Synthesis and receptor-binding examinations of the normal and 13-epi-D-homoestrones and their 3-methyl ethers

Article abstract of DOI:10.1016/S0039-128X(02)00181-2

An effective epimerization of the normal estrone 3-methyl and 3-benzyl ethers by using o-phenylenediamine and AcOH made the possibility for facile entry into the 13α-estrone series. Combination of this synthetic methodology with an isolation step carried out by means of the Girard-P reagent, the corresponding ethers of 13-epi-estrone were obtained in excellent yields. The 3-hydroxy and 3-methoxy D-homoestrone derivatives in both the normal and the 13α-estrone series were then synthesized and tested in vitro in a radioligand-binding assay. The estrogen receptor recognizes these compounds, but their relative binding affinities (RBAs) are lower than that of the reference compound 3,17β-estradiol. The progesterone receptor-binding affinities of the four D-homo derivatives were also tested showing low values for 13α-D-homoestrone and its 3-methyl ether. Pharmacologically, these 13α-D-homoestrone derivatives are estrogen receptor-selective molecules.

Full text of DOI:10.1016/S0039-128X(02)00181-2

Steroids 68 (2003) 277–288
Synthesis and receptor-binding examinations of the
normal and 13-epi-D-homoestrones and their
3-methyl ethers
János Wölflinga , Erzsébet Mernyáka, Éva Franka , George Falkayb,
Árpád Márkib, Renáta Minoricsb, Gyula Schneidera,∗
Department of Organic Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary
a
b
Institute of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary
Received 2 July 2002; received in revised form 11 September 2002; accepted 17 September 2002
Abstract
An effective epimerization of the normal estrone 3-methyl and 3-benzyl ethers by using o-phenylenediamine and AcOH made the
possibility for facile entry into the 13␣-estrone series. Combination of this synthetic methodology with an isolation step carried out by
means of the Girard-P reagent, the corresponding ethers of 13-epi-estrone were obtained in excellent yields. The 3-hydroxy and 3-methoxy
D-homoestrone derivatives in both the normal and the 13␣-estrone series were then synthesized and tested in vitro in a radioligand-binding
assay. The estrogen receptor recognizes these compounds, but their relative binding affinities (RBAs) are lower than that of the reference
compound 3,17␤-estradiol. The progesterone receptor-binding affinities of the four D-homo derivatives were also tested showing low values
for 13␣-D-homoestrone and its 3-methyl ether. Pharmacologically, these 13␣-D-homoestrone derivatives are estrogen receptor-selective
molecules.
© 2002 Elsevier Science Inc. All rights reserved.
Keywords: D-Homosteroids; 13-epi-Estrone derivatives; Estrogen receptor-binding
1. Introduction
Besides the substitution of different side-chains and func-
tional groups onto the sterane skeleton [4–6], other struc-
tural modifications, such as D-ring expansion [7] or/and
13-epimerization, might alter the receptor site affinity of the
sex hormone analogs appropriately. The total synthesis of
D-homoestrone in the normal series was first reported by
Ananchenko et al. [8]. Complex, multistep methods are also
known for the formation of the six-membered ring D, for
which expansion of the five-membered ring D of estrone
analogs is often used [9–11], although these transformations
exhibit low efficiency.
We have developed an effective route for production of
the D-homo derivative of the natural estrone 19b and its
3-methyl ether 18b, applying the same structural alteration
in the 13␣ series for the synthesis of 19a and 18a. These
four compounds were subjected to receptor-binding exami-
nations in order to answer the following questions: (1) What
is the difference between the receptor-binding processes of
the normal and 13-epi analogs? (2) Is the receptor-binding
process influenced by the potentially flexible conformation
of rings C and D of the 13␣ derivatives, caused by the
13␣-methyl group [12]?
Breast cancer is the most common of all malignant dis-
eases in women worldwide: one woman in eight develops
this disease in her lifetime [1].
It is now well recognized that a proportion of breast
cancers carry cellular receptors, or binding sites, for estro-
gens and other steroid hormones (including progesterone)
in both their cell nuclei and the cytoplasm. Cells containing
these binding sites are known as hormone receptor-positive
cells. Since estrogens have been proved to play a role in
the development of many breast cancers, a logical approach
to the treatment of estrogen-sensitive breast cancer is the
use of anti-estrogens that prevent the estrogens from reach-
ing cancerous cells and from interacting with their specific
receptors, thereby halting the growth of the tumor cells [2].
A number of estrone derivatives have been synthesized
and their relative binding affinities (RBAs) for the estrogen
receptor have been evaluated during the past few years [3].
∗
Corresponding author. Tel.: +36-62-544276; fax: +36-62-544200.
E-mail address: schneider@chem.u-szeged.hu (G. Schneider).
0039-128X/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.
doi:10.1016/S0039-128X(02)00181-2

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J. Wölfling et al. / Steroids 68 (2003) 277–288
2. Experimental
2.1. General
5ꢀ-H) and 7.40 (d, 2H, J = 7.3 Hz, 2ꢀ-H and 6ꢀ-H). 13C
NMR (125 MHz, CDCl3, δ [ppm]): 21.0, 25.1 (C-18), 28.2,
28.4, 30.3, 32.1, 33.4, 41.4, 41.5, 49.4, 50.1 (C-13), 70.0
(3-benzyl-CH2), 112.6 (C-2), 114.7 (C-4), 126.8 (C-4ꢀ),
127.4 (2C, C-2ꢀ and C-6ꢀ), 127.8 (C-1), 128.5 (2C, C-3ꢀ and
C-5ꢀ), 132.2 (C-10), 137.3 (C-1ꢀ), 138.0 (C-5), 156.8 (C-3)
and 121.4 (C-17). EI-MS (70 eV) m/z (%): 360 (100) [M+],
91 (84). Analysis calculated for C25H28O2: C, 83.29; H,
7.83; Found: C, 83.41; H, 7.95%.
Melting point (m.p.) was determined on a Kofler block
and is uncorrected. Specific rotations were measured in
CHCl3 (c 1) at 25 ◦C with a POLAMAT-A (Zeiss-Jena)
polarimeter and are given in units of 10−1 degree cm2 g−1
.
Elemental analysis was carried out in the analytical lab-
oratory of the University of Szeged with a Perkin-Elmer
CHN analyzer model 2400. The reactions were moni-
tored by TLC on Kieselgel-G (Merck Si 254 F) layers
(0.25 mm thick); solvent systems (ss): (A) tert-butyl methyl
ether (MTBE)/light petroleum (50:50 v/v), (B) ethylac-
etate/chloroform (2.5:97.5 v/v), (C) dichloromethane, (D)
dichloromethane/ethylacetate (30:70 v/v). The spots were
detected by spraying with 5% phosphomolybdic acid in
50% aqueous phosphoric acid. The Rf values were deter-
mined for the spots observed by illumination at 254 and
365 nm. Flash chromatography: silica gel 60, 40–63 ␮m.
2.3. 3-Benzyloxy-16-hydroxymethylene-13α-estra-1,3,5
(10)-trien-17-one (6a)
Compound 5 (10 g, 28 mmol) was dissolved in anhydrous
benzene (65 ml) and NaOMe, free from alcohol of crys-
tallization (3 g, 57 mmol), and freshly distilled HCOOEt
(32 ml) were added. The mixture was stirred for 6 h at 50 ◦C
and allowed to stand overnight at room temperature. It
was then diluted with water (500 ml) and carefully poured
onto a mixture of ice (50 g) and concentrated HCl (11 ml).
The white precipitate that separated out was filtered off,
washed thoroughly with water and dried. Recrystalliza-
tion from CHCl3/light petroleum gave 6a (9.7 g, 89%), as
white crystals. Melting point 180–182 ◦C; Rf = 0.26 (ss
1
All solvents were distilled prior to use. H NMR spectra
were obtained in CDCl3 or DMSO-d6 solution at 500 MHz
(Bruker DRX 500), and 13C NMR spectra were recorded
at 125 MHz on the same instrument under the same condi-
tions. Chemical shifts (δ) are reported relative to TMS and
are given in ppm; the coupling constants (J) are given in
Hz. 13C NMR spectra are 1H-decoupled. For determina-
tion of the multiplicities, the J-MOD pulse sequence was
used. Mass spectra were measured on a Varian MAT 311A
spectrometer.
C); [α]2D5 = −34.1 (c 1). H NMR (500 MHz, CDCl3, δ
1
[ppm]): 1.11 (s, 3H, 18-H3), 2.81 (m, 2H, 6-H2), 5.02 (s,
2H, 3-benzyl-H2), 6.68 (d, 1H, J = 2.4 Hz, 4-H), 6.77 (dd,
1H, J = 8.6 Hz, J = 2.4 Hz, 2-H), 7.09 (s, 1H, 16a-H),
7.18 (d, 1H, J = 8.6 Hz, 1-H), 7.30 (t, 1H, J = 7.3 Hz,
4ꢀ-H), 7.37 (t, 2H, J = 7.3 Hz, 3ꢀ-H and 5ꢀ-H) and 7.41
(d, 2H, J = 7.3 Hz, 2ꢀ-H and 6ꢀ-H). 13C NMR (125 MHz,
DMSO-d6, δ [ppm]): 25.0 (C-18), 25.1, 26.6 (2C), 29.7,
31.5, 40.6, 42.1, 46.3, 49.6 (C-13), 68.9 (3-benzyl-CH2),
111.3 (C-16), 112.3 (C-2), 114.2 (C-4), 126.5 (C-4ꢀ), 127.3
(2C, C-2ꢀ and C-6ꢀ), 127.5 (C-1), 128.2 (2C, C-3ꢀ and C-5ꢀ),
131.9 (C-10), 137.2 (C-1ꢀ), 137.6 (C-5), 151.4 (C-16a),
156.0 (C-3) and 207.6 (C-17). EI-MS (70 eV) m/z (%): 388
(74) [M+], 91 (100). Analysis calculated for C26H28O3: C,
80.38; H, 7.26; Found: C, 80.25; H, 7.37%.
2.2. 3-Benzyloxy-13α-estra-1,3,5(10)-trien-17-one (5)
A mixture of a solution of 2 (10 g, 28 mmol) in AcOH
(100 ml) and o-phenylenediamine (4.6 g, 42 mmol) was
stirred for 3 h at 117 ◦C. After cooling, the mixture was
carefully poured onto ice and the precipitate was filtered
off, washed with water and dried. The solid was dissolved
in CHCl3 (100 ml), washed with water (3 × 100 ml), dried
over Na2SO4 and evaporated in vacuo. The crude product
was then dissolved in MeOH (100 ml) and Girard-P reagent
(6 g, 32 mmol) and AcOH (6 ml) were added. The mixture
was heated under reflux for 3 h and then allowed to stand
overnight at room temperature. After neutralization with
dilute Na2CO3, the precipitate was filtered off. The precip-
itate was dissolved in CHCl3 (100 ml) and the solution was
washed with water (3×100 ml) to remove the water-soluble
derivative of the residual normal estrone-3-benzyl ether.
After drying over Na2SO4, the solvent was evaporated in
vacuo to give 5 (9.9 g, 98%) as white crystals. Melting
point 134–136 ◦C; Rf = 0.38 (ss C); [α]2D5 = −31.1 (c
1). 1H NMR (500 MHz, CDCl3, δ [ppm]): 1.05 (s, 3H,
18-H3), 2.81 (m, 2H, 6-H2), 5.01 (s, 2H, 3-benzyl-H2),
6.68 (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), 7.30 (t,
1H, J = 7.3 Hz, 4ꢀ-H), 7.36 (t, 2H, J = 7.3 Hz, 3ꢀ-H and
2.4. 16-Acetoxymethylene-3-benzyloxy-13α-estra-1,3,5
(10)-trien-17-one (6b)
Compound 6a (389 mg, 1.00 mmol) was dissolved in a
mixture of pyridine (1.5 ml) and Ac2O (1.5 ml). The solu-
tion was allowed to stand at room temperature until com-
plete conversion was achieved (TLC), and then poured onto
a mixture of concentrated H2SO4 (5 ml) and ice (20 g). The
precipitate that separated out was filtered off, dissolved in
CHCl3 (10 ml) and washed with water (3 × 10 ml). The
organic layer was dried over Na2SO4 and evaporated in
vacuo. After purification by column chromatography (sil-
ica gel, MTBE/light petroleum = 20:80), compound 6b
(421 mg, 98%) was obtained as white crystals. Melting point
125–128 ◦C; Rf = 0.28 (ss C). 1H NMR (500 MHz, CDCl3,
δ [ppm]): 1.06 (s, 3H, 18-H3), 2.23 (s, 3H, Ac-H3), 2.80

J. Wölfling et al. / Steroids 68 (2003) 277–288
279
(m, 2H, 6-H2), 5.00 (s, 2H, 3-benzyl-H2), 6.67 (d, 1H, J =
2.4 Hz, 4-H), 6.75 (dd, 1H, J = 8.6 Hz, J = 2.4 Hz, 2-H),
7.16 (d, 1H, J = 8.6 Hz, 1-H), 7.30 (t, 1H, J = 7.4 Hz,
4ꢀ-H), 7.36 (t, 2H, J = 7.4 Hz, 3ꢀ-H and 5ꢀ-H), 7.40 (d,
2H, J = 7.4 Hz, 2ꢀ-H and 6ꢀ-H) and 8.18 (s, 1H, 16a-H).
13C NMR (125 MHz, CDCl3, δ [ppm]): 20.6 (Ac-CH3),
25.3 (C-18), 26.1, 28.1, 28.3, 30.2, 31.9, 41.3, 42.7, 46.9,
51.1 (C-13), 70.0 (3-benzyl-CH2), 112.5 (C-2), 114.6 (C-4),
119.6 (C-16), 126.8 (C-4ꢀ), 127.4 (2C, C-2ꢀ and C-6ꢀ), 127.8
(C-1), 128.5 (2C, C-3ꢀ and C-5ꢀ), 132.2 (C-10), 137.3 (C-1ꢀ),
137.9 (C-5), 141.2 (C-16a), 156.8 (C-3), 167.1 (Ac-CO) and
209.2 (C-17). Analysis calculated for C28H30O4: C, 78.11;
H, 7.02; Found: C, 78.05; H, 6.96%.
18-H3), 2.78 (m, 2H, 6-H2), 3.61 (t, 1H, J = 9.2 Hz) and
3.81 (dd, 1H, J = 9.2 Hz, J = 5.5 Hz): 16a-H2, 3.96 (d,
1H, J = 7.8 Hz, 17-H), 5.02 (s, 2H, 3-benzyl-H2), 6.69
(d, 1H, J = 2.4 Hz, 4-H), 6.78 (dd, 1H, J = 8.7 Hz,
J = 2.4 Hz, 2-H), 7.21 (d, 1H, J = 8.7 Hz, 1-H), 7.30 (t,
1H, J = 7.3 Hz, 4ꢀ-H), 7.37 (t, 2H, J = 7.3 Hz, 3ꢀ-H and
5ꢀ-H) and 7.41 (d, 2H, J = 7.3 Hz, 2ꢀ-H and 6ꢀ-H). 13C
NMR (125 MHz, CDCl3, δ [ppm]): 23.4 (C-18), 26.1, 27.8,
28.0, 29.9, 32.8, 41.8, 42.1, 43.3, 44.9, 48.4, 63.6 (C-16a),
68.9 (3-benzyl-CH2), 73.0 (C-17), 112.3 (C-2), 114.2 (C-4),
126.6 (C-4ꢀ), 127.3 (2C, C-2ꢀ and C-6ꢀ), 127.5 (C-1), 128.2
(2C, C-3ꢀ and C-5ꢀ), 132.4 (C-10), 137.3 (C-1ꢀ), 137.8
(C-5) and 156.0 (C-3). Analysis calculated for C26H32O3:
C, 79.56; H, 8.22; Found: C, 79.71; H, 8.15%.
2.5. 3-Benzyloxy-16α-hydroxymethyl-13α-estra-1,3,5
(10)-trien-17β-ol (7a) and 3-benzyloxy-16β-hydroxymethyl-
13α-estra-1,3,5(10)-trien-17α-ol (8a)
2.6. 3-Benzyloxy-16α-acetoxymethyl-13α-estra-1,3,5
(10)-trien-17β-ol (7b) and 3-benzyloxy-16β-acetoxymethyl-
13α-estra-1,3,5(10)-trien-17α-ol (8b)
Compound 6a (10 g, 26 mmol) was suspended in MeOH
(200 ml), and KBH4 (7 g, 130 mmol) was added in small por-
tions at room temperature. The mixture was allowed to stand
for 4 h, then poured onto ice (500 g) and acidified with dilute
HCl to pH 3. The precipitate that separated out was filtered
off, washed until free from acid and dried. In this case, a mix-
ture of 7a and 8a (10 g, 98%) was obtained in a ratio of 6:1.
In order to obtain the two isomers in pure form, the mixture
was selectively acetylated to give 7b and 8b (Section 2.6).
The two acetates were separated by column chromatogra-
phy and alkaline methanolysis of the two monoacetates was
carried out as follows. Compound 7b (5.6 g, 13 mmol) or
8b (1 g, 2 mmol) was dissolved in MeOH (150 ml for 7b,
and 30 ml for 8b), and KOH (1.5 g, 27 mmol, for 7b, and
0.4 g, 7.0 mmol, for 8b) was added. After standing for 24 h
at room temperature, the mixture was neutralized with di-
lute HCl and diluted with water. The precipitate was then
filtered off and recrystallized from CH2Cl2/light petroleum
to give 7a (5.4 g, 53% relative to 6a) or 8a (0.9 g, 8.8%
relative to 6a) as white crystals.
A mixture of 7a and 8a (10 g, 25.5 mmol) was dissolved in
pyridine (35 ml), and a solution of Ac2O (2.4 ml) in pyridine
(50 ml) was added dropwise with cooling in ice. The mixture
was allowed to stand for 5 h at room temperature, and then
poured onto a mixture of concentrated H2SO4 (42 ml) and
ice (300 g). The precipitate that separated out was filtered
off and dissolved in CHCl3 (50 ml), and the CHCl3 solu-
tion was washed with water (3 ×50 ml), dried over Na2SO4
and evaporated in vacuo. The mixture of products was sub-
jected to chromatographic separation (silica gel, MTBE/light
petroleum = 30:70) to give 7b (5.6 g, 51%) as a colorless
oil, 8b (1.0 g, 9%) as white crystals, a residual mixture of 7a
and 8a (2.0 g, 20%) and a mixture of 7c and 8c (2.4 g, 20%).
After alkaline methanolysis of the mixture of diacetates (7c
and 8c), the mixture of diols (7a and 8a) was recycled.
7b: Rf = 0.43 (ss A); [α]25 = +74.9 (c 1). H NMR
1
(500 MHz, CDCl3, δ [ppm]): D1.06 (s, 3H, 18-H3), 2.06 (s,
3H, Ac-H3), 2.78 (m, 2H, 6-H2), 3.49 (d, 1H, J = 7.4 Hz,
17-H), 4.12 (dd, 1H, J = 11.0 Hz, J = 6.1 Hz) and 4.19
(dd, 1H, J = 11.0 Hz, J = 6.1 Hz): 16a-H2, 5.01 (s, 2H,
3-benzyl-H2), 6.67 (d, 1H, J = 2.4 Hz, 4-H), 6.77 (dd, 1H,
J = 8.5 Hz, J = 2.4 Hz, 2-H), 7.14 (d, 1H, J = 8.5 Hz,
1-H), 7.29 (t, 1H, J = 7.2 Hz, 4ꢀ-H), 7.36 (t, 2H, J = 7.2 Hz,
3ꢀ-H and 5ꢀ-H) and 7.40 (d, 2H, J = 7.2 Hz, 2ꢀ-H and 6ꢀ-H).
13C NMR (125 MHz, CDCl3, δ [ppm]): 20.9 (Ac-CH3), 27.9,
28.9, 29.0 (2C), 29.1 (C-18), 30.3, 38.6, 40.9, 43.6 (C-13),
45.0, 50.3, 66.9 (C-16a), 70.0 (3-benzyl-CH2), 84.6 (C-17),
112.8 (C-2), 114.3 (C-4), 127.4 (2C, C-2ꢀ and C-6ꢀ), 127.5
(C-4ꢀ), 127.8 (C-1), 128.5 (2C, C-3ꢀ and C-5ꢀ), 134.0 (C-10),
137.4 (C-1ꢀ), 137.9 (C-5), 156.5 (C-3) and 171.2 (Ac-CO).
EI-MS (70 eV) m/z (%): 434 (100) [M+], 91 (75). Analysis
calculated for C28H34O4: C, 77.39; H, 7.89; Found: C, 77.28;
H, 7.95%.
7a: m.p. 130–135 ◦C; Rf = 0.41 (ss D); [α]2D5 = +107.2
1
(c 1). H NMR (500 MHz, CDCl3, δ [ppm]): 1.10 (s, 3H,
18-H3), 2.78 (m, 2H, 6-H2), 3.57 (d, 1H, J = 8.2 Hz, 17-H),
3.66 (t, 1H, J = 10.1 Hz) and 3.81 (dd, 1H, J = 10.1 Hz,
J = 5.5 Hz): 16a-H2, 5.02 (s, 2H, 3-benzyl-H2), 6.67 (d,
1H, J = 2.3 Hz, 4-H), 6.78 (dd, 1H, J = 8.7 Hz, J =
2.3 Hz, 2-H), 7.14 (d, 1H, J = 8.7 Hz, 1-H), 7.30 (t, 1H,
J = 7.3 Hz, 4ꢀ-H), 7.37 (t, 2H, J = 7.3 Hz, 3ꢀ-H and 5ꢀ-H)
and 7.41 (d, 2H, J = 7.3 Hz, 2ꢀ-H and 6ꢀ-H). 13C NMR
(125 MHz, DMSO-d6, δ [ppm]): 27.9, 28.1, 29.2 (C-18),
29.3, 29.6, 29.7, 38.9, 40.2, 43.0, 48.3, 50.5, 63.7 (C-16a),
68.9 (3-benzyl-CH2), 82.3 (C-17), 112.5 (C-2), 113.9 (C-4),
127.1 (C-4ꢀ), 127.3 (2C, C-2ꢀ and C-6ꢀ), 127.5 (C-1), 128.2
(2C, C-3ꢀ and C-5ꢀ), 133.6 (C-10), 137.3 (C-1ꢀ), 137.5
(C-5) and 155.8 (C-3). Analysis calculated for C26H32O3:
C, 79.56; H, 8.22; Found: C, 79.68; H, 8.18%.
8b: m.p. 65–67 ◦C; Rf = 0.30 (ss A); [α]2D5 = +17.0
1
(c 1). H NMR (500 MHz, CDCl3, δ [ppm]): 0.96 (s, 3H,
8a: m.p. 112–116 ◦C; Rf = 0.41 (ss D); [α]2D5 = +23.8
18-H3), 2.00 (s, 3H, Ac-H3), 2.78 (m, 2H, 6-H2), 3.88 (d,
1H, J = 7.5 Hz, 17-H), 4.13 (m, 2H, 16a-H2), 5.00 (s, 2H,
1
(c 1). H NMR (500 MHz, CDCl3, δ [ppm]): 0.99 (s, 3H,

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J. Wölfling et al. / Steroids 68 (2003) 277–288
3-benzyl-H2), 6.68 (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), 7.29 (t, 1H, J = 7.2 Hz, 4ꢀ-H), 7.35 (t, 2H, J = 7.2 Hz,
3ꢀ-H and 5ꢀ-H) and 7.40 (d, 2H, J = 7.2 Hz, 2ꢀ-H and 6ꢀ-H).
13C NMR (125 MHz, CDCl3, δ [ppm]): 20.9 (Ac-CH3),
22.9 (C-18), 26.5, 28.1, 28.3, 30.5, 33.0, 42.2, 42.4, 43.2,
43.9 (C-13), 48.7, 67.6 (C-16a), 70.0 (3-benzyl-CH2), 76.1
(C-17), 112.6 (C-2), 114.6 (C-4), 126.9 (C-4ꢀ), 127.3 (2C,
C-2ꢀ and C-6ꢀ), 127.7 (C-1), 128.4 (2C, C-3ꢀ and C-5ꢀ), 132.5
(C-10), 137.3 (C-1ꢀ), 138.0 (C-5), 156.5 (C-3) and 171.1
(Ac-CO). EI-MS (70 eV) m/z (%): 434 (100) [M+], 91 (69).
Analysis calculated for C28H34O4: C, 77.39; H, 7.89; Found:
C, 77.45; H, 7.92%.
2H, J = 7.3 Hz, 3ꢀ-H and 5ꢀ-H) and 7.41 (d, 2H, J = 7.3 Hz,
2ꢀ-H and 6ꢀ-H). 13C NMR (125 MHz, CDCl3, δ [ppm]): 11.8
(C-16a), 27.8, 28.8, 29.2 (C-18), 29.3, 30.3, 33.7, 38.5, 41.0,
44.1 (C-13), 47.7, 50.2, 70.0 (3-benzyl-CH2), 86.8 (C-17),
112.8 (C-2), 114.3 (C-4), 127.4 (2C, C-2ꢀ and C-6ꢀ), 127.5
(C-4ꢀ), 127.8 (C-1), 128.5 (2C, C-3ꢀ and C-5ꢀ), 133.9 (C-10),
137.9 (2C, C-1ꢀ and C-5) and 156.5 (C-3). EI-MS (70 eV)
m/z (%): 502 (78) [M+], 377 (10) and 91 (100). Analy-
sis calculated for C26H31IO2: C, 62.15; H, 6.22; Found: C,
62.32; H, 6.18%.
8d: m.p. 113–116 ◦C; Rf = 0.25 (ss C). 1H NMR
(500 MHz, CDCl3, δ [ppm]): 0.98 (s, 3H, 18-H3), 2.81 (m,
2H, 6-H2), 3.29 (dd, 1H, J = 9.4 Hz, J = 6.9 Hz) and
3.40 (dd, 1H, J = 9.4 Hz, J = 5.6 Hz): 16a-H2, 3.75 (d,
1H, J = 8.5 Hz, 17-H), 5.02 (s, 2H, 3-benzyl-H2), 6.70
(d, 1H, J = 2.3 Hz, 4-H), 6.79 (dd, 1H, J = 8.5 Hz, J =
2.3 Hz, 2-H), 7.21 (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-H and 5ꢀ-H)
and 7.41 (d, 2H, J = 7.4 Hz, 2ꢀ-H and 6ꢀ-H). 13C NMR
(125 MHz, CDCl3, δ [ppm]): 14.0 (C-16a), 23.4 (C-18),
26.6, 28.3, 30.5, 32.8, 33.4, 42.2, 43.4, 44.6 (C-13), 45.4,
48.3, 70.0 (3-benzyl-CH2), 79.1 (C-17), 112.7 (C-2), 114.7
(C-4), 127.0 (C-4ꢀ), 127.4 (2C, C-2ꢀ and C-6ꢀ), 127.8 (C-1),
128.5 (2C, C-3ꢀ and C-5ꢀ), 132.5 (C-10), 137.4 (C-1ꢀ), 138.3
(C-5) and 156.8 (C-3). EI-MS (70 eV) m/z (%): 502 (86)
[M+] and 91 (100). Analysis calculated for C26H31IO2: C,
62.15; H, 6.22; Found: C, 62.38; H, 6.10%.
2.7. 3-Benzyloxy-16α-iodomethyl-13α-estra-1,3,5
(10)-trien-17β-ol (7d) and 3-benzyloxy-16β-iodomethyl-
13α-estra-1,3,5(10)-trien-17α-ol (8d)
2.7.1. Method A
In sequence, Ph3P (6.5 g, 25 mmol), imidazole (1.7 g,
25 mmol), I2 (3.2 g, 25 mmol) and a mixture of 7a and
8a in a ratio of 6:1 (5 g, 13 mmol) were added to CH2Cl2
(150 ml). The mixture was stirred for 24 h at room temper-
ature and then poured into water (150 ml). After separation,
the organic layer was washed with water (2 × 150 ml) to
remove the Ph3PO, dried over Na2SO4 and evaporated to
dryness. The residue obtained was subjected to chromato-
graphic separation (silica gel, CH2Cl2) to furnish a mixture
of 7d and 8d (6.2 g, 95%). The mixture of the products
were separated by repeated column chromatography (silica
gel, CH2Cl2/light petroleum 50:50 → 100:0) to give 7d
(5.2 g, 80%) and 8d (0.9 g, 14%).
2.8. 16,17-seco-3-Benzyloxy-13α-estra-1,3,5(10),
16-tetraen-17-al (4a) and 3-benzyloxy-16-
methylene-13α-estra-1,3,5(10)-trien-17β-ol (9)
A 6:1 mixture of compounds 7d and 8d (5 g, 10 mmol)
was dissolved in methanol (150 ml) and heated under re-
flux with KOH (2.8 g, 0.05 mol) for 3 h. The mixture was
then poured into water and neutralized with 10% HCl and
the precipitate was filtered off. After dissolution in CH2Cl2
(100 ml), the organic phase was washed with water (2 ×
100 ml), dried over Na2SO4 and evaporated. Purification by
column chromatography (silica gel, CH2Cl2) and recrys-
tallization from CH2Cl2/light petroleum yielded 4a (3.1 g,
83%) and 9 (0.4 g 10%).
2.7.2. Method B
In sequence, diphenyl-2-pyridylphosphine (6.6 g, 25
mmol), imidazole (1.7 g, 25 mmol), I2 (3.2 g, 25 mmol) and
a mixture of 7a and 8a in a ratio of 6:1 (5 g, 13 mmol) were
added to CH2Cl2 (150 ml). The mixture was stirred for 24 h
at room temperature and then poured into water (150 ml).
After separation, the organic layer was washed with HCl
(2 M, 150 ml) to remove the diphenyl-2-pyridylphosphine
oxide, washed with water (2 × 150 ml), dried over Na2SO4
and evaporated to dryness. The residue was purified by flash
chromatography (silica gel, CH2Cl2) to afford a mixture
of 7d and 8d (6.3 g, 96%). The mixture of products was
separated by repeated column chromatography (silica gel,
CH2Cl2/light petroleum 50:50 → 100:0) to give 7d (5.4 g,
83%) and 8d (0.8 g, 12%). 7d was crystallized from light
petroleum, and 8d was crystallized from MeOH.
4a: m.p. 63–64 ◦C; Rf = 0.67 (ss C); [α]2D5 = +68.9
1
(c 1). H NMR (500 MHz, CDCl3, δ [ppm]): 1.17 (s, 3H,
18-H3), 2.82 (m, 2H, 6-H2), 4.97–5.05 (m, 2H, 16a-H2),
5.00 (s, 2H, 3-benzyl-H2), 5.82 (m, 1H, 16-H), 6.69 (d, 1H,
J = 2.6 Hz, 4-H), 6.75 (dd, 1H, J = 8.6 Hz, J = 2.6 Hz,
2-H), 7.14 (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-H and 5ꢀ-H), 7.39 (d, 2H,
J = 7.3 Hz, 2ꢀ-H and 6ꢀ-H) and 9.71 (s, 1H, formyl-H).
13C NMR (125 MHz, CDCl3, δ [ppm]): 23.1 (C-18), 27.5
(2C), 30.2, 33.4, 36.5, 42.2, 43.4, 49.5, 50.1 (C-13), 69.9
(3-benzyl-CH2), 112.5 (C-2), 114.5 (C-4), 115.2 (C-16a),
126.4 (C-4ꢀ), 127.4 (2C, C-2ꢀ and C-6ꢀ), 127.7 (C-1), 128.5
(2C, C-3ꢀ and C-5ꢀ), 132.2 (C-10), 137.3 (C-1ꢀ), 137.8 (C-5),
139.2 (C-16), 156.8 (C-3) and 206.8 (formyl-C). EI-MS
7d: m.p. 88–89 ◦C; Rf = 0.40 (ss C); [α]2D5 = +61.0 (c 1).
1H NMR (500 MHz, CDCl3, δ [ppm]): 1.08 (s, 3H, 18-H3),
2.77 (m, 2H, 6-H2), 3.24 (dd, 1H, J = 9.5 Hz, J = 7.1 Hz,
16a-H), 3.39 (overlapping multiplets, 2H, 16a-H and 17-H),
5.01 (s, 2H, 3-benzyl-H2), 6.67 (d, 1H, J = 2.5 Hz, 4-H),
6.78 (dd, 1H, J = 8.6 Hz, J = 2.5 Hz, 2-H), 7.14 (d, 1H,
J = 8.6 Hz, 1-H), 7.30 (t, 1H, J = 7.3 Hz, 4ꢀ-H), 7.36 (t,

J. Wölfling et al. / Steroids 68 (2003) 277–288
281
(70 eV) m/z (%): 375 (18) [M+], 374 (62) and 91 (100).
Analysis calculated for C26H30O2: C, 83.38; H, 8.07; Found:
C, 83.47; H, 8.18%.
(C-3). EI-MS (70 eV) m/z (%): 470 (6), 298 (72), 228 (17),
160 (20), 147 (100) and 91 (20). Analysis calculated for
C27H34O5S: C, 68.91; H, 7.28; Found: C, 68.77; H, 7.32%.
9: m.p. 102–105 ◦C; Rf = 0.45 (ss C). 1H NMR
(500 MHz, CDCl3, δ [ppm]): 0.93 (s, 3H, 18-H3), 2.77 (m,
2H, 6-H2), 3.92 (s, 1H, 17-H), 4.99 (s, 2H, 3-benzyl-H2),
5.05 (s, 1H) and 5.17 (s, 1H): 16a-H2, 6.66 (d, 1H, J =
2.5 Hz, 4-H), 6.75 (dd, 1H, J = 8.6 Hz, J = 2.5 Hz, 2-H),
7.18 (d, 1H, J = 8.6 Hz, 1-H), 7.27 (t, 1H, J = 7.4 Hz,
4ꢀ-H), 7.34 (t, 2H, J = 7.4 Hz, 3ꢀ-H and 5ꢀ-H) and 7.39
(d, 2H, J = 7.4 Hz, 2ꢀ-H and 6ꢀ-H). 13C NMR (125 MHz,
CDCl3, δ [ppm]): 28.9, 29.2, 29.6 (C-18), 30.4, 32.3, 34.1,
40.6, 42.4, 43.8 (C-13), 50.6, 70.0 (3-benzyl-CH2), 84.0
(C-17), 109.9 (C-16a), 112.6 (C-2), 114.5 (C-4), 127.2
(C-4ꢀ), 127.4 (2C, C-2ꢀ and C-6ꢀ), 127.7 (C-1), 128.4 (2C,
C-3ꢀ and C-5ꢀ), 133.4 (C-10), 137.5 (C-1ꢀ), 138.2 (C-5),
155.7 (C-3) and 156.6 (C-16). Analysis calculated for
C26H30O2: C, 83.38; H, 8.07; Found: C, 83.47; H, 7.98%.
2.9.2. 17aβ-Hydroxy-3-methoxy-D-homoestra-1,3,5
(10)-trien-16β-yl p-toluenesulfonate (14b)
After evaporation in vacuo, the crude product was pu-
rified by column chromatography (silica gel, MTBE/light
petroleum 30:70 → 100:0) and recrystallized from CH2Cl2/
light petroleum to give 14b (409 mg, 87%) as white crystals.
Melting point Rf = 0.14 (ss A); [α]2D5 = +80.8 (c 1). H
1
NMR (500 MHz, CDCl3, δ [ppm]): 0.78 (s, 3H, 18-H3), 2.46
(s, 3H, Tos-H3), 2.72 (m, 1H, 17a␣-H), 2.79 (m, 2H, 6-H2),
3.77 (s, 3H, 3-OMe), 4.44 (m, 1H, 16␣-H), 6.61 (d, 1H, J =
2.1 Hz, 4-H), 6.71 (dd, 1H, J = 8.5 Hz, J = 2.1 Hz, 2-H),
7.18 (d, 1H, J = 8.5 Hz, 1-H), 7.37 (d, 2H, J = 7.9 Hz,
3ꢀ-H and 5ꢀ-H) and 7.84 (d, 2H, J = 7.9 Hz, 2ꢀ-H and 6ꢀ-H).
13C NMR (125 MHz, CDCl3, δ [ppm]): 11.5 (C-18), 21.6
(Tos-CH3), 26.0, 26.5, 29.9 (2C), 32.7, 37.1, 37.9, 38.1, 43.6,
45.1, 55.2 (3-OMe), 79.5 and 80.2 (C-16 and C-17a), 111.7
(C-2), 113.5 (C-4), 126.2 (C-1), 127.7 (2C, C-2ꢀ and C-6ꢀ),
129.8 (2C, C-3ꢀ and C-5ꢀ), 132.4 (C-10), 134.8 (C-1ꢀ), 137.5
(C-5), 144.6 (C-4ꢀ) and 157.6 (C-3). Analysis calculated for
C27H34O5S: C, 68.91; H, 7.28; Found: C, 68.75; H, 7.25%.
2.9. General procedure for preparation of the 3-methyl
and 3-benzyl ethers of the 17α-hydroxy-16-tosyloxy
derivatives
A total of 298 mg (1.00 mmol) of 13␣- or 13␤-secoes-
trone-3-methyl ether (3a or 3b) or 375 mg (1.00 mmol) of
13␣- or 13␤-secoestrone-3-benzyl ether (4a or 4b) was
dissolved in 10 ml of ice-cold CH2Cl2, and 190 mg of an-
hydrous p-toluenesulfonic acid (1.1 mmol) was added to the
solution in portions. The temperature was allowed to rise
to room temperature and the mixture was stirred overnight.
The reaction was carried out until complete conversion
(TLC) was achieved. The solution was then diluted with
water (10 ml) and neutralized with NaHCO3, the aqueous
phase was extracted with CH2Cl2 (3 × 10 ml) and the com-
bined organic phases were washed with brine and dried
over Na2SO4.
2.9.3. 17aα-Hydroxy-3-benzyloxy-13α-D-homoestra-1,3,5
(10)-trien-16α-yl p-toluenesulfonate (15a)
After evaporation in vacuo, the crude product was pu-
rified by column chromatography (silica gel, MTBE/light
petroleum 30:70 → 100:0) and recrystallized from light
petroleum to give 15a (476 mg, 87%) as white crystals.
Melting point 103–106 ◦C; Rf = 0.17 (ss A); [α]2D5
=
1
+28.1 (c 1). H NMR (500 MHz, CDCl3, δ [ppm]): 0.97
(s, 3H, 18-H3), 2.45 (s, 3H, Tos-H3), 2.72 (m, 2H, 6-H2),
3.86 (m, 1H, 17a␤-H), 4.38 (m, 1H, 16␤-H), 5.03 (s, 2H,
3-benzyl-H2), 6.70 (d, 1H, J = 2.4 Hz, 4-H), 6.78 (dd, 1H,
J = 8.5 Hz, J = 2.4 Hz, 2-H), 7.15 (d, 1H, J = 8.5 Hz,
1-H), 7.35 (overlapping multiplets, 7H, 3ꢀ-H, 5ꢀ-H, 2ꢀꢀ-H,
3ꢀꢀ-H, 4ꢀꢀ-H, 5ꢀꢀ-H and 6ꢀꢀ-H) and 7.77 (d, 2H, J = 8.2 Hz,
2ꢀ-H and 6ꢀ-H). 13C NMR (125 MHz, CDCl3, δ [ppm]): 21.7
(Tos-CH3), 25.9, 26.5, 26.9 (C-18), 28.3, 30.0, 34.7, 37.1,
37.2, 37.8, 43.1, 47.2, 66.7 (C-16), 70.0 (3-benzyl-CH2),
76.7 (C-17a), 112.6 (C-2), 114.5 (C-4), 126.4 (C-1), 127.4
(2C, C-2ꢀꢀ and C-6ꢀꢀ), 127.8 (2C, C-2ꢀ and C-6ꢀ), 127.9
(C-4ꢀꢀ), 128.6 (2C, C-3ꢀꢀ and C-5ꢀꢀ), 129.9 (2C, C-3ꢀ and
C-5ꢀ), 132.5 (C-10), 134.2 (C-1ꢀ), 137.3 (C-5), 137.6
(C-1ꢀꢀ), 144.7 (C-4ꢀ) and 156.9 (C-3). Analysis calculated
for C33H38O5S: C, 72.50; H, 7.01; Found: C, 72.35; H,
7.12%.
2.9.1. 17aα-Hydroxy-3-methoxy-13α-D-homoestra-1,3,5
(10)-trien-16α-yl p-toluenesulfonate (14a)
After evaporation in vacuo, the crude product was pu-
rified by column chromatography (silica gel, MTBE/light
petroleum 30:70
→
100:0) and recrystallized from
CH2Cl2/light petroleum to give 14a (419 mg, 89%) as
white crystals. Melting point 120–124 ◦C; Rf = 0.2 (ss A);
[α]2D5 = +35.7 (c 1). 1H NMR (500 MHz, CDCl3, δ [ppm]):
0.96 (s, 3H, 18-H3), 2.46 (s, 3H, Tos-H3), 3.77 (s, 3H,
3-OMe), 3.86 (dd, 1H, J = 11.0 Hz, J = 4.0 Hz, 17a␤-H),
4.37 (m, 1H, 16␤-H), 6.60 (d, 1H, J = 2.6 Hz, 4-H), 6.69
(dd, 1H, J = 8.5 Hz, J = 2.6 Hz, 2-H), 7.15 (d, 1H, J =
8.5 Hz, 1-H), 7.34 (d, 2H, J = 8.3 Hz, 3ꢀ-H and 5ꢀ-H) and
7.77 (d, 2H, J = 8.3 Hz, 2ꢀ-H and 6ꢀ-H). 13C NMR (125
MHz, CDCl3, δ [ppm]): 21.6 (C-18), 25.9, 26.4, 26.9, 28.2,
29.9, 34.6, 37.1 (2C), 37.2, 43.0, 47.1, 55.2 (3-OMe), 66.5
(C-16), 76.7 (C-17a), 111.7 (C-2), 113.3 (C-4), 126.3 (C-1),
127.7 (2C, C-2ꢀ and C-6ꢀ), 129.8 (2C, C-3ꢀ and C-5ꢀ), 132.2
(C-10), 134.0 (C-1ꢀ), 137.4 (C-5), 144.7 (C-4ꢀ) and 157.5
2.9.4. 17aβ-Hydroxy-3-benzyloxy-D-homoestra-1,3,5
(10)-trien-16β-yl p-toluenesulfonate (15b)
After evaporation in vacuo, the crude product was pu-
rified by column chromatography (silica gel, MTBE/light
petroleum 30:70 → 100:0) and recrystallized from light
petroleum to give 15b (465 mg, 85%) as white crystals.

282
J. Wölfling et al. / Steroids 68 (2003) 277–288
Melting point 68–70 ◦C; Rf = 0.40 (ss A); [α]2D5 = +66.36
2.76 (m, 2H, 6-H2), 5.00 (s, 2H, 3-benzyl-H2), 5.88 (dd, 1H,
J = 7.9 Hz, J = 2.1 Hz, 17-H), 6.66 (d, 1H, J = 2.3 Hz,
4-H), 6.69 (m, 1H, 16-H), 6.75 (dd, 1H, J = 8.6 Hz, J =
2.3 Hz, 2-H), 7.14 (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-H and 5ꢀ-H)
and 7.39 (t, 2H, J = 7.3 Hz, 2ꢀ-H and 6ꢀ-H). 13C NMR
(125 MHz, CDCl3, δ [ppm]): 24.7, 25.6 (C-18), 27.5 (2C),
29.8, 34.4, 38.7, 42.5, 46.8 (C-13), 48.2, 69.9 (3-benzyl-C),
112.4 (C-2), 114.3 (C-4), 126.2 (C-4ꢀ), 127.3 (2C, C-2ꢀ and
C-6ꢀ), 127.8 (C-1), 128.5 (2C, C-3ꢀ and C-5ꢀ), 128.6 (C-17),
133.0 (C-10), 137.3 (C-1ꢀ), 137.7 (C-5), 145.5 (C-16), 156.8
(C-3) and 203.1 (C-17a). Analysis calculated for C26H28O2:
C, 83.83; H, 7.58; Found: C, 83.91; H, 7.44%.
1
(c 1). H NMR (500 MHz, CDCl3, δ [ppm]): 0.89 (s, 3H,
18-H3), 2.45 (s, 3H, Tos-H3), 2.72 (m, 1H, 17a␣-H), 2.77 (m,
1H, 6-H2), 4.45 (m, 1H, 16␣-H), 5.00 (s, 2H, 3-benzyl-H2),
6.69 (d, 1H, J = 2.5 Hz, 4-H), 6.75 (dd, 1H, J = 8.5 Hz,
J = 2.5 Hz, 2-H), 7.15 (d, 1H, J = 8.5 Hz, 1-H), 7.36 (over-
lapping multiplets, 7H, 3ꢀ-H, 5ꢀ-H, 2ꢀꢀ-H, 3ꢀꢀ-H, 4ꢀꢀ-H, 5ꢀꢀ-H
and 6ꢀꢀ-H) and 7.75 (d, 2H, J = 8.0 Hz, 2ꢀ-H and 6ꢀ-H).
13C NMR (125 MHz, CDCl3, δ [ppm]): 12.3 (C-18), 21.7
(Tos-CH3), 26.2, 26.5, 29.8 (2C), 32.5, 36.9, 37.7, 38.0, 42.6,
44.9, 70.1 (3-benzyl-CH2), 78.5 and 80.0 (C-16 and C-17a),
112ꢀ.ꢀ5 (C-2), 114.3 (C-4), 126.3 (C-1), 127.3 (2C, C-2ꢀꢀ and
C-6 ), 127.5 (2C, C-2ꢀ and C-6ꢀ), 127.9 (C-4ꢀꢀ), 128.5 (2C,
C-3ꢀꢀ and C-5ꢀꢀ), 129.7 (2C, C-3ꢀ and C-5ꢀ), 132.5 (C-10),
134.0 (C-1ꢀ), 137.5 (C-5), 137.7 (C-1ꢀꢀ), 144.6 (C-4ꢀ) and
157.2 (C-3). Analysis calculated for C33H38O5S: C, 72.50;
H, 7.01; Found: C, 72.40; H, 7.25%.
2.12. General procedure for the preparation of 13α- and
13β-D-homoestrones and their methyl ethers
A suspension of unsaturated ketone 16a or 16b (296 mg,
1.00 mmol) or 17a or 17b (372 mg, 1.00 mmol) and Pd/C
(0.60 g, 10%) in EtOAc (20 ml) was subjected to 20 bar
of H2 pressure at room temperature for 24 h. The cata-
lyst was then removed by filtration through a short pad of
silica gel. After evaporation of the solvent in vacuo, the
crude product was purified by column chromatography (sil-
ica gel, CH2Cl2/light petroleum 70:30 for 18a and 18b, and
MTBE/light petroleum 30:70 → 100:0 for 19a and 19b) to
give 18a (263 mg, 88%), 18b (275 mg, 92%), 19a (242 mg,
85%) or 19b (233 mg, 82%), respectively, as a white solid.
2.10. 3-Methoxy-13α-D-homoestra-1,3,5
(10),16-tetraen-17a-one (16a)
Jones reagent (8N, 0.5 ml) was added dropwise to a so-
lution of 14a (500 mg, 1.1 mmol) in ice-cold Me2CO (5 ml)
and the solution was stirred for 1 h until complete conversion
(TLC) was achieved. The solution was next poured into wa-
ter and extracted with CH2Cl2 (3×10 ml). The combined or-
ganic layers were washed with water, dried over Na2SO4 and
concentrated in vacuo, and the crude product was purified by
column chromatography (silica gel, CH2Cl2) to afford 16a
(316 mg, 97%) as a white solid. Melting point 86–88 ◦C;
Rf = 0.24 (ss C); [α]25 = +24.0 (c 1). 1H NMR (500 MHz,
CDCl3, δ [ppm]): 1.2D0 (s, 3H, 18-H3), 2.79 (m, 2H, 6-H2),
3.75 (s, 3H, 3-OMe), 5.88 (dd, 1H, J = 9.9 Hz, J = 2.2 Hz,
17-H), 6.58 (d, 1H, J = 2.6 Hz, 4-H), 6.68 (overlapping
multiplets, 2H, 2-H and 16-H) and 7.15 (d, 1H, J = 8.6 Hz,
1-H). 13C NMR (125 MHz, CDCl3, δ [ppm]): 24.7, 25.6
(C-18), 27.4 (2C), 29.8, 34.4, 38.6, 42.4, 46.7 (C-13), 48.0,
55.1 (3-OMe), 111.4 (C-2), 113.2 (C-4), 126.2 (C-1), 128.4
(C-17), 132.6 (C-10), 137.6 (C-5), 145.6 (C-16), 157.4 (C-3)
and 203.2 (C-17a). Analysis calculated for C20H24O2: C,
81.04; H, 8.16; Found: C, 80.95; H, 8.10%.
2.12.1. 3-Methoxy-13α-D-homoestra-1,3,5(10)-trien-
17a-one (18a)
Melting point 87–89 ◦C; Rf = 0.53 (ss B); [α]D25
=
1
+139.7 (c 1). H NMR (500 MHz, CDCl3, δ [ppm]): 1.26
(s, 3H, 18-CH3), 2.77 (m, 2H, 6-H2), 3.74 (s, 3H, 3-OMe),
6.57 (d, 1H, J = 2.4 Hz, 4-H), 6.68 (dd, 1H, J = 8.6 Hz,
J = 2.4 Hz, 2-H), 7.15 (d, 1H, J = 8.5 Hz, 1-H). 13C NMR
(125 MHz, CDCl3, δ [ppm]): 20.8, 21.1, 25.8, 27.8 (C-18),
28.0, 29.9, 35.2, 37.3, 39.1, 42.4, 49.4, 49.7, 55.0 (3-OMe),
111.5 (C-2), 113.1 (C-4), 126.5 (C-1), 132.5 (C-10), 137.4
(C-5), 157.3 (C-3), 214.8 (C-17a). EI-MS (70 eV) m/z (%):
298 (100) [M+], 199 (10), 188 (16), 111 (10). Analysis cal-
culated for C20H26O2: C, 80.50; H, 8.78; Found: C, 80.37;
H, 8.85%.
2.11. 3-Benzyloxy-13α-D-homoestra-1,3,5
(10),16-tetraen-17a-one (17a)
2.12.2. 3-Hydroxy-13α-D-homoestra-1,3,5
(10)-trien-17a-one (19a)
Jones reagent (8N, 0.6 ml) was added dropwise to a so-
lution of 15a (600 mg, 1.1 mmol) in ice-cold Me2CO (6 ml)
and the solution was stirred for 1 h until complete conver-
sion (TLC) was achieved. The solution was next poured into
water and extracted with CH2Cl2 (3×10 ml). The combined
organic layers were washed with water, dried over Na2SO4
and concentrated in vacuo, and the crude product was pu-
rified by column chromatography (silica gel, CH2Cl2) to
furnish 17a (389 mg, 95%) as a white solid. Melting point
Melting point 262–264◦C; Rf = 0.38 (ss A); [α]2D5
=
+74.8 (c 1, DMSO). 1H NMR (500 MHz, DMSO-d6, δ
[ppm]): 1.21 (s, 3H, 18-CH3), 2.65 (m, 2H, 6-H2), 6.41 (d,
1H, J = 2.4 Hz, 4-H), 6.50 (dd, 1H, J = 8.5 Hz, J =
2.4 Hz, 2-H), 7.01 (d, 1H, J = 8.5 Hz, 1-H), 8.97 (s, 1H,
3-OH). 13C NMR (125 MHz, DMSO-d6, δ [ppm]): 20.4,
20.6, 25.3, 27.2 (C-18), 27.9, 34.4, 37.1, 38.8, 41.8, 48.8,
48.9, 112.8 (C-2), 114.3 (C-4), 126.2 (C-1), 130.2 (C-10),
136.9 (C-5), 154.7 (C-3), 213.9 (C-17a). EI-MS (70 eV) m/z
(%): 284 (100) [M+], 213 (13), 174 (32), 111 (29). Analy-
25
1
163–165 ◦C; Rf = 0.58 (ss B); [α] = +25.5 (c 1). H
NMR (500 MHz, CDCl3, δ [ppm]):D1.20 (s, 3H, 18-H3),

J. Wölfling et al. / Steroids 68 (2003) 277–288
283
sis calculated for C19H24O2: C, 80.24; H, 8.51; Found: C,
80.07; H, 8.63%.
against the concentration of unlabeled steroid. The molar
concentration of the steroid competitor that reduced the
radioligand binding by 50% (IC50) and the Ki value (inhibi-
tion constant) were calculated with GraphPad 2.0 software.
All assays were carried out at least three times in duplicate.
2.12.3. 3-Methoxy-D-homoestra-1,3,5(10)-trien-17a-one
(18b)
Melting point 137–138 ◦C, [32]: m.p. 140–141 ◦C. 18b is
identical with the compound described in [32]. 13C NMR
(125 MHz, CDCl3, δ [ppm]): 16.8 (C-18), 22.9, 25.9, 26.6,
30.1, 32.5, 37.2, 38.8, 43.0, 48.4, 50.3, 55.2 (3-OMe), 111.6
(C-2), 113.4 (C-4), 126.3 (C-1), 132.4 (C-10), 137.6 (C-5),
157.5 (C-3), 216.3 (C-17a). EI-MS (70 eV) m/z (%): 298
(100) [M+], 227 (9), 199 (15), 173 (7). Analysis calculated
for C20H26O2: C, 80.50; H, 8.78; Found: C, 80.41; H, 8.80%.
3. Results and discussion
3.1. Synthetic studies
In preliminary experiments, we synthesized the 3-methyl
and 3-benzyl ethers of the secoestrone aldehyde 3b and
4b via a multistep pathway from estrone 3-methyl and
benzyl ether in the normal series [15,16]. In order to
achieve the similar fragmentation in the 13a series to pro-
duce 3a and 4a, we had to apply an effective method for
the epimerization of estrone (Scheme 1). The 3-methoxy
and 3-benzyloxy-1,3,5(10)-trien-17-one (1b and 2b) were
epimerized by the method of Yaremenko and Khvat
[17], which was extended to the 13␣-estrone-3-methyl
ether by Schönecker et al. [12]. We obtained 92% of
13␣-estrone-3-methyl ether 1a in one step by using
o-phenylenediamine and AcOH [42] when the product
was separated from the starting material by means of the
Girard-P reagent [18]. The same transformation was also
carried out with the corresponding 3-benzyl ether of estrone
to give 98% of 13␣-estrone 3-benzyl ether 2a. The reason
for application of the 3-benzyl ether is that the benzyloxy
group can easily be removed by catalytic hydrogenation to
give the potentially more active 3-hydroxy derivative.
Formylation of the 3-benzyloxy-13␣-estra-17-one (5)
with HCOOEt in the presence of NaOMe in benzene gave
the 16-hydroxymethylene-17-ketone (6a) in high yield
(Scheme 2). The hydroxy function could readily be acety-
lated by Ac2O in pyridine to furnish 6b. Treatment of 6a
with KBH4 in MeOH resulted in two diastereomeric diols
(7a and 8a) in an overall yield of 98%. In contrast with the
similar reaction in the normal estrone series, we obtained
only the two trans diols (7a and 8a) in a ratio of nearly 6:1.
The mixture of diols could be separated by flash chromatog-
raphy after selective acetylation of their hydroxy groups
2.12.4. 3-Hydroxy-D-homoestra-1,3,5(10)-trien-17a-one
(19b)
Melting point 265–268 ◦C, [13]: m.p. 269 ◦C. 19b is
identical with the compound described in [13,33]. 13C
NMR (125 MHz, DMSO-d6, δ [ppm]): 16.4 (C-18), 22.2,
25.2, 25.5, 26.1, 29.4, 32.4, 36.6, 38.4, 42.4, 47.6, 49.5,
112.7 (C-2), 114.5 (C-4), 125.9 (C-1), 130.1 (C-10), 136.8
(C-5), 154.9 (C-3), 214.8 (C-17a). Analysis calculated for
C19H24O2: C, 80.24; H, 8.51; Found: C, 80.15; H, 8.58%.
2.13. Receptor-binding assay
The newly synthesized compounds were evaluated
for their ability to competitively inhibit the binding of
[3H]estra-1,3,5(10)-triene-3,17␤-diol (85 Ci mmol−1) to the
estrogen receptor, and of [3H]ORG2058 (34 Ci mmol−1
)
to the progesterone receptor in cytosol prepared from im-
mature rabbit uteri. Rabbits were treated with 25 ␮g of
3,17␤-estradiol in 0.5 ml of oil s.c. 96 h before the uteri
were excised. All of the tritiated ligands were purchased
from Amersham, UK. All subsequent steps were carried out
at 4 ◦C. The minced tissues were homogenized in 6–10 vol.
of 10 mM Tris–HCl buffer (pH 7.4) to measure the binding
abilities of the compounds to the estrogen receptor or in
10 mM Tris–HCl buffer (pH 7.4) containing 0.25 M sucrose
and 15 mM Na2MoO4 to measure the binding abilities of
the compounds to the progesterone receptor [14]. The ho-
mogenate was centrifuged at 105,000 × g for 60 min and
the supernatant (cytosol) was used for competitive binding.
Aliquots of 100 ␮l of cytosol were incubated for 4 h at
4 ◦C with the appropriate tritiated steroids in the presence
of the synthesized molecules in 11 different concentrations
(from 10−12 to 10−5 M). To determine the total binding of
the radiolabeled ligand, 100 ␮l of cytosol was incubated
with 100 ␮l of isotope and 100 ␮l of assay buffer. Unbound
steroids were removed by treatment with dextran-coated
charcoal (10% Norit-A and 0.1% dextran T-70 in the assay
buffer). After incubation for 20 min at 4 ◦C, the samples
were centrifuged at 3000 rpm for 10 min, and the radioac-
tivity of the supernatant was determined in a Wallac 1409
liquid scintillation counter. The percentage of the radioli-
gand bound in the presence of the competitor was plotted
Scheme 1.

284
J. Wölfling et al. / Steroids 68 (2003) 277–288
appropriate starting materials for the fragmentation process,
because no side-reaction (the disadvantageous disubstitu-
tion) had to be considered and the yield was 95–96%. The
method of Appel is an effective procedure for the conver-
sion of alcohols to alkyl iodides [19]. The mixture of diols
7a and 8a was reacted with Ph3P (Section 2.7.1) [20,21]
or diphenyl-2-pyridylphosphine (Section 2.7.2), which are
also often used in the Mitsunobu esterification reactions
[22,23], and imidazole and I2 in CH2Cl2 to result in an
isomeric mixture of 7d and 8d, in the same 6:1 ratio as for
the starting material.
The structures of the iodohydrins 7d and 8d were con-
1
1
firmed by H and 13C NMR measurements. The H NMR
spectra demonstrated that the singlet of the 18-methyl group
of the 17b-substituted main product (7d) appears 0.2 ppm
upfield from that in the spectrum of the other diastereomer
(8d). The multiplet of the 17-hydrogen of 7d can be identi-
fied at 3.39 ppm, while the signal of the same hydrogen of
8d is at 3.75 ppm, as a doublet (J = 8.5 Hz).
The following step was the fragmentation of the iodo
derivatives by alkaline solvolysis. Not only hydroxy tosy-
lates [24,25], but ␣,␥-halohydrins [26] are often used to pre-
pare different secosteroids via a fragmentation mechanism.
The reactions were carried out with KOH in MeOH under
reflux for 3 h with a mixture of the isomers (7d and 8d) or
with one of the isomers (7d or 8d), to give the secoaldehyde
4a in good yield (83%). When the mixture of iodohydrins
was used, the fragmentation was accompanied by a side-
reaction to give an elimination product (9) in 10%. The for-
mation of this compound was not observed on solvolysis of
the pure minor compound 8d. The structure of the aldehyde
4a was identified by NMR spectroscopy, which showed the
signals of the newly formed formyl group in the 13␤ position
(formyl-H at 9.71 ppm, as a singlet) and the propenyl side-
chain (multiplets of the vinyl protons at 5.01 and 5.82 ppm).
The presence of the olefinic moiety and the formyl group
in the precursors 3a, 3b, 4a and 4b makes the molecules
suitable for intramolecular Prins-type cycloadditon. The ring
closure of 3a and 4a is particularly favorable, since the two
functional groups that take part in the reaction have the
same spatial orientation. Treatment of the aldehyde 3a, 3b,
4a or 4b with 1.1 equivalents of p-toluenesulfonic acid in
CH2Cl2 gave the corresponding 16-tosylates 14a, 14b, 15a
or 15b, containing a six-membered ring D (Scheme 3). The
formyl oxygen was first protonated by the applied Brønsted
acid to give the mesomeric structures 10 and 11, which was
followed by the attack of the unsaturated side-chain on the
positively charged carbonyl-C to give the secondary carbo-
cation 12 or 13. The cation could be further transformed by
the nucleophilic addition of a tosyloxy group. The two chiral
centers at C-16 and C-17a theoretically permit the formation
of four isomers. However, all the transformations proceeded
in a stereospecific manner; only one of the four possible iso-
mers was isolated in each case. The cyclizations proceeded
to furnish products with cis functional groups at C-16 and
C-17a: 16␣,17a␣ isomers (14a and 15a) were obtained for
Scheme 2. Reagents and conditions: (i) o-phenylenediamine, AcOH,
117 ◦C, 3 h; (ii) HCOOEt, NaOMe, anhydrous benzene, 50 ◦C, 6 h → r.t.,
24 h; (iii) Ac2O, pyridine, r.t.; (iv) KBH4, MeOH, r.t., 4 h; (v)
Ac2O, pyridine, 0 ◦C
→
r.t., 5 h; (vi) Ph3P (Section 2.7.1) or
diphenyl-2-pyridylphosphine (Section 2.7.2), imidazole, I2, CH2Cl2, r.t.,
24 h; (vii) KOH, MeOH, 65 ◦C, 3 h.
to give 7b and 8b, and could be regained in pure form by
subsequent alkaline methanolysis. During the acetylation,
the corresponding diacetates 7c and 8c were also produced
in 20% yield, but after solvolysis they were recycled to the
reaction. With the aim of preparing the 13␣-D-secoestrone
aldehyde (4a), the primary hydroxy groups of the diols
were converted into good leaving groups. Earlier studies
revealed that the 16-iodomethyl derivatives were the most

J. Wölfling et al. / Steroids 68 (2003) 277–288
285
C-16 from the 16␣-H multiplet at around δ = 4.44, which
is in accord with two diaxial and two axial-equatorial cou-
plings. Verification of the structures of the 13-epimers (14a
and 15a) was complicated by the fact that the 13␣-estrones
may have different stable conformations as a consequence of
the flexibility of rings C and D. The characteristic signals of
17a-H and 16-H displayed very similar shapes to those for
the normal derivatives 14b and 15b: the doublet-like multi-
plet appears at around δ = 3.8, with the 16-H multiplet at
around δ = 4.4. Nevertheless, X-ray crystal structure anal-
ysis of a structurally, and therefore spectroscopically simi-
lar 16-fluorinated,17a-ethoxy compound in the 13-epi series
confirmed the cis anellation of rings C and D in 14a and 15a,
with both in the chair conformation, and the ␤,␤ orientation
of the protons on C-16 and C-17a.
Since the tosyloxy group at position C-16 is known to be
a good leaving group, oxidation of the tosylates 14a, 14b,
15a and 15b with the Jones reagent (8N) in acetone led
to the conjugated ketones 16a, 16b [30], 17a and 17b [31]
by simultaneous oxidation and ␤-elimination. The 1H NMR
spectra of the 13-epi derivatives 16a and 17a indicate the
disappearance of the 17a-H signal, while the 17-H signal
appears at δ = 5.88, as a double doublet.
After hydrogenation of the ␣,␤-unsaturated ketones 16a,
16b, 17a and 17b in the presence of a catalytic amount
of Pd/C in EtOAc, the desired homoestrone derivatives
18a, 18b [32,33], 19a and 19b [34] were obtained in high
yield. On saturation of 17a and 17b, hydrogenolysis of the
3-benzyloxy group also occurred, to give the 3-hydroxy
compounds 19a and 19b.
3.2. Radioligand-binding assays
3.2.1. Results
The binding affinities of 3-methoxy-D-homoestra-1,3,5
(10)-trien-17a-one (18b) and its derivatives for the es-
trogen and progesterone receptors were determined by
radioligand-binding assay.
Scheme 3. Reagents and conditions: (i) p-TosOH, CH2Cl2, 0 ◦C → r.t.,
24 h; (ii) Jones reagent (8N), Me2CO, 0 ◦C, 1 h; (iii) H2 (20 bar), Pd/C,
EtOAc, 24 h.
Specifically bound [3H]estra-1,3,5(10)-triene-3,17␤-diol
was displaced from the immature rabbit uterine estrogen re-
ceptor by all the examined molecules, though their affinities
are at least three orders of magnitude lower than the Ki value
of 17␤-estradiol, the endogenous ligand of the receptor.
The inhibition constant (Ki) values and RBAs are listed in
Table 1. 3-Hydroxy-13␣-D-homoestra-1,3,5(10)-trien-17a-
one (19a) is the most potent molecule in this series.
3-Hydroxy-D-homoestra-1,3,5(10)-trien-17a-one (19b), the
epimeric counterpart of the previous compound, has a Ki
value of the same order of magnitude. The remaining two
derivatives, 18a and 18b, exhibit Ki values higher than
1000 nM (1131 and 3394 nM, respectively).
3a and 4a, and 16␤,17a␤ isomers (14b and 15b) for 3b and
4b [27–29].
The favored formation of the 16␤,17a␤ diastereomers can
be explained by addition of the alkene moiety to the oxo-
nium ion anti to the angular methyl group in 3b or 4b, while
the subsequent addition of the nucleophile to the cation 12b
or 13b takes place syn to the angular methyl group. The en-
try of the nucleophile from the ␤ direction is not surprising
since the large tosyloxy group probably prefers an equa-
torial orientation. Accordingly, the converse consideration
predominates in the case of 3a or 4a.
The configuration of the substituents was determined by
NMR spectroscopy in the normal series and by X-ray crys-
tal analysis of an analogous 13␣-D-homo derivative in the
13␣ series. The stereochemistry at C-17a in 14b and 15b
follows from the 17a␣-H multiplet at δ = 2.72 and that at
The tested compounds were bound to the progesterone
receptor labeled by [3H]ORG2058 with different affinities.
18a possesses the highest Ki value (25,640 nM) whereas the
most potent molecule is seen to be 19b (Ki = 196.1 nM). A
nearly one order of magnitude difference can be observed

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J. Wölfling et al. / Steroids 68 (2003) 277–288
Table 1
Inhibition constant (Ki) values of estra-1,3,5(10)-triene-3,17␤-diol (reference compound) and some synthesized derivatives (18b, 19b, 18a and 19a) on
the uterine estrogen (ER) and progesterone (PR) receptors
Compound
Ki [nM]/ER
Ki [nM]/PR
RBA
100
SR
3,17␤-Estradiol
0.3917
3394
0.1087
1532
1303
102
292
3911
18b
19b
18a
19a
1131
167.5
17.68
10.39
0.0115
0.38
0.67
22.7
43.7
293.7
1131
196.1
25,640
7372
77.64
1612
3516
0.133
0.0346
0.232
168.8
The following radioligands were used: 2.5 nM [3H]estra-1,3,5(10)-triene-3,17␤-diol (ER) and 4.0 nM [3H]ORG2058 (PR). The RBA values for the ER
were calculated according to the equation RBA =Kiestradiol/Ki∗. The selectivity ratio (SR) for each compound was calculated according to the equation
SR =KiPR/KiER
.
between the Ki values of the other two compounds 19a (Ki =
7372 nM) and 18b (Ki = 1303 nM). The Ki value of 18a
is significantly lower than that of 3,17␤-estradiol. The Ki
values for the progesterone receptor and the selectivity ratios
are listed in Table 1.
these functional groups. Two of our four molecules con-
tain a 3-hydroxy group and their Ki values as concerns the
estrogen receptor were determined to be higher than those
of the analogs with a 3-methoxy function. This 3-methoxy
function in 18a and 18b weakens the strength of the in-
teraction as a consequence of its lower hydrogen-donating
ability (Fig. 1).
3.2.2. Discussion
It is well known that the estrane skeleton is not essential
for molecules that strongly bind to the estrogen receptor.
This recognition led to the discovery of diethylstilbestrol
[35] or tamoxifen and its analogs [36]and also the recently
synthesized tetrahydrochrysene [37] and the furan deriva-
tives [38].
The present study has revealed that four new compounds
containing the D-homoestrane skeleton have been prepared,
which belong in one chemical series. Examination of the
binding of these molecules to the estrogen receptor proved
that the receptor recognizes them. The differences between
the Ki values can be explained by the different modes of
binding to the estrogen receptor. The estrogen receptor
ligand-binding domain (ERLBD) binds the ligand at the
end of its structure. The 3-hydroxy group in ring A forms
a hydrogen-bond with Glu353, as does the 17-hydroxy
group in ring D with His524 [39]. Compounds exhibiting
a high binding affinity for the estrogen receptor possess
Ring D in the tested compounds contains six C atoms,
which cannot influence the receptor-binding ability signif-
icantly, because this part of the estrogen receptor-binding
pocket becomes wider, making room for bulky molecules
[40].
The binding affinities for the progesterone receptor are
of different orders of magnitude for the various compounds
(Fig. 2). The progesterone receptor ligand-binding domain
(PRLBD) makes contact with progesterone at its ends:
Gln725 donates a hydrogen-bond to the 3-keto group, and
Thr894 seems to form a hydrogen-bond with the 20-keto
oxygen. The Thr894 residue is present only in receptors that
bind hormones containing a 17-alkyl-keto group (proges-
terone receptor, glycocorticoid receptor and mineralocorti-
coid receptor) [41]. In this case, the second requirement was
only partially satisfied, because the examined molecules
contain only one keto group, not at C-20, but at C-17a, so
the measured Ki values are lower than that of progesterone.
Fig. 1. Competitive inhibition of [3H]estra-1,3,5(10)-triene-3,17␤-diol in rabbit uterus cytosol by 3-methoxy-D-homoestra-1,3,5(10)-trien-17a-one (18b)
and its derivatives (18a, 19a and 19b).

J. Wölfling et al. / Steroids 68 (2003) 277–288
287
Fig. 2. Competitive inhibition of [3H]ORG2058 in rabbit uterus cytosol by 3-methoxy-D-homoestra-1,3,5(10)-trien-17a-one (18b) and its derivatives (18a,
19a and 19b).
The Ki values (for the progesterone receptor) of the two
epimers which display higher affinity for the estrogen recep-
tor (19a and 19b) differ by one order of magnitude. These
two compounds differ only in the position of the methyl
group on C-13. This functional group is fixed by Met909
of the PRLBD through a van der Waals contact [41]. If this
group is in the ␣ position, this is unfavorable according to our
measurements, because the selectivity ratio of the molecule
is lower. A similar affinity difference is observed between
the other two epimers (18a and 18b), which supports our
previous conclusion.
In summary, two epimer pairs containing the D-homoe-
strane skeleton were prepared and tested in the radioligand-
binding assay. The estrogen receptor recognizes these
compounds, but their Ki values are at least three orders
of magnitude lower than that of the reference molecule
3,17␤-estradiol. Two compounds, 18␣ and 19␣, are con-
sidered estrogen receptor-selective molecules by virtue of
their selectivity ratios.
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Acknowledgments
The authors thank the Hungarian Scientific Research
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