Design, Synthesis, and Structure-Activity Relationship Studies of Novel 3CLpro Inhibitors.

The SARS-CoV-2 3C-like protease (3CLpro) is an essential enzyme for coronavirus replication and remains an attractive antiviral target due to its high sequence conservation among coronaviruses. Based on a previously identified catechol-containing covalent 3CLpro inhibitor (P1-E11), structural optimization was carried out using a linker-replacement strategy to systematically modify the central linker moiety. Two series comprising 20 analogues were designed, synthesized, and evaluated for enzymatic and antiviral activities. Among them, compound B2, featuring a direct amide linkage between the catechol warhead and the 4-(trifluoromethoxy)phenyl group, exhibited improved enzymatic inhibition (IC50 = 0.83 ± 0.06 μM) and favorable covalent inhibition kinetics (kinact/Ki = 380.01 M-1 s-1). In cell-based assays (A549-hACE2-TMPRSS2), B2 showed antiviral activity (EC50 = 15.03 ± 1.13 μM) with acceptable cytotoxicity (CC50 = 79.87 ± 6.85 μM). Structure-activity relationship analysis revealed that sulfur-containing linkers enhanced antiviral potency in certain cases but were associated with increased cytotoxicity. Collectively, this study expands the structural framework of covalent 3CLpro inhibitors and offers a rational strategy for their further optimization.