Organometallic cyclic polyphenols derived from 1,2-(α-keto tri or tetra methylene) ferrocene show strong antiproliferative activity on hormone-independent breast cancer cells

Article abstract of DOI:10.1039/c0dt00169d

We have previously shown that achiral ferrocenyl diphenol butene derivatives are strong antitumor agents against both hormone-dependent and -independent breast cancer cell lines. We report now examples of a new series of two planar chiral diphenol derivatives, namely 1,2-[1-[1,1-bis(4-hydroxyphenyl) methylidene]trimethylene] ferrocene (4), and 1,2-[1-[1,1-bis(4-hydroxyphenyl) methylidene]tetramethylene]ferrocene (5). They were prepared under racemic form from a McMurry coupling reaction with 30% and 16% yields, respectively. Compound 5 gave crystals suitable for X-ray structural analysis. Compounds 4 and 5 were tested for ERα and ERβ affinity, lipophilicity, and proliferative/antiproliferative effects against the hormone-dependent breast cancer cell line MCF-7, and the hormone-independent breast cancer cell line MDA-MB-231. Both compounds exhibit better affinity for ERβ (16.4 ± 0.1, and 7.0 ± 0.4, respectively) than for ERα (6.4 ± 0.2, and 6.6 ± 0.2). The test on hormone-independent breast cancer cell line MDA-MB-231 showed that 4 with a 5-membered ring gives an IC₅₀ value of 2.7 μM while with 5 in which the ring has 6 carbons, the value is reduced to IC₅₀ = 1.23 μM.

Full text of DOI:10.1039/c0dt00169d

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/dalton | Dalton Transactions
Organometallic cyclic polyphenols derived from 1,2-(a-keto tri or tetra
methylene) ferrocene show strong antiproliferative activity on
hormone-independent breast cancer cells†
Damian Plazuk,a,b Siden Top,a Anne Vessie`res,a Marie-Aude Plamont,a Michel Huche´,a Janusz Zakrzewski,b
Anna Makal,c Krzysztof Woz´niakc and Ge´rard Jaouen*a
Received 18th March 2010, Accepted 20th May 2010
First published as an Advance Article on the web 8th July 2010
DOI: 10.1039/c0dt00169d
We have previously shown that achiral ferrocenyl diphenol butene derivatives are strong
antitumor agents against both hormone-dependent and -independent breast cancer cell lines.
We report now examples of a new series of two planar chiral diphenol derivatives, namely
1,2-[1-[1,1-bis(4-hydroxyphenyl)methylidene]trimethylene] ferrocene (4), and
1,2-[1-[1,1-bis(4-hydroxyphenyl)methylidene]tetramethylene]ferrocene (5). They were prepared under
racemic form from a McMurry coupling reaction with 30% and 16% yields, respectively. Compound 5
gave crystals suitable for X-ray structural analysis. Compounds 4 and 5 were tested for ERa and ERb
affinity, lipophilicity, and proliferative/antiproliferative effects against the hormone-dependent breast
cancer cell line MCF-7, and the hormone-independent breast cancer cell line MDA-MB-231. Both
compounds exhibit better affinity for ERb (16.4 0.1, and 7.0 0.4, respectively) than for ERa (6.4
0.2, and 6.6 0.2). The test on hormone-independent breast cancer cell line MDA-MB-231 showed that
4 with a 5-membered ring gives an IC50 value of 2.7 mM while with 5 in which the ring has 6 carbons,
the value is reduced to IC50 = 1.23 mM.
In addition, it is known that a number of molecules, e.g.
genistein, apigenin, isoflavone, cyanidin, theaflavin, enterolactane,
curcumin, resveratrol 1 etc., all coming under the general heading
of dietary polyphenols, provide an important source of antioxi-
Introduction
One of the most promising developments in bioorganometallic
chemistry1 is the field of organometallic medicinal chemistry.2,3
dants in the human diet.15–26 These polyphenols exhibit a variety
of biological activities, ranging from anti-aging and anticancer to
lowering of blood cholesterol levels and increasing bone strength.
Dietary polyphenols are derived from plants and are consumed in
the form of fruit, vegetables, spices, and herbs.
Recently, we have combined the two above notions and
described a new approach redirecting the natural protective
properties of some stilbene-like polyphenols, enhanced via a
ferrocene redox antenna.9–10
One example is ferroquine, an antimalarial derived from chloro-
quine but effective on chloroquine-resistant malaria strains, cur-
rently in phase II clinical trials at Sanofi-Aventis.4 In this context,
ferrocene plays an important role in providing access to new
structures of potential medical importance.5 Several reasons may
account for this. One is based on the similarities in aromaticity,
lipophilicity, and morphology between this metallocene and a
simple organic aryl radical, taking into account of course the
obvious discrepancies in the analogy such as the volume it
occupies. The concept of bioisosterism in particular has recently
been discussed in this context.6 Another reason resides in the
unusual redox characteristics of this metallocene. The relevance
of this property was shown for the ferrocifens,7–11 ferrocene
derivatives of the standard treatment for hormone-dependent
breast cancers, tamoxifen. The ferrocifens may be adaptable to
an extended range of applications, as we recently showed for
melanoma,12 glioma,13 and mesothelial.14
aChimie ParisTech (Ecole Nationale Supe´rieure de Chimie de Paris),
Laboratoire Charles Friedel, UMR CNRS 7223, 11 rue P. et M. Curie,
75231, Paris cedex 05, France. E-mail: gerard-jaouen@chimie-paristech.fr;
Fax: 33-143260061; Tel: 33-143269555
In particular we have shown that in favorable cases, the bio-
logical properties of organic polyphenols can undergo substantial
modification by substitution of a ferrocenyl entity in place of an
aryl group of the skeleton. For example, 2 behaves as an estrogen
with no cytotoxicity on MCF-7 hormone-dependent breast cancer
cells, producing a proliferative effect, while 3, which bears a
ferrocenyl entity, produces a powerful antiproliferative effect
on both MCF-7 cells and MDA-MB-231 hormone-independent
bDepartment of Organic Chemistry, Faculty of Chemistry, University of
Ło´dz´, Tamka 12, 90-403, Ło´dz´, Poland
cStructural Research Laboratory, Faculty of Chemistry, University of
˙
Warsaw, Zwirki i Wigury Street 101, 02-089, Warszawa, Poland
† CCDC reference numbers 757145. For crystallographic data in CIF or
other electronic format see DOI: 10.1039/c0dt00169d
7444 | Dalton Trans., 2010, 39, 7444–7450
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
breast cancer cells.9 In the latter case, IC50 values are found to be
on the order of 0.6 mM. It has been observed that the presence of
a ferrocene antenna with a strong, reversible oxidation potential
favors the generation of quinone methide with cancer cells10–27
and leads to cell death. We note that resveratrol 1 has an IC50
of only 20 mM on MDA-MB-231.26 This makes a non-genomic
route for this organic product inoperable at the therapeutic level.
Modification via organometallics changes the scale and ipso facto
opens up new perspectives since we pass from chemoprevention
to potential therapy.
The achiral compound 3 proves to be one of the best examples
of this surprising, scale-changing effect on the cytotoxic activity
of organometallic polyphenols against cancer cells. Many other
entities have been shown to be inactive,28,11 but anilines are
also capable of replacing phenols in terms of efficacity.29,30 We
have looked for equally effective new structures, and present
here our first results with chiral rigid and constrained ring
structures in which a 5- or 6-carbon ring is attached to one
of the cyclopentadienyl rings of ferrocene as in 4 and 5; only
the Sp configuration is shown.31 This series of 1,2-[1-[1,1-bis(4-
hydroxyphenyl)methylidene]tri or tetramethylene] ferrocene has
never been explored from this perspective.
Fig. 1 ORTEP representation of 5. Thermal ellipsoids are drawn
at the 50% probability level. The hydrogen atom labels were omit-
˚
ted for the sake of clarity. Selected bond lengths [A] and va-
lence angles [◦]: C(6)–C(14), 1.4791 (18); C(7)–C(11), 1.4985 (19);
C(14)–C(15), 1.3517 (17); C(15)–C(16), 1.4972 (17); C(15)–C(22), 1.5008
(17); O(1)–C(19), 1.3799 (15); O(2)–C(25), 1.3700 (15); C(15)–C(14)–C(6),
124.68 (11); C(15)–C(14)–C(13), 121.45 (12); C(6)–C(14)–C(13), 113.83
(11); C(14)–C(15)–C(16), 123.18 (11); C(14)–C(15)–C(22), 124.37
(11); C(16)–C(15)–C(22), 112.44 (10); O(1)–C(19)–C(18), 117.61 (12);
O(2)–C(25)–C(26), 122.95 (12).
Results and discussion
Compounds 4 and 5 were prepared by a McMurry reaction which
we had already used for 3 and related compounds32–34 including
an efficient achiral cyclic series called ferrocenophanes.35 The
reaction induces coupling between the 4,4¢-dihydrobenzophenone
and the 1,2(a-ketotrimethylene) ferrocene or the racemic 1,2(a-
ketotetramethylene)ferrocene (Scheme 1). They are obtained in
yields of 30% for 4 and 16% for 5.
Fig. 2 The closed network of strong hydrogen bonds – indicated as dashed
lines – formed in the structure of 5. View of the structure along the 100
crystallographic direction.
The ferrocene moiety possesses geometrical features common to
the iron sandwich complexes. The two Cp rings are nearly eclipsed,
with a distortion of -5.97(12)◦. In the case of the C5H3 ring,
the carbon C(11) lies in the plane of the substituted ring and
˚
C(13) is elevated above that plane by only 0.14 A, while C(12)
is inclined below the plane. The following C(14)–C(15) bond is
˚
clearly double (1.3517(17) A), as indicated by a very short C–C
distance in contrast with the surrounding bond lengths, suggesting
single bond character. The double bond lies nearly exactly in the
˚
plane of the adjacent Cp. The C(15) carbon is located only 0.05 A
below that plane.
Scheme 1 Preparation of compounds 4 and 5.
The two phenol moieties are inclined at different angles towards
the C5H3 ring plane. The O(1) oxygen, connected with the ring
containing carbons C(16) to C(21), labelled Ph-1, participates in
two hydrogen bonds, both as donor and as acceptor, while the
O(2), connected with the second phenyl ring, Ph-2, acts only as a
donor in a single hydrogen bond. Table 1 contains details of the H-
bond geometrical parameters. The formation of short H-bonds by
both hydroxyl groups yields a closed net of interactions, encircling
a channel along the 100 direction in which the acetone molecules
are located and thus constituting the main packing motif. The
X-Ray structure of 5†
Molecule 5 gave crystals suitable for X-ray structural analysis. The
ORTEP representation of the crystallographic asymmetric unit is
presented in Fig. 1. One molecule of 5 together with one solvent
acetone molecule constitutes the asymmetric unit in the unit cell.
Strong hydrogen bonds connect the bisphenol with the solvent, as
well as with the symmetry-related molecules in the crystal (Fig. 2).
This journal is
The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 7444–7450 | 7445
©

View Article Online
◦
˚
Table 3 Enthalpy variation values for the bioligand docked into ERa
and ERb
Table 1 Hydrogen bonds and angles for structure 5 [A and
]
D–H ◊ ◊ ◊ A
d(D–H) d(H ◊ ◊ ◊ A) d(D ◊ ◊ ◊ A) <(DHA) <Ph–DH
DrH◦/kcal mol-1
O(1)–
H(1O) ◊ ◊ ◊ O(3)#1
O(2)–
0.75(2) 1.94(2)
0.79(2) 2.02(2)
2.6928(16) 180(3)
2.8005(15) 169(2)
-9(2)
Compound and configuration
ERa
ERb
-17(3)
(Sp)-4
(Rp)-4
(Sp)-5
(Rp)-5
-23.7
-16.5
-26.8
-7.3
-26.8
-15.2
-16.7
-13
H(2O) ◊ ◊ ◊ O(1)#1
Table 2 Relative binding affinity values (RBA); log Po/w and effect on
the growth of cancer cells of compounds 3, 4, 5
RBA (%)
Log Po/w
IC50/mMa
(for planar) form as defined in ref. 31 is always better adapted to
the cavity.
Compounds
ERa
ERb
MDA-MB-231
Molecular docking experiments were performed based on
the crystal structure of human ERa crystallized with di-
ethylstilbestrol (DES) (pdb erd.ent)44 and of ERb crystal-
lized with 5,11-cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol
(THC) (pdb 12j.ent),45 retaining only the amino acids that
constitute the wall of the cavity. The DES or THC molecules
were removed from the cavity and replaced successively with
different bioligands. Energy minimization was then carried out
using Merck molecular force field (MMFF). All the heavy atoms
of the amino acids of the cavity wall were then immobilized and
the side chain of His524 for ERa and His475 for ERb were
liberated. This allowed the ideal positions of the bioligands to be
determined. Quantum mechanical semi-empirical PM3 methods
were then used to determine the affinity of the bioligands for the
cavity. This requires calculation of the energies of the bioligand-
cavity group, of the cavity itself, and of the ligand, the latter
two in the conformations they had in the molecular assemblies
to give the DrH◦ enthalpy variations of the reactions: bioligand
+ cavity → molecular assembly (Table 3). For all compounds,
binding with both isoforms of the ER is thermodynamically
favored, as evidenced by the negative enthalpy of formation for the
ligand-receptor complex. In each case there is a marked difference
between the values of the two optical isomers of a compound
(Table 3).
The classification order for the formation of the ligand-receptor
complex is for ERa: (Sp)-5 > (Sp)-4 > (Rp)-4 > (Rp)-5. There is a
pronounced selectivity between (Sp)-5 and (Rp)-5 (19.5 kcal mol-1)
for ERa. The b cavity is less selective for the enantiomers (Sp)-5,
and (Rp)-5 than the a cavity.
Fig. 3 shows (Sp)-5 in the cavity ERa. The OH group forms a
hydrogen bond with the residues Glu353 and Arg394. On the other
side of the molecule the iron atom of the ferrocene interacts with
His524. This triple anchorage demonstrates the good association
of the isomer with the cavity. A similar design can be obtained for
(Sp)-4.
3
4
5
9.6 0.9
6.4 0.2
6.6 0.8
16.3 1.5
16.4 0.1
7.0 0.4
5.95
4.93
5.21
0.64 0.06
2.7 0.02
1.23 0.08
a Measured after 5 days of culture (mean of two independent experiments).
Non-treated cells are used as the control.
presence of the hydrogen bonds and the solvent in structure 5
proves that the moiety is quite capable of producing intermolecular
interactions, possibly also in the protein environment.
These new compounds were tested for ERa and ERb affinity,
lipophilicity, and proliferative/antiproliferative effects against
the hormone-dependent breast cancer cell line MCF-7, and
the hormone-independent breast cancer cell line MDA-MB-231
(Table 2). Also shown are the values obtained for the non-cyclic
compound 3, for comparison. For these studies, we followed the
protocol described earlier.32
As indicated in Table 2, cyclic compounds 4 and 5 recognize
the estrogen receptors ERa and ERb, present in varying degrees
in hormone-dependent MCF-7 cells. We observed two distinct
effects with these molecules: an estrogenic one at low concen-
tration (around 10 nM) and the manifestation of a cytotoxic
behavior at higher concentration (10-7 M). The data (prolifera-
tive/antiproliferative with respect to the control taken as 100%)
obtained at 1 mM, 87.6% for 4, 145% for 5 (72.4% for 4 at 10-5
M) are the result of this competition. According to these results,
it seems that 5 is more estrogenic than 4. This difference could
not be predicted from the RBA values which encompass several
parameters, but are in agreement with the modeling studies of the
ER binding site.
Compounds 4 and 5 are racemic, with a planar met-
allocene chirality. The use of pure enantiomers of 1,2-
(a-ketotrimethylene)ferrocene or 1,2-(a-ketotetramethylene)-
ferrocene would allow compounds 4 and 5 to be obtained
as pure enantiomers, but we believe that the results obtained
with the racemic molecules are sufficiently clear for this study,
considering that the synthesis of the two optically pure ketones
would require asymmetric synthetic reactions and would be very
time-consuming.36–38 In the racemic form, both 4 and 5 show better
recognition for the estrogen receptor b (ERb), discovered more
recently39 and found in lower quantities in the breast,40,41 than
for receptor a (ERa), the form first identified, most abundant42
(Table 2).
It is of interest to compare the structure of the enantiomer (Sp)-4
with that of the conformer of 3 providing the best association with
ERa.46 The virtual splitting of the corresponding C–C bond of
the cyclopentene moiety in the (Sp)-4 enantiomer would generate
exactly conformer-1 in 3 (Scheme 2). The same process applied to
(Rp)-4 would give rise to conformer-2 in 3. In addition the rotation
of the methyl group in the ethyl substituent to a greater distance
from the cyclopentadienyl group generates two other conformers,
namely (conformer-3)-3 and (conformer-4)-3. We have calculated
the association energies for these 4 conformers of 3. The values
are listed in Table 4.
Molecular modelling studies, carried out using the programs
Trident and Titan,43 show that the two enantiomers, whether for 4
or 5, do not adapt in the same way to the receptor pocket. The Sp
7446 | Dalton Trans., 2010, 39, 7444–7450
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
Table 4 Enthalpy variation values for 3 docked into ERa and ERb
Compound and configuration
DrH◦ (kcal mol-1)
ERa
-20.6
-14.7
-13.4
-8.7
ERb
-33.6
-20.0
-7.6
(Conformer-1)-3a
(Conformer-2)-3
(Conformer-3)-3
(Conformer-4)-3
-13.6
a Ref. 46.
Table 5 Crystal data and structure refinement for 5
Identification code
Empirical formula
Formula weight
Temperature/K
Wavelength/A
Crystal system
Fe2989
C30H30FeO3
494.39
100(2)
˚
0.71073
Triclinic
¯
P1
Space group
˚
a/A
9.0572(12)
10.5343(14)
14.155(2)
111.543(3)
103.732(3)
95.834(2)
1193.3(3)
2
˚
b/A
˚
c/A
a/◦
b/◦
g /◦
Fig. 3 Molecular modelling representation of (Sp)-5 in the ligand binding
domain of the hERa.
3
˚
Volume/A
Z
Calculated density/Mg m-3
Absorption coefficient/mm-1
F(000)
1.376
0.662
520
Crystal size/mm
0.21 ¥ 0.16 ¥ 0.08
1.62 to 29.13
-12 ≤ h ≤ 12
-14 ≤ k ≤ 8
-19 ≤ l ≤ 19
Theta range for data collection/◦
Limiting indices
Reflections collected/unique
Completeness to theta = 29.13
Absorption correction
Max. and min. transmission
Refinement method
28 164/6403 [R(in) = 0.0320]
99.5%
Semi-empirical from equivalents
0.9456 and 0.8419
Full-matrix least-squares on F2
6403/0/317
Data/restraints/parameters
Goodness-of-fit on F2
1.042
Final R indices [I > 2s(I)]
R1 = 0.0304
wR2 = 0.0752
R indices (all data)
R1 = 0.0363
wR2 = 0.0788
-3
˚
Largest diff. peak and hole/e A
0.456 and -0.315
receptor is not unprecedented as we have previously demonstrated
it in the steroid series.31
In order to create a compound more active against MCF-7 cells,
one could envision eliminating the estrogenic effect by replacing
one hydroxyl group with the lateral chain O(CH2)xN(CH3)2 (x = 3,
5) as we have previously shown for the hydroxyferrocifen series.47,48
The combination of the two contradictory effects seen on
MCF-7 did not produce an effect on the non-hormone-dependent
breast cancer cell line MDA-MB-231 (Table 2). The difference in
antiproliferative effect between 4 and 5 is clear, being in the order
of two-fold. Compound 4 with a 5-membered ring gives an IC50
value of 2.7 mM while with 5 in which the ring has 6 carbons, the
value is reduced to IC50 = 1.23 mM. It is quite possible that the
greater flexibility of the ring with 6 carbons compared to 5 may
need to be taken into account to explain this behavior.
Scheme 2 Relationship between the enantiomers of 4 with the conformers
of 3 after a virtual splitting of C–C bond in 4.
These data show clearly that (conformer-1)-3 is the most
favorable for a good association with the ERa and ERb binding
sites. This result corresponds to the good association obtained for
the cyclic enantiomer (Sp)-4.
Ferrocene tetralone, in optically active form, permits an addi-
tional approach to improve the results further still.37 Selectivity
in recognition of a metallocene planar chirality in a biological
A mechanism that we have suggested to explain the cytotoxic
effect of 3 invokes the reversible oxidation of the ferrocene antenna
(Fe2+) by ROS (reactive oxygen species) present in certain cancer
This journal is
The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 7444–7450 | 7447
©

View Article Online
cells10,11 and which are augmented by 3. As a result of this
intramolecular activation of the phenols by the radical ferricinium
cation (Fe3+), an electrophilic quinone methide is produced that is
responsible for the cancer cell death.27 To establish the extent to
which this mechanism can be retained here with the ring structures
4 and 5, we have used the Hartree–Fock method to calculate
the energy differences of the frontier orbitals between these two
compounds. The results found are 1.1 eV for 4 (FeIII), and 1.3 for
5 (FeIII), thus proving their high activity.
dH(300 MHz, acetone-d6) 8.27 (1H, s, OH); 8.24 (1H, s, OH), 7.15
(d, J = 8.7 Hz, 2H, Ar), 7.07 (2H, d, J = 8.7 Hz, Ar), 6.86 (7H,
d, J = 8.7 Hz, Ar), 6.78 (2H, d, J = 8.7 Hz, Ar), 4.14 (1H, m,
Cp), 4.03 (5H, s, Cp), 3.95 (1H, m, Cp), 3.58 (1H, m, CH2), 3.16
(1H, m, Cp), 2.90 (1H, m, CH2), 2.70 (1H, m, CH2), 2.49 (1H, m,
CH2). dC(75.48 MHz, acetone-d6) 156.9, 156.5, 137.7, 135.6, 131.9,
131.6, 130.8, 130.4, 115.7, 115.5, 98.1, 91.6, 71.4, 69.4, 62.9, 62.7,
37.1, 25.5. m/z (EI) 422.0953 (M+ C26H22FeO2 requires 422.0969).
1,2-[1-[1,1-Bis(4-hydroxyphenyl)methylidene]
tetramethylene]ferrocene (5)
Conclusion
Compound 5 is a good candidate for further studies with a
view to develop, especially as the optical form is accessible. It
must however be remembered that the bio-availability of the
polyphenols is generally low. A solution to this problem is a
challenge currently being addressed.16 We have already found an
answer to this question for 3, for which we have reported two
effective formulations in the form of lipoid nanocapsules.13,49,50
The value of the logPo/w distribution coefficient, an indicator
of lipophilicity, is 5.21 for 5 compared to 4.95 for 3. This is
favourable for increasing the load of product in the nanocapsule
and indicates that the same type of approach via nanocapsules
could be envisaged to achieve a slow-release formulation effective
in vivo. This work will be the subject of future studies.
Compound 5 was prepared from the same McMurry cou-
pling reaction procedure. 0.508
ketotetramethylene)ferrocene, 0.428
g
(2 mmol) of 1,2-(1-
(2 mmol) of 4,4¢-
g
dihydroxybenzophenone, 1.13 g (0.66 mL, 6 mmol) of TiCl4,
0.784 g (11.7 mmol) of zinc powder, 0.944 g (0.97 mL, 12.0 mmol)
of anhydrous pyridine, and anhydrous THF (28 mL + 10 mL). The
crude product was purified on a silica gel column using pentane–
diethyl ether 2 : 1 as an eluent. Compound 5 was obtained as
an orange powder in 16% yield (Found C 74.15, H 5.34. Calc.
for C27H24FeO2: C 74.32, H 5.54). dH(700 MHz, acetone-d6) 8.31
(1H, s, OH), 8.35 (1H, s, OH), 7.05 (4H, d, J = 8.4 Hz, Ar), 6.87
(2H, m, Ar), 6.78 (2H, d, J = 8.8 Hz, Ar), 4.15 (1H, s, Cp), 4.02
(5H, s, Cp), 3.85 (1H, t, J = 2.2 Hz, Cp), 3.15 (1H, m, Cp), 2.80–
2.75 (2H, m, CH2), 2.41–2.34 (2H, m, CH2), 2.03–1.99 (1H, m,
CH2), 1.67–1.63 (1H, m, CH2). dC(75.45 MHz, acetone-d6) 157.0,
156.5, 137.0, 136.5, 135.9, 133.2, 131.7, 131.4, 116.0, 115.5, 87.5,
82.8, 70.8, 67.4, 66.8, 66.1, 32.1, 26.1, 25.3. m/z (EI) 436 (M+).
Experimental
General remarks
All reactions were carried out under argon. All reagents used in this
work are commercially available (Aldrich) and were used without
further purification. Solvents were dried over appropriate drying
agents and distilled before use. Chromatographic separations were
carried out using Silica gel 60 (Merck, 230–400 mesh ASTM).
The NMR spectra were run on Varian Gemini 200 BB (200 MHz
for 1H) and Bruker Avance 300 (300 MHz for 1H) and Bruker
Avance II Plus 700 (700 MHz for 1H) spectrometers. Mass spectra
were measured on a Finnigan MAT 95 spectrometer. Elemental
analyses were performed by Analytical Services of the Center of
Molecular and Macromolecular Studies of the Polish Academy
of the Sciences (Ło´dz´). 1,2-(1-Ketotrimethylene)ferrocene and
1,2-(1-ketotetramethylene)ferrocene were prepared following the
literature procedures.51,52
X-Ray crystallography†
The data for 5 were collected using the BRUKER KAPPA APEXII
ULTRA controlled by APEXII software53 system and equipped
with Mo-Ka rotating anode X-ray source, APEX-II CCD detector
and graphite monochromator. The experiments were carried out at
liquid nitrogen temperature using an Oxford Cryostream cooling
device. The crystal was mounted on a thin glass rod with a droplet
of Paratone-N oil and immediately cooled.
Indexing, integration and initial scaling were performed with
SAINT and SADABS software.54 The unit cell parameters were
obtained and refined based on whole datasets. The absorption
correction from crystal faces was applied before scaling. The
average mosaicity was refined to the value of 0.5◦. Finally the
datasets were merged with SORTAV (Blessing R., 1995). The data
collection and processing statistics are given in Table 5.
1,2-[1-[1,1-Bis(4-hydroxyphenyl)methylidene]trimethylene]
ferrocene (4)
Compound 4 was prepared from the McMurry coupling reaction
according to the literature procedure for the synthesis of 3,33,34
using 0.120 g (0.5 mmol) of 1,2-(1-ketotrimethylene)ferrocene,
0.107 g (0.5 mmol) of 4,4¢-dihydroxybenzophenone, 0.282 g
(0.16 mL, 1.5 mmol) of TiCl4, 0.192 g (2,93 mmol) of zinc
powder, 0.236 g (0.24 mL, 3.0 mmol) of anhydrous pyridine,
and anhydrous THF (6 mL + 1.8 mL). The crude product was
first chromatographed on silica gel column using pentane–diethyl
ether 3 : 4 as eluent. The compound obtained was purified again
on silica gel column using pentane–diethyl ether 1 : 1 as eluent.
Compound 4 was obtained as an orange powder in 30% yield
(Found: C 73.61, H 5.77; Calc. for C26H22FeO2: C 73.95, H 5.25).
The structure was solved by the direct methods approach using
SHELXS-9755 and then refinements based on F2, except reflections
with negative intensities, were carried out with SHELXL-97.
Weighted R factors wR and all goodness-of-fit S values were
based on F2, whereas conventional R factors were based on the
amplitudes, with F set to zero for negative F2. Scattering factors
were taken from Tables 4.2.6.8 and 6.1.1.4 from the International
Crystallographic Tables Vol. C.56 In the final refinement all non-
hydrogen atoms for both structures were refined with anisotropic
thermal displacement parameters. Hydrogen atoms were located
directly from the Fourier map and then their positions and
isotropic thermal displacement parameters were refined using a
7448 | Dalton Trans., 2010, 39, 7444–7450
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
riding model. No extinction correction was needed. The molecular
graphics were generated with the program ORTEP.
655 nm with a Biorad microplate reader. The results are expressed
as the percentage of proteins vs. the control. Experiments were
performed at least in duplicate.
Biochemical studies
Acknowledgements
Materials: stock solutions (10-3 M and 10-2 M) of the com-
pounds to be tested were prepared in DMSO and were kept
at -20 ◦C. Under these conditions, they are stable for at least
2 weeks. Serial dilutions in DMSO were prepared just prior
to use. Dulbecco’s Modified Eagle Medium (DMEM), fetal
calf serum, glutamine and kanamycine were purchased from
Invitrogen (France), estradiol and protamine sulfate were from
Sigma-Aldrich (France). Breast cancer cells MCF-7 (hormone-
dependent) and MDA-MB231 (hormone-independent) were from
the American Type Culture Collection (ATCC–LGC standards).
Sheep uteri weighing approximately 7 g, were obtained from the
slaughterhouse at Mantes-la-Jolie, France. They were immediately
frozen and kept in liquid nitrogen prior to use. ERb PanVera were
purchased from Invitrogen (France).
We thank Barbara McGlinchey for helpful discussions, and the
Agence Nationale de la Recherche for financial support (No.
ANR-06-BLAN-0384-01, “FerVect). D. P.’s postdoctoral stay was
financed by Foundation for Polish Science (FNP) within the
framework of KOLUMB Fellowship.
References
1 G. Jaouen, Bioorganometallics, Wiley-VCH, Weinheim Germany, 2006.
2 G. Jaouen, S. Top, A. Vessie`res and R. Alberto, J. Organomet. Chem.,
2000, 600, 23–36.
3 C. G. Hartinger and P. J. Dyson, Chem. Soc. Rev., 2009, 38, 391–401.
4 D. Dive and C. Biot, ChemBioChem, 2008, 3, 383–391.
5 G. Jaouen, S. Top and A. Vessie`res, in Bioorganometallics, ed.
G. Jaouen, Wiley-VCH, Weinheim, 2006, pp. 65–95.
6 M. Salmain and N. Metzler-Nolte, in Ferrocenes, ed. P. Stepnicka,
Wiley, West Sussex, 2008, pp. 499–541.
7 G. Jaouen, S. Top, A. Vessie`res, G. Leclercq and M. J. McGlinchey,
Curr. Med. Chem., 2004, 11, 2505–2517.
8 A. Vessie`res, S. Top, W. Beck, E. A. Hillard and G. Jaouen, Dalton
Trans., 2006, 529–541.
9 A. Vessie`res, S. Top, P. Pigeon, E. A. Hillard, L. Boubeker, D. Spera
and G. Jaouen, J. Med. Chem., 2005, 48, 3937–3940.
10 E. A. Hillard, A. Vessie`res, L. Thouin, G. Jaouen and C. Amatore,
Angew. Chem., Int. Ed., 2006, 45, 285–290.
11 A. Nguyen, E. A. Hillard, A. Vessie`res, S. Top, P. Pigeon and G. Jaouen,
Chimia, 2007, 61, 716–725.
Determination of the relative binding affinity (RBA) of the
compounds for ERa and ERb. RBA values were measured on
ERa from lamb uterine cytosol and on purified ERb PanVera
(Invitrogen). Sheep uterine cytosol prepared in buffer A (0.05 M
Tris-HCL, 0.25 M sucrose, 0.1% b-mercaptoethanol, pH 7.4 at
◦
25 C) as previously described48 was used as a source of ERa.
For ERb, 10 mL of the solution containing 3500 pmol mL-1 were
added to 10 mL of buffer B (10% glycerol, 50 mM Bis-Tris-propane
pH 9400 mM KCl, 2 mM DTT, 1 mM EDTA, 0.1% BSA). Aliquots
(200 mL) of ERa in glass tubes or ERb in polypropylene tubes
12 Q. Michard, G. Jaouen, A. Vessie`res and A. Bernard, J. Inorg. Biochem.,
2008, 102, 1980–1985.
◦
were incubated for 3 h 30 min at 0 C with [6,7-3H]-estradiol
(2 ¥ 10-9 M, specific activity 1.62 TBq mmol-1, NEN Life Science
Product) in the presence of nine concentrations of the compounds
to be tested (between 6 ¥ 10-7 M and 6 ¥ 10-9 M) or of non-
radioactive E2 (between 8 ¥ 10-8 M and 8 ¥ 10-10 M). At the end of
the incubation period, the free and bound fractions of the tracer
were separated by protamine sulfate precipitation. The percentage
reduction in binding of [3H]-estradiol (Y) was calculated using the
logit transformation of Y (logitY: ln[y/1 - Y] vs. the log of the
mass of the competing steroid. The concentration of unlabelled
steroid required to displace 50% of the bound [3H]-estradiol was
calculated for each steroid tested, and the results expressed as
RBA. The RBA value of estradiol is by definition equal to 100%.
Culture conditions: cells were maintained in a monolayer
culture in DMEM with phenol red/Glutamax I supplemented
with 9% fetal bovine serum at 37 ◦C in a 5% CO2/air-humidified
incubator. For proliferation assays, MCF-7 and MDA-MB-231
cells were plated in 1 mL of DMEM without phenol red,
supplemented with 9% decomplemented and hormone-depleted
fetal bovine serum, 0.9% kanamycin, 0.9% Glutamax I and
incubated. The following day (D0), 1 mL of the same medium
containing the compounds to be tested was added to the plates.
After 3 days (D3) the incubation medium was removed and 2 mL
of the fresh medium containing the compounds was added. After
5 days the total protein content of the plate was analyzed as
follows: cell monolayers were fixed for 1 h at room temperature
with methylene blue (1 mg mL-1 in 50 : 50 water–MeOH mixture),
then washed with water. After addition of HCl (0.1 M, 2 mL),
the plate was incubated for 1 h at 37 ◦C and then the absorbance
of each well (three wells for each concentration) was measured at
13 E. Allard, C. Passirani, E. Garcion, P. Pigeon, A. Vessie`res, G. Jaouen
and J.-P. Benoit, J. Controlled Release, 2008, 130, 146–153.
14 I. Zanellato, J.-M. Heldt, A. Vessie`res, G. Jaouen and D. Osella, Inorg.
Chim. Acta, 2009, 362, 4037–4042.
15 C. A. Rice-Evans, N. J. Miller and G. Paganga, Free Radical Biol. Med.,
1996, 20, 933–956.
16 M. Hu, Mol. Pharmaceutics, 2007, 4, 803–806.
17 B. A. Craf, P. E. Milbury and J. B. Blumberg, J. Med. Food, 2005, 8,
281–290.
18 M. Jang, L. Cai, G. O. Udeani, K. V. Slowing, C. F. Thomas, C. W. W.
Beecher, H. H. S. Fong, N. R. Farnsworth, A. D. Kinghorn, R. G.
Mehta, R. C. Moon and J. M. Pezzuto, Science, 1997, 275, 218–220.
19 C. S. Yang, J. D. Lamber, Z. Hou, J. Ju, G. Lu and X. Hao, Mol.
Carcinog., 2006, 45, 431–435.
20 J. A. Baur, K. J. Pearson, N. L. Price, H. A. Jamieson, C. Lerin, A.
Kalra, V. V. Prabhu, J. S. Allard, G. Lopez-Lluch, K. Lewis, P. J. Pistell,
S. Poosala, K. G. Becker, O. Boss, D. Gwinn, M. Wang, S. Ramaswamy,
K. W. Fishbein, R. G. Spencer, E. G. Lakatta, D. Le Couteur, R. J. Shaw,
P. Navas, P. Puigserver, D. K. Ingram, R. de Cabo and D. A. Sinclair,
Nature, 2006, 444, 337–342.
21 J. Jankun, S. Selman, R. Swiercz and E. Skrzypczak-Jankun, Nature,
1997, 387, 561.
22 J. Slavin, Proc. Nutr. Soc., 2003, 62, 129–134.
23 B. B. Aggarwal, C. Sundaram, N. Malani and H. Ichikawa, Adv. Exp.
Med. Biol., 2007, 595, 1–75.
24 V. M. Adhami and H. Mukhtar, Free Radical Res., 2006, 40, 1095–1104.
25 D. L. Bemis, J. L. Capodice, J. E. Costello, G. C. Vorys, A. E. Katz and
R. Buttyan, Curr. Urol. Rep., 2006, 7, 166–174.
26 F. Minutolo, G. Sala, A. Bagnacani, S. Bertini, I. Carboni, G. Placanica,
G. Prota, S. Rapposelli, N. Sacchi, M. Macchia and R. Ghidoni, J. Med.
Chem., 2005, 48, 6783–6786.
27 D. Hamels, P. M. Dansette, E. A. Hillard, S. Top, A. Vessie`res, P.
Herson, G. Jaouen and D. Mansuy, Angew. Chem., Int. Ed., 2009, 48,
9124–9126.
28 A. Vessie`res, D. Spera, S. Top, B. Misterkiewicz, J. M. Heldt, E. A.
Hillard, M. Hiuche´, M.-A. Plamont, E. Napolitano, R. Fiaschi and G.
Jaouen, ChemMedChem, 2006, 1, 1275–1281.
This journal is
The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 7444–7450 | 7449
©

View Article Online
29 P. Pigeon, S. Top, O. Zekri, E. A. Hillard, A. Vessie`res, M.-A. Plamont,
O. Buriez, E. Labbe´, M. Huche´, S. Boutamine, C. Amatore and G.
Jaouen, J. Organomet. Chem., 2009, 694, 895–901.
30 O. Zekri, E. A. Hillard, S. Top, A. Vessie`res, P. Pigeon, M.-A. Plamont,
M. Huche´, S. Boutamine, M. J. McGlinchey, H. Mu¨ller-Bunz and G.
Jaouen, Dalton Trans., 2009, 4318–4326.
43 WavefunctionCo., Irvine CA 92612, USA.
44 A. K. Shiau, D. Barstad, P. M. Loria, L. Cheng, P. J. Kushner, D. A.
Agard and G. L. Greene, Cell, 1998, 95, 927–937.
45 A. K. Shiau, D. Barstad, J. T. Radek, J. L. Meyers, K. W. Nettles, B. S.
Katzenellenbogen, J. A. Katzenellenbogen, D. A. Agard and G. L.
Greene, Nat. Struct. Biol., 2002, 9, 359.
31 B. Ferber, S. Top, R. Welter and G. Jaouen, Chem.–Eur. J., 2006, 12,
2081–2086.
32 E. A. Hillard, A. Vessie`res, F. Le Bideau, D. Plazuk, D. Spera, M.
Huche´ and G. Jaouen, ChemMedChem, 2006, 1, 551–559.
33 S. Top, B. Dauer, J. Vaissermann and G. Jaouen, J. Organomet. Chem.,
1997, 541, 355–361.
34 G. Jaouen, S. Top, A. Vessie`res, G. Leclercq, J. Quivy, L. Jin and A.
Croisy, C. R. Acad. Sci. Paris, 2000, Se´rie IIc, 89–93.
35 D. Plazuk, A. Vessie`res, E. A. Hillard, O. Buriez, E. Labbe´, P. Pigeon,
M.-A. Plamont, C. Amatore, J. Zakrzewski and G. Jaouen, J. Med.
Chem., 2009, 52, 4964–4967.
36 T. Shirafuji, A. Odaira, Y. Yamamoto and H. Nozaki, Bull. Chem. Soc.
Jpn., 1972, 45, 2884–2887.
46 E. A. Hillard, A. Vessie`res, S. Top, P. Pigeon, K. Kowalski, M. Huche´
and G. Jaouen, J. Organomet. Chem., 2007, 692, 1315–1326.
47 S. Top, A. Vessie`res, C. Cabestaing, I. Laios, G. Leclercq, C. Provot and
G. Jaouen, J. Organomet. Chem., 2001, 637–639, 500–506.
48 S. Top, A. Vessie`res, G. Leclercq, J. Quivy, J. Tang, J. Vaissermann, M.
Huche´ and G. Jaouen, Chem.–Eur. J., 2003, 9, 5223–5236.
49 A. Nguyen, V. Marsaud, C. Bouclier, S. Top, A. Vessie`res, P. Pigeon,
R. Gref, P. Legrand, G. Jaouen and J.-M. Renoir, Int. J. Pharm., 2008,
347, 128–135.
50 E. Allard, D. Jarnet, A. Vessie`res, S. Vinchon-Petit, G. Jaouen, J.-P.
Benoit and C. Passirani, Pharmacol. Res., 2010, 27, 56–64.
51 P. Bickert, B. Hildebrandt and K. Hafner, Organometallics, 1984, 3,
653–657.
37 M. E. Kuehne, W. Dai and Y.-L. Li, J. Org. Chem., 2001, 66, 1560–1566.
38 S. Malfait, L. Pe´linski and J. Brocard, Tetrahedron: Asymmetry, 1998,
9, 2595–2610.
52 K. L. Rinehart, R. J. Curby, D. H. Gustafson, K. G. Harrison,
R. E. Bozak and D. E. Bublitz, J. Am. Chem. Soc., 1962, 84, 3263–
3269.
53 APEXII-2007v1.0, Bruker Nonius, 2007.
54 SADABS-2004/1, Bruker Nonius, 2007.
39 G. G. Kuiper, E. Enmark, M. Pelto-Huikko, S. Nilsson and J.-A.
Gustafsson, Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 5925–5930.
40 J. A. Gustafsson, Trends Pharmacol. Sci., 2003, 24, 479–485.
41 P. Pigeon, S. Top, A. Vessie`res, M. Huche´, E. A. Hillard, E. Salomon
and G. Jaouen, J. Med. Chem., 2005, 48, 2814–2821.
55 G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr., 2008,
64, 112–122.
56 A. J. C. Wilson, International Tables for Crystallography, Kluwer,
Dordrecht, 1992.
42 V. C. Jordan, J. Med. Chem., 2003, 46, 1081–1111.
7450 | Dalton Trans., 2010, 39, 7444–7450
This journal is
The Royal Society of Chemistry 2010
©