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 IC 50 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 IC 50 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.