Copper(ii) complexes based on quinoline-derived Schiff-base ligands: synthesis, characterization, HSA/DNA binding ability, and anticancer activity

Article abstract of DOI:10.1039/C8MD00223A

Three copper(ii) complexes, [Cu(L1)(NO₃)₂] (C1), [Cu(L2)Cl₂] (C2) and [Cu(L2)SO₄]₂·H₂O (C3), were designed and synthesized by the reaction of Cu(NO₃)₂·3H₂O,

Full text of DOI:10.1039/C8MD00223A

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Copper(II) complexes based on quinoline derived Schiff-base ligands: synthesis,
characterization, HSA/DNA binding ability, and anticancer activity
Received 00th January 20xx,
Accepted 00th January 20xx
Kun Hu, *a Chensi Liu,a Jingui Lia and Fupei Liang*a,b
DOI: 10.1039/x0xx00000x
Three copper(II) complexes[Cu(L1)(NO3)2] (C1), [Cu(L2)Cl2] (C2) and [Cu(L2)SO4]2·H2O (C3) were designed and synthesized
by reaction of Cu(NO3)2·3H2O, CuCl2·2H2O and CuSO4·5H2O with a quinoline derived Schiff base ligand L1 or L2 prepared by
condensation of quinoline-8-carbaldehyde with 4-amino-benzoic acid methyl ester or 4-amino-benzoic acid ethyl ester
(benzocaine). The efficient bindings of the C1-C3 with human serum albumin (HSA) and calf thymus DNA (CT-DNA) were
analyzed by spectroscopy and molecular docking. These complexes could significantly quench the fluorescence of HSA
through static quenching process, and hydrophobic interaction with HSA through sub domain IIA and IIIA cavity.
Meanwhile, the complexes could bind to DNA via intercalative mode, and they fit well into the curved contour of the DNA
target into the minor groove region. Furthermore, the interaction ability of the Cu(II) complexes with HSA/DNA was more
than their corresponding ligand. Interestingly, C1-C3, especially C3 exhibited more cytotoxic to HeLa cells compared to
normal HL-7702 cells and other three tumor cell lines (Hep-G2, NCI-H460, and MGC80-3). Moreover, their cytotoxicity
toward the HeLa cell lines was 1.9-3.5-fold more potent than cisplatin. Further studies indicated that these complexes
arrested cell cycle in G0/G1 phase and promoted tumor cell apoptosis via reactive oxygen species (ROS)-mediated
www.rsc.org/
mitochondrial
pathway.
based anticancer agents was the effect of the ligand
framework. Because the organic ligand could regulate
metal absorption, distribution, and metabolism in
biological systems, and thus reduce toxic side effects,
improve efficiency, enhance delivery, and provide better
activities in drug resistance cells.6 Thus, the ligand
introduced was crucial for the metal-based anticancer
agents. As an important class of compounds, Schiff bases
have drawn considerable attention due to their
bioactivities, pharmacological activities and good
coordination ability with metal ion.7-9 Some Schiff bases
and their copper complexes have been designed and
employed as very effective metal-based multiple drug
resistance modulators.10-12 Moreover, Schiff base copper
complexes can also bind DNA or cause DNA damage,
promote intracellular reactive oxygen species (ROS)
levels, as well as the activation on the mitochondrial
pathway and induce caspase-dependent apoptosis.13, 14
Quinoline and its derivatives, as an important class of
biologically active heterocyclic compounds, exhibited
various pharmaceutical activities.15-18 In particular,
quinoline scaffolds play a key role in the development of
anticancer drugs because they exhibit excellent results
by different mechanisms of action such as growth
inhibitors by apoptosis induction, cell cycle arrest, and
disruption of cell migration and inhibition of
angiogenesis.16 Copper complex based on quinoline
Schiff base has been found to show significant
1. Introduction
With the development of medicinal inorganic chemistry,
especially the anticancer activity of cisplatin being found,
metal-based drugs have been placed in the frontline in
the fight toward cancer.1 Among these bio-essential
metals, the homeostasis and metabolism of copper are
important to all kinds of human cancers, and copper
levels in tumor tissue of cancer patients significantly
elevated compared to normal tissue.2 So, cancer cells
may represent a suitable and selective target for copper-
based agents.3 The copper also alters cancer cells
metabolism and exhibits differential response to tumor
cells compared with normal cells, which endowed copper
complexes
essential antineoplastic
characteristics.
Therefore, copper complexes showed encouraging
perspectives and are considered as promising
alternatives to platinum drugs in this field.1,4,5
Recent literature revealed that the activity of metal-
a.State Key Laboratory Cultivation Base for Chemistry and Molecular Engineering of
Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal
University, 15 Yucai Road, Guilin 541004, P. R. China., Fax, 086-773-2120958 ; E-
mail: hukun216@163.com, fliangoffice@yahoo.com.
b.Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional
Materials, College of Chemistry and Bioengineering, Guilin University of
Technology, Guilin 541004, China.
Electronic Supplementary Information (ESI) available: CCDC 1587628, 1587630 and
1587631.For ESI and crystallographic data in CIF or other electronic format. See
DOI: 10.1039/x0xx00000x
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perspectives for anticancer therapies. For example,
several quinoline-2-carboxaldehyde Schiff base copper
complexes were reported and showed antiproliferative
activity toward prostate cancer cells and cervical cancer
cell.19-22 Following these report, aminoquinoline Schiff
base copper complexes were also synthesized and
evaluated their anticancer activity.22,23 A series of 2-oxo-
2.1 Synthesis and structures of Cu(II)
complexes
2.1.1 Synthesis
Synthesis of L1 and L2. L1 and L2 ligand were synthesized
32
In brief, a
1,
2-dihydroquinoline-3-carbaldehyde
(2′-
according to the available methods.31,
hydroxybenzoyl,24
benzoyl,25
2′-methylbenzoyl,26
mixture of quinoline-8-carbaldehyde (1.57 g, 10 mmol)
and 4-amino-benzoic acid methyl ester (1.51 g, 10
mmol,) or benzocaine (1.65 g, 10 mmol) in 50 mL
methanol was stirred overnight (60 °C). And then the
resulting yellow solid products suitable for structural
analysis were obtained.
semicarbazone27) hydrazone copper complexes have
been found to demonstrate low cytotoxic activity toward
normal mouse embryonic fibroblasts cell line (NIH 3T3)
(IC50 > 200 µM). However, they showed significantly less
activity against HeLa cell lines than cisplatin at the same
time. Moreover, transition metal complexes with
quinoline Schiff base ligands have kindled many
researchers’ interest in continuing the subject of many
studies, especially as anticancer chemotherapeutic
agents.21, 28 And in previous studies we found that the
Cu(II) quinolin hydrazone Schiff base complex showed
L1. Yield: 93%. Anal. Calcd for C18H14N2O2: C, H and N
of 74.47, 4.86 and 9.65, respectively. Found: C, H and N
of 74.35, 4.90 and 9.51, respectively. FT-IR (KBr, cm-1):
1704, 1599, 1578, 1274, 1105, 779, 706 and 652. HRMS
(ESI): m/z=291.1127 [M+H]+, and calcd for: 291.1134
[M+H+]+. 1H NMR (500 MHz, DMSO-d6) δ 9.84 (s, 1H, -
CH=N), 9.05 (d, J=3.8 Hz, 1H, ArH), 8.55 (d, J=7.2 Hz, 1H,
ArH), 8.51 (d, J=8.2 Hz, 1H, ArH), 8.24 (d, J=8.0 Hz, 1H,
ArH), 8.04 (d, J=8.2 Hz, 2H, ArH), 7.79 (t, J=7.7 Hz, 1H,
ArH), 7.67 (dd, J=8.3, 4.1 Hz, 1H, ArH), 7.41 (d, J=8.3 Hz,
2H, ArH), 3.87 (s, 3H, CH3O).
high anticancer activity against HeLa and other cancer
30
Although these quinoline Schiff base
cell lines.29,
copper complexes exhibit good biological activities, their
cytotoxicity to normal cell lines is still unsatisfactory
which may restrict their application. So it is very
interesting to design and synthesize some novel
quinoline Schiff base copper complexes with high
efficacy and less toxicity.
L2. Yield: 94%. Anal. Calcd for C19H16N2O2: C, H and N
of 74.73, 5. 61 and 9.17, respectively. Found: C, H and N
of 74.80, 5.72 and 9.01, respectively. FT-IR (KBr, cm-1):
1704, 1599, 1578, 1274, 1105, 779, 706 and 652. HRMS
(ESI): m/z=305.1280 [M+H]+, and calcd for: 305.1290
[M+H+]+. 1H NMR (500 MHz, DMSO-d6) δ 9.85 (s, 1H, -
CH=N-), 9.05 (dd, J=4.1, 1.8 Hz, 1H, ArH), 8.57 (dd, J=7.3,
1.2 Hz, 1H, ArH), 8.52 (dd, J=8.4, 1.7 Hz, 1H, ArH), 8.26
(dd, J=8.1, 1.2 Hz, 1H, ArH), 8.07-8.03 (m, 2H, ArH), 7.81
(t, J=7.7 Hz, 1H, ArH), 7.68 (dd, J=8.3, 4.2 Hz, 1H, ArH),
7.44-7.39 (m, 2H, ArH), 4.34 (q, J=7.1 Hz, 2H,-CH2CH3),
1.35 (t, J=7.1 Hz, 3H, -CH2CH3).
Taking the significance mentioned above into
consideration, we designed and synthesized three
copper(II) complexes[Cu(L1)(NO3)2] (C1), [Cu(L2)Cl2] (C2
)
.
and [Cu(L2)SO4]2 H2O (C3) with quinoline derived Schiff
base ligands (L1=(E)-methyl 4-((quinolin-8-ylmethylene)
amino)
benzoate,
L2=(E)-ethyl
4-((quinolin-8-
ylmethylene) amino) benzoate). The interactions of
these complexes with HSA/DNA were evaluated through
spectrum and molecular docking method. The
antiproliferative activities of ligands and C1-C3 in the cell
Synthesis of C1, C2 and C3. Copper salt (0.15 mmol,
.
Cu(NO3)2 3H2O (36.3 mg) for C1, CuCl2 2H2O (25.5 mg) for
.
lines were investigated, and the anticancer mechanisms
were further surveyed.
.
C2 and CuSO4 5H2O (37.5 mg) for C3) and ligand (0.15
mmol, L1 for C1, L2 for C2 and C3) were put in a thick
Pyrex tube, and then the Pyrex tube was quenched in
liquid N2 and sealed. They were heated for four days at
80 °C, and programmed cooling to room temperature (5
°C/h). Crystals suitable for X-ray structure analysis were
obtained.
2. Experimental section
Antibody, CT-DNA, HSA, quinoline-8-carboxaldehyde,
benzocaine and 4-amino-benzoic acid methyl ester were
purchased from Sigma Chemical Co. (USA). Hep-G2, NCI-
H460, MGC80-3, HeLa cell lines and normal liver cells
(HL-7702) were obtained from ATCC.
C1. Green blocks crystal. Yield: 82%. Anal. Calcd for
C18H14CuN4O8 (477.87): C, H and N of 45.24, 2.95 and
11.72, respectively. Found: C, H and N of 45.05, 3.11 and
11.42, respectively. FT-IR (KBr, cm−1): 3440, 1704, 1637,
1589, 1409, 1378, 1285, 1184, 1116, 1015, 775, 705, 610,
521, 479 and 430. HRMS (ESI): C18H14CuN3O5 [M-NO3]+,
calcd for: 415.0230, found 415.0227.
Flow cytometric analysis was carried out using a FACS
Aria II flow cytometer
.
Fluorescence spectra were
measured on PE LS55 spectrometer. UV-Vis spectra were
detected using an Aglient Carry 100. 1H NMR spectra
were got on Bruker Avance 500 spectrometer. High-
C2. Green blocks crystals. Yield: 80%. Anal. Calcd for
C19H16N2O2CuCl2 (403.34): C, H and N of 52.01, 3.68 and
6.38, respectively. Found: C, H and N of 51.87, 3.55 and
6.59, respectively. FT-IR (KBr, cm-1): 3444, 1715, 1595,
resolution mass spectra were recorded on
a
Thermofisher
spectrometer.
Scientific
Exactive
LC-MS
mass
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1511, 1279, 1184, 1100, 1022, 843, 775, 695, 606, 521,
479 and 433. HRMS (ESI): C19H16N2O2CuCl [M-Cl]+, calcd
for: 402.0196, found 402.0183.
2.4. Molecular docking analysis
Docking studies were performed in order to analyze
interactions of test complex to HSA /DNA. The AutoDock
Tools with 1.5.6 docking programmers and Autodock
Vina were employed to analyze the molecular docking.39,
40 The HSA and B-DNA crystal structures were got from
the bank of Protein Data (HSA: PDB ID: 1E7H; DNA: PDB
ID: 1BNA; (CGCGAATTCGCG)2). The structure of complex
C3. Bright green blocks crystals. Yield: 78%. Anal. Calcd
for C38H32N4O14Cu2S2 (959.88): C, H and N of 47.55, 3.36
and 5.84, respectively. Found: C, H and N of 47.15, 3.81
and N 5.65, respectively. FT-IR (KBr, cm-1): 3440, 1715,
1637, 1384, 1274, 1142, 838, 780, 659, 574, 490 and 427.
HRMS (ESI): C19H17N2O6CuS [M-H2O+H]-, calcd for:
464.0103, found 464.0048.
and HSA/DNA was prepared according to reference
method.41,
42
Briefly, the Cu(II) complexes structures
were converted into PDB format from cif format using
Mercury software. The structure of DNA and protein was
modified to include polar hydrogen atoms and water
molecules and the ligands were removed, and Gasteiger
charges were added to the complex by Autodock Tools
(ADT) before performing docking calculations.
2.1.2 X-Ray crystallography
The crystal structure data of complexes C1-C3 were
collected using a SuperNova CCD Area Detector (Mo-Ka,
λ=0.71073 Å). Data collection and refinement were made
using Apex2 software.33 SADABS program was used for
absorption corrections.34 The structures of complexes
C1
-
C3 were solved through SHELX-97 programs. The
2.5. MTT assays
complexes C1-C3 crystallographic information is given in
The cytotoxic of all compounds against cell lines was
screened through MTT standard method. In brief, in a
96-well plate, the cells were seeded (5×103cells/well) and
cultured for 24 h in 5% CO2. And test compounds (0-100
μM) were added and further incubated for 48 h. In the
each well, 10 μL, 5 mg/mL MTT solution was put into and
cultured for 5 h, and then DMSO (100 μL) was injected
after removal of the supernatant. Finally, we used the
ELISA microplate reader to measure the absorbance of
each well and calculate the IC50 values.
Table S1 and Table S2.
2.2. HSA binding studies
Fluorescence method. A 2.0 mL HSA aqueous solution (1
µM) was titrated by the C1-C3 and L1, L2. The changes of
fluorescence of HSA were recorded at excitation
wavelength =275 nm. By using the Scatchard equation
(Eq. (1)):35 log[(F0 -F)/F]=logKbin+n×log(Q), the binding
constant (Kbin) of test compounds to HSA were analyzed.
Furthermore, the quenching rate constant (Kq) and
quenching constant (Ksv) can be calculated as (Eq. (2)):
F0/F=1+Kqτ0[Q]=1+Ksv[Q].Where F0 is fluorescence of HSA;
F is the fluorescence of HSA after adding test compound;
[Q] is the test compound concentration; τ0 =4.43 ns.36
And the synchronous fluorescence spectra of HSA were
also scanned when ∆λ=60, 15 nm, respectively.
2.6. Cell cycle distribution analysis and cellular
uptake
After HeLa cells were cultured, the C3 was added at
indicated concentration. The mixture was kept in 5 %
CO2 at 37 °C for 24 h. And then, we collected and fixed
the cells with ice-cold ethanol (70 %, -20 °C) overnight.
The 100 µg/ml Rnase A was put into the cells and further
cultured (30 min, 37 °C). In the dark, the cells were
stained using PI for 15 min. Finally, the above solution
was tested and analyzed immediately.
UV-visible absorption method. The concentration of
the compound and HSA was kept at 2.5 µM. The
absorption spectra are scanned from 250 to 350 nm at
room temperature.
HeLa cell was treated with C3 (10 µM) and cultured for
24 h. The cells were washed five times with ice-cold
buffer and treated with concentrated HNO3. The amount
of Cu(II) compound in cells was monitored by ICP-MS.
2.3. DNA binding studies
CT-DNA/EB system (10 µM) was titrated by the test
compounds ranging from
0 to 30 μM and their
fluorescence spectra were measured. And the quenching
constant (Kq) and the apparent DNA binding constant
2.7. Cells apoptosis
(Kapp) were given using the Eq. (3): I0/I = 1+Kq [Q] and Eq.
37, 38
(4): KEB [EB] = Kapp [complex],
respectively. Where I0
HeLa cells were cultured with C3 at different
concentrations for 48 h, and then harvested and
suspended in buffer of annexin-binding (200 µL 1×). We
used the annexin V (5 µL) and PI (5 µL) to stain the cell
suspension in the dark (15 min). And binding buffer (300
µL 1×) was follow put into the while gently mixing.
is the fluorescence of CT-DNA/EB system. And I is the
fluorescence of CT-DNA/EB after adding test compounds.
[Q] is the compound concentration; KEB = 1.0×107 M-1;
[complex] is the compound concentration at 50 %
fluorescence of CT-DNA/EB system.
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Finally, the mixtures were measured immediately using
FACS Calibur flow cytometer (BD).
3.1. Characterization of L1, L2, C1-C3
L1, L2, C1-C3 were synthesized and characterized via the
above
synthetic method (Fig.S1-S12). The
2.8. ROS level
characterization results indicated that the structure of
these compounds is consistent with the compositions.
The crystal structures of C1 and C2 are shown in Fig.1.
The HeLa cells were treated with C3 (0-20 μM) and
incubated in CO2 incubator (24 h, 37 °C) after
attachment. HeLa cells were then collected and
incubated for 30 min at 37 ºC with H2DCFDA (10 µM) in
serum-free medium. Afterward, HeLa cells were washed
and then the fluorescence intensity was recorded.
The C1 and C2 belong to the triclinic (
P-1) and monoclinic
(P21/n) crystal system, respectively. In complex C1, the
copper atom is four-coordinate, bonded to two N (N1
-
and N2) donors from L1 ligand and two terminals NO3 .
All Cu-X bond lengths are in good consistent with
previously literature of Cu(II) complex.43 In complex C2
,
copper atoms are four coordinated by two nitrogen
atoms (N1 and N2) of L2 ligand and two terminal chlorine
(Cl1 and Cl2).
2.9. Mitochondrial membrane potential
After HeLa cells were seeded and incubated with
complete DMEM for 24 h, the C3 was added and further
cultured (24 h). The cells were collected and re-
suspended in PBS buffer contains pre-warmed JC-1 (5
µg/mL) and kept incubation at 37 °C (30 min).
Subsequently, we used PBS buffer to wash the cells three
times. Finally, the mixture was measured immediately by
FACS Calibur flow cytometer (BD).
Compared to C1 and C2, the C3 is a dinuclear neutral
2-
complex, [Cu(L2)SO4]2, with SO4 anion bridges (Fig. 1).
Every central copper(II) in the dimer is five-coordinated
in the form of a slightly distorted square pyramidal
geometry with the τ=0.01.41 Interestingly, the Cu-
N(quinolin) lengths (Cu1-N1 = 1.985 (4) Å in C1, Cu1-N1 =
1.987 (2) Å in C2 and Cu1-N2 = 1.979 (5) Å in C3) of
complexes C1-C3 are longer than the Cu-N(imine) lengths
(Cu1-N2=1.972 (4) Å in C1, Cu1-N2 = 1.9855 (19) in C2
and Cu1-N1=1.958 (5) Å in C3) of these complexes,
respectively, which is possibly due to weaker π-back-
bonding in the Cu-N (quinolin) bond than in the Cu-N
(imine) bond.
2.10. Western blot assay
Briefly, after the HeLa cells were cultured overnight, C3
was added and further incubated (24 h). The total
proteins were extracted after cell-lysing. And the extract
was loaded onto SDS polyacrylamide gel, and follow
transferred to PVDF membranes. 5 % BSA in TBST
buffer was used to block the membrane. The primary
antibodies were added and incubated overnight (4 °C).
After washing, the secondary antibody was also added
and cultured (120 min). Finally, the signal of immuno
reactive was detected.
3.2. Stability of C1-C3
The metal complexes stability has an effect on their
antitumor activity. Therefore, the hydrolytic stabilities of
the Cu(II) complexes were investigated. The UV-Vis
absorption spectra of C1-C3 in aqueous solutions (TBS,
pH 7.4, 1 % DMSO) at different times (0, 24 and 48 h) are
shown in Fig. S13. The UV-Vis spectra do not differ
significantly from those observed even after 48 h at
3. Results and Discussion
laboratory temperature, which indicated that the C1-C3
Fig. 1. ORTEP drawing of the complex C1-C3 with atom numbering scheme. Color scheme: Cu, aqua; O, red; N, blue; C, gray; Cl, green.
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were stable in TBS buffer within 48 h.
C1-C3 (Fig. S17). The addition of test compounds
gradually leads to hypochromic effect of HSA. Moreover,
when Δλ=60 nm, the maxima emission wavelength of
HSA not only decreased gradually but also accompanied
by a slight blue shift (1 nm for C1 and C2, 2 nm for C3).
However, the maximum emission wavelength has no
3.3 HSA binging ability
The binding ability of anticancer agents with HSA affects
their distribution, uptake, transfer, metabolism and
mechanism of action to cancer cells.44-46 First, we studied
shift at Δλ=15 nm. The results demonstrated that the C1
-
C3 bind effectively with HSA, and thus quench the
fluorescence of tyrosine and tryptophan residues of HSA.
And the fluorescence changes also indicate that the
interaction of them may occur in domain II because the
Tyr and Trp residues are located in domain II of HSA.42,49
These observed changes of fluorescence are different
from the results obtained for cisplatin which induce the
emission wavelength of HSA red-shifted and it bound to
HSA mainly via covalent bonds.49,50,51 Therefore, the
the nature of the C1-C3 binding with HSA using
fluorescence and UV-Vis absorption method.
The fluorescence spectral changes were investigated
before and after titration using the Cu(II) complexes (Fig.
2-A, B, C) and ligands (Fig. S14). Addition of these
compounds to HSA resulted in essential fluorescence
quenching of HSA with small blue shift (59 %, 1 nm, C1
;
60 %, 3 nm, C2; 62 %, 7 nm, C3; 43 %, 1 nm, L1; 47 %, 2
nm, L2). And the quenching efficiency of these
complexes for HSA was more than free ligands. These
facts indicated that the binding of the test compounds to
HSA did happen and the binding sites of HSA are buried
in a hydrophobic environment.
results suggest that the interaction of C1-C3 with HSA
should be noncovalent and reversible.49
The high-performance liquid chromatographic
(HPLC)52 was often used to study the interaction mode of
guest molecules and HSA. The feature of
chromatographic spectrum of mixture of C3 or L2 and
HSA are similar to the pure HSA, indicating that binding
of C3 and L2 induces slight structural changes in the
protein (Fig. S18). These experiment results imply that
To further study the quenching process, the Kq, Ksv and
Kbin were assessed using the Eq(1) and Eq(2) (Fig. S15)
(Table 1). As can be seen from Table 1, the higher Ksv and
Kbin values of C3 than the C1 and C2 may be due to its
higher solubility in aqueous medium. The Kq values of
these complexes are 1000-fold higher than the maximum
collisional quenching constant (2.0×1010 mol L-1 s-1).34 So
the fluorescence of HSA are quenched by these Cu(II)
complexes through static quenching processes. And
these larger values of Ksv and Kbin demonstrated strong
the nature of the interaction of C1-C3 and their
respective ligands with HSA is proposed to be
noncovalent.42, 52
The above results confirmed that the C1-C3 exhibit
prominent HSA binding ability via noncovalent
interactions. And the binding ability of these complexes
for HSA was more than free ligands. Meanwhile, these
complexes simultaneously weakened the fluorescence of
tyrosine and tryptophan, but their interaction affected
the micro-region of tryptophan more than the tyrosine.
The strong binding ability of the complexes to HSA
interaction of C1-C3 with HSA.
UV-vis absorption spectroscopy is a simple method to
explore the type of quenching mechanism of HSA. So, we
chose C3 as an example for further study of the
quenching mechanism. The absorption intensity of HSA
at 280 nm was enhanced and a slight blue shift of about
2 nm was observed with the addition of C3 (Fig. S16),
which demonstrated that the quenching of the test
compounds to the fluorescence of HSA is mainly via
static processes.
suggested that the C1-C3 can be stored in protein easily,
then be carried to the objective area and be released
from the protein to the desired targets.29
Furthermore, synchronous fluorescence spectra were
used for the analysis of changes in microenvironment
around the fluorophore functional groups. The
information about microenvironment for tryptophan and
3.4. DNA Binding
DNA is one of the main molecular targets for cancer
therapeutics.53, 54 Hence, the fluorescence method based
on the EB competitive experiment is used to investigate
tyrosine was reflected when Δλ=60 nm and 15 nm,
respectively.47,
48
Synchronous fluorescence spectra of
the nature of the interaction of C1-C3 (Fig.2-D, E,F) and
HSA were obtained with different concentration of the
ligand (Fig.S19) with CT-DNA. The emission band of CT-
DNA/EB was gradually decreased when these
compounds was titrated. And the effect of
hypochromism of complex to CT-DNA/EB system is more
than corresponding ligand. Furthermore, the Kq and Kapp
values are analyzed (Table 2) according to the Eq (3) and
(4) (Fig. S20) for evaluating the binding ability of
complexes and free ligand. As can be seen from the
Table 1. Stern-Volmer quenching constants and binding parameters of
the HSA-complexes drug system at 295 K.
Kq (M-1 s -1
)
Ksv (M-1)
Kbin (M-1
)
n
C1
C2
C3
L1
L2
6.36±0.31×1013
6.49±0.25×1013
7.42±0.27×1013
3.12±0.18×1013
4.02±0.20×1013
2.83±0.14×105
2.89±0.11×105
3.30±0.12×105
1.38±0.08×105
1.78±0.09×105
0.94±0.09×108
1.15±0.04×108
1.60±0.07×108
0.47±0.05×106
1.20±0.09×106
1.37
1.49
1.51
1.29
1.38
Table 2, the binding of C1-C3 with CT-DNA is stronger
than that of the free ligand. The higher DNA binding
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Fig. 2. The emission spectrum of HSA (1μM; λex=280 nm) in the absence and presence of C1 (A), C2 (B) and C3 (C) with increasing concentrations (0, 1.0, 2.0, 3.0, 4.0 and 5.0 μΜ).
And fluorescence quenching spectra of DNA-EB (10 µM) in the presence of C1 (D), C2 (E) and C3 (F) (0, 5.0, 10, 15, 20, 25 and 30 µM).
affinity of C3 may be due to its solubility in aqueous
medium.55
and IIA, but mainly in IIA. And it was surrounded by
Lys199, Ser202, Phe211, Ala210, Trp214, Val344, Leu453,
Leu457, Leu481, Arg485, Ser454, Glu450 and Ser342.
Molecular models show that complexes C2 (Fig. S23-B)
and C3 are almost in the same position. The docking
results showed potential Trp214. C1 (Fig. S23-A) is
located in subdomain IIA hydrophobic cavity, and it is
surrounded by Leu260, Leu219, Leu238, Lys199, Arg218,
Arg257, His242, Val241, π-π stacking interactions of C2
and C3 with the residue Ala291, Glu153, Gln196, Ser192,
His288 and Ser287. The methyl 4-aminobenzoate
fragment of the C1 interacts with Gln196 and Glu153
amino acid residues by hydrogen bonds. The lipophilic
quinoline fragment of the C1 is coiled into a hydophobic
The complexes have no UV-vis absorption (Fig. S13)
and fluorescence excitation spectra in 550-700 nm range
(Fig. S21), which indicated that these compounds cannot
quench the fluorescence of EB through using the inner
effect or energy transfer mechanism. It may be inferred
that the EB is being replaced efficiently from the binding
sites of CT-DNA by these test compounds. The above
results demonstrated that the C1-C3 can interact with
CT-DNA via an intercalative mode and their binding
ability is greater than the free ligand.56
3.5 Molecular docking studies
Docking studies were investigated in order to provide
complement of the spectroscopic results and more
insight into complex-HSA interaction. The resulting
docked pattern of C3 with the lowest energy and the
entire protein structure is respectively shown in Fig.3
and Fig. S22. The results reveal that the complex C3
bound at hydrophobic cavities of HSA subdomains IIIA
Table 2. Stern-Volmer constant (Kq) and the apparent binding constant
(Kapp) for the Cu(II) complexes.
Kq (M-1 s -1
)
Kapp (M-1)
C1
C2
C3
L1
L2
0.93±0.04×104
0.92±0.05×104
1.10±0.06×104
0.22±0.02×104
0.44±0.03×104
0.95±0.05×104
0.94±0.06×104
1.11±0.06×104
0.23±0.02×104
0.45±0.02×104
Fig. 3. Molecular docked structure of the C3 interacting with HSA (PDB ID:
1E7H).
6 | J. Name., 2012, 00, 1-3
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Table 3. IC50a (µM) values of L1, L2 and C1-C3 on the selected cells for 48 h.
channel lined by Leu260, Leu219, Leu238, Ala291 and
Val241. We can fount that their binding sites with HSA
are different from cisplatin.57 The lowest binding
Compound
Hep-G2
NCI-H460
>100
MGC80-3
>100
HeLa
>100
HL-7702
>100
energies of C1, C2, and C3 respectively are -9.3, -10.8,
4-amino-benzoic >100
acid methyl ester
and -11.8 kcal mol-1, which suggested that their
bindingaffinity ability with HSA is more than the other
copper(II) complex.58
Benzocaine
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
L1
The interaction of C1-C3 with B-DNA (PDB ID: 1BNA)
L2
were also studied by docking studies. Energetically
favourable docked poses obtained from the molecular
docking of the C3 to the B-DNA is shown in Fig. 4, and
C1
>100 18.72 ± 1.03 87.70 ± 4.01
C2
35.45± 2.35 16.87 ± 2.01 15.76± 1.19 92.81± 6.03
>100 17.52± 1.81 9.98± 0.87 60.39± 5.86
C3
the C1, C2 is demonstrated in Fig. S24. And their lowest
Cisplatin
27.31± 2.01 47.68± 1.98 21.28± 2.81 35.25± 2.80 23.02± 5.68
>100 >100 >100 >100 >100
binding energies are -8.1 (C1), -8.0 (C2), and -8.4 (C3) kcal
mol-1, respectively, and those are lower than that for the
other copper(II) Schiff base complexes.59 Meanwhile, the
Copper(II) salt
cells. The enhanced antiproliferative activity of C1-C3
may result from the synergistic effect of Cu(II) ion and
ligand. Despite this potency, the Cu(II) complexes were
much less toxic toward human normal cell (Hl-7702),
with IC50 values ranging from 60.39 to 92.81 μM, which
are significantly higher than that of cisplatin (23.02 μM).
C1-C3 fitted well into the curved contour of the B-DNA
target into the minor groove region, which brings about
hydrogen bonds, hydrophobic interactions and van der
Waals with DNA functional groups that define the groove.
And their interaction region with DNA is different from
the cisplatin which binding to the DNA major groove
faces inward.60 Furthermore, the energy minimized
And the cytotoxicity of C1-C3 on the normal HL-7702
cells respectively was 4.7, 5.9 and 6.1 times lower than
the activity on HeLa cells, which suggested that the C1
-
structures suggest that the C1-C3 stabilized by π-π
C3 might selectively inhibition activity against HeLa cell.
stacking (edge to face interactions). Therefore, these
data suggested that the docking studies results
corroborated well with spectroscopic results.
In comparison, the cytotoxic activity toward HeLa cell
follows the order C3 C2 C1, which is consistent with
>
>
DNA/HSA binding results. These results implied that the
introduction of the drug molecule of benzocaine was
more effective for enhancing the anticancer activity than
that of 4-amino-benzoic acid methyl ester.
3.6. IC50 level
The ligand, metal salt, C1-C3 and cisplatin cytotoxicities
Moreover, the IC50 values of these complexes,
especially C3 to HeLa cell are comparable or lower than
of in vitro toward the tested cells were examined via
MTT method. And the IC50 values obtained are
summarized in Table 3.
most of the previously reported quinoline derived Schiff
20, 21, 23-30
base complexes.
Meanwhile, the cytotoxic
The C1-C3 showed a potent inhibition activity toward
activity toward HL-7702 was clearly decreased
comparing with our previously reported quinoline-8-
carboxaldehyde hydrazone copper complexes,29, 30 which
may be that introduction of the benzocaine into
quinoline scaffolds induces a synergistic cytotoxicity.
Hence, the results suggested that the Cu(II) complexes
possess well selectivity between normal and cancer cells,
and thus show application potential in human cervical
cancer chemotherapy.
Hela, with IC50 in the micromolar range and obviously
lower than cisplatin. In contrast, the free ligand, and
metal salt displayed relatively low activity toward HeLa
Base on the above results, it can be inferred that C3
possesses higher cytotoxicity toward HeLa cell lines
compared to the free ligand as well as C1 and C2. Thus,
C3 was selected to investigate the anticancer
mechanism.
3.7. Possible anticancer mechanism of C3
Because complex C3 exhibits the highest cytotoxicity to
HeLa, the complex was used for further cellular studies
to understand its mechanism of action. Most previously
reported cell apoptosis induction and cell cycle arrest
closely correlated to cancer cells death,61, 62 we therefore
monitored features related to these pathways.
Fig. 4. Molecular docked model of complex C3 with DNA (PDB ID:
1BNA).
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The cellular uptake properties affect metal-based
Journal Name
anticancer drug antiproliferative performance, so we first
assessed the uptake of C3 in HeLa cells using ICP-MS
assay. The accumulated amount of C3 was 9.6
±2.5
nM/106 cells in the HeLa cells after 24 h of treatment.
Subsequently, the cell cycle in HeLa cells induced by C3
(5, 10 and 20 μM) was examined by flow cytometry (Fig.
5). With the increase of the complex C3, the S-phase and
G2/M-phase cells population reduced gradually.
However, a dramatically increase in G0/G1-phase was
obtained from 53.79 % to 83.66 %. The results indicated
that C3 stalled the cell cycle at the G0/G1-phase in a
concentration-dependent manner. It is known that
cyclin-dependent kinases (CDKs) and cyclins complexes
affect the cell cycle progression at each phase.63
Therefore, the expression of cell cycle regulatory
proteins of G0/G1-phase was determined using western
blotting assay to study the molecular mechanisms of the
complex C3 induced G0/G1-phase block (Fig. 6). As can
be seen from Fig. 6, the progression of CDK2, cyclin D1
and E reduced in a dose-dependent manner. And the
expression of CDK4 has almost no changes. The observed
phenomenon revealed that inhibition of the activity of
G0/G1-phase-promoting cyclin-Cdk complexes by
limiting the supply of CDK2, cyclins D1 and E1 may be a
mechanism by which the C3 mediated growth G0/G1-
phase arrest.
Fig. 6. Western blot was used to determine the expression of CDK2, CDK4,
cyclin E, cyclin D1 in HeLa cells treated with C3 (5 and 10 μM).
It has been reported that most metal anticancer
agents generally kill tumor cells through induction of
apoptosis,64,
65
we therefore monitored the ability of
complex C3 to promote apoptosis in HeLa cells (Fig. 7). It
was found that the populations of the apoptotic HeLa
cells (both at the early stage and the late stage of
Fig. 7. Annexin-V/propidium iodide assay of HeLa cells treated by C3 (5, 10 and
apoptosis, Q2+Q3) distinctly increased in
a dose-
20 µM) measured by flow cytometry.
dependent manner when incubating with the C3. And
the apoptosis populations of HeLa cells in early-and late-
stage were 16.7% and 9.28%, respectively, which
suggested that cell apoptosis in HeLa cells can be
effectively induced by complexes.
Mitochondrial dysfunction plays an importance in
triggering various apoptotic pathways.66 To confirm
whether mitochondrial dysfunction induces cells
apoptosis, we used flow cytometry to measure the
mitochondrial membrane potential (MMP) (Fig. 8). The
C3 led to reduce in MMP compared with the control
cells, and exhibited in a dose-dependent manner, which
implies that the mitochondrially-mediated apoptosis was
activated.
In apoptosis pathway, reactive oxygen species (ROS)
elevation closely associated with mitochondrial
dysfunction.67 Thus, the complex C3 catalytic ability of
the intracellular ROS elevation in HeLa cells was
monitored by flow cytometry and the fluorescent 2’,7’-
dichlorofluorescein (DCF) probe. As shown in Fig. 9A,
comparing with the control groups, incubation of HeLa
cells with the complex C3 can increase the levels of DCF
fluorescence and lead to the red shift of the fluorescence
Fig. 5. Cell cycle analysis by flow cytometry for HeLa cells treated by
C3 (5, 10 and 20 µM
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The western blot data reveal that the complex
induced expression significantly down-regulation of Bcl-2
protein and slightly up-regulation of Bax protein in a
concentration-dependent manner. These results
demonstrated that C3 could activate the Bcl-2 and Bax
proteins and decrease the ratio of Bcl-2 protein to Bax
protein, which lead to the mitochondrial release of
apoptogenic factors in HeLa cells and ultimately induced
cells apoptosis.
4. Conclusions
In conclusion, three Cu(II) complexes of quinoline
derived Schiff base were designed, synthesized and
characterized. The C1-C3 display high quenching ability
of HSA fluorescence and strong binding activity by
noncovalent interactions, and hydrophobic interaction
with HSA through sub domain IIA and IIIA cavity.
Moreover, the complexes effectively bind to DNA by
intercalative mode, and they fit well into the curved
contour of the DNA target into the minor groove region.
Cytotoxicity assay reveal that these complexes enhanced
significantly the cytotoxic activity toward HeLa cell lines
with their IC50 values being markedly lower than those
of cisplatin, the ligands and metal salt. The in vito
antiproliferative activities of C2 and C3 against the HeLa
Fig. 8. Effects of C3 (5, 10 and 20 µM) on MMP analyzed by JC-1 staining and
flow cytometry.
cell are greater than that of C1, suggesting that the
introduction of the drug molecule of benzocaine was
more effective for enhancing the anticancer activity than
that of 4-amino-benzoic acid methyl ester. Meanwhile,
the complexes show low cytotoxic against normal HL-
7702 cell lines, which demonstrate that these complexes
have more selective for HeLa cell than for normal HL-
7702 cells. The mechanism studies indicate that C3 can
regulate the expression of CDKs along with cyclins, and
arrest cell cycle at the G0/G1 phase. At the same time,
the C3 can effectively induce HeLa cell lines apoptosis via
the ROS-mediated mitochondrial pathways, and activate
the Bcl-2 family proteins. In general, the introduction of
active medicament into ligand could improve the biologic
effects of the copper complexes with quinoline derived
Schiff base in the respect of DNA/HSA affinity and cell
cytotoxicity. The present work implies that it may be
helpful for the design and development of the potential
anticancer complex by using the modified clinical drug as
ligand.
Fig. 9. A) Analysis of ROS levels by flow cytometry after HeLa cells were treated
with vehicle, C3 at indicated concentrations for 12
h and stained with
H2DCFDA. B) Quantification of the flow cytometric results in (A) showing the
percentage of cells with increased intracellular DCF oxidation compared to
control cells. Results are the mean ±SD (n=5): (**) p<0.01.
peak, which infers that the C3 increased oxidative
damage to the HeLa cell. The level of ROS shows a dose-
dependent manner (Fig. 9B).
The Bcl-2 family proteins have been verified as key
regulators of apoptosis.68 To further study the
mechanism of cells apoptosis, we tested the Bcl-2 family
proteins expression through C3 treated with cells (Fig.
10).
Conflicts of interest
The authors declare no competing interests.
Acknowledgements
This work was support by National Natural Science Foun
dation of China (No. 21565007,21271050 and 51572050),
Guangxi Natural Science Foundation (No. 2015GXNSFDA
Fig. 10. Western blot was used to determine the expression of Bcl-2
and Bax in HeLa cells treated with C3 (5 and 10 μM).
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22 T. Thirunavukkarasu, H. A. Sparkes, K. Natarajan
and V. G. Gnanasoundari, Inorg Chim Acta., 2018,
473, 255-262.
23 J. Lu, Q. Sun, J.L. Li, L. Jiang, W. Gu, X. Liu, J.L. Tian
and S.P. Yan, J. Inorg. Biochem., 2014, 137, 46-56.
24 [23] D. S. Raja, N. S.P. Bhuvanesh and K.
Natarajan, Inorg Chim Acta., 2012, 385, 81-93.
25 D. S. Raja, N. S.P. Bhuvanesh and K. Natarajan,
Eur. J. Med. Chem., 2012, 47, 73-85.
139007), State Key Laboratory Cultivation Base for the
Chemistry and Molecular Engineering of Medicinal
Resources, Ministry of Science and Technology of China
(CMEM2014-A06,
CMEMR2012-A08),
IRT_16R15,
Talent’s Small Highland Project of Guangxi Medicine
Industry (No. 1509).
26 D. S. Raja, E. Ramachandran, N. S.P. Bhuvanesh
and K. Natarajan, Eur. J. Med. Chem., 2013, 64,
148-159.
27 D. S. Raja, N. S. P. Bhuvanesh and K. Natarajan,
Inorg. Chem., 2011, 50, 12852-12866.
References
1
C. Santini, M. Pellei, V. Gandin, M. Porchia, F.
Tisato and C. Marzano, Chem. Rev., 2014, 114,
815-862.
28 T. R. Todorović, J. Vukašinović, G. Portalone, S.
Suleiman, N. Gligorijević, S. Bjelogrlić, K.
Jovanović, S. Radulović, K. Anđelković, A. Cassar,
N. R. Filipović and P. Schembri-Wismayer, Med.
Chem. Commun., 2017, 8, 103-111.
29 K. Hu, F.Y. Li, Z. Zhang and F.P. Liang, New J.
Chem., 2017, 4, 2062-2072.
30 K. Hu, G.M. Zhou, Z. Zhang, F.Y. Li, J.G. Li and F.P.
Liang, RSC Adv., 2016, 6, 36077-36084.
2
3
4
5
A. Gupte and R.J. Mumper, Cancer Treat. Rev.,
2009, 35, 32-46.
F. Tisato, C. Marzano, M. Porchia, M. Pellei and C.
Santini, Med. Res. Rev., 2010, 30, 708-749.
L. Ruiz-Azuara and M.E. Bravo-Gomez, Curr. Med.
Chem., 2010, 17, 3606-3615.
J. Y. Hu, C.L. Liaob R.N. Mao, J.S. Zhang, J.A. Zhao
and Z.Z. Gu, Med. Chem. Commun., 2018, 9, 337-
343.
31 B.S. Creaven, B. Duff, D.A. Egan, K. Kavanagh, G.
Rosair, V.R. Thangella and M. Walsh, Inorg. Chim.
Acta, 2010, 363, 4048-4058.
6
C. Santini, M. Pellei, V. Gandin, M. Porchia, F.
Tisato and C. Marzano, Chem. Rev., 2014, 114,
815-862.
32 M.-F. Zaltariov, M. Cazacu, M. Avadanei, S.
Shova, M. Balan, N. Vornicu, A. Vlad, A. Dobrov
and C.-D. Varganici, Polyhedron, 2015, 100, 121-
131.
33 E.C.P. APEX2 “Program for Data Collection and
Integration on Area Detectors” BRUKER AXS Inc.,
Madison, WI 53711-5373 USA.
7
8
I. Kostova and L. Saso, Curr. Med. Chem., 2013,
20, 4609-4632.
K. Sztanke, A. Maziarka, A. Osinka and M.
Sztanke, Bioorg. Med. Chem., 2013, 21, 3648-
3666.
9
P. H. Wang, J. G. Keck, E. J. Lien and M. M. Lai, J.
Med. Chem., 1990, 33, 608-614.
34 G.M. Sheldrick, SADABS: Program for Empirical
Absorption Correction of Area Detector Data,
University of Göttingen, Germany, 1996.
35 O.K. Abou-Zied and O.I. Al-Shihi, J. Am. Chem.
Soc., 2008, 130, 10793-10801.
10 A. Ganguly, P. Chakraborty, K. Banerjee and S. K.
Choudhuri, Eur.J. Pharm. Sci., 2014, 51, 96-109.
11 R. D. Ghosh, P. Chakraborty, K. Banerjee, A.
Adhikary, A. Sarkar, M. Chatterjee, T. Das and S.
K. Choudhuri, Mol. Cell. Biochem., 2012, 364,
309-320.
12 A. Ganguly, S. Basu, K. Banerjee, P. Chakraborty,
A. Sarkar, M. Chatterjee and S. K. Chaudhuri,
Mol. BioSyst., 2011, 7, 1701-1712.
36 J.R. Lakowicz and G. Weber, Biochemistry, 1973,
12, 4161-4170.
37 M. Lee, A.L. Rhodes, M.D. Wyatt, S. Forrow and
J.A. Hartley, Biochemistry, 1993, 32, 4237-4245.
38 M. V. Lunagariya, K. P. Thakor, R. R. Varma, B. N.
Waghela,C. Pathak and M. N. Patel, Med. Chem.
Commun., 2018, 9, 282-298.
13 X.Q. Zhou, Y. Li, D.Y. Zhang, Y. Nie, Z.J. Li, W.
Gu, X. Liu, Ji.L. Tian and S.P. Yan, Eur. J. Med.
Chem., 2016, 114, 244-256.
39 G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner,
R.K. Belew, D.S. Goodsell and A.J. Olson, J.
Comput. Chem., 2009, 30, 2785-2791.
14 C.X. Lu, A. Eskandari, P. B. Cressey and K.
Suntharalingam, Chem. Eur. J., 2017, 23, 1366-
1374.
40 O.Trott and A. J. Olson, J. Comput. Chem., 2010,
31, 455-461.
41 D. P. Yeggoni, M. Gokara, D. M. Manidhar, A.
Rachamallu, S. Nakka, C. S. Reddy and R.
Subramanyam, Mol. Pharmaceutics, 2014, 11,
1117-1131.
15 Y.Q. Hu, C. Gao, S. Zhang, L. Xu, Z. Xu, L.S. Feng,
X. Wu and F. Zhao, Eur. J. Med. Chem., 2017, 139,
22-47.
16 O. Afzal, S. Kumar, M.R. Haider, M.R. Ali, R.
Kumar, M. Jaggi and S. Bawa, Eur. J. Med. Chem.,
2015, 97, 871-910.
42 Y. R. Zheng, K. Suntharalingam, T. C. Johnstone, H.
Yoo, W. Lin, J. G.Brooks and S. J. Lippard, J. Am.
Chem. Soc., 2014, 136: 8790-8798.
43 M.F. Iskander, T.E. Khalil, W. Haase, R. Werner, I.
Svoboda and H. Fuess, Polyhedron, 2001, 20,
2787-2798.
44 S. Tabassum, M. Zaki, M. Ahmad, M. Afzal, S.
Srivastav, S. Srikrishna and F. Arjmand, Eur. J.
Med. Chem., 2014, 83, 141-154.
45 A.M. Merlot, S. Sahni, D.J. Lane, A.M. Fordham,
N. Pantarat, D.E. Hibbs, V. Richardson, M.R.
Doddareddy, J.A. Ong, M.L. Huang, D.R.
Richardson and D.S. Kalinowski, Oncotarget, 2015,
6, 10374-10398.
17 R. Musiol, Expert Opin Drug Discov., 2017, 12,
583-597.
18 N. Srimongkolpithak, S. Sundriyal, F.L. Li, M.
Vedadi and M. J. Fuchter, Med. Chem. Commun.,
2014,5, 1821-1828.
19 S. Adsule, V. Barve, D. Chen, F. Ahmed, Q.P. Dou,
S. Padhye and F.H. Sarkar, J. Med. Chem., 2006,
49, 7242-7246.
20 Saswati, A. Chakraborty, S. P. Dash, A. K. Panda,
R. Acharyya, A. Biswas, S.Mukhopadhyay, S. K.
Bhutia, A. Crochet, Y. P. Patil, M. Nethajie and R.
Dinda, Dalton Trans., 2015, 44, 6140-6157.
21 Y.P. Du, W. Chen, X. Fu, H. Deng and J.G. Deng,
RSC Adv., 2016, 6, 109718-109725.
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 11 of 12
View Article Online
DOI: 10.1039/C8MD00223A
Journal Name
46 Y. Gou, Y. Zhang, J. Qi, S. Chen, Z. Zhou, X. Wu, H.
ARTICLE
Liang and F. Yang, Oncotarget, 2016, 7, 67004-
67019.
47 B.K. Sahoo, K.S. Ghosh and S. Dasgupta,
Biopolymers, 2009, 91, 108-119.
48 N. Ibrahim, H. Ibrahim, S. Kim, J.P. Nallet and F.
Nepveu, Biomacromolecules, 2010, 11, 3341-
3351.
49 Y. Q. Wang, X.Y. Wang, J. Wang, Y. M. Zhao, W. J.
He and Z. J. Guo, Inorg. Chem. 2011, 50, 12661-
12668.
50 B. P. Espósito and Najjar, R. Coord. Chem. Rev.
2002, 232, 137-149.
51 W. Hu, Q. Luo, K. Wu, X. Li, F. Wang, Y. Chen, X.
Ma, J. Wang, J. Liu, S. Xiong and P. J. Sadler,
Chem. Commun. 2011, 47, 6006-6008.
52 R. J. Williams, M. Lipowska, G.Patonay and L.
Strekowski, Anal. Chem. 1993, 65, 601-605.
53 Y. Gou, J. Li, B. Fan, B. Xu, M. Zhou and F. Yang,
Eur. J. Med. Chem., 2017, 134, 207-217.
54 K. Suntharalingam, T.C. Johnstone, P.M. Bruno,
W. Lin, M.T. Hemann and S.J. Lippard, J. Am.
Chem. Soc., 2013, 135, 14060-14063.
55 Saswati, A. Chakraborty, S. P. Dash, A. K. Panda,
R. Acharyya, A. Biswas, S. Mukhopadhyay, S.K.
Bhutia, A. Crochet, Y. P. Patil, M. Nethaji and R.
Dinda, Dalton Trans., 2015, 44, 6140-6157.
56 M.J. Li, T.Y. Lan, X.H. Cao, H.H. Yang, Y. Shi, C. Yi
and G.N. Chen, Dalton Trans., 2014, 43, 2789-
2798.
57 G. Ferraro, L. Massai, L. Messori and A. Merlino,
Chem. Commun., 2015, 51, 9436-9439.
58 F. Shen, Y.X. Liu, S.M. Li, C.K. Jiang, B.F. Wang,
Y.H. Xiong, Z.W. Mao and X.Y. Le, New J. Chem.,
2017, 41, 12429-12441.
59 S. Kathiresan, S. Mugesh, J. Annaraj and M.
Murugan, New J. Chem., 2017, 41, 1267-1283.
60 B. Wu, P. Dröge and C. A.Davey, Nat. Chem. Biol.,
2008, 4(2): 110-112.
61 T. Chen and Y.S. Wong, Cell. Mol. Life Sci., 20018,
65, 2763-2775.
62 P.L. Toogood, Curr. Opin. Chem. Biol., 2002, 6,
472-478.
63 J. Bloom and F.R. Cross, Nat. Rev. Mol. Cell Biol.,
2007, 8, 149-160.
64 W. Ginzinger, G. Muhlgassner, V.B. Arion, M.A.
Jakupec, A. Roller, M. Galanski, M. Reithofer, W.
Berger and B.K. Keppler, J. Med. Chem., 2012, 55,
3398-3413.
65 A. Kisova, L. Zerzankova, A. Habtemariam, P.J.
Sadler, V. Brabec and J. Kasparkova, Mol. Pharm.,
2011, 8, 949-957.
66 C. Wang and R.J. Youle, Annu. Rev. Genet., 2009,
43, 95-118.
67 L. He, S.Y. Liao, C.P. Tan, R.R. Ye, Y.W. Xu, M.
Zhao, L.N. Ji and Z.W. Mao, Chemistry (Easton),
2013, 19, 12152-12160.
68 M.F. van Delft and D.C. Huang, Cell Res., 2006,
16, 203-213.
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DOI: 10.1039/C8MD00223A
Three Cu(II) complexes (C1-C3), especially C3 with the ligand derived from
benzocaine exhibited more selective for HeLa cells, and arrested cell cycle, promoted
tumor cell apoptosis.