Copper(II) N, N, O-Chelating Complexes as Potential Anticancer Agents

Article abstract of DOI:10.1021/acs.inorgchem.0c02932

Three novel dinuclear Cu(II) complexes based on a N,N,O-chelating salphen-like ligand scaffold and bearing varying aromatic substituents (-H, -Cl, and -Br) have been synthesized and characterized. The experimental and computational data obtained suggest that all three complexes exist in the dimeric form in the solid state and adopt the same conformation. The mass spectrometry and electron paramagnetic resonance results indicate that the dimeric structure coexists with the monomeric form in solution upon solvent (dimethyl sulfoxide and water) coordination. The three synthesized Cu(II) complexes exhibit high potentiality as ROS generators, with the Cu(II)/Cu(I) redox potential inside the biological redox window, and thus being able to biologically undergo Cu(II)/Cu(I) redox cycling. The formation of ROS is one of the most promising reported cell death mechanisms for metal complexes to offer an inherent selectivity to cancer cells. In vitro cytotoxic studies in two different cancer cell lines (HeLa and MCF7) and in a normal fibroblast cell line show promising selective cytotoxicity for cancer cells (IC50 about 25 μM in HeLa cells, which is in the range of cisplatin and improved with respect to carboplatin), hence placing this N,N,O-chelating salphen-like metallic core as a promising scaffold to be explored in the design of future tailor-made Cu(II) cytotoxic compounds.

Full text of DOI:10.1021/acs.inorgchem.0c02932

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Copper(II) N,N,O‑Chelating Complexes as Potential Anticancer
Agents
̃
Quim Pena, Giuseppe Sciortino, Jean-Didier Maréchal, Sylvain Bertaina, A. Jalila Simaan, Julia Lorenzo,
Merce Capdevila, Pau Bayón, Olga Iranzo,* and Oscar Palacios*
̀
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ABSTRACT: Three novel dinuclear Cu(II) complexes based on a N,N,O-chelating salphen-like ligand scaffold and bearing varying
aromatic substituents (−H, −Cl, and −Br) have been synthesized and characterized. The experimental and computational data
obtained suggest that all three complexes exist in the dimeric form in the solid state and adopt the same conformation. The mass
spectrometry and electron paramagnetic resonance results indicate that the dimeric structure coexists with the monomeric form in
solution upon solvent (dimethyl sulfoxide and water) coordination. The three synthesized Cu(II) complexes exhibit high potentiality
as ROS generators, with the Cu(II)/Cu(I) redox potential inside the biological redox window, and thus being able to biologically
undergo Cu(II)/Cu(I) redox cycling. The formation of ROS is one of the most promising reported cell death mechanisms for metal
complexes to offer an inherent selectivity to cancer cells. In vitro cytotoxic studies in two different cancer cell lines (HeLa and
MCF7) and in a normal fibroblast cell line show promising selective cytotoxicity for cancer cells (IC50 about 25 μM in HeLa cells,
which is in the range of cisplatin and improved with respect to carboplatin), hence placing this N,N,O-chelating salphen-like metallic
core as a promising scaffold to be explored in the design of future tailor-made Cu(II) cytotoxic compounds.
Tumors contain a more reducing environment with respect to
healthy tissues. This is based on what is known as “the
Warburg effect” and consequence of the fact that cancer cells
do primarily generate energy by an atypical aerobic glycolysis
pathway.10,11 This abnormal metabolic process (instead of the
usual oxidative phosphorylation) induces an imbalanced redox
homeostasis inside cancer cells, leading to an enhanced
intracellular ROS production.12 Consequently, the interference
with cellular redox homeostasis arises as an attractive and
promising target for chemotherapy. Cancer cells exhibit
abnormal levels of ROS, and they show higher vulnerability
INTRODUCTION
■
Metals and their inorganic complexes show an enormous
versatility in front of strictly organic compounds for the
development of therapeutic agents. The possibility of having
several oxidation states, different coordination numbers, and
diverse geometries gives rise to a broader spectrum of tuneable
properties.1 Among them, Cu complexes have become
promising alternatives for cancer treatment during the two
last decades.2−5 Copper is a physiological metal, being widely
present in many biomolecules and playing a remarkable role in
a diversity of biochemical processes because of its interesting
Cu(II)/Cu(I) redox pair.6 In fact, one of the main
potentialities of Cu as an antiproliferative agent lies in its
capability to form reactive oxygen species (ROS) inside the
cells. The generation of these entities (H2O2, O2•−, HO•, etc.)
is not only reported to damage DNA but also to offer a
putative discrimination between healthy and cancer cells.7,8
The lack of selectivity in cancer therapy has always been a
downside in this field, giving rise to severe side effects.9
Received: October 2, 2020
Published: February 17, 2021
https://dx.doi.org/10.1021/acs.inorgchem.0c02932
© 2021 American Chemical Society
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Scheme 1. General Synthesis of the N,N,O-Chelating Salphen-like Ligands H2L1 (R = H), H2L2 (R = Cl), and H2L3 (R = Br)
and of Their Corresponding Cu(II) Complexes C1−C3
Figure 1. SQUID data obtained for complex C3: (A) susceptibility measured at 1 T. Red curve is the best fit for a dimeric complex, (B)
magnetization measured at 2 K showing a fitting with the theoretical model for 2 coupled spins, and (C) ACmagnetic susceptibility. (D)
Comparison of the magnetic susceptibility of C1−C3 complexes.
to ROS level changes than healthy cells do; therefore, the
alteration of those levels may be a unique opportunity to
selectively target cancer cells.7,8 There is, hence, high potential
for the development of bioreducible metal complexes.
Up to date, several Cu(II) complexes have been reported to
be redox-active,5,13−15 and indeed, some structure−activity
relationships have been reported between the redox behavior
of N-donor aromatic Cu(II) complexes and their ROS-
mediated cytotoxicity.16−18 In particular, Schiff-based Cu(II)
complexes have attracted attention from researchers in this
field and have been reported to show interesting cytotoxicity
toward cancer cells and DNA cleavage.15,19−21 Not many
Cu(II) complexes with N,N,O-chelating Schiff base ligands
have been evaluated in cancer cells,22 in contrast to Cu(II)
N,N,S-chelated thiosemicarbazone and bis(thiosemicarbazone)
complexes.23−27
be able to generate high ROS levels in cells. This should lead
to enhanced toxicity toward cancer cells with respect to healthy
ones. The impact of halogenated substituents has also been
evaluated and is discussed here. Speciation and the putative
active species in solution are discussed hereby from a
theoretical approach, and the mechanism of action is
thoroughly evaluated and related to the redox behavior of
the Cu(II) complexes.
RESULTS AND DISCUSSION
■
This work is based on the design of a basic scaffold ((E)-N-(2-
(2-hydroxybenzylideneamino)phenyl)acetamide, H2L1),
which specifically intends to chelate Cu(II) in a tridentate
fashion having a fourth labile in-plane coordination position,
with the idea of biologically attaining a fast Cu(II)/Cu(I)
redox cycle.
Here, we describe the synthesis, characterization, and
evaluation of the biological activity of three novel Cu(II)
complexes bearing N,N,O-chelating salphen-like ligands as
potential antitumoral agents. The aim is to obtain biologically
accessible Cu(II)/Cu(I) redox cycling systems, which would
Synthesis and Characterization of the Ligands and
Their Corresponding Copper(II) Complexes. The three
N,N,O-chelating salphen-like ligands (H2L1, H2L2, and H2L3,
Scheme 1) were synthesized based on a condensation reaction
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Table 1. Experimental JCu−Cu (cm−1) and g-Factors
Obtained from SQUID Measurements in the Solid State for
Complexes C1−C3
between the mono-protected benzene-1,2-diamine precursor
(1) and the corresponding salicylaldehyde precursor. Pure
ligands were obtained by column chromatography purification.
Characterization data are reported in the experimental section
and Supporting Information (Figures S1−S3).
JCu−Cu (cm−1
)
g
C1
C2
C3
3.5
5.7
8.7
2.19
2.15
2.19
Mono-protected diamine (1) was obtained following the
procedures reported in the literature.28 For H2L1, commer-
cially available 2-hydroxybenzaldehyde was used as the starting
material. To obtain H2L2 and H2L3, halogen derivatization
was carried out on the para-hydroxy position of the starting
material 2-hydroxybenzaldehyde following the reported
procedures. Chlorination was carried out under mild
conditions using N-chlorosuccinimide (NCS) and an acid
catalyst.29 This reaction provided lower yields than those
found with common chlorinating agents (Cl2 or sulfuryl
chloride), but a cleaner reaction.30 The final 4-chloro-2-
hydroxybenzaldehyde (2) precursor was purified through
column chromatography. Alternatively, bromination of the
starting material was carried out using standard procedures
with Br2, to obtain compound 3.
Complexation of pure H2L1−H2L3 was carried out using
Cu(OAc)2 as the metal precursor salt (Scheme 1). The use of
the Cu(II) acetate salt allowed deprotonating −OH and −NH
at once. Complexes (C1−C3, Scheme 1) were isolated as
brownish powders by precipitation from the reaction media. In
all the cases, the solubility of the complexes was very poor in
the common organic solvents, especially for C2 and C3, even if
this can be improved by the use of coordinating solvents such
as dimethyl sulfoxide (DMSO) and dimethylformamide.
The data recorded for the complexes (Experimental Section,
Figure S4 and Table S1) suggest a 1:1 ligand to metal
stoichiometry in the solid state (elemental analysis, see
Experimental Section), with both −OH and −NH groups
deprotonated and in the absence of any additional ligand or
counterion. The IR bands of H2L (Figures S1C−S3C)
assigned to the stretching of both O−H (phenol) and N−H
(amide) bonds at 3500−3300 cm−1 as well as those related to
the bending mode of the N−H bond (about 1660 cm−1, scissor
bending) and of the O−H (1500 cm−1) bond have disappeared
in the corresponding Cu(II) complexes (Figure S4A). This
confirms the deprotonation of both the amide (−NH) and the
phenol (−OH) groups upon metalation. No peaks for
additional counterions or ligands have been observed. The
Cu(II) coordination sphere in the solid state is composed of
the N,N,O-chelating salphen-like ligand and a fourth oxygen
from the carbonyl of the amide group (CO) of the second
entity of the dimer.
obtained points to an analogous conformation for all three
complexes. Isothermal magnetization (T = 2 K) confirms the
presence of two coupled spins (Figures 1B and S5). Finally,
AC-susceptibility measurements (Figures 1C and S5) indicate
the absence of long-range order that would be due to the
presence of polymers.
The integrity of the species was evaluated in the DMSO
solution. The dimeric structure of C1−C3 has been confirmed
by the observation of the corresponding peaks in HR-ESI-MS
(m/z 631.0456, 700.9661, and 786.8678, respectively, Figure
S4A). These data indicate that the nuclearity is at least partially
maintained in solution. At the same time, peaks attributed to
the corresponding mononuclear species were also found
(Figure S4C), suggesting a solvent-dependent process
involving partial breakage of the dinuclear species and
coordination of the DMSO solvent in the fourth binding site
of the metal coordination sphere. Electron paramagnetic
resonance (EPR) shows the presence of a single EPR-active
Cu(II) species in solution for all the three complexes (Figure
S4B and Table S1). The observed EPR signals for C1−C3 in
the DMSO solution are typical for Cu(II) monomeric species
in square−pyramidal-derived geometries with the single
2
2
electron in dx −y orbitals (g∥ > g⊥ > ge). From the analysis of
the EPR parameters derived from simulations (A and g-
tensors), it appears that the three complexes mainly exist in a
nonsignificantly distorted square-planar or square-pyramidal
geometries with the N2O2 coordination in the equatorial plane,
as expected based on the N,N,O-chelating ligands and on
solvent coordination. In order to provide further insights into
the monomer/dimer coexistence of the complexes in DMSO,
quantification of the Cu(II) mononuclear species through
double integration of the EPR spectra of C1, as the model
compound, was carried out at different time points (Table S2).
EPR spin quantification data demonstrate that the dissolution
of C1 in DMSO gives rise to 30% of the mononuclear Cu(II)
signal, thereby pointing to the presence of EPR-silent
magnetically coupled dinuclear species. The Cu(II) signal
evolves over time reaching about 50% of the mononuclear
species after 24 h. This confirms the dimeric cleavage process
in a solvent and time-dependent manner. In addition,
increasing the ionic strength by the addition of salts seems
to slightly contribute to the cleavage of the dimeric form
(Table S2) reaching up to 60% after several days. The overall
data are thus in concordance with those of ESI-MS analysis
(Figure S4C), and suggest the coexistence of both dimer and
monomer species in solution.
The magnetic properties of the complexes C1−C3 were
studied using a conventional SQUID magnetometer. The
results for the three complexes are very similar. The results for
C3 are presented in Figure 1, while those for C1 and C2 are
given in the Supporting Information (Figure S5). Figure 1A
shows the isofield (H = 1 T) χT as a function of temperature
(T). The red curve is the best fit using the Bleaney−Bowers
equation of a coupled S = 1/2 dimer.31 The fit shows a
ferromagnetic coupling for the three complexes, with small
exchange coupling constant (J) values ranging from 3.5 to 8.7
cm−1 and a g-factor of 2.15 to 2.2 coherent with the presence
of Cu(II) (Table 1) and the absence of monomers. The
increase in the J values (Table 1) with the increase of the size
of the R substituent (Scheme 1) suggests that the
functionalization influences the Cu(II)−Cu(II) distance in
the dinuclear structure. Additionally, the very similar g values
DFT Studies for the Evaluation of the Active Species
in Solution. Density functional theory (DFT) computational
studies of the parent ligand H2L1 and its corresponding Cu(II)
complex C1 have been carried out to model and rationalize the
speciation in solution of C1−C3 complexes. Two solvents
were chosen to be computed: DMSO to compare with the
beforehand obtained experimental values and water because of
its biological relevance. Dimeric and monomeric Cu(II)
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Figure 2. (A−C) Optimized geometry of the three main conformations of the dimeric complex [CuII(L1)]2, and the monomeric species (D)
[CuII(L1)(H2O)] and (E) [CuII(L1)(DMSO)]. Cu−Cu distances are reported in angstrom. The deviation from square-planar toward tetrahedral
geometry is also reported in degrees as the dihedral angle (θ) between the fourth equatorial donor atom and the donors of the tridentate chelating
L1 ligand (D−Ncis−O−Ntrans).
Table 3. Simulated JCu−Cu (cm−1) and ΔG Values for the
complexes formed by the ligand H2L1 (Scheme 1) were
Different Conformations of the Dimeric [CuII(L1)]2
Species
examined and their proposed structures were simulated:
a
[CuII(L1)]2, [CuII(L1)(H2O)], and [CuII(L1)(DMSO)]
(Figure 2). Concerning the dimeric species, among the seven
different conformations considered with relative orientation of
Cu−Cu
JCu−Cu
[CuII(L1)]2 (cm−1
distance
(Å)
ΔGDMSO/ΔGaq
b
(kcal·mol−1
)
L1 ligands and the coordination position (axial or equatorial)
of their donors, only three have been characterized as minima
in the potential energy surface (Figure 2A−C).
In all the cases, the Cu(II) centers present a square-planar
arrangement with different grades of distortion, ranging from
almost pure square-planar geometries for [CuII(L1)(H2O)]
(170.0°, Figure 2D) to highly distorted for [CuII(L1)]2
(114.8° in conformation C, Figure 2C).
)
coupling
conf. A
conf. B
conf. C
12.0
−43.2
22.3
ferromagnetic
antiferromagnetic
ferromagnetic
2.741
4.128
2.766
0.0/0.0
0.7/7.4
5.3/9.7
a
b
J has been determined with a reported method.33,34 DFT level using
B3LYP-D3 combined with the basis-set def2-TZVP for the main
group elements and the quadruple-ζ def2-QZVP basis set for Cu.
The Gibbs energy calculations for the dissociation reactions
of the dimeric forms to monomeric species (Table 2) suggest a
experimentally (Table 1). Computed values show that only
conformation B (Figure 2B) has an antiferromagnetic coupling
(J = −43.2 cm−1). In contrast, for conformations A and C
(Figure 2A,C), the predicted interaction is ferromagnetic (12.0
and 22.3 cm−1, respectively). The reason behind the different
magnetic behaviors can be found from the Cu−Cu distances
between the two radical spins, that is, ∼2.7 Å in conformations
A and C allowing a ferromagnetic coupling, versus 4.1 Å in
conformation B, in which the cores are well separated (Figure
2).32 According to the experimental ferromagnetic exchange
couplings for the three complexes (JCu−Cu, Table 1), the most
probable structure of C1−C3 would fit with conformation A,
which shows the lowest Gibbs energy and JCu−Cu values (Table
3).
The EPR parameter simulations for the monomeric species
in DMSO, [CuII(L1)(DMSO)], are in the range of the
experimental results (Table 4). The relative deviation of the
calculated gz and Az values from the experimental ones is
−16.0% for Az and −1.9% for gz. The larger deviation of Az for
[CuII(L1)(DMSO)] must be related to the significant
distortion of the equatorial plane of the Cu(II) ion because
of the coordination of DMSO (θ = 138.4°), and these
differences are common between the computed and exper-
imental parameters, especially on the A tensor.35
Table 2. ΔG Values for the Dissociation of the Dimeric
Species [CuII(L1)]2 to the Monomeric Complex
a
[CuII(L1)(solv)] in Different Solvents
[CuII(L1)]2
solv
ΔEsolv
ΔGsolv
conformation A
H2O
DMSO
H2O
DMSO
H2O
DMSO
−1.8
9.2
−9.5
6.9
−12.6
2.8
−1.8
4.4
−9.2
3.7
−11.4
−1.0
conformation B
conformation C
a
Values reported in kcal mol−1. The corrections of RT ln V (1.89
kcal/mol) and RT ln([solv]/n) were applied. Values computed in the
SMD continuum model for H2O or DMSO.
different behavior depending on the solvent. In water, the
dissociation reaction appears to be favored with ΔGaq from
−1.8 up to −12.6 kcal·mol−1, while in DMSO, data are
consistent with the coexistence of the dimeric and monomeric
forms with ΔGaq from 4.4 to −1.0 kcal·mol−1.
To corroborate the structures obtained and to discriminate
between the three dimeric forms, the J values were computed
in each case to determine the ferro or antiferromagnetic nature
of the interaction between the two unpaired electrons on the
Cu(II) centers (Table 3), and compared with those obtained
The UV−vis vertical excitation has also been computed for
both dimeric [CuII(L1)]2 (conformation A) and monomeric
[CuII(L1)(H2O)] and [CuII(L1)(DMSO)] species and
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Table 4. EPR Parameters Computed (Calc) for the Monomeric [CuII(L1)(DMSO)] Species in the DMSO Medium, and
Comparison with the Experimental (Exp) Values [Relative Deviation (RD) Related to the Experimental Value], Extracted
from Figure S4B and Table S1
a
Aczalc,
a
Aezxp,
species
ARz D
gczalc
gezxp
gRz D
b
b
[CuII(L1)(DMSO)]
154.0
183.4
16.0%
2.201
2.244
1.9%
a
b
Values in 10−4 cm−1. Values recorded in DMSO.
Figure 3. (A) Cyclic voltammograms vs Fc+/Fc (Fc) of C1−C3 in DMSO with 0.1 M TBAP at a scan rate of 100 mV/s. (B) Representation of E1/2
values obtained for C1−C3 placed in the biological redox window at pH 7.36
compared with experimental values (Figures S6 and S7 and
Table S3). Computed MLCT transition bands are in the range
of the experimental ones for the monomeric [CuII(L1)(solv)]
species (Figure S6 and Table S3), while computed Cu(II) d−d
transitions could indeed fit with both forms (dimer and
monomer, Figure S7 and Table S3). The overall results are in
concordance with the EPR values compared previously. Even if
the presence of the dimeric form has been widely
demonstrated, all data point to a significant role of the
monomeric Cu(II) form in the final activity of C1 in solution.
Evaluation of the Potentiality of the Complexes as
ROS Generators. The redox properties of the Cu(II)
complexes were evaluated by cyclic voltammetry (CV)
experiments. CV was carried out with both the ligands
(H2L1−H2L3) and the complexes (C1−C3) in DMSO.
Taking into account that the biological redox window
approximately ranges from −1.1 to 0.2 V versus Fc+/Fc
(values arising from the oxidation and reduction of water at pH
7,36 respectively), we specifically analyzed in detail this region
(Figure 3). H2L1−H2L3 do not show any kind of redox
activity in this specific range. On the contrary, all the Cu(II)
complexes are redox active and the signals observed on the
cyclic voltammograms of C1−C3 have been ascribed to the
Cu(II) ⇄ Cu(I) redox process (Figure 3).
The redox potentials (Table S4) were assigned to the redox
couple Cu(II)/Cu(I) based on bulk electrolysis and EPR
experiments (data not shown). The difference between
cathodic and anodic peaks in C1−C3 cyclic voltammograms
is higher than the theoretical 0.060 V for fully reversible redox
processes (ΔEp ranging from 0.11 to 0.16 V), but in the range
of the ΔEp (0.10−0.12 V) obtained for the ferrocene (Fc+/Fc)
reference compound under the same experimental conditions.
Successive scans were performed, and the lack of signal change
upon the successive collected scans indicates that no
disproportion occurred after cycling between Cu(II) and
Cu(I) in none of the three Cu(II) complexes. The Ipa/Ipc ratio
close to 1 and the calculated ΔEp values (Table S4) suggest a
quasireversible one-electron process. The linear dependence of
the peak currents Ipc and Ipa versus the square root of the scan
rate (ν1/2) is indicative of a diffusion-controlled process
(Figure S8).37
The determined Cu(II)/Cu(I) redox potentials (E1/2 =
−1.07 V for C1 and −1.03 for C2−C3 vs Fc+/Fc, Table S4)
are within the biological range of −1.1 to 0.2 V versus Fc+/Fc
(Figure 3B). The presence of electrowithdrawing groups in C2
and C3 slightly favors the Cu(II) reduction to Cu(I) (Ered
−1.09 and −1.08 V, respectively) compared to C1 (Ered
=
=
−1.15 V) (Table S4). Both the chloro- and bromo-derivatives
have the reduction potential 60 and 70 mV higher than C1.
Despite the fact that halogen groups make Cu(II) more prone
to be reduced to Cu(I), the final E1/2 values for the three
complexes are similar (Figure 3B and Table S4).
In order to characterize the center of the redox process, the
Gibbs energy of the product of the monomeric C1 reduction
process was calculated at the DFT theory level for two
different spin multiplicities: S = 1, corresponding to [CuI(L1)-
(DMSO)]−, and S = 3, accounting for the L1 reduction
forming [CuII(L1•−)(DMSO)]− (Figure S9).18 The obtained
Gibbs free energy value of the reduction on the ligand is 32.7
kcal·mol−1 higher than that on the metal center (Table S5).
This difference highlights that the ligand participation in the
redox process is negligible and the oxidation state of Cu in the
two minima can be described as +II and +I.
The CV results suggest that the C1−C3 complexes can be
thermodynamically reduced by biological redox buffers, and
perform a quasireversible redox process. Therefore, they seem
to be capable of undergoing Cu(II)/Cu(I) redox cycling under
biological conditions. In order to confirm their capability to
biologically undergo Cu(II)/Cu(I) redox cycling, ascorbate
consumption at pH 7.2 was monitored by UV−vis (Figure 4).
Cu(II), in the presence of ascorbate and under aerobic
conditions, catalyzes the generation of ROS.38 Measuring the
consumption of ascorbate at its maximum absorbance (265
nm) in the presence of the Cu(II) complexes provides an idea
of their capability to generate ROS inside cells. In the absence
of any Cu catalyst (DMSO control), no decrease in the
absorbance at 265 nm can be observed (Figure 4), thus
indicating that ascorbate (100 μM) is stable and the medium
does not consume it. In contrast, the presence of a catalytic
amount of free Cu(II) ions (2 μM of CuCl2 addition) clearly
shows a rapid decrease in the absorbance, and ascorbate has
been almost totally consumed after just 20 min. Complex C1
(2 μM concentration added) is able to consume it at similar
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show significant antiproliferative activity, yet bearing a
nontoxic ligand (H2L1). The toxicity of the latter can then
only be attributed to a conjoint contribution between the
ligand H2L1 and the Cu(II) ion, that is, to the entire complex;
and not solely to the simple addition of the Cu(II) ion plus the
ligand toxicities. In the case of C1, this feature may imply an
advantage in terms of drug metabolism because none of the
frameworks that constitute the complex (H2L1 and Cu(II)
ion) do separately exhibit cytotoxicity.
Because of the low solubility in the biological culture
medium exhibited by C2 and, at lesser extent, C3, and
considering the similar IC50 values in both cancer cell lines
with that of C1, the latter was chosen as the model scaffold to
evaluate the cytotoxicity toward normal embryotic fibroblasts
(NIH 3T3), selected as nontumoral cell lines. As observed in
the dose−response cell viability diagram (Figure 5), complex
Figure 4. Consumption of ascorbate (100 μM) mediated by CuCl2
and complexes C1, C2, and C3 in NaCl and Tris−HCl buffer at pH
7.2 (5% DMSO). The four Cu(II) compounds were at a
concentration of 2 μM.
rates than free copper(II) ions do, while C2 and C3 (at the
same concentration as C1) exhibit a slower consumption rate
than that of C1. One possible explanation for the different
consumption rates could be related to solubility issues because
C2 and C3 are less soluble in aqueous media than C1.
Consequently, and based on the overall ascorbate consumption
data, it is expected that C1−C3 can exert some kind of redox-
mediated cytotoxicity through the generation of ROS inside
cells.
Cytotoxicity Assays in Cancer and Normal Cell Lines.
The in vitro antiproliferative activity of the complexes C1−C3
and their corresponding free ligands were determined on
somatic HeLa and MCF7 cancer cell lines (Table 5 and Figure
S10).
Figure 5. Comparison of the dose−response cell viability diagrams of
C1 in HeLa, MCF7, and NIH 3T3 (fibroblasts) cell lines (0−100
μM) at 72 h. The obtained values average at least three independent
experiments.
Table 5. IC50 (μM) Values at 72 h of Complexes C1, C2, C3
and Their Corresponding Ligands in HeLa, MCF7, and NIH
3T3 Cultures, Using CuCl2·2H2O as the Reference
C1 exhibits lower toxicity toward normal fibroblasts with
respect to both HeLa and MCF7 cancer cells. This is
interesting in terms of selective chemotherapy because it
might provide less side effects.
Evaluation of the Interactions of the Complexes
toward DNA. In order to evaluate the effect and interaction of
the complexes C1−C3 with DNA, traditionally considered as
one of the main targets of chemotherapy, several experiments
have been carried out, namely gel electrophoresis, UV−vis,
and/or circular dichroism (CD).
First of all, the cleaving properties of complexes C1, C2, and
C3 were investigated by gel electrophoresis because many
Cu(II) complexes have been reported to induce cell death
through DNA cleavage.5,19,43,44 The conversion of supercoiled
circular plasmid DNA to open DNA forms was followed
(Figure 6) and the obtained results indicate that the three
complexes are only able to partially cleave supercoiled plasmid
DNA (ScdsDNA), leading into a minor band corresponding to
its open circular form (ocDNA, form II). This confirms that
they do not possess prominent cleaving capacity by themselves.
In contrast, the presence of a reductant species, such as
ascorbic acid (a biological reductant), enhances their cleaving
capacity (Figure 6, colored lines), and they are then able to
practically transform all the ScdsDNA into ocDNA and, to a
lesser extent, into its linear form (form III). As already
mentioned, the generation of Cu(I) stimulates the potential
formation of ROS, which have DNA-cleaving abilities.3 In our
particular case, the results clearly point to a redox-dependent
mechanism, triggered by the presence of ascorbic acid, which
a
Compound
compound
HeLa
MCF7
NIH 3T3
C1
C2
C3
H2L1
H2L2
26
25
23 10
≥200
≥50
4
2
30
6
≥100
b
b
b
29
5
≥150
≥200
b
b
b
H2L3
CuCl2·2H2O18,39
≥50
≥200
≥50
≥200
≥200
a
The results shown are representative of at least three independent
b
experiments (N = 3). Experiments were not carried out because of
poor solubility in the cell culture medium. In the case of the
nonassayed complexes, their corresponding ligands were not assayed
either.
The IC50 values obtained in HeLa cancer cells (Table 5 and
Figure S10) for the ligands show that while ligand H2L1
presents poor or negligible toxicity, H2L2 and H2L3 display
significant cytotoxicity. This difference might be attributed to
the presence of the halogen substituents.40,41 Complexes C1,
C2, and C3 exhibit remarkable and dose-dependent cytotox-
icity in both HeLa and MCF7 cells (IC50 about 25 μM, Table
5) when compared to CuCl2 and to the two commercially
available Pt-drugs cisplatin (IC50,72h of 15 μM in HeLa39) and
carboplatin (IC50,72h of 39 μM in HeLa42). Both C2 and C3
are bearing toxic ligands (H2L2 and H2L3), whereas C1 does
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Ao
A − Ao
εG
εG
εH−G − εG Kb[DNA]
1
=
+
·
εH−G − εG
(1)
The plot of the relative variation of the absorbance (Ao/(A
− Ao)) versus the inverse of the DNA concentration (1/
[DNA]) (Figure S11) allows the determination of Kb (Table
6). The Kb values obtained for complexes C1, C2, and C3 are
in the order of 104 M−1, indicating a moderate interaction and
lower than the values around 106 to 107 known for classical and
strong mtallointercalators (DAPI, HOECHST, etc.).48,51,52
In Vitro ROS Generation and Induction of Apoptosis.
The results obtained from the CV studies (Figure 3), ascorbate
consumption experiments (Figure 4), and DNA-cleaving
activity (Figure 6) strongly indicate an oxidative dependent
mechanism of action. In order to confirm the formation of
intracellular ROS in HeLa cancer cells, the 2′,7′-dichloro-
fluorescin diacetate (DCFDA) assay was performed.14,53
DCFDA is a nonfluorescent and permeable dye that, after
cleavage by intracellular esterases and subsequent oxidation by
ROS, generates dichlorofluorescein (DCF), a fluorescent and
nonpermeable compound.
The experiment was performed with C1 as the main scaffold,
and to serve as a proof-of-concept to understand the
mechanism of action of the N,N,O-chelating metallic core.
After 4 h treatment, strong DCF fluorescence, of up to 3-fold
respect to control cells, was observed for C1 (Figure 8),
highlighting the ROS production capabilities of this Cu(II)
complex. The ROS levels of C1 are equivalent to those
produced by the positive control H2O2. On the contrary, H2L1
was not able to increase the ROS levels (Figure 8) with respect
to the control group. This is in concordance with the Cu(II)/
Cu(I) redox potential of C1 (Figure 3) and with the results
obtained for the toxicity of H2L1 and C1 (Table 5).
These results confirm the relationship inferred between the
Cu(II)/Cu(I) redox potential of C1, its ROS production
inside the cells, and the exerted biological activity.
Furthermore, this ROS cell death pathway might explain the
different toxicity profiles observed for C1 in HeLa and MCF7
cancer cells with respect to normal cell lines (NIH 3T3)
(Figure 5). Taking into account that cancer cells have higher
radical levels than healthy ones, the production of ROS might
appear as a differentiating feature. Accordingly, C1 displays a
lower toxicity profile in fibroblasts than in the two tested
cancer cell lines (Figure 5).
Finally, the evaluation of the mechanism of cell death in
HeLa cancer cells by C1 was carried out by using the standard
propidium iodide (PI)/Annexin V-Alexa Fluor 488 assay
(Table S6). The induction of ROS has been related to the
mechanism of apoptosis,54 and many research efforts have
been devoted to the synthesis of potential anticancer agents
that induce an apoptotic cell death pathway.55,56 The results
indicate that C1 is able to partially trigger apoptosis in HeLa
cancer cells, where at least about 12% of cells are in the early
apoptotic stage (Table S6). This value is in the range of
cisplatin, which is well known to induce an apoptotic
pathway.57,58 The rest of death cells (24%) have high
fluorescence values of PI, indicating that the membrane is
not intact. This might point to a necrosis, with loss of
membrane integrity, or to an apoptotic necrosis (late
apoptosis). This last mechanism involves an early apoptosis,
which ends up (with time and in the absence of phagocytosis)
in the membrane lysis of the already formed apoptotic bodies
and in the organelle breakdown.
Figure 6. Agarose gel electrophoresis of a BlueScript Supercoiled
DNA (ScdsDNA) treated with complexes C1, C2, and C3.
Incubation time of 24 h at 37 °C. Some samples were incubated
for an additional 1.5 h in the presence of ascorbic acid.
promotes the Cu(I) generation, the potential formation of
ROS, and the concomitant DNA damage.
Next, the binding of C1−C3 with calf thymus DNA (ct-
DNA) was studied. Covalent interactions with DNA are highly
important in the case of cisplatin and Pt compounds,45,46
whose mechanism of action is usually conceived through the
formation of Pt-DNA adducts. In the case of Cu(II)
complexes, covalent adducts with DNA are less common and
normally they do not show this kind of binding.18 CD and
UV−vis spectroscopies have been used to enlighten the
putative DNA-complex binding modes of C1, C2, and C3
(Figure 7).
CD spectroscopy allows assessing the possible structural
alterations of the characteristic bands of ct-DNA (a positive
band around 280 nm and a negative band around 245 nm)47
upon complex interaction. ct-DNA (50 μM) was incubated
with C1, C2, or C3 (from 0 to 2 equivalents) overnight and
analyzed by CD spectroscopy (Figure 7A−C). In all cases, only
minor modifications of the initial CD signals were observed,
pointing to slight structural changes in the helicity of the ct-
DNA. This suggests some kind of noncovalent interaction, but
without significant structural DNA modifications.
Three main classes of noncovalent binding have been
proposed for metal complexes: intercalation, groove binding,
and electrostatic interactions with the negatively charged
phosphate backbone of DNA. In order to assess the nature of
the complex−DNA interactions, UV−vis spectroscopy has
been used to monitor the changes in the absorbance of C1−
C3 complexes upon increasing additions of ct-DNA to a
solution of the corresponding metal complexes. Absorption
spectra in the range of 225−550 nm were recorded at a
constant complex concentration (30 μM) with increasing
amounts of DNA. The results for complexes C1−C3 (Figure
7D−F) clearly show a hypochromic effect upon ct-DNA
addition, but no significant bathochromism is observed in any
spectra. This points to an interaction with DNA via groove
binding or electrostatic interactions rather than via inter-
calation.48,49 Compounds displaying high DNA-intercalating
capabilities usually induce a bathochromic shift because of
their π−π interactions with the aromatic bases of DNA, a
phenomenon that has not been observed in this case.
Quantitative data, that is, the intrinsic binding constant Kb,
can be obtained from the recorded absorption spectra using
the Benesi−Hildebrand equation (eq 1).50 Ao is the
absorbance of the complex in the absence of DNA, A is the
absorbance at any given DNA concentration, and εG and εH−G
are the extinction coefficients of the complex and the
complex−DNA, respectively.
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Figure 7. DNA-binding studies. On the left, the results of CD studies for C1 (A), C2 (B), and C3 (C) at 50 μM of ct-DNA and at 1:1 and 1:2
(DNA/complex) ratios in NaCl/Tris−HCl at pH 7.2. Samples were previously incubated overnight at 37 °C. On the right, the results of UV−vis
studies for complexes C1 (D), C2 (E), and C3 (F) at 30 μM of each complex upon ct-DNA titration from 0 to 60 μM in NaCl/Tris−HCl at pH
7.2. Each spectrum was recorded after 15 min of stabilization time. The arrows indicate change upon increasing concentrations of ct-DNA.
dimeric form coexists with the monomeric one in DMSO and
water solutions, as clearly observed in ESI-MS and EPR, and
supported by computational studies. The computational data
(together with the SQUID results) also suggest that for the
three complexes (C1, C2, and C3), the most probable
dinuclear structure adopts conformation A, with a Cu−Cu
distance of about ∼2.7 Å, and a dihedral angle in the metal
coordination plane of 154°.
CONCLUSIONS
■
In summary, three novel Cu(II) complexes have been
synthesized with different N,N,O-chelating salphen-like ligands
bearing varying substituents (−H, −Cl, and −Br) on the
aldehyde aromatic scaffold. Synthesis and characterization have
been carried out, and a dimeric structure was found in the solid
state. Magnetic measurements indicated that there is a
ferromagnetic coupling between both Cu centers in the
dimeric state and that similar conformations can be expected
for the three complexes. For the model complex C1, the
The three Cu(II) complexes have the Cu(II)/Cu(I) redox
potential inside the biological redox window, and therefore
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Table 6. Intrinsic Binding Constants (Kb) and
Hypochromism for the Interaction of ct-DNA with
Complexes C1, C2, and C3
EXPERIMENTAL SECTION
■
Chemicals. Reagents like copper(II) chloride, copper(II) acetate,
DCFDA, calf thymus DNA (ct-DNA) sodium salt, benzene-1,2-
diamine, N-chlorosuccinimide (NCS), 2-hydroxybenzaldehyde, p-
toluenesulfonic acid (p-TsOH), bromine, and 2-amino-2-
(hydroxymethyl)propane-1,3-diol (TRIS) were obtained from
Sigma-Aldrich and Thermo Fisher. Solvents such as acetonitrile
(ACN), methanol (MeOH), ethanol (EtOH), ether, chloroform
(CHCl3), dimethyl sulfoxide (DMSO), ethyl acetate (EtOAc),
dichloromethane (DCM), acetic anhydride, and hexane were used
at synthesis grade purity and directly from commercial sources
(Scharlab, Panreac and VWR).
Synthesis of Ligand Precursors. N-(2-Aminophenyl)acetamide
(1).59 Acetic anhydride (5.12 mL, 51.2 mmol) was added dropwise at
0 °C under a N2 atmosphere to a solution of benzene-1,2-diamine
(5.54 g, 51.2 mmol) in anhydrous DCM (75 mL). The mixture was
stirred for 2 h at 0 °C and then stored at −35 °C overnight. The
precipitate was filtered off and washed with cold DCM (3 × 5 mL)
and ether (3 × 5 mL) to yield 1.01 g of a white solid. The filtrate was
further concentrated to the half of its volume and stored at −35 °C
for 48 h more. The new precipitate was filtered off to render
additional 1.56 g of the product. Yield: 38% (2.57 g). 1H NMR (360
MHz, DMSO-d6): δ 9.12 (s, 1H), 7.14 (d, J = 7.8 Hz, 1H), 6.88 (t, J =
7.4 Hz, 1H), 6.70 (d, J = 8.0 Hz, 1H), 6.52 (t, J = 7.3 Hz, 1H), 4.85
(s, 2H), 2.03 (s, 3H).
a
Complex
Kb (M−1
)
log Kb
% hypochromism (λ in nm)
C1
C2
C3
2.2 × 104
6.2 × 104
7.2 × 104
4.34
4.79
4.86
25 (397)
27 (424)
28 (438)
a
Kb is obtained from the ratio of the intercept to the slope, according
to the Benesi−Hildebrand equation (eq 1),48 after the fitting of the
UV−vis data (Figure 7D−F). The calculated Kb values arise from a
DNA−drug interactions according to the Benesi−Hildebrand model
(which gives approximated Kb values), and hence they should be
compared in orders of magnitude, rather than with the exact numbers.
5-Chloro-2-hydroxybenzaldehyde (2).60 Water (4 mL) was added
to 2-hydroxybenzaldehyde (488 mg, 4.0 mmol). Under magnetic
stirring, NCS (536 mg, 4.01 mmol, 1 equiv), p-TsOH (764 mg, 4.0
mmol), and NaCl (355 mg, 6.1 mmol, 1.5 equiv) were added at room
temperature. The final solution was stirred at 40 °C for 1 h. Water (3
mL) was added and the formed precipitate was filtered off and washed
with water (2 × 2 mL). Then, the solid was extracted with DCM and
dried with sodium sulphate to afford an off-white solid. Titled
compound was obtained after column chromatography (hexane/
Figure 8. In vitro ROS production measured with the DCFDA assay
in HeLa cancer cells for complex C1 (25 μM), H2L1 (50 μM), and
H2O2 (100 μM) as positive control after treatment for 4 h.
1
EtOAc 6:1). Yield: 14% (85 mg). H NMR (360 MHz, CDCl3): δ
10.94 (s, 1H), 9.87 (s, 1H), 7.56 (s, 1H), 7.49 (d, J = 8.4 Hz, 1H),
6.99 (d, J = 8.2 Hz, 1H).
5-Bromo-2-hydroxybenzaldehyde (3).61 To a solution of 2-
hydroxybenzaldehyde (0.5 g, 4.0 mmol) in chloroform (10 mL),
bromine (0.65 g, 4.0 mmol) in chloroform (5 mL) was added
dropwise over a period of 15 min at 0 °C. The resulting mixture was
stirred overnight at 50 °C. Then, the reaction was diluted in water (20
mL) and extracted with chloroform (3 × 8 mL). The organic phases
were combined, extracted with water (8 mL) and brine (8 mL), dried
over Na2SO4, and the solvent removed under reduced pressure. The
crude solid was powdered and washed with hexane (2 × 2 mL) and
ether (2 × 3 mL) and the solvents were decanted. 3 was obtained
they are thermodynamically able to biologically undergo a
Cu(II)/Cu(I) redox cycling. Their similar ascorbate con-
sumption rates compared to free Cu(II) confirms the
potentiality of the complexes as ROS generators. The presence
of the electrowithdrawing substituents on the aromatic ring (Cl
or Br) shifts the Cu(II)/Cu(I) redox potential, slightly
favoring the reduction from Cu(II) to Cu(I). The three
complexes exhibited significant cytotoxicity in HeLa and
MCF7 cancer cells, in the range of cisplatin and improved
values with respect to carboplatin. The most interesting feature
relies on the higher toxicity displayed by C1 in cancer cells
with respect to normal cells, most likely owing to its
demonstrated high in vitro ROS production capabilities. In
terms of the biological target, the studies with DNA suggest
that complexes C1−C3 show a moderate noncovalent binding
to the double-strand DNA, but an interesting redox-dependent
cleaving capacity.
The results altogether place C1 as a promising cytotoxic
agent to be further explored, whose ROS-mediated mechanism
of action might produce some inherent selectivity toward
cancer cells against healthy cells, giving rise to less undesired
effects. Unfortunately, the nature of the halogen substituent
shows no influence on the in vitro cytotoxicity of the
complexes, and it also results in some solubility issues.
Nonetheless, the promising in vitro outcome observed for
C1 encourage us to keep working on improving the properties
of this metallic core to position it as a promising anticancer
candidate.
1
without further purification. Yield: 55% (430 mg). H NMR (250
MHz, DMSO-d6): δ 10.95 (s, 1H), 10.22 (s, 1H), 7.72 (d, J = 2.6 Hz,
1H), 7.65 (dd, J = 8.8, 2.6 Hz, 1H), and 6.99 (d, J = 8.8 Hz, 1H).
Synthesis of Ligands H2 L1−H2 L3. (E)-N-(2-(2-
Hydroxybenzylideneamino)phenyl)acetamide (H2L1). 2-Hydroxy-
benzaldehyde (43.5 mg, 0.36 mmol) in absolute EtOH (6 mL) was
added dropwise to a solution of 1 (58.4 mg, 0.39 mmol, 1.1 equiv) in
absolute EtOH (22 mL) at 0 °C and under strong agitation. The final
mixture was stirred for 15 min at 0 °C and then overnight (12 h) at
room temperature. The solution was filtered and the solvent of the
filtrate removed under vacuum to afford a yellowish crude. Pure H2L1
was obtained by silica gel column chromatography using a gradient
elution (from DCM/hexane 1:1 to EtOAc/hexane 1:1). Yield: 39%
(36 mg). Rf (EtOAc/hexane, 2:1) = 0.7. HR-MS (ESI+, MeOH): for
1
[H2L1+H]+, 255.1104 (theoretical, 255.1128). H NMR (360 MHz,
DMSO-d6): δ 12.76 (s, 1H), 9.54 (s, 1H), 8.87 (s, 1H), 7.77−7.57
(m, 2H), 7.49−7.33 (m, 2H), 7.27 (s, 2H), 6.97 (d, J = 7.7 Hz, 2H),
2.05 (s, 3H). 13C NMR (400 MHz, DMSO-d6): δ 168.8, 163.7, 160.7,
142.3, 133.8, 132.9, 132.6, 127.3, 126.2, 125.4, 120.2, 119.6, 119.4,
117.1, 23.8. FTIR−ATR (wavenumber, cm−1): 3294.31, 3055.63,
1662.29, 1613.70, 1589.52, 1573.64, 1515.92, 1443.20, 1365.24,
1304.59, 1278.20, 1225.08, 1180.92, 1150.51, 1108.70, 1033.13,
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1005.30, 965.08, 939.91, 909.25, 854.86, 829.27, 779.77, 752.58,
723.25, 674.22, 642.06.
mg, 0.12 mmol, 1 equiv) in ACN/DCM (1:1, 12 mL) at room
temperature. The same procedure as for C1 was followed to obtain
pure C3. Yield: 74% (35 mg). HR-MS (ESI+, DMSO−MeOH): for
[C3 + H]+, 786.8678 (theoretical, 786.8673). Elemental Analysis
Calcd for C3 (C30H22Br2Cu2N4O4): C, 45.64; H, 2.81; N, 7.10.
Found: C, 45.41; H, 2.81; N, 6.82. FTIR−ATR (wavenumber, cm−1):
1613.76, 1492.89, 1475.85, 1454.37, 1436.29, 1408.34, 1374.91,
1317.00, 1281.65, 1241.56, 1203.98, 1159.97, 1133.05, 1070.82,
1028.23, 988.61, 961.31.
Physical Measurements. Instruments and Experimental
Procedures. SQUID Data. Magnetic characterization has been
performed using a conventional SQUID magnetometer MPMS-XL
from Quantum Design working at a magnetic field up to 5 T and
temperature down to 2 K. The samples (powder) are filled in
polypropylene sleeves then sealed in order to remove the maximum of
dioxygen, which give the signal around 50 K (antiferromagnetic
transition). However, despite such care, the oxygen signal is visible in
the C1 sample, but C2 and C3 are rather clean. Diamagnetic
contribution of the sample holder was removed. The susceptibility
was fitted using the Bleaney−Bowers formula of two coupled S = 1/
2.31
(E)-N-(2-(5-Chloro-2-hydroxybenzylideneamino)phenyl)-
acetamide (H2L2). 2 (20 mg, 0.13 mmol) in absolute EtOH (4 mL)
was added dropwise to a solution of 1 (20 mg, 0.13 mmol) in absolute
EtOH (10 mL) at 0 °C and under strong agitation. The final mixture
was stirred for 15 min at 0 °C and at room temperature overnight.
The solution was filtered, and the solvent of the filtrate was removed
to afford a yellowish crude. Pure H2L2 was obtained by silica gel
column chromatography using a gradient elution (from DCM/hexane
3:4 to hexane/DCM:EtOAc, 1:1:0.5). Yield: 43% (16 mg). Rf
(EtOAc/hexane, 2:1) = 0.7. HR-MS (ESI+, MeOH): for [H2L2 +
H]+, 289.0708 (theoretical, 289.0738). 1H NMR (360 MHz, DMSO-
d6): δ 12.66 (br s, 1H), 9.55 (s, 1H), 8.86 (s, 1H), 7.83 (s, 1H), 7.68
(d, J = 7.4 Hz, 1H), 7.45 (d, J = 8.7 Hz, 1H), 7.35 (d, J = 7.2 Hz, 1H),
7.28 (m, 2H), 7.01 (d, J = 8.8 Hz, 1H), 2.05 (s, 3H). 13C NMR (400
MHz, DMSO-d6): δ 168.8, 161.9, 159.3, 142.2, 133.2, 132.7, 131.3,
127.7, 126.1, 125.2, 123.0, 121.8, 119.2, 119.1, 23.9. FTIR−ATR
(wavenumber, cm−1): 3274.78, 2361.39, 1662.26, 1515.30, 1593.63,
1529.51, 1479.28, 1452.05, 1358.29, 1303.45, 1280.16, 1220.51,
1176.62, 1109.38, 1090.29, 1048.24, 1011.05, 959.80, 922.81, 870.39,
819.90, 760.15, 739.44, 697.78, 654.92, 641.38.
(E)-N-(2-(5-Bromo-2-hydroxybenzylideneamino)phenyl)-
acetamide (H2L3). Compound 3 (150 mg, 0.75 mmol, 1 equiv) in
absolute EtOH (5 mL) was added dropwise to a solution of 1 (123
mg, 0.76 mmol, 1 equiv) in absolute EtOH (10 mL) at 0 °C and
under stirring. The final mixture was kept under the same conditions
for 15 min at 0 °C and at room temperature for additional 24 h. The
solution was filtered, the precipitate was washed with DCM (2 × 3
mL), and the solvent of the filtrate was removed to afford the crude
H2L3. Titled compound was obtained after purification by flash silica
gel column chromatography (DCM/EtOAc, 1:1). Yield: 52% (125
mg). Rf (EtOAc/hexane, 2:1) = 0.7. HR-MS (ESI+, MeOH): for
[H2L3 + H]+, 333.0193 (theoretical, 333.0233). 1H NMR (250 MHz,
DMSO-d6): δ 12.67 (s, 1H), 9.53 (s, 1H), 8.85 (s, 1H), 7.95 (d, J =
2.5 Hz, 1H), 7.67 (d, J = 6.9 Hz, 1H), 7.56 (dd, J = 8.8, 2.5 Hz, 1H),
7.40−7.22 (m, 3H), 6.96 (d, J = 8.8 Hz, 1H), 2.07 (s). 13C NMR
(400 MHz, DMSO-d6): δ 171.0, 164.1, 161.9, 144.2, 138.2, 136.5,
134.9, 129.9, 128.4, 127.4, 124.5, 121.7, 121.4, 112.6, 26.1. FTIR−
ATR (wavenumber, cm−1): 3295.33, 1661.38, 1614.48, 1587.78,
1566.65, 1528.15, 1472.56, 1451.73, 1368.30, 1355.88, 1307.60,
1277.36, 1219.00, 1175.25, 1130.46, 1111.42, 1076.79, 1038.90,
1016.55, 960.34, 937.67, 914.61.
Synthesis of Cu(II) Complexes. Complex C1 ([Cu(L1)]2).
Cu(OAc)2·2H2O (15.7 mg, 0.08 mmol, 1 equiv) in ACN (3 mL)
was slowly added to a solution of H2L1 (20 mg, 0.08 mmol, 1 equiv)
in ACN (8 mL) at room temperature. The final mixture was stirred
for 2 h and the formed precipitate was filtered off and washed with
ACN (2 × 3 mL) and with Et2O (2 × 3 mL). The solid so obtained
was identified as C1. Yield: 68% (17 mg). HR-MS (ESI+, DMSO−
MeOH): for [C1 + H]+, 631.0456 (theoretical, 631.0462); for [C1 +
Na]+, 653.0194 (theoretical, 653.0282). Elemental Analysis Calcd for
C1 (C30H24Cu2N4O4): C, 57.05; H, 3.83; N, 8.87. Found: C, 56.61;
H, 3.81; N, 8.56. FTIR−ATR (wavenumber, cm−1): 2363.09,
1610.64, 1477.82, 1458.40, 1429.20, 1401.49, 1376.22, 1353.19,
1326.39, 1281.82, 1244.19, 1217.10, 1173.53, 1145.83, 1126.54,
1026.08, 961.53, 922.74, 849.42, 793.31, 747.55, 679.15, 649.79,
620.37.
Complex C2 ([Cu(L2)]2). Cu(OAc)2·2H2O (6.0 mg, 0.03 mmol, 1
equiv) in ACN (2 mL) was slowly added to a solution of H2L2 (9 mg,
0.03 mmol, 1 equiv) in ACN (5 mL) at room temperature. The same
procedure as for C1 was followed to obtain pure C2. Yield: 73% (8
mg). HR-MS (ESI+, DMSO−MeOH): for [C2 + H]+, 700.9661
(theoretical, 700.9839). Elemental Analysis Calcd for C2
(C30H22Cl2Cu2N4O4): C, 51.44; H, 3.17; N, 8.00. Found: C, 51.47;
H, 3.18; N, 7.66. FTIR−ATR (wavenumber, cm−1): 1614.62,
1492.27, 1475.85, 1406.65, 1375.34, 1318.32, 1281.53, 1240.53,
1201.84, 1159.66, 1129.77, 1027.64, 988.20, 960.72, 932.29.
Complex C3 ([Cu(L3)]2). Cu(OAc)2·2H2O (24.0 mg, 0.12 mmol, 1
equiv) in ACN (3 mL) was slowly added to a solution of H2L3 (40
The isothermal (T = 2 K) magnetization was fitted using the
Brillouin function with one S = 1 (equivalent to two coupled S = 1/2
at T < 2J) and two uncoupled S = 1/2
NMR Spectrometry. NMR experiments were recorded on
BRUKER DPX-250, 360, and 400 MHz instruments at the Servei
̀
̀
de Ressonancia Magnetica Nuclear (UAB). Deuterated solvents were
directly purchased from commercial suppliers. All spectra have been
recorded at 298 K. The abbreviations used to describe signal
multiplicities are: s (singlet), bs (broad singlet), d (doublet), dd
(double doublet), and m (multiplet). All 13C NMR acquired spectra
are proton decoupled.
ESI-MS Measurements. HR ESI-MS measurements were recorded
after diluting the corresponding solid complexes using a MicroTOF-Q
(Brucker Daltonics GmbH, Bremen, Germany) instrument equipped
with an electrospray ionization source (ESI) in positive mode at the
́
̀
Servei d’Analisi Quimica (UAB). The nebulizer pressure was 1.5 bar,
the desolvation temperature was 180 °C, flow rate of dry gas was 6 L
min−1, the capillary counter electrode voltage was 5 kV, and the
quadrupole ion energy was 5.0 eV.
EPR Experiments. EPR measurements were carried out on a
BRUKER ELEXSYS 500 X-band CW-ESR spectrometer, with an
ELEXSYS Bruker instrument equipped with a BVT 3000 digital
temperature controller. The spectra were recorded at 120 K in frozen
DMSO solutions otherwise noticed. Typical parameters were: a
microwave power of 10−20 mW, a modulation frequency of 100 kHz,
and a modulation gain of 3 G. EPR spectra were simulated using the
EasySpin toolbox developed for Matlab.62 Copper spin quantification
has been carried out for C1 in frozen DMSO solutions (0.5 mM, e.g.,
1 mM copper concentration, with or without 0.1 M [NBu4][PF6]
(TBAP) electrolyte) through double integration of the EPR derivative
signal, using standardized Cu(NO3)2 solutions as an external
calibrator.
Cyclic Voltammetry. Cyclic voltammograms were taken on a
BioLogic SP-150 potentiostat and using EC-Lab 5,40 software.
DMSO was used as a solvent with 0.1 M of [NBu4][PF6] (TBAP) as a
supporting electrolyte. Measurements were carried out with a three-
electrode configuration cell: glassy carbon electrode as the working
electrode, Ag wire in a 0.1 M TBAP solution in DMSO
(semielectrode) as the reference electrode, and Pt as the counter
electrode. The ferrocene (Fc+/Fc) system was used as the internal
standard. The scan rate (ν) varied between 300 and 25 mV·s−1. All
the experiments were recorded under an argon atmosphere.
Elemental Analysis. C, H, and O analyses were performed at the
́
̀
Servei d’Analisi Quimica (UAB) on a Flash EA 2000 CHNS Thermo
Fisher Scientific equipment, with a TCD and a MAS 200 R
autosampler for solid samples.
IR Spectroscopy. Attenuated total reflectance (ATR)−FTIR
spectra were recorded on a PerkinElmer spectrometer, equipped
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with a universal ATR accessory, with a diamond window in the range
4000−650 cm−1.
(FITC) by using an Annexin V−FITC apoptosis detection kit
(Roche). Exponentially growing HeLa cells in 6-well plates (3 × 105
cells/well) were exposed to concentrations equal to the IC50 for 24 h
(70 μM), determined prior to the experiment. After the cells had been
stained with the Annexin V−FITC and propidium iodide, the
percentage of apoptotic cells was analyzed by flow cytometry (FACS
Calibur).
Computational Details. The geometry of the monomeric
[CuII(L1)(DMSO)] and dimeric [CuII(L1)]2 complexes was
optimized with Gaussian 0963 at the DFT theory level using the
hybrid B3LYP functional combined with Grimme’s D3 correction64
for dispersion and the split-valence plus polarization function 6-
31g(d,p) basis-set for the main group elements, SDD plus f-
functions65 and pseudopotential were applied for copper. The effect
of solvation was taken into account using the SMD continuum model
of Marenich et al.66 For all the structures, minima were verified
through frequency calculations.
UV−Vis Characterization. All the spectra were recorded at room
temperature either on an Agilent HP 8453, Varian Cary 50 Bio, a
Varian Cary 60 Bio, or a PerkinElmer Lambda 650 spectropho-
tometer, using 1 cm quart-cuvettes. Noncovalent DNA−complex
interactions were studied by UV−vis measurements. Solutions of
complexes C1−C3 were prepared in 50 mM NaCl/5 mM Tris−HCl
buffer (pH 7.2), containing a maximum of 5% DMSO to solubilize
them. ct-DNA stock solutions were prepared from their correspond-
ing sodium salt and the concentration was determined from its
absorbance at 260 nm (ε = 6600 cm−1). Blank and dilution effects
were corrected. Ascorbate consumption experiments were monitored
by UV−vis at the maximum absorption band of the ascorbic acid (100
μM) at 265 nm for about 45 min. CuCl2 and the assayed complexes
C1−C3 were added at a final concentration of 2 μM in 50 mM NaCl/
5 mM Tris−HCl buffer (pH 7.2), with a maximum of 5% of DMSO.
Circular Dichroism. CD experiments were acquired on a JASCO
715 spectropolarimeter. Measurements were carried out at a constant
temperature of 20 °C. CD spectra were measured in 50 mM NaCl/5
mM Tris−HCl buffer (pH 7.2). The ct-DNA concentration was 50
μM. Different samples with increasing amounts of the complexes to
study (0, 50, 100 μM) were incubated at 37 °C for 24 h, containing a
maximum of 5% DMSO to solubilize them. Ct-DNA stock solutions
were prepared from their corresponding sodium salt (Sigma-Aldrich)
and the concentration was determined from their absorbance at 260
nm (ε = 6600 cm−1).
The thermodynamic stability in solution was estimated computing
the Gibbs free energy change using the implicit solvent continuum
model.67 Concerning the 1e− reduction products, the previously
optimized geometry of the Cu(II) complexes [CuII(L1)(DMSO)]
was reoptimized at the same level of theory imposing the multiplicity
relative to the [CuI(L1)(DMSO)]− and [CuII(L1•−)(DMSO)]−
forms. The Gibbs free energy values were obtained by the addition
of the thermal and entropic corrections (Gtherm), obtained in the
optimization stage, to the potential energy value of single point
calculations with the extended basis-set def2-TZVP for the main
group elements67 and the quadruple-ζ def2-QZVP basis set for
Cu.68,69
DNA-Cleaving Experiments. Gel electrophoresis experiments were
performed on agarose gel (1% in Tris−acetate EDTA (TAE) buffer),
using a BIORAD horizontal tank connected to a variable potential
power supply. Samples were stained with EB and revealed with a
Super GelDoc PlusImager. Complexes C1−C3 were incubated with
the plasmid DNA (200 ng of BlueScript plasmid per well) in 20 mM
NaCl/40 mM Tris−HCl buffer (pH 7.20) medium for 24 h at 37 °C
(<10% DMSO in the final mixture to solubilize the complexes).
Samples containing the reducing agent ascorbic acid were incubated
for 1.5 extra hours in the presence of ascorbic acid (100 μM).
Cell Viability Assays. The IC50 values were evaluated using the
PrestoBlue Cell Reagent (Life Technologies) assay. Working
concentrations of complexes C1−C3 (final amount <0.1% DMSO
in biological experiments) were prepared in the corresponding MEM
(modified Eagle’s medium, Invitrogen) for each cell. Human cancer
cells (HeLa and MCF7) and nontumoral NIH 3T3 cells were
obtained from American Type Culture Collection (ATCC, Manassas,
VA, USA). HeLa cells were routinely cultured with MEM; MCF7,
with DMEN-F12 (Dulbecco’s MEM/Nutrient Mixture F-12 Ham);
and NIH 3T3, with DMEM (Dulbecco’s MEM), all containing 10%
heat-inactivated fetal bovine serum at 37 °C in a humidified CO2
atmosphere. Cells were plated at a density of 3 × 103 cells/well in 100
μL of culture medium and allowed to grow overnight. After the
required incubation time with different concentrations (0, 1, 5, 10, 25,
50, 100, or 200 μM) of each complex, 10 μL of PrestoBlue were
added following the standard protocol. The fluorescence of each well
was measured at 572 nm with a Microplate Reader Victor3
(PerkinElmer). The relative cell viability (%) for each sample related
to the control well was calculated. Each complex was tested per
triplicate and averaged from three independent sets of experiments.
Blank and complex controls were also considered.
The g and A tensors of the 63Cu center for each complex were
obtained using the method implemented into the Orca package.70,71
The A tensor is obtained as a sum of the three contributions: the
isotropic Fermi contact (AFC), the anisotropic dipolar (ADx,y,z), and the
spin−orbit coupling term (ASx,Oy,z). A tensors were computed using the
functional B3LYP, while for g PBE0 was used, both coupled with a
triple-ζ basis set 6-311g(d,p).35
The exchange coupling constants J for the dinuclear C1 complex
were calculated with the functional B3LYP and the 6-311g basis set
with the software ORCA,70 according to the method reported in the
literature.72 Using S1 = S1 = 1/2 in the Heisenberg Hamiltonian H =
̂
̂
̂
S1·S2, the value of J can be expressed as: J = EBS − EHS, where EBS and
EHS are the energies of the broken-symmetry solution and the triplet
state.
UV−vis vertical excitations were simulated on the time-dependent
DFT framework using the solvent continuum model.73 The
simulations were carried out on the previously optimized geometries
in solvent using BH and HLYP functionals and the triple-ζ type def2-
TZVP basis set, according to the method established previously.74
The predicted electronic spectrum of C1 was generated using Gabedit
software.75
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c02932.
NMR, ESI-MS, FT-IR, EPR, UV−vis absorbance, and
SQUID data of ligands and complexes; cell viability
results; EPR parameters obtained; and Gibbs energy
values computationally calculated and flow cytometry of
HeLa cells (PDF)
Intracellular ROS Production Assays. HeLa cells were plated and
allowed to adhere overnight in a 96-well plate (2 × 104 cells/well).
The DCFDA reagent (25 μM in DMSO) was then added and the
cells incubated at 37 °C in the dark for 30 min. The DCFDA solution
was removed and cells were treated with the compounds at the
corresponding IC50 values (at 72 h) and incubated for 4 h. The
experiments were run in triplicate. H2O2 was used as a positive
control at 100 μM. The fluorescence of each well was measured at
535 nm with a Microplate Reader Victor3 (PerkinElmer) after
excitation at 485 nm.
AUTHOR INFORMATION
■
Corresponding Authors
Olga Iranzo − Aix Marseille Univ., CNRS, Centrale Marseille,
iSm2, 13397 Marseille, France; orcid.org/0000-0001-
8542-2429; Email: olga.iranzo@univ-amu.fr
In Vitro Apoptosis Assays. Induction of apoptosis was determined
by a flow cytometric assay with Annexin V−fluorescein isothiocyanate
2949
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Article
̀
(II) Complexes of Phenanthroline Bases Showing DNA Photo-
cleavage Activity and Cytotoxicity. Inorg. Chem. 2011, 50, 8452−
8464.
Oscar Palacios − Departament de Química, Facultat de
Ciències, Universitat Autònoma de Barcelona, 08193
Cerdanyola del Vallès, Barcelona, Spain; orcid.org/0000-
0002-2987-7303; Email: oscar.palacios@uab.cat
(3) Leite, S. M. G.; Lima, L. M. P.; Gama, S.; Mendes, F.; Orio, M.;
Bento, I.; Paulo, A.; Delgado, R.; Iranzo, O. Copper(II) Complexes of
Phenanthroline and Histidine Containing Ligands: Synthesis,
Characterization and Evaluation of Their DNA Cleavage and
Cytotoxic Activity. Inorg. Chem. 2016, 55, 11801−11814.
(4) Santini, C.; Pellei, M.; Gandin, V.; Porchia, M.; Tisato, F.;
Marzano, C. Advances in Copper Complexes as Anticancer Agents.
Chem. Rev. 2014, 114, 815−862.
Authors
Quim Pena − Departament de Química, Facultat de Ciències,
̃
Universitat Autònoma de Barcelona, 08193 Cerdanyola del
Vallès, Barcelona, Spain; Aix Marseille Univ., CNRS,
Centrale Marseille, iSm2, 13397 Marseille, France;
orcid.org/0000-0001-6477-8127
(5) Maheswari, P. U.; Roy, S.; den Dulk, H.; Barends, S.; van Wezel,
Giuseppe Sciortino − Departament de Química, Facultat de
Ciències, Universitat Autònoma de Barcelona, 08193
Cerdanyola del Vallès, Barcelona, Spain; Institute of
Chemical Research of Catalonia (ICIQ), 43007 Tarragona,
Spain; orcid.org/0000-0001-9657-1788
̌
G.; Kozlevcar, B.; Gamez, P.; Reedijk, J. The Square-Planar Cytotoxic
[Cu(II)(pyrimol)Cl] Complex Acts as an Efficient DNA Cleaver
without Reductant. J. Am. Chem. Soc. 2006, 128, 710−711.
(6) Kraatz, H.-B.; Metzler-Nolte, N. Concepts and Models in
Bioinorganic Chemistry; Wiley-VCH, 2006.
Jean-Didier Maréchal − Departament de Química, Facultat
de Ciències, Universitat Autònoma de Barcelona, 08193
Cerdanyola del Vallès, Barcelona, Spain; orcid.org/0000-
0002-8344-9043
Sylvain Bertaina − Aix Marseille Univ., CNRS, IM2NP,
13397 Marseille, France; orcid.org/0000-0002-6466-
8830
A. Jalila Simaan − Aix Marseille Univ., CNRS, Centrale
Marseille, iSm2, 13397 Marseille, France; orcid.org/
0000-0003-2537-0422
Julia Lorenzo − Institut de Biotecnologia i Biomedicina,
Departamento de Bioquímica i Biologia Molecular,
Universitat Autònoma de Barcelona, 08193 Cerdanyola del
Vallès, Barcelona, Spain; orcid.org/0000-0001-5659-
6008
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and Therapeutic Implications. Drug Resist. Updat. 2004, 7, 97−110.
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Platinum-Based Chemotherapy Drugs: A Review for Chemists. Dalton
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Oxindole-Schiff Base Copper(II) Complexes with Potential Anti-
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̀
Merce Capdevila − Departament de Química, Facultat de
Ciències, Universitat Autònoma de Barcelona, 08193
Cerdanyola del Vallès, Barcelona, Spain
Pau Bayón − Departament de Química, Facultat de Ciències,
Universitat Autònoma de Barcelona, 08193 Cerdanyola del
Vallès, Barcelona, Spain
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.0c02932
Notes
́
(16) Verduzco-Ramírez, A.; Graciela Manzanilla-Davila, S.; Eugenia
Morales-Guillén, M.; Carlos García-Ramos, J.; Toledano-Magana, Y.;
The authors declare no competing financial interest.
̃
́
Marin-Becerra, A.; Flores-Alamo, M.; Antonio Ortiz-Frade, L.;
Fernando Olguín-Contreras, L.; Ruiz-Azuara, L. Essential Metal-
Based Drugs: Correlation between Redox Potential and Biological
Activity of M2+ with a N2O2 Ligand. J. Mex. Chem. Soc. 2017, 2017,
109−119.
(17) Bravo-Gómez, M. E.; García-Ramos, J. C.; Gracia-Mora, I.;
Ruiz-Azuara, L. Antiproliferative Activity and QSAR Study of
copper(II) Mixed Chelate [Cu(N-N)(acetylacetonato)]NO3 and
ACKNOWLEDGMENTS
Experimental support of Sergi Montané and Sergi Rodriguez-
Calado (Institut de Biotecnologia i Biomedicina, Dept.
■
̀
Bioquímica i Biologia Molecular, Universitat Autonoma de
Barcelona) is kindly acknowledged. This work was supported
by the Spanish Ministerio de Ciencia e Innovación and FEDER
to the projects BIO2015-67358-C2-2-P, CTQ2015-70371-
REDT, and RTI2018-098027-B-C22. Q.P. acknowledges the
Ministerio de Educación, Cultura y Deporte the financial
support of a FPU grant. The authors from the UAB are
members of the “Grup de Recerca de la Generalitat de
Catalunya” ref. 2017SGR-864. The Servei d’Analisi Química
(UAB) and Spectropole facilities (AMU) are also kindly
acknowledged for allocating instrument time.
́
[Cu(N-N)(glycinato)]NO3 Complexes, (Casiopeinas). J. Inorg.
Biochem. 2009, 103, 299−309.
(18) Pena, Q.; Lorenzo, J.; Sciortino, G.; Rodríguez-Calado, S.;
̃
Maréchal, J.-D.; Bayón, P.; Simaan, A. J.; Iranzo, O.; Capdevila, M.;
̀
Palacios, O. Studying the Reactivity of “old” Cu(II) Complexes for
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“novel” Anticancer Purposes. J. Inorg. Biochem. 2019, 195, 51−60.
(19) Chakravarty, A. R.; Anreddy, P. A. N.; Santra, B. K.; Thomas, A.
M. Copper Complexes as Chemical Nucleases. Proc.-Indian Acad. Sci.,
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Cytotoxic DNA-Interacting copper(II) Coordination Compounds.
Metallomics 2014, 6, 1853−1868.
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