Ferrocenyl flavonoid-induced morphological modifications of endothelial cells and cytotoxicity against B16 murine melanoma cells

Article abstract of DOI:10.1016/j.jorganchem.2012.12.031

With the aim of improving the cytotoxic and vascular disrupting activities of flavonoids, several classes of ferrocenyl-modified flavonoids were prepared and tested on cancer and endothelial cells. Three ten-member series of ferrocenyl flavonoids: chalcones ((E)-1-(R-2′-hydroxyphenyl)-3- ferrocenylprop-2-en-1-ones), aurones ((Z)-R-2-(ferrocenylidene)benzofuran-3- ones) and flavones (R-2-ferrocenyl-chromen-4-ones) were synthesized by recently reported methods. Three ferrocenyl flavonols (R-3-hydroxy-2-ferrocenyl-chromen- 4-ones) and four ferrocenyl flavanones (3-ferrocenylmethylidenyl-R-2- phenylchroman-4-ones) were also obtained. All compounds were evaluated for their cytotoxic effects on a cancer cell line (B16 murine melanoma) and for their morphological effects on endothelial cells (EAhy 926). Some interesting structure-activity relationships were disclosed: of all the compounds, the halogen-substituted aurones showed the best cytotoxic activity, with IC ₅₀ values ranging between 12 and 18 μM. Ferrocenyl flavonols and ferrocenyl flavanones with substitution in the 3-position (-OH and C-Fc respectively) were not active against cancer or endothelial cells. Some of the ferrocenyl flavones caused the endothelial cells to adopt a round shape ("rounding up") at submicromolar concentrations, which can be predictive of vascular disrupting activity. The most morphologically active flavones showed only moderate cytotoxicity against cancer cells, indicating that they may primarily act as antivascular agents.

Full text of DOI:10.1016/j.jorganchem.2012.12.031

Journal of Organometallic Chemistry 734 (2013) 78e85
Contents lists available at SciVerse ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Ferrocenyl flavonoid-induced morphological modifications of
endothelial cells and cytotoxicity against B16 murine melanoma cells
,
b
,
,
b
,
,
d
,
e
,
,b
Jean-Philippe Monserrat a 1, Keshri Nath Tiwari a 1, Lionel Quentin c
f, Pascal Pigeon a
,
Gérard Jaouen a b, Anne Vessières a b, Guy G. Chabot c
a ENSCP Chimie ParisTech, Laboratoire Charles Friedel (LCF), 75005 Paris, France
f, Elizabeth A. Hillard a
,
,
,
d,
e,
,b,
*
b CNRS, UMR 7223, 75005 Paris, France
c ENSCP Chimie ParisTech, Unité de Pharmacologie Chimique et Génétique et Imagerie (UPCGI), 75005 Paris, France
d Université Paris Descartes, Faculty of Pharmacy, 75006 Paris, France
e CNRS, UMR 8151, 75006 Paris, France
f INSERM, U 1022, 75006 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
With the aim of improving the cytotoxic and vascular disrupting activities of flavonoids, several classes of
ferrocenyl-modified flavonoids were prepared and tested on cancer and endothelial cells. Three ten-
member series of ferrocenyl flavonoids: chalcones ((E)-1-(R-20-hydroxyphenyl)-3-ferrocenylprop-2-en-
1-ones), aurones ((Z)-R-2-(ferrocenylidene)benzofuran-3-ones) and flavones (R-2-ferrocenyl-chromen-4-
ones) were synthesized by recently reported methods. Three ferrocenyl flavonols (R-3-hydroxy-
2-ferrocenyl-chromen-4-ones) and four ferrocenyl flavanones (3-ferrocenylmethylidenyl-R-2-phenyl-
chroman-4-ones) were also obtained. All compounds were evaluated for their cytotoxic effects on a cancer
cell line (B16 murine melanoma) and for their morphological effects on endothelial cells (EAhy 926). Some
interesting structureeactivity relationships were disclosed: of all the compounds, the halogen-substituted
Received 16 October 2012
Received in revised form
24 December 2012
Accepted 26 December 2012
Keywords:
Flavones
Aurones
Ferrocene
aurones showed the best cytotoxic activity, with IC50 values ranging between 12 and 18 mM. Ferrocenyl
Cytotoxicity
flavonols and ferrocenyl flavanones with substitution in the 3-position (eOH and ]CeFc respectively)
were not active against cancer or endothelial cells. Some of the ferrocenyl flavones caused the endothelial
cells to adopt a round shape (“rounding up”) at submicromolar concentrations, which can be predictive of
vascular disrupting activity. The most morphologically active flavones showed only moderate cytotoxicity
against cancer cells, indicating that they may primarily act as antivascular agents.
Ó 2013 Elsevier B.V. All rights reserved.
Antivascular agents
Bioorganometallic chemistry
1. Introduction
as angiogenesis inhibitors. Flavonoids are an important class of
vascular disrupting agents, an experimental treatment involving
Flavonoids are small molecules, widespread in the plant king-
dom, consisting of two phenyl rings linked by a 3-carbon atom
bridge, and represent the most common polyphenols in the human
diet. They are currently enjoying enormous interest as nutraceut-
icals and drug candidates for diabetes, cognitive function, blood
pressure, cancer, obesity, and other diseases [1]. Appearing with the
first plants on earth and thus having accompanied the evolution of
animal life, flavonoids interact with many biological pathways. In
cancer research, flavonoids have been shown to inhibit various
pathways related to drug resistance [2], apoptosis [3], division [4]
and metastasis [5]. Currently, there is great interest in flavonoids
rapid destruction of the tumor vasculature and thus preventing
oxygen and nutrient flow to the tumor [6]. One flavonoid, ASA404
(vadimezan), is currently undergoing Phase II clinical trials as
a vascular disrupting agent [7]. ASA404 acts by disrupting the actin
cytoskeleton of tumor vascular endothelial cells, making the vessels
more permeable. When used in combination, this molecule has been
shown to enhance the efficacy of carboplatin and paclitaxel [8].
Organometallic medicinal chemistry is one of the most mature
and active areas in bioorganometallic chemistry [9], and related
applications in enzyme inhibition, bioimaging, immunoassay, small
molecule delivery, and in cellulo catalysis are growing topics [10].
The use of organometallic compounds in anticancer drug discovery
continues to receive a great deal of attention [11], as such molecules
are often more stable to hydrolysis than coordination compounds.
The introduction of organometallics can alter the physicochemical
characteristics of a bioactive compound by acting as a structural
* Corresponding author. Present address: CNRS, CRPP, UPR 8641, F-33600 Pessac,
France. Tel.: þ33 (0)5 56 84 56 24; fax: þ33 (0)5 56 84 56 00.
E-mail address: hillard@crpp-bordeaux.cnrs.fr (E.A. Hillard).
1
These authors contributed equally to the work.
0022-328X/$ e see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jorganchem.2012.12.031

J.-P. Monserrat et al. / Journal of Organometallic Chemistry 734 (2013) 78e85
79
element [12], controlling hydrolysis kinetics, or modifying its lip-
ophilicity [13]. Organometallic units can also add supplementary
reactivity to the biomolecule by imparting redox properties [14] or
acting as an alkylating function to proteins [15] or DNA [16]. From
another point of view, bioligands can be used to vectorize cytotoxic
metal moieties, bringing them into the mitochondria or nucleus,
and allowing them to interact with receptors [17]. Ferrocene in
particular has gained considerable interest as a substituent in
medicinal chemistry due to its stability in aqueous and aerobic
media, its lipophilicity, its ease of derivation, and its redox activity
[18]. Some ferrocene derivatives have shown promising activity
against proteins, cells or organisms associated with diseases
including fungal and bacterial infections [19], malaria [20], HIV
[21], and cancer [22].
While there are several examples of biologically active flavonoid
metal complexes [23], few examples of organometallic flavonoids,
apart from ferrocenyl chalcones [24], can be found in the literature
[25]. Notably, however, ruthenium arenes coordinated to flavonols
recently displayed particularly interesting cytotoxic activity against
CH1 ovarian cells, possibly due to binding to DNA accompanied by
topoisomerase IIa inhibitory activity [26]. As part of our ongoing
project studying ferrocene-modified biomolecules, we have
recently obtained access to a variety of ferrocenyl flavonoids [27].
We now present our studies on the antiproliferative effects on B16
murine melanoma and the potential vascular disrupting effects on
the EAhy 926 endothelial cell model of a number of ferrocenyl
chalcones, aurones, flavones, flavanols and flavanones. Some
interesting structureeactivity relationships were observed and
a clear differential activity between the cytotoxic effects on cancer
cells and the morphological effects on endothelial cells was
observed for certain compounds, suggesting that these activities
are not correlated.
solution was cooled to rt and poured into an aqueous solution of
1 M NaOH (100 mL). The mixture was extracted with EtOAc
(3 ꢀ 50 mL) and washed with water. The organic phase was dried
over MgSO4, filtered, and evaporated. The crude product was pu-
rified using a silica gel column, with 60/40 petroleum ether/ethyl
acetate as an eluent and was isolated as an orange solid. Charac-
terization of 3a, 3c and 3h has been previously reported [27].
2.2.1.1. 6-chloro-2-ferrocenyl-chromen-4-one, 3b. Yield: 72%; mp
202 ꢁC, nmax/cmꢂ1 1639 (C]O), 1608 (C]C), dH (300 MHz; CDCl3)
4.17 (s, 5H, C5H5), 4.54 (s, 2H, C5H4), 4.85 (s, 2H, C5H4), 6.45 (s, 1H,
vinyl), 7.45 (d,1H, J ¼ 8.5 Hz, C6H3), 7.59 (d, 1H, J ¼ 8.5 Hz, C6H3), 8.16
(s,1H, C6H3). dC (75 MHz; CDCl3) 67.5 (C5H4), 70.3 (C5H5), 71.6 (C5H4),
74.5 (C5H4 ipso),105.6 (C]C), 111.6 (C6H3),119.4 (C6H3),125.1 (C6H3),
130.9 (C6H3), 133.4 (C6H3), 154.5 (C6H3), 168.7 (C]C), 176.2 (C]O).
HRMS (ESI) calcd. for C19H14ClFeOþ2 : 365.0032, found: 365.0027.
2.2.1.2. 6,8-difluoro-2-ferrocenyl-chromen-4-one, 3d. Yield: 60%;
mp 155 ꢁC, nmax/cmꢂ1 1639 (C]O), 1585 (C]C), dH (400 MHz;
CDCl3) 4.18 (s, 5H, C5H5), 4.56 (t, 2H, J ¼ 1.9 Hz, C5H4), 4.88 (t, 2H,
J ¼ 1.9 Hz, C5H4), 6.44 (s, 1H, vinyl), 7.18 (dd, 1H, J ¼ 8.8 Hz,
J ¼ 2.4 Hz, C6H2), 7.63 (d, 1H, J ¼ 8.8 Hz, C6H2). dC (100 MHz; CDCl3)
67.7 (C5H4), 70.5 (C5H5), 71.9 (C5H4), 74.2 (C5H4 ipso), 105.4 (C]C),
106.0 (dd, 2J ¼ 23.7 Hz, 4J ¼ 4.0 Hz, C6H2) 108.7 (dd, 3J ¼ 20.1 Hz,
3J ¼ 28.5 Hz, C6H2), 126.8 (s, C6H2), 141.9 (d, 2J ¼ 9.4 Hz, C6H2), 151.3
(dd, 1J ¼ 256 Hz, 3J ¼ 11.6 Hz, C6H2), 158.3 (dd, 1J ¼ 249 Hz,
3J ¼ 9.7 Hz, C6H2), 168.7 (C]C), 176.0 (C]O). HRMS (ESI) calcd. for
C19H13F2FeOþ2 : 367.0233, found: 367.0244.
2.2.1.3. 6,8-dichloro-2-ferrocenyl-chromen-4-one, 3e. Yield: 60%;
mp 193 ꢁC, nmax/cmꢂ1 1646 (C]O), 1612 (C]C), dH (300 MHz;
CDCl3) 4.18 (s, 5H, C5H5), 4.58 (t, 2H, J ¼ 1.9 Hz, C5H4), 4.88 (t, 2H,
J ¼ 1.9 Hz, C5H4), 6.44 (s,1H, vinyl), 7.71 (d,1H, J ¼ 1.8 Hz, C6H2), 8.07
(d, 1H, J ¼ 1.8, C6H2). dC (75 MHz; CDCl3); 67.7 (C5H4), 70.4 (C5H5),
71.9 (C5H4), 74.1 (C5H4 ipso), 105.3 (C]C), 123.5 (C6H2), 123.9
(C6H2), 125.9 (C6H2), 130.5 (C6H2), 133.3 (C6H2), 150.4 (C6H2), 168.9
(C]C), 175.4 (C]O). HRMS (ESI) calcd. for C19H13Cl2FeOþ2 :
398.9642, found: 398.9642.
2. Material and methods
2.1. General remarks
All reactions were carried out under argon. THF was distilled over
sodium/benzophenone. All other chemical reagents and solvents
were used without further purification. Silica gel chromatography
2.2.1.4. 6,8-dibromo-2-ferrocenyl-chromen-4-one, 3f. Yield: 55%;
mp 194 ꢁC, nmax/cmꢂ1 1643 (C]O), 1612 (C]C), dH (300 MHz;
CDCl3) 4.22 (s, 5H, C5H5), 4.59 (t, 2H, J ¼ 1.9 Hz, C5H4), 4.93 (t, 2H,
J ¼ 1.9 Hz, C5H4), 6.44 (s, 1H, vinyl), 8.01 (d, 1H, J ¼ 2.4 Hz, C6H2),
8.27 (d, 1H, J ¼ 2.4 Hz, C6H2). dC (75 MHz; CDCl3), 67.8 (C5H4), 70.4
(C5H5), 71.8 (C5H4), 74.1 (C5H4 ipso), 105.2 (C]C), 112.5 (C6H2), 118.1
was performed with Merck 60 (40e63
spectra were recorded with a 300 or 400 MHz Bruker Avance
m
m) silica. 1H and 13C NMR
spectrometer, and
d
are given in ppm and referenced to the residual
solvent peaks (1H,
d
7.26 and 13C{1H}, 77.1 for CDCl3 and 1H,
d
d
2.05
and 13C{1H},
d
29.7 for acetone d6). Mass spectra were measured on
a Thermoscientific ITQ1100 spectrometer using the direct exposure
probe method by the mass spectrometry service at ENSCP Chimie
ParisTech. High resolution mass spectra were obtained by ESI/ESCI-
TOF using a Waters LCT Premier XE or a Thermo Fischer LTQOrbitrap
XL at the Institut Parisien de Chimie Moléculaire (IPCM). Melting
points were determined using an Electrothermal 9100 apparatus. IR
data were collected on a JASCO FT/IR-4100 using a KBr pellet. The
purity of all compounds (>98%) was confirmed using a Shimadzu
HPLC LC20 series equipped with LC-20AB pump and SPD-20A UV
detector prior to biological testing.
(C6H2), 127.8 (C6H2), 136.6 (C6H2), 138.8 (C6H2), 151.7 (C6H2), 169.0
79
(C]C), 175.3 (C]O). HRMS (ESI) calcd. for C19
H
Br2FeOþ2 :
13
486.8632, found: 486.8629.
2.2.1.5. 7-methoxy-2-ferrocenyl-chromen-4-one, 3g. Yield: 70%; mp
174 ꢁC, nmax/cmꢂ1 1631 (C]O), 1604 (C]C), dH (300 MHz; CDCl3)
3.93 (s, 3H, OMe) 4.17 (s, 5H, C5H5), 4.50 (t, 2H, J ¼ 1.8 Hz, C5H4),
4.83 (t, 2H, J ¼ 1.8 Hz, C5H4), 6.40 (s, 1H, vinyl), 6.89 (d, 1H,
J ¼ 2.3 Hz, C6H3), 6.95 (dd, 1H, J ¼ 2.3 Hz, J ¼ 8.9 Hz, C6H3), 8.10 (d,
1H, J ¼ 8.9 Hz, C6H3). dC (75 MHz; CDCl3) 55.6 (OMe), 67.1 (C5H4),
70.0 (C5H5), 71.0 (C5H4), 75.1 (C5H4 ipso), 100.1 (C6H3), 105.4 (C]C),
113.7 (C6H3), 117.7 (C6H3), 126.7 (C6H3), 157.7 (C6H3), 163.6 (C6H3),
167.4 (C]C), 176.8 (C]O). HRMS (ESI) calcd. for C20H17FeOþ3 :
361.0520, found: 361.0527.
2.2. Synthesis
The synthesis and characterization of the ferrocenyl chalcones
1aej and ferrocenyl aurones 2aej have been previously
reported [28].
2.2.1.6. 5-methoxy-2-ferrocenyl-chromen-4-one, 3i. Yield: 68%; mp
165 ꢁC, nmax/cmꢂ1 1635 (C]O), 1608 (C]C), dH (300 MHz) 3.99
(s, 3H, OMe) 4.16 (s, 5H, C5H5), 4.49 (t, 2H, J ¼ 1.8 Hz, C5H4), 4.82 (t,
2H, J ¼ 1.8 Hz, C5H4), 6.38 (s, 1H, vinyl), 6.80 (d, 1H, J ¼ 8.2 Hz, C6H3),
7.06 (d, 1H, J ¼ 8.4 Hz, C6H3), 7.54 (t, 1H, J ¼ 8.4 Hz, C6H3). dC
2.2.1. General synthesis of ferrocenyl flavones 3aej
Ferrocenyl aurone (30 mg) and potassium cyanide (1.5 equiv)
were dissolved in ethanol (25 mL) and stirred at reflux for 2 h. The

80
J.-P. Monserrat et al. / Journal of Organometallic Chemistry 734 (2013) 78e85
(75 MHz; CDCl3) 55.3 (OMe), 67.0 (C5H4), 69.9 (C5H5), 70.9 (C5H4),
74.8 (C5H4 ipso), 106.1 (C6H3), 107.7 (C6H3), 109.7 (C6H3), 114.4 (C]
C), 133.0 (C6H3), 158.1 (C6H3), 159.5 (C6H3), 165.4 (C]C), 177.4 (C]
O). HRMS (ESI) calcd. for C20H17FeOþ3 : 361.0527, found: 361.0527.
before being purified using a silica gel column using CH2Cl2/pe-
troleum ether (3:2) as an eluent. Yield ¼ 18%; mp 163 ꢁC,
nmax/cmꢂ11461, 1596, 1662, dH (300 MHz; CDCl3) 4.16 (s, 5H, C5H5),
4.22 (s, 1H, C5H4), 4.47 (s, 2H, C5H4), 4.55 (s, 1H, C5H4), 6.62 (s, 1H),
6.90 (m, 2H), 7.27e7.37 (m, 4H), 7.46 (m, 2H), 7.94 (m, 2H). dC
(75 MHz; CDCl3) 68.5.2 (C5H5), 69.5 (C5H4), 71.8 (C5H4), 72.0 (C5H4,
C ipso) 74.0,118.5,121.6, 122.4, 127.6,127.8,128.5, 128.6, 135.5,138.2,
140.5, 158.8, 181.3. MS (APEI) m/z 420.18 (M)þ.
2.2.1.7. 5,7-dimethoxy-2-ferrocenyl-chromen-4-one, 3j. Yield: 67%;
mp 179 ꢁC, nmax/cmꢂ1 1643 (C]O), 1608 (C]C), dH (300 MHz;
CDCl3) 3.90 (s, 3H, OMe), 3.93 (s, 3H, OMe), 4.14 (s, 5H, C5H5), 4.45
(t, 2H, J ¼ 1.8 Hz, C5H4), 4.77 (t, 2H, J ¼ 1.8 Hz, C5H4), 6.30 (s, 1H,
vinyl), 6.34 (d, 1H, J ¼ 2.3 Hz, C6H2), 6.48 (d, 1H, J ¼ 2.3 Hz, C6H2). dC
(75 MHz; CDCl3) 55.4 (OMe), 56.1 (OMe),66.8 (C5H4), 69.8 (C5H5),
70.6 (C5H4), 74.6 (C5H4 ipso), 92.4 (C6H2), 95.6 (C6H2), 106.9 (C6H2),
109.0 (C]C), 159.5 (C6H2), 160.5 (C6H2), 163.4 (C6H2), 164.5 (C]C),
176.6 (C]O). HRMS (ESI) calcd. for C21H19FeOþ4 : 391.0622, found:
391.0633.
2.2.4.2. 3-ferrocenylmethylidenyl-7-methoxy-2-phenylchroman-4-
one, 6b. 7-methoxy-2-phenylchroman-4-one (50 mg, 0.20 mmol)
and ferrocene carboxaldehyde (43 mg, 0.20 mmol) were dissolved
in 10 mL EtOH in a round bottom flask and a spatula of MgSO4 was
added. HCl gas was bubbled into the flavanone solution for 1 h and
the solution was stirred at rt for 48 h. The solution was extracted
with CH2Cl2 and washed with a saturated solution of NaCl before
being purified using a silica gel column with 50/50 CH2Cl2/petro-
leum ether as an eluent. Yield ¼ 6%; dH (300 MHz; acetone d6): 4.05
(s, 3H, OMe), 4.40 (s, 5H, C5H5), 4.50 (d, J ¼ 1.6 Hz, 1H, C5H4), 4.75 (t,
J ¼ 1.6 Hz, 2H, C5H4), 4.87 (d, J ¼ 1.6 Hz,1H, C5H4), 6.72 (d, J ¼ 2.4 Hz,
1H), 6.8 (dd, J ¼ 2.2 Hz, 8.8 Hz, 1H), 6.91 (s, 1H), 7.58 (d, J ¼ 7.3 Hz,
1H), 7.65 (t, J ¼ 1.6 Hz, 2H), 7.75 (d, J ¼ 7.3 Hz, 2H), 8.01 (d, J ¼ 8.8 Hz,
1H), 8.1 (s, 1H). MS (APCI) m/z 451.04 (M þ H)þ.
2.2.2. General synthesis of ferrocenyl flavonols 3a, 3b and 3h
Carbonate buffer (15 mL) was added to a solution of ferrocenyl
flavone (40 mg) in CH2Cl2/acetone (2:1,10 mL) and stirred vigorously
at rt. A 5 mL aqueous solution of oxone (0.6 g) was added in four
portions every 10 min, and the pH of the solution was monitored to
ensure that it remained alkaline. The consumption of starting ma-
terial was followed by TLC. After the reaction was complete, the two
phases were separated and the aqueous phase was extracted with
CH2Cl2 (3 ꢀ 50 mL). The combined organic layer was washed with
sodium thiosulfate solution, dried with MgSO4, filtered, and evapo-
rated. The crude product was purified using a silica gel column with
80/20 petroleum ether/ethyl acetate as eluent. Characterization of 3a
and 3b has previously been reported [27].
2.2.4.3. 3-ferrocenylmethylidenyl-6-methoxy-2-phenylchroman-4-
one, 6c. 6-methoxy-2-phenylchroman-4-one (25 mg, 0.1 mmol)
and ferrocene carboxaldehyde (22 mg, 0.1 mmol) were dissolved in
15 mL EtOH in a round bottom flask and a spatula of MgSO4 was
added. HCl gas was bubbled into the flavanone solution for 5 min
and the solution was stirred at 50 ꢁC for 20 h. The solution was
extracted with CH2Cl2 and washed with a saturated solution of NaCl
before being purified using a silica gel column with 50/50 CH2Cl2/
petroleum ether as an eluent. Yield ¼ 4%; dH (300 MHz; acetone d6)
4.06 (s, 3H, OMe), 4.16 (s, 5H, C5H5), 4.41 (t, J ¼ 1.8 Hz, 1H, C5H4),
4.50 (t, J ¼ 1.8 Hz, 2H, C5H4), 4.67 (t, J ¼ 1.8 Hz, 1H, C5H4), 6.52 (s,
1H), 6.81 (s, 1H), 7.16 (d, J ¼ 2.8 Hz, 2H), 7.31 (t, J ¼ 1.2 Hz, 3H), 7.38
(d, J ¼ 7.3 Hz, 2H), 7.77 (s, 1H). MS (APCI) m/z 451.04 (M þ H)þ.
2.2.2.1. 7-methoxy-3-hydroxy-2-ferrocenyl-chromen-4-one,
5h.
Yield: 78%; mp 208 ꢁC, nmax/cmꢂ1 3255 (OH),1716 (C]O),1608 (C]
C), dH (400 MHz; CDCl3) 3.95 (s, 3H, OMe), 4.16 (s, 5H, C5H5), 4.50 (t,
3J ¼ 1.9 Hz, 2H, C5H4), 5.15 (t, 3J ¼ 1.9 Hz, 2H, C5H4), 6.70 (s, 1H, OH),
6.91 (d, 4J ¼ 2.3 Hz, 1H, C6H3), 6.95 (dd, 3J ¼ 8.9 Hz, 1H, C6H3), 8.08
(d, 3J ¼ 8.9 Hz, 1H, C6H3). dC (100 MHz; CDCl3) 55.9 (OMe), 68.4
(C5H4), 70.0 (C5H5), 70.7 (C5H4), 74.5 (C5H4eC ipso), 100.0 (C6H3),
114.5 (C6H3), 115.2 (C6H3), 126.7 (C6H3), 136.5 (C]C), 149.4 (C]C),
157.3 (C6H3), 163.8 (C6H3), 171.2 (C]O). HRMS (ESI) calcd. for
C20H16FeOþ4 : 376.03980, found: 376.03939.
2.2.4.4. 3-ferrocenylmethylidenyl -6-chloro-2-phenylchroman-4-one,
6d. 6-chloro-2-phenylchroman-4-one (150 mg, 0.58 mmol) and
ferrocenecarboxaldehyde (124 mg, 0.58 mmol) were dissolved in
20 mL EtOH in a round bottom flask. HCl gas was bubbled into the
flavanone solution for 1 h and the solution was stirred at 40ꢁ for
72 h. The solution was extracted with CH2Cl2 and washed with
a saturated solution of NaCl before being purified using a silica gel
column with 10/90 ethyl acetate/petroleum ether as an eluent.
Yield ¼ 15%; mp 192 ꢁC, nmax/cmꢂ1 1727, 1662, 1585, 1461; dH
(300 MHz; CDCl3) 4.13 (t, J ¼ 4.0 Hz, 5H, C5H5), 4.18 (d, J ¼ 1.5 Hz,1H,
C5H4), 4.47 (t, J ¼ 1.5 Hz, 2H, C5H4), 4.53 (dd, J ¼ 1.5 Hz, 3.8 Hz, 1H,
C5H4), 6.60 (s,1H), 6.87 (d, J ¼ 8.8 Hz,1H), 7.29e7.34 (m, 3H), 7.36 (s,
1H), 7.43e7.46 (m, 2H), 7.88 (d, J ¼ 2.6 Hz, 1H), 7.97 (s, 1H). dC
(75 MHz; CDCl3) 68.4, 69.7, 72.1, 72.3, 74.2, 78.2, 120.2, 123.3, 126.9,
127.6, 128.7, 128.7, 135.3, 137.7, 141.5, 157.2, 180.2. MS (APCI) m/z
454.94 (M þ H)þ.
2.2.3. Synthesis of organic flavones 4aej
Organic flavones 4aej were synthesized by treating chalcones
with I2 in DMSO according to a literature procedure [29]. The 1H
NMR spectra of 4ec [30], 4e [31], 4f [32], 4g, 4h [33], 4i [34]and 4j
[35] were consistent with those found in the literature. Compound
4d was not found in the literature, and its structure was deduced
from its 1H NMR spectrum: dH (300 MHz; CDCl3) 6.84 (s, 1H, vinyl),
7.23e7.30 (m, 2H, C6H5), 7.54e7.56 (m, 1H, C6H5), 7.65e7.69 (m, 1H,
C6H2), 7.93e7.96 (m, 2H, C6H5/C6H2).
2.2.4. Synthesis of ferrocenyl flavanones
Flavanone was purchased from Sigma Aldrich. Starting materials 7-
methoxy-2-phenylchroman-4-one, 5-methoxy-2-phenylchroman-4-
one and 6-chloro-2-phenylchroman-4-one were prepared via treat-
ment of the chalcone with pyridine in MeOH and water according to
a reported procedure [36].
2.3. X-ray crystallography
Intensity data were collected at 200 K with an Enraf-Nonius
Kappa-CCD diffractometer equipped with a CCD two-dimensional
detector. Data reduction was performed with DENZO/SCALEPACK.
Data were corrected for Lorentz and polarization effects, and
2.2.4.1. 3-ferrocenylmethylidenyl-2-phenylchroman-4-one,
6a.
2-phenylchroman-4-one (100 mg, 0.45 mmol) and ferrocene car-
boxaldehyde (96 mg, 0.45 mmol) were dissolved in 15 mL EtOH in
a round bottom flask. HCl gas, generated from concentrated sulfuric
acid addition to NaCl, was bubbled into the flavanone solution for
1 h and the solution was stirred at 0ꢁ for 72 h. The solution was
extracted with CH2Cl2 and washed with a saturated solution of NaCl
a
semi-empirical absorption correction based on symmetry
equivalent reflections was applied using SADABS. Lattice parame-
ters were obtained from least-squares analysis of 188 reflections.
The structures were solved by direct methods and refined by full

J.-P. Monserrat et al. / Journal of Organometallic Chemistry 734 (2013) 78e85
81
matrix least squares (5725 reflections/263 parameters), based on
F2, using the Crystals software package. All non-hydrogen atoms
were refined with anisotropic displacement parameters. All
hydrogen atoms were located with geometrical restraints in riding
mode.
(rounding up) for more than 20% of cells after a 2 h exposure time
(EC20).
3. Results and discussion
3.1. Synthesis of compounds
2.4. Biological assays
Although ferrocenyl chalcones, where ferrocene replaces the
phenyl ring in the chalcone skeleton, have been studied since the
1950s [39], only recently have ferrocene-substituted higher flavo-
noids been obtained. Access to these molecules was accomplished by
the synthesis of the ferrocenyl aurone via an oxidative 1,5-cyclization
of 2-hydroxychalcone. The latter being obtained via a condensation
of ferrocenecarboxaldehyde and 2-hydroxyacetophenone, a variety
of substitution patterns can be acquired by selecting appropriate
commercially-available 2-hydroxyacetophenones. A series of ten
ferrocene-substituted chalcones, 1aej, with halogen and methoxy
substitution were synthesized (Scheme 1) and subsequently treated
with Hg(OAc)2 in pyridine to form the corresponding series of fer-
rocenyl aurones 2aej (cf. Table 1) [28]. Isomerization of the aurones
by refluxing the latter in EtOH with KCN gave ferrocenyl flavones,
3aej, inyields ranging from 55% to 88% (cf. Table 1) [27]. Flavonols 5a,
5c and 5g were obtained from the corresponding flavones by treat-
ment with oxone and acetone in a biphasic CH2Cl2/carbonate buffer
solution as previously reported [27].
2.4.1. Cytotoxicity on cancer cells
Murine B16 melanoma cells were grown in Dulbecco’s modified
essential medium (DMEM) containing 2 mM L-glutamine, 10% fetal
bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin, in
an incubator set at 37 ꢁC with a humidified atmosphere containing
5% CO2. Exponentially growing cancer cells were plated onto 96-
well plates at 5000 cells per well in 200 mL DMEM; 24 h later, the
cells were exposed for 48 h to the solvent alone (1% DMSO final
concentration for the controls) or to the flavonoid at the indicated
concentrations. Viability was assessed using the MTT (1-(4,5-
dimethylthiazol-2-yl)-3,5-diphenyltetrazolium) test, and absorb-
ance was read at 562 nm in a microplate reader (BioKinetics Reader,
EL340, Fisher Bioblock Scientific, Illkirch, France). Experiments
were run in triplicate and repeated 3e6 times. Results are
presented as the inhibitory concentrations for 50% (IC50) of cells
for a 48 h exposure time.
2.4.2. Morphologic effects on EAhy 926 endothelial cells
Ferrocenyl flavanones 6aed were synthesized by coupling
organic flavanones e formed by base-catalyzed cyclization of the
corresponding chalcone e with ferrocene carboxaldehyde in an
ethanolic solution of HCl. The yields of these reactions were poor,
due to decomposition of the desired adduct over the long reaction
times necessary. Attempts to synthesize the condensation products
using neat piperidine, sulfuric acid or NaOH in EtOH or diethyl-
amine or piperidine [40] in DMF yielded no traces of the desired
compounds (Scheme 2).
One of the ferrocenyl flavanones, 6a, gave crystals suitable for
X-ray analysis by slow diffusion of pentane into a saturated
dichloromethane solution (Table 1). The structure of 6a (Fig. 1) is
essentially comprised of a quasi-planar arrangement of the C5H4
ring of the ferrocene and the chromanone, while the FeCp and
phenyl moieties extend below this plane. The ferrocene group is
slightly twisted out of the quasi-plane of the chromanone moiety
with a torsion angle of 15.5ꢁ for C2eC1eC11eC12. All of the atoms
of the chromanone share one plane, except for C16 and O1, where
torsion angles of between 13.7ꢁ (C14eC13eC12eC16) and 176.7ꢁ
(C20eC14eC15eO1) are observed. The phenyl ring is almost
To assess the effects of flavonoids on the morphology of endo-
thelial cells, we used the EAhy 926 endothelial cell line which is
derived from the fusion of human umbilical vein endothelial cells
(HUVEC) with the permanent human cell line A549 [37]. The EAhy
926 cell line is considered as one of the best immortalized HUVEC
cell lines because these cells express most of the biochemical
markers of parental HUVEC EAhy 926 cells [38]. EAhy 926 cells
were grown in DMEM containing 2 mM
bovine serum, 100 U/mL penicillin and 100
(37 ꢁC, 5% CO2). Exponentially growing EAhy 926 cells were plated
onto 96-well plates at 5000 cells/100 L/well. Twenty-four hours
L-glutamine, 10% fetal
mg/mL streptomycin
m
after plating, the medium was aspirated, and 100 mL of medium
containing the test compound was added to the well containing the
cells (in triplicate) in 10-fold dilutions, and incubated for 2 h. After
the 2 h incubation period, digital photographs were taken of rep-
resentative centre areas of each well at a magnification of 100ꢀ and
200ꢀ. The antivascular agent combretastatin A4 was routinely
included in the experiments as internal standard. Results are pre-
sented as the efficacious concentration for the change in cell shape
Scheme 1. Synthesis of ferrocenyl aurones, flavones and flavonols. a: R ¼ H; b: R3 ¼ Cl; c: R3 ¼ Br; d: R1 ¼ R3 ¼ F; e: R1 ¼ R3 ¼ Cl; f: R1 ¼ R3 ¼ Br; g: R2 ¼ OMe; h: R3 ¼ OMe; i:
R4 ¼ OMe; j: R2 ¼ R4 ¼ OMe.

82
J.-P. Monserrat et al. / Journal of Organometallic Chemistry 734 (2013) 78e85
Table 1
MDA-MB-231 breast cancer cells found IC50 values in the low
micromolar range (1e4 M) for methoxylated aurones 2gej.
Cell and refinement parameters for compound 6a.
m
Sum formula
Molecular weight
C26H20FeO2
420.29
3.3. Morphological effects of ferrocenyl chalcones, aurones and
flavones
Crystal system
Space group
orthorhombic
Pcab
a, Å
b, Å
c, Å
V, Å3
Z
14.706(1)
15.632(2)
17.146(3)
3941.5(8)
8
200(2)
0.71073
1.416
0.785
1744
2.95e30.00
23,720
5725/0/263
0.9286
The effect of these compounds on the morphology of endothe-
lial cells (EAhy 926), which could be predictive of antivascular ac-
tivity, was assessed. Fig. 2 shows the characteristic “rounding up” of
endothelial cells after a brief 2-h exposure time to a representative
active flavonoid compared to controls. It can be observed that
compound 3b causes a rounding up of endothelial cells for 53% of
T, K
l
, Å
Dcalc, g/cm3
mMoKa, mmꢂ1
cells at a concentration of 3
adopted a round shape.
mM, whereas 14% of control cells
F(000)
ꢁ
q
range,
Reflections collected
Remarkably, many of the compounds showed morphological
activity after a 2 h exposure time at non-cytotoxic concentrations.
The exposure time was deliberately chosen to be short to simulate
more closely the acute effects of these flavonoids upon in vivo
administration. Because most of these compounds have a short
half-life the endothelial cells are therefore exposed to high con-
centrations for a short time in vivo.
The ferrocenyl flavonoids show a wide range in morphological
activity, and the geometry of the flavonoid skeleton has an
important impact on their potency. The ferrocenyl chalcones have
little effect on the endothelial cells, with only two molecules, 1d
Refinement reflections/restraints/parameters
GooF on F2
Final R indices [I > 2
R indices (all data)
s
(I)]
R1 ¼ 0.0406a
wR2 ¼ 0.0924b
R1 ¼ 0.0848a
wR2 ¼ 0.1116b
R1
w ¼ 1/[
¼
aSjjF0j
ꢂ
jFCjj/SjFoj, and wR2
¼
b[Sw(F20
ꢂ
F2C)2/Sw(F02)2]1/2
,
s
2(F2o) þ (aP)2 þ bP] and P ¼ (Fo2 þ 2F2C)/3.
perpendicular to the plane of the molecule, with an angle of 88ꢁ
between the plane formed by the phenyl ring and that formed by
the C5H4eCechromanone.
and 1f, showing activity at concentrations below 50
rocenyl aurones are more effective, with three compounds chang-
ing the cell morphology at concentrations below 50 M, and two,
2h and 2i, showing activity at 6.25 M. The flavones show by far the
mM. The fer-
m
m
3.2. Cytotoxicity of ferrocenyl chalcones, aurones and flavones
best morphological activity of all of the ferrocenyl flavonoid classes
examined. Indeed, most of the compounds are active at concen-
trations below 25 mM. Most notably, the halogenated compounds
3b, 3c and 3f, are active at low or submicromolar concentrations,
which compare favorably to compounds in the combretastatin se-
ries, which are considered as the reference compounds in the
antivascular series [41].
The antiproliferative effects, expressed as IC50 values, of the
ferrocenyl chalcones, aurones and flavones against B16 melanoma
cells for a 48 h exposure time, are presented in Table 2. For the
compounds where R ¼ H or OMe, the geometry of the flavonoid and
the number or position of the substituents have only a modest ef-
fect on the cytotoxicity. For example, the unsubstituted compounds
1a, 2a and 3a presented similar activity, with an average IC50 value
of 44 ꢃ 5
all have IC50 values ranging within a factor of 2 (between 36 and
63 M), except for the bismethoxychalcone 1j, which is not active at
100 M.
The compounds comprising the halogen series have a more
diverse profile. The halogenated chalcones and flavones show low
m
M. The methoxylated compounds 1gej, 2gej and 3gej,
3.4. Comparison of the biological effects of ferrocenyl and organic
flavones
m
m
As shown above, some of the ferrocenyl flavones can induce
changes in the morphology of endothelial cells at low concentrations.
To investigate the influence of the ferrocenyl group on morphological
effects and cytotoxicity, the corresponding series of organic flavones
was synthesized by a 1,6-oxidative cyclization of chalcones with I2 in
DMSO [24]. We observed that the presence of a ferrocene in lieu of
a phenyl group generally enhanced the cytotoxicity on cancer cells, as
presented in Table 3. This enhanced cytotoxicity against cancer cells
has already been observed by us and by others in various ferrocene
functionalized biomolecules [12,13,18b,42].
activity, with IC50 values ranging between 30
However, the aurones 2bef all show cell growth inhibition at
concentrations below 20 M. The IC50 values are similar, regardless
of the type of halogen substitution, ranging between 12 M and
18 M.
Indeed, it is the presence of the active halogenated aurones
mM and 91 mM.
m
m
m
which drive down the mean IC50 value for the aurone class. The
ferrocenyl chalcones and flavones, on the other hand, have a similar
Concerning the morphological effects on endothelial cells, the
comparison of ferrocenyl flavones to their organic counterparts did
not allow us to draw clear structureeactivity relationships. For the
compound pairs 3a/4a, 3d/4d, 3g/4g and 3j/4j, the presence of
mean and range of activity (55.5 ꢃ 44.5 and 50.6 ꢃ 40.6
mM,
respectively). We should mention that B16 cells seem to be par-
ticularly resistant to these compounds. Previous studies [28] on
Scheme 2. Synthesis of ferrocenyl flavanones. 6a ¼ unsubstituted; 6b: R2 ¼ OMe; 6c: R3 ¼ OMe; 6d: R3 ¼ Cl.

J.-P. Monserrat et al. / Journal of Organometallic Chemistry 734 (2013) 78e85
83
Fig. 1. Ortep diagram of 6a.Thermal ellipsoids shown at 50% probability, hydrogen atoms omitted for clarity. Selected bond distances (Å): C13eO2 ¼ 1.224(2); Fe1eCCp ¼ 2.027(2)e
2.049(2) (range), 2.036(2) (avg); C1eC11 ¼ 1.439(3); C11eC12 ¼ 1.339(3); and angles (ꢁ): CpcentroideFeeCpcentroid ¼ 178.10ꢁ; C1eC11eC12 ¼ 131.05(18)ꢁ; C13eC12eC16e
O1 ¼ ꢂ41.13ꢁ.
ferrocene inhibited the morphological activity of compound com-
pared to its phenyl analogue. For example, the simple unsubstituted
flavone 4a shows surprisingly good morphological activity on
endothelial cells, whereas its ferrocenyl counterpart 3a was active at
a 32-fold higher concentration. On the other hand, for the pairs of
compounds 3c/4c, 3e/4e, 3f/4f, 3h/4h and 3i/4i the ferrocenyl de-
rivative clearly showed a stronger morphological effect on the
endothelial cells compared to its unsubstituted counterpart. There-
fore, in summary, while the addition of ferrocene to the ferrocenyl
flavones tends to enhance the compound’s cytotoxicity, there does
not seem to be a clear effect on their morphological activity.
interest. It is therefore rather surprising that none of the three
flavonols tested in this study had any effect on cell viability or cell
morphology below 100 mM (Table 4). This is in contradiction with
the behavior of natural flavonoids. From a biosynthesis point of
view, natural 3-substituted flavonoids, such as flavonols and
isoflavones, possessing respectively a hydroxyl and an aromatic
ring in this position, are more “evolved” than the others, since
their biosynthetic enzymes appeared last [43]. Currently, the
majority of natural flavonoids possess substitutions in the 3-
position. Moreover, flavonols and flavanonols possess a 1,2-
ketol motif, which allows chelation of certain metals, such as
iron or zinc [44], known to have an important role in many
biological functions.
3.5. Ferrocenyl flavonoids with 3-substitution
Likewise, no antitumoral or antivascular effects were observed
for the flavanones possessing a ferrocenylmethylidenyl group in the
3-position. This result was also unexpected, as several
3-benzylidenyl flavanones have recently exhibited IC50 values in
Of the many studies undertaken on the biological properties of
flavonoids, those compounds with substitution in the 3-position
of the ketone, whether OH or a glycoside, have raised the most
Table 2
Cytotoxicity on B16 murine melanoma cells (IC50 in
compounds.
mM) and lowest concentration causing morphological changes on EAhy 926 endothelial cells (EC20 in mM) of ferrocenyl
IC50
(
m
M)a
EC20
(
m
M)b
IC50
(
m
M)a
EC20
25
>100
>100
>100
>100
50
(m
M)b
IC50
(
m
M)a
EC20 (m
M)b
Unsubstituted
R3 ¼ Cl
1a
1b
1c
1d
1e
1f
1g
1h
1i
50 ꢃ 1
30 ꢃ 2
45 ꢃ 3
49 ꢃ 1
74 ꢃ 3
84 ꢃ 5
43 ꢃ 2
39 ꢃ 4
42 ꢃ 2
>100
>100
>100
>100
12.5
50
25
50
50
50
2a
2b
2c
2d
2e
2f
2g
2h
2i
45 ꢃ 4
16 ꢃ 1
16 ꢃ 1
18 ꢃ 1
12 ꢃ 1
14 ꢃ 1
48 ꢃ 1
46 ꢃ 2
53 ꢃ 2
46 ꢃ 1
31 ꢃ 22
3a
3b
3c
3d
3e
3f
3g
3h
3i
39 ꢃ 1
41 ꢃ 1
41 ꢃ 1
54 ꢃ 2
55 ꢃ 4
91 ꢃ 8
36 ꢃ 1
38 ꢃ 1
63 ꢃ 2
49 ꢃ 2
51 ꢃ 40
25
1.56
0.8
100
6.25
0.8
50
12.5
6.25
R3 ¼ Br
R1 ¼ R3 ¼ F
R1 ¼ R3 ¼ Cl
R1 ¼ R3 ¼ Br
R2 ¼ OMe
100
R3 ¼ OMe
6.25
6.25
50
R4 ¼ OMe
R2 ¼ R4 ¼ OMe
Mean IC50
1j
50
2j
3j
>100
56 ꢃ 44
a
IC50 is the cytotoxicity for 50% of cells and was assessed using the MTT viability test for a 48 h exposure time.
EC20 is the lowest concentration (mM) showing more than 20% rounded cells (negative controls with 1% DMSO z15% rounded cells).
b

84
J.-P. Monserrat et al. / Journal of Organometallic Chemistry 734 (2013) 78e85
Fig. 2. Representative photographs depicting the morphological effects of compound 3b on endothelial cells (EAhy 926). Cells in exponential growth were exposed for 2 h to either
1% DMSO (control) or compound 3b at 3
mM. Original magnification was 360ꢀ.
Table 3
cancer cells, whereas for the morphological effects on endothelial
cells, the presence of the ferrocenyl substituent could not be linked
to a definite structure activity relationship. Surprisingly, the pres-
ence of substitution in the 3-position of ferrocenyl flavonoids
yielded biologically inactive compounds.
Although the mechanism of action of these novel compounds is
not presently known, they possibly act in a similar fashion as other
flavonoids. For their cytotoxic action, flavonoids can act through
a myriad of signaling pathways. For example, flavonoids and chal-
cones can regulate expression of VEGF, matrix metalloproteinases
(MMPs), EGFR and inhibit NFkappaB, PI3-K/Akt, ERK1/2 signaling
pathways [46]. Regarding the flavonoids morphological activity on
endothelial cells, it appears that interaction with the tubulin is
needed and results in a disorganization of the cytoskeleton which
ultimately cause the endothelial cell to adopt a round shape. This
cell morphology ultimately may translate into an antitumoral
antivascular action in vivo [47].
Overall, there was no correlation observed between cytotoxic
effects and potential antiangiogenic effects. For example, the most
cytotoxic compounds, the halogenated ferrocenyl aurones, show
only mild morphological effects, while the most active compounds,
the chloro and bromo ferrocenyl flavones, are not very cytotoxic.
Thus we expect that these ferrocenyl flavones will act as pure
antivascular agents in vivo, that is, destroying tumor vasculature
without directly interfering with cancer cell growth or division.
Ideally, however, the most promising drug leads will possess both
cytotoxic and antivascular properties, which do not seem to be
correlated in this class of molecules. Thus future work will focus on
optimizing the ferrocenyl flavones possessing both increased
cytotoxic and morphologic activities compared to their organic
analogues, such as 3c, 3e, 3f, 3h and 3i.
Cytotoxicity on B16 murine melanoma cells and lowest concentration causing
morphological changes on EAhy 926 endothelial cells of organic flavones 4aej
compared to ferrocenyl flavones 3aej.
IC50
M)a
EC20
M)b
IC50
M)a
EC20
(m
M)b
(
m
(
m
(m
Unsubstituted 3a (Fc) 39 ꢃ 1 25
4a (Ph) 49 ꢃ 3 0.78
R1 ¼ R3 ¼ Br
3f (Fc) 91 ꢃ 8 0.8
4f (Ph) >100 6.3
3g (Fc) 36 ꢃ 1 50
4g (Ph) >100 1.5
3h (Fc) 38 ꢃ 1 12.5
4h (Ph) >100 25
R3 ¼ Cl
3b (Fc) 41 ꢃ 1 1.56 R2 ¼ OMe
4b (Ph) >100 n.d.
R3 ¼ Br
3c (Fc) 41 ꢃ 1 0.8
R3 ¼ OMe
4c (Ph) >100 3.1
R1 ¼ R3 ¼ F
3d (Fc) 54 ꢃ 2 100 R4 ¼ OMe
4d (Ph) >100 50
3i (Fc) 63 ꢃ 2 6.25
4i (Ph) 89 ꢃ 9 12.5
R1 ¼ R3 ¼ Cl 3e (Fc) 55 ꢃ 4 6.25 R2 ¼ R4 ¼ OMe 3j (Fc) 49 ꢃ 2 >100
4e (Ph) >100 25
4j (Ph) 98 ꢃ 11 12.5
a
IC50 is the cytotoxicity for 50% of cells and was assessed using the MTT viability
test for a 48 h exposure time.
b
EC20 is the lowest concentration (mM) showing more than 20% rounded cells
(negative controls with 1% DMSO z15% rounded cells).
Table 4
Cytotoxicity on B16 murine melanoma cells (IC50 in mM) and lowest concentration
causing morphological changes on EAhy 926 endothelial cells (EC20 in
rocene flavonols 5a, 5b and 5h, and of ferrocenyl flavanones (6aec).
m
M) of fer-
IC50
M)a
EC20
M)b
IC50
M)a
EC20
(m
(
m
(
m
(m
M)b
5a
5c
5h
Unsub.
>100
>100
>100
>100
>100
>100
6a
6b
6c
6d
Unsub.
36 ꢃ 2
56 ꢃ 8
52 ꢃ 7
44 ꢃ 1
50
R3 ¼ Br
R2 ¼ OMe
R3 ¼ OMe
R3 ¼ Cl
>100
100
25
R2 ¼ OMe
a
Cytotoxicity was assessed using the MTT viability test for a 48 h exposure time.
Lowest rounding up concentration (mM) showing more than 20% rounded cells
b
Acknowledgements
(negative controls with 1% DMSO ¼ 15% rounded cells).
We thank L. Etienne and P. Rosa (Institute de Chimie de la
Matière Condensée de Bordeaux) for ICP-AES measurements, X.
Tournaire for synthetic assistance, M.N. Rager for NMR assistance,
C. Fosse for MS measurements and P. Herson (Université Pierre et
Marie Curie, Paris) for the X-ray structure. This study was supported
by the CNRS, INSERM, the French Ministry of Research, and a grant
from the French National Institute of Cancer (INCa, Boulogne Bill-
ancourt, France). KNT thanks the “Association pour la recherche
the low to submicromolar range against cancer cells [45]. In this
case the ferrocene does not seem to act as a good mimic of a phenyl
ring. Moreover, the absence of activity of the 3-substituted ferro-
cenyl flavonoids (whether OH or ]CeFc) suggest an entirely dif-
ferent mode of action than that of the organic compounds.
4. Conclusions
contre le cancer” (ARC, Villejuif, France) for
a postdoctoral
fellowship.
The cytotoxic and morphological effects of some three dozen
ferrocenyl flavonoids have been reported. Some broad structuree
activity relationships can be discerned. Overall, the halogenated
aurones showed the best cytotoxic activity, although their mor-
phological activity was modest. For the flavones, the presence of
a ferrocenyl group led to a clear increase in cytotoxic activity on
Appendix A. Supplementary data
CCDC 906156 contains the supplementary crystallographic data for
this paper. These data can be obtained free of charge from The

J.-P. Monserrat et al. / Journal of Organometallic Chemistry 734 (2013) 78e85
85
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G. Jaouen, N. Neamati, E.A. Hillard, Bioorg. Med. Chem. Lett. 20 (2011)
6195e6197.
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
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Appendix B. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.jorganchem.2012.12.031.
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