Synthesis, characterization, and evaluation of novel diphenylpyrazole-based hydrazine-1-carboximidamides as broad-spectrum antibacterial leads with antibiofilm and virulence-modulating activities

Abstract

The emergence of multidrug-resistant bacterial pathogens and biofilm-associated infections highlights the urgent need for antibacterial agents with novel chemical architectures and complementary modes of action. In the present study, a new series of diphenylpyrazole-based hydrazine-1-carboximidamide derivatives (5a–i) was designed, synthesized, and evaluated as potential antibacterial and antibiofilm agents. The target compounds were obtained through a three-step synthetic sequence involving phenylhydrazone formation, Vilsmeier–Haack cyclization/formylation, and subsequent condensation with aminoguanidine bicarbonate. Structural characterization was established using spectroscopic and mass spectrometric techniques. The synthesized derivatives exhibited variable antibacterial activity against representative Gram-positive and Gram-negative bacteria, demonstrating a pronounced structure-dependent response. Among the series, compound 5g bearing a nitrophenyl substituent emerged as the most promising derivative, displaying MIC values of 8, 4, 4, and 16 µg mL−1 against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa, respectively. Compound 5g also showed substantial antibiofilm activity at 0.5 × MIC, with inhibition values of 75.95%, 53.33%, 77.57%, and 63.73% against the corresponding bacterial strains. Time-kill studies demonstrated a concentration- and time-dependent bactericidal effect against E. coli ATCC 25922, while potassium leakage and scanning electron microscopy provided complementary evidence of bacterial membrane perturbation and cellular-envelope damage. Furthermore, compound 5g exhibited promising anti-quorum-sensing activity against P. aeruginosa, significantly reducing pyocyanin production by 26.80% and 54.33% at ¼ and ½ MICs, respectively. Importantly, 5g exhibited low cytotoxicity toward WI-38 normal human cells (IC50 = 255 ± 22.1 µg mL−1) and a favorable selectivity index of 32. Collectively, these findings identify diphenylpyrazole carboximidamide 5g as a promising multifunctional antibacterial lead with direct antibacterial, antibiofilm, membrane-perturbing, and anti-quorum-sensing activities, warranting further optimization and mechanistic investigation.