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 IC ₅₀ values in the low nanomolar range, k _inact / K _i values of >100,000 M ^-1 s ^-1 and high antiviral activity. High-resolution X-ray cocrystal structures indicated an important role of ortho -fluorobenzoyl substitution, forming a water network that stabilizes the inhibitor-bound enzyme. The most potent antiviral compound was the p -ethoxy- o -fluorobenzoyl chloropyridyl ester (PSB-21110, 29b , MW 296 g/mol; EC ₅₀ 2.68 nM), which may serve as a lead structure for broad-spectrum anticoronaviral therapeutics.