Neighboring group participation: Part 16. Stereoselective synthesis and receptor-binding examination of the four stereoisomers of 16-bromomethyl-3,17- estradiols

Article abstract of DOI:10.1016/j.steroids.2005.09.008

The four possible isomers of 3-benzyloxy-16-hydroxymethylestra-1,3,5(10)- trien-17-ol (1a-4a) with proven configurations were converted into the corresponding 3-benzyloxy-16-bromomethylestra-1,3,5(10)-triene-3,17-diols (5e-8e). Depending on the reaction c

Full text of DOI:10.1016/j.steroids.2005.09.008

steroids 7 1 ( 2 0 0 6 ) 141–153
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journal homepage: www.elsevier.com/locate/steroids
Neighboring group participation
Part 16. Stereoselective synthesis and receptor-binding
examination of the four stereoisomers of
16-bromomethyl-3,17-estradiolsଝ
a
a,∗
a
b
b
´
´
Agota Sza´jli , Ja´nos Wo¨lfling , Erzse´bet Mernya´k , Rena´ta Minorics , Arpa´d Ma´rki ,
George Falkayb, Gyula Schneidera
a
´
´
Department of Organic Chemistry, University of Szeged, Dom ter 8, H-6720 Szeged, Hungary
b
¨
¨
Institute of Pharmacodynamics and Biopharmacy, University of Szeged, Eotvos u. 6, H-6720 Szeged, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
The four possible isomers of 3-benzyloxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol
(1a–4a) with proven configurations were converted into the corresponding 3-benzyloxy-16-
bromomethylestra-1,3,5(10)-triene-3,17-diols (5e–8e). Depending on the reaction conditions
the cis isomers of 3-benzyloxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol (1a and 2a) were
transformed into 3-benzyloxy-16-bromomethylestra-1,3,5(10)-trien-17-yl acetate (5b and
6b) or 16-bromomethyl-3-hydroxyestra-1,3,5(10)-trien-17-yl acetate (5c and 6c) on treatment
with HBr and acetic acid. The mechanism of the process can be interpreted as involv-
ing front-side neighboring group participation. Under similar experimental conditions, the
trans isomers (3a and 4a) yielded only 3-benzyloxy-16-acetoxymethylestra-1,3,5(10)-trien-
17-yl acetates (3b and 4b) or 16-acetoxymethylestra-1,3,5(10)-triene-3,17-diyl diacetates (3d
and 4d). Both the cis (1a and 2a) and the trans (3a, and 4a) isomers were transformed into
16-bromomethylestra-1,3,5(10)-trien-17-ol (5a–8a) by the Appel reaction on treatment with
CBr4/Ph3P. Debenzylation of 5a–8a was carried out with HBr and acetic acid to yield 5e–8e.
The debenzylation process in the presence of acetic anhydride produces the diacetates
5d–8d. The structures of the compounds were determined by means of MS, 1H NMR and
13C NMR spectroscopic methods. Compounds 5c–8c and 5e–8e were tested in a radioligand-
binding assay. Except for the affinity of 7e for the estrogen receptor (Ki = 2.55 nM), the
affinities of the eight compounds (5c–8c and 5e–8e) for the estrogen, androgen and pro-
gesterone receptors are low (Ki > 0.55, 0.52 and 0.21 ␮M, respectively).
Received 23 May 2005
Received in revised form 31 August
2005
Accepted 8 September 2005
Available online 17 November 2005
Dedicated to Professor Dr. Andra´s
Lipta´k on the occasion of his 70th
birthday.
Keywords:
16-Bromomethylestra-1,3,5(10)-
triene-3,17-diol
isomers
Front-side neighboring group
participation
Debenzylation
Wagner–Meerwein rearrangement
Estrogen
© 2005 Elsevier Inc. All rights reserved.
Androgen
Progesterone receptor binding
1.
Introduction
On the basis of the observations that more than two-thirds
of breast cancers occur in post-menopausal women, and that
ଝ
For a preceding paper in this series, see ref. [1].
Corresponding author. Tel.: +36 62 544199; fax: +36 62 544200.
∗
E-mail address: wolfling@chem.u-szeged.hu (J. Wo¨lfling).
0039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2005.09.008

142
steroids 7 1 ( 2 0 0 6 ) 141–153
their proliferation and survival depend on the estrogens, there
are now two major options for the treatment of estrogen-
dependent breast cancers [2]. One is the use of aromatase
inhibitors, which reduces the local in situ formation of estro-
gens. The other is the use of anti-estrogens, which block the
interaction of estrogens with their specific receptor (estro-
gen receptor, ER) [3]. Synthetic non-steroidal compounds are
significant anti-estrogens which are routinely used in the
endocrine therapy of breast cancer [2]. Tamoxifen behaves
as a mixed agonist/antagonist of estrogen action, thus lim-
iting its therapeutic potential [4]. Attempts to improve the
therapeutic efficacy have been focused on creating estradiol
analogs with anti-estrogenic activity. It is known from the lit-
erature that halogen-containing estrogens are of great impor-
tance in the diagnosis [5,6] and chemotherapy [4] of estrogen
receptor-positive human breast cancers [7]: the strongly elec-
tronegative halogens onto a sterane skeleton can enhance the
receptor affinity, while at the same time it may reduce the
tumorigenicity, too. Earlier examinations suggested that the
ER tolerates reasonably large substituents at positions C-7␣,
C-11␤, C-16␣ and C-17␣ in estradiol [8,9]. Moreover, Poirier
and co-workers reported that 16␣-(3-halopropyl)estradiol
and 16␣-bromoalkylamidoestradiol derivatives inhibit 17␤-
hydroxysteroid dehydrogenase, which is responsible for the
interconversion of the less potent estrone into the most potent
estrogen, estradiol, in the target tissues [10,11]. On the other
hand, Heiman et al. [12] and Katzenellenbogen and co-workers
[13] reported that halogenation of ring D of the steroid nucleus,
especially at position C-16␣, results in compounds with higher
affinities for lamb and rat uterine ERs than that of 17␤-
estradiol itself. In consequence of the above observations,
efforts have been directed toward the synthesis of estradiol
derivatives with a halomethyl group at C-16, which may be
hormonally inactive or at least may have a favorable ratio of
tumor inhibitory to hormonal activity.
acetate/CHCl3 (5:95, v/v); (C) ethyl acetate/CHCl3 (10:90, v/v).
The spots were detected by spraying with 5% phosphomolyb-
dic acid in 50% aqueous phosphoric acid. The Rf values were
determined for the spots observed by illumination at 254 and
365 nm. Flash chromatography: silica gel 60, 40–63 ␮m (Merck).
All solvents were distilled prior to use. Specific rotations were
measured in CHCl3 (c 1.00) or ethyl acetate (c 1.00) at 25 ◦C with
a Polamat-A (Zeiss-Jena) polarimeter and are given in units
of 10−1◦ cm2 g−1. Elemental analyses were performed with a
Perkin-Elmer CHN analyzer model 2400. 1H NMR spectra were
obtained in CDCl3 or DMSO-d6 solution at 500 MHz (Bruker DRX
500), and the 13C NMR spectra were recorded at 125 MHz on
the same instrument under the same conditions. Chemical
shifts (ı) are reported relative to TMS, and are given in ppm;
the coupling constants (J) are given in Hz. 13C NMR spectra are
1H-decoupled. Mass spectra were measured on a Varian MAT
311A spectrometer.
2.2.
Conversion of 3-benzyloxy-16-
hydroxymethylestra-1,3,5(10)-trien-17-ol (1a–4a) and
3-benzyloxy-16-bromomethylestra-1,3,5(10)-trien-17-ol
isomers (7a and 8a) with HBr in acetic acid
2.2.1. General procedures
2.2.1.1. Method A. A solution of 1.18 g (3 mmol) of 3-benzy-
loxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol (1a–4a) [14]
in 24 ml of anhydrous acetic acid was cooled to 10 ◦C and 2.4 ml
of 33% HBr (in acetic acid) was added. The reaction mixture
was diluted with water after stirring for 1 h. The precipitate
was collected by filtration and dissolved in CH2Cl2, and the
solution was washed with water and dried over Na2SO4. After
evaporation in vacuo, the crude product was purified by col-
umn chromatography (silica gel, CHCl3 or ethyl acetate/CHCl3:
2.5:97.5).
Herein, we report the synthesis of the four possible 16-
2.2.1.2. Method B. According to procedure A, 1.18 g (3 mmol) of
compound 1a–4a, or 1.78 g (3 mmol) of compounds 7a–8a and
2.4 ml of 33% HBr (in acetic acid) were allowed to react for 12 h.
The reaction mixture was diluted with water, the precipitate
was collected by filtration and dissolved in CH2Cl2, and the
solution was washed with water and dried over Na2SO4. After
evaporation in vacuo, the crude product was purified by col-
umn chromatography (silica gel, ethyl acetate/CHCl3: 2.5:97.5).
bromomethyl-3-hydroxyestra-1,3,5(10)-trien-17-yl
acetates
(5c–8c) and the four possible 16-bromomethylestradiol
derivatives (5e–8e) from the corresponding 3-benzyloxy-16-
hydroxymethylestra-1,3,5(10)-triene-3,17-diols (1a–4a). The
starting materials (1a–4a) were synthesized from estrone
according to a recently reported method [14]. The receptor-
binding examinations on the synthesized new compounds
were carried out on the ERs, androgen receptors (ARs) and
progesterone receptors (PRs). We wished to obtain answers
to the following questions: (1) How do the receptor binding
properties/processes of these eight derivatives (5c–8c and
5e–8e) differ? (2) How is the receptor-binding influenced by
the bromomethyl group at position C-16 and the acetoxy
group at position C-17 in ring D of the estrane skeleton?
2.2.1.3. Method C. According to procedure A, 1.18 g (3 mmol)
of compounds 1a–4a, or 1.78 g (3 mmol) 7a–8a and 2.4 ml of
33% HBr (in acetic acid) were allowed to react for 12 h. Next,
1 ml of acetic anhydride was added, and the reaction mixture
was diluted with water after stirring for 6 h. The precipitate
was collected by filtration and dissolved in CH2Cl2, and the
solution was washed with water and dried over Na2SO4. After
evaporation in vacuo, the crude product was purified by col-
umn chromatography (silica gel, ethyl acetate/CHCl3: 2.5:97.5).
2.
Experimental
2.1.
General
2.2.2. Reaction of 3-benzyloxy-16ˇ-hydroxymethylestra-
1,3,5(10)-trien-17ˇ-ol (1a) with HBr in acetic acid
2.2.2.1. Method A.
2.2.2.1.1. 3-Benzyloxy-16ˇ-bromomethylestra-1,3,5(10)-
trien-17ˇ-yl acetate (5b) and 3-benzyloxy-16ˇ-acetoxy-
methylestra-1,3,5(10)-trien-17ˇ-yl acetate (1b). Compound
Melting points were determined with a Kofler hot-stage appa-
ratus and are uncorrected. The reactions were monitored by
TLC on Kieselgel-G (Merck Si 254 F) layers (0.25 mm thick); sol-
vent system (ss): (A) ethyl acetate/CHCl3 (2.5:97.5, v/v); (B) ethyl

steroids 7 1 ( 2 0 0 6 ) 141–153
143
5b (1.24 g, 83%). Mp 144–146 ◦C; Rf = 0.65 (ss A); [˛]D20 + 54 (c
[M+], 266 (4), 213 (12), 172 (12), 159 (16), 133 (12), 106 (13), 43
(48).
1.00 in CHCl3). (Found: C, 67.55; H, 6.75. C28H33O3Br requires:
C, 67.60; H, 6.69%). 1H NMR (CDCl3) ı, ppm: 0.84 (s, 3H,
18-H3), 2.10 (s, 3H, OAc-CH3), 2.84 (m, 2H, 6-H2), 3.27 (t, 1H,
J = 10.0 Hz) and 3.46 (dd, 1H, J = 10.0 Hz, J = 5.8 Hz): 16a-H2,
4.86 (d, 1H, J = 10.0 Hz, 17-H), 5.02 (s, 2H, 3-benzyl-CH2), 6.71
(d, 1H, J = 2.5 Hz, 4-H), 6.76 (dd, 1H, J = 8.6 Hz, J = 2.5 Hz, 2-H),
7.17 (d, 1H, J = 8.6 Hz, 1-H), 7.31 (t, 1H, J = 7.3 Hz, 4ꢀ-H), 7.36
(t, 2H, J = 7.3 Hz, 3ꢀ,5ꢀ-H), 7.41 (d, 2H, J = 7.3 Hz, 2ꢀ,6ꢀ-H); 13C
NMR (CDCl3) ı, ppm: 13.2 (C-18), 20.9 (OAc-CH3), 26.1, 27.3,
29.7, 32.6, 35.2, 37.5, 38.0, 41.5, 43.7, 44.0 (C-13), 48.2, 70.0
(3-benzyl-CH2), 82.3 (C-17), 112.4 (C-2), 114.9 (C-4), 126.3 (C-1);
127.4 (2C, C-2ꢀ,6ꢀ), 127.8 (C-4ꢀ), 128.5 (2C, C-3ꢀ,5ꢀ), 132.6 (C-10),
137.4 (C-1ꢀ), 137.8 (C-5), 156.9 (C-3), 170.6 (OAc-CO). EI-MS
(70 eV) m/z (%): 498 (24) [M+ + 2], 496 (23) [M+], 91 (100), 43 (8).
1b (157 mg 11%). Mp 100–102 ◦C ([14], Mp 100–101 ◦C); Rf = 0.40
(ss A); [˛]2D0 + 37 (c 1.00 in CHCl3). (Found: C, 75.48; H, 7.52.
2.2.2.3. Method C.
2.2.2.3.1. 16ˇ-Bromomethylestra-1,3,5(10)-triene-3,17ˇ-
diyl diacetate (5d) and 16ˇ-acetoxymethylestra-1,3,5(10)-
triene-3,17ˇ-diyl diacetate (1d). Compound 5d (1.02 g, 76%).
Mp 122–123.5 ◦C; Rf = 0.45 (ss A); [˛]D20 + 58 (c 1.00 in CHCl3).
(Found: C, 61.58; H, 6.67. C23H29O4Br requires: C, 61.47; H,
6.50%). 1H NMR (CDCl3) ı, ppm: 0.84 (s, 3H, 18-H3), 2.10 (s, 3H,
17-OAc-CH3), 2.27 (s, 3H, 3-OAc-CH3), 2.86 (m, 2H, 6-H2), 3.26 (t,
1H, J = 10.0 Hz) and 3.46 (dd, 1H, J = 10.0 Hz, J = 5.8 Hz): 16a-H2,
4.86 (d, 1H, J = 10.1 Hz, 17-H), 6.79 (d, 1H, J = 2.2 Hz, 4-H), 6.84
(dd, 1H, J = 8.5 Hz, J = 2.2 Hz, 2-H), 7.25 (t, 1H, J = 8.5 Hz, 1-H);
13C NMR (CDCl3) ı, ppm: 13.2 (C-18), 20.8 (17-OAc-CH3), 21.0
(3-OAc-CH3), 25.9, 27.1, 29.4, 32.6, 35.1, 37.4, 37.6, 41.5, 43.9,
44.0 (C-13), 48.2, 82.2 (C-17), 118.6 (C-2), 121.5 (C-4), 126.3 (C-1);
137.6 (C-10), 138.0 (C-5), 148.6 (C-3), 169.6 (3-OAc-CO), 170.6
(17-OAc-CO); EI-MS (70 eV) m/z (%): 450 (8) [M+ + 2], 448 (8) [M+],
408 (100), 406 (100), 267 (12), 172 (9), 159 (14), 133 (14), 43 (41).
1d (103 mg, 8%). Mp 150–152 ◦C; Rf = 0.50 (ss B); [˛]D20 + 44 (c
1.00 in CHCl3). (Found: C, 69.95; H, 7.45. C25H32O6 requires: C,
70.07; H, 7.53%). 1H NMR (CDCl3) ı, ppm: 0.86 (s, 3H, 18-H3),
2.02 and 2.07 (2 × s, 2 × 3H, 16a- and 17-OAc-CH3), 2.27 (s,
3H, 3-OAc-CH3), 2.86 (m, 2H, 6-H2), 4.02 (dd, 1H, J = 11.0 Hz,
J = 7.5 Hz) and 4.12 (dd, 1H, J = 11.0 Hz, J = 6.9 Hz): 16a-H2, 4.89
(d, 1H, J = 10.1 Hz, 17-H), 6.79 (d, 1H, J = 1.8 Hz, 4-H), 6.83 (dd, 1H,
J = 8.3 Hz, J = 1.8 Hz, 2-H), 7.26 (d, 1H, J = 8.3 Hz, 1-H); 13C NMR
(CDCl3) ı, ppm: 12.8 (C-18), 20.8 (2C, 16a- and 17-OAc-CH3),
21.0 (3-OAc-CH3), 25.9, 27.1, 29.3, 29.4, 37.4, 37.6, 37.7, 43.5
(C-13), 43.8, 48.8, 65.1 (C-16a), 81.5 (C-17), 118.6 (C-2), 121.4
(C-4), 126.3 (C-1); 137.6 (C-10), 137.9 (C-5), 148.5 (C-3), 169.6
(3-OAc-CO), 170.7 (2C, 16a- and 17-OAc-CO); EI-MS (70 eV) m/z
(%): 428 (11) [M+], 386 (100), 172 (4), 159 (6), 133 (5), 43 (16).
C30H
36O5 requires: C, 75.60; H, 7.61%). 1H NMR (CDCl3) ı, ppm:
0.85 (s, 3H, 18-H3), 2.02 and 2.06 (2 × s, 2 × 3H, 2 × OAc-CH3),
2.84 (m, 2H, 6-H2), 4.02 (dd, 1H, J = 11.0 Hz, J = 7.5 Hz) and 4.12
(dd, 1H, J = 11.0 Hz, J = 6.9 Hz): 16a-H2, 4.89 (d, 1H, J = 10.1 Hz,
17-H), 5.01 (s, 2H, 3-benzyl-CH2), 6.70 (d, 1H, J = 2.3 Hz, 4-H),
6.76 (dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H), 7.17 (d, 1H, J = 8.5 Hz,
1-H), 7.29 (t, 1H, J = 7.3 Hz, 4ꢀ-H), 7.35 (t, 2H, J = 7.3 Hz, 3ꢀ,5ꢀ-H),
7.41 (d, 2H, J = 7.3 Hz, 2ꢀ,6ꢀ-H); 13C NMR (CDCl3) ı, ppm: 12.9
(C-18), 20.8 (2C, 2 × OAc-CH3), 26.1, 27.3, 29.3, 29.6, 37.4, 37.7,
38.0, 43.6 (C-13), 43.7, 48.8, 65.2 (C-16a), 70.0 (3-benzyl-CH2),
81.6 (C-17), 112.4 (C-2), 114.9 (C-4), 126.2 (C-1); 127.4 (2C,
C-2ꢀ,6ꢀ), 127.8 (C-4ꢀ), 128.5 (2C, C-3ꢀ,5ꢀ), 132.6 (C-10), 137.3 (C-1ꢀ),
137.8 (C-5), 156.8 (C-3), 170.7 (2C, 2 × OAc-CO); EI-MS (70 eV)
m/z (%): 476 (60) [M+], 91 (100), 43 (8).
2.2.2.2. Method B.
2.2.2.2.1. 16ˇ-Bromomethyl-3-hydroxyestra-1,3,5(10)-
trien-17ˇ-yl acetate (5c) and 16ˇ-acetoxymethyl-3-hydro-
xyestra-1,3,5(10)-trien-17ˇ-yl acetate (1c). Compound 5c
(1.16 g, 75%). Mp 194–198 ◦C; Rf = 0.45 (ss C); [˛]2D0 + 63 (c 1.00
in CHCl3). (Found: C, 62.07; H, 6.54. C21H27O3Br requires: C
61.92; H 6.68%). 1H NMR (CDCl3) ı, ppm: 0.84 (s, 3H, 18-H3),
2.11 (s, 3H, OAc-CH3), 2.81 (m, 2H, 6-H2), 3.27 (t, 1H, J = 10.0 Hz)
and 3.46 (dd, 1H, J = 10.0 Hz, J = 5.9 Hz): 16a-H2, 4.86 (d, 1H,
J = 10.0 Hz, 17-H), 6.56 (d, 1H, J = 2.4 Hz, 4-H), 6.62 (dd, 1H,
J = 8.4 Hz, J = 2.4 Hz, 2-H), 7.12 (d, 1H, J = 8.4 Hz, 1-H); 13C NMR
(CDCl3) ı, ppm: 13.2 (C-18), 20.9 (OAc-CH3), 26.1, 27.2, 29.5,
32.6, 35.2, 37.4, 38.0, 41.4, 43.7, 44.0 (C-13), 48.2, 82.3 (C-17),
112.8 (C-2), 115.3 (C-4), 126.4 (C-1); 132.3 (C-10), 138.0 (C-5),
153.5 (C-3), 170.8 (OAc-CO); EI-MS (70 eV) m/z (%): 408 (99)
[M+ + 2], 406 (100) [M+], 267 (18), 213 (14), 172 (16), 159 (28),
133 (28), 43 (52). 1c (157 mg, 12%). Mp 184 ◦C; Rf = 0.40 (ss C);
[˛]2D0 + 46 (c 1.00 in CHCl3). (Found: C, 71.54; H, 7.65. C23H30O5
requires: C 71.48; H 7.82%). 1H NMR (CDCl3) ı, ppm: 0.84 (s,
3H, 18-H3), 2.04 and 2.08 (2 × s, 2 × 3H, 2 × OAc-CH3), 2.81 (m,
2H, 6-H2), 4.02 (dd, 1H, J = 11.0 Hz, J = 7.6 Hz) and 4.13 (dd, 1H,
J = 11.0 Hz, J = 6.9 Hz): 16a-H2, 4.89 (d, 1H, J = 10.0 Hz, 17-H),
6.57 (d, 1H, J = 2.2 Hz, 4-H), 6.64 (dd, 1H, J = 8.4 Hz, J = 2.2 Hz,
2-H), 7.12 (d, 1H, J = 8.4 Hz, 1-H); 13C NMR (CDCl3) ı, ppm:
12.9 (C-18), 21.0 (2C, 2 × OAc-CH3), 26.1, 27.3, 29.5 (2C), 37.4,
37.6, 38.0, 43.6 (C-13), 43.7, 48.7, 65.4 (C-16a), 81.8 (C-17), 112.8
(C-2), 115.3 (C-4), 126.4 (C-1); 132.0 (C-10), 138.0 (C-5), 153.8
(C-3), 171.3 (2C, 2 × OAc-CO); EI-MS (70 eV) m/z (%): 386 (100)
2.2.3. Conversion of 3-benzyloxy-16˛-hydroxymethyl-
estra-1,3,5(10)-trien-17˛-ol (2a) with HBr in acetic acid
2.2.3.1. Method A.
2.2.3.1.1. 3-Benzyloxy-16˛-bromomethylestra-1,3,5(10)-
trien-17˛-yl acetate (6b) and 3-benzyloxy-16˛-acetoxy-
methylestra-1,3,5(10)-trien-17˛-yl acetate (2b). Compound
6b was eluted with CHCl3 (1.27 g, 85%). Mp 107–108 ◦C; Rf = 0.65
(ss A); [˛]2D0 + 75 (c 1.00 in CHCl3). (Found: C, 67.52; H, 6.75.
C28H
33O3Br requires: C, 67.60; H, 6.69%). 1H NMR (CDCl3) ı,
ppm: 0.86 (s, 3H, 18-H3), 2.11 (s, 3H, 17-OAc-H3), 2.85 (m, 2H, 6-
H2), 3.36 (dd, 1H, J = 9.3 Hz, J = 7.6 Hz) and 3.44 (t, 1H, J = 9.3 Hz):
16a-H2, 5.02 (s, 2H, 3-benzyl-CH2), 5.08 (d, 1H, J = 5,5 Hz, 17-H),
6.70 (d, 1H, J = 2.4 Hz, 4-H), 6.77 (dd, 1H, J = 8.5 Hz, J = 2.4 Hz,
2-H), 7.17 (d, 1H, J = 8.5 Hz, 1-H), 7.30 (t, 1H, J = 7.4 Hz, 4ꢀ-H), 7.37
(t, 2H, J = 7.4 Hz, 3ꢀ,5ꢀ-H), 7.41 (d, 2H, J = 7.4 Hz, 2ꢀ,6ꢀ-H); 13C NMR
(CDCl3) ı, ppm: 16.7 (C-18), 20.8 (17-OAc-CH3), 25.9, 27.9, 29.8,
31.2, 31.9, 33.4, 38.9, 42.7, 43.5, 46.3 (C-13), 48.3, 70.0 (3-benzyl-
CH2), 81.5 (C-17), 112.4 (C-2), 114.9 (C-4), 126.3 (C-1); 127.4
(2C, C-2ꢀ,6ꢀ), 127.8 (C-4ꢀ), 128.5 (2C, C-3ꢀ,5ꢀ), 132.6 (C-10), 137.3
(C-1ꢀ), 137.8 (C-5), 156.8 (C-3), 170.3 (OAc-CO). EI-MS (70 eV)
m/z (%): 498 (28) [M+ + 2], 496 (28) [M+], 91 (100), 43 (7). Con-
tinued elution with ethyl acetate/CHCl3 (2.5:97.5) yielded 2b
(170 mg, 12%). Mp 106–108 ◦C ([14], Mp 106–108); Rf = 0.30 (ss A);
[˛]2D0 + 67 (c 1.00 in CHCl3). (Found: C, 75.42; H, 7.56. C30H36O5
requires: C 75.60%, H 7.61%). 1H NMR (CDCl3) ı, ppm: 0.85 (s,

144
steroids 7 1 ( 2 0 0 6 ) 141–153
3H, 18-H3), 2.01 and 2.07 (2 × s, 2 × 3H, 2 × OAc-H3), 2.84 (m,
2H, 6-H2), 4.03–4.12 (overlapping multiplets, 2H, 16a-H2), 5.02
(d, 2H, 3-benzyl-CH2), 5.09 (d, 1H, J = 5.7 Hz, 17-H), 6.71 (d, 1H,
J = 2.5 Hz, 4-H), 6.77 (dd, 1H, J = 8.6 Hz, J = 2.5 Hz, 2-H), 7.18 (d, 1H,
J = 8.6 Hz, 1-H), 7.30 (t, 1H, J = 7.3 Hz, 4ꢀ-H), 7.36 (t, 2H, J = 7.3 Hz,
3ꢀ,5ꢀ-H), 7.41 (d, 2H, J = 7.3 Hz, 2ꢀ,6ꢀ-H); 13C NMR (CDCl3) ı, ppm:
16.8 (C-18), 20.8 (2C, 2 × OAc-CH3), 25.9, 27.9, 28.1, 29.7, 31.6,
38.6, 38.9, 43.5, 46.0 (C-13), 48.1, 64.0 (C-16a), 70.0 (3-benzyl-
CH2), 80.7 (C-17), 112.3 (C-2), 114.9 (C-4), 126.2 (C-1); 127.4 (2C,
C-2ꢀ,6ꢀ), 127.8 (C-4ꢀ), 128.5 (2C, C-3ꢀ,5ꢀ), 132.6 (C-10), 137.3 (C-1ꢀ),
137.8 (C-5), 156.8 (C-3), 170.2 and 170.9 (2C, 2 × OAc-CO); EI-MS
(70 eV) m/z (%): 476 (16) [M+], 91 (100), 43 (74).
83 (14), 43 (39). 2d (102 mg, 8%). Mp 67–68 ◦C; Rf = 0.45 (ss B);
[˛]2D0 + 64 (c 1.00 in CHCl3). (Found: C, 69.92; H, 7.61. C25H32O6
requires: C 70.07; H 7.53%). 1H NMR (CDCl3) ı, ppm: 0.85 (s,
3H, 18-H3), 2.01 and 2.07 (2 × s, 2 × 3H, 16a- and 17-OAc-CH3),
2.27 (s, 3H, 3-OAc-CH3), 2.86 (m, 2H, 6-H2), 4.03–4.13 (m, 2H,
16a-H2), 5.10 (d, 1H, J = 5.2 Hz, 17-H), 6.79 (d, 1H, J = 2.3 Hz, 4-H),
6.83 (dd, 1H, J = 8.1 Hz, J = 2.3 Hz, 2-H), 7.26 (d, 1H, J = 8.1 Hz,
1-H); 13C NMR (CDCl3) ı, ppm: 16.7 (C-18), 20.8 (2C, 16a- and
17-OAc-CH3), 21.0 (3-OAc-CH3), 25.7, 27.7, 28.1, 29.4, 31.6, 38.5,
38.6, 43.6, 45.9 (C-13), 48.1, 64.0 (C-16a), 80.6 (C-17), 118.6 (C-2),
121.4 (C-4), 126.2 (C-1); 137.6 (C-10), 138.0 (C-5), 148.5 (C-3),
169.6 (3-OAc-CO), 170.2 and 170.9 (2C, 16a- and 17-OAc-CO);
EI-MS (70 eV) m/z (%): 428 (14) [M+], 386 (100), 172 (5), 159 (12),
133 (8), 43 (41).
2.2.3.2. Method B.
2.2.3.2.1. 16˛-Bromomethyl-3-hydroxyestra-1,3,5(10)-
trien-17˛-yl acetate (6c) and 16˛-acetoxymethyl-3-hydro-
xyestra-1,3,5(10)-trien-17˛-yl acetate (2c). Compound 6c
(953 mg, 78%). Mp 201–203 ◦C; Rf = 0.30 (ss A) [˛]2D0 + 85 (c 1.00
2.2.4. Reaction of 3-benzyloxy-16˛-hydroxymethyl-
estra-1,3,5(10)-trien-17ˇ-ol (3a) with HBr in acetic acid
2.2.4.1. Method A.
in CHCl3). (Found: C, 62.05; H, 6.72. C21
H
27O3Br requires: C,
2.2.4.1.1.3-Benzyloxy-16˛-acetoxymethylestra-1,3,5(10)-
trien-17ˇ-yl acetate (3b). Compound 3b (1.36 mg, 95%). Mp
132–134 ◦C ([14] Mp 131–132 C); Rf = 0.30 (ss A); [˛]2D0 + 7 (c 1.00
in CHCl3). (Found: C, 75.58; H, 7.78. C30H36O5 requires: C, 75.60;
H, 7.61%). 1H NMR (CDCl3) ı, ppm: 0.85 (s, 3H, 18-H3), 2.03 and
2.06 (2 × s, 2 × 3H, 2 × OAc-CH3), 2.83 (m, 2H, 6-H2), 4.06 (dd,
1H, J = 10.9 Hz, J = 6.9 Hz) and 4.12 (dd, 1H, J = 10.9 Hz, J = 6.4 Hz):
16a-H2, 4.72 (d, 1H, J = 7.7 Hz, 17-H), 5.01 (s, 2H, 3-benzyl-H2),
6.70 (d, 1H, J = 2.4 Hz, 4-H), 6.76 (dd, 1H, J = 8.6 Hz, J = 2.4 Hz,
2-H), 7.17 (d, 1H, J = 8.6 Hz, 1-H), 7.29 (t, 1H, J = 7.3 Hz, 4ꢀ-H), 7.35
(t, 2H, J = 7.3 Hz, 3ꢀ,5ꢀ-H), 7.41 (d, 2H, J = 7.3 Hz, 2ꢀ,6ꢀ-H); 13C NMR
(CDCl3) ı, ppm: 12.7 (C-18), 20.8 and 21.0 (2C, 2 × OAc-CH3),
26.1, 27.1, 27.4, 29.6, 36.9, 38.4, 40.3, 43.7, 44.3 (C-13), 48.7,
66.7 (C-16a), 70.0 (3-benzyl-CH2), 83.8 (C-17), 112.4 (C-2), 114.9
(C-4), 126.3 (C-1); 127.4 (2C, C-2ꢀ,6ꢀ), 127.8 (C-4ꢀ), 128.5 (2C,
C-3ꢀ,5ꢀ), 132.6 (C-10), 137.4 (C-1ꢀ), 137.8 (C-5), 156.8 (C-3), 170.7
and 170.9 (2C, 2 × OAc-CO); EI-MS (70 eV) m/z (%): 476 (48), 91
(100), 84 (20), 49 (52), 43 (11).
61.92; H, 6.68%). 1H NMR (CDCl3) ı, ppm: 0.86 (s, 3H, 18-H3),
2.12 (s, 3H, OAc-CH3), 2.81 (m, 2H, 6-H2), 3.36 (dd, 1H, J = 9.4 Hz,
J = 7.5 Hz) and 3.44 (t, 1H, J = 9.4 Hz): 16a-H2, 4.76 (s, 1H, 3-OH),
5.08 (d, 1H, J = 5.6 Hz, 17-H), 6.56 (d, 1H, J = 2.5 Hz, 4-H), 6.62
(dd, 1H, J = 8.4 Hz, J = 2.5 Hz, 2-H), 7.12 (d, 1H, J = 8.4 Hz, 1-H);
13C NMR (CDCl3) ı, ppm: 16.8 (C-18), 20.9 (OAc-CH3), 25.9,
27.8, 29.6, 31.2, 31.9, 33.4, 38.9, 42.8, 43.5, 46.3 (C-13), 48.3,
81.6 (C-17), 112.8 (C-2), 115.3 (C-4), 126.5 (C-1); 132.4 (C-10),
138.1 (C-5), 153.5 (C-3), 170.5 (OAc-CO); EI-MS (70 eV) m/z
(%): 408.1 (69) [M+ + 2], 406 (67) [M+], 267 (16), 172 (26), 160
(100), 146 (47), 133 (50), 107 (22), 43 (87). 2c (115 mg, 10%) as a
colorless oil. Rf = 0.45 (ss C); [˛]20 + 57 (c 1.00 in CHCl3). (Found:
D
C, 71.36; H, 7.95. C23H30O5 requires: C, 71.48; H, 7.82%). 1H
NMR (CDCl3) ı, ppm: 0.85 (s, 3H, 18-H3), 2.01 and 2.08 (2 × s,
2 × 3H, 2 × OAc-CH3), 2.81 (m, 2H, 6-H2), 4.04–4.13 (overlapping
multiplets, 2H, 16a-H2), 5.10 (d, 1H, J = 5.7 Hz, 17-H), 6.56 (d,
1H, J = 2.4 Hz, 4-H), 6.63 (dd, 1H, J = 8.5 Hz, J = 2.4 Hz, 2-H), 7.13
(d, 1H, J = 8.5 Hz, 1-H); 13C NMR (CDCl3) ı, ppm: 16.8 (C-18),
20.8 (2C, 2 × OAc-CH3), 25.9, 27.9, 28.2, 29.6, 31.7, 38.6, 38.9,
43.5, 46.0 (C-13), 48.2,64.1 (C-16a), 80.8 (C-17), 112.8 (C-2), 115.3
(C-4), 126.5 (C-1); 132.4 (C-10), 138.1 (C-5), 153.6 (C-3), 170.4 and
171.2 (2C, 2 × OAc-CO); EI-MS (70 eV) m/z (%): 386 (100) [M+],
266 (12), 213 (7), 172 (12), 159 (26), 133 (16), 106 (16), 43 (74).
2.2.4.2. Method B.
2.2.4.2.1. 16˛-Acetoxymethyl-3-hydroxyestra-1,3,5(10)-
trien-17ˇ-yl acetate (3c). Compound 3c (1.00 g, 87%). Mp
145–147 ◦C; Rf = 0.40 (ss C); [˛]D20 − 3 (c 1.00 in CHCl3). (Found: C,
71.57; H, 7.88. C23H30O5 requires: C, 71.48; H, 7.82%). 1H NMR
(CDCl3) ı, ppm: 0.85 (s, 3H, 18-H3), 2.05 and 2.08 (2 × s, 2 × 3H,
2 × OAc-CH3), 2.80 (m, 2H, 6-H2), 4.08 (dd, 1H, J = 10.9 Hz,
J = 6.8 Hz) and 4.12 (dd, 1H, J = 10.9 Hz, J = 6.5 Hz): 16a-H2, 4.74
(d, 1H, J = 7.7 Hz, 17-H), 5.74 (s, 1H, 3-OH), 6.56 (d, 1H, J = 2.4 Hz,
4-H), 6.63 (dd, 1H, J = 8.4 Hz, J = 2.4 Hz, 2-H), 7.11 (d, 1H, J = 8.4 Hz,
1-H); 13C NMR (CDCl3) ı, ppm: 12.6 (C-18), 20.8 and 21.0 (2C,
2 × OAc-CH3), 26.0, 27.1, 27.4, 29.4, 36.8, 38.4, 40.2, 43.6, 44.3
(C-13), 48.7, 66.9 (C-16a), 84.0 (C-17), 112.8 (C-2), 115.3 (C-4),
126.4 (C-1); 132.0 (C-10), 137.9 (C-5), 153.7 (C-3), 171.0 and 171.3
(2C, 2 × OAc-CO); EI-MS (70 eV) m/z (%): 386 (95) [M+], 266 (12),
213 (12), 172 (19), 159 (34), 159 (34), 133 (27), 10 (8), 43 (100).
2.2.3.3. Method C.
2.2.3.3.1. 16˛-Bromomethylestra-1,3,5(10)-triene-3,17˛-
diyl diacetate (6d) and 16˛-acetoxymethylestra-1,3,5(10)-
triene-3,17˛-diyl diacetate (2d). Compound 6d (990 mg,
74%). Mp 120–122 ◦C; Rf = 0.50 (ss A); [˛]D20 + 72 (c 1.00 in CHCl3).
(Found: C, 61.66; H, 6.35. C23H29O4Br requires: C 61.47; H
6.50%). 1H NMR (CDCl3) ı, ppm: 0.86 (s, 3H, 18-H3), 2.12 (s, 3H,
17-OAc-CH3), 2.27 (s, 3H, 3-OAc-CH3), 2.87 (m, 2H, 6-H2), 3.36
(dd, 1H, J = 9.2 Hz, J = 7.5 Hz) and 3.44 (t, 1H, J = 9.2 Hz): 16a-H2,
5.08 (d, 1H, J = 5.6 Hz, 17-H), 6.79 (d, 1H, J = 2.1 Hz, 4-H), 6.84
(dd, 1H, J = 8.4 Hz, J = 2.1 Hz, 2-H), 7.26 (d, 1H, J = 8.4 Hz, 1-H);
13C NMR (CDCl3) ı, ppm: 16.7 (C-18), 20.8 (17-OAc-CH3), 21.1
(3-OAc-CH3), 25.7, 27.7, 29.5, 31.2, 31.8, 33.3, 38.5, 42.7, 43.6,
46.2 (C-13), 48.4, 81.4 (C-17), 118.6 (C-2), 121.5 (C-4), 126.3 (C-1),
137.6 (C-10), 138.0 (C-5), 148.5 (C-3), 169.7 (3-OAc-CO) and
170.3 (17-OAc-CO); EI-MS (70 eV) m/z (%): 450 (10) [M+ + 2], 448
(10) [M+], 408 (96), 406 (100), 267 (87), 172 (7), 160 (26), 133 (12),
2.2.4.3. Method C.
2.2.4.3.1. 16˛-Acetoxymethylestra-1,3,5(10)-triene-
3,17ˇ-diyl diacetate (3d). Compound 3d (1.25 g, 98%). Mp
85–88 ◦C; Rf = 0.40 (ss B); [˛]D20 − 6.0 (c 1.00 in CHCl3). (Found:
C, 69.95; H, 7.65. C25H32O6 requires: C, 70.07; H, 7.53%). 1H
NMR (CDCl3) ı, ppm: 0.85 (s, 3H, 18-H3), 2.04 and 2.07 (2 × s,

steroids 7 1 ( 2 0 0 6 ) 141–153
145
2 × 3H, 16a- and 17-OAc-CH3), 2.27 (s, 3H, 3-OAc-CH3), 2.85
(m, 2H, 6-H2), 4.06 (dd, 1H, J = 10.8 Hz, J = 6.8 Hz) and 4.12 (dd,
1H, J = 10.8 Hz, J = 6.4 Hz): 16a-H2, 4.74 (d, 1H, J = 7.7 Hz, 17-H),
6.79 (d, 1H, J = 2.2 Hz, 4-H), 6.83 (dd, 1H, J = 8.3 Hz, J = 2.2 Hz,
2-H), 7.25 (d, 1H, J = 8.3 Hz, 1-H); 13C NMR (CDCl3) ı, ppm: 12.6
(C-18), 20.8, 20.9 and 21.0 (3C, 3 × OAc-CH3), 25.9, 26.9, 27.3,
29.3, 36.8, 38.0, 40.2, 43.8, 44.2 (C-13), 48.7, 66.7 (C-16a), 83.7
(C-17), 118.6 (C-2), 121.4 (C-4), 126.3 (C-1); 137.6 (C-10), 137.9
(C-5), 148.5 (C-3), 169.6 (3-OAc-CO), 170.6 and 170.9 (2C, 16a-
and 17-OAc-CO); EI-MS (70 eV) m/z (%): 428 (11) [M+], 386 (100),
172 (8), 159 (14), 133 (10), 43 (54).
17), 112.4 (C-2), 114.9 (C-4), 126.3 (C-1), 127.4 (2C, C-2ꢀ,6ꢀ), 127.8
(C-4ꢀ), 128.5 (2C, C-3ꢀ,5ꢀ), 132.7 (C-10), 137.4 (C-1ꢀ), 137.9 (C-5),
156.8 (C-3), 170.3 and 171.0 (2C, 2 × OAc-CO); EI-MS (70 eV) m/z
(%): 476 (39) [M+], 91 (100), 43 (9).
2.2.5.2. Method B.
2.2.5.2.1. 16ˇ-Acetoxymethyl-3-hydroxyestra-1,3,5(10)-
trien-17˛-yl acetate (4c). Compound 4c (560 mg, 48%). Mp
155–157 ◦C; Rf = 0.40 (ss C); [˛]D20 + 57 (c 1.00 in CHCl3). (Found:
C, 71.55; H, 7.96. C23H30O5 requires: C, 71.48; H, 7.82%).
1H NMR (CDCl3) ı, ppm: 0.84 (s, 3H, 18-H3), 2.07 and 2.08
(2 × s, 2 × 3H, 2 × OAc-CH3), 2.81 (m, 2H, 6-H2), 4.18 (dd, 1H,
J = 11.0 Hz, J = 7.2 Hz) and 4.22 (dd, 1H, J = 11.0 Hz, J = 7.5 Hz):
16a-H2, 4.72 (d, 1H, J = 1.9 Hz, 17-H), 5.70 (s, 1H, 3-OH), 6.57 (d,
1H, J = 2.4 Hz, 4-H), 6.64 (dd, 1H, J = 8.4 Hz, J = 2.4 Hz, 2-H), 7.13
(d, 1H, J = 8.5 Hz, 1-H); 13C NMR (CDCl3) ı, ppm: 17.0 (C-18),
20.8 and 21.0 (2C, 2 × OAc-CH3), 25.9, 27.8, 29.2, 29.5, 32.1, 38.5,
43.3, 44.5 (C-13), 45.6, 49.9, 66.6 (C-16a), 83.7 (C-17), 112.8 (C-2),
115.2 (C-4), 126.3 (C-1); 132.0 (C-10), 137.9 (C-5), 153.7 (C-3),
170.6 and 171.3 (2C, 2 × OAc-CO); EI-MS (70 eV) m/z (%): 386
(100) [M+], 266 (10), 213 (6), 172 (10), 159 (23), 133 (15), 106 (13),
43 (10).
2.2.5. Conversion of 3-benzyloxy-16ˇ-
hydroxymethylestra-1,3,5(10)-trien-17˛-ol (4a)
with HBr in acetic acid
2.2.5.1. Method A.
2.2.5.1.1. 16ˇ-Acetoxymethyl-3-benzyloxy-17-methyl-
18-nor-estra-1,3,5(10),13(17)-tetraene (16b), 3-benzyloxy-
16ˇ-hydroxymethylestra-1,3,5(10),13(17)-tetraene (16a) and
16ˇ-acetoxymethyl-3-benzyloxyestra-1,3,5(10)-trien-17˛-yl
acetate (4b). Compound 16b (290 mg, 23%) as colorless oil;
Rf = 0.80 (ss A); [˛]20 + 85 (c 1.00 in CHCl3). (Found: C, 80.56;
D
H, 7.83. C28H32O3 requires: C, 80.73; H, 7.74%). 1H-NMR ı,
ppm 1.61 (s, 3H, 18-H3), 2.03 (s, 3H, Ac-Me), 2.81 (m, 2H,
6-H2), 3.98 (dd, 1H, J = 10.8 Hz, J = 6.9 Hz) and 4.22 (dd, 1H,
J = 10.8 Hz, J = 4.8 Hz): 16a-H2, 5.02 (s, 2H, benzyl-H2), 6.70 (d,
1H, J = 2.3 Hz, 4-H), 6.78 (dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H), 7.24
(d, 1H, J = 8.5 Hz, 1-H), 7.30 (t, 1H, J = 7.5 Hz, 4ꢀ-H), 7.37 (t, 2H,
J = 7.5 Hz, 3ꢀ-, 5ꢀ-H), 7.42 (d, 2H, J = 7.5 Hz, 2ꢀ-, 6ꢀ-H). 13C-NMR
ı, ppm: 12.3 (C-18), 21.4 (Ac-Me), 26.4, 28.1, 30.8, 31.8, 32.8,
42.8, 48.1, 49.6, 51.3, 68.0, 70.4, 113.0, 115.1, 127.6, 127.9, 128.2,
128.9, 132.8, 137.8, 138.8, 139.3, 157.1 (C-3), 171.7 (Ac-CO).
The white oily 16b (104 mg, 0.25 mmol) obtained in
the chromatographic separation was dissolved in methanol
(10 ml) containing NaOCH3 (5 mg, 0.092 mmol) and the solu-
tion was allowed to stand for 24 h. It was diluted with water,
and the white precipitate that separated out was filtered off
and recrystallized from a mixture of acetone/hexane; 16a
(76 mg, 81%). Mp 128–129 ◦C; Rf = 0.65 (ss A); [˛]2D0 + 52 (c 1.00 in
CHCl3). (Found: C, 83.57; H, 7.92. C26H30O2 requires: C, 83.38;
H, 8.07%). 1H-NMR ı, ppm: 1.67 (s, 3H, 18-H3), 2.84 (m, 2H,
6-H2), 3.72 (m, 2H, 16a-H2), 5.06 (s, 2H, benzyl-H2), 6.74 (d,
1H, J = 2.6 Hz, 4-H), 6.82 (dd, 1H, J = 8.6 Hz, J = 2.6 Hz, 2-H), 7.27
(d, 1H, J = 8.6 Hz, 1-H), 7.35 (t, 1H, J = 7.0 Hz, 4ꢀ-H), 7.41 (t, 2H,
J = 7.0 Hz, 3ꢀ-, 5ꢀ-H), 7.46 (d, 2H, J = 7.0 Hz, 2ꢀ-, 6ꢀ-H). 13C-NMR ı,
ppm: 12.14 (C-18), 26.5, 28.0,28.1,28.2, 30.8, 31.7, 31.9,42.8,49.5,
51.3, 51.4,65.2, 70.4,113.0,115.2, 127.6, 127.9, 128.3, 129.0, 132.9,
137.8, 138.9,140.4, 157.1.
2.2.5.3. Method C.
2.2.5.3.1. 16ˇ-Acetoxymethylestra-1,3,5(10)-triene-
3,17˛-diyl diacetate (4d). Compound 4d (530 mg, 41%)
as a colorless oil; Rf = 0.30 (ss A); [˛]20 + 45 (c 1.00 in CHCl3).
D
(Found: C, 69.91; H, 7.45. C25H32O6 requires: C, 70.07; H, 7.53%).
1H NMR (CDCl3) ı, ppm: 0.85 (s, 3H, 18-H3), 2.06 and 2.07 (2 × s,
2 × 3H, 16a- and 17-OAc-CH3), 2.27 (s, 3H, 3-OAc-CH3), 2.87
(m, 2H, 6-H2), 4.17 (dd, 1H, J = 10.9 Hz, J = 7.1 Hz) and 4.23 (dd,
1H, J = 10.9 Hz, J = 7.4 Hz): 16a-H2, 4.71 (d, 1H, J = 1.64 Hz, 17-H),
6.79 (d, 1H, J = 1.9 Hz, 4-H), 6.84 (dd, 1H, J = 8.4 Hz, J = 1.9 Hz,
2-H), 7.27 (d, 1H, J = 8.4 Hz, 1-H); 13C NMR (CDCl3) ı, ppm: 16.9
(C-18), 20.8 (3-OAc-CH3) and 21.0 (2C, 16a- and 17-OAc-CH3),
25.7, 27.7, 29.2, 29.4, 32.1, 38.1, 43.5, 44.5 (C-13), 45.7, 50.0, 66.4
(C-16a), 83.5 (C-17), 118.6 (C-2), 121.4 (C-4), 126.2 (C-1); 137.6
(C-10), 138.0 (C-5), 148.5 (C-3), 169.6 (3-OAc-CO), 170.3 and
171.0 (2C, 16a- and 17-OAc-CO); EI-MS (70 eV) m/z (%): 428 (12)
[M+], 386 (100), 172 (6), 159 (12), 133 (9), 43 (57).
2.3.
Conversion of 3-benzyloxy-16-
hydroxymethylestra-1,3,5(10)-trien-17-ol isomers
(1a–4a) to 3-benzyloxy-16-bromomethylestra-
1,3,5(10)-trien-17-ol (5a–8a) by Appel bromination
2.3.1. Method D., General procedure
Compounds 1a–3a or 4a (1.18 g, 3 mmol) was suspended in
40 ml of anhydrous CH2Cl2, and 4.97 g (15 mmol) of CBr4 was
added. After cooling the suspension to 0 ◦C, 3.94 g (15 mmol)
of Ph3P in 20 ml anhydrous CH2Cl2 was added dropwise and
the reaction mixture was allowed to become warm and stirred
until it became a yellow solution. After evaporation in vacuo
the crude product was purified on a silica gel column with
ethyl acetate/CHCl3 2.5:97.5.
5a (1.13 g, 83%). Mp 109–110 ◦C; Rf = 0.60 (ss A); [˛]D20 + 66 (c
1.00 in CHCl3). (Found: C, 68.35; H, 6.92. C26H31O2Br requires:
C, 68.57; H, 6.86%). 1H NMR (CDCl3) ı, ppm: 0.79 (s, 3H, 18-
H3), 2.84 (m, 2H, 6-H2), 3.33 (t, 1H, J = 9.7 Hz) and 3.72 (dd, 1H,
J = 9.7 Hz, J = 6.4 Hz): 16a-H2, 3.82 (d, 1H, J = 9.8 Hz, 17-H), 5.01
4b (850 mg, 59%). Mp 80–82 ◦C ([14], Mp 79–82 ◦C); Rf = 0.35
(ss A); [˛]2D0 + 51 (c 1.00 in CHCl3). (Found: C, 75.82; H, 7.51.
C30H
36O5 requires: C, 75.60; H, 7.61%). 1H NMR (CDCl3) ı, ppm:
0.85 (s, 3H, 18-H3), 2.06 and 2.07 (2 × s, 2 × 3H, 2 × OAc-CH3),
2.85 (m, 2H, 6-H2), 4.17 (dd, 1H, J = 10.9 Hz, J = 7.1 Hz) and 4.22
(dd, 1H, J = 10.9 Hz, J = 7.4 Hz): 16a-H2, 4.71 (d, 1H, J = 1.7 Hz, 17-
H), 5.03 (s, 2H, 3-benzyl-H2), 6.71 (d, 1H, J = 2.2 Hz, 4-H), 6.77
(dd, 1H, J = 8.5 Hz, J = 2.2 Hz, 2-H), 7.19 (d, 1H, J = 8.5 Hz, 1-H),
7.30 (t, 1H, J = 7.3 Hz, 4ꢀ-H), 7.37 (t, 2H, J = 7.3 Hz, 3ꢀ,5ꢀ-H), 7.42 (d,
2H, J = 7.3 Hz, 2ꢀ,6ꢀ-H); 13C-NMR (CDCl3) ı, ppm: 17.1 (C-18), 20.9
and 21.1 (2C, 2 × OAc-CH3), 25.9, 27.9, 29.3, 29.6, 32.2, 38.5, 43.4,
44.6 (C-13), 45.8, 50.0, 66.6 (C-16a), 70.0 (3-benzyl-CH2), 83.6 (C-

146
steroids 7 1 ( 2 0 0 6 ) 141–153
celite. After evaporation of the filtrate, the crude product was
purified by column chromatography with ethyl acetate/CHCl3
10:90).
(s, 2H, benzyl-H2), 6.71 (d, 1H, J = 2.3 Hz, 4-H), 6.77 (dd, 1H,
J = 8.6 Hz, J = 2.3 Hz, 2-H), 7.18 (d, 1H, J = 8.6 Hz, 1-H), 7.30 (t,
1H, J = 7.3 Hz, 4ꢀ-H), 7.36 (t, 2H, J = 7.3 Hz, 3ꢀ,5ꢀ-H), 7.41 (d, 2H,
J = 7.3 Hz, 2ꢀ,6ꢀ-H). 13C NMR (CDCl3) ı, ppm: 12.3 (C-18), 26.2,
27.4, 29.7, 32.6, 36.4, 37.6, 38.1, 43.3, 43.9, 44.5 (C-13), 48.2, 70.0
(benzyl-CH2), 81.9 (C-17), 112.4 (C-2), 114.9 (C-4), 126.2 (C-1);
127.4 (2C, C-2ꢀ,6ꢀ), 127.8 (C-4ꢀ), 128.5 (2C, C-3ꢀ,5ꢀ), 132.8 (C-10),
137.4 (C-1ꢀ), 137.9 (C-5), 156.8 (C-3); EI-MS (70 eV) m/z (%): 456
(42) [M+ + 2], 454 (41) [M+], 91 (100).
5e (542 mg, 96%). Mp 168–169 ◦C; Rf = 0.40 (ss B); [˛]D20 + 114
(c 1.00 in ethyl acetate). (Found: C, 62.55; H, 7.02. C19H25O2Br
requires: C 62.47; H 6.90%). 1H NMR (DMSO-d6) ␦, ppm: 0.68
(s, 3H, 18-H3), 2.70 (m, 2H, 6-H2), 3.28 (t, 1H, J = 10.9 Hz, 17-H),
3.63 (m, 1H) and 3.76 (dd, 1H, J = 9.6 Hz, J = 4.2 Hz): 16a-H2, 4.84
(d, 1H, J = 5.5 Hz, 17-OH), 6.43 (s, 1H, 4-H), 6.50 (d, 1H, J = 7.9 Hz,
2-H), 7.02 (d, 1H, J = 8.5 Hz, 1-H), 8.96 (s, 1H, 3-OH). 13C NMR
(DMSO-d6) ␦, ppm: 12.4 (C-18), 25.9, 27.0, 29.1, 32.4, 37.1, 37.9,
39.3, 43.1, 43.4, 43.8, 47.4, 80.6 (C-17), 112.7 (C-2), 114.9 (C-4),
125.9 (C-1); 130.3 (C-10), 137.0 (C-5), 154.8 (C-3); EI-MS (70 eV)
m/z (%): 366 (34) [M+ + 2], 364 (36) [M+], 267 (23), 213 (12), 185 (9),
159 (17), 145 (10), 133 (24), 83 (11), 43 (100).
6a (1.16 g, 85%). Mp 119–120 ◦C; Rf = 0.80 (ss A); [˛]D20 + 89 (c
1.00 in CHCl3). (Found: C, 68.67; H, 6.79. C26H31O2Br requires:
C, 68.57; H, 6.86%). 1H NMR (CDCl3) ı, ppm: 0.78 (s, 3H, 18-H3),
2.83 (m, 2H, 6-H2), 3.48 (dd, 1H, J = 9.4 Hz, J = 6.5 Hz) and 3.61 (t,
1H, J = 9.4 Hz): 16a-H2, 3.85 (t, 1H, J = 4.4 Hz, 17-H), 5.02 (s, 2H,
benzyl-H2), 6.70 (d, 1H, J = 2.5 Hz, 4-H), 6.77 (dd, 1H, J = 8.5 Hz,
J = 2.5 Hz, 2-H), 7.19 (d, 1H, J = 8.5 Hz, 1-H), 7.30 (t, 1H, J = 7.3 Hz,
4ꢀ-H), 7.36 (t, 2H, J = 7.3 Hz, 3ꢀ,5ꢀ-H), 7.41 (d, 2H, J = 7.3 Hz, 2ꢀ,6ꢀ-
H). 13C NMR (CDCl3) ı, ppm: 17.2 (C-18), 26.0, 27.9, 29.8, 31.1,
31.2, 34.9 (C-16a), 39.0, 43.5, 44.0, 46.3 (C-13), 47.3, 70.0 (benzyl-
CH2), 79.9 (C-17), 112.3 (C-2), 114.9 (C-4), 126.3 (C-1); 127.4 (2C,
C-2ꢀ,6ꢀ), 127.8 (C-4ꢀ), 128.5 (2C, C-3ꢀ,5ꢀ), 132.9 (C-10), 137.4 (C-1ꢀ),
137.9 (C-5), 156.8 (C-3); EI-MS (70 eV) m/z (%): 456 (23) [M+ + 2],
454 (23) [M+], 91 (100).
6e (550 mg, 98%). Mp 181–183 ◦C; Rf = 0.50 (ss B); [˛]D20 + 70
(c 1.00 in ethyl acetate). (Found: C, 62.55; H, 7.02. C19H25O2Br
requires: C 62.47; H 6.90%). 1H NMR (DMSO-d6) ı, ppm: 0.68 (s,
3H, 18-H3), 2.70 (m, 2H, 6-H2), 3.41 (t, 1H, J = 8.6 Hz) and 3.63
(t, 1H, J = 8.6 Hz): 16a-H2, 3.60 (d, 1H, J = 5.0 Hz, 17-H), 6.43 (d,
1H, J = 2.0 Hz, 4-H), 6.50 (dd, 1H, J = 8.4 Hz, J = 2.0 Hz, 2-H), 7.04
(d, 1H, J = 8.4 Hz, 1-H), 8.96 (s, 1H, 3-OH). 13C NMR (DMSO-d6)
ı, ppm: 17.0 (C-18), 25.8, 27.6, 29.2, 30.6, 31.2, 36.7, 38.8, 43.2,
43.6, 46.1, 46.3, 78.5 (C-17), 112.7 (C-2), 114.8 (C-4), 125.9 (C-1);
130.4 (C-10), 137.1 (C-5), 154.8 (C-3); EI-MS (70 eV) m/z (%): 366
(99) [M+ + 2], 364 (100) [M+], 267 (15), 213 (21), 198 (15), 185 (11),
172 (22), 159 (35), 146 (20), 133 (37), 107 (13).
7e (547 mg, 97%). Mp 188–189 ◦C; Rf = 0.30 (ss B); [˛]D20 + 37
(c 1.00 in ethyl acetate). (Found: C, 62.36; H, 6.84. C19H25O2Br
requires: C 62.47; H 6.90%). 1H NMR (DMSO-d6) ı, ppm: 0.71 (s,
3H, 18-H3), 2.70 (m, 2H, 6-H2), 3.24 (m, 1H, 17-H), 3.55 (t, 1H,
J = 9.6 Hz) and 3.73 (dd, 1H, J = 9.6 Hz, J = 3.4 Hz): 16a-H2, 4.87 (d,
1H, J = 4.8 Hz, 17-OH), 6.43 (s, 1H, 4-H), 6.50 (d, 1H, J = 8.4 Hz,
2-H), 7.01 (d, 1H, J = 8.4 Hz, 1-H), 9.00 (s, 1H, 3-OH). 13C NMR
(DMSO-d6) ı, ppm: 12.0 (C-18), 25.9, 26.8, 28.9, 29.1, 36.5, 38.3,
40.6, 43.4, 44.0, 45.6, 47.7, 83.4 (C-17), 112.7 (C-2), 114.9 (C-4),
125.9 (C-1); 130.3 (C-10), 137.1 (C-5), 154.8 (C-3); EI-MS (70 eV)
m/z (%): 366 (87) [M+ + 2], 364 (89) [M+], 267 (31), 213 (34), 198
(43), 185 (32), 172 (72), 159 (89), 145 (44), 133 (100), 107 (27), 43
(19).
8e (540 mg, 96%). Mp 186–188 ◦C; Rf 0.30 (ss B); [˛]D20 + 61
(c 1.00 in ethyl acetate). (Found: C, 62.55; H, 7.08. C19H25O2Br
requires: C 62.47, H 6.90%). 1H NMR (DMSO-d6) ı, ppm: 0.67 (s,
3H, 18-H3), 2.69 (m, 2H, 6-H2), 3.38 (d, 1H, J = 4.2 Hz, 17-H), 3.50
(t, 1H, J = 9.2 Hz) and 3.66 (t, 1H, J = 9.2 Hz): 16a-H2, 4.67 (d, 1H,
J = 4.6 Hz, 17-OH), 6.43 (s, 1H, 4-H), 6.50 (d, 1H, J = 8.4 Hz, 2-H),
7.03 (d, 1H, J = 8.4 Hz, 1-H), 8.95 (s, 1H, 3-OH). 13C NMR (DMSO-
d6) ı, ppm: 17.8 (C-18), 25.8, 27.7, 29.2, 31.6, 32.0, 38.4, 38.9, 43.0,
44.6, 48.7, 51.7, 82.6 (C-17), 112.7 (C-2), 114.9 (C-4), 125.9 (C-1);
130.4 (C-10), 137.0 (C-5), 154.8 (C-3); EI-MS (70 eV) m/z (%): 366
(100) [M+ + 2], 364 (98) [M+], 267 (23), 213 (17), 198 (11), 172 (18),
159 (21), 146 (12), 133 (23).
7a (1.08 g, 79%). Mp 90–91 ◦C; Rf = 0.30 (ss A); [˛]2D0 + 50 (c 1.00
in CHCl3). (Found: C, 68.63; H, 6.95. C26H31O2Br requires: C,
68.57; H, 6.86%). 1H NMR (CDCl3) ı, ppm: 0.83 (s, 3H, 18-H3), 2.84
(m, 2H, 6-H2), 3.47 (d, 1H, J = 7.6 Hz, 17-H), 3.54–3.61 (overlap-
ping multiplets, 2H, 16a-H2, 5.02 (s, 2H, benzyl-H2), 6.71 (d, 1H,
J = 2.3 Hz, 4-H), 6.77 (dd, 1H, J = 8.6 Hz, J = 2.3 Hz, 2-H), 7.18 (d, 1H,
J = 8.6 Hz, 1-H), 7.30 (t, 1H, J = 7.3 Hz, 4ꢀ-H), 7.36 (t, 2H, J = 7.3 Hz,
3ꢀ,5ꢀ-H), 7.41 (d, 2H, J = 7.3 Hz, 2ꢀ,6ꢀ-H). 13C NMR (CDCl3) ı, ppm:
12.0 (C-18), 26.1, 27.2, 29.7, 29.8, 36.6, 38.3, 38.9 (C-16a), 43.9,
44.6 (C-13), 45.8, 48.3, 69.9 (benzyl-CH2), 85.8 (C-17), 112.3 (C-
2), 114.8 (C-4), 126.2 (C-1); 127.4 (2C, C-2ꢀ,6ꢀ), 127.8 (C-4ꢀ), 128.5
(2C, C-3ꢀ,5ꢀ), 132.7 (C-10), 137.3 (C-1ꢀ), 137.9 (C-5), 156.7 (C-3);
EI-MS (70 eV) m/z (%): 456 (24) [M+ + 2], 454 (25) [M+], 91 (100).
8a (1.09 g, 80%). Mp 51–53 ◦C; Rf = 0.35 (ss A); [˛]2D0 + 35 (c 1.00
in CHCl3). (Found: C, 68.62; H, 6.94. C26H31O2Br requires: C,
68.57; H, 6.86%. 1H NMR (CDCl3) ı, ppm: 0.76 (s, 3H, 18-H3),
2.84 (m, 2H, 6-H2), 3.51 (m, 2H, 16a-H2),), 3.64 (s, 1H, 17-H),
5.02 (s, 2H, benzyl-H2), 6.71 (d, 1H, J = 2.4 Hz, 4-H), 6.77 (dd,
1H, J = 8.6 Hz, J = 2.4 Hz, 2-H), 7.19 (d, 1H, J = 8.6 Hz, 1-H), 7.30
(t, 1H, J = 7.3 Hz, 4ꢀ-H), 7.36 (t, 2H, J = 7.3 Hz, 3ꢀ,5ꢀ-H), 7.41 (d, 2H,
J = 7.3 Hz, 2ꢀ,6ꢀ-H). 13C NMR (CDCl3) ı, ppm: 17.9 (C-18), 25.9,
27.9, 29.7, 31.9, 32.1, 37.3 (C-16a), 38.6, 43.3, 45.1 (C-13), 49.3,
52.2, 70.0 (benzyl-CH2), 84.1 (C-17), 112.3 (C-2), 114.9 (C-4), 126.2
(C-1); 127.4 (2C, C-2ꢀ,6ꢀ), 127.8 (C-4ꢀ), 128.5 (2C, C-3ꢀ,5ꢀ), 132.8 (C-
10), 137.3 (C-1ꢀ), 137.9 (C-5), 156.8 (C-3); EI-MS (70 eV) m/z (%):
456 (6) [M+ + 2], 454 (6) [M+], 91 (100).
2.4.
Debenzylation of 3-benzyloxy-16-
bromomethylestra-1,3,5(10)-trien-17-ol isomers (5a–8a)
with Pd/C to 16-bromomethylestra-1,3,5(10)-
triene-3,17-diols (5e–8e)
2.5.
Reaction of 3-benzyloxy-16˛-
bromomethylestra-1,3,5(10)-trien-17ˇ-ol (7a)
2.4.1. General procedure
with HBr in acetic acid
Compounds 5a–7a or 8a (700 mg, 1.54 mmol) was dissolved
in 50 ml of ethyl acetate and 250 mg of Pd/C was added to
the solution, which was stirred at room temperature for 6 h
at 30 bar H2 pressure. The reaction mixture was filtered over
2.5.1. Method B
2.5.1.1. 16˛-Bromomethyl-3-hydroxyestra-1,3,5(10)-trien-17ˇ-
yl acetate (7c). Compound 7c (950 mg, 78%). Mp 181–182 ◦C,

steroids 7 1 ( 2 0 0 6 ) 141–153
147
[˛]2D0 − 3 (c 1.00 in CHCl3); Rf = 0.60 (ss C). (Found: C, 62.05; H,
6.50. C21H27O3Br requires: C, 61.92; H, 6.68%). 1H-NMR (CDCl3)
ı, ppm: 0.85 (s, 3H, 18-H3), 2.09 (s, 3H, OAc-CH3), 2.81 (m,
2H, 6-H2), 3.43 (dd, 1H, J = 10.0 Hz, J = 7.6 Hz) and 3.57 (dd, 1H,
J = 10.0 Hz, J = 5.0 Hz): 16a-H2, 4.71 (d, 1H, J = 7.7 Hz, 17-H), 6.56
(d, 1H, J = 2.4 Hz, 4-H), 6.63 (dd, 1H, J = 8.4 Hz, J = 2.5 Hz, 2-H),
7.11 (d, 1H, J = 8.4 Hz, 1-H). 13C-NMR (CDCl3) ı, ppm: 12.9 (C-18),
21.1 (OAc-CH3), 26.0, 27.0, 29.5, 29.8, 36.8, 37.6, 38.3, 43.6 (2C),
44.8 (C-13), 48.4, 85.3 (C-17), 112.8 (C-2), 115.3 (C-4), 126.4 (C-1);
132.2 (C-10), 138.0 (C-5), 153.5 (C-3), 171.4 (OAc-CO); EI-MS
(7 eV) m/z (%): 408 (100) [M+ + 2], 406 (99) [M+], 267 (9), 172 (16),
159 (18), 146 (11), 133 (16), 107 (4), 43 (11).
32.2 (2C), 36.6, 38.1, 43.4, 44.7 (C-13), 49.5, 50.1, 85.1 (C-17),
118.6 (C-2), 121.5 (C-4), 126.3 (C-1), 137.6 (C-10), 138.0 (C-5),
148.4 (C-3), 169.8 (3-OAc-CO), 170.4 (17-OAc-CO); EI-MS (7 eV)
m/z (%): 450 (16) [M+ + 2], 448 (16) [M+], 408 (17), 406 (19), 160
(6), 85 (15), 83 (26), 57 (10), 43 (100).
2.6.
3-Benzyloxy-16-bromomethylestra-
1,3,5(10)-trien-17-yl acetate (7b and 8b)
2.6.1. General procedure
Compound 7a or 8a (455 mg, 1 mmol) was dissolved in 2 ml
of pyridine and 2 ml of acetic anhydride. The solution was
allowed to stand at room temperature for 6 h. The mixture
was then diluted with water. The precipitate was collected
by filtration and dissolved in CH2Cl2, and the solution was
washed with water and dried over Na2SO4. After evaporation
in vacuo, the product was chromatographed on silica gel with
ethyl acetate/CHCl3 (2.5:97.5).
2.5.2. Method C
2.5.2.1. 16˛-Bromomethylestra-1,3,5(10)-triene-3,17ˇ-diyl
diacetate (7d). Compound 7d (1.01 g, 75%) as a colorless oil.
[˛]2D0 − 1 (c 1.00 in CHCl3); Rf = 0.50 (ss A). (Found: C, 61.32; H,
6.68. C23H29O4Br requires: C 61.47, H 6.50%). 1H NMR (CDCl3)
ı, ppm: 0.85 (s, 3H, 18-H3), 2.09 (s, 3H, 17-OAc-CH3), 2.27 (s, 3H,
3-OAc-CH3), 2.86 (dd, 2H, J = 7.6 Hz, J = 3.2 Hz, 6-H2), 3.43 (dd,
1H, J = 10.0 Hz, J = 7.7 Hz) and 3.58 (dd, 1H, J = 10.0 Hz, J = 4.8 Hz):
16a-H2, 4.70 (d, 1H, J = 7.7 Hz, 17-H), 6.78 (d, 1H, J = 1.8 Hz, 4-H),
6.83 (dd, 1H, J = 8.4 Hz, J = 2.2 Hz, 2-H), 7.25 (d, 1H, J = 8.4 Hz,
1-H). 13C NMR (CDCl3) ı, ppm: 12.8 (C-18), 21.1 (2C, OAc-CH3),
25.8, 26.8, 29.3, 29.7, 36.7, 37.7, 37.9, 43.6, 43.8, 44.7 (C-13), 48.4,
84.9 (C-17), 118.6 (C-2), 121.4 (C-4), 126.3 (C-1), 137.6 (C-10),
138.0 (C-5), 148.4 (C-3), 169.7 (3-OAc-CO), 171.0 (17-OAc-CO);
EI-MS (7 eV) m/z (%): 450 (8) [M+ + 2], 448 (8) [M+], 408 (100), 406
(98), 267 (9), 172 (11), 159 (12), 133 (10), 83 (30), 57.1 (39), 43
(27).
7b (483 mg, 97%). Mp 120–121 ◦C; Rf = 0.65 (ss A); [˛]D20 + 12 (c
1.00 in CHCl3). (Found: C, 67.51; H, 6.75. C28H33O3Br requires:
C, 67.60; H, 6.69%). 1H NMR (CDCl3) ı, ppm: 0.85 (s, 3H, 18-H3),
2.08 (s, 3H, OAc-CH3), 2.83 (m, 2H, 6-H2), 3.42 (dd, 1H, J = 9.8 Hz,
J = 7.7 Hz) and 3.57 (dd, 1H, J = 9.8 Hz, J = 5.1 Hz): 16a-H2, 4.70 (d,
1H, J = 7.7 Hz, 17-H), 5.01 (s, 2H, Bzl-H2), 6.70 (d, 1H, J = 2.2 Hz, 4-
H), 6.76 (dd, 1H, J = 8.5 Hz, J = 2.5 Hz, 2-H), 7.17 (d, 1H, J = 8.6 Hz,
1-H), 7.31 (t, 1H, J = 7.3 Hz, 4ꢀ-H), 7.36 (t, 2H, J = 7.3 Hz, 3ꢀ,5ꢀ-H),
7.41 (d, 2H, J = 7.3 Hz, 2ꢀ,6ꢀ-H). 13C NMR (CDCl3) ı, ppm: 12.8 (C-
18), 21.1 (Ac-CH3), 26.0, 27.1, 29.6, 29.8, 36.8, 37.7, 38.3, 43.6
(2C), 44.7 (C-13), 48.4, 69.9 (C-Bzl-CH2), 85.0 (C-17), 112.3 (C-2),
114.8 (C-4), 126.2 (C-1); 127.4 (2C, C-2ꢀ,6ꢀ), 127.8 (C-4ꢀ), 128.5 (2C,
C-3ꢀ,5ꢀ), 132.5 (C-10), 137.3 (C-1ꢀ), 137.8 (C-5), 156.7 (C-3), 171.0
(OAc-CO); EI-MS (7 eV) m/z (%): 498 (25) [M+ + 2], 496 (25) [M+],
91 (100), 43 (11).
2.5.3. Reaction of 3-benzyloxy-16ˇ-bromomethylestra-
1,3,5(10)-trien-17˛-ol (8a) with HBr in acetic acid
2.5.3.1. Method B.
8b (478 mg, 96%) as a colorless oil; Rf = 0.70 (ss A); [˛]20 + 37 (c
D
2.5.3.1.1. 16ˇ-Bromomethyl-3-hydroxyestra-1,3,5(10)-
trien-17˛-yl acetate (8c). Compound 8c (940 mg, 77%). Mp
210–212 ◦C; Rf = 0.60 (ss C); [˛]D20 + 38 (c 1.00 in CHCl3). (Found:
C, 61.25; H, 6.81. C21H27O3Br requires: C 61.92, H 6.68%). 1H
NMR (CDCl3) ı, ppm: 0.86 (s, 3H, 18-H3), 2.09 (s, 3H, OAc-CH3),
2.83 (m, 2H, 6-H2), 3.43 (t, 1H, J = 9.7 Hz) and 3.74 (dd, 1H,
J = 9.7 Hz, J = 6.6 Hz): 16a-H2, 4.63 (d, 1H, J = 2.0 Hz, 17-H), 4.75
(s, 1H, 3-OH), 6.57 (d, 1H, J = 2.5 Hz, 4-H), 6.63 (dd, 1H, J = 8.4 Hz,
J = 2.5 Hz, 2-H), 7.14 (d, 1H, J = 8.4 Hz, 1-H). 13C-NMR (CDCl3)
ı, ppm: 17.5 (C-18), 21.1 (OAc-CH3), 25.9, 27.8, 29.6, 32.2 (2C),
36.7, 38.5, 43.3, 44.8 (C-13), 49.5, 50.1, 85.2 (C-17), 112.7 (C-2),
115.2 (C-4), 126.5 (C-1); 132.4 (C-10), 138.1 (C-5), 153.5 (C-3),
170.6 (OAc-CO); EI-MS (7 eV) m/z (%): 408 (96) [M+ + 2], 406 (100)
[M+], 267 (17), 172 (21), 160 (78), 146 (39), 133 (50), 43 (100).
1.00 in CHCl3). (Found: C, 67.48; H, 6.75. C28H33O3Br requires: C,
67.60; H, 6.69%). 1H NMR (CDCl3) ı, ppm: 0.86 (s, 3H, 18-H3), 2.08
(s, 3H, OAc-CH3), 2.85 (m, 2H, 6-H2), 3.42 (t, 1H, J = 9.7 Hz) and
3.74 (dd, 1H, J = 9.7 Hz, J = 6.5 Hz): 16a-H2, 4.62 (d, 1H, J = 2.1 Hz,
17-H), 5.02 (s, 2H, Bzl-H2), 6.71 (d, 1H, J = 2.5 Hz, 4-H), 6.77 (dd,
1H, J = 8.6 Hz, J = 2.5 Hz, 2-H), 7.19 (d, 1H, J = 8.6 Hz, 1-H), 7.31 (t,
1H, J = 7.3 Hz, 4ꢀ-H), 7.37 (t, 2H, J = 7.3 Hz, 3ꢀ,5ꢀ-H), 7.42 (d, 2H,
J = 7.3 Hz, 2ꢀ,6ꢀ-H). 13C NMR (CDCl3) ı, ppm: 17.44 (C-18), 21.1
(OAc-CH3), 25.8, 27.8, 29.7, 32.2 (2C), 36.7, 38.5, 43.3, 44.8 (C-13),
49.5, 50.1, 69.9 (Bzl-CH2), 85.1 (C-17), 112.3 (C-2), 114.8 (C-4),
126.3 (C-1); 127.4 (2C, C-2ꢀ,6ꢀ), 127.8 (C-4ꢀ), 128.5 (2C, C-3ꢀ,5ꢀ),
132.6 (C-10), 137.3 (C-1ꢀ), 137.8 (C-5), 156.8 (C-3), 170.5 (OAc-
CO); EI-MS (7 eV) m/z (%): 498 (9) [M+ + 2], 496 (13) [M+], 91 (100),
43 (10).
2.5.3.2. Method C.
2.7.
Receptor-binding assay
2.5.3.2.1. 16ˇ-Bromomethylestra-1,3,5(10)-triene-3,17˛-
diyl diacetate (8d). Compound 8d (1.03 g, 76%) as a colorless
Eight synthesized compounds (5c–8c and 5e–8e) were evalu-
ated for their ability to inhibit competitively the binding of
[3H]estra-1,3,5(10)-triene-3,17␤-diol ([3H]E2; 110 Ci mmol−1) to
ERs, of [3H]ORG2058 (34 Ci mmol−1) to PRs in cytosol prepared
from ovariectomized rat uteri, and of [3H]dihydrotestosterone
([3H]DHT; 110 Ci mmol−1) to ARs in castrated rat prostate
cytosol [15].
oil; Rf = 0.50 (ss A); [˛]20 + 36 (c 1.00 in CHCl3). (Found: C,
D
61.58; H, 6.82.C23H29O4Br requires: C, 61.47; H, 6.50%). 1H
NMR (CDCl3) ı, ppm: 0.86 (s, 3H, 18-H3), 2.08 (s, 3H, 17-OAc-
CH3), 2.27 (s, 3H, 3-OAc-CH3), 2.86 (m, 2H, 6-H2), 3.43 (t, 1H,
J = 9.6 Hz) and 3.74 (dd, 1H, J = 9.6 Hz, J = 6.6 Hz): 16a-H2, 4.62 (d,
1H, J = 1.8 Hz, 17-H), 6.79 (d, 1H, J = 1.8 Hz, 4-H), 6.83 (dd, 1H,
J = 8.4 Hz, J = 1.8 Hz, 2-H), 7.27 (d, 1H, J = 8.4 Hz, 1-H); 13C NMR
(CDCl3) ı, ppm: 17.4 (C-18), 21.1 (2C, OAc-CH3), 25.6, 27.6, 29.4,
Mature female (200–300 g) and male (300–400 g) rats
were gonadectomized under general anesthesia (sodium

148
steroids 7 1 ( 2 0 0 6 ) 141–153
tion counter. The percentage of the bound radioligand in the
presence of competitor was plotted against the concentration
of unlabeled steroid. The molar concentration of the steroid
pentobarbital 1 g/kg i.p.) 48 or 24 h before the tissue prepara-
tion, respectively.
The tritiated ligands were purchased from Perkin-Elmer,
USA, except for [3H]ORG2058, which was obtained from Amer-
sham, UK. All subsequent steps were carried out at 4 ◦C.
The minced uterine tissues were homogenized in 10 volume
(w/v) of buffer A (25 mM Tris–HCl, 1.5 mM EDTA disodium salt,
10 mM ␣-monothioglycerol, 10% glycerol and 10 mM sodium
molybdate, pH 7.4) to measure the abilities of the compounds
to bind to the ERs or PRs [16]. The ventral prostates tissues were
homogenized in 3 volume (v/v) of buffer I (20 mM Tris–HCl,
1 mM EDTA disodium salt, 1 mM dithiothreitol and 10% glyc-
erol, pH 7.5) to measure the abilities of the compounds to
bind to the ARs [17]. The homogenates were centrifuged at
105,000 × g for 60 min and the supernatants (cytosol) were
used for competitive binding. Aliquots of 100 ␮l of cytosol
were incubated with 5 nM [3H]E2, 4 nM [3H]ORG2058 or 5 nM
[3H]DHT in the presence of synthesized molecules in 11 dif-
ferent concentrations (from 10−12 to 10−5 M) for 3 h at 25 ◦C for
ER, for 24 h at 4 ◦C for PRs, and for 4 h at 4 ◦C for ARs. To deter-
mine the total bound radiolabeled ligand, we incubated 100 ␮l
of cytosol with 100 ␮l of isotope and 100 ␮l of assay buffer.
Unbound steroids were separated by incubation for 15 min
at 4 ◦C with dextran-coated charcoal (0.5% Norit-A and 0.05%
dextran T-70 in buffer B (1.5 mM EDTA disodium salt, 10 mM
␣-monothioglycerol and 10 mM Tris–HCl, pH 7.4) for ERs and
PRs, and for 30 min at 4 ◦C with 1.25% Norit-A and 0.625% dex-
tran T-70 in buffer II (10 mM Tris–HCl and 1 mM EDTA disodium
salt, pH 7.5) for ARs. After incubation, the samples were cen-
trifuged at 3000 rpm for 10 min, and the radioactivity of the
supernatant was determined in a Wallac 1409 liquid scintilla-
competitor that reduced the radioligand binding by 50% (IC50
)
and the Ki value (inhibition constant) were calculated with
GraphPad 2.0 software. To compare the ER-binding abilities of
the investigated molecules with the Ki value of the reference
compound, we calculated the relative binding affinity (RBA)
according to the following equation: RBA = (Ki,E2/Ki,x) × 100. All
assays were carried out at least three times.
3.
Results and discussion
3.1.
Synthetic studies
In preliminary experiments, the four possible isomers of
3-benzyloxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol iso-
mers (1a–4a) were synthesized via a multistep pathway [14] in
order to obtain the 16-bromomethyl-3-hydroxyestra-1,3,5(10)-
trien-17-yl acetates (5c–8c) and the parent 16-bromomethyl-
diols (5e–8e) derivatives.
First we studied the behavior of the four starting materi-
als 1a–4a in the presence of HBr and acetic acid. From the
cis diols 1a, and 2a, we obtained different 16-bromomethyl-
17-acetates (5b–d and 6b–d) which, depending on the reaction
time or conditions, differed from each other in the substituent
at position C-3 (Schemes 1 and 2). Reactions for 1 h led to 5b
and 6b, which have benzyl protecting groups at position C-3.
After 12 h, the bromo substitution was at C-16a and deben-
zylation also occured. By this method, the cis diols furnished
Scheme 1

steroids 7 1 ( 2 0 0 6 ) 141–153
149
Scheme 2
uration occurs, since ring cleavage in the ambidentate cations
11 and 14 takes place at the sterically favored site C-16a [19].
Furthermore, the absence of 16-bromomethylestradiol deriva-
tives in the case of the trans diols 3a and 4a can be explained
by the rigidity of the estrane skeleton and the unfavorable
configuration of the substituents, which should take part in
a formation of six-membered acyloxonium cation interme-
diates. The formation of 16b from 4a can be explained by
a Wagner–Meerwein rearrangement: the secondary hydroxy
function at C-17 is protonated and a carbocation 15 is gener-
ated after the elimination of water under the appropriate reac-
tion conditions. This carbocation (15) furnishes 16b through
migration of the angular methyl group and subsequent depro-
tonation. The isolated double bond of the tetraene of type 16b
during a prolonged reaction time with HBr addition produced
very sensitive undefined compounds.
3-deprotected 16-bromomethyl derivatives (5c and 6c), which
were subjected to receptor-binding examinations directly. In
the following experiments, the 3-hydroxy function was trans-
formed into an acetoxy group by adding acetic anhydride
to the reaction mixture, yielding 5d and 6d. This procedure
allowed us to replace the benzyl protecting group with the
acetate group, which is easily removable and convenient. The
16-bromomethylestradiol derivatives were accompanied by
16-acetoxymethylestradiol derivatives (1b–d and 2b–d) as by-
products in low yields.
In contrast, from the trans diols (3a and 4a), 16-
bromomethyl products were not formed (Schemes 3 and 4).
Instead of bromination, acetylation occurred, yielding differ-
ent diacetoxyestradiol derivatives (3b–d and 4b–d), depending
on the reaction conditions. In the case of 3-benzyloxy-16␤-
hydroxymethylestra-1,3,5(10)-trien-17␣-ol (4a), compound
The above experimental findings led us to develop a con-
venient method for the synthesis of trans 16-bromomethyl-
17-acetate derivatives (7 and 8). First we brominated the
trans diols 3a and 4a by the Appel reaction and obtained the
trans 3-benzyloxy-16-bromomethylestra-1,3,5(10)-trien-17-ols
(7a and 8a). These compounds (7a and 8a) permitted direct
synthesis of the trans 16-bromomethyl-3,17-diacetates 7d
and 8d by conversion of the substituent at C-3. The acety-
lation of 7a and 8a with acetic anhydride yielded 17-
acetoxy compounds 7b and 8b in good yields. The transpro-
tected derivatives 7d and 8d were synthesized from 7b and
8b by debenzylation with HBr and the subsequent addi-
tion of acetic anhydride to the reaction mixture in one
step.
16b was formed as
rearrangement.
a by-product via Wagner–Meerwein
The experimental results reveal that the four diols (1a–4a)
take part in stereoselective substitution in the presence of HBr
and acetic acid. The formation of 16-bromomethyl-estradiol
derivatives 5b and 6b can easily be explained in terms of
front-side neighboring group participation [18]. According to
the earlier observations the 16-hydroxymethyl group of 1a
and 2a undergoes acetylation and generates 9 and 12, which
are formed under the appropriate reaction conditions. These
monoacetates (9 and 12) cyclized into an ortho acid–ortho ester
(10 and 13) in the acid medium, which are transformed via the
ambidentate 2ꢀ-methyl-1ꢀ,3ꢀ-dioxenium ions 11 and 14 into 5b
and 6b. During the formation of 5b and 6b, no change in config-

150
steroids 7 1 ( 2 0 0 6 ) 141–153
Scheme 3
After the synthesis of 17-acetoxy-16-bromomethyl-3-
higher ppm values than for the analogous signals of the cis
isomers.
hydroxy compounds (5c–8c) we set out to produce the
appropriate 17-hydroxy derivatives, too. This was reason-
able in view of their potential biological activities, while
17-hydroxyestradiol derivatives presumably have higher
RBAs than their 17-acetoxy counterparts. The cis 16-
bromomethylestradiol derivatives 5a and 6a could also be
obtained by application of the Appel reaction. The 16-
bromomethyl-17-hydroxy compounds 5a–8a were depro-
tected by hydrogenolysis, which yielded the corresponding
estradiols (5e–8e). The synthesized products could be sub-
jected to receptor-binding examinations directly.
The NMR spectra demonstrated certain interesting fea-
tures. In the 1H NMR spectra of the 16-bromomethyl-17-
acetates (5b–8b, d) and 16-bromomethylestradiol deriva-
tives (5e–8e), the double doublet or triplet of the 16a-H2
appears at ı = 3.26–3.76 ppm. The analogous signals of the
16-acetoxymethyl-17-acetates (1b–4b, d) are found at higher
chemical shifts, around 4.0–4.2 ppm). They also occur at higher
ppm values than the similar signals of the cis isomers.
Further, the doublet assigned to 17-H in 16-bromo-
methyl,17-acetates (5b–8b, d) and 16-acetoxymethyl-17-
acetates (1b–4b, d) appears at ı = 4.62–5.10 ppm. For 16-
bromomethylestradiol derivatives (5e–8e), the signal of 17-
H is observed at higher frequencies, ı = 3.47–3.82 ppm, as
compared with the 17-acetates. As opposed to the 16a-H
signals, the doublets of 17-H of the corresponding cis iso-
mers lie at higher ppm values than in the spectra of the
trans isomers. In agreement with the literature [20], the
coupling constants J16,17 display the following sequence:
J16␣H,17␤H < J16␤H,17␤H < J16␤H,17␣H < J16␣H,17␣H. The 13C NMR res-
onance of C-17 is usually observed at ı = 79.9–85.8 ppm and
this signal of the corresponding trans isomer is found at
For the products containing a benzyl protecting group
at position C-3 (1a–8a, b, d), the singlet of the benzyl-CH2
appears in the 1H NMR spectra at ı = 5.02 ppm and in the 13C
NMR spectra at ı = 70.0 ppm. The resonance of C-3 in the 13C
NMR spectra is observed at ı = 148.5–156.8 ppm. The singlet
at ı = 156.8 ppm can be assigned to bearing the C-3 benzyloxy
group, that at ı = 153.5 ppm to the C-3 hydroxy function, and
that at ı = 148.5 ppm to the C-3 acetoxy group. Additionally, the
OAc-CH3 at position C-3 in 1d–8d resonates at ı = 2.27 ppm in
1H NMR spectra and at 5 = 21.0 ppm in the 13C NMR spectra.
Finally, the signal from C O (3-OAc) is found at ı = 169.9 ppm.
3.2.
Radioligand-binding assay
The RBAs of 16-bromomethyl-3-hydroxyestra-1,3,5(10)-trien-
17-yl 17-acetates (5c–8c) and 16-bromomethylestra-1,3,5(10)-
triene-3,17-diols (5e–8e) and their derivatives for the ERs, PRs
and ARs were determined by radioligand-binding assay.
Our reference molecule for the ERs was estra-1,3,5(10)-
triene-3,17␤-diol (E2), the inhibition constant of which under
our experimental conditions (Ki = 2.31 0.76 nM) was in good
agreement with the literature Ki value (2.48 nM) [16].
Specifically bound [3H]E2 was displaced from the ovariec-
tomized rat uterine ERs by 5c and its stereoisomers (6c–8c).
16␣-Bromomethylestra-1,3,5(10)-triene-3,17-diyl 17␤-acetate
(7c) displays the highest binding affinity to the ERs in this
series, though its Ki is two orders of magnitude lower than
that of E2, the endogenous ligand of the ER. The other three
molecules (5c, 6c and 8c) have Ki values one order of magni-
tude higher than that for 7c, and there is no major difference
between the RBAs of the compounds. The Ki values are listed

steroids 7 1 ( 2 0 0 6 ) 141–153
151
Scheme 4
in Table 1, and representative curves from the ER displacement
analysis are depicted in Fig. 1.
The reference molecule for the PR was ORG2058, with
Ki = 7.40 0.98 nM under our experimental circumstances. All
of the 17-acetate derivatives of estradiol (5c–8c) bind weakly
to the PRs, with Ki values in excess of 3000 nM, and thus this
receptor does not recognize them. Three compounds in this
series exhibit a higher affinity to the ARs than to the ERs. The
Table 1 – Inhibition constants (Ki) of estra-1,3,5(10)-triene-3,17␤-diol (E2), ORG2058 and dihydrotestosterone (DHT) as
reference compounds and currently synthesized derivatives for the uterine ERs and PRs and for the prostate ARs
Investigated compounds
Ki (nM) ER
RBA (%)
Ki (nM) PR
Ki (nM) AR
E2
ORG2058
DHT
2.31 0.76
100
–
–
–
–
–
–
–
7.40 0.98
–
9.17 0.71
5c
7c
8c
6c
3855 2350
553.8 198.3
5254 1040
4143 2269
0.06
0.42
0.04
0.09
≥3000
≥3000
≥3000
≥3000
Not measurablea
475.5 7.28
2963 1528
237.2 23.76
5e
7e
8e
6e
461.4 145.6
2.55 0.64
781.1 149.1
684.4 154.0
0.50
90.59
0.29
≥3000
≥3000
525.2 142.7
≥3000
≥3000
≥3000
0.34
≥3000
214.7 10.96
The following radioligands were used: 5 nM [3H]estra-1,3,5(10)-triene-3,17␤-diol for the ERs, 4 nM [3H]ORG2058 for the PRs and 5 nM
[3H]dihydrotestosterone for the ARs. The relative binding affinities (RBAs) on the ERs were calculated according to the following equation:
RBA = (Ki;E2/Ki;x) × 100.
a
Ki > 10,000 nM; the ligand is not able to displace the radioligand from the receptor-binding site.

152
steroids 7 1 ( 2 0 0 6 ) 141–153
16 and C-17 to the ERs, PRs and ARs have been char-
acterized. The molecules belong in two different sub-
stituted stereoisomer series: 16-bromomethylestra-1,3,5(10)-
triene-3,17-diyl 17-acetates (5c–8c) and 16-bromomethylestra-
1,3,5(10)-triene-3,17-diols (5e–8e).
Examination of the binding abilities of these compounds
to the ERs proved that this receptor recognizes them. Consid-
erable differences are observed between the Ki values, which
can be explained by the different modes of receptor binding.
Tanenbaum et al. [21] suggest that estrogens adopt a well-
defined orientation in the ligand-binding domain (LBD) of the
ERs: ring D is in contact with helix-11, the 17␤-hydroxy group
is H-bonded to His 524, ring A is projected toward helix-3, the
3-phenolic hydroxy forms a water-mediated H-bond with Glu
353, and the molecule is fixed by van der Waals contact around
the binding pocket. The biggest divergence in the Ki values is
exhibited between the members of the 17-acetate epimers and
the 3,17-diols, suggesting that a hydroxy group at position 17␤
of the estradiol ring is necessary to form the H-bond with the
His 524 residue. Highly polar and bulky groups, such as acetate,
are poorly tolerated by ER LBD at positions 17␣ and ␤ [22].
Several research groups have investigated the effects of C16
substitution on the affinity of estradiol derivatives for the ERs
[13,23]. They concluded that ER tolerance for substituents at
position 16␣ is mixed, although groups of moderate size and
polarity, such as halogens or haloalkyl, are generally toler-
ated, but position 16␤ is sterically less permissive, even the
smallest functional group (e.g. fluorine) decreasing the RBA
substantially. Our experimental work with the four estradiol
epimer pairs lend support to these findings, because the tested
17␤ derivatives with 16␣ substituents (7c and 7e) possess Ki
values at least one order of magnitude lower than those of
16␤-substituted molecules. The RBA of 7e is 90.95%, and this
epimer has a two-orders of magnitude lower Ki than that of its
cis counterpart 5e. Ring D of estradiol derivatives makes non-
polar contacts with the Ile 424, Gly 521 and Leu 525 residues,
and the Ile 424 residue can be found close to C-16 of the ligand
in the ERLBD [24]. Our results suggest that the position 16␤ is
sterically unfavorable because of the side-chain of the Ile 424
residue, which is close to the ligand.
Fig. 1 – Competitive inhibition of [3H]estra-1,3,5(10)-triene-
3,17␤-diol binding in rat uterine cytosol by 16-bromometh-
yl-3-hydroxyestra-1,3,5(10)-trien-17-yl 17-acetate (A) and
16-bromomethylestra-1,3,5(10)-triene-3,17-diol (B) and
their stereoisomers. The reference molecule is
estra-1,3,5(10)-triene-3,17b-diol (E2).
most marked difference is observed in the case of 6c, which
binds one order of magnitude more strongly to the ARs than
to the ERs. Its Ki value for the ARs is the lowest in this group.
The isomer (5c) could not displace specifically bound [3H]DHT
from the castrated rat prostate ARs.
The 16-bromomethylestrane derivatives of 3,17-estradiol
(5e–8e) recognize the ERs well. Compound 7e exhibits the
highest affinity to this receptor (Ki = 2.55 0.64 nM), and this
compound therefore binds to the ERs as strongly as E2 does.
The Ki values of the other molecules (5e, 6e and 8e) are at least
200-fold higher than that for 7e.
The investigated molecules are able to bind to the ARs and
PRs, because the C-3 hydroxy group can act not only as an H-
donor, but also as an H-acceptor [22], and in the AR and PR
there is a good H-donor Glu residue instead of the Gln 353 in
the ERs [21,25].
Compound 7e displays the highest affinity for the PRs, but
this interaction is 200-fold weaker than that with the ERs.
The remaining three derivatives have Ki values higher than
3000 nM. The members of this series display weak affinity for
the ARs (Ki > 3000 nM), except for the 16␣-bromomethylestra-
1,3,5(10)-triene-3,17␣-diol substituent (6e). This latter exhibits
the strongest RBA to the ARs. It binds 3-fold more strongly to
the ARs than to the ERs, but its interactions with the ARs are
23-fold weaker than that of to the reference compound DHT
(Ki = 9.17 0.71 nM).
In summary, four substituted estradiol epimer pairs were
tested in the radioligand-binding assay. The ER recognizes
them, but only 7e exhibits a really high binding affinity for
the ERs. The selectivities of the investigated compounds are
variable: only three derivatives (5e, 7e and 8e) are considered
ER-selective. The others, (7c, 8c and 6c and 6e) bind slightly
more strongly to the ARs than to the ERs, and 5c does not
show any affinity to the ARs.
Acknowledgments
3.3.
Discussion
This work was supported financially by the Hungarian Sci-
entific Research Fund (OTKA T-042673 and M-041532). The
authors thank Pe´ter Forgo´ (University of Szeged, Hungary) for
In the present study, the binding affinities of eight estra-
diol derivatives containing substituents at positions C-

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