Synthesis and estrogenic activities of novel 7-thiosubstituted estratriene derivatives

Article abstract of DOI:10.1016/S0960-894X(99)00648-4

A diastereomerically pure series of 7α-thioestratrienes was prepared and evaluated for its affinity for both the human estrogen receptor α and the more recently discovered estrogen receptor β. The functional estrogenic activities of the compounds were measured in a MCF-7 ERE-tk-luciferase assay. The activities and selectivities of the compounds were sensitive to the nature of the thioether side chain.

Full text of DOI:10.1016/S0960-894X(99)00648-4

Bioorganic & Medicinal Chemistry Letters 10 (2000) 147±151
Synthesis and Estrogenic Activities of Novel 7-Thiosubstituted
Estratriene Derivatives
Chris P. Miller, a,* Ivo Jirkovsky, b Bach D. Tran, a,y Heather A. Harris,c
Robert A. Moran c and Barry S. Komm c
aChemical Sciences, Wyeth-Ayerst Research, 145 King of Prussia Road, Radnor, PA 19087, USA
bWyeth-Ayerst Research & Development, Pearl River, NY 10965, USA
cWomen's Health Research Institute, Wyeth-Ayerst Research, 145 King of Prussia Road, Radnor, PA 19087, USA
Received 9 August 1999; accepted 15 November 1999
AbstractÐA diastereomerically pure series of 7a-thioestratrienes was prepared and evaluated for its anity for both the human
estrogen receptor a and the more recently discovered estrogen receptor b. The functional estrogenic activities of the compounds
were measured in a MCF-7 ERE-tk-luciferase assay. The activities and selectivities of the compounds were sensitive to the nature of
the thioether side chain. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
been reported (6 in Fig. 1). Interestingly, the 6-thiosub-
stituted estratriene compounds were reported to show
no antiestrogenic activity in an estrogen receptor posi-
tive ZR-75-1 cell line.9 So far, with respect to antiestro-
gens having the steroidal estratriene core, only the
attachment of groups at the 7a-position and the 11b-
position have been conducive to the generation of com-
pounds with `pure antiestrogen' characteristics.
Recent years have witnessed the introduction of anti-
estrogenic compounds that are signi®cantly devoid of
any estrogenic activity.1 It has been speculated that
these compounds may o€er an advantage over mixed
estrogen agonists, such as tamoxifen, for the treatment
of breast cancer. The partial agonism of tamoxifen has
been implicated in the development of tamoxifen-resis-
tant breast cancer as well as in the development of
uterine cancer.2,3 Compounds that have reduced estro-
genic agonist pro®les could provide a more complete
estrogen withdrawal and thus might avoid the putative
pitfalls of tamoxifen therapy. The prototypical, `purely
antiestrogenic' compounds were introduced by ICI and
consist of estratrienes with long carbon chains con-
nected to the steroid backbone at the 7a-position and
possessing either amide (typi®ed by ICI 164384 (1 in
Fig. 1)) or sulfoxide groups (typi®ed by ICI 182780 (2 in
Fig. 1)).4,5 Since then a number of compounds have
been reported where similar functionality was attached
to various positions of the estratrienes, including the
11b-position (4 and 5 in Fig. 1)6,7 as well as the 15 and
17 positions (not shown).8 More recently, the connec-
tion of various alkanamide chains to the estratriene
backbone at the 6 position through a sulfur atom has
We were interested in investigating the e€ect of con-
necting various antiestrogenic functionalities by a sulfur
atom at the 7-position of the estratriene backbone. We
envisioned the synthesis of compounds that contained
various moieties connected to the sulfur atom that
would resemble the functionality of other known ster-
oidal and antisteroidal antiestrogens (Fig. 2). It has
been suggested that nonsteroidal antiestrogens bind to
the estrogen receptor through their stilbene like cores,
and project a functionality (usually a 4-aminoethoxy
phenyl group (i.e. tamoxifen, raloxifene, centchroman,
etc.)) into a region of space that corresponds to the 7a-
position or 11b-position of an estratriene nucleus.10
Indeed, X-ray crystal structures of ERa with 17b-estra-
diol and raloxifene or tamoxifen show that the amino-
ethoxy side chains of the nonsteroidal compounds
project from the region of space which corresponds to
the 11b-position of 17b-estradiol.11,12
*Corresponding author. Tel.: +1-610-341-2168; fax: +1-610-989-
4588.
yCurrent address: Guilford Pharmaceuticals, 6611 Tributary Street,
Baltimore, MD 21224, USA.
Estratrienes with a phenylaminoethoxy group placed in
the 7a-position or the 11b-position have been synthe-
sized and reported (compounds 3 and 4 in Fig. 1).13 We
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(99)00648-4

148
C. P. Miller et al. / Bioorg. Med. Chem. Lett. 10 (2000) 147±151
Figure 1. Structures of some steroidal and nonsteroidal antiestrogens.
Figure 2. 7a-Thiosteroids with various antiestrogenic functionalities attached.
thought it would be informative to probe the 7a-region
of the estratriene nucleus by connecting the phenyl
group to the steroid backbone by a sulfur atom and
thus provide a `hinging' atom to change the orientation
of the phenyl group in the antiestrogenic e€ector space
of the estrogen receptor. Compound 9 shown in Figure
2 contains the 4-(piperidinoethoxy)phenyl group present
in the tissue selective estrogen, raloxifene (compound 8
in Fig. 1). In the ovariectomized rat model, raloxifene
(Evista1 for osteoporosis in humans) is antiestrogenic
on uterine and breast tissue while showing estrogen
agonist-like e€ects in bone and the cardiovascular sys-
tem.14 The tissue selective estrogenicity/antiestrogenicity
of this benzothiophene template is very sensitive to the
nature of the side chain appended to the 3-position. It
appears that the 4-piperidinoethoxy side chain is opti-
mal for reducing estrogen agonism on the uterus.15
an ovariectomized rat assay. Compounds 10 and 11
(Fig. 2) were synthesized in order to explore the e€ects
of the acrylamide or acrylic acid substituents when
placed at the 4-position of the thiophenol substituted at
the 7-position of the estratriene. Finally, we investigated
the e€ect of an ICI 164384-like undecamide side chain
attached to the estratriene nucleus through the tethering
sulfur atom (compound 12 in Fig. 2).
Chemistry
The synthesis of the various analogues shown in Figure
2 is demonstrated in Schemes 1±3. Analogue 9 shown in
Scheme 1 was prepared by substituting the bromo-
ketone 13a17 with 4-OH-thiophenol (only alpha isomer
detected) followed by reduction of the ketone with
NaBH4 and subsequent deoxygenation with TFA/
Et3SiH. The observation of a single diastereomer can
probably best be explained by substitution followed by
full epimerization under the reaction conditions.18 The
basic side chain was attached by reaction of 14 with b-
chloroethylpiperidine and 9 was obtained after base
hydrolysis of the 17-acetyl group.
Analogues of tamoxifen have been reported where the
aminoethoxy group is replaced by an acrylamide or
acrylic acid moiety (compounds 7a and 7b in Fig. 1).16
These compounds were reported to show reduced uter-
ine activity relative to tamoxifen while still displaying
the tissue selective, bone sparing activity of tamoxifen in
Scheme 1. Synthesis of 7a-thiophenol-40-piperidinoethoxy estratriene. (a) 4-OH thiophenol, NaH, DMF; (b) NaBH4, EtOH; (c) TFA, Et3SiH;
(d) b-chloroethylpiperidine, K2CO3, DMF; (e) NaOH, MeOH/H2O.

C. P. Miller et al. / Bioorg. Med. Chem. Lett. 10 (2000) 147±151
149
Scheme 2. Synthesis of 7a-thiophenyl-40-acrylamide and 7a-thiophenyl-40-acrylic acid estratrienes. (a) 4-Bromothiophenol, NaH, DMF; (b) NaBH4,
EtOH; (c) TFA, Et3SiH; (d) ethyl acrylate, Pd(OAc)2, P(o-tol)3, Et3N; (e) NaOH, MeOH/H2O; (f) N,N0 dimethyl acrylamide, Pd(OAc)2, P(o-tol)3,
Et3N.
Scheme 3. Synthesis of 7a-thioundecamide estratrienes. (a) HS-(CH2)10CONBuMe, NaH, DMF; (b) NaOH, MeOH/H2O; (c) NaBH4, EtOH;
(d) TFA, Et3SiH.
The analogues containing the acrylic acid and dimethyl
acrylamide moiety (compounds 10 and 11 in Scheme 2)
were prepared by substitution of the bromoketone 13b17
with 4-bromothiophenol and deoxygenation at the 6-
position as previously described. The acrylic acid 10 was
prepared by Heck coupling of the substrate 16 with
methyl acrylate followed by saponi®cation. The di-
methyl acrylamide (11) was prepared by an analogous
reaction of 16 with N,N0-dimethylacrylamide followed
by saponi®cation. The bromo compounds 19 and 20
were prepared as shown and tested as well. Finally, the
thioundecamide analogues 12, 17 and 18 were prepared
as shown in Scheme 3. The thioundecamide side chain
was prepared as previously described by Labrie.19
was achieved when the 6-position was unsubstituted
(compare 12 with 17 and 18). This is consistent with the
reported data for 6-oxo-estradiol when compared to
17b-estradiol.21 The lack of a free phenol in compound
9 is a likely explanation for its poor receptor anity.
The functional estrogen receptor a activity of these
compounds was determined using the MCF-7 ERE-tk-
luciferase assay and was ®rst tested in an antagonist
mode (by cotreating with 0.1 nM 17b-estradiol) to gen-
erate an IC50.22
Compounds that did not block the e€ect of 17b-estra-
diol were tested alone to generate an EC50. Interestingly,
the compounds that contained a phenyl linker failed to
function as antagonists and demonstrated only estrogen
agonist-like activity in the MCF-7 cells. This is despite
the fact that they contain similar functionality to known
nonsteroidal antiestrogens shown in Figure 1 (compare
steroid analogue 10 to tamoxifen analogue 7a and
steroid analogue 9 to raloxifene 8). Despite showing no
activity in the binding assay, compounds 9 and 19
showed agonist activity in the MCF-7 cells. Presumably
this is due to metabolic activation at the 3 or 17 posi-
tion. Compounds that contain the thioundecamide side
chain behaved as estrogen antagonists (compounds 12,
17 and 18). This demonstrates that the sulfur atom
located at the 7a-position is not per se a barrier to
achieving high receptor anity and potent estrogen
antagonism. This is in contrast to the report that no
signi®cant estrogen antagonism was displayed when
similar thio-alkanamide side chains were placed at the
Discussion of Biological Evaluation of Estrogenic
Activity
The structures of the compounds synthesized and tested
are shown in Table 1. The compounds were ®rst tested
for their ability to displace [3H]17b-estradiol from
estrogen receptor ligand binding domain constructs of
both the alpha and beta receptors.20 The binding results
are listed in Table 2 as IC50s and a value for unlabelled
17b-estradiol is included for comparison. None of the
steroids tested were particularly selective for one recep-
tor over the other although compound 20 showed an
approximately 6-fold preference for the beta receptor.
Some of the compounds displayed reasonably high a-
nities for both receptors. Optimal binding in the series

150
C. P. Miller et al. / Bioorg. Med. Chem. Lett. 10 (2000) 147±151
Table 1. Compound numbers and structures
Compound
R
X
R0
R00
Compound
R
H
X
R0
R00
9
CH3
CH2
OH
17
CˆO
OH
10
11
12
H
H
H
CH2
CH2
CH2
OH
OH
OH
18
19
20
H
H
H
CH±OH
CˆO
OH
OAc
OH
CH±OH
Table 2. Activity of compounds in binding and reporter gene assays
Radioligand binding assaya
ERE/MCF-7
Transient transfection assay (ER-a)b
Compound
ER-a IC50 (nM)
ER-b IC50 (nM)
Antagonist IC50 (nM)
Agonist EC50 (nM)
17b-estradiol
ICI-182780
3‹1
6‹1
2‹1
39
Not active
46‹23
94‹55
9‹4
33‹18
32‹21
Not active
397‹143
4‹2
7‹3
43‹13
56
Not active
21‹7
59‹40
9‹4
37‹8
Not active
0.47‹0.2 (100)
0.72‹0.2 (100)
654
Not active
Not active
Not active
3.5‹2 (99)
519‹9 (97)
316‹52 (98)
Not active
Not active
0.007‹0.007 (100)
Not active
Not active
Not active
336‹20 (116)
13‹1 (104)
173‹6 (104)
Not active
28‹8 (51)
ꢀ40 (23)
Raloxifene
7ac
9
10
11
12
17
18
19
20
32‹21
Not active
63‹17
5‹0 (105)
7‹1 (78)
aMean‹S.D.; nꢁ2 unless noted.
bMean‹S.D.; nꢁ2 unless noted; ecacy in parentheses.
cn=1; Not active means no e€ect or competition at <1000 nM.
6-position of the estratriene nucleus.9 When compound
12 was dosed alone in a 3-day immature rat uterine
assay, it did not stimulate uterine wet weight gain.
When dosed with 17b-estradiol, compound completely
and potently blocked the uterine weight gain normally
seen when dosing 17b-estradiol alone.23 This is con-
sistent with the supposition that compound 12 behaves
as a pure antiestrogen in the same way as its progenitor,
ICI 182780 (compound 1 in Fig. 1), does in these assays.
References and Notes
1. Nicholson, R. I.; Gee, J. M. W.; Bryant, S.; Francis, A. B.;
McClelland, R. A.; Knowlden, J.; Wakeling, A. E.; Osborne,
C. K. Ann. N.Y. Acad. Sci. 1996, 784, 325.
2. Furr, B. J. A.; Jordan, V. C. Pharmac. Ther. 1984, 25, 127.
3. Black, L. J.; Goode, R. L. Life Sci. 1980, 26, 1453.
4. Wakeling, A. E.; Bowler, J. J. Steroid Biochem. 1988, 31,
645.
5. Wakeling, A. E.; Dukes, M.; Bowler, J. Cancer Res. 1991,
51, 3867.
6. Claussner, A.; Nedelec, L.; Nique, F.; Philibert, D.;
Teutsch, G.; Van De Velde, P. J. Steroid Biochem. Mol. Biol.
1992, 41, 609.
7. Van de Velde, P.; Nique, F.; Planchon, P.; Prevost, G.;
Bremaud, J.; Hameu, M. C.; Magnien, V.; Philibert, D.;
Teutsch, G. J. Steroid Biochem. Mol. Biol. 1996, 59, 449.
8. Poirer, D.; Merand, Y.; Labrie, C.; Labrie, F. Bioorg. Med.
Chem. Lett. 1996, 6 (21), 2537.
Acknowledgements
The authors acknowledge Dr. P. Bodine and Dr. J.
Yardley for valuable editorial oversight, Dr. S.-M.
Chang and M. Blume for the preparation of constructs
and protein for the binding assays and S. Jenkins and
M. Bookler for cell culture.

C. P. Miller et al. / Bioorg. Med. Chem. Lett. 10 (2000) 147±151
151
9. Auger, S.; Merand, Y.; Pelletier, J. D.; Porier, D.; Labrie, F.
J. Steroid Biochem. Mol. Biol. 1995, 52, 547.
10. Bouhoute, A.; Leclercq, G. Biochemical Pharmacology
1994, 47, 748.
11. Brzozowski, A. M.; Pike, A. C. W.; Dauter, Z.; Hubbard,
R. E.; Bonn, T.; Engstrom, O.; Ohman, L.; Greene, G. L.;
Gustafsson, J.-A.; Carlquist, M. Nature (London) 1997, 389
(6652), 753.
12. Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.; Kush-
ner, P. J.; Agard, D. A.; Greene, G. L. Cell 1998, 95 (7), 927.
13. Nedelec, L.; Nique, F.; Bouton, M. M.; Van De Velde, P.
Proc. 8th Int. Congress on Hormonal Steroids, The Hague, The
Netherlands, 1990, Abst. No. 302.
14. Black, L. J.; Sato, M.; Rowley, E. R.; Magee, D. E.;
Dekele, A.; Williams, D. C.; Cullinan, G. J.; Bendele, R.;
Kau€man, R. F.; Bensch, W. R.; Frolik, C. A.; Termine, J.
D.; Bryant, H. U. J. Clin. Invest. 1994, 93, 63.
15. Jones, C. D.; Jevnikar, M. G.; Pike, A. J.; Peters, M. K.;
Black, L. J.; Thompson, A. R.; Falcone, J. F.; Clemens, J. A.
J. Med. Chem. 1984, 27, 1057.
ERa or ERb was prepared. Both receptors and compounds
were diluted in 1X Dulbecco's PBS (DPBS) supplemented with
1 mM EDTA. Using a high binding masked microtiter plate,
100 mL of receptor (1 mg/well) was combined with 2 nM [3H]-
17b-estradiol and various concentrations of compound. After
between 5 and 15 hours at room temperature, the plates were
washed with DPBS/1mM EDTA and bound radioactivity
determined by liquid scintillation counting. The IC50 is de®ned
as the concentration of compound which decreases total [3H]-
17b-estradiol by 50%.
21. Katzenellenbogen, J. A.; Carlson, K. E.; Johnson, H. J. Jr.;
Myers, H. N. J. Toxicol. Environ. Health 1976, (Suppl 1), 205.
22. Bodine, P. V.; Harris, H. A.; Komm, B. S. Endocrinology
1999, 140 (6), 2439.
23. Procedure: Sprague±Dawley rats (n=6, 18 day old) were
treated by daily subcutaneous injection with compounds or
vehicle (50% DMSO/50% DPBS) for three days. On day 4 the
animals were euthanized by CO2 asphyxiation. Uteri were
weighed after removing external fat and expressing any lumi-
nal ¯uid.
16. Willson, T. M.; Henke, B. R.; Momtahen, T. M.; Clar-
ifson, P. S.; Batchelor, K. W.; Lubahn, D. B.; Moore, L. B.;
Oliver, B. B.; Sauls, H. R.; Trianta®llou, J. A.; Wolfe, S. G.;
Baer, P. G. J. Med. Chem. 1994, 37, 1550.
17. Pearson, A. J.; Han, G. R. J. Org. Chem., 1985, 50 (15),
2791. The 6-ketoestratrienes produced were subsequently bro-
minated by treating with 1.1 eq. Br2 in ether.
18. Tedesco, J. A.; Katzenellenbogen, J. A.; Napolitano, E.
Tetrahedron Lett. 1997, 38, 7997.
19. Poirier, D.; Auger, S.; Merand, Y.; Labrie, F. J. Med.
Chem. 1994, 37, 1115.
Compound dose
(mg/rat/day)
Compound 12
Mean uterine weight
(mg) ‹ stdev
Tamoxifen
Mean uterine weight
(mg) ‹ stdev
1
10
31.8‹3.4
33.6‹3.5
41.6‹2.1
46.0‹4.1
53.3‹1.5
69.6‹1.9
71.4‹1.6
Not tested
100
100+estradiol
(1mg)
veh=43.1‹4.7;
estradiol (1mg)=
82.1‹4.1
veh=42.7‹4.4;
estradiol (1mg)=
98.2‹10.4
20. Binding material and methods: A crude lysate of E. coli
expressing the ligand binding domain (D, E, & F) of human