A series of 2-amino-4-hydroxy-quinazolines was synthesized and evaluated as inhibitors of
colon adenocarcinoma and the
folate-dependent
enzymes,
thymidylate synthase and
dihydrofolate reductase. Of the
quinazolines tested,
5,8-dideazaisopteroylglutamate, (
IAHQ), when administered at 85 mg/kg on days 2 and 10 after
tumor implantation delayed the growth of colon
tumor No. 38, and resulted in 6 of 20
tumor-free animals at 90 days. In contrast,
methotrexate had no effect on the growth of colon
tumor No. 38 at maximally tolerated doses.
IAHQ was also active against human
colon adenocarcinoma cells (HCT-8) in tissue culture, requiring a concentration of 5 X 10(-7) M to inhibit cell growth 50% after 72 hours continuous exposure. Since
IAHQ was an effective substrate for
folylpolyglutamate synthetase, we examined the effects of
IAHQ and its possible tri-gamma-glutamyl metabolite, 5,8-dideazaisoPteGLu3, on
thymidylate synthase and
dihydrofolate reductase. Neither
IAHQ nor 5,8-dideazaisoPteGlu3 stimulated significant binding of 5-fluorodeoxyuridylate to
thymidylate synthase. This was consistent with the observation that
IAHQ antagonized the killing of HCT-8 cells by
5-fluorouracil. 5,8 DideazaisoPteGlu3 bound more tightly to
thymidylate synthase than
dihydrofolate reductase as indicated by Kis of 0.09 and 0.7 microM when deoxyuridylate and dihydropteroylglutamate, respectively, were the variable substrates. Inhibition studies also revealed that binding of
IAHQ and 5,8-dideazaisoPteGlu3 to
thymidylate synthase is promoted and not antagonized by deoxyuridylate. The data suggests that the biochemical basis for the antitumor effects of
IAHQ is the intracellular conversion of
IAHQ to poly-gamma-glutamyl metabolites, which inhibit
thymidylate synthase via formation of an inhibitor-deoxyuridylate-
enzyme complex.