Synthesis and biochemical characterization of a series of 17α-perfluoroalkylated estradiols as selective ligands for estrogen receptor α

Article abstract of DOI:10.1021/jm100563h

Despite intensive research efforts, the distinct biological roles of two closely related estrogen receptors, ERα and ERβ, are only partially understood. Therefore, ligands selective for either of two isotypes are useful research tools because they allow for exerting a desired subset of biological effects mediated by only one of the receptors. Here we report on the synthesis of a new class of potent and selective ligands for ERα represented by a series of 17α-substituted estradiols bearing lipophilic perfluoroalkyl chains. These 17α-perfluoroalkylated estradiols were synthesized by Ru-catalyzed cross metathesis reactions of 17α-allyl- or 17α-vinylestradiols with perfluoroalkylpropenes. Compounds were tested in both agonistic and antagonistic modes using a panel of stable steroid receptor reporter cell lines established in U2OS cells and consisting of ERα-LBD, ERβ-LBD, GR-LBD, and MR-LBD reporters. Some of the compounds are potent and selective agonists of ERα, exhibiting weak partial to no detectable agonistic activity on ERβ. Notably, 11c is the most ERα selective ligand of the prepared compounds because it activates ERα but inhibits ERβ. In addition, some compounds are pure agonists on ERα but show mixed agonistic/antagonistic profile on ERβ which is a typical pattern observed for selective estrogen receptor modulators (SERMs).

Full text of DOI:10.1021/jm100563h

J. Med. Chem. 2010, 53, 6947–6953 6947
DOI: 10.1021/jm100563h
Synthesis and Biochemical Characterization of a Series of 17r-Perfluoroalkylated Estradiols as
Selective Ligands for Estrogen Receptor r
§,
David Sedlak, Martin Dracı
,§
nsky, Petr Bartunek,* and Martin Kotora*
,†,‡
ꢀ †,‡,
Barbara Eignerova,
ꢀ
ꢁ
ꢀ ‡
ꢁ
´
†Department of Organic and Nuclear Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43 Prague 2, Czech Republic,
‡
§
Institute of Organic Chemistry and Biochemistry AS CR, Flemingovo nam. 2, 166 10 Prague 6, Czech Republic, and Center for Chemical
ꢀ
´
ꢁ ꢀ
Genetics, Institute of Molecular Genetics AS CR, Vıdenska 1083, 142 20 Prague 4, Czech Republic. B.E. and D.S. contributed to the work equally.
B.E. did the synthetic part and D.S. the biochemical testing.
Received May 10, 2010
Despite intensive research efforts, the distinct biological roles of two closely related estrogen receptors,
ERR and ERβ, are only partially understood. Therefore, ligands selective for either of two isotypes are
useful research tools because they allow for exerting a desired subset of biological effects mediated by
only one of the receptors. Here we report on the synthesis of a new class of potent and selective ligands
for ERR represented by a series of 17R-substituted estradiols bearing lipophilic perfluoroalkyl chains.
These 17R-perfluoroalkylated estradiols were synthesized by Ru-catalyzed cross metathesis reactions
of 17R-allyl- or 17R-vinylestradiols with perfluoroalkylpropenes. Compounds were tested in both ago-
nistic and antagonistic modes using a panel of stable steroid receptor reporter cell lines established in
U2OS cells and consisting of ERR-LBD, ERβ-LBD, GR-LBD, and MR-LBD reporters. Some of the
compounds are potent and selective agonists of ERR, exhibiting weak partial to no detectable agonistic
activity on ERβ. Notably, 11c is the most ERR selective ligand of the prepared compounds because it
activates ERR but inhibits ERβ. In addition, some compounds are pure agonists on ERR but show
mixed agonistic/antagonistic profile on ERβ which is a typical pattern observed for selective estrogen
receptor modulators (SERMs).
Introduction
The attachment of highly lipophilic moieties onto the steroid
framework has various beneficial effects. A typical example is
the synthesis of antiprogestine (ZK 230211, Figure 1) (a potent
progesterone receptor antagonist).10 In case of compounds
with the estradiol framework the best known derivatives are
fulvestrant11 and RU5866812 (steroidal antiestrogen) bearing
the pentafluoroethyl moiety. The introduction of perfluoroalkyl
chains onto the estrone skeleton has attracted the attention of
Blazejewski et al., who synthesized a series of perfluoroalky-
lated estrone derivatives such as 7R-perfluoroalkylestradiols I
(perfluorohexyl and trifluoromethyl groups),13 11β-perfluoro-
alkylestradiols II (perfluorohexyl,14 perfluorobutylethyl, and
perfluorooctylethyl15 groups), and 11β isomers of fulvestrant
III.16 In general, compounds I and II retained their affinity for
ERs and compound III also retained strong antiestrogenic
properties. Similarly, we have recently shown that the presence
of perfluoroalkylated chain in the brassinosteroid series had a
positive effect on the metabolic stability while preserving the
biological activity of compounds.17
In this regard substituted estradiol derivatives and 17R-
arylestradiols have attracted considerable attention because
of their interesting activity on ERR and ERβ.18-24 Poirier et
al. reported a synthesis of a new class of reversible inhibitors
of steroid sulfatase represented by a series of 17R-alkyl- and
17R-alkenylestradiol derivatives.18 We have also recently
shown that 17R-arylestradiols bearing a relatively bulky lipo-
philic indanyl moiety surprisingly retain estrogenic properties
and affinity to ERs.25 Moreover, our results show that different
substituents at the 17R position can alter the selectivity of
the ligand for either ERR or ERβ. In one particular case,
The estrogen receptor R (ERR,a NR3A1), estrogen recep-
tor β (ERβ, NR3A2), glucocorticoid receptor (GR, NR3C1),
and mineralocorticoid receptor (MR, NR3C2) belong to a
steroid hormone receptor family of ligand inducible transcrip-
tion factors. These receptors bind to hydrophobic ligands and
modulate the transcription of target genes. ERR and ERβ are
products of two separate genes and mediate the effect of the
main and the most potent natural estrogen: 17β-estradiol
(E2). E2 binds to both receptors with a similar affinity.1
Although ERR and ERβ are very similar proteins, the expres-
sion distribution is different in various tissues. ERR mediates
the action of estrogens in classical tissues like uterus and
mammary gland. ERR is also an important marker and a
traditional target for a therapy of breast cancer,2 and it
promotes a proliferation of certain healthy and cancer tissues.
On the other hand, the role for ERβ was established in the
brain, ovary, cardiovascular system,3 prostate, and several
animal models of inflammation.4 Numerous studies report
about the antiproliferative effect of the increasing expression
level of ERβ on the prostatic tissue5,6 or cell lines derived from
different breast7 or colon cancers.8,9
*To whom correspondence should be addressed. For P.B.: phone,
þ420 241 063 117; fax, þ420 241 063 586; e-mail, petr.bartunek@
img.cas.cz. For M.K.: phone, þ420 221 951 334; fax, þ420 221 951 326;
e-mail, kotora@natur.cuni.cz.
a Abbreviations: ER, estrogen receptor; GR, glucocorticoid receptor;
MR, mineralocorticoid receptor; E2, 17β-estradiol; LBD, ligand-binding
domain; RTC, relative transactivation capacity; SERM, selective estrogen
receptor modulator.
r
2010 American Chemical Society
Published on Web 09/02/2010
pubs.acs.org/jmc

6948 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Eignerovaꢀ et al.
Scheme 2. Cross-Metathesis of Estradiol Derivatives with
Perfluoroalkylpropenes
bromide was sluggish and gave rise to the vinyl derivative 4 in
a poor yield of 45%. Moreover, the course of the reaction
was accompanied by the formation of the nonalkylated
product of the reduction and unreacted starting material.
29
The next step, deprotection of 3 and 4 with BBr3 to get
unprotected derivatives 6 and 7 with the free hydroxyl group,
was not successful. The analysis of reaction mixtures showed
extensive decomposition and no formation of any major
product. This issue was solved by carrying out the whole
alkenylation process with a substrate bearing a different
protecting group. The 3-hydroxyl group of estrone 2 was
readily converted into the THP ether 5 in high 92% isolated
yield. Subsequently, two reactions with Grignard reagents
(allyl and vinyl) were carried out and followed by the
deprotection under acidic conditions (p-TsOH). This proce-
dure gave compounds 7 and 8 in 82% and 47% yields,
respectively (Scheme 1).
Figure 1. Perfluoroalkylated steroid derivatives.
Scheme 1. Synthesis of Allyl- and Vinylestradiols 3, 4, 6, and 7
The attachment of side chains by cross-metathesis was accom-
plished via our previously reported procedure by using Hoveyda-
Grubbs second generation catalyst between the estradiol deriva-
tives 3, 4, 6, and 7 and perfluoroalkylpropenes 8.17,26,30 Thus,
estrone derivatives 9-12 were obtained (Scheme 2), and the
results are summarized in Table 1. Initially, a metathesis of the
substrate 3 and (perfluorohexyl)propene 8a afforded the deriva-
tive 9a in acceptable 53% yield. Then the reaction between 8b
and 3 was carried out and compound 9b was obtained in good
68% yield. Lower efficiency was achieved with the substrate 4,
where products 10a and 10b were obtained in low 36% and 29%
yields, respectively. This fact could be attributed to a larger steric
hindrance of the double bond in the vinyl derivative 4. The same
conditions were also used for metathesis of compound 6 with
three (perfluoroalkyl)propenes 8a, 8b, and 8c, and the corre-
sponding metathesis products 11a-c were isolated in reasonable
yields of 58-67%. The metathesis of compound 7 with 8a, 8b,
and 8c afforded the corresponding products 12a, 12b, and 12c in
rather low yields for reasons like in the case of compound 4. The
product 12a was obtained in 39% yield. The synthesis of 12b and
12c was even less effective and gave corresponding compounds in
25% and 12% isolated yields, respectively.
Biological Testing. We tested the ability of newly synthe-
sized compounds to modulate the activity of ERR, ERβ, GR,
and MR in the U2OS reporter cell-based assays that we
established recently in our laboratory. The agonistic proper-
ties are summarized in Table 2 and the antagonistic proper-
ties in Table 3. In order to estimate to what extent new
ligands activate ERs and to see the relation between efficacy
and potency of ligands, Figure 2 shows dose response curves
from ERR and ERβ reporter assays. All new compounds
activate ERR and ERβ with lower potency than the natural
ligand 17β-estradiol. The methoxy group in the position 3
considerably decreases the ability of the compound to acti-
vate either of two receptors, which is well illustrated by a
comparison of 10b and 12b or 9a and 11a. Compound 12b
exhibits the highest potency of all new compounds for the
substitution of the hydrogen atom at the 17R position gave
rise to a ligand that was 13ꢀ more selective for ERR.25
The above-mentioned results, coupled with the fact that
adding of perfluoroalkyl chains to the steroid framework
introduces interesting and often desired properties to these
compounds, sparked our interest in synthesizing a series of
17R-alkylestradiol derivatives bearing highly lipophilic chains
represented by perfluoroalkyl moieties.
Results and Discussion
Synthesis of 17r-Perfluoroalkylestradiols. We envisioned
that the synthetically easiest and most flexible approach for
the introduction of perfluoroalkylated chains would be based
on the use of cross-metathesis between 17R-allyl- or 17R-
vinylestradiols with perfluoroalkylpropenes. We have recently
developed this method26 and applied it in the synthesis of
perfluoroalkylated analogues of brassinosteroids.17
The starting 17-alkenyl derivatives were synthesized from
the commercial estrone 1 and 3-methoxyestrone 2 via stan-
dard procedures using organomagnesium reagents.27 Since
the methyl-18 on the β-face of the steroid directs the nucleo-
philic attack at the less hindered steroidal R-face, such
alkylations of a C17-keto steroid are known to be stereo-
selective.28 Starting from 3-methoxyestrone 2, the 17R-alkyla-
tion products were then obtained exclusively (Scheme 1). The
first reaction with allylmagnesium bromide proceeded
exceptionally cleanly and furnished compound 3 in high
94% isolated yield. The second one using vinylmagnesium

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6949
Table 1. Synthesis of 17R-Perfluoroalkylated Estradiols 9-12
a Isolated yields.
Table 2. Agonistic Effect of Compounds on ERR, ERβ, GR, and MR Transactivation in Whole Cells
log(EC50 (M))a
RTC (%)b
compd
ERR
ERβ
GR
MR
ERR
ERβ
cLogP c
controld
9a
9b
-8.557 ( 0.057
>-4.8
-5.781 ( 0.045
>-4.8
-5.395 ( 0.037
-5.988 ( 0.050
-6.314 ( 0.069
-6.395 ( 0.069
-5.682 ( 0.046
-7.450 ( 0.080
-5.002 ( 0.054
-9.339 ( 0.058
>-4.902
>-4.817
-8.172 ( 0.117
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
-9.471 ( 0.045
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
100
100
3.78
NDe
NDe
NDe
NDe
7.46
7.34
6.36
7.14
6.43
6.04
0.17
10a
10b
11a
11b
11c
12a
12b
12c
>-5.079
-5.285 ( 0.133
-7.284 ( 0.178
-5.620 ( 0.144
>-4.8
>-5.561
-6.888 ( 0.299
>-4.713
0.07
0.27
0.57
0.69
0.13
7.82
0.03
0.01
0.88
0.02
>-4.8
>-4.8
>-4.8
>-4.8
0.35
a Transcriptional response of ERR, ERβ, GR and MR to tested compounds was assessed in U2OS reporter cell lines stably expressing fusion of the Gal4
DNA binding domain and the ligand binding domain (LBD) of the corresponding steroid receptor and the reporter vector with 9xUAS response element
followed by the coding sequence for the luciferase. Cells were incubated with compounds at the indicated concentrations for 18 h. EC50 values were
generatedby fitting datafromthe luciferase reporterassaybynonlinear regressionfunction. Datashown are representative oftwo independentexperiments
performed in triplicate. Values are reported as the mean ( standard error of the mean (SEM). b Relative transactivation capacity (RTC) of each compound
was calculated as the ratio of EC50 of the tested compound to that of E2. c Computed partion coefficient (cLopP) was calculated as described in the
Experimental Section. d E2 was used as a control ligand for ERR and ERβ, dexamethasone for GR, and aldosterone for MR. e ND = not calculated.
ERR and 11a the highest potency for the ERβ, but the
introduction of the methoxy group in position 3 resulting
in 10b and 9a, respectively, completely eliminates the ago-
nistic property of the compound or it decreases it by more
than 2 logs. On the other hand 9b, which also carries the
3-methoxy group, activates ERR with only slightly higher
EC50=1.66 μM (log EC50 = -5.78) than when the methoxy
group is replaced by the hydroxy group (11b, EC50 = 0.49
μM, log EC50 = -6.31).
been essential to prepare new ligands binding and activating
ERR or ERβ. Using selective ligands, one can selectively
exert a desired set of biological effects mediated by only one
of the receptors. Out of the new compounds, 11c is a highly
selective compound for ERR. It activates ERR with EC50=
400 nM (log EC50 = -6.40) but is completely inactive on
ERβ. Furthermore, this compound antagonizes the effect of
E2 on ERβ but not on ERR, which means that it acts as the
agonist for ERR and antagonist for ERβ (Table 3, Figure 3).
Since the biological effects exerted by ERR and ERβ only
partially overlap and given that E2 is a very potent ligand but
does not differentiate between two receptors, it has always
Similarly 12b shows high affinity for ERR with EC50 =
35.5 nM (log EC50 = -7.45) and is 22ꢀ selective for ERR
over ERβ. It is almost a full ERR agonist reaching 80% of the

6950 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Eignerovaꢀ et al.
Table 3. Antagonistic Effect of Compounds on ERR, ERβ, GR, and
MR transactivation in Whole Cells
log(IC50 (M)) a
compd
ERR
ERβ
GR
MR
controlb -7.677 ( 0.104 -7.969 ( 0.066 -9.294 ( 0.067 -8.033 ( 0.087
9a
9b
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-4.8
>-5.393
>-4.926
>-4.906
>-4.805
>-4.8
>-4.8
>-4.8
>-5.034
>-4.8
>-5.260
>-4.870
>-4.8
-5.505 ( 0.168 -5.419 ( 0.346
-4.868 ( 0.061 >-5.177
>-4.8
>-4.8
>-4.8
>-4.841
>-5.317
>-4.901
-5.289 ( 0.464
>-5.271
10a
10b
11a
11b
11c
12a
12b
12c
a Antagonistic properties of the tested compounds on ERR, ERβ,
GR, and MR were tested in U2OS stable reporter cell lines. Cells were
incubated with compounds at the indicated concentrations together
with 5 nM E2, 5 nM E2, 1 nM dexamethasone, and 1 nM aldosterone for
ERR, ERβ, GR, and MR, respectively, for 18 h. IC50 values were gene-
rated by fitting data from the luciferase reporter assay by nonlinear
regression function using GraphPad Prism 5.0 software. Data shown are
representative of two independent experiments performed in triplicate.
Values are reported as the mean ( standard error of the mean (SEM).
b ICI 182780 was used as a control for ERR and ERβ, RU-486 for GR,
and spironolactone for MR.
full receptor activation by E2 but a weak partial agonist for
ERβ reaching only 23% of the receptor activation by E2.
Interestingly, 12a acts as a partial agonist on both ERR
and ERβ, but in addition, it also has an antagonistic effect on
ERβ. Similar mixed agonistic/antagonistic patterns can be
observed in selective estrogen receptor modulators (SERMs),
which are frequently used drugs in the therapy of different
cancers and disorders. Such a mixed pattern can be explained
by the ability of the liganded receptor to recruit to some extent
both coactivators and corepressors. In this particular case, more
detailed study would be necessary to clarify the mechanism of
the receptor activation by this compound.
Although E2 is natural and the most potent activator of
ERR and ERβ, it can also affect the activity of the remain-
ing steroid receptors another two steroid receptors with
reduced potency and efficacy. Since the introduction of per-
fluoroalkylated chains to the 17R position of E2 considerably
changes its properties, we speculated that these changes might
also change its ability to modulate function of the two re-
maining steroid hormone receptors GR and MR that are not
activated by E2. Therefore, we tested the effect of new
compounds for the transactivation by GR and MR in the
U2OS reporter assay in both agonist (Table 2, Figure 2)
and antagonist mode (Table 3, Figure 3). None of the tested
compounds were able to activate any of these receptors. On
the other hand, a subset of compounds (10b, 11b, 11c, 12b,
12c) showed antagonistic properties on both receptors with
various potencies and 9a, 9b, and 10a were completely inactive
in the concentration range used in the experiments. Compound
12b is the most potent antagonist from the tested compounds of
both GR and MR with IC50=3.13 μM (logIC50=-5.51) and
IC50 =3.81 μM (logIC50 =-5.42), respectively. At the same
time, 12b is the most potent agonist of ERR and exhibits weak
mixed agonistic/antagonistic properties on ERβ.
Figure 2. Transactivation by ERR-LBD and ERβ-LBD in
response to an increasing concentration of tested compounds (b)
and E2 (O). Cells were incubated with compounds at the indicated
concentrations for 18 h, and at the end of this time luciferase activity
was measured. Maximal stimulation of the receptor by E2 was
arbitrarily set to 100%. Data shown are representative of two
independent experiments performed in triplicate. Dose response
curves were generated by fitting data from the reporter assay by
nonlinear regression function using GraphPad Prism 5.0 software.
Values are reported as the mean ( standard error of the mean
(SEM).
Conclusion
A ruthenium complex catalyzed cross-metathesis between
17R-vinyl- and 17R-allylestradiols with perfluoroalkylpro-
penes constitutes a convenient synthetic pathway for the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6951
ligand-induced change in the conformation of ERR. From
this perspective, further study of 12b, 11c, and related deriva-
tives can bring new light to our understanding of how the
specific ligand-induced conformational changes of ERs trans-
late into the transcription of target genes.
Experimental Section
General Procedure for Cross-Metathesis of Terminal Alkenes
with (Perfluoroalkyl)propenes. To a mixture of a terminal alkene
4-7 (1 equiv) and (perfluoroalkyl)propene 1a-c (2 equiv)
in CH2Cl2 was added Hoveyda-Grubbs second generation
catalyst (10 mol %) under an argon atmosphere. The result-
ing solution was stirred at 42 °C for 4 h. Removal of the sol-
vent in vacuo gave brown oils, which were purified by flash
chromatography.26
20-(E)-[17-(50,50,60,60,70,70,70-Heptafluorohept-20-en-10-yl)estra-
3,17β-diol] (11b). The reaction was carried out with 6 (130 mg, 0.42
mmol) and (perfluoropropyl)propene 8b (176 mg, 0.84 mmol)
according to the general procedure. Column chromatography on
silica gel (1/1 hexane/Et2O) and on fluorinated silica gel (first elution
of 7/3 MeOH/water for washing of the nonfluorinated starting
material, second elution of Et2O for washing of the product)
afforded 122 mg (58%) of the title compound 11b as a white foam:
[R]D þ29.7 (c 0.20, CHCl3); 1H NMR (600 MHz, CDCl3) δ 0.92
(s, 3H, 3 ꢀ H-18), 1.88 (m, 1H, H-7b), 1.99 (m, 1H, H-16a), 2.14
0
0
0
(m, 1H, H-9), 2.27 (dd, Jgem=14.1 Hz, J1 a,2 =8.1 Hz, 1H, H-1 a),
0
0
2.32 (m, 1H, H-11b), 2.41 (dd, Jgem=14.2 Hz, J1 b,2 =6.4 Hz, 1H,
0
Figure 3. Transactivation by ERβ-LBD, GR-LBD, and MR-LBD
in response to an increasing concentration of the selected com-
pounds (b) and control ligand (O) in antagonist mode. Cells were
incubated with compounds at the indicated concentrations in the
presence of 5 nM E2, 1 nM dexamethasone, and 1 nM aldosterone
for ERβ-LBD, GR-LBD, and MR-LBD, respectively, for 18 h, and
at the end of this time luciferase activity was measured. Maximal
inhibition of the receptor by the control ligand was arbitrarily set to
0%. Data shown are representative of two independent experiments
performed in triplicate. Dose response curves were generated by
fitting data from the reporter assay by nonlinear regression function
using GraphPad Prism 5.0 software. Values are reported as the
mean ( standard error of the mean (SEM).
H-10b), 2.83 (m, 2H, 2 ꢀ H-6), 2.86 (td, J4 ,F=18.4 Hz, J4 ,3 =7.0 Hz,
0
0
2H, 2 ꢀ H-40), 4.94 (bs, 1H, 3-OH), 5.55 (dm, J3 ,2 =15.4 Hz, 1H,
0
0
H-30), 5.94 (dm, J2 ,3 =15.4 Hz, 1H, H-2 ), 6.56 (d, J4,2=2.7 Hz, 1H,
H-4), 6.63 (dd, J2,1=8.4 Hz, J2,4=2.8 Hz, 1H, H-2), 7.15 (d, J1,2=
0
0
0
8.6 Hz, 1H, H-1); 13C NMR (150.9 MHz, CDCl3) δ14.28 (CH3-18),
23.39 (CH2-15), 26.25 (CH2-11), 27.40 (CH2-7), 29.60 (CH2-6),
31.68 (CH2-12), 34.71 (t, J4 ,F=22.6 Hz, CH2-40), 34.80 (CH2-16),
0
39.56 (CH-8), 40.41 (CH2-10), 43.76 (CH-9), 46.55 (C-13), 49.59
(CH-14), 82.94 (C-17), 112.67 (CH-2), 115.24 (CH-4), 120.37
(t, J3 ,F = 4.2 Hz, CH-30), 126.45 (CH-1), 132.52 (C-10), 135.19
0
(CH-20), 138.23 (C-5), 153.40 (C-3); IR (CHCl3) ν 3598, 3388, 1610,
1585, 1499, 1380, 1353, 1228, 975 cm-1; MS (EI, m/z (rel %)) 494
(Mþ, 86), 476 (5), 312 (10), 294 (11), 271 (94), 213 (53), 159 (67);
HR-MS (EI) calcd for C25H29O2F7 [Mþ] 494.2056, found 494.2048.
Rf(1/1 hexane/Et2O)=0.26.
synthesis of 17R-substituted estradiol derivatives bearing per-
fluoroalkylated side chains in good isolated yields. We sub-
jected compounds to biochemical testing in ERR, ERβ, GR,
and MR reporter cell-based assays and measuredthe ability to
induce transactivation or transrepression by these receptors.
Some of the derivatives showed activity on ERR and ERβ,
and in addition, some compounds were selective for ERR
(11b, 11c, 12b). The most ERR selective compounds were 11c
and 12b that not only strongly activate ERR but also inhibit
ERβ. A few other compoundsshowingsimilar properties were
described recently; R,R-THC (R,R-tetrahydrochrysene)31 is
one well-known example, but in contrast to 11c and 12b, R,R-
THC is a partial agonist on ERR and a full antagonist on ERβ.
Compounds with similar properties are very useful research tools
because they allow for separating biological effects mediated by
each of the ERs, given that the natural ligand E2 is unselective.
Furthermore, 12a and 12b have unique properties. 12b has
the highest potency from all tested compounds for ERR,
activates ERR with almost full efficacy, and at the same time
and together with 12a shows mixed partial agonistic/antag-
onistic properties on ERβ, which is often observed in SERMs
like raloxifene. Interestingly, unlike 12a and 12b, classical
SERMs only act as both agonists and antagonists on ERR
while they are mostly full antagonists on ERβ (data not
shown). The mixed agonist/antagonist profile of classical
SERMs is often attributed to the involvement of AF-1 in
the recruitment of coactivators or corepressors as a result of
20-(E)-[17-(50-(Trifluoromethyl)-50,60,60,60-tetrafluorohex-
20-en-10-yl)estra-3,17β-diol] (11c). The reaction was carried out
with 6 (150 mg, 0.48 mmol) and (perfluoroisopropyl)propene 8c
(210 mg, 1.00 mmol) according to the general procedure. Column
chromatography on silica gel (1/1 hexane/Et2O) and on fluori-
nated silica gel (first elution of 7/3 MeOH/water for washing
of the nonfluorinated starting material, second elution of Et2O
for washing of the product) and crystallization (4/1 hexane/
CH2Cl2) afforded 158 mg (67%) of the title compound 11c as
white crystals: mp 180-181 °C; [R]D þ32.2 (c 0.17, CHCl3); 1H
NMR (600 MHz, CDCl3) δ 0.92 (s, 3H, 3 ꢀ H-18), 1.88 (m, 1H,
H-7b), 1.97 (m, 1H, H-16a), 2.14 (m, 1H, H-9), 2.25 (dd, Jgem
0
14.2 Hz, J1 b,2 =8.0 Hz, 1H, H-1 b), 2.31 (m, 1H, H-11a), 2.39
=
0
0
0
0
0
(dd, Jgem=14.3 Hz, J1 a,2 =6.2 Hz, 1H, H-1 a), 2.83 (m, 2H, 2 ꢀ
0
0
0
0
H-6), 2.88 (dd, J4 ,F=20.0 Hz, J4 ,3 =7.0 Hz, 2H, 2 ꢀ H-4 ), 4.80
0
0
(bs, 1H, 3-OH), 5.54 (dm, 1H, J3 ,2 =15.1 Hz, H-3 ), 5.92 (dm,
0
0
0
2 ,3 =15.3 Hz, 1H, H-2 ), 6.56 (d, J4,2=2.8 Hz, 1H, H-4), 6.63
0
J
(dd, J2,1=8.4 Hz, J2,4=2.8 Hz, 1H, H-2), 7.15 (d, J1,2=8.4 Hz,
1H, H-1); 13C NMR (150.9 MHz, CDCl3) δ 14.26 (CH3-18),
23.38 (CH2-15), 26.25 (CH2-11), 27.40 (CH2-7), 29.61 (CH2-6),
31.67 (CH2-12), 32.68 (d, J4 ,F = 20.9 Hz, CH2-40), 34.77
0
(CH2-16), 39.55 (CH-8), 40.32 (CH2-10), 43.76 (CH-9), 46.53
(C-13), 49.59 (CH-14), 82.93 (C-17), 112.65 (CH-2), 115.23
(CH-4), 121.39 (d, J3 ,F = 5.8 Hz, CH-30), 126.47 (CH-1),
0
132.56 (C-10), 134.56 (CH-20), 138.25 (CH-5), 153.36 (C-3);
IR (CHCl3) ν 3598, 3370, 1611, 1585, 1500, 1380, 1353, 1163,
979 cm-1; MS (EI, m/z (rel %)) 494 (Mþ, 89), 476 (7), 271 (100),

6952 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Eignerovaꢀ et al.
253 (61), 228 (24), 213 (82); HR-MS (EI) calcd for C25H29O2F7
[Mþ] 494.2056, found 494.2062. Rf(1/1 hexane/Et2O)=0.26.
10-(E)-[17-(50,50,60,60,70,70,80,80,90,90,100,100,100-Tridecafluoronon-
10-en-10-yl)estra-3,17β-diol] (12a). The reaction was carried out
with 7 (100 mg, 0.34 mmol) and (perfluorohexyl)propene 8a
(242 mg, 0.68 mmol) according to the general procedure. Column
chromatography on silica gel (1/1 hexane/Et2O) and on fluori-
nated silica gel (first elution of 7/3 MeOH/water for washing
of the nonfluorinated starting material, second elution of Et2O
for washing of the product) and crystallization (4/1 hexane/
CH2Cl2) afforded 39 mg (19%) of the title compound 12a as
white crystals: mp 167-168 °C; [R]D þ32.1 (c 0.08, CHCl3); 1H
NMR (600 MHz, CDCl3) δ 0.94 (s, 3H, 3 ꢀ H-18), 1.74 (m, 1H,
H-15a), 1.89 (m, 2H, H-7a and H-16b), 2.01 (m, 1H, H-16a),
(2) Palmieri, C.; Cheng, G. J.; Saji, S.; Zelada-Hedman, M.; Warri, A.;
Weihua, Z.; Van Noorden, S.; Wahlstrom, T.; Coombes, R. C.;
Warner, M.; Gustafsson, J. A. Estrogen receptor beta in breast
cancer. Endocr.-Relat. Cancer 2002, 9, 1–13.
(3) Zhu, Y.; Bian, Z.; Lu, P.; Karas, R. H.; Bao, L.; Cox, D.; Hodgin,
J.; Shaul, P. W.; Thoren, P.; Smithies, O.; Gustafsson, J. A.;
Mendelsohn, M. E. Abnormal vascular function and hyperten-
sion in mice deficient in estrogen receptor beta. Science 2002, 295,
505–508.
(4) Harris, H. A. Estrogen receptor-beta: recent lessons from in vivo
studies. Mol. Endocrinol. 2007, 21, 1–13.
(5) Weihua, Z.; Makela, S.; Andersson, L. C.; Salmi, S.; Saji, S.;
Webster, J. I.; Jensen, E. V.; Nilsson, S.; Warner, M.; Gustafsson,
J. A. A role for estrogen receptor beta in the regulation of growth of
the ventral prostate. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 6330–
6335.
(6) Neubauer, B. L.; McNulty, A. M.; Chedid, M.; Chen, K.; Goode,
R. L.; Johnson, M. A.; Jones, C. D.; Krishnan, V.; Lynch, R.;
Osborne, H. E.; Graff, J. R. The selective estrogen receptor
modulator trioxifene (LY133314) inhibits metastasis and extends
survival in the PAIII rat prostatic carcinoma model. Cancer Res.
2003, 63, 6056–6062.
(7) Omoto, Y.; Inoue, S.; Ogawa, S.; Toyama, T.; Yamashita, H.;
Muramatsu, M.; Kobayashi, S.; Iwase, H. Clinical value of the
wild-type estrogen receptor beta expression in breast cancer.
Cancer Lett. 2001, 163, 207–212.
(8) Campbell-Thompson, M.; Lynch, I. J.; Bhardwaj, B. Expression of
estrogen receptor (ER) subtypes and ERbeta isoforms in colon
cancer. Cancer Res. 2001, 61, 632–640.
(9) Foley, E. F.; Jazaeri, A. A.; Shupnik, M. A.; Jazaeri, O.; Rice, L. W.
Selective loss of estrogen receptor beta in malignant human colon.
Cancer Res. 2000, 60, 245–248.
(10) Fuhrmann, U.; Hess-Stumpp, H.; Cleve, A.; Neef, G.; Schwede,
W.; Hoffmann, J.; Fritzemeier, K.-H.; Chwalisz, K. Synthesis and
biological activity of a novel, highly potent progesterone receptor
antagonist. J. Med. Chem. 2000, 43, 5010–5016.
(11) (a) DeFriend, D. J.; Howell, A.; Nicholson, R. F.; Anderson, E.;
Dowsett, M.; Mansel, R. E.; Blamey, R. W.; Bundred, N. J.;
Robertson, J. F.; Saunders, C.; Baum, M.; Walton, P.; Sutcliffe,
F.; Wakeling, A. E. Investigation of a new pure antiestrogen
(ICI 182,780) in women with primary breast cancer. Cancer Res.
1994, 54, 408–14.(b) Stevenson, R.; Kerr, F. W.; Anthony, R.; Brazier,
E. J.; Hogan, P. J.; David, D. D. P. Process for Preparing Intermediates
for the Production of 7-Substituted Antiestrogens. PCT Int Appl.
20022032922, 2002 (AstraZeneca).
(12) Van de Velde, P. F.; Nique, F.; Bouchoux, F.; Bremaud, J.;
Hameau, M. C.; Lucas, D.; Moratille, C.; Viet, S.; Philibert, D.;
Teutsch, G. RU 58668, a new potent antiestrogen inducing regres-
sion of human mammary carcinoma implanted in nude mice.
J. Steroid Biochem. Mol. Biol. 1994, 48, 187–196.
(13) Blazejewski, J.-C.; Wilmshurst, M. P.; Popkin, M. D.; Wakselman,
C.; Laurent, G.; Nonclercq, D.; Cleeren, A.; Ma, Y.; Seoc, H.-S.;
Leclercq, G. Synthesis, characterization and biological evaluation
of 7alpha-perfluoroalkylestradiol derivatives. Bioorg. Med. Chem.
2003, 11, 335–345.
(14) Agouridas, V.; Blazejewski, J.-C.; Manier, E.; Popkin, M. D.
Synthesis of 11beta-perfluorohexylestradiol. J. Org. Chem. 2005,
70, 8907–8912.
(15) Agouridas, V.; Blazejewski, J.-C.; Cleeren, A.; Laos, I.; Leclerq, G.;
Manier, E. Fluorous tolerance of the estrogen receptor alpha as
probed by 11beta-polyfluoroalkylestradiol derivatives. Steroids
2008, 73, 320–327.
(16) Agouridas, V.; Manier, E.; Blazejewski, J.-C.; Laos, I.; Cleeren, A.;
Nonclercq, D.; Laurent, G.; Leclercq, G. Effect of fluorination on
the pharmacological profile of 11beta isomers of fulvestrant in
breast carcinoma cells. J. Med. Chem. 2009, 52, 883–887.
2.11 (m, 1H, H-9), 2.27 (m, 1H, H-11a), 2.83 (m, 2H, 2 ꢀ H-6),
0
0
0
0
2.91 (dt, J3 ,F=18.2 Hz, J3 ,2 =7.2 Hz, 2H, 2 ꢀ H-3 ), 4.58 (s, 1H,
0
0
0
0
0
3-OH), 5.62 (dt, J2 ,1 =15.60Hz, J2 ,3 =7.1 Hz, 1H, H-2 ), 6.01
6.62 (dd, J2,1=8.5 Hz, J2,4=2.8 Hz, 1H, H-2), 7.13 (d, J1,2=
0
(bd, J1 ,2 =15.7 Hz, 1H, H-1 ), 6.56 (bd, J4,2=2.8 Hz, 1H, H-4),
0
8.5 Hz, 1H, H-1); 13C NMR (150.9 MHz, CDCl3) δ 14.01
(CH3-18), 23.22 (CH2-15), 26.25 (CH2-11), 27.35 (CH2-7),
0
29.58 (CH2-6), 32.20 (CH2-12), 34.67 (t, J3 ,F=23.1 Hz, CH2-
30), 36.86 (CH2-16), 39.40 (CH-8), 43.68 (CH-9), 46.92 (C-13),
0
49.15 (CH-14), 83.89 (C-17), 112.65 (CH-2), 114.54 (t, J2 ,F
=
4.0 Hz, CH-20), 115.21 (CH-4), 126.50 (CH-1), 132.60 (C-10),
138.22 (C-5), 143.32 (CH-10), 153.29 (C-3); IR (CHCl3) ν 3600,
1611, 1585, 1499, 1381, 1357, 1243, 979 cm-1; MS (EI, m/z (rel
%)) 630 (Mþ, 38), 612 (33), 597 (17), 437 (9), 387 (10), 213 (100);
HR-MS (EI) calcd for C27H27O2F13 [Mþ] 630.1803, found
630.1800. Rf(1/1 hexane/Et2O)=0.26.
10-(E)-[17-(40,40,50,50,60,60,60-Heptafluorohex-10-en-10-yl)estra-
3,17β-diol] (12b). The reaction was carried out with 7 (150 mg,
0.50 mmol) and (perfluoropropyl)propene 8b (210 mg, 1.00 mmol)
according to the general procedure. Column chromatography on
silica gel (1/1 hexane/Et2O) and on fluorinated silica gel (first
elution of 7/3 MeOH/water for washing of the nonfluorinated
starting material, second elution of Et2O for washing of the
product) and crystallization (4/1 hexane/CH2Cl2) afforded 61 mg
(25%) of the title compound 12b as white crystals: mp 185-186 °C;
[R]D þ26.3 (c 0.17, CHCl3); 1H NMR (600 MHz, CDCl3) δ 0.94
(s, 3H, 3 ꢀ H-18), 1.74 (m, 1H, H-15a), 1.89 (m, 2H, H-7a and
H-16b), 2.01 (m, 1H, H-16a), 2.10 (m, 1H, H-9), 2.27 (m, 1H,
0
0
0
=
H-11a), 2.81 (m, 2H, 2 ꢀ H-6), 2.89 (td, J3 ,F=17.6 Hz, J3 ,2
7.1 Hz, 2H, 2 ꢀ H-30), 4.70 (bs, 1H, 3-OH), 5.62 (dt, J2 ,1 =15.5 Hz,
0
0
0 0
0 0 0 0
2 ,3 =7.2 Hz, 1H, H-2 ), 6.00 (bd, J1 ,2 =15.6 Hz, 1H, H-1 ), 6.56
J
(d, J4,2=2.8 Hz, 1H, H-4), 6.62 (dd, J2,1=8.4 Hz, J2,4=2.8 Hz, 1H,
H-2), 7.13 (bd, J1,2=8.5 Hz, 1H, H-1); 13C NMR (150.9 MHz,
CDCl3) δ 14.01 (CH3-18), 23.21 (CH2-15), 26.23 (CH2-11), 27.35
0
(CH2-7), 29.58 (CH2-6), 32.17 (CH2-12), 34.40 (t, J3 ,F=22.3 Hz,
CH2-30), 36.79 (CH2-16), 39.38 (CH-8), 43.68 (CH-9), 46.90 (C-
0
13), 49.10 (CH-14), 83.91 (C-17), 112.65 (CH-2), 114.56 (t, J2 ,F
=
4.4 Hz, CH-20), 115.22 (CH-4), 126.50 (CH-1), 132.56 (C-10),
138.20 (C-5), 143.26 (CH-10), 153.31 (C-3); IR (CHCl3) ν 3599,
3399, 1611, 1585, 1499, 1353, 1232, 979 cm-1; MS (FAB, m/z (rel
%)) 680 (Mþ, 65), 462 (9), 265 (17), 228 (38), 213 (100); HR-MS
(EI) calcd for C24H27O2F7 [Mþ] 480.1899, found 480.1905. Rf(1/1
hexane/Et2O)=0.26.
ꢀ
ꢀ
ꢁꢁ ꢁ
ꢀ ꢀ
(17) Eignerova, B.; Slavı
´
kova, B.; Budesınsky, M.; Dracınsky,
´
´
ꢁ
ꢀꢁ
ꢀ
ꢀ
M.; Klepetarova, B.; St’astna, E.; Kotora, M. Synthesis of fluori-
nated brassinosteroids based on alkene cross-metathesis and pre-
liminary biological assessment. J. Med. Chem. 2009, 52, 5753–
5757.
Acknowledgment. This work was supported by the Research
Projects AV0Z50520514, Z40550506, LC06070, LC06077,
IAA40055069, and MSM0021620857 from the Ministry of
Education of the Czech Republic.
(18) Boivin, R. P.; Luu-The, V.; Lachance, R.; Labrie, F.; Poirier, D.
Structure-activity relationships of 17alpha-derivatives of estradiol
as inhibitors of steroid sulfatase. J. Med. Chem. 2000, 43, 4465–
4478.
(19) Salman, M.; Reddy, B. R.; Delgado, P.; Stotter, P. L.; Fulcher,
L. C.; Chamness, G. C. 17-alpha-Substituted analogs of estradiol
for the development of fluorescent estrogen receptor ligands.
Steroids 1991, 56, 375–387.
Supporting Information Available: Additional synthesis infor-
mation, compound characterization data, and spectra. This material
is available free of charge via the Internet at http://pubs.acs.org.
References
(20) Peters, R. H.; Crowe, D. F.; Avery, M. A.; Chong, W. K.; Tanabe,
M. 17-Desoxy estrogen analogues. J. Med. Chem. 1989, 32, 1642–
1652.
(1) Gustafsson, J. A. Estrogen receptor beta, a new dimension in
estrogen mechanism of action. J. Endocrinol. 1999, 163, 379–383.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6953
(21) el Amouri, H.; Vessieres, A.; Vichard, D.; Top, S.; Gruselle, M.;
Jaouen, G. Syntheses and affinities of novel organometallic-labeled
estradiol derivatives: a structure-affinity relationship. J. Med.
Chem. 1992, 35, 3130–3135.
(22) Vichard, D.; Gruselle, M.; Amouri, H.; Jaouen, G.; Vaissermann,
J. Controlled and selective introduction of a Cp-asterisk-Ruþ
(Cp-asterisk = C5Me5) fragment to the 17-alpha-substituent of
17-alpha-phenylestradiol. Organometallics 1992, 11, 2952–2956.
(23) Foy, N.; Stephan, E.; Jaouen, G. Synthesis of 17 alpha-4-amino-
and 4-iodophenylestradiols. Tetrahedron Lett. 2000, 41, 8089–8092.
(24) Foy, N.; Stephan, E.; Vessieres, A.; Salomon, E.; Heldt, J. M.;
Huche, M.; Jaouen, G. Synthesis, receptor binding, molecular model-
ing, and proliferative assays of a series of 17alpha-arylestradiols.
ChemBioChem 2003, 4, 494–503.
(28) (a) Salman, M.; Reddy, B. R.; Suprabhat, R.; Stotter, P. L.;
Chamness, G. C. 17R-Allyl estradiol analogues as candidates for
development of high-affinity fluorescein-estradiol conjugates.
J. Steroid. Biochem. 1986, 24, 539–548. (b) Salman, M.; Reddy, B. R.;
Delgado, P.; Stotter, P. L.; Fulcher, L. C.; Chamness, G. C. 17R-Sub-
stituted analogues of estradiol for the development of fluorescent estrogen
receptor ligants. Steroids 1991, 56, 375–385. (c) Anstead, G. M.; Carlson,
K. E.; Katzenellenbogen, J. A. The estradiol pharmacophore: ligand
structure-estrogen receptor binding affinity relationships and a model
for the receptor binding site. Steroids 1997, 62, 268–303.
(29) Aloui, F.; El Abed, R.; Marinetti, A.; Ben Hassine, B. Synthesis
and characterization of new hexahelicene derivatives. Tetrahedron
Lett. 2007, 48, 2017–2020.
(30) For attachment of the steroid side chain by using 15-allylestrone,
ꢀ
ꢀ
ꢁ
(25) Sedlak, D.; Novak, P.; Kotora, M.; Bartunek, P. Synthesis and
evaluation of 17R-arylestradiols as ligands for estrogen receptor R
and β. J. Med. Chem. 2010, 53, 4290–4294.
see the following: Kirschning, A.; Harmrolfs, K.; Mennecke, K.;
€
Messinger, J.; Schon, U.; Grela, K. Homo- and heterogeneous
Ru-based metathesis catalysts in cross-metathesis of 15-allylestrone;
towards 17beta-hydroxysteroid dehydrogenase type 1 inhibitors.
Tetrahedron Lett. 2008, 49, 3019–3022.
ꢀ
ꢁ
ꢀ
(26) Eignerova, B.; Dracınsky, M.; Kotora, M. Perfluoroalkylation via
´
cross-metathesis of alkenes with (perfluoroalkyl)propenes. Eur. J.
Org. Chem. 2008, 4493–4499.
(31) Harrington, W. R.; Sheng, S.; Barnett, D. H.; Petz, L. N.;
Katzenellenbogen, J. A.; Katzenellenbogen, B. S. Activities of
estrogen receptor alpha- and beta-selective ligands at diverse
estrogen responsive gene sites mediating transactivation or trans-
repression. Mol. Cell. Endocrinol. 2003, 206, 13–22.
(27) Gupta, A. K.; Yadav, K. M.; Patro, B.; Ila, H.; Junjappa, H.
Synthesis of D-ring functionalized and D-benzo/hetero-annulated
estrone derivatives via R-oxoketene dithioacetal. Synthesis 1995,
841–844.