Design, Synthesis,and Unprecedented Interactionsof Covalent Dipeptide-Based Inhibitors of SARS-CoV‑2 Main Proteaseand Its Variants Displaying Potent Antiviral Activity

The main protease (M^pro) 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 M^pro inhibitors, including compounds <b>8n</b> (IC₅₀ = 0.0752 μM), <b>8p</b> (IC₅₀ = 0.0887 μM), <b>8r</b> (IC₅₀ = 0.0199 μM), <b>10a</b> (IC₅₀ = 0.0376 μM), <b>10c</b> (IC₅₀ = 0.0177 μM), and <b>10f</b> (IC₅₀ = 0.0130 μM). Most of them additionally inhibited cathepsin L and were also active against SARS-CoV-1 and MERS-CoV M^pro. In Calu-3 cells, several inhibitors, including <b>8r</b>, <b>10a</b>, and <b>10c</b>, displayed high antiviral activity in the nanomolar range without showing cellular toxicity. The cocrystal structure of SARS-CoV-2 M^pro in complex with <b>8p</b> revealed covalent binding to the enzyme’s catalytic residue Cys145 and showed specific, unprecedented interactions within the substrate binding pocket. Compounds <b>10c</b> and especially <b>8n</b> 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.