Three new mixed-ligand copper(II) complexes containing glycyl-l-valine and N,N-aromatic heterocyclic compounds: Synthesis, characterization, DNA interaction, cytotoxicity and antimicrobial activity

Article abstract of DOI:10.1002/aoc.4126

Three novel copper(II) complexes, [Cu(Gly-l-Val)(HPBM)(H₂O)]·ClO₄·H₂O (1), [Cu(Gly-l-Val)(TBZ)(H₂O)]·ClO₄ (2) and [Cu(Gly-l-Val)(PBO)(H₂O)]·ClO₄ (3) (Gly-l-Val?=?glycyl-l-valine anion, HPBM?=?5-methyl-2-(2′-pyridyl)benzimidazole, TBZ?=?2-(4′-thiazolyl)benzimidazole, PBO?=?2-(2′-pyridyl)benzoxazole), have been prepared and characterized with elemental analyses, conductivity measurements as well as various spectroscopic techniques. The interactions of these copper complexes with calf thymus DNA were explored using UV–visible, fluorescence, circular dichroism, thermal denaturation, viscosity and docking analyses methods. The experimental results showed that all three complexes could bind to DNA via an intercalative mode. Moreover, the cytotoxic effects were evaluated using the MTT method, and the antimicrobial activity of these complexes was tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The results showed that the activities are consistent with their DNA binding abilities, following the order of 1 > 2 > 3.

Full text of DOI:10.1002/aoc.4126

Received: 3 August 2017
DOI: 10.1002/aoc.4126
Revised: 6 September 2017
Accepted: 9 September 2017
F U L L P A P E R
Three new mixed‐ligand copper(II) complexes containing
glycyl‐L‐valine and N,N‐aromatic heterocyclic compounds:
Synthesis, characterization, DNA interaction, cytotoxicity
and antimicrobial activity
Yong‐Yu Qi1 | Ya‐Xian Liu1 | Qian Gan1 | Ya‐Hong Xiong1 | Zong‐Wan Mao1,2 | Xue‐Yi Le1
1 Department of Applied Chemistry, South
Three novel copper(II) complexes, [Cu(Gly‐L‐Val)(HPBM)(H2O)]·ClO4·H2O (1),
China Agricultural University, Guangzhou
510642, People's Republic of China
2 School of Chemistry and Chemical
[Cu(Gly‐L‐Val)(TBZ)(H2O)]·ClO4 (2) and [Cu(Gly‐L‐Val)(PBO)(H2O)]·ClO4 (3)
(Gly‐L‐Val = glycyl‐L‐valine anion, HPBM = 5‐methyl‐2‐(2′‐pyridyl)benzimid-
Engineering, Sun yat‐sen University,
Guangzhou 510275, People's Republic of
azole, TBZ = 2‐(4′‐thiazolyl)benzimidazole, PBO = 2‐(2′‐pyridyl)benzoxazole),
China
have been prepared and characterized with elemental analyses, conductivity
measurements as well as various spectroscopic techniques. The interactions of
these copper complexes with calf thymus DNA were explored using UV–visible,
fluorescence, circular dichroism, thermal denaturation, viscosity and docking
analyses methods. The experimental results showed that all three complexes
could bind to DNA via an intercalative mode. Moreover, the cytotoxic effects
were evaluated using the MTT method, and the antimicrobial activity of these
complexes was tested against Bacillus subtilis, Staphylococcus aureus,
Escherichia coli and Pseudomonas aeruginosa. The results showed that the
activities are consistent with their DNA binding abilities, following the order
of 1 > 2 > 3.
Correspondence
Xue‐Yi Le, Department of Applied
Chemistry, South China Agricultural
University, Guangzhou 510642, People's
Republic of China.
Email: lexyfu@163.com
Funding information
Natural Science Fund, Grant/Award
Number: 2015A030313423; College
Students' Innovation and Entrepreneur-
ship Training Program in Guangdong,
Grant/Award Number: 1056413042
KEYWORDS
antimicrobial activity, copper(II) complexes, cytotoxicity, DNA interaction, Gly‐L‐Val
1 | INTRODUCTION
In these copper(II) complexes, chemical and biological
activities are greatly affected by a ligand. A change of the
During recent years metal complexes as DNA molecule
probes and chemotherapeutic reagents have attracted
considerable attention.[1–4] Under most circumstances,
metal ions act as an inorganic modifier of the organic
backbone of bioactive molecules and ligands endowing
DNA affinity and specificity.[5] Among transition metals,
copper has good coordination properties and its com-
plexes have shown encouraging potential, given that
many of them display favorable photo‐cleavage activity
and various bioactivities such as antibacterial, antitumor
and anti‐inflammatory activities.[3,6,7]
substituent properties and binding site of the ligand can
cause certain differences in the spatial configuration and
electron cloud density distribution of the complexes,
resulting in differences in DNA binding properties or bio-
activities.[8–12] Very recently, Kumar and co‐workers have
observed that copper(II) complexes exhibit effective anti-
cancer and antimicrobial activity via strongly binding
and cleaving DNA, and the activity of the complexes varied
with the ligand.[13–15] Thus, studies of the differences can
be favorable for thoroughly understanding the binding
mechanism of copper complexes to DNA. So far, particular
Appl Organometal Chem. 2017;e4126.
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Copyright © 2017 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/aoc.4126

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QI ET AL.
attention has been primarily focused on copper(II) com-
plexes containing N,N‐aromatic heterocyclic compounds
due to their high DNA binding ability[16] and antiphlogis-
tic,[17] antibacterial[18] and antitumor activities.[19,20]
Moreover, numerous copper complexes with various mod-
ified N,N‐aromatic heterocyclic compounds have been
synthesized, and their DNA binding and cleavage activities
have also been explored.[21,22]
Dipeptides, acting as structural units of proteins and
related metabolites, have been commonly used as hor-
mones, immunomodulators, enzyme inhibitors and neu-
rotransmitters in living systems.[23,24] Based on the
above‐mentioned properties, copper complexes with
dipeptides have been investigated as models for both pro-
tein–DNA and antitumor agent–DNA interactions.[25–27]
Consequently, it is meaningful to gain some insight into
the DNA interaction, antibacterial and antitumor proper-
ties of copper complexes with N,N‐aromatic heterocyclic
compounds and dipeptides.
In our previous works, we have reported a series of
copper(II) peptide complexes containing N,N‐aromatic
heterocyclic compounds,[28–34] and also studied their
DNA interactions, antioxidation properties and cytotoxic-
ity. It was found that the ligands have significant influ-
ence on the activities. Among them, copper(II)
complexes containing glycyl‐L‐valine (Gly‐L‐Val) and 2‐
(2′‐pyridyl)benzimidazole exhibited high nucleobase
affinity and nuclease activity and had potential to serve
as anticancer agents.[34] However, the antibacterial activ-
ity of these complexes has been neglected and little sys-
tematic investigation has been made into the effect of
the structure of N,N‐aromatic heterocyclic ligands on
the activities.
(MTT) assay and the antibacterial activities of the com-
plexes were measured using the agar diffusion method
and double broth dilution method. The results showed
that the structures of the N,N‐aromatic heterocyclic
ligands have an important impact on the activities of
the complexes.
2 | EXPERIMENTAL
2.1 | Materials and methods
The ligands HPBM and PBO were synthesized based on
methods reported in the literature.[35,36] TBZ, Gl‐L‐Val,
CT‐DNA, ethidium bromide (EB), dimethylsulfoxide
(DMSO), RPMI‐1640 and DMEM were purchased from
Sigma. pBR322 DNA was provided by MBI Fermentas
(Lithuania). Cancer cell lines A549, PC‐3 and HeLa and
normal cell line 3 T3 were purchased from the American
Type Culture Collection. MTT was obtained from
Sinopharm Chemical Reagent Co. Ltd (Shanghai, China).
All reagents and chemicals were of analytical reagent
grade and were used as commercially purchased without
any further purification.
DNA stock solution was prepared in buffer containing
5 mM Tris–HCl and 50 mM NaCl at pH = 7.2, and kept at
4 °C for no more than three days. The solution of CT‐
DNA gave a ratio of UV absorbance at 260 and 280 nm
of about 1.8–1.9, indicating that the DNA was fully free
of protein, and its concentration was determined by UV
absorbance at 260 nm using an extinction coefficient of
6600 M−1 cm−1 [37]
Elemental analyses (C, H and N con-
.
tents) were performed with a Vario EL elemental analyzer
(Elementar, Germany). Fourier transfer infrared (FT‐IR)
spectra were recorded with samples as KBr pellets using
a VERTEX 70 FT‐TR spectrometer (Bruker, Germany) in
the spectral range 400–4000 cm−1. UV–visible, fluores-
cence, electrospray ionization (ESI) mass and electron
spin resonance (ESR) spectra were obtained with a
Pharmacia 2550 spectrophotometer (Shimadzu, Japan),
Hitachi F‐4500 fluorescence spectrophotometer (Hitachi,
Japan), API4000 triple quadrupole mass spectrometer
(AB Sciex, USA) and Bruker EMX A300 spectrometer
(Bruker, Germany), respectively. The molar conductivities
were measured using a DDS‐11A digital conductance
meter (LeiCi, Shanghai).
As an extension of these previous works, we have syn-
thesized three new complexes of similar type: [Cu(Gly‐L‐
Val)(HPBM)(H2O)]·ClO4·H2O (1), [Cu(Gly‐L‐Val)(TBZ)
(H2O)]·ClO4 (2) and [Cu(Gly‐L‐Val)(PBO)(H2O)]·ClO4
(3) (HPBM
=
5‐methyl‐2‐(2′‐pyridyl)benzimidazole,
TBZ = 2‐(4′‐thiazolyl)benzimidazole, PBO = 2‐(2′‐pyri-
dyl)benzoxazole). UV–visible and fluorescence spectros-
copies were applied to determine the strength of
interaction between the complexes and calf thymus
DNA (CT‐DNA). Further experiments, including viscos-
ity, circular dichroism (CD), thermal denaturation and
docking analyses, were carried out to assess the binding
modes. In order to evaluate the biological properties of
the complexes, we investigated their DNA cleavage abil-
ities via gel electrophoresis measurements. Furthermore,
the cytotoxicities against A549 (human lung carcinoma),
HeLa (human cervical carcinoma) and PC‐3 (human
prostate carcinoma) cell lines and 3 T3 (mouse embry-
onic fibroblast) cells were determined using 3‐(4,5‐
dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide
2.2 | Preparation of complexes
The complexes were prepared by a method similar to that
reported in the literature.[34] Cu(ClO4)·6H2O (0.5 mmol)
aqueous solution was added into 5 ml of an aqueous solu-
tion containing Gly‐L‐Val (0.5 mmol) and NaOH
(0.5 mmol) with stirring, and then HPBM/TBZ/PBO

QI ET AL.
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(0.5 mmol) in 20 ml of CH3OH was added dropwise into
the mixture. The resultant solutions were stirred thor-
oughly, and the pH was adjusted to 4.83 with perchloric
acid solution. These mixtures were further stirred for
about 1 h at 50 °C and slowly evaporated to afford the
complexes. The products were filtered, dried under vac-
uum and further purified by recrystallization from meth-
anol–water (80% v/v).
wavelength set at 550–660 nm. The effect of the addition
of each complex to a sample including EB and CT‐DNA
(8 and 10 μM, respectively) in Tris buffer (pH = 7.2) was
investigated by recording the variation in fluorescence
spectra.
2.3.3 | Viscosity measurements
Viscosity experiments were performed with an Ostwald
viscometer immersed in a water bath maintained at
29 0.1 °C for EB (standard) and complexes 1, 2 and 3.
The concentration of CT‐DNA was 200 μM, and the con-
centration of the complexes and EB was varied from 0 to
70 μM with 10 μM intervals. The flow time for every sam-
ple was determined three times with a digital stopwatch
and then averaged. Relative viscosity values (η) were cal-
culated based on the relation η = (t − t0)/t0, where t is
the flow time of DNA‐containing solutions and t0 is the
flow time of free buffer. The obtained data are presented
as (η/η0)1/3 versus [complex]/[DNA], where η and η0 are
the viscosity of the CT‐DNA solution in the absence and
presence of the complexes, respectively.
Complex 1. Blue; yield: 70 %. Anal. Calcd for
C20H28N5O9ClCu (MW = 582.91) (%): C, 41.31; H,
4.85; N, 12.05. Found (%): C, 41.23; H, 4.63; N, 11.98.
FT‐IR (KBr, cm−1): ν(―OH) 3425; νas(―NH2) 3104;
νs(―NH2) 2964; νas(―COO–) 1615; νs(―COO–) 1379;
ν(C―N) 1458; ν(Cu―O) 621; ν(Cu―N) 433. UV–visi-
ble (MeOH; λnm/nm, ε/M−1 cm−1): 323, 2.28 × 104;
666, 66.16.
Complex 2. Bluish‐green; yield: 78 %. Anal. Calcd for
C17H22N5O8SClCu (MW = 556.94) (%): C, 36.76; H,
3.99; N, 12.61. Found (%): C, 36.58; H, 4.17; N, 12.43.
FT‐IR (KBr, cm−1): ν(―OH) 3409; νas(―NH2) 3090;
νs(―NH2) 2962; νas(―COO–) 1613; νs(―COO–) 1326;
ν(C―N) 1435; ν(Cu―O) 648; ν(Cu―N) 434. UV–visi-
ble (MeOH; λnm/nm, ε/M−1 cm−1): 234, 2.88 × 104;
300, 3.68 × 104; 666, 87.45.
2.3.4 | CD spectral measurements
Complex 3. Blue; yield: 72 %. Anal. Calcd for
C19H23N4O9ClCu (MW = 551.91) (%): C, 41.46; H,
4.21; N, 10.18. Found (%): C, 41.42; H, 4.01; N, 9.99.
FT‐IR (KBr, cm−1): ν(―OH) 3422; νas(―NH2) 3099;
νs(―NH2) 2962; νas(―COO–) 1613; νs(―COO–) 1376;
ν(C―N) 1480; ν(Cu―O) 624; ν(Cu―N) 428. UV–visi-
ble (MeOH; λnm/nm, ε/M−1 cm−1): 231, 1.41 × 104;
304, 2.65 × 104; 634, 60.07.
CD spectra of CT‐DNA (100 μM) with [complex]/[DNA]
ratio of 0.6 were recorded from 220 to 320 nm using a
10 mm path quartz cuvette and a 100 nm min−1 scan-
ning rate. Each spectrum was obtained by averaging
three scans and subtracting the background signal of
the buffer.
2.3.5 | Thermal denaturation studies
DNA thermal denaturation experiments were carried out
with a Chirascan using CT‐DNA (50 μM) by varying the
temperature from 70 to 100 °C at a heating rate of 2 °C
min−1, both in the absence and presence of each complex
(30 μM). The absorption changes at 260 nm were contin-
uously monitored and the melting temperature (Tm) was
defined as half of the total unbound base pairs, which
was confirmed from the mid‐point of the melting curves.
The data were presented as (A − A1)/(Af − A1) versus tem-
perature, where A, A1 and Af are the observed, initial and
final absorbance, respectively.
2.3 | Interaction with CT‐DNA
2.3.1 | UV–visible absorption spectroscopy
Absorption spectra were determined in the range 210–
500 nm by maintaining a constant complex concentration
in the presence of CT‐DNA for various r ([complex]/[CT‐
DNA] mixing ratio) values at room temperature. Mean-
while, the reference solution was used to remove the
absorption of CT‐DNA itself, and Tris buffer was
subtracted through baseline correction. Every sample
solution was permitted to equilibrate for 6 min before
recording its spectrum.
2.3.6 | DNA excision measurements
Electrophoresis experiments were performed using
pBR322 plasmid DNA according to established proce-
dures. The samples were incubated at 37 °C for a period
of 1 h in the dark. Then the reactions were quenched by
addition of loading buffer and the analysis involved
2.3.2 | Fluorescence spectroscopy
Fluorescence quenching measurements were carried out
at an excitation wavelength of 525 nm and emission

4 of 14
QI ET AL.
loading of the solutions onto 0.8% agarose gels contain-
ing 5 μl of GoldView. The final solutions were subjected
to gel electrophoresis (100 V for 40 min in standard
Tris–boric acid–EDTA buffer, pH = 8.3). The gel bands
obtained were visualized and photographed with a
BIO‐RAD Laboratories Segrate Gel Imaging System.
Control experiments were carried out using 50 μM
ascorbic acid (VC), 20 μM Cu(ClO4)2·6H2O and 20 μM
free ligands.
In order to explore the mechanism, the cleavage of
supercoiled DNA was carried out in the presence of typi-
cal reactive oxygen species scavengers such as DMSO, eth-
anol and tert‐butyl alcohol (hydroxyl radical), superoxide
dismutase (SOD; superoxide anion radical) and sodium
azide (singlet oxygen). Each sample was incubated at
37 °C for 1 h and analyzed according to the procedure
described above.
inhibits cell growth by 50%) were calculated to evaluate
the cytotoxic effects of the complexes.
2.5 | Antimicrobial studies
2.5.1 | Test microorganisms
The antimicrobial activities of the compounds (complexes
and ligands) were determined against four strains of bac-
teria. Reference strains were Gram‐positive bacteria Bacil-
lus subtilis and Staphylococcus aureus, and Gram‐negative
bacteria Escherichia coli and Pseudomonas aeruginosa.
These microorganisms were generously provided by the
Key Laboratory of Plant Molecular Breeding of Guang-
dong Province, College of Agriculture, South China Agri-
cultural University.
2.5.2 | Agar diffusion method
The antimicrobial activities of the prepared ligands and
complexes were determined using the Oxford cup
method. Each compound was dissolved at a concentration
of 2 mg ml−1 in a mixed solvent of aseptic water with 5%
DMSO. Under aseptic conditions Oxford cups were placed
in inoculated agar media, and 100 μl of samples was
inhaled into the cups. Then the agar plates were incu-
bated for 24 h at 37 °C. After this period, the diameter of
the inhibition zone formed around each cup was
observed, and measured in millimeters. Each test was car-
ried out in triplicate.
2.3.7 | Molecular docking studies
The molecular docking studies were performed using the
AutoDock Vina1.1.2 set of programs[38] with the
Lamarckian genetic algorithm. The molecular structures
of the complexes were sketched via Gaussian viewer,
while the crystal structure of DNA d(5′‐G‐Diu‐TGCAAC‐
3′) (PBD ID: 454D) was downloaded from the protein data
bank. For docking, the water molecules and the substrate
DNA were deleted, the active site was defined and
selected, and a grid box was constructed to enclose the
whole DNA molecule, setting the grid size to 60, 60, 60
with a spacing of 0.375 Å. Subsequently, the complexes
were docked to the DNA under other default settings.
Visualization of the docked conformation was produced
with PyMol software.
2.5.3 | Microdilution method
Screening was performed by determining the minimum
inhibitory concentration (MIC) and the minimum bacteri-
cidal concentration (MBC) using the microdilution
method with MTT.[39,40] The compounds were dissolved
in sterile water containing 5% DMSO with concentrations
of 1024, 800 and 640 μg ml−1. Under twofold serial
dilutions the concentration in the tubes was 512–1.00 μg
ml−1 (400–1.81 and 320–1.25 μg ml−1). Then, 10 μl of
bacterial suspension (106 CFU ml−1) was added to appro-
priate tubes. All cultures were incubated for 24 h in a
shock incubator at 37 °C. Finally, 10 μl of phosphate‐buff-
ered saline including 5 mg ml−1 MTT, as an indicator of
microbial growth, was added to each tube and incubated
at 37 °C for 30 min. MTT is a light yellow dye that turns
blue‐violet when it is reduced to formazan by dehydroge-
nase within viable cells. The lowest concentrations of the
test substances that prevented MTT color change from
yellow to blue were considered as the MIC values. Then,
20 μl of each solution was extracted from the test tubes
of the yellow solutions and coated on nutrient agar
2.4 | In Vitro cytotoxicity assays
The cytotoxic activities of the complexes and cisplatin
were studied against cancer cell lines A549, PC‐3 and
HeLa and normal cell line 3 T3 using the MTT method,
where cancer cell lines and normal cell line were cultured
in RPMI‐1640 and DMEM, respectively. All the cells were
seeded into 96‐well plates (1 × 104 per well) and cultured
in a CO2 incubator (37 °C, 5% CO2). Once the cells
adhered and reached 70–80% confluency, complex
(3.125–200 μM) was added to each well, and incubated
for 48 h. Then MTT reagent (20 μl, 5 mg ml−1) was added.
After 4 h, DMSO (100 μl) was added to solubilize the MTT
formazan. Subsequently, the optical densities were mea-
sured at 490 nm with a microplate spectrophotometer.
These data were obtained from triplicate independent cell
passages and the IC50 values (drug concentration that

QI ET AL.
5 of 14
medium. After 24 h incubation, the lowest concentration
with no growth was defined as the MBC value. All the
equipment and culture media were sterilized.
νs(―COO−) stretching vibrations of the carboxylate group
of the coordinated dipeptide ligand, respectively. The Δν
(νas(―COO−) − νs(―COO−)) values in the range 236–
287 cm−1 indicated the monodentate coordination mode
of the carboxylate group of the dipeptide.[42] Moreover,
the vibrations ν(C―N) and ν(C―C) were, respectively,
at 1458 and 788 cm−1 for HPBM, at 1435 and 749 cm−1
for TBZ and at 1480 and 758 cm−1 for PBO, suggesting
that HPBM/TBZ/PBO was coordinated to the central cop-
per ion. Furthermore, the peaks at 433 and 621 cm−1 for 1,
434 and 749 cm−1 for 2 and 428 and 624 cm−1 for 3 were
assigned to the stretching vibrations ν(Cu―N) and
ν(Cu―O), respectively. These overall characteristics of
the FT‐IR spectra further verified that the ligands were
coordinated to the central copper(II) ion.[43–46]
3 | RESULTS AND DISCUSSION
3.1 | Synthesis and spectroscopy
The complexes were synthesized in high yields (70–80%)
via the reaction of equal molar ratio of Gly‐L‐Val with
Cu(ClO4)2·6H2O in the presence of HPBM/TBZ/PBO
(Scheme 1). The structure of the complexes was con-
firmed using elemental analyses, molar conductivity mea-
surements and various spectroscopies, namely FT‐IR,
UV–visible, ESI‐MS and ESR. The results of elemental
analyses corresponded with the theoretically expected
values, which confirmed the compositions for all the com-
plexes. The values of the molar conductivity of 1 mM
MeOH solution of the three complexes are within the
range 90–98 S cm2 mol−1, indicating 1:1 type electro-
lytes.[41] Furthermore, ESI‐MS studies of the complexes
were carried out in MeOH solution. The results showed
peaks of mass‐to‐charge ratio m/z at 446.2, 437.1 and
432.0, which matched accurately with [Cu(Gly‐L‐Val)
(HPBM)]+, [Cu(Gly‐L‐Val)(TBZ)]+ and [Cu(Gly‐L‐Val)
(PBO)]+, respectively.
The combining mode of Gly‐L‐Val was investigated
using FT‐IR spectroscopy. In the FT‐IR spectra, the very
broad peak at about 3420 cm−1 was assigned as ν(―OH)
stretching vibration of the water molecules. The absorp-
tion peaks at 3104 and 2964 cm−1 for 1, at 3090 and
2962 cm−1 for 2 and at 3099 and 2966 cm−1 for 3 were
ascribed to the stretching vibrations νas(―NH2) and
νs(―NH2), respectively, while the peaks at 1615 and
1379 cm−1 for 1, 1613 and 1326 cm−1 for 2 and 1630 and
1376 cm−1 for 3 were assigned to νas(―COO−) and
The electronic spectra of the complexes in the UV–
visible region were measured in methanol solution. The
bands at 323 nm (ε = 22 766 M−1 cm−1) for 1, 234 nm
(ε = 28 758 M−1 cm−1) and 300 nm (ε = 36 769 M
−1
cm−1) for 2 and 231 nm (ε = 14 084 M−1 cm−1) and
304 nm (ε = 26 460 M−1 cm−1) for 3 were observed.
These bands correspond to π → π* transitions of ligand
HPBM/TBZ/PBO. Furthermore, the broad and weak
absorption bands observed in the range 634–666 nm
(ε = 60–88 M−1 cm−1) were attributed to the d → d tran-
sition of the copper(II) ion in a distorted square pyrami-
dal environment.[47]
The X‐band ESR spectra at 9.46 GHz were obtained
using tetracyanoethylene as field marker under a mag-
netic field strength of 3000
1000 gauss in MeOH
(100 K). The optimal simulating parameters were
obtained as follows: g|| = 2.2618, g⊥ = 2.0509 and
A|| = 157.120 for 1, g|| = 2.2585, g⊥ = 2.0386 and
A|| = 155.843 for 2 and g|| = 2.2865, g⊥ = 2.0627 and
A|| = 165.777 for 3. The order of g|| > g⊥ > ge (2.0023)
showed that the unpaired electron in copper(II) was
.
R1
ClO4 yH2O
X
X
NH2
PPA / PPA+P2O5
N
N
HOOC
+
R2
N
N
R2
180 °C 2h
OH2
N
Cu
O
H2N
MeOH 50°C 1h
R2
H
N
O
O
HO
NH
2H
N
O
.
Cu(ClO4)2 6H 2O+NaOH
Complex 1: R1=NH2, R2=CH3, X=NH, y=1
Complex 3: R1=OH, R2=H, X=O, y=0
O
H2O
H
H
N
ClO4
N
S
S
N
N
N
N
OH2
Cu
O
MeOH 50°C 1h
H2N
H
N
O
O
Complex 2
SCHEME 1 Synthesis of ligands and corresponding complexes

6 of 14
QI ET AL.
2
2
localized in the dx −y orbital, revealing the nearly square
pyramidal coordination geometry of the copper(II)
ion.[48,49]
of slope to intercept was obtained in a plot of [DNA]/(εa
− εf) versus [DNA]. The obtained Kb values for complexes
1, 2 and 3 were 3.78 × 105, 7.47 × 104 and 2.28 × 104 M−1
,
respectively, following the order 1 > 2 > 3. The Kb values
showed that 1 and 2 exhibit stronger DNA binding ability
than 3, which may be because the NH group on the imid-
azole ring of HPBM and TBZ can more easily form hydro-
gen bonds than the O atom on the thiazole ring of PBO,
which was verified by the molecular docking studies (Sec-
tion 3.2.7). In addition, the DNA binding ability of 1 was
significantly stronger than that of 2, which was due to
HPBM with large planarity and high lipophilicity.
Therefore, based on the above experimental results
and relevant literature,[32–34] the preliminary structures
of the three copper(II) complexes could be proposed with
an approximate square pyramidal geometry in which four
equatorial positions were occupied by Gly‐L‐Val (N, O)
and aromatic heterocycle (N, N) and the axial position
was occupied by an O atom of H2O as shown in Scheme 1.
3.2 | Interaction with CT‐DNA
3.2.1 | Electronic absorption spectra
3.2.2 | Fluorescence spectra
Electronic absorption spectroscopy is an effective method
for investigating the binding pattern and strength of metal
complexes with DNA. As the intercalative mode involves
a stacking interaction between the aromatic chromophore
of a complex and the base pairs of DNA, hypochromism
and red‐shift are usually associated with the insertion
ability of complexes to DNA.[50] The absorption spectra
of complexes 1–3 with DNA were recorded for a constant
complex concentration (5.0 × 10−5 M) with various [com-
plex]/[DNA] ratios (r). Representative spectra of the com-
plexes with DNA derived for various mixing ratios are
shown in Figure 1. The intensity of the absorption bands
was gradually decreased, with maximum hypochromism
of 40.35, 22.39 and 11.94%, respectively, for complexes 1,
2 and 3. In order to compare the DNA binding affinities
of 1–3 quantitatively, the binding constants (Kb) were cal-
culated using the following equation:[51]
To further confirm the binding mode of the complexes
with DNA, competitive binding experiments using EB as
a probe were carried out (Figure 2). The DNA–EB fluores-
cence intensity decreased on addition of the complexes,
showing that the complexes could displace EB from the
DNA–EB system and interact with DNA through an
intercalative mode.[52] Additionally, the Stern–Volmer
constant quenching constants (Ksv), a measure of the
binding propensity of complexes to DNA, were calculated
using the following equation:[53]
I0
¼ 1 þ Ksv½Qꢀ
(2)
I
in which I0 and I are the fluorescence intensities of
DNA + EB in the absence and presence of quencher,
respectively, Ksv is the Sterm–Volmer quenching constant
and [Q] is the concentration of the quencher. The Ksv
values of the complexes at 300 K were obtained as
1.774 × 104, 1.532 × 104 and 4.350 × 103 M−1, respectively,
following the order 1 > 2 > 3, which was consistent with
the electronic absorption spectroscopy results.
½DNAꢀ ½DNAꢀ
1
¼
þ
(1)
εa−εf
εb−εf
Kbðεb−εf Þ
in which [DNA] is the concentration of DNA and εa, εb
and εf denote, respectively, Aobs/CCu, extinction coeffi-
cients of complex in bound form and free. Also, the ratio
FIGURE 1 Absorption spectra of complexes (a) 1, (b) 2 and (c) 3. Arrows indicate the change upon increasing DNA concentration. Insets:
Plots of [DNA]/(εf − εa) versus [DNA] for the titration of DNA with complexes

QI ET AL.
7 of 14
FIGURE 2 Emission spectra of CT DNA–EB system upon titration of complexes (a) 1, (b) 2 and (c) 3. The arrows show the change upon
increasing concentration of complexes. (d) Plot of F0/F versus [Q] for the titration of the complexes
3.2.3 | Viscosity measurements
The viscosity of DNA, which is sensitive enough to DNA
length changes, can offer reliable evidence for the interac-
tive binding mode of complexes with DNA. If the interac-
tion mode is intercalative, DNA base pairs will separate to
contain the bound compound, resulting in an increase of
the DNA helix length, and thus an increase in DNA vis-
cosity. On the other hand, if a complex binds to DNA by
a partial or non‐classic intercalative mode, DNA helix will
bend or kink, thus causing a reduction of its valid length,
with the DNA viscosity keeping invariant or displaying a
slight decrease.[54–56] In order to further illustrate the
essence of the interaction between the complexes and
DNA, viscosity measurements were performed and the
results are shown in Figure 3. The relative viscosity of
DNA increased continuously with the addition of the
complexes, indicating that the complexes bind to DNA
through an intercalative mode. The increased degree of
the viscosity follows the order 1 > 2 > 3, which was in
agreement with the results obtained from electronic
absorption spectra and fluorescence spectra.
FIGURE 3 Relative viscosities of CT‐DNA (50 μM) in the
presence of the complexes and EB at 302 K
molecule–DNA interactions, so we can monitor these
very smalls DNA changes by using this technique and
then obtain useful conformation information. The CD
spectrum of free DNA consists of two CD signals: a pos-
itive band due to base stacking (274 nm) and a negative
one due to right‐hand helicity (246 nm) of B‐DNA,
while changes in these signals are usually ascribed to
corresponding changes in DNA structure.[22,57,58] The
3.2.4 | CD spectra
The technique of CD spectroscopy is quite sensitive to
DNA conformation changes which are caused by small

8 of 14
QI ET AL.
CD spectra of DNA with complexes 1–3 are shown in
Figure 4. On addition of the complexes to DNA solu-
tion, the strength of the DNA bands was decreased
and the strength of the positive band was obviously
higher than that of the negative band. This phenome-
non is generally observed when a complex is interca-
lated into the bases of DNA, since it unwinds the
double helix of DNA, and thus decreases the strength
of the CD bands.[59] The results showed that the
decreasing extent of these bands kept the order 1 > 2
> 3, which was in accordance with the binding abilities
of the complexes to DNA.
FIGURE 5 Temperature melting curves of CT‐DNA (50 μM) in
the absence and presence of complexes (30 μM) at 260 nm
3.2.5 | DNA thermal denaturation
DNA melting experiment is an important tool for study-
ing the interaction of small molecules with DNA. A melt-
ing temperature (Tm) change of about 5–8 °C is usually
observed upon the intercalation of complexes into
DNA, whereas non‐intercalation of complexes leads to
no marked increase in melting point.[58] Under our
experimental conditions, the melting curves of DNA in
the absence and presence of complexes 1–3 are provided
in Figure 5. The Tm of DNA was 84.5 °C and that in the
presence of complexes 1, 2 and 3 was 91.6, 91.1 and
88.9 °C, respectively. Therefore, the ΔTm values (7.1,
6.6 and 4.4 °C) indicated that complexes 1 and 2 could
be strongly inserted into DNA, but complex 3 could only
be slightly inserted. Moreover, the ΔTm changes revealed
the bonding strength still follows the order of 1 > 2 > 3,
which was consistent with the results of studies
discussed above.
3.2.6 | pBR322 DNA excision
The DNA cleavage ability of complexes 1–3 was moni-
tored using the agarose gel electrophoresis technique by
reacting supercoiled pBR322 plasmid DNA (20 μM)
with 1–3 (5, 10, 15 and 20 μM) in the absence and
presence of VC (50 μM) (Figure 6; Table 1). When
DNA alone, VC, Cu(ClO4)·6H2O, HPBM/TBZ/PBO,
Gly‐L‐Val and complexes 1–3 were used for control
experiment, no marked cleavage was observed, but
the complexes could cleave DNA more efficiently in
the presence of VC, and the cleavage exhibited a con-
centration‐dependent manner. Among the complexes, 1
possessed the highest DNA cleavage activity (Table 1),
which was closely linked to its highest DNA binding affin-
ity (as discussed above).
In order to investigate the DNA cleavage mechanism
of the complexes, various additives, namely hydroxyl rad-
ical scavengers (DMSO, tert‐butyl alcohol and EtOH), a
singlet oxygen scavenger (NaN3) and a superoxide anion
scavenger (SOD), were used to determine the nature of
the reactive species involved in the DNA oxidative cleav-
age reactions (Figure 7; Table 2). When a hydroxyl radical
scavenger (DMSO, tert‐butyl alcohol and EtOH) was
added to the reaction mixture of the complexes and
pBR322 DNA, the DNA cleavage activity was significantly
•
inhibited (lanes 4–6), suggesting the involvement of OH
radicals in the DNA cleavage process. Upon adding a sin-
glet oxygen (1O2) quencher (NaN3) to the reaction mix-
ture, the cleavage activity was not clearly inhibited (lane
1
7), indicating that O2 was not involved in the cleavage
process. Furthermore, upon addition of superoxide anion
scavenger (SOD) to the reaction mixture, cleavage reac-
•−
tions were promoted (lane 8), revealing that O2 was
FIGURE 4 CD spectra of complexes 1, 2 and 3 upon titration of
CT‐DNA at a molar ratio of 3:5 (complex to DNA)
likely to be indirectly involved in the cleavage process.

QI ET AL.
9 of 14
FIGURE 6 Cleavage of pBR322 DNA (250 ng) with (a) 1, (b) 2 and (c) 3. Lane 1, DNA control; lane 2, DNA + VC (50 μM); lane 3,
DNA + Cu(ClO4)·6H2O (20 μM); lane 4, DNA + HPBM/TBZ/PBO (20 μM); lane 5, DNA + Gly‐L‐Val (20 μM); lane 6, DNA + 1/2/3 (20
μM); lanes 7–10, DNA + VC (50 μM) + 1/2/3 (5, 10, 15 and 20 μM, respectively)
TABLE 1 Cleavage of pBR322 DNA by complexes after incubation for 1 h at 37 °C
Form (%)
Lane number
Reaction conditions
I
II
III
1
DNA (1/2/3)
88.4/91.2/94.9
11.6/8.8/5.1
0/0/0
6
DNA + 1/2/3 (20 μM)
88.1/90.0/92.0
33.5/61.5/74.4
27.1/46.4/49.7
0/0/39.3
11.9/10.0/8.0
66.5/38.5/25.6
72.9/53.6/50.3
68.1/82.7/60.7
28.6/40.6/78.8
0/0/0
7
DNA + VC + 1/2/3 (5 μM)
DNA + VC + 1/2/3 (10 μM)
DNA + VC + 1/2/3 (15 μM)
DNA + VC + 1/2/3 (20 μM)
0/0/0
8
0/0/0
9
31.9/17.3/0
71.4/59.4/0
10
0/0/21.2
FIGURE 7 Cleavage of pBR322 DNA (250 ng) by (a) 1, (b) 2 and (c) 3 at 37 °C with incubation time of 1 h in the presence of various typical
reactive oxygen species scavengers. Lane 1, DNA control; lane 2, DNA + VC (50 μM) + SOD (15 units); lane 3, DNA + VC (50 μM) + 1/2/3 (10
μM); lanes 4–8, DNA + VC (50 μM) + 1/2/3 (10 μM) + [DMSO (0.2 M), tert‐butyl alcohol (0.2 M), EtOH (0.2 M), NaN3 (0.2 M) and SOD
(15 units), respectively]
All the results suggested that the complexes oxidatively
cleaved DNA through the OH mechanism.
biomolecules like DNA towards small molecules or drug
molecules with the aid of a computer.[58,60] For the pur-
pose of this work, we chose double‐strand DNA with the
sequence d(5′‐G‐dIU‐TGCAAC‐3′) (PDB ID: 454D). The
results of analyses of docking between the complexes
and DNA are shown in Figure 8. The resulting docked
models indicated that complexes 1–3 were likely to insert
•
3.2.7 | Molecular docking
The molecular docking technique, as a tool to assist in
biophysics research, can simulate the binding of

10 of 14
QI ET AL.
TABLE 2 Oxidative cleavage of pBR322 DNA by complexes in presence of various typical reactive oxygen species scavengers after incu-
bation for 1 h at 37 °C
Form (%)
Lane
number
Reaction conditions
I
II
III
1
DNA (1/2/3)
90.7/87.8/92.7
9.3/12.3/7.3
0/0/0
3
4
5
6
7
8
DNA + VC + 1/2/3
24.5/48.2/52.2
69.9/64.4/77.0
71.3/65.3/75.8
61.4/68.8/70.2
30.4/45.1/48.2
0/23.9/38.4
75.5/51.8/47.8
30.1/35.6/23.0
28.7/34.7/24.2
38.6/31.2/29.8
69.6/54.9/51.8
55.3/76.1/61.6
0/0/0
0/0/0
0/0/0
0/0/0
0/0/0
44.7/0/0
DNA + VC + 1/2/3 + DMSO
DNA + VC + 1/2/3 + tert‐butyl alcohol
DNA + VC + 1/2/3 + EtOH
DNA + VC + 1/2/3 + NaN3
DNA + VC + 1/2/3 + SOD
FIGURE 8 Molecular docked models of (a) complexes (spheres representation) with DNA. (b) Hydrogen bonds of the complexes (stick
representation) and DNA (cartoon form) are expressed as red dashed lines. (a1, b1: Complex 1; a2, b2: Complex 2; a3, b3: Complex 3)
into the empty cavity within GC/GC base pair‐rich region
from the DNA central position through the aromatic het-
erocyclic ligands. Thus, hydrophobic interactions were
the main force. Moreover, different amounts of hydrogen
bonds were formed between the complexes and DNA. The
hydrogen‐bonding interaction had an important effect on
the binding affinity of the complexes with DNA, in which
complex 1 showed the best binding affinity due to it
forming more hydrogen bonds with DNA nucleobases
G4, G12, C5 and C13, but complex 3 had the lowest
binding affinity owing to a lack of interaction. The
resulting relative binding energies of the docked com-
plexes 1–3 with DNA were found to be −37.24, −32.46
and −30.06 kJ mol−1, respectively. The more negative
the relative binding energy, the stronger the binding abil-
ity of the complex to DNA. Therefore, the docking results
revealed that the binding affinities of the complexes with
DNA followed the order 1 > 2 > 3, which was positively
related to the experimental results of DNA binding
discussed above.

QI ET AL.
11 of 14
3.3 | In Vitro cytotoxicity
The cytotoxic activity of the complexes against cell lines
A549, PC‐3, HeLa and 3 T3 was studied by using the
MTT method with cisplatin as a positive control. All cell
lines were incubated for 48 h with an increasing con-
centration (3.125–200 μM) of each of 1, 2 and 3. The
results were analyzed by cell inhibition expressed as
IC50 values (Table 3). The results showed that the order
of cytotoxicity was 1 > cisplatin > 2 > 3, which was in
good agreement with their DNA binding and oxidative
DNA cleavage abilities. The copper complex with HPBM
was more active than that with TBZ/PBO, which may
be due to the higher lipid solubility of HPBM. However,
1 exhibited higher cytotoxic effects against the normal
cell line (3 T3) than tumor cell lines (HeLa, A549 and
PC‐3). In addition, compared with the results of our
previous research,[28] it was found that the copper(II)
complexes with Gly‐L‐Val were more cytotoxic than
those with Gly‐gly, and the influence of N,N‐aromatic
heterocyclic ligands was more noticeable than that of
dipeptides.
FIGURE
9
Antibacterial activities of the complexes,
Cu(ClO4)2·6H2O, Gly‐L‐Val, HPBM, TBZ and PBO against B.
subtilis, S. aureus, E.coli and P. aeruginosa. Inset: (a) B. subtilis; (b) S.
aureus; (c) E.coli; (d) P. aeruginosa
the microorganisms was B. subtilis > S. aureus > E. coli
> P. aeruginosa. The complexes showed higher antimi-
crobial activities than the corresponding ligands,
which might be mainly due to the structural changes
caused by coordination. In the complexes, the polarity
of the metal ion was reduced due to the partial shar-
ing of the negative charge of the heterocyclic ligands,
thereby causing an increase of the lipophilic nature of
the central metal ion which could penetrate into lipid
layers of microorganisms more efficiently and then
destroy them.
The higher antimicrobial activities of 1 and 2 com-
pared with 3 may be attributed to the better biocompati-
bility of the benzimidazole‐like ligands.[18,20] The
antimicrobial activity of complex 1 was significantly
greater than that of complex 2, which may be mainly
due to the relatively strong lipophilic property of HPBM
compared with TBZ, having been verified by experiment.
Furthermore, the results using the dilution method also
proved the above results of the agar diffusion method. It
Of note, complex 2 exhibited lower cytotoxic effect
compared with cisplatin, but it had no injurious effect
on normal cells, indicating that 2 might be suitable as
an effective anticancer drug against PC‐3.
3.4 | In Vitro antibacterial activity
In order to explore the antibacterial properties of the
complexes, Cu(ClO4)2·6H2O, Gly‐L‐Val, HPBM, TBZ,
PBO and complexes 1, 2 and 3 were evaluated against
microorganisms B. subtilis, S. aureus, E. coli and P.
aeruginosa through the agar diffusion and double dilu-
tion methods.
The diameters of inhibition zones as well as the MIC
and MBC values are presented in Figure 9 and Tables 4
and 5. From Figure 9, it can be seen that both the com-
plexes and some ligands showed certain inhibition for all
bacterial strains, but TBZ ligand did not show any
inhibition. The sensitivity rank of the complexes against
TABLE 3 IC50 values of complexes and cisplatin against various cell lines
IC50 (μM)
Compound
A549
PC‐3
HeLa
3 T3
9.33 0.5
Complex 1
10.08 0.3
13.09 0.7
9.93 0.3
Complex 2
61.73 3.7
66.10 2.6
24.77 1.7
68.14 1.8
38.04 1.8
101.93 11.2
20.93 1.4
93.44 5.8
56.84 5.6
81.02 9.5
17.39 3.7
98.63 6.9
138.04 1.4
83.18 2.0
29.51 1.9
—
Complex 3
Cisplatin
[Cu(PBO)(Gly‐gly)(H2O)]·ClO4·1.5H2O[24]

12 of 14
QI ET AL.
TABLE 4 Antibacterial activities (μg ml−1) of complexes and Cu(ClO4)2·6H2O
Complex 1
Complex 2
Complex 3
Cu(ClO4)2·6H2O
Species
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
320
B. subtilis
13
16
100
128
150
160
300
S. aureus
E. coli
18
26
64
20
150
200
320
320
320
320
400
320
320
320
512
400
32
256
512
512
P. aeruginosa
512
>512
>512
>512
TABLE 5 Antibacterial activities (μg ml−1) of ligands (HPBM, TBZ, PBO, Gly‐L‐Val)
HPBM
TBZ
PBO
Gly‐L‐Val
Species
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
B. Subtilis
64
512
320
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
S. Aureus
E. Coli
320
64
>512
>512
>512
>512
400
>512
>512
>512
>512
>512
>512
>512
>512
>512
P. Aeruginosa
512
512
is evident (Table 4) that the antimicrobial activity of com-
plex 1 is much greater than that of the other two com-
plexes, indicating that the complex has more potential
for clinical application.
the rational design of potential novel antitumor and anti-
bacterial agents in the future.
ACKNOWLEDGMENTS
This work was supported by the Natural Science Fund
(no. 2015A030313423) and College Students’ Innova-
tion and Entrepreneurship Training Program (no.
1056413042) in Guangdong.
4 | CONCLUSIONS
Three new copper(II) complexes containing Gly‐L‐Val and
N,N‐aromatic heterocyclic ligand were synthesized and
characterized. DNA binding, DNA cleavage, cytotoxicity
and antibacterial activities were investigated. The interac-
tion of the complexes with CT‐DNA has been studied
with various spectroscopic techniques, and it has been
revealed that the complexes bind to CT‐DNA through
the intercalative mode. In subsequent experiments,
pBR322 DNA cleavage verified that the complexes could
induce DNA damage in the presence of VC. The cytotox-
icity of the complexes was investigated against three dif-
ferent cancer cell lines (A549, PC‐3 and HeLa) and
normal cell line (3 T3). The IC50 values indicated that
complex 1 exhibited high cytotoxicity. In addition, the
cytotoxic effect of 2 was lower than that of 1 and cisplatin,
but it has no injurious effect on normal cell line, indicat-
ing that 2 has the potential as an effective anticancer drug
against PC‐3. Furthermore, the antibacterial activity of
the complexes against four different microorganisms was
enhanced compared with the free ligands, and the struc-
tures of the N,N‐aromatic heterocyclic ligands influenced
the activity in the order of HPBM > TBZ > PBO. These
encouraging studies may provide valuable insights into
ORCID
Xue‐Yi Le http://orcid.org/0000-0002-5703-336X
REFERENCES
[1] C. H. Leung, H. Z. He, L. J. Liu, M. Wang, S. H. Chan, D. L. Ma,
Coord. Chem. Rev. 2013, 257, 3139.
[2] U. Ndagi, N. Mhlongo, M. E. Soliman, Drug Des. Dev. Ther.
2017, 11, 599.
[3] C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato, C.
Marzano, Chem. Rev. 2014, 114, 815.
[4] K. M. Deo, B. J. Pages, D. L. Ang, C. P. Gordon, J. R. Aldrich‐
Wright, Int. J. Mol. Sci. 2016, 17, 1818.
[5] K. H. Reddy, J. Indian Chem. Soc. 2003, 80, 69.
[6] A. T. Chaviara, P. C. Christidis, A. Papageorgiou, E.
Chrysogelou, D. J. Hadjipavlou‐Litina, C. A. Bolos, J. Inorg.
Biochem. 2005, 99, 2102.

QI ET AL.
13 of 14
[7] A. Meenongwa, R. F. Brissos, C. Soikum, P. Chaveerach, P.
Gamez, Y. Trongpaniche, U. Chaveerach, New J. Chem. 2016,
40, 5861.
[30] X. B. Fu, Z. H. Lin, H. F. Liu, X. Y. Le, Spectrochim. Acta A
2014, 122, 22.
[31] X. B. Fu, G. T. Weng, D. D. Liu, X. Y. Le, J. Photochem.
Photobiol. A 2014, 276, 83.
[8] A. Jayamani, V. Thamilarasan, N. Sengottuvelan, P.
Manisankar, S. K. Kang, Y. I. Kim, V. Ganesan, Spectrochim.
Acta A 2014, 122, 365.
[32] Q. Gan, Y. Y. Qi, Y. H. Xiong, Y. L. Fu, X. Y. Le, J. Fluoresc.
2017, 27, 701.
[9] T. Kiran, V. G. Prasanth, M. M. Balamurali, C. S. Vasavi, P.
Munusami, K. I. Sathiyanarayanan, M. Pathak, Inorg. Chim.
Acta 2015, 433, 26.
[33] Q. Gan, X. B. Fu, W. J. Chen, Y. H. Xiong, Y. L. Fu, S. Chen, X.
Y. Le, J. Fluoresc. 2016, 26, 905.
[34] Q. Gan, C. L. Zhang, B. F. Wang, Y. H. Xiong, Y. L. Fu, Z. W.
Mao, X. Y. Le, RSC Adv. 2016, 6, 35952.
[10] R. A. Steiner, D. Foreman, H. X. Lin, B. K. Carney, K. M. Fox, L.
Cassimeris, J. M. Tanski, L. A. Tyler, J. Inorg. Biochem. 2014,
137, 1.
[35] R. Schiffmann, A. Neugebauer, C. D. Klein, J. Med. Chem. 2006,
49, 511.
[11] M. J. Li, T. Y. Lan, X. H. Cao, H. H. Yang, Y. P. Shi, C. Q. Yi, G.
[36] A. W. Addison, T. N. Rao, C. G. Wahlgen, J. Heterocycl. Chem.
1983, 20, 1481.
N. Chen, Dalton Trans. 2014, 43, 2789.
[12] B. L. Fei, W. S. Xu, H. W. Tao, W. Li, Y. Zhang, J. Y. Long, Q. B.
[37] M. E. Reichmann, S. A. Rice, C. A. Thomas, P. Doty, J. Am.
Chem. Soc. 1954, 76, 3047.
Liu, B. Xia, W. Y. Sun, J. Photochem. Photobiol. B 2014, 132, 36.
[38] O. Trott, A. J. Olson, J. Comput. Chem. 2010, 31, 455.
[13] R. Kumar, S. Obrai, A. Kaur, G. Hundal, H. Meehnian, A. K.
Jana, Polyhedron 2013, 56, 55.
[39] A. M. Bukonjic, D. L. Tomovic, M. V. Nikolic, M. Z. Mijajlovic,
V. V. Jevtic, Z. R. Ratkovic, S. B. Novakovic, G. A. Bogdanovic,
I. D. Radojevic, J. Z. Maksimovic, S. M. Vasic, L. R. Comic, S. R.
Trifunovic, G. P. Radic, J. Mol. Struct. 2017, 1128, 330.
[14] R. Kumar, S. Obrai, A. Kaur, M. S. Hundal, J. Mitra, S. Sharma,
J. Coord. Chem. 2015, 68, 2130.
[15] R. Kumar, S. Obrai, A. Sharma, A. K. Jassal, M. S. Hundal, J.
[40] S. Indoria, T. S. Lobana, H. Sood, D. S. Arora, G. Hundal, J. P.
Mitra, J. Mol. Struct. 2014, 1075, 43.
Jasinski, New J. Chem. 2016, 40, 3642.
[16] P. Singla, V. Luxami, R. Singh, V. Tandon, K. Paul, Eur. J. Med.
Chem. 2017, 126, 24.
[41] W. J. Geary, Coord. Chem. Rev. 1971, 7, 81.
[42] K. Nakamoto, Appl. Inorg. Chem. 2008, 77, 1122.
[17] P. Vicini, M. Incerti, L. Amoretti, V. Ballabeni, M. Tognolini, E.
[43] R. S. Kumar, S. Arunachalam, Eur. J. Med. Chem. 2009, 44,
Barocelli, Farmaco 2002, 57, 363.
1878.
[18] H. Z. Zhang, S. C. He, Y. J. Peng, H. J. Zhang, L. Gopala, V. K.
R. Tangadanchu, L. L. Gan, C. H. Zhou, Eur. J. Med. Chem.
2017, 136, 165.
[44] A. Garcıa‐Raso, J. J. Fiol, B. Adrover, V. Moreno, I. Mata, E.
Espinosa, E. Molins, J. Inorg. Biochem. 2003, 95, 77.
[45] P. R. Reddy, N. Raju, Polyhedron 2012, 44, 1.
[19] J. Akhtar, A. A. Khan, Z. Ali, R. Haider, M. S. Yar, Eur. J. Med.
Chem. 2017, 125, 143.
[46] F. Arjmand, A. Jamsheera, D. K. Mohapatra, J. Photochem.
Photobiol. B 2013, 121, 75.
[20] N. Shrivastava, M. J. Naim, M. J. Alam, F. Nawaz, S. Ahmed, O.
[47] E. Prenesti, P. G. Daniele, S. Berto, S. Toso, Polyhedron 2006, 25,
Alam, Arch. Pharm. 2017, 350, e201700040.
2815.
[21] J. Y. Hu, C. L. Liao, Y. Guo, F. Yang, W. Sang, J. A. Zhao, Poly-
hedron 2017, 132, 28.
[48] S. K. Tadavi, J. D. Rajput, S. D. Bagul, A. A. Hosamani, J. N.
Sangshetti, R. S. Bendre, Res. Chem. Intermed. 2017, 43, 4863.
[22] A. Saswati, S. Chakraborty, P. Dash, A. K. Panda, R. Acharyya,
A. Biswas, S. Mukhopadhyay, S. K. Bhutia, A. Crochet, Y. P.
Patil, M. Nethajie, R. Dinda, Dalton Trans. 2015, 44, 6140.
[49] B. Annaraj, M. A. Neelakantan, Eur. J. Med. Chem. 2015, 102, 1.
[50] G. W. Zhang, P. Fu, L. Wang, M. M. Hu, J. Agric. Food Chem.
2011, 59, 8944.
[23] S. Mandal, G. Das, H. Askari, New J. Chem. 2015, 39, 5208.
[51] P. Sinha, N. Kumari, K. Singh, K. Singh, L. Mishra, Inorg. Chim.
Acta 2015, 432, 71.
[24] M. Yagasaki, S. I. Hashimoto, Appl. Microbiol. Biotechnol. 2008,
81, 13.
[52] S. Tabassum, M. Ahmad, M. Afzal, M. Zaki, P. K. Bharadwaj, J.
Photochem. Photobiol. B 2014, 140, 321.
[25] G. Facchin, M. H. Torre, E. Kremer, O. E. Piro, E. E. Castellano,
E. J. Baran, J. Inorg. Biochem. 2002, 89, 174.
[53] F. J. Fard, Z. M. Khoshkhoo, H. Mirtabatabaei, M. R.
Housaindokht, R. Jalal, H. E. Hosseini, M. R. Bozorgmehr, A.
A. Esmaeili, M. J. Khoshkholgh, Spectochim. Acta A 2012, 97,
74.
[26] R. Pogni, M. C. Baratto, E. Busi, R. Basosi, J. Inorg. Biochem.
1999, 73, 157.
[27] S. Iglesias, N. Alvarez, M. H. Torre, E. Kremer, J. Ellena, R. R.
Ribeiro, R. P. Barroso, A. J. Costa‐Filho, M. G. Kramer, G.
Facchin, J. Inorg. Biochem. 2014, 139, 117.
[54] J. W. Liang, Y. Wang, K. J. Du, G. Y. Li, R. L. Guan, L. N. Ji, H.
Chao, J. Inorg. Biochem. 2014, 141, 17.
[28] X. B. Fu, D. D. Liu, Y. Lin, W. Hu, Z. W. Mao, X. Y. Le, Dalton
Trans. 2014, 43, 8721.
[55] H. L. Wu, H. Wang, X. L. Wang, G. L. Pan, F. R. Shi, Y. H.
Zhang, Y. C. Bai, J. Kong, New J. Chem. 2014, 38, 1052.
[29] X. B. Fu, J. J. Zhang, D. D. Liu, Q. Gan, H. W. Gao, Z. W. Mao,
[56] L. H. Abdel‐Rahman, A. M. Abu‐Dief, H. Moustafa, S. K.
Hamdan, Appl. Organometal. Chem. 2017, 31, e3555.
X. Y. Le, J. Inorg. Biochem. 2015, 143, 77.

14 of 14
QI ET AL.
[57] J. Lu, Q. Sun, J. L. Li, L. Jiang, W. Gu, X. Liu, J. L. Tian, S. P.
Yan, J. Inorg. Biochem. 2014, 137, 46.
How to cite this article: Qi Y‐Y, Liu Y‐X, Gan Q,
Xiong Y‐H, Mao Z‐W, Le X‐Y. Three new mixed‐
ligand copper(II) complexes containing glycyl‐L‐
valine and N,N‐aromatic heterocyclic compounds:
Synthesis, characterization, DNA interaction,
cytotoxicity and antimicrobial activity. Appl
Organometal Chem. 2017;e4126. https://doi.org/
10.1002/aoc.4126
[58] P. Haris, V. Mary, M. Haridas, C. Sudarsanakumar, J. Chem. Inf.
Model. 2015, 55, 2644.
[59] M. Zaki, M. Afzal, M. Ahmad, S. Tabassum, J. Photochem.
Photobiol. B 2016, 161, 318.
[60] M. A. Rizvi, M. Zaki, M. Afzal, M. Mane, M. Kumar, B. A. Shah,
S. Srivastav, S. Srikrishna, G. M. Peerzada, S. Tabassum, Eur. J.
Med. Chem. 2015, 90, 876.