Design, synthesis and antitubercular evaluation of novel 5,6-diphenyl-1,2,4-triazine-piperazine derivatives targeting mycobacterial dihydrofolate reductase

Background and purpose: Tuberculosis (TB), a serious global health concern, continues to contribute to the global health burden, underscoring the urgent need to discover potent antitubercular agents. In the present study, a series of novel 5,6-diphenyl-1,2,4-triazine-piperazine derivatives were synthesized, and their potential for anti-tubercular activity was assessed. Experimental approach: The antitubercular potential of the synthesized compounds was assessed using the microplate alamar blue assay (MABA). Cytotoxicity studies were carried out on RAW 264.7 macrophages. To assess the mode of action of the synthesized compounds, docking analysis of the active compounds was performed on two key enzymes of Mycobacterium, decaprenylphosphoryl-β-D-ribose 2′-epimerase and Mycobacterium tuberculosis-dihydro­folate reductase (Mtb-DHFR). Furthermore, molecular dynamics simulations and density functional theory calculations were performed to validate the stability of the ligand–protein complexes and the electronic properties of the lead compounds. Key results: The results revealed promising anti-tubercular activity for the synthesized compounds. Among these, compounds 2-((5,6-Diphenyl-1,2,4-triazin-3-yl)thio)--1-(4-(4-fluoro­benzoyl)pi­perazin-1-yl)ethan-1-one (FP3) and 2-((5,6-diphenyl-1,2,4-triazin-3-yl)thio)-1-(4-(4-methylbe­nzoyl) piperazin-1-yl)ethan-1-one (FP8) exhibited potent activity with an minimum inhibitory concentration of 1.6 µg mL⁻¹, along with promising cytotoxicity profiles against RAW 264.7 macrophage cells. Docking studies revealed potent docking scores compared to the standard, thereby confirming the involvement of the DHFR enzyme. Interaction analysis revealed stable hydrogen-bond interactions, π-π stacking, and hydrophobic interactions with the active-site residues of the DHFR enzyme. Additionally, molecular dynamics simulation validated the stability of the interactions and the structural integrity of the complexes, while density functional theory calculations supported the desirable electronic properties of the lead compounds. Conclusion: The combined results of the experiments and calculations validated the potential of FP3 and FP8 as DHFR-targeted antitubercular agents. The study highlights the potential of 5,6-diphenyl-1,2,4-triazine-piperazine derivatives as promising candidates for further anti-tubercular drug development.