. The overall goal of this continuation project is the discovery of new agents against Pneumocystis carinii and Toxoplasma gondii, two of the opportunistic pathogens known to be major causes of morbidity and mortality in patients with acquired immunodeficiency syndrome (AIDS) and AIDS related complex (ARC). More specifically, the project will focus on the design and synthesis of di and tricyclic diaminopyrimidine derivatives that are expected to have not only the high potency of trimetrexate (TMQ) and piritrexim (PTX) but also the favorable binding selectivity of tremethoprim (TMP) and pyrimethamine (PM) for P. carinii and T. gondii dihydrofolate reductase (DHFR) as opposed to mammalian DHFR. Compounds proposed for synthesis include six general types of 6/6 and 6/5 fused diaminopyrimidine systems, with emphasis being placed initially on compounds with a short bridge (no more than two atoms) and either 3,4,5 trimethoxy substitution (as in TMQ and TMP) or 2,5 dimethoxy substitution (as in PTX). The synthetic schemes are general enough to allow preparation of congeners with other ring substituents, including for example halogens. Target compounds will be evaluated for the ability to inhibit DHFR from P. carinii, T. gondii, and mammalian (rat liver) cells, and those that show a promising selectivity and potency will be tested as inhibitors or human DHFR and the growth of CCRF CEM human lymphoblasts. Compounds may also be tested, when appropriate, for the ability to inhibit P. carinii and T. gondii growth in feeder cultures of rat lung fibroblasts. Compounds with sufficient activity and selectivity in these in vitro assays to justify further preclinical development will be identified as candidates for scaled up synthesis, so as to eventually enable in vivo pharmacological and toxicological studies in mice and other animals to be carried out. The in vitro studies using P. carinii, T. gondii, and rat liver DHFR, as well as the assays using intact parasites in culture, will continue to be done collaboratively with Dr. Sherry Queener, of the Department of Pharmacology & Toxicology, Indiana University School of Medicine. The in vitro assays using human DHFR and human cells in culture will be performed at the Dana Farber as part of the applicant's other currently funded work in the antifolate area.
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