FXR AGONIST COMPOUNDS, THEIR USE AND PHARMACEUTICAL COMPOSITION

专利摘要:
fxr agonist compounds, their use and pharmaceutical composition the present invention relates to compounds that bind to the nr1h4 receptor (fxr) and act as fxr agonists. the invention further relates to the use of the compounds for the preparation of a medicament for the treatment of diseases and / or conditions by linking said nuclear receptor by said compounds and a process for the synthesis of said compounds (1). z is selected from (a), (b), (c) or (d).
公开号:BR112014000260B1
申请号:R112014000260-6
申请日:2012-07-12
公开日:2020-08-11
发明作者:Olaf Kinzel；Christoph Steeneck；Claus Kremoser
申请人:Gilead Sciences, Inc；
IPC主号:

专利说明:

[0001] The present invention relates to compounds that bind to the NR1H4 receptor (FXR) and act as agonists or modulators of FXR. The invention further relates to the use of the compounds for the treatment and / or prophylaxis of diseases and / or conditions by binding said nuclear receptor to said compounds.
[0002] Multicellular organisms are dependent on advanced mechanisms of information transfer between cells and compartments of the body. The information that is transmitted can be highly complex and can result in the alteration of genetic programs involved in cell differentiation, proliferation, or reproduction. The signals or hormones are often molecules of low molecular weight, such as peptides, fatty acid, or cholesterol derivatives.
[0003] Many of these signals produce their effects by finally changing the transcription of specific genes. A well-studied group of proteins that mediates a cell's response to a variety of signals is the family of transcription factors known as nuclear receptors, hereinafter often referred to as "NR". Members of this group include receptors for steroid hormones, vitamin D, ecdysone, cis and trans retinoid acid, thyroid hormone, bile acids, cholesterol derivatives, fatty acids (and other peroxisomal proliferators), as well as so-called orphan receptors, proteins that are structurally similar to other members of this group, but for which no ligand is known. Orphan receptors can be indicative of unknown signaling paths in the cell or they can be nuclear receptors that function without ligand activation. Activation of transcription by some of these orphan receptors can occur in the absence of an exogenous ligand and / or through signal transduction pathways that originate from the cell surface (DJ Mangelsdorf et al., Cell 1995, 83, 835; RM Evans, Mol. Endocrinol. 2005, 19, 1429).
[0004] In general, three functional domains have been defined in NRs. An amino termination domain is believed to have some regulatory function. It is followed by a DNA binding domain hereinafter referred to as "DBD" which normally comprises two zinc finger elements and recognizes a specific Hormone Responsive Element hereinafter referred to as "HRE" in the responsive gene promoters. Specific amino acid residues in the "DBD" have been shown to confer DNA sequence binding specificity (M. Schena and K. R. Yamamoto, Science 1988, 241, 965). A linker-binding domain hereinafter referred to as "LBD" is in the carboxy terminating region of known NRs.
[0005] In the absence of a hormone, LBD appears to interfere with the interaction of DBD with its HRE. Hormone binding appears to result in a conformational change in NR and thus opens up this interference (A. M. Brzozowski et al., Nature 1997, 389, 753). An NR without the LBD constitutively activates transcription, but at a low level.
[0006] It is proposed that transcriptional activators or activators link sequence-specific transcription factors, the basal transcription mechanism and, furthermore, influence the chromatin structure of a target cell. Several proteins like SRC-1, ACTR, and Gripl interact with NRs in an improved way of the ligand (DM Heery et al., Nature 1997, 387, 733; T. Heinzel et al., Nature 1997, 387, 43; KW Nettles and GL Greene, Annu. Rev. Physiol. 2005, 67, 309).
[0007] Nuclear receptor modulators such as steroid hormones affect the growth and function of specific cells by binding to intracellular receptors and forming nuclear receptor-ligand complexes. Nuclear-hormone receptor complexes then interact with an HRE in the region of specific gene control and alter specific gene expression (A. Aranda and A. Pascual, Physiol. Rev. 2001,81, 1269).
[0008] The Farnesoid X alpha Receptor (hereinafter also often referred to as NR1H4 when referring to the human receptor) is a prototypical type 2 nuclear receptor that activates genes by binding to promote region of target genes in a heterodimeric mode with Retinoid X Receiver (BM Forman et al., Cell 1995, 81, 687). The relevant physiological ligands of NR1H4 are bile acids (D. J. Parks et al., Science 1999, 284, 1365; M. Makishima et al., Science 1999, 284, 1362). The most potent one is chenodeoxolic acid (CDCA), which regulates the expression of several genes that participate in bile acid homeostasis. Farnesol and derivatives, together called farnesides, are originally described to activate the rat orthologist at high concentration 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. In addition to controlling intracellular gene expression, FXR also appears to be involved in paracrine and endocrine signaling, suppressing the expression of cytokine fibroblast growth factor 15 (rodents) or 19 (monkeys, humans, JA Holt et al., Genes Dev. 2003, 17, 1581; T. Inagaki et al., Cell Metab. 2005, 2, 217).
[0009] Small molecule compounds that act as FXR modulators have been disclosed in the following publications: WO 2000/037077, WO 2003/015771, WO 2004/048349, WO 2007/076260, WO 2007/092751, WO 2007/140174, WO 2007/140183, WO 2008/051942, WO 2008/157270, WO 2009/005998, WO 2009/012125, WO 2008/025539 and WO 2008/025540. In addition, small molecule FXR modulators have recently been reviewed (M. L. Crawley, Expert Opin Ther. Pat. 2010, 20, 1047; D. Merk et al., Future Med. Chem. 2012, 4, 1015).
[00010] In WO 2011/020615 we disclose chiral cyclopropylidene compounds of the following general formula

[00011] in which the variables are defined similarly in this application.
[00012] The fundamental problem of the present invention is to generate FXR agonists with better physicochemical properties in general, and less hydrophobicity, greater aqueous solubility and better membrane permeability, in particular, compared to compounds claimed in WO 2011/020615.
[00013] Said problem was solved by a compound according to the following formula (1), an enantiomer, diastereomer, tautomer, solvate, promedication or acceptable pharmaceutical salt of these

[00014] where
[00015] R is selected from the group consisting of COORe, CONR7R8, tetrazolyl, SO2NR7R8, C1-6 alkyl, SO2-C1-6 alkyl and H, with R6 independently selected from the group consisting of H or C1-6 alkyl, and R7 and Rs independently of each other selected from the group consisting of H, C1-6 alkyl, C1-6 halo-alkyl, C1-6-R9 alkylene, Sθ2-C1-6-alkyl, where RΘis selected from the group consisting of COOH, OH and SO3H;
[00016] A is selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyrazolyl, indolyl, thienyl, benzothienyl, indazolyl, benzisoxazolyl, benzofuranyl, benzotriazolyl, furanyl, benzothiazolyl, thiazolyl, oxadiazolyl, each of which optionally substituted with one or two groups independently selected from the group consisting of OH, O-C1-6 alkyl, O-halo-C1-6 alkyl, C1-6 alkyl, C1-6 halo-alkyl, C3-6 cycloalkyl and halogen;
[00017] Q is selected from the group consisting of phenyl, pyridyl, thiazolyl, thiophenyl, pyrimidyl, each optionally substituted with one or two groups independently selected from the group consisting of C1-6 alkyl, C1-6 halo-alkyl, halogen and CF3;
[00018] Y is selected from N or CH;
[00019] Ze selected from

[00020] where
[00021] X = CH, N, NO;
[00022] Ri is selected from the group consisting of hydrogen, C1-3 alkyl, C3-6 cycloalkyl, C4-5 alkylcycloalkyl, where C1-3 alkyl is optionally substituted with 1 to 3 substituents independently selected from halogen, hydroxy or alkoxy Ci-β;
[00023] R2 and R3 are independently selected from the group consisting of hydrogen, C1-3 alkyl, C1-3 haloalkyl, C1-3 alkoxy, C1-3 haloalkoxy θ halogen.
[00024] In another modality, in combination with any of the previous or following modalities, RA in the compound according to formula (1) is selected from

[00025] In another embodiment, in combination with any of the previous or following embodiments, Q in the compound according to formula (1) is

[00026] In another embodiment, in combination with any of the previous or following embodiments, Z in the compound according to formula (1) is

[00027] In another modality, in combination with any of the previous or following modalities, the compound according to formula (1) is selected from



[00028] In another embodiment, in combination with any of the previous or following embodiments, the compound according to formula (1) is

[00029] where R is selected from the group consisting of CO2H, CONHSO2Me, and tetrazolyl.
[00030] In another embodiment, the present invention relates to a compound according to formula (1) for use as a medicament.
[00031] In another embodiment, the present invention relates to a compound according to formula (1) for use in the prophylaxis and / or treatment of diseases mediated by FXR.
[00032] In another embodiment, the present invention relates to the use of a compound according to formula (1) for the preparation of a medicament for the prophylaxis and / or treatment of diseases mediated by FXR.
[00033] In another modality, in combination with any of the previous or following modalities, the disease is selected from chronic intrahepatic conditions or some forms of extrahepatic cholestatic conditions; hepatic fibrosis; chronic obstructive or inflammatory disorders of the liver; hepatical cirrhosis; hepatic steatosis and associated syndromes, cholestatic or fibrotic effects that are associated with alcohol-induced cirrhosis or with viral forms of hepatitis; liver failure or liver ischemia after major liver resection; steatohepatitis associated with chemotherapy (CASH); acute liver failure; and / or inflammatory bowel diseases.
[00034] In another modality, in combination with any of the previous or following modalities, the disease is selected from disorders of lipid and lipoprotein; Type II diabetes and clinical complications of Type I and Type II diabetes, including diabetic nephropathy, diabetic neuropathy, diabetic retinopathy and other observed effects of diabetes that clinically manifests itself in the long term; conditions and diseases that result from chronic fatty and fibrotic organ degeneration due to the accumulation of forced lipid and specifically triglycerides and subsequent activation of profibrotic pathways, such as non-alcoholic fatty liver disease (NAFLD), or non-alcoholic steatohepatitis (NASH) ; obesity or metabolic syndrome (combined conditions of dyslipidemia, diabetes or abnormally high body mass index); and / or acute myocardial infarction, acute stroke or thrombosis that occurs as an end point of chronic obstructive atherosclerosis.
[00035] In another modality, in combination with any of the previous or following modalities, the disease is selected from non-malignant hyperproliferative disorders and malignant hyperproliferative disorders, specifically from hepatocellular carcinoma, colon adenoma and polyposis, colon adenocarcinoma, cancer of the breast, adenocarcinoma of the pancreas, Barrett's esophagus or other forms of neoplastic diseases of the gastrointestinal tract and liver.
[00036] The best physicochemical properties were obtained by introducing a polar hydroxyl group in a 1,3-cyclobutylidene or 1,3-azetidinylidene group replacing the first 1,2-cyclopropylidene ring.

[00037] surprisingly, the resulting compounds maintained their activity at the FXR receptor but demonstrated better physicochemical properties, such as greater aqueous solubility and / or membrane permeability.
[00038] The compounds of the present invention share a common chemical structure according to formula (1) in claim 1.
[00039] In a preferred embodiment in combination with any of the foregoing or following embodiments, the present invention relates to an enantiomer, diastereomer or pharmaceutically acceptable salt of a compound according to formula (1).
[00040] In a preferred embodiment in combination with any of the previous or following embodiments, R in formula (1) is selected from the group consisting of COORΘ, CONRyRs, SO2NR7R8, the Sθ2-C1-6 alkyl.
[00041] In a preferred embodiment in combination with any of the previous or following embodiments, Re in formula (1) is H.
[00042] In a preferred embodiment in combination with any of the foregoing or following embodiments, R7 and Re in formula (1) are independently selected from the group consisting of H and Sθ2-C1-6 alkyl.
[00043] In a preferred embodiment in combination with any of the previous or following embodiments, R7 in formula (1) is H.
[00044] In a preferred embodiment in combination with any of the foregoing or following embodiments, Rs in formula (1) is Sθ2-C1-6 alkyl.
[00045] In a preferred embodiment in combination with any of the foregoing or following embodiments, A is selected from the group consisting of phenyl, pyridyl, pyrimidyl, pyrazolyl, indazolyl, and oxadiazolyl.
[00046] In a preferred embodiment in combination with any of the foregoing or following embodiments, A is substituted with one or two groups independently selected from C1-6 alkyl, more preferably C1-3 alkyl. In another preferred embodiment in combination with any of the foregoing or following embodiments, A is unsubstituted.
[00047] In a preferred embodiment in combination with any of the foregoing or following embodiments, Q is phenyl.
[00048] In a preferred embodiment in combination with any of the foregoing or following embodiments, Q is replaced with one or two independently selected halogen groups, more preferably a selected halogen group, in particular Cl.
[00049] In a preferred embodiment in combination with any of the previous or following embodiments, Z is

[00050] In a preferred embodiment in combination with any of the previous or following embodiments, X = CH.
[00051] In a preferred embodiment in combination with any of the foregoing or following embodiments, Ri is C3-6 cycloalkyl, in particular cyclopropyl.
[00052] In a preferred embodiment in combination with any of the preceding or following embodiments, R2 and R3 are independently selected from halogen, in particular Cl. BRIEF DESCRIPTION OF THE FIGURES
[00053] Figure 1: NOEs detected for example 8 with trans-annular 1,3-trans configuration of aromatic fractions.
[00054] Figure 2: NOEs detected for example 8A with 1,3-cis transanular configuration of aromatic fractions.
[00055] The compounds of the present invention can be in the form of a promising compound. "Promising compound" means a derivative that is converted to a compound according to the present invention, by a reaction with an enzyme, gastric acid or the like in a physiological condition in the living body, for example, by oxidation, reduction, hydrolysis or similar, each of which is carried out enzymatically. Examples of the promedicament are compounds, in which the amino group in a compound of the present invention is acylated, alkylated or phosphorylated to form, for example, eicosanoylamino, alanylamino, pivaloyloxymethylamino or in which the hydroxyl group is acylated, alkylated, phosphorylated or converted to borate , for example, acetyloxy, palmitoyloxy, pivaloyloxy, succinyloxy, fumaryloxy, alanyloxy or in which the carboxyl group is esterified or amidated. Such compounds can be produced from the compounds of the present invention according to well-known methods. Other examples of the promedicament are compounds, wherein the carboxylate in a compound of the present invention is, for example, converted to an alkyl-, aryl-, choline-, amino, acyloxymethylester, linolenoylester.
[00056] Metabolites of the compounds of the present invention are also within the scope of the present invention.
[00057] Where tautomerism, for example, keto-enol type tautomerism, of the compounds of the present invention or its prominences can occur, the individual forms, for example, keto and enol type, are each within the scope of the invention, as well like their mixtures for any reason. The same applies to stereoisomers, for example, type enantiomers, cis / trans isomers, shapers and the like.
[00058] If desired, isomers can be separated by methods well known in the art, for example, by liquid chromatography. The same applies to enantiomers using, for example, chiral stationary phases. Additionally, enantiomers can be isolated by converting them to diastereomers, that is, coupling with an enantiomerically pure auxiliary compound, subsequent separation of the resulting diastereomers and dividing of the auxiliary residue. Alternatively, any enantiomer of a compound of the present invention can be obtained from stereoselective synthesis using optically pure starting materials. Another way to obtain pure enantiomers from racemic mixtures is to use enantioselective crystallization with chiral counterions.
[00059] The compounds of the present invention can be in the form of a pharmaceutically acceptable salt or a solvate. The terms "pharmaceutically acceptable salts" refer to salts prepared from non-toxic bases or pharmaceutically acceptable acids, including inorganic bases or acids and organic bases or acids. In the event that the compounds of the present invention contain one or more acidic or basic groups, the invention also comprises their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically usable salts. Thus, compounds of the present invention that contain acid groups can be present in those groups and can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts or ammonium salts. More precise 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. The compounds of the present invention that contain one or more basic groups, that is, groups that can be protonated, can be present and can be used according to the 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, naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaric acid, lactic acid, silicic acid , formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pyelic acid, fumaric acid, maleic acid, malic acid, sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to those skilled in the art. If the compounds of the present invention contain both acidic and basic groups in the molecule, the invention also includes, in addition to the salt forms mentioned, salts or internal betaines (zwiterions). The respective salts can be obtained by usual methods that are known to those skilled in the art, for example, by placing these in contact with an organic or inorganic acid or base in a solvent or dispersant, or by ion or cation exchange with other salts. The present invention also includes all the salts of the compounds of the present invention which, due to low physiological compatibility, are not directly suitable for use in pharmaceutical products, but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.
[00060] Additionally, the compounds of the present invention can be present in the form of solvates, such as those that include water as a solvate, or pharmaceutically acceptable solvates, such as alcohols, in particular ethanol.
[00061] In addition, the present invention relates to pharmaceutical compositions comprising at least one compound of the present invention, or a promising compound thereof, or a pharmaceutically acceptable salt or solvate thereof as an active ingredient together with a pharmaceutically acceptable carrier.
[00062] "Pharmaceutical composition" means one or more active ingredients, and one or more inert ingredients that constitute the carrier, as well as any product that results, directly or indirectly, from the combination, complexation or aggregation of any two or more of the ingredients, or dissociation of one or more of the ingredients, or other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by mixing at least one compound of the present invention and a pharmaceutically acceptable carrier.
[00063] The pharmaceutical composition of the present invention can additionally comprise one or more other compounds as active ingredients such as a promising compound or other nuclear receptor modulators.
[00064] The compositions are suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (nasal or buccal inhalation) or nasal administration, although the most suitable route in any given case it will depend on the nature and severity of the conditions being treated and the nature of the active ingredient. They can conveniently be presented in unit dosage form and prepared by any of the methods well known in the pharmacy art.
[00065] The compounds of the present invention can be prepared by a combination of methods described in schemes I to III. As shown in Scheme I, a 4-membered cyclic ketone substituted with substituent A in position 3 can react with an MQO-CH2Z metallized aromatic or heteroaromatic ring (M = metal, for example, Li) in aprotic solvents and preferably at low temperatures for provide a hydroxyl-substituted 4-membered ring carrying substituents A and Q. In the case where Y is CH, two isomers can form (A and Q transanular cis or trans with each other). Under optimized conditions, the formation of mainly one of the two isomers can be obtained. The two isomers can be separated by appropriate methods known in the art, for example, like silica gel chromatography or preparative RP-HPLC. Scheme I

[00066] In Scheme II, methods that are used to prepare the 4-membered cyclic ketone necessary for the synthesis of the compounds of this invention are summarized. In option a), a vinyl carrying intermediate, for example, prepared by vinylation of a starting material containing corresponding halogen RAX (X = halogen) can react with α, α-dichloro ketone formed in situ to form a 2,2-dichlorocyclobutanone. After dehalogenation, for example, with Zn in reflux acetic acid, the desired 3-substituted cyclobutanones are obtained. Alternatively, the vinyl intermediates can react with unsubstituted ketone generated in situ to make the desired cyclobutanone intermediates available in one step. In option b) 3-methylenocyclobutanecarbonitrile is used as a starting material. Substituted heterocycles can be constructed from the cyano group in several stages by methods known to those skilled in the art. The desired cyclobutanones can be obtained by oxidizing divination of the exocyclic double bond using conditions and reagents known to those skilled in the art, for example, by using Osθ4, ozone or RhCh / NalCU as oxidants. Option c) shows the methods used to prepare the substituted azetidinones. Cu or Pd catalyzed CN cross coupling between 3-hydroxy-azetidine and halo-aromatic or halo-heteroaromatic rings provides the corresponding 3-hydroxy-azetidines / V-substituted that can be transformed into the desired azetidinones by oxidation. Scheme II

[00067] Scheme III illustrates some possibilities of making changes to the substituents in group A after the formation of the rings carrying 4-membered hydroxy. For example, a leaving group X (for example, bromide) can be replaced by a cyano group, a carboxylic ester, methylsulfonyl or thioether by transition metal catalyzed cross-coupling reactions. The derivatives obtained can be further transformed into other derivatives by methods known to those skilled in the art. For example, the cyano group and the ester can be hydrolyzed under basic conditions to make a carboxylic acid available which in turn can be converted to acyl sulfonamides. A benzyl thioether can be chlorinated to provide the chlorosulfonyl intermediate which reacts with ammonia in the corresponding sulfonamides. Scheme III

[00068] As a result, the present invention relates to compounds according to general formula (1) that bind to FXR and act as agonists or modulators of FXR.
[00069] The invention additionally relates to the use of said compounds for the treatment and / or prophylaxis of diseases and / or conditions by linking said nuclear receptor by said compounds. In addition, the present invention relates to the use of said compounds for the preparation of a medicament for the treatment and / or prophylaxis of diseases and / or conditions by linking said nuclear receptor by said compounds. Specifically, the present invention relates to the use of the compounds according to formula (1) in the preparation of a medicament for the prophylaxis and / or treatment of chronic intrahepatic conditions or some forms of extrahepatic cholestatic conditions, of hepatic fibrosis , from acute intraheptic cholestatic conditions, from obstructive or chronic inflammatory disorders that arise from improper bile composition, from gastrointestinal conditions with a lower intake of dietary fat and fat-soluble dietary vitamins, from inflammatory bowel diseases, from lipid disorders and lipoprotein, Type II diabetes and clinical complications of Type I and Type II diabetes, conditions and diseases that result from chronic fatty and fibrotic organ degeneration due to forced lipid and specifically triglyceride accumulation and subsequent activation of profibrotic pathways, obesity and metabolic syndrome (combined conditions of dyslipidemia, diabetes and and abnormally high body mass), acute myocardial infarction, acute stroke, thrombosis that occurs as an end point of chronic obstructive atherosclerosis, persistent infections by intracellular bacteria or parasitic protozoa, non-malignant hyperproliferative disorders, disorders malignant hyperproliferatives, colon adenocarcinoma and hepatocellular carcinoma in particular, liver steatosis and associated syndromes, liver failure or liver malfunction as a result of chronic liver diseases or surgical liver resection, hepatitis B infection, infection of hepatitis C and / or cholestatic and fibrotic effects that are associated with alcohol-induced cirrhosis or with viral forms of hepatitis.
[00070] Medicines as referred to herein can be prepared by conventional processes, including the combination of a compound according to the present invention and a pharmaceutically acceptable carrier.
[00071] FXR is proposed to be a nuclear bile acid sensor. As a result, it modulates both the synthetic production of bile acids in the liver and their recycling in the intestine (regulating bile acid binding proteins). But in addition to the physiology of bile acid, FXR appears to be involved in the regulation of many diverse physiological processes that are relevant in the etiology and for the treatment of diseases as diverse as cholesterol gallstones, metabolic disorders such as Type II diabetes, dyslipidemia or obesity , chronic inflammatory diseases such as inflammatory bowel diseases or chronic intrahepatic forms of cholestasis and many other diseases (T. Claudel et al., Arterioscler. Thromb. Vase. Biol. 2005, 25, 2020; YD Wang et al., Cell Res. 2008, 18, 1087).
[00072] FXR regulates a complex pattern of response genes in the liver and gastrointestinal tract. Genetic products have an impact on different physiological processes. In the course of functional analysis of FXR, the first regulatory network that was analyzed was the regulation of bile acid synthesis. While LXRs induce the key enzyme of the conversion of cholesterol to bile acids, Cyp7A1, by inducing the regulatory nuclear receptor LRH-1, FXR suppresses the induction of Cyp7A1 through the suppression of mRNA encoding SHP, an additional nuclear receptor that it is dominant repressive with respect to LRH-1. Since FXR binds the end products of this pathway, primary bile acids such as cholic acid (CA) or CDCA, can be considered as an example of feedback inhibition at the level of gene expression (B. Goodwin et al., Mol. Cell 2000, 6, 517; TT Lu et al., Mol. Cell 2000, 6, 507). Parallel to the repression of bile acid synthesis by means of SHP, FXR induces a range of so-called ABC transporters (for ATP binding cassettes) that are responsible for exporting toxic bile acids from the cytosol hepatocyte in the canaliculi, the small branches of the bile duct where bile originates. This hepatoprotective FXR function is first apparent with the analysis of FXR in knockout mice (C. J. Sinal et al., Cell 2000, 102, 731), where it has been shown to be under or overexpressed by various ABC transporters in the liver. In addition, detailed analysis revealed that the main BSEP or ABCB11 bile salt excretory pump (M. Ananthanaraioanan et al., J. Biol. Chem. 2001, 276, 28857; JR Plass et al., Hepatology 2002, 35, 589) as well as the key enzyme that mediates lipid transfer from lipoproteins to phospholipids, PLTP (NL Urizar et al., J. Biol. Chem. 2000, 275, 39313), and the two key canalicular membrane transporters for phospholipids, MRP-2 (ABCC4) (HR Kast et al., J. Biol. Chem. 2002, 277, 2908) and MDR-3 (ABCB4); L. Huang et al., J. Biol. Chem. 2003, 278, 51085) are targets for targeted transcriptional activation of the ligand by FXR (summarized in: M. Miyata, J. Pharmacol. Exp. Ther. 2005, 312, 759; G. Rizzo et al., Curr. Drug Immune Endocr Targets, Metabol, Disord, 2005, 5, 289).
[00073] The fact that FXR appears to be the primary metabolite sensor and regulator for the synthesis, export and recirculation of bile acids has suggested the use of FXR ligands to induce bile flow and change the composition of bile acid to a more hydrophilic composition. With the development of the first synthetic FXR linker GW4064 (PR Maloney et al., J. Med. Chem. 2000, 43, 2971; TM Willson et al., Med. Res. Rev. 2001, 21, 513) as a compound tool and the semi-synthetic artificial bile acid ligand 6-alpha-ethyl-CDCA, the effects of FXR over-stimulation by potent agonists can be analyzed. Both ligands have been shown to induce bile flow in animals bound in the bile duct. Furthermore, in addition to choleretic effects, hepatoprotective effects can also be demonstrated (R. Pellicciari et al., J. Med. Chem. 2002, 45, 3569; Y. Liu et al., J. Clin. Invest. 2003, 112, 1678). This hepatoprotective effect was further narrowed to an antifibrotic effect that results from the suppression of matrix metalloproteinase tissue inhibitors, TIMP-1 and 2, the induction of collagen deposition resolving matrix 2 metalloproteinase in liver stellate cells and the subsequent reduction of alpha mRNA -collagen and growth transforming beta factor (TGF-beta) mRNA which are both propibrotic factors by FXR agonists (S. Fiorucci et al., Gastroenterology 2004, 127, 1497; S. Fiorucci et al., J. Pharmacol Exp. Ther. 2005, 314, 584). In addition, anti-cholestatic activity has been demonstrated in animal models linked to the bile duct as well as in animal models of estrogen-induced cholestasis (S. Fiorucci et al., J. Pharmacol. Exp. Ther. 2005, 313, 604).
[00074] Genetic studies demonstrate that, in hereditary forms of cholestasis (progressive familial interhepatic colasease = PFIC, Type I - IV), both nuclear localization of FXR by itself is reduced as a result of a mutation in the FIC1 gene (in PFIC Type I, also called Byler disease) (F. Chen et al., Gastroenterology 2004, 126, 756; L. Alvarez et al., Hum. Mol. Genet. 2004, 13, 2451) regarding levels of the FXR target gene that encode MDR-3 phospholipid export pump are reduced (in PFIC Type III). Taken together, there is a growing body of evidence that FXR-binding compounds will demonstrate substantial clinical utility in the therapeutic regimen of chronic cholestatic conditions such as primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC) (reviewed in: G. Rizzo et al ., Curr. Drug Targets Immune Endocr. Metabol. Disord. 2005, 5, 289; G. Zollner et al., Mol. Pharm. 2006, 3, 231; SY Cai et al., Expert Opin. Ther. Targets 2006, 10, 409).
[00075] The profound impact that activation of FXR has on bile acid metabolism and excretion is not only relevant for cholestatic syndromes, but even more directly for a therapy against gallstone formation. Cholesterol gallstones form due to the low solubility of cholesterol that is actively pumped out of the liver cell into the lumen of the canaliculi. It is the relative percentage of content of the three main components, bile acids, phospholipids and free cholesterol, which determines the formation of mixed micelles and, consequently, the apparent solubility of free cholesterol in bile. Map of FXR polymorphisms as quantitative tract loci as a contributing factor to gallstone disease (H. Wittenburg, Gastroenterology 2003, 125, 868). Using the compound of the synthetic FXR tool GW4064 it was possible to demonstrate that activation of FXR leads to an improvement in the cholesterol saturation index (CSI) and directly to an abolition of gallstone formation in mice susceptible to gallbladder C57L while treatment of drug in knockout mice with FXR shows no effect on gallstone formation (A. Moschetta et al., Nature Medicine 2004, 10, 1352).
[00076] These results qualify FXR as a good target for the development of small molecule agonists that can be used to prevent cholesterol gallstone formation or to prevent gallstone reform after surgical removal or shock wave lithotripsy (discussed in: SA Doggrell, Curr. Opin. Investig. Drugs 2006, 7, 344).
[00077] Thus, in an embodiment of the invention, the compound according to formula (1) and pharmaceutical compositions comprising said compound are used for the prophylaxis and / or treatment of obstructive or chronic inflammatory disorders arising from the inappropriate bile composition such like cholelithiasis also known as cholesterol gallstones.
[00078] In addition to its strong hepatoprotective and chloretic effect as well as antifibrotics that FXR shows through small molecule activation stimulated in the liver, FXR seems to have a role in protecting the intestine from neoplastic transformation and the development of polyps and their transition in intestinal adenocarcinoma ( S. Modica et al., Cancer Res. 2008, 68, 9589 and RR Maran et al., J. Pharmacol. Exp. Ther. 2009, 328, 469). Similar to the situation in the intestine, the absence of FXR leads to a greater increase in the formation of hepatocellular carcinone (HCC), the most prominent form of liver cancer (I. Kim et al., Carcinogenesis 2007, 28, 940 and F. Yang et al., Cancer Res. 2007, 67, 863). While a functional FXR prevents the formation of colon adenocarcinoma and hepatocellular carcinoma, activation of FXR induces liver regeneration after hepatectomy (W. Huang et al., Science 2006, 312, 233).
[00079] The combined hepatoprotective, antineoplastic and regenerative effects of the liver associated with FXR activation can be therapeutically exploited for the use of FXR agonists in the treatment of severe liver diseases. In one embodiment, the compounds according to the invention and pharmaceutical compositions comprising said compounds are used in the treatment of liver diseases such as HCC, stimulating liver regrowth and ameliorating side effects associated with major liver resection, liver cirrhosis regardless of the etiology and preventing or treating liver ischemia in the course of liver transplantation or major liver surgery.
[00080] Since the discovery of the first synthetic FXR agonist and its administration to rodents it has become evident that FXR is a key regulator of serum triglycerides (P. Maloney et al., J. Med. Chem. 2000, 43, 2971; T Willson et al., Med. Res. Rev. 2001, 21, 513). In the last six years evidence of accumulation has been published that activation of FXR by synthetic agonists leads to significant reduction of serum triglycerides, mainly in the form of low VLDL, but also to low cholesterol in the total serum (HR Kast et al., Mol. Endocrinol. 2001, 15, 1720; NL llrizar et al., Science 2002, 296, 1703; G. Lambert et al., J. Biol. Chem. 2003, 278, 2563; M. Watanabe et al., J. Clin Invest. 2004, 113, 1408; A. Figge et al., J. Biol. Chem. 2004, 279, 2790; S. Bilz et al., Am. J. Physiol. Endocrinol. Metab. 2006, 290, E716 ).
[00081] But, the reduction of triglycerides in serum is not an independent effect. Treatment of db / db or ob / ob mice with synthetic FXR agonist GW4064 resulted in a remarkable and combined reduction in serum triglycerides, total cholesterol, free fatty acids, ketone bodies such as 3-OH butyrate. Furthermore, activation of FXR fits with the intracellular insulin signaling pathway in hepatocytes, resulting in low glucose production from liver gluconeogenesis, but a concomitant increase in liver glycogen. Insulin sensitivity as well as glucose tolerance were positively impacted by FXR treatment (KR Stayrook et al., Endocrinology 2005, 146, 984; Y. Zhang et al., PNAS 2006, 103, 1006; B. Cariou et al., J. Biol. Chem. 2006, 281, 11039; K. Ma et al., J. Clin. Invest. 2006, 116, 1102; D. Duran-Sandoval et al., Biochimie 2005, 87, 93). An effect on body weight reduction has also recently been observed in mice overfed with a high lipid diet (C. Lihong et al., American Diabetes Association (ADA) 66th annual scientific sessions, June 2006, Abstract Number 856-P). This weight loss effect may result from the FXR induction of FGF-19, a fibroblast growth factor that is known to lead to weight loss and an athletic phenotype (J. Holt et al., Genes Dev. 2003, 17, 1581; E. Tomlinson et al., Endocrinology 2002, 143, 1741). In recent patent applications, the effect of FXR agonist on reducing body weight has been demonstrated (WO 2004/087076; WO 2003/080803).
[00082] Taken together, these pharmacological effects of FXR agonists can be explored in different therapeutic modes: FXR-binding compounds are considered good candidates for the treatment of Type II diabetes because of their insulin sensitization, glycogenogenic effects, and reduction of lipid.
[00083] In one embodiment, the compounds according to the invention and pharmaceutical compositions comprising said compounds are used in the prophylaxis and / or treatment of Type II diabetes, which can be overcome by FXR-mediated suppression of systemic insulin sensitivity and signaling intracellular insulin in the liver, greater peripheral glucose uptake and metabolism, greater storage of glycogen in the liver, less production of glucose in the serum from gluconeogenesis of liver infection.
[00084] In an additional embodiment, said compounds and pharmaceutical compositions are used for the prophylaxis and / or treatment of chronic intrahepatic conditions, such as PBC, PSC, progressive family cholestasis (PFIC), alcohol-induced cirrhosis and associated cholestasis , and some forms of extrahepatic cholestatic conditions, or liver fibrosis.
[00085] The invention also relates to a compound of formula (1) or a pharmaceutical composition comprising said compound for the prophylaxis and / or treatment of gastrointestinal conditions with less uptake of dietary fat and fat-soluble dietary vitamins that can be overcome by higher intestinal levels of bile acids and phospholipids.
[00086] In an additional 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 such as hypercholesterolemia, hypertriglyceridemia, and atherosclerosis as a clinically manifested condition that can be improved by the beneficial effect of FXR in lowering total plasma cholesterol, reducing serum triglycerides, increasing the conversion of cholesterol in the liver to bile acids and increased release and metabolic conversion of VLDL and other lipoproteins in the liver.
[00087] In an additional modality, said compound and pharmaceutical composition are used for the prophylaxis and / or treatment of diseases where the lipid reduction, anti-cholestatic and anti-fibrotic effects combined of drugs targeted by FXR can be exploited for the treatment of hepatic steatosis and associated syndromes such as NASH, or for the treatment of cholestatic and fibrotic effects that are associated with alcohol-induced cirrhosis, or with viral forms of hepatitis.
[00088] Along with the hypolipidemic effects it has also been shown that loss of functional FXR leads to increased atherosclerosis in ApoE knockout mice (E. A. Hanniman et al., J. Lipid Res. 2005, 46, 2595). Therefore, FXR agonists may have clinical utility as anti-atherosclerotic and cardioprotective drugs. The infra-regulation of endothelin-1 vascular soft muscle cells can also contribute to such beneficial therapeutic effects (F. He et al., Circ. Res. 2006, 98, 192).
[00089] The invention also relates to a compound according to formula (1) or a pharmaceutical composition comprising said compound for preventive and post-traumatic treatment of cardiovascular disorders such as acute myocardial infarction, acute stroke, or thrombosis that occur as an end point of chronic obstructive atherosclerosis.
[00090] In addition to controlling the formation of intestinal and colonic polyp, FXR appears to be expressed in breast cancer tissue and cell lines, but not in healthy breast tissue and appears to interact with the estrogen receptor in breast cancer cells ER positive (KE Swales et al., Cancer Res. 2006, 66, 10120 and F. Journe et al., Breast Cancer Res. Treat. 2009, 115, 523).
[00091] This would allow FXR to also be considered as a potential target for the treatment of proliferative diseases, especially metastatic forms of cancer that express a small molecule responsive form of FXR.
[00092] In an additional embodiment, said compounds and pharmaceutical compositions are used for the prophylaxis and / or treatment of malignant hyperproliferative disorders such as different forms of cancer, specifically certain forms of breast, liver or colon cancer where interference with a FXR binder will have a beneficial impact.
[00093] Finally, FXR also appears to be involved in the control of antibacterial defense in the intestine (T. Inagaki et al., PNAS. 2006, 103, 3920) although an exact mechanism is not provided. From these published data, however, it can be concluded that treatment with FXR agonists can have a beneficial impact on the therapy of inflammatory bowel disorders (IBD), in particular those forms where the upper part (ileum) of the intestine is affected ( for example, ileal Crohn's disease) because it appears to be the site of FXR control action on bacterial growth. In IBD, desensitization of the adaptive immune response is in any way impaired in the intestinal immune system. Bacterial overgrowth can then be the trigger for establishing a chronic inflammatory response. Consequently, dampening bacterial growth by mechanisms carried by FXR may be a key mechanism for preventing acute inflammatory episodes.
[00094] Thus, the invention also relates to a compound according to formula (1) or a pharmaceutical composition comprising said compound to prevent and / or treat a disease related to inflammatory bowel diseases such as Crohn's disease or colitis ulcerative. FXR-mediated restoration of function and reduction of the intestinal barrier in non-commensal bacterial load is believed to be useful in reducing the exposure of bacterial antigens to the intestinal immune system and can therefore reduce inflammatory responses.
[00095] The invention additionally relates to a compound or pharmaceutical composition for the prophylaxis and / or treatment of obesity and associated disorders such as metabolic syndrome (combined conditions of dyslipidemia, diabetes and abnormal high body mass index) that can be overcome by FXR-mediated reduction of serum triglycerides, blood glucose and increased insulin sensitivity and FXR-mediated weight loss.
[00096] In an additional embodiment, the compounds or pharmaceutical composition of the present invention are used to prevent and / or treat clinical complications of type I and type II diabetes. Examples of such complications include diabetic neuropathy, diabetic retinopathy, diabetic neuropathy, or peripheral arterial occlusive disease (PAOD). Other clinical complications of diabetes are also encompassed by the present invention.
[00097] In addition, conditions and diseases that result from chronic fatty and fibrotic organ degeneration due to forced lipid and specifically accumulation of triglycerides and subsequent activation of profibrotic pathways can also be prevented and / or treated by applying the compounds or pharmaceutical composition of this invention. Such conditions and diseases include NASH and chronic cholestatic conditions in the liver, Glomerulosclerosis and diabetic neuropathy in the kidney, macula degeneration and diabetic retinopathy in the eye and neurodegenerative diseases such as Alzheimer's disease in the brain, or diabetic neuropathy in the peripheral nervous system.
[00098] In practical use, the compounds of the present invention can be combined as the active ingredient in admixture with a pharmaceutical carrier according to conventional pharmaceutical composition techniques. The carrier can take a wide variety of 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 of the usual pharmaceutical means can be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like, in the case of liquid oral preparations such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of solid oral preparations such as, for example, hard and soft powders, capsules and tablets, with oral preparations solids being preferred over liquid preparations.
[00099] Due to their ease of administration, tablets and capsules represent the most advantageous unitary oral dosage form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets can be coated by standard aqueous or non-aqueous techniques. Such compositions and preparations must contain at least 0.1 percent of the active compound. The percentage of active compound in these compositions can, of course, be varied and can conveniently be between about 2 percent to about 60 percent of the weight of the unit. The amount of the active compound in such therapeutically used compositions is such that an effective dosage will be obtained. The active compounds can also be administered intranasally such as, for example, liquid drops or spray.
[000100] Tablets, pills, capsules, and the like may also contain binders such as tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin. When the unit dosage form is a capsule, it may contain, in addition to materials of the type mentioned, a liquid carrier such as a fatty oil.
[000101] Various other materials may be present as coatings or to modify the physical form of the dosage unit. For example, tablets can be coated with shellac, sugar, or both. A syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry or orange flavor.
[000102] Since the compounds of the present invention basically represent carboxylic acids or anionic isosteres similar to these and, since it is well known that salt forms of the ionic drug compounds can substantially affect the bioavailability of the drug compounds, the compounds of the present invention can also be used as salts with various contractions to produce an orally available formulation. Such pharmaceutically acceptable cations can be, among others, mono or divalent ions such as ammonium, sodium or potassium of alkali metals or magnesium or calcium of alkaline earth metals, certain pharmaceutically acceptable amines such as tris (hydroxymethyl) aminomethane, ethylenediamine, diethylamine , piperazine or others, or certain cationic amino acids such as lysine or arginine.
[000103] 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 glycerol, liquid polyethylene glycols and mixtures of these in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[000104] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
[000105] Any suitable route of administration can be employed to provide a mammal, especially a human, with an effective dose of a compound of the present invention. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, and the like can be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like. Preferably, compounds of the present invention are administered orally.
[000106] The effective dosage of active ingredient employed may vary depending on the particular compound employed, the mode of administration, the condition being treated and the severity of the condition being treated. Such a dosage can be easily determined by those skilled in the art.
[000107] During the treatment or prevention of FXR-mediated conditions for which the compounds of the present invention are indicated, generally satisfactory results are obtained when the compounds of the present invention are administered at a daily dosage of about 0.1 milligram to about of 100 milligrams per kilogram of the animal's body weight, preferably given as a single daily dose or in doses divided into two to six times a day, or in the form of sustained release. For most large mammals, the total daily dosage is from about 1.0 milligram to about 1,000 milligrams, preferably from about 1 milligram to about 50 milligrams. In the case of a 70 kg adult human, the total daily dose will generally be about 7 milligrams to about 350 milligrams. This dosage regimen can be adjusted to provide the optimal therapeutic response.
[000108] The compounds of the present invention can be prepared according to the procedures of the following schemes and examples, using appropriate materials and are further exemplified by the following specific examples. Furthermore, using the procedures described herein, along with those skilled in the art, additional compounds of the present invention claimed here can be easily prepared. The compounds illustrated in the examples should not however be interpreted as forming only the genus that is considered in the invention. The examples further illustrate details for the preparation of the compounds of the present invention. Those skilled in the art will readily understand that known variations in the conditions and processes of the following preparatory procedures can be used to prepare these compounds. The present compounds are generally isolated in the form of their pharmaceutically acceptable salts, such as those described above.
[000109] The free amine bases corresponding to the isolated salts can be generated by neutralization with a suitable base, such as aqueous sodium hydrogen carbonate, sodium carbonate, sodium hydroxide and potassium hydroxide, and extraction of the released free amine base in an organic solvent, followed by evaporation. The free amine base isolated in this way can be further converted to another pharmaceutically acceptable salt by dissolving it in an organic solvent, followed by addition of the appropriate acid and subsequent evaporation, precipitation or crystallization. The free carboxylic acids corresponding to the isolated salts can be generated by neutralization with a suitable acid, such as aqueous hydrochloric acid, sodium hydrogen sulfate, sodium dihydrogen phosphate, and extraction of the free carboxylic acid released in an organic solvent, followed by evaporation. The carboxylic acid isolated in this way can be further converted to another pharmaceutically acceptable salt by dissolving it in an organic solvent, followed by the addition of the appropriate base and subsequent evaporation, precipitation or crystallization.
[000110] An illustration of the preparation of the compounds of the present invention is shown below. Unless otherwise indicated in the diagrams, the variables have the same meaning as described above. The examples presented below are intended to illustrate particular embodiments of the invention. Suitable starting materials, building blocks and reagents used in the synthesis described below are commercially available from Sigma-Aldrich or Acros Organics, for example, or can be routinely prepared by procedures described in the literature, for example, in "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 organics Synthesis" John Wiley & Sons 2000. Examples Example 1: 3 - ((1s, 3s) -3- (2-chloro-4 - ((5-cyclopropyl-3- (2,6-dichlorophenyl ) methyl isoxazol-4-yl) methoxy) phenyl) -3-hydroxycyclobutyl) benzoate (1)
Step 1: 4 - (((4-bromo-3-chlorophenoxy) methyl) -5-cyclopropyl-3- (2,6-dichloropheniD-isoxazole (1a)
[000111] To a solution of (5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methanol (13 g, 45.8 mmol) in CH2Cl2 (DCM) (200 ml_) was added SOCI2 drop the drop (40 ml, 336 mmol). The resulting mixture was stirred at room temperature for 2 hours and the solvents were removed under low pressure. The residue was dissolved in / V, / V-dimethylformamide (DMF) (200 ml) and 4-bromo-3-chlorophenol (9.7 g, 47 mmol), K2CO3 (40 g, 290 mmol) and Nal (12 g , 80 mmol) were added to this solution. The mixture was stirred at 60 ° C overnight, then cooled to room temperature, diluted with water (1,000 ml) and extracted with ethyl acetate (EA) (500 ml x 3). The combined organic phases were washed with brine (500 ml_3), dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (CC) to give the title compound 1a (19 g, 88%) in the in the form of a white solid. Step 1: Methyl 3- (2,2-dichloro-3-oxocyclobutyl) benzoate (1b)
[000112] A 3-necked round-bottom flask, under a nitrogen atmosphere, fitted with a condenser, a suspended stirrer and pressure equalized drip funnel was dissolved methyl 3-vinylbenzoate (5 g, 31 mmol) in Et2θ dry (150 mL). To this flask, zinc powder (6 g, 3 eq) was added and the reaction was sonicated for 30 minutes. After this time a solution of trichloroacetylchloride (8.7 ml, 2.5 eq) in dry E2 O (50 ml) was added dropwise while sonicating for the next 30 minutes. During the process the reaction mixture was heated to 35 ° C. The sonication continued for 2.5 hours at reflux and the reaction appeared to be complete by 1H MRI analysis. The reaction was cooled naturally to room temperature and ended with water (~ 50 ml). This was done in a drop-by-drop fashion interspersed several times for a few minutes once a delayed exothermic reaction occurred. After 20 minutes of stirring in water, the reaction mixture was filtered through a pad of celite and rinsed completely with Et2θ. The organic layer was washed with portions of water (2 x 250 ml), saturated sodium bicarbonate (2 x 250 ml) and brine (1 x 250 ml), dried over sodium sulfate, filtered and concentrated under low pressure to provide the crude product 1b as a thick dark yellow oil (crude 8.7 g). Step 2: methyl 3- (3-oxocyclobutyl) benzoate (1c)
[000113] Crude compound 1b (8.7 g) was dissolved in glacial acetic acid (55 mL) in a round-bottomed flask under an atmosphere of nitrogen. To this flask, zinc powder (4.6 g, 2.2 eq) was added and the reaction was stirred and heated to 120 ° C for 3 hours. After cooling to room temperature, the mixture was filtered through a pad of celite, which was washed with portions of EA. The combined solution was concentrated under low pressure before being dissolved in EA (500 ml), washed with brine (150 ml x 2) and then dried over sodium sulfate, filtered and concentrated again. The crude mixture was stirred for 5 minutes in chloroform (250 ml) and filtered through a sintered funnel. The filtrate was concentrated to give the crude product as a faded yellow oil. The crude product was purified by CC in (PE / EA = 9: 1, PE = petroleum ether) to give the desired product 1c (2.5 g, 38% for 2 steps) as a faded yellow oil. Step 3: Methyl 3- (3- (2-chloro-4 - (((5-cyclopropyl-3- (216-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3-hydroxycyclobutyl) benzoate (1)
[000114] To a stirring solution of compound 1a (1.67 g, 3.5 mmol) in dry THF (30 ml) was added n-uuLi (2.5 M in hexane, 1.2 eq, 1.69 ml_) drop by drop for 10 minutes at -78 ° C under a nitrogen atmosphere. This was stirred for 1 hour at this temperature before adding a solution of compound 1c (0.72 g, 1 eq) in dry THF (10 ml) dropwise and stirred for 1 hour at this temperature. The reaction mixture was naturally warmed to room temperature slowly and left to stir overnight. The reaction was terminated with a solution of saturated ammonium chloride solution (50 ml) and EA (250 ml). The organic layer was separated and the aqueous layer was washed with EA (2 x 100 ml). The combined organic extracts were dried over sodium sulfate, filtered and concentrated to give the crude product as a brown oil. The product was isolated after CC with PE / EA (19: 1 to 3: 1). The reaction and purification were repeated twice on the same scale and the combined product (3.13 g) was repurified under the same conditions to make final product 1 available (1.7 g, 19%). 1H NMR (CDCh): 7.93 (m, 1H), 7.90-7.85 (m, 1H), 7.50-7.30 (m, 5H), 6.88 (s, 1H), 6.75-6.72 (m, 1H), 4.80 (s, 2H), 3.88 (s, 3H), 3.20-3.10 (m, 1H), 3.00-2, 91 (m, 2H), 2.60-2.49 (m, 2H), 2.15-2.08 (m, 1H), 1.30-1.25 (m, 2H), 1.15- 1.10 (m, 2H). Example 2: 3 - ((1s, 3s) -3- (2-chloro-4 - (((5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3- acid hydroxycyclobutyl) benzoic (2)

[000115] Compound 1 (1.7 g, 2.84 mmol) was dissolved in THF (100 ml) at room temperature. A solution of LiOH (285 mg, 4.2 eq) in water (20 ml) was added and the solution was stirred and heated to 35 ° C for three days. After this time the THF was removed under low pressure. The remaining aqueous solution was diluted with water (25 ml) and washed with Et2Ü (2 x 50 ml). The aqueous layer was then transferred to a round bottom flask and acidified to pH 6 using 1 N HCI. The white precipitate formed was filtered off and dried under low pressure at 50 ° C to give the title compound 2 (1.3 g , 78%, single isomer by 1H RNM and LC-mS) as a white solid. 1H RNM (400 MHz, CD3OD) δ: 7.98 (s, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.58-7.46 (m, 5H), 7, 41 (t, J = 7.6 Hz, 1H), 6.91 (d, J = 2.4 Hz, 1H), 6.80 (dd, J = 8.8, 2.4 Hz, 1H), 4.95 (s, 2H), 3.29-3.25 (m, 2H), 2.96 (m, 1H), 2.55-2.49 (m, 2H), 2.37 (m, 1H), 1.24-1.22 (m, 4H), MS (ESI ') m / z: 584 (582) [M-1] ~.
[000116] NOEs of relevant intensives (obtained from the ROESY spectrum; arrows below) indicate that the two aromatic fractions are 1,3-trans oriented in Example 2.
Alternative route to Example 2 Step 1: 3- (3-bromophenyl) cyclobutanone (2a)
[000117] N, N-dimethylacetamide (9.0 g, 103 mmol) was dissolved in 1,2-dichloroethane (200 ml). the solution was cooled to 0 ° C before trifluoromethanesulfonic anhydride (63 g, 223 mmol) was added. The reaction was stirred for another 60 minutes at 0 ° C. Then 1-bromo-3-vinylbenzene (15 g, 81.9 mmol) and 2,4,6-collidine (10.5 g, 86.6 mmol) were added. The reaction was heated to reflux overnight, terminated by adding water (300 ml) and stirred for 2 hours at room temperature. The mixture was extracted with DCM (300 ml x 3). The combined organic layers were dried over Na2SO4 and concentrated in vacuo. CC purification (EA / PE = 1:20) gave the title compound 2a (5.0 g, 27%) as a faded yellow solid. Step 2: 3- (3-bromophenyl) -1- (2-chloro-4 - (((5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) cyclobutanol (2b)
[000118] To a solution of compound 1a (14 g, 29.6 mmol) in dry THF (500 ml) at -78 ° C was added n-buLi dropwise (18.5 ml_, 1.6 M in hexane , 29.6 mmol). The mixture was stirred for an additional 1 hour at - 78 ° C and a solution of compound 2a (6.5 g, 28.9 mmol) in dry THF (50 ml) was added dropwise. The resulting mixture was stirred at - 78 ° C for 1 hour and then warmed to room temperature and finished with saturated aqueous NH4 Cl (500 ml). The mixture was extracted with EA (500 ml_2). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by CC (EA / PE = 1: 5) to give the title compound 2b (6.5 g, 37%) as a white solid. Step 3: 3- (3-cyanophenyl) -1- (2-chloro-4 - (((5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) cyclobutanol (2c)
[000119] To a solution of compound 2b (3.1 g, 5 mmol) in DMF (50 ml_) was added under an atmosphere of argon Zn (CN) 2 (500 mg, 4.3 mmol), Pd2 (dba) 3 (300 mg, 0.33 mmol) and xanthophos (150 mg, 0.31 mmol). The mixture was stirred for 10 hours at 115 ° C under microwave irradiation. After cooling to room temperature the reaction mixture was diluted with water (250 ml_) and extracted with EA (250 ml_2). The combined organic layers were washed with brine (100 ml x 3) and dried over Na2SO4. The residue was purified by CC (EA / PE) to give the title compound 2c (1.2 g, 42%) as a faded yellow solid. Step 4: Acid 3 - ((1s, 3s) -3- (2-chloro-4 - ((5-cyclopropyl-3- (2.6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3-hydroxycyclobutyl) benzoic (2)
[000120] To a solution of compound 2c (15 g, 24.2 mmol) in EtOH (750 ml) was added aqueous NaOH (40 g in 100 ml of water). The resulting mixture was heated to reflux overnight and then cooled to room temperature. The reaction was concentrated in vacuo to remove the volatile solvent, diluted with water (1,000 ml) and the pH was adjusted to 2 with diluted aqueous HCl (1 N). The formed precipitate was collected by filtration to give the crude product as a yellow solid (13.8 g). Purification by preparative reverse phase HPLC (RP-HPLC) provided the title compound 2 (8.0 g, 56%, single isomer by 1H RNM) as a white solid. Preparative Example 3
Step 1: methyl 3- (3-hydroxyazetidin-1-yl) benzoate (3a)
[000121] To a solution of methyl 3-iodobenzoate (4.5 g, 17.2 mmol) in DMSO (30 mL) was added 3-azetidin-3-ol chloride hydrogen salt (1.3 g, 11, 8 mmol), CS2CO3 (9.5 g, 29.2 mmol), Cul (446 mg, 2.3 mmol) and L-proline (540 mg, 4.7 mmol) and then the mixture was heated to 90 ° C for 18 hours under an argon atmosphere. The solution was diluted with EA and water and the organic layer was washed with brine three times, concentrated under low pressure and purified by CC (PE / EA = 2: 1) to give compound 3a (1.6 g, 66%) in the form of a yellow solid. Step 2: methyl 3- (3-oxoazetidin-1-yl) benzoate (3)
[000122] To a solution of compound 3a (1.60 g, 7.7 mmol) in dry DCM (30 ml) was added Dess-martin periodinane (6.5 g, 15.4 mmol) at 0 ° C and the mixture was stirred at room temperature for 2 hours under N2 atmosphere. The mixture was finished with saturated sodium bicarbonate solution and diluted with EA. The organic portion was washed with brine, dried over Na2SO4, filtered, concentrated under low pressure and purified by CC (PE / EA = 4: 1) to give compound 3 (1.2 g, 75%) as a solid White. Example 4: 3 - ((1 s, 3s) -3- (2-chloro-4 - (((5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3- hydroxycyclobutyl) -N- (methylsulfonyl) benzamide (4)

[000123] To the solution of compound 2 (100 mg, 0.17 mmol) in DCM (5 ml), EDCI HCI (100 mg, 0.52 mmol), DMAP (100 mg, 0.81 mmol) and MeSO2NH2 ( 40 mg, 0.42 mmol). The mixture was stirred at 30 ° C overnight and then diluted with EA and washed with H2O, brine and dried over Na2SO4. Concentration and purification in vacuo by preparative TLC gave crude target compound as a light yellow solid. Purification by RP-HPLC provided the title compound 4 (38 mg, 33%) as a white solid. 1H RNM (400 MHz, CD3OD) δ: 7.87 (s, 1H), 7.74 (d, J = 7.6 Hz, 1H), 7.61- 7.53 (m, 4H), 7, 50-7.46 (m, 2H), 6.91 (d, J = 2.4 Hz, 1H), 6.80 (dd, J = 8.8, 2.4 Hz, 1H), 4.95 (s, 2H), 3.38 (s, 3H), 3.30-3.26 (m, 2H), 3.01 (m, 1H), 2.57-2.51 (m, 2H), 2.37 (m, 1H), 1.25-1.23 (m, 4H). MS (ESI-) m / z: 659 [M-1] -. Example 5: 3- (3- (2-chloro-4 - (((5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3-hydroxycyclobutyl) benzenesulfonamide (5)
Step 1: 3- (3- (benzylthio) phenyl) -1- (2-chloro-4 - (((5-cyclopropyl-3- (2l6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) cyclobutanol (5a)
[000124] To a solution of compound 2b (619 mg, 1 mmol) in toluene (20 mL) under an argon atmosphere were added K2CO3 (276 mg, 2 mmol), phenylmethanethiol (125 mg, 1 mmol), Pd2 (dba) 3 (200 mg, 0.22 mmol) and xanthophos (75 mg, 0.16 mmol). Then the mixture was stirred at 115 ° C for 4 hours. After being cooled to room temperature, the reaction was diluted with water (100 ml) and extracted with EA (100 ml x 2). The combined organic layers were washed with brine (100 ml x 2), dried over Na2SÜ4 and concentrated to dryness. CC purification gave compound 5a (200 mg; 30%) as a faded yellow solid. 1H RNM (400 MHz, CDCI3) ó: 7.36-7.32 (m, 3H), 7.28-7.07 (m, 9H), 7.01 (d, J = 7.2 Hz, 1H ), 6.82 (d, J = 2.0 Hz, 1H), 6.66 (dd, J = 8.8, 2.0 Hz, 1H), 4.75 (s, 2H), 4.04 (s, 2H), 3.06-3.00 (m, 2H), 2.84-2.78 (m, 2H), 2.44-2.38 (m, 3H), 2.09 (m , 1H), 1.24-1.18 (m, 2H), 1.11-1.08 (m, 2H). MS (ESI +) m / z: 662 [M + 1] +. Step 2: 3- (3- (2-chloro-4 - ((5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3-hydroxycyclobutyl) benzene-1 chloride - sulfonyl (5b)
[000125] To a solution of compound 5a (34 mg, 0.05 mmol) in CH3CN / HOAC / H2O (1 ml / 37 pL / 25 pL) was added 2,4-dichloro-5,5-dimethylidantoin (20 mg , 0.1 mmol). The mixture was stirred at 0-5 ° C for 2 hours. The reaction was diluted with water and extracted with CH2 Cl2. The combined organic layers were washed with a 5% NaHCOa solution, brine and dried over Na2SO4. Concentration to dryness provided the crude product 5b (30 mg) as a colorless oil, which was used directly in the next step. Step 3: 3- (3- (2-chloro-4 - (((5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3-hydroxycyclobutyl) benzenesulfonamide (5)
[000126] To the solution of compound 5b (30 mg) in CH3CN (2 ml) was added NH4OH (0.3 ml). The mixture was stirred at room temperature for 1 hour. Concentration to dryness and purification by preparative RP-HPLC gave the title compound 5 (3.5 mg, 10% for two steps) as a white solid. 1H RNM (400 MHz, CDCh) δ: 7.85 (s, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.54-7.41 (m, 5H), 7, 35 (d, J = 8.4 Hz, 1H), 6.90 (s, 1H), 6.75 (d, J = 8.4 Hz, 1H), 4.83 (s, 2H), 4, 77 (s, broad, 2H), 3.20 (t, J = 10.4 Hz, 2H), 3.04 (m, 1H), 2.58 (t, J = 10.6 Hz, 2H), 2.17 (m, 1H), 1.31-1.30 (m, 2H), 1.20-1.16 (m, 2H). MS (ESI-) m / z: 617 [M-1] '. Example 6: 1- (2-chloro-4 - ((5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3- (3- (methylsulfonyl) phenyl) cyclobutanol ( 6)

[000127] To the solution of compound 2b (200 mg, 0.32 mmol) in DMSO, sodium methanesulfinate (50 mg, 0.46 mmol), Cul (20 mg, 0.1 mmol), L-proline (37 mg , 0.32 mmol) and diisopropylethylamine (DIEA) (41 mg, 0.32 mmol) were added. The mixture was stirred at 95 ° C overnight and then diluted with water and extracted with EA. The combined organic layers were washed with water and dried over Na2SO4. Concentration to dryness under low pressure and purification by preparative RP-HPLC gave the title compound 6 as a white solid (35 mg, 21%, single isomer by 1M RNM and LC-mS). 1H RNM (400 MHz, CDCh) δ: 7.84 (s, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H) , 7.53 (t, J = 7.6 Hz, 1H), 7.44-7.41 (m, 3H), 7.34 (t, J = 7.2 Hz, 1H), 6.90 ( d, J = 2.8 Hz, 1H), 6.75 (dd, J = 8.4, 2.0 Hz, 1H), 4.83 (s, 2H), 3.24-3.19 (m , 2H), 3.08-3.04 (m, 4H), 2.62-2.56 (m, 2H), 2.17 (m, 1H), 1.31-1.29 (m, 2H ), 1.20-1.16 (m, 2H). MS (ESI +) m / z: 618 (620) [M + 1] +, 600 (602) [M-H2O + 1] +. Example 7: 5 - ((1s, 3s) -3- (2-chloro-4 - (((5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3-hydroxycyclobutyl ) Methyl -1-isopropyl-1H-pyrazol-3-carboxylate (7)
Step 1: Methyl 1-isopropyl-5-vinyl-1H-pyrazol-3-carboxylate (7a)
[000128] A suspension of methyltriphenylphosphonium bromide (2.69 g, 7.52 mmol) in dry THF (40 ml) was cooled to -78 ° C and n-butyllithium (1.6 M solution in hexane, 3.7 ml, 5.91 mmol) was added dropwise. The yellowish orange suspension was stirred at -78 ° C for 50 minutes and then a solution of methyl 5-formyl-1-isopropyl-1H-pyrazol-3-carboxylate (prepared as described in WO 2011/020615, 1.05 g , 5.37 mmol) in dry THF (10mL) was added dropwise. The mixture was stirred at -78 ° C for 1.75 hours, the cooling bath was removed and the mixture (ice-white suspension) was stirred at room temperature for 1 hour. The mixture was then partitioned between diluted aqueous NaHCOs solution (150 ml) and EA (150 ml). The aqueous layer was extracted twice with EA (50 ml each) and the combined organic layer was washed twice with water (50 ml each) and concentrated without drying to give 2.74 g of a yellow oil which slowly crystallized. The crude product was purified by CC (pre-adsorption with CH2 Cl2, hexane / EA 4: 1) to give alkene 7a (590 mg, 57%) as a colorless oil. 1H NMR (DMSO-de) δ: 7.02 (s, 1H), 6.87 (dd, J = 17.3, 11.2 Hz, 1H), 5.94 (dd, J = 17.3, 1.3 Hz, 1H), 5.45 (dd, J = 11.2, 1.3 Hz, 1H), 4.80 (sept, J = 6.6 Hz, 1H), 3.79 (s, 3H), 1.38 (d, J = 6.6 Hz, 6H). C10H14N2O2 (194.23). LC-mS (ESI): 195 [M + H] +. Step 2: Methyl 1-isopropyl-5- (3-oxocyclobutyl) -1H-pyrazol-3-carboxylate (7b)
[000129] The reaction was carried out in two dry sealed tubes (two batches of equal quantity). The batches were combined for manufacturing and purification. Single batch procedure: To a solution of A /, / V-dimethylacetamide (0.22 mL, 2.34 mmol) in 1,2-dichloroethane (12 mL) under nitrogen at -15 to -20 ° C was added dropwise a drop of trifluoromethanesulfonic anhydride (0.43 mL, 2.57 mmol), forming an opaque suspension. The mixture was stirred at -15 ° C for 10 minutes, and a solution of alkene 7a (151 mg, 0.78 mmol) and sym.-collidine (0.42 mL, 3.12 mmol) in 1,2-dichloroethane (3 mL) was added dropwise (yellow solution formed). Upon completion of the addition, the cooling was removed from the bath, the mixture was naturally heated to room temperature (turbid orange solution) and the tube was sealed. The mixture was then stirred at 90 ° C for 15 hours (brown mixtures). Water (5 mL) was added at room temperature and the mixtures were stirred at 100 ° C for 2 hours (turbid two-phase solutions). After cooling to room temperature, the mixtures were combined and partitioned between dilute aqueous NaHCOs and CH2 Cl2 solution and the aqueous layer was extracted three times with CH2 Cl2 (30 ml each). The combined organic layer was dried (Na2SO4), filtered and concentrated to give a brown oil (2.2 g). Purification by CC (6x13 cm, pre-adsorption with CH2 Cl2, toluene / EA 3: 1) gave cyclobutanone 7b (115.5 mg, 31%) as a yellow oil. 1H NMR (DMSO-de) δ: 6.81 (s, 1H), 4.58 (sept, J = 6.5 Hz, 1H), 3.78 (s, 3H), 3.85-3.73 (m , 1H), 3.59-3.45 (m, 2H), 3.37-3.24 (m, 2H, partially overlapped by water signal), 1.39 (d, J = 6.6 Hz, 6H), C12H16N2O3 (236.27). LC-mS (ESI): 237 [M + H] +. Step 3: 5 - ((1 s, 3s) -3- (2-chloro-4 - (((5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3- hydroxycyclobutyl) methyl-1-isopropyl-1H-pyrazol-3-carboxylate (7)
[000130] A solution of bromide 1a (368 mg, 0.78 mmol) in dry THF (6 mL) was cooled to -78 ° C and a 1.6 M n-butyllithium solution in hexanes (0.48 mL, 0 , 76 mmol) was added dropwise. The mixture was stirred at -78 ° C for 20 minutes and a solution of cyclobutanone 7b (164 mg, 0.69 mmol) in dry THF (4 ml) was added dropwise. The mixture was stirred at -78 ° C for 2.5 hours and saturated aqueous NH4 Cl solution (1 ml) was added dropwise at this temperature. The cooling bath was removed and the mixture was naturally heated to room temperature and stirred at room temperature for 0.5 hour. The mixture was then added to the diluted aqueous NH4 Cl solution and extracted three times with EA. The combined organic layer was dried (Na2SO4), filtered and concentrated to give 516 mg of an almost colorless oil. Purification by CC (4.5x23 cm, pre-adsorption with CH2Cl2, eluent hexane / acetone = 2: 1) provided recovered cyclobutanone 7b (31.3 mg, 19%, slightly yellow oil) and impure product (333 mg). Repurification by CC (4x22 cm, hexane / EA = 1: 1) or preparative TLC gave pure product 7 (210 mg, 48%) as white foam. 1H NMR (DMSO-de) δ: 7.65 (d, J = 2.1 Hz, 1H), 7.62 (s, 1H), 7.59-7.48 (m, 2H), 6.92 (d, J = 2.4 Hz, 1H), 6.76 (dd, J = 8.6, 2.6 Hz, 1H), 6.66 (s, 1H), 5.49 (s, 1H) , 4.92 (s, 2H), 4.42 (quint-like m, J = 6.5 Hz, 1H), 3.78 (s, 3H), 3.24-3.11 (m, 2H, partially overlapped by water signal), 3.04-2.90 (m, 1H), 2.54-2.33 (m, 3H, partially overlapped by DMSO signal), 1.32 (d, J = 6, 5 Hz, 6H), 1.26 - 1.08 (m, 4H). C31H30CI3N3O5 (630.95). LC-mS (ESI): 630, 632 [M + H] +. Example 8: 5 - ((1s, 3s) -3- (2-chloro-4 - ((5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3 -hydroxycyclobutyl) -1-isopropyl-1H-pyrazole-3-carboxylic (8)

[000131] Ester 7 (98.3 mg, 0.156 mmol) was dissolved in a mixture of THF (7.5 ml), MeOH (2.5 ml) and water (2.5 ml) and LOH H2O (65 mg , 1.56 mmol) were added at room temperature. The mixture was stirred at room temperature for 18 hours. The mixture was partitioned between diluted aqueous NH4 Cl solution and EA and the organic layer was washed once with water. The combined aqueous layer was extracted twice with EA. The combined organic layer was dried (Na2SO4), filtered and concentrated to give 103 mg of an almost white solid. The product was purified by CC (3x3.5 cm, EA / EtOH = 10: 1 to 1: 4) to make 8 (94.8 mg, 99%) available as a white solid. 1H NMR (DMSO-d6) δ: 7.66-7.60 (m, 1H), 7.62 (s, 1H), 7.59- 7.49 (m, 2H), 6.91 (d, J = 2.5 Hz, 1H), 6.76 (dd, J = 8.6, 2.4 Hz, 1H), 6.38 (s, 1H), 5.51 (s, 1H, interchangeable with D2O ), 4.92 (s, 2H), 4.31 (quint-like m, J = 6.5 Hz, 1H), 3.25-3.08 (m, 2H, partially overlapped by a water signal), 2.93-2.77 (m, 1H), 2.57-2.43 (m, 1H, hidden by DMSO signal), 2.43-2.29 (m, 2H, partially overlapped by DMSO signal) ), 1.29 (d, J = 6.5 Hz, 6H), 1.26-1.08 (m, 4H). The CO2H signal does not appear in the spectrum. C30H28CI3N3O5 (616.92). LC-mS (ESI): 616, 618 [M + H] +. Alternative route to Example 8 Step 1: 1- (3-methylenocyclobutyl) ethanone (8a)
[000132] Methylene cyclobutane carbonitrile (5.0 g, 53.7 mmol) was dissolved in dry diethyl ether (25 mL), cooled in an ice bath and MeMgBr (26.8 mL, 80.5 mmol, 3 M in ether) was added dropwise. The mixture was left stirring overnight at room temperature, cooled to 0 ° C, carefully finished with 15% aqueous NaHSO4 solution (100 ml). The mixture was stirred at room temperature for 30 minutes and the layers were separated. The aqueous phase was extracted with pentane (50 ml) and diethyl ether (50 ml). The combined organic layers were washed with brine and dried over Na2SO4. The solvents were removed under vacuum at room temperature and the crude product was obtained as a yellowish liquid. Step 2: Ethyl 4- (3-methylenocyclobutyl) -2,4-dioxobutanoate (8b)
[000133] Sodium (1.15 g, 49.9 mmol) was dissolved in dry EtOH (30 mL, denatured with 5% diethyl ether). Compound 8a (5.5 g, 49.9 mmol, crude) was dissolved in dry EtOH (45 ml) and the prepared sodium ethoxide solution was added. This mixture was stirred at room temperature for 15 minutes and then diethyl oxalate (6.8 ml, 49.9 mmol) was added dropwise. The reaction mixture was placed in a preheated oil bath (at 67 ° C) and stirred at this temperature for 4.5 hours. The mixture was left at room temperature overnight. The solvent was removed, EA (100 ml) and 1M HCI (70 ml) were added and the organic phase was separated. The aqueous phase was re-extracted with EA (50 ml). The combined organic phases were washed with water, brine and dried over anhydrous Na2SO4. The solvent was removed under low pressure and the residue was purified on silica using hexanes / MTBE 9: 1 as eluent giving pure product 8b. Yield: 6.29 g, 56% in both stages. 1H NMR (CDCh), δ (ppm): 6.36 (s, 1H), 4.85-4.80 (m, 2H), 4.34 (q, J = 8.0 Hz, 2H), 3 , 35-3.25 (m, 1H), 3.05-2.85 (m, 4H), 1.36 (t, J = 8.0 Hz, 3H). Step 3: Ethyl 1-isopropyl-5- (3-methylenocyclobutyl) -1H-pyrazol-3-carboxylate (8c)
[000134] Compound 8b (6.29 g, 29.9 mmol) was dissolved in dry EtOH (65 mL, denatured with 5% MeOH) and hydrazine isopropyl hydrochloride (3.97 g, 35.9 mmol) added. The reaction mixture was stirred for 3 hours at room temperature. The solvent was removed and to the oily residue EA (100 ml), water (50 ml) and saturated NaHCOa (50 ml) were added sequentially. The layers were separated and the aqueous phase was re-extracted with EA (50 ml). The combined organic phases were washed with brine (70 ml) and dried over anhydrous Na2SO4. The solvent was removed in vacuo and the residue was dried under low pressure. Yield: 7.23 g (contains 3.4% EtOAc per MRI, pure recalculated yield: 6.98 g, 94%). Crude product 8c is 98% pure by HPLC and RNM. 1H NMR (CDCh), δ (ppm): 6.62 (s, 1H), 4.88-4.82 (m, 2H), 4.42-4.32 (m, 3H), 3.56- 3.45 (m, 1H), 3.17-3.07 (m, 2H), 2.88-2.79 (m, 2H), 1.49 (d, J = 8.0 Hz, 6H) , 1.37 (t, J = 8.0 Hz, 3H). Step 4: ethyl 1-isopropyl-5- (3-oxocyclobutyl) -1H-pyrazol-3-carboxylate (8d)
[000135] Compound 8c (6.55 g, 26.0 mmol) was dissolved in a mixture of MeCN (77 mL) and water (13 mL) and cooled in an ice bath. To this solution RuChxH2O (0.19 g, 0.86 mmol) was added, followed by addition in portions of NalCU (19.35 g, 90.9 mmol). An exotherm was observed during this addition. The thick sludge obtained was stirred at room temperature for 45 minutes. The reaction mixture was diluted with aqueous Na2S20s solution (10%, 260 ml), water (50 ml) and DCM (100 ml). The phases were separated and the aqueous phase was extracted with DCM (2x70 ml). The combined organic phases were washed with aqueous Na2S2θ3 solution (10%, 50 ml), water (100 ml), brine (100 ml) and dried over anhydrous Na2SO4. The crude product (6.5 g) was purified on silica, eluting with hexanes / MTBE to give the pure product as an oil which solidified upon storage at -20 ° C. Yield: 5.8 g (78% in both stages). 1H NMR (DMSO-dδ), δ (ppm): 6.78 (s, 1H), 4.57 (h, J = 8.0 Hz, 1H), 4.26 (q, J = 8.0 Hz , 2H), 3.85-3.75 (m, 1H), 3.58-3.45 (m, 2H), 3.35-3.25 (m, 2H), 1.39 (d, J = 8.0 Hz, 6H), 1.28 (t, J = 8.0 Hz, 3H). Step 5: 4 - (((4-bromo-3-chlorophenoxy) methyl) -5-cyclopropyl-3- (2,6-dichloropheniDisoxazole (8e)
[000136] 3-chloro-4-bromophenol (3.8 g, 18.3 mmol) was mixed with (5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methanol (3.47 g , 12.2 mmol) and triphenylphosphine (6.51 g, 24.4 mmol) in toluene (150 mL). The mixture was cooled in an ice bath and DIAD (4.8 ml, 24.4 mmol) as a solution in toluene (10 ml) was added dropwise. The reaction was stirred at room temperature for 21 hours and the solvents were removed on a rotavap leaving a yellow oily residue. This was dissolved in DCM (200 ml), silica (~ 20 g) was added and the mixture was evaporated to dryness. This material was loaded on top of a silica column and purified eluting with hexanes / MTBE 9: 1. The product containing fractions was pooled and the solvent removed under low pressure, leaving the pure product 8e as a colorless oil which crystallized by drying under vacuum overnight. Yield: 5.07 g (88%). 1H NMR (CDCh), δ (ppm): 7.45-7.30 (m, 4H), 6.90 (s, 1H), 6.60-6.55 (m, 1H), 2.15- 2.07 (m, 1H), 1.32-1.25 (m, 2H), 1.20-1.11 (m, 2H). Step 6: 5 - ((1 s, 3s) -3- (2-chloro-4 - (((5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3- hydroxycyclobutyl) ethyl-1-isopropyl-1H-pyrazol-3-carboxylate (8f)
[000137] LiCI (0.684 g, 16.15 mmol) was dissolved in THF (20 mL) at room temperature and / 'PrMgCI (2.0 M in THF, 8.1 mL, 16.15 mmol) was added. The mixture was stirred for 10 minutes at room temperature, cooled in an ice bath and a solution of compound 8e (2.55 g, 5.38 mmol) in THF (20 ml) was added for 5 minutes. The cooling bath was removed and the mixture was stirred at room temperature for 4 hours. The mixture was cooled to -10 ° C and a solution of compound 8d (1.48 g, 5.92 mmol) in THF (16 ml) was added quickly. The mixture was stirred at room temperature for 90 minutes and then aqueous 0.5 M NaHSO4 (35 ml) and EA (50 ml) were added. The resulting mixture was stirred for 10 minutes, the layers were separated and the aqueous layer was extracted with EA (30 ml). The combined organic phases were washed with aqueous NaHCOs (50 ml), brine (50 ml) and dried over anhydrous Na2SO4. The crude product (3.79 g) was obtained after removing the solvent as a white foam. 3.6 g of this crude was purified on a silica column, eluting with 3: 2 hexanes / EA to give the pure product 8f as a solid foam. Yield: 1.62 g (49%). 1H NMR (DMSO-de), δ (ppm): 7.65-7.47 (m, 4H), 6.93.6.91 (m, 1H), 6.79-6.72 (m, 1H), 6.65 (s, 1H), 5.48 (s, 1H), 4.92 (s, 2H), 4.42 (h, J = 8.0 Hz, 1H), 4.26 (q, J = 8.0 Hz, 2H), 3.32 (s, 2H), 3.22-3.14 (m, 2H), 3.05-2.90 (m, 1H), 2.45-2, 35 (m, 2H), 1.35-1.10 (m, 14H). Step 7: 5 - ((1s, 3s) -3- (2-chloro-4 - ((5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3 acid -hydroxycyclobutyl) -1-isopropyl-1H-pyrazole-3-carboxylic (8)
[000138] Compound 8f (1.60 g, 2.48 mmol) was dissolved in THF (100 ml), then MeOH (50 ml), water (50 ml) and LiOH * H2O (1.04 g, 24 , 8 mmol) were added sequentially. The mixture was stirred for 4.5 hours at room temperature and then concentrated under low pressure to remove MeOH and THF. The remaining aqueous solution was acidified by adding 1M aqueous HCI (24 mL) to reach a pH of 4.55 (pH electrode control). At pH 7, a precipitate started to form. The formed solid was filtered off, washed on the filter with water and dried under vacuum at room temperature to give the product 8 as a white powder. Yield: 1.40 g (92%). 1H NMR (CDCh), δ (ppm): 7.44 - 7.32 (m, 4H), 6.91 (d, J = 4.5 Hz, 1H), 6.78 (s, 1H), 6 , 75 (dd, J = 4.5 Hz, J = 8.0 Hz, 1H), 4.83 (s, 2H), 4.35-4.20 (m, 1H), 3.25-3, 14 (m, 2H), 3.04-2.90 (m, 1H), 2.62-2.54 (m, 2H), 2.21-2.11 (m, 1H), 1.46 ( d, J = 8.0 Hz, 6H), 1.34-1.28 (m, 2H), 1.20-1.14 (m, 2H), 13C-RNM (CDCh), δ (ppm): 172.7, 164.8, 159.2, 158.4, 147.2, 141.3, 135.8, 134.1, 132.8, 131.3, 128.1, 127.6, 127, 3, 117.7, 113.3, 110.0, 106.3, 73.1, 59.8, 51.1, 41.7, 22.6, 22.0, 8.5, 7.8. MS (ESI +) m / z: 616.5 [M + 1] +. Example 8A: 5 - ((1r, 3r) -3- (2-chloropropyl-3- ((5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3-hydroxycyclobutyl ) -1-isopropyl-1H-pyrazol-3-carboxylate (8A)

[000139] Example 8A can be prepared by subjecting crude product 8f to ester hydrolysis as described for 8 and isolation of crude product 8 as a secondary isomer by preparative RP-HPLC. 1H NMR (CDCh), δ (ppm): 7.42-7.30 (m, 2H), 7.11 (d, J = 8.0 Hz, 1H), 6.75-6.65 (m, 1H), 6.57 (s, 1H), 4.79 (s, 2H), 4.50-4.41 (m, 1H), 3.96-3.85 (m, 1H), 2.98 -2.90 (m, 2H), 2.67-2.57 (m, 2H), 2.20-2.09 (m, 1H), 1.51 (d, J = 8.0 Hz, 6H ), 1.32-1.14 (m, 4H). 13M RNC (CDCh), õ (ppm): 172.6, 166.2, 159.2, 158.4, 147.4, 141.2, 135.7, 134.6, 132.8, 131.3 , 128.1, 127.7, 127.5, 116.8, 113.5, 110.0, 105.8, 75.1, 59.8, 51.2, 41.8, 25.4, 22 , 6, 8.5, 7.8, MS (ESI +) m / z: 616.3 [M + 1] +.
[000140] The transanular configuration of the main isomer (compound 8) and the secondary isomer (compound 8A) was confirmed by NOE experiments. The indicative NOEs detected between protons are indicated in the following figures by double arrows:
[000141] NOEs detected for example 8 with 1,3-trans-annular configuration of aromatic fractions are shown in Figure 1.
[000142] NOEs detected for example 8A with 1,3-cis transanular configuration of the aromatic fractions are shown in Figure 2. Example 9: 6- (3- (2-chloro-4 - ((5-cyclopropyl-3- ( Methyl 2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3-hydroxycyclobutyl) -1-methyl-1 H-indazol-3-carboxylate (9)
Step 1: Methyl 1-methyl-6-vinyl-1H-indazole-3-carboxylate (9a)
[000143] To the solution of methyl 6-bromo-1-methyl-1H-indazol-3-carboxylate (60 mg, 0.22 mmol) in DMF (10 mL), tributyl (vinyl) tin (99 pL, 0, 34 mmol), Pd (Ph3) 4 (11 mg, 9 pmol) was added. After the addition was completed, the mixture was stirred at 90 ° C for 4 hours under Ar. Then the solvent was removed under low pressure. CC purification provided compound 9a (52 mg, 88%). Step 2: methyl 1-methyl-6- (3-oxocyclobutyl) -1H-indazole-3-carboxylate (9b)
[000144] Following the procedure described in example 7 / Step 2, compound 9b was obtained from 9a in 57% yield. 1H RNM (400 MHz, CDCh) δ: 8.14 (d, J = 8.4 Hz, 1H), 7.31 (s, 1H), 7.23 (d, J = 8.8 Hz, 1H) , 4.13 (s, 3H), 3.99 (s, 3H), 3.87-3.79 (m, 1H), 3.58-3.51 (m, 2H), 3.33-3 , 26 (m, 2H). m / z: 259 [M + 1] +. Step 3: 6- (3- (2-chloro-4 - (((5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3-hydroxycyclobutyl) -1 -methyl- 1 Methyl H-indazole-3-carboxylate (9)
[000145] Following the procedure described in example 7 / Step 3, compound 9 was obtained from 9b in 40% yield. Example 10: 6- (3- (2-Chloro-4 - ((5-cyclopropyl-3- (2,6-dichlorophenyl) isoxazol-4-yl) methoxy) phenyl) -3-hydroxycyclobutyl) -1- methyl-1H-indazole-3-carboxylic (10)

[000146] Following the procedure described in example 8, compound 10 was obtained from compound 9 in 45% yield as a white solid. 1H RNM (400 MHz, CDCh) δ: 8.14 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 8.8 Hz, 1H), 7.43-7.32 ( m, 4H), 7.29 (m, 1H), 6.92 (d, J = 2.4 Hz, 1H), 6.76 (dd, J = 7.2 Hz, 2.4 Hz, 1H) , 4.84 (s, 2H), 4.18 (s, 3H), 3.45-3.40 (m, 1H), 3.28-3.23 (m, 2H), 3.19-3 , 10 (m, 1H), 2.68-2.63 (m, 2H), 2.21-2.14 (m, 1H), 1.33-1.29 (m, 2H), 1.20 -1.15 (m, 2H). m / z: 638 [M + 1] +. Preparative Example 11
Step 1: Methyl 5- (3-hydroxyazetidin-1-yl) nicotinate (11a)
[000147] A mixture of methyl 5-bromonicotinate (2.00 g, 9.26 mmol), azetidin-3-ol (1.01 g, 9.26 mmol), CS2CO3 (9.06 g, 27.8 mmol), BINAP (1.15 g, 1.85 mmol) and Pd (OAc) 2 (0.44 g, 1.85 mmol) in dry dioxane (115 mL) was heated overnight at 85 ° C under N2 atmosphere. The resulting mixture was filtered, concentrated under low pressure and purified by preparative HPLC to give compound 11a (250 mg, 13%) as a yellow solid.
[000148] Step 2: methyl 5- (3-oxoazetidin-1-yl) nicotinate (11)
[000149] To a solution of compound 11a (250 mg, 1.20 mmol) in dry DCM (15 mL) was added Dess-martin periodinane (1.014 g, 2.40 mmol) at 0 ° C under N2 atmosphere and at The solution was stirred at room temperature for 2 hours. The resulting solution was finished with saturated sodium bicarbonate solution and diluted with EA. The organic portion was washed with brine, dried over Na2SO4, filtered, concentrated under low pressure and purified by CC (DCM / MeOH = 150: 1) to give compound 11 (140 mg, 57%) as a yellow solid. Preparative Example 12
[000150] Using a procedure similar to that described in Preparative Example 11 the following compound was prepared:
Example 13/1 to 13/9
[000151] The following table additionally lists examples prepared according to the preparatory examples and examples mentioned above. All compounds listed were prepared as single isomers.



Example 14/1 and 14/2
[000152] Using a procedure similar to that described in examples 1 to 13 and previous schemes, the following compounds were obtained using the appropriate building blocks.


[000153] The following compound can be prepared in the same way using similar procedures described previously:
Essay FRET activity test
[000154] Determination of a ligand-mediated cofactor peptide interaction to quantify ligand binding at the nuclear receptor FXR was performed as follows: Preparation of the human FXR alpha ligand binding domain: The human FXRalpha LBD was expressed in E. coli BL21 (DE3) strain as a GST fusion protein labeled at the N-terminus. The DNA encoding the FXR binding ligand domain was cloned into the pDEST15 vector (Invitrogen). The expression was under the control of an IPTG-inducible T7 promoter. The amino acid limits of the ligand binding domain were amino acids 187-472 from database entry NM_005123 (RefSeq). FXR-LBD expression and purification: An overnight preculture of a transformed E.coli strain was diluted 1:20 in LB-ampicillin medium grown at 30 ° C at an optical density of OD6oo = 0.4-0, 6. Gene expression was then induced by adding 0.5 mM IPTG. Cells were incubated for another 6 hours at 30 ° C, 180 rpm. Cells were collected by centrifugation (7,000 x g, 7 minutes, room temperature). Per liter of original cell culture, 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) and left on ice for 30 minutes. Cells were then sonicated and cell fragments removed by centrifugation (22,000 x g, 30 minutes, 4 ° C). Per 10 mL of supernatant, 0.5 mL of pre-washed Glutathione sepharose 4B slurry (Qiagen) was added and the suspension was kept spinning slowly for 1 hour at 4 ° C. Microspheres of Glutathione sepharose 4B were pelleted by centrifugation (2,000 x g, 15 s, 4 ° C) and washed twice in wash buffer (25 mM Tris, 50 mM KCI, 4 mM MgCh and 1M NaCI). The precipitate was resuspended in 3 ml elution buffer per liter of original culture (elution buffer: 20 mM Tris, 60 mM KCI, 5 mM MgCh and 80 mM Glutathione added immediately before use as a powder). The suspension was left spinning for 15 minutes at 4 ° C, the microspheres were precipitated and eluted again with half the volume of elution buffer as the first time. The eluates were pooled and dialyzed overnight in 20 mM Hepes buffer (pH 7.5) containing 60 mM KCI, 5 mM MgCh as well as 1 mM dithiothreitol and 10% (v / v) glycerol. The protein was analyzed by SDS-Page.
[000155] The method measures the ability of putative ligands to modulate the interaction between the purified bacterial expressed FXR binding ligand (LBD) domain and a synthetic biotinylated peptide based on residues 676-700 of SRC-1 (LCD2, 676- 700). The peptide sequence used was B-CPSSHSSLTERHKILHRLLQEGSPS-COOH where the N-terminus was biotinylated (B). The FXR ligand binding domain (LBD) was expressed as a GST fusion protein in BL-21 cells using the vector pDEST15. Cells were lysed by sonication, and the fusion proteins purified with Glutathione sepharose (Pharmacia) according to the manufacturer's instructions. To classify the compounds according to their influence on the FXR-peptide interaction, Perkin Elmer LANCE technology was applied. This method is based on the transfer of energy dependent on a donor's attachment to an acceptor fluorophore attached to the attachment partner of interest. For ease of handling and reduced background of the compound's fluorescence, LANCE technology makes use of generic fluorophore brands and detection resolved over time. Assays were performed in a final volume of 25 pL in a 384-well plate, in a Tris-based buffer (Tris 20 mM-HCI pH 7.5; 60 mM KCI, MgCI25 mM; BSA 35 ng / pL), containing 20-60 ng / well of recombinantly expressed FXR-LBD fused to GST, biotinylated peptide at the N 200- 600 nM termination, SRC1 representing amino acids 676-700, 200 ng / well of Streptavidin-xIAPC conjugate (Prozyme) and 6-10 ng / well Eu W1024 - antiGST (Perkin Elmer). The DMSO content of the samples was maintained at 1%. After generating the assay mixture and potentially diluting the FXR modulating binders, the assay was equilibrated for 1 hour in the dark at room temperature on 384-well FIA (Greiner) black plates. The LANCE signal was detected by a Perkin Elmer VICTOR2VTM Multilabel Counter. The results were visualized by plotting the ratio between the light emitted at 665 and 615 nm. A baseline level of FXR-peptide formation is observed in the absence of added ligand. Ligands that promote complex formation induce a concentration-dependent increase in the time-resolved fluorescent signal. It would be expected that compounds that bind equally well to both the monomeric FXR and the FXR-peptide complex would not give any change in the signal, whereas it would be expected that ligands that preferentially bind to the monomeric receptor would induce a concentration-dependent decrease in the observed signal. .
[000156] To assess the inhibitory potential of the compounds, ECso values were determined, for example, compounds listed below in table 1 (A = ECso <25 nM; B = 25 <ECso <100 nM; C = ECso 100 nM). Table 1
Assay a mammalian hybrid (M1H)
[000157] Determination of a ligand-mediated Gal4 promoter triggered transactivation to quantify ligand-mediated FXR activation was performed as follows: The part of cDNA encoding the FXR-ligand domain was cloned into the pCMV-bD vector (Stratagen) as a fusion with the yeast GAL4 DNA binding domain under the control of the CMV promoter. The amino acid limits of the ligand binding domain were amino acids 187-472 from database entry NM_005123 (RefSeq). The plasmid pFR-Luc (Stratagen) was used as the reporter plasmid, containing a synthetic promoter with five tandem replications of the yeast GAL4 binding sites, triggering the expression of the firefly Photinus (American firefly) luciferase gene as the reporter gene. In order to increase the experimental precision the plasmid pRL-CMV (Promega) was cotransfected. pRL-CMV contains the constitutive CMV promoter, which controls the expression of reniform luciferase from the sea pansy. All Gal4 reporter gene assays were performed on HEK293 cells (obtained from DSMZ, Braunschweig, Germania) growth in MEM with L-Glutamine and Earle's BSS supplemented with 10% fetal bovine serum, 0.1 non-essential amino acids mM, mM sodium pyruvate, and 100 Penicillin / Streptavidin units per mL at 37 ° C in 5% CO2. Medium and supplements were obtained from Invitrogen. For the assays, 5 x 105 cells were plated per well in 96-well plates in 100 pL per MEM well without phenol red and L-Glutamine and with Earle's BSS supplemented with 10% charcoal / FBS treated with dextran (HyClone, South Logan, Utah), 0.1 mM non-essential amino acids, 2 mM glutamine, 1 mM sodium pyruvate, and 100 units of Penicillin / Streptavidin per mL, incubated at 37 ° C in 5% CO2. The next day the cells had> 90% confluence. The medium was removed and cells were transiently transfected using 20 µl per well of an OptiMEM - polyethyleneimine-based transfection reagent (OptiMEM, Invitrogen; Polyethyleneimine, Aldrich Cat No. 40,827-7) including the three plasmids described above. MEM with the same composition used for plating cells was added 2-4 hours after adding the transfection mixture. Then stocks of the compound, prediluted in MEM were added (final vehicle concentration not exceeding 0.1%). Cells were incubated for an additional 16 hours before firefly luciferase and sea pansy activities were measured sequentially in the same cell extract using a Dual-Light-Luciferase-Assay system (Dyer et al., Anal. Biochem. 2000, 282 , 158-161). All experiments were done in triplicate.
[000158] To assess the agonistic FXR potency of the compounds of the example, potency ranges were determined in the M1H assay listed below in table 2 (A = EC50 <25 nM; B = 25 <EC50 <100 nM; C = ECÕO ^ 100 nM). Table 2
Aqueous solubility test
[000159] The aqueous solubility in PBS, pH 7.4 was determined as follows. A stock solution of the 10 mM compound in DMSO was added to PBS (pH 7.4) to achieve a theoretical final concentration of 200 pM. The resulting solution / suspension was stirred at 1,250 rpm for 1 hour and then stored in the dark at room temperature for 23 hours. At this time, any precipitate is separated from the solution by centrifugation at 3,900 rpm for 30 minutes. Aqueous solubility was determined by comparing the peak area of the main peak in a calibration standard (200 pM) in an organic solvent (methanol / water 60:40, v / v) with the corresponding peak peak area in the buffer sample . As detection method, HPLC-UV / VIS at 230 nm was used. Parallel Artificial Membrane Permeation Assay (PAMPA)
[000160] For PAMPA, 5 mM stock solutions of test items were prepared in DMSO. Stock solutions of 5 mM of reference items were prepared in EtOH (carbamazepine, guanabenz) or in EtOH: H2O 1: 1 (v / v) (ceftriaxone), respectively. Compounds were diluted in PBS (pH 7.4) to obtain the starting solutions containing 5% of the respective organic solvent and 250 pM of reference compounds or 10 pM of test items, respectively. For the assay, a modified PAMPA procedure described by Kansy et al. Kansy et al. (J. Med. Chem. 1998, 41, 1007) was used. Reference compounds for low (ceftriaxone), medium (guanabenz) and high permeation (carbamazepine) were included as internal controls.
[000161] Permeation experiments were performed in a 96-well Multiscreen tray (donor) covered by a 96-well Multiscreen Immobilon (acceptor). The hydrophobic filter material on the Immobilon plate was pre-wetted with 70% ethanol and treated with a lipid solution (lecithin dissolved in dodecane). The donor plate was filled with test compounds and reference compounds and both plates were inserted into each other and placed on an orbital shaker for 15 minutes at 100 rpm. The transport study was initiated by applying 150 pL of PBS buffer containing the test and reference compounds to the donor plate. After 15 - 16 hours of diffusion at room temperature, the contents of the acceptor and donor plate were collected and quantified using LC / MS detection (test items) or by UV spectroscopy using a Spectramax Plus384 (Molecular Devices) (reference items) . The maximum absorption for ceftriaxone, guanabenz and carbamazepine in the reference items were 240 nm, 270 nm and 286 nm, respectively. Recovery samples were prepared as described for the permeation test samples and were incubated in representative flasks during the permeation period under the same conditions.
[000162] For LC / MS analysis of the test items, 100 incubated pL were removed from the acceptor and donor compartment and processed for acetonitrile (ACN) precipitation as described below. In addition, lipid layer test intern samples were extracted by flushing each well twice with 150 pL EA. The solutions were collected in 1.5 ml reaction tubes and the solvent was evaporated. The dry residues were resuspended in a mixture of PBS / DMSO / ACN reflecting the composition of the acceptor and donor samples (that is, 100 pL of buffer supplemented with 5% DMSO, 200 pL of ACN + ISTD). The final solvent content of each sample was 66% ACN.
[000163] Samples from the donor and acceptor compartments and calibration standards were precipitated by adding 200 pL of ACN / ISTD or 400 pL of ACN / ISTD, respectively. After vigorous stirring (10 seconds) and centrifugation (5 minutes at 4,800 x g, room temperature), the free particle supernatants were subjected to LC-mS / MS. Membrane compartments were extracted as previously described. After reconstitution, the samples were shaken vigorously (10 seconds) and centrifuged (5 minutes at 4,800 x g, room temperature). The free particle supernatants were subjected to LC-mS / MS.
[000164] For analysis of the compounds in the present invention, the HPLC system consisted of an Accela U-HPLC pump and an Accela autosampler (Thermo Fisher Scientific, USA). Mass spectrometry was performed on an Exactive mass spectrometer (orbitrap technology with exact mass) equipped with a heated electrospray interface (H-ESI2) (Thermo Fisher Scientific, USA) connected to a PC running on the standard Xcalibur 2.1 software.
[000165] LC was performed in gradient mode (Table 3) using ACN / 0.1% formic acid as organic phase (A) and 10mM ammonium formate / 0.1% formic acid as aqueous phase (B); and the pump flow rate was adjusted to 500 pL / minutes. Separation was performed on a Gemini C6-Phenila analytical column, 3 pm, 50x2.0 mm (Phenomenex, Germania) with a pre-column (Gemini C6-Phenila, 3 pm, 4x2.0 mm). Table 3: HPLC gradients

[000166] As a MS tune file, a generic tune file was used for all analytes using positive or negative ion mode. As the blocking mass for internal mass calibration, the ion [M + H] + of diisooctyl phthalate (m / z 391.28429) was used, which is omnipresent in the solvent system.
[000167] Analyte was acquired by scanning ± 1 Thomson around the expected mass of the ion [M + H] + or [M-H] - monoisotopic. The mass resolution of Orbitrap has been adjusted to 50,000. The exact mass of each analyte was used for peak integration. In addition, instrument settings were as follows: HCD-process gas, AGCHigh dynamic range, maximum trapping injection time 100 ms, shielding gas 30, auxiliary gas 8, sweeping gas 2, spray voltage 4 kV, capillary temperature 250 ° C, ESI 2 heater temperature 250 ° C.
[000168] The purpose of the present invention was to generate FXR agonists with better physicochemical properties compared to compounds claimed in WO 2011/020615. This was achieved by introducing a polar hydroxyl group into a 1,3-cyclobutylidene or 1,3-azetidinylidene group replacing the first 1,2-cyclopropylidene ring.

[000169] Surprisingly, the resulting compounds maintained their activity at the FXR receptor, but demonstrated better physicochemical properties, such as greater solubility / or aqueous membrane permeability. A direct comparison of the corresponding compounds from the two series is given in Table 4. Table 4


* Flow (%) = (acceptor well c) I sum (donor well c + acceptor well c) x 100 x 2 ** nd = not determined
[000170] In each case, both the aqueous solubility and the permeability of the PAMPA membrane or both are significantly improved by the introduction of the hydroxy-cyclobutyl or hydroxy-azetidyl fraction. Like most active nuclear receptor molecules, FXR agonists are generally very lipophilic (M. L. Crawley, Expert Opin. Ther. Patents 2010, 20, 1047). Therefore, it is assumed that better aqueous solubility and membrane permeability result in greater oral bioavailability and in general a better suitability for the clinical development of these compounds as drugs (L. Huang, J. Dong, S. Karki in Evaluation of drug candidates for preclinical development (Eds. C. Han, CB Davis, B. Wang), Wiley & Sons, Hoboken 2010, 187-217).

权利要求:
Claims (4)
[0001]
1. Composite, characterized by the fact that it is selected from
[0002]
2. Compound, according to claim 1, characterized by the fact that it has the following structure
[0003]
3. Compound, according to claim 1, characterized by the fact that it has the following structure
[0004]
Pharmaceutical composition, characterized in that it comprises the compound as defined in any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient (s) thereof.

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法律状态:
2018-03-06| B07D| Technical examination (opinion) related to article 229 of industrial property law|

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2018-03-27| B25A| Requested transfer of rights approved|Owner name: GILEAD SCIENCES, INC. (US) |

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2019-02-19| B65X| Notification of requirement for priority examination of patent application|

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2019-03-12| B65Y| Grant of priority examination of the patent application (request complies with dec. 132/06 of 20061117)|

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2019-08-20| B07E| Notice of approval relating to section 229 industrial property law|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2020-02-18| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application according art. 36 industrial patent law|

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2020-06-23| B09A| Decision: intention to grant|

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2020-08-11| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 12/07/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161507153P| true| 2011-07-13|2011-07-13|

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EP11005722.1|2011-07-13|

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EP11005722A|EP2545964A1|2011-07-13|2011-07-13|Novel FXRbinding and activity modulating compounds|

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US61/507153|2011-07-13|

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PCT/EP2012/002941|WO2013007387A1|2011-07-13|2012-07-12|Novel fxrbinding and activity modulating compounds|BR122019026062-1A| BR122019026062B1|2011-07-13|2012-07-12|COMPOUNDS 3-ISOXAZOL-4-IL) METOXI) PHENYL) -3-HYDROXY-AZETIDIN-1-IL, 3-4-IL) -1H-1,2,3-TRIAZOL-5-IL) METOXI) PHENYL) -3-HYDROXY-AZETIDIN-1-IL, 3--1H-1,2,3-TRIAZOL-5-IL) METOXI) PHENYL) -3-HYDROXY-AZETIDIN-1-IL, E 3--1H -PIRAZOL-5-IL) METOXI) PHENYL) -3-HYDROXY-AZETIDIN- 1-IL REPLACED AND OXIDES OF THE SAME|