Intoplicine (RP 60475, NSC 645008) is an antitumor derivative in the 7H-benzo[e]pyrido[4,3-b]indole series which is now being tested in clinical trials. Intoplicine strongly binds DNA (KA = 2 x 10(5) M-1) and thereby increases the length of linear DNA. These properties are consistent with DNA unwinding by intoplicine. Intoplicine was found to be a dual topoisomerase I and II inhibitor, with DNA sites of enzyme inhibition being different for these two enzymes. In this study, 22 analogues of intoplicine were evaluated for their effects on topoisomerase I- and II-mediated DNA cleavage reactions by using enzymes purified from calf thymus. Site-specific DNA cleavage mediated by topoisomerase I was observed with 7H-benzo[e]pyrido[4,3-b]indole derivatives but not with 11H-benzo[g]-pyrido[4,3-b]indole derivatives. Site-specific DNA cleavage mediated by topoisomerase II occurred with derivatives having hydroxyl groups at the 3-position on the 7H-benzo[e]pyrido[4,3-b]indole ring or at the 4-position on the 11H-benzo[g]pyrido[4,3-b]indole ring. Study of the relationships between the in vivo antitumor activity on P388 leukemia and the topoisomerase I- and/or II-mediated DNA cleavage activity revealed that the most highly active antitumor compounds possessed both topoisomerase I-and II-inhibitory properties. Compounds selectively inhibiting either topoisomerase I or II were less active. These results suggest that dual topoisomerase I and II inhibition is critical for the antitumor activity of this new series of antitumor compounds.