With the identification of small nonpolar substituents

With the identification of small nonpolar substituents at R3 of the phenylpropanoic acid ring providing improved activity at FFA4, exploration of the benzyl SAR was undertaken (). Small substituents were well tolerated at both the and positions (–) with the 2-bromo- (), 2-isopropoxy- (), and 3-methyl-substituents () providing potent and selective FFA4 agonists. In select cases incorporation of --disubstitution (2,5-orientation) and --disubstitution (3,5-orientation) afforded equipotent agonists with the mono-substituted comparators ( vs. ; vs. & ) while for the majority of exemplars improved potency was realized ( vs. ; vs. ; vs. ; vs. ) at FFA4. Benzyl disubstitution provided weak activity at FFA1. Unmasking polar functionality ( vs. ) provided a dramatic decrease in activity at FFA4, consistent with the lipophilic nature of the binding pocket and structures of the natural ligands. Interestingly, combination of the biphenyl ether moiety of with the -methyl () provided the most potent FFA4 agonist of the series (). With a series of FFA4 agonists with varying degrees of selectivity over FFA1 in hand, examination of the pharmacokinetic (PK) properties of select compounds in C57Bl6 mice was initiated (). In general, the compounds were characterized with low to moderate IV clearance, low volumes of distribution (V<2 L/kg), and rapid oral masitinib (T values of 0.25–0.5h). Compound showed good oral exposure and high oral bioavailability with moderate half-life (t½=1.7h). Examination of the protein binding of the compounds revealed all to be >99% bound in human plasma with 99.9% protein binding measured for compound . Despite high protein binding, compound was chosen as a tool molecule for in vivo studies based upon its superior oral exposure (C=6779ng/mL), potency at FFA4 (EC=299nM), and selectivity over human FFA1 (EC=11.8μM). Prior to in vivo studies, the in vitro selectivity of compound was evaluated in a panel of targets in both full curve binding and functional assays including the rodent ortholog assays for FFA1 and FFA4 and additional members of the free fatty acid receptor family (). Increased selectivity (>85-fold) for both rat and mouse FFA4 over FFA1 was realized relative to the human receptors (40-fold). With respect to activity on the remaining members of the free fatty acid family, compound showed weak partial agonism of the FFA2 receptor and profiled as inactive against FFA3 in FLIPR based assays. The remainder of the panel included a mixture of GPCR, ion channel, and enzyme assays with compound showing robust selectivity against the panel. Investigation of the anti-diabetic properties of compound was undertaken in Zucker Diabetic Fatty (ZDF) rats, an established preclinical model of type 2 diabetes. Compound was administered to 8-week old male ZDF rats at doses of 10 and 100mg/kg twice daily for 14days with plasma glucose concentrations determined at baseline, 7 and 14days. All groups of ZDF rats had comparable levels of plasma glucose, insulin, and HbA prior to treatment. As illustrated in , the vehicle-treated animals showed a progressive hyperglycemia with plasma glucose rising from baseline values of 188 to 245mg/dL. In contrast, plasma glucose for compound treated animals was reduced to 151 and 139mg/dL at the 10 and 100mg/kg doses, respectively, from a starting average of 192mg/dL. With positive effects on plasma glucose measured in the ZDF rat, compound was progressed into a second rodent model of type 2 diabetes, the db/db mouse. In this model fed db/db mice were orally administered either vehicle or compound (100mg/kg) twice a day and blood glucose was measured before and after 1 and 2weeks of treatment. In addition, HbA levels were measured before and after 2weeks of treatment. As shown in , compound gave a robust response, normalizing blood glucose after 1week of treatment. Glucose lowering was maintained through the end of the study with levels for the compound treated animals finishing at 106mg/dL versus 276mg/dL for vehicle controls. Compound also robustly lowered HbA levels by 1.6% to a final value of 5.8% with the vehicle control group finishing the study at 7.8%.