Discovery of baicalein derivatives as novel covalent inhibitors of SARS CoV-2 Mpro: Structure-activity relationships and inhibitory mechanisms.

The SARS-CoV-2 main protease (Mpro) has been validated as a promising target for the development of anti-SARS-CoV-2 agents. This work aims to explore the structure-activity relationships (SARs) of flavonoids as Mpro inhibitors, and to develop more potent Mpro inhibitors. Firstly, the anti-Mpro activity of 109 natural flavonoids were evaluated, identifying baicalein as a potent lead compound. Guided by SARs, 55 baicalein derivatives were designed and synthesized, while the C-8 bromine-substituted baicalein (BA-21) was found as the most potent Mpro inhibitor. Further investigations showed that BA-21 potently inactivate Mpro in a time-dependent manner (IC50 = 0.35 μM). Inactivation kinetics showed that BA-21 was a potent Mpro inactivator, its inactivation efficacy (555.56 M-1 s-1) was about 7.26-fold higher than that of baicalein (76.50 M-1 s-1). Both chemoproteomics and molecular docking simulations demonstrated that baicalein could covalently modify four cysteine residues of Mpro, but BA-21 could covalently modify more functional cysteines of Mpro (such as Cys44, Cys145) via forming at least three reactive intermediates. Collectively, this work uncovers several essential structural features of flavonoids responsible for Mpro inhibition and devises a novel bromine-substituted flavonoid as a more efficacious covalent inhibitor of Mpro.