To develop antagonists selective for the mouse EP receptor
To develop antagonists selective for the mouse EP1 receptor, we started with compound (), synthesized as previously described (). Diethyl dipicolinic Glucose Uptake Fluorometric Assay Kit () was reduced with NaBH to . Parikh–Doering oxidation of with sulfur trioxide–pyridine complex and DMSO produced the unstable aldehyde . 4-Chlorophenoxide was then reacted with , followed by neutralization with HCl to form . Reduction of the secondary alcohol of under H and Pd/C with the addition of HSO and ZnBr gave . Alkylation of with 2-fluoro-4-chlorobenzyl bromide and cleavage of the ester by refluxing with NaOH produced the sodium salt () of the lead () which was formed by protonation of . The lead compound was shown to have good affinity for the human EP1 receptor and was stable in microsomes and S9 fractions of several species. However, was previously reported to have a high-affinity interaction with the human thromboxane (TP) receptor. We evaluated the molecular pharmacology of at the mouse EP receptors as well as the mouse TP receptor. Compound was confirmed to be a functional antagonist of mEP1 in vitro and to have submicromolar affinity for the mouse EP1 receptor by Schild Analysis (). had no detectable affinity for mouse EP3 or EP4 receptors by radioligand binding assays. had poor, but detectable affinity at mouse EP2, and suppressed signaling through mouse TP receptor at concentrations 100-fold higher than at the human receptor (), confirming the off-target activity of at mouse TP. Results from in vivo pharmacokinetics experiments () revealed compound to possess a moderate systemic plasma clearance (CL) and volume of distribution predicted at steady-state (), subsequently displaying a short half-life (, >60min) in mice receiving a parenteral administration of the EP1 receptor antagonist. We observed a bioavailability (%F) of approximately 14% following the oral administration (10mg/kg) of to mice. Recently, Ostenfeld have shown that in rats is cleared primarily by glucuronidation and sequestration into the bile. With the goal of inhibiting glucuronidation while improving molecular pharmacology of , a series of carboxylic acid bioisosteres of were pursued. -acylsulfonamides are common carboxylic acid bioisosteres that have been successfully implemented in antagonists of angiotensin II AT1 receptors as well as EP3 receptors. A series of analogs (–) resulting from the amidation of was prepared (). Tertiary amide analogues of ( and ) were included to evaluate a structure–activity role of an acidic proton in ligand-receptor interactions. Each was synthesized by coupling to a series of primary and secondary amines (–) and sulfonamides (–) employing common activators such as EDC·HCl, HOBt, and DIPEA in DMF (). The molecular pharmacology observed for – was determined at mEP1–mEP4 and mTP (). Generally, -acylsulfonamides retained mEP1 affinity similar to (representative data for , ) while the amide series had reduced affinity for mEP1. Each analog displayed reduced affinity for mEP2 and mTP. Interestingly, four analogs (, , , and ) displayed enhanced affinity for mEP3, a potential therapeutic target for hypertension- and diabetes mellitus-related ESRD. EP3 is of particular interest as it shares signaling pathways and endogenous ligands with EP1 and may represent a compensatory signaling pathway in the event of EP1 blockade., , , , These dual-selectivity compounds were confirmed to be functional antagonists of mEP3 by Schild analysis (data not shown). We subsequently determined the intrinsic clearance (Cl) of several potent amide and -acylsulfonamide analogs (). Results indicated an exceptional instability to metabolism in vitro, displaying estimated predicted hepatic clearance (CL) values that approached the hepatic blood flow in mice (, 90mL/min/kg). Results from metabolite identification studies in hepatic subcellular fractions indicated extensive biotransformation of the amide and the -acylsulfonamide , including NADPH-independent hydrolysis (i.e., esterases) and NADPH-dependent oxidation (i.e., P450) of these analogs. depicts the metabolism of , including the hydrolysis of the sulfonamide (), and P450-mediated oxidation of the methylene linker () and benzylic oxidation (). The extent of plasma protein binding (fraction unbound, ) in mouse was determined to be extensive for three compounds assessed (: =0.005, =0.010, =0.004).