Unravelling the antibacterial potential and mechanistic insights of spirocyclic naphthalimide analogues against drug-resistant bacteria.

The pervasive and uncontrolled use of antibiotics has dramatically expedited the evolution of drug resistance, prompting an urgent demand for effective antibacterial agents to combat this growing challenge. Herein, we report a series of spiroacenaphthylene- and spiroindenoquinoxaline-based naphthalimides, designed as antibacterial scaffolds that diverge from conventional drug frameworks to mitigate cross-resistance concerns. Initial screening suggests that some of the synthesized analogues possess appreciable antibacterial efficacy. Notably, the methylthioethyl-functionalized spiroindenoquinoxaline naphthalimide analogue 12f (MIC = 1.56 μg/mL) exhibited good activity against E. faecalis, surpassing the efficacy of standard antibiotics such as amoxicillin and tetracycline. It demonstrated rapid bactericidal effects, inhibited biofilm formation, showed minimal cytotoxicity toward HEK293 cells, and displayed favorable ADME predictions, supporting its potential for further development. Its drug combination with tetracycline exhibited synergism, further enhancing its antibacterial efficacy. Moreover, the active analogue induced bacterial death by increasing intracellular reactive oxygen species (ROS), leading to the oxidation of GSH to GSSG, resulting in the depletion of intracellular GSH levels, and consequent oxidative damage. Importantly, it also induced multi-targeting effects, notably membrane disruption and reduced metabolic activity. Additionally, 12f binds efficiently to DNA, blocking its replication and forming supramolecular complexes, thereby contributing to its antibacterial activity. By integrating multiple antibacterial mechanisms within a single framework, these spiro naphthalimide analogues emerge as promising scaffolds for future class of antibacterial agents, poised to reshape treatment strategies against drug-resistant and persistent bacterial infections, thereby substantiating their progression toward clinical investigation.