GW was also the starting point for a study

GW4064 was also the starting point for a study by an independent research group. The co-crystal structure of GW4064 with FXR suggested the potential for favorable hydrogen bond interactions between the isoxazole 3-aryl group and several receptor residues such as Tyr373 and Ser336. Replacing the 2,6-dichlorophenyl with a 2,6-dichloro-4-pyridyl moiety attenuated both FXR binding and functional activity. However, combination of this pyridine moiety with an N-methyl indole ring, an optimized stilbene replacement, afforded 7 with good FXR binding affinity (94nM) in a human scintillation proximity binding assay compared to 64nM for GW4064 [20]. Oxidation of the pyridine to the corresponding N-oxide gave 8, the most potent Actinomycin D in this series, with a FXR binding affinity of 45nM. Compared to other analogs reported in this study, 8 had the best permeability (PAMPA, 5.87×10cm/s). Molecular docking suggested that the N-oxide oxygen most likely participates in an H-bond acceptor interaction with Tyr373 on Helix 7 and/or Ser336 on Helix 5 [20]. From a separate screening effort, benzimidazolyl acetamide 9 was discovered as a novel FXR agonist with binding affinity of 70nM [21], [22]. Attempts to improve the physical properties of this compound by replacing the cyclohexyl groups with more polar moieties proved unsuccessful. This result is consistent with the co-crystal structure of compound 9 and hFXR, where the cyclohexyl groups are oriented within highly lipophilic pockets. The lead molecule from this series, 10 (IC50 SPA=13nM), was evaluated in LDLR−/− mice. It significantly reduced total cholesterol (45%), LDL (48%), and TG (52%) when orally administered at a dose of 30mg/kg/d for 5 days [21]. The poor physiochemical properties of 10, namely high lipophilicity and low aqueous solubility, limited its potential for further development. In addition, this molecule inhibited the hERG potassium channel in vitro (IC50=1.6μM). Further structural analysis revealed a more polar and yet unexplored pocket consisting of Gln267, Asn297, His298, Arg335, and three water molecules near the region where the N-cyclohexyl group binds [23]. Subsequent SAR efforts to replace this cyclohexyl group with a 4-carboxyphenyl ring yielded compound 11 without loss of receptor binding activity (FXR IC50 SPA=50nM) but with significantly improved solubility (88 vs. <1μg/mL for 10) and reduced hERG inhibition (IC50>20μM) [23], [24], [25]. Compound 12, a fluoro analog of 11, showed further enhancement of FXR binding (IC50 SPA=37nM) and solubility (115μg/mL) with no significant hERG activity (IC50>20μM). As a result of its good murine in vitro potency (IC50=290nM, EC50=870nM, eff=38%) and PK properties in mice (Cl=10mL/min/kg, F=33%), 12 was evaluated in LDLR−/− mice. After 5 days of treatment (10mg/kg/d, po), statistically significant decreases in plasma total cholesterol (41%), LDLc (33%), and TG (59%) were observed [23]. There have been numerous recent patent applications disclosing FXR agonists with novel chemical scaffolds. In a 2009 U.S. patent application, hexahydropyrroloazepines exemplified by 13 were claimed as FXR agonists [26]. Using a Gal4/hFXR fusion protein expressed in the HEK293 cell line, compound 13 showed an EC50 of 280nM. A related tetrahydropyrroloazepine series of FXR agonists was disclosed in a separate U.S. patent application [27]. Representative compound 14 showed an EC50 of 3nM in the HEK293 cell assay. A class of benzofurane/benzothiophene/benzothiazole derivatives was described as FXR modulators. Representative compound 15 showed an EC50 of 1.95 μM in a hFXR transactivation assay in CV-1 cells [28], [29]. Novel biaryl carboxylates were described in a 2009 patent application [30]. Compound 16 was exemplified and had an EC50 of <100nM in a hFXR SRC-1 cofactor recruitment assay. Another application described benzimidazole analogs represented by 17 which had an IC50 of 20nM in a FXR binding assay [31], [32]. A series of benzoic acid analogs were reported to display FXR agonist activity. Compound 18 provided an example from this class and had an EC50 of 7.7nM in a FRET functional assay [33].