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 (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 such as nirmatrelvir as M Pro 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 M Pro inhibitors and characterized them on enzymatic inhibition potency, structures of their complexes with M Pro, cellular M Pro inhibition potency, antiviral potency, cytotoxicity, and in vitro metabolic stability. Our results indicated that M Pro 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.