Configurational analysis and relative binding affinities of 16-methyl-5α-androstane derivatives

Article abstract of DOI:10.1016/S0039-128X(01)00113-1

The four possible isomers 16β-hydroxymethyl-5α-androstane-3β,17β-diol 1, 16α-hydroxymethyl-5α-androstane-3β,17β-diol 2, 16β-hydroxymethyl-5α-androstane-3β,17α-diol 3 and 16α-hydroxymethyl-5α-androstane-3β,17α-diol 4 with proven configuration were converte

Full text of DOI:10.1016/S0039-128X(01)00113-1

Steroids 66 (2001) 833–843
Configurational analysis and relative binding affinities of
16-methyl-5␣-androstane derivatives
Pa´l Tapolcsa´nyia, Ja´nos Wo¨lflinga, Istva´n To´thb, Miha´ly Sze´csib, Pe´ter Forgo´a,
Gyula Schneidera,*
aDepartment of Organic Chemistry, University of Szeged, Do´m te´r 8, H-6720 Szeged, Hungary
bEndocrine Unit and Research Laboratory, University of Szeged, Kora´nyi fasor 8, H-6720 Szeged, Hungary
Received 20 November 2000; received in revised form 25 January 2001; accepted 8 February 2001
Abstract
The four possible isomers 16␤-hydroxymethyl-5␣-androstane-3␤,17␤-diol 1, 16␣-hydroxymethyl-5␣-androstane-3␤,17␤-diol 2, 16␤-
hydroxymethyl-5␣-androstane-3␤,17␣-diol 3 and 16␣-hydroxymethyl-5␣-androstane-3␤,17␣-diol 4 with proven configuration were con-
verted into the corresponding 16␤-methyl-5␣-androstane-3␤,17␤-diol 5, 16␣-methyl-5␣-androstane-3␤,17␤-diol 6, 16␤-methyl-5␣-andro-
stane-3␤,17␣-diol 7, 16␣-methyl-5␣-androstane-3␤,17␣-diol 8, furthermore into the 16␤-methyl-17␤-hydroxy-5␣-androstane-3-one 13,
16␣-methyl-17␤-hydroxy-5␣-androstan-3-one 14, 16␤-methyl-17␣-hydroxy-5␣-androstan-3-one 15 and 16␣-methyl-17␣-hydroxy-5␣-an-
drostan-3-one 16. The steric structures of the resulting epimers were determined by means of 1H-, and 13C-NMR spectroscopy. In this way,
comparison was possible with the C-16 epimers 5, 6 and 13, 14 prepared earlier by a different route, and the series of isomers could be
completed with the steric structures of 16␤-methyl-17␣-hydroxy-5␣-androstan-3␤-ol 7 and 16␣-methyl-17␣-hydroxy-5␣ 8 and with their
3-keto derivatives 15 and 16. The relative binding affinities of the 16-methyl-5␣-androstane-3␤,17-diols 5, 6, 7, 8 and 17-hydroxy-16-
methyl-5␣-androstan-3-ones 13, 14, 15, 16 were studied. The introduction of a 16-methyl substituent into 5␣-androstane molecules
substantially decreases the binding affinity to the androgen receptor and 16␣-methyl derivatives were always bound more weakly than the
16␤-methyl isomers. © 2001 Elsevier Science Inc. All rights reserved.
Keywords: 16-Methyl steroids; Configurational analysis; Androgen receptor binding
1. Introduction
substitution of this group follows unequivocally from the
method of synthesis, but the literature reveals uncertainties
as concerns the steric situation of the alkyl group.
The presence of a C-16 alkyl group in steroids often
enhances, sometimes significantly, the biological properties
of the parent compound. In the corticoid series, the intro-
duction of a C-16 methyl group enhances the activity of the
parent compound [1,2]. In other steroids, such as andro-
stane, estrane and the aldosterone antagonists, alkyl substi-
tution at a similar site causes a reduction in hormone activ-
ity [3,4]. This observation assumed importance when the
antihormone effects of 16-alkylestrene [5,6,7] and 16,16-
dimethyl-4-estren-3-one were recognized [8]. The literature
provides a large number of methods for the introduction of
a 16-methyl group onto the sterane skeleton. The site of
Ruggieri et al. [9,10] prepared 16␣- and 16␤-methyl-3␤-
hydroxypregn-5-en-20-one with confirmed configuration by
effecting a Grignard reaction between 3␤-hydroxypregna-
5,16-dien-20-one and methyl iodide, and 1,3-dipolar cy-
cloaddition with diazomethane with subsequent decompo-
sition. Side-chain isomerization of 16␤-methyl-3␤-
hydroxypregn-5-en-20-one in alkaline medium yielded
16␤-methyl-17-iso-3␤-hydroxypregn-5-en-20-one. Baeyer-
Villiger oxidation of pregnane derivatives substituted in
different manners yielded three of the four possible isomers
of 16-methylandrost-5-ene-3␤,17-diol. At the same time,
Beckmann rearrangement of 16␣- and 16␤-methyl-3␤-hy-
droxypregn-5-en-20-one resulted in 16␣- and 16␤-methyl-
3␤-hydroxyandrost-5-en-17-one, which were hydrogenated
on Pd-CaCO3 to furnish the two 16-methyl-3␤-hydroxy-5␣-
* Corresponding author. Tel.: ϩ36-62-544-276; fax: ϩ36-62-544-200.
E-mail: schneider@chem.u-szeged.hu (G. Schneider).
0039-128X/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(01)00113-1

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P. Tapolcsa´nyi et al. / Steroids 66 (2001) 833–843
F254; layer thickness 0.2 mm (Merck); solvent system (ss):
(A) chloroform, (B) ethyl acetate/chloroform (5:95 v/v), (C)
ethyl acetate/chloroform (10:90 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 chromatogra-
phy: silica gel 60, 40–63 ␮m. Column chromatography:
Al2O3 (standardized according to Brockmann) with activity
1
of III-IV. H-NMR spectra were recorded with a Bruker
DRX-400 instrument at 400 MHz in CDCl3 solution (if not
otherwise given), using Me4Si as internal standard. 13C-
NMR spectra were recorded with the same instrument at
100 MHz under the same conditions.
2.1. 3␤,17␣-Diacetoxy-16␤-hydroxymethyl-5␣-androstane
(3a) and 3␤-acetoxy-16␣-hydroxymethyl-5␣-androstan-
17␣-ol (4a)
Scheme 1.
Alkaline alumina (25 g) was added to a solution of 16␤-
or 16␣-acetoxymethyl-5␣-androstane-3␤,17␣-diacetate (3c
or 4c) [12] (448 mg, 1 mmol) dissolved in dichloromethane
(5 ml) and the solvent was evaporated off in vacuo. The
air-dried material, in a small beaker, was placed inside a
microwave oven. After 6 min (in the case of 3c) or 10 min
(in the case of 4c) of irradiation at 90 W, the product was
extracted into chloroform (5 ϫ 20 ml). Following evapora-
tion, the residue was subjected to chromatographic separa-
tion on silica gel. During the column chromatography of the
3c mixture, ethyl acetate/chloroform (10:90) first eluted 3c,
and then 3a (244 mg, 60%). Mp of 3a 154–156°C, Rf ϭ
androstan-17-ones 11a, 12a. In the reduction with NaBH4,
their acetylated derivatives afforded the 16-methyl-3␤-ace-
toxy-5␣-androstan-17-ol isomers 5b, 6b [9].
The above synthesis of the 16-methyl-3␤-acetoxy-17␤-
hydroxy-5␣-androstane epimers 5b, 6b were achieved by
the reduction at C-17 of 16-methyl-17-oxo steroids 11b,
12b. Neef et al. [11] have pointed out that the 16-methyl-
17-ketosteroids undergo interconversion in equilibrium re-
actions under both acidic and alkaline conditions. The ste-
reochemical homogeneity of 17-hydroxy-16-methyl epimers
obtained by the reduction of 16-methyl-17-oxo steroids is
therefore strongly in doubt because of the possible equilibrium
isomerization of the starting compounds (Scheme 1).
The present work aimed at the preparation of the four
possible isomers of 16-methyl-5␣-androstane-3␤,17-diol
5a, 6a, 7a, 8a and 16-methyl-17-hydroxy-5␣-androstan-3-
one 13a, 14a, 15a, 16a with confirmed configurations, in-
dependently from the reaction path described in the litera-
ture. This would allow a configurational comparison of the
isomers 5a, 6a and 13a, 14a prepared earlier by other
methods, and also completion of the isomer series with
compounds 7a, 8a and 15a, 16a. In possession of the dif-
ferent isomers, it seemed of interest to study their binding to
the androgen receptor, the specific binding protein in target
cells mediating androgen steroid action (Schemes 2 and 3).
20
0.30 (ss C); [␣]D Ϫ42 (c 1 in chloroform). (Found: C,
70.82; H, 9.50. C24H38O5 requires C, 70.90; H, 9.42%);
1H-NMR ␦ ppm 0.70(m, 1H, 9-H), 0.76(s, 3H, 18-H),
0.80(s, 3H, 19-H), 2.00(s, 3H) and 2.05(s, 3H): 3- and
17-CH3CO, 3.60(m, 2H, 16-CH2), 4.44(d, 1H, Jϭ1.6 Hz,
17-H), 4.68(m, 1H, 3-H). 13C-NMR ␦ ppm 12.2(C-19),
17.5(C-18), 20.4(C-11), 21.3 and 21.4(2C, CH3CO), 27.4,
28.4, 29.5, 32.1, 32.2, 34.0, 35.2(C-8), 35.6, 36.8, 44.3(C-
13), 44.6(C-5), 50.4 and 50.5(2C, C-14 and C-16), 53.8(C-
9), 66.1(C-16), 73.6(C-3), 84.8(C-17), 170.7 and 172.1(2C,
CH3CO).
Chromatography with ethyl acetate/chloroform (10:90)
of the 4c,4d mixture resulted first in 4c, and then in 4d (40
20
mg, 10%). Mp of 4d 189–192°C, Rf ϭ 0.35 (ss C); [␣]D
Ϫ4 (c 1 chloroform). (Found: C, 70.97; H, 9.45. C24H38O5
requires C, 70.90; H, 9.42%); 1H-NMR ␦ ppm 0.68(m, 1H,
9-H), 0.82(s, 3H) and 0.83(s, 3H):18-H and 19-H, 2.02(s,
3H, 3-CH3CO), 2.12(s, 3H, 17-CH3CO), 2.60(m, 1H),
3.52(m, 2H, 16-CH2), 4.68(m, 1H, 3-H), 4.96(d, 1H, Jϭ5.5
Hz, 17-H). 13C-NMR ␦ ppm 12.9(C-19), 17.7(C-18),
21.1(C-11), 21.7 and 22.1(2C, CH3CO), 28.1, 28.6, 29.1,
32.2, 32.8, 34.6, 36.2(C-10), 36.3(C-8), 37.4, 43.5(C-16),
45.3(C-5), 46.4(C-13), 49.9(C-14), 54.6(C-9), 63.3(C-16Ј),
74.3(C-3), 83.3(C-17), 171.4 and 172.3(2C, CH3CO). Con-
tinued elution with ethyl acetate/chloroform (30:70) gave 4a
2. Experimental
Melting points (mps) were determined with a Kofler
hot-stage apparatus and are uncorrected. Specific rotations
were measured with a POLAMAT-A (Zeiss-Jena) polarim-
eter in chloroform, methanol or acetic acid solutions (c 1)
and are given in units of 10Ϫ1 deg cm2 gϪ1. Elemental
analyses were performed with a Perkin-Elmer CHN anal-
yser model 2400. Thin-layer chromatography: silica gel 60
20
(183 mg, 51%). Mp 203–205°C, Rf ϭ 0.20 (ss C); [␣]D
ϩ6 (c 1 chloroform). (Found: C, 72.55; H 9.82. C22H36H4

P. Tapolcsa´nyi et al. / Steroids 66 (2001) 833–843
835
Scheme 2.

836
P. Tapolcsa´nyi et al. / Steroids 66 (2001) 833–843
Scheme 3.
requires C, 72.49; H, 9.95%); 1H-NMR ␦ ppm 0.66(m, 1H,
9-H), 0.70(s, 3H, 18-H), 0.81(s, 3H, 19-H), 2.00(s, 3H,
CH3CO), 2.44(m, 1H), 3.66(m, 1H) and 3.80(m, 1H):16-
CH2, 3.83(d, 1H, Jϭ5.7 Hz, 17-H), 4.68(m, 1H, 3-H).
13C-NMR ␦ ppm 12.2(C-19), 17.3(C-18), 20.5(C-11),
21.5(CH3CO), 27.4, 27.8, 28.5, 31.2, 32.2, 34.0, 35.5,
35.7(C-8), 36.8, 41.7(C-16), 44.6(C-5), 46.1(C-13), 48.4(C-
14), 53.9(C-9), 63.6(C-16Ј), 73.8(C-3), 81.7(C-17),
170.9(CH3CO).
2.2. 16␤-Toluene-p-sulfonyloxymethyl-5␣-androstane-
3␤,17␣-diacetate (3b)
Compound 3a (2.03 g, 0.005 mol) was dissolved in
pyridine (20 ml) and a solution of toluene-p-sulfonyl chlo-
ride (1.9 g, 0.01 mol) dissolved in pyridine (10 ml) was
added dropwise. The reaction mixture was allowed to stand
at room temperature for 24 h. It was then poured onto a
mixture of sulfuric acid (12 ml) and ice (200 g), and the

P. Tapolcsa´nyi et al. / Steroids 66 (2001) 833–843
837
precipitate was filtered off, washed and dried. The product
was subjected to chromatographic separation on silica gel
with chloroform as eluent, and crystallized from methanol.
stirred at 70°C for 6 h. Aqueous ethanol was added to the
cooled reaction mixture, which was then acidified with
dilute hydrochloric acid and diluted with an equal volume of
water, and the organic fraction was evaporated under re-
duced pressure. The residual precipitate was filtered off,
dissolved in chloroform and subjected to chromatographic
separation on alumina with chloroform as eluent. The sub-
stance obtained was crystallized from a mixture of chloro-
form-petroleum ether.
20
3b (2.45 g, 89%) mp 147–149°C, Rf ϭ 0.80 (ss C); [␣]D
ϩ4 (c 1 chloroform). (Found: C, 66.32; H, 8.02. C31H44O7S
requires C, 66.40; H, 7.91%); 1H-NMR ␦ ppm 0.69(m, 1H,
9-H), 0.70(s, 3H, 18-H), 0.81(s, 3H, 19-H), 2.01(s, 3H) and
2.02(s, 3H):3- and 17-CH3CO), 2.15(m, 1H), 2.45(s, 3H,
Ts-CH3), 4.09(m, 1H) and 4.18(s, 1H):16-CH2, 4.43(d, 1H,
Jϭ2.2 Hz, 17-H), 4.68(m, 1H, 3-H), 7.35(d, 2H, Jϭ8.1 Hz,
3Ј- and 5Ј-H), 7.79(d, 2H, Jϭ8.1 Hz, 2Ј- and 6Ј-H). 13C-
NMR ␦ ppm 12.9(C-19) and 17.8(C-18), 21.1(C-11), 21.7
and 22.1(2C, CH3CO), 22.4(Ts-CH3), 28.1, 29.1, 30.2,
32.7(2C), 34.6, 35.8(C-8), 36.2(C-10), 37.4, 45.0(C-13),
45.2(C-5), 46.5(C-16), 51.6(C-14), 54.4(C-9), 73.0(C-16Ј),
74.2(C-3), 83.7(C-17), 128.6(2C, C-2Ј and C-6Ј), 130.5(2C,
C-3Ј and C-5Ј), 134.3(C-1Ј), 145.3(C-4Ј), 171.2 and
171.4(2C, CH3CO).
5a (1.20 g, 78%) mp 198–199°C, Rf ϭ 0.15 (ss B);
20
[␣]D ϩ13 (c 1 chloroform) ([9] mp 194–196°C, [␣]D
ϩ10.9). (Found: C, 78.42; H, 11.25. C20H34O2 requires: C,
78.38; H, 11.18%); 1H-NMR ␦ ppm 0.66(m, 1H, 9-H),
0.73(s, 3H, 18-H), 0.81(s, 3H, 19-H), 0.99(d, 3H, Jϭ7.35
Hz, 16-CH3), 3.54–3.65(2H, m, 1H, 3-H and d, 1H, Jϭ10.4
Hz, 17-H), 3.60(d, 1H, Jϭ10.4 Hz, 17-H).
6a (1.05 g, 68%) mp 182–184°C, Rf ϭ 0.10 (ss B);
20
[␣]D Ϫ12 (c 1 chloroform) ([9] mp 179–181°C, [␣]D
Ϫ15). (Found: C, 78.30; H, 11.30. C20H34O2 requires: C,
78.38; H, 11.18%); 1H-NMR ␦ ppm 0.64(m, 1H, 9-H),
0.75(s, 3H, 18-H), 0.81(s, 3H, 19-H), 1.09(d, 3H, Jϭ7.0 Hz,
16-CH3), 3.10(d, 1H, Jϭ7.5 Hz, 17-H), 3.6(m, 1H, 3-H).
7a (0.98 g, 64%) mp 243–245°C, Rf ϭ 0.10 (ss B);
2.3. 3␤-Acetoxy-16␣-toluene-p-sulfonyloxymethyl-5␣-
androstan-17␣-ol (4b)
Compound 4a (3.64 g, 0.01 mol) was dissolved in an-
hydrous pyridine (30 ml) and a solution of toluene-p-sulfo-
nyl chloride (2.85 g, 0.015 mol) in anhydrous pyridine (15
ml) was added dropwise during cooling with ice. The reac-
tion mixture was allowed to stand for 24 h and was then
poured onto a mixture of ice (500 g) and sulfuric acid (10
ml). The precipitate that separated out was filtered off and
recrystallized from a mixture of benzene/light petroleum.
20
[␣]D ϩ3 (c 1 methanol). (Found: C, 78.40; H, 11.02.
C20H34O2 requires: C, 78.38; H, 11.18%); 1H-NMR
(DMSO-d6) ␦ ppm 0.56(m, 1H, 9-H), 0.63(s, 3H, 18-H),
0.74(s, 3H, 19-H), 1.07(d, 3H, Jϭ7.3 Hz, 16-CH3), 4.24(d,
1H, Jϭ4.7 Hz, 17-H), 4.39(d, 1H, Jϭ4.6 Hz, 3-H).
8a (1.00 g, 65%) mp 265–267°C, Rf ϭ 0.15 (ss B);
20
[␣]D ϩ5 (c 1 in acetic acid). (Found: C, 78.51; H, 11.06.
20
4b (4.80 g, 92%) mp 142–144°C, Rf ϭ 0.60 (ss C); [␣]D
C20H34O2 requires: C, 78.38; H, 11.18%). 1H-NMR
(DMSO-d6) ␦ ppm 0.56(m, 1H, 9-H), 0.64(s, 3H, 18-H),
0.73(s, 3H, 19-H), 0.87(d, 3H, Jϭ7.2 Hz, 16-CH3), 4.12(d,
1H, Jϭ5.2 Hz, 17-H), 4.39(d, Jϭ4.7 Hz, 3-H).
Ϫ6 (c 1 chloroform). (Found: C, 67.22; H, 8.20. C29H42O6S
requires C, 67.15; H, 8.16%); 1H-NMR ␦ ppm 0.63(m, 1H,
9-H), 0.68(s, 3H, 18-H), 0.81(s, 3H, 19-H), 2.01(s, 3H,
CH3CO), 2.45(s, 3H, Ts-CH3), 2.57(m, 1H), 3.73(s, 1H,
17-H), 3.98(m, 1H) and 4.21(s, 3H): 16-CH2, 4.68(m, 1H,
3-H), 7.34(d, 2H, Jϭ8.0 Hz, 3Ј- and 5Ј-H), 7.79(d, 2H,
Jϭ8.0 Hz, 2Ј- and 6Ј-H). 13C-NMR ␦ ppm 12.6(C-19),
17.4(C-18), 20.8(C-11), 21.8 and 22.0(2C, CH3CO and Ts-
CH3), 27.8, 28.3, 28.8(2C), 31.4, 34.4, 35.8(C-10), 35.9(C-
8), 37.2, 40.5(C-16), 45.0(C-5), 46.4(C-13), 48.0(C-14),
54.3(C-9), 71.6(C-16Ј), 74.0(C-3), 79.5(C-17), 128.3(2C,
C-2Ј and C-6Ј), 130.2(2C, C-3Ј and C-5Ј), 133.5(C-1Ј),
148.5(C-4Ј), 171.1(CH3CO).
2.5. 16-Methyl-3␤-acetoxy-5␣-androstan-17-ols (5b, 6b,
7b, 8b)
2.5.1. General procedure
Compound 5a, 6a, 7a, or 8a (3.00 g, 0.01 mol) was
dissolved in anhydrous pyridine (20 ml), and a solution of
acetic anhydride (2 ml, 0.02 mol) in pyridine (10 ml) was
then added dropwise while cooling in ice under continuous
stirring. The progress of the reaction was monitored by
TLC. The reaction mixture was poured onto a mixture of
sulfuric acid (10 ml) and ice (100 g) and extracted with
chloroform. The chloroform phase was evaporated to dry-
ness and subjected to chromatographic separation on silica
gel in ethyl acetate/chloroform (5:95). The product was
crystallized from methanol.
2.4. 16-Methyl-5␣-androstane-3␤-17-diols (5a, 6a, 7a,
8a)
2.4.1. General procedure
LiAlH4 (1.0 g) was suspended in anhydrous tetrahydro-
furan (50 ml) cooled in a salt-ice bath, and a solution of
16-toluene-p-sulfonyloxymethyl-3␤-acetoxy-5␣-androstan-
17-ol (1b, 2b [12] or 4b) (2.60 g, 0.005 mol) or 16␤-
toluene-p-sulfonyloxymethyl-5␣-androstane-3␤, 17␣-diac-
etate (3b) (2.80, 0.005 mol) in anhydrous tetrahydrofuran
(50 ml) was added dropwise. The reaction mixture was
5b (3.10 g, 89%) mp 161–163°C, Rf ϭ 0.35 (ss B);
20
[␣]D ϩ2 (c 1 in chloroform) ([9] mp 159–161°C, [␣]D
ϩ3). (Found: C, 75.76; H, 10.55. C22H36O3 requires: C,
75.82; H, 10.41%). 1H-NMR ␦ ppm 0.66(m, 1H, 9-H),
0.73(s, 3H, 18-H), 0.83(s, 3H, 19-H), 0.99(d, 3H, Jϭ7.5 Hz,
16-CH3), 2.02(s, 3H, CH3CO), 3.61(d, 1H, Jϭ9.9 Hz, 17-

838
P. Tapolcsa´nyi et al. / Steroids 66 (2001) 833–843
H), 4.68 (m, 1H, 3-H). 13C-NMR ␦ ppm 12.4 and 12.6 (2C,
C-18 and C-19), 16.6(C-16Ј), 20.9(C-11), 21.6(CH3CO),
27.6, 28.6, 31.9, 34.1, 34.2 and 35.1 (2C, C-8 and C-16),
34.6, 35.7(C-10), 36.9, 37.9, 44.1(C-13), 44.9(C-5), 49.7(C-
14), 54.6(C-9), 73.8(C-3), 82.4(C-17), 170.9(CH3CO).
6b (2.95 g, 84%) mp 168–171°C, Rf ϭ 0.30 (ss B);
13C-NMR ␦ ppm 12.2(C-19), 13.4(C-18), 16.8(C-16’),
20.6(C-11), 20.9(17-CH3CO), 21.4(3-CH3CO), 27.4, 28.5,
31.7, 32.9 and 34.8(2C, C-8 and C-16), 34.0, 34.3, 35.6,
36.7, 37.7(C-12), 43.2(C-13), 44.6, 49.8(C-14), 54.2, 73.6,
83.7(C-17), 170.6(3-CH3CO), 171.1(17-CH3CO).
6d (3.60 g, 92%) mp 140–142°C, Rf ϭ 0.65 (ss B);
20
20
[␣]D Ϫ20 (c 1 in chloroform) ([9] mp 159–161°C, [␣]D
[␣]D Ϫ49 (c 1 in chloroform) ([9] mp 141–143°C, [␣]D
Ϫ21). (Found: C, 75.68; H, 10.58. C22H36O3, requires: C,
75.82; H, 10.41%). 1H-NMR ␦ ppm 0.66(m, 1H, 9-H),
0.75(s, 3H, 18-H), 0.83(s, 3H, 19-H), 1.09(d, 3H, Jϭ6.9 Hz,
16-CH3), 2.02(s, 3H, CH3CO), 3.10(d, 1H, Jϭ7.7 Hz, 17-
H), 4.68(m, 1H, 3-H). 13C-NMR ␦ ppm 11.9 and 12.2 (2C,
C-18 and C-19), 20.4(C-16Ј), 20.6(C-11), 21.4(CH3CO),
27.4, 28.4, 31.5, 32.3, 34.0, 35.2 and 38.2(2C, C-8 and
C-16), 35.6(C-10), 36.8(2C), 44.0(C-13), 44.7(C-5),
Ϫ49). (Found: C, 73.75; H, 9.90. C24H38O4 requires: C,
73.81; H, 9.81%). 1H-NMR ␦ ppm 0.66(m, 1H, 9-H),
0.77(s, 3H, 18-H), 0.82(s, 3H, 19-H), 1.04(d, 3H, Jϭ7.0 Hz,
16-CH3), 2.02(s, 3H, 3-CH3CO), 2.05(s, 3H, 17-CH3CO),
4.42(d, 1H, Jϭ7.6 Hz, 17-H), 4.68(m, 1H, 3-H). 13C-NMR
␦ ppm 12.2(C-19), 12.8(C-18), 20.3(C-16Ј), 20.6(C-11),
21.2(17-CH3CO), 21.4(3-CH3CO), 27.4, 28.4, 31.4, 32.4(C-
15), 34.0, 35.1 and 35.4(2C, C-8 and C-16), 35.6, 36.7, 37.1,
44.3(C-13), 44.7, 49.2(C-14), 54.2, 73.6, 89.2(C-17),
170.6(3-CH3CO), 171.2(17-CH3CO).
49.1(C-14),
54.5(C-9),
73.6(C-3),
89.7(C-17),
170.7(CH3CO).
7b (3.00 g, 86%) mp 178–180°C, Rf ϭ 0.25 (ss B);
7d (3.45 g, 88%) mp 114–117°C, Rf ϭ 0.65 (ss B);
20
[␣]D20 Ϫ8 (c 1 in chloroform). (Found: C, 75.74; H, 10.34.
[␣]D ϩ8 (c 1 in chloroform). (Found: C, 73.86; H, 9.75.
1
1
C22H36O3 requires: C, 75.82; H, 10.41%). H-NMR ␦ ppm
C24H38O4 requires: C, 73.81; H, 9.81%). H-NMR ␦ ppm
0.68(m, 1H, 9-H), 0.72(s, 3H, 18-H), 0.83(s, 3H, 19-H),
1.16(d, 3H, 16-CH3), 2.02(s, 3H, CH3CO), 3.34(d, 1H,
Jϭ1.2 Hz, 17-H), 4.68(m, 1H, 3-H). 13C-NMR ␦ ppm 12.2
(C-19), 17.9(C-18), 20.5(C-11), 21.1(C-16Ј), 21.4(CH3CO),
27.5, 28.5, 32.1, 32.3, 34.0, 34.7, 35.4(C-8), 35.6(C-10),
36.8, 43.3(C-16), 44.6(C-5), 45.1(C-13), 50.6(C-14),
53.9(C-9), 72.1(C-3), 87.2(C-17), 170.6(CH3CO).
0.67(m, 1H, 9-H), 0.80(s, 3H, 18-H), 0.82(s, 3H, 19-H),
1.19(d, 3H, Jϭ7.1 Hz, 16-CH3), 2.02(s, 3H, 3-CH3CO),
2.04(s, 3H, 17-CH3CO), 4.42(d, 1H, Jϭ1.5 Hz, 17-H),
4.68(m, 1H, 3-H). 13C-NMR ␦ ppm 12.6(C-19), 17.8(C-18),
20.8(C-11), 21.2, 21.7, 21.9, 27.9, 28.9, 32.6, 32.7, 34.4,
35.0, 35.7(C-8), 35.9, 37.1, 41.1(C-16), 45.0(C-5), 45.0(C-
13), 51.9(C-14), 54.2(C-9), 74.1(C-3), 88.9(C-17), 171.1
and 171.2(2C, CH3CO).
8b (3.05 g, 87%) mp 159–161°C, Rf ϭ 0.38 (ss B);
[␣]D20 Ϫ27 (c 1 in chloroform). (Found: C, 75.69; C, 10.30.
8d (3.70 g, 94%) mp 155–157°C, Rf ϭ 0.68 (ss B);
1
20
C22H36O3 requires: C, 75.82; H, 10.41%). H-NMR ␦ ppm
[␣]D ϩ2 (c 1 in chloroform). (Found: C, 73.72; H, 9.90.
1
0.66(m, 1H, 9-H), 0.72(s, 3H, 18-H), 0.82(s, 3H, 19-H),
0.99(d, 3H, Jϭ7.2 Hz, 16-CH3), 2.02(s, 3H, CH3CO),
3.53(d, 1H, Jϭ4.7 Hz, 17-H), 4.68(m, 1H, 3-H). 13C-NMR
␦ ppm 12.2(C-19), 15.4(C-16Ј), 17.4(C-18), 20.6(C-11),
21.4(CH3CO), 27.5, 28.6, 31.5, 32.2, 33.5, 34.0, 34.4 and
35.7(2C, C-8 and C-16), 35.6(C-10), 36.8, 44.7(C-5),
46.3(C-13), 48.0(C-14), 54.1(C-9), 73.7(C-3), 81.7(C-17),
170.6(CH3CO).
C24H38O4 requires: C, 73.81; H, 9.81%). H-NMR ␦ ppm
0.67(m, 1H, 9-H), 0.79(s, 3H, 18-H), 0.82(s, 3H, 19-H),
0.87(d, 3H, Jϭ7.3 Hz, 16-CH3), 2.02(s, 3H, 3-CH3CO),
2.08(s, 3H, 17-CH3CO), 2.47(m, 1H, 16-H), 4.68(m, 1H,
3-H), 4.85(d, 1H, Jϭ5.8 Hz, 17-H). 13C-NMR ␦ ppm
12.6(C-19), 16.0(C-16Ј), 17.5(C-18), 20.8(C-11), 21.3 and
21.8(2C, CH3CO), 27.8, 28.9, 32.2, 32.5, 34.06, 34.11(C-
16), 34.4, 35.9, 36.0(C-8), 37.1, 45.1(C-5), 46.3(C-13),
49.4(C-14), 54.4(C-9), 74.0(C-3), 83.7(C-17), 171.1 and
171.2(2C, CH3CO).
2.6. 16-Methyl-5␣-androstane-3␤,17-diacetates (5d, 6d,
7d, 8d)
2.7. 16-Methyl-17-acetoxy-5␣-androstan-3␤-ols (5c, 6c,
2.6.1. General procedure
7c, 8c)
Compound 5a, 6a, 7a, or 8a (3.00 g, 0.01 mol) was
dissolved in a mixture of pyridine (10 ml) and acetic anhy-
dride (5 ml, 0.05 mol) and allowed to stand for 24 h. It was
then poured onto a mixture of sulfuric acid (4 ml) and ice
(50 g). The precipitate was filtered off, washed and dried.
The product was crystallized from methanol.
5d (3.50 g, 89%) mp 119–120°C, Rf ϭ 0.70 (ss B);
[␣]D20 ϩ16 (c 1 in chloroform) ([9] mp 127–129°C, [␣]D ϩ
12). (Found: C, 73.92; H, 9.75. C24H38O4 requires: C,
73.81; H, 9.81%). 1H-NMR ␦ ppm 0.68(m, 1H, 9-H),
0.80(s, 3H, 18-H), 0.83(s, 3H, 19-H), 0.87(d, 3H, Jϭ7.25
Hz, 16-CH3), 2.02(s, 3H, 3-CH3CO), 2.08(s, 3H, 17-
CH3CO), 4.57(d, 1H, Jϭ10.0 Hz, 17-H), 4.68(m, 1H, 3-H).
2.7.1. General procedure
Compound 5d, 6d, 7d, or 8d (3.90 g, 0.01 mol) was
dissolved in methanol (200 ml), the solution was cooled to
0°C, and a solution of KOH (0.280 g, 0.005 mol) in meth-
anol (100 ml) was added. The progress of the selective
hydrolysis was monitored by TLC. After 24 h at 0°C, the
solution was poured onto ice and acidified with dilute hy-
drochloric acid. The precipitate was filtered off and washed
with water. It was subjected to column chromatography on
silica gel in ethyl acetate/chloroform (5:95) and crystallized
from a mixture of methanol and water.
5c (2.90 g, 83%) mp 172–175°C, Rf ϭ 0.25 (ss B);

P. Tapolcsa´nyi et al. / Steroids 66 (2001) 833–843
839
[␣]D20 ϩ37 (c 1 in chloroform). (Found: C, 75.74; H, 10.58.
C22H36O3 requires: C, 75.82; H, 10.41%). H-NMR ␦ ppm
76.26; H, 9.89%). 1H-NMR ␦ ppm 0.76(m, 1H, 9-H),
0.83(s, 3H, 18-H), 0.89(d, 3H, Jϭ7.5 Hz, 16-CH3), 1.02(s,
3H, 19-H), 2.08(s, 3H, CH3CO), 4.58(d, 1H, Jϭ10.0 Hz,
17-H). 13C-NMR ␦ ppm 11.5(C-19), 13.4(C-18), 16.8(C-
16Ј), 20.8(C-11), 20.9(CH3CO), 28.8(C-6), 31.4(C-7), 32.9
and 34.7(2C, C-8 and C-16), 34.3, 35.8(C-10), 37.7, 38.1(C-
2), 38.5(C-1), 43.2(C-13), 44.7(C-4), 46.7(C-5), 49.7(C-
14), 53.8(C-9), 83.6(C-17), 171.1(CH3CO), 211.8(C-3).
14b (1.60 g, 92%) mp 176–179°C, Rf ϭ 0.28 (ss A);
[␣]D20 Ϫ28 (c 1 in chloroform) ([9] 173–174°C, [␣]D ϩ18).
(Found: C, 76.14; H, 9.98. C22H34O3 requires: C, 76.26; H,
1
0.66(m, 1H, 9-H), 0.80 and 0.81(2s, 6H, 18-H and 19-H),
0.87(d, 3H, Jϭ7.5 Hz, 16-CH3), 2.07(s, 3H, 17-CH3CO),
3.59(m, 1H, 3-H), 4.57(d, 1H, Jϭ9.8 Hz, 17-H). 13C-NMR
␦ ppm 12.2 and 13.4(2C, C-18 and C-19), 16.8(C-16Ј),
20.6(C-11), 20.9(CH3CO), 28.6, 31.5, 31.8, 32.9 and 34.8
(2C, C-8 and C-16), 34.3, 35.6(C-10), 37.0, 37.8, 38.2,
43.2(C-13), 44.9(C-5), 49.9(C-14), 54.4(C-9), 71.2(C-3),
83.7(C-17), 171.2(CH3CO).
6c (3.10 g, 84%) mp 134–136°C, Rf ϭ 0.20 (ss B);
[␣]D20 Ϫ45 (c 1 in chloroform). (Found: C, 75.71; H, 10.35.
9.89%). H-NMR ␦ ppm 0.76(m, 1H, 9-H), 0.80(s, 3H,
1
1
C22H36O3 requires: C, 75.82; H, 10.41%). H-NMR ␦ ppm
18-H), 1.01(s, 3H, 19-H), 1.05(d, 3H, Jϭ6.95 Hz, 16-CH3),
2.06(s, 3H, CH3CO), 4.43(d, 1H, Jϭ7.4 Hz, 17-H). 13C-
NMR ␦ ppm 11.5(C-19), 12.5(C-18), 20.3(C-16Ј), 20.8(C-
11), 21.2(CH3CO), 28.8(C-6), 31.2(C-7), 32.4, 35.1 and
35.4 (2C, C-8 and C-16), 35.7(C-10), 37.0, 38.1(C-2),
38.5(C-1), 44.3(C-13), 44.6(C-4), 46.6(C-5), 49.1(C-14),
53.8(C-9), 89.1(C-17), 171.2(CH3CO), 211.8(C-3).
0.66(m, 1H, 9-H), 0.77 and 0.80(2s, 6H, 18-H and 19-H),
1.04(d, 3H, Jϭ7.1 Hz, 16-CH3), 2.05(s, 3H, 17-CH3CO),
3.59(m, 1H, 3-H), 4.42(d, 1H, Jϭ7.7 Hz, 17-H). 13C-NMR
␦ ppm 12.3 and 12.7(2C, C-18 and C-19), 20.3(C-16Ј),
20.6(C-11), 21.1(CH3CO), 28.5, 31.4, 31.5, 32.4, 35.1 and
35.3(2C, C-8 and C-16), 35.5(C-10), 36.9, 37.1, 38.1,
44.3(C-13), 44.8(C-5), 49.2(C-14), 54.3(C-9), 71.2(C-3),
89.2(C-17), 171.3(CH3CO).
15b (1.48 g, 85%) mp 148–152°C, Rf ϭ 0.32 (ss A);
[␣]D20 ϩ42 (c 1 in chloroform). (Found: C, 76.41; H, 9.77.
1
7c (2.85 g, 81%) mp 174–175°C, Rf ϭ 0.22 (ss B);
C22H34O3 requires: C, 76.26; H, 9.89%). H-NMR ␦ ppm
[␣]D20 ϩ16 (c 1 in chloroform). (Found: C, 75.69; H, 10.48.
0.76(m, 1H, 9-H), 0.83(s, 3H, 18-H), 1.01(s, 3H, 19-H),
1.21(d, 3H, Jϭ7.1 Hz, 16-CH3), 2.04(s, 3H, CH3CO),
4.44(d, 1H, Jϭ1.5 Hz, 17-H). 13C-NMR ␦ ppm 11.4(C-19),
17.4(C-18), 20.7(C-11), 20.8(C-16Ј), 21.2(CH3CO),
28.9(C-6), 32.0, 32.3, 34.6, 35.3(C-8), 35.7(C-10), 38.1(C-
2), 38.6(C-1), 40.8(C-16), 44.6(2C, C-4 and C-13), 46.7(C-
5), 51.4(C-14), 53.4(C-9), 88.4(C-17), 170.7(CH3CO),
211.8(C-3).
1
C22H36O3 requires: C, 75.82; H, 10.41%). H-NMR ␦ ppm
0.65(m, 1H, 9-H), 0.80 and 0.81(2s, 6H, 18-H and 19-H),
1.18(d, 3H, Jϭ7.1 Hz, 16-CH3), 2.03(s, 3H, CH3CO),
3.59(m, 1H, 3-H), 4.42(d, 1H, Jϭ1.5 Hz, 17-H). 13C-NMR
␦ ppm 12.3(C-19), 17.4(C-18), 20.5(C-11), 20.8(C-16Ј),
21.2(CH3CO), 28.6, 31.5, 32.3(2C), 34.6, 35.4(C-8),
35.6(C-10), 37.0, 38.2, 40.8(C-16), 44.6(C-13), 44.9(C-5),
51.6(C-14),
170.7(CH3CO).
54.0(C-9),
71.3(C-3),
88.6(C-17),
16b (1.40 g, 81%) mp 137–140°C, Rf ϭ 0.32 (ss A);
[␣]D20 ϩ25 (c 1 in chloroform). (Found: C, 76.30; H, 9.97.
1
8c (3.25 g, 93%) mp 172–173°C, Rf ϭ 0.22 (ss B);
C22H34O3 requires: C, 76.26; H, 9.89%). H-NMR ␦ ppm
[␣]D20 ϩ3 (c 1 in chloroform). (Found: C, 75.73; H, 10.32.
0.76(m, 1H, 9-H), 0.82(s, 3H, 18-H), 0.89(d, 3H, Jϭ7.2 Hz,
16-CH3), 1.01(s, 3H, 19-H), 2.08(s, 3H, CH3CO), 4.86(d,
1H, Jϭ5.6 Hz, 17-H). 13C-NMR ␦ ppm 11.4(C-19), 15.6(C-
16Ј), 17.0(C-18), 20.7(C-11), 20.8(CH3CO), 28.9(C-6),
31.8, 31.9, 33.6, 33.7 and 35.5(2C, C-8 and C-16), 35.7(C-
10), 38.1(C-2), 38.5(C-1), 44.6(C-4), 45.9(C-13), 46.7(C-
5), 49.0(C-14), 53.6(C-9), 83.2(C-17), 170.8(CH3CO),
211.8(C-3).
1
C22H36O3 requires: C, 75.82; H, 10.41%). H-NMR ␦ ppm
0.67(m, 1H, 9-H), 0.79 and 0.80(2s, 6H, 18-H and 19-H),
0.88(d, 3H, Jϭ7.3 Hz, 16-CH3), 2.07(s, 3H, CH3CO),
3.60(m, 1H, 3-H), 4.85(d, 1H, Jϭ5.8 Hz, 17-H). 13C-NMR
␦ ppm 12.3(C-19), 15.6(C-16Ј), 17.1(C-18), 20.5(C-11),
20.9(CH3CO), 28.7, 31.5, 31.9, 32.3, 33.6, 33.7 and 35.7
(2C, C-8 and C-16), 35.6(C-10), 37.0, 38.2, 44.9(C-5),
45.9(C-13), 49.1(C-14), 54.1(C-9), 71.3(C-3), 83.4(C-17),
170.9(CH3CO).
2.9. 16-Methyl-17-hydroxy-5␣-androstan-3-ones (13a,
14a, 15a, 16a)
2.8. 16-Methyl-17-acetoxy-5␣-androstan-3-ones (13b, 14b,
15b, 16b)
2.9.1. General procedure
Compounds 13b, 14b, 15b or 16b (1.73 g, 0.005 mol)
was dissolved in methanol (50 ml) and KOH (0.6 g, 0.01
mol) was added. After 24 h of standing at room temperature,
the mixture was neutralized with dilute hydrochloric acid
and diluted with water. The precipitate was then filtered off,
and crystallized from acetone/light petroleum
2.8.1. General procedure
Jones reagent (2 ml) was added dropwise to a solution of
compounds 5c, 6c, 7c, or 8c (1.75 g, 0.005 mol) in acetone
(10 ml) during cooling with ice. The mixture was diluted
with ice-water and the precipitate was filtered off, and
recrystallized from aqueous methanol.
13a (1.35 g, 90%) mp 186–189°C, Rf ϭ 0.22 (ss B);
20
13b (1.65 g, 95 %) mp 185–187°C, Rf ϭ 0.30 (ss A);
[␣]D ϩ35 (c 1 in chloroform) ([9] mp 184–186°C, [␣]D
20
[␣]D ϩ47 (c 1 in chloroform) ([9] mp 180–182°C, [␣]D
ϩ33). (Found: C, 79.02; H, 10.45. C20H32O2 require: C,
ϩ47). (Found: C, 76.35; H, 9.94. C22H34O3 requires: C,
78.90; H, 10.59%). 1H-NMR ␦ ppm 0.76(s, 3H, 18-H),

840
P. Tapolcsa´nyi et al. / Steroids 66 (2001) 833–843
1
1.00(d, 3H, Jϭ7.6 Hz, 16-CH3), 1.02(s, 3H, 19-H), 3.62(d,
1H, Jϭ10.0 Hz, 17-H). 13C-NMR ␦ ppm 11.5 and 12.5(2C,
C-18 and C-19), 16.4(C-16Ј), 21.0(C-11), 28.8(C-6),
31.5(C-7), 34.1 and 34.9(2C, C-8 and C-16), 34.5, 35.8(C-
10), 37.8, 38.1(C-2), 38.6(C-1), 43.9(C-13), 44.7(C-4),
46.7(C-5), 49.4(C-14), 54.1(C-9), 82.1(C-17), 211.8(C-3).
14a (1.25 g, 83%) mp 150–153°C, Rf ϭ 0.20 (ss B);
79.42; H, 10.10 %). H-NMR ␦ ppm 0.81(m, 1H, 9-H),
0.92(s, 3H, 18-H), 1.04(s, 3H, 19-H), 1.10(d, 3H, Jϭ7.7 Hz,
16-CH3). 13C-NMR ␦ ppm 11.5(C-19), 14.4(C-18), 16.7(C-
16Ј), 20.7(C-11), 28.6(C-6), 30.2, 30.5, 31.8, 34.8(C-8),
35.8(C-10), 38.1(C-2), 38.4(C-1), 39.2(C-16), 44.6(C-4),
46.6(C-5), 48.3(C-13), 48.5(C-14), 53.9(C-9), 211.6(C-3),
222.7(C-17).
20
[␣]D ϩ11 (c 1 in chloroform). ([9] mp 156–157°C, [␣]D
ϩ9.2). (Found: C, 78.86; H, 10.67. C20H32O2 requires: C,
78.90; H, 10.59%). 1H-NMR ␦ ppm 0.78(s, 3H, 18-H),
1.01(s, 3H, 19-H), 1.09(d, 3H, Jϭ6.9 Hz, 16-CH3), 3.11(d,
1H, Jϭ7.6 Hz, 17-H). 13C-NMR ␦ ppm 11.4 and 11.8 (2C,
C-18 and C-19), 20.4(C-16Ј), 20.9(C-11), 28.8(C-6),
31.2(C-7), 32.3, 35.2 and 38.2(2C, C-8 and C-16), 35.7(C-
10), 36.8, 38.1(C-2), 38.5(C-1), 44.0(C-13), 44.6(C-4),
46.8(C-5), 49.0(C-14), 54.0(C-9), 89.6(C-17), 211.9(C-3).
15a (1.40 g, 93%) mp 194–197°C, Rf ϭ 0.18 (ss B);
2.11. 17␤,16␤-(Epoxymethano)-5␣-androstan-3␤-ol (9a)
and 17␣,16␣-(epoxymethano)-5␣-androstan-3␤-ol (10a)
2.11.1. General procedure
LiA1H4 (1.0 g, 0.03 mol) was suspended in anhydrous
tetrahydrofuran (50 ml) cooled in a salt-ice bath and a
solution of 1b or 2b (2.60 g, 0.005 mol) in anhydrous
tetrahydrofuran (50 ml) was added dropwise. The reaction
mixture was stirred at room temperature for 2 h. Aqueous
ethanol (50 ml) and 20% aqueous NH4Cl solution (50 ml)
were added to the cooled reaction mixture. The organic
fraction was evaporated under reduced pressure. The resid-
ual precipitate was filtered off, and subjected to chromato-
graphic separation on alumina with chloroform/light petro-
leum (1:1) as eluent. The substance obtained was
crystallized from acetone/light petroleum.
20
[␣]D ϩ33 (c 1 chloroform). (Found: C, 78.92; H, 10.63.
1
C20H32O2 requires: C, 78.90; H, 10.59%). H-NMR ␦ ppm
0.74(s, 3H, 18-H), 1.02(s, 3H, 19-H), 1.17(d, 3H, Jϭ7.3 Hz,
16-CH3), 3.36(d, 1H, Jϭ1.6 Hz, 17-H). 13C-NMR ␦ ppm
11.5(C-19), 17.9(C-18), 20.8(C-11), 21.1(C-16Ј), 28.9(C-
6), 32.0, 32.1, 34.7, 35.3(C-8), 35.7(C-10), 38.1(C-2),
38.6(C-1), 43.4(C-16), 44.7(C-4), 45.1(C-13), 46.6(C-5),
50.5(C-14), 53.5(C-9), 87.1(C-17), 211.9(C-3).
9a (1.10 g, 72%) mp 185–187°C, Rf ϭ 0.45 (ss B);
16a (1.38 g, 92%) mp 224–225°C, Rf ϭ 0.18 (ss B);
[␣]D20 ϩ2 (c 1 in chloroform). (Found: C, 78.88; H, 10.65.
20
1
[␣]D ϩ9 (c 1 chloroform). (Found: C, 79.05; H, 10.47.
C20H32O2 requires: C, 78.90; H, 10.59%). H-NMR ␦ ppm
1
C20H32O2 requires: C, 78.90; H, 10.59%). H-NMR ␦ ppm
0.60(m, 1H, 9-H), 0.82(s, 3H, 19-H), 1.04(s, 3H, 18-H),
3.12(m, 1H, 16-H), 3.56(m, 1H, 3-H), 4.21(dd, 1H, Jϭ6.3
Hz, 6.1 Hz) and 4.73(dd, 1H, Jϭ8.1 Hz, 6.3 Hz): 16Ј-H,
4.54(d, 1H, Jϭ8.0 Hz, 17-H). 13C-NMR ␦ ppm 12.3(C-19),
12.8(C-18), 21.5(C-11), 28.6, 31.2, 31.5, 32.3, 35.1 and
37.1(2C, C-8 and C-16), 35.5(C-10), 37.0, 37.5, 38.1,
44.5(C-13), 44.8(C-5), 54.2(C-14), 55.4(C-9), 71.2 (C-3),
76.9(C-16Ј), 95.7(C-17).
0.76(s, 3H, 18-H), 1.00(d, 3H, Jϭ7.6 Hz, 16-CH3), 1.01(s,
3H, 19-H), 3.55(d, 1H, Jϭ5.2 Hz, 17-H). 13C-NMR ␦ ppm
11.4(C-19), 15.3(C-16Ј), 17.4(C-18), 20.8(C-11), 28.9(C-
6), 31.5(C-7), 31.9, 33.5, 34.4 and 35.6(2C, C-8 and C-16),
35.7(C-10), 38.1(C-2), 38.5(C-1), 44.6(C-4), 46.3(C-13),
46.7(C-5), 47.9(C-14), 53.6(C-9), 81.6(C-17), 211.9(C-3).
2.10. 16-Methyl-5␣-androstane-3,17-diones (17, 18)
10a (1.0 g, 65%) mp 196–198°C, Rf ϭ 0.50 (ss B);
[␣]D20 ϩ34 (c 1 in chloroform). (Found: C, 79.02; H, 10.47.
1
2.10.1. General procedure
C20H32O2 requires: C, 78.90; H, 10.59%). H-NMR ␦ ppm
Compound 5a or 6a (1.50 g, 0.005 mol) was dissolved in
acetone (10 ml). Jones reagent (2 ml) was added during
cooling with ice. The mixture was diluted with ice-water,
and the precipitate was filtered off, and crystallized from
acetone-light petroleum.
0.50(s, 3H, 18-H), 0.83(s, 3H, 19-H), 3.04(m, 1H, 16-H),
3.61(m, 1H, 3-H), 3.95(dd, 1H, Jϭ5.5 Hz, 4.1 Hz) and
4.80(dd, 1H, Jϭ6.6 Hz, 5.5 Hz): 16Ј-H, 4.62(d, 1H, Jϭ4.8
Hz, 17-H). 13C-NMR ␦ ppm 12.2(C-19), 14.6(C-18),
20.6(C-11), 28.7, 30.0, 31.0, 31.5, 32.7, 34.9 and 36.1(2C,
C-8 and C-16), 35.6(C-10), 37.1, 38.2, 44.6(C-13), 44.9(C-
5), 48.4(C-14), 54.3(C-9), 71.3(C-3), 75.2(C-16Ј), 93.5(C-
17).
17 (1.25 g, 82%) mp 160–162°C, Rf ϭ 0.30 (ss A);
20
[␣]D ϩ115 (c 1 in chloroform) ([9] mp 163–164°C, [␣]D
ϩ115). (Found: C, 79.54; H, 10.08. C20H30O2 requires: C,
79.42; H, 10.00%). 1H-NMR ␦ ppm 0.81(m, 1H, 9-H),
0.85(s, 3H, 18-H), 1.04(s, 3H, 19-H), 1.20(d, 3H, Jϭ6.7 Hz,
16-CH3). 13C-NMR ␦ ppm 11.4(C-19), 14.1(C-18), 16.9(C-
16Ј), 20.6(C-11), 28.7(C-6), 30.7, 30.8, 31.9, 34.6(C-8),
35.8(C-10), 38.1(C-2), 38.4(C-1), 43.7(C-16), 44.6(C-4),
46.6(C-5), 48.0(C-13), 49.9(C-14), 54.1(C-9), 211.4(C-3),
223.0(C-17).
2.12. 3␤-Acetoxy-17␤,16␤-(epoxymethano)-5␣-androstane
(9b) and 3␤-acetoxy-17␣,16␣-(epoxymethano)-5␣-
androstane (10b)
2.12.1. General procedure
Compound 9a or 10a (304 mg, 1 mmol) was dissolved in
a mixture of pyridine (3 ml) and acetic anhydride (3 ml) and
the solution was allowed to stand at room temperature for
6 h. The mixture was then diluted with water and the
18 (1.30 g, 86%) mp 159–161°C, Rf ϭ 0.28 (ss A);
[␣]D20 ϩ105 (c 1 in chloroform) ([9] mp 156–158°C, [␣]D
ϩ90). (Found: C, 79.35, H, 10.12. C20H30O2 requires: C,

P. Tapolcsa´nyi et al. / Steroids 66 (2001) 833–843
841
precipitate that separated out was filtered off and crystal-
lized from methanol.
5), 49.0(C-13), 49.2(C-14), 55.0(C-9), 74.3(C-3),
171.3(CH3CO), 223.6(C-17).
9b (310 mg, 89%), mp 108–111 °C, Rf ϭ 0.50 (ss A);
[␣]D20 Ϫ13 (c 1 in chloroform). (Found: C, 76.35, H, 9.78.
C22H34O3 requires: C, 76.26; H, 9.89%). H-NMR ␦ ppm
2.14. In vitro binding of 16-methyl-5␣-androstane
derivatives to the androgen receptor
1
0.60(m, 1H, 9-H), 0.85(s, 3H, 19-H), 1.05(s, 3H, 18-H),
2.01(s, 3H, CH3CO), 3.13(m, 1H, 16-H), 4.21(dd, 1H,
Jϭ6.4 Hz, 6.1 Hz) and 4.73(dd, 1H, Jϭ8.0 Hz, 6.4 Hz):
16Ј-H, 4.55(d, 1H, Jϭ8.0 Hz, 17-H), 4.67(m, 1H, 3-H).
13C-NMR ␦ ppm 12.2(C-19), 12.8(C-18), 21.4(C-11),
21.5(CH3CO), 27.5, 28.5, 31.2, 32.2, 33.8, 35.0 and 36.8
(2C, C-8 and C-16), 35.6(C-10), 36.4, 37.2, 44.5(C-13),
44.7(C-5), 54.1(C-14), 55.4(C-9), 73.6(C-3), 76.8(C-16Ј),
95.7(C-17), 170.6(CH3CO).
The androgen-binding experiments were carried out with
cytosol of castrated rat prostate, with [3H]methyltrienolone
(R1181; 17␤-hydroxy-17␣-methylestra-4,9,11-trien-3-one)
as radioligand. The competitive receptor assays were per-
formed as previously described [14]. The abilities of the
currently synthetized 16-methyl-5␣-androstane derivatives
(5a, 6a, 7a, 8a, 13a, 14a, 15a, 16a, 17, 18) to inhibit the
specific binding of the radioligand are characterized quan-
titatively by their IC50 values (the concentration of inhibitor
at which 50% of the specific radioligand binding is inhib-
ited). Relative binding affinities (RBAs) are defined by
10b (300 mg, 86%), mp 183–185°C, Rf ϭ 0.55 (ss A);
[␣]D20 ϩ15 (c 1 in chloroform). (Found: C, 76.20; H, 9.94.
1
C22H34O3 requires: C, 76.26; H, 9.89%). H-NMR ␦ ppm
0.50(s, 3H, 18-H), 0.85(s, 3H, 19-H), 2.02(s, 3H, CH3CO),
3.04(m, 1H, 16-H), 3.95(dd, 1H, Jϭ5.6 Hz, 4.1 Hz) and
4.80(dd, 1H, Jϭ6.6 Hz, 5.6 Hz): 16Ј-H, 4.62(d, 1H, Jϭ5.0
Hz, 17-H), 4.70(m, 1H, 3-H). 13C-NMR ␦ ppm 12.2(C-19),
14.6(C-18), 20.5(C-11), 21.4(CH3CO), 27.5, 28.6, 29.9,
31.0, 34.9 and 36.1(2C, C-8 and C-16), 35.6(C-10), 36.8,
44.6(C-13), 44.7(C-5), 48.3(C-14), 54.2(C-9), 73.7(C-3),
75.2(C-16Ј), 93.4(C-17), 170.6(CH3C).
IC50͓͑3H͔R1881͒
RBA(%) ϭ
100
IC50 ͑inhibitor͒
3. Results and discussion
3.1. Synthetic studies
In an earlier publication we reported on the preparation
of the four possible isomers of the 16-hydroxymethyl-5␣-
androstane-3␤,17-diol [12]. 3␤-Acetoxy-16␤-hydroxy-
methyl-5␣-androstan-17␤-ol (1a) and 3␤-acetoxy-16␣-hy-
droxymethyl-5␣-androstan-17␤-ol (2a) were obtained
directly from the reduction of 3␤-acetoxy-16-acetoxymeth-
ylene-5␣-androstan-17-one. 16␤-Acetoxymethyl-5␣-andro-
stan-3␤,17␣-diacetate (3c) and 16␣-acetoxymethyl-5␣-an-
drostan-3␤,17␣-diacetate (4c) were prepared by utilizing
the neighboring group participation characterized by the
general symbol (AcO-6) occurring during the acetolysis of
16␤-toluene-p-sulfonyloxymethyl-5␣-androstane-3␤,17␤-
diacetate and 3␤-acetoxy-16␣-acetoxymethyl-17␤-toluene-
p-sulfonyloxy-5␣-androstane, respectively. Whereas the
two isomers prepared by the solvolysis method were ob-
tained in their triacetate forms (3c, 4c), their selective
deacetylation was carried out at the primary acetoxy group
according to an earlier developed method [13]. 3c and 4c
were subjected to microwave irradiation at 90 W on an
alumina surface in household microwave equipment. After
irradiation of 3c at 90 W, the 16␤-hydroxymethyl-5␣-an-
drostane-3␤,17␣-diacetate (3a) was obtained in 60% yield.
Irradiation of 4c for 10 min gave 51% of 3␤-acetoxy-16␣-
hydroxymethyl-5␣-androstan-17␣-ol (4a) This is explained
by the selective deacetylation of the triacetate 4c at the
primary acetoxy group. Subsequent acyl migration was ob-
served for the compounds containing the 16,17 functional
groups in the cis orientation, and further deacetylation at the
primary C-16 position.
2.13. 16␤-Methyl-3␤-acetoxy-5␣-androstan-17-one (11b)
and 16␣-methyl-3␤-acetoxy-5␣-androstan-17-one (12b)
2.13.1. General procedure
Compound 5b or 6b (350 mg, 1 mmol) dissolved in
acetone (3 ml). Jones reagent (1 ml) was added during
cooling with ice. The mixture was diluted with ice-water,
and the resulting precipitate was filtered off, and crystallized
from acetone-light petroleum.
11b (320 mg, 92%) mp 107–110°C, Rf ϭ 0.45 (ss B),
[␣]D20 ϩ73 (c 1 in chloroform) ([10] mp 108–110°C, [␣]D
ϩ56). (Found: C, 76.18; H, 9.95. C22H34O3 requires: C,
76.26; H, 9.89%). 1H-NMR ␦ ppm 0.70(m, 1H, 9-H),
0.82(s, 3H) and 0.85(s, 3H): 18-H and 19-H, 1.19(d, 3H,
Jϭ7.0 Hz, 16-CH3), 2.02(s, 3H, CH3CO), 2.12(m, 2H),
4.69(m, 3H, 3-H). 13C-NMR ␦ ppm 12.9(C-19), 14.7(C-18),
17.6(C-16Ј), 21.1(C-11), 21.3(CH3CO), 28.1, 29.0, 31.4,
31.7, 32.6, 34.6, 35.3(C-8), 36.3, 37.3, 44.4(C-16), 45.3(C-
5), 48.8(C-13), 50.6(C-14), 55.1(C-9), 74.1(C-3),
171.3(CH3CO), 224.1(C-17).
12b (300 mg, 86%) mp 151–153°C, Rf ϭ 0.42 (ss B);
20
[␣]D ϩ61 (c 1 in chloroform) ([10] mp 143–145°C,
[␣]D ϩ 46). (Found: C, 76.32; H, 9.70. C22H34O3 requires:
1
C, 76.26; H, 9.89%). H-NMR ␦ ppm 0.70(m, 1H, 9-H),
0.85(s, 3H) and 0.89(s, 3H): 18-H and 19-H, 1.09(d, 3H,
Jϭ7.7 Hz, 16-CH3), 2.02(s, 3H, CH3CO), 2.50(m, 1H),
4.69(m, 3H, 3-H). 13C-NMR ␦ ppm 12.9(C-19), 15.1(C-18),
17.3(C-16Ј), 21.1(C-11), 21.3(CH3CO), 28.1, 28.9, 30.8,
31.4, 32.5, 34.6, 35.6(C-8), 36.3, 37.4, 39.8(C-16), 45.3(C-
The three 3␤-acetoxy-16-hydroxymethyl-17-hydroxy

842
P. Tapolcsa´nyi et al. / Steroids 66 (2001) 833–843
Table 1
isomers 1a, 2a, 4a and the 16␤-hydroxymethyl-3␤,17␣-
diacetate isomers 3a were converted into their 16-toluene-
p-sulfonyloxymethyl derivatives 1b, 2b, 3b, 4b. The next
step was LiAlH4 reduction in tetrahydrofuran, which fur-
nished the desired 16-methyl-5␣-androstane-3␤,17-diol iso-
mers 5a, 6a, 7a, 8a. Our earlier results [15] indicated that
the reduction of 16,17-cis isomers 1a and 4a proceeded via
oxetanes [16] condensed to ring D in the ␤ and ␣ positions,
as in 9a and 10a. Whereas the reduction did not involve any
chiral center, the configurations of the compounds obtained
agreed with those of the starting materials, which had con-
firmed configurations. The 16-methyl-5␣-androstane-
3␤,17-diacetate isomers 5d, 6d, 7d, 8d were then selec-
tively deacetylated. Jones oxidation of the resulting 17-
acetoxy-16-methyl-5␣-androstan-3␤-ols 5c, 6c, 7c, 8c gave
the 17-acetoxy-16␤-methyl-5␣-androstan-3-one isomers
13b, 14b, 15b, 16b. Since the oxidation did not affect the
chiral centers C-16 and C-17, the configurations of the
products and the starting compounds were identical.
Relative binding affinities (RBAs) of 16-methyl-5␣-androstane
derivatives and reference compounds for androgen receptor in rat
prostate cytosol
Compounds
tested
Range of concentration
(nM)
IC50 (nM) Ϯ SD
RBA
(%)
R1881a
19b
20c
5a
6a
7a
0.5–5.0
1.0–10
50–500
200–2000
500–2000
500–2000
500–2000
10–200
1.53 Ϯ 0.12
2.11 Ϯ 0.70
115 Ϯ 15
460 Ϯ 65
993 Ϯ 50
Ͼ1000
100
72.5
1.3
0.33
0.15
Ͻ0.15
Ͻ0.15
6.4
8a
Ͼ1000
13a
14a
15a
16a
17
24 Ϯ 3.0
83 Ϯ 16
Ͼ1000
Ͼ1000
Ͼ1000
10–200
1.8
500–2000
500–2000
500–2000
200–2000
Ͻ0.15
Ͻ0.15
Ͻ0.15
Ͻ0.15
18
Ͼ1000
a 17␤-Hydroxy-17␣-methylestra-4,9,11-trien-3-one.
b 17␤-Hydroxy-5␣-androstan-3-one.
c 5␣-Androstane-3␤,17␤-diol.
In agreement with the literature, the coupling constants
J
J
16,17 display the following sequence [17,18]: J16␣H,17␣H Ͼ
16␤H,17␣H Ͼ J16␤H,17␤H Ͼ J16␣H,17␤H 5d: 10.00 6d: 7.6 8d:
introduction of a 16␤-methyl substituent into 5␣-androstan-
3-on-17␤-ol (13a) decreases RBA from 72.5% to 6.4%,
while introduction of a 16␣-methyl group (14a) leads to a
more significant decreased, to 1.8%. In the other reference
compound, 20, which is a major metabolite of 5␣-dihy-
drotestosterone (19) in living organisms, the 3-oxo function
is changed to 3␤-ol and this compound has a relatively low
binding affinity for the androgen receptor (RBA ϭ 1.3%).
The 16␤-methyl and 16␣-methyl derivatives (5a, 6a) of this
compound exhibit lower binding affinities, with RBA
0.33% and 0.15%, respectively.
All 16-methyl-5␣-androstane molecules containing a
17␣-hydroxy (7a, 8a, 15a, 16a), or 17-oxo group (17, 18)
do not possess the structural requirement essential androgen
receptor binding a (17␤-hydroxy group), and they therefore
practically lack binding affinity (RBA Ͻ 0.5%) (Table 1).
In conclusion, in vitro receptor binding studies and the
determination of IC50 and RBA data are important in inves-
tigations relating to biological activity, but they do not
reveal the pharmacological type (agonist or antagonist) of
the currently synthetized competitor molecules. The intro-
duction of a 16-methyl substituent into 5␣-androstane mol-
ecules substantially decreases the binding affinity to the
androgen receptor and 16␣-methyl derivatives were always
bound more weakly than the 16␤-methyl isomers.
5.8 7d: 1.5 13b: 10.00 14b: 7.4 16b: 5.6 15b: 1.5.
The configurational correlation with compounds pub-
lished in the literature [9,10] was carried out by comparing
their physical data. We found that the stereochemical as-
signments of substituents on the basis of empirical rules
were correct except in the case of 16␣-methyl-17␤-acetoxy-
5␣-androstan-3-one 14b. The specific rotation of this com-
pound, which was prepared by the reduction and hydrolysis
of 16␣-methyl-3-N-pyrrolidyl-5␣-androst-3-en-17-one, was
ϩ18 [9]. Compound 14b which we prepared by another
method had a specific rotation of Ϫ47 (c 1 chloroform). The
structure was confirmed via the NMR spectrum, and the
negative value confirmed the empirical rule that a 16␣
substituent in the androstane series is generally more neg-
ative than a 16␤ substituent [19,20]. On the other hand,
NaBH4 reduction of 3␤-acetoxy-16␤-methyl-5␣-androstan-
17-one (11b) by the procedure of Ruggieri et al. [9] led to
a mixture. We detected the presence of two isomers by
TLC. 13C-NMR spectroscopy allowed determination of
their stereochemistry without separation. Besides the
16␤,17␤ derivative 5b, the only isomer prepared by Rug-
gieri et al., the 16␣,17␤-epimer 6b was also formed in the
reaction.
3.2. Receptor-binding studies
The IC50 values and the RBAs of the 16-methyl-5␣-
androstane derivatives (5a, 6a, 7a, 8a, 13a, 14a, 15a, 16a,
17, 18) and the reference compounds 17␤-hydroxy-5␣-an-
drostan-3-one (19) and 5␣-androstane-3␤,17␤-diol (20) ob-
tained in competitive binding receptor assays are illustrated
in Table 1. The natural androgen hormone 17␤-hydroxy-
5␣-androstan-3-one (19) is a powerful competitor and binds
strongly to the androgen receptor (RBA ϭ 72.5%). The
Acknowledgments
Financial support by the Hungarian National Research
Foundation (Grant OTKA T 032265) and by the Hungarian
Board of Education (FKFP 0110/2000) is gratefully ac-
knowledged.

P. Tapolcsa´nyi et al. / Steroids 66 (2001) 833–843
843
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