Synthesis, antiproliferative activity and mechanism of copper(II)-thiosemicarbazone complexes as potential anticancer and antimicrobial agents

Article abstract of DOI:10.1080/00958972.2020.1768378

Malignant tumors have become one of the challenges to global human health today, and the development of new drugs has become a research hotspot for tumor treatment. In order to prove that copper(II)-thiosemicarbazone complexes can be used as antibacterial and antitumor agents, we have synthesized and characterized three copper(II) complexes (C1-C3). These Cu(II) complexes exhibited excellent anticancer and antimicrobial activity, and greatly exceed the corresponding metal-free ligands. C3 has the highest anti-proliferative activity (0.20 ± 0.04 μM) against A549 (human lung carcinoma cell lines) and the best inhibitory zone diameter (25.78 ± 0.18 mm) against E. coli. The lipophilicity of the ligand is closely related to the anti-proliferative and antibacterial activities of these Cu(II) complexes. The study of the cellular mechanism demonstrates that these Cu(II) complexes promoted cell apoptosis by catalyzing hydrogen peroxide to produce intracellular reactive oxygen species (ROS). Antibacterial experiments showed that these Cu(II) complexes exhibited potent antibacterial activities, especially against gram-negative bacteria. These Cu(II) complexes may initially cause outer membrane/lipopolysaccharide (LPS) instability, disrupt cell membranes, and ultimately lead to bacterial cell lysis.

Full text of DOI:10.1080/00958972.2020.1768378

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: https://www.tandfonline.com/loi/gcoo20
Synthesis, antiproliferative activity and
mechanism of copper(II)-thiosemicarbazone
complexes as potential anticancer and
antimicrobial agents
Jinxu Qi, Xuejiao Wang, Taichen Liu, Martha Kandawa-Schulz, Yihong Wang &
Xinhua Zheng
To cite this article: Jinxu Qi, Xuejiao Wang, Taichen Liu, Martha Kandawa-Schulz, Yihong
Wang & Xinhua Zheng (2020): Synthesis, antiproliferative activity and mechanism of copper(II)-
thiosemicarbazone complexes as potential anticancer and antimicrobial agents, Journal of
Coordination Chemistry, DOI: 10.1080/00958972.2020.1768378
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JOURNAL OF COORDINATION CHEMISTRY
https://doi.org/10.1080/00958972.2020.1768378
Synthesis, antiproliferative activity and mechanism of
copper(II)-thiosemicarbazone complexes as potential
anticancer and antimicrobial agents
Jinxu Qia#, Xuejiao Wangb#, Taichen Liua, Martha Kandawa-Schulzc, Yihong
Wangb and Xinhua Zhenga
b
aSchool of Medicine, Pingdingshan University, Pingdingshan, China; School of Chemistry and
c
Chemical Engineering, Southeast University, Nanjing, China; Department of Chemistry and
Biochemistry, University of Namibia, Windhoek, Namibia
ABSTRACT
ARTICLE HISTORY
Received 16 February 2020
Accepted 1 May 2020
Malignant tumors have become one of the challenges to global
human health today, and the development of new drugs has
become a research hotspot for tumor treatment. In order to prove
that copper(II)-thiosemicarbazone complexes can be used as anti-
bacterial and antitumor agents, we have synthesized and charac-
terized three copper(II) complexes (C1-C3). These Cu(II) complexes
exhibited excellent anticancer and antimicrobial activity, and
greatly exceed the corresponding metal-free ligands. C3 has the
highest anti-proliferative activity (0.20 0.04 mM) against A549
(human lung carcinoma cell lines) and the best inhibitory zone
diameter (25.78 0.18 mm) against E. coli. The lipophilicity of the
ligand is closely related to the anti-proliferative and antibacterial
activities of these Cu(II) complexes. The study of the cellular
mechanism demonstrates that these Cu(II) complexes promoted
cell apoptosis by catalyzing hydrogen peroxide to produce intra-
cellular reactive oxygen species (ROS). Antibacterial experiments
showed that these Cu(II) complexes exhibited potent antibacterial
activities, especially against gram-negative bacteria. These Cu(II)
complexes may initially cause outer membrane/lipopolysaccharide
(LPS) instability, disrupt cell membranes, and ultimately lead to
bacterial cell lysis.
KEYWORDS
Copper complex;
thiosemicarbazone; anti-
cancer activity;
antimicrobial activ-
ity; apoptosis
CONTACT Yihong Wang
Yihongwang@Seu.edu.cn; Xinhua Zheng
zhengxinhua70@163.com
#Jinxu Qi and Xuejiao Wang equally contributed to this study.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

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J. QI ET AL.
1. Introduction
Malignant tumors are common and frequently-occurring diseases that seriously endan-
ger human health in modern life [1–3]. After Rosenberg et al. [4] found that
cisplatin(II) is a better drug for cancer, a series of new platinum drugs have been
developed one after another, but they still show strong renal toxicity, ototoxicity and
neurotoxicity in clinical trials [5–8]. Therefore, this has also led to research interest in
new metal-based antitumor drugs [9–13]. At the same time, many patients with malig-
nant tumors have low immune function or long-term use of chemotherapeutic drugs,
resulting in neutrophil reduction or even deficiency, which causes pathogenic bacterial
infections in the body [14, 15]. In order to take into account the complications of
microbial infection caused by the treatment of malignant tumors, the development of
new drugs with strong antitumor activity and strong antibacterial activity is also a
new direction for the research of antitumor drugs.
Copper is an important part of human proteins and enzymes. Many important
enzymes require a small amount of copper to participate and activate [16, 17]. Due to
their higher proliferation rate, tumor cells usually require more copper than normal
cells [18]. In recent years, the high activity of copper-based complexes in cancer cells
has been studied in depth [13, 19–21]. Thiosemicarbazone derivatives and their com-
plexes with metals have significant antiviral, antituberculous, antibacterial and antitu-
mor activities [22–25]. Therefore, it is of great theoretical and practical significance to
design and synthesize thiosemicarbazone ligands and copper complexes of various
compositions and structures, and explore the anticancer, antibacterial activity, and
mechanism of action of these compounds.
Herein, we synthesized and characterized three Cu(II) thiosemicarbazone com-
plexes. To evaluate the antitumor mechanism of copper(II) complexes, we studied
their antiproliferative activity, apoptosis analysis, intracellular reactive oxygen species
(ROS), and cell cycle. Meanwhile, the antibacterial mechanism of Cu(II) complexes was
studied by antibacterial activity, inhibition zone diameters and cell membrane
permeability.

JOURNAL OF COORDINATION CHEMISTRY
3
Scheme 1. Synthesis routes for C1-C3.
2. Experimental
2.1. Materials and methods
All chemical reagents were supplied by Innochem Company (Shanghai, China) and
were of analytical grade as well as were used without additional purification. Water
used in the reactions was distilled prior to use. Human lung carcinoma cell lines A549
and human colon cancer cell lines Caco-2 were obtained from Chinese Academy
of Sciences.
2.2. Synthesis and characterization of Cu(II) complexes
The ligands (L1-L3) were synthesized according to the description in our published ref-
erence [26]. 2-Acetylpyridine (1 mole equivalent) and acetic acid glacial (5 drops) were
placed in a round-bottom flask to which ethanol (10 mL) and thiosemicarbazide
(1 mM) were added. The solution was heated under refluxed for 4 h. The precipitate
formed was collected by vacuum filtration, washed three times with cold ethanol and
dried in a vacuum desiccator.
Cu(II) complexes (C1, C2, and C3) were synthesized following the previous descrip-
tion [20, 27, 28]. One mole equivalent of CuCl2 was added to methanol solution of thi-
osemicarbazone (10 mL, 1 mM) and stirred, stand and allow the solvent to evaporate
to gradually precipitate the Cu(II) complex single crystals. The precipitate was collected
by filtration and washed with ice-cold ethanol and dried in vacuo overnight. The syn-
thesis process is shown in Scheme 1.
[2-[1-(2-Pyridinyl)ethylidene]hydrazinecarbothioamide-N,N,S-copper(II)] chloride (C1):
yield 74%. Anal. Calcd for C8H10Cl2CuN4S: C, 29.23; H, 3.07; N, 17.04. Found: C, 29.18;
H, 3.11; N, 17.09. MS m/z (%) 291.95 (M–Cl, 100).
[N-methyl-2-[1-(2-pyridinyl)ethylidene]hydrazinecarbothioamide-N,N,S-copper(II)]
chloride (C2): yield 78%. Anal. Calcd for C9H11ClCuN4S: C, 35.29; H, 3.62; N, 18.29.
Found: C, 35.22; H, 3.66; N, 18.21. MS m/z (%) 270.0 (M–Cl, 100).
[N,N-dimethyl-2-[1-(2-pyridinyl)ethylidene]hydrazinecarbothioamide-N,N,S-copper(II)]
chloride (C3): yield 76%. Anal. Calcd for C10H13ClCuN4S: C, 37.50; H, 4.09; N, 17.49.
Found: C, 37.42; H, 4.02; N, 17.41. MS m/z (%) 320.29 (M–Cl, 100).

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J. QI ET AL.
Table 1. Crystal data and structure refinement for C1-C3.
Complexes
C1
C2
C3
Empirical formula
Formula weight
C8H10Cl2CuN4S
328.7
C9H11ClCuN4S
306.27
C10H13ClCuN4S
320.29
Temperature/K
296.15
296.15
296.15
Crystal system
Space group
Monoclinic
P21/c
Monoclinic
P21/n
Monoclinic
P21/n
a/Å
b/Å
11.0147(9)
9.5131(4)
12.3311(10)
90
8.029(8)
15.917(17)
9.133(10)
90
92.378(19)
90
1166(2)
11.338(7)
9.512(6)
12.350(8)
90
c/Å
a/ꢀ
b/ꢀ
112.051(9)
104.611(10)
c/ꢀ
90
90
Volume/Å3
Z
1197.59(14)
4
1288.8(14)
4
4
F(000)
Index ranges
660
620
652
ꢂ13 ꢃ h ꢃ 13
ꢂ11 ꢃ k ꢃ 11
ꢂ15 ꢃ l ꢃ 14
1.078
ꢂ10 ꢃ h ꢃ 10
ꢂ19 ꢃ k ꢃ 19
ꢂ11 ꢃ l ꢃ 11
1.019
ꢂ14 ꢃ h ꢃ 14
ꢂ11 ꢃ k ꢃ 12
ꢂ15 ꢃ l ꢃ 15
1.074
Goodness-of-fit on F2
Final R indexes [I ꢄ 2r (I)] R1 ¼ 0.0281, wR2 ¼ 0.0703 R1 ¼ 0.0332, wR2 ¼ 0.0722 R1 ¼ 0.0270, wR2 ¼ 0.0715
Final R indexes [all data]
CCDC NO.
R1 ¼ 0.0370, wR2 ¼ 0.0749 R1 ¼ 0.0540, wR2 ¼ 0.0807 R1 ¼ 0.0344, wR2 ¼ 0.0759
1976432 969493 1055568
2.3. Determination of structure of Cu(II) complexes
Crystallographic data were collected at 296.15 K on a Bruker SMART Apex II CCD dif-
fractometer employing Mo-Ka radiation (k ¼ 0.71073 Å) source. Data reduction and
empirical absorption corrections (multiscan) were performed with Oxford Diffraction
CrysAlisPro software. All structures were solved by direct methods with Olex2 and
refined by full-matrix least-squares methods with Olex2 software [29]. All non-H atoms
were refined anisotropically using the riding model to fix the H atom in a geometric-
ally ideal position. Molecular structure diagrams were produced with Olex2 software
[29]. The crystal data of Cu(II) complexes are exhibited in Table 1. Selected bond
lengths and angles are shown in Tables S1–S6.
2.4. Cytotoxicity assay (MTT)
In this study, MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)
assay [30] was used to examine the cytotoxicity of cells. At 37 ꢀC, the cell lines were
grown in an incubator (ThermoFisher) with 95% air and 5% CO2. Thiosemicarbazide
ligands and Cu(II) complexes were dissolved in DMSO as 10 mM stock solutions. Stock
solutions in DMSO were diluted to a concentration of <0.1% in cell culture medium
to avoid the solvent effect on cell proliferation. Cells were seeded in 96-well plates
and allowed to adhere overnight in an incubator at 37 ꢀC. The seeding densities used
were 5 ꢁ 104 cells/well. Different concentrations (0, 1, 2.5, 5, 10 lM) of ligands and
their Cu(II) complexes were added to the plates and incubated for 48 h/37 ꢀC. 20 lL
MTT (5 mg/mL) was added to the cells and incubated for another 4 h. Finally, DMSO
(100 lL) was added, and the optical density (OD) of living cells was recorded at a
wavelength of 570 nm. MTT color formation was directly proportional to the number
of viable cells, validating its use in these studied. The half-maximum inhibitory

JOURNAL OF COORDINATION CHEMISTRY
5
concentration (IC50) values were processed and analyzed according to the nonlinear
multi-purpose curve fitting program GraphPad Pris.
2.5. Cell cycle distribution analysis
The A549 cells were seeded in 70-mm culture dishes at a density of 1 ꢁ 105 cells/mL
and incubated at 37 ꢀC for 24 h. Then, the medium was removed and freshly prepared
solutions of the tested compounds, C1, C2 and C3, at a 1 mM concentration were
added. After 24-h treatment, cells were harvested and washed 3 times with ice-cold
PBS. Then, the cells were fixed in ice cold 75% ethanol and stored at -20 ꢀC for 12 h,
as well as rinsed with cold PBS. The RNase (Ribonuclease, 2.5 mg/ml) was added to the
cells for culture and stained with pyridinium iodide (PI, 50 mg/mL) for 0.5 h, followed
by flow cytometry (BD, AccuriTM C6) analysis. The 10,000 events for each sample were
recorded and the data was resolved by MFLT32 software.
2.6. Cell apoptosis assay
The A549 cell apoptotic events induced by C1, C2 and C3 were determined by
Annexin V and PI staining according to the manufacturer’s protocol for the Annexin V-
FITC Apoptosis Detection Kit (Bection Dickinson). At 5% CO2/95% air and 37 ꢀC, 2 mL
1 ꢁ 106 cells/mL were used for incubation with C1, C2 and C3 (1 lM) for 24 h. The
A549 cells were suspended in 300 lL of mixed buffer (140 mM NaCl, 10 mM Hepes/
NaOH, 2.5 mM CaCl2, pH 7.4), and then 5 lL each of PI and Annexin V were added to
these samples. Then, samples incubated at room temperature for 20 min were
determined by flow cytometry analysis (BD, AccuriTM C6), and the rate of apoptosis
was analyzed by FlowJo software.
2.7. Intracellular ROS measurements
Evaluation of intracellular ROS was performed by the procedure of active oxygen assay
kit (Beyotime, Suzhou) and flow cytometry. The A549 cells were treated with C1, C2 or
C3 (5 lM) for 30 min at 37 ꢀC. 20,70-Dichlorodihydro-fluorescein diacetate (H2DCF-DA)
was added to the cells for 0.5 h and then washed twice with ice-cold PBS. Cells were
collected for flow cytometry assessment (BD, AccuriTM C6). Data analysis was per-
formed using FlowJo software.
2.8. Antimicrobial assays
In vitro antimicrobial activity of the compounds was tested by the standard serial dilu-
tion method against four bacterial strains i.e. Gram-positive bacteria Staphylococcus
aureus and Staphylococcus epidermidis (S. aureus ATCC 29213, S. epidermidis ATCC
14990), Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa (E. coli
ATCC 25922, P. aeruginosa ATCC 27853), which were cultured on nutrient broth and
potato dextrose broth as nutrient medium.

6
J. QI ET AL.
Stock solutions of thiosemicarbazide ligands and Cu(II) complexes were serially
diluted two-fold from 512 to 1 lM/mL in Mueller-Hinton (MH) broth. 100 lL of each
dilution and 100 lL of bacterial suspension at 1 ꢁ 106 cells/mL (OD540 value of 1.0 is
equivalent to the density of 109 cells/mL) were transferred into the wells of a micro-
plate, respectively. The negative control (media only) and positive control (bacteria
without inhibitors) on the same plate were used as references to determine the
growth inhibition of bacteria. All the plates were covered and incubated at 37 ꢀC for
18 h without shaking. After incubation, the minimal inhibitory concentrations (MICs)
were determined by the lack of turbidity in the wells. The minimum bactericidal con-
centrations (MBCs) were measured by spreading 100 lL of solution from each clear
well (ꢄ MIC) onto agar plates. The agar plates were kept in an incubator at 37 ꢀC for
24 h. the MBC was determined as the lowest concentration with no colonies had
formed corresponded.
2.9. Inhibition zone tests
Antibacterial studies are revealed by analyzing thiosemicarbazone ligands and Cu(II)
complexes by disk diffusion method. 100 lL of cell suspension was spread over the steri-
lized agar plate with the use of a sterilized spreader, and pasted a filter paper containing
2MICs antibacterial drug C1-C3 on the surface of the agar inoculated with the test
ꢀ
bacteria. The plates were then incubated at 37 C/16 h in the incubator. Following the
incubation period, the inhibition zone was considered in millimeter (mm) [31, 32].
2.10. Membrane permeability study
As described above, the Sytox green uptake assay was performed [33]. S. aureus and
E. coli were incubated for 12 h, washed twice with cold PBS, centrifuged and resus-
pended to form a 2 ꢁ 107 cfu/mL solution. Cells were fixed on glass slides coated with
poly (L-lysine) by incubation with Cu(II) complexes at 2 MICs for 0.5 h at 30 ꢀC. The
slides were washed with PBS and incubated with 5 lM Sytox green dye in a dark
environment at 30 ꢀC for 30 minutes. Finally, the slide was washed again with PBS and
analyzed with a confocal microscope (Olympus FV3000) at an excitation wavelength of
485 nm. Samples containing only bacteria and Sytox green dye as blank controls.
2.11. Statistical analysis
Experiment data were compared using Student’s test. Results were expressed as
mean SD (number of experiments) and considered to be statistically significant when
p < 0.05. All experiments were repeated at least three times.
3. Results and discussion
3.1. Synthesis and crystal structure description
According to the method reported in the literature, the ligands L1-L3 were directly
prepared by Schiff base condensation reaction, and the purity of the obtained ligand

JOURNAL OF COORDINATION CHEMISTRY
7
Figure 1. Molecular structure of Cu(II) complexes showing the environment about the Cu(II) atom.
(A) C1; (B) C2; (C) C3.
product was high without further purification [26, 34, 35]. The coordination of the lig-
and with copper chloride produced a ligand/Cu(II) complex of 1:1 (Figure 1). All Cu(II)
complexes were crystallized from methanol solution, and the structures were identified
by single-crystal X-ray diffractometer.
The crystal data of the three Cu(II) complexes are shown in Table 1, and the bond
lengths and angles are listed in Tables S1–S6. Although the ligands are similar in struc-
ture, the Cu(II) complexes are not. Complex C1 contains neutral thiosemicarbazone lig-
and and adopts a square-pyramidal geometry, while the other compounds contain
anionic deprotonated thiosemicarbazone ligands and crystallize in square-planar geome-
tries. Certainly, the apical chlorido Cl2 ligand in C1 exhibits a bond length with Cu(II) lon-
ger than the Cu-Cl1 due to the Jahn-Teller effect, but the bond remains. C1-C3 are all
monoclinic, but the space group to which C1 belongs is P21/c, and the space group to
which C2 and C3 belong is P21/n. The Cu(II) metal center of C1 is coordinated by two N
atoms (N1 and N2), one Cl atom (Cl1) and one S atom forming an approximately planar
structure (Figure 1). Due to coordination with Cu(II), most of the ligand atoms (N1-N4 and
C1-C9) are fixed on the approximate plane. The bond angles of N1-Cu1-Cl1, Cl1-Cu1-S1,
S1-Cu1-N2 and N1-Cu1-N2 with Cu(II) as the center are 99.41ꢀ, 96.41ꢀ, 84.35ꢀ and 79.11ꢀ,
respectively. The lengths of Cu1-N1 (¼ 2.029(2) Å) and Cu1-N2 (¼ 1.9783(18) Å) are simi-
lar. There is no significant difference in the length of Cu1-Cl1 (¼ 2.2864(7) Å) and Cu1-S2
(¼ 2.2243(7) Å) bonds. The bond angles and bond lengths in C2 and C3 are similar to C1.
In C2 and C3, the anion Cl and tridentate thiosemicarbazone coordinate with the central
copper(II) ion to form an approximately planar structure. The bond length, bond angle
and coordination environment of C2 match the published results [36, 37].
3.2. In vitro cytotoxic studies of ligands and Cu(II) complexes
Scientists have deeply studied thiosemicarbazone analogs and proved that these ligands
and complexes have significant antitumor activity [37–49]. Their research focuses on SK-
N-MC Neuroepithelioma Cells, SK-Mel-28 melanoma, etc.; our experimental results are
complementary to their experimental results. Numerous reports indicate that thiosemi-
carbazone ligands have significant activity against tumors, such as breast cancer, liver
cancer, lung cancer, colon cancer, etc. [18, 34, 50]. The antiproliferative properties of the
thiosemicarbazone ligands and Cu(II) complexes on two malignant cancer cell lines
(human lung cancer cell lines A549, human colon cancer cell lines Caco-2) were eval-
uated, as shown in Table 2. The anticancer activity of L3 was significantly higher than that
of L1 and L2. The results showed that the increased lipophilicity of the terminal-N

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J. QI ET AL.
Table 2. IC50 Values of ligands and Cu(II) complexes toward
cell lines for 48 h.
IC50 SD (lM)
Compounds
A549
Caco-2
L1
L2
L3
C1
C2
C3
11.77 0.78
8.04 0.22
1.07 0.38
0.22 0.06
0.42 0.15
0.20 0.04
86.89 0.24
1.36 0.27
0.75 0.17
0.74 0.51
1.07 0.29
0.68 0.14
substitution can enhance the antitumor activity of the compound. After 48h of incubation,
the antiproliferative activity of the Cu(II) complexes was significantly higher than that of the
corresponding individual ligands, and the antitumor proliferative activity of C1 was similar
to C3. Results indicated that the antiproliferative activity of copper complexes is deter-
mined by the corresponding ligands, and the antitumor activity of the ligands is enhanced
by coordination with copper. Since the Cu(II) complexes C1-C3 exhibited better antitumor
activities than the ligands, they were selected for further analyses.
3.3. Cell cycle analysis
Cell cycle arrest plays a vital role in preventing the malignant proliferation of tumor
cells, of which G1/S and G2/M transitions are the most important stages of the cell
cycle [51]. The effect of Cu(II) complexes on A549 cell cycle was evaluated by staining
the DNA of A549 cells with pyridine iodide (PI) and analyzing them by flow cytometry
(Figure 2). From the figure we can see that the percentage of G2 phase of cells treated
with C1, C2 and C3 were reduced by 3.38%, 2.62% and 12.41%, respectively, com-
pared with the control group. Data demonstrated the Cu(II) complexes can inhibit cell
proliferation by inhibiting the G2/M phase transition in the cell cycle.
3.4. Cell apoptosis assay
Programmed cell death refers to the orderly death of cells controlled by genes in
order to maintain internal environment stability [52]. However, apoptosis is usually
suppressed in tumor cells, so activating apoptosis is an effective anticancer pathway
[53]. After treating A549 cells with Cu(II) complexes for 12 h, staining with Annexin V-
FITC/PI and analyzing the effect of complexes on promoting apoptosis by flow cytom-
etry (Figure 3). According to the data C1, C2 and C3 induced early apoptosis of cells,
which increased by 33.77%, 19.07% and 44.67% compared with the control group,
respectively. The results of cell apoptosis assay proved that C3 has a stronger ability
to promote apoptosis, which was consistent with the results of MTT assay.
3.5. Intracellular ROS measurements
The intervention of some exogenous drugs will cause excessive production of reactive
oxygen species (ROS) in the cell or reduce the ability to clear them, leading to apop-
tosis [54]. To assess whether Cu(II) complexes can induce increased intracellular ROS,

JOURNAL OF COORDINATION CHEMISTRY
9
Figure 2. Effect of the cell cycle of A549 treated with (a) control, (b) C1, (c) C2, and (d) C3.
after co-incubating A549 with Cu(II) complexes for 48 h, they were stained with fluor-
escent probes (DCFH-DA) and analyzed by flow cytometry (Figure 4). Compared with
the control, the intracellular ROS of the three drug-treated cells were significantly
increased, and C3 had the strongest ability to promote the production of ROS.
Excessive intracellular ROS can inhibit G1, S, and G2 phase proliferation and combined
with the above-mentioned cell cycle analysis, it can be found that G2/M phase block
is related to antioxidative damage and antiapoptotic processes.
3.6. Antibacterial activity assays
The antibacterial activity of synthesized ligands and Cu(II) complexes were screened
against two Gram-positive bacteria (S. aureus and S. epidermidis) and two Gram-negative
bacteria (E. coli and P. aeruginosa). MIC and MBC are given in Table 3. Antibacterial activity
data showed that under similar conditions, the antibacterial activity of metal complexes
is higher than that of the corresponding ligands, and the ligands have little inhibition
activity. Cu(II) complexes exhibited more antimicrobial activity to Gram-negative bacteria
than Gram-positive bacteria. This is mainly due to the complexity of the cell wall structure
of Gram-positive bacteria and Gram-negative bacteria. The lipid membrane facilitates the
passage of any fat-soluble substance, and liposolubility is known to be an important fac-
tor in controlling antibacterial activity [55].

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J. QI ET AL.
Figure 3. Effect of the cell apoptosis. Quantification of Annexin V and PI double-stained A549 cells
after treatment with (a) control, (b) C1, (c) C2, and (d) C3, for 24 h at the indicated concentrations
by flow cytometry.
Figure 4. Intracellular ROS was detected in A549 cells after treatment with (a) control, (b) C1, (c)
C2, and (d) C3 for 24 h.

JOURNAL OF COORDINATION CHEMISTRY
11
Table 3. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of
ligands and Cu(II) complexes.
MIC (MBC) (lM)
Compounds
S. aureus 29213
S. epidermidis 14990
P. aeruginosa 27853
E. coli 25922
L1
L2
L3
C1
C2
C3
>256(>256)
>256(>256)
128(>256)
128(256)
32(32)
>256(>256)
>256(>256)
256(>256)
128(256)
32(32)
>256(>256)
>256(>256)
256(>256)
128(256)
16(16)
>256(>256)
256(>256)
128 (>256)
128(256)
16(32)
16(32)
16(32)
8(16)
8(16)
Table 4. Zone of inhibition (in mm) against microorganisms
(antimicrobial agent dose per disk is 2MICs).
Compounds
S. aureus 29213
E. coli 25922
L1
L2
L3
C1
C2
C3
0
0
0
0
12.22 0.11
13.74 0.30
19.62 0.07
21.68 0.15
25.78 0.18
14.00 0.14
14.94 0.03
15.52 0.21
Figure 5. Zone of inhibition against microorganisms (a, c) S. aureus; (b, d) E. coli.
In order to further clarify the antibacterial effects of ligands and Cu(II) complexes
on different bacteria, zone of inhibition tests were performed on S. aureus and E. coli.
As shown in Table 4 and Figure 5, for S. aureus, the ligands did not show antibacterial
activity over the considered strains since no inhibition zones were observed. For E.
coli, the diameters of the inhibition zones of L2 and L3 were 12.22 0.11 mm and
13.74 0.30 mm, respectively. The diameters of the corresponding Cu(II) complexes

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J. QI ET AL.
Figure 6. Complex-induced influx of Sytox Green into (a) S. aureus and (b) E. coli.
were 19.62 0.07 mm, 21.68 0.15 mm and 25.78 0.18 mm. It has excellent antibacter-
ial properties against Gram-negative bacteria.
3.7. Membrane permeabilization
The ability of Cu(II) complexes to disrupt and increase cell membrane permeability
was evaluated by using Sytox green into S. aureus and E. coli cells. Sytox green is a
green nucleic acid dye that easily penetrates the damaged plasma membrane and
cannot penetrate the plasma membrane of living cells [33]. As shown in Figure 6,
when treated with copper complexes, a significant fluorescent signal was present in S.
aureus and E. coli, while the untreated control did not. The results indicate that copper
complexes may destroy the integrity of bacterial cell membranes and induce the
absorption of Sytox green.
4. Conclusion
Herein, we report three thiosemicarbazide and Cu(II) complexes with potential antipro-
liferative and antibacterial activity. The structure-activity relationship showed that the
modification of lipophilic groups on the ligand significantly increased the biological
resistance of Cu(II) complexes. The intervention of Cu(II) complexes greatly shortened
the G2 phase in the cell cycle. Studies on the mechanism of apoptosis have shown
that copper complexes promote early cell apoptosis by increasing the release of intra-
cellular ROS. The inhibition zone experiment proved that the Cu(II) complexes had bet-
ter inhibitory effect on Gram-negative bacteria. In addition, this study suggested that
Cu(II) complexes may initially cause instability of the outer membrane/LPS, destroying
the cell membrane, leading to bacterial cell lysis. These results may be beneficial for
the development of new Cu(II) complexes as candidates for antitumor drugs.

JOURNAL OF COORDINATION CHEMISTRY
13
Disclosure statement
The authors declare that they have no conflict of interests.
Funding
This work was supported by National Natural Science Foundation of China (81571812),
Pingdingshan College PhD Startup Fund under Grant PXY-BSQD-202003; key specialized research
and development breakthrough in Henan province (202102310476 and 182102310181); and A
Project Funded by the Priority Academic Program Development of Jiangsu Higher Education
Institutions under Grant 1107047002.
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