Synthesis, characterization, DNA/HSA interactions, and anticancer activity of two novel copper(II) complexes with 4-chloro-3-nitrobenzoic acid ligand

Article abstract of DOI:10.3390/molecules26134028

The purpose of this study was to identify new metal-based anticancer drugs; to this end, we synthesized two new copper(II) complexes, namely [Cu(ncba)₄ (phen)] (1) and [Cu(ncba)₄ (bpy)] (2), comprised 4-chloro-3-nitrobenzoic acid as the main ligand. The single-crystal XRD approach was employed to determine the copper(II) complex structures. Binding between these complexes and calf thymus DNA (CT-DNA) and human serum albumin (HSA) was explored by electronic absorption, fluorescence spectroscopy, and viscometry. Both complexes intercalatively bound CT-DNA and statically and spontaneously quenched DNA/HSA fluorescence. A CCK-8 assay revealed that complex 1 and complex 2 had substantial antiproliferative influences against human cancer cell lines. Moreover, complex 1 had greater antitumor efficacy than the positive control cisplatin. Flow cytometry assessment of the cell cycle demonstrated that these complexes arrested the HepG2 cell cycle and caused the accumulation of G0/G1-phase cells. The mechanism of cell death was elucidated by flow cytometry-based apoptosis assays. Western blotting revealed that both copper(II) complexes induced apoptosis by regulating the expression of the Bcl-2(Bcl-2, B cell lymphoma 2) protein family.

Full text of DOI:10.3390/molecules26134028

molecules
Article
Synthesis, Characterization, DNA/HSA Interactions, and
Anticancer Activity of Two Novel Copper(II) Complexes with
4-Chloro-3-Nitrobenzoic Acid Ligand
Zhen-Fang Zeng 1,
*
, Qiu-Ping Huang 1, Jie-Hui Cai 1, Guang-Jin Zheng 1, Qiu-Chan Huang 1, Zi-Lu Liu 1,
Zi-Lu Chen 2,
*
and You-Huan Wei 1,
*
1
School of Chemical and Biological Engineering, Guangxi Normal University for Nationalities, 23 Fozi Road,
Chongzuo 532200, China; huangqiuping@gxnun.edu.cn (Q.-P.H.); caijiehui@gxnun.edu.cn (J.-H.C.);
zhengguangjin0624@163.com (G.-J.Z.); huangqiuchan@163.com (Q.-C.H.); liuzilu0624@163.com (Z.-L.L.)
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, School of
Chemistry and Pharmacy, Guangxi Normal University, 15 Yucai Road, Guilin 541004, China
Correspondence: zengzhenfang@gxnun.edu.cn (Z.-F.Z.); zlchen@mailbox.gxnu.edu.cn (Z.-L.C.);
weiyouhuan@gxnun.edu.cn (Y.-H.W.); Tel./Fax: +86-771-787-0799 (Z.-F.Z.)
2
*
Abstract: The purpose of this study was to identify new metal-based anticancer drugs; to this end,
we synthesized two new copper(II) complexes, namely [Cu(ncba)4(phen)] (1) and [Cu(ncba)4(bpy)]
(2), comprised 4-chloro-3-nitrobenzoic acid as the main ligand. The single-crystal XRD approach
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Zeng, Z.-F.; Huang, Q.-P.;
Cai, J.-H.; Zheng, G.-J.; Huang, Q.-C.;
Liu, Z.-L.; Chen, Z.-L.; Wei, Y.-H.
Synthesis, Characterization,
DNA/HSA Interactions, and
Anticancer Activity of Two Novel
Copper(II) Complexes with
4-Chloro-3-Nitrobenzoic Acid Ligand.
Molecules 2021, 26, 4028.
was employed to determine the copper(II) complex structures. Binding between these complexes
and calf thymus DNA (CT-DNA) and human serum albumin (HSA) was explored by electronic
absorption, fluorescence spectroscopy, and viscometry. Both complexes intercalatively bound CT-
DNA and statically and spontaneously quenched DNA/HSA fluorescence. A CCK-8 assay revealed
that complex
cell lines. Moreover, complex
1
and complex
2
1
had substantial antiproliferative influences against human cancer
had greater antitumor efficacy than the positive control cisplatin.
Flow cytometry assessment of the cell cycle demonstrated that these complexes arrested the HepG2
cell cycle and caused the accumulation of G0/G1-phase cells. The mechanism of cell death was
elucidated by flow cytometry-based apoptosis assays. Western blotting revealed that both copper(II)
complexes induced apoptosis by regulating the expression of the Bcl-2(Bcl-2, B cell lymphoma 2)
protein family.
https://doi.org/10.3390/
molecules26134028
Academic Editors: Bartosz Tylkowski,
Anna Bajek and
Keywords: anticancer; apoptosis; copper(II) complex; cytotoxicity; DNA/HSA
Krzysztof Roszkowski
Received: 7 May 2021
Accepted: 25 June 2021
Published: 1 July 2021
1. Introduction
Carcinogenesis transforms normal cells into cancer cells in response to tissue injury
or the influence of neighboring aging cells. The latter promote inflammation and tissue
remodeling, and use secretome and paracrine regulation to reprogram differentiated cells
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with regard to jurisdictional claims in
published maps and institutional affil-
iations.
into pluripotent cells associated with malignant growth [1]. In order to eliminate cancer
cells, we must understand their characteristics and identify the factors that cause them
to overproliferate. Cancers sustain proliferative signaling, resist cell death, deregulate
cellular energetics, evade growth repressors, stimulate angiogenesis, activate infiltration
and metastasis, enable replicative immortality, avoid immune destruction, support tumor-
promoting inflammation, and cause genome instability and mutation [2]. The relationship
between apoptosis and cancer forms the basis of innovative cancer therapy.
Metal-based anticancer agents are among the most successful chemotherapeutic drugs.
Cisplatin was the first metal-based drug administered to treat various solid tumors includ-
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
ing testicular and ovarian cancers [
cell lung cancers [ ]. In clinical practice, however, the toxicity profile of cisplatin includes
nausea and vomiting, renal dysfunction, neurotoxicity, and ototoxicity [ ]. Analogs such
as carboplatin and oxaliplatin are well tolerated in comparison to cisplatin but may cause
3], bladder cancer and melanoma [4], and non-small
5
6
Molecules 2021, 26, 4028. https://doi.org/10.3390/molecules26134028
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 4028
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innate, as well as acquired, resistance, neurotoxicity, nausea, and nephrotoxicity [
Hence, novel non-Pt (II/IV) anticancer complexes with fewer side effects are urgently
required [ 10]. Copper(II) complexes have attracted attention in medicinal inorganic chem-
istry research [11 16]. Copper(II) is an essential micronutrient and a structural and catalytic
cofactor in numerous biochemical pathways. It has demonstrated anticancer activity and
could be a formulant in innovative anticancer drugs [17 19]. Copper(II) complexes, such
7,8].
9,
–
–
as hydroxytryptophan copper(II) [20], substituted derivatives of pregnenolone acetate cop-
per(II) [21], thiosemicarbazone copper(II) [22], substituted carboxylic acid copper(II) [23],
1,10-phenanthroline derivative copper(II) [24], pyridine derivative copper(II) [25], thiosemi-
carbazone copper(II) [26], piperazine copper(II) [27], and schiff copper(II) [28], have been
synthesized, and their anticancer efficacy has been assessed.
Although substituted benzoic acids are often used as ligands to synthesize metal com-
plexes [29
order to obtain new possible metal anticancer drugs, herein, we synthesized the copper(II)
complexes [Cu(ncba)4(phen)] ( ) and [Cu(ncba)4(bpy)] ( ) and characterized them via car-
,30], 4-chloro-3-nitrobenzoic acid copper(II) complexes have not been reported, in
1
2
bon hydrogen nitrogen sulfur (CHNS) analysis, infrared spectroscopy, and single-crystal
XRD. We investigated their interactions with CT-DNA/HSA and used a Cell Counting
Kit 8 (CCK-8) assay to evaluate their cytotoxicity in vitro against human hepatocellular
carcinoma (HepG2), human cervical carcinoma (HeLa), and human lung carcinoma (A549)
cancer cell lines. We employed flow cytometry to explore HepG2 cell cycle distribution
and induction of apoptosis. We also assayed caspase and Bcl-2 protein family expression in
response to copper(II) complex exposure. Both complexes intercalatively bound CT-DNA
and statically and spontaneously quenched DNA/HSA fluorescence. In vitro cytotoxicity
assays demonstrated that complex
cells than complex or cisplatin. Complex
and induced G0/G1.
1
was more cytotoxic to HepG2, HeLa, and A549 cancer
2
1
and complex arrested the HepG2 cell cycle
2
2. Materials and Methods
2.1. Chemicals and Cell Lines
All reagents were used as purchased without any purification. Ethidium bromide (EB),
CT-DNA, and HSA were supplied by Sigma-Aldrich Corp. (Shanghai, China). The human
hepatocellular carcinoma HepG2, human cervical carcinoma HeLa, and the human lung
carcinoma A549 cells were commercially provided by the Chinese Academy of Sciences Cell
Bank/Stem Cell Bank and Council Cell Bank of Typical Culture Preservation (Shanghai,
China). The other chemicals were provided by Sinopharm Chemical Reagent Co. Ltd.
(Shanghai, China). All reagents were analytical grade. We prepared a stock CT-DNA
solution in 5 mM Tris-HCl/50 mM NaCl buffer (pH 7.2) and determined its concentration
by measuring absorbance at 260 nm using the UV-8000 spectrophotometer (Yuanxi Corp.,
Shanghai, China) with a molar absorption coefficient of 6600 M−1 cm−1
[31]. A stock
·
solution of HSA was prepared in Tris-HCl/NaCl; to determine the concentration of HAS,
the UV-8000 spectrophotometer (Yuanxi Corp., Shanghai, China) was used to measure
absorbance at 280 nm, and a molar absorption coefficient of 35,700 M−1 cm−1 was used
·
to calculate the concentration [32]. All solutions were kept at 4 ◦C and used within 4 d.
Double-distilled H2O was the solvent in all experiments.
The infrared (IR) spectra of their ligands a−n1d of their respective copper(II) complexes
were recorded within the limits of 4000–400 cm using a Spectrum 65 FT/IR spectrometer
(PerkinElmer, Waltham, MA, USA) with KBr pellets. Carbon, nitrogen, as well as hydrogen
were quantitated in a Vario EL cube elemental analyzer (Elemantar, Langenselbold, Hesse,
GER). The UV-8000 spectrophotometer (Yuanxi Corp., Shanghai, China) in the 200–1100 nm
range was employed to read the electronic spectra. The fluorescence experiment was
performed on the RF-5301PC spectrofluorometer (Shimadzu Corp., Kyoto, Japan) fitted
with a 1.0 cm quartz cell. Viscosity was determined with the Ubbelohde viscometer
(0.7–0.8-mm; Shenyi Glass Products Co. Ltd., Shanghai, China). In vitro cytotoxicity
assays and Western blot analyses were conducted on a multifunctional microplate reader

Molecules 2021, 26, 4028
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(Multiskan Spectrum, Thermo Fisher Scientific, Waltham, MA, USA). Cell cycle along with
apoptosis assays were performed in a CyAn-ADP flow cytometry platform (Beckman
Coulter, Brea, CA, USA).
2.2. Preparation
Complex 1 and complex 2 were prepared at high yield as depicted in Scheme 1.
Scheme 1. Synthetic routes of the ligand and complex 1 and complex 2.
2.2.1. 4-Chloro-3-Nitrobenzoic Acid (ncba) Synthesis
To a 100 mL three-port flask, 25.◦0 mL of 98% (w/v) concentrated H2SO4 was added
and the temperature was set to <15 C. Then, 0.1 mol p-chlorobenzoic acid was slowly
added with constant stirring. After the solid dissolved, the temperature was set to <15 ◦C,
20.0 mL of 68% (w/v) nitric acid was introduced, and the temperature was increased to
50–65 ◦C and maintained for five hours. Thereafter, the mixture was allowed to cool to
20–25 ◦C, poured into ice H2O, stirred, precipitated, filtered, and rinsed using water to
obtain the unrefined product. The latter was recrystallized with ethanol and dried to obtain
the finished product with the following physicochemical characteristics: melting point
◦
range, 180.0–182.0 C; 1H NMR (400 MHz, D2O):
δ 8.43 (s, 1H), 8.06 (d, J = 8.1 Hz, 1H), and
7.67 (d, J = 8.5 Hz, 1H).
2.2.2. [Cu(ncba)4(phen)] (Complex 1) Synthesis
A mixture of 0.45 mmol ncba (0.091 g) and 0.027 g (0.15 mmol) 1,10-phenanthroline
(phen) was dissolved in absolute methanol (10.0 mL) and a methanolic solution of copper(II)
nitrate trihydrate (0.024 g [0.10 mmol] in 15.0 mL) was introduced dropwise to the mixture.
We stirred the solution for 30 min and left it to evaporate in a beaker at 20–25 ◦C. After
several days, we collected well-formed blue crystals suitable for XRD and rinsed them with
cold methanol. The final product (C40H20Cl4CuN6O16) yield was 63.4%, and its molecular
weight was 1045.96. Its calculated and measured C, H, and N values were 45.93%, 1.93%,
and 8.03% and 46.15%, 2.18%, and 8.12%, respectively. Its infrared (IR) (KBr, cm−1) data
were v(=CH) 3438, v(C=O) 1695, v(C=C) 1541 and 1358, v(C=N) 1282, and v(C-N-C) 1047
and 776.
2.2.3. [Cu(ncba)4(bpy)] (Complex 2) Synthesis
Synthesis of complex
2
was performed in the same manner as complex
1
except 0.023 g
0
(0.15 mol) 2,2 -bipyridine (bpy) was used instead of 1,10-phenanthroline. The final product
(C38H20Cl4CuN6O16) yield was 52.5%, and its molecular weight was 1021.94. Its calculated
and measured C, H, and N values were 44.66%, 1.97%, and 8.22% and 44.64%, 2.32%, and

Molecules 2021, 26, 4028
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8.12%, respectively. Its IR (KBr, cm−1) data were v(=CH) 3426 and 2995, v(C=O) 1597,
v(C=C) 1455, v(C=N) 1316, and v(C-N-C) 1115, 758 and 621.
2.3. X-ray Structure Determination
Appropriate individual crystals of complex
1 and complex 2 were selected and
mounted onto thin glass fibers. We collected the data at 296(2) K in a Bruker Smart ApexII
CCD instrument (Bruker Corp., Karlsruhe, Germany) fitted with graphite monochromatic
Mo-Ko radiation (λ = 0.71073 Å). The absorption effects were semi-empirically calibrated.
Olex2 [33] was employed to solve the structures and refined with SHELXL-97 crystallo-
graphic software [34]. Non-hydrogen atoms were anisotropically refined. Hydrogen atoms
bound to carbons were placed in geometrical idealized positions, followed by refinement
with a riding model. All other hydrogens were localized in the final difference Fourier map.
The full-matrix least-squares refinement final cycle was based on the observed reflections
along with variable parameters. Crystallographic as well as structural refinement data for
complex
(◦) are listed in Table S1. CCDC 2013056 and CCDC 2013057 for complex
harbor supplementary crystallographic data were provided freely by the Cambridge
1
and complex
2
are listed in Table 1. Selected bond lengths (Å) as well as angles
1
and complex
2
Crystallographic Data Centre (Cambridge, UK).
Table 1. Crystallographic data for complex 1 and complex 2.
Complex
1
2
CCDC
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
a (Å)
2013056
C40H20Cl4CuN6O16
1045.96
2013057
C38H20Cl4CuN6O16
1021.94
296(2)
0.71073
Monoclinic
C2/c
21.8874(6)
296(2)
0.71073
Monoclinic
C2/c
21.7978(6)
13.7262(4)
13.8337(4)
b (Å)
14.1778(5)
14.0676(4)
c (Å)
◦
α ( )
90
90
◦
β ( )
101.336(3)
100.120(2)
◦
γ ( )
90
90
Volume (Å3)
4,176.4(2)
4,176.0(2)
Z
4
4
−3
1.664
0.862
1.625
0.860
Dcalcd. (Mg·m
)
−1
µ (mm
)
F (000)
2108
2060
Crystal size (mm)
0.31 × 0.25 × 0.14
1.90–25.00
−25 ≤ h ≤ 26, −15 ≤ k ≤ 16,
−16 ≤ l ≤ 16
16,512/3692
[Rint = 0.0223]
3692/0/311
1.104
0.24 × 0.16 × 0.14
1.90–25.00
−25 ≤ h ≤ 23, −16 ≤ k ≤ 15,
−16 ≤ l ≤ 15
14,725/3682
[Rint = 0.0191]
3682/0/301
1.081
◦
θ ( )
Limiting indices
Reflections collected/unique
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2sigma(I)]
R indices (all data)
R1 = 0.0385, wR2 = 0.1157
R1 = 0.0434, wR2 = 0.1240
R1 = 0.0420, wR2 = 0.1194
R1 = 0.0484, wR2 = 0.1253
Largest diff. peak and hole
0.758 and −0.618
0.669 and −0.414
(e/Å3)
2.4. DNA Binding Studies
Electronic absorption titration was employed to explore binding of DNA with metal
complexes. We carried out the absorption titration tests via introducing 80 L of 2 mM
CT-DNA to 2.5 mL of 50 M complex and 3.0 mL of 50 M complex . The solutions
µ
µ
1
µ
2

Molecules 2021, 26, 4028
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were kept at 20 ◦C for 5 min to ensure complete reaction. The UV-8000 spectrophotometer
(Yuanxi Corp., Shanghai, China) was used to measure the absorption spectra at 200–450 nm.
Fluorescence was measured using the RF-5301PC spectrofluorometer (Shimadzu
Corp., Kyoto, Japan) fitted with a 1 cm quartz cuvette. Fluorescence spectra of both
complexes were measured in the 500–700 nm range. The excitation wavelength was 565 nm
for complex
We conducted the scanning at 240 nm/min at a constant 4.0
CT-DNA. Three milliliters buffer solution was used for complex
1
and 491 nm for complex
2
. The excitation/emission slits were set to 5 nm.
M EB and a constant 5.0
, and 2.5 mL buffer
µ
µM
1
solution was used for complex 2. The quantities of complex 1 or complex 2 (20 µL; 1 mM)
were increased several folds. The complex-DNA solutions were left to stand 5 min before
each measurement.
CT-DNA viscosity was measured in the Ubbelohde viscometer (0.7–0.8-mm; Shenyi
Glass Products Co. Ltd., Shanghai, China) with and without complex
EB. The samples were maintained at 29.0
0.1 ◦C, and a digital electronic stopwatch was
employed to measure the flow time. Viscosity was calculated based on the DNA-containing
1 or complex 2 or
±
solution flow times corrected for buffer flow time (t0),
η
= (t
−
t0)/t0. Data were reported as
1/3
(
η
/
η0
)
relative to (Ccomplex/CDNA), where η is the DNA viscosity in the presence of the
complex, η0 is the DNA viscosity, Ccomplex is the quantity of the complex, and CDNA is the
DNA quantity. The latter was maintained at a constant 200 M, and the complex quantities
µ
were varied to create [complex]/[DNA] ratios in the 0.00–0.30 range. The average time was
computed from triplicate assessments.
2.5. HSA Binding
Absorption titration was performed by adding 5.0
µL of 0.30
µ
M complex to 3.0 mL
1
of 200
µ
M HSA and by adding 50
µ
L◦of 1.2 mM complex
2
to 2.5 mL of 200 µM HSA.
The solutions were left to stand at 20 C for 5 min for complete reaction. The UV-8000
spectrophotometer (Yuanxi Corp., Shanghai, China) was employed to record the absorption
in the 200–400 nm range.
Fluorescence spectra were measured from 250–500 nm at an excitation wavelength of
280 nm. Both the excitation and emission slits were set to 10 nm. Scanning was conducted
at 240 nm/min and a constant 5.0
was used for complex , and 2.5 mL buffer solution was used for complex
of complex (10 L, 1.2 mM), along with complex (2 L, 1.2 mM) were increased several
µM HSA quantity. An amount of 3 mL of buffer solution
1
2. The quantities
1
µ
2
µ
folds. Complex–HSA solutions were left to stand for 5 min before each measurement.
2.6. In Vitro Cytotoxicity Assays
The cytotoxicity levels of complex 1 and complex 2 against HepG2, HeLa, and A549
cells were determined with a CCK-8 kit (Biosharp, Beijing, China) according to the manu-
facturer’s instructions. Briefly, log-phase HepG2, HeLa, and A549 cells were harvested,
dissociated, and inoculated in triplicate on 96-well plates at 5
×
103/well. The cells were
then allowed to grow overnight at 37 ◦C under a 5% CO2/95% O2 atmosphere. Cells with
or without the copper(II) complex were allowed to grow for 24 h, 48 h, 72 h, or 96 h and
then inoculated in 10 µL CCK-8 + 90 µL medium in 10% (v/v) FBS in the dark for 3 h.
Absorbance was read at 450 nm in a Multiskan Spectrum multifunctional microplate reader
(Thermo Fisher Scientific, Waltham, MA, USA).
2.7. Cell Cycle Assays
Anticancer agents interfere with various cell cycle phases. The cancer cell cycle
may help elucidate the DNA-binding copper(II) complex mechanisms. HepG2 cells were
◦
inoculated onto six-well plates at 1
×
105/well at 37 C overnight under a 5% CO2/95% O2
conditions. The cells were left untreated or subjected to complex 1 or complex 2 for 24 h,
trypsinized, rinsed with cold PBS (phosphate-buffered saline, Hyclone Laboratories Inc.,
Logan, UT, USA), fixed using 70% (v/v) ethanol, and kept at 4 ◦C. Afterwards, the pellets
were washed twice with 1.0 mL PBS. Then, 10.0 mL of 20 µg/mL RNase (Sigma-Aldrich

Molecules 2021, 26, 4028
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Corp., Shanghai, China) and 10
µL of 50
µg/mL PI (propidium iodide, Sigma-Aldrich
Corp., Shanghai, China) were introduced, and the cell suspensions allowed to grow for
◦
0.5 h at 37 C. The samples were then analyzed with a Beckman CyAn-ADP flow cytometry
platform (Beckman Coulter, Brea, CA, USA). Ten thousand cells were analyzed per sample.
A similar procedure was used to assay the HepG2 cell cycles after 48 h treatment with
complex 1 or complex 2.
2.8. Cell Apoptosis Assay
Cell culture was performed as described for the cell cycle assay. The cells were left
untreated or subjected to complex
rinsing twice with 1.0 mL PBS. One hundred microliters of binding buffer, 5
1
or complex
2
for 24 h, and then harvested, followed by
L Annexin
µL PI were introduced to the cell suspensions, and the latter were
µ
V-FITC along with 5
◦
incubated at 25 C in the dark for 15 min. Fluorescence was measured at 488 nm excitation
in a Beckman CyAn-ADP flow cytometry platform (Beckman Coulter, Brea, CA, USA). Ten
thousand cells were analyzed per sample. A similar procedure was used to assay HepG2
cell apoptosis after 48 h treatment with complex 1 or complex 2.
2.9. Western Blot
HepG2 cells were lysed, and their protein contents were purified with RIPA lysis
buffer (Beyotime, Shanghai, China) in 1% (w/v) phenylmethanesulfonyl fluoride (PMSF;
Sigma-Aldrich Corp., Shanghai, China). Equivalent amounts of protein from each extract
were fractionated on an SDS-PAGE gel and transfer-embedded onto PVDF membranes
(EMD Millipore, Billerica, MA, USA). The membranes were then inoculated overnight
with primary antibodies at 4 ◦C and rinsed thrice with Tris-buffered saline-Tween (TBST).
The primary antibodies consisted of anti-caspase-3 (Abcam, Cambridge, UK; 1:1000), anti-
Bcl-2 (Cell Signaling Technology, Danvers, MA, USA; 1:1000), anti-Bax (Cell Signaling
Technology, Danvers, MA, USA; 1:1000), and anti-GAPDH (Cell Signaling Technology,
Danvers, MA, USA; 1:5000) diluted in TBST. Goat immunoglobulin G (IgG) labeled with
HRP (MultiSciences, Hangzhou, China; 1:2000) was introduced, and the membranes were
◦
incubated at 20 C for 1 h. ECL detection reagent (Thermo Fisher Scientific, Waltham, MA,
USA) was applied to detect the signals.
3. Results and Discussion
3.1. Synthesis and Characterization
Here, we prepared two new copper(II) complexes using 4-chloro-3-nitrobenzoic acid
ligand. Both are stable in air and soluble in dimethylformamide (DMF). Single-crystal X-ray
analysis illustrated that complex
1 and complex 2 crystallized in the monoclinic crystal
system with the C2/c space group (Figure 1). The Cu(II) ions in both complexes were
hexacoordinated by four oxygen (O) atoms from the 4-chloro-3-nitrobenzoic acid ligand for
complex
1 and by two nitrogen (N) atoms from phen and 2,2’-bpy for complex 2. Table S1
shows that the Cu(II)-O and Cu(II)-N distances were in the ranges of 1.9354(17)–1.9380(18)
Å and 1.999(2)–2.010(2) Å, respectively. The O/N-Cu-O/N bond angles were in the ranges
of 81.90(11)–168.87(7)◦ for complex 1 and 81.25(13)–169.97(8)◦ for complex 2.

Molecules 2021, 26, 4028
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Figure 1. Crystalline structures of Cu(II) complex 1 (a) and Cu(II) complex 2 (b).
3.2. DNA Binding Experiments
3.2.1. Electronic Absorption
Electronic absorption spectroscopy reveals the binding characteristics of the metal com-
plexes with DNA [35]. Intercalative binding of a complex to DNA causes hypochromism
and redshift due to the remarkable stacking intercalation of the aromatic chromophore of
the ligand with the base pairs of DNA. The magnitude of the hypochromism is proportional
to the intercalative binding strength [36]. Figure 2 indicates the absorption spectra of the
complexes with and without CT-DNA. The absorption band intensities exhibited negligible
redshift, 23.90% hypochromism for complex 2, and 35.83% hypochromism for complex 1,
in response to increasing DNA quantity. Hence, intercalative binding occurred between
the DNA and each complex. The intrinsic binding constant (Kb) was computed as shown
in Equation (1) below [37]:
[DNA]/(εa − εf) = [DNA]/[(εb − εf) + 1/Kb(εb − εf)]
(1)
where εa designates the apparent complex extinction coefficient, εf designates free complex
extinction coefficient, εb designates the bound complex extinction coefficient, and [DNA]
designates the CT-DNA quantity. In the [DNA]/(εa − εf) vs. [DNA] plot, Kb is the slope:
−1
−1
103 M
intercept ratio. The calculated binding constants were 1.20
×
104 M and 5.06
×
for complex 1 and complex 2, respectively. Complex 1 had larger binding constants and
greater hypochromism than complex 2. The calculated intrinsic equilibrium binding
constants (Kb) (103–105 M−1) were consistent with literature values [38]. Hence, there was
strong affinity between the complexes and the DNA.
3.2.2. Fluorescence Spectroscopy
EB complexes with DNA and strongly fluoresces because of the interaction of the
planar phenanthridine rings with the neighboring DNA base pairs. A competitive reaction
takes place when the complex co-exists with EB-DNA. The EB complex dissociates from
the DNA double helix and decreases fluorescence. Therefore, EB is a fluorescent probe for
the DNA [39].
Figure 3 illustrates the alterations in the fluorescence emission spectra of the complexes
with CT-DNA. Emission intensity decreased in response to the addition of complex
1, as
well as complex . The magnitude of quenching resembled that of externally binding
2
molecules [40]. Complex fluorescence quenching by CT-DNA is quantitated by the classical
Stern–Volmer Equation (2) [41]:
F0/F = 1 + Kqτ0[C] = 1 + Ksv[C]
(2)

Molecules 2021, 26, 4028
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where F0 designates the intensity of fluorescence without the quencher, and F designates
the intensity of fluorescence with the quencher: τ0 (~10−8 s) is the fluorophore lifespan, [C]
is the quencher quantity, Ksv designates the Stern–Volmer quenching, and Kq designates
the quenching rate constant. The F0/F vs. [C] plot is linear, and Ksv is calculated from its
slope. The quenching constants (Ksv) for complex 1−a1nd complex
2
were 5.49
×
103 M−1
−1 −1
.
and 3.79
×
103 M−1, respectively. Kq1 = 5.49
×
1011
M
·
s
−1 and Kq2 = 3.79
×
1011
M
·
s
Therefore, quenching efficiency was high [42]. The foregoing Kq we−re1 markedly higher
relative to the biomolecule limiting diffusion constant Kdif (2.0
reason, the fluorescence quenching was probably static [44]. In that case, the relationship
×
10l0
M
·
s
−1) [43]. For this
of the intensity of fluorescence with [C] is described by Equation (3) [45,46]:
log[(F0 − F)/F] = log Ka + n log[C]
(3)
where n designates the number of binding sites, and Ka designates the binding constant.
Figure 4 shows that the Ka were acquire−d1from the intercepts and slopes of the log[(F0
respectively, for complex 1 and complex 2.
−
−1
F)/F] vs. log[C] plots. Ka = 1.30
×
104 M and 6.99
×
103 M while n = 1.0984 and 1.0716,
Figure 2. The electronic absorption spectra of complex
1 (a) and complex 2 (b) with or without CT-DNA. Arrows indicate
changes in absorbance with escalating DNA quantity. Inset: [DNA]/(εa − εf) vs. DNA] plots.
Figure 3. EB-DNA emission spectra in the absence or the presence of complex
1 (a) and complex 2 (b). Inset: F0/F vs. [C]
plots. Arrows indicate the changes in intensity of fluorescence with escalating complex quantity.

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Figure 4. Double-log plots of DNA fluorescence quenching by complex 1 (a) and complex 2 (b).
3.2.3. Viscosity Measurements
We evaluated the changes DNA solution viscosity upon the addition of the complexes
and the EB to clarify the interactions between CT-DNA and the complexes. The viscosity
was measured to assess the complex–DNA binding mode because DNA viscosity increases
with DNA length [47]. Intercalation elongates the DNA helices and increases DNA viscosity.
The base pairs are disassociated to accommodate the binding ligands. On the contrary, non-
classical or partial intercalation bends the DNA helices and lowers DNA viscosity. Neither
grooving nor electrostatic binding affects DNA viscosity [48]. Figure 5 shows that DNA
viscosity increases with the [complex]/[DNA] ratio. Therefore, EB and both complexes
bind intercalatively to DNA, and this finding was corroborated by the absorption and
emission spectra.
Figure 5. Impacts of increasing complex
viscosity at 29.0 ± 0.1 C.
1/complex
2
numbers, as well as EB on relative DNA
◦
3.3. HSA Binding Experiments
The procedure used to study the interactions between HSA and the complexes was
the same as that used to evaluate the interactions between DNA and the complexes. In the
former case, however, no fluorophore (EB) was required. The absorption bond intensities
became hypochromic with increasing complex
show that the quenching constants (Ksv) were 1.06
105 M−1 for complex 1013 M−1
1.45 . Kq1 = 1.06
103 M−1 while n were 1.42 and 0.60 for complex
1
1
and complex
105 for complex
−1 and Kq2 = 1.45
2
quantity. Figures 6 and 7
, as well as
1013 M−1 s−1
and for
×
1
×
2
×
×
·
s
×
·
.
Ka were 6.63
complex 2.
×
106 and 1.84

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Figure 6. HSA electronic absorption spectra with and without complex
1 (a). HSA platform emission spectra with and
without complex 1 (b). Inset: F0/F vs. [C] plot. Arrows illustrate the intensity changes with increasing complex quantity.
Figure 7. HSA electronic absorption spectra with and without complex
2 (a). HSA platform emission spectra with and
without complex
the complex.
2
(b). Inset: F0/F vs. [C] plot. Arrows illustrate the changes in intensity with increasing quantity of
3.4. Cytotoxicity Assay In Vitro
Complex cytotoxicity was tested on A549, HeLa, and HepG2 cell lines by the CCK-8
cell survival assay. A549, HeLa, and HepG2 were subjected to various complex
complex quantities for 24 h, 48 h, 72 h, as well as 96 h. Complex had IC50 = 8.99
11.32 M, and 12.19 M against HeLa, A549, and HepG2 cells, respectively, after 48 h
(Figure 8a). Figure 9a shows that complex had IC50 = 24.46 M, 35.92 M, and 44.85
against HepG2, HeLa, and A549 cells, respectively. The IC50 of complex and complex
against A549, HeLa, as well as HepG2 cells at various time points are listed in Table 2.
Both complexes displayed strong antiproliferative influence against A549, HeLa, as well
as HepG2 cells. However, complex had stronger activity against all three cell lines than
cisplatin and exhibited the highest activity against HeLa cells at 48 h. Cell viability was
1
and
2
1
µM,
µ
µ
2
µ
µ
µM
1
2
1
both dose- and time-dependent. Hence, complex
the cells, inducing cell death.
1 and complex 2 gradually penetrated

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Figure 8.
48 h exposure. (
and proliferation.
(
a
) Effects of complex
1
on HepG2, HeLa, and A549 cell viability and proliferation after
b–d) Time course of effects of complex 1 on A549, HeLa, and HepG2 cell viability
Figure 9.
48 h exposure. (
and proliferation.
(
a
) Effects of complex
2
on HepG2, HeLa, and A549 cell viability and proliferation after
on A549, HeLa, and HepG2 cell viability
b
–d
) Time course of effects of complex 2

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Table 2. IC50 of complex 1 and complex 2 against A549, HeLa, and HepG2 at various time points.
IC50 (µM)
Sample
t (h)
A549
HeLa
HepG2
24
48
72
96
24
48
72
96
48
48
41.29 ± 1.03
11.60 ± 0.94
6.91 ± 0.82
4.88 ± 0.79
145.01 ± 0.81
45.03 ± 1.02
34.59 ± 1.01
26.08 ± 0.90
>10,000
28.50 ± 1.22
9.93 ± 1.36
8.00 ± 1.29
5.89 ± 1.81
92.16 ± 0.91
37.58 ± 0.85
29.96 ± 0.77
22.14 ± 0.68
>10,000
31.40 ± 0.63
11.86 ± 0.78
7.89 ± 0.87
6.46 ± 0.87
58.25 ± 1.02
27.99 ± 0.94
23.59 ± 0.80
17.97 ± 0.83
>1000
1
2
ncba
cisplatin
36.50 ± 1.76
37.37 ± 2.01
43.81 ± 1.62
3.5. Cell Cycle Arrest
Cell cycle arrest plays an indispensable role in cancer cell apoptosis [49]. We used
flow cytometry and PI staining to analyze HepG2 distribution at various cell stages.
Figures 10 and 11 show that 52.45% and 51.32% of all HepG2 cells were at G0/G1 at
24 h and 48 h, respectively. After inoculating HepG2 cells with complex
for 24 h or 48 h, the proportions of HepG2 cells in the G0/G1 phase escalated, while
the proportions of HepG2 cells in G2/M and S decreased. In the control, complex and
complex induced arrest of G0/G1 phase in HepG2 cells, and the fraction of HepG2 cells
at G0/G1 was dose- and time-dependent.
1 and complex
2
1
2
Figure 10. HepG2 cell cycle distributions after exposure to 5 µM, as well as 20 µM complex 1 for 24 h and 48 h.

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Figure 11. HepG2 cell cycle distributions after exposure to 5 µM and 20 µM complex 2 for 24 h and 48 h.
3.6. Induced Cell Apoptosis
Morphological apoptosis studies documented that both complexes triggered apoptosis
in HepG2 cells. Apoptosis was evaluated by flow cytometry (Figures 12 and 13). Untreated
HepG2 cells were employed as the control. After the HepG2 cells were inoculated with
5
µ
M, as well as 20
cells were 16.5% and 45.1% for complex
In the control, the proportions of living and apoptotic or necrotic cells were 99.6% and
0.4%, respectively. After the HepG2 cells were inoculated with 5 M and 20 M of each
complex for 48 h, the proportions of apoptotic or necrotic cells were 33.6% and 71.3% for
complex and 18.3% and 62.5% for complex , respectively. In the control, the proportions
µ
M of each complex for 24 h, the proportions of apoptotic or necrotic
1
and 7.1% and 43.2% for complex , respectively.
2
µ
µ
1
2
of living and apoptotic cells were 99.5% and 0.5%, respectively. Relative to the control, the
proportion of living cells had decreased, and the fraction of apoptotic or necrotic cells had
increased in the complex treatments. Hence, the effects of the complexes on apoptosis and
necrosis in HepG2 cells were dose- and time-dependent.

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Figure 12. HepG2 cells inoculated with 0 µM, 5 µM, or 20 µM of complex 1 for 24 h or 48 h.
Figure 13. HepG2 cells inoculated with 0 µM, 5 µM, or 20 µM complex 2 for 24 h or 48 h.

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3.7. Caspase and Bcl-2 Protein Family Expression
Figure 14 shows that when the HepG2 cells were inoculated with 5 µM or 10 µM
complex
1
or complex
2
for 48 h, caspase 3 was upregulated compared with the control.
Moreover, complex
1
and complex 2 decreased the expression of the antiapoptotic protein
Bcl-2 and elevated the expression of the proapoptotic protein Bax. Therefore, complex
1
and complex 2 trigger apoptosis by modulating Bcl-2 protein family expression.
Figure 14. Western blot of Bax, caspase 3, and Bcl-2 in HepG2 cells inoculated with 5
complex 1 or complex 2. GAPDH was the internal control.
µM or 10 µM
4. Conclusions
Here, two novel copper(II) complexes were synthesized using 4-chloro-3-nitrobenzoic
acid, phen, and bpy as the ligands. Single-crystal XRD showed that both complexes
were monoclinic crystals with a C2/c space groups and hexacoordinated copper(II) center
ions. We investigated their interactions with DNA/HSA via electronic absorption assays,
fluorescence spectroscopy, and viscosity tests. Electronic absorption revealed that both
copper(II) complexes intercalatively bound DNA, but complex
constant than complex . Complex and complex statically and spontaneously quenched
DNA/HSA fluorescence. In vitro cytotoxicity assays demonstrated that complex was
relatively more cytotoxic to HepG2, HeLa, and A549 cancer cells than complex or cisplatin.
1 had a higher binding
2
1
2
1
2
This finding was concordant with the binding strength of the complexes with DNA and
suggests that their anticancer activity might be associated with their potential to bind DNA.
Both complex 1 and complex 2 caused HepG2 cell cycle arrest and induced G0/G1. The
Annexin V-FITC/PI apoptosis assay showed that both complexes induced apoptosis or
necrosis in cancer cells by modulating antiapoptotic Bcl-2 protein family expression. This
study evidences the foregoing copper(II) complexes as potential anticancer drugs, which
could serve in the rational development of innovative copper(II)-based antitumor agents.
1
Supplementary Materials: The following are available online. Figure S1: H NMRspectrum of cnba,
Figure S2: IR spectrum of the complex 1, Figure S3: IR spectrum of the complex 2, Table S1: Bond
lengths and bond angles parameters for complexes 1 and 2.
Author Contributions: Writing—original draft preparation, Z.-F.Z.; methodology, Q.-P.H.; data
curation, J.-H.C., G.-J.Z. and Q.-C.H.; investigation, Z.-L.L.; writing—review and editing, Z.-L.C.;
funding acquisition, Y.-H.W. All authors have read and agreed to the published version of the
manuscript.
Funding: This research was funded by the National Natural Science Foundation of China, grant
number 21661009; the Guangxi Natural Science Foundation, grant number 2016GXNSFAA380113;
the Guangxi Universities Research Foundation of Young and Middle-Aged Teachers Foundation,
grant numbers 2017KY0846 and 2019KY0769; the Guangxi Normal University for Nationalities
Foundation, grant numbers 327003070414, 327003070416, 2019YB018, and 2020GP008; and the State

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Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources of Guangxi Normal
University, grant numbers CMEMR2018-C8 and CMEMR2017-A11.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: CCDC 2013056 and 2013057 contain the supplementary crystallo-
graphic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/
data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge
Crystallographic Data Centre.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of Complex 1 and complex 2 are available from the authors.
Abbreviations
CHNS
EB
carbon hydrogen nitrogen sulfur
ethidium bromide
ncba
phen
bpy
4-chloro-3-nitrobenzoic acid
1,10-phenanthroline
2,2 -bipyridine
0
CCK-8
cell counting kit 8
CT-DNA calf thymus DNA
HeLa
HepG2
HSA
A549
Bax
BSA
Bcl-2
PBS
human cervical carcinoma
human hepatocellular carcinoma
human serum albumin
human lung carcinoma
Bcl-2-associated X protein
bovine serum albumin
B cell lymphoma 2
phosphate-buffered saline
propidium iodide
PI
References
1.
Mosteiro, L.; Pantoja, C.; Alcazar, N.; Marión, R.M.; Chondronasiou, D.; Rovira, M.; Fernandez-Marcos, P.J.; Muñoz-Martin, M.;
Blanco-Aparicio, C.; Pastor, J.; et al. Tissue damage and senescence provide critical signals for cellular reprogramming in vivo
.
Science 2016, 354, aaf4445. [CrossRef]
2.
3.
Hanahan, D.; Weinberg, R.A. Hallmarks of Cancer: The Next Generation. Cell 2011, 144, 646–674. [CrossRef]
Muggia, F.M.; Bonetti, A.; Hoeschele, J.D.; Rozencweig, M.; Howell, S.B. Platinum Antitumor Complexes: 50 Years Since Barnett
Rosenberg’s Discovery. J. Clin. Oncol. 2015, 33, 4219–4226. [CrossRef] [PubMed]
4.
5.
Dasari, S.; Tchounwou, P.B. Cisplatin in cancer therapy: Molecular mechanisms of action. Eur. J. Pharmacol. 2014, 740, 364–378.
[CrossRef] [PubMed]
Westendorf, A.F.; Woods, J.A.; Korpis, K.; Farrer, N.J.; Salassa, L.; Robinson, K.; Appleyard, V.; Murray, K.; Grünert, R.; Thompson,
A.M.; et al. Trans, trans, trans-[PtIV(N3)2(OH)2(py)(NH3)]: A Light-Activated Antitumor Platinum Complex That Kills Human
Cancer Cells by an Apoptosis-Independent Mechanism. Mol. Cancer Ther. 2012, 11, 1894–1904. [CrossRef] [PubMed]
Ayres, L.S.; Berger, M.; Durli, I.C.L.D.O.; Kuhl, C.P.; Terraciano, P.B.; Garcez, T.N.A.; dos Santos, B.G.; Guimarães, J.A.; Passos,
E.P.; Cirne-Lima, E.O. Kallikrein-kinin system and oxidative stress in cisplatin-induced ovarian toxicity. Reprod. Toxicol. 2020, 93,
1–9. [CrossRef]
6.
7.
8.
9.
Mok, T.; Wu, Y.; Thongprasert, S.; Yang, C.; Chu, D.T.; Saijo, N.; Sunpaweravong, P.; Han, B.; Margono, B.; Ichinose, Y. Gefitinib or
carboplatin–paclitaxel in pulmonary adenocarcinoma. N. Engl. J. Med. 2009, 361, 947–957. [CrossRef] [PubMed]
Cunningham, D.; Starling, N.; Rao, S.; Iveson, T.; Nicolson, M.; Coxon, F.; Middleton, G.; Daniel, F.; Oates, J.; Norman, A.R.
Capecitabine and Oxaliplatin for Advanced Esophagogastric Cancer. N. Engl. J. Med. 2008, 358, 36–46. [CrossRef]
Meng, T.; Qin, Q.-P.; Chen, Z.-L.; Zou, H.-H.; Wang, K.; Liang, F.-P. Cyclometalated Ir(III)-8-oxychinolin complexes acting as
red-colored probes for specific mitochondrial imaging and anticancer drugs. Eur. J. Med. Chem. 2020, 192, 112192. [CrossRef]
10. Meng, T.; Qin, Q.-P.; Chen, Z.-L.; Zou, H.-H.; Wang, K.; Liang, F.-P. High in vitro and in vivo antitumor activities of Ln(III)
complexes with mixed 5,7-dichloro-2-methyl-8-quinolinol and 4,4’-dimethyl-2,2’-bipyridyl chelating ligands. Eur. J. Med. Chem.
2019, 169, 103–110. [CrossRef] [PubMed]

Molecules 2021, 26, 4028
17 of 18
11. Zhang, D.-Y.; Zheng, Y.; Zhang, H.; Sun, J.-H.; Tan, C.-P.; He, L.; Zhang, W.; Ji, L.-N.; Mao, Z.-W. Delivery of Phosphorescent
Anticancer Iridium(III) Complexes by Polydopamine Nanoparticles for Targeted Combined Photothermal-Chemotherapy and
Thermal/Photoacoustic/Lifetime Imaging. Adv. Sci. 2018, 5, 1800581. [CrossRef]
12. S¸en, B.; Kalhan, H.K.; Demir, V.; Güler, E.E.; Kayalı, H.A.; Subas¸ı, E. Crystal structures, spectroscopic properties of new
cobalt(II), nickel(II), zinc(II) and palladium(II) complexes derived from 2-acetyl-5-chloro thiophene thiosemicarbazone: Anticancer
evaluation. Mater. Sci. Eng. C 2019, 98, 550–559. [CrossRef]
13. Mu, C.; Prosser, K.E.; Harrypersad, S.; MacNeil, G.A.; Panchmatia, R.; Thompson, J.R.; Sinha, S.; Warren, J.J.; Walsby, C.J.
Activation by Oxidation: Ferrocene-Functionalized Ru(II)-Arene Complexes with Anticancer, Antibacterial, and Antioxidant
Properties. Inorg. Chem. 2018, 57, 15247–15261. [CrossRef]
14. Khan, T.; Azad, I.; Ahmad, R.; Raza, S.; Dixit, S.; Joshi, S.; Khan, A.R. Synthesis, characterization, computational studies and
biological activity evaluation of Cu, Fe, Co and Zn complexes with 2-butanone thiosemicarbazone and 1,10-phenanthroline
ligands as anticancer and antibacterial agents. EXCLI J. 2018, 17, 331–348. [CrossRef]
15. De Camargo, M.S.; De Grandis, R.A.; Da Silva, M.M.; Da Silva, P.B.; Santoni, M.M.; Eismann, C.E.; Menegário, A.A.; Cominetti,
M.R.; Zanelli, C.; Pavan, F.R.; et al. Determination of in vitro absorption in Caco-2 monolayers of anticancer Ru(II)-based
complexes acting as dual human topoisomerase and PARP inhibitors. BioMetals 2018, 32, 89–100. [CrossRef] [PubMed]
16. Cao, J.-J.; Zheng, Y.; Wu, X.-W.; Tan, C.-P.; Chen, M.-H.; Wu, N.; Ji, L.-N.; Mao, Z.-W. Anticancer Cyclometalated Iridium(III)
Complexes with Planar Ligands: Mitochondrial DNA Damage and Metabolism Disturbance. J. Med. Chem. 2019, 62, 3311–3322.
[CrossRef] [PubMed]
17. Yaqub, A.; Malkani, N.; Shabbir, A.; Ditta, S.A.; Tanvir, F.; Ali, S.; Naz, M.; Kazmi, S.A.R.; Ullah, R. Novel Biosynthesis of Copper
Nanoparticles Using Zingiber and Allium sp. with Synergic Effect of Doxycycline for Anticancer and Bactericidal Activity. Curr.
Microbiol. 2020, 77, 2287–2299. [CrossRef] [PubMed]
18. Yang, P.; Zhang, D.-D.; Wang, Z.-Z.; Liu, H.-Z.; Shi, Q.-S.; Xie, X.-B. Copper(ii) complexes with NNO ligands: Synthesis, crystal
structures, DNA cleavage, and anticancer activities. Dalton Trans. 2019, 48, 17925–17935. [CrossRef] [PubMed]
19. Pilon, A.; Lorenzo, J.; Rodriguez-Calado, S.; Adao, P.; Martins, A.M.; Valente, A.; Alves, L.G. New Cyclams and Their Copper(II)
and Iron(III) Complexes: Synthesis and Potential Application as Anticancer Agents. ChemMedChem 2019, 14, 770–778. [CrossRef]
20. Medina, J.J.M.; Naso, L.G.; Pérez, A.L.; Rizzi, A.; Okulik, N.B.; Valcarcel, M.; Salado, C.; Ferrer, E.G.; Williams, P.A. Synthesis,
characterization, theoretical studies and biological (antioxidant, anticancer, toxicity and neuroprotective) determinations of a
copper(II) complex with 5-hydroxytryptophan. Biomed. Pharmacother. 2019, 111, 414–426. [CrossRef]
21. Zafar, A.; Singh, S.; Ahmad, S.; Khan, S.; Siddiqi, M.I.; Naseem, I. Interaction of C20-substituted derivative of pregnenolone
acetate with copper(II) leads to ROS generation, DNA cleavage and apoptosis in cervical cancer cells: Therapeutic potential of
copper chelation for cancer treatment. Bioorganic Chem. 2019, 87, 276–290. [CrossRef] [PubMed]
22. Kallus, S.; Uhlik, L.; van Schoonhoven, S.; Pelivan, K.; Berger, W.; Enyedy, É.A.; Hofmann, T.; Heffeter, P.; Kowol, C.R.; Keppler,
B.K. Synthesis and biological evaluation of biotin-conjugated anticancer thiosemicarbazones and their iron(III) and copper(II)
complexes. J. Inorg. Biochem. 2019, 190, 85–97. [CrossRef]
23. Zhu, K.; Chen, L.; Jin, X.; Qu, C. Two Cu(II) coordination polymers based on benzene-1,3,5-tricarboxylate and 1,2,4-triazolide
ligands: Their crystal structures and application of nanoparticles in anti-esophageal cancer activity evaluation. Struct. Chem.
2019, 30, 1485–1494. [CrossRef]
24. Ng, P.Y.; Chye, S.M.; Tiong, Y.L.; Chan, C.W.; Tan, K.W.; Ooi, I.H.; Ng, C.H. Enantiomeric pairs of copper(II) polypyridyl-alanine
complex salts: Anticancer studies. Transit. Met. Chem. 2018, 43, 479–496. [CrossRef]
25. Deng, J.; Yu, P.; Zhang, Z.; Wang, J.; Cai, J.; Wu, N.; Sun, H.; Liang, H.; Yang, F. Designing anticancer copper(II) complexes by
optimizing 2-pyridine-thiosemicarbazone ligands. Eur. J. Med. Chem. 2018, 158, 442–452. [CrossRef] [PubMed]
26. Milunovic´, M.N.M.; Palamarciuc, O.; Sirbu, A.; Shova, S.; Dumitrescu, D.; Dvoranová, D.; Rapta, P.; Petrasheuskaya, T.V.; Enyedy,
E.A.; Spengler, G.; et al. Insight into the Anticancer Activity of Copper(II) 5-Methylenetrimethylammonium-Thiosemicarbazonates
and Their Interaction with Organic Cation Transporters. Biomolecules 2020, 10, 1213. [CrossRef] [PubMed]
27. Kettenmann, S.D.; Nossol, Y.; Louka, F.R.; LeGrande, J.R.; Marine, E.; Fischer, R.C.; Mautner, F.A.; Hergl, V.; Kulak, N.; Massoud,
S.S. Copper(II) Complexes with Tetradentate Piperazine-Based Ligands: DNA Cleavage and Cytotoxicity. Inorganics 2021, 9, 12.
[CrossRef]
28. Padnya, P.; Shibaeva, K.; Arsenyev, M.; Baryshnikova, S.; Terenteva, O.; Shiabiev, I.; Khannanov, A.; Boldyrev, A.; Gerasimov, A.;
Grishaev, D.; et al. Catechol-Containing Schiff Bases on Thiacalixarene: Synthesis, Copper (II) Recognition, and Formation of
Organic-Inorganic Copper-Based Materials. Molecules 2021, 26, 2334. [CrossRef]
29. Özgül, G.; Tas¸temel, A.; Özkaya, A.R.; Bulut, M. Synthesis, characterization and comparative electrochemistry of beta and
alpha tetra-[4-oxy-3-methoxybenzoic acid]-substituted Zn(II), Co(II) and Cu(II) phthalocyanines. Polyhedron 2015, 85, 181–189.
[CrossRef]
30. Shmelev, M.A.; Gogoleva, N.V.; Kuznetsova, G.N.; Kiskin, M.A.; Voronina, Y.K.; Yakushev, I.A.; Ivanova, T.M.; Nelyubina, Y.V.;
Sidorov, A.A.; Eremenko, I. Cd(II) and Cd(II)–Eu(III) Complexes with Pentafluorobenzoic Acid Anions and N-Donor Ligands:
Synthesis and Structures. Russ. J. Coord. Chem. 2020, 46, 557–572. [CrossRef]
31. Zhang, C.-L.; Zhang, X.-M.; Liu, W.; Chen, S.; Mao, Z.-W.; Le, X.-Y. Synthesis, crystal structures and DNA/human serum albumin
binding of ternary Cu(II) complexes containing amino acids and 6-(pyrazin-2-yl)-1,3,5-triazine-2,4-diamino. Appl. Organomet.
Chem. 2017, 32, e3994. [CrossRef]

Molecules 2021, 26, 4028
18 of 18
32. Zhang, C.-L.; Zhang, X.-M.; Liu, W.; Chen, S.; Le, X.-Y. A copper(II) complex of 6-(pyrazin-2-yl)-1,3,5-triazine-2,4-diamine and
L-serinate: Synthesis, crystal structure, DNA-binding and molecular docking studies. Transit. Met. Chem. 2018, 43, 201–209.
[CrossRef]
33. Dolomanov, O.V.; Bourhis, L.J.; Gildea, R.J.; Howard, J.A.K.; Puschmann, H. OLEX2: A complete structure solution, refinement
and analysis program. J. Appl. Cryst. 2009, 42, 339–341. [CrossRef]
34. Sheldrick, G.M. A short history of SHELX. Acta Crystallogr. Sect. A Found. Crystallogr. 2008, 64, 112–122. [CrossRef]
35. Ding, P.; Wang, Y.; Kou, H.; Li, J.; Shi, B. Synthesis of heterobinuclear Cu(II)-Ni(II) complex: Structure, CT-DNA interaction,
hydrolytic function and antibacterial studies. J. Mol. Struct. 2019, 1196, 836–843. [CrossRef]
36. Chao, H.; Mei, W.-J.; Huang, Q.-W.; Ji, L.-N. DNA binding studies of ruthenium(II) complexes containing asymmetric tridentate
ligands. J. Inorg. Biochem. 2002, 92, 165–170. [CrossRef]
37. Na, N.; Zhao, D.-Q.; Li, H.; Jiang, N.; Wen, J.-Y.; Liu, H.-Y. DNA Binding, Photonuclease Activity and Human Serum Albumin
Interaction of a Water-Soluble Freebase Carboxyl Corrole. Molecules 2015, 21, 54. [CrossRef] [PubMed]
38. Draksharapu, A.; Boersma, A.J.; Leising, M.; Meetsma, A.; Browne, W.R.; Roelfes, G. Binding of copper(ii) polypyridyl complexes
to DNA and consequences for DNA-based asymmetric catalysis. Dalton Trans. 2014, 44, 3647–3655. [CrossRef] [PubMed]
39. Jiang, W.; Zhou, Q.; Liu, M.; Zhang, F.; Kuang, D.; Tan, Y. Microwave assisted synthesis of disubstituted benzyltin arylformylhy-
drazone complexes: Anticancer activity and DNA-binding properties. Appl. Organomet. Chem. 2019, 33, e5092. [CrossRef]
40. Huang, J.-T.; Wang, X.-L.; Zhang, Y.; Mahmood, M.H.; Huang, Y.-Y.; Ying, X.; Ji, L.-N.; Liu, H.-Y. DNA binding and nuclease
activity of a water-soluble sulfonated manganese(III) corrole. Transit. Met. Chem. 2013, 38, 283–289. [CrossRef]
41. Wang, C.-X.; Yan, F.-F.; Zhang, Y.-X.; Ye, L. Spectroscopic investigation of the interaction between rifabutin and bovine serum
albumin. J. Photochem. Photobiol. A Chem. 2007, 192, 23–28. [CrossRef]
42. Zhang, Y.; Wen, J.-Y.; Mahmood, M.H.; Wang, X.-L.; Lv, B.-B.; Ying, X.; Wang, H.; Ji, L.-N.; Liu, H.-Y. DNA/HSA interaction and
nuclease activity of an iron(III) amphiphilic sulfonated corrole. Luminescence 2015, 30, 1045–1054. [CrossRef]
43. Zhang, G.; Ma, Y. Mechanistic and conformational studies on the interaction of food dye amaranth with human serum albumin
by multispectroscopic methods. Food Chem. 2013, 136, 442–449. [CrossRef] [PubMed]
44. Kandagal, P.; Ashoka, S.; Seetharamappa, J.; Shaikh, S.; Jadegoud, Y.; Ijare, O. Study of the interaction of an anticancer drug with
human and bovine serum albumin: Spectroscopic approach. J. Pharm. Biomed. Anal. 2006, 41, 393–399. [CrossRef] [PubMed]
45. Fernández-Sainz, J.; Liñán, P.J.P.; Granadino-Roldan, J.M.; Bravo, I.; Garzón, A.; Martinez, J.R.; Albaladejo, J. Binding of the
anticancer drug BI-2536 to human serum albumin. A spectroscopic and theoretical study. J. Photochem. Photobiol. B Biol. 2017, 172,
77–87. [CrossRef]
46. Hindie, E.; Morgat, C.; Zanotti-Fregonara, P.; Haissaguerre, M.; Bordenave, L.; Tabarin, A. Advantages and Limits of Targeted
Radionuclide Therapy with Somatostatin Antagonists. J. Nucl. Med. 2017, 59, 546–547. [CrossRef] [PubMed]
47. Gurumoorthy, P.; Mahendiran, D.; Prabhu, D.; Arulvasu, C.; Rahiman, A.K. Mixed-ligand copper(II) phenolate complexes:
Synthesis, spectral characterization, phosphate-hydrolysis, antioxidant, DNA interaction and cytotoxic studies. J. Mol. Struct.
2015, 1080, 88–98. [CrossRef]
48. El-Deen, I.; Shoair, A.; El-Bindary, M. Synthesis, characterization and biological properties of oxovanadium(IV) complexes. J. Mol.
Struct. 2019, 1180, 420–437. [CrossRef]
49. Liang, Z.-H.; Wang, Y.-N.; Xiong, Z.-W.; Chen, X.-Y.; Tong, L. Studies of the anticancer activities of ruthenium(II) polypyridyl
complexes toward human hepatocellular carcinoma BEL-7402 cells. Transit. Met. Chem. 2019, 44, 585–594. [CrossRef]