Methotrexate analogs. 20. Replacement of glutamate by longer-chain amino diacids: Effects on dihydrofolate reductase inhibition, cytotoxicity, and in vivo antitumor activity

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Abstract
Chain-extended analogs of methotrexate were synthesized by condensation of 4-amino-4-deoxy-N10-methylpteroic acid with esters of L-.alpha.-aminoadipic, L-.alpha.-aminopimelic, and L-.alpha.-aminosuberic acids, followed by ester hydrolysis with acid or base. Coupling was accomplished in up to 85% yield by the use of the peptide bond forming reagent diethyl phosphorocyanidate at room temperature. The products bound bacterial (Lactobacillus casei) and mammalian (L1210 mouse leukemia) dihydrofolate reductase with an affinity comparable to methotrexate and were also equitoxic to L1210 cells in culture. Cytotoxicity increased up to 3-fold as the number of CH2 groups in the amino acid side chain was extended from 2 to 5. The .alpha.-aminoadipate and .alpha.-aminopimelate analogs were poor substrates for caboxypeptidase G1, confirming that this enzyme has a strict requirement for a C-terminal L-glutamic acid residue. The in vivo antitumor activity of the chain-extended analogs against L1210 leukemia in mice was comparable to that of the parent drug on the qd .times. 9 [9 times/day] schedule, but higher doses were required to achieve the same increase in survival. The results were consistent with findings, reported separately, that these compounds are poor substrates for folate polyglutamate synthetase and therefore would not be expected to form .gamma.-polyglutamates once they enter a cell. This distinctive property has potential therapeutic implications for the treatment of certain MTX-resistant tumors whose resistance may be associated with a lower than normal capacity to form .gamma.-polyglutamates in comparison with proliferative tissues such as intestinal mucosa or marrow.