9-fluorenone-based synthetic sulfonamide compounds as dual inhibitors of SARS-CoV-2 Main-Protease and Papain-like Protease.

Tilorone, a 9-fluorenone scaffold-based molecule, is a known broad-spectrum antiviral with an IC₅₀ of 180 nM against SARS-CoV-2, but its mechanism is not known. In the present study, we found it to have weak activity against PLpro (IC₅₀ = 30.7 ± 7.5 μM) and was inactive against Mpro. Several sulfonamide derivatives of 9-fluorenone, having the same scaffold as tilorone, were synthesized based on our in-silico studies to enhance their protease activity. They were evaluated for their inhibitory potential for targeting SARS-CoV-2 Mpro (main protease) and PLpro (papain-like protease) using FRET-based high-throughput screening and gel-based assays. Among the derivatives, 3e exhibited dual inhibition against Mpro (IC₅₀ = 23 ± 3.4 μM) and PLpro (IC₅₀ = 6.33 ± 0.5 μM), while 3h selectively inhibited PLpro (IC₅₀= 5.94 ± 1.0 μM). Both 3e and 3h suppressed SARS-CoV-2 replication with IC₅₀ values of 13.4 ± 0.28 μM and 18.2 ± 3.2 μM, respectively. Molecular docking and dynamics studies revealed that the NO₂ group in 3h enhances the rigidity of the BL2 loop of PLpro, contributing to its higher PLpro activity. Both 3e and 3h showed antiviral activity comparable with standard alpha-ketoamide inhibitor (13b-K) in cell-based assays and were non-cytotoxic with acceptable selectivity indices (S.I. > 5.5). These findings suggest that 9-fluorenone-based sulfonamides, particularly 3e and 3h, may be promising candidates as dual or selective protease inhibitors against SARS-CoV-2.