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 mainprotease(M-Pro) for replication and pathogenesis. M-Pro is a demonstrated target for the development of antivirals for SARS-CoV-2.Past studies have systematically explored tripeptidyl inhibitors suchas nirmatrelvir as M-Pro inhibitors. However, dipeptidylinhibitors especially those with a spiro residue at their P2 positionhave not been systematically investigated. In this work, we synthesizedabout 30 dipeptidyl M-Pro inhibitors and characterized themon enzymatic inhibition potency, structures of their complexes withM(Pro), cellular M-Pro inhibition potency, antiviralpotency, cytotoxicity, and in vitro metabolic stability.Our results indicated that M-Pro has a flexible S2 pocketto accommodate inhibitors with a large P2 residue and revealed thatdipeptidyl 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-carboxylatedisplayed high antiviral potency, low cellular cytotoxicity, and high in vitro metabolic stability.