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 ana logue, DL-gamma,gamma-difluoromethotrexate (DL-gamma,gamma-F(2)MTX), w ere synthesized and evaluated as alternate substrates or Inhibitors of folate-dependent enzymes. Synthesis of DL-4,4-F(2)Glu involved the ni troaldol reaction of ethyl nitroacetate with a difluorinated aldehyde ethyl hemiacetal as a key step. Attempted ligation of DL-4,4-F(2)Glu t o methotrexate (MTX), catalyzed by human folylpoly-gamma-glutamate syn thetase (FPGS), revealed that DL-4,4-F(2)Glu is a poor alternate subst rate. DL-gamma,gamma-F(2)MTX was synthesized by a route proceeding thr ough N-[4-(methylamino)benzoyl]-4,4-dinuoroglutamic acid di-tert-butyl ester followed by alkylation with 6-(bromomethyl)-2,4-pteridinediamin e hydrobromide. DL-gamma,gamma-F(2)MTX was found to be neither a subst rate nor an inhibitor of human FPGS. The fluorinated analogue of MTX, however, inhibits DHFR and cell growth with the same potency as MTX.