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

ABSTRACT 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 –1 s –1 . The chloracetohydrazide derivative 12 exhibited antiviral activity (EC 50 = 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.