Synthesis and receptor-binding examination of 16-hydroxymethyl-3,17-estradiol stereoisomers

Article abstract of DOI:10.1016/S0039-128X(01)00191-X

The four 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers were synthesized and tested in a radioligand-binding assay. The estrogen receptor recognizes these compounds, but their relative binding affinities are lower than 2.0% relative to that of the reference molecule estra-1,3,5(10)-triene-3,17β-diol. The affinities of the tested compounds for the androgen and progesterone receptors are very low (K_i> 100 μm and 1 μM, respectively). The prepared 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers are therefore estrogen receptor-selective molecules.

Full text of DOI:10.1016/S0039-128X(01)00191-X

Steroids 67 (2002) 371–377
Synthesis and receptor-binding examination of 16-hydroxymethyl-3,17-
estradiol stereoisomers
a
a
b
b
b
´
Pa´l Tapolcsa´nyi , Ja´nos Wo¨lfling , George Falkay , Arpa´d Ma´rki , Rena´ta Minorics ,
Gyula Schneidera,*
aDepartment of Organic Chemistry, University of Szeged, Do´m te´r 8, H-6720 Szeged, Hungary
bInstitute of Pharmacodynamics and Biopharmacy, University of Szeged, Eo¨tvo¨s u 6, H-6720 Szeged, Hungary
Received 27 March 2001; received in revised form 6 September 2001; accepted 12 September 2001
Abstract
The four 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers were synthesized and tested in a radioligand-binding assay. The
estrogen receptor recognizes these compounds, but their relative binding affinities are lower than 2.0% relative to that of the reference
molecule estra-1,3,5(10)-triene-3,17␤-diol. The affinities of the tested compounds for the androgen and progesterone receptors are very low
(KiϾ 100 ␮m and 1 ␮M, respectively). The prepared 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers are therefore estrogen
receptor-selective molecules. © 2002 Elsevier Science Inc. All rights reserved.
Keywords: 16-Hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers; Estrogen receptor binding
1. Introduction
been evaluated over the years [11]. The sites of the sub-
stituents are unequivocal; the steric structure often depends
on the method of synthesis. As concerns the 16-substituted
estrogens, usually the 16␣-substituted-17␤-hydroxy com-
pounds have been studied. The biologic activity has gener-
ally not been studied for the whole isomer series.
In the 16 substituted 17-hydroxysteroids, the two chiral
centres permit four stereochemical modifications. We set
out to prepare the four possible isomers of 16-hydroxymeth-
yl-3,17-estradiol and to subject them to receptor-binding
examinations to obtain answers to the following questions:
(1) How do the receptor-binding processes of the four iso-
mers 3d, 4d, 5c and 6d differ? (2) How is the receptor-
binding process influenced by the relative steric position of
the 16-hydroxymethyl and 17-hydroxy groups?
Breast cancer is the most frequently diagnosed cancer in
women worldwide [1]. Since estrogens are known to play a
role in the development of many breast cancers, a logical
approach to the treatment of estrogen-sensitive breast can-
cer is the use of anti-estrogens that block the interaction of
estrogens with their specific receptor. A new generation of
steroidal compounds have recently been described as anti-
estrogens. These compounds generally contain a long alkyl-
amide side-chain at position 7␣ or 11␤ of an estradiol
nucleus, this side-chain playing an essential role in the
anti-estrogenic properties of the compounds [2–6]. Earlier
examinations suggested that the estrogen receptor tolerates
reasonably large substituents at positions 7␣, 11␤, 16␣ and
17␣ in estradiol [9,10]. Poirier et al. demonstrated that
16␣-halogenopropylestradiol and 16␣-bromoalkylamide-
estradiol derivatives inhibit 17␤-hydroxysteroid dehydroge-
nase, which is responsible for the transformation of the less
potent estrone to the most potent estrogen, estradiol [7,8]. A
number of estradiol derivatives have been synthesized and
their relative binding affinities for estrogen receptors have
2. Experimental
2.1. General
Melting points were determined with a Kofler hot-stage
apparatus and are uncorrected. Specific rotations were mea-
sured with a POLAMAT-A (Zeiss-Jena) polarimeter on
solutions in chloroform (c 1) and are given in units of 10Ϫ1
deg cm2 gϪ1. Elemental analyses were performed with a
* Corresponding author. Tel.: ϩ36-62-544276; fax: ϩ36-62-544200.
E-mail address: schneider@chem.u-szeged.hu (G. Schneider).
0039-128X/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.
PII: S0039-128X-(01)00191-X

372
P. Tapolcsa´nyi et al. / Steroids 67 (2002) 371–377
Perkin-Elmer CHN analyser model 2400. Thin-layer chro-
matography: silica gel 60, layer thickness 0.2 mm (Merck);
solvent system (ss): (A) ethyl acetate/chloroform (10:90
v/v), (B) ethyl acetate/chloroform (15:85 v/v), (C) ethyl
acetate/chloroform (50:50 v/v); detection with iodine or UV
(365 nm) after spraying with 50% phosphoric acid and
heating at 100–120°C for 10 min. Flash chromatography:
silica gel 60, 40–63 ␮m. Column chromatography: Al2O3
(standarized according to Brockmann) with an activity of
III-IV. The NMR spectra were recorded with a Bruker
AMX-400 instrument. Chemical shifts (␦) are given in ppm,
and coupling constants (J) in Hz.
2.4. 16-Acetoxymethylene-3-benzyloxyestra-1,3,5(10)-
trien-17-one (2b)
Crude 2a (7.76 g, 0.02 mol) was dissolved in a mixture
of pyridine (10 ml) and acetic anhydride (10 ml), and the
solution was allowed to stand at room temperature for 12 h.
The reaction mixture was then poured onto a mixture of ice
(300 g) and 5 ml of concentrated H2SO4. The precipitate
was collected by filtration and recrystallized from a mixture
of methanol and water (8.1 g, 94%). Mp 215–218°C, Rf ϭ
0.90 (ss A); [␣]D20 ϩ98 (c 1 in chloroform) (Found: C, 78.25;
1
H, 6.95. C28H30O4 requires: C, 78.11; H, 7.02%). H-NMR
(400 MHz, CDCl3): ␦ ppm 0.93(s, 3H, H-18), 2.23(s, 3H,
CH3CO), 2.90(m, 2H, H-6), 5.03(s, 2H, C6H5CH2), 6.73(d,
1H, J ϭ 2.6 Hz, H-4), 6.79(dd, 1H, J ϭ 8.6 Hz, 2.6 Hz, H-2),
7.19(d, 1H, J ϭ 8.6 Hz, H-1), 7.31–7.43(m, 5H, C6H5). 13C-
NMR (100 MHz, CDCl3): ␦ ppm 14.4(C-18), 20.6(CH3CO),
25.1, 25.9, 26.7, 29.6, 31.4, 37.8, 38.3, 44.0, 48.0, 48.9(C-
13), 69.9(C6H5CH2), 112.4(C-2), 114.9(C-4), 121.2(C-16),
126.2(C-1), 127.4(2C, C-2Ј and C-6Ј), 127.8(C-4Ј), 128.5(2C,
C-3Ј and C-5Ј), 132.2(C-10), 137.2(C-1Ј), 137.6(C-5),
139.9(CHOAc), 156.9(C-3), 167.1(CH3CO), 209.2(C-17).
2.2. 3-Benzyloxyestra-1,3,5(10)-trien-17-one (1b)
Sodium (5 g, 0.22 mol) was dissolved in methanol
(250 ml) and 3-hydroxy-1,3,5(10)-trien-17-one (27 g, 0.1
mol) was added. The mixture was stirred and warmed
slightly until the steroid had dissolved. After the addition
of benzyl chloride (25 ml, 0.15 mol), the reaction mixture
was heated at reflux for 6 h, and then poured into water.
The precipitate was collected by filtration, washed with
water and dried in a vacuum desiccator over P2O5. Crys-
tallization from acetone gave 1b as white crystals (32.4 g,
2.5. 16-Hydroxymethyl-3-benzyloxyestra-1,3,5(10)-trien-
17-ol isomers (3a, 4a and 5a)
20
90%). Mp 127–128°C, [␣]D ϩ132 (c 1 in chloroform)
([12] Mp 132–134°C, [␣]D ϩ 122) (Found C, 83.15; H,
7.92. C25H28O2 requires C, 83.29; H, 7.83%). H-NMR
Compound 2a (19.4 g, 0.05 mol) was suspended in
ethanol (500 ml), and KBH4 (8.1 g, 0.15 mol) was added in
small portions during cooling in ice. The reduction mixture
was allowed to stand for 24 h, and was then acidified with
dilute HCl. The resulting solution was poured onto ice
(1000 g) and the precipitate was collected by filtration and
dried in a vacuum desiccator over P2O5. The dried isomeric
mixture of 3a, 4a and 5a (18 g, 92%) was suspended in a
mixture of dichloromethane (500 ml) and acetone (100 ml)
in the presence of a catalytic amount of p-toluenesulfonic
acid. The reaction mixture was kept at boiling temperature
for 1 h, and was next neutralized with morpholine and
evaporated to dryness. The residue obtained was then dis-
solved in chloroform (50 ml) and chromatographed on
Al2O3. Chloroform/light petroleum (1:3) eluted 3c (9.75 g;
49%). Mp 133–134°C, Rf ϭ 0.80 (ss A); [␣]D20 ϩ56 (c 1 in
chloroform). (Found: C, 80.40; H, 8.32. C29H36O3 requires:
1
(400 MHz, CDCl3): ␦ ppm 0.90(s, 3H, H-18), 2.89(m,
2H, H-6), 5.03(s, 2H, C6H5CH2), 6.73(d, 1H, J ϭ 2.6 Hz,
H-4), 6.78(dd, 1H, J ϭ 8.6 Hz, 2.6 Hz, H-2), 7.19(d, 1H,
J ϭ 8.6 Hz, H-1), 7.31–7.43(m, 5H, C6H5). 13C-NMR
(100 MHz, CDCl3): ␦ ppm 13.8(C-18), 21.5(C-15), 25.9,
26.5, 29.6, 31.6, 35.8(C-16), 38.3, 43.9, 47.9(C-13), 50.4(C-
14), 69.9(C6H5CH2), 112.3(C-2), 114.9(C-4), 126.3(C-1),
127.3(2C, C-2Ј and C-6Ј), 127.8(C-4Ј), 128.5(2C, C-3Ј and
C-5Ј), 132.3(C-10), 137.2(C-1Ј), 137.7(C-5), 156.8(C-3),
220.6(C-17).
2.3. 16-Hydroxymethylene-3-benzyloxyestra-1,3,5(10)-
trien-17-one (2a)
Sodium (2.5 g, 0.11 mol) was dissolved in methanol
(120 ml), and the solvent was removed by distillation
under vacuum to obtain NaOCH3 free from methanol.
3-Benzyloxyestra-1,3,5(10)-trien-17-one (18 g, 0.05 mol)
dissolved in anhydrous benzene (100 ml) was added to
the homogenized NaOCH3 powder. Freshly distilled
ethyl formate (60 ml) was added dropwise and the mix-
ture was stirred at room temperature for 2 h, and then at
50°C for 4 h. The reaction mixture was poured onto ice,
and the aqueous phase was separated and acidified with
dilute HCl to pH 3. The precipitate was collected by
filtration, washed with water and dried in a vacuum
desiccator over P2O5 (18.5 g, 95%).
1
C, 80.52; H, 8.39%). H-NMR (400 MHz, CDCl3): ␦ ppm
0.90(s, 3H, H-18), 1.36 an d 1.37(two s, 6H, (CH3)2C),
2.86(m, 2H, H-6), 3.56(dd, 1H, J ϭ 11.9 Hz, 10.6 Hz,
H-16), 3.69(dd, 1H, J ϭ 10.6 Hz, 9.3 Hz, H-16), 3.78(d, 1H,
J ϭ 9.3 Hz, H-17), 5.05(s, 2H, C6H5CH2), 6.73(d, 1H, J ϭ
2.6 Hz, H-4), 6.79(dd, 1H, J ϭ 8.6 Hz, 2.6 Hz, H-2), 7.21(d,
1H, J ϭ 8.6 Hz, H-1), 7.32–7.45(m, 5H, C6H5). 13C-NMR
(100 MHz, CDCl3): ␦ ppm 13.3(C-18), 25.1 and 27.4(2C,
(CH3)2C), 26.4, 27.7, 27.9, 29.8, 37.8, 38.3, 38.4, 43.9,
44.3(C-13), 49.7, 63.5(C-16Ј), 70.0(C6H5CH2), 79.2(C-17),
98.7((CH3)2C), 112.3(C-2), 114.9(C-4), 126.2(C-1), 127.4(2C,
C-2Ј and C-6Ј), 127.8(C-4Ј), 128.5(2C, C-3Ј and C-5Ј),
133.0(C-10), 137.4(C-1Ј), 137.9(C-5), 156.8(C-3). Continued

P. Tapolcsa´nyi et al. / Steroids 67 (2002) 371–377
373
elution with chloroform yielded 4a together with 5a. The
chloroform solution was evaporated to dryness, and the residue
was dissolved in hot acetone. From this solution, the bulk of
the 4a crystallized out as hard crystals (6.7 g, 37%). Mp
151–153°C, Rf ϭ 0.35 (ss B); [␣]D20 ϩ56 (c 1 in chloroform).
(Found: C, 79.65; H, 8.14. C26H32O3 requires: C, 79.56; H,
2.6. 16-Acetoxymethyl-3-benzyloxyestra-1,3,5(10)-trien-17-
acetate isomers (3b, 4b and 5b)
2.6.1. (General procedure)
Compound 3a, 4a or 5a (3.9 g, 0.01 mol) was dissolved
in a mixture of pyridine (10 ml) and acetic anhydride (10
ml) and the solution was allowed to stand at room temper-
ature for 12 h. The mixture was then diluted with water and
the precipitate was collected by filtration and recrystallized
from methanol. 3b (4.7 g, 98%). Mp 100–101°C, Rf ϭ 0.80
1
8.22%). H-NMR (400 MHz, CDCl3): ␦ ppm 0.90(s, 3H,
H-18), 1.36 and 1.37(two s, 6H, (CH3)2C), 2.86(m, 2H, H-6),
3.56(dd, 1H, J ϭ 11.9 Hz, 10.6 Hz, H-16), 3.69(dd, 1H, J ϭ
10.6 Hz, 9.3 Hz, H-16), 3.78(d, 1H, J ϭ 9.3 Hz, H-17), 5.05(s,
2H, C6H5CH2), 6.73(d, 1H, J ϭ 2.6 Hz, H-4), 6.79(dd, 1H, J ϭ
8.6 Hz, 2.6 Hz, H-2), 7.21(d, 1H, J ϭ 8.6 Hz, H-1), 7.32–
7.45(m, 5H, C6H5) 13C-NMR (100 MHz, CDCl3): ␦ ppm
13.3(C-18), 25.1 and 27.4(2C, (CH3)2C), 26.4, 27.7, 27.9,
29.8, 37.8, 38.3, 38.4, 43.9, 44.3(C-13), 49.7, 63.5(C-16Ј),
70.0(C6H5CH2), 79.2(C-17), 98.7((CH3)2C), 112.3(C-2),
114.9(C-4), 126.2(C-1), 127.4(2C, C-2Ј and C-6Ј), 127.8(C-
4Ј), 128.5(2C, C-3Ј and C-5Ј), 133.0(C-10), 137.4(C-1Ј),
137.9(C-5), 156.8(C-3). The mother liquor was evaporated and
subjected to chromatographic separation on a silica gel column
with tert-butyl methyl ether. 4a eluted first (1.3 g, 7%). Con-
tinued elution resulted in 5a (0.85 g, 4.7%). Mp 128–130°C,
20
(ss A); [␣]D ϩ 37. (Found: C, 75.78; H, 7.55. C30H36O5
requires C, 75.60; H, 7.61%). 1H-NMR (400 MHz, CDCl3):
␦ ppm 0.86(s, 3H, H-18), 2.03 and 2.08(two s, 6H, two
CH3CO), 2.88(m, 2H, H-6), 4.03(dd, 1H, J ϭ 11.1 Hz, 7.5
Hz) and 4.13(dd, 1H, J ϭ 11.1 Hz, 7.0 Hz): H-16a, 4.90(d,
1H, J ϭ 10.1 Hz, H-17), 5.03(s, 2H, C6H5CH2), 6.72(d, 1H,
J ϭ 2.6 Hz, H-4), 6.78(dd, 1H, J ϭ 8.6 Hz, 2.6 Hz, H-2),
7.19(d, 1H, J ϭ 8.6 Hz, H-1), 7.31–7.44(m, 5H, C6H5).
13C-NMR (100 MHz, CDCl3): ␦ ppm 12.9(C-18), 20.9(2C,
CH3CO), 26.1, 27.3, 29.3, 29.7, 37.4, 37.6, 37.9, 43.6(C-
13), 43.7, 48.7, 65.3(C-16Ј), 69.9(C6H5CH2), 81.6(C-17),
112.3(C-2), 114.8(C-4), 126.3(C-1), 127.4(2C, C-2Ј and
C-6Ј), 127.8(C-4Ј), 128.5(2C, C-3Ј and C-5Ј), 132.6(C-10),
137.3(C-1Ј), 137.8(C-5), 156.8(C-3), 170.9(2C, CH3CO).
20
Rf ϭ 0.30 (ss B); [␣]D ϩ46. (Found: C, 79.60; H, 8.10.
C26H32O3 requires: C, 79.56; H, 8.22%). 1H-NMR (400 MHz,
CDCl3): ␦ ppm 0.75(s, 3H, H-18), 2.85(m, 2H, H-6), 3.68(d,
1H, J ϭ 1.6 Hz, H-17), 3.66(dd, 1H, J ϭ 10.5 Hz, 8.8 Hz) and
3.75(dd, 1H, J ϭ 10.5 Hz, 6.5 Hz): H-16a, 5.03(s, 2H,
C6H5CH2), 6.72(d, 1H, J ϭ 2.7 Hz, H-4), 6.78(dd, 1H, J ϭ 8.6
Hz, 2.7 Hz, H-2), 7.22(d, 1H, J ϭ 8.6 Hz, H-1), 7.31–7.44(m,
5H, C6H5) 13C-NMR (100 MHz, CDCl3): ␦ ppm 17.7(C-18),
26.0, 28.0, 29.0, 29.8, 32.0, 38.6, 43.4, 44.9(C-13), 48.8, 52.0,
66.5(C-16Ј), 70.0(C6H5CH2), 82.4(C-17), 112.3(C-2),
114.9(C-4), 126.3(C-1), 127.4(2C, C-2Ј and C-6Ј), 127.8(C-
4Ј), 128.5(2C, C-3Ј and C-5Ј), 132.9(C-10), 137.3(C-1Ј),
137.9(C-5), 156.8(C-3).
20
4b (4.65 g, 97%). Mp 131–132°C, Rf ϭ 0.75 (ss A); [␣]D
ϩ 3. (Found: C, 75.72; H, 7.66. C30H36O5 requires: C,
1
75.60; H, 7.61%). H-NMR (400 MHz, CDCl3): ␦ ppm
0.86(s, 3H, H-18), 2.05 and 2.08(two s, 6H, two CH3CO),
2.86(m, 2H, H-6), 4.07(dd, 1H, J ϭ 10.9 Hz, 6.7 Hz) and
4.13(dd, 1H, J ϭ 11.3 Hz, 6.7 Hz): H-16a, 4.75(d, 1H, J ϭ
8.1 Hz, H-17), 5.03(s, 2H, C6H5CH2), 6.72(d, 1H, J ϭ 2.6
Hz, H-4), 6.78(dd, 1H, J ϭ 8.6 Hz, 2.6 Hz, H-2), 7.19(d,
1H, J ϭ 8.6 Hz, H-1), 7.31–7.44(m, 5H, C6H5). 13C-NMR
(100 MHz, CDCl3): ␦ ppm 12.6(C-18), 20.9 and 21.0(2C,
CH3CO), 26.0, 27.1, 27.4, 29.6, 36.8, 38.3, 40.2, 43.7,
44.3(C-13), 48.6, 66.7(C-16Ј), 69.9(C6H5CH2), 83.7(C-17),
112.3(C-2), 114.8(C-4), 126.3(C-1), 127.4(2C, C-2Ј and
C-6Ј), 127.8(C-4Ј), 128.5(2C, C-3Ј and C-5Ј), 132.5(C-10),
137.3(C-1Ј), 137.8(C-5), 156.7(C-3), 170.8 and 171.0(2C,
CH3CO). 5b (4.55 g, 95%). Mp 79–82°C, Rf ϭ 0.50 (ss A);
[␣]D20 ϩ 51. (Found: C, 75.55; H, 7.78. C30H36O5 requires:
Compound 3c (8.64 g, 0.02 mol) was dissolved in 96%
ethanol (150 ml) in the presence of a catalytic amount of
p-toluenesulfonic acid. The reaction mixture was allowed to
stand at room temperature for 6 h, and was then diluted with
water. The crystalline substance was collected by filtration
and was recrystallized from chloroform/light petroleum to
obtain 3a (7.5 g, 95%). Mp 151–153°C, Rf ϭ 0.40 (ss B);
[␣]D20 ϩ 45. (Found: C, 79.31; H, 8.35. C26H32O3 requires
1
C, 75.60; H, 7.61%). H-NMR (400 MHz, CDCl3): ␦ ppm
0.85(s, 3H, H-18), 2.06 and 2.07(two s, 6H, two CH3CO),
2.85(m, 2H, H-6), 4.17(dd, 1H, J ϭ 11.0 Hz, 7.2 Hz) and
4.22(dd, 1H, J ϭ 11.0 Hz, 7.3 Hz): H-16a, 4.71(d, 1H, J ϭ
2.0 Hz, H-17), 5.03(s, 2H, C6H5CH2), 6.72(d, 1H, J ϭ 2.7
Hz, H-4), 6.78(dd, 1H, J ϭ 8.6 Hz, 2.7 Hz, H-2), 7.19(d,
1H, J ϭ 8.6 Hz, H-1), 7.30–7.43(m, 5H, C6H5). 13C-NMR
(100 MHz, CDCl3): ␦ ppm 17.0(C-18), 20.9 and 21.1(2C,
CH3CO), 25.9, 27.9, 29.3, 29.7, 32.2, 38.5, 43.4, 44.6(C-
13), 45.7, 49.9, 66.5(C-16Ј), 69.9(C6H5CH2), 83.5(C-17),
112.3(C-2), 114.8(C-4), 126.3(C-1), 127.4(2C, C-2Ј and
C-6Ј), 127.8(C-4Ј), 128.5(2C, C-3Ј and C-5Ј), 132.6(C-10),
137.3(C-1Ј), 137.8(C-5), 156.8(C-3), 170.4 and 171.1(2C,
CH3CO).
1
C, 79.56; H, 8.22%). H-NMR (400 MHz, CDCl3): ␦ ppm
0.85(s, 3H, H-18), 3.65(m, 1H) and 3.84(m, 1H): H-16a,
3.94(d, 1H, J ϭ 9.8 Hz, H-17), 5.02(s, 2H, C6H5CH2),
6.71(d, 1H, J ϭ 2.6 Hz, H-4), 6.77(dd, 1H, J ϭ 8.6 Hz, 2.6
Hz, H-2), 7.19(d, 1H, J ϭ 8.6 Hz, H-1), 7.30–7.42(m, 5H,
C6H5). 13C-NMR (100 MHz, CDCl3): ␦ ppm 12.2(C-18),
26.3, 27.5, 27.8, 29.7, 37.7, 38.1, 41.9, 43.9, 44.3(C-13),
49.1, 64.7(C-16Ј), 70.0(C6H5CH2), 83.0(C-17), 112.3(C-2),
114.9(C-4), 126.3(C-1), 127.4(2C, C-2Ј and C-6Ј), 127.8(C-
4Ј), 128.5(2C, C-3Ј and C-5Ј), 132.8(C-10), 137.3(C-1Ј),
137.9(C-5), 156.8(C-3).

374
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2.7. 16␣-Acetoxymethyl-3-benzyloxyestra-1,3,5(10)-trien-
17␤-ol (4c)
2.9. 16␣-Acetoxymethyl-3-benzyloxyestra-1,3,5(10)-trien-
17␣-ol (6c)
LiAlH4 (3.8 g, 0.1 mol) was suspended in anhydrous
diethyl ether (200 ml), and tert-butanol (28.25 ml, 0.3 mol)
was added carefully during stirring and cooling with salt-
ice. Compound 7 (8.64 g, 0.02 mol) dissolved in diethyl
ether (150 ml) was added dropwise to the suspension. The
reaction mixture was stirred for 1 h and was then decom-
posed by the careful addition of water (200 ml) during
stirring and cooling. It was next acidified with dilute HCl.
The organic phase was separated and the aqueous phase was
extracted with diethyl ether. The substance obtained on
washing, drying and evaporation of the solvent was sub-
jected to chromatographic separation on silica gel with
tert-butyl methyl ether/light petroleum (40:60). 6c eluted
Compound 4a (20 g, 0.05 mol) was dissolved in pyridine
(80 ml), and acetic anhydride (5 ml, 0.05 mol) in pyridine
(40 ml) was added dropwise during cooling with ice. The
reaction mixture was allowed to warm up to room temper-
ature, stirred for 3 h, then poured onto a mixture of ice and
H2SO4, and extracted with chloroform. The chloroform
solution was washed with NaHCO3 solution and then with
water, dried and evaporated. The residual oil was chromato-
graphed on silica gel with ethyl acetate/chloroform (10:90),
yielding pure 4b (2.4 g, 10%). Continued elution resulted in
4c (14.4 g, 65%). Mp 91–93°C, Rf ϭ 0.50 (ss B); [␣]D20 ϩ
49. (Found: C, 77.25; H, 7.95. C28H34O4 requires: C, 77.39,
H, 7.89%). 1H-NMR (400 MHz, CDCl3): ␦ ppm 0.84(s, 3H,
H-18), 2.09(s, 3H, CH3CO), 2.85(m, 2H, H-6), 3.46(d, 1H,
J ϭ 7.6 Hz, H-17), 4.14(dd, 1H, J ϭ 10.7 Hz, 7.0 Hz) and
4.18(dd, 1H, J ϭ 10.7 Hz, 6.7 Hz): H-16a, 5.04(s, 2H,
C6H5CH2), 6.72(d, 1H, J ϭ 2.6 Hz, H-4), 6.78(dd, 1H, J ϭ
8.6 Hz, 2.6 Hz, H-2), 7.20(d, 1H, J ϭ 8.6 Hz, H-1), 7.30–
7.44(m, 5H, C6H5). 13C-NMR (100 MHz, CDCl3): ␦ ppm
11.8(C-18), 21.0(CH3CO), 26.1, 27.2, 27.4, 29.7, 36.7,
38.5, 42.9, 43.9, 44.1(C-13), 48.7, 67.7(C-16Ј),
69.9(C6H5CH2), 84.7(C-17), 112.3(C-2), 114.8(C-4),
126.3(C-1), 127.4(2C, C-2Ј and C-6Ј), 127.8(C-4Ј),
128.5(2C, C-3Ј and C-5Ј), 132.7(C-10), 137.3(C-1Ј),
137.9(C-5), 156.7(C-3), 171.3(CH3CO). Methanol eluted
the unchanged starting compound 4a (3.8 g, 19%).
20
first (1.42 g, 16%). Mp 58–60°C, Rf ϭ 0.55 (ss B); [␣]D
ϩ 40. (Found: C, 77.51; H, 7.71. C28H34O4 requires: C,
1
77.39; H, 7.89%). H-NMR (400 MHz, CDCl3): ␦ ppm
0.78(s, 3H, H-18), 2.08(s, 3H, CH3CO), 2.88(m, 2H, H-6),
3.76(d, 1H, J ϭ 5.0 Hz, H-17), 4.15(dd, 1H, J ϭ 11.0 Hz,
6.1 Hz) and 4.33(dd, 1H, J ϭ 11.0 Hz, 9.5 Hz): H-16a,
5.04(s, 2H, C6H5CH2), 6.72(d, 1H, J ϭ 2.6 Hz, H-4),
6.78(dd, 1H, J ϭ 8.6 Hz, 2.6 Hz, H-2), 7.22(d, 1H, J ϭ 8.6
Hz, H-1), 7.32–7.44(m, 5H, C6H5). 13C-NMR (100 MHz,
CDCl3): ␦ ppm 17.3(C-18), 21.1(CH3CO), 26.0, 27.9(2C),
29.8, 31.1, 38.9, 40.4, 43.5, 46.3(C-13), 46.8, 64.8(C-16Ј),
69.9(C6H5CH2), 79.3(C-17), 112.3(C-2), 114.8(C-4),
126.3(C-1), 127.4(2C, C-2Ј and C-6Ј), 127.8(C-4Ј),
128.5(2C, C-3Ј and C-5Ј), 133.0(C-10), 137.3(C-1Ј),
138.0(C-5), 156.7(C-3), 171.5(CH3CO). Further elution
gave 4c (5.34 g, 61%).
2.8. 16␣-Acetoxymethyl-3-benzyloxyestra-1,3,5(10)-trien-
17-one (7)
2.10. 16-Acetoxymethyl-3-benzyloxyestra-1,3,5(10)-trien-
17␣-acetate (6b)
Compound 4c (10.8 g, 0.025 mol) was dissolved in
acetone (200 ml) and Jones reagent was added dropwise
during cooling with ice. The reaction mixture was poured
onto ice (500 g) and extracted with chloroform. The chlo-
roform solution was washed with water, dried and evapo-
rated. The substance obtained was crystallized from ace-
tone/light petroleum to give pure 7 (10.2 g, 94%). Mp
Compound 6c (2.17 g, 0.005 mol) was dissolved in a
mixture of pyridine (5 ml) and acetic anhydride (5 ml) and
the solution was allowed to stand at room temperature for
12 h. The mixture was then diluted with water and the
precipitate was collected by filtration and recrystallized
from methanol to obtain 6b (2.30 g, 96%). Mp 106–108°C,
20
111–113°C, Rf ϭ 0.70 (ss B); [␣]D ϩ 98. (Found: C,
20
77.64; H, 7.28. C28H32O4 requires: C, 77.75; H, 7.46%).
1H-NMR (400 MHz, CDCl3): ␦ ppm 0.97(s, 3H, H-18),
2.05(s, 3H, CH3CO), 2.89(m, 2H, H-6), 4.21(dd, 1H, J ϭ
11.0 Hz, 6.8 Hz) and 4.34(dd, 1H, J ϭ 11.0 Hz, 4.5 Hz):
H-16a, 5.04(s, 2H, C6H5CH2), 6.74(d, 1H, J ϭ 2.6 Hz,
H-4), 6.79(dd, 1H, J ϭ 8.6 Hz, 2.6 Hz, H-2), 7.20(d, 1H,
J ϭ 8.6 Hz, H-1), 7.32–7.44(m, 5H, C6H5). 13C-NMR (100
MHz, CDCl3): ␦ ppm 14.4(C-18), 20.9(CH3CO), 25.7 (2C),
26.5, 29.5, 31.4, 38.2, 44.0, 44.1, 48.5(C-13), 48.6, 64.1(C-
16Ј), 69.9(C6H5CH2), 112.3(C-2), 114.9(C-4), 126.3(C-1),
127.4(2C, C-2Ј and C-6Ј), 127.8(C-4Ј), 128.5(2C, C-3Ј and
C-5Ј), 132.1(C-10), 137.2(C-1Ј), 137.7(C-5), 156.8(C-3),
170.9(CH3CO), 218.2(C-17).
Rf ϭ 0.85 (ss A); [␣]D ϩ 67. (Found: C, 75.58; H, 7.72.
C30H36O5 requires: C, 75.60; H, 7.61%). NMR (400 MHz,
CDCl3): ␦ ppm 0.86(s, 3H, H-18), 2.02 and 2.08(two s, 6H,
two CH3CO), 2.85(m, 2H, H-6), 4.05(dd, 1H, J ϭ 10.6 Hz,
7.1 Hz) and 4.10(dd, 1H, J ϭ 10.6 Hz, 9.0 Hz): H-16a,
5.03(s, 2H, C6H5CH2), 5.10(d, 1H, J ϭ 5.5 Hz, H-17),
6.72(d, 1H, J ϭ 2.5 Hz, H-4), 6.78(dd, 1H, J ϭ 8.6 Hz, 2.5
Hz, H-2), 7.19(d, 1H, J ϭ 8.6 Hz, H-1), 7.31–7.44(m, 5H,
C6H5). 13C-NMR (100 MHz, CDCl3): ␦ ppm 16.7(C-18),
20.9 and 21.0(2C, CH3CO), 25.8, 27.9, 28.1, 29.8, 31.6,
38.5, 38.9, 43.4, 46.0(C-13), 48.1, 64.0(C-16aЈ),
69.9(C6H5CH2), 80.7(C-17), 112.3(C-2), 114.8(C-4),
126.3(C-1), 127.4(2C, C-2Ј and C-6Ј), 127.8(C-4Ј),

P. Tapolcsa´nyi et al. / Steroids 67 (2002) 371–377
375
128.5(2C, C-3Ј and C-5Ј), 132.6(C-10), 137.3(C-1Ј),
137.8(C-5), 156.8(C-3), 170.9(2C, CH3CO).
3.18(dd, 1H, J ϭ 7.6 Hz, 5.1 Hz, H-17), 3.32(m, 1H) and
3.54(m, 1H): H-16a, 4.39(t, 1H, J ϭ 5.0 Hz, 4.6 Hz,
16-CH2OH), 4.43(d, 1H, J ϭ 5.1 Hz, 17-OH), 6.43(d, 1H,
J ϭ 2.5 Hz, H-4), 6.50(dd, 1H, J ϭ 8.4 Hz, 2.5 Hz, H-2),
7.03(d, 1H, J ϭ 8.4 Hz, H-1), 8.94(s, 1H, 3-OH). 6d (420
mg, 92%). Mp 255–258°C, Rf ϭ 0.45. (Found: C, 75.40;
2.11. 16␣-Hydroxymethyl-3-benzyloxyestra-1,3,5(10)-
trien-17␣-ol (6a)
1
Compound 6b (0.47 g, 0.001 mol) was dissolved in
methanol (25 ml) containing NaOCH3 (54 mg, 1 mmol) and
the solution was allowed to stand for 24 h. It was then
diluted with water, and the white precipitate was collected
by filtration and recrystallized from ethanol to obtain 6a
(0.36 g, 91%). Mp 139–141°C, Rf ϭ 0.45 (ss B); [␣]D20 ϩ
81. (Found: C, 79.68; H, 8.12. C26H32O3 requires: C, 79.56;
H, 8.22%). 1H-NMR (400 MHz, CDCl3): ␦ ppm 0.78(s, 3H,
H-18), 2.85(m, 2H, H-6), 3.74(dd, 1H, J ϭ 11.0 Hz, 8.3 Hz)
and 3.89(dd, 1H, J ϭ 11.0 Hz, 4.5 Hz): H-16a, 3.94(d, 1H,
J ϭ 5.5 Hz, H-17), 5.04(s, 2H, C6H5CH2), 6.72(d, 1H, J ϭ
2.6 Hz, H-4), 6.78(dd, 1H, J ϭ 8.6 Hz, 2.6 Hz, H-2), 7.22(d,
1H, J ϭ 8.6 Hz, H-1), 7.32–7.44(m, 5H, C6H5). 13C-NMR
(100 MHz, CDCl3): ␦ ppm 17.3(C-18), 26.0, 27.5, 28.0,
29.9, 31.3, 39.1, 41.8, 43.5, 46.3(C-13), 47.6, 63.6(C-16Ј),
69.9(C6H5CH2), 81.9(C-17), 112.3(C-2), 114.8(C-4), 126.3(C-
1), 127.4(2C, C-2Ј and C-6Ј), 127.8(C-4Ј), 128.5(2C, C-3Ј and
C-5Ј), 132.9(C-10), 137.3(C-1Ј), 138.0(C-5), 156.7(C-3).
H, 8.93. C19H26O3 requires: C, 75.46; H, 8.67%). H-
NMR (400 MHz, DMSO-d6): ␦ ppm 0.67(s, 3H, H-18),
2.68(m, 2H, H-6), 3.36 and 3.53(two m, 2H, H-16a),
3.60(dd, 1H, J ϭ 5.6 Hz, 5.1 Hz, H-17), 4.14(t, 1H, J ϭ 5.3
Hz, 16-CH2OH), 4.30(d, 1H, J ϭ 5.1 Hz, 17-OH), 6.41(d, 1H,
J ϭ 2.5 Hz, H-4), 6.48(dd, 1H, J ϭ 8.6 Hz, 2.5 Hz, H-2),
7.03(d, 1H, J ϭ 8.6 Hz, H-1), 8.92(s, 1H, 3-OH).
2.13. Receptor binding assay
The new compounds were evaluated for their ability to
competitively inhibit the binding of [3H]estra-1,3,5(10)-
triene-3,17␤-diol (155 Ci mmolϪ1
) to estrogen, of
[3H]ORG-2058 (33 Ci mmolϪ1) to progesterone and of
[3H]dihydrotestosterone (112 Ci mmolϪ1) to androgen re-
ceptors in cytosol prepared from rabbit uteri (estrogen and
progesterone receptors) and rat prostate (androgen recep-
tors). All of the tritiated ligands were purchased from Am-
ersham, UK. All subsequent steps were carried out at 4°C.
The minced tissues were homogenized in 6–10 volumes of
10 mM Tris-HCl buffer (pH 7.4) containing 1.5 mM EDTA
(ethylenediaminetetraacetic acid), 0.5 mM DTT (dithiothre-
itol (DTT)), 10 mM Na2MoO4 (estrogen and progesterone
receptors), or in 10 mM Tris-HCl buffer (pH: 7.4) contain-
ing 0.25 M sucrose and 15 mM Na2MoO4 (androgen recep-
tors) [16]. The homogenate was centrifuged at 105 000ϫ g
for 60 min and the final supernatant (cytosol) was used for
competitive binding. Aliquots of 100 109 ml cytosol were
incubated overnight at 4°C with tritiated steroids in the
presence or absence of synthetic derivatives. Unbound ste-
roids were removed by treatment with dextran-coated char-
coal (10% charcoal-Norit A and 0.1% dextran T-70 in the
assay buffer). After incubation for 10 min at 4°C, the sam-
ples were centrifuged at 1500ϫ g for 10 min, and the
radioactivity of the supernatant was determined in a Wallac
1409 scintillation counter. The percentage of radioligand
bound in the presence of competitor as compared with that
bound in its absence was plotted 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 (inhibition constant) were calculated
with GraphPad 2.0 software. All assays were carried out at
least three times in duplicates.
2.12. 16-Hydroxymethylestra-1,3,5(10)-triene-3,17-diol
isomers (3d, 4d, 5c and 6d)
2.12.1. (General procedure)
Compound 3a, 4a, 5a or 6a (590 mg, 1.5 mmol) was
dissolved in ethanol (50 ml) in an autoclave, and Pd/C
(50 mg) was added to the solution, which was then stirred
at room temperature for 3 h at 20 bar H2 pressure. The
reaction mixture was filtered, the filtrate was evaporated
and the product obtained was crystallized from a mixture
of ethanol/water. 3d (430 mg, 94%). Mp 279–282°C, Rf
ϭ 0.45 (ss C). (Found: C, 75.38; H, 8.72. C19H26O3
requires: C, 75.46; H, 8.67%). 1H-NMR (400 MHz,
DMSO-d6): ␦ ppm 0.69(s, 3H, H-18), 2.70(m, 2H, H-6),
3.30(m, 1H, H-16a), 3.67(m, 2H, H-16a and H-17),
4.13(dd, 1H, J ϭ 5.9 Hz, 4.6 Hz, 16-CH2OH), 4.59(d,
1H, J ϭ 4.4 Hz, 17-OH), 6.43(d, 1H, J ϭ 2.4 Hz, H-4),
6.50(dd, 1H, J ϭ 8.4 Hz, 2.4 Hz, H-2), 7.03(d, 1H, J ϭ
8.4 Hz, H-1), 8.97(s, 1H, 3-OH). 4d (425 mg, 93%). Mp
297–298°C, ([13] 277°C dec.), Rf ϭ 0.45 (ss C). (Found:
C, 75.53; H, 8.58. C19H26O3 requires: 75.46; H, 8.67%).
1H-NMR (400 MHz, DMSO-d6): ␦ ppm 0.71(s, 3H,
H-18), 2.70(m, 2H, H-6), 3.18(dd, 1H, J ϭ 7.6 Hz, 5.1
Hz, H-17), 3.32(m, 1H) and 3.54(m, 1H): H-16a, 4.39(t,
1H, J ϭ 5.0 Hz, 4.6 Hz, 16-CH2OH), 4.43(d, 1H, J ϭ 5.1
Hz, 17-OH), 6.43(d, 1H, J ϭ 2.5 Hz, H-4), 6.50(dd, 1H,
J ϭ 8.4 Hz, 2.5 Hz, H-2), 7.03(d, 1H, J ϭ 8.4 Hz, H-1),
8.94(s, 1H, 3-OH). 5c (410 mg, 90%). Mp 235–238°C, Rf
ϭ 0.40 (ss C). (Found: C, 75.38; H, 8.50. C19H26O3
requires: C, 75.46; H, 8.67%). 1H-NMR (400 MHz,
DMSO-d6): ␦ ppm 0.71(s, 3H, H-18), 2.70(m, 2H, H-6),
3. Results and discussion
3.1. Synthetic studies
The hydroxy group of estrone (1a) was protected with a
benzyl group, which is easily removed by hydrogenolysis at

376
P. Tapolcsa´nyi et al. / Steroids 67 (2002) 371–377
Table 1
the end of the reaction sequence. Treatment of 3-benzyl-
oxyestra-1,3,5(10)-trien-17-one (1b) with NaOMe and ethyl
formate gave 2a in a yield of 94%. The reduction of 2a with
KBH4 in ethanol led to 3a, 4a and 5a in a ratio of 50:45:5
in 92% yield.
Inhibition constant (Ki) values of estra-1,3,5(10)-triene-3,17␤-diol
(reference compound) and newly synthesized estradiol derivatives (3d,
4d, 5c and 6d) on uterine estrogen (ER), androgen (AR) and
progesterone (PR) receptors. The following radioligands were used: 5
nM [3H]estra-1,3,5(10)-triene-3,17␤-diol (ER), 5 nM [3H]ORG 2058
(PR) and 5 nM [3H]dihydrotestosterone (AR). The relative binding
affinity values on the ER were calculated according to the following
equation: RBA ϭ Kiestradiol/Ki*
The simultaneous development of the two chiral centers
permits formation of the four isomers 3a, 4a, 5a and 6a.
However, only three isomers (3a, 4a and 5a) were detected
on thin-layer chromatography. The mixture of the isomers
was reacted with acetone in dichloromethane in the pres-
ence of a catalytic amount of p-toluenesulfonic acid. 3a was
converted to the corresponding cyclic acetonide derivative
3c, whereas the mixture of 4a and 5a could easily be
separated by flash chromatography on Al2O3. The mixture
of 4a and 5a was dissolved in acetone, and 4a crystallized
as a hard crystalline product. 5a was separated from the
mother liquor by flash chromatography on silica gel with
tert-butyl methyl ether. Since reduction of the 17-ketone
function in steroids normally yields a 17␤-hydroxy group
(with a few exceptions [14]), isomers 3a and 4a, present in
larger quantities, are expected to have different configura-
tions at C-16. In the NMR spectrum of the diacetate 3b, the
doublet at 4.90 ppm, due to coupling of the protons on C-16
and C-17 with a coupling constant of 10.1 Hz, confirms the
␤,␤ arrangement of the substituents on C-16 and C-17. The
cis arrangement is supported by the selective acetonide
building. The ␣,␤ arrangement of the C-16 and C-17 sub-
stituents in 4b is indicated by the lower coupling constant
(8.1 Hz) of the doublet at 4.75 ppm. The debenzylated
compound 4d is known in the literature [13]. The ␤,␣
arrangement of the C-16 and C-17 substituents in 5b is
indicated by the lowest coupling constant (2.0 Hz) of the
doublet appearing at 4.71 ppm; these values are in good
agreement with earlier observations on 16-hydroxymethyl-
3-methoxyestra-1,3,5(10)-trien-17-ol isomers [15].
Ki [nM]
Compound ER
AR
PR
RBA
100
Estradiol
1.19 Ϯ 0.53
Ͼ100 000
1532 Ϯ 102
3398 Ϯ 249.5
Ͼ100 000
Ͼ100 000
6242 Ϯ 567
3d
4d
5c
6d
82.32 Ϯ 6.53
215.3 Ϯ 47.6
814.4 Ϯ 79.5
213.3 Ϯ 35.7
660.9 Ϯ 58.9
834.2 Ϯ 127.3
Ͼ100 000
1.44
0.62
0.14
0.44
Ͼ100 000
by hydrogenolysis in the presence of Pd/C to afford the
triols 3d, 4d, 5c and 6d, which are suitable for receptor-
binding examinations.
3.2. Radioligand-binding assays
The binding affinities of estra-1,3,5(10)-triene-3,17␤-
diol and its derivatives 3d, 4d, 5c and 6d for the estrogen,
progesterone and androgen receptors were determined by
radioligand-binding assay. Specifically bound [3H]estra-
1,3,5(10)-triene-3,17␤-diol was readily displaced from the
rabbit uterine estrogen receptor by estra-1,3,5(10)-triene-
3,17␤-diol and 3d.
The remaining three derivatives (4d, 5c and 6d) exhibit
lower affinities for the estrogen receptor, but only the Ki
value of 5c is significantly lower than the others. The inhi-
bition constant (Ki) values and relative binding affinities
(RBA) for the estrogen receptor are listed in Table 1. 16␤-
Hydroxymethylestra-1,3,5(10)-triene-3,17␤-diol (3d) is the
most potent estradiol derivative in this series (Ki: 82.32
nM), but its affinity is about two orders of magnitude lower
than that measured for estradiol. Fevig et al. reported that
16␣-hydroxymethylestra-1,3,5(10)-triene-3,17␤-diol (4d)
binds to the estrogen receptor with low affinity as compared
to estra-1,3,5(10)-triene-3,17␤-diol and has an RBA value
of 2.4 [13]. Our study indicated that this compound has a Ki
value of 191.7 nM and an RBA of 0.62 on rabbit uterine
cytosol, relative to estra-1,3,5(10)-triene-3,17␤-diol. The
differences between the RBA values stem from the fact that
the two groups used different uterine cytosol from different
species (rat or lamb [13] and rabbit in the present work).
The Ki values of 16␣-hydroxymethylestra-1,3,5(10)-triene-
3,17␣-diol (6d) and 16␤-hydroxymethylestra-1,3,5(10)-
triene-3,17␣-diol (5c) are higher than 200 nM (268.2 nM
and 863.3 nM, respectively). The 16-hydroxymethylestra-
1,3,5(10)-triene-3,17-diol isomers 3d, 4d, 5c and 6d possess
less affinity toward the estrogen receptor than estra-
1,3,5(10)-triene-3,17␤-diol, because this region of the re-
ceptor tolerates groups of moderate size and polarity [13,
Since availability of the complete series of isomers
would permit a number of interesting comparative exami-
nations, we wished to prepare the unknown fourth isomer
6a, which contains functional groups with the configuration
16␣, 17␣.
The primary hydroxy group in 16␣-hydroxymethyl-3-
benzyloxyestra-1,3(10)-trien-17␤-ol (4a) was acetylated
with acetic anhydride, and the product 4c was oxidized by
the Jones method to 16␣-acetoxymethyl-3-benzyloxyestra-
1,3(10)-trien-17-one (7). Reduction of 7 with lithium tri-
tert-butoxyaluminum hydride afforded the isomers 16␣-
acetoxymethyl-3-benzyloxyestra-1,3,5(10)-trien-17␤-ol
(4c) and 16␣-acetoxymethyl-3-benzyloxyestra-1,3,5(10)-
trien-17␣-ol (6c) in a ratio of 4:1. These isomers (4c and 6c)
are separable by flash chromatography. The repeated Jones
oxidation of 4c yields 7, which returns to the reduction
process. The deacetylation of 4c by the Zemplen method
produces the 16␣-hydroxymethyl-3-benzyloxyestra-
1,3,5(10)-trien-17␣-ol (4a).
The four 16-hydroxymethyl-3-benzyloxyestra-1,3,5(10)-
triene-17-ol isomers (3a, 4a, 5a and 6a) were deprotected

P. Tapolcsa´nyi et al. / Steroids 67 (2002) 371–377
377
17]. The tested compounds inhibit [3H]ORG 2058 binding
to the progesterone receptor only in the micromolar range.
The finding, that the tested compounds bind to the androgen
and progesterone receptors with very low affinity is surely
because these compounds contain a phenolic function and
not a 3-keto function, which forms hydrogen bonds with
Gln711 and Arg752 in the case of the androgen receptor
[18,19] and with Gln725 and Arg766 in the case of the
progesterone receptor [19,20]. It is interesting that 3d and
4d bind to the androgen receptor with affinities (Ki: 660.9
nM and 834.2 nM, respectively) more than 150 times higher
than those for the other compounds (Ki Ͼ 100 109 mM). In
this case, the 17␤-hydroxy group in estra-1,3,5(10)-triene-
3,17␤-diol, 3d and 4d forms hydrogen bonds with the side-
chain of Asn705 and Thr877 while the 17␣-hydroxy group in
5c and 6d can not. Moreover, the 16-hydroxymethyl group
in 3d and 4d can form another hydrogen bond, which
increases the affinity of these compounds toward the andro-
gen receptor, while estra-1,3,5(10)-triene-3,17␤-diol, which
lacks a 16-hydroxymethyl group possesses a much lower
affinity than those of 3d and 4d, but significantly higher
than those of 5c and 6d, which contain a 17␣-hydroxy
group.
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compared with that of the reference molecule estradiol. The
affinities of the tested agents for the androgen and progesterone
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the prepared 16-hydroxymethyl-3,17-estradiol isomers are
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1
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Acknowledgments
Financial support by the Hungarian National Research
Foundation (OTKA grant T 032265) and by the Hungarian
Board of Education (FKFP grant 0110/2000) is gratefully ac-
knowledged.
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