A Systematic Survey of Reversibly Covalent Dipeptidyl Inhibitors of the SARS-CoV‑2 Main Protease

SARS-CoV-2, the COVID-19 pathogen, relies on its main
protease
(MPro) for replication and pathogenesis. MPro is a demonstrated target for the development of antivirals for SARS-CoV-2.
Past studies have systematically explored tripeptidyl inhibitors such
as nirmatrelvir as MPro inhibitors. However, dipeptidyl
inhibitors especially those with a spiro residue at their P2 position
have not been systematically investigated. In this work, we synthesized
about 30 dipeptidyl MPro inhibitors and characterized them
on enzymatic inhibition potency, structures of their complexes with
MPro, cellular MPro inhibition potency, antiviral
potency, cytotoxicity, and in vitro metabolic stability.
Our results indicated that MPro has a flexible S2 pocket
to accommodate inhibitors with a large P2 residue and revealed that
dipeptidyl inhibitors with a large P2 spiro residue such as (S)-2-azaspiro [4,4]­nonane-3-carboxylate and (S)-2-azaspiro­[4,5]­decane-3-carboxylate have favorable characteristics.
One compound, MPI60, containing a P2 (S)-2-azaspiro­[4,4]­nonane-3-carboxylate
displayed high antiviral potency, low cellular cytotoxicity, and high in vitro metabolic stability.