Sequential Optimization Approach Toward an Azapeptide‐Based SARS‐CoV‐2 Main Protease Inhibitor

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative agent of the coronavirus disease 2019 (COVID-19), is still circulating and posing a health threat to the global population. Its main protease (M^pro) constitutes an excellent target for the development of antivirals due to its indispensable role in the viral replication cycle. In this work, we employed a sequential approach to identify a potent azapeptide-based M^pro inhibitor. Starting from a series of small-molecule peptidomimetics, identical in their scaffold but equipped with different cysteine-reactive groups, we identified auspicious warheads. The combination of selected moieties with an optimized, previously described P1-P4 azapeptide structure resulted in a potent M^pro inactivator (12) with a k_inac/K_i value of 78,900 M^-1s^-1. The chloracetohydrazide derivative 12 exhibited antiviral activity (EC₅₀ = 0.47 µM), no cytotoxicity, and plasma stability. The molecular interaction of 12 with M^pro was elucidated by an X-ray crystal structure. A thioether linkage was generated through a nucleophilic substitution of chloride by the active-site thiolate, giving rise to irreversible inhibition.