Substituted Dibenzo[c,h]cinnolines: Topoisomerase I-Targeting Anticancer Agents Younong Yu, a Sudhir K. Singh, a Angela Liu, b Tsai-Kun Li, b Leroy F. Liu b,c and Edmond J. LaVoie a,c, * a Department of Pharmaceutical Chemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA b Department of Pharmacology, The University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA c The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA Received 18 July 2002; accepted 24 October 2002 Abstract—Several substituted dibenzo[c,h]cinnolines were synthesized and evaluated for their potential to target topoisomerase I and for their relative cytotoxic activity. Select benzo[i]phenanthridines are capable of stabilizing the cleavable complex formed with topoisomerase I and DNA. This study was initiated to examine whether dibenzo[c,h]cinnolines, which are in essence aza analogues of benzo[i]phenanthridines, possess similar pharmacological properties. 2,3-Dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine is one of the more potent benzo[i]phenanthridine derivatives in regard to topoisomerase I-targeting activity and cytotoxicity. The structure–activity relationship observed with these substituted dibenzo[c,h]cinnolines parallels that observed for benzo[i]phenan- thridine derivatives. Compared to similarly substituted benzo[i]phenanthridines, the dibenzo[c,h]cinnoline analogues exhibit more potent topoisomerase I-targeting activity and cytotoxicity. The relative IC 50 values obtained in assessing the cytotoxicity of 2,3- dimethoxy-8,9-methylenedioxydibenzo[c,h]cinnoline and 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine in the human lymphoblastma cell line, RPMI8402, are 70 and 400 nM, respectively. In tumor cell lines selected for resistance to camptothecin and known to express mutant topoisomerase I, benzo[i]phenanthridine derivatives were not cross-resistant. In contrast, similarly sub- stituted dibenzo[c,h]cinnolines with significant topoisomerase I-targeting activity did exhibit cross-resistance in these camptothecin- resistant cell lines. The cytotoxicity of these dibenzo[c,h]cinnolines was not diminished in cells overexpressing the efflux transporter, MDR1. These data indicate that substituted dibenzo[c,h]cinnolines can exhibit potent topoisomerase I-targeting activity and are capable of overcoming the multi-drug resistance associated with this efflux transporter. # 2003 Elsevier Science Ltd. All rights reserved. Introduction The topological state of DNA is regulated by DNA topoisomerases, which perform this function through the breaking and rejoining of DNA strands. 1 4 Studies have also demonstrated that these enzymes are involved in controlling template supercoiling during RNA tran- scription. 5,6 There are two major subtypes of topo- isomerases based upon differences in their initial mechanisms. While the mechanism associated with topoisomerase I (TOP1) involves the formation of a single-strand DNA break, topoisomerase II (TOP2) functions by creating a double-strand DNA break. The antitumor activity of topoisomerase-targeting agents is associated with their ability to stabilize the enzyme- DNA cleavable complex. This drug-induced stabili- zation of the enzyme–DNA cleavable complex effectively converts these enzymes into cellular poisons. Camptothecin and its structurally-related analogues are among the more extensively studied agents that target TOP1. Bi- and terbenzimidazoles, 7 10 certain benzo[c]- phenanthridine and protoberberine alkaloids and their synthetic analogues, 11 15 indolocarbazoles, 16 the fungal metabolites bulgarein 17 and saintopin, 18 and indenoiso- quinolines, 19,20 phenazines, 21 and benzophenazines, 22 have been identified as TOP1-directed agents. Recently, several benzo[i]phenanthridines have been identified that exhibit potent activity as TOP1-targeting agents and significant cytotoxicity. 23,24 2,3-Dimethoxy-8,9- methylenedioxybenzo[i]phenanthridine, 1 (Fig. 1), is 0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. PII: S0968-0896(02)00604-1 Bioorganic & Medicinal Chemistry 11 (2003) 1475–1491 *Corresponding author at: Ernest Mario School of Pharmacy, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA. Tel.: +1-732-445- 2674; fax: +1-732-445-6312; e-mail: elavoie@rci.rutgers.edu http://www.rci.rutgers.edu/  layla/Faculty/LaVoie.htm