Isoxazole analogs as FXR agonists and methods of use thereof

The present invention provides compounds of Formula I: pharmaceutical compositions comprising these compounds and methods of using these compounds to treat or prevent a disease or disorder mediated as FXR modulators. Specifically, the present invention relates to isoxazole derivatives useful as agonists for FXR, and methods for their preparation and use.

TECHNICAL FIELD

The present invention relates generally to compounds and pharmaceutical compositions useful as FXR modulators. Specifically, the present invention relates to isoxazole derivatives useful as agonists for FXR, and methods for their preparation and use.

BACKGROUND OF THE INVENTION

Farnesoid X Receptor (FXR) is an orphan nuclear receptor initially identified from a rat liver cDNA library (B M. Forman, et al.,Cell,1995, 81(5), 687-693) that is most closely related to the insect ecdysone receptor. FXR is a member of the nuclear receptor family of ligand-activated transcription factors that includes receptors for the steroid, retinoid, and thyroid hormones (D J. Mangelsdorf, et al.,Cell,1995, 83(6), 841-850). The relevant physiological ligands of FXR are bile acids (D. Parks et al., Science, 1999, 284(5418), 1362-1365). The most potent one is chenodeoxycholic acid (CDCA), which regulates the expression of several genes that participate in bile acid homeostasis. Farnesol and derivatives, together called farnesoids, are originally described to activate the rat orthologue at high concentration but they do not activate the human or mouse receptor. FXR is expressed in the liver, throughout the entire gastrointestinal tract including the esophagus, stomach, duodenum, small intestine, colon, ovary, adrenal gland and kidney. Beyond controlling intracellular gene expression, FXR seems to be also involved in paracrine and endocrine signaling by upregulating the expression of the cytokine Fibroblast Growth Factor (J. Holt et al.,Genes Dev.,2003, 17(13), 1581-1591; T. Inagaki et al.,Cell Metab.,2005, 2(4), 217-225).

There is a need for the development of FXR modulators for the treatment and prevention of disease.

SUMMARY OF THE INVENTION

In one aspect, the invention provides compounds represented by Formula I, or pharmaceutically acceptable salts thereof:

is selected from the group consisting of:

wherein R3is independently selected from the group consisting of halogen, optionally substituted —C1-C6alkyl, optionally substituted —C1-C6alkoxy, optionally substituted —C3-C6cycloalkyl, optionally substituted —C3-C6cycloalkenyl, optionally substituted 3- to 6-membered heterocycloalkyl, optionally substituted aryl, and optionally substituted heteroaryl; n is 0, 1, 2, or 3; and m is 0, 1 or 2.

Z is selected from the group consisting of:

wherein:
R5and R6are independently selected from the group consisting of:

R7is selected from the group consisting of:

11) NR9R10; wherein R9and R10are each independently selected from hydrogen, optionally substituted —C1-C8alkyl, optionally substituted —C2-C8alkenyl, optionally substituted —C2-C8alkynyl, optionally substituted —C3-C8cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted 3- to 8-membered heterocycloalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; alternatively, R9and R10are taken together with the nitrogen atom to which they are attached to form an optionally substituted heterocyclic ring.

R8is selected from the group consisting of:

11) NR12R13; wherein R12and R13are each independently selected from hydrogen, optionally substituted —C1-C8alkyl, optionally substituted C2-C8alkenyl, optionally substituted C2-C8alkynyl, optionally substituted —C3-C8cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted 3- to 8-membered heterocycloalkyl, optionally substituted heteroaryl and optionally substituted heteroarylalkyl, alternatively 102and R13are taken together with the nitrogen atom to which they are attached to form an optionally substituted heterocyclic ring; provided that at least one of R12and R13is not hydrogen.

In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound or combination of compounds of the present invention, or a pharmaceutically acceptable salt form, stereoisomer, solvate, hydrate or combination thereof, in combination with a pharmaceutically acceptable carrier or excipient.

In another embodiment, the present invention provides a method for the prevention or treatment of an FXR mediated disease or condition. The method comprises administering a therapeutically effective amount of a compound of Formula (I). The present invention also provides the use of a compound of Formula (I) for the preparation of a medicament for the prevention or treatment of an FXR mediated disease or condition.

DETAILED DESCRIPTION OF THE INVENTION

A first embodiment of the invention is a compound represented by Formula I as described above, or a pharmaceutically acceptable salt thereof.

In certain embodiments of the invention is a compound represented by Formula I as described above, or a pharmaceutically acceptable salt thereof, wherein R1is optionally substituted isopropyl, optionally substituted tert-butyl or optionally substituted cyclopropyl.

In certain embodiments of the invention is a compound represented by Formula I as described above, or a pharmaceutically acceptable salt thereof, wherein R2is selected from the groups:

each of which can be optionally further substituted with halogen, optionally substituted —C1-C6alkyl, optionally substituted —C1-C6alkoxy, optionally substituted —C3-C6cycloalkyl, optionally substituted —C3-C6cycloalkenyl, optionally substituted aryl, or optionally substituted heteroaryl.

In certain embodiments of the invention is a compound represented by Formula I as described above, or a pharmaceutically acceptable salt thereof, whereinselected from:

wherein R3, m and n are as previously defined.

In certain embodiments of the invention is a compound represented by Formula I as described above, or a pharmaceutically acceptable salt thereof, wherein is selected from:

wherein each of these groups is optionally substituted.

In certain embodiments of the invention is a compound represented by Formula I as described above, or a pharmaceutically acceptable salt thereof, whereinis selected from:

and wherein each of these groups is optionally substituted.

In certain embodiments, the compounds of the invention is represented by Formula (II), or (III), and pharmaceutically acceptable salts thereof:

In certain embodiments, the compounds of the invention are represented by Formula (IV) or (V), and pharmaceutically acceptable salts thereof:

In certain embodiments, the compounds of the invention are represented by Formula (VI) or (VII), and pharmaceutically acceptable salts thereof:

In certain embodiments, the compounds of the invention are represented by Formula (VIII) or (IX), and pharmaceutically acceptable salts thereof:

In certain embodiments, the compounds of the invention is represented by Formula (X) or (XI), and pharmaceutically acceptable salts thereof:

Representative compounds of the invention include, but are not limited to, the following compounds (compound 1 to compound 102 in Table 1) according to Formula X, and pharmaceutically acceptable salts thereof, wherein R7andare delineated for each compound in Table 1.

Representative compounds of the invention include, but are not limited to, the following compounds (compound 103 to compound 174 in Table 2) according to Formula XI, and pharmaceutically acceptable salts thereof, wherein R8andare delineated for each compound in Table 2.

In certain embodiments, the compounds of the invention are represented by Formula (XII) or (XIII), and pharmaceutically acceptable salts thereof:

Representative compounds of the invention include, but are not limited to, the following compounds (compound 175 to compound 276 in Table 3) according to Formula XII, wherein R7andare delineated for each compound in Table 3.

Representative compounds of the invention include, but are not limited to, the following compounds (compound 277 to compound 348 in Table 4) according to Formula XIII, wherein R8andare delineated for each compound in Table 4.

In certain embodiments, the compounds of the invention are represented by Formula (XIV) or (XV), and pharmaceutically acceptable salts thereof:

Representative compounds of the invention include, but are not limited to, the following compounds (compound 349 to compound 450 in Table 5) according to Formula XIV, wherein R7andare delineated for each compound in Table 5.

Representative compounds of the invention include, but are not limited to, the following compounds (compound 451 to compound 522 in Table 6) according to Formula XV, wherein R8andare delineated for each compound in Table 6.

In certain embodiments, the compounds of the invention are represented by Formula (XVI) or (XVII), and pharmaceutically acceptable salts thereof:

Representative compounds of the invention include, but are not limited to, the following compounds (compound 523 to compound 624 in Table 7) according to Formula XVI, wherein R7andare delineated for each compound in Table 7.

Representative compounds of the invention include, but are not limited to, the following compounds (compound 625 to compound 696 in Table 8) according to Formula XVII, wherein

R8andare delineated for each compound in Table 8.

In certain embodiments, the present invention provides a method for the prevention or treatment of an FXR mediated disease or condition in a subject. The method comprises administering a therapeutically effective amount of a compound of Formula (I) to the subject. The present invention also provides the use of a compound of Formula (I) for the preparation of a medicament for the prevention or treatment of an FXR mediated disease or condition.

In certain embodiments, the FXR-mediated disease or condition is cardiovascular disease, atherosclerosis, arteriosclerosis, hypercholesterolemia, or hyperlipidemia chronic liver disease, gastrointestinal disease, fibrotic diseases such as primary biliary cirrhosis, primary sclerosing cholangitis, pulmonary fibrosis, renal fibrosis, liver fibrosis, renal disease, metabolic disease, cancer (i.e., colorectal cancer), or neurological indications such as stroke.

In certain embodiments, the renal disease is diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), hypertensive nephrosclerosis, chronic glomerulonephritis, chronic transplant glomerulopathy, chronic interstitial nephritis, or polycystic kidney disease.

In certain embodiments, the cardiovascular disease is atherosclerosis, arteriosclerosis, dyslipidemia, hypercholesterolemia, or hypertriglyceridemia.

In certain embodiments, the metabolic disease is insulin resistance, Type I and Type II diabetes, or obesity.

In one aspect, the compound is a selective FXR agonist over TGR5 activator.

In another aspect, the invention provides a method of treating a disease selected from an inflammatory disease, an autoimmune disease and a gastrointestinal disease in a subject in need thereof. The method comprises the step of administering to the subject a therapeutically effective amount of a compound of Formula I. In another aspect, the invention provides for the use of a compound of Formula I in the manufacture of a medicament for treating an inflammatory disease, an autoimmune disease or a gastrointestinal disease.

In certain embodiments, the inflammatory disease is selected from allergy, osteoarthritis, appendicitis, bronchial asthma, pancreatitis, allergic rash, and psoriasis.

In certain embodiments, the autoimmune disease is selected from rheumatoid arthritis, multiple sclerosis, and type I diabetes.

In certain embodiments, the gastrointestinal disease is selected from inflammatory bowel disease (Crohn's disease, ulcerative colitis), short bowel syndrome (post-radiation colitis), microscopic colitis, irritable bowel syndrome (malabsorption), and bacterial overgrowth.

Yet a further aspect of the present invention is a process of making any of the compounds delineated herein employing any of the synthetic means delineated herein.

Definitions

The term “alkenyl”, as used herein, denote a monovalent group derived from a hydrocarbon moiety by the removal of a single hydrogen atom wherein the hydrocarbon moiety has at least one carbon-carbon double bond. Preferred alkenyl groups include C2-C6alkenyl and C2-C8alkenyl groups. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl, octenyl and the like.

The term “alkynyl”, as used herein, denotes a monovalent group derived from a hydrocarbon moiety by the removal of a single hydrogen atom wherein the hydrocarbon moiety has at least one carbon-carbon triple bond. Preferred alkynyl groups include C2-C6alkynyl and C2-C8alkynyl groups. Representative alkynyl groups include, but are not limited to, for example, ethynyl, 1-propynyl, 1-butynyl, heptynyl, octynyl and the like.

The term “cycloalkyl”, as used herein, denotes a monovalent group derived from a monocyclic or polycyclic saturated carbocyclic ring, wherein the said polycyclic saturated carbocyclic ring is bi or tri cyclic group fused, bridged or spiro system, and one or more carbon atoms may be optionally oxo-substituted. Preferred cycloalkyl groups include C3-C8cycloalkyl and C3-C12cycloalkyl groups. Examples of C3-C8-cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl and cyclooctyl; and examples of C3-C12-cycloalkyl include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl, bicyclo[3.1.0]hexyl, spiro[2.5]octyl, spiro[4.4]nonanyl.

The term “cycloalkenyl” as used herein, denote a monovalent group derived from a monocyclic or polycyclic carbocyclic ring having at least one carbon-carbon double bond, wherein the said polycyclic cycloalkenyl ring is bi or tri cyclic group fused, bridged or spiro system, and one or more carbon atoms may be optionally oxo-substituted. Preferred cycloalkenyl groups include C3-C8cycloalkenyl and C3-C12cycloalkenyl groups. Examples of C3-C8-cycloalkenyl include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like; and examples of C3-C12-cycloalkenyl include, but not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cycloheptenyl, bicyclo[2.2.1]hept-2-enyl, bicyclo[3.1.0]hex-2-enyl, spiro[2.5]oct-4-enyl, spiro[4.4]non-1-enyl, and the like.

The term “aryl,” as used herein, refers to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl and the like.

The term “arylalkyl,” as used herein, refers to a C1-C3alkyl or C1-C6alkyl residue attached to an aryl ring. Examples include, but are not limited to, benzyl, phenethyl and the like.

The term “heteroaryl,” as used herein, refers to a mono-, bi-, or tri-cyclic aromatic radical or ring having from five to ten ring atoms of which at least one ring atom is selected from S, O and N; wherein any N or S contained within the ring may be optionally oxidized. Preferred heteroaryl groups are monocyclic or bicyclic. Heteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.

The term “heteroarylalkyl,” as used herein, refers to a C1-C3alkyl or C1-C6alkyl residue attached to a heteroaryl ring. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl and the like.

As used herein, the term “alkoxy” employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers. Preferred alkoxy are (C1-C3) alkoxy.

In accordance with the invention, any of the aryls, substituted aryls, heteroaryls and substituted heteroaryls described herein, can be any aromatic group. Aromatic groups can be substituted or unsubstituted.

It is understood that any alkyl, alkenyl, alkynyl, cycloalkyl and cycloalkenyl moiety described herein can also be an aliphatic group, an alicyclic group or a heterocyclic group. An “aliphatic group” is non-aromatic moiety that may contain any combination of carbon atoms, hydrogen atoms, halogen atoms, oxygen, nitrogen or other atoms, and optionally contain one or more units of unsaturation, e.g., double and/or triple bonds. An aliphatic group may be straight chained, branched or cyclic and preferably contains between about 1 and about 24 carbon atoms, more typically between about 1 and about 12 carbon atoms. In addition to aliphatic hydrocarbon groups, aliphatic groups include, for example, polyalkoxyalkyls, such as polyalkylene glycols, polyamines, and polyimines, for example. Such aliphatic groups may be further substituted. It is understood that aliphatic groups may be used in place of the alkyl, alkenyl, alkynyl, alkylene, alkenylene, and alkynylene groups described herein.

The term “alicyclic,” as used herein, denotes a monovalent group derived from a monocyclic or polycyclic saturated carbocyclic ring compound by the removal of a single hydrogen atom. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl, and bicyclo[2.2.2]octyl. Such alicyclic groups may be further substituted.

The term “heterocycloalkyl” and “heterocyclic” can be used interchangeably and refer to a non-aromatic ring or a bi- or tri-cyclic group fused, bridged, or spiro system, where: (i) each ring contains between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, (ii) each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (iii) the nitrogen and sulfur heteroatoms may optionally be oxidized, (iv) the nitrogen heteroatom may optionally be quaternized, (v) any of the above rings may be fused to a benzene ring, and (vi) the remaining ring atoms are carbon atoms which may be optionally oxo-substituted. Representative heterocycloalkyl groups include, but are not limited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, 2-azabicyclo[2.2.1]heptyl, 8-azabicyclo [3.2.1]octyl, 5-azaspiro[2.5]octyl, 1-oxa-7-azaspiro[4.4]nonanyl, and tetrahydrofuryl.

Such heterocyclic groups may be further substituted to give substituted heterocyclic. Heteroaryl or heterocyclic groups can be C-attached or N-attached (where possible).

It will be apparent that in various embodiments of the invention, the substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, arylalkyl, heteroarylalkyl, and heterocycloalkyl are intended to be monovalent or divalent. Thus, alkylene, alkenylene, and alkynylene, cycloaklylene, cycloalkenylene, cycloalkynylene, arylalkylene, heteroarylalkylene and heterocycloalkylene groups are to be included in the above definitions, and are applicable to provide the Formulas herein with proper valency.

The term “hydrogen” includes hydrogen and deuterium. In addition, the recitation of an atom includes other isotopes of that atom so long as the resulting compound is pharmaceutically acceptable.

In certain embodiments, the compounds of each formula herein include isotopically labelled compounds. An “isotopically labelled compound” is a compound in which at least one atomic position is enriched in a specific isotope of the designated element to a level which is significantly greater than the natural abundance of that isotope. For example, one or more hydrogen atom positions in a compound can be enriched with deuterium to a level which is significantly greater than the natural abundance of deuterium, for example, enrichment to a level of at least 1%, preferably at least 20% or at least 50%. Such a deuterated compound may, for example, be metabolized more slowly than its non-deuterated analog, and therefore exhibit a longer half-life when administered to a subject. Such compounds can synthesized using methods known in the art, for example by employing deuterated starting materials. Unless stated to the contrary, isotopically labelled compounds are pharmaceutically acceptable.

The term “subject” as used herein refers to a mammal. A subject therefore refers to, for example, dogs, cats, horses, cows, pigs, guinea pigs, and the like. Preferably the subject is a human. When the subject is a human, the subject may be referred to herein as a patient.

As used herein, the term “pharmaceutically acceptable salt” refers to those salts of the compounds formed by the process of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art.

The term “amino protecting group,” as used herein, refers to a labile chemical moiety which is known in the art to protect an amino group against undesired reactions during synthetic procedures. After said synthetic procedure(s) the amino protecting group as described herein may be selectively removed. Amino protecting groups as known in the art described generally in T. H. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis,3rd edition, John Wiley & Sons, New York (1999). Examples of amino protecting groups include, but are not limited to, t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, and the like.

The term “pharmaceutically acceptable prodrugs” as used herein refers to those prodrugs of the compounds formed by the process of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the present invention. “Prodrug”, as used herein means a compound, which is convertible in vivo by metabolic means (e.g. by hydrolysis) to afford any compound delineated by the Formulae of the instant invention. Various forms of prodrugs are known in the art, for example, as discussed in Bundgaard, (ed.),Design of Prodrugs, Elsevier (1985); Widder, et al. (ed.),Methods in Enzymology, Vol.4, Academic Press (1985); Krogsgaard-Larsen, et al., (ed). “Design and Application of Prodrugs,Textbook of Drug Design and Development, Chapter 5, 113-191 (1991); Bundgaard, et al.,Journal of Drug Deliver Reviews,8:1-38(1992); Bundgaard, J. ofPharmaceutical Sciences,77:285 et seq. (1988); Higuchi and Stella (eds.) Prodrugs as Novel Drug Delivery Systems,American Chemical Society(1975); and Bernard Testa & Joachim Mayer, “Hydrolysis In Drug And Prodrug Metabolism: Chemistry, Biochemistry And Enzymology,” John Wiley and Sons, Ltd. (2002).

The term “treating”, as used herein, means relieving, lessening, reducing, eliminating, modulating, or ameliorating, i.e. causing regression of the disease state or condition. Treating can also include inhibiting, i.e. arresting the development, of an existing disease state or condition, and relieving or ameliorating, i.e. causing regression of an existing disease state or condition, for example when the disease state or condition may already be present.

The term “preventing”, as used herein means, to completely or almost completely stop a disease state or condition, from occurring in a patient or subject, especially when the patient or subject is predisposed to such or at risk of contracting a disease state or condition.

The term “aprotic solvent,” as used herein, refers to a solvent that is relatively inert to proton activity, i.e., not acting as a proton-donor. Examples include, but are not limited to, hydrocarbons, such as hexane and toluene, for example, halogenated hydrocarbons, such as, for example, methylene chloride, ethylene chloride, chloroform, and the like, heterocyclic compounds, such as, for example, tetrahydrofuran and N-methylpyrrolidinone, and ethers such as diethyl ether, bis-methoxymethyl ether. Such solvents are well known to those skilled in the art, and individual solvents or mixtures thereof may be preferred for specific compounds and reaction conditions, depending upon such factors as the solubility of reagents, reactivity of reagents and preferred temperature ranges, for example. Further discussions of aprotic solvents may be found in organic chemistry textbooks or in specialized monographs, for example:Organic Solvents Physical Properties and Methods of Purification,4th ed., edited by John A. Riddick et al., Vol. II, in theTechniques of Chemistry Series, John Wiley & Sons, NY, 1986.

The terms “protogenic organic solvent” or “protic solvent” as used herein, refer to a solvent that tends to provide protons, such as an alcohol, for example, methanol, ethanol, propanol, isopropanol, butanol, t-butanol, and the like. Such solvents are well known to those skilled in the art, and individual solvents or mixtures thereof may be preferred for specific compounds and reaction conditions, depending upon such factors as the solubility of reagents, reactivity of reagents and preferred temperature ranges, for example. Further discussions of protogenic solvents may be found in organic chemistry textbooks or in specialized monographs, for example:Organic Solvents Physical Properties and Methods of Purification,4th ed., edited by John A. Riddick et al., Vol. II, in theTechniques of Chemistry Series, John Wiley & Sons, NY, 1986.

The synthesized compounds can be separated from a reaction mixture and further purified by a method such as column chromatography, high pressure liquid chromatography, or recrystallization. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. In addition, the solvents, temperatures, reaction durations, etc. delineated herein are for purposes of illustration only and variation of the reaction conditions can produce the desired isoxazole products of the present invention.

Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein include, for example, those described in R. Larock,Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis,2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser,Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995).

The compounds of this invention may be modified by appending various functionalities via synthetic means delineated herein to enhance selective biological properties. Such modifications include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.

Pharmaceutical Compositions

Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one with ordinary skill in the art. All publications, patents, published patent applications, and other references mentioned herein are hereby incorporated by reference in their entirety.

Abbreviations

Abbreviations which have been used in the descriptions of the schemes and the examples that follow are:

CDI for carbonyldiimidazole;

DBU for 1,8-diazabicycloundec-7-ene;

DCC for N,N-dicyclohexylcarbodiimide;

DCM for dichloromethane;

EtOAc for ethyl acetate;

HCl for hydrochloric acid;

THF for tetrahydrofuran.

Synthetic Methods

As shown in Scheme 1, novel isoxazole sulfonyl urea analogs of the compound of formula (1-4) are prepared from the compound of formula (1-1), wherein R1, R2, Z,andare previously defined. Thus, the compound of formula (1-1) is converted to the acyl azide compound of formula (1-2) using a suitable reagent such as, but not limited to, DPPA. The reaction solvent can be, but not limited to, THF, DCM and toluene. The reaction temperature is from −20° C. to 80° C. Alternatively, the acid (1-1) could be transformed to the acyl azide (1-2) via activated acid derivative such as acyl chlorides or anhydrides in presence of azide source. The reagents for activation of acid includes, but not limited to, tetramethylfluoroformadinium hexafluorophosphate, phenyl dichlorophosphate, SOC12-DMF, triphosgene, cyanuric chloride, NCS-Ph3P and Cl3CCN-Ph3P. The azide source includes, but not limited to, sodium azide, tetrabutylammonium azide, trimethylsilyl azide and N,N,N′,N′-tetramethylguanidinium azide. Curtius rearrangement of the compound of formula (1-2) at elevated temperature preferably from 50° C. to 120° C. lead to the isocyanate intermediate, which then can react with sulfonamide compound of formula (1-3) to afford the compound of formula (1-4). wherein R1, R2, Z,,and R7are previously defined.

As shown in Scheme 2, novel isoxazole acylsulfonamide analogs of the compound of Formula (2-1) are prepared from the compound of Formula (1-1), wherein R1, R2, Z,and, are as previously defined. The compound of Formula (1-1) is coupled with a sulfonamide using suitable coupling reagents in presence of suitable bases to give the compound of Formula (2-3), wherein R1, R2, Z,,, and R8are as previously defined. The coupling reagent can be selected from, but not limited to, DCC, EDC, CDI, diisopropyl carbodiimide, BOP-Cl, PyBOP, PyAOP, TFFH and HATU. Suitable bases include, but are not limited to, triethylamine, diisopropylethylamine, DBU, N-methylmorpholine and DMAP. The coupling reaction is carried out in an aprotic solvent such as, but not limited to, DCM, DMF or THF. The reaction temperature can vary from 0° C. to 80° C.

In the reactions described, reactive functional groups such as hydroxyl, amino, imino, thio or carboxy groups, may be protected to avoid unwanted participation in the reactions. These protecting groups may be removed at suitable steps via solovolysis, reduction, photolysis. The protection and deprotection are common practices in organic synthesis (see T. W. Greene and P. G. M Wuts,Protective Groups in Organic Chemistry,4thEd., Wiley-Interscience, 2006).

Examples

The following preparations and examples further illustrate the invention.

To the acid 1 (111 mg, 0.2 mmol) in DCM (2 mL) and DMF (1 mL) was added EDCI (77 mg, 0.4 mmol), DMAP (48.9 mg, 0.4 mmol) and N,N-dimethylsulfonamide (49.7 mg, 0.4 mmol) and the resulting mixture was stirred at room temperature for 16 hrs. The reaction was quenched with brine, extracted with ethyl acetate. The organic layers were combined, washed with brine, dried (Na2SO4), filtered and the filtrate was concentrated. The resulting residue was chromatographed with CombiFlash eluting with hexane to 50% acetone in hexane to give example 1a (117 mg, 88%). LC/MS observed [M-H], 660.07.

To the acid 1 (300 mg, 0.541 mmol) in toluene (3 mL) was added triethylamine (0.151 mL, 1.081 mmol), and diphenyl phosphorazidate (0.128 mL, 0.595 mmol) dropwise and the resulting mixture was stirred at room temperature for 1 hr, then at 85° C. for 3.5 hrs and 95° C. for 1 hr. The reaction was cooled down to room temperature and divided into two portions equally. To one of the portion was added a solution of N,N-dimethylsulfamide (40.2 mg, 0.324 mmol) in THF (0.5 mL) and DBU (48.8 μL, 0.324 mmol) and the resulting mixture was stirred at room temperature for 14 hrs. The mixture was quenched with 1N HCl and extracted with ethyl acetate (2×). The combined organic layers was dried (Na2SO4), filtered and the filtrate was concentrated in vacuo. The resulting residue was chromatographed with silica gel eluting with hexane to 30% acetone in hexane which is further purified with C18column eluted with 50% MeCN in water to 90% MeCN in water to give example 1b (5 mg). LC/MS observed [M-H], 675.08; 673.08.

To the acid 1 (85 mg, 0.4 mmol) in DCM (2 mL) and DMF (1 mL) was added EDCI (77 mg, 0.4 mmol) DMAP (48.9 mg, 0.4 mmol) and 4-(tert-butyl)benzenesulfonamide (85 mg, 0.4 mmol) and the resulting mixture was stirred at room temperature for 16 hrs. The reaction was quenched with brine, extracted with ethyl acetate. The organic layers were combined, washed with brine, dried (Na2SO4), filtered and the filtrate was concentrated. The resulting residue was chromatographed with CombiFlash eluting with hexane to 55% acetone in hexane to give example 2a (102 mg, 68%). LC/MS observed [M-H], 751.13.

To the acid 1 (111 mg, 0.2 mmol) in DCM (2 mL) and DMF (1 mL) was added EDCI (77 mg, 0.4 mmol), DMAP (48.9 mg, 0.4 mmol) and piperidine-1-sulfonamide (65.7 mg, 0.4 mmol) and the resulting mixture was stirred at room temperature for 16 hrs. The reaction was quenched with brine, extracted with ethyl acetate. The organic layers were combined, washed with brine, dried (Na2SO4), filtered and the filtrate was concentrated. The resulting residue was chromatographed with CombiFlash eluting with hexane to 50% acetone in hexane to give example 2b (120 mg, 86%). LC/MS observed [M-H], 700.11.

To the acid 1 (111 mg, 0.2 mmol) in DCM (2 mL) and DMF (1 mL) was added EDCI (77 mg, 0.4 mmol), DMAP (48.9 mg, 0.4 mmol) and cyclopropanesulfonamide (48.5 mg, 0.4 mmol) and the resulting mixture was stirred at room temperature for 16 hrs. The reaction was quenched with brine, extracted with ethyl acetate. The organic layers were combined, washed with brine, dried (Na2SO4), filtered and the filtrate was concentrated. The resulting residue was chromatographed with CombiFlash eluting with hexane to 60% acetone in hexane to give example 2c (79 mg, 60%). LC/MS observed [M-H], 657.06.

To the acid 1 (300 mg, 0.541 mmol) in toluene (3 mL) was added triethylamine (0.151 mL, 1.081 mmol), and diphenyl phosphorazidate (0.128 mL, 0.595 mmol) dropwise and the resulting mixture was stirred at room temperature for 1 hr, then at 85° C. for 3.5 hrs and 95° C. for 1 hr. The reaction was cooled down to room temperature and divided into two portions equally.

To one of the portion was added a solution of 4-(tert-butyl)benzenesulfonamide (69.1 mg, 0.324 mmol) in THF (0.5 mL) and DBU (48.8 uL, 0.324 mmol) and the resulting mixture was stirred at room temperature for 14 hrs. The mixture was quenched with 1N HCl and extracted with ethyl acetate (2×). The combined organic layers was dried (Na2SO4), filtered and the filtrate was concentrated in vacuo. The resulting residue was chromatographed with silica gel eluting with hexane to 30% acetone in hexane which is further purified with C18column eluted with 50% MeCN in water to 90% MeCN in water to give example 2d (5.4 mg). LC/MS observed [M-H], 764.13; 762.14.

Assays

Determination of a ligand mediated Gal4 promoter driven transactivation to quantify ligand binding mediated activation of FXR. FXR Reporter Assay kit purchased from Indigo Bioscience (Catalogue number: 1B00601) to determine the potency and efficacy of compound developed by Enanta that can induce FXR activation. The principle application of this reporter assay system is to quantify functional activity of human FXR. The assay utilizes non-human mammalian cells, CHO (Chinese hamster ovary) cells engineered to express human NR1H4 protein (referred to as FXR). Reporter cells also incorporate the cDNA encoding beetle luciferase which catalyzes the substrates and yields photon emission. Luminescence intensity of the reaction is quantified using a plate-reading luminometer, Envision. Reporter Cells include the luciferase reporter gene functionally linked to an FXR responsive promoter. Thus, quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in FXR activity. EC50and efficacy (normalize to CDCA set as 100%) is determined by XLFit. The assay is according to the manufacturer's instructions. In brief, the assay was performed in white, 96 well plates using final volume of 100 ul containing cells with different doses of compounds. Retrieve Reporter Cells from −80° C. storage. Perform a rapid thaw of the frozen cells by transferring a 10 ml volume of 37° C. cell recovery medium into the tube of frozen cells. Recap the tube of Reporter Cells and immediately place it in a 37° C. water bath for 5-10 minutes. Retrieve the tube of Reporter Cell Suspension from the water bath. Sanitize the outside surface of the tube with a 70% alcohol swab, and then transfer it into the cell culture hood. Dispense 90 μl of cell suspension into each well of the 96-well Assay Plate. Transfer the plate into 37° C. incubator, allowing the cells adherent to the bottom of the well. Dilute compounds in Dilution Plate (DP), and administrate to cells at Assay Plate (AP). DMSO content of the samples was kept at 0.2%. Cells were incubated for additional 22 hours before luciferase activities were measured. Thirty minutes before intending to quantify FXR activity, remove Detection Substrate and Detection Buffer from the refrigerator and place them in a low-light area so that they may equilibrate to room temperature. Remove the plate's lid and discard all media contents by ejecting it into an appropriate waste container. Gently tap the inverted plate onto a clean absorbent paper towel to remove residual droplets. Cells will remain tightly adhered to well bottoms. Add 100 μl of luciferase detection reagent to each well of the assay plate. Allow the assay plate to rest at room temperature for at least 5 minutes following the addition of LDR. Set the instrument (Envision) to perform a single 5 second “plate shake” prior to reading the first assay well. Read time may be 0.5 second (500 mSec) per well. EC50and Efficacy (normalize to CDCA set as 100%) is determined by XLFit.

To assess the FXR agonistic potency of the example compounds as well as for reference compound, potency ranges were determined in the Human FXR (NR1H4) Assay as listed below in Table 9. The efficacy was normalized to CDCA set as 100%. (A=EC50<0.1 μM; B=0.1 μM<EC50<1.0 μM; C=1.0 μM<EC50<10 μM, D=EC50>10 μM).