Development of (E)-2-((1,4-Dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide, ML336: Novel 2-Amidinophenylbenzamides as Potent Inhibitors of Venezuelan Equine Encephalitis Virus

[Image: see text] Venezuelan equine encephalitis virus (VEEV) is an emerging pathogenic alphavirus that can cause significant disease in humans. Given the absence of therapeutic options available and the significance of VEEV as a weaponized agent, an optimization effort was initiated around a quinazolinone screening hit 1 with promising cellular antiviral activity (EC(50) = 0.8 μM), limited cytotoxic liability (CC(50) > 50 μM), and modest in vitro efficacy in reducing viral progeny (63-fold at 5 μM). Scaffold optimization revealed a novel rearrangement affording amidines, specifically compound 45, which was found to potently inhibit several VEEV strains in the low nanomolar range without cytotoxicity (EC(50) = 0.02–0.04 μM, CC(50) > 50 μM) while limiting in vitro viral replication (EC(90) = 0.17 μM). Brain exposure was observed in mice with 45. Significant protection was observed in VEEV-infected mice at 5 mg kg(–1) day(–1) and viral replication appeared to be inhibited through interference of viral nonstructural proteins.