br FXR Agonists Azepinol b indole hEC nM
FXR Agonists Azepinol[4,5-b]indole 1 (hEC50=600nM, efficacy (eff)=100%) was identified as a FXR agonist lead from a high-throughput screening effort. Structure–activity relationship (SAR) studies around the azepine ring demonstrated that dialkyl substitution at C-1 led to a 30-fold improvement in potency. In addition, incorporation of an isopropyl ester yielded another ∼3-fold boost in potency. Compound 2 represented the most potent FXR agonist within the series (hEC50=4nM, eff=149%) . A rat pharmacokinetic (PK) study showed that sc9 synthesis 2 had good oral bioavailability (F=38%) and a long half-life (t1/2=24h). Oral treatment of normal C57BL/6 mice with 2 administered at a dose of 10mg/kg/d for 7 days yielded statistically significant reductions of triglycerides (TG, 24%) and total cholesterol (22%) levels. When administered to low-density lipoprotein receptor knockout (LDLR−/−) mice fed a Western diet for 8 weeks, 2 lowered both TG (19% and 39% at doses of 1 and 3mg/kg, respectively) and total cholesterol (23% and 50% at doses of 1 and 3mg/kg, respectively). However, this molecule was poorly soluble. Guided by crystallographic data, the appended morpholine analogs 3a and 3b were identified and showed dramatic 400-fold improvements in equilibrium solubility measured in 0.5% methylcellulose/2% Tween-80 in water. However, bioavailability of 3a and 3b in rats was not improved compared to that of 2 (F=38% and 25% for 3a and 3b, respectively) which is likely due to their high clearance (Cl=52 and 64mL/min/kg for 3a and 3b, respectively). Both compounds showed potencies (3a: mEC50=52nM, eff=117%; 3b: mEC50=188nM, eff=110%) at mouse FXR similar to that of compound 2 (mEC50=152nM, eff=174%). Oral administration of 3a and 3b to LDLR−/− mice caused a dose-dependent reduction of low-density lipoprotein cholesterol (LDLc). In female rhesus monkeys, 3a given at a dose of 60mg/kg/d po for 4 weeks resulted in a significant lowering of TG (∼50%; absolute value was not reported), very low-density lipoprotein cholesterol (VLDLc∼50%; absolute value was not reported), and LDLc (63%) . The potent FXR agonist, 4a (GW4046, FXR transient transfection (TT) assay EC50=65nM, eff=100%), was unsuitable for further development due to several issues. These liabilities include poor rat PK (high clearance and low bioavailability), a potentially toxic stilbene pharmacophore, and stilbene-mediated UV light instability. SAR development at the 3- and 5-positions of the isoxazole ring revealed a preference for hydrophobic substituents . In addition, some polarity was tolerated in the tether at the 3-position of the isoxazole linked to the phenyl group, for example, 4b (FXR TT EC50=89nM, eff=89%). A rat PK study demonstrated that 4b had an improved t1/2 (2h) and clearance (Cl=20mL/min/kg). Unfortunately, low oral bioavailability (F=9%) was observed. It was hypothesized that the stilbene moiety could be the predominating detrimental structural feature irrespective of modifications elsewhere in these molecules. In an attempt to address the perceived liability of the stilbene functional group, a series of conformationally constrained analogs were explored . Benzothiophene analog 5 (FXR TT EC50=32nM, eff=87%) was equipotent with 4a. Indole analog 6a showed a slight attenuation of FXR activity (FXR TT EC50=210nM, eff=84%). In rat PK studies, compound 5 had very high clearance (Cl=66mL/min/kg), a short half-life (t1/2=15min), and poor bioavailability (F=9%). Indole 6a had significantly lower clearance (Cl=6.7mL/min/kg); however, the half-life was modest (t1/2=45min) and the oral bioavailability was low (F=12%). It was suggested that poor solubility of 6a may contribute to its poor oral bioavailability. To improve solubility, a second nitrogen atom was incorporated into the indole ring to give benzimidazole 6b. Although this compound was significantly less active at FXR (FRX TT EC50=5μM, eff=40%), a twofold improvement in bioavailability was achieved (F=26%) with little alteration of clearance or half-life suggesting that low solubility of these GW 4064 analogs may limit their absorption.