Study of the structure, prooxidative, and cytotoxic activity of some chelate copper(II) complexes

Article abstract of DOI:10.1007/s11696-017-0200-1

The six chelate N,O-copper(II) complexes were synthesised starting from salicylaldehyde anil Schiff bases, as ligands. Their structure is elucidated using experimental and theoretical tools. In vitro biological activities, i.e. cytotoxic and prooxidative

Full text of DOI:10.1007/s11696-017-0200-1

Chem. Pap.
DOI 10.1007/s11696-017-0200-1
ORIGINAL PAPER
Study of the structure, prooxidative, and cytotoxic activity of some
chelate copper(II) complexes
1
Vladimir P. Petrovic Marko N. Zivanovic Dusica Simijonovic
2
1
•
ˇ
•
•
´
´
ˇ
´
1,3
1
2
•
•
´
Jelena Ðorovic
ˇ
´ ´
Zorica D. Petrovic Snezana D. Markovic
Received: 20 February 2017 / Accepted: 11 May 2017
Ó Institute of Chemistry, Slovak Academy of Sciences 2017
Abstract The six chelate N,O-copper(II) complexes were
synthesised starting from salicylaldehyde anil Schiff bases,
as ligands. Their structure is elucidated using experimental
and theoretical tools. In vitro biological activities, i.e.
cytotoxic and prooxidative effects against human epithelial
mammary gland/breast metastatic carcinoma MDA-MB-
231, epithelial colorectal carcinoma HCT-116, and foetal
lung fibroblast healthy MRC-5 cell lines of investigated
compounds are also determined. Complexes Cu-1, Cu-6,
and especially Cu-7 showed significant cytotoxic effects,
with IC50 values comparable with effects of positive con-
trol CisPt. In addition, investigated complexes induced
extreme oxidative and nitrosative stress in all treated cell
lines. The most prominent effect is observed on HCT-116
cells, and on MRC-5 cells, while MDA-MB-231 cells
showed higher resistance to the investigated cell lines,
giving us direction towards the substances with more
specific selectivity.
Keywords Chelate copper(II)–Schiff bases complexes Á
Structure elucidation Á Cytotoxic activity Á Oxidative
stress Á Prooxidative activity
Introduction
Schiff bases are organic compounds known for almost two
centuries (Da Silva et al. 2011; Schiff 1864; Vigato and
Tamburini 2004; Abu-Dief and Mohamed 2015). They
found utilisation in almost every part of chemistry,
including organic synthesis, catalysis, analytical chemistry,
food industry, as well as industry of pigments and dyes
(Genin et al., 2000; Santwana, 2003; Supuran et al., 1996).
The main structural fragment of these compounds is imine
group, which is considered to be responsible for a broad
range of biological activities (Bringmann et al. 2004; Guo
et al. 2007; Souza et al. 2007; Abdel-Rahman et al.
2014, 2015). Some of them are antibacterial, antifungal,
antimalarial, anti-inflammatory, antiviral, antiproliferative,
and antipyretic properties (Guo et al. 2007).
In addition to this, if Schiff bases consist of some
additional heteroatom to nitrogen from imine group, they
can serve as chelators for numerous transition metal ions
(Krishnapriya and Kandaswamy 2005; Panda and
Chakravorty 2005). Various Schiff base complexes have
been described, as well as their chemical and biological
applications (Bian et al. 2003; Faniran et al. 1974;
Mohamed et al. 2006; Abdel Rahman et al. 2016, 2017).
It is known that coordination of ligands to metal can
Electronic supplementary material The online version of this
article (doi:10.1007/s11696-017-0200-1) contains supplementary
material, which is available to authorized users.
´
& Vladimir P. Petrovic
vladachem@kg.ac.rs
1
Department of Chemistry, Faculty of Science, University of
´
Kragujevac, Radoja Domanovica 12, Kragujevac 34000,
Serbia
´
induce increase of biological activity (Petrovic et al.
2
3
Department of Biology and Ecology, Faculty of Science,
´
University of Kragujevac, Radoja Domanovica 12,
Kragujevac 34000, Serbia
2015a, b; Sharma and Khar 1998). Numerous articles
studied biological activity of transition metal complexes
ˇ
´
(Canovic et al. 2016; Sun et al. 2007; Xu et al. 2002).
Bioengineering Research and Development Center,
Kragujevac 34000, Serbia
Schiff base-derived complexes with transition metal ions,
123

Chem. Pap.
such as Cu(II), Mn(II), Co(II), Ni(II), and Pd(II), have also
been reported (Ispir et al. 2008; Kavitha and Reddy 2013;
Kondaiah et al. 2013; Prakash and Adhikari 2011). Among
others, copper(II) complexes showed important effects
such as antifungal (Creaven et al. 2010), antiviral (Wang
et al. 1990), and antibacterial (Zhang 2016). For copper–
Schiff base complexes it is shown that they possess sig-
nificant effects against cancer cells, e.g. Joseyphus et al.
pointed IC50 25.8 lM and Rama et al. IC50 54 lM against
HCT-116, and 6.5 lM against MDA-MB-231 cancer cells
(Rama and Selvameena 2015; Joseyphus et al. 2014).
Similarly, we chose to examine the impact of Cu(II)–Schiff
bases complexes on epithelial colorectal carcinoma HCT-
116 and human epithelial mammary gland/breast meta-
static carcinoma MDA-MB-231 cells, as well as on foetal
lung fibroblast healthy MRC-5 cell lines.
split valence basis set 6-311 ? G(d,p) was used for all
atoms (Lee et al. 1988; Vosko et al. 1980). The structures
of investigated compounds were fully optimised in the gas
phase. Frequency calculations were carried out to confirm
that all structures are local minima (all positive eigenval-
ues). The gas phase structures were used for the exami-
nation of geometrical parameters and predicting IR spectra.
The computed frequencies were scaled by the factor of
0.97. The natural bond orbital analysis (Gaussian NBO
version) was performed.
Experimental
Synthesis of copper(II) complexes
Our previous studies were based on the influence of
Schiff bases and their palladium(II) complexes on cancer
cell lines. In continuation of our examinations, we wanted
to synthesise complexes with a biometal/chelating agent.
The choice was the life-important copper. Although this
metal is essential for a lot of life processes, there are many
questions concerning copper’s influence on dysregulation
of Cu homeostasis, which leads to diseases (Festa and
Thiele 2008). In addition, copper is one of the participants
in the Fenton’s reaction whose radical products may cause
cell damage or antioxidant/prooxidative response. There-
fore, we focused our efforts towards the in vitro investi-
gation of the prooxidative and cytotoxic effects of the
Cu(II) complexes, obtained in the reactions with previously
Copper(II) acetate (0.5 mmol) was added to a solution of
corresponding Schiff base (1, 3, 4, 5, 6, 7) (1 mmol) of
ethanol (5 mL) and heated at reflux for 3 h. After com-
pletion of the reaction, the solvent was evaporated and
remaining powder was washed with ethanol (3 9 2 mL).
Obtained powder was dissolved in a mixture of dichlor-
omethane:ethanol 1:2, recrystallized, and the resulting
crystals/powder was dried under vacuum. Unfortunately,
obtained crystals were not suitable for X-ray analysis.
Complexes were obtained in 80–85% yield. All complexes
were characterised with elemental microanalysis and IR
(Fig. 1). It is worth pointing out that obtained compounds
were not suitable for NMR spectroscopy due to their dia-
magnetic properties.
´
studied Schiff bases, Scheme 1 (Markovic et al. 2015;
´
Petrovic et al. 2015a, b) on the cancer and healthy cell
Cu-1:
(FW = 487.99): C, 63.99; N, 5.74; H, 4.13%; found: C,
63.45; N, 5.81; H, 4.16%.
dark
brown
powder—C26H20CuN2O4
lines.
Cu-3: brown crystals—C28H24CuN2O2 (FW = 484.06):
C, 69.48; N, 5.79; H, 5.00%; found: C, 69.18; N, 5.85; H,
5.04%.
Computational methods
Cu-4: brown crystals—C26H20CuN2O2 (FW = 456.00):
C, 68.48; N, 6.14; H, 4.42%; found: C, 68.05; N, 6.21; H,
4.38%.
All calculations were performed with the Gaussian 09
software package (Frisch et al. 2009), B3LYP functional
with D3 dispersion term using Becke–Johnson damping
function (Grimme et al. 2010) in combination with triple
Cu-5:
(FW = 491.98): C, 63.47; N, 5.69; H, 3.69%; found: C,
63.51; N, 5.76; H, 3.65%.
red-brown
crystals—C26H18CuF2N2O2
Cu-6: brown crystals—C26H20CuN2O4 (FW = 487.99):
C, 63.99; N, 5.74; H, 4.13%; found: C, 63.54; N, 5.8; H,
4.16%.
Cu-7: green powder—C26H20CuN2O4 (FW = 487.99):
C, 63.99; N, 5.74; H, 4.13%; found: C, 63.48; N, 5.83; H,
4.17%.
Cell preparation and culturing
The epithelial colorectal carcinoma HCT-116, human
epithelial mammary gland/breast metastatic carcinoma
Scheme 1 Schiff bases used as ligands
123

Chem. Pap.
Fig. 1 Optimised structures, calculated and experimental IR spectra of trans-square planar and tetrahedral complexes Cu-1, Cu-3, Cu-4, Cu-5,
Cu-6, and Cu-7
123

Chem. Pap.
MDA-MB-231 cells, and foetal lung fibroblast healthy
MRC-5 cell lines were purchased from the American Tis-
sue Culture Collection (Manassas, VA, USA). The cells
were propagated in controlled laboratory conditions in
DMEM supplemented with 10% foetal bovine serum,
100 IU/mL penicillin, and 100 lg/mL streptomycin in
humidified atmosphere with 5% CO2 at 37 °C.
2015a, b). These compounds, as well as their respective Pd
complexes, were tested in vitro on cancer cell lines (Pet-
´
rovic et al. 2015a, b). It was found that Pd complexes exert
noticeable higher cytotoxic activity, than Schiff bases by
themselves. Taking into account induced increase in
cytotoxic activity by complexation with palladium, we
explored the effects of complexations with copper. Here we
present six copper(II) complexes, prepared from N-sali-
´
cylidene aniline Schiff bases (Scheme 1 (Markovic et al.
´
2015; Petrovic et al. 2015a, b), Fig. 1) and copper(II)
Biological in vitro assays
Determinations of cell viability, concentration of super-
oxide anion radical, nitrites and reduced glutathione were
performed following standard procedures briefly described
acetate (molar ratio 2:1). The obtained complexes were
structurally characterised using experimental (IR) and
theoretical tools (Density Functional Theory), and sub-
jected to biological testings. For complexes (Cu-3–6),
crystal structures are known (Bindlish et al. 1976; Burgess
et al. 2001; Ren et al. 2016; Shibuya et al. 2008; Wei et al.
1964; Xu and Pei 2012; Xu et al. 2012; Yildirim et al.
2002), and they were used as starting points in theoretical
optimizations. For Cu-7, structural characterization is
given for the first time here, and to the best of our
knowledge, this kind of characterization for copper com-
plexes has not been reported elsewhere until now.
´
in our previous studies (Petrovic et al. 2014, 2015a, b).
Statistics
All experiments have been performed in three individual
experiments performed in triplicates for each dose and all data
were expressed as mean standard error (SE). Statistical
significance was determined using the Student’s t test or the
one-way ANOVA test for multiple comparisons.
A
p value \0.05 was considered as significant. The magnitude
of correlation between variables was done using SPSS (Chi-
cago, IL, USA) statistical software package (SPSS for Win-
dows, version 17, 2008). The IC50 values were calculated
from the dose curves by a computer program (CalcuSyn).
Structural characterization of the investigated
complexes
The optimised geometries of investigated Cu-1, Cu-3, Cu-
4, Cu-5, Cu-6, and Cu-7 complexes, as well as experi-
mental and simulated IR spectra are presented in Fig. 1.
Bond lengths, angles, and dihedral angles of all complexes
calculated are listed in Tables S1–S6, while corresponding
atoms’ labellings are depicted in Figs. S1–S6. In all
investigated cases, copper is forming two six-membered
chelate rings. Each of them is formed via coordination of
nitrogen from azomethine group and oxygen originating
from salicylaldehyde part of Schiff base, with copper(II)
ion. In the case of Cu-7 complex, chelation in five-mem-
bered fashion is also explored, Fig. S7.
Chemicals
The compounds salicylaldehyde, aniline, 4-fluoroaniline,
4-nitroaniline, toluidine, 2-hydroxyaniline, 3-hydroxyani-
line, 4-hydroxyaniline, copper(II) acetate, and 5,50-dithio-
bis(2-nitrobenzoic acid) were obtained from Aldrich
Chemical Co. Dulbecco’s Modified Eagle Medium
(DMEM) and PBS were obtained from GIBCO, Invitrogen,
USA. Foetal bovine serum (FBS) and trypsin–EDTA were
from PAA (The Cell Culture Company, Pasching, Austria).
Dimethyl sulfoxide (DMSO), 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide (MTT), and nitro blue
tetrazolium (NBT) were obtained from SERVA, Heidelberg,
Germany. N-1-Naphthylethylenediamine dihydrochloride
was purchased from Fluka chemie GMBH, Buchs,
Switzerland. Sulphanilamide and sulphosalicylic acid were
purchased from MP Hemija Belgrade, Serbia. All solvents
and chemicals were of analytical grade.
Possibility of the formation of different geometric iso-
mers of the investigated complexes is explored (Fig. 1;
Figs. S1–S8; Tables S1–S7). It is assumed that the com-
plexes exhibit square planar coordination (either trans or
cis). Yet, it is found that, after the optimisations, some of
the structures become distorted. In these cases, it was not
obvious whether the coordination is square planar or
tetrahedral. Hence, the geometry of chelate complexes is
determined on the basis of the plane–plane angle defined
by the two chelate rings (Shibuya et al. 2008). Angles 0°,
90°, and 180° correspond to the ideal trans-square planar,
tetrahedral, and cis-square planar geometries, respectively.
On the basis of this, structures with angles of the chelate
planes close to 0° (Fig. 1; Tables S1–S6) are characterised
with trans-square planar. In all of structures delineated this
Results and discussion
In our previous studies we examined in detail antioxidative
properties of some salicylaldehyde- and vanillin-derived
´ ´
Schiff bases (Markovic et al. 2015; Petrovic et al.
123

Chem. Pap.
way, copper(II) exhibits nearly ideal square planar coor-
dination, with N–Cu–O, and N–Cu–N/O–Cu–O bond
angles close to 90° and 180°, respectively. The only
deviation is in the case of Cu-7, where chelating plane
angle is close to 25°. This distortion is most probably due
to favourable position of OH substituent in aniline ring
towards ligating oxygen atom from the other chelating ring.
In that way hydrogen OHÁÁÁO bond is formed. On the other
hand, structures with plane–plane angles close to 125°
possess geometry which is closer to tetrahedral than cis-
square planar.
vibrations. It is worth pointing out that our IR data, both
experimental and the calculated, are in accordance with the
literature available (Abdel-Latif et al. 2007; Campos-Val-
lette et al. 1993; Kusmariya et al. 2016; Vafazadeh et al.
2010).
Based on the calculated spectra, one cannot conclude
which isomer prevails. Comparison of the relative free
energies among corresponding isomeric structures revealed
that, except in the case of Cu-7, tetrahedral isomers are
somewhat more stable, but one should note that differences
are not pronounced (up to about 6 kJ/mol, Table S7). On
the other hand, in the case of Cu-7, trans-square planar
structure is by about 30 kJ/mol more stable than corre-
sponding tetrahedral geometry. In addition, this six-mem-
bered complex is far more stable than any of equivalent
five-membered chelates, Table S7. Taking into account that
the crystal packing of Cu-3 is built of both isomeric
structures (Xu and Pei 2012), it is possible that in all cases,
apart from Cu-7, both isomers are present.
In all cases, the NBO analysis revealed that, in place of
formed bonds between copper(II) and ligands, there is
donation of electron density from the donor atoms to the
metal centre. Oxygen lone pairs from pure p orbitals, and
nitrogen lone pairs from the sp2 orbitals delocalize to the
half empty d orbital and formally empty s orbital of Cu(II).
As a result, occupancies of mentioned copper orbitals are
higher than before, with consequently lowered occupancies
in the orbitals of donor atoms.
To confirm or to negate that calculated structures cor-
respond to the experimentally obtained complexes, theo-
retical spectra obtained by means of density functional
theory are compared to experimentally acquired IR spectra.
Biological evaluation
Cytotoxic effects
Standardised MTT assay was used for the determination of
the cytotoxicity of investigated complexes and results are
expressed by IC50 values presented in Table 1 and
Figs. S10–S12. When compared with positive control
CisPt, the strongest effect was observed with Cu-7, which
showed very high cytotoxicity to all three cell lines. Cu-1
and Cu-6 expressed significant activity on HCT-116 and
MRC-5 cells. Unlike these complexes, Cu-3, Cu-4, and
Cu-5 did not express such prominent cytotoxic effect, but
these substances expressed noticeable cytotoxic activity
only towards HCT-116 cells. This is important since these
complexes showed selectivity towards one cancer cell line
versus healthy cells. On the other hand, Cu(OAc)2 served
as negative control and we observed no cytotoxicity on all
three cell lines, Table 1. These results might suggest that
investigated thermodynamically stable six-membered che-
late Cu(II) complexes are stable and that they hydrolyse
very slowly in cell. Our previous results showed that pal-
ladium complex analogues of Cu-1 and Cu-6 expressed
significant cytotoxicity on these three investigated cell
lines.24 Similar to our previous findings (Kosaric et al.
IR spectral characterization
In all investigated cases, good agreement between experi-
mental and calculated spectra is achieved, Fig. 1. Similar
´
to our previous work with Pd complexes (Petrovic et al.
2015a, b), deviations are observed in the region above
3000 cm-1. Namely, in the cases where OH group is still
present, these bands are overestimated, apart from the
trans-Cu-7, where OH stretches are matching experimental
ones. This can be rationalised on the basis of that in this
case there is intramolecular OHÁÁÁO hydrogen bonding
(Shibuya et al. 2008). On the other hand, this was not
repeated in other calculated structures. This is most prob-
ably due to excluded possibility of intramolecular hydro-
gen bonding, as well as due to the intermolecular hydrogen
bonding present in the solid state but not in the optimised
´
structures. Compared to the spectra of ligands (Petrovic
et al. 2015a, b) (Fig. S9) bands belonging to OH stretching
vibrations are changed in cases for Cu-1, Cu-6, and Cu-7,
while in others these bands are missing, implying that Cu–
O coordination occurred.
´
2014; Petrovic et al. 2014, 2015a, b), HCT-116 cells are
In the spectra of corresponding complexes, the C=N
bands are present at lower wavenumbers 1605–1612 cm-1
more sensitive to investigated Cu(II) complexes than
MDA-MB-231 cells. This could be explained by the nature
and origin of these cells. MDA-MB-231 cells are meta-
static and thus more resistant cells, while HCT-116 cells
have been derived from primary tumour.
,
than in corresponding ligands, implying that N coordina-
tion occurred (Abdel-Rahman et al. 2016, 2017a, b). In
addition, there are new bands in the regions of 520–550 and
450–470 cm-1 originating from Cu–O and Cu–N
123

Chem. Pap.
Table 1 IC50 values (lM) of
the investigated compounds
HCT-116
24 h
MDA-MB-231
24 h
MRC-5
24 h
72 h
72 h
72 h
IC50, lM
Cu-1
25.49
[500.00
53.69
18.55
[500.00
[500.00
[500.00
[500.00
[500.00
67.30
155.0
51.78
[500.00
125.38
225.85
12.17
31.03
237.21
105.53
470.47
11.42
Cu-3
38.27
21.84
[500.00
[500.00
[500.00
108.2
Cu-4
Cu-5
58.77
37.89
Cu-6
35.15
13.98
Cu-7
9.31
2.15
27.80
12.51
11.66
Cu(OAc)2
CisPt
[500.00
219.70
[500.00
19.40
[500.00
322.40
[500.00
38.60
[500.00
188.10
[500.00
22.10
Inhibitory activity was expressed as the mean of 50% inhibitory concentration of triplicate experiments
Superoxide anion radical (OÁ22) content changes
of aniline moiety, as well as Schiff base which was used as
ligand and which showed significant cytotoxic effect on
´
HCT-116 cells (Petrovic et al. 2015a, b). According to the
It is well known that redox equilibrium in cells is very
important. Thus, we examined the influence of Cu(II)
Schiff base complexes on the level of superoxide radical
anion OÁ2- as an important indicator of reactive oxygen
species (ROS). Results representing the level of measured
OÁ2- are presented in tables S8–S10 for HCT-116, MDA-
MB-231, and MRC-5 cells, respectively. These data rep-
resent the level of measured OÁ2- in whole plate well. From
the obtained data, one can notice that there is significant
increase of OÁ2- content for investigated Cu(II) complexes.
Considering that treatment influenced significant lowering
in cell number (cytotoxic effect), we recalculated all data to
be related to the number of survived cells (from MTT data)
fact that our complexes are Cu(II) based, one of the
questions from where such high amount of OÁ2- comes
could be explained by Fenton reaction. In mitochondria,
produced H2O2 reacts with cytochrome iron yielding water
and OÁ2-. It is proved that Fenton reaction could also be
catalysed by transition metals such as copper (Pham et al.
2013). It is known that many of the anti-cancer drugs are
designed to induce overproduction of ROS and, therefore,
prooxidative (Barrera 2012). Our results showed that
increase in OÁ2- amount is larger in HCT-116 cells and in
MRC-5 cells than in MDA-MB-231 cells.
Nitrite (NO22) content changes
´
(Petrovic et al. 2015a, b). This way, we obtained another
point of view of the level of OÁ2-. Such recalculated data
revealed very significant increase of OÁ2- for all complexes
on all three cell lines, i.e. cells were under significant
oxidative stress, Figs. S13–S15. Similar to the effects on
cell viability, Cu-1, Cu-6, and especially Cu-7 induced
huge increase in OÁ2- content in dose- and time-dependent
Besides ROS-induced oxidative stress, the cell redox
equilibria could also be altered by reactive nitrogen species
(RNS). Nitrites could be considered as an indicator of NO
which is one of the most important RNS factors. Results
representing nitrite levels are presented in Tables S11–S13
and nitrite levels related to the number of viable cells in
Figs. S19–21 for HCT-116, MDA-MB-231 and MRC-5
cells, respectively. It was observed that investigated com-
plexes induced increase in nitrite level. The most signifi-
cant effect was in cases of Cu-1, Cu-6, and Cu-7 in the
dose- and time-dependent manner. Cu-4 increased nitrites
only in the highest concentration. Similarly, as in NBT
assay, when recalculated to the number of survived cells,
´
manner (Petrovic et al. 2015a, b). Increased content of ROS
could lead to the irreversible damage of cellular compart-
ments and biomacromolecules (including DNA), leading to
enhanced cytotoxicity. Thus, it is expected that if some
substance leads to the enhanced production of ROS, it
could also be cytotoxic (Deavall et al. 2012). When the
results of OÁ2- and MTT are compared, significant com-
patibility can be found. Cu-1 induced the highest produc-
tion of OÁ2- and the highest cytotoxic effect on HCT-116
cells. Cu-6 induced similar compatibility on MRC-5 cells.
Cu-7 showed the highest cytotoxic effect on HCT-116
cells, while the increase of OÁ2- was largest in healthy
MRC-5 cells. Comparison of the structures of phenolic
complexes Cu-1, Cu-6, and Cu-7 revealed that the most
active complex Cu-7 contains free OH group in o-position
-
the NO2 increase is significant and related to the cell
viability. Thus, our results again suggest that cells are
under high nitrosative stress. The origin of nitrites is quite
complicated for explanation because it could be derived
from many sources, e.g. from catabolism of investigated
substances (our substances contain nitrogen atoms), and/or
from the induction of enzymes that could be involved in
123

Chem. Pap.
NO production such as iNOS and many others. NO (and
thus nitrite) level is greatly influenced by O2Á- production.
According to Ferrer-Sueta and Radi (2009), O2Á- possess
great affinity towards NO, forming peroxynitrites, which
exert great oxidative potential. NOÁ and O2Á- react rapidly
in vivo, and the formed ONOO- also quickly reacts with
thiol groups of GSH and many proteins (Lancaster 2006)
inducing non-controlled and non-desired posttranslational
modifications. The largest production of NO2- is observed
in healthy MRC-5 cells.
Pd(II) Schiff complexes, Cu-1, Cu-6, and Cu-7 induced
significant oxidative/nitrosative stress followed by
enhanced cytotoxicity of healthy and cancer cells. Com-
pared to CisPt, investigated Cu-7 showed higher cytotoxic
effect against treated cells with no selectivity. Such cyto-
toxic effect is a consequence of increased production of
superoxide radical anions and nitrites as a result of
prooxidative action of these complexes. It is important to
emphasise the effect of Cu-3, Cu-4, and Cu-5. They
exhibited higher cytotoxicity against HCT-116 cells than
against healthy MRC-5 cells, with no effect against MDA-
MB-231 cells. Compared to the results of analogue Pd(II)
complexes, Cu(II) complexes exerted higher selectivity to
healthy MRC-5 cells.
Reduced glutathione (GSH) content changes
Glutathione is
a tripeptide, c-L-glutamyl-L-cysteinyl-
glycine, responsible for preventing the influence of ROS
and RNS that could cause damage to the cell structures and
affect cellular processes and cell viability. Tables S14–S16
and Figs. S16–S18 represent results obtained for GSH
measurements on HCT-116, MDA-MB-231 and MRC-5
cells, respectively. Considering the level of GSH related to
the number of survived cells, these results suggest signifi-
cant increase in GSH level induced by investigated sub-
stances, especially after 72 h from treatment. The highest
production is observed in healthy MRC-5 cells. On HCT-
116 cells all investigated substances induced increase of
GSH in similar extent. On MDA-MB-231 cells, Cu-7
induced the most significant increase, while in MRC-5 cells
Cu-1, Cu-6, and Cu-7 induced the largest increase. Such a
denominated increase in GSH level could be related to the
increased oxidative and nitrosative stress as a positive
feedback of cell. This GSH production could be related to
previous intensive production of ROS/RNS. Interaction of
GSH with redox changed active moieties of proteins
includes oxidation to GSSG with subsequent NADPH-de-
pendent reduction to GSH by glutathione reductase
Acknowledgements This work was supported by the Ministry of
Education, Science and Technological Development of the Republic
of Serbia (Projects No. 172016, III41010).
References
Abdel Rahman LH, Abu-Dief AM, Newair EF, Hamdan SK (2016)
Some new nano-sized Cr(III), Fe(II), Co(II), and Ni(II) com-
plexes incorporating
napthalen-1-ol ligand:
2-((E)-(pyridine-2-ylimino)methyl)-
structural characterization,
electrochemical, antioxidant, antimicrobial, antiviral assessment
and DNA interaction. J Photoch Photobio B 160:18–31. doi:10.
1016/j.jphotobiol.2016.03.040
Abdel-Latif SA, Hassib HB, Issa YM (2007) Studies on some
salicylaldehyde Schiff base derivatives and their complexes with
Cr(III), Mn(II), Fe(III), Ni(II) and Cu(II). Spectrochim Acta A
67:950–957. doi:10.1016/j.saa.2006.09.013
Abdel-Rahman LH, El-Khatib RM, Nassr LAE, Abu-Dief AM,
Ismael M, Seleem AA (2014) Metal based pharmacologically
active agents: synthesis, structural characterization, molecular
modeling, CT-DNA binding studies and in vitro antimicrobial
screening of iron(II) bromosalicylidene amino acid chelates.
Spectrochim Acta A 117:366–378. doi:10.1016/j.saa.2013.07.
056
Abdel-Rahman LH, Abu-Dief AM, Ali Hashem N, Seleem AA (2015)
Recent advances in synthesis, characterization and biological
activity of nano sized schiff base amino acid M(II) Complexes.
Inter J Nano Chem 1:79–95. doi:10.12785/ijnc/010205
Abdel-Rahman LH, Abu-Dief AM, Adam MSS, Hamdan SK (2016)
Some new nano-sized mononuclear Cu(II) Schiff base com-
plexes: design, characterization, molecular modeling and cat-
alytic potentials in benzyl alcohol oxidation. Catal Let
146:1373–1396. doi:10.1007/s10562-016-1755-0
Abdel-Rahman LH, Nabawia MI, Mohamed I, Abu-Dief AM, Ebtehal
Abdel-Hameed A (2017a) Synthesis, characterization, DFT
calculations and biological studies of Mn(II), Fe(II), Co(II) and
Cd(II) complexes based on a tetradentate ONNO donor Schiff
base ligand. Jour Mol Struc 1134:851–862. doi:10.1016/j.
molstruc.2017.01.036
´
(Nordberg and Arner 2001). In addition, this might be
achieved in interaction of GSH with tested substances, or
with some reactive species originating from phenolic
complexes Cu-1, Cu-6, and Cu-7 (Radu et al. 2010).
Conclusions
The results presented in this paper include the synthesis of
the six chelate N,O-copper(II) complexes, investigation of
their structures using experimental and theoretical tools, as
well as in vitro biological evaluation on cancer and healthy
cell lines. On the basis of obtained experimental and the-
oretical data, it is found that the complexes Cu-1-6 exhibit
either trans-square planar or tetrahedral geometry, while
Cu-7 exclusively adopts trans-square planar geometry.
In vitro study of biological activity of synthesised Cu(II)
complexes revealed that, similar to our earlier findings with
Abdel-Rahman LH, Abu-Dief AM, Mostafa H, Hamdan SK (2017b)
Ni(II) and Cu(II) complexes with ONNO asymmetric tetraden-
tate Schiff base ligand: synthesis, spectroscopic characterization,
theoretical calculations, DNA interaction and antimicrobial
studies. Appl Organomet Chem 31:e3555. doi:10.1002/aoc.3555
123

Chem. Pap.
Abu-Dief AM, Mohamed IMA (2015) A review on versatile
applications of transition metal complexes incorporating Schiff
bases. Beni-Suef Univ J Basic Appl Sci 4:119–133. doi:10.1016/
j.bjbas.2015.05.004
Barrera G (2012) Oxidative stress and lipid peroxidation products in
cancer progression and therapy. ISRN Oncol. doi:10.5402/2012/
137289
Bian HD, Xu JY, Gu W, Yan SP, Liao DZ, Jiang ZH, Cheng P (2003)
Synthesis, structure and properties of terephthalate-bridged
copper (II) polymeric complex with zigzag chain. Inorg Chem
Commun 6:573–576. doi:10.1016/S1387-7003(03)00042-X
Bindlish JM, Bhatia SC, Jain PC (1976) Crystallographic studies of
(2009) Gaussian 09, Revision A1 [computer software] Walling-
ford. CT, USA
Genin MJ, Biles C, Keiser BJ, Poppe SM, Swaney SM, Tarpley WG,
Romero DL (2000) Novel 1,5-diphenylpyrazole nonnucleoside
HIV-1 reverse transcriptase inhibitors with enhanced activity
versus the delavirdine-resistant P236L mutant: lead identification
and SAR of 3- and 4-substituted derivatives. J Med Chem
43:1034–1040. doi:10.1021/jm990383f
Grimme S, Antony J, Ehrlich S, Krieg H (2010) A consistent and
accurate ab initio parametrization of density functional disper-
sion correction (DFT-D) for the 94 elements H-Pu. The J Chem
Phys 132:154104. doi:10.1063/1.3382344
bis-(N-p-tolylsalicylaldiminato)copper(II).
B32:2848–2851
Bringmann G, Dreyer M, Faber JH, Dalsgaard PW, Stærk D,
Jaroszewski JW, Christensen SB (2004) Ancistrotanzanine C and
related 5,10- and 7,30-coupled naphthylisoquinoline alkaloids
from Ancistrocladus tanzaniensis. J Nat Prod 67:743–748
Acta
Crystal
Guo Z, Xing R, Liu S, Zhong Z, Ji X, Wang L, Li P (2007) Antifungal
properties of Schiff bases of chitosan, N-substituted chitosan and
quaternized chitosan. Carbohyd Res 342:1329–1332. doi:10.
1016/j.carres.2007.04.006
ˇ
Ispir E, Toroglu S, KayraldIz A (2008) Syntheses, characterization,
antimicrobial and genotoxic activities of new Schiff bases and
their complexes. Transition Met Chem 33:953–960. doi:10.1007/
s11243-008-9135-2
Burgess J, Fawcett J, Palma V, Gilani SR (2001) Fluoro derivatives of
bis(salicylidene-aminato-N, O)copper(II) and -oxovana-
dium(IV). Acta Crystal
S0108270100020059
Campos-Vallette M, Figueroa K, Latorre RO, Diaz Fleming G,
Costamagna J, Canales JC, Derouault J (1993) Vibrational study
of N-phenyl-substituted hydroxynaphthylaldiminate copper com-
C
57:277–280. doi:10.1107/
Joseyphus SR, Shiju C, Joseph J, Justin Dhanaraj C, Arish D (2014)
Synthesis and characterization of metal complexes of Schiff base
ligand derived from imidazole-2-carboxaldehyde and
4-aminoantipyrine. Spectrochimic Acta A 133:149–155. doi:10.
1016/j.saa.2014.05.050
plexes.
2031(93)87019-P
Vib
Spectrosc
6:25–35.
doi:10.1016/0924-
Kavitha P, Laxma Reddy K (2013) Pd(II) complexes bearing
chromone based Schiff bases: Synthesis, characterisation and
biological activity studies. Arab J Chem 9:640–648. doi:10.
1016/j.arabjc.2013.06.018
ˇ
ˇ
´
ˇ
´
´
´
Canovic P, Bogojeski J, Kosaric JV, Markovic SD, Zivanovic MN
(2016) Pt(IV), Pd (II), and Rh (III) complexes induced oxidative
stress and cytotoxicity in the HCT-116 colon cancer cell line.
Turk J Biol 40:1–7. doi:10.3906/biy-1605-77
Kondaiah S, Reddy GNR, Rajesh D, Joseph J (2013) Characteriza-
tion, and antibacterial activity of the Schiff base derived from
P-toluic hydrazide and o-vanilin (OVPTH Ligand) and its
Mn(II), Co(II), Ni(II) and Cu. Indian Adv Chem Sci 1:228–235
Kosaric JV, Cvetkovic DM, Zivanovic MN, Curcic MG, Seklic DS,
Bugarcic ZM, Markovic SD (2014) Antioxidative and antipro-
liferative evaluation of 2-(phenylselenomethyl)tetrahydrofuran
and 2-(phenylselenomethyl)tetrahydropyran. J Buon 19:283–290
Krishnapriya KR, Kandaswamy M (2005) Coordination properties of
a dicompartmental ligand with tetra- and hexadentate coordina-
tion sites towards copper(II) and nickel(II) ions. Polyhedron
24:113–120. doi:10.1016/j.poly.2004.10.010
Kusmariya BS, Tiwari A, Mishra AP, Naikoo GA (2016) Theoretical
and experimental studies of Cu(II) and Zn(II) coordination
compounds with N, O donor bidentate Schiff base ligand
containing amino phenol moiety. J Mol Struct 1119:115–123.
doi:10.1016/j.molstruc.2016.04.056
Creaven BS, Duff B, Egan DA, Kavanagh K, Rosair G, Thangella
VR, Walsh M (2010) Anticancer and antifungal activity of
copper(II) complexes of quinolin-2(1H)-one-derived Schiff
bases. Inorg Chim Acta 363:4048–4058. doi:10.1016/j.ica.
2010.08.009
Da Silva CM, Da Silva DL, Modolo LV, Alves RB, De Resende MA,
ˆ
´
Martins CVB, De Fatima A (2011) Schiff bases: a short review
of their antimicrobial activities. J Adv Res 2:1–8. doi:10.1016/j.
jare.2010.05.004
Deavall DG, Martin EA, Horner JM, Roberts R (2012) Drug-induced
oxidative stress and toxicity. J Toxicol. doi:10.1155/2012/
645460
Faniran JA, Patel KS, Bailar JC Jr (1974) Infrared Spectra of N, N0-
bis(salicylidene)-1,10-(dimethyl)ethylene-diamine and its metal
complexes. J Inorg Nucl Chem 36:1547–1551. doi:10.1016/
0022-1902(74)80621-4
Ferrer-Sueta G, Radi R (2009) Chemical biology of peroxynitrite:
kinetics, diffusion, and radicals. ACS Chem Biol 4:161–177.
doi:10.1021/cb800279q
Lancaster JR Jr (2006) Nitroxidative, nitrosative, and nitrative stress:
kinetic predictions of reactive nitrogen species chemistry under
biological conditions. Chem Res Toxicol 19:1160–1174. doi:10.
1021/tx060061w
Festa RA, Thiele DJ (2008) Copper: an essential metal in biology.
Curr Biol 144:724–732. doi:10.1016/j.cub.2011.09.040
Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA,
Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson
GA, Nakatsuji H, Caricato M, Li X, Hratchian HP, Izmaylov AF,
Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M, Toyota K,
Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao
O, Nakai H, Vreven T, Montgomery JA Jr, Peralta JE, Ogliaro F,
Bearpark M, Heyd JJ, Brothers E, Kudin KN, Staroverov VN,
Kobayashi R, Normand J, Raghavachari K, Rendell A, Burant
JC, Iyengar SS, Tomasi J, Cossi M, Rega N, Millam JM, Klene
M, Knox JE, Cross JB, Bakken V, Adamo C, Jaramillo J,
Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R,
Pomelli C, Ochterski JW, Martin RL, Morokuma K, Zakrzewski
VG, Voth GA, Salvador P, Dannenberg JJ, Dapprich S, Daniels
AD, Farkas O, Foresman JB, Ortiz JV, Cioslowski J, Fox DJ
Lee C, Yang W, Parr RG (1988) Development of the Colle-Salvetti
correlation-energy formula into a functional of the electron
density. Phys Rev B 37:785–789. doi:10.1103/PhysRevB.37.785
´
´
´
´
´
Markovic Z, Ðorovic J, Petrovic ZD, Petrovic VP, Simijonovic D
(2015) Investigation of the antioxidant and radical scavenging
activities of some phenolic Schiff bases with different free
radicals. J Mol Mod 21:293. doi:10.1007/s00894-015-2840-9
Mohamed GG, Omar MM, Hindy AM (2006) Metal complexes of
Schiff bases: preparation, characterization, and biological activ-
ity. Tur J Chem 30:361–382
´
Nordberg J, Arner ESJ (2001) Reactive oxygen species, antioxidants,
and the mammalian thioredoxin system. Free Radical Biol Med
31:1287–1312. doi:10.1016/S0891-5849(01)00724-9
Panda BK, Chakravorty A (2005) Carbonylation of four-membered
ruthenium and osmium metallacycles incorporating an orthomet-
allated phenolic function: new acylruthenium and arylosmium
123

Chem. Pap.
complexes. J Organomet Chem 690:3169–3175. doi:10.1016/j.
jorganchem.2005.04.012
Andrade-Neto M, de Oliveira MCF (2007) Antimycobacterial
and cytotoxicity activity of synthetic and natural compounds.
ˇ
Petrovic VP, Simijonovic D, Zivanovic MN, Kosaric JV, Petrovic
´
´
´
ˇ
´
´
´
Quım
40422007000700012
Nova
30:1563–1566.
doi:10.1590/S0100-
´
´
ZD, Markovic S, Markovic SD (2014) Vanillic Mannich bases:
synthesis and screening of biological activity. Mechanistic
insight into the reaction with 4-chloroaniline. RSC Adv
4:24635–24644. doi:10.1039/c4ra03909b
Sun RWY, Ma DL, Wong ELM, Che C-M (2007) Some uses of
transition metal complexes as anti-cancer and anti-HIV agents.
Dalton T 43:4884–4892. doi:10.1039/b705079h
ˇ
Petrovic VP, Zivanovic MN, Simijonovic D, Ðorovic J, Petrovic ZD,
´
Markovic SD (2015a) Chelate N, O-palladium complexes:
´
´
´
´
Supuran CT, Barboiu M, Luca C, Pop E, Brewster ME, Dinculescu A
(1996) Carbonic anhydrase activators. Part 14. Syntheses of
mono and bis pyridinium salt derivatives of 2-amino-5-(2-
aminoethyl)- and 2-amino-5-(3-aminopropyl)-1,3,4-thiadiazole
and their interaction with isozyme II. Eur J Med Chem
31:597–606. doi:10.1016/0223-5234(96)89555-9
Vafazadeh R, Hayeri V, Willis AC (2010) Synthesis, crystal structure
and electronic properties of bis(N-2-bromophenyl-salicydenam-
inato)copper(II) complex. Polyhedron 29:1810–1814. doi:10.
1016/j.poly.2010.02.030
Vigato PA, Tamburini S (2004) The challenge of cyclic and acyclic
Schiff bases and related derivatives. Coordin Chem Rev
248:1717–2128. doi:10.1016/j.cct.2003.09.003
Vosko SH, Wilk L, Nusair M (1980) Accurate spin-dependent
electron liquid correlation energies for local spin density
calculations: a critical analysis. Can J Phys 58:1200–1211.
doi:10.1139/p80-159
Wang PH, Keck JG, Lien EJ, Lai MM (1990) Design, synthesis,
testing, and quantitative structure-activity relationship analysis
of substituted salicylaldehyde Schiff bases of 1-amino-3-hy-
droxyguanidine tosylate as new antiviral agents against coron-
avirus. J Med Chem 33:608–614
Wei L, Stogsdill RM, Lingafelter EC (1964) The crystal structure of
bis (N-phenylsalicylaldiminato) copper(II). Acta Cryst
17:1058–1062. doi:10.1107/S0365110X64002663
Xu SP, Pei Y (2012) Microwave synthesis and crystal structure of
2-(p-tolylimino-methyl)-phenol-copper (II). J Chem Crystallogr
42:330–332. doi:10.1007/s10870-011-0247-8
Xu W, Liu LZ, Loizidou M, Ahmed M, Charles IG (2002) The role of
nitric oxide in cancer. Cell Res 12:311–320. doi:10.1038/sj.cr.
7290133
´
synthesis, characterization and biological activity. RSC Adv
5:86274–86281. doi:10.1039/C5RA10204A
´
´
´
´
´
Petrovic ZD, Ðorovic J, Simijonovic D, Petrovic VP, Markovic Z
(2015b) Experimental and theoretical study of antioxidative
properties of some salicylaldehyde and vanillic Schiff bases.
RSC Adv 5:24094–24100. doi:10.1039/C5RA02134K
Pham AN, Xing G, Miller CJ, Waite TD (2013) Fenton-like copper
redox chemistry revisited: Hydrogen peroxide and superoxide
mediation of copper-catalyzed oxidant production. J Catal
301:54–64. doi:10.1016/j.jcat.2013.01.025
Prakash A, Adhikari D (2011) Application of Schiff bases and their
metal complexes-A review. Int J ChemTech Res 3:1891–1896
Radu M, Munteanu MC, Petrache S, Serban AI, Dinu D, Hermenean
A, Sima C, Dinischiotu A (2010) Depletion of intracellular
glutathione and increased lipid peroxidation mediate cytotoxicity
of hematite nanoparticles in MRC-5 cells. Acta Biochim Pol
57:355–360
Rama I, Selvameena R (2015) Synthesis, structure analysis, anti-
bacterial and in vitro anti-cancer activity of new Schiff base and
its copper complex derived from sulfamethoxazole. J Chem Sci
127:671–678. doi:10.1007/s12039-015-0824-z
Ren K, Shang X, Fu J, Zhao P, Zhang J (2016) Copper complex based
on 2-(phenylimino-methyl)-phenol as a high selective fluores-
cent probe for hydrogen sulfide. Polyhedron 104:99–105. doi:10.
1016/j.poly.2015.11.022
Santwana G (2003) Physico-chemical and biological properties of
Mn(II), Co(II), Ni(II) and Cu(II) chelates of Schiff bases. Asian J
Org Chem 15:250–254
Schiff H (1864) Mitteilungen aus dem universitats laboratorium in
Pisa: eineneue reihe organischer basen. Justus Liebigs Ann
Chem 131:118–119. doi:10.1002/jlac.18641310113
Sharma RC, Khar VK (1998) Synthesis, characterization and
antimicrobial studies of some macrocyclic ligands and their
Nickel (II) complexes. Asian J Chem 10:467–471
Xu SP, Tang JF, Liu JT, Ruan BF, Zhu HL (2012) Microwave
synthesis and crystal structure of 2-(p-tolyliminomethyl)phenol-
copper(II). Russ J Coord Chem 38:426–429. doi:10.1134/
S1070328412050107
Yildirim T, Emregul KC, Kurtaran RK, Atakol O (2002) Structure
and electrochemical behaviour of Bis [N-(4-methylphenyl)sali-
cylaldimine] copper (II) N, N0 dimethylformamide solvate. Cryst
Res Technol 37:1344–1351
Zhang XL (2016) Copper(II) complexes with bis-Schiff bases:
synthesis, crystal structures, and antibacterial activities. Synth
Shibuya Y, Nabari K, Kondo M, Yasue S, Maeda K, Uchida F,
Kawaguchi
H (2008) The Copper(II) Complex with two
Didentate Schiff base ligands. The unique rearrangement that
proceeds under alcohol vapor in the solid state to construct
noninclusion structure. Chem Lett 37:78–79. doi:10.1246/cl.
2008.78
React Inorg
1137069
M
46:1848–1853. doi:10.1080/15533174.2015.
Souza AODE, Galetti FCS, Silva CL, Bicalho B, Parma MM, Fonseca
SF, Marsaioli AJ, Trinidade ACLB, Freitas Gil RP, Bezzera FS,
123