The object of this application is the synthesis of analogues and homologues of trimethoprim (TMP) and its P. carinii dihydrofolate reductase (DHFR) selective analogue epiroprim as potent and selective inhibitors of DHFR from P. carinii (pc), T. gondii (tg) and M. avium (ma) and as potential clinical candidates for the treatment of often fatal infections caused by these organisms in patients with acquired immunodeficiency syndrome (AIDS). The target compounds were designed using molecular modeling based on the X-ray crystal structure of TMP and piritrexim (PTX) with P. carinii DHFR and as hybrid molecules of TMP and epiroprim [which are selective but poor inhibitors of DHFR and trimetrexate (TMQ)] and PTX (which are potent but nonselective inhibitors of DHFR), in an attempt to preserve both selectivity and potency in single drug entities for pcDHFR, tgDHFR and/or maDHFR. Compounds recently synthesized in the P.I.'s laboratory support this hypothesis. The compound syntheses are proposed via suitable modifications of procedures carried out by the P.I. and via established literature methods. Initially, two analogues of each series (total of 18 compounds) will be synthesized and evaluated as inhibitors of pcDHFR, tgDHFR, and maDHFR, with rat liver (rl)DHFR and human (h)DHFR serving as mammalian reference enzymes to determine selectivity. Depending upon the biological results of these initial analogues other compounds in the same series will be synthesized or not synthesized. Thus, the biological activity obtained will drive the synthetic targets. Based on the enzyme inhibitory studies, selected analogues will be evaluated against P. carinii and T. gondii cells in culture and against human cells for cytotoxicity and subsequently in whole animal (murine) models of P. carinii and T. gondii. These biological evaluations will be carried out on a collaborative basis. In addition, X-ray crystal structures of selected analogues with DHFRs will be determined on a collaborative basis. The results of this study will provide a molecular basis for selectivity and potency in single drug entities as it pertains to DHFR binding to pcDHFR, tgDHFR, and maDHFR. Further, these compounds may provide selective, less toxic, clinically useful agents against P. carinii, T. gondii, and M. avium infections in AIDS patients. Selected analogues will also be submitted to the in vitro preclinical tumor cell culture program of the National Cancer Institute.
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