Synthesis, crystal structure, antioxidant activities and DNA-binding studies of the Ln(III) complexes with 7-methoxychromone-3-carbaldehyde-(4′-hydroxy) benzoyl hydrazone

Article abstract of DOI:10.1016/j.ejmech.2008.10.023

A novel chromone Schiff base, 7-methoxychromone-3-carbaldehyde-(4′-hydroxy) benzoyl hydrazone (L) and its Ln(III) complexes (Ln = La, Eu) were synthesized and characterized. The crystal structure of the La(III) complex was determined by single-crystal X-r

Full text of DOI:10.1016/j.ejmech.2008.10.023

European Journal of Medicinal Chemistry 44 (2009) 2425–2433
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, crystal structure, antioxidant activities and DNA-binding studies
of the Ln(III) complexes with 7-methoxychromone-3-carbaldehyde-(40-hydroxy)
benzoyl hydrazone
*
Qian Wang, Zheng-Yin Yang , Gao-Fei Qi, Dong-Dong Qin
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
A novel chromone Schiff base, 7-methoxychromone-3-carbaldehyde-(40-hydroxy) benzoyl hydrazone (L)
and its Ln(III) complexes (Ln ¼ La, Eu) were synthesized and characterized. The crystal structure of the
La(III) complex was determined by single-crystal X-ray diffraction: crystallized in the orthorhombic
system, space group Pbcn, Z ¼ 8, a ¼ 14.595(2) Å, b ¼ 20.055(3) Å, c ¼ 30.078(5) Å, R1 ¼0.0657. Fluores-
cence titration spectra, electronic absorption titration spectra, EB displacement and viscosity measure-
ment indicated that both the ligand and the complexes can bind to DNA via the intercalation mode, and
that the binding affinity of the La(III) complex is higher than that of the Eu(III) complex and of the ligand
(L). The antioxidant activity experiments show that these compounds also exhibit good antioxidant
Article history:
Received 2 September 2008
Received in revised form
19 October 2008
Accepted 23 October 2008
Available online 30 October 2008
Keywords:
7-Methoxychromone-3-carbaldehyde-(40-
hydroxy) benzoyl hydrazone
DNA binding
ꢀ
activities against OH and Oꢀ2ꢁ. Moreover, the Eu(III) complex exhibits characteristic fluorescence of
europium ion in different organic solvents.
Antioxidant activity
Fluorescence property
Lanthanide complex
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
and coordination chemistry [12,13]. Lanthanide complexes with
tetracycline, phenanthroline [14], adriamycin and pyridine [15–18]
Binding of small molecules with DNA has been studied exten-
sively since DNA is the material of inheritance and controls the
structure and function of cells [1]. Small molecules possessing
DNA-binding abilities include metal complexes, porphyrins, natural
antibiotics, simple aromatic hydrocarbons and some heterocyclic
cations [2–7]. The process, denoted as intercalation, can produce
deep alterations in the nucleotide secondary structure [8,9] with
major consequences for DNA replication and transcription [10].
Actually, the activity of many anticancer, antimalarial and anti-
bacterical agents, and that of aromatic carcinogens finds its origin
in intercalative interactions [11]. Therefore, in order to generate
new drugs for novel applications, many efforts have been devoted
to design and synthesize small molecules that could specifically
target polynucleotide sites or sequences through intercalating to
DNA. The tested molecules are usually concentrated on condensed
aromatic compounds, since the presence of a planar hydrophobic
residue is an essential requirement for intercalation. Moreover,
lanthanide ions are subjects of increasing interest in bioinorganic
have been synthesized as a probe to study nucleic acids.
Of particular interests, chromone derivatives which have
aromatic planar structure show wide variety of biological activities
[19–21]. Based on the above consideration, we suppose that
complexes of chromone with lanthanides could present interesting
biological activities. In our previous work, the lanthanide
complexes derived from 6-hydroxy-3-carbaldehyde chromone
Schiff base have been reported and all the complexes can strongly
interact with DNA through intercalation mode [22–24]. It is well
known that the different substitute and its different situation can
affect the pharmacological activities and DNA-binding abilities of
compound. More importantly, a recent epidemiological study
provides strong support that the methoxylated chromones have
potential cancer preventive properties [25]. In this regard, as
a further research, the Schiff base ligand, 7-methoxychromone-3-
carbaldehyde-(40-hydroxy) benzoyl hydrazone (L), and its Ln(III)
complexes (Ln ¼ La, Eu) were synthesized and their DNA-binding
modes were investigated systematically. In addition, the antioxi-
dant activities of the metal complexes were studied by the hydroxyl
radical (ꢀOH) and superoxide anion (Oꢀ2ꢁ) which are highly reactive
species implicated in cell membrane disintegration, membrane
protein damage, DNA mutation [26,27]. Furthermore, the Eu(III)
* 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 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.10.023

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Q. Wang et al. / European Journal of Medicinal Chemistry 44 (2009) 2425–2433
Table 1
complex can emit intrinsic spectra under excitation both in solid
and organic solvents. That may provide more advantages to apply
the Eu(III) complex as a candidate of anticancer drugs and nucleic
acid probe.
Crystal data and experimental data.
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
a (Å)
C39H35.31$LaN7O21
1076.96
296(2) K
0.71073 Å
Orthorhombic
Pbcn
14.595(2)
20.055(3)
30.078(5)
90
90
90
8804(2)
8
1.62
1.062
2. Results and discussion
The complexes were prepared by direct reaction of ligand with
the appropriate mole ratios of Ln(III) nitrate in ethanol. The yields
were good to moderate. The complexes were air stable for extended
periods and soluble in methanol, DMF (N,N-dimethylformamide)
and DMSO (dimethylsulfoxide); slightly soluble in ethanol,
acetone, acetonitrile, THF (tetrahydrofuran) and water; insoluble in
benzene, ethyl acetate. The elemental analyses show that formula
of the complexes in powder conform to [LnL2(NO3)3] (Ln ¼ La, Eu),
while the single-crystal structure of La(III) complex was [LaL2
(NO3)3]$C2H5OH$CH3OH according to the single-crystal X-ray
analysis.
b (Å)
c (Å)
a
b
g
(ꢂ)
(ꢂ)
(ꢂ)
Volume (Å3)
Z
Calculated density (Mg/m3)
Absorption coefficient (mmꢁ1
F(000)
)
4347
1.35–28.29
q
Range (ꢂ)
Reflections collected/unique
50,933/10,742 [Rint ¼ 0.1304]
Goodness of-fit on F2
1.015
R indices [I > 2
s(I)]
R1 ¼0.0657, wR2 ¼ 0.1241
2.1. IR spectra
and La(1)–O(9). The average Ln–O carbonyl distance (2.475 Å) was
significantly shorter than the average hydrazonic Ln–O distance
(2.564 Å), which suggested that the carbonyl bond was stronger
than the hydrazonic bond. The La–imine distances were found to be
2.841(5) and 2.951(5) Å for La(1)–N(2) and La(1)–N(4), which was
unusually longer than the other La–O bond lengths [30]. The nitrate
anion had proved to be a very useful ligand for the construction of
coordination complex. In the crystal structure of the La(III)
complex, three nitrates had two coordination types, monodentate
and bidentate; the monodentate nitrate bond lengths of La–O(11)
and La–O(14) were 2.648(5) and 2.625(5) Å, respectively, while the
bidentate nitrate bond lengths of La–O(17) and La–O(18) were
2.683(5) and 2.591(5) Å, respectively. In [LaL2(NO3)3]$C2H5OH$-
CH3OH complex, the bond lengths of C(8)–N(2) and C(26)–N(4)
(mean 1.286(7) and 1.275(7) Å, respectively) were consistent with
double bond while bond lengths of C(7)–N(1) and C(25)–N(3)
(mean 1.369(7) and 1.350(7) Å, respectively) were corresponding to
a single bond. Take the crystal structure of the [LaL02(CH3OH)(-
H2O)(NO3)]$[NO3]$H2O (L02 ¼ 6-hydroxychromone-3-carbaldehyde
benzoyl hydrazone) [24] for comparison, observe their construc-
tions of coordination in the two crystal structures, both La(III) ions
were ten coordinated and both the structures have two tridentate
ligands. However, in [LaL2(NO3)3]$C2H5OH$CH3OH, all the three
nitrates took part in coordination, while in [LaL02(CH3OH)(H2O)(-
NO3)]$[NO3]$H2O only one nitrate took part in coordination. The
reason may be the difference of substitute and its situation.
Since the characteristic absorption peaks of the La(III) complex
and the Eu(III) complex were very similar, the structure of the
Eu(III) complex could be talked about in the same way. Take the
La(III) complex for example, in the La(III) complex, n(hydrazonic) C]O
n(carbonyl) C]O
nC]N vibrations were at 1667, 1626 and 1613 cmꢁ1
respectively, while for the free ligand, these peaks were at 1643,
1623 and 1589 cmꢁ1, and
,
,
,
n
(ligand ꢁ complex) was equal to 24, 3
and 24 cmꢁ1. These shifts demonstrated that the above three
groups have taken part in coordination to La(III) ion. The absorption
bands of the coordinated nitrates were observed at about 1452,
1289, 1022, 783, 733 cmꢁ1 suggesting that the nitrate group took
part in coordination [28,29]. The above speculations were consis-
tent with the crystal structure of the La(III) complex by X-ray
analysis.
2.2. Crystal structure of the La(III) complex
On the basis of the single-crystal X-ray analysis of the complex
[LaL2(NO3)3]$C2H5OH$CH3OH, each La(III) ion was deca-coordi-
nated by two tridentate ligand, two monodentate nitrate and one
bidentate nitrate. The complex crystallized in the orthorhombic
lattice with a space group Pbcn. Each unit cell contained eight
molecules. The crystal structure is shown in Fig. 1. The X-ray
diffraction data for the La(III) complex are given in Table 1 and the
selected bond lengths and angles are summarized in Table 2.
The La(1)–O(2) and La(1)–O(7) hydrazonic interactions were
found to be 2.577(4) and 2.541(3) Å, respectively, the carbonyl
distances were found to be 2.501(4) and 2.449(4) Å for La(1)–O(4)
2.3. DNA-binding studies
2.3.1. Fluorescence spectra
The fluorescence titration spectra have been confirmed to be
effective for characterizing the binding mode of the metal
complexes to DNA [31,32]. The enhancements in the emission
intensity of the ligand and its Ln(III) complexes (Ln ¼ La, Eu) with
increasing CT-DNA concentrations are shown in Fig. 2. The ligand
and its Ln(III) complexes (Ln ¼ La, Eu) could emit luminescence in
Tris–HCl buffer with
a
maximum appearing at 438 nm
(lex ¼ 360 nm). This phenomenon was related to the extent to
which the compounds penetrate into the hydrophobic environment
inside the DNA, thereby avoiding the quenching effect of solvent
water molecules. The marked increase in emission intensity of these
three compounds were also in accordance with those observed in
the fluorescence titration spectra studies of other intercalators
[33,34]. Consequently, it might be concluded that the ligand and the
La(III) and Eu(III) complexes bound DNA via intercalation mode and
Fig. 1. The structure of the La(III) complex.

Q. Wang et al. / European Journal of Medicinal Chemistry 44 (2009) 2425–2433
2427
Table 2
Selected bond lengths (Å) and angles (ꢂ) for [LaL2(NO3)3]$C2H5OH$CH3OH complex.
Selected bond
Bond length or angle
Selected bond
Bond length or angle
Selected bond
Bond length or angle
La(1)–O(2)
La(1)–O(9)
La(1)–O(17)
La(1)–N(4)
2.577(4)
2.449(4)
2.683(5)
2.951(5)
1.429(9)
La(1)–O(4)
La(1)–O(11)
La(1)–O(18)
C(7)–O(2)
2.501(4)
2.648(5)
2.591(5)
1.228(6)
1.225(6)
La(1)–O(7)
La(1)–O(14)
La(1)–N(2)
C(13)–O(4)
2.541(4)
2.625(5)
2.841(5)
1.239(6)
1.245(7)
C(18)–O(5)
C(25)–O(7)
C(31)–O(9)
C(36)–O(10)
1.406(9)
C(7)–N(1)
1.369(7)
C(8)–N(2)
1.286(7)
C(25)–N(3)
1.350(7)
C(26)–N(4)
1.275(7)
N(1)–N(2)
1.376(6)
N(3)–N(4)
1.395(6)
N(5)–O(11)
1.249(7)
N(5)–O(12)
1.223(6)
N(5)–O(13)
1.220(6)
N(6)–O(14)
1.278(7)
N(6)–O(15)
1.232(7)
N(6)–O(16)
1.220(7)
N(7)–O(17)
1.245(7)
N(7)–O(18)
1.287(7)
N(7)–O(19)
1.206(7)
O(9)–La(1)–O(4)
O(9)–La(1)–O(2)
O(9)–La(1)–O(18)
O(2)–La(1)–O(18)
O(7)–La(1)–O(14)
O(9)–La(1)–O(11)
O(2)–La(1)–O(11)
O(9)–La(1)–O(17)
O(2)–La(1)–O(17)
O(11)–La(1)–O(17)
O(7)–La(1)–N(2)
O(14)–La(1)–N(2)
O(9)–La(1)–N(4)
O(2)–La(1)–N(4)
O(11)–La(1)–N(4)
165.53(16)
67.22(13)
70.95(16)
72.64(14)
148.98(15)
90.76(16)
146.25(15)
116.11(16)
77.13(14)
136.62(14)
63.85(10)
101.16(11)
116.16(10)
65.91(8)
O(9)–La(1)–O(7)
O(4)–La(1)–O(2)
O(4)–La(1)–O(18)
O(9)–La(1)–O(14)
O(2)–La(1)–O(14)
O(4)–La(1)–O(11)
O(18)–La(1)–O(11)
O(4)–La(1)–O(17)
O(18)–La(1)–O(17)
O(9)–La(1)–N(2)
O(2)–La(1)–N(2)
O(11)–La(1)–N(2)
O(4)–La(1)–N(4)
O(18)–La(1)–N(4)
O(17)–La(1)–N(4)
115.73(13)
121.00(13)
121.89(15)
84.42(16)
67.58(15)
76.29(15)
125.62(15)
78.20(14)
48.08(14)
74.09(11)
126.51(9)
67.86(8)
O(4)–La(1)–O(7)
O(7)–La(1)–O(2)
O(7)–La(1)–O(18)
O(4)–La(1)–O(14)
O(18)–La(1)–O(14)
O(7)–La(1)–O(11)
O(14)–La(1)–O(11)
O(7)–La(1)–O(17)
O(14)–La(1)–O(17)
O(4)–La(1)–N(2)
O(18)–La(1)–N(2)
O(17)–La(1)–N(2)
O(7)–La(1)–N(4)
O(14)–La(1)–N(4)
N(2)–La(1)–N(4)
66.59(14)
140.55(15)
72.00(14)
88.15(15)
138.92(15)
71.41(14)
85.77(15)
66.41(14)
127.81(15)
83.40(10)
72.69(10)
168.15(10)
115.31(9)
71.13(10)
49.34(9)
158.40(10)
118.57(10)
125.78(8)
69.63(9)
according to the previous literature [22–24], we can also find that
the fluorescence spectra of other Ln(III) complexes reported by
Wang [22–24] do not differ significantly with the substitutions on
the chromone hydrazone ligand.
compound at the DNA/compounds molar ratios varying from0 to3.5
induced large hypochromic responses about 39.3% at 295 nm for
ligand, 66.6% at 296 nm for La(III) complex and 60.9% at 296 nm for
Eu(III) complex, respectively, and the bathochromism responses
about 8, 8, 10 nm. The phenomenon of large hypochromism and
appreciable bathochromism which were similar to our previously
reported complexes [22–24] were both attributed to the strong
stacking interaction between aromatic group and the base pairs of
CT-DNA, when the compound intercalate to the CT-DNA [37]. The
spectroscopic changes suggested that both the ligand and the
complexes interacted with DNA most likely through an intercalation
2.3.2. Electronic absorption titration
Electronic absorption spectroscopy is one of the most useful
techniques for DNA-binding studies of metal complexes [35,36]. The
absorption spectra of the ligand and its Ln(III) complexes (Ln ¼ La,
Eu) in presence of CT-DNA are given in Fig. 3(a), (b) and (c),
respectively. The addition of CT-DNA to the solutions of the
the La(III) complex
225
the ligand
60
150
75
0
30
0
400
450
500
550
400
450
500
550
Wavelength /nm
Wavelength /nm
the Eu(III) complex
200
150
100
50
0
400
450
500
550
Wavelength /nm
Fig. 2. Emission enhancement spectra of the ligand, the La(III) and Eu(III) complexes in the presence of increasing amounts of CT-DNA.

2428
Q. Wang et al. / European Journal of Medicinal Chemistry 44 (2009) 2425–2433
0.6
the ligand
the La(III) complex
0.10
0.05
0.00
a
b
0.4
0.2
0.0
250
300
350
400
250
300
350
400
Wavelength /nm
Wavelength /nm
1
the Eu (III) complex
the La(III) complex
Kb= 3.54×105M-1
c
d
0.6
0.4
0.2
0.0
0.8
0.6
0.4
0.2
0
250
300
350
400
0
10
20
30
40
Wavelength /nm
[DNA]×106
Fig. 3. Electronic absorption spectra of (a) the ligand, (b) the La(III) complex, (c) the Eu(III) complex in the presence of increasing amounts of CT-DNA. (d) Plot of [DNA]/(3a
versus [DNA] for the titration of the La(III) complex with CT-DNA.
ꢁ
3f)
mode. To further illustrate the binding affinity of the compounds
quantitatively, the intrinsic DNA-binding constants Kb of the
compounds were determined by monitoring the changes in absor-
bance at 300 nm with increasing concentrations of DNA, according
to Eq. (1). As an example of the La(III) complex, Fig. 3(d) showed the
mathematic method of evaluating Kb value, which was calculated
DNA [44–46]. Therefore, the binding of the compounds in title were
evaluated using competitive binding studies involving EB. Fig. 4(a–
c) showed the emission spectra of DNA–EB system with increasing
amounts of the ligand, the La(III) and Eu(III) complex. It was clear
that the fluorescence intensity of DNA–EB system greatly decreases
upon the addition of the ligand, La(III) and Eu(III) complex and
there were more significant decreases (>50%) in the emission
spectra of DNA–EB system of the La(III) and Eu(III) complex. These
results indicated that EB was partially replaced by the compounds
intercalate into the DNA. As an example of the La(III) complex,
Fig. 4(d) showed the mathematic method of evaluating Kq (the
quenching constant) value, which was calculated from the plot of
F0/F versus [Q]. According to linear Stern–Volmer equation, Kq was
given by the ratio of the slope to the intercept. The Kq values for the
ligand, the La(III) and Eu(III) complexes were 2.81 ꢃ104, 7.02 ꢃ 104
and 5.55 ꢃ104 Mꢁ1, respectively. These data suggested that the
interaction of the La(III) complexes with CT-DNA was strongest and
the interaction of the Eu(III) complexes with CT-DNA was stronger
then that of the free ligand, which were consistent with the above
absorption spectral results. Compare their Kq values with those of
other known DNA-intercalative complexes which possess analog-
ical structure, [LaL02(CH3OH)(H2O)(NO3)][NO3]$H2O 5.28 ꢃ 104
from the plot of [DNA]/(3a
ꢁ
3f) versus [DNA]. Take this method, the
intrinsic DNA-binding constants Kb were found to be the La(III)
complex, 3.54 ꢃ105 Mꢁ1; the Eu(III) complex, 5.11 ꢃ104 Mꢁ1; the
ligand, 3.89 ꢃ 104 Mꢁ1, in the order of the La(III) complex > the
Eu(III) complex > the ligand.
Furthermore, comparing the Kb values of the compounds in title
with those of other known DNA-intercalative complexes, (for
instance, [(bpy)2Ru(
m
-bipp)Ru(bpy)2](ClO4)4, 2.6 ꢃ 105 Mꢁ1 [38];
[Ru(NH3)4dppz]2þ, 1.24 ꢃ105 [39]; [Ru(phen)2(PPIP)]2þ, 1.1 ꢃ105;
[Ru(bpy)2(PPIP)]2þ, 4.3 ꢃ104 [40]; [Pd2(
-bzta)4]$1.5DMSO, 1.2 ꢃ
m
104 [41]; [Sr(Phen)2(DMF)2] [Fe(NO)(CN)5], 1.6 ꢃ 104 [42]; [Ru
(dmb)2(NMIP)]2þ, 5.46 ꢃ 103 Mꢁ1; [Ru(bpy)2(NMIP)]2þ, 1.15 ꢃ104
M
ꢁ1 [43]), it was obviously that the Kb value of the La(III) complex
was much higher than all of the above DNA intercalators. However,
the Kb values of the Eu(III) complex and the ligand were only higher
than some of them. In spite of that, the results implied that the
three compounds had good ability to bind to DNA.
Mꢁ1; [SmL02(NO3)2]$[NO3]$0.5H2O$2CH3OH 1.37 ꢃ 104 Mꢁ1 (L02
¼
6-hydroxychromone-3-carbaldehyde benzoyl hydrazone) [24], the
La(III) and Eu(III) complexes in our paper have stronger affinities
with CT-DNA. The reason may be the difference of substitute and its
situation.
2.3.3. EB displacement experiment
The results obtained from absorption titration and spectro-
fluorimetric titration experiments indicated that the ligand, the
La(III) and Eu(III) complexes could effectively bind to DNA. In order
to confirm the binding mode and compare their binding affinities,
EB displacement experiment was carried out. EB (ethidium
bromide) is a conjugate planar molecule. Its fluorescence intensity
is very weak, but it is greatly increased when EB is specifically
intercalated into the base pairs of double stranded DNA [1]. In
previous studies, the fluorescent light could be quenched by the
addition of the complex which can compete with EB to bind with
DNA. This is a proof that the complexes intercalate to base pairs of
2.3.4. Viscosity measurement
Optical photophysical probes provide necessary but not suffi-
cient clues to support a binding mode and viscosity experiment is
considered as one of the least ambiguous and the most critical tests
of the binding modes of complexes to DNA [47]. Therefore, the
viscosity experiments were performed to investigate the binding
mode of these complexes to DNA. In general, a classical intercala-
tion mode might increase the relative viscosity of DNA, because the

Q. Wang et al. / European Journal of Medicinal Chemistry 44 (2009) 2425–2433
2429
150
120
90
60
30
0
the ligand
the La(III) complex
a
b
120
90
60
30
0
550
575
600
625
550
575
600
625
Wavelength /nm
Wavelength /nm
c
2.4
2.2
2
the Eu(III) complex
the La(III) complex
d
120
90
60
30
0
K
= 7.02×104
q
1.8
1.6
1.4
1.2
1
550
575
600
625
0
5
10
20
25
Wavelength /nm
Concentration (μM)
Fig. 4. The emission spectra of DNA–EB system in the presence of (a) the ligand, (b) the La(III) complex, (c) the Eu(III) complex. (d) Plot of F0/F versus [Q] for the titration of the La(III)
complex with DNA–EB.
binding compounds would separate base pairs of double helix DNA,
and thus lengthened the DNA helix. On the contrary, a partial and/
or non-classical intercalation of compounds would reduce the
relative viscosity of DNA, since the binding ligand might bent (or
kinked) the DNA helix and reduce its effective length [48]. As
shown in Fig. 5, the specific viscosity of the DNA sample increased
obviously with the addition of the compounds, which provided
a further support that the binding modes of ligand and the
complexes were intercalative in the interactions with CT-DNA.
chromones have attracted increased interest and consequently
have been extensively investigated, mainly in the in vitro systems
[49,50]. It has been reported that their free radical-scavenging
ability is excellently directed toward hydroxyl radical (ꢀOH) [51] and
superoxide radical (Oꢀ2ꢁ) [52,53] 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 [54]. Consequently, in
this paper, the ligand and its Ln(III) complexes (Ln ¼ La, Eu) studied
for its antioxidant activity by comparing their scavenging effect on
hydroxyl radical (ꢀOH) and superoxide radical (Oꢀ2ꢁ).
2.4. Antioxidant activity
As shown in Fig. 6(a) and (b), the inhibitory effect of the tested
ꢀ
compounds on OH and Oꢀ2ꢁ was concentration related and the
Since the ligand and its lanthanide complexes exhibit good
DNA-binding affinity, it is considered worthwhile to study other
potential aspects of these compounds, such as antibacterial, anti-
oxidant and antiradical activity. The antiradical properties of
suppression ratio increased with the increasing of sample
concentration in the range of 1.0–8.0 mM. The suppression ratio
against ꢀOH valued from 4.85 ꢄ1.63% to 30.30 ꢄ 1.13% for the ligand,
6.17 ꢄ 2.06% to 47.31 ꢄ1.87% for the La(III) complex and
16.17 ꢄ 0.99% to 50.82 ꢄ1.56% for the Eu(III) complex. Mannitol is
nearly no scavenging effect at the same concentration lift. Mannitol
was employed as a standard since it is known to selectively inhibit
the ligand
1.6
the La(III) complex
ꢀ
the OH radical [55,56]. According to previous literature [57], the
the Eu(III) complex
1.5
experiment was carried out again at higher concentration. The
suppression ratio of mannitol was valued from 24.10 ꢄ 0.95% to
71.79 ꢄ 1.78% at a concentration from 5 to 40
mM. The results clearly
1.4
1.3
1.2
suggest that the ligand and its the La(III) and Eu(III) complex have
stronger scavenging activity against hydroxyl radical than
mannitol. The suppression ratio against Oꢀ2ꢁ valued from about
1.98 ꢄ 1.54% to 17.39 ꢄ 1.52% for ligand, 10.08 ꢄ 1.92% to
45.38 ꢄ 0.99% for La(III) complex and 5.93 ꢄ1.09% to 33.20 ꢄ 1.42%
for the Eu(III) complex. The results show that the three compounds’
ability against superoxide radical is significantly higher than that of
nitroxide Tempo which has been recently used in biological system
for its capacity to mimic superoxide dismutase [58].
1.1
1
0
0.2
0.4
0.6
0.8
It could be seen clearly that the Eu(III) complex was the most
efficient scavenger against ꢀOH, and the suppression ratio took the
order of the Eu(III) complex > the La(III) complex > the ligand at
the same concentration. However, in the experiment of
[Compounds]/[DNA]
Fig. 5. Effect of increasing amounts of the ligand, the La(III) and Eu(III) complexes on
the relative viscosity of CT-DNA at 25.0 ꢂC.

2430
Q. Wang et al. / European Journal of Medicinal Chemistry 44 (2009) 2425–2433
60
50
40
30
20
10
0
7F2
→
600
the ligand
the La(III) complex
the Eu(III) complex
5D0
a
a
400
200
0
5D0
7F1
→
5D0
7F0
→
7F3
5D0
→
550
600
650
0
2
4
6
8
Wavelength /nm
Concentration (μM)
50
40
30
20
10
0
7F2
5D0
b
→
the ligand
b
100
50
0
1
the La(III) complex
the Eu(III) complex
2
5D0
7F1
→
3
4
5
600
625
Wavelength /nm
0
2
4
6
8
Concentration (μM)
Fig. 7. (a) The fluorescence emission spectrum of the Eu(III) complex in solid state at
room temperature; (b) the fluorescence emission spectrum of the Eu(III) complex in
different solutions (1, ethanol; 2, acetone; 3, methanol; 4, cyclohexane; 5, THF).
Fig. 6. (a) Scavenging effect of the tested compounds on ꢀOH by the Fenton system; (b)
scavenging effect of the tested compounds on Oꢀ2ꢁ by MET/VitB2/NBT system.
investigating superoxide radical (Oꢀ2ꢁ) scavenging activity, the
La(III) complex showed excellent activity. The order of the
suppression ratio for Oꢀ2ꢁ was the La(III) complex > the Eu(III)
complex > the ligand. The results suggested that the scavenging
effect on ꢀOH and Oꢀ2ꢁ could be enhanced by the formation of metal–
ligand coordination complexes and the lanthanide ions had
differential and selective nature for scavenging Oꢀ2ꢁ and ꢀOH. It was
believed that the information obtained from the present work
would ultimately be helpful to develop new potential antioxidants
and new therapeutic reagents for some diseases.
the excitation and emission slit widths were 5.0 nm, Fig. 7(b)) were
recorded at room temperature.
It could be seen from Fig. 7(a) that the Eu(III) complex showed
strong emission when excited with 349 nm in the solid state. This
indicated that the ligand in title was a good organic chelator to
absorb energy and transfer it to Eu(III) ion. The most intensity ratio
value
h
(5D0 / 7F1/5D0 / 7F2) was 2.9, indicating a low symmetry
for the Eu(III) site [61]. The result was in agreement with the crystal
structure analysis.
As shown in Fig. 7(b), the emission spectra of the Eu(III) complex
in different solvents were similar. They all showed characteristic
emission bands of an Eu(III) ion at about 590 and 614 nm, corre-
sponding to 5D0 / 7F1 and 5D0 / 7F2 transitions, respectively.
From Fig. 7(b), it could be found that the Eu(III) complex had the
strongest fluorescence in ethanol, and then in acetone, methanol,
cyclohexane and THF. This was probably due to the coordinating
effects of solvents, namely solvate effect [62]. The energy transfer
from the triplet excited state of the ligand to the emitting level of
the europium ion could not be carried out perfectly because the
oscillatory motions of the entering solvent molecules may consume
more energy. It was clear that excellent luminescence properties
made the Eu(III) complex to be a good candidate for biological use.
2.5. Fluorescence studies
As shown by DNA-binding and antioxidant activity studies, the
lanthanide complexes in title possess outstanding biological
properties. Furthermore, luminescence of several Ln(III) ions has
been a useful probe of metal ion binding sites in biomolecules. The
Eu(III) ion, in particular, is valuable for use in determination of
specific features of binding, since the non-degenerate 5D0 excited
state results in relatively simple emission spectra [59]. Excitation of
the 7F0 / 5D0 transition results in a characteristic and unique
fluorescence in the red, with its strongest lines associated with
5D0 / 7FJ transitions at 580, 593, 614, 650 and 700 nm, for J ¼ 0–4.
The hypersensitive 5D0 / 7F2 transition at 614 nm is typically
monitored because each chemically distinct Eu(III) species gives
rise to a unique excitation spectrum, the intensity of which is
directly related to its concentration [60].
3. Conclusion
It is well documented that 7-methoxychromone-3-carbalde-
hyde-(40-hydroxy) benzoyl hydrazone (Schiff base ligand, L) and its
Ln(III) complexes (Ln ¼ La, Eu) can interact with calf thymus DNA by
intercalation mechanism, and the La(III) complex has a greater
affinity. Additionally, the ligand and its Ln(III) complexes (Ln ¼ La,
The emission spectra of the Eu(III) complexes in solid state (the
excitation and emission slit widths were 3.0 nm, Fig. 7(a)) and in
several organic solvents solutions (concentration: 1.0 ꢃ 10ꢁ4 mol Lꢁ1
,

Q. Wang et al. / European Journal of Medicinal Chemistry 44 (2009) 2425–2433
2431
Eu) also show antioxidant activities. In particular, it is observed the
4.3. Preparation of the ligand and its complexes
4.3.1. Synthesis of the ligand
ligand is a good organic chromophore to absorb and transfer energy
to Eu(III) ion. The outstanding luminescence properties of the
Eu(III) complex provide more advantages to apply it in probing
metal-binding properties of some biomolecules. These results
suggest that the combination of a chromone and a hydrazone has
formed a good ligand. It is not only a good ligand to form biological
lanthanide complexes but also a good organic chromophore to
absorb and transfer energy to Eu(III) ions.
As shown in Scheme 1, Compound 1 and Compound 2 were
prepared according to the literature [65]. Synthesis of the ligand
was based on the following method: an ethanol solution (25 ml)
which contained 4-hydroxy benzoyl hydrazine (0.76 g, 5 mmol)
was added dropwise to compound 2 (1.02 g, 5 mmol) of chloroform
solution (10 ml). After stirring at 70–80 ꢂC for 6 h, the light yellow
precipitates were collected then recrystallized from 1:1 (V/V) DMF/
H2O. Yield: 80%. 1H NMR (DMSO-d6, ppm):
d 11.72 (1H, s, –NH),
10.12 (1H, s, –OH), 8.72 (1H, s, 2-H), 8.56 (1H, s, CH]N), 8.02 (1H, d,
J ¼ 8.9 Hz, H-5), 7.81 (2H, d, J ¼ 8.7 Hz, ph-H(2,4)), 7.21 (1H, d,
J ¼ 2.3 Hz, 8-H), 7.11 (1H, dd, J ¼ 2.3 and J ¼ 8.9 Hz, H-6), 6.84 (2H, d,
J ¼ 8.7 Hz, ph-H(1,5)), 3.90 (3H, s, –CH3). The labelled numbers of H-
4. Experimental
4.1. Instrumentation
atoms are shown in Fig. 7. IR (KBr) cmꢁ1
: n(hydrazonic) C]O: 1667;
The metal contents of the complexes were determined by
titration with EDTA. Elemental analyses were carried out on an
Elemental Vario EL analyzer. The IR spectra were obtained in KBr
discs on a Therrno Mattson FTIR spectrometer in the 4000–
400 cmꢁ1 region.1H NMR spectra were recorded on a Bruker Avance
Drx 200-MHz spectrometer with TMS as an internal standard. The
melting points of the compounds were determined on a Beijing
XT4-100ꢃ microscopic melting point apparatus. Fluorescence
spectra were obtained on a Shimadzu RF-5301 spectrophotometer
at room temperature. The UV–vis spectra were recorded on a Per-
kin–Elmer Lambda-35 UV–vis spectrophotometer. The antioxidant
activities were performed in DMF with a 721E spectrophotometer
(Shanghai Analytical Instrument factory China).
n(carbonyl)C]O: 1626; nC]N: 1613.
4.3.2. Synthesis of lanthanide complexes
The ligand (1 mmol, 0.34 g) was dissolved in anhydrous ethanol
(20 ml) and another ethanol solution (10 ml) containing La(N-
O3)3$6H2O (1 mmol, 0.45 g) was then added dropwise with stirring.
Then the mixture solution was refluxed on an oil-bath at 80 ꢂC for
4 h with stirring. After cooling to room temperature, a large amount
of light yellow precipitate appeared. It was separated from the
solution by suction filtration, purified by washing several times
with ethanol, and dried for 48 h in vacuum. The Eu(III) complex was
prepared by the same method.
[LaL2(NO3)3]. Yield: 50%. IR (KBr) cmꢁ1
: n(hydrazonic) C]O: 1643;
n(carbonyl)C]O: 1623; nC]N: 1589; nC]N: 1589; nNO : 1452, 1289, 1022,
3
783, 733. Anal. Calcd for C28H36N7O21La: C, 43.17; H, 2.82; N, 9.79;
La, 13.87. Found: C, 43.36; H, 3.029; N, 9.680; La, 13.53.
4.2. Materials
[EuL2(NO3)3]. Yield: 55%. IR (KBr) cmꢁ1
: n(hydrazonic) C]O: 1643;
CT-DNA (Calf thymus DNA), ethidium bromide (EB), NBT
(nitroblue tetrazolium), MET (methionine), and VitB2 (vitamin B2)
were purchased from Sigma Chemical Co. EDTA, Safranin, BF3$Et2O,
POCl3, and Ln(NO3)3$6H2O (Ln ¼ La, Eu) were produced in China. All
the chemicals used were of analytical grade. Tris–HCl buffer,
KH2PO4–Na2HPO4 buffers and EDTA–Fe(II) were prepared with
twice distilled water.
n(carbonyl)C]O: 1623; nC]N: 1572; nNO : 1452, 1287, 1024, 781, 748.
3
Anal. Calcd for C21H25N6O15Eu: C, 42.62; H, 2.78; N, 9.66; Eu, 14.98.
Found: C, 42.71; H, 3.104; N, 9.533; Eu, 14.62.
4.4. X-ray crystallography
All the experiments involving the interaction of the ligand and
the complexes with CT-DNA were carried out in doubly distilled
water buffer containing 5 mM Tris and 50 mM NaCl and adjusted to
pH ¼ 7.1 with hydrochloric acid. Solution of CT-DNA gave ratios of
absorbance at 260 and 280 nm of about 1.8–1.9, indicating that the
CT-DNA was sufficiently free of protein [63,64]. The CT-DNA
concentration per nucleotide was determined spectrophotometri-
cally by employing an extinction coefficient of 6600 Mꢁ1 cmꢁ1 at
260 nm.
A yellow crystal of [LaL2(NO3)3]$C2H5OH$CH3OH obtained from
a methanol and ethanol mixture solution at room temperature of
dimensions 0.23 ꢃ 0.25 ꢃ 0.29 mm was centered on a Bruker
Smart-1000 CCD diffractometer operating in
u/2q scan mode with
graphite monochromated Mo K
a radiation (0.71073 Å). The struc-
ture 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
H3CO
OH
F
H3CO
O
B
O
F
POCl3
DMF
F3BH Et2O (CH3CO)2O
100 °C, 1h
O
100 °C, 1h cool and pour
into ice water
Compound 1
O
1
8
CH3O
O
O
H3CO
CNHNH2
9
OH
2
7
6
1'
2'
4'
4
70-80°C, 1h
3'
3
CH
CHO
10
NNHC
O
OH
5
O
O
5'
Compound 2
The ligand
Scheme 1. The preparation of the ligand.

2432
Q. Wang et al. / European Journal of Medicinal Chemistry 44 (2009) 2425–2433
non-hydrogen atoms were refined on F2 by full-matrix least-
squares techniques with the SHELX-97 program package. Absorp-
tion correction was employed using semi-empirical methods from
equivalents.
CT-DNA solution (5.0 mM) present in the viscometer. Data were
1/3
presented as (
compound and CT-DNA, where
h
/
h0
)
versus the ratio of the concentration of the
was the viscosity of CT-DNA in the
h
presence of the compound and h0 was the viscosity of CT-DNA
alone [68,69].
4.5. DNA-binding study methods
4.6. Scavenger measurements of hydroxyl radical (ꢀOH) and
superoxide radical (O2ꢀꢁ
)
4.5.1. Fluorescence spectroscopy
The ligand and the complexes were dissolved in a mixture
solvent of 1% methanol and 99% Tris–HCl buffer (5 mM Tris–HCl;
50 mM NaCl, pH ¼ 7.1). The example solution of fixed amounts
The hydroxyl radical (ꢀOH) in aqueous media was generated by
the Fenton system [34]. The solution of the tested compound was
prepared with DMF (N,N-dimethylformamide). The 5 ml assay
compounds (2 mM) were titrated with increasing amounts of DNA,
mixture contained following reagents: safranin (11.4
mM), EDTA–
over a range of DNA concentrations from 0.5 to 6.0
tion wavelength of 360 nm was used.
mM. An excita-
Fe(II) (40 M), H2O2 (17.6 M), the tested compound (1.0–8.0 m
m
m
M)
and a phosphate buffer (67 mM, pH ¼ 7.4). The assay mixtures were
incubated at 37 ꢂC for 30 min in a water bath. After which, the
absorbance was measured at 520 nm. All the tests were run in
triplicate and expressed as the mean and (ꢄ) standard deviation
(S.D.). Ai was the absorbance in the presence of the tested
compound; A0 was the absorbance in the absence of the tested
compound; Ac was the absorbance in the absence of the tested
compound, EDTA–Fe(II), H2O2. The suppression ratio (ha) was
calculated on the basis of (Ai ꢁ A0)/(Ac ꢁ A0) ꢃ 100%.
4.5.2. Electronic absorption spectroscopy
In order to affirm quantitatively the affinity of the compounds
binding to DNA, the intrinsic binding constant Kb of the ligand and
its Ln(III) complexes (Ln ¼ La, Eu) were obtained by the electronic
absorption spectroscopy method. The ligand and the complexes
were dissolved in a mixture solvent of 1% methanol and 99% Tris–
HCl buffer (5 mM Tris–HCl; 50 mM NaCl, pH ¼ 7.1) at concentration
10
fixed concentration of drugs (10
concentration of CT-DNA with the range from 5 to 35
m
M. Absorption titration experiments were performed with
M) while gradually increasing the
M. The
The superoxide radical (Oꢀ2ꢁ) was produced by the system of
MET/VitB2/NBT and determined spectrophotometrically by nitro-
blue tetrazolium (NBT) photoreduction method with a little
modification in the method adopted elsewhere [70,71]. The
amount of Oꢀ2ꢁ and suppression ratio for Oꢀ2ꢁ could be calculated by
measuring the absorbance at 560 nm. Solution of VitB2 and NBT
were prepared under the condition of avoiding light. The tested
compounds were dissolved in DMF. The assay mixture, in a total
m
m
reference solution was the corresponding Tris–HCl buffer solution.
While measuring the absorption spectra, equal amount of DNA was
added to both the compound solution and the reference solution to
eliminate the absorbance of DNA itself. Each sample solution was
scanned in the range of 210–450 nm. From absorption data, Kb was
determined using the following Eqs. (1) [66] through a plot of
volume of 5 ml, contained MET (10 mM), NBT (46
mM), VitB2
[DNA]/(3a
ꢁ
3f) versus [DNA]
(3.3 M), the tested compound (1.0–8.0 M) and a phosphate buffer
m
m
h
i ꢀ
ꢁ
ꢀ
ꢁ
h
ꢀ
ꢁi
(67 mM, pH ¼ 7.8). After illuminating with a fluorescent lamp at
30 ꢂC for 10 min, the absorbance of the samples (Ai) was measured
at 560 nm. The sample without the tested compound was used as
control and its absorbance was A0. All experimental results were
expressed as the mean and (ꢄ) standard deviation (S.D.) of tripli-
cate determinations. The suppression ratio for Oꢀ2ꢁ was calculated
from the following expression. The suppression ratio ha ¼ (A0 ꢁ Ai)/
A0 ꢃ 100%.
DNA = 3a
ꢁ
3f ¼ ½DNAꢅ= 3a
ꢁ
3f þ 1= Kb 3b
ꢁ
3f
(1)
where [DNA] is the concentration of DNA; 3a
,
3f and 3b are the
apparent extinction coefficient (Aobsd/[compound]), the extinction
coefficient for free compound and the extinction coefficient for
compound in the fully bound form, respectively. In plots of [DNA]/
(
3a
ꢁ
3f) versus [DNA], Kb is given by the ratio of the slope to the
intercept.
4.5.3. EB displacement experiment
5. Supplementary data
Further support for the ligand and its Ln(III) complexes (Ln ¼ La,
Eu) binding to DNA via intercalation is given through the emission
Crystallographic data for the structural analysis have been
deposited with the Cambridge Crystallographic Data Centre, CCDC
(695464). Copy of this information may be obtained free of charge
from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK
(fax: þ44-1223-336033; e-mail: deposit@ccdc.cam.ac.uk or http://
www.ccdc.cam.ac.uk/deposit).
quenching experiment. A 2 ml solution of 50
EB was titrated against 10–40
mM DNA and 0.4 mM
M of compounds (lex ¼ 525 nm,
m
lem ¼ 540.0–650.0 nm, slit ¼ 10 nm). According to the classical
Stern–Volmer equation [67]:
F0=F ¼ Kq½Qꢅ þ 1
(2)
Acknowledgements
where F0 is the emission intensity in the absence of quencher, F is
the emission intensity in the presence of quencher, Kq is the
quenching constant, and [Q] is the quencher concentration. The
shape of Eq. (2) plots can be used to characterize the quenching as
being predominantly dynamic or static. Plots of F0/F versus [Q]
appear to be linear.
This work is supported by the National Natural Science Foun-
dation of China (20475023) and Gansu NSF (0710RJZA012).
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