5H-Dibenzo[c,h]1,6-naphthyridine-6-ones can exhibit potent antitumor activity. The effect of varied substituents at the 5-position of 5H-8,9-dimethoxy-2,3-methylenedioxydibenzo[c,h]1,6-naphthyridine on relative cytotoxicity and topoisomerase I-targeting activity was evaluated. Potent TOP-1-targeting activity is observed when the 5-position is substituted with either a 2-(N,N-dimethylamino)ethyl group, as in 3a, or a 2-(pyrrolidin-1-yl)ethyl substituent, 3c. In contrast, the addition of a beta-methyl group or a beta-hydroxymethyl group to compound 3a, as in 3b and 3j, results in a loss of significant TOP1-targeting activity. While the presence of a 3-(N,N-dimethylamino)propyl substituent at the 5-position or a methyl(2-tetrahydrofuranyl) group allows for retention of TOP1-targeting activity, the 2-(4-methyl-1-piperazinyl)ethyl analogue, 3d, did not exhibit significant activity. Replacement of the N,N-dimethylamino group of 3a with either C(2)H(5) or OH, as in 3f and 3h, respectively, also had a negative impact on both cytotoxicity and TOP1-targeting activity. Treatment of 3a with LAH gave the 5,6-dihydrodibenzo[c,h]naphthyridine, 4a. This dihydro derivative has approximately 2/3 the potency of 3a as a TOP1-targeting agent. Compounds 3a, 3b, 3h, 3i, and 4a were evaluated for antitumor activity in the human tumor xenograft model using athymic nude mice. The non-estrogen responsive breast tumor cell line, MDA-MB-435, was used in these assays. Compound 3a proved to be effective in regressing tumor growth in vivo when administered either by ip injection or orally 3x week at a dose of 2.0mg/kg. Compound 4a when administered orally 5x weekly at a dose of 40 mg/kg also suppressed tumor growth.