Ferrocenyl compounds possessing protected phenol and thiophenol groups: Synthesis, X-ray structure, and in vitro biological effects against breast cancer

Article abstract of DOI:10.1016/j.jorganchem.2007.12.011

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 metabolic quinone methide (QM) formation. To fur

Full text of DOI:10.1016/j.jorganchem.2007.12.011

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Journal of Organometallic Chemistry 693 (2008) 1716–1722
www.elsevier.com/locate/jorganchem
Note
Ferrocenyl compounds possessing protected phenol and
thiophenol groups: Synthesis, X-ray structure, and in vitro
biological effects against breast cancer
Julia B. Heilmann a, Elizabeth A. Hillard a, Marie-Aude Plamont a, Pascal Pigeon a,
b
a
a,
*
`
´
Michael Bolte , Gerard Jaouen , Anne Vessieres
a Laboratoire 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
b Institut fu¨r Anorganische Chemie, J.W. Goethe-Universita¨t Frankfurt, Max von Laue-Strasse 7, D-60438 Frankfurt (Main), Germany
Received 31 October 2007; received in revised form 6 December 2007; accepted 6 December 2007
Available online 15 December 2007
Abstract
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 metabolic quinone methide (QM) formation. To
further evaluate this proposed mechanism, we have created a series of ferrocenyl prodrugs containing methyl and acetyl-protected
thio- and oxo-phenols: 2-ferrocenyl-1,1-bis(4-acetoxyphenyl)-but-1-ene (5), 2-ferrocenyl-1,1-bis(4-thioacetylphenyl)-but-1-ene (6),
2-ferrocenyl-1,1-bis(4-methoxyphenyl)-but-1-ene (7), and 2-ferrocenyl-1,1-bis(4-thiomethylphenyl)-but-1-ene (8), which might be acti-
vated by hydrolysis enzymes in situ. Only the acetoxy 5 displayed antiproliferative effects (IC50 on MDA-MB-231 of 0.5 lM) while
6–8 act as pure estrogens (proliferative on MCF-7 and little to no effect on MDA-MB-231). The behaviour of 5 is similar to that pre-
viously found for the free phenol 2-ferrocenyl-1,1-di(4-hydroxyphenyl)-but-1-ene (2), indicating that 5 is metabolized in situ to 2, which
could then undergo oxidative QM formation. The observation that the thioacetyl 6 is not cytotoxic suggests that the in situ oxidative
chemistry of the putative ferrocenyl thiophenol is different from that of 2. Because p-thioquinone methides are practically unknown,
the negative results for 6 further implicate the bioformation of the QM in the case of 2 and related compounds. The lack of cytotoxicity
of 7 and 8 can be attributed to lack of efficient hydrolysis in situ. Estrogen receptor binding affinity studies for the compounds and the
X-ray structure of 8 are also reported.
Ó 2008 Elsevier B.V. All rights reserved.
Keywords: Ferrocene; Breast cancer; Bioorganometallic chemistry; Iron
1. Introduction
quine, a ferrocenyl analogue of chloroquine, has shown
activity against chloroquine-resistant strains of malaria,
and is currently in phase II clinical trials with Sanofi-Aven-
tis [2], while two ruthenium arene complexes have entered
clinical trials as antimetastatic candidates [3].
Our work is focused on the use of organometallic nano-
vectors in oncology, especially cancers of the breast and
prostate [4–7]. We have found that compounds possessing
a ferrocenyl moiety linked to a p-phenol group by a
conjugated system show considerable potency against
breast cancer cells, with IC50 values ranging from 0.44 to
Bioorganometallic chemistry is a growing multidisciplin-
ary field which encompasses the synthesis and study of
organometallic complexes of biological importance [1].
One of the most promising applications of bioorganome-
tallic chemistry is in the area of drug development. Ferro-
*
Corresponding author. Tel.: +33 01 44 27 67 29; fax: +33 01 43 26 00
61.
`
E-mail address: a-vessieres@enscp.fr (A. Vessieres).
0022-328X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2007.12.011

J.B. Heilmann et al. / Journal of Organometallic Chemistry 693 (2008) 1716–1722
1717
1.1 lM against the hormone-independent MDA-MB-231
cell line, Chart 1. We have posited that these compounds
can be activated by in situ oxidation to form a cytotoxic
quinone methide (QM) species. Electrochemical experi-
ments have suggested that this transformation can occur
at low potentials, due to the role of the ferricenium
cation as an intramolecular oxidant of the phenol group
in basic conditions [8–10]. We now wish to under-
stand how the replacement of the oxygen atom by a sulfur
atom will influence the cytotoxicity of these types of
molecules.
The formation of QMs is an important pathway for the
metabolism of many compounds, including natural prod-
ucts [12–14], selective estrogen receptor modulators
[15–18], and drugs [19–22]. However, to our knowledge,
only one example of an isolable p-thioquinone methide
has been reported, 10-(dicyanomethylene)thioanthrone,
which is stabilized by electron withdrawing cyano groups
and an annelated phenyl ring [23]. Thioquinone methides
do not seem to be important in biology, because oxidation
of aromatic thiols immediately leads to dimerization or
reaction with other thiols, thiolates, or molecular oxygen
[24]. Therefore, if QM formation is indeed an important
mechanism of cytotoxicity in the series of ferrocenyl phenol
compounds, one would expect their sulfur congeners to be
non-cytotoxic.
However, other mechanisms of cytotoxicity could also
be relevant. It should be mentioned that phenolic com-
pounds [25] and ferricenium salts [26] can produce reactive
oxygen species through phenol-quinone redox cycling and
Fenton–Haber–Weiss reactions, leading to cell damage.
The oxidation of aromatic thiols has also been shown to
generate H2O2 and provoke oxidative stress [24,27]. There-
fore, if the cytotoxicity observed in ferrocenyl phenols
arises mainly through oxidative stress instead of QM for-
mation, the substitution of the oxygen atom by a sulfur
atom would be expected to retain the compounds’ ability
to undergo redox cycling, Fenton chemistry, and observed
cytotoxic properties.
Chart 2. New compounds 5–8. Compound 4 is a reaction intermediate
that was isolated and chemically characterized, although biological results
are not reported here.
the estrogen receptor positive MCF-7 and estrogen recep-
tor negative MDA-MB-231 cell line. Protecting groups
are often used in drug formulation to enhance the stability
and bioavailability of molecules [28,29]. The protected
compounds can be considered as prodrugs; only after
hydrolysis by appropriate enzymes is the active drug
released. In terms of this study, the use of two different pro-
tecting groups is expected to provide information about the
hydrolysis of these compounds to the free phenol or thio-
phenol in vitro. Finally, we were also interested in deter-
mining the influence that the change from oxygen to
sulfur has on estrogen receptor binding, and report here
the affinity for the estrogen receptors a and b.
2. Experimental
2.1. General remarks
The synthesis of all compounds was carried out using
standard Schlenk techniques, under an argon atmosphere.
THF and diethyl ether were dried and distilled over
sodium/benzophenone, while CH2Cl2 was dried and dis-
tilled over CaH2, prior to use. Thin layer and column chro-
matography was performed on silica gel 60 GF254 and
60 M, respectively. 1H and 13C NMR spectra were acquired
on a Bruker 300 MHz spectrometer. EI-MS was performed
on a Nermag R 10-10C spectrometer; ESI-MS using a tri-
ple quadrupole mass spectrometer API 3000 LC–MS/MS
system (Applied Biosystems, PE Sciex) in positive-ion
mode; and high resolution mass spectrometry (HRMS)
with a Joel MS 7000 instrument. Elemental analyses were
performed at the microanalysis laboratory of the CNRS
(Gif sur Yvette, France). Melting points were measured
with a Kofler device.
We have synthesized both oxo- and thio-analogues of
compound 2, protected by methyl or acetyl groups,
Chart 2, and tested their effects on the proliferation of
4-Bromothioanisole, 4-bromothiophenol and 4,4-di-
methoxybenzophenone were purchased from Aldrich and
used without further purification. (4-Bromo)phenyl-t-
butylthioether [30], propionyl ferrocene [31], 4,40-bis
(t-butylthio)benzophenone [32], and 4,40-bis(methylthio)
benzophenone [32] were synthesized according to proce-
dures found in the literature.
Chart 1. Previously reported cytotoxic ferrocenyl phenolic compounds 1
[6], 2 [11], and 3 [5].

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J.B. Heilmann et al. / Journal of Organometallic Chemistry 693 (2008) 1716–1722
2.2. 2-Ferrocenyl-1,10-bis(4-thio-t-butylphenyl)-but-1-ene, 4
2.4. 2-Ferrocenyl-1,1-bis(4-thioacetylphenyl)-but-1-ene, 6
TiCl4 (3.35 mmol, 0.636 g) was added dropwise to a slurry
of zinc dust (5.02 mmol, 0.328 g) in THF (15 ml). The result-
ing black mixture was heated under reflux for 2 h and then
cooled to r.t. Propionyl ferrocene (1.42 mmol, 0.344 g) and
4,40-bis(t-butylthio)benzophenone (0.837 mmol, 0.300 g)
were dissolved in THF (15 ml) and added to the Zn/TiCl4
mixture. The reaction mixture was heated under reflux for
5 h (reaction monitored by TLC). After cooling to r.t. the
mixture was poured onto H2O/HCl/ice, whereupon the col-
our changed from dark brown to orange. The aqueous phase
was extracted with CH2Cl2 (4 ꢀ 25 ml) and the combined
organic layers were washed with H2O (3 ꢀ 20 ml), dried over
MgSO4, and concentrated. Purification by flash chromatog-
raphy (CH2Cl2/hexane 1:1) yielded the pure product as a red
solid (120 mg, 25%). 1H NMR (300 MHz, CDCl3): d = 7.50
(d, J = 8.1 Hz, 2H, C6H4), 7.37 (d, J = 8.1 Hz, 2H, C6H4),
7.19 (d, J = 8.1 Hz, 2H, C6H4), 7.02 (d, J = 8.1 Hz, 2H,
C6H4), 4.12 (s, 5H, C5H5), 4.08 (t, J = 1.8 Hz, 2H, C5H4),
3.86 (t, J = 1.8 Hz, 2H, C5H4), 2.60 (q, J = 7.5 Hz, 2H,
CH2), 1.31 (s, 9H, C(CH3)3), 1.27 (s, 9H, C(CH3)3), 1.01 (t,
J = 7.5 Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3):
d = 144.9, 144.5, 138.9 (Cquat), 137.3, 137.2 (C6H4), 136.5,
130.6, 130.4 (Cquat), 130.1, 129.5 (C6H4), 86.5 (Fcipso), 69.3
(C5H4), 69.2 (C5H5), 68.2 (C5H4), 45.8, 45.7 (C(CH3)3),
31.0, 30.9 (C(CH3)3), 28.1 (CH2), 15.3 (CH3). MS (EI,
70 eV) m/z: 568 [M]+Å, 121 [CpFe]+. HRMS calcd for
C34H40FeS2: 568.1921; found: 568.1929 [M]+Å. Elemental
Anal. Calc. for C34H40FeS2: C, 71.81; H, 7.09; S, 11.28.
Found: C, 71.80; H, 7.01; S, 11.31. m.p. = 150 °C.
Compound 4 (0.341 mmol, 0.194 g) was dissolved in
CH2Cl2 (25 ml) and acetyl chloride (1.4 ml) was added at
r.t. To the resulting mixture, a 1.0 M solution of BBr3 in
CH2Cl2 (2.73 ml, 2.73 mmol) was added and the dark
brown solution was stirred at r.t. for 6 days. The mixture
was poured into an aqueous solution of NaHCO3 and
CH2Cl2 (20 ml) was added. The phases were separated
and the aqueous phase was extracted with CH2Cl2
(4 ꢀ 25 ml). The combined organic layers were washed
with H2O (3 ꢀ 20 ml), dried over MgSO4, and concen-
trated. Purification by flash chromatography (CH2Cl2)
1
yielded the pure product as a red oil (113 mg, 61%). H
NMR (300 MHz, CDCl3): d = 7.38 (d, J = 8.4 Hz, 2H,
C6H4), 7.27–7.23 (m, 4H, C6H4), 7.09 (d, J = 8.4 Hz, 2H,
C6H4), 4.13–4.11 (m, 7H, C5H4/C5H5), 3.91 (t, J = 1.8
Hz, 2H, C5H4), 2.61 (q, J = 7.5 Hz, 2H, CH2), 2.42 (s,
3H, C(O)CH3), 2.39 (s, 3H, C(O)CH3), 1.03 (t, J = 7.5
Hz, 3H, CH3). 13C NMR (75 MHz, CDCl3): d = 194.1,
194.0 (C@O), 145.3, 145.0, 139.7, 135.7 (Cquat), 134.2,
134.1, 130.9, 130.3 (C6H4), 125.7, 97.0 (Cquat), 86.1 (Fcipso),
69.5 (C5H4), 69.2 (C5H5), 68.5 (C5H4), 30.2, 30.1
(C(O)CH3), 28.2 (CH2), 15.5 (CH3). MS (ESI) m/z: 540.3
[M]+Å. HRMS calcd for C30H28FeO2S2: 540.0880; found:
540.0880 [M]+Å. m.p. = 111–113 °C.
2.5. 2-Ferrocenyl-1,1-bis(4-methoxyphenyl)-but-1-ene, 7
Same procedure as 4 using TiCl4 (32.8 mmol, 6.22 g),
zinc dust (61.2 mmol, 4.00 g) in THF, propionyl ferrocene
(10.0 mmol, 2.42 g) and 4,40-dimethoxybenzophenone
(20.0 mmol, 4.84 g). Yield = 3.890 g, 86%. 1H NMR
(300 MHz, CDCl3): d = 7.16 (d, J = 7.0 Hz, 2H, C6H4),
7.01 (d, J = 7.0 Hz, 2H, C6H4), 6.90 (d, J = 7.0 Hz, 2H,
C6H4), 6.79 (d, J = 7.0 Hz, 2H, C6H4), 4.15 (s, 5H,
C5H5), 4.12 (t, J = 1.9 Hz, 2H, C5H4), 3.95 (t, J = 1.9 Hz,
2H, C5H4), 3.85 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 2.62
(q, J = 7.4 Hz, 2H, CH2), 1.07 (t, J = 7.4 Hz, 3H, CH3).
13C NMR (75 MHz, CDCl3): d = 157.9, 137.5, 137.3,
137.2, 136.7 (Cquat), 131.0, 130.4, 113.6, 113.5 (C6H4),
87.2 (Fcipso), 69.3 (C5H4), 69.2 (C5H5), 68.0 (C5H4), 55.2,
55.1 (OCH3), 28.0 (CH2), 15.5 (CH3). MS (EI, 70 eV) m/z:
452 [M]+Å, 423 [MꢁEt]+, 387 [MꢁCp]+, 121 [FeCp]+. Ele-
mental Anal. Calc. for C28H28FeO2: C, 74.34; H, 6.24.
Found: C, 73.99; H, 6.26%. m.p. = 94 °C.
2.3. 2-Ferrocenyl-1,1-bis(4-acetoxyphenyl)-but-1-ene, 5
Acetyl chloride (1 ml) was added dropwise to a suspen-
sion of 2 (0.471 mmol, 0.200 g) in CH2Cl2 (15 ml) at r.t.
The reaction mixture was stirred at r.t. overnight, yielding
a clear orange solution. The solution was then poured into
H2O (20 ml) and the phases were separated. The aqueous
phase was extracted with CH2Cl2 (3 ꢀ 20 ml) and the com-
bined organic layers were washed with H2O (2 ꢀ 20 ml),
dried over MgSO4, and concentrated. Flash chromatogra-
phy (CH2Cl2) yielded the pure product as an orange solid
(216 mg, 90 %). 1H NMR (300 MHz, CDCl3): d = 7.20
(d, J = 8.7 Hz, 2H, C6H4), 7.06 (d, J = 8.7 Hz, 2H,
C6H4), 7.05 (d, J = 8.7 Hz, 2H, C6H4), 6.94 (d, J =
8.7 Hz, 2H, C6H4), 4.11 (s, 5H, C5H5), 4.10 (t,
J = 1.8 Hz, 2H, C5H4), 3.90 (t, J = 1.8 Hz, 2H, C5H4),
2.59 (q, J = 7.5 Hz, 2H, CH2), 2.30 (s, 3H, C(O)CH3),
2.27 (s, 3H, C(O)CH3), 1.03 (t, J = 7.5 Hz, 3H, CH3). 13C
NMR (75 MHz, CDCl3): d = 169.3, 169.2 (C@O), 149.0,
149.0, 141.8, 141.6, 138.6, 135.9 (Cquat), 130.9, 130.3,
121.3, 121.1 (C6H4), 86.3 (Fcipso), 69.3 (C5H4), 69.1
(C5H5), 68.3 (C5H4), 28.0 (CH2), 21.2 (C(O)CH3), 15.4
(CH3). MS (EI, 70 eV) m/z: 508 [M]+Å, 465 [MꢁCH3CO]+,
121 [CpFe]+, 43 [CH3CO]+. HRMS calcd for C30H28FeO4:
508.1337; found 508.1337. m.p. = 119 °C.
This compound had previously been obtained by a dif-
ferent route [33]; this is the first communication of its
characterization.
2.6. 2-Ferrocenyl-1,1-bis(4-thiomethylphenyl)-but-1-ene, 8
Same procedure as 4 using TiCl4 (3.28 mmol, 0.623 g),
zinc dust (5.91 mmol, 0.386 g), propionyl ferrocene
(1.09 mmol, 0.265 g) and 4, 40-bis(methylthio)benzophe-
none (0.729 mmol, 0.200 g). Yield = 106 mg, 30 %. 1H
NMR (300 MHz, CDCl3): d = 7.21 (d, J = 8.4 Hz, 2H,

J.B. Heilmann et al. / Journal of Organometallic Chemistry 693 (2008) 1716–1722
1719
C6H4), 7.10 (d, J = 8.4 Hz, 4H, C6H4), 6.96 (d, J = 8.4 Hz,
2H, C6H4), 4.11 (s, 5H, C5H5), 4.09 (t, J = 1.8 Hz, 2H,
C5H4), 3.92 (t, J = 1.8 Hz, 2H, C5H4), 2.59 (q,
J = 7.5 Hz, 2H, CH2), 2.49 (s, 3H, SCH3), 2.45 (s, 3H,
SCH3), 1.02 (t, J = 7.5 Hz, 3H, CH3). 13C NMR
(75 MHz, CDCl3): d = 141.4, 141.3, 137.9, 136.6, 136.0,
135.9 (Cquat), 130.4, 129.9, 126.4, 126.2 (C6H4), 86.6
(Fcipso), 69.3 (C5H4), 69.1 (C5H5), 68.2 (C5H4), 28.0
(CH2), 15.8, 15.7, 15.5 (CH3). MS (EI, 70 eV) m/z: 484
[M]+Å, 419 [MꢁCp]+, 121 [FeCp]+. Elemental Anal. Calc.
for C28H28FeS2: C, 69.41; H, 5.83; S, 13.24. Found: C,
69.35; H, 5.75; S, 13.23%. m.p. = 118–120 °C.
observed = 33343/5079/4639 (2h = 54.26); 284 parameters;
R1, wR2 (all data) = 0.0311, 0.0725.
2.8. Biochemical experiments
Procedures for the determination of the relative binding
affinity (RBA) for ERa and ERb and cell culture condi-
tions have been previously reported [38].
3. Results and discussion
3.1. Synthesis
2.7. X-ray crystal structure of compound 8
Compounds 4 and 8 were synthesized by McMurry
cross-coupling reactions between propionyl ferrocene and
4,40-bis(t-butylthio)benzophenone or 4,40-bis(methylthio)
benzophenone, respectively, as shown in Scheme 1. The
reaction of the t-butylthioether 4 with an excess of BBr3
and acetyl chloride gave us the expected thioacetyl 6 in
moderate yield. Although this deprotection/acetylation
process proceeded smoothly in the case of 4, the methyl-
thioether 8 was unreactive under these conditions. Even a
mixture of HNEt2/NaH/HMPT, which has been described
as a dealkylation reagent for alkyl aryl sulfides [39], was
not strong enough to demethylate 8.
We then attempted to deacetylate 6 to obtain the free
thiol by reaction with aqueous NH4OH. Although traces
of the desired compound could be detected in the mass
spectrum, the free thiol has not yet been isolated. Free aro-
matic thiols are known to be unstable to oxidation, and
thus can be difficult to isolate [40].
A stable orange plate of 0.41 ꢀ 0.32 ꢀ 0.19 mm3 was
mounted directly into the coldstream of an Oxford Cryo-
stream crystal cooling apparatus installed on a STOE
IPDS-II two-circle diffractometer, using perfluoropolyether
oil. Data were collected using the x-scan technique. An
empirical absorption correction was performed using the
MULABS option [34] in PLATON [35]. The structure was solved
by direct methods [36] and refined based on F2 using the
SHELXL-97 program [37]. All atoms were refined with aniso-
tropic displacement parameters. Hydrogen atoms were
placed in idealized positions and allowed to ride on the rel-
evant carbon atom.
Crystallographic data for 8: C28H28FeS2; triclinic,
˚
˚
˚
ꢀ
P1, a = 7.6480(4) A, b = 9.4223(6) A, c = 17.491(1) A,
a = 103.537(5)°, b = 90.396(5)°, c = 107.989(5)°; k =
˚
0.71073 A; Z = 2; Number of reflections total/unique/
t
n
Scheme 1. (i) 1 – BuCl, AlCl3, r.t., 1 h and 2 – H2O; (ii) 1 – BuLi, Et2O ꢁ30 °C ? ꢁ10 °C, 3 h, 2 – Me2NC(O)Cl, Et2O, ꢁ 50 °C ? 0 °C, 3 h, and 3 –
NH4Cl, H2O; (iii) 1 – TiCl4/Zn, THF, reflux, 3–6 h and 2 – HCl, H2O; and (iv) 1 – exc. BBr3, exc. H3CC(O)Cl, CH2Cl2, r.t., 6 days and 2 – NaHCO3(aq)
.

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J.B. Heilmann et al. / Journal of Organometallic Chemistry 693 (2008) 1716–1722
Scheme 2. (i) 1 – TiCl4/Zn, THF, reflux, 3 h and 2 – HCl, H2O and (ii) 1 – exc. H3CC(O)Cl, CH2Cl2, r.t., 12 h and 2 – H2O.
Fig. 1. ORTEP diagram of 8. Ellipsoids shown at 50% probability.
Compound 7 was synthesized by McMurry cross-cou-
able twisted (torsion angle C37–S1–C34–C33 – 29.87(15)°)
out of the plane of the aromatic ring to which it is attached.
The two cyclopentadienyl rings are coplanar (dihedral
angle 2.05(14)°). There are no short C–Hꢂ ꢂ ꢂX contacts.
pling reaction between propionyl ferrocene and 4,40-
dimethoxybenzophenone, as shown in Scheme 2. The acet-
oxy derivative 5 was synthesized by the reaction of the
known compound 2 with an excess of acetyl chloride at
ambient temperature.
3.3. Biochemical results
3.2. X-ray crystal structure of 8
3.3.1. Receptor affinity
The affinities for the estrogen receptor of the newly syn-
thesized complexes were measured on the two isoforms of
the receptor, ERa and ERb, and are reported as relative
binding affinity (RBA) values in Table 1. The RBA values
for both isoforms of the receptor are very low for all of the
compounds, except for 5, which showed a moderate affinity
for ERa. In general, the poor affinity can be explained by
the fact that the hydroxyl groups are protected with methyl
or acetyl groups in this series of molecules, thus preventing
stabilizing bioligand-receptor hydrogen bonding interac-
tions. It is also interesting to note that the affinity of the
The X-ray crystal structure of 8 is shown in Fig. 1. All
atoms connected to the central double bond are located
˚
in a common plane (r.m.s. deviation 0.02 A). The cyclopen-
tadienyl ring attached to the double bond is almost copla-
nar with the latter plane (dihedral angle 3.7(1)°) whereas
the two phenyl rings are almost perpendicular to that plane
(dihedral angles 70.21(4)° and 89.37(4)°). Whereas one of
the thiomethyl groups (S2–C47) is almost coplanar (torsion
angle C47–S2–C44–C43 – 1.89(15)°) with the phenyl ring
to which it is attached, the other one (S1–C37) is consider-

J.B. Heilmann et al. / Journal of Organometallic Chemistry 693 (2008) 1716–1722
1721
Table 1
Relative binding affinity (RBA) of the compounds for the two isoforms of the estrogen receptor (ERa and ERb) and effect of 1 lM (1 nM of 17b-estradiol)
on cell growth after 5 days of culture of hormone-dependent (MCF-7) and hormone-independent (MDA-MB-231) breast cancer cells
Compound
RBA (%)a
MCF-7a,* (%)
MDA-MB-231a,*(%)
ERa
ERbb
17b-Estradiol
100c,d
100d
0.8 0.1
0.03e
0.26 0.02
0.037 0.005
16.3 1.5
222 8f
No effect
22.5 0.9
5
6
7
8
2h
*
6.5 0.5c
0.270 0.005b
0.20 0.1c
0.04 0.01c
9.6 0.6c
37.7 2g
187 32f
151 15f
169.5 20f
37 5g
99
5
101
99
1
4
29 1.6
Values compared to control, defined as 100%.
Mean of two experiments range.
Purified receptor.
Receptor from lamb uterine cytosol.
Value by definition.
a
b
c
d
e
One determination.
f
Medium without phenol red.
Medium with phenol red.
Values from Ref. [5].
g
h
known ferrocene diphenol 2 for ERb is much higher than
for ERa, while this trend is not observed with the new com-
pounds 5–8. This indicates that neither 5 nor 7 were hydro-
lysed to 2 under the experimental conditions which involve
purified ER instead of living cells.
At 1 lM, all of the compounds show a strong prolifera-
tive effect on the hormone-dependent breast cancer cell line
MCF-7, except compound 5, which shows an antiprolifera-
tive effect. Again, this antiproliferative effect is similar to that
of previously reported compound 2, suggesting in situ
hydrolysis and possible QM formation. The strong estro-
genic effects exhibited by 6–8 are surprising, considering
the low affinity the compounds have for ERa. However,
we have already noticed that some compounds with low
RBA values are estrogenic at this high concentration [38].
3.3.2. Effect of the compounds on the growth of breast cancer
cells
The effect of these complexes at a concentration of 1 lM
was studied on hormone-independent (MDA-MB-231) and
hormone-dependent (MCF-7) breast cancer cells and the
results are displayed in Table 1. Any antiproliferative effect
observed on the hormone-independent breast cancer cells
can be attributed only to a cytotoxic effect. On these cells,
only compound 5 showed a antiproliferative effect, which is
of comparable magnitude to that shown by the reference
4. Conclusions
We have synthesized four new ferrocene compounds
containing protected phenol and thiophenol functions to
determine how the replacement of an oxygen atom by a
sulfur atom and the presence of two types of protecting
groups influence the compounds’ proliferative/antiprolifer-
ative effects against breast cancer. Compounds 6–8 acted as
pure estrogens, while compound 5 showed cytotoxic effects
similar in magnitude to those observed with previously
described ferrocenyl diphenol 2. These results suggest that
the thiophenol analogue of 6 is non-cytotoxic, further
implicating the production of QM in the cytotoxicity of 2
and related compounds.
compound ferrocenyl diphenol
2
(IC50(5) = 0.5 lM;
IC50(2) = 0.6 lM). This strongly suggests that enzymes
in the living cells hydrolysed the ester function of 5 to gen-
erate the dihydroxyl derivative 2 in situ, as observed in
other systems such as ester-estrogen cleavage [41–43] and
the activation of fluorescent probes [44]. Compound 2
can then engage in QM formation to give rise to cytotoxic
effects. As thioesterases are also known to exist in breast
cancer cells [45], one might therefore expect hydrolysis to
proceed in the case of the analogous sulfur compound 6
as well. However, no antiproliferative effect was observed
for 6, suggesting that the putative ferrocenyl thiophenol
is not cytotoxic.
Neither of the ether compounds 7 nor 8 had any signif-
icant effect against the MDA-MB-231 cell line (IC50(7) =
82.5 lM), likely because these compounds are simply not
hydrolysed in situ. Eukaryotic etherase enzymes have
been detected in liver cells [46] but not yet in breast cancer
cells, and thus hydrolysis of the methyl protected com-
pounds does not proceed in vitro. Finally, as expected,
estradiol has no effect on these cells lacking ERa (see
Table 1).
Acknowledgements
We thank A. Cordaville for technical assistance, and the
Agence Nationale de la Recherche for financial support
(No. ANR-06-BLAN-0384-01, ‘‘FerVect”). J.B.H. acknow-
ledges a fellowship within the Postdoc Programme of the
German Academic Exchange Service (DAAD).
Appendix A. Supplementary material
CCDC 665513 contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free

1722
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