Synthesis, crystal structures, and biological evaluation of manganese(II) and nickel(II) complexes of 4-cyclohexyl-1-(1-(pyrazin-2-yl)ethylidene) thiosemicarbazide

Article abstract of DOI:10.1016/j.ejmech.2011.07.009

4-Cyclohexyl-1-(1-(pyrazin-2-yl)ethylidene)thiosemicarbazide (HL) and its transition metal complexes formulated as [Mn(L)₂] (1) and [Ni(L) ₂] (2) have been prepared in 55-75% yield and characterized by elemental analysis, IR, MS, NMR

Full text of DOI:10.1016/j.ejmech.2011.07.009

European Journal of Medicinal Chemistry 46 (2011) 4383e4390
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journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, crystal structures, and biological evaluation of manganese(II)
and nickel(II) complexes of 4-cyclohexyl-1-(1-(pyrazin-2-yl)ethylidene)
thiosemicarbazide
a
,
b
,
, Li Zhi Zhang , Dong Zhang , Bian Sheng Ji ,**, Jun Wei Zhao a
a
a
b
*
Ming Xue Li
a
Institute of Molecular and Crystal Engineering, College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, PR China
Key Laboratory of Natural Medicines and Immune Engineering, Henan University, Kaifeng 475004, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 February 2011
Received in revised form
4-Cyclohexyl-1-(1-(pyrazin-2-yl)ethylidene)thiosemicarbazide (HL) and its transition metal complexes
formulated as [Mn(L) ] (1) and [Ni(L) ] (2) have been prepared in 55e75% yield and characterized by
2 2
elemental analysis, IR, MS, NMR and single-crystal X-ray diffraction studies. Biological activities of the
synthesized compounds have been evaluated against selected Gram positive bacteria Bacillus subtilis,
Gram negative bacteria Pseudomonas aeruginosa and the K562 leukemia cell line, respectively. The
cytotoxicity data suggest that these compounds may be endowed with important biological properties,
29 June 2011
Accepted 4 July 2011
Available online 8 July 2011
especially the nickel complex 2 with MIC ¼ 31.2
m
g/mL and IC50 ¼ 0.53
mM, respectively. Effect of the free
Keywords:
Thiosemicarbazone
Complex
Crystal structure
Cytotoxicity
ligand and its two complexes on Mitochondria membrane potential (MMP) and PI-associated fluores-
cence intensity as well as their effect on cell apoptosis in K562 leukemia cell line was also studied. The
tested compounds may exert their cytotoxicity activity via induced loss of MMP.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
decrease upon complexation [8]. Their mechanism of action is still
controversial in many respects, but it is known that TSCs act by
Heterocyclic thiosemicarbazones (TSCs) and their transition
metal complexes have received considerable attention due to their
coordination chemistry and the beneficial pharmacological prop-
erties, notable for antiparasital, antibacterial and antitumor
activities [1e6]. Currently the most promising drug candidate
of this class of compounds is Triapine (3-aminopyridine-2-
carboxaldehyde thiosemicarbazone, 3-AP), which entered several
phase I and II clinical trials as an antitumor chemotherapeutic agent
inhibiting ribonucleotide reductase, a key enzyme in the biosyn-
thesis of DNA precursors [9].
Our previous studies on
a variety of substituted thio-
semicarbazones and their diverse metal complexes showed that
both 2-acetylpyrazine thiosemicarbazone and 2-acetylpyrazine
N(4)-methylthiosemicarbazone exhibited remarkable biological
activity in vitro against K562 leukemia cell lines [10]. In particular,
2-acetylpyrazine N(4)-methylthiosemicarbazone shows a lower
[4]. The biological activities of thiosemicarbazones often showed
IC50 value (25.9
mM) than the 2-acetylpyrazine thiosemicarbazone
a high dependence on their substituents. Minor modifications in
the thiosemicarbazones can lead to widely different biological
activities. Earlier reports on N(4)-substituted thiosemicarbazones
have concluded that the presence of a bulky group at the terminal
nitrogen considerably increases biological activity [7]. Moreover,
the biological properties of thiosemicarbazones are often modu-
lated by metal ion coordination. In some cases, the highest bio-
logical activity is associated with a metal and some side effects may
(47.5 M), which indicates that the presence of bulky group at
m
position N(4) of the thiosemicarbazone moiety enhanced the
antitumor activities [10b]. On the other hand, their metal
complexes also exhibited significant antitumor activity [10]. Stim-
ulated by these encouraging and promising results, it seemed
useful and desirable to us to initiate systematic investigation of 2-
acetylpyrazine N(4)-substituted thiosemicarbazones and their
metal complexes. Particularly, information on the mechanism of
these compounds is sparse and valuable.
In the present work, with the main aim of comparison, we have
tested the biological activity of 4-cyclohexyl-1-(1-(pyrazin-2-yl)
ethylidene)thiosemicarbazide (HL) (Scheme 1) and its transition
*
Corresponding author. Institute of Molecular and Crystal Engineering, College of
Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, PR China.
** Corresponding author.
2 2
metal complexes formulated as [Mn(L) ] (1) and [Ni(L) ] (2) against
E-mail addresses: limingxue@henu.edu.cn (M. X. Li), jibiansheng@yahoo.com.cn
(
B. S. Ji).
selected Gram positive bacteria Bacillus subtilis, Gram negative
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.009

4384
M.X. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 4383e4390
The tentative assignments of the significant IR spectral bands of
HL and complexes 1 and 2 are presented in Table 1. The infrared
spectral bands most useful for determining the mode of coordi-
nation of the ligand are the
n(C]N), n(NeN) and n(C]S) vibrations.
The (C]N) band of thiosemicarbazone in complexes 1 and 2
n
undergoes a negative shift of wavenumber compared to that of the
ligand, a clear sign of coordination via the imine nitrogen atom
[
11b]. The increase in the frequency of n(NeN) band of the thio-
Scheme 1. 4-Cyclohexyl-1-(1-(pyrazin-2-yl)ethylidene)thiosemicarbazide, HL.
semicarbazone in the spectra of complexes is due to the increase in
the bond strength, again confirms the coordination via the imine
nitrogen [12]. The thioamide band, which contains considerable
Table 1
IR spectral assignments (cmꢀ1) of HL and complexes 1 and 2.
n
(C]S) character, is less intense in the complexes and is found at
a lower frequency, suggesting coordination of the metal through
sulfur [13]. The breathing motion of the pyrizine ring is shifted to
a higher frequency upon complexation and is consistent with
pyrizine ring nitrogen coordination. These observations have also
been confirmed by X-ray single-crystal structure analysis.
Compound
n
(C]N)
n
(NeN)
n
(C]S)
r(pz)
HL
1
2
1568
1523
1538
1109
1148
1144
866
827
832
620
639
687
bacteria Pseudomonas aeruginosa and the K562 leukemia cell line,
respectively. It is worth noting that cytotoxic agents can induce cell
death through various pathways that include necrosis and
apoptosis [11a]. Apoptosis is a common process of programmed cell
death and is the focus of current oncology research. To explore their
biochemical mechanism of cytotoxicity initially, effect of the title
three compounds on Mitochondria membrane potential (MMP)
and PI-associated fluorescence intensity as well as their effect on
cell apoptosis in K562 leukemia cell line are also studied. In addi-
tion, we also describe the synthesis and single-crystal X-ray crystal
structures of complexes 1 and 2 here.
3. Results and discussion
3.1. X-ray crystallography
Table 2 summarizes crystal and refinement data for 1 and 2. The
molecular structures of 1 and 2 along with the atomic numbering
scheme are shown in Figs. 1 and 2, respectively. Selected bond
lengths and angles are listed in Table 3.
2 2
In view of the structural similarity of [Mn(L) ] (1) and [Ni(L) ]
(2), only complex 1 was described in some detail. As shown in Fig. 1,
the manganese(II) ion is in a slightly distorted octahedral envi-
ronment, where two 4-cyclohexyl-1-(1-(pyrazin-2-yl)ethylidene)
2
. Chemistry
2
thiosemicarbazide units deprotonated act as N S tridentate ligands
coordinated to the central manganese atom via the pyrazine
nitrogen, imine nitrogen and sulfur atoms. One sulfur atom, one
imine nitrogen atom and one pyrazine nitrogen atom from one
ligand and one imine nitrogen atom from another ligand occupy
the basal positions, the two remaining positions in the octahedral
geometry are the axial ones which are occupied by one sulfur atom
and one pyrazine nitrogen atom from the second ligand. The
4
-Cyclohexyl-1-(1-(pyrazin-2-yl)ethylidene)thiosemicarbazide
HL) and its transition metal complexes formulated as [Mn(L) ] (1)
and [Ni(L) ] (2) have been synthesized. The complexes 1 and 2 were
(
2
2
characterized by elemental analysis, IR, MS and single-crystal X-ray
diffraction studies. Biological activities of these compounds have
been investigated.
Table 2
Summary of crystal data and refinement results for complexes 1 and 2.
Compound
1
2
Empirical formula
Formula weight
Crystal size (mm)
Crystal system
Space group
a (Å)
C
26
H
36 Mn N10
S
2
26 36 2
C H N10 Ni S
611.48
607.71
0.37 ꢁ 0.19 ꢁ 0.17
0.37 ꢁ 0.18 ꢁ 0.15
Monoclinic
Monoclinic
C2/c
17.4629 (19)
C2/c
29.395
b (Å)
13.0277 (14)
13.743
c (Å)
14.3027 (15)
3183.1 (6)
101.970 (2)
1.268
21.170
6155.2
133.97
1.320
3
V (Å )
ꢂ
b
/
(g cm 3
ꢀ
)
D
Z
m
q
c
4
8
(mmꢀ1)
0.578
1.97e25.00
0.800
1.77e25.00
ꢂ
( )
F (000)
hkl Range
T (K)
1276
2576
ꢀ20 ꢃ h ꢃ 14, ꢀ14 ꢃ k ꢃ 15, ꢀ16 ꢃ l ꢃ 16
ꢀ34 ꢃ h ꢃ 30, ꢀ16 ꢃ k ꢃ 16, ꢀ17 ꢃ l ꢃ 25
296 (2)
293 (2)
Refl. measured
Refl. unique
2796
2306
5261
3882
R
int
0.0243
0.0412
Parameters
177
352
R
R
1
,wR
,wR
2
[I ꢄ 2
s
(I)]
0.0580, 0.1591
0.0688, 0.1670
1.089
1.491, ꢀ1.0840
0.0420, 0.1217
0.0585, 0.1283
0.967
1.572, ꢀ0.276
1
2
(all date)
2
Goodness-of-fit on F
Drmax, min(e Å
ꢀ
3
)

M.X. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 4383e4390
4385
Fig. 1. Structure of complex 1 with atomic numbering scheme.
pseudo-macrocyclic coordination mode of each ligand affords two
3.2. Antibacterial activity
five-membered chelate rings.
The C(7)eS(1) bond length of 1.743 (4) is within the normal
range of CeS single bonds, indicating that the thiosemicarbazone
moieties adopt the thiol tautomeric form [14]. The CeN and NeN
In view of the antimicrobial activity of thiosemicarbazone [6],
we have tested the inhibition ability of the obtained compounds
against selected Gram positive bacteria B. subtilis and Gram nega-
tive bacteria P. aeruginosa by the disc diffusion method. Based on
the minimum inhibitory concentration (Table 5), generally, the title
three compounds display more inhibitory properties against Gram
positive bacteria B. subtilis than against Gram negative bacteria P.
aeruginosa and HL and 2 are more active than complex 1. It should
be emphasized that HL and 2 show higher activities against Gram
negative bacteria P. aeruginosa than positive control antibiotics
ampicillin (Amp), streptomycin (Str), kanamycin sulfate (Kan),
respectively. In addition, although the MIC values of the parent
ligand and its complexes did not reveal meaningful differences
which is consistent with the previously reported that of 2-
acetylpyridine N(4)-cyclohexylthiosemicarbazone and its man-
ganese(II) and nickel(II) complexes, 2-acetylpyrazine N(4)-
phenylthiosemicarbazone and its diorganotin(IV) complexes [16],
in general these results are still very gratifying which are encour-
aging further screening in vitro and/or in vivo as well as evaluation
of mechanism.
ꢀ
bond lengths in L are intermediate between formal single and
double bonds, pointing to an extensive electron delocalization over
the entire molecular skeleton. The two thiosemicarbazone ligands
have slightly different MneN(pyrizine) bond distances and they are
longer than the MneN(imine) distances, this may be attributed to
the fact that the imine nitrogen is a stronger base compared with
the pyrizine nitrogen [15].
Complex 1 is stabilized by intermolecular hydrogen bonds (see
Fig. 3, Table 4). The hydrogen bond involves the terminal nitrogen
atom N(1) and the uncoordinated pyrazine nitrogen atom N(5) with
ꢂ
N(1)/N(5) 3.055 (5) Å and the angle N(1)eH(1A)/N(5) being 173.6
(
symmetrycode: xꢀ1/2,yꢀ1/2,z). Similarly, intermolecularhydrogen
bonds of complex 2 also link the different components to stabilize the
crystal structure (see Fig. 4, Table 4), differing only in the hydrogen
bond donors and acceptors. These intermolecular hydrogen bonds
include: terminal nitrogen atoms N(1) and N(6), hydrazine nitrogen
atom N(2) and the coordinated sulfur atom S(2), respectively.
Fig. 2. Structure of complex 2 with atomic numbering scheme.

4386
M.X. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 4383e4390
Table 3
Selected bond lengths (Å) and angles ( ) of complexes 1 and 2.
Table 4
ꢂ
ꢂ
Hydrogen bond lengths (Å) and bond angles ( ) in complexes 1 and 2.
1
2
DeH/A
d(H/A)
d(D/A)
:(DHA)
Mn(1)eN(3)
Mn(1)eN(4)
Mn(1)eS(1)
S(1)eC(7)
N(3)eC(8)
N(3)eN(2)
N(1)eC(7)
2.257 (3)
2.350 (3)
2.531 (1)
1.743 (4)
1.303 (4)
1.362 (4)
1.343 (4)
Ni(1)eN(3)
Ni(1)eN(4)
Ni(1)eS(1)
S(1)eC(7)
N(3)eC(8)
N(3)eN(2)
N(1)eC(7)
2.010 (2)
2.120 (2)
2.411 (1)
1.709 (3)
1.311 (3)
1.351 (2)
1.343 (3)
1
a
N(1)eH(1A)/N(5)
2.20
3.055 (5)
173.6
2
N(1)eH(1A)/S(2)b
2.64
2.17
3.484 (2)
3.029 (3)
167.1
178.9
c
N(6)eH(6A)/N(2)
Symmetry transformation used to generate the equivalent atoms is given in the
footnotes listed below.
a
xꢀ1/2, yꢀ1/2, z.
N(3)eMn(1)eN(3A)
N(3)eMn(1)eN(4)
N(4)eMn(1)eN(4A)
N(3)eMn(1)eS(1)
N(4)eMn(1)eS(1A)
145.51 (14)
70.14 (10)
98.55 (16)
76.13 (7)
N(3)eNi(1)eN(8)
N(3)eNi(1)eN(4)
N(4)eNi(1)eN(9)
N(3)eNi(1)eS(1)
N(4)eNi(1)eS(2)
173.80 (7)
78.78 (8)
92.03 (7)
81.96 (5)
90.41 (6)
b
ꢀ
ꢀ
xþ3/2, yꢀ1/2, ꢀzþ1/2.
xþ3/2, yþ1/2, ꢀzþ1/2.
c
90.60 (8)
indicated stronger growth inhibition activities than that of cisplatin.
Therefore, the title complexes may be endowed with important
antitumor properties and merit further investigation for further
development as anticancer agents.
3.3. Antitumor evaluation
Mitochondria was considered to be a major site of antitumor
agents through electron leakage from the electron transport chain
Intermsofthecytotoxicactivityofthiosemicarbazones[17,18], we
have tested the ability of HL, 1 and 2 to inhibit tumor cell growth
against the K562 leukemia cell line. In our experiments, IC50 values
compound concentration that produces 50% of cell death) in micro
molar units were calculated. The investigation has clearly shown that
-cyclohexyl-1-(1-(pyrazin-2-yl)ethylidene)thiosemicarbazide and
its metal complexes show significant antitumor activityagainst K562
leukemia cell line (Fig. 5), due to the NNS tridentate system [18,19].
[21,22], the decreased MMP may open the mitochondrial perme-
ability transition (MPT) and trigger the release of cytochrome c
which activate caspase cascade, causing the cell death. In the
present report, rhodamine 123 (Rh123), a mitochondrial specific
stain that is dependent on the transmembrane potential, and
another one, Propidine iodide (PI), were applied for the determi-
nation of MMP and apoptosis cells as well as necrosis cells,
respectively. As shown in Figs. 6e8, the number of apoptosis cells
and PI-associated fluorescence intensity in K562 leukemia cells
were increased in a concentration-dependent manner companying
decreased MMP after incubation with the tested compounds for
(
4
Furthermore, HL shows much lower IC50 value (9.92
previously reported cases of the 2-acetylpyrazine N(4)-
methylthiosemicarbazone (25.9 M) and the 2-acetylpyrazine thio-
semicarbazone (47.5 M) which indicate that the presence of bulky
mM) than the
m
m
group at position N(4) of the thiosemicarbazone moiety greatly
enhanced the antitumor activities [10b]. In addition, it is clearly
observed that complexation with metals has a synergetic effect on
theantitumoractivityof thesecompoundsandtheantitumoractivity
depends upon the type of metal ion. As can be expected, coupling of
4
8 h, indicating the remarkable antitumor activity in vitro. The
present results also revealed that the complexes 1 and 2 exhibited
more potent effects than their parent ligand, which was consistent
with their actions in MTT assay.
HL to Mn(II) (2.78 mM) or Ni(II) (0.53 mM) increases the antitumor
activity of their parent ligand, in a similar way to that observed other
Mn(II) and Ni(II) compounds [11b,16a,20]. This confirms the
conclusion that the antitumor activities of thiosemicarbazones can
be enhanced by the linkage to metal ions. Importantly, in our
previous unpublished studies, cisplatin only exhibited the inhibitory
4. Conclusions
In summary, 4-cyclohexyl-1-(1-(pyrazin-2-yl)ethylidene)thio-
semicarbazide and its manganese(II) and nickel(II) complexes were
synthesized and characterized. Biological studies showed that the
title three compounds exhibited significant and different biological
activities. The title compounds may exert their cytotoxicity activity
percentage of 9.46% at a concentration of 50
mM for 24 h against K562
leukemia cell line, suggesting that these studied compounds
Fig. 3. Hydrogen bond indicated by dashed lines in complex 1.

M.X. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 4383e4390
4387
Fig. 4. Hydrogen bond indicated by dashed lines in complex 2.
via induced loss of MMP. These promising results will be essential
5.2.2. Synthesis of complex 1
for antibacterial and anticancer drug discovery and medical
practice.
A methanol solution containing Mn(ClO
0.2 mmol) was added dropwise to a solution of 4-cyclohexyl-1-(1-
pyrazin-2-yl)ethylidene)thiosemicarbazide (0.111 g, 0.4 mmol)
4
)
2
$6H
2
O (0.072 g,
(
dissolved in 20 mL methanol. The solution immediately turned
deep red. After stirring for 0.5 h, the resultant solution was filtered.
Deep-red crystals suitable for X-ray studies were obtained by slow
evaporation of its ethanol solution. Yield: 58% eC26 H36 Mn N10 S2
5
. Experimental
5.1. Materials and chemicals
(
607.71): calcd. C 51.34, H 5.92, N 23.04; found C 51.76, H 5.71, N
þ
All chemicals were of reagent grade quality obtained from
23.38. ESI-MS (m/z): 608.3 ¼ [Mn(L)(HL)] , calc. mass ¼ 608.2.
commercial sources and used without further purification. Instru-
mentation: Elemental analysis of C, H and N was performed with
a PerkineElmer240 analyzer. The infrared spectra were recorded
5.2.3. Synthesis of complex 2
Complex 2 was prepared by a similar procedure to that of
complex 1 using Ni(ClO ) $6H O in place of Mn(ClO ) $6H O. Red
crystals suitable for X-ray studies were obtained by slow evapora-
tion of its ethanol solution. Yield: 55% eC26 H36 N10 Ni S (611.48):
calcd. C 51.02, H 5.89, N 22.90; found C 51.68, H 5.64, N 22.51. ESI-
MS (m/z): 611.3 ¼ [Ni(L)(HL)] , calc. mass ¼ 611.2.
1
from KBr discs with a Nicolet 170 FT infrared spectrophotometer. H
4
2
2
4 2
2
NMR spectra were recorded in DMSO-d
6
using a BrukerAV-400
spectrometer. The mass spectra were carried out on an Esquire
000 LCeMS mass spectrophotometer.
2
3
þ
5
5
.2. Synthesis
5.3. X-ray crystallography
.2.1. Synthesis of ligand
Cyclohexyl isothiocyanate (1.41 g, 10 mmol) and hydrazine
Intensities of the title complexes were collected on a Siemens
hydrate (0.50 g, 10 mmol), each dissolved in 20 mL methanol were
mixed with constant stirring. The stirring was continued for 1 h and
the white product, N(4)-cyclohexyl thiosemicarbazide formed was
filtered, washed, dried and recrystallized from methanol. A meth-
anolic solution of N(4)-cyclohexyl thiosemicarbazide (0.52 g,
mmol) was then refluxed with 2-acetylpyrazine (0.37 g, 3 mmol)
in 10 mL methanol continuously for 4 h after adding a few drops of
acetic acid. Yellow crystals separated on cooling were washed and
SMART-CCD diffractometer equipped with
chromatic Mo-K
a graphite-mono-
¼ 0.71073 Å) radiation using the SMART and
a
(l
SAINT programs. The structure was solved by direct method and
2
refined on F by full-matrix least-squares techniques with SHELXTL
version 5.1 [23,24]. All of the non-hydrogen atoms were refined
with anisotropic thermal displacement parameters. The hydrogen
3
recrystallorized from methanol. Yield: 75% eC13
calcd. C 56.32, H 6.86, N 25.27; found C 55.74, H 6.71, N 25.58. H
NMR (DMSO-d
19 5
H N S (277):
1
6
,
d
ppm): 10.37 (s, 1H, NH), 9.58 (d, J ¼ 7.2Hz, 1H,
C
C
3
4
N
N
2
H
H
3
), 8.62e8.61(m, 2H, C
), 1.86e1.13 (m, 11H, C
00.1 ¼ [HL þ Na ], calc. mass ¼ 300.1.
4
N
2
H
3, NH), 8.30 (d, J ¼ 7.2Hz, 1H,
4
2
3
6
H
11), 2.35(s, 3H, CH ). ESI-MS (m/z):
3
þ
Table 5
Antibacterial activities of the tested compounds.
Microorganisms
MIC (
HL
m
g/mL)
1
2
Amp
5
Str
Kan
B. subtilis
P.aeruginosa
15.6
31.2
31.2
62.5
31.2
31.2
20
e
10
40
a
e
a
No inhibition or MIC >200
m
g/mL.
Fig. 5. The antitumor activity of HL, 1 and 2 against K562 leukemia cell line.

4388
M.X. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 4383e4390
Fig. 6. Effect of HL, 1 and 2 on MMP in K562 eukaemia cell line. Each value represents the mean ꢅ S.D. from four experiments. #: p < 0.05 vs control.
Fig. 7. Effect of HL, 1 and 2 on cell apoptosis in K562 leukemia cell line.

M.X. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 4383e4390
4389
Fig. 8. Effect of HL, 1 and 2 on PI-associated fluorescence intensity in K562 leukemia line. Each value represents the mean ꢅ S.D. from four experiments. #: p<0.05 vs control.
ꢂ
atoms were positioned according to theoretical models. Crystallo-
graphic data of 1 and 2 are summarized in Table 2.
CCDC 798836 and 798837 contain the supplementary crystal-
lographic data for 1 and 2, respectively. These data can be obtained
free of charge from the Cambridge Crystallographic Centre via
www.ccdc.cam.ac.uk/data_request/cif.
Rh123 (2
m
M) and Hoechst 33342 (3 mg/ml) for 30 min at 37 C, and
then rinsed with freshly prepared PBS. The fluorescence intensity
was measured at emission wavelength of 530 nm and excitation
wavelengths of 480 nm by High-Content Screening Reader (Arrary
Scan VTI 600, USA).
Statistical analysis: All the data were expressed as mean ꢅ S.D.
and analyzed using analysis of ariance (ANOVA) followed by
Student’s t-test. Differences were considered statistically significant
at p < 0.05.
5.4. Antibacterial experiments
The in vitro antibacterial activity of the title compounds was
investigated against selected Gram positive bacteria B. subtilis and
Gram negative bacteria P. aeruginosa. The minimal inhibitory
5.7. PI staining
After incubation with the tested compounds with different
concentrations (MIC,
method. The final concentration of bacteria in MuellereHinon agar
mg/mL) were estimated by the disk diffusion
concentration for 48 h, the K562/DOX cells were preloaded with PI
6
ꢂ
(
100
m
g/ml) and Hoechst 33342 (3 mg/ml) for 30 min at 37 C, and
(
MHA) was adjusted to 10 cfu/mL and used for inoculation in the
then rinsed with freshly prepared PBS. The fluorescence intensity
was measured at emission wavelength of 620 nm and excitation
wavelengths of 488 nm by High-Content Screening Reader (Arrary
Scan VTI 600, USA), the stained cells were observed and taken
photographs.
Statistical analysis: All the data were expressed as mean ꢅ S.D.
and analyzed using analysis of ariance (ANOVA) followed by
Student’s t-test. Differences were considered statistically significant
at p < 0.05.
MIC test. Serial dilutions of the test compounds, previously dis-
solved in dimethyl sulfoxide (DMSO) were prepared at concentra-
tions of 0e2000 mg/mL. To each plate was inoculated with 0.1 mL of
the prepared bacterial cultures. Similarly, each plate carried a blank
disc, with solvent DMSO only in the center to serve as negative
control, as well as positive control antibiotics ampicillin (Amp),
streptomycin (Str), kanamycin sulfate (Kan). The inoculated plates
ꢂ
were then incubated at 37 C for 18e20 h. The minimal inhibitory
concentration (MIC) was detected as the lowest concentration of
drug in plate for which no visible growth took place by macroscopic
evaluation. All determinations were performed in triplicate and
confirmed by three separate experiments.
Acknowledgments
This work was financially supported by the National Natural
Science Foundation of China (21071043), the China Postdoctoral
Science Foundation (20090460847), the Foundation for University
Young Key Teacher by Henan Province (2009GGJS-025) and the
Natural Science Foundation of the Educational Department of
Henan Province (2010B150003).
5
.5. Antitumor experiments
K562 leukemia cell line (purchased from the Institute of
Biochemistry and Cell Biology, SIBS, CAS) was cultured in RPMI-
ꢀ1
1640 medium supplemented with 10% FBS, 100 U mL of peni-
ꢂ
cillin, 100
atmosphere of 5% CO
mg (200
mL per well) of streptomycin at 37 C in humid air
2
. Cell cytotoxicity was assessed by the MTT
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