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

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 <b>1</b> with promising cellular antiviral activity (EC₅₀ = 0.8 μM), limited cytotoxic liability (CC₅₀ > 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 <b>45</b>, which was found to potently inhibit several VEEV strains in the low nanomolar range without cytotoxicity (EC₅₀ = 0.02–0.04 μM, CC₅₀ > 50 μM) while limiting in vitro viral replication (EC₉₀ = 0.17 μM). Brain exposure was observed in mice with <b>45</b>. 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