Structure-based design of selective inhibitors of dihydrofolate reductase:Synthesis and antiparasitic activity of 2,4-diaminopteridine analogues with a bridged diarylamine side chain

As part of a larger search for potent as well as selective inhibitors of di hydrofolate reductase (DHFR) enzymes from opportunistic pathogens found in patients with AIDS and other immune disorders, N-[(2,4-diaminopteridin-6-yl )methyl] dibenz [b,f]azepine (4a) and the corresponding dihydrodibenz[b,f]a zepine, dihydroacridine, phenoxazine, phenothiazine, carbazole, and dipheny lamine analogues were synthesized from 2,4-diamino-6-(bromomethyl)pteridine in 50-75% yield by reaction with the sodium salts of the amines in dry tet rahydrofuran at room temperature. The products were tested for the ability to inhibit DHFR from Pneumocystis carinii (pcDHFR), Toxoplasma gondii (tgDH FR), Mycobacterium avium (maDHFR), and rat liver (rlDHFR). The member of th e series with the best combination of potency and species selectivity was 4 a, with IC50 values against the four enzymes of 0.21, 0.043, 0.012, and 4.4 mu M, respectively. The dihydroacridine, phenothiazine, and carbazole anal ogues were also potent, but nonselective. Of the compounds tested, 4a was t he only one to successfully combine the potency of trimetrexate with the se lectivity of trimethoprim. Molecular docking simulations using published 3D structural coordinates for the crystalline ternary complexes of pcDHFR and hDHFR suggested a possible structural interpretation for the binding selec tivity of 4a and the lack of selectivity of the other compounds. According to this model, 4a is selective because of a unique propensity of the seven- membered ring in the dibenz[b,f]azepine moiety to adopt a puckered orientat ion that allows it to fit more comfortably into the active site of the P. c arinii enzyme than into the active site of the human enzyme. Compound 4a wa s also evaluated for the ability to be taken up into, and retard the growth of, P. carinii and T. gondii in culture. The IC50 of 4a against P. carinii trophozoites after 7 days of continuous drug treatment was 1.9 mu M as com pared with previously observed IC50 values of >340 mu M for trimethoprim an d 0.27 mu M for trimetrexate. In an assay involving [H-3]uracil incorporati on into the nuclear DNA of T. gondii tachyzoites as the surrogate endpoint for growth, the IC50 Of 4a after 5 h of drug exposure was 0.077 mu M. The f avorable combination of potency and enzyme selectivity shown by 4a suggests that this novel structure may be an interesting lead for structure-activit y optimization.