Conclusion

In conclusion, the current study aims to report the successful design, synthesis, and extensive biological and computational investigation of a series of phenyl-substituted analogues as prospective anti-tubercular agents. The in vitro screening against M. tuberculosis H37Rv has led to the identification of promising inhibitory activities, among which FP3 and FP8 were identified as the most potent analogues, possessing low micromolar MIC values. The structure-activity relationship has been established, showing that small hydrophobic substituents on the phenyl ring significantly increase anti-tubercular activity, whereas bulky halogens and highly electron-donating substituents have a detrimental effect on activity. Molecular docking was carried out against both DprE1 and Mtb-DHFR, proving that the synthesized compounds have a consistently higher affinity towards Mtb-DHFR, surpassing even the reference drug methotrexate, thus identifying DHFR as the most prospective molecular target. The detailed interaction analysis has been carried out, demonstrating that the active compounds bound effectively within the DHFR binding pocket via hydrogen bonding, π-π stacking, and hydrophobic interactions with key catalytic residues. Finally, molecular dynamics simulations were carried out on the most active complexes, demonstrating the structural stability of the ligand-protein complexes, as evidenced by stable RMSD and RMSF profiles and the maintenance of key intermolecular interactions throughout the simulation. Complementary DFT calculations were carried out, providing valuable insight into the electronic properties of the lead compounds, thus proving their highly favourable reactivity profiles, consistent with their biological activities.