Synthesis and Biological Evaluation of dl-4,4-Difluoroglutamic Acid and dl-γ,γ-Difluoromethotrexate

dl-4,4-Difluoroglutamic acid (dl-4,4-F2Glu) and its methotrexate analogue, dl-γ,γ-difluoromethotrexate (dl-γ,γ-F2MTX), were synthesized and evaluated as alternate substrates or inhibitors of folate-dependent enzymes. Synthesis of dl-4,4-F2Glu involved the nitroaldol reaction of ethyl nitroacetate with a difluorinated aldehyde ethyl hemiacetal as a key step. Attempted ligation of dl-4,4-F2Glu to methotrexate (MTX), catalyzed by human folylpoly-γ-glutamate synthetase (FPGS), revealed that dl-4,4-F2Glu is a poor alternate substrate. dl-γ,γ-F2MTX was synthesized by a route proceeding through N-[4-(methylamino)benzoyl]-4,4-difluoroglutamic acid di-tert-butyl ester followed by alkylation with 6-(bromomethyl)-2,4-pteridinediamine hydrobromide. dl-γ,γ-F2MTX was found to be neither a substrate nor an inhibitor of human FPGS. The fluorinated analogue of MTX, however, inhibits DHFR and cell growth with the same potency as MTX.