Discovery of piperazine-based multi-site occupying coronavirus Mpro inhibitors with potent and broad-spectrum antiviral activity.

The continuous evolution of SARS-CoV-2 and the emergence of drug-resistant variants underscore the urgent need for broad-spectrum antiviral agents targeting conserved viral proteins. The main protease (Mpro) represents a promising target due to its essential role in coronavirus replication. In this study, we report the discovery and optimization of a novel series of piperazine-based Mpro inhibitors using a multi-site binding strategy guided by analysis of conserved residues within the coronavirus Mpro active sites. Starting from the noncovalent lead GC-14, systematic optimization of substituents occupying the S1', S1, S2, and S4 subsites of Mpro led to the development of the noncovalent inhibitor GY-e2, which showed improved inhibitory efficacy against both SARS-CoV-2 and SARS-CoV Mpro. To further enhance its antiviral efficacy in cellular models, reactive warheads targeting C145 were incorporated into the scaffold to generate covalent inhibitors. This strategy yielded the isomeric compounds Y-U0-R and Y-U0-S, which displayed potent Mpro inhibition and markedly enhanced antiviral activity in SARS-CoV-2-infected Calu-3 cells. Moreover, both compounds exhibited broad-spectrum antiviral activity against other human coronaviruses, and notably remained effective against the two major Nirmatrelvir-resistant strains evaluated in this study. Mechanistic studies further confirmed kinetically stable binding and time-dependent inhibition of Y-U0-R, supporting the rationale of covalent inhibitor design. These findings highlight the utility of structure-based design for the development of promising broad-spectrum anti-coronavirus agents.