Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1401
(14) Boos, G.; Stopper, H. Genotoxicity of several clinically used
topoisomerase II inhibitors. Toxicol. Lett. 2000, 116, 7-16.
(15) Strick, R.; Strissel, P. L.; Borgers, S.; Smith, S. L.; Rowley, J .
D. Dietary bioflavonoids induce cleavage in the MLL gene and
may contribute to infant leukemia. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 4790-4795.
(16) Von Angerer, E.; Biberger, C.; Leichtl, S. Studies on heterocycle-
based pure estrogen antagonists. Ann. N. Y. Acad. Sci. 1995,
761, 176-191.
(17) Takeuchi, K.; Kohn, J . T.; Sall, D. J .; Denney, M. L.; McCowan,
J . R.; Smith, G. F.; Gifford-Moore, D. S. Dibasic benzo[b]-
thiophene derivatives as a novel class of active site directed
thrombin inhibitors: 4. SAR studies on the conformationally
restricted C3-side chain of hydroxybenzo[b]thiophenes. Bioorg.
Med. Chem. Lett. 1999, 9, 759-764.
(18) Graham, S. L.; Shepard, K. L.; Anderson, P. S.; Baldwin, J . J .;
Best, D. B.; Christy, M. E.; Freedman, M. B.; Gautheron, P.;
Habecker, C. N.; Hoffman, J . M.; Lyle, P. A.; Michelson, S. R.;
Ponticello, G. S.; Robb, C. M.; Schwam, H.; Smith, A. M.; Smith,
R. L.; Sondey, J . M.; Strohmaier, K. M.; Sugrue, M. F.; Varga,
S. L. Topically active carbonic anhydrase inhibitors. 2. Benzo-
[b]thiophenesulfon-amide derivatives with ocular hypotensive
activity. J . Med. Chem. 1989, 32, 2548-2554. 4,6-Dimethoxy-
benzthiophene was prepared using 2-bromo-1,1-dimethoxy-
ethane for steric reasons.
an EC50 comparable to what was previously observed
in HeLa cells.
In summary, we have synthesized and tested a series
of aryl benzthiophene derivatives and have found ago-
nists with selectivity for ERâ. These findings could lead
to drugs that, by targeting ERâ in the brain, are able
to separate the beneficial effects of estrogens on mood,
learning, and memory from unwanted side effects such
as the stimulation of endometrial and breast cancer.
Ack n ow led gm en t. We thank W. Huber, J . Zadro-
bilek, I. Bobirnac, P. Martin, and N. Reymann for expert
technical assistance.
Su p p or tin g In for m a tion Ava ila ble: Analytical and spec-
tral characterization data of key compounds. This material is
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(19) [3H] binding assay: The radioreceptor assay was performed by
using 96-well microtiterplates (Picoplates, Packard) in volumes
of 0.2 mL of incubation buffer (50 mM Tris, pH 7.4). The
incubation mixture contained 5 nM ERR or 6 nM ERâ long form
receptors, 8 nM [3H]17â-estradiol (≈180 000 total counts), the
compound to be tested, and 0.25 mg/well SPA-beads. After
incubation at room temperature for 240 min, the reaction was
terminated by centrifugation at room temperature (10 min at
1000g). The radioactivity was counted at least 3 h after comple-
tion of the experiment in
a Packard Topcount scintillation
counter. Nonspecific binding was defined as the remaining
radioactivity in the presence of 10 µM nonradioactive 17â-
estradiol (Sigma). Assays were performed in triplicate.
(20) Reporter gene assay in HeLa cells: Cells were obtained from P.
Balaguer, Montpellier: Balaguer, P.; Franc¸ois, F.; Comunale,
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C. Reporter cell lines to study the estrogenic effects of xenoestro-
gens. Sci. Total Environ. 1999, 233, 47-56. Cells were seeded
at a density of 75 000 cells/well in 96-well microtitre plates. After
5 h, 17â-estradiol, test compounds, or vehicle (DMSO 0.1% final
concentration) were added and left in contact with the cells for
20 h. After aspiration of the medium, the cells were subjected
to the luciferase assay using the LucLite reagent kit (Packard).
Luminescence activity was counted for 6 s in
a TopCount
(Packard). Cells were maintained in medium deprived of
a
phenol red and containing charcoal stripped serum to keep
steroid levels as low as possible. Compounds were tested in
duplicate over the concentration range of 1 pM-1 µM, in parallel
with 17â-estradiol (0.1 pM-0.1 µM). Concentration-response
curves were fitted to the nonlinear logistic function of the
Microcal Origin software package to yield EC50 values. The Emax
parameter was constrained to the value obtained for 17â-
estradiol in the same experiment.
(21) Paech, K.; Webb, P.; Kuiper, G. G.; Nilsson, S.; Gustafsson, J .
A.; Kushner, P. J .; Scanlan, T. S. Differential ligand activation
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(22) Kraichely, D. M.; Sun, J .; Katzenellenbogen, J . A.; Katzenellen-
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(23) Reporter gene assay in SH-SY5Y cells: the assay was performed
essentially as described in ref 20.
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