Design and Synthesis of Novel Pyrazole-1,3,4-thiadiazine-Isoxazole Hybrids as Potent Antibacterial and Antibiofilm Agents: An Integrated In Vitro and In Silico Study

Abstract

This study aimed to develop and synthesize new pyrazole-1,3,4- thiadiazine-linked isoxazole derivatives and assess their efficacy as antibacterial and antibiofilm agents. The synthesized compounds were characterized and assessed for their in vitro antibacterial activity against Bacillus subtilis, Staphylococcus aureus, and Staphylococcus epidermidis. In addition, antibiofilm assays were conducted, and the findings were supported by in silico molecular docking studies targeting penicillin-binding proteins. (PDB: 1MWT). Several compounds exhibited significant antibacterial activity, with 6i, 6n, and 6o demonstrating particularly strong activities. These compounds achieved minimum inhibitory concentrations (MICs) of 1.56 ± 0.12 and 6.25 ± 0.34 μg/mL) against Bacillus subtilis, Staphylococcus aureus, and Staphylococcus epidermidis. Compound 6n was notably effective in reducing biofilm formation in all the examined strains. Molecular docking studies indicated that these potent compounds had superior binding affinities compared to the reference, primarily owing to key interactions with the THR600 residue of the target protein. The increased biological activity is attributed to the advantageous structural features of the pyrazole-thiadiazine-isoxazole framework, which facilitates efficient target interaction and biofilm inhibition in bacteria. This study identified pyrazole-1,3,4-thiadiazine-linked isoxazole derivatives, specifically 6n and 6o, as potential candidates for the development of novel antibacterial and antibiofilm drugs.