Identification and exploration of a series of SARS-Cov-2 M^Pro cyano-based inhibitors revealing ortho-substitution effects within the P3 biphenyl group

Starting from a simple scaffold hopping exercise based on our previous exploration of cysteine protease inhibitors against legumain, compound 6a was identified as a starting point for the development of a SARS-CoV-2 main protease (M^Pro) inhibitor. Compound 6a displayed submicromolar biochemical potency in the ultrasensitive assay developed by Drag and co-workers. Through an iterative structure–activity relationship campaign, we discovered an unexpected improvement in both biochemical and cellular potency through the incorporation of an ortho substituent within the P3 benzamide. X-ray crystallography revealed that incorporation of the ortho substituent caused a subtle but important binding enhancement of the P1 glutamate group within the M^Pro S1 pocket. While incorporation of the ortho substituent improved the potency, the off-target selectivity against a panel of cysteine proteases and cell activity remained suboptimal. Further scanning of the P2 core revealed that incorporation of the 3.1.0 proline could address these issues and afford compound 22e, a highly potent and cellularly active M^Pro inhibitor.