Design and synthesis of isatin derivatives as effective SARS-CoV-2 3CL protease inhibitors

SARS-CoV-2 chymotrypsin-like cysteine protease (3CL^pro ) is one of the most widely developed drug targets for COVID-19. This study aimed to design and synthesize isatin derivatives to target SARS-CoV-2 3CL^pro in a covalent binding manner. Through the process, a potent 3CL^pro inhibitor (5g) was discovered with an IC₅₀ value of 0.43 ± 0.17 μM. To understand the binding affinity and specificity of 5g as a candidate inhibitor of SARS-CoV-2 3CL^pro , several assays were conducted, including FRET enzyme activity assays, thermodynamic-based and kinetic-based validation of inhibitor-target interactions, and cell-based FlipGFP assays. The interaction mechanism between 3CL^pro -5g was characterized by docking. Overall, these findings suggest that 5g is a new potent SARS-CoV-2 3CL^pro inhibitor for the treatment of COVID-19.