The influence of phenolic hydroxy substitution on the electron transfer and anti-cancer properties of compounds based on the 2-ferrocenyl-1-phenyl-but-1- ene motif

Article abstract of DOI:10.1039/b705030e

The ferrocenyl compound 2-ferrocenyl-1,1-bis(4-hydroxyphenyl)-but-1-ene (3), is very cytotoxic against breast cancer cells (IC₅₀ = 0.44 M against MDA-MB-231). We now report the synthesis of a new series of para- and meta- substituted mono- and di- ferrocenyl phenols [2-ferrocenyl-1-(3- hydroxyphenyl)-1-phenyl-but-1-ene (6), 2-ferrocenyl-1-(3-hydroxyphenyl)-1-(4- hydroxyphenyl)-but-1-ene (7), 1,2-di-ferrocenyl-1-(4-hydroxyphenyl)-but-1-ene (8), and 1,2-di-ferrocenyl-1-(3-hydroxyphenyl)-but-1-ene (9)] and their electrochemical and biochemical properties, especially in comparison to the previously reported standard compounds [2-ferrocenyl-1-(4- hydroxyphenyl)-1-phenyl-but-1-ene (2) and (3)]. We also report the synthesis and characterization of the diphenyl analogue, 2-ferrocenyl-1,1-diphenyl-but-1-ene (5). This structure-activity relationship study was motivated by our hypothesis that the cytotoxicity of 3 is related to its ability to form a quinone methide structure after two in situ 1-electron oxidations, a process which requires the presence of at least one p-phenol. The mono-ferrocenyl compounds (including those previously reported) are reasonably well recognized by the oestrogen receptors ?? (RBAs = 0.9-9.6%) and β (RBAs = 0.28-16.3%), although the bulkier di-ferrocenyl compounds show very little affinity. In vitro, the cytotoxic effects of the phenolic complexes are related to the positioning of the hydroxyl group (para- superior to meta-), and to the number of ferrocenyl groups (one superior to two), with IC₅₀ values against the MDA-MB-231 cell line ranging from 0.44-3.5 M. On the hormone-dependent breast cancer cell line MCF-7, the observed effect seems to be the result of two components, one cytotoxic (antiproliferative) and one estrogenic (proliferative). Electrochemical studies show that only the compounds with a p-phenol engage in proton-coupled intramolecular electron transfer. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b705030e

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/dalton | Dalton Transactions
The influence of phenolic hydroxy substitution on the electron
transfer and anti-cancer properties of compounds based on the
2-ferrocenyl-1-phenyl-but-1-ene motif†
Elizabeth Anne Hillard,a Pascal Pigeon,a Anne Vessie`res,a Christian Amatoreb and Ge´rard Jaouen*a
Received 3rd April 2007, Accepted 25th May 2007
First published as an Advance Article on the web 20th September 2007
DOI: 10.1039/b705030e
The ferrocenyl compound 2-ferrocenyl-1,1-bis(4-hydroxyphenyl)-but-1-ene (3), is very cytotoxic against
breast cancer cells (IC50 = 0.44 lM against MDA-MB-231). We now report the synthesis of a new series
of para- and meta- substituted mono- and di- ferrocenyl phenols [2-ferrocenyl-1-(3-hydroxyphenyl)-1-
phenyl-but-1-ene (6), 2-ferrocenyl-1-(3-hydroxyphenyl)-1-(4-hydroxyphenyl)-but-1-ene (7),
1,2-di-ferrocenyl-1-(4-hydroxyphenyl)-but-1-ene (8), and 1,2-di-ferrocenyl-1-(3-hydroxyphenyl)-
but-1-ene (9)] and their electrochemical and biochemical properties, especially in comparison to the
previously reported “standard” compounds [2-ferrocenyl-1-(4-hydroxyphenyl)-1-phenyl-but-1-ene (2)
and (3)]. We also report the synthesis and characterization of the diphenyl analogue, 2-ferrocenyl-
1,1-diphenyl-but-1-ene (5). This structure–activity relationship study was motivated by our hypothesis
that the cytotoxicity of 3 is related to its ability to form a quinone methide structure after two in situ
1-electron oxidations, a process which requires the presence of at least one p-phenol. The
mono-ferrocenyl compounds (including those previously reported) are reasonably well recognized by
the oestrogen receptors a (RBAs = 0.9–9.6%) and b (RBAs = 0.28–16.3%), although the bulkier
di-ferrocenyl compounds show very little affinity. In vitro, the cytotoxic effects of the phenolic
complexes are related to the positioning of the hydroxyl group (para- superior to meta-), and to the
number of ferrocenyl groups (one superior to two), with IC50 values against the MDA-MB-231 cell line
ranging from 0.44–3.5 lM. On the hormone-dependent breast cancer cell line MCF-7, the observed
effect seems to be the result of two components, one cytotoxic (antiproliferative) and one estrogenic
(proliferative). Electrochemical studies show that only the compounds with a p-phenol engage in
proton-coupled intramolecular electron transfer.
activity. Cytotoxic pathways involving DNA have been suggested
Introduction
for the activity of ferrocenyl compounds.12
Although the anti-cancer properties of ferrocene-containing
Our laboratory has been studying the effects on the proliferation
molecules were first studied in the late 1970s,1 systematic in-
of breast cancer cells of ferrocenyl phenols, especially those based
on the 1,1-diphenyl-but-1-ene motif.13, 14 Some small organic phe-
nols have been shown to possess oestrogen receptor modulating
properties,15 and, at higher concentrations, cytotoxic properties.16
We have been trying to enhance the cytotoxicity of these types of
compounds by the addition of ferrocene, which we hope will be
oxidized to ferricenium within the cell. We routinely test the prolif-
erative/antiproliferative effects of our new compounds on MCF-7
(oestrogen receptor positive) and MDA-MB-231 (oestrogen recep-
tor negative) breast cancer cell lines, and have had varying degrees
of success. The most promising compounds to date are shown
in Chart 1, all of which exhibit IC50 values at low micromolar
or submicromolar concentrations for the MDA-MB-231 cell line.
The MCF-7 cell line is the standard for hormone dependant breast
cancers, and the proliferative/antiproliferative effects connected to
the estrogenicity/antiestrogenicity of molecules at concentrations
of 10−5–10−7 M are primarily mediated by the oestrogen receptor
alpha (ERa), a nuclear receptor present in these cells. It is known
that the dimethyl amino chain of the active metabolite of the breast
cancer drug tamoxifen, and also present in compound 1, interacts
with ERa in such a way as to prevent DNA transcription and
vestigations were not carried out until Ko¨pf-Meyer and Neuse
established anti-tumour activity for ferricenium salts in 1984.2
This work led to the proposal that ferrocenyl compounds could be
activated in the cell by biooxidation, and that both ferricenium-
and ferrocene- (in a water soluble form) containing compounds
could give rise to cytotoxic effects.3 To this end ferrocene has
been incorporated into water soluble polymers,4 tethered to a
DNA intercalator,5 phosphino compounds,6 vitamin B1,7 and
other biomolecules.8 Diferrocene compounds9 and ferrocene-
bearing transition metal ligands,10 and a variety of other small
ferrocenyl molecules11 have also been investigated for anti-cancer
aLaboratoire de Chimie et Biochimie des Complexes Mole´culaires,
UMR CNRS 7576, Ecole Nationale Supe´rieure de Chimie de Paris,
11 rue Pierre et Marie Curie, 75231, Paris, Cedex 05, France.
E-mail: gerard-jaouen@enscp.fr; Fax: +33 1 43 26 00 61; Tel: +33 1 43
26 95 55
bEcole Normale Supe´rieure, De´partement de Chimie, UMR CNRS-ENS-
UPMC 8640 ‘PASTEUR’, 24 rue Lhomond, F-75231, Paris, Cedex 05,
France
† Based on the presentation given at Dalton Discussion No. 10, 3rd–5th
September 2007, University of Durham, Durham, UK.
This journal is
The Royal Society of Chemistry 2007
Dalton Trans., 2007, 5073–5081 | 5073
©

View Article Online
Chart 1 Previously reported cytotoxic ferrocenyl phenolic compounds 1,14 2,19 3,13 4.20
cell replication.17 The other compounds, lacking the amino chain,
would be expected to be estrogenic and proliferative on MCF-
7 cells. However, compounds 2–4 have shown antiproliferative
effects in both the MCF-7 (ER+, hormone dependent) and MDA-
MB-231 (ER−, hormone independent) cell lines, which can be
attributed only to the innate cytotoxicity of the molecule. It is
important to note that 1–4 conform to a particular structural
motif, where the ferrocenyl group is located on carbon 2 of
the but-1-ene group, the phenol group(s) resides on carbon 1,
and a conjugated p-system exists between the ferrocenyl and
phenol groups. It appears that this motif is directly related to the
cytotoxic effects shown by these compounds. In a previous study
we discovered that compounds 1–4 exhibit cyclic voltammograms
characteristic of an interesting structural rearrangement due to
an intramolecular electron transfer from the phenolic donor to
the electrochemically generated ferricenium acceptor in basic
conditions.18 We have proposed that the final outcome of this
process is the formation of a reactive quinone methide-type (QM)
structure after two one-electron oxidations and the loss of two
protons.
Chart 2 New compounds studied in this report.
Donor–acceptor assemblies possessing a ferrocene donor have
been extensively studied, especially in terms of their non-linear
optical properties,21 and structural rearrangements as a result
of proton- or metal ion-coupled intramolecular electron transfer
processes.22 However, the possibility of a ferricenium moiety acting
as an acceptor has only recently been explored.23, 24 In particular,
a paper by Nishihara and co-workers in Tokyo described the
rearrangement of 2-(2-ferrocenylvinyl)hydroquinone to a novel
allene quinonoid structure via two one-electron oxidations and
intramolecular electron transfer to a ferricenium acceptor.25
In order to test our hypothesis that QM-generation is related
to the observed cancer cell death, and to further study this novel
mechanism of formation, we have synthesized a number of new
compounds, including those where the hydroxyl group of the
phenol is in the meta-position. For example, in compound 6, the
ferrocene–p-system–phenol motif is maintained, but it carries a
m-phenol, so that a QM structure is not accessible. Compound
7 is similar to 6, with the addition of a p-phenol group; in this
case we expect that the m-phenol will act as a spectator, and the p-
phenol might engage in QM formation. We have also synthesized
the diferrocenyl p-phenol (8), and m-phenol (9) to evaluate the
importance of the steric effect on the biological efficacy of the
compounds. We here report the synthetic, electrochemical, and
biochemical results for the new compounds 5–9 shown in Chart
2, and compare these results to those of previously reported
compounds 2 and 3 as appropriate.
Results and discussion
Synthesis
The formation of the new compounds 5–9 was generally ac-
complished by a Friedel–Craft acylation of ferrocene, followed
by a McMurry cross-coupling of the ferrocenyl ketone with
the appropriate benzophenone. Thus, to synthesize compounds
8 and 9, we first prepared the known ketone 1026 (yield 62%)
and the new ketone 11 (yield 82%) by a Friedel–Craft reaction
of the corresponding acyl chlorides with ferrocene, as shown in
Scheme 1. The ketones were demethyled with boron tribromide in
dichloromethane to give the phenolic compounds 12 and 13 with
yields of 74% and 76%, respectively. The yield for the formation of
12 using boron tribromide was higher than the previously reported
Scheme 1
5074 | Dalton Trans., 2007, 5073–5081
This journal is
The Royal Society of Chemistry 2007
©

View Article Online
◦
ꢀ
demethylation of 10 with aluminium trichloride (32%),26 but the
reaction time was longer (unoptimized 12 h vs. 1.25 h). The
McMurry reaction of these ketones with propionyl ferrocene27
gave the cross-coupled diferrocenyl compounds 8 and 9 with a
yield of 39% for each, Scheme 2.
The separation of observed redox potentials (DE ) for the two
waves generated by compounds 8 and 9 was not significantly
different: 162 and 159 mV, respectively. These values are slightly
=
lower than that of trans-Fc(CH CH)Fc, which has been reported
as 170 mV in CH2Cl2,30 although the disparity can be accounted
for by the decrease in Coulombic repulsion due to the solvent cage.
The presence of two one-electron oxidation waves instead of one
two-electron oxidation wave signifies a stabilization of the mixed
valence species (FcII, FcIII), the extent of which is often expressed
as the comproportionation constant, Kc. For 8 and 9, Kc = 550
and 490, respectively, (using the equation DE◦ = (RT/F)lnKc)30
ꢀ
and thus these compounds can be considered Robin–Day Class II
mixed-valent complexes, with moderate electron coupling between
the oxidized and reduced centres.31 Oxidation potentials vs. SCE
are given in Table 1.
In terms of the interaction of the electrochemically generated
cations with the added pyridine, the compounds can be divided
into two categories. For compounds 5, 6 and 9, which do not
possess p-phenols, no significant difference in the electrochemical
behaviour was observed upon the addition of pyridine, Fig. 1a,b.
Conversely, for those compounds carrying a p-phenol, 7 and 8, the
addition of pyridine altered the CV substantially, especially in view
of the reversibility of the ferrocene redox couple and the emergence
of a new peak slightly higher in potential than the ferrocene oxida-
tion. We have previously observed this electrochemical behaviour
for compounds 1–4, and have attributed it to an intramolecular
electron transfer from the organic skeleton to the ferricenium
moiety coupled with deprotonation of the phenol by pyridine,
eventually resulting in a QM structure.18 While the electrochemical
behaviour of compound 7 is similar to that of compounds 1–4, that
of 8 is complicated by the presence of a second ferrocenyl group.
The voltammetry of 8 in the presence and absence of pyridine
shows that the intramolecular electron transfer leading ultimately
to the quinone methide formation occurs at the level of the first
oxidation wave. Since the ferricinium group attached to the C1
Scheme 2
In the same manner, the McMurry reaction of propionyl
ferrocene with dibenzophenone gave compound 5 with a yield
of 25%, Scheme 3. This compound was impossible to separate
by silica gel column chromatography from the two homo-coupled
compounds, due to the similar (low) polarities of these compounds
lacking any phenol group. Compound 5 was directly isolated by
preparative HPLC of the crude mixture.
Table 1 Formal oxidation and redox potentials for compounds 2, 5–9 vs.
SCE.a
Scheme 3
Electron
transfer?
ꢀ
Solvent
MeOH
Ep,o (Fc) Ep,o (other) E◦ (Fc/Fc+)c
The mono-ferrocenyl compounds 6 and 7 were synthesized
from propionyl ferrocene by a McMurry reaction using the
corresponding phenolic ketones (commercially available for 6, and
known from the literature for 728,29) to give a mixture of Z and E
isomers in an approximately 1 : 1 ratio.
Compounds 6 and 7 were purified on a silica gel column with
dichloromethane or a dichloromethane–acetone solution as the
eluent, and then were re-purified via HPLC. Z and E isomers of
compounds 6 and 7 were impossible to separate, while isomers of
compounds 8 and 9 were partially separated but rapidly (within a
few hours) isomerised to give a 1 : 1 mixture of Z and E isomers.
2b
0.397(2)
0.357(2)
Yes
0.97(1)d
d
MeOH–py 0.423(4)d,e
0.510(3)c
0.421(3)
5b
6
MeOH
MeOH–py 0.442(3)
MeOH 0.403(3)
MeOH–py 0.408(3)
0.380(3)
0.400(3)
0.365(3)
0.375(3)
0.386(3)
No
No
Yes
7
MeOH
0.418(3)
1.01(1)d
d
MeOH–py 0.43(1)d,e
0.528(3)d
8
9
MeOH
0.334(3) 0.504(3)
0.300(3) 0.462(3) Yes
d
MeOH–py 0.34(1)d,e
0.444(3)d
0.649(3)
Cyclic voltammetry.
0.703(3)
0.344(3) 0.503(3)
MeOH
0.311(3) 0.470(3) No
0.313(3) 0.488(3)
Compounds 5–9 were studied by cyclic voltammetry in methanol
and methanol–pyridine solutions. In methanolic solutions, all
of the compounds gave rise to the expected reversible fer-
rocene/ferricenium redox waves, with 5, 6, and 7 giving rise to one
wave, and compounds 8 and 9 displaying 2 one-electron waves.
MeOH–py 0.346(3) 0.523(3)
a Scan rate = 0.1 V s−1
.
b Values reported from reference 18. c E◦ꢀ/V is
the average of the anodic and cathodic peak potentials. d Irreversible.
e Shoulder.
This journal is
The Royal Society of Chemistry 2007
Dalton Trans., 2007, 5073–5081 | 5075
©

View Article Online
comparison of the ferrocene oxidation potentials in MeOH for 2
(0.397 V), 5 (0.421 V), and 6 (0.403 V), which show little difference,
and hence little stabilization of the radical by the phenol group.
The answer may be found instead in considering the stability
of the neutral radical species which is generated after electron
transfer and deprotonation. By drawing the canonical structures
contributing to the delocalization of the radical, Chart 3, we
can see that the p-phenol is able to delocalize the radical onto
the C1,C2-alkene, while the radical in the m-phenol can only be
delocalized over the phenol itself. Thus, complexes with a para-
substituted phenol benefit from greater resonance stabilization
and are therefore energetically more accessible intermediates than
their m-phenol analogues.
Fig. 1 Cyclic voltammograms of 6 (a), 9 (b), 7 (c), and 8 (d) in 0.1 M
Bu4NBF4/MeOH in absence (solid line) and presence (dashed line) of
pyridine in a 1 : 6 volume ratio. Scan rate 0.1 V s−1. Pt electrode of
0.5 mm diameter. The CV of compound 5 has been previously published
in reference 18.
Chart 3 Electron delocalization on a) p-phenol and b) m-phenol.
carbon atom of the central double-bond (see Scheme 4) cannot be
conjugated directly to the phenol moiety, one must conclude that
the intramolecular phenol oxidation occurs via the C2 ferricinium.
Yet since both ferrocene units give rise to a significant electron
transfer interaction, one cannot strictly differentiate between the
two under electrochemical conditions. Nevertheless, this suggests
that the C1 ferrocene moiety is oxidized after the electron transfer
to the C2 ferricenium moiety and rearrangement to the quinonoid,
as shown in Scheme 4.
Thus, the electronic environment around the C1 ferrocene is
expected to be substantially changed when it is finally oxidized.
This is confirmed by the large anodic shift of its oxidation
potential in MeOH–py compared to that in MeOH (0.703 vs.
0.504 V, respectively). The influence of nearby quinonoids on
the destabilization of the ferricenium cation has been previously
observed, and has been attributed to partial electron donation
from the ferrocene to the quinonoid moiety.24
We asked ourselves why only the compounds possessing a p-
phenol engaged in electron transfer, given the ostensible similar-
ities in molecular and electronic structure between these com-
pounds. We have previously shown in these types of compounds
that in the initial cation the radical is localized on the ferricenium
group, and that there is little electronic delocalization with the
phenol group prior to deprotonation.18 This is supported by the
Biochemistry
RBA and lipophilicity values. The affinities for the oestrogen
receptor of the newly synthesized complexes were measured on
the two isoforms of the oestrogen receptor, ERa and ERb, and
are reported as relative binding affinity (RBA) values in Table 2.
All the compounds were recognized by both forms of the ER but
the RBA values are quite different ranging from high (about 5%)
to low values (less than 1%). In the mono-ferrocenyl series the
change of the OH substituent from the para- to the meta-position
induced only a slight decrease of the RBA value for the alpha form
of the estrogen receptor (4.6% versus 3.6% for the monophenols,
9.6% versus 5.4% for the diphenols). On the contrary, the presence
of an m-OH substituent dramatically decreased the affinity of the
complexes for the beta form of the oestrogen receptor with an
RBA ratio ERb/ERb of 21 for the monophenols 2 and 6, and 6.8
for the diphenols 3 and 7. Quite surprisingly, the diphenyl complex
5, i.e. the compound with no OH, has a non-zero RBA value of
0.9%. A similar RBA value (0.8%) was previously reported for
the corresponding organic molecule, 1,1,2-triphenylbut-1-ene.32
Finally, the RBA values found for the di-ferrocenyl derivatives
are quite low for both forms of the oestrogen receptor. This is
probably due to the presence of the two ferrocenyl units which are
bulkier than a phenyl substituent. Regarding the log Po/w values,
Scheme 4
5076 | Dalton Trans., 2007, 5073–5081
This journal is
The Royal Society of Chemistry 2007
©

View Article Online
Table 2 Relative binding affinity (RBA) for the alpha form of the oestrogen receptor (ERa from cytosol) and ERb (purified), IC50 values on MDA-MB-
231 hormone-independent breast cancer cells and lipophilicity (log Po/w) of the complexes
RBA (%)a
Compound
ERa (cytosol)
ERb
IC50/lM on MDA-MB-231a
log Po/w
17b-Estradiol
2 (Z + E)
3
100b
100b
—
3.2
4.6 0.1c
9.6 0.6d
0.9 0.1
3.6 0.1
5.4 1.2
0.24 0.15
0.15 0.01
11 1c
1.13 0.07c
0.44 0.08
—
6.0 (Z)c6.13 (E)c
16.3 1.5d
0.28 0.03
0.53 0.06
2.4 0.5
0.06 0.03
0.01 0.01
5.0d
6.43
5.80
5.08
6.4
5
6 (Z + E)
7 (Z + E)
8 (Z + E)
9 (Z + E)
2.7 0.1
1.03 0.01
2.8 0.1
3.5 0.2
6.4
a Mean of two experiments range. b Value by definition. c Value from reference 19. d Value from reference 13.
the change of the OH group from the para- to the meta- position
plays no role, and, as expected, the complexes with no hydroxyl
groups or with two ferrocenyl substituents are more hydrophobic
with log Po/w values around 6.4.
compounds 2 and 7 are more cytotoxic (IC50 values around 1 lM);
while 3 with an IC50 of 0.44 lM is the most cytotoxic of the series.
These results show that the repositioning of one OH group from
the para- to the meta-position significantly lowers the cytotoxicity
of the complex (ratio of IC50 values of 6 versus 2 and 7 versus 3 being
respectively 2.4 and 2.3). The presence of a second ferrocenyl unit
decreases the cytotoxicity of the complexes (IC50 of 2.8 lM for 8
and 1.13 lM for 2; 3.5 lM for 9 and 2.7 lM for 6); here also the
complex with a m-OH group is less cytotoxic than the one with a
p-OH. Finally, as expected, estradiol has no effect on these cells
with no ERa.
On the contrary, the effect observed on hormone-dependent
breast cancer cells is the result of the estrogenic (proliferative)
effect expected for these compounds that all show an affinity for
the alpha form of the ER minus its cytotoxic component observed
on the hormone-independent cancer cells. At a concentration of
1 lM and in a medium without phenol red, which is best suited
to the expression of the estrogenic component of a molecule, only
the most cytotoxic complexes 2 and 3 are able to reverse the strong
estrogenic effect shown by estradiol. As expected complexes 5 and
6 which are the less cytotoxic on the MDA-MB-231 cells show a
clear proliferative effect.
Effect of the compounds on the growth of breast cancer cells.
The effect of these complexes at a concentration of 1 × 10−6 M was
studied on hormone-independent (MDA-MB-231) and hormone-
dependent (MCF-7) breast cancer cells and the results are
displayed in Fig. 2. The antiproliferative effect observed on the
hormone-independent breast cancer cells can be attributed only
to a cytotoxic effect potentially induced by the ferrocenyl unit. On
these cells, complexes 6, 8, and 9 show the lowest antiproliferative
effects with IC50 values between 2.7 and 3.5 lM (Table 2);
Experimental
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.
Infrared spectra were obtained on an IRFT 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
spectrometer. 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 of 2 cm, particles size of 10 lm).
Fig. 2 Effect of 1 lM of the compounds and of 1 nM of estradiol (E2)
on cell growth after 5 days of culture in medium (without phenol red) of
MDA-MB-231 (hormone-independent breast cancer cells) and of MCF-7
(hormone-dependent breast cancer cells). C = control.
This journal is
The Royal Society of Chemistry 2007
Dalton Trans., 2007, 5073–5081 | 5077
©

View Article Online
Cyclic voltammograms were obtained utilizing an Autolab PG-
Stat20 potentiostat, driven by GPES software (General Purpose
Electrochemical System, Version 4.8, EcoChemie B.V., Utrecht,
the Netherlands), a platinum wire counter electrode, a 500 lM
platinum disc working electrode, and an aqueous standard calomel
reference electrode. Analyte solutions were 1–2 mM in MeOH with
0.1 M Bu4NBF4 supporting electrolyte. Solvent ratios were 6 : 1
MeOH : py, except for compound 5, where the ratio was 3 : 1
MeOH : py. Unfortunately, the insolubility of these compounds
in water prevented the preparation of aqueous samples at suitable
concentrations. Solvents were spectrometric grade and used as
received.
Formation of compound 13. This compound was obtained as
an orange dark solid (2.3 g, 76% yield). H NMR (300 MHz,
1
CDCl3) d 4.12 (s, 5 H, Cp), 4.53 (s, 2 H, C5H4), 4.87 (s, 2 H, C5H4),
7.02 (d, J = 6.8 Hz, 1 H, Harom), 7.12–7.41 (m, 3 H, Harom), 7.45 (s,
1 H, OH). 13C NMR (CDCl3) d 70.4 (5 CH Cp), 71.7 (2 CH C5H4),
73.0 (2 CH C5H4), 77.7 (C C5H4), 115.1 (CHarom), 119.2 (CHarom),
120.3 (CHarom), 129.4 (CHarom), 140.8 (C), 156.4 (C), 200.3 (CO).
IR: 3423 (OH), 2928, 2856 (CH3), 1614 (CO) cm−1. MS (CI, NH3)
m/z: 307 [MH]+• , 324 [M + NH4]+. Anal. Calcd for C17H14FeO2:
C, 66.69; H, 4.60. Found: C, 66.83; H, 4.57.
General procedure for formation of compounds 5, 6, 7, 8, 9.
Titanium tetrachloride (3.6 mL, 33 mmol) was added dropwise
to a suspension of zinc powder (4 g, 61 mmol) in 80 mL of
dry THF at 0 ◦C. The mixture was heated at reflux for 2 h. A
second solution was prepared by dissolving propionyl ferrocene
(2.42 g, 10 mmol) and the corresponding ketones (10 mmol) in
50 ml of dry THF. This latter solution was added dropwise to
the first solution and then the reflux was continued for 2 h. After
cooling to room temperature, the mixture was stirred with water
and dichloromethane. The mixture was acidified with diluted
hydrochloric acid until the dark colour disappeared and was
decanted. The aqueous layer was extracted with dichloromethane
and the combination of organic layers was dried over magnesium
sulfate. After concentration under reduced pressure, the crude
product was chromatographed on a silica gel column with
dichloromethane or a solution of dichloromethane–acetone 95 : 5
as the eluent (5 excepted). For the biological tests, each products
were re-purified on semi-preparative HPLC with acetonitrile–
water or acetonitrile as the eluent to give pure 5, 6, 7, 8, and 9.
The isomers, if any, were either inseparable, or partially separated
but remixed in the same flask (because of rapid isomerisation)
before evaporation of acetonitrile under reduced pressure. The
mixture was extracted with dichloromethane and water, decanted,
dried over magnesium sulfate, and concentrated under reduced
pressure. The mixture of isomers, if any, was recrystallized in the
appropriate solvent.
Synthesis and characterization
Compound 10. The synthesis is described in reference 26.
Compound 11. Ferrocene (10.2 g, 54.8 mmol) was dissolved
in dry dichloromethane (400 mL). Aluminium trichloride (7.30 g,
54.8 mmol) was added in portions over 15 min. Then, m-anisoyl
chloride (7.79 g, 45.7 mmol) was added slowly over 20 min and
the stirring was continued overnight. The solution was slowly
poured into a mixture of water and ice and decanted. The aqueous
layer was extracted with dichloromethane and the combined
organic layers were washed with water, dried over magnesium
sulfate and concentrated under reduced pressure. The mixture
was chromatographed on a silica gel column with a solution
of dichloromethane–petroleum ether 50 : 50 as the eluent. The
pure product 11 was obtained as an oil (11.92 g, 82%). 1H NMR
(300 MHz, CDCl3) d 3.81 (s, 3 H, OCH3), 4.14 (s, 5 H, Cp), 4.51 (t,
J = 1.9 Hz, 2 H, C5H4), 4.85 (t, J = 1.9 Hz, 2 H, C5H4), 7.02 (ddd,
J = 8.0, 3.6, 1.0 Hz, 1 H, Harom), 7.30 (t, J = 8.0 Hz, 1 H, Harom),
7.35 (m, 1 H, Harom), 7.42 (dt, J = 8.0, 1.0 Hz, 1 H, Harom). 13C
NMR (CDCl3) d 55.5 (OCH3), 70.2 (5 CH Cp), 71.5 (2 CH C5H4),
72.6 (2 CH C5H4), 78.1 (C C5H4), 113.1 (CHarom), 117.6 (CHarom),
120.6 (CHarom), 129.2 (CHarom), 141.1 (C), 159.5 (C), 198.9 (CO).
IR: 3098, 2938, 2836 (CH3), 1638 (CO) cm−1. MS (CI, NH3) m/z:
321 [MH]+• .
2-Ferrocenyl-1,1-di-phenyl-but-1-ene, 5. The reaction was per-
formed with 1.82 g (10 mmol) of commercially available benzophe-
none. The crude product was directly purified on HPLC with
acetonitrile as eluent to yield 5 as an orange solid (0.96 g, 25%
yield). The compound was recrystallized from acetonitrile. Mp:
160 C. H NMR (300 MHz, CDCl3) d 0.97 (t, J = 7.5 Hz, 3
H, CH3), 2.50 (q, J = 7.5 Hz, 2 H, CH2), 3.80 (t, J = 1.9 Hz,
2 H, C5H4), 3.99 (t, J = 1.9 Hz, 2 H, C5H4), 4.04 (s, 5 H, Cp),
6.95–7.36 (m, 10 H, Harom). 13C NMR (CDCl3) d 15.5 (CH3), 27.8
(CH2), 68.2 (2 CH C5H4), 69.2 (5 CH Cp), 69.3 (2 CH C5H4),
86.5 (C C5H4), 126.2 (2 CHarom), 128.2 (2 CHarom), 128.3 (2 CHarom),
129.3 (2 CHarom), 129.8 (2 CHarom), 137.4 (C), 138.0 (C), 144.5 (C),
144.7 (C). IR: 3078, 3044, 2963, 2930, 2869 (CH2, CH3) cm−1. MS
(EI, 70 eV) m/z: 392[M]+• , 363 [M − Et]+, 327 [M − Cp]+, 121
[CpFe]+. HRMS (EI, 70 eV, C26H24Fe: M+) calcd: 392.1228, found:
392.1218. Anal. Calcd for C26H24Fe: C, 79.59; H, 6.16. Found: C,
79.37; H, 6.11.
Demethylation of compounds 10 and 11. Compound 10 or 11
(3.2 g, 10 mmol), was dissolved in dry dichloromethane (60 mL)
at 0 ◦C and boron tribromide (2.84 mL, 30 mmol) was added.
The mixture was stirred overnight at room temperature. The
solution was slowly poured into a mixture of water and ice and
extracted with ethyl acetate. The combined organic layers were
washed with water, dried over magnesium sulfate and concentrated
under reduced pressure. The mixture was chromatographed on
a silica gel column with dichloromethane as the eluent to yield
the phenol compounds which were recrystallized from an ether–
pentane solution.
◦
1
Formation of compound 12, anisoylferrocene. This compound
is described in the literature and was synthesized via the demethy-
lation of 10 using AlCl3 (yield 32%) in place of BBr3, the latter
being used in our case. Compound 12 was obtained as a dark
orange solid (2.24 g, 74% yield). The characteristics were identical
to those described in the literature (mp 186 ◦C, 186–188 ◦C,26 190–
191 ◦C27). Anal. Calcd for C17H14FeO2: C, 66.69; H, 4.60. Found:
C, 66.77; H, 4.45.
2-Ferrocenyl-1-(3-hydroxyphenyl)-1-phenyl-but-1-ene, 6. The
reaction was performed with 1.98 g (10 mmol) of commer-
cially available 3-hydroxybenzophenone. The crude product was
5078 | Dalton Trans., 2007, 5073–5081
This journal is
The Royal Society of Chemistry 2007
©

View Article Online
chromatographed with CH2Cl2 as eluent. The compound was
purified on HPLC with acetonitrile–water 80 : 20 as eluent to yield
6 as an orange solid (0.753 g, 19% yield). The mixture of isomers
and 68.3 (2 CH C5H4), 68.9 and 69.0 (2 CH C5H4), 69.1 (2 × 5
CH Cp), 69.6 and 70.1 (2 CH C5H4), 86.8 and 89.6 (C C5H4), 88.8
and 89.0 (C C5H4), 114.6 and 115.2 (2 CHarom), 130.2 and 131.1 (2
CHarom), 132.5 and 134.8 (C), 136.5 and 136.6 (C), 137.2 and 137.3
(C), 154.0 and 154.3 (C). IR: 3438 (OH), 3092,•2967, 2929, 2872
(CH2, CH3) cm−1. MS (EI, 70 eV) m/z: 516 [M]+ , 451 [M − Cp]+,
121 [CpFe]+. HRMS (EI, 70 eV, C30H28Fe2O: M+) calcd: 516.0840,
found: 516.0820. Anal. Calcd for C30H28Fe2O: C, 69.79; H, 5.46.
Found: C, 69.91; H, 5.39.
1
was recrystallized from heptane. H NMR (300 MHz, CDCl3) d
1.03 and 1.07 (t, J = 7.4 Hz, 3 H, CH3), 2.56 and 2.60 (q, J =
7.4 Hz, 2 H, CH2), 3.87 and 3.94 (t, J = 1.9 Hz, 2 H, C5H4), 4.07
and 4.09 (t, J = 1.9 Hz, 2 H, C5H4), 4.11 and 4.12 (s, 5 H, Cp),
4.53 and 4.62 (s, 1 H, OH), 6.54–6.88 (m, 3 H, Harom), 7.06–7.28
(m, 5 H, Harom), 7.33 (t, J = 7.5 Hz, 1 H, Harom). 13C NMR (CDCl3)
d 15.5 and 15.6 (CH3), 27.8 (CH2), 68.2 and 68.3 (2 CH C5H4),
69.2 (5 CH Cp), 69.3 (2 CH C5H4), 86.3 (C C5H4), 113.2 (CHarom),
116.2 and 116.6 (CHarom), 121.9 and 122.5 (CHarom), 126.2 (CHarom),
128.2 and 128.3 (2 CHarom), 129.3 and 129.8 (2 CHarom), 129.4 and
129.5 (CHarom), 137.4 (C), 137.5 and 137.6 (C), 144.3 and 144.4 (C),
146.2 and 146.3 (C), 155.4 (C). IR: 3498, 3422 (OH), 3078, 3052,
3021, 2959, 2927, 2870 (CH2, CH3) cm−1. MS (EI, 70 eV) m/z:
408 [M]+• , 379 [M − Et]+, 343 [M − Cp]+, 121 [CpFe]+. HMRS
(EI, 70 eV, C26H24FeO: M+) calcd: 408, 1177; found: 408.1172.
Anal. Calcd for C26H24FeO: C, 76.48; H, 5.92. Found: C, 76.39;
H, 5.81.
1,2-di-Ferrocenyl-1-(3-hydroxyphenyl)-but-1-ene, 9. The reac-
tion was performed with 3.06 g (10 mmol) of compound 13. The
crude product was chromatographed with CH2Cl2 as eluent. The
compound was purified on HPLC with acetonitrile–water 90 :
10 as eluent to yield 9 as an orange solid (2.00 g, 39% yield).
The mixture of isomers was recrystallized from an ether–pentane
1
solution. H NMR (300 MHz, CDCl3) d 1.14 and 1.37 (t, J =
7.4 Hz, 3 H, CH3), 2.32 and 2.81 (q, J = 7.4 Hz, 2 H, CH2),
3.69 and 3.74 (t, J = 1.9 Hz, 2 H, C5H4), 3.82 and 4.00 (s, 5 H,
Cp), 4.04 and 4.09 (s, 5 H, Cp), 4.00–4.25 (m, 6 H, C5H4), 4.84
and 4.87 (s, 1 H, OH), 6.65–6.95 (m, 3 H, Harom), 7.30 (t, J =
7.9 Hz, 1 H, Harom). 13C NMR (CDCl3) d 14.5 (CH3), 25.5 and
29.6 (CH2), 66.4 and 67.1 (2 CH C5H4), 66.5 and 67.4 (2 CH
C5H4), 67.9 and 68.0 (2 CH C5H4), 68.2 (2 × 5 CH Cp), 68.6
and 69.0 (2 CH C5H4), 85.6 and 88.3 (C C5H4), 87.4 and 88.5 (C
C5H4), 112.0 and 112.4 (CHarom), 114.9 and 116.0 (CHarom), 120.3
and 121.3 (CHarom), 127.7 and 128.2 (CHarom), 131.7 and 133.5 (C),
135.3 and 135.4 (C), 145.2 and 145.3 (C), 154.5 and 155.0 (C). IR:
3407 (OH), 3094, 2967, 2975, 2927, 2866 (CH2, CH3) cm−1. HRMS
(CI, NH3, C30H29Fe2O: MH+) calcd: 517.0918, found: 517.0922.
Anal. Calcd for C30H28Fe2O: C, 69.79; H, 5.46. Found: C, 69.67;
H, 5.48.
2-Ferrocenyl-1-(3-hydroxyphenyl)-1-(4-hydroxyphenyl)-but-1-
ene, 7. The reaction was performed with 2.14 g (10 mmol) of
the known 3,4ꢀ-dihydroxybenzophenone.29 The crude product was
chromatographed with a solution of CH2Cl2–acetone 95 : 5 as
eluent. The compound was purified on HPLC with acetonitrile–
water 70 : 30 as eluent to yield 7 as an orange solid (2.43 g, 58%
yield). The mixture of isomers was recrystallized from ethanol. 1H
NMR (300 MHz, CD3COCD3) d 1.07 and 1.09 (t, J = 7.4 Hz, 3
H, CH3), 2.64 and 2.65 (q, J = 7.4 Hz, 2 H, CH2), 3.96 and 3.97 (t,
J = 2.0 Hz, 2 H, C5H4), 4.10 and 4.11 (t, J = 2.0 Hz, 2 H, C5H4),
4.15 and 4.16 (s, 5 H, Cp), 6.59–6.65 (m, 1 H, Harom), 6.66–7.05 (m,
5 H, Harom), 7.05–7.25 (m, 2 H, Harom), 8.12 and 8.21 (s, 1 H, OH),
8.24 and 8.27 (s, 1 H, OH). 13C NMR (CD3COCD3) d 15.9 and
16.0 (CH3), 28.2 and 28.3 (CH2), 68.8 (2 CH C5H4), 69.9 (5 CH
Cp), 70.0 (2 CH C5H4), 87.2 and 87.4 (C C5H4), 113.8 and 113.9
(CHarom), 115.8 and 115.9 (2 CHarom), 116.9 and 117.3 (CHarom),
121.2 and 121.7 (CHarom), 129.9 and 130.0 (CHarom), 131.0 and
131.5 (2 CHarom), 136.8 and 136.9 (C), 137.2 and 137.4 (C), 138.7
(C), 147.4 and 147.6 (C), 156.7 and 156.8 (C), 158.1 and 158.2 (C).
IR: 3284, 3397 (OH), 2872, 2931, 2977, 3092 (CH2, CH3) cm−1.
MS (EI, 70 eV) m/z: 424 [M]+• , 395 [M − Et]+, 359 [M − Cp]+,
121 [CpFe]+. HRMS (EI, 70 eV, C26H24FeO2: M+) calcd: 424.1126,
found: 424.1129. Anal. Calcd for C26H24FeO2: C, 73.59; H, 5.70.
Found: C, 73.72; H, 5.85.
Biochemical experiments
Materials
Stock solutions (1 × 10−3 M) of the ferrocenyl complexes 2, 3,
and 5–9 to be tested were prepared in DMSO and were kept
at 4 ◦C in the dark; under these conditions they are stable at
least two months. Serial dilutions in DMSO were prepared just
prior to use. A stock solution (1 × 10−3 M) of 17b-estradiol was
prepared in ethanol. Dulbecco’s modified eagle medium (DMEM)
was purchased from Gibco BRL, fetal calf serum from Dutscher,
Brumath, France, glutamine, estradiol and protamine sulfate were
from Sigma. MCF-7 and MDA-MB-231 cells were from the
Human Tumor Cell Bank. 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.
1,2-di-Ferrocenyl-1-(4-hydroxyphenyl)-but-1-ene, 8. The reac-
tion was performed with 3.06 g (10 mmol) of the known compound
12.26 The crude product was chromatographed with CH2Cl2 as
eluent. The compound was purified on HPLC with acetonitrile–
water 90 : 10 as eluent to yield 8 as an orange solid (1.98 g, 39%
yield). The mixture of isomers was recrystallized from an ether–
pentane solution. 1H NMR (300 MHz, CDCl3) d 1.12 and 1.38 (t,
J = 7.4 Hz, 3 H, CH3), 2.32 and 2.81 (q, J = 7.4 Hz, 2 H, CH2),
3.65 and 3.69 (t, J = 1.9 Hz, 2 H, C5H4), 3.83 and 4.00 (s, 5 H,
Cp), 4.02 and 4.09 (s, 5 H, Cp), 4.00–4.25 (m, 6 H, C5H4), 5.10 and
5.14 (s, 1 H, OH), 6.89 and 6.90 (d, J = 8.5 Hz, 2 H, Harom), 7.08
and 7.17 (d, J = 8.5 Hz, 2 H, Harom). 13C NMR (CDCl3) d 15.4 and
15.5 (CH3), 26.6 and 30.9 (CH2), 67.3 and 68.1 (2 CH C5H4), 67.4
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 ERb purchased in solution
from Pan Vera (Madison, WI, USA). 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 described previously,14
was used as a source of ERa. For ERb, 10 ll of the solution
containing 3500 pmol ml−1 were added to 16 ml of buffer B (10%
glycerol, 50 mM Bis-Tris-Propane pH = 9, 400 mM KCl, 2 mM
This journal is
The Royal Society of Chemistry 2007
Dalton Trans., 2007, 5073–5081 | 5079
©

View Article Online
DTT, 1 mM EDTA, 0.1% BSA) in a silanized flask. Aliquots
(200 ll) of ERa in glass t◦ubes or ERb in polypropylene tubes were
incubated for 3 h at 0 C with [6,7–3H]-estradiol (2 × 10−9M,
specific activity 1.62 TBq mmol−1, NEN Life Science, Boston
MA) in the presence of nine concentrations of the ferrocenyl
complexes 2, 3, and 5–9 to be tested (between 6 × 10−7 M and
6 × 10−9 M for the complexes with RBA values higher than 5%
and between 6 × 10−6 M and 6 × 10−8 M for the compounds
with RBA values lower than 5%) or of 17b-estradiol (between 8 ×
10−8 M and 7.5 × 10−10 M). At the end of the incubation period,
the fractions of [3H]-estradiol bound to the estrogen receptors
(Y values) were precipitated by addition of a 200 ll of a cold
solution of protamine sulfate (1 mg mL−1 in water). After a 10 min
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. One 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.
Conclusions
Our results show that the presence of a p-phenol has a significant
influence on both the electrochemistry and on the biological
efficacy of these ferrocenyl phenol compounds, supporting our
hypothesis that oxidative activation to a QM structure may be
a key to their biological activity. Compounds 5 and 6 which
do not possess a p-phenol also did not show intramolecular
electron transfer and rearrangement to a QM structure in the
electrochemical experiments, as expected. Compound 5, lacking
any kind of phenolic group, showed no appreciable cytotoxicity,
while 6, possessing only a m-phenol, was much less efficacious than
its para-substituted analogue 2. Similarly, compound 7, carrying
one m- and one p-phenol did not show nearly as strong an activity
as that of 3, which possesses two p-phenols. In fact, the biological
activity of 7 is much closer to that of 2 (with IC50 values of 1.13 vs.
1.03 lM, respectively) than that of 3 (0.44 lM).
The diferrocenyl compounds 8 and 9 follow the same trend,
with higher biological activity observed for p-phenol 8, than
m-phenol 9. As expected, electron transfer from the phenol to
the ferricenium moiety is only electrochemically observed for
compound 8. However, the activity of these compounds cannot be
solely attributed to their electronic and structural rearrangements.
There also exists the steric effect of the additional ferrocene group,
which has a negative effect on the biological activity of 8 and 9.
Whether carrying a p- or m- phenol, the diferrocenyl compounds
are less efficacious than their mono-ferrocenyl analogues 2 and 6.
Although each of the compounds carrying a p-phenol behaves
similarly from an electrochemical perspective, the steric effect
suggests that the QMs are formed, or react, via one or several
specific biological pathways. For example, QMs are alkylating
agents which can react with O-, N-, or S- nucleophiles, espe-
cially glutathione, via a Michael 1,4-addition.35 The depletion
of glutathione can then lead to the alkylation of proteins by
an excess of quinone. Alternatively, quinonoids are known to
undergo redox cycling in cells resulting in the production of
ROS, via such enzymes as NADPH:cytochrome P450 reductase,
NADPH:cytochrome b5 reductase, NADPH:ubiquinone oxidore-
ductase, and NAD(P)H:quinone oxidoreductase, among others,36
and iron-containing compounds are known to produce ROS via
the Fenton reaction and H2O2. We are currently investigating the
ROS production of these types of compounds, and we hope that
future use of specific ROS probes and enzyme-inhibitors will allow
us to begin to unravel the metabolic fate of this new class of
cytotoxic ferrocenyl phenolic compounds.
◦
period of incubation at 4 C, the precipitates were recovered by
filtration on 25 mm circle glass microfibre filters (GF/C) using a
Millipore 12 well filtration ramp. The filters were rinsed twice with
cold phosphate buffer and then transferred in 20 ml plastic vials.
After addition of 5 ml of scintillation liquid (BCS Amersham)
the radioactivity of each fraction was counted in a Packard tri-
carb 2100TR liquid scintillation analyzer. The concentration of
unlabeled steroid required to displace 50% of the bound [3H]-
estradiol was calculated for 17b-estradiol and for each complex
by plotting the logit values of Y (logit Y = ln(Y/100 − Y) versus
the mass of the competing complex. The RBA was calculated
as follows: RBA of a compound = concentration of estradiol
required to displace 50% of [3H]-estradiol × 100/concentration
of the compound required to displace 50% of [3H]-estradiol. The
RBA value of estradiol is by definition equal to 100%.
Measurement of octanol/water partition coefficient (log Po/w
)
of the compounds. The log Po/w values of the compounds were
determined by reverse-phase HPLC on a C-8 column (Nucleosil
5 C8, from Macherey Nagel, France) according to the method
previously described by Minick33 and Pomper.34 Measurement of
the chromatographic capacity factors (kN) for each compounds
was performed at various concentrations in the range 85%–
60% methanol (containing 0.25% octanol) and an aqueous
phase consisting of 0.15% n-decylamine in 0.02 M MOPS (3-
morpholinopropanesulfonic acid) buffer pH 7.4 (prepared in 1-
octanol–saturated water). These capacity factors (kN) are extrap-
olated to 100% of the aqueous component given the value of
kꢀw. log Po/w(y) is then obtained by the formula: y = 0.13418 +
0.98452 × log kꢀw.
Culture conditions
Cells were maintained in monolayer in DMEM with phenol red
supplemented with 8–9% fetal calf serum and 2 mM glutamine
at 37 ◦C in a 5% CO2 air humidified incubator. For proliferation
assays, cells were plated in 24-well sterile plates at a density of
1.1 × 104 cells for MDA-MB-231 and of 3 × 104 cells for MCF-7
in 1 mL of DMEM medium without phenol red, supplemented
with 10% decomplemented and hormone-depleted fetal calf serum
and 2 mM glutamine and incubated. 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%; 4 wells
for each conditions). After 3 days (D3) the incubation medium
was removed and fresh medium containing the compounds was
added. After 5 days (D5) the total protein content of the plate was
analyzed by methylene blue staining as follows. Cell monolayers
Acknowledgements
We thank A. Cordaville and M. A. Plamont for technical
assistance, and the Agence Nationale de la Recherche for financial
support (No. ANR-06-BLAN-0384–01, “FerVect”. E. A. H.
thanks the NSF (grant no. 0302042) for financial support.
5080 | Dalton Trans., 2007, 5073–5081
This journal is
The Royal Society of Chemistry 2007
©

View Article Online
16 G. Galati and P. J. O’Brien, Free Radical Biol. Med., 2004, 37,
287–303; M. Kawase, N. Motohashi, J. Molnar and H. Sakagami,
in Functional Polyphenols and Carotenes with Antioxidative Action,
Research Signpost, Trivandrum, India, 2005, pp. 83–109.
17 A. M. Brzozowski, A. C. Pike, Z. Dauter, R. E. Hubbard, T. Bonn, O.
Engstrom, L. Ohman, G. L. Greene, J.-A. Gustafsson and M. Carlquist,
Nature, 1997, 389, 753–758; A. C. Pike, A. M. Brzozowski, R. E.
Hubbard, T. Bonn, A. G. Thorsell, O. Engstrom, J. Ljunggren, J.-A.
Gustafsson and M. Carlquist, EMBO J., 1999, 18, 4608–4618; A. C. W.
Pike, A. M. Brzozowski, J. Walton, R. E. Hubbard, A. G. Thorsell,
Y. L. Li, J.-A. Gustafsson and M. Carlquist, Structure (Cambridge,
MA, US), 2001, 9, 145–153; 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.
References
1 V. J. Fiorina, R. J. Dubois and S. Brynes, J. Med. Chem., 1978, 21,
393–395; G. N. Yashchenko, A. A. Shashmurina, G. M. Anoshina,
L. A. Gorelova, N. G. Evstigneeva, L. V. Alekseeva and L. B. Radina,
Khim.-Farm. Zh., 1978, 12, 68–72; M. Wenzel, M. Schneider and E.
Liss, Z. Naturforsch, C: Biosci., 1979, 34, 670–676.
2 P. Ko¨pf-Maier, H. Ko¨pf and E. W. Neuse, Angew. Chem., Int. Ed. Engl.,
1984, 23, 456–457; P. Ko¨pf-Meyer, H. Ko¨pf and E. W. Neuse, J. Cancer
Res. Clin. Oncol., 1984, 108, 336–340.
3 E. W. Neuse and F. Kanzawa, Appl. Organomet. Chem., 1990, 4,
19–26.
4 J. C. Swarts, E. W. Neuse and G. J. Lamprecht, J. Inorg. Organomet.
Polym., 1994, 4, 143–153; E. W. Neuse, Macromol. Symp., 2001, 172,
127–138; J. C. Swarts, D. M. Swarts, D. M. Maree, E. W. Neuse, C. La
Madeleine and J. E. Van Lier, Anticancer Res., 2001, 21, 2033–2037;
M. T. Johnson, E. Kreft, D. D. N’Da, E. W. Neuse and C. E. J. van
Rensburg, J. Inorg. Organomet. Polym., 2003, 13, 255–267; G. Caldwell,
M. G. Meirim, E. W. Neuse and C. E. J. van Rensburg, Appl. Organomet.
Chem., 1998, 12, 793–799.
18 E. A. Hillard, A. Vessie`res, L. Thouin, G. Jaouen and C. Amatore,
Angew. Chem., Int. Ed., 2006, 45, 285–290.
19 S. Top, A. Vessie`res, C. Cabestaing, I. Laios, G. Leclercq, C. Provot and
G. Jaouen, J. Organomet. Chem., 2001, 637, 500–506.
20 E. A. Hillard, A. Vessie`res, S. Top, P. Pigeon, K. Kowalski, M. Huche´
and G. Jaouen, J. Organomet. Chem., 2007, 692, 1315–1326.
21 S. Barlow, H. E. Bunting, C. Ringham, J. C. Green, G. U. Bublitz,
S. G. Boxer, J. W. Perry and S. R. Marder, J. Am. Chem. Soc., 1999,
121, 3715–3723; S. Barlow and S. R. Marder, Chem. Commun., 2000,
1555–1562; S. Barlow, Inorg. Chem., 2001, 40, 7047–7053.
22 S. Fukuzumi, K. Okamoto, Y. Yoshida, H. Imahori, Y. Araki and O.
Ito, J. Am. Chem. Soc., 2003, 125, 1007–1013; M. Murata, T. Fujita, M.
Yamada, M. Kurihara and H. Nishihara, Chem. Lett., 2000, 29, 1328–
1329; M. Murata, M. Yamada, T. Fujita, K. Kojima, M. Kurihara,
K. Kubo, Y. Kobayashi and H. Nishihara, J. Am. Chem. Soc., 2001,
123, 12903–12904; M. Kurihara and H. Nishihara, Coord. Chem. Rev.,
2002, 226, 125–135.
23 L. Cuffe, R. D. A. Hudson, J. F. Gallagher, S. Jennings, C. J. McAdam,
R. B. T. Connelly, A. R. Manning, B. H. Robinson and J. Simpson,
Organometallics, 2005, 24, 2051–2060.
24 S. B. Colbran, S. T. Lee, D. G. Lonnon, F. J. D. Maharaj, A. M.
McDonagh, K. A. Walker and R. D. Young, Organometallics, 2006,
25, 2216–2224.
5 C. W. Ong, J. Y. Jeng, S. S. Juang and C. F. Chen, Bioorg. Med. Chem.
Lett., 1992, 2, 929–932.
6 V. Scarcia, A. Furlani, B. Longato, B. Corain and G. Pilloni, Inorg.
Chim. Acta, 1988, 153, 67–70; D. T. Hill, G. R. Girard, F. L. McCabe,
R. K. Johnson, P. D. Stupik, J. H. Zhang, W. M. Reiff and D. S.
Eggleston, Inorg. Chem., 1989, 28, 3529–3533; G. Pilloni, R. Graziani,
B. Longato and B. Corain, Inorg. Chim. Acta, 1991, 190, 165–167; C.
Bincoletto, I. L. S. Terariol, C. R. Oliveira, S. Dreher, D. M. Fausto,
M. A. Soufen, F. D. Nascimento and A. C. F. Caires, Bioorg. Med.
Chem., 2005, 13, 3047.
7 D. A. Oparin, V. D. Makhaev, V. D. Vilchevskaya, T. I. Zimatkina, Z. V.
Motylevich, S. M. Zimatkin, S. V. Zabrodskaya, A. I. Krylova and Y. Y.
Gorelikova, Khim.-Farm. Zh., 1996, 30, 11–13.
8 A. Ferle-Vidovic, M. Poljak-Blazi, V. Rapic and D. Skare, Cancer
Biother. Radiopharm., 2000, 15, 617–624; C. G. Hartinger, A. A.
Nazarov, V. B. Arion, G. Giester, M. Jakupec, M. Galanski and B. K.
Keppler, New J. Chem., 2002, 26, 671–673.
25 M. Kurihara, H. Sano, M. Murata and H. Nishihara, Inorg. Chem.,
2001, 40, 4–5.
26 A. C. Benyei, C. Glidewell, P. Lightfoot, B. J. L. Royles and D. M.
Smith, J. Organomet. Chem., 1997, 539, 177–186.
9 L. V. Popova, V. N. Babin, Y. A. Belousov, Y. S. Nekrasov, A. E.
Snegireva, N. P. Borodina, G. M. Shaposhnikova, O. B. Bychenko,
P. M. Raevskii, N. B. Morozova, A. I. Iiyina and K. G. Shitkov, Appl.
Organomet. Chem., 1993, 7, 85–94.
27 S. Top, B. Dauer, J. Vaissermann and G. Jaouen, J. Organomet. Chem.,
1997, 541, 355–361.
28 P. Nussbaumer, M. Bilban and A. Billich, Bioorg. Med. Chem. Lett.,
2002, 12, 2093–2095.
29 S. Gauthier, J. Y. Sanceau, J. Mailhot, B. Caron and J. Cloutier,
Tetrahedron, 2000, 56, 703–709.
30 A.-C. Ribou, J.-P. Launay, M. L. Sachtleben, H. Li and C. W. Spangler,
Inorg. Chem., 1996, 35, 3735–3740.
10 W. C. M. Duivenvoorden, Y. N. Liu, G. Schatte and H. B. Kraatz,
Inorg. Chim. Acta, 2005, 358, 3183–3189; E. W. Neuse, M. G. Meirim
and N. F. Blom, Organometallics, 1988, 7, 2562–2565.
11 P. N. Kelly, A. Preˆtre, S. Devoy, I. O’Rielly, R. Devery, A. Goel, J. F.
Gallagher, A. J. Lough and P. T. M. Kenny, J. Organomet. Chem., 2007,
692, 1327–1331; X. Wu, E. R. T. Tiekink, I. Kostetski, N. Kocherginsky,
A. L. C. Tan, S. B. Khoo, P. Wilairat and M.-L. Go, Eur. J. Pharm.
Sci., 2006, 27, 175–187.
31 M. B. Robin and P. Day, Adv. Inorg. Chem. Radiochem., 1967, 10,
247–423.
32 M. R. Schneider, E. Von Angerer, H. Scho¨nenberger, R. T. Michel and
H. P. Fortmeyer, J. Med. Chem., 1982, 25, 1070–1077.
33 D. J. Minick, J. H. Frenz, M. A. Patrick and D. A. Brent, J. Med. Chem.,
1988, 31, 1923–1933.
12 D. Osella, M. Ferrali, P. Zanello, F. Laschi, M. Fontani, C. Nervi and G.
Cavigiolio, Inorg. Chim. Acta, 2000, 306, 42–48; G. Tabbi, C. Cassino,
G. Cavigiolio, D. Colangelo, A. Ghiglia, I. Viano and D. Osella, J. Med.
Chem., 2002, 45, 5786–5796; H. Tamura and M. Miwa, Chem. Lett.,
1997, 1177–1178; G. Y. N. Vashisht, D. Jayaraju and A. K. Kondapi,
Arch. Biochem. Biophys., 2000, 376, 229–235.
34 M. G. Pomper, H. VanBrocklin, A. M. Thieme, R. D. Thomas, D. O.
Kiesewetter, K. E. Carlson, C. J. Mathias, M. J. Welch and J. A.
Katzenellenbogen, J. Med. Chem., 1990, 33, 3143–3155.
35 P. W. Fan, F. Zhang and J. L. Bolton, Chem. Res. Toxicol., 2000, 13,
45–52.
13 A. Vessie`res, S. Top, P. Pigeon, E. A. Hillard, L. Boubeker, D. Spera
and G. Jaouen, J. Med. Chem., 2005, 48, 3937–3940.
14 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.
15 J. A. Katzenellenbogen and R. Muthyala, Pure Appl. Chem., 2003, 75,
1797–1817; J. A. Dodge, Pure Appl. Chem., 1998, 70, 1725–1733.
36 C. Asche, Mini-Rev. Med. Chem., 2005, 5, 449–467.
This journal is
The Royal Society of Chemistry 2007
Dalton Trans., 2007, 5073–5081 | 5081
©