Synthesis, characterization, cytotoxic activity and DNA binding Ni(II) complex with the 6-hydroxy chromone-3-carbaldehyde thiosemicarbazone

Article abstract of DOI:10.1016/j.jorganchem.2009.08.024

A new ligand L, 6-hydroxy chromone-3-carbaldehyde thiosemicarbazone, and its Ni(II) complex have been synthesized and characterized. The crystal structure of Ni(II) complex was determined by single crystal X-ray diffraction. Ni(II) complex and ligand L were subjected to biological tests in vitro using THP-1, Raji and Hela cancer cell lines. Compared with the ligand, Ni(II) complex showed significant cytotoxic activity against these three cancer cell lines. The interactions of Ni(II) complex and ligand L with calf thymus DNA were then investigated by spectrometric titration, ethidium bromide displacement experiments and viscosity measurements methods. The experimental results indicated that Ni(II) complex bound to DNA by intercalative mode via the ligand L. The intrinsic binding constants of Ni(II) complex and ligand L with DNA were (1.10 ± 0.65) × 10⁶ M^-1 and (1.48 ± 0.57) × 10⁵ M^-1, respectively.

Full text of DOI:10.1016/j.jorganchem.2009.08.024

Journal of Organometallic Chemistry 694 (2009) 4069–4075
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis, characterization, cytotoxic activity and DNA binding Ni(II) complex
with the 6-hydroxy chromone-3-carbaldehyde thiosemicarbazone
a
a,*
b
b
b
c
Bao-dui Wang , Zheng-Yin Yang , Ming-hua Lü , Jun Hai , Qin Wang , Zhong-Ning Chen
a
College of Chemistry and Chemical Engineering and State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, PR China
College of life science, Lanzhou University, Lanzhou 730000, PR China
Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 June 2009
Received in revised form 3 August 2009
Accepted 18 August 2009
Available online 21 August 2009
A new ligand L, 6-hydroxy chromone-3-carbaldehyde thiosemicarbazone, and its Ni(II) complex have
been synthesized and characterized. The crystal structure of Ni(II) complex was determined by single
crystal X-ray diffraction. Ni(II) complex and ligand L were subjected to biological tests in vitro using
THP-1, Raji and Hela cancer cell lines. Compared with the ligand, Ni(II) complex showed significant cyto-
toxic activity against these three cancer cell lines. The interactions of Ni(II) complex and ligand L with calf
thymus DNA were then investigated by spectrometric titration, ethidium bromide displacement experi-
ments and viscosity measurements methods. The experimental results indicated that Ni(II) complex
bound to DNA by intercalative mode via the ligand L. The intrinsic binding constants of Ni(II) complex
Keywords:
6
-hydroxy chromone-3-carbaldehyde
thiosemicarbazone
Ni(II) complex
Crystal structure
DNA binding
6
ꢁ1
5
ꢁ1
and ligand L with DNA were (1.10 ± 0.65) ꢀ 10 M and (1.48 ± 0.57) ꢀ 10 M , respectively.
Ó 2009 Elsevier B.V. All rights reserved.
Cytotoxic activity
1
. Introduction
Metal complexes of sulphur containing Schiff bases has been
the above mentioned findings and as continuation of our effort to
identify new potent, selective and less toxic antitumor agents,
we report in the present work the synthesis of a new ligand, 6-hy-
droxy chromone-3-carbaldehyde thiosemicarbazone (Fig. 1), and
its transition metal complex. We described a comparative study
of the interactions of Ni(II) complex and ligand with CT-DNA using
UV-visible, fluorescence and viscosity measurements for the first
time.
the subject of current and growing interest because it has been
shown that many of these complexes possess anticancer activity.
In particular, thiosemicarbazones, as a class of compounds, exhibit
several interesting physical, chemical and wide range of biological
properties [1]. Thiosemicarbazones with different chemical struc-
tures have been investigated but few works deal with chromone
skeleton derivatives. Compounds containing the chromone skele-
ton (4H-benzopyran-4-one) (flavones and chromones) are an
important class of compounds belonging to the flavonoid group
that occur naturally in plants. They are minor constituents of the
human diet and have been reported to exhibit a wide range of
biological effects [2,3]. These biological properties include anti-
inflammatory, antibacterial, antitumor [2], antioxidant [4], anti-
HIV [5], vasodilator, antiviral and antiallergenic [6].
2
. Result and discussion
2.1. Chemistry
6
-hydroxy chromone-3-carbaldehyde thiosemicarbazone was
carried out by stirring the mixture of 3-formyl chromone (1)
1 mmol) with thiosemicarbazide (1 mmol) in ethanol. The pro-
(
gress of reaction was monitored by TLC. The resulting product
was recrystallized from ethanol.
In our research, we have found that the rare earth complexes of
flavane benzoyl hydrazone have certain antioxidant and cytotoxic
activity, and can bind to CT-DNA by intercalation [7–9]. In view of
The Ni(II) complex was prepared by reacting chromone thio-
semicarbazone (1 mmol) with Ni(II) nitrate (1 mmol) in ethanol.
The mixtures were heated to 60 °C about 5 min, and then the mix-
tures was filtrated to move residue and continued stirring for 24 h
at room temperature. The product was purified by washing several
times with ethanol.
Abbreviations: CT-DNA, calf thymus DNA; L, 6-hydroxy chromone-3-carbalde-
hyde thiosemicarbazone; Tris, tris(hydroxymethyl)-aminomethane; NMR, nuclear
magnetic resonance; EB, ethidium bromide; CDC, 6-hydroxy-3-carboxaldehydes
chromone; UV–visible, ultraviolet and visible.
The structures of the synthesized compounds were determined
*
Corresponding author. Fax: +86 931 8912582.
1
E-mail address: yangzy@lzu.edu.cn (Z.-Y. Yang).
by using spectroscopic techniques which include H NMR and IR
0
022-328X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2009.08.024

4
070
B.-d. Wang et al. / Journal of Organometallic Chemistry 694 (2009) 4069–4075
OH
OCOCH3
AlCl
OH
POCl3
(
CH CO) CO
3
COCH3
3
2
9
8% H SO4
DMF
2
OCOCH3
OH
3
OH
1
2
8
5
S
O
2
7
O
NH CNHNH2
2
S
CH NNHCNH2
HO
HO
CHO
O
O
4
5
Fig. 1. Scheme of the synthesis of the ligand.
spectroscopy. The crystal structure of Ni(II) complex was deter-
mined by single crystal X-ray diffraction. Elemental analysis re-
sults were also found to be satisfactory.
2.2. IR spectra studies
The (C@O) vibration of the free ligand is observed at
m
Fig. 2. The structure of complex 1.
ꢁ
1
1
1
5
623 cm ; for the complex this peak high frequency shift to
631 cmꢁ1;
91 cm is assignable to
D
m
(ligand ꢁ complex) is equal to 8 cm . The band at
ꢁ1
ꢁ1
m
(M–O) [10,11]. These demonstrate that
Table 1
Select bond lengths [A] and angles [deg] for Ni(II) complex.
the oxygen of carbonyl has formed a coordinative bond with the
ꢁ1
nickel ion. The band at 1592 cm for the free ligand is assignable
Ni–O(3)
Ni–N(1)
Ni–O(6)
Ni–O(4)
Ni–O(5)
Ni–S
O(3)–Ni–N(1)
O(3)–Ni–O(6)
O(3)–Ni–O(4)
N(1)–Ni–O(4)
2.033(4)
2.049(5)
2.064(5)
2.066(5)
2.076(5)
2.3724(18)
89.65(19)
90.9(2)
O(6)–Ni–O(4)
O(3)–Ni–O(5)
N(1)–Ni–O(5)
O(6)–Ni–O(5)
O(4)–Ni–O(5)
O(3)–Ni–S
88.6(2)
84.1(2)
92.0(2)
171.6(2)
88.5(2)
173.78(13)
84.38(15)
93.76(15)
95.28(16)
94.
ꢁ1
to the
its complex. Weak bands at 427 cm are assignable to
10,11]. These further confirm that the nitrogen of the imino-group
m
(C@N) stretch [7,12], which shifts to about 1571 cm for
ꢁ1
m
(M–N)
[
ꢁ1
bonds to the metal ion. Two bands at 1310 and 820 cm are as-
signed to thiocarbonyl (C@S) stretching and bending modes of
m
N(1)–Ni–S
O(6)–Ni–S
vibrations [13], which are shifted towards lower energy in the
90.7(2)
O(4)–Ni–S
spectrum of Ni(II) complex thus indicating coordination of thiona-
179.4(2)
O(5)–Ni–S
0
ꢁ1
to sulphur. The
3
m (E ) free nitrates appear at 1384 cm in the
spectra of the complex.
about 9 and 2 nm, respectively. Generally, large hypochromism
of an aromatic dye in presence of double helical CT-DNA is charac-
teristic of intercalation into CT-DNA base-pairs for the dye, due to
the strong stacking interaction between the aromatic chromophore
and the base-pairs [14,15]. So, the above phenomena imply that
the Ni(II) complex and ligand interact with DNA by intercalating
mode. Compared to ligand, the Ni(II) complex shows somewhat
more hypochromicity, indicating that the association strength of
the Ni(II) complex is much stronger than that of the free ligand.
2
.3. Description of the crystal structures
The structure of Ni(II) complex is shown in Fig. 2. Selected bond
lengths and angles are given in Table 1. This compound crystallizes
in the monoclinic P2 (1)/n space group with Z = 4. The structure
shows that the thiosemicarbazone ligand is coordinated to nickel
in the expected tridentate fashion, forming a six- and a five-mem-
bered chelate ring with O–Ni–N and N–Ni–S bite angles of
8
9.65(19) and 84.38(15), respectively. In Ni(II) complex, six types
of Ni-ligand interactions were found. The Ni–O (3) carbonyl dis-
tances were found to be 2.033(4) Å. The Ni-imine distances were
found to be 2.049(5) Å, for Ni–N (1). The coordinated water was
found to be 2.066(5), 2.076(5) and 2.064(5) Å, for Ni–O (4), Ni–O
2.5. Steady-state emission titration
The ligand and Ni(II) complex exhibit strong emission bands
around 450 nm when excited at 332 nm, as shown in Fig. 5. Titra-
tion of CT-DNA led to a remarkable increase of the intensity of the
emission for Ni(II) complex and ligand, as illustrated in Fig. 4. The
emission behavior strongly supports that the two compounds bind
to double-stranded CT-DNA by classical intercalation, because pe-
netrating into the hydrophobic environment inside the CT-DNA
can avoid the quenching effect of solvent water molecules. Accord-
(
5) and Ni–O (6), respectively.
2.4. Electronic absorption spectra
The electronic absorption peak wavelengths of ligand are 207
and 296 nm, while the Ni(II) complex presents three absorption
bands at 205, 263 and 323 nm, respectively. As seen in Fig. 3, titra-
tion of CT-DNA into above Tris buffer solution containing Ni(II)
complex and ligand caused some spectral changes. The absorption
bands of Ni(II) complex at 205, 263 and 323 nm exhibited hypo-
chromism of about 47, 29 and 23% and bathochromism of about
ing to the Scathchard equation [16], a plot of r/C
f
versus r gave
6
ꢁ1
the binding constants (1.10 ± 0.65) ꢀ 10 M and (1.48 ± 0.57) ꢀ
5
ꢁ1
10 M from the fluorescence data for Ni(II) complex and ligand,
respectively. These results show that the Ni(II) complex binds
more strongly than the free ligand. A similar phenomenon was
1
3, 3 and 2 nm, respectively. The ligand at 207 and 298 nm exhib-
previously noticed to be caused by the extension of the
p system
ited hypochromism of about 45% and 19% and bathochromism of
of the intercalated ligand due to the coordination of the metal, such

B.-d. Wang et al. / Journal of Organometallic Chemistry 694 (2009) 4069–4075
4071
as chromone-3-carbaldehyde benzoyl hydrazone and its lantha-
nide complexes [7,9].
0
0
0
0
0
0
.5
.4
.3
.2
.1
.0
2.6. Emission quenching titration
EB is a common fluorescent probe for DNA structures and has
been employed in examinations of the mode and process of metal
complex binding to DNA. The fluorescent emission of EB bound to
DNA in the presence of Ni(II) complex and ligand is shown in Fig. 5.
The emission intensity of the DNA–EB system (k = 595 nm) de-
creased apparently as the concentration of each compound in-
creased, which indicate that the two compounds replace EB from
the DNA–EB system. The resulting decrease in fluorescence was
caused by EB changing from a hydrophobic environment to an
aqueous environment.
2
00
250
300
350
400
450
Wavelength (nm) (a)
1
1
0
0
0
0
0
.2
.0
.8
.6
.4
.2
.0
According to the classical Stern–Volmer equation [17]:
F
0
=F ¼ K
q
½Qꢂ þ 1
ð1Þ
Where F
0
is the emission intensity in the absence of quencher, F is
the emission intensity in the presence of quencher, K is the
q
quenching constant, and [Q] is the quencher concentration. The
quenching plots of F0 = F vs [Q] are in good agreement with the lin-
q
ear Stern–Volmer equation. The K values for the Ni(II) complex and
3
ꢁ1
3
ꢁ1
ligand are 7.57 ꢀ 10 M and 1.16 ꢀ 10 M , respectively.
200
250
300
350
400
450
2.7. Viscosity measurements
Wavelength (nm) (b)
Fig. 3. (a) Electronic spectra of ligand (10
amounts of CT-DNA. [DNA] = 0–25 M. The arrow indicates the absorbance changes
upon increasing DNA concentration (the concentration of DNA is expressed per
nucleotide). (b) Electronic spectra of Ni(II) complex (10 M) in the presence of
increasing amounts of CTDNA. [DNA] = 0–25 M. The arrow indicates the absor-
bance changes upon increasing DNA concentration.
lM) in the presence of increasing
The interaction mode of Ni(II) complex and ligand with DNA is
l
further confirmed via viscosity study. Intercalation is expected to
increase the DNA viscosity; in contrast, a partial, nonclassical
intercalation of the ligand can reduce its effective length, and con-
comitantly, its viscosity by bending the DNA helix [16]. In this
l
l
6
5
4
3
2
1
000
000
000
000
000
000
0
2
1.8
1
1
1
.6
.4
.2
1
.8
.6
0.4
.2
0
0
0
0
0
3
80 400 420 440 460 480 500 520
.15
0.35
0.55
0.75
Wavelength λ / nm (a)
r (b)
1
1
1
400
200
000
3.5
3
2.5
2
8
6
4
2
00
00
00
00
0
1.5
1
0.5
0
3
80 400 420 440 460 480 500 520
0.15
0.25
r (d)
0.35
Wavelength λ / nm (c)
Fig. 4. (a) The emission enhancement spectra of ligand (10
changes upon increasing DNA concentration. Inset (b): Scatchard plot of the fluorescence titration data of ligand, K = (1.48 ± 0.57) 105 M . (c) The emission enhancement
spectra of Ni(II) complex (10 M) in the presence of 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20 and 22.5 M CT-DNA. Arrow shows the emission intensities changes upon increasing
DNA concentration. Inset (d): Scatchard plot of the fluorescence titration data of Ni(II) complex, K = (1.10 ± 0.65) ꢀ 106 M
lM) in the presence of 0, 2.5, 5, 7.5, 10, 12.5, 15 and 17.5 lM CT-DNA. Arrow shows the emission intensities
ꢁ
1
l
l
ꢁ
1
.

4
072
B.-d. Wang et al. / Journal of Organometallic Chemistry 694 (2009) 4069–4075
3
2
2
1
1
000
500
000
500
000
1.045
3
-1
1
.04
Kq = 1.16x10 M
1
.035
1.03
.025
1
1
.02
1.015
.01
5
00
0
1
5
20 540 560 580 600 620 640
0
1
2
3
-
5
Wavelength λ / nm (a)
[M] (10 M)(a')
2
2
1
1
500
000
500
000
1
1.2
1
0.8
0
.4
3
-1
Kq = 7.57x10 M
.6
5
00
0.4
0
.2
0
0
5
0
1
2
3
4
20 540 560 580 600 620 640
-
5
Wavelength λ / nm (b)
[M] (10 M) (b')
Fig. 5. (a) The emission spectra of DNA–EB system (10 M DNA and 0.32 M EB), kex = 500 nm, kem = 520.0–650.0 nm, in the presence of 0, 5, 10, 15, 20, 25 M ligand. Arrow
0
ꢁ1
shows the emission intensities changes upon increasing ligand concentration. Inset (a ): Stern–Volumer plot of the flouresence titration data of ligand, Kq = 1.16 ꢀ 103 M
.
(
b) The emission spectra of DNA–EB system (10 M and 0.32 MEB), kex = 500 nm, kem = 520.0–650.0 nm, in the presence of 0, 5, 10, 15, 20, 25 M Ni(II) complex. Arrow shows
the emission intensities changes upon increasing Ni(II) complex concentration. Inset (b ): Stern–Volumer plot of the flouresence titration data of Ni(II) complex,
0
Kq = 7.57 ꢀ 105 Mꢁ
1
.
experiment, we used the EB as a reference. As can be seen from the
Fig. 6, EB can increase the relative viscosity of DNA greatly. Upon
increasing the amounts of Ni(II) complex and ligand, the relative
viscosity of DNA increases steadily similarly to the behavior of
EB, and the extent of the viscosity increase caused by EB is more
obvious. The increased degree of viscosity, which may depend on
the binding affinity to DNA, follows the order of EB > Ni(II) com-
plex > ligand. These results suggest that the binding mode of Ni(II)
complex and ligand involves base-pair intercalation, and the bind-
ing affinity of Ni(II) complex is higher than that of ligand, which is
consistent with the above experimental results.
2.8. Cytotoxic study
Two compounds were evaluated for their cytotoxic activity
in vitro against three human cancer cell lines (THP-1, Raji and Hela
cells). The results are summarized in Figs. 7–9. The IC50 values of
two compounds were listed in Table 2.
As shown in Figs. 7–9, the Ni(II) complex and ligand exhibited
the same inhibitions on the growth of selected tumor cell lines,
especially on Raji and Hela cells in the low concentration. But the
Ni(II) complex exhibited higher cytotoxic effects than that of the li-
gand in the high concentration. The IC50 values of Ni(II) complex
against three cell lines were lower than that of ligand (Table 2).
4 3 2
In the range of the tested concentration, the NiSO and Ni(NO )
exhibited little cytotoxic activity. It is clear that the coordination
of metal ion with ligand can enhance the anticancer activity of li-
1.034
1.032
1.030
1.028
1.026
1.024
1.022
1.020
1.018
1.016
1.014
1.012
1.010
1.008
1.006
Ni (III) complex
Ligand
EB
0.1
0.2
0.3
M/[DNA]
0.4
0.5
Fig. 6. Effect of increasing amounts of complexes, ligand and EB on the relative
viscosity of calf thymus DNA at 25.0 °C.
Fig. 7. Cytotoxic activity of compounds against Hela cancer cell lines.

B.-d. Wang et al. / Journal of Organometallic Chemistry 694 (2009) 4069–4075
4073
Fig. 8. Cytotoxic activity of compounds against Raji cancer cell lines.
Fig. 9. Cytotoxic activity of compounds against Thp-1 cancer cell lines.
Table 2
In vitro cytotoxicity.
gand to the cancer cell lines. We also observed that the Ni(II) com-
plex exhibited different cytotoxic activity in vitro against three hu-
IC50 (uM)
man cancer cell lines (Hela, IC50 = 181
, IC50 = 89 M).
lM; Raji, IC50 = 97 lM; Thp-
Type of cells
Hela
1
l
Raji
240.2
97
Thp-1
>300
89
Ligand
Ni(II) complex
>300
181
2.9. Changes in cell morphological features
To validate the induction of apoptosis by identifying morpho-
logical features, phase-contrast microscopy was used to show that
the dose-dependent loss in cell viability in cultures of Hela treated
with ligand and Ni(II) complex was accompanied by morphological
changes. Two days after treatment with Ni(II) complex, visible cell
numbers decreased as Ni(II) complex concentration increased from
20 to 160 M. Moreover, the distinctive morphological features of
cells include detachment and cell shrinkage. Hela cell death in-
l
duced by Ni(II) was more obvious than ligand (Fig. 10).
Fig. 10. Morphological characteristics of Hela cells treated with ligand or Ni(II) complex. Hela cells were seeded into tissue culture flask and treated with increasing
concentrations of compounds. (a) Control, (b)–(e) 20, 40, 80 and 160 mM of ligand, (f)–(i) 20, 40, 80 and 160 mM of Ni(II) complex. Cells were photographed after 48 h drug
treatment.

4
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B.-d. Wang et al. / Journal of Organometallic Chemistry 694 (2009) 4069–4075
3
. Conclusion
(1.90 g, 10 mmol). The mixture was stirred for two hours at room
temperature and a yellow precipitate formed. The precipitate
was collected by filtration and washed with ethanol. Recrystallista-
A novel chromone thiosemicarbazone and its Ni(II) complex
were synthesized and characterized. The DNA binding mode of
complex and ligand with CT-DNA were also studied via spectra
and viscosity measurement. The experiment results suggest that
the ligand and its complex bind to DNA via intercalation mode,
and the complex has higher binding ability than free ligand. The
inhibition experiment results of the complex and ligand to Hela,
Raji and Thp-1 cancer cell lines suggested that the complex has
high anticancer activity, and the complex has obvious selectivity
against different cancer cell lines.
tion from 1:1 (V/V) DMF/H
2
O gave the ligand (L), which was dried
in a vacuum. Yield: 90% m.p. 221–222 °C. FAB-MS: m/z = 264
+
[M+H] . Anal. Calc. for C11
9 3 3
H N O S: C, 50.45; H, 3.45; N, 15.96.
1
Found: C, 50.98; H, 3.68; N, 16.08%.
H
NMR (DMSO-d
6
300 MHz): d 8.43 (1H, s, CH @ N), 8.10 (1H, s, 2-H), 7.62 (1H, s,
5-H) 7.13 (1H, d, J = 6 Hz, 7-H), 7.09 (1H, d, J = 6 Hz, 8-H). IR for li-
ꢁ
1
ꢁ1
ꢁ1
gand (cm ):
mC@O: 1623,
m
C@N: 1591 cm
,
m
C@S: 1320 cm . Umax
(nm): 207, 296.
4.5. Preparation of complex
4
. Experimental section
The ligand (1.0 mmol, 0.263 g) and the Ni(II) nitrate (1.0 mmol,
.290 g) were added to the methanol (10 mL). The mixtures were
0
4.1. Chemicals
stirred at 60 °C. After 5 min, the mixtures solution was filtrated
to move residue and continued stirring for 24 h at room tempera-
ture. After that, the mixtures solution was concentrated and
cooled, a light blue solid separated out. The light blue precipitate
was separated from the solution by suction filtration, purified by
washing several times with ethanol, and dried for 24 h in a vac-
Acetic anhydride, hydroquinone and thiosemicarbazide were
produced in China.
4.2. DNA sample
uum. Anal. Calc. for Ni(II) complex C11
H
9
N
5
O
9
SNi: C, 29.35; H,
Calf thymus DNA (CT-DNA) was obtained from Sigma Chemicals
2
1
1
.36; N, 15.37; Ni, 13.12. Found: C, 29.61; H, 2.02; N, 15.70; Ni,
Co. (USA) and used as received. A stock solution of CT-DNA was
prepared and stored in 5 mM Tris–HCl buffer at pH 7.1. The con-
centration of CT-DNA solutions was determined spectrophotomet-
ꢁ1
ꢁ1
3.16%. IR (cm ):
m
C@O: 1631,
mC@N: 1571,
m
C@S: 1168 cm
NO3
, m :
480, 1380, 1325, 1068, 838. Umax (nm): 205, 264, 325.
rically using the reported molar absorptivity of
e
259 nm=1.31 ꢀ
[18] and the results were expressed in terms of
base-pair equivalents per cubic decimeter. A solution of CT-DNA
4
.6. Biological and pharmacological test
4
ꢁ1
ꢁ1
1
0 M cm
The interactions of Ni(II) complex and ligand with CT-DNA use
ꢁ5
(
ca. 10 M in base-pair, bp) in Tris–HCl buffer gave a ratio of UV
UV-visible, fluorescence and viscosity measurements. Absorption
titration experiment was performed with fixed concentrations of
the drugs (10 lm) while gradually increasing concentration of
absorbance at 260 and 280 nm, A260/A280 P 1.9 [19], indicating
that the CT-DNA was sufficiently free from protein.
CT-DNA. Viscosity experiments were conducted on an Ubbdlodhe
viscometer, immersed in a thermostated water-bath maintained
to 25 °C.
4
.3. Instrumentation
Carbon, hydrogen, and nitrogen were analyzed on an Elemental
Three different cell lines, uterine cervix carcinoma cell (Hela),
Vario EL analyzer. The metal contents of the complex were deter-
mined by titration with EDTA. Infrared spectra (4000–400 cm
were determined with KBr disks on a Therrno Mattson FTIR spec-
trometer. The UV-visible spectra were recorded on a Varian Cary
1
leukemic cells (THP-1 and Raji), were plated in 96-well plates.
ꢁ1
)
4
The adherent cells, hela, was plated at a density of 5 ꢀ 10 cells/
5
mL, nonadherent cells, THP-1 and Raji, 1 ꢀ 10 cells/mL, then trea-
ted with varied concentration (20, 40, 80, 160 and 320 lM) of the
00 Conc spectrophotometer. 1H NMR spectra were measured on
compounds. The culture medium was removed from the plates
a Varian VR 300-MHz spectrometer, using TMS as a reference in
DMSO-d . Mass spectra were performed on a VG ZAB-HS (FAB)
instrument and electrospray mass spectra (ESI-MS) were recorded
on a LQC system (Finngan MAT, USA) using CH OH as mobile
phase. The fluorescence spectra were recorded on a Hitachi RF-
500 spectrofluorophotometer.
after 48 h of culture, and each well was washed once with
6
Table 3
3
Crystal data and structure refinement for Ni(II) complex.
4
Ni(II) complex
Empirical formula
Formula weight
Crystal system
space group
a (A°)
11 19 5
C H N Ni O14 S
536.08
Monoclinic
P2(1)/n
10.3949(2)
17.13070(10)
12.24160(10)
90
106.7040(10)
90
2087.90(5)
4
1.705
4
.4. Preparation of ligand (L)
Organic 2 and 3 were prepared according to the literature meth-
ods [20].
The 6-hydroxy chromone-3-carbaldehyde (4): Over an ice bath,
the POCl (10 mL) was slowly added to a solution of 3 (1.52 g,
mmol) in 20 mL dry DMF. The resulting mixture was stirred at
°C for 1 h and then at room temperature for overnight. The reac-
b (A°)
c (A°)
a
(°)
b (°)
(°)
3
1
0
c
3
V (A°)
tion mixture was poured into ice water. The solid was collected by
vacuum filtration, washed with water, dried in vacuum, and Yield
Z
D
calc (g/cm3)
F(000)
h min and max (°)
Reflections collected/unique
Refinement method
Final R indices [I > 2sigma(I)]
1104
2.10–25.00
5
0%. Recrystallistation from 1:1 (V/V) DMF/H
2
O gave the organic 4.
Yield: 50 m.p. 133–135 °C. H NMR (DMSO-d 300 MHz): d 9.65
1H, s, CH @ O), 8.15 (1H, s, 2-H), 7.11 (1H, d, J = 6 Hz, 5-H) 6.95
1
6
6839/3628 (R(int) = 0.0264)
(
(
Full-matrix least-squares on F2
+
1H, d, J = 6 Hz, 7-H), 6.75 (1H, d, 8-H). FAB-MS: m/z = 191[M+H] .
-hydroxy chromone-3-carbaldehyde thiosemicarbazone: An
R1 = 0.0804
wR2 = 0.2034
R1 = 0.0880
wR2 = 0.2095
6
R indices (all data)
ethanol solution containing thiosemicarbazide (0.91 g, 10 mmol)
was added dropwise to another ethanol solution containing 4

B.-d. Wang et al. / Journal of Organometallic Chemistry 694 (2009) 4069–4075
4075
2
1
00
00
l
l
L phosphate-buffered saline (PBS, pH 7.2). To each well,
L of buffer containing 0.1 M sodium acetate (pH 5.0), 0.1% Tri-
Appendix A. Supplementary material
ton X-100, and 5 mM p-nitrophenyl phosphate (p-NPP) was added.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jorganchem.2009.08.024.
The reaction was stopped with the addition of 10 L of 1 M NaOH,
l
and color development was assayed at 405 nm using a microplate
reader (BIO-RAD 680). The nonenzymatic hydrolysis of the p-NPP
substrate was determined for each assay by including wells that
did not contain cells as blank wells. Cell survival was expressed
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