Novel Fluorinated Chlorhexidine Analogues Overcome Resistance in Gram-Negative Bacteria

Antimicrobial resistance (AMR) is a critical global health challenge that compromises the effectiveness of widely used biocides such as chlorhexidine (CHX). The emergence of CHX-resistant bacterial strains, often mediated by efflux mechanisms and membrane adaptations, necessitates the development of new chemical entities capable of overcoming these resistance pathways. Here, we report the rational design, synthesis, and biological evaluation of structurally modified CHX analogues. These analogues were designed by substituting the terminal chlorophenyl rings of CHX with strategically selected aromatic groups bearing fluorine and methyl substituents to enhance membrane permeability, modulate physicochemical properties, and reduce efflux susceptibility. A library of 13 compounds was prepared using biscyanoguanidine chemistry and aniline-based substitutions via microwave-assisted synthesis, and was fully characterized by LC–MS, HRMS, and NMR. Antibacterial activity was assessed using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays against a panel of Gram-positive and Gram-negative bacteria, including CHX-resistant Klebsiella pneumoniae and Pseudomonas aeruginosa strains harboring smvR, phoQ, and pmrB mutations. Selected analogues, particularly compounds 8 and 11, demonstrated potent antibacterial activity. Compound 11 showed MIC values predominantly in the range of 4–8 μg/mL across wild-type strains and retained activity in resistant isolates, with MIC values of 4–8 μg/mL in P. aeruginosa and 64 μg/mL in CHX-resistant K. pneumoniae. MBC values were generally comparable to MIC values for the fluorinated analogues, consistent with a biocidal mode of action, while chlorhexidine showed elevated MBC values in resistant strains. Molecular modeling suggests that these compounds form favorable interactions within hydrophobic regions of the SmvA efflux pump in K. pneumoniae, potentially reducing efflux susceptibility. Structure–activity relationship analysis highlights the importance of combining fluorine and methyl substituents to optimize physicochemical properties associated with antibacterial activity and resistance bypass. Collectively, these findings establish a foundation for the development of next-generation CHX-based biocides with improved efficacy against multidrug-resistant pathogens and support further translational evaluation.