Novel 2-pyridinecarboxaldehyde thiosemicarbazones Ga(III) complexes with a high antiproliferative activity by promoting apoptosis and inhibiting cell cycle

Article abstract of DOI:10.1016/j.ejmech.2017.04.009

Two types of 2-pyridinecarboxaldehyde thiosemicarbazones Ga(III) complexes, which are 2:1 and 1:1 ligand/Ga(III) complexes, were synthesized and determined by X-ray single crystal diffraction. The antiproliferative activity of these Ga(III) complexes have

Full text of DOI:10.1016/j.ejmech.2017.04.009

Accepted Manuscript
Novel 2-pyridinecarboxaldehyde thiosemicarbazones Ga(III) complexes with a high
antiproliferative activity by promoting apoptosis and inhibiting cell cycle
Jinxu Qi, Jungang Deng, Kun Qian, Liang Tian, Jiaming Li, Kunhuan He, Xueren
Huang, Zhen Cheng, Yunyun Zheng, Yihong Wang
PII:
S0223-5234(17)30264-7
10.1016/j.ejmech.2017.04.009
EJMECH 9356
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 December 2016
Revised Date: 4 April 2017
Accepted Date: 5 April 2017
Please cite this article as: J. Qi, J. Deng, K. Qian, L. Tian, J. Li, K. He, X. Huang, Z. Cheng, Y.
Zheng, Y. Wang, Novel 2-pyridinecarboxaldehyde thiosemicarbazones Ga(III) complexes with a high
antiproliferative activity by promoting apoptosis and inhibiting cell cycle, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.009.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel 2-pyridinecarboxaldehyde thiosemicarbazones Ga(III) complexes with a high antiproliferative
activity by promoting apoptosis and inhibiting cell cycle
a
a
b
b
b
Jinxu Qi a,b, Jungang Deng c, Kun Qian , Liang Tian , Jiaming Li , Kunhuan He , Xueren Huang ,
Zhen Cheng d, Yunyun Zheng b* and Yihong Wang a*
a School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China.
b Qinzhou University, 12 Binhai Avenue, Qinzhou, Guangxi, China.
c Affiliated Hospital, Guilin Medical College, 15 Lequn Road, Guilin, Guangxi, China.
d Stanford Cancer Institute, Member of Academic Council, Stanford University, USA.
*Corresponding author: Yihong Wang
Address: 2 Southeast Road, Nanjing, China. Zip code: 211189
Email: yihongwang@seu.edu.cn
Phone: 159-9631-5308
*Corresponding author: Yunyun Zheng
Address: 12 Binhai Avenue, Qinzhou, Guangxi, China. Zip code: 535011
Email: yunyunjinxu@163.com
Keywords: Gallium (III); thiosemicarbazone; anti-cancer mechanism; cell cycle; cell apoptosis.
Abstract
Two types of 2-pyridinecarboxaldehyde thiosemicarbazones Ga(III) complexes, which are 2:1 and 1:1
ligand/Ga(III) complexes, were synthesized and determined by X-ray single crystal diffraction. The
antiproliferative activity of these Ga(III) complexes have been examined to illuminate the
structure-activity relationships essential to form Ga(III) complexes with remarkable anticancer activity.
In addition, Ga(III) complexes where the metal/ligand ratio was 1:1 (C4) had observably higher
antiproliferative activity than 1:2 (C3). Ga(III) complexes caused a marked increase of caspase-3 and 9
activity in NCI-H460 cells compared to the metal free ligand. Caspase activation was somewhat
mediated by the release of Cyt C from mitochondria after incubation with selected agents. Both types of
Ga(III) complexes showed more effective in inhibition of the G1/S transition than the ligand alone.
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1 Introduction
Iron plays an important role in cell proliferation, ribonucleotide reductase, DNA synthesis,
mitochondrial electron transport, redox reaction, etc. [1, 2]. The more rapid proliferation of tumor cells
than normal cells requires more irons [3]. In fact, tumor cells can express more transferrin receptor 1
(TfR1) than normal cells, and can quickly absorb iron from transferrin (Tf), so the iron chelator on the
tumor cells have a certain selectivity [1, 4]. Ga(III) shares certain chemical properties with Fe(III) with
respected to its ion radius and its binding to transferrin (Tf) in the circulation, so an important therapeutic
effect factor of gallium is disrupt the intracellular iron absorption and metabolism[5, 6]. Therefore,
development of Ga(III) complexes has become a chemotherapy strategy showing significant promise
[7-9].
Gallium, the second metal ion that has been used in cancer therapy after platinum, has been proven to
be effective in clinical trials against a rage of malignancies [10-14]. As a simple gallium salt, gallium
nitrate was first applied to the treatment of lymphoma and bladder cancer and has achieved an exciting
therapeutic effect [13, 15, 16]. Recent studies have shown that a series of gallium compounds have
significant
antitumor
activity,
such
as
gallium
chloride,
gallium
maltolate,
tris(8-quinolonato)gallium(III), pyridine and phenolate ligand complexes of gallium, gallium
thiosemicarbazones and other gallium complexes [11, 13, 15-18]. However, the bioavailability of simple
gallium salts is relatively low, affecting its application in the anti-tumor chemotherapy [18, 19]. To
overcome this deficiency more gallium complexes anchored by multi-dentate chelators were evaluated
[8, 17].
The ternary thiosemicarbazide chelating agents can coordinate with the metal atom to form a stable
complexes, such as Fe, Cu, Mn, Zn, Ga, et al.[17, 20, 21]. Because of the potential biological activity that
includes antitumor, antimicrobial and antimalarial activity, thiosemicarbazones have extensive and
long-term
biomedical
applications
[22-25].
Previous
studies
demonstrated
that
3-aminopyridinecarbaldehyde thiosemicarbazone (3-AP) showed significant antitumor activity both in
vivo and in vitro, which is currently in phase II trials to treat various kinds of malignancies [4, 26-28].
Forming a stable coordination bond of NNS of ligands with the metal is the key to its anti-tumor
proliferation efficacy in vivo and in vitro [1, 29-31]. The phenomenon that thiosemicarbazone-metal
complexes had markedly higher antiproliferative activity than the ligand alone is becoming a research
hotspot reported by many scientists [32-36].
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In order to develop new thiosemicarbazone compounds and gallium complexes as antitumor agents,
we carried out the following studies: (1) design and synthesis of the 2:1 and 1:1 ligand/Ga (III)
complexes (Scheme 1); (2) investigation of their antiproliferative activity on several cell lines
(NCI−H460 human lung cancer cell line, BEL-7402 human hepatoma cell line and T24 human bladder
cancer cell line); (3) comparing antiproliferative activity of the 2:1 and 1:1 ligand/Ga (III) complexes; (4)
determination of the potential inhibition of cell cycle mechanism of the 2:1 and 1:1 ligand/Ga (III)
complexes; (5) investigating the potential promote cell apoptosis mechanism of the 2:1 and 1:1 ligand/Ga
(III) complexes.
2. Results and Discussion
2.1 Synthesis and Crystal structure description
In this article, we successfully synthesized and determined the 2:1 and 1:1 ligand/Ga (III) complexes by
single-crystal X-ray diffraction. Ligand L1 and L2 were prepared straightly forward via Schiff base
condensation reactions and did not require further purification. 2:1 ligand/Ga(III) complexes were
generated by the coordination of ligand with Gallium(III) nitrate, while 1:1 ligand/Ga(III) complexes
were produced by the coordination of ligand with Gallium(III) chloride. All of Gallium(III) complexes
were crystallized from the methanol solution with high purity without further purification.
The crystal data of four Ga(III) complexes was showed in Table 1, and the selected bond lengths was
shown in Table S1-4. The results showed that these four structures are different from each other. C1 and
C3 in the triclinic system, C2 in the monoclinic system, C4 in the orthorhombic system. Interestingly, the
hydroxyl was substituted for nitrate as an anion in C3 relative to C1. The Ga(III) metal center of C1 and
C3 is coordinated by four N atoms (N1, N2, N5 and N6) and two S atoms (S1 and S2) forming a
deformation of octahedral configuration (Fig.1). However, the Ga(III) metal center of C2 and C4 is
coordinated by two N atoms (N1 and N2), S atom (S1) and two Cl atoms (Cl1 and Cl2), leading to a
deformation of trigonal bipyramidal geometry (Fig.1). The asymmetry of the electrons in the degenerate
orbital will lead to the distortion of the geometry of the molecule, which is characteristic of the
Jahn-Teller effect [37]. There is a slight vibration for Ga-N1(6) bond (2.102Å − 2.157Å), Ga–N2(5)
bond (2.037Å − 2.067Å) and Ga-S bond (2.037Å − 2.157Å). The lengths of the coordinating bond are
Ga–S bond, Ga–Cl bond (2.198Å – 2.214Å) and Ga–N bond in descending order. N3 or N7
deprotonated in the crystal structure of Ga(III) complexes, which was also present in other known
compounds [26].
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1
In the H NMR spectra of L1 and L2 the signals of N-NH-C were found in the δ 11.20 and 11.27,
respectively, which is in accordance with hydrogen bonding to the oxygen of DMSO and with the
presence of the ligands in the E configuration [38]. However, no signal was observed for N-NH-C in
the spectra of Ga(III) complexes, indicating that the ligands were protonated after coordination. The
pyridine hydrogens shifted significantly upon coordination and formation of Ga(III) complexes. The
signals of the pyridine carbons, C=N and C=S underwent significant shifts in the 13C NMR spectrum,
indicating the formation of Gallium complex. Infrared (IR) spectroscopy showed that the strong
absorption peak near 1500 nm indicates that the stretching vibration of carbon-nitrogen double bond in
these ligands and Ga(III) complexes. Ultraviolet Visible (UV-Vis) spectrophotometric absorption
analysis confirmed that the ligand and complexes were stable within 48 h in in 0.1% DMSO aqueous
solution (Fig.S1).
2.2 Anticancer properties of Ga complexes
We evaluated the anticancer properties of thiosemicarbazide ligands and Ga(III) complexes on three
malignant tumor cell lines: T24 (human bladder cancer cell line), BEL-7402 (human hepatoma cell line)
and NCI−H460 (human lung cancer cell line). The corresponding ligands and a relevant positive control
(3−AP) were compared with these Ga(III) complexes. All of the thiosemicarbazide ligands and Ga(III)
complexes have a higher anti-proliferation activity than 3-AP for these selected cell lines, while no
pronounced anti-proliferative effect was observed with Ga(NO3)3 (100 µM) alone (Table 2).
Table 2 showed that the anti-proliferative activity of L1 (IC50, 1.85 ± 0.21 µM) was similar to that of
L2 (IC50, 1.04 ± 0.06 µM) after a 48 h incubation with NCI−H460 cell. From Table 2 we can observed
that the Ga(III) complexes (C1-C4) had a significantly (p<0.05) increased anti-proliferative activity after
a 48 h incubation in comparison with their free ligands (L1 and L2). This phenomenon of the antitumor
activity of C1 (IC50, 0.61 ± 0.05 µM) was lower than that of C2 (IC50, 0.52 ± 0.03 µM) and the antitumor
activity of C3 (IC50, 0.72 ± 0.05 µM) was lower than that of C4 (IC50, 0.43 ± 0.02 µM) for NCI−H460
cell means that a molar ratio of 1:1 demonstrated significantly (p<0.05) more activity than the Ga(III)
complexes at a 2:1 ligand to Ga(III) molar ratio. As L2 has higher anti-proliferative activity in the ligands
while C4 presented better activity in Ga(III) complexes on NCI−H460 cells, these compounds were
selected for further investigation.
2.3 Cell cycle analysis
Cells can complete self-replication through the cell cycle, which is the basic process of all life. To further
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evaluate the influence of the ligands and Ga(III) complexes on cell cycle, PI was used for staining
NCI−H460 cell and analyzed by flow cytometry (Fig.2). Quantification of the result revealed that the
percentage of G1 phase cells were found to be significantly increased 21.37%, 23.75%, 25.25% and
27.89% than vehicle treated control after the 24h incubation of 3-AP, L2, C3 and C4, respectively (Fig.2).
The outcome showed that the selected agents could inhibit the G1/S transition in the cell cycle.
2.4 Cell apoptosis assay
To quantify the effect of ligands and Ga(III) complexes on promoting NCI−H460 cell apoptosis, we
stained the cells with Annexin V−FITC/PI and analyzed by flow cytometry. Quantification of the result
revealed that 3-AP, L2, C3 and C4 were found to be 4.67%, 5.42%, 9.63% and 15.4% more effective than
vehicle treated control, inducing cell early apoptosis (Fig.3). The result of apoptosis assay demonstrated
that the ligand/Ga(III) ratio of 1:1 had more activity of promoting cell apoptosis than 2:1 molar ratio,
which was consistent with MTT assay.
2.5 Intracellular ROS measurements
Free radicals, such asreactive oxygen species (ROS), produce too much or antioxidant capacity decline
will make the balance disorders, biological macromolecular damage, affecting cell function, and then
induceing cell apoptosis [39, 40]. To examine the effects of thiosemicarbazide ligands and Ga(III)
complexes on intracellular ROS in NCI−H460 cells, the fluorescent 2,7-di-chlorofluorescein (DCF)
probe and flow cytometry were employed (Fig. 4). The DCF fluorescence intensity of cells incubated
with the 3-AP, L2, C3 and C4 showed that the peak was shifted to the right relative to the control cells,
indicating that these cell samples suffered increased oxidative damage by the above agents (Fig.4A).
Quantification of the DCF fluorescence peaks in parts A of Fig.4 demonstrated that the H2DCF oxidation
of 3-AP, L2, C3 or C4 significant (p < 0.001) increase than control cells (Fig.4B). Intracellular reactive
oxygen species (ROS) overload can inhibit the proliferation of cells in G1, S and G2 phase, and G1 phase
arrest is related to the process of defense against oxidative damage and anti-apoptosis [41-43].
2.6 Mitochondrial membrane potential assay
When the production of ROS is too high and oxygen stress reaches a certain level, the mitochondrial
membrane permeability might be changed, resulting in the release of Cyt C and other apoptosis factors
[25, 44]. JC−1 fluorescent probe (5, 5, 6, 6′-tetrachloro-1, 1', 3, 3′-tetraethyl-imidacarbocyanineiodide)
was used to detect the effect of ligands and Ga(III) complexes on mitochondrial membrane potential
(∆ψm). When the mitochondrial membrane potential is high, JC−1 accumulates in the matrix of the
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mitochondrion to form a polymer, which can produce red fluorescence. However, when the
mitochondrial membrane potential is low, JC−1 cannot accumulate in the matrix of mitochondria, and
produced green fluorescence. From the fluorescence inverted microscope photographs (Fig.5) we can
observed that the NCI−H460 cells treated with 3-AP, L2, C3 or C4 showed a green fluorescence signal
while vehicle treated controls presented red, which meant that Ga(III) complexes could lead to the
collapse of the mitochondrial transmembrane potential (∆ψm) (Fig.5). Especially, the mitochondrial
membrane potential (∆ψm) of the cells incubated with C4 was almost completely lost.
2.7 The expression of pro-apoptotic proteins assay
The changes in mitochondrial membrane potential may contribute to the release of Cyt C and other
apoptosis factors [45, 46]. The release of pro-apoptotic factors may affect the expression of
anti-apoptotic proteins [47]. Western blot analysis was take out to examine effect of ligands and Ga(III)
complexes on the expression of pro-apoptotic and anti-apoptotic protein in NCI−H460 cells. Incubating
NCI−H460 cells with L2, C3 and C4 raised the levels of Cyt C to 160%, 166% and 172% relative to the
control, respectively (Fig.6B). After incubation with L1, C3 and C4, the activated caspase 9 protein
levels increased to 139%, 142%, and 167% of the control, and the activated caspase 3 protein levels
increased to 151%, 168%, and 221% of the control, respectively (Fig.6B). The results demonstrated that
the intrinsic mitochondrial signal pathway was involved in Ga(III) complexes induced apoptosis. L2, C3
and C4 could decrease Bcl-2 (anti-apoptotic) protein expression and promote Bax (pro-apoptotic protein)
expression compared with control (Fig.6). The significant increase of Bax/Bcl-2 ratio resulting in
enhanced permeability of the mitochondrial membrane for the release of pro-apoptotic proteins, such as,
Cyt C (Fig.6) [36]. Mitochondrial pathway induced apoptosis could be described as follows: firstly,
upstream mitochondrial signaling molecules affect the mitochondrial membrane, and activate Bcl-2
family proteins to form the protein channels, which would open the mitochondrial permeability transition
pore (PTP) to release apoptotic active substances (such as, Cyt C); secondly, these substances would
cause the activation of the downstream caspase family protein and act on the corresponding substrate to
induce cells apoptosis [25, 48-50].
The foregoing results demonstrated that coordination of Ga(III) to thiosemicarbazones might be a
feasible strategy for enhancing both ligand and metal activities. We observed that the coordination of
Ga(III) and ligand had a direct influence on the bioavailability properties of the compounds. Obviously,
Ga(III) played an important role in enhancing the antiproliferative activity of the Ga(III) complexes.
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Moreover, the type of 1:1 ligand/Ga complex is better than 2:1 ligand/Ga complex in various
bioactivities (antitumor activity, promoting cell apoptosis, inhibiting cell cycle, intracellular ROS). So a
simple synthesis method is needed to prepare the type of 1:1 ligand/Ga complex. The study results are
meaningful in the development of gallium compounds in medicinal chemistry.
3. Conclusion
In this study we demonstrated an increase of cytotoxicity by complexation of Thiosemicarbazone ligands
to gallium(III). In general, the type of 1:1 ligand/Ga complex showed higher antitumor activity than the
type of 2:1 ligand/Ga complex. Anticancer mechanism assays illustrated that the increased cytotoxicity
of Ga(III) complexes than ligands not only partly depended on the ability of these complexes to promote
NCI-H460 cell apoptosis, but also inhibited the G1/S transition in the cell cycle. Ga(III) complexes
promoting cancer cell apoptosis might involve the intrinsic ROS-mediated mitochondrial pathway
accompanied by the regulation of Bcl-family proteins and the activation of caspase family proteins. In
conclusion, these experimental results may be beneficial in the development of new antineoplastic
agents.
4 Materials and Methods
4.1 Material
2-Pyridinecarboxaldehyde, Thiosemicarbazide, Ga(NO3)3 and GaCl3 were purchased from Innochem
Company (Shanghai, China). Other chemicals and solvents were analytically pure. Water was reaction
distillated before use. The NCI-H460, BEL-7402 and T24 cell lines were purchased from Chinese
academy of sciences.
4.2 Synthesis and characterization of Ga(III) complexes
4.2.1 Synthesis and characterization of ligands (L1 and L2).
The ligands L1 and L2 were prepared by previous published methods [35, 36]. In brief,
2-pyridinecarboxaldehyde (10 mmol) was dissolved in methanol (10 mL), and thiosemicarbazide was
added while stirred, followed by 5 drops of glacial acetic acid, and the mixture solution was refluxed for
4–6 h to form a bright yellow solution. When the mixture solution was cooled to 0 , the precipitate was
filtered, ice-cold water washed, and dried in a vacuum desiccator.
N,N-dimethyl-2-(2-pyridinylmethylene)hydrazinecarbothioamide (L1): yield 63%. Anal. Calcd for
C9H12N4S: C, 51.90; H, 5.81; N, 26.90. Found: C, 51.88; H, 5.84; N, 26.92. IR (main peaks): 2919 (vs,
amide), 1537 (s, pyridine), 1427 (s, C=N), 1377s, 1307s, 1217s, 1156vs, 1098vs, 1054vs, 879 (m, C=S),
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768vs, 720vs, 574 m. MS m/z (%) 207.08 (M − H, 100). 1H NMR (400 MHz, DMSO-d6) δ 11.20 (s, 1H,
N-NH-C), 8.60 – 8.55 (m, 1H, Py-H), 8.24 (s, 1H, N=CH), 7.90 (d, J = 8.0 Hz, 1H, Py-H), 7.87 – 7.82
(m, 1H, Py-H), 7.37 (d, J = 1.0 Hz, 1H, Py-H), 3.31 (s, 6H, N(CH3)2). 13C NMR (101 MHz, DMSO-d6
)
δ 180.97 (s, C=S), 154.05 (s, C=N), 149.86 (s, Py–C), 144.35 (s, Py–C), 137.19 (s, Py–C), 124.35 (s,
Py–C), 119.95 (s, Py–C), 42.72 (s, CH3).
1-pyrrolidinecarbothioic acid 2-(2-pyridinylmethylene)hydrazide (L2): yield 69%. Anal. Calcd for
C11H14N4S: C, 56.38; H, 6.02; N, 23.91. Found: C, 56.41; H, 6.04; N, 23.89. IR (main peaks): 3061vs,
2966 (vs, amide), 2875m, 1555 (s, Py), 1439 (s, C=N), 1350s, 1274s, 1165vs, 911 (vs, C=S), 782vs,
567m. MS m/z (%) 243.10 (M − H, 100). 1H NMR (400 MHz, DMSO-d6) δ 11.27 (s, 1H, N-NH-C),
8.57 (d, J = 4.7 Hz, 1H, Py-H), 8.20 (s, 1H, N=CH), 7.84 (ddd, J = 10.5, 9.3, 4.7 Hz, 2H, Py-H), 7.40 –
7.32 (m, 1H, Py-H), 3.76 (s, 4H, Py-H), 1.89 (s, 4H, Pyrrolidine-H). 13C NMR (101 MHz, DMSO-d6) δ
176.86 (s, C=S), 154.13 (s, C=N), 149.87 (s, Py–C), 143.59 (s, Py–C), 137.19 (s, Py–C), 124.27 (s, Py–
C), 119.89 (s, Py–C), 52.69 (s, Pyrrolidine-C), 26.45 (s, Pyrrolidine-C).
4.2.2
Synthesis
and
characterization
of
[bis(N,N-dimethyl-2-(2-pyridinylmethylene)hydrazinecarbothioamide)-N,N,S-gallium(III)]
Nitrate (C1).
L1 (1 mmol) was dissolved in 10 mL methanol, and then Ga(NO3)3 (0.5 mmol) dissolved in methanol
(15 mL) was added. The mixture was gently refluxed for 10 min. Upon cooling, the yellow solution was
volatilized slowly. A few days later, single yellow crystals of Ga complexes were obtained from the
filtrate. The Ga complexes were isolated, washed three times with EtOH and dried in a vacuum
desiccator. Yield 85%. Anal. Calcd for C18H24GaN9O4S2: C, 39.58; H, 4.06; N, 23.08; O, 8.79. Found: C,
39.57; H, 4.05; N, 23.06; O, 8.78. IR (main peaks): 2926 (m, amide), 1514 (vs, Py), 1377 (s, C=N),
1310vs, 1252vs, 1139vs, 1054m, 911 (vs, C=S), 775m. MS m/z (%) 482.03 (M−, 100). 1H NMR (400
MHz, DMSO-d6) δ 9.06 (s, 1H, Py-H), 8.84 (d, J = 5.1 Hz, 1H, Py-H), 8.49 (s, 1H, Py-H), 8.28 – 8.19
(m, 2H, N=CH), 8.00 (d, J = 7.4 Hz, 2H, Py-H), 7.89 (d, J = 7.8 Hz, 1H, Py-H), 7.78 – 7.74 (m, 1H,
Py-H), 7.63 – 7.51 (m, 1H, Py-H), 3.27 (d, J = 26.1 Hz, 12H, N(CH3)2). 13C NMR (101 MHz,
DMSO-d6) δ 177.30 (s, C=S), 146.53 – 146.31 (m, C=N), 145.92 (d, J = 31.0 Hz, C=N), 145.51 –
145.33 (m, Py–C), 145.12 (d, J = 33.0 Hz, Py–C), 142.80 (s, Py–C), 138.24 (s, Py–C), 133.69 (s, Py–
C), 127.61 (s, Py–C), 126.38 (d, J = 13.2 Hz, Py–C), 125.51 (s, Py–C), 40.49 (s, CH3).
4.2.3
Synthesis
and
characterization
of
[N,N-dimethyl-2-(2-pyridinylmethylene)hydrazinecarbothioamide-N,N,S-gallium(III)]
bis(Chloride) (C2).
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The procedure of C2 is same as C1 except that Ga(NO3)3 (0.5 mmol) was replaced by GaCl3 (1 mmol).
Yield 78%. Anal. Calcd for C9H11Cl2GaN4S: C, 31.07; H, 3.19; N, 16.10. Found: C, 31.10; H, 3.17; N,
16.11. IR (main peaks): 2929 (vs, amide), 1603m, 1514 (vs, pyridine), 1368 (s, C=N), 1305s, 1248s,
1136s, 1054s, 903 (s, C=S), 775vs, 666m, 624m. MS m/z (%) 275.00. (M − 2Cl, 100). 1H NMR (400
MHz, DMSO-d6) δ 9.04 (s, 1H, Py-H), 8.20 (d, J = 1.4 Hz, 1H, N=CH), 7.99 (t, J = 6.6 Hz, 2H, Py-H),
7.57 (s, 1H, Py-H), 3.31 – 3.21 (m, 6H, N(CH3)2). 13C NMR (101 MHz, DMSO-d6) δ 177.30 (s, C=S),
145.27 (s, C=N), 144.95 (s, Py–C), 142.81 (s, Py–C), 138.23 (s, Py–C), 127.61 (s, Py–C), 126.44 (s,
Py–C), 40.58 (s, CH3).
4.2.4
Synthesis
and
characterization
of
[bis(1-pyrrolidinecarbothioic
acid
2-(2-pyridinylmethylene)hydrazide) -N,N,S-gallium(III)] hydroxide (C3).
The procedure of C3 is same as C1 except that L1 was replaced by L2. Yield 82%. Anal. Calcd for
C22H24GaN8OS2: C, 47.75; H, 4.92; N, 20.25; O, 2.89. Found: C, 47.73; H, 4.95; N, 20.22 O, 2.32. IR
(main peaks): 2963 (vs, amide), 1605 (vs, pyridine), 1450 (s, C=N), 1368 s, 1242s, 1164s, 913 (vs, C=S),
1
776vs, 650m, 516m. MS m/z (%) 534.09 (M−, 100). H NMR (400 MHz, DMSO-d6) δ 9.04 (s, 1H,
Py-H), 8.84 (d, J = 5.2 Hz, 1H, Py-H), 8.47 (s, 1H, Py-H), 8.27 – 8.17 (m, 2H, N=CH), 7.99 (t, J = 6.8
Hz, 2H, Py-H), 7.87 (d, J = 7.8 Hz, 1H, Py-H), 7.77 – 7.73 (m, 1H, Py-H), 7.61 – 7.51 (m, 1H, Py-H),
3.81 – 3.55 (m, 8H, Pyrrolidine-H), 1.98 – 1.88 (m, 8H, Pyrrolidine-H). 13C NMR (101 MHz,
DMSO-d6) δ 174.13 (s, C=S), 145.36 (s, C=N), 144.89 (s, C=N), 142.76 (s, Py–C), 137.63 (s, Py–C),
127.50 (s, Py–C), 126.31 (s, Py–C), 50.78 (s, Pyrrolidine-C), 49.06 (s, Pyrrolidine-C), 25.05 (s,
Pyrrolidine-C), 24.51 (s, Pyrrolidine-C).
4.2.5
Synthesis
and
characterization
of
[1-pyrrolidinecarbothioic
acid
2-(2-pyridinylmethylene)hydrazide-N,N,S-gallium(III)] bis(Chloride) (C4).
The procedure of C4 is same as C3 except that Ga(NO3)3 (0.5 mmol) was replaced by GaCl3 (1 mmol).
Yield 83%. Anal. Calcd for C11H13Cl2GaN4S: C, 35.33; H, 3.50; N, 14.98. Found: C, 35.30; H, 3.53; N,
14.96. IR (main peaks): 2958 (vs, amide), 2866vs, 1604 (vs, pyridine), 1457 (s, C=N), 1282s, 1179s,
1118s, 907 (vs, C=S), 774vs, 647vs, 577m. MS m/z (%) 301.00 (M − 2Cl, 100). 1H NMR (400 MHz,
DMSO-d6) δ 9.02 (s, 1H, Py-H), 8.20 (td, J = 7.8, 1.4 Hz, 1H, N=CH), 7.98 (dd, J = 15.8, 6.5 Hz, 2H,
Py-H), 7.61 – 7.51 (m, 1H, Py-H), 3.68 (dd, J = 28.5, 21.6 Hz, 4H, Pyrrolidine-H), 1.96 – 1.84 (m, 4H,
Pyrrolidine-H). 13C NMR (101 MHz, DMSO-d6) δ 174.15 (s, C=S), 145.97 (d, J = 18.1 Hz, C=N),
144.88 (s, Py–C), 142.75 (s, Py–C), 137.63 (s, Py–C), 127.50 (s, Py–C), 126.34 (s, Py–C), 48.59 (s,
Pyrrolidine-C), 25.02 (d, J = 6.9 Hz, Pyrrolidine-C).
4.3 Determination of structure of Ga(III) complexes
X-ray crystallographic data of Ga(III) complexes (C1-C4) were collected by a Bruker SMART Apex II
CCD diffractometer using graphite-monochromated Mo-Kα (λ = 0.71073 Å) radiation. The SADABS
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was used for empirical adsorption corrections. The structures were solved by Olex2 methods and refined
against F2 by full-matrix least-squares methods using the Olex2 [51]. All of the atoms except H were
refined anisotropically. Hydrogen atoms were placed in geometrically ideal positions and constrained to
ride on their parent atoms by Olex2. The crystallographic data for Ga(III) complexes are showed in Table
1. Selected bond lengths and angles are given in Table S1-4. Crystal structure analysis data has been
deposit in Cambridge crystal data center. Crystal data can get free from the Cambridge Crystallographic
Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.
4.4 Cytotoxicity assay (MTT)
Ligands and Ga complexes were dissolved in DMSO as 10mM stock solutions and diluted in cell culture
medium to different concentration (10, 5, 2.5 1, 0 µM). This DMSO concentration (< 0.1%) had no effect
on cell proliferation. The NCI-H460, BEL-7402 and T24 cell lines were grown at 37 , 5% CO2/95% air
in a ThermoFisher incubator. The cell cytotoxicity was determined by MTT method according to the
published methods. In brief, 5 × 104 cells per well were seeded in triplicate in 96 well plates and
incubated for 24 h at 37 , 5% CO2/95%. Then the medium was replaced with the respective medium
containing ligands or Ga complexes at different concentrations and incubated for 48 h. The final DMSO
concentration in all experiments was less than 0.1% in medium. 10 µL of MTT was added and the
medium was removed after 4 h of incubation. The absorbance of the converted dye in living cells was
measured at a wavelength of 570 nm after 100 µL of DMSO add. IC50 values were determined by the
nonlinear multipurpose curve-fitting program GraphPad Prism. All of the tests were repeated at least
triplicate.
4.5 Cell Cycle Distribution Analysis
2 mL of 1 × 105 cells/ mL NCI−H460 cells were cultured in 70 mm culture dishes, cultivated at least 12 h,
and treated with 10 µM of 3-AP, L2, C3 or C4 for 24h. Cells were collected and three times with cold
PBS, and then fixed with 75% ethanol overnight at -20 . The samples washed with cold PBS, stained
with PI for 30 min, and then analyzed by flow cytometry (FACScan, Becton Dickinson, San Jose, CA).
For each sample, 10,000 events were recorded. Data analysis was performed by MFLT32 software.
4.6 Cell apoptosis assay
The NCI-H460 cell apoptotic events induced by 3-AP, L2, C3 and C4 were determined by Annexin V
and PI staining according to the manufacturer’s protocol for the Annexin V: FITC Apoptosis Detection
Kit (Bection Dickinson). 2 mL of 1 × 105 cells/ mL were incubated at 37 , 5% CO2/95% with 5 µM of
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above agents for 12 h. The BEL-7402 cells were suspended in 100 µL annexin V−binding buffer (2.5
mM CaCl2, 140 mM NaCl, 10 mM Hepes/NaOH, pH 7.4), and 5µL each of annexin V and PI were added
to these samples. Next, these samples were incubated for 15 min at room temperature and then assayed
by flow cytometric analysis (FACScan, Bection Dickinson, San Jose, CA). The rate of cell apoptosis was
analyzed by FlowJo software. All of the tests were repeated at least triplicate.
4.7 Intracellular ROS measurements
Intracellular ROS generation of NCI-H460 cells was measured using Reactive oxygen species assay kit.
2 mL of 1 × 106 cells/ mL cells were induced with 20 µM of 3-AP, L2, C3 and C4 at 37 for 1 h, serum
free medium washed twice. DCFH-DA could pass through the cell membrane to produce DCFH by
hydrolysis of the intracellular esterase. Intracellular reactive oxygen species (ROS) can oxidize DCFH to
produce fluorescent DCF. Therefore, the detection of DCF fluorescence could inform us of the level of
intracellular reactive oxygen species. The cells were incubated in 2 mL of serum free medium containing
H2DCF−DA (2 µM) for 30 min at 37 , serum free medium washed twice. Cells were collected and
assayed by flow cytometric analysis (FACScan, Bection Dickinson, San Jose, CA), and ten thousand
events were collected for every sample. The DCF fluorescence peaks for these cell samples were
evaluated and quantified by FlowJo software (Fig.2B).
4.8 The mitochondrial membrane potential assay
The change of mitochondrial membrane potential of NCI-H460 cells induced by 3-AP, L2, C3 and C4
were determined by JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolcarbocyanine iodide)
staining according to the manufacturer’s protocol for the JC-1 assay kit (Beyotime, Suzhou). Briefly, 2
mL of 1 × 106 cells/ mL cells were treated with 20 µM of 3-AP, L2, C3 or C4 in 6-well plates for 1 h. The
samples were washed three times with cold PBS and incubated with 1 µg/mL of JC-1 at 37 for 20 min
in the dark. The supernatant was removed washed three times with cold PBS, and then imaged under
fluorescence microscope.
4.9 Western blot analysis
5 mL of 5×105 cells/mL NCI−H460 cells were cultivated at least 12 h in 10 cm plates. Cells were
incubated with 5 µM of 3-AP, L2, C3 or C4 in cell culture medium for 12 h at 37 in a humidified
atmosphere of 5% CO2/95% air in an incubator. Cells were collected and cracked in lysis buffer.
Insoluble jelly were removed by centrifuge for 30 min at 10,000g under 4 . BCA assay kit (Beyotime,
Suzhou) was used to determine protein concentration. The protein samples were assayed by SDS–
polyacrylamide gel electrophoresis. Proteins were then metastasized to poly (vinylidene difluoride)
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(PVDF) membranes (Millipore) in ice bath for 3 h, and then blocked with 5% defatted milk in TBST
buffer for 1 h. The membranes were incubated with 1:1000 dilutions in 5% defatted milk of primary
antibodies for 12h at 4 . After washed three times with TBST for 30 min, the membrane were incubated
with secondary antibodies at 1:1000 dilutions for 1 h at 25 . After washed three times with TBST, the
immunoreactivity was imaged by Amersham ECL Plus (Amersham) western blotting detection. β−actin
was used for detect amount of proteins of each lane.
4.10 Statistical analysis
All experiments were repeated three to six times. Student's t test was applied to evaluate the significance
of differences measured. Results were expressed as mean ± SD and considered to be significant when P <
0.05.
Acknowledgements
This work was supported by National Nature Science Foundations of China (81571812), Priority
Academic Program Development of Jiangsu Higher Education Institutions (1107047002) and the
Hydron contact lens co., LTD (8507040152).
Conflict of interest
The authors declare that they have no conflict of interests.
Supplementary Information
All relative data of the experiment have been deposited into Supplementary Information.
Figure legends
Scheme 1. Synthesis routes for C1-C4.
Fig. 1. The molecular structures of C1-C4 showing the environment around the Ga(III) atom.
Fig. 2. Effect of the cell cycle of NCI−H460 treated with (A) control, (B) 3-AP, (C) L2, (D) C3 and (E)
C4.
Fig. 3. Representative dot plots of PI and annexin V double staining on the NCI−H460 cells treated by
Ga(III) complexes. (A) control, (B) 3-AP, (C) L2, (D) C3 and (E) C4.
Fig. 4. (A) Intracellular ROS was detected in NCI−H460 cells; (B) quantification of the flow cytometric
results in (A) showing the percentage of cells with increased intracellular DCF oxidation compared to
control cells effects on ROS generation induced by different concentrations of Ga(III) complexes.
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Results are the mean ± SD (n = 6−7): *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 5. Assay of NCI−H460 cells mitochondrial membrane potential with JC−1 as fluorescence probe
staining method. (A) control, (B) 3-AP, (C) L2, (D) C3 and (E) C4.
Fig. 6. (A) Western blot analysis of Bcl−2, Bax, Cyt C, Caspase 9 and 3 levels after 3-AP, L2 or Ga(III)
complexes treatment. β−actin was used as internal control. (B) Densitometric analysis of the expression
of Bcl−2, Bax, Cyt C, Caspase 9 and 3. The percentage values are those relative to the control. Results
are the mean ± SD (n = 6−7): *p < 0.05, **p < 0.01, ***p < 0.001.
Table 1 Crystal Data of four Ga(III) complexes.
Table 2 IC50 values of ligands and Ga(III) complexes toward cell lines for 48 h.
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Table 1 Crystal Data of four Ga(III) complexes.
Identification code C1 C2
Empirical formula C18H24GaN9O4S2 C9H11Cl2GaN4S C22H24GaN8OS2 C11H13Cl2GaN4S
C3
C4
Formula weight
564.30
347.90
550.33
373.93
Temperature/K
296.15
296.15
296.15
296.15
Crystal system
triclinic
P-1
monoclinic
P21/c
triclinic
P-1
orthorhombic
P212121
9.709(2)
11.162(3)
13.603(3)
90.00
Space group
a/Å
9.056(5)
11.817(7)
12.301(7)
79.227(9)
88.733(9)
70.142(9)
1215.1(12)
2
9.5625(12)
10.7575(14)
13.7562(18)
90.00
8.7685(16)
11.229(2)
15.974(3)
99.683(3)
98.058(3)
111.840(3)
1403.4(5)
2
b/Å
c/Å
α/°
β/°
109.221(2)
90.00
90.00
γ/°
90.00
Volume/Å3
1336.2(3)
4
1474.1(6)
4
Z
-11 ≤ h ≤ 11,
-15 ≤ k ≤ 15,
-16 ≤ l ≤ 16
0.872
-13 ≤ h ≤ 13,
-15 ≤ k ≤ 15,
-19 ≤ l ≤ 19
0.806
-10 ≤ h ≤ 10,
-13 ≤ k ≤ 13,
-18 ≤ l ≤ 18
1.251
-11 ≤ h ≤ 11,
-12 ≤ k ≤ 13,
-15 ≤ l ≤ 16
1.004
Index ranges
GooF
Final R indexes [allR1 = 0.0837,
R1 = 0.0431,
wR2 = 0.1101
1488979
R1 = 0.0847,
wR2 = 0.2820
1488977
R1 = 0.0352,
wR2 = 0.1160
1488978
data]
wR2 = 0.1437
1488976
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IC50 values of ligands and Ga(III) complexes toward cell lines for 48 h.
Table 2
NCI-H460
4.19 ± 0.32
1.85 ± 0.21
1.04 ± 0.06
0.61 ± 0.05
0.52 ± 0.03
0.72 ± 0.05
0.43 ± 0.02
BEL-7402
4.54 ± 0.33
T24
3-AP
L1
4.34 ± 0.43
2.74 ± 0.34
1.39 ± 0.12
2.56 ± 0.52
2.12 ± 0.45
1.22 ± 0.09
L2
C1
1.83 ± 0.19
1.11 ± 0.23
1.19 ± 0.18
0.89 ± 0.05
C2
1.07 ± 0.28
C3
1.09 ± 0.17
0.79 ± 0.04
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(1) We have design and synthesis of the 2:1 and 1:1 ligand/Ga (III)
complexes;
(2) Ga(III) complexes where the metal/ligand ratio was 1:1 (C4) had
observably higher antiproliferative activity than 1:2 (C3) and ligand
alone;
(3) The increased cytotoxicity of Ga(III) complexes than ligands not only
partly depended on the ability of these complexes to promote NCI-H460
cell apoptosis, but also inhibited the G1/S transition in the cell cycle.