Steroidal affinity labels of the estrogen receptor. 3. Estradiol 11β- n-alkyl derivatives bearing a terminal electrophilic group: Antiestrogenic and cytotoxic properties

Article abstract of DOI:10.1021/jm970019l

With the aim of developing a new series of steroidal affinity labels of the estrogen receptor, six electrophilic 11β-ethyl (C₂), 11β-butyl (C₄), or 11β-decyl (C₁₀) derivatives of estradiol bearing an 11β-terminal electroph

Full text of DOI:10.1021/jm970019l

J . Med. Chem. 1997, 40, 2217-2227
2217
Ster oid a l Affin ity La bels of th e Estr ogen Recep tor . 3. Estr a d iol 11â-n -Alk yl
Der iva tives Bea r in g a Ter m in a l Electr op h ilic Gr ou p : An tiestr ogen ic a n d
Cytotoxic P r op er ties
Carole Lobaccaro,† J ean-Franc¸ois Pons,‡ Marie-J ose`phe Duchesne,† Gilles Auzou,‡ Michel Pons,† Franc¸ois Nique,§
Georges Teutsch,§ and J ean-Louis Borgna*,†
INSERM Unite´ 439, 70 rue de Navacelles, 34090 Montpellier, France, INSERM Unite´ 300, Faculte´ de Pharmacie, 15 avenue
Charles Flahault, 34060 Montpellier Cedex, France, and ROUSSEL-UCLAF, 102 route de Noisy, 93230 Romainville, France
Received J anuary 10, 1997X
With the aim of developing a new series of steroidal affinity labels of the estrogen receptor, six
electrophilic 11â-ethyl (C2), 11â-butyl (C4), or 11â-decyl (C10) derivatives of estradiol bearing
an 11â-terminal electrophilic functionality, i.e. bromine (C4), (methylsulfonyl)oxy (C2 and C4),
bromoacetamido (C2 and C4), and (p-tolylsulfonyl)oxy (C10), were synthesized. The range of
their affinity constants for binding the estrogen receptor was 0.4-37% that of estradiol; the
order of increasing affinity (i) relative to the 11â-alkyl arm was ethyl < butyl and (ii) relative
to the electrophilic functionality was bromoacetamido < bromine < (methylsulfonyl)oxy.
Regardless of the conditions used, including prolonged exposure of the receptor to various pH
levels (7-9) and temperatures (0-25 °C), the extent of receptor affinity labeling by the 11â-
ethyl and 11â-butyl compounds, if any, was under 10%. This was in sharp contrast to results
obtained using 11â-((tosyloxy)decyl)estradiol which labeled from 60% to 90% of the receptor
hormone-binding sites with an EC50 of ∼10 nM. Estrogenic and antiestrogenic activities of
the compounds were determined using the MVLN cell line, which was established from the
estrogen-responsive mammary tumor MCF-7 cells by stable transfection of a recombinant
estrogen-responsive luciferase gene. The two 11â-ethyl compounds were mainly estrogenic,
whereas the three 11â-butyl and the 11â-decyl compounds essentially showed antiestrogenic
activity. The fact that the chemical reactivities of 11â-ethyl and 11â-butyl compounds were
not compromised by interaction with the estrogen receptor made the synthesized high-affinity
compounds potential cytotoxic agents which might be able to exert either (i) a specific action
on estrogen-regulated genes or (ii) a more general action in estrogen-target cells. Therefore
the ability of the compounds (1) to irreversibly abolish estrogen-dependent expression of the
luciferase gene and (2) to affect the proliferation of MVLN cells were determined. All
electrophiles were able to irreversibly suppress expression of the luciferase gene; the
antiestrogenic electrophiles were more potent than the estrogenic ones but less efficient than
4-hydroxytamoxifen, a classical and chemically inert triphenylethylene antiestrogen. Only the
antiestrogenic electrophiles decreased cell proliferation; however, they were less potent than
4-hydroxytamoxifen. In conclusion, the synthesized electrophilic estradiol 11â-ethyl and 11â-
butyl derivatives (i) were not efficient affinity labels of the estrogen receptor and (ii) did not
display significant cytotoxicity in estrogen-sensitive mammary tumor cells. However, since
these derivatives displayed high affinity for the estrogen receptor, they could be used to prepare
potential cytotoxic agents which might be selective for tumors affecting estrogen-target tissues,
by coupling them with a toxic moiety.
In tr od u ction
liganded retinoic receptor γ (RARγ),7 and rat thyroid
hormone receptor R1 (TRR1)8 were recently proposed
from crystallographic data. Using information obtained
from the crystal structure of RARγ, and through a
general sequence alignment of ligand-binding domains
of nuclear receptors, Wurtz et al.9 inferred a putative
general structure of the ligand-binding pocket of nuclear
receptors.
The nuclear receptor superfamily comprises1,2 more
than 100 structurally related proteins which act as
transcriptional regulators. Members of this family
possess two main functional domains: the DNA-binding
domain and the hormone-binding domain which include
∼70 amino acids and ∼250 amino acids, respectively,
and are the most conserved sequences in these proteins.
The DNA-binding domain structures of estrogen and
glucocorticoid receptors were established in the early
1990s on the basis of NMR3,4 and crystallographic
studies.5 Structures of hormone-binding domains of
human unliganded retinoic X receptor R (RXRR),6
Affinity labeling of receptors provides a means to
directly identify amino acids of the hormone-binding
pocket which are in contact with or in close proximity
to the ligands. Concerning the estrogen receptor, such
an approach was first used by J . Katzenellenbogen’s
group who developed several series of nonsteroidal
affinity- and photoaffinity-labeling agents of the
receptor.10-14 Recently, we described affinity labeling
of the estrogen receptor by electrophilic estradiol 17R-
derivatives.15,16 Covalent attachment sites of tamoxifen
* Corresponding author: J ean-Louis Borgna. Tel: (33) 467 04 37
14. Fax: (33) 467 52 06 77.
† INSERM Unite´ 439.
‡ INSERM Unite´ 300.
§ ROUSSEL-UCLAF.
X Abstract published in Advance ACS Abstracts, J une 1, 1997.
S0022-2623(97)00019-8 CCC: $14.00 © 1997 American Chemical Society

2218 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Lobaccaro et al.
Sch em e 1
aziridine, a nonsteroidal antiestrogen, and 17R-[(halo-
acetamido)alkyl]estradiols were identified in the human
estrogen receptor. Three out of the four invariant
cysteines (C381, C417, C447, and C530 in the human
estrogen receptor) of the hormone-binding domain were
found to be the covalent attachment sites of these
electrophiles: C381 and C530 were alkylated by tamox-
ifen aziridine,17,18 whereas C417 and C530 were alky-
lated by the estradiol 17R-derivatives.19 These results
were in agreement with the general structure proposed
for the ligand-binding pocket of nuclear receptors since
the three cysteines were located at the extreme border
or in structural elements involved in delineation of the
hormone-binding pocket. Moreover, they suggested that
the conformation of the antiestrogen-liganded receptor
differs from those of unfilled and estrogen-filled recep-
tors. Finally, they enabled development of a selective
mode of superimposition of tamoxifen-class antiestro-
gens with estradiol, which can account for the relative
positioning of the two types of ligands in the hormone-
binding pocket.19
their hormonal activity, their ability to irreversibly
abolish the expression of a recombinant estrogen-
responsive gene, and to affect the growth of estrogen-
sensitive mammary tumor cells.
Resu lts
Syn th esis of Electr op h ilic Estr a d iol 11â-Der iva -
tives. To obtain the projected estradiol 11â-derivatives,
we used the regio- and stereospecific synthesis pathway
first described by Belanger et al.,20 whereby a 5R,10R-
epoxyestr-9(11)-ene is submitted to the action of Grig-
nard reagents in the presence of a catalytic amount of
copper chloride. 3,3-[(2,2-dimethyltrimethylene)dioxy]-
estra-5(10),9(11)-dien-17â-ol (1) and 3,3-(ethylenedioxy)-
estra-5(10),9(11)-dien-17-one (15) were used as starting
materials for the preparation of electrophilic estradiol
derivatives, including bromoacetamido compounds 12a
and 12b (Scheme 1), (methylsulfonyl)oxy compounds
13a and 13b, bromobutyl compound 14 (Scheme 2), and
tosylate 20 (Scheme 3). Preparation of epoxy 5R,10R-
derivatives 2 and 16 from 1 and 15 were previously
described.21,22 In the presence of a catalytic amount of
copper chloride, the addition of vinyl- or butenylmag-
nesium chloride to epoxide 2 only afforded the 11â-
substituted steroid 3a or 3b (Scheme 1). The action of
p-toluenesulfonic acid, through simultaneous deketal-
ization and dehydration of the compound, converted
3a ,b into dienone 4a ,b. The A ring of the compound
was then aromatized by treatment with a mixture of
acetyl bromide and acetic anhydride. This treatment
acylated the phenol function, which was regenerated by
saponification.
Studies performed by Belanger et al.20 evinced, for the
first time, the very high affinity of 11â-aliphatic or
aromatic derivatives of 17R-ethynyl estradiol for the
estrogen receptor. These results suggested that large
11â-substituents of steroidal ligands are tolerated by
the hormone-binding pocket of the estrogen receptor.
Therefore, with the aim of developing 11â-derived
steroidal affinity labels which display high affinity for
the estrogen receptor and would be useful for gaining
further information concerning the positioning of ligands
in the hormone-binding pocket of the receptor, we
prepared six new estradiol 11â-derivatives in which an
11â-ethyl, 11â-butyl, or 11â-decyl chain was substituted
with various electrophilic functionalities: bromine, me-
syloxy, tosyloxy, or bromoacetamido. We then deter-
mined their binding affinity and ability to become
irreversibly bound to the receptor. Finally, we report
The phenolic and 17â-hydroxy functions of 5a ,b were
protected as TBDMS ethers to give 6a ,b. The terminal
vinyl or butenyl function was submitted to hydrobora-
tion-oxidation conditions (9-BBN and basic hydrogen
peroxide) to afford the primary alcohol 7a ,b. The
primary hydroxyl group was esterified by methanesulfo-

Steroidal Affinity Labels of the Estrogen Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2219
Ta ble 1. Reversible and Irreversible Binding of Electrophilic
Estradiol 11â-Derivativesa to the Estrogen Receptor
Sch em e 2
11â-alkyl
chain
covalent
binding
compound
estradiol
apparent RAC
100
butene 5b
C4
C2
C4
C2
C4
C4
C10
38.60 ( 0.80
0.38 ( 0.05
1.64 ( 0.28
8.15 ( 0.10
36.80 ( 0.98
11.00 ( 3.70
2.59 ( 0.12
bromoacetamide 12a
bromoacetamide 12b
mesylate 13a
mesylate 13b
bromide 14
-
-
-
-
-
+
tosylate 20
a
Apparent RACs (relative affinity constants) of compounds for
the cytosolic estrogen receptor were determined by competitive
binding (20 h, 20 °C) radiometric assay using [3H]estradiol as
tracer, as described in the Materials and Methods; data given are
means of duplicate determinations ( SD in two to four separate
experiments. Covalent binding of compounds to the estrogen
receptor was established from their ability to irreversibly inhibit
binding of [3H]estradiol (cf. Results, irreversible binding to the
estrogen receptor) symbol (-) means that the decrease in estradiol-
binding sites inactivated by the compound, if any, was <10%.
nyl chloride to give 8a ,b. Action of potassium phthal-
imide on mesylate 8a ,b afforded phthalimide 9a ,b,
which was hydrolyzed into primary amine 10a ,b. The
corresponding bromoacetamide 11a ,b was obtained by
condensation with bromoacetic acid in the presence of
carbodiimide. Removal of the TBDMS group from 11a ,b
with a mixture of acetic acid and water in THF afforded
11â-[(bromoacetamido)alkyl]estradiol 12a ,b. Removal
of the TDMS groups from 8a ,b gave 11â-[[(methylsul-
fonyl)oxy]alkyl]estradiol 13a ,b (Scheme 2). Action of
LiBr on 13b gave 11â-(bromobutyl)estradiol 14.
In the presence of copper chloride, addition of a
TBDMS derivative of (hydroxydecyl)magnesium bro-
mide to epoxide 16 afforded the silylated 11â-hydroxy-
decyl steroid 17 (Scheme 3). The sequential action of
NaBH4 and acetic anhydride converted the 17-keto
function of compound 17 into a 17â-acetoxy function,
whereas action of hydrochloric acid through simulta-
neous deprotection of the primary alcohol and elimina-
tion of the tertiary alcohol gave dienone 18. The
primary alcohol was esterified with p-toluenesulfonyl
chloride, and the A ring of the compound was aroma-
tized with a mixture of acetic bromide and acetic
anhydride to give 19. Tosylate 20 with free 3- and 17â-
hydroxy functions was obtained from 19 by saponifica-
tion.
Estr ogen Recep tor Bin d in g Affin ity. The appar-
ent affinity of synthesized compounds for the lamb
estrogen receptor was determined by competitive bind-
ing radiometric assay using [3H]estradiol as tracer. At
20 °C the competition level between [3H]estradiol and
the compounds did not significantly change after 20 h
incubation, suggesting that binding equilibrium was
reached under these conditions. Table 1 gives the
corresponding apparent affinity constants of the com-
pounds, relative to that of estradiol (100%), which were
calculated according to Korenman.23 Affinities of the
compounds (from 0.4% to 37%) were much lower than
those reported for other 11â-derivatives of estradiol
including both small-group-substituted, e.g. chlorom-
ethyl,24 vinyl,25 or ethyl25,26 molecules, or large-group-
substituted, e.g. N-propyl- or N-butylundecanamide,
molecules.27 Affinities of the electrophilic estradiol
derivatives markedly varied according to the length of
the 11â-n-alkyl arm and to the substituent on the ꢀ
carbon of the alkyl chain. Affinities increased when the
ethyl chain was changed to the n-butyl chain (compare
12a with 12b and 13a with 13b ) and when the
NHCOCH2Br group was changed to Br and then to the
Sch em e 3

2220 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Lobaccaro et al.
F igu r e 1. Time course of inactivation of specific estradiol-
binding sites in lamb uterine cytosol by 11â-[(tosyloxy)decyl]-
estradiol. Uterine cytosol (4 mg of protein/mL, pH 8.5) was
incubated at 0 or 25 °C without steroid, with 50 nM estradiol
or 50 nM tosylate 20. After various periods of time, aliquots
were removed and treated for 30 min at 0 °C with an equal
volume of charcoal suspension. Charcoal was pelleted by
centrifugation, then the total and nonspecific binding of [3H]-
estradiol occurring in the supernatant under exchange condi-
tions were determined by the standard irreversible binding
assay, as described in the Materials and Methods. The total
binding of [3H]estradiol in supernatants corresponding to
cytosol incubated at 0 °C (open symbols) or 25 °C (close
symbols) without steroid (O, b), with estradiol (4, 2), or
tosylate 20 (3, 1) and the nonspecific binding of [3H]estradiol
(×) which did not significantly vary according to the compound
incubated with cytosol, are represented as functions of the
incubation time. Values are means of duplicate determina-
tions; experimental variation was under 10%.
F igu r e 2. Concentration-dependent inactivation of specific
estradiol-binding sites by 11â-[(tosyloxy)decyl]estradiol. Uter-
ine cytosol was incubated for 4 h at 25 °C without steroid or
with increasing concentrations of estradiol or tosylate 20. After
charcoal treatment and then centrifugation, supernatant
aliquots were incubated under exchange conditions with [3H]-
estradiol. The total binding of [3H]estradiol in supernatant
corresponding to cytosol incubated with estradiol (2) or
tosylate 20 (b) and the nonspecific binding of [3H]estradiol (×)
which did not significantly vary according to the steroid
incubated with the cytosol, are represented as functions of the
steroid concentration. Values are means of triplicate determi-
nations; error bars indicate standard deviations.
dependent decrease in the capacity of cytosol to bind
[3H]estradiol. However, a substantial decrease occurred
at 25 °C, and the concentration of binding sites reached
a plateau from 4 h, which accounted for 50% and 40%
of the concentration of binding sites in the controls
(cytosol incubated without steroid, in which unprotected
binding sites were progressively heat-inactivated at 25
°C, and also cytosol incubated with 50 nM unlabeled
estradiol, in which protected binding sites were stable
at 25 °C). The effect of compound 20, not dependent
on the cytosol pH, increased with the compound con-
centration, reaching a quasi-optimal level (70-90% of
binding sites inactivated) for 10 nM 20 (Figure 2).
Moreover, inactivation of estrogen-binding sites by
compound 20 was not prevented by methyl methaneth-
iosulfonate, an SH group specific reagent, suggesting
that the covalent attachment site of the compound was
not a cysteine residue. Conversely, none of the other
electrophilic estradiol 11â-derivatives (at a concentra-
tion equivalent to 20 nM estradiol) was able to induce,
at 0 or 25 °C and within the 7-9 pH range, a marked
decrease in the capacity of cytosol to bind estradiol, even
after a prolonged exposure (20 h) of cytosol to the
compound. The potential decrease in the concentration
of binding sites, if any, was always under 10% (Table
1). Therefore, in the estradiol 11â-alkyl-substituted
series, only compounds with a long electrophile-bearing
arm appeared to be able to specifically alkylate the
estrogen receptor.
OSO2CH3 group (compare 12b, 13b, and 14). Due to
irreversible binding of the compound to the estrogen
receptor (cf. following section), the 2.6% value deter-
mined for the 11â-decyl derivative 20 affinity was
probably an overestimate of the true affinity constant
of the compound.
Ir r ever sible Bin d in g to th e Estr ogen Recep tor .
The estrogen receptor-alkylating activities of electro-
philic estradiol derivatives were evaluated from their
ability to irreversibly inhibit estradiol specific binding
in cytosol.15 Affinity labeling of the receptor is usually
a concentration- and time-dependent process which,
depending of the type of electrophile, could be influenced
by temperature and pH. To determine the alkylation
level of the receptor, the cytosol was thus incubated at
various pH values (from 7 to 9) and at either 0 or 25 °C
for increasing times with the electrophilic estradiol 11â-
derivatives, at a concentration usually equivalent to 20
nM estradiol; excess compound was removed and the
concentration of estrogen binding sites was determined
by incubating the cytosol with [3H]estradiol under
exchange conditions. Cytosol was concomittantly incu-
bated without steroid, with unlabeled estradiol or with
the high-affinity, but chemically inert, compound 5b,
to obtain reference values for the concentration of
estrogen binding sites in cytosol. Any decrease from
these values elicited by one of the electrophiles would
characterize an estrogen receptor affinity labeling agent.15
Results obtained with tosylate 20 (Scheme 3) indicated
that at 25 °C, but not at 0 °C, this compound was an
efficient affinity labeling agent of the receptor. Figure
1 shows variations in the concentration of binding sites
for [3H]estradiol in cytosol incubated for increasing
times at pH 8.5 and 0 or 25 °C with 50 nM tosylate 20.
At 0 °C, the compound did not elicit any marked time-
Estr ogen ic a n d An tiestr ogen ic P r op er ties. To
determine the estrogen agonist/antagonist activity of
estradiol 11â-derivatives, we used the MVLN cell line,28
which was derived from estrogen-responsive MCF7 cells
by stable transfection of the p-Vit-tk-Luc plasmid. For
a large series of estrogenic or antiestrogenic compounds,
it was previously shown that the bioluminescent re-
sponse of these cells was qualitatively similar to natural

Steroidal Affinity Labels of the Estrogen Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2221
Ta ble 2. Estrogenic/Antiestrogenic and Antiproliferative Activities of Electrophilic Estradiol 11â-Derivatives
estrogen
agonisma
EC50 (nM)
antagonisma
IC50 (nM)
11â-alkyl
luciferase inactivationb
t1/2 (days)
antiproliferative
activityc (%)
compound
butene 5b
bromoacetamide 12a
bromoacetamide 12b
mesylate 13a
mesylate 13b
bromide 14
tosylate 20
4-hydroxytamoxifen
chain
%
%
C4
C2
C4
C2
C4
C4
C10
80
90
<10
110
>30
>30
20
>30
22
26
13
10
20
5
90
320
90
10
90
80
85
100
95
6
95
85
95
6
3
4
7
100
a
The estrogen agonism or antagonism of compounds was determined from experiments similar to those described in the Figure 3
legend. For estrogen agonists, the percent value indicates the maximal luciferase specific activity induced by the compound relative to
that induced by 0.1 nM estradiol (100%); the EC50 value is the compound concentration required to induce a luciferase specific activity
equal to 50% of that induced by 0.1 nM estradiol. For estrogen antagonists, the percent value indicates the maximal inhibition of luciferase
specific activity induced by 0.1 nM estradiol; the IC50 value is the compound concentration required to inhibit by 50% the luciferase
specific activity induced by 0.1 nM estradiol. Values are means of two independent determinations; experimental variation was under
b
5% (percent values) or 30% (EC50 or IC50 values). Experiments similar to those described in the Figure 4 legend were performed for 7,
14, 21, and 28 days of treatment to determine the kinetics of luciferase irreversible inhibition of expression. The time for half-irreversible
inhibition was obtained by interpolation or extrapolation of experimental curves (logarithm of luciferase specific activity percent plotted
against the treatment time); experimental variation was under 30%. c MVLN cells were grown for 7 days without compound, in the
presence of 0.1 nM estradiol or in the presence of 100 nM estradiol 11â-derivatives or 4-hydroxytamoxifen. Cells were processed as
described in the Materials and Methods. Antiproliferative activity was calculated from the difference between DNA amounts measured
in wells exposed to estradiol and in wells exposed to estradiol 11â-derivatives. It was expressed as a percentage of antiproliferative
activity determined for 4-hydroxytamoxifen. Values are means of triplicate determinations; experimental variation was under 10%.
ficiency of compounds to induce estrogenic expression
of the luciferase gene was in line with their relative
affinities for the estrogen receptor, whereas the ef-
ficiency of antiestrogenic compounds to inhibit expres-
sion of the gene was not correlated with their affinity
for the receptor (compare affinities and activities of 12b
and 20 for instance).
Ir r ever sible In a ctiva tion of th e Lu cifer a se Gen e.
Long-term treatment of MVLN cells with antiestrogens
was shown to progressively abolish the ability of cells
to express the luciferase gene in response to estrogens.30
This irreversible effect, which seems to result from an
epigenetic mechanism, such as methylation or chroma-
tin remodeling rather than gene mutation (Badia et al.,
submitted), progressively affected the whole cell popula-
tion and occurred by a first-order process, with a half-
inactivation time of ∼10 days in the case of 4-hydrox-
philic estradiol 11â-derivatives in MVLN cells. MVLN cells
F igu r e 3. Estrogenic and antiestrogenic activities of electro-
ytamoxifen. Since the electrophilic estradiol 11â-
derivatives 12a -14 displayed high affinity and did not
react in vitro with the estrogen receptor, they could be
concentrated (via the receptor) in the nucleus of estrogen
target cells where, by virtue of their chemical reactivity,
they could exert general cytotoxicity or targetted cyto-
toxicity toward for instance estrogen-regulated genes
involved in cellular proliferation (i.e. genes coding for
growth factors, growth factor receptors, protooncogenes,
etc.). To clarify this possibility, we first investigated
the ability of the compounds to inactivate the luciferase
gene of MVLN cells. All of the electrophilic estradiol
11â-derivatives decreased the ability of cells to express
luciferase in response to estradiol. During a 7-day cell
treatment, only the antiestrogenic compounds 12b, 13b,
and 14 elicited a significant loss (g20%) in luciferase
inducibility (not shown); whereas no marked inactiva-
tion was observed with the estrogenic compounds 5b,
12a , and 13a . During a 21-day treatment, all of the
compounds were found to be efficient; there was 20-
75% less luciferase inducibility depending on the com-
pound (Figure 4). The results indicated that (i) the
compounds were less efficient than 4-hydroxytamoxifen
(Table 2) and (ii) estrogen antagonism or the electro-
were incubated for 24 h in FCS/C medium with 1 µM
4-hydroxytamoxifen (2) or with increasing concentrations of
mesylate 13a (A), bromoacetamide 12a (B), mesylate 13b (C)
or bromoacetamide 12b (D) in the absence (b) and presence
(O) of 0.1 nM estradiol. Luciferase activity and protein
concentration of cell homogenates were then determined.
Specific luciferase activity, expressed as a percentage of the
specific luciferase activity corresponding to cells incubated with
estradiol alone, is plotted against the compound concentra-
tions. Values are means of duplicate determinations; error bars
indicate standard deviations.
estrogenic responses such as progesterone receptor
induction.29
Estrogenic and antiestrogenic activities of compounds
were determined separately by incubating the cells for
24 h with increasing concentrations of compounds in the
absence and in the presence of 0.1 nM estradiol,
respectively. Activity of the electrophilic compounds
(Figure 3, Table 2) was not correlated with the whole
11â-substituent, but rather with the alkyl part of the
substituent, since the two estradiol 11â-ethyl derivatives
12a and 13a mainly displayed estrogenic activity,
whereas the three estradiol 11â-butyl derivatives 12b,
13b , and 14 and the 11â-decyl derivative 20 showed
almost pure antiestrogenic activity. The relative ef-

2222 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Lobaccaro et al.
F igu r e 4. Irreversible inhibition of estrogen-induced expres-
sion of luciferase in MVLN cells by estradiol 11â-derivatives.
MVLN cells were grown for 21 days in FCS/C medium in the
absence of compounds (0) or in the presence of 100 nM
4-hydroxytamoxifen (OHT), butene 5b, bromoacetamide 12a
or 12b, mesylate 13a or 13b, bromide 14, or tosylate 20. Cells
were then rinsed and incubated for 7 days in FCS/C medium
containing 100 nM estradiol. Luciferase activity and protein
concentration of cell homogenates were then determined.
Specific luciferase activities (expressed as percents of the
specific luciferase activity corresponding to cells not exposed
to 4-hydroxytamoxifen or estradiol 11â-derivatives) are rep-
resented. Values are means of duplicate determinations; error
bars indicate standard deviations.
F igu r e 5. Proliferative and antiproliferative activities of
estradiol 11â-derivatives. MVLN cells were grown for 7 days
in FCS/C medium in the absence of compounds (0) or in the
presence of 0.1 nM estradiol (E2), 100 nM 4-hydroxytamoxifen
(OHT), butene 5b, bromoacetamide 12a or 12b, mesylate 13a
or 13b, bromide 14, or tosylate 20. The well DNA contents
were then determined. Values are means of triplicate deter-
minations; error bars indicate standard deviations.
all five new synthesized steroidal electrophiles, related
either to 11â-ethylestradiol or 11â-butylestradiol, dis-
played no significant estrogen receptor affinity labeling
activity, since any of these compounds was able to elicit
a significant decrease in the concentration of estradiol
binding sites in cytosol. Conversely tosylate 20 induced
a strong decrease in the estradiol-binding site concen-
tration. This effect very probably reflected the covalent
attachment of the compound in the receptor hormone-
binding site. Indeed, another 11â-derivative, i.e. 11â-
(chloromethyl)estradiol was initially thought to be an
affinity-labeling agent of the receptor31-33 based on its
apparent inability to be displaced by estradiol. It was
later demonstrated that this compound was in fact a
reversible ligand;24 however, due to its very high affinity
for the receptor, 10-30-fold higher than that of estra-
diol, its dissociation rate and therefore exchange rate
under typical assay conditions were negligible. In
contrast with the high affinity of such ligands as 11â-
(chloromethyl)estradiol, the binding affinity constant of
tosylate 20 appeared at least 38-fold lower than that of
estradiol. Since the value of the affinity constant mainly
reflects the compound dissociation rate from receptor,
the decrease of estradiol binding-site concentration
following incubation of the receptor with tosylate 20
very probably did not result from low dissociation rate
of noncovalently receptor-bound compound but rather
from covalent attachment of the compound in the
receptor hormone-binding pocket. Therefore we con-
cluded that tosylate 20, like electrophilic estradiol 17R-
derivatives, was a very efficient receptor affinity label-
ing agent. The fact that none of the five compounds
12a -14, whose electrophilic carbon is two to seven C-C
or C-N bonds away from C-11, was able to alkylate the
receptor suggests that there was no reactive nucleophilic
amino acid residue in the close vicinity of the C ring
when the steroidal ligands were in the hormone-binding
pocket. Conversely, affinity labeling of the receptor by
compound 20, whose electrophilic centre is 10 C-C
bonds away from C-11, suggests that at least one
nucleophilic amino acid residue was in the remote
vicinity of the C ring. The length of the decyl substitu-
ent in compound 20 allowed the electrophilic carbon to
philic character of the compound was not required to
induce the inactivation process since the mainly estro-
genic and nonelectrophilic compound 5b was able to
inactivate the gene. We then tested the electrophilic
compounds using the ETL cell line, obtained from
MCF-7 cells by stable transfection of a plasmid includ-
ing an ERE-tk-Luc sequence (P. Balaguer et al., unpub-
lished results) instead of the p-Vit-tk-Luc sequence used
to generate the MVLN cell line. Contrary to MVLN,
expression of the luciferase gene of ETL cells was not
profoundly suppressed by antiestrogens such as 4-hy-
droxytamoxifen; therefore this cell line could be useful
for determining whether estrogen receptor-targetted,
chemically reactive molecules could inactivate estrogen-
regulated genes. After a 14-day treatment with the
different electrophiles, the ability of ETL cells to express
luciferase in response to estradiol was not markedly
changed (not shown). We therefore concluded that
estrogen receptor ligands bearing a single electrophilic
group on a 11â-alkyl substituent of estradiol-related
molecules did not seem to be able to inactivate estrogen
target genes.
An tip r olifer a tive Activity. The proliferative or
antiproliferative activity of compounds was evaluated
from their ability to promote or prevent DNA accumula-
tion from MVLN cells grown for 7 days in “steroid-free”
medium. Contrary to estradiol, the four antiestrogenic
derivatives 12b, 13b, 14, and 20 did not promote MVLN
cell proliferation (Figure 5); however, the decrease in
DNA accumulation they elicited was significantly lower
than that induced by 4-hydroxytamoxifen. Conversely,
the three estrogenic derivatives 5b, 12a , and 13a were
almost as efficient as estradiol in stimulating cell
proliferation.
Discu ssion
Despite a much higher affinity for the estrogen
receptor than estradiol 17R-electrophilic derivatives,15,16

Steroidal Affinity Labels of the Estrogen Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2223
its antiestrogenicity. It is thus difficult to determine
whether part of the observed effects were due to the
chemical reactivity of the compounds. The fact that
luciferase gene expression in ETL cells was not mark-
edly abolished by the compounds is another argument
favoring the absence of specific cytotoxicity of the
compounds. Nevertheless, the two interesting charac-
teristics of 11â-ethyl- or 11â-butylestradiol derivatives,
i.e. high affinity for the estrogen receptor and lack of
neutralizing chemical reactivity by the receptor, make
them a choice support for grafting a cytotoxic moiety
such as nitrosoureas, nitrogen mustards, intercalating
drugs, platinum complexes, or enediyne structures,
which are used in antitumoral therapy but whose
general toxicity is high due to their lack of selectivity.
Such hybrid estradiol-cytotoxic molecules, which would
display both high affinity for the estrogen receptor and
strong cytotoxic activity, might be useful agents for the
treatment of estrogen receptor-containing mammary
tumors. Work is presently in progress in our laboratory
to develop such potential selective antitumor agents.
potentially scan a large volume centered at C-11. When
compound 20 was in the receptor hormone-binding
pocket, the location of the electrophilic carbon relative
to the steroid nucleus therefore could not be a priori
ascertained. For instance, although the original orien-
tation of the substituent at C-11 is above and roughly
perpendicular to the median plane of the steroid, the
terminal carbon of the decyl chain could still be located
under the median plane of the steroid. The uncertainty
concerning the relative location of the electrophilic
carbon reduces the interest of the compound for analysis
of the hormone-binding pocket of the receptor. Never-
theless, the inability of methyl methanethiosulfonate to
prevent affinity labeling of the receptor and pH-
independence of the process suggest that, contrary to
tamoxifen aziridine, ketononestrol aziridine, and 17R-
[(bromoacetamido)alkyl]estradiols, the covalent attach-
ment site(s) of tosylate 20 did not occur at cysteine
residues. In the hormone-binding pocket of the receptor,
the environment of the electrophilic carbon of compound
20 thus differed from that of electrophilic centers of
tamoxifen aziridine, ketononestrol aziridine, and 17R-
[(bromoacetamido)alkyl]estradiols.
Ma ter ia ls a n d Meth od s
Ch em ica l. Syn th esis a n d Ch a r a cter iza tion of Estr a -
d iol Der iva tives. Starting materials were 3,3-[(2,2-dimeth-
yltrimethylene)dioxy]estra-5(10),9(11)-dien-17â-ol (1, ZK 37
875) (obtained from Professor Wiechert of Schering) and 3,3-
(ethylenedioxy)estra-5(10),9(11)-dien-17-one (15).21 Starting
reactions were carried out under an argon atmosphere with
dry solvents used under anhydrous conditions. Melting points
(mp) were determined on a Bioblock melting point apparatus
and were uncorrected. Infared spectra (IR) were recorded on
a Perkin-Elmer 983 spectrophotometer by means of potassium
disks. Proton magnetic resonance (1H NMR) spectra were
recorded at 32 °C on a Bruker WM 360 WB spectrometer.
Except for compounds 5a and 12b (which were solubilized in
DMSO-d6), compounds were dissolved in CDCl3 from which
The estradiol electrophilic 11â-derivatives proved to
be either estrogenic (11â-ethyl compounds) or anties-
trogenic (11â-butyl and 11â-decyl compounds). The fact
that the type of hormonal/antihormonal activity was
related to the alkyl part of the 11â-substituent but not
to the size of the whole substituent (which was for
estrogenic compounds 12a and 13a similar to or higher
than that of antiestrogenic compound 14) or to the type
of terminal electronegative group (12a and 12b on one
hand and 13a and 13b on the other displayed opposite
hormonal activities, even though they had the same
terminal function) was astonishing. This could result
(i) from intrinsic but still unexplained properties of the
compounds or (ii) from hydrolysis of the compounds in
MVLN cells. In cytosolic extracts, all electrophiles
appeared to be fairly stable, with only a decrease in the
amount of extractible compounds according to time or
temperature, which was probably due to reaction of the
compounds with cytosolic nucleophiles. However, since
no such radioactive compounds were available, it was
difficult to study their potential metabolism or hydroly-
sis within MVLN cells at the concentrations used. Note
that hydrolysis could convert the bulky 11â-substituents
of 12a and 13a into smaller ones, i.e. aminoethyl and
hydroxyethyl, respectively. For homologous compounds
12b and 13b, the relative decrease in size resulting from
hydrolysis would be less pronounced. In any case, with
estradiol 11â-substituted derivatives, it appears that the
threshold which separates estrogenic from antiestro-
genic compounds is rapidly reached when the size of the
11â-alkyl chain increases, with a threshold between C-2
and C-4.
1
the residual signal was taken as reference at 7.24 ppm for H.
Except when otherwise stated, column chromatography was
performed with silica gel (0.063-0.2 mm). Thin-layer chro-
matography (TLC) was performed using 0.25 mm silica gel
plates with F-254 indicator. Elemental analyses were deter-
mined for C, H, and O and were within 0.4% of theoretical
values. Standard product isolation involved quenching the
reaction mixture in water or aqueous solution, followed by
exhaustive extraction with ether or ethyl acetate, washing
extracts with aqueous solutions when necessary, drying or-
ganic extracts over Na2SO4, filtration, and evaporation of the
solvent under reduced pressure. The solvents and the aqueous
solutions used for quenching the reaction are mentioned in
parentheses after “product isolation”.
[3,3-(2,2-Dim eth yltr im eth ylen e)d ioxy]-11â-vin ylestr -9-
en e-5r,17â-d iol (3a ). A 1.73 M solution of vinylmagnesium
chloride (8.8 mL, 15.2 mmol) in THF was cooled to -30 °C,
and a solution of copper(I) chloride (80 mg, 0.8 mmol) in 6 mL
of THF was added. The reaction mixture was stirred for 0.5
h, a solution of epoxide 2 (0.95 g, 2.33 mmol) in 3 mL of THF
was added dropwise, and the mixture was stirred for 3 h while
the temperature rose from -30 to -10 °C. The reaction
mixture was poured into cold, saturated aqueous ammonium
chloride. Product isolation (EtOAc, NaCl, Na2SO4) and then
chromatography on aluminum 90 basic oxide (EtOAc-hexane,
2:8) gave 850 mg (84%) of 3a which was recrystallized from
diethyl ether: mp 121-123 °C; IR 3451, 1638 cm-1; NMR
(CDCl3) 0.87 (s, 3H, ketal-CH3), 0.95 (s, 3H, 18-CH3), 1.06 (s,
3H, ketal-CH3), 3.55 (m, 4H, 2CH2O), 3.67 (t, 1H, J ) 6 Hz,
17-CH), 3.69 (m, 1H, 11-CH), 4.76-4.96 (dd, 2H, J 1 ) 10.3
Hz, J 2 ) 17.3 Hz, 2′-CH2), 5.86 (m, 1H, 1′-CH) ppm. Anal.
(C25H38O4) C, H, O.
According to the two criteria used, i.e. inactivation of
the luciferase gene and inhibition of MVLN cell growth,
the six electrophiles did not appear to exert cytotoxic
activity in these cells. All electrophiles, whether they
were estrogen agonists or antagonists, abolished expres-
sion of the luciferase gene, and those displaying anti-
estrogenic activity also decreased cell proliferation.
However, to induce these two effects, the compounds
were less efficient than 4-hydroxytamoxifen, a chemi-
cally inert compound whose effects are probably due to
[3,3-(2,2-Dim eth yltr im eth ylen e)dioxy]-11â-(3′-bu ten yl)-
estr -9-en e-5r,17â-d iol (3b). The 11â-butenyl derivative 3b
was prepared from epoxide 2 by the procedure described for

2224 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Lobaccaro et al.
compound 3a using butenylmagnesium chloride instead of
vinylmagnesium chloride. Compound 3b (85%) was purified
by chromatography on aluminium 90 basic oxide (EtOAc-
3,17â-Bis[(t er t -b u t yld im e t h ylsilyl)oxy]-11â-(2′-h y-
d r oxyeth yl)estr a -1,3,5(10)-tr ien e (7a ). 9-BBN (0.5 M solu-
tion in THF, 10.22 mmol) under argon was added dropwise to
a solution of 770 mg (1.46 mmol) of 6a in 5 mL of THF at 60
°C. After 1 h, the solution was cooled to -5 °C, 1.5 mL of water
was added, and stirring was continued for 5 min. Sodium
hydroxide (3 M, 1.5 mL) was added, and after an additional
10 min, 1.5 mL of hydrogen peroxide (30%) was added
dropwise. After 30 min of stirring at 0 °C, product isolation
(NaHCO3, EtOAc, Na2SO4) and then chromatography (EtOAc-
hexane, 5:95) afforded 750 mg of the primary alcohol 7a
(94%): mp 148.1-149.9 °C; IR 3437, 1570 cm-1; NMR (CDCl3)
0.03-0.06 (2s, 12H, 2Si(CH3)2), 0.84 (s, 3H, 18-CH3), 0.87-
0.94 (2s, 18H, 2SiC(CH3)3), 3.40 (m, 2H, 2′-CH2), 4.00 (m, 1H,
11-CH), 6.50 (d, 1H, J ) 2.6 Hz, 4-CH), 6.70 (m, 1H, 2-CH),
7.02 (d, 1H, J ) 8.2 Hz, 1-CH) ppm. Anal. (C32H56O3Si2) C,
H, O.
hexane, 4:6): mp 153.3-154.4 °C; IR 3419, 1736, 1655 cm-1
;
NMR (CDCl3) 0.90 (s, 6H, ketal-2CH3), 0.95 (s, 3H, 18-CH3),
3.45 (m, 4H, 2CH2O), 3.65 (t, 1H, J ) 6.1 Hz, 17-CH), 4.98
(dd, 2H, J 1 ) 10.3 Hz, J 2 ) 17 Hz, 4′-CH2), 5.76 (m, 1H, 3′-
CH) ppm. Anal. (C27H42O4) C, H, O.
11â-Vin yl-17â-h yd r oxyestr a -4,9-d ien -3-on e (4a ). A so-
lution of 410 mg (1.02 mmol) of 3a and 20 mg (0.11 mmol) of
p-toluenesulfonic acid in acetone (20 mL) was stirred for 2 h
at room temperature. Product isolation (EtOAc, NaHCO3, Na2-
SO4) and then chromatography (EtOAc-hexane, 5:5) gave 270
mg (90%) of 4a which was recrystallized from diethyl ether:
mp 143.1-145.3 °C; IR 3419, 1736, 1655 cm-1; NMR (CDCl3)
0.85 (s, 3H, 18-CH3), 3.61 (t, 1H, J ) 6 Hz, 17-CH), 3.72 (m,
1H, 11-CH), 4.80-4.96 (m, 2H, 2′-CH2), 5.66 (s, 1H, 4-CH),
5.90 (m, 1H, 1′-CH) ppm. Anal. (C20H26O2) C, H, O.
3,17â-Bis[(t er t -b u t yld im e t h ylsilyl)oxy]-11â-(4′-h y-
d r oxybu tyl)estr a -1,3,5(10)-tr ien e (7b). The 11â-hydroxy-
butyl derivative 7b was prepared from 6b by the procedure
described for compound 7a . Product 7b (97%) was purified
by chromatography (EtOAc-hexane, 1:3) and recrystallizated
from pentane: mp 114.1-115.2 °C; IR 3455, 1585 cm-1; NMR
(CDCl3) 0.04-0.17 (2s, 12H, 2Si(CH3)2), 0.84 (s, 3H, 18-CH3),
0.88-0.94 (2s, 18H, 2SiC(CH3)3), 3.58 (t, 1H, J ) 6.1 Hz, 17-
CH), 6.51 (d, 1H, J ) 2.6 Hz, 4-CH), 6.59 (m, 1H, 2-CH), 6.95
(d, 1H, J ) 8.2 Hz, 1-CH) ppm. Anal. (C34H60O3Si2) C, H, O.
3,17â-Bis[(ter t-bu tyld im eth ylsilyl)oxy]-11â-[2′-[(m eth -
ylsu lfon yl)oxy]eth yl]estr a -1,3,5(10)-tr ien e (8a ). Methane-
sulfonyl chloride (0.4 mL, 5 mmol) was added to a solution of
600 mg (1.1 mmol) of primary alcohol 7a in 1.5 mL of
triethylamine. The solution was stirred for 1 h at room
temperature. Product isolation (NaHCO3, EtOAc, Na2SO4) and
chromatography (EtOAc-hexane, 1:9) afforded 550 mg of 8a
(80%): mp 148.9-150.3 °C; IR 1608, 1550 cm-1; NMR (CDCl3)
0.03-0.07 (2s, 12H, 2Si(CH3)2), 0.85 (s, 3H, 18-CH3), 0.88-
0.92 (2s, 18H, 2SiC(CH3)3), 2.90 (s, 3H, SCH3), 3.45 (m, 2H,
2′-CH), 3.95 (m, 1H, 11-CH), 6.45 (d, 1H, J ) 2.6 Hz, 4-CH),
6.70 (m, 1H, 2-CH), 6.95 (d, 1H, J ) 8.2 Hz, 1-CH) ppm. Anal.
(C33H58O5Si2S) C, H, O.
11â-(3′-Bu ten yl)-17â-h yd r oxyestr a -4,9-d ien -3-on e (4b).
The derivative 4b was prepared from 3b by the procedure
described for compound 4a . Compound 4b (84%) was purified
by chromatography (EtOAc-CH2Cl2, 2:8) as a colorless oil: IR
3427, 1710, 1655 cm-1; NMR (CDCl3) 0.85 (s, 3H, 18-CH3), 3.49
(t, 1H, J ) 6 Hz, 17-CH), 4.85 (dd, 2H, J 1 ) 10.3 Hz, J 2 ) 17.3
Hz, 4′-CH2), 5.52 (s, 1H, 4-CH), 5.63 (m, 1H, 3′-CH) ppm. Anal.
(C22H30O2) C, H, O.
11â-Vin ylestr a -1,3,5(10)-tr ien e-3,17â-d iol (5a ). Acetic
anhydride (2.4 mL, 25.4 mmol) and acetyl bromide (1.3 mL,
7.6 mmol) were added, with stirring, to an ice-cooled solution
of compound 4a (1.91 g, 6.4 mmol) in methylene chloride (15
mL). After the mixture was stirred for 2 h at 0 °C, a saturated
aqueous sodium bicarbonate solution was added. Extraction
with methylene chloride, followed by the usual workup, gave
the phenolic acetate which was treated with 10 N sodium
hydroxide in methanol (120 mL) for 4 h at room temperature.
After acidification with 0.5 N hydrochloric acid, product
isolation (EtOAc, Na2SO4) and then chromatography (EtOAc-
hexane, 3:7) gave 1.63 g of 5a (85%): mp 204.9-206.1 °C (lit.25
196-198 °C); IR 3380, 1620, 1586 cm-1; NMR (DMSO-d6) 0.70
(s, 3H, 18-CH3), 3.22 (m, 1H, 11-CH), 3.50 (t, 1H, J ) 6 Hz,
17-CH), 4.86-4.96 (dd, 2H, J 1 ) 10.3 Hz, J 2 ) 17.3 Hz, 2′-
CH2), 5.67 (m, 1H, 1′-CH), 6.40 (d, 1H, J ) 2.6 Hz, 4-CH), 6.46
(m, 1H, 2-CH), 6.85 (d, 1H, J ) 8.2 Hz, 1-CH) ppm. Anal.
(C20H26O2) C, H, O.
11â-(3′-Bu t en yl)est r a -1,3,5(10)-t r ien e-3,17â-d iol (5b ).
Compound 5b was prepared from 4b by the procedure de-
scribed for compound 5a . The product (85%) was purified by
chromatography (EtOAc-hexane, 3:7): mp 182.4-183.8 °C; IR
3410, 1625, 1590 cm-1; NMR (CDCl3) 0.92 (s, 3H, 18-CH3), 3.7
(dd, 1H, J 1 ) 7.3 Hz, J 2 ) 8.2 Hz, 11-CH), 4.91 (m, 2H, 4′-
CH2), 5.71 (t, 1H, J ) 6 Hz, 3′-CH), 6.52 (d, 1H, J ) 2.6 Hz,
4-CH), 6.62 (dd, 1H, J 1 ) 2.7 Hz, J 2 ) 8.2 Hz, 2-CH), 6.98 (d,
1H, J ) 8.2 Hz, 1-CH) ppm. Anal. (C22H30O2) C, H, O.
3,17â-Bis[(ter t-bu tyld im eth ylsilyl)oxy]-11â-vin ylestr a -
1,3,5(10)-tr ien e (6a ). A mixture of 500 mg (3.3 mmol) of
TBDMS chloride and 400 mg (5.9 mmol) of imidazole in 1.3
mL of DMF was added to 300 mg (1.01 mmol) of 5a . After
the mixture was stirred for 2 h at room temperature, product
isolation (CH2Cl2, NaHCO3, Na2SO4) and recrystallization from
ethyl acetate gave 510 mg of 6a (96%): mp 204.9-206.1 °C;
IR 1609,1571 cm-1; NMR (CDCl3) 0.10-0.15 (2s, 12H, 2Si-
(CH3)2), 0.75 (s, 3H, 18-CH3), 0.87-0.95 (2s, 18H, 2SiC(CH3)3),
3.26 (m, 1H, 11-CH), 3.60 (t, 1H, J ) 6.1 Hz, 17-CH), 4.90-
4.97 (dd, 2H, J 1 ) 10.3 Hz, J 2 ) 17.3 Hz, 2′-CH2), 5.70 (m, 1H,
1′-CH), 6.50 (d, 1H, J ) 2.6 Hz, 4-CH), 6.56 (m, 1H, 2-CH),
6.92 (m, 1H, 1-CH) ppm. Anal. (C32H54O2Si2) C, H, O.
3,17â-Bis[(ter t-bu tyldim eth ylsilyl)oxy]-11â-(3′-bu ten yl)-
estr a -1,3,5(10)-tr ien e (6b). Compound 6b was prepared from
5b by the procedure described for compound 6a . The product
(77%) was purified by recrystallization from ethyl acetate: mp
109.8-111.2 °C; IR 1630, 1585 cm-1; NMR (CDCl3) 0.05-0.17
(2s, 12H, 2Si(CH3)2), 0.86 (s, 3H, 18-CH3), 0.89-0.96 (2s, 18H,
2SiC(CH3)3), 3.60 (t, 1H, J ) 6.1 Hz, 17-CH), 4.91 (m, 2H, 4′-
CH2), 5.72 (m, 2H, 3′-CH), 6.51 (d, 1H, 4-CH), 6.60 (m, 1H,
2-CH), 6.96 (d, 1H, J ) 8.2 Hz, 1-CH) ppm. Anal. (C34H58O2-
Si2) C, H, O.
3,17â-Bis[(ter t-bu tyld im eth ylsilyl)oxy]-11â-[4′-[(m eth -
ylsu lfon yl)oxy]bu tyl]estr a -1,3,5(10)-tr ien e (8b). The 11â-
(mesyloxy)butyl derivative 8b was prepared from 7b by the
procedure described for compound 8a . The product (90%) was
purified by chromatography (EtOAc-hexane, 1:9): mp 76.1-
77.1 °C; IR 1608, 1570 cm-1
; NMR (CDCl3) 0.03-0.10 (2s, 12H,
2Si(CH3)2), 0.84 (s, 3H, 18-CH3), 0.88-0.96 (2s, 18H, 2SiC-
(CH3)3), 3.60 (t, 1H, J ) 6.1 Hz, 17-CH), 4.14 (m, 2H, 4′-CH),
6.51 (d, 1H, J ) 2.6 Hz, 4-CH), 6.60 (m, 1H, 2-CH), 6.94 (d,
1H, J ) 8.2 Hz, 1-CH) ppm. Anal. (C35H62O5Si2S) C, H, O.
11â-[2′-(Br om oa ceta m id o)eth yl]estr a -1,3,5(10)-tr ien e-
3,17â-d iol (12a ). Potassium phthalimide (490 mg, 2.64 mmol)
was added to a solution of 1.41 g (2.26 mmol) of mesylate 8a
in 20 mL of DMF. The mixture was stirred for 20 h at 45 °C.
Product isolation (H2O, NaHCO3, EtOAc, Na2SO4) and recrys-
tallization from ethanol gave phthalimide 9a (52%), mp 110-
111.5 °C. A solution of 200 mg (0.29 mmol) of 9a and 80.8 µL
of hydrazine hydrate (1.63 mmol) in 12 mL of EtOH was
stirred for 10 h at 30 °C. Product isolation (EtOAc, 0.5 N
NaOH, Na2SO4) afforded 150 mg of crude primary amine 10a
(90%). Bromoacetic acid (99 mg, 0.78 mmol) and 1-ethyl-3-
[3-(dimethylamino)propyl]carbodiimide (CDI) hydrochloride
(149 mg, 0.78 mmol) were added to a solution of 10a (195 mg,
0.36 mmol) in THF (3 mL). The mixture was stirred for 5 min,
and then 6.5 µL of pyridine was added. After 30 min of stirring
at 40 °C, the mixture was poured in H2O. Product isolation
(H2O, NaHCO3, EtOAc, Na2SO4) afforded the crude 11â-
(bromoacetamido)ethyl steroid 11a . Removal of the TBDMS
group was performed by stirring a solution of 11a in 30 mL of
THF, 10 mL of AcOH, and 10 mL of H2O for 20 h at room
temperature. Product isolation (H2O, NaHCO3, EtOAc, Na2-
SO4) then chromatography (CH2Cl2-AcOEt, 3:7) and recrys-
tallization from ethyl ether afforded 80 mg (51%) of 12a as an
amorphous solid: mp 170-172 °C; IR 3520, 1685 cm-1; NMR
(CDCl3) 0.85 (s, 3H, 18-CH3), 3.20 (m, 2H, 2′-CH2), 3.50 (m,
1H, 17-CH), 4.00 (s, 2H, CH2Br), 6.45 (d, 1H, J ) 2.6 Hz,

Steroidal Affinity Labels of the Estrogen Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2225
4-CH), 6.50 (m, 1H, 2-CH), 6.95 (m, 1H, 1-CH) ppm. Anal.
(C22H30NO3Br) C, H, O.
1654, 1601 cm-1; NMR (CDCl3), 1.00 (s, 3H, 18-CH3), 1.27
(wide s, 18H, 11-C(CH2)9), 2.07 (s, 3H, CH3CO), 2.96 (q, 1H, J
) 7 Hz, 11-CH), 3.63 (t, 2H, J ) 6.5 Hz, CH2-OH), 4.57 (dd,
1H, J 1 ) 7.5 Hz, J 2 ) 9 Hz, 17-CH), 5.68 (s, 1H, 4-CH) ppm.
3,17â-Dia cetoxy-11â-[10′-[[(4-m eth ylp h en yl)su lfon yl]-
oxy]d ecyl]estr a -1,3,5(10)-tr ien e (19). 4-Methylbenzene-
sulfonyl chloride (2.55 g, 12.4 mmol) was added to a solution
of 2.53 g (5.36 mmol) of dienone 18 in pyridine (12 mL). The
suspension obtained was stirred for 2 h at room temperature.
Product isolation (NaHCO3, AcOEt, Na2SO4) gave 3.41 g of an
oil which was dissolved in methylene chloride (32 mL) and
cooled in an ice bath. A mixture of acetic anhydride (3.2 mL,
33.8 mmol) and acetyl bromide (1.6 mL, 21.6 mmol) was slowly
added under argon while maintaining the flask in ice. The
solution was then stirred at room temperature for 1.5 h.
Methanol (5 mL) was cautiously added, and then product
isolation (NaHCO3, CH2Cl2, Na2SO4) followed by chromatog-
raphy (AcOEt-cyclohexane, 2:8) afforded 1.82 g (56%) of
diacetate 19 as an oil: IR 1747, 1727, 1630, 1611, 1609, 1599,
1581, 1499, 1118 cm-1; NMR (CDCl3) 0.95 (s, 3H, 18-CH3), 1.18
(wide s, 18H, 11-C(CH2)9), 2.07 (s, 3H, 17-COCOCH3), 2.28 (s,
3H, 3-COCOCH3), 2.44 (s, 3H, Ph-CH3), 4.01 (t, 2H, J ) 6.5
Hz, CH2OSO2), 4.63 (dd, 1H, J 1 ) 7 Hz, J 2 ) 9 Hz, 17-CH),
11â-[4′-(Br om oa ceta m id o)bu tyl]estr a -1,3,5(10)-tr ien e-
3,17â-d iol (12b). The 11â-(bromoacetamido)butyl derivative
12b was prepared from 8b by the procedure described for
compound 12a . The product (54%) was purified by chroma-
tography (CH2Cl2-AcOEt, 2:3) and recrystallized from ethyl
ether: mp 240 °C dec; IR 3535, 1675 cm-1; NMR (DMSO-d6)
0.85 (s, 3H, 18-CH3), 2.95 (m, 2H, 4′-CH2), 3.50 (t, 1H, J ) 6
Hz, 17-CH), 3.80 (s, 2H, CH2-Br), 6.50 (d, 1H, J ) 2.6 Hz,
4-CH), 6.60 (m, 1H, 2-CH), 6.95 (m, 1H, 1-CH) ppm. Anal.
(C24H34NO3Br) C, H, O.
11â-[2′-[(Meth ylsu lfon yl)oxy]eth yl]estr a-1,3,5(10)-tr ien e-
3,17â-d iol (13a ). Silylated 3- and 17â-hydroxy groups of
mesylate 8a were deprotected by exposing the compound (530
mg, 0.85 mmol) solubilized in AcOEt (4 mL) to p-toluene-
sulfonic acid (72 mg, 0.4 mmol). The mixture was stirred for
1 h at 40 °C. Product isolation (H2O, EtOAc, Na2SO4) and then
chromatography (EtOAc-CH2Cl2, 2:8) afforded 335 mg of 13a
(65%): mp 159.4-162.1 °C; IR 3385, 1580 cm-1; NMR (CDCl3)
0.85 (s, 3H, 18-CH3), 2.85 (s, 3H, SCH3), 3.45 (m, 2H, 2′-CH2),
3.85 (m, 1H, 11-CH), 6.50 (d, 1H, J ) 2.6 Hz, 4-CH), 6.65 (dd,
1H, J 1 ) 2.7 Hz, J 2 ) 8.2 Hz, 2-CH), 7.00 (d, 1H, J ) 8.2 Hz,
1-CH) ppm. Anal. (C21H30O5S) C, H, O.
6.78 (d, 1H, J ) 2.5 Hz, 4-CH), 6.84 (dd, 1H, J 1 ) 2.5 Hz, J 2
)
8.5 Hz, 2-CH), 7.13 (d, 1H, J ) 8.5 Hz, 1-CH), 7.34 and 7.79
(AA′BB′, 4H, C6H4) ppm. A small amount of the corresponding
10-chlorodecyl derivative (0.42 g, 16%) was also isolated.
11â-[10′-[[(4-Met h ylp h en yl)su lfon yl]oxy]d ecyl]est r a -
1,3,5(10)-tr ien e-3,17â-d iol (20). A solution of 379 mg (0.57
mmol) of diacetate 19 in methanol (9 mL) and 2 N sodium
hydroxyde (3.5 mL) was stirred for 2 h at room temperature.
Hydrochloric acid (2 N, 3.7 mL) was then added. Product
isolation (NaHCO3, AcOEt, Na2SO4) followed by chromatog-
raphy on silica gel (AcOEt-cyclohexane, 3:7) and then on
Lichrosorb RP 18 (water-methanol, 10:90) afforded 114 mg
(34%) of tosylate 20 as an off-white amorphous powder: IR
11â-[4′-[(Meth ylsu lfon yl)oxy]bu tyl]estr a-1,3,5(10)-tr ien e-
3,17â-d iol (13b). Compound 8b (550 mg, 0.84 mmol) was
deprotected by the procedure described for compound 8a .
Product isolation and then chromatography (EtOAc-CH2Cl2,
2:8) afforded 180 mg of 13b (50%): mp 145.1-146.5 °C; IR
3385, 1580 cm-1; NMR (CDCl3) 0.90 (s, 3H, 18-CH3), 2.95 (s,
3H, S-CH3), 3.70 (t, 1H, J ) 6 Hz, 17-CH), 4.41 (m, 2H, 4′-
CH2), 6.53 (m, 1H, 4-CH), 6.61 (m, 1H, 2-CH), 6.96 (d, 1H, J
) 8.2 Hz, 1-CH) ppm. Anal. (C23H34O5S) C, H, O.
11â-(4′-Br om obu tyl)estr a-1,3,5(10)-tr ien e-3,17â-diol (14).
A mixture of 260 mg (3 mmol) of LiBr and 120 mg (0.28 mmol)
of mesylate 13b in 15 mL of acetone was heated to reflux for
5 h. Product isolation (H2O, EtOAc, Na2SO4) and then
chromatography (EtOAc-CH2Cl2, 2:8) afforded 90 mg (80%)
of the 11â-bromobutyl derivative 14: mp 117.1-118.5 °C; IR
3400, 1575 cm-1; NMR (CDCl3) 0.90 (s, 3H, 18-CH3), 3.40 (m,
2H, 4′-CH2), 3.65 (t, 1H, J ) 6 Hz, 17-CH), 6.50 (d, 1H, J )
2.6 Hz, 4-CH), 6.58 (m, 1H, 2-CH), 6.90 (d, 1H, J ) 8.2, Hz,
1-CH) ppm. Anal. (C22H31O2Br) C, H, O.
3602, 1621, 1607, 1599, 1581, 1497, 1358, 1189, 1170 cm-1
;
NMR (CDCl3) 0.92 (s, 3H, 18-CH3), 1.25 (wide s, 11-C(CH2)9),
2.45 (s, 3H, Ph-CH3), 3.71 (t, 1H, J ) 8 Hz, 17-CH), 4.02 (t,
2H, J ) 6.5 Hz, CH2OSO2), 6.55 (d, 1H, J ) 2.5 Hz, 4-CH),
6.64 (dd, 1H, J 1 ) 2.5 Hz, J 2 ) 8 Hz, 2-CH), 7.00 (d, 1H, J )
8 Hz, 1-CH), 7.34 and 7.79 (AA′BB′, 4H, C6H4) ppm. Anal.
(C35H50O5S) C, H, S.
Biologica l. Ma ter ia ls. [6,7-3H]Estradiol (specific activity
1.89 PBq/mol, radiochemical purity >98%), was purchased
from Amersham International (Amersham, England). Estra-
diol, luciferin, and 4′,6-diamidino-2-phenylindole were pur-
chased from Sigma Chemical Co. (St. Louis, MO). 4-Hydrox-
ytamoxifen was given by Dr. A. E. Wakeling (Zeneca,
Macclesfield, England). Estradiol, 4-hydroxytamoxifen, and
estradiol 11â-derivatives used for binding studies and cell
experiments were solubilized in absolute ethanol. Solutions
were stored at -20 °C in the dark. The purity of solubilized
compounds was checked before use by TLC. Cell culture
materials came from Life-Technologies (Cergy-Pontoise, France).
Cytosolic Estr ogen Recep tor . Cytosol was prepared in
20 mM Tris-HCl buffer (T20), pH 7 (competitive binding
experiments) or 8.5 (irreversible binding experiments), from
immature lamb uteri as described previously.15 The protein
concentration, determined according to Layne,34 was adjusted
to 2 mg/mL (competitive binding experiments) or 4 mg/mL
(irreversible binding experiments).
Com p etitive Bin d in g Assa y: Ap p a r en t Rela tive Af-
fin ity Con sta n ts. Competition between 5 nM [3H]estradiol
and increasing concentrations of nonradioactive estradiol or
11â-derivatives for binding to lamb uterine cytosol (2 mg
protein/mL, pH 7) were performed for 20 h at 20 °C, as
described previously.15 Apparent relative affinity constants
(RACs) of competitors were calculated according to Koren-
man23 using concentrations of unbound and specifically bound
[3H]estradiol at 50% specific binding inhibition and concentra-
tions of unlabeled estradiol and 11â-derivatives which inhib-
ited 50% of the specific binding of [3H]estradiol.15
3,3-(E t h ylen ed ioxy)-5r-h yd r oxy-11â-[10′-[(ter t-b u t yl-
d im eth ylsilyl)oxy]d ecyl]estr -9-en -17-on e (17). A 0.59 M
suspension of the Grignard reagent prepared from 31.2 g (88.8
mmol) of 10-bromodecanol, protected as TBDMS ether, and
2.6 g (107 mmol) magnesium in 70 mL of THF was cooled in
an ice bath, Copper(I) chloride (875 mg, 8.85 mmol) was added,
and then the mixture was cooled to -20 °C. A solution of 19.5
g (59 mmol) of epoxide 1621 in 100 mL of THF was added
dropwise over 15 min; the temperature rose to -15 °C. The
mixture was stirred for 1 h at that temperature; it was then
poured in cold, saturated aqueous ammonium chloride solu-
tion. Product isolation (AcOEt, Na2SO4) followed by chroma-
tography (AcOEt-cyclohexane, 3:7) gave 18.5 g (52%) of ketone
17 as an oil: IR 3508, 1733 cm-1; NMR (CDCl3) 0.05 (s, 6H,
Si(CH3)2), 0.89 (s, 9H, SiC(CH3)3), 1.01 (s, 3H, 18-CH3), 1.25
(wide s, 18H, 11-C(CH2)9), 3.60 (t, 2H, J ) 6.5 Hz, CH2OSi),
3.94-4.02 (m, 4H, 2 CH2O) ppm. A 7.4 g (30%) sample of
starting material was also recovered.
11â-(10′-H yd r oxyd ecyl)-17â-a cet oxyest r a -4,9-d ien -3-
on e (18). Sodium borohydride (1.45 g, 38.3 mmol) was slowly
added to an ice-cooled solution of 23.1 g (38.3 mmol) of
compound 17 in 285 mL of methanol. The mixture was stirred
for 45 min at 0 °C, acetone (30 mL) was added, and the solution
was concentrated under vacuum. Product isolation (NaCl,
AcOEt, Na2SO4) yielded 22.3 g (96%) of an oil which was
immediately dissolved in pyridine (45 mL). Acetic anhydride
(22.5 mL) was added, and the solution was left at room
temperature for 72 h. Product isolation (NH4Cl, AcOEt, Na2-
SO4) gave an oil (23.7 g) which was dissolved in methanol (200
mL) and 2 N hydrochloric acid (100 mL). After 1 h of stirring
at room temperature, product isolation (NaHCO3, AcOEt, Na2-
SO4) followed by chromatography (AcOEt-cyclohexane, 1:1)
afforded 15.2 g (84%) of dienone 18 as an oil: IR 3624, 1729,
Sta n d a r d In d ir ect Ir r ever sible Bin d in g Assa y. Cytosol
(4 mg of protein/mL, pH 8.5) was first incubated for 20 h at
25 °C with estradiol, 11â-derivatives, or without any steroid.

2226 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Lobaccaro et al.
Samples were then treated with an equal volume of charcoal
suspension (1% charcoal, 0.1% dextran T70 in T20, pH 7) for
30 min at 0 °C, and charcoal was removed by centrifugation.
The total and nonspecific [3H]estradiol binding formed under
exchange conditions (16 h, 20 °C) in supernatant aliquots were
determined by charcoal assay, as previously described,15 using
10 nM [3H]estradiol in the absence and presence of 5 µM
nonradioactive estradiol.
Me´dicale” and the “Association pour la Recherche sur
le Cancer”.
Refer en ces
(1) Evans, R. M. The steroid and thyroid hormone receptor super-
family. Science 1986, 240, 889-895.
(2) Mangelsdorf, D. J .; Thummel, C.; Beato, M.; Herrlich, P.; Schutz,
G.; Umesono, K.; Blunberg, G.; Kastner, P.; Mark, M.; Chambon,
P.; Evans, R. M. The nuclear receptor superfamily: the second
decade. Cell 1995, 83, 835-839.
(3) Hard, T.; Kellenbach, E.; Boelens, R.; Maler, B. A.; Dahlman,
K.; Freedman, L.P.; Carstedt-Duke, J .; Yamamoto, K. R.;
Gustafsson, J .-A. Solution structure of the glucocorticoid receptor
DNA binding domain. Science 1990, 249, 157-160.
(4) Schwabe, J . W. R.; Neuhaus, D.; Rhodes, D. Solution structure
of the DNA-binding domain of the oestrogen receptor. Nature
1990, 348, 458-461.
(5) Luisi, B. F.; Xu, W. X.; Otwinowski, Z.; Freedman, L. P.;
Yamamoto, K. R.; Sigler, B. P. Crystallographic analysis of the
interaction of the glucocorticoid receptor with DNA. Nature 1991,
352, 497-505.
(6) Bourguet, W.; Ruff, M.; Chambon, P.; Gronemeyer, H.; Moras,
D. Crystal structure of the ligand-binding domain of the human
nuclear receptor RXRR. Nature 1995, 375, 377-382.
(7) Renaud, J .-P.; Rochel, N.; Ruff, M.; Vivat, V.; Chambon, P.;
Gronemeyer, H.; Moras, D. Crystal structure of the RAR-γ ligand
binding domain bound to all-trans retinoic acid. Nature 1995,
378, 681-689.
(8) Wagner, R. L.; Apriletti, J . W.; Mc Grath, M. E.; West, B. L.;
Baxter, J . D.; Fletterick, R. J . A structural role for hormone in
the thyroid hormone receptor. Nature 1995, 378, 690-697.
(9) Wurtz, J .-M.; Bourguet, W.; Renaud, J .-P.; Vivat, V.; Chambon,
P.; Moras, D.; Gronemeyer, H. A canonical structure for the
ligand-binding domain of nuclear receptors. Nature Struct. Biol.
1996, 3, 87-94.
(10) Katzenellenbogen, J . A.; Carlson, K. E.; Heiman, D. F.; Robert-
son, D. W.; Wei, L. L.; Katzenellenbogen, B. S. Efficient and
highly selective covalent labeling of the estrogen receptor with
[3H] tamoxifen aziridine. J . Biol. Chem. 1983, 258, 3487-3495.
(11) Zablocki, J . A.; Katzenellenbogen, J . A.; Carlson, K. E.; Norman,
M. J .; Katzenellenbogen, B. S. Estrogenic affinity labels: syn-
thesis, irreversible receptor binding, and bioactivity of aziridine
substituted hexestrols derivatives. J . Med. Chem. 1987, 30, 829-
838.
(12) Pinney, K. G.; Carlson, K. E.; Katzenellenbogen, B. S.; Katzenel-
lenbogen, J . A. Efficient and selective photoaffinity labeling of
the estrogen receptor using two nonsteroidal ligands that
embody aryl azide and tetrafluoroaryl azide photoreactive func-
tions. Biochemistry 1991, 30, 2421-2431.
Ra d ioa ctivity Deter m in a tion s. The radioactivity of the
various cytosol samples (100 or 200 µL) was counted in 4 mL
of Emulsifier Safe (Packard).
Cell Lin es a n d Cell Cu ltu r e Con d ition s. MVLN cells
were established28 by transfecting MCF-7 cells (mammary
tumor cells expressing the estrogen receptor and whose growth
and expression of specific genes are stimulated by estrogens)
with the pVit-tk-Luc plasmid. In this reporter gene, the
fragment -331/-87 of the 5′ flanking region of Xenopus vitel-
logenin A2 gene, which contains a palindromic estrogen
response element (ERE), was inserted in front of the Herpes
simplex virus promoter for thymidine kinase as previously
reported for pVit-tk-CAT.35 This regulatory part controls
expression of the firefly luciferase structural gene. Cells were
cultured either in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 3% fetal calf serum (FCS medium) or in
DMEM without phenol red supplemented with 3% of a
“steroid-free”, dextran-coated, charcoal-treated fetal calf serum
(FCS/C medium). Medium was replaced every other day.
Lu cifer a se Assa y. Cells were rinsed twice in luminescence
buffer (1.5 mM potassium phosphate, 8 mM MgCl2, pH 7.4),
suspended in 0.5 mL of luminescence buffer containing 2 mM
ATP and kept frozen until the luciferase assay was performed,
as described elsewhere,28 in the presence of 0.1 mM luciferin,
0.1 mM CoA, and 0.3% Triton X-100, using an LKB Wallac
1251 luminometer (Sundyberg, Sweden). The protein assay
was performed on 0.1 mL of cellular homogenate according to
Lowry’s method.36 The experimental values were expressed
as arbitrary luminescence units per milligram of protein.
Estr ogen ic a n d An tiestr ogen ic Assa y. Cells grown in
FCS/C medium for at least 3 days were seeded in 6-well tissue
culture plates (105 cells/well). One day later, increasing
concentrations of 4-hydroxytamoxifen or estradiol 11â-deriva-
tives in ethanol (0.1% final concentration) without or with 0.1
nM estradiol were added to the medium. One day later, cells
were rinsed twice with medium alone and then processed for
luciferase activity determination.
Lu cifer a se Gen e In a ctiva tion Assa y. Cells grown in
FCS/C medium for at least 3 days were seeded in T25 tissue
culture flasks (∼5 × 105 cells/flask). One day later, medium
was supplemented with 0.1% ethanol with or without 100 µM
4-hydroxytamoxifen or estradiol 11â-derivatives. Medium was
replaced every other day, and cells were grown for various
periods of time (to prevent confluency, when necessary cells
were trypsinized and seeded at lower concentration in new
wells). Cells were rinsed twice with medium alone, followed
by the addition of medium with or without 100 nM estradiol.
Cells were grown for 7 days, rinsed twice with medium alone,
and then processed for luciferase activity determination.
(13) Pinney, K. G.; Carlson, K. E.; Katzenellenbogen, J . A. Nonste-
roidal estrogens bearing acyl azide functions: potential electro-
philic and photoaffinity labeling agents for the estrogen receptor.
Steroids 1992, 57, 222-232.
(14) Bergmann, K. E.; Carlson, K. E.; Katzenellenbogen, J . A.
Hexestrol diaziridine photoaffinity labeling reagent for the
estrogen receptor. Bioconjugate Chem. 1994, 5, 141-150.
(15) El Garrouj, D.; Aumelas, A.; Borgna, J .-L. Steroidal affinity
labels of the estrogen receptor. 1. 17R-(bromoacetoxy)alkyl/
alkynylestradiols. J . Med. Chem. 1993, 36, 2973-2983.
(16) El Garrouj, D.; Aliau, S.; Aumelas, A.; Borgna, J .-L. Steroidal
affinity labels of the estrogen receptor. 2. 17R-[(haloacetamido)-
alkyl]estradiols. J . Med. Chem. 1995, 38, 2339-2348.
(17) Harlow, K. W.; Smith, D. N.; Katzenellenbogen, J . A.; Greene,
G. L.; Katzenellenbogen, B. S. Identification of cysteine 530 as
the covalent attachment site of an affinity-labeling estrogen
(ketononestrol aziridine) and antiestrogen (tamoxifen aziridine)
in the human estrogen receptor. J . Biol. Chem. 1989, 264,
17476-17485.
(18) Reese, J . C.; Katzenellenbogen, B. S. Mutagenesis of cysteines
in the hormone binding domain of the human estrogen receptor.
J . Biol. Chem. 1991, 266, 10880-10887
(19) Aliau, S.; El Garrouj, D.; Yasri, A.; Katzenellenbogen, B. S.;
Borgna, J .-L. 17R-[(haloacetamido)alkyl]estradiols alkylate the
human estrogen receptor at cysteine residues 417 and 530.
Biochemistry, 1997, in press.
Cell P r olifer a tion Assa y. Cells grown in FCS/C medium
for at least 3 days were seeded in 24-well tissue culture plates
(5 × 103 cells/well). One day later, medium was supplemented
with 0.1% ethanol containing or not estradiol (100 nM),
4-hydroxytamoxifen, or estradiol 11â-derivatives (100 µM).
Cells were grown in triplicate for 7 days. The DNA contents
of the wells were determined according to a method using
fluorescence enhancement of 4′,6-diamidino-2-phenylindole
complexed with DNA.37 Doubling time was determined by
semilogarithmic analysis of DNA content.
(20) Belanger, A.; Philibert, D.; Teutsch, G. Regio and stereospecific
synthesis of 11â-substituted 19-norsteroids. Steroids 1981, 37,
361-382.
(21) Teutsch, G.; Costerousse, G.; Philibert, D.; Deraedt, R. Steroid
derivatives. U.S. Patent 4 447 424, 1984; Chem. Abstr. 1984, 101,
130975m.
(22) Faraj, H.; Claire, M.; Rondot, A.; Aumelas, A.; Auzou, G.
Synthesis of new steroidal 11â-substituted spirolactones. J .
Chem. Soc., Perkin Trans. 1 1990, 3045-3048.
(23) Korenman, S. G. Relation between estrogen receptor inhibitory
activity and binding to cytosol of rabbit and human uterus.
Endocrinology 1970, 87, 1119-1123.
Ack n ow led gm en t. We are grateful to R. Wiechert
(Schering) and A. Wakeling (Zeneca) for donations of
ZK 37 875 and 4-hydroxytamoxifen, respectively. We
thank A. Aumelas for expert assistance in obtaining and
interpreting the NMR data. This work was supported
by the “Institut National de la Sante´ et de la Recherche

Steroidal Affinity Labels of the Estrogen Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2227
(24) Bindal, R. D.; Carlson, K. E.; Reiner, G. A. C.; Katzenellenbogen,
J . A. 11â-chloromethyl-[3H]estradiol-17â: a very high affinity,
reversible ligand for the estrogen receptor. J . Steroid Biochem.
1987, 28, 361-370.
(25) Hanson, R. N.; Napolitano, E.; Fiaschi, R. Synthesis and estrogen
receptor binding of novel 11â-substituted estra-1,3,5(10)-triene-
3,17â-diols. J . Med. Chem. 1990, 33, 3155-3160.
(26) Pomper, M. G.; VanBrocklin, H.; Thieme, A. M.; Thomas, R. D.;
Kiesewetter, D. O.; Carlson, K. E.; Mathias, C. J .; Welch, M. J .;
Katzenellenbogen, J . A. 11â-methoxy-, 11â-ethyl- and 17R-
ethynyl-substituted 16R-fluoroestradiols: receptor-based imag-
ing agents with enhanced uptake efficiency and selectivity. J .
Med. Chem. 1990, 33, 3143-3155
(27) Claussner, A.; Nedelec, L.; Nique, F.; Philibert, D.; Teutsch, G.;
Van de Velde, P. 11â-amidoalkylestradiols, a new series of pure
antiestrogens. J . Steroid Biochem. Mol. Biol. 1992, 41, 609-
614.
(28) Pons, M.; Gagne, D.; Nicolas, J .-C.; Mehtali, M. A new cellular
model of response to estrogen: a bioluminescent test to char-
acterize (anti)estrogen molecules. Biotechniques 1990, 9, 450-
459.
(29) Demirpence, E.; Duchesne, M.-J .; Badia, E.; Gagne, D.; Pons,
M. MVLN cells: a bioluminescent MCF-7-derived cell line to
study the modulation of estrogenic activity. J . Steroid Biochem.
Mol. Biol. 1993, 46, 355-364.
(30) Badia, E.; Duchesne, M.-J .; Fournier-Bidoz, S.; Simar-Blanchet,
A. E.; Terouanne, B.; Nicolas, J .-C.; Pons, M. Hydroxytamoxifen
induces a rapid and irreversible inactivation of an estrogenic
response in an MCF-7-derived cell line. Cancer Res. 1994, 54,
5860-5866.
(31) Van den Broek, A.; Leemhuis, J .; de Winter, M. S.; Zeelen, F. J .
Org-4333, a potent irreversibly binding estrogen agonist. Week-
lab. Sci. Edit. 1983, 5, 182-183.
(32) Leclercq, G.; Devleeschouwer, N.; Legros, N.; Heuson, J . In vitro
screening of cytotoxic estrogens of potential therapeutic activity.
In Cytotoxic Estrogens in Hormone Receptive Tumors; Raus, J .,
Martens, H., Leclercq, G., Eds.; Academic Press: London, 1980;
pp 165-181.
(33) Ratajczak, T.; Sheppard, P. N.; Capon, R. J .; Hahnel, R. The
synthesis and study of some potential affinity labelling reagents
for estrogens receptors. Steroids 1981, 38, 537-555.
(34) Layne, E. Spectrophotometric and turbidimetric methods for
measuring proteins. Methods Enzymol. 1957, 3, 444-454.
(35) Klein-Hitpass, L.; Schorpp, M.; Wagner, U.; Ryffel, G. U. An
estrogen-responsive element derived from the 5′-flanking region
of the Xenopus vitellogenin A2 gene functions in transfected
human cells. Cell 1986, 46, 1053-1061.
(36) Lowry, O. H.; Rosenbrough, N. I.; Farz, A. L.; Randall, R. G.
Protein measurement with the Folin phenol reagent. J . Biol.
Chem. 1951, 193, 265-275.
(37) Brunk, C. F.; J ones, K. C.; J ames, T. W. Assay for nanogram
quantities of DNA in cellular homogenates. Anal. Biochem. 1979,
92, 497-500.
J M970019L