Second-generation inhibitors for the metalloprotease neprilysin based on bicyclic heteroaromatic scaffolds: Synthesis, biological activity, and X-ray crystal-structure analysis

A new class of nonpeptidic inhibitors of the Zn-II-dependent metalloprotease neprilysin with IC50 values in the nanomolar activity range (0.034-0.30 mu m) were developed based on structure-based de novo design (Figs. I and 2). The inhibitors feature benzimidazole and imidazo[4,5-c]pyridine moieties as central scaffolds to undergo H-bonding to Asn542 and Arg717 and to engage in favorable pi-pi stacking interactions with the imidazole ring of His711. The platform is decorated with a thiol vector to coordinate to the Zn-II ion and an aryl residue to occupy the hydrophobic S1' pocket, but lack a substituent for binding in the S2' pocket, which remains closed by the side chains of Phe106 and Arg110 when not occupied. The enantioselective syntheses of the active compounds (+)-1, (+)-2, (+)-25, and (+)-26 were accomplished using Evans auxiliaries (Schemes 2, 4, and 5). The inhibitors (+)-2 and (+)-26 with an imidazo[4,5-c]pyridine core are ca. 8 times more active than those with a benzimidazole core ((+)-1 and (+)-25) (Table 1). The predicted binding mode was established by X-ray analysis of the complex of neprilysin with (+)-2 at 2.25-angstrom resolution (Fig. 4 and Table 2). The ligand coordinates with its sulfanyl residue to the Zn-II ion, and the benzyl residue occupies the S1' pocket. The 1H-imidazole moiety of the central scaffold forms the required H-bonds to the side chains of Asn542 and Arg717. The heterobicyclic platform additionally undergoes pi-pi stacking with the side chain of His711 as well as edge-to-face-type interactions with the side chain of Trp693. According to the X-ray analysis, the substantial advantage in biological activity of the imidazopyridine inhibitors over the benzimidazole ligands arises from favorable interactions of the pyridine N-atom in the former with the side chain of Arg102. Unexpectedly, replacement of the phenyl group pointing into the deep S1' pocket by a biphenyl group does not enhance the binding affinity for this class of inhibitors.