Design, Synthesis, and Unprecedented Interactions of Covalent Dipeptide-Based Inhibitors of SARS-CoV-2 Main Protease and its Variants Displaying Potent Antiviral Activity

The main protease (Mpro) of SARS-CoV-2 is a key drug target for the development of antiviral therapeutics. Here, we designed and synthesized a series of small-molecule peptidomimetics with various cysteine-reactive electrophiles. Several compounds were identified as potent SARS-CoV-2 Mpro inhibitors, including compounds 8n (IC50 = 0.0752 µM), 8p (IC50 = 0.0887 µM), 8r (IC50 = 0.0199 µM), 10a (IC50 = 0.0376 µM), 10c (IC50 = 0.0177 µM), and 10f (IC50 = 0.0130 µM). Most of them additionally inhibited cathepsin L and were also active against SARS-CoV-1 and MERS-CoV Mpro. In Calu-3 cells, several inhibitors, including 8r, 10a, and 10c, displayed high antiviral activity in the nanomolar range without showing cellular toxicity. The co-crystal structure of SARS-CoV-2 Mpro in complex with 8p revealed covalent binding to the enzyme’s catalytic residue Cys145 and showed specific, unprecedented interactions within the substrate binding pocket. Compounds 8n and 10c, especially 8n, were effective against a panel of naturally occurring nirmatrelvir-resistant mutants, particularly E166V, and showed metabolic stability and additional favorable pharmacokinetic properties, making it a suitable candidate for further preclinical development.