H), 7.74 - 7.66 (m, 3H), 7.39 (d, J=8.7 Hz, 1H), 6.90 (d, J=2.6 Hz, 1H), 6.75 (dd, J=8.7, 2.6 Hz, 1H), 6.26 (s, 1H), 4.87 (s, 2H), 4.69 (d, J=9.8 Hz, 2H), 4.34 (d, J=9.8 Hz, 2H), 2.57 (s, 3H).

Example 5: 6-(3-(2-chloro-4-((4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazol-5-yl)methoxy)phenyl)-3- Hydroxyazetidin-1-yl)-5-fluoronicotinic acid

- 29 043973

Step 1: (2,6-dichloro-4-fluorophenyl)hydrazine hydrochloride.

To a -5°C cooled solution (internal temperature, ice/acetone bath) of 2,6-dichloro-4fluoroaniline (3.0 g, 17 mmol) in 37% hydrochloric acid (30 ml) and trifluoroacetic acid (20 ml) an aqueous solution of sodium nitrite (1.4 g, 20 mmol, 6 ml water) was added dropwise. The reaction mixture was stirred for 90 minutes and then a solution of tin chloride dihydrate (5.6 g, 25 mmol) in 37% hydrochloric acid (16 ml) was added over 15 minutes, maintaining the internal temperature <2°C.

The mixture was stirred overnight at room temperature. The mixture was filtered and the collected solid was washed with isopropyl alcohol and dried using a household vacuum pump to obtain the title compound. LCMS-ESI+ (m/z): [M+H] ⁺ calculated for C ₆ H ₆ Cl ₂ FN ₂ : 195.0; established: 194.9.

Step 2: ethyl 4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazole-5-carboxylate.

N,N-Dimethylformamide dimethyl acetal (2.7 ml, 20 mmol) was added to ethyl 3-cyclopropyl-2oxopropanoate (Synnovator, 1.6 g, 10 mmol) and the mixture was stirred overnight at room temperature. The mixture was then concentrated to dryness under reduced pressure. An excess amount of ethanol (40 ml), (2,6-dichloro-4-fluorophenyl)hydrazine hydrochloride (2.6 g, 11 mmol) and 37% hydrochloric acid (150 μl) was added to the residue. The reaction mixture was stirred at room temperature for four hours and then for 2 days at reflux temperature. The cooled mixture was purified by flash chromatography (silica gel) to give the title compound. LCMS-ESI+ (m/z): [M+H] ⁺ calculated for C ₁₅ H ₁₄ Cl ₂ FN ₂ O ₂ : 343.0; set: 343.1.

Step 3: (4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazol-5-yl)methanol.

A solution of ethyl 4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-III-pyrazole-5-carboxylate (1.5 g, 4.4 mmol) in tetrahydrofuran (50 ml) was cooled to a temperature of - 12 to -10°C. A solution of lithium aluminum hydride (Aldrich, 2M solution in tetrahydrofuran, 2.6 ml, 5.2 mmol) was added dropwise. The mixture was stirred for 35 minutes. The reaction was quenched (Fieser procedure) and the mixture was purified by flash chromatography (silica gel) to give the title compound. LCMS-ESI+ (m/z): [M+H] ⁺ calculated for C ₁₃ H ₁₂ Cl ₂ FN ₂ O: 301.0; set: 301.1.

Step 4: 5-(chloromethyl)-4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazole.

Thionyl chloride (110 μl, 1.5 mmol) was added to a solution of (4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazol-5-yl)methanol (0.15 g, 0.51 mmol) in dichloromethane (2.5 ml). The mixture was heated at 60°C for 40 min and then concentrated under reduced pressure to obtain crude cele

- 30 043973 of the product, which was used without additional purification. LCMS-ESI+ (m/z): [M+H] ⁺ calculated for C ₁₃ H ₁₁ Cl ₃ FN2: 319.0; established: 319.1.

Step 5: methyl-6-(3-(2-chloro-4-((4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazol-5-yl)methoxy)phenyl)- 3-hydroxyazetidin-1-yl)-5-fluoronicotinate.

A solution of 4-(chloromethyl)-5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole (0.16 g, 0.51 mmol) in DMF (3 ml) was treated with methyl 6-(3- (2-chloro-4-hydroxyphenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinate (0.20 g, 0.56 mmol), sodium iodide (0.13 g, 0.86 mmol) and potassium carbonate (0.14 g, 1.0 mmol). The mixture was heated at 65°C overnight and then purified by flash chromatography (silica gel) to obtain the target material. LCMS-ESI+ (m/z): [M+H] ⁺ calculated for C ₂₉ H ₂₄ Cl ₃ F ₂ N ₄ O ₄ : 635.1; installed: 635.2.

Step 6: 6-(3-(2-chloro-4-((4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazol-5-yl)methoxy)phenyl)-3- Hydroxyazetidin-1-yl)-5-fluoronicotinic acid (example 5).

A mixture of methyl 6-(3-(2-chloro-4-((4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazol-5-yl)methoxy)phenyl)-3- Hydroxyazetidin-1-yl)-5-fluoronicotinate (0.35 g, 0.39 mmol) and lithium hydroxide monohydrate (49 mg, 1.2 mmol) were dissolved in a 1:1 aqueous solution of tetrahydrofuran (6 ml) and the mixture was stirred at room temperature. After completion of the reaction, the mixture was acidified with glacial acetic acid and concentrated. The residue was purified by flash chromatography (silica gel) to give 6-(3(2-chloro-4-((4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazol-5-yl) methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinic acid (example 5). LCMS-ESI+ (m/z): [M+H] ⁺ calculated for C28H22CI3F2N4O4: 621.1; set: 621.2. 1H-NMR (400 MHz, DMSO^) δ 12.85 (s, 1H), 8.44 (t, J=1.6 Hz, 1H), 7.76 (d, J=8.3 Hz, 2H ), 7.70 (dd, J=12.7, 1.7 Hz, 1H), 7.49 (s, 1H), 7.40 (d, J=8.7 Hz, 1H), 7.00 (d, J=2.6 Hz, 1H), 6.80 (dd, J=8.7, 2.6 Hz, 1H), 6.28 (s, 1H), 5.01 (s, 2H) , 4.69 (d, J=9.8 Hz, 2H), 4.34 (d, J=9.6 Hz, 2H), 1.89 (tt, J=8.4, 5.1 Hz, 1H), 0.93 (m, 2H), 0.65 (m, 2H).

Example 6: 5-((1S,3S)-3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl) -3-hydroxycyclobutyl)-6-methoxynicotinic acid

Preparation of intermediate compound 5.

Step 1: (5-bromo-6-methoxypyridin-3-yl)methanol

To a solution of methyl 5-bromo-6-methoxynicotinate (52.8 g, 215.0 mmol) in THF (500 ml) was added DIBAL-H (1.0 M solution in toluene) (344 ml, 344 mmol) at -20 °C. The mixture was then stirred at RT for 2 hours. The reaction was quenched by us. NH ₄ Cl solution and the mixture was diluted with ethyl acetate. The organic portion was washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure and purified by silica gel flash chromatography (PE/EtOAc=4/1) to give the title compound.

Step 2: 3-bromo-5-(((tert-butyldimethylsilyl)oxy)methyl)-2-methoxypyridine

To a solution of (5-bromo-6-methoxypyridin-3-yl)methanol (42.2 g, 194 mmol) and tert-butyldimethyl

- 31 043973 silyl chloride (35.0 g, 232 mmol) in CH ₂ Cl ₂ (500 ml) was added imidazole (19.8 g, 291 mmol). The mixture was stirred at RT for 8 hours. The reaction was quenched with water and the mixture was diluted with ethyl acetate. The organic portion was washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure and purified by silica gel flash chromatography (PE/EAAc=10/1) to give the title compound.

Step 3: 3-(benzyloxy)-1-(5-(((tert-butyldimethylsilyl)oxy)methyl)-2-methoxypyridin-3-yl)cyclobutan-1-ol

3-Bromo-5-(((tert-butyldimethylsilyl)oxy)methyl)-2-methoxypyridine (61.2 g, 184 mmol) was dissolved in absolute THF (500 ml) in an argon atmosphere, 1.6 M nbutyllithium solution was added dropwise (138 ml, 221 mmol) in THF at -78°C. The mixture was stirred for 30 minutes at the same temperature. A solution of 3-(benzyloxy)cyclobutan-1-one (35.7 g, 202 mmol) in THF (100 ml) was then added at -78° C. and the mixture was then stirred at the same temperature for 30 minutes. Then, a saturated aqueous ammonium chloride solution was added and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated sodium chloride solution, dried over magnesium sulfate and filtered. After removal of the solvent by rotary evaporation, the residue was purified by flash chromatography on silica gel (PE/EAAc=2/1) to give the title compound.

Step 4: 3-(benzyloxy)-1-(5-(hydroxymethyl)-2-methoxypyridin-3-yl)cyclobutan-1-ol

To a solution of 3-(benzyloxy)-1-(5-(((tert-butyldimethylsilyl)oxy)methyl)-2-methoxypyridin-3yl)cyclobutan-1-ol (31.6 g, 73.6 mmol) in THF ( 300 ml) TBAF (88 ml, 1 mol/l) was added. The mixture was stirred at RT for 6 hours and then immersed in water and extracted with ethyl acetate. The organic phase was washed with water and saturated sodium chloride solution, dried over magnesium sulfate and filtered. The organic phase was concentrated to give the title compound.

Step 5: 5-(3-(benzyloxy)-1-hydroxycyclobutyl)-6-methoxynicotinic acid

To a solution of 3-(benzyloxy)-1-(5-(hydroxymethyl)-2-methoxypyridin-3-yl)cyclobutan-1-ol (23.2 g, 73.6 mmol) in MeCN (300 ml) and H2O ( 100 ml), iodobenzene diacetate (64.4 g, 200 mmol) and TEMPO (7.86 g, 50 mmol) were added and the solution was stirred at room temperature for 2 hours. The reaction was quenched. sodium bicarbonate solution and the mixture was diluted with ethyl acetate. The organic part was washed with saline, dried over anhydrous sodium sulfate, filtered; the organic phase was concentrated to give the title compound.

Step 6: Methyl 5-(3-(benzyloxy)-1-hydroxycyclobutyl)-6-methoxynicotinate

To a solution of 5-(3-(benzyloxy)-1-hydroxycyclobutyl)-6-methoxynicotinic acid (17.5 g, crude) in THF/MeOH (200/50 ml) was added TMSN ₂ CH ₃ (50 ml, 20 mol /l) at 0°C. The mixture was stirred at room temperature for 3 hours and then immersed in water and extracted with ethyl acetate. The organic phase was washed with water and saturated sodium chloride solution, dried over magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure and purified by flash silica gel chromatography (PE/EA=10:1) to give the title compound.

Step 7: Methyl 5-(3-(benzyloxy)-1-fluorocyclobutyl)-6-methoxynicotinate

- 32 043973

To a cooled solution of methyl 5-(3-(benzyloxy)-1-hydroxycyclobutyl)-6-methoxynicotinate (15.2 g, 44.3 mmol) in DCM (200 ml), DAST (8.0 ml) was added dropwise through a syringe ) at -78°C. After stirring for 5 min at -78°C, the reaction mixture was allowed to warm to -20°C and stirred for 75 min before the reaction was quenched with H ₂ O (100 ml), the mixture was diluted with EtOAc and the phases were separated. The organic phase was washed for us. water NaHCO ₃ solution and saline solution, and then dried over MgSO ₄ , filtered and concentrated. The crude product was purified by chromatography (PE:EtOAc=4:1) to give the title compound.

Step 8: Methyl 5-(3-hydroxycyclobutyl)-6-methoxynicotinate

The catalyst Pd black ( 3.0 g). The reaction mixture was stirred vigorously under N2 atmosphere. After about 1.5 hours, more Pd black catalyst (1.5 g) was added and the reaction mixture was stirred overnight. The reaction mixture was filtered and concentrated.

The residue was dissolved in EtOAc and washed with us. Na2CO3 solution. The organic phase was dried over MgSO ₄ , filtered and concentrated to an oily residue. The residue was purified by chromatography (MeOH:CH ₂ Cl ₂ =1:20) to obtain the title compound.

Step 9: Methyl 6-methoxy-5-(3-oxocyclobutyl)nicotinate (intermediate 5)

Oh Oh

Odbenzene diacetate (16.1 g, 50 mmol) was added to a solution of methyl 5-(3-hydroxycyclobutyl)-6-methoxynicotinate (4.0 g, 16.9 mmol) in MeCN (100 ml) and H2O (30 ml) and TEMPO (2.92 g, 18.6 mmol) and the solution was stirred at room temperature for 2 hours. The reaction was quenched. Na ₂ CO ₃ solution, and then the mixture was diluted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous sodium sulfate and filtered, the organic phase was concentrated and purified by chromatography (PE:EA=5:1) to obtain intermediate 5.

Step 10: 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 (prepared as described in Example 2, Step 2; 0.42 g, 1.3 mmol) in N ,N-dimethylformamide (DMF, 6 ml) was treated with 4-bromo-3-chlorophenol (0.27 g, 1.3 mmol), sodium iodide (0.34 g, 2.2 mmol) and potassium carbonate (0.37 g, 2.6 mmol). The mixture was heated at 60°C for 35 minutes and then cooled and purified by flash chromatography (silica gel) to obtain the target material. LCMS-ESI+ (m/z): [M+H] ⁺ calculated for C ₁₉ H ₁₃ BrCl ₃ FNO ₂ : 491.9; installed: 492.0.

Step 11: Methyl 5-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl )-6-methoxynicotinate.

Under argon, a solution of 4-((4-bromo-3-chlorophenoxy)methyl)-5-cyclopropyl-3-(2,6-dichloro-4fluorophenyl)isoxazole (0.83 g, 1.7 mmol) in 2-methyltetrahydrofuran (2 mL) was treated with isopropylmagnesium chloride/lithium chloride solution (Aldrich, 1.3 M solution in tetrahydrofuran, 1.3 mL, 1.7 mmol) by dropwise addition through a syringe. After four hours, an additional volume of isopropylmagnesium chloride/lithium chloride solution (1.3 ml) was added. In a separate vessel under argon, a solution of methyl 6-methoxy-5-(3-oxocyclobutyl)nicotinate (intermediate 5, 0.21 g, 0.90 mmol) in tetrahydrofuran (5 ml) was treated with a solution of lanthanum(III) chloride/2 parts lithium chloride (Aldrich, 0.6 M solution in tetrahydrofuran, 1.5 ml, 0.9 mmol). The mixture was stirred for one hour at room temperature and then cooled in a -8° C. liquid ice/acetone bath. The above Grignard solution was added dropwise through a syringe to the ketone solution. The reaction mixture was stirred overnight under argon. The reaction was quenched with a saturated aqueous solution of ammonium chloride. The aqueous phase was extracted three times with ethyl acetate. The combined organic layers were washed once with saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The crude residue was purified by flash chromatography (silica gel) to give the title compound. LCMS-ESI+ (m/z): [M+H] ⁺ calculated for C ₃₁ H ₂₇ Cl ₃ FN ₂ O ₆ : 647.1; established: 647.1.

Step 12: 5-((1S,3S)-3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl) -3-hydroxycyclobutyl)-6-methoxynicotinic acid (example 6).

A mixture of methyl 5-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)- 6-methoxynicotinate (0.26 g, 0.40 mmol) and hydroxide monohydrate

- 33 043973 lithium (33 mg, 0.79 mmol) was dissolved in a 1:1 aqueous solution of tetrahydrofuran (10 ml) and the mixture was stirred overnight at room temperature. Volatile compounds were preferentially removed under reduced pressure. The aqueous mixture was diluted with water and a 10% aqueous solution of hydrochloric acid was added dropwise. The resulting mixture was extracted with ethyl acetate three times. The combined organic layers were washed with saturated aqueous sodium chloride (with a small amount of hydrochloric acid added). The combined organic layers were dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified first by flash chromatography (silica gel) and then by preparative HPLC (acetonitrile/water, TFA). The pooled fractions collected by HPLC were neutralized with saturated aqueous sodium hydrogen carbonate, saturated sodium chloride, and extracted three times with ethyl acetate. The combined organic layers were dried over anhydrous magnesium sulfate, filtered and concentrated. The residue was dissolved in ethyl acetate, treated with anhydrous magnesium sulfate, filtered and concentrated. The residue was again dissolved in ethyl acetate and filtered through a pad of celite, diatomaceous earth. The filtrate was concentrated to give 5-((1S,3S)-3-(2-chloro-4-((5-cyclopropyl-3(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl) -3-hydroxycyclobutyl)-6-methoxynicotinic acid (example 6). LCMS-ESI+ (m/z): [M+H] ⁺ calculated for C30H25Cl3FN2O6: 633.1; established: 633.1. ¹ H-NMR (400 MHz, DMSO-d6) δ 13.00 (br s, 1H), 8.58 (d, J=2.2 Hz, 1H), 8.13 (dd, J=2, 3, 0.8 Hz, 1H), 7.72 (d, J=8.5 Hz, 2H), 7.51 (d, J=8.7 Hz, 1H), 6.94 (d, J= 2.6 Hz, 1H), 6.79 (dd, J=8.7, 2.6 Hz, 1H), 4.94 (s, 2H), 3.90 (s, 3H), 3.15 - 3.03 (m, 2H), 2.91 (p, J=8.8 Hz, 1H), 2.49 - 2.41 (m, 1H), 2.41 - 2.30 (m, 2H) , 1.21 1.09 (m, 4H).

Example 7: 2-(6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3 - hydroxyazetidin-1-yl)-5-fluoronicotinamido)ethane-1-sulfonic acid\-o

FOHJN

I/pi g p

ABOUT

ΗΝ ^F o ^z he

Solution of 6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)3-hydroxyazetidin-1-yl) -5-fluoronicotinic acid (example 3, 0.11 g, 0.18 mmol) in DMF (4 ml) was treated with HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4, 5-b]pyridinium-3-oxide hexafluorophosphate, 0.10 g, 0.27 mmol), followed by taurine (34 mg, 0.27 mmol) and N,N-diisopropylethylamine (90 μl, 0.54 mmol). The mixture was stirred overnight at room temperature and then purified by preparative HPLC (water/acetonitrile/TFA). The combined fractions were treated with ammonium hydroxide solution and concentrated to give 2-(6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro4-fluorophenyl)isoxazol-4-yl)methoxy) phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinamido)ethane-1 sulfonic acid (example 7) in the form of ammonium salt. LCMS-ESI+ (m/z): [M+H] ⁺ calculated for C30H26Cl3F2N4O7S: 729.1; set: 729.2. ¹ H-NMR (400 MHz, DMSO-d6) δ 8.34 (m, 2H), 7.73 - 7.61 (m, 3H), 7.37 (d, J=8.6 Hz, 1H) , 7.29 - 6.95 (m, 4H), 6.92 (d, J=2.5 Hz, 1H), 6.75 (dd, J=8.6, 2.5 Hz, 1H), 6.22 (s, 1H), 4.90 (s, 2H), 4.63 (d, J=9.6 Hz, 2H), 4.29 (d, J=9.6 Hz, 2H), 3.46 (q, J=6.5 Hz, 2H), 2.63 (t, J=7.3 Hz, 2H), 2.45 - 2.38 (m, 1H), 1.16 (dt , J=8.5, 3.1 Hz, 2H), 1.10 (dt, J=5.4, 2.9 Hz, 2H).

Example 8: (6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin- 1-yl)-5-fluoronicotinoyl)glycine

Step 1: methyl-(6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3 - hydroxyazetidin-1-yl)-5-fluoronicotinoyl)glycinate.

Solution of 6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl )-5-fluoronicotinic acid (example 3, 0.12 g, 0.19 mmol) in DMF (4 ml) was treated with HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4 ,5-b]pyridinium-3-oxide hec

- 34 043973 saffluorophosphate, 0.11 g, 0.29 mmol), followed by glycine methyl ester hydrochloride (36 mg, 0.29 mmol) and N,N-diisopropylethylamine (100 μl, 0.58 mmol). The mixture was stirred overnight at room temperature, and then the reaction was quenched with saturated aqueous sodium hydrogen carbonate. The aqueous phase was extracted twice with ethyl acetate. The combined extracts were washed once with a 1:1 mixture of saturated aqueous sodium chloride/saturated aqueous sodium hydrogen carbonate, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure to obtain the expected product, which was used without further purification. LCMS-ESI+ (m/z): [M+H] ⁺ calculated for C ₃₁ H ₂₆ Cl ₃ F ₂ N ₄ O ₆ : 693.1; installed: 693.2.

Step 2: (6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin- 1-yl)-5-fluoronicotinoyl)glycine (example 8).

A mixture of crude methyl (6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol4-yl)methoxy)phenyl)-3-hydroxyazetidine-1 -yl)-5-fluoronicotinoyl)glycinate (about 0.19 mmol) and lithium hydroxide monohydrate (38 mg, 0.91 mmol) in aqueous tetrahydrofuran (2:1, 3 ml) were stirred at room temperature for 3.5 h. The volatile solvent was removed under reduced pressure. The residue was diluted with water and acidified to pH 1 with a 10% aqueous solution of hydrochloric acid. The acidic aqueous mixture was extracted with ethyl acetate three times. The combined organic extracts were washed once with saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, filtered and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel) to give (6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4fluorophenyl)isoxazol-4-yl)methoxy)phenyl )-3-hydroxyazetidin-1-yl)-5-fluoronicotinoyl)glycine (example 8). LCMS-ESI+ (m/z): [M+H] ⁺ calculated for C30H24Cl3F2N4O6: 679.1; established: 679.3. ¹ H-NMR (400 MHz, DMSO-d6) δ 12.68 (s, 1H), 8.68 (t, J=5.8 Hz, 1H), 8.44 (t, J=1.7 Hz , 1H), 7.80 (dd, J=13.2, 1.8 Hz, 1H), 7.71 (d, J=8.5 Hz, 2H), 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), 6.26 (s, 1H) , 4.93 (s, 2H), 4.66 (d, J=9.5 Hz, 2H), 4.32 (d, J=9.3 Hz, 2H), 3.87 (d, J= 5.8 Hz, 2H), 2.48 - 2.42 (partially hidden by DMSO, m, 1H), 1.16 (m, 4H).

Example 9: FRET activity assay.

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: The human FXR-alpha LBD 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-LBD: a pre-cultured overnight culture of the transformed E. coli strain was diluted 1:20 in LB medium with ampicillin and grown at 30°C to an optical density 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, RT). 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. Then the cells were treated with ultrasound and cell debris was removed by centrifugation (22000xg, 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 continued to rotate slowly for 1 h at 4°C. Glutathione-4B-Sepharose beads were pelleted by centrifugation (2000xg, 15 s, 4°C) and washed twice in wash buffer (25 mM Tris, 50 mM KCl, 4 mM MgCl ₂ 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 pelleted 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 , and 1 mM dithiothreitol and 10% v/v. glycerin. 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 (LBD) of bacterially expressed FXR and a synthetic 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 target of interest.

- 35 043973 to the binding partner. For ease of processing and reduced background from compounds, LANCE fluorescence technology uses universal fluorophore tags and time-resolved detection. Assays were performed in a final volume of 25 μl in a 384-well plate in Tris-based buffer (20 mM Tris-HCl pH 7.5; 60 mM KCl, 5 mM MgCl ₂ ; 35 ng/μl BSA) containing 20-60 ng/μl well of recombinantly expressed FXR-LBD fused to GST, 200-600 nM N-terminal biotinylated peptide representing amino acids 676-700 of SRC1, 200 ng/well streptavidinxlAPC 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 RT in black 384-well FIA 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. Basal levels of FXR-peptide complex formation were 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, the EC ₅₀ values for the compounds given in the examples and presented below in Table 2 (FRET EC ₅₀ ) were determined.

Example 10. Analysis of the 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.

The DNA portion encoding the FXR ligand-binding domain was cloned into the pCMV-BD vector (Stratagene) as a hybrid with the yeast GAL4 DNA-binding domain 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 MEM with L-glutamine and Earle's CCP 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 37°C in a 5% CO ₂ atmosphere. Media and supplements were obtained from Invitrogen. For analysis, 5x105 cells per well were seeded in 96-well plates in 100 μl per well of MEM without phenol red and L-glutamine and with Earl's SSR supplemented with FBS (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 37°C in a 5% CO ₂ 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. 408277), including the three plasmids described above. MEM with the same composition as for cell seeding was added 2–4 h after adding the transfection mixture. Then, stock solutions of the compounds, previously diluted in MEM, 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.

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. 2 (M1H EC ₅₀ ).

table 2

Example FRET EU ₅ o (nM) M1H EC ₅ o (nM) 1 263 3000 2 25 831 3 7.4 3.8 4 35 176 5 18 8.6 6 49 353 7 6.9 1696 8 8.1 1264

- 36 043973

Example 11. Identification of metabolites in human liver microsomes.

The metabolic stability study of Example 3 and Comparative Example 1 in human liver microsomes was carried out according to the following procedure. Human liver microsomes (35 μl with a protein concentration of 20 mg/ml), 350 μl of 100 mM potassium phosphate buffer (pH 7.4), 245 μl of deionized water and 0.7 μl of compound stock solution (5 mM) were combined into 1. 5 ml microcentrifuge tube.

The tube was sealed and gently mixed on a vertex for 10 s, and then placed in an Eppendorf ThermoMixer C and preheated to 37°C with shaking at 1100 rpm for 5 min.

NADP solution (70 μl; 10 mM solution in water) was added with shaking, the mixture was aspirated several times with a pipette, and 200 μl was transferred to another 1.5 ml microcentrifuge tube placed on ice and containing 200 μl of cold acetonitrile. This aliquot was mixed on a vertex at high speed for 10 s and then placed in ice. After 30 and 60 min, an additional 200 μl aliquot was removed and transferred to another 1.5 ml microcentrifuge tube placed on ice and containing 200 μl of cold acetonitrile. The tubes were mixed on a vertex at high speed for 10 s and then placed in ice.

Cooled aliquots were centrifuged at 14,300 rpm in a microcentrifuge for 10 min at 10°C, and then the supernatant was transferred to a 96-well deep well plate (1 ml) and sealed with a silicone mat. The sample was transferred to the cooling compartment of the autosampler (temperature set at 10°C) and 20 μL was injected into the Thermo Elite Orbitrap mass spectrometer. 20 µl samples were analyzed by UHPLC-MS for identification and quantification of metabolites (UHPLC with Agilent 1290 G4220 binary pump and Agilent G1316 TCC column oven; Waters Acquity UPLC BEH C18 column (pore size 130 A, particle size 1.7 µm, 2. 1x50 mm), maintained at 40°C; Agilent 1290 G4212 DAD diode array with a wavelength range from 190 to 400 nm; Thermo Electron Orbitrap Elite mass spectrometer in FTMS mode with registration of positive ions).

Final concentration of microsomal protein: 1 mg/ml.

Final concentration of NADP: 1 mM.

Final substrate concentration: 5 µM.

Control time points: 0, 30, 60 min.

Incubation volume at each control time point: 200 µl.

Comparative Example 1, a direct comparator of Example 3 lacking the 4fluorophenyl substituent present in the compounds described herein, was found to be metabolized to the diol compound (M1) under the conditions described above (Scheme 1). The inclusion of a 4-fluoro substituent inhibits the formation of the M1 metabolite under the same conditions.

Scheme 1

He

Comparison example 1 ^M1 m/z 604.0610 m/z 604.0610

UV content 23%

Example 3 Ml m/z 622.0512 m/z 638.0664

UV content <1%

Example 12. In vivo pharmacodynamics assessment in cynomolgus monkeys.

The in vivo pharmacodynamics of a representative compound of formula (I) and a comparative example compound were determined as follows.

Test sample and its preparation.

Oral dosage suspensions of a representative compound of Formula (I) (Example 3) and Comparative Example 2 (Example 13/9 in US Pat. No. 9,139,539) were prepared at concentrations of 2, 6, 20, and 60 mg/ml

- 37 043973 in aqueous suspensions of 0.5% sodium carboxymethylcellulose (Na-CMC), 1% ethanol and 98.5% 50 mM Tris buffer at pH 8.

Animals.

Each dose group consisted of three male cynomolgus macaques. At dosing, the animals weighed from 2.5 to 4.4 kg.

Dosing.

The test samples were administered to monkeys through a gastric tube at a dose of 5 ml/kg. Before removal, the gastric tube was flushed with approximately 10 ml of water.

Sampling.

Venous blood samples from each animal were collected at predetermined control time points after dosing. Blood samples were collected and transferred into tubes containing the anticoagulant potassium salt (K2) 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 A 50 μl aliquot of each of the plasma samples obtained in the 10 and 30 mg/kg dose groups, and samples at t=0 obtained in the 100 and 300 mg/kg groups, were treated with 200 μl of acetonitrile (ACN), containing an internal standard. A 25 μl aliquot of the remaining samples obtained in the 100 mg/kg group was combined with 25 μl of control plasma for a 1:2 dilution and treated with 200 μl of acetonitrile (ACN) containing the internal standard. A 10 μl aliquot of the remaining samples obtained in the 300 mg/kg group was combined with 40 μl of control plasma for a 1:5 dilution and treated with 200 μl of acetonitrile (ACN) containing the internal standard. The above solutions were 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. Samples exceeding the instrument calibration range were diluted and reanalyzed.

HPLC conditions.

A Zorbax Extend C18 HPLC column (50 x 2.1 mm, 3.5 μm) from Agilent Technologies (product no. 735700-902) was used. Mobile phase A contained an aqueous solution of 1% acetonitrile in 10 mM ammonium formate, adjusted to pH 3.0 with formic acid. Mobile phase B contained a 10% solution of 10 mM ammonium formate in acetonitrile adjusted to pH 5.2 with 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 program used is given in the following table. 3.

Table 3

Time (sec) Stage Consumption (ml/min) Mobile phase A (%) Mobile phase B (%) thirty Loading a sample 0.50 85 15 180 Gradual change 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

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.

-

Claims (3)

Table 4 Gas 1 Gas 2 Gas for Ion source Voltage (prod. (prod. collisions Spray drying temperature units units (produced units) chambers (°C) (V) change) change) change) Turbo ion atomizer 5500 70 50 6 550 Results. FGF19 levels were compared following oral administration of increasing doses of Example 3 or Comparative Example 2 (3 to 300 mg/kg). Dose-dependent plasma concentrations were observed for both compounds and the maximum AUC values achieved for each compound at a dose of 300 mg/kg were comparable (Fig. 1). Example 3 demonstrated a dose-dependent increase in plasma FGF19 levels, reaching a Cmax of 16,000 pg/ml at the highest dose (Fig. 2). Administration of Comparative Example 2 also caused an increase in plasma FGF19 levels, but the maximum level of FGF19 was significantly lower (Cmax 3000 ng/ml) compared to Example 3. In addition, the maximum induction of FGF19 by Comparative Example 2 was achieved at a dose of 5 mg/kg; higher doses did not provide further increases despite higher plasma drug levels (Fig. 2). This example demonstrates that intravenous or oral administration of Example 3 can provide higher levels of FGF19 compared to Comparative Example 2. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention relates. 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 herein 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 herein represent preferred embodiments, are provided by way of example and do not limit the scope of the present invention. The present invention has been described broadly and generally herein. 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 stated herein 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 is understood that although the present invention has been described in connection with the above-mentioned embodiments, the above description and examples are intended to be illustrative and not limiting 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. Compound formula or its salt. 2. Compound formula 3. Compound formula -