Multifaceted antibacterial action of dihydrofurocoumarins against drug-resistant <i>Escherichia coli</i> : biofilm inhibition, membrane disruption, metabolic dysfunction, and oxidative stress damage

The alarming rise in antibiotic resistance necessitates the urgent development of novel therapeutic agents. Herein, we report a bifunctional approach to synthesize two series of dihydrofurocoumarins (DHFCs), one incorporating naphthalimide and the other featuring coumarin analogues, designed to explore their antibacterial potential and ability to combat antibiotic resistance through structural diversification. Preliminary assessments reveal that some synthesized analogues exhibit significant antibacterial potency. Notably, analogues with electron-withdrawing substituents, particularly 16b and 21e (MIC = 1.56 µg mL^-1), display outstanding activity against <i>E. coli</i>, demonstrating a higher potency than the marketed antibiotic amoxicillin. The low-frequency resistance observed for analogues 16b and 21e, as evidenced by stable MIC values even after extended passages, may be attributed to their rapid bactericidal action. Additionally, both analogues strongly inhibit biofilm formation, disrupting a critical pathway involved in the development of drug resistance. Mechanistic investigations revealed that both analogues effectively disrupt bacterial membranes, triggering cytoplasmic leakage and a significant loss of metabolic activity. They also induce reactive oxygen species (ROS) generation, catalyzing the oxidation of GSH to GSSG, thereby diminishing cellular GSH activity and weakening the bacterial antioxidant defense system, ultimately leading to oxidative damage and cell death. Active analogues were evaluated for their binding affinity to human serum albumin (HSA), demonstrating a balanced binding profile with optimal binding constants, indicative of their potential to facilitate targeted delivery without compromising drug release at the intended site. Site marker drug displacement studies further identified their binding sites, showing that 16b exhibited a preference for Sudlow site I, while 21e selectively associated with the heme site on HSA. Molecular docking studies further corroborated these findings, revealing perfect alignment with experimental results. Further investigations indicated that both active analogues intercalated into DNA, forming DNA-16b/21e complexes that disrupted essential biological functions, leading to bacterial death. Quantum chemical insights revealed a narrower HOMO-LUMO energy gap, facilitating electronic transitions and enhancing molecular reactivity, which may be pivotal for their antibacterial effectiveness. Amidst the limitations of conventional antibiotics, these findings underscore the potential of dihydrofurocoumarins as potent multitarget, broad-spectrum antibacterial agents. Their ability to impair bacterial defense mechanisms and combat persistent pathogens presents a promising avenue for advancing antibacterial therapeutics, paving the way for further clinical exploration and the development of novel antibacterial analogues.