Synthesis of (19E)-3β,7α-dihydroxy-17-oxoandrost-5-en-19-al 19-(O-carboxymethyl)oxime, a new hapten for 7α-hydroxydehydroepiandrosterone (3β,7α-dihydroxyandrost-5-en-17-one)

Article abstract of DOI:10.1016/S0039-128X(98)00047-6

The title compound was prepared in 11 steps from 17,17-ethylenedioxy-19-hydroxyandrost-5-en-3β-yl acetate. After tert-butyldimethylsilyl protection of the 19-hydroxyl group, a 7-oxo group was introduced by oxidation with 3,5-dimethylpyrazole-chromium trioxide complex, and then selectively reduced with L-Selectride to give a 7α-hydroxy derivative. This partially protected trial was acetylated and desilylated to 3,7-diacetate. Subsequent oxidation with pyridine-chromium trioxide complex gave 19-aidehyde, which was transformed into the corresponding protected 19-(O-carboxymethyl)oxime. Successive ketal cleavage, deacetylation, and methyl ester splitting gave the final (19E)-3β,7α-dihydroxy-17-oxoandrost-5-en-19-al 19-(O-carboxymethyl)oxime, designed as a hapten for 7α-hydroxydehydroepiandrosterone immunoassays.

Full text of DOI:10.1016/S0039-128X(98)00047-6

Papers
Synthesis of (19E)-3␤,7␣-dihydroxy-
17-oxoandrost-5-en-19-al 19-(O-
carboxymethyl)oxime, a new hapten
for 7␣-hydroxydehydroepiandrosterone
(3␤,7␣-dihydroxyandrost-5-en-17-one)
ˇ
Vladim´ır Pouzar, Tereza Slav´ıkova´, and Ivan Cerny´
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
166 10 Prague 6, Czech Republic
The title compound was prepared in 11 steps from 17,17-ethylenedioxy-19-hydroxyandrost-5-en-3␤-yl acetate.
After tert-butyldimethylsilyl protection of the 19-hydroxyl group, a 7-oxo group was introduced by oxidation with
3,5-dimethylpyrazole-chromium trioxide complex, and then selectively reduced with L-Selectride௡ to give a
7␣-hydroxy derivative. This partially protected triol was acetylated and desilylated to 3,7-diacetate. Subsequent
oxidation with pyridine-chromium trioxide complex gave 19-aldehyde, which was transformed into the corre-
sponding protected 19-(O-carboxymethyl)oxime. Successive ketal cleavage, deacetylation, and methyl ester
splitting gave the final (19E)-3␤,7␣-dihydroxy-17-oxoandrost-5-en-19-al 19-(O-carboxymethyl)oxime, designed
as a hapten for 7␣-hydroxydehydroepiandrosterone immunoassays. (Steroids 63:454–458, 1998) © 1998 by
Elsevier Science Inc.
Keywords: synthesis; 7␣-hydroxy DHEA; hapten; oxime
cross-reactivity of generated antisera with 7␣-
hydroxypregnenolone.
For the preparation of a hapten with better conservation
of the original structure of 7␣-hydroxy DHEA 2, a position
which does not interfere with characteristic functional
groups has to be used. Consequently, more complex chem-
istry is necessary.
From this point of view, compound 4, with its 19-(O-
carboxymethyl)oxime (19-CMO) group as the connecting
point for further conjugation, is potentially very promising.
The synthesis of its epimer 5 was recently published,5 and
the preliminary immunological results have shown its suit-
ability for the 7␤-hydroxy DHEA 3 assays. A different
strategy for the chemical synthesis of 4 was needed, and this
is the subject of our present paper.
Introduction
Of the possible metabolic transformations of dehydroepi-
androsterone 1 (DHEA), the hydroxylation in position 71,2
has attracted considerable interest, as it is relevant to the
body’s immune system regulation.3 Isomers 2 and 3, which
differ in configuration at position 7, have been isolated. The
major isomer 2 was found to be more biologically active.
With respect to the assays for 7␣-hydroxy DHEA 2, a
hapten for radioimmunoassay based on a derivative with the
17␤-carboxyl group (3␤,7␣-dihydroxyandrost-5-ene-17␤-
carboxylic acid) is known.4 The drawback of the use of this
acid, when conjugated to bovine serum albumin, is the high
Tereza Slav´ıkova´ died on July 6, 1997 at the age of 25.
Presented in part at the conference “Advances in organic, bioorganic and
pharmaceutic chemistry” (in Czech), Liblice, Czech Republic, November
10–12, 1997.
Address reprint requests to Dr. Vladim´ır Pouzar, Institute of Organic
Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
Flemingovo n. 2, 166 10 Prague 6, Czech Republic.
Experimental
Melting points were determined on a micro melting point appara-
tus Boetius (Jena, Germany). Optical rotations were measured at
25°C on a Perkin-Elmer 141 MC polarimeter, and [␣]D values are
given in degrees [10Ϫ1 deg cm2 gϪ1]. Infrared spectra (wavenum-
Received October 10, 1997; accepted April 6, 1998.
Steroids 63:454–458, 1998
© 1998 by Elsevier Science Inc. All rights reserved.
655 Avenue of the Americas, New York, NY 10010
0039-128X/98/$19.00
PII S0039-128X(98)00047-6

New hapten for 7␣-hydroxydehydroepiandrosterone: Pouzar et al.
bers in cmϪ1) were recorded on a Bruker IFS 88 spectrometer. 1H
19-(tert-Butyldimethylsiloxy)-17,17-ethylenedioxy-7␣-
hydroxyandrost-5-en-3␤-yl acetate (9)
NMR spectra were taken on a Varian UNITY-200 (200 MHz, FT
mode) spectrometer at 23°C in deuteriochloroform with tetrameth-
ylsilane as the internal standard, unless stated otherwise. Chemical
shifts are given in ppm (␦-scale), coupling constants (J) and width
of multiplets (W) in Hz. Thin-layer chromatography was per-
formed on silica gel G (ICN Biochemicals), with detection by
spraying with concentrated sulfuric acid followed by heating.
Preparative TLC was done on 200 ϫ 200 mm plates with 0.4 mm
layer thickness, and detected by spraying with morin (0.2% solu-
tion in methanol) followed by UV visualization (365 nm). For
column chromatography, silica gel 60–120 ␮m was used. Prior to
evaporation on a rotary evaporator in vacuo (bath temperature
50°C), solutions in organic solvents were dried over anhydrous
magnesium sulfate.
O-(Carboxymethyl)hydroxylamine hemihydrochloride (98%
purity), tert-butyldimethylsilyl chloride (97% purity), 3,5-
dimethylpyrazole (99% purity), L-Selectride௡ (lithium tri-sec-
butylborohydride 1.0 M solution in tetrahydrofuran), and morin
hydrate (2Ј,3,4Ј,5,7-pentahydroxyflavone) were obtained from
Sigma-Aldrich, Czech Republic.
L-Selectride௡ (1 M solution in tetrahydrofuran, 7.8 mL) was added
under argon at Ϫ78°C to a solution of ketone 8 (3.38 g, 6.52
mmol) in tetrahydrofuran (70 mL). The mixture was stirred at
Ϫ78°C for 5 h, then water (4 mL) was added, and the mixture was
warmed to 0°C. An aqueous solution of sodium hydroxide (6 M, 9
mL) was added, followed by hydrogen peroxide (30%, 9 mL).
After stirring for 30 min at room temperature, the mixture was
diluted with ether (1.2 L), and the organic layer was washed
successively with ice cold 5% aqueous citric acid (3 times), water,
saturated aqueous potassium hydrogen carbonate (twice), and wa-
ter (twice). The solvent was evaporated, and the residue was
chromatographed on a column of silica gel (150 g) with a mixture
of petroleum ether-ethyl acetate (80:20). The yield of 9 was 2.43 g
(72%), m.p. 174–176°C (ether), [␣]D Ϫ119° (c 1.4, chloroform).
IR (chloroform): 3 603, 3 490 (O™H); 1 728 (C¢O); 1 669 (C¢C);
1 252 (C™O, acetate). 1H NMR: 5.84 (1 H, dd, J ϭ 5.2, JЈ ϭ 1.5,
6-H); 4.67 (1 H, m, W ϭ 32, 3␣-H); 3.90 (5 H, m, OCH2CH2O and
7␤-H); 3.77 and 3.62 (2 H, AB system, J(AB) ϭ 10.7, 19-H2);
2.04 (3 H, s, CH3COO); 0.89 (3 H, s, 18-H3); 0.86 (9 H, s,
(CH3)3Si); 0.04 (3 H, s, CH3Si); 0.03 (3 H, s, CH3Si). Analysis
calculated for C29H48O6Si (520.8): C, 66.88; H, 9.29. Found: C,
66.54; H, 9.08.
19-(tert-Butyldimethylsiloxy)-17,17-
ethylenedioxyandrost-5-en-3␤-yl acetate (7)
tert-Butyldimethylsilyl chloride (4.52 g, 30 mmol) was added at
0°C to a solution of hydroxy derivative6,7 6 (3.90 g, 10.0 mmol)
and imidazole (4.08 g, 60 mmol) in N,N-dimethylformamide (80
mL). The reaction mixture was allowed to stand at room temper-
ature overnight, diluted with ether (1.2 L), and washed succes-
sively with 5% aqueous citric acid (3 times), water (twice), satu-
rated aqueous potassium hydrogen carbonate (twice), and water
(twice). The solvent was evaporated, and the residue was chro-
matographed on a column of silica gel (200 g) with a mixture of
petroleum ether-ethyl acetate (96:4). The yield of 7 was 4.64 g
(92%), m.p. 150–152°C (ether), [␣]D Ϫ80° (c 1.5, chloroform). IR
(tetrachloromethane): 1 734 (C¢O); 1 246, 1 034 (C™O, acetate); 1
103 (C™OSi). 1H NMR: 5.57 (1 H, m, 6-H); 4.63 (1 H, m, W ϭ 32,
3␣-H); 3.87 (4 H, m, OCH2CH2O); 3.74 and 3.59 (2 H, AB
system, J(AB) ϭ 10.5, 19-H2); 2.04 (3 H, s, CH3COO); 0.88 (12
H, s, (CH3)3Si and 18-H3); 0.05 (3 H, s, CH3Si); 0.04 (3 H, s,
CH3Si). Analysis calculated for C29H48O5Si (504.8): C, 69.00; H,
9.58. Found: C, 68.73; H, 9.35.
17,17-Ethylenedioxy-19-hydroxyandrost-5-ene-
3␤,7␣-diyl diacetate (11)
Acetic anhydride (2.2 mL, 23.3 mmol) was added at 0°C to a
solution of hydroxy derivative 9 (1.5 g, 2.88 mmol) in pyridine (10
mL). After 10 h at room temperature, the reaction mixture was
poured on ice (200 g). The separated oily product was dissolved in
ether (300 mL), and the solution was washed successively with ice
cold 5% aqueous citric acid (3 times), water (twice), saturated
aqueous potassium hydrogen carbonate (twice), and water (twice).
1
Evaporation of the solvent gave the crude acetate 10 (1.62 g). H
NMR: 5.81 (1 H, dd, J ϭ 5.2, JЈ ϭ 1.2, 6-H); 5.02 (1 H, bt, J Ϸ
5, 7␤-H); 4.70 (1 H, m, W ϭ 32, 3␣-H); 3.91 (4 H, m,
OCH2CH2O); 3.81 and 3.64 (2 H, AB system, J(AB) ϭ 10.7,
19-H2); 2.04 (3 H, s, CH3COO); 2.05 (3 H, s, CH3COO); 0.88 (3
H, s, 18-H3); 0.86 (9 H, s, (CH3)3Si); 0.04 (3 H, s, CH3Si); 0.03 (3
H, s, CH3Si).
19-(tert-Butyldimethylsiloxy)-17,17-ethylenedioxy-7-
oxoandrost-5-en-3␤-yl acetate (8)
Tetrabutylammonium fluoride in tetrahydrofuran (1 M, 19
mL) was added to the solution of 10 (1.62 g) in tetrahydrofuran
(9 mL). After 24 h of stirring at room temperature, the mix-
ture was diluted with ethyl acetate (0.5 L), and washed succes-
sively with ice cold 10% aqueous sulfuric acid (3 times), water
(twice), saturated aqueous potassium hydrogen carbonate
(twice), and water (twice). The solvent was evaporated, and
the residue was chromatographed on a column of silica gel
(100 g) with a mixture of petroleum ether-ethyl acetate (92:8).
The yield of 11 was 1.13 g (87% calculated on the hydroxy
derivative 9), m.p. 167–170°C (ether), [␣]D Ϫ215°C (c 1.6,
chloroform). IR (chloroform): 3 620, 3 595 shoulder, 3 502
broad (O™H); 1 724 (C¢O); 1 252 (C™O acetate); 1 034, 1 018
(C™O). 1H NMR (CDCl3 ϩ CD3COOD; 5:1): 5.81 (1 H, d,
J ϭ 5, 6-H); 4.96 (1 H, t, J ϭ 5, 7␤-H); 4.61 (1 H, m, W ϭ 32,
3␣-H); 3.82 (4 H, m, OCH2CH2O); 3.81 and 3.61 (2 H,
AB system, J(AB) ϭ 11.9, 19-H2); 1.97 (3 H, s, CH3COO);
1.94 (3 H, s, CH3COO); 0.79 (3 H, s, 18-H3). Analysis calcu-
lated for C25H36O7 (448.6): C, 66.94; H, 8.09. Found: C, 66.78;
H, 8.08.
A suspension of chromium (VI) oxide (18.57 g, 185.7 mmol) in
dichloromethane (110 mL) was stirred at Ϫ25°C with 3,5-
dimethylpyrazole (18.39 g, 191.3 mmol). After 15 min, a solution
of olefin 7 (5.34 g, 10.6 mmol) in dichloromethane (40 mL) was
added dropwise. Stirring was continued at Ϫ20°C for 4.5 h, then
after dilution with a mixture of benzene-ethyl acetate (100 mL,
7:3), the reaction mixture was filtered through a short column of
silica gel (50 g) layered with Celite௡. The column was washed
with the same solvent mixture, and the solvents were evaporated.
Chromatography on a column of silica gel (260 g) with a mixture
of petroleum ether-ethyl acetate (95:5) gave 3.49 g (64%) of
ketone 8, m.p. 155–158°C (ether-hexane), [␣]D Ϫ139° (c 2.6,
chloroform). IR (tetrachloromethane): 1 738 (C¢O, acetate); 1 675
(C¢O, ketone); 1 638 (C¢C); 1 238, 1 034 (C™O, acetate). 1H
NMR: 5.87 (1 H, d, J ϭ 1.8, 6-H); 4.74 (1 H, m, W ϭ 32, 3␣-H);
3.87 (6 H, m, OCH2CH2O and 19-H2); 2.06 (3 H, s, CH3COO);
0.89 (3 H, s, 18-H3); 0.85 (9 H, s, (CH3)3Si) 0.07 (3 H, s, CH3Si);
0.04 (3 H, s, CH3Si). Analysis calculated for C29H46O6Si (518.8):
C, 67.14; H, 8.94. Found: C, 66.93; H, 9.07.
Steroids, 1998, vol. 63, September 455

Papers
for C26H35NO8 (489.6): C, 63.79; H, 7.21; N, 2.86. Found: C,
63.54; H, 7.35; N, 2.92.
(19E)-17,17-Ethylenedioxy-19-oxoandrost-5-ene-
3␤,7␣-diyl 3,7-diacetate 19-(O)-
methoxycarbonylmethyl)oxime (14)
Pyridine (3.57 mL, 44.1 mmol) was added dropwise at 0°C under
an argon atmosphere to a stirred suspension of chromium (VI)
oxide (1.47 g, 14.7 mmol) and anhydrous magnesium sulfate (2.0
g) in dichloromethane (50 mL), and the stirring continued at 0°C
for 20 min. Subsequently, a solution of hydroxy derivative 11
(1.10 g, 2.45 mmol) in dichloromethane (10 mL) was added, and
the reaction mixture was stirred under argon at 0°C for 3 h. After
dilution with ether (50 mL), the mixture was filtered through a
column of alumina (50 g), which was washed with an ether-
dichloromethane (1:1) mixture. The filtrate was concentrated, and
pyridine was removed from the residue by coevaporation with
toluene. The yield of the crude aldehyde 12 was 1.02 g (93%). 1H
NMR: 9.65 (1 H, d, J ϭ 0.9, 19-H); 6.09 (1 H, dd, J ϭ 5, JЈ ϭ 2,
6-H); 5.17 (1 H, bt, J ϭ 5, 7␤-H); 4.69 (1 H, m, W ϭ 32, 3␣-H);
3.92 (4 H, m, OCH2CH2O); 2.09 (3 H, s, CH3COO); 2.03 (3 H, s,
CH3COO); 0.80 (3 H, s, 18-H3).
A mixture of aldehyde 12 (1.02 g, 2.28 mmol), O-(carboxy-
methyl) hydroxylamine hemihydrochloride (749 mg, 6.85 mmol),
and pyridine (15 mL) was stirred at room temperature for 60 h.
Toluene (30 mL) was added, and the solvents were evaporated.
The residue was dissolved in ethyl acetate (300 mL) and 5% citric
acid (150 mL), the aqueous phase was extracted with ethyl acetate
(300 mL), and the combined organic phase was washed with 5%
citric acid (twice) and then with water (twice). The solvent was
evaporated, and the residue (crude acid 13) was dissolved in ether
(70 mL) and methanol (30 mL) and treated with an ethereal
solution of diazomethane at 0°C for 5 min. Excess diazomethane
and solvents were evaporated, and the residue was chromato-
graphed on a column of silica gel (30 g) in a mixture of
dichloromethane-ether (50:50). The yield of the methyl ester 14
was 1.21 g (92% calculated on hydroxy derivative 11), m.p.
183–185°C (ether), [␣]D Ϫ248°C (c 1.5, chloroform). IR (tetra-
chloromethane): 1 765 (C¢O, OCH2COOCH3); 1 734 (C¢O, ace-
tate); 1 240 (C™O, acetate); 1 037, 1 023 (C™O). 1H NMR: 7.37 (1
H, s, 19-H); 5.83 (1 H, d, J ϭ 5, JЈ ϭ 2, 6-H); 5.07 (1 H, bt, J Ϸ
5, 7␤-H); 4.70 (1 H, m, W ϭ 32, 3␣-H); 4.63 (2 H, s, OCH2COO);
3.90 (4 H, m, OCH2CH2O); 3.76 (3 H, s, COOCH3); 2.06 (3 H, s,
CH3COO); 2.02 (3 H, s, CH3COO); 0.82 (3 H, s, 18-H3). Analysis
calculated for C28H39NO9 (533.6): C, 63.02; H, 7.37; N, 2.62.
Found: C, 63.24; H, 7.25; N, 2.75.
(19E)-17,19-Dioxo-3␤-hydroxyandrost-5-en-7␣-yl
7-acetate 19-(O-methoxycarbonylmethyl)oxime (16)
and (19E)-3␤,7␣-dihydroxy-17-oxoandrost-5-en-19-
al 19-(O-methoxycarbonylmethyl)oxime (17)
Compound 15 (475 mg, 0.97 mmol) was dissolved in tetrahydro-
furan (12 mL) and methanol (3 mL). After addition of 0.4 M
aqueous sodium hydroxide (10 mL), the mixture was stirred at
room temperature for 5 days. Excess alkali was neutralized with
5% hydrochloric acid, and the solvents were evaporated. The
residue was partitioned between ethyl acetate (100 mL) and water
(50 mL), and the aqueous layer was extracted with ethyl acetate
(4 ϫ 100 mL). The combined extract was washed with water, and
the solvent was evaporated. The residue was dissolved in ether (40
mL) and methanol (10 mL) and treated with an ethereal solution of
diazomethane for 5 min at 0°C. Excess diazomethane and solvents
were evaporated, and the residue was chromatographed on 6
preparative silica gel plates with a mixture of benzene-acetone
(50:50). Zones containing less polar compound were collected and
eluted with acetone-dichloromethane (50:50). The yield of 16 was
45 mg (10%), m.p. 131–134°C (dichloromethane-hexane), [␣]D
Ϫ191° (c 1.4, chloroform). IR (chloroform): 3 608, 3 506 broad,
3 459 broad (O™H); 1 756 shoulder (C¢O), OCH2COOCH3); 1 732
(C¢O, ketone and acetate); 1 246 (C™O, acetate); 1 093, 1 058,
1 050, 1 026, 1 016 (C™O). 1H NMR: 7.38 (1 H, s, 19-H); 5.83 (1
H, dd, J ϭ 5.3, JЈ ϭ 1.7, 6-H); 5.18 (1 H, bt, J Ϸ 5.5, 7␤-H); 4.62
(2 H, s, OCH2COO); 3.75 (3 H, s, COOCH3); 3.63 (1 H, m, W ϭ
32, 3␣-H); 2.05 (3 H, s, CH3COO); 0.86 (3 H, s, 18-H3). Analysis
calculated for C24H33NO7 (447.5): C, 64.41; H, 7.43; N, 3.13.
Found: C, 64.57; H, 7.65; N, 2.87.
Zones containing more polar compound were collected and
eluted with acetone-dichloromethane (50:50). The yield of 17 was
265 mg (67%), m.p. 201–204°C (ether), [␣]D Ϫ138°C (c 1.2,
chloroform). IR (chloroform): 3 606, 3 500 shoulder, 3 459 broad
(O™H); 1 755 shoulder (C¢O, OCH2COOCH3); 1 735 (C¢O,
ketone); 1 109, 1 091, 1 050, 1 026, 1 011 (C™O). 1H NMR: 7.37
(1 H, s, 19-H); 5.87 (1 H, dd, J ϭ 5.5, JЈ ϭ 1.8, 6-H); 4.62 (2 H,
s, OCH2COO); 4.07 (1 H, bt, J ϭ 5.5, 7␤-H); 3.75 (3 H, s,
COOCH3); 3.62 (1 H, m, W ϭ 32, 3␣-H); 0.86 (3 H, s, 18-H3).
Analysis calculated for C22H31NO6 (405.5): C, 65.17; H, 7.71; N,
3.45. Found: C, 65.27; H, 7.55; N, 3.34.
(19E)-17,19-Dioxoandrost-5-ene-3␤,7␣-diyl 3,7-
diacetate 19-(O-methoxycarbonylmethyl)oxime (15)
Water (10 mL) and 4-toluenesulfonic acid monohydrate (80 mg,
0.42 mmol) were added to a solution of acetal 14 (1.15 g, 2.16
mmol) in acetone (50 mL). The reaction mixture was stirred at
room temperature for 5 days, the solvents were evaporated, and the
residue was dissolved in ether (200 mL), and water (50 mL). The
aqueous phase was extracted with ether (200 mL), and the com-
bined organic phase was washed with aqueous potassium hydro-
gen carbonate solution and water (twice). The solvent was evap-
orated, and the residue was chromatographed on a column of silica
gel (70 g) with a mixture of benzene-ether (90:10). The yield of 15
was 798 mg (76%), m.p. 173–175°C (ether), [␣]D Ϫ199° (c 1.6,
(19E)-3␤,7␣-Dihydroxy-17-oxoandrost-5-en-19-al
19-(O-carboxymethyl)oxime (4)
Aqueous sodium hydroxide (0.4 M, 3.2 mL) was added to a stirred
solution of compound 17 (265 mg, 0.65 mmol) in a mixture of
tetrahydrofuran (5 mL) and methanol (1 mL), and the stirring
continued at room temperature for 40 h. Then, the solvents were
evaporated, and the residue was partitioned between ethyl acetate
(100 mL) and water (50 mL). The aqueous layer was acidified with
5% hydrochloric acid, the separated product was collected on a
filter, washed with water, and dried over phosphorus pentoxide in
vacuo to give 152 mg (59%) of acid 4, m.p. 256–258°C (decom-
position), [␣]D Ϫ88°C (c 0.5, dimethyl sulfoxide). IR (KBr pellet):
3 237 (O™H); 2 550 (COOH); 1 729 (C¢O, ketone); 1 241, 1 084
(C™O). 1H NMR (CD3SOCD3): 7.39 (1 H, s, 19-H); 5.66 (1 H, bd,
J Ϸ 5, 6-H); 4.48 (2 H, s, OCH2COO); 3.80 (1 H, bs, 7␤-H); 0.74
(3 H, s, 18-H3). Analysis calculated for C21H29NO6 (391.5): C,
64.43; H, 7.47; N, 3.58. Found: C, 64.33; H, 7.65; N, 3.41.
chloroform). IR (chloroform):
1
756 shoulder (C¢O,
OCH2COOCH3); 1 730 (C¢O, ketone and acetate); 1 248 (C™O,
acetate); 1 112, 1 093, 1 038, 1 028, 1 015 (C™O). 1H NMR: 7.39
(1 H, s, 19-H); 5.85 (1 H, dd, J ϭ 5.3, JЈ ϭ 1.7, 6-H); 5.18 (1 H,
bt, J Ϸ 5, 7␤-H); 4.71 (1 H, m, W ϭ 32, 3␣-H); 4.63 (2 H, s,
OCH2COO); 3.76 (3 H, s, COOCH3); 2.06 (3 H, s, CH3COO);
2.03 (3 H, s, CH3COO); 0.86 (3 H, s, 18-H3). Analysis calculated
456 Steroids, 1998, vol. 63, September

New hapten for 7␣-hydroxydehydroepiandrosterone: Pouzar et al.
performed10 by 3,5-dimethylpyrazole and chromium (IV)
oxide complex in dichloromethane. The resulting ketone 8
was reduced8 by L-Selectride௡ to a 7␣-hydroxy derivative 9.
Subsequent acetylation gave diacetate 10 from which the
tert-butyl-dimethylsilyl group was cleaved by tetrabutylam-
monium fluoride in tetrahydrofuran. The 1H NMR spectrum
of the obtained derivative 11 displayed signals characteris-
tic11 for the 7␣-acetoxy group on the androst-5-ene skele-
ton, i.e. doublet at ␦ 5.81 (J ϭ 5 Hz, 6-H) and triplet at ␦
4.96 (J ϭ 5 Hz, 7␤-H), besides signals of 19-H2 (␦ 3.81 and
3.61, AB system, J ϭ 11.9 Hz) and 3␣-H (␦ 4.61, m, W ϭ
32 Hz).
The hydroxy derivative 11 was oxidized12 by pyridine
and chromium (VI) oxide complex in dichloromethane to
aldehyde 12. Reaction with O-(carboxymethyl)hydrox-
ylamine in pyridine and subsequent diazomethane esterifi-
cation gave a protected 19-(O-carboxymethyl)oxime deriv-
ative 14. At this stage, the ketal protecting group in position
17 was cleaved by prolonged treatment13 with 4-toluene-
sulfonic acid in aqueous acetone. The presence of an intact
19-CMO group was proved by the 1H NMR spectrum of the
obtained 17-oxo derivative 15. Three singlets at ␦ 7.39
(19-H), ␦ 4.63 (OCH2COO), and ␦ 3.76 (COOCH3) are
characteristic14 for protected 19-CMO derivatives with
(19E)-configuration.
Due to limited solubility of functionalized steroid oximes
and very slow and incomplete deacetylation in the present
case, the final deprotection of derivative 15 was performed
in two successive steps. The first involved deacetylation by
aqueous sodium hydroxide in a mixture of tetrahydrofuran
and methanol. Since the methyl ester was cleaved during the
reaction, the resulting mixture was reesterified with dia-
zomethane, and products were separated by preparative
thin-layer chromatography. The less polar product (yield
10%) was monoacetate 16, with a protecting acetyl group in
1
position 7 as follows from the H NMR shift of 7␤-H (␦
5.18). The more polar product (yield 67%) was fully
deacetylated 17. The protecting methyl ester group was
removed from 17 by aqueous sodium hydroxide in a mix-
ture of tetrahydrofuran and methanol, and the final product
4 was obtained in 59% yield. Its IR spectrum in a KBr pellet
contained characteristic bands of hydroxyl groups (3 237
Scheme 1
Results and discussion
The most feasible method for the preparation of 7␣-hydroxy
derivatives in the steroid series8 is the reduction of the
corresponding 7-oxo derivatives with L-Selectride௡
(Scheme 1). Originally, we intended to apply this method to
cmϪ1), carboxylic acid (2 550 cmϪ1, broad), and carbonyl
groups (1 729 cmϪ1). The 1H NMR spectrum in
CD3SOCD3 contained two characteristic singlets of the
19-CMO group (␦ 7.39 and ␦ 4.48), signals of 7␤-H (␦
3.80), 6-H (␦ 5.66), and 18-H3 (␦ 0.74). The signal of 3␣-H
was overlapped with the signal of residual water in the
solvent.
(19E)-7,19-dioxoandrost-5-ene-3␤,17␤-diyl
17-acetate
3-benzoate 19-(O-methoxycarbonylmethyl)oxime, an inter-
mediate from the synthesis of 5; however, the model exper-
iment revealed instability of the 19-CMO group during the
reaction. The 1H NMR spectrum of the product contained a
signal at ␦ 5.2 ppm, corresponding to the presence of a
7␣-hydroxy group, but it lacked the singlet at ␦ 7.4 ppm,
characteristic for 19-H in 19-CMO derivatives. This forced
us to change the strategy and to move onto the construction
of the 19-CMO group only after reduction of the 7-oxo
group in the suitable precursor.
Immunological properties of 19-CMO derivative 4 will
be published separately.
Acknowledgments
The authors are indebted to Dr. P. Fiedler for performing
and interpreting the IR spectra. Our thanks are due to M.
As a starting compound, we used the known6,7 19-
hydroxy derivative 6. Its reaction9 with tert-butyldimethyl-
silyl chloride and imidazole in N,N-dimethylformamide
gave a protected derivative 7. Oxidation in position 7 was
1
Snopkova´ for measurements of the H NMR spectra. The
elemental analyses were carried out in the Analytical Lab-
oratory (Dr. V. Pechanec, head of this institute).
Steroids, 1998, vol. 63, September 457

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