The object of this proposal is two fold. The first is to develop compounds (previously) identified as selective and potent inhibitors to provide more effective in vivo-active compounds in animal models of T. gondii and P. carinii as a prelude for clinical trials. This will be accomplished by the synthesis of multigram amounts of the potential drugs in various salt forms and will include preformulation and formulation studies to afford the most appropriate route of administration and doses, to be used in therapy studies along with relapse and toxicity studies in murine models. Two entities each in P. carinii and T. gondii will be tested each year. The second object of this proposal is the synthesis of analogues of compounds identified via an iterative process of synthesis, biological evaluation and X-ray crystal structure determinations (for P. carinii DHFR) as potent and selective inhibitors of dihydrofolate reductase (DHFR) from P. carinii (pc), T. gondii (tg) and M. avium (ma) and as potential clinical candidates for the treatment of infections caused by these organisms in patients with AIDS. The analogues in this study were designed on the crystal structures of previous compounds (pcDHFR) and their activity and selectivity against these DHFR, which showed them to be superior to TMP and epiroprim. The synthesis of the analogues are proposed via suitable modifications of procedures from our laboratory and by established literature methods. The analogues will be synthesized and evaluated as inhibitors of pcDHFR, tgDHFR and maDHFR with rat liver (rl) and human DHFR as reference enzymes. The biological activity and selectivity of initial 18 analogues will determine if other analogues in the series will be synthesized or not synthesized. Thus biological activity will determine the synthetic targets. Based on the enzyme studies selected analogues will be evaluated against P. carinii, T. gondii, M. avium and human foreskin fibroblast cells in culture (for cytotoxicity) and subsequently in whole animal models (mouse) of P. carinii and T. gondii. The biological evaluations and preformulation and formulation studies will be carried out on a collaborative basis. X-ray crystal structures of selected analogues with DHFRs will be determined collaboratively. This study should afford more effective in vivo active compounds and will also provide the molecular basis for selectivity and potency as it pertains to DHFR binding to pcDHFR, tgDHFR and maDHFR. Further, these compounds may provide selective, less toxic, clinically useful agents which overcome the drawbacks of currently used regimens against these opportunistic infections in patients with HIV. The analogues synthesized will also be submitted for screening against tuberculosis (NIAID) parasitic diseases (WHO) and tumor cells in culture (NCI).
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