A Refined Search of SARS-CoV-2 Main Protease Inhibitors as COVID-19 Antivirals

Abstract MPI8 was among one of the first SARS-CoV-2 main protease (MPro) inhibitors with strong antiviral potency. Building on its peptidomimetic aldehyde scaffold, we retained the P2 3-cyclohexylalanine and P3 O-tert-butylthreonine elements, which support S2 binding, S-subsite engagement, and likely cellular permeability, and varied the S4/S1′ substituents and covalent warheads. SAR and co-crystal structures identified compact N-terminal carbamates or amides bearing cyclopropane, isopropyl, or trifluoromethyl groups as favorable S4 binders. Benzoxazole/benzothiazole-carbonyl groups and acyloxymethyl ketones were explored as S1′-engaging motifs, with one benzoxazole-containing structure confirming tight S1′ packing by crystal structure. Nitrile and 2-amino-2-oxyacetyl warheads provided viable alternatives to the parent aldehyde. Combining optimized S4 groups with alternative warheads or S1′ motifs yielded multiple inhibitors more potent than MPI8. Notably, MPI-131 showed an IC50 of 1.7 nM, strong antiviral potency, and CC50 of 60.2 μM, placing it among the most potent MPro inhibitors reported.