Design, Synthesis, and Structure–Activity Relationship Studies of Novel 3CL pro Inhibitors

The SARS‐CoV‐2 3C‐like protease (3CL pro ) 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 3CL pro 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 (IC 50 = 0.83 ± 0.06 μM) and favorable covalent inhibition kinetics ( k inact / K i = 380.01 M −1 s −1 ). In cell‐based assays (A549‐hACE2‐TMPRSS2), B2 showed antiviral activity (EC 50 = 15.03 ± 1.13 μM) with acceptable cytotoxicity (CC 50 = 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 3CL pro inhibitors and offers a rational strategy for their further optimization.