Four copper(II) compounds synthesized by anion regulation: Structure, anticancer function and anticancer mechanism

Article abstract of DOI:10.1016/j.ejmech.2016.05.021

Copper (Cu) compounds are a promising candidate for next generation metal anticancer drugs. Therefore, we regulated anions to synthesize four mononuclear and binuclear Cu(II) compounds derived from thiosemicarbazone Schiff base ligands and characterized them. Four of these compounds showed very high cytotoxicity to cancer cell lines in vitro. These Cu(II) compounds strongly promoted the apoptosis of BEL-7404 cells and had a capacity to arrest the cell cycle at S phase of those cells. Furthermore, reactive oxygen species (ROS), mitochondrial membrane potential and Western blot analyses revealed that these Cu(II) compounds exert their cytotoxicity through an ROS-mediated intrinsic mitochondrial pathway accompanied by the regulation of Bcl-2 family proteins.

Full text of DOI:10.1016/j.ejmech.2016.05.021

Accepted Manuscript
Four copper(II) compounds synthesized by anion regulation: Structure, anticancer
function and anticancer mechanism
Zhenlei Zhang, Yi Gou, Jun Wang, Kun Yang, Jinxu Qi, Zuping Zhou, Shichu Liang,
Hong Liang, Feng Yang
PII:
S0223-5234(16)30401-9
10.1016/j.ejmech.2016.05.021
EJMECH 8613
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 February 2015
Revised Date: 4 May 2016
Accepted Date: 6 May 2016
Please cite this article as: Z. Zhang, Y. Gou, J. Wang, K. Yang, J. Qi, Z. Zhou, S. Liang, H. Liang,
F. Yang, Four copper(II) compounds synthesized by anion regulation: Structure, anticancer function
and anticancer mechanism, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.05.021.
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ACCEPTED MANUSCRIPT

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Four copper(II) compounds synthesized by anion regulation:
structure, anticancer function and anticancer mechanism
Zhenlei Zhang1§, Yi Gou2§, Jun Wang2§, Kun Yang2, Jinxu Qi2, Zuping Zhou3, Shichu
Liang1*, Hong Liang2*, Feng Yang1, 2*
1
Key Laboratory of Ecology of Rare an Endangered species and Environmental
Protection, Ministry of education of the People's Republic of China, Guangxi Normal
University, Guilin, Guangxi, China
2
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal
Resources, Ministry of Science and Technology of China. Guangxi Normal University,
Guilin, Guangxi, China.
3
Guangxi Universities Key Laboratory of Stem Cell and Pharmaceutical Biotechnology,
Guangxi Normal University, Guilin, Guangxi, China.
*To whom correspondence should be addressed: Shichu Liang, Hong Liang,
Feng Yang
Address: 15 Yucai Road, Guilin, Guangxi, China. Zip code: 541004
Email: fyang@mailbox.gxnu.edu.cn
Tel: 86-773-212-0958; Fax: 86-773-212-0958.
§ZL Zhang, Y Gou and J Wang pay the same contribution to the article.
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Abstract:
Copper (Cu) compounds are a promising candidate for next generation metal
anticancer drugs. Therefore, we regulated anions to synthesize four
mononuclear and binuclear Cu(II) compounds derived from
thiosemicarbazone Schiff base ligands and characterised them. Four of these
compounds showed very high cytotoxicity to cancer cell lines in vitro. These
Cu(II) compounds strongly promoted the apoptosis of BEL-7404 cells and
had a capacity to arrest the cell cycle at S phase of those cells. Furthermore,
reactive oxygen species (ROS), mitochondrial membrane potential and
Western blot analyses revealed that these Cu(II) compounds exert their
cytotoxicity through an ROS-mediated intrinsic mitochondrial pathway
accompanied by the regulation of Bcl-2 family proteins.
Keywords:
copper compound, thiosemicarbazone Schiff base ligands, mitochondrial
membrane potential, reactive oxygen species, Bcl-2 family proteins.
Abbreviations:
reactive oxygen species (ROS); salicylaldehyde thiosemicarbazone ligand
(HL); cytochrome c (Cyt c).
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1. Introduction
Although cisplatin has been successfully applied to treat cancer for
several decades, multifactorial resistance and inherent limitations, such as
serious side effects and general toxicity, has limited its use. These
disadvantages have spurred investigators to find better alternatives for
cancer chemotherapy [1, 2]. With this aim, various classes of metal
compounds have been synthesized, and their anticancer activity has been
successfully evaluated both in vitro and in vivo [3–6]. Among them, copper
compounds have attracted more attention given that many of them display
promising anticancer activity because copper possesses excellent
pharmacological properties associated with its oxidative nature [7–10].
Recent investigations have demonstrated that high levels of copper have
been detected in various types of human cancer cells [11, 12]. Therefore, the
differential response of normal and tumor cells to copper may be a
mechanism to selectively kill tumor cells without causing significant damage
to normal cells.
Thiosemicarbazones are versatile ligands that adopt various binding
modes with transition metal ions [13]. In the 1950s, the first
thiosemicarbazone 2-formylpyridine thiosemicarbazone with antitumor
properties was reported [14]. Subsequently, some thiosemicarbazones, such
as 5-hydroxy-2-formylpyridine thiosemicarbazone and Triapine, have
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entered phase I clinical trials [15–18]. Several studies have shown that the
biological activities of a variety of metal compounds with
thiosemicarbazones are higher than those of metal-free ligands [19–22]. To
elevate the effect of complexation on the cytotoxicity of thiosemicarbazones,
several thiosemicarbazone metal compounds were synthesized [23–29].
These compounds play a major role in several enzymatic activities,
particularly redox reactions that have an influence on the antioxidant system
of the organism [30–32]. Particularly, thiosemicarbazone copper compounds
have been widely investigated because of their excellent anticancer effects
[33–39]. Although many copper complexes have been proposed as
promising anticancer agents on the basis of in vitro assays, their mechanisms
of action remain unclear in many cases [40].
With this background in mind and considering that copper is a crucial
trace element in redox chemistry specific to growth and development in all
living organisms [11], we synthesized Cu(II) compounds with a
salicylaldehyde thiosemicarbazone ligand (HL1) and salicylaldehyde
4-methylthiosemicarbazone
ligand
(HL2).
The
four
copper
2−
thiosemicarbazone compounds, namely, [(CuII)(HL1)2]SO4 ·2.5H2O·1/2
−
CH3CH2OH
(C1),
[(CuII)2(HL1)2(H2O)]·2NO3
(C2),
[(CuII)(HL2)(H2O)]·SO4 (C3) and [(CuII)2(HL2)2(MeOH)(NO3 )]·NO3
2−
−
−
(C4) were synthesized and characterized by single crystal x-ray diffraction,
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elemental analysis and infrared spectroscopic analysis (Scheme 1, Figs 1 and
2) Herein, we not only studied the biological activities of these four
.
compounds but also determined their potential anticancer mechanism(s).
2. Results and Discussion
2.1 Crystal structure of the four copper compounds
Anion templates are a good method for synthesizing novel metal
compounds because anions can be a part of the coordination polymer that
has a great influence on the formation of the network structure [41–43].
Compared with conventional solvent-based methods, anion template
synthesis can avoid large quantities of solvent and high temperatures and
sometimes gives products that are different from those produced by solution
reactions [44, 45]. Therefore, synthesis of Cu(II) anticancer compounds
based on an anion template may be a good choice. To synthesize novel Cu(II)
anticancer compounds, we attempted to use different anions to regulate their
−
2−
synthesis, such as NO3 and SO4 . As a result, we obtained four novel Cu(II)
compounds (C1–C4) with different structures.
The crystal structure of C1 was solved in the space group C2/c, and the
building block is shown in Fig 1A. The Cu(II) ion has a square-planar
coordination geometry resulting from the O, N and S donor atoms from one
dianionic tridentate ligand and the S donor atom from one neutral ligand,
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which is similar to the coordination mode reported in the literature [46]. The
Cu−N/S/O distances are consistent with the bond distances of other similar
complexes [47–49]. The phenolate oxygen O1 and O2 acts as a double
proton acceptor shaping both intramolecular hydrogen bonds
(N4−H4B···O1 and O2−H4A···N6) and intermolecular hydrogen bond
(N3−H3B···O2i, symmetry code: i = 0.5 + x, 0.5 + y, z). In the solid state,
the discrete Cu(II) dimers of C1 were further linked into a one-dimensional
(1D)-polymeric chain by an intermolecular hydrogen bond (Fig 1B).
In contrast to C1, the complex C2 crystallized in the triclinic space
group P−1 with one discrete dimeric Cu(II) molecule in the unit cell. As
−
shown in Fig 1C, it contains two Cu atoms, two NO3 anions, two Schiff
base ligands and one H2O. The phenolic O atoms from a neighboring
molecule act as a bridge, offering the phenolato-bridged binuclear Cu(II)
centres the usual Cu···Cu bond distance of about 3.006 Å. The Cu1 are
four-coordinated by two O atoms from two different Schiff base ligands as
well as one N and one S atom from one Schiff base ligand. However, the
coordination polyhedron around the Cu2 centre could be described as a
slightly distorted square-pyramidal (τ = 0.03) [50], with the metal displaced
from the O1/O2/N4/S2 basal plane (maximum displacement of 0.015 Å for
atom O2) and O3 atom at the apex, with the metal displaced by 0.272 Å
towards O3 from the mean basal plane. Moreover, the coordination of H2O
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is stabilized by two hydrogen bonds between the hydrogens of the H2O and
−
two NO3 anions (O3−H3C···O8 and O3−H3D···O6). In addition, the
discrete Cu(II) dimers can be extended into a 3D-network by the hydrogen
−
bond between the NO3 anions and Schiff base ligands (Fig 1D). Successful
isolation of C1 and C2 indicates that anions play an important role in their
geometries. Anion-controlled self-assembly has been reported in several
recent reports [51, 52].
To investigate the influence of ligand modification on Cu(II) compound
anticancer activity, we modified H atom of amino group in HL1 into -CH3
(HL2) (Scheme 1). We obtained the other two novel Cu(II) compounds
derived from salicylaldehyde 4-methylthiosemicarbazone ligand (C3 and C4)
−
2−
by NO3 and SO4 regulation (Fig 2). Surprisingly, C3 structure is totally
different from C1. X-ray analysis of complex C3 revealed that it crystallized
in the orthorhombic Fdd2 space group. There is one Cu(II), one H2O, one
−
Schiff base ligand and one SO4 anion. As illustrated in Fig 2A, the Cu(II)
centre has a square-planar coordination geometry. The multidentate Schiff
base ligand uses only, in part, its donor capacity; it acts in the complex as a
tridentate ligand, binding to Cu(II) via S1, N1 and O1. The fourth position in
−
the coordination polyhedron is occupied by a H2O molecule, while the SO4
has proton acceptor ability. Then, it is extended to a 3D-supramolecular
2−
framework by the hydrogen bond between the SO4 anions and Schiff base
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ligands or the H2O (Fig 2B).
Compared with C3, x-ray analysis of complex C4 revealed that it
crystallized in the triclinic P−1 space group resulting from anion control. The
two Cu atoms of the dimer separated by 3.018 Å are doubly bridged by two
phenolic O atoms of two Schiff base ligands to form a strictly planar
four-membered Cu2O2 core by symmetry requirements (Fig 2C). The Cu1
atom is five-coordinated by two O atoms from two different Schiff base
ligands, one N and one S atom from a Schiff base ligand and one O atom
−
from NO3 . The coordination polyhedron around the Cu1 center could be
described as a slightly distorted square pyramid (τ = 0.008) [50], with the
metal displaced from the O1/O2/N4/S2 basal plane (maximum displacement
of 0.067 Å for the O2 atom) and the O4 atom at the apex, with the metal
displaced by 0.241 Å towards O4 from the mean basal plane. The
coordination geometry around Cu2 in this complex is very similar to that
around Cu1; the only difference is the presence of a methanol molecule at
the apical position. Although methanol is not a good ligand, the metal-bound
−
methanol has been observed in C4 with solvated NO3 stabilized by
hydrogen bonding similar to analogous metal-bound methanol(s) in other
known cases [53–55]. The τ-value calculated for Cu2 has a value of 0.066,
indicating a much more distorted structure compared with that of Cu1. The
longer Cu−O apical bond lengths (Cu1−O4 = 2.357 Å; Cu2−O3 = 2.311 Å)
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to the basal Cu−O distances (weighted mean value of 1.957 Å) are
associated with the pseudo-Jahn–Teller effect on the Cu(II) ion. Moreover,
the C−S bond distances (C1 = 1.724/1.706 Å; C2 = 1.704/1.701 Å; C3 =
1.715 Å; C4 = 1.713/1.700 Å) of all complexes were close to a single bond,
which support thiolate formation in the ligands upon complexation. In the
solid state, the discrete Cu(II) dimers can be extended into a 2D-network by
−
the hydrogen bond between NO3 anions and Schiff base ligands or
methanol (Fig 2D).
2.2 Anticancer properties of Cu(II) compounds in vitro
Table 1 demonstrates that complexes C1–C4 have much higher
cytotoxicity against cancer cells than ligands and Cu(II) salts alone,
implying that chelation of Cu(II) ions with ligands is responsible for the
observed high cytotoxic activity of C1–C4. Among them, C4 has the highest
cytotoxic activity, whereas C1 shows the lowest cytotoxicity. Furthermore,
for the same thiosemicarbazone Schiff base ligands, cytotoxicity of the
binuclear Cu(II) complexes C2 and C4 are promising compared with the
corresponding mononuclear Cu(II) complexes C1 and C3.
Whether Cu(II) compounds affect apoptosis and cell cycle arrest is a
matter of great interest. Therefore, we investigated cellular apoptosis by
flow cytometric analysis. The results of Annexin V-FITC/PI staining showed
that the percentage of apoptotic BEL-7404 cells at Cu(II) complex treatment
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concentrations of 3 ꢀM were 14.8% for C1, 18.3% for C2, 17.4% for C3
and 24.7% for C4 (Figs 3B-E). In contrast, the percentage of apoptotic cells
without treatment (control) was only 3.9% (Fig 3A). Apoptosis is commonly
characterized by certain distinct morphological features, such as cytoplasmic
shrinkage, membrane blebbing, chromatin condensation and apoptotic body
formation [56]. To further understand the cell death induced by these Cu(II)
complexes, the apoptosis-inducing properties of these complexes were
examined by AO/EB and DAPI double staining. As shown in Fig 4 and Fig
S1, untreated cells displayed a homogenous morphology, whereas cells
treated with C1–C4 had distinct morphological changes characteristic of
apoptosis (e.g. cytoplasmic shrinkage and apoptotic bodies) similar to that
characterized by Annexin V-FITC/PI staining. As shown in Fig 5F, flow
cytometry results revealed that treatment with compounds for 48 h caused
marked accumulation of cells in S phase in a concentration-dependent
manner. Compared with the vehicle-treated control (29.94%, Fig 5A), the
percentage of cells in S phase approximately were increased by 48.08% for
C1, 54.34% for C2, 50.94% for C3 and 59.53% for C4 when treated with
2-ꢀM C1–C4 for 48 h (Figs 5B-E).
The above results indicate that C2 and C4 have stronger anticancer
property than C1 and C3. These may be more active mononuclear Cu(II)
species formed by the dissociation of binuclear Cu(II) complexes in cells.
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On the other hand, compared with the HL1 ligand, the overall lipophilicity
of the HL2 ligand increased because of methylation of the terminal N,
leading to an increase in antiproliferative activity [57], which offers the
possibility that the anticancer property of Cu(II) agents could be optimized
through modifying coordination ligands in metal compounds.
2.3 Potential anticancer mechanism of Cu(II) compounds
One important anticancer pathway is mitochondrial apoptosis, which
depends on the control of mitochondrial outer membrane permeabilisation to
activate proapoptotic molecules, such as initiator caspase-9. Loss of
mitochondrial transmembrane potential (ꢁψm) is crucial to apoptotic
processes and can lead to the release of apoptogenic factors and a decrease
in ATP generation, finally resulting in cellular apoptosis [58, 59]. Out of the
apoptogenic factors, the Bcl-2 family of proteins plays an important role.
Therefore, we analyzed their potential role in anticancer mechanism(s) by
fluorescence microscopy, flow cytometry and Western blot.
Previous studies have revealed that the loss of ꢁψm is directly related to
hydroxyl radicals and reactive oxygen species (ROS) produced in cancer
cells [29, 30]. To assess the ability of our Cu(II) compounds to catalyze the
production of intracellular ROS in BEL-7404 cells, fluorescence microscopy
and
flow
cytometry
was
performed
using
a
fluorescent
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2′,7′-dichlorofluorescein (DCF) probe. As shown in Fig 6A, no significant
fluorescence was found in control cell. After BEL-7404 cells were exposed
to 25 ꢀM of compounds C1–C4 for 30 minutes, the fluorescence images are
observed (Figs 6B-E). These results demonstrate that ROS in BEL-7404 cell
lines can be generated and C1–C4 can increase the levels of ROS. To
quantitatively compare the levels of the ROS induced by C1–C4, we also
measured the fluorescent intensity of DCF. Flow cytometry results showed
that the incubation of BEL-7404 cells with C1–C4 induced a red shift of the
DCF in fluorescence peak relative to control cells, which reflects increased
oxidative damage to these cells by our Cu(II) compounds (Fig 7A). These
results showed that C1–C4 produced a higher level of ROS in cancer cells
than in ligands and Cu(II) salts alone; the level of ROS in BEL-7404 cells
was approximately six times higher than that in controls (Fig 7B).
Mitochondria play a vital role in apoptosis triggered by chemical reagents.
Chemical-induced apoptosis mediated by mitochondrial apoptotic cascades
is often associated with the collapse of ꢁψm as a result of proapoptotic factor
leakage and/or activation. The lipophilic fluorescent probe
5,5,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) was used
to determine changes in ꢁψm [60]. As shown in Fig 8, red fluorescence
gradually changed to green when each of the four Cu(II) compounds were
incubated with BEL-7404 cells, indicating C1–C4 influenced ꢁψm; this
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resulted in its eventual collapse. The intrinsic mitochondrial apoptotic
pathway is evidently induced by these Cu(II) compounds.
The Bcl-2 family of proteins plays an important role in cellular apoptosis,
and it includes the antiapoptotic members Bcl-xl and Bcl-2 and the
proapoptotic members Bax and Bak [61–63]. These proteins are key
regulators of mitochondrial-mediated apoptosis [64–68]. To examine the
mitochondrial release of apoptogenic factors, the effect of the four Cu(II)
compounds of our study on the levels of Bcl-2 family proteins was
determined by Western blot. As shown in Fig 9, the level of the antiapoptotic
proteins Bcl-2 and Bcl-xl were down-regulated in BEL-7404 cells. However,
the level of the proapoptotic Bax was up-regulated. On the other hand, the
ratio of Bax/Bcl-2 was increased, indicating that Bcl-2 family proteins
regulated the loss of ꢁψm in these cancer cells. Furthermore, Western blot
results showed that cytochrome c (Cyt c) and caspase family proteins
(caspase-3 and capsese-9) were upregulated, indicating that Cyt c caused an
activation of caspase-3 and capsase-9 [69, 70].
3. Conclusion
Since the chelation of Cu(II) with ligands results in a synergistic effect,
Cu(II) compounds derived from salicylaldehyde benzoylhydrazone possess
high anticancer activity. Furthermore, the binuclear Cu(II) compounds (C2
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and C4) had stronger anticancer activity than the mononuclear Cu(II)
compounds (C1 and C3). Interestingly, the geometry of Cu(II) compounds
can be regulated by different anions, which can confer different anticancer
activities. Apoptosis of cancer cells induced by our Cu(II) compounds likely
occurred via the mitochondrial production of hydroxyl radicals and ROS.
Furthermore, Cu(II) compounds had a strong influence on pro- and
antiapoptotic Bcl-2 family proteins. In conclusion, our results may be
helpful to design potentially novel chemotherapeutic agents based on
apoptotic pathways.
4. Experimental section
4.1 Reagents and instrumentation
All chemicals and reagents were purchased from commercial sources
and were all used as received without further purification unless noted
specifically. RNA polymerase, AO/EB (Acridine orange/Ethidium bromide),
DAPI bromide and 3-(4, 5-dimathylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT) were purchased from Sigma (USA). JC-1 assay kit and ROS
assay kit were purchased from Beyotime Biotech. Annexin V–FITC/PI assay
kit is purchased from Jiamay Biotech. Human hepatoma cell lines
BEL–7404, liver hepatocellular carcinoma cell lines HepG2, human lung
adenocarcinoma cell A549, human gastric carcinoma cells MGC-803 were
purchased from ATCC (Manassas, VA). Cells were maintained in a humid
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atmosphere containing 5% CO2 at 37 ꢀ in GIBCO DMEM medium
supplemented with 100 units mL−1 penicillin, 100 µgmL–1 streptomycin and
10% fetal bovine serum. Fetal bovine serum (FBS) was obtained from
Hyclone. GIBCO DMEM was obtained from GIBCO. Elemental analyses (C,
H, N) was carried out on a Perkin Elmer Series II CHNS/O 2400 elemental
analyzer. Bruker Smart Apex II CCD diffractometer equipped with graphite
monochromated Mo-Kα radiation.
4.2 General method for the synthesis of compounds C1–C4.
As the following was described, Schiff base ligands (HL1 and HL2) were
prepared by condensation reaction between salicylaldehyde and
thiosemicarbazides (hydrazinecarbothioamide and N-methylhydrazine-
carbothioamide). The key step in the formation of the Schiff base ligands
backbone was achieved by a mild reaction in the basic conditions. The Cu(II)
compounds were prepared by direct reaction of ligands dissolved in
dichloromethane with Cu(NO3)2·3H2O or CuSO4·5H2O in 20 mL MeOH or
ETOH. Thereafter, the beaker was covered and kept for crystal solid
precipitation. The solid was filtered and dried in air.
HL1: C8H9N3OS (195.2): calcd. C, 49.23; H, 4.62; N, 21.54; found: C, 49.02;
1
H, 4.49; N, 21.43. Η NMR ([D6]DMSO): δ = 11.36 (s, 1H), 9.86 (s, 1H),
8.37 (s, 1H), 8.09 (s, 1H), 7.90 (d, J = 7.9 Hz, 2H), 7.21 (t, J = 7.7 Hz, 1H)
and 6.90 – 6.76 (m, 2H). 13C NMR ([D6]DMSO): δ = 178.16, 156.88, 140.14,
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131.56, 127.24, 120.82, 119.74 and 116.51 ppm.
HL2: C9H11N3OS (209.3): calcd. C, 51.65; H, 5.30; N, 20.08; found: C,
51.26; H, 5.41; N, 20.27. 1Η NMR ([D6]DMSO): δ = 11.40 (s, 1H), 9.86 (s,
1H), 8.54 – 8.28 (m, 2H), 7.93 (d, J = 7.6 Hz, 1H), 7.21 (dd, J = 11.2, 4.2 Hz,
1H), 6.95 – 6.70 (m, 2H), 3.01 (d, J = 4.5 Hz, 3H). 13C NMR ([D6]DMSO):
δ = 178.04, 156.78, 139.48, 131.42, 127.08, 120.95, 119.65, 116.51 and
31.29 ppm.
C1: Dark green crystals (Yield: 56.1%). Elemental analysis (%): calc. for
C17H24CuN6O6.75S2.5: C, 36.19; H, 4.28; N, 14.89. Found: C, 36.01; H, 4.41;
N, 14.97. IR (KBr, cm−1): 3744.1, 3432.1, 1637.4, 1612.9ꢁ1549.8ꢁ1465.6,
1427.0, 1311.4, 1185.2, 939.8, 894.3, 792.6, 750.54.
C2: Dark green crystals (Yield: 68.1%). Elemental analysis (%): calc. for
C16H18N8O9S2Cu2: C, 29.22; H, 2.76; N, 17.04. Found: C, 29.11; H, 2.54; N,
17.21. IR (KBr, cm−1): 3425.1, 3001.0, 1637.4, 1602.3, 1497.2, 1336, 1279.4,
1192.2, 1038.0, 936.3, 813.6.
C3: Dark green crystals (Yield: 67.1%). Elemental analysis (%): calc. for
C18H24N6O8S3Cu2: C, 31.99; H, 3.58; N, 12.44. Found: C, 31.75; H, 3.64; N,
12.65. IR (KBr, cm−1): 3439.1, 3211.3, 1616.4, 1528.7, 1469.1, 1350.0,
1248.3, 1202.7, 852.2, 768.1.
C4: Dark green crystals (Yield: 76.1%). Elemental analysis (%): calc. for
C19H24N8O9S2Cu2: C, 32.61; H, 3.46; N, 16.01. Found: C, 32.25; H, 3.37; N,
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15.87. IR (KBr, cm−1): 3432.1, 3246.3, 3036.0, 1605.8, 1549.8, 1472.6,
1381.5, 1213.2, 1034.5, 1039.5, 820.6, 715.5, 757.6.
4.3 X-ray crystallography
Crystal data were collected at 291-297 K on a Bruker Smart Apex II
CCD diffractometer equipped with graphite monochromated Mo–Kα
radiation (λ = 0.71073 Å). The structures were solved by direct methods and
refined with SHELX-97 programs. The non–hydrogen atoms were located in
successive difference Fourier synthesis. The final refinement was carried out
by full–matrix least–squares methods with anisotropic thermal parameters
for non–hydrogen atoms on F2 [71]. The hydrogen atoms were added
theoretically and riding on the concerned atoms. The details of
crystallographic data and structure refinement parameters are summarized in
Table 2.
4.4 Anticancer mechanism of Cu(II) compounds in vitro cytotoxicity
4.4.1 MTT assay
The drug sensitivities of cells were determined in growth inhibition assays
with exposure to drugs [72]. 1 × 104 cells per well were seeded in triplicate
in 96–well plates and incubated for 12 h at 37 ꢀ and 5% CO2. Then graded
amounts of HL, Cu(NO3)2, C1–C4 were added to the wells in 10 ꢀL of FBS
free culture medium and the plates were incubated in a 5% CO2 humidified
atmosphere for 48 h. Six replica wells were used for controls. As shown in
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Table S1, after incubation of cells with each compound at a concentration of
20 ꢀM for 48 h as identical conditions, these compounds exhibit different
antitumor activity and certain selectivity. Then compounds were tested at
concentrations ranging between 0 and 40 ꢀM/L. After 48 h of culture, 0.1
mg of MTT (in 20 ꢀL of PBS) was added to each well, and cells were
incubated at 37 ꢀ for 4 h. The formed formazan crystals were then
dissolved in 100 ꢀL DMSO and the absorbance was read at 570 nm using an
Enzyme–linked Immunosorbent Assay (ELISA) reader. The sensitivity of
cells to compounds treatment was expressed in terms of IC50 (drug
concentration producing 50% inhibition of cell growth, calculated on the
regression line in which absorbance values at 570 nm were plotted against
the logarithm of drug concentration).
4.4.2 Measurement of apoptosis by Annexin V-FITC/PI analysis for
Cu(II) compounds
Annexin V-FITC/PI staining was used to detect apoptotic cells. The
staining single–positive for Annexin V–FITC mostly reflected early
apoptotic cells and the staining single positive for PI mostly reflected
necrotic cells. So cells that were stained double-positive could be either
necrotic or apoptotic cells. BEL-7404 cells were treated with copper
compounds at 0 ꢀM and 2 ꢀM for 12 h, and stained with Annexin V-FITC/PI
double staining. The distribution of the apoptosis was measured by a FAC
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Star Plus flow cytometer, and data analysis was carried out with Flowjo
software.
4.4.3 AO/EB and DAPI double staining for Cu(II) compounds
Characteristic of apoptosis can be identified by visualizing nuclear
changes and apoptotic body formation; BEL-7404 cells were plated at a
density of 5 × 105 well in 6-well plate, treated with Cu(II) compounds at 0
ꢀM, 3 ꢀM for 12 h. Then cells were washed with PBS, and were stained
with DAPI and AO/EB double staining, respectively, for 10 minutes at room
temperature. After a wash in PBS, samples were observed under an inverted
fluorescence microscope and photographed.
4.4.4 Cell cycle analysis for Cu(II) compounds
Cell cycle analysis was carried out by flow cytometry [73]. Briefly, 5 ×
105 BEL–7404 cells were incubated with Cu(II) compounds at 0 ꢀM, 1.0 ꢀM,
2.0 ꢀM at 37 ꢀ for 48 h. Cells were harvested in cold PBS, centrifuged,
resuspended and fixed in 70% EtOH, overnight at 4 ꢀ. After a further
washing steps with cold PBS, cells were digested by RNase A (0.25 mg
mL−1) at 37 ꢀ for 30 minutes, and stained with PI (50 mg mL−1) in the
dark at room temperate for 30 minutes. The distribution of the cell cycle was
measured by a FAC Star Plus flow cytometer, and data analysis was carried
out with ModFit LT software.
4.5 Anticancer mechanism of Cu(II) compounds
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4.5.1 ROS assay
BEL-7404 cells were seeded into six-well plates at a density of 1 × 106
cells per well and incubated for 12 h. The cells were cultured in GIBCO
DMEM medium supplemented with 10% of fetal bovine serum (FBS) and
incubated at 37 ꢀ and 5% CO2. The medium was removed and replaced
with medium (final DMSO concentration 0.05% v/v) containing C1–C4 (25
ꢀM) for 24 h. The medium was removed again. The fluorescent dye
DCFH-DA was added to the medium with a final concentration of 10 ꢀM to
cover the cells. The treated cells were then washed with cold PBS-EDTA
twice (PBS = phosphatebuffered saline), collected by trypsinization and
centrifugation at 1500 rpm for 5 min, and resuspended in PBS-EDTA.
Fluorescence was imaged with fluorescent microscope at an excitation
wavelength of 488 nm and emission at 525 nm. To quantitatively compare
the levels of the ROS induced by C1–C4, BEL-7404 cells (1 × 106
cells/well) were incubated with either HL1 (25 ꢀM), HL2 (25 ꢀM), Cu(II)
salts (25 ꢀM) and Cu(II) compounds (25 ꢀM) for 30 minutes at 37 °C. Cells
were collected for flow cytometric assessment. The fluorescence intensity
was monitored with excitation wavelength at 488 nm and emission
wavelength at 525 nm.
4.5.2 Measurement of mitochondrial transmembrane potential for Cu(II)
compounds
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The cells were stained with JC-1 to determine the mitochondrial
membrane potential. First, BEL-7404 cells (about 5 × 105) were seeded in
6-well plates and treated with concentrations of Cu(II) compounds (3 ꢀM)
for 12 h. Then the medium was removed and the cells were washed with
PBS twice, stained with JC-1. After 20 minutes of incubation at 37 ꢀ in a
5% CO2 humidified atmosphere, the cells were washed with PBS twice and
observed with an inverted fluorescence microscope.
4.5.3 Western blot analysis
BEL-7404 cells were seeded in 3.5 cm dishes for 24 h and incubated with
different concentrations of the compounds in the presence of 10% FBS.
Then cells were harvested in lysis buffer. After sonication, the samples were
centrifuged for 20 min at 13,000g. The protein concentration of the
supernatant was determined by BCA (bicinchoninic acid) assay. Sodium
dodecyl sulfate–polyacrylamide gel electrophoresis was done loading equal
amount of proteins per lane. Gels were then transferred to poly (vinylidene
difluoride) membranes (Millipore) and blocked with 5% non-fat milk in
TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 8.0) buffer
for 1 h. The membranes were incubated with primary antibodies at 1:5000
dilutions in 5% non–fat milk overnight at 4 ꢀ, and after being washed for
four times with TBST for a total of 30 min, then the secondary antibodies
conjugated with horseradish peroxidase at 1:5000 dilution for 1 h at room
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temperature and washed for four times with TBST. The blots were visualized
with the Amersham ECL Plus western blotting detection reagents according
to the manufacturer's instructions. To assess the presence of comparable
amount of proteins in each lane, the membranes were strippedfinally to
detect the β-actin.
4.5.4 Statistical Analysis
All experiments were repeated five 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 the Natural Science Foundation of China (31460232 and
21431001), the Natural Science Foundation of Guangxi (2014GXNSFDA118016),
National Basic Research Program of China (IRT1225), Ministry of Education of China
(CMEMR2015-A01), Technology division of Guilin (20130403-1) and Guangxi ‘Bagui’
scholar program to ZP Zhou.
Supplementary data
All relative data of the experiment have been deposited into Supplementary
Material. CCDC NO. 1030943, 1030944, 1030945 and 1021208 contains the
supplementary crystallographic data for C1–C4. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
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Reference
[1]
[2]
[3]
B.V. Rosenberg, L. Camp, T. Krigas, Nature 205 (1965) 698−699.
M.D. Hall, T.W. Hambley, Coordination Chemistry Reviews 232 (2002) 49−67.
J. Easmon, G. Purstinger, G. Heinisch, T. Roth, H.H. Fiebig, W. Holzer, W. Jager, M.
Jenny, J. Hofmann, Journal of Medicinal Chemistry 44 (2001) 2164−2171.
M. Jiang, Y.T. Li, Z.Y. Wub, Z.Q. Liu, C.W. Yan, Journal of Inorganic Biochemistry
103 (2009) 833−844.
[4]
[5]
D. Palanimuthu, S.V. Shinde, K. Somasundaram, A.G. Samuelson, Journal of
Medicinal Chemistry 56 (2013) 722−734.
[6]
S. Tabassum, M. Zaki, M. Ahmad, M. Afzal, S. Srivastav, S. Srikrishna, F. Arjmand,
European Journal of Medicinal Chemistry 83 (2014) 141−154.
F. Liang, C. Wu, H. Lin, T. Li, D. Gao, Z. Li, J. Wei, C. Zheng, M. Sun, Bioorganic
& Medicinal Chemistry Letters 13 (2003) 2469−2472.
[7]
[8]
G. Barone, A. Terenzi, A. Lauria, A.M. Almerico, J.M. Leal, N. Busto, B. García,
Coordination Chemistry Reviews 257 (2013) 2848−2862.
[9]
D. Lovejoy, P. Jansson, U. Brunk, J. Wong, P. Ponka, D. Richardson. Cancer
Research 71 (2011) 5871−5880.
[10]
M. Alagesana, N.S.P. Bhuvanesh, N. Dharmaraj, European Journal of Medicinal
Chemistry 78 (2014) 281−293.
[11]
[12]
[13]
[14]
[15]
F. Tisato, C. Marzano, M. Porchia, M. Pellei, C. Santini, Medicinal Research
Reviews 30 (2010) 708−749.
C. Marzano, M. Pellei, F. Tisato, C. Santini, Anti–Cancer Agents in Medicinal
Chemistry 9 (2009) 185−211.
J. S. Casas, M. S. Garcia-Tasende, Sordo, journal of coordination chemistry Reviews
209 (2000) 197−261.
R. W. Brockman, J. R. Thomson, M. J. Bell, H. E. Skipper, Cancer Research 16
(1956) 167−170.
Y. Yu, J. Wong, D.B. Lovejoy, D.S. Kalinowski, D.R. Clin. Richardson, Cancer
Research 12 (2006) 6876−6883.
23

ACCEPTED MANUSCRIPT
[16]
[17]
[18]
B. Ma, B.C. Goh, E.H. Tan, K.C. Lam, R. Soo, S.S. Leong, L.Z. Wang, F. Mo, A.T.C.
Chan, B. Zee, T. Mok, Invest. New Drugs 26 (2008) 169−173.
M.J. Mackenzie, D. Saltman, H. Hirte, J. Low, C. Johnson, G. Pond, M.J. Moore,
Investigational New Drugs 25 (2007) 553−558.
C.R. Kowol, R. Trondl, P. Heffeter, V.B. Arion, M.A. Jakupec, A. Roller, M.
Galanski, W. Berger, B.K. Keppler, Journal of Medicinal Chemistry 52 (2009)
5032−5043.
[19]
P.J. Jansson, P.C. Sharpe, P.V. Bernhardt, D.R. Richardson, Journal of Medicinal
Chemistry 53 (2010) 5759−5769.
[20]
[21]
J. Yuan, D.B. Lovejoy, D.R. Richardson, Blood 104 (2004) 1450−1458.
P.V. Bernhardt, P.C. Sharpe, M. Islam, D.B. Lovejoy, D.S. Kalinowski, D.R.
Richardson, Journal of Medicinal Chemistry 52 (2009) 407−415.
D.S. Kalinowski, Y. Yu, P.C. Sharpe, M. Islam, Y.T. Liao, D.B. Lovejoy, N. Kumar,
P.V. Bernhardt, D.R. Richardson, Journal of Medicinal Chemistry 50 (2007)
3716−3729.
[22]
[23]
[24]
[25]
P. Kalaivani, S. Saranya, P. Poornima, R. Prabhakaran, F. Dallemer, V. Vijaya Padma,
K. Natarajan, European Journal of Medicinal Chemistry 82 (2014) 584−599.
C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato, C. Marzano, Chemical
Reviews 114 (2013) 815−862.
A.T. Chaviara, P.J. Cox, K.H. Repana, A.A. Pantazaki, K.T. Papazisis, A.H. Kortsaris,
D.A. Kyriakidis, G.S. Nikolov, C.A. Bolos, Journal of Inorganic Biochemistry 99
(2005) 467−476.
[26]
[27]
Z. Debebe, T. Ammosova, D. Breuer, D.B. Lovejoy, D.S. Kalinowski, P.K. Karla,
K.Kumar, M. Jerebsova, P. Ray, F. Kashanchi, V.R. Gordeuk, D.R. Richardon, S.
Nekhai, Molecular Pharmacology 79 (2011) 185−196.
G.L. Parrilha, K.S.O. Ferraz, J.A. Lessa, K.N. Oliveira, B.L. Rodrigues, J.P. Ramos,
E.M. Souza–Fagundes, I. Ott, H. Beraldo, European Journal of Medicinal Chemistry
24

ACCEPTED MANUSCRIPT
84 (2014) 537−544.
[28]
K. Kumar, S. Schniper, A. González–Sarrías, A.A. Holder, N. Sanders, D. Sullivan,
W.L. Jarrett, K. Davis, F. Bai, N.P. Seeram, V. Kumar, European Journal of
Medicinal Chemistry 86 (2014) 81−86.
[29]
[30]
[31]
J. Shao, Z.Y. Ma, A. Li, Y.H. Liu, C.Z. Xie, Z.Y. Qiang, J.Y. Xu, Journal of Inorganic
Biochemistry 136 (2014) 13−23.
Y. Gou, Y. Zhang, J.X. Qi, Z.P. Zhou, F. Yang, H. Liang, Journal of Inorganic
Biochemistry 136 (2014) 13−23.
A.G. Majouga, M.I. Zvereva, M.P. Rubtsova, D.A. Skvortsov, A.V. Mironov, D.M.
Azhibek, O.O. Krasnovskaya, V.M. Gerasimov, A.V. Udina, N.I. Vorozhtsov, E.K.
Beloglazkina, L. Agron, L.V. Mikhina, A.V. Tretyakova, N.V. Zyk, N.S. Zefirov, A.V.
Kabanov, O.A. Dontsova, Journal of Medicinal Chemistry 57 (2014) 6252−6258.
D.B. Lovejoy, D.M. Sharp, N. Seebacher, P. Obeidy, T. Prichard, C. Stefani, Basha,
M.T. Basha, P.C. Sharpe, P.J. Jansson, D.S. Kalinowski, P.V. Bernhardt, D.R.
Richardson, Journal of Medicinal Chemistry 55 (2012) 7230−7244.
X.Y. Qin, L.C. Yang, F.L. Le, Q.Q. Yu, D.D. Sun, Y.N. Liu, J. Liu, Dalton
Transactions 42 (2013) 14681−14684.
[32]
[33]
[34]
[35]
D. Palanimuthu, A.G. Samuelson, Inorganica Chimica Acta 408 (2013) 152−161.
C.N. Hancock, L.H. Stockwin, B. Han, R.D. Divelbiss, J.H. Jun, S.V. Malhotra, M.G.
Hollingshead, D.L. Newton, Free Radical Biology and Medicine 50 (2011) 110−121.
P. Kalaivani, S. Saranya, P. Poornima, R. Prabhakaran, F. Dallemer, V.V. Padma, K.
Natarajan, European Journal of Medicinal Chemistry 82 (2014) 584−599.
R.W. Chen, C. Yao, X.C.M. Lu, Z.G. Jiang, R. Whipple, Z. Liao, H.A. Ghanbari, B.
Almassian, F.C. Tortella, J.R. Dave, Neuroscience 135 (2005) 191−201.
D.S. Kalinowski, P. Quach, D.R. Rchardson, Future Medicinal Chemistry 1 (2009)
1143−1151.
[36]
[37]
[38]
[39]
S. Medici, M. Peana, V.M. Nurchi, J.I. Lachowicz, G. Crisponi, M.A. Zoroddu,
25

ACCEPTED MANUSCRIPT
Coordination Chemistry Reviews 284 (2015) 329−350.
[40]
[41]
[42]
C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato, C. Marzano, Chemical
Reviews 114 (2014) 815−862.
X.C. Huang, J.P. Zhang, X.M. Chen, Journal of the American Chemical Society 126
(2004) 13218−13219.
Y.F. Zhou, F.L. Jiang, D.Q. Yuan, B.L. Wu, R.H. Wang, Z.Z. Lin, M.C. Hong,
Angewandte Chemie International Edition 43 (2004) 5665−5668.
P. Ballester, Chemical Society Reviews 39 (2010) 3810−3830.
S.T. Wu, L.S. Long, R.B. Huang, Crystal Growth & Design 7 (2007) 1746−1752.
W. Yuan, T. Friscic, D. Apperley and S.L. James, Angewandte Chemie International
[43]
[44]
[45]
Edition 49 (2010) 3916−3919.
[46]
[47]
M. Cindrić, M. Uzelac, D. Cinčić, I. Halasz, G. Pavlović, T. Hrenar, M. Ćurićc, D.
Kovačević, CrystEngComm 14 (2012) 3039−3045.
M.N.M. Milunovic, E.A. Enyedy, N.V. Nagy, T. Kiss, R. Trondl, M.A. Jakupec, B.K.
Keppler, R. Krachler, G. Novitchi, V.B. Arion, Inorganic Chemistry 51 (2012)
9309−9321.
[48]
[49]
[50]
[51]
[52]
[53]
[54]
M. Zimmer, G. Schulte, X.L. Luo, R.H. Crabtree, Angewandte Chemie International
Edition 30 (1991) 193−194.
J. Valdes–Martinez, R.A. Toscano, A. Zentella–Dehesa, M.M. Salberg, G.A. Bain,
D.X. West, Polyhedron 15 (1996) 427−431.
A. Pal, B. Biswas, S.K. Mondal, C.H. Lin, R. Ghosh, Polyhedron 31 (2012)
671−675.
C.Q. Wan, Z.J. Wang, G. Wang, H. Liu, Y.H. Deng, Q.H. Jin, Crystal Growth &
Design 12 (2012) 376−386.
F.J. Liu, D. Sun, H.J. Hao, R.B. Huang, L.S. Zheng, Crystal Growth & Design 12
(2012) 354−361.
P.H. Walton, C.J. Boxwell, Journal of the Chemical Cociety- Chemical
Communications 1999, 1647−1648.
L. Cronin, B. Greener, M.H. Moore, P.H. Walton, Journal of the Chemical Cociety-
Dalton Transactions 1996, 3337−3339.
26

ACCEPTED MANUSCRIPT
[55]
[56]
[57]
K.M. Barkigia, M.D. Berber, J. Fajer, C.J. Medforth, M.W. Renner, K.M. Smith,
Journal of the American Chemical Society 112 (1990) 8851−8857.
R.C. Taylor, S.P. Cullen, S.J. Martin, Nature Reviews Molecular Cell Biology 9
(2008) 231−241.
C. Stefani, G. Punnia–Moorthy, D.B. Lovejoy, P.J. Jansson, D.S. Kalinowski, P.C.
Sharpe, P.V. Bernhardt, D.R. Richardson. Journal of Medicinal Chemistry 54 (2011)
6936−6948.
[58]
[59]
J.D. Ly, D.R. Grubb, A. Lawen, Apoptosis. 8 (2003) 115−128.
X.H. Liu, J. Wang, C.Q. Lu, CY. Zhu, B. Qian, Z.W. Li, C. Liu, J. Shao, J.S. Yan,
Chemosphere 124 (2015) 10−21.
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
F. Ding, Z.W. Shao, S.H. Yang, Q. Wu, F. Gao, L.M. Xiong, Apoptosis 17 (2012)
579−590.
L.L. Li, Y.S. Wong, T.F. Chen, C.D. Fan, W.J. Zheng, Dalton Transactions 41 (2012)
1138−1141.
T.F. Chen, Y.N. Liu, W.J. Zheng, J. Liu, Y.S. Wong, Inorganic Chemistry 49 (2010)
6366−6368.
X.X. Yang, L.M. Chen, Y.N. Liu, Y.G. Yang, T.F. Chen, W.J. Zheng, J. Liu, Q.Y. He,
Biochimie 94 (2012) 345−353.
M. Certo, D.G. Moore V, M. Nishino, G. Wei, S. Korsmeyer, S.A. Armstrong, et al.
Cancer Cell 9 (2006) 351−365.
M.C. Wei, W.X. Zong, E.H. Cheng, T. Lindsten, V. Panoutsakopoulou, A.J. Ross, et
al. Science 292 (2001) 727−730.
L. Chen, S.N. Willis, A. Wei, B.J. Smith, J.I. Fletcher, M.G. Hinds, et al. Molecular
Cell 17 (2005) 393−403.
D.J. Silva, H. Wang, N.M. Allanson, R.K. Jain, M.J. Sofia. Journal of Organic
Chemistry 64 (1999) 5926−5929.
G. Lessene, P.E. Czabotar, P.M. Colman, Nat. Rev. Drug Discovery 7 (2008)
989−1000.
M.F. Rega, B. Wu, J. Wei, Z. Zhang, J.F. Cellitti, M. Pellecchia, Journal of Medicinal
Chemistry 54 (2011) 6000−6013.
27

ACCEPTED MANUSCRIPT
[70]
[71]
J. Wei, S. Kitada, J.L. Stebbins, W. Placzek, D. Zhai, B. Wu, M.F. Rega, Z. Zhang, J.
Cellitti, L.Yang, R. Dahl, J.C. Reed, M. Pellecchia, Journal of Medicinal Chemistry
53 (2010) 8000−8011.
G.M. Sheldrick, SHELXS-97, Program of the Solution of Crystal Structure,
University of Göttingen, Germany, 1997.
[72]
[73]
T. Mosmann, J. Immunol. Methods 65 (1983) 55−63.
D. Jain, N. Patel, M. Shelton, A. Basu, R. Roque, W. Siede, Chemother Pharmacol 66
(2010) 945−952.
28