Design, synthesis and antibacterial evaluation of bis-pyridinium cationic antimicrobial peptide mimics with low hemolytic toxicity.

The rampant overuse of antibiotics has triggered widespread bacterial resistance, further exacerbated by biofilm-forming pathogens and non-mutating persisters, necessitating urgent development of novel antibiotics. Here, a series of bis-pyridinium cationic amphiphilic antimicrobial small molecules were designed and synthesized. Structure-activity relationships indicated that the hydrophobic tail chain length is crucial for the antibacterial activity. In vitro bioassay demonstrated that compound 3g exerted potent antibacterial activity (MIC = 1 μg/mL and MIC = 2 μg/mL) while also exhibiting low hemolytic activity (HC > 1000 μg/mL), capability for rapid and complete bacterial sterilization, a longer post-antibiotic effect (PAE), and hardly induce bacterial resistance. Encouragingly, when 3g was used to combat persisters and biofilms in vitro, it demonstrated powerful bactericidal effects. Mechanistic studies indicated that 3g exerted its antimicrobial action through related membrane activity and was associated with membrane components phosphatidylglycerol (PG) and cardiolipin (CL), inducing content leakage, and ultimately causing bacterial death. Notably, 3g demonstrates superior in vivo antibacterial activity compared to vancomycin. Overall, these findings deliver important insights into the development of amphiphilic antimicrobial peptide mimics while validating the potential of 3g as an effective antimicrobial.