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Chem. Res. Toxicol. 1998, 11, 408-411
Articles Large-Scale Synthesis of the Catechol Metabolites of Diethylstilbestrol and Hexestrol Shyi-Tai Jan, Eleanor G. Rogan, and Ercole L. Cavalieri* Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-6805 Received August 11, 1997
Diethylstilbestrol (DES) and hexestrol (HES) are carcinogenic synthetic estrogens. The major metabolites of these compounds are their catechol derivatives, 3′-OH-DES and 3′-OH-HES. Oxidation of these metabolites leads to the electrophilic quinones, which are presumably involved in the tumor-initiating process. A synthetic route based on the McMurry coupling reaction was developed for the synthesis of 3′-OH-DES. Using commercially inexpensive starting materials, this compound was synthesized in four steps, and the cis and trans isomers were separated and identified. Following the same synthetic route, 3′-OH-HES was synthesized in five steps.
Introduction (DES)1
Diethylstilbestrol and hexestrol (HES) are carcinogenic synthetic estrogens (1, 2). The major metabolites of these phenolic synthetic estrogens are their corresponding catechol derivatives, 3′-OH-DES (1) and 3′-OH-HES (2) (Scheme 1) (2-5). Oxidation of these catechols leads to the corresponding quinones, which are presumably the electrophilic intermediates responsible for tumor initiation. Analogously to the natural estrogen3,4-quinones, these should form depurinating adducts when reacted with DNA (6, 7). Because we are interested in studying the mechanism of tumor initiation by synthetic estrogens, the availability of their catechol metabolites in large quantity became essential to our research. Although synthesis of 3′-OH-DES (1) in eight steps was reported in 1981 (8), we developed a shorter synthetic route (four steps) based on the McMurry coupling reaction (9, 10). Furthermore, for the first time the cis and trans isomers of 3′-OH-DES have been isolated and characterized. 3′-OH-HES (2) was previously synthesized by treating HES with Fremy salt, with a yield of 50% (2). Chemical separation, however, was required in this method because a substantial amount of starting material, HES, was left in the crude product. Separation by silica gel column chromatography was ruled out because the catechol was easily oxidized during the separation process. Preparative HPLC (reverse-phase using CH3OH and H2O as mobile phase) produced pure 3′-OH-HES (2), but only in limited amounts. Therefore, a new approach to synthesize cis- and trans-3′-OH-DES * To whom correspondence should be addressed. 1 Abbreviations: DES, diethylstilbestrol; DME, dimethoxyethane; HES, hexestrol; MS, mass spectrometry; 3′-OH-DES, 3′-hydroxydiethylstilbestrol; 3′-OH-HES, 3′-hydroxyhexestrol; PDC, pyridinium dichromate; THF, tetrahydrofuran.
(1) and 3′-OH-HES (2) was developed (Scheme 1).
Experimental Procedures Caution: 3′-OH-DES and 3′-OH-HES may be carcinogenic and should be handled according to NIH guidelines (11). Chemicals. All chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI) and were used without further purification. HPLC. Preparative HPLC (reverse-phase) was conducted by using a YMC ODS-AQ 5-µm, 120-Å column (20 × 250 mm). It was conducted on a Waters (Milford, MA) 600E solvent delivery system equipped with a 484 tunable absorbance detector operating at 254 nm. NMR. NMR spectra were recorded on samples at room temperature in CDCl3, CD3OD, or Me2SO-d6 on a Varian XL300 spectrometer at 299.938 MHz or on a Varian Unity 500MHz instrument (Palo Alto, CA), with chemical shifts reported relative to CDCl3 at 7.24 ppm, CD3OD at 3.31 ppm, or Me2SOd6 at 2.49 ppm. Coupling constants (J) are given in hertz. Mass Spectrometry. Electron ionization mass spectrometry (MS) was conducted on a Kratos MS-50 instrument at the Nebraska Center for Mass Spectrometry (Department of Chemistry, University of Nebraska-Lincoln), and accurate mass measurements were made using the same instrument with peak-matching techniques at a mass resolving power of 10 000. Synthetic Methods. 1-(3′,4′-Dimethoxyphenyl)propanol, 4. To a solution of 3,4-dimethoxybenzaldehyde (3) (10.0 g, 60.2 mmol) (Scheme 1) in anhydrous tetrahydrofuran (THF) (100 mL) under nitrogen was added ethylmagnesium bromide (72 mL, 1.0 M solution in THF). The mixture was heated to reflux under nitrogen for 10 h. The reaction mixture was cooled to room temperature, quenched with saturated NH4Cl (60 mL), and extracted with ethyl acetate (3 × 80 mL). The combined ethyl acetate portion was washed sequentially with H2O (3 × 80 mL) and brine, dried over anhydrous Mg2SO4, and concentrated to give 4 (11.4 g, 97%) (Scheme 1): Rf ) 0.30 (ethyl acetate/hexane, 3:7); 1H NMR (300 MHz, CDCl3) 6.89 (bs, 1H), 6.86-6.79 (m, 2H), 4.52 (m, 1H), 3.87 (s, 3H), 3.85 (s, 3H), 2.15 (bs, 1H), 1.85-1.58 (m, 2H), 0.89 (t, J ) 7.5 Hz, 3H); 13C NMR
S0893-228x(97)00140-9 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/09/1998
Synthesis of Catechol Metabolites of DES and HES
Chem. Res. Toxicol., Vol. 11, No. 5, 1998 409
Scheme 1. Synthesis of 3′-OH-DES and 3′-OH-HES
(75 MHz, CDCl3) 148.97, 148.32, 137.23, 118.21, 110.78, 108.91 (aromatic carbons), 75.90, 55.88, 55.81 (oxygen-bonded carbons), 31.80, 10.23 (aliphatic carbons); MS m/z 196 (33, M•+), 178 (20, [M - H2O]+), 167 (100, [M - C2H5]+), 139 (45, [M - C2H5 CO]+); exact mass calcd for C11H16O3 196.1099, obsd 196.1100. 1-(3′,4′-Dimethoxyphenyl)propanone, 5. To a solution of 4 (10.0 g, 51.0 mmol) in CH2Cl2 (200 mL) at room temperature was added pyridinium dichromate (PDC; 21.1 g, 56.1 mmol). The resulting suspension was stirred at room temperature for 2 h and then heated to reflux for 2 h. The cooled reaction mixture was diluted with hexane (600 mL) and filtered through Celite. The filtrate was dried and redissolved in ethyl acetate (200 mL). The ethyl acetate solution was sequentially washed with 5% HCl (3 × 100 mL), H2O (3 × 100 mL), and brine. The ethyl acetate solution was dried over anhydrous Mg2SO4 and evaporated to afford 5 (8.81 g, 89%): Rf ) 0.24 (ethyl acetate/ hexane, 1:9); 1H NMR (300 MHz, CDCl3) 7.57 (dd, J ) 8.5, 2.0 Hz, 1H), 7.52 (d, J ) 2.0 Hz, 1H), 6.86 (d, J ) 8.5 Hz, 1H), 3.92 (s, 3H), 3.91 (s, 3H), 2.95 (q, J ) 7.5 Hz, 2H), 1.20 (t, J ) 7.5 Hz, 3H); 13C NMR (75 MHz, CDCl3) 199.45 (carbonyl carbon), 152.96, 148.87, 130.05, 122.45, 109.98, 109.86 (aromatic carbons), 55.95, 55.86 (methoxy carbons), 31.21, 8.48 (aliphatic carbons); MS m/z 194 (30, M•+), 165 (100, [M - C2H5]+); exact mass calcd for C11H14O3 194.0943, obsd 194.0943. 3-(p-Methoxyphenyl)-4-(3′,4′-dimethoxyphenyl)-3-hexene, 7. Lithium wire (6.0 g, 864.6 mmol) was added to a stirred slurry of TiCl3 (38.2 g, 247.4 mmol) in 400 mL of dimethoxyethane (DME) under a nitrogen atmosphere, and the mixture was heated to reflux for 1 h. The black slurry was then cooled to room temperature, and a solution of 5 (2.0 g, 10.3 mmol) and 6 (8.45 g, 51.5 mmol) in DME (35 mL) was added. The mixture was stirred for 2 h at room temperature and then heated to reflux for 16 h. After cooling to room temperature, the reaction mixture was diluted with hexane and filtered through a small pad of Florisil. Evaporation of the solvent from the filtrate gave the crude mixture of products. The crude product was separated by silica gel column chromatography using CH2Cl2 and hexane as solvent to afford 7 (1.85 g, 55%): Rf ) 0.37 (ethyl acetate/ hexane, 1:9); 1H NMR (300 MHz, CDCl3) 6.85 (m, 2H), 6.59 (m, 4H), 6.37 (d, J ) 2.0 Hz, 1H), 3.78 (s, 3H), 3.69 (s, 3H), 3.56 (s, 3H), 2.51 (m, 4H), 0.94 (m, 6H); 13C NMR (75 MHz, CDCl3) 157.24, 147.59, 146.54, 138.21, 138.18, 135.81, 135.74, 130.60 (2C’s), 121.36, 113.97, 112.86 (2C’s), 110.07 (aromatic and double-bond carbons), 55.53, 55.49, 54.98 (methoxy carbons), 27.33, 27.10, 13.40, 13.25 (aliphatic carbons); MS m/z 326 (100,
M•+), 311 (6, [M - CH3]+), 297 (9, [M - C2H5]+); exact mass calcd for C21H26O3 326.1882, obsd 326.1881. trans-3′-OH-DES (1a) and cis-3′-OH-DES (1b). The trimethoxy olefin 7 (355 mg, 1.09 mmol) was dissolved in CH2Cl2 (25 mL) and cooled to -78 °C under a nitrogen atmosphere. The cooled solution was treated dropwise with boron tribromide (3.81 mL of 1.0 M solution in CH2Cl2, 3.81 mmol). The reaction mixture was stirred at -78 °C for 2 h and then allowed to warm to room temperature overnight. The reaction mixture was treated with ice water (20 mL) and then extracted with ethyl acetate (3 × 35 mL). The combined ethyl acetate portion was washed with H2O (3 × 25 mL) and brine. Evaporation of solvent, followed by recrystallization from ether/hexane, gave pure 3′-OH-DES as a mixture of trans 1a and cis 1b isomers (217 mg, 70%). For compound characterization, 1a,b were separated by preparative reverse-phase HPLC. The column was initially eluted with H2O/CH3OH (50:50) for 5 min, followed by a linear gradient to 100% CH3OH in 50 min. The retention times of 1a,b were 42 and 50 min, respectively. 1a (trans): 1H NMR (300 MHz, CD3OD) 6.97 (m, 2H), 6.76 (m, 2H), 6.74 (d, J ) 8.0 Hz, 1H), 6.62 (d, J ) 2.0 Hz, 1H), 6.49 (dd, J ) 8.0, 2.0 Hz, 1H), 2.11 (m, 4H), 0.75 (m, 6H); 13C NMR (75 MHz, CD3OD) 156.80, 145.86, 144.67, 140.24, 139.86, 135.84, 135.15, 130.77 (2C’s), 121.22, 116.99, 115.99, 115.80 (2C’s) (aromatic and double-bond carbons), 29.58, 29.51, 13.83, 13.80 (aliphatic carbons). 1b (cis): 1H NMR (300 MHz, CD3OD) 6.72 (m, 2H), 6.48 (m, 2H), 6.46 (d, J ) 8.0 Hz, 1H), 6.40 (d, J ) 2.0 Hz, 1H), 6.24 (dd, J ) 8.0, 2.0 Hz, 1H), 2.50 (m, 4H), 0.92 (m, 6H); 13C NMR (75 MHz, CD3OD) 155.97, 145.30, 143.99, 139.55, 139.09, 136.69, 135.96, 131.85 (2C’s), 122.72, 118.20, 115.49, 115.22 (2C’s) (aromatic and double-bond carbons), 28.35, 28.17, 13.66 (2C’s) (aliphatic carbons). 3-(p-Methoxyphenyl)-4-(3′,4′-dimethoxyphenyl)hexane, 8. A solution of 7 (725 mg, 2.22 mmol), 5% Pd/C (30 mg), and ethyl acetate (25 mL) was stirred for 10 h under a hydrogen atmosphere (using a balloon filled with the gas). The Pd/C was removed by filtration through a pad of Celite. The filtrate was dried to afford 8 (656 mg, 90%): Rf ) 0.32 (ethyl acetate/hexane, 1:9); 1H NMR (300 MHz, CDCl3) 7.05 (m, 2H), 6.84 (m, 2H), 6.80 (d, J ) 8.0 Hz, 1H), 6.70 (dd, J ) 8.0, 2.0 Hz, 1H), 6.62 (d, J ) 2.0 Hz, 1H), 3.86 (s, 3H), 3.86 (s, 3H), 3.79 (s, 3H), 2.45 (m, 2H), 1.41-1.31 (m, 2H), 1.30-1.19 (m, 2H), 0.53 (m, 6H); 13C NMR (75 MHz, CDCl3) 157.77, 148.64, 147.09, 137.14, 136.47, 129.19 (2C’s), 120.44, 113.49 (2C’s), 111.19, 110.78 (aromatic carbons), 55.83, 55.79, 55.18, 54.03, 53.53 (3 methoxy and 2
410 Chem. Res. Toxicol., Vol. 11, No. 5, 1998
Jan et al.
Scheme 2. Proposed Mechanism of Cis-Trans Isomerization of 3′-OH-DES
allylic tertiary carbons), 27.29, 27.22, 12.28 (2C’s) (aliphatic carbons); MS m/z 328 (5, M•+), 179 (100, [M - CH3O - C6H4 C3H6]+), 149 (47, [M - (CH3O)2 - C6H3 - C3H6]+); exact mass calcd for C21H28O3 328.2038, obsd 328.2040. 3′-Hydroxy-HES, 2. Removal of the methyl-protecting groups in 8 was carried out under the same conditions used in the conversion of 7 to 1. The crude product was recrystallized from hexane/ether to give pure 2 with a chemical yield of 72%: 1H NMR (500 MHz, Me SO-d ) 8.00 (bs, 1H, Ar-OH), 7.59 (bs, 2 6 1H, Ar-OH), 7.54 (bs, 1H, Ar-OH), 6.92 (d, J ) 8.5 Hz, 2H, Ar-H), 6.68 (d, J ) 8.5 Hz, 2H, Ar-H), 6.65 (d, J ) 8.0 Hz, 1H, 5′-H), 6.60 (d, J ) 2.0 Hz, 1H, 2′-H), 6.44 (dd, J ) 8.0, 2.0 Hz, 1H, 6′-H), 2.30 (m, 2H, CH), 1.32-1.09 (m, 4H, CH2), 0.39 (m, 6H, CH3); MS m/z 286 (4, M•+), 151 (89, [M - HOC6H4 - C3H6]+), 135 (100, [M - (HO)2C6H3 - C3H6]+); exact mass calcd for C18H22O3 286.1569, obsd 286.1567.
Results and Discussion The key step in the new route shown in Scheme 1 is the synthesis of 7 by using the McMurry reaction to couple the two carbonyl components, 5 and 6 (9, 10). Compound 7 is an important synthetic intermediate because it can be converted to cis- and trans-3′-OH-DES (1) in one step and to 3′-OH-HES (2) in two steps. Compound 6 is commercially available, and compound 5 can be easily synthesized from 3. Treatment of 3 with ethylmagnesium bromide gave 4. Alcohol 4 was converted to ketone 5 by PDC oxidation. Because the desired compound 7 is not a symmetric olefin, the McMurry coupling reaction was carried out under conditions in which 1 equiv of 5 and 5 equiv of 6 were used to ensure that most of 5 was coupled with 6, instead of with itself (10). The crude product from the reductive carbonyl coupling reaction was separated by silica gel column chromatography to give pure olefin 7 with unknown double-bond geometry. Compound 7 can be seen as a hydroxyl-protected form of 3′-OH-DES and is stable during long-term storage. Removal of the methyl-protecting groups in 7 with BBr3 followed by recrystallization in ether/hexane afforded a mixture of cis and trans isomers of 3′-OH-DES, 1a,b. The formation of 1a,b isomers is related to cis-trans isomerization of DES. According to the mechanism of cis-trans isomerization of DES proposed by Winkler et al. (12), it is clear that 3′-OH-DES is capable of undergoing the same isomerization process (see below). Because a mixture of cis- and trans-3′-OH-DES was produced from isomerically pure 7, the isomerization must have occurred after formation of 3′-OH-DES. The isomers 1a,b were the coexisting forms of 3′-OH-DES. We were able, however, to separate the two isomers, 1a,b, by using reverse-phase HPLC with CH3OH and H2O as the mobile phase. This was possible because of the slow cis-trans isomerization in polar solvents (see below). It was observed by 1H NMR that pure 1a (major peak in HPLC)
and 1b (minor peak in HPLC) rapidly isomerized to a 30:70 cis:trans mixture in CDCl3. We attributed the major isomer to the trans (1a) because this isomer is thermodynamically more stable than the cis isomer 1b. In contrast, the isomerization process was significantly slowed by using CD3OD as the NMR solvent. Therefore, we were able to obtain pure 1H and 13C NMR spectra for both 1a,b with CD3OD as the NMR solvent. When the NMR samples of the pure 1a,b isomers were allowed to sit overnight, isomerization of the pure samples was observed. Furthermore, a small amount of 3′-OH-DES was oxidized to its quinone over the course of 1 week. The observation of different rates of isomerization of 3′-OH-DES in various solvents was consistent with the isomerization of the parent compound, DES, and can be explained by the rationale used by Winkler et al. (12). Kinetic studies revealed that conversion of trans-DES to a cis-trans equilibrium mixture is a reversible, secondorder reaction. From these studies it was concluded that isomerization of DES in solution was bimolecular (12). On this basis, it was proposed that the isomerization process involves proton shifts from the phenolic groups of one molecule of DES to the stilbene double bond of the second molecule of DES. The same mechanism of isomerization is illustrated in Scheme 2 for 3′-OH-DES. The isomerization of trans-3′-OH-DES to cis-3′-OH-DES involves formation of an intermediate quinone methide structure that allows rotation of one benzene ring with respect to the other. Rates of isomerization are controlled by the phenolic protons, and their availability is a function of the solvent. A more polar solvent will form hydrogen bonds more strongly to the phenolic protons, making them less available for the proton transfer necessary in the isomerization process (Scheme 2) (12). For the preparation of 3′-OH-HES (2), compound 7 was hydrogenated at the stilbene double bond to form compound 8. The removal of the methyl-protecting groups in 8 with BBr3, followed by recrystallization in ether/ hexane, afforded the pure 3′-OH-HES (2). In conclusion, the research reported here leads to the large-scale formation of the major metabolites of DES, namely, trans-3′-OH-DES (1a) and cis-3′-OH-DES (1b). These two isomers can be obtained pure and used in biological studies. In addition, intermediate 7 is converted in two steps to the major metabolite of HES, 3′OH-HES. DNA adducts formed after oxidation of this compound are reported in the accompanying paper.
Acknowledgment. This research was supported by a U.S. PHS grant from the National Cancer Institute (P01 CA49210) and a grant from the Nebraska Department of Health. Dr. Jan was supported by a postdoctoral fellowship from the Eppley Institute Cancer Research Training Program Grant T32 CA09476, and core support
Synthesis of Catechol Metabolites of DES and HES
at the Eppley Institute was funded by NCI Laboratory Cancer Research Center Support (Core) Grant CA36727. We thank Dr. Ronald L. Cerny for mass spectral analysis conducted at the Nebraska Center for Mass Spectrometry, Department of Chemistry, University of NebraskaLincoln.
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Chem. Res. Toxicol., Vol. 11, No. 5, 1998 411 (6) Stack, D. E., Byun, J., Gross, M. L., Rogan, E. G., and Cavalieri, E. (1996) Molecular characteristics of catechol estrogen quinones in reactions with deoxyribonucleosides. Chem. Res. Toxicol. 9, 851-859. (7) 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. (8) Hill, K. A., Peterson, D. M., Damodaran, K. M., and Rao, P. N. (1981) Synthesis of metabolic intermediates of diethylstilbestrol. Steroids 37, 327-343. (9) McMurry, J. E., and Fleming, M. P. (1974) A new method for the reductive coupling of carbonyls to olefins. Synthesis of β-carotene. J. Am. Chem. Soc. 96, 4708-4709. (10) McMurry, J. E., Fleming, M. P., Kees, K. L., and Krepski, L. R. (1978) Titanium-induced reductive coupling of carbonyls to olefins. J. Am. Chem. Soc. 100, 3255-3266. (11) NIH Guidelines for the Laboratory Use of Chemical Carcinogens (1981), NIH Publications No. 81-2385, U.S. Government Printing Office, Washington, DC. (12) Winkler, V. W., Nyman, M. A., and Egan, R. S. (1971) Diethylstilbestrol cis-trans isomerization and estrogen activity of diethylstilbestrol isomers. Steroids 17, 197-207.
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