Biochemical and cellular pharmacology of 1843U89, a novel benzoquinazoline inhibitor of thymidylate synthase.

Studies on a series of benzoquinazoline folate analogues as inhibitors of human thymidylate synthase led to the selection of 1843U89 for further evaluation. This compound had a Ki of 90 pM versus human thymidylate synthase and was noncompetitive with (6R,S)-5,10-methylenetetrahydrofolate. It was a good substrate for the addition of the second glutamate by hog liver folylpolyglutamate synthetase, having a Vmax/Km value 7.8-fold higher than (6R,S)-tetrahydrofolate. The data indicate that 1843U89 was transported into cells via the reduced folate carrier. The Kt for 1843U89 in MOLT-4 cells was 0.33 microM, which was 3-fold lower than that for methotrexate and 16-fold lower than that for (6S-5-formyltetrahydrofolate. V/K values were 20.3 for 1843U89 versus 1.2 and 1.9 for methotrexate and (6S)-5-formyltetrahydrofolate, respectively. It was a potent inhibitor of the growth of human cells, having 50% inhibitory concentrations below 1 nM for all cell lines tested. Growth inhibition was reversed by thymidine alone, indicating that thymidylate synthase was the only site of action of this compound. Growth inhibition was not affected by (6R,S-5-formyltetrahydrofolate at concentrations below 5 microM. However, the 50% inhibitory concentration increased when the concentration in the medium was increased to 100 microM, presumably due to competition for transport. Relative to the human cell lines used, murine cell lines were 80-1300-fold less sensitive to 1843U89 and the other benzoquinazolines tested. This decreased sensitivity appeared to be due, at least in part, to decreased transport or accumulation in murine cells. Ki values for inhibition of methotrexate transport for the benzoquinazolines were 5-17-fold higher in L1210 cells than in MOLT-4 cells. 1843U89, the benzoquinazoline which was transported most efficiently and which was the most potent inhibitor of the growth of human cells, exhibited the largest difference between binding to the MOLT-4 human and L1210 murine transporter. The V/K for L1210 transport was 80-fold less than that for MOLT-4. Initial antitumor studies, using the human thymidine kinase-deficient line GC3TK- to circumvent problems associated with murine transport as well as the high circulating thymidine levels in mice, indicated that 1843U89 had marked in vivo antitumor activity.