DNA Cleavage by the Antitumor Agent 3-Amino-1,2,4-benzotriazine 1,4-Dioxide (SR4233): Evidence for Involvement of Hydroxyl Radical

3-Amino-1,2,4-benzotriazine 1,4-dioxide (SR4233, WIN59075, tirapazamine, 1) is a clinically promising antitumor agent that requires bioreductive activation, selectively kills oxygen-deficient cells, and is believed to derive its biological activity from DNA cleavage. Using a xanthine-xanthine oxidase enzyme system as a one-electron reductant to activate 1 for DNA cleavage, it has been found that radical scavengers such as mannitol, dimethyl sulfoxide, ethanol, methanol, and tert-butyl alcohol significantly inhibit drug-dependent DNA cleavage. Compound 1, in concert with the xanthine-xanthine oxidase system, converts DMSO to methanesulfinic acid, a reaction characteristic of hydroxyl radical. In addition, treatment of a 32P-labeled restriction fragment with reductivelyactivated 1 results in cleavage at every base pair, with little sequence specificity, consistent with involvement of a highly reactive, nonselective agent such as hydroxyl radical. These results strongly support the involvement of radicals in the cleavage of DNA by 1 and are consistent with hydroxyl radical as the major DNA-cleaving species generated by reduction of 1. Compounds that damage DNA play an important role in cancer chemotherapy.1 In the pursuit of improved cancer chemotherapeutic agents, one approach involves identification of features unique to cancer cells that can be used to direct the cytotoxic action of DNA-damaging agents specifically toward these cells. One such feature that may be exploited in the treatment of certain cancers is the oxygen-deficient (hypoxic) nature of solid tumor cells relative to normal cells.2 Due to the fact that hypoxic cells are resistant to radiation therapy3 and a number of common chemotherapeutic agents,4 tumor cell hypoxia is often a problem rather than an advantage in cancer treatment; however, several promising or clinically useful antitumor agents are thought to obtain some therapeutic advantage by causing DNA damage more efficiently in hypoxic tumor cells as compared to normally oxygenated cells.5 1,2,4-Benzotriazine 1,4-dioxides are a novel class of anticancer agents whose remarkable antitumor properties are thought to stem from their selective toxicity toward the hypoxic cells found in solid tumors.6-8 One member of this class of molecules, 3-amino-1,2,4-benzotriazine 1,4-dioxide (SR4233, WIN 59075, tirapazamine, 1), is currently in phase II and III clinical trials for the treatment of certain cancers.8 Because it is thought that 1 derives its biological activity from the cleavage of cellular DNA,6-8 we undertook an investigation of the mechanism of DNA cleavage by 1 with the expectation that a detailed understanding of this chemistry might ultimately facilitate the design of new therapeutic agents with improved antitumor properties. It is believed that in ViVo DNA cleavage by 1 is due to a radical species generated by enzymatic one-electron reduction of the heterocycle (Scheme 1).8 This theory is supported by several observations. In the absence of reducing systems, 1 alone does not damage DNA.9,10 In mammalian cells under anaerobic conditions, 1 is ultimately reduced to 3, which is not highly cytotoxic,6,11 and the rates of reduction parallel cytotoxicity in several different cell lines.12,13 Furthermore, Brown and co-workers have shown that addition of the radical scavenger dimethyl sulfoxide (DMSO) to hypoxic cell cultures significantly * Author to whom correspondence should be addressed. † Department of Chemistry. ‡ Department of Biochemistry. X Abstract published in AdVance ACS Abstracts, March 15, 1996. (1) (a) Remers, W. A. In Textbook of Organic, Medicinal and Pharmaceutical Chemistry; Delgado, J. W., Remers, W. A., Eds.; Lippincott: Philadelphia, PA, 1991; pp 313-358. (b) Kane, S. A.; Hecht, S. M. Prog. Nucleic Acids Res. Mol. Biol. 1994, 49, 313-352. (c) Henderson, D.; Hurley, L. H. Nature (Med) 1995, 1, 525-527. (2) (a) Hockel, M.; Schlenger, K.; Knoop, C.; Vaupel, P. Cancer Res. 1991, 51, 6098-6102. (b) Mueller-Klieser, W.; Vaupel, P.; Manz, R.; Schimdseder, R. Int. J. Radiat. Oncol. Biol. Phys. 1981, 7, 1397-1404. (c) Vaupel, P.; Schlenger, K.; Knoop, C.; Hockel, M. Cancer Res. 1991, 51, 3316-3322. (3) (a) Gatenby, R. A.; Kessler, H. B.; Rosenblum, J. S.; Coia, L. R.; Moldofsky, P. J.; Hartz, W. H.; Broder, G. J. Int. J. Radiat. Oncol. Biol. Phys. 1988, 14, 831-838. (b) Overgaard, J.; Hansen, H. S.; Jorgenson, K.; Hansen, M. H. Int. J. Radiat. Oncol. Biol. Phys. 1986, 12, 515-521. (c) Bush, R. S.; Jenkin, R. D.; Allt, W. E. C.; Beale, F. A.; Bean, H.; Denbo, A. J.; Pringle, J. F. Br. J. Cancer 1978, 37, Suppl. III, 302-306. (d) Hockel, M.; Knoop, C.; Schlenger, K.; Vorndran, B.; Baubmann, E.; Mitze, M.; Knapstein, P. G.; Vaupel, P. Radiother. Oncol. 1993, 26, 45-50. (4) (a) Kennedy, K. A. Anticancer Drug Res. 1987, 2, 181-194. (b) Sartorelli, A. C. Cancer Res. 1988, 48, 775-778. (c) Tannock, I.; Guttman, P. Br. J. Cancer 1981, 42, 245-248. (5) (a) Adams, G. E. Radiation Res. 1992, 132, 129-139. (b) Palmer, B. D.; Wilson, W. R.; Atwell, G. J.; Schultz, D.; Xu, X. Z.; Denny, W. A. J. Med. Chem. 1994, 37, 2175-2184. (6) Zeman, E. M.; Brown, J. M.; Lemmon, M. J.; Hirst, V. K.; Lee, W. W. Int. J. Radiat. Oncol. Biol. Phys. 1986, 12, 1239-1242. (7) Zeman, E. M.; Baker, M. A.; Lemmon, M. J.; Pearson, C. I.; Adams, J. A.; Brown, J. M.; Lee, W. W.; Tracy, M. Int. J. Radiat. Onc. Biol. Phys. 1989, 16, 977-981. (8) Brown, J. M. Br. J. Cancer 1993, 67, 1163-70. (9) Fitzsimmons, S. A.; Lewis, A. D.; Riley, R. J.; Workman, P. Carcinogenesis 1994, 15, 1503-1510. (10) Studies involving electrochemical reduction of 1: (a) Tocher, J. H.; Edwards, D. I. Free Radical Res. 1994, 21, 277-283. (b) Tocher, J. H.; Virk, N. S.; Edwards, D. I. Biochem. Pharmacol. 1990, 39, 781-786. (11) Baker, M. A.; Zeman, E. M.; Hirst, V. K.; Brown, J. M. Cancer Res. 1988, 48, 5947-5952. (12) Biedermann, K. A.; Wang, J.; Graham, R. P.; Brown, J. M. Br. J. Cancer 1991, 63, 358-362. (13) Costa, A. K.; Baker, M. A.; Brown, J. M.; Trudell, J. R. Cancer Res. 1989, 49, 925-929. 3380 J. Am. Chem. Soc. 1996, 118, 3380-3385 0002-7863/96/1518-3380$12.00/0 © 1996 American Chemical Society reduces the cytotoxicity of 1.8,14 A radical species resulting from the incubation of 1 with rat liver microsomes has been observed by ESR;15 however, no relation between this radical and DNA cleavage has been established. The identity of the enzyme(s) responsible for in ViVo reductive activation of 1 remains a subject of investigation.9,16 The specific toxicity of 1 toward hypoxic cells may result from the fact that the “activated” radical form of the drug (2) is destroyed by reaction with molecular oxygen.17 Such a back-oxidation would regenerate the drug (1) and produce superoxide radical, a species whose in ViVo toxicity is mitigated by the cellular enzymes superoxide dismutase and catalase.18 Here we report evidence strongly supporting the notion that radical species are involved in the cleavage of DNA by reductively-activated 1. In addition, we provide evidence that hydroxyl radical, rather than a radical form of 1, may be the major DNA-cleaving species in these reactions.