Synthesis, crystal structure and biological evaluation of a main group seven-coordinated bismuth(III) complex with 2-acetylpyridine N 4-phenylthiosemicarbazone

Article abstract of DOI:10.1016/j.bmcl.2013.02.097

Up to now, bismuth(III) complexes with thiosemicarbazones have been comparatively rare. Here, a main group seven-coordinated bismuth(III) complex [Bi(L)(NO₃)₂(CH₃CH₂OH)] (1) (HL = 2-acetylpyridine N(4)-phenylthiosemicarbazone) has been synthesized and characterized by elemental analysis, IR, ¹H NMR and single-crystal X-ray diffraction studies. The cytotoxicity data suggest that 1 exhibits higher in vitro antiproliferative activity in four human cancer cells tested. Its possible apoptotic mechanism has been evaluated in HepG2 cells. Compound 1 promotes a dose-dependent apoptosis in HepG2 cells and the apoptosis is associated with an increase in intracellular reactive oxygen species (ROS) production and reduction of mitochondrial membrane potential (MMP).

Full text of DOI:10.1016/j.bmcl.2013.02.097

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2288–2292
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, crystal structure and biological evaluation of a main group
seven-coordinated bismuth(III) complex with 2-acetylpyridine
N4-phenylthiosemicarbazone
a
a
b,
Yan-Ke Li a, Min Yang a, Ming-Xue Li a, , Han Yu , He-Chen Wu , Song-Qiang Xie
⇑
⇑
a Henan Key Laboratory of Polyoxometalates, College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, PR China
b Institute of Chemical Biology, Henan University, Kaifeng 475004, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Up to now, bismuth(III) complexes with thiosemicarbazones have been comparatively rare. Here, a main
group seven-coordinated bismuth(III) complex [Bi(L)(NO3)2(CH3CH2OH)] (1) (HL = 2-acetylpyridine N(4)-
phenylthiosemicarbazone) has been synthesized and characterized by elemental analysis, IR, 1H NMR and
single-crystal X-ray diffraction studies. The cytotoxicity data suggest that 1 exhibits higher in vitro anti-
proliferative activity in four human cancer cells tested. Its possible apoptotic mechanism has been eval-
uated in HepG2 cells. Compound 1 promotes a dose-dependent apoptosis in HepG2 cells and the
apoptosis is associated with an increase in intracellular reactive oxygen species (ROS) production and
reduction of mitochondrial membrane potential (MMP).
Received 8 January 2013
Revised 18 February 2013
Accepted 21 February 2013
Available online 1 March 2013
Keywords:
Thiosemicarbazone
Bismuth(III)
Crystal structure
Cytotoxicity
Ó 2013 Elsevier Ltd. All rights reserved.
Apoptosis
Heterocyclic thiosemicarbazones and their metal complexes
have received considerable attention in chemistry and biology, pri-
marily because of their marked and different biological proper-
ties.14 It has been reported that biological activities of the
thiosemicarbazones are closely related to the parent aldehyde or
ketone group, metal chelation ability and amino-terminal substitu-
tion.5 The biological properties of thiosemicarbazones by the link-
age to metal ions can be modified and differ from those of either
the ligand or the metal ion itself6–9 In some cases the highest activ-
ity is associated with a metal complex rather than the parent li-
gand and some side effects may decrease upon complexation.10,11
Bismuth(III) compounds have been widely used in the clinic for
centuries because of their high effectiveness and low toxicity in the
treatment of a variety of microbial infections, including syphilis,
diarrhea, gastritis and colitis.12–15 Apart from antimicrobial
activity, bismuth compounds exhibit anticancer and antiviral
activities, 212Bi and 213Bi compounds have also been used as
targeted radio-therapeutic agents for cancer treatment.16–23
Furthermore, bismuth(III) ion with a larger ionic radius (1.16 Å)
has one inert electron pair (6s2) and forms the complexes with
higher coordination numbers which makes their structural charac-
erization interesting and meaningful.
In recent years we have been working on the structural and bio-
logical properties of heterocyclic thiosemicarbazones and their
transition metal complexes.24,25a–c These results reveal that thio-
semicarbazones derived from 2-acetylpyridine and their transition
metal complexes show significant cytotoxicity. After a careful liter-
ature search, we can affirm that 2-acetylpyridine N4-substituted
thiosemicarbazones and their bismuth(III) complexes are scar-
ce.5,25a–c One reason may be that the crystals suitable for X-ray dif-
fraction study of these compounds have been difficult to obtain. It
is envisaged that they are likely to exhibit interesting properties
structurally and biologically. Particularly, information on the
mechanism of these compounds is sparse and valuable. Therefore,
it seems important for us to obtain their bismuth(III) complexes as
a strategy of preparation of new drug candidates in which the me-
tal and ligand could act synergistically.
As a part of our ongoing studies, in the present Letter, we have
synthesized and characterized a main group seven-coordinated
complex [Bi(L)(NO3)2(CH3CH2OH)] (1), where HL = 2-acetylpyri-
dine N(4)-phenylthiosemicarbazone (Scheme 1). The main aim of
this work is to study anticancer activity and possible apoptotic
mechanism of 1. Apoptosis is a common process of programmed
cell death and is the focus of current oncology research. Reactive
oxygen species (ROS), which are pro-oxidants, play a critical role
in regulating cell death.5 Therefore, we examine whether ROS are
critical mediators of 1-induced tumor cell death. In addition, effect
of 1 on mitochondria membrane potential (MMP) is also studied. In
this report, we present evidence that 1 promotes a dose-dependent
⇑
Corresponding authors. Tel./fax: +86 378 3881589.
E-mail addresses: limingxue@henu.edu.cn (M.-X. Li), kfxsq@yahoo.com.cn
(S.-Q. Xie).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.097

Y.-K. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2288–2292
2289
experiments, IC50 values (compound concentration that produces
50% of cell death) in micro molar units were calculated. For compar-
ison purposes the cytotoxicity of cisplatin (cis-DDP) and the free li-
gand as well as the starting compound Bi(NO3)3Á5H2O has been
evaluated under the same experimental conditions. It is clearly ob-
served that complexation with metal has a synergistic effect on the
cytotoxicity (Fig. 2A). The comparison of cytotoxicity indicates that
Scheme 1. The reaction scheme for the synthesis of 1.
1 shows much lower IC50 value (5.22
lM) than both HL (94.7 lM)
alone and Bi(NO3)3Á5H2O (41.2
l
M)24c alone. Therefore, the chela-
tion of the free ligand with bismuth(III) ion is essential for antican-
cer activities of 1. These results are consistent with the cases of
many other analogues of thiosemicarbazones.5,25a,25b,32 Impor-
tantly, it should be emphasized that 1 shows excellent activity sim-
apoptosis in HepG2 cells and the apoptosis is associated with an
increase in intracellular ROS production and reduction of MMP.
The ligand HL has been prepared by refluxing condensation of
2-acetylpyridine and thiosemicarbazide (1:1 molar ratio) with ace-
tic acid as catalyst in methanol,26 whereas complex 1 was synthe-
sized by reacting 2-acetylpyridine N(4)-phenylthiosemicarbazone
and Bi(NO3)3Á5H2O (1:1 ligand–metal molar ratio) in methanol.27
Single-crystal X-ray analysis reveals that 1 crystallizes in mono-
clinic system, with space group P21/c. As shown in Figure 1,28 the
molecular structure of 1 is depicted as a monomeric, seven-coordi-
nated complex formed with one electron pair (6s2) of the bis-
muth(III) ion (Fig. 1A), two nitrogen and one sulfur atoms from
one tridentate N2S thiosemicarbazone ligand and three oxygen
atoms from two nitrate ions and one ethanol molecule. The O(3)
atom was coordinated to the bismuth(III) ion from one side of
the plane formed by S(1), N(3), N(4), O(7) and an electron pair
(the sum of N(3)–Bi(1)–N(4), N(3)–Bi(1)–S(1), S(1)–Bi(1)–O(7)
and N(4)–Bi(1)–O(7) is 353.15°), while the O(4) atom was coordi-
nated from the opposite side. The bond distances around the bis-
muth(III) ion (Bi(1)–S(1) 2.585(2), Bi(1)–N(3) 2.392(1), Bi(1)–N(4)
2.484(1), Bi(1)–O(3) 2.444(1), Bi(1)–O(4) 2.362(1), Bi(1)–O(7)
2.712(1) Å) are compared with other data in related bismuth(III)
complexes.24c,25a–c,29 The longer Bi–N bonds and Bi–S bond are
resulting from the larger ionic radius of Bi3+ compared to that of
Mn2+, Zn2+ and Ni2+, respectively.24 Formation of seven-coordi-
nated structure of the bismuth(III) ion is unusual and interesting.30
The molecules of 1 are held together in the crystal packing through
intramolecular and intermolecular hydrogen bonds involving the
oxygen atom O(7) of ethanol molecule, the terminal nitrogen
N(1) atom, the oxygen atoms O(3) and O(5) of the nitrate ions
(Fig. 1B).The O(7) and N(1) act as hydrogen bond donors, while
the O(3) and O(5) act as acceptors. The separation for N(1)Á Á ÁO(3)
(symmetry code: x, y-1, z) is 3.181(5) Å with the N–HÁ Á ÁO angle
being 143.1° and the separation for O(7)Á Á ÁO(5) is 3.247(6) Å with
the O–HÁ Á ÁO angle being 154.0°, respectively.
ilar to that of cisplatin (1.2 l
M).24c These gratifying results are
encouraging its further screening in vitro. Later on, upon further
analysis, 1 also exhibits considerable cell growth inhibition activity
against human colorectal cancer HCT-116 cells, human cervical car-
cinoma Hela cells, human liver hepatocellular carcinoma HepG2
cells (Fig. 2B), respectively. Therefore, its further biological evalua-
tion in vivo as well as studies of mechanism of action is necessary.
In order to confirm that the cell death in HepG2 cells is due to
apoptosis, the apoptotic effect of 1 has been assessed with acridine
orange (AO) and ethylene dibromide (EB) staining.33 Mitoxantone
is a kind of antibiotic antitumor drugs, used as the reference com-
pound for comparison. Green live cells with normal morphology
were observed in control group (Fig. 3A). Green early apoptotic
cells with nuclear margination and chromatin condensation were
observed after 1 treatment in a dose-dependent manner, while or-
ange later apoptotic cells with fragmented chromatin and apopto-
tic bodies were seen after the treatment of mitoxantrone (Fig. 3A).
Furthermore, the apoptosis percentage of HepG2 cells significantly
increases after 1 treatment, compared with untreated cells and
mitoxantrone treated cells (Fig. 3B). These data suggested that 1
could induce liver cancer cells apoptosis in vitro.
The intracellular redox levels are important and play a crucial
role in driving the cellular apoptosis.5,34 A large body of evidence
suggests that the intracellular levels of ROS are associated with
the cell death processes and the apoptosis is associated with an in-
crease in intracellular production. In this report, the production of
ROS was investigated at that time by fluorescence microscope with
the use of DCFH-DA.35 An oxidation of the intracellular DCFH to
fluorescent DCF was observed in 1-treated cells as indicated by
the mean fluorescence value, which was higher in 1-treated cells
than in untreated cells (Fig. 4). This data confirmed the production
of ROS during 1-induced HepG2 cells apoptosis.
In view of the biological activity of thiosemicarbazones, we
firstly have evaluated the ability of the title complex to inhibit
cancer cell growth against human leukemia K562 cells.31 In our
Mitochondria is considered to be a major site of antitumor
agents through electron leakage from the electron transport
Figure 1. (A) Structure of complex 1 with atomic numbering scheme. (B) Hydrogen bond in dashed lines in complex 1.

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Y.-K. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2288–2292
Figure 2. (A) The cytotoxicity of HL and 1 against human leukemia K562 cells. (B) The cytotoxicity of complex 1 on tumor cells. Mitoxantone (Mito) were used as a positive
control.
Figure 3. HepG2 cell apoptosis was determined by AO/EB staining with the indicated concentrations of 1 for 48 h. (A) Morphologic observation. (B) Percentages of apoptotic
cells were determined using fluorescence microscopy after staining with AO/EB. Mitoxantone (Mito) were used as a positive control.
ꢀ
Figure 4. The effects of 1 on intracellular ROS content in HepG2 cells, (x s, n = 4). (A) The representative pictures of ROS generation in 1-mediated HepG2 cells by DCFH-DA
staining, Images were acquired on the ArrayScanÒ HCS Reader using Cellomics’ Target Activation BioApplication. Scale bar = 10
lm. (B) The percentage of ROS in HepG2 cells
induced by 1. Mitoxantone (Mito) were used as a positive control.
Figure 5. Effect of 1 on MMP in HepG2 cells. (A) The change of MMP was detected by Rh123 and Hoechst 33342 double staining using HCS in HepG2 cells with the indicated
concentrations of 1. (B) Representative pictures are from one of three independent experiments with similar results (magnification, 20Â). Each value represents the
mean SD (n = 4). Mitoxantone (Mito) were used as a positive control.

Y.-K. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2288–2292
2291
chain,36,37 the decreased MMP may open the mitochondrial
permeability transition (MPT) and trigger the release of cyto-
chrome c which activate caspase cascade, causing the cell apopto-
sis. In the present report, rhodamine 123 (Rh123), a mitochondrial
specific stain, which can bind to the inner and outer membrane of
mitochondria, and their degrees of fluorescence are proportional to
the MMP.38 As shown in Figure 5, the green fluorescence was sig-
nificant decrease after treatment with 1 for 48 h, suggesting 1 in-
duced the MMP decrease in cells apoptosis.
In summary, the bismuth(III) complex 1 has been synthesized
and structurally characterized. It indicates remarkable cytotoxicity
and promotes a dose-dependent apoptosis in HepG2 cells and the
apoptosis is associated with an increase in intracellular ROS pro-
duction and reduction of MMP. These promising results are
encouraging its further screening in vivo which will provide useful
clues in the design of even more effective agents for cancer
treatment.
23. Kondo, Y.; Himeno, S.; Satoh, M.; Naganuma, A.; Nishimura, T.; Imura, N. Cancer
Chemother. Pharmacol. 2004, 53, 33.
24. (a) Li, M. X.; Zhang, D.; Zhang, L. Z.; Niu, J. Y.; Ji, B. S. Inorg. Chem. Commun.
2010, 13, 1572; (b) Li, M. X.; Chen, C. L.; Zhang, D.; Niu, J. Y.; Ji, B. S. Eur. J. Med.
Chem. 2010, 45, 3169; (c) Zhang, L. Z.; An, G. Y.; Yang, M.; Li, M. X.; Zhu, X. F.
Inorg. Chem. Commun. 2012, 20, 37; (d) Li, M. X.; Zhang, L. Z.; Zhang, D.; Ji, B. S.;
Zhao, J. W. Eur. J. Med. Chem. 2011, 46, 4383; (e) Li, M. X.; Zhang, L. Z.; Chen, C.
L.; Niu, J. Y.; Ji, B. S. J. Inorg. Biochem. 2012, 106, 117.
25. (a) Li, M. X.; Yang, M.; Niu, J. Y.; Zhang, L. Z.; Xie, S. Q. Inorg. Chem. 2012, 51,
12521; (b) Li, M. X.; Lu, Y. L.; Yang, M.; Li, Y. K.; Zhang, L. Z.; Xie, S. Q. Dalton
Trans. 2012, 41, 12882; (c) Li, M. X.; Zhang, L. Z.; Yang, M.; Niu, J. Y.; Zhou, J.
Bioorg. Med. Chem. Lett. 2012, 22, 2418; (d) Näslund, J.; Persson, I.; Sandström,
M. Inorg. Chem. 2000, 39, 4012.
26. Synthesis of HL: N(4)-phenyl thiosemicarbazide (0.50 g, 3 mmol) was added
dropwise to a methanol solution (30 mL) of 2-acetylpyridine (0.37 g, 3 mmol)
with five drops of acetic acid as catalyst. After refluxed for 2 h, the resultant
solution was filtered. Yellow powder separated on cooling were washed with
methanol and dried over P4O10 in vacuo. Yield: 81%-C14H14N4S (%). Calcd C
62.20, H 5.22, N 20.72. Found C 62.46, H 5.35, N 20.90. Mp 182–184 °C. IR (KBr,
cmÀ1): (C@S) 894. 1H NMR (DMSO-d6, d ppm):
m(C@N) 1587, m(N–N) 1189, m
10.70 (s, 1H, NH), 10.22 (s, 1H, NH), 8.59 (d, J = 4.8 Hz, 1H, Py), 8.54 (d, J = 8 Hz,
1H, Py), 7.81 (t, J = 7.6 Hz, 1H, Py), 7.54 (d, J = 8 Hz, 2H, Ph), 7.42–7.31 (m, 2H,
Ph), 7.23 (t, J = 7.2 Hz, 1H, Ph), 7.15 (t, J = 7.2 Hz, 1H, Py), 3.42 (s, 3H, CH3).
27. Synthesis of 1: Bi(NO3)3Á5H2O (0.097 g, 0.2 mmol) solution dissolved in ethanol
with the help of a few drops of nitrate acid was added dropwise to an ethanol
solution (20 mL) of 2-acetylpyridine N(4)-phenylthiosemicarbazone (0.034 g,
0.2 mmol). After refluxing with stirring for 2 h, the resultant solution was
filtered. The solid product formed was recrystallized from ethanol and dried
over P4O10 in vacuo. Yield: 60%-C16H19BiN6SO7 (%). Calcd C 29.64, H 2.95, N
Acknowledgment
This work was financially supported by the National Natural
Science Foundation of China (21071043).
12.96. Found C 29.55, H 2.79, N 14.20. M.p. 212–214 °C. IR (KBr, cmÀ1):
m(C=N)
1544, (N–N) 1258,
m
m
(C@S) 847. 1H NMR(DMSO-d6, d ppm): 9.60 (s, 1H, NH),
Supplementary data
9.05 (t, J = 6 Hz, 1H, Py), 8.29–8.17 (m, 2H, Py), 7.80–7.63 (m, 3H, Ph), 7.39–7.28
(m, 2H, Ph), 7.08–6.97 (m, 1H, Py), 3.43 (s, 3H, CH3). Orange crystals suitable for
X-ray studies were obtained by slow evaporation of its ethanol solution.
28. Crystal data for HL: M = 648.41, monoclinic, space group P21/c, a = 17.962(1) Å,
b = 7.7290(1) Å, c = 17.699(1) Å, b = 114.60 (1)°, V = 2234.1(7) Å3, Z = 2,
CCDC 851899 contains the supplementary crystallogphic data
of 1. These data can be obtained free of charge from the Cambridge
Crystallographic Centre via www.ccdc.cam.ac.uk/data_request/cif.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.02.
097.
Dc = 1.928 g cmÀ1 = 8.034 cmÀ1, F(000) = 1248, T = 296(2) K. A crystal with
, l
approximate dimensions of 0.47 Â 0.25 Â 0.17 mm3 was mounted on a glass
fiber in a random orientation. Crystallographic data were collected with a
Siemens Smart-CCD diffractometer with graphite-monochromated MoK
a
radiation (l = 0.71073 Å). A total of 11628 reflections was measured by w
scan technique at 296(2) K within 2.31 6 h 6 26.00°, of which 2979 were
independent with Rint = 0.0412, and 4387 were observed with I P 2
r (I). The
References and notes
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lL of medium with 10
lL of 5 mg/mL MTT
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l
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cell suspension. EB-negative cells with nuclear shrinkage, blebbing, and
apoptotic bodies were counted as apoptotic cell. The percentage of apoptotic
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Elmer, USA).
lM DCFH-DA, using fluorescence microplate reader (Perkin
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Y.-K. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2288–2292
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779.
38. After incubation with 1 with different concentration for 48 h, the HepG2 cells
were preloaded with Rh123 (0.25 nM) and Hoechst 33342 (1
lM) for 30 min at
37 °C, and then rinsed with freshly prepared PBS. The fluorescence intensity