TARGETING DIHYDROFOLATE REDUCTASE (DHFR) ENZYME: SYNTHESIS, IN-VITRO BIOLOGICAL EVALUATION, AND MOLECULAR MODELLING OF NOVEL TETRAHYDROPYRIMIDINE AND DIHYDROPYRIDINE DERIVATIVES

The increasing prevalence of multidrug-resistant (MDR) pathogens has severely undermined the efficacy of conventional antibiotics, necessitating the development of novel antimicrobial agents. Dihydrofolate reductase (DHFR), a crucial enzyme in bacterial DNA synthesis, presents a validated target for the design of new antimicrobial therapies. In this study, a new series of pyrazolyl-pyrimidine and pyridine-based derivatives was synthesized and systematically evaluated for DHFR inhibition and antimicrobial activity. The compounds were screened against a panel of pathogenic microorganisms, including Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Candida albicans. Notably, compound 4c emerged as a lead molecule, displaying excellent antimicrobial potency with MIC values of 16 µg/mL against S. aureus and C. albicans. Additionally, it demonstrated a potent inhibitory effect on DHFR (IC50 = 5.53 ± 0.26 µM) and achieved a docking score of –12.114 kcal/mol, indicating strong binding affinity. Structure-activity relationship (SAR) analysis revealed that the presence of an imino group at position 2 and a small methyl group at position 6 conferred superior binding characteristics, while bulkier substitutions diminished efficacy due to steric hindrance. Among the pyridine-based analogs, compound 11 showed notable activity, particularly against K. pneumoniae (MIC = 32 µg/mL), with an IC50 of 5.60 ± 0.27 µM and a favorable docking profile. Molecular docking simulations highlighted the critical interactions within the DHFR active site and validated the observed in-vitro trends. Overall, compounds 4c and 11 represent promising scaffolds for the development of next-generation DHFR-targeting antimicrobial agents.