Ferrocenyl quinone methides as strong antiproliferative agents: Formation by metabolic and chemical oxidation of ferrocenyl phenols

Article abstract of DOI:10.1002/anie.200903768

Ferrocenyl quinone methides, potentially cytotoxic species, are formed by metabolic and chemical oxidation of ferrocenyl phenols (see scheme). These species display strong antiproliferative properties.

Full text of DOI:10.1002/anie.200903768

Communications
DOI: 10.1002/anie.200903768
Antitumor Agents
Ferrocenyl Quinone Methides as Strong Antiproliferative Agents:
Formation by Metabolic and Chemical Oxidation of Ferrocenyl
Phenols**
Didier Hamels, Patrick M. Dansette, Elizabeth A. Hillard, Siden Top, Anne Vessiꢀres,
Patrick Herson, Gꢁrard Jaouen,* and Daniel Mansuy*
[
12]
The modification of the biological effects of some biologically
under heightened oxidative stress suggests that this class of
redox-activated compounds may show an interesting selec-
tivity profile.
[
1,2]
active molecules by ferrocene is an active field of study.
The addition of a ferrocenyl moiety to selected polyaromatic
[3]
[4]
phenols, amines, and amides can potentiate their antipro-
liferative effects against breast and prostate cancer cells. For
example, hydroxytamoxifen, the active metabolite of the
breast cancer drug tamoxifen, shows limited toxicity against
hormone-refractory breast cancer cells (IC for MDA-MB-
The formation of ferrocenyl QMs has been supported only
by electrochemical experiments until now. We herein show
that QMs do form upon metabolism of 1–4 by rat liver
[7]
5
0
[5]
2
31: 29 mm). However, when a phenyl group is replaced by a
ferrocene moiety, the resulting “hydroxyferrocifen” displays a
[
6]
dramatic improvement in toxicity (IC = 0.5 mm). In 2006,
5
0
we proposed in this journal a novel mechanism of activation
of ferrocene phenols: their ferrocene-mediated oxidation to
[
7]
quinone methide (QM) metabolites. This work was sub-
[
8]
sequently featured as part of a Highlight article. Quinone
[
9–11]
metabolites are potentially cytotoxic species,
and, when
ferrocene is present, QM formation takes place at compara-
tively mild (i.e. biologically relevant) oxidation potentials.
Furthermore, the observation that some cancer cells are
microsomes and can also be prepared by chemical oxidation
of 1–4 with Ag O. These new organometallic QMs have been
2
1
completely characterized by mass spectrometry and H and
C NMR spectroscopy, and one crystal structure has been
1
3
[*] D. Hamels, Dr. E. A. Hillard, Dr. S. Top, Dr. A. Vessiꢀres,
Prof. G. Jaouen
obtained. These compounds were also found to exhibit
marked antiproliferative behavior against hormone-inde-
pendent breast cancer cells. This study thus prefigures the
preclinical development of ferrocenyl phenols currently
underway.
Chimie ParisTech (Ecole Nationale Supꢁrieure de Chimie de Paris)
Laboratoire Charles Friedel, UMR CNRS 7223
11 rue P. et M. Curie, 75231 Paris cedex 05 (France)
Fax: (+33)1-43-25-79-75
E-mail: gerard-jaouen@enscp.fr
To establish whether QMs could be formed during
metabolism of ferrocene phenols 1–4 in mammals, we studied
the transformation of these complexes by liver microsomes
containing the main enzymes responsible for xenobiotics
metabolism. Incubation of 100 mm 3 with an aerobic suspen-
sion of rat liver microsomes (1 mm cytochrome P450) in
phosphate buffer (pH 7.4) containing 1 mm nicotinamide
adenine dinucleotide phosphate (NADPH) for 30 min at
Dr. P. M. Dansette, Dr. D. Mansuy
Universitꢁ Paris Descartes, UMR CNRS 8601
45 rue des Saints-Pꢀres, 75270 Paris Cedex 06 (France)
Fax: (+33)1-42-86-21-69
E-mail: daniel.mansuy@parisdescartes.fr
Dr. P. Herson
Universitꢁ Pierre et Marie Curie
Institut Parisien de Chimie Molꢁculaire, UMR CNRS 7201
4
place Jussieu, 75252 Paris cedex 05 (France)
3
78C led to the formation of a new major product that was
[
**] This work was supported by the Ministꢀre de la Recherche, the
Centre National de la Recherche Scientifique, and the Agence
Nationale de la Recherche (No. ANR-06-BLAN-0384-01, “FerVect”).
We thank M.-N. Rager for the NOESY NMR experiments, C. Fosse
and C. Fleurant-Keil for the mass spectroscopy experiments, and
M.-A. Plamont for the cell culture experiments.
detected by HPLC–MS and purified by preparative HPLC. Its
+
mass spectrum (ESI ) exhibited a molecular ion at m/z 422
+
[M ] corresponding to the QM derived from two-electron
oxidation of 3, whose mass spectrum was characterized by a
+
molecular ion at m/z 424 [M ]. Fragmentation of the ion at
Supporting information for this article (experimental conditions,
m/z 422 by MS–MS led to an ion at m/z 356 that results from
the loss of 66 u, which could correspond to a cyclopentadiene
moiety.
Identical microsomal incubations with monophenolic
compounds 1 and 4 led to the formation of the corresponding
QM metabolites as major products. These metabolites were
1
13
synthesis, H and C NMR spectroscopy and mass spectrometry
for 1a, 2a, 4, and 4a and intermediates; melting points and
elemental analysis data for 4 and 4a; NOESY, HMQC, and COSY
studies for 1a and 2a, and ORTEP diagrams for 4 and 4a) is
available on the WWW under http://dx.doi.org/10.1002/anie.
200903768.
9
124
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Angew. Chem. Int. Ed. 2009, 48, 9124 –9126

Angewandte
Chemie
+
also identified by HPLC–MS: a molecular ion [M ] at m/z 406
and 434 for the metabolite of 1 and 4, respectively, and MS–
MS fragments corresponding to the loss of 15 and 66 u (loss of
CH3 and C H , respectively) were observed for the two
5
6
metabolites. Finally, incubation of the ferrocifen compound
related to tamoxifen (2) also led to the formation of a QM
metabolite that exhibited a MS molecular ion at m/z 508
+
[
M+H ], whereas the mass spectrum of 2 showed a molecular
ion at m/z 510.
These data strongly suggest that QM metabolites are
generally formed upon oxidation of the studied ferrocenyl
phenols by liver microsomes. As these QMs appeared to be
stable in the conditions used for their identification, we
decided to prepare them by chemical oxidation. It is note-
worthy that the chemically prepared QMs exhibited HPLC
retention times and MS spectra identical to those of the
corresponding QM metabolites formed in microsomal incu-
bations.
Compounds 1, 2, and 4 were obtained as 50/50 mixtures of
Z and E isomers. With respect to the double bond of the
terminal alkene function, the QM could exist as a Z or E
isomer. Surprisingly, only one isomer was visible in the NMR
spectra of 1a, 2a, and 4a. NOESY NMR experiments of 1a
and 2a showed that the methyl allylic protons point away
from the ferrocenyl group towards either the quinone (1a) or
the aromatic ring (2a), and that the vinylic proton points
towards the ferrocene moiety, giving the compounds E confi-
guration.
[
13]
[14]
Chemical oxidation was performed by dissolving 1, 2,
and newly synthesized 4 (1 mmol; Scheme 1) in a small
[
1]
3,
volume of distilled acetonitrile and adding freshly prepared
Compound 4a proved to be stable enough to afford X-ray
Ag O (5 equiv). After filtration and evaporation of the
quality red plates from
a
CH Cl /pentane mixture
2
2 2
[15]
(Figure 1). The QM structure of 4a
was confirmed by comparison of its
bond lengths with those of 4 and
literature values for those of the QM
[
16]
2
,5-dimethylfuchsone.
The C18ÀO1
bond is significantly shorter for 4a than
for 4, suggesting C=O bond character.
Similarly, the C1ÀC15 and C2ÀC3
bonds are shorter in 4a than in 4,
indicating C=C bond character. In
contrast, the C1ÀC2 bond is longer in
4
a than in 4, which is consistent with a
CÀC bond. Another feature is the
alternating long and short bonds in
the quinone group in 4a, which is
characteristic of a 1,4-unsaturated ring.
The methide double bond in 4a is
slightly distorted (dihedral angle of
Scheme 1. Synthesis of 4 and 4a.
7
.938); this distortion may be explained
by the bulkiness of the different groups
involved, which try to minimize steric interactions with one
another. The molecule is quite strained; the ferrocene moiety
is in an eclipsed configuration, and an angle of 5.868 can be
observed between the two cyclopentadienyl (Cp) rings. The
obtained E configuration is explained by the observation that
the C2ÀC3 double bond and the upper cyclopentadiene ring
solvent under reduced pressure, the corresponding QMs 1a
(
88% yield), 2a (79%), and 4a (84%) were obtained in
greater than 95% purity after 45, 90, and 10 min, respectively.
For all compounds, the color rapidly changed from orange to
dark red, and NMR spectroscopy confirmed a structural
change to a QM; the appearance of a doublet and a quartet in
1
the H NMR spectra at around d = 1.68 and 6.44 ppm,
are nearly in the same plane (torsion angle of 18.978), which
means that if the C=C bond had Z configuration, the methyl
respectively, indicated the formation of a double bond on
1
3
the ethyl group. Furthermore, C NMR spectroscopy indi-
cated the formation of a C=O bond as the peak corresponding
to the CÀOH bond in 1, 2, and 4 shifted from around d =
protons would strongly interact with the Cp ring. As free
rotation is possible around the C1ÀC2 bond, steric interac-
tions with both rings can thus be minimized.
1
53 ppm to d = 187 ppm. As observed above, the mass spectra
Parent molecules 1 and 2 show strong antiproliferative
(cytotoxic) effect on hormone-independent breast cancer
cells (MDA-MB-231) and yield IC values of 1.1 and 0.5 mm,
of 1a, 2a, and 4a showed the loss of two protons from their
parent molecules. For compound 3, however, although we
were able to observe the appearance of characteristic QM
peaks in the NMR spectra, QM 3a was unstable and started
decomposing before oxidation was complete.
5
0
[
14,17]
respectively.
Freshly synthesized 1a and 2a were also
toxic against MDA-MB-231 cells (IC values of 21.7 Æ 4.3 and
5
0
4.2 Æ 1 mm, respectively). These higher values for 1a and 2a,
Angew. Chem. Int. Ed. 2009, 48, 9124 –9126
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
9125

Communications
[
4] P. Pigeon, S. Top, O. Zekri, E. A. Hillard, A. Vessiꢀres, M.-A.
Plamont, E. Labbꢁ, O. Buriez, M. Huchꢁ, S. Boutamine, C.
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5.
[
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4
[
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Jeurissen, M. E. Schutte, G. M. Alink, Mutat. Res. 2005, 574, 124.
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15] Intensity data were collected at 200 K with a Enraf–Nonius
Kappa CCD diffractometer equipped with a CCD two-dimen-
sional detector [l MoKa = 0.71073 ꢂ]. Data were collected in 236
frames (4) and 345 frames (4a·CH Cl ) with f and w scans
2
2
(width of 1.58 and exposure time of 75 s per frame for 4 and 28
and 40 s for 4a·CH Cl ). Data reduction was performed with
2
2
EVALCCD software (Duisenberg & Scheurs 1990 – 2000). Data
were corrected for Lorentzian and polarization effects, and a
semi-empirical absorption correction based on symmetry equiv-
alent reflections was applied by using the SADABS program
[G. M. Sheldrick, SADABS, Program for Scaling and Correction
of Area Detector Data, University of Gꢃttingen, Gꢃttingen,
Germany, 1997, R. H. Blessing, Acta Crystallogr. Sect. A 1995,
51, 33]. Lattice parameters were obtained from least-squares
analysis of 91 (4) and 143 (4a·CH Cl ) reflections. The structure
Figure 1. ORTEP views of a) (Z)-4 and b) (E)-4a with thermal ellipsoids
presented at 50% probability. Selected bond lengths [ꢂ] for 4: C18–O1
.402(4), C15–C16 1.401(4), C16–C17 1.399(4), C17–C18 1.407(5),
C15–C1 1.510(4), C1–C2 1.361(4), C2–C3 1.536(4); for 4a: C18–O1
.246(3), C15–C16 1.459(4), C16–C17 1.361(4), C17–C18 1.481(4),
1
1
C15–C1 1.381(4), C1–C2 1.512(4), C2–C3 1.343(4).
2
2
was solved by direct methods and refined by full matrix least-
2
squares analysis, based on F , using the Crystals software
package [Betteridge et al., 2003]. All non-hydrogen atoms
were refined with anisotropic displacement parameters. All
hydrogen atoms were located with geometrical restraints in the
riding mode.
relative to those 1 and 2, could be due to their chemical
instability, as strong electrophiles, in the incubation medium.
These IC50 values nonetheless suggest that 1a and 2a should
have remarkable intrinsic antiproliferative properties. More-
over, since 1a and 2a are formed as major metabolites of 1
and 2 inside the cells, at the level of the endoplasmic
reticulum, they should play a key role in the anticancer
properties of ferrocenyl phenols. The previously proposed
mechanism of oxidative activation of such ferrocenyl phenols
is therefore established.
4
: C H FeO; M = 436.38; monoclinic; P21/n; a = 9.319(1), b =
28 28
3
12.066(1), c = 20.305(1) ꢂ, b = 94.717(9)8, V= 2275.4(4) ꢂ ; Z =
4; 0.21 ꢄ 0.18 ꢄ 0.15 mm ; 1calcd = 1.27; m = 0.679; 2Vmax = 608;
3
total reflections: 22754; independent reflections: 6597; R-
(
I>2s(I)) = 0.0546, wR2(all) = 0.1387, S = 0.9016; highest resid-
À3
ual electron density 1.11 eꢂ
.
ꢀ
4
8
1
a·CH Cl : C H Cl FeO; M = 519.29; triclinic, P1; a =
2 2 29 28 2
.904(1), b = 9.6247(8), c = 15.404(2) ꢂ, a = 92.208(7), b =
3
01.598(8), g = 90.581(7)8. V= 1291.99(18) ꢂ ; Z = 2; 0.26 ꢄ
3
Received: July 9, 2009
Published online: October 28, 2009
0.19 ꢄ 0.12 mm ; 1
= 1.33; m = 0.810; 2V= 608; total reflec-
calcd
tions: 26816; independent reflections: 7517; R (I > 2s(I)) =
0
.0562, wR2 (all) = 0.1392, S = 1.0357; highest residual electron
À3
Keywords: antitumor agents · ferrocene · metabolism ·
density 1.10 eꢂ . CCDC 736717 (4) and 736718 (4a) contain
the supplementary crystallographic data for this paper. These
data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif.
.
quinones
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Angew. Chem. Int. Ed. 2009, 48, 9124 –9126