Antibacterial profiling, mechanistic characterization and in silico studies of pyrrolo[3,4-d]isoxazolidine-naphthalimide hybrids targeting Staphylococcus aureus.

Abstract

In an effort to diverge from the molecular framework of conventional drug molecules, a series of pyrrolo[3,4-d]isoxazolidine-naphthalimide hybrids have been synthesized via a 1,3-dipolar cycloaddition reaction between azomethine N-oxides and N-substituted maleimide, with the aim of exploring moieties capable of lessening bacterial infections that continue to burden human health and the livestock industry. Several of the synthesized analogues demonstrate potent antibacterial activity at low concentrations. Notably, 9k and 9l, featuring indole and N,N-dimethylaniline moieties, respectively, outperform chloramphenicol and amoxicillin against Staphylococcus aureus, as evidenced by their low MIC values (1.56 µg mL-1). Mechanistic studies reveal that both analogues effectively inhibit biofilm formation and disrupt bacterial cell membrane integrity, as confirmed by confocal laser scanning microscopy and SEM imaging, significantly reducing bacterial metabolic activity. These compounds also induce reactive oxygen species (ROS) generation and diminish cellular GSH activity, thereby weakening the bacterial antioxidant defense system and leading to oxidative damage and cell death. Additionally, these compounds show low cytotoxicity toward HEK293 cells, indicating good biocompatibility and a favorable safety profile. Both analogues exhibit strong affinity toward human serum albumin (HSA), as reflected by favorable binding constants, suggesting their potential suitability for transport in biological systems. This observation is further supported by molecular docking studies, which reveal stable binding orientations within the HSA binding cavity mediated by multiple non-covalent interactions. Furthermore, both analogues exhibit DNA intercalation behavior, which may hinder DNA replication and thereby contribute to bacterial cell death. Moreover, in silico ADME predictions indicate that the analogues possess a balanced drug-likeness profile, while quantum chemical calculations reveal a narrow HOMO-LUMO energy gap, suggesting enhanced electronic reactivity that may contribute to their antibacterial activity. Collectively, these findings highlight the potential of these multitarget antibacterial hybrids as promising leads for the development of new antibacterial agents against Staphylococcus aureus, a persistent threat to both human health and the livestock industry.