Synthesis, characterization, DNA binding properties and antioxidant activity of Ln(III) complexes with hesperetin-4-one-(benzoyl) hydrazone

Article abstract of DOI:10.1016/j.ejmech.2009.06.027

Novel Ln(III) complexes with hesperetin-4-one-(benzoyl) hydrazone (H₄L) have been synthesized and characterized. Electronic absorption spectroscopy, fluorescence spectra, ethidium bromide displacement experiments, iodide quenching experiments,

Full text of DOI:10.1016/j.ejmech.2009.06.027

European Journal of Medicinal Chemistry 44 (2009) 4585–4595
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, characterization, DNA binding properties and antioxidant activity of
Ln(III) complexes with hesperetin-4-one-(benzoyl) hydrazone
*
Yong Li, Zheng-Yin Yang , Ming-Fang Wang
College of Chemistry and Chemical Engineering and 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:
Novel Ln(III) complexes with hesperetin-4-one-(benzoyl) hydrazone (H4L) have been synthesized and
characterized. Electronic absorption spectroscopy, fluorescence spectra, ethidium bromide displacement
experiments, iodide quenching experiments, salt effect and viscosity measurements indicate that the
ligand and Ln(III) complexes, especially the Nd(III) complex, strongly bind to calf thymus DNA,
presumably via an intercalation mechanism. The intrinsic binding constants of the Nd(III) complex and
Received 9 April 2009
Received in revised form
25 June 2009
Accepted 25 June 2009
Available online 1 July 2009
ligand with DNA were 2.39 ꢀ 106 and 2.70 ꢀ 105
M
ꢁ1, respectively. Furthermore, the antioxidant activity
of the ligand and Ln(III) complexes was determined by superoxide and hydroxyl radical scavenging
Keywords:
ꢂ
method in vitro, which indicate that the ligand and Ln(III) complexes have the activity to suppress O2ꢁ
Hesperetin hydrazone
Ln(III) complexes
DNA binding studies
Antioxidant activity
and HOꢂ and the Ln(III) complexes exhibit more effective antioxidant activity than the ligand alone.
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
Some hydrazones and their metal complexes have diverse bio-
logical and pharmaceutical activities, such as antimicrobial, anti-
It is well known that deoxyribonucleic acid (DNA) is an impor-
tant genetic substance in organism. The regions of DNA involved
vital processes, such as gene expression, gene transcription,
mutagenesis and carcinogenesis [1]. Since DNA is an important
cellular receptor, many chemicals exert their antitumor effects
through binding to DNA thereby changing the republication of DNA
and inhibiting the growth of the tumor cells, which is the basis of
designing new and more efficient antitumor drugs and their
effectiveness depends on the mode and affinity of the binding
[2–5]. Therefore, the qualitative and quantitative analyses of the
nucleic acids as the material base of genetic inheritance are
becoming more and more important. Binding studies of small
molecules to DNA are very important in the development of DNA
molecular probes and new therapeutic reagents [6–8]. During the
past decades, identifying small molecules that are capable of
binding to DNA through an intercalation mode has attracted
considerable interests [9]. Small molecule compounds would
potentially be valuable tools in biotechnology, nanotechnology,
therapeutic approaches and the study of nucleic acid conforma-
tions and then a large number of metal complexes with small
molecule compounds as ligands are being used at the forefront of
many of these efforts [10,11].
tuberculostatic, anticancer, antioxidant properties and so on [12–
15]. Our previous studies indicate that naringenin hydrazones and
their metal complexes possess better antitumor, antioxidant and
cytotoxicity activities [16–18]. Furthermore, Schiff bases are an
important class of ligands in coordination chemistry and are found
to possess extensive applications in different fields, such as phar-
macological field [19], which are able to inhibit the growth of
several animal tumors [20]. Hesperetin (5,7,30-trihydroxyl-40-
methoxyl-flavanone) is a kind of flavonoid which occurs ubiqui-
tously in plants, fruits, flowers and foods of plant origin [21]. As an
important bioactive Chinese traditional medicine, hesperetin has
multiple biological and pharmacological activities, including anti-
oxidant properties [22,23], inhibition of cancer development
[24,25], effects on the blood-brain barrier [26,27], signal trans-
duction pathways [28], etc. However, hesperetin hydrazones and
their complexes have not been extensively investigated. Moreover,
Our previous work clearly indicates that the lanthanide complexes
possess better bioactivities, such as antioxidant activity, cytotoxic
activity, DNA binding affinity and so on [16–18]. In addition,
lanthanide ions are subjects of increasing interest in bioinorganic
and coordination chemistry [29]. Lanthanide complexes with
tetracycline, phenanthroline [30], adriamycin and pyridine [31,32]
have been already synthesized as a probe to study nucleic acids.
In order to give a deep research to hesperetin ramifications and
their lanthanide complexes, in this paper, we synthesized and
characterized a novel ligand, hesperetin-4-one-(benzoyl) hydrazone
* Corresponding author. Tel.: þ86 931 8913515; fax: þ86 931 8912582.
E-mail address: yangzy@lzu.edu.cn (Z.-Y. Yang).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.06.027

4586
Y. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4585–4595
(H4L, Fig. 1) and its Ln(III) complexes. We described a comparative
study of the interaction of novel Ln(III) complexes and the ligand
with calf thymus DNA (CT DNA) using electronic absorption spec-
troscopy, fluorescence spectra, ethidium bromide experiments,
iodide quenching experiments, salt effect and viscosity measure-
ments for the first time. The antioxidant activity of the ligand and its
Ln(III) complexes was also investigated. Information obtained from
our study would be helpful to understand the mechanism of inter-
actions of hesperetin hydrazones and their complexes with nucleic
acid and should be useful in the development of potential probes of
DNA structure and conformation, and new therapeutic reagents for
some certain diseases.
J ¼ 2.2 Hz, 6-H), 5.93 (1H, d, J ¼ 2.2 Hz, 8-H), 6.93 (3H, br, 20, 50, 60-
H), 7.52 (2H, dd, J ¼ 1.8, 7.4 Hz, 300, 500-H), 7.54 (1H, dd, J ¼ 7.4,
11.6 Hz, 400-H), 7.88 (2H, dd, J ¼ 1.8, 11.6 Hz, 200, 600-H), 9.09 (1H, s, 30-
OH), 9.99 (1H, s, 7-OH),11.12 (1H, s, –NH–C]O), 13.06 (1H, s, 5-OH).
[Ln(H4L)2(NO3)2]NO3: (DMSO-d6, 200 MHz),
d (ppm) 2.74 (2H, dd,
J ¼ 8.0, 16.2 Hz, 3(a)-H), 2.90 (2H, dd, J ¼ 2.2, 16.2 Hz, 3(e)-H), 3.85
(6H, s, –OCH3), 5.50 (2H, dd, J ¼ 2.2, 8.0 Hz, 2-H), 5.58 (2H, d,
J ¼ 2.2 Hz, 6-H), 5.87 (2H, d, J ¼ 2.2 Hz, 8-H), 6.92 (6H, br, 20, 50, 60-
H), 7.45 (4H, dd, J ¼ 2.0, 7.6 Hz, 300, 500-H), 7.85 (2H, dd, J ¼ 7.6,
13.4 Hz, 400-H), 7.96 (4H, dd, J ¼ 2.0, 13.4 Hz, 200, 600-H), 9.06 (2H, s,
30-OH), 10.00 (2H, s, 7-OH), 11.12 (2H, s, –NH–C]O), 13.07 (2H, s, 5-
OH). In the complex, the –OH-5 hydrogen still exists. The hydrogen
of the ]NNH– group is also detected and there are no hydrogens
replaced by the metal ion, which is also supported by the IR spectra.
The complexes begin to decompose at 298 ꢃC or so and there are
three exothermic peaks appear around 298–646 ꢃC. The corre-
sponding TG curves show a series of weight loss. Under 200 ꢃC, there
are no endothermic peaks and no weight loss on the corresponding
curves. It indicates that there are no crystal or coordinate solvent
molecules. While being hated to 800 ꢃC, the complexes become their
corresponding oxides and the residues are in accordance with
calculations.
The study of the electronic spectra in the ultraviolet and visible
(UV–vis) ranges for the Ln(III) complexes and ligand was carried out
in Tris–HCl buffer solution. The electronic spectra of the free ligand
have a strong band at lmax ¼ 328 nm and a medium band at
lmax ¼ 285 nm. The complexes also yield two bands, and the two
bands are shifted to 343 and 291 nm or so. These indicate that the
Ln(III) complexes are formed.
The electrospray ionization (ESI) mass spectra of Sm(III) and
Dy(III) complexes were made. The mass spectra of Sm(III) and
Dy(III) complexes show peaks at 991 and 1002 which can be
assigned to the fragments [Sm(III) complex–3NO3–H]2þ and [Dy(III)
complex–3NO3–2H]þ, respectively.
Since the crystal structures of the Ln(III) complexes have not
been obtained yet, we characterized the complexes and determined
its possible structure by elemental analyses, molar conductivities, IR
spectra, 1H NMR, TG/DTA, mass spectra and UV–vis spectra. The
ligand and lanthanide ions can form mononuclear ten-coordination
[Ln(H4L)2(NO3)2]NO3 [Ln(III) ¼ La, Sm, Dy, Yb and Nd] complexes
with 1:2 metal-to-ligand stoichiometry at the Ln(III) centers (Fig. 2).
2. Results and discussion
2.1. Structure of the Ln(III) complexes
The complexes were prepared by direct reaction of ligand with
the appropriate mole ratios of Ln(III) nitrate in acetone. The yields
were good to moderate. The desired Ln(III) complexes were sepa-
rated from the solution by suction filtration, purified by washing
several times with ethanol. The complexes are air-stable for
extended periods and soluble in DMSO and DMF; slightly soluble in
ethanol and methanol; insoluble in benzene, water and diethyl
ether. The molar conductivities of the complexes are around 73.9–
88.1 S cm2 molꢁ1 in DMF solution, showing that all complexes are
1:1 electrolytes [33]. The elemental analyses and molar conduc-
tivities show that formulas of the Ln(III) complexes conform to
[Ln(H4L)2(NO3)2]NO3 [Ln(III) ¼ La, Sm, Dy, Yb and Nd].
The IR spectra of the complexes are similar. IR spectra usually
provide a lot of valuable information on coordination reactions. All
the spectra are characterized by vibrational bands mainly due to the
NH, C]O, C]N and NO3 groups. The
for the ligand, and this peak shifts at 3386 cmꢁ1 or so for the
complexes. The
(C]O) vibration of the free ligand is at 1642 cmꢁ1
for the complexes, the peak shifts to 1610 cmꢁ1
Dn(ligand–complexes) is
equal to 32 cmꢁ1. In the complexes, the band at 555 cmꢁ1 or so is
assigned to (M–O). It demonstrates that the oxygen of carbonyl has
formed a coordinative bond with the lanthanide ions [34]. The band
at 1604 cmꢁ1 for the free ligand is assigned to the
(C]N) stretch,
which shifts to 1570 cmꢁ1 for its complexes, Dn(ligand–complexes) is
equal to 34 cmꢁ1. Weak bands at 410 cmꢁ1 or so are assigned to
n
(N–H) appears at 3362 cmꢁ1
n
;
,
n
n
n
(M–N) in the complexes. These shifts and new bands further
confirm that the nitrogen of the imino-group bonds to the lantha-
nide ions [35]. The absorption bands of the coordinated nitrates
were observed at about 1479 (nas) and 870 (ns) cmꢁ1. The n3 free
nitrates appear at 1384 cmꢁ1 or so in the spectra of the Ln(III)
complexes. In addition, the separation of the two highest frequency
2.2. Magnetic properties
Effective magnetic moments were calculated from the molar
magnetic susceptibilities [49]. Dy(III) complex (Dn ¼ 42.0 Hz) has
a magnetic moment of 12.60 B.M., as predicted for a high spin f9
system with five unpaired electrons. However, La(III) complex
bands rn4
ꢁ
n
1r is approximately 160 cmꢁ1, and accordingly the
coordinated NOꢁ3 ion in the complexes is a bidentate ligand [36].
The 1H NMR spectra of the ligand and its La(III) complex are
(
Dn ¼ 0) is diamagnetic as expected for a f0 configuration. Magnetic
moments for Nd(III) complex
(
Dn ¼ 4.5 Hz), Sm(III) complex
assigned as follows: H4L (DMSO-d6, 200 MHz),
d
(ppm) 2.51 (1H, dd,
(
Dn ¼ 1.5 Hz) and Yb(III) complex (Dn ¼ 7.2 Hz) are 0, 3.84, 1.85 and
J ¼ 12.3, 17.0 Hz, 3(a)-H), 2.88 (1H, dd, J ¼ 3.0, 17.0 Hz, 3(e)-H), 3.77
4.65 B.M., respectively. These results are consistent with the calcu-
lated values.
(3H, s, –OCH3), 5.06 (1H, dd, J ¼ 3.0, 12.3 Hz, 2-H), 5.87 (1H, d,
Fig. 1. Scheme of the synthesis of the ligand (H4L).

Y. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4585–4595
4587
hypochromism of 40.0% and 32.6%, respectively. For the Nd(III)
complex, the absorption bands at 343 and 291 nm appeared with
hypochromism of 33.7% and 38.0%. The hypochromisms observed
for the bands of the free ligand and its Nd(III) complex are
accompanied by a small red shift by less than 4 nm.
The electronic absorption spectroscopy is one of the most useful
techniques in DNA binding studies of metal complexes [37]. A
compound binding to DNA through intercalation usually results in
hypochromism and bathochromism due to the intercalation mode
involving a strong
p–p stacking interaction between the aromatic
chromophore and the DNA base pairs. It seems to be generally
accepted that the extent of the hypochromism in the UV–vis band is
consistent with the strength of intercalative interaction [38]. So the
above phenomena imply that the compounds interact with CT DNA
quite probably by intercalating the compounds into the DNA base
pairs. From the results of electronic absorption spectroscopy, we
can conclude that the free ligand and Nd(III) complex can interact
with CT DNA through the same mode (intercalation).
In order to further test if the complexes could bind to CT DNA via
the mode of intercalation, ethidium bromide (EB) was employed,
because EB interacts with DNA as a typical indicator of intercalation
[39]. Fig. 4 shows the electronic absorption spectra when EB was
employed. The absorbance of Tris–HCl buffer solution is highest
when there is only EB alone. After adding CT DNA into Tris–HCl
buffer solution, the absorbance decreases. However, it increases
when the ligand and Nd(III) complex was added dropwise into the
buffer solution. Fig. 4 shows that the maximal absorption of EB at
479 nm decreased and shifted to 494 nm in the presence of DNA,
which was characteristic of intercalation mode. Curve (B) in Fig. 4
was the absorption of a mixture solution of EB, DNA and the free
ligand or Nd(III) complex. It was found that the absorption at
495 nm increased comparing with curve (C). It could result from
two reasons: (1) EB bound to the free ligand or Nd(III) complex
strongly, resulting in the decreased amount of EB intercalated into
DNA; (2) there exists a competitive intercalation between the free
ligand or Nd(III) complex and EB with DNA, so releasing some free
EB from DNA–EB system. However, the former reason could be
precluded because there were no new absorption peaks appearing.
Fig. 2. The suggested structure of the lanthanide complexes.
2.3. DNA binding studies
2.3.1. Electronic absorption titration
Before reacting the ligand and its Ln(III) complexes interacting
with CT DNA, its solution behavior in Tris–HCl buffer solution at
room temperature was monitored by UV–vis spectroscopy for 24 h.
Liberation of the ligand and its Ln(III) complexes was not observed
under these conditions. These suggest that the ligand and its Ln(III)
complexes are stable under the conditions studied.
The electronic absorption spectra of the free ligand and Nd(III)
complex in the absence and presence of CT DNA are given in Fig. 3.
As seen from Fig. 3a, there exist two bands at 328 and 285 nm for
the free ligand, and in Fig. 3b two bands are at 343 and 291 nm for
the Nd(III) complex. With increasing concentrations of CT DNA, the
absorption bands of the free ligand at 328 and 285 nm exhibited
2.3.2. Fluorescence spectra
The ligand and its Ln(III) complexes can emit weak lumines-
cence in Tris–HCl buffer with a max wavelength of about 445 nm.
The results of the emission titrations for the Nd(III) complex and
free ligand with DNA are illustrated in the titration curves (Fig. 5).
0.18
a
0.25
0.20
0.15
0.10
0.05
0.00
b
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
250
300
350
Wavelength (nm)
400
450
250
300
350
400
Wavelength (nm)
450
500
Fig. 3. (a) Electronic absorption spectra of the free ligand (10
20.0 mM; subsequent spectra). Arrow shows the absorbance changes upon increasing DNA concentration. (b) Electronic absorption spectra of the Nd(III) complex (10 m
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 and 20.0
upon increasing DNA concentration.
m
M) 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 and
M) in the
M; subsequent spectra). Arrow shows the absorbance changes
m

4588
Y. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4585–4595
0.038
a
b
A
A
C
0.05
0.04
0.03
0.02
0.01
0.00
0.036
0.034
0.032
0.030
0.028
0.026
0.024
0.022
B
B
C
400
500
Wavelength (nm)
600
400
500
Wavelength (nm)
600
Fig. 4. (a) The visible absorption spectra of 1 ꢀ10ꢁ5 M EB (A); (A) þ 2.5 ꢀ10ꢁ5 M DNA (C); (C) þ 2.5 ꢀ10ꢁ5 M free ligand (B) in Tris–HCl buffer (5 mM tris–HCl, 50 mM NaCl, pH 7.1)
solution. (b) The visible absorption spectra of 1 ꢀ10ꢁ5 M EB (A); (A) þ 2.5 ꢀ10ꢁ5 M DNA (C); (C) þ 2.5 ꢀ10ꢁ5 M Nd(III) complex (B) in Tris–HCl buffer (5 mM tris–HCl, 50 mM NaCl,
pH 7.1) solution.
Compared to the Nd(III) complex and free ligand alone, the emis-
sion intensity increases with increasing concentrations of CT DNA.
The results of the emission titrations suggest that both the
Nd(III) complex and free ligand are protected from solvent water
molecules by the hydrophobic environment inside the DNA helix
and that the Nd(III) complex can be protected more efficiently than
the free ligand. This implies that both the Nd(III) complex and free
ligand can insert between DNA base pairs deeply and the Nd(III)
complex can bind to DNA more strongly than the free ligand. Since
the hydrophobic 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 significantly increase
in emission intensity also agrees with those observed for other
intercalators [40]. According to the Scatchard equation, a plot of r/Cf
200
8
a
Kb=2.70X105M-1
K =2.70X105M-1
150
b
6
100
4
2
50
0
400
450
500
550
600
0.5
1.0
1.5
2.0
2.5
r
Wavelength λ/nm
800
700
600
500
400
300
200
100
0
b
20
15
10
5
Kb=2.39X106M-1
Kb=2.39X106M-1
0
350
400
450
500
550
600
0.6
0.8
1.0
1.2
1.4
1.6
r
Wavelength λ/nm
Fig. 5. (a) The emission enhancement spectra of free ligand (10 mM) in the presence of 0, 2.5, 5, 7.5, 10, 12.5 and 15 mM DNA. Arrow shows the emission intensity changes upon
increasing DNA concentration. Inset: Scatchard plot of the fluorescence titration data of ligand, Kb ¼ 2.70 ꢀ 105
M
ꢁ1. (b) The emission enhancement spectra of Nd(III) complex
(10
m
M) in the presence of 0, 2.5, 5, 7.5, 10, 12.5 and 15
m
M DNA. Inset: Scatchard plot of the fluorescence titration data of ligand, Kb ¼ 2.39 ꢀ 106 Mꢁ1
.

Y. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4585–4595
4589
versus
r
gave the binding constants (Kb) 2.39 ꢀ 106 and
addition of the Nd(III) complex and free ligand to the DNA-bound EB
solutions caused marked reduction in emission intensities. The
quenching plots illustrated that the quenching of EB bound to DNA
by the complexes and free ligand is in good agreement with the
linear Stern–Volmer equation. In the plots of F0/F versus [Q], Kq is
given by the ratio of the slope to the intercept. The Kq values for the
2.70 ꢀ 105 Mꢁ1 from the fluorescence data for the Nd(III) complex
and the free ligand, respectively. Kb values for the La(III) complex,
Sm(III) complex, Dy(III) complex and Yb(III) complex were
3.23 ꢀ105, 1.65 ꢀ106, 9.66 ꢀ 105 and 1.25 ꢀ106 Mꢁ1, respectively,
which were obtained according to the same method. These results
show that the ligand and its Ln(III) complexes can bind to DNA,
especially the Nd(III) complex binds more strongly than the free
ligand. The higher binding affinity of the Nd(III) complex is prob-
Nd(III) complex and ligand are 1.20 ꢀ 105 and 4.73 ꢀ104 Mꢁ1
,
respectively. Kq values for the La(III) complex, Sm(III) complex, Dy(III)
complex and Yb(III) complex were 6.67 ꢀ 104, 1.17 ꢀ 105, 1.09 ꢀ 105
and 1.11 ꢀ105 Mꢁ1, respectively, which were obtained according to
the same method. The data show that the interaction of the Ln(III)
complexes with DNA is stronger than that of the free ligand, which is
consistent with the electronic absorption spectral results. Since
these changes indicate only one kind of quenching process, it may be
concluded that the Ln(III) complexes and free ligand bind to DNA via
the same mode (intercalation mode).
ably attributed to the extension of the
p system of the intercalated
ligand and the coordination of Nd(III), which leads to a planar area
greater than that of the free ligand and that the coordinated ligand
penetrates more deeply into and stacks more strongly with DNA
base pairs.
The DNA binding modes of the compounds were further moni-
tored bya fluorescent EB displacement assay. It is well known that EB
can intercalate nonspecifically into DNA which causes it to fluoresce
strongly. Competitive binding of other drugs to DNA and EB will
result in displacement of DNA and a decrease in the fluorescence
intensity. The fluorescence-based competition technique can
provide indirect evidence for the DNA binding mode. The emission
band of the DNA–EB system decreased in intensity with an increase
of the compounds concentrations, indicating that the compounds
can displace EB from DNA–EB system [41]. Such characteristic
change is usually observed in intercalative DNA interactions [42].
Steady-state emission quenching experiments are used to observe
the binding mode of the Nd(III) complex and free ligand to CT DNA.
Fig. 6 shows the emission spectra of the DNA–EB system with
increasing amounts of the Nd(III) complex and free ligand. The
2.3.3. Iodide quenching experiments
The results of the emission titrations for the Nd(III) complex and
potassium iodide with absence and presence of CT DNA are illus-
trated in the titration curves (Fig. 7). Compared to the Nd(III)
complex alone, the emission intensity decreases with increasing
concentrations of CT DNA. The quenching plots illustrated that the
quenching studies of the Nd(III) complex are in good agreement
with the linear Stern–Volmer equation.
In the aqueous solution, iodide and ferrocyanide anions quench
the fluorescence of Nd(III) complex very efficiently, so we used
potassium iodide as the quencher to determine the relative
accessibilities of the free and bound Nd(III) complexes. From Fig. 7,
100
a
2.0
1.9
1.8
80
Kq = 4.73X104 M-1
Kq = 4.73X104 M-1
1.7
1.6
1.5
1.4
1.3
1.2
1.1
60
40
20
0
550
600
Wavelength (nm)
650
700
0
2
4
6
8
[Compound] X 106M
10 12 14 16 18 20
600
b
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
500
400
300
200
100
0
Kq = 1.20X105 M-1
Kq = 1.20X105 M-1
600
650
Wavelength (nm)
700
0
5
10 15 20 25 30 35 40
[Compound] X 106M
Fig. 6. (a) The emission spectra of DNA–EB system, in the presence of 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20 and 22.5
m
M ligand. Arrow shows the emission intensity changes upon
increasing ligand. Inset: Stern–Volmer plot of the fluorescence titration data of ligand. Kq ¼ 4.73 ꢀ 104
M
ꢁ1. (b) The emission spectra of DNA–EB system, in the presence of 0, 2.5, 5,
7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 32.5 and 35 mM Nd(III) complex. Arrow shows the emission intensity changes upon increasing Nd(III) complex. Inset: Stern–Volmer plot of
the fluorescence titration data of Nd(III) complex. Kq ¼ 1.20 ꢀ 105 Mꢁ1
.

4590
Y. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4585–4595
900
800
700
600
500
400
300
200
100
0
800
a
b
Ksv = 4.7 M-1
600
Ksv = 15.3 M-1
400
200
0
400
450
500
550
Wavelength number (nm)
600
400
450
Wavelength number (nm)
500
550
600
1.35
1.30
1.25
1.20
1.15
1.10
1.05
1.00
c
Ksv (1) = 15.3 M-1
Ksv (2) = 4.7 M-1
1
2
0.00
0.01
0.02
0.03
0.04
[KI]
Fig. 7. (a) Fluorescence spectra of Nd(III) complex (10 mM) with increasing concentration of KI (5, 10, 20, 30, 40, 50, 60, 70 and 80 mM). (b) Fluorescence spectra of Nd(III) complex
(10 mM) and DNA (20 mM) system with increasing concentration of KI (5, 10, 20, 30, 40, 50, 60, 70 and 80 mM). (c) Inset: Stern–Volmer plot of the fluorescence titration data of Nd(III)
complex. Effect of KI concentration (1: Nd(III) complex þ KI; 2: Nd(III) complex þ KI þ CT DNA). Ksv (1) ¼ 15.3 Mꢁ1, Ksv (2) ¼ 4.7 Mꢁ1
.
we can find that the addition of Nd(III) complex and CT DNA results
in extensive quenching of the fluorescence intensity. The quench-
ing data were plotted according to the Stern–Volmer equation and
the slopes were calculated by the linear least-squares method. The
observed quenching constants were 15.3 and 4.7 Mꢁ1 with and
without CT DNA, respectively. The quenching of the Nd(III) complex
was in fact enhanced by a factor of more than 3 when the Nd(III)
complex was bound to the DNA helix. We can conclude that the
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
1.8
1.5
1
1.2
2
0.9
0.6
0.3
0.0
0.0
0.1
0.2
0.3
0.4
0.5
[M]/[DNA]
0.6
0.7
0.8
0.9
1.0
H4L
Nd(III) complex
Dy(III) complex
Yb(III) complex
Sm(III) complex
La(III) complex
0
2
4
6
8
10
12
14
[NaCl]
Fig. 8. Effect of NaCl concentration (1: Nd(III) complex þ NaCl; 2: Nd(III) complex -
þ NaCl þ CT DNA).
Fig. 9. Effect of increasing amounts of the ligand and its Ln(III) complexes on the
relative viscosity of DNA at 25.0 ꢃC.

Y. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4585–4595
4591
H4L
La(III) complex
Sm(III) complex
Nd(III) complex
Dy(III) complex
Yb(III) complex
90
80
70
60
50
40
30
20
10
Fig. 10. Molecular mode for Ln(III) complexes (in an intercalative mode).
Ln(III) complexes are intercalated into the DNA helix and they
should be protected from the anionic quencher, owning to the base
pairs above and below the intercalators [43].
2.3.4. Salt effect
0
1
2
3
4
5
6
7
8
9
10
The effect of the ionic strength on the compounds fluorescence
was tested by the addition of a strong electrolyte, NaCl. As seen from
Fig. 8, there are almost no changes happening to the fluorescence
emission intensities with increasing concentrations of CT DNA.
The effect of the ionic strength on the Nd(III) complex fluores-
cence intensity was tested by the addition of a strong electrolyte,
such as NaCl, instead of potassium iodide. Cations of the salts can
neutralize the negatively charged phosphate. If the compound
binds to DNA through an electrostatic interaction mode, the surface
of DNA will be surrounded by the sodium ions with the increase of
ionic strength. Then the compound is difficult to approach DNA and
the strength of interaction with DNA decreases, the degree of
fluorescence quenching also falls [44]. Addition of NaCl to the
Nd(III) complex in the absence and presence of CT DNA has little or
no influence on the fluorescence intensity, showing that the
interaction of the Nd(III) complex with CT DNA is not electrostatic
interaction.
Concentration (μM)
ꢂ
Fig. 11. Scavenging effect of the ligand and Ln(III) complexes on O2ꢁ
.
as [Ru(bpy)3]2þ, which interacts with DNA by an electrostatic
binding mode, have no influence on DNA viscosity [46]. The effects
of all the compounds on the viscosity of CT DNA are shown in Fig. 9.
The viscosities of the DNA increase steadily with increasing
concentrations of ligand and Ln(III) complexes and the extent of the
increase observed for the ligand is smaller than that for the Ln(III)
complexes with an increasing concentration of the complexes.
Viscosity measurements clearly show that all the compounds can
intercalate between adjacent DNA base pairs, causing an extension
in the helix and thus increase the viscosity of DNA, and that the
complexes can intercalate more strongly and deeply than the free
ligand, leading to the greater increase in viscosity of the DNA. The
results obtained from the viscosity experiments validate those
obtained from the spectroscopic studies.
2.3.5. Viscosity measurements
In order to further clarify the interaction nature between the
compounds and DNA, viscosity measurements were carried into
execution. Optical photophysical probes provide necessary but not
sufficient evidence to support a binding mode. Hydrodynamic
measurements that are sensitive to length change (i.e. viscosity and
sedimentation) are regarded as the least ambiguous and the most
critical tests of a binding mode in solution in the absence of crys-
tallographic structural data [45]. A classical intercalation mode
demands that the DNA helix must lengthen as base pairs are sepa-
rated to accommodate the binding complexes, leading to the
increase of DNA viscosity, as for the behaviors of the known DNA
intercalators [38]. In contrast, a partial and/or non-classical inter-
calation of the complex could bend (or kink) the DNA helix, reducing
its viscosity concomitantly [45]. In addition, some complexes such
On the basis of all the spectroscopic studies together with the
viscosity measurements, we find that the Ln(III) complexes and free
ligand can bind to CT DNA via an intercalative mode (Fig. 10) and
the Ln(III) complexes bind to CT DNA more strongly and deeply
than the free ligand.
2.4. Antioxidant activity
Owning to the ligand and its Ln(III) complexes exhibit good DNA
binding affinity, it is considered worthwhile to study the antioxi-
dant activity of these compounds. The antioxidant properties of
hesperetin derivatives have attracted a lot of interests and have
been extensively investigated, mainly in the in vitro systems [21,22].
Table 1
ꢂ
The influence of investigated compounds for O2ꢁ
.
ꢂ
Compound
Average inhibition (%) for O2ꢁ
(m
M)
Equation
IC50
(m
M)
R2
0.4
8.41
27.85
23.25
29.67
24.98
12.59
1.0
2.0
4.0
6.0
8.0
H4L
15.11
37.14
31.46
35.42
30.22
23.46
22.06
48.71
42.59
39.97
35.13
32.13
28.54
59.62
54.67
50.72
47.21
43.25
34.17
70.14
62.43
59.36
52.89
49.67
37.89
82.54
72.14
62.91
58.52
54.28
y ¼ 22.67x þ 16.10
y ¼ 40.30x þ 39.65
y ¼ 37.06x þ 34.25
y ¼ 26.25x þ 36.78
y ¼ 26.17x þ 31.96
y ¼ 32.28x þ 24.14
31.287
1.806
2.661
3.189
4.890
6.236
0.989
0.955
0.970
0.945
0.948
0.995
La(III) complex
Sm(III) complex
Nd(III) complex
Dy(III) complex
Yb(III) complex
IC50 values were calculated from regression lines where: x was log of the tested compound concentration and y was percent inhibition of the tested compounds. When the
percent inhibition of the tested compounds was 50%, the tested compound concentration was IC50. R2 ¼ correlation coefficient.

4592
Y. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4585–4595
Table 2
The influence of investigated compounds for HOꢂ.
Compound
Average inhibition (%) for HOꢂ (
m
M)
Equation
IC50
(m
M)
R2
1.0
1.17
20.03
23.31
18.80
13.53
6.83
2.0
8.61
3.0
4.0
5.0
6.0
H4L
14.03
64.66
48.87
39.30
32.25
25.22
24.63
17.54
72.18
57.14
48.50
40.12
35.43
32.91
21.05
81.95
65.41
57.14
48.14
42.01
43.09
23.79
92.53
74.26
61.20
54.63
51.53
43.82
y ¼ 29.06x þ 0.52
y ¼ 88.97x þ 21.30
y ¼ 66.00x þ 18.92
y ¼ 55.88x þ 15.88
y ¼ 52.56x þ 10.31
y ¼ 55.51x þ 3.41
y ¼ 57.26x ꢁ 0.73
50.429
2.102
2.957
4.079
5.690
6.907
10.190
0.995
0.994
0.961
0.973
0.963
0.952
0.958
La(III) complex
Sm(III) complex
Nd(III) complex
Dy(III) complex
Yb(III) complex
Mannitol
50.38
33.08
30.01
23.35
18.03
12.52
2.30
IC50 values were calculated from regression lines where: x was log of the tested compound concentration and y was percent inhibition of the tested compounds. When the
percent inhibition of the tested compounds was 50%, the tested compound concentration was IC50. R2 ¼ correlation coefficient.
Their free radical scavenging ability is always excellently directed
From Tables 1 and 2, we can easily find that lanthanide
complexes exhibit considerable scavenging activity due to the
chelation of organic molecules to the lanthanide ions which exert
different and selective effects on scavenging radicals of the bio-
logical system. We also find that the five lanthanide complexes are
more effective inhibitor than mannitol which is usually used as
special scavenger for HOꢂ. It is reported that IC50 value of ascorbic
acid (Vc, a standard agent for non-enzymatic reaction) for HOꢂ is
ꢂ
toward superoxide radical (Oꢁ2 ) and hydroxyl radical (HOꢂ) which
are highly reactive products of reactive oxygen species (ROS)
implicated with pathogenic processes including carcinogenesis
through direct effects on DNA directly and by acting as a tumor
promoter [47]. In this paper, the antioxidant activity of the ligand
and its Ln(III) complexes was studied by comparing their scavenging
ꢂ
effects on superoxide radical (Oꢁ2 ) and hydroxyl radical (HOꢂ) in
ꢂ
KH2PO4–Na2HPO4 buffer solution.
1.537 mg mLꢁ1 (8.727 mmol), and the scavenging effect for Oꢁ2 is
The data of the suppression ratio for Oꢁ2 ꢂ are listed in Table 1. We
only 25% at 1.75 mg mLꢁ1 (9.94 mmol) [48]. Notably, the investi-
gated ligand and lanthanide complexes have much stronger scav-
ꢂ
find that the inhibitory effect of the compounds tested on Oꢁ2 is
ꢂ
concentration dependent and the suppression ratio increases with
enging abilities for HOꢂ and O2ꢁ radicals. It was believed that the
increasing sample concentrations in the range tested (Fig. 11). The
information obtained form present work would be useful to
develop new potential antioxidants and therapeutic agents for
some diseases.
ꢂ
average suppression ratio of thefreeligand (IC50 ¼ 31.287
is the least in all compounds. The La(III) complex (IC50 ¼ 1.806
the most effective in the five lanthanide complexes and the Nd(III)
m
M) for Oꢁ2
mM) is
complex (IC50 ¼ 3.189
mM) is also a more effective inhibitor than the
3. Conclusion
free ligand.
The data of the suppression ratio for HOꢂ are listed in Table 2.
We find that the inhibitory effect of the compounds tested on HOꢂ
is also concentration dependent and the suppression ratio
increases with increasing sample concentrations in the range
tested (Fig. 12). The average suppression ratio of the free ligand
Taken together, a novel hesperetin Schiff base ligand, hesperetin-
4-one-(benzoyl) hydrazone (H4L), and its La(III), Sm(III), Nd(III),
Dy(III) and Yb(III) complexes were reported. The structure of the
Ln(III) complexes was determined on the basis of elemental analyses,
molar conductivities, IR spectra, 1H NMR, TG/DTA, mass spectra and
UV–vis spectra. It is notable that the ligand and its Ln(III) complexes
have good planar property, which results in the higher DNA binding
constants of compounds. Experimental results indicate that the
ligand and lanthanide complexes bind to DNA via an intercalation
mode and the lanthanide complexes can bind to DNA more strongly
than the free ligand alone. Moreover, lanthanide complexes have
better antioxidant activity than the ligand. Results obtained from our
present work would be useful to understand the mechanism of
interactions of the small molecule compounds binding to DNA and
helpful in the development of their potential biological, pharma-
ceutical and physiological implications in the future.
(IC50 ¼ 50.429
m
M) for HOꢂ is the least and the La(III) complex
M) is the most effective in all the compounds. The
(IC50 ¼ 2.102
m
order of the suppression ratio of the tested compounds for HOꢂ is
La(III) complex > Sm(III) complex > Nd(III) complex > Dy(III)
complex > Yb(III) complex > mannitol > H4L.
H4L
Nd(III) complex
Dy(III) complex
Yb(III) complex
La(III) complex
Sm(III) complex
100
80
60
40
20
0
4. Experimental
4.1. Materials
Nitroblue tetrazolium (NBT), methionine (MET), vitamin B2
(VitB2), EB and CT DNA were purchased from Sigma Chemical Co.
Ethylenediaminetetraaceticacid (EDTA), safranin, benzoyl hydra-
zine, acetic acid, hesperetin, NaCl, potassium iodide, Ln(NO3)3$6H2O
[Ln(III) ¼ La, Sm, Dy, Yb and Nd] were produced in China. All
chemicals used were of analytical grade. EDTA–Fe(II) and KH2PO4–
Na2HPO4 buffers were prepared with doubly distilled water.
All theexperiments involved with the interaction of theligandand
its lanthanide complexes with CT DNA were carried out in doubly
distilled water buffer containing 5 mM Tris [Tris(hydroxymethyl)-
aminomethane] and 50 mM NaCl and adjusted to pH 7.1 with
1
2
3
4
5
6
7
Concentration (μM)
Fig. 12. Scavenging effect of the ligand and Ln(III) complexes on HOꢂ.

Y. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4585–4595
4593
hydrochloric acid. Solution of CT DNA in Tris–HCl buffer gave ratios of
UV absorbance of about 1.8–1.9:1 at 260 and 280 nm, indicating that
the CT DNA was sufficiently free of protein [22]. The CT DNA
concentration per nucleotide was determined spectrophotometri-
cally by employing an extinction coefficient of 6600 Mꢁ1 cmꢁ1 at
260 nm [23]. The ligand and its complexes were dissolved in a solvent
mixture of 1% DMF and 99% Tris–HCl buffer (pH 7.1) at the concen-
tration 1.0 ꢀ 10ꢁ5 M.
(cmꢁ1):
1383, 1313, 1187, 711 cmꢁ1
n
(C]O): 1608,
n
(C]N): 1569,
n
(N–H): 3382,
n
(NO3): 1479,
.
lmax (nm): 287, 330 cmꢁ1
. Lm
(S cm2 molꢁ1): 77.2. Thermal analyses: TDecomp. (ꢃC): 298, 307, 647.
Residue Calcd. (%) 14.84 (14.93). Anal Calcd. for Dy(III) complex
C46H40N7O21Dy requires: C, 46.45; H, 3.39; N, 8.24; Dy, 13.66;
Found: C, 46.54; H, 3.32; N, 8.26; Dy, 13.80. IR nmax (cmꢁ1):
n
(C]O):
(NO3): 1480, 1384, 1316, 1185,
lmax (nm): 287, 331 cmꢁ1 Lm (S cm2 molꢁ1): 88.1.
1609,
n (C]N): 1569, n (N–H): 3386, n
712 cmꢁ1
.
.
Thermal analyses: TDecomp. (ꢃC): 299, 308, 646. Residue Calcd. (%)
15.62 (15.79). Anal Calcd. for Yb(III) complex C46H40N7O21Yb
requires: C, 46.05; H, 3.36; N, 8.17; Yb, 14.42; Found: C, 46.10; H,
4.2. Instrumentation
Carbon, hydrogen and nitrogen were determined using an
Elemental Vario EL analyzer. The metal contents of the complexes
were determined by titration with EDTA (xylenol orange tetraso-
dium salt used as an indicator and hexamethylenetetramine 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 4000–400 cmꢁ1
region. 1H NMR spectra were recorded on a Varian VR 200 MHz
spectrometer in DMSO-d6 with TMS (tetramethylsilane) as internal
standard. Mass spectra were performed on an APEX II FT-ICR MS
instrument using methanol as mobile phase. All conductivity
measurements were performed in DMF solution with a DDS-11C
conductometer at 25.0 ꢃC. Thermal behavior (TG/DTA) was moni-
tored on a PCT-2 differential thermal analyzer. The UV–vis spectra
were recorded on a Shimadzu UV-240 spectrophotometer. Fluo-
rescence measurements were recorded on a Hitachi RF-4500 spec-
trofluorophotometer at room temperature. Magnetic susceptibility
was determined in deuterium oxide solution (2% t-butyl alcohol) at
305 K by JEOL PMX-60SI NMR spectrometer (Japan) according to the
literature method [49]. The antioxidant activity was performed in
DMF solution with a 721-E spectrophotometer.
3.27; N, 8.25; Yb, 14.39. IR nmax (cmꢁ1):
1571, (N–H): 3382,
(nm): 289, 336 cmꢁ1
n
(C]O): 1610,
(NO3): 1480, 1383, 1321, 1185, 712 cmꢁ1
Lm (S cm2 molꢁ1): 73.9. Thermal analyses:
n
(C]N):
n
n
.
lmax
.
TDecomp. (ꢃC): 298, 308, 646. Residue Calcd. (%) 16.21 (16.35). Anal
Calcd. for Nd(III) complex C46H40N7O21Nd requires: C, 47.18; H,
3.44; N, 8.37; Nd, 12.32; Found: C, 47.30; H, 3.38; N, 8.42; Nd, 12.24.
IR nmax (cmꢁ1):
n (C]O): 1610, n (C]N): 1570, n (N–H): 3390, n
(NO3): 1479, 1386, 1316, 1184, 709 cmꢁ1 lmax (nm): 291, 343 cmꢁ1
. .
Lm (S cm2 molꢁ1): 84.9. Thermal analyses: TDecomp. (ꢃC): 298, 308,
646. Residue Calcd. (%) 14.03 (14.20).
4.5. Electronic absorption titration
Electronic absorption titration experiments were performed
with fixed concentration drugs (10 mM), while gradually increasing
the concentration of CT DNA. When measuring the absorption
spectra, an equal amount of CT DNA was added to both the complex
solutions and the reference solution to eliminate the absorbance of
CT DNA itself. Each sample solution was scanned in the range of
190–500 nm.
4.6. Fluorescence spectra
4.3. Preparation of ligand (H4L)
To compare quantitatively the affinity of the compounds bound
to DNA, Kb values of the compounds to DNA were obtained by the
luminescence titration method. Fixed amounts of compound were
titrated with increasing amounts of DNA, over a range of DNA
Scheme of the synthesis of the ligand is shown in Fig. 1. Hes-
peretin (3.02 g, 10 mmol) and benzoyl hydrazine (1.50 g, 11 mmol)
were dissolved in methanol (40 mL). Acetic acid (1.0 mL) was added
to this solution and the solution was refluxed on a water bath for
one day with stirring. After that, the brown solution was concen-
trated and cooled, a grey solid separating out and the grey
precipitate was filtered, washed with massive water, and recrys-
tallized from DMF and water to give the ligand. Yield: 72.14%. Mp
264–266 ꢃC. Anal Calcd. for C23H20N2O6 requires C, 65.71; H, 4.79;
concentrations from 2.5 to 15 mM. Excitation wave lengths of
331 nm for Nd(III) complex and 323 nm for H4L were used, and
fluorescence emission intensities were monitored at 443 and
440 nm, respectively. The concentration of the bound compound
was calculated using equation [44].
ÂÀ
ÁꢀÀ
ÁÃ
Cb ¼ Ct F ꢁ F0
Fmax ꢁ F0
;
N, 6.66. Found: C, 65.51; H, 4.92; N, 6.76. IR nmax (cmꢁ1):
1642, (C]N): 1605, lmax (nm): 328, 285 nm.
(N–H): 3362 cmꢁ1
n (C]O):
where Ct is the total compound concentration, F is the observed
fluorescence emission intensity at given DNA concentration, F0 is
the intensity in the absence of DNA, and Fmax is the fluorescence of
the totally bound compound. Binding data were cast into the form
of a Scatchard plot of r/Cf versus r, where r is the binding ratio
Cb/[DNA]t and Cf is the free compound concentration. All experi-
ments were conducted at room temperature in a buffer containing
5 mM Tris–HCl (pH 7.1) and 50 mM NaCl concentrations.
n
n
.
4.4. Preparation of Ln(III) complexes
The H4L ligand (84 mg, 0.20 mmol) was dissolved in acetone
(20 mL). After 5 min, La(III) nitrate (87 mg, 0.20 mmol) was added
quickly and the solution was refluxed on a water bath for 10 h with
stirring. A yellow precipitate, the La(III) complex, was separated
from the solution by suction filtration, purified by washing several
times with ethanol and dried for 24 h in a vacuum. The Sm(III),
Dy(III), Yb(III) and Nd(III) complexes were prepared by the same
way. Anal Calcd. for La(III) complex C46H40N7O21La requires: C,
47.39; H, 3.46; N, 8.41; La, 11.92; Found: C, 47.48; H, 3.29; N, 8.43;
Further support for the ligand and complexes binding to DNA via
intercalation is given through the emission quenching experiment.
EB is a common fluorescent probe for DNA structure and has been
employed in the examinations of the mode and process of metal
complexes binding to DNA [41]. A 2.0 mL solution of 4
mM DNA and
La, 11.94. IR nmax (cmꢁ1):
3384,
(NO3): 1479, 1384, 1314, 1189, 707 cmꢁ1
333 cmꢁ1 Lm (S cm2 molꢁ1): 82.1. Thermal analyses: TDecomp. (ꢃC):
n
(C]O): 1610,
n
(C]N): 1561,
n (N–H):
lmax (nm): 287,
0.32 M EB (at saturating binding levels) was titrated by 2.5–35 mM
m
complexes and ligand. Quenching data were analyzed according to
the Stern–Volmer equation which could be used to determine the
fluorescent quenching mechanism:
n
.
.
298, 308, 646. Residue Calcd. (%) 13.82 (13.98). Anal Calcd. for
Sm(III) complex C46H40N7O21Sm requires: C, 46.93; H, 3.42; N, 8.33;
Sm, 12.77; Found: C, 47.08; H, 3.39; N, 8.43; Sm, 12.94. IR nmax
Fo=F ¼ 1 þ Kq½Qꢄ;

4594
Y. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4585–4595
where Fo 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 quencher concentration. Plots of Fo/F versus [Q]
appear to be linear and Kq depends on temperature [50].
The solution of the tested compounds was prepared with DMF. The
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
mM safranin, 1 mL 945 mM EDTA–Fe(II), 1 mL
m
concentration: Ci(i¼1–6) ¼ 1.0, 2.0, 3.0, 4.0, 5.0, 6.0
m
4.7. Iodide quenching experiments
without the tested compound was used as the control. The reaction
mixtures were incubated at 37 ꢃC for 60 min in a water bath.
Absorbances (Ai, A0, Ac) at 520 nm were measured. The suppression
ratio for HOꢂ was calculated from the following expression:
The fluorescence quenching efficiency is evaluated by Stern–
Volmer Ksv, which varies with the experimental conditions. The
values of Ksv were used to deduce the interaction mode of the
fluorescence probe with DNA. High binding constants should
correspond to better protection by the DNA and a stronger inhibi-
tion of quenching by anionic species. Quenching plots were con-
structed according to the following Stern–Volmer equation.
Suppression ratioð%Þ ¼ ½ðAi ꢁ A0Þ=ðAc ꢁ A0Þꢄ ꢀ 100%
where Ai ¼ the absorbance in the presence of the tested compound;
A0 ¼ the absorbance in the absence of the tested compound;
Ac ¼ the absorbance in the absence of the tested compounds,
EDTA–Fe(II) and H2O2.
The antioxidant activity was expressed as the 50% inhibitory
concentration (IC50). IC50 values were calculated from regression
Â
Ã
Fo=F ¼ 1 þ Ksv Iꢁ
;
where Fo and F are the fluorescence intensity in the absence and
presence of iodide at [Iꢁ] concentration, respectively; Ksv is the
quenching constant and [Iꢁ] is the concentration of iodide. Plots of
Fo/F versus [Iꢁ] appear to be linear and Ksv was evaluated by linear
least-squares analysis of the data according to the equation [51].
lines where: x was the tested compound concentration in mM and y
was percent inhibition of the tested compounds.
Acknowledgements
This work is supported by the National Natural Science Foun-
dation of China (20475023) and Gansu NSF (0710RJZA012).
4.8. Salt effect
Fluorescence intensities were recorded in the absence and
presence of DNA in the mixture solution of each compound and
NaCl at room temperature.
References
[1] E.C. Miller, J.A. Miller, Cancer 47 (1981) 1055–1064.
[2] A.E. Friedman, C.V. Kumar, N.J. Turro, J.K. Barton, Nucleic Acids Res. 19 (1991)
2595–2602.
4.9. Viscosity measurements
[3] A.M. Pyle, T. Morri, J.K. Barton, J. Am. Chem. Soc. 112 (1990) 9432–9434.
[4] J.K. Barton, J.M. Goldberg, C.V. Kumar, N.J. Turro, J. Am. Chem. Soc. 108 (1986)
2081–2088.
[5] P.H. Proctor, E.S. Reynolds, Physiol. Chem. Phys. Med. NMR 16 (1984) 175–195.
[6] K.E. Erkkila, D.T. Odom, J.K. Barton, Chem. Rev. 99 (1999) 2777–2795.
[7] M. Mrksich, P.B. Dervan, J. Am. Chem. Soc. 115 (1993) 9892–9899.
[8] B.N. Trawick, A.T. Danihe, J.K. Bashkin, Chem. Rev. 98 (1998) 939–960.
[9] K. Dhara, J. Ratha, M. Manassero, X.Y. Wang, S. Gao, P. Banerjee, J. Inorg.
Biochem. 101 (2006) 95–103.
Viscosity experiments were conducted on an Ubbelohde
viscometer, immersed in a thermostated water bath maintained to
25.0 ꢃC. Titrations were performed for the complexes (0.5–4
m
M),
M)
versus
is
and each compound was introduced into a DNA solution (5
present in the viscometer. Data were presented as (h/h0)
the ratio of the concentration of the compound and DNA, where
m
1/3
h
[10] D.M. Kong, J. Wang, L.N. Zhu, Y.W. Jin, X.Z. Li, H.X. Shen, H.F. Mi, J. Inorg.
Biochem. 102 (2008) 824–832.
[11] F. Firdaus, K. Fatma, M. Azam, S.N. Khan, A.U. Khan, M. Shakir, Transition Met.
Chem. 33 (2008) 467–473.
the viscosity of DNA in the presence of the compound and h0 is the
viscosity of DNA alone [52,53].
[12] B.K. Kaymakcioglu, S. Rollas, Farmaco 57 (2002) 595–599.
[13] S. Ku¨ çu¨kgu¨ zel, S. Rollas, I. Ku¨çu¨kgu¨zel, M. Kiraz, Eur. J. Med. Chem. 34 (1999)
1093–1100.
[14] S.R. Zhang, A.D. Sherry, J. Solid State Chem. 171 (2003) 38–43.
[15] Z.Y. Yang, Synth. React. Inorg. Met.-Org. Chem. 30 (2000) 1265–1272.
[16] B.D. Wang, Z.Y. Yang, Q. Wang, T.K. Cai, P. Crewdson, Bioorg. Med. Chem. 14
(2006) 1880–1888.
4.10. Antioxidant activity
ꢂ
In antioxidant activity experiments the superoxide radicals (O2ꢁ
)
were produced by the system MET/VitB2/NBT [54]. The amount of
ꢂ
ꢂ
Oꢁ2 and suppression ratio for Oꢁ2 can be calculated by measuring
the absorbance at 560 nm, because NBT can be reduced quantita-
[17] B.D. Wang, Z.Y. Yang, D.D. Qin, Z.N. Chen, J. Photochem. Photobiol., A: Chem.
194 (2008) 49–58.
ꢂ
tively to blue formazan by O2ꢁ . The solution of MET, VitB2 and NBT
[18] B.D. Wang, Z.Y. Yang, T.R. Li, Bioorg. Med. Chem. 14 (2006) 6012–6021.
[19] F. Yakuphanoglu, M. Sekerci, J. Mol. Struct. 751 (2005) 200–203.
[20] T. Miyadera, E.M. Kosower, J. Med. Chem. 15 (1972) 339–340.
[21] J.V. Formica, W. Regelson, Food Chem. Toxicol. 33 (1995) 1061–1080.
[22] Y.Z. Cai, Q. Luo, M. Sun, H. Corke, Life Sci. 74 (2004) 2157–2184.
[23] K.E. Heim, A.R. Tagliaferro, D.J. Bobilya, J. Nutr. Biochem. 13 (2002) 572–584.
[24] H.C. Cooray, T. Janvilisri, H.W. Veen, S.B. Hladky, M.A. Barrand, Biochem.
Biophys. Res. Commun. 317 (2004) 269–275.
[25] F.V. So, N. Guthrie, A.F. Chambers, K.K. Carroll, Cancer Lett. 112 (1997) 127–133.
[26] Y. Mitsunaga, H. Takanaga, H. Matsuo, M. Naito, T. Tsuruo, H. Ohtani,
Y. Sawada, Eur. J. Pharmacol. 395 (2000) 193–201.
[27] K.A. Youdim, M.S. Dobbie, G. Kuhnle, A.R. Proteggente, N.J. Abbott, C. Rice-
Evans, J. Neurochem. 85 (2003) 180–192.
was prepared with 0.067 M phosphate buffer (pH ¼ 7.8) at the
condition of avoiding light. The tested compounds were dissolved
in DMF. The reaction mixture contained MET (0.01 mol Lꢁ1), NBT
(4.6 ꢀ 10ꢁ5 mol Lꢁ1), VitB2 (3.3 ꢀ10ꢁ6 mol Lꢁ1), phosphate buffer
solution (0.067 mol Lꢁ1) and the tested compound (the final
concentration: Ci(i¼1–6) ¼ 0.4, 1.0, 2.0, 4.0, 6.0, 8.0
mM). After incu-
bating at 30 ꢃC for 10 min and illuminating with a fluorescent lamp
for 3 min, the absorbance (Ai) of the samples was measured at
560 nm. The sample without the tested compound and avoiding
ꢂ
light was used as the control. The suppression ratio for Oꢁ2 was
[28] J.O. Jim, J. Brown, J. Fleming, P.R. Harrison, Biochem. Pharmacol. 66 (2003)
2075–2088.
calculated from the following expression:
[29] I. Kostova, N. Trendafilova,G. Momekov, J. TraceElem. Med. Biol. 22(2008)100–111.
[30] C.C. Bisi, O. Carugo, Inorg. Chim. Acta 159 (1989) 157–161.
[31] D.Z. Horne, P.B. Dervan, J. Am. Chem. Soc. 112 (1990) 2435–2437.
[32] J.M. Gottesfield, L. Nealy, J.W. Trauger, E.E. Baird, P.B. Dervan, Nature 387
(1997) 202–205.
[33] W.J. Geary, Coord. Chem. Rev. 7 (1971) 81–122.
[34] F. Maarchetti, C. Pettinari, R. Pettinari, D. Leonesi, A. Lorenzotti, Polyhedron 18
(1999) 3041–3050.
Suppression ratioð%Þ ¼ ½ðA0 ꢁ AiÞ=A0ꢄ ꢀ 100%
where Ai ¼ the absorbance in the presence of the ligand or its
complexes, A0 ¼ the absorbance in the absence of the ligand or its
complexes.
In antioxidant activity experiments the hydroxyl radical (HOꢂ) in
aqueous media was generated through the Fenton reaction [55].
[35] K. Narang, V.P. Singh, Transition Met. Chem. 18 (1993) 287–290.

Y. Li et al. / European Journal of Medicinal Chemistry 44 (2009) 4585–4595
4595
[36] B.D. Wang, Z.Y. Yang, D.W. Zhang, Y. Wang, Spectrochim. Acta, Part A 63 (2006)
213–219.
[47] S.S. Leonard, D. Keil, T. Mehlmanb, S. Proper, X. Shi, G.K. Harris, J. Ethno-
pharmacol. 103 (2006) 288–296.
[37] J.K. Barton, A. Danishefsky, J. Goldberg, J. Am. Chem. Soc. 106 (1984)
2172–2176.
[48] R.G. Xing, H.H. Yu, S. Liu, W.W. Zhang, Q.B. Zhang, Z.E. Li, P.C. Li, Bioorg. Med.
Chem. 13 (2005) 1387–1392.
[38] J.G. Liu, Q.L. Zhang, X.F. Shi, L.N. Ji, Inorg. Chem. 40 (2001) 5045–5050.
[39] W.D. Wilson, L. Ratmeyer, M. Zhao, L. Stekowshi, D. Boykin, Biochemistry 32
(1993) 4098–4104.
[40] H. Xu, K.C. Zheng, L.J. Lin, H. Li, Y. Gao, L.N. Ji, Inorg. Chem. 98 (2004) 87–97.
[41] Y.B. Zeng, N. Yang, W.S. Liu, N. Tang, J. Inorg. Biochem. 97 (2003) 258–264.
[42] C.V. Kumar, J.K. Barton, N.J. Turro, J. Am. Chem. Soc. 107 (1985) 5518–5523.
[43] L.S. Lerman, J. Mol. Biol. 3 (1961) 18–30.
[44] G.M. Howe, K.C. Wu, W.R. Bauer, Biochemistry 15 (1976) 4339–4346.
[45] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 32 (1993) 2573–2584.
[46] J.G.Liu,B.H.Ye, H.Li, Q.X. Zhen, L.N.Ji, Y.H. Fu,J.Inorg.Biochem.76(1999)265–271.
[49] Z.Y. Yang, X.H. Xu, R.D. Yang, Q. Li, J. Rare Earths 13 (1995) 89–93.
[50] M.R. Efink, C.A. Ghiron, Anal. Biochem. 114 (1981) 199–206.
[51] C.V. Kumar, R.S. Turner, E.H. Asuncion, J. Photochem. Photobiol., A: Chem. 74
(1993) 231–238.
[52] M. Eriksson, M. Leijon, C. Hiort, B. Norden, A. Gradsland, Biochemistry 33
(1994) 5031–5040.
[53] Y. Xiong, X.F. He, X.H. Zou, J.Z. Wu, X.M. Chen, L.N. Ji, R.H. Li, J.Y. Zhou, R.B. Yu, J.
Chem. Soc., Dalton Trans. 1 (1999) 19–24.
[54] C.C. Winterbourn, Biochem. J. 182 (1979) 625–628.
[55] C.C. Winterbourn, Biochem. J. 198 (1981) 125–131.