Nonpeptidic\nIrreversible Inhibitors of SARS-CoV‑2\nMain Protease with Potent Antiviral Activity

SARS-CoV-2 infections pose a high risk for vulnerable\npatients.\nIn this study, we designed benzoic acid halopyridyl esters bearing\na variety of substituents as irreversible inhibitors of the main viral\nprotease (M<sup>pro</sup>). Altogether, 55 benzoyl chloro/bromo-pyridyl\nesters were synthesized, with broad variation of the substitution\npattern on the benzoyl moiety. A workflow was employed for multiparametric\noptimization, including M<sup>pro</sup> inhibition assays of SARS-CoV-2\nand related pathogenic coronaviruses, the duration of enzyme inhibition,\nthe compounds’ stability versus glutathione, cytotoxicity,\nand antiviral activity. Several compounds showed IC<sub>50</sub> values\nin the low nanomolar range, <i>k</i><sub>inact</sub>/<i>K</i><sub>i</sub> values of >100,000 M<sup>–1</sup> s<sup>–1</sup> and high antiviral activity. High-resolution\nX-ray\ncocrystal structures indicated an important role of <i>ortho</i>-fluorobenzoyl substitution, forming a water network that stabilizes\nthe inhibitor-bound enzyme. The most potent antiviral compound was\nthe <i>p</i>-ethoxy-<i>o</i>-fluorobenzoyl chloropyridyl\nester (PSB-21110, <b>29b</b>, MW 296 g/mol; EC<sub>50</sub> 2.68\nnM), which may serve as a lead structure for broad-spectrum anticoronaviral\ntherapeutics.