Synthesis of four binuclear copper(II) complexes: Structure, anticancer properties and anticancer mechanism

Article abstract of DOI:10.1016/j.ejmech.2015.04.031

Four binuclear Cu(II)-Schiff base complexes were synthesized and characterized.Cu complexes have high anticancer activity.Cu complexes induce cells apoptosis possible via intrinsic ROS-mediated mitochondrial pathway.Cu complexes regulate Bcl-2 family prot

Full text of DOI:10.1016/j.ejmech.2015.04.031

European Journal of Medicinal Chemistry 96 (2015) 360e368
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of four binuclear copper(II) complexes: Structure, anticancer
properties and anticancer mechanism
Jinxu Qi a , Shichu Liang , Yi Gou , Zhenlei Zhang , Zuping Zhou , Feng Yang
,
b
a
,
*
b
b
a
a, b, *
,
a, b, *
Hong Liang
a
Key Laboratory of Ecology of Rare an Endangered Species and Environmental Protection, Ministry of Education of the People's Republic of China, Guangxi
Normal University, Guilin, Guangxi, China
State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Science and Technology of China,
b
Guanxi Normal University, Guilin, Guangxi, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 February 2015
Received in revised form
Copper (Cu) compounds are a promising candidate for next generation metal anticancer drugs and have
been extensively studied. Therefore, four binuclear copper(II) compounds derived from Schiff base thi-
osemicarbazones (L1eL4), namely [CuCl(L1)]
2 3 2 2
(C1), [CuNO (L2)] (C2), [Cu(NCS) (L3)] (C3) and
12 April 2015
[
Cu(CH COO) (L4)] (C4) were synthesized and characterized. Four of these compounds showed very
3
2
Accepted 13 April 2015
Available online 14 April 2015
high cytotoxicity to cancer cell lines in vitro. These Cu(II) compounds strongly promoted the apoptosis of
BEL-7404 cells. The formation of reactive oxygen species (ROS), change in mitochondrial membrane
potential and western blot analysis revealed that Cu compounds could induce cancer cell apoptosis
through the intrinsic ROS-mediated mitochondrial pathway accompanied by the regulation of Bcl-2
family proteins.
Keywords:
Copper(II) complex
Anticancer activity
Cell apoptosis
© 2015 Elsevier Masson SAS. All rights reserved.
Anticancer mechanism
Bcl-2 family proteins
1. Introduction
such as the role of Cu in neurodegenerative disorders and
Cu-radioisotopebased imaging [14,15].
Although Cis-platin is a promising anticancer agent [1], multi-
factorial resistance, serious side effects and general toxicity have
limited the use of Cisplatin in cancer chemotherapy [2]. Therefore,
considerable effort is being made to replace this drug with more
efficacious, less toxic and target specific metal-based anticancer
drugs [3,4].
The previous studies revealed that cancer cells take up greater
amounts of copper (Cu) than normal cells, and Cu metabolism has
been linked to angiogenesis and metastasis [5e8]. Cu complexes
can interact with molecular oxygen and cellular oxidants and re-
ductants, allowing Cu to redox-cycle between its mono- and diva-
lent oxidation states [9,10]. Therefore, how to develop compounds
that chelate Cu has become a therapeutic strategy, and showing
significant promise [8,11,12]. Cu complexes derived from a great
variety of ligands, such as thiosemicarbazones, imidazoles, and
phosphines, have been proposed as potential anticancer agents
Owing to potential biological activities, thiosemicarbazones
(e.g. the HDp44 mT series) have been brought to focus for their
anticancer activity [11,16e18]. In addition, suitable modification at
C5 and/or N4 position of thiosemicarbazone can effectively in-
crease its anticancer potency [19]. Interestingly, many studies have
shown that the biological activity of a variety of metals in complex
with thiosemicarbazone is higher than that of the ligand alone and
this is particularly relevant for antitumour and anti-HIV activity
[20,21]. Liver cancer has become very common worldwide and is
responsible for causing a significant number of cancer deaths [22].
Copper containing anticancer agents are promising leads for next
generation metal-based anticancer agents because Cu plays a sig-
nificant role in biological systems [3,23]. In addition, the current
studies showed that the binuclear Cu(II) compounds possess high
anticancer activities [24e30]. Therefore, we attempt to build model
on binuclear copper(II) complexes' anticancer properties to liver
cancer cell (BEL-7404) in vitro. Herein, the four new binuclear Cu(II)
complexes derived from thiosemicarbazone Schiff bases as ligands
were synthesized and denoted by C1, C2, C3 and C4 respectively
(Scheme 1 and Figs. 1e3). We not only evaluated and characterized
[13]. More recently, Cu complexes have expanded into other areas
*
Corresponding authors. 15 Yucai Road, Guilin, Guangxi 541004, China.
E-mail address: fyang@mailbox.gxnu.edu.cn (F. Yang).
http://dx.doi.org/10.1016/j.ejmech.2015.04.031
223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
0

J. Qi et al. / European Journal of Medicinal Chemistry 96 (2015) 360e368
361
Scheme 1. Synthesis routes for C1eC4.
their biological activities but also determined their mechanism of
action for liver cancer cell (BEL-7404) in vitro. Thus, our study
might provide new useful insights for designing metal containing
drugs for anticancer therapy.
dimers of 1 were further linked into a one-dimensional (1D)
polymeric chain by the NeH$$$N hydrogen bonds involving a ni-
iii
trogen atom (N4) from a Schiff base ligand and the N3 from the
iii
adjacent dimeric molecule (N4$$$N3
¼
3.07 Å and the
iii
ꢁ
N4eH4A$$$N3 angle was 167.2 , symmetry code: (iii) 2 ꢀ x,
2
. Results
1 ꢀ y, ꢀz) (Fig. 1C).
C2 has been shown in Fig.1B. The coordination geometry around
2.1. Crystal structure description
Cu (II) in this complex was very similar to that in 1; the only dif-
ꢀ
ference was the presence of a NO
3
at the apical position. Here also,
The single-crystal X-ray diffraction analysis for C1eC4 revealed
two Cu atoms and two S atoms formed a strictly planar four-
membered Cu core and the Cu (II)eS distances [2.268 Å/
that their structures are different from each other. C1 and C2 in the
triclinic system, feature a butterfly structure, and C3 and C4 in the
monoclinic system.
C1 crystallized in the triclinic space group P-1, with one discrete
dimeric Cu (II) molecule in the unit cell. As illustrated in (Fig. 1A), it
contains two Schiff base ligands, two Cu atoms and two Cl per unit.
The two Cu atoms of the dimer were separated by 3.504 Å and
doubly bridged by two S atoms of two Schiff base ligands to form a
2 2
S
2.765 Å] were very similar to the Cu (II)eS distances found in 1. The
Cu atom was displaced from the N1/N2/S1/O1 basal plane
(maximum displacement 0.043 Å for the N1 atom) by 0.129 Å in the
i
direction of S1 . The Cu atom of the dimer was basically penta-
coordinated (
t
¼ 0.08), but with the second O atom, O2, of the
ꢀ
NO
3
group occupied a sixth-coordinate position at a distance
greater than 2.6 Å, namely 2.785 Å, to give (4 þ 1 þ 1*) type coor-
2 2
strictly planar four-membered Cu S core. Both Cu atoms (Cu1 and
dination [33] (Fig. 1B).
i
Cu1 , symmetry code: (i) 2 ꢀ x, 2 ꢀ y, 1 ꢀ z) were penta-coordinated
by two S atoms from two different Schiff base ligands, two nitrogen
atoms from one Schiff base ligand and one terminal Cl atom. The
distances of CueN/S bonds were in the range of 1.99e2.75 Å and
were in good agreement with the corresponding distances reported
in literature [31,32]. The coordination polyhedron surrounding Cu1
at the centre could be described as a slightly distorted square
C3 crystallized in a monoclinic system with space group P21/n.
ꢀ
As shown in Fig. 2A, it contained two Cu(II), two SCN ions and two
Schiff base ligands. Each Cu(II) at the centre exhibited a slightly
distorted square pyramidal geometry (
t
¼ 0.09) with equatorial
ligation formed by the pendant pyridine arm of a second Cu(II)
monomer. The mode of ligand sharing by di-2-pyridyl unit of thi-
osemicarbazone Schiff base was similar to that noted with a ligand
with bis 2-imidazole moiety, which facilitated formation of ligand-
shared Cu(II) dimer [34]. The bridging arrangement in C3 complex
was very different from that of other dimeric Cu (II) complexes such
as NNS thiosemicarbazone [35] analogues 1 and 2. The Cu(II)eN
pyramid (
t
¼ 0.18) with the metal displaced from the N1/N2/S1/Cl1
basal plane (maximum displacement 0.366 Å for the N2 atom), and
i
the S1 atom at the apex with the metal displaced by 0.275 Å toward
i
S1 from the mean basal plane. In the solid state, the discrete Cu (II)

362
J. Qi et al. / European Journal of Medicinal Chemistry 96 (2015) 360e368
Fig. 1. (A) The local coordination environment of C1. Hydrogen atoms are omitted. Symmetry code: i ¼ 2 ꢀ x, 2 ꢀ y, 1 ꢀ z. (B) The local coordination environment of C2. Hydrogen
atoms are omitted for the sake of clarity. Symmetry code: i ¼ 1 ꢀ x, 1 ꢀ y, 1 ꢀ z. (C) The zigzag chains are interlinked by the NeH$$$N interactions (Symmetry code: ii ¼ 1 þ x, y, z;
iii ¼ ꢀ1 ꢀ x, ꢀy, ꢀz).
N4$$$O2iv ¼ 2.774 Å and the N4eH4$$$O2 angle was 164.7 ,
symmetry code: (ii) 1 ꢀ x, ꢀ0.5 þ y, 1.5 ꢀ z), resulting in 2D pleated
nets based on the sql topology with rectangular grid-like corru-
iv
ꢁ
and Cu(II)eS distances in 3 were comparable to analogous dis-
tances in other known complexes [36,37]. Dimerization of the
[
Cu(L3)] units introduced significant strain in 3 as evidenced by the
ꢁ
2
short N1eCu1eS1 angle 163.08 . In solid state, the dimeric units of
gated sheets showing dimensions of 8.48 ꢂ 11.85 Å (Fig. 3B).
3
were arranged in a chain-like fashion by the NeH$$$N hydrogen
bonds such that the N4 of one dimeric unit stacks on the next one
2.2. Anticancer properties of Cu compounds
ii
ii
N3 from the adjacent dimeric molecule (N4$$$N3 ¼ 3.02 Å and
ii
ꢁ
the N4eH4B$$$N3
angle was 173.1 , symmetry code:
The results indicated that Cu(II) and HL were not that much
potent against cancer cells, but the C1eC4 compounds showed high
cytotoxicity against cancer cells. This implied that the chelation of
(
ii) ꢀx, ꢀy, ꢀz) (Fig. 2B).
X-Ray analysis of C4 revealed that it also formed a dimeric
ꢀ
2þ
structure consisting of two Cu(II), two CH
3
COO ions and two Schiff
Cu with HL was responsible for the observed high cytotoxicity of
base ligands; it crystallized in a monoclinic system with space
group P21/n. As shown in Fig. 3A, the coordination geometry
around Cu(II) in the dimeric complex was structurally very similar
the Cu compounds. Among them, C4 had the highest cytotoxic
activity, whereas C1 showed the lowest cytotoxicity (Table 1).
Acridine orange/ethidium bromide double staining revealed
that Cu(II) compounds induced BEL-7404 cell apoptosis (Fig. 4A).
The degree of apoptosis induced by Cu compounds co-related with
the cytotoxic effect of Cu compounds on cancer cells. The results
from Annexin V-FITC/PI staining showed that the percentage of
apoptosis in BEL-7404 cells were 9.42% for C1, 25.4% for C2, 25.3%
for C3 and 36.0% for C4 (Fig. 4B). In contrast, the percentage of
apoptotic cells without treatment (control) was only 4.69% (Fig. 4B).
to that of complex 3; the only difference was the presence of
ꢀ
CH
3
COO ion at the outer axial sites on Cu. There was a weak
interaction between the Cu(II) at the centres and the oxygen of the
ꢀ
CH
3
COO anions [Cu(II) 1eO
2
, distance 2.670 Å] in the complex
structure. Hence the geometry was best described as a square py-
ramidal distorted octahedral (4 þ 1 þ1*). The Cu1eN5 distances in
3
and 4 (2.377 and 2.414 Å respectively) indicated that the dimeric
[
Cu(L3)]
Interestingly, the CeS bond distances (1.747 Å for 1, 1.760 Å for 2,
.713 Å for 3 and 1.741 Å for 4) of all complexes were closer to a
single bond distance (single bond [ca. 1.81 Å] and double bond [ca.
.58 Å]) [36] thereby indicating thiolate formation on complexa-
tion. In solid state, there are NeH$$$O hydrogen bond interactions,
2
unit of 3 was slightly more compact in comparison with 4.
2.3. Anti-tumour mechanism
1
The mitochondrial apoptotic signalling pathway is one impor-
tant route of cell apoptosis. Apoptosis is usually induced by the loss
of mitochondrial transmembrane potential (Dj ) marked by the
m
1

J. Qi et al. / European Journal of Medicinal Chemistry 96 (2015) 360e368
363
release of apoptogenic factors from the mitochondria and decrease
of ATP generation [32]; the Bcl-2 family proteins play an important
role in the release of cell apoptogenic factors. In this study, we
investigated the mechanism by which the Cu compounds induced
cell apoptosis using fluorescence microscopy, flow cytometry and
western blot techniques.
2.3.1. Intracellular ROS measurements and GSH/GSSG assay
Fluorescence intensity produced by C4 obviously higher than
that of C1eC3 as observed under fluorescence microscope (Fig. 5A).
The DCF fluorescence peaks for Cu compounds were evaluated and
quantified (Fig. 5B and C). However, no significant increase in
H DCF oxidation could be observed with high concentration of
2
Cu(II) alone over 1 h incubation (Fig. 5B). Notably, these results
indicated that Cu compounds produced intracellular ROS in BEL-
7404 cells (Fig. 5). Furthermore, the intracellular GSH/GSSG ratio
was significantly decreased after incubation with C1eC4 complexes
thereby implying that Cu compounds had a stronger intracellular
oxidation potential (Fig. 6) to catalyse the intracellular oxidation of
2
H DCF/DCF than thiosemicarbazones ligands.
2.3.2. Mitochondrial membrane potential assay
Chemically induced apoptosis mediated by the mitochondria/
Fig. 2. (A) A molecular structure of C3 showing the environment about the Cu(II) atom.
Symmetry code: i ¼ ꢀ x, ꢀy, ez. (B) The zigzag chains are interlinked by the NeH$$$N
interactions (Symmetry code: ii ¼ 1 þ x, y, z; iii ¼ ꢀ1 ꢀ x, ꢀy, ꢀz).
apoptotic cascades is often associated with the collapse of the
mitochondrial membrane potential as a result of the leakage of pro-
0
apoptotic factors [38]. The lipophilic fluorescent probe (5, 5, 6, 6 -
Fig. 3. (A) A molecular structure of C4 showing the environment about the Cu(II) atom. Symmetry code: i ¼ 1 ꢀ x, ꢀy, 1 e z. (B) The zigzag chains are interlinked by the NeH$$$N
interactions (Symmetry code: ii ¼ 1 þ x, y, z; iii ¼ ꢀ1 ꢀ x, ꢀy, ꢀz).
Table 1
IC50
(
m
M) values of ligands (L1ꢀL4) and Cu(II) complexes (C1eC4) toward the cell lines.
Compounds IC50 M)
(
m
BEL-7404
A549
NCIeH460
>60
HL-7702
CuCl
2
>60
>60
>60
L1
L2
L3
L4
C1
C2
C3
C4
18.756 ± 0.751
6.266 ± 0.452
5.266 ± 0.264
3.256 ± 0.020
2.532 ± 0.052
1.232 ± 0.011
1.261 ± 0.010
0.486 ± 0.010
10.324 ± 0.520
14.614 ± 0.233
8.267 ± 0.524
6.255 ± 0.745
3.152 ± 0.544
2.665 ± 0.214
1.256 ± 0.064
1.612 ± 0.010
0.507 ± 0.021
17.364 ± 0.125
17.753 ± 0.551
5.262 ± 0.594
6.287 ± 0.512
3.294 ± 0.554
3.462 ± 0.112
2.236 ± 0.222
1.424 ± 0.221
0.235 ± 0.010
8.235 ± 0.010
19.561 ± 0.122
7.126 ± 0.245
7.26 ± 0.425
5.134 ± 0.254
4.216 ± 0.145
2.256 ± 0.123
3.241 ± 0.045
0.754 ± 0.011
9.166 ± 0.010
Cis-platin

364
J. Qi et al. / European Journal of Medicinal Chemistry 96 (2015) 360e368
Fig. 4. (A) Comet assay of EB-stained of BELꢀ7404 cells treated by Cu compounds with 12 h incubation. (a) control; (bee) C1eC4. The red (white/gray in the gray-scale print
version) well-formed comets were observed. (B) Representative dot plots of PI and annexin V double staining on the BELꢀ7404 cells treated by Cu compounds with 12 h incubation.
(
a) control; (bee) C1eC4.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Intracellular production of reactive oxygen species of BELꢀ7404 cells treated by 20
cells: control (a); (bee) C1eC4; (B) control cells or cells treated with C1eC4 and CuCl
m
M CuCl
2
and C1eC4 with 1 h incubation. (A) intracellular ROS was detected in BEL-7404
ꢁ
2
for 1 h at 37 C; (C) quantification of the flow cytometric results in (A) and (B) showing the
percentage of cells with increased intracellular DCF oxidation compared to control cells effects on ROS generation induced by different concentrations of C1eC4 and CuCl
are the mean (SD (n ¼ 6), p < 0.01).
2
. Results

J. Qi et al. / European Journal of Medicinal Chemistry 96 (2015) 360e368
365
ꢁ
Fig. 6. Incubation for 1 h at 37 C with 20
2
mM of C1eC4 and CuCl decreases the GSH/
GSSG ratio in cultured BEL-7404 cells. Results are the mean (SD (n ¼ 6), p < 0.01).
0
0
tetrachloro-1, 1 , 3, 3 -tetraethyl-imidacarbocyanineiodide, JC-1)
was used to detect changes in the mitochondrial membrane po-
tential. As depicted in (Fig. 7), the red fluorescence was gradually
replaced by green (in the web version) fluorescence on addition of
Cu compounds into BEL-7404 cells. Clearly, compounds C1eC4
exerted an influence that eventually led to the collapse of the
m
mitochondrial transmembrane potential (Dj ). Thus it could be
confirmed that the intrinsic mitochondrial pathway was involved
in apoptosis induced by Cu compounds.
2.3.3. The assay of the expression of caspase, antiapoptotic and
proapoptotic proteins
The Bcl-2 family proteins constitute a critical intracellular
checkpoint in the intrinsic mitochondrial pathway of apoptosis
[39]. To examine mitochondrial release of apoptogenic factors, we
evaluated the effect of the Cu compounds on the expression levels
of proteins in BEL-7404 cells by western blot analysis. We found
that the expressions of antiapoptotic proteins (Bcl-2 and Bcl-xl)
were downregulated, whereas the level of pro-apoptotic protein
Fig. 8. (A) Western blot analysis of Bcl-2, Bcl-xl, Bax, Cyt C, Caspase-3 and Caspase-9 in
BEL-7404 cells treated with C1eC4 for 24 h b-actin was used as internal control. (B)
Percentage expression levels of Bcl-2, Bcl-xl and Bax in panel. (C) Percentage expres-
sion levels of Cyt C, Caspase-3 and Caspase-9 in panel. The percentage values are those
relative to the control.
(
Bax) was upregulated. The ratio of Bax/Bcl-2 was significantly
increased (Fig. 8A and B) resulting in enhanced permeability of the
mitochondrial membrane for the release of pro-apoptotic proteins.
In addition, cytochrome C (Cyt C) and caspase family proteins
3. Discussion
(
caspase-3 and caspase-9) were upregulated (Fig. 8A and C).
Therefore, our results demonstrated that Cu compounds were able
to induce BEL-7404 cell apoptosis by promoting Cyt C release fol-
lowed by activation of caspase family proteins (caspase-3 and
caspase-9).
Our results demonstrated that antiproliferative activity of a
ligand could be enhanced by pre-coordination with Cu probably
because of their intracellular redox activity. Additionally compared
2
þ
Fig. 7. Assay of BEL-7404 cells mitochondrial membrane potential with JC-1 as fluorescence probe staining method. (A) control; (BeE) C1eC4.

366
J. Qi et al. / European Journal of Medicinal Chemistry 96 (2015) 360e368
with the ligands alone, Cu complexes were more lipophilic and
therefore could cross the cell membrane more easily [40]. We
observed that the nature of the ligand bound to Cu had a direct
influence on the anticancer properties of the compounds. Obvi-
ously, lipophilic groups attached to the ligand played an important
role in enhancing the anticancer activity of the Cu complexes. Thus
when H atom of a ligand was replaced by more lipophilic functional
moieties such as methyl or pyridine, C2 and C3 showed a two fold
increase in anticancer activity in comparison with C1. Interestingly,
the anticancer activity of the Cu compounds was proportional to
the degree of lipophilicity of the ligand (Table S3); this explains
why the anticancer activity of C4 was about four fold more than
that of C2 and C3 [41,42]. Therefore, by keeping the basic phar-
macophore of the ligand attached to Cu intact we could manipulate
the lipophilic groups of the ligand to regulate the anticancer ac-
tivity of the compounds.
2
5.2.1. Synthesis of [CuCl(L1)] (C1)
C1 was synthesized by the following procedure: 2-
Pyridinecarboxaldehyde (1 mmol) was dissolved in 15 mL meth-
anol/acetonitrile mixed solution (v:v ¼ 3:2) and subsequently the
appropriate thiosemicarbazide (1 mmol) was dissolved in 10 mL
methanol/acetonitrile mixed solution (v:v ¼ 3:2) and the two so-
lutions then refluxed 24 h to give an orange solution. CuCl
2
(1 mmol) was added and the mixture gently refluxed for 30 min.
Upon cooling, the celadon solution was filtered off and covered by
plastic wrap. A few weeks later, single brown crystals of Cu com-
pound suitable for X-ray diffraction were obtained from the filtrate.
The crystals were isolated, washed three times with distilled water
and dried in a vacuum desiccator. Yield (0.396 g, 71%). Anal. Calcd
for C14
16 2 8 2
H Cl2Cu N S (558.46): C, 30.11; H, 2.89; N, 20.06 and S,
11.48. Found: C, 30.56; H, 2.63; N, 20.35 and S, 11.99.
The Bcl-2 family proteins have an important role in cell
apoptosis; for instance, the antiapoptotic family members such as
Bcl-xl and Bcl-2 are the key regulators of apoptosis in the
mitochondria-mediated pathway. Therefore, we may design drugs
that can inhibit the expression of antiapoptotic proteins to promote
cancer cell apoptosis. Clearly the Cu compounds possess this
function. Importantly by modifying the functional groups linked to
the coordination ligand in the Cu compounds we could regulate
their inhibitory potency for inhibiting the expression of anti-
apoptotic proteins. This was the reason why C2eC4 were stronger
inhibitors of antiapoptotic proteins compared with C1 (Fig. 8).
Meanwhile, Fig. 8 showed that C4 had stronger capacity of inhib-
iting antiapoptotic proteins than C2eC3; this clearly demonstrated
that by increasing lipophilic groups in the Cu compound we could
enhance its inhibitory potential.
5.2.2. Synthesis of [Cu(NO
3 2
) (L2)] (C2)
The procedure of C2 is same as C1 except that 2-
Pyridinecarboxaldehyde and CuCl were replaced by 4-
Methylthiosemicarbazide and Cu(NO ) , respectively. The brown
3 2
crystals suitable for X-ray diffraction analysis were harvested. Yield
(0.422 g, 66%). Anal. Calcd for C16 (639.62): C, 30.04;
H, 3.15; N, 21.90; O, 15.01; S, 10.03. Found: C, 30.64; H, 2.55; N,
21.86; O, 15.64; S, 9.67.
2
2 10 6 2
H20Cu N O S
5.2.3. Synthesis of [Cu(NCS) (L3)]
The procedure of C3 is similar to C1 except that 2-
Pyridinecarboxaldehyde and CuCl were replaced by Di(2-pyridyl)
ketone and Cu(CH COO) , respectively. The brown crystals suitable
for X-ray diffraction analysis were harvested. Yield (0.348 g, 46%).
Anal. Calcd for C26 (757.89): C, 41.20; H, 2.93; N, 22.18;
S, 16.92. Found: C, 41.55; H, 2.03; N, 22.46; S, 16.66.
2
(C3)
2
3
2
2 12 4
H22Cu N S
4
. Conclusion
5
.2.4. Synthesis of [Cu(CH
The procedure of C4 is similar to C1 except that Thio-
semicarbazide and CuCl were replaced by
ꢀPhenylꢀ3ꢀthiosemicarbazide and Cu(CH COO) , respectively.
The brown crystals suitable for Xꢀray diffraction analysis were
harvested. Yield (0.876 g, 69%). Anal. Calcd for C40
912.00): C, 52.68; H, 3.98; N,15.36; O, 7.02; S, 7.03. Found: C, 52.38;
H, 3.26; N, 15.99; O, 7.53; S, 7.34.
3 2
COO) (L4)] (C4)
Our results showed that the four binuclear Cu(II) compounds
2
with thiosemicarbazone possess higher antitumour activity than
corresponding thiosemicarbazone ligands. The apoptosis of cancer
cells induced by Cu compounds might occur through the intrinsic
ROS-mediated mitochondrial pathway. Furthermore, modification
of functional groups in the ligand attached to Cu could regulate
their anticancer activity. In conclusion, our results may be useful in
designing novel Cu(II) anticancer agents.
4
3
2
H
2 10 4 2
36Cu N O S
(
5
.3. X-ray crystallography
Crystallographic data were acquired at 293 K on an Bruker Smart
000 CCD diffractometer employing graphite-monochromate
5
. Experimental
1
MoK
range 3.2 <
a
radiation (
l
¼ 0.071073 nm) and operating within the
5.1. Material
ꢁ
ꢁ
q
< 25.0 . Data reduction and empirical absorption
corrections (multiscan) were performed with Oxford Diffraction
CrysAlisPro software. Structures were solved by direct methods
with SHELXS-97 and refined by fullꢀmatrix least squares analysis
with SHELXL-97 [44]. All nonꢀH atoms were refined with aniso-
tropic thermal parameters. Molecular structure diagrams were
produced with ORTEP3 [45]. The data in CIF format have been
deposited at the Cambridge Crystallographic Data Centre (Table 2).
Crystal data and selected bond lengths were shown in Table 2 and
Table S1, respectively.
All chemical reagents are analytically pure and available from
commercial sources. Ultrapure MilliQ water was used in all ex-
periments. Elemental analyses (C, N, and H) were carried out on a
PerkineElmer 2400 analyzer.
5
.2. The synthesis of four binuclear copper(II) compounds
Compounds L1-L4 were all prepared by high yielding,
straightforward Schiff base condensation reactions leading to
crystalline compounds that typically did not require further puri-
fication [43]. The synthesis method of four compounds is almost
5.4. Cytotoxicity assay in vitro
same. The ethyl alcohol solution of CuCl
2
was added into the so-
Chelators were dissolved in DMSO as 10 mM stock solutions and
diluted in PBS so that the final [DMSO] < 0.5% (v/v). At this DMSO
concentration there was no effect on proliferation. The BEL-7404
lution of the Schiff base ligands in methanol, and then the mixed
solution was stirred well. The single crystals of compounds suitable
for X-ray diffraction were obtained from the solution in several
days.
cell line (Chinese academy of sciences) was grown as previously
ꢁ
described at 37 C in a humidified atmosphere of 5% CO
2
/95% air in

J. Qi et al. / European Journal of Medicinal Chemistry 96 (2015) 360e368
367
Table 2
Crystal Data of four Cu(II) complexes.
Cu complex
C1
C2
C3
C4
formula
formula weight
crystal system
space group
colour
C14H14Cl2Cu2N8S2
556.45
triclinic
P-1
C16H18Cu2N10O6S2
637.6
triclinic
P-1
C26H20Cu2N12S4
755.86
monoclinic
P21/n
C40H34Cu2N10O4S2
909.97
monoclinic
P21/c
Brown
Brown
Brown
Brown
a, Ẳ
b, Ẳ
c, Ẳ
R, deg
7.644(2)
8.332(2)
8.843(2)
111.104(4)
93.545(4)
108.472(4)
488.4(2)
296.15
7.523(3)
8.709(3)
9.916(4)
72.687(5)
86.576(5)
70.289(5)
583.3(4)
296.15
8.6722(11)
16.635(2)
10.9362(14)
90
95.799(2)
90
10.8044(7)
15.9236(10)
12.3628(8)
90
111.405(7)
90
b
g
, deg
, deg
V, Ẳ3
T, K
1569.6(3)
296.15
1980.2(2)
298.15
Z
1
1
2
2
R1 (obs data)
wR2 (all data)
GOOF
0.0197
0.0629
1.102
0.0267
0.0979
0.779
0.0449
0.1495
0.998
0.0816
0.2407
1.579
CCDC
993235
993280
993283
993285
an incubator (ThermoꢀFisher). The MTT assay was performed ac-
[12,47]. H
DCF, which leads to it becoming trapped within the cytosol.
Cellular oxidants localized to the cytosol oxidize nonfluorescent
2
DCFꢀDA is hydrolyzed by intracellular esterases to
4
cording to the literature method [46]. Briefly, 5 ꢂ 10 cells per well
H
2
were seeded in triplicate in 96ꢀwell plates and incubated for
ꢁ
2
4 h at 37 C in 5% CO
2
. Then the medium was replaced with the
H
2
DCF to the fluorescent product DCF. BELꢀ7404 cells were treated
ꢁ
respective medium with 1% FBS containing the compounds at
with 20
m
M of C1eC4 or 20
m
M of CuCl
2
1 h at 37 C, and then
ꢁ
various concentrations and incubated at 37 C under conditions of
washed twice with serum free medium. The cells were incubated
ꢁ
5
% CO
2
for 48 h. The final DMSO concentration in all experiments
with 2
mM H
2
DCFꢀDA for 30 min at 37 C, and then washed twice
was <0.5% in medium. The absorbance of the converted dye in
living cells was measured at a wavelength of 570 nm. Values shown
are the mean values of at least three measurements. IC50 values (the
drug concentrations that reduced the number of viable cells to 50%
of the control level) were determined by the nonlinear multipur-
pose curve-fitting program GraphPad Prism.
with serum free medium. Cells were collected for flow cytometric
assessment. Intracellular ROS was detected as an increase in green
cytosolic DCF fluorescence with an FACS Canto flow cytometer
(Beyotime Institute of Biotechnology, China), and 10 000 events
were acquired for every sample. Data analysis was performed using
Flow Jo software.
5.5. Cell apoptosis
5.7. Intracellular GSH/GSSG assay
Apoptosis studies were performed with a staining method uti-
The GSH/GSSG ratio reflects the cellular redox state [20,48], and
this was assessed using an GSH and GSSG assay kit (Beyotime
Institute of Biotechnology, China) based on the enzymatic recycling
method using glutathione reductase. BEL-7404 cells were seeded in
lizing acridine orange (AO) and ethidium bromide (EB). According
to the difference in membrane integrity between necrotic and
apoptosis. AO can pass through cell membrane, but EB can not.
Under fluorescence microscope, living cells appear green. Necrotic
cells stain red but have a nuclear morpholopy resembling that of
viable cells. Apoptosis cells appear green, and morphological
changes such as cell blebbing and formation of apoptotic bodies
6
10 cm tissue culture plates at 2.5 ꢂ 10 cells/plate and incubated at
ꢁ
least 12 h before treatment. Cells were treated for 1 h at 37 C with
20
mM of C1eC4 or 20
2
mM of CuCl .
Cells were washed with PBS once. Then detached with EDTA
will be observed. BEL-7404 cells was incubated in the absence and
(1mMin calcium/magnesiumfree PBS) and prepared according to
the instructions obtained with the GSH/GSSG ratio assay kit
(Beyotime Institute of Biotechnology, China). Briefly, keep cells on
ice for 5 min and centrifuge the samples at 10,000g for 10 min at
ꢁ
presence of C1eC4 at concentration of 5
m
M at 37 C and 5% CO
2
for
12 h, then each cell culture was stained with AO/EB solution
(
100 g/mL AO, 100 g/mL EB). Samples were observed under a
m
m
ꢁ
fluorescence microscope.
4 C to remove insoluble material. For the GSSG sample, GSH Clear
The apoptotic events induced by the copper(II) compound
C1eC4) were determined by annexin V staining and PI according
auxiliary liquid and GSH Clear reagent working solution was added.
The cell lysates were added to GSSG or GSH assay buffers, respec-
(
0
0
to the manufacturer's protocol for the Annexin VꢀFITC Apoptosis
tively. Samples were mixed sequentially with the chromogen 5 ,5 -
Dithiobis 2-nitrobenzoic acid (DTNB) and GSH to form TNB, which
exhibits maximum absorbance at 412 nm. A standard curve was
constructed using a known quantity of GSH. The GSH and GSSG
concentrations were calculated by linear regression against the
standard curve. The GSH/GSSG ratio was obtained from the equa-
tion [ratio ¼ (GSH ꢀ 2GSSG)/GSSG]. The results were expressed as a
percentage of the control.
5
Detection Kit (Abcam). For these analyses, we used 1 ꢂ 10 cells/mL,
ꢁ
which were incubated at 5% CO
2
and 37 C with the C1eC4 (5
mM)
for 12 h. The BEL-7404 cells were resuspended in 100
mL
1
2
ꢂ annexin Vꢀbinding buffer (10 mM Hepes/NaOH, 140 mM NaCl,
.5 mM CaCl , pH 7.4), and 5 L each of annexin V and PI were
2
m
added to each sample. Next, we incubated the cells for 15 min at
room temperature and then subjected them to flow cytometric
analysis (FACScan, Bection Dickinson, San Jose, CA). The rate of cell
apoptosis was analyzed.
5.8. The change of mitochondrial membrane potential assay
5
.6. Intracellular ROS measurements
BELꢀ7404 cells were treated with 20
M of CuCl for 1 h in 6-well plates and were then washed three
times with cold PBS. The cells were then detached with trypsin-
mM of L1-L4, C1eC4 or
20
m
2
Intracellular ROS generation was measured using H
2
DCFꢀDA

368
J. Qi et al. / European Journal of Medicinal Chemistry 96 (2015) 360e368
EDTA solution. Collected cells were incubated for 20 min with 1
m
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total of 30 min, then the secondary antibodies conjugated with
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This work was supported by the Natural Science Foundation of
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