17β-Hydroxy-11α-(3'-sulfanylpropyl)oxy-estra-1,3,5(10)-trien-3-yl sulfamate - A novel hapten structure: Toward the development of a specific enzyme immunoassay (EIA) for estra-1,3,5(10)-triene-3-yl sulfamates

Article abstract of DOI:10.1016/S0039-128X(99)00020-3

The title compound 17 has been synthesized for the use as hapten in the development of a competitive enzyme immunoassay for estrogen sulfamates. The synthesis started from estradiol diacetate 2. Oxyfunctionalization at C-11 to give 11α-hydroxy steroid 8 was accomplished by hydroboration/alkaline hydrogen peroxide oxidation of the 9(11)-dehydro derivative 7, which was obtained from compound 2 via 9-hydroxylation with dimethyldioxirane. After transformation of compound 8 into the allyl ether 9, the side chain was thio- functionalized at the ω-position affording the thioate 11 in two steps. Selective silylether deprotection at position 3 followed by sulfamoylation gave the sulfamate 19, which in turn was demasked at position 17 and treated with sodium borohydride/aluminum chloride to liberate the side chain thiol. Alternatively, title compound 17 was synthesized via the disulfides 13-16. For the preparation of the immunogen the title compound 17 was coupled to bovine gamma globulin in a two-step procedure using an amine and thiol specific bifunctional crosslinker. The immunization of rabbits resulted in the formation of antibodies which clearly discriminated the sulfamoylated estrogens from the non-esterified estrogens. The use of a biotinylated hapten derivative as a tracer in combination with a streptavidin-peroxidase- tetramethylbenzidine based detection system allowed the measurement of estradiol 3-sulfamate (1) in the range of about 1 to 1000 pg/well.

Full text of DOI:10.1016/S0039-128X(99)00020-3

Steroids 64 (1999) 460–471
17␤-Hydroxy-11␣-(3Ј-sulfanylpropyl)oxy-estra-1,3,5(10)-trien-3-yl
sulfamate – a novel hapten structure: Toward the development of a
specific enzyme immunoassay (EIA) for estra-1,3,5(10)-triene-3-yl
sulfamates
Sigfrid Schwarza,*, Matthias Schumacherb, Sven Ringa, Anita Nanningab, Gisela Webera,
Ina Thiemea, Bernd Undeutscha, Walter Elgerc
aDivision of Research and Development, Jenapharm GmbH & Co.KG, Jena, Germany
bInstitute for Hormone and Fertility Research, Hamburg, Germany
cEnTec GmbH Hamburg/Jena, Jena, Germany
Received 11 September 1998; accepted 16 December 1998
Abstract
The title compound 17 has been synthesized for the use as hapten in the development of a competitive enzyme immunoassay for estrogen
sulfamates. The synthesis started from estradiol diacetate 2. Oxyfunctionalization at C-11 to give 11␣-hydroxy steroid 8 was accomplished
by hydroboration/alkaline hydrogen peroxide oxidation of the 9(11)-dehydro derivative 7, which was obtained from compound 2 via
9-hydroxylation with dimethyldioxirane. After transformation of compound 8 into the allyl ether 9, the side chain was thio-functionalized
at the ␻-position affording the thioate 11 in two steps. Selective silylether deprotection at position 3 followed by sulfamoylation gave the
sulfamate 19, which in turn was demasked at position 17 and treated with sodium borohydride/aluminum chloride to liberate the side chain
thiol. Alternatively, title compound 17 was synthesized via the disulfides 13–16. For the preparation of the immunogen the title compound
17 was coupled to bovine gamma globulin in a two-step procedure using an amine and thiol specific bifunctional crosslinker. The
immunization of rabbits resulted in the formation of antibodies which clearly discriminated the sulfamoylated estrogens from the
non-esterified estrogens. The use of a biotinylated hapten derivative as a tracer in combination with a streptavidin-peroxidase-tetrameth-
ylbenzidine based detection system allowed the measurement of estradiol 3-sulfamate (1) in the range of about 1 to 1000 pg/well. © 1999
Elsevier Science Inc. All rights reserved.
Keywords: Steroid thiols; Steroid disulfides; Estrogen sulfamates; Steroid hapten; Immunoassay for estrogens; Hydroboration; Alkaline hydrogen peroxide
oxidation
1. Introduction
estradiol-3-sulfamate (1, J 995) (Scheme 1) as a first se-
lected estrogen sulfamate.
Pharmacokinetic studies in rats with H-labeled sulfa-
mate 1 have shown that almost 98% of H-levels in the
3
Steroidal estrogens esterified at position 3 by sulfamic
acid (estrogen sulfamates), have a potential to overcome the
liver barrier without alteration of important hepatic func-
tions if administered orally. A confirmation of the current
animal experiments by clinical trials may lead to new drugs
with fundamental benefits in the hormone replacement ther-
apy in women or in the therapy of prostate cancer [1,2].
Recently, clinical Phase I studies have been started, using
3
blood were detected in the erythrocyte fraction, whereas
only 2% were found in the plasma. Upon oral administra-
tion, the main part of the erythrocyte-bound steroid proved
to be estrone 3-sulfamate (2, J 994). However, after intra-
venous (i.v.) administration, estradiol 3-sulfamate (1) was
the dominant species [3].
Sulfamate 1 has been found to be incapable of specific
binding to uterine cytosol and of displacing estradiol from
the estrogen receptor at high concentrations. Thus, estradiol
3-sulfamate (1) and its metabolite estrone sulfamate (2)
*Corresponding author. Tel.: ϩ49-36-41-64-6218; fax: ϩ49-36-41-64-
6085.
E-mail address: sigfrid.schwarz@jenapharm.de (S. Schwarz)
0039-128X/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.
PII: S00039-128X(99)00020-3

S. Schwarz et al. / Steroids 64 (1999) 460–471
461
defined coupling of the hapten molecule to the carrier pro-
tein which is a prerequisite for obtaining sulfamate specific
antibodies.
We report here on our efforts in the synthesis of the
11␣-(3Ј-sulfanylalkyl)oxy substituted compound 1 (17) [9]
and in the characterization of specific antibodies raised
against a bovine ␥-globulin conjugate derived from hapten
17.
Scheme 1. Estrone sulfamate (J 994) and estradiol 3-sulfamate (J 995).
2. Experimental
appear to be prodrugs which exert estrogenic effects only
after cleavage of the sulfamate moiety. As yet, the site(s)
and the mechanism of the estradiol/estrone liberation from
sulfamates 1/2 are unknown. However, to fully understand
the pharmacokinetic behavior of estradiol 3-sulfamate (1), a
detailed insight into the complex bioactivating process is
essential. As a result, pharmacokinetic studies as part of our
ongoing clinical trials of compound 1 called for a highly
sensitive and specific immunoassay allowing us to quanti-
tatively detect picomolar amounts of estra-1,3,5 (10)-trien-
3-yl sulfamates in the presence of the corresponding estra-
1,3,5 (10)-trien-3-ols.
2.1. Reagents and equipment
All reactions were run under argon protection, and, if
essential, with strict protection from moisture. Solvents and
chemicals (Sigma-Aldrich Chemie GmbH, Deisenhofen/
Germany, Merck KGaA, Darmstadt/Germany) were reagent
grade or analytical grade and were used without further
purification. Estradiol 3-sulfamate, estrone sulfamate, 17␣-
estradiol 3-sulfamate, and estriol 3-sulfamate were prepared
as described previously [10,11], as were estra-1,3,5 (10)-
trien-3,17␤-diyl 17-pentanoate, 3-sulfamate [11], estrone
(N-acetyl)sulfamate, estradiol 3-(N-acetyl)sulfamate, and
estriol 3-(N-acetyl)sulfamate [12]. Estradiol 3-pentanoate,
estradiol, 17␣-estradiol, ethinylestradiol, sodium estradiol
3-sulfate, and estradiol 3-glucuronide were purchased from
Sigma-Aldrich Chemie GmbH, Deisenhofen (Germany).
Organic solutions were dried with anhydrous sodium sulfate
or magnesium sulfate monohydrate. Evaporation in vacuo
was carried out with a rotary evaporator. Chromatography
means flash chromatography on Kieselgel 60 (Merck-Darm-
stadt, 0.04–0.063 mm); eluents were given in volume pro-
portions. Melting points (m.p.) were measured with a Boe-
tius equipment and are uncorrected. Optical rotations were
taken with a Jasco DIP-1000 equipment. Unless otherwise
stated, chloroform was used as solvent, c ϭ 1 g/100 ml, t ϭ
Immunogens obtained by conjugating estra-1,3,5 (10)-
trienes with a protein by means of a 6-(O)-carboxymethyl
oxime moiety often fail to produce antisera with the desired
specificity which may be due to E-/Z-isomerism of the
oxime and conformational distortion of the B-ring by the
sp2 hybridized C-6 [4,5]. Antisera directed against the es-
tradiol-6-(O)-carboxymethyl oxime, in general, are specific
to the D-ring of the steroid molecule (only little cross-
reaction with estrone- and estriol-derivatives), but less spe-
cific to the A-ring showing substantial cross-reaction with
C-3 conjugated estradiol or other A-ring substituted deriv-
atives (Schumacher M, unpublished results and Refs. [6]
and [7]). This also proved to be true for some antisera used
in commercially available radioimmunoassays of estradiol
which displayed a considerable cross-reactivity with sulfa-
mate 1 (Elger W, unpublished results). However, coupling
A-ring aromatic steroids to the protein through non-rigid
carboxyalkyl ether linkages was shown to give immunogens
capable of generating highly specific antisera [5] (Schuma-
cher M, unpublished results and Ref. [8]). With this back-
ground, we decided to couple sulfamate 1 to the protein
carrier through a (3Ј-sulfanyl) propanoxy spacer attached at
11␣-position. On three counts, the hapten 17 (Scheme 4)
was thought to meet the requisites for generating highly
selective and sensitive antibodies upon conjugation with a
suitable carrier protein. First, the spacer being arranged far
from position 3 was assumed to facilitate the differentiation
between the 3-sulfamate group and the phenolic hydroxy
function. Second, the highly flexible spacer bound to the sp3
hybridized C-11 in an equatorial arrangement was consid-
ered to closely mimic unmodified sulfamate 1. Third, the
␻-sulfanyl group of the spacer offered the possibility of a
1
ϩ20°C. H NMR spectra were recorded on a Varian 300.
Unless otherwise stated, deuteriochloroform was used as
solvent. Chemical shifts were reported as ␦-values in ppm
downfield from tetramethylsilane as internal standard, J was
given in Hz. The spectral data were recorded as ␦ (coupling
pattern, J, proton number). Mass spectra (EI) were taken
with an MS AMD 402 (BE configuration) of AMD- Intec-
tra, Harpstedt. Mass spectrometry by electrospray ionization
(ESI) was run with a VG-Quattro of Pfizens, VG Biotech,
Altricham. Acronyms in the text were used according to
Ref. [13].
2.2. 9␣-Hydroxy-estra-1,3,5 (10)-triene-3,17␤-diyl
diacetate (4)
To a solution of estra-1,3,5 (10)-triene-3,17␤-diyl 3,17-
diacetate 3 (10 g, 28 mmol) in dichloromethane (200 ml)
were added water (220 ml), sodium hydrogen carbonate (64
g), acetone (177 ml), and tetrabutyl ammonium hydrogen

462
S. Schwarz et al. / Steroids 64 (1999) 460–471
sulfate (0.1 g). The suspension was cooled to ϩ12°C and
oxone (133 g) was added over a period of 2.5 h with
vigorous stirring. The suspension was then stirred for an-
other 3.5 h at 15°C–20°C. The reaction mixture was filtered
and the separated organic layer was evaporated in vacuo.
Purification of the residue by chromatography (eluent: tol-
uene/ethyl acetate 10:1) and crystallization from methanol
afforded compound 4 (8.36 g, 80%) identical in all respects
with reported data [14].
2.5. 3,17␤-Bis-(t-butyldimethylsilyl)oxy-estra-1,3,5 (10)-
trien-11␣-ol (8)
At 50°C, a stirred solution of compound 7 (21.8 g,
43.7 mmol) in tetrahydrofuran (100 ml) was treated with
borane-dimethyl sulfide complex (12.5 ml, 130 mmol) for
1 h. Upon cooling to 0°C, the reaction was then carefully
quenched with water (20 ml). Subsequently, a cold (5°C)
aqueous mixture of sodium hydroxide (9.6 g, 240 mmol)
and hydrogen peroxide (30%, 40 ml) in water (80 ml)
was added. The mixture was stirred for another 1 h at
room temperature and then extracted several times with
n-hexane. The combined organic fractions were washed
with water, dried and evaporated in vacuo to dryness. The
residue was purified by chromatography (eluent: toluene/
cyclohexane 1:1), followed by crystallization from ethyl
acetate/methanol to yield compound 8 (16.87 g, 74.5%).
2.3. Estra-1,3,5(10),9(11)-tetraene-3,17␤-diol (6)
Compound 4 (10 g, 27 mmol) was dissolved in dichlo-
romethane (200 ml). The solution was cooled to Ϫ20°C and
stirred with 70% aqueous sulfuric acid (1 ml) for 2 h. The
mixture was then treated with saturated sodium hydrogen
carbonate solution until neutral. The organic phase was
dried and evaporated in vacuo. The resulting estra-1,3,5
(10)9(11)-tetraene-3,17␤-diyl diacetate 5 (9 g, 25.4 mmol)
was dissolved in methanol (133 ml), potassium hydroxide
(6.65 g, 118.5 mmol) was added and the solution was stirred
at 40°C for 4 h. The main part of the methanol was distilled
off in vacuo and to the resulting solution 1 N aqueous
hydrochloric acid was added until neutral. On addition of
water (300 ml), the precipitated crystals were filtered off
and recrystallized from methanol to give compound 6 (4 g,
65%). The substance retained water tenaciously (1.7%),
even upon prolonged drying in vacuo. m.p. 186–191°C.
1
m.p. 141°C-146°C. [␣]D-46°. H nmr: 7.74 (d, 8.7, H-1),
6.64 (dd, 8.7, 2.8, H-2), 6.57 (d, 2.8, H-4), 4.21 (m,
H-11), 3.67 (t, 8.1, H-17), 2.76 (t, 6.7, H-6), 2.23 (dd,
11.6, 5.0, H-12), 2.11 (t, 9.5, H-9), 0.97 (s, -C(CH3)3),
0.89 (s, -C(CH3)3), 0.73 (s, H-18), 0.19 (s, Si-CH3), 0.04
(s, Si-CH3), 0.02 (s, Si-CH3). ms: (EI) m/z 516.34582
(M)ϩ. C30H52O3Si2 (516.92) calculated C 69.71 H 10.14
found C 69.75 H 10.15.
2.6. 3,17␤-Bis-(t-butyldimethylsilyl)oxy-11␣-(prop-2Ј-
enyl)oxy-1,3,5 (10)-triene (9)
1
[␣]D ϩ 132°. H nmr (d6-DMSO): 9.22 (s, 3-OH), 7.41 (d,
At 0°C, a solution of compound 8 (62.9 g, 122 mmol)
and allyl bromide (88.5 g, 730 mmol) in tetrahydrofuran
(400 ml) was treated portionwise with potassium t-butoxide
(41 g, 365 mmol). Two min after completion of addition, the
reaction was quenched with saturated aqueous ammonium
chloride solution (100 ml). The mixture was extracted with
toluene, the combined extracts were washed with water,
dried, evaporated in vacuo, and the residue was purified by
crystallization from methanol to give compound 9 (43 g,
63.5%). m.p. 121°C–124°C. [␣]D-56°. 1H nmr: 7.53 (d, 8.8,
H-1), 6.60 (dd, 8.8, 2.8, H-2), 6.54 (d, 2.8, H-4), 6.03 (m,
H-3Ј), 5.34 (dq, 17.1, 1.6, H-4Ј), 5.18 (dq, 10.3, 1.7, H-4Ј),
4.25 (ddt, 12.7, 5.9, 1.1, H-2Ј), 4.25 (ddt, 12.7, 5.4, 1.6,
H-2Ј), 3.86 (td, 10.1, 4.7, H-11), 3.66 (t, 8.4, H-17), 2.76
(dd, 6.8, 6.3, H-6), 2.39 (dd, 12.2, 5.5, H-12), 2.30 (t, 10.1,
H-9), 1.12 (dd, 11.2, 10.5, H-12), 0.97 (s, -C(CH3)3), 0.90
(s, -C(CH3)3), 0.71 (s, H-18), 0.18 (s, Si-CH3), 0.05 (s,
Si-CH3), 0.03 (s, Si-CH3). ms: m/z 556.37500 (M)ϩ.
C33H56O3Si2 (556.98) calculated C 71.16 H 10.13 found C
71.20 H 10.15.
8.8, H-1) 6.54 (dd, 8.8, 2.6, H-2), 6.45 (d, 2.6, H-4), 6.03 (d,
4.9, H-11), 4.57 (d, 4.7, 17-OH), 3.63 (td, 8.5, 4.9, H-17),
2.72 (m, H-6), 0.68 (s, H-18). ms: (EI) m/z 270.0 (M)ϩ.
C18H22O2 (270.37) calculated: with 1.7% water C 79.19 H
8.20 found C 78.81 H 8.28.
2.4. 3,17␤-Bis-(t-butyldimethylsilyl)oxy-estra-
1,3,5(10),9(11)-tetraene (7)
To a solution of compound 6 (20 g, 74 mmol) in
dimethylformamide (200 ml) and pyridine (200 ml) was
added t-butyl-dimethylsilylchloride (55.76 g, 370 mmol).
The mixture was stirred at 60–70°C for 3 h, then cooled
to ϩ10°C, poured into saturated aqueous sodium hydro-
gen carbonate solution (1-l), and extracted with toluene.
The combined extracts were concentrated in vacuo and
the residue was purified by chromatography (eluent: tol-
uene), followed by crystallization from methanol to af-
ford compound 7 (31.45 g, 85%). m.p. 130–132°C. [␣]D
1
ϩ 79°. H nmr: 7.46 (d, 8.9, H-1), 6.62 (dd, 8.9, 2.8,
H-2), 6.54 (d, 2.8, H-4), 6.11 (m, H-11), 3.72 (t, 8.4,
H-17), 2.79 (m, H-6), 0.97 (s, -C(CH3)3), 0.90 (s,
-C(CH3)3), 0.77 (s, H-18), 0.18 (s, Si-CH3), 0.05 (s,
Si-CH3), 0.03 (s, Si-CH3). ms: (EI) m/z 498.33661 (M)ϩ.
C30H50O2Si2 (498.90) calculated C 72.23 H 10.10 found
C 72.31 H 10.08.
2.7. 3,17␤-Bis-(t-butyldimethylsilyl)oxy-11␣-(3Ј-
hydroxypropyl)oxy-estra-1,3,5 (10)-triene (10)
Compound 9 (43 g, 77 mmol), dissolved in tetrahydrofuran
(300 ml), was allowed to react with 9-borabicyclo
[3.3.1]nonane (37.7 g, 154.4 mmol) for 1 h at 65°C with

S. Schwarz et al. / Steroids 64 (1999) 460–471
463
1
stirring. After that, the solution was cooled to 0°C and the
reaction was quenched with water (30 ml). 3 N aqueous so-
dium hydroxide solution (200 ml) and aqueous hydrogen per-
oxide solution (200 ml, 30%) were added and the mixture was
stirred at room temperature for 30 min. The product was
isolated by extraction with n-hexane, chromatography (eluent:
cyclohexane/methyl t-butyl ether 10:1) and crystallization
from methanol/water to give compound 10 (42.6 g, 96%). m.p.
88°C–91°C. [␣]D-57°. 1H NMR: 7.40 (d, 8.3, H-1), 6.63 (dd,
8.3, 2.7, H-2), 6.55 (d, 2.7, H-4), 3.89 (m, H-1Ј), 3.80 (m,
H-3Ј), 3.80 (m, H-11), 3.68 (m, H-17), 3.68 (m, H-1Ј), 2.76 (t,
7.1, H-6), 2.42 (dd, 11.9, 4.9, H-12), 0.98 (s, -C(CH3)3), 0.90
(s, -C(CH3)3), 0.70 (s, H-18), 0.19 (s, Si-CH3), 0.06 (s, Si-
CH3), 0.03 (s, Si-CH3). ms: (EI) m/z 574.38702 (M)ϩ.
C33H58O4Si2 (575.00) calculated C 68.93 H 10.17 found C
68.80 H 10.12.
[␣]D-53°. H nmr: 7.39 (d, 8.6, H-1), 6.62 (dd, 8.6, 2.7,
H-2), 6.55 (d, 2.7, H-4), 3.80 (m, H-1Ј), 3.80 (m, H-11),
3.66 (t, 8.0, H-17), 3.58 (dt, 9.3, 6.1, H-1Ј), 2.75 (t, 6.7,
H-6), 2.69 (d, 6.7, H-3Ј), 2.65 (d, 6.7, H-3Ј), 2.40 (dd,
11.8, 5.1, H-12), 2.23 (t, 9.6, H-9), 1.06 (t, 11.8, H-12),
0.98 (s, -C(CH3)3), 0.90 (s, -C(CH3)3), 0.70 (s, H-18),
0.19 (s, Si-CH3), 0.06 (s, Si-CH3), 0.03 (s, Si-CH3). ms:
(EI) m/z 590.36621 (M)ϩ. C33H58O3S Si2 (591.06) cal-
culated C 67.06 H 9.89 S 5.42 found C 66.83 H 9.89 S
5.43.
2.10. Bis-3-[3Ј,17Ј␤-bis-(t-butyldimethylsilyl)oxy-estra-
1Ј,3Ј,5Ј(10Ј)-trien-11Ј␣-yl]oxypropyl-disulfane (13)
A stirred suspension of compound 12 (14 g, 23.7 mmol)
in dichloromethane (150 ml) and saturated aqueous sodium
hydrogen carbonate solution (150 ml) was treated dropwise
with a solution of iodine (3.18 g, 2.45 mmol) in ethanol (250
ml) until the iodine color did no longer disappear. The
organic phase was separated, dried, and evaporated in
vacuo. After trituration with methanol compound 13 (14 g,
nearly quantitative yield) was obtained. m.p. 83°–90°C.
2.8. S-3-[3Ј,17Ј␤-Bis-(t-butyldimethylsily)oxy-estra-
1Ј,3Ј,5Ј(10Ј)-trien-11Ј␣-yl]oxypropyl ethanethioate (11)
A 0°C cold solution of triphenylphosphine (20.69 g, 78.6
mmol) and diisopropyl azo-dicarboxylate (15.89 g, 78.6
mmol) in tetrahydrofuran (200 ml) was stirred for 30 min.
Then, in turn, a solution of compound 10 (22.6 g, 39.3
mmol) in tetrahydrofuran (150 ml) and thioacetic S-acid
(5.97 g, 78.6 mmol) in tetrahydrofuran (15 ml) were added.
The resulting mixture was stirred for another 45 min and,
after that, evaporated in vacuo. The residue was treated with
n-hexane/methyl t-butyl ether and the crystals (triph-
enylphosphane oxide) were filtered off. The filtrate was
evaporated in vacuo and the residue was purified by chro-
matography (eluent:toluene) followed by crystallization
from methanol to afford compound 11 (22.5 g, 90.4%). m.p.
1
[␣]D-46°. H nmr: 7.38 (d, 8.3, H-1), 6.62 (dd, 8.3, 2.9,
H-2), 6.55 (d, 2.9, H-4), 3.80 (m, H-1Ј), 3.80 (m, H-11),
3.65 (t, 8.5, H-17), 3.56 (m, 9.3, 6.1, H-1Ј), 2.80 (t, 6.6,
H-6), 2.75 (t, 6.6, H-3Ј), 2.40 (dd, 12.2, 5.0, H-12), 2.23 (t,
9.7, H-9), 1.06 (t, 12, H-12), 0.97 (s, -C(CH3)3), 0.90 (s,
-C(CH3)3), 0.70 (s, H-18), 0.19 (s, Si-CH3), 0.06 (s, Si-
CH3), 0.03 (s, Si-CH3). ms: (EI) m/z 1180.1 (Mϩ).
C66H114O6S2Si4 (1180.11) calculated C 67.18 H 9.74 S 5.43
found C 66.95 H 9.70 S 5.62.
1
77°–80°C. [␣]D-44°. H nmr: 7.37 (d, 8.6, H-1), 6.62 (dd,
2.11. Bis-3-[17Ј␤-(t-butyldimethylsilyl)oxy-3Јhydroxy-
estra-1Ј,3Ј,5Ј(10Ј)-trien-11Ј␣-yl]oxypropyl-disulfane (14)
8.6, 2.8, H-2), 6.55 (d, 2.8, H-4), 3.76 (m, H-1Ј), 3.76 (m,
H-11), 3.66 (t, 8.4, H-17), 3.52 (dt, 9.1, 5.7, H-1Ј), 2.99 (t,
7.1, H-3Ј), 2.75 (t, 7.0, H-6), 2.38 (dd, 12.1, 5.1, H-12), 2.34
(s, -CO-CH3), 0.98 (s, -C(CH3)3), 0.91 (s, -C(CH3)3), 0.70
(s, H-18), 0.19 (s, Si-CH3), 0.06 (s, Si-CH3), 0.03, (s,
Si-CH3). ms: m/z (EI) 632.37347 (M)ϩ. C35H60O4S Si2
(633.10) calculated C 66.40 H 9.55 S 5.06 found C 66.38 H
9.55 S 5.20.
A mixture of compound 13 (14 g, 11.8 mmol) in a
solution of tetrabutylammonium fluoride (7.6 g, 24
mmol) in tetrahydrofuran (200 ml) was stirred at room
temperature for 30 min. Saturated sodium hydrogen car-
bonate solution (50 ml) was then added, the organic
solvent was evaporated in vacuo and the suspension ex-
tracted with n-hexane. The organic fractions were com-
bined, washed with water, dried, and evaporated in
vacuo. Purification of the residue by chromatography
(eluent:toluene/ethyl acetate 10: 1) followed by crystal-
lization from methanol gave compound 14 (6 g, 93.2%).
m.p. 154°C–157°C. [␣]D-42°. 1H nmr: 7.40 (d, 8.3, H-1),
6.64 (dd, 8.3, 2.6, H-2), 6.55 (d, 2.6, H-4), 5.25 (s,
3-OH), 3.80 (m, H-1Ј), 3.80 (m, H-11), 3.65 (t, 8.2,
H-17), 3.56 (m, H-1Ј), 2.80 (t, 7.0, H-6), 2.73 (t, 6.6,
H-3Ј), 2.39 (dd, 12.0, 5.0, H-12), 2.23 (t, 9.9, H-9), 1.07
(t, 11.0, H-12), 0.90 (s, -C(CH3)3), 0.69 (s, H-18), 0.06 (s,
Si-CH3), 0.03 (s, Si-CH3). ms: (EI) m/z 952.5 (M)ϩ.
C54H86O6S2Si2 (951.58) calculated C 68.16 H 9.11 S
6.74 found C 68.01 H 9.07 S 6.95.
2.9. 3,17␤-Bis-(t-butyldimethylsilyl)oxy-11␣-(3Ј-
sulfanylpropyl)oxy-estra-1,3,5 (10)-triene (12)
To a stirred solution of compound 11 (13 g, 20.5
mmol) in tetrahydrofuran (150 ml) 1 N lithium alumi-
nium hydride solution in tetrahydrofuran (50 ml, 50
mmol) was added dropwise. After 2 h, the reaction was
quenched with ethyl acetate (100 ml) and water. The
organic phase was separated, washed with water, dried,
and evaporated in vacuo. The residue was purified by
chromatography (eluent:cyclohexane/methyl tert-butyl
ether 10:1) followed by crystallization from methanol to
give compound 12 (10 g, 82.5%). m.p. 102°C–103°C.

464
S. Schwarz et al. / Steroids 64 (1999) 460–471
2.12. Bis-3-[17Ј␤-(t-butyldimethylsilyl)oxy-3Ј-
sulfamoyloxy-estra-1Ј,3Ј,5Ј (10Ј)-trien-11Ј␣-yl]oxypropyl-
disulfane (15)
ate solution (20 ml) at 0°C and the resulting mixture ex-
tracted with ethyl acetate. The combined fractions were
washed with saturated aqueous sodium chloride solution,
dried, and evaporated in vacuo. The residue was purified by
twofold chromatography (eluent:cyclohexane/ethyl acetate
1:2) followed by precipitating the compound with n-hexane
from an acetone solution to give compound 17 (0.2 g, 49%)
as a white amorphous powder. Purity by HPLC: 99.1 area
%. [␣]D-17° (pyridine). 1H nmr (d6-DMSO): 7.88 (s, -NH2),
7.53 (d, 8.4, H-1), 7.03 (dd, 8.4, 2.7, H-2), 6.98 (d, 2.7,
H-4), 4.60 (d, 4.9, -OH), 2.91 (t, 7.4, H-3Ј), 2.77 (t, 7.3,
H-6), 2.44 (dd, 11.6, 4.8, H-12), 1.01 (t, 11.1, H-12), 0.63 (s,
H-18). ms: (EI) m/z 441.16668 (M)ϩ; (ESIϪ) m/z 440.5
(M-H)Ϫ, 881.0 (2 M-H)Ϫ; (ESIϩ) m/z 442.5 (MϩH)ϩ,
459.6 (MϩNH4)ϩ, 464.5 (MϩNa)ϩ. C21H31NO5S2
(441.60) calculated C 57.12 H 7.08 N 3.17 found C 57.11 H
7.35 N 3.17. From compound 20 by reduction with borane-
dimethyl sulfide complex: To a 0°C cold solution of com-
pound 20 (0.62 g, 1.28 mmol) in tetrahydrofuran (3 ml) was
added borane-disulfide complex (2.5 ml, 26.4 mmol). The
solution was stirred for 3 h at room temperature, followed
by cautious addition of water (15 ml) at 0°C. The mixture
was then extracted with ethyl acetate. The combined
fractions were washed with water until neutral, dried, and
evaporated in vacuo. The residue was purified by column
chromatography (eluent:cyclohexane/chloroform/methanol
45:45:10) and precipitated from an acetone solution with
n-hexane to yield compound 17 (0.32 g, 57%). Purity by
HPLC: 93.2 area %. The spectra proved to be identical in all
respects with those of compound 17 prepared by sodium
borohydride/aluminum chloride reduction. From disulfane
16: Compound 16 (0.1 g, 0.11 mmol) dissolved in diglyme
(1.7 ml) was reduced with sodium borohydride (0.035 g,
0.92 mmol) in the presence of anhydrous aluminum chlo-
ride (57 mg, 0.43 mmol) as described for the reduction of
thioacetate 20. The product was purified by chromatography
(eluent:cyclohexane/ethyl acetate 1:4) followed by precipi-
tation with n-hexane from an acetone solution to yield
compound 17 (0.050 g, practically quantitative yield). Pu-
rity by HPLC: 92 area %. The spectra proved to be identical
in all respects with those of compound 17 prepared from
thioacetate 20.
To a stirred solution of compound 14 (0.48 g, 0.5 mmol)
in dichloromethane (15 ml) were added in turn 2,6-di-tert-
butyl-pyridine (1.2 ml, 5.44 mmol) and sulfamoyl chloride
(1.74 g, 15.06 mmol) at room temperature. Stirring was
continued for 3 h. The reaction was then quenched with
water, the organic phase was separated, and the aqueous
phase re-extracted with dichloromethane. The combined
organic phases were washed with saturated aqueous sodium
hydrogen carbonate solution and water until neutral, dried,
and evaporated in vacuo to give an oil which was purified
by chromatography (eluent:cyclohexane/ethyl acetate 5:2)
1
to give semi-crystalline compound 15 (0.145 g, 26%). H
nmr: 7.54 (d, 8.8, H-1), 7.08 (dd, 8.8, 2.6, H-2), 7.02 (d, 2.6,
H-4), 5.08 (s, -NH2), 3.80 (m, H-1Ј), 3.80 (m, H-11), 3.66
(t, 8.0, H-17), 3.51 (m, H-1Ј), 2.79 (t, 6.4, H-6), 2.40 (dd,
11.9, 4.9, H-12), 2.26 (t, 9.7, H-9), 1.07 (t, 10.4, H-12), 0.90
(s, -C(CH3)3), 0.68 (s, H-18), 0.05 (s, Si-CH3), 0.03 (s,
Si-CH3). ms: (ESIϪ) m/z 1107.9 (M-H)Ϫ; (ESIϩ) m/z
1126.7 (MϩNH4), 1131.3 (MϩNa).
2.13. Bis-3-(17Ј␤-hydroxy-3Ј-sulfamoyloxy-estra-
1Ј,3Ј,5Ј(10Ј)-trien-11Ј␣-yl)oxypropyl-disulfane (16)
Compound 15 (0.43 g, 0.39 mmol) was dissolved in a
mixture of acetic acid, tetrahydrofuran, and water (3:2:1, 60
ml). The solution was allowed to stand 6 days at room
temperature. Afterwards, the solvents were distilled off in
vacuo and the resulting oil was partitioned between water
and ethyl acetate. The organic phase was washed with
saturated aqueous sodium hydrogen carbonate solution and
water, dried, and evaporated in vacuo. Purification of the
residue by chromatography (eluent:cyclohexane/ethyl ace-
1
tate 1:2) gave compound 16 (0.2 g, 58.6%) as a foam. H
nmr (d6-DMSO): 7.89 (s, -NH2), 7.52 (d, 8.4, H-1), 7.01
(dd, 8.4, 2.5, H-2), 6.98 (d, 2.5, H-4), 4.61 (d, 4.5, -OH),
3.74 (m, H-1Ј), 3.74 (m, H-11), 3.53 (m, H-17), 3.53 (m,
H-1Ј), 2.77 (t, 6.7, H-6), 2.42 (dd, 11.4, 4.8, H-12), 2.19 (t,
9.1, H-9), 1.00 (t, 10.4, H-12), 0.62 (s, H-18). ms: (ESIϪ)
m/z 879.2 (M-H)Ϫ; (ESIϩ) m/z 898.8 (MϩNH4)ϩ, 903
(MϩNa)ϩ.
2.15. S-3-[17Ј␤ -(t-butyldimethylsilyl)oxy-3Ј-hydroxy-
estra-1Ј,3Ј,5Ј(10Ј)-trien-11Ј␣-yl]oxypropyl ethanethioate
(18)
2.14. 17␤-Hydroxy-11␣-(3Ј-sulfanylpropyl)oxy-estra-1,3,5
(10)-trien-3-yl sulfamate (17)
Following the same procedure as used to prepare com-
pound 14, compound 11 (5.51 g, 8.7 mmol) was reacted
with 1 eq. tetrabutylammonium fluoride in tetrahydrofuran
solution to afford compound 18 (2.8 g, 62%) as a foam.
From compound 20 by reduction with sodium borohy-
dride / aluminum chloride: To a 0°C cold solution of com-
pound 20 (0.45 g, 0.93 mmol) and sodium borohydride (0.3
g, 7.93 mmol) in diglyme (15 ml) anhydrous aluminum
chloride (0.473 g, 3.55 mmol) was added portionwise. After
the addition was finished, the reaction mixture was stirred
for an additional h at 23°C. Afterwards, the reaction was
quenched with saturated aqueous sodium hydrogen carbon-
1
[␣]D-46°. H nmr: 7.40 (d, 8.6, H-1), 6.63 (dd, 8.6, 2.9,
H-2), 6.55 (d, 2.9, H-4), 3.76 (m, H-1Ј), 3.76 (m, H-11),
3.66 (t, 8.1, H-17), 3.51 (dt, 9.3, 6.1, H-1Ј), 3.01 (t, 6.8,
H-3Ј), 2.75 (t, 6.7, H-6), 2.38 (dd, 11.9, 5.3, H-12), 2.34 (s,
-Ac), 1.07 (dd, 11.5, 11.2, H-12), 0.91 (s, -C(CH3)3), 0.70

S. Schwarz et al. / Steroids 64 (1999) 460–471
465
(s, H-18), 0.06 (s, Si-CH3), 0.03 (s, Si-CH3). ms: (EI) m/z
518.29052 (M)ϩ. C29H46O4S Si (518.84) calculated C 67.14
H 8.94 S 6.18 found C 67.15 H 8.95 S 6.33.
1 h at room temperature followed by centrifugation
(ϳ10 000 g, 5 min). The clear supernatant was passed
through a Sephadex G 50 column (15 ϫ 90 mm), equili-
brated in phosphate buffer (vide supra) and the absorbance
of the effluent was monitored at 280 nm. The protein peak
(ϳ4 ml) was collected and after dilution with dioxane (1.4
ml), compound 17 (10 mg, 22.6 ␮mol) in dioxane (150 ␮l)
was slowly added with fast stirring. The cloudy mixture was
stirred for 5 h at room temperature and then dialyzed once
against 25% dioxane in phosphate buffer (1 l) and twice
against phosphate-buffered saline (PBS) (1 l). As control,
cysteine was coupled to BGG in a similar procedure (5 mg
were added in 150 ␮l pf buffer), followed by identical
treatment. The amount of steroid coupled to the BGG was
determined by comparing the UV spectra of the conjugate,
the control and of a defined concentration of compound 17.
The mole ratio calculated for the coupling was 32 mol
steroid to 1 mol BGG.
2.16. S-3-[17Ј␤-(t-butyldimethylsilyl)oxy-3Ј-sulfamoyloxy-
estra-1Ј,3Ј,5Ј(10Ј)-trien-11Ј␣-yl]oxypropyl ethanethioate
(19)
To a solution of compound 18 (1.1 g, 2.12 mmol) in a
mixture of dichloromethane (30 ml) and 2,6-di-tert-butyl-
pyridine (2.3 ml, 10.43 mmol) was added sulfamoyl chlo-
ride (1.3 g, 11.3 mmol). After stirring for 1 h at 22°C, the
solution was diluted with water (30 ml) and extracted with
ethyl acetate. The combined organic phases were washed
with saturated aqueous sodium hydrogen carbonate solution
and water and dried. After evaporation in vacuo, the residue
was purified by column chromatography (eluent:toluene:
ethyl acetate 9:1) to give compound 19 (1.03 g, 84%) as an
amorphous powder. [␣]D-49° (pyridine). 1H nmr (d6-
DMSO): 7.89 (s, -NH2), 7.53 (d, 8.9, H-1), 7.02 (dd, 8.9,
2.5, H-2), 6.98 (d, 2.5, H-4), 2.91 (t, 7.4, H-3Ј), 2.77 (t, 6.8,
H-6), 2.32 (s, -Ac), 1.05 (t, 11.5, H-12), 0.89 (s, -C(CH3)3),
0.65 (s, H-18), 0.06 (s, Si-CH3), 0.04 (s, Si-CH3). ms: (EI)
m/z 597.25873 (M)ϩ. C29H47NO6S2Si (597.90) calculated
C 58.26 H 7.92 N 2.34 S 10.72 found C 58.37 H 7.95 N 2.57
S 10.86.
2.19. Production of antisera
Six male rabbits (ϳ3 kg) were subcutaneously (s.c.)
immunized with conjugate 21 (1 mg) in emulgate (1.5 ml)
containing complete Freund’s Adjuvant and boosted there-
after with conjugate (0.5 mg) in incomplete adjuvant at 6 to
8 weekly intervals. Animals were bled 2 weeks after the
third boost.
2.17. S-3-(17Ј␤-Hydroxy-3Ј-sulfamoyloxy-estra-
1Ј,3Ј,5Ј(10Ј)-trien-11Ј␣-yl)oxypropyl ethanethioate (20)
2.20. Biotinylated compound 22
Following the procedure described for the formation of
compound 16, compound 19 (1.0 g, 1.67 mmol) was treated
with a mixture of acetic acid, tetrahydrofuran, and water
(3:2:1, 115 ml) for 6 days at room temperature to give
compound 20 (0.69 g, 85%) as a foam. [␣]D-69° (pyridine).
1H nmr (d6-DMSO): 7.90 (s, -NH2), 7.52 (d, 8.2, H-1), 7.02
(dd, 8.2, 2.7, H-2), 6.98 (d, 2.7, H-4), 4.62 (d, 4.7, -OH),
2.90 (t, 7.1, H-3Ј), 2.77 (t, 7.0, H-6), 2.42 (dd, 12.0, 4.9,
H-12), 2.34 (s, -Ac), 1.00 (t, 10.9, H-12), 0.62 (s, H-18). ms:
(ESI)Ϫ m/z 482.5 (M-H)Ϫ, 965.9 (2 M-H)Ϫ, 1448.8 (3
M-H)Ϫ; (ESI)ϩ m/z 484.8 (MϩH)ϩ, 506.5 (MϩNa)ϩ,
989.9 (2MϩNa)ϩ. C23H33NO6S2 (483.64) calculated C
57.12 H 6.88 N 2.9 S 13.26 found C 57.46 H 7.31 N 2.87
S 13.13.
Compound 17 (1 ␮mol) dissolved in dimethylformamide
(20 ␮l) was added to a solution of N-biotinoyl-NЈ-[6-ma-
leidohexanoyl]-hydrazide (Sigma) (1 ␮mol) in dimethylfor-
mamide (150 ␮l). The mixture was incubated for 2 h at
room temperature with stirring. The biotinylated compound
22 was purified by thin-layer chromatography using silica G
60 coated aluminium sheets and the solvent mixture ethyl
acetate/ methanol/acetic acid (70/30/2) as mobile phase.
The coupling product (Rf ϭ 0.77) was localized under UV
light, cut off and extracted with aqueous methanol (80%).
On the basis of UV absorption, a yield of about 50% of
compound 22 was estimated.
2.21. Enzyme immunoassay protocol
2.18. 17␤-Hydroxy-11␣-(3Ј-sulfanylpropyl)oxy-estra-1,3,5
(10)-trien-3-yl sulfamate bovine ␥- globulin conjugate
(21)
Microtiter plates (NUNC 460348) were coated with af-
finity purified goat-anti-rabbit ␥-globulin (Scantibodies,
USA) by pipetting 150 ␮l antibody solution (6.6 ␮g/ml 50
mM sodium carbonate pH 9.6) into each well, followed by
an overnight incubation at room temperature. Then, 250 ␮l
blocking buffer (0.16 M sodium phosphate pH 7.0, 1.44%
sodium chloride, 0.8% bovine serum albumin (BSA), 0.08%
Tween 20, 0.02% Thimerosal) were added to each well and
the incubation was continued until next day. The plates were
emptied by decantation and patted dry onto absorbent paper.
They were washed once with 375 ␮l wash solution (0.02%
Hapten 17 was coupled to bovine ␥-globulin (BGG) in a
two-step procedure. BGG (25 mg) was dissolved in 0.1 M
sodium phosphate (0.9 ml, pH 7.2) containing 0.9% sodium
chloride. ␤-Maleimido propionic acid N-hydroxysuccinim-
ide ester (Sigma) (13 mg, 48.9 ␮mol) was dissolved in
dimethylformamide (0.3 ml) and immediately added to the
protein solution with stirring. The mixture was stirred for

466
S. Schwarz et al. / Steroids 64 (1999) 460–471
Scheme 2. 11␣-Oxyfunctionalization of estradiol diacetate. Reaction conditions: (i) Oxone, acetone, CH2Cl2, H2O, NaHCO3, TBAHS, 15°C, 80%. (ii) H2SO4,
CH2Cl2, Ϫ30°C; 95%. (iii) MeOH, KOH, 45°C, 65%. (iv) TBSCl, DMF, Py, 60°C, 92%. (v) BH3.Me2S, THF, 50°C. (vi) H2O2, NaOH, 40°C, (v)ϩ(vi) 74%.
Tween 20, 0.5% sodium chloride) and then stored at Ϫ20°C
for up to 6 months. Prior to assay, the frozen plates were
brought to room temperature and washed once with assay
buffer (0.1 M sodium phosphate pH 7.0 containing 0.15 M
sodium chloride, 0.005 M EDTA, 0.2% BSA, 0.01%
Thimerosal, 0.005% Tween 20). Estrogen sulfamate stan-
dards or test compounds for cross-reaction studies were
added in assay buffer (100 ␮l) followed by the addition of
the biotinylated tracer (ϳ1 fmol) and the antiserum (K4;
dilution 1:80 000) in assay buffer (50 ␮l each). The covered
plates were incubated overnight in a humidity chamber at
4°C and then emptied and patted dry. Two hundred ␮l of
streptavidine-horse radish peroxidase conjugate (Camon;
0.15 ␮g/ml assay buffer, 4°C) were added, and after incu-
bation for 30 min at 4°C, the wells were emptied and
washed four times with 375 ␮l cold wash solution. Finally,
the emptied wells were brought to room temperature and
filled with 250 ␮l freshly prepared substrate solution (90
mM sodium acetate, 4.5 mM citric acid, 0.004% H2O2,
0.01% 3,3Ј,5,5Ј-tetramethylbenzidine, 2% DMSO) adjusted
to room temperature. The enzyme reaction was run for 40
min at room temperature and stopped by the addition of 50
␮l 2 M sulfuric acid. The absorption was measured at 450
nm in a microplate photometer.
compound 17. In previous work on the large-scale synthesis
of the progestin desogestrel we presented a novel efficient
approach to the synthesis of an 11␣-hydroxylated A-ring
aromatic 18a-homo steroid involving as key steps (i) a C-9
hydroxylation of the corresponding aromatic steroid by in
situ formed dimethyldioxirane (DMD) and (ii) a very effec-
tive hydroboration/alkaline hydrogen peroxide oxidation of
the 9(11)-dehydro compound formed upon regioselective
water elimination of the 9-hydroxy group [15]. Adapting
our protocol to estradiol diacetate (3), 9-hydroxy steroid 4
was obtained in 80% yield using DMD generation in a
phase transfer catalyzed two-phase system (Scheme 2).
Water elimination by sulfuric acid transformed hydroxy
compound 4 into the 9(11)-olefin 5. By HPLC, formation of
the undesired 8-dehydro isomer was detected in less than
3%. The diol 6, prepared from diacetate 5, was converted
into the bis-silyl ether 7. Hydroboration/alkaline hydrogen
peroxide oxidation of compound 7 then furnished the bis-
protected 11␣-hydroxy estradiol 8.
Initially, alkylation of the 11-hydroxy group in compound 8
was attempted by reaction of compound 8 with 2-trimethylsi-
lyloxy bromoethane [16] and 3-trimethylsilyloxy bromopro-
pane [17] using various bases and solvents. A trimethylsilyl
(TMS) ether group at the ␻-position of the spacer was thought
to undergo chemoselective cleavage in the presence of the 3-
and 17-tert-butyl-dimethylsilyl (TBS) ethers, thus opening a
route for a subsequent thiofunctionalization of the side chain.
However, in all cases studied, only marginal alkylation of the
11-hydroxy group was observed. The reactions suffered from
cleavage of the phenolic 3-TBS ether by the bases used and
resilylation by the C2- and C3-building blocks to give com-
pounds with a TMS group in position 3 or 11 or 3 and 11 as
3. Results and discussion
3.1. Synthesis of hapten 17␤-hydroxy-11␣-(3Ј-
sulfanylpropyl)oxy-estra-1,3,5 (10)-trien-3-yl sulfamate
(17)
Effective oxyfunctionalization of estradiol at 11␣-posi-
tion was an essential prerequisite to the synthesis of title

S. Schwarz et al. / Steroids 64 (1999) 460–471
467
Scheme 3. Introduction of the 11␣-(␻-functionalized) side chain. Reaction conditions: (i) Allylbromide, KOtBu, THF, 0°C, 74%. (ii) 9-BBN, THF, 65°C.
(iii) H2O2, NaOH, (ii)ϩ(iii) 96%. (iv) PPh3, DIAD, AcSH, THF, 0°C, 90%.
well. C2- or C3-alkylation at position 3 instead of position 11
was also observed.
Thioate 11, when subjected to lithium aluminum hydride,
afforded thiol 12. Subsequent iodine oxidation of the thiol
12 achieved the disulfide 13. The disulfide bridging pro-
vided a valuable thiol group protection with regard to the
following sulfamate formation. Treatment of disulfide 13
with tetrabutylammonium fluoride (2 equiv., 30 min)
chemo- and regioselectively removed the 3-TBS ether and
gave phenol 14. Sulfamoylation of the exposed phenolic
hydroxy group in compound 14 with sulfamoyl chloride/
2,6-di-tert-butyl 4-methylpyridine/dichloromethane [10] gave
a modest yield of ester 15 which was deprotected at C-17 by
acetic acid in THF/water (6 days, room temperature), thus
providing compound 16. As described before [10], this
protocol, though requiring a rather long reaction time, suc-
ceeded in a clean silyl ether cleavage rigorously avoiding a
concomitant hydrolysis of the phenolic sulfamate group.
Finally, title compound 17 was obtained by reductive
cleavage of the disulfide bridge in compound 16. Sodium
As outlined in Scheme 3, switching to allyl bromide as a
C3-building block was more successful, after the reaction
had been carefully optimized. When, at 0°C, a solution of
potassium tert-butoxide (3 equiv.) in tetrahydrofuran was
added to a mixture of compound 8 and allyl bromide (6
equiv.) in tetrahydrofuran, and, thereupon, the mixture was
allowed to react for a strictly limited time of 2 min, allyl
ether 9 was isolated in 63% yield after work-up. Subsequent
allyl ether functionalization was realized by hydroboration/
alkaline hydrogen peroxide oxidation of compound 9 with
9-BBN to give alcohol 10, which was transformed into
compound 11 by thioacetic S-acid using Mitsunobu condi-
tions [18]. Attempted hydroboration of compound 9 using
the borane–THF complex or the borane–dimethyl sulfide
complex suffered from formation of various by-products.
The synthesis was continued as depicted in Scheme 4:
Scheme 4. Synthesis of title compound 17 via disulfide formation. Reaction conditions: (i) LAH, THF, 82%. (ii) I2, EtOH, CH2Cl2, nearly quantitative yield.
(iii) TBAF (1 equiv.), THF, 53%. (iv) H2N-SO2Cl, CH2Cl2, DTBP (26%). (v) AcOH, H2O, THF (3:1:1), 58%. (vi) NaBH4, AlCl3, diglyme, nearly
quantitative yield.

468
S. Schwarz et al. / Steroids 64 (1999) 460–471
fide 14 and the moderate purity of title compound 17 ob-
tained from compound 15 prompted us to look for an alter-
native approach to hapten 17. As outlined in Scheme 5, this
was realized from thioate 11 via the intermediates 18, 19,
and 20. Analogously to the disulfide bridge reduction of
compound 16, the thioate moiety of compound 20 was
readily removed by sodium borohydride/aluminum chloride
to yield title compound 17 in very high purity as demon-
strated by HPLC measurement. Borane–dimethyl sulfide
reduction of thioate 20 was also effective, however, gave
some less pure compound 17.
Scheme 5. Synthesis of title compound 17 via ethane thioate 18 and 19.
Reaction conditions: (i) TBAF (1 equiv.), THF, 62%. (ii) H2N-SO2Cl,
CH2Cl2, DTBP, 84%. (iii) AcOH, H2O, THF (3:1:1), 85%. (iv) NaBH4,
AlCl3, diglyme, 48% or BH3.Me2S, THF, 56%.
3.2. Synthesis of 17␤-hydroxy-11␣-(3Ј-sulfanylpropyl)oxy-
estra-1,3,5 (10)-trien-3-yl sulfamate bovine ␥-globulin
conjugate (21)
To exclude a possible coupling between the carrier pro-
tein and hapten 17 through its sulfamate moiety, 3-maleido
propionic acid N-hydroxysuccinimide ester, a heterobifunc-
tional cross-linker carrying a thiol-reactive maleimide group
at the one end and an amino group-specific N-hydroxysuc-
cinimide ester at the other, has been used as coupling re-
agent (see Scheme 6). In a two-step procedure the cross-
linker was first reacted with the amino groups of bovine
␥-globulin (which lacks any thiol groups), and second, after
removal of unbound cross-linker, with the ␻-thiol group of
the hapten 17.
borohydride which was reported to reduce disulfides to the
corresponding thiols [19], failed completely. On the other
hand, lithium aluminum hydride [19] and diisobutyl alumi-
num hydride (tetrahydrofuran, 0°C) led to disulfide and
sulfamate reduction as well. Finally, we found that in the
presence of aluminum chloride a diethylene glycol dimethyl
ether solution of sodium borohydride [20,21] resulted in
disulfide cleavage without attacking the phenolic sulfamate
group of compound 16 (0°C, then 2 h at room temperature).
Sodium borohydride and aluminum chloride apparently
form aluminum borohydride to some extent [20,22]. How-
ever, we speculated that, additionally, borane should be a
player. Thereupon, various boranes have been studied for
being capable of reducing disulfide 16. Among them, the
borane–dimethyl sulfide complex (0°C, then 2 h at room
temperature) was found to successfully reduce disulfide 16
as well.
3.3. Immunoassay
All 6 rabbits responded to the immunization against
conjugate 21 with the development of antibodies against
estradiol sulfamate (1). Using a tracer concentration of ϳ5
pM (1 fmol/well) it was possible to dilute the antisera
The low yield of sulfamate 15 from the phenolic disul-
Scheme 6. Synthesis of BGG conjugate 21 and biotinylated compound 22. Reaction conditions: (i) a) BGG, 3-maleido propionic acid N-hydroxy-succinimide
ester, buffer, DMF b) 17, dioxane c) dialysis. (ii) 17, DMF, N-biotinoyl-NЈ-(6-maleidohexanoyl) hydrazide, 50%.

S. Schwarz et al. / Steroids 64 (1999) 460–471
469
Table 1
Cross reactivities of antiserum No. 4
Compound
Cross-reaction (%)
Estradiol sulfamate
Estrone sulfamate
17␣-Estradiol sulfamate
Estriol sulfamate
Estriol 3-(N-acetyl)sulfamate
Estra-1,3,5(10)-triene-3,17␤-diyl 17-
Pentanoate, 3-sulfamate
Estrone (N-acetyl)sulfamate
Estradiol 3-(N-acetyl)sulfamate
Estradiol 3-pentanoate
Estradiol
100.0
100.0
100.0
33.3
1.7
1.0
0.1
0.1
0.01
0.01
17␣-Estradiol
Ethinyl-estradiol
Estradiol-3-sulfate
Estradiol-3-glucuronide
Ͻ0.005
Ͻ0.005
Ͻ0.005
Ͻ0.005
Preliminary results have shown that estrone sulfamate is
easily converted into the 2,4-dinitrophenylhydrazone deriv-
ative according to Knapp [23], and that this modification
decreases its cross-reaction to a value below 0.2%. Under
the reaction conditions used, estradiol sulfamate (1) appar-
ently remains unchanged as indicated by the retention of its
immunoreactivity (data not shown).
Fig. 1. Typical standard curve for estradiol sulfamate (1) obtained with
antiserum No. 4. The assay was performed in duplicate as described under
Experimental. Antiserum dilution (final): 1:320 000; Tracer concentration:
ϳ1 fmol/well. Non-Specific Binding (NSB): OD ϭ 0.068. Zero standard
binding (Bo): OD ϭ 1.748.
In a previous work by Webb et al. [24], a specific radio-
immunoassay for estradiol was developed showing very
little cross-reaction with estrone and estriol. For the prepa-
ration of the immunogen the authors coupled 11␤-hemisuc-
cinyl-estradiol to BSA, and as tracer they used estradiol-
11␣-succinyl-tyrosinemethylester-125I. The high specificity
of their antiserum for the D-ring suggests that the 11␤-
configuration of the hapten in the immunogen might be
advantageous over the 11␣-configuration, perhaps, by leav-
ing the D-ring of the steroid molecule more unmasked.
Although our estradiol sulfamate assay shows a very low
detection limit of ϳ1 pg/well, the large assay range up to
1000 pg/well is disadvantageous in terms of precision. The
large assay range is presumably due to the homologous
design of the assay, in which the immunogen and the bio-
tinylated tracer possess the same steroid structure and
bridge configuration (11␣). As a consequence, the tracer is
more tightly bound by the polyclonal antibodies than the
ligand to be measured. In this case relatively large amounts
of ligand would be required to displace the tracer from the
antibody binding sites, resulting in flat standard curves.
Webb et al. [24] circumvented this problem of ‘bridge
recognition’ by using the 11␤-configuration for the prepa-
ration of the immunogen and the 11␣-configuration for the
synthesis of the tracer. The advantage of this heterologous
assay design became significant in a steeper slope of the
standard curve and an increased sensitivity in comparison to
the homologous system (both configurations 11␤).
between 1:140 000 and 1:1 000 000 (final dilutions) to
produce an optical density (OD) of about 1.5.
The most sensitive standard curve was obtained with
antiserum No. 4. As illustrated in Fig. 1, the curve covers a
concentration range from about 1 to 1000 pg/well (ED50 ϭ
27 pg/well), which appears to satisfy future demands with
respect to sensitivity.
Concerning the specificity of antiserum No. 4, the cross-
reactions of various sulfamoylated and non-sulfamoylated
estrogens listed in Table 1 indicate that this antiserum is
very specific to the intact sulfamate moiety, and does not
tolerate any changes at the C-3 position. On the other hand,
the specificity to the D-ring appears to be low, since the
sulfamates of estrone, estriol and 17␣-estradiol show high
cross-reactions (100%, 33%, and 100%, respectively). An-
other antiserum (No. 5) showed a higher specificity for the
D-ring with cross-reactions of 10%, 1.5%, and 10%, respec-
tively; however, its sensitivity was about five times lower in
comparison to antiserum No. 4, which makes it unsuitable
for future measurements of low plasma levels.
The low specificity at the D-ring appears not to be a great
disadvantage, since only estrone sulfamate will be formed
from estradiol sulfamate in substantial amounts in the blood
circulation. Of course, it is desirable to differentiate be-
tween these two estrogen sulfamates, and therefore we have
started to establish a simple routine derivatization step,
which selectively modifies estrone sulfamate at the 17-keto
group, thereby decreasing drastically its cross-reactivity.
The disadvantage of homologous hapten immunoassays
with respect to sensitivity is also well recognized in other
competitive immunoassay systems. In this context, the kind

470
S. Schwarz et al. / Steroids 64 (1999) 460–471
of labeling (e.g. 125I-iodotyrosine, biotin, enzyme) appears
to play a minor role. There are in general two ways to
circumvent this problem:
i) The ligand to be measured is modified by a chemical
derivatization step prior to assay in a way that makes it more
similar to the hapten structure, as it is shown for the cAMP
and cGMP RIAs (Harper and Brooker [25]) or for the
serotonin RIA (Gow et al. [26]). Such an assay design,
however, cannot be adapted to the assay of estradiol sulfa-
mate described here, since the latter is not easily derivatized
at 11␣-position.
reduced hepatic estrogenicity at oral application. J Steroid Biochem
Mol Biol 1995;55:395–403.
[2] Schwarz S, Elger W. Estrogen sulfamates, a novel approach to oral
contraception and hormone replacement therapy. Drugs Future 1996;
21:49–61.
[3] Elger W, Palme H-J, Schwarz S. Novel oestrogen sulfamates: a new
approach to oral hormone therapy. Exp Opin Invest Drugs 1998;7:
575–89.
[4] Adamczyk M, Mattingly PG, Reddy RE. Synthesis of 6␤-aminoestra-
diol and its biotin, acridinium, and fluorescein conjugates. Steroids
1998;63:130–4.
[5] Rao PN, Wang Z, Cessac JW, Moore PH Jr. Synthesis of new
immunogens for estrone and estradiol-17␤ and antisera evaluation.
Steroids 1998;63:141–5.
[6] Franek M, Bursa J, Hruska K. Characteristics of antibodies against
17␤-oestradiol in homologous and heterologous systems of radioim-
munoassay. J Steroid Biochem 1983;19:1371–4.
[7] Meyer HHD, Sauerwein H, Mutayoba BM. Immunoaffinity chroma-
tography and a biotin-streptavidin amplified enzyme immunoassay
for sensitive and specific estimation of estradiol-17␤. J Steroid Bio-
chem 1990;35:263–9.
[8] Droescher P, Schwarz S, Ring S, Menzenbach B. Synthesis of estra-
diol derivatives functionalized at C-6 by substituted aryl and alkyl
groups mediated by transition metals. Organo Metallic Chemistry
Directed towards Organic Synthesis 8 (OMCOS-8), Santa Barbara
(USA), Poster 1995:100.
[9] Part of this work: Ring S, Weber G, Thieme I, Schwarz S. 11␣-(␻Ј-
Sulfanylalkanoxy) estra-1,3,5(10)-triene - a basic hapten structure.
Synthesis of 17␤-hydroxy 11␣-(3Ј-sulfanylpropanoxy)estra-1,3,5(10)-
trien-3-yl sulfamate. Poster MC-B9, 36th IUPAC Congress, Geneva,
August 1997:17–22.
[10] Schwarz S, Thieme I, Richter M, Undeutsch B, Henkel H, Elger W.
Synthesis of estrogen sulfamates: compounds with a novel endocri-
nological profile. Steroids 1996;61:710–7.
[11] Schwarz S, Elger W, Siemann H-J, Reddersen G, Schneider B,
inventors; Jenapharm GmbH, Co. KG, Germany, assignee. Estra-
1,3,5(10)-triene derivatives, process for their preparation and phar-
maceutical compositions containing these compounds. German
Patent Applic. P 344 29 397.6. 1994 Aug 9.
[12] Schwarz S, Elger W, Reddersen G, Schneider B, Thieme I, Richter
M, inventors; Jenapharm GmbH, Co. KG, Germany, assignee. Sul-
famate derivatives of 1,3,5(10)-estratriene derivatives, methods for
their production and pharmaceuticals containing these compounds.
US Patent 5, 705, 495. 1996 Oct 18.
ii) A tracer molecule with a heterologous structure in
comparison to the hapten structure in the immunogen is
used, which still binds to the antibody, but is more easily
displaced by the ligand thereby increasing the sensitivity.
Besides Webb et al. [24], numerous other authors have
followed such assay designs, some of which being exem-
plified below. Franec et al. [27] drastically increased the
sensitivity of an estradiol RIA by using an anti-estradiol-2
(4)-azo-serum in combination with estradiol-6-O-(CMO)-
[
125I]iodohistamine as tracer. Tiefenauer et al. [28] used
antibodies against estradiol-6-CMO-albumin in combina-
tion with a tracer, in which 6␣-aminoestradiol was labeled
with [125I]iodophenyl propionic acid N-hydroxysuccinimide
ester. Meyer et al. [7] developed an ELISA using an anti-
serum directed against estradiol-17␤-hemisuccinate-albu-
min, and a tracer in which a biotin derivative was coupled
to estradiol-17␤-D-glucuronic acid. Allen and Redshaw
[29] compared eight homologous and heterologous 125I-
tracers in a progesterone RIA using an antiserum raised to
progesterone-11␣-hemisuccinate-albumin. They found that
all tracers with the 11␣-hemisuccinate structure or similar
structures produced very unsensitive standard curves com-
pared to the tritiated tracer (reference tracer), whereas 3- or
12-(O-carboxymethyl)oxime tracer derivatives resulted in
standard curves equal to, or even more sensitive than, that
obtained with the tritiated tracer.
At present, our efforts are ongoing to synthesize a suit-
able heterologous tracer derivative which is more easily
displaced by the unlabeled ligand than the one described
here.
[13] Rhodes PH. The Organic Chemist’s Desk Reference. London: Chap-
man & Hall, 1995. pp. 90–105.
[14] Marples BA, Brown DS, Muxworthy JP, Baggaley KH. Preparation
of 9␣-hydroxyestra-1,3,5(10)-trienes by direct benzylic oxidation
with dimethyldioxirane. J Chem Res 1992:28–9.
[15] Schwarz S, Ring S, Weber G, Teichmu¨ller G, Palme H-J, Pfeiffer C,
Undeutsch B, Erhart B, Grawe D. Synthesis of 13-ethyl-11-methyl-
ene-18,19-dinor-17␣-pregn-4-en-20-yn-17-ol (desogestrel) and its
main metabolite 3-oxo desogestrel. Tetrahedron 1994;50:10709–20.
[16] Kricheldorf HR. Ringo¨ffnung von Lactonen und cyclischen Carbon-
aten mit Brom- oder Jodtrimethylsilan. Angew Chem 1979;91:749.
[17] Kizlink J, Harustiak M, Siles B. Preparation of hydroxyalkyltin com-
pounds. Ropa Uhlie 1990;32:298–300.
[18] Volante RP. A new, highly efficient method for the conversion of
alcohols to thiolesters and thiols. Tetrahedron Lett 1981;22:3119–22.
[19] Greene TW, Wuts PGM. Protective Groups in Organic Synthesis.
New York: John Wiley & Sons, Inc., 1991. pp. 302–3.
[20] Brown HC, Rao BC. A new powerful reducing agent—sodium boro-
hydride in the presence of aluminum chloride and other polyvalent
metal halides. J Am Chem Soc 1956;78:2582–8.
Acknowledgments
We are grateful to Harry Henkel for HPLC analysis, Ms
Sabine Mann, Ms Andrea Triller, Ms Sabine Matz, and Mr
Richard Werner for their excellent technical assistance, and
Ms Sonja Ha¨gele and Ms Ute Zo¨rner for their help to
prepare this manuscript.
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