Febuxostat analogs as anti-quorum sensing and antibacterial agents

Febuxostat, a marketed anti-gout drug, has been reported to inhibit quorum sensing (QS), biofilm formation, and virulence in Gram-negative bacteria Pseudomonas aeruginosa PAO1 and Chromobacterium violaceum CV026. However, no structure-activity relationship (SAR) studies have been reported to date. To address this gap, we synthesized 27 novel febuxostat analogs and evaluated their QS inhibitory activity using wild-type C. violaceum and the mutant strain CV026. This dual-strain assay enabled differentiation between quorum sensing inhibition and quorum quenching mechanisms, allowing determination of whether QS interference occurred at the level of signal synthesis or signal reception. Here, we demonstrated that febuxostat acts as a quorum quencher at concentrations lower than previously reported, strongly inhibiting violacein production in CV026 without affecting bacterial viability. At 400 µM, several analogs showed comparable or stronger activity than febuxostat in one or both strains. At 40 µM, trifluoromethoxyphenyl analog 49 emerged as the most effective QS inhibitor in the wild-type strain, whereas cyclopentyl analog 24 was most active in CV026, with a few other derivatives also retaining notable activity. We also assessed whether febuxostat and its analogs exhibit antibacterial activity. While febuxostat showed no antibacterial activity, seven analogs (26-30, 33, and 49) exhibited minimum inhibitory concentrations (MICs) ranging from 2.5 to 40 µM against Gram-positive bacteria Staphylococcus aureus, S. epidermidis, Enterococcus faecalis, and E. faecium. On the other hand, no activity was observed against Gram-negative bacteria. In follow-up studies, the two most potent antibacterial compounds 29 (MIC = 5 µM) and 30 (MIC = 10 µM), showed no cytotoxicity against the mammalian cell line A549, while they were less tolerated by THP-1 cells. In summary, our results provide SAR insights into febuxostat analogs and highlight their potential as novel anti-QS and antibacterial agents against multidrug-resistant bacteria.