The replacement of a phenol group by an aniline or acetanilide group enhances the cytotoxicity of 2-ferrocenyl-1,1-diphenyl-but-l-ene compounds against breast cancer cells

Article abstract of DOI:10.1016/j.jorganchem.2008.11.035

We have previously shown that conjugated ferrocenyl p-phenols show strong cytotoxic effects against both the hormone-dependent MCF-7 and hormone-independent MDA-MB-231 breast cancer cell lines, possibly via oxidative quinone methide formation. We now pres

Full text of DOI:10.1016/j.jorganchem.2008.11.035

Journal of Organometallic Chemistry 694 (2009) 895–901
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Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
The replacement of a phenol group by an aniline or acetanilide group enhances
the cytotoxicity of 2-ferrocenyl-1,1-diphenyl-but-l-ene compounds against
breast cancer cells
a
Pascal Pigeon a, Siden Top a, Ouardia Zekri a,c, Elizabeth A. Hillard a, Anne Vessières a, , Marie-Aude Plamont ,
*
Olivier Buriez b, Eric Labbé b, Michel Huché a, Sultana Boutamine c, Christian Amatore b, Gérard Jaouen a
a Laboratoire de Chimie et Biochimie des Complexes Moléculaires, UMR CNRS 7576, Ecole Nationale Supérieure de Chimie de Paris, 11 rue Pierre et Marie Curie,
75231 Paris Cedex 05, France
b Ecole Normale Supérieure, Département de Chimie, UMR CNRS-ENS-UPMC 8640, 24 rue Lhomond, 75231 Paris Cedex 05, France
c Université des Sciences et de la Technologie Houari Boumediene, Faculté de Chimie, BP32, El Alia Bab Ezzouar, Alger, Algeria
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 August 2008
Received in revised form 12 November 2008
Accepted 13 November 2008
Available online 25 November 2008
We have previously shown that conjugated ferrocenyl p-phenols show strong cytotoxic effects against
both the hormone-dependent MCF-7 and hormone-independent MDA-MB-231 breast cancer cell lines,
possibly via oxidative quinone methide formation. We now present a series of analogous amine and acet-
amide compounds: 2-ferrocenyl-1-(4-aminophenyl)-1-phenyl-but-1-ene (Z+E-2), 2-ferrocenyl-1-(4-N-
acetylaminophenyl)-1-phenyl-but-1-ene (Z-3), and their corresponding organic molecules 1-(4-amino-
phenyl)-1,2-bis-phenyl-but-1-ene (Z+E-4) and 1-(4-N-acetamidophenyl)-1,2-bis-phenyl-but-1-ene
(Z+E-5). All of the compounds have adequate relative binding affinity values for the estrogen receptor;
Keywords:
Bioorganometallic chemistry
Ferrocene
Anti-cancer drugs
Breast cancer
Iron
between 2.8% and 5.7% for ER
ing in in silico ER docking experiments. Compounds 2 and 3 show dual estrogenic/cytotoxic activity on the
MCF-7 cell line; they are proliferative at low concentrations (0.1 M) and antiproliferative at high con-
centrations (10 M). On the MDA-MB-231 cell line, the ferrocenyl complexes 2 and 3 are antiproliferative
with IC50 values of 0.8 M for 2 and 0.65 M for 3, while the purely organic molecules 4 and 5 show no
a, and between 0.18% and 15.5% for ERb, as well as exothermic ligand bind-
l
l
l
l
effect. Electrochemical experiments suggest that both 2 and 3 can be transformed to oxidized quinoid-
type species, analogous to what had previously been observed for the ferrocene phenols.
Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction
system linking the two moieties [14], for a strong cytotoxic activity
in vitro. Electrochemical experiments have suggested that the ac-
Compounds that can be activated by the redox environment of a
cellular target to produce reactive oxygen species (ROS) [1] or reac-
tive intermediates [2] have recently gained much attention, partic-
ularly in cancer treatment [3]. For example, compounds containing
phenol moieties, such as resveratrol, can modify the oxidative
stress level of the cell to cause necrosis and apoptosis [4,5]. Tamox-
ifen (Chart 1), a leading treatment for all phases of hormone-
dependent breast cancer [6], also shows a mild non-genomic
cytotoxic activity, which has been attributed to its aromatic
hydroxylation and biooxidation to radical or electrophilic species
[7]. We have been studying a series of conjugated ferrocenyl phe-
nols, which show antiproliferative effects in vitro on both hor-
mone-independent (ERÀ) and hormone-dependent (ER+) breast
cancer cells [8,9] and in vivo against glioma [10]. Structure activity
relationship studies have demonstrated the importance of the
tive species in cell cultures could be a quinone methide, which
may be formed via an intramolecular oxidation of the phenol moi-
ety by the in situ-generated ferricenium cation [15]. Other investi-
gators have also shown a correlation between the toxicity of a
tamoxifen–ferrocene conjugate and the production of ROS [16].
Ferricenium-promoted intramolecular electron transfer in pheno-
lic systems has been recognized [17–21], although our work is
the first application of this mechanism to oxidation-activated drug
development.
According to the proposed mechanism of activation [15], we ex-
pect that the substitution of the hydroxyl group by other protic and
oxidizable functionalities should retain the molecules’ cytotoxic
properties and specific electrochemical signature. However, in a
previous study, we found that the dithioacetyl prodrug analog 1a
(Chart 1) displayed only proliferative effects on hormone-depen-
dent breast cancer cells (MCF-7), and no (cytotoxic) effect on hor-
mone-independent breast cancer cells (MDA-MB-231) [22]. This
was in marked contrast to the diacetoxy compound, 1b, which
showed cytotoxic effects similar to that of the diphenol 1c, sug-
presence of a ferrocene group [11,12], a p-phenol [13], and a
p-
* Corresponding author. Tel.: +33 01 44 27 67 29; fax: +33 01 43 26 00 61.
E-mail address: a-vessieres@enscp.fr (A. Vessières).
0022-328X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2008.11.035

896
P. Pigeon et al. / Journal of Organometallic Chemistry 694 (2009) 895–901
R
NH2
1a: R = SC(O)CH3
1b: R = OC(O)CH3
1c: R = OH
1d: R= H
Fe
Fe
Fe
O
N
R
HN
Tamoxifen
O
1
(Z+E)-2
(Z)-3
NH2
OH
NH2
HN
O
Fe
O
N
(Z+E)-6
(Z+E)-4
(Z+E)-5
NH2-tamoxifen
Chart 1. The breast cancer drug tamoxifen, the amino derivative of tamoxifen, previously studied ferrocene complexes 1a–1d and 6 and new compounds 2–5.
gesting that intracellular hydrolysis of the compound to the active
hydroxylated species takes place. As thioesterases have been iso-
lated from breast cancer cells [23], we attributed this lack of activ-
ity not to the protection of the thiol, but to the poor overlap
between the 3p S and 2p C atomic orbitals, which disfavors thio-
quinone methide formation.
activity of aminobenzophenone in McMurry reactions [29]. The Z
and E isomers of 2 could not be separated using preparative HPLC,
and thus the mixture of (Z+E)-2 was used in all further experi-
ments. Similarly, its organic analog 4 was synthesized by a reaction
between propiophenone and aminobenzophenone as a mixture of
Z and E isomers in 38% yield (see Scheme 1).
We now investigate the synthesis, cell proliferation effects, and
electrochemical behavior of the ferrocenyl aniline 2 and acetani-
lide 3 shown in Chart 1, particularly as compared to the cytotoxic
monophenolic analog 6 [8]. We have also studied their purely or-
ganic counterparts, 4 and 5 to evaluate the importance of the ferr-
ocenyl moiety. These later compounds are relevant, in that the
literature on the biological effects of aryl amines or amides based
on the tamoxifen or triphenylethylene skeleton is scarce; to the
best of our knowledge, only one study on the uterotrophic effects
of the amino derivative of tamoxifen (Chart 1) has been carried
out [24].
An alternative pathway via a coupling reaction between propi-
onyl ferrocene or propiophenone and 4-nitrobenzophenone also
gave access to 2 and 4. Originally we used this reaction in an at-
tempt to synthesize the corresponding nitro species, but found that
an in situ reduction of the nitro group to an amino group furnished
2 and 4. However, depending on the batch of 4-nitrobenzophenone
used, the yield of the reaction varied significantly from high (>50%)
to poor.
Treating 2 with acetyl chloride and pyridine gave 3 as a mixture
of Z and E isomers (60/40) in 91% yield, as shown in Scheme 2. The
Z isomer was isolated by fractional crystallization and all further
tests were performed with the pure isomer. Compound 5 was sim-
ilarly synthesized from 4 in 81% yield as a mixture of Z and E iso-
mers (85/15).
2. Results and discussion
2.1. Preparation of compounds
2.2. Biological studies
The synthesis of the organometallic compounds was based on a
McMurry cross-coupling reaction, the method we commonly use
to form alkenes from the reaction of two ketones in the presence
of TiCl4/Zn [25–27]. Compound 2 was synthesized as a mixture
of Z and E isomers via the reaction between propionyl ferrocene
and 4-aminobenzophenone, as previously reported [28]. The mod-
erate yield (26%) is consistent with the previously observed low
2.2.1. Determination of the relative binding affinity (RBA) values of the
compounds for the two forms of estrogen receptors (ER
a and ERb)
The RBA values obtained for the new compounds are given in
Table 1. The values found for the ferrocenyl aniline and acetanilide
derivatives 2 and 3 on ER
a
were only slightly lower than that
NH
2
NH
NH
HN
2
2
O
CH
O
Cl
3
TiCl /Zn
4
Pyridine
O
O
R
THF, reflux
THF
R
R
R
R = Fc, Ph
(
(
Z
Z
+
E
)-2; R = Fc
)-4; R = Ph
(
(
Z
Z
+
E
)-2; R = Fc
)-4; R = Ph
(
(
Z
Z
+
E
)-3; R = Fc
)-5; R = Ph
+
E
+
E
+
E
Scheme 1. Synthesis of the ferrocenyl and phenyl aniline derivatives 2 and 4.
Scheme 2. Synthesis of the ferrocenyl and phenyl acetanilide derivatives 3 and 5.

P. Pigeon et al. / Journal of Organometallic Chemistry 694 (2009) 895–901
897
Table 1
Relative binding affinity (RBA) of the compounds on the two isoforms of the estrogen
receptor (ER and ERb) and IC50 values for the compounds on hormone-independent
breast cancer cells, MDA-MB-231.
a
Compound
RBA (%)
ER
IC50 [lM]
a
ERb
(Z+E)-2
(Z)-3
(Z+E)-4
(Z+E)-5
(Z+E)-6
2.8 0.1
3.5 0.3
1.08 0.07
0.18 0.02
15.5 2.5
7.7 0.8
0.8 0.1
0.65 0.03
Not toxic at 1
Not toxic at 1
1.13 0.07b
7
0.3
l
l
M
M
5.7 0.1
4.6 0.1a
11 1a
a
Value from Ref. [8].
Value from Ref. [13].
b
found for the ferrocenyl monophenol 6 (RBA = 4.6%). This is quite
surprising for molecules having no phenol group, which is consid-
ered to be essential for ER recognition [30]. Although all com-
pounds have RBA values on the same order of magnitude, the
purely organic compounds 4 and 5, lacking the bulky ferrocene
group, have slightly higher affinities for ERa. Interestingly, both 2
and 4 showed a greater affinity for ER than that found for the
a
Fig. 1. (E)-2 in the cavity of ERa.
amino derivative of tamoxifen (0.2%), evidently due to the absence
of the amino side-chain [24]. The binding affinities for ERb are
much more variable than for ERa, with the organic compounds
with His524. This triple anchorage demonstrates the good associa-
tion of this isomer with the cavity.
showing significantly higher values than the ferrocenyl com-
pounds. Nonetheless, all compounds have non-zero RBA values,
and thus would be expected to interact with the ERs.
2.2.3. Effect on hormone-dependent breast cancer cells MCF-7
The effect of 2 and 3 on MCF-7 cells is shown on Fig. 2. At the
2.2.2. Molecular modeling of receptor interactions
low concentration of 0.1
fect while 3 was only slightly proliferative. At the high concentra-
tion of 10 M 2 had almost no effect while 3 gave rise to a
lM, 2 showed a marked proliferative ef-
Molecular docking experiments using the crystal structure of
ER
a crystallized with DES (pdb erd.ent) [31], and of ERb crystal-
l
lized with 5,11-cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol
(THC) (pdb 112j.ent) [32] were performed. Only the amino acids
that constitute the wall of the cavity have been retained. The DES
or THC molecules were removed from the cavity and replaced suc-
cessively with the 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
moderate antiproliferative effect. It should be mentioned that the
experimental conditions used (cell medium without phenol red,
an agent normally used for MCF-7 cell culture, but containing an
impurity known to be estrogenic) was chosen to favor the expres-
sion of the estrogenic component of the tested molecules. How-
ever, the estrogenic effects are not consistent with the observed
immobilized and the side chain of His524 for ERa and His475 for
ERb were liberated. This allowed the ideal positions of the bioli-
gands to be determined. Quantum mechanical semi-empirical
PM3 methods were then used to determine the affinity of the bioli-
gands for the cavity. This requires calculation of the energies of
bioligand-cavity group, of the cavity itself, and of the ligand, the
latter two in the conformations they had in the molecular assem-
blies to give the DrH° enthalpy variations of the reactions: bioli-
gand + cavity ? molecular assembly (Table 2). For all compounds,
binding with both isoforms of the ER is thermodynamically fa-
vored, as evidenced by the negative enthalpy of formation for the
ligand–receptor complex.
300
MCF-7
200
100
0
Fig. 1 shows (E)-2 in the cavity of ERa. The NH2 group forms a
hydrogen bond with the residues Glu353 and Arg394. On the other
side of the molecule the iron atom of the ferrocene group interacts
Table 2
Enthalpy variation values for the bioligand docked into ER
a and ERb.
Compound
DrH° (kcal molÀ1
ER
)
a
ERb
3
2
(Z)-2
(E)-2
(Z)-3
(Z)-4
(Z)-5
(E)-5
À16.4
À17.5
À16.5
À13.3
À13.5
À8.8
À17.3
À31.1
À16.7
À8.4
Fig. 2. Effect of estradiol (E2, 10 nM), ferrocenyl aniline 2 and ferrocenyl acetanilide
3 at low (0.1 M) and high (10 M) concentrations on MCF-7 cells (hormone-
dependent breast cancer cells) after 5 days of culture. Non-treated cells are used as
the control (C). Representative data of one experiment performed twice with
similar results (six measurements confidence limit; P = 0.1, t = 1.895).
l
l
À13.2
À10.3

898
P. Pigeon et al. / Journal of Organometallic Chemistry 694 (2009) 895–901
RBA values, which suggest that 3, with a stronger binding affinity,
should be more estrogenic than 2. This is because the observed ef-
fect is actually the net result of the estrogenic (proliferative) effect
which is expressed at low concentrations, combined with the cyto-
toxic (antiproliferative) effect which begins to appear at higher
concentrations. The observed results are consistent with the higher
cytotoxicity of 3 compared with 2 (vide infra).
2.2.4. Effect on hormone-independent breast cancer cells MDA-MB-
231
On MDA-MB-231 2 and 3 showed a strong antiproliferative ef-
fect (Fig. 3) with IC50 values of 0.8 and 0.65 lM, respectively (Table
1). Remarkably, these compounds are more cytotoxic than the ferr-
ocenyl monophenol 6 and approach the antiproliferative potency
of the previously reported ferrocenyl diphenol 1c (IC50 = 0.6
[33]. Consequently, they join the group of the ferrocenyl deriva-
tives having the lowest IC50 values (<1 M) found so far [9]. It
should be noted that the unsubstituted ferrocenyl compound 1d
showed no effect even at concentrations as high as 10 M, demon-
lM)
l
l
strating the participation of the functional groups in the cytotoxic
activity [13]. Interestingly, neither the organic amine 4 nor the ace-
tylamide 5 was cytotoxic at 1 lM, although the oxidative metabo-
lism of aromatic amines is known to produce toxic species [34,35],
including in breast cancer cells [36]. Nonetheless, in our experi-
ments, the presence of the ferrocenyl group was essential for a
strong cytotoxic effect.
Xenobiotics are metabolized in vivo, generally to more polar,
water-soluble products [37]. Hydrolyzing enzymes as esterases
and amidases, are ubiquitous, [38] including in breast cancer cells
[39–44]. These enzymes compete with arylamine N-acetyltransfer-
ase enzymes (NATs), which catalyze the acetylation of arylamines
as a detoxification mechanism, and are also expressed in breast
cancer cells [45,46]. Thus it is difficult to predict whether the active
form of the ferrocenyl compound is the amine, the amide, or both.
Fig. 4. Cyclic voltammograms for (a) 2 and (b) 3, in DMF. Pt (0.5 mm) working
electrode, platinum mesh counter electrode, SCE reference electrode, scan
rate = 100 mV/s. For 2, the first wave corresponds to the ferrocene oxidation, while
the second corresponds to the amine oxidation.
demonstrating electron transfer from the organic part of the mol-
ecule to the electrochemically-generated ferricenium moiety. The
addition of imidazole enhanced the intensity and irreversibility of
this wave for both compounds. The base-promoted electron
transfer in compound 2 is further evidenced by the decrease in
intensity and potential of the oxidation wave of the amino group
at 0.7 V upon addition of a base. This reveals the transition of an
amino group to an aminyl radical triggered by the oxidation of
the ferrocene at the first wave. In Fig. 4b, this is not apparent, be-
cause the amide oxidation wave lies outside the displayed
window.
2.3. Electrochemistry
Electrochemical results suggest that both 2 and 3 can be trans-
formed into potentially toxic oxidized products via the previously
proposed ferricenium-mediated proton-coupled electron transfer
mechanism; the cyclic voltammograms in DMF and DMF/imidaz-
ole are shown in Fig. 4. A characteristic feature of this mechanism
is a base-dependent enhancement of the wave appearing at the
ferrocene oxidation potential, coupled with loss of reversibility,
Electronic communication between a ferrocene group and an
electrode via a conjugated system has been previously demon-
strated by electron exchange when such compounds are anchored
by a sulfur atom to a Au electrode [47]. This communication is very
fast (t1/2 ca. 0.5
ls), compared to the scan rates we use in this re-
port, and thus an equilibrium between the ferrocene-centered rad-
ical cation and the phenol-centered radical cation is reached
essentially immediately. When the substituted phenyl radical cat-
ion can react (for example with a base present in the system), this
equilibrium acts as an activation barrier for the overall reaction,
such that, according to the Hammond postulate, the rate can be de-
D
Go/RT), where k0 is the intrinsic rate con-
*
scribed as k = k0 exp(À
stant of the deprotonation step and
D
Go >> 0, the free Gibbs energy
difference between the two forms of the cation radical of the mol-
ecule, as shown in Scheme 3.
The oxidation potential of the functional group is related to the
position of the equilibrium and the height of the activation barrier,
D
Go. Due to the electron withdrawing character of the acetyl
Fig. 3. Effect of 1 lM of the ferrocenyl aniline 2 and acetanilide 3 and of their
corresponding purely organic molecules 4 and 5 on the growth of MDA-MB-231
(hormone-independent breast cancer cells) after 5 days of culture. Non-treated cells
are used as the control (C). Representative data of one experiment performed twice
with similar results (six measurements confidence limit; P = 0.1, t = 1.895).
group, the oxidation of anilines occurs at less positive potentials
than those of acetanilides [48–50], thus
Go would be expected
to be larger in the case of 3 compared to 2. In our experiments,
D

P. Pigeon et al. / Journal of Organometallic Chemistry 694 (2009) 895–901
899
k
NHR
NHR
NHR
NR
NR
-e-
-e-
k
0
-H+
-BH
Fe
Fe
Fe
Fe
Fe
ΔGo is the difference in energy
between these species
Scheme 3. Proposed mechanism of activation of 2 and 3.
we found that the irreversible oxidation wave of the amino group
in the organic molecule 4 occurred at +0.84 V (vs. SCE) and that of
the amido group in 5 occurred irreversibly at +1.40 V. On the other
hand, the value of k0 will be influenced by the acidity of the amine
or acetamide radical cation. The radical cation of phenylamine is
very reactive [51], and that of acetanilide likely even more so, con-
sidering the difference in their pKa values (aniline = 30.6, aniline
radical cation = 6.4 [52] and acetanilide = 21.45, acetanilide radical
cation = À5 [53]). A recent electrochemical study demonstrated
that 2 could be covalently bonded to an electrode surface through
the nitrogen atom by electrolysis at the ferrocene oxidation poten-
tial (+0.4 V) in the presence of 2,4,6-pyridine [28]. This clearly
demonstrates an intramolecular electron transfer for 2 even in
the presence of a weak base. Due to the similarity of the CVs, it ap-
pears that the high k0 value for [3]+ may be enough to compensate
4. Experimental
4.1. General remarks
The synthesis of all compounds was performed under an argon
atmosphere, using standard Schlenk techniques. Anhydrous THF
was obtained by distillation from sodium/benzophenone. Thin
layer chromatography was performed on silica gel 60 GF254. Infra-
red spectra were obtained on an IR-FT BOMEM Michelson-100
spectrometer equipped with a DTGS detector as a KBr plate. 1H
and 13C NMR spectra were recorded on a 300 MHz Bruker spec-
trometer. Mass spectrometry was performed with a Nermag R
10-10C spectrometer. High resolution mass spectrometry (HRMS)
was performed on a JEOL MS 700 instrument. Melting points were
measured with a Kofler device. Elemental analyses were performed
by the microanalysis service of CNRS at Gif sur Yvette. The semi-
preparative HPLC separations were performed on a Shimadzu
apparatus with a Kromasil C18 column (length of 25 cm, diameter
for the much smaller value of exp(À
D
Go/RT), compared to 2,
although further experiments will have to be carried out to address
this point.
In terms of the putative active agent, imino methides are known
[54], and indeed have been implicated in cytotoxic processes [55–
60]. However, while acetylated quinone imines are also important
toxic metabolites, for example of the Parkinson’s disease drug tol-
capone [48] and the analgesic acetaminophen [61], acetylated
imine methides are rare. To our knowledge, the only evidence of
an acetylated p-imine methide was a short lived species (1 ms)
generated from laser flash photolysis of 4-acetylaminostilbene
[62]. These compounds are probably less stable due to the electron
withdrawing acetyl group, because donation of the nitrogen atom
of 2 cm, and particle size of 10 lm). Cyclic voltammograms were
obtained utilizing a Princeton Applied Research potentiostat. Ana-
lyte solutions were 1 mM in DMF with 0.1 M Bu4NBF4 supporting
electrolyte. Cyclic voltammetry experiments were performed at
room temperature under an argon atmosphere in a three-electrode
cell. The reference electrode was an SCE (Tacussel), which was sep-
arated from the solution by a bridge compartment filled with the
same solvent/supporting electrolyte solution as used in the cell.
The counter electrode was a platinum mesh (Goodfellow). The
platinum working electrode was home-made (0.5 mm diameter;
Goodfellow).
p-electrons into the ring favors the participation of the quinoid
form over the aromatic form [63]. We are currently attempting
to isolate the oxidation products of 2 and 3 and examine their reac-
tivity with nucleophiles such as glutathione.
4.2. Synthesis and characterization of compounds
4.2.1. 2-Ferrocenyl-1-(4-aminophenyl)-1-phenyl-but-1-ene, (Z+E)-2
The synthesis of 2 via the reaction between propionyl ferrocene
and 4-aminobenzophenone was reported in Ref. [28].
3. Conclusion
We have demonstrated that the oxidative activation of ferroce-
nyl compounds to toxic species is not limited to the series of ferr-
ocenyl phenols. The ferrocenyl aniline 2 and acetanilide 3 showed
estrogenic activity on the ER + MCF-7 breast cancer cell line, due to
their affinity for the estrogen receptor, and a pronounced cytotox-
icity against the ER-MDA-MB-231 breast cancer cell line. Notably,
the purely organic aniline 4 and acetanilide 5 did not give rise to
cytotoxic effects. The mechanism of activation seems to follow
the intramolecular electron transfer process previously observed
for the series of conjugated ferrocenyl p-phenols. This study thus
broadens the perspectives for useful functional groups in the de-
sign of redox activated cancer drug candidates.
4.2.1.1. Alternative pathway. Zn powder (3.92 g, 60 mmol) was sus-
pended in 30 mL of THF at 5–10 °C in a Schlenk tube under argon.
While stirring, TiCl4 (5.69 g, 30 mmol) was added slowly via a syr-
inge. The reaction mixture was removed from the cold bath and re-
fluxed for 1h30. To the reaction mixture was added 15 mL of a THF
solution containing propionyl ferrocene (2.42 g, 10 mmol) and 4-
nitrobenzophenone (2.27 g, 10 mmol), and reflux conditions were
maintained for 4 h. The reaction mixture was poured into 100 mL
of water, and extracted with 3 Â 100 mL of dichloromethane. The
organic phase was washed with 100 mL of water, dried over mag-
nesium sulfate, filtered, and the solvent was evaporated. The

900
P. Pigeon et al. / Journal of Organometallic Chemistry 694 (2009) 895–901
brown oil was purified on a silica gel column using dichlorometh-
ane as an eluent. Compound 2 (1.31 g, 54% yield) was recrystallized
from ethanol to give a mixture of Z and E isomers (45/55, major
isomer was not identified). The yield varies significantly from
one batch of 4-nitrobenzophenone to another.
1H NMR (300 MHz, CDCl3): d 0.84 and 0.87 (t, J = 7.4 Hz, 3H,
CH3), 2.37 and 2.45 (q, J = 7.4 Hz, 2H, CH2), 4.29 (s broad, 2H,
NH2), 6.34 and 6.65 (d, J = 8.5 Hz, 2H, Harom), 6.60 and 6.95 (d,
J = 8.5 Hz, 2H, Harom), 6.70–7.35 (m, 10H, Harom); 13C NMR
(75 MHz, CDCl3): d 13.6 (CH3), 29.0 (CH2), 114.4 and 114.9 (2 CHarom),
125.5 and 125.9 (CHarom), 125.9 and 126.4 (CHarom), 127.2 and
127.8 (2 CHarom), 127.7 and 128.0 (2 CHarom), 129.5 and 129.7 (2
CHarom), 129.7 and 130.5 (2 CHarom), 130.9 and 131.8 (2 CHarom),
133.8 and 134.3 (C), 138.5 and 138.7 (C), 140.6 and 141.5 (C),
4.2.2. 2-Ferrocenyl-1-(4-acetylaminophenyl)-1-phenyl-but-1-ene, 3
In a Schlenk flask under argon, 2 (407 mg, 1 mmol) was dis-
solved in 15 mL of anhydrous THF. Acetyl chloride (78 mg, 1 mmol)
and pyridine (79 mg, 1 mmol) were added and the reaction mix-
ture was stirred for 3 h. Water (50 mL) was added and the product
was extracted with dichloromethane (3 Â 50 mL). The organic
phase was washed with 50 mL of water, dried over magnesium sul-
fate, filtered, and the solvent was evaporated. The product was
purified on a silica gel column with ether/pentane (1/1) as the elu-
ent. Compound 3 was obtained as a brown oil (372 mg, 91% yield)
consisting of a mixture of Z and E isomers (60/40, respectively). The
identity of the major (Z) isomer was determined by 2D NMR.
(Z)-3: 1H NMR (300 MHz, DMSO-d6): d 1.00 (t, J = 7.5 Hz, 3H,
CH3), 2.02 (s, 3H, CH3CO), 2.47 (q, J = 7.5 Hz, 2H, CH2), 3.82 (t,
J = 1.0 Hz, 2H, C5H4), 4.11 (t, J = 1.0 Hz, 2H, C5H4), 4.13 (s, 5H,
C5H5), 6.99 (d, J = 8.5 Hz, 2H, Harom), 7.22 (m, 3H, Harom), 6.99 (t,
J = 8.5 Hz, 2H, Harom), 7.46 (d, J = 8.5 Hz, 2H, Harom), 9.89 (s, 1H,
NH); 13C NMR (75 MHz, DMSO-d6): d 15.3 (CH3), 23.9 (CH3CO),
27.0 (CH2), 68.0 (C5H4), 68.7 (C5H4), 69.0 (C5H5), 85.4 (C C5H4),
118.9 (2 CHarom), 126.1 (CHarom), 128.3 (2 CHarom), 128.7 (2 CHarom),
129.4 (2 CHarom), 136.6 (C), 136.9 (C), 137.5 (C), 139.0 (C), 144.3 (C),
168.1 (CO).
142.6 and 142.7 (C), 143.6 (C), 144.0 and 144.6 (C); IR (KBr,
m
cmÀ1): 3474, 3380 (NH2), 3077, 3025, 2959, 2929, 2870 (CH2,
CH3); MS (EI, 70 eV) m/z: 299 [M]+., 284 [M-CH3]+; Anal. Calc. for
C22H21N: C, 88.25; H, 7.06; N, 4.67. Found: C, 88.09; H, 7.09; N,
4.66%.
4.2.4. 1-(4-N-acetamidophenyl)-1,2-bis-phenyl-but-1-ene, (E+Z)-5
The synthesis was identical to that of 3, except that compound
(Z+E)-4 (0.299 g, 1 mmol) was used in place of compound (Z+E)-2,
to furnish a mixture of Z and E isomers (85/15). After recrystalliza-
tion from ethanol, the ratio was 95/5; Yield 81%.
1H NMR (300 MHz, acetone-d6): d 0.95 and 0.97 (t, J = 7.4 Hz,
3H, CH3), 2.03 and 2.12 (s, 3H, CH3CO), 2.50 and 2.55 (q,
J = 7.4 Hz, 2H, CH2), 6.77–7.75 (m, 14H, Harom), 9.0 and 9.22 (s
broad, 1H, NH); 13C NMR (75 MHz, acetone-d6): d 13.7 (CH3),
24.2 (CH3CO), 29.5 (CH2), 118.7 and 118.8 (2 CHarom), 126.6 and
127.0 (CHarom), 127.0 and 127.5 (CHarom), 128.2 and 128.7 (2 CHarom),
128.6 and 129.0 (2 CHarom), 130.1 and 130.4 (2 CHarom), 130.5 (2
CHarom), 131.4 and 131.7 (2 CHarom), 138.4 (C), 138.7 (C), 139.5
(E)-3: 1H NMR (300 MHz, DMSO-d6): d 1.00 (t, J = 7.5 Hz, 3H,
CH3), 2.06 (s, 3H, CH3CO), 2.47 (q, J = 7.5 Hz, 2H, CH2), 3.80 (t,
J = 1.0 Hz, 2H, C5H4), 4.10 (t, J = 1.0 Hz, 2H, C5H4), 4.13 (s, 5H,
C5H5), 7.08 (d, J = 8.5 Hz, 2H, Harom), 7.15 (d, J = 8.5 Hz, 2H, Harom),
7.26 (m, 3H, Harom), 7.54 (d, J = 8.5 Hz, 2H, Harom), 9.93 (s, 1H,
NH); 13C NMR (75 MHz, DMSO-d6): d 15.3 (CH3), 23.9 (CH3CO),
27.0 (CH2), 68.0 (C5H4), 68.7 (C5H4), 69.0 (C5H5), 85.4 (C C5H4),
118.9 (2 CHarom), 126.1 (CHarom), 128.3 (2 CHarom), 128.9 (2 CHarom),
129.1 (2 CHarom), 136.6 (C), 136.9 (C), 137.5 (C), 139.0 (C), 144.3 (C),
168.1 (CO).
(C), 142.5 (C), 143.2 (C), 144.5 (C), 168.6 (CON); IR (KBr, m
cmÀ1):
3458, 3294, 3251 (NH), 3103, 3045, 2969, 2929, 2865 (CH2, CH3),
1663 (CON); HRMS (EI, 70 eV, C24H23NO: M+) Calc.: 341.1780.
Found: 341.1776.
4.3. Biochemical studies
4.3.1. Materials
Stock solutions (10À3 M and 10À2 M) of the compounds to be
tested were prepared in DMSO and were kept at À20 °C. Under
these conditions, they are stable 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
MCF7 (hormone-dependent) and MDA-MB231 (hormone-indepen-
dent) were from the American Type Culture Collection (ATCC – LGC
Promochem). Sheep uteri weighing approximately 7 g, were ob-
tained 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).
The product was recrystallized from a mixture of ether/pentane
to give the pure Z isomer: M.p.: 206 °C; Rf: 0.35 (ether/pentane = 1/
1); IR (KBr,
m
cmÀ1): 1656 (CON); MS (EI, 70 eV) m/z: 449 [M]+, 407,
384 [MÀC5H5]+, 342, 326, 121 [FeC5H5]+; Anal. Calc. for C28H27FeNO:
C, 74.84; H, 6.06; N, 3.12. Found: C, 74.82; H, 5.85; N, 3.02%.
4.2.3. 1-(4-Aminophenyl)-1,2-bis-phenyl-but-1-ene, (Z+E)-4
Zinc powder (3.92 g, 60 mmol) was suspended in 30 ml of THF
at 5–10 °C in a Schlenk tube under argon. While stirring, titanium
tetrachloride (5.69 g, 30 mmol) was added slowly via a syringe. The
reaction mixture was removed from the cold bath and refluxed for
1h30. To the reaction mixture was added 15 mL of a THF solution
containing propiophenone (1.34 g, 10 mmol) and 4-aminobenzo-
phenone (1.97 g, 10 mmol), and reflux conditions were maintained
for three days. The reaction mixture was poured into 100 mL of
water, and extracted with 3 Â 100 mL of dichloromethane. The or-
ganic phase was washed with 100 mL of water, dried over magne-
sium sulfate, filtered, and the solvent was evaporated. The oil was
purified on a silica gel column using dichloromethane as an eluent.
Compound 4 was obtained as an 85/15 mixture of Z and E isomers
(major isomer was not identified) and was recrystallized from eth-
anol; Yield 38%.
4.3.2. Determination of the relative binding affinity (RBA) of the
compounds for ER
RBA values were measured on ER
and on purified ERb (PanVera). Sheep uterine cytosol prepared in
buffer (0.05 M Tris–HCL, 0.25 M sucrose, 0.1% b-mercap-
toethanol, pH 7.4 at 25 °C) as previously described [64] was used
as a source of ER . For ERb, 10 L of the solution containing
a and ERb
a
from lamb uterine cytosol
A
a
l
3500 pmol/mL 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 lL) of ERa in glass tubes or ERb
in polypropylene tubes were incubated for 3h30 at 0 °C with
[6,7-3H]-estradiol (2 Â 10À9 M, specific activity 1.62 TBq/mmol,
NEN Life Science Product) in the presence of nine concentrations
of the compounds to be tested (between 6 Â 10À7 M and 6 Â
4.2.3.1. Alternative pathway. The synthesis was identical to the
alternative pathway of 2, except that propiophenone (0.134 g,
1 mmol) was used in place of propionyl ferrocene, to furnish a mix-
ture of Z and E isomers (85/15); Yield 55%. As for 2 the yield varies
greatly with the batch of 4-nitrobenzophenone used.
10À9 M) or of non-radioactive E2 (between 8 Â 10À8
M and
8 Â 10À10 M). At the end of the incubation period, the free and

P. Pigeon et al. / Journal of Organometallic Chemistry 694 (2009) 895–901
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precipitation. The percentage reduction in binding of [3H]-estradiol
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