Membrane-targeted schiff base derivatives overcome MRSA resistance through phosphatidylglycerol binding and ROS-mediated killing

Introduction The urgent need for novel antibacterial agents against drug-resistant Gram-positive pathogens, particularly Methicillin-resistant Staphylococcus aureus (MRSA), drives this research. This study aimed to synthesize and evaluate a series of N’-substituted methylene-4-chlorobenzohydrazide derivatives as potential anti-MRSA agents. Methods Sixteen target compounds (C1–C16) were synthesized from commercial ethyl 4-chlorobenzoate via ester aminolysis and condensation. Their structures were confirmed by 1 H NMR, 13 C NMR, and HRMS. Biological evaluations included in vitro antibacterial assays against a panel of bacteria, cytotoxicity (VERO cells), hemolytic activity, mechanistic studies (membrane targeting, depolarization, permeability, content leakage, ROS generation), biofilm inhibition, and resistance development assessment. Drug-likeness properties were also analyzed. Results Two novel (C1, C16) and fourteen known analogues were obtained. The series showed weak activity against Gram-negative bacteria but potent inhibition against various Gram-positive bacteria, including MRSA. Compound C12 emerged as the optimal derivative, exhibiting the strongest broad-spectrum anti-Gram-positive activity (MIC = 26 μM) and high selectivity. C12 showed no significant cytotoxicity or hemolysis at effective concentrations. It specifically targeted phosphatidylglycerol (PG) in the bacterial membrane, causing rapid membrane depolarization, increased permeability, leakage of intracellular proteins/DNA, ROS burst, and bactericidal effects. Furthermore, C12 inhibited S. aureus biofilm formation and displayed a very low propensity for spontaneous resistance development. It demonstrated moderate metabolic stability and suitable lipophilicity. Discussion Compound C12 represents a promising anti-MRSA lead compound. It combines potent antibacterial activity with a unique multi-mechanistic action targeting the cell membrane, a favorable biosafety profile, and a low resistance risk. These merits warrant further in-depth investigation and development.