Synthesis of Equine Estrogen Metabolites
Chem. Res. Toxicol., Vol. 12, No. 2, 1999 201
was purified by preparative TLC (silica gel, 250 µm) with
hexane/acetone/methanol (2:1:0.03) as the eluent (6 mg, 42%
yield). The remaining material is 2-hydroxyequilenin which has
chromatographic properties very similar to those of 2-hydroxy-
equilin, making purification difficult. As a result, the synthesis
can only be accomplished on a small scale: 1H NMR (acetone-
d6) δ 0.75 (s, 3H, 18-CH3), 1.44 (m, 1H, H11), 1.60 (m, 1H, H12),
1.84 (m, 1H, H12), 1.93 (m, 2H, 2 x H15), 2.16 (m, 1H, H16), 2.26
(m, 1H, H11), 2.44 (m, 1H, H14), 2.51 (m, 2H, H16), 3.13 (m, 1H,
H9), 3.35 (m, 2H, 2 x H6), 5.53 (m, 1H, H7), 6.57 (s, 1H, ArH),
6.76 (s, 1H, ArH), 7.61 (s, 1H, exchangeable with D2O, OH), 7.65
(s, 1H, exchangeable with D2O, OH); UV (CH3OH) 288 nm; CI-
MS (positive ion, methane) m/z 285 (MH+, 100% relative
intensity).
Sch em e 1. Syn th esis of 2-Hyd r oxyequ ilin a n d
2-Hyd r oxyequ ilen in
(3) 2-Meth oxyequ ilen in (6). 2-Methoxyequilin (280 mg,
0.939 mmol) was dissolved in tert-butyl alcohol (18 mL), followed
by the addition of pyridine (0.19 mL) and selenium dioxide (128
mg, 1.2 mmol). The mixture was refluxed for 1.5 h and cooled
to room temperature. The solvent was removed en vacuo, and
the residue was purified by flash chromatography (silica gel)
using hexane/ethyl acetate (3:2) as the eluent, giving 2-meth-
oxyequilenin which was further purified by crystallization from
methanol/acetone (110 mg, 39.5% based on 2-methoxyequilin).
Complete characterization was accomplished by 1D and 2D
NMR experiments, including 1H, 13C, HMQC, HMBC, and
COSY: 1H NMR (CDCl3) δ 0.81 (s, 3H, CH3), 1.92 (m, 1H, H12),
2.00 (m, 1H, H15), 2.21 (m, 1H, H12), 2.39 (m, 1H, H16), 2.54 (m,
1H, H15), 2.68 (m, 1H, H16), 3.18 (m, 1H, H14), 3.26 (m, 2H, 2 x
H11), 4.03 (s, 3H, OCH3), 5.89 (s, D2O exchangeable, 1H, OH3),
7.1 (d, J ) 9.0 Hz, 1H, H7), 7.2 (s, 1H, H1), 7.26 (s, 1H, H4), 7.55
(d, J ) 9.0 Hz, 1H, H6); 13C NMR (CDCl3) δ 13.1 (C18), 21.8 (C15),
24.3 (C11), 29.1 (C12), 36.6 (C16), 46.8 (C14), 47.5 (C13), 55.8
(OCH3), 101.4 (C1), 110.1 (C4), 122.2 (C7), 125.0 (C6), 127.3 (C10),
128.5 (C9), 129.4 (C3), 132.4 (C8), 144.9 (C5), 147.5 (C2), 220.6
(C17); UV (CH3OH) 244, 268, 278, 286, 298, 316, 330 nm; CI-
MS (positive ion, methane) m/z 297 (MH+, 100% relative
intensity).
(4) 2-Hyd r oxyequ ilen in . 2-Hydroxyequilenin was synthe-
sized using a procedure analogous to that used for 2-hydroxy-
equilin: 1H NMR (acetone-d6) δ 0.75 (s, 3H, CH3), 1.80 (m, 1H,
H12), 1.96 (m, 1H, H15), 2.15 (m, 1H, H12), 2.35 (m, 1H, H16),
2.61 (m, 1H, H15), 2.67 (m, 1H, H16), 3.17 (m, 3H, 2 x H11, H14),
7.1 (d, J ) 9.0 Hz, 1H, H7), 7.2 (s, 1H, H1), 7.3 (s, 1H, H4), 7.5
(d, J ) 9.0 Hz, 1H, H6), 8.39 (s, D2O exchangeable, 2H, OH2,
OH3); 13C NMR (acetone-d6) δ 13.0 (C18), 22.5 (C15), 24.7 (C11),
29.5 (C12), 36.8 (C16), 47.5 (C14), 47.9 (C13), 106.5 (C1), 111.2 (C4),
122.2 (C7), 125.5 (C6), 128.8 (C10), 129.3 (C9), 129.5 (C5), 133.1
(C8), 146.2 (C2), 147.3 (C3), 219.0 (C17); UV (CH3OH) 242, 270,
284, 292, 304, 318, 332 nm; CI-MS (positive ion, methane) m/z
283 (MH+, 100% relative intensity).
as the starting material. The synthesis of 2-hydroxyequi-
lin has not been reported previously, although our
scheme does include a modification of the procedure used
to synthesize one of the intermediates, 2-methoxyequilin
(21; Scheme 1).
Ma ter ia ls a n d Meth od s
Ca u tion : 2-Hydroxyequilin and 2-hydroxyequilenin may be
carcinogenic and should be handled according to NIH guidelines
for the Laboratory Use of Chemical Carcinogens (22).
Ma ter ia ls. All chemicals were purchased from Aldrich
Chemical Co. (Milwaukee, WI), Fisher Scientific (Itasca, IL), or
Sigma (St. Louis, MO) unless stated otherwise.
Syn th etic Meth od s. (1) 2-Meth oxyequ ilin (5). 2-Methoxy-
equilin was synthesized as described in the literature (20;
Scheme 1). Briefly, equilin (1 g) was converted into 2,4-
diiodoequilin (1) with iodine in NH4OH. The 4-iodo substituent
in
1 was removed using formic acid/ascorbic acid, giving
2-iodoequilin (2). The 17-keto group in 2 was protected by
reaction with ethylene glycol to produce 3. Treatment of 3 with
sodium methoxide exchanged the 2-iodo substituent for
a
methoxy group 4, and acid hydrolysis followed by purification
by flash chromatography on silica gel with hexane/ethyl acetate
(3:2) as the eluent gave 2-methoxyequilin (5) as an impure solid.
Further purification was accomplished using preparative TLC
(silica gel) with hexane/ethyl acetate/triethylamine (10:12:3, Rf
) 0.7) as the eluant to yield 150 mg of 5 (13.3% overall yield).
Complete characterization was accomplished by one-dimen-
sional (1D) and two-dimensional (2D) NMR experiments, in-
cluding 1H, 13C, HMQC, HMBC, and COSY: 1H NMR (acetone-
d6) δ 0.75 (s, 3H, 18-CH3), 1.49 (m, 1H, H11), 1.61 (m, 1H, H12),
1.83 (m, 1H, H12), 1.91 (m, 2H, 2 x H15), 2.17 (m, 1H, H16), 2.25
(m, 1H, H11), 2.43 (m, 1H, H14), 2.50 (m, 1H, H16), 3.14 (m, 1H,
H9), 3.45 (m, 2H, 2 x H6), 3.82 (s, 3H, OCH3), 5.54 (m, 1H, H7),
6.57 (s, 1H, H4), 6.83 (s, 1H, H1), 7.38 (bs, 1H, OH3); 13C NMR
(acetone-d6) δ 14.0 (C18), 20.3 (C15), 29.7 (C6), 32.7 (C11), 33.1
(C12), 36.0 (C16), 41.6 (C9), 49.9 (C13), 51.2 (C14), 56.2 (OCH3),
111.3 (C1), 114.9 (C4), 116.7 (C7), 126.1 (C5), 129.2 (C10), 136.8
(C8), 145.7 (C3), 147.0 (C2), 219.1 (C17); UV (CH3OH) 286 nm;
CI-MS (positive ion, methane) m/z 299 (MH+, 100% relative
intensity).
(2) 2-Hyd r oxyequ ilin . Under an inert atmosphere, 2-meth-
oxyequilin (15 mg, 0.053 mmol) was dissolved in anhydrous CH2-
Cl2 (4 mL) and the solution cooled to -15 °C. BBr3 (0.5 mL of a
1 M solution in CH2Cl2) was added to the cooled solution, and
the mixture was stirred for 9 h. Water (10 mL) was added to
quench the reaction, and the solution was extracted with ethyl
acetate (2 × 20 mL). The combined organic layers were washed
with 10% sodium bicarbonate (1 × 10 mL), 1 N HCl (2 × 10
mL), and water (1 × 10 mL) and dried over sodium sulfate. After
filtration, the solvent was removed en vacuo and the residue
In str u m en ta tion . NMR spectra were obtained with a Bruk-
er Avance DPX300 spectrometer at 300 MHz, and CI mass
spectra were obtained with a Finnigan MAT 90 magnetic sector
mass spectrometer.
Resu lts a n d Discu ssion
The major advantage of the new synthetic route shown
in Scheme 1 is that we utilize the commercially available
optically pure equilin as the starting material and use a
synthetic route much shorter then that of Ikegawa et al.
(20) to synthesize optically active 2-hydroxyequilin and
2-hydroxyequilenin which are potential metabolites of
equilin and equilenin, respectively, in vivo. The key steps
for the synthesis of 2-hydroxyequilin are the synthesis
of 2-methoxyequilin 5 and its demethylation. For the
synthesis of 2-methoxyequilin, we use equilin (optically
pure) as the starting material; after aromatic substitu-
tion, reduction, and nucleophilic substitution of the A ring
of equilin, 2-methoxyequilin was obtained with the ster-
eochemistry of equilin. The purification of 2-methoxy-