Exploring Novel Tetrahydropyrimidine and Fused Pyrimidine‐Based Compounds as DHFR Inhibitors and Antimicrobial Agents: Synthesis, In Vitro, In Vivo, and In Silico Studies

New series of 2-imino/oxo-tetrahydropyrimidines (4a-4j), and fused pyrimidines (5a-5i and 6a-6h) were designed and synthesized as attractive scaffolds to be investigated in vitro and in vivo for antimicrobial activity against gram-positive Staphylococcus aureus, gram-negative Escherichia coli and Klebsiella pneumoniae, and fungus Candida albicans. In the in vitro antimicrobial screening using agar diffusion method, compounds 4 d, 4 f, 6a and 6 d showed broad-spectrum antimicrobial activity against all the tested strains when compared to levofloxacin as a reference drug. Moreover, compound 4 f showed higher antibacterial activity against all the tested microorganisms with MIC = 22-45 µM compared with levofloxacin with MIC = 50- > 708 µM. Compound 5 g exhibited lower IC₅₀ than that of reference trimethoprim (TMP) towards the DHFR enzyme inhibition. Additionally, compounds 4 d, 4 f, 4 g, 6 d and 6 f had kept the superiority over the reference drug with IC₅₀ ranging from 4.10 to 4.77 µM. Compounds 4 f and 6a were subjected to in vivo evaluation for their antibacterial activity. They caused a significant reduction in abscess volume and area in the skin of mice inoculated with S. aureus. Moreover, compound 4 f had reduced the immune-expression of interleukin-1β in the isolated tissues of the infected skin. Molecular docking results were in a good agreement with the DHFR enzyme assay results and justified the binding profiles and affinities profile of all tested compounds. Conclusively, compounds 4 d, 4 f, 5 g, 6a and 6 d are very promising candidates for further antimicrobial studies.