NZ714082B2 - 11-hydroxyl-derivatives of bile acids and amino acid conjugates thereof as farnesoid x receptor modulators - Google Patents

Abstract

The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof, wherein R1 is a hydroxy group and R2, R3, R4, R5, and R6 are as described herein. The present invention relates generally to selective FXR agonists and to methods of making and using them. of making and using them.

Description

-HYDROXYL-DERIVATIVES OF BILE ACIDS AND AMINO ACID CONJUGATES THEREOF AS FARNESOID X RECEPTOR TORS CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to, and the benefit of, U.S.S.N. 61/823,169, filed on May 14, 2013, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION 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 83:841-850 (1995)). Northern and in situ analysis show that FXR is 1O most ntly expressed in the liver, intestine, kidney, and l (B.M. Forman, et al, Cell 81:687-693 (1995) and W. Seol, et al, Mal. Endocrinnol. 9:72-85 (1995)). FXR binds to DNA as a heterodimer with the 9-cis retinoic acid receptor (RXR). The rat FXR is activated by micromolar concentrations of famesoids such as famesol and le hormone (B.M. Forman, et al, Cell 81 93 (1995)). However, these compounds failed to activate the mouse and human FXR, leaving the nature of the endogenous FXR ligands in doubt.

Several naturally-occurring bile acids (e. g., chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), and the taurine and glycine ates thereof) serve as FXR s and bind to and activate FXR at physiological concentrations (WO 00/37077).

Bile acids are terol metabolites that are formed in the liver and secreted into the duodenum of the intestine, where they have important roles in the solubilization and absorption of y lipids and vitamins. Most bile acids (~95%) are subsequently reabsorbed in the ileum and returned to the liver via the enterohepatic circulatory system.

The conversion of cholesterol to bile acids in the liver is under feedback regulation: bile acids down-regulate the transcription of cytochrome P450 7a (CYP7a), which encodes the enzyme that catalyzes the rate limiting step in bile acid biosynthesis. It is suggested that FXR is involved in the repression of CYP7a expression by bile acids (D.W. Russell, Cell 97:539-542 ). In the ileum, bile acids induce the expression of the intestinal bile acid binding protein (IBABP), which binds bile acids with high affinity and may be involved in their cellular uptake and king. It is demonstrated that bile acids mediate their effects on IBABP sion through activation of FXR, which binds to an IR-l type response element that is conserved in the human, rat, and mouse IBABP gene promoters. Thus, FXR is involved in both the stimulation (IBABP) and the sion (CYP7a) of target genes ed in bile acid and cholesterol homeostasis. Accordingly, there is a need for FXR modulators suitable for drug development. The present invention addresses this need.

SUMMARY OF THE INVENTION The invention es compounds and s of preparing these compounds.

Specifically, the invention provides a compound of a I: COZH (I), or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof, wherein R1, R2, R3, R4, R5 and R6 are as described herein. The compounds of the invention are useful for 1O treating and preventing diseases and conditions.

The invention also provides a pharmaceutical composition comprising a compound of the invention or a ceutically acceptable salt, e, or amino acid conjugate thereof, and a pharmaceutically acceptable carrier or excipient.

The ion also provides a method for the treatment or prevention of a disease and condition, comprising administering to the subject in need thereof an effective amount of a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof. In one aspect, the disease or condition is FXR-mediated.

The invention also provides for the manufacture of a medicament for treating or preventing a disease or condition (e.g., a disease or ion mediated by FXR), wherein the medicament comprises a nd of the invention or a pharmaceutically able salt, solvate, or amino acid conjugate thereof.

The invention also provides a composition for use in a method for treating or preventing a disease or condition (e.g., a disease or condition mediated by FXR), n the ition comprises a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. Although methods and als similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and als are described below. All ations, patent applications, patents, and other references mentioned herein are incorporated by reference. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure l is a graph showing the activity of a compound of the invention and a ison compound in a transactivation assay in HEK293T cells Figure 2 is a series of graphs showing the lack of TGRS activity of a compound of the invention in human enteroendocrine cells expressing TGRS at physiological level (A) and in human Chinese hamster ovary (CHO) cells over-expressing TGRS (B).

Figure 3 is a series of graphs g the activity of a compound of the invention and other comparison compounds in regulating expression of OSTd (A), OSTB (B), BSEP (C), MRP2 (D), CYP7A1 (E), SHP (F), FGF—l9 (G), and UGT2B4 (H).

Figure 4 is a series of graphs showing the activity of a compound of the invention and other comparison compounds in regulating PLTP involved in lipid lism (A), SREBP- lC (B), APOCII (C), and PPARy (D).

Figure 5 is a graph showing the regulation of a compoundd of the ion and other comparison nds on PEPCK gene.

Figure 6 is a graph showing the measurement ofATP in HepG2 cells, treated with the indicated concentrations of a compound of the invention for 4 h.

Figure 7 is a series of graphs showing the choleretic effect of Compound 100 for id and iv administration (A), the secretion of nd 100 over time for id and iv administration (B), and the plasma concentration of Compound 100 over time (C).

DETAILED DESCIRPTION OF THE INVENTION Compounds of the invention The t invention relates to a nd of formula I: (I), or a pharmaceutically able salt, solvate, or amino acid ate thereof, wherein: R1 is hydroxyl; R2 is hydrogen, hydroxyl, alkyl, or n, n said alkyl is unsubstituted or substituted with one or more Ra; R3 is hydrogen, yl, alkyl, or halogen, wherein said alkyl is unsubstituted or substituted with one or more Rb; R4 is hydrogen, alkyl, alkenyl, alkynyl, or halogen, wherein said alkyl is unsubstituted or substituted with one or more R“; 1O Ra, Rb, and R0 are each independently halogen or hydroxyl; R5 is hydroxyl, osogH, osog', OCOCHg, opogH, opof', or hydrogen; and R6 is hydroxyl, osogH, osog‘, OCOCHg, opogH, opogz‘, or hydrogen; or taken together R5 and R6 with the carbon atom to which they are attached form a carbonyl.

In one aspect, the present invention relates to a compound formula II: COZH (II), or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof.

In one aspect, the present invention relates to a compound of formula III: (111), or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof.

In one aspect, the present invention s to a compound of formula IV: 2014/059896 (IV), or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof.

In one aspect, the present invention s to a compound of a I, II, III, or IV, wherein the compound is the compound (ag. the native compound, or the compound in the lt, unsolvateed, and non-conjuated form).

In one aspect, the present invention relates to a compound of formula I, II, III, or IV, wherein the compound is the pharmaceutically acceptable salt.

In one aspect, the present invention relates to a compound of formula I, II, III, or IV, wherein the compound is the amino acid conjugate. In one aspect, the amino acid conjugate 1O is a glycine conjugate. In one aspect, the amino acid ate is a taurine conjugate.

In one aspect, the t invention relates to a compound of formula I, wherein one of R2 or R3 is hydroxyl or halogen and the remaining R2 or R3 is hydrogen or unsubstituted alkyl. In one aspect, one of R2 or R3 is hydroxyl and the remaining R2 or R3 is hydrogen.

In one aspect, the present invention relates to a compound of formula I, wherein one of R5 or R6 is hydroxyl and the remaining R5 or R6 is hydrogen.

In one aspect, the present invention s to a compound of formula I, II, III, or IV, wherein R2 is hydroxyl or halogen. In one aspect, R2 is hydroxyl. In another aspect, R2 is halogen.

In one aspect, the present invention relates to a compound of formula I, II, III, or IV, wherein R3 is hydrogen or unsubstituted alkyl. In one aspect, R3 is hydrogen. In another aspect, R3 is methyl.

In one aspect, the t invention relates to a compound of formula I, II, III, or IV, wherein R2 is hydroxyl and R3 is hydrogen.

In one aspect, the present invention relates to a compound of formula I, II, III, or IV, n R5 is hydroxyl.

In one aspect, the present invention relates to a compound of formula I, II, III, or IV, wherein R6 is hydrogen.

In one aspect, the t invention s to a compound of formula I, II, III, or IV, wherein R2 and R5 are each hydroxyl and R3 and R6 are each hydrogen.

In one aspect, the present invention relates to a compound of formula I, II, III, or IV, wherein R4 is alkyl or hydrogen. In one aspect, the present invention relates to a compound of formula I, II, III, or IV, wherein R4 is unsubstituted alkyl. In one aspect, R4 is methyl, ethyl, propyl, or butyl. In one , R4 is methyl or ethyl. In one aspect, R4 is methyl. In one aspect, R4 is ethyl.

In one aspect, the present invention relates to compound ’I COZH Ho‘“ "’OH H /.... (l 00), or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof.

In one aspect, the t invention relates to a compound of a I, II, III, or IV, wherein the compound is an FXR agonist. In one , the compound of the invention is a highly potent FXR agonist. For example, the compound of the ion tes FXR at a tration below 1 uM, below 0.8 uM, below 0.6 uM, below 0.4 uM, or below 0.2 uM (e.g., as measured by an AlphaScreen assay), as compared to 15 uM for CDCA. For example, the compound of the invention activates FXR at a concentration below 0.2 uM (e.g., as measured by an AlphaScreen assay). For example, the compound of the invention activates FXR with an EC50 below 1 uM, below 0.8 uM, below 0.6 uM, below 0.4 uM, or below 0.2 uM (6.3., as measured by an AlphaScreen assay), as compared to 8.9 uM for CDCA. For e, the compound of the invention activates FXR with an EC50 below 0.2 uM (e.g., as measured by an AlphaScreen assay).

In one aspect, the present invention relates to a compound of formula I, II, III, or IV, wherein the compound is not active against other nuclear receptors. In one aspect, the present invention relates to a compound of formula I, II, III, or IV, wherein the compound does not activate TGRS (6.57., as measured by an HTR—FRET TGRS assay, where the TGRS is either expressed at a physiological level or overexpressed).

In one , the present invention relates to a compound of a I, II, III, or IV, wherein the compound induces apoptosis.

In one aspect, the present invention relates to a compound of a I, II, III, or IV, wherein the compound shows no cytotoxic effect on human HepG2 liver cells (e.g., as measured by an LDH release assay or an intracellular ATP assay). 2014/059896 In one , the present ion relates to a compound of formula I, II, III, or IV, wherein the nd does not inhibit one or more CYP450 isoforms selected from CYP1A2, CYP3A4 (green substrate), CYP3A4 (blue ate), CYP2C9, CYP2C19, , and CYP2El. For example, the compounds of the invention have an IC50 greater than 10 uM as measured by CPY450 inhibition assay.

In one aspect, the present invention relates to a compound of formula I, II, III, or IV, wherein the compound does not inhibit the human ERG potassium channel.

In one aspect, the t invention relates to a method of synthesizing a compound of the ion, or a ceutically acceptable salt, solvate, or amino acid conjugate thereof.

In one aspect, the t ion relates to a kit containing one or more nds of the invention, or a pharmaceutically acceptable salt, solvate, or amino acid conjutate thereof. In one aspect, the kit further contains a pharmaceutically acceptable ingredient.

In one aspect, the present invention relates to a pharmaceutical composition sing a compound of the invention and a pharmaceutically acceptable excipient.

One technical problem to be solved by the present invention is the identification of novel compounds that are agonists of the nuclear hormone famesoid X receptor (FXR), which represents an tive target for the treatment of metabolic and chronic liver diseases.

It is well known that natural bile acids modulate not only several nuclear hormone receiptors, but are also agonists for the G protein-coupled receptor (GPCR) TGRS. Selectivity can be a problem for drug compounds directed to modulating a nuclear hormone receptor. It is therefore an objective of the present invention to provide a compound that is a specific FXR agonist, for example, a compound that shows no activity against other nuclear receptors or a compound that does not activate the bile acid GPCR TGRS. Other problems in the pment of a drug compound include a non-suitable cokinetic profile, safety issues such as toxicity (e.g., liver) and undesirable drug-drug interactions. Accordingly, further objectives of the present invention are to provide compounds that do not suffer from the aforementioned technical problems, i. e. a compound that has a suitable pharmacokinetic profile, a compound that does not exert a cytotoxic effect on cells, a compound that does not inhibit cytochrome P450 enzymes, and/or a compound that does not inhibit hERG.

The patent and scientific literature referred to herein establishes knowledge that is available to those with skill in the art. The issued patents, ations, and references that are cited herein are hereby incorporated by nce to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of inconsistencies, the present disclosure will prevail.

For purposes of the present invention, the following definitions will be used (unless expressly stated ise).

The general chemical terms used throughout have their usual meanings. For example, the term alkyl refers to a branched or unbranched saturated hydrocarbon group. The term “n- alkyl” refers to an unbranched alkyl group. The term "CK-Cy alkyl” refers to an alkyl group having between x and y carbon atoms, inclusively, in the branched or unbranched hydrocarbon group. By way of illustration, but without tion, the term “C1-C8 alkyl” refers to a straight chain or branched hydrocarbon moiety having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “C1-C6” refers to a ht chain or branched hydrocarbon moiety having 1, 2, 3, 4, 5, or 6 carbon atoms. “C1-C4 alkyl” refers to a ht chain or branched hydrocarbon moiety having 1, 2, 3, or 4 carbon atoms, including methyl, ethyl, n-propyl, isopropyl, nbutyl , isobutyl, sec-butyl, and tert-butyl. The term “C1-C4 n-alkyl” refers to straight chain arbon moieties that have from 1, 2, 3, or 4 carbon atoms including , ethyl, npropyl , and n-butyl. The term “C3-C6 cycloalkyl” refers to cyclopropyl, utyl, cyclopentyl, and cyclohexyl. The term “C3-C7 cycloalkyl” also includes cycloheptyl. The term “C3-C8 cycloalkyl” also es ctyl. Cycloalkylalkyl refers to cycloalkyl moieties linked through an alkyl linker chain, as for example, but without limitation, cyclopropylmethyl, ropylethyl, cyclopropylpropyl, cyclopropylbutyl, cyclobutylmethyl, cyclobutylethyl, cyclobutylpropyl, cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl, cyclohexylmethyl, cyclohexylethyl, and cyclohexylpropyl. Each alkyl, cycloalkyl, and cycloalkylalkyl group may be optionally substituted as specified herein.

The term “C4-C8 cycloalkenyl” refers cyclobutenyl, entyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl rings having one or more sites of unsaturation, e.g., one or more double bonds.

The term “halogen” refers to fluoro, chloro, bromo, or iodo.

The term “hydroxyl” means OH.

It will be understood that “substitution” or “substituted with” es the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is plated to include all permissible substituents of organic compounds unless otherwise specified. In a broad aspect, the permissible substituents include c and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible tuents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

The term “pharmaceutical” or “pharmaceutically acceptable” when used herein as an adjective, means substantially non-toxic and substantially leterious to the recipient.

By “pharmaceutical formulation” it is further meant that the carrier, solvent, excipient, and salt must be compatible with the active ingredient of the formulation (e.g., a compound of the invention). It is understood by those of ordinary skill in this art that the terms “pharmaceutical formulation” and “pharmaceutical composition” are generally hangeable, and they are so used for the es of this application.

Suitable ceutically acceptable salts according to the invention will be y determined by one skilled in the art and will include, for example, basic salts such as alkali or alkaline—earth metallic salts made from aluminium, m, lithium, magnesium, potassium, sodium, and zinc or organic salts made from N,N'-dibenzylethylenediamine, chlorprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine.

Salts with pharmaceutically acceptable amines such as lysine, arginine, tromethamine, triethylamine and the like can also be used. Such salts of the compounds ofthe invention may be prepared using conventional techniques, from the compound of the invention by reacting, for example, the appropriate base with the compound of the invention.

When used in medicine, the salts of a compound of the invention should be pharmaceutically acceptable, but pharmaceutically unacceptable salts may conveniently be used to prepare the corresponding free base or pharmaceutically able salts thereof As used herein, the term “amino acid conjugate” refers to a conjugate of a compound of the invention with any suitable amino acid. In one , such suitable amino acid conjugate of a compound of the invention will have the added advantage of enhanced integrity in bile or intestinal fluids. The present invention asses the glycine and e conjugates of any of the compounds of the invention. For e, the glycine and the taurine conjugates of a nd of formula I have the following formula: N/Vsogn (glycine) (taurine) In one aspect, the glycine and taurine conjuates of a compound of the invention may be a pharmaceutically acceptable salt thereof. The amino acid conjugates of compounds of the invention can be prepared according to methods known in the art. For example, the free acid can be coupled to the glycine or taurine amino acid using standard peptide coupling conditions.

In one aspect, the sodium salt of the taurine conjugate of Compound 100 can be ed as follows. taurine conjugate of compound 100 1O nd 100 Compound 100 is treated with a base (e.g., Eth) and taurine in a polar protic solvent (e.g. , EtOH). The resulting mixture can be d with a coupling reagent (ag. , DMT-MM (4—(4,6-Dimethoxy-l,3,5—triazinyl)—4-methylmorpholinium chloride)). The reaction mixture can be concentrated and dissolved in a base (e.g., 3% w/v aqueous solution of NaOH). The ing reaction mixture can be extracted with an organic solvent (e.g., AcOEt). The aqueous phase can be trated and filtered on a silica pad, eluting first with, e.g., H20 (until neutral pH) and then with, e.g., HZO/MeOH 80:20 v/v to give the taurine conjugate of Compound 100. Suitable amino acids include but are not d to glycine and taurine.

Some of the compounds of the present invention may exist in ated as well as ed forms such as, for example, hydrates.

The present invention provides s for the synthesis of the compounds of invention described herein. The present invention also provides detailed methods for the WO 84271 synthesis of various disclosed compounds of the invention according to the following schemes as shown in the examples.

The synthetic processes of the invention can tolerate a wide variety of functional groups, therefore various substituted starting materials can be used. The processes generally provide the d final compound at or near the end of the overall process, although it may be desirable in certain instances to further convert the compound to a pharmaceutically acceptable salt, ester or prodrug thereof.

Compounds of the invention can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates, by employing standard tic methods and procedures either known to those skilled in the art, or which will be apparent to the skilled artisan in light of the teachings herein. Standard synthetic s and procedures for the ation of organic molecules and functional group ormations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field. Although not limited to any one or several sources, classic texts such as Smith, M. 3, March, J March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition, John Wiley & Sons: New York, 2001; and , T.W., Wuts, P.G. M., Protective Groups in c Synthesis, 3rd edition, John Wiley & Sons: New York, 1999, incorporated by reference herein, are useful and recognized reference oks of organic synthesis known to those in the art. The following ptions of synthetic s are designed to illustrate, but not to limit, general procedures for the preparation of compounds of the present invention.

All the iations used in this application are found in “Protective Groups in Organic Synthesis” by John Wiley & Sons, Inc, or the MERCK INDEX by MERCK & Co., Inc, or other chemistry books or chemicals catalogs by chemicals vendor such as Aldrich, or according to usage know in the art.

Scheme 1 COZMe 7 compound 100 COZH Ho‘“ "’OH compound 101 Reagents and conditions: a) 1) MeOH, p-TSA, ultrasound, 3 h, quantitative; 2) Ac20, NaHCOg, THF, reflux 12 h, 85%; b) PCC, CHzClz, 6 h, 62%; c) ACZO, Bi(OTf)3, CH2C12, 1 h, 91%; d) Brz, Benzene, 30 0C overnight, 74%; e) NaBH4, NaOAc, Pyr, rt. 2 days, 80%; f) H1 57%, ACOH, rt. 30 min; g) CI'Og, ACOH, rt. 45 min; h) Zn dust, NaOAc, reflux 20 min; i) NaOH 2M, MeOH, r.t. overnight, 65% from compound 5; l) NaBH4, THF/H20 4:1, 70%; m) Na(s), sec-BuOH, 50 0C, 70%.

The synthesis is based on the use of 60t-ethyl-cholic acid (6-ECA, 1) as ng material which was ed using methods known in the art. 6-ECA (1) was treated with p- TSA in MeOH under ultrasound irradiation to give the corresponding methyl ester, which was selectively protected at the C3 position by refluxing with AczO in the present of NaHC03 in THF to afford compound 2. ng compound 2 with PCC in CH2C12 at room temperature followed by treatment with AczO, Bi(OTf)3 in CHzClz at room temperature ed the intermediate methyl 30L,7u-diacetoxyoxo-SB-cholanoate (compound 3; about 48% from compound 2).

Treatment of compound 3 with Brz in benzene for 6.57., 12 h yielded compound 4. on of compound 4 with NaBH4 and NaOAc in freshly distilled pyridine gave the corresponding B e (compound 5), in about 59% yield after silica gel ation. The reaction of compound 5 with H1 in AcOH at room temperature ed the halohydrine intermediate which were then oxidized at Cll position with Cr03 in AcOH to generate compound 6. Reaction of compound 6 with Zn dust in boiling AcOH and alkali hydrolysis (NaOH/MeOH) afforded 3u,7oi-hydroxyketo-5l3-cholanoic acid und 7; about 65% yield from compound 5).

Compound 7 was selectively reduced at the Cl l-carbonyl using NaBH4 in a mixture ofTHF/H20= (4:1, V/V) to give 30L,70t,l lB-trihydroxy-60t-ethyl-5B-cholan—24-oic acid (Compound 100; about 27% from nd 3), after tographic purification to afford Compound 100. Alternatively, compound 7 was reduced with sodium in sec-BuOH at 50 0C to give Compound 101 (about 70% yield), after purification.

“Solyate” means a t addition form that contains either a stoichiometric or non stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the t is water the solvate formed is a hydrate; when the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one of the substances in which the water retains its molecular state as H20, such combination being able to form one or more hydrate.

The term “suitable solvent” refers to any t, or mixture of solvents, inert to the ongoing reaction that sufficiently solubilizes the reactants to afford a medium within which to effect the desired reaction.

The compounds described herein can have asymmetric centers. Compounds of the present invention containing an asymmetrically substituted atom can be isolated in optically active or racemic forms. It is well known in the art how to e optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials.

Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the nds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and can be ed as a mixture of isomers or as separate isomeric forms. All chiral, diastereomeric, racemic, and geometric isomeric forms of a structure are intended, unless specific stereochemistry or isomeric form is specifically indicated. All processes used to prepare compounds of the present ion and intermediates made therein are considered to be part of the present ion. All tautomers of shown or described compounds are also considered to be part of the present invention. Furthermore, the invention also es metabolites of the nds described herein.

The ion also comprehends isotopically-labeled compounds, which are identical to those recited in the formulae of the invention, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature. Examples of isotopes that can be incorporated into compounds of the invention e isotopes of hydrogen, carbon, nitrogen, fluorine, such as 3H, 11C, 14C, 2H, and 18F.

Compounds of the t invention and pharmaceutically acceptable salts, solvates or amino acid ates thereof that contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of the present invention. Isotopically-labeled compounds of the present invention, for example those into which ctive isotopes such as 3H and 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3H, and carbon—l4, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. 11C and 18F isotopes are particularly usefial in PET (positron emission tomography). PET is useful in brain imaging. Further, substitution with r isotopes such as deuterium, i.e., 2H, can afford certain therapeutic ages resulting from greater metabolic stability, for example increased in viva half-life or reduced dosage ements and, hence, may be preferred in some circumstances. Isotopically labeled compounds of this ion can generally be prepared through techniques known in the art, such as by carrying out the procedures disclosed in the Schemes and/or in the Examples below, by substituting a readily ble isotopically labeled reagent for a non—isotopically labeled reagent. In one embodiment, the compounds of the invention, salts, hydrates, solvates, or amino acid conjugates thereof are not isotopically labelled.

When any variable (e.g. occurs more than one time in any constituent or formula , RX) for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with one or more RX moieties, then RX at each occurrence is selected independently from the definition of RX.

Also, combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds within a designated atom’s normal valency.

As used herein, the term ,9) “treating,” or “treatment” is meant sing the symptoms, markers, and/or any ve effects of a condition in any appreciable degree in a subject who currently has the ion. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the condition for the e of decreasing the risk of developing the disease or condition.

As used , the term “prevent,3, “prevention,” or “preventing” refers to any method to partially or completely prevent or delay the onset of one or more symptoms or features of a disease, disorder, and/or condition. Prevention may be administered to a subject who does not exhibit signs of a disease or condition.

As used herein, “subject” means a human or animal (in the case of an animal, more typically a mammal). In one aspect, the subject is a human. Such subject can be considered to be in need of treatment with an FXR agonist.

As used herein, “unsaturated” refers to compounds or structures having at least one degree of unsaturation (e.g., at least one double or triple bond).

As used , the term “a compound of the invention” includes a compound of any of formulae I, II, III, or IV, or any compound explicitly sed herein.

As used herein, farnesoid X receptor or FXR refers to all mammalian forms of such receptor including, for example, alternative splice isoforms and naturally occurring ms (see, e.g., Huber er 611., Gene 290:35-43 (2002)). Representative FXR species include, without limitation rat FXR (Gen Bank Accession No. NM_021745), mouse FXR nk Accession No. NM_009108), and human FXR (GenBank ion No. NM_005123).

As used , Compound A is which is also known as obeticholic acid, INT-747, 6ECDCA, 6-alpha-ethyl chenodeoxycholic acid, or 60t-ethyl-3 0t,70t-dihydroxy-5 B-cholan oic acid.

As used herein, Compound B is 0803‘ Na‘" HO“. . "’OH which is also known as INT-767 or 60t-ethyl-30t,70t,23— trihydroxy—24-nor—5B-cholan-23—sulfate sodium salt.

As used herein, nd C is , cow which is also known as INT-777 or 6d-ethyl-23(S)-methyl- 30L,70L,120L trihydroxy-SB-cholanoic acid.

As used herein, Compound D is COZH HO“. which is also known as 60L-ethyl-23(R)—methyl chenodeoxycholic acid, and S-EMCDCA.

As used herein, Compound E is cogH HO‘“ "’OH 1 0 As used herein, cholic acid is 002H which is also known as CA.

As used herein, chenodeoxycholic acid is which is also known as CDCA.

As used herein, ursodeoxycholic acid is which is also known as UDCA.

As used herein, taurochenodeoxycholic acid is 0 00 u‘ I: NNS‘OH which is also known asn TCDCA.

As used herein, tauroursodeoxycholic acid is which is also known as TUDCA.

As used herein, lithocholic acid is which is also known as LCA.

Methods of the Invention Compounds of the invention are useful in therapy in subjects such as s, including humans. In particular, nds of the invention are usefill in a method of treating or preventing a disease or condition in a subject comprising administering to the subject in need thereof an effective amount of a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof. In one aspect, the disease or ion is FXR-mediated (e.g. FXR plays a role in the initiation or progress of the disease or ion). In one aspect, the disease or condition is mediated by decreased FXR activity. In one , the disease or condition is selected from cardiovascular disease, 2014/059896 chronic liver disease, lipid disorder, gastrointestinal disease, renal disease, metabolic disease, cancer, and neurological disease.

In one aspect, the invention relates to a method of treating or preventing cardiovascular disease in a subject, comprising administereing to the subject in need thereof an effective amount of a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate f. In one aspect, the invention relates to a method of treating cardiovascular disease. In one aspect, the invention relates to a method of ting cardiovascular disease. In one aspect, cardiovascular disease selected from atherosclerosis, arteriosclerosis, dyslipidemia, hypercholesteremia, hyperlipidemia, ipoproteinemia, and hypertriglyceridemia.

The term “hyperlipidemia” refers to the presence of an abnormally elevated level of lipids in the blood. Hyperlipidemia can appear in at least three forms: (1) hypercholesterolemia, i. 6., an elevated cholesterol level; (2) hypertriglyceridemia, i.e., an ed triglyceride level; and (3) combined hyperlipidemia, i.e., a combination of hypercholesterolemia and hypertriglyceridemia.

The term “dyslipidemia” refers to abnormal levels of lipoproteins in blood plasma including both depressed and/or elevated levels of oteins (e.g., elevated levels of LDL, VLDL and depressed levels of HDL).

In one aspect, the invention relates to a method selected from reducing terol levels or modulating cholesterol metabolism, catabolism, absorption of dietary cholesterol, and reverse cholesterol transport in a subject, comprising administering to the t in need thereof an effective amount of a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof.

In one , the invention relates to a method of treating or preventing a disease ing cholesterol, triglyceride, or bile acid levels in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the ion or a pharmaceutically able salt, solvate, or amino acid conjugate thereof.

In one aspect, the invention relates to a method of lowering triglycerides in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the invention or a pharmaceutically able salt, solvate, or amino acid conjugate thereof.

In one aspect, the invention s to a method of treating or preventing a disease state ated with an elevated cholesterol level in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the invention or a pharmaceutically able salt, e, or amino acid conjugate thereof. In one aspect, the invention relates to a method of treating a e state associated with an elevated cholesterol level in a subject. In one aspect, the invention relates to a method of preventing a disease state associated with an elevated cholesterol level in a subject. In one aspect, the disease state is selected from coronary artery disease, angina pectoris, carotid artery e, strokes, cerebral arteriosclerosis, and xanthoma.

In one aspect, the invention relates to a method of treating or ting a lipid disorder in a subject, comprising administereing to the subject in need thereof an effective amount of a compound of the ion or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof. In one aspect, the invention relates to a method of treating a lipid disorder. In one aspect, the invention relates to a method of preventing a lipid disorder.

Lipid disorders are the term for abnormalities of cholesterol and cerides. Lipid abnormalities are associated with an increased risk for vascular disease, and especially heart attacks and strokes. Abnormalities in lipid disorders are a combination of genetic predisposition as well as the nature of dietary intake. Many lipid disorders are associated with being overweight. Lipid disorders may also be associated with other diseases including diabetes, the metabolic me (sometimes called the insulin resistance syndrome), underactive thyroid or the result of certain medications (such as those used for anti—rej ection regimens in people who have had transplants).

In one , the invention relates to a method of treating or ting one or more symptoms of e affecting lipid metabolism (i.e., strophy) in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof. In one aspect, the invention relates to a method of treating one or more symptoms of a disease affecting lipid metabolism. In one aspect, the invention relates to a method of preventing one or more symptoms of a disease affecting lipid metabolism.

In one aspect, the invention s to a method of decreasing lipid accumulation in a subject, comprising administering to the subject in need f an effective amount of a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof.

In one aspect, the ion s to a method of treating or preventing c liver disease in a subject, comprising administereing to the sufj ect in need thereof an effective amount of a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof. In one aspect, the invention relates to a method of treating c liver disease. In one aspect, the invention s to a method of preventing chronic 2014/059896 liver disease. In one aspect, the chronic liver disease is selected from primary biliary cirrhosis (PBC), cerebrotendinous xanthomatosis (CTX), primary sclerosing cholangitis (PSC), drug induced tasis, epatic cholestasis of pregnancy, parenteral nutrition associated cholestasis (PNAC), ial overgrowth or sepsis associated cholestasis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver transplant associated graft versus host disease, living donor transplant liver regeneration, congenital hepatic fibrosis, choledocholithiasis, granulomatous liver disease, intra— or epatic malignancy, Sjogren's syndrome, dosis, Wilson's disease, r' s e, hemochromatosis, and alpha 1- antitrypsin deficiency.

In one aspect, the invention relates to a method of ng or preventing one or more symptoms of cholestasis, including complications of cholestasis in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the invention or a pharmaceutically able salt, solvate, or amino acid conjugate thereof. In one aspect, the invention relates to a method of treating one or more symptoms of cholestasis.

In one aspect, the invention relates to preventing one or more symptoms of cholestasis.

Cholestasis is typically caused by factors within the liver (intrahepatic) or outside the liver (extrahepatic) and leads to the lation of bile salts, bile t bilirubin, and lipids in the blood stream instead of being eliminated normally. Intrahepatic cholestasis is characterized by widespread blockage of small ducts or by disorders, such as tis, that impair the body's ability to eliminate bile. Intrahepatic cholestasis may also be caused by alcoholic liver e, primary biliary cirrhosis, cancer that has spread (metastasized) from another part of the body, primary sclerosing cholangitis, gallstones, biliary colic and acute cholecystitis. It can also occur as a complication of surgery, serious injury, cystic fibrosis, infection, or enous feeding or be drug induced. Cholestasis may also occur as a complication of pregnancy and often develops during the second and third trimesters.

Extrahepatic cholestasis is most often caused by choledocholithiasis (Bile Duct Stones), benign biliary strictures (non-cancerous narrowing of the common duct), cholangiocarcinoma (ductal oma) and pancreatic carcinoma. Extrahepatic cholestasis can occur as a side effect of many tions.

A compound of the invention may be used for treating or preventing one or more symptoms of intrahepatic or extrahepatic cholestasis, including without limitation, biliary artesia, obstetric cholestasis, neonatal cholestasis, drug induced tasis, cholestasis arising from Hepatitis C infection, chronic cholestatic liver disease such as primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC).

In one aspect, the invention relates to a method of enhancing liver regeneration in a subject, sing administering to the subject in need thereof an effective amount of a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof. In one aspect, the method is enhancing liver regeneration for liver transplantation.

In one aspect, the invention relates to a method of treating or preventing fibrosis in a subject, comprising administering to the t in need thereof an effective amount of a compound of the invention or a pharmaceutically acceptable salt, e, or amino acid conjugate thereof In one aspect, the invention relates to a method of treating fibrosis. In one , the invention relates to a method of preventing fibrosis. ingly, as used herein, the term fibrosis refers to all recognized fibrotic disorders, including fibrosis due to ogical conditions or diseases, fibrosis due to physical trauma (“traumatic fibrosis”), fibrosis due to radiation damage, and fibrosis due to exposure to herapeutics. As used herein, the term “organ s” includes but is not limited to liver fibrosis, fibrosis of the kidneys, fibrosis of lung, and fibrosis of the intestine. atic s” es but is not limited to fibrosis ary to surgery (surgical scarring), accidental physical trauma, burns, and hypertrophic scarring.

As used herein, “liver fibrosis” includes liver fibrosis due to any cause, including but not limited to virally-induced liver fibrosis such as that due to hepatitis B or C virus; exposure to alcohol (alcoholic liver disease), certain pharmaceutical compounds including but not limited to methotrexate, some chemotherapeutic agents, and chronic ingestion of arsenicals or vitamin A in megadoses, oxidative stress, cancer radiation therapy or certain industrial chemicals including but not limited to carbon tetrachloride and dimethylnitrosamine; and diseases such as y biliary sis, primary sclerosing colangitis, fatty liver, obesity, non-alcoholic steatohepatitis, cystic fibrosis, romatosis, mmune hepatitis, and steatohepatitis. Current therapy in liver fibrosis is primarily directed at removing the causal agent, e.g., removing excess iron (e.g., in the case of hemochromatosis), decreasing Viral load (e.g., in the case of chronic Viral hepatitis), or eliminating or decreasing exposure to toxins (e.g., in the case of alcoholic liver disease).

Anti-inflammatory drugs such as osteroids and colchicine are also known for use in treating inflammation that can lead to liver fibrosis.

As is known in the art, liver fibrosis may be clinically classified into five stages of severity (SO, S1, S2, S3, and S4), usually based on histological examination of a biopsy en. SO indicates no fibrosis, s S4 indicates cirrhosis. While various criteria for staging the severity of liver fibrosis exist, in general early stages of fibrosis are identified by discrete, localized areas of scarring in one portal (zone) of the liver, whereas later stages of fibrosis are identified by bridging fibrosis ing that crosses zones of the liver).

In one aspect, the invention relates to a method of treating or preventing organ fibrosis in a subject, comprising administering to the subject in need f an effective amount of a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof. In one aspect, the fibrosis is liver fibrosis.

In one aspect, the ion relates to a method of treating or preventing intestinal disease in a subject, comprising administereing to the sufj ect in need thereof an effective amount of a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof. In one , the ion relates to a method of treating gastrointestinal disease. In one aspect, the invention relates to a method of preventing gastrointestinal disease. In one aspect, the gastrointestinal disease is selected from inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), bacterial overgrowth, malabsorption, post-radiation colitis, and microscopic colitis. In one aspect, the inflammatory bowel disease is selected from Crohn’s disease and ulcerative colitis.

In one aspect, the invention relates to a method of treating or preventing renal e in a subject, comprising administereing to the sufj ect in need thereof an ive amount of a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof. In one aspect, the invention relates to a method of treating renal disease.

In one aspect, the invention relates to a method of preventing renal disease. In one aspect, the renal disease is selected from ic nephropathy, focal tal ulosclerosis (FSGS), hypertensive sclerosis, chronic glomerulonephritis, chronic transplant glomerulopathy, chronic interstitial tis, and polycystic kidney disease.

In one aspect, the invention relates to a method of treating or preventing metabolic disease in a subject, comprising administereing to the sufj ect in need thereof an effective amount of a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof. In one , the invention s to a method of treating renal disease. In one aspect, the ion relates to a method of preventing renal disease. In one aspect, the metabolic disease is selected from insulin resistance, hyperglycemia, diabetes mellitus, ity, and obesity. In one aspect, the es mellitus is type I diabetes. In one aspect, the diabetes us is type II diabetes.

Diabetes mellitus, commonly called diabetes, refers to a disease or condition that is generally characterized by metabolic defects in production and utilization of glucose which result in the failure to maintain appropriate blood sugar levels in the body.

In the case of type II diabetes, the e is characterized by insulin resistance, in which insulin loses its ability to exert its biological effects across a broad range of concentrations. This resistance to insulin siveness results in insufficient insulin activation of glucose uptake, oxidation and storage in muscle and inadequate n repression of lipolysis in adipose tissue and of glucose production and secretion in liver. The resulting ion is elevated blood glucose, which is called “hyperglycemia”. Uncontrolled hyperglycemia is associated with increased and ure mortality due to an increased risk for microvascular and macrovascular diseases, including retinopathy (the impairment or loss of Vision due to blood vessel damage in the eyes); neuropathy (nerve damage and foot problems due to blood vessel damage to the s system); and nephropathy (kidney disease due to blood vessel damage in the kidneys), hypertension, cerebrovascular disease and coronary heart disease. Therefore, control of glucose homeostasis is a critically ant ch for the treatment of diabetes.

Insulin resistance has been hypothesized to unify the clustering of hypertension, glucose intolerance, hyperinsulinemia, increased levels of triglyceride and decreased HDL cholesterol, and central and overall obesity. The association of insulin resistance with glucose intolerance, an increase in plasma triglyceride and a decrease in high-density otein cholesterol concentrations, hypertension, ricemia, smaller denser lowdensity lipoprotein particles, and higher circulating levels of plasminogen activator inhibitor- 1, has been referred to as “Syndrome X”. Accordingly, methods of treating or preventing any disorders related to insulin resistance including the cluster of disease states, conditions or disorders that make up “Syndrome X” are provided. In one aspect, the invention relates to a method of treating or preventing metabolic syndrome in a subject, comprising stering to the subject in need thereof an effective amount of a compound of the invention or a aceutically acceptable salt, solvate, or amino acid conjugate thereof. In one aspect, the invention s to a method of treating metabolic syndrome. In one , the ion s to a method of preventing metabolic syndrome.

In one aspect, the invention relates to a method of treating or preventing cancer in a subject, comprising administereing to the sufj ect in need thereof an effective amount of a compound of the ion or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof. In one aspect, the invention relates to a method of treating cancer. In one aspect, the invention relates to a method of preventing cancer. In one aspect, the cancer is colorectal .

In one aspect, the invention relates to a method of treating or preventing gallstones in a subject, comprising administering to the t in need thereof an effective amount of a compound of the invention or a ceutically acceptable salt, solvate, or amino acid conjugate thereof. In one aspect, the invention relates to a method of treating gallstones. In one aspect, the invention s to a method ofpreventing gallstones.

A gallstone is a crystalline concretion formed within the gallbladder by ion of bile ents. These calculi are formed in the gallbladder but may distally pass into other parts of the y tract such as the cystic duct, common bile duct, pancreatic duct, or the ampulla of Vater. Rarely, in cases of severe inflammation, gallstones may erode through the gallbladder into adherent bowel potentially causing an obstruction termed one ileus.

Presence of gallstones in the gallbladder may lead to acute ystitis, an atory condition characterized by retention of bile in the gallbladder and often secondary infection by intestinal microorganisms, predominantly Escherichia coli and Bacteroides species.

Presence of gallstones in other parts of the biliary tract can cause obstruction of the bile ducts, which can lead to serious conditions such as ascending cholangitis or pancreatitis.

In one aspect, the ion relates to a method of treating or preventing cholesterol gallstone disease in a subject, comprising administering to the subject in need thereof an effective amount of a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate f. In one aspect, the invention relates to a method of ng cholesterol gallstone disease. In one aspect, the invention relates to a method of preventing cholesterol gallstone disease.

In one aspect, the invention relates to a method of treating or preventing neurological disease in a subject, sing administering to the t in need thereof an effective amount of a compound of the invention or a pharmaceutically able salt, solvate, or amino acid conjugate thereof. In one aspect, the invention relates to a method of treating neurological disease. In one aspect, the invention relates to a method of preventing neurological e. In one aspect, the neurological disease is stroke.

In one aspect, the invention relates to a method as decribed herein and further wherein, the compound is administered by a route selected from oral, parenteral, WO 84271 intramuscular, intranasal, sublingual, intratracheal, inhalation, ocular, l, , and intracerebroventricular. In one aspect, the route is oral.

In one , the compound zed in one or more of the methods described herein is an FXR agonist. In one aspect, the nd is a selective FXR agonist. In one aspect, the compound does not activate TGRS. In one aspect, the compound does not activate other nuclear receptors involved in metabolic pathways (e.g., as measured by an creen assay). In one aspect, such other nuclear receptors involved in metabolic pathways are selected from LXRB, PXR, CAR, PPAROL, PPARS, RAROL, VDR, TR, PR, RXR, GR, and ER.

In one aspect, the compound induces apoptosis.

In one aspect, the invention relates to a method of regulating the expression level of one or more genes involved in bile acid homeostasis.

In one aspect, the invention relates to a method of down regulating the expression level of one or more genes selected from CYP7Q1 and SREBP-lC in a cell by administering to the cell a compound of the ion. In one aspect, the invention relates to a method of up regulating the expression level of one or more genes selected from OSTOL, OSTB, BSEP, SHP, UGT2B4, MRPZ, FGF-l9, PPARy, PLTP, APOCII, and PEPCK in a cell by administering to the cell a compound of the invention.

The invention also relates to the manufacture of a medicament for treating or preventing a disease or ion (e.g., a disease or condition ed by FXR), wherein the medicament comprises a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof. In one aspect, the invention relates to the manufacture of a medicament for treating or preventing any one of the diseases or conditions described herein above, wherein the medicament comprises a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof.

The invention also relates to a composition for use in a method for treating or ting a disease or condition (e.g., a disease or condition mediated by FXR), wherein the composition comprises a compound of the invention or a pharmaceutically acceptable salt, solvate, or amino acid conjugate thereof. In one aspect, the invention relates toa composition for use in a method for treating or preventing any one of the diseases or conditions described herein above, n the ition ses a compound of the invention or a pharmaceutically acceptable salt, e, or amino acid conjugate thereof. 2014/059896 Formulations The methods of the invention comprise the step of administering an effective amount of a compound of the invention. As used herein, the term an “effective amount” refers to an amount of a compound of the ion which is sufficient to achieve the stated effect.

Accordingly, an effective amount of a compound of the invention used in a method for the prevention or treatment of FXR ed diseases or conditions will be an amount sufficient to prevent or treat the FXR mediated disease or condition.

Similarly, an effective amount of a compound of the invention for use in a method for the prevention or treatment of a cholestatic liver disease or increasing bile flow will be an amount sufficient to increase bile flow to the ine.

The amount of the nd of the ion which is required to achieve the desired biological effect will depend on a number of factors such as the use for which it is intended, the means of administration, and the recipient, and will be ultimately at the discretion of the attendant physician or veterinarian. In general, a typical daily dose for the treatment of a FXR mediated disease and ion, for instance, may be expected to lie in the range of from about 0.01 mg/kg to about 100 mg/kg. This dose may be administered as a single unit dose or as several separate unit doses or as a continuous infiasion. Similar dosages would be applicable for the treatment of other diseases, conditions and therapies including the prevention and treatment of cholestatic liver diseases.

Thus, in a further aspect, the t invention provides a pharmaceutical composition comprising, as active ingredient, a compound of the invention together, and/or in admixture, with at least one pharmaceutical carrier or diluent. These pharmaceutical compositions may be used in the prevention or treatment of the foregoing diseases or conditions.

The carrier must be pharmaceutically acceptable and must be compatible with, i. e. not have a deleterious effect upon, the other ingredients in the ition. The carrier may be a solid or liquid and is preferably formulated as a unit dose formulation, for example, a tablet which may contain from 0.05 to 95% by weight of the active ingredient. If desired, other physiologically active ingredients may also be incorporated in the pharmaceutical compositions of the invention. le formulations include those suitable for oral, sublingual, buccal, parenteral (for example subcutaneous, intramuscular, or intravenous), rectal, l including transdermal, intranasal and inhalation stration. Most le means of administration for a particular patient will depend on the nature and severity of the disease or condition being treated or the nature of the therapy being used and on the nature of the active compound, but where possible, oral administration is preferred for the prevention and treatment of FXR mediated diseases and conditions. Formulations suitable for oral administration may be provided as discrete units, such as tablets, capsules, s, lozenges, each containing a predetermined amount of the active compound; as powders or granules; as solutions or suspensions in s or non-aqueous liquids; or as oil-in-water or water-in-oil emulsions.

Formulations suitable for sublingual or buccal administration include lozenges comprising the active compound and, typically a ed base, such as sugar and acacia or tragacanth and pastilles comprising the active nd in an inert base, such as gelatine and glycerine or sucrose .

Formulations suitable for parenteral administration typically comprise sterile aqueous solutions containing a predetermined tration of the active nd; the solution is preferably ic with the blood of the intended recipient.

Additional formulations suitable for parenteral administration include formulations containing physiologically le co—solvents and/or complexing agents such as surfactants and cyclodextrins. Oil-in—water emulsions are also suitable formulations for parenteral formulations. Although such solutions are preferably administered intravenously, they may also be administered by subcutaneous or intramuscular injection.

Formulations le for rectal administration are preferably provided as unit-dose suppositories comprising the active ingredient in one or more solid carriers forming the suppository base, for example, cocoa butter.

Formulations le for topical or intranasal application include nts, creams, lotions, pastes, gels, sprays, ls and oils. Suitable carriers for such formulations include petroleum jelly, n, polyethyleneglycols, alcohols, and combinations thereof Formulations of the invention may be prepared by any suitable method, typically by uniformly and intimately admixing the active compound with liquids or finely divided solid carriers or both, in the required proportions and then, if necessary, shaping the resulting e into the desired shape.

For example a tablet may be prepared by compressing an intimate mixture comprising a powder or granules of the active ingredient and one or more optional ingredients, such as a binder, ant, inert diluent, or surface active dispersing agent, or by moulding an intimate mixture ofpowdered active ingredient and inert liquid diluent.

Suitable formulations for stration by inhalation e fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulisers, or lators.

For pulmonary administration via the mouth, the particle size of the powder or droplets is typically in the range 0.5-10 um, preferably 1-5 um, to ensure ry into the ial tree. For nasal administration, a particle size in the range 10-500 um is red to ensure retention in the nasal cavity.

Metered dose inhalers are pressurised aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquefied propellant. During use, these devices discharge the formulation h a valve adapted to deliver a metered volume, lly from 10 to 150 ul, to produce a fine particle spray containing the active ingredient. Suitable propellants e certain chlorofluorocarbon compounds, for example, rodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation may additionally n one or more vents, for example, ethanol surfactants, such as oleic acid or sorbitan trioleate, anti-oxidants and suitable flavouring agents.

Nebulisers are commercially available devices that transform solutions or suspensions of the active ingredient into a therapeutic aerosol mist either by means of acceleration of a compressed gas typically air or oxygen, through a narrow venturi orifice, or by means of ultrasonic agitation. le ations for use in nebulisers consist of the active ingredient in a liquid carrier and comprise up to 40% w/w of the formulation, preferably less than 20% w/w. The carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride.

Optional additives include preservatives if the formulation is not prepared sterile, for example, methyl hydroxy-benzoate, anti-oxidants, flavouring agents, volatile oils, ing agents and tants.

Suitable formulations for administration by insufflation include finely comminuted powders which may be delivered by means of an insufflator or taken into the nasal cavity in the manner of a snuff. In the insufflator, the powder is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually- operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional tant. The active ingredient typically comprises from 0.1 to 100 w/w of the formulation.

In addition to the ients cally mentioned above, the formulations of the present invention may include other agents known to those skilled in the art of pharmacy, having regard for the type of formulation in issue. For example, formulations suitable for oral administration may e flavouring agents and formulations suitable for intranasal administration may include perfumes.

The following Examples are illustrative and should not be interpreted in any way so as to limit the scope of the ion.

EXAMPLES In general, the potential of a compound of the invention as a drug candidate can be tested using various assays known in the art. For example, for in vitro validation for FXR: its activity and selectivity can be ted using AlphaScreen (biochemical assay); gene sion can be evaluated using RT—PCR (FXR target gene); and cytotoxicity (e.g. , HepG2) can be evaluated using ATP content, LDH release, and Caspase-3 activation. For the in vitro validation for TGRS: its activity and selectivity can be evaluated using HTR-FRET (cell—based assay); gene expression can be evaluated using RT—PCR (TGRS target gene (i.e., cFOS)); and cytotoxicity (e.g., HepG2) can be evaluated using ATP t, LDH release, and Caspase-3 activation. The ADME (absorption, distribution, metabolism, and excretion) /pharmacokinetic properties and in vivo validation of compounds of the invention can also be studied using methods known in the art.

Example 1: Synthesis of compounds 100 and 101 Compounds 100 and 101 were synthesized according to the scheme below.

Scheme 1 COZMe 7 compound 100 COZH Ho‘“ "’OH compound 101 ts and conditions: a) 1) MeOH, p-TSA, ultrasound, 3 h, quantitative; 2) Ac20, NaHCOg, THF, reflux 12 h, 85%; b) PCC, CHzClz, 6 h, 62%; c) ACZO, Bi(OTf)3, , 1 h, 91%; d) Brz, Benzene, 30 0C overnight, 74%; e) NaBH4, NaOAc, Pyr, rt. 2 days, 80%; f) H1 57%, ACOH, rt. 30 min; g) CI'Og, ACOH, rt. 45 min; h) Zn dust, NaOAc, reflux 20 min; i) NaOH 2M, MeOH, r.t. overnight, 65% from compound 5; l) NaBH4, THF/H20 4:1, 70%; m) Na(s), sec-BuOH, 50 0C, 70%.

The synthesis is based on the use of 60t-ethyl-cholic acid (6-ECA, 1) as starting material which was prepared using methods known in the art. 6-ECA (1) was treated with p- TSA in MeOH under ultrasound irradiation to give the corresponding methyl ester, which was ively protected at the C3 position by refluxing with AczO in the present of NaHC03 in THF to afford compound 2. Treating compound 2 with PCC in CH2C12 at room temperature followed by treatment with AczO, Bi(OTf)3 in CHzClz at room temperature ed the intermediate methyl 30L,7u-diacetoxyoxo-SB-cholanoate (compound 3; about 48% from compound 2).

Treatment of compound 3 with Brz in benzene for e.g., 12 h yielded compound 4.

Reaction of compound 4 with NaBH4 and NaOAc in freshly distilled pyridine gave the ponding 1113-12B epoxide (compound 5), in about 59% yield after silica gel purification. The reaction of compound 5 with H1 in AcOH at room temperature ed the halohydrine intermediate which were then ed at C11 position with Cr03 in AcOH to generate compound 6. Reaction of compound 6 with Zn dust in boiling AcOH and alkali hydrolysis (NaOH/MeOH) afforded hydroxyketocholanoic acid (compound 7; about 65% yield from compound 5).

Compound 7 was stereoselectively reduced at the C11-carbonyl using NaBH4 in a mixture ofTHF/H20= (4:1, V/V) to give 30L,7ot,11B-trihydroxy-60L-ethyl-5B—cholan—24-oic acid (Compound 100; about 27% from compound 3), after chromatographic purification to afford Compound 100. Alternatively, compound 7 was reduced with sodium in sec-BuOH at 50 0C to give Compound 101 (about 70% , after purification.

Example 2: Compound 100 is a potent, specific FXR agonist In the nucleus, ligand-bound nuclear receptors (NRs) modulate initiation of transcription by directly interacting with the basal transcriptional machinery or by contacting bridging factors called coactivators (Onate SA, er al., Science 1995; 270: 1354-1357; Wang JC, et al., JBiol Chem 1998; 273:30847-30850; Zhu Y, et al., Gene Expr 1996; 6:185-195).

The ligand-dependent interaction ofNRS with their coactivators occurs between activation function 2 (AF-2), d in the receptor ligand-binding domain (LED) and the nuclear or boxes (NR box) located on the coactivators (Nolte RT, et al., Nature 1998; 395:137- 143). Several lines of evidence have demonstrated that the LXXLL peptide ce present in the NR box ents a signature motif that tates the interaction of different proteins with the AF-2 region (Heery DM, et al., Nature 1997; 387:733-736; Torchia J, et al., Nature 1997; 387:677-684). creen was used with the aim of fy novel modulators by taking advantage of the bimolecular interaction ling between FXR and the LXXLL motif present in the NR box of the steroid receptor coactivator 1 (SRC-l).

Human FXR-LBD—GST was incubated with increasing concentrations of the indicated ligands in the presence of biotinylated LXXLL SRC-l peptide. The AlphaScreen signal ses when the complex receptor-coactivator is formed. EC50 values were 8.9 uM for chenodeoxycholic acid (CDCA; which is a positive control), 0.16 uM for Compound A, and 0.16 uM for Compound 100. These results are the mean :: SD. of triplicate samples from a representative experiment of three performed. The AlphaScreen assay is a very robust and reproducible assay, as shown by the 2' factor of 0.84 (Zhang JH, et al., J Biomol Screen 1999; 4:67-73). Thus, Compound 100 is a highly potent FXR agonist.

Further, the data in the table below show that Compound 100 is ive for human FXR and is not active for human TGRS.

Table l HTR-FRET (cAMP) HTR-FRET (CAMP) AlphaScreen Assay compound Human TGR5 Human TGR5 Human FXR CI-H716 cells overex oression Ref. CDCA = 15:: Ref. LCA = 7:: Ref. LCA = 0.9::0.luM Compound 100 0.02 No activity N0 actiVitY Compound 101 3:2 415 nd A 0.220.018 15::5 compound B 0.03 0.63 Compound C 175 09 Additionally, using the AlphaScreen assay, it was demonstrated that Compound 100 specifically activates FXR and does not activate 13 other nuclear ors involved in the metabolic pathways. 2014/059896 Table 2 FXR LXRB PXR CAR PPARo. PPAR6 PPARy Compound Activation Activation Activation Activation Activation tion Activation ence (CDCA = (T0901317 (SF-12183 (CITCO = (GW_7647 2 (ERR/1929 _ 0062 _ _ 0‘004 _ 0‘0” Standard) 10—20 ”M) = 0 08 ' MM) 0 005 ”M) M ' 0.003 M M M RARo. VDR TR PR RXR GR ER Compound Activation Activation Activation Activation Activation Activation Activation (Di- (Corticoste. . .

(Reference (ATRA . (Budenosrd (Estradiol = (901sRA = rone = e = 0.0002 2 0001 standard) 0.001 uM) tD3 — 0.004 uM) 0050 MM) HM) HM) nd No activity No activity No activity No ty No activity No activity Comgound No activity No activity No activity No activity No activity No ty *: inverse agonist.

Values for compound B taken from Rizzo G., et al., M01. Pharm, 2010; 78: 617-630.

FXR activation by Compound 100 was also tested in cell-based transactivation assays with the use of HEK293T cell line transiently transfected with Gal4-FXR-LBD chimera and the (UAS)5-Luc system (Figure 1). FXR activation by Compound 100 was comparable to that induced by compound A indicating that these compounds are potent FXR agonists in cell-based assays. Figure l is a graph showing the activity of Compound 100 in comparison to compound A in a transactivation assay in HEK293T cells. NT is FXR vector-transfected cells t exposure to compound A or Compound 100. Values are represented in uM.

Bile acids (BAs) modulate not only several nuclear hormone receptors, but are also agonists for the G protein-coupled receptor (GPCR) TGRS ( Makishima M, et al., Science 1999; 62-1365; Parks DJ, et al., Science 1999; 284: 1365-1368; Maruyama T, et al., Biochem Biophys Res Commun 2002; 298:714—719; Kawamata Y, et al., JBiol Chem 2003; 278:9435-9440). Signalling via FXR and TGRS modulates l metabolic pathways, regulating not only BA sis and enterohepatic recirculation, but also triglyceride, terol, glucose, and energy homeostasis. To evaluate the capacity of a compound of the invention to activate TGRS, Compound 100 and other comparison compounds were screened for an increase of intracellular CAMP as a read—out for TGRS activation. Human enteroendocrine NCI-H716 cells constitutively expressing TGRS were exposed to sing concentrations of Compound 100, and ellular cAMP levels were measured by TR- FRET. Lithocholic acid (LCA) was used as positive control.

As shown in Figure 2A, Compound 100 does not induce TGRS activity in cells expressing the or physiologically as no change in the level of intracellular cAMP was observed. To further assess if Compound 100 could bind TGRS, a clonal cell line over- expressing TGRS was exposed to different trations of Compound 100. The results illustrated in Figure 2B show that even with the over-expression of the TGRS receptor, Compound 100 had no relevant effect. Figure 2A is a graph showing the TGRS activity of Compound 100 (no activity) and LCA in human enteroendocrine cells expressing TGRS at physiological level. Results are shown as the mean i S. D. of triplicate samples from a representative experiment of three performed. Figure 2B is a graph showing the TGRS activity of Compound 100 (no activity) and LCA in human Chinese hamster ovary (CHO) cells over—expressing TGRS.

Example 3: FXR target genes modulated by Compound 100 To te the capacity of Compound 100 to te FXR target genes, quantitative RT-PCR assays were performed. HepG2 cells were selected as a relevant cell line to determine whether a compound of the invention can regulate the endogenous FXR c network. The ability of a compound of the invention to induce FXR target genes was assessed by isolating total RNA from cells treated overnight with luM of compounds A, B, and 100. Compound A is established as a potent FXR selective t and compound B is ished as a dual potent FXIVTGRS agonist. Compound 100’s gene activation profile in HepG2 cells was compared to the s of compounds A and B. (Pellicciari, R, et al., J Med Chem. 2002; Aug 15; 45: 3569-72; Rizzo, G, et al., Mal. Pharm, 2010; 78: 617-630).

FXR regulates the expression of several target genes involved in BA homeostasis.

Briefly, FXR plays a l role in several lic pathways, including i.e. , lipid metabolism, bile—acids metabolism, and carbohydrate metabolism. Regarding gene expression profiling, the genes encoding proteins involved in lipid metabolism include, e.g., APOCII, APOE, APOAI, SREBP-lC, VLDL-R, PLTP, and LPL; the genes encoding proteins involved in bile-acids metabolism include, e.g., OSTa/B, BSEP, MRP2, SHP, CYP7A1, FGF19, SULT2A1, and UGT2B4; and the genes encoding proteins involved in ydrate metabolism include, e.g. , PGCla, PEPCK, and GLUT2.

As shown in Figures 3A—3H, nd 100 activation of FXR indirectly represses the expression of the BA biosynthetic enzymes CYP7A1 by increasing the levels ofthe nuclear receptor SHP in the liver and intestine and increasing the level of FGF19 (Goodwin, B, et al., Mol. Cell 2000; 6: 517-526). nd 100 activated FXR also positively regulates the expression of genes encoding proteins involved in the transport of BA, including, BSEP, and OSTd and OSTB. Newly synthesized BAs are ated with taurine or glycine and then ly secreted in the gall bladder, FXR regulates both of these critical ses. Monoanionic— and nic—conjugated BAs are then actively secreted in the gall bladder by BSEP and the multidrug related protein 2 (MRP2), respectively. These transporters belonging to the ABC transporter family and are both induced by FXR at the transcriptional level. The regulation of these ABC transporters is of critical importance in order to avoid BA accumulation in the liver and consequent hepatic injury (Schinkel AH, et al., Mammalian drug efflux transporters of the ATP binding cassette (ABC) : an overview. Adv Drug Deliv Rev. 2012; Sep 13).

Figure 3 are a series of graphs showing the activity of Compound 100 and other comparison compounds in regulating expression of OSTu (A), OSTB (B), BSEP (C), MRP2 (D), CYP7A1 (E), SHP (F), FGF-19 (G), and UGT2B4 (H). Note in the Figures 3A-3H, the y-axis displays folds change in expression relative to untreated cells. The data were ized relative to B2M. The error bars display the standard error of the three replicates.

FXR activation contributes to reverse cholesterol transport, a process that results in the delivery of cholesterol from peripheral tissues to the liver for biliary al and consequent fecal elimination (Lambert, G, et al., J Biol Chem 2003; 278, 2563-70). In this metabolic scenario, FXR tes the expression of phospholipids transfer protein , responsible for the transfer of phospholipids and cholesterol from LDL to HDL, hepatic lipoproteins, such as ApoE, ApoC-I, ApoC-IV, and scavenger receptor B1(SRB1), which is involved in the hepatic uptake of HDL.

FXR controls triglyceride (TG) metabolism by regulating hepatic de novo lipogenesis and triglyceride clearance. Upon activation by Compound 100, FXR down regulates the expression of SREBP-lc, a transcription factor that plays a critical role in stimulating fatty acid sis and lipogenesis (Figures 4A-4D) (Landrier, JF, et al., J Clin Invest 2004; 113, 1408-18). In addition to the reduction of de novo lipogenesis, FXR activation also tes TG clearance. This additional TG-lowering effect of FXR is explained at the molecular level by the induction of key genes, such as Apo-C-Il LPL and VDL receptor (Kast, HR, et al., Mol Endocrinol 2001; 15, 1720-8).

Figure 4 are a series of graphs showing the activity of Compound 100 and other comparison compounds in regulating PLTP involved in lipid metabolism (A), SREBP-lC (B), APOCII (C), and PPARy (D). Note in the Figures 4A-4D, the y-axis displays folds change in expression relative to not treated cells. The data were ized relative to B2M.

The error bars display the standard error of the three replicates.

FXR may also have a role in carbohydrate lism. (Ma K, et al., J Clin Invest. 2006; 116:1102-9). PEPCK gene regulation was studied e 5) using Compound 100.

Figure 5 is a graph showing the regulation of Compound 100 and other comparison compounds on PEPCK gene. The y-axis displays folds change in expression relative to not treated cells. The data were normalized relative to B2M. The error bars display the standard error of the three replicates.

Collectively the gene expression studies showed that Compound 100 modulates the same FXR target genes as compound A or B (also see Table 3).

Table 3 nd B nd 100 Example 4: Compound 100 does not exert cytotoxic effects in HepG2 cells.

To evaluate in vitro cytotoxicity of Compound 100, two different assay methods were employed. The assays evaluated cell Viability by measuring ATP levels and cytotoxicity by ing LDH release. Adenosine Triphosphate (ATP) tide represents the source of energy at the basic molecular level, as it is a multifunctional molecule that is used in every cell as a coenzyme and is an integral part of the ondrial DNA (Kangas L, et (11., Medical Biology, 1984; 62, 338—343; Crouch SPM, et al., J. Immunol. Methods, 1993; 160, 81 — 88; Petty RD, et al., J. in. Chemilumin. 1995; 10, 29 — 34). It has been called the “molecular unit of currency” when it comes to intracellular energy transfer. This is to ensure the important role ofATP in metabolism and a drop in ATP content is the first step in 2014/059896 revealing cellular damage (Storer RD, et al., Mutation Research, 1996; 368, 59 — 101; Cree IA, Andreotti PE., Toxicology in Vitro, 1997; 11, 553 — 556).

Cell Viability was determined as measure of intracellular ATP related to the time of exposure and concentration of the test compounds (Sussman, NL.; Promega Cell Notes, Issue 3. 2002).

Figure 6 is a graph g the measurement ofATP in HepG2 cells, treated with the indicated concentrations of compounds for 4 h. It demonstrated that all cells in presence of different concentrations of Compound 100 were viable as cells treated with the vehicle alone, i. e., all cells treated with nd 100 remain viable . LCA, a well-known cytotoxic bile acid, was used as comparator and Tamoxifen was used as positive controls for the assays.

An additional method to determine the Viability of cells is to detect the integrity of the membrane that defines the cellular compartmentalization. Measuring the leakage of components out of the cytoplasm, in damaged cell membranes, indicates loss of membrane ity, and LDH release is the method used to determine common toxicity in cells. HepG2 cells were treated with Compound 100, serial dilutions were performed, LCA dilutions were added to the plated cells as assay control together with no-cell and untreated cells. The assay was performed in triplicate for each test compound concentration.

The results show that Compound 100 does not induce any cytotoxic effect on HepG2 cells. Lithocolic Acid increased LDH release at 70 uM whilst the l fen exerted the cytotoxic effects at approximately 25 uM (see Table 4).

Table 4 Membrane integrity EC50 (uM) (LDHmeasm) :10 Compound A 1 90:3 0 Compound 100 No toxicity 100% 1ivin_ cells nd 101 No toxicity 100% livin; cells * Rizzo et al., Mol. Pharm. 2010 Example 5: nd 100 does not inhibit cytochrome P450 enzymes.

To evaluate the ial of Compound 100 for drug-drug interactions, the six main CYP450 ms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4) were investigated. (Obach, RS, er al., JPharmcol Exp Ther, 2006; 316(1): p. 336-48).

To determine interaction between Compound 100 and cytochrome P450 enzymes, Compound 100 was analyzed by its capacity to inhibit (or not) the production of a fluorescent signal, using inant CYP450 proteins (baculosomes; Invitrogen), ates and inhibitors (Bidstrup, TB, et al., Br J Clin Pharmacol, 2003; 56(3): p. 305-14). As a positive control, a selective inhibitor for each CYP450 isoform was tested in the same plate (Table 5).

Table 5 1C50( M) ICso( M) ICso( M) CYP1A2 >10 >10 >10 Fura lline = 0.5 M CYP3A4 (Green Substrate) >10 >10 >10 Reference: Ketoconazole = 0.044 M CYP3A4 (Blue Substrate) Reference: Ketoconazole = 0.04 M CYP2C9 Reference: Sulfaphenazole = 0.4uM Reference: Miconazole = 0.06 M CYP2D6 Reference: Quinidine = 0.01 M CYP2E1 Reference: DCC = 0.4 M IC50 > 10 uM means that the compound does not inhibit the CYP450. The results obtained demonstrated that Compound 100, like compounds A and B, does not inhibit the Cytochrome P450 enzymes tested, showing that Compound 100 is not likely to be influenced by rug interaction effects. , G, et al., M01 Pharm, 2010; 78: 617-630).

Example 6: Compound 100 does not inhibit human ERG potassium channel To determine ion channel function, torTM hERG Fluorescence Polarization assay was employed as it provides an nt method for an initial determination of the propensity of test compounds to block the hERG channel (Dom, A, et al. J Biomol Screen, 2005; 10(4): 339-47). The assay is based on the assumption that the hERG potassium channel activity contributes to the resting membrane potential in permanently ected cells, and thus a block ofhERG ls should result in a depolarization of the cell membrane. The assay was designed to identify potential hERG channel blockers by producing data that accurately correlates with clamp electrophysiology studies. Results from the Predictor assay demonstrate a high correlation with those obtained from patch clamp techniques (Table 6) (Dom, A, et al. J Biomol Screen, 2005; 10(4): ).

Table 6 Patch-Clam n * Radioli_and* Compound ———m ——M- Amitripyline 10000 f2440 l 1200 Table 6 show the comparison of IC50 values generated with the PredictorTM hERG Fluorescence Polarization assay with ed IC50 values from patch-clamp and radioligand displacrnent assays.

Membrane preparations from Chinese hamster ovary cells stably transfected with hERG potassium channel were used to evaluate the potential inhibitory effect of Compound 100 on this channel using the Predictor fluorescence polarization assay. Reduction of membrane polarization as a result of inhibition of the hERG potassium channel is directly ated with a reduction of the fluorescence polarization (PP). The results show that like compounds A and B, Compound 100 does not block or inhibit the hERG potassium channel.

The assay was performed in triplicate by using a 16-point dose-response of test compound and the ve controls E-403l and Tamoxifen. An IC50 of 15 nM (AmP = 163) for E-4031 and 1.4 uM (AmP = 183) for Tamoxifen were obtained. The assay window more than 100 mP (millipolarization) is considered good. 2' value was 0.78 indicates an excellent assay. The near regression curves were obtained by ad Prism (GraphPad Software Inc.) analysis, to calculate the IC50 values.

Briefly, signalling through FXR modulates a variety of metabolic pathways, so selective FXR modulators are tive ates for the treatment of a range of chronic diseases ing liver, kidney, as well as metabolic diseases. Results in the examples described herein characterize Compound 100, as a potent and specific FXR agonist.

Remarkably, although it potently ted FXR, Compound 100 showed no activity against other nuclear receptors and did not active the bile acid GPCR TGRS. In addition to high nuclear receptor selectivity, Compound 100 possesses a pharmacological profile suitable for a drug ate. nd 100 shows no cytotoxic effect on human HepG2 liver cells, ting a lack of liver toxicity, and does not inhibit any of the CYP450 enzymes tested, indicating that Compound 100 is devoid of significant drug-drug interaction risk. Further, Compound 100 does not inhibit the human ERG potassium channel.

The combined selectivity and potency of Compound 100 together with its favorable drug-like properties, in particular an ent safety profile, make Compound 100 an attractive candidate for ng and preventing disease.

Example 7: Physiochemical properties of Compound 100 Physiochemical properties of Compound 100 such as water solubility, critical micellular concentration, surface tension and LogPA were determined using methods known in the art. These properties of Compound 100 were compared with natural and synthetic analogues (Table 7).

Table 7 . . WS a) CMCGj) STCMC(C) d W) (Dyna/cm) ——-_ ——_-_ -———— -———— -__— -———— a Ws: water solubility refers to BA as protonated s and ore not evaluated for Compound B, TCDCA and TUDCA which are highly soluble (hs); b CMC: Critical Micellar tration determined in 0.15 M NaCl water on; C STcmc: Surface Tension at CMC in 0.15 M NaCl water solution; LogPA': 1-octanol-water partition coefficient of the studied bile acids as ionized species; Example 8: Pharmacokinetics and metabolism in bile fistula rat after id and iv stration: in—vivo The in-vivo models, rats, were administered single dose of nd 100 at l umol/min/Kg.l hour (see Figures 7A, 7B, and 7C). Figure 7A is a graph showing the choleretic effect of Compound 100 for id and iv administration. Figure 7B is a graph showing the secretion of Compound 100 over time for id and iv administration. Figure 7C is a graph showing the plasma concentration of Compound 100 over time.

LENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many lents to the specific embodiments and methods described herein.

Such equivalents are intended to be encompassed by the scope of the present invention.

All patents, patent applications, and literature nces cited herein are hereby expressly incorporated by reference.

Definitions of the specific embodiments of the invention as claimed herein follow.

According to a first embodiment of the invention, there is provided a compound of formula (I): (I), or a pharmaceutically acceptable salt or amino acid ate thereof, n: R1 is beta-hydroxyl; R2 is hydrogen, hydroxyl, alkyl, or halogen, wherein said alkyl is unsubstituted or substituted with one or more Ra; R3 is hydrogen, hydroxyl, alkyl, or halogen, wherein said alkyl is unsubstituted or substituted with one or more Rb; R4 is alkyl, alkenyl, alkynyl, or halogen, wherein said alkyl is unsubstituted or substituted with one or more Rc; Ra, Rb, and Rc are each independently halogen or hydroxyl; R5 is hydroxyl, OSO3H, OSO3-, O(CO)CH3, OPO3H2, , or hydrogen; and R6 is hydroxyl, OSO3H, OSO3-, O(CO)CH3, OPO3H2, OPO32-, or hydrogen; or taken together R5 and R6 with the carbon atom to which they are attached form a carbonyl. ing to a second embodiment of the invention, there is ed a compound of the following formula: or a pharmaceutically acceptable salt or amino acid conjugate thereof.

According to a third embodiment of the invention, there is provided a pharmaceutical composition comprising the compound of the first or second embodiment or a pharmaceutically acceptable salt or amino acid conjugate thereof, and a pharmaceutically acceptable excipient.

According to a fourth embodiment of the invention, there is provided use of an effective amount of the compound of the first or second embodiment or a pharmaceutically acceptable salt or amino acid conjugate thereof in the manufacture of a ment for treating a chronic liver disease or condition in a subject, wherein the chronic liver disease or condition is ed from the group consisting of primary biliary cirrhosis (PBC), cerebrotendinous xanthomatosis (CTX), y sclerosing cholangitis (PSC), drug induced cholestasis, intrahepatic cholestasis of pregnancy, parenteral nutrition associated cholestasis (PNAC), ial overgrowth or sepsis associated tasis, autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis , liver transplant associated graft versus host disease, living donor transplant liver regeneration, congenital hepatic fibrosis, ocholithiasis, granulomatous liver disease, intra- or extrahepatic malignancy, Sjogren's syndrome, Sarcoidosis, Wilson's disease, Gaucher's disease, hemochromatosis, and alpha 1-antitrypsin deficiency.

According to a fifth embodiment of the invention, there is provided use of an effective amount of the compound of the first embodiment in the manufacture of a ment for ng a chronic liver disease or condition in a t, wherein the chronic liver e or condition is nonalcoholic hepatitis (NASH).

Claims (24)

CLAIMS :

1. A compound of formula (I): (I), or a pharmaceutically acceptable salt or amino acid conjugate thereof, wherein: R1 is beta-hydroxyl; R2 is hydrogen, hydroxyl, alkyl, or halogen, wherein said alkyl is tituted or substituted with one or more Ra; R3 is hydrogen, yl, alkyl, or halogen, wherein said alkyl is unsubstituted or substituted with one or more Rb; R4 is alkyl, alkenyl, alkynyl, or halogen, wherein said alkyl is unsubstituted or substituted with one or more Rc; Ra, Rb, and Rc are each ndently halogen or hydroxyl; R5 is hydroxyl, OSO3H, OSO3-, O(CO)CH3, OPO3H2, OPO32-, or hydrogen; and R6 is hydroxyl, OSO3H, OSO3-, O(CO)CH3, OPO3H2, OPO32-, or hydrogen; or taken together R5 and R6 with the carbon atom to which they are attached form a carbonyl.

2. The compound of claim 1, n the compound of formula (I) is (IV), or a pharmaceutically acceptable salt or amino acid ate thereof.

3. The compound of claim 1, wherein one of R2 or R3 is hydroxyl or halogen and the remaining R2 or R3 is hydrogen or unsubstituted alkyl.

4. The compound of claim 3, wherein one of R2 or R3 is yl and the remaining R2 or R3 is hydrogen.

5. The compound of claim 1, wherein one of R5 or R6 is hydroxyl and the remaining R5 or R6 is hydrogen.

6. The compound of claim 1, wherein R2 is hydroxyl or n.

7. The compound of claim 1, wherein R3 is hydrogen or unsubstituted alkyl.

8. The compound of claim 7, wherein R3 is methyl.

9. The compound of claim 1, wherein R2 is hydroxyl and R3 is hydrogen.

10. The compound of claim 1, wherein R5 is hydroxyl.

11. The compound of claim 1, wherein R6 is hydrogen.

12. The compound of claim 1, wherein R2 and R5 are each hydroxyl and R3 and R6 are each hydrogen.

13. The compound of claim 1, wherein R4 is alkyl.

14. The compound of claim 13, wherein R4 is unsubstituted alkyl.

15. The compound of claim 14, wherein R4 is ethyl.

16. A nd of the ing formula: or a pharmaceutically acceptable salt or amino acid ate thereof.

17. A pharmaceutical composition comprising the compound of any one of claims 1 to 16 or a pharmaceutically acceptable salt or amino acid conjugate thereof, and a pharmaceutically acceptable excipient.

18. Use of an effective amount of the compound of any one of claims 1 to 16 or a pharmaceutically acceptable salt or amino acid conjugate thereof in the manufacture of a medicament for treating a c liver disease or condition in a subject, wherein the chronic liver disease or condition is selected from the group consisting of primary biliary cirrhosis (PBC), cerebrotendinous xanthomatosis (CTX), primary sclerosing cholangitis (PSC), drug induced cholestasis, epatic cholestasis of pregnancy, parenteral nutrition associated cholestasis (PNAC), bacterial overgrowth or sepsis associated cholestasis, autoimmune tis, c viral hepatitis, lic liver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver transplant associated graft versus host disease, living donor transplant liver regeneration, congenital hepatic fibrosis, choledocholithiasis, granulomatous liver disease, intra- or extrahepatic malignancy, Sjogren's syndrome, Sarcoidosis, Wilson's disease, r's disease, hemochromatosis, and alpha 1- antitrypsin deficiency.

19. Use of an effective amount of the compound of any one of claims 1 to 15 in the manufacture of a ment for treating a c liver e or condition in a t, wherein the chronic liver disease or condition is nonalcoholic steatohepatitis (NASH).

20. The use of claim 19, wherein the compound of formula (I) is (IV), or a pharmaceutically acceptable salt or amino acid conjugate thereof.

21. The use of claim 20, wherein R4 is alkyl.

22. The use of claim 20, wherein R4 is ethyl.

23. The use of claim 19, wherein the compound of formula (I) is compound 100 (100), or a pharmaceutically acceptable salt or amino acid conjugate thereof.

24. The use of claim 23, n the compound of formula (I) is compound 100 in free acid form.