Ferrocene-mediated proton-coupled electron transfer in a series of ferrocifen-type breast-cancer drug candidates

Article abstract of DOI:10.1002/anie.200502925

A double-barreled approach: The addition of ferrocene to an antiestrogen drug skeleton was found to induce cytotoxicity towards breast-cancer cells that are resistant to the common antiestrogen drug. The efficacy of this drug has been related to the proton-coupled electron transfer observed in the presence of pyridine as a base (see picture). (Chemical Equation Presented)

Full text of DOI:10.1002/anie.200502925

Angewandte
Chemie
Antitumor Agents
DOI: 10.1002/anie.200502925
Ferrocene-Mediated Proton-Coupled Electron
Transfer in a Series of Ferrocifen-Type Breast-
Cancer Drug Candidates**
Elizabeth Hillard, Anne Vessi›res, Laurent Thouin,
GØrard Jaouen,* and Christian Amatore*
The archetypical selective estrogen receptor modulator
(SERM) tamoxifen, (Z)-2-[4-(1,2-diphenyl-1-butenyl)phen-
oxy]-N,N-dimethylethanamine, is widely prescribed for
patients diagnosed with hormone-dependent breast cancer,
that is, cancer in which the estrogen receptor (ER) is present
(ER(+)). The antiproliferative action in the breast of the
hydroxylated form of tamoxifen (OH-Tam) arises primarily
from an antiestrogenic effect caused by competitive binding
to the ER, which represses estradiol-mediated DNA tran-
scription.[1] Unfortunately, some breast-cancer cells are resist-
ant to tamoxifen because they either do not express ER
(classified as ER(À)) or have developed resistance following
prolonged exposure to the drug. To fight both ER(+) and
ER(À) breast cancer, we have focused on the creation of
dual-function drugs that combine antiestrogenicity and estro-
gen-independent cytotoxicity in the same molecule.
The discovery of the inorganic complex cisplatin (cis-
[PtCl2(NH3)2]) has revolutionized the treatment of testicular
cancer[2] and has led to increased research into organometallic
complexes as antitumor agents.[3] Our strategy, therefore, has
been to incorporate a potentially cytotoxic moiety, ferrocene,
by grafting it onto the tamoxifen skeleton. A series of these
molecules, called “hydroxyferrocifens” by analogy, have been
prepared (Scheme 1) and their antiproliferative effects have
been studied in ER(+) and ER(À) breast-cancer cell lines.[4]
In the ER(+) MCF7 cells, the hydroxyferrocifens behave
similarly to OH-Tam in that they express an antiestrogenic
effect, although they also possess a cytotoxic component.[5]
However, the hydroxyferrocifens present a remarkable anti-
[*] Dr. E. Hillard, Dr. A. Vessi›res, Prof. Dr. G. Jaouen
Laboratoire de Chimie et Biochimie des Complexes MolØculaires
Ecole Nationale SupØrieure de Chimie de Paris
11, Rue Pierre et Marie Curie, 75231 Paris Cedex 05 (France)
Fax: (+33)1-4326-0061
E-mail: gerard-jaouen@enscp.fr
Dr. L. Thouin, Prof. Dr. C. Amatore
Ecole Normale SupØrieure
DØpartement de Chimie
24 rue Lhomond, 75231 Paris Cedex 05 (France)
Fax: (+33)1-4432-3863
E-mail: christian.amatore@ens.fr
[**] This work was supported by the CNRS (UMR 7576 and UMR 8640),
the French Ministry of Research, ENSCP, and ENS. E.A.H. thanks
the National Science Foundation (USA) for a Postdoctoral Grant
(No. 0302042) and acknowledges Dr. J. Wadhawan for useful
discussions. The authors also thank K. Kowalski, F. Le Bideau, P.
Pigeon, and D. Plazuk for providing the compounds.
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Scheme 1. Hydroxyferrocifens.
proliferative behavior on the ER(À) MDA-MB231 cell line,
with IC50 values on the order of 0.5 mm, whereas OH-Tam is
completely inactive at this concentration range; this effect can
be attributed only to cytotoxicity of the ferrocenyl complexes.
Thus, the observed antiproliferative effect of the hydroxyfer-
rocifens can be divided into two components: antiestrogenic
in ER(+) cells and cytotoxic in both ER(+) and ER(À) cells;
the cytotoxicity likely arises from the oxidation in situ of Fe2+
to Fe3+ ions.[6]
Although hydroxytamoxifen does not show cytotoxic
activity towards MCF7 or MDA-MB231 breast-cancer cell
lines at sub-micromolar concentrations, a cytotoxic effect has
been observed at higher concentrations (IC50 = 34 mm for the
ER(À) MDA-MB231 cell line).[7] The fact that hydroxyferro-
cifens show cytotoxic activity at much lower concentrations
than hydroxytamoxifen suggests that the ferrocene group
somehow modulates this effect. We have synthesized several
compounds based on the hydroxyferrocifen structure
(Scheme 2) and assessed their antiproliferative effects in vitro
to further understand the role of ferrocene and the structure–
activity relationship in the cytotoxicity of these molecules in
both hormone-dependent (MCF7) and hormone-independ-
ent (MDA-MB231) breast-cancer cell lines. In the MDA-
MB231 cell line, an antiproliferative effect was observed for 1,
2, 3, and 4, whereas the remaining compounds showed little or
no effect towards the MDA-MB231 cells. These results are
compiled in Table 1.
As cell death has been linked with the oxidation in situ of
phenol groups and the ferrocenium cation, we have used
electrochemistry to monitor the reactivity of these com-
pounds in a model environment. First, the cyclic voltammo-
grams were obtained in methanol, and then pyridine was
added to determine the reactivity of the electrochemically
generated cations towards nucleophiles; the standard poten-
tials (E8) are shown in Table 2. In pure MeOH, the
compounds exhibited voltammograms that are essentially
due to the ferrocene/ferrocenium (Fc/Fc+) redox couple,
often followed by the irreversible oxidation of the phenolic
moiety.[8] However, two distinct types of electrochemical
behavior were observed when pyridine was added. Very little
change was observed in the cyclic voltammograms upon the
addition of pyridine for the compounds that showed slight or
no cytotoxic effects in vitro. However, the addition of
pyridine caused two major changes to the voltammograms
of the biologically active compounds. Firstly, the Fc/Fc+
couple became irreversibile at low scan rates, which indicates
Scheme 2. Hydroxyferrocifen-type molecules used for electrochemical
and bioactivity screening.
Table 1: Biological and electrochemical results for the ferrocenyl deriv-
atives.
Compound Antiproliferative effect on
MDA-MB231 cells
Cytotoxic
effect?[b]
Electron
transfer?[c]
[% of inhibition][a]
OH-Tam
1a
1b
1c
2
none[g]
53[g]
77[g]
80[g]
35[h]
71[i]
À
n.a.
Y
Y
Y
Y
+
+
+
+
+
+
3
Y
Y
4[d]
49[j]
6a[e]
6b[e]
6c[e]
7[d]
26[k]
18[k]
16[k]
16[j]
slight
slight
slight
slight
À
N
N
N
Y
N
N
8[d]
3[j]
9[f]
none[j]
À
[a] Defined as the effect of 1 mm of the compound after 6 days of
culturing relative to the control, set by definition at 100%. [b] The
cytotoxic effect is considered positive (+) for an antiproliferative effect
(% inhibition) greater than 30%, slight for an effect 10–30%, and
negative (À) for an effect less than 10%. [c] Observed by the loss of the
ferrocenium reduction wave in the presence of pyridine. [d] Prepared as
described in Ref. [17]. [e] Prepared as described in Ref. [18]. [f] Prepared
as described in Ref. [19]. [g] Ref. [4c]. [h] Ref. [4b]. [i] Ref. [16].
[j] Ref. [17]. [k] Ref. [20].
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Table 2: Standard oxidation potentials in methanol and methanol/pyridine (6:1 v/v).
compounds displaying significant
cytotoxic effects in vitro also
showed reactivity with pyridine in
the electrochemical experiments
(Table 1) validates the use of this
electrochemical model.
Compound
Solvent
E8, (Fc/Fc+)
Second-wave peak potential
[V vs. SCE][a]
[V vs. SCE][a]
ferrocene
MeOH
MeOH/py
MeOH
MeOH/py
MeOH
MeOH/py
MeOH
MeOH/py
MeOH
MeOH/py
MeOH
MeOH/py
MeOH
MeOH/py
MeOH
MeOH/py
MeOH
MeOH/py
MeOH
MeOH/py
MeOH
MeOH/py
MeOH
MeOH/py
MeOH
MeOH/py
MeOH
0.417(2)
0.432(2)
0.395(3)
0.393(3)
0.401(2)
0.420(3)
0.385(2)
0.383(3)
0.397(2)
0.423(4)
0.373(2)
0.387(3) (sh)
0.413(2)
0.460(2)
0.421(3)
0.442(3)
0.379(3)
0.408(3)
0.404(3)
0.428(3)
0.391(2)
0.414(2)
0.387(2)
0.407(2) (sh)
0.39(2)
–
–
1a
1b
1c
2
very broad
0.482(3)
0.92(3)
0.513(3)
0.93(4)
0.476(2)
0.96(3)
0.510(3)
0.88(3)
0.480(2)
0.87(4)
0.584(3)
–
The ferrocenium group appears
to play the role of an intramolec-
ular electron acceptor in the cyto-
toxicity in vitro, as evidenced by
the transience of the electrochemi-
cally produced Fe3+ center in the
presence of pyridine. The inertness
of the non-phenolic compound 5 to
pyridine in our electrochemical
model system shows that a phe-
nolic group is necessary for elec-
tron transfer to take place
(Figure 2). Furthermore, the fact
that chemical reduction of the Fe3+
centers was not observed for the
nonconjugated compounds 6a–c
suggests that the electron-transfer
process is intramolecular and
occurs through a slight coupling in
the molecular p system. A further
electrochemical experiment to sup-
port this interpretation was per-
formed using a mixture of ferro-
cene and 1,1-di-p-hydroxyphenyl-
2-phenylbut-1-ene in pyridine/
3
4
5
–
6a
6b
6c
7
not observed
not observed
not observed
not observed
not observed
not observed
0.93(3)
0.513(3)
not observed
1.03(4)
8
0.461(2)
0.153(3)
0.164(2)
9
not observed
0.83(4)
MeOH/py
[a] The uncertainty of last digit given in parentheses.
that the ferrocenium cation was scavenged chemically prior to
the reverse sweep. The loss of reversibility was accompanied
by an increase of the Fc oxidation wave, which is indicative of
secondary electron transfer following a chemical reaction of
the primary cation radical. Secondly, any phenol-oxidation
waves present in the cyclic voltammogram in MeOH under-
went a dramatic cathodic shift. An example of this phenom-
enon for 3 is shown in Figure 1. The fact that all of the
MeOH. The Fc/Fc+ couple was conventional and reversible,
and the phenol-oxidation wave was observed at a potential
typical of the hydroxyferrocifens when pyridine is not present.
These results confirm the conclusion that the electron transfer
from the phenol moiety to the ferrocenyl group is intra-
molecular and relies on weak electron delocalization within
the p system. It is important to note, however, that the
p electron delocalization in the initial cation radical is quite
weak. This is attested to by the observation that the standard
oxidation potentials of the Fc/Fc+ couple of the hydroxyfer-
Figure 1. Cyclic voltammograms of 3 (2 mm in 0.1m Bu4NBF4/MeOH)
in the absence (solid line) and presence (dashed line) of pyridine in
1:6 volume ratio. Scan rate 0.5 VsÀ1. Pt electrode of 0.5 mm diameter.
The oxidations of the phenol groups in MeOH occur at 0.88 and
1.17 V versus SCE (not shown).
Figure 2. Cyclic voltammograms of 5 in the absence (solid line) and
presence (dashed line) of added pyridine. Same conditions as for
Figure 1.
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Communications
rocifens 2 (0.397 V), 3 (0.373 V), and 7 (0.387 V) are not very
much lower than that of the non-hydroxylated analogue 5
(0.421 V). The standard oxidation potentials of the non-
conjugated compounds 6a (0.379 V), 6b (0.404 V), and 6c
(0.391 V) suggest that any stabilization of the radical species
in the hydroxyferrocifens is due to inductive effects, rather
than resulting from extended electron-delocalization effects,
and that the cation radical is primarily ferrocene-centered.
However, weak delocalization is sufficient to allow significant
interaction between the phenolic and ferrocenium substitu-
ents during intramolecular electron transfer. Thus, as soon as
an adequate base is present, a relatively fast intramolecular
electron transfer may occur, thereby leading to the oxidation
of the phenolic moiety becoming easier because of its
displacement by the reaction of the phenoxy cation with the
pyridine base.[9–11]
Pyridine could conceivably contribute to the irreversible
electron-transfer process either by proton abstraction or
nucleophilic attack. To rule out a nucleophilic mechanism, the
nucleophilic, but nonbasic, electrolyte Me4NCl was substi-
tuted for pyridine and Bu4NBF4. In this experiment, the
ferrocene moiety displayed a reversible redox event, which
suggests that pyridine acts as a base, not as a nucleophile.
Further experiments have shown that the pKa value of the
added base influences the reaction pathway. For example,
dimethylformamide (DMF) and pyrazole are too weak to
deprotonate the phenol moiety in the activated molecule, as
the cyclic voltammograms are not qualitatively different from
those in MeOH, whereas pyridine (pKa = 5.14)[12a] and
imidazole (pKa = 6.95)[12b] are sufficiently basic. In this
respect, it may be envisioned that DNA bases act as proton
scavengers in vitro. Alternatively, the relatively high
pKa values (> 7) of a-NH2 groups present in most peptides
suggest that these groups could also act as bases in vitro in
proteinic environments.[12c] However, this point was not tested
in this work because the low solubility of the ferrocenyl
compounds precludes electrochemical experiments in aque-
ous solution.
Finally, the importance of the position of the ethyl group
with respect to the ferrocene moiety was assessed by
comparing the electrochemical results for the geometric
isomers 7 and 8. Only the compound in which the ethyl
group and the ferrocene group are attached to the same
carbon atom (7) showed the characteristic irreversible Fc
oxidation and shift of the phenol oxidation wave upon
addition of pyridine (Figure 3). These results strongly suggest
the participation of the ethyl group in the mechanism.
In view of the role of the ferrocene moiety as an
intramolecular hole reservoir,[10] the conjugated p system,
the basic action of pyridine, and the interesting constraints on
the placement of the ethyl group, we propose the mechanism
shown in Scheme 3 for the generation of the quinone methide
species most likely responsible for the cytotoxicity in the
MDA-MB231 cell line. The ferrocene moiety is electrochemi-
cally oxidized, and the electron may to a small extent be
delocalized over the p system. This imparts a partial positive
charge to the hydroxy group, thus acidifying the proton, which
may then be easily abstracted by pyridine.[11] The small
delocalization is almost not observable in the absence of
Figure 3. Cyclic voltammograms of A) 8 and B) 7 in the absence (solid
line) and presence (dashed line) of added pyridine. Same conditions
as for Figure 1.
pyridine, as evidenced by the small change in the oxidation
potential of the ferrocene moiety. In the presence of pyridine,
the transient existence of an adduct may encourage a strong
coupling in the deprotonated transition state. The resulting
phenoxy radical species can be described by many mesomeric
structures, one of the most stable being the quinoid in which
the radical is positioned on the a carbon atom with respect to
the ferrocene moiety. This species may then be oxidized,
observed as the second oxidation wave at slightly higher
potential than the ferrocene oxidation wave. This second
oxidation may then be followed by another proton abstraction
from the ethyl group, which results in a quinone methide
structure. This mechanism is the only possibility that we can
envision to account for the difference in reactivity between 7
and 8. In the latter case, there is no possibility to stabilize the
a-carbenium radical through back-bonding into the iron
d orbital, and simultaneously abstract an adjacent proton to
form a double bond (see bottom of Scheme 3).[13]
The potential cell-damaging pathways for tamoxifen
include metabolism to electrophilic o-quinones, quinone
methides, or carbocations, which may form adducts with
DNA, GSH, or proteins.[14] We believe that the biologically
active ferrocifen-type compounds undergo similar oxidative
metabolism, which is enhanced by the easier oxidation of
ferrocene in comparison to phenol. Thus, the ferrocene
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in contrast to an antihormonal effect, is not reversed by the
addition of estradiol.
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[8] The wave of the phenol moiety was shifted to higher potential, as
expected, because of the positive charge on the Fc+ moiety.
[9] In our system, phenol oxidation waves occurred at potentials
higher than 0.8 V vs. SCE.
[10] a) C. Amatore, A. ThiØbault, J.-N. Verpeaux, J. Chem. Soc.
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Scheme 3. Proposed mechanism for transformation of 2 to a quinone
methide species in the presence of pyridine. As indicated in the lower
part of the Scheme, compound 8 cannot follow the same mechanism.
[11] C. J. Schlesener, C. Amatore, J. K. Kochi, J. Am. Chem. Soc.
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moiety may be oxidized far from the biological target and
may thus serve as an intramolecular carrier of the hole while
the activated drug finds its way to its target. Furthermore, the
small degree of coupling between the ferrocenium-centered
radical and the oxo radical yields a relatively high energy
transition state, which may stabilize the ferrocenium species
on its path to the target.[15] In this way the ferrocene acts as a
kind of intramolecular oxidation “antenna” and may oxidize
the phenol group through a intramolecular pathway, thus
producing cytotoxic species in milder oxidizing conditions.
Only when an adequate base is present will the electron
transfer proceed, presumably in a concerted fashion with
deprotonation, to yield the neutral phenoxy radical. DMF, for
example, is not basic enough to allow such electron transfer.
Therefore, the drug will only be activated to form the quinone
methide species in the presence of basic species such as DNA
nucleobases or peptides, the harm of which may lead to cell
death.
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[13] The involvement of a quinone methide species subsequent to the
electron-transfer activation is further supported by the observa-
tion that the meta-OH-substituted analogues that have been
tested did not show any reactivity following their initial one-
electron oxidation in the presence of base (comparable to
Figure 3a), whereas intramolecular electron transfer was
observed electrochemically when a para-phenol was added to
the meta-substituted derivative, (comparable to Figure 3b). (P.
Pigeon, A. Vessi›res, E. Hillard, G. Jaouen, unpublished results).
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90, 3747.
Received: August 17, 2005
Published online: November 28, 2005
[16] A. Vessi›res, S. Top, P. Pigeon, E. A. Hillard, L. Boubeker, D.
Spera, G. Jaouen, J. Med. Chem. 2005, 48, 3937.
Keywords: antitumor agents · electrochemistry ·
electron transfer · hormones · metallocenes
.
[17] A. Vessi›res, E. A. Hillard, P. Pigeon, S. Top, K. Kowalski, J.
Zakrzewski, G. Jaouen, unpublished results. Compounds 4, 7,
and 8 were synthesized by McMurry cross-coupling reactions
between 3-chloropropionylferrocene and 4,4’-dihydroxybenzo-
phenone, ferrocene ethyl ketone and 4-hydroxy,4’-methoxyben-
zophenone, and 4-hydroxyphenylethyl ketone and 4-methoxy-
phenylferrocenyl ketone, respectively, and characterized by
NMR spectroscopy and HRMS.
[1] a) S. Bardon, F. Vignon, D. Derocq, H. Rochefort, Mol. Cell.
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Communications
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[18] D. Plazuk, A. Vessi›res, F. Le Bideau, G. Jaouen, J. Zakrzewski,
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[19] K. Kowalski, A. Vessi›res, S. Top, G. Jaouen, J. Zakrzewski,
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