Macrocyclic Azapeptide Nitriles: Structure-Based Discovery of Potent SARS-CoV-2 Main Protease Inhibitors as Antiviral Drugs

Given the crucial role of the main protease (M^pro) in the replication cycle of SARS-CoV-2, this viral cysteine protease constitutes a high-profile drug target. We investigated peptidomimetic azapeptide nitriles as auspicious, irreversibly acting inhibitors of M^pro. Our systematic approach combined an M^pro active-site scanning by combinatorially assembled azanitriles with structure-based design. Encouraged by the bioactive conformation of open-chain inhibitors, we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization. This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent M^pro inhibitor 84 with an IC₅₀ value of 3.23 nM and a second-order rate constant of inactivation, k_inac/K_i, of 448,000 M^-1s^-1. The open-chain M^pro inhibitor 58, along with the macrocyclic compounds 83 and 84, a broad-spectrum anticoronaviral agent, demonstrated the highest Antiviral activity with EC₅₀ values in the single-digit micromolar range. Our findings are expected to promote the future development of peptidomimetic M^pro inhibitors as anti-SARS-CoV-2 agents.