The clonal cytotoxic effects and mechanism of action of a new series of 2-amino-4-hydroxyquinazoline
folate analogues (5,8-dideazafolates) have been assessed using the human colon tumor cell line HCT-8. Of these compounds only 5-methyl-5,8-dideazafolate was potentially more effective than a compound previously identified, 5,8-dideazaisofolate (H-338, NSC 289517). HCT-8 sublines resistant to
methotrexate,
5-fluorodeoxyuridine, and H-338 were either minimally or not cross-resistant to the other agents. The cytotoxicity of H-338 was strongly dependent on the time of exposure; at exposure times shorter than 8 h it was essentially nontoxic.
Thymidine alone, as well as
leucovorin or
folic acid, protected against the cytotoxic effects of H-338. This is consistent with
thymidylate synthase (TS) as its only locus of action. Studies with
dihydrofolate reductase and TS isolated from HCT-8 cells indicated that these
quinazolines were weaker inhibitors of
dihydrofolate reductase than was
methotrexate, but they were not particularly potent TS inhibitors. However, synthetic
poly-gamma-glutamate derivatives of
quinazolines showed dramatically increased TS, but not
dihydrofolate reductase, inhibition. TS inhibition increased as the
polyglutamate chain length increased. Using isolated HCT-8
folylpolyglutamate synthetase, all the parent
quinazolines containing
L-glutamate were found to be substrates. With H-338, the results indicated that tetraglutamate or longer derivatives could be synthesized intracellularly. These results are consistent with our hypothesis that cytotoxicity by such
quinazolines necessarily involves "lethal synthesis" from a
prodrug; i.e., the nontoxic parent
drug must be converted to polyglutamates before TS inhibition and subsequent cytotoxicity can occur.