Several N(1)-substituted polyamines containing various spacer units between nitrogen centers were synthesized as their respective HCl salts. The N(1)-substituents included benzyl, naphthalen-1-ylmethyl, anthracen-9-ylmethyl, and pyren-1-ylmethyl. The polyamine spacer units ranged from generic (4,4-triamine, 4,3-triamine, and diaminooctane) spacers to more exotic [2-(ethoxy)ethanoxy-containing diamine, hydroxylated 4,3-triamine, and cyclohexylene-containing triamine] spacers. Two control compounds were also evaluated: N-(anthracen-9-ylmethyl)-butylamine and N-(anthracen-9-ylmethyl)-butanediamine. Biological activities in L1210 (murine leukemia), alpha-difluoromethylornithine (DFMO)-treated L1210, and Chinese hamster ovary (CHO) and its polyamine transport-deficient mutant (CHO-MG) cell lines were investigated via IC(50) cytotoxicity determinations. K(i) values for spermidine uptake were also determined in L1210 cells. Of the series studied, the N(1)-benzyl-4,4-triamine system 6 had significantly higher IC(50) values (lower cytotoxicity) in the L1210, CHO, and CHO-MG cell lines. A cellular debenzylation process was observed in L1210 cells with 6 and generated "free" homospermidine. The size of the N(1)-arylmethyl substituent had direct bearing on the observed cytotoxicity in CHO-MG cells. The N(1)-naphthalenylmethyl, N(1)-anthracenylmethyl, and N(1)-pyrenylmethyl 4,4-triamines had similar toxicity (IC(50)s: approximately 0.5 microM) in CHO cells, which have an active polyamine transporter (PAT). However, this series had IC(50) values of >100 microM, 66.7 microM, and 15.5 microM, respectively, in CHO-MG cells, which are PAT-deficient. The observed lower cytotoxicity in the PAT-deficient CHO-MG cell line supported the premise that the conjugates use PAT for cellular entry. In general, moderate affinities for the polyamine transporter were observed for the N-arylmethyl 4,4-triamine series with their L1210 K(i) values all near 3 microM. In summary, the 4,4-triamine motif was shown to facilitate entry of polyamine conjugates into cells containing active polyamine transporters.