In this grant application we propose to investigate the syntheses and antifolate activities of two analogs of the vitamin, folic acid, which are altered in the pteridine ring system. Specifically, we will replace the nitrogen atom at the 8th position of folic acid with an oxygen. This compound, 8-oxa-dihydrofolic acid, is an analog of 7, 8-dihydrofolic acid. We will also replace the pterdine ring of folic acid with a dihyropyrimido-diazepine ring system by expanding the pyrazine ring of the pteridine moiety by introducing a methylene group between carbon #6 and 7. These two compounds are envisioned to be synthetic substrates of dihydrofolate reductase. We expect that these enzymatic reduction products might interfere with tetrahydrofolate utilization or inhibit the enzyme thymidylate synthase. In this regard, the dihydro derivatives of these two analogs (analogs of tetrahydrofolate) will be investigated as potential inhibitors of thymidylate synthase in vitro. In addition, their antifolate activities will be examined with the use of folate requiring microorganisms and mammalian cells in culture.