1843u89 and Breast-Neoplasms

Syntheses of several new inhibitors of thymidylate synthase (TS) structurally related to folic acid are described in which the pterin portion of the folate molecule is replaced by a benzo[f]quinazoline moiety, but which retain the natural methyleneamino link to the benzoylglutamate side chain. The effect on enzyme activity and cytotoxicity of various changes in the structure of the (p-aminobenzoyl)glutamate side chain are reported. Replacement of the benzamide portion of the (p-aminobenzoyl)glutamate moiety with 2-fluorobenzamido, 2-isoindolinyl, 1,2-benzisothiazol-2-yl, and 2-thenamido moieties varied in effect from a 9-fold diminution of TS activity to a 5-fold enhancement, while cytotoxic potency on SW-480 and MCF-7 tumor lines showed increases ranging from 3.6- to 450-fold. The detrimental effect on enzyme activity and cytotoxicity of alkyl substitution on the PABA nitrogen is confirmed for these compounds, in contrast with several series of previously reported quinazoline antifolates (2). Substitution of a C3-methyl substituent for 3-amino had little effect on TS activity but was beneficial in terms of solubility and cytotoxicity. The excellent combination of TS inhibitory activity, FPGS substrate activity, and affinity for the reduced folate transport system in the most potent of these derivatives, 3e, resulted in IC50 values of 0.2-0.8 nM against these tumor lines.

Topics: Adenocarcinoma; Antineoplastic Agents; Breast Neoplasms; Colonic Neoplasms; Glutamates; Humans; Indoles; Isoindoles; Quinazolines; Structure-Activity Relationship; Thymidylate Synthase; Tumor Cells, Cultured

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.

Topics: Animals; Binding, Competitive; Breast Neoplasms; Cell Division; Colonic Neoplasms; Female; Humans; Indoles; Isoindoles; Leucovorin; Leukemia L1210; Leukemia, T-Cell; Methotrexate; Quinazolines; Structure-Activity Relationship; Subrenal Capsule Assay; Tetrahydrofolates; Thymidylate Synthase