Nonpeptidic Irreversible Inhibitors of SARS-CoV-2 Main Protease with Potent Antiviral Activity

SARS-CoV-2 infections pose a high risk for vulnerable patients. In this study, we designed benzoic acid halopyridyl esters bearing a variety of substituents as irreversible inhibitors of the main viral protease (M^pro). Altogether, 55 benzoyl chloro/bromo-pyridyl esters were synthesized, with broad variation of the substitution pattern on the benzoyl moiety. A workflow was employed for multiparametric optimization, including M^pro inhibition assays of SARS-CoV-2 and related pathogenic coronaviruses, the duration of enzyme inhibition, the compounds' stability versus glutathione, cytotoxicity, and antiviral activity. Several compounds showed IC₅₀ values in the low nanomolar range, <i>k</i>_inact/<i>K</i>_i values of >100,000 M^-1 s^-1 and high antiviral activity. High-resolution X-ray cocrystal structures indicated an important role of <i>ortho</i>-fluorobenzoyl substitution, forming a water network that stabilizes the inhibitor-bound enzyme. The most potent antiviral compound was the <i>p</i>-ethoxy-<i>o</i>-fluorobenzoyl chloropyridyl ester (PSB-21110, <b>29b</b>, MW 296 g/mol; EC₅₀ 2.68 nM), which may serve as a lead structure for broad-spectrum anticoronaviral therapeutics.