Designing anticancer copper(II) complexes by optimizing 2-pyridine-thiosemicarbazone ligands

Article abstract of DOI:10.1016/j.ejmech.2018.09.020

To develop potential next-generation metal anticancer agents, we designed and synthesised five Cu(II) 2-pyridine-thiosemicarbazone complexes by modifying the hydrogen atom at the N-4 position of ligands, and then investigated their structure-activity rela

Full text of DOI:10.1016/j.ejmech.2018.09.020

Accepted Manuscript
Designing anticancer copper(II) complexes by optimizing 2-pyridine-
thiosemicarbazone ligands
Jungang Deng, Ping Yu, Zhenlei Zhang, Jun Wang, Jinhua Cai, Na Wu, Hongbin
Sun, Hong Liang, Feng Yang
PII:
S0223-5234(18)30792-X
10.1016/j.ejmech.2018.09.020
EJMECH 10731
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 July 2018
Revised Date: 4 September 2018
Accepted Date: 8 September 2018
Please cite this article as: J. Deng, P. Yu, Z. Zhang, J. Wang, J. Cai, N. Wu, H. Sun, H. Liang, F.
Yang, Designing anticancer copper(II) complexes by optimizing 2-pyridine-thiosemicarbazone ligands,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.09.020.
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ACCEPTED MANUSCRIPT

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Designing anticancer copper(II) complexes by optimizing
2-pyridine-thiosemicarbazone ligands
Jungang Deng1, Ping Yu1, Zhenlei Zhang1, Jun Wang1, Jinhua Cai2, Na Wu1, Hongbin Sun1,
3, Hong Liang1*, Feng Yang1*
1
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal
Resources, Ministry of Science and Technology of China. Guangxi Normal University,
Guilin, Guangxi, China.
2
College of Chemistry & Chemical Engineering, Jinggangshan University, Jian, Jiangxi,
China.
3
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, Nanjing, Jiangsu, China.
*Corresponding author:
Hong Liang, hliang@mailbox.gxnu.edu.cn
Feng Yang, fyang@mailbox.gxnu.edu.cn
Phone/Fax: 86-773-584-8836
Address: 15 Yucai Road, Guilin, Guangxi, China.
Zip code: 541004
Keywords: Cu(II) complexe; thiosemicarbazone; anticancer; p53;
telomerase.

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Abstract
To develop potential next-generation metal anticancer agents, we
designed and synthesised five Cu(II) 2-pyridine-thiosemicarbazone
complexes by modifying the hydrogen atom at the N-4 position of ligands,
and then investigated their structure-activity relationships and anticancer
mechanisms. Modification of the N-4 position with different groups caused
significant differences in cellular uptake and produced superior antitumor
activity. Cu complexes arrested the cell cycle at S phase, leading to
down-regulation of levels of cyclin and cyclin-dependent kinases and
up-regulation of expression of cyclin-dependent kinase inhibitors. Cu
complexes exerted chemotherapeutic effects via activating p53 and inducing
production of reactive oxygen species to regulate expression of the B-cell
lymphoma-2 family of proteins, causing a change in the mitochondrial
membrane potential and release of cytochrome c to form a dimer with
apoptosis protease activating factor-1, resulting in activation of caspase-9/3
to induce apoptosis. In addition, Cu complexes inhibited telomerase by
down-regulating the c-myc regulator gene and expression of the human
telomerase reverse transcriptase.
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1 Introduction
Since cisplatin began being used as an anticancer drug in the 1970s, most
chemotherapy drugs in clinical and preclinical studies are platinum-based
complexes [1-8]. However, drug resistance and adverse side effects were
common in cisplatin chemotherapy, therefore, scientists focused on
developing novel metal complexes with better antitumor activity, fewer side
effects, and reduced drug resistance to replace cisplatin therapy [9]. In recent
years, several transition-metal complexes have been developed as antitumor
treatments, and the most promising of these complexes are based on copper
[10, 11], ruthenium [12, 13], and gallium [14-16]. The coordination
complexes of ruthenium and gallium have also entered clinical trials [17-19].
Copper, as the active centre of many enzymes, plays an important role in
development and function of human organs and tissues [20-22]. To date,
copper-based complexes have been extensively synthesised and investigated
as anticancer agents because of their high antitumor activity and advantages
relative to other metal anticancer complexes [23-38]. Thiosemicarbazones
are formed by a condensation reaction of thiosemicarbazide and aldehydes
or ketones. Pharmacological studies have demonstrated that
thiosemicarbazones present a variety of biological activities [39, 40].
Thiosemicarbazones contain nitrogen and sulphur atoms with strong
coordination ability, and can form various complexes with novel structures.
Metal complexes derived from thiosemicarbazone ligands exhibit high
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anticancer activity [41-44]. Currently, copper complexes derived from
thiosemicarbazides have become a potential research hotspot [45-47].
Copper thiosemicarbazone complexes can produce reactive oxygen species,
and act against topoisomerase II, DNA, and other potential targets in the
nucleus [48]. Hence, the copper thiosemicarbazone complex is a promising
candidate for developing ideal non-platinum and multi-target antitumor
drugs.
Considering above factors, we synthesised five Cu(II) complexes with
ligand/Cu at a ratio of 1:1 via modification at the N-4 position (Scheme 1),
and characterised their structures using X-ray crystallography, elemental
analysis, infrared spectroscopic analysis, and electrospray ionization-mass
spectrometry.
Subsequently,
we
investigated
their
associated
structure-activity relationship. The five complexes identified demonstrated
marked toxicity. Finally, we confirmed their multi-target anticancer
mechanisms. The selected complex C4 caused apoptosis via the
p53-mediated mitochondrial pathway and telomerase inhibition by
regulating the expression of c-myc regulator gene and hTERT.
2 Results
2.1 Design and structure of five Cu complexes
Previous research showed that aldehyde-thiosemicarbazones were more
advantageous than ketone-thiosemicarbazones, the latter being less active
and toxic [39]. These compounds present a variety of biological activities,
and these activities can increase after the N-4 position is modified to obtain
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different derivatives, such as those containing hexatomic or heptatomic rings,
or monocyclic or bicyclic derivatives. Based on these findings, we selected
2-pyridine-thiosemicarbazone to design and synthesise five novel Cu(II)
complexes by modification of the N-4 position.
The complexes C1-C5 were prepared via the copper salt reaction with
L1-L5, respectively, in methanol, stirred, and refluxed at 65 °C for 6 h. The
solvent evaporation method was used to prepared crystals. Furthermore, the
structures of the Cu(II) complexes were identified by X-ray crystallography
as shown in Table 1. The crystal structure identified showed that the copper
centres of the C1, C2, and C3 complexes were tetra-coordinated by two
nitrogen atoms, one sulphur atom of chelating Schiff base ligand, and one
halogen atom or one nitrate ion. Moreover, the copper centres of C4 and C5
were penta-coordinated by two nitrogen atoms, one sulphur atom of
chelating thiosemicarbazone, one halogen atom or one nitrate ion, and one
methanol molecule (Figure 1). The five complexes crystallise with different
space groups, P-1, C2/c, P21/c, P-1, and P-1, respectively. The selected
bond lengths are shown in Table S1.We found no obvious vibration for the
Cu1-N1 bond (2.017Å-2.048Å), Cu1-N2 bond (1.959Å-1.994Å), and
Cu1-S1 bond (2.2366Å-2.2744Å).The angle of N-Cu-N was in the range of
80.39-81.19˚(C1-C5). The angle of N(1)-Cu-S was in the range of
161.46-164.10˚ (C1-C5). The angle of N(2)-Cu-S was in the range of
83.33-83.68˚, and there were no major changes in the bond angles for each
complex.
2.2 Stability of Cu complexes
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UV-Vis spectroscopy was employed to study the stability of complexes
[49]. Briefly, the complexes were dissolved in the PBS for 0, 24, and 48 h,
and detected by UV-Vis spectroscopy. The results demonstrated that the
UV-Vis spectra of the five complexes show no significant changes in
spectral characteristics and peak absorption over time (Figure S1), which
suggested that the five complexes are stable in PBS for 48 hours at room
temperature.
2.3 Structure-activity relationships of Cu complexes in vitro
Six cell lines including a normal liver cell line (MGC80-3, HepG2,
SK-OV-3, NCI-H460 and HL-7702) were employed to study the antitumor
activity of the 2-pyridine-thiosemicarbazone ligands and their complexes in
vitro.IC50 values were determined using the colourimetric assay, MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium).
As shown in Table 2, Cu(II) complexes are more cytotoxic to cancer cells
relative to the corresponding ligand because of chelation of ligand to Cu2+.
All Cu(II) complexes exhibited considerable antitumor activity with IC50
values from 0.84 to 12.11 µM. Compared with C1, the activity of the
complexes increased by 9-, 11- and 10- fold via introduction of a benzene
ring (C2),formation of a piperidine ring with N-4 (C3), or apyrrolidine ring
(C5) at the N-4 position in MGC80-3 cells. Introduction of two methyl
groups at N-4 carrying a lead group increased the activity by more than
14-fold, which showed that the modified complexes have better activity
against selected tumour cells.
Inductively coupled plasma (ICP-MS) results demonstrated that the total
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intracellular copper contents were 11.94-20.81nmol (Figure 2). Relative to
C1, the total concentration of the N4-modified complexes that were
absorbed into cells increased to 51.21%, 40.59%, 74.28%, and 41.89%,
respectively. However, there were no significant differences in the
concentration of each complex distributed to the mitochondria (3.24-3.73
nmol), whereas the concentration in the nucleus increased to 31.85%,
28.38%, and 27.91% after the cells were treated with C3, C4, andC5,
respectively, for 24 h. Similarly, the concentration of C2-C5 in the
cytoplasm increased significantly resulting from modification at the N-4
position (Figure 2). These results showed that modifications at the N-4
position affect the amount of complex absorbed into cells and can lead to
differences in antitumor activity.
2.4 Confirmation of anticancer mechanisms of Cu complexes
Apoptosis is a complex process with multiple pathways regulated by
multiple proteins. The p53 protein, a nuclear transcription factor, plays an
important role in regulating a wide variety of genes [50-52].A downstream
response factor of p53, p21 expression is significantly increased following
p53 activation, leading to cell cycle arrest. In the mitochondria-induced
apoptosis pathway, p53 can activate the apoptosis regulator, Bcl-2-like
protein 4, Bax, to form a heterodimer with Bcl-2 as an apoptotic activator
[53, 54]. Bax increases the anion channel open probability, and induces loss
of mitochondrial membrane potential (∆ψm), ultimately promotes release of
cytochrome c (Cyt c). In addition, p53 can activate the Bcl-2-associated
death promoter. Bad, by linking the protein with dephosphorylated-Badto
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form a complex, and promote release of Cyt c [55, 56]. In the nucleus, p53
can bind the promoter of c-myc and promote histonedeacetylation, thereby
inducing the repression of c-myc [57].
2.4.1 P53-regulates the levels of cell cycle regulators and promotes the cell
cycle
Cell cycle was examined by flow cytometry. Cell cycle analysis clearly
shows that the C1 complex blocks the cell cycle in the G1 phase (Figure 3A).
The cell population in the G1 phase increased to 11% and 12% after
treatment with 6 µM and 12 µM of the C1 complex, respectively. The C4
complex arrested the cells in the G1/S phase when used at a concentration of
0.4 µM and in the S phase with a concentration of 0.8 µM. In comparison
with control cells, the G1/S populations increased 8.55% and 1.36% after the
cells were treated with C4 at 0.4µM, and S populations increased 14.07%
after the cells were treated with C4 at 0.8 µM.
The cell cycle is regulated by cyclin and cyclin-dependent kinases
(CDKs); cyclin A, cdc25 A, and CDK2, which are closely associated with S
phase. Cdc25 A activates CDK2 by dephosphorylation and promotes cell
cycle progression [58, 59]. Western blot analysis using anti-cdc25A, cyclin
A, and CDK2 antibodies was used to determine whether the selected
complexes affect expression of related proteins in MGC80-3 cells. As shown
in Figures 3B and C, expression of these three proteins decreased at varying
degrees. Therefore, we further examined expression of p21 and p27 protein
levels, which act as cyclin-dependent kinase inhibitors. As showed in
Figures 3B and C, expression levels of p21 and p27 increased significantly
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after treatment with the C4 complex for 24 h. In particular, p21
demonstrated a 6-fold increase compared to the control group.
2.4.2 P53-regulates the levels of c-myc, hTERT, and telomerase inhibition
Telomerase is over-expressed in most tumour cells, while little to no
expression is found in normal cells [60, 61]. C-myc is a member of the myc
family of proteins, which are strong proto-oncogenes and regulate a range of
genes including those mediating cell proliferation and apoptosis. C-myc is
also the first transcription factor found to regulate the expression of hTERT,
an enzyme considered to act as a rate limiting factor for telomerase
activation. As such, inhibition of telomerase activity by regulating c-myc
and hTERT gene expression is considered a new direction in antitumor
research [62-65]. Western blot results showed that levels of c-myc were
reduced by 88.08% with 12 µM C1 complex relative treatment relative to
control levels (Figures 4A and B). However, there were no changes when
the cells were treated with 6 µM of the C1 complex. In contrast, c-myc
levels were significantly reduced by 77.63% and 80.57%when cells were
treated with the C4 complex at 0.4 and 0.8 µM, respectively. In addition,
expression levels of hTERT significantly decreased by 72.95% and 78.27%
after the cells were treated with the C4 complex at 0.4 and 0.8 µM,
respectively. In contrast, this reduction was not particularly obvious after
treatment with the C1 complex (6 or 12 µM). Measurement of telomerase
inhibition using the polymerase chain reaction (PCR)-based telomeric repeat
amplification protocol (TRAP) assay revealed that the complexes C1 and C4
inhibited telomerase activity at rates of 17.17% and 33.73%, respectively
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(Figure 4C).
2.4.3 P53-mediates anticancer mechanisms via mitochondrial pathways
Proteins belonging to the Bcl-2 family play a key role in mediating the
mitochondrial apoptosis pathway by controlling mitochondrial membrane
permeability [66, 67]. The p53 protein can up-regulate the pro-apoptotic
proteins Bad and Bax, and down-regulate expression of Bcl-2, resulting in
the dissipation of ∆ψm and release of Cyt c [68, 69], which is a key step in
apoptosis. Moreover, Cyt c forms a dimer with apoptosis-related factor 1
(Apaf-1) and activates caspase-3/9. Flow cytometry results showed that cells
treated with C1 and C4 complexes can lead to collapse of the ∆ψm
.
Compared with the 3.24% ∆ψm of control cells, the ∆ψm decreased to
18.60% and 54.90% after treatment with the C1 complex at 6 µM and 12
µM, respectively. Similarly, the ∆ψm decreased to 12.30% and 52.90% after
treatment with the C4 complex at0.4µM and 0.8 µM, respectively (Figure
S3). Importantly, the decrease in ∆ψm was positively correlated with the dose
of complex administered. Western blot results showed that levels of Apaf-1,
Bad, Bax, and Cyt c increased significantly after MGC80-3 cells were
treated with complex C4, while Bcl-2 levels decreased (Figures 5A and B).
Meanwhile, we used the fluorescent 2,7-dichlorofluorescein (DCF) probe to
evaluate the ability of the selected complexes to catalyse production of
intracellular ROS in MGC80-3 cells, which can promote expression of
apoptotic proteins as second messengers and induce opening of the
mitochondrial permeability transition pore [70-73]. After cells were exposed
to the C1 complex (6 or 12 µM) and the C4 complex (0.4 or 0.8 µM) for 24
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h, cells produced a green fluorescence characteristic of activation (Figure
S3).
Downstream of the mitochondrial apoptotic pathway, caspase 3 acts as
an apoptotic signalling molecule as caspase 9 activation leads to caspase 3
activation [74, 75]. In this experiment, flow cytometry was used to examine
activation of caspase-9/3. As shown in Figure S4, activated caspase-9 levels
increased 24.93% and 31.19% after treatment with the C1 (12 µM) and C4
(0.8 µM) complexes. Similarly, activated caspase-3 levels increased 5.96%
and 10.56%, respectively.
Our results showed that cells treated with two doses of the selected
complexes underwent dose-dependent late apoptosis, with the percentages of
the apoptotic MGC80-3 cells increasing with dose by 5.04% and 18.85%
when treated with the C1 complex at6 µM and 12 µM, respectively, and by
2.65% and 29.84% when treated with the C4 complex (0.4 or 0.8 µM) as
compared to the control group (Figure 5C).
2.4.4 The action of DNA cleavage studies
Many studies have shown that Schiff base copper complexes can target
DNA [76, 77]. Therefore, we investigated the effects of C1 and C4 on
Pbr322 plasmid DNA by gel electrophoretic separations. The results showed
that a relatively high concentration of C1 did not significantly cleave DNA.
In contrast, C4 relaxed Form I (supercoiled form) of DNA into Form II at
250 µM (Figure S5), implying that C4 may act against DNA in cancer cells.
3 Discussion
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A common approach in drug discovery is to find a promising lead drug
and modify the structure of the compound to improve performance.
Structural modification can change absorption, extend duration of effect,
reduce toxicity, and increase solubility, among other potential benefits.
Discovery of novel high-efficiency and low-toxicity antitumor agents
from thiosemicarbazone derivatives and their complexes is a field receiving
increasing amounts of attention. Studies have shown that modification at the
N-4 position of thiosemicarbazides with moieties such as introducing
carboxyl groups, phenyl group, forming a 5- or 6-membered aromatic
heterocycle, etc. can improve anti-tumour potential of these molecules. Our
experiments evaluating cellular uptake showed that complexes modified at
the N-4 position were more readily absorbed into cells and distributed to the
nucleus than the original complex, which may indicate the mechanisms
underlying the increase in activity of the N-4 modified complexes in our
studies.
Based on the complexity of tumour development, there are crossovers
and compensations between signalling pathways for most tumours to
maintain their growth and survival. In clinical studies, the effects of targeted
therapy against a single pathway are often unsatisfactory, with activation of
alternative pathways as one of the mechanisms for this effect. Multi-target
drug development can achieve synergistic effects and overcome drug
resistance by inhibiting multiple signalling pathways or multiple molecules
upstream and/or downstream of one pathway, providing therapeutic action
against multiple targets in cancer cells simultaneously. Our results showed
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that the Cu N-4 modified 2-pyridine-thiosemicarbazone complexes could
significantly up-regulate p21 and arrest MGC80-3 cells at S phase, and
induce apoptosis by regulating ∆ψm via down-regulation of p53 to regulate
expression of Bcl-2 family proteins. In addition, since telomerase is
considered a promising target for novel anticancer drugs, C4 was shown to
inhibit telomerase activity by significantly inhibiting the expression of
c-myc and hTERT. Therefore, the thiosemicarbazone-based Cu complexes
show promise as an avenue for development of multi-target anticancer metal
pro-drugs.
4 Conclusion
To develop the next-generation metal-based anticancer agents, we
designed and optimised Cu(II) tridentate 2-pyridine-thiosemicarbazone
complexes by modifying the N-4 position in thiosemicarbazones. The Cu
complexes not only confer marked anticancer activity, but can also directly
induce cancer cell apoptosis through multiple mechanisms, including
regulation of proteins related to the cell apoptosis pathway, and inhibition of
telomerase activity. Our results suggested that the Cu(II) tridentate
2-pyridine-thiosemicarbazone complexes have potential as promising
multi-target anticancer metal lead drugs.
5 Materials and Methods
All chemicals were provided by Energy Chemical Company (Shanghai,
China).All cell lines (MGC80-3, SK-OV-3, NCI-H460, HepG2 and
HL-7702) were provided by the Shanghai Institute for Biological Science
(Shanghai, China). The mitochondrial membrane potential assay kit with
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JC-1, the reactive oxygen species assay kit, the mitochondria isolation kit,
and nuclei isolation kit were purchased from Beyotime (Jiangsu, China).
Fluorescein active caspase-3 and -9 staining kits were purchased from
BioVision (Milpitas, CA). The telomerase activity assay kit was provided by
Genmed (Minneapolis, MN). The antibodies used in western blot were
provided by Abcam (Cambridge, UK).
5.1 Synthesis of ligands
The L1-L5 ligands were prepared using previously published methods
[78]. In brief, 2-pyridinecarboxaldehyde (10 mmol) MeOH solution was
added to the corresponding equimolar thiosemicarbazides (10 mmol) and
stirred for 6 h at 60℃, and the ligands were precipitated and filtered to
obtain the L1−L5 ligands. The ligands were purified by re-crystallization.
All ligands were characterised by elemental analysis, infrared spectral
1
analysis, H-NMR and electrospray ionization-mass spectrometry (ESI-MS;
Supporting Information, Figures S6−S15).
N-methyl-2-(pyridin-2-ylmethylene)hydrazinecarbothioamide(L1):
yield
(1.52 g, 78%). Anal. Calcd (%) for C8H10N4S: C, 49.46; H, 5.19; N, 28.84; S,
16.51. Found: C, 49.23; H, 5.02.; N, 28.68, S, 16.43. IR (KBr, cm-1): 3450,
3288, 3138, 2943, 1528, 1433, 1319, 1267, 1247, 1151, 1109, 1088, 1038,
997, 928, 885, 779, 675, 623, 608, 520, 414. 1H NMR (400 MHz, DMSO)
δ
11.70 (s, 1H), 8.66 (d, J= 4.4 Hz, 1H), 8.56 (d, J = 4.7 Hz, 1H), 8.26 (d, J =
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8.0 Hz, 1H), 8.08 (s, 1H), 7.87–7.82 (m, 1H), 7.40–7.34 (m, 1H), 3.32 (s,
3H).ESI-MS: m/z =177.0[C8H10N4S-2H-CH3]－.
N-phenyl-2-(pyridin-2-ylmethylene)hydrazinecarbothioamide(L2):
yield
(1.81 g, 70%). Anal. Calcd (%) for C13H12N4S:C, 60.91; H, 4.72; N, 21.86; S,
12.51. Found: C, 60.77; H, 4.61; N, 21.73; S, 12.58.IR (KBr, cm-1):3307,
3124, 2952, 1597, 1551, 1524, 1468, 1326, 1287, 1256, 1189, 1160, 1109,
1
1077, 997, 942, 923, 754, 692, 622, 506, 480. H NMR (400 MHz, DMSO)
δ
12.03 (s, 1H), 10.25 (s, 1H), 8.59 (ddd, J = 4.9, 1.6, 0.9 Hz, 1H), 8.45 (d, J
= 8.0 Hz, 1H), 8.20 (s, 1H), 7.85 (td, J = 7.7, 1.4 Hz, 1H), 7.59–7.53 (m, 2H),
7.42–7.34 (m, 3H), 7.24 (dd, J = 10.5, 4.2 Hz, 1H).ESI-MS: m/z=
179.00[C13H12N4S−C6H5]+.
N'-(pyridin-2-ylmethylene)piperidine-1-carbothiohydrazide(L3): yield (1.86
g, 75%). Anal. Calcd (%) for C12H16N4S: C, 58.04; H, 6.49; N, 22.56; S,
12.91. Found: C, 58.04; H, 6.49; N, 22.56; S, 12.91. IR (KBr, cm-1): 3431,
3141, 2929, 1497, 1349, 1281, 1010, 997, 894, 774, 619, 536, 515, 446,
411.1H NMR (400 MHz, DMSO)
δ 11.25 (s, 1H), 8.58 (dt, J =4.8, 1.2 Hz,
1H), 8.17 (s, 1H), 7.87–7.83 (m, 2H), 7.37 (d, J =4.1 Hz, 1H), 3.88 (s, 4H),
1.66–1.60 (m, 6H).ESI-MS: m/z = 249.12 [C12H16N4S+H]+.
N,N-dimethyl-2-(pyridin-2-ylmethylene)hydrazinecarbothioamide(L4):
yield (1.73 g, 83%). Anal. Calcd (%) for C9H12N4S: C, 51.90; H, 5.81; N,
26.90; S, 15.39. Found: C, 51.76; H, 5.88; N, 26.81; S, 15.31. IR (KBr, cm-1):
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3431, 3125, 2972, 1553, 1410, 1238, 1154, 1103, 1078, 1056, 900, 875, 778,
737, 620, 583, 513. 1H NMR (400 MHz, DMSO)
δ 11.71 (s, 1H), 8.67 (d, J
= 4.3 Hz, 1H), 8.57 (d, J = 4.6 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.09 (s,
1H), 7.85 (td, J = 7.8, 1.3 Hz, 1H), 3.04 (d, J = 4.5 Hz, 6H).ESI-MS: m/z =
207.07[C9H12N4S−H]－.
N'-(pyridin-2-ylmethylene)pyrrolidine-1-carbothiohydrazide(L5):yield (1.77
g, 75%). Anal. Calcd (%) for C11H14N4S: C, 56.38; H, 6.02; N, 23.91; S,
13.68. Found: C, 56.22; H, 6.11; N, 23.83; S, 13.62.IR (KBr, cm-1): 3435,
3018, 2927, 2856, 2768, 1604, 1567, 1498, 1435, 1360, 1308, 1274, 1249,
1222, 1192, 1164, 1134, 1014, 951, 918, 885, 781, 714, 624, 518,
419.1HNMR (400 MHz, DMSO)
δ 11.24 (s, 1H), 8.60–8.54 (m, 1H), 8.20 (s,
1H), 7.90–7.81 (m, 2H), 7.36 (ddd, J = 6.7, 4.9, 1.4 Hz, 1H), 3.76 (s, 4H),
1.90 (s, 4H).ESI-MS: m/z = 233.09 [C11H14N4S−H]－.
5.2 Synthesis and characterization of Cu(II) complexes
The ligands (0.1 mmol) and the copper salts solution (0.1 mmol) were
added into 20ml MeOH solution and refluxed for 6 h at 65 °C. The reaction
solution was placed at room temperature to obtain crystals, and the
complexes were purified by re-crystallization. The new complexes were
[Cu(P4mT)Cl] (C1), [Cu(P4pT)NO3] (C2), [Cu(P3piT)Br] (C3),
[Cu(P44mT)(CH3OH)Br] (C4), and [Cu(P3pyT)(CH3OH)Cl] (C5).
Elemental analysis, infrared spectral analysis, and ESI-MS were employed
to verify the structures of the complexes (Supporting Information, Figures
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S6−10, S21−25).
[Cu(P4mT)Cl] (C1): yield (23.96 mg, 82%). Anal. Calc. for C8H9ClCuN4S:
C, 32.88; H, 3.10; N, 19.17; S, 10.97. Found: C, 32.71; H, 3.01; N, 19.29; S,
10.81. IR (KBr, cm-1): 3451, 1637, 1595, 1537, 1469, 1357, 1306, 1283,
1226, 1121, 1043, 608, 520. MS m/z: 334.0 [M+H+C2H3N]－.
[Cu(P4pT)NO3] (C2): yield (27.04 mg, 71%). Anal. Calc. for C13H11Cu
N5O3S: C, 41.00; H, 2.91 N, 18.39; S, 8.42. Found: C, 41.13; H, 2.82 N,
18.25; S, 8.31. IR (KBr, cm-1): 3328, 1600, 1544, 1455, 1433, 1383, 1284,
1128, 747. MS m/z: 318.00 [M−NO3]+.
[Cu(P3piT)Br] (C3): yield (28.92 mg, 74%). Anal. Calc. for
C12H15BrCuN4OS: C, 36.88; H, 3.87; N, 14.34. Found: C, 36.76; H, 3.95; N,
14.26. IR (KBr, cm-1) 3435, 1629, 1584, 1563, 1463, 1432, 1285, 1219,
11084, 1041, 991, 953, 863, 777, 618, 500, 405. MS m/z: 390.04[M−H]－.
[Cu(P44mT)(CH3OH)Br] (C4): yield (29.09 mg, 76%). Anal. Calc. for
C10H15BrCuN4OS: C, 31.46; H, 3.70; N, 14.68; S, 8.40. Found: C, 31.34; H,
3.83; N, 14.58; S, 8.31. IR (KBr, cm-1) 3433, 2897, 1581, 1519, 1466, 1415,
1280, 1149, 906, 782, 644, 613. MS m/z : 350.01 for [M−H−CH3OH]－.
[Cu(P3pyT)(CH3OH)Cl] (C5): yield (28.78 mg, 79%). Anal. Calc. for
C12H17ClCuN4OS: C, 39.67; H, 4.44; N, 15.42; S, 8.82. Found: C, 39.54; H,
4.32; N, 15.56; S, 8.73. IR (KBr, cm-1): 3434, 1631, 1381, 1160, 773. MS
m/z: 331.94[M−H−CH3OH]－.
5.3X-ray crystallography
A diffractometer was used to collect X-ray diffraction data for the five
copper complexes. Olex2 software was employed to resolve the structure of
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the complexes [79].The atoms excluding hydrogen atom are refined
anisotropically. The hydrogen atom is theoretically added at the appropriate
position by Olex2.The crystal data of all copper complexes are listed in
Table 1. Selected bond lengths (Ẳ) and angles (degrees) are given in Tables
S1-S2.
5.4 In vitro cytotoxicity assay
Stock solutions (10 mM) of the ligands and copper complexes were
prepared by dissolving the complexes in DMSO, and diluted to various
concentrations with PBS. The MTT assay was employed to investigate cell
toxicity [80]. Briefly, all cells were seeded in 96-well plates. After
incubation for 24 h, the complexes were added to the culture medium at
various concentrations and the cells were incubated for 48 h. MTT solution
(10µL) was added to each well and incubated for 4 h. One-hundred
microliters of DMSO was added to each well after the culture medium was
removed. The absorbance was measured by an enzyme-labelling instrument
and IC50 values were calculated.
5.5 Cellular uptake
To investigate the underlying mechanisms of the enhanced activity of the
modified complexes, ICP-MS was performed to detect distribution of the
complexes in cancer cells [81]. Briefly, the selected cells were treated with
the five complexes at the same dose (0.8 µM) for 24 h, the mitochondria
isolation kit and nuclei isolation kit were used to separate and extract the
mitochondrial membrane fraction, nuclear fraction, and cytoplasm.
Meanwhile, copper content in the control cells was also measured.
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5.6 TRAP assay
To investigate inhibition of telomerase activity by the selected
complexes, a TRAP-Silver stain assay was performed [82]. Briefly, 1×106
MGC80-3 cells were treated with C1 (12 µM) and C4 (0.8 µM) for 24 h.
The cells were collected and telomerase was extracted according to the kit
instructions. Five microliters of telomerase reaction mixture added to a
final volume of 50 µL for PCR amplification. The sample was incubated at
30 °C for 30 minutes, followed by 30 cycles of 92 °C for 30 seconds, 52 °C
for 30 seconds, and 72 °C for 30 seconds. Non-denaturing acrylamide gel
electrophoresis (12%; 19:1) was used to isolate proteins at 180 V for 40
minutes and 220 V for 80 minutes. Gels were stained with 0.2 %
AgNO3andbands developed with sodium bicarbonate.
5.7 Cell cycle analysis
To study the effects of the complexes on the cell cycle of tumours, we
used flow cytometry to detect changes in cell cycle distribution before and
after drug treatment. Briefly, MGC80-3 cell suspension (1×105 cells/mL)
was plated in 10-mm culture dishes and incubated for 12 h, then treated with
C1 (6 or 12 µM) and C4 (0.4 or 0.8 µM) for 48 h. The cells were harvested
and washed with cool PBS solution. Cells were fixed in ice-cold 70%
ethanol solution and stored at -25 °C for 24 h. The cells were centrifuged
and incubated with RNase A for 30 min. The cell cycle distribution was
detected with a flow cytometer.
5.8 Intracellular measurements of ROS
Intracellular ROS generation was investigated using the fluorescent
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probe dichloro-dihydro-fluorescein diacetate (H2DCFH-DA) and visualised
by fluorescence microscopy. The cells seeded in 6-well plates were exposed
to C1 (6 or 12 µM) and C4 (0.4 or 0.8 µM) for 24 h. Cool PBS solution was
used to wash the cells twice, and the cells were incubated in serum-free
medium containing H2DCFDA (2 mM), and finally washed twice using
serum-free medium. A fluorescence microscope was used to observe the cell
fluorescence.
5.9 Measurement of the ∆ψm
A flow cytometer employing JC-1 probe was used to detect changes in
∆ψm in MGC80-3 cells. The cells in 6-well plates were exposed to C1 (6 or
12 µM) and C4 (0.4 or 0.8 µM) for 24 h, harvested, and washed twice with
cool PBS solution. JC-1 stock solution was added to the cells and incubated
for 30 minutes. A flow cytometer was used to detect the fluorescence of
separated cells.
5.10 DNA cleavage studies
Briefly, supercoiled plasmid pBR322 DNA, Tris-HCl buffer and
different concentrations of C1 and C4 (25, 100, and 250 µM) were mixed
and incubated in a tube at 37 °C for 3 h. Subsequently, the loading buffer
was added into the mixture. The mixture was loaded onto 1% agarose gel
containing 1% (v/v) goldView II, and analysed by gel electrophoresis in 1X
TBE buffer at 80 V.
5.11 Western blot analysis
The selected cells were seeded in the plate and exposed toC1 (6 or 12
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µM) and C4 (0.4 or 0.8 µM) for 24 h. A lysis buffer was employed to
lysethe harvested cells. 12% SDS–polyacrylamide gel electrophoresis was
employed to separate proteins. Proteins were transferred to polyvinylidene
difluoride (PVDF) membranes, and the membranes were incubated with
different antibodies. A western blot detection system was used. Gray scale
analysis was used for quantitative analysis of bands.
5.12 Determination of caspase-3/9 activity
Activation of caspase-3/9 was measured by flow cytometry employing a
Fluoresce in Active Caspase-3/9 Staining Kit. Appropriate cells in six well
plates were exposed for 24 h to C1and C4 at 12 µM and 0.8µM, respectively.
Cells were harvested and washed with cool PBS solution, plus wash buffer
containing FITC-DEVD-FMK, and incubated for 30 minutes. A flow
cytometer was employed to detect caspase-3/9 activity.
5.13 Cell apoptosis assay
Annexin V/PI double staining was employed to investigate cell apoptosis,
which was analysed by flow cytometry. Briefly, after treatment with C1 (6
or 12 µM) and C4 (0.4 or 0.8 µM) for 24 h, the cells were harvested and
re-suspended. Annexin V-FITC and PI were added and the cells were
incubated for 15 minutes and analysed by flow cytometry. The data were
then analysed using FlowJo software.
5.14 Statistical analysis
Student's t-test was employed to evaluate significant differences. The
data are showed as mean ± standard deviation (SD). A p-value < 0.05 was
considered significant.
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Acknowledgements
This work received financial support from the Natural Science Foundation
of China (31460232, 21431001, 21561017, 21462004), the Natural Science
Foundation of Guangxi (2017GXNSFEA198002, AD17129007),
IRT_16R15, Guangxi “Bagui” scholar program to HB Sun, and High-Level
Innovation Team and Distinguished Scholar program of Guangxi universities
to F Yang.
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