title: ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that is a potent inhibitor of L1210 tumor cell growth in vitro and in vivo: a new agent for clinical study.
abstract: N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2-thenoyl)-L-glutamic acid (ICI D1694) is a water-soluble, folate-based thymidylate synthase (TS) inhibitor designed to be a less toxic and more potent analogue of the clinically tested N10-propargyl-5,8-dideazafolic acid. Inhibition of isolated L1210 TS by ICI D1694 is mixed noncompetitive (although tending toward competitive), with a Ki of 62 nM (Kies = 960 nM). The synthetic gamma-polyglutamates are up to 2 orders of magnitude more potent as inhibitors of TS; e.g., the tetraglutamate (glu4) has a Ki of 1.0 nM (Kies = 15 nM). Although inhibitory activity of ICI D1694 toward rat liver dihydrofolate reductase was similar to that of TS (Ki = 92 nM; competitive inhibition) the polyglutamate derivatives did not show enhanced activity. ICI D1694 was also a very potent inhibitor of L1210 cell growth (50% inhibitory activity = 8 nM). L1210 growth inhibition was not observed in the presence of thymidine, consistent with TS being the locus of action. Folinic acid antagonized L1210 growth inhibition in a competitive fashion such that the highest folinic acid concentration used (25 microM) increased the 50% inhibitory activity 6000-fold. When given as a 4-h delayed "rescue", folinic acid was much less effective in antagonizing growth inhibition. These observations are consistent with folinic acid competing with ICI D1694 for uptake into the cell and/or intracellular polyglutamation. The L1210:1565 cell line, which has greatly impaired reduced-folate/methotrexate transport and thus is resistant to methotrexate, was significantly cross-resistant to ICI D1694 (121-fold), suggesting that ICI D1694 is dependent on this uptake mechanism for good cytotoxic potency in L1210 cells. L1210 cells that were incubated for 4 h with 0.1 microM 3H-ICI D1694 accumulated approximately 1.5 microM intracellular 3H, and the high performance liquid chromatography analysis of the cell extracts demonstrated that 96% of the 3H was associated with the ICI D1694 polyglutamate fractions (principally glu4). Upon resuspension in drug-free medium for 24 h, approximately 75% of the cellular 3H was retained, this being the higher polyglutamate pool (glu4-6). In mice, after a single bolus injection of 10 mg/kg of ICI D1694, TS was inhibited greater than 80% for 24 h in ascitic L1210:NCI cells (as measured by the rate of 3H release from [5-3H]deoxyuridine). ICI D1694 cured the L1210:ICR ascitic tumor in mice at 0.4 mg/kg daily for 5 days (maximum tolerated dose, approximately 50 mg/kg).(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Drug Development Section, Institute of Cancer Research, Sutton, Surrey, United Kingdom.
authors.forename: A L
authors.initials: AL
authors.lastname: Jackman
authors.forename: G A
authors.initials: GA
authors.lastname: Taylor
authors.forename: W
authors.initials: W
authors.lastname: Gibson
authors.forename: R
authors.initials: R
authors.lastname: Kimbell
authors.forename: M
authors.initials: M
authors.lastname: Brown
authors.forename: A H
authors.initials: AH
authors.lastname: Calvert
authors.forename: I R
authors.initials: IR
authors.lastname: Judson
authors.forename: L R
authors.initials: LR
authors.lastname: Hughes
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000494:pharmacology; D005971:Glutamates / Q000378:metabolism; D007939:Leukemia L1210 / Q000201:enzymology / Q000473:pathology*; D008297:Male; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D008811:Mice, Inbred DBA; D011799:Quinazolines / Q000378:metabolism / Q000494:pharmacology*; D011956:Receptors, Cell Surface; D013876:Thiophenes / Q000378:metabolism / Q000494:pharmacology*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*
chemical_list: D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D005971:Glutamates; D011799:Quinazolines; D011956:Receptors, Cell Surface; D013876:Thiophenes; D005492:Folic Acid; D013940:Thymidylate Synthase; C068874:raltitrexed; D008727:Methotrexate