In vitro inhibitory properties of ferrocene-substituted chalcones and aurones on bacterial and human cell cultures

Article abstract of DOI:10.1039/c2dt12180h

Two series of ten chalcones and ten aurones, where ferrocene replaces the C ring and with diverse substituents on the A ring were synthesized. The compounds were tested against two antibiotic-sensitive bacterial strains, E. coli ATCC 25922 and S. aureus ATCC 25923, and two antibiotic-resistant strains, S. aureus SA-1199B and S. epidermidis IPF896. The unsubstituted compound and those with methoxy substitution showed an inhibitory effect on all bacterial strains at minimum inhibitory concentrations ranging between 2 and 32 mg L ^-1. For four of these compounds, the effect was bactericidal, as opposed to bacteriostatic. The corresponding organic aurones did not show growth inhibition, underscoring the role of the ferrocene group. The methoxy-substituted aurones and the unsubstituted aurone also showed low micromolar (IC₅₀) activity against MRC-5 non-tumoral lung cells and MDA-MB-231 breast cancer cells, suggesting non-specific toxicity.

Full text of DOI:10.1039/c2dt12180h

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PAPER
In vitro inhibitory properties of ferrocene-substituted chalcones and aurones
on bacterial and human cell cultures
a
a
a
a
Keshri Nath Tiwari†, Jean-Philippe Monserrat†, Arnaud Hequet, Carine Ganem-Elbaz,
b
a
a
a
a
Thierry Cresteil, Gérard Jaouen, Anne Vessières, Elizabeth A. Hillard* and Claude Jolivalt
Received 14th November 2011, Accepted 9th December 2011
DOI: 10.1039/c2dt12180h
Two series of ten chalcones and ten aurones, where ferrocene replaces the C ring and with diverse
substituents on the A ring were synthesized. The compounds were tested against two antibiotic-sensitive
bacterial strains, E. coli ATCC 25922 and S. aureus ATCC 25923, and two antibiotic-resistant strains,
S. aureus SA-1199B and S. epidermidis IPF896. The unsubstituted compound and those with methoxy
substitution showed an inhibitory effect on all bacterial strains at minimum inhibitory concentrations
−1
ranging between 2 and 32 mg L . For four of these compounds, the effect was bactericidal, as opposed
to bacteriostatic. The corresponding organic aurones did not show growth inhibition, underscoring the
role of the ferrocene group. The methoxy-substituted aurones and the unsubstituted aurone also showed
low micromolar (IC ) activity against MRC-5 non-tumoral lung cells and MDA-MB-231 breast cancer
50
cells, suggesting non-specific toxicity.
properties, some of which have been recently reviewed in the lit-
Introduction
4
erature. Aurones play a primarily protective role in plants, pos-
5
6
7
Bacterial infection and disease is a ubiquitous health hazard and
despite having clinically efficacious antibiotics the issue of bac-
terial resistance poses a strong necessity for the design and
development of novel antibacterial agents. In 2004, methicillin-
resistant Staphylococcus aureus (MRSA) was one of the most
common causes of nosocomial infections accounting for more
than 60% of S. aureus infections in intensive care units in the
sessing insect antifeedent, antiparasitic and antifungal activity.
In humans, there is interest in developing aurones for anti-
8,9
10,11
cancer and antileishmanial
applications.
Aurones have demonstrated in vitro antibacterial activity and
some structural aspects have been identified by structure–activity
relationship (SAR) studies. Recently, QSAR analysis of a class
of
(Z)-2-(5-nitrofuran-2-yl-methylene)-3(2H)-benzofuranones
1
2
USA, and from 40% to 70% in India. Of particular concern,
these nosocomial MRSA infections commonly have reduced
susceptibility to classes of antibiotics other than the β-lactams.
Furthermore, the rising incidence of community-associated
MRSA is alarming since infections have occurred in otherwise
(
Chart 1, 1a) identified the benzofuranic core as a key structural
12
feature contributing to antibacterial properties, and a recent
publication concluded that Z stereochemistry around the double
bond is preferred. The modification of ring A has also been
demonstrated to influence the antibacterial activity of aurones.
For example, compounds in series 1a possessing electron-donat-
ing groups showed better antibacterial activity than those with
13
3
healthy individuals with no known health risks. There is thus a
critical need for new therapeutics to combat these evolving
pathogens. In the search for biologically active compounds, one
potential strategy is to look for unnatural molecules. These are
less likely to induce bacterial resistance than biosynthetized mol-
ecules like some penicillins or streptogramins, providing their
medical use remains under control, as shown by the worldwide
spread of fluoroquinolone resistance.
12
electron-withdrawing groups.
In another study, minimum
Aurones, 2-benzylidenebenzofuran-3(2H)-ones, are naturally
occurring yellow-colored flavonoids, structurally isomeric to the
more well-studied flavones. Besides their role in pigmentation,
aurones have also been found to possess variety of biological
†
These authors contributed equally to the work.
a
ENSCP Chimie ParisTech, Laboratoire Charles Friedel (LCF), 75005
Paris, FranceCNRS, UMR 7223, 75005 Paris, France.
E-mail: elizabeth-hillard@chimie-paristech.fr
Chart 1 Aurones from the literature showing antibacterial activity
(1a–1d); ferrocenyl aurones studied in this report (3).
b
CNRS, Inst. Chim. Subst. Nat., F-91198 Gif Sur Yvette, France
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 6451–6457 | 6451

inhibitory concentration (MIC) values ranged between 0.8 to
80 °C for 3–5 h cleanly yielded 3a–j in good yield (60–80%)
after purification on silica gel. The configuration of the aurones
was found to be Z as confirmed by 2D NMR studies along with
100 mg L− against Gram positive bacteria in a series of substi-
1
>
1
4
tuted (Z)-2-arylidene-3(2H)-benzofuranones
possessing 1-
26
methyl-5-nitroimidazole (Chart 1, 1b). On the other hand, the
effect of replacement of the phenyl group C by another function
cannot be simply generalized. For instance, for 2,2-bisamino-
methylated aurone analogues (Chart 1, 1c) the activity varied
the X-ray crystallographic characterization of 3b. The organic
analogs of 3a and 3g–j were similarly obtained in 70–80% yield
13
after purification on silica gel. In all cases, a single Z geometric
isomer was obtained as confirmed by proton and carbon NMR
7
28
only by a factor of 2 within the series. However, in the case of
spectra and IR spectra.
series 1b, the changing of ring C from a 5-nitroimidizole to a 4-
nitroimidazole completely suppressed the antibacterial properties
14
Antibacterial activities
of the former. Increased lipophilicity also may improve anti-
15
bacterial activity; prenyloxy-substituted aurones (Chart 1, 1d)
Ferrocenyl chalcones 2a–j, ferrocenyl aurones 3a–j and the
organic aurones 5a, 5g–j were tested against four bacterial
strains (Table 1). Two strains are antibiotic-sensitive: Escherichia
coli ATCC 25922, a Gram negative strain, and Staphylococcus
aureus ATCC 25923, a Gram positive strain. Two laboratory
showed enhanced activity probably due to better interaction with
cellular membranes.
Our group is interested in how the modification of natural pro-
ducts or drugs with organometallic units can influence their bio-
logical properties. Ferrocene, in particular, has gained
considerable interest as a substituent in medicinal chemistry due
to its stability in aqueous and aerobic media, its lipophilicity, the
ease of substitution and its redox activity. The literature on ferro-
cene in medicinal chemistry is vast and diverse; highlights
1
6
17
include work on cancer and malaria. Some of the first appli-
cations of ferrocene in medicinal chemistry were in the context
of antibacterial studies, when Edwards et al. demonstrated an
improvement in the therapeutic spectrum of penicillin and cepha-
1
8
Scheme 1 Synthesis of aurones from precursor chalcones; a: unsubsti-
tuted; b: 5-Cl; c: 5-Br; d: 5,7-diF; e: 5,7-diCl; f: 5,7-diBr; g: 6-OMe; h:
losporin by ferrocene substitution. Similarly, ferrocene deriva-
tized penem antibiotics showed increased activity vs. the
5
-OMe; i: 4-OMe; j: 4,6-diOMe.
19
standard faropenem. Ferrocene itself, as well as (dimethylami-
nomethyl)ferrocene have shown moderate inhibitory activities
2
0
against E. coli ATCC 25922. In other work, ferrocene-substi-
Table 1 MIC values for 2a–j, 3a–j and 5a, 5g–j against E. coli, S.
aureus and S. epidermidis strains
2
1
22
23
tuted tamoxifen derivatives, pyrazoles antibiotics and acyl-
24
hydrazones have been studied for their antibacterial effects.
Taking into consideration the antibacterial properties of
aurones and the often favorable effects of ferrocene substitution,
we reasoned that aurones, where the phenyl group C is replaced
by ferrocene, may show promising antibacterial properties, par-
ticularly against antibiotic-resistant strains. As part of our
ongoing project studying ferrocene-modified biomolecules, we
have recently obtained access to such ferrocenyl aurones in high
Minimum Inhibitory Concentration (MIC, mg L−
)
1 a
E. coli
ATCC 25922
S. aureus
ATCC 25923
S. aureus
SA-1199B
S. epidermidis
IPF 896
2
2
a
b
>32
>32
>32
>32
>32
>32
>32
>32
>100
>32
>100
>100
8–16
>32
>32
>32
>32
>32
2–4
8–32
2–8
2–8
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>100
>32
>100
>100
8–16
>32
>32
>32
>32
>32
2–4
8–32
4–8
4–8
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32c
n.d.
>32c
n.d.
2c >32
>32
2e >32
>32
2
d
25,26
yields.
We here report the synthesis of a series of ten ferro-
2
f
b
b
b
cene aurones and their chalcone precursors and their antibacterial
effects on two antibiotic-sensitive bacterial cell strains, E. coli
ATCC 25922 and S. aureus ATCC 25923, and two resistant
strains, S. aureus SA-1199B and S.epidermidis IPF 896 as com-
pared to selected organic aurones. To judge their utility for
further development in animals, we also tested selected com-
pounds’ cytotoxicity against MRC-5 non-tumoral lung cells and
MDA-MB-231 breast cancer cells.
2g >100
2h >32
2
2
3
b
b
b
b
i
j
a
>100
>100
32
b
b
c
n.d.
8–16
>32
>32
>32
>32
>32
4
3b >32
3
3
3
3
3
3
3
3
5
5
5
5
5
c
d
e
f
g
h
i
>32
>32
>32
>32
8
32
8
16–32
4–8
4–8c
n.d.
Results and discussion
j
8
a
g
h
i
>32
>32
>32
>32
>32
Synthesis
c
n.d.c
n.d.c
Ferrocene chalcones 2a–j were obtained by a standard Claisen-
Schmidt condensation of ten different hydroxyacetophenones
and ferrocenecarboxaldehyde with NaOH in refluxing EtOH, as
n.d.
n.d.
c
j
a
27
When only one value is given, results for the two experiments were
b c
previously reported. All the chalcones were found to have an E
−1
identical. Tests carried out using 10 mg mL stock solution. Not
determined.
1
configuration based on their H NMR coupling constants (J ≈
1
5 Hz). Treatment of 2a–j with mercury(II) acetate in pyridine at
6452 | Dalton Trans., 2012, 41, 6451–6457
This journal is © The Royal Society of Chemistry 2012

a
Table 2 Time kill assay: Reduction in pathogen concentration following a 24 h antibiotic exposure at the MIC
log10 CFU/mL (t = 0) − log10 CFU/mL (t = 24 h)
3
a
3g
3h
3i
3j
E. coli ATCC 25922
S. aureus ATCC 25923
S. aureus SA-1199B
S. epidermidis IPF896
>3.5
>3.8; 1.2 ± 0.1
>3.9
>3.5
>3.7
>3.8
3.0 ± 0.0
>3.5
>3.5
>3.8
>3.9
3.5 ± 0.2
>3.5
1.5 ± 0.3;>3.8
1.2 ± 0; 3.4 ± 0.3
0.2 ± 0.3; >3.5
2.9 ± 0.3
3.2 ± 0.2
>3.6
>3.6
a
Molecules were tested at their MIC (from Table 1) in a time-kill endpoint assay. Bactericidal activity is defined as a 1000-fold or greater reduction in
bacterial concentration, expressed as colony forming unit (CFU)/mL. When [log10 CFU/mL (t = 0) − log10 CFU/mL (t = 24 h)] ≥ 3, the compound is
considered bactericidal. Experimental error is mentioned only for counting above the detection limit. When only one value is given, results for the
two experiments were identical, except for 3h where only one experiment was performed.
strains with known resistance mechanisms were also used. The
fluoroquinolone resistant strain SA-1199B of S. aureus harbors
the norA gene that encodes a membrane-associated protein that
mediates the active efflux of fluoroquinolones. S. epidermidis
IPF 896 is resistant to erythromycin, by overexpressing Msr(A),
an ATP-binding cassette (ABC) protein involved in macrolide
efflux, which is one of the two most prevalent mechanisms of
resistance to this widely used antibiotic in Streptoccoci. NorA,
the most studied pump among the major facilitator superfamily
efflux system, is involved in the so-called multidrug resistant
that a role of the redox properties of ferrocene in the inhibitory
effects of the ferrocenyl aurones cannot be excluded.
In order to further assess the role of the ferrocenyl group in
the antimicrobial activity, the organic aurones representative of
the most active ferrocenyl derivatives were tested (Scheme 1).
Their MIC was found to be higher than 32 mg L− against three
bacterial strains (Table 1), demonstrating that the organometallic
group is required for the antibacterial activity.
2
9
1
The antibacterial activities of the most active aurones 3a, 3g–
j, were characterized using 24 h-time-kill assays (Table 2). Time-
kill studies allow the quantification of bacterial death over a
specified amount of time. This type of methodology has an
advantage over traditional MIC testing in that it allows for
typing the antibiotic activity as bactericidal or bacteriostatic.
Bactericidal activity (99.9% kill) is defined as a ≥3-log10 colony
forming unit (CFU)/mL reduction in colony count from the
(MDR) bacterial phenotype and exports an extensive range of
structurally unrelated compounds while Msr(A) is specific to 14
and 15 membered macrolides and steptogramins B. Both efflux
3
0
pumps are widely found in resistant Staphyloccoci.
None of the ferrocenyl chalcones showed activity at the
maximum concentration of 32 mg L−1 or even at 100 mg L−1
for 2g and 2i–j. This result suggests that the benzofuranic core is
one of the key structural features contributing to antibacterial
properties since, unlike the chalcones, some of the tested
aurones efficiently inhibited bacterial growth. For the ferrocenyl
aurones, the halogenated-substituted derivatives were inactive.
However, the unsubstituted compound 3a as well as all of the
methoxy-substituted compounds 3g–j showed significant
31
initial inoculum. That is, after 24 h, if there is more than a
1000-fold reduction in bacterial CFU, the compound is con-
sidered bactericidal.
Bacterial counting after 24 h incubation showed that 3a and
3h–j were bactericidal against all strains at their MIC, having
values of log10 CFU/mL greater than 3. In a first test, compound
3g demonstrated bacteriostatic activity against Gram positive
bacteria, while the compound was found to be bactericidal
against Staphylococci in a second test. A similar situation for 3a
was observed for antibiotic-sensitive S. aureus. Such variability
in the obtained results was clearly due to the fact that the time-
kill assays were performed at the MIC, that is, at the lowest con-
centration where the compounds show inhibition of bacterial
growth. The bactericidal activity evidenced for the aurone com-
pounds is noteworthy from a clinical perspective. When treating
severe infections, a rapid clearance of a pathogen may be necess-
ary to achieve a better prognosis, and therefore a bactericidal
agent is preferred.
−1
activity, ranging from 2–32 mg L against both Staphyloccoci
and E. coli, Gram positive and Gram negative bacteria, respect-
ively, demonstrating activity on a wide bacterial spectrum. The
compounds were generally slightly more active on Gram positive
bacteria and showed a similar activity against antibiotic-sensitive
−1
and antibiotic-resistant strains. A 10 mg L MIC is generally
recognized as a maximal value in the pharmaceutical industry
for a valuable hit to be further developed as a potential drug.
The methoxy substituted ferrocenyl aurones thus show an inter-
esting potential in this regard.
It should be mentioned that these results are consistent with
previous observations that electron-donating groups on ring A
1
2,14
tend to enhance antibacterial effects.
Here, however, a pos-
Cytotoxic effects on MRC-5 and MDA-MB-231 cells
ition effect is noted; the unsubstituted ferrocenyl compound 3a
and that with methoxy substitution in position 5 (3h) exhibited
In order to evaluate the selectivity of the most active aurones
against bacterial cell strains, compounds 3a, 3g–3j were evalu-
ated against MRC-5 human fetal lung cells and estrogen receptor
negative MDA-MB-231 breast carcinoma (Table 3). Cell growth
−1
lower activities (MIC = 8–32 mg L ) than those methoxy-sub-
stituted aurones in position 4 and/or 6 (3g, 3i, 3j, MIC = 2–8 mg
−1
L ). We recorded the redox potential for the ferrocenyl group
on each of the molecules by cyclic voltammetry in CH Cl . The
inhibition was determined by the MTT test at 10− M and 10
5
−6
2
2
most active compounds, 3g, 3i and 3j had the lowest E (0.118,
M in triplicate, followed by the evaluation of the IC50 values at
concentrations ranging from 100 to 0.005 μM on a 96-well plate.
Stock solutions were freshly prepared 30 min before cell testing.
1
/2
+
0
.125 and 0.103 ± 0.003 V vs. Fc /Fc respectively) compared to
3
a and 3h (0.144 and 0.147 ± 0.003 V respectively), suggesting
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 6451–6457 | 6453

Table 3 Cell growth inhibition for non-tumoral and cancer cells for 3a
and 3g–j
(Z)-5-chloro-2-(ferrocenylidene)benzofuran-3-one, 3b
65%); mp 159 °C; Found: 61.65;
(
C
H
3.67%.
MRC-5 IC50 (μM)
MDA-MB-231 IC50 (μM)
C H O FeCl·0.25 H O requires: C 61.66, H 3.69%; UV/VIS
1
9
13
2
2
−1
−1
(
CH Cl ): λ
= 394 nm (ε = 16620 L.mol .cm ), 565 nm
max
2
2
3
3
3
3
3
a
g
h
i
3.5 ± 0.0
3.5 ± 0.1
1.0 ± 0.3
1.0 ± 0.3
3.7 ± 0.1
2.8 ± 0.5
4.1 ± 0.2
1.8 ± 0.0
1.1 ± 0.2
3.8 ± 0.1
−1
−1
(5490 L.mol .cm ); IR (KBr): 1700 (CvO), 1639 (CvC)
cm ; H NMR (300 MHz; CDCl ) 4.24 (s, 5H, C H ), 4.63 (s,
−
1 1
3
5 5
2
H, C H ), 4.91 (s, 2H, C H ), 6.92 (s, 1H, Hvinyl), 7.24 (m, 1H,
5 4 5 4
j
4
3
Harom), 7.57 (dd, J = 2.1 Hz, J = 8.7 Hz, 1H, Harom), 7.77 (d, J
=
13
2.1 Hz, 1H, Harom); C NMR (75 MHz; CDCl ) 70.0 (C H ),
3 5 5
7
1.6 (C H ), 72.2 (C H ), 74.7 (C H ipso), 114.2 (Carom),
5 4 5 4 5 4
All compounds showed almost complete inhibition of cell
growth at 10− M against both cell lines, while no significant
5
117.9 (Cvinyl), 123.8 (Carom), 124.0 (Carom), 128.8 (Carom), 135.8
−6
(Carom), 146.1 (C
), 163.5 (Carom), 181.4 (CvO); MS (APCI)
vinyl
cell growth inhibition was observed at 10 M (data not shown).
IC50 values differed only slightly between compounds, with IC50
values ranging from 1 to 4 μM. The difference between cell lines
was even smaller (±1 μM), suggesting non-specific toxicity. It
should be mentioned however that the SAR observed in the anti-
bacterial tests was not observed here. Nonetheless, due to the
relatively high value of the ratio MIC (μM)/IC50 (μM) of these
compounds their future development as therapeutic agents
appears to be therefore rather in jeopardy.
+
m/z 364.91 [M+H] .
(Z)-5,7-difluoro-2-(ferrocenylidene)benzofuran-3-one, 3d
(66%); mp 171 °C; Found: 61.04;
C
(CH
(5630 L.mol .cm ); IR (KBr): 1701 (CvO), 1654 (CvC)
cm ; H NMR (400 MHz; CDCl ) 4.22 (s, 5H, C H ), 4.65 (s,
C
H
3.22%.
·0.5 H O requires: C 60.83; H 3.49%; UV/VIS
2 2
H
O
FeF
): λmax = 395 nm (ε = 16940 L.mol .cm ), 575 nm
2
19
12
2
−1
−1
Cl
2
−1
−1
−1 1
3
5 5
2
H, C H ), 4.91 (s, 2H, C H ), 7.02 (s, 1H, Hvinyl), 7.22 –7.15
5 4 5 4
Experimental
13
(m, 1H, Harom), 7.31–7.29 (m, 1H, Harom); C NMR (100 MHz;
CDCl ) 70.2 (C H ), 71.9 (C H ), 72.6 (C H ), 74.3 (C H
3
5
2
5
5
4
4
5
4
5
4
General remarks
2
ipso), 105.5 (dd, J = 23.8 Hz, J = 4.1 Hz, C
), 110.9 (dd, J
arom
2
3
All reactions were carried out under argon. THF was distilled
over sodium/benzophenone and all other chemical reagents and
solvents were used as received without further purification.
Silica gel chromatography was performed with Merck 60
= 19.7 Hz, J = 24.3 Hz, Carom), 119.8 (C
), 125.6 (d, J =
vinyl
1
3
7.7 Hz, Carom), 145.9 (C
), 148.1 (dd, J = 254.7 Hz, J =
vinyl
2
1
11.2 Hz, C
), 148.8 (d, J = 11.3 Hz, C
), 157.9 (dd, J =
arom
arom
3
246.5 Hz, J = 7.3 Hz, Carom), 180.7 (CvO); MS (APCI) m/z
1
13
+
(40–63 μm) silica. H and C NMR spectra were recorded with
367.07 [M+H] .
a 300 or 400 MHz Bruker Avance spectrometer, and δ are given
1
in ppm and referenced to the residual solvent peaks ( H, δ 7.26
(Z)-5,7-dichloro-2-(ferrocenylidene)benzofuran-3-one, 3e
1
3
1
1
13
1
and C{ H}, δ 77.1 for CDCl and H, δ 2.05 and C{ H}, δ
3
2
9.7 for acetone-d ). Mass spectra were measured on a Thermo-
6
(64%); mp 181 °C; Found: 56.05; 3.31%.
C
H
scientific ITQ1100 spectrometer using the direct exposure probe
method by the mass spectrometry service at the École Nationale
Supérieure de Chimie de Paris. High resolution mass spectra
were determined by ESI/ESCI-TOF using a Waters LCT Premier
XE or a Thermo Fischer LTQ-Orbitrap XL. Melting points were
determined using an Electrothermal 9100 apparatus. IR data
were collected on a JASCO FT/IR-4100 using a KBr pellet.
C H O FeCl ·0.5 H O requires: C 55.93; H 3.21%; UV/VIS
19
12
2
2
2
−1
−1
(
(
CH Cl ): λ
= 397 nm (ε = 15700 L.mol .cm ), 581 nm
max
2
2
−1
−1
12380 L.mol .cm ); IR (KBr): 1697 (CvO), 1635 (CvC)
−1 1
cm ; H NMR (300 MHz; CDCl ) 4.22 (s, 5H, C H ), 4.66 (s,
3
5 5
2
2
H, C H ), 4.93 (s, 2H, C H ), 7.06 (s, 1H, H
.1 Hz, 1H, Harom), 7.69 (d, J = 2.1 Hz, 1H, Harom); C NMR
), 7.64 (d, J =
vinyl
5
4
5
4
13
(
(
(
(
75 MHz; CDCl ) 69.2 (C H ), 71.0 (C H ), 71.7 (C H ), 73.3
3 5 5 5 4 5 4
C H ipso), 118.4 (Carom), 118.9 (Cvinyl), 121.4 (Carom), 124.1
5
4
Carom), 127.9 (Carom), 134.0 (Carom), 144.8 (C
Carom), 179.3 (CvO); MS (APCI) m/z 398.96 [M+H] .
), 158.2
vinyl
+
Synthesis of ferrocenyl chalcones
Ferrocenyl chalcones 2a–2j were synthesized by combining
equimolar quantities of the appropriate 2-hydroxyacetophenone
with ferrocenecarboxaldehyde in EtOH with 3 equivalents of
NaOH. The synthetic procedure and their structural characteriz-
(
Z)-5,7-dibromo-2-(ferrocenylidene)benzofuran-3-one, 3f
(
60%); mp 122 °C; Found: 46.47;
C
H
2.60%.
2
7
ation has been reported.
C H O FeBr ·0.25 H O requires: C 46.34; H 2.56%; UV/VIS
(
(
1
9
12
2
2
2
−1
−1
CH Cl ): λ
= 394 nm (ε = 14400 L.mol .cm ), 570 nm
max
2
2
−
1
−1
4400 L.mol .cm ); IR (KBr): 1697 (CvO), 1639 (CvC)
Synthesis of ferrocenyl aurones
−
1 1
cm ; H NMR (300 MHz; CDCl ) 4.22 (s, 5H, C H ), 4.67 (s,
3
5 5
Ferrocenyl aurones 3a–3j were synthesized by refluxing the
2H, C H ), 4.93 (s, 2H, C H ), 7.03 (s, 1H, H
1.5 Hz, 1H, Harom), 7.92 (d, J = 1.5 Hz, 1H, Harom); C NMR
), 7.86 (d, J =
vinyl
5
4
5
4
13
appropriate chalcone in pyridine with 2 eq. Hg(OAc) according
2
2
6
to the reported procedure. The aurone synthesis was performed
on a 200 mg scale (in chalcone). The characterization of ferroce-
nyl aurones 3a, 3c and 3h has been previously reported.
Elemental analyses for 3a–3f reprinted from ref. 25.
(75 MHz; CDCl ) 69.2 (C H ) 71.0 (C H ), 71.7 (C H ), 73.3
3
5
5
5
4
5 4
(C H ipso), 115.0 (Carom), 119.0 (Cvinyl), 124.5 (Carom), 125.0
5
4
26
(Carom), 129.8 (Carom), 139.2 (Carom), 144.4 (Cvinyl), 159.9
(Carom), 179.3 (CvO); MS (EI) m/z 485.99 [M] .
+
6454 | Dalton Trans., 2012, 41, 6451–6457
This journal is © The Royal Society of Chemistry 2012

(Z)-6-methoxy-2-(ferrocenylidene)benzofuran-3-one, 3g
(Z)-6-methoxy-benzofuran-3-one, 5g
1 1
(73%); mp 140 °C; IR (KBr) 1697, 1650, 1604, 1496 cm− ; H
(
75%); mp 142 °C; HRMS (ESI) Found: 360.04434.
+
C H FeO requires: 360.04489; UV/VIS (CH Cl ): λ
3
cm ); IR (KBr): 1685 (CvO), 1639 (CvC) cm ; H NMR
(
=
.
NMR (300 MHz; CDCl ) 3.92 (s, 3H, OMe), 6.75 (m, 2H,
2
0
16
3
2
2
max
−1
3
−1
−1
65 nm (ε = 16560 L.mol .cm ), 534 nm (3810 L.mol
Harom), 6.82 (s, 1H, Hvinyl), 7.35–7.47 (m, 3H, Harom), 7.70 (dd,
J = 8.2 Hz, 0.9 Hz, 1H, Harom), 7.88 (dd, J = 8.2 Hz, 0.9 Hz,
−
1
−1 1
13
300 MHz; CDCl ) 3.93 (s, 3H, OMe), 4.19 (s, 5H, C H ), 4.53
2H, Harom). C NMR (75 MHz; CDCl ) 56.0, 96.6, 111.9,
3
5
5
3
(
6
s, 2H, C H ), 4.85 (s, 2H, C H ), 6.74 (s, 1H, H
.72–6.79 (m, 2H, Harom), 7.71 (d, J = 8.1 Hz, 1H, Harom);
),
C
112.2, 114.8 (C ), 125.8, 128.8, 129.6, 131.3, 132.4 (C ), 147.8
5
4
5
4
vinyl
q
q
1
3
(C ), 167.4 (C ), 168.6 (C ), 183.0 (CvO); MS (APCI) m/z
q q q
+
NMR (100 MHz; CDCl ) 55.7 (OMe), 69.5 (C H ), 70.9
253.08 [M+H] .
3
5 5
(
(
(
C H ), 71.2 (C H ), 74.8 (C H ipso), 96.3 (Carom), 111.8
5 4 5 4 5 4
Carom), 114.4 (Cvinyl), 115.4 (Carom), 125.2 (Carom), 146.5
Cvinyl), 166.6 (C ), 167.5 (Carom), 181.1 (CvO); MS (APCI)
(
Z)-5-methoxy-benzofuran-3-one, 5h
arom
+
m/z 361.15 [M+H] .
76%); mp 122 °C; IR (KBr) 1704, 1646, 1596, 1488 cm−1; H
1
(
NMR (300 MHz; CDCl ) 3.83 (s, 1H, OMe), 6.87 (s, 1H,
3
(
Z)-4-methoxy-2-(ferrocenylidene)benzofuran-3-one, 3i
Hvinyl), 7.20–7.24 (m, 3H, Harom), 7.36–7.48 (m, 3H, Harom),
13
7
.90 (dd, J = 8.2 Hz, 1.5 Hz, 2H, Harom); C NMR (75 MHz;
(
66%); mp 174 °C; HRMS (ESI) Found: 383.03411.
CDCl ) 55.9, 105.2, 113.1, 113.8, 121.7 (C ), 126.2, 128.9,
3
q
+
C H FeO Na requires: 383.03411; UV/VIS (CH Cl ): λ
3
cm ); IR (KBr): 1700 (CvO), 1643 (CvC) cm ; H NMR
=
.
2
0
16
3
2
2
max
−1
129.9, 131.5, 132.3 (C ), 147.7 (C ), 156.1 (C ), 161.3 (C ),
q q q q
−1
−1
90 nm (ε = 17350 L.mol .cm ), 536 nm (4550 L.mol
+
1
85.0 (CvO); MS (APCI) m/z 253.08 [M+H] .
−
1
−1 1
(
300 MHz; Acetone d ) 4.00 (s, 3H, OMe), 4.24 (s, 5H, C H ),
6 5 5
4
6
8
.59 (t, J = 1.8 Hz, 2H, C H ), 4.95 (t, J = 1.8 Hz, 2H, C H ),
(Z)-4-methoxy-benzofuran-3-one, 5i
5
4
5 4
.68 (s, 1H, Hvinyl) 6.78 (d, J = 8.4 Hz, 1H, Harom) 6.98 (d, J =
(71%); mp 144 °C. IR (KBr) 1704, 1654, 1596, 1488 cm−1;1H
1
3
.4 Hz, 1H, Harom) 7.66 (t, J = 8.4 Hz, 1H, Harom); C NMR
NMR (300 MHz; CDCl ) 4.00 (s, 3H, OMe), 6.61 (d, J = 8.2
3
(
(
(
(
[
100 MHz; CDCl ) 55.8 (OMe), 69.5 (C H ), 70.9 (C H ), 71.2
C H ), 74.8 (C H ipso), 104.2 (Carom), 104.4 (Carom), 110.8
Carom), 114.4 (Cvinyl), 137.3 (Carom), 145.4 (Cvinyl), 157.9
Carom), 165.9 (Carom), 180.3 (CvO); MS (APCI) m/z 361.15
3 5 5 5 4
Hz, 1H, Harom), 6.83 (s, 1H, Hvinyl), 6.86 (dd, J = 8.2 Hz, 0.6
Hz, 1H, Harom), 7.34–7.46 (m, 3H, Harom), 7.56 (t, J = 8.2 Hz,
5
4
5 4
13
1
H, Harom), 7.89 (dd, J = 8.2 Hz, 1.5 Hz, 2H, Harom); C NMR
+
(75 MHz; CDCl ) 56.2, 104.7, 105.1, 110.8 (C ), 111.9, 128.8,
3 q
M+H] .
1
29.6, 131.3, 132.4 (C ), 138.4, 146.9 (C ), 158.5 (C ), 167.0
q
q
q
+
(
C ), 182.4 (CvO); MS (APCI) m/z 253.08 [M+H] .
q
(Z)-4,6-dimethoxy-2-(ferrocenylidene)benzofuran-3-one, 3j
(
68%); mp 166 °C; HRMS (ESI) Found: 413.04467.
Bacterial strains and media
+
C H FeO Na requires: 413.04467; UV/VIS (CH Cl ): λ
3
cm ); IR (KBr): 1693 (CvO), 1643 (CvC) cm ; H NMR
(
(
1
(
(
(
(
=
2
0
16
3
2
2
max
−1
−1
−1
Two sensitive strains (Escherichia coli ATCC 25922 and Staphy-
lococcus aureus ATCC 25923) and two resistant strains (Staphy-
lococcus aureus SA-1199B and Staphylococcus epidermidis IPF
68 nm (ε = 17300 L.mol .cm ), 522 nm (3600 L.mol .
−
1
−1 1
300 MHz; CDCl ) 3.90 (s, 3H, OMe), 3.93 (s, 3H, OMe), 4.15
3
8
96) were used. The strain SA-1199B is resistant to fluoroquino-
s, 5H, C H ), 4.47 (s, 2H, C H ), 4.78 (s, 2H, C H ), 6.11 (s,
5
5
5
4
5 4
1
3
lones due notably to the overexpression of the NorA efflux
H, Harom), 6.35 (s, 1H, Harom), 6.72 (s, 1H, Hvinyl); C NMR
pump, a membrane-associated protein that mediates the active
75 MHz; CDCl ) 56.0 (OMe), 56.1 (OMe), 69.7 (C H ), 70.9
3 5 5
2
8,30
efflux of fluoroquinolones.
S. epidermidis IPF 896 is resist-
C H ), 71.1 (C H ), 75.3 (C H ipso), 89.0 (Carom), 93.7
5
4
5
4
5 4
ant to 14- and 15-membered macrolides, including erythromycin,
and steptogramins B by overexpressing Msr(A), an ATP-binding
Carom), 105.9 (Carom), 113.2 (Cvinyl), 117.1 (Carom), 146.8
Cvinyl), 159.2 (C ), 168.3 (Carom), 179.3 (CvO); MS (APCI)
arom
+
31
cassette (ABC) protein. All strains were grown at 37 °C in
Mueller Hinton Broth (MH, Bio Rad) or spread on MH agar
plates for counting. In case of S. epidermidis IPF 896, 10 μg
m/z 391.19 [M+H] .
Synthesis of organic chalcones
−1
mL of chloramphenicol was added in the overnight culture
used to prepare the antibacterial susceptibility test inoculum.
Organic chalcones 4a and 4g–j were synthesized by the same
method as for the ferrocenyl chalcones 2a–j.
Test compound preparation
Synthesis of organic aurones
Ferrocenyl chalcones (2a–j) and aurones (3a–j) are colored
(violet for those with the most electron-donating substituents to
blue for those with the most electron-withdrawing substituents),
thus interfering with the optical density measured at 620 nm to
assess the bacterial growth. In addition, their solubility assess-
ment in DMSO was difficult at high concentration (10 mg
Organic aurones 5a and 5g–j were synthesized by refluxing the
appropriate chalcone (100 mg) in pyridine with 2 eq. Hg(OAc)
according to a literature procedure. For the aurones 5a and 5j,
2
1
3
the structural characterization was found in good agreement with
32,33
the reported values.
The aurones were found to be the Z-
−1
isomer based on the chemical shift of the vinylic proton (<7 ppm).
mL ). As a compromise, compounds were prepared at
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 6451–6457 | 6455

1
1
0
mg mL− in DMSO (corresponding to a concentration range of
performed in duplicate, compounds were added in the range 5
nM–100 μM in a fixed volume of DMSO.
.03 to 32 mg L− in the MIC test) except for 2g and 2i–j which
were tested from stock solutions at 10 mg mL . The organic
molecules (5a, 5g–j) were tested in the same conditions as their
corresponding ferrocenyl derivatives.
1
−1
Electrochemistry
Cyclic voltammograms (CVs) were obtained using a three elec-
trode cell with a 0.5 mm Pt working electrode, stainless steel rod
counter electrode, and Ag/AgCl ethanol reference electrode, with
an μ-Autolab 3 potentiostat driven by GPES software (General
Purpose Electrochemical System, Version 4.8, EcoChemie B.V.,
Determination of antibacterial susceptibility
Flavonoid derivatives were dispensed in a 96-wells microplate
by two fold serial dilutions in Muller-Hinton medium using a
Biomek 2000 (Beckman) handling robot. 100 μL of an overnight
bacterial culture diluted to achieve a bacterial concentration of 5
Utrecht, the Netherlands). Solutions consisted of 10 mL CH
Cl
NPF supporting
6
,
2
2
approximately 1 mM analyte, and 0.1 M Bu
electrolyte.
4
5
6
×
10 –1 × 10 CFU/mL (as checked by colony counting) in each
well was then added. The final volume in each well was 200 μL.
Growth was assayed with a microplate reader by monitoring
absorption at 620 nm at 0, 2, 4, 6 and 24 h incubation at 37 °C.
In addition, the plates were read visually after 24 h incubation.
The MIC was defined as the minimal concentration of the
flavonoid compound that completely inhibited cell growth
during 24 h incubation at 37 °C. 3.2% DMSO was used as a
negative control. MIC determination of reference antibiotics -
Conclusions
Two series of ten chalcones and ten aurones possessing a ferro-
cene group in lieu of the phenyl ring were synthesized and eval-
uated for their antibacterial activity against E. coli, antibiotic-
sensitive and antibiotic-resistant strains of S. aureus and a resist-
ant strain of S. epidermidis. The unsubstituted aurone 3a and the
methoxy-substituted aurones 3g–j showed minimum inhibitory
−1
ampicillin for E. coli ATCC 25922 (MIC 4 mg L ), ciprofloxa-
−
1
−1
cin for S. aureus ATCC 25923 and 1199B (MIC 0.25 mg L
concentrations between 2 and 32 mg L , with higher activity
against Gram positive bacteria and similar activities against anti-
biotic-sensitive and antibiotic-resistant strains, while their
organic analogs showed no effect at concentrations of 32 mg
and 8 mg L− , respectively), and erythromycin for S. epidermidis
IPF89 (MIC 64-128 mg L ) was used as a positive control, in
the same run as the flavonoids MIC measurement.
1
−1
−1
The accepted variance on MIC value can be estimated to a 2-
fold difference. All experiments were performed in duplicate.
L . The most active compounds, 3g, 3i and 3j have methoxy
substitution in the 4 and/or 6 position and all showed a bacteri-
cidal effect from time-kill assays. All of the active compounds
were also active in vitro against human fetal lung cells and
human breast carcinoma, with IC50 values in the low micromolar
range.
Determining time-kill endpoint at 24 h
CFU monitoring was carried out by enumerating colonies in
1
0 μl of a serial log dilutions of the tested bacterial culture
spotted in duplicate on MH agar plates. Plates were examined
for growth after 24 h of incubation at 37 °C. This methodology
has a lower limit of detection of 2 log10 CFU/mL. Bacterial
counting was performed in duplicate, except for 3h.
Acknowledgements
We thank Marie-Noelle Rager for NMR and Céline Fosse for
MS expertise. We are grateful to Olivier Chesneau (Institut
Pasteur, Paris) for kindly providing us with the S. epidermidis
IPF896 strain. KNT thanks the Association pour la recherche sur
le cancer for a postdoctoral fellowship.
Cytotoxicity assay
The human cell line MRC5-SV2 (fetal lung), purchased from
ECACC, was grown in complete D-MEM medium, sup-
plemented with 10% fetal calf serum, in the presence of penicil-
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Dalton Trans., 2012, 41, 6451–6457 | 6457