Methotrexate analogues—XVII: Antitumor activity of 4-amino-4-deoxy- N10-methylpteroyl- d, l-homocysteic acid and its dual inhibition of dihydrofolate reductase and folyl polyglutamate synthetase

Abstract

A new analogue of methotrexate was synthesized from 4-amino-4-deoxy- N 10-methylpteroic acid and d, l-homocysteic acid. The product (mAPA-HCysA) was bound tightly to L1210 mouse leukemia dihydrofolate reductase ( ic 50 = 1 nM ), inhibited L1210 cell proliferation in culture ( ic 50 = 0.3 μ M ), and prolonged the survival of L1210 leukemic mice (98% increase in lifespan at 120 frso|mg/kg, qdx9). Studies on the interaction of mAPA-HCysA with partially purified mouse liver folyl polyglutamate synthetase revealed that mAPA-HCysA was not a substrate. Hence, the increased dose of mAPA-HCysA required to inhibit tumor growth in vitro and in vivo relative to methotrexate may reflect, in part, the inability of this compound to form non-effluxing polyglutamates. Folyl polyglutamate synthetase was competitively inhibited by mAPA-HCysA ( K i = 190 ± 70 μM) when folate was the variable substrate. Thus, mAPA-HCysA is the first known compound to inhibit both mammalian dihydrofolate reductase and mammalian folyl polyglutamate synthetase.