Pyridine-3-Carboxamide–1,3,4-Thiadiazole Hybrids as Antimicrobial Agents: Synthesis, Biological Evaluation, In Vivo Toxicity, and Computational Studies

The rapid emergence of antimicrobial resistance necessitates the development of novel antimicrobial agents with improved efficacy and favorable safety profiles. In this study, a new series of pyridine-3-carboxamide–1,3,4-thiadiazole hybrids and their corresponding N-Mannich bases bearing structurally diverse piperazine substituents were designed, synthesized, and characterized by FT-IR, 1H/13C NMR, and HR-MS. Their antimicrobial activity was evaluated against reference and clinical bacterial and fungal strains using broth microdilution and agar diffusion assays. The compounds exhibited selective activity against Gram-positive bacteria, with minimum inhibitory concentrations of 0.034–0.069 mg/mL against Staphylococcus aureus ATCC 33591 and bactericidal effects reflected by MBC values comparable to MICs. No activity was observed against Pseudomonas aeruginosa or Candida albicans, and the compounds were ineffective in eradicating preformed staphylococcal biofilms. In vivo toxicity studies using the Galleria mellonella model indicated no-to-low apparent acute toxicity under the applied experimental conditions for most derivatives. DFT and QTAIM analyses revealed substituent-dependent electronic and conformational differences, while molecular docking identified DNA gyrase as the most likely molecular target, with compounds 6 and 7 showing the strongest predicted binding. These results identify pyridine-3-carboxamide–1,3,4-thiadiazole Mannich bases as promising leads for developing new antibacterial agents against Gram-positive pathogens.