Discovery, Synthesis, and in vitro Evaluation of West Nile Virus Protease Inhibitors Based on the 9,10‐Dihydro‐3 H ,4a H ‐1,3,9,10a‐tetraazaphenanthren‐4‐one Scaffold

West Nile virus (WNV), a member of the Flaviviridae family, is a mosquito‐borne pathogen that causes a great number of human infections each year. Neither vaccines nor antiviral therapies are currently available for human use. In this study, a WNV NS2B–NS3 protease inhibitor with a 9,10‐dihydro‐3 H ,4a H ‐1,3,9,10a‐tetraazaphenanthren‐4‐one scaffold was identified by screening a small library of non‐peptidic compounds. This initial hit was optimized by solution‐phase synthesis and screening of a focused library of compounds bearing this scaffold. This led to the identification of a novel, uncompetitive inhibitor ( 1a40 , IC 50 =5.41±0.45 μ M ) of WNV NS2B–NS3 protease. Molecular docking of this chiral compound onto the WNV protease indicates that the S  enantiomer of 1a40 appears to interfere with the productive interactions between the NS2B cofactor and the NS3 protease domain; ( S )‐ 1a40 is a preferred isomer for inhibition of WNV NS3 protease.