The biological activities of novel analogues of methotrexate (MTX) and aminopterin (AMT) in which the γ-carboxyl was replaced by a 1H-tetrazol-5-yl ring, an isosteric group with acidic properties similar to a carboxyl group, were investigated. The tetrazolyl analogues of MTX and AMT were more potent inhibitors of the growth of CCRF-CEM and K562 human leukemia cell lines during continuous (120 h) and 24-h pulse exposure than were the respective parent drugs; only when the exposure time was reduced to 6 h were the parent drugs more potent. These inhibitory effects on growth correlated with the onset of and recovery from inhibition of de novo thymidylate biosynthesis. Growth inhibition by the analogues was protectable by leucovorin. MTX-resistant CCRF-CEM sublines with decreased transport or increased dihydrofolate reductase (DHFR) levels were cross-resistant to the analogues. The analogues were as potent as their parent drugs in inhibiting DHFR activity in vitro and at displacing [3H]MTX from intracellular DHFR. Each analogue was more effective than its parent drug at inhibiting uptake of [3H]MTX into CCRF-CEM cells. The tetrazole analogue of AMT was a linear competitive inhibitor (Kis = 50 µm) of CCRF-CEM folylpolyglutamate synthetase, while the tetrazole analogue of MTX, unlike all other inhibitors, was linear noncompetitive (Kis = 51 µm, Kii = 321 µm). The data suggest that, compared with MTX or AMT, the tetrazole substituent, in place of the γ-carboxyl group, allows more efficient transport into cells via the reduced folate/MTX carrier and the resulting greater uptake of the analogues leads to inhibition of DNA synthesis and cell death at lower extracellular concentrations during long exposures. The mechanism of cell death could involve inhibition at folypolyglutamate synthetase, but DHFR is the primary target. The low potency of the analogues during short exposure is presumably related to the inability to form the poly-γ-glutamyl metabolites required for intracellular retention.

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This work was supported by Grants CA43500 and CA24538 (J. J. M.) and CA35212 (T. I. K.) from the National Cancer Institute, USPHS, Department of Health and Human Services, and CH-192 (T. I. K.) from the American Cancer Society. J. J. M. was a Leukemia Society Scholar during completion of this work. C. M. F. was supported by the Dave Fellowship from the Grace Cancer Drug Center as a participant in the 1989 Roswell Park Summer Research Program. A preliminary account of this work was presented at the 80th Annual Meeting of the American Association for Cancer Research, San Francisco, CA, May 1989 (1).

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