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)