Chapter 9
Fluoroamino Acid Containing Analogues of Folic Acid and Methotrexate 1
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Takashi Tsukamoto , James K. Coward , and John J . McGuire Biomedical Frontiers of Fluorine Chemistry Downloaded from pubs.acs.org by COLUMBIA UNIV on 12/05/18. For personal use only.
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Departments of Chemistry and Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109-1055 Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, NY 14263
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Various fluorinated analogues of folic acid and methotrexate have been synthesized fromfluorine-containingglutamic acids, ornithine, and dipeptides. These analogues were evaluated as substrates or inhibitors of three folate-dependent enzymes, folylpoly-γ-glutamate synthetase (FPGS), γ-glutamyl hydrolase (γ-GH) and dihydrofolate reductase (DHFR), and also for their effects on intact mammalian cells; e.g., transport and cytotoxicity. The most marked effects of fluorine substitution were observed with FPGS and γ-GH which catalyze the ligation or hydrolysis of glutamyl moieties, respectively. In the biosynthesis of amino acids and nucleotides in mammalian cells and tissues, several derivatives of tetrahydrofolic acid (THF, lUPteGlu), the metabolically significant forms of the vitamin folic acid (FA, PteGlu), act as carriers of one-carbon units. Several antifolate drugs have as their primary target dihydrofolate reductase (DHFR). For example, methotrexate (MTX, AMPteGlu), one of the most potent inhibitors of DHFR, is extremely cytotoxic and is used extensively as a chemotherapeutic anticancer agent. Dihydrofolic acid (DHF, H2PteGlu) is the oxidation product arising from reductive methylation of deoxyuridylate to thymidylate. DHFR catalyzes the reduction of H2PteGlu to tUPteGlu, a reaction that is required to maintain thymidylate biosynthesis. Thus, the inhibition of the enzyme causes a deficiency in thymidylate, ultimately leading to cell death. The reduced folates are present in cells as poly-y-glutamate conjugates. These polyglutamates, the physiological substrates for folate-dependent enzymes (7,2), are polyanionic and do not readily cross the cell membrane, and thus are also involved in cellular retention of folates. Folylpoly-y-glutamate synthetase (FPGS) is responsible for the biosynthesis of poly-y-glutamyl conjugates of folates (Scheme I). In contrast, γ-glutamyl hydrolase (γ-GH) catalyzes the hydrolysis of poly^glutamates, resulting in regeneration of a pteroylmonoglutamate. Cells are believed to maintain a balance of the poly^-glutamates through the activities of these two enzymes. Interestingly both enzymes utilize not only naturally occurring folates but also antifolates such as MTX (3). In order to distinguish the two glutamate species involved in the FPGS reaction, the terminal glutamate residue of the folate substrate is referred to as the 'accepting' glutamate. Similarly, the glutamate to be incorporated in the growing folate polyglutamate is defined as die 'incoming' glutamate. In our studies of FPGS and γ-GH, fluorine-containing glutamic acids have been essential materials from two perspectives. The fluorinated amino acids can be evaluated as incoming substrates 0097-6156/96/0639-0118$15.00/0 © 1996 American Chemical Society
9. TSUKAMOTO ET AL.
Analogues of Folic Acid and Methotrexate
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9 Η C0 H N-^^-C-N' 2
CQ H, 2
Pte (Pteroyl) L-Glu Folic acid (FA = Re- L-Glu) P
^JT\
/ °2
H
C
H
9 ^-o-N>-
