Exploring diverse reactive warheads for the design of SARS-CoV-2 main protease inhibitors

SARS-CoV-2 main protease (M^pro) is a validated antiviral drug target of nirmatrelvir, the active ingredient in Pfizer's oral drug Paxlovid. Drug-drug interactions limit the use of Paxlovid. In addition, drug-resistant M^pro mutants against nirmatrelvir have been identified from cell culture viral passage and naturally occurring variants. As such, there is a need for a second generation of M^pro inhibitors. In this study, we explored several reactive warheads in the design of M^pro inhibitors. We identified Jun11119R (vinyl sulfonamide warhead), Jun10221R (propiolamide warhead), Jun1112R (4-chlorobut-2-ynamide warhead), Jun10541R (nitrile warhead), and Jun10963R (dually activated nitrile warhead) as potent M^pro inhibitors. Jun10541R and Jun10963R also had potent antiviral activity against SARS-CoV-2 in Calu-3 cells with EC₅₀ values of 2.92 and 6.47 μM, respectively. X-ray crystal structures of M^pro with Jun10541R and Jun10221 revealed covalent modification of Cys145. These M^pro inhibitors with diverse reactive warheads collectively represent promising candidates for further development.