Spectroscopic, antiproliferative and antiradical properties of Cu(II), Ni(II), and Zn(II) complexes with amino acid based Schiff bases

Article abstract of DOI:10.1007/s00044-013-0826-7

Novel six Cu(II), Ni(II), and Zn(II) complexes with Schiff bases derived from 4-aminobenzoic acid with terephtaldehyde and amino acids (glycine, β-alanine). Structures have been proposed from elemental analysis, UV-Vis, IR, NMR, TGA, DTA, and magnetic mea

Full text of DOI:10.1007/s00044-013-0826-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2476–2485
DOI 10.1007/s00044-013-0826-7
ORIGINAL RESEARCH
Spectroscopic, antiproliferative and antiradical properties
of Cu(II), Ni(II), and Zn(II) complexes with amino acid
based Schiff bases
•
Zuhal Karagoz Genc Sibel Selcuk Suleyman Sandal Naki Colak
•
•
•
•
Serhat Keser Memet Sekerci Mustafa Karatepe
•
Received: 24 January 2013 / Accepted: 3 October 2013 / Published online: 23 October 2013
ꢀ Springer Science+Business Media New York 2013
Abstract Novel six Cu(II), Ni(II), and Zn(II) complexes
with Schiff bases derived from 4-aminobenzoic acid with
terephtaldehyde and amino acids (glycine, b-alanine).
Structures have been proposed from elemental analysis,
UV–Vis, IR, NMR, TGA, DTA, and magnetic measure-
ments. Spectroscopic studies suggest that coordination
occurs through azomethine nitrogen, hydroxyl group, and
carbonyl oxygen of the ligands to the metal ions. The
elemental analyses of the complexes where L is Schiff base
ligands, are confined to the stoichiometry of the type
M2L2(CH3COO)2 [M = Cu(II)]; and M2L(CH3COO)2
[M = Ni(II) and Zn(II)]. The cytotoxicity activities of the
compounds against human breast carcinoma MCF-7 cell
line have been studied. Ligands and their Zn(II) com-
pounds inhibited cell proliferation of MCF-7 cancer cell
lines in a dose- and time-dependent manner. The free
radical scavenging activity was measured by 1,1-diphenyl-
2-picryl-hydrazil. Our results show that the synthesized
compounds induced oxidative damage by increasing the
lipid peroxidation in yeast since MDA formation was
increased, and it could be concluded that the synthesized
compounds caused oxidative stress. In addition, the
antioxidant activities of the synthesized compounds were
very much lower than those of standard antioxidants.
Keywords Spectroscopy ꢀ Schiff base ꢀ
Antiproliferative activity ꢀ DPPH
Introduction
Schiff bases form an interesting class of ligands that have
enjoyed popular use in the coordination chemistry of tran-
sition, innertransition, and main group elements (Etaiw et al.,
2011; Collinson and Fenton, 1996). Schiff bases have been
reported to show a variety of biological actions by virtue of
the azomethine linkage which is responsible for various
antibacterial, antifungal, herbicidal, and clinical activities
(Sharma and Chandra, 2011; Abd El-Halim et al., 2011;
Ravoof et al., 2010). Transition metal ions play vital roles in
a vast number of widely different biological processes (Priya
et al., 2009). It is well known that some drugs exhibit
increased activity when administered as metal complexes
(Castillo-Blum and Barba-Behrens, 2000; El-Sherif and
Eldebss, 2011; Campbell, 1975; Williams, 1972), and sev-
eral metal chelates have been shown to inhibit tumor growth
(Furst and Haro, 1969). Metal complexes of Schiff bases
derived from heterocyclic compounds containing nitrogen,
sulfur, and/or oxygen as ligand atoms are of interest as
simple structural models of more complicated biological
systems (Sakiyan et al., 2004).There have been several
reports on metal complexes of the Schiff base ligands having
a variety of applications including biological, clinical, ana-
lytic, and industrial fields in addition to their important roles
in catalysis and organic synthesis (Dwyer et al., 1965;
Ouyang et al., 2002; Raman et al., 2001; Jayabalakrishnan
and Natarajan, 2002; Sharghi and Nasser, 2003; Gao and
Z. Karagoz Genc (&) ꢀ S. Selcuk ꢀ S. Keser ꢀ M. Sekerci ꢀ
M. Karatepe
Department of Chemistry, Faculty of Science, Firat University,
Elazig, Turkey
e-mail: zuhalkaragoz23@gmail.com
S. Sandal
Department of Physiology, Faculty of Medicine, Inonu
University, Malatya, Turkey
N. Colak
Department of Chemistry, Faculty of Science and Arts,
Hitit University, Corum, Turkey
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Med Chem Res (2014) 23:2476–2485
2477
Zheng, 2002). Moreover; the activity can be enhanced, when
the biologically active ligand is coordinated to a transition
metal ion.
soluble in strong coordinating solvents like DMF and
DMSO. Therefore, the crystals were unsuitable for single
crystal X-ray structure determination. The analytic data
showed that the complexes had stoichiometry the type
M2L2(CH3COO)2 [M = Cu(II)], M2L(CH3COO)2 [M = Ni(II)
and Zn(II)] respectively, where L is of base ligands
(Table 1).
In the present article, we report the synthesis and char-
acterization of a series of copper(II), nickel(II), and zinc(II)
complexes obtained from {4-[(4-{[4-(carboxymethyl-car-
bamoyl)-phenylimino]-methyl}-benzylidene)-amino]-ben-
zoylamino}-acetic acid (L1) and {4-[(4-{[4-(carboxymethyl-
carbamoyl)-phenylimino]-methyl}-benzylidene)-amino]-
benzoylamino}-propionic acid (L2).
1
The H-NMR spectra of ligands (L1 and L2) have been
carried out in DMSO-d6 at room temperature. Broad peaks
at 13.00–13.06 were attributed to the –OH protons of car-
boxylic acid for the L1 and L2, respectively. The broad
multiplets at 7.43–8.86 ppm were assigned to the aromatic
ring protons. The singlet at 8.86 ppm was attributed to the
proton of the azomethine group, and the multiplets at
3.66–4.15 ppm were assigned to the N–CH2 group. The 1H-
NMR spectrum of the HL2 showing a peak at 2.92 ppm was
assigned to the protons of methyl group. In comparison with
In addition, in vitro antitumoral activity and antioxidant
effects of the ligands and their complexes have been
studied, and the free radical scavenging activity of com-
pounds was measured by 1,1-diphenyl-2-picryl-hydrazil
(DPPH•). Malondialdehyde (MDA) levels were measured
by high-performance liquid chromatography (HPLC) in
Saccharomyces cerevisiae yeast cells (Fig. 1).
1
the H-NMR spectrum of the L1 and L2 and their Zn(II)
complexes, the mode of coordination between the ligands
and metal ions was clarified. Through the discussion of 1H-
NMR spectrum of Zn(II) complexes, it was found that the
peak (signals)-specialized OH proton of carboxylic acid
disappeared. This behavior suggests participation of
hydroxyl group in the ligand. For the CONH protons, a
triplet was observed in the 8.63–8.91 ppm, which is shifted
to 8.27–8.62 ppm in the Zn(II) complexes with a dramatic
decrease in intensity, suggesting that the azomethine
nitrogen was involved in coordination with Zn(II) in the
complexes. The multisignals within the range of 2.5–2.7 are
assigned to the protons of the acetate groups in the complex.
In order to clarify the mode of bonding and the effect of
the metal ion on the ligand, the IR spectra of the free ligand
and their Cu(II), Ni(II), and Zn(II) complexes were com-
pared and assigned on the basis of the careful compari-
son (Table 2).
Results and discussion
All the Cu(II), Ni(II), and Zn(II) complexes were colored,
stable at room temperature for extended periods, and
decompose on heating. These complexes are insoluble in
water and many common organic solvents but are readily
f
N
N
e
e
d
c
b
a
2
CONH-R
2
CONH-R
1
1
3
All complexes show similar IR spectral features,
R: -CH2COOH
R: -CH2CH2COOH
exhibiting
a medium-to-strong bands at 1615 and
Fig. 1 Structures of the ligand (L1 and L2)
1631 cm-1 regions corresponding to t(C=N) stretch of
Table 1 Elemental analyses of the ligands and their compounds
Compounds
FW (g/mol) Color
M.P. (ꢁC) Yield (%) leff (B.M.) Elemental analyses/(%) calculated (Found)
C
H
N
C26H20N4O6 (L1)
484
514
Yellow 265–267
67
64
56
64
66
42
51
69
–
64.19 (63.68) 4.13 (4.25) 11.57 (10.52)
65.36 (65.22) 5.09 (4.98) 10.89 (11.01)
C
28H26N4O6 (L2)
Yellow 298–299
Brown 335
Green 325
–
[Cu2L12(CH3COO)2(H2O)2]ꢀ2H20 1356.7
773.2
1.40
2.46
–
49.53 (49.38) 4.56 (4.18)
46.66 (46.59) 4.14 (4.16)
49.14 (48.56) 3.54 (3.59)
56.56 (56.89) 4.56 (4.30)
45.85 (46.71) 4.77 (4.67)
50.35 (50.24) 4.19 (3.93)
8.25 (8.22)
7.25 (7.14)
7.64 (7.54)
8.80 (8.92)
6.68 (6.65)
7.34 (7.02)
[Ni2L1(CH3COO)2]ꢀ3H2O
[Zn2L1(CH3COO)2]
732.6
Yellow 330
Brown 345
Green 315
[Cu2L22(CH3COO)2(H2O)2] H20 1322.7
1.53
2.76
–
[Ni2L2(CH3COO)2]ꢀ3.5H2O
830.2
762.6
[Zn2L2(CH3COO)2]
Yellow 330
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Med Chem Res (2014) 23:2476–2485
Table 2 Important IR spectral bands (cm-1) of the ligands and their metal complexes
Compound
m(OH)
m(CON)
m(COOH)
m(C=N)
d(N–H)
m(M–N)
m(M–O)
mas(OCO)
ms(OCO)
C26H20N4O6 (L1)
3380–3100
3550–3240
3300–3210
3410–3210
3450–3120
3340–3210
3300–3100
3430–3190
1645
1660
1610
1617
1610
1635
1625
1620
1708
1710
1695
1690
1687
1689
1691
1685
1615
1631
1617
1615
1621
1635
1636
1639
1567
1565
1510
1520
1515
1540
1545
1530
–
–
–
–
–
–
–
C
28H26N4O6 (L2)
–
[Cu2L12(CH3COO)2(H2O)2]ꢀ2H20
[Ni2L1(CH3COO)2]ꢀ3H2O
[Zn2L1(CH3COO)2]
[Cu2L22(CH3COO)2(H2O)2]ꢀH20
[Ni2L2(CH3COO)2]ꢀ3.5H2O
[Zn2L2(CH3COO)2]
528
521
515
509
506
510
487–480
497–490
466–470
496–469
490–470
495–464
1552
1641
1635
1555
1550
1610
1430
1463
1350
1445
1310
1320
azomethine groups. These bands shifted slightly to higher
wave number side relative to the free ligands, indicating
noncoordination of ‘‘N’’ of C=N group in bonding. The
observed strong bands at 1665, 1640 cm-1 and 1708,
1710 cm-1, in the free ligands were attributed to t(CO)
stretches of the CONH and COOH groups, respectively.
These bands shifted slightly to lower wave number side in
all complexes indicating the participation of the carboxyl
oxygen in bonding with metal ions (Zaki et al., 1998;
Bellamy, 1964). Coordination of the Schiff base ligands to
the metal ions through the nitrogen atom is expected to
reduce electron density in the CONH link and lower the
(NH) absorption frequency. These bands that shifted to a
lower wave number side in all complexes indicate the
coordination of the NH group to the metal ions. The IR
spectra of the ligands reveal broad bands in the
3380–3100 cm-1 and 3550–3240 cm-1 regions, and are
assigned to the t(OH) groups. These bands must have
disappeared in the spectra of the complexes, indicating
deprotonation of hydroxyl group and coordinating through
deprotonated OH groups, but the OH stretching frequency
appearing in the spectra of complexes, are attributed to the
presence of water of hydration and coordination. In addi-
tion, thermal analysis data have further confirmed the
results of elemental analyses.
certain transition, which suggests the geometry of the
complex compounds.
The electronic spectra of the L1Ni and L2Ni complexes
showed two strong broad bands at 645–630 nm and
3
385–380 nm, respectively, attributed to 3T1 ? T1 (P) (m3)
and charge transfer in a tetrahedral geometry. (Sonmez
et al., 2004; Satyaranayana and Nagasundura, 2004). The
observed magnetic moments (2.46 and 2.76 B.M.) confirm
the tetrahedral geometry.
Electronic spectra of six-coordinate copper complexes
have either D4h or C4v symmetry, and eg and t2g levels of 2D
free ion term will split into B1g, A1g, B2g, and Eg levels. Thus,
three spin-allowed transitions are expected in the visible and
near IR region. However, only a few complexes are known,
in which such bands are resolved by single crystal polari-
zation studies (Mohan et al., 1990; Dubey and Sangwan,
1994). These bands may be assigned to following transitions:
2B1g ? A1g (dx2-y ? dz2), B1g ? B2g(dx2-y ? dxy), and
2
2
2
2
2B1g ? Eg(d2x-y ? dxz dyz) in the order of increasing
energy. The electronic spectra of complexes having molec-
ular formulae L1Cu and L2Cu show broad bands in the ranges
2
of 600–849 nm and 580–700 nm assigned to the 2B1g ? Eg
transition, respectively. More intense bands at 438 and
450 nm are assigned to the LMCT transitions for L2Cu
complex (Dogan et al., 1998). These band positions are in
agreement with those generally observed for octahedral
copper (II) complexes. The magnetic moments of the copper
complexes are 1.40 and 1.53 B.M. (Table 1) corresponding
to one unpaired electron, which are lower than spin-only
value, i.e., 1.73 BM for one unpaired electron for complexes.
This reveals that these complexes are dimeric in nature and
also show the presence of metal–metal interaction along the
axial positions (Figs. 2, 3).
The new weak nonligand bands in the spectral regions
528–506 cm-1 and 464–497 cm-1 of the complexes are
assigned to the stretching frequencies of t(M–N) and
t(M–O) bonds, respectively. These indicate that azome-
thine nitrogen and carbonyl oxygen atoms are involved in
coordination.
The electronic spectra of the two free ligands exhibit
bands in the region of 350–250 nm, which could be
attributed to n ? p* and p ? p* transitions, respectively.
The magnetic moments are reported at the room tempera-
ture and shown in Table 1. The electronic spectra of the
ligands and their complexes were recorded in DMSO. The
electronic spectra of metal complexes show different bands
at different wavelengths, with each one corresponding to
The thermogravimetric analysis (TGA) and differential
thermal analysis (DTA) curves were obtained at a heating
rate of 15 ꢁC/min in nitrogen atmosphere over a tempera-
ture range of 25–1000 ꢁC.
The [Cu2L12(AcO)2(H2O)2]ꢀ2H2O complex was stable up
to 35 ꢁC, and their decomposition started at this
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Med Chem Res (2014) 23:2476–2485
2479
Fig. 2 Suggested structure of
the Cu(II) complex of the ligand
(L1)
N
O
N
O
NH
OH2
COOH
O
Cu
NH
O
.2H2O
OOCCH3
OH2
H3CCOO
O
NH
HOOC
Cu
O
HN
O
N
O
N
Fig. 3 Suggested structure of
the Cu(II) complex of the ligand
(L2)
N
O
N
COOH
O
NH
OH2
O
NH
Cu
O
.H2O
OOCCH3
OH2
H3CCOO
O
NH
Cu
O
COOH
NH
O
N
O
N
temperature. 2.73 % weight loss was observed at 195 ꢁC
corresponding to 2 mol of water of the crystallization, and
11.66 % weight loss was observed at 230 ꢁC. These weight
losses corresponded to the 2 mol of coordinated water
molecule and two acetate groups. The [Cu2L22(A-
cO)2(H2O)2] H2O complex was stable up to 55 ꢁC, and its
decomposition started at this temperature. 1.36 % weight
loss was observed at 110 ꢁC corresponding to a mole of
water of the crystallization, and 11.64 % weight loss was
observed at 270 ꢁC; the weight losses corresponded to
2 mol of coordinated water molecule and two acetate
groups. The decomposition processes of the Cu(II) com-
plexes are irreversible.
The [Ni2L1(AcO)2]ꢀ3H2O and [Ni2L2(AcO)2]ꢀ3.5H2O
complexes were stable up to 43 and 40 ꢁC, and their
decompositions started at these temperatures. In the
decomposition process of the complexes, 6.38 and 7.69 %
weight losses were observed at 170 and 150 ꢁC, corre-
sponding to 3 and 3.5 mol lattice water, respectively. In the
second stage, 14.06 and 13.59 % weight losses were
observed at 360 and 340 ꢁC, respectively. These weight
losses corresponded to two acetate groups. Also, coordi-
nated water should exhibit frequency at 850, 575, and
500 cm-1 in the IR spectrum. The absence of spectral
bands in these regions in the spectra of Ni(II) complexes
indicates that the water molecules in these complexes are
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Med Chem Res (2014) 23:2476–2485
Fig. 4 Suggested structures of
the Zn(II) and Ni(II) complexes
of the ligand (L1)
N
N
O
OOCCH3
M
N
O
.nH2O
O
N
O
M
O
H3CCOO
O
Fig. 5 Suggested structures of
the Zn(II) and Ni(II) complexes
of the ligand (L2)
N
N
N
O
M
O
O
OOCCH3
.nH2O
N
O
H3CCOO
M
O
O
not coordinated but are present in the form of lattice water
(Fig. 4).
in cell proliferation was clearly observed in the cell lines
studied (Tables 3, 4). Treated samples were significantly
different from the control in MCF-7 cells.
The Zn(II) complexes were stable up to 315 and 330 ꢁC,
respectively. Their decomposition started at these temper-
atures. In the decomposition process of the Zn(II) com-
plexes, the mass losses corresponded to two acetates in the
first stage of the decomposition of the Zn(II) complexes.
The higher dehydration temperatures of Zn(II) complexes
suggest that the water molecule is not coordinated to the
metal ions (Fig. 5).
It is shown in MCF-7 cell lines that all compounds
inhibit cell viability that may induce lipid peroxidation.
Our study shows that L1 and its compounds induced loss of
cell proliferation in concentration- and time-dependent
manner for cell lines. L1Zn possess greater antitumoral
activity against MCF-7 in comparison with L1, Cu(II), and
Ni(II) complexes.
L2 and its metal complexes showed only time-dependent
antitumoral activity in MCF-7 cell cultures. It was seen that
cytotoxicity activities of the L2 and L2Zn complexes on
MCF-7 cells were greater than those of the Cu(II) and
Ni(II) complexes. The results show that the Zn(II) com-
plexes have significant cytotoxic activity toward cell line
(Turan et al., 2011).
Bioactivity
Cytotoxicity assays were carried out by means of trypan
blue test. Cultures grown in each MCF-7 cell line were
exposed to an higher concentration of test compounds
(7.5–60 lM) for 24 and 48 h. A dose-dependent decrease
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Med Chem Res (2014) 23:2476–2485
2481
Table 3 Dose- and time-dependent cell viability results in MCF-7 cells after exposure to L1 ligand and its complexes
Groups (N = 6) 24 h 7.5 lM 24 h 15 lM
24 h 30 lM
24 h 60 lM
48 h 7.5 lM 48 h 15 lM
48 h 30 lM
48 h 60 lM
Control
L1
L1–Ni
L1–Cu
L1–Zn
a
93.00 1.06 90.33 0.56 82.83 1.51 74.50 0.76 83.83 1.05 79.83 1.37 83.16 1.42 71.66 0.76
68.00 2.13c 49.00 2.57c 50.66 1.33c 18.17 2.59c 49.33 2.82c 36.50 2.23c 34.66 1.89c 11.00 4.16c
56.33 3.01c 50.83 4.38c 34.50 1.43c 15.33 1.87c 34.17 1.22c 27.66 3.36c 13.83 1.54c 0.83 0.54c
81.33 1.05c 77.00 1.39c 60.00 2.37c 29.66 1.20c 69.83 1.25c 37.17 5.01c 45.66 0.95c 5.00 2.25c
39.16 1.95c 30.50 1.18c 30.00 0.82c 9.17 2.68c 16.83 1.26c 6.66 2.12c 3.00 1.91c 2.83 1.56c
p \ 0.05
b
c
p \ 0.01
p \ 0.001
Table 4 Dose- and time-dependent cell viability results in MCF-7 cells after exposure to L2 ligand and its complexes
Groups (N = 6) 24 h 7.5 lM 24 h 15 lM 24 h 30 lM 24 h 60 lM 48 h 7.5 lM 48 h 15 lM 48 h 30 lM
48 h 60 lM
Control
L2
L2–Ni
L2–Cu
L2–Zn
a
88.83 0.6
86.00 0.97 81.00 0.68 77.33 0.67 87.17 0.6
85.67 0.88 79.83 0.31 75.33 0.42
63.33 1.52c 46.33 0.76c 50.83 3.02c 42.00 0.82c 34.00 2.05c 24.50 1.54c 28.17 1.19c 8.50 2.17c
74.50 0.84c 42.83 2.01c 58.00 2.11c 34.17 1.11c 31.17 2.32c 24.17 1.35c 24.83 2.27c 3.00 1.15c
77.00 0.58c 63.17 0.87c 51.50 1.94c 34.67 1.41c 41.67 2.45c 36.50 1.50c 21.50 1.28c 0.50 0.50c
57.50 0.88c 46.67 1.52c 54.50 0.96c 33.33 1.36c 29.33 1.09c 6.67 1.56c 22.50 2.31c 2.00 1.48c
p \ 0.05
b
c
p \ 0.01
p \ 0.001
Lipid peroxidation level
Table 5 Saccharomyces cerevisiae yeast cells’ mean MDA levels
(mg/2 9 106 cells) for 24 h, according to the doses, treated with L1,
L2, and their complexes
ROS are formed and degraded by all aerobic organisms,
leading to either physiological concentrations required for
normal cell function or excessive quantities, the state of
which is called oxidative stress (Nordberg and Arner,
2001). ROS-mediated oxidation of membrane lipids results
in the formation of lipid peroxidation products such as
MDA (Maccarrone et al., 1997) and isoprostanes (Morrow,
2003). The cellular antioxidant systems can be divided into
two major groups: enzymatic, and nonenzymatic. Some
nonenzymatic low molecular weight antioxidant com-
pounds such as ascorbic acid, a-tocopherol, and carote-
noids are consumed and may fall below normal ranges.
Determinations of enzymatic and/or nonenzymatic anti-
oxidant levels and MDA levels in the samples are impor-
tant in the evaluation of oxidative stress in biological
systems.
MDA
(50 lM)
(100 lM)
Control
L1
L1–Ni
L1–Cu
L1–Zn
L2
L2–Ni
L2–Cu
L2–Zn
a
0.515 0.02
1.05 0.08b
1.02 0.06a
1.01 0.03a
1.13 0.00a
0.561 0.05
0.615 0.01
0.63 0.00
0.55 0.15
0.515 0.02
1.15 0.00b
1.04 0.00b
1.10 0.03b
1.16 0.02b
0.587 0.03
0.803 0.04b
0.88 0.02a
0.63 0.03
p \ 0.05
b
c
p \ 0.01
p \ 0.001
To assess lipid peroxidation induced by the compounds
tested, we determined MDA levels, an end product of lipid
peroxidation considered as a late biomarker of oxidative
stress, and cellular damage. Lipid degradation and conse-
quently MDA production alter the structure and function of
the cellular membrane and block cellular metabolism
leading to cytotoxicity (Ennamany et al., 1995). MDA is
generally considered to be an excellent indicator of lipid
oxidation (Bird and Draper, 1984; Wendel and Reiter,
1984). Our results show that the compounds tested induced
oxidative damage by increasing lipid peroxidation in yeast
since MDA formation was increased (Table 5). In sum-
mary, it may be concluded that the compounds tested
caused oxidative stress.
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Med Chem Res (2014) 23:2476–2485
trypsin, penicillin, streptomycin. Infrared (IR) spectrum
was determined on a Perkin-Elmer Spectrum One Fourier
transform-infrared (FT-IR) spectrometer. Electronic spec-
tral studies were conducted on a Shimadzu model UV-1700
Spectrophotometer in the wavelength range of
1000–200 nm. NMR spectra were recorded on Bruker
300 MHz spectrometer. Thermal analyses (TGA and
DTA) of compounds were carried out in nitrogen atmo-
sphere at a heating rate of 20 ꢁC/min using Shimadzu TA
60WS thermal analyzer. Elemental analyses were per-
formed on a LECO model 932 instrument. The cells were
incubated under 5 % CO2 air at 37 ꢁC conditions in Nuaire
humidified carbon dioxide incubator (Playmouth, MN,
USA). Cells’ conditions were checked by means of inver-
ted microscope (Soif Optical Inc. China). Results are
expressed as mean SE. Statistical analysis and compar-
ison between mean values for cytotoxicity were performed
by Tukey variance analysis (SPSS 10.0 for Windows;
Chicago, IL, USA).
Table 6 Free radical scavenging activities of samples, trolox, and
tocopherol by DPPH radicals (%)
Samples (1,000 lg/mL)
DPPH• scavenging activity (%)
Control
L1
L1–Zn
L1–Cu
L1–Ni
L2
L2–Zn
L2–Cu
L2–Ni
Trolox
Tocopherol
0
29.53
33.43
31.76
31.33
32.99
54.72
34.94
34.29
94.00
89.45
DPPH radical scavenging activity
The model of scavenging the stable DPPH radical is a
widely used method to evaluate antioxidant activities in a
relatively short time in comparison with the other methods.
The effect of antioxidants on DPPH radical scavenging was
thought to be because of their hydrogen-donating ability.
DPPH• is a stable, free radical and accepts an electron or
hydrogen radical to become a stable diamagnetic molecule
(Talaz et al., 2009). The reduction capability of DPPH
radicals was determined by the decrease in its absorbance
at 517 nm induced by antioxidants. The decrease in the
absorbance of DPPH radical was caused by antioxidants,
because of the reaction between antioxidant molecules and
radical progresses, which results in the scavenging of the
radical by hydrogen donation. It is visually noticeable in
the form of a discoloration from purple to yellow. Hence,
DPPH• is usually used as a substrate to evaluate antioxi-
dative activity of antioxidants. DPPH scavenging activities
of all complexes were lower than those of the standard
antioxidants, tocopherol and trolox (Table 6).
Synthesis of the ligands (L1 and L2)
Ligands (L1 and L2) were synthesized according to the lit-
erature (Colak and Yildirir, 2008). Ligand (L1): IR (KBr,
cm-1): m(NH) 3,319, m(Ar–H) 3,075, m(CON) 1,708,
m(COOH) 1,645, m(C=N) 1,615. 1H-NMR (300 MHz, DMF-
d7/TMS): d (ppm) 4.15 (d, 2H, H-1), 7.45 (d, 2H, H-b), 8.12
(d, 2H, H-c), 8.21 (s, 2H, H-e), 8.86 (s, 1H, CH=N), 8.91 (t,
1H, CONH), 12.95 (b, 1H, COOH). 13C APT (75 MHz,
DMF-d7/TMS): d (ppm): Positive amplitude: 41.19, 129.95,
141.21, 154.42, 166.41 (CONH), 171.83 (COOH), Negative
amplitude: 121.09, 126.76, 129.95, 161.20 (CH=N).
Ligand (L2): IR (KBr, cm-1): m(NH) 3326, m(Ar–H)
3,063, m(C–H) 2,930, m(CO) 1,708, m(C=N) 1,631. 1H-NMR
(300 MHz, DMF-d7/TMS): d (ppm) 2.92 (t, 2H, CH2), 3.66
(q, 2H, NCH2), 7.43 (d, 2H, H-b), 8.12 (d, 2H, H-c) 8.24 (s,
2H, H-e), 8.63 (t, 1H, CONH), 8.86 (s, 1H, CH=N), 12.60 (b,
1H, COOH). 13C APT (75 MHz, DMF-d7/TMS): d (ppm):
Positive amplitude: 33.88, 36.05, 129.96, 141.22, 154.21,
166.11 (CONH), 173.06 (COOH), Negative amplitude:
121.02, 128.77, 129.52, 161.27 (CH=N) (Scheme 1).
According to lipid peroxidation and antiradical activity
results, while the synthesized compounds showed
increased MDA levels in yeast cells, these compounds
showed lower antiradical activities in DPPH radical scav-
enging assay. In fact, these results are complementary to
each other.
Synthesis of the Ni(II), Cu(II), and Zn(II) Complexes
of Ligand (L1) and Ligand (L2)
Experimental
The proposed formula of the ligand is found to be in good
agreement with the stoichiometry results as concluded
from its analytic data (Table 1). Schiff base complexes
under investigation were prepared by mixing 20 mL hot
DMF of the Schiff base (1.02 g; 2.1 mmol for L1 and
1.07 g; 2.1 mmol for L2) with 25 mL ethanolic solution of
the metal salts [Cu(Ac)2ꢀH2O (0.419 g, 2.1 mmol),
Ni(Ac)2ꢀ4H2O (1.045 g, 4.1 mmol), and Zn(Ac)2 (0.770 g,
All reagents were of commercial quality, and solvents were
used without further purification. The human breast carci-
noma MCF-7 cell line was obtained from the American
type culture collection (ATCC). Dulbecco’s modified
eagle’s medium (DMEM) and newborn calf serum were
purchased from Hyclone (Waltham, MA, USA); and
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Med Chem Res (2014) 23:2476–2485
2483
N
CH
COOH
+
O
O
Et-OH
reflux
+
NH2
HOOC
H3N-R
Cl
H
H
COOH
N
CH
N
CH
CONH-R
1. DCC / CH2Cl2
CH2CH2COOH
for L2
=
R
1 and
for L
CH2COOH
.
2 NaOH
.
3 H3O
CONH-R
N
CH
Scheme 1 General procedure of the synthesized ligands
Determination of MDA level
4.2 mmol)]. The obtained mixture was refluxed for about
3 h, and then kept overnight to insure the complete reac-
tions. Then, the formed complexes were filtered, collected,
and washed several times with hot ethanol until the filtrate
becomes colorless. The yield ranged from 42 to 69 %. The
dried complexes were subjected to elemental and spectro-
scopic analyses.
MDA levels in S. cerevisiae yeast cells in this study were
measured by means of high-performance liquid chroma-
tography (HPLC) using the previously described methods
for MDA (Karatepe, 2004).
DPPH• free radical scavenging activity
Cell culture and treatment
The free radical scavenging activities of complexes were
measured by DPPH• according to the method described by
Shimada et al. (1992). In brief, 0.1 mM solution of DPPH•
in ethanol was prepared. 3 mL of the solution was added to
1000 lg/mL of complexes solution. The mixture was
allowed to remain at room temperature for 30 min. Then,
the absorbance was measured at 517 nm by means of a
spectrophotometer (Shimadzu UV-1700 Spectrophotome-
ter). Lower absorbance of the reaction mixture indicated
higher free radical scavenging activity. The percent DPPH•
scavenging effect can be calculated using the following
equation (Keser et al., 2012):
MCF-7 cancer cell line was grown as monolayered culture
in a high glucose (4.5 g/l). DMEM medium supplemented
with 10 % fetal calf serum (FCS), 1 % L-glutamine
(200 mM), and 1 % of penicillin/streptomycin incubated at
37 ꢁC in an air-humidified incubator at 5 % CO2.
Cell viability
For the cytotoxicity analysis of test compounds, cells were
seeded at 1 9 106 cell/mL of MCF-7 cells per eppendorf
tubes in six replicates. The test compounds were dissolved
in DMSO. Proliferative MCF-7 cells were seeded in flasks
and incubated for 24 h. After preincubation, the cell-cul-
ture medium was replaced with fresh medium. Each test
compound was added to the medium within the concen-
tration range of 7.5, 15, 30, and 60 lM, and the cells were
incubated under 5 % CO2 air at 37 ꢁC conditions. After
24 h and 48 h of incubation, cell viability was measured
using Trypan blue exclusion method. Vehicle-treated tubes
served as controls. The final concentration of DMSO in
culture medium did not exceed 0.5 %.
% DPPHꢁ scavenging activity ¼ ½AC ꢂ AS=ACꢃ ꢄ 100
where AC is the absorbance of the control reaction, and AS
is the absorbance in the presence of the synthesized
compounds.
Statistical analysis
Results are expressed as mean SE Statistical analysis,
and comparison between mean values for cytotoxicity were
123

2484
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