Single-crystal structure and intracellular localization of Zn(II)-thiosemicarbazone complex targeting mitochondrial apoptosis pathways

Article abstract of DOI:10.1016/j.bmcl.2020.127340

Tracking of drugs in cancer cells is important for basic biology research and therapeutic applications. Therefore, we designed and synthesised a Zn(II)-thiosemicarbazone complex with photoluminescent property for organelle-specific imaging and anti-cancer proliferation. The Zn(AP44eT)(NO₃)₂ coordination ratio of metal to ligand was 1:1, which was remarkably superior to 2-((3-aminopyridin-2-yl) methylene)-N, N-diethylhydrazinecarbothioamide (AP44eT·HCl) in many aspects, such as fluorescence and anti-tumour activity. Confocal fluorescence imaging showed that the Zn(AP44eT)(NO₃)₂ was aggregated in mitochondria. Moreover, Zn(AP44eT)(NO₃)₂ was more effective than the metal-free AP44eT·HCl in shortening the G2 phase in the MCF-7 cell cycle and promoting apoptosis of cancer cells. Supposedly, the effects of these complexes might be located mainly in the mitochondria and activated caspase-3 and 9 proteins.

Full text of DOI:10.1016/j.bmcl.2020.127340

Bioorganic&MedicinalChemistryLetters30(2020)127340
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Single-crystal structure and intracellular localization of Zn(II)-
thiosemicarbazone complex targeting mitochondrial apoptosis pathways
Jinxu Qi⁎, Wei Zhao, Yunyun Zheng, Ruiya Wang, Qiu Chen, Fu-An Wang, Weiwei Fan,
Huashan Gao, Xichao Xia⁎
Medcine College of Pingdingshan University, Pingdingshan, Henan 467000, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Tracking of drugs in cancer cells is important for basic biology research and therapeutic applications. Therefore,
we designed and synthesised a Zn(II)-thiosemicarbazone complex with photoluminescent property for organelle-
specific imaging and anti-cancer proliferation. The Zn(AP44eT)(NO3)2 coordination ratio of metal to ligand was
1:1, which was remarkably superior to 2-((3-aminopyridin-2-yl) methylene)-N, N-diethylhy-
drazinecarbothioamide (AP44eT·HCl) in many aspects, such as fluorescence and anti-tumour activity. Confocal
fluorescence imaging showed that the Zn(AP44eT)(NO3)2 was aggregated in mitochondria. Moreover, Zn
(AP44eT)(NO3)2 was more effective than the metal-free AP44eT·HCl in shortening the G2 phase in the MCF-7
cell cycle and promoting apoptosis of cancer cells. Supposedly, the effects of these complexes might be located
mainly in the mitochondria and activated caspase-3 and 9 proteins.
Zn(II) complex
Thiosemicarbazone
Antiproliferative
Photoluminescent
Imaging
Mitochondria play a key role in most eukaryotic cells, from the
production of energy to the regulation of apoptosis1–3. Under normal
conditions, cell proliferation is controlled by a variety of signalling
mechanisms3–5. For example, cellular structure and molecular damage
can lead to programmed cell death or apoptosis2. However, the me-
chanism of uncontrolled cell division and the formation of tumours are
abnormal in cancer cells6. Mutations in mitochondrial genes and high
levels of intracellular reactive oxygen species (ROS) could induce tu-
mour2,7. The ROS that were generated and accumulated during mi-
tochondrial respiration induced the impairment of electron transport
chain4,8. As the research progressed, researchers designed and synthe-
sised a series of metal-based drugs targeting mitochondria, but these
drugs are only in the basic research stage9–12..
hydroxide and improve their anti-cancer activity and bioavail-
ability19–23. The coordination with metals also increased the membrane
affinity and anti-tumour activity of thiosemicarbazones24–26
.
The interesting photophysical properties of Zn complexes can be
applied to biotechnology, biology and potential pharmaceuticals27
.
Therefore, AP44eT·HC (la thiosemicarbazone ligand) and Zn(AP44eT)
(NO3)2 were designed and synthesised as imaging and therapeutic
agents (Scheme 1). Their targets were researched by laser confocal
microscopy. The effects of AP44eT·HCl and Zn(AP44eT)(NO3)2 on cy-
totoxicity and anti-tumour mechanisms were specifically studied.
Design and structure of Zn(AP44eT)(NO3)2
Zinc is an essential trace element in organisms which is involved in
many biological processes. The thiosemicarbazide chelator is mainly
responsible for disrupting the iron metabolism of tumour cells against
The thiosemicarbazide ligand (AP44eT·HCl) which was synthesised
by the classical Schiff base reaction has a high purity and does not re-
quire further purification23,28. AP44eT·HCl is slightly soluble in water
but soluble in organic solvents, including ethanol, methanol and DMSO.
AP44eT·HCl and zinc (II) complex crystallise for suitable x-ray single-
crystal diffraction are crystallised from a slowly volatilised ethanol
solution. The crystal structure of the X-ray diffraction analysis is pre-
sented in Fig. 1, the structure refinement and crystal data for
AP44eT·HCl and Zn(AP44eT)(NO3)2 are shown in Table S1, and the
bond length and angle of the crystal are shown in Table S2-3.
AP44eT·HCl and Zn(AP44eT)(NO3)2 were crystallised in the monoclinic
cell proliferation13–15
. 3-aminopyridinecarbaldehyde thiosemicarba-
zone (3-AP), one of the most representatives, is applied to clinical trials
and achieved a remarkable therapeutic effect14,16. Recent studies
showed that its biological and anti-tumour activities are considerably
enhanced by modifying lipophilic groups on the N4 groups of thiose-
micarbazones17,18. The inherent NNS tridentate coordination scaffolds
leading to organic thiosemicarbazones are excellent chelators to metal
ion (Fe, Cu, Mn, Zn, Ga, etc.), which prevents the formation of
⁎ Corresponding authors at: ChongWen Road, Pingdingshan 467000, China.
E-mail addresses: jinxuqi@pdsu.edu.cn (J. Qi), 5966@pdsu.edu.cn (X. Xia).
https://doi.org/10.1016/j.bmcl.2020.127340
Received 4 April 2020; Received in revised form 17 May 2020; Accepted 6 June 2020
Availableonline09June2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

J. Qi, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127340
Scheme 1. Synthesis routes for AP44eT·HCl and Zn(AP44eT)(NO3)2.
surrounding the nucleus, MitoDeedRed (mitochondria) was used for
staining the MCF-7 cell line to locate mitochondria (Fig. 3). After the
cells were incubated with AP44eT·HCl and Zn(AP44eT)(NO3)2, peri-
nuclear punctuated green fluorescence was seen under 465 nm excita-
tion (Fig. 3), which was consistent with the location of the Mito-
DeedRed. The correlation coefficients of Pearson were 0.79 and 0.85 for
AP44eT·HCl and Zn(AP44eT)(NO3)2, indicating that AP44eT·HCl and
Zn(AP44eT)(NO3)2 were localised in mitochondria.
Fig. 1. (A) Molecular structure of AP44eT·HCl; (B) Molecular structure of Zn
Anti-cancer activity
(AP44eT)(NO3)2.
Studies showed that 3-aminopyridinecarbaldehyde thiosemicarba-
zone (3-AP) has a strong anti-tumour activity against a variety of tu-
mour cells, and the anti-tumour activity of the N4 group lipoprotein
(alkyl) modification is remarkably improved14,29,30. More so, the anti-
tumour activity of thiosemicarbazones could be increased by co-
ordination with metal ions (Cu2+, Ga3+, Zn2+, etc.)16. The colorimetric
method of 3- (4, 5-dimethylthiazole-2-yl) −2, 5-diphenyltetrazolium
ammonium bromide (MTT) was used to detect the cytotoxicity of li-
gands and complexes. The antiproliferative of AP44eT·HCl and Zn
(AP44eT)(NO3)2 were examined by using MCF-7 Cells (human breast
adenocarcinoma cell line), and half-maximal inhibitory concentration
(IC50) was evaluated. Therefore, we designed and synthesised a thio-
semicarbazone ligand (AP44eT·HCl) which was modified at the N4
position of 3-AP to form a new ligand. The Zn(II) nitrate (60 μΜ) did
not show notable antiproliferative activity against the MCF-7 cells. As a
vehicle control, cisplatin showed a relatively low anti-tumour activity
space group P21/n. AP44eT•HCl adopted an E-isomeric to form a hy-
drogen bond between N2 and N5, and a free chloride ion formed hy-
drogen bond with C6, N3 and C8 simultaneously. In the Zn(AP44eT)
(NO3)2, the coordination polyhedron was a deformed octahedron in
which AP44eT·HCl was coordinated to Zn by two nitrogen atoms of
pyridine, a thiosemicarbazide, sulfur donor and two nitrates were co-
ordinated from the sides of the metal. The CeS bond and the CeN bond
of AP44eT·HCl did not change notably after coordination with the zinc
atom, but the configuration changed from the E-configuration to the Z-
isomeric form.
Spectroscopic properties
AP44eT·HCl and Zn(AP44eT)(NO3)2 were dissolved in DMSO as
10 mM stock solutions and diluted in cell culture medium to different
concentrations for the involved biological experiment. Therefore, the
UV–Vis spectra of AP44eT·HCl and Zn(AP44eT)(NO3)2 were tested in
0.1% DMSO PBS solution (pH 7.4), and the results are shown in Fig. S1
and S2. The results showed that the Zn(AP44eT)(NO3)2 was stable in
0.1% DMSO PBS solution (pH 7.4) for 48 h (Fig. S2). The AP44eT·HCl
had a strong absorption in the 300–400 nm region and the maximum
absorption band (λmax) was 352 nm while Zn(AP44eT)(NO3)2 showed a
stronger absorption bands around 340–500 nm and the maximum ab-
sorption band (λmax) was 426 nm (Fig. S1). In the PBS solution,
AP44eT·HCl emitted a blue emission centred at 435 nm upon excitation
at 320 nm (Fig. 2A) while the Zn(AP44eT)(NO3)2 emitted a blue
emission centred at 461 nm upon excitation at 355 nm (Fig. 2B).
(IC50 = 15.16
(IC50 = 0.68
1.75 μΜ). The anti-tumour activity of AP44eT·HCl
0.04 μΜ) was significantly (p < 0.001) higher than
that of 3-AP (IC50 = 4.88
0.31 μΜ). The IC50 values of Zn(AP44eT)
(NO3)2 was 0.23
0.01 μΜ, meaning that the Zn(AP44eT)(NO3)2
showed considerably (p < 0.05) higher anti-cancer activity than that
of the free AP44eT·HCl. The Zn (II) thiosemicarbazone complexes tend
to be more cytotoxic than thiosemicarbazone ligands alone, and this
experimental result is consistent with studies from other labora-
tories22,27,31,32. It is known that the toxicity of Zn (II) complex of dif-
ferent cell lines depends on the distribution of sub-cellular layers in
tumour cells27
.
Promoting apoptosis mechanism
Zn(AP44eT)(NO3)2 localised in mitochondria
Mitochondria are structures that provide energy in cells and the
main places for cells to breathe aerobically33. Therefore, it is important
to study the mechanism of mitochondria-targeted drugs in promoting
apoptosis34–36. Mitochondria-mediated apoptosis is usually associated
with mitochondrial membrane changes that cause the leakage of
apoptotic factors37. The lipophilic fluorescent probe JC-1 (5, 5, 6, 6′-
The good cell permeability and excellent photo-stability of Zn
(AP44eT)(NO3)2 made it easier to know their location within the cell,
providing vital information about their potential mechanism of action.
The blue AP44eT·HCl fluorescence at 435 nm and Zn(AP44eT)(NO3)2 at
461 nm were again seen in MCF-7 cells (Fig. 3). As the mitochondria are
2

J. Qi, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127340
Fig. 2. (A) Photoluminescence (PL) emission spectra of AP44eT·HCl; (B) Photoluminescence (PL) emission spectra of the Zn(AP44eT)(NO3)2.
tetrachloro-1, 1′, 3, 3′-tetraethyl-imidacarbocyanine iodide) was used
to analyse the mitochondrial membrane potential changes (Δψm). The
reduction of JC-1 aggregates (red fluorescence) and increment of JC-1
monomers (green fluorescence) showed the decrease of Δψm. The JC-1
probe in the control cell was mainly in the form of an aggregated state,
indicating that the mitochondrial membrane potential of the cells was
at a normal level (Fig. 4A). After MCF-7 cells incubated with 10 μM of
AP44eT·HCl and Zn(AP44eT)(NO3)2 for 30 mins, more red fluorescence
was replaced by the green fluorescence compared to the control
(Fig. 4A). Mitochondria appeared orange after AP44eT·HCl treatment
(suggesting a partial loss of mitochondrial membrane potential) while
the JC-1 aggregates (red fluorescence) almost completely disappeared
after treatment with the Zn(AP44eT)(NO3)2 (which means the complete
loss of the mitochondrial membrane potential). Importantly, the mi-
tochondrial membrane potential completely collapsed and the mor-
phology of the cells changed from shuttle to round after the incubation
with Zn(AP44eT)(NO3)2 (Fig. 4A). The collapse of mitochondrial
membrane potential caused irreversible release of pro-apoptotic factors.
After the MCF-7 cells were exposed to 2 μM of AP44eT·HCl and Zn
(AP44eT)(NO3)2 for 24 h, the expression levels of p53, Bax and Cyt C
proteins increased observably while the expression of anti-apoptotic
protein (Bcl-2) decreased (Fig. 5).
permeability transition pore (PT pore) is necessary and sufficient for
apoptosis, such as activated caspase-3 and 919. The pro-apoptotic factor
released by the opening of the PT pore leads to apoptosis while PT pore
closure prevents apoptosis. The effect of AP44eT·HCl and Zn(AP44eT)
(NO3)2 on promoting caspase-3 and 9 activities were assayed by flow
cytometry. After incubation with the control, AP44eT·HCl and Zn
(AP44eT)(NO3)2, the activated caspase-3 protein levels increased to
11.29%, and 30.59% as per the control, and the activated caspase-9
protein levels increased to 15.29%, and 37.09% as per the control,
respectively (Fig. 4B and C).
Zn(AP44eT)(NO3)2 significantly promotes apoptosis
The activation of the caspase family proteins may lead to cell
apoptosis. We used Annexin V FITC/PI staining to analyse the effect of
AP44eT·HCl and Zn(AP44eT)(NO3)2 on promoting the MCF-7 cell
apoptosis. After MCF-7 cells were incubated with 2 μM of AP44eT·HCl
and Zn(AP44eT)(NO3)2 for 24 h, the treated cells were collected and
analysed by flow cytometry. Quantification of the result showed that
the MCF-7 cell induced cell early apoptosis of 7.68% for AP44eT·HCl
and 29.46% for Zn(AP44eT)(NO3)2 which were more effective than the
vehicle-treated control (Fig. 6A). The results of the apoptosis assay
showed that the coordination with the Zn atom considerably increased
the activity of the AP44eT·HCl.
In the process of apoptosis, the mitochondrial transmembrane po-
tential dissipation is the major manifestation of increased mitochon-
drial membrane permeability38. Studies showed that mitochondrial
Fig. 3. The intracellular localisation of AP44eT·HCl (5 μM) and Zn(AP44eT)(NO3)2 (5 μM) in the MCF-7 cell line.
3

J. Qi, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127340
Fig. 4. (A) Assay of the MCF-7 cells mitochondrial membrane potential with JC-1 as fluorescence probe staining method; (B) The effect of the caspase-3 activation of
the MCF-7 cells; (C) The effect of the caspase-9 activation of the MCF-7 cells. The cell line was treated with the control, AP44eT·HCl and Zn(AP44eT)(NO3)2.
Zn(AP44eT)(NO3)2 significantly inhibits the cell cycle
control, respectively; and G1 increased by 9.8% and 19.62% compared
with the vehicle-treated control (Fig. 6B). These results showed that the
Zn(AP44eT)(NO3)2 anti-cell proliferation property was via inhibiting or
delaying cell cycle progression in the G1 and S phases. The results of
apoptosis and cell cycle analysis were line with the cytotoxicity ex-
periments.
The relationship between the cell cycle and tumour has always been
one of the hot topics in life science research. Tumour is a clonal po-
pulation formed by the infinite proliferation of cells that have lost cell
cycle control39,40. Thus, we used the PI staining of MCF-7 cell DNA to
examine the effect of AP44eT·HCl and Zn(AP44eT)(NO3)2 on the cell
cycle (Fig. 6B). After treated with 2 μM of AP44eT·HCl and Zn(AP44eT)
(NO3)2 for 24 h, the percentage of the MCF-7 cells in the G2 phase
decreased by 4.39% and 13.85% compared with the vehicle-treated
In summary, we have synthesised
a novel thiosemicarbazone
AP44eT·HCl with fluorescent properties and high anti-tumour activity.
The fluorescence emission wavelength (435 nm) of AP44eT·HCl pre-
sented
a redshift (461 nm) after the coordination with Zn(II).
Fig. 5. Western blot analysis of p53 and apoptosis-related proteins (Bax, Bcl-2 and Cyt C) in the MCF-7 cells treated by the control, AP44eT·HCl and Zn(AP44eT)
(NO3)2 for 12 h.
4

J. Qi, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127340
Fig. 6. (A) The representative dot plots of PI and Annexin V double staining on the MCF-7; (B) The effect of the cell cycle of MCF-7. The cell line was treated with the
control, AP44eT·HCl and Zn(AP44eT)(NO3)2.
AP44eT·HCl and Zn(AP44eT)(NO3)2 were localised in mitochondria
organelles. Compare to AP44eT·HCl, Zn(AP44eT)(NO3)2 was more ef-
fective in causing mitochondrial membrane potential collapse, pro-
moting caspase-3 and 9 activation, resulting in cell cycle arrest and
apoptosis. Briefly, Zn(AP44eT)(NO3)2 has a higher advantage in the
fluorescence emission wavelength, mitochondrial-targeted anti-tumour
activity, the collapse of mitochondrial membrane potential, promotion
of apoptosis and inhibition of cell cycle, etc. These experimental results
may be useful in the development of novel Zn(AP44eT)(NO3)2 as tu-
mour therapy and imaging agents.
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Funding
This work was supported by Science and Technology projects of
Henan Province (202102310476, 202102310478), Pingdingshan
University PhD Startup Fund under Grant (PXY-BSQD-202003) and
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Declaration of Competing Interest
14. Kowol CR, Miklos W, Pfaff S, et al. Impact of Stepwise NH2 -Methylation of Triapine
on the Physicochemical Properties, Anticancer Activity, and Resistance
Circumvention. J Med Chem. 2016;59:6739–6752.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
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