The goal of the proposed research is the identification of new agents that exert greater effectiveness in the antifolate therapy of human cancer than agents now available, particularly in regard to achieving a broader spectrum of antitumor response. The proposed work is concerned with design and synthesis of potential antifolates which will be subjected to biochemical-pharmacologic evaluation. Proposed syntheses are based mainly two premises. One premise concerns differences in the cell membranes transport systems that mediate folate analogue uptake, not only among different tumors but between tumor and drug-limiting normal proliferative tissue. The second premise involves findings that suggest effective nhibition of folylpolyglutamate synthetase could exert a regulatory effect on folate metabolism. Most of the proposed structures may be placed in two categories: (1) analogues of methotrexate (MTX) designed as potential inhibitors of dihydrofolate reductase and (2) analogues of folic acid designed as potential inhibitors of folylpolyglutamate synthetase. Structural variants in the methotrexate analogs are intended to alter transport without adversely affecting binding to dihydrofolate reductase or to effect tighter or irreversible binding to the reductase. The MTX analogues are of four types: (a) modifications of the MTX metabolite MTX-Lamdba-glu in which the peptide linkage has been replaced by functional grops stable to in vivo processes such that the analogue resembles MTX-Lambda-Glu but cannot be converted in vivo to MTX, (b) compounds in which the glutamate part of the side chain has been modified to include reactive groupings that might react irreversibly with the enzyme, (c) compounds modified in the C9-N10 bridge, and (d) compounds in which the pteridine ring is replaced by substituted pyrido [2, 3-b] pyrimidine ring (5-deazapteridine) bearing a 5-methyl substituent. The MTX analogs will be evaluated as inhibitors of dihyerofolate reductase, for cytotoxicty, and for their transport characteristics in cancer and gut epithelial cells. Compounds of interest will then be studied for in vivo activity in animal tumor systems. Further synthetic efforts will be guided by these results. All of the folic acid analogues proposed bear modified glutamate groupings; several have reactive groupings in the side chain positioned such that irreversible inhibition of the synthetase enzyme might result. Studies beyond the enzyme level will be guided by early results.
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