Synthesis and biological evaluation of DL-4,4-difluoroglutamic acid and DL-gamma,gamma-difluoromethotrexate

DL-4,4-Difluoroglutamic acid (DL-4,4-F(2)Glu) and its methotrexate analogue, DL-gamma,gamma-difluoromethotrexate (DL-gamma,gamma-F(2)MTX), were synthesized and evaluated as alternate substrates or Inhibitors of folate-dependent enzymes. Synthesis of DL-4,4-F(2)Glu involved the nitroaldol reaction of ethyl nitroacetate with a difluorinated aldehyde ethyl hemiacetal as a key step. Attempted ligation of DL-4,4-F(2)Glu to methotrexate (MTX), catalyzed by human folylpoly-gamma-glutamate synthetase (FPGS), revealed that DL-4,4-F(2)Glu is a poor alternate substrate. DL-gamma,gamma-F(2)MTX was synthesized by a route proceeding through N-[4-(methylamino)benzoyl]-4,4-dinuoroglutamic acid di-tert-butyl ester followed by alkylation with 6-(bromomethyl)-2,4-pteridinediamine hydrobromide. DL-gamma,gamma-F(2)MTX 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.