Synthesis and multitarget inhibitory effect of indole-based ethyl cinnamate derivatives against SARS-CoV-2 Mpro and cathepsins for broad-spectrum anti-coronavirus activity

The experience of the SARS-CoV-2 pandemic has highlighted the urgent need to develop broad-spectrum antivirals (BSA) agents capable of targeting viral and/or host factors essential for infection as undeniable weapons against future coronavirus threats with potential pandemic perspectives. In this study, we report the synthesis and biological evaluation of a novel series of indole-based ethyl cinnamate derivatives designed as multitarget inhibitors of both SARS-CoV-2 Main Protease (M^pro) and human cathepsin, namely CatL and CatS, key enzymes involved in viral replication and entry. Enzymatic assays showed that several compounds exert significant inhibition on multiple targets. Structure-activity relationship (SAR) studies highlighted the critical role of the tert-leucine (Tle) residue at P2 position of these (pseudo)dipeptides, which proved to be essential for multitarged enzyme inhibition and antiviral activity. In antiviral assays against representative α- (hCoV-229E) and β-coronavirus (hCoV-OC43), 12 (EC₅₀ = 4.09 µM for hCoV-OC43, 0.77 µM for hCoV-229E), 20 (EC₅₀ = 6.68 µM for hCoV-OC43, 0.62 µM for hCoV-229E), and 3 (EC₅₀ = 2.96 µM for hCoV-OC43, 0.64 µM for hCoV-229E) restricted viral replication with high selectivity values (SI = 98, 56, and 101, respectively). Time-of-drug-addition experiments then revealed that 12 primarily inhibited viral entry by targeting CatL, whereas 20 affected both entry and post-entry stages of hCoV replication cycle likely thanks to its dual-inhibitory activity against both CatL and M^pro. These findings support the development of multitarget inhibitors as promising antivirals able to inhibit both α- and β-coronavirus, so as to reduce the risk of resistance associated with single-target agents. Furthermore, the demonstrated effectiveness of Tle-containing indole-based cinnamates as BSA highlights their potential in the context of the strategic framework for pandemic preparedness.