Synthesis and Reactivity of Potential Toxic Metabolites of Tamoxifen Analogues: Droloxifene and Toremifene o-Quinones Dan Yao, Fagen Zhang, Linning Yu, Yanan Yang, Richard B. van Breemen, and Judy L. Bolton* Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612-7231 Received August 15, 2001 Tamoxifen remains the endocrine therapy of choice in the treatment of all stages of hormone- dependent breast cancer. However, tamoxifen has been shown to increase the risk of endometrial cancer which has stimulated research for new effective antiestrogens, such as droloxifene and toremifene. In this study, the potential for these compounds to cause cytotoxic effects was investigated. One potential cytotoxic mechanism could involve metabolism of droloxifene and toremifene to catechols, followed by oxidation to reactive o-quinones. Another cytotoxic pathway could involve the oxidation of 4-hydroxytoremifene to an electrophilic quinone methide. Comparison of the amounts of GSH conjugates formed from 4-hydroxytamoxifen, droloxifene, and 4-hydroxytoremifene suggested that 4-hydroxytoremifene is more effective at formation of a quinone methide. However, all three substrates formed similar amounts of o-quinones. Both the tamoxifen-o-quinone and toremifene-o-quinone reacted with deoxynucleo- sides to give corresponding adducts. However, the toremifene-o-quinone was shown to be considerably more reactive than the tamoxifen-o-quinone in terms of both kinetic data as well as the yield and type of deoxynucleoside adducts formed. Since thymidine formed the most abundant adducts with the toremifene-o-quinone, sufficient material was obtained for characterization by 1 H NMR, COSY-NMR, DEPT-NMR, and tandem mass spectrometry. Cytotoxicity studies with tamoxifen, droloxifene, 4-hydroxytamoxifen, 4-hydroxytoremifene, and their catechol metabolites were carried out in the human breast cancer cell lines S30 and MDA-MB-231. All of the metabolites tested showed cytotoxic effects that were similar to the parent antiestrogens which suggests that o-quinone formation from tamoxifen, droloxifene, and 4-hydroxytoremifene is unlikely to contribute to their cytotoxicity. However, the fact that the o-quinones formed adducts with deoxynucleosides in vitro implies that the o-quinone pathway might contribute to the genotoxicity of the antiestrogens in vivo. Introduction Tamoxifen 1 is currently the standard treatment for hormone dependent breast cancer in postmenopausal women (1-4). However, tamoxifen has been shown to cause an increased incidence of hepatocellular tumors in rats (5, 6) and an increase in the risk of endometrial cancer in women (7). A potential genotoxic mechanism might involve metabolic activation by oxidative enzymes in vivo to an electrophile(s) that binds irreversibly to DNA (8). Concerns about the side effects of tamoxifen have stimulated a search for new effective agents that do not form such genotoxic species. Toremifene has been recently approved by the United States Food and Drug Administration for the treatment of advanced breast cancer in postmenopausal women (9). There have been several recent reports comparing the efficacy and metabolism of tamoxifen and toremifene (10-19). Although toremifene is structurally similar to tamoxifen, differing only by a single chlorine atom in the ethyl side chain, toremifene does not cause hepatocar- cinogenesis in rats in vivo and it produces considerably fewer DNA adducts (20). Droloxifene (3-hydroxytamox- ifen) is another antiestrogen, which has shown promise in the treatment of breast cancer (21-23). There is no evidence that droloxifene produces DNA adducts or hepatocelluar carcinoma in rats (7, 24). There are several metabolic activation pathways of tamoxifen and toremifene leading to DNA adducts such as formation of carbocations (20, 25) and electrophilic quinone methides (26-28). In addition, an o-quinone has been shown to be a potential reactive intermediate involved in the metabolism of both droloxifene and toremifene (29, 30). In the present study, we compared * To whom correspondence should be addressed. Phone: (312) 996- 5280. Fax: (312) 996-7107. E-mail: judy.bolton@uic.edu. 1 Abbreviations: tamoxifen, Z-2-[4-(1,2-diphenyl-1-butenyl)-phenoxy]- N,N-dimethylethanamine; toremifene, Z-2-[(4-chloro-1,2-diphenyl-1- butenyl)-phenoxy]-N,N-dimethylethanamine; 4-OHTAM, 4-hydroxyta- moxifen; 3,4-di-OHTAM, 3,4-dihydroxytamoxifen; 4-OHTAM-SG, glutathione conjugates of 4-hydroxytamoxifen; 3,4-di-OHTAM-diSG, di-glutathione conjugates of 3,4-dihydroxytamoxifen; 4-OHTOR, 4-hy- droxytoremifene; 3,4-di-OHTOR, 3,4-dihydroxytoremifene; 4-OHTOR- diSG, di-glutathione conjugates of 4-hydroxytoremifene; 3,4-di-OHTOR- diSG, di-glutathione conjugates of 3,4-dihydroxytoremifene; 3,4-di- OHTOR-triSG, tri-glutathione conjugates of 3,4-dihydroxytoremifene; 3,4-di-OHTOR-T, thymidine adducts of 3,4-di-OHTOR; 3,4-di-OHTOR- dG, deoxyguanosine adducts of 3,4-di-OHTOR; 3,4-di-OHTOR-dA, deoxyadenosine adducts of 3,4-di-OHTOR; 3,4-di-OHTOR-dC, deoxy- cytosine adducts of 3,4-di-OHTOR; P450, cytochrome P450; GSH, glutathione; electrospray-MS, electrospray mass spectrometry; MS- MS, tandem mass spectrometry; ER, estrogen receptor. 1643 Chem. Res. Toxicol. 2001, 14, 1643-1653 10.1021/tx010137i CCC: $20.00 © 2001 American Chemical Society Published on Web 11/27/2001