Design, Synthesis, and Biological Evaluations of Constrained Ugi Adducts as Anti-JEV Agents

Japanese encephalitis virus (JEV) remains a major global health threat, with no approved antiviral therapeutics currently available. In this study, we exploited the versatility of the Ugi four-component reaction (U4CR) to design and synthesize a library of more than 70 novel conformationally constrained Ugi adducts. By systematically incorporating carbocyclic and heterocyclic ketones, we restricted the conformational flexibility of the Ugi scaffold to optimize interactions with viral replication machinery. Comprehensive structure–activity relationship studies led to the identification of 5g, 5h, 6b, 6q, and 7an as potent inhibitors of JEV replication, exhibiting submicromolar antiviral activity (5g, IC50 = 1.79 ± 0.13 μM; 5h, IC50 = 0.76 ± 0.01; 6b, IC50 = 1.37 ± 0.15 μM; 6q, IC50 = 1.02 ± 0.89; 7an, IC50 = 1.45 ± 0.17 μM) with good selectivity indexes (>72) and a favorable safety profile in Vero cells. Importantly, phospholipidosis assays demonstrated that the antiviral activity of 6b is not associated with phospholipid accumulation, indicating that its antiviral effects are unlikely to result from nonspecific phospholipidosis. A preliminary molecular docking study against conserved JEV nonstructural proteins, including RNA-dependent RNA polymerase (RdRp), helicase, and protease, suggested that these conformationally constrained Ugi adducts represent a promising chemotype targeting RdRp for the development of potent antiviral agents against JEV.