Discovery of novel SERMs with a ferrocenyl entity based on the oxabicyclo[2.2.1]heptene scaffold and evaluation of their antiproliferative effects in breast cancer cells

Article abstract of DOI:10.1039/c2ob26226f

We have synthesized a series of novel SERMs bearing a ferrocenyl unit based on a three-dimensional oxabicyclo[2.2.1]heptene core scaffold. These compounds displayed high receptor binding affinities as well as ERα or ERβ selectivity. In cell proliferation

Full text of DOI:10.1039/c2ob26226f

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Organic &
Biomolecular
Chemistry
PAPER
Discovery of novel SERMs with a ferrocenyl entity
based on the oxabicyclo[2.2.1]heptene scaffold and
evaluation of their antiproliferative effects in breast
cancer cells†
Cite this: Org. Biomol. Chem., 2012, 10,
9689
Yangfan Zheng,‡a Caihua Wang,‡b Changhao Li,b Jinxia Qiao,a Feng Zhang,a
Minjian Huang,a Wenming Ren,a Chune Dong,a Jian Huang*b and Hai-Bing Zhou*a
We have synthesized a series of novel SERMs bearing a ferrocenyl unit based on a three-dimensional oxa-
bicyclo[2.2.1]heptene core scaffold. These compounds displayed high receptor binding affinities as well
as ERα or ERβ selectivity. In cell proliferation assays, we found that these ligands were cytotoxic at micro-
molar concentrations in both ER-positive and ER-negative breast cancer cells. On further examination, we
found that the antiproliferative effects of compounds 9b, 10h and 11b on MCF-7 cells line does not arise
from antiestrogenicity, but rather proceeds through a cytotoxic pathway. Possible mechanisms for the
unique activities of these ligands were also investigated by molecular modeling. These new ligands could
act as scaffolds for the development of novel anti-breast cancer agents.
Received 27th June 2012,
Accepted 23rd October 2012
DOI: 10.1039/c2ob26226f
www.rsc.org/obc
cells.7–11 The antiproliferative properties of ferrocene appeared
to be related to distinct properties of the ferrocenyl
Introduction
The development of novel selective estrogen receptor modu- moiety.12–15 Ferrocene is chemically stable in various non-oxi-
lators (SERMs) against breast cancer has been a major objec- dizing milieu, but the ferrocenium ion, produced by oxidation,
tive for medicinal chemistry.1,2 Two-thirds of breast cancers is recognised as having antitumour activity.16–18 Tamoxifen
belong to the hormone-positive (ER+) type, making them good (1, TAM, Fig. 1) is the front-line therapeutic agent for patients
candidates for hormone therapy by SERMs such as tamoxifen with hormone-dependent breast cancer, and 4-hydroxy-tam-
and affording these patients significantly greater chances for oxifen (2, 4OHT, Fig. 1) is an active metabolite of TAM. The
successful treatment with endocrine therapies compared to group of Jaouen has investigated analogues of 3 and 4, in which
the hormone-negative (ER−) group.3,4 While tamoxifen is not a ferrocenyl unit has replaced one of the phenyl rings of TAM
effective in ER− breast cancer patients, it also is ineffective in a or 4OHT, giving compounds named ferrocifens.7,11 Conjugates
significant fraction of ER+ patients, and even when initially like 4 maintained good binding affinity towards the ER and
effective, resistance to tamoxifen therapy often develops with had antiproliferative effects in ER+ MCF-7 cells; surprisingly,
time. For these reasons, there is great interest in the discovery however, they were also active against the ER− MDA-MB-231
of new molecules that would be effective on ER− and tamoxi- cells, on which SERMs are not normally active. Interestingly,
fen non-responsive or resistant ER+ tumors.
use of other organometallic fragments, like ruthenocene
Recently, derivatives of ferrocene, discovered originally as in place of the ferrocene group gave compounds that were
the most prototypical organometallic compound,5,6 have been
reported to have antiproliferative activity on breast cancer
aLaboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University),
Ministry of Education, State Key Laboratory of Virology, Wuhan University School of
Pharmaceutical Sciences, Wuhan, 430071, China. E-mail: zhouhb@whu.edu.cn;
Fax: +86-27-68759850; Tel: +86-27-68759586
bCollege of Life Sciences, Wuhan University, Wuhan, 430072, China.
E-mail: jianhuang@whu.edu.cn; Tel: +86 27 68753582
†Electronic supplementary information (ESI)
10.1039/c2ob26226f
available. See DOI:
‡These two authors contributed equally to this work.
Fig. 1 Structures of tamoxifen and ferrocifens.
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indirect mechanism. This contrasts with the mechanism of
action of tamoxifen, which directly interacts with helix 12,
pushing it out of the conformation required for gene
activation.31
Our understandings in the crystal structure of ERα-OBHS
and the unique feature of the ferrocenyl entity led us to
prepare a new family of ferrocene complexes based on the
OBHS core scaffold which might retain ER antagonist activity,
but more importantly might, in addition, have antitumor
activity against hormone-independent breast cancer cell lines,
such as MDA-MB-231 cells. We chose ligand cores that exhib-
ited excellent binding affinity and good transcription potency
to evaluate the activities of analogs into which a ferrocenyl
group was introduced. We followed three strategies for introdu-
cing the ferrocenyl group into the OBHS skeleton, as shown in
Fig. 3: (1) substitute one phenol ring on C-5 or C-6 in OBHS
scaffold with the ferrocenyl group (compound series 9); (2)
append the ferrocenyl group onto one of these phenols (com-
pound series 10); (3) add the ferrocenyl group onto the phenyl
sulfonate moiety (compounds 11). To make a systematic and
logical examination of the effects of ferrocenyl group addition
on the biological activity of OBHS derivatives, we synthesized
an array of 17 new compounds according to these design prin-
ciples. In ligand binding assays, these new analogues showed
moderate binding affinity, and in the breast cancer cell-based
assays, we identified several compounds that have a moderate
antiproliferative effect on both hormone-dependent (MCF-7)
breast cancer cells, and more importantly, also on hormone-
independent (MDA-MB-231) breast cancer cells.
Fig. 2 Structures of tamoxifen derivatives with ferrocenyl side chains and ferro-
cenophane polyphenol derivatives.
Fig. 3 Three new ferrocenyl ER ligand derivatives based on OBHS scaffold.
antiestrogenic on ER+ breast cancer cells but were inactive on
ER− cells.19,20
Because the MDA-MB-231 breast cell line lacks ER and
hence is not responsive to treatment with tamoxifen, the
activity of ferrocifens on these ER− cells suggests that the
metallocyclic systems have a novel, dual-mode mechanism of
action: besides the tamoxifen-like antiestrogenic activity that
results from its binding to the ER, a second, ER-independent
activity might derive from the redox activity of the ferrocene
unit. This redox activity of the metallocene could be the mech-
anism accounting for the fact that the biological activity of the
ferrocifens exceeds that of a purely organic analogue, such as
tamoxifen.17 A mechanism that could underlie this second,
more general cytotoxic effect of ferrocenyl phenols on various
cancer cells, is based on the in situ transformation to an
electrophilic quinone methide (QM), which is mediated by the
ferrocenyl group, a transformation that can be followed by
electrochemistry.21–23
Recently, the same group has presented work on tamoxifen
derivatives with ferrocenyl side chains and ferrocenophane
polyphenol derivatives (Fig. 2) that also have good antiproli-
ferative activity.24,25
As part of our ongoing study of selective estrogen receptor
modulators,26–29 we have recently described a new series of
analogues based on novel three-dimensional core scaffold that
have good binding affinity. The best compound, exo-5,6-bis-(4-
Results and discussion
Synthesis
The synthesis of the oxabicyclic bridged ferrocenyl compounds
9 was accomplished by a Diels–Alder reaction of the 3-ferro-
cenyl-4-(4-hydroxyphenyl) furan 17 with a variety of dienophiles
18. The dienophiles 18 were prepared, as previously reported,
by the reaction of 2-chloroethanesulfonyl chloride with substi-
tuted phenols in the presence of Et3N.32 The synthesis of
the important intermediate, 3-ferrocenyl-4-(4-hydroxyphenyl)
furan 17, was accomplished in 4 steps, as shown in Scheme 1.
α-Chloroacetylferrocene 12 reacted with 4-methoxyl phenyl-
acetic acid 13 in the presence of NaI and Et3N to give the con-
densation product 14.33,34 Treatment of ester 14 with NaH in
anhydrous DMSO gave the 2(5H)-furanone 15, which was
demethylated with BBr3 to afford the butenolide 16.35 Diisobutyl-
aluminum hydride reduction of 16 at −78 °C gave, after a
careful, low-temperature acidic workup, the 3-ferrocenyl-4-(4-
hydroxyphenyl) furan 17. Finally, a Diels–Alder reaction of 17
with the dienophiles 18 produced compounds 9a–e (regio-
isomers, ca. 1 : 1).
hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonic
acid
phenyl ester 8 (OBHS, Fig. 3),30 acted as an indirect antagonist
on both ER subtypes in transcriptional assays. From X-ray
crystal structures of complexes of the ERα-LBD (ligand binding
domain) with OBHS we noticed that these ligands act entirely
by interactions with helix 11 and modulate the position of the
critical helix 12 in the ER ligand-binding domain by an
The isomers, however, could not be separated by prepara-
tive HPLC; thus a mixture of isomers was used in all tests. It
is noteworthy that, as we had observed previously, high stereo-
selectivity was observed in the cycloaddition reaction of the
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Scheme 3 Synthesis of derivatives 11a and 11b.
chloride 22a and 4-methoxybenzyl alcohol 22b with different
acid catalysts (AlCl3 for 22a, CF3COOH for 2b) to afford the cor-
responding ferrocenyl substituted products 23a and 23b;36,37
which were demethylated with BBr3 to produce phenolic pro-
ducts 24a and 24b; these were then reacted with 2-chloro-
ethanesulfonyl chloride in the presence of Et3N to give the
dienophiles 25a and 25b.37 Finally, 25a and 25b reacted with
3,4-bis-(4-hydroxyphenyl) furan, which was prepared using an
improved Diels–Alder reaction procedure we reported, to afford
the target compounds 11a and 11b in high yields (90% and
85%) (Scheme 3).
Scheme 1 Synthesis of ferrocenyl derivatives 9a–e.
Binding affinity
We examined the binding affinity of compounds 9–11 for both
purified human ERα and ERβ LBDs, by a competitive binding
assay based on fluorescence polarization,38–41 which is
described in detail in the Experimental section. The binding
affinities of all of the ligands in this study were expressed as
relative binding affinity (RBA) values, where estradiol was set
as 100%. All of the newly synthesized 17 compounds were
tested, and results are summarized in Table 1.
Scheme 2 Synthesis of derivatives 10a–j.
phenolic furans with the dienophiles; the exo product was
obtained nearly exclusively, while, the endo products were
observed in only trace amounts.30 This exo selectivity is
presumed due to the high rate, and ready reversibility of
the Diels–Alder reaction of furans, which allows the thermo-
dynamically favored exo product to become dominant.
From the Table 1 we noticed that the disposition of the ferro-
cenyl substituent on OBHS scaffolds proves to be an impor-
tant factor in determining the binding affinity and selectivity
of these new compounds. Some of the ligands showed moder-
ate to good binding affinity to both ERα and ERβ. Compounds
9a–9e in which a ferrocenyl group displaced one of the pheno-
lic ring in the C-5 or C-6 in OBHS scaffold, had RBA values of
1.4%–12.2% for ERα, with the selectivity in favor of ERα
(Table 1, entries 1–5). Among these five analogs, compound 9d
possessing a para-bromine substituent on phenyl of sulfonate
gave the highest RBA value of 12.2% for ERα (Table 1, entry 4).
When the ferrocenyl group was introduced as a side chain,
a trend towards ERβ-selectivity was observed for most of the
derivatives (10a–10j, Table 1, entries 6–15). In this series, com-
pounds that had shorter side chains exhibited higher RBA
values than those with longer ones. Compounds 10b and 10g,
which possessed an ortho-chlorine substituent on phenyl ring
of sulfonate, showed a high RBA value of 36.4% and 25.1% for
ERβ, respectively (Table 1, entries 7 and 12). Analogues 11a
and 11b, in which the ferrocenyl groups are appended on the
phenyl of sulfonate, show RBAs of 3.8% and 1.9% for ERα,
and 2.1% and less than 0.1% for ERβ, respectively (Table 1,
entries 16 and 17). It is notable that the ERβ affinities of the
ferrocenyl ligands 10 (except 10c and 10f) were comparable
with or even higher than those of the corresponding OBHS
Compounds 10a–j with a ferrocenyl group appended on the
phenol substituents at the C-5 or C-6 positions of the bicyclic
core unit were prepared by a Mitsunobu reaction (Scheme 2).
The OBHS 8 and its derivatives 19a–d were conveniently pre-
pared by a Diels–Alder reaction of 3,4-bis-(4-hydroxyphenyl)-
furan with a variety of dienophiles, as previously described.31
A Mitsunobu reaction of the OBHS derivatives with 1.2 equiva-
lents of the ferrocenyl alcohol, in the presence of triphenyl-
phosphine and diisopropyl azodicarboxylate (DIAD) in THF for
24–48 h, surprisingly, afforded one regioisomer 10a–j (yield
25–42%). As shown below, only traces, if any, of another regio-
isomer were observed, but about 15–25% of disubstituted
byproducts were produced.24 As has been described previously,
the isolated products are the exo diastereomers, and they are
also racemic (see ESI† for details of 2D 1H NMR and assign-
ments of exo 10g).30
To examine the effect of the ferrocenyl group when posi-
tioned on the phenyl ring of sulfonate, we prepared com-
pounds 11a and 11b using the Diels–Alder reaction of 3,4-bis-
(4-hydroxyphenyl) furan 26 with dienophiles 25a and 25b
under neat conditions. 25a and 25b were prepared by three
steps: first, the ferrocene reacted with 4-methoxybenzoyl
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Table 1 Relative binding affinity (RBA) values of ferrocenyl ligands 9–11 and
representative analogues 8 and 19 without ferrocene moietya
Table 2 Effect on cancer cell growth of ferrocenyl ligands 9–11 and represen-
tative analogues 19 without ferrocene moiety
Entry
Cmpdb
ERα (%)
ERβ (%)
Effect on the growth of cancer
cellsa (IC50 [μM])
1
2
3
4
5
6
7
8
9a
9b
9c
9d
9e
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
11a
2.5 0.3
1.4 0.2
4.6 0.2
12.2 0.4
2.5 0.5
1.9 0.4
8.3 0.2
2.8 0.7
<0.1
1.9 0.1
<0.1
<0.1
1.2 0.0
<0.1
4.6 1.1
3.8 1.1
1.9 0.3
14.4 1.7
26.2 6.2
10.1 0.3
2.0 0.02
8.19 0.19
13.8 0.2
—
<0.1
<0.1
<0.1
<0.1
Entry
Cmpd
MCF-7
MDA-MB-231
1
2
3
4
5
6
7
8
(Z)-4-OH-Tamoxifen
9a
9b
9c
9d
9e
10a
10b
10c
10d
10e
10f
10g
10h
10i
16.8 0.3
>100
>100
<0.1
7.5 0.8
5.6 0.7
9.8 0.9
3.1 0.5
7.9 0.9
48.2 5.9
8.7 1.1
9.8 1.2
11.2 0.9
21.2 1.8
63.8 7.8
6.7 0.8
10.4 1.1
54.3 7.1
39.5 4.9
13.9 1.2
7.5 1.0
20.9 1.0
17.8 0.4
30.8 4.4
29.9 3.9
31.1 1.6
5.5 0.3
36.4 1.8
<0.1
20.7 1.3
11.2 1.9
<0.1
25.1 2.3
1.6 0.4
1.5 0.5
2.4 0.0
2.1 0.2
<0.1
6.93 0.05 15
3.83 0.19 16
2.51 0.06 17
2.41 0.18 18
3.66 0.23 19
12.2 0.9
28.7 2.3
17.5 2.1
>100
>100
43.5 2.8
>100
63.3 5.2
>100
>100
18.4 2.2
12.6 1.2
>100
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
9
10
11
12
13
14
11b
8
19a
19b
19c
19d
10j
>100
11a
11b
8
19a
19b
19c
19d
Ferrocene
10.3 0.3
7.8 0.6
57.1 12.2
88.4 11.7
>100
58.8 0.8
52.0 3.8
>100
(Z)-4-OH-Tamoxifen
Ferrocene
12.6 1.7
—
20
21
22
23
24
a Relative binding affinity (RBA) values were determined by competitive
binding assay based on fluorescence polarization and expressed as
>100
IC50 estradiol/IC50 compound × 100
the range (RBA, estradiol =
100%). b Mean of three experiments range.
a Mean of two experiments range.
derivatives, but the whole of series 10 compounds showed
deceased affinities toward the ERα (Table 1, 8, 19a–d, entries
18–22). (Z)-Tamoxifen show moderate RBAs of 13.8% and
12.6% for ERα and ERβ, respectively (Table 1, entry 23). As
expected, ferrocene did not show any affinity for either ER
(Table 1, entry 24).
When the ferrocenyl group was appended onto the phenyl
of the sulfonate (analogues 11a and 11b), affinities were lower,
with RBA values of 3.8% and 1.9% for ERα, respectively
(Table 1, entries 16–17). It seems that, for binding to ERβ, the
analogue with a carbonyl linkage (11a) is much better than the
one with a methylene linkage.
In terms of binding affinity, we can conclude that replace-
ment of a phenolic ring with a ferrocenyl group (as in com-
pounds 9) gave selectivity for ERα, while introduction of
ferrocenyl group as side chain (as compounds 10), in most
cases, afforded the selectivity for ERβ.
Overall, there are some interesting results. Compared with
the effect of OH-TAM (Table 2, entry 1), most of the com-
pounds were effective in inhibiting the hormone-dependent
MCF-7 cells, except for 10a, 10f, 10i and 10j (Table 2, entries 7,
12, 15 and 16). In addition, at least 7 compounds (9b–d, 10g–h
and 11a–b) effectively inhibited the proliferation of the
hormone-independent MDA-MB-231 breast cancer cells.
We noticed that the analogues 9a–9e, in which one pheno-
lic ring of OBHS was replaced by a ferrocenyl group, were
effective in inhibiting the hormone-dependent MCF-7 breast
cancer cells, with low IC50 values of 3.1–9.8 μM (Table 2,
entries 2–6). However, only three of them (9b–9d) inhibited the
proliferation of the hormone-independent MDA-MB-231 breast
cancer cells.
Compared to the 9a–9e, most of the derivatives 10a–10j in
which the ferrocenyl group was introduced as a side chain,
were less effective in inhibiting MDA-MB-231 cell proliferation,
and they displayed only modest cytotoxicity to MCF-7 cells.
Two members of this series, 10g and 10h, which have ortho or
meta-chlorine substituents on phenyl of sulfonate, did show
Cell proliferation assays
The influence of the compounds on the proliferation of cancer promising cytotoxicity on both MCF-7 and MDA-MB-231 cells
cells has been tested on both hormone-dependent (MCF-7) (Table 2, entries 13 and 14). The parent compounds 8 and
breast cancer cells and hormone-independent (MDA-MB-231) 19a–d showed moderate inhibitory activities on MCF-7 cells,
breast cancer cells. Four different concentrations (0.1, 0.5, 1 however, with no obvious inhibition for MDA-MB-231 cells
and 10 μM) were used to determine from semi-log plots IC50 (Table 2, entries 19–23).
values (concentration causing 50% inhibition of cell prolifer-
Surprisingly, the analogues 11a and 11b with ferrocenyl
ation). The indicated values were the mean
independent experiments, as given in Table 2.
range of two groups appended onto the phenyl of sulfonate, were cytotoxic
on both MCF-7 and MDA-MB-231 cells, and with similar
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Fig. 4 Effect of E2, 9b, 10h and 11b on the proliferation of hormone-depen-
dent (MCF-7) breast cancer cells after 5 days of culture. Non-treated MCF-7 cells
are used as the control set at 100%. Mean of two separate experiments
range.
activities in both cases (IC50 values between 7.5 and 13.9 μM)
(Table 2, entries 17 and 18). This suggested that binding
affinity and anti-proliferative potency are independent.
Notably, compound 11b displayed the best antiproliferative
effect on the hormone-independent MDA-MB-231 breast
cancer cells, although it showed low binding affinity to both
ERs. It was observed that in terms of antiproliferative activity,
11b was a little bit better than 11a, which could result from
the fact that a methylene linkage is more flexible than a carbo-
nyl linkage and might enable 11b to stabilize a strongly antag-
onistic conformation ER conformation. Ferrocene itself was
not active in either cell type (Table 2, entry 24).
It is noteworthy that compounds 9b, 10h and 11b showed
significant and quite similar antiproliferative effects on both
MCF-7 and MDA-MB-231 cells (Table 2). To determine whether
the antiproliferative effect of these compounds on MCF-7 cells
was the result of ER binding, the cells were incubated with
10 μM of 9b, 10h and 11b in the presence and absence of 10
nM E2. As shown in Fig. 4, the activity of 9b, 10h and 11b on
MCF-7 cell proliferation seems to be cytotoxic at high concen-
trations (>1 μM), while the addition of E2 did not reverse the
antiproliferative effect of 9b, 10h and 11b, which indicates that
their antiproliferative effect is cytotoxic and not antiestrogenic.
Fig. 5 The binding models of exo1 and exo2 of 9a with ERα (2ouz). In both
cases, a hydrogen bond formed between phenolic hydroxyl group and Glu 353,
the ferrocenyl group is forced into a hydrophobic pocket (colored in yellow).
only the exo1s of 9a–9e gave positive results upon docking in
the antagonist conformation of ERβ.
It is challenging to determine the orientation of the com-
pounds in the ligand binding pocket because the ferrocenyl
group may adopt different orientations. OBHS binds to ERα
mainly through hydrogen bonds between the two phenolic
hydroxyl groups and two amino acids, Glu 353 and Thr 347, in
helix 3.31 The phenyl sulfonate ring is close to helix 11 and
indirectly affects the conformation of the critical helix 12. Both
regioisomers of 19 (exo1, exo2) can similarly form a hydrogen
bond between a phenolic hydroxyl group and Glu 353, which
simulates the interaction of the A ring of steroidal estrogens in
ERα complexes, but unlike OBHS, the ferrocenyl groups are
forced into a hydrophobic pocket and the phenyl sulfonate
ring may directly interfere with helix 12 adopting an agonist
conformation in ERα (Fig. 5A showed the binding model of
exo1 9a with ERα-2ouz). Similar to the exo1s, the exo2s of com-
pounds 9a–9e can form a hydrogen bond between the phenolic
hydroxyl group and Glu 353; the ferrocenyl groups are also
forced into a hydrophobic pocket, but the sulfonate phenyl
ring interferes with helix 12 in ERα in different way (Fig. 5B
showed the binding model of exo2 9a with ERα-2ouz).
In this study, compounds 10a–10j formed largely as exo1
regioisomers, so only these isomers were used in the mole-
cular modeling. The results indicated that the compounds
10a–10j bind to ERα through hydrogen bonds of the hydroxyl
groups of the phenols with the Thr 347 and Glu 353, and the
ferrocenyl group near helix 12 induces the latter to adopt an
antagonist conformation (Fig. 6 show a model of 10a bound to
ERα).
Molecular modeling indicated that for both of compounds
9 and 10 a portion of the three-dimensional oxabicyclic core
occupies space in the 11β region of the binding pocket that
is typically void of substituents with steroidal estrogens. This
Molecular-docking and structure-activity relationships
We undertook a molecular modeling study of the ferrocenyl
compounds, hoping to better understand which regioisomers
were dominant and how they might be oriented in the ligand
binding pocket. We performed docking with agonist and
antagonist conformations of ERα and ERβ, respectively (PDB
code 1l2i, 2ouz, 1 × 7b, 1l2j).42–44 Compounds 9a–9e are able
to orient in both agonist and antagonist conformations of
ERα, and the exo2s are dominant regioisomers based on the
docking free energy of two exo regioisomers of 9a–9e with ERα
and ERβ (see ESI† for details). Compounds 10a–10j can only
dock well in the antagonist conformation of ERα; no positive
result in agonist conformation of ERα was obtained. After
docking with agonist and antagonist conformation of ERβ,
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Biospin AV400 (400 MHz) instrument. The chemical shifts are
reported in ppm and are referenced to either tetramethylsilane
or the solvent. High resolution MS data were obtained on
IonSpec 4.7 Tesla FTMS. Tetrahydrofuran and toluene were
dried over Na and distilled prior to use. Dichloromethane was
dried over CaH2 and distilled prior to use. Glassware was
oven-dried, assembled while hot, and cooled under an inert
atmosphere. Unless otherwise noted, all reactions were
conducted in an inert atmosphere. Reaction progress was
monitored using analytical thin-layer chromatography (TLC).
Visualization was achieved by UV light (254 nm). Flash chromato-
graphy was performed with silica gel (0.040–0.063 mm)
packing.
SYNTHESIS OF 2-OXO-2-FERROCENYL ETHYL 2-(4-METHOXYPHENYL)ACETATE
(14). A solution of 2-chloro-1-ferrocenyl ethanone 12 (2.62 g,
10 mmol, 1 equiv.) and 4-methoxyphenylacetic acid 13 (1.66 g,
10 mmol, 1 equiv.) in acetone was stirred at room temperature,
NaI (1.80 g, 12 mmol, 1.2 equiv.) and Et3N (1.67 mL, 1.21 g,
12 mmol, 1.2 equiv.) were added sequentially to the solution,
the mixture was refluxed for another 4 h and then solvent was
evaporated and the residue was extracted with EtOAc, washed
sequentially with 2 N HCl (30 mL), saturated aqueous NaHCO3
(2 × 30 mL), and brine (30 mL). The organic layer was dried
over sodium sulfate. Filtration and evaporation of the solvent
gave the crude 14, which was purified by column chromato-
graphy using 20% EtOAc/petroleum ether as eluent to afford
pure product as a red solid (88%). 1H NMR (400 MHz,
Acetone-d6) δ 7.28 (d, J = 8.7 Hz, 2H), 6.88 d, J = 8.7 Hz, 2H),
5.07 (s, 2H), 4.78 (m, 2H), 4.54 (m, 2H), 4.27 (s, 5H), 3.79 (s,
3H), 3.76 (s, 2H). 13C NMR (101 MHz, Acetone-d6), δ 196.33,
171.85, 159.71, 131.36, 127.23, 114.62, 76.57, 73.11, 70.88,
69.44, 67.46, 55.50, 40.23.
SYNTHESIS OF 3-(4-METHOXYPHENYL)-4-FERROCENYLFURAN-2(5H)-ONE
(15). A solution of 14 (3.45 g, 8.8 mmol, 1 equiv.) in dimethyl-
sulfoxide was added dropwise to a stirred suspension of
sodium hydride (70% in oil, 0.604 g, 17.6 mmol, 2 equiv.) in
dimethylsulfoxide at 25 °C. The reaction was allowed to
proceed for 4 h at 25 °C prior to pouring into water. Extraction
with ethyl acetate, washing the combined ethyl acetate extracts
with water, drying the organic fraction with Na2SO4, and
removal of the solvent in vacuo afforded a residue. Purification
of this residue by silica-gel column chromatography using
20% EtOAc/petroleum ether as eluent furnished 15 as dark red
needles (75%). 1H NMR (400 MHz, CDCl3) δ 7.40 (d, J = 8.7 Hz,
2H), 6.99 (d, J = 8.7 Hz, 2H), 5.09 (s, 2H), 4.40 (dt, J = 19.5, 1.7
Hz, 4H), 4.13 (s, 5H), 3.86 (s, 3H). 13C NMR (101 MHz, CDCl3),
δ 174.40, 159.71, 157.99, 130.63, 123.27, 121.91, 114.06, 72.75,
71.09, 70.51, 70.15, 68.32, 55.34, 29.70.
Fig. 6 A model of 10a binding with ERα (2ouz). Hydrogen bonds formed
through the phenolic hydroxyl group, the Thr 347 and Glu 353, the ferrocenyl
group near to the helix 12.
suggests the potential advantage of using this rigid bicyclic
scaffold compared to the use of less rigid structures.
Compared to ERα, the active pocket of ERβ is short and
narrow. The inward motion of helixes 8, 9 and 11 and an
inward turn of the loop13 eliminates some of the sub-pocket
space and reduces the size of the active pocket of ERβ con-
siderably. Therefore accommodating large ligands by ERβ is a
great challenge.
Conclusion
A small library of analogues having ferrocenyl substituents at
various positions on the OBHS scaffold were prepared and
evaluated for their binding affinities for ERα and ERβ as well
as for their effects on breast cancer cell proliferation. These
compounds displayed high receptor binding affinities as well
as ERα or ERβ binding selectivity. Interestingly, replacement of
the phenolic ring with a ferrocenyl group (9a–9e) gave the
selectivity of ERα, whereas introduction of a ferrocenyl group
as side chain (10a–10j) afforded ERβ selectivity. In cell prolifer-
ation assays, we were pleased to find that some of these
ligands were cytotoxic at micromolar concentrations in both
MCF-7 and MDA-MB-231 breast cancer cells; compound 11b
has the lowest IC50 value (7.8 μM) for the ER-negative
MDA-MB-231 cells. Through molecular modeling studies of
these ligands complexed with ERα, we explored possible mech-
anisms for their unique activities. Thus, it is possible that
these new ligands could act as candidates for the development
of anti-breast cancer agents.
SYNTHESIS OF 3-(4-HYDROXYPHENYL)-4-FERROCENYLFURAN-2(5H)-ONE
(16). A solution of 15 (2.47 g, 6.6 mmol, 1 equiv.) dissolved in
dry CH2Cl2 was cooled to −78 °C. Then a solution of BBr3
(8.25 g, 33 mmol, 5 equiv.) in CH2Cl2 was added, and the reac-
tion mixture was allowed to warm to room temperature over-
Experimental section
Synthesis materials and methods
Melting points were obtained on X-4 melting point apparatus night. The brown solution was cooled on ice and water was
(Beijing TECH Instruments, Co., Ltd.) and are uncorrected. slowly added. The two layers were separated, and the water
1H NMR and 13C NMR spectra were obtained on Bruker layer was washed twice with CH2Cl2. The collected organic
9694 | Org. Biomol. Chem., 2012, 10, 9689–9699
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layers were dried with MgSO4 and evaporated to dryness. After
5-(4-HYDROXYPHENYL)-6-FERROCENYL-7-OXABICYCLO[2.2.1]HEPT-5-ENE-
purification via column chromatography using 30% EtOAc/ 2-SULFONIC ACID 2-CHLOROPHENYL ESTER (9B). 1H NMR (400 MHz,
petroleum ether as eluent, pure 16 was obtained as a red CDCl3) δ 7.60–7.17 (m, 8H), 6.82 (d, J = 8.1 Hz, 2H), 5.74 (d, J =
1
solid (85%). H NMR (400 MHz, DMSO-d6) δ 9.80, (s, 1H), 7.30 3.2 Hz, 1H), 5.46 (s, 1H), 5.36 (s, 1H), 4.38–4.08 (m, 9H),
(d, J = 8.6 Hz, 2H), 6.97 (d, J = 8.6 Hz, 2H), 5.29 (s, 2H), 4.59 (d, 3.82–3.74 (m, 0.5H), 3.63 (dd, J = 8.3, 4.3 Hz, 0.5H), 2.67–2.51
J = 8.0 Hz, 4H), 4.30 (s, 5H). 13C NMR (101 MHz, DMSO-d6) (m, 1H), 2.25–2.10 (m, 1H). 13C NMR (101 MHz, Acetone-d6)
δ 173.75, 158.64, 157.36, 130.48, 121.66, 120.94, 115.19, 72.43, δ 158.43, 158.31, 146.32, 146.18, 141.92, 141.43, 141.39,
70.77, 70.22, 69.98, 68.10. HRMS calcd for C20H16O3Fe 136.53, 131.85, 131.79, 131.74, 131.28, 130.63, 130.19, 129.49,
[M + 1]+, 361.05272, found, 361.05206.
129.42, 129.34, 129.24, 129.14, 127.74, 125.13, 124.16, 116.40,
SYNTHESIS OF 4-(4-FERROCENYLFURAN-3-YL)PHENOL (17). To a cooled 116.31, 116.21, 85.37, 85.24, 84.04, 83.86, 77.26, 70.17, 70.12,
(−78 °C) solution of 16 (2.02 g, 5.60 mmol, 1 equiv.) in dry 70.03, 68.99, 68.84, 68.31, 67.61, 67.52, 63.65, 63.11, 32.07,
THF, a solution of 1 M DIBAl-H in toluene was added dropwise 31.97. HRMS calcd for C28H23O5SCl54Fe (M+), 560.03509,
(22.4 mL, 22.4 mmol, 4 equiv.). The resulting solution was found, 560.03454.
stirred for 10 h at −20 °C and quenched by slow addition of
5-(4-HYDROXYPHENYL)-6-FERROCENYL-7-OXABICYCLO[2.2.1]HEPT-5-ENE-
4% H2SO4. After 30 min, the solution was diluted with ether, 2-SULFONIC ACID 3-CHLOROPHENYL ESTER (9C). 1H NMR (400 MHz,
and the organic layer was washed with water and brine. After Acetone-d6) δ 7.48–7.32 (m, 6H), 6.93–6.85 (m, 2H), 5.64 (dd,
drying over sodium sulfate and evaporation of the solvent, the J = 6.6, 1.2 Hz, 1H), 5.49–5.37 (m, 1H), 4.43–4.15 (m, 9H), 3.92
crude furan was purified by column chromatography using (dd, J = 8.3, 4.4 Hz, 0.4H), 3.75 (dd, J = 8.3, 4.4 Hz, 0.6H),
20% EtOAc/petroleum ether as eluent to obtain 17 as a red 2.47–2.35 (m, 1H), 2.28 (dd, J = 12.1, 8.4 Hz, 1H). 13C NMR
1
solid (70%). Mp = 104–106 °C. H NMR (400 MHz, DMSO-d6) (101 MHz, Acetone-d6) δ 158.56, 158.45, 150.93, 136.50, 135.33,
δ 9.43 (s, 1H), 7.83 (s, 1H), 7.56 (s, 1H), 7.08 (d, J = 8.2 Hz, 2H), 132.15, 131.98, 131.28, 130.56, 130.09, 128.30, 128.20, 123.56,
6.72 (d, J = 8.2 Hz, 2H), 4.14 (d, J = 16.0 Hz, 4H), 3.69 (s, 5H). 123.54, 121.95, 116.42, 116.23, 85.21, 85.16, 84.11, 83.92,
13C NMR (101 MHz, DMSO-d6) δ 156.68, 140.46, 140.16, 77.56, 70.37, 70.14, 70.09, 70.03, 70.00, 69.93, 68.93, 67.60,
130.09, 125.90, 122.63, 122.12, 114.93, 77.22, 69.05, 67.68, 67.46, 62.84, 61.95, 32.64, 31.94. HRMS calcd for
67.53. HRMS calcd for C20H16O254Fe (M+), 342.05464, found, C28H23O5SCl54Fe (M+), 560.03508, found, 560.03497.
342.05413.
5-(4-HYDROXYPHENYL)-6-FERROCENYL-7-OXABICYCLO[2.2.1]HEPT-5-ENE-
2-SULFONIC ACID 4-BROMOPHENYL ESTER (9D). 1H NMR (400 MHz,
SYNTHESIS OF ETHENESULFONIC ACID PHENYL ESTERS (18). 2-Chloro-
ethanesulfonyl chloride (2.1 mL, 2 mmol) dissolved in anhy- Acetone-d6) δ 7.66 (dd, J = 6.8, 2.1 Hz, 1H), 7.59–7.53 (m, 1H),
drous dichloromethane (5 mL) and substituted phenols 7.44–7.32 (m, 3H), 7.23–7.19 (m, 1H), 6.89 (t, J = 8.5 Hz, 2H),
(2 mmol) were treated at 0 °C with triethylamine (0.58 mL, 5.62 (s, 1H), 5.47–5.37 (m, 1H), 4.42–4.14 (m, 9H), 3.88 (dd, J =
4 mmol). After 1 h, the reaction mixtures were concentrated, 8.3, 4.4 Hz, 0.4H), 3.70 (dd, J = 8.3, 4.5 Hz, 0.6H), 2.40 (ddd,
and the crude residues were purified by flash chromatography J = 12.0, 5.9, 2.8 Hz, 1H), 2.24 (td, J = 12.0, 8.4 Hz, 1H).
on silica gel to provide 18 (72–88% yields).
13C NMR (101 MHz, Acetone-d6) δ 158.41, 158.30, 149.72,
145.11, 141.70, 141.31, 136.47, 133.95, 133.87, 133.79, 133.67,
131.18, 130.65, 130.10, 125.43, 125.36, 125.30, 124.11, 120.78,
116.39, 116.23, 115.66, 85.19, 84.08, 83.91, 77.53, 70.14, 70.11,
Synthesis of derivatives 9a–e
GENERAL PROCEDURE FOR THE DIELS–ALDER REACTION. 3-(4-Hydroxy- 70.04, 70.01, 69.94, 68.94, 67.61, 67.45, 61.77, 32.65, 31.92.
phenyl)-4-ferrocenyl furans 17 (0.4 mmol) and 5 various dieno- HRMS calcd for C28H23O5SBr54Fe (M+), 603.98457, found,
philes 18 (0.5 mmol) were placed in a round flask, and the 603.98403.
mixture was stirred under argon atmosphere at 90 °C for
5-(4-HYDROXYPHENYL)-6-FERROCENYL-7-OXABICYCLO[2.2.1]HEPT-5-ENE-
8–12 h. The crude product was purified by flash chromato- 2-SULFONIC ACID 1-NAPHTHYL ESTER (9E). 1H NMR (400 MHz,
graphy (10–25% EtOAc/petroleum ether), yield 75–88%, com- Acetone-d6) δ 8.34–8.16 (m, 1H), 8.04–7.87 (m, 2H), 7.68–7.36
pounds are ca. 1 : 1 isomer mixtures.
(m, 6H), 6.90 (t, J = 11.4 Hz, 2H), 5.73 (d, J = 1.0 Hz, 0.5H), 5.71
5-(4-HYDROXYPHENYL)-6-FERROCENYL-7-OXABICYCLO[2.2.1]HEPT-5-ENE- (d, J = 1.0 Hz, 0.5H), 5.49 (d, J = 4.3 Hz, 0.5H), 5.46 (d, J =
2-SULFONIC ACID PHENYL ESTER (9A). 1H NMR (400 MHz, Acetone-d6) 4.4 Hz, 0.5H), 4.45–4.11 (m, 9H), 4.11–4.04 (m, 0.5H), 3.95 (dd,
δ 7.50–7.35 (m, 6H), 7.24 (d, J = 7.8 Hz, 1H), 6.89 (t, J = 7.8 Hz, J = 8.3, 4.4 Hz, 0.5H), 2.64–2.50 (m, 1H), 2.41–2.23 (m, 1H).
2H), 5.61 (d, J = 3.8 Hz, 1H), 5.45 (d, J = 4.2 Hz, 0.6H), 5.39 (d, 13C NMR (101 MHz, Acetone-d6) δ 158.40, 158.36, 158.29,
J = 4.2 Hz, 0.4H), 4.36–4.15 (m, 9H), 3.84 (dd, J = 8.3, 4.4 Hz, 158.25, 146.33, 141.81, 141.32, 136.65, 135.93, 135.83, 131.35,
0.4H), 3.65 (dd, J = 8.3, 4.4 Hz, 0.6H), 2.40 (dt, J = 9.2, 4.4 Hz, 131.25, 130.57, 130.16, 128.91, 128.83, 128.31, 128.23, 128.03,
1H), 2.29–2.21 (m, 1H). 13C NMR (100 MHz, Acetone-d6) 127.99, 127.95, 127.88, 127.85, 126.52, 126.39, 124.21, 122.71,
δ 157.60, 157.50, 148.91, 144.30, 140.89, 140.50, 135.66, 122.67, 119.52, 119.25, 116.43, 116.35, 116.29, 116.20,
133.14, 133.07, 132.99, 132.86, 130.37, 129.84, 129.29, 124.63, 85.48, 85.33, 84.76, 84.43, 84.13, 83.92, 77.58, 77.05,
124.56, 124.50, 123.30, 120.09, 119.97, 115.59, 115.43, 114.85, 70.45, 70.16, 70.11, 70.04, 69.98, 69.93, 69.26, 68.92, 68.79,
84.38, 83.28, 83.11, 76.72, 69.33, 69.30, 69.24, 69.20, 69.13, 68.10, 67.58, 67.52, 67.40, 63.12, 62.51, 61.56, 32.19, 32.14.
68.13, 66.80, 66.64, 61.89, 60.97, 31.85, 31.11. HRMS calcd for HRMS calcd for C32H26O5S54Fe (M+), 576.08971, found,
C28H24O5S54Fe (M+), 526.07406, found, 526.07351.
576.08916.
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Synthesis of derivatives 10a–10j
129.52, 129.37, 125.24, 120.87, 116.46, 115.87, 85.25,
83.69, 69.53, 69.23, 67.31, 61.83, 60.62, 32.64, 32.64, 31.43.
General procedure for the Mitsunobu reaction. To a solution HRMS calcd for C36H32O6SBr54Fe (M+), 710.02644, found,
of ferrocenyl alcohol 20 (1.2 equiv.) in dry THF, the resulting 710.02600.
compounds of Diels–Alder reaction 19 (1 equiv.) and triphenyl-
5-(4-HYDROXYPHENYL)-6-(4-(FERROCENY-METHOXY)-PHENYL)-7-OXABICYCLO-
phosphine (1.5 equiv.) in dry THF were added. Then a solution [2.2.1]HEPT-5-ENE-2-SULFONIC ACID 1-NAPHTHYL ESTER (10E). Mp =
of DIAD (1.5 equiv.) in dry THF was added dropwise at 0 °C. 147–150 °C. 1H NMR (400 MHz, Acetone-d6) δ 8.23–8.17 (m,
The reaction was stirred at room temperature for 24–48 h. The 1H), 7.99 (d, J = 9.4 Hz, 1H), 7.90 (s, 1H), 7.63–7.57 (m, 3H),
solvent was evaporated under vacuum, and the residue was 7.52 (dt, J = 15.4, 4.8 Hz, 2H), 7.35–7.30 (m, 2H), 7.25 (dd, J =
purified by chromatography to give 10 as red viscous oil or 8.8, 2.4 Hz, 2H), 6.97 (dd, J = 8.8, 3.1 Hz, 2H), 6.82 (d, J =
solids, yield 28–40%.
5-(4-HYDROXYPHENYL)-6-(4-(FERROCENY-METHOXY)-PHENYL)-7-OXABICYCLO- 4.90 (d, J = 1.3 Hz, 2H), 4.38 (d, J = 1.2 Hz, 2H), 4.22 (d, J =
[2.2.1]HEPT-5-ENE-2-SULFONIC ACID PHENYL ESTER (10A). Mp 8.3 Hz, 7H), 4.07–4.02 (m, 1H), 2.59 (dt, J = 12.1, 4.4 Hz, 1H),
8.6 Hz, 2H), 5.79 (d, J = 5.7 Hz, 1H), 5.51 (t, J = 3.4 Hz, 1H),
=
152–154 °C. 1H NMR (400 MHz, Acetone-d6) δ 7.42–7.22 (m, 2.37 (ddd, J = 12.1, 8.4, 1.9 Hz, 1H). 13C NMR (101 MHz,
10H), 7.01–6.79 (m, 4H), 5.67 (s, 1H), 5.45 (d, J = 3.8 Hz, 1H), Acetone-d6) δ 159.80, 157.29, 146.32, 142.95, 135.86, 129.96,
4.90 (d, J = 3.6 Hz, 2H), 4.40 (s, 2H), 4.24 (s, 7H), 3.79 (dd, J = 129.78, 129.64, 129.48, 128.82, 128.26, 127.89, 126.40, 122.68,
8.1, 4.3 Hz, 1H), 2.43 (dt, J = 12.0, 4.2 Hz, 1H), 2.28 (dd, J = 119.12, 116.49, 115.85, 115.72, 85.40, 85.38, 83.79, 83.79,
12.1, 8.5 Hz, 1H). 13C NMR (101 MHz, Acetone-d6) δ 159.85, 70.43, 69.52, 69.23, 67.29, 62.56, 31.74, 31.67. HRMS calcd for
157.26, 150.57, 142.78, 130.82, 130.13, 129.93, 129.54, 129.36, C39H32O6S54Fe (M+), 682.13157, found, 682.13102.
127.98, 123.14, 116.45, 116.36, 115.87, 115.69, 85.25, 83.69,
5-(4-HYDROXYPHENYL)-6-(4-(FERROCENY-ETHOXY)-PHENYL)-7-OXABICYCLO-
69.58, 69.19, 67.29, 61.62, 32.65, 31.48. HRMS calcd for [2.2.1]HEPT-5-ENE-2-SULFONIC ACID PHENYL ESTER (10F). Mp
1
=
C35H30O6S54Fe (M+), 632.11592, found, 632.11547.
143–145 °C. H NMR (400 MHz, CDCl3) δ 7. 35–7.23 (m, 8H),
5-(4-HYDROXYPHENYL)-6-(4-(FERROCENY-METHOXY)-PHENYL)-7-OXABICYCLO- 6.80 (dd, J = 8.6, 4.0 Hz, 4H), 5.70 (d, J = 3.2 Hz, 1H), 5.40 (s,
[2.2.1]HEPT-5-ENE-2-SULFONIC ACID 2-CHLOROPHENYL ESTER (10B). Mp = 1H), 4.99–4.91 (m, 2H), 4.11 (d, J = 13.8 Hz, 9H), 3.60 (dd, J =
142–144 °C. 1H NMR (400 MHz, Acetone-d6) δ 7.58–7.22 8.4, 4.6 Hz, 1H), 2.82 (t, J = 6.8 Hz, 2H), 2.57 (d, J = 12.2 Hz,
(m, 8H), 6.96 (dd, J = 8.4, 6.4 Hz, 2H), 6.86–6.79 (m, 2H), 5.74 1H), 2.16 (dd, J = 12.1, 8.5 Hz, 1H). 13C NMR (101 MHz,
(d, J = 1.1 Hz, 1H), 5.48 (d, J = 4.4 Hz, 1H), 4.89 (s, 2H), 4.38 (s, Acetone-d6) δ 159.91, 159.83, 158.46, 150.55, 142.78, 137.67,
2H), 4.22 (d, J = 9.0 Hz, 7H), 3.99–3.93 (m, 1H), 2.53 (d, J = 130.82, 130.30, 130.10, 130.00, 129.95, 129.53, 129.42, 129.39,
12.2 Hz, 1H), 2.36 (d, J = 8.5 Hz, 1H). 13C NMR (101 MHz, 128.00, 125.25, 124.83, 124.06, 123.14, 116.66, 116.47, 115.71,
Acetone-d6) δ 159.84, 146.24, 131.77, 130.03, 129.84, 129.64, 115.63, 115.52, 115.44, 85.28, 83.69, 69.28, 64.14, 64.10, 61.66,
129.46, 129.36, 129.15, 127.79, 125.01, 116.49, 115.85, 115.72, 61.56, 32.66, 31.48, 31.43. HRMS calcd for C32H32O6S54Fe (M+),
85.31, 83.71, 70.45, 69.54, 69.22, 67.30, 63.12, 60.61, 32.65, 646.13157, found, 646.13103.
31.54, 23.35, 22.29, 14.53, 14.40. HRMS calcd for
C35H29O6SCl54Fe (M+), 666.07695, found, 666.07640.
5-(4-HYDROXYPHENYL)-6-(4-(FERROCENY-ETHOXY)-PHENYL)-7-OXABICYCLO-
[2.2.1]HEPT-5-ENE-2-SULFONIC ACID 2-CHLOROPHENYL ESTER (10G). Vis-
1
5-(4-HYDROXYPHENYL)-6-(4-(FERROCENY-METHOXY)-PHENYL)-7-OXABICYCLO- cous oil. H NMR (400 MHz, Acetone-d6) δ 7.58–7.48 (m, 2H),
[2.2.1]HEPT-5-ENE-2-SULFONIC ACID 3-CHLOROPHENYL ESTER (10C). Vis- 7.42–7.31 (m, 4H), 7.25 (dd, J = 8.4, 4.2 Hz, 2H), 6.94 (t, J =
1
cous oil. H NMR (400 MHz, Acetone-d6) δ 7.42–7.23 (m, 8H), 7.9 Hz, 2H), 6.82 (dd, J = 8.3, 6.3 Hz, 2H), 5.73 (s, 1H), 5.48 (d,
7.00–6.93 (m, 2H), 6.86–6.78 (m, 2H), 5.70 (d, J = 2.7 Hz, 1H), J = 3.4 Hz, 1H), 4.21 (s, 2H), 4.14 (s, 5H), 4.08 (s, 2H), 3.97 (dd,
5.49–5.45 (m, 1H), 4.90 (d, J = 5.6 Hz, 2H), 4.37 (d, J = 1.5 Hz, J = 8.5, 4.4 Hz, 1H), 2.82 (t, J = 6.7 Hz, 2H), 2.58–2.48 (m, 1H),
2H), 4.21 (dd, J = 8.1, 1.6 Hz, 7H), 3.87 (dd, J = 8.3, 4.4 Hz, 1H), 2.36 (d, J = 8.2 Hz, 1H). 13C NMR (101 MHz, Acetone-d6)
2.47–2.41 (m, 1H), 2.31 (ddd, J = 12.1, 8.3, 1.6 Hz, 1H). δ 159.85, 146.36, 143.04, 137.58, 131.76, 130.04, 129.92,
13C NMR (101 MHz, Acetone-d6) δ 159.91, 158.50, 157.26, 129.64, 129.52, 129.34, 129.13, 127.78, 125.38, 125.00, 124.77,
150.91, 142.93, 138.54, 135.34, 132.03, 130.05, 129.88, 129.57, 116.57, 116.43, 115.69, 115.54, 85.89, 85.32, 83.69, 69.35,
129.40, 128.21, 126.05, 125.23, 123.50, 121.93, 116.66, 116.45, 69.29, 68.12, 63.12, 60.58, 32.64, 31.53. HRMS calcd for
115.88, 115.71, 85.21, 83.73, 70.35, 69.43, 69.34, 69.19, 67.30, C36H31O6SCl54Fe (M+), 680.09260, found, 680.09215.
62.05, 32.65, 31.44. HRMS calcd for C35H29O6SCl54Fe (M+),
666.07695, found, 666.07640.
5-(4-HYDROXYPHENYL)-6-(4-(FERROCENY-ETHOXY)-PHENYL)-7-OXABICYCLO-
[2.2.1]HEPT-5-ENE-2-SULFONIC ACID 3-CHLOROPHENYL ESTER (10H). Vis-
1
5-(4-HYDROXYPHENYL)-6-(4-(FERROCENY-METHOXY)-PHENYL)-7-OXABICYCLO- cous oil. H NMR (400 MHz, Acetone-d6) δ 7.45–7.39 (m, 2H),
[2.2.1]HEPT-5-ENE-2-SULFONIC ACID 4-BROMOPHENYL ESTER (10D). Vis- 7.30 (dd, J = 18.8, 8.5 Hz, 4H), 7.23 (d, J = 8.6 Hz, 2H), 6.95 (d, J
1
cous oil. H NMR (400 MHz, Acetone-d6) δ 7.59–7.56 (m, 2H), = 8.7 Hz, 2H), 6.80 (d, J = 8.6 Hz, 2H), 5.70 (s, 1H), 5.46 (d, J =
7.33–7.21 (m, 7H), 6.98 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 8.6 Hz, 4.0 Hz, 1H), 4.22 (s, 2H), 4.19–4.14 (m, 7H), 4.08 (s, 2H), 3.88
2H), 5.67 (s, 1H), 5.62 (s, 1H), 5.46 (d, J = 3.8 Hz, 1H), 4.91 (dd, J = 8.4, 4.5 Hz, 1H), 2.82 (d, J = 6.4 Hz, 2H), 2.46–2.41
(s, 2H), 4.40 (d, J = 0.5 Hz, 2H), 4.23 (d, J = 7.4 Hz, 7H), 3.83 (m, 1H), 2.34–2.28 (m, 1H). 13C NMR (101 MHz, Acetone-d6)
(dd, J = 8.3, 4.4 Hz, 1H), 2.42 (dt, J = 12.1, 4.4 Hz, 1H), 2.29 δ 160.14, 158.55, 151.05, 135.47, 132.03, 130.02, 129.93,
(dd, J = 12.1, 8.4 Hz, 1H). 13C NMR (101 MHz, Acetone-d6) 129.89, 129.54, 128.21, 125.32, 124.82, 123.97, 123.48, 121.92,
δ 159.85, 158.48, 149.71, 142.97, 137.58, 133.83, 129.94, 116.67, 116.46, 115.71, 115.63, 115.44, 85.21, 83.72, 69.72,
9696 | Org. Biomol. Chem., 2012, 10, 9689–9699
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64.09, 63.37, 62.08, 61.52, 32.65, 31.43. HRMS calcd for 1H NMR (400 MHz, Acetone-d6) δ 8.76 (d, J = 24.5 Hz, 1H), 8.00
C36H31O6SCl54Fe (M+), 680.09260, found, 680.09215.
(d, J = 8.5 Hz, 2H), 7.43 (d, J = 8.5 Hz, 2H), 7.26 (dd, J = 12.9,
5-(4-HYDROXYPHENYL)-6-(4-(FERROCENY-ETHOXY)-PHENYL)-7-OXABICYCLO- 8.5 Hz, 4H), 6.83 (dd, J = 18.4, 8.5 Hz, 4H), 5.70 (s, 1H), 5.47 (d,
[2.2.1]HEPT-5-ENE-2-SULFONIC ACID 4-BROMOPHENYL ESTER (10I). Mp = J = 4.2 Hz, 1H), 4.86 (s, 2H), 4.68 (s, 2H), 4.26 (s, 5H), 3.87 (dd,
156–160 °C. 1H NMR (400 MHz, Acetone-d6) δ 7.57 (d, J = J = 8.3, 4.3 Hz, 1H), 2.45 (s, 1H), 2.37–2.28 (m, 1H). 13C NMR
8.9 Hz, 2H), 7.30 (d, J = 5.7 Hz, 2H), 7.24 (dd, J = 12.9, 8.7 Hz, (101 MHz, Acetone-d6) δ 158.13, 152.50, 139.17, 137.82, 130.88,
4H), 6.95 (d, J = 8.6 Hz, 2H), 6.80 (d, J = 8.5 Hz, 2H), 5.66 130.13, 129.51, 123.02, 116.61, 116.41, 116.32, 85.28, 83.69,
(s, 1H), 5.45 (d, J = 4.1 Hz, 1H), 4.22 (d, J = 1.4 Hz, 2H), 4.14 73.58, 72.17, 72.07, 71.03, 61.96, 55.03, 31.85. HRMS calcd for
(s, 7H), 4.08 (s, 2H), 3.84 (dd, J = 8.4, 4.5 Hz, 1H), 2.82 (d, J = C35H28O7S54Fe (M+), 646.09519, found, 646.09464.
6.6 Hz, 2H), 2.44–2.39 (m, 1H), 2.28 (dd, J = 12.1, 8.4 Hz, 1H).
13C NMR (101 MHz, Acetone-d6) δ 149.75, 133.80, 129.98,
129.54, 125.36, 125.21, 124.79, 120.78, 116.45, 115.71, 85.89,
Biochemical experiments
MATERIALS. Coumestrol (CS), bovine gamma globulin (BGG)
were purchased from Sigma-Aldrich, DMSO, potassium
phosphate were obtained from Guoyao company. ERα/
β-LBD protein were expressed in E. coli BL21, and purified
by GSH resin. GSH resin was purchased from Genscript
Company.
85.27, 83.69, 69.33, 69.28, 68.12, 61.93, 32.63, 31.44. HRMS
calcd for C36H31O6SBr54Fe (M +), 724.04209, found, 724.04154.
5-(4-HYDROXYPHENYL)-6-(4-(FERROCENY-ETHOXY)-PHENYL)-7-OXABICYCLO-
[2.2.1]HEPT-5-ENE-2-SULFONIC ACID 1-NAPHTHYL ESTER (10J). Viscous
1
oil. H NMR (400 MHz, Acetone-d6) δ 8.23–8.17 (m, 1H), 7.98
(d, J = 3.7 Hz, 1H), 7.90 (d, J = 7.9 Hz, 1H), 7.61–7.50 (m, 4H),
7.35–7.23 (m, 4H), 6.93 (dd, J = 8.7, 2.6 Hz, 2H), 6.82 (d, J = 8.4
Hz, 2H), 5.77 (s, 1H), 5.62 (s, 1H), 5.51 (d, J = 3.3 Hz, 1H), 4.24
(s, 2H), 4.16 (s, 7H), 4.10 (s, 2H), 4.08–4.04 (m, 1H), 2.80 (t, J =
6.3 Hz, 2H), 2.62–2.56 (m, 1H), 2.37 (ddd, J = 11.9, 8.4, 3.2 Hz,
1H). 13C NMR (101 MHz, Acetone-d6) δ 159.84, 158.57,
146.32, 142.95, 137.75, 135.86, 129.98, 129.86, 129.65,
129.54, 128.82, 128.25, 127.93, 127.87, 126.39, 125.38,
124.85, 122.67, 119.10, 116.64, 116.48, 116.39, 115.69, 115.55,
85.39, 83.78, 69.54, 69.30, 69.20, 68.32, 62.56, 32.64, 31.66.
HRMS calcd for C40H34O6S54Fe (M+), 696.14722, found,
696.14668.
DETERMINATION OF THE RELATIVE BINDING AFFINITY (RBA) OF THE COM-
POUNDS FOR ERα AND ERβ. Protein (ERα or ERβ) and fluorescence
trace drug (CS) were diluted in 100 mM potassium phosphate
buffer (pH 7.4), containing 100 μg mL−1 BGG, to a concen-
tration of 0.8 μM and 40 nM respectively. All of the tested com-
pounds were prepared as a stock in DMSO at a concentration
of 100 mM and were diluted in potassium phosphate/BGG
buffer to different concentration before use (9 molarities:
2 nM, 6.32 nM, 20 nM, 63.2 nM, 200 nM, 632 nM, 2 μM,
6.32 μM, 20 μM for IC50 determination). Compounds mixed
with ERα or ERβ and CS were added to each well (20 μL
respectively), and the plate was incubated at room temperature
(25 °C) for 2 h. The competing ligand displaced CS from ERs
as the concentration of the compounds increased. The large
receptor–ligand complex tumbles slowly and has a high
polarized fluorescence (PF) value, while free CS molecule
tumbles more rapidly and therefore has a low PF value. So the
measured polarization is the weighted average of the bond and
free CS molecules, which can be directly used to calculate
the percentage of inhibition. The polarization was measured
on SpectraMax M5 using the recommended fluorescent polar-
ization filter set (ex 365 nm, em 440 nm). The polarization
values (in millipolarization45) was plotted against the in-
creasing concentrations of the test compounds. IC50 values
were calculated according the following equation using Origin
software.
Synthesis of derivatives of 11a and 11b
25a and 25b were prepared in three steps: first, the ferrocene
reacted with 4-methoxybenzoyl chloride 22a and 4-methoxy-
benzyl alcohol 22b with different acid catalysts (AlCl3 for 22a,
CF3CO2H for 2b) to afford the corresponding ferrocenyl substi-
tuted products 23a and 23b,36,37 which were demethylated
with BBr3 to produce the phenolic products 24a and 24b;
then 24a and 24b reacted with 2-chloroethanesulfonyl
chloride in the presence of Et3N gave the dienophiles 25a
and 25b. Finally, 25a and 25b (1.0 equiv.) reacted with 3,4-bis-
(4-hydroxyphenyl)-furan (1.0 equiv.) via
a
Diels–Alder
reaction to afford the target compounds 11a and 11b in high
yields.
5,6-BIS-(4-HYDROXYPHENYL)-7-OXABICYCLO[2.2.1]HEPT-5-ENE-2-SULFONIC
ACID (4-FERROCENY-BENZYL) ESTER (11A). Mp = 76–79 °C. 1H NMR
(400 MHz, Acetone-d6) δ 8.77 (d, J = 21.9 Hz, 1H), 7.26–7.11
Y ¼ mP100% þ ðmP0% ꢀ mP100%Þ=ð1 þ 10ððLogIC50ꢀXÞꢁHillslopeÞÞ
(m, 8H), 6.81 (dd, J = 13.0, 8.4 Hz, 4H), 5.61 (s, 2H), 5.42 (d, J = where: Y = mP, X = Log [inhibitor], mP100% = 100% inhi-
4.0 Hz, 1H), 4.20 (d, J = 19.8 Hz, 9H), 3.72 (dd, J = 8.2, 4.3 Hz, bition, and mP0% = 0% inhibition.
1H), 2.40 (dt, J = 11.6, 4.1 Hz, 1H), 2.25 (dd, J = 11.9, 8.4 Hz,
Relative binding affinity (RBA) values expressed as IC50
1H). 13C NMR (101 MHz, Acetone-d6) δ 158.36, 148.65, 142.22, estradiol/IC50 compound × 100 the range or standard devi-
141.98, 137.92, 130.58, 130.10, 129.50, 124.99, 124.22, 122.83, ation (RBA, estradiol = 100%).
116.62, 116.53, 116.41, 116.33, 85.24, 83.65, 70.28, 61.38,
THE EFFECTS OF THE COMPOUNDS ON THE PROLIFERATION OF THE
55.04, 35.83, 31.46. HRMS calcd for C35H28O6S54Fe (M+), HORMONE-DEPENDENT AND THE HORMONE-INDEPENDENT CANCER CELLS.
632.11592, found, 632.11538.
Cells were maintained in monolayer in DMEM with phenol red
5,6-BIS-(4-HYDROXYPHENYL)-7-OXABICYCLO[2.2.1]HEPT-5-ENE-2-SULFONIC (Gibco) supplemented with 9% fetal calf serum (Gibco) and
ACID (4-FERROCENY METHANONE-PHENYL) ESTER (11B). Viscous oil. 2 mM glutamine (Gibco) at 37 °C in a 5% CO2 air humidified
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incubator. For proliferation assays, cells were plated and incu-
bated in 1 mL of DMEM medium without phenol red sup-
plemented with 10% decomplemented and hormone-depleted
fetal calf serum and glutamine. The following day (D0) 1 mL of
the same medium containing the compounds to be tested was
added to the plates (final volumes of DMSO: 0.1%; 8 wells
for each conditions, one plate per day, 4 molarities: 0.1 μM,
0.5 μM, 1 μM and 10 μM for IC50 determination). After 3 days
(D3) the incubation medium was removed and fresh medium
containing the compounds was added. After 5 days (D5) the
total protein contents of the plate was analyzed by methylene
blue staining as follows. Cell monolayers were fixed for 1 h in
methanol, stained for 1 h with methylene blue (1 mg mL−1) in
PBS, then washed thoroughly with water. 1 mL of HCl (0.1 M)
was then added and the absorbance of each well was measured
at 620 nm with a Biorad spectrophotometer. The results are
expressed as the percentage of proteins versus the control. Four
different concentrations (1 × 10−7 M, 5 × 10−7 M, 1 × 10−6 M,
1 × 10−5 M) were used to determine graphically IC50 values
(concentration causing 50% inhibition of cell proliferation).
The indicated value was the mean of two independent
experiments.
Notes and references
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