A series of heretofore unknown lipophilic bisamide derivatives (la-m) of the antitumor agent methotrexate (MTX) was synthesized from MTX diethyl ester (2) by reaction with various amines. The amines were used in large excess, generally without solvent, at temperatures ranging from 50 to 100 °C and for periods of 24-72 h. Yields were in excess of 60% in most instances, and the products were stable and easily purified. The MTX bisamides proved significantly less active than MTX or MTX esters against human lymphoblastic leukemia (CCRF-CEM) cells in vitro (IDso > 1.0 lg/mL vs. <0.05 pg/mL). However, some enhancement of activity was observed in two instances against rat basophilic leukemia (RBL) cells, which are myeloid rather than lymphoid in character. The bis(npropylamide) lb was inactive in vivo against L1210 mouse leukemia even at doses of 525 mg/kg (q3d 1,4,7), but the bis(benzy1amide) lh gave a +77% increase in median survival at 100 mg/kg (q3d 1,4,7). Neither compound was cleaved even after 24 h of incubation in whole rat serum at 37 °C, but the bis(benzy1amide) lh was cleaved substantially in vivo, as evidenced by the detection of free MTX in the liver and plasma of mice 6 and 24 h after treatment with a single 100 mg/kg dose of "prodrug". These results suggest that MTX bis(benzy1amide) (lh) may owe its in vivo activity against L1210 leukemia to the release of free MTX at sites other than the serum.