We report here the design, synthesis, DHFR affinity, and X-ray crystallographic binding analysis of a series of 3'-carboxyalkoxy analogues of trimethoprim (TMP). Using the unrefined three-dimensional structure of E. coli dihydrofolate reductase (DHFR)-methotrexate (MTX) complex, we aimed to create TMP analogues with higher affinity for E. coli DHFR and investigate binding interactions. Synthesized compounds 3-13 showed that acids 5-8 had significantly higher affinity than TMP and corresponding esters 10-13, implying ionic interactions with DHFR's basic residues. X-ray crystallography of compounds 4 and 7 confirmed that their carboxy groups interacted with Arg-57. In summary, we used DHFR models to design TMP analogues with higher affinity, verified the binding mode via X-ray crystallography, and demonstrated the potential of this structure-based inhibitor design approach, though these analogues were less effective as broad-spectrum antibacterials. Additionally, the present communication reports on the hypotensive activities of 2-[(2,3,6-trichlorophenyl)imino]imidazolidine (2) and 2-[(2,3-dichloro-6-methylphenyl)imino]imidazolidine (3), which were synthesized based on structure-hypotensive activity studies of clonidine, demonstrating the potential of this newly substituted class of centrally acting, hypotensive imidazolidine compounds.