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

The SARS-CoV-2 main protease (M) 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 M inhibitors, and to develop more potent M inhibitors. Firstly, the anti-M 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 M inhibitor. Further investigations showed that BA-21 potently inactivate M in a time-dependent manner (IC = 0.35 μM). Inactivation kinetics showed that BA-21 was a potent M inactivator, its inactivation efficacy (555.56 M s) was about 7.26-fold higher than that of baicalein (76.50 M s). Both chemoproteomics and molecular docking simulations demonstrated that baicalein could covalently modify four cysteine residues of M, but BA-21 could covalently modify more functional cysteines of M (such as Cys44, Cys145) via forming at least three reactive intermediates. Collectively, this work uncovers several essential structural features of flavonoids responsible for M inhibition and devises a novel bromine-substituted flavonoid as a more efficacious covalent inhibitor of M.