Synthesis and cytotoxic mechanism study of aziridinyl spermidine analogues

Abstract

In this study, the three-membered alkylating moiety, aziridine, was chosen to replace a terminal amino group of the polyamine, spermidine, to generate N{dollar}\sp1{dollar}- and N{dollar}\sp8{dollar}-aziridinyl spermidine analogues. Cytotoxicity was tested in vitro against L1210 murine lymphoblastic leukemic and HL60 human leukemic cell lines. Aminoguanidine, a serum amine oxidase inhibitor, reduced the cell growth inhibitory effect of spermidine approximately 6- or 20-fold in HL60 or L1210 cells, respectively, but did not show any effect on the cytotoxicity induced by aziridinyl spermidines. N{dollar}\sp1{dollar}-Aziridinyl spermidine was slightly more potent than the N{dollar}\sp8{dollar}-isomer (IC{dollar}\sb{lcub}50{rcub}{dollar} values 0.15 {dollar}\mu{dollar}M vs 0.3 {dollar}\mu{dollar}M for L1210 cells and 0.14 {dollar}\mu{dollar}M vs 0.28 {dollar}\mu{dollar}M for HL60 cells). Assessment of cell viability with a flow cytometric technique using fluorescein diacetate and propidium revealed that both N{dollar}\sp{lcub}1{rcub}{dollar}- and N{dollar}\sp8{dollar}-aziridinyl spermidines were cytotoxic. Both aziridinyl spermidine analogues inhibited (H{dollar}\sp3{dollar}) -thymidine and (H{dollar}\sp3{dollar}) -uridine incorporation into L1210 cells in a dose-dependent fashion. This effect was observed after an exposure time as short as one hour. The fact that the cytotoxicity induced by these two aziridinyl derivatives of spermidine was potentiated by 24 hour pretreatment of L1210 cells with 100 {dollar}\mu{dollar}M {dollar}\alpha{dollar}-diflouromethylornithine (DFMO), and were prevented by coincubation with 3.7 {dollar}\mu{dollar}M exogenous spermidine suggests a mechanistic relationship of the aziridinyl spermidine analogues with the polyamine system. Under the same conditions, pretreatment with DFMO and coincubation with spermidine had no effect on the cytotoxicity induced by thiotepa. The cell growth inhibitory effect induced by DFMO (100 {dollar}\mu{dollar}M for 24 hours) was overcome by washing away the test compound and replenishment with 3.7 {dollar}\mu{dollar}M spermidine. However, the cytotoxicity induced by aziridinyl spermidines was not affected at all. These observations suggest that the cytotoxic effect of aziridinyl compounds is irreversible. The accumulation of both N{dollar}\sp1{dollar}- and N{dollar}\sp8{dollar}-aziridinyl spermidine increased proportionally with increasing extracellular concentrations. Enhancement of cellular accumulation of both aziridinyl compounds by DFMO pretreatment provided evidence to support the argument that the aziridinyl spermidine analogues might utilize the polyamine transport system to enter cells. Other evidence which strengthens this argument is the fact that both N- and N{dollar}\sp8{dollar}-aziridinyl spermidines inhibited the uptake of natural spermidine in a dose-dependent manner. The perturbation of polyamine biochemistry by the test compounds was characterized by their ability to deplete cellular putrescine, as well as spermidine and spermine. Finally, results from DNA alkylation studies showed the formation of aziridinyl spermidine-DNA adducts, suggesting that DNA might be one target of the test compounds. (Abstract shortened by UMI.)