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

[Image: see text] 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 IC(50) of 120 nM against the M(pro) and EC(50) 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.