Diastereomeric Resolution Yields Highly Potent Inhibitor of SARS-CoV-2 Main Protease

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic. The main protease (M^pro, 3CL^pro) of SARS-CoV-2 is a key enzyme that processes polyproteins translated from the viral RNA. M^pro is therefore an attractive target for the design of inhibitors that block viral replication. We report the diastereomeric resolution of the previously designed SARS-CoV-2 M^pro α-ketoamide inhibitor 13b. The pure (S,S,S)-diastereomer, 13b-K, displays an IC₅₀ of 120 nM against the M^pro and EC₅₀ values of 0.8-3.4 μM for antiviral activity in different cell types. Crystal structures have been elucidated for the M^pro complexes with each of the major diastereomers, the active (S,S,S)-13b (13b-K), and the nearly inactive (R,S,S)-13b (13b-H); results for the latter reveal a novel binding mode. Pharmacokinetic studies show good levels of 13b-K after inhalative as well as after peroral administration. The active inhibitor (13b-K) is a promising candidate for further development as an antiviral treatment for COVID-19.