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Activity of the Thymidylate Synthase Inhibitor 2-Desamino-N¹⁰-propargyl-5,8-dideazafolic Acid and Related Compounds in Murine (L1210) and Human (W1L2) Systems in Vitro and in L1210 in Vivo¹

Drug Development Section, Institute of Cancer Research, 15, Cotswold Road, Belmont, Sutton, Surrey, SM25PX United Kingdom [A. L. J., G. A. T., B. M. O., J. A. B., A. H. C.], and Laboratory of Cellular and Biochemical Pharmacology, Children's Hospital of Los Angeles, Los Angeles, California 90027 [R. G. M.]

We examined the in vitro activity of 2-desamino-5,8-dideazafolate and 2-desamino-N¹⁰-propargyl-5,8-dideazafolate (desamino-CB3717), the more water-soluble 2-desamino analogues of 5,8-dideazafolate and N¹⁰-propargyl-5,8-dideazafolic acid (CB3717). We report K₁ values for the inhibition of L1210 thymidylate synthase (TS) of 2 and 0.027 µM for 2-desamino-5,8-dideazafolate and desamino-CB3717, respectively, indicating a 30- and 10-fold loss in TS-inhibitory activity compared with the corresponding 2-NH₂ compounds. The synthetic tri- and tetrapolyglutamate derivatives of desamino-CB3717 were 66- and 101-fold more potent than the monoglutamate form as inhibitors of TS. Both desamino compounds were more potent as inhibitors of L1210 and W1L2 cell growth than were their 2-amino counterparts. 2-Desamino-5,8-dideazafolate retains quite good activity against both the TS-overproducing W1L2:C1 line and the L1210 cell line grown in the presence of thymidine, suggesting that a secondary locus of action may be involved. This other target is a folate-dependent enzyme as evidenced by the protection of the inhibition of cell growth by the addition of hypoxanthine or folinic acid together with thymidine. The methotrexate-resistant, dihydrofolate reductase-overproducing L1210:R7A cell line is cross-resistant to 2-desamino-5,8-dideazafolate, which suggests that dihydrofolate reductase is the other target. An L1210 subline (1565) unable to transport reduced folates is 10-fold resistant to desamino-CB3717 and 2-desamino-5,8-dideazafolate but is not cross-resistant to CB3717 or 5,8-dideazafolate. The removal of the 2-amino function of CB3717 did not affect folylpolyglutamate synthetase substrate activity (CB3717 K_m = 48 µM, desamino-CB3717 K_m = 40 µM). However, both 5,8-dideazafolate and its desamino analogue were about 10-fold better substrates for folylpolyglutamate synthase than were the N¹⁰-propargyl compounds, and this may contribute to their good growth-inhibitory properties. In vivo, desamino-CB3717 cured 75% of mice bearing the L1210:ICR tumor at doses of 50 mg/kg daily for 5 days and above (maximum tolerated dose > 1000 mg/kg daily for 5 days). This activity is comparable to that observed for CB3717 (maximum tolerated dose = 100 mg/kg daily for 5 days) despite the much more rapid plasma clearance of desamino-CB3717. Considerable TS inhibition (>80%) was observed in the refractory L1210:NCI tumor (as measured by the release of ³H from 5-[³H] 2' deoxyuridine) removed 2 h after the i.v. injection of 100 mg/kg of desamino-CB3717 and, after a dose of 1000 mg/kg, TS remained inhibited in cells removed 12 h after drug administration. These data are consistent with the rapid formation of an intracellular, noneffluxable pool of desamino-CB3717 which allows the rapid and sustained inhibition of the target enzyme, TS, in tumor cells.

¹ This work was supported by grants from the Cancer Research Campaign and Medical Research Council of the United Kingdom as well as by a grant from the North Atlantic Treaty Organization that enabled collaboration between Dr. A. L. Jackman, Dr. A. H. Calvert, and Professor R. G. Moran.

² To whom requests for reprints should be addressed.

³ Present address: Cancer Research Laboratory, University of Southern California, Los Angeles, CA 90027.

Received 9/18/89. Revised 5/ 9/90.

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