Biocompatible Guanidine-Functionalized Compounds with Biofilm and Membrane Disruptive Activity Against MRSA

Three guanidine-functionalized 3,4-dihydropyrimidin-2(1<i>H</i>)-imine compounds (<b>5a</b>, <b>5b</b>, <b>5c</b>) were synthesized from 3,5-diaryldiene-4-piperidone and evaluated for antibacterial and antibiofilm activity against <i>Staphylococcus aureus</i>, CA-MRSA and HA-MRSA. The compounds showed bacteriostatic effects (MICs: 2.34-4.68 μg/mL). In vitro antibiofilm potential was demonstrated by significant reductions in biomass and metabolic activity, and structural analyses via SEM and fluorescence microscopy. Ex vivo antibiofilm activity was confirmed in porcine skin model. RT-qPCR revealed downregulation of biofilm associated virulence genes, indicating a multifactorial mechanism. Confocal microscopy showed increased levels of extracellular DNA and proteins, suggesting disruption of the biofilm matrix. Membrane interaction assays demonstrated time- and dose-dependent effects, suggesting a complementary mechanism of action. Compounds <b>5a</b> and <b>5c</b> exhibited synergistic and additive effects with oxacillin. The compounds were stable intracellularly, and resistance studies revealed low induction potential. Biocompatibility was confirmed by lack of mutagenicity, hemolysis, or cytotoxicity. Moreover, in vivo efficacy was demonstrated by survival of <i>Tenebrio molitor</i> larvae infected with <i>S. aureus</i> and treated. These guanidine-based compounds are promising candidates for new MRSA drug development.