Novel Angular Benzophenazines: Dual Topoisomerase I and Topoisomerase II Inhibitors as Potential Anticancer Agents Nigel Vicker, ² Luke Burgess, ² Irina S. Chuckowree, ² Rory Dodd, ² Adrian J. Folkes, ² David J. Hardick, ² Timothy C. Hancox, ² Warren Miller, ² John Milton, ² Sukhjit Sohal, ² Shouming Wang, ² Stephen P. Wren, ² Peter A. Charlton,* ,‡ Wendy Dangerfield, ‡ Chris Liddle, ‡ Prakash Mistry, ‡ Alistair J. Stewart, ‡ and William A. Denny § Medicinal Chemistry and Pharmacology Departments, Xenova Ltd., 957 Buckingham Avenue, Slough, Berkshire, SL1 4NL, U.K., and Auckland Cancer Society Research Centre, Faculty of Medicine and Health Science, The University of Auckland, Private Bag 92019, Auckland 1000, New Zealand Received July 17, 2001 A series of substituted angular benzophenazines were prepared using a new synthetic route via a novel regiocontrolled condensation of 1,2-naphthoquinones and 2,3-diaminobenzoic acids. The synthesis and biological activity of this new series of substituted 8,9-benzo[a]phenazine carboxamide systems are described. The analogues were evaluated against the H69 parental human small cell lung carcinoma cell line and H69/LX4 resistant cell line which overexpresses P-glycoprotein. Selected analogues were evaluated against the COR-L23 parental human non small cell lung carcinoma cell line and the COR-L23/R resistant cell line which overexpresses multidrug resistance protein. This series of novel angular benzophenazines were potent cytotoxic agents in these cell lines and may be able to circumvent multidrug resistance mechanisms which result in the lack of efficacy of many drugs in cancer chemotherapy. These compounds show dual inhibition of topoisomerase I and topoisomerase II and thus target two key enzymes responsible for the topology of DNA that are active at different points in the cell cycle. The introduction of chirality into the carboxamide side chain of these novel benzophenazine carboxamides has resulted in the discovery of a potent enantiospecific series of cytotoxic agents, exemplified by 4-methoxy-benzo[a]phenazine-11-carboxylic acid (2-(dimethylamino)-1-(R)- methyl-ethyl)-amide, XR11576 ((R)-4j′′). In vivo activity has been demonstrated for 4-methoxy- benzo[a]phenazine-11-carboxylic acid (2-(dimethylamino)-1-(R)-methyl-ethyl)-amide, XR11576, after intravenous administration to female mice, and this compound has been selected as a development candidate for further evaluation. Introduction The topoisomerases are essential enzymes in the regulation of DNA topology, which is required if cells are to divide and proliferate, 1 and are important cellular targets for a number of successful chemotherapeutic agents. 1 Drugs that target topoisomerase II, for example doxorubicin and etoposide, have been widely used in cancer chemotherapy, 2 while those that specifically target topoisomerase I, principally the camptothecin analogues, have made an important impact more re- cently for the treatment of colon cancer. 3 A shortfall of many of these specific inhibitors of either topoisomerase I or topoisomerase II is their inability to overcome multidrug resistance (MDR). 4,5 Several dual inhibitors of topoisomerase I and II have been identified; these include intoplicine, 6 XR5000 (DACA), 7 and TAS-103, 8 all of which are in clinical evaluation. Recently it has been suggested that TAS-103 predominantly acts as a topoisomerase IIR inhibitor. 9 An advantage of both XR5000 and TAS-103 is their ability to circumvent MDR and to target two key enzymes that affect the topology of DNA which are active at different points in the cell cycle. XR5000 (1) is an acridine derivative which acts as a DNA intercalating agent, and the steric and electronic effects that determine its cytotoxicity have recently been discussed in detail. 10 To find a novel structural class of second generation dual topoisomerase inhibitors, a program of work was initiated to discover an orally active dual inhibitor of topoisomerase I and II that avoided MDR. The phenazines are different chemically and structur- ally from the acridines but have similar shape and have been shown to fulfill the fundamental physicochemical requirements for DNA intercalation. 11 A strategy of modifying the phenazine template to give structurally novel dual topoisomerase inhibitors was adopted. The * To whom correspondence should be addressed. Ph: +44 1753 706600. Fax: +44 1753 706607. E-mail: Peter_Charlton@Xenova.co.uk. ² Medicinal Chemistry Department, Xenova Ltd. ‡ Pharmacology Department, Xenova Ltd. § Auckland Cancer Society Research Centre. 721 J. Med. Chem. 2002, 45, 721-739 10.1021/jm010329a CCC: $22.00 © 2002 American Chemical Society Published on Web 01/08/2002