Discovery and Mechanism Study of SARS-CoV-2 3C-like Protease Inhibitors with a New Reactive Group.

3CL is an attractive target for the treatment of COVID-19. Using the scaffold hopping strategy, we identified a potent inhibitor of 3CL () that contains a thiocyanate moiety as a novel warhead that can form a covalent bond with Cys145 of the protein. Tandem mass spectrometry (MS/MS) and X-ray crystallography confirmed the mechanism of covalent formation between and the protein in its catalytic pocket. Moreover, several analogues of compound were designed and synthesized. Among them, compound shows the best inhibition of 3CL with an IC of 0.322 μM and a / value of 1669.34 M s, and it exhibits good target selectivity for 3CL against host proteases. Compound inhibits SARS-CoV-2 in Vero E6 cells (EC = 2.499 μM) with low cytotoxicity (CC > 200 μM). These studies provide ideas and insights to explore and develop new 3CL inhibitors in the future.