Three structurally related Copper complexes with two isomers: DNA/BSA binding ability, DNA cleavage activity and excellent cytotoxicity

Article abstract of DOI:10.1016/j.ica.2016.12.002

Three Cu(II) complexes of [Cu(L¹)(Br)₂]·CH₃CN (1), [Cu(L²)₂(OAc)](PF₆)·2C₂H₅OH (2), and [Cu(L¹)(L²)](PF₆)₂(3) (L¹

Full text of DOI:10.1016/j.ica.2016.12.002

Inorganica Chimica Acta 457 (2017) 7–18
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Research paper
Three structurally related Copper complexes with two isomers: DNA/
BSA binding ability, DNA cleavage activity and excellent cytotoxicity
Dong-Yan Zhang a,c,1, Yan Nie b,1, Hui Sang b, Jing-Jing Suo a,c, Zong-Jin Li b, Wen Gu a,c, Jin-Lei Tian a,c,
,
⇑
Xin Liu a,c, Shi-Ping Yan a,c
a Department of Chemistry, Nankai University, Tianjin 300071, PR China
b Medical School of Nankai University, Nankai University, Tianjin 300071, PR China
c Key Laboratory of Advanced Energy Materials Chemistry (MOE), Nankai University, Tianjin 300071, PR China
a r t i c l e i n f o
a b s t r a c t
Three Cu(II) complexes of [Cu(L1)(Br)2]ꢀCH3CN (1), [Cu(L2)2(OAc)](PF6)ꢀ2C2H5OH (2), and [Cu(L1)(L2)]
Article history:
Received 5 June 2016
Received in revised form 25 November 2016
Accepted 1 December 2016
Available online 2 December 2016
(PF6)2 (3) (L1 = 2-(6-(pyridine-2-yl)-4-p-tolylpyridin-2-yl)pyridine,
L
2 = 2-(4-(pyridine-2-yl)-6-p-tolyl-
pyridin-2-yl)pyridine) were designed and synthesized as anti-cancer drugs. All complexes were struc-
turally characterized by X-ray crystallography showing that three complexes were the mononuclear
ꢀ
compounds with the triclinic crystal system p1 space group. The interaction between each of these three
complexes and calf thymus DNA (CT-DNA) was investigated via spectroscopic techniques and viscosity
measurement, which indicated that these complexes could bind to CT-DNA by intercalation. Moreover,
DNA cleavage experiment showed that, in the natural light under an aerobic environment, all complexes
could cleave DNA in the absence of exogenous oxidant agent, and that singlet oxygen (1O2) might serve as
the major cleavage active species. Protein binding experiment indicated that 1–3 could bind to bovine
serum albumin (BSA) with moderate bonding via the static quenching mechanism. In addition, the
IC50 values of these three complexes were significantly lower which denoted highly anti-cancer activities
in comparison to cisplatin. And each of these complexes could individually inhibit proliferation potential
of cancer cells by promoting G1-phase cell cycle arrest (G1 arrest) and inducing apoptosis through the
production of reactive oxygen species (ROS) and the activation of caspase-3
Keywords:
Copper complexes
DNA cleavage
Anti-cancer ability
ROS
Caspase-3
Ó 2016 Elsevier B.V. All rights reserved.
1. Introduction
metal complexes and DNA is of the most importance in under-
standing the mechanism of the treatment for cancer. Moreover,
According to statistics, the number of people diagnosed with
cancer and people died from cancer has been considerably
increased during the past several decades [1,2]. However, the
emergence of different kinds of anti-cancer regimens and potential
anti-cancer drugs, such as cisplatin, have many drawbacks like
general toxicity, nonspecific targeting and acquired drug resistance
[3–6]. Thus, researchers now face their biggest challenge of devel-
oping novel anti-cancer therapies. Metal-based complexes served
as anti-cancer drugs have attracted a great deal of attention of
researchers because the metal ions play important roles in biolog-
ical processes [7]. DNA is the primary pharmacological target of
many metal-based drugs, hence exploring the interaction between
studies showed that transition metal complexes with DNA cleav-
age activity could be promising DNA structural probes and thera-
peutic agents [8–10].
Transition metal complexes have been synthesized and devel-
oped rapidly for chemotherapeutic applications attributing to their
various coordination modes, redox behaviors, feasible substitution
kinetic pathways, and highly anti-cancer activity [11]. Many tran-
sition metal complexes (such as Ru, Ln, V, Co, Zn, Cu, etc.) have
been reported for their DNA cleavage activity, DNA/BSA binding
ability and cytotoxicity [12–17]. Among those transition metal
compounds, copper complexes have shown broad prospects as
the anti-cancer drugs for the following reasons: copper is an essen-
tial element related to life process and an active component in var-
ious bio-enzymes; Cu(II) ion is easy to form complex with ligands;
Cu(II) ion itself own redox potential and can produce ROS when
they participate in the physiological reaction [18–21].
⇑
Corresponding author at: Department of Chemistry, Nankai University, Tianjin
300071, PR China.
E-mail addresses: tiant@nankai.edu.cn (J.-L. Tian), liuxin64@nankai.edu.cn (X.
In the field of Cu(II) compounds acting as anti-cancer agent,
complexes combined with polypyridine ligands which have aro-
Liu).
1
Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ica.2016.12.002
0020-1693/Ó 2016 Elsevier B.V. All rights reserved.

8
D.-Y. Zhang et al. / Inorganica Chimica Acta 457 (2017) 7–18
maticity and strong coordination ability with Cu(II) ions played the
most important roles [22,23]. One of the most widely studied poly-
pyridine ligands are terpyridyl and its derivatives. According to the
reported literatures, terpyridine ligands can inhibit protein kinase
activity and have cytotoxicity against several human tumor cell
lines because the terpyridine could partly form a conjugated aro-
matic ring system with a relatively planar structure [24,25]. In
recent years, a series of Cu(II) complexes based on terpyridine
and its derivatives have been reported as potent DNA cleavage
agents, and these derivatives with different substituent groups
on the 4 site of the middle pyridine ring of terpyridine have been
designed and synthesized in order to enhance the selectivity
towards tumor cell as well as improve the anti-cancer activity
[26,27]. Guo and co-workers reported a mitochondrion-targeting
copper complex ([Cu(ttpy-tpp)Br2]Br, ttpy-tpp = 40-p-tolyl-
(2,20:60,200-terpyridyl)triphenyl phosphonium bromide). As a triph-
enyl phosphate (TPP) group was added to the 4 site of the middle
pyridine ring of terpyridine, the complex could specifically identify
the mitochondrion when it accessed to the tumor cells [28].
All the above facts stimulated our interest in the present work
on describing the synthesis of two isomers: L1 = 2-(6-(pyridine-2-
yl)-4-p-tolylpyridin-2-yl)pyridine and L2 = 2-(4-(pyridine-2-yl)-6-
p-tolylpyridin-2-yl)pyridine suitable to coordinate metal ions.
The two molecules act as tridentate ligands to the Copper(II) ion
through the three heterocycle N atoms. Three Cu(II) compounds
were synthesized and characterized by spectroscopic techniques
and X-ray diffraction, which showed that they were mononuclear
compounds with five-coordinated ligands, and were similar to
the reported complexes with tripyridine ligands [29,30]. And there
is a significant difference between the ligands used in our study
and previously investigation. The main objective of this work is
to determine the cytotoxicity of these new complexes and study
the possible mechanisms action as anti-cancer drugs. UV-visible
spectroscopy, fluorescence spectral titration and viscosity mea-
surement showed that the three complexes had a strong binding
affinity to DNA than previously reported Copper(II) complexes.
Spectrum experiments revealed that complexes could combine
with BSA. Three Copper(II) compounds also showed good DNA
cleavage activity in the absence of exogenous oxidants, and they
could generate singlet oxygen (1O2) in the natural light under an
aerobic environment, which was consisted with previous reports
[31–36]. All these complexes exhibited excellent anti-cancer
potency with considerably lower IC 50 values against three human
tumor cell lines in comparison to cisplatin. Further tests with the
aim to explore the mechanisms behind the enhanced anti-cancer
activity were also performed.
1H NMR spectra were recorded on a Bruker 400 (1H/400 MHz)
spectrometer. Infrared spectroscopy on KBr pellets was performed
on a Bruker Vector 22 FT-IR spectrophotometer in the 4000–
400 cmꢁ1 range. Electrospray ionization mass spectrometry (ESI-
MS) was obtained on Agilent 6520 top of flight liquid chromatog-
raphy/mass spectrometry. In our work, ESI-MS experiment was
operated in DMF solution which was attenuated by C2H5OH. UV
absorption was measured on a JASCO V-570 spectrophotometer.
Fluorescence spectral data were acquired on a MPF-4 fluorescence
spectrophotometer at room temperature. Viscosity measurements
were experimented on an Ubbelodhe viscometer at a constant
temperature (37.0 0.1 °C) in a thermostatic water-bath. The Gel
Imaging and documentation DigiDoc-It System of DNA cleavage
were assessed using Labworks Imaging and Analysis Software
(UVI, UK). MTT assay was performed on Glomax-multi detection
system (Promega, USA). HE and Hoechst 33342 staining assay kit
were purchased from KeyGEN Biotech (Nanjing, China). ROS detec-
tion experiment was performed using reactive oxygen species kit
(Beyotime Biotech, Shanghai, China). Flow cytometry analysis
was carried out with LSR Fortessa and CellQuest software (BD,
USA).
2.2. Synthesis of L1, L2 and the Copper(II) complexes of 1–3
2.2.1. Synthesis of L1 and L2
The syntheses of both L1 and L2 were based on literature proce-
dure [24,37,38]. A mixture of acetamide (36.6 g, 0.6 mmol), ammo-
nium acetate (23.6 g, 0.3 mmol), p-tolualdehyde (2.48 g,
20.6 mmol) and 2-acetylpyridine (5 g, 41.2 mmol) were heated at
160 °C with stirring for 2 h in 250 ml round-bottom flask, then
the resulting solution was cooled to 120 °C, and the aqueous
sodium hydroxide (10% solution, 20 ml) was added over 20 min
with heating to 120 °C for 2 h. The black brown paste was formed
when the solution was cooled to room temperature and filtered
out, and then washed with water and cool C2H5OH. The obtained
solid was dissolved into CHCl3 and separated using column chro-
matography (Al2O3, n-hexane: ethyl acetate = 8:1, v/v), rotary
evaporating and the white crystalline solid L1 and L2 were
obtained, individually. The structure of L1 and L2 are shown in
Scheme 1.
L1 1.9 g, 20%, 1H NMR (400 MHz, CDCl3, 300 K): d 8.78 (2H, s),
8.73 (2H, m), 8.69(2H, d), 7.88 (2H, td), 7.84 (2H, d), 7.39 (2H,
dd), 7.36 (2H, d), 2.43 (3H, s) (Fig. S1A). Selected FT-IR (KBr, m,
cmꢁ1): 1584, 1466, 1392, 1113, 789, 619, 504. ESI-MS: m/z
324.15 for [L1 + H]+.
L2: 1.1 g 12%, 1H NMR (400 MHz, CDCl3, 300 K): d 8.83 (1H, s),
8.74 (1H, d), 8.72 (1H, s), 8.69 (1H, s), 8.66 (1H, s), 7.91 (1H, s),
7.86 (2H, d) 7.83 (1H, d), 7.36 (2H, q), 7.34 (1H, s), 7.31 (1H, s),
7.29 (1H, s), 2.43 (3H, s) (Fig. S1B). Selected FT-IR (KBr, m
, cmꢁ1):
2. Experimental
1583, 1549, 1471, 1113, 779, 619, 501. ESI-MS: m/z 324.15 for
[L2 + H]+.
2.1. Materials and measurements
2.2.2. Synthesis of [Cu(L1)(Br)2]ꢀCH3CN (1)
Cisplatin, Cu(OAc)2ꢀ2H2O, NH4PF6 and all solvents were pur-
chased from Aldrich Co. Solution of the Cu(II) complexes and other
reagents used for strand scission were prepared freshly in triple-
distilled water before use. Solvents used in this research were puri-
fied by standard procedures. Ultra-pure MilliQ water
To a solution of L1 (64.6 mg, 0.2 mmol) in CH3CN (10 ml) was
added a solution of CuBr2 (44.7 mg, 0.2 mmol) in CH3OH (10 ml).
The mixture solution was refluxed with stirring for 4 h, and then
the reaction solution was cooled to room temperature and filter.
Blue and bulk crystals suitable for X-ray diffraction were obtained
by slow evaporation of the filtrate after several weeks. Yield: 55%.
(18.24 MX cm) was used in all experiments. Ethidium bromide
(EB), agarose, bovine serum albumin (BSA), calf thymus DNA (CT-
DNA), pBR322 plasmid DNA and biological stains were purchased
from Sigma. Tris–HCl buffer solution was prepared using deionized
sonicated triple-distilled water. Human breast cancer cell line
(MDA-MB-231), human epithelial cervical cancer cell line (HeLa),
and human lung cancer cell line (A549) were obtained from the
American Type Culture Collection (Rockville, MD, USA).
Selected FT-IR (KBr, m
, cmꢁ1): 1602, 1113, 619, 447. ESI-MS: m/z
466.99 for [Cu(L1)(Br)]+.
2.2.3. Synthesis of [Cu(L2)2(OAc)](PF6)ꢀ2C2H5OH (2)
To a solution of L2 (129.2 mg, 0.4 mmol) in C2H5OH (10 ml) was
added a solution of Cu(CH3COO)2ꢀ2H2O (39.8 mg, 0.2 mmol) in
C2H5OH (10 ml). The mixture solution was refluxed with stirring

D.-Y. Zhang et al. / Inorganica Chimica Acta 457 (2017) 7–18
9
Table 1
Crystal data and structure refinement for complexes 1–3.
Complex
1
2
3
Formula
C
24H20Br2CuN4 C50H49CuF6N6O4P C44H34CuF12N6P2
Mr
587.80
1006.46
Triclinic
p1
1000.25
Triclinic
p1
Crystal system
Space group
Triclinic
ꢀ
ꢀ
ꢀ
p1
T/K
a/Å
b/Å
c/Å
113 K
293(2) K
11.797(2)
14.778(3)
15.549(3)
108.49(3)
109.75(3)
100.14(3)
2294.6(8)
2
113.1500 K
10.472(2)
14.319(3)
14.895(3)
84.14(3)
69.76(3)
77.01(3)
2041.4(7)
2
8.0291(16)
12.058(3)
12.756(3)
115.147(3)
91.128(4)
96.029(3)
1108.8(4)
2
L1
L2
a
/°
b/°
Scheme 1. Schematic molecular structures of L1 and L2.
c
/°
V/Å3
Z
for 4 h, and then the reaction solution was cooled to room temper-
ature and filter. Dark blue and clubbed crystals suitable for X-ray
diffraction were obtained by slow evaporation of the filtrate after
D/g/cm3
l
1.761
4.608
5024
1.457
0.589
8094
1.627
0.712
9636
/mmꢁ1
Reflections
measured
Independent
reflections
Rint
several weeks. Yield: 52%. Selected FT-IR (KBr,
m
, cmꢁ1): 1613,
1472, 1113, 840, 619, 447. ESI-MS: m/z 709.21 for [Cu(L2)2ꢁH]+.
14,307
16,289
21,297
0.0268
0.0275
0.0979
0.0320
0.1005
0.824
0.0383
0.0489
0.1149
0.0659
0.1274
0.999
0.0412
0.0491
0.1238
0.0708
0.1426
0.840
2.2.4. Synthesis of [Cu(L1)(L2)](PF6)2 (3)
R1(I > 2
r (I))
wR(F2)(I > 2
r (I))
To a solution of L1 (64.6 mg, 0.2 mmol) in CH3OH (10 ml) was
added a solution of Cu(CH3COO)2ꢀ2H2O (39.8 mg, 0.2 mmol) in
CH3OH (5 ml). The mixture solution was refluxed with stirring
for 2 h and a solution of L2 (64.6 mg, 0.2 mmol) in CH3OH (10 ml)
was added, then the reaction solution was refluxed with stirring
for another 2 h and cooled to room temperature and filter. Dark
blue and rhomboic crystals suitable for X-ray diffraction were
obtained by slow evaporation of the filtrate after several weeks.
R1(all data)
wR(F2) (all data)
Goodness of fit on
F2
CT-DNA stock solution was added to the two pools with equal vol-
ume of scanning measurement of the absorption peak until the
solution was saturated.
Viscosity experiments were carried out by using an Ubbelodhe
viscometer maintained at a constant temperature at 37.0 0.1 °C
in a thermostatic water-bath. Flow time was measured with a dig-
ital stopwatch. Each sample was measured three times, and an
average flow time was calculated. According to Cohen and Eisen-
Yield: 58%. Selected FT-IR (KBr, m
, cmꢁ1): 1608, 1109, 832, 619,
447. ESI-MS: m/z 709.21 for [Cu(L1)(L2)ꢁH]+.
2.3. X-ray crystallography
Diffraction data for 1–3 at 293 K, with a Bruker Smart 1000 CCD
diffractometer using Mo-K
a radiation (k = 0.71073Å) with the x-
berg [43,44],
in the presence of complex, and
alone in 5 mM Tris buffer medium), the viscosity values were cal-
culated from the observed flow time of CT-DNA containing solu-
tions (t), duly corrected for that of the buffer alone (t0), and then
data were presented as (
g
= (t ꢁ t0)/t0 (where
g
0
is the viscosity of CT-DNA
is the viscosity of CT-DNA
2h scan technique. An empirical absorption correction was applied
to raw intensities [39]. The structures were analyzed by Olex2-
1.2.3 and refined with full-matrix least-squares technique on F2
using the Olex2-1.2.3 [40]. Hydrogen atoms were added according
to the theory, and refined with intrinsic thermal factors. The crys-
tallographic data and the parameters of structure refinement were
summarized in Table 1. Selected bond angles and distances were
listed in Table S1.
g
1/3
g
/g0
)
versus [complex]/[DNA].
2.4.2. Competitive binding experiments
Through fluorescence spectral method, the relative bindings of
complexes to CT-DNA were studied with an EB-bound CT-DNA
solution in 5 mM Tris–HCl/50 mM NaCl buffer (pH = 7.2). The fluo-
rescence spectra intensity was at 602 nm (excite at 510 nm), which
was carried out by titrating complexes into EB-DNA solution con-
2.4. DNA-binding experiments
2.4.1. Absorption spectrophotometric and viscosity studies
taining 2.4 lM EB and 48 lM CT-DNA.
The interaction of complexes 1–3 with CT-DNA was studied by
UV spectroscopy, and the binding of DNA with complexes were
operated at room temperature. The CT-DNA stock solutions were
prepared in 5 mM Tris–HCl/50 mM NaCl buffer (pH = 7.2), showed
a ratio of UV absorbance at 260 nm and 280 nm, (A260/A280, 1.8–
1.9) indicating that the DNA was sufficiently free of protein [41].
The exact concentration of CT-DNA was determined according to
its absorption intensity at 260 nm with a molar extinction coeffi-
cient of 6600 Mꢁ1cmꢁ1 [42]. Then the solution was stored at 4 °C
and used within 4 days. Specific methods were as follows: At room
temperature, setting various parameters required (excitation
wavelength 260 nm), 2 ml TE1 solution was added to the sample
pool and reference pool respectively, followed by scanning base-
line and deducting background; then the appropriate amount of
volume of complex solution was added to the sample pool (the
maximum absorption peak the complexes should be around 1),
the same volume of DMF solution was added to the reference pool,
and the absorption peak of the complex was scanned; after that
2.4.3. DNA cleavage and mechanism studies
The implementation of DNA cleavage experiments was per-
formed by agarose gel electrophoresis at 37.0 °C as follows:
pBR322 DNA (0.1 lg/lL) was treated with complexes 1–3 in
50 mM Tris-HCl/18 mM NaCl buffer (pH = 7.2), and incubated for
3 h at 37.0 °C. Then the samples were electrophoresed for 2 h at
0.9% agarose gel into Tris-boric acid-EDTA buffer. After elec-
trophoresis, the different DNA forms were visualized under UV
light and photo-graphed. The cleavage efficiency was measured
by determining the ability of the complex to convert the super-
coiled (SC) DNA (Form I) to nicked circular (NC) DNA (Form II)
and linear (LC) DNA (Form III) [45].
To investigate the cleavage mechanistic of pBR322DNA, differ-
ent reagents such as hydroxyl radical scavengers (DMSO, KI), sin-
glet oxygen quenchers (NaN3,
L-histidine), superoxide scavenger
(SOD), hydrogen peroxide scavenger (catalase), chelating agent

10
D.-Y. Zhang et al. / Inorganica Chimica Acta 457 (2017) 7–18
(EDTA), minor groove scavenger (Methyl Green), and major groove
scavenger (SYBR Green) were added into pBR322DNA solution
prior to the addition of the complexes.
2.10. Colony-forming assay
For colony-forming assays, single cell suspension of MDA-MB-
231 cells were seeded into 6-well tissue culture plates at a density
of 3000 cells/well and allowed to adhere overnight. Afterward,
FBS-free culture medium containing Cu(II) compounds at their
IC50 values was added to each well. After expose to these com-
plexes for 24 h, cells were washed twice with sterile PBS and incu-
bated at 37 °C in drug-free medium containing 10% FBS for 10 days.
On the 10th day, cells were fixed with 4% paraformaldehyde and
stained with 2% crystal violet in methanol.
2.5. Protein binding studies
The investigation of interaction between BSA and complexes 1–
3 were carried out by fluorescence quenching experiments. BSA
solution (1.5 mM) were prepared in 10 mM phosphate buffer
(pH = 7.0). The spectra of fluorescence quenching experiments
were recorded at room temperature with the excitation wave-
length of BSA at 280 nm (the emission wavelength at 342 nm) by
keeping the concentration of BSA on a constant (29.4
varying the concentration of complex.
lM) while
2.11. Cell cycle assays
Cell cycle distributions at different stages were determined
using cell cycle kit (BestBio). MDA-MB-231 cells were seeded to
6-well plate at density of 2 ꢂ 105 cells/well allowed to adhere
overnight. Then cells were treated with 1–3 separately at IC50 value
for 24 h. Then cells were harvested including the floating cells in
the medium and fixed in 70% ethanol at 4 °C for 3 h. Ethanol was
removed from the fixed cell through centrifugation at 1000 rpm
for 5 min. Cell pellets were re-suspended in 1 ml 1–3 Banding Buf-
2.6. Cytotoxicity assessment by MTT assay
The influence of 1–3 and Cisplatin on the proliferation of cancer
cells was measured using the MTT assay kit (Roche Diagnostics
GmbH). Cells were seeded in 96-well plates at a density of 3000
cells/well and incubated for 24 h to allow the attachment. Then,
these cells were incubated with Cu(II) complexes or DMF vehicle
control with a gradient concentration in FBS-free medium for
fer containing 100
lg/ml RNAase and incubated with 1 ml PI stain-
24 h, then 50 ll of the MTT reagent was added to each well. After
ing solution (50 g/ml) for 30 min at 4 °C in the dark according to
l
incubation for 3 h at 37 °C, the absorbance was measured at
560 nm using an enzyme linked immunosorbent assay reader (Pro-
mega, USA).
the manufacture’s instruction. Cell cycle distribution was investi-
gated using BD AccuriR C6 Flow Cytometer. Data, in the form of a
DNA histogram, were analyzed by using FlowJo 7.6 software.
2.7. Morphological changes and nuclear fragmentation
2.12. Caspase-3 activation assay
Copper complexes induced morphological changes and nuclear
chromatin condensation in MDA-MB-231 cells were evaluated by
Hematoxyline-Eosin (HE) staining and Hoechst 33342 staining
(KeyGEN Biotech, Nanjing, China). MDA-MB-231 cells were treated
with 1–3 for 24 h at the concentration of IC50. Then, cells were
fixed with 4% paraformaldehyde for 20 min, washed in PBS, and
stained with HE or Hoechst 33342 according to the manufacturer’s
protocol separately. After washing twice with PBS, HE staining
manifested the morphological changes which were observed under
a microscope, Hoechst 33342 staining showed the nuclear frag-
mentation which was observed under a fluorescent microscope
(Olympus).
The Caspase-3 activation was measured using Caspase-3 colori-
metric assay kit (KeyGEN, China) following the manufacturer’s pro-
tocol. Briefly, the MDA-MB-231 cells were incubated with
compound 1, 2 and 3 (1.5
were harvested and lysates using lysis buffer containing DTT. Each
sample of these cell lysates (200 g per sample) were mixed with
lM) for 24 h, respectively. Then cells
l
2 ꢂ reaction buffer and probe for Caspase-3 at 37 °C for 4 h. D400
values were measured using an enzyme-linked immunosorbent
assay reader (Promega, USA).
3. Result and discussion
3.1. Synthesis and characterization
2.8. Measurement of ROS
Ligands L1 and L2 were prepared with the same method, and
they were the isomeride (Scheme 1) and separated using column
chromatography (Al2O3, n-hexane: ethyl acetate = 8:1, v/v). 1H
NMR spectra showed that they had no obvious difference
(Fig. S1), and the value of ESI-MS was same: to L1, m/z 324.15 for
[L1 + H]+, to L2, m/z 324.15 for [L2 + H]+. All the Cu(II) complexes
were synthesized by reflux and slow evaporation at room temper-
ature, and they all were mononuclear compounds with the triclinic
To measure ROS generation, reactive oxygen species assay kit
(Beyotime, Cat S0033, China) was used. MDA-MB-231 cells were
seeded to 6-well plate at density of 2 ꢂ 105 cells/well. The follow-
ing day (at ꢃ 80% confluence), cells were exposed to 1–3 at concen-
tration of 1 lM for 24 h. Then cells were washed twice with sterile
PBS. To each test sample, the ROS probe, DCFH-DA, was added
according to the manufacture’s instruction. After incubation for
20 min at 37 °C, cells were washed twice with sterile PBS. Follow-
ing, those samples were observed under a fluorescent microscope
(Olympus) at 100ꢂ magnification.
ꢀ
crystal system p1 space group. The results of ESI-MS analysis indi-
cated these copper complexes kept stable in DMF solution, to 1,
466.99 for [Cu(L1)(Br)]+; to 2, 709.21 for [Cu(L2)2-H]+ and to 3,
709.21 for [Cu(L1)(L2)-H]+.
2.9. Flow cytometry analysis for apoptosis quantification
MDA-MB-231 cells seeded to 35 mm dishes at density of
2 ꢂ 105 cells/dish. The following day (at ꢃ 80% confluence), cells
were treated with 1–3 at concentration of IC50 and were harvested
at 24 h. Annexin V-FITC/propidium iodide (PI) double staining
apoptosis detection kit (Bioscience, Mountain View, CA, USA) was
used following the manufacture’s protocol. Then, cells were ana-
lyzed immediately by flow cytometry (BD Biosciences, San Diego,
CA, USA). The data were analyzed by using FlowJo 7.6 software.
3.2. X-ray crystal structure of complexes 1–3
Complexes 1–3 have been structurally characterized by X-ray
crystallography (Fig. 1), details of data collection conditions and
parameters of refinement process were given in Table 1, the
selected bond lengths and angles were given in Table S1, respec-
tively, and the distances of
were given in Table 2.
p-p interaction (Fig. 2) about 1 and 2

D.-Y. Zhang et al. / Inorganica Chimica Acta 457 (2017) 7–18
11
Fig. 1. The ORTEP diagram of coordination sphere for 1–3, the solvent molecules and all hydrogen atoms are omitted for clarity. A is for 1, B is for 2, C is for 3.
Fig. 2. A is the 1D lain structure of 1 with p-p interactions (purple) and hydrogen bond (green), B is the structure of 2 with p-p interactions (purple). (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)
Table 2
Intermolecular
ligand, two bromide ions and one acetonitrile molecules. 1 was a
mononuclear structure, as shown in Fig. 1A, Cu1 located in a
five-coordinated environment completed by three nitrogen atoms
(N1, N2 and N3) from L1 ligand and two bromide atoms (Br1 and
Br2). The distance between metal and coordination atoms were:
p-
p
interaction distances (Å).
Plane A
p-
p
Plane B
Distances
3.8537(16)
3.5220(18)
interactions
1
Cg(A)—Cg(B)
N2—C6—C7—C8—
C9—C10
N3—C11—C12—
C13—C14—C15
Cu(1)-N(1) = 2.049(2)Å,
Cu(1)-N(2) = 1.963(2)Å,
Cu(1)-N(3)
= 2.054(2)Å, Cu(1)-Br(1) = 2.3914(6)Å and Cu(1)-Br(2) = 2.6163
(6)Å. The selected angles between metal and coordination atoms
were: Br(1)-Cu(1)-Br(2) = 107.069(16)°, N(2)-Cu(1)-Br(2) = 101.19
(6)°, N(2)-Cu(1)-Br(1) = 151.73(6)° and N(1)-Cu(1)-N(3) = 156.88
(8)°. The space configuration of five-coordinated complexes could
2
Cg(A)—Cg(B)
N4—C23—C24—
C25—C26—C27
C16—C17—C18—
C19—C20—C21
be well defined by the
the two largest coordination angles),
mid as well as = 1 for an ideal triangular bipyramid [46,47]. For
[Cu(L1)(Br)2], the value of
were equal to 0.09. So in the case of
1, the Cu(II) square-pyramid were defined by the three nitro-gen-
s
value (
s
= (b ꢁ
a)/60 and a and b being
= 0 for an ideal square pyra-
3.2.1. Crystal structure of [Cu(L1)(Br)2]ꢀCH3CN (1)
s
Complex 1 was the mononuclear compounds with the triclinic
s
ꢀ
crystal system p1 space group. The asymmetric unit of 1 was com-
s
posed of one crystallographically independent Cu(II) ion, one L1

12
D.-Y. Zhang et al. / Inorganica Chimica Acta 457 (2017) 7–18
donors of the L1 ligand and two bromide ligands. As shown in
Fig. 2A, 1 could form two-double 1D chain structure with wake
C-Hꢀ ꢀ ꢀBr, and the distance of C12-Hꢀ ꢀ ꢀBr2A and C (CH3CN)-Hꢀ ꢀ ꢀBr1
were 2.817Å and 2.864Å, respectively [48]. Moreover, the
p-p
interactions were existed between the two-double, as shown in
Fig. 2A. The center distance between the ring obtain N3 and the
ring obtain N2A was 3.8537(16) Å (Table 2) and their dihedral
angel was 7.143(102)°, suggesting the two rings obtain N3 and
N2A exist p-p interactions [49].
3.2.2. Crystal structure of [Cu(L2)2(OAc)](PF6)ꢀ2C2H5OH (2)
Single crystal X-ray analysis revealed that complex 2 crystal-
ꢀ
lized in the monoclinic space group p1 with one Cu(II) ion, two
L2 ligands, one CH3COOꢁ ion, one PFꢁ6 counter ion and two ethanol
molecules in the asymmetric unit. As shown in Fig. 1B, 2 was a
mononuclear structure and Cu1 located in a five-coordinated envi-
ronment completed by four nitrogen atoms (N1, N2, N3 and N4)
from two L2 ligands and one oxygen atom (O1) from CH3COOꢁ
ion. The distance between metal and coordination atoms were:
Fig. 3. Absorption spectra of complex 1 (37.5
presence (colored line) of increasing amounts of CT-DNA (9.5, 19, 28.5, 38, 47.5, 57,
66.5, 76 M) in 5 mM Tris–HCl/50 mM NaCl buffer (pH = 7.2). The insert shows the
least-squares fit of (ea f)/(eb f) vs [DNA] for complex 1.
lM) in the absence (black line) and
l
ꢁ
e
ꢁ e
Cu(1)-N(1) = 2.078(2)Å,
Cu(1)-N(2) = 1.981(2)Å,
Cu(1)-N(3)
= 2.274(2)Å, Cu(1)-N(4) = 1.997(2)Å and Cu(1)-O(1) = 1.972(2)Å.
The selected angles between metal and coordination atoms were:
while increasing concentration of CT-DNA stock solution into com-
plex solution. As shown in Fig. 3 (Fig. S2), when increasing the con-
centration of CT-DNA to the solution of complex, the absorption
dramatically decreased and the hypochromism of 1–3 was
between 53% and 56%. In order to further confirm the binding
strength of complex with CT-DNA, the intrinsic binding constants
Kb were calculated from a nonlinear fitting according to the blow
equation [52,53].
N(2)-Cu(1)-N(4) = 163.97(10)°, N(2)-Cu(1)-N(3) = 117.20(10)°,
N
(1)-Cu(1)-N(3) = 111.93(9)° and O(1)-Cu(1)-N(1) = 160.48(9)°. For
the five-coordinated environment of [Cu(L2)2(OAc)], the value of
s
was equal to 0.06. So in the case of 2, the square-pyramid of Cu
(II) complex was defined by the two nitro-gendonors of the two
L2 ligands and one oxygen atom by the acetic acid ligand. In addi-
tion, as shown in Fig. 2B, the center distance between the rings
obtain N4 and C16-C21 was 3.5220(18) Å (Table 2) and their dihe-
ðea
ꢁ
ebÞ=ðeb
ꢁ
ef Þ ¼ fb ꢁ ðb2 ꢁ 2K2Ct½DNAꢄ=sÞ1=2g=2KbCt
ð1aÞ
ð1bÞ
b
dral angel is 10.979(105)°, thus, the two rings exist
p-p interac-
tions [50].
b ¼ 1 þ KbCt þ Kb½DNAꢄ=2s
3.2.3. Crystal structure of [Cu(L1)(L2)](PF6)2 (3)
Where Ct is the concentration of complex; [DNA] is the concentra-
tion of DNA; s is the binding site size of DNA which combine with
complex; ea, eb and ef are the molar absorption coefficient and ea
is the extinction coefficient observed for the charge-transfer
absorption band at a given DNA concentration; eb is the extinction
The structure of 3 was sample and easy, single crystal X-ray
analysis revealed that complex 3 crystallized in mononuclear
ꢀ
structure with monoclinic space group p1 with one Cu(II) ion,
one L1 ligand, one L2 ligand, and two PFꢁ6 counter ions in the asym-
metric unit. As shown in Fig. 1C, Cu1 located in a five-coordinated
environment completed by five nitrogen atoms: N1, N2 and N3
from L1 ligand, and N4, N5 from L2 ligand. The distance between
metal and five nitrogen atoms were: Cu(1)-N(1) = 2.047(2)Å, Cu
(1)-N(2) = 1.930(2)Å, Cu(1)-N(3) = 2.031(2)Å, Cu(1)-N(4) = 2.001
(2)Å and Cu(1)-N(5) = 2.277(2)Å. The selected angles between Cu
(II) and N atoms were: N(2)-Cu(1)-N(5) = 118.72(9)°, N(3)-Cu(1)-
N(1) = 158.53(9)° and N(2)-Cu(1)-N(4) = 162.77(9)°. For the five-
coefficient of the complex when fully bound to DNA;
tion coefficient of the complex free in solution. The data of Kb and s
were presented as (ea f)/(eb f) vs [DNA] in Fig. 3 (Fig. S2) and
ef is the extinc-
-
e
-e
Table 3. As shown in Table 3, the binding constant of complexes
to DNA as follows: Kb = 6.36 ꢂ 105 for 1, Kb = 3.82 ꢂ 105 for 2 and
Kb = 1.26 ꢂ 104 for 3. When compared the Kb values of the three
Cu complexes to the reported Copper(II) complexes [54,55] with
the same structure (Kb = 2.02 ꢃ 6.04 ꢂ 104, Kb = 2.3 ꢃ 3.3 ꢂ 105),
results showed that although all the Cu(II) compounds were the
mononuclear compounds with five-coordinated, owing to the major
ligand had the difference in the structures and the small molecule
ions were different. And the interaction between the complexes
and DNA suggested this difference. Moreover, the magnitude of
the Kb values of 1–3 were between 104 and 105, the value of DNA
binding constant about 1–3 was less than the classical binding con-
stant (107 Mꢁ1) of EB, which suggested that the interaction of three
complexes with DNA was a moderate strength intercalative mode.
And 1 exhibited a slightly stronger DNA binding affinity in compar-
coordinated environment of [Cu(L1)(L2)], the value of
to 0.07. So in the case of 3, the square-pyramid of Cu(II) complex
was defined by the three nitro-gendonors of the L1 ligand and
two nitro-gendonors of the L2 ligand.
s was equal
3.3. DNA-binding experiments
3.3.1. Absorption spectrophotometric and viscosity studies
DNA-binding experiment of metal complexes to DNA is consid-
ered to be an essential step in the field of DNA cleavage study. UV-
visible spectroscopy is known as a basic and reliable method to
evaluate the binding ability of metal complexes with DNA. The
absorption spectra of three complexes were characterized by
Table 3
Fluorescence spectral properties of complex 1–3 bound to CT–DNA.
Complex
Kb(Mꢁ1
)
s
D
e
(%)
Kapp (Mꢁ1
)
intense
charge transfer (MLCT) transition in the visible region. After com-
plex interacts with the base pairs of DNA [51], the
energy would decrease and the transition probabilities are
decreased as well as resulting in hypochromism. The CT-DNA bind-
ing ability of complexes 1–3 were conformed on the UV absorption
p
-
p
⁄ ligand transitions in the UV and metal-to-ligand
1
2
3
6.36 ꢂ 105
3.82 ꢂ 105
1.26 ꢂ 104
0.06
0.05
0.11
54.7
53.7
55.7
1.25 ꢂ 1012
5.98 ꢂ 1011
2.04 ꢂ 1012
p-
p⁄ transition
Kb: intrinsic equilibrium DNA binding constant; s: binding site size value;
trend in hypochromism; Kapp: apparent DNA binding constant; estimated errors for
the constants are 5%.
De: the

D.-Y. Zhang et al. / Inorganica Chimica Acta 457 (2017) 7–18
13
ison with 2 and 3. The reason might be that 1 owned one ligand of
better planarity than other compounds. Thus, the space hindrance
of 1 was small than other compounds when interacted with DNA.
Compared with the UV-visible spectroscopy, viscosity measure-
ment is an effective method to study the small molecule interac-
tion between complexes and DNA in the form of a more accurate
and more effective method of fluid mechanics [56]. When com-
plexes bound to DNA via insertion, the distance between DNA base
pairs will increase, which leads the increased length of DNA chain
together with increased viscosity [57]. The ratio of three complexes
1/3
relative specific viscosity (
g/g0)
versus [complex]/[DNA] for 1, 2
and 3 were given in Fig. 4. When added the complex concertration
into DNA solution, the relative viscosity of DNA increased effi-
ciently, suggesting that compounds could bind to DNA by
intercalation.
Fig. 5. Emission spectra of EB bound to CT-DNA in the absence (dashed line) and
presence (solid lines) of complex 1 (0–22.5 lM) in 50 mM Tris–HCl/18 mM NaCl
3.3.2. Competitive binding experiments
Fluorescence spectral titration measurement was used to fur-
ther investigate the mode of binding of the complexes with DNA,
via intercalation or groove binding. Fluorescence quenching assay
using EB-bound DNA as probe is an effective method to study
the mode of binding of the complexes to DNA. EB, a planar cationic
dye, is a typical indicator of intercalation, which could form soluble
complexes with CT-DNA and then emit intense fluorescence [58].
When add a second DNA intercalate agent that could replace EB
from DNA-bound EB system into EB-DNA solution, the fluorescence
of system will be quenched as the fact that free EB molecules are
readily quenched by surrounding water molecules [59]. The fluo-
rescence quenching spectra of 1–3 with EB-bound DNA were
shown in Fig. 5 (Fig. S3). Emission band at 610 nm displayed more
obvious hypochromism when the increasing concentrations of 1–3.
The quenching tendency illustrated that the quenching of EB sys-
tem by complex was in agreement with the classical linear
Stern–Volmer equation [60].
buffer (pH = 7.2).Inset: the plot of I0/I versus [complex].
nitude of the Kapp values of 1–3 ware between 1011 and 1012. As
expected, the competitive binding of three complexes to DNA with
EB could result in displacement of EB as well as dramatic decrease
of emission intensity.
3.3.3. DNA cleavage and mechanism studies
In order to detect the DNA cleavage activities of the complexes,
agarose gel electrophoresis for the cleavage of pBR322 plasmid
DNA was performed in the buffer (50 mM Tris-HCl/18 mM NaCl,
pH = 7.2) under the natural light in an aerobic environment with-
out the exogenous oxidant agent. All these complexes could relax
the supercoiled circular (SC) form of plasmid DNA into nicked cir-
cular (NC) form and/or linear form. Fig. 6 showed the cleavage
activity of 1–3 to pBR322 DNA at different concentrations, and dif-
ferent cleavage patterns (Form I, Form II and Form III) were
observed after the samples were subjected to electrophoresis.
Form I is the supercoiled structure without cleaving by complex
which migrates at the fastest rate, Form II has only one incision
on the one strand cleaved by complex which moves at the slowest
rate, and Form III is a linear structure with the rate of migration
I0=I ¼ 1 þ Ks ½Qꢄ
ð2aÞ
v
KEB½EBꢄ ¼ Kapp½complexꢄ
ð2bÞ
Where I0 and I represent the fluorescence intensities in the absence
and presence of quencher, [Q] is the concentration of quencher, Ksv
is the Stern-Volmer dynamic quenching constant. Here KEB is the
binding constant of EB to DNA [KEB = 1.0 ꢂ 107 Mꢁ1
, ([EB]
= 2.4 lM)], [complex] is the concentration value of complex at a
50% reduction of fluorescence intensity of EB and Kapp is the appar-
ent DNA binding constant. The data of Ksv was presented as I0 /I vs
[Q] in the Fig. 5 (Fig. S3) and Table 3. As shown in Table 3, the mag-
Fig. 6. Cleavage of plasmid pBR322 DNA (0.1
complex 1 (A), 2 (B) and 3 (C) after 3 h incubation at 37 °C; Line 1: DNA control; Line
2–6: DNA + complex (5, 20, 35, 50, 65, 80 M).
lg/lL) at different concentrations of
Fig. 4. Effects of increasing amount of complex on relative viscosity of CT-DNA at
37.0 0.1 °C conditions: [DNA] = 1 mM, [complex] = 0–0.01 mM.
l

14
D.-Y. Zhang et al. / Inorganica Chimica Acta 457 (2017) 7–18
(EDTA), big groove scavenger (Methyl Green), and small groove
scavenger (SYBR Green) were added into pBR322DNA solution
prior to the addition of the complexes [61]. As showed in Fig. 7
(Fig. S4), singlet oxygen quenchers (L-histidine) (lane 6 of all)
markedly inhibited the cleavage activity of all complexes, suggest-
ing that singlet oxygen (1O2) was the major cleavage active species
in the cleavage reaction. In addition, the chelating agent (EDTA)
(lane 9 of all) had the ability to inhibit DNA cleavage efficiently,
which indicated Cu2+ played a key role in the DNA destruction.
Fig. 7. Agarose (1%) gel electrophoresis showing cleavage of pBR322 DNA (200 ng)
by complex 2 (65
Lane 1, DNA control; Lane 2, DNA + 2; Lane 3, DNA + 2 + KI (1 mM); Lane 4, DNA + 2
+ DMSO (1 mM); Lane 5, DNA + 2 + NaN3 (20 mM); Lane 6, DNA + 2 + -histidine
lM) in different conditions for anincubation time of 3.0 h at 37 °C.
L
(20 mM); Lane 7, DNA + 2 + SOD (20U/mL); Lane 8, DNA + 2 + Catalase (20U/mL);
Lane 9, DNA + 2 + EDTA (10 mM); Lane 10, DNA + 2 + Methyl Green; Lane 11, DNA
+ 2 + SYBR Green.
3.4. Protein binding studies
In blood plasma, Serum albumin (SA) is the main protein acting
as an important role in drug transport and drug metabolism and
constitutes ꢃ 55% of the total protein. The study of the interaction
between metal complexes and proteins is the basis for the investi-
gation of how the metal drugs affect the cancer cells. In our study,
bovine serum albumin (BSA) was used because its structure is sim-
ilar to human serum albumin (HSA) and it can be easily obtained
[62,63]. BSA has three fluorophores: tryptophan, tyrosine and
phenylalanine, and the intrinsic fluorescence of BSA is mainly
attributed to tryptophan [64]. Fluorescence quenching measure-
ments have been widely used to study the interaction of metal
complexes or small molecules with proteins [65]. Brustein etc
[66] believed that the greatest fluorescence emission peak position
of tryptophan was sensitive to the environment. Fig.S5 showed the
effect of 1–3 on fluorescent intensity of BSA at room temperature, a
Table 4
The quenching constant, binding constant and number of binding sites for the
interactions of complex 1–3 with BSA.
Complex
Ksv (Mꢁ1
)
Kq (Mꢁ1 sꢁ1
)
Kbin (Mꢁ1
)
n
1
2
3
6.1 ꢂ 104
4.5 ꢂ 104
3.9 ꢂ 104
6.1 ꢂ 1012
4.5 ꢂ 1012
3.9 ꢂ 1012
5.25 ꢂ 104
2.4 ꢂ 107
3.8 ꢂ 103
0.99
1.61
0.79
Ksv: quenching constant; Kq: quenching rate constant; Kbin: binding constant of
compound with DNA; n: the number of binding sites; estimated errors for the
constants are 5%.
between the rate of Form I and Form II. As shown in Fig. 6, obvious
DNA cleavage could be detected at the concentration of 35
1, 5 M for 2 and 20 M for 3. Moreover, when the concentration
was increased to 65 M for 2, the Form III appeared, suggesting 2
lM for
l
l
l
solution of BSA (29.4 lM) was titrated with varying concentrations
of complexes, and fluorescence intensity was recorded in the range
of 290–500 nm upon excitation at 280 nm. A gradual decrease in
fluorescence intensity as well as a blue shift in wavelength were
observed after increasing concentration of compounds. To further
study quenching process, fluorescence quenching data were ana-
lyzed with Stern-Volmer equation (3a) and Scatchard equation
(3b) [67].
had the best cleavage activity.
In order to authenticate the cleavage mechanism of pBR322
DNA by complexes 1–3, several ROS inhibiting agents, including
hydrogen radical scavenger (KI, DMSO), singlet oxygen quenchers
(NaN3,
L-histidine), superoxide scavenger (SOD, superoxide dismu-
tase), peroxide anion scavenger (catalase) and chelating agent
Fig. 8. MTT assay of 1–3 with MDA-MB-231 (A), A549 (B) and HeLa (C) cells.

D.-Y. Zhang et al. / Inorganica Chimica Acta 457 (2017) 7–18
15
I0=I ¼ 1 þ Ks ½Qꢄ ¼ Kqs0½Qꢄ
ð3aÞ
Fig. 8. The results from MTT assay indicated that these metal
chelating compounds significantly reduced the viability of cancer
cells within 24 h in a dose-dependent manner. The IC50 values of
these three complexes on different cells at 24 h were showed in
Table 5. The cytotoxicity of 1–3 on MDA-MB-231, A549 and HeLa
cells were similar and the range of IC50 values were about 1.7–
v
lg½ðI0 ꢁ IÞ=Iꢄ ¼ lg Kbin þ n lg½Qꢄ
ð3bÞ
Where I0 and I are the fluorescence intensities of the fluorophore in
the absence and presence of quencher, respectively, Kq is the
quenching rate constant, s0 the average life-time of the biomolecule
2.1 lM. In addition, the cytotoxic effects of cisplatin on these can-
without quencher (about 10ꢁ8 s), Kbin is binding constant of com-
pound with DNA and n is the number of binding sites. As showed
in Fig. S6, Ksv was observed by using the plot of I0/I versus [Q],
and the number of binding sites (n) and binding constant (Kbin) were
obtained from the plot of lg(I0-I)/I versus lg[Q]. In Table 4, the mag-
nitude of the Kbin values of 1–3 were between 103 and 107, and the
magnitude of the Kq was 1012, suggesting the complexes had mod-
erate binding activities to BSA at room temperature.
cer cells in comparison to 1–3 were detected and showed in
Table 5. The result suggested that the cytotoxic effects of 1–3 were
more efficient than cisplatin. Further, the anti-cancer effects of
these complexes were proved by the colony-forming assay, as
shown in Fig. 9D, compared with the control cell, the number of
live cell was decreased obviously when cells were treated with
1–3 for 24 h. Thus, these three cooper complexes may have the
potential to develop into anti-cancer agents.
3.5. Cytotoxicity Assessment by MTT assay
3.6. Cell apoptosis analysis and attestation
The cytotoxicity of 1–3 on the growth of cancer cells were mea-
sured using the MTT assay. Cells in the control group and treat-
ment groups were seeded in 96-well plates at a density of 3000
cells/well and incubated for 24 h to allow the attachment. Cyto-
toxic effects of these complexes on different cancer cell lines
(MDA-MB-231, A549, and HeLa) were determined and showed in
Based on HE staining, morphological changes, such as the con-
densed and marginalized chromatin, could be observed after cells
were exposing to the cooper complexes for 24 h, suggesting the
initial apoptosis in MDA-MB-231 cells [68,69]. Hoechst 33342
staining further confirmed the nuclear chromatin condensation
in MDA-MB-231 cells using fluorescence microscopy. As shown
in Fig. 9 (A and B), compared with the control cells, cells treated
with 1–3 for 24 h showed stronger blue fluorescence under a fluo-
rescent microscope at 100X magnification, which suggested three
cooper complexes had the ability of inducing the cell apoptosis.
Several reports indicated that the cell apoptosis pathway
induced by complex might be dependent on ROS generation
[70,71]. Besides, according to the previous study in our work, cop-
per compounds could generate singlet oxygen (1O2) in the DNA
cleavage reaction. Therefore, the experiment on measurement of
ROS in the cell apoptosis pathway induced by complexes was
Table 5
IC50 values of 1–3 and Cisplation in MDA-MB-231 (A), A549 (B) and HeLa (C) cancer
cells.
Complex
MDA-MB-231
IC50 M)
A549
IC50
HeLa
IC50
(
l
(
lM)
(lM)
1
2
3
1.9 0.1
1.8 0.2
1.7 0.1
5.2 0.6
2.1 0.2
2.1 0.2
1.9 0.1
8.9 0.5
2.0 0.3
1.8 0.1
1.9 0.1
3.4 0.8
Cisplation
Fig. 9. A and B are morphological changes in the nuclei (apoptosis) of MDA-MB-231 cells treated with 1–3 for 24 h, respectively, followed with HE and Hoechst 33342
staining. C is ROS generation assay in MDA-MB-231 cancer cells after treatment with 1–3 for 24 h respectively. D is the multiplication inhibition of MDA-MB-231 cell treated
with 1–3 for 24 h respectively.

16
D.-Y. Zhang et al. / Inorganica Chimica Acta 457 (2017) 7–18
applied. As shown in Fig. 9C, cells were exposed to 1–3 at concen-
tration of 1 M for 24 h, samples were observed under a fluores-
from these Cu(II) compounds could be the possible reason for cell
apoptosis.
l
cent microscope (Olympus) at 100X magnification. Strong green
fluorescence indicating the generation of ROS was observed com-
pared with the control group, which suggested that ROS generated
Annexin V-FITC/PI dual staining was carried out to detect com-
plexes induced apoptosis in MDA-MB-231 cells. As shown in
Fig. 10 (A-D), the complexes treatments resulted in apoptosis rates
Fig. 10. Annexin V-FITC/PI staining (A–D) detected apoptosis in MDA-MB-231 cells after treatment with 1–3 for 24 h at the selected concentration by MTT assay. E showed
the Caspase-3 activity about 1–3.
Fig. 11. (1) Cell cycle analysis using flow cytometry was carried on MDA-MB-231 cells treated with 1–3 for 24 h at the selected concentration by MTT assay. (2) The
percentages of MDA-MB-231 cancer cells in the different phases of cell cycle were presented.

D.-Y. Zhang et al. / Inorganica Chimica Acta 457 (2017) 7–18
17
Fig. 12. Proposed anti-cancer mechanisms of 1–3.
of 10.52 0.23% for 1, 7.61 0.17% for 2, 8.61 0.34% for 3, respec-
4. Conclusion
tively, all of which were statistically different from the control. The
complexes induced apoptosis was further analyzed by examining
the mRNA expression of caspase family using quantitive PCR
shown in Fig. S7. Increased caspase 9 and caspase 3 expression
were positively correlated with the apoptotic rate when treated
with 1–3 for 24 h, respectively, while caspase 8 was downregu-
lated. And the Caspase-3 activity of MDA-MB-231 cells was further
detected using Caspase-3 colorimetric assay. As shown in Fig. 10
(E), the three complexes upregulated the caspase-3 activities in
MDA-MB-231 cells. Among these three complexes, 1 showed
strongest ability of Caspase-3 activation compared with 2 and 3
at the same concentration. Thus, Caspase-3-induced apoptosis
might be the possible mechanism of these complexes induced cell
apoptosis and death.
Cell cycle distributions of MDA-MB-231 cells at different stages
were determined using flow cytometry (FCM). Cells were treated
with these three compounds at IC50 value for 24 h, individually.
As shown in Fig. 11(1), although a lot of parallel experiments had
been done, signals for sub-G1 peaks related to the presence of
apoptosis were rarely observed, which was different from the pre-
viously reported literature [72]. However, as shown in Fig. 11(2),
the population in S and G2/M phases declined significantly, com-
bined with a dramatic increase in G0/G1 phase, respectively, sug-
gesting that these three copper complexes arrested the MDA-MB-
231 cell at the G0/G1 phase of cell cycle.
Three Cu(II) complexes with two isomer ligands obtain N atoms
were synthesized and characterized. X-ray crystallography struc-
ture showed that three Cu(II) ions had the same five-coordinated
mode and they were all mononuclear compounds with monoclinic
ꢀ
p1 space group. The space configuration of three complexes was
square-pyramid because the value of
s was equal to 0.09 for 1,
0.06 for 2 and 0.07 for 3. Those three complexes were characterized
in DMF solution by using ESI-MS: to 1, 466.99 for [Cu(L1)(Br)]+; to
2, 709.21 for [Cu(L2)2-H]+ and to 3, 709.21 for [Cu(L1)(L2)-H]+. The
result of ESI-MS showed that the small molecule ion (Brꢁ in 1 and
CH3COOꢁ in 2) leaved from complex in DMF solution. The binding
ability of complexes to DNA was measured by UV-visible spec-
troscopy, fluorescence spectral titration and viscosity measure-
ments, which showed three complexes could bind to DNA in a
slightly strong binding affinity with Kb = 6.36 ꢂ 105 for 1,
Kb = 3.82 ꢂ 105 for 2 and Kb = 1.26 ꢂ 104 for 3. A series of spectrum
experiments of three Cu(II) complexes with BSA showed that com-
plexes could combine with BSA very well. Gel electrophoresis
experiment showed that 1–3 had excellent DNA cleavage activities
in the absence of exogenous oxidants, and 2 exhibited the best
DNA cleavage activity among all complexes. Moreover, three
cooper complexes could generate singlet oxygen (1O2) as the ROS
during the DNA cleavage process in the natural light under an aer-
obic environment, the more accurate DNA cleavage mechanism of
the three complexes needs further exploration, such as: anaerobic
experiment, light avoidance test and so on. MTT assay gave a result
that 1–3 had the lower IC50 value than cisplatin, and HE, Hoechst
33342 and Annexin V-FITC/PI staining assays demonstrated their
anti-cancer effects of promoting G1 arrest and inducing apoptosis.
Further studies indicated that the inhibited proliferation ability on
According to data mentioned above, we speculated the pro-
posed anti-cancer mechanisms of 1–3, as shown in Fig. 12. When
treated with these complexes, DNA damage would be induced
which led to the G1 arrest in cancer cells. At the same time, the for-
mation of ROS activated the caspase family mediated mitochon-
drial apoptotic pathway.

18
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Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (21171101, 21071083 and 21471085) and MOE
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