WO2004046162A2

WO2004046162A2 - Non-steroidal fxr agonists

Info

Publication number: WO2004046162A2
Application number: PCT/US2003/036195
Authority: WO
Inventors: Kyriacos C. Nicolaou; Anthony J. Roecker; Robert Hughes; Jeffrey A. Pfefferkorn
Original assignee: The Scripps Research Institute
Priority date: 2002-11-14
Filing date: 2003-11-14
Publication date: 2004-06-03
Also published as: AU2003290796A8; AU2003290796A1; WO2004046162A3; WO2004046162A8

Abstract

ABSTRACT Potent non-steroidal farnesoid X receptor (FXR) agonists are N-aryl-N-arylmethyl amido and ureido compounds having the chemical structure represented by the following formula (I): INSERT FORMULA wherein E1 is (C1-C8)alkyl, cyclohexyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, phenyl, or NH(C1-C8)alkyl; L1 and L2 are both H, or together form a pi-bond; X1 is C(O), or CH2; Y1 is H, NHZ1, NH(Z2)Z3, or OZ4; aryl moiety A1 is selected from the group of radicals consisting of: INSERT FORMULA A2 and G1 - G11 are as defined in the specification; and T1 and T2 are each independently O, S, NH, or N(C1-C8)alkyl. The FXR agonists are useful as therapeutic agents for the treatment of diseases linked to cholesterol, bile acids, and their metabolism and homeostasis.

Description

NON-STEROID AL FXR AGONISTS

Cross-reference to Related Applications

This application claims the benefit of U.S. Provisional Application Serial No. 60/426,456, filed on November 14, 2002, and U.S.

Provisional Application Serial No. 60/491,185, filed on July 29, 2003, the disclosures of which are incorporated herein by reference. Statement of Government Interest

This invention was made with government support under contract number C A -54418 from the National Institutes of Health. The U. S . government has certain rights in this invention. Field of the Invention

The invention relates to agonists of farnesoid X receptor (FXR). More particularly, the invention relates to non-steroidal agonists of FXR, which are N-aryl-N-arylmethyl amido and ureido compounds. The agonists are useful for the regulation of cholesterol and related biological molecules. Background

The efficient regulation of cholesterol biosynthesis, metabolism, acquisition, and transport is an essential component of lipid homeostasis. The farnesoid X receptor (FXR) is a transcriptional sensor for bile acids, the primary product of cholesterol metabolism. To date, only one class of high affinity, non-steroidal agonists for FXR has been reported, viz., the class exemplified by GW4064 (compound 3, Figure 1), as reported by Maloney, P. R, et al. (J. Med. Chem. 2000, 43, 2971-297 '4). Potent steroid- derived agonists of FXR have been reported by Pellicciari, R., et al. (J. Med. Chem. 2002, 45, 3569-3572).

Potent, selective, non-steroidal small molecule FXR agonists are powerful tools for exploring the biological function of the farnesoid X receptor and would have many other useful applications (Willson, T. M., et al.,

Med. Res. Rev. 2001, 21, 513-522). For example, such compounds facilitate the analysis of FXR physiology in vivo, and in conjunction with DNA arraying technology facilitate discovery of new gene products under the control of FXR. FXR modulators also are useful in the treatment of cholestasis and other disease states associated with aberrant levels, flow, and release of bile acids. The crystal structure of FXR has not yet been reported. There is an unfulfilled need for a thorough structure/activity relationship (SAR) study of materials that modulate FXR activity, and for novel and potent ligands and therapeutic agents targeted to FXR. The non-steroidal FXR agonists of the present invention fulfill these needs. Summary of the Invention

The non-steroidal FXR agonists of the present invention are N- aryl-N-arylmethyl amido and ureido compounds having the chemical structure represented by the following formula (I):

wherein electrophile-derived moiety E¹ is (C_rC₈)alkyl, cyclohexyl, 2- furyl, 3-furyl, 2-tlιienyl, 3-thienyl, phenyl, or NFJ^ -C^alkyl;

L¹ and L² are both H, or together form a pi-bond;

X¹ is C(O), or CH₂;

Y¹ is H, NHZ¹, NH(Z²)Z³, or OZ⁴; aryl moiety A¹ is selected from the group of radicals consisting of:

A² is a radical selected from the group consisting of:

substituent group G¹ is H or OCH₃;

G² and G³ are each independently H, (C₁-C₈)alkyl, F, Cl, Br, I, OH, O(C_rC₈)alkyl, SH, S(C_rC₈)alkyl, C(O)H, C(O)(C_rC₈)alkyl, N((C_rC₈)alkyl)₂, CO₂CH₃, or together form a 5 or 6-member carbocyclic ring or OCH₂O;

G⁴ is H or OCH₃;

G⁵ is (C_rC₄)alkyl or C(O)(C_rC₈)alkyl;

G⁶ is H, or together with G⁸ fonris a pi-bond, an epoxide, a cyclopropyl ring, a dichlorocyclopropyl ring, or a dibromocyclopropyl ring;

G⁷ is H, CH₃, or OZ⁵, with the proviso that G⁷ is H or CH₃ when G⁶ and G⁸ together form a pi-bond, an epoxide, a cyclopropyl ring, a dichlorocyclopropyl ring, or a dibromocyclopropyl ring; when G⁶ is H, G⁸ is H, or together with G⁹ forms a moiety selected from the group consisting of =NOZ⁶ and =O;

G⁹ is H, OH, OZ⁷, CN, C(O)O(C_rC₈)alkyl, SPh, S(C_rC₈)alkyl, NHZ⁸, NH(Z⁹)Z¹⁰, or together with G¹⁰ forms a moiety selected from the group consisting of =NOZ⁶ and =O;

G¹⁰ and G¹¹ are each independently H, (C_rC₈)alkyl, SCH₃, C(O)(C_rC₈)alkyl, or C(O)O(C_rC₈)alkyl; and

G¹² and G¹³ are each independently H or F; G¹⁴ and G¹⁶ are each independently (C_rC₈)alkyl, phenyl, or benzyl;

G¹⁵ is phenyl,

hydroxyphenyl,

chlorophenyl, bromophenyl, or fluorophenyl; and

G¹⁷ and G¹⁸ are each independently H, (C_rC₈)alkyl, SCH₃, C(O)(C_rC₈)alkyl, or C(O)O(C_rC₈)alkyl;

T¹ and T² are each independently O, S, NH, or N(C₁-C₈)alkyl;

Z¹ is H, phenyl, (C_rC₈)alkyl, benzyl, C(O)Ph, C(O)(C₁-C₈)alkyl, C(O)OCH₂Ph, or C(O)NH(C_rC₈)alkyl;

Z² and Z³ are each independently (C_rC₈)alkyl or together form a (C₁-C₈)cycloalkyl ring;

Z⁴, Z⁵, Z⁶, and Z⁷are each independently H or an oxygen protecting group;

Z⁸ is H, phenyl, (C_rC₈)alkyl, benzyl, C(O)Ph, C(O)(C₁-C₈)alkyl, C(O)OCH₂Ph, or C(O)NH(C_rC₈)alkyl; Z⁹ and Z¹⁰ are each independently ( -C^alkyl, or together form a (C₃-C₈)cyclic amine ring.

Typical examples of the FXR agonists of the present invention are illustrated in Figure 2. Several of these compounds are among the most potent FXR activators reported to date. Preferred examples of the FXR agonists of the present invention are fexaramate (105), fexarene (121), fexaramine (259), fexarine (244), and fexarchloramide (149) as shown in Figure 2. The FXR agonists of the present invention can be employed as therapeutic agents for the treatment of diseases linked to cholesterol, bile acids, and their metabolism and homeostasis, and are useful as tools for elucidation of FXR biological function.

Brief Description of Drawings

Figure 1 illustrates structures of some natural and synthetic agonists of FXR (farnesoid X receptor) and their activity in a cell based assay.

Figure 2 illustrates structures of 9 non-steroidal FXR agonists of the present invention and their EC₅₀ values obtained from a cell-based assay.

Figure 3(a) illustrates structures of selected hits from a high throughput screen for FXR agonism of a 10,000-memberbenzopyran-based natural product-like library (EC50 = 5-10 mM). Figure 3(b) shows the structures of selected low affinity FXR agonists from a follow-up solid phase benzopyran library (EC50 = 5-10 mM). The boxed compounds represent the most potent FXR agonists in this group.

Figure 4 schematically illustrates the solid-phase synthesis of a focused library of benzopyran-containing small molecules as potential FXR agonists. Figure 4(a) shows the solid-phase protocol. Figure 4(b) shows O-prenylated phenols employed as scaffolds. Figure 4(c) shows the structures of the electrophiles utilized in the acylation step in the transformation of S-2 to S-3 shown in Figure 4(a). Figure 4(d) shows the structures of the amines employed in the reductive animation step in the transformation of S-2 to S-3 shown in Figure 4(a). The reagents and conditions for these reactions are well known in the art and have been reported in Nicolaou, K. C; et al. J. Am. Chem.

Soc. 2000, 122, 9939-9953, the relevant disclosures of which are incorporated herein by reference.

Figure 5 illustrates selected regions of interest for SAR evaluation of lead compound 26. Region I: Right-hand aromatic system; Region II: Acyl group region; Region III: Left-hand benzopyran ring system. Figure 6 illustrates the structural variants of Region I that were examined in a SAR study. See Figures 7, 8, 9 and 10 for a schematic illustration of the synthesis of these compounds. In compound 46 the benzopyran double bond was hydrogenated. The boxed compounds represent the most potent FXR agonists within this group of compounds.

Figure 7 schematically illustrates the representative procedure for the preparation of Region I-modified compounds: synthesis of methyl acrylate 29. Reagents and conditions: (a) (Glass, C. K.; et al. Curr. Opin. Cell Biol. 1997, 9, 222-232; (b) 1.5 equivalents of 2-methyl-3-butyn-2-ol, 1.5 of DBU, 1.7 equivalents trifluoroacetic anhydride, 0.1 equivalents of CuCl₂,

CH₃CN, 0 → 25 °C,12 h, 75%; (c) N,N-diethylaniline, 190 °C, 0.5 h, 90%; (d) 1.5 equivalents of 3-bromoaniline, THF, 70 °C, 4h; then 2.0 equivalents of NaCNBH₃, 10% MeOH, 70 °C, 4h, 83%; (e) 1.3 equivalents of cyclopropanecarbonyl chloride, 1.3 equivalents of Et₃N, 0.1 equivalents of 4-DMAP, CH₂C1₂, 25 °C, 12 h, 90%; (f) 4.0 equivalents of methyl acrylate, 0.2 equivalents of Pd₂(dba)₃, 0.5 equivalents of P(o-tol)₃, 5.0 equivalents of Et₃N, DMF, 90 °C, 24 h, 80%. DBU = l,8-diazabicyclo[5.4.0]undec-7-ene, P(o-tol)₃ = tris-(2-methylphenyl)phosphine, 4-DMAP = 4-dimethylaminopyridine, Pd₂(dba)₃ = tris(dibenzylidineacetone)dipalladium(0). Figure 8 schematically illustrates the solution phase synthesis of ester and acid containing compounds (SAR region I). Reagents and conditions: (a) (Glass, C. K.; et al. Curr. Opin. Cell Biol. 1997, 9, 222-232; (b) 1.5 equivalents of 2-methyl-3-butyn-2-ol, 1.5 equivalents of DBU, 1.7 equivalents of trifluoroacetic anhydride, 0.1 equivalents of CuCl₂, CH₃CN, 0 - 25 °C,12 h, 75%; (c) N,N-diethylaniline, 190 °C, 0.5 h, 90%; (d) 1.5 equivalents of methyl 4-aminobenzoate, THF, 70 °C,4 h; then 2.0 equivalents of NaCNBH₃, 10% MeOH, 70 °C, 4 h, 82%; (e) 1.3 equivalents of cyclopropanecarbonyl chloride, 1.3 equivalents of Et₃N, 0.1 equivalents of 4-DMAP, CH₂C1₂, 25 °C, 12 h, 85-95%; (f) 1.5 equivalents of ethyl 3-aminobenzoate, THF , 70 °C, 4 h; then 2.0 equivalents of NaCNBH₃, 10% MeOH, 70 °C, 4h, 77%; (g) 1.5 equivalents of methyl 4-(aminomethyl)benzoate, THF, 70 °C, 4h; then 2.0 equivalents of NaCNBH₃,10% MeOH, 70 °C, 4h, 80% ; (h) 4.0 equivalents of LiOH, THF:H₂O (10:1), 25 °C, 12 h, 75-98%; (i) 1.5 equivalents of 4-bromoaniline, THF, 70 °C, 4h; then 2.0 equivalents of NaCNBH₃, 10% MeOH, 70 °C, 4h,

78%; (j) 4.0 equivalents of methyl acrylate, 0.2 equivalents of Pd₂(dba)₃, 0.5 equivalents of P(ø-tol)₃, 5.0 equivalents of Et₃N, DMF, 90 °C, 24 h, 71-80%; (k) 1.5 equivalents of 3-bromoaniline, THF, 70 °C, 4h; then 2.0 equivalents of NaCNBH₃, 10% MeOH, 70 °C, 4h, 83%; (1) 1.3 equivalents of cyclohexanecarbonyl chloride, 1.3 equivalents of Et₃N, 0.1 equivalents of

4-DMAP, CH₂C1₂, 25 °C, 12 h, 95%.

Figure 9 schematically illustrates the solution phase synthesis of various ester and vinyl cyanide containing compounds via palladium catalyzed reaction manifolds (SAR region I). Reagents and conditions: (a) 2.0 equivalents of penta-2,4-dienoic acid methyl ester, 0.2 equivalents of Pd₂(dba)₃,

0.6 equivalents of P(o-tol)₃, 5.0 equivalents of Et₃N, DMF, 90 °C, 24 h, 70%; (b) 5.0 equivalents of 3-(methoxycarbonylphenyl)boronic acid, toluene:MeOH:lM Na₂CO₃ (10:3:1), 90 °C, 24 h, 75%; (c) 5.0 equivalents of 4-(methoxycarbonylphenyl)boronic acid, toluene:MeOH:lM Na₂CO₃ (10:3:1), 90 °C, 24 h, 78%; (d) 2.0 equivalents of 3-vinylbenzaldehyde, 0.2 equivalents of Pd₂(dba)₃, 0.6 equivalents of P(o-tol)₃, 5.0 equivalents of Et₃N, DMF, 90 °C, 24 h, 85%; (e) 1.5 equivalents of NaClO₂ , 4.0 equivalents of NaH₂PO₄, 10.0 equivalents of 2-methyl-2-butene, THF:t-BuOH:H2O (3:1:1), 25 °C, 3 h, 98% ; (f) 10 equivalents of CH₂N₂, Et₂O, 0 °C, 1 h, 100%; (g) 2.0 equivalents of acrylonitrile, 0.2 equivalents ofPd₂(dba)₃, 0.6 equivalents ofP(σ-tol)₃, 5.0 equivalents of Et₃N, DMF, 90 °C, 24h, 55%. q

Figure 10 schematically illustrates the solution phase synthesis of ester modifications (SAR region I). Reagents and conditions: (a) 0.5 equivalents of n-Bu₂Sn=O, EtOH or /-PrOH, 25 °C, 48 h, 50% and 34%, respectively; (b) 1.2 equivalents of diisobutylaluminum hydride, toluene, -78 °C, 0.5 h, 52%; (c) 2.0 equivalents of NaH, 3.0 equivalents of Mel, 0 °C, 1 h, 95%; (d) 1.2 equivalents ofMeOC(O)Cl, 2.0 equivalents of Et₃N, 0.1 equivalents of 4-DMAP, CH₂C1₂, 25 °C, 24 h, 88%; (e) 1.2 equivalents of MeC(O)Cl, 2.0 equivalents of Et₃N, 0.1 equivalents of 4-DMAP, CH₂C1₂, 25 °C, 24 h, 90%; (f) 4.0 equivalents of LiOH, THF:H₂O (10:1), 25 °C, 12h,

90%; (g) 1.2 equivalents of EtOC(O)Cl, 1.5 equivalents of Et₃N, CH₂C1₂, 25 °C,1 h, then 3.0 equivalents of amine, CH₂C1₂, 25 °C,12 h, 85-95%; (h) 10.0 equivalents of CH₂N₂, 0.2 equivalents of Pd(OAc)₂, Et₂O, 25 °C, 12 h, 95%.

Figure 11 depicts the structures of compounds in which the acyl group of region II was varied. See Figure 12 for a schematic representation of the synthesis of these compounds. Boxed compounds are the most active FXR agonists within this group.

Figure 12 schematically illustrates the solution phase synthesis of ester modifications (SAR region II). Reagents and conditions: (a) 1.0 equivalents of 60, 2.0 equivalents of 130, THF, 70 °C, 4 h, then 2.0 equivalents of NaCNBH₃, 10% MeOH, 70 °C, 4 h, 70%; (b) 1.5 equivalents of methyl acrylate, 0.2 equivalents of Pd₂(dba)₃, 0.5 equivalents of P(ø-toι)₃, 5.0 equivalents of Et₃N, DMF, 90 °C, 12 h, 65%; (c) 5.0 equivalents of NaHCO₃, 5.0 equivalents of alkyl halide, EtOH, 80 °C, 24 h, 70-85%; (d) 5.0 equivalents of acid chloride, 5.0 equivalents of Et₃N, 0.2 equivalents of 4-DMAP, CH₂C1₂,

25 °C, 24 h, 55-100%; (e) 5.0 equivalents of isocyanate, 5.0 equivalents of Et₃N, CH₂C1₂, 25 °C, 24 h, 75-85%; (f) 5.0 equivalents of thioacid chloride or thioisocyanate, 5.0 equivalents of Et₃N, CH₂C1₂, 25 °C, 24h, 50-70%.

Figure 13 depicts the structures of compounds in which the acyl group of region II was varied. See Figures 14, 15, and 16 for a schematic representation of the synthesis of these compounds. Boxed compounds are the most active FXR agonists in this group.

Figure 14 schematically illustrates the solution phase synthesis of benzopyran olefin modifications (SAR region III). Reagents and conditions: (a) 2.0 equivalents of benzoyl chloride, 2.0 equivalents of Et₃N, 0.2 equivalents of 4-DMAP, CH₂C1₂, 25 °C, 24 h, 95%; (b) 10 equivalents of DMDO, acetone, 0 °C, 1 h, 100%; (c) 5.0 equivalents of PhSH, Amberlyst-15 (cat.), CH₂C1₂ , 25 °C, 24 h, 95%; (d) 2.0 equivalents of acetic anhydride, 2.0 equivalents of Et₃N, 0.2 equivalents of 4-DMAP, CH₂C1₂, 25 °C, 24 h, 90%; (e) 2.0 equivalents of methyl acrylate, 0.2 equivalents of Pd₂(dba)₃, 0.6 equivalents of P(o-tol)₃, 5.0 equivalents of Et₃N, DMF, 90 °C, 24 h, 70-84%; (f) 5.0 equivalents of piperidine, CH₂C1₂, 25 °C, 48h, 65%; (g) 5.0 equivalents of H₂O, Amberlyst-15 (cat.), THF , 25 °C, 24 h, 95%; (h) 2.0 equivalents of Et₂AlCN, CH₂C1₂, 0 °C, 1 h, 83%; (i) 40% KOH:MeOH (1:2), 25 °C,24 h, 90%. DMDO = dimethyldioxirane.

Figure 15 schematically illustrates the solution phase synthesis of benzopyran olefin modifications (SAR region III). Reagents and conditions: (a) 0.02 equivalents of OsO₄, 2.0 equivalents ofNMO, acetone:H₂O (10:1), 25 °C, 24 h, 85%; (b) 5.0 equivalents of acetic anhydride, 10.0 equivalents of Et₃N, 0.2 equivalents of 4-DMAP, CH₂C1₂, 25 °C, 24 h, 90%; (c) 2.0 equivalents of methyl acrylate, 0.2 equivalents of Pd₂(dba)₃, 0.6 equivalents of P(o-tol)₃, 5.0 equivalents of Et₃N, DMF, 90 °C, 24 h, 65-80%; (d) 10% Pd/C, EtOAc, 25 °C, 0.5 h, 100%; (e) CHC1₃ : 2.0 NNaOH (7:1), adogen 464 (cat.) 25 °C, 6 h, 85%. NMO = 4-methylmo holine N-oxide. Figure 16 schematically illustrates the synthesis of compound

102 (Modifications of region III SAR). Reagents and conditions: (a) CHC1₃ : 2.0 NNaOH (7:1), adogen 464 (cat.) 25 °C, 6 h, 85%; (b) 2.0 equivalents of methyl acrylate, 0.2 equivalents of Pd₂(dba)₃, 0.6 equivalents of P(o-tol)₃, 5.0 equivalents of Et₃N, DMF, 90 °C, 24 h, 75%. DMF = N,N-dimethylformamide.

Figure 17 depicts the structures utilized for examining the region III benzopyran replacement SAR study. See Figures 21, 18, 19, and 25 for a schematic representation of the synthesis of these compounds.

Figure 18 schematically illustrates the solution phase synthesis of region III analogs in which the benzopyran group has been replaced. Reagents and conditions: (a) 1.1 equivalents of C₆H_nCOCl, 1.3 equivalents of Et₃N, 0.05 equivalents of 4-DMAP, CH₂C1₂, 25 °C, 3 h, 95%; (b) 4.0 equivalents of methyl acrylate, 5.0 equivalents of Et₃N, 0.2 equivalents of Pd₂(dba)₃, 0.6 equivalents of P(o-tol)₃, DMF, 90 °C, 12 h, 80% ; (c) 1.1 equivalents of NaH, THF, 0 °C, 30 min; then 1.3 equivalents of benzyl bromides, THF, 0 °C, 2 h, 60 - 90%. R-X = methyl iodide, benzyl bromide, 2-bromobenzyl bromide, 3-bromobenzyl bromide, 4-bromobenzyl bromide, 4-tert-butylbenzyl bromide, 3-methoxybenzyl bromide, 3,5-dimethoxybenzyl bromide, 3 -(trifluromethyl)benzyl bromide, 2-napthyl bromide. Figure 19 schematically illustrates the solution phase synthesis of region III derivatives. Reagents and conditions: (a) 4.0 equivalents of tert-butyl acrylate, 5.0 equivalents of Et₃N, 0.05 equivalents of Pd₂(dba)₃, 0.15 equivalents of P(o-tol)₃, DMF, 90 °C, 12 h, 80%; (b) 20% TFA in CH₂C1₂, 25 °C, 1 h, 95%. Figure 20 shows the structures of compounds from the SAR studies. Figure 20(a) illustrates highlights of the region I SAR. Figure 20(b) illustrates highlights of the region II SAR for bis-cinnamate compounds. Figure 20(c) illustrates effects of benzopyran substitution. Figure 20(d) illustrates highlights of the region III SAR, including bis-cinnamate, styryl and biaryl compounds. The EC₅₀ values represent the mean of at least four measurements. RE = relative efficacy of the indicated compound at 1 mM to 100 mM CDC A.

Figure 21 schematically illustrates the preparation of the bis-cinnamate compound 105. Reagents and conditions: (a) 1.1 equivalents of C₆H_πCOCl, 1.3 equivalents of Et₃N, 0.05 equivalents of 4-DMAP, CH₂C1₂, 25

°C, 3 h, 95%; (b) 4.0 equivalents of methyl acrylate, 5.0 equivalents of Et₃N, 0.2 equivalents of Pd₂(dba)₃, 0.6 equivalents of P(o-tol)₃, DMF, 90 °C, 12 h, 80%; (c) 1.1 equivalents of NaH, THF, 0 °C, 30 min; then 1.3 equivalents of 4-bromobenzylbromide, THF, 0 °C, 2 h, 90%; (d) 4.0 equivalents of acrylate, 5.0 equivalents of Et₃N, 0.05 equivalents of Pd₂(dba)₃, 0.15 equivalents of P(o-tol)₃, DMF, 90 °C, 12 h, 75%.

Figure 22 schematically illustrates the synthesis of analogs with region III modifications and cinnamate substitutions. Reagents and conditions:

(a) 4.0 equivalents of styrene, 5.0 equivalents of Et₃N, 0.05 equivalents of Pd₂(dba)₃, 0.15 equivalents of P(ø-tol)₃, DMF, 90 °C, 12 h, 65 - 80% ; (b) 2.5 equivalents of boronic acid, 0.2 equivalents of Pd(PPh₃)₄, toulene:MeOH:l M Na₂CO₃ (10:3:1), 80 °C, 12 h, 60 - 80%.

Figure 23 schematically illustrates the synthesis of analogs having region I/region III cinnamate modifications. Reagents and conditions: (a) 4.0 equivalents of tert-butyl acrylate, 5.0 equivalents of Et₃N, 0.05 equivalents of Pd₂(dba)₃, 0.15 equivalents of P(o-tol)₃, DMF, 90 °C, 12 h, 85%;

(b) 1.5 equivalents of 3-bromoaniline, 0.05 equivalents of AcOH, MeOH, 25 °C, 30 min; then 1.7 equivalents of NaCNBH₃, 1 h, 90%; (c) 1.1 equivalents of C₆H„COCl, 1.3 equivalents of Et₃N, 0.05 equivalents of 4-DMAP, CH₂C1₂, 25 °C, 3 h, 90%; (d) 4.0 equivalents of acrylate, 5.0 equivalents of Et₃N, 0.05 equivalents of Pd₂(dba)₃, 0.15 equivalents of P(o-tol)₃, DMF, 90 °C, 12 h, 60 - 85%; (e) 4.0 equivalents of alkene, 5.0 equivalents of Et₃N, 0.05 equivalents of Pd₂(dba)₃, 0.15 equivalents of P(o-tol)₃, DMF, 90 °C, 12 h, 35 - 80%; (f) 0.05 equivalents of Pd/C, H₂ (1 ami), EtOAc, 25 °C, 30 min, 100 %. Figure 24 schematically illustrates the synthesis of acyl group analogs of the bis-cinnamate compounds. Reagents and conditions: (a) 1.0 equivalents of S-24, 1.0 equivalents of S-27, 0.05 equivalents of AcOH, MeOH, 25 °C, 30 min; then 1.2 equivalents of NaCNBH₃, 25 C, 1 h, 85%; (b) 2.0 equivalents of acid chloride, 3.0 equivalents of Et₃N, 0.05 equivalents of 4-DMAP, CH₂C1₂, 25 C, 1 h, 80 - 95%; (c) 2.0 equivalents of isocyanate, 3.0 equivalents of Et₃N, 0.05 equivalents of 4-DMAP, CH₂C1₂, 25 °C, 1 h, 60 - 80%.

Figure 25 schematically illustrates the synthesis of region III cinnamate modifications. Reagents and conditions: (a) 4.0 equivalents of acrylate, 5.0 equivalents of Et₃N, 0.05 equivalents of Pd₂(dba)₃, 0.15 equivalents of P(o-tol)₃, DMF, 90 °C, 12 h, 50 - 80% ; (b) 20% TFA in CH₂C1₂, 1 h, 25 C, 95%; (c) 1.2 equivalents of DCC, 10.0 equivalents of z-PrOH, 0.2 equivalents of 4-DMAP, DMF, 25 °C, 12 h, 60%; (d) 1.2 equivalents of DCC, 10.0 equivalents of BnOH, 0.2 equivalents of 4-DMAP, DMF, 25 °C, 12 h, 60%; (e) 4.0 equivalents of alkene, 5.0 equivalents of Et₃N, 0.05 equivalents of

Pd₂(dba)₃, 0.15 equivalents of P(o-tol)₃, DMF, 90 °C, 12 h, 35 - 75%; (f) 0.05 equivalents of Pd/C, H₂ (1 arm), EtOAc, 25 °C, 30 min, 100 %. DCC = 1 ,3-dicyclohexylcarbodiimide.

Figure 26 schematically illustrates the synthesis of region III ring analogs. Reagents and conditions: (a) 1.0 equivalents of SEMC1, 1.2 equivalents of Et₃N, CH₂C1₂, 25 °C, 12 h, 75%; (b) 1.05 equivalents of Tf₂O, 1.2 equivalents of Et₃N, CH₂C1₂, -78 °C, 1 h, 95%; (c) 4.0 equivalents of tert-butyl acrylate, 5.0 equivalents of Et₃N, 0.05 equivalents of Pd₂(dba)₃, 0.15 equivalents of P(o-tol)₃, 90 °C, 12 h, 76%; (d) 1.2 equivalents of S-27, 0.05 equivalents of AcOH, MeOH, 25 °C, 1 h; then 1.5 equivalents of NaCNBH₃, 2 h, 80%; (e) 1.2 equivalents of C₆H„COCl, 1.5 equivalents of Et₃N, 0.05 equivalents of 4-DMAP, CH₂C1₂, 25 °C, 4 h, 90%; (f) 7.0 equivalents of TBAF, THF:HMPA (9:1), 55 °C, 12 h, 65%; (g) 3.0 equivalents of Mel, 5.0 equivalents of K₂CO₃, DMF, 80 °C, 12 h, 90%; (h) 3.0 equivalents of BnBr, 5.0 equivalents of K₂CO₃, DMF, 80 °C, 12 h, 65%; (i) 3.0 equivalents of

BrCH₂COOEt, 5.0 equivalents of K₂CO₃, DMF, 80 °C, 12 h, 85%; 0) 3.0 equivalents of AcCl, BzCl orMsCl, 5.0 equivalents of Et₃N, CH₂C1₂, 25 °C, 2 h, 70-90%. HMPA = hexamethylphosphoramide, Tf₂O = trofluoroacetic anhydride, TBAF = tetrabutylammonium fluoride, SEMC1 = 2-(trimethylsilyl)ethoxymethyl chloride.

Figure 27 schematically illustrates the solid phase synthesis of focused libraries of biaryl and stilbene cinnamates. Reagents and conditions: (a) 2.0 equivalents of 168, 1.0 equivalents of Merrifield Resin (0.91 mrnol/g), 2.0 equivalents of Cs₂CO₃, 0.5 equivalents of TBAI, DMF, 55 °C, 24 h; (b) 20% TFA in CH₂C1₂, 25 °C, 1 h; (c) 10.0 equivalents of 4-bromobenzaldehyde, 0.05 equivalents of AcOH, THF MeOH (2:1), 25 °C, 1 h; then, 8.0 equivalents ofNaCNBH₃, THF MeOH (2:1), 25 °C, 2 h; (d) for ^0(0)01: 30.0 equivalents of /-PrC(O)Cl or C₆H_nC(O)Cl, 40.0 equivalents of Et₃N, 1.0 equivalents of 4-DMAP, CH₂C1₂, 25 °C, 12 h; for RμNTCO, 30.0 equivalents of i-PrNCO, 40.0 equivalents of Et₃N, 1.0 equivalents of 4-DMAP, DMF, 65 °C,

60 h; (e) 8.0 equivalents of styrene, 10.0 equivalents of Et₃N, 0.5 equivalents of Pd₂(dba)₃, 1.5 equivalents of P(o-tol)₃, DMF, 90 °C, 48 h; (f) 5.0 equivalents of boronic acid, 3.0 equiv Cs₂CO₃, 0.5 equivalents of Pd(PPh₃)₄, DMF, 90 °C, 24 h; (g) 10.0 equivalents of NaOMe, Et₂OMeOH (10:1), 25 °C, 20 min. AcOH = acetic acid, TB AI = tetrabutylammonium iodide, Pd(PPh₃)₄ = tetrakis(triphenylphosphine)palladium(0), TFA = trifluoroacetic acid.

Figure 28 depicts the structures and activities of stilbene and biaryl compounds. RE = relative efficacy of the indicated compound at 1 mM to 100 mM CDC A. Figure 29 illustrates a summary of structural parameters of compounds of formula (I) that are important for potent FXR activation.

Figure 30 illustrates structures of styrenes and boronic acids used in library construction illustrated in Figure 27.

Figure 31 illustrates structures of prefened FXR agonist compounds of formula (II).

Figure 32 illustrates structures of prefened FXR agonist compounds of formula (III).

Figure 33 illustrates structures of preferred FXR agonist compounds of formulas (II), (IN) and (N). Detailed Description of Preferred Embodiments

The non-steroidal FXR agonists of the present invention are Ν- aryl-Ν-arylmethyl amido and ureido compounds represented by the following formula (I):

wherein electrophile-derived moiety E¹ is (C_rC₈)alkyl, cyclohexyl, 2- fiiryl, 3-furyl, 2-thienyl, 3-thienyl, phenyl, or NH(C_rC₈)alkyl;

L¹ and L² are both H, or together form a pi-bond;

X¹ is C(O), or CH₂;

A² is a radical selected from the group consisting of:

substituent group G¹ is H or OCH₃;

G² and G³ are each independently H, (C_rC₈)alkyl, F, Cl, Br, I, OH, O(C,'-C₈)alkyl, SH, S(C_rC₈)alkyl, C(O)H, C(O)(C_rC₈)alkyl,

N((C₁-C₈)alkyl)₂, CO₂CH₃, or together form a 5 or 6-member carbocyclic ring or OCH₂O, preferably G² and G³ are each independently H, F, Cl, OCH₃, SCH₃, CH₃, N(CH₃)₂, or together form OCH₂O;

G⁴ is H or OCH₃; G⁵ is (C_rC₄)alkyl or C(O)(C_rC₈)alkyl;

G⁶ is H, or together with G⁸ forms a pi-bond, an epoxide, a cyclopropyl ring, a dichlorocyclopropyl ring, or a dibromocyclopropyl ring;

G⁷ is H, CH₃, or OZ⁵, with the proviso that G⁷ is H or CH₃ when G⁶ and G⁸ together form a pi-bond, an epoxide, a cyclopropyl ring, a dichlorocyclopropyl ring, or a dibromocyclopropyl ring; when G⁶ is H, G⁸ is H, or together with G⁹ forms a moiety selected from the group consisting of =NOZ⁶ and =O (otherwise, as indicated above, G⁸ together with G⁶ forms a moiety selected from the group consisting of a pi-bond, an epoxide, a cyclopropyl ring, a dichlorocyclopropyl ring, and a dibromocyclopropyl ring);

G¹⁰ and G" are each independently H, (C_rC₈)alkyl, SCH₃, C(O)(C_rC₈)alkyl, or C(O)O(C_rC₈)alkyl; and G¹² and G¹³ are each independently H or F;

G¹⁴ and G¹⁶ are each independently (Ci-C₈)alkyl, phenyl, or benzyl;

G¹⁵ is phenyl,

hydroxyphenyl, (C₁-C₈)alkoxyphenyl, chlorophenyl, bromophenyl, or fluorophenyl; and

G¹⁷ and G¹⁸ are each independently H, (C_rC₈)alkyl, SCH₃, C(O)(C_rC₈)all yl, or C(O)O(C_rC₈)alkyl;

T¹ and T² are each independently O, S, NH, or N(C_rC₈)alkyl;

Z¹ is H, phenyl, (C_rC₈)alkyl, benzyl, C(O)Ph, C(O)(C,.C₈)alkyl, C(O)OCH₂Ph, or C(O)NH(C_rC₈)alkyl;

Z² and Z³ are each independently (C_rC₈)alkyl or together form a (C_rC₈)cycloalkyl ring;

Z⁴, Z⁵, Z⁶, and Z⁷are each independently H or an oxygen protecting group, preferably an oxygen protecting group selected from the group consisting of phenyl, (C_rC₈)alkyl, benzyl, C(O)Ph, C(O)(C₁-C₈)alkyl,

C(O)OCH₂Ph, and C(O)NH(C_rC₈)alkyl;

Z⁸ is H, phenyl, (C_rC₈)alkyl, benzyl, C(O)Ph, C(O)(C₁.C₈)alkyl, C(O)OCH₂Ph, or C(O)NH(C_rC₈)alkyl;

Z⁹ and Z¹⁰ are each independently (C_rC₈)alkyl, or together form a (C₅-C₈)cyclic amine ring.

One aspect of the present invention is a biaryl subclass of FXR agonists represented by formula (II):

wherein

E² is isopropyl or cyclohexyl;

A³ is an aryl moiety selected from the group consisting of:

G¹⁹ is H or OCH₃;

G²⁰ and G²¹ are each independently H, (C_rC₈)alkyl, F, Cl, Br, I, OH, O(C_rC₈)alkyl, SH, S(C_rC₈)alkyl, C(O)H, C(O)(C_rC₈)alkyl, N((C₁-C₈)alkyl)₂, CO₂CH₃, or together fonn a 5 or 6-member carbocyclic ring or OCH₂O; preferably, G²⁰ and G²¹ are each independently H, F, Cl, OCH₃, SCH₃, CH₃, N(CH₃)₂, or together are OCH₂O;

G²² is H or OCH₃.

G²³ and G²⁴ are each independently H, (C_rC₈)alkyl, SCH₃, C(O)(C_rC₈)alkyl, or C(O)O(C₁-C₈)alkyl; and

T³ and T⁴are each independently O, S, NH, or N(C_rC₈)aιkyl.

Prefened embodiments of FXR agonists of the present invention represented by formula (II) are illustrated in Figure 31 and Figure 33. Another aspect of the present invention is a stilbene subclass of FXR agonists represented by fomiula (III):

wherein

E³ is isopropyl or cyclohexyl; and

G²⁵ and G²⁶ are each independently H or F.

Prefereed embodiments of FXR agonists of the present invention represented by formula (III) are illustrated in Figure 32.

Yet another aspect of the present invention is a benzopyran subclass of FXR agonists represented by formula (IN):

wherein

E⁴ is (C₁-C₈)alkyl, cyclohexyl, 2-furyl, 3-furyl, 2-thienyl,

3-thienyl, phenyl, or ΝH(C₁-C₈)alkyl;

L³ and L⁴ are both H, or together form a pi-bond;

X² is C(O), or CH₂;

Y² is H, NHZ¹¹, NH(Z¹²)Z¹³, or OZ¹⁴;

G²⁷ is (C_rC₄)alkyl or C(O)(C_rC₈)alkyl; G²⁸ is H, or together with G³⁰ forms a pi-bond, an epoxide, a cyclopropyl ring, a dichlorocyclopropyl ring, or a dibromocyclopropyl ring;

O²⁹ is H, CH₃, and OZ¹⁵, with the proviso that when G²⁸ and G³⁰ together form a pi-bond, an epoxide, a cyclopropyl ring, a dichlorocyclopropyl ring, or a dibromocyclopropyl ring, G²⁹ is H or CH₃; when G²⁸ is H, G³⁰ is H, or together with G²⁶ forms a moiety selected from the group consisting of =NOZ¹⁶ and =O;

G³¹ is H, OH, OZ¹⁷, CN, C(O)O(C_rC₈)alkyl, SPh, S(C_rC₈)alkyl, NHZ¹⁸, NH(Z¹⁹)Z²⁰, or together with G³⁰ forms a moiety selected from the group consisting of =NOZ¹⁶ and =O;

Z" is H, phenyl, (C_rC₈)alkyl, benzyl, C(O)Ph, C(O)(C_r C₈)alkyl, C(O)OCH₂Ph, or C(O)NH(C_rC₈)alkyl;

Z¹² and Z¹³ are each independently (C C₈)alkyl or together form a (C_rC₈)cycloalkyl ring; Z¹⁴, Z¹⁵, Z¹⁶, and Z¹⁷ are each independently H, or an oxygen protecting group, preferably an oxygen protecting group selected from the group consisting of phenyl, (Cι.C₈)alkyl, benzyl, C(O)Ph, C(O)(C_!.C₈)alkyl, C(O)OCH₂Ph, and C(O)NH(C₁-C₈)alkyl;

Z¹⁸ is H, phenyl, (C_rC₈)alkyl, benzyl, C(O)Ph, C(O)(Cj.C₈)alkyl, C(O)OCH₂Ph, and C(O)NH(C_rC₈)alkyl; and

Z¹⁹ and Z²⁰ are each independently

or together form a (C₅-C₈)cyclic amine ring.

A preferred embodiment of a FXR agonist of the present invention represented by formula (IN) is illustrated in Figure 33. An additional aspect of the present invention is a subclass of FXR agonists represented by formula (N):

wherein

E⁵ is isopropyl or cyclohexyl;

Z²¹ is a radical selected from the group consisting of:

G³² and G³⁴ are each independently

phenyl, or benzyl;

G³³ is phenyl, hydroxyphenyl,

chlorophenyl, bromophenyl, or fluorophenyl; and

G³⁵ and G³⁶ are each independently H, (C_rC₈)alkyl, SCH₃,

C(O)(C_rC₈)alkyl, or C(O)O(C_rC₈)alkyl.

Preferred embodiments of FXR agonists of the present invention represented by formula (N) are illustrated in Figure 33.

The FXR agonists represented by formula (I), including compounds of formulas (II), (HI), (IN), and (N), are useful as therapeutic agents for the treatment of diseases linked to cholesterol, and bile acid metabolism and homeostasis. The present FXR agonists are also useful tools for selectively activating FXR in vivo.

Initial screening of a diversity-orientated library of 10,000 benzopyran containing small molecules for FXR activation utilizing a cell-based reporter assay led to the identification of several lead compounds possessing low micromolar activity (EC₅₀'s = 5-10 mM). These compounds were systematically optimized employing parallel solution-phase synthesis and solid-phase synthesis to provide compounds that potently activate FXR. Two series of compounds, bearing stilbene or biaryl moieties, contain members that are among the most potent FXR agonists reported to date in cell-based assays.

These compounds are useful in studies aimed at further defining the physiological role of FXR and as potential agents for the treatment of diseases linked to cholesterol and bile acid metabolism and homeostasis. In addition to the discovery of potent compounds, the complete investigation of the structure- activity relationship (SAR) of the present agonists provide a valuable knowledge base for development of even more potent FXR agonists. In contrast to the previously reported FXR agonists (see Figure 1), none of the compounds of the present invention contain a carboxylic acid moiety.

Previous screening technologies for identifying small molecule activators of FXR utilized a fluorescence resonance energy transfer (FRET) assay to detect the ligand-dependent recruitment of the coactivator SRC-1 to FXR (Fraser, G. J., et al., J. Biol. Screen. 2002, 7, 3-10). The association of FXR with a coactivator is a necessary event for transcriptional activation. In the present investigations, however, a cell-based transcription assay was employed in which an FXR responsive promoter is linked to a luciferase reporter as a primary screen. In addition to ensuring that only cell permeable compounds were selected for further optimization, this approach allows for the detection of FXR activation in a natural system (i.e. conect folding of the protein and in the presence of a complete compliment of co-activators and co-repressors) (Xu, L., et al., Curr. Opin. Gen. Develop. 1999, 9, 140-147; and Glass, C. K., et al., Curr. Opin. Cell Biol. 1997, 9, 222-232).

Initial screening of a 10,000-member combinatorial library of benzopyran-based small molecules in this high-throughput, cell-based assay for FXR activation produced several lead compounds whose structures are listed in Figure 3(a) (4-15). Guided by the preliminary SAR gained from the evaluation of this initial library, a follow-up focused library of ca. 200 benzopyran-based compounds was designed and synthesized on solid support (see Figure 4). A selection of the most active compounds, possessing EC₅₀ values between 5 and 10 μM, obtained from this second round of screening is shown in Figure 3(b) (16-27). Compounds 26 and 27 proved to be among the most active FXR agonists at this stage and were the subject of further optimization as described below.

With initial lead compounds identified and validated, the stage was set for the systematic optimization of the three regions of the lead structure shown in Figure 5. As described in detail in the following sections, focused libraries were synthesized and screened in the cell-based assay in order to evaluate the structural requirements of each region of the molecule for potent FXR agonism. At this point, parallel solution-phase chemistry was utilized for the construction of additional focused libraries. This shift away from ^• solid-phase chemistry provided maximum flexibility, enabling rapid and systematic optimizations of each region of the lead molecules using relatively small designed libraries.

Evaluation of Benzopyran Region I SAR.

Most of the FXR agonists reported to date including CDCA (1), TTNPB (2) and GW4064 (3) (see Figure 1) contain a carboxylic acid moiety.

Based on these prior studies, materials having a variety of substituents within regions I, II and III of structure 26 (Figure 5) were prepared and the SAR of region I was evaluated. Several compounds displaying a carboxylic acid unit in various positions were synthesized (e.g. compounds 28, 36, 52, 54 and 56, Figure 6) and tested. Surprisingly, none of these compounds exhibited improved activation of FXR. Interestingly, compound 29, bearing a meta methyl acrylate moiety, was a substantially better activator of FXR than compound 26.

The preparation of compound 29 is representative of the methods employed to construct these compounds and is described in Figure 7

(see Figures 8-10 for further experimental procedures): aldehyde 59 was selectively methylated (Boger, D. L., et al., J Am. Chem. Soc. 1999, 121, 2471-2477). (NaH, Mel), alkylated (2-methyl-3-butyn-2-ol, TFAA, DBU, CuCl₂), reduced (Lindlar, H₂) and thermally cyclized to yield benzopyran 60. Reductive amination of aldehyde 60 with 3-bromoaniline (NaCNBH₃) followed by acylation with cyclopropanecarbonyl chloride (C₃H₅COCl, Et₃N) and palladium-mediated Heck coupling (Pd₂(dba)₃, P(o-tol)₃, Et₃N) with methyl acrylate provided compound 29.

Significantly, the location of the methyl acrylate moiety at the meta position led to potent activation of FXR, whereas, compound 53 (Figure

6), which bears a para methyl acrylate group did not activate FXR. The additional compounds shown in Figure 6 were synthesized to further examine which functional groups could be tolerated at the metα position. From biological screening of these compounds in the cell-based assay described herein, it is likely that the length and rigidity of the tether between the aromatic core and the interacting functionality (either methyl ester or methyl ether) are significant factors for FXR agonist activity. For instance, compounds 41 and 45 possess either too short or too long of a tether, respectively, for potent activity; compounds 35 and 46-49 apparently cannot adopt the correct orientation for potent activation; and compounds 30, 31, 34, 38, 39, 40 and 50 apparently do not present active functional groups to the receptor, since they are inactive. Indeed, of all the analogs designed to probe the SAR of region I, only compounds 29 and 33 significantly activated FXR in the cell-based assay. Due to relative ease of synthesis of compound 29, this analog was chosen as a starting point for the optimization of region II. Evaluation of Benzopyran Region II SAR.

Figure 11 illustrates the effect of numerous substitution patterns in this region of the molecule (see Figure 12 for a schematic representation of the preparation of these compounds). Only compounds 65 (EC₅₀ = 358 nM) and 68 (EC₅₀ = ca. 1 μM) were more effective than compound 29 in activating

FXR in this series of compounds. Substituted aromatic amide derivatives such as 69-77 were all less active than the parent compound 68, although they did exhibit significant activity. Alkyl derivatives 78 and 79 were inactive, as were sulfonamide 82, thiourea 84, and thioamide 83, suggesting the importance of N-acylation in region II. These results indicate that region II requires moderately bulky alkyl and cycloalkyl amide moieties for good FXR agonist activity.

Evaluation of Benzopyran Region III SAR.

Region III was optimized after regions I and II were thoroughly examined. Figure 13 shows structures of the compounds prepared for the region III SAR investigation. Figures 14 and 15 schematically illustrate preparation of the compounds. Incorporation of a polar hydrogen-bond donating functional group, such as those present in compounds 86, 93, 94, 98 and 100, and a hydrogen-bond acceptor group, such as those present in compounds 89, 90, 95, 99 and 101, did not improve FXR agonist activity over that of the parent compound 68. Similarly, the addition of a bulky lipophilic group to the benzopyran moiety afforded compounds that only weakly activated FXR. Surprisingly, however, replacement of the double bond in the benzopyran unit by a dichlorocyclopropane unit provided analog 102 (EC₅₀ = 333 nM), which was highly active. The synthesis of this potent compound is depicted in Figure 16: benzopyran 103 was cyclopropanated under phase transfer conditions (adogen 464 (cat), NaOH, CHC1₃) and converted to the conesponding cinnamate via a Heck coupling (Pd₂(dba)₃, P(o-tol)₃, Et₃N) with methyl acrylate to yield 102. Replacement of the benzoyl group in region II of compound 102 with the cyclohexanecarbonyl moiety afforded the even more potent compound 149 (EC₅₀ = 188 nM). Compound 149 (EC₅₀ = 188 nM) represents a significant improvement in potency over compound 65 (EC₅₀ = 358 nM).

The effect of replacing the benzopyran moiety with other ring systems was then examined to gain further insight into the structural requirements for optimal FXR agonist activity. Figure 17 shows a set of compounds in which the benzopyran moiety was replaced with groups of varying molecular diversity (see Figures 18 and 19 for a schematic representation of the synthesis of theses compounds). Results of cell-based reporter assays of the compounds indicated that replacement of the benzopyran with a small aromatic unit generally had a detrimental effect on activity. For instance, compounds 110 and 112-117 in Figure 20(c) were inactive, while compounds 111 and 118 showed only moderate activation of FXR (EC₅₀ = 680 nM and 606 nM, respectively). Replacement of the benzopyran with an aromatic ring bearing a substituent at the para position produced compounds with improved activity over that of compound 68. For example, 4-tert-butyl cinnamate 105 (EC₅₀ = 127 nM), stilbenes 121 and 122 (EC₅₀ = 36 and 208 nM, respectively), biaryls 124-127 (EC₅₀ = 510, 69, 77, 227 nM, respectively) and aryl thiophenes 128 and 129 (EC₅₀ = 206 and 256 nM, respectively) were all potent activators of FXR in the cell-based reporter assay. The synthesis of compound 105 is outlined in Figure 21 (see Figure 22 for the preparation of compounds 121-129). Acylation of 3-bromoaniline (C₆H_πCOCl, Et₃N) provided cyclohexylamide 131. Subsequent reaction of 131 under Heck coupling conditions (Pd₂(dba)₃, P(o-tol)₃, Et₃N) with methyl acrylate gave compound 132. Finally, alkylation (4-bromobenzyl bromide, NaH) of cinnamate 132 followed by a second Heck coupling (Pd₂(dba)₃, P(o-tol)₃, Et₃N) with tert-butyl acrylate afforded compound 105.

This initial survey of the three regions of SAR outlined in Figure 5 led to the identification of a number of potent FXR agonists for further evaluation. The compounds can be classified into four subclasses of formula (I), supra, namely compounds of formulas (II), (III), (IN), and (N), described above. Benzopyran dichlorocyclopropane compound 149 (EC₅₀ = 188 nM) is representative of compounds of formula (IN). Bis-cinnamate compound 105 (EC₅₀ = 127 nM) is a member of the formula (N) compounds. Stilbene compound 121 (EC₅₀ = 36 nM) and biaryl compound 124 (EC₅₀ = 69 nM) are examples of the foraiula (III) and formula (II) compounds, respectively. Based on data presented in Figures 6, 11, and 13, compound 149 appeared to represent the highest potency compound among the benzopyran compounds of formula (IN). The compounds of formula (N), formula (II), and fonnula (III) appeared to possess considerable potential for further development and rigorous SAR analysis, the results of which are presented below.

Examination of the bis-cinnamate and related compounds of formula (V). Similar to the results described above, the meta substituted methyl cinnamate moiety on the "right-hand" region of the molecule remained an important factor for good activity in the bis-cinnamate series (see Figure 20(a) and Figure 23). Replacement of the methyl acrylate unit with either a methyl or ethyl allylic ether (compounds 136 and 137) caused only a slight decrease in activity (EC₅₀ = 243 and 220 nM, respectively) compared to compound 105. A marked decline in potency accompanied substitution of the methyl acrylate by a sterically bulkier ether or ester (133 and 134) or amide (135). Interestingly, saturation of the acrylate olefin (139) afforded only a two-fold decrease in potency, EC₅₀ = 274 nM, which supports the hypothesis that conformational rigidity is a factor contributing to, but not essential for, high affinity ligands of FXR. Importantly, compound 139 suggests that the methyl acrylate moiety is not simply functioning as a latent electrophile.

The activity of Region II variants also closely minored the preceding data, in that cycloalkyl amides remained the optimal substituents (105 and 140-142: EC₅₀ = 127-250 nM) in the bis-cinnamate series (see Figure 20(b) and Figure 24). Aromatic and heterocychc amides as well as alkyl ureas led to moderate potency (143-145: EC₅₀ = 205-236 nM), whereas incorporation of a bulky urea such as present in compound 146 rendered compounds of only marginal efficacy.

As mentioned above, replacement of the benzopyran moiety with a benzyl group bearing a tert-butyl acrylate moiety in the jcαr -position yielded compound 105 with dramatically increased efficacy (EC₅₀ = 127 nM). Interestingly, placement of the same tert-butyl acrylate group in either the meta or ortho positions of the aromatic ring (107 and 109, Figure 17 and Figure 19 for synthesis) in Region III led to only micromolar potency. Further investigation of the "left-hand" region in this series of compounds demonstrated that a decrease in ester group size yielded a conesponding decrease in efficacy (EC₅₀ of t-butyl > z-propyl > ethyl > methyl; compounds 105, 150-152, Figure 20(d) and Figure 25). Similarly, substitution of the ester with either a carboxylic acid or an amide group provided less effective compounds with EC₅₀ values in the micromolar range. Compounds in which the tert-butyl acrylate moiety was substituted with a methyl or ethyl allylic ether (156 and 157) retained considerable potency (EC₅₀ = 233 and 198 nM, respectively). The bulkier phenyl allylic ether 158 possessed only micromolar activity, however, hi addition, saturation of the acrylate moiety, as in compound 159, resulted in a two-fold decrease in potency from the parent compound (105). Finally, substitution at the ortho position of the aromatic ring of the tert-butyl acrylate series with oxygenated functionality (e.g., compounds 161-167, see Figure 20(d) and Figure 26 for synthesis) afforded compounds with very low biological activity. Construction of Focused Libraries of Biaryl Compounds of Formula (II) and Stilbene Compounds of Formula (III).

In an effort to further optimize the biaryl and stilbene series, a 93-member library of such compounds was constructed employing a split-and-pool solid phase strategy. Individual library members were identified via radio frequency encoding using LRORI™ tags and MarcroKan™ technologies (Nicolaou, K. C, et al., J. Am. Chem. Soc. 2000, 122, 9939-9953; Nicolaou, K. C, et al., J. Am. Chem. Soc. 2000, 122, 9954-9967; and Nicolaou, K. C, et al, J. Am. Chem. Soc. 2000, 122, 9968-9976). As shown in Figure 27, Boc protected cinnamic acid 168 was immobilized on Merrifield resin (Cs₂CO₃) to afford resin 169. The Boc group of this resin was removed by treatment with 20% TFA in CH₂C1₂ and the resultant resin-bound amine was reductively alkylated with 4-bromobenzaldehyde (NaCNBH₃) to yield amino resin 170. Resin 170 was acylated with one of three acyl groups to give amide or urea resins 171. The acylated resins (171) were subjected to either Heck coupling (Pd₂(dba)₃, P(o-tol)₃, Et₃N) with thirteen substituted styrenes or Suzuki coupling (Pd(PPh₃)₄, Cs₂CO₃) with eighteen boronic acids to yield stilbene resins 172 and biaryl resins 173, respectively.

In selecting appropriate styrenes and boronic acids as inputs into this combinatorial library, guidance was obtained by initial comparisons of tert-butyl stilbene (123, EC₅₀ = > 1000 nM) to the unsubstituted stilbene 102

(EC₅₀ = 36 nM), and biaryl compound 124 (EC₅₀ = 510 nM) to 125 (EC₅₀ = 69 nM) as shown in Figure 20. Without being bound by theory, it is likely that both the stilbene and the biaryl ligands needed to fit into the same region of space within the receptor site for potent activation. Hence, stilbenes in which the aromatic nucleus is removed two carbon atoms further away from the core of the molecule compared to biaryls, are more potent when adorned with small substituents. In contrast, biaryl compounds are generally more potent when substituted with larger functional groups. Cleavage of resins 172 and 173 with NaOMe yielded methyl acrylates 121, 125, 126 and 174-263. Analysis of the library by liquid chromatograpy / mass spectrometry (LCMS) after purification using preparative thin-layer chromatography (PTLC), mdicated that the average purity of these compounds was generally greater than 95%.

Screening of this compound library in the cell-based reporter assay led to some intriguing results as summarized in Figure 28. For example, in both the stilbene and biaryl series, analogs bearing the cyclohexyl amide moiety were generally the most potent followed by those bearing the isopropyl amide or isopropyl urea groups. Stilbenes bearing relatively small substituents were more potent than those carrying larger substituents. For instance, unsubstituted stilbene 121 and mono-fluoro stilbenes 192, 200, and 203 were among the most active, while the mono-methyl derivative 174 and tri-methyl derivative 195 were among the least active. Interestingly, the heterocychc compounds 207 and 210 retained good potency (EC₅₀ = 309 and 227 nM, respectively).

In the biaryl series, compounds which presented bulkier substituents at the terminus of the structure were the most active, particularly compounds 259 (EC₅₀ = 25 nM) and 244 (EC₅₀ = 38 nM). Overall, most of the compounds synthesized in tins follow-up study were efficient activators of FXR, providing further support for the structural requirements for the FXR binding pocket described above. This model provides a solid basis for further development of FXR activators .

A summary of the molecular requirements of compounds of formula (I) that are important for potent FXR activation is shown in Figure 29. In region I, the presence of the metα methyl acrylate unit or an allylic methyl ether is important for potent activation, as only a few modifications retained good activity. The most potent compounds possessed a cycloalkylamide group in region II. Finally, region III is the most tolerant toward structural variations and several structural elements were found to provide a good fit within the pocket of the receptor.

In order to determine how selectively the compounds of the present invention activated FXR, some of the most active compounds were screened against a panel of nuclear receptors to look for cross-activation. The lead compounds were analyzed for their ability to modulate the activity of the following nuclear receptors: RXRαa, PPARα, PPARγ, PPARδ, PXR, SXR, LXRcc, TRβ, RARβ, CARR, ERR3, NDR. Most of these compounds were found to be highly selective, activating only FXR. Notably, however, compound 149 also potently activated SXR (FXR: EC₅₀ = 188 nM, SXR: EC₅₀ = 77 nM). This result may ultimately lead to compounds having utility in the treatment of diseases linked to the accumulation of toxic bile acids (Willson, T. M., et al., J. Lipid. Res. 2002, 43, 359-364; and Wen, X., et al., Proc. Nat. Acad. Sci. 2001, 98, 3375-3380).

General Techniques

Reagents and resins were purchased at highest commercial quality and used without further purification, unless otherwise stated. Anhydrous solvents were obtained by passing them through commercially available alumina column. All reactions were carried out under an argon atmosphere with dry solvents under anhydrous conditions, unless otherwise noted. Solution phase reactions were monitored by thin layer chromatography carried out on 0.25 mm E. Merck silica gel plates (60F-254) using UN light as visualizing agent and 7% ethanolic phosphomolybdic acid or -anisaldehyde solution and heat as developing agents. E. Merck silica gel (60, particle size

0.040-0.063 mm) was used for flash column chromatography. Preparative thin-layer chromatography (PTLC) separations were carried out on 0.25 mm E. Merck silica gel plates (60F-254). All final products cleaved from solid support were characterized by LCMS. ΝMR spectra were recorded on Bruker DRX-600, AMX-500 or AMX-400 instruments and calibrated using residual undeuterated solvent as an internal reference. High resolution mass spectra (HRMS) were recorded on a NG ZAB-ZSE mass spectrometer under MALDI-FTMS conditions with NBA as the matrix. Representative procedures for each region of SAR and the final combinatorial library synthesized are provided below. Compounds were screened for their ability to activate FXR expression in vivo. Solutions of the compounds at varying concentrations were added to cultures of cells that were cotransfected with human FRX gene linked to a luciferase reporter gene (see Dowries, et al, Mol. Cell, 2003; 11 : 1079- 1092; Nicolaou et al. Org. Biomol. Chem., 2003; 1: 908-920). Expression of FXR in the cells was designed to drive luciferase expression. FXR activation was determined by photometric measurement of luciferase luminescence. This approach allows for the detection of FXR activation in a natural system, i.e., correct folding of the protein and in the presence of a complete compliment of coactivators and corepressors (see Xu et al. Curr. Opin. Gen. Dev., 1999; 9:140-147; and Glass et al. Curr. Opin. Cell Biol., 1997; 9:222-232).

Representative procedure for synthesis Region I/II analogues; Synthesis of acrylate 29 (Figure 7):

To a solution of aldehyde 60 (50.0 mg, 0.229 mmol, 1.0 equivalent) in THF (1.0 mL) at 25°C was added 3-bromoaniline (59.0 mg, 0.344 mmol, 1.5 equivalents) and the reaction mixture was heated to 70°C. The solution was stirred for 4 hours and cooled to ambient temperature. To the resulting mixture was added methanol (0.2 mL) andNaCNBH₃ (28.8 mg, 0.458 mmol, 2.0 equivalents) and heated to 70 "C for 4 hours. The reaction mixture was then cooled and quenched with brine (5 mL). The reaction mixture was then concentrated and extracted with EtO Ac (3 x 5 mL). The combined organic phase was dried over MgSO₄, filtered and concentrated and used without further purification (90% yield by crude ^lR NMR analysis). To a solution of the resulting secondary amine (0.206 mmol, 1.0 equivalent) in CH₂C1₂ (1.0 mL) was added triethylamine (0.038 mL, 0.268 mmol, 1.3 equivalents), 4-DMAP (2.6 mg, 0.021 mmol, 0.1 equivalent), and cyclopropanecarbonyl chloride (28.0 mg, 0.268 mmol, 1.3 equivalents). The reaction mixture was stirred at 25°C for 12 hours and quenched with the addition of brine (5 mL). The aqueous phase was then extracted with CH₂C1₂ (3 x 5 mL). The combined organic phase was dried over MgSO₄, filtered and concentrated and used without further purification (95% yield by crude *H

NMR analysis). To the resulting amide (0.196 mmol, 1.0 equivalent) in N,N-dimethylformamide (1.0 mL) was added triethylamine (0.137 mL, 0.980 mmol, 5.0 equivalents), methyl acrylate (0.071 mL, 0.784 mmol, 4.0 equivalents), tri-o-tolylphosphine (30.0 mg, 0.098 mmol, 0.5 equivalent), and tris(dibenzylidineacetone)dipalladium(0) (35.9 mg, 0.039 mmol, 0.2 equivalent) sequentially and heated to 90°C. The reaction mixture was stirred for 24 hours and then cooled to ambient temperature. The reaction mixture was then diluted with EtOAc (10 mL) and washed with water (3 5 mL) and brine (1 x 5 mL). The combined organic phase was dried over MgSO₄, filtered, concentrated and purified by column cliromatography (silica, 0 (30% EtOAc in hexanes) to afford 29 (70.1 mg, 80%). 29: Ry= 0.42 (silica, 25% ethyl acetate in hexane); FT-IR (neat) v_max 2943, 1720, 1642, 1596, 1443, 1414, 1314, 1267, 1202 cm^"1; HRMS calcd for C₂₇H₂₉NO₅ [M + H⁺] 448.2118, found 448.2117.

Representative procedure for synthesis Region III benzopyran containing analogues; Synthesis of dichlorocyclopropane 102 (Figure 16):

To a solution of 103 (50.0 mg, 0.089 mmol, 1.0 equivalent) in CHC1₃ (2.0 mL) at 25°C was added NaOH (2.0 N, 0.3 mL) and adogen 464 (5.0 mg, ca. 0.1 equivalent). The resulting reaction mixture was stirred for 6 hours and quenched with water (5 mL). The aqueous phase was then extracted with

CH₂C1₂ (3 x 5 mL). The combined organic phase was dried over MgSO₄, filtered and concentrated, and used without further purification (85% yield by crude ^XH NMR analysis). To the resulting dichlorocyclopropane (0.073 mmol, 1.0 equivalent) in N,N-dimethylformamide (2.0 mL) was added triethylamine (0.051 mL, 0.366 mmol, 5.0 equivalents), methyl acrylate (0.026 mL, 0.292 mmol, 4.0 equivalents), tri-ø-tolylphosphine (11.1 mg, 0.037 mmol, 0.5 equivalent), and tris(dibenzylidineacetone)dipalladium(0) (13.4 mg, 0.015 mmol, 0.2 equivalent) sequentially and heated to 90°C. The reaction mixture was stirred for 24 hours and then cooled to ambient temperature. The reaction mixture was then diluted with EtOAc (10 mL) and washed with water (3 x 5 mL) and brine (1 x 5 mL). The combined organic phase was dried over MgSO₄, filtered, concentrated and purified by column chromatography (silica, 0 (30% EtOAc in hexanes) to afford 102 (30.9 mg, 75%). 102: Ry= 0.38 (silica, 25% ethyl acetate in hexane); FT-LR (neat) v_max 2943, 1719, 1640, 1590, 1443, 1378, 1314, 1226, 1161 cm^"1; HRMS calcd for C₃₁H₂₉Cl₂NO₅ [M + Na⁺] 588.1315, found 588.1323.

Representative procedure for synthesis Region III non-benzopyran containing analogues; Synthesis of bis-cinnamate 105 (Figure 21): To a solution of 3-bromoaniline (130, 60.0 mg, 0.349 mmol,

1.0 equivalent) in CH₂C1₂ (1.0 mL) at 25°C was added triethylamine (0.064 mL, 0.453 mmol, 1.3 equivalents), 4-DMAP (2.1 mg, 0.017 mmol, 0.05 equivalent), and cyclohexanecarbonyl chloride (56.3 mg, 0.384 mmol, 1.1 equivalents). The reaction mixture was stirred for 3 hours and quenched with brine (5 mL). The aqueous phase was extracted with CH₂C1₂ (3 5 mL) and subsequently dried over MgSO₄, filtered, concentrated to afford amide 131 (95% yield by crude *H NMR analysis) which was utilized without further purification. To a solution of amide 131 (0.332 mmol, 1.0 equivalent) in N,N-dimethylformamide (2.0 mL) was added triethylamine (0.232 mL, 1.66 mmol, 5.0 equivalents), methyl acrylate (0.119 mL, 1.33 mmol, 4.0 equivalents), tri-o-tolylphosphine (60.8 mg, 0.199 mmol, 0.6 equivalent), and tris(dibenzylidineacetone)dipalladium(0) (60.8 mg, 0.066 mmol, 0.2 equivalent) and heated to 90°C. The reaction mixture was stirred for 24 hours and then cooled to ambient temperature. The reaction mixture was then diluted with EtOAc (15 mL) and washed with water (3 x 5 mL) and brine (1 x 5 mL).

The combined organic phase was dried over MgSO₄, filtered, concentrated and purified by column cliromatography (silica, 0 ( 50% EtOAc in hexanes) to afford 132 (71.6 mg, 75%). To a solution of acrylate 132 (60.0 mg, 0.209 mmol, 1.0 equivalent) in THF (1.0 mL) at 0°C was added ΝaH (9.2 mg, 0.230 mmol, 60% dispersion in mineral oil, 1.1 equivalents) followed by

4-bromobenzyl bromide (67.9 mg, 0.272 mmol, 1.3 equivalents). The reaction mixture was stirred for 2 hours and quenched with saturated ΝH₄C1 (5 mL). The aqueous phase was extracted with EtOAc (3 5 mL) and the combined organic phase was dried over MgSO₄, filtered, concentrated and purified by column cliromatography (silica, 0 ( 30% EtOAc in hexanes) to afford 112 (85.8 mg, 90%). To a solution of amide 112 (50.0 mg, 0.110 mmol, 1.0 equivalent) inNN-dimethylformamide (2.0 mL) was added triethylamine (0.077 mL, 0.550 mmol, 5.0 equivalents), tert-butyl acrylate (0.064 mL, 0.440 mmol, 4.0 equivalents), tri-o-tolylphosphine (5.0 mg, 0.017 mmol, 0.15 equivalent), and tris(dibenzylidineacetone)dipalladium(0) (5.0 mg, 0.006 mmol, 0.05 equivalent) and heated to 90°C. The reaction mixture was stirred for 12 hours and then cooled to ambient temperature. The reaction mixture was then diluted with EtOAc (5 mL) and washed with water (3 x 5 mL) and brine (1 x 5 mL). The combined organic phase was dried over MgSO₄, filtered, concentrated and purified by column chromatography (silica, 0 ( 50% EtOAc in hexanes) to afford 105 (fexaramate, 41.5 mg, 75%), a representative member of the formula (N) compounds.

105: R₇= 0.40 (silica, 25% ethyl acetate in hexane); FT-LR (neat) v_max 2977, 2931, 2855, 1713, 1640, 1483, 1446, 1393, 1367, 1323, 1279, 1209 cm^"1; HRMS calcd for C₃₁H₃₇ΝO₅ [M + H⁺] 504.2744, found 504.2764.

General procedure for the solid phase synthesis of 93-membered library of biaryl and stilbene cinnamates (formula (II) compounds 125, 126, and 213-264, and formula (III) compounds 121, and 174-212, Figure 27): This library was constructed via directed split-and-pool techniques using IRORI MacroKans™. The microreactors were initially filled with commercially available Merrifield resin (110 mg, 0.91 mmol/g). After encoding, all 93 microreactors were suspended in N,N-dimethylformamide (900 mL) and treated with Boc-protected cinnamic acid 168 (4.94 g, 18.8 mmol, 2.0 equivalents), CsCO₃ (6.13 g, 18.8 mmol, 2.0 equivalents) and TBAI

(1.73 g, 4.7 mmol, 0.5 equivalent), and heated to 55°C. After 24 hours, the reaction mixture was cooled to ambient temperature and the reaction solvent was decanted prior to washing the microreactors with MeOH (3 x 500 mL), CH₂C1₂ (3 x 500 mL), and Et₂O (3 x 500 mL). Subsequently all microreactors were pooled and suspended in CH₂C1₂ (1000 mL) at 25 °C and treated with trifluoroacetic acid (200 mL). After 1 hour, the reaction mixture was quenched with Et₃N (200 mL) and the reaction solvent was decanted prior to washing the microreactors with MeOH (3 x 500 mL), CH₂C1₂ (3 x 500 mL), and Et₂O (3 x 500 mL). The microreactors were then pooled and resuspended in THFMeOH (2: 1, 1000 mL) at 25^βC and treated with 4-bromobenzaldehyde (17.4 g, 94.0 mmol, 10.0 equivalents) and acetic acid (30 mg, 0.47 mmol, 0.05 equivalent). After 1 hour, NaCNBH₃ (4.72 g, 75.2 mmol, 8.0 equivalents) was added and the resulting reaction was stirred a further 2 hours. The reaction solvent was then decanted and the microreactors were washed with MeOH (3 x 500 mL), CH₂C1₂ (3 x 500 mL), and Et₂O (3 x 500 mL).

At this point the microreactors were sorted into one of three reaction vessels and subjected to one of two acylation protocols. The microreactors of two of the reaction vessels were suspended in CH₂C1₂ (500 mL) at 25 °C and treated with either cyclohexanecarbonyl or isobutyryl chloride (94.0 mmol, 30.0 equivalents), Et₃N (17.4 mL, 124 mmol, 40.0 equivalents), and 4-DMAP (380 mg, 3.1 mmol, 1.0 equivalent) and stirred for 12 hours. The microreactors of the remaining reaction vessel were suspended in N,N-dimethylformamide (350 mL) and treated with isopropyl isocyanate (8.0 g, 94.0 mmol, 30.0 equivalents), Et₃Ν (17.4 mL, 124 mmol, 40.0 equivalents), and 4-DMAP (380 mg, 3.1 mmol, 1.0 equivalent), heated to 60°C and stirred for 60 hours. The microreactors were then cooled and the reaction solvent was decanted prior to washing the microreactors with MeOH (3 x 500 mL), CH₂C1₂ (3 x 500 mL), and Et₂O (3 x 500 mL). The microreactors were then sorted into one of 31 reaction vessels to be treated with either one of 13 commercially available styrenes or one of 18 commercially available boronic acids. For Heck couplings: The microreactors were suspended in NJV-dimethylformamide (100 mL) and treated with a stryrene (2.4 mmol, 8.0 equivalents, see Figure 30 for the identities of styrenes), Et₃Ν (0.42 mL, 3.0 mmol, 10.0 equivalents), tri-o-tolylphosphine (138 mg, 0.45 mmol, 1.5 equivalents), and tris(dibenzylidineacetone)dipalladium(0) (138 mg, 0.15 mmol, 0.5 equivalent) and heated to 90°C for a period of 48 hours. For Suzuki couplings: The microreactors were suspended in N,N-dimethylformamide (100 mL) and treated with a boronic acid (2.4 mmol, 8.0 equivalents, see Figure 30 for the identities of boronic acids), CsCO₃ (293 mg, 0.9 mmol, 3.0 equivalents), and tetrakis(triphenylphosphine)palladium(0) (173 mg, 0.15 mmol, 0.5 equivalent) and heated to 90°C for a period of 24 hours. The microreactors were then pooled and the reaction solvent was decanted prior to washing the microreactors with MeOH (3 x 500 mL), CH₂C1₂ (3 x 500 mL), and Et₂O (3 x 500 mL). Finally, each microreactor was sorted into an individual reaction vessel and cleaved upon suspension in Et₂O and subsequent treatment with a solution of ΝaOMe in MeOH (approx. 10 equivalents) at 25"C for a period of 20 min. The reactions were quenched with brine, extracted with Et₂O, concentrated and each compound was purified by preparatory thin layer chromatography (PTLC). Each compound was analyzed using LCMS which gave an average purity of 95% for the library and 93/93 parent mass peaks found.

Full characterization of representative members of the four classes of potent, selective FXR agonists (Figure 2):

Compounds of Formula (II):

125: Ry= 0.33 (silica, 25% ethyl acetate in hexane); FT-LR (neat) v_max 3025,

2926, 2854, 1714, 1643, 1597, 1580, 1488, 1435, 1394, 1359, 1318, 1269, 1231, 1202 cm^"1; HRMS calcd for C₃₁H₃₃ΝO₃S [M + Na⁺] 522.2073, found 522.2053.

128: R_/= 0.43 (silica, 25% ethyl acetate in hexane); FT-IR (neat) v_max 3058,

2927, 2854, 1715, 1642, 1598, 1588, 1483, 1435, 1398, 1318, 1271, 1230, 1201 cm^"1; HRMS calcd for C₂₉H_3INO₃S [M + Na⁺] 496.1917, found 496.1924. 244: R_f= 0.25 (silica, 25% ethyl acetate in hexane); FT-LR (neat) v_max 2930, 2855, 1716, 1652, 1599, 1579, 1504, 1486, 1445, 1398, 1318, 1226 cm^"1; HRMS calcd for C₃₁H₃₁NO₅ [M + H⁺] 498.2275, found 498.2269.

259: R_f= 0.27 (silica, 25% ethyl acetate in hexane); FT-IR (neat) v_max 2928, 1716, 1646, 1609,1579, 1539, 1504, 1446, 1396, 1357, 1319, 1268 cm^"1; HRMS calcd for C₃₂H₃₆N₂O₃ [M + H⁺] 496.2720, found 496.2715.

Compounds of Formula (III):

121: R_f= 0.35 (silica, 25% ethyl acetate in hexane); FT-LR (neat) v_max 2928, 2854, 1717, 1646, 1597, 1578, 1508, 1489, 1448, 1397, 1318, 1268, 1200 cm^"1; HRMS calcd for C₃₂H₃₃NO₃ [M + H⁺] 480.2533, found 480.2534.

192: R_f= 0.35 (silica, 25% ethyl acetate in hexane); FT-LR (neat) v_max 2928,

2854, 1716, 1652, 1508, 1485, 1449, 1397, 1318, 1268, 1231, 1200 cm^"1; HRMS calcd for C₃₂H₃₂FNO₃ [M + H⁺] 498.2439, found 498.2450.

Compound of Formula (TV): 149: R_f= 0.25 (silica, 25% ethyl acetate in hexane); FT-IR (neat) v_max 2932, 1720,

1642, 1580, 1454, 1399, 1341, 1318, 1269, 1202 cm^'1; HRMS calcd for C_3IH_3SCl₂NO₅ [M + Na⁺] 594.1784, found 594.1790.

Compound of Formula (V): 210: Ry= 0.10 (silica, 25% ethyl acetate in hexane); FT-LR (neat) v_max 2930, 1715,

1644, 1530, 1446, 1398, 1318, 1174 cm^"1; HRMS calcd for C₃₀H₃₂N₂O₃S [M + H⁺] 501.2206, found 501.2202.

Claims

We claim:

1. A non-steroidal farnesoid X receptor (FXR) agonist having the chemical structure represented by the following foraiula (I):

wherein electrophile-derived moiety E¹ is (C₁-C₈)alkyl, cyclohexyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, phenyl, or NH(C_rC₈)alkyl;

L¹ and L² are both H, or together form a pi-bond;

X¹ is C(O), or CH₂; Y¹ is H, NHZ¹, NH(Z²)Z³, or OZ⁴; aryl moiety A¹ is selected from the group of radicals consisting of:

A² is a radical selected from the group consisting of:

substituent group G¹ is H or OCH₃;

G² and G³ are each independently H, (C_rC₈)alkyl, F, Cl, Br, I, OH, O(C_rC₈)alkyl, SH, S(C_rC alkyl, C(O)H, C(O)(C_rC₈)alkyl, N((C_rC₈)alkyl)₂, CO₂CH₃, or together form a 5 or 6-member carbocyclic ring or OCH O;

G⁴ is H or OCH₃;

G⁵ is (C_rC₄)alkyl or C(O)(C_rC₈)allcyl;

G⁶ is H, or together with G⁸ forms a pi-bond, an epoxide, a cyclopropyl ring, a dichlorocyclopropyl ring, or a dibromocyclopropyl ring; G⁷ is H, CH₃, or OZ⁵, with the proviso that G⁷ is H or CH₃ when G⁶ and G⁸ together form a pi-bond, an epoxide, a cyclopropyl ring, a dichlorocyclopropyl ring, or a dibromocyclopropyl ring; when G⁶ is H, G⁸ is H, or together with G⁹ fomis a moiety selected from the group consisting of =NOZ⁶ and =O; G⁹ is H, OH, OZ⁷, CN, C(O)O(C_rC₈)alkyl, SPh, S(C_rC₈)alkyl,

NHZ⁸, NH(Z⁹)Z¹⁰, or together with G¹⁰ forms a moiety selected from the group consisting of =NOZ⁶ and =O;

G¹⁰ and G¹¹ are each independently H, (C_rC₈)alkyl, SCH₃, C(O)(C_rC₈)alkyl, or C(O)O(C_rC₈)alkyl; and G¹² and G¹³ are each independently H or F;

G¹⁴ and G¹⁶ are each independently (C_rC₈)alkyl, phenyl, or benzyl; G¹⁵ is phenyl, (CrC^alkylphenyl; hydroxyphenyl, (Ci-C₈)alkoxyphenyl, chlorophenyl, bromophenyl, or fluorophenyl; and

G¹⁷ and G¹⁸ are each independently H, (C_rC₈)alkyl, SCH₃, C(O)(C_rC₈)aUcyl, or C(O)O(C_rC₈)alkyl; T¹ and T² are each independently O, S, NH, or N(C_rC^a]kyl;

Z¹ is H, phenyl, (C_rC₈)alkyl, benzyl, C(O)Ph, C(O)(C_rC₈)alkyl, C(O)OCH₂Ph, or C(O)NH(C_rC₈)alkyl;

2. The non-steroidal FXR agonist of claim 1 wherein Z⁴, Z⁵, Z⁶, and Z⁷ are each independently H, or an oxygen protecting group selected from the group consisting of phenyl, (C_rC₈)alkyl, benzyl, C(O)Ph, C(O)(C_!-C₈)alkyl, C(O)OCH₂Ph, and C(O)NH(C_rC₈)alkyl.

3. The non-steroidal FXR agonist of claim 1 represented by formula (II):

wherein

E² is isopropyl or cyclohexyl; A³ is an aryl moiety selected from the group consisting of:

G¹⁹ is H or OCH₃;

G²⁰ and G²¹ are each independently H, (C_rC₈)alkyl, F, Cl, Br, I, OH, O(C_rC₈)alkyl, SH, S(C_rC₈)alkyl, C(O)H, C(O)(C_rC₈)alkyl, N((C_rC₈)alkyl)₂,

CO₂CH₃, or together fonn a 5 or 6-member carbocyclic ring or OCH₂O; G²² is H or OCH₃;

G²³ and G²⁴ are each independently H, (C_rC₈)alkyl, SCH₃, C(O)(C_rC₈)alkyl, or C(O)O(C_rC₈)alkyl; and T³ and T⁴ are each independently O, S, NH, or N(C₁-C₈)alkyl.

4. The non-steroidal FXR agonist of claim 3 wherein G²⁰ and G²¹ are each independently H, F, Cl, OCH₃, SCH₃, CH₃, N(CH₃)₂, or together are OCH₂O.

5. The non-steroidal FXR agonist of claim 3 represented by the formula:

6. The non-steroidal FXR agonist of claim 3 represented by the formula:

7. The non-steroidal FXR agonist of claim 3 represented by the formula:

The non-steroidal FXR agonist of claim 3 represented by the formula:

9. The non-steroidal FXR agonist of claim 3 represented by the formula:

10. The non-steroidal FXR agonist of claim 3 represented by the formula:

11. The non-steroidal FXR agonist of claim 3 represented by the formula:

12. The non-steroidal FXR agonist of claim 3 represented by the formula:

13. The non-steroidal FXR agonist of claim 3 represented by the formula:

14. The non-steroidal FXR agonist of claim 3 represented by the formula:

15. The non-steroidal FXR agonist of claim 3 represented by the formula:

16. The non-steroidal FXR agonist of claim 3 represented by the formula:

17. The non-steroidal FXR agonist of claim 3 represented by the formula:

18. The non-steroidal FXR agonist of claim 3 represented by the formula:

19. The non-steroidal FXR agonist of claim 1 represented by formula (III):

wherein

E³ is isopropyl or cyclohexyl; and

G²⁵ and G²⁶ are each independently H or F.

20. The non-steroidal FXR agonist of claim 19 represented by the formula:

21. The non-steroidal FXR agonist of claim 19 represented by the formula:

22. The non-steroidal FXR agonist of claim 19 represented by the formula:

23. The non-steroidal FXR agonist of claim 19 represented by the formula:

24. The non-steroidal FXR agonist of claim 19 represented by the formula:

25. The non-steroidal FXR agonist of claim 19 represented by the formula:

26. The non-steroidal FXR agonist of claim 1 represented by formula (IN):

wherein

E⁴ is (CrC₈)alkyl, cyclohexyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, phenyl, or ΝH(C_rC₈)alkyl;

L³ and L⁴ are both H, or together form a pi-bond;

X² is C(O), or CH₂;

Y² is H, NHZ¹¹, NH(Z¹²)Z¹³, or OZ¹⁴;

G²⁷ is (C_rC₄)alkyl or C(O)(C_rC₈)alkyl;

G²⁸ is H, or together with G³⁰ forms a pi-bond, an epoxide, a cyclopropyl ring, a dichlorocyclopropyl ring, or a dibromocyclopropyl ring;

G²⁹ is H, CH₃, and OZ¹⁵, with the proviso that when G²⁸ and G³⁰ together form a pi-bond, an epoxide, a cyclopropyl ring, a dichlorocyclopropyl ring, or a dibromocyclopropyl ring, G²⁹ is H or CH₃; when G²⁸ is H, G³⁰ is H, or together with G²⁶ forms a moiety selected from the group consisting of =NOZ¹⁶ and =O;

Z¹¹ is H, phenyl, (C_rC₈)alkyl, benzyl, C(O)Ph, C(O)(C_rC₈)alkyl, C(O)OCH₂Ph, or C(O)NH(C_rC₈)alkyl; Z¹² and Z¹³ are each independently (C₁-C₈)alkyl or together form a

(Cι-C₈)cycloalkyl ring;

Z¹⁴, Z^1S, Z¹⁶, and Z¹⁷ are each independently H, or an oxygen protecting group;

Z¹⁸ is H, phenyl, (C_rC₈)alkyl, benzyl, C(O)Ph, C(O)(C₁-C₈)aUcyl, C(O)OCH₂Ph, and C(O)NH(C_rC₈)alkyl; and

Z¹⁹ and Z²⁰ are each independently (C_rC₈)alkyl, or together form a (C₅-C₈)cyclic amine ring.

27. The non-steroidal FXR agonist of claim 26 wherein Z¹⁴, Z¹⁵, Z¹⁶, and Z¹⁷ are each independently H, or an oxygen protecting group selected from the group consisting of phenyl, (C_rC₈)alkyl, benzyl, C(O)Ph, C(O)(C₁-C₈)alkyl,

C(O)OCH₂Ph, and C(O)NH(C_rC₈)alkyl.

28. The non-steroidal FXR agonist of claim 26 represented by the formula:

29. The non-steroidal FXR agonist of claim 1 represented by formula (V):

wherein

E⁵ is isopropyl or cyclohexyl;

Z²¹ is a radical selected from the group consisting of:

G³² and G³⁴ are each independently (C_rC₈)alkyl, phenyl, or benzyl;

G³³ is phenyl, (C₁-C₈)alkylphenyl; hydroxyphenyl, (C₁.C₈)alkoxyphenyl, chlorophenyl, bromophenyl, or fluorophenyl; and

G³⁵ and G³⁶ are each independently H, (C_rC₈)alkyl, SCH₃, C(O)(C_rC₈)alkyl, or C(O)O(C_rC₈)alkyl.

30. The non-steroidal FXR agonist of claim 29 represented by the formula:

31. The non-steroidal FXR agonist of claim 29 represented by the formula:

32. The non-steroidal FXR agonist of claim 29 represented by the formula: