Substituent effects on the biological properties of Zn-salophen complexes

Article abstract of DOI:10.1021/ic4004356

The synthesis, characterization, and luminescent properties of a series of 5,5′-X-substituted salophen ligands, X= OCH₃, Br, and NO ₂, and the corresponding Zn(II) complexes are reported here. Their biological activity has been analy

Full text of DOI:10.1021/ic4004356

Article
pubs.acs.org/IC
Substituent Effects on the Biological Properties of Zn-Salophen
Complexes
Ilaria Giannicchi, Rosa Brissos, David Ramos, Joaquin de Lapuente, Joao
Carlos Lima,∥
†
‡
§
§
̃
,†
,‡
Antonella Dalla Cort,* and Laura Rodríguez*
†Dipartimento di Chimica and IMC-CNR Sezione Meccanismi di Reazione, Universita
̀
La Sapienza, Box 34 Roma 62, 00185 Roma,
Italy
‡
Departament de Química Inorgan
̀
ica, Universitat de Barcelona, Martí i Franques
̀
1-11, 08028 Barcelona, Spain
§
Unitat de Toxicologia Experimental i Ecotoxicologia, Parc Científic de Barcelona, c/Baldiri i Reixach, 10-12, 08028 Barcelona, Spain
∥
̂
REQUIMTE, Departamento de Química, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte de
Caparica, Portugal
*
S Supporting Information
ABSTRACT: The synthesis, characterization, and luminescent properties of a
series of 5,5′-X-substituted salophen ligands, X= OCH , Br, and NO , and the
3
2
corresponding Zn(II) complexes are reported here. Their biological activity has
been analyzed and related to the different Lewis acid character of the complexes.
In vitro studies (AFM and absorption and emission titrations) show that the
strongest interaction with free plasmid DNA is observed for 5,5′-dinitro-
substituted Zinc-salophen complex 3b. Semiempirical theoretical calculations
together with redox potential measurements suggest that this might be
interpreted as a direct consequence of this compound having the hardest
Lewis acid character. Cellular uptake and cytotoxicity studies undertaken with
these metal complexes show that they enter the cells but are not cytotoxic.
INTRODUCTION
abundant transition metal in living organisms after iron, and it
■
15
is the only metal that appears in all enzyme classes. It has
been observed that in general the lack of redox activity of
Zn(II) normally leads to Zn-salen/salophen compounds that
Metallo-salen/salophen (metal complexes of bis(salicylidene)-
ethylenediamine and bis(salicylidene)diphenyldiamine) com-
plexes are well established efficient catalysts in organic
chemistry because of their rich redox chemistry and ease of
1
5,19,20
do not show cytotoxic properties.
This, together with the
10
1
−4
closed-shell d configuration featured by the metal, make such
complexes ideal candidates for the generation of biological
fluorescence probes and ligand-dependent fluorescence materi-
manipulation.
Furthermore, biochemists have utilized the
oxidative nature of metallo-salen complexes to develop novel
DNA/RNA modifiers and biomolecular probes.
ultimate cytotoxicity of such metal complexes depends on a
number of factors, including the mode of cellular interaction,
transport/cellular-uptake potential, intrinsic structure, and
reactivity of the compounds.
apoptotic activity of the metallo-salen complexes appears to be
highly dependent on the nature of the central metal ion and the
substituents present on the salen moiety.
5
−7
The
21−23
als.
It has been established that the nature and position of the
substituents on the ligand skeleton, solubility, and cellular
permeability play a critical role in determining their cytotoxic
8
−11
The cellular-interaction and
1
2,24
behavior.
apoptotic activity displayed by Mn(III)-salen/salophen deriv-
S. Mandal and co-workers described the diverse
17
1
2−14
atives decorated with methoxy or hydroxyl groups. However,
no straightforward correlation with the nature of the
substituent was clearly established. However, the bridging
group, which is ethylenediamine in the case of salen and an o-
phenylenediamine unit for salophen derivatives, also seems to
have an effect.
The redox properties of sal(oph)en-containing coordination
compounds have been used in biological studies that revealed
their involvement in metal-dependent DNA-damage pro-
1
5,16
cesses.
Indeed, Fe(III)- and Mn(III)-salen/salophen
complexes show cytotoxicity and effective DNA damage,
1
7
Recently, we reported the biological study of two Zn(II)-
inducing apoptosis in cultured human cells. Some lantha-
nide-salophen complexes are able to bind neutral sugars and
lipids, including lysophosphatidic acid (a biomarker for several
19
salophen complexes that differ in their bridging unit. They
both display high IC50 values, but they partially differ for their
18
pathological conditions including ovarian cancer).
The nonredox metal-ion zinc(II) salen/salophen-type
complexes are also relevant because zinc is the second most
Received: February 19, 2013
Published: July 29, 2013
©
2013 American Chemical Society
9245
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Inorganic Chemistry
Article
cytotoxicity in cells. Thanks to their intrinsic luminescent
properties, we proposed them to be potentially good candidates
for use as fluorescent markers in cells.
2H, N=C−H), 7.86 (m, 2H, CH), 7.53−7.39 (m, 6H, CH), 6.91 (m,
13
2
1
1
3
H, CH), 3.67 (s, 6H, CH ). C NMR (75 MHz, DMSO-d ) δ:
3 6
63.27, 154.88, 152.26, 142.91, 128.19, 121.14, 119.96, 119.89, 118.00,
15.10, 55.97. HRMS-ESI-TOF: calcd, 376.15; found, [M + H ]
+
In this study, we focused our attention on a series of 5,5′-X-
77.15 . Anal. Calcd for C H N O : C, 70.20; H, 5.36; N, 7.44.
22
20
2
4
substituted Zn(II)-salophen derivatives, X =OCH , Br, and
3
Found: C, 70.22; H, 5.37; N, 7.46.
NO (Chart 1), monitoring their biological behavior with
2
N,N′-(o-Phenylene)bis(5-bromosalicylideneimine), 2a. A solution
of 2-hydroxy-5-bromobenzaldehyde (0.487 g, 2.4 mmol) and 1,2-
diaminobenzene (0.133 g, 1.2 mmol) in MeOH (10 mL) was stirred
for 24 h. The mixture was then filtered, and the final product was
obtained as an orange solid in 84% yield. 1H NMR (300 MHz,
Chart 1
DMSO-d ) δ: 12.90 (bs, 2H, OH), 8.89 (s, 2H, N=C−H), 7.40−7.25
6
1
3
(
(
m, 6H, CH), 7.02−6.84 (m, 4H, CH), 3.71 (s, 6H, CH ). C NMR
75 MHz, DMSO-d ) δ: 162.66, 159.90, 142.46, 136.10, 134.20,
3
6
1
4
28.58, 121.88, 120.09, 119.60, 110.32. HRMS-ESI-TOF: calcd,
+
71.95; found, [M + H ] 472.95. Anal. Calcd for C H Br N O :
2
0
14
2
2
4
C, 50.66; H, 2.98; N, 5.91. Found: C, 50.68; H, 2.99; N, 5.93.
N,N′-(o-Phenylene)bis(5-nitrosalicylideneimine), 3a. A solution of
-hydroxy-5-nitrobenzaldehyde (0.403 g, 2.4 mmol) and 1,2-
2
diaminobenzene (0.136 g, 1.2 mmol) in MeOH (25 mL) was stirred
for 24 h. The mixture was then filtered, and the final product was
obtained as an orange solid in 92% yield. 1H NMR (300 MHz,
DMSO-d ) δ: 10.17 (bs, 2H, OH), 9.16 (s, 2H, N=C−H), 9.16 (s, 2H,
6
CH), 8.73 (m, 2H, CH), 8.28−8.16 (m, 2H, CH), 7.71−7.47 (m, 4H,
CH), 7.09−7.01 (m, 2H, CH). HRMS-ESI-TOF: calcd, 406.09; found,
+
[
M + H ] 407.09. Anal. Calcd for C H N O : C, 59.12; H, 3.47; N,
20
14
4
6
1
3.79. Found: C, 59.18; H, 3.50; N, 13.81.
N,N′-(o-Phenylene)bis(5-methoxysalicylideneiminato)zinc(II), 1b.
A solution of 2-hydroxy-5-methoxybenzaldehyde (0.395 g, 2.6 mmol),
,2-diaminobenzene (0.140 g, 1.3 mmol), and Zn(OAc) ·2H O (0.307
1
2
2
g, 1.4 mmol) in MeOH (14 mL) was stirred for 24 h. The mixture was
then filtered, and the final product was obtained as a yellow solid in
1
7
7
6
3% yield. H NMR (300 MHz, DMSO-d ) δ: 8.98 (s, 2H, N=C−H),
6
.85 (m, 2H, CH), 7.33 (m, 2H, CH), 6.94−6.90 (m, 4H, CH), 6.64−
.61 (m, 2H, CH), 3.67 (s, 6H, CH ). C NMR (75 MHz, DMSO-d )
respect to the different nature of the substituent. Analogous
studies with the uncomplexed ligands were performed for
comparison purposes.
13
3
6
δ: 168.12, 162.30, 147.56, 139.53, 127.25, 124.81, 124.20, 117.78,
16.52, 116.06, 55.67. HRMS-ESI-TOF: calcd, 438.06; found, [M +
1
+
H ] 439.06. Anal. Calcd for C H N O Zn: C, 60.08; H, 4.13; N,
22
18
2
4
EXPERIMENTAL SECTION
■
6
.37. Found: C, 60.12; H, 4.15; N, 6.38.
General Information. The best commercially available chemicals
were obtained and used without further purification unless otherwise
stated. pBR322 plasmid DNA was obtained from Boehringer-
Mannheim (Mannheim, Germany). HEPES (N-2-hydroxyethylpiper-
azine-N′-2-ethanesulfonic acid) was obtained from ICN (Madrid).
N,N′-(o-Phenylene)bis(5-bromosalicylideneiminato)zinc(II), 2b. A
solution of 2-hydroxy-5-bromobenzaldehyde (0.265 g, 1.3 mmol), 1,2-
diaminobenzene (0.076 g, 0.7 mmol), and Zn(OAc) ·2H O (0.344 g,
2
2
1
.6 mmol) in MeOH (15 mL) was stirred for 24 h. The mixture was
then filtered, and the final product was obtained as a yellow solid in
Physical Measurements. NMR spectra were recorded in DMSO-
d with either a 200 or 300 MHz spectrometer. ES mass spectra were
1
6
7
2
1
1
5% yield. H NMR (300 MHz, DMSO-d ) δ: 8.97 (s, 2H, N=C−H),
+
6
6
.84 (m, 2H, CH), 7.58 (m, 2H, CH), 7.38 (m, 2H, CH), 7.27 (m,
H, CH), 6.63 (m, 2H, CH). C NMR (75 MHz, DMSO-d ) δ:
71.11, 162.33, 139.36, 137.46, 136.62, 127.98, 125.66, 121.13, 116.92,
02.88. HRMS-ESI-TOF: calcd, 533.86; found, [M + H ] 534.86.
recorded on a Fisons VG Quatro spectrometer at the Servei
d’Espectrometria de Masses (Universitat de Barcelona). Elemental
13
6
analyses of C, H, N, and S were carried out at the Serveis Cientifi
Tecnics (Universitat de Barcelona). The atomic-force microscopy
AFM) images were obtained with a NanoScope III MultiMode AFM
́
co-
+
̀
Anal. Calcd for C H Br N O Zn: C, 44.69; H, 2.25; N, 5.21. Found:
20
12
2
2
2
(
C, 44.72; H, 2.26; N, 5.24.
system from Digital Instruments, Inc. operating in tapping mode.
Fluorescence microscopy was carried out with an Olympus E800 and a
Nikon Eclipse E600 equipped with a UV2A filter. Electrochemical data
were obtained by cyclic voltammetry under nitrogen at 25 °C using
dry acetonitrile as the solvent and tetrabutylammonium hexafluor-
ophosphate (0.1 M) as the supporting electrolyte. The redox half-wave
potentials were referenced to an Ag/AgCl (in 3 M NaCl) electrode
separated from the solution by a medium-porosity fritted disk. A
platinum-wire auxiliary electrode was used in conjunction with a
platinum-disk working electrode, Tacussel-EDI rotatory electrode
N,N′-(o-Phenylene)bis(5-nitrosalicylideneiminato)zinc(II), 3b. A
solution of 2-hydroxy-5-nitrobenzaldehyde (0.372 g, 2.2 mmol), 1,2-
diaminobenzene (0.119 g, 1.1 mmol), and Zn(OAc) ·2H O (0.295 g,
2
2
1
.2 mmol) in MeOH (10 mL) was stirred for 24 h. The mixture was
then filtered, and the final product was obtained as a yellow solid in
1
7
7
(
0% yield. H NMR (300 MHz, DMSO-d ) δ: 8.39 (s, 2H, N=C−H),
6
.79 (m, 2H, CH), 7.29−7.13 (m, 4H, CH), 6.67 (m, 2H, CH), 5.95
m, 2H, CH). 13C NMR (75 MHz, DMSO-d ) δ: 177.15, 163.08,
6
1
56.97, 139.46, 134.82, 134.46, 128.89, 124.17, 119.00, 117.69.
+
2
−4
HRMS-ESI-TOF: calcd, 468.00; found, [M + H ] 469.0. Anal.
Calcd for C H N O Zn: C, 51.14; H, 2.57; N, 11.93. Found: C,
(
3.14 mm ). Cyclic voltammograms of 5 × 10 M solutions of the
samples in CH Cl2 were recorded with a BioLogic Science
20 12
4
6
2
5
1.17; H, 2.59; N, 11.96.
Instruments SP-150 potentiostat.
Molecular Modeling and Semiempirical Calculations. The
Synthesis and Characterization. N,N′-(o-Phenylene)bis(5-me-
thoxysalicylideneimine), 1a. A solution of 2-hydroxy-5-methoxyben-
zaldehyde (0.6 g, 3.9 mmol) and 1,2-diaminobenzene (0.213 g, 2.0
mmol) in MeOH (8 mL) was stirred for 24 h. The mixture was then
electronic structure and electronic transitions were calculated with the
AM1 semiempirical method in structures previously optimized with
the MM+ molecular mechanics method. Both of these methods were
included in the software package HyperChem 7.0. (Hypercube,
filtered, and the final product was obtained as orange solid in 72%
1
25
yield. H NMR (300 MHz, DMSO-d ) δ: 12.26 (bs, 2H, OH), 8.87 (s,
Inc.).
6
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Inorganic Chemistry
Article
Atomic Force Microscopy Experiments. pBR322 DNA was
heated before use at 60 °C for 10 min to obtain the open circular
then electrophoresed for 30 min at 25 V and 300 mA at 4 °C. After
neutralization with 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 μL of DAPI at 14.28 μM. Nondamaged cells exhibit a
spherical shape, whereas fragmented nucleoids migrate, forming a tail
(“comet” shape). The nucleoid damage was quantified as the
percentage of fluorescence in the tail. In the assay, the percentage of
DNA in the tail was determined with respect to the intensity of the
total DNA for 50 cells. The determination was obtained with the
Comet Assay IV software, and the corresponding statistics were
performed using the SPSS program (version 15 for Windows) by
applying Mann−Whitney U statistics.
Preliminary Cellular-Uptake Studies. 3T3 Balb/c cells were
exposed to a single concentration of compounds 1b−3b of ca. 71.17,
58.54, and 66.63 μM, respectively, and they were observed on an
inverted fluorescence microscope with two different filters (B2A and
UV2A). The observations were carried out at different times: 15 and
30 min as well as 1, 1.5, 2, and 24 h after exposure.
DNA-Binding Studies in Cells. Preliminary experiments were
carried out with free nucleoids in an agarose matrix. Fixed nucleotides
were stained with concentrations of compounds 1b−3b of ca. 113.88,
93.66, and 106.61 μM, respectively. Staining with DAPI (4′,6-
diamidino-2-phenylindole, 14.28 μM), a well-known fluorescent stain
that acts as a DNA minor-groove binder and DNA intercalator, at a
concentration of 14.28 μM allowed us to obtain better microscopy
images.
For the internalization of compounds, 3T3 cells were seeded onto a
coverslip in a 24-well plate and grown overnight under normal growth
conditions followed by incubation with 1b at 30.29 μM, 2b at 37.46
μM, and 3b at 53.3 μM for an additional 3 h. Control cells were
treated with an equivalent amount of DMSO. Cells were fixed with 4%
formaldehyde in PBS for 30 min, washed (2×) with PBS, and
permeabilized with 0.2% Triton X-100 in PBS for 5 min. The cells
were washed 3× with cold PBS followed by incubation with Draq5 (10
μM) for 30 min at room temperature. Stained cells were washed 3×
with cold PBS, mounted on a microscope slide with Mowiol mounting
media, and visualized under a Leica TCS SP2 AOBS system (405 nm
excitation laser diode for the complexes and 688 nm for Draq5). To
detect molecules inside the cell, we performed a lambda scan with a
wide range of wavelengths. With the z plain fixed inside the nucleus
thanks to the Draq5 colocalization stain, pictures were taken at
different wavelengths ranging from 405 to 600 nm. We selected
different points within the cell and measured the intensity of the light
emitted from each region.
(
OC) form. The stock solution is 1 mg/mL in a HEPES buffer
solution. Each sample contains 1 μL of DNA pBR322 at a
concentration of 0.25 μg/μL and a final volume of 50 μL. The
amount of drug added was expressed as r (i.e., the ratio between the
i
drug’s molar concentration to the number of base pairs). All of the
stock solutions were freshly prepared in milli-Q water with 2% DMSO.
UV−Vis Spectrophotometric Studies. The absorption titrations
were performed by adding increasing amounts of ct-DNA (calf thymus
DNA) to the complexes in Tris-HCl buffer (5 mM) and NaCl (50
mM) at pH 7.2. The concentration of ct-DNA was determined from
the absorption intensity at 260 nm with a molar extinction coefficient
−1
−1
of 6600 M cm . After the addition of DNA to the metal complex,
the resulting solution was allowed to equilibrate at 25 °C for 2 min,
after which time the absorption spectra were recorded.
Fluorescence Spectrophotometric Titrations. The relative
binding affinities of the complexes to ct-DNA were studied with an
ethidium bromine (EB)-bound ct-DNA solution in a 5 mM Tris-HCl
and 50 mM NaCl buffer (pH 7.2). The fluorescence spectra were
recorded at room temperature with excitation at 514 nm. The titration
experiments were carried out with an EB−DNA solution containing
−
5
−5
−3
−1
2
.5 × 10 M EB, 1 × 10 M ct-DNA, and 75 × 10 μg μL of the
compounds.
Cytotoxicity Studies. For the cytotoxicity assays, 3T3 Balb/c cells
were cultured in Dulbecco’s modified Eagle’s medium (DMEM,
Sigma-Aldrich) containing 10% fetal calf serum (Hyclone), 2 mM
glutamine (Sigma-Aldrich), and antibiotics (Sigma-Aldrich, 50 μg/mL
of penicillin and 50 μg/mL of streptomycin). Thus, the cells were
incubated for 24 h for correct adhesion. Afterward, solutions of the
salophen were added to some wells, whereas a 0.2% solution of sodium
dodecylsulfate (SDS) was added to the others for a positive control.
The incubation was prolonged for a further 24 h. The cells were
exposed to a range of concentrations from 2277 to 8.91 mM for 1b,
1873 to 7.32 mM for 2b, and 2132 to 8.34 mM for 3b under 5% CO2
at 37 °C. The viability was assessed by the MTT assay on the basis of
the mitochondrial dehydrogenase enzyme’s capability of hydrolyzing a
bromide chromogen (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide). The cells were washed with PBS (3×), and fresh
medium was added together with a 5 mg/mL MTT solution and
incubated for 2 h at 37 °C under a 5% CO2 atmosphere. This
compound shows a pale-yellow color in solution, and its conversion by
the enzyme reduces the MTT to a formazan in the mitochondria of
living cells, which is a crystallized, dark-blue compound that cannot
cross the cell membrane.
Because of the low solubility of the compounds in water, a second
cytotoxicity assay was carried out using DMSO (1%) as the solvent
with the same concentrations of compounds that were previously
assayed.
RESULTS AND DISCUSSION
Synthesis and Characterization. Compounds 1a−3a
were prepared according to the standard procedure for salen
■
The data were statistically analyzed to calculate the 50% inhibition
Table 1. Absorption and Emission Data of Compounds 1a−
concentrations, IC , using the SPSS program (version 15 for
5
0
a
Windows) and applying the Probit regression.
Genotoxicity Comet Assay. The genotoxicity study of the 3T3
3a and 1b−3b
‑
3
−1
a
compound
absorption (nm) (ε × 10 , M cm−1
)
emission (nm)
Balb/c fibroblast cell line was performed via the comet assay according
26
to the ASTM-E2186 guideline.
1a (OMe)
2a (Br)
274 (4.9), 354 (3.1)
268 (21.4), 341 (14.2)
279 (27.7), 404 (9.3)
300 (10.4), 432 (7.2)
290 (24.9), 400 (19.7)
295 (37.1), 380 (39.6)
Cells were cultured in 6-well flat-bottomed microtiter plates
5
containing 2 mL of the cell suspension (∼2 × 10 cells). After 24 h
3a (NO2)
1b (OMe)
of incubation at 37 °C with 5% CO , the compound to be investigated
2
551
507
476
was added. Only two doses were used for compound 1b, 8.91 and
2
3
b (Br)
1
3
7.81 μM. For 2b, 7.32, 14.65, 29.28, and 58.54 μM were used, and for
b, 16.67 and 33.33 μM were used. After 24 h of exposure, the 3T3
b (NO2)
a
Excitation at the lowest energy absorption band.
fibroblast cells were detached from the well with a 0.05% trypsin
solution and collected by centrifugation. The cells were embedded in
low-melting point 0.9% agarose (LMP-Gibco) prepared in Milli-Q
water (18 MΩ) and layered on precoated slides. The slides were
27
and salophen ligands using the appropriate substituted
salicylaldehyde and 1,2-diaminobenzene as starting materials.
The reactants were refluxed in methanol for 1 h, during which
time the desired products precipitated as orange solids in pure
form. A similar procedure was used for the synthesis of the
corresponding Zn-salophen complexes 1b−3b in the presence
placed in lysing buffer (2.5 M NaCl, 100 mM Na EDTA, 10 mM Tris
2
pH 10, and N-lauryl-sarcosine 1% (w/v) (Sigma) with 1% Triton X-
00) for 1 h at 4 °C. The isolated DNA from the nuclei in the agarose
gels was unwound for 40 min in electrophoresis buffer (1 mM
1
Na EDTA and 300 mM NaOH, pH > 13). The SCGE slides were
2
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Figure 1. Normalized absorption (left) and emission spectra (right) of compounds 1b (dotted line), 2b (solid line), and 3b (dashed line).
Figure 2. AFM image of pBR322 plasmid DNA (A) and pBR322 plasmid DNA incubated with 1b (B), 2b (C), and 3b (D).
Table 2. Calculated Energy of the HOMO and LUMO Frontier Orbitals of Salophen Complexes 1b−3b and the DNA
Nucleobases and HOMO−LUMO Energy Differences (ΔE = E
(salophen) − E
(nucleobase))
LUMO
HOMO
compound
HOMO (eV)
LUMO (eV)
ΔE, G (eV)
ΔE, C (eV)
ΔE, A (eV)
ΔE, T (eV)
1
2
3
b (OMe)
b (Br)
−7.95
−8.43
−9.20
−8.62
−9.38
−8.77
−9.61
−1.24
−1.47
−2.00
−0.29
−0.10
−0.11
−0.29
7.38
7.15
6.62
8.14
7.91
7.38
7.53
7.3
8.37
8.14
7.61
b (NO2)
6.77
guanine
cytosine
adenine
thymine
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Article
Figure 6. Microscopy image (20×) of cells in a DAPI-staining
solution in the presence of compound 1b at 17.81 μM using a UV2A
fluorescence filter. The comet shape is not observed. The inset shows
the comet shape observed for the MMS positive control as a reference.
Figure 3. Electronic absorption spectra of complex 3b in the absence
and presence of increasing amounts of DNA. The inset shows a plot of
[
DNA]/(ε − ε ) vs [DNA].
a
f
Figure 7. Histogram of the genotoxicity assay results for compounds
1
=
b−3b with 3T3 cells. Medium = vehicle control (1% DMSO) and C+
positive control (MMS).
of a stoichiometric quantity of zinc acetate dihydrate. All of the
compounds were obtained in moderate−good yields (ca. 70−
9
0%).
Figure 4. Emission spectra of ethidium bromide (EB) bound to DNA
in the absence and presence of increasing amounts of 3b. The inset
Characterization by different spectroscopic techniques and
spectrometry verified the formation of the ligands and
complexes. H NMR shows the expected signals for the
shows the Stern−Volmer plot I /I vs [3b].
1
0
salophen derivatives. The protons are clearly affected by metal
coordination (e.g., whereas the signal of phenolic protons
obviously disappears, imine and aromatic protons are ca. 0.5−1
ppm upfield shifted, respectively). HRMS-ESI-TOF led us to
identify unequivocally the products because they display the
corresponding protonated molecular peak in all cases.
Optical Properties. Absorption and emission spectra of the
1
a−3a ligands and respective Zn(II) complexes 1b−3b were
studied, and the relevant photophysical data are summarized in
Table 1 and Figures 1 and S1.
Figure 5. Fluorescence-microscopy images of 3T3 cells incubated for
24 h with 14.61 (left) and 29.28 μM (right) of compound 2b. The
Uncomplexed salophen compounds 1a−3a show broad,
unstructured absorption bands in the 270−400 nm region with
a similar pattern for the reported salophen derivatives (Figure
S1). We have performed theoretical semiempirical calculations,
pink tint represents the culture medium and green areas represent
fluorescent compound 2b.
a
28
Table 3. Estimated IC50 Values of 1b−3b in DMSO
and as already observed for similar derivatives the observed
transitions are predominantly π−π* (Figures S2−S4). No
compound
IC50 (μM, DMSO)
(LCI, UCI)
significant luminescence was observed for any of them.
1
2
3
b
b
b
71
NP
Coordination to Zn(II) metal (complexes 1b−3b) gives
absorption patterns similar to those observed for the
corresponding free ligands but generally shifted to lower
energies. Excitation at the lower-energy band gives rise to
fluorescence broad bands centered in the 475−550 nm region.
The donor/acceptor electronic character of the substituents on
89
(76, 102)
(943, 1236)
1073
a
LCI: lower confidence interval. UCI: upper confidence interval (95%
confidence). NP: not possible to calculate.
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Figure 8. Fluorescence microscopy image of 3T3 fibroblast cells incubated with 3b at 66.63 μM for different amounts of time (20× and UV2A
filter).
DNA and all the three salophen ligands (Figure S5). The open
circle (OC) form of DNA is just slightly perturbed upon
incubation with nitro-salophen ligand 3a, and no effect is
observed with 1a and 2a. Instead, incubation with zinc
complexes induces much more significant effects in the DNA
structure, in particular in the presence of 3b (Figure 2). The
initial OC form of the DNA disappears almost completely in
the presence of this complex, giving rise to the formation of
mixed toroidal and plectonemic supercoils as well as to
complete plectonemic supercoiling. It is likely that such
behavior is due to the intercalation of the molecules into the
DNA, a phenomenon already observed by us with similar zinc-
19
salophen complexes. However, the OC form of the DNA is
almost unaffected by the presence of 1b and 2b (Figure 2).
Because the only difference in the three complexes is the
nature of the substituents, the degree of the interaction with
DNA might be related to their electron-withdrawing/electron-
donating character. The complex that visibly interacts with
DNA is the one containing the strongest electron-withdrawing
substituent, 3b. Consequently, the observed effect can be
ascribed to the Lewis acid−base interaction that occurs
between the electron-poor salophen (electronic density is
withdrawn by the presence of the nitro substituent) and the
DNA nucleobases (most likely guanine and adenine).
Figure 9. Fluorescence microscopy image (20×) of nucleoids
incubated with compound 1b (green dots, 113.88 μM) in the
presence of DAPI with the UV2A filter.
the salophen ligands is reflected in the energy of both the
absorption and emission bands, showing the energy trend OMe
<
Br < NO (i.e., the higher the electron-donating character,
2
the lower the energy of the observed transition).
AFM Studies. Recently, the AFM technique has been
shown to be a very useful tool to explore the interactions of
19
similar Zn(II)-salophen complexes with DNA. In the present
work, we used this technique to investigate the effect of
substituents with different electronic characteristics on the
interaction with DNA.
To support such a hypothesis, the compounds have been
modeled using the AM1 method in structures previously
optimized with MM+ molecular mechanics (both are included
25
Free pBR322 plasmid DNA was incubated for 24 h with
aqueous solutions (2% DMSO) of 1a−3a and 1b−3b. At the
end, no significant interactions were observed between the
in the software package HyperChem 7.0). The energies of the
HOMO and LUMO orbitals of 1b−3b were analyzed with
respect to those of the frontier orbitals of the DNA nucleobases
Figure 10. Fluorescence confocal microscopy image of 3T3 cells incubated with compound 3b (106.61 μM, blue color, left), Draq5 (red-color
stained nuclear DNA, middle), and the superimposed images (right). The incubation time was 3 h.
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and reported in Table 2. The energy differences between the
HOMO of the different nucleobases and the LUMO of the
different salophen complexes, ΔE, are also included for clarity.
We should point out that this is an approximation because
DNA contains stacks of nucleobases rather than individual
nucleobases, and the HOMO and LUMO energies could be
modified accordingly. In fact, stacks of guanine bases have been
found to have an even lower ionization potential than individual
guanine bases, and the presence of metal complexes could have
an important electronic role of pulling electrons from the
coordinated aromatic ligands, making them increasingly
electron-deficient and therefore much more likely to be
involved in substantial π−π interactions with G-quartets and
intercalation between adjacent DNA base pairs. When a second
species is added, it can compete with EB for DNA binding sites,
causing a decrease in the fluorescence intensity. The quenching
is due to the decrease of the number of available DNA binding
sites. Therefore, EB is used as a common fluorescent probe for
DNA structure that is able to provide information about the
31
mode and process of single species binding to DNA.
Fluorescence titrations showed that only 3b can replace
some of the EB binding sites, inducing a quenching effect
(Figures 4, S8, and S9). The observed quenching of DNA−EB
in the presence of 3b follows the Stern−Volmer equation
I0
1
1
= 1 + K [Q]
q
nucleobases.
I
As shown in Table 2, the lowest energy HOMO and LUMO
orbitals of the salophen complexes are those of nitro derivative
where I
and I represent the emission intensity in the absence
and presence of quencher, respectively, K is a linear Stern−
q
0
3
b. The complex acts as a Lewis acid; therefore, the suggested
interaction with DNA can be associated to a charge-transfer
process from the DNA bases to the salophen complex. From
the data in Table 2, it appears that purine nucleobases favor this
process because the ΔE (HOMO (nucleobase) → LUMO
Volmer quenching constant, and [Q] is the quencher
concentration (i.e., the concentration of the zinc-salophen
derivatives in our study). This equation is considered for static
quenching because this is the expected process for the
displacement of EB by the salophen complexes. Hence, the
(3b)) are the lowest (ΔE values G and A for 3b in Table 2). In
particular, guanine should provide the lowest transition barrier,
which is in agreement with the fact that its ionization potential
obtained I /I versus [Q] plot follows a linear pattern that
0
3
−1
allowed us to calculate a K value of 9.4 × 10 M , which is the
q
29
is the lowest among the four nucleobases. Therefore, it is the
complex that is more susceptible to involvement in the charge-
transfer interaction that contributes to the stabilization of the
same value previously reported for similar Zn-salophen
19
complexes.
Absorption and emission experiments are indicative of a
likely intercalation mode of the complex into double helix ct-
DNA. Nevertheless, the association constant between DNA and
EB is two orders of magnitude higher than the one calculated
for our complex, showing that EB is a stronger intercalating
agent.
3
b−nucleobase adduct. Consequently, the calculations are in
agreement with the AFM data that indicate that 3b was the
only complex that strongly interacts with DNA.
We also measured the redox potentials of complexes 1b−3b.
0
The values are E = −0.71, −0.59, and −0.35 V, respectively.
From these data, it suggests that the strongest oxidant is nitro-
The different values obtained for K and K are due to the
b
q
derivative 3b, which should be involved in the highest net redox
different chemical processes under study. From UV−vis
spectroscopic titrations we can calculate the association
constants between the compounds and DNA, whereas
spectrofluorimetric titrations let us to obtain the quenching
constant for the EB-displacement process. Thus, the process
under study is characterized by an apparent constant defined by
the equation Kapp = K /K [EB]. By taking into consideration
0
0
potential value of these electronic processes, E (E
E (nucleobase) + E red(complex)).
ox
=
0
0
DNA-Binding Studies by Absorption and Emission
Titrations. Absorption spectra of the complexes were recorded
after the addition of increasing amounts of DNA (Figures 3, S6,
and S7). A decrease in molar absorptivity (hypochromism) was
observed for the three complexes, and a slight bathochromism
ass
EB
7
−1
that KEB is 1 × 10 M , the corresponding K values obtained
b
5
−1
(
ca. 4 nm) was observed for 3b.
The binding affinity constants, K , of the complexes with
from the luminescence experiments are ca. 1.5 × 10 M ,
which is exactly the same value calculated by the absorption
experiment.
b
DNA could be determined quantitatively using the following
3
0
equation
The experiments were not carried out with the ligands
because no significant interactions were observed with any of
them in the AFM studies.
[
DNA]/(ε − ε ) = [DNA]/(ε − ε ) + 1/K (ε − ε )
a
f
b
f
b
b
f
where [DNA] is the concentration of ct-DNA in base pairs, and
Cytotoxicity Studies. Before studying the in vitro
internalization of the compounds using 3T3 cells and the
interactions of the complexes with DNA in these assays, it was
necessary to assess the toxicity of the compounds. Cytotoxicity
was evaluated by MTT experiments following a protocol
detailed in the Experimental Section. Incubated cells were
ε , ε , and ε are the apparent extinction coefficients
a
f
b
corresponding to A /[complex], which is the extinction
obs
coefficient for the unbound compound and the extinction
coefficient for the compound in the fully bound form,
respectively.
5
The binding constants for 1b−3b were calculated to be 8.8 ×
seeded in 96-well plates at a 10 cell/mL density for 24 h.
4
5
5
−1
1
0 , 1.3 × 10 , and 1.5 × 10 M , respectively. The values are
Seeded cell wells without MTT were used as blank experiments
to evaluate the effect (cell binding) of the complexes on the
3T3 cells. After 24 h of incubation, the cells were analyzed by
fluorescence microscopy using a UV2A filter.
Fluorescence-microscopy images of the cells incubated with
two different concentrations of the complexes show that an
increase in the concentration of the salophen compound does
not seem to reduce the number of incubated cells; therefore, no
significant cytotoxicity was expected for any of them (Figure 5).
comparable with those reported recently by us for similar Zn-
5
5
−1 19
salophen complexes (1.7 × 10 and 1.6 × 10 M ).
Further experiments were carried out using EB, a known
intercalating agent, to establish if the interactions with DNA
(especially in the case of 3b) can be due to an intercalation
mode. EB is nonemissive in phosphate buffer (pH 7.2) because
of fluorescence quenching of free EB by solvent molecules, but
it emits intensely in the presence of DNA as the result of its
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The cytotoxicity has been evaluated with respect to negative
control DMSO (1%) using SDS as a positive control (with a
growth-inhibition value above 90%). It is most likely that above
a 70 μM concentration the cytotoxicity does not originate
exclusively from the interaction between the metal-containing
compounds and the DNA but also from the excess of 1b and
of the substituents can be used to modulate molecular
recognition processes. Work along this line is in progress.
Preliminary Cellular-Uptake Studies. Cellular uptake
studies were therefore carried out using 3T3 fibroblasts
(connective tissue cells). After cells were incubated with 1b−
3b (71.17, 58.54, and 66.63 μM, respectively), the cultures were
washed, and the cells were observed under an inverted
fluorescence microscopy. Internalization seems to become
evident at 1.5 h (Figure 8), but this observation is not
homogenously extended to all of the cells.
2b that covers the cells (outside the cell membrane, Figure
S10), which does not allow for proper growth. A very large
amount (>500 μM) of 3b is required to induce cell death. By
considering that for concentrations higher than ca. 60 μM the
compounds do not seem to induce cells death, the growth-
inhibition effects observed may be directly due to a cell-
recovery effect by the compounds (outside the cell membrane)
and not to interactions between the complexes and organelles
or DNA. The growth-inhibition percentages obtained at
different concentrations allowed the calculation of the 24 h
IC50 values using a Probit regression method included in the
SPSS (Statistical Package for the Social Sciences) program,
version 15 for Windows (Table 3 and Figure S11). These
values are calculated from 19 independent repetitions.
DNA Binding. Compounds 1b−3b were incubated with
fixed nucleoids in an agarose matrix in at concentrations of
113.88, 93.66, 106.61 μM, respectively. The use of lower-
concentration solutions seems to indicate nucleoid−com-
pounds interaction, although it was difficult to localize them
within the cells. DAPI (4′,6-diamidino-2-phenylindole) was
also included in each sample in a clearly lower concentration (5
μg/mL) because it is a well-known fluorescent stain that acts as
a DNA minor-groove binder and DNA intercalator (exhibiting
an emission at 461 nm when it is excited at 358 nm), and its
presence allowed us to identify the presence of the complexes
in the cells and to obtain better microscopy images because
more intensely DAPI-stained regions indicate more condensed
The estimated IC50 values are lower (mainly for 1b and 2b)
than those previously obtained for similar Zn-salophen
derivatives containing two isopropyl groups in the 3,3′
19
33
positions (1.29 and 1.85 mM in water). An increase in the
electron-withdrawing effect (poorer electronic character of the
salophen complex ligand) leads to an increase of the calculated
IC50 (less cytotoxicity).
DNA and vice versa. Although these experiments let us to
detect the presence of all three salophen complexes, they do
not interact with the nucleus in the same way. Fluorescence
microscopy images allowed us to identify the presence of 1b
and 3b in the nucleus as small green dots (Figures 9 and S11),
but the localization of 2b is not so clear (Figure S12).
However, the IC values of the parent ligands (1a−3a) have
5
0
been also calculated for comparison purposes, and the
corresponding values are 158.71, 160.99, and 595.96 μM,
respectively. All of these values are clearly different from those
of the corresponding complexes, indicating the relevant role of
the metal on the cytotoxic activity and the different effect of the
nitro substituent with respect to that of the methoxy and
bromide substituents.
To analyze further these data, colocalization living-cell
internalization was investigated using Draq5 as the fluoro-
chrome. This is a far-red emitting fluorescent DNA dye is used
for permeabilized and fixed live-cell analysis that can be used in
combination with other common fluorophores. In the present
case, the Draq5 staining proved to be appropriate because 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 fluorescence confocal microscopy at 688 nm for Draq5 and
at 500 nm for the complexes. Interestingly and in agreement
with previous data (e.g., AFM, luminescence, redox potentials,
etc.), we observed the presence of complex 3b in the nucleus
Genotoxicity: Comet Assays. Comet assays were carried
out with 3T3 fibroblast cells to detect the interaction of the
complexes with DNA in the cells (experiments have been
undertaken at different concentrations that are always below
the calculated IC ).
50
This experiment is commonly applied to detect DNA
fragmentation in individual cells, which allows the assessment
of genotoxic effects or antigenotoxic (protective) activity
(
Figures 10 and S13). In contrast, no traces of compounds 1b
and 2b were detected. Images of cells stained by zinc complex
b (Figure 10, left) and with Draq5 (Figure 10, middle) are
32
against a clastogenic challenge (i.e., chromosome breakage).
Nondamaged cells will exhibit a spherical shape, whereas
fragmented nucleoids will migrate, forming a tail with a comet
shape. The nucleoid damage was quantified as the percentage
of a cells fluorescence observed in the tail. A comparison of the
results with a genotoxic positive control (400 μM solution of
methyl methanesulfonate, MMS) and with DMSO (1%) as a
vehicle control revealed that the nucleoid is clearly not
damaged because the spherical shape of the nucleoid is not
modified (Figures 6 and 7).
3
superimposed in Figure 10, right and indicate that the red
staining resulting from Draq5 is colocalized with that
corresponding to salophen complex 3b, which is a definitive
proof that this is the only complex of this series that interacts
with DNA.
CONCLUSIONS
■
The corresponding images displayed in Figures 6 and 7
clearly show that the nucleus is stained by DAPI and that the
zinc complexes seem to enter the cells but without binding
specifically to the nucleus. The observed effect is slightly
inferior to that previously obtained for similar Zn-salophen
The present study of the biological properties of a series of
salophen Zn(II) complexes clearly shows that the Lewis acid
character of the complexes strongly affects the interaction with
DNA in in vitro studies. Variation in the electronic properties
of the substituents at the 5,5′ positions seems to modulate the
strength of such interactions. In particular, the presence of the
electron-withdrawing nitro substituents, increasing the electro-
philic character of the metal center, generates the strongest
interaction with plasmid DNA. Similar studies performed on
the uncomplexed salophen ligands do not evidence any
1
9
complexes.
All of these results demonstrate a clear effect of the electronic
character of the substituent in in vitro studies, but according to
the first studies carried out in cells, this effect does not seem to
be observed in this biological medium. Nevertheless, the effect
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Inorganic Chemistry
Article
significant interaction even in the case of the nitro derivative,
further highlighting the role played by zinc.
(7) Muller, J. G.; Paikoff, S. J.; Rokita, S. E.; Burrows, C. J. J. Inorg.
Biochem. 1994, 54, 199.
(
8) Routier, S.; Vezin, H.; Lamour, E.; Bernier, J. L.; Catteau, J. P.;
Bailly, C. Nucleic Acids Res. 1999, 27, 4160.
9) Gust, R.; Ott, I.; Posselt, D.; Sommer, K. J. Med. Chem. 2004, 47,
837.
10) Reed, J. E.; Arola Arnal, A.; Neidle, S.; Vilar, R. J. Am. Chem. Soc.
006, 128, 5992.
11) Campbell, N. H.; Abd Karim, N. H.; Parkinson, G. N.;
Semiempirical calculations together with cyclic voltammetry
measurements suggest that this interaction can be also related
to the lowest ΔE (HOMO (nucleobase) → LUMO (3b)) and
thus a more favored charge-transfer process. Hence, our studies
indicated that interaction with DNA can be modulated by
changing the substituents of the salophen skeleton.
However, it has been observed that none of the complexes
present significant cytotoxicity in cells. They are able to enter
the cells, and 3b in particular goes into the nucleus. Thus, they
can be regarded as potential candidates for use as fluorescence
markers.
(
5
(
2
(
Gunaratnam, M.; Petrucci, V.; Todd, A. K.; Vilar, R.; Neidle, S. J. Med.
Chem. 2012, 55, 209.
(12) Ansari, K. I.; Grant, J. D.; Woldemaram, G. A.; Kasiri, S.;
Mandal, S. S. Org. Biomol. Chem. 2009, 7, 926.
(
13) Woldemaram, G. A.; Mandal, S. S. J. Inorg. Biochem. 2008, 102,
40.
14) Ansari, K. I.; Grant, J. D.; Kasiri, S.; Woldemaram, G. A.;
7
(
ASSOCIATED CONTENT
Supporting Information
■
Shrestha, B.; Mandal, S. S. J. Inorg. Biochem. 2009, 103, 818.
(15) Dalla Cort, A.; De Bernadin, P.; Forte, G.; Yafteh Mihan, F.
Chem. Soc. Rev. 2010, 39, 3863.
*
S
Absorption spectra of compounds 1a−3a in ethanol; calculated
orbitals involved in the lowest energy absorption transitions of
compounds 1a−3a; AFM image of pBR322 plasmid DNA
alone and incubated with 1a−3a; electronic absorption spectra
of complexes 1b and 2b in the absence and presence of
increasing amounts of DNA; emission spectra of ethidium
bromide (EB) bound to DNA in the absence and presence of
increasing amounts of 1b and 2b; fluorescence-microscopy
images of 3T3 cells incubated with compound 2b; cytotoxicity
dose responses of metal complexes in 3T3 cells for compounds
(16) Hille, A.; Ott, I.; Kitanovic, A.; Kitanovic, I.; Alborzinia, H.;
Lederer, E.; Wo
Bischof, C.; Schatzschneider, U.; Gust, R. J. Biol. Inorg. Chem. 2009, 14,
11.
17) Ansari, K. I.; Kasiri, S.; Grant, J. D.; Mandal, S. S. Dalton Trans.
009, 8525.
18) Alpturk, O.; Rusin, O.; Fakayode, S. O.; Wang, W.; Escobedo, J.
O.; Warner, I. M.; Crowe, W. E.; Kral, V.; Pruet, J. M.; Strongin, R. M.
Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 9756.
(19) Brissos, R.; Ramos, D.; Lima, J. C.; Yafteh Mihan, F.; Borras
̈ ̈
lfl, S.; Metzler-Nolte, N.; Schafer, S.; Sheldrick, W. S.;
7
(
2
(
̈
́
̀
, M.;
1
b−3b; fluorescence microscopy image of nucleoids incubated
de Lapuente, J.; Dalla Cort, A.; Rodríguez, L. New J. Chem. 2013, 37,
with compound 2b and 3b; fluorescence confocal microscopy
image of 3T3 incubated cells with Draq5 and compounds 1b
and 2b. This material is available free of charge via the Internet
at http://pubs.acs.org.
1046.
(
20) Arola-Arnal, A.; Benet-Buchholz, J.; Neidle, S.; Vilar, R. Inorg.
Chem. 2008, 47, 11910.
21) Hai, Y.; Chen, J.-J.; Zhao, P.; Lv, H.; Yu, Y.; Xu, P.; Zhang, J.-L.
Chem. Commun. 2011, 47, 2435.
22) Pascu, S. I.; Waghorn, P. A.; Conry, T. D.; Lin, B.; Betts, H. M.;
(
(
AUTHOR INFORMATION
Corresponding Author
E-mail: antonella.dallacort@uniroma1.it (A.D.C.); E-mail:
■
*
Dilworth, J. R.; Sim, R. B.; Churchill, G. C.; Aigbirhio, F. I.; Warren, J.
E. Dalton Trans. 2008, 2107.
(
23) Gao, Y.; Wu, J.; Li, Y.; Sun, P.; Zhou, H.; Yang, J.; Zhang, S.; Jin,
B.; Tian, Y. J. Am. Chem. Soc. 2009, 131, 5208.
24) Ansari, K. I.; Kasiri, S.; Grant, J. D.; Mandal, S. S. J. Biomol.
Screening 2011, 16, 26.
25) HyperChem, version 7.5; Hypercube, Inc.: Gainsville, FL.
laura.rodriguez@qi.ub.es, Tel: +34 934039130, Fax: +34
34907725 (L.R.).
(
9
Notes
(
The authors declare no competing financial interest.
(26) Standard Guide for Determining DNA Single-Strand Damage in
Eukaryotic Cells using the Comet Assay; ASTM Technical Bulletin
E2186; ASTM International: West Conshohocken, PA, 2003.
ACKNOWLEDGMENTS
The support and sponsorship provided by COST Action
CM1005 is acknowledged. We are also grateful to the
■
(27) Singer, A. L.; Atwood, D. A. Inorg. Chim. Acta 1998, 277, 157.
(28) Cano, M.; Rodríguez, L.; Lima, J. C.; Pina, F.; Dalla Cort, A.;
Pasquini, C.; Schiaffino, L. Inorg. Chem. 2009, 48, 6229.
Ministerio de Ciencia e Innovacio
́
n of Spain (project
cia e Tecnologia
(29) Mc Govern, D. A.; Doorley, G. W.; Whelan, A. M.; Parker, A.
CTQ2012-31335) and Fundaca para a Cien
̧
o
̃
̂
W.; Towrie, M.; Kelly, J. M.; Quinn, S. J. Photochem. Photobiol. Sci.
2009, 8, 542.
(30) Wolfe, A.; Shimer, G. H., Jr; Meehan, T. Biochemistry 1987, 26,
6392.
of Portugal (PTDC/QUI-QUI/112597/2009). We thank Drs.
Lidia Bardia and Julien Collombelli from IRB-Barcelona for
fruitful discussions.
(
31) Lippard, S. J. Acc. Chem. Res. 1978, 11, 211.
32) Barenys, M.; Macia, N.; Camps, L.; de Lapuente, J.; Gom
ez-Linares, J.; Borras, M.; Rodamilans, M.; Llobet, J.
M. Toxicol. Lett. 2009, 191, 40.
33) Ansari, K. I.; Mishra, B. P.; Mandal, S. S. Biochim. Biophys. Acta
008, 1779, 66.
(
́
ez-
REFERENCES
■
Catalan, J.; Gonzal
́
̀
(
1
1) Jacobsen, E. N.; Zhang, W.; Guler, M. L. J. Am. Chem. Soc. 1991,
13, 6703.
(
2
(
2) Haak, R. M. S.; Wezenberg, J.; Kleij, A. W. Chem. Commun. 2010,
6, 2713.
3) Venkataramanan, N. S.; Kuppuraj, G.; Rajagopal, S. Coord. Chem.
Rev. 2005, 249, 1249.
4) (a) Dalla Cort, A.; Mandolini, L.; Schiaffino, L. Chem. Commun.
005, 3867. (b) Dalla Cort, A.; Pasquini, C.; Schiaffino, L. Supramol.
4
(
(
2
Chem. 2007, 19, 79. (c) Dalla Cort, A.; Mandolini, L.; Schiaffino, L. J.
Org. Chem. 2008, 73, 9439.
(
(
5) Bhattacharya, S.; Mandal, S. S. Chem. Commun. 1996, 1515.
6) Czlapinski, J. L.; Sheppard, T. L. J. Am. Chem. Soc. 2001, 123,
8618.
9
253
dx.doi.org/10.1021/ic4004356 | Inorg. Chem. 2013, 52, 9245−9253