Synthesis, characterization, in vitro antimicrobial efficacy and cytotoxicity of substituted benzylidene-based pyrazole motifs

The need to discover new antimicrobial agents remains pressing, given the emerging and persistent resistance of microorganisms to existing drugs. This study investigated the synthesis, characterization, and biological evaluation of a series of benzylidene-based pyrazoles (BBP, 3a–i ) as potential antimicrobial agents. The precursors, benzylidene-based malononitriles (2a–i) , were synthesized by condensing malononitrile with substituted aromatic benzaldehydes and subsequently reacting with hydrazine hydrate to afford the target BBP compounds (3a-i) in diverse yields. Structural characterization was achieved using UV–Vis, FT-IR, 1 H NMR, 13 C NMR, and DEPT-135 spectroscopy. Vibrational frequencies around 3500–3200 (amine N H), 3145–3034, (aromatic C H), 2998–2814 (aliphatic C H), 1659–1595 cm −1 (C=C) and 1576–1528 cm −1 (C=N) confirmed synthesis, while singlet peaks at δ H 11.67–11.91 (2H singlet of 2(C=NH)) and δ H 4.09–4.35 (2H singlet of 2(NH)) further supported formation of the compounds (3a-i) . In vitro antimicrobial assays revealed that compound 3b exhibited the strongest antibacterial activity with a minimum inhibitory concentration (MIC) of 3.13 μg/mL against Staphylococcus aureus 25,923, Streptococcus pyogene , Enterococcus faecalis , Salmonella typhimurium , and Shigella sp. It also showed potential antifungal activity (MIC = 3.13 μg/mL) against Candida albicans and Aspergillus fumigatus . Cytotoxicity screening confirmed low toxicity across all compounds. In silico docking demonstrated favourable binding affinities (−6.599 to −5.692 kcal/mol), binding free energies (−32.56 to −22.3 kcal/mol), and good ADMET profiles. Molecular dynamics simulation confirmed structural stability, with 3b performing comparably to the reference ligand 629. Overall, compound 3b (4-(2-chlorobenzylidene) pyrazolidine-3,5-diimine) was identified as a promising antimicrobial drug candidate.