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 potential antifolates are designed with the aim of exploiting differences in two biochemical parameters of antifolate action in tumors and normal proliferative tissue. These differences, which appear to be determining factors for selective antitumor activity, are in cellular membrane transport and in intracellular polyglutamylation. Earlier biochemical studies identified the 5 and 10 positions of the classical antifolate molecular structure as sites where modifications affect transport and polyglutamylation. During the current program, we began use of computer- aided drug design (molecular modelling) based on the three-dimensional structure of the antifolate target enzyme dihydrofolate reductase (DHFR) determined by X-ray crystallography. Our plan is to combine biochemical- structure activity information on tissue specificity of antifolates with molecular modelling and organic synthesis to produce improved antitumor folate analogues of greater selectivity. The multiple effects of antifolates and their intracellular polyglutamate metabolites on purine and thymidylate synthesis also influence our planning.
Our specific aims are to (a) identify improved novel antifolates through modelling, synthesis, and testing of MTX/AMT analogues in both the pteridine and 5-deazapteridine series bearing side chains modified throughout depending on the molding to the DHFR cavity; (b) to convert selected MTX/AMT analogues to folic acid analogues (2-amino-4-oxo types) which might inhibit folate-utilizing enzymes other than DHFR (such as TS, GAR TFase or AICAR TFase); (c) to extend (with the aid of molecular modelling) recent findings by other investigators on the antitumor properties of 2,4-diaminopyrrolo[2,3-d]pyrimidine antifolates by preparing analogues with modified side chains which might favorably influence cell membrane transport, DHFR inhibition and intracellular polyglutamylation toward greater selectivity; (d) to study 7-substituted guanines as possible inhibitors of folate metabolism at sites other than DHFR; (e) to supply these compounds to our biological collaborator, Dr. F. M. Sirotnak of Memorial Sloan-Kettering Cancer Center, in order to have these compounds evaluated in preclinical test systems that will determine their efficacy. The testing of these candidates will be done in biological and biochemical test systems appropriate for their 'design systems to define their antifolate activity, their cellular pharmacology and their efficacy as antitumor agents. Results from these tests will guide the program.
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