5-(l-Adamantyl)pyrimidines as Inhibitors of Folate Metabolism1

Seven pyrimidines with a lipophilic substituent at position 5 have been investigated with respect to inhibition of both dihydrofolate reductases of Sarcoma 180 and Escherichia coli and site of inhibitory action in mammalian cells in culture. Of the 7 pyrimidines, 5 were competitive inhibitors of both dihydrofolate reductases, 1 was noncompetitive, and 1 produced no effect. The Sarcoma 180 enzyme was 10 to 20 times more sensitive to the active inhibitors than was the E. coli enzyme. The 2,4-diaminopyrimidine with an adamantyl group attached directly to the ring carbon at position 5 was a superior inhibitor of the mammalian enzyme (Kj, 6 nM), as compared to the corresponding pyrimidine with the group separated from the ring by NHCO bridge (2000 times less affinity) or by CH2NHCOCH2 bridge (no inhibition). A methyl group at position 6 of 2,4-diamino-5-adamantylpyrimidine increased the affinity to the enzyme by 30-fold, and a hydroxy substituent in place of amino at position 4 decreased the affinity by 150-fold. These dihydrofolate reducÃ-aseinhibitors interfered with the folate metabolism of mammary adenocarcinoma cells (TA3) in culture, as suggested by the prevention of growth inhibition by thymidine, hypoxanthine, and glycine, as well as by the cross-resistance of amethopterin-resistant subline of Sarcoma 180 cells. There was also a secondary, far less sensitive site of inhibition for 3 of the compounds; this site was not related to folate metabolism (50% inhibition at 30 to SOjuM). 2,4-Diamino-5-(l -adamantyl)-6-methylpyrimidine was equal to or more potent than amethopterin in cell culture, although its affinity to dihydrofolate reducÃ-asewas 100 times lower lhan lhat of amethopterin. Thus, il appears lhal the adamantyl group in position 5 greatly facililales Ihe passage of pyrimidines through Ihe plasma membrane of the cells, thereby increasing the inhibitory potency of these compounds in cellular systems over and above lhal expecled from enzyme inhibition analysis.