Eleven novel spermidine (SPD) derivatives were synthesized as potential anticancer agents and evaluated for their ability to compete with [3H]SPD for cellular uptake, to inhibit cell growth, to affect polyamine biosynthesis, to suppress enzyme activity, and to substitute for SPD in supporting growth of cultured L1210 leukemia cells. The compounds included a series of N4-SPD derivatives (N4-methyl-SPD, N4-ethyl-SPD, N4-acetyl-SPD, N4-hexyl-SPD, N4-hexanoyl-SPD, N4-benzyl-SPD, and N4-benzoyl-SPD) and a series of N1,N8-SPD derivatives [N1,N8-bis(ethyl)-SPD, N1,N8-bis(acetyl)-SPD, N1,N8-bis(propyl)-SPD, and N1,N8-bis(propionyl)-SPD]. Uptake studies revealed N4-alkyl derivatives to be the most effective competitive inhibitors of [3H]SPD uptake (Ki, 26 to 43 µm) followed by N1,N8-alkyl derivatives (Ki, 71 to 115 µm), then N4-acyl derivatives (Ki, 115 to >500 µm), and N1,N8-acyl derivatives (Ki, >500 µm). The data indicate the relative importance of the terminal amines and of charge as determinants of cellular uptake. Of the 11 derivatives, only N4-hexyl-SPD, N1,N8-bis(ethyl)-SPD, and N1,N8-bis(propyl)-SPD demonstrated antiproliferative activity at 0.1 mm with 50%-inhibitory concentration values at 48 h of 30, 40, and 50 µm, respectively. In the case of the N1,N8-SPD derivatives, recovery from growth inhibition was enhanced considerably by exogenous SPD, suggesting involvement of polyamine depletion. At 10 to 30 µm, both N1,N8-bis(ethyl)-SPD and N1,N8-bis(propyl)-SPD (but not N4-hexyl-SPD) inhibited polyamine biosynthesis as indicated by significant reductions in polyamine pools and in biosynthetic enzyme activities. The more effective of the two, N1,N8-bis(ethyl)-SPD, depleted intracellular putrescine and SPD and reduced spermine by ∼50% at 96 h and decreased ornithine and S-adenosylmethionine decarboxylase activities by 98 and 62%, respectively. Since neither derivative (at 5 mm) directly inhibited these enzymes from untreated cell extracts by significantly more than SPD itself, it is suspected that they act by regulating enzyme levels. As a measure of regulatory potential of the derivatives, ornithine decarboxylase was assayed in cells treated for 24 h and compared to the effects of 10 µm SPD which reduced the enzyme activity by 80%. None of the N4-SPD derivatives affected ornithine decarboxylase activity, while N1,N8-bis(ethyl)- and (propyl)-SPD were nearly as effective as SPD. Apparently, the central amine of the molecule is critical for regulatory function. Neither of the N1,N8-SPD derivatives was capable of functionally substituting for the depleted SPD pools as determined by their inability to prevent α-difluoromethylornithine-mediated cytostasis. By contrast, several of the N4-SPD derivatives were effective in this activity, suggesting that the N1,N8-terminal amines are critical for SPD function. It is concluded that inhibition of cell growth via polyamine depletion occurs with those SPD derivatives [i.e., N1,N8-bis(ethyl)- or (propyl)-SPD] which regulate biosynthetic enzyme activities in a manner similar to polyamines but which, unlike the natural polyamines, are incapable of performing in functions essential for cell growth.

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This investigation was supported in part by Grants CA-33321, CA-22153, and CA-24538 from the National Cancer Institute, Department of Health, Education, and Welfare.

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