Methotrexate analogs. 19. Replacement of the glutamate side-chain in classical antifolates by L-homocysteic acid and L-cysteic acid: effect on enzyme inhibition and antitumor activity

Abstract

Methotrexate (MTX) and aminopterin (AMT) analogs containing L-homocysteic acid or L-cysteic acid in place of L-glutamic acid were synthesized and tested as inhibitors of dihydrofolate reductase from L1210 cells and folyl polyglutamate synthetase from mouse liver. The ID50 against dihydrofolate reductase was comparable for the MTX and AMT analogs (0.04-0.07 .mu.M), whereas the ID50 against folyl polyglutamate synthetase was 3- to 4-fold (over for the AMT analogs (40-60 .mu.M) than for the MTX analogs (100-200 .mu.M). Thus, N10-substitution has a greater effect on binding to folyl polyglutamate synthetase than dihydrofolate reductase. The cytotoxicity of these compounds was assayed in vitro against L1210 cells, and the AMT analogs again proved more potent (ID50 = 0.03-0.05 .mu.M) than the MTX analogs (ID50 = 0.1-0.4 .mu.M). A similarly increased potency was observed for the AMT analogs against L1210 leukemia in vivo. Though differential cell uptake cannot be ruled out as the basis of increased potency, it is possible that part of the activity of the AMT analogs involves interference with the intracellular polyglutamation of reduced folate cofactors, i.e., that they are self-potentiating antifolates. Of the 4 compounds reported, the most active was N-(4-amino-4-deoxypteroyl)-L-homocysteic acid, which reduced a 138% increase in life span (ILS) in L1210 leukemic mice when given on a modified bid times .times. 10 schedule at a dose of 2 mg/kg. A comparable ILS was obtained with AMT itself at 0.24 mg/kg. Thus, replacement of .gamma.-CO2H by .gamma.-SO3H in the side chain does not decrease therapeutic effect. However, a higher dose is required, presumably to offset pharmacological differences reflecting the inability of the sulfonate group to be polyglutamated.