Phthalazine-based quaternary ammonium salts: synthesis, biological evaluation and membrane-targeting mechanism against Staphylococcus aureus

Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), poses a severe threat to human health due to the limited efficacy of traditional antibiotics and the rapid emergence of drug resistance. Quaternary ammonium compounds (QACs) are promising antibacterial agents with a membrane-disruptive mechanism that is less prone to inducing resistance. Herein, a series of novel phthalazine-derived QACs (2a–2v) were designed, synthesized, and evaluated for their antibacterial activities. The structure-activity relationship (SAR) analysis revealed that the length of N-alkyl substituent significantly affected antibacterial potency, with compound 2n emerging as the lead compound. Compound 2n exhibited broad-spectrum antibacterial activity against S. aureus, MRSA, Streptococcus pneumoniae, and Klebsiella pneumoniae, with MIC values of 2 μg/mL. It showed rapid bactericidal activity, low hemolysis (HC50 > 64 μg/mL), minimal resistance evolution, and excellent stability in physiological salts and against proteolytic enzymes, with certain concentration-dependent effects on biofilm. Mechanistic studies revealed that compound 2n exerts antibacterial activity via triggering bacterial membrane damage, which was verified by multiple phenotypic results, including Gram staining alteration, increased extracellular AKP activity, cell membrane depolarization, intracellular protein and nucleic acid leakage, as well as morphological observations from SEM and DAPI/PI staining. Molecular docking and competitive displacement assays indicated that compound 2n may bind to bacterial DNA, which is presumed to be a potential auxiliary antibacterial mechanism. These findings suggest phthalazine-derived QACs are promising and compound 2n has great potential for further development to combat drug-resistant bacterial infections.