Synthesis and Reactivity of the Catechol Metabolites from the Equine

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Synthesis and Reactivity of the Catechol Metabolites from the Equine Estrogen, 8,9-Dehydroestrone Fagen Zhang, Dan Yao, Yousheng Hua, 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 March 1, 2001

The risk factors for women developing breast and endometrial cancers are all associated with a lifetime of estrogen exposure. Estrogen replacement therapy in particular has been correlated with an increased cancer risk. Previously, we showed that the equine estrogens equilin and equilenin, which are major components of the widely prescribed estrogen replacement formulation Premarin, are metabolized to highly cytotoxic quinoids which caused oxidative stress and alkylation of DNA in vitro [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. Chem. Res. Toxicol. 1998, 11, 1113-1127]. In this study, we have synthesized 8,9-dehydroestrone (a third equine estrogen component of Premarin) and its potential catechol metabolites, 4-hydroxy-8,9-dehydroestrone and 2-hydroxy-8,9-dehydroestrone. Both 2-hydroxy8,9-dehydroestrone and 4-hydroxy-8,9-dehydroestrone were oxidized by tyrosinase or rat liver microsomes to o-quinones which reacted with GSH to give one mono-GSH conjugate and two di-GSH conjugates. Like endogenous estrogens, 8,9-dehydroestrone was primarily converted by rat liver microsomes to the 2-hydroxylated rather than the 4-hydroxylated o-quinone GSH conjugates; the ratio of 2-hydroxy-8,9-dehydroestrone versus 4-hydroxy-8,9-dehydroestrone was 6:1. Also in contrast to experiments with equilin, 4-hydroxyequilenin was not observed in microsomal incubations with 8,9-dehydroestrone or its catechols. The behavior of 2-hydroxy8,9-dehydroestrone was found to be more complex than 4-hydroxy-8,9-dehydroestrone as GSH conjugates resulting from 2-hydroxy-8,9-dehydroestrone were detected even without oxidative enzyme catalysis. Under physiological conditions, 2-hydroxy-8,9-dehydroestrone isomerized to 2-hydroxyequilenin to form the very stable 2-hydroxyequilenin catechol; however, 4-hydroxy8,9-dehydroestrone was found to be stable under similar conditions. Finally, preliminary studies conducted with the human breast tumor S-30 cell lines demonstrated that the catechol metabolites of 8,9-dehydroestrone were much less toxic than 4-hydroxyequilenin (20-40-fold). These results suggest that the catechol metabolites of 8,9-dehydroestrone may have the ability to cause cytotoxicity in vivo primarily through formation of o-quinones; however, most of the adverse effects of Premarin estrogens are likely due to formation of 4-hydroxyequilenin o-quinone from equilin and equilenin.

Introduction There are many benefits of estrogen replacement therapy including relief of menopausal symptoms, prevention of osteoporosis, and cardiovascular disease (1). In addition, there is some evidence that estrogen replacement therapy can protect women from Alzheimer’s disease (2-5) and stroke (6, 7), and improve motor disability in Parkinsonian postmenopausal women with motor fluctuations (8). Equine estrogens make up approximately 50% of the estrogens in the widely prescribed estrogen replacement therapy marketed under the name of Premarin (Wyeth-Ayerst). One of these equine estrogens, 8,9-dehydroestrone differs from endogenous human estrogens in that it contains a 8,9-double bond in the B ring. The presence of 8,9-dehydroestrone1 in Premarin is the primary reason that there has never been a generic replacement for Premarin (9). Recent studies have shown that 8,9-dehydroestrone has estrogenic activity, novel * To whom correspondence should be addressed. Fax: (312) 9967107. E-mail: [email protected].

tissue selectivity in postmenopausal women, and a distinct pharmacological profile that results in significant clinical activity in vasomotor, neuroendocrine, and bone preservation parameters (10, 11). Despite the numerous benefits of estrogen replacement therapy, there have been troubling, controversial reports concerning the increase in risk of developing breast and endometrial cancers, particularly for women on long-term high dose estrogen replacement therapy (12-18). The exact mechanism(s) by which estrogens cause these hormone-dependent cancers is unknown. One mechanism 1 Abbreviations: 8,9-dehydroestrone, 3-hydroxy-1,3,5(10),8,9-estratetraen-17-one; 2-hydroxy-8,9-dehydroestrone, 2,3-dihydroxy-1,3,5(10),8,9-estratetraen-17-one; 4-hydroxy-8,9-dehydroestrone, 3,4-dihydroxy-1,3,5(10),8,9-estratetraen-17-one;equilenin,3-hydroxy-1,3,5(10),6,8estrapentaen-17-one; 4-hydroxyequilenin, 3,4-dihydroxy-1,3,5(10),6,8estrapentaen-17-one; 2-hydroxyequilenin, 3,4-dihydroxy-1,3,5(10),6,8estrapentaen-17-one; 4-hydroxyequilin, 3,4-dihydroxy-1,3,5(10),7(8)estratetraen-17-one; 2-hydroxyequilin, 2,3-dihydroxy-1,3,5(10),7(8)estratetraen-17-one; 4-hydroxyestrone, 3,4-dihydroxy-1,3,5(10)-estratrien17-one; estrone, 3-hydroxy-1,3,5(10)-estratrien-17-one; equilenin, 1,3,5(10),6,8-estrapentaen-3-ol-17-one; equilin, 1,3,5(10),7-estratetraen3-ol-17-one; LC-MS, liquid chromatography-mass spectrometry; ER, estrogen receptor.

10.1021/tx010049y CCC: $20.00 © 2001 American Chemical Society Published on Web 05/18/2001

Catechol Metabolites from 8,9-Dehydroestrone

Chem. Res. Toxicol., Vol. 14, No. 6, 2001 755

Scheme 1. Synthesis of 8,9-Dehydroestrone

Scheme 2. Synthesis of 2-Hydroxy-8,9-dehydroestrone

might involve metabolism of estrogens to catechols, which are then oxidized to redox active/electrophilic o-quinones that could initiate the carcinogenic process by binding to cellular macromolecules. For example, it is well established that the endogenous estrogens, estrone and 17β-estradiol, are metabolized by P450 enzymes to 2- and 4-hydroxylated catechols (19, 20). The o-quinones formed from peroxidase/P450-catalyzed oxidation of these catechols have previously been implicated as the ultimate carcinogens. Redox cycling between the catechols and their quinones generates reactive hydroxyl radicals which cause oxidation of the purine/pyrimidine residues of DNA (21). The o-quinones could also alkylate DNA to form adducts which have been detected both in vitro (22-24) and in vivo (25-27). There have also been a few studies on the potential for equine estrogens to be metabolized to reactive catechol intermediates (28-31). It has been demonstrated that increasing unsaturation in the B ring leads to a change in product distribution from primarily

2-hydroxylation as observed for endogenous estrogens to mainly 4-hydroxylation for equilenin. Recently, we have found that the equine catechol estrogens, 4-hydroxyequilenin and 4-hydroxyequilin, form unusual cyclic adducts with DNA in vitro (32-34). If the similar adducts are formed in vivo, which are not repaired efficiently, mutations could result leading to initiation of the carcinogenic process in hormone sensitive tissues. There has only been one report on the metabolism of 8,9-dehydroestrone which suggested that the only metabolite was 17β-hydroxy-8,9-dehydroestradiol (35). To accurately study the potential for catechol formation from 8,9-dehydroestrone, we synthesized 8,9-dehydroestrone (Scheme 1), 4-hydroxy-8,9-dehydroestrone (Scheme 2), and 2-hydroxy-8,9-dehydroestrone (Scheme 3). We then examined the metabolism of 8,9-dehydroestrone in rat liver microsomes and showed that both 2-hydroxylated and 4-hydroxylated GSH conjugates were formed (Scheme 4). Finally, we tested the relative toxicity of 8,9-dehy-

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Zhang et al.

Scheme 3. Synthesis of 4-Hydroxy-8,9-dehydroestrone

Scheme 4. Metabolism of 8,9-Dehydroestrone to o-Quinones and Reaction with GSH

droestrone and its catechol metabolites in breast cancer cells. The data suggest that although catechols were formed from 8,9-dehydroestrone, they were considerably less toxic then 4-hydroxyequilenin which implies that quinoids formed from equilin and equilenin are more likely to contribute to the adverse effects of Premarin.

Materials and Methods Caution: The catechol estrogen o-quinones were handled in accordance with NIH guidelines for the Laboratory Use of Chemical Carcinogens (36). All chemicals were purchased from Aldrich (Milwaukee, WI), Fisher Scientific (Itasca, IL), or Sigma (St. Louis, MO) unless stated otherwise. (3H)-GSH (glycine-23H) was obtained from NEN Life Science Products, Inc. (Boston, MA) and diluted to a specific activity of 40 mCi/mmol. 2-Hydroxyequilenin was synthesized as described previously (37). Instrumentation. HPLC experiments were performed on a Shimadzu (Columbia, MD) LC-10A gradient HPLC equipped with a SIL-10A auto injector, SPD-M10AV UV-vis photodiode array detector, and SPD-10AV detector. Peaks were integrated with Shimadzu EZ-Chrom software. UV spectra were measured

on Hewlett-Packard (Palo Alto, CA) UV-vis spectrophotometer. NMR spectra were obtained with a Bruker (Billerica, MA) Avance DPX300 spectrometer at 300 MHz. Positive ion electrospray mass spectra were obtained using a Hewlett-Packard 5989B MS Engine quadrupole mass spectrometer equipped with a ChemStation data system and high-flow pneumatic nebulizerassisted electrospray LC-MS interface. The mass spectrometer was interfaced to a Hewlett-Packard 1050 gradient HPLC system. The quadrupole analyzer was maintained at 120 °C and unit resolution was used for all measurements. Nitrogen at a pressure of 80 psi was used for nebulization of the HPLC effluent, and nitrogen bath gas at 300 °C and a flow rate of 10 L/min were used for evaporation of solvent from the electrospray. A mass range from 300 to 1050 mass units were scanned every 1.5 s (1.5 s/scan). Tandem MS (MS-MS) spectra were obtained using a Micromass (Manchester, U.K.) Quattro II triple quadrupole mass spectrometer equipped with an electrospray ionization source. Collision-induced dissociation was carried out using a range of collision energy from 25 to 70 eV and argon collision gas pressure of 2.7 µbar. 1H

HPLC Methodology. Two general methods were used to analyze and separate the various metabolites and GSH conju-



Scheme 5. Mechanism of Isomerization of 2-Hydroxy-8,9-dehydroestrone to 2-Hydroxyequilenin

gates. All retention times reported in the text were obtained using Method A. Method A. Analytical HPLC analysis was performed using a 4.6 × 250 mm Ultrasphere C-18 column (Beckman) on the Shimadzu HPLC system described above. The mobile phase consisted of 20% acetonitrile in 0.25% perchloric acid/0.25% acetic acid (pH 3.5) at a flow rate of 1.0 mL/min for 5 min, increased to 18% acetonitrile over 5 min, isocratic for 50 min, increased to 45% over the next 10 min, and increased to 90% acetonitrile over the last 5 min of the run. Method B. For LC-MS-MS analysis of GSH conjugates of catechol metabolites from 8,9-dehydroestrone, analytical HPLC analysis was performed using a 4.6 × 250 mm Ultrasphere C-18 column (Beckman) on the above-mentioned Hewlett-Packard HPLC system, and tandem mass spectra were obtained using the Micromass system interfaced with the Hewlett-Packard HPLC system. The mobile phase consisted of 20% acetonitrile in 0.5% ammonium acetate (pH 3.5) at a flow rate of 1.0 mL/ min for 5 min, increased to 18% acetonitrile over 5 min, isocratic for 50 min, increased to 45% over the next 10 min, and increased to 90% acetonitrile over the last 5 min of the run. Synthesis of 8,9-Dehydroestrone (Scheme 1). (1) 6-tertButyldimethylsiloxytetralone, 2. 6-tert-Butyldimethylsiloxytetralone was synthesized as described in the literature (38). Briefly, to a stirred mixture of 6-hydroxy-1-tetralone 1 (2.5 g, 15.4 mmol) and imidazole (2.6 g, 38.5 mmol) in DMF (14 mL) under a stream of nitrogen at room temperature was added tertbutyldimethylsilyl chloride (2.9 g, 18.7 mmol). After stirring for 18 h, the reaction mixture was diluted with diethyl ether (280 mL). The resulting mixture was then washed with 1 M HCl (200 mL) and brine (2 × 50 mL), dried over MgSO4, filtered, and the solvent removed en vacuo. The residue obtained was purified by flash chromatography (silica gel, hexane/ethyl acetate, 3:1) to give 2 as a pale yellow liquid (4.6 g, 96% yield): 1H NMR (CDCl3) δ 0.23 (s, 6H, Si(CH3)2), 0.97 (s, 9H, C(CH3)3), 2.09 (m, 2H, CH2), 2.61 (t, 2H, J ) 6.3 Hz, CH2), 2.90 (t, 2H, J ) 6.3 Hz, CH2), 6.65 (s, 1H, ArH), 6.74 (d, 1H, J ) 8.4 Hz, ArH), 7.96 (d, 2H, J ) 8.4 Hz, ArH); 13C NMR (CDCl3) δ -4.09, 18.4, 23.6, 25.8, 30.1, 39.1, 118.9, 119.3, 127.1, 129.7, 147.0, 160.5, 197.5; CI-MS m/z 277 (100%) [M + H]+. (2) 6-tert-Butyldimethylsiloxy-1-vinyl-3,4-dihydronaphathalene, 3. To a flame dried flask, 6-tert-butyldimethylsiloxytetralone 2 (4.6 g, 16.6 mmol) and anhydrous THF (50 mL) were added. Vinylmagnesium bromide (50 mL, 1 M in THF) was added to this mixture through a dropping funnel at room temperature. After stirring for 16 h, the mixture was refluxed for 40 min, cooled to room temperature, and HCl (60 mL, 1 M) was added through a dropping funnel. The final reaction mixture was extracted with diethyl ether (2 × 100 mL). The combined organic layers were washed with saturated NaHCO3 (2 × 60 mL) and brine (2 × 60 mL), dried over Na2SO4, filtered,

and the solvent removed in vacuo to give a yellow liquid which was purified by column chromatography (silica gel) using ethyl acetate/hexane (1:8 v/v) as eluent to give 3 (2.5 g, 52% yield): 1H NMR (CDCl ) δ 0.23 (s, 6H, Si(CH ) ), 0.99 (s, 9H, C(CH ) ), 3 3 2 3 3 2.30 (m, 2H, CH2), 2.69 (t, 2H, CH2), 5.15 (dd, 1H, Jcis ) 10.6 Hz, Jgem ) 1.8 Hz, Hcis of CH2dCH), 5.50 (dd, 1H, Jtrans ) 10.6 Hz, Jgem ) 1.8 Hz, Htrans of CH2dCH), 6.08 [m, 1H, H-C(2)], 6.56 (m, 1H, CH2dCH), 6.66 (m, 2H, ArH), 7.20 (d, 1H, J ) 9 Hz, ArH); CI-MS m/z 287 (100%) [M + H]+. (3) 3-tert-Butyldimethylsiloxy-8,9-dehydroestrone, 4. The cycloaddition reaction procedure described in the literature was followed (39). To a 100 mL, three-necked, round-bottom flask, a solution of TiCl4 (1 mL, 9.15 mmol) in dry CH2Cl2 (15 mL) was added to a solution of 2-methylcyclopent-2-en-1-one (0.32 g, 3.33 mmol) at -78 °C. The yellow mixture was stirred for 15 min and a solution of 6-tert-butyldimethylsiloxy-1-vinyl-3,4dihydronaphathalene 3 (2.32 g, 8.40 mmol) in dry CH2Cl2 (15 mL) was added dropwise over 1 h. The mixture was stirred for another 5 h at -78 °C until 2-methylcyclopent-2-en-1-one could not be detected by TLC (silica gel, hexane/ethyl acetate ) 8:1). The mixture was warmed to 0 °C and 1 M HCl (70 mL) was added. The final mixture was extracted with ethyl ether (2 × 250 mL), washed with brine (2 × 250 mL), dried over sodium sulfate, filtered, and evaporated to give a deep yellow liquid which was further purified by flash column chromatography (silica gel) using ethyl acetate/hexane (1:8 v/v) as eluent to give 4 as a yellow power (0.66 g, 7.2% yield): 1H NMR (acetone-d6) δ 0.21 (s, 6H, Si(CH3)2), 0.99 (s, 9H, C(CH3)3), 1.02 (s, 3H, CH3), 1.41 (m, 1H), 1.77 (m, 2H), 2.12 (m, 1H), 2.36 (m, 5H), 2.47 (m, 1H), 2.70 (m, 2H), 2.80 (m, 1H), 6.63 (s, 1H), 6.68 (d, 1H, J ) 8.3 Hz, ArH), 6.88 (d, 1H, J ) 8.3 Hz, ArH); 13C NMR (acetoned6) δ -4.25 [Si(CH3)2], 18.7 [C(CH3)3], 20.7, 22.5, 26.2, 26.4, 27.4, 28.1 29.5, 37.0, 47.4, 49.2, 118.2, 119.8, 123.9, 126.8, 130.6, 133.4, 137.9, 154.8, 221.7 (C17); CI-MS (positive ion, methane) m/z 383 (100%) [M + H]+. (4) 8,9-Dehydroestrone, 5. To a flame dried flask, 6-tertbutyldimethylsiloxy-8,9-dehydroestrone 4 (300 mg, 0.785 mmol), dry THF (15 mL), and Bu4NF (1.5 mL, 1 M in THF) were added. The mixture was stirred for 20 min at room temperature, followed by addition of THF (50 mL). This solution was washed with brine (2 × 50 mL), dried over Na2SO4, filtered, and the solvent was removed en vacuo to give a yellow residue which was further purified by flash chromatography (silica gel, hexane: acetone ) 2:1 + 1% MeOH) to give 8,9-dehydroestrone 5 (160 mg, 76% yield). Complete characterization was accomplished by 1D and 2D NMR experiments including 1H, 13C, HMQC, HMBC, and COSY: 1H NMR (acetone-d6) δ 1.01 (s, 3H, 18-CH3), 1.41 (m, 1H, H11), 1.74 (m, 2H, H12), 2.12 (m, 1H, H12), 2.27 (m, 5H, 2 × H15), 2.49 (m, 1H, H16), 2.4 (m, 2H, H11), 2.67 (m, 1H, H14), 6.63 (m, 2H, H4), 7.10 (s, 1H, J ) 8.3 Hz, H1), 8.18 (bs, 1H, OH3); 13C NMR (acetone-d6) δ 20.7 (C18), 22.6 (C15), 26.2

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(C6), 27.5 (C11,), 28.1 (C12), 29.4 (C16), 37.0 (C9), 47.4 (C13), 49.2 (C14), 113.4 (OCH3), 115.1 (C1), 124.1 (C4), 126.9 (C7), 128.8 (C5), 132.2 (C10), 137.9 (C8), 146.3 (C2), 156.7(C3), 221.8 (C17); UV (CH3OH), 220, 286 nm; EI-MS m/z 268 (100%) [M]+•. Synthesis of 2-Hydroxy-8,9-dehydroestrone (Scheme 2). (1) 6,7-Dihydroxy-1-tetralone, 7. To a flame dried flask, 6,7dimethoxy-1-tetralone 6 (5.0 g, 24 mmol), anhydrous AlCl3 (25.5 g, 192 mmol), and benzene (100 mL) were added under a stream of nitrogen. The mixture was refluxed for 2.5 h, cooled to room temperature, and 3 N HCl (100 mL) was added at 0 °C. After filtration, the deep yellow solid was collected and recrystallized from hot water to gave 6,7-dihydroxy-1-tetralone 7 (1.5 g, 35% yield): 1H NMR (acetone-d6) δ 2.04 (m, 2H, CH2), 2.44 (t, 2H, J ) 6.3 Hz, CH2), 3.11 (t, 2H, J ) 6.3 Hz, CH2), 6.71 (s, 1H, ArH), 7.35 (s, 1H, ArH), 8.44 (b, 2H, OH); 13C NMR (CDCl3) δ 23.8, 25.8, 39.1, 119.1, 119.7, 127.3, 129.9, 147.2, 152.5, 196.5; CIMS m/z 179 (100%) [M + H]+. (2) 6,7-Di-tert-butyldimethylsiloxytetralone, 8. 6,7-Di-tertbutyldimethylsiloxytetralone 8 (3.2 g, 94% yield) was prepared by reacting 6,7-dihydroxy-1-tetralone 7 (1.5 g, 8.42 mmol) with tert-butyldimethylsilyl chloride (3.17 g, 20.5 mmol) in DMF (20 mL) and imidazole (2.84 g, 42.1 mmol) according to the same procedure described above: 1H NMR (acetone-d6) δ 0.24 (s, 6H, Si(CH3)2), 0.27 (s, 6H, Si(CH3)2), 1.01 (s, 18H, C(CH3)3), 2.05 (m, 2H, CH2), 2.50 (t, 2H, J ) 7.0 Hz, CH2), 2.84 (t, 2H, J ) 7.0 Hz, CH2), 6.81 (s, 1H, ArH), 7.46 (d, 1H, ArH); 13C NMR (acetone-d6) δ -4.50, 18.3, 18.4, 23.6, 25.5, 25.6, 118.2, 120.3, 139.3, 145.7, 151.8,195.6; CI-MS m/z 407 (100%) [M + H]+. (3) 6,7-Di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaphathalene, 9. 6,7-Di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaphathalene 9 (1.08 g, 31% yield) was obtained by reacting 6,7-ditert-butyldimethylsiloxytetralone 8 (3.5 g, 8.6 mmol) with vinylmagnesium bromide (50 mL, 1 M in THF) following the same procedure as described above for 6-tert-butyldimethylsiloxy-1vinyl-3,4-dihydronaphathalene 3: 1H NMR (CDCl3) δ 0.23 (s, 12H, Si(CH3)2), 0.99 (s, 18H, C(CH3)3), 2.21 (m, 2H, CH2), 2.62 (m, 2H, CH2), 5.15 (dd, 1H, Jcis ) 10.6 Hz, Jgem ) 1.8 Hz, Hcis of CH2dCH), 5.51 (dd, 1H, Jtrans ) 10.6 Hz, Jgem ) 1.8 Hz, Htrans of CH2dCH), 6.07 [m, 1H, H-C(2)], 6.56 (m, 1H, CH2dCH), 6.64 (s, 1H, ArH), 6.89 (d, 1H, ArH); CI-MS m/z 417 (100%) [M + H]+. (4) 2,3-Di-tert-butyldimethylsiloxy-8,9-dehydroestrone, 10. According to the same cycloaddition reaction procedure described above, 6,7-di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaphathalene 9 (1.8 g, 4.3 mmol) was converted into 2,3-di-tertbutyldimethylsiloxy-8,9-dehydroestrone 10 (250 mg, 11% yield) by reaction with 2-methylcyclopent-2-en-1-one (0.21 g, 2.18 mmol) in the presence of TiCl4 (6.4 mmol): 1H NMR (acetoned6) δ 0.22 (s, 12H, Si(CH3)2), 0.99 (s, 18H, C(CH3)3), 1.02 (s, 3H, CH3), 1.41 (m, 1H), 1.73 (m, 2H), 2.15 (m, 1H), 2.30 (m, 5H), 2.40 (m, 1H), 2.65 (m, 2H), 2.81 (m, 1H), 6.67 (s, 1H), 6.74 (d, 1H, ArH); 13C NMR (acetone-d6) δ -3.69, 19.14, 20.8, 22.6, 26.3, 27.4, 28.1, 28.4, 29.5, 37.0, 47.4, 49.4, 116.3, 121.0, 126.7, 129.6, 130.6, 133.8,145.6, 147.5, 221.7 (C17); APCI-MS (positive ion, methane) m/z 513 (100%) [M + H]+. (5) 2-Hydroxy-8,9-dehydroestrone, 11. 2-Hydroxy-8,9-dehydroestrone 11 (8 mg, 37% yield) was obtained by deprotection of 2,3-di-tert-butyldimethylsiloxy-8,9-dehydroestrone 10 (50 mg, 0.097 mmol) with Bu4NF (0.5 mL, 1.0 M in THF) according to the same procedure described above: 1H NMR (acetone-d6) δ 1.01 (s, 3H, CH3), 1.41 (m, 1H), 1.74 (m, 2H), 2.15 (m, 1H), 2.26 (m, 5H), 2.40 (m, 1H), 2.55 (m, 2H), 2.91 (m, 1H), 6.61 (s, 1H, ArH), 6.72 (s, 1H, ArH), 7.60 (b, 2H, OH); CI-MS m/z 285 (100%) [M + H]+. Synthesis of 4-Hydroxy-8,9-Dehydroestrone (20) (Scheme 3). (1) 5,6-Dihydroxy-1-tetralone, 16. 2-Carboxyethyltriphenylphosphonium bromide was obtained by refluxing triphenylphosphine with 3-bromopropanoic acid in the presence of benzene (40). 2,3-Dimethoxybenzaldehyde 12 was treated with 2-carboxyethyltriphenylphosphonium bromide in the presence of NaH and DMSO to give 4-(3′,5′-dimethoxyphenyl)-3-butenoic acid bromide 13 (41). Hydrogenation of 13 using 5% Pd/C gave

Zhang et al. 4-(2′,3′-dimethoxyphenyl)butanoic acid 14 (42). Compound 14 was cyclized with polyphosphoric acid (PPA) to produce 5,6dimethoxy-1-tetralone 15 (42). Demethylation of 5,6-dimethoxy1-tetralone 15 using anhydrous AlCl3 /benzene gave 5,6dihydroxy-1-tetralone 16: 1H NMR (acetone-d6) δ 2.07 (m, 2H, CH2), 2.49 (t, 2H, J ) 6.3 Hz, CH2), 2.97 (t, 2H, J ) 6.3 Hz, CH2), 6.81 (d, 1H, J ) 8.4 Hz, ArH), 7.46 (d, 1H, J ) 8.4 Hz, ArH), 9.17 (b, 2H, OH); 13C NMR (CDCl3) δ 23.5, 23.7, 39.1, 113.8, 120.2, 126.8, 132.9, 142.3, 149.9, 197.1; CI-MS m/z 179 (100%) [M + H]+. (2) 5,6-Di-tert-butyldimethylsiloxytetralone, 17. 5,6-Di-tertbutyldimethylsiloxytetralone 17 was prepared by reacting 5,6dihydroxy-1-tetralone 16 (2.6 g, 14.5 mmol) with tert-butyldimethylsilyl chloride (7.2 g, 46.44 mmol) in DMF (50 mL) and imidazole (6.8 g, 100.8 mmol) according to the same procedure described above (6.4 g, 94% yield): 1H NMR (acetone-d6) δ 2.05 (m, 2H, CH2), 2.52 (m, 2H, J ) 7.0 Hz, CH2), 2.93 (m, 2H, CH2), 6.92 (d, 1H, J ) 8.4 Hz, ArH), 7.58 (d, 1H, J ) 8.4 Hz, ArH); CI-MS m/z 407 (100%) [M + H]+. (3) 5,6-Di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaphathalene, 18. 5,6-Di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaphathalene 18 (1.1 g) was obtained by reacting 5.6-di-tert-butyldimethylsiloxytetralone 17 (4.21 g, 10.4 mmol) with vinylmagnesium bromide (70 mL, 1 M in THF) following the same procedure as described above for 6-tert-butyldimethylsiloxy-1vinyl-3,4-dihydronaphathalene. (4) 3,4-Di-tert-butyldimethylsiloxy-8,9-dehydroestrone, 19. According to the same cycloaddition reaction procedure described above, 5,6-di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaphathalene 18 (1.1 g, 2.64 mmol) was converted into 3,4-di-tertbutyldimethylsiloxy-8,9-dehydroestrone 19 (30 mg, 2.5% yield) by reaction with 2-methylcyclopent-2-en-1-one (0.16 g, 1.66 mmol) in the presence of TiCl4 (6.4 mmol): 1H NMR (acetone-d6) δ 6.71 (d, 1H, J ) 8.4 Hz, ArH), 6.76 (d, 1H, J ) 8.4 Hz, ArH); APCI-MS (positive ion) m/z 513 (100%) [M + H]+. (5) 4-Hydroxy-8,9-dehydroestrone, 20. 3,4-Di-tert-butyldimethylsiloxy-8,9-dehydroestrone 19 (25 mg, 1.95 mmol) was deprotected using Bu4NF to give 4-hydroxy-8,9-dehydroestrone 20 (7 mg, 51% yield): 1H NMR (acetone-d6) δ 1.02 (s, 3H, CH3), 1.42 (m, 1H), 1.80 (m, 2H), 2.10 (m, 1H), 2.30 (m, 5H), 2.45 (m, 1H), 2.55 (m, 2H), 2.80 (m, 1H), 6.61 (d, 1H, J ) 8.4 Hz, ArH), 6.66 (d, 1H, J ) 8.4 Hz, ArH), 7.11 (b, 1H, OH), 8.24 (b, 1H, OH), Negative APCI-MS m/z 283 (100%) [M - H]-. GSH Conjugates of 2-Hydroxy-8,9-dehydroestrone-oquinone and 4-Hydroxy-8,9-dehydroestrone-o-quinone. The o-quinone GSH conjugates of 2-hydroxy-8,9-dehydroestrone and 4-hydroxy-8,9-dehydroestrone were prepared by incubating the corresponding catechol (0.5 mM) with 5.0 mM GSH and tyrosinase (0.4 µg/mL) in 2 mL of 50 mM sodium phosphate buffer (pH 7.4), 37 °C for 30 min. The conjugates were isolated from the aqueous phase using solid-phase extraction cartridges (Oasis; Waters Corporation, Milford, MA) and eluted with methanol. The methanol extract was concentrated to 150 µL and aliquots (25 µL) were analyzed directly by HPLC. Two diGSH conjugates and one mono-GSH conjugates (Scheme 4) were obtained with both 2-OHDHES and 4-OHDHES. Since the tandem mass spectrometry of the di-SG and mono-SG conjugates showed a similar pattern for each GSH conjugate, MSMS data are only reported for the 4-OHDHES-diSG and 4-OHDHES-SG (method A): 4-OHDHES-diSG1, UV (CH3OH/ H2O) 256 nm, positive ion electrospray MS m/z 895 [M + H]+ (100%); MS-MS with CID of m/z 895 gave m/z 877 [MH - H2O]+ (10%), 820 [MH - Gly]+ (5%), 766 [MH - Glu]+ (30%), 637 [MH - 2Glu]+ (20%), retention time 20 min; 4-OHDHES-diSG2, UV (CH3OH/H2O) 262 nm, positive ion electrospray MS m/z 895 [M + H]+ (100%); CID MS-MS pattern same as 4-OHDHES-diSG1, retention time 21 min; 4-OHDHES-SG, UV (CH3OH/H2O) 255 nm, positive ion electrospray MS m/z 590 [M + H]+ (100%); MSMS with CID of m/z 590 gave m/z 515 [MH - Gly]+ (20%), 461 [MH - Glu]+ (40%), 358 [MH - 2Glu]+ (60%), 315 [MH - SG + 32]+, retention time 55 min; 2-OHDHES-diSG1, UV (CH3OH/

Catechol Metabolites from 8,9-Dehydroestrone H2O) 260, 334 nm; positive ion electrospray MS m/z 895 [M + H]+ (100%); MS-MS pattern same as 4-OHDHES-diSG1, retention time 24 min; 2-OHDHES-diSG2, UV (CH3OH/H2O) 257, 335 nm, positive ion electrospray MS m/z 895 [M + H]+ (100%), retention time 25 min; 2-OHDHES-SG, UV (CH3OH/H2O) 272, 337 nm, positive ion electrospray MS m/z 590 [M + H]+ (100%); retention time 51 min. Kinetic Studies. The rate of formation 2-hydroxyequilenin from the autoxidation of 2-hydroxy-8,9-dehydroestrone (0.5 mM) in potassium phosphate buffer (pH 7.4, 37 °C) was determined by monitoring product formation by UV-visible spectroscopy using a Hewlett-Packard HP8452 diode array spectrophotometer. After 1.5 h, the pH was adjusted to 4 and the solution was extracted with CHCl3 (2 mL). The organic layer was concentrated and analyzed by positive ion CI-MS (methane). The [M + H]+ ion of 2-hydroxy-8,9-dehydroestrone (285) was reduced 95% compared to the starting material and the [M + H]+ ion of 2-hydroxyequilenin (283) was the base peak in the spectrum. Incubations. Female Sprague-Dawley rats (180-200 g) were obtained from Sasco Inc. (Omaha, NE). The rats were pretreated with dexamethasone to induce P450 3A isozymes (43). Dexamethasone was administered by i.p. injections of 100 mg/kg in corn oil daily for 4 days and the animals were sacrificed on day 5. Microsomes were prepared from rat liver, and protein and P450 concentrations determined as described previously (44). Incubations containing microsomal protein were conducted for 10-30 min at 37 °C in 50 mM phosphate buffer (pH 7.4, 500 µL total volume). Substrates were added as solutions in dimethyl sulfoxide, and (3H)-GSH (specific activity of 40 mCi/ mmol) was added in phosphate buffer to achieve final concentrations of 0.5 and 1.0 mM, respectively. A NADPH generating system consisting of 1.0 mM NADP+, 5 mM isocitric acid, and 0.2 units of isocitric dehydrogenase/mL were used together with 5.0 mM MgCl2. For control incubations NADP+ was omitted. The reactions were initiated by the addition of NADP+, and terminated by chilling in an ice bath followed by the addition of perchloric acid (25 µL). Adduct Quantification. The reaction mixtures were centrifuged at 13 000 rpm for 6 min to remove precipitated microsomal protein. Aliquots of the supernatant (100 µL) were analyzed directly by HPLC as described above for the GSH conjugates. For quantification of GSH conjugates, 0.3 mL aliquots of the column effluent were collected every 18 s during each run, and radioactivity was measured with a Beckman model LS 5801 liquid scintillation counter. Concentrations of the GSH conjugates were calculated by summing the radioactivity associated with each peak and converting the data to nanomolar amounts using the specific activity of the (3H)-GSH. Each analysis was performed immediately after the incubation in order to limit degradation of the o-quinone GSH conjugates. Cytotoxicity Experiments in S-30 Breast Cancer Cells. Cell viability was determined by Trypan blue exclusion (45, 46). The S-30 cell line was a generous gift from V. C. Jordan (Northwestern University, Evanston, IL). Briefly, S-30 cells were maintained in MEME supplemented with 1% penicillin, streptomycin, fungizome, 10 mM nonessential amino acids, and 5% 2× stripped bovine serum. The medium was changed 24 h before beginning cytotoxicity assays to maintain logarithmic growth. The cells were treated with 2-OHDHES, 4-OHDHES, 4-OHEN, 8,9-dehydroestrone, and equilenin for 18 h. The test samples were assayed in triplicate, and final concentrations ranged from 1.6 to 500 µM. Each assay included negative controls (cells treated with DMSO only) that were used to define 100% cell viability. After treatment, floating cells were collected by centrifugation at 3000 rpm for 5 min, and attached cells were first trypsinized and then harvested by centrifugation. Floating and attached cells were combined, washed with PBS, and stained with 0.4% Trypan blue. A drop of cell suspension was placed on a hemocytometer and the cell numbers were determined under a microscope. The dead cells were stained blue while viable cells remained unstained. The LC50 values were

Chem. Res. Toxicol., Vol. 14, No. 6, 2001 759 obtained by regression and linear estimation analysis. The data represent the mean ( SD of triplicate determinations.

Results and Discussion Synthesis of 8,9-Dehydroestrone. There are reports in the literature on the synthesis of 8,9-dehydroestrone and 3-methoxy-8,9-dehydroestrone (39, 47-49). One method involved isomerization of equilin to 8,9-dehydroestrone in the presence of glacial acetic acid and hydrochloric acid (47). However, this study did not report sufficient spectral data in order to unequivocally confirm the structure of 8,9-dehydroestrone. When this method was attempted, only a mixture of products resulted which could not be separated by chromatography. Other literature synthetic methods included multistep reactions (39, 48, 49). However, all of these methods concentrated on the synthesis of 3-methoxy-8,9-dehydroestrone which could not be easily converted into 8,9-dehydroestrone. Demethylation of 3-methoxy-8,9-dehydroestrone was attempted by using mild demethylation agents, such as BBr3 (50), BBr3‚SMe2 (51), AlCl3/C2H5SH (52), and BCl3 (53); however, these experiments were not successful due to the instability of the conjugated 8,9-double bond in the B ring. Therefore, an alternative method for the synthesis of 8,9-dehydroestrone was developed (Scheme 1). In this methodology, the commercially available 6-hydroxy-1tetralone 1 was used as starting material. After protection of the 6-hydroxy group with tert-butyldimethylsilyl chloride, 6-tert-butyldimethylsiloxy-1-tetralone 2 was obtained. The protected tetralone was converted into 1-vinyl-6-tert-butyldimethylsiloxy-3,4-dihydronaphathalene 3 by reacting compound 2 with commercially available vinylmagnesium bromide. Catalytic cycloaddition of 1-vinyl-6-tert-butyldimethylsiloxy-3,4-dihydronaphathalene with 2-methylcyclopent-2-en-1-one in the presence of TiCl4 gave 3-tert-butyldimethylsiloxy-8,9-dehydroestrone 4. Removal of the protecting group under mild conditions with Bu4NF gave 8,9-dehydroestrone 5. On the basis of this methodology, the potential catechol metabolites of 8,9-dehydroestrone, 4-hydroxy-8,9-dehydroestrone (Scheme 3) and 2-hydroxy-8,9-dehydroestrone (Scheme 2), were also synthesized. GSH Conjugates of 2-Hydroxy-8,9-dehydroestrone- and 4-Hydroxy-8,9-dehydroestrone-o-quinone. Previous studies have shown that enzymatic oxidation of endogenous catechol estrogens generated o-quinones which could be trapped in situ by GSH (54, 55). In addition, we have shown that 4-hydroxyequilenin and 4-hydroxyequilin readily autoxidized to o-quinones which reacted with GSH to form the corresponding GSH conjugates at physiological pH and temperature (50, 56). In contrast, 4-hydroxy-8,9-dehydroestrone was found to be very stable under physiological conditions; however, it could be oxidized to an o-quinone by oxidative enzymes. This o-quinone was trapped with GSH to form one monoGSH conjugate (retention time 55 min) and two di-GSH conjugates eluting at 20 and 21 min (HPLC method A). Similar to other equine catechol estrogen GSH conjugates, these GSH conjugates were unstable and we could not obtain sufficient material for NMR characterization, and as a result their structures could not be determined unambiguously. However, based on the LC-MS and MSMS data, the structures of the GSH conjugates (mono or di) are consistent with reaction of the o-quinone of 4-hydroxy-8,9-dehydroestrone with one or two GSH

760


Figure 1. Positive ion electrospray MS/MS with CID of the protonated molecule (m/z 590) of 4-hydroxy-8,9-dehydroestrone mono-GSH conjugate.

Figure 2. Positive ion electrospray MS/MS with CID of the protonated molecule (m/z 895) of 4-hydroxy-8,9-dehydroestrone di-GSH conjugate.

molecules (Figure 1, ref 2). The molecule at m/z 590 was consistent with the protonated molecule of the monoGSH conjugate (Figure 1). The product ion spectrum of this ion of m/z 590 gave abundant fragment ions at m/z 515 [MH - 75]+ (loss of glycine), m/z 461 [MH - 129]+ (loss of pyroglutamic acid), and m/z 315. The two GSH conjugates of 4-hydroxy-8,9-dehydroestrone had similar MS-MS patterns. The MS-MS pattern of 4-hydroxy-8,9dehydroestrone-diSG 1 is shown in Figure 2. The product ion spectrum of the ion at m/z 895 gave abundant fragment ions at m/z 820 [MH - 75]+ (loss of glycine), m/z 766 [MH - 129]+ (loss of pyroglutamic acid), and m/z 637 [MH - 258]+ (loss of two pyroglutamic acid moieties). Loss of 129 units (pyroglutamic acid) from the protonated molecule is a characteristic fragment ion of GSH conjugates formed during MS-MS (57, 58). The behavior of 2-hydroxy-8,9-dehydroestrone was found to be different from 4-hydroxy-8,9-dehydroestrone under similar conditions. This catechol formed one mono-GSH conjugate at a retention time of 51 min and two di-GSH conjugates at 24 and 25 min. These conjugates were formed either in the absence or in the presence of oxidative enzymes (P450 or tyrosinase). Like the GSH conjugates of 4-hydroxy-8,9-dehydroestrone, these GSH conjugates were also unstable and only LC-MS-MS spectra were obtained. The mono- and di-GSH conjugates showed the same MS-

Zhang et al.

MS pattern as that of the 4-hydroxy-8,9-dehydroestrone GSH conjugates. However, incubation of 2-hydroxy-8,9dehydroestrone in phosphate buffer (pH 7.4) gave a distinctive time-dependent UV absorbance change (Figure 3). The new species formed at the end of the incubation had the same UV absorbance pattern as that of 2-hydroxyequilenin (Figure 3). This transformation was further confirmed by HPLC with a co-injection of an authentic sample 2-hydroxyequilenin. In addition, extraction of the final incubation solution with CHCl3 and analysis by CI-MS showed an ion at 283 corresponding to the protonated molecule of 2-hydroxyequilenin. It is likely that the transformation of 2-hydroxy-8,9-dehydroestrone to 2-hydroxyequilenin under these conditions was accomplished through a analogous o-quinone methide mechanism (Scheme 5) by which 4-hydroxyequilin isomerized to 4-hydroxyequilenin (34). The GSH conjugates of 2-hydroxy-8,9-dehydroestrone and 4-hydroxy-8,9dehydroestrone imply that quinones were formed prior to nucleophilic GSH addition. These o-quinones might produce toxicity through depletion of cellular GSH. Once GSH is depleted, the o-quinones could alkylate cysteine residues on cellular proteins, resulting in toxic effects (59, 60). Oxidation of Catechol Estrogens and 8,9-Dehydroestrone by Rat Liver Microsomes. We examined the oxidation of 2-hydroxy-8,9-dehydroestrone, 4-hydroxy-8,9-dehydroestrone, and 8,9-dehydroestrone to quinoid metabolites in rat liver microsomes by trapping these reactive species with [3H]GSH (Table 1). The trapping reaction should be very efficient because of the high concentration of GSH in the incubation medium and the relatively fast rate of GSH addition as compared to amino and hydroxyl groups (61, 62). However, a small amount of binding to the microsomal protein by the quinoids is possible and as a result conjugate formation shown in Table 1 should be considered a lower limit for the generation of quinoids. Microsomal incubations with the catechols showed that 2-hydroxy-8,9-dehydroestrone was a slightly better substrate than 4-hydroxy-8,9dehydroestrone in terms of o-quinone formation (Table 1). Incubations with 8,9-dehydrodroestrone also showed production of mono-GSH conjugates from both catechols although considerably less o-quinone derived GSH conjugates were produced in incubations with the parent phenol as compared to similar experiments with the catechols. These data differ from what has been reported for the metabolism of 8,9-dehydroestrone in female dogs where only the 17β-hydroxy-8,9-dehydroestradiol was detected (35). It is possible that these catechols were formed in the female dogs which were not detected because of the instability of the catechols and o-quinones. Regioselectivity was observed in the P-450-catalyzed hydroxylation of 8,9-dehydroestrone since 2-hydroxy-8,9dehydroestrone GSH conjugates predominated over 4-hydroxy-8,9-dehydroestrone GSH conjugates by a factor of 6. Previously, we studied the metabolism of estrone, equilin, and equilenin to catechol o-quinone GSH conjugates in rat liver microsomes (50, 54). It was shown that estrone was primarily metabolized to 2-hydroxyestrone (2:4 hydroxylation ratio ) 6:1), equilin was primarily metabolized to 4-hydroxyequilin (2:4 hydroxylation ratio ) 1:6), and equilenin was exclusively metabolized to 4-hydroxyequilenin. The present data showed that the ratio of 2:4 hydroxylation of 8,9-dehydroestrone was 6:1



Table 1. Conversion of 8,9-Dehydroestrone and Its Catechol Metabolites to o-Quinone-Derived GSH Conjugates by Rat Liver Microsomesa substrate

conjugate

retention time (min)

rate of formation (nmol/nmol of P450‚10 min) 0.037 ( 0.003 0.007 ( 0.001 total ) 0.044 ( 0.004 0.17 ( 0.02 0.23 ( 0.01 4.50 ( 0.03 total ) 4.90 ( 0.06 0.31 ( 0.01 0.48 ( 0.02 2.07 ( 0.03 total ) 2.86 ( 0.06

8,9-dehydroestrone

2-OHDHES-SG 4-OHDHES-SG

51 55

2-hydroxy-8,9-dehydroestrone

2-OHDHES-diSG 1 2-OHDHES-diSG 2 2-OHDHES-SG

24 25 51

4-hydroxy-8,9-dehydroestrone

4-OHDHES-diSG 1 4-OHDHES-diSG 2 4-OHDHES-SG

20 21 55

a Incubations were conducted for 30 min with 0.5 mM substrate, rat liver microsomes (1.0 nmol P450/mL) in the presence of an NADPHgenerating system and [3H]GSH, at 37 °C. Radioactivity eluting from the HPLC column was measured in fractions collected at 18-s intervals. Results are the average ( SD of three incubations. Background radioactivity in the control (-NADP+) samples have been subtracted from each peak.

Table 2. Cytotoxicity of Equine Estrogens and Their Catechol Metabolites in S-30 Breast Cancer Cellsa substrate

LC50 (µM)

2-hydroxy-8,9-dehydroestrone 4-hydroxy-8,9-dehydroestrone 4-hydroxyestrone 4-hydroxyequilenin 8,9-dehydroestrone equilenin

84 ( 7 147 ( 7 251 ( 9 4.0 ( 0.1b 126 ( 7 155 ( 8

a Cell viability was assessed by Trypan blue exclusion as described in Materials and Methods. Values are expressed as the mean ( SD of at least three determinations. b From ref 63.

Figure 3. (A) UV-vis spectral analysis of the conversion of 2-hydoxy-8,9-dehydroestrone to 2-hydroxyequilenin. Incubations contained 2-hydroxy-8,9-dehydroestrone (0.15 mM) in potassium phosphate buffer (50 mM, pH 7.4, 37 °C). Scans were taken every 5 min for 1.5 h. (B) UV spectrum of 2-hydroxyequilenin.

which was quite similar to that of the endogenous estrogen, estrone. Cytotoxicity of 8,9-Dehydroestrone and Catechol Metabolites in Breast Cancer Cells (S-30). Preliminary studies conducted with the ERR stably transfected human breast tumor cell line S-30, demonstrated that the cytotoxicity of the catechol metabolites from 8,9dehrdroestrone were much higher than that of 4-hydroxyestrone, but less than that of 4-hydroxyequilenin (63). The cytotoxicity of 2-hydroxy-8,9-dehydroestrone is almost double that of 4-hydroxy-8,9-dehydroestrone which had similar cytotoxic effects as 8,9-dehrydroestrone and equilenin (Table 2). The higher cytotoxicity of 2-hydroxy8,9-dehydroestrone might result from the fact that this catechol can autoxidize to 2-hydroxy-8,9-dehydroestrone o-quinone under physiological conditions without oxidative enzymatic catalysis (see above). In addition, the microsomal incubations suggest that the amount of quinoids formed in the S30 cells is likely higher for 2-hydroxy-8,9-dehydroestrone as compared to 4-hydroxy8,9-dehydroestrone. In conclusion, data have been presented on the formation of GSH conjugates from the catechol metabolites of

8,9-dehydroestrone in rat liver microsomes and the relative cytotoxicity of these catechols in breast cancer cells. We have found that 8,9-dehydroestrone was primarily metabolized to 2-hydroxy-8,9-dehydroestrone in rat liver microsomes and that this catechol can autoxidize, isomerize, and aromatize giving 2-hydroxyequilenin. In contrast, 4-hydroxy-8,9-dehydroestrone is a stable catechol under physiological conditions. These data might explain the enhanced toxicity of 2-hydroxy-8,9-dehydroestrone compared to 4-hydroxy-8,9-dehydroestrone in breast cancer cells. In should be noted that both catechol metabolites of 8,9-dehydroestrone were much less toxic (20-40-fold) than 4-hydroxyequilenin which is the major metabolite of both equilin and equilenin (34, 50). These results may suggest that the catechol metabolites of 8,9dehydroestrone might have the ability to cause cytotoxicity in vivo primarily through formation of catechol quinoids; however, most of the adverse effects of Premarin estrogens are likely due to formation of quinoids from equilin and equilenin.

Acknowledgment. This research was supported by NIH Grant CA73638 (J.L.B.) and CA83124 (R.B.v.B.). We are grateful to Dr. V. C. Jordan (Northwestern University) for the gift of the S-30 cell line.

References (1) Grodstein, F., Stampfer, M. J., Colditz, G. A., Willett, W. C., Manson, J. E., Joffe, M., Rosner, B., Fuchs, C., Hankinson, S. E., Hunter, D. J., Hennekens, C. H., and Speizer, F. E. (1997) Postmenopausal hormone therapy and mortality. New Eng. J. Med. 336, 1769-1775. (2) Wickelgren, I. (1997) Estrogen: A new weapon against Alzheimer’s. Science 276, 676-677. (3) Henderson, V. W. (1997) The epidemiology of estrogen replacement therapy and Alzheimer’s disease. Neurology 48, S27-S35.

762


(4) Henderson, V. W., Paganini-Hill, A., Miller, B. L., Elble, R. J., Reyes, P. F., Shoupe, D., McCleary, C. A., Klein, R. A., Hake, A. M., and Farlow, M. R. (2000) Estrogen for Alzheimer’s disease in women: randomized, double-blind, placebo-controlled trial. Neurology 54, 295-301. (5) Mulnard, R. A., Cotman, C. W., Kawas, C., van Dyck, C. H., Sano, M., Doody, R., Koss, E., Pfeiffer, E., Jin, S., Gamst, A., Grundman, M., Thomas, R., and Thal, L. J. (2000) Estrogen replacement therapy for treatment of mild to moderate Alzheimer disease: a randomized controlled trial. Alzheimer’s Disease Cooperative Study. J. Am. Med. Assoc. 283, 1007-1015. (6) Paganinihill, A. (1995) Estrogen replacement therapy and stroke. Prog. Cardio. Dis. 38, 223-242. (7) Clark, K. E., Baker, R. S., and Lang, U. (2000) Premarin-induced increases in coronary and uterine blood flow in nonpregnant sheep. Am. J. Obstet. Gynecol. 183, 12-17. (8) Tsang, K. L., Ho, S. L., and Lo, S. K. (2000) Estrogen improves motor disability in Parkinsonian postmenopausal women with motor fluctuations. Neurology 54, 2292-2298. (9) Thayer, A. (1997) Thumbs-down on generic estrogen. Chem. Eng. News 75, 13. (10) Baracat, E., Haidar, M., Lopez, F. J., Pickar, J., Dey, M., and Negro-Vilar, A. (1999) Estrogen activity and novel tissue selectivity of delta 8,9-dehydroestrone sulfate in postmenopausal women. J. Clin. Endocrinol. Metab. 84, 2020-2027. (11) Dey, M., Lyttle, C. R., and Pickar, J. H. (2000) Recent insights into the varying activity of estrogens. Maturitas. 34 (Suppl. 2), S25-S33. (12) Steinberg, K. K., Smith, S. J., Thacker, S. B., and Stroup, D. F. (1994) Breast cancer risk and duration of estrogen use - The role of study design in meta-analysis. Epidemiology 5, 415-421. (13) Davidson, N. E. (1995) Hormone-replacement therapy - Breast versus heart versus bone. New Engl. J. Med. 332, 1638-1639. (14) Colditz, G. A., Hankinson, S. E., Hunter, D. J., Willett, W. C., Manson, J. E., Stampfer, M. J., Hennekens, C., Rosner, B., and Speizer, F. E. (1995) The use of estrogens and progestins and the risk of breast cancer in postmenopausal women. New Eng. J. Med. 332, 1589-1593. (15) Lupulescu, A. (1995) Estrogen use and cancer incidence: A review. Cancer Invest. 13, 287-295. (16) Zumoff, B. (1998) Does postmenopausal estrogen administration increase the risk of breast cancer? Contributions of animal, biochemical, and clinical investigative studies to a resolution of the controversy. Proc. Soc. Exp. Biol. Med. 217, 30-37. (17) Service, R. F. (1998) New role for estrogen in cancer? Science 279, 1631-1633. (18) Henderson, B. E., and Feigelson, H. S. (2000) Hormonal carcinogenesis. Carcinogenesis 21, 427-433. (19) Martucci, C. P., and Fishman, J. (1993) P450 enzymes of estrogen metabolism. Pharmacol. Ther. 57, 237-257. (20) Liehr, J. G. (1994) Mechanism of metabolic activation and inactivation of catecholestrogens: A basis of genotoxicity. Polycycl. Aromat. Compd. 6, 229-239. (21) Yager, J. D., and Liehr, J. G. (1996) Molecular mechanisms of estrogen carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 36, 203232. (22) Dwivedy, I., Devanesan, P., Cremonesi, P., Rogan, E., and Cavalieri, E. (1992) Synthesis and characterization of estrogen 2,3- and 3,4-quinones. Comparison of DNA adducts formed by the quinones versus horseradish peroxidase-activated catechol estrogens. Chem. Res. Toxicol. 5, 828-833. (23) Stack, D. E., Byun, J., Gross, M. L., Rogan, E. G., and Cavalieri, E. L. (1996) Molecular characteristics of catechol estrogen quinones in reactions with deoxyribonucleosides. Chem. Res. Toxicol. 9, 851-859. (24) Akanni, A., and Abul-Hajj, Y. J. (1997) Estrogen-nucleic acid adducts: reaction of 3,4-estrone-o-quinone radical anion with deoxyribonucleosides. Chem. Res. Toxicol. 10, 760-766. (25) Liehr, J. G., Avitts, T. A., Randerath, E., and Randerath, K. (1986) Estrogen-induced endogenous DNA adduction: possible mechanism of hormonal cancer. Proc. Natl. Acad. Sci. U.S.A. 83, 53015305. (26) Han, X., Liehr, J. G., and Bosland, M. C. (1995) Induction of a DNA adduct detectable by 32P-postlabeling in the dorsolateral prostate of NBL/Cr rats treated with estradiol-17 beta and testosterone. Carcinogenesis 16, 951-954. (27) Cavalieri, E. L., Stack, D. E., Devanesan, P. D., Todorovic, R., Dwivedy, I., Higginbotham, S., Johansson, S. L., Patil, K. D., Gross, M. L., Gooden, J. K., Ramanathan, R., Cerny, R. L., and Rogan, E. G. (1997) Molecular origin of cancer: Catechol estrogen3,4,-quinones as endogenous tumor initiators. Proc. Natl. Acad. Sci. U.S.A. 94, 10937-10942.

Zhang et al. (28) Purdy, R. H., Moore, P. H., Williams, M. C., Goldzheher, H. W., and Paul, S. M. (1982) Relative rates of 2- and 4-hydroxyestrogen synthesis are dependent on both substrate and tissue. FEBS Lett. 138, 40-44. (29) Li, S. A., Klicka, J. K., and Li, J. J. (1985) Estrogen 2- and 4-hydroxylase activity, catechol estrogen formation, and implications for estrogen carcinogenesis in the hamster kidney. Cancer Res. 45, 181-185. (30) Li, J. J., Li, S. A., Oberley, T. D., and Parsons, J. A. (1995) Carcinogenic activities of various steroidal and nonsteroidal estrogens in the hamster kidney: relation to hormonal activity and cell proliferation. Cancer Res. 55, 4347-4351. (31) Sarabia, S. F., Zhu, B. T., Kurosawa, T., Tohma, M., and Liehr, J. G. (1997) Mechanism of cytochrome P450-catalyzed aromatic hydroxylation of estrogens. Chem. Res. Toxicol. 10, 767-771. (32) Shen, L., Qiu, S., van Breemen, R. B., Zhang, F., Chen, Y., and Bolton, J. L. (1997) Reaction of the Premarin metabolite 4-hydroxyequilenin semiquinone radical with 2′-deoxyguanosine: Formation of unusual cyclic adducts. J. Am. Chem. Soc. 119, 1112611127. (33) Shen, L., Qiu, S., Chen, Y., Zhang, F., van Breemen, R. B., Nikolic, D., and Bolton, J. L. (1998) Alkylation of 2′-deoxynucleosides and DNA by the Premarin metabolite 4-hydroxyequilenin semiquinone radical. Chem. Res. Toxicol. 11, 94-101. (34) Zhang, F., Chen, Y., Pisha, E., Shen, L., Xiong, Y., van Breemen, R. B., and Bolton, J. L. (1999) The major metabolite of equilin, 4-hydroxyequilin autoxidizes to an o-quinone which isomerizes to the potent cytotoxin 4-hydroxyequilenin-o-quinone. Chem. Res. Toxicol. 12, 204-213. (35) Chandrasekaran, A., Osman, M., Raveendranath, P., Chan, K., Scatina, J. A., and Sisenwine, S. F. (1995) Characterization of metabolites of delta(8,9)-dehydroestrone in dogs. J. Mass Spectrom. 30, 1505-1511. (36) NIH Guidelines for the Laboratory Use of Chemical Carcinogens. Washington, DC, U.S. Government Printing Office, 1981. (37) Zhang, F., and Bolton, J. L. (1999) Synthesis of the equine estrogen metabolites 2-hydroxyequilin and 2- hydroxyequilenin. Chem. Res. Toxicol. 12, 200-203. (38) Woo, L. W., Howarth, N. M., Purohit, A., Hejaz, H. A. M., Reed, M. J., and Potter, B. V. L. (1998) Steroidal and nonsteroidal sulfamates as potent inhibitors of steroid sulfatase. J. Med. Chem. 41, 1068-1083. (39) Quinkert, G., Grosso, M. D., Doring, A., Doring, W., Schenkel, R. I., Bauch, M., Dambacher, G., Bats, J. W., Zimmermann, G., and Durner, G. (1995) Total synthesis with a chrogenic opening move demonstrated on steroids with estrane or 18a-homoestrane skeleton. Helv. Chim. Acta 78, 1345-1391. (40) Findlay, J., and Kwan, D. (1973) Metabolites from a scytalidium species. Can. J. Chem. 51, 3299-3301. (41) Beetz, T., Meuleman, D. G., and Wieringa, J. H. (1982) Inhibitors of blood platelet aggregation. Activity of some 1H-benz[de]isoquinolinecarboximidamides on the in vivo blood platelet aggregation induced by collagen. J. Med. Chem. 25, 714-719. (42) Oka, Y., Motohash, M., Sugihara, H., Miyashita, O., Itoh, K., Nishikawa, M., and Yurugi, S. (1977) Syntheses of conformationally rigid catecholamine derivatives. Chem. Pharm. Bull. 25, 632639. (43) Okey, A. B. (1990) Enzyme induction in the cytochrome P-450 system. Pharmac. Ther. 45, 241-298. (44) Thompson, J. A., Malkinson, A. M., Wand, M. D., Mastovich, S. L., Mead, E. W., Schullek, K. M., and Laudenschlager, W. G. (1987) Oxidative metabolism of butylated hydroxytoluene by hepatic and pulmonary microsomes from rats and mice. Drug Metab. Dispos. 15, 833-840. (45) Andrews, D. L., Williams, G. S., Manoney, J. C., and West, J. R. (1999) DNA fragmentation during exposure of rat cerelella to ethanol under hypoxia imposed in vitro. J. Neurobiol. 7, 82-92. (46) Sandoval, M., Zhang, X. J., Liu, X., Mannick, E. E., Clark, D. A., and Miller, M. J. (1997) Peroxynitrite-induced apoptosis in T84 and RAW 264.7 cells: attenuation by L-ascorbic acid. Free Radical Biol. Med. 22, 489-495. (47) Hirschmann, H., and Wintersteiner, O. (1938) Isoequilin A. J. Biol. Chem. 126, 737-748. (48) Jung, M. E., and Halweg, K. M. (1984) Stereoselectivity in intramolecular Diels-Alder reactions: A short total synthesis of estrone. Tetrahedron Lett. 25, 2121-2124. (49) Stein, R. P., Buzby, G. C., and Smith, H. (1970) Totally synthetic steroid hormones - XX. Novel total synthesis of equilin and equilenin. Tetrahedron. 26, 1917-1933. (50) Chang, M., Zhang, F., Shen, L., Pauss, N., Alam, I., van Breemen, R. B., Blond-Elguindi, S., and Bolton, J. L. (1998) Inhibition of glutathione S-transferase activity by the quinoid metabolites of equine estrogens. Chem. Res. Toxicol. 11, 758-765.

Catechol Metabolites from 8,9-Dehydroestrone (51) Williard, P. G., and Fryhle, C. B. (1980) Boron trihalide-methyl sulfide complexes as convenient reagents for dealkylation of aryl ethers. Tetrahedron Lett. 21, 3731-3734. (52) Node, M., Nishide, K., Fuji, K., and Fujita, E. (1980) Hard acid and soft nucleophile system. 2. Demethylation of methyl ethers of alcohol and phenol with an aluminum halide-thiol system. J. Org. Chem. 45, 4275-4277. (53) Teitel, S., and O’Brien, J. P. (1976) Selective removal of an aromatic methylenedioxy group. J. Org. Chem. 41, 1657-1658. (54) Iverson, S. L., Shen, L., Anlar, N., and Bolton, J. L. (1996) Bioactivation of estrone and its catechol metabolites to quinoidglutathione conjugates in rat liver microsomes. Chem. Res. Toxicol. 9, 492-499. (55) Butterworth: M., Lau, S. S., and Monks, T. J. (1996) 17β-Estradiol metabolism by hamster hepatic microsomes: Implications for the catechol-O-methyl transferase-mediated detoxication of catechol estrogens. Drug Metab. Dispos. 24, 588-594. (56) Shen, L., Pisha, E., Huang, Z., Pezzuto, J. M., Krol, E., Alam, Z., van Breemen, R. B., and Bolton, J. L. (1997) Bioreductive activation of catechol estrogen-ortho-quinones: Aromatization of the B ring in 4-hydroxyequilenin markedly alters quinoid formation and reactivity. Carcinogenesis 18, 1093-1101. (57) Kassahum, K., Davis, M., Hu, P., Martin, B., and Baillie, T. (1997) Biotransformation of the naturally occurring isothiocyanate sulforaphane in the rat: Indentification of phase I metabolitea and glutathione conjugates. Chem. Res. Toxcol. 10, 1228-1233. (58) Ramanathan, R., Cao, K., Cavalieri, E., and Gross, M. L. (1998) Mass spectrometric methods for distinguishing structural isomers


(59)

(60)

(61)

(62)

(63)

of glutathione conjugates of estrone and estradiol. J. Am. Soc. Mass Spectrom. 9, 612-619. Dehal, S. S., and Kupfer, D. (1996) Evidence that the catechol 3,4-dihydroxytamoxifen is a proximate intermediate to the reactive species binding covalently to proteins. Cancer Res. 56, 12831290. Dehal, S. S., and Kupfer, D. (1999) Cytochrome P-450 3A and 2D6 catalyze ortho hydroxylation of 4-hydroxytamoxifen and 3-hydroxytamoxifen (droloxifene) yielding tamoxifen catechol: involvement of catechols in covalent binding to hepatic proteins. Drug Metab. Dispos. 27, 681-688. Bolton, J. L., Valerio, L. G. J., and Thompson, J. A. (1992) The enzymatic formation and chemical reactivity of quinone methides correlate with alkylphenol-induced toxicity in rat hepatocytes. Chem. Res. Toxicol. 5, 816-822. Kalyanaraman, B., Premovic, P. I., and Sealy, R. C. (1987) Semiquinone anion radicals from addition of amino acids, peptides, and proteins to quinones derived from oxidation of catechols and catecholamines: An ESR stabilization study. J. Biol. Chem. 262, 11080-11087. Chen, Y., Liu, X., Pisha, E., Constantinou, A. I., Hua, Y., Shen, L., van Breemen, R. B., Elguindi, E. C., Blond, S. Y., Zhang, F., and Bolton, J. L. (2000) A metabolite of equine estrogens, 4-hydroxyequilenin, induces DNA damage and apoptosis in breast cancer cell lines. Chem. Res. Toxicol. 13, 342-350.

TX010049Y

Synthesis and Reactivity of the Catechol Metabolites from the Equine

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