Mammalian folypoly-gamma-glutamate synthetase (FPGS) converts folylmonoglutamates, the transport form of folates, into their polyglutamate congeners by the successive addition of glutamic acid monomers using, ATP a source of energy. The underlying thesis of this work is that inhibitors of FPGS will be a unique new class of antimetabolites that will be useful as cancer chemotherapeutic drugs. The folylpolyglutamates are retained in cells, in contrast to the effluxable monoglutamates, and are the preferred form of cofactor for the folate-dependent enzymes. FPGS has been shown to be an essential function for a dividing cell population. The goal of this proposal is to design and synthesize potent and selective inhibitors of mammalian FPGS and to study the cellular and biochemical effects of this unique new class of folate antimetabolites. The synthesis of some folate analogs predicted to be analogs of the transition states and of the intermediate product of the FPGS reaction will be undertaken and the interaction of these compounds with purified FPGS will be studied. Second generation analogs of the potent FPGS inhibitor 4-amino-4- deoxypteroyl-L-ornithine will be prepared that penetrate the cell membrane efficiently or that are prodrugs converted to inhibitors in the cell. Other compounds to be made would be more potent analogs of this lead compound or would have no activity as dihydrofolate reductase. Finally, the interaction of the side chain of folate analogs with the active site of FPGS would be further investigated. The effects of these new compounds will be studied on purified mammalian FPGS and on the function of FPGS in whole cells. Inhibition of folate metabolism by existing antagonists of folate metabolism (which, until recently, have been uniformly dihydrofolate reductase inhibitors) has been very useful for treatment of human neoplasms. Antifolates are active against a spectrum of malignancies arising in different tissues, have managable toxicities, have low or non-existent mutagenic and carcinogenic threat and are reversible by reduced folates. We propose that inhibitors of FPGS will share these beneficial characteristics, yet will have a mechanism distinct from that of classical antifolates such as methotrexate.