BiologicalPropertiesof A/4-and A/1,A/8-Spermidine Derivativesin Cultured L1210 LeukemiaCells1

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 A/4-SPD derivatives (W-methyl-SPD, A/4-ethyl-SPD, A/4-acetyl-SPD, A/4hexyl-SPD, A/4-hexanoyl-SPD, A/4-benzyl-SPD, and N4-benzoylSPD) and a series of A/1,A/8-SPD derivatives [A/1,A/8-bis(ethyl>SPD, A/1,A/8-bis(acetyl)-SPD, A/1,A/8-bis(propyl)-SPD, and N\NBbis(propionyl)-SPD]. Uptake studies revealed A/4-alkyl derivatives to be the most effective competitive inhibitors of [3H]SPD uptake (K,, 26 to 43 MM)followed by A/1,A/8-alkyl derivatives (K„ 71 to 115 MM),then A/4-acyl derivatives (K,, 115 to >500 MM),and A/1, A/8-acyl derivatives (K¡, >500 MM).The data indicate the relative importance of the terminal amines and of charge as determinants of cellular uptake. Of the 11 derivatives, only A/4-hexyl-SPD, N\ A/8-bis(ethyl)-SPD, and A/1,A/8-bis(propyl)-SPD demonstrated antiproliferative activity at 0.1 mw with 50%-inhibitory concentration values at 48 h of 30, 40, and 50 MM,respectively. In the case of the A/1,A/8-SPD derivatives, recovery from growth inhibition was enhanced considerably by exogenous SPD, suggesting involve ment of polyamine depletion. At 10 to 30 MM, both A/1,A/8bis(ethyl)-SPD and N1,A/8-bis(propyl)-SPD (but not A/4-hexyl-SPD) inhibited polyamine biosynthesis as indicated by significant re ductions in polyamine pools and in biosynthetic enzyme activities. The more effective of the two, A/1,A/8-bis(ethyl)-SPD, depleted intracellular putrescine and SPD and reduced spermine by ~50% at 96 h and decreased omithine and S-adenosylmethionine decarboxylase activities by 98 and 62%, respectively. Since neither derivative (at 5 mM) directly inhibited these enzymes from un treated 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, omithine decarboxylase was assayed in cells treated for 24 h and compared to the effects of 10 MMSPD which reduced the enzyme activity by 80%. None of the A/4-SPD derivatives affected omithine decarboxylase activity, while A/1,A/8-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 A/1,A/8-SPD derivatives was capable of functionally substituting for the depleted SPD pools as determined by their inability to prevent a-difluoromethylornithine-mediated cytostasis. By con trast, several of the A/4-SPD derivatives were effective in this 1This 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. * To whom requests for reprints should be addressed. 3 Recipient of American Cancer Society Postdoctoral Fellowship Grant PF-2425. Received 10/11/83; revised 12/27/84; accepted 1/30/85. activity, suggesting that the A/1,A/8-terminal amines are critical for SPD function. It is concluded that inhibition of cell growth via polyamine depletion occurs with those SPD derivatives [;'.e., A/1,A/8-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.