Development of pyrimidone derivatives as nonpeptidic and noncovalent 3-chymotrypsin-like protease (3CLpro) inhibitors with anti-coronavirus activities.

3CLPro is crucial to the life cycle of SARS-CoV-2 and exhibits high sequence similarity with other coronaviruses, while being absent in human proteases. This makes it an ideal target for developing broad-spectrum antiviral drugs. Ensitrelvir (S-217622) is the only launched non-covalent, non-peptidomimetic 3CLPro inhibitor, offering certain advantages in terms of dosage and metabolism. Using S-217622 as the lead, we designed and synthesized 43 pyrimidone derivatives and conducted a systematic evaluation of their structure-activity relationships. Among them, A36 exhibited strong inhibitory activity against several β-coronaviruses and demonstrated low cytotoxicity. A36 also displayed moderate stability in mouse liver microsomes. Co-crystal structure analysis of 3CLPro in complex with A36 revealed the similar binding mode with S-217622. A36 shows strong potential as a promising lead for broad-spectrum anti-coronavirus therapy, warranting further investigation.