Influence of the introduction of a triphenylphosphine group on the anticancer activity of a copper complex

Article abstract of DOI:10.1016/j.jinorgbio.2020.111102

Aiming at obtaining new copper complexes with good cytotoxicity against cancer cells, triphenylphosphine (TPP) was introduced to obtain insight into the influence of the co-ligands. In this paper, two copper complexes, Cu(2-pbmq)(CH₃OH)Br₂ (1) and [Cu(2-pbmq)(TPP)Br]₂ (2) were designed, synthesized, and characterized by X-ray crystallography, 2-((2-(pyrazin-2-yl)-1H-benzo[d]imidazol-1-yl)methyl))quinolone (2-pbmq), to investigate the influence of the TPP group on the anticancer activity of the metal complex. Although the presence of the TPP group diminished the intensity of the interaction properties of the complex with DNA, the in vitro anticancer activity and cellular uptake of the TPP-containing complex were markedly superior to those of its TPP-lacking counterpart. Detailed studies on the more potently cytotoxic complex 2 revealed that it accumulated in nucleus, arrested the cell cycle at the G0-G1 phase, causing mitochondrial dysfunction, involving the potential simultaneous mitochondrial membrane collapse, cellular ATP level depletion, and Ca²⁺ leakage, eventually inducing cell apoptosis. In summary, the introduction of a TPP group enhances the biological activity and cytotoxicity of the complex.

Full text of DOI:10.1016/j.jinorgbio.2020.111102

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Influence of the introduction of a mitochondrion-targeting group
on the anticancer activity of a copper complex
Sen Li, Jin'an Zhao, Yan Guo, Yameng Mei, Bangpeng Yuan,
Ning Gan, Junshuai Zhang, Jiyong Hu, Hongwei Hou
PII:
S0162-0134(20)30130-6
DOI:
https://doi.org/10.1016/j.jinorgbio.2020.111102
JIB 111102
Reference:
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
15 January 2020
29 April 2020
30 April 2020
Please cite this article as: S. Li, J. Zhao, Y. Guo, et al., Influence of the introduction of a
mitochondrion-targeting group on the anticancer activity of a copper complex, Journal of
Inorganic Biochemistry (2018), https://doi.org/10.1016/j.jinorgbio.2020.111102
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Influence of the Introduction of a Mitochondrion-targeting Group
on the Anticancer Activity of a Copper Complex
Sen Lib, Jin'an Zhaoa,*, Yan Guoa, Yameng Meib, Bangpeng Yuanb, Ning Gana, Junshuai
Zhanga, Jiyong Hua and Hongwei Houb
aCollege of Material and Chemical Engineering, Henan University of Urban Construction,
Pingdingshan 467036 Henan, P. R. China.
bCollege of Chemistry, Zhengzhou University, Zhengzhou 450001 Henan, P. R. China.
Corresponding Author
Jin'an Zhao
Address: College of Material and Chemical Engineering, Henan University of Urban
Construction, Pingdingshan 467036 Henan, P. R. China.
Email: zjinan@zzu.edu.cn
Abstract
Mitochondria participate in multiple cellular functions, including in particular energy
production and cell apoptosis, and targeting mitochondria might enhance the efficiency of
chemotherapeutic
agents.
Triphenylphosphine
(TPP)
is
a
commonly
used
mitochondrion-targeting group, so two copper complexes, Cu(2-pbmq)(CH3OH)Br2 (1) and
[Cu(2-pbmq)(TPP)Br]2 (2) were designed, synthesized, and characterized by X-ray
crystallography, (2-pbmq = 2-((2-(pyrazin-2-yl)-1H-benzo[d]imidazol-1-yl)methyl))quinoline),
to investigate the influence of the mitochondrion-targeting group on the anticancer activity of
the metal complex. Although the presence of the TPP group diminished the intensity of the
interaction properties of the complex with DNA, the in vitro anticancer activity and cellular
uptake of the TPP-containing complex were markedly superior to those of its TPP-lacking
counterpart. Detailed studies on the more potently cytotoxic complex 2 revealed that it arrested

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the cell cycle at the G0-G1 phase, causing mitochondrial dysfunction, involving the potential
simultaneous mitochondrial membrane collapse, cellular ATP leave depletion, and Ca2+ leakage
from the mitochondria, eventually inducing cell apoptosis. In summary, the introduction of a
mitochondrion-targeting group enhances the biological activity and cytotoxicity of the complex.
Keywords: Mitochondria-targeting; Triphenylphosphine; Copper complexes; Anticancer;
Apoptosis
1. Introduction
Mitochondria are considered prime targets of metal complexes with anticancer activity [1].
These subcellular organelles are present in most eukaryotic cells and are generally recognized as
“the cell powerhouses” [2]. Mitochondria also have a key role in regulating cell death [3].
Mitochondrion-targeting metal complexes can disturb the function of mitochondria and may
trigger extensive ATP depletion, leading to the death of cancer cells [4].
Currently, the TPP group is regarded as a pharmacophore of interest, as its induction is regard
as an effective approach to delivering agents to mitochondria [5]. As a functional targeting
group, TPP has been extensively studied in the context of the design and synthesis of metal
complexes, most of which have excellent potential in chemotherapy, as they influence
mitochondrial metabolism. For instance, Guo’s research group recently reported a
triphenylphosphonium-modified terpyridine platinum(II) complex, TTP, which exhibited
marked cytotoxicity and exerted strong inhibition of both mitochondrial and glycolytic
bioenergetics, inducing cancer cells to enter a hypometabolic state [6]. In addition, platinum,
ruthenium, and gold complexes containing TPP were designed and synthesized by other group
to investigate the effect that the TPP targeting group had on metal complexes’ bioactivity and
cytotoxicity [7–11]. In contrast to the mentioned metallic elements, copper is an endogenous and
readily available metal. It is also physiologically essential and characterized by low toxicity. In
fact, copper is involved in many biological processes taking place in the human body [12–14].
Some copper complexes comprising TPP have been reported to target mitochondria and

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adversely affect mitochondrial metabolism [15–18]. Notably, the rational choice of the axial
organic scaffold offers an approach to the fine-tuning of the pharmacological properties of metal
complexes with high anticancer activity. The benzimidazole and quinolone organic scaffolds are
a good choice for synthesizing anticancer chemotherapeutics, given their extensive biological
properties, including their antiviral, anticancer, antibacterial, and antifungal activities [19–21].
Our group investigated two copper complexes, dinuclear [Cu(qbm)Cl2]2 and mononuclear
[Cu(qbm)Br(TPP)] (qbm=2-(2'-quinolyl)benzimidazole), and found that the complex bearing
the TPP ligand is superior to the other one in terms of reactivity and cytotoxicity. Additionally,
[Cu(qbm)Br(TPP)] was observed to cause dual mitochondrial and nuclear DNA damage [22].
Our persisting research interest in metal complexes with anticancer activity led us to explore
the effect that the TPP targeting group has the cytotoxicity and mitochondrial damage properties
of the said complex. In the present study, complexes 1 and 2 were synthesized, and their in vitro
anticancer activities were investigated. The insight we garnered through these investigations
may provide the basis for a promising strategy for the functional modification of metal
complexes aimed at enhancing their anticancer activity.
2. Experimental section
2.1. Materials and instruments
GelRed (Biotium, USA), pBR322 DNA (TaKaRa Biotechnology Ltd, China), calf thymus
DNA (CT-DNA, Sigma Aldrich, USA), dimethyl sulfoxide (DMSO, Sigma Aldrich, USA)
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma Aldrich, USA),
Fluo-3 acetoxymethyl ester (Fluo-3/AM, Sigma Aldrich, USA), ethidium bromide (EB, Sigma
Aldrich, USA), cisplatin (Shanghai Energy Chemical Ltd, China), fetal bovine serums (FBS,
Hyclone Laboratoreis Inc, USA). All the experimental reagents involved in this study were
purchased from commercial sources and used as received without further purification treatment.
CuBr2, CuBr, 2-(bromomethyl)isoquinoline, 1,2-diaminobenzene, 2-pyrazinecarboxylicacid
were purchased from Bide Pharmatech Ltd (China). TPP was purchased from J&K Scientific

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Ltd (China). Mitochondrial membrane potential assay kit, enhanced ATP assay kit, and the cell
mitochondria isolation kit were purchased from Beyotime (China). Annexin V-FITC apoptosis
detection kit was purchased from BD Bioscience (USA). Buffer solutions (5 mM Tris-HCl/50
mM NaCl; pH = 7.4 and 50 mM Tris-HCl/50 mM NaCl; pH = 7.4; TAE buffer (40 mM Tris, 20
mM acetic acid, and 1 mM EDTA, pH = 8.3).) for the related biological experiments was
prepared using double distilled water.
Infrared spectra (IR) with KBr discs were recorded on a PerkinElmer Fourier transform-IR
spectrometer (Perkin Elmer, Waltham, MA, USA) in the 400–4000 cm–1 range. Electrospray
ionization mass spectra (ESI-MS) were measured on a Thermo Scientific LCQ fleet ESI-MS
spectrometer. Microanalysis (C, H, and N) was carried out using Flash EA 1112 elemental
analyzer (Thermo Fisher Scientific, Waltham, MA, USA). UV-Vis absorption spectra were
measured by a Specord 200 UV-Vis spectrophotometer (Analytik Jena, Germany). MOS-500
instrument (Bio-Logic Science Instruments, Seyssinet-Pariset, France) was used in circular
dichroism spectral analysis. Tecan Infnite M1000 Pro microplate reader (Tecan, Morrisville, NC,
USA) was used in MTT assay, adenosine triphosphate (ATP) production assay, and EB replacing
experiment to read the chemiluminescence or absorbance. ICP-MS Nex-ION 300X instrument
(PerkinElmer, USA) was used in cell uptake assay. Double distilled water was obtained using a
Milli-Q® water purification system (Millipore) for all related experiments. Flow cytometric
analysis was done using a BD LSRFortessaTM Cell Analyzer (BD Biosciences, USA). DNA
images were captured on a G: BOX F3 gel imaging system (Syngene, UK).
2.2. Synthesis of ligand and its corresponding complexes
The route for the synthesis of the ligand and its counterpart complexes was presented in Scheme
1.
2.2.1. Synthesis of ligand (2-pbmq)
2-((2-(pyrazin-2-yl)-1H-benzo[d]imidazol-1-yl)methyl)quinoline (2-pbmq) was prepared by
the substitution reaction between 2-(pyrazin-2-yl)-1H-benzo[d]imidazole (1 mmol, 0.196 g) and

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2-(bromomethyl)isoquinoline (1 mmol, 0.221 g) in acetone solution. After the reaction heated to
60 °C and refluxed 12 h and slowly cooled to room temperature, ashen powder-shaped ligands
were obtained. IR (KBr/pellet, cm–1): 3400 (w), 3053 (m), 1598 (m), 1505 (m), 1445 (w), 1414
(s), 1168 (m), 1117 (s), 1015 (s), 977 (m), 742 (m), 616 (w).
2.2.2. Synthesis of the complex Cu(2-pbmq)(CH3OH)Br2 (1)
2-pbmq (0.02 mmol, 0.0067 g) dissolved in methanol (3 mL) was added to a solution of
CuBr2 (0.02 mmol, 0.0045 g) in ethanol (2 mL), which placed together in a sealed glass
container at 25 °C. After one week, the brown crystals were isolated to give complex 1. Yield:
Elemental Analysis (%) calcd. for C H Br CuN O: C, 44.58; H, 3.23; N,
81%, based on Cu.
22
2
5
19
11.81; found (%): C, 44.63; H, 3.24; N, 11.93.
ESI-MS (Found, m/z): 558.83 [Cu(2-pbmq) Br2 +
1616 (m), 1566 (w), 1506
IR (KBr/pellet, cm–1): 3434 (m), 3062 (m),
+
H] , (Calcd. m/z: 558.90).
(m), 1479 (s), 1453 (s), 1389 (m), 1180 (m), 1125 (m), 1038 (s), 753 (s), 517 (w), 478 (w).
2.2.3. Synthesis of the complex [Cu(2-pbmq)(TPP)Br]2 (2)
A mixture solution (7 mL) containing CuBr (0.03 mmol, 0.0043 g,), 2-pbmq (0.01 mmol,
0.0034 g), TPP (0.02 mmol, 0.0052 g), acetonitrile (4 mL), and dichloromethane (3 mL) stirred
at room temperature to obtain a yellow solution. The resulting mixture solution was left to stand
at 25 °C about two weeks, and the yellow crystals were obtained. Yield: 61%, based on Cu.
Elemental Analysis (%) calcd. for C H Br Cu N
: C, 63.03; H, 4.07; N, 9.42; found (%):
ESI-MS (found, m/z): 662.25 [Cu(2-pbmq)(TPP)]+, (calcd. m/z:
10P2
78 60
2
2
C, 62.89; H, 4.06; N, 9.68.
662.13). IR (KBr/pellet, cm–1): 3434 (w), 3061 (m), 1598 (m), 1503 (m), 1479 (m), 1433 (s),
1331 (s), 1150 (s), 1017 (m), 824 (m), 748 (s), 696 (s), 608 (w).
2.3. X-ray crystallography determination
Crystals of complexes 1 and 2 qualified for X-ray analyses were obtained by solvent
evaporation method. X-ray diffraction measurements were performed on a Super Nova
diffractometer with Cu-Kα radiation (λ = 1.54184 Å) at 293 K or 160 K. The structures were

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solved and expanded utilizing both direct method and Fourier technology. The analyses of
complexes were obtained with SHELXL 2018 and the OLEX2 crystallographic programs [23–
24]. All the non-hydrogen atoms were refined anisotropically [25]. The structures were plotted
by using crystal maker. The crystallographic and structure parameters of the complexes were
shown in the table 1. The data related to the main bond lengths and bond angles were shown in
the table 2.
2.4. Thermogravimetric assay and solution stability determination
A model TGA/SDTA 851 MOD thermal analyzer was used to record simultaneous
thermogravimetric curves at a heating rate of 10 °C min–1 over the temperature ranging from
room temperature to 900 °C in a flowing nitrogen atmosphere of 20 mL/min using platinum
crucibles. Complexes 1 and 2 were dissolved in buffer solutions (5 mM Tris-HCl/50 mM NaCl;
pH = 7.4) at 5×10–5 M concentration. UV-Vis absorption spectra of the complexes 1 and 2 were
recorded on a Specord 200 UV-Vis spectrophotometer (Analytik Jena, Germany) in different
time intervals.
2.5. DNA interaction properties assays
2.5.1. Circular dichroism spectral analysis
CT-DNA concentration was measured by UV-Vis at 260 nm and 280 nm with a molar
absorption coefficient 6600 M−1 cm−1 [26]. The value of A260/A280 was located in 1.8–1.9, which
illustrated that the DNA solution was free of protein sufficiently [27–28]. CT-DNA (50 µM) was
incubated with various concentrations of complexes 1 and 2 (10–50 µM) in the buffer solution
(5 mM Tris-HCl/50 mM NaCl buffer pH = 7.4) at room temperature. The mixture solutions were
measured immediately in MOS-500 instrument (Bio-Logic Science Instruments,
Seyssinet-Pariset, France) and recorded scanning wavelength ranging from 220 nm to 320 nm.
The background of the buffer solution was deducted before testing.

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2.5.2. EB replacing experiment
CT-DNA (100 µM)/EB (10 µM) system was incubated in a 96-well cell culture plate for 1 h
at 37 ºC. And then the mixture solutions were titrated by complexes 1 and 2 ranging from 0 to
90 μM in constant temperature incubator at 37 ºC for 4 h. The fluorescence spectra were
measured on Tecan Infnite M1000 Pro microplate reader (Tecan, Morrisville, NC, USA) (λex =
490 nm; λem = 510–800 nm).
2.5.3. DNA cleavage
pBR322 DNA was incubated with various concentrations of complexes 1 and 2 (10–50 µM)
in buffer solution (50 mM Tris-HCl/50 mM NaCl; pH = 7.4) at 37 °C for 4 h. DNA samples
were analyzed by electrophoresis (85 V, 1 h) on a 1% agarose gel in 1×TAE buffer (40 mM Tris,
20 mM acetic acid, and 1 mM EDTA, pH = 8.3). The image was captured on a G: BOX F3 gel
imaging system (Syngene, UK).
2.6. In vitro anticancer activities
2.6.1. Cell lines and culture conditions
Human cancer and normal cells: BGC823 (gastric cancer cell line), TFK-1 (cholangio
carcinoma cell line), SMMC7721 (liver cancer cell line), L-02 (normal liver cell line). L-02 and
BGC823 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) at 37 °C
under 5% CO2, 95% air in a humidified incubator. TFK-1 and SMMC7721 cells were
maintained in Roswell Park Memorial Institute (RPMI) 1640 medium at 37 °C under 5% CO2,
95% air in a humidified incubator.
2.6.2. Cytotoxicity assay
The cytotoxicity of the tested complexes towards BGC823, TFK-1, SMMC7721, L-02 cell
lines was determined by MTT assay. When cells cultured in 96-well cell culture plate were
grown to confluence, various concentrations of the complexes, cisplatin, and 2-pbmq were

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added for 48 h at 37 °C under 5% CO2, 95% air in a humidified incubator. The treated cells were
labeled with 20 μL MTT dye (5 mg/mL) for 4 h. The culture medium was sucked out and
DMSO was added (150 μL per well). The 96-well cell culture plate was shaken for 10 min. The
absorbance values at 492 nm were measured using a Tecan Infnite M1000 Pro microplate reader
(Tecan, Morrisville, NC, USA).
2.6.3. Lipophilicity and cellular uptake
The lipophilicity (log PO/W) of complexes 1 and 2 was measured in a water/1-octanol system
using the shake-flask method. Solutions of complexes 1 and 2 (30, 40, and 50 μM) were
prepared in water presaturated with 1-octanol. Equal volumes (2.0 mL) of the solution and
1-octanol presaturated with water were mixed and shaken at room temperature for 24 h and then
separated into two phases by centrifugation. The concentration of the solute in the aqueous
phase was determined by spectrophotometry. The log P values were calculated from the average
of three independent measurements. The log PO/W value was calculated using the equation
log PO/W = log[([C]initial−[C]final)/[C]final
]
Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure the
intracellular Cu contents. SMMC7721 cells were seeded in 100 mm cell culture plate to welt.
After 24 h, SMMC7721 cells were treated with complexes 1 and 2 at 10 µM for 8 h. And then
cells were collected with trypsin and washed with PBS in centrifuge tubes. The cytoplasm,
nucleus, and mitochondria of SMMC7721 cells were isolated by a cell mitochondria isolation
kit according to the manufacturer's instruction. The collected extractions were digested with
HNO3 (100 μL) at 95 °C for 1 h, H2O2 (30%, 50 μL) at 95 °C for 1 h, and HCl (100 μL) at 95 °C
for 1 h, sequentially. The solutions were then diluted to a final volume of 4 mL with double
distilled water. The Cu contents were calculated as ng Cu per 106 cells.
2.7. Cell death mechanism studies

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2.7.1. Annexin V-PI assay
SMMC7721 cells were cultured in 6-well cell culture plate to welt for 24 h and then treated
with complex 2 at the indicated concentrations for 24 h. The SMMC7721 cells were collected
and stained using an Annexin V-FITC apoptosis detection kit (BD) according to the
manufacturer's instruction. SMMC7721 cells were collected by using flow cytometry. Cells at
different stages were analysed by FlowJo software.
2.7.2. Cell cycle assay
SMMC7721 cells were cultured in 6-well cell culture plate to welt for 24 h. Then culture
mediums were replaced by the fresh mediums containing various concentrations of complex 2
for 8 h. The cells were washed with PBS and digested with trypsin and immobilized with 70%
ice-cold ethanol, and eventually preserved at 4 ºC. The immobilized cells were disposed
according to the manufacturer's instruction of KeyGEN Cycle Test Plus DNA reagent kit. The
cells were collected by flow cytometry and data were analyzed by ModFit LT Software.
2.7.3. Measurement of mitochondrial membrane potential
The mitochondrial membrane potential assay was performed using mitochondrial membrane
potential assay kit according to the manufacturer’s instruction (Beyotime, China). SMMC7721
cells were cultured in 6-well cell culture plate to welt for 24 h. After incubated with specified
concentrations of complex 2 at 37 °C for 8 h, the cells were washed with PBS and digested with
trypsin and collected in different centrifuge tubes. The cells were further incubated with 1 mL
Rh123 working solution for 20 min at 37 ºC in dark before rinsed and centrifuged three times
with RPMI 1640 medium without FBS. The cells were measured by flow cytometry. Data were
analyzed by FlowJo Software.
2.7.4. ATP production assay
The ATP concentrations were performed using enhanced ATP assay kit (Beyotime, China)
according to the manufacturer's instruction. The standard curve was obtained by the known

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concentrations of standard ATP sample. SMMC7721 cells were incubated in 6-well cell culture
plate to welt for 24 h. The cells were incubated with various concentrations of complex 2 at
37 °C for 24 h. After incubated, the cells were lysed and collected in centrifuge tubes and
centrifuged in 12000 g for 5 min at 4 °C and only reserved supernatant. 100 μL ATP working
solutions were added to the 96 round black well plate for 5 min before reserved supernatant
were added to the 96 round black well plate. The luminescence was measured using a Tecan
Infinite M1000 Pro microplate reader (Tecan, Morrisville, NC, USA).
2.7.5. Intracellular Ca2+generation assay
Fluo-3/AM (Sigma Aldrich, USA) staining method was used to measure the intracellular Ca2+
concentration. SMMC7721 cells were cultured in 6-well cell culture plate to welt for 24h. After
SMMC7721 cells treated with complex 2 at indicated concentrations for 8 h, the cells were
washed with PBS and incubated with 1 mL Fluo-3/AM working solution for 30 min at 37 ºC in
dark. Lastly, the cells were washed with PBS and collected, and fleetly analyzed by flow
cytometry. Data were analyzed by FlowJo Software.
3. Results and discussion
3.1. Crystal structure
Complex 1 crystallizes in a triclinic crystal system and P-1 space group. As illustrated by the
structure depicted by Fig. 1, the complex’s asymmetric unit consists of one Cu2+ ion, one neutral
ligand, one coordinated methanol molecule and two bromine atoms. In five-coordinated
complexes, the geometry index, τ5, can be used to discriminate between structures characterized
by the square pyramidal and trigonal bipyramidal geometries. The τ5 value for complex 1 was
determined to be 0.27, by performing a calculation utilizing the values for the N(2)–Cu(1)–Br(2)
(163.22(8)°) and N(1)–Cu(1)–Br(1) (147.03(7)°) angles. Importantly, a τ5 value of 1.00
identifies a structure with an ideal trigonal bipyramidal geometry, whereas a value of 0.00
identifies a structure with an ideal square pyramidal geometry [29]. In complex 1, the

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coordination geometry around the Cu center can be described as distorted tetragonal pyramidal.
The Cu–N bond lengths are similar to each other: the Cu(1)–N(1) = 2.071(3) Å and Cu(1)–N(2)
= 1.991(3) Å. Although the crystal structures of complexes 2 and 1 are characterized by the
same crystal system and space group, the coordination mode of the Cu center of complex 2 is
different from its counterpart of complex 1. In four-coordinated complexes, the geometry index
τ4 can be used to discern between a square planar and a tetrahedral geometry. The τ4 value for
complex 2 was determined to be 0.92, by performing a calculation utilizing the values for the
P(1)–Cu(1)–Br(1) (118.019(16)°) and Br(1)–Cu(1)–Br(1a) (112.387(15)°) angles. Notably, with
regard to τ4, a value of 1.00 identifies a structure with an ideal tetrahedral geometry, a value of
0.00 identifies a structure with an ideal square planar geometry and a value of 0.85 identifies a
structure with an ideal trigonal pyramidal geometry [30]. The distorted tetrahedral coordination
around the Cu center of complex 2 comprises two bromine atoms, the N atom of a terminally
bound ligand, and the P atom of TPP. Additionally, a μ2-Br group acts as bridge between Cu(1)
and Cu(1a). The distance between Cu(1) and Cu(1a) is 2.7896(5) Å. In complex 2, the ligands
coordinating
Cu(1)
and
Cu(1a)
lie
on
two
essentially
parallel planes
(2-(pyrazin-2-yl)-1H-benzo[d]imidazole and isoquinoline planes). The values for the bond
lengths and bond angles determined for complex 2 are within the normal range for similar Cu(I)
complexes [31].
3.2. Stability determination
As illustrated by the data reported in Fig. S1, complexes 1 and 2 are relatively stable, and the
integrity of their crystals is maintained up to at least 200 °C. In complex 1 the unit cell includes
disorder, which was observed from the difference Fourier maps. In the case of complex 1, a 5.69%
weight loss observed to occur as the temperature rose from 50 to 200 °C, which was ascribed
to the loss of solvent molecules.
In order to conduct investigations on the anticancer activity of a complex, the stability of the
said complex under physiological conditions is necessary. The UV-Vis absorption spectra of
complexes 1 and 2 recorded at different time intervals are reported in Fig. S2, no obvious

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change was observed in the spectra of complexes 1 and 2 over a 48 h period, indicating that
these complexes were almost completely stable in solution.
3.3. DNA interactions
3.3.1. Circular dichroism spectral analysis
DNA is the primary intracellular target for anticancer metal complexes [32]. The interaction
of the complexes with CT-DNA was investigated by circular dichroism spectrometry. The
characteristic peaks of B-DNA consist of a positive and a negative band at 275 nm and 245 nm,
which are due to base stacking and the B-conformation of DNA, respectively [33]. As can be
evinced from the spectra reported in Fig. 2, in the presence of complexes 1 and 2, the typical
bands of B-DNA underwent red-shifts and their intensities were significantly reduced, indicating
that both complexes can interact with DNA. Furthermore, the spectral modifications associated
with the presence of complex 2 were stronger than their counterparts observed for complex 1,
indicating that complex 2 causes a stronger perturbation of DNA’s secondary structure.
3.3.2. EB competition binding assay
The DNA binding properties of complexes 1 and 2 were further studied by conducting EB
competition binding assays to quantify the complexes’ binding affinity for DNA. As can be
evinced from the data in Fig. 3, as increasing amounts of complexes 1 and 2 are added to the
EB-DNA solution, a dramatic hypochromism effect was observed at 612 nm. The classical
linear Stern-Volmer equation: I0/I = 1 + Ksv[Q], where I0 and I are the fluorescence intensities in
the absence and presence of a quencher, respectively, and [Q] is the quencher concentration. Ksv,
the linear fit plot of I0/I versus [Q], represents the Stern-Volmer dynamic quenching constant
[34]. Kapp, the apparent DNA binding constant, was calculated via the following equation: KEB
[EB] = Kapp [complex]. Herein KEB = 1.0×107 M−1 and [EB] = 10 μM. Notably, KEB is the
binding constant of EB to DNA, and [complex] is the concentration of the complex in the
situation whereby a 50% reduction in the fluorescence intensity of EB is observed. The

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approximate values for Ksv and Kapp were calculated to be 1.10×104 M–1 and 1.79×107 M–1, in
the case of complex 1 and 1.26×103 M–1 and 1.73×106 M–1, respectively, in the case of complex
2, demonstrating that complex 1 displayed a stronger affinity for DNA than complex 2. All in all,
the results of EB competition binding assays in combination with circular dichroism
spectrometry data indicated that complexes 1 and 2 may bind to DNA via intercalation.
3.3.3. Agarose gel electrophoresis assays
The interaction of complexes 1 and 2 with pBR322 DNA was investigated conducting agarose
gel electrophoresis assays. pBR322 DNA can present itself in three forms: supercoiled (Form I),
nicked/open circular (Form II) and linear (Form III). As can be evinced from the electrophoresis
assay results reported in Fig. 4, in the presence of an endogenous biological oxidation-reduction
agent (H2O2 or ascorbic acid), the treatment of pBR322 DNA with complexes 1 and 2 resulted in
the gradual conversion of the Form I into Forms II and III. Complexes 1 and 2 displayed strong
DNA cleavage activity, and DNA was cleaved by complex 1 to a greater extent than by complex
2.
3.4. In vitro anticancer activities
3.4.1. Cytotoxicity analysis
The in vitro toxicity of complex 1, complex 2, and 2-pbmq against SMMC7721, BGC823,
TFK-1, and L-02 cells were determined performing the MTT assay over a 48 h period. The IC50
values thus determined are reported in Table 3. Treatment of cells with 2-pbmq alone does not
affect cell viability, even at 100 μM concentration [35]. Complex 1 exhibited medium
cytotoxicity against the tested cancer cell lines. Complex 2, on the other hand, showed marked
cytotoxicity against the same cancer cell lines. The in vitro anticancer activity of the tested
compounds decreased in the following order: 2 > cisplatin > 1 > 2-pbmq. The enhanced
anticancer activity of complex 2 may result from the introduction of the ancillary ligand-TPP,
which, as indicated by the results of the experiments described in the next section, increased the

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relative lipophilicity of the copper complex [36–37].
3.4.2. Cellular uptake and lipophilicity
The cellular uptake properties of metal-based anticancer agents have an effect on the
anticancer activity of the said agents. Consequently, the cellular uptake ability of complexes 1
(10 μM) and 2 (10 μM) in SMMC7721 cells were assessed in vitro implementing the ICP-MS
assay. As can be evinced from the data reported in Fig. 5, under the same conditions, the cellular
uptake of complex 2 (109.28 ± 5.46 ng per 106 cells) was greater than that of complex 1 (18.48
± 0.80 ng per 106 cells). Furthermore, compared to complex 1, complex 2 was mainly
distributed in the nucleus. Additionally, the copper content of the mitochondria following
treatment with complex 2 (8.34 ± 0.34 ng per 106 cells) was approximately three times higher than
that observed after the treatment with complex 1 (2.64 ± 0.07 ng per 106 cells). This difference
may be the result of the introduction of the mitochondrion-targeting group. The lipophilicity
(log Po/w) of complex 2 was determined by the shake–flask method to be 1.24 in a
water/1-octanol system (Table 4.), a substantially larger value than that determined for complex
1 (0.46), which suggests that complex 2 has a greater tendency to penetrate the phospholipid
bilayers than complex 1. The results of the cellular uptake and cytotoxicity experiments detailed
above may be positive correlation with lipophilicity of the complexes. Since complex 2
exhibited significant cytotoxicity and high uptake by SMMC7721 cells, we were prompted to
further explore the possible anticancer mechanism of this complex.
3.5. Cell death mechanism studies
3.5.1. Cell apoptosis
The Annexin V-PI assay was implemented to test whether apoptosis was involved in complex
2-induced cell death. As can be evinced from the data reported in Fig. 6, the percentage of early
and late apoptotic cells increased in a dose-dependent manner after SMMC7721 cells were
treated with complex 2. After being treated with various concentrations of complex 2 (0, 1.5, 3,

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and 4.5 μM) for 24 h, the percentage of cells in the early and late apoptotic phases was
measured to be 2.69%, 10.29%, 16.92%, and 51.6%, respectively, which suggests that complex
2 can indeed induce apoptosis in SMMC7721 cells.
3.5.2. Cell cycle arrest
The cycle of SMMC7721 cells was investigated by flow cytometry after the said cells were
treated with various concentrations of complex 2 (0, 1.5, and 3 μM) for 8 h. As can be evinced
from the data reported in Fig. 7, with respect to the control (0 μM of complex 2), the percentage
of SMMC7721 cells in the G0-G1 phase increased from 38.73% to 42.28% and 53.24%, after
treatment with complex 2 (1.5 and 3 μM, respectively). Meanwhile, as the concentration of the
complex 2 used to treat the cells increased, the percentage of cells in the G2-M phase and in the
S phase decreased. These results indicate that complex 2 can disturb DNA synthesis and
ultimately arrest the cell cycle at the G0-G1 phase in a concentration-dependent manner.
3.5.3. Detection of mitochondrial membrane potential
Dissipation of the electrostatic potential across the mitochondrial membrane is one of the key
hallmarks of mitochondrial dysfunction [38]. Rh123 is widely used to detect mitochondrial
membrane potential. As can be evinced from the data in Fig. 8, the Rh123 fluorescence intensity
decreased when cells were treated with complex 2, indicating that the mitochondrial membrane
potential had decreased and that mitochondrial activity had been impaired.
3.5.4. Cellular ATP production and Ca2+ level
Mitochondria are the cell’s energy factories, which can participate in the synthesis of ATP, the
primary energy source of cellular processes in living organisms [39–40]. Mitochondria also
mediate Ca2+ uptake into the matrix to regulate metabolism, cell death, and cytoplasmic Ca2+
signaling. Mitochondrial dysfunction is always accompanied by ATP depletion in the cell and
the Ca2+ leakage from the mitochondria into the cell cytoplasm [41]. As can be evinced from the
data in Fig. 9, complex 2 induces a substantial dose-dependent decrease in intracellular ATP

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levels. The intracellular Ca2+ level was determined using a fluorescent probe, Fluo-3/AM, by
flow cytometry. As indicated by the data reported in Fig. 10, complex 2 treated cells displayed
higher fluorescence intensity than control cells, indicating that vast amounts of Ca2+ ions had
leaked out of the mitochondria.
4. Conclusions
Complexes 1 and 2 were designed, synthesized, and characterized. The complex containing
the mitochondrion-targeting TPP group displayed enhanced cytotoxicity and cellular uptake
compared with its TPP-free counterpart. Additionally, although complex 2, which comprises
TPP, was observed to bind DNA by intercalation thus disturbing DNA’s secondary structure, its
binding affinity toward DNA was observed to be marginally lower than that of complex 1,
which does not have a TPP moiety. The anticancer properties of the TPP-containing complex 2
were then investigated. Evidence indicated that complex 2 arrested the cell cycle at the G0-G1
phase. This complex disrupted mitochondrial structure, which potentially led to the
simultaneous mitochondrial membrane collapse, ATP depletion in the cell, and Ca2+ leakage
from the mitochondria into the cell cytoplasm, and eventually inducing cell apoptosis. Results
thus indicated that the biological activity and cytotoxicity of the copper complex were markedly
influenced by the presence of TPP acting as a mitochondrion-targeting group.
Acknowledgements
We gratefully acknowledge financial support by the National Natural Science Foundation of
China (Nos. 21371046 and 21401041), and Key scientific research project plan of Colleges and
Universities in He'nan province (19A150017), the Funding Program for Academic Technology
Leaders of the He'nan University of Urban Construction (No. YCJXSJSDTR201705).
A. supplementary data
Crystallographic data for structural analysis have been deposited with the Cambridge
Crystallographic Data Center, CCDC reference numbers 1959672–1959673. These data can be

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obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.htm (or from the Cambridge
Crystallographic Data Center, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223
336033).
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Conflict of Interest
The authors declare no conflict of interest.

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Figure
Scheme 1 Synthesis route to the ligand and complexes.
Fig. 1 Crystal structures of complexes 1 and 2. Hydrogen atoms are omitted for clarity.

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Fig. 2 The Circular dichroism spectral of CT-DNA in the absence and presence various
concentrations of complexes. r = [complex]/[DNA] = 0, 0.2, 0.4, 0.6, 0.8, and 1 for complex 1 (a)
and complex 2 (b), respectively.
Fig. 3 The emission spectrum of EB-DNA in the absence and presence of various
concentrations of complexes (0–90 μM). Conditions: [DNA] = 100 μM, [EB] = 10 μM for
complex 1 (a) and complex 2 (b), respectively.

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Fig. 4 The cleavage patterns of the agarose gel electrophoresis for pBR322 plasmid DNA by
complex at 37 °C for 4 h. Lane 1, DNA control; lane 2, DNA + ascorbic acid (1 mM) or H2O2
(1 mM); lane 3, DNA + complex (50 µM); lanes 4–8, DNA + ascorbic acid (1 mM) or H2O2 (1
mM) + complex (10, 20, 30, 40, and 50 µM, respectively) for complex 1 (a) and complex 2 (b),
respectively.
Fig. 5 Copper content (ng per106 cells) in SMMC7721 cells treated with complexes 1 (10 µM)
and 2 (10 µM) for 8 h. Data are expressed as mean ± SD (n = 3), significant at the *p<0.05,
**p<0.001, ***p<0.0001 levels using t-test, ns = not significant.

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Fig. 6 Annexin V-PI assay of SMMC7721 cells treated with complex 2 at the indicated
concentrations for 24 h.
Fig. 7 The percentage of cell populations in different phases (G0-G1, G2-M, and S phase) in
SMMC7721 cells treated by complex 2 for 8 h.

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Fig. 8 Mitochondrial membrane potential changes in SMMC7721 cells after treatment with
complex 2 at indicated concentrations for 8 h.
Fig. 9 Measurement of the intracellular level of ATP in the SMMC7721 cells after treatment
with complex 2 at indicated concentrations for 24 h. Data are expressed as mean ± SD (n = 3),
significant at the *p<0.05, **p<0.001, ***p<0.0001 levels using t-test.

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Fig. 10 Measurement of the intracellular level of Ca2+ in the SMMC7721 cells after treatment
with complex 2 at indicated concentrations for 8 h using Fluo-3/AM as a fluorescent probe.