Luminescent zinc salophen derivatives: Cytotoxicity assessment and action mechanism studies

Article abstract of DOI:10.1039/c3nj41125g

The biological activity of two fluorescent Zn(ii)-salophen derivatives has been evaluated. In vitro studies (AFM, emission and UV-vis titration with ethidium bromide and cell growth inhibition) show different mechanisms of interaction with DNA. It has been observed that these compounds enter the cells. Comet assays (with cultured fibroblast cells) have revealed that cellular uptake occurs without damaging the DNA strands. Preliminary studies carried out with living cells have shown IC₅₀ values in a millimolar range, indicative of a non-cytotoxic behaviour. This fact could be understood by confocal microscopy co-localization studies with living cell internalization that have shown that, in fact, the compounds seem to enter the cells but not the nucleus under in vivo conditions.

Full text of DOI:10.1039/c3nj41125g

NJC
View Article Online
View Journal | View Issue
PAPER
Luminescent zinc salophen derivatives: cytotoxicity
assessment and action mechanism studies†
Cite this: NewJ.Chem., 2013,
37, 1046
Rosa Brissos,a David Ramos,b Joa˜o Carlos Lima,c Francesco Yafteh Mihan,d
b
b
d
`
Miquel Borras, Joaquin de Lapuente,* Antonella Dalla Cort* and
Laura Rodr´ıguez*a
The biological activity of two fluorescent Zn(II)–salophen derivatives has been evaluated. In vitro studies
(AFM, emission and UV-vis titration with ethidium bromide and cell growth inhibition) show different
mechanisms of interaction with DNA. It has been observed that these compounds enter the cells.
Comet assays (with cultured fibroblast cells) have revealed that cellular uptake occurs without
damaging the DNA strands. Preliminary studies carried out with living cells have shown IC50 values in a
millimolar range, indicative of a non-cytotoxic behaviour. This fact could be understood by confocal
microscopy co-localization studies with living cell internalization that have shown that, in fact, the
compounds seem to enter the cells but not the nucleus under in vivo conditions.
Received (in Montpellier, France)
1st August 2012,
Accepted 16th January 2013
DOI: 10.1039/c3nj41125g
www.rsc.org/njc
a
1. Introduction
´
`
´
`
Departament de Quımica Inorganica, Universitat de Barcelona, Martı i Franques
1-11, 08028 Barcelona, Spain. E-mail: laura.rodriguez@qi.ub.es;
Fax: +34 934907725; Tel: +34 934039130
Real-time tracking of (bio-)molecules is a highly valuable tool to
investigate cellular processes that implicate molecular motions.1,2
Hence, molecular imaging can provide fundamental information
regarding dynamic biological processes in living systems.3–5 In
this context, the breakthroughs achieved during the past five years
in fluorescence microscopy,6 thanks to the improvement of image
resolution,7,8 have boosted the emerging field of cell imaging, and
generated remarkable advances in cell and molecular biology.9,10
Accordingly, fluorescence probes have become some of the most
powerful elements for molecular-imaging studies as a result of
their high sensitivity and selectivity.11–15
Luminescent lanthanide-based compounds are the most
prominent class of inorganic complexes used in bio-imaging.16
However, transition-metal luminescent complexes, like zinc
derivatives, are increasingly reported in the literature.17
Metal salen (salen: N,N0-bis(salicylidene)ethylenediamine)
complexes are a long-standing, well-known family of coordina-
tion compounds that have found numerous applications in
chemistry, depending on the nature of the metallic centre.
Salen/salophen ligands (which act as planar tetradentate
ligands with a cis-configuration around the metal ion) are
capable of stabilizing various oxidation states of metal ions,
hence controlling their performance in a large variety of useful
catalytic transformations.18–26 The redox properties of salen-
containing coordination compounds have been used in biologi-
cal studies in which their involvement in metal-dependent DNA-
damage processes has been revealed.27–35 For example, iron- and
b
c
´
Unitat de Toxicologia Experimental i Ecotoxicologia. Parc Cientıfic de Barcelona,
c/Baldiri i Reixach, 10-12, 08028 Barcelona, Spain. E-mail: jlapuente@pcb.ub.cat;
Fax: +34 934037109; Tel: +34 934039710
´
ˆ
REQUIMTE. Departamento de Quımica, Faculdade de Ciencias e Tecnologia,
Universidade Nova de Lisboa, 2829-516 Monte de Caparica, Portugal
d Dipartimento di Chimica and IMC-CNR Sezione Meccanismi di Reazione,
`
Universita La Sapienza, Box 34 Roma 62, 00185 Roma, Italy.
E-mail: Antonella.Dallacort@uniroma1.it; Fax: +39 06490421;
Tel: +39 0649913087
† Electronic supplementary information (ESI) available: AFM images of pBR322
plasmid DNA incubated with 1 for 24 h (Fig. S1); AFM images of pBR322 plasmid
DNA incubated with 2 for 24 h (Fig. S2); electronic absorption spectra of complex
1 in the absence and presence of increasing amounts of ct-DNA. Inset: plot of
[DNA] vs. [DNA]/(ea ꢀ ef) (Fig. S3); emission spectra of ethidium bromide (EB)
bound to DNA in the absence and presence of increasing amounts of 2. Inset:
Stern–Volmer plot: I0/I vs. [2] (Fig. S4); normalized absorption spectra of 1
recorded at different pH values (Fig. S5); normalized absorption spectra of 2
recorded at different pH values (Fig. S6); microscopy images of 3T3 cells
incubated for 24 h with concentrations of compound 2 above 70 mM. White light
is used for co-localized cells. Green area represents fluorescent compound 2
(Fig. S7); 20ꢁ microscopy image of cells in DAPI-staining solution, using a UV2A
fluorescence filter. Image of the negative effect on nucleotide in the presence of
compound 2 (left) compared to positive control with MMS (right) (Fig. S8);
binding-time studies of 1 with 3T3 cells. Pictures taken with a fluorescence
microscope equipped with a UV2A filter (Fig. S9); binding-time studies of 2 with
3T3 cells. The pictures were taken with a fluorescence microscope equipped with
a
UV2A filter (Fig. S10); 20ꢁ fluorescence microscopy image of nucleoids
incubated with 1 (concentration of 50 mg mLꢀ1) in the absence (left) and in the
presence of 10% DAPI (right) (Fig. S11); fluorescence confocal microscopy image
of 3T3 incubated cells with Draq5 (left), salophen complex 2 (middle) and
superimposed images (right) (Fig. S12). See DOI: 10.1039/c3nj41125g
c
1046 New J. Chem., 2013, 37, 1046--1055
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013

View Article Online
Paper
NJC
copper–salen derivatives may generate reactive oxygen species
(ROS) in reducing environments, leading to oxidative DNA
damage. For this reason they have been applied for drug–DNA
foot-printing.36 Recently, it has been shown that iron(III)–salophen
(salophen = N,N0-bis(salicylidene)1,2-phenylenediamine) compounds
can act as potent growth-supressing agents for ovarian-cancer cell
lines in vitro, as well as a potential therapeutic drug for tumor
treatment in vivo.35,37 In addition, iron(III) complexes of salophen
Schiff bases can undergo electron-transfer reactions mimicking
the catalytic functions of peroxidases.38 The formation of
DNA adducts using Ni(II) complexes of redox-active ligands by
covalent modification of nucleobases is also of great interest.39
Some lanthanide–salophen complexes are able to bind neutral
sugars and lipids, including lysophosphatidic acid that is a
biomarker for several pathological conditions (like ovarian
cancer).40 A number of manganese–salophen and manganese–
salen derivatives display cytoprotective features in fibroblast
cultures via dihydrogen peroxide scavenging, and therefore are
synthetic mimics of superoxide dismutase.41,42 Very recent
examples of new applications of salen derivatives haven been
found such as liquid crystals, with a Mn(III) salen complex,43 in
the detection of Cu2+ in water and living cells with sulfonato–
salen derivatives44 and second order NLO properties.45 More-
over, the possibility of salen/salophen derivatives to behave as
excellent binders towards non-canonical DNA structures such as
quadruplexes makes their use in this field very promising.46–50
Among such derivatives, the salen–salophen complexes of
the non-redox metal ion zinc(II) are also quite important.50–62
Their relevance is due to the fact that zinc is the second most
abundant transition metal in living organisms after iron, and is
the only metal which appears in all enzyme classes.33 The
molecular structure of these complexes features the metal
centre in a five-coordinate square-pyramidal geometry with
the ligand occupying the basal plane and a solvent molecule
in the apical position. The closed-shell, d10 configuration of
Zn(II) represents a key feature that can be exploited to design
and produce ligand-dependent fluorescence materials50,52,53
and luminescence chemosensors.54–61 The lack of redox activity
of Zn(II) normally generates Zn–salen compounds with low
cytotoxicity.53 So this, in addition to their potential intrinsic
luminescence properties, makes them ideal candidates for the
generation of biological fluorescence probes.50,52,53
Chart 1
(Mannheim-Germany). HEPES (N-2-hydroxyethylpiperazine-N0-2-
ethanesulfonic acid) was obtained from ICN (Madrid). 1,2-Diamino-
benzene and 2,3-diaminonaphthalene were obtained from Aldrich.
3-Isopropylsalicylaldehyde was prepared from 2-isopropyl phenol
(Aldrich) according to a general procedure.64
2.2. Physical measurements
The Atomic-Force Microscope (AFM) images were obtained
with a nanoscope III multimode AFM system from Digital
Instruments Inc. operating in tapping mode.
Fluorescence microscopy was carried out on an Olympus
E800 equipped with a UV2A filter. The fluorescence emission
spectra were carried out on a Horiba-Jobin-Yvon SPEX Nanolog-
TM at 25 1C.
2.3. Synthesis and characterization
In the study reported herein, the potential biological applica-
Synthesis of salophen-ligand L1. A solution of 3-isopropyl-
tions of two Zn(II)–salophen complexes, namely compounds 1 and salicylaldehyde (0.355 g, 2.16 mmol) and 1,2-diaminobenzene
2, are explored and their cytotoxicity is clearly assessed. Similar (0.117 g, 1.08 mmol) in MeOH (2 mL) was stirred for 24 h at
studies were performed also on the corresponding free ligands. 60 1C. After some time an orange slurry precipitates from the
To the best of our knowledge, this is the first investigation cold solution. It was washed twice with cold MeOH and the
of this type regarding Zn(II)–salophen derivatives, while relevant final product was obtained as orange oil in 57% yield. 1H NMR
work has been done instead with Zn–salen compounds (200 MHz, CDCl3) d: 13.38 (s, 2H, OH), 8.64 (s, 2H, NH),
(Chart 1).63
7.51–7.11 (m), 6.89 (t, J = 7.7 Hz, 2H, CH), 3.41 (hept, J =
6.9 Hz, 2H, CH), 1.26 (d, J = 6.9 Hz, 2H, CH3) ppm. 13C NMR
(75 MHz, CDCl3) d: 164.33, 163.93, 159.16, 142.74, 136.86,
130.12, 127.64, 120.05, 118.77, 118.71, 26.79, 22.41, 22.11 ppm.
2. Experimental section
2.1. General
Compounds 1 and 2 have been synthesized as previously reported.55 401.2250. Elemental analysis: calc. (%) for C26H28N2O2: C,
+
HRMS-ESI-TOF for C26H29N2O2 calc.: 401.2229; found:
pBR322 plasmid DNA was obtained from Boehringer-Mannheim 77.97; H, 7.05; N, 6.99%; found: C, 77.98; H, 7.06; N, 6.99%.
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem., 2013, 37, 1046--1055 1047

View Article Online
NJC
Paper
Synthesis of salophen-ligand L2. A solution of 3-isopropyls- 24 h under 5% CO2 at 37 1C. The viability was assessed by the MTT
alicylaldehyde (0.208 g, 1.23 mmol) and 2,3-diaminonaphthalene assay based on the mitochondrial dehydrogenase enzyme capability
(0.100 g, 0.632 mmol) in MeOH (3 mL) was stirred for 24 h. The to hydrolyze chromogen bromide (3-(4,5-dimethylthiazol-2-yl)-
mixture was then filtered and the final product was obtained as a 2,5-diphenyltetrazolium bromide). The cells were washed
pale yellow solid in 69% yield. 1H NMR (300 MHz, CDCl3) d 13.38 with PBS (3ꢁ) and fresh medium was added together with a
(s, 2H, OH), 8.74 (s, 2H, NH), 7.86 (dd, J = 6.2, 3.3 Hz, 2H, CH), 5 mg mLꢀ1 MTT solution and incubated for 2 h at 37 1C, under
7.49 (dd, J = 6.2, 3.2 Hz, 2H, CH), 7.40–7.19 (m, 6H, CH), 6.92 a 5% CO2 atmosphere This compound shows a pale-yellow
(t, J = 7.6 Hz, 2H, CH), 3.43 (hept, J = 7.0 Hz, 2H, CH), 1.27 (d, J = color in solution; by the action of enzymes, MTT is reduced to
6.9 Hz, 12H, CH3) ppm. 13C NMR (75 MHz, CDCl3) d: 164.42, formazan in the mitochondria of living cells, a crystallized,
159.28, 143.02, 136.91, 132.86, 130.24 130.19, 127.82, 126.39, dark blue compound that cannot cross the cell membrane.
118.85, 118.77, 116.89, 26.83, 22.42 ppm. HRMS-ESI-TOF
A second cytotoxicity assay was carried out using DMSO as
for [C30H30N2O2K]+ calc.: 489.1944; found: 489.1954. Elemental a solvent with the same concentrations of compounds that
analysis: calc. (%) for C30H30N2O2: C, 79.97; H, 6.71; N, 6.22%; previously assayed. The maximum dose of DMSO and compounds
found: C, 79.98; H, 6.72; N, 6.22%.
used was 1%.
The data were statistically analyzed to calculate the 50%
inhibition concentrations, IC50, using the SPSS program, version
2.4. Atomic-force microscopy experiments
pBR322 DNA was heated before use at 60 1C for 10 min to 15 for Windows applying Probit Regression.
obtain the open circular (OC) form. Stock solution is 1 mg mLꢀ1
in a buffer solution of HEPES. Each sample contains 1 mL of
2.8. Genotoxicity-comet assay
DNA pBR322 of concentration 0.25 mg mLꢀ1 for a final volume of The genotoxicity study on the 3T3 Balb/c fibroblast cell line was
50 mL. All the Stock solutions were freshly prepared before use performed via the Comet Assay that was carried out according to
in milli-Q water with 2% DMSO.
the guideline ASTM-E2186.65
Cells were cultured in 6-well flat-bottom microtiter plates
containing 2 mL of cell suspension (2 ꢁ 105 cells approxi-
2.5. UV-vis spectrophotometric studies
The absorption titrations were performed by adding increasing mately). After 24 h of incubation at 37 1C with 5% CO2, the
amounts of ct-DNA DNA (calf thymus DNA) to the complexes in compound to be investigated was added. Due the data obtained
Tris–HCl buffer (5 mM) and NaCl (50 mM) at pH = 7.2. The in cytotoxicity assay, only 2 doses were needed. For compound 1
concentration of ct-DNA was determined from the absorption we exposed 16.86, 8.43 mM and for compound 2, 583, 71 and
intensity at 260 nm with a molar extinction coefficient of 291.86 mM with a solvent control with 1% DMSO. After 24 hours
6600 Mꢀ1 cmꢀ1. After addition of DNA to the metal complex, of exposition, the 3T3 fibroblast cells were detached from the
the resulting solution was allowed to equilibrate at 25 1C for well with 0.05% trypsin solution and collected by centrifuga-
2 min, after which absorption spectra were recorded.
tion. Cells were embedded in low-melting point 0.9% agarose
(LMP-Gibco) prepared in MilliQ water (18 MO), and layered on
pre-coated slides. The slides were placed in lysing buffer (2.5 M
2.6. Fluorescence spectrophotometric titrations
The relative binding affinities of the complexes to ct-DNA were NaCl, 100 mM Na2EDTA, 10 mM Tris pH 10, N-lauryl-sarcosine
studied with an EB-bound (EB stands for ethidium bromide) 1% (w/v) (Sigma)) with 1% Triton X-100 for 1 h at 4 1C. The
ct-DNA solution in 5 mM Tris–HCl/50 mM NaCl buffer (pH = 7.2). isolated DNA of the nuclei in the agarose gels was unwinded for
The fluorescence spectra were recorded at room temperature with 40 min in electrophoresis buffer (1 mM Na2EDTA and 300 mM
excitation at 514 nm. The titration experiments were carried out NaOH, pH 4 13). The SCGE slides were then electrophoresed
with an EB–DNA solution containing 2.5 ꢁ 10ꢀ5 M EB, 1 ꢁ 10ꢀ5 M for 30 min at 25 V and 300 mA, at 4 1C. After neutralization with
ct-DNA and 75 ꢁ 10ꢀ3 mg mLꢀ1 of the compounds.
400 mM Tris buffer (pH 7.5), the slides were dried at room
temperature. For image analysis, the slides were hydrated and
stained with 10 mL of DAPI at 14.28 mM. Non-damaged cells
2.7. Cytotoxicity studies
For the cytotoxicity assays, 3T3 Balb/c cells were cultured in exhibit a spherical shape, while fragmented nucleoids migrate,
Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) forming a tail (‘‘comet’’ shape). The nucleoid damage was
containing 10% fetal calf serum (Hyclone), 2 mM glutamine quantified as fluorescence percentage in the tail. In the assay,
(Sigma-Aldrich) and antibiotics (Sigma-Aldrich, 50 mg mLꢀ1 the percentage of DNA in the tail was determined in respect to
penicillin and 50 mg mLꢀ1 streptomycin). Thus cells were the intensity of the total DNA, in 50 cells. The determination
incubated for 24 h for correct adhesion and then, decreasing was obtained with the software Comet Assay IV and the corres-
solutions of the salophen compounds at 10% were applied in ponding statistical using the SPSS program version 15 for
some wells together with a solvent control (sterile water at 10%) Windows applying Mann–Whitney U statistics.
and a positive control (0.2% sodium dodecylsulfate, i.e. SDS,
2.9. Preliminary cellular uptake studies
which is a known cytotoxic agent), and they were incubated for
24 h. The cells were exposed to a range of concentrations from 3T3 Balb/c cells were exposed to two different concentrations of
2155.73 to 8.43 mM for compound 1 and 1945.75 to 4.55 mM compounds: 1 (4.31 and 8.62 mM) and 2 (3.89, 7.78 mM) both in
for compound 2, both with a dilution of 2-fold each time for DMSO and they were observed on an inverted fluorescence
c
1048 New J. Chem., 2013, 37, 1046--1055
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013

View Article Online
Paper
NJC
microscope with two different filters (B2A and UV2A) co-stained Moreover, the luminescent properties displayed by 1 and 2
with acridine orange and 40,6-diamidino-2-phenylindole (DAPI). encouraged us to investigate their potential use as markers in
Observations were carried out at different times: 15, 30 minutes, cellular uptake processes. Obviously to do this the cytotoxic
1 h, 1.5 h, 2 h and 24 h after exposition.
effects should be evaluated and compared with those of the
corresponding metal-free ligands L1 and L2.
2.10. DNA binding studies in cells
DNA-binding processes and mechanisms have been firstly
Preliminary experiments were carried out with free nucleoids in investigated by the interaction of 1 and 2 with isolated DNA
an agarose matrix. Fixed nucleoids were stained with concen- using Atomic Force Microscopy (AFM) and UV-vis and fluores-
trations of compounds 1 and 2 of ca. (107.78 mM, 97.29 mM) (see cence titration experiments.
comet assay protocol). Staining with DAPI (40,6-diamidino-2-
phenylindole, 14.28 mM), a well-known fluorescent stain that
3.1. AFM studies
acts as a DNA minor-groove binder and DNA intercalator, at a
concentration of 5 mg mLꢀ1 allowed us to obtain better micro- Free pBR322 plasmid DNA was incubated for 5 h and 24 h with
scopy images.
DMSO solutions of 1 and 2 and AFM images were taken
For living internalization of compounds, 3T3 cells were subsequently (see Fig. 1, 2 and Fig. S1 and S2, ESI†). After an
seeded onto a cover slip in a 24-well plate and grown overnight incubation time of 5 h with 1 (Fig. 1), the initial OC form of
under normal growth conditions followed by incubation of 1 at DNA disappears completely, giving rise to the formation of
28.02 mM or 2 at 97.29 mM for an additional 3 h. Control cells mixed toroidal and plectonemic supercoils, as well as to
were treated with an equivalent amount of DMSO. Cells were complete plectonemic supercoiling, most likely due to the
fixed with 4% formaldehyde in PBS for 30 min, washed twice intercalation of the complexes into the DNA molecules. In the
with PBS, and permeabilized with 0.2% Triton X-100 in PBS for case of 2, the OC form of DNA is comparatively much less
5 min. The cells were washed three times with cold PBS affected (Fig. 2) but plectonemic supercoiled regions are still
followed by incubation with Draq5 (10 mM) for 30 min at room observed (although sparsely), which again suggest intercalation
temperature. Stained cells were washed three times with cold of the salophen compound. After an incubation time of 24 h,
PBS, mounted on a microscope slide with mounting media the intercalating properties of both compounds are further
Mowiol and visualized under a Leica TCS SP2 AOBS system corroborated, as a strong clustering of DNA is observed, producing
(405 nm excitation laser diode for 2 molecules and 688 nm for rod-like aggregates (see Fig. S1 and S2, ESI†).
Draq5). To detect molecules inside the cell, we performed a
lambda scan with a wide range of wavelengths. With Z plain
fixed inside the nucleus thanks to Draq5 co-localization stain,
pictures were taken at different wavelengths, from 405 to 600.
We selected different points of the cell and measured the
intensity of the light emitted from each region.
3. Results and discussion
L1 and L2 were prepared using the appropriately substituted
salicylaldehyde and 1,2-diaminobenzene as starting materials.
The reactants were stirred in methanol for 24 h and the
products precipitated upon cooling the solution. The orange
crystals were collected by vacuum filtration and washed with
ice-cold methanol. Characterization by different spectroscopic
techniques and mass spectrometry verifies the formation of the
desired compounds. As stated above, the corresponding Zn
derivatives 1 and 2 were prepared according to the literature.55
Fluorescence spectrophotometric measurements, previously
reported by some of us, showed that 1 and 2 recognize anions
of biological relevance, i.e. the nucleotides AMP2ꢀ, ADP3ꢀ, and
Fig. 1 AFM image of pBR322 plasmid DNA (left) and pBR322 plasmid DNA
(0.10 mM) incubated with 1 (0.33 mM) for 5 h (right).
ATP4ꢀ 56
The electronic absorption spectra of the complexes
.
display broad unstructured bands in the 240–500 nm
range.55,56 Excitation at both low energy bands led to a broad
emission band centred at 530 and 532 nm for 1 and 2
respectively.56
The risk of cytotoxic effects from exposure to Zn(II)-containing
derivatives is lower (zinc is indeed an essential trace element)50,52,53
when compared to other transition metals or lanthanides.
Fig. 2 AFM image of pBR322 plasmid DNA (left) and pBR322 plasmid DNA
(0.10 mM) incubated with 2 (0.15 mM) for 5 h (right).
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem., 2013, 37, 1046--1055 1049

View Article Online
NJC
As mentioned above, Zn-based compounds are not expected
Paper
to present cytotoxic properties. However with the perspective of
using our derivatives as biomarkers, we decided to analyze their
potential cytotoxicity (see Section 3.4).
3.2. DNA-binding studies by absorption and emission
titrations
Absorption spectra of the complexes with increasing amounts
of added DNA were recorded (Fig. 3 and Fig. S3, ESI†). A
decrease in molar absorptivity (hypochromism) and slight
bathochromism (2–5 nm) which is larger for 2, probably due
to the planarity of the naphthalene ring, is observed for the two
complexes. Such variations are indicative of a likely intercalation
mode of the two complexes into ct double helix DNA, most likely
similar to that obtained with ethidium bromide, EB (see
below).66 Nevertheless, the association constant between DNA
and ethidium bromide is two orders of magnitude higher than
the one calculated for our complexes showing that EB is a
stronger intercalating agent.
Fig. 4 Emission spectra of ethidium bromide (EB) bound to DNA in the absence
and presence of increasing amounts of 1. Inset: Stern–Volmer plot: I0/I vs. [1].
non-emissive in phosphate buffer (pH 7.2), due to fluorescence
quenching of free EB by solvent molecules, but emits intensely
in the presence of DNA as a result of its intercalation between
adjacent DNA base pairs. When a second species is added, this
can compete with EB for DNA binding sites causing the
decrease in the fluorescence intensity. The quenching is due
to the reduction of the number of DNA binding sites available
to the EB. Therefore EB is used as a common fluorescent probe
for DNA structure able to provide information about the mode
and the process of single species binding to DNA.70
Binding affinity of the complexes with DNA, Kb, could be
determined quantitatively by using the following equation:67
[DNA]/(ea ꢀ ef) = [DNA]/(eb ꢀ ef) + 1/Kb(eb ꢀ ef)
where [DNA] is the concentration of ct DNA in base pairs, ea, ef,
and eb are the apparent extinction coefficient corresponding
to Aobs/[complex], the extinction coefficient for the unbound
compound and the extinction coefficient for the compound in
the fully bound form, respectively.
The binding constants Kb for 1 and 2 were found to be 1.7 ꢁ
105 Mꢀ1 and 1.6 ꢁ 105 Mꢀ1 respectively, therefore suggesting a
similar strength interaction of both complexes with DNA. These
values are similar to those reported recently in the literature
for DNA metallointercalators containing N-donor ligands
(e.g. ethylenediamine, hydrazone).68,69
The observed quenching of DNA–EB in the presence of 1 and
2 is shown in Fig. 4 and Fig. S4 (ESI†) and follows the Stern–
Volmer equation:
I0
¼ 1 þ Kq½Qꢂ
I
where I0 and I represent the emission intensity in the absence
and presence of a quencher, respectively, Kq is a linear Stern–
Volmer quenching constant and [Q] is the quencher concen-
tration, i.e. the concentration of the zinc–salophen derivatives
in our study. This equation is considered for a static quenching,
since this is the expected process for the displacement of EB by
the salophen complexes. Hence, the obtained I0/I vs. [Q] plots
follow a linear pattern that let us to calculate Kq that is 9.4 ꢁ
103 Mꢀ1 and 8.1 ꢁ 103 Mꢀ1 for 1 and 2 respectively. Accordingly,
the competitive process is favorable in a similar way for both
complexes.
Further support for the binding of the complexes to DNA by
intercalation mode was provided by competitive binding experi-
ments with ethidium bromide (EB). This planar compound is
The different values obtained for Kb and Kq are due to the
different chemical processes under study. In the UV-vis spectro-
scopic titrations we can calculate association constants
between the compounds and DNA, while spectrofluorimetric
titrations let us to calculate the quenching constant from the
EB displacement process. The Kb constant could be also calcu-
lated by the luminescence quenching experiments using the
representation log[(I0 ꢀ I)/I] vs. log[Q] where apparent binding
constants 1.8 ꢁ 103 Mꢀ1 and 1.6 ꢁ 103 Mꢀ1 could be calculated
for 1 and 2 respectively. Thus, the particular process under
study gives an apparent constant value, Kapp = Kass/KBE[EB].
Fig. 3 Electronic absorption spectra of complex 1 in the absence and presence
of increasing amounts of DNA. Inset: Plot of [DNA] vs. [DNA]/(ea ꢀ ef).
Taking into consideration that KEB is 1 ꢁ 107
M
ꢀ1, the
c
1050 New J. Chem., 2013, 37, 1046--1055
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013

View Article Online
Paper
NJC
corresponding Kb values obtained from luminescence experi-
Most likely, above 70 mM concentration, the cytotoxicity does
ments are ca. 4.1 ꢁ 105 Mꢀ1 for both 1 and 2, which fit quite well not originate exclusively from the interaction between the
with those previously calculated by absorption experiments.
metal-containing compounds and the DNA, but from the excess
of 1 and 2 that covers the cells (outside the cell membrane, see
Fig. S7, ESI†) and does not allow proper growth.
3.3. pH dependence stability of the complexes
Cytotoxicity has been evaluated with respect to the solvent
control using SDS as a positive control (with a growth-inhibition
value above 90%). A very large amount of the salophen deriva-
tives (4500 mM) is required to induce cell death as shown in the
dose–response curves depicted in Fig. 6.
Considering that for concentrations higher than 70 mM the
compounds do not seem to interact with DNA, the growth-
inhibition effects observed may be due to the cell-recovery
effect by the compounds and not to the interaction with
organelles and/or DNA. The growth-inhibition percentages
obtained at different concentrations allowed the calculation
of the 24 h IC50 values using a Probit regression method (that is
the quantile function, i.e., the inverse cumulative distribution
function (CDF), associated with the standard normal distribu-
tion) included in the SPSS (Statistical Package for the Social
Sciences) program version 15 for Windows (see Fig. 7 and
Table 1).
Absorption spectra in DMSO/buffer solutions of 1 and 2 have
been recorded in the pH range 6–9, corresponding to pH values
that may be found in cellular media. The complexes are stable
within this range since no significant changes are observed
in UV-vis spectra. Compounds decompose at lower pH values
(pH = 2 and 5) (see Fig. S5 and S6, ESI†).
3.4. Cytotoxicity studies
Evaluation of the toxicity of the compounds was necessary
previously to start the study of the in vitro internalization on
3T3 cells and interactions of the complexes with DNA in assays.
Cytotoxicity was evaluated by MTT experiments following
preparation detailed in Experimental Section. Seeded cell wells
without MTT were used as blank experiments to evaluate the
effect (cell binding) of 1 and 2 on the 3T3 cells. After 24 h of
incubation, the cells were analyzed by fluorescence microscopy,
using a UV2A filter. The best concentration of Zn complexes
that seemed to interact with cells (not covering them on the
outside) was ca. 70 mM (67.4 mM for 1 and 60.8 mM for 2). The
cytotoxic effect is exemplified in Fig. 5, where a decrease in the
number of cells is observed with higher concentrations of
compound 2.
Similar studies were carried out with the free ligands L1 and
L2. The obtained IC50 values after 24 h, 1.21 [461.588, 1.687]
mM and 1.38 [1.21, 1.595] mM, respectively, are comparable
to those of the corresponding Zn-complexes. Moreover, L1
and L2 are not luminescent and they hydrolyze rapidly in
solution.
Fig. 5 Microscopy images of 3T3 fibroblast cells incubated for 24 h with 15 mM (left) and 60.8 mM of compound 2 (right). White light is used for co-localized cells. Pink
tint represents culture medium; green area represents fluorescent compound 2.
Fig. 6 Cytotoxicity dose responses of metal complexes in 3T3 cells for compound 1 (left) and 2 (right).
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem., 2013, 37, 1046--1055 1051

View Article Online
NJC
Paper
Fig. 7 Probit plot for compounds 1 and 2 at different concentrations in water and DMSO. Interception of two axes corresponds to IC50
Table 1 IC50 values of 1 and 2 in water. LB: lower bound. UB: upper bound
.
Compound
IC50 (mM)
[LB, UB]
1
2
1.29
1.85
[0.953, 1.453]
[1.214, 2.456]
The potential cytotoxic properties of the zinc compounds
and ligands were also examined at a longer incubation time
(72 h) but no significant changes were noticed.
3.5. Genotoxicity: comet assays
Comet assays, as genotoxicity experiments, were carried out
with 3T3 fibroblast cells to investigate whether our zinc
compounds interact with the DNA strands (a polyphosphate
macromolecule that may indeed bind 1 and/or 2) and produce
damages. This well-established and extremely sensitive method
is commonly applied to detect DNA fragmentation in individual
cells, thus allowing the assessment of genotoxic effects, or
the anti-genotoxic (protective) activity against a clastogenic
challenge (i.e. chromosome breakage71).
Non-damaged cells exhibit a spherical shape, while frag-
mented nucleoids migrate, forming a tail (‘‘comet’’ shape). The
nucleoid damage was quantified as fluorescence percentage in
the tail. Comparison of the results with a positive control
(methyl methanesulfonate, MMS) reveals that the nucleoid is
not damaged by the two zinc–salophen complexes (see Fig. 8
and Fig. S7, ESI†).
Fig. 9 Histogram of the genotoxicity assay results for compounds 1 and 2 with
3T3 cells. Control: culture medium; solvent control 10% water; C1: compound 1;
C2: compound 2; C+: positive control (MMS).
As shown in Fig. 9, the genotoxicity of the salophen deriva-
tives is clearly lower than that observed for the MMS control.
This feature is consistent with a lack of DNA (nucleoid)-damage
activity in cells.
3.6. Preliminary cellular uptake studies
First of all, cellular uptake studies were therefore carried out
using 3T3 fibroblasts (connective tissue cells). After incubation
with compound 1 or 2, the cultures were washed and the cells
were observed under an inverted fluorescence microscope with
an ultraviolet filter. Interestingly, as it is evidenced in Fig. 10,
fluorescence is observed for cells incubated with the
two compounds, suggesting that both 1 and 2 can interact
with cells.
The optimal time required for the cell uptake/binding was
analyzed by taking pictures at 5 min, 30 min, 1 h, 1.5 h, 2 h and
2.5 h. The corresponding microscopy images indicate that the
binding of the compounds appears optimal after an incubation
time of about 1 h. Above 1 h, 1 and 2 are uptaken by the cells,
but their binding effect does not seem to increase significantly
(see Fig. S8 and S9, ESI†). The strong fluorescence observed at
Fig. 8 40ꢁ microscopy image of cells in DAPI-staining solution, using a UV2A
fluorescence filter. Image of the cells in the presence of compound 2 (left) and in
the presence of the MMS positive control (right).
c
1052 New J. Chem., 2013, 37, 1046--1055
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013

View Article Online
Paper
NJC
Fig. 10 Microscopy image of 3T3 fibroblast cells with white light for localized cells (left), fluorescent image of 3T3 fibroblast cells incubated with 1 (70 mM), 20ꢁ and
UV2A filter (middle) and 3T3 fibroblast cells incubated with 2 (70 mM), 20ꢁ and UV2A filter (right). Incubation time: 1 h.
incubation times of 1.5 h, 2 h and 2.5 h most likely arises from assay suggest that the metal-coordination compound could be
an excess of the fluorophores covering the cells, namely located located inside the nucleus, without affecting its integrity
outside the cellular membrane.
(Fig. 11, right). Well-defined nucleoids are observed, i.e. the
internalization appears to occur without damaging the nucleus
(in vitro studies).
However, analysis of the same samples after 3 days reveals an
inhibiting effect on cell growth in the presence of compounds 1
and 2. Therefore, the interaction of the zinc derivatives with the
DNA strands appears to prevent cell division. It should be noticed
as well that the compounds seem to bind at specific DNA sites.
3.7. DNA binding and interaction within the cells
After a quick exposure to compounds 1, 2 (107.78, 97.29 mM
concentrations respectively) over fixed nucleoids, it is possible
to see how the compounds interact with DNA in a specific
region (see Fig. 11, right and Fig. S11, right, ESI†). DAPI stains
cell nuclei blue as a result of its emission at 461 nm (when Hence, the DNA/compound interaction is not uniform (see Fig. 11
excited at 358 nm); hence, this technique is routinely used to and Fig. S11, ESI†) and appears to be present in different organelles
visualize nuclear morphologies. Usually, more intensely DAPI- (not only in the nucleoid). To analyze further these data, co-
stained regions indicate more condensed DNA and vice versa.72
localization of living cell internalization was investigated by using
The presence of Zn(II)–salophen complexes seems to be
detected in the cell nucleus (yellow-green dots in Fig. 11, left).
Moreover, the DAPI-staining experiments and genotoxicity
Draq5 as fluorochrom. This is a far-red emitting fluorescent DNA
dye used for permeabilized and fixed live cell analysis that can be
used in combination with other common fluorophores. In the
present case, the utilization of Draq5 proved to be appropriate since
its red emission could be clearly separated from that of the zinc–
salophen derivatives (observed at higher energies). Thus, 3T3 cells
were stained with Draq5 and the emissions were observed by a
fluorescence confocal microscope at 688 nm for Draq5 and at
500 nm for the complexes. Lambda scans from 405 nm to 600 nm
were performed.
The corresponding images displayed in Fig. 12 and Fig. S12
(ESI†) clearly show that the nucleus is stained by Draq5 (Fig. 12,
left), and that the zinc complexes seem to enter the cells
(Fig. 12, middle), but without binding specifically to the
nucleus. Superimposition of the images (see Fig. 12, right)
indeed indicates that the red staining due to Draq5 is not
mixed with that corresponding to the salophen compounds.
Fig. 11 20ꢁ Fluorescence microscopy image of nucleoids incubated with
compound 2 (concentration of 97.29 mM) and in the presence of DAPI. Left:
with a B2A filter without DAPI co-staining (see the staining of compound 2);
right: with a UV2A filter (see the staining of DAPI). Incubation time: 1 h.
Fig. 12 Fluorescence confocal microscopy image of 3T3 incubated cells with Draq5 (red color stain nucleus DNA, left), salophen complex 1 (concentration of
107.78 mM green color, middle) and superimposed images (right). Incubation time: 3 h.
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem., 2013, 37, 1046--1055 1053

View Article Online
NJC
Accordingly, these first co-localization experiments demon-
strate that although the zinc–salophen complexes 1 and 2 enter
the living cells, they do not enter the nucleus, since no mixture
of colours could be observed in the nucleus region. This
is perfectly in agreement with the lack of nucleoid damage
previously observed with the in vivo comet assay.
Paper
6 J. R. Lakowicz, Principles of Fluorescence Spectroscopy,
Plenum, New York, 2nd edn, 1999.
7 S. W. Hell, Science, 2007, 316, 1153.
8 M. J. Rust, M. Bates and X. Zhuang, Nat. Methods, 2006,
3, 793.
9 J. Jaiswal and S. M. Simon, Nat. Chem. Biol., 2007, 3, 92.
10 X. S. Xie, J. Yu and W. Y. Yang, Science, 2006, 312, 228.
11 D. J. Stephens and V. J. Allan, Science, 2003, 300, 82.
12 G. U. Nienhaus and J. Wiedenmann, ChemPhysChem, 2009,
10, 1369.
4. Conclusions
Two luminescent zinc–salophen compounds, 1 and 2, have
been assessed as potential DNA-intercalator agents with the
prospective of using them as bio-markers for cell imaging. AFM
studies together with emission and UV-vis titration experiments
with ethidium bromide are indicative of interaction with DNA.
Cell-growth inhibition investigations are also in agreement
with in vitro studies. Actually, both compounds can enter the
cells and remain inside. Comet assays clearly show that this
cellular uptake does not lead to nucleoid damage. The observed
interaction of the complexes with DNA in vitro encouraged us to
investigate their activity in a living biological system. Staining
experiments with Draq5 on 3T3 fibroplast cells have indeed
demonstrated that the compounds enter the cells but do not
reach the nucleus. These features, together with the very high
IC50 24 hours values obtained for 1 and 2, indicate their non-
cytotoxic character and encourage their use as fluorescence
markers in cells. Their high energy emission properties would
allow them to be easily followed by two-photon microscopy
imaging. To complete the investigation, the cytotoxicity of the
metal-free ligands L1 and L2 has been evaluated in a similar
way. The obtained IC50 values are comparable to those of the
corresponding Zn-complexes, but their lack of luminescence
and low stability precludes their use as fluorescence markers.
13 L. Kurzawa and M. C. Morris, ChemBioChem, 2010, 11, 1037.
14 A. Paganin-Gioanni, E. Bellard, L. Paquereau, V. Ecochard,
´
M. Golzio and J. Teissie, Radiol. Oncol., 2010, 44, 142.
15 H. Hong, S. Goel, Y. Zhang and W. Cai, Curr. Med. Chem.,
2011, 18, 4195.
16 F. Wang, W. B. Tan, Y. Zhang, X. Fan and M. Wang,
Nanotechnology, 2006, 17, R1.
17 J. A. Drewry and P. T. Gunning, Coord. Chem. Rev., 2011,
255, 459.
18 R. M. Haak, S. J. Wezenberg and A. W. Kleij, Chem.
Commun., 2010, 46, 2713.
19 B. Bahramian, V. Mirkhani, M. Moghadam and A. H. Amin,
Appl. Catal., A, 2006, 315, 52.
20 N. S. Venkataramanan, G. Kuppuraj and S. Rajagopal,
Coord. Chem. Rev., 2005, 249, 1249.
21 K. Omura, T. Uchida, R. Irie and R. Katsuki, Chem. Commun.,
2004, 2060.
22 J. D. McGilvra and V. H. Rawal, Synlett, 2004, 2440.
23 A. Dalla Cort, L. Mandolini and L. Schiaffino, Chem. Commun.,
2005, 3867.
24 C. K. Shin, S. J. Kim and G. J. Kim, Tetrahedron Lett., 2004,
45, 7429.
25 T. Maeda, T. Takeuchi, Y. Furusho and T. J. Takata, J. Polym.
Sci., Part A: Polym. Chem., 2004, 42, 4693.
26 S. S. Kim and G. Rajagopal, Synthesis, 2003, 2461.
27 J. L. Czlapinski and T. L. Sheppard, J. Am. Chem. Soc., 2001,
123, 8618.
Acknowledgements
The support and sponsorship provided by COST Action
CM1005 are acknowledged. Authors are also grateful to the
28 S. Bhattacharya and S. S. Mandal, Chem. Commun., 1996,
1515.
´
Ministerio de Ciencia e Innovacion of Spain (Projects Project
˜
CTQ2012-31335 and CTQ2011-27929-C02-01) and Fundaçao para
29 J. G. Muller, S. J. Paikoff, S. E. Rokita and C. J. Burrows,
J. Inorg. Biochem., 1994, 54, 199.
ˆ
a Ciencia e Tecnologia of Portugal (PTDC/QUI-QUI/112597/2009).
Authors would like to thank Drs Lidia Bardia and Julien
Collombelli from IRB-Barcelona for fruitful discussions.
30 S. Routier, J. L. Bernier, M. J. Waring, P. Colson, C. Houssier
and C. Bailly, J. Org. Chem., 1996, 61, 2326.
31 D. J. Gravert and J. H. Griffin, J. Org. Chem., 1993, 58, 820.
32 H. Y. Shrivastava, S. N. Devaraj and B. U. Nair, J. Inorg.
Biochem., 2004, 98, 387.
References
1 X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay,
S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir
and S. Weiss, Science, 2005, 307, 538.
33 A. Dalla Cort, P. De Bernadin, G. Forte and F. Yafteh Mihan,
Chem. Soc. Rev., 2010, 39, 3863.
34 K. I. Ansari, S. Kasiri, J. D. Grant and S. S. Mandal, Dalton
Trans., 2009, 8525.
35 A. Hille, I. Ott, A. Kitanovic, I. Kitanovic, H. Alborzinia,
2 V. Fernandez-Moreira, F. L. Thorp-Greenwood and
M. P. Coogan, Chem. Commun., 2010, 46, 186.
3 H. Kobayashi, M. R. Longmire, M. Ogawa and P. L. Choyke,
Chem. Soc. Rev., 2011, 40, 4626.
4 D. A. Mankoff, J. Nucl. Med., 2007, 48, 18N.
5 R. D. Goldman, J. R. Swedlow and D. L. Spectror, Live Cell
Imaging, Cold Spring Harbor Laboratory Press, New York,
2010.
¨
¨
E. Lederer, S. Wolfl, N. Metzler-Nolte, S. Schafer,
W. S. Sheldrick, C. Bischof, U. Schatzschneider and
R. Gust, J. Biol. Inorg. Chem., 2009, 14, 711.
36 S. Routier, H. Vezin, E. Lamour, J. L. Bernier, J. P. Catteau
and C. Bailly, Nucleic Acids Res., 1999, 27, 4160.
c
1054 New J. Chem., 2013, 37, 1046--1055
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013

View Article Online
Paper
NJC
37 T. S. Lange, K. K. Kim, R. K. Singh, R. M. Strongin, 55 A. Dalla Cort, L. Mandolini, C. Pasquini, K. Rissanen,
C. K. McCourt and L. Brard, PLoS One, 2008, 3, e2303, 1. L. Russo and L. Schiaffino, New J. Chem., 2007, 31, 1633.
38 Y. W. Liou and C. M. Wang, J. Electroanal. Chem., 2000, 481, 102. 56 M. Cano, L. Rodrıguez, J. C. Lima, F. Pina, A. Dalla Cort,
39 J. G. Muller, L. A. Kayer, S. J. Paikoff, V. Duarte, N. Tang, C. Pasquini and L. Schiaffino, Inorg. Chem., 2009, 48, 6229.
´
´
´
R. J. Perez, S. E. Rokita and C. J. Burrows, Coord. Chem. Rev., 57 S. J. Wezenberg, E. C. Escudero-Adan, J. Benet-Buchholz and
1999, 185–186, 761. A. W. Kleij, Chem.–Eur. J., 2009, 15, 5695.
40 O. Alptu¨rk, O. Rusin, S. O. Fakayode, W. Wang, 58 A. Dalla Cort, P. De Bernardin and L. Schiaffino, Chirality,
´
J. O. Escobedo, I. M. Warner, W. E. Crowe, V. Kral,
2009, 21, 104.
J. M. Pruet and R. M. Strongin, Proc. Natl. Acad. Sci. U. S. A., 59 I. P. Oliveri and S. Di Bella, Tetrahedron, 2011, 67, 9446.
2006, 103, 9756.
60 G. Consiglio, S. Failla, P. Finocchiaro, I. P. Oliveri and S. Di
41 S. R. Doctrow, K. Huffman, C. B. Marcus, G. Tocco,
Bella, Dalton Trans., 2012, 41, 387.
E. Malfroy, C. A. Adinolfi, H. Kruk, K. Baker, 61 I. P. Oliveri, G. Maccarrone and S. Di Bella, J. Org. Chem.,
N. Lazarowych, J. Mascarenhas and B. Malfroy, J. Med.
Chem., 2002, 45, 4549.
42 Y. Rong, S. R. Doctrow, G. Tocco and M. Baudry, Proc. Natl.
Acad. Sci. U. S. A., 1999, 96, 9897.
2011, 76, 8879.
62 G. Consiglio, S. Failla, I. P. Oliveri, R. Purrello and S. Di
Bella, Dalton Trans., 2009, 10426.
63 J. Jing, J. J. Chen, Y. Hai, J. Zhan, P. Xu and J.-L. Zhang,
Chem. Sci., 2012, 3, 3315.
´
43 R. Chico, C. Domınguez, B. Donnio, S. Coco and P. Espinet,
Dalton Trans., 2011, 40, 5977.
44 L. Zhou, P. Cai, Y. Feng, J. Cheng, H. Xiang, J. Liu, D. Wu
and X. Zhou, Anal. Chim. Acta, 2012, 735, 96.
45 S. Di Bella, I. P. Oliveri, A. Colombo, C. Dragonetti,
S. Righetto and D. Roberto, Dalton Trans., 2012, 41, 7013.
64 E. Verner, B. A. Katz, J. R. Spencer, D. Allen, J. Hataye,
W. Hruzewicz, H. C. Hui, A. Kolesnikov, Y. Li, C. Luong,
A. Martelli, K. Radika, R. Rai, M. She, W. Shrader,
P. A. Sprengeler, S. Trapp, J. Wang, W. B. Young and
R. L. Mackman, J. Med. Chem., 2001, 44, 2753.
46 J. E. Reed, A. Arola Arnal, S. Neidle and R. Vilar, J. Am. Chem. 65 ASTM-E2186, Standard Guide for Determining DNA Single-
Soc., 2006, 128, 5992.
Strand Damage in Eukaryotic Cells using the Comet Assay,
47 A. Arola-Arnal, J. Benet-Buchholz, S. Neidle and R. Vilar,
Inorg. Chem., 2008, 47, 11910.
48 N. H. Campbell, N. H. Abd Karim, G. N. Parkinson,
June 2003.
66 H. Wang, R. Shen, J. Wu and N. Tang, Chem. Pharm. Bull.,
2009, 57, 814.
M. Gunaratnam, V. Petrucci, A. K. Todd, R. Vilar and 67 A. Wolfe, G. H. Shimer Jr and T. Meehan, Biochemistry, 1987,
S. Neidle, J. Med. Chem., 2012, 55, 209. 26, 6392.
49 P. Wu, D.-L. Ma, C.-H. Leung, S.-C. Yan, N. Zhu, R. Abagyan 68 P. Krishnamoorthy, P. Sathyadevi, A. H. Cowley,
and C.-M. Che, Chem.–Eur. J., 2009, 15, 13008.
50 S. I. Pascu, P. A. Waghorn, T. D. Conry, B. Lin, H. M. Betts,
R. R. Butorac and N. Dharmaraj, Eur. J. Med. Chem., 2011,
46, 3376.
J. R. Dilworth, R. B. Sim, G. C. Churchill, F. I. Aigbirhio and 69 M. Shilpa, P. Nagababu, Y. P. Kumar, J. N. Lavanya Latha,
J. E. Warren, Dalton Trans., 2008, 2107.
M. R. Reddy, K. S. Karthikeyan, N. Md. Gabra and
51 A. L. Singer and D. A. Atwood, Inorg. Chim. Acta, 1998, 277, 157.
S. Satyanarayana, J. Fluoresc., 2011, 21, 1155.
52 Y. Hai, J.-J. Chen, P. Zhao, H. Lv, Y. Yu, P. Xu and 70 S. J. Lippard, Acc. Chem. Res., 1978, 11, 211.
´
71 M. Barenys, N. Macia, L. Camps, J. de Lapuente, J. Gomez-
J.-L. Zhang, Chem. Commun., 2011, 47, 2435.
53 Y. Gao, J. Wu, Y. Li, P. Sun, H. Zhou, J. Yang, S. Zhang, B. Jin
and Y. Tian, J. Am. Chem. Soc., 2009, 131, 5208.
´
`
Catalan, J. Gonzalez-Linares, M. Borras, M. Rodamilans and
J. M. Llobet, Toxicol. Lett., 2009, 191, 40.
54 M. E. Germain, T. R. Vargo, B. A. McClure, J. J. Rack, P. G. Van 72 K. I. Ansari, B. P. Mishra and S. S. Mandal, Biochim. Biophys.
Patten, M. Odoi and M. J. Knapp, Inorg. Chem., 2008, 47, 6203.
Acta, 2008, 1779, 66.
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2013
New J. Chem., 2013, 37, 1046--1055 1055