Synthesis and Biological Activity of Folic Acid and Methotrexate Analogues Containing l-threo-(2S,4S)-4-Fluoroglutamic Acid and dl-3,3-Difluoroglutamic Acid

Abstract
The stereospecific syntheses of l-threo-γ-fluoromethotrexate (1 t) and l-threo-γ-fluorofolic acid (3 t) are reported. Compounds 1 t and 3 t have no substrate activity with folylpoly-γ-glutamate synthetase isolated from CCRF-CEM human leukemia cells, and compound 1 t inhibits human dihydrofolate reductase at similar levels as methotrexate. The synthesis of dl-3,3-difluoroglutamic acid (6) and its incorporation into dl-β,β-difluorofolic acid (4) are also reported. Compound 4 acts as a better substrate for human CCRF-CEM folylpoly-γ-glutamate synthetase than folic acid (V/K = ca. 7-fold greater). Thus, replacement of the glutamate moiety of methotrexate and folic acid with 4-fluoroglutamic acid and 3,3-difluoroglutamic acid results in folates and antifolates with altered polyglutamylation activity.

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