Synthesis, crystal structures, biological activities and fluorescence studies of transition metal complexes with 3-carbaldehyde chromone thiosemicarbazone

Article abstract of DOI:10.1016/j.ejmech.2010.09.025

3-Carbaldehyde chromone thiosemicarbazone (L) and its transition metal complexes were synthesized and characterized systematically. Crystal structures of Zn(II) and Ni(II) complexes were determined by single crystal X-ray diffraction analysis. Zn(II) complex exhibits blue fluorescence under UV light and its fluorescent property in solid state was investigated. Interactions of ligand and Cu(II), Zn(II) and Ni(II) complexes with DNA were investigated by spectral and viscosity studies, indicating the compounds bind to DNA via intercalation and Zn(II) complex binds to DNA most strongly. Antioxidant tests in vitro show the compounds possess significant antioxidant activity against superoxide and hydroxyl radicals, and the scavenging effects of Cu(II) complex are stronger than Zn(II), Ni(II) complexes and some standard antioxidants, such as mannitol and vitamin C.

Full text of DOI:10.1016/j.ejmech.2010.09.025

European Journal of Medicinal Chemistry 45 (2010) 5692e5701
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, crystal structures, biological activities and fluorescence studies of
transition metal complexes with 3-carbaldehyde chromone thiosemicarbazone
*
Yong Li, Zheng-Yin Yang , Jin-Cai Wu
College of Chemistry and Chemical Engineering, State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2010
Received in revised form
6 September 2010
Accepted 8 September 2010
Available online 17 September 2010
3-Carbaldehyde chromone thiosemicarbazone (L) and its transition metal complexes were synthesized
and characterized systematically. Crystal structures of Zn(II) and Ni(II) complexes were determined by
single crystal X-ray diffraction analysis. Zn(II) complex exhibits blue fluorescence under UV light and its
fluorescent property in solid state was investigated. Interactions of ligand and Cu(II), Zn(II) and Ni(II)
complexes with DNA were investigated by spectral and viscosity studies, indicating the compounds bind
to DNA via intercalation and Zn(II) complex binds to DNA most strongly. Antioxidant tests in vitro show
the compounds possess significant antioxidant activity against superoxide and hydroxyl radicals, and the
scavenging effects of Cu(II) complex are stronger than Zn(II), Ni(II) complexes and some standard anti-
oxidants, such as mannitol and vitamin C.
Keywords:
3-Carbaldehyde chromone
thiosemicarbazone
Crystal structures
Ó 2010 Elsevier Masson SAS. All rights reserved.
Fluorescence studies
DNA binding properties
Antioxidant activity
1. Introduction
electronic effects [5]. Many experiments indicated that the inves-
tigated anticancer drugs possess certain anticancer activity since
Cancer has overtaken heart disease as the world’s top killer by
2010, part of a trend that should be more than double global can-
cer cases and deaths by 2030 [1]. Cisplatin (cis-diamminedi-
chloroplatinum(II)) is one of the most effective anticancer drugs in
the treatment of a variety of tumors and it has been clinically used
widely [2]. However, its limited usefulness in the development of
resistance in tumor cells and the significant side effects have
generated new areas of research, which mainly focused on
searching for new metal-based complexes with low toxicity and
improved therapeutic properties [3]. Despite the recent excitement
about personalized anticancer therapies, such drugs currently
represent a small fraction of anticancer agents. A great number of
chemotherapeutic anticancer drugs are those compounds inter-
acting with DNA directly or preventing the proper relaxation of
DNA. Moreover, DNA-targeting cancer drugs continue to be devel-
oped, as evidenced by the approval of belotecan [4]. Given the
applications of DNA-targeting drugs for cancer and beyond, the
investigation of drugeDNA interactions is of considerable interest
and drugeDNA interactions can be classified into three major
categories, i.e., intercalation, groove binding and external static
their DNA binding mode is the same as cisplatin, whereas cisplatin
interacts with DNA via the intercalation binding mode, thus
searching for drugs binding to DNA via intercalation rouse many
scientists’ interest during the past several decades [6].
Chromones (1-benzopyran-4-one) are a group of naturally and
widely distributed compounds which are ubiquitous in nature,
especially in the plant kingdom [7]. They are oxygen-containing
heterocyclic compounds with a benzo-annelated
g-pyrone ring.
Molecules containing the chromone structure (such as flavonoids
and chromones) receive considerable attention in the literatures
recently, mainly due to their biological and physiological activities
including antimycobacterial, antifungal, anticonvulsant, antimi-
crobial, mushroom tyrosinase inhibition activities, intermediates to
many products of fine chemical industries [8e13] and so on.
Furthermore, chromone hydrazones and their metal complexes
often possess diverse biological and pharmaceutical activities, such
as antimicrobial, antituberculostatic, anticancer, antioxidant prop-
erties and so on [14e16]. On the other hand, over production of
ꢀꢁ
activated oxygen species in the forms of superoxide anion (O2
)
and hydroxyl radical (HOꢀ), generated by normal metabolic process,
is considered to be the main contributor to oxidative damages to
biomolecules such as DNA, lipids and proteins, thus accelerating
cancer, aging, inflammation, cardiovascular and neurodegenerative
diseases [17e22]. The potential value of antioxidants has already
* Corresponding author. Tel.: þ86 931 8913515; fax: þ86 931 8912582.
E-mail address: yangzy@lzu.edu.cn (Z.-Y. Yang).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.025

Y. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 5692e5701
5693
prompted investigators to search for the cooperative effects of
2.1.3. Mass spectra
metal complexes and natural compounds for improving antioxi-
dant activity and cytotoxicity [23].
The electrospray ionization (ESI) mass spectra of Cu(II) complex
were made. The mass spectra of Cu(II) complex show a peak at 527
which can be assigned to the fragment [Cu(II) complex þ K]þ.
In our previous work, the metal complexes derived from chro-
mones Schiff bases have been reported and all the compounds can
strongly interact with DNA via an intercalation binding mode
[24e27]. It is notable that different metal ions and substitutes can
affect the binding mode and affinity. In this regard, as a further
research, a novel Schiff base ligand, 3-carbaldehyde chromone
thiosemicarbazone (L) and its Copper (II), Zinc (II) and Nickel (II)
complexes were synthesized and their DNA binding modes were
investigated systematically. In addition, the antioxidant activities of
the ligand and its mꢁetal complexes were determined by the
2.1.4. Crystal structure of Zn(II) complex
The Zn(II) complex crystallized in the monoclinic lattice with
a space group P21/c. Each unit cell contains four molecules. The X-
ray diffraction data for the Zn(II) complex were given in Table 1 and
the selected bond lengths and angles were summarized in Table 2.
The crystal structure in Fig. 1a showed the composition of the Zn(II)
complex was [Zn2L2 NO3 3H2O]$3NO3$C2H5OH (L ¼ 3-carbaldehyde
chromone thiosemicarbazone). In the Zn(II) complex, there exist
two coordination styles. For one style, Zn atom was five coordinated
with one ligand and two water molecules, whereas for the other
style, Zn atom was coordinated with one ligand, one nitrate and one
water molecule. In the Zn(II) complex, each ligand acted as a tri-
dentate ligand, binding to Zn atom through the nitrogen atom from
eCH]Ne group of the Schiff base, oxygen atom from 3-carbalde-
hyde chromone unit and eC]S of the thiosemicarbazone side
chain. In Zn(II) complex, ten types of Zn-ligand interactions were
found. In one unit, the Zn(1)eO(1) carbonyl distance was found to
be 2.090(3) Å. The Zn-imine distance was found to be 2.138(4) Å, for
Zn(1)eN(1), and the distance was the same as Zn(2)eN(3). The
coordinated water and nitrate were found to be 2.021(4) and 1.993
(14) Å for Zn(1)eO(5) and Zn(1)eO(6), respectively. In the other
unit, the Zn(2)eO(3) carbonyl and Zn(2)eS(2) distances were found
to be 2.050(3) and 2.3358(14) Å respectively. The two coordinated
ꢀ
superoxide anion (O2 ) and hydroxyl radical (HOꢀ) scavenging
methods in vitro. This continuing research on designing new
ligands and complexes may help us investigate DNA binding
mechanism of chromone derivatives and more efficient antioxi-
dants, and we hope that our research will be widely applied in
biology and medicine areas in the future.
2. Results and discussion
2.1. Characterization of complexes
The complexes were prepared by direct reaction of the ligand (L)
with appropriate mole ratios of M(II) (M ¼ Cu, Zn, and Ni) nitrate in
ethanol. The yields were good to moderate. The transition metal
complexes were stable in atmospheric conditions for extended
periods, and are easily soluble in dimethylsulfoxide (DMSO) and
N,N-dimethylformamide (DMF); slightly soluble in ethanol, meth-
anol, acetone and chloroform; insoluble in water, benzene and
ethylacetate. The molar conductivities of Cu(II) and Ni(II)
complexes are 142 and 156 S cm2 molꢁ1 in DMF solution and in
accord with them being formulated as 1:2 electrolytes [28].
Table 1
Crystal data and experimental data of Zn(II) and Ni(II) complexes.
Complex
[Zn2L2NO3
[NiL(H2O)3]$2NO3
3H2O]$3NO3$C2H5OH
Chemical formula
Formula weight
Crystal color
Crystal size (mm)
Temperature (K)
Wavelength (Å)
Radiation
Crystal system
Space group
Z
Zn2C24H30N10O20S2
973.44
Yellow
NiC11H15N5O11
484.05
Green
S
2.1.1. IR spectra
The IR spectra of the complexes are similar and usually provide
a lot of valuable information on coordination reaction. The n(C]O)
0.43 ꢂ 0.42 ꢂ 0.31
0.35 ꢂ 0.33 ꢂ 0.31
vibration of the free ligand is observed at 1642 cmꢁ1; for the metal
complexes, this peak low frequency shifts to 1615 cmꢁ1 or so;
Dn(ligand-complexes) is equal to 27 cmꢁ1. In the complexes, there exist
296(2)
296(2)
0.71073
0.71073
Mo Ka
Triclinic
Mo K
a
Monoclinic
P2(1)/c
4
19.9695(10)
10.1747(5)
19.3720(9)
90.00
107.713(2)
90.00
bands at 620 cmꢁ1 assigned to
n(MeO), indicating that the oxygen
P ꢁ 1
of carbonyl has formed a coordinative bond with the transition
2
metal ions [29]. The band at 1600 cmꢁ1 for the free ligand is
a (Å)
b (Å)
c (Å)
9.0958(16)
10.371(2)
11.1553(19)
99.394(10)
93.464(8)
115.227(7)
929.1(3)
1.730
1.226
496
2.50e27.04
ꢁ8 ꢄ h ꢄ 11
ꢁ12 ꢄ k ꢄ 12
ꢁ13 ꢄ l ꢄ 13
5180
assignable to
the complexes. Weak bands at about 453 cmꢁ1 are assigned to
n
(C]N) stretch, which shifts to 1564 cmꢁ1 or so for
n
a
b
g
(ꢃ)
(ꢃ)
(ꢃ)
(MeN) in the complexes. The new bands further confirm that the
nitrogen of the imino-group bonds to the metal ions [30]. The
Volume (Å3)
3749.5(3)
1.724
1.486
aqueous
n(H2O) bands of complexes appear at about
D (Calc) (g/cm3)
m
3221e3430 cmꢁ1. This shows that there are some crystal water
(mmꢁ1
)
molecules in the complexes [31]. The n(C]S) stretch of the free
F (000)
qmin/max (ꢃ)
Index ranges
1984
ligand appears at 1342 cmꢁ1, which shifts towards lower energy for
the complexes and indicates the coordination of thꢁio1na-to sulphur
[32]. The n3 (E0) free nitrates appear at 1384 cm or so in the
spectra of the complexes, showing that there exist nitrate mole-
cules of the complexes.
2.57e23.75
ꢁ24 ꢄ h ꢄ 19
ꢁ10 ꢄ k ꢄ 12
ꢁ23 ꢄ l ꢄ 23
20482
Reflections collected
Independent reflections
Max. and min.
7359 (Rint ¼ 0.0411)
3565 (Rint ¼ 0.0165)
0.6559 and 0.5676
0.7025 and 0.6737
transmission
Refinement method
2.1.2. UVevis spectra
Full matrix
Full matrix
least-squares on F2
3565/6/281
1.064
The study of the electronic spectra in the ultraviolet and visible
(UVevis) ranges for the ligand and metal complexes was carried
out in TriseHCl buffer solution. The electronic spectra of the free
ligand have a strong band at Umax ¼ 282 nm and a medium band at
Umax ¼ 310 nm. The complexes also yield two bands and the two
bands which are shifted to 277 and 303 nm or so. These indicate
that the metal complexes are formed.
least-squares on F2
7359/11/545
1.059
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2
s
(I)]
R1 ¼ 0.0521,
R1 ¼ 0.0285,
u
R2 ¼ 0.1253
u
R2 ¼ 0.0692
R indices (all data)
R1 ¼ 0.0926,
R2 ¼ 0.1446
R1 ¼ 0.0355,
R2 ¼ 0.0733
u
u

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Y. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 5692e5701
Table 2
Selected bond lengths (Å) and angles (ꢃ) for Zn(II) complex.
Selected bond
Bond lengths or angles
Selected bond
Bond lengths or angles
Selected bond
Bond lengths or angles
Zn(1)eO(1)
Zn(1)eO(5)
Zn(2)eO(3)
Zn(2)eO(9)
2.090(3)
2.021(4)
2.050(3)
1.995(4)
30.7(5)
Zn(1)eS(1)
Zn(1)eO(6)
Zn(2)eS(2)
Zn(2)eO(10)
2.3529(15)
1.993(14)
2.3358(14)
2.036(3)
112.2(6)
95.9(5)
127.6(4)
117.7(4)
157.92(11)
92.16(15)
141.80(15)
98.03(11)
16.0
Zn(1)eN(1)
2.138(4)
1.972(12)
2.137(4)
1.3740
Zn(1)eO(60)
Zn(2)eN(3)
Zn(2)eO(10D)
O(60)eZn(1)eO(6)
O(60)eZn(1)eO(1)
O(60)eZn(1)eN(1)
O(1)eZn(1)eN(1)
O(5)eZn(1)eS(1)
O(9)eZn(2)eO(10)
O(9)eZn(2)eN(3)
O(9)eZn(2)eS(2)
N(3)eZn(2)eS(2)
O(3)eZn(2)eH(10D)
O(60)eZn(1)eO(5)
O(6)eZn(1)eO(1)
O(6)eZn(1)eN(1)
O(60)eZn(1)eS(1)
O(1)eZn(1)eS(1)
O(9)eZn(2)eO(3)
O(10)eZn(2)eN(3)
O(10)eZn(2)eS(2)
O(9)eZn(2)eH(10D)
N(3)eZn(2)eH(10D)
O(6)eZn(1)eO(5)
O(5)eZn(1)eO(1)
O(5)eZn(1)eN(1)
O(6)eZn(1)eS(1)
N(1)eZn(1)eS(1)
O(10)eZn(2)eO(3)
O(3)eZn(2)eN(3)
O(3)eZn(2)eS(2)
O(10)eZn(2)eH(10D)
S(2)eZn(2)eH(10D)
86.8(4)
81.8(4)
98.9(6)
83.92(14)
144.45(17)
106.2(5)
82.75(11)
82.86(14)
84.10(14)
160.92(11)
85.5
83.92(14)
96.92(12)
101.39(15)
114.86(16)
106.23(12)
83.60(11)
89.0
130.0
110.1
water molecules were found to be 1.995(4) and 2.036(3) Å for Zn
(2)eO(9) and Zn(2)eO(10), respectively.
coordinated number is six. That was may be due to the different
chelation styles between the ligand and the transition metals.
On the basis of single crystal structures, elemental analyses,
molar conductivities, mass spectra, UVevis spectra and IR spectra,
the structures of the Cu(II), Zn(II) and Ni(II) complexes were [CuL
(H2O)3]$2NO3, [Zn2L2$NO3$3H2O]$3NO3$C2H5OH and [NiL(H2O)3]$
2NO3 (L is 3-carbaldehyde chromone thiosemicarbazone),
respectively.
2.1.5. Crystal structure of Ni(II) complex
On the basis of the single crystal X-ray analysis, the composi-
tion of the Ni(II) complex was [NiL(H2O)3]$2NO3, where L was
3-carbaldehyde chromone thiosemicarbazone. The Ni(II) complex
crystallized in a triclinic lattic with a space group P ꢁ 1. Each unit
cell contained two molecules. The crystal structure is shown in
Fig. 1b. The X-ray diffraction data for Ni(II) complex were given in
Table 1 and the selected bond lengths and angles were summarized
in Table 3. In the Ni(II) complex, each Ni atom was six coordination
with a ligand and three water molecules. The thiosemicarbazone
ligand is coordinated to nickel atom in the expected tridentate
fashion, forming a six- and a five- membered chelate ring with
OeNieN and NeNieS bite angles of 89.80(6) and 84.17(5),
respectively. The distances of coordinated water were found to be
2.0671(17), 2.0204(16) and 2.0822(17) Å for Ni(1)eO(3), Ni(1)eO(4)
and Ni(1)eO(5), respectively. In addition, the Ni(1)eO(2) carbonyl
distance was found to be 2.3697(7) Å. The Ni(1)eN(1) and Ni(1)eS
(1) distances were found to be 2.0488(17) and 2.3697(7) Å,
respectively. Comparing the above data, the bond length Ni(1)eO
(2) was shorter than any other bond lengths, indicating that the
coordinated ability of carbonyl bond was the strongest.
Observing the crystal structures of Zn(II) and Ni(II) complexes,
we found that, in the complexes, the ligand acted as a tridentate
ligand, binding to the metal atom through the nitrogen atom from
eCH]Ne group of the Schiff base, oxygen atom from 3-carbalde-
hyde chromone unit and eC]S of the thiosemicarbazone side
chain. In other words, the binding sites of the ligand in the
two complexes were the same. However, there still exist some
differences. In the Zn(II) complexes, there were two coordination
styles, and the coordinated solvent molecules were different. In the
Ni(II) complex, there was only one coordination style and the
2.2. Solid fluorescence
The emission spectra of the ligand and Zn(II) complex in solid
state (the excitation and emission slit widths were 3 nm) were
recorded at room temperature. The Zn(II) complex exhibits blue
fluorescence and the emission is readily observed with naked eyes
under UV light, and the ligand exhibits quite weak fluorescence
with naked eyes under UV light. Moreover, the fluorescence
intensities of the Cu(II) and Ni(II) complexes are much too weak in
the solid state, which are not discussed in our present work.
Fluorescence spectra of the ligand and Zn(II) complex in solid state
are shown in Fig. 2. The Zn(II) complex and ligand in the solid state
at ambient temperature have different emission bands at 467 and
455 nm at the same excitation band (305 nm), respectively. Because
Zn(NO3)2 possesses no fluorescence, the observed blue fluores-
cence is attributed to the coordination of the ligand and zinc ion.
Thus, Zn(II) complex may be used as an advanced material for blue
light emitting diode devices.
2.3. DNA binding properties
2.3.1. Electronic absorption spectroscopy
The binding ability of the ligand and its metal complexes with
CT DNA is characterized by measuring their effects on UVevis
spectroscopy. Compounds binding to DNA through intercalation
Fig. 1. ORTEP view of Zn(II) complex (a) and Ni(II) complex (b) showing the atom numbering of scheme and 30% thermal ellipsoids probability for the non-hydrogen atoms.

Y. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 5692e5701
5695
Table 3
Selected bond lengths (Å) and angles (ꢃ) for Ni(II) complex.
Selected bond
Bond lengths or angles
Selected bond
Bond lengths or angles
Selected bond
Bond lengths or angles
Ni(1)eO(2)
Ni(1)eO(3)
2.0251(15)
2.0671(17)
87.58(6)
90.06(7)
88.25(7)
Ni(1)eS(1)
Ni(1)eO(4)
2.3697(7)
2.0204(16)
175.81(7)
86.13(7)
86.24(7)
98.61(5)
90.96(5)
Ni(1)eN(1)
Ni(1)eO(5)
2.0488(17)
2.0822(17)
89.80(6)
93.04(7)
88.30(7)
O(4)eNi(1)eO(2)
O(4)eNi(1)eO(3)
O(4)eNi(1)eO(5)
O(3)eNi(1)eO(5)
N(1)eNi(1)eS(1)
O(4)eNi(1)eN(1)
O(2)eNi(1)eO(3)
O(2)eNi(1)eO(5)
O(4)eNi(1)eS(1)
O(3)eNi(1)eS(1)
O(2)eNi(1)eN(1)
N(1)eNi(1)eO(3)
N(1)eNi(1)eO(5)
O(2)eNi(1)eS(1)
O(5)eNi(1)eS(1)
172.24(7)
84.17(5)
173.17(5)
96.78(5)
usually results in hypochromism and bathochromism due to
intercalative mode involving a strong stacking interaction between
an aromatic chromophore and DNA base pairs [33]. In the present
investigation, the interaction of prepared compounds in TriseHCl
buffer solution with CT DNA has been studied. The binding of
complexes to DNA led to decrease in the absorption intensities with
a small amount of red shifts in the UVevis absorption spectra. After
intercalating into the DNA base pairs, the p* orbital of the inter-
free ligand and its metal complexes can interact with CT DNA via
the same mode (intercalation).
2.3.2. Fluorescence spectra
The fluorescence titration spectra of the ligand and its Zn(II)
complex in the absence and presence of CT DNA were given in
Fig. 4. The ligand and Zn(II) complex can emit fluorescence in
TriseHCl buffer solution with a maximum wavelength appearing at
430 nm or so. According to the Scatchard equation, a plot of r/Cf
versus r gave Kb values of (5.02 ꢅ 0.45) ꢂ 105, (6.68 ꢅ 0.92) ꢂ 105,
(9.68 ꢅ 0.94) ꢂ 105, (8.10 ꢅ 0.11) ꢂ 105 Mꢁ1 for the ligand, Cu(II), Zn
(II) and Ni(II) complexes, respectively. Results obtained from fluo-
rescence titration spectra suggest that all the compounds are pro-
tected from solvent water molecules by the hydrophobic
environment inside the DNA helix and that the metal complexes
can be protected more efficiently than the free ligand alone. This
implies that the metal complexes and free ligand can insert
between DNA base pairs deeply and the metal complexes can bind
to DNA more strongly than the free ligand alone. Since the hydro-
phobic environment inside the DNA helix reduces the accessibility
of solvent water molecules to the compound and the compound
mobility is restricted at the certain binding sites, a decrease of the
vibrational modes of relaxation results. The binding of the
compounds to DNA leading to a significant increase in emission
intensity also agrees with those observed for other intercalators
[35]. The higher binding affinities of the complexes are probably
calated ligand can couple with the
thus, decreasing the
p* transition energy and resulting in the
bathochromism. On the other hand, the coupling orbital is
p orbital of the DNA base pairs,
p
/
p
partially filled by electrons, thus, decreasing the transition proba-
bilities and concomitantly resulting in the hypochromism [34].
Therefore, in order to obtain evidence for the possibility of binding
mode of each compound to CT DNA, spectroscopic titration of
compounds solutions with CT DNA should be performed. The
electronic spectra of the free ligand and Zn(II) complex in the
presence and absence of CT DNA are shown in Fig. 3. The appre-
ciable decrease in absorption density and slight red shift of the
p
/
p* band of the compounds is slightly similar. As shown, in the
presence of DNA, the absorption bands of the ligand, Cu(II), Zn(II)
and Ni(II) complexes at about 282 nm exhibited hypochromism of
about 31.22%, 10.36%, 58.29%, and 12.17%, and at about 310 nm
exhibited hypochromism of about 10.78%, 44.2%, 42.39%, and
13.02%, respectively. The hypochromism observed for the ligand
and metal complexes are all accompanied by a small red shift by
about 3 nm. All these results suggest that the ligand and metal
complexes can interact with CT DNA quite probably by intercalating
the compounds into DNA base pairs, so we can conclude that the
attributed to the extension of the
p system of the intercalated
ligand due to the coordination of transition metal ions, which also
leads to a planar area greater than that of the free ligand and the
coordinated ligand penetrating more deeply into and stacking
more strongly with the DNA base pairs.
2.3.3. Competitive studies with EB
250
Competitive EB binding studies have been undertaken with
UVevis and fluorescence experiments in order to investigate if the
studied compounds could displace EB from its EBeDNA system. The
molecular fluorophore EB, a phenenthridine fluorescence dye, is
a typical indicator of intercalation [36], which forms soluble
complexes with nucleic acids and emits intense fluorescence in the
presence of CT DNA due to the intercalation of the planar phe-
nenthridinium ring between adjacent base pairs on the double
helix [37]. The spectroscopic changes of EB on its binding to DNA
are often utilized to study the interactions between DNA and other
substances such as small molecules and metal complexes [38].
Fig. 5 shows the electronic absorption spectra of the ligand and
Zn(II) complex when EB was employed. The absorbance of
TriseHCl buffer solution is highest when there is EB alone. After
adding CT DNA into the buffer solution, the absorbance decreases.
However, it increases when the ligand and metal complexes were
added dropwise into the buffer solution. In addition, the maximal
absorption of EB at 479 nm decreases and shifts to 494 nm in the
presence of CT DNA, which is characteristic of intercalation mode.
Curve (B) in Fig. 5 shows the absorption of a mixture solution of EB,
Zn(II) Complex
200
150
100
50
L
0
350
400
450
500
550
600
Wavelength (nm)
Fig. 2. Solid state emission spectra of the ligand (lex ¼ 304 nm, lem ¼ 455 nm) and Zn
(II) complex (lex ¼ 305 nm, lem ¼ 467 nm).

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Y. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 5692e5701
Zn(II) complex
1.2
L
0.35
0.28
0.21
0.14
0.07
0.00
1.0
0.8
0.6
0.4
0.2
0.0
400
350
250
300
350
400
250
300
Wavelength (nm)
Wavelength (nm)
Fig. 3. Electronic absorption spectra of the ligand and Zn(II) complex (10
mM) in the absence (top spectrum) and presence of increasing amounts of CT DNA (2.5, 5.0, 7.5, 10.0, 12.5,
15.0, 17.5, 20.0, 22.5, 25, 27.5, 30.0, 32.5, 35, 37.5 and 40.0 M; subsequent spectra). Arrows show the absorbance changes upon increasing DNA concentration.
m
DNA and the investigated compounds. It is also found that the
absorption at 494 nm has increased comparing with curve (C).
These may result from two reasons: (1) EB bound to the free
ligand and metal complexes strongly, resulting in the decrease of
the amount of EB intercalated into DNA; (2) there exists competi-
tive intercalation between the free ligand and metal complexes
and EB with DNA, so releasing some free EB from DNAeEB
system. However, the former reason could be precluded because
there were no new absorption peaks appearing. Thus, we can come
to a conclusion that the investigated compounds can displace EB
from its DNAeEB system. Quite similar results are also observed for
other metal complexes.
Complexes intercalating into the DNA base pairs leads to
a decrease in the binding sites of DNA available for EB and results in
the decrease in the fluorescence intensity of the DNAeEB system. The
reduction of the fluorescence emission intensity gives criteria to
investigate the DNA binding propensity of the compounds and the
stacking interaction (intercalation) between the adjacent DNA base
Fig. 4. The emission enhancement spectra of the free ligand (lex ¼ 435 nm, lem ¼ 301 nm) and Zn(II) (lex ¼ 422 nm, lem ¼ 298 nm) complex (10
spectrum) and presence of increasing amounts of CT DNA (2.5, 5.0, 7.5, 10.0, 12.5, 15.0 and 17.5 M; subsequent spectra). Arrows show the emission intensity changes upon
(5.02 0.45) Kb (Zn(II)
105 Mꢁ1
mM) in the absence (bottom
m
increasing DNA concentration. Inset: Scatchard plot of the fluorescence titration data of ligand and Zn(II) complex, Kb (L)
¼
ꢅ
ꢂ
;
complex) ¼ (9.68 ꢅ 0.94) ꢂ 105 Mꢁ1
.

Y. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 5692e5701
5697
Zn(II) complex
0.07
L
0.07
A
B
A
0.06
0.05
0.04
0.03
0.02
0.01
0.00
0.06
0.05
0.04
0.03
0.02
0.01
0.00
C
B
C
400
450
650
350
400
450
500
550
600
650
500
600
350
550
Wavelength (nm)
Wavelength (nm)
Fig. 5. The ultraviolet visible absorption spectra of 1 ꢂ 10ꢁ5 M EB (A); (A) þ2.5 ꢂ 10ꢁ5 M DNA (C); (C) þ2.5 ꢂ 10ꢁ5 M ligand and Zn(II) complex (B) in TriseHCl buffer (5 mM
TriseHCl, 50 mM NaCl, pH 7.1) solution.
pairs [39]. Fig. 6 shows the emission spectra of the DNAeEB system
with increasing amounts of the free ligand and Zn(II) complex. The
addition of the compounds to the DNA-bound EB solutions caused
greatreductioninemissionintensities andthequenchingofEBbound
to DNA by the compounds is in good agreement with the linear
SterneVolmer equation. In the plots of F0/F versus [Q], Kq is given
by the ratio of the slope to the intercept and Kq values for the ligand,
the compounds can bind to DNA and the Zn(II) complex bind to DNA
more strongly than the free ligand and other metal complexes, which
also validate the electronic absorption spectral results. Since these
changes indicate only one kind of quenching process, it may be
concluded that the complexes and free ligand can bind to DNA via the
same mode (intercalation).
Cu(II), Zn(II), and Ni(II) complexes are (1.38 ꢅ 0.04) ꢂ 104 Mꢁ1
,
2.3.4. Viscosity measurements
Viscosity titration measurements were carried out to clarify the
interaction modes between the investigated compounds and CT
(1.88 ꢅ 0.01) ꢂ 104
M
ꢁ1, (2.74 ꢅ 0.01) ꢂ 104 Mꢁ1 and (2.70 ꢅ
0.02) ꢂ 104 Mꢁ1, respectively. The experimental results show that all
Fig. 6. The emission spectra of DNAeEB system lex ¼ 525 nm, lem ¼ 550e650 nm, in the presence of 0,2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20.0, 22.5, 25, 27.5, 30.0, 32.5, 35.0, 37.5, 40, 42.5,
45, 47.5 and 50.0 mM ligand and Zn(II) complex. Arrows show the emission intensity changes upon increasing ligand. Inset: SterneVolmer plot of the fluorescence titration data of
ligand and Zn(II) complex, Kq(L) ¼ (1.38 ꢅ 0.04) ꢂ 104 Mꢁ1; Kq(Zn(II) complex) ¼ (2.74 ꢅ 0.01) ꢂ 104 Mꢁ1
.

5698
Y. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 5692e5701
1.32
1.28
1.24
1.20
1.16
1.12
1.08
1.04
1.00
L
Cu(II) Complex
Zn(II) Complex
Ni(II) Complex
Fig. 8. Molecular mode for the ligand and its metal complexes (via an intercalative
mode).
Cu(II) and Ni(II) complexes are octahedron, so the Zn(II) complex
binds to DNA more strongly than the free ligand and other
complexes, which is consistent with the above spectral results.
On the basis of all the spectroscopic studies together with the
viscosity measurements, we find that the free ligand and metal
complexes can bind to CT DNA via an intercalative binding mode
(Fig. 8) and the Zn(II) complex bind to CT DNA more strongly and
deeply than the free ligand alone.
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
[Compound]/[DNA]
Fig. 7. Effects of increasing amounts of the ligand and its metal complexes on the
relative viscosity of CT DNA at 25.0 ꢅ 0.1 ꢃC.
DNA. It is greatly sensitive to length changes and regarded as the
least ambiguous and the most critical tests of a binding model in
solution in the absence of crystallographic structural data [39]. A
classical intercalation model demands that the DNA helix must
lengthen as base pairs are separated to accommodate the binding
complexes, leading to the increase of DNA viscosity, as for the
behaviors of the known DNA intercalators [40]. In contrast, a partial
and/or non-classical intercalation of the complex could bend (or
kink) the DNA helix, reducing its viscosity concomitantly [39]. In
addition, some compounds such as [Ru(bpy)3]2þ, which interacts
with DNA by an electrostatic binding mode, have no influence on
DNA viscosity [41]. To further elucidate the binding mode of the
present complexes, viscosity measurements were carried out by
2.4. Antioxidant activity
Since the synthesized ligand and its transition metal complexes
exhibit good DNA binding affinity, it is considered worthwhile to
investigate their other biological activities, such as antioxidant
activity. It has been reported tꢁhat reactive oxygen species (ROS),
such as superoxide anion (O2 ) and hydroxyl radical (HOꢀ), are
ꢀ
involved in the pathogenesis of various diseases through direct
effects on DNA directly and by acting as a tumor promoter [42,43].
In this paper, the antioxidant activity of the ligand and its transition
metal complexes was studied by comparing their scavenging
effects on superoxide and hydroxyl radicals.
Fig. 9 depictsꢁthe inhibitory effects of the ligand and metal
ꢀ
complexes on O2 (a) and HOꢀ (b). It caꢁn be seen that the inhibitory
keeping [DNA] ¼ 5
mM and varying the concentration of the
effects of the tested compounds on O2 and HOꢀ are concentration
ꢀ
compounds.
The effects of the ligand and metal complexes on the viscosities
of CT DNA are shown in Fig. 7. With the ratios of the investigated
compounds to CT DNA increasing, the relative viscosities of DNA
increase steadily, indicating that there exist intercalations between
the compounds with DNA helix. The increased degree of viscosity,
which may depend on the binding affinity of compounds to DNA,
follows the order of Zn(II) complex > Ni(II) complex > Cu(II)
complex > L. The crystal and other structural analyses indicate that
Zn(II) complex have two parallel planes, whereas the structures of
related and the suppression ratio increases with the increasing
sample concentration in the range of the tested concentration. The
antioxidant activity of the compounds is expressed as 50% inhibi-
tory concentration (IC50 in
the ligand, Cu(II), Zn(II) and Ni(II) complexes are 263.028, 0.799,
6.485 and 9.076 M, respectively. These compounds clearly
show higher superoxide dismutase activity compared with that of
standard antioxidants like vitamin C (IC50: 852 M). In comparison
with the prepared compounds, Cu(II) complex shows the higher
mM). As seen from Fig. 9a, IC50 values of
m
m
a
100
b
90
75
60
45
30
15
0
L
L
Cu(II) Complex
Cu(II) Complex
Zn(II) Complex
Ni(II) Complex
Zn(II) Complex
Ni(II) Complex
80
60
40
20
0
0
1
2
3
4
5
6
2
4
6
8
10
Concentration (μM)
Concentration (μM)
ꢀꢁ
Fig. 9. Scavenging effects of the ligand and its metal complexes on O2 (a) and HOꢀ (b).

Y. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 5692e5701
5699
scavenging effect than the other compounds. As seen from Fig. 9b,
IC50 values of the ligand, Cu(II), Zn(II) and Ni(II) complexes are
Carbon, hydrogen and nitrogen were determined using an
Elemental Vario EL analyzer. The metal contents of the metal
complexes were determined by titration with EDTA (xylenol orange
tetrasodium salt used as an indicator and hexamethylidynete-
traimine as buffer). The melting point of the ligand was determined
on a Beijing XT4-100X microscopic melting point apparatus (the
thermometer was not corrected). The IR spectra were obtained in
KBr discs on a Therrno Mattson FTIR spectrophotometer in the
4000e400 cmꢁ1 region. 1H NMR spectra were recorded on a Varian
VR 200 MHz spectrometer in DMSO-d6 with tetramethylsilane (TMS)
as an internal standard. Mass spectra were performed on an APEX II
FT-ICR MS instrument using CH3OH as the mobile phase. Conduc-
tivity measurements were performed in DMF solution with a DDS-
11C conductometer at 25.0 ꢃC. The UVevis spectra were recorded on
a Shimadzu UV-240 spectrophotometer. Fluorescence spectra were
recorded on a Hitachi RF-4500 spectrofluorophotometer at constant
room temperature.
31.017, 3.013, 6.198 and 9.872
mM, respectively. The metal
complexes are better antioxidants than the ligand. Usually,
mannitol and vitamin C are used as the standard antioxidant to
assess other compounds’ antioxidant activities. When arriving at
the same inhibitory values, IC50 values of the investigated ligand
and metal complexes are far less than those of mannitol and
vitamin C [44], indicating that the prepared compounds are more
efficient antioxidants. In addition, we can find that the prepared
compounds exert differential and selective effects on scavenging
radicals of the biological system due to the chelation of organic
molecules to the metal ions and the effects of metal ions. It was
believed that the information obtained form present work would
be useful to develop new potential antioxidants and therapeutic
agents for some diseases.
3. Conclusion
4.2. Synthesis of the ligand and complexes
In summary, 3-carbaldehyde chromone thiosemicarbazone (L)
and its Copper (II), Zinc (II) and Nickel (II) complexes were
synthesized and characterized based on X-ray diffraction and other
structural methods. The Zn(II) complex can emit blue fluorescence
under UV light in solid state and may be used as an advanced
material for blue light emitting dioxide devices. However, we
almost can not find the solid fluorescence of Cu(II) and Ni(II)
complexes, so it is not necessary to discuss the fluorescence
properties of Cu(II) and Ni(II) complexes. DNA binding studies
indicate that the investigated compounds bind to DNA via an
intercalation binding mode. The metal complexes can insert and
stack between the DNA base pairs more easily and deeply than the
free ligand alone, especially the Zn(II) complex can bind to DNA
more strongly than the free ligand, Cu(II) and Ni(II) complexes. This
is due to the chelating effects (metal ions to free ligand) which can
enhance the planar functionality of the metal complexes. Antioxi-
dant activities of the compounds were studied by the scavenging
superoxide anions and hydroxyl radicals in vitro. Experimental
results indicate that the Cu(II) complex possesses better antioxi-
dant activity than the ligand, Zn(II) and Ni(II) complexes. Our
present work would be helpful to understand the interaction
mechanism of small molecule compounds binding to DNA and
useful in the development of potential probes of DNA structure and
conformation, new therapeutic reagents and antioxidants.
4.2.1. Synthesis of the ligand
Synthesis of the ligand is shown in Scheme 1. 3-Carbaldehyde
chromone was prepared according to the literature methods [47].
An ethanol solution containing thiosemicarbazide (0.91 g,
10 mmol) was added dropwise to another ethanol solution con-
taining 3-carbaldehyde chromone (1.74 g, 10 mmol). The mixture
solution was refluxed on an oil bath for 24 h with stirring and then
a yellow precipitate formed. The precipitate was collected by
filtration and washed several times with ethanol. Recrystallistation
with 1:1 (V:V) DMF/H2O gave the ligand (L), which was dried in
a vacuum. Yield: 75.4%. Mp: 237e239 ꢃC. Anal. Calc. for C11H9N3O2S:
C, 53.43; H, 3.67; N, 16.99%. Found: C, 53.47; H, 3.63; N, 17.04%. 1H
NMR (DMSO-d6, ppm):
d 11.55 (1H, s,]NNH), 9.18 (1H, s, 2-H), 8.28
(1H, s, eCH]N), 8.18 (1H, dd, J ¼ 0.6, 8.0 Hz, 5-H), 8.11 (2H, bs,
eNH2), 7.85 (1H, dd, J ¼ 1.5, 8.0 Hz, 6-H), 7.72 (1H, dd, J ¼ 1.5, 8.0 Hz,
8-H), 7.54 (1H, dd, J ¼ 0.6, 8.0 Hz, 7-H). The label numbers of H-
atoms were shown in Fig. 1. IR (KBr) cmꢁ1
: n(C]O): 1642; n(C]N):
1600;
n
(C]S): 1342 cmꢁ1. Umax (nm): 282, 310 nm.
4.2.2. Synthesis of the metal complexes
The ligand (1.0 mmol, 0.247 g) and the Cu(II) nitrate (1.2 mmol,
0.342 g) were added to the ethanol solution (30 mL). The mixture
solution was refluxed and became clear, and then it was stirred and
refluxed on an oil bath for 24 h. After that, the mixture solution was
concentrated and cooled, a large amount of brown solids were
separated out from the solution by suction filtration, purified by
washing several times with ethanol and dried for 24 h in a vacuum.
The Zn(II) and Ni(II) complexes were prepared by the same method.
Anal. Calc. for Cu(II) complex CuC11H15N5O11S: C, 27.02; H, 3.09; N,
14.33; Cu, 13.00%. Found: C, 27.07; H, 3.13; N, 14.29; Cu, 13.06%.
4. Experimental section
4.1. Instrumentation and materials
Ethidium bromide (EB) and calf thymus DNA (CT DNA) were
purchased from Sigma Chemical Co. (USA). All chemicals and
solvents used were of analytical reagent grade and used without
further purification unless otherwise noted.
All the experiments involved with the interactions of the ligand
and complexes with CT DNA were carried out in doubly distilled
water buffer containing 5 mM Tris [Tris(hydroxymethyl)-amino-
methane] and 50 mM NaCl and adjusted to pH 7.1 with hydro-
chloric acid. Solution of CT DNA in TriseHCl buffer gave ratios of UV
absorbance of about 1.8e1.9:1 at 260 and 280 nm, indicating that
the CT DNA was sufficiently free of protein [45]. The CT DNA
concentration per nucleotide was determined spectrophotometri-
cally by employing an extinction coefficient of 6600 Mꢁ1 cmꢁ1 at
260 nm [46]. The ligand and complexes were dissolved in a solvent
mixture of 1% DMF and 99% TriseHCl buffer at the concentration of
1.0 ꢂ 10ꢁ5 M.
Scheme 1. The preparation route of the ligand (L).

5700
Y. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 5692e5701
Yield: 62%. IR (KBr) cmꢁ1
:
n
(H2O): 3242e3427;
n
(C]O): 1616;
n
(C]
a given DNA concentrations CDNA (nucleotides); F0 is the intensity in
the absence of DNA; and Fmax is the fluorescence of the totally
bound for DNA at a titration end point. The intrinsic binding data
were cast into the form of a Scatchard plot of r/Cf versus r.
N): 1561;
n(NO3): 1384; n(C]S): 1178; n(MeO): 620; n(MeN):
459 cmꢁ1. Umax (nm): 274, 303 nm L (S cm2 molꢁ1): 142. Anal.
m
Calc. for Zn(II) complex Zn2C24H30N10O20S2: C, 29.61; H, 3.11; N,
14.39; Zn, 13.43%. Found: C, 29.59; H, 3.16; N, 14.46; Zn, 13.36%.
Yield: 57%. IR (KBr) cmꢁ1
:
n
(H2O): 3257e3415;
n
(C]O): 1616;
n
(C]
4.4.3. Competitive studies with EB
N): 1571;
n
(NO3): 1383;
n
(C]S): 1169;
n
(MeO): 617;
n
(MeN):
Further support for the mode of the ligand and complexes
binding to DNA is given through the emission quenching experi-
421 cmꢁ1. Umax (nm): 276, 306 nm L (S cm2 molꢁ1): 121. Anal.
m
Calc. for Ni(II) complex NiC11H15N5O11S: C, 27.30; H, 3.12; N, 14.47;
Ni, 12.13%. Found: C, 27.27; H, 3.16; N, 14.49; Ni, 12.08%. Yield: 73%.
ments. 4
mM CT DNA solution was added incrementally to
0.32 M EB solution, until the rise in the fluorescence and then
m
IR (KBr) cmꢁ1
:
n
(H2O): 3215e3435;
n
(C]O): 1614;
n
(C]N): 1568;
n
small aliquots of concentrated compound solutions (1.0 mmol)
were added till the drop of fluorescence intensity (lex ¼ 489 nm,
lem ¼ 525 nm) attained a saturation. Measurements were made at
constant room temperature in TriseHCl buffer solution. Quenching
data were analyzed according to the SterneVolmer equation which
could be used to determine the fluorescent quenching mechanism:
(NO3): 1383; n(C]S): 1161; n(MeO): 620; n
(MeN): 450 cmꢁ1. Umax
(nm): 277, 307 nm Lm (S cm2 molꢁ1): 156.
4.3. Determination of crystal structures
The crystal of the Zn(II) complex was obtained from an ethanol
solution before concentrating at room temperature. X-ray diffrac-
tion data for the crystal were performed with graphite-mono-
F0=F ¼ 1 þ Kq½Qꢆ;
where F0 and F are the fluorescence intensity in the absence and
presence of drug at [Q] concentration respectively; Kq is the
quenching constant and [Q] is the quencherconcentration. Plotsof F0/
F versus [Q] appear to be linear and Kq depends on temperature [49].
chromated Mo-K
CCD diffractometer and collected by the
a
radiation (0.71073 Å) on a Bruker Smart-1000
/2 scan technique at 296
u
q
(2) K. The crystal structure was solved by direct methods. The
positions of non-hydrogen atoms were determined from successive
Fourier syntheses. The hydrogen atoms were placed in their
geometrically calculated positions. The positions and anisotropic
thermal parameters of all non-hydrogen atoms were refined on F2
by full matrix least-squares techniques with the SHELX-97 program
package. Absorption correction was employed using Semi-empirical
methods from equivalents. Taking the similar way, the crystallog-
raphy data of the Ni(II) complex were obtained.
4.4.4. Viscosity measurements
Viscosity experiments were conducted on an Ubbelodhe
viscometer, immersed in a thermostated water-bath maintained to
25.0 ꢅ 0.1 ꢃC. Titrations were performed for an investigated
compound (0.5e4 mM), and each compound was introduced into
a DNA solution (50
m
M) present in the viscometer. Data were pre-
versus the ratio of the concentration of the
is the viscosity of DNA in the presence
1/3
sented as (h/h0)
compound and DNA, where
h
of the compound and h0 is the viscosity of DNA alone. Relative
viscosities for DNA in either the presence or absence of investigated
compounds were calculated from the following equation [50,51]:
4.4. DNA binding properties
4.4.1. Electronic absorption spectroscopy
The ligand and complexes were dissolved in a mixture solvent of
1% DMF and 99% TriseHCl buffer (5 mM TriseHCl; 50 mM NaCl, pH
h
¼ ðt ꢁ t0Þ=t0;
where t is the observed flow time of the DNA containing solution,
and t0 is the flow time of TriseHCl buffer solution.
7.1) at concentration of 10
experiments were performed with fixed concentration drugs
(10 M), while gradually increasing the concentration of CT DNA
with the range from 2.5 to 40 M. When measuring the absorption
mM. Electronic absorption titration
m
4.5. Antioxidant activity
m
spectra, an equal amount of CT DNA was added to both the
compounds solutions and the reference solution to eliminate the
absorbance of CT DNA itself. Each sample solution was scanned in
the range of 190e500 nm.
In antioxidant activity experiments the superoxide radicals
(O2 ꢁ) were produced by the system MET/VitB2/NBT [52]. The
ꢀ
amount of O2 ꢁ and suppression ratio for O2 ꢁ can be calculated by
measuring the absorbance at 560 nm, because NBT can be reduced
ꢀ
ꢀ
quantitatively to blue formazan by O2 ꢁ. The solution of MET, VitB2
ꢀ
4.4.2. Fluorescence spectra
and NBT was prepared with 0.067 M phosphate buffer (pH ¼ 7.8) at
the condition of avoiding light. The tested compounds were dis-
solved in DMF. The reaction mixture contained MET (0.01 mol Lꢁ1),
NBT (4.6 ꢂ 10ꢁ5 mol Lꢁ1), VitB2 (3.3 ꢂ 10ꢁ6 mol Lꢁ1), phosphate
buffer solution (0.067 mol Lꢁ1) and the tested compound (the final
Fixed amounts (10
increasing amounts of CT DNA, over a range of DNA concentrations
from 2.5 to 30 M. Excitation wavelengths of the samples were
mM) of compounds were titrated with
m
about 300 nm, scan speed was 480 nm/min, and both of excitation
and emission slit widths were 10 nm. All experiments were con-
ducted at a constant room temperature in TriseHCl buffer solution.
The intrinsic binding constants (Kb) of the compounds with DNA
were obtained by the fluorescence titration methods and Scatchard
equation [48]:
concentration: Ci(i¼1e5) ¼ 0.4, 1.0, 2.0, 4.0, 6.0
mM). After incubating
at 30 ꢃC for 10 min and illuminating with a fluorescent lamp for
3 min, the absorbances (Ai) of the samples were measured at
560 nm. The sample without the tested compound and avoiding
ꢀꢁ
light was used as the control. The suppression ratio for O2 was
calculated from the following expression:
r/Cf ¼ Kb ꢂ (n ꢁ r),
Cf ¼ Ct ꢁ Cb,
ÁÃ
Suppression ratio ð%Þ ¼ ½ðA0 ꢁ AiÞ=ðA0 ꢂ100%
Cb ¼ Ct[(F ꢁ F0)/(Fmax ꢁ F0)],
where Ai is the absorbance in the presence of the ligand or its
complexes, and A0 is the absorbance in the absence of the ligand or
its complexes.
r ¼ Cb=CDNA
;
where Ct is the total compound concentration, Cb is the molar
concentration of compound bound for DNA; n is the binding site
number; F is the observed fluorescence emission intensity at
In antioxidant activity experiments the hydroxyl radical (HOꢀ) in
aqueous media was generated through the Fenton reaction [53].

Y. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 5692e5701
5701
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reaction mixture contained 2.5 mL 0.15 M phosphate buffer
(pH ¼ 7.4), 0.5 mL 114
3% H2O2 and 30 L the tested compound solution (the final
M). The sample
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m
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Suppression ratio ð%Þ ¼ ½ðAi ꢁ A0Þ=ðAc ꢁ A0 ꢂ100%
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