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 IC 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 (IC = 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 (IC = 23 ± 3.4 μM) and PLpro (IC = 6.33 ± 0.5 μM), while 3h selectively inhibited PLpro (IC= 5.94 ± 1.0 μM). Both 3e and 3h suppressed SARS-CoV-2 replication with IC values of 13.4 ± 0.28 μM and 18.2 ± 3.2 μM, respectively. Molecular docking and dynamics studies revealed that the NO 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.