Investigations on estrogen receptor binding. The estrogenic, antiestrogenic, and cytotoxic properties of C2-alkyl-substituted 1,1-bis(4-hydroxyphenyl)-2-phenylethenes

Article abstract of DOI:10.1021/jm0209230

C2-Alkyl-substituted 1,1-bis(4-hydroxyphenyl)-2-phenylethenes were synthesized and assayed for estrogen receptor binding in a competition experiment with radiolabeled estradiol ([³H]-E2) using calf uterine cytosol. The relative binding affinity decreased with the length of the side chain R = H (3a: 35.2%) > Me (3b: 32.1%) < Et (3c: 6.20%) ≈ CH₂CF₃ (3d: 5.95%) > n-Pr (3e: 2.09%) > Bu (3f: 0.62%). Agonistic and antagonistic effects were evaluated in the luciferase assay with MCF-7-2a cells stably transfected with the plasmid ERE_wtcluc. All compounds showed high antiestrogenic activity without significant agonistic potency. The comparison of the IC₅₀ values for the inhibition of E2 (1 nM) documented the dependence of the antagonistic effects on the kind of the side chain: 3a (IC₅₀ = 150 nM), 3b (IC₅₀ = 30 nM), and 3f (IC₅₀ = 500 nM) were weak antagonists, while 3c (IC₅₀ = 15 nM), 3d (IC₅₀ = 9 nM), and 3e (IC₅₀ = 50 nM) were full antiestrogens and antagonized the effect of E2 completely. The most active compound 3d possessed the same antagonistic potency as 4-hydroxytamoxifen (40HT: IC₅₀= 7 nM) without bearing a basic side chain. 3d as well as all other 1,1-bis(4-hydroxyphenyl)-2-phenylalkenes were not able to influence the proliferation of hormone dependent MCF-7 cells despite the antagonistic mode of action. In this assay tamoxifen (TAM) and 40HT reduced the cell growth concentration dependent up to T/C_corr = 15% and 25%, respectively.

Full text of DOI:10.1021/jm0209230

5358
J . Med. Chem. 2002, 45, 5358-5364
In vestiga tion s on Estr ogen Recep tor Bin d in g. Th e Estr ogen ic, An tiestr ogen ic,
a n d Cytotoxic P r op er ties of C2-Alk yl-Su bstitu ted
1,1-Bis(4-h yd r oxyp h en yl)-2-p h en yleth en es
Veronika Lubczyk, Helmut Bachmann, and Ronald Gust*
Institute of Pharmacy, Free University of Berlin, Ko¨nigin-Luise Strasse 2+4, D-14195 Berlin, Germany
Received April 30, 2002
C2-Alkyl-substituted 1,1-bis(4-hydroxyphenyl)-2-phenylethenes were synthesized and assayed
for estrogen receptor binding in a competition experiment with radiolabeled estradiol ([3H]-
E2) using calf uterine cytosol. The relative binding affinity decreased with the length of the
side chain R ) H (3a : 35.2%) > Me (3b: 32.1%) > Et (3c: 6.20%) ≈ CH2CF3 (3d : 5.95%) >
n-Pr (3e: 2.09%) > Bu (3f: 0.62%). Agonistic and antagonistic effects were evaluated in the
luciferase assay with MCF-7-2a cells stably transfected with the plasmid EREwtcluc. All
compounds showed high antiestrogenic activity without significant agonistic potency. The
comparison of the IC50 values for the inhibition of E2 (1 nM) documented the dependence of
the antagonistic effects on the kind of the side chain: 3a (IC50 ) 150 nM), 3b (IC50 ) 30 nM),
and 3f (IC50 ) 500 nM) were weak antagonists, while 3c (IC50 ) 15 nM), 3d (IC50 ) 9 nM), and
3e (IC50 ) 50 nM) were full antiestrogens and antagonized the effect of E2 completely. The
most active compound 3d possessed the same antagonistic potency as 4-hydroxytamoxifen
(4OHT: IC50) 7 nM) without bearing a basic side chain. 3d as well as all other 1,1-bis(4-
hydroxyphenyl)-2-phenylalkenes were not able to influence the proliferation of hormone
dependent MCF-7 cells despite the antagonistic mode of action. In this assay tamoxifen (TAM)
and 4OHT reduced the cell growth concentration dependent up to T/Ccorr ) 15% and 25%,
respectively.
In tr od u ction
in both secondary and tertiary structural organization
from that driven by E2 binding. Especially the orienta-
tion of helix 12 is different in both structures. While
helix 12 protects the LBD nearly completely from the
environment after binding of agonists, it is reoriented
in the 4OHT complex and occupies the part of the
coactivator binding groove formed by the residues of
helices 3, 4, and 5 and the turn connecting helices 3 and
4. The binding of the antiestrogens 4OHT and raloxifen
(RAL)9 in the LBD is very similar and indicates that
the orientation of the basic side chains in a narrow side
pocket of the LBD effects the alternative conformation
of helix 12, which inhibits coactivator recruitment and
ultimate transcription regulation and leads to anti-
estrogenic effects.
Interestingly, Schneider and co-workers10-13 showed
that, depending on the number and position of O-acyl
groups, the 1,1,2-triarylethenes induce in the immature
mouse uterine weight test low but significant anti-
estrogenic effects up to 50%, however, in the low dose
of 1 µg. In higher dosage, the estrogenic potency was
dominant. To get more information about the antago-
nistic effects in hormone-dependent tumor cells, we
synthesized 1,1-bis(4-hydroxyphenyl)-2-phenylethene
analogues and studied their agonistic and antagonistic
potency on the molecular level in a luciferase assay on
MCF-7-2a cells stably transfected with the plasmid
EREwtcluc.
The estrogen receptor (ER) as a ligand inducible
transcription factor mediates the physiological effects
of endogenous estrogens.1 Besides diverse positive ef-
fects (e.g., on bone density maintenance,2 regulation of
blood lipid profile,3 and brain function4) estrogens play
a predominant role in breast cancer growth and devel-
opment.5 Therefore, many efforts have been devoted to
the blockade of estrogen formation and action and led
to the introduction of antiestrogens in the therapy of
breast cancer. Tamoxifen (TAM), as a nonsteroidal
antiestrogen,6 has become a widely used drug for a first
line endocrine therapy for all stages of breast cancer in
pre- and postmenopausal women.7 The estrogen-like
effects in certain tissues led to the classification as a
selective estrogen receptor modulator (SERM). Its phar-
macological properties are related to the ability to
compete in target tissues with estradiol (E2) for binding
sites in the ligand-binding domain (LBD) of the ER.
TAM itself acts as a prodrug, which is activated by
hydroxylation of the para-position of the 1-phenyl ring.
The resulting 4-hydroxytamoxifen (4OHT) possesses an
8 times higher binding affinity to the ER and acts as a
pure antiestrogen in hormone dependent tumor cells.
Ligand recognition is achieved in both cases by a
combination of specific hydrogen bonds and van der
Waals interactions leading to a reorientation of the 12
helical units of the LBD.8 Nevertheless, binding of
4OHT induces a conformation of the LBD that differs
Ch em istr y
The 1,1-bis(4-hydroxyphenyl)-2-phenylethenes 3a -f
were synthesized according to the method of Dodds et
* To whom correspondence should be addressed. Phone: (030) 838
53272. Telefax: (030) 838 56906. E-mail: rgust@zedat.fu-berlin.de.
10.1021/jm0209230 CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/23/2002

Estrogen Receptor Binding
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24 5359
Ta ble 1. Biological Properties of Compounds 3a -f
Sch em e 1. Synthesis of
1,1-Bis(4-hydroxyphenyl)-2-phenylethenes
phenyl)-2-phenylethenes 2a -f by using either phospho-
ric acid or hydrobromic acid in THF (Scheme 1).
Compounds 2a -f were converted to the hydroxy deriva-
tives 3a -f by ether cleavage with BBr3 (Scheme 1,
method D). The characterization of all compounds was
1
performed by H NMR, IR, and mass spectroscopy.
Resu lts a n d Discu ssion
The affinity to the ER was determined in a competi-
tion experiment with [3H]-E2 using calf uterine cyto-
sol.15 As listed in Table 1, all 1,1,2-triarylethenes
effectively displaced E2 from its binding site. The
binding curves were parallel to that of E2 so that a
competitive inhibition can be assumed. The relative
binding affinity (RBA) showed a clear dependence on
the C2-alkyl chain and decreased with the length of the
side chain in the series H (35.2%) < Me (32.1%) < Et
(6.20%) ≈ CH2CF3 (5.95%) < n-Pr (2.09%) < Bu (0.62%).
Not only the C-2 substituents but also the substitu-
ents on the C-1 aryl rings influenced the ER binding.
4OHT as 4-OH derivative of TAM possesses an 8 times
higher affinity to the ER (4OHT: RBA ) 15.6%; TAM:
RBA ) 1.8%). The comparison of the RBA of 4OHT with
that of 3c (RBA ) 6.20%) shows that the dimethylami-
noethoxy side chain increases the RBA by the factor 3.
Because the RBA value does not directly reflect
biological effects such as estrogenic or antiestrogenic
activity, the compounds 3a -f were evaluated in more
detail in a luciferase assay using MCF-7-2a cells.16
These ER positive human breast cancer cells are stably
transfected with the reporter plasmid EREwtcluc. After
binding of a hormonally active drug, the ER/drug
conjugates dimerize and are able to interact with the
estrogen response elements (ERE) of the plasmid,
leading to the activation of the luciferase gene. There-
fore, the quantification of the luciferase expression
allows not only a prediction of the agonistic but also of
the antagonistic potencies of drugs.
al.14 starting with 1-(4-methoxyphenyl)-2-phenylethan-
1-one 1a which was obtained by a Friedel-Crafts
acylation of phenylacetyl chloride and anisole (Scheme
1, method A). Subsequently, 1a was reacted under the
influence of potassium tert-butanolate with the ap-
propriate alkyl bromide to obtain the C2-alkyl-substi-
tuted 1-(4-methoxyphenyl)-2-phenylethanones 1b -f
(Scheme 1, method B). The following Grignard reaction
of 1a -f with 4-methoxyphenylmagnesium bromide
yielded the corresponding carbinols, which were dehy-
drated to the C2-alkyl-substituted 1,1-bis(4-methoxy-
Among the assayed 1,1,2-triarylethenes (3a to 3f),
only 3a and 3b exerted low estrogenic effects in the
luciferase assay (relative activation at 1 µM: 3a 25%;
3b 10%, see Table 1). On the other hand, all compounds
were antagonists and inhibited the effect of 1 nM E2
(see Table 1) dependent on the length of C2-alkyl chain
(IC50 for the inhibition of 1nM E2: R ) H (150 nM) >
Me (30 nM) > Et (15 nM) > CH2CF3 (9 nM) < n-Pr (50
nM) < Bu (500 nM)).
The concentration activation curves (see Figure 1)
document that 3a , 3b , and 3f are weak antagonists,

5360 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24
Lubczyk et al.
F igu r e 1. Luciferase expression in MCF-7-2a cells, stably transfected with the reporter plasmid EREwtcluc treated with a
combination of E2 (1nM) and the 1,2-bis(4-hydroxyphenyl)-2-phenylalkenes 3a -f, tamoxifen (TAM), or 4-hydroxytamoxifen (4OHT).
while 3c, 3d , and 3e are true antiestrogens and an-
tagonized the E2 effect completely (see Table 1 and
Figure 1). As most active compound 3d (IC50 ) 9 nM)
showed the same antagonistic potency as 4OHT (IC50
) 7 nM) and was almost 50 times more active than
TAM (IC50 ) 500 nM). Interestingly, none of the 1,1-
bis(4-hydroxyphenyl)-2-phenylethenes bears a basic side
chain which is held responsible for antagonistic effects.
nitrogen of the dimethylamino group of 4OHT. This
binding displaces helix 12 from the binding cavity and
prevents the formation of a transcriptionally competent
conformation of the activation function 2 (AF2).
The group of J ordan also determined the relevance
of an H-bridge between the basic side chain of 4OHT
and RAL and Asp351.17 They studied the TGFR expres-
sion in MDA-MB-231 cells stably transfected with cDNA
for wild-type ER (Asp351) as well as for the mutants
Asp351Tyr and Asp351Gly. The exchange of Asp351 by
Shiau et al.8 detected in their X-ray studies a direct
hydrogen bond between the amino acid Asp351 and the

Estrogen Receptor Binding
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24 5361
F igu r e 2. Effects of 4-hydroxytamoxifen (4OHT), tamoxifen (TAM), and the 1,1-bis(4-hydroxyphenyl)-2-phenyl-4,4,4-trifluorobut-
1-ene (3d ) on the MCF-7 breast cancer cell line.
Asp351Tyr enhanced the estrogenic properties of 4OHT
and changed also the pharmacology of RAL by convert-
ing it from antiestrogen to estrogen. Substitution of
glycine for aspartate (Asp351Gly) resulted in the con-
version of the ER/4OHT complex from estrogen-like to
antiestrogen. The authors propose that the side chain
of an antiestrogen either neutralizes or displaces the
charge at Asp351 thereby removing a charged site for
the opportunistic binding of a novel coactivator.
Previously, Shiau et al.18 have suggested that it is not
just the side chain that is responsible for the antago-
nistic effect of TAM. The bulk causes also a conforma-
tional change that is inappropriate for full coactivator
binding. This is in accordance with our results because
none of the antiestrogenically active 1,1,2-triarylethenes
bears a side chain. The antiestrogenic properties depend
only on the C2-alkyl chain.
The structural analogy of the compounds to 4OHT
investigated in this study allows the assumption of an
analogous orientation in the LBD. One of the 4-hy-
droxyphenyl rings is H-bound to Glu351 and Arg394
and the other is positioned in the narrow side pocket
and is oriented toward Asp351. In a structure activity
study we determined a similar orientation of the phar-
macophoric 1,2-diarylethane/ethene moiety in estrogeni-
cally active 2,3-diarylpiperazines, 4,5-diarylimidazolines
and 4,5-diarylimidazoles and suppose that Asp351 is
also an appropriate anchor for estrogens.19 Therefore,
the missing agonistic properties and the antagonistic
effects of 1,1,2-triarylethenes must be the consequence
of the interaction of the C2-phenyl and the C2-alkyl
chain in the LBD.
In this test, 4OHT showed high antiestrogenic po-
tency and turned out to be a partial agonist (3c) with
antiestrogenic properties after deletion of the side chain.
Interestingly, the effects did not change after O-acyla-
tion. A similar relationship was found for cyclophenyl,
so it can be deduced that 1,1-bis(4-hydroxyphenyl)-
alkene derivatives are partial agonists in pituitary cells.
The antitumor activity and the hormonal profile of
3c and its O-acyl derivative were tested in vivo by
Schneider et al.13,23,24 Both compounds caused a growth
inhibition of T/C ) 10-15% on the hormone-dependent
MXT-M 3.2 mammary tumor of the BDF1 mouse in a
dose of 20 µM/kg. In the immature uterine weight test,
however, they possessed significant estrogenic but no
significant antiestrogenic properties. Therefore, the
mammary tumor-inhibiting effects cannot be caused by
the antagonism of tumor growth stimulating endog-
enous estrogens. Rather a mode of action must be taken
into consideration described by Schlemmer et al.25-28
They proposed an indirect mode of action for an estro-
genic active platinum complex due to its hormonal
properties, which involves other cells of the host as well,
e.g., cells of the immune system.
This is supported by the results on the MCF-7 cell
line. 3c and its derivatives were inactive, independent
of their antiestrogenic properties determined in these
cells (Table 1). Only the CF3 derivative 3d caused low
antiproliferative effects (T/C ) 70%, see Figure 2). On
the other hand, TAM with only low antiestrogenic
potency inhibited the cell proliferation to T/Ccorr) 15%
in a concentration of 5µM, while 4OHT was less active
(T/Ccorr ) 25%).
The structural requirements for the pharmacological
activity of nonsteroidal antiestrogens were already
studied two decades ago. The inhibition of the E2-
stimulated prolactin synthesis in primary cells of im-
mature rat pituitary glands by antiestrogens were used
as in vitro parameter.20-22 Three categories of active
compounds were identified: full estrogens, partial ago-
nists with antiestrogenic actions against the effects of
0.1nM E2, and full antagonists which antagonize the
effect of E2 completely.
Con clu sion
In this study we demonstrated that the removal of
the dimethylaminoethoxy side chain of 4-hydroxy-
tamoxifen did not decrease the antagonistic effects on
the MCF-7-2a cell line. All 1,1-bis(4-hydroxyphenyl)-2-
phenylethenes derived from 4OHT showed antiestro-
genic potency without estrogenic side effects. These
properties do not influence in vitro the proliferation of

5362 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24
Lubczyk et al.
3
(s, 3H, OCH3); 6.54 (AA′BB′, J ) 8.8 Hz, 2H, ArH-3, ArH-5);
hormone dependent tumor cells. The antitumor effects
of 3c and related compounds should follow the mecha-
nism proposed by Schlemmer et al., because growth-
inhibiting properties are only observed in vivo.
3
6.76 (AA′BB′, J ) 8.8 Hz, 2H, ArH-2, ArH-6); 6.88 (AA′BB′,
3J ) 8.6 Hz, 2H, ArH-3, ArH-5); 7.07-7.18 (m, 7H; ArH). IR
(KBr, cm-1): 3056 m (ArH); 3000 m (ArH); 2956 s (CH2); 2930
s (CH2); 2870 m (OCH3); 2835 m (OCH3); 1606 s (CdC); 1574
m (CdC); 1508 s (CdC). MS (EI, 80 °C): m/z (%) ) 372 [M]+•
(54.38); 135 (100).
Exp er im en ta l Section
Meth od D: Eth er Clea va ge w ith BBr 3. A solution of 1
equiv of the appropriate methyl ether in dry dichloromethane
was cooled to -52 °C. BBr3 (6 equiv) in dry dichloromethane
was added dropwise under nitrogen atmosphere. The mixture
was warmed to room temperature and stirred for 3 days.
Subsequently, dry methanol was added under cooling three
times, and the solvent was removed respectively under re-
duced pressure to yield the 1,1-bis(4-hydroxyphenyl)-2-
phenylethene.
Gen er a l P r oced u r es. IR spectra (KBr pellets): Perkin-
Elmer Model 580 A. H NMR: ADX 400 spectrometer at 400
1
MHz (internal standard: TMS). Elemental analyses: Micro-
laboratory of Free University of Berlin; on the basis of the C,
H, and N analyses, all compounds were of acceptable purity
(within 0.4% of the calculated values). Liquid Scintillation
Counter: 1450 Microbeta Plus (Wallac, Finland). Microplate
Photometer: Labsystems Multiscan Plus (Labsystems, Fin-
land). Microlumat: LB 96 P (EG & G Berthold, Germany).
Syn th eses. Methods A to D are representatives for the
compounds reported in Scheme 1. The compounds 1b to 1e
and 2a to 2e were already described by Schneider et al.13
Meth od A: 1-(4-Meth oxyph en yl)-2-ph en yleth an on e (1a).
Phenylacetyl chloride (9.28 g, 6.0 mmol) was added dropwise
to a suspension of aluminum chloride (9.71 g, 7.28 mmol) and
anisole (6.5 g, 6.01 mmol) in 20 mL of dry 1,2-dichloroethane
under cooling. After heating to reflux for 2 h, 50 mL of water
was added, and the organic layer was separated, washed with
water, and dried over Na2SO4. After removal of the solvent,
the crude product was recrystallized from diethyl ether/ligroine
to give 1a as colorless crystals (mp 68-69 °C). Yield: 10.43 g
1,1-Bis(4-h yd r oxyp h en yl)-2-p h en yleth en e (3a ). From
2a (0.15 g, 0.47 mmol). Purification was carried out by
crystallization with chloroform/ligroine. Yield: 0.08 g (0.29
mmol, 61%) of colorless crystals; mp 134-137 °C. 1H NMR
(DMSO-d6): δ 6.7-6.76 (m, 4H, ArH); 6.81 (s, 1H, CH); 6.89
3
3
(AA′BB′, J ) 8,5 Hz, 2H, Ar′H-3, Ar′H-5); 6.99 (AA′BB′, J )
7.2 Hz, 2H, Ar′H-2, Ar′H-6); 7.06-7.17 (m, 5H, (C2)ArH); 9.5
(d, 2H, OH). IR (KBr; cm-1): 3377 s (Ar-OH); 3077 (ArH);
3023 (ArH); 1606 m (CdC); 1512 s (CdC). MS (EI, 140 °C):
m/z (%) ) 288 [M]+• (100). Anal. (C20H16O2) C H.
1,1-Bis(4-h ydr oxyph en yl)-2-ph en ylpr op-1-en e (3b). From
2b (0.053 g, 0.16 mmol). Purification was performed by column
chromatography on silica gel with ether and recrystallization
from methanol/diethyl ether. Yield: 0.04 g (0.13 mmol, 81.3%)
of a colorless powder (mp 135 °C). 1H NMR (DMSO-d6): δ 2.04
(s, 3H, CH3); 6.41 (AA′BB′, 3J ) 8.4 Hz, 2H, ArH-3, ArH-5);
6.60 (AA′BB′, 3J ) 8.4 Hz, 2H, ArH-2, ArH-6); 6.74 (AA′BB′
3J ) 8.4 Hz, 2H, Ar′H-3, Ar′H-5); 6.97 (AA′BB′ 3J ) 8.4 Hz,
2H, Ar′H-2, Ar′H-6); 7.03-7.17 (m, 5H, (C2)ArH). IR (KBr;
cm-1): 3442 br (Ar-OH); 3073 m (ArH); 3026 m (ArH); 2976
m (CH2); 1608 s (CdC); 1510 s (CdC). MS (EI, 90 °C): m/z
(%) ) 302.3 [M]+• (6.5). Anal. (C21H18O2‚H2O) C H.
1,1-Bis(4-h yd r oxyp h en yl)-2-p h en ylbu t-1-en e (3c). From
2c (0.75 g, 2.18 mmol). The crude product was purified by
crystallization from chloroform/ligroine. Yield: 0.43 g (1.36
mmol, 62.4%) of light yellow crystals; mp 130-135 °C. 1H NMR
(MeOD-d4): δ 0.9 (t, 3H, CH3); 2.47 (q, 2H, CH2); 6.39 (AA′BB′
3J ) 8.5 Hz, 2H, ArH-3, ArH-5); 6.65 (AA′BB′ 3J ) 8.5 Hz,
2H, ArH-2, ArH-6); 6.76 (AA′BB′ 3J ) 8.6 Hz, 2H, Ar′H-3,
Ar′H-5); 7.01 (AA′BB′, 3J ) 8.6 Hz, 2H, Ar′H-2, Ar′H-6); 7.06-
7.17 (m, 5H, (C2)ArH). IR (KBr, cm-1): 3400 bs (OH); 3028 m
(ArH); 2966 m (ArH); 2928 m (CH2); 2870 m (CH2); 1608 s
(CdC); 1508 s (CdC). MS (EI, 130 °C): m/z (%) ) 316 [M]+•
(100); 301 (38.9). Anal. (C22H20O2‚0.5H2O) C H.
1
(4.61 mmol, 76.8%). H NMR (CDCl3): δ 3.86 (s, 3H, OCH3);
4.23 (s, 2H, CH2); 6.92 (AA′BB′, 3J ) 8.9 Hz, 2H, ArH-3, ArH-
3
5); 7.27 (m, 5 H, Ar′H), 7.99 (AA′BB′, J ) 8,9 Hz, 2H, ArH-2,
ArH-6). IR (KBr, cm-1): 3063 w (ArH); 3029 w (ArH); 2973 w
(CH2); 2932 w (CH2); 2904 w (OCH3); 1678 s (CdO); 1602 s
(CdC); 1576 m (CdC); 1507 m (CdC). MS (EI, 50 °C): m/z
(%) ) 226 (1.6) [M]+•; 135 (100).
Meth od B: 1-(4-Meth oxyp h en yl)-2-p h en ylh exa n on e
(1f). Butyl bromide (0.49 g, 3.5 mmol) was added carefully to
a suspension of 1a (1.0 g, 3.5 mmol) and potassium tert-
butylate (0.39 g, 3.5 mmol) in dry diethyl ether and heated to
reflux for 6 h. The organic layer was separated after addition
of 20 mL of water and was washed with 0.5 N sodium
thiosulfate solution and water, dried over Na2SO4, and evapo-
rated. The crude product was purified by column chromatog-
raphy on silica gel with diethyl ether/ligroine 1:2 to obtain a
colorless oil. Yield: 0.79 g (2.79 mmol, 79.7%) of a colorless
1
oil. H NMR (CDCl3): δ 0.85 (t, 3H, CH3); 1.14-1.38 (m; 4H,
2 × CH2); 1.81 (m, 1H, R2CdCRCH2); 2.17 (m; 1H; R3C-
CHPhCH2); 3.82 (s, 3H, OCH3); 4.49 (t, 1H, CH); 6.86 (AA′BB′,
3J ) 8.9 Hz, 2H, ArH-3, ArH-5); 7.18 (m, 1H, Ar′H-4); 7.29
3
(m, 4H, ArH-2, 3, 5, 6); 7.96 (AA′BB′, J ) 8.9 Hz, 2H, ArH-2,
ArH-6). IR (Film; cm-1): 3061 w (ArH); 3025 w (ArH); 2955 s
(CH2); 2930 s (CH2); 2869 m (OCH3); 1672 s (CdO); 1601 m
(CdC); 1575 m (CdC); 1509 s (CdC). MS (EI, 80 °C): m/z (%)
) 282 [M]+• (0.4); 135 (100).
1,1-Bis(4-h yd r oxyp h en yl)-2-p h en yl-4,4,4-tr iflu or obu t-
1-en e (3d ). From 2d (0.40 g, 1 mmol). Yield: 0.204 g (0.55
mmol, 55%) of a reddish powder; mp 157-161 °C. 1H NMR
3
(CDCl3): δ 3.5 (q, J ) 10.7 Hz, 2H, CH2); 6.5-6.7 (m, 13H,
Ar-H); 8.2 (s, 1H, ArOH); 8.4 (s, 1H, Ar-OH). IR (KBr, cm-1):
3600-3200 s (ArH); 2900 s (CH); 1600 m (CdO); 1510 s
(CdO); 1205 s (C-F); 840 s. Anal. (C22H17F3O2) C H.
Meth od C: 1,1-Bis(4-m eth oxyp h en yl)-2-p h en ylh ex-1-
en e (2f). A solution of 1f (0.5 g, 1.77 mmol) in 5 mL of dry
THF was added dropwise to a solution of 4-methoxyphenyl-
magnesium bromide in THF, which was generated before from
4-bromoanisole (0.5 g, 2.66 mmol) and Mg (0.07 g, 2.66 mmol).
The mixture was refluxed for 12 h and was then decomposed
with ice and 6 N acetic acid. The solvent was narrowed down
under reduced pressure, and the remaining aqueous solution
was extracted with ether. The organic layers were combined,
washed with saturated NaHCO3 solution and water, dried over
Na2SO4, and evaporated to dryness to obtain the carbinol. After
dissolution in dry THF, it was added dropwise to ice cold HBr
(47%). The mixture was stirred for 2 h, poured onto ice, and
extracted with dichloromethane. The organic layer was washed
with NaHCO3 solution and water. After drying over Na2SO4,
the solvent was evaporated, and the remaining product was
purified by column chromatography on silica gel with diethyl
ether/ligroine 1:5. Yield: 0.22 g (0.6 mmol, 33%) of a colorless
1,1-Bis(4-h ydr oxyph en yl)-2-ph en ylpen t-1-en e (3e). From
2e (0.1 g, 0.29 mmol). The crude product was purified by
column chromatography on silica gel with ether and subse-
quent crystallization from chloroform/ligroine. Yield 0.075 g
1
(0.23 mmol, 80.7%) of an orange powder; mp 142-145 °C. H
NMR (DMSO-d6): δ 0.75 (t, 3H, CH3); 1.45 (m, 2H, CH2CH3);
2.34 (t, 2H, CdCPh-CH2); 6.39 (AA′BB′, 3J ) 8.5 Hz, 2H, ArH-
3, ArH-5); 6.60 (AA′BB′, 3J ) 8.5 Hz, 2H, ArH-2, ArH-6); 6.74
3
3
(AA′BB′, J ) 8.4 Hz, 2H, Ar′H-3, Ar′H-5); 6.96 (AA′BB′, J )
8.5 Hz, 2H, Ar′H-2, Ar′H-6); 7.06-7.18 (m, 5H, (C2)ArH); 9.12
(s, 1H, OH); 9.37 (s, 1H, OH). IR (KBr, cm-1): 3400 bs (OH);
2957 m (ArH); 2927 m (ArH); 2868 m (CH2); 1608 s (CdC);
1508 s (CdC). MS (EI, 150 °C): m/z (%) ) 330 [M]+• (100);
301 (79.4). Anal. (C23H22O2‚0.5H2O) C H.
1,1-Bis(4-h yd r oxyp h en yl)-2-p h en ylh ex-1-en e (3f). From
2e (0.16 g, 0.41 mmol). The brown crude product was re-
crystallized from 2-propanol/dichloromethane. Yield: 0.1 g
1
oil. H NMR (CDCl3): δ 0.78 (t, 3H, CH3); 1.17-1.34 (m, 4H,
2 × CH2); 2.43 (t, 2H, R2CdCR-CH2); 3.68 (s, 3H, OCH3); 3.83

Estrogen Receptor Binding
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24 5363
(0.29 mmol, 71.5%) of a pale orange powder; mp 158 °C. 1H
NMR (CDCl3): δ ) 0.78 (t, 3H, CH3); 1.23 (m, 4H, 2 × CH2);
2.43 (t, 2H, CdCPh-CH2); 6.46 (AA′BB′, 3J ) 8.7 Hz, 2H, ArH-
3, ArH-5); 6.72 (AA′BB′, 3J ) 8.7 Hz, 2H, ArH-2, ArH-6); 6.80
concentration of the competitor (log-axis) is plotted. At least
six concentrations of each compound were chosen to estimate
its binding affinity. From this plot those molar concentrations
of unlabeled estradiol and of the competitors were determined
which reduced the binding of the radioligand by 50%.
3
(AA′BB′, J ) 8.5 Hz, 2H, Ar′H-3, Ar′H-5); 7.08-7.2 (m, 7H,
ArH). IR (KBr, cm-1): 3401 br s (OH); 3059 w (ArH); 3026 w
(ArH); 2956 s (ArH); 2926 s (CH2); 2857 (CH2); 1608 s (CdC);
1508 (CdC). MS (EI, 140 °C): m/z (%) ) 344 [M]+• (100); 301
(83.9). Anal. (C24H24O2‚0.5H2O) C H.
IC50 Estradiol
RBA )
µL 100 %
IC50 Sample
Lu cifer a se Assa y. Estr ogen ic Activity. The pertinent in
vitro assay was described earlier by Hafner et al.16 One week
before starting the experiment MCF-7-2a cells were cultivated
in DMEM supplemented with L-glutamine, antibiotics, and
dextran-charcoal-treated FCS (ct-FCS, 50 mL/L). Cells from
an almost confluent monolayer were removed by trypsinization
and suspended to approximately 2.2 × 105 cells/mL in the
growth medium mentioned above. The cell suspension was
then cultivated in six-well flat-bottomed plates (0.5 mL cell
suspension and 2 mL medium per well) at growing conditions
(see above). After 24 h, 25 µL of a stock solution of the test
compounds was added to achieve concentrations ranging from
10-5 to 10-10 M, and the plates were incubated for 50 h. Before
harvesting, the cells were washed twice with PBS, and then
200 µL of cell culture lysis reagent was added into each well.
After 20 min of lysis at room-temperature, cells were trans-
ferred into reaction tubes and centrifuged. Luciferase was
assayed using the Promega luciferase assay reagent. Fifty
microliters of each supernatant was mixed with 50 µL of
substrate reagent. Luminescence (in relative light units, RLU)
was measured for 10 s using a microlumat. Measurements
were corrected by correlating the quantity of protein (quanti-
fied according to Bradford30) of each sample with the mass of
luciferase. Estrogenic activity was expressed as % activation
of a 10-8 M estradiol control (100%).
Bioch em ica ls, Ch em ica ls, An d Ma ter ia ls. Dextran, 17â-
estradiol, L-glutamine (L-glutamine solution: 29.2 mg/mL
phosphate-buffered saline (PBS), and Minimum Essential
Medium Eagle (EMEM) were purchased from Sigma (Munich,
Germany); Dulbecco’s Modified Eagle Medium without phenol
red (DMEM) was from Gibco (Eggenstein, Germany); fetal calf
serum (FCS) was from Bio whittaker (Verviers, Belgium);
N-hexamethylpararosaniline (crystal violet), and gentamicin
sulfate were from Fluka (Deisenhofen, Germany); glutardial-
dehyde (25%) was from Merck (Darmstadt, Germany); trypsin
(0.05%) in ethylenediaminetetraacetic acid (0.02%) (trypsin/
EDTA) was from Boehringer (Mannheim, Germany); penicil-
lin-streptomycin gold standard (10000 IE penicillin/mL, 10
mg streptomycin/mL), and geneticin disulfate (geneticin solu-
tion: 35.71 mg/mL PBS) were from ICN Biomedicals GmbH
(Eschwege, Germany); norit A (charcoal) was from Serva
(Heidelberg, Germany); cell culture lysis reagent (5×) (diluted
1:5 with purified water before use) and the luciferase assay
reagent were from Promega (Heidelberg, Germany); optiphase
HiSafe3 scintillation liquid was from Wallac (Turku, Finland);
NET-317-estradiol[2,4,6,7-3H(N)] (17â-[3H]estradiol) was from
Du Pont NEN (Boston, MA); PBS was prepared by dissolving
8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4‚2H2O, and 0.2
g of KH2PO4 (all purchased from Merck or Fluka) in 1000 mL
of purified water. TRIS-buffer (pH ) 7.5) was prepared by
dissolving 1.211 g of trishydroxymethylaminomethane, 0.3722
g of Titriplex III, and 0.195 g of sodium azide (all from Merck
or Fluka) in 1000 mL of purified water. Deionized water,
produced by means of a Millipore Milli-Q Water System,
resistivity > 18 MΩ. T-75 flasks, reaction tubes, 96-well plates,
and six-well plates were purchased from Renner GmbH
(Dannstadt, Germany).
Cell Lin es a n d Gr ow th Con d ition s. The MCF-7-2a cell
line and the MCF-7cell line were kindly provided by Prof. Dr.
E. v. Angerer, University of Regensburg (Germany). Both cell
lines were maintained as a monolayer culture at 37 °C in a
humidified atmosphere (95% air, 5% CO2) in T-75 flasks. Cell
line banking and quality control were performed according to
the seed stock concept reviewed by Hay.29
Growth media: MCF-7-2a cell line: phenol red free DMEM
with penicillin-streptomycin 1%, L-glutamine 1%, FCS 5%,
and geneticin solution 0.5%. MCF-7 cell line: L-glutamine
containing EMEM supplemented with NaHCO3 (2.2 g/L),
sodium pyruvate (110 mg/L), gentamicin sulfate (50 mg/L), and
FCS (100 mL/L).
Estr ogen Recep tor Bin d in g Assa y. The applied method
was already described by Hartmann et al.15 and used with
some modifications. The relative binding affinity (RBA) of the
test compounds to the ER was determined by the displacement
of 17â-[3H]estradiol from its binding site. For this purpose the
test compounds were dissolved in ethanol and diluted with
TRIS-buffer to 6-8 appropriate concentrations (300 µL) and
were incubated while shaking with calf uterine cytosol (100
µL) and 17â-[3H]estradiol (0.723 pmol in TRIS-buffer (100 µL);
activity: 2249.4 Bq/tube) at 4 °C for 18-20 h. To stop the
reaction, 500 µL of a dextran-charcoal-suspension in TRIS-
buffer was added to each tube. After shaking for 90 min at 4
°C and centrifugation, 500 µL of HiSafe3 was mixed with 100
µL of supernatant of each sample, and the reactivity was
determined by liquid scintillation spectroscopy. The same
procedure was used to quantify the binding of 17â-[3H]estradiol
(0.723 pmol - control). Nonspecific binding was calculated
using 2 nmol of 17â-estradiol as the competing ligand. On a
semilog plot the percentage of maximum bound labeled steroid
corrected by the nonspecifically bound 17â-[3H]estradiol vs
An tiestr ogen ic Activity. The MCF-7-2a cells were treated
as mentioned above. The cells were incubated with the test
compounds in concentrations from 10-6 to 10-11 M along with
a constant concentration of estradiol (10-9 M). The concentra-
tion of the compound, which is necessary to reduce the effect
of estradiol by 50%, is IC50
.
Deter m in a tion of Cytosta tic Activity in MCF -7 Hu m a n
Br ea st Ca n cer Cells. The cytotoxicity assay in MCF-7 cells
had been described previously by Ruenitz et al.31 Fixation,
staining, and quantitation of the cells were carried out
according to Gillies32 and Kueng et al.33 Cells from an almost
confluent monolayer were harvested by trypsinization and
suspended to approximately 7 × 104 cells/mL. At the beginning
of the experiment, the cell suspension was transferred to 96-
well microplates (100 µL/well). After cultivating them for 3
days at growing conditions the medium was removed and
replaced by one containing the test compounds. Control wells
(16/plate) contained 0.1% of DMF, which was used for the
preparation of the stock solutions. The initial cell density was
determined by addition of glutaric dialdehyde (1% in PBS; 100
µL/well). After incubation for 4-7 days, the medium was
removed, and glutaric dialdehyde (1% in PBS; 100 µL/well)
was added for fixation. After 15 min, the solution of the
aldehyde was decanted and 180 µL PBS/well added. The plates
were stored at 4 °C until staining. Cells were stained by
treating them for 25 min with 100 µL of an aqueous solution
of crystal violet (0.02%). After decanting, cells were washed
several times with water to remove the adherent dye. After
addition of 180 µL of ethanol (70%), plates were gently shaken
for 4 h. Optical density of each well was measured in a
microplate autoreader at 590 nm.
Ack n ow led gm en t . The technical assistance of S.
Bergemann and I. Schnautz is acknowledged. The
presented study was supported by Grant Gu285/3-1
from the Deutsche Forschungsgemeinschaft.
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