New copper complexes inducing bimodal death through apoptosis and autophagy in A549 cancer cells

Article abstract of DOI:10.1016/j.jinorgbio.2020.111260

Two copper complexes, Cu1 (CuL¹Cl₂, L¹ = 2-(6,7-dimethoxyisoquinolin-1-yl) aniline) and Cu2 (CuL²Cl₂, L² = 2-(6-methoxyisoquinolin-1-yl) aniline), were synthesized and characterized. These

Full text of DOI:10.1016/j.jinorgbio.2020.111260

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New copper complexes inducing bimodal death through apoptosis
and autophagy in A549 cancer cells
Noor Shad Gul, Taj-Malook Khan, Ming Chen, Ke-Bin Huang,
Cheng Hou, Muhammad Iqbal Choudhary, Hong Liang, Zhen-
Feng Chen
PII:
S0162-0134(20)30288-9
DOI:
https://doi.org/10.1016/j.jinorgbio.2020.111260
JIB 111260
Reference:
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
11 July 2020
13 September 2020
21 September 2020
Please cite this article as: N.S. Gul, T.-M. Khan, M. Chen, et al., New copper complexes
inducing bimodal death through apoptosis and autophagy in A549 cancer cells, Journal of
Inorganic Biochemistry (2018), https://doi.org/10.1016/j.jinorgbio.2020.111260
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New copper complexes inducing bimodal death through apoptosis and autophagy in A549
cancer cells
1
,#
1,#
1
1
1
Noor Shad Gul , Taj-Malook Khan , Ming Chen , Ke-Bin Huang , Cheng Hou , Muhammad
2
1
1,
Iqbal Choudhary , Hong Liang , and Zhen-Feng Chen *
1
State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School
of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004, P. R. China
2
International Center for Chemical and Biological Sciences, University of Karachi, Karachi-
7
4270, Pakistan
Correspondence to:
Professor Zhen-Feng Chen
State Key Laboratory 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: chenzf@gxnu.edu.cn (Z.-F. Chen)
1

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Abstract
1
1
Two copper complexes, Cu1 (CuL Cl , L = 2-(6,7-dimethoxyisoquinolin-1-yl) aniline) and Cu2
2
2
2
(
CuL Cl , L = 2-(6-methoxyisoquinolin-1-yl) aniline), were synthesized and characterized.
2
These complexes exhibited high cytotoxic activity toward different cancer cell lines, including
the A549 lung cancer cell line, and low cytotoxicity toward normal human cells. Mechanistic
studies have shown that these complexes induce bimodal death of cancer cells through apoptosis
and autophagy, including the activation of apoptotic and autophagic cell signaling pathways. In
addition, Cu1 and Cu2 interacted with calf thymus DNA (ct-DNA) via an intercalative binding
mode. The different biological behaviors of these copper complexes could be attributed to the
presence of electron-donating methoxy groups on the ligands. Cu1 and Cu2 effectively inhibited
tumor growth in a xenografted mouse model bearing A549 cells but exhibited lower in vivo
toxicity than cisplatin. Thus, Cu1 and Cu2 can be developed as potential anticancer agents.
Keywords: copper agent; isoquinoline; anticancer activity; apoptosis; autophagy; cell cycle.
2

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1
. Introduction
Transition metal complexes have been used for the treatment of many diseases such as cancer,
bacterial infection, and inflammation during the past few decades [1]. In the field of cancer
chemotherapy, platinum complexes have attained notable achievements for their clinical
applications [2-4]. However, acquired resistance and toxicity cause severe side effects and limit
the clinical application of these drugs [5,6]. The side effects are owing to the low selectivity for
cancer cells [7].
The metal complexes showing single anticancer mechanism are typically not effective in the
treatment of tumors, and they produce drug resistance and toxicity because cancer is a
multigenetic and complicated disease [8]. Therefore, to overcome these side effects and multi-
drug resistance, the development of multi-targeted anticancer agents is most desirable [10]. At
present, drugs with multitargeting capability are highly sought to overcome the drawbacks of
single targeting drugs. Transition metal complexes can be designed as multi-targeted anticancer
agents [9]. In this regard, copper could be an alternative to platinum because it plays a vital role
in cancer treatment [11] as numerous copper complexes have been developed and investigated
for anticancer activities. In addition, most of the copper complexes produce cytotoxicity by the
generation of reactive oxygen species (ROS) inside the cells that initiated various apoptotic
pathways [12-14].
During the past two decades, many copper complexes with different ligands have been
synthesized and have shown promising anticancer activities against various cancerous cells. For
example, several studies have reported some Cu(ІІ) and Pt(ІІ) complexes with high anticancer
and pharmacological activity, having isoquinolines and thiosemicarbazones as ligands [15-22].
Some of these complexes kill cells via multiple action mechanisms, which suggest that the
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copper complexes could be developed as multitargeting anticancer agents [23-25]. As these
complexes damage the cells through various mechanisms, drug resistance in cells, which is
typically shown in drugs with single anticancer mechanism, is evaded [26].
Currently, the design and synthesis of transition metal complexes with organic ligands
possessing anticancer activity has been the focus of cancer chemotherapy. This is considered a
promising strategy to develop potential anticancer agents because the metal complexes usually
possess higher biological activities than the corresponding free organic ligands, and exert a
synergistic effect after formation of coordination compounds [27,28]. Isoquinoline alkaloids are
commonly found in nature, and they exhibit different kinds of biological and pharmacological
activities, such as the inhibition of cellular proliferation and cancer cell growth [29].
Berberrubine, a compound of protoberberine alkaloids, is active against tumors in an animal
model [30]. Another isoquinoline alkaloid, [1,3]-dioxolo-[4,5-g]-isoquinoline (papraline), which
was isolated from the aerial part of Fumaria indica, is considered as a mild analgesic, laxative,
and diuretic agent and beneficial for skin scrofulous infection and dyspepsia [31]. Previously, we
have reported gold and rhodium complexes with isoquinoline derivatives as ligands which
exhibited high anticancer activity [32,33].
Herein, we synthesized and determined the crystal structure and in vitro and in vivo activity
of two copper(ІІ) complexes with isoquinoline derivatives as ligands. Bimodal death through
apoptosis and autophagy in A549 cells was also investigated. These complexes interacted
through various pathways such as apoptosis via the mitochondrial-mediated pathway, apoptosis
via the mitogen-activated protein kinase mediated (MAPK) pathway, light chain3 protein II
mediated (LC3-II) autophagy, and cell damage through the endoplasmic reticulum stress-
mediated pathway.
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2
2
2
. Results and discussion
.1. Chemistry
.1.1. Synthesis of ligands
1
2
Ligands ―L ‖ 2-(6,7-dimethoxyisoquinolin-1-yl) aniline and ―L ‖ 2-(6-methoxyisoquinolin-1-yl)
aniline were synthesized using the Bischler–Napieralski method of cyclization of acyl products
[
34] (Scheme S1).
.1.2. Preparation and crystal structures of Cu1 and Cu2
Copper complexes were synthesized in sealed glass tubes by mixing L or L with CuCl 2H O
2
1
2
2
2
in a solvent of dichloromethane and methanol (v:v = 1:1) (Scheme S1). Elemental analysis and
infrared spectroscopy (IR) were used for structural characterization. Further confirmation of
structures was acquired by single crystal X-ray diffraction analysis. The data of crystallographic
analysis and refinement details are listed in Table S1 (Supporting Information). The selected
bond lengths and bond angles are shown in Tables S2 and S3 (Supporting Information).
1
2
Isoquinoline is a bidentate ligand having two nitrogen donors (L -N^N and L -N^N) bound with
copper to form the distorted square planar structure in the geometry of Cu1 and Cu2. The Cu1
crystallized in the P2 /c space group, whereas Cu2 crystallized in the C2/c space group. In Cu1,
1
the bond lengths of CuN1 and CuN2 are 1.9980(19) Å and 2.0993(18) Å, respectively;
whereas the bond lengths of CuCl1 and CuCl2 are 2.3400(10) Å and 2.3437(9) Å,
respectively. In Cu2, the bond lengths of CuN1 and CuN2 are 2.007(3) Å and 2.061(3) Å,
respectively, whereas the bond lengths of CuCl1 and CuCl2 are 2.2679(10) Å and 2.2658(9)
Å, respectively (Figs. 1, 2).
[
Fig. 1]
Fig. 2]
[
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2
.2. Stability of Cu1 and Cu2
The stability of the copper complexes and ligands in phosphate buffer saline (PBS) solution
containing 1% DMSO at pH 7.4 were determined at different time intervals of 0, 4, 12, and 24 h
by UV-Vis spectroscopy. Furthermore, purity and stability were also checked by high
performance liquid chromatography (HPLC). The data showed that Cu1 and Cu2 were stable at
the given conditions for 24 h. No blue or red shift was observed in the absorption region of
spectra, and there was no new peak either (Figs. S1S8, Supporting Information).
2
.3. In vitro cytotoxicity
To evaluate the in vitro cytotoxicity of Cu1 and Cu2 ligands and copper salt against various
cancer cell lines, MGC80-3, T-24, SKOV-3, HeLa, A549, BEL-7704, and MDA-MB216 and
human normal liver cells HL-7702, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide colorimetric assay (MTT) was performed with cisplatin as the positive control. Table 1
shows the IC values of these compounds against the tested cancer cells. The copper complexes
5
0
exhibited higher cytotoxicity than corresponding ligands and copper salt. The IC value of Cu1
5
0
was 3.05 ± 0.3 M, whereas that of Cu2 was 6.04 ± 0.5 M against the A549 cell lines. It was
observed that the cytotoxicity of the copper complexes increased by the coordination of
1
isoquinoline derivative ligands with the copper center. In addition, Cu1 and L were found to be
2
more cytotoxic than Cu2 and L likely because of the differences in the position and number of
methoxy groups, respectively. As reported by Nussbaum [35] the cytotoxicity of complexes
mostly depends on the basicity of amine ligands attached to the metal center. For example, the
methoxy group is an electron donor that donates electrons to the ring as a result of which
electron density becomes high and affords the most effective complexes. Consequently, in the
case of Cu1, the substitution with two methoxy groups made this complex more basic than Cu2
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with one methoxy group. The results were found to be consistent with those obtained for the
cellular uptake of both copper(II) complexes. Amongst the tested tumor cell lines, Cu1 and Cu2
showed the highest cytotoxicity against A549 cells; therefore, we used A549 to investigate their
anticancer mechanisms.
[Table 1]
2
.4. Distribution and uptake of Cu1 and Cu2
The effect of a drug can be evaluated by its uptake and distribution in a cell [36]. To examine the
distribution of Cu1 and Cu2 in different parts of the cell including cytosolic,
membrane/particulate, cytoskeletal and nucleic fractions, the cells were incubated, based on the
concentration of IC values with Cu1 (3 µM) and Cu2 (6 µM) for 20 h and the content of the
5
0
metals was measured using inductively coupled plasma mass spectrometry (Fig. 3). With the
6
same quantity of A459 cells (10 cells), accumulated content of copper complexes was several
times greater than that of the control especially in the cytosolic fraction (Cu1 3570 ng, Cu2 1830
ng) and membrane fraction (Cu1 1850 ng, Cu2 1450 ng). In addition, in the cytoskeletal
fraction, the accumulation of Cu1 content was higher than that of Cu2 (1700 ng and 500 ng,
respectively). Comparatively little copper was found in the nucleic fraction. This distribution and
uptake of metals is critical for the mechanism of actions in different cellular pathways which
control the induction of apoptosis [37]. A more detailed evaluation of the possible action
mechanisms of Cu1 and Cu2 was subsequently conducted.
[Fig. 3]
2
2
.5. Anticancer mechanism of Cu1 and Cu2
.5.1. Induction of apoptosis by arresting the cell cycle
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The process of apoptosis is exploited as an antitumor mechanism because it is dysregulated in
cancer cells. Apoptosis is a highly controlled and conserved process which removes unwanted
and abnormal cells during the growth of multicellular organisms [38,39]. To investigate the
effects of Cu1 (1.5, 3.0, 6.0 µM) and Cu2 (3.0, 6.0, 12.0 µM) on the triggering of apoptosis in
A549 cells, after 24-h treatment, the cells were stained with annexin V and propidium iodide and
analyzed using flow cytometry. As shown in Figs. 4, S9, S10, the induction of early and late
apoptosis showed a dose-dependent increase after treatment with Cu1 and Cu2. The proportions
of cells in early and late apoptosis were 1.8% and 4.3%, respectively, in the control. However,
this increased to 14.2% and 26.1%, respectively, after treatment with 6.0 M Cu1 and 14.4%
and 24.3%, respectively, after treatment with 12.0 M Cu2. The extent of apoptosis induction by
Cu1 was greater than that by Cu2. As expected, we found that pro-apoptotic proteins, such as
Bcl-2-associated X protein (Bax) and Bcl-2 antagonist / killer protein (Bak), were upregulated as
compared to that in the control, whereas anti-apoptotic proteins, such as B-cell lymphoma 2
(Bcl-2) and B-cell leukemia xL (Bcl-xL), were down-regulated (Fig. S11).
We further examined the activation of caspases. To this end, A549 cells were exposed to
Cu1 (3.0 µM) and Cu2 (6.0 µM), and analyzed using flow cytometry after 24 h. As shown in
Fig. 5, the levels of caspase-3 were increased from 0.01% in the control to 47.1% and 32.2%
after treatment with Cu1 and Cu2 respectively. In addition, increased activation of caspase-8/9
was observed from 2.8%/1.8% in the control to 21.1%/50.6% and 22.6%/40.0% after treatment
with Cu1 and Cu2, respectively (Fig S12). All these observations demonstrated that the copper
complexes were potent in the activation of caspases. Therefore, Cu1 and Cu2 are excellent
activators of all three caspases and can trigger apoptosis in A549 cells.
[Fig. 4]
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[Fig. 5]
To determine the anticancer mechanism, we further analyzed the cell cycle of A549 cells
after treatment with Cu1 (1.5, 3.0 and 6.0 µM) and Cu2 (3.0, 6.0, and 12.0 µM). The flow
cytometry data exhibited an increase in % population of cells in the G1-phase of Cu1 (45.7%,
5
3
6.5%, and 74.5%) and Cu2 (48.3%, 56.4%, and 64.8%) compared with that in the control (G1:
8.9%) after treatment with three different concentrations (1.5, 3.0, 6.0 µM and 3.0, 6.0, 12 µM)
of the copper complexes, which clearly showed the arrest of the G0/G1-phase in A549 cells by
both the copper complexes. These results showed that Cu1 and Cu2 arrested the cell cycle at the
G1-phase in A549 cells; thus, no replication of DNA occurred, and the cell cycle was blocked at
the G1-phase (Figs. 6, S13, S14). In addition, the extent of cell cycle arrest by Cu1 was greater
than that by Cu2. Indeed, western blot results further indicated that the expression of cell cycle-
related proteins, such as cyclin D, cyclin E, and cyclin A, as well as cyclin dependent kinases
(CDK2 and CDK6) were down-regulated after treatment with the copper complexes for 24 h.
However, the levels of p21, p27, and p53 were significantly upregulated. The decrease in the
expression of cyclins and increase in the expression of cyclin dependent kinase inhibitors
(
CDKIs) confirmed cell cycle arrest at the G1-phase and induction of apoptosis. These
observations are consistent with the flow cytometry results (Fig. S15).
Fig. 6]
.5.2. Activation of mitochondrial/ROS/ER-stress pathway
[
2
The mitochondria are considered an important cellular organelle in the induction of apoptosis.
Therefore, targeting mitochondria is considered a new strategy for the discovery of novel
anticancer drugs. The mitochondrial membrane potential is well-regulated and highly controlled
[
40]. Apoptotic pathways are activated when there is damage to the mitochondrial membrane
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potential, which releases pro-apoptotic factors and ultimately leads to cell death [41]. To
examine the effects of copper complexes, A549 cells were incubated with Cu1 (1.5, 3.0, 6.0 µM)
and Cu2 (3.0, 6.0, 12.0 µM) for 24 h. A fluorescent dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-
tetraethylbenzimi-dazolylcarbocyanine iodide) was used for analyzing mitochondrial membrane
polarization using flow cytometry. A dose-dependent increase was observed in Cu1- and Cu2-
treated cells from 1.36% cells in the control to 54.8% and 46.8%, respectively, at maximum
concentration. These results indicate that apoptosis was induced by the copper complexes in
A549 through mitochondrial-mediated pathways. The possibility of involvement of other
pathways also existed (Figs. 7, S16, S17). Moreover, Cu1 showed more effective damage on the
membrane potential of mitochondria.
ROS is mainly produced in mitochondria, and the excessive production of ROS is harmful
to mitochondria, disturbing its membrane potential and genetic stability and resulting in
apoptotic cell death [42]. Therefore, we aimed to determine the ROS production in A495 cells
after treatment with Cu1 and Cu2. To this end, the A549 cells were treated with Cu1 (3.0 µM,
very close to the IC value) and Cu2 (6.0 µM, very close to the IC value), for 24 h and
5
0
50
analyzed using flow cytometry. As shown in Fig. 8A, we found that a high level of ROS was
generated by the Cu1 and Cu2 complexes compared to that with the control. In addition, the
density functional theory (DFT) calculation results show that the coordination geometrical
structure and charge distribution of the two copper complexes are not much different, but they
show a difference in the energy level of frontier molecular orbital due to the electron donating
character of methoxy group. The energy level of the single-occupied molecular orbital (SOMO)
of Cu1 with two methoxy groups is slightly higher (0.184 hartree) than Cu2 with one methoxy
groups (0.187 hartree). Therefore, it may lead to a better activity in the generation of reactive
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oxygen species for Cu1 than Cu2. We further analyzed ROS-induced endoplasmic reticulum
stress. To this end, A549 cells were treated with the copper complexes and analyzed based on the
oxidation of H DCFDA (2ʹ,7-dichlorodihydrofluorescein diacetate) to DCF (2ʹ,7ʹ-
2
dichlorofluorescein) by ROS and (endoplasmic reticulum stress) ER-stress was analyzed using
ER-Tracker Red. The results clearly showed colocalization, but with an increase in concentration,
the ROS exhibited less colocalization and more diffusion with higher permeabilization of the
endoplasmic reticulum membrane which led to the induction of ER-mediated apoptosis (Fig. 8B).
We further confirmed the ER-stress pathway using western blot analysis. As shown in Fig. 9,
Cu1 and Cu2 can induce the over production of CCAAT-enhancer-binding homologous proteins
(CHOP) and the activation of Eukaryotic initiation factor-2 (eIF2α) and Protein kinase RNA-like
endoplasmic reticulum kinase (PERK) in A549 cells. Together, these results confirmed the ER-
stress mediated apoptosis by the copper complexes in A549 cells.
[Fig. 7]
[Fig. 8]
[Fig. 9]
2
.5.3. Induction of autophagy by activating the MAPK signaling pathway
LC3 is a cytosolic protein (light chain-3 protein) linked with autophagy. This protein is
conjugated with phosphatidylethanolamine in the cytosolic form to prepare the LC3-II complex
which helps the induction of cell autophagy [43]. Another autophagic receptor protein
SQSTM1/p62 (sequestosome1) interacts with LC3, which is continuously degraded during
autophagy, and is associated with autophagy-dependent eradication of several substances [44].
Furthermore, an autophagy related Bcl-2-interacting protein (Beclin-1) is essential in the
regulation of autophagy and apoptosis. Apoptosis and autophagy are interconnected by the
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Beclin-1 caspase-mediated cleavage [45]. Interestingly, we found that treatment of A549 cells
with Cu1 and Cu2 resulted in the upregulation of LC3-II and Beclin-1, whereas down-regulation
of p62 and LC3-I (Fig. 10A). The results revealed the activation of autophagy as well as the link
between autophagy and apoptosis [46]. To further confirm the autophagy pathway, western blot
analysis of related proteins was performed. P38MAPK proteins are involved in the direction and
regulation of cellular responses, autophagy, and apoptotic pathways [47]. The expression level of
p38MAPKs was upregulated in A549 cells treated with Cu1 and Cu2, which indicated that the
p38MAPK pathway was activated and p38MAPK-mediated apoptosis occurred. In addition, cell
survival proteins, such as protein kinase B also known as AKT (Activation of the
phosphatidylinositol 3-kinase/protein kinase B) promote cell survival and inhibit apoptosis. Bcl-
2
death associated (BAD) protein is phosphorylated by AKT, which is isolated as a Bcl-2/Bcl-xL
complex, and inhibits proapoptotic capability. The targeting of AKT by the anticancer drugs
results in tumor growth inhibition and apoptosis [48]. The western blot results showed the level
of expression of AKT was down-regulated. The results provide evidence of induction of
apoptosis and autophagy, which are associated with AKT and p38MAPK signaling pathways, in
A549 cells by Cu1 and Cu2, (Fig. 10B).
[Fig. 10]
2
.5.4. Competitive binding studies
Comparatively less amount of copper was found in the nucleic fraction, during the investigation
of cellular uptake and distribution of complexes in different parts of A549 cells. It implies that
DNA may be act as a potential target. Therefore, ethidium bromide (EB) was used as a
competitive intercalative probe for the investigation of binding abilities of Cu1, Cu2 and
cisplatin to the Calf Thymus-DNA (ct-DNA). When the EB-DNA system as [EB] / [DNA] = 1 :
1
0 ( [EB] = 2 mM, [DNA] = 2 mM was excited by 350 nm light in the competitive binding
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study, a characteristic strong emission was observed at 610 nm, depicted that the EB intercalated
molecule has been significantly protected from the quenching caused by water in the DNA by
the adjacent base pairs of the DNA. When the ratios of the concentration of [EB] / [DNA] /
[
Cu1] and [EB] / [DNA] / [Cu2] were increased from 1:10:1 to 1:10:10, a significant quenching
in the characteristic emission of EB has been observed, as shown in Fig. 11. The KSV constants
3
4
were calculated by Stern-Volmer equation as 5.518×10 for Cu1 and 4.07×10 for Cu2,
respectively. It was found that both the metal complexes could moderately quench the
fluorescence emission of EB, which strongly suggests that they could competitively interact with
DNA via an intercalative binding mode at the similar binding sites of DNA with EB, whereas the
cisplatin has no intercalative binding mode with DNA as shown (Fig. S18). The cisplatin
covalently binds to the deoxyribonucleic acid (DNA) at the N-7 position of guanine (G) or
adenine (A) sequences of GG and AG, to form an inter and intra-strand cross-linkages
respectively [49].
[Fig. 11]
2
.6. In vivo anticancer activity
The in vivo anticancer activity of Cu1 and Cu2 was investigated using a xenograft model of
mice bearing A549 cells. Four groups of randomly divided mice were treated with vehicle
(negative control), cisplatin (positive control), Cu1, and Cu2. The mice were treated
intraperitoneally with low and high doses of Cu1 and Cu2 (7.5 and 15 mg/kg, respectively).
Injection of the positive control with cisplatin (2 mg/kg) and negative control with 5% DMSO in
3
saline was performed when the volume of the tumor reached 100130 mm (Figs. 12, 13). There
was no obvious change observed in the body weight of Cu1-, Cu2-, and negative control-treated
groups. A significant weight loss was observed in the cisplatin-treated group during the 15-day-
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treatment (Figs. 12B, 13B). The tumor growth was inhibited by Cu1 (21.1% and 41.3%) and
Cu2 (19.5% and 41.7%) to a higher extent than that with the control, but lower than that by
cisplatin which was 57.5 % (Figs. 12C, 13C). These experiments showed that Cu1 and Cu2
exhibited a higher in vivo safety profile than cisplatin and effectively inhibited tumor growth.
[
Fig. 12]
Fig. 13]
[
3
. Conclusion
Two new copper agents, Cu1 and Cu2, displayed high selective cytotoxicity against human
tumor cells by a multi-targeted anticancer mechanism. Importantly, the complexes exhibited less
toxicity against human normal cells (HL-7702). A mechanistic study showed that the agents
induced bimodal death through apoptosis and autophagy in A549 cells. Cu1 and Cu2 induced
cytotoxicity via mitochondria-mediated apoptosis which initiated the caspase cascade and ROS
generation that produced ER stress and the MAPK apoptotic pathway that collectively caused
cell death. Furthermore, Cu1 and Cu2 regulated the pro-death autophagy proteins such as
Beclin1, p62, LC3-1 and LC3-II and induced autophagy. Both Cu1 and Cu2 induced
mitochondrial-mediated apoptosis and pro-death autophagy (Fig. 14). Cu1 and Cu2 interacted
with ct-DNA via an intercalative binding mode. The slightly different biological behavior of Cu1
and Cu2 could be attributed to the position and number of the methoxy group. Experiments also
showed that Cu1 and Cu2 effectively inhibited the tumor growth in a xenografted mouse model
bearing A549 cells but exhibited lower in vivo toxicity than cisplatin. Cu1 and Cu2 could be
developed as potential anticancer agents.
[Fig. 14]
4
. Experimental methods
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4
.1. Materials
Chemical reagents were purchased from Alfa Aesar (Ward Hill, Massachusetts, USA), Sigma
Aldrich (Saint Louis, Missouri, USA), and Xilong Scientific Co., Ltd. (China) and used as
received without further purification. Dimethyl sulfoxide (DMSO) of analytical grade was used
for the preparation of stock solutions of the copper complexes. The working solutions of
different concentrations used for the various biological experiments were prepared by dilution of
stock solution in PBS. Compounds of more than 97% purity were used for the pharmacological
studies and spectroscopic and biological experiments. Kits used for performing different
biological assays such as the MTT assay, cell cycle determination, detection of apoptosis,
determination of mitochondrial membrane potential, ROS Detection Assay Kit, and Caspases
Colorimetric Assay Kit were purchased from BD Biosciences (San Jose, CA, USA) and
BioVision (Milpitas, California, USA). The primary and secondary antibodies were purchased
from Abcam (Cambridge, MA, USA). The A549 xenograft mouse model was purchased from
Beijing Biosciences Co., Ltd. (Beijing, China).
4
.2. Instruments
Electrospray ionization mass spectrometry (ESI-MS) spectra were obtained on a Bruker HCT
mass spectrometer. A Bruker AV-500 NMR spectrometer was used for obtaining (Nuclear
magnetic resonance spectroscopy) NMR spectra. Crystallographic data were recorded on a
Bruker smart APEX ІІ machine. UV absorption spectra were recorded on a TU-1901 UV-
spectrometer. Elite P230ІІ (Dalian, China) was used for HPLC analysis. A Tecan Microplate
reader M1000 T (Shanghai, China) was used for performing the MTT assay. A FACS Arial ІІ
flow cytometer BD Biosciences (San Jose, CA, USA) was used for performing the flow
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cytometry analysis. The ECL western blotting system was used for the detection and
confirmation of protein expressions.
1
2
4
.3. Synthesis of ligands (L and L )
Both ligands and intermediate products were synthesized according to reported procedure [34].
1
13
The NMR data, including H-NMR and C-NMR, and ESI-MS are given in supporting
information.
4
.3.1. N-(4-methoxyphenethyl)-2-nitrobenzamide and N-(3,4-dimethoxyphenethyl)-2-
nitrobenzamide (1a-b) general procedure A: Nitrobenzoic acid (0.1 mol) and thionyl chloride
were treated in dry dichloromethane (CH Cl ). The resulting mixture was stirred for 12 h at 70
2
2
℃. The excess thionyl chloride was discarded under reduced pressure and the resulting solution
was dissolved in benzene. This solution was added dropwise to the corresponding 0.1 mol
amines, [(2-(3,4-dimethoxyphenyl)ethan-1-amine) and 2-(4-methoxyphenyl)ethan-1-amine], in
dry benzene. The reaction mixture was stirred continuously for 1216 h at 25 ℃. This mixture
was washed with 500 mL water, then separated and the organic layer collected. The product was

recrystallized in n-hexane/methanol. ESI-MS: m/z (1a) = 329.11 [M ‒ H] , (1b) = 301.12 [M +
+
H] . (Figs. S19, S20).
4
.3.2. 6,7-Dimethoxy-1-(2-nitrophenyl)-3,4-dihydroisoquinoline and 6-methoxy-1-(2-
nitrophenyl)-3,4-dihydroisoquinoline (2a-b) general procedure B: To a stirred solution of
(a) 200 mmol and 1(b) 200 mmol dissolved in acetonitrile (800 mL), POCl (150 mL) was
1
3
added and continuously stirred for 1 h at room temperature. Then, the mixture was refluxed for
0 h and monitored by thin layer chromatography (TLC), until completion of the reaction and all
1
the starting material was consumed. Water (500 mL) and dichloromethane (500 mL) were added
after the removal of the solvent by vacuum. The pH of the aqueous phase was adjusted to 10 by
16

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the addition of 10 N NaOH. The organic phase was washed with an aqueous solution of NaHCO3
(
500 mL), dried with MgSO , and concentrated with reduced pressure. The target product was
4
+
precipitated after sonication with methanol. ESI-MS: m/z (2a) = 313.11 [M + H] , (2b) = 283.10
+
[
M + H] . (Figs. S21, S22).
4
.3.3.
6,7-Dimethoxy-1-(2-nitrophenyl)isoquinoline
and
6-methoxy-1-(2-
nitrophenyl)isoquinoline (3a-b) general procedure C: Activated MnO (0.6 g) was added to
2
the refluxed solution of (2a) 10 mmol and (2b) 10 mmol in dry CHCl once every 3040 min,
3
and the reaction was completed within 30 h. The reaction was monitored by TLC. On completion
of the reaction, the hot mixture was filtered, and the filter cake was washed with
dichloromethane (500 mL). The black particles of MnO were removed through filtration on
2
Celite, and the filtrate was dried using MgSO . The target product was obtained after
4
+
+
recrystallization in methanol. ESI-MS: m/z (3a) = 311.09 [M + H] , (3b) = 281.08 [M + H] .
Figs. S23, S24).
.3.4. 2-(6,7-Dimethoxyisoquinolin-1-yl) aniline (L ) and 2-(6-methoxyisoquinolin-1-yl)
(
1
4
2
aniline (L ) general procedure D: SnCl .2H O (26.4 mmol) was added to the stirred solution of
2
2
8
mmol each of (3a) and 3(b) dissolved in dry ethyl acetate (150 mL) and refluxed for 24 h. The
reaction was monitored by TLC. After completion, water (30 mL) and 10 N NaOH (5 mL) were
added. The resulting solution was mixed with NaHCO (100 mL), extracted with ethyl acetate
3
(
150 mL), dried using MgSO , and the solvent evaporated. The target compound was obtained
4
after recrystallization with ethyl acetate/n-hexane.
1
1
4
7
5
.3.5. L : H-NMR (500 MHz, (CD ) SO) δ 8.43 (d, J = 4.6 Hz, 1H), 7.68 (d, J = 4.6 Hz, 1H),
3 2
.44 (s, 1H), 7.22 (m, 2H), 7.18 (s, 1H), 6.91 (d, J = 6.3 Hz, 1H), 6.75 (td, J = 0.9, 6.0 Hz, 1H),
1
3
.11 (s, 2H), 3.98 (s, 3H), 3.75 (s, 3H). C-NMR (100 MHz, (CD ) SO) δ 157.1, 157.0, 152.9,
3
2
17

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1
50.1, 146.9, 141.1, 131.2, 129.5, 123.3, 122.7, 119.0, 116.4, 116.2, 105.9, 105.5, 56.2, 55.6.
+
ESI-MS: m/z = 281.12 [M + H] . (Figs. S25S27).
2
1
4
.3.6. L : H-NMR (500 MHz, (CD ) SO) δ 8.50 (d, J = 4.7 Hz, 1H), 7.77 (m, 3H), 7.44 (d, J =
3 2
2
1
1
2
4
.1 Hz, 1H), 7.26 (dd, J = 2.1, 8.0 Hz, 1H), 7.22 (m, 1H), 7.12 (dd, J = 1.1, 6.2 Hz, 1H), 6.74 (m,
1
3
H), 5.09 (s, 2H), 3.96 (s, 3H). C-NMR (100 MHz, (CD ) SO) δ 160.6, 159.1, 146.9, 142.9,
3
2
39.2, 131.3, 129.6, 123.1, 122.6, 120.3, 119.5, 116.2, 116.1, 108.1, 105.4, 56.0. ESI-MS: m/z =
+
51.11 [M + H] . (Figs. S28S30).
.4. Formation of copper complexes
Cu1 and Cu2 complexes were prepared in a Pyrex glass tube. Briefly, the copper salt, ligands,
and solvent were placed in a glass tube and chilled with liquid nitrogen. The ice-chilled tube was
evacuated and sealed using a torch flame and placed at 80 ºC for 72 h. Block crystals were
formed by slow cooling to room temperature. Elemental analysis IR and ESI-MS were
performed, and the acquired data are presented in Supporting Information.
4
.4.1. Cu1: (Yield 6070%), Anal. Calc. (%) for C H Cl CuN O : C, 49.23; H, 3.89; N, 6.75;
17
16
2
2
2
-1
O, 7.71. Found (%): C, 49.35; H, 3.98; N, 6.80; O, 7.81. IR (KBr) νmax cm : 3271 (N–H
stretching), 1610 (aromatic ring vibration), 10771265 (C–O), 200300 (Cu–Cl), 300600 very
weak (Cu–N). ESI-MS m/z: 412.9. (Figs. S31, S32).
4
.4.2. Cu2: (Yield 6070 %), Anal. Calc. (%) for C H Cl CuN O: C, 49.95; H, 3.67; N, 7.28;
16
14
2
2
-1
O, 4.16. Found (%): C, 50.08; H, 3.92; N, 7.40; O, 4.45. IR (KBr) νmax cm : 3251 (N–H
stretching), 1618 (δ (NH bending)), 1473 (C–N), 1265 (C–O), 200300 (Cu–Cl). ESI-MS m/z:
2
3
4
4
82.9. (Figs. S33, S34).
.5. In vitro experiments
.5.1 Culturing of cells
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Cells, including T-24, MGC-803, HeLa, A549, SKOV-3, and MDA-MB216 and human normal
liver cells HL-7702 were purchased from the Shanghai Cell Bank of the Chinese Academy of
Sciences. They were grown at a constant temperature of 37 °C and under humidified air of 5%
CO with Dulbecco’s modified eagle medium containing 5% fetal bovine serum. Dimethyl
2
sulfoxide was used for the dissolution of copper complexes. The stock solution was 2 mM, and
the working solution was prepared by dilution with PBS to the required concentration. The
cisplatin stock solution was prepared by dissolving 24 mg powder in 10 mL of 0.9% saline, and
the solution was kept at 4 ℃. On each day of the experiment, a quality control sample was freshly
prepared.
4
.5.2. MTT assay
6-Well plates were used for this assay. The cells were seeded at a density of 6 × 10 /180 µL in
3
9
each well for 24 h. To prepare the required concentration, 20 µL medium having different
concentrations of each compound was added to the wells and the cells were incubated for 48 h.
To the control well, 0.5% DMSO was added. Subsequently, 10 µL MTT (5 mg/mL) was added
and incubated for another 6 h under the same conditions. The medium was discarded, and the
crystalized form of formazan was dissolved in 100 µL DMSO. A microplate reader with the
double wavelength of 490/630 nm was used for measuring absorption. This assay was
independently performed thrice in triplicate. The Bliss method (n = 5) was used for the
calculation of IC values.
5
0
4
.5.3. Morphological analysis of cells
Cells were cultivated at a suitable temperature (37 °C) and under CO (5%). After attachment,
2
the cells were exposed to Cu1 and Cu2 and incubated for 24 h in the same environment. Cell
morphology was examined under a bright-field microscope.
19

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4
.5.4. Cell cycle analysis
6
Cells at the density of 57 × 10 were cultured in a 50 mm plate for 24 h. The cells were
incubated with various concentrations of Cu1, Cu2, and cisplatin for 24 h. After 24-h treatment,
the cells were fixed with 75% prechilled ethanol and placed for one night at -20 °C. Cells were
washed with PBS and resuspended. The cells were incubated with PBS containing RNAs (100
µg/mL) and propidium iodide (PI) (50 µg/mL) for 30 min in the dark. BD FACS Calibur Flow
cytometer was used for analysis. Mod FIT LT version 3.3 (Variety Software House) was used for
the calculation of cell cycle distribution.
4
.5.5. Apoptosis analysis
6
A 6-well plate was used for overnight culturing of cells at a density of 2 × 10 at 37 °C. The cells
were treated with the copper complexes for 24 h. After incubation, the cells were collected and
washed with PBS. An apoptosis detection kit was used according to the manufacturer’s
instructions. A BD FACS flow cytometer was used for analysis.
4
.5.6. ROS generation
5
Cells were cultured at a density of 1 × 10 in a 6-well plate for 24 h at 37 °C. The cells were
treated with the copper complexes for 24 h. After treatment, the cells were collected and washed
to remove the medium and then incubated with 2′,7′-Dichlorodihydrofluorescein diacetate
(DCFH-DA) for 25 min in the dark. The cells were washed again with 1 mL of PBS. A BD
FACS flow cytometer was used for the detection of generated ROS and measurement of 2ʹ, 7ʹ –
dichlorofluorescein (DCF) fluorescence. DCFH-DA (non-fluorescent) was converted to DCFH
by the enzymatic action of esterase and as DCFH is not very stable it was quickly converted to
DCF (a fluorescent compound). The fluorescence intensity of DCF depends on the production of
ROS.
20

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4
.5.7. Mitochondrial membrane potential
The JC-1 staining kit was used for the determination of mitochondrial membrane potential. A549
5
cells were cultured at a density of 1 × 10 in a 6-well plate for 24 h at 37 °C. The cells were
treated with different concentrations of copper complexes for 24 h. After treatment, the cells
were harvested and washed with PBS to remove the medium and incubated with JC-1 for 25 min
in the dark. To prepare the sample for BD FACS flow cytometer, 0.5 mL PBS was added to the
samples to determine the mitochondrial membrane potential.
4
.5.8. Determination of activated caspases 3, 8, and 9
6
Cells were placed in a 6-well plate for 24 h at a density of 1 × 10 at 37 °C. After 24-h treatment
with the copper complexes the cells were washed with PBS to remove the medium and incubated
with 1 µL of caspase 3, 8, and 9 inhibitors, fluorescein isothiocyanate-Asp-Glu-Val-Asp-
fluoromethyl ketone (FITC-D(OMe)-E(OMe)-VD(OMe)-FMK) for 1 h in the dark. The BD
FACS flow cytometer was used for the determination of caspases activation in copper
complexes-treated cells compared with that in the negative control.
4
.5.9. ROS visualization of endoplasmic reticulum
Cells were placed on coverslips coated with poly-L-lysin in a 6-well plate for 24 h at the density
6
of 2 × 10 cells in each well. The cells were exposed to copper complexes for 7 h, and then, cells
were washed with prewarmed PBS. The cells were then incubated with 25 mM H DCFDA (2ʹ,7-
2
dichlorodihydrofluorescein diacetate) for 25 min. After addition of ER-Tracker Red (1 mM), the
cells were washed with PBS and mounted cells were visualized using confocal microscopy. Zeiss
FLUOVIEW was used for processing images acquired using a 20× objective lens.
4
.5.10. Western blotting analysis
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Cells were placed in a 100 mm culture plate for 24 h. When the cells were ready for treatment,
the copper complexes were added at various concentrations and incubated for more than 24 h at
3
7 °C. After 24 h, the cells were harvested, washed with PBS, and lysed with lysis buffer (149
µL) and 1 µL phenylmethylsulfonyl fluoride (PMSF). Total proteins were extracted by
centrifugation (15000G g) at 4 °C for 20 min before the samples were put on ice for 40 min. A
mixture of purified proteins (50 µg) and an equal volume of electrophoresis sample buffer was
prepared and boiled for 58 min including the controls. The samples were run on 12% SDS-
PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoreses) and then transferred to a
PVDF (polyvinylidene fluoride) layer. The membrane was blocked using TBS buffer (tris-
buffered saline) of 5% BSA for 2 h, after the complete transfer of proteins from the gel to the
membrane. The PVDF was washed with PBS and incubated overnight at 4 °C with suitable
primary antibodies. After incubation with primary antibodies, the membrane was incubated with
secondary antibodies for 2 h at room temperature. The membrane was washed with PBS before
and after incubation with antibodies. Immunoreactive signals were detected using a chemo-
luminance kit according to the procedure described by the manufacturer.
4
.5.11. In vivo experiments
BALB/c nude mice (male, aged 3040 days, 1822 g) were provided by the Guangxi Medical
University Laboratory Animal Centre (Guanxi, China). The mice were placed in individually
ventilated cages with a humidified atmosphere of 4550% at 2530 °C with a constant duration
of light and dark (12 h light, 12 h dark). For the development of tumor, A549 cells at a density of
6
5
× 10 were injected into the right flank of mice. The mice were sacrificed when the xenograft
3
3
tumor reached 1000 mm . The tumor tissue was cut into pieces of 1.6 mm and then translocated
3
to the nude male mice. When the tumor tissue reached 100160 mm , all the mice were divided
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randomly into groups including solvent controls and complexes-treated groups. Cisplatin was
used as a positive control and was administrated at 2 mg/kg per two days. The negative control
comprised treatment with 5% DMSO v/v per saline. Cu1 and Cu2 were administered in low and
high doses of various concentrations (7.5 and 15 mg/kg, respectively). For the systemic safety of
complexes, the body weight was taken as a parameter and was recorded every three days. The
tumor size was also recorded at three-day intervals. The mice were sacrificed after 15 days and
2
the tumor weight was recorded. The formula V = L × W /2 was used for the calculation of tumor
volume (V) using the measured width (W) and length (L) of the tumor. The rate of tumor growth
was determined using the formula (L-TW /TW ) × 100, where TWt represents the complex-
t
c
treated mice and TW represents the vehicle-treated mice [50-52].
c
4
.5.12. Computational details for DFT
All calculations were performed using Gaussian 16 program Rev. A.03 [53]. Geometry
optimizations were carried out with M06-L/def2-SVP level of theory [54,55]. Frequency analysis
calculations were performed to characterize the structures to be the minima (no imaginary
frequency). The 3D optimized structure figures in this paper were displayed by IBOview
visualization program [56]. Additional computational information and the Cartesian coordinates
(xyz) of the optimized structures and figure are given in the supporting information (Fig. S35).
4
.5.13. Fluorescence emission titration
A solution of DNA (2 mM) and EB (2 mM) with 10:1 ([DNA] / [EB]) was prepared for
competitive binding studies. Using Stern-Volmer equation (I / I = 1 + Ksv × [Q]) the quenching
0
constant were calculated of the tested compounds. In this equation the I and I represented the
0
highest emission intensities of the EB-DNA system with and without the presence of quencher.
Ksv represented the Stern-Volmer quenching constant, that is obtained by the linear fitting of the
23

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plot I / I against [Q], and to determine the quenching ability of small molecules on EB indicated
0
as a DNA intercalative probe.
4
.6. Statistical analysis
The experiments were conducted in 36 replicates. The Student’s t-test was used to measure
significant differences and the results are reported as the mean ± SD. The difference was
considered statistically significant when the p-value was less than 0.05.
Abbreviations
DMEM
FBS
Dulbecco’s modified eagle medium
Fetal bovine serum
MMP
Bax
Mitochondrial membrane potential
Bcl-2-associated X protein
Bak
Bcl-2 antagonist / killer protein
B-cell lymphoma 2
Bcl-2
Bcl-xl
ROS
B-cell leukemia xl
Reactive oxygen species
MAPK
LC3
Mitogen-activated protein kinases
Light chain3 proteins
PERK
Protein kinase RNA-like endoplasmic reticulum kinase
Eukaryotic initiation factor-2
CCAAT-enhancer-binding homologous proteins
Sequestosome1
eIF2α
CHOP
SQSTM1
MTT
Microculture tetrazolium assay
24

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IC50
The half maximal inhibitory concentration
Propidium iodide
PI
PBS
Phosphate buffer saline
DCFH-DA
ER-stress
SDS-PAGE
MDA-MB216
CDKs
Dichloro-dihydro-fluorescein diacetate
Endoplasmic reticulum stress
Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis
Invasive ductal carcinoma
Cyclin dependent kinases
Caspases
Acknowledgment
Cysteine-dependent aspartate-directed proteases
This work was supported by the National Natural Science Foundation of China (Grant
1431001), IRT_16R15, and the Natural Science Foundation of Guangxi Province of China
Grants: 2016GXNSFGA380005 and AD17129007), and the ―BAGUI Scholar‖ program of the
2
(
Guangxi Province of China.
Supporting Information
Vendor codes for Cu1 and Cu2 crystal data, HPLC, ESI-MS, and IR data. The supplementary
crystallographic data of this paper can be seen under the CCDC No. 19811393, 19811396.
Author Contributions
#
#
Noor Shad Gul and Taj-Malook Khan contributed equally to this work denoted by a superscript
#).
(
References
25

Journal Pre-proof
[
1] S. Rafique, M. Idrees, A. Nasim, H. Akbar, A. Athar, Biotechnol. Mol. Biol. Rev. 5 (2010)
3
845.
[
[
[
2] L. Kelland, Nat. Rev. Cancer 7 (2007) 573584.
3] L. Amable, Pharmacol. Res. 106 (2016) 2736.
4] S.M. Dencic, J. Poljarevic, A.M. Isakovic, T. Sabo, I. Markovic, V. Trajkovic, Curr. Med.
Chem. 27 (2020) 380410.
[
[
[
[
[
5] S. Ghosh, Bioorg. Chem. 88 (2019) 203208.
6] R. Oun, Y.E. Moussa, N.J. Wheat, Dalton Trans. 47 (2018) 66456653.
7] D.E. Gerber, Am. Fam. Physician. 77 (2008) 311319.
8] D. Hanahan, R.A. Weinberg, Cell 144 (2011) 646674.
9] W. Zheng, Y. Zhao, Q. Luo, Y. Zhang, K. Wu, F. Wang, Curr. Topic Med. Chem. 17 (2017)
3
0843098.
[
[
[
10] S. Banerjee, Y. Li, Z. Wang, F. H. Sarkar, Cancer Lett. 269 (2008) 226242.
11] U.N. dagi, N. M. hlongo, M. E. Soliman, Drug Des. Devel. Ther. 11 (2017) 599616.
12] C.R. Kowol, P. Heffeter, W. Miklos, L. Gille, R. Trondl, L. Cappellacci, W. Berger, B.K.
Keppler, J. Biol. Inorg. Chem. 17 (2012) 409423.
[
[
[
13] A.S. Potapov, E.A. Nudnova, G.A. Domina, L.N. Kirpotina, M.T. Quinn, A.I.
Khlebnikov, I.A. Schepetkin, Dalton Trans. 23 (2009) 44884498.
14] I. Schepetkin, A. Potapov, A. Khlebnikov, E. Korotkova, A. Lukina, G. Malovichko, L.
Kirpotina, M.T. Quinn, J. Biol. Inorg. Chem. 11 (2006) 499513.
15] J.A. Ahsan, A.A. Khan, Z. Ali, R. Haider, M.S. Yar, Eur. J. Med. Chem. 125 (2017)
1
43189.
26

Journal Pre-proof
[
[
[
16] S.S. Gu, P. Yu, J.N. Hu, Y. Liu, Z.W. Li, Y. Qian. Y. Wang, Y. Gou, F. Yang, Eur. J.
Med. Chem. 164 (2019) 654664.
17] M. Wehbe, A.W.Y. Leung, M.J. Abrams, C. Orvig, M.B. Bally, Dalton Trans. 46 (2017)
1
075810773.
18] F.G. Parsa, M.A.H. Feizi, R.S. Alizadeh, S.A.H Yazdi, M. Mahdavi, J. Biol. Inorg. Chem.
2
5 (2020) 383394.
[
[
19]
S. Monro, K.L. Colón, H. Yin, J. Roque, Chem. Rev. 119 (2019) 797–828.
20] A. Kellett, Z. Molphy, C. Slator, V. McKee, N.P. Farrell, Chem. Soc. Rev. 48 (2019)
71988.
D. Colangelo, A.L. Ghiglia, I. Viano, G. Cavigiolio, D. Osella, Biometals 16 (2003)
53560.
9
[
21]
5
[
[
22] J. Malina, N.P. Farrell, V. Brabec, Angew. Chem. Int. Ed. 57 (2018) 8535 –8539.
23] C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato, C. Marzano, Chem. Rev. 114
(2013) 815862.
[
[
24] D.B. Lovejoy, P.J. Jansson, U.T. Brunk, J. Wong, P. Ponka, D.R. Richardson, Cancer
Res. 71 (2011) 58715880.
25] F. Tisato, C. Marzano, M. Porchia, M. Pellei, C. Santini, Med. Res. Rev. 30 (2010)
7
08749.
[
[
26] I. R. Canelon, P. J. Sadler, Inorg. Chem. 52 (2013) 1227612291.
27] N.S. Moorthy, N.M. Cerqueira, M.J. Ramos, P.A. Fernandes, Anticancer Drug Discov. 8
(2013) 168182.
[
28] A. Bergamo, G. Sava, Chem. Soc. Rev. 44 (2015) 88188835.
27

Journal Pre-proof
[
[
[
[
[
29] M. Saxena, J. Saxena, R. Nema, D. Singh, A. Gupta, J. Pharmacogn. Phytochemistry 1
(
2013) 168182.
30] M. Ferraroni, C. Bazzicalupi, A.R. Bilia, P. Gratteri, Chem. Commun. 47 (2011)
9174919.
4
31] K.B. Huang, Z.F. Chen, Y.C. Liu, M. Wang, J.H. Wei, X.L. Xie, J.L. Zhang, K. Hu, H.
Liang, Eur. J. Med. Chem. 70 (2013) 640648.
32] T.M. Khan, N.S. Gul, X. Lu, J. H. Wei, Y.C. Liu, H. Sun, H. Liang, C. Orvig, Z.F. Chen,
Eur. J. Med. Chem. 163 (2019) 333343.
33] T. M. Khan, N. S. Gul, X. Lu, R. Kumar, M. I. Choudhary, H. Liang, Z. F. Chen, Dalton
Trans. 48 (2019) 1146911479.
[
[
34] N. Sotomayor, E. Domínguez, E. Lete, J. Org. Chem. 61 (1996) 40624072.
35] F.V. Nussbaum, B. Miller, S. Wild, C.S. Hilger, S. Schumann, H. Zorbas, W. Beck, W.
Stiglich, J. Med. Chem. 42 (1999) 34783485.
[
[
36] Y.P. Ho, K.K. To, S.C. A. Yeung, X. Wang, G. Lin, X. Han, J. Med. Chem. 44 (2001)
2
0652068.
37] Z.Q. Deng, P. Gao, L.L. Yu, B. Ma, Y.Y. You, L. Chan, C. Mei, T.F. Chen, Biomaterials
29 (2017) 111126.
1
[
[
[
[
38] R. Gerl, D.L. Vaux, 26 (2005) 263270.
39] L.H. Aung, R. Li, B.S. Prabhakar, A.V. Maker, P. Li, Oncotarget 8 (2017) 5658256597.
40] J. Armstrong, Br. J. Pharmacol. 151 (2007) 11541165.
41] J. Chipuk, L.B. Hayes, D. Green, Cell Deat. Differ. 13 (2006) 13961402.
28

Journal Pre-proof
[
42] P. Nagakannan, P. Tabeshmehr, E. Eftekharpour, Free Radical Biol. Med.
https://doi.org/10.1016/j.freeradbiomed.2020.04.001.
[
[
43] I. Tanida, T. Ueno, E. Kominami, Int. J. Biochem. Cell Biol. 36 (2004) 25032518.
44] S. Pankiv, T.H. Clausen, T. Lamark, A. Brech, J. A. Bruun, H. Outzen, A. Øvervatn, G.
Bjørkøy, T. Johansen, J. Biol. Chem. 282 (2007) 2413124145.
[
[
[
45] S. Pattingre, A. Tassa, X. Qu, R. Garuti, X.H. Liang, N. Mizushima, M. Packer, M.D.
Schneider, B. Levine, Cell 122 (2005) 927939.
46] W.W. Li, J.Y. Yu, H.L. Xu, J.K. Bao, Biochem. Biophys. Res. Commun. 414 (2011)
2
82286.
47] X. Sui, N. Kong, L. Ye, W. Han, J. Zhou, Q. Zhang, C. He, H. Pan, Cancer Lett. 344
2014) 174179.
(
[
[
[
48] M.E. Hardie, H.W. Kava, V. Murray, Curr. Pharm. Des. 22 (2016) 66456664.
49] G. Song, G. Ouyang, S. Bao, J. Cell Mol. Med. 9 (2005) 5971.
50] H.Y. Lee, A. C. Tsai, M.C. Chen, P.J. Shen, Y.C. Cheng, C.C. Kuo, S.L. Pan, Y.M. Liu,
J.F. Liu, T.K. Yeh, J. Med. Chem. 57 (2014) 40094022.
[
[
[
51] P.H. Thaker, L.Y. Han, A.A. Kamat, J.M. Arevalo, R. Takahashi, C. Lu, N.B. Jennings,
G.A. Pena, J.A. Bankson, M. Ravoori, Nat. Med. 12 (2006) 939944.
52] A. Ventura, D.G. Kirsch, M.E. McLaughlin, D.A. Tuveson, J. Grimm, L. Lintault, J.
Newman, E.E. Reczek, R. Weissleder, T. Jacks, Nature 445 (2007) 661665.
53] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A Robb., J.R. Cheeseman, G.
Scalmani, V. Barone, G.A Peterson, H. Nakatsuji, X. Li, M. Caricato, Gaussian. 16 Rev. A.03.
2
016.
[
54] Y. Zhao, D.G. Truhlar, Accounts Chem. Res. 41 (2008) 157-167.
29