Synthesis and In Vitro Biological Evaluation of Amphiphilic Derivatives of Carvacrol as Potent Antibacterial Agents.

BACKGROUND/OBJECTIVES: The rapid emergence and spread of antibiotic-resistant bacterial strains represent a major global health threat, requiring the development of novel broad-spectrum antimicrobial agents. This study aims to design, synthesize, and evaluate a novel series of multitarget amphiphilic carvacrol derivatives to combat molecular resistance mechanisms by combining a natural monoterpene with polycationic ribonuclease scaffolds. METHODS: A series of 16 dimeric carvacrol-1,4-diazabicyclo[2.2.2]octane (DABCO) hybrids, including both novel and previously reported derivatives, with varying central and lateral linkers were synthesized. In vitro antibacterial activity was evaluated against Gram-positive (S. aureus) and Gram-negative (E. coli, S. enterica, C. freundii, P. aeruginosa, A. baumannii) strains. Antibacterial action was examined through a time-kill kinetic assay against S. aureus at minimum inhibitory concentrations (MICs). RNA-hydrolyzing efficiency (using a FAM-labeled 17-mer ribooligonucleotide), hemolytic activity on rabbit red blood cells, and cytotoxicity (MTT assay) against RPMI8226, LMTK, and MEF cell lines were assessed. RESULTS: Several compounds exhibited potent antibacterial activity, with MIC values ranging from 4 to 63 µM against most strains. The most active hybrid, DL4CAR-6, demonstrated broad-spectrum efficacy (MIC 4-16 µM against most strains) and outperformed the reference amphiphile DL4-12 against P. aeruginosa eightfold. Bacterial growth inhibition kinetic assay revealed that the cationic amphiphiles eradicate S. aureus faster than the reference antibiotic, indicating rapid membrane disruption. Most hybrids demonstrated pronounced RNA-hydrolyzing activity (up to 60% cleavage). The hemolytic assay showed less than 10% hemolysis for most compounds even at 500 µM. Furthermore, DL4CAR-6 displayed low cytotoxicity (IC50 114-250 µM), with safety thresholds exceeding effective antibacterial doses. CONCLUSIONS: The newly developed carvacrol-DABCO amphiphilic hybrids represent a promising class of multitarget antibacterial agents. Their broad-spectrum efficacy, bactericidal kinetics, RNA-cleaving activity, low hemolytic toxicity, and favorable selectivity indices support their potential for further in vivo evaluation.