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

3CLpro is an attractive target for the treatment of COVID-19. Using the scaffold hopping strategy, we identified a potent inhibitor of 3CLpro (3a) 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 3a and the protein in its catalytic pocket. Moreover, several analogues of compound 3a were designed and synthesized. Among them, compound 3h shows the best inhibition of 3CLpro with an IC50 of 0.322 μM and a kinact/Ki value of 1669.34 M–1 s–1, and it exhibits good target selectivity for 3CLpro against host proteases. Compound 3c inhibits SARS-CoV-2 in Vero E6 cells (EC50 = 2.499 μM) with low cytotoxicity (CC50 > 200 μM). These studies provide ideas and insights to explore and develop new 3CLpro inhibitors in the future.