Synthesis and crystal structure of a Schiff base derived from two similar pyrazolone rings and its rare earth complexes: DNA-binding and antioxidant activity

Article abstract of DOI:10.1080/00958972.2012.727208

A Schiff base derived from two similar pyrazolone derivatives 4-(1-phenyl-3-methyl-5-hydroxypyrazol-4-yl)methyleneiminophenazone and its Tb(III) and Dy(III) complexes were synthesized and characterized. The molecular structures of the ligand and Dy(III) c

Full text of DOI:10.1080/00958972.2012.727208

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Synthesis and crystal structure
of a Schiff base derived from two
similar pyrazolone rings and its rare
earth complexes: DNA-binding and
antioxidant activity
Ming-Fang Wang a , Zheng-Yin Yang a , Zeng-Chen Liu a , Yong Li a ,
Tian-Rong Li a , Mi-Hui Yan a & Xiao-Ying Cheng a
a State Key Laboratory of Applied Organic Chemistry and College
of Chemistry and Chemical Engineering , Lanzhou University ,
Lanzhou 730000 , PR China
Accepted author version posted online: 04 Sep 2012.Published
online: 17 Sep 2012.
To cite this article: Ming-Fang Wang , Zheng-Yin Yang , Zeng-Chen Liu , Yong Li , Tian-Rong Li , Mi-
Hui Yan & Xiao-Ying Cheng (2012) Synthesis and crystal structure of a Schiff base derived from two
similar pyrazolone rings and its rare earth complexes: DNA-binding and antioxidant activity, Journal
of Coordination Chemistry, 65:21, 3805-3820, DOI: 10.1080/00958972.2012.727208
To link to this article: http://dx.doi.org/10.1080/00958972.2012.727208
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Journal of Coordination Chemistry
Vol. 65, No. 21, 10 November 2012, 3805–3820
Synthesis and crystal structure of a Schiff base derived from
two similar pyrazolone rings and its rare earth complexes:
DNA-binding and antioxidant activity
MING-FANG WANG, ZHENG-YIN YANG*, ZENG-CHEN LIU, YONG LI,
TIAN-RONG LI, MI-HUI YAN and XIAO-YING CHENG
State Key Laboratory of Applied Organic Chemistry and College of Chemistry and
Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China
(Received 20 June 2012; in final form 10 August 2012)
A Schiff base derived from two similar pyrazolone derivatives 4-(10-phenyl-30-methyl-50-
hydroxypyrazol-40-yl)methyleneiminophenazone and its Tb(III) and Dy(III) complexes were
synthesized and characterized. The molecular structures of the ligand and Dy(III) complex
were determined by X-ray crystal diffraction. The DNA-binding properties of the compounds
were investigated by electronic absorption spectroscopy, fluorescence spectra, and viscosity
measurements. The compounds interact with DNA through intercalation. The compounds also
exhibit potential antioxidant activities in vitro and the antioxidant activity of the Ln(III)-
complexes was stronger than that of the ligand alone and some standard antioxidants, such as
mannitol and vitamin C.
Keywords: 4-(10-Phenyl-30-methyl-50-hydroxypyrazol-40-yl)methyleneiminophenazone;
Spectroscopy; Crystal structure; DNA-Binding mode; Antioxidant activity
1. Introduction
Experiments indicate that the binding study between small molecules and DNA can
help design more effective anti-cancer drugs [1, 2] and provide new insights into
designing anticancer drugs and developing highly sensitive diagnostic agents [3, 4].
Generally, small molecules bind to DNA in non-covalent modes, such as intercalation,
groove-binding, and external electrostatic binding [5, 6]. Applications of metal
complexes require that the complexes bind to DNA via intercalation, which could
induce cellular degradation [7]. Intercalation ability correlates to coordination
geometry, ligand donor type, metal ion type and its valence [8, 9]. Lanthanide
complexes with tetracycline, phenanthroline, adriamycin, and pyridine have already
been synthesized as probes to study nucleic acids [10, 11]; lanthanide ions have much
higher coordination numbers and more flexible coordination geometry, which lead to
the formation of unusual multidimensional architectures.
*Corresponding author. Email: yangzy@lzu.edu.cn
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/00958972.2012.727208

3806
M.-F. Wang et al.
Schiff bases and their coordination complexes have attracted attention because of
facile syntheses, wide application, and the diverse structural modifications [12–16].
Antipyrine (2,3-dimethyl-1-phenyl-3-pyrazolin-5-one) is the first pyrazolone derivative
used in the management of pain and inflammation, and their derivatives have many
potential activities including anticancer activity and antimicrobial activity [17, 18].
Schiff bases of 4-aminoantipyrine and their coordination complexes possess applica-
tions in biological, clinical, pharmacological, analytical, and materials fields [19].
In this article, the synthesis, characterization, and biological activities of Tb and Dy
with 4-(10-phenyl-30-methyl-50-hydroxypyrazol-40-yl) methyleneiminophenazone Schiff
base are presented. The structure of the ligand and Dy(III) complex have been
investigated by X-ray crystallography. Some rare earth metal complexes exhibit
potential antioxidant activities [20–22]. Since free radicals like hydroxyl are relevant to
diseases such as Alzheimers and Parkinsons [23], the in vitro antioxidative activities of
hydroxyl radical scavenging by metal complexes is of interest.
2. Experimental
2.1. Materials and instrumentation
All materials and solvents were of analytical grade and used without purification.
1-Phenyl-3-methyl-5-pyrazole, ethylenediaminetetraaceticacid (EDTA), safranin,
4-aminophenazone, Dy(NO3)3 ꢀ 6H2O, and Tb(NO3)3 ꢀ 6H2O were produced in China.
Calf thymus DNA (DNA) and ethidium bromide (EB) were purchased from Sigma
Chemical Co. All experiments involved with interaction of the ligand and complexes
with DNA were carried out in Tris-HCl buffer (pH ¼ 7.2) containing 5 mmol Lꢁ1 Tris
[Tris(hydroxymethyl)-aminomethane] and 50 mmol Lꢁ1 NaCl. Solution of DNA gave
ratios of absorbance at 260 and 280 nm of about 1.8–1.9 : 1, indicating that the DNA
was sufficiently free of protein [24]. DNA concentration per nucleotide was determined
by
(6600 (mol L
absorption
)
spectroscopy
using
the
molar
absorption
coefficient
ꢁ1 ꢁ1 cmꢁ1) at 260 nm [25]. The ligand and complexes were dissolved in a
solvent mixture of 1% methanol and 99% Tris-HCl buffer (pH ¼ 7.2) at
1.0 ꢂ 10ꢁ5 mol Lꢁ1
.
Melting point of the ligand was determined on a Beijing XT4-100X microscopic
melting point apparatus. IR spectra were obtained in KBr discs on a Therrno Mattson
FTIR spectrophotometer from 4000 to 400 cmꢁ1. 1H NMR spectra were recorded on a
Varian VR 400 MHz spectrometer in DMSO-d6 with TMS as an internal standard.
Conductivity measurements were performed in methanol solution with a DDS-11C
conductometer at 25ꢃC. UV–Vis spectra were obtained on a Perkin-Elmer Lambda-35
UV–Vis spectrophotometer. Fluorescence measurements were recorded on a Shimadzu
RF-5301 spectrofluorophotometer at room temperature. The antioxidant activities
were performed in methanol solution with a 721-E spectrophotometer.
2.2. Preparation of the ligand and its Ln(III) complexes
Synthesis of L. The synthetic route to L is shown in figure 1. 1-Phenyl-3-methyl-4-
formyl-2pyrazolin-5-one (PMFP) was prepared according to the literature [26].

Rare earth complexes
3807
O
O
H2N
H
O
O
O
N POCl3/DMF
Heat
O
+
N
N
N
N
N
N
N
PMFP
Ethanol
Reflux
9
9
N
N
N
N
N
N
N
N
N
N
10
10
O
11
11
OH
O
O
keto form (II)
enol form (I)
Figure 1. Scheme for synthesis of L.
An ethanol solution (20 mL) of 4-aminophenazone (2.131 g, 10.5 mmol) was added to
an ethanol solution (20 mL) of PMFP (2.020 g, 10 mmol). The reaction mixture was
refluxed on an oil bath for 8 h with stirring, where upon a yellow precipitate is
separated. The precipitate was washed with massive water and ethanol and then
recrystallized from ethanol to give the ligand, which was dried under a vacuum. Yield:
2.90 g, 75.15%, m.p.: 222–224ꢃC. IR ꢀmax (cmꢁ1): ꢀ(C(11)¼O) [ꢀ(C¼O)4-aminophenazone]:
1
1672, ꢀ(C¼N):1632, ꢀ(C(10)¼O) [ꢀ(C¼O)PMFP]: 1653. H NMR (400 MHz, DMSO-d6,
0
ppm): 8.98 (s, 1H, H9), 7.97 (d, 2H, H1, H1 ), 7.48 (t, 2H, H2, H2 ), 7.39 (overlap, 5H,
0
0
0
0
H5, H5 , H4, H4 , H3), 7.15 (t, 1H, H3 ), 3.11 (s, 3H, –C(6)H3), 2.38 (s, 3H, –C(8)H3), 2.26
(s, 3H, –C(7)H3).
2.3. Preparation of the complexes
The ligand (77.4 mg, 0.20 mmol) was dissolved in methanol (20 mL). After 5 min,
Tb(III) nitrate (90.6 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 Tb(III) complex, was
separated from the solution by suction filtration, purified by washing several times with
methanol and dried for 24 h under vacuum. The Dy(III) complex was prepared in the
same way. For the Tb(III) complex: IR ꢀmax (cmꢁ1): ꢀ(11)C¼O: 1657, ꢀC¼N: 1594,
ꢀ
(10)C¼O: 1460, ꢀNO3: 1383, ꢀM–O: 620, ꢀM–N: 454 cmꢁ1. Umax (nm): 255, 360 nm.
Lm (S cm2 molꢁ1): 101 and for Dy(III) complex: IR ꢀmax (cmꢁ1): ꢀ(11)C¼O: 1658, ꢀC¼N
:
1594, ꢀ(10)C¼O: 1456, ꢀNO3: 1382, ꢀM–O: 627, ꢀM–N: 442 cmꢁ1. Umax (nm): 257, 350 nm.
Lm (S cm2 molꢁ1): 103.
2.4. Crystal structure determination
The single crystals of the ligand and Dy(III) complex were obtained in methanol using
the diffusing method. A yellow crystal of L (0.34 ꢂ 0.25 ꢂ 0.20 mm3) was measured on
a Bruker Smart-1000 CCD diffractometer with graphite monochromated Mo-Ka
radiation (ꢁ ¼ 0.71073 A) at 296(2) K. 1.98ꢃ < ꢂ < 25.16ꢃ for hkl (ꢁ9 ꢄ h ꢄ 10,
˚

3808
M.-F. Wang et al.
ꢁ14 ꢄ k ꢄ 14, ꢁ22 ꢄ l ꢄ 20) in the monoclinic system. Yellow crystal of Dy(III) complex
(0.36 ꢂ 0.32 ꢂ 0.27 mm3) was measured on a Bruker Smart-1000 CCD diffractometer
˚
with graphite monochromated Mo-Kꢃ radiation (ꢁ ¼ 0.71073 A) at 296(2) K.
2.12ꢃ < ꢂ < 28.24ꢃ for hkl (ꢁ20 ꢄ h ꢄ 20, ꢁ20 ꢄ k ꢄ 30, ꢁ38 ꢄ l ꢄ 21) in the orthorhom-
bic system. The positions and anisotropic thermal parameters of all non-hydrogen
atoms were refined on F2 by full-matrix least-squares techniques with the SHELX-97
program package. Absorption corrections were employed using semi-empirical methods
from equivalents.
2.5. DNA binding procedures
2.5.1. UV absorption measurement. Electronic absorption titration experiments were
performed with fixed concentration (10 mmol Lꢁ1), while gradually increasing the
concentration of DNA. When measuring the absorption spectra, equal amounts of
DNA were added to both the sample and reference solutions to eliminate the
absorbance of DNA itself. Each sample solution was scanned from 190 to 500 nm.
2.5.2. Fluorescence spectral titration. Fixed amount (10 mmol Lꢁ1) were titrated with
increasing amounts of DNA over a range of DNA concentrations from 2.5 to
30 mmol Lꢁ1. The fluorescence spectra from 350 to 600 nm of the above solutions were
collected with excitation wavelength of 320 nm. The concentration of the bound
compound was calculated using the following equation [27]:
Cb ¼ Ct½ðF ꢁ F0Þ=ðFmax ꢁ F0Þꢅ,
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 put into 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 experiments were conducted at room temperature in a
Tris-HCl (pH ¼ 7.2) buffer.
2.5.3. Effect of the ligand and complexes on the binding of EB to DNA. Further
support for the ligand and complexes binding to DNA via intercalation is given through
emission quenching experiments. EB is a common fluorescent probe for DNA structure
and has been employed in examinations of the mode and process of metal complexes
binding to DNA [28]. The solution (2.0 mL) of 5 mmol Lꢁ1 DNA and 0.4 mmol Lꢁ1 EB
(at saturating binding levels) was titrated by 2.5–75 mmol Lꢁ1 lanthanide complexes and
ligand. The fluorescence spectra from 540 to 700 nm of the solution were collected with
excitation wavelength of 525 nm. Quenching data were analyzed according to the
SternꢁVolmer equation which could be used to determine the fluorescent quenching
mechanism:
F0=F ¼ 1 þ Kq½Qꢅ,
where F0 and F are the fluorescence intensity in the absence and the presence of complex
at [Q] concentration, respectively, Kq is the quenching constant, and [Q] is the quencher

Rare earth complexes
3809
concentration. Plots of F0/F versus [Q] appear to be linear and Kq depends on
temperature [29].
2.5.4. Effect of ionic strength on the fluorescence spectra. Fluorescence intensities were
recorded in the absence and the presence of DNA in solution of each compound and
NaCl to investigate salt effects of compounds to DNA.
Iodide quenching experiments were done according to the below methods: fluores-
cence intensities were recorded in the absence and the presence of DNA in solution of
each compound and KI. The fluorescence quenching efficiency is evaluated by Stern–
Volmer Ksv, which varies with the experimental conditions. Quenching plots were
constructed according to the following Stern–Volmer equation:
F0=F ¼ 1 þ Ksv½Iꢁꢅ,
where F0 and F are the fluorescence intensity in the absence and the presence of iodide
at [Iꢁ] concentration, respectively, Ksv is the quenching constant, and [Iꢁ] is the
concentration of iodide. Plots of F0/F versus [Iꢁ] appear to be linear and Ksv was
evaluated by linear least-squares analysis of the data [30].
2.5.5. Viscosity measurements. Viscosity experiments were conducted on an
Ubbelodhe viscometer immersed in a thermostated water bath maintained at
25.0 ꢆ 0.1ꢃC. Titrations were performed for the ligand and complexes (0.5–
4 mmol Lꢁ1), and each compound was introduced into a DNA solution (5 mmol Lꢁ1
)
present in the viscometer. Data were analyzed as (ꢄ/ꢄ0)1/3 versus the ratio of the
concentration of the compound and DNA, where ꢄ is the viscosity of DNA in the
presence of the compound and ꢄ0 is the viscosity of DNA alone [31, 32]. Viscosities were
calculated from the observed flow time of solutions containing DNA corrected for the
flow time of buffer alone (t0), ꢄ ¼ t ꢁ t0.
2.6. Scavenger measurements of hydroxyl radical (OH. )
Hydroxyl radicals (OH. ) in aqueous media were generated through the Fenton reaction
[33]. Solutions of the test compounds were prepared with DMF. The reaction mixture
contained 2.0 mL of 0.15 mol Lꢁ1 phosphate buffer (pH ¼ 7.4), 1.0 mL of 114 mmol Lꢁ1
safranin, 1 mL of 945 mmol Lꢁ1 EDTA–Fe(II), 1 mL of 3% H2O2 and 30 mL of the test
compound solution (the final concentration: Ci(i¼1–6) ¼ 1.0, 2.0, 3.0, 4.0, 5.0,
6.0 mmol Lꢁ1). Sample without the test compound was used as the control. Reaction
mixtures were incubated at 37ꢃC for 60 min in a water bath. Absorbances (Ai, A0, Ac) at
520 nm were measured. The scavenging ratio is defined as
Suppression ratio ð%Þ ¼ ½ðAi ꢁ A0Þ=ðAc ꢁ A0Þꢅ ꢂ 100%,
where Ai ¼ absorbance in the presence of the test compound; A0 ¼ absorbance of the
blank in the absence of the test compound; Ac ¼ absorbance in the absence of the test
compound, EDTA–Fe(II) and H2O2. The antioxidant activity was expressed as the
50% inhibitory concentration (IC50). IC50 values were calculated from regression lines
where x was the test compound concentration in mmol Lꢁ1 and y was percent inhibition
of the test compounds.

3810
M.-F. Wang et al.
3. Results and discussion
3.1. Characterization and structures of the complexes
The ligand and its complexes are stable in atmospheric conditions for extended periods
and easily soluble in DMF and DMSO; slightly soluble in ethanol, methanol, and
acetone; insoluble in benzene, water, and diethyl ether. Crystals of the ligand and
Dy(III) complex were obtained from methanol and diethyl ether diffusing method. The
molar conductivities of the complexes are 102 S cm2 molꢁ1 in methanol solution and in
accord with them being formulated as 1 : 1 electrolytes [34].
The IR bands of the ligand at 1672, 1653, and 1632 cmꢁ1 are ꢀ(C¼O) of the
4-aminoantipyrine, ꢀ(C¼O) of the PMFP and ꢀ(C¼N), respectively. Thus the ligand
exists in the keto form in the solid state. However, in spectra of the complexes, these are
replaced by bands at 1657, 1594, and 1458 cmꢁ1, respectively, and a new band is
observed at 1458 cmꢁ1 due to ꢀ(C–Oꢁ). It is concluded that the ligand reacts in the enol
form (I) when coordinated to the metal. Weak bands at 448 and 628 cmꢁ1 are allocated
as ꢀ(M–N) and ꢀ(M–O). The shifts and new bands further confirmed that nitrogen of
imino and oxygen of carbonyl coordinated with the metal [35, 36]. Additionally, an
intense band associated with the asymmetric stretch of nitrate is observed at 1384 cmꢁ1
establishing that the complexes contain free nitrate (C2ꢀ).
,
3.2. Description of the crystal structure
The crystallographic data for the ligand and Dy(III) complex are given in table 1 and
selected bond lengths and angles are listed in tables 2 and 3, respectively. The ligand
crystallizes in the monoclinic lattice with a space group of P21/n. Each unit cell contains
four molecules. The distances for C(11)–O, C(11)–C, C(10)–O, and C(10)–C are 1.229(2),
˚
1.430(2), 1.250(2), and 1.429(2) A, respectively, so the ligand is in the keto form (II) in
figure 1. An ORTEP representation of the ligand and Dy(III) complex are shown in
figure 2. The Dy(III) complex crystallizes into an orthorhombic lattice with the space
group Pbca. The complex is mononuclear and coordination of ligand to Dy(III) results
in formation of two five-membered (DyOCCN) and six-membered (DyNCCCO)
chelate rings. The Dy(III) is coordinated by two ONO tridentate ligands with two
nitrogen atoms of azomethine nitrogen (N4, N7), two PMFP-pyrazolone oxygen atoms
(O39, O91), and another two 4-antipyrine pyrazolone oxygen atoms (O3, O12). There
are also coordinated water O1 and methanol O5. The distances in the Dy(III) complex
˚
for C(10)–O, C(10)–C are 1.253(9), 1.401(9), 1.272(9), and 1.403(11) A. Compared to the
ligand, the C(10)–O distance lengthened and the C(10)–C distance is shorter. The
˚
distances in the Dy(III) complex for C(10)–O are between C–O (1.41–1.44 A) and C¼O
˚
(1.19–1.23 A) [37], suggesting that the oxygen of pyrazole ring take part in coordination
˚
in the enolic form. Distances for C(10)–C are between C–C (1.47–1.53 A) and C¼C
˚
(1.32–1.38 A), supporting this conclusion. In the complex, the ligand in the enol form (I)
loses one proton and is a tridentate chelating agent. The analytical results indicate that
the Dy(III) complex has 1 : 2 metal to ligand stoichiometry by coordination at the
Dy(III) center. However, in papers reported in our group, the general formulas of
complexes were Ln(HL)3, with tricapped trigonal prism coordination polyhedra. The
difference might be because geometric constraints were applied to these molecules to
ensure stable refinement.

Rare earth complexes
3811
Table 1. Crystal data and experimental data of the ligand and Dy(III) complex.
Compound
L
[DyL2(CH3OH)(H2O)] ꢀ [NO3(CH3OH)2]
Empirical formula
Formula weight
Crystal color
C22H21N5O2
387.44
Yellow
0.34 ꢂ 0.25 ꢂ 0.20
296(2)
0.71073
Mo-Kꢃ
Monoclinic
P21/n
C47H46DyN11O11
1103.50
Yellow
Crystal size (mm3)
Temperature (K)
0.36 ꢂ 0.32 ꢂ 0.27
296(2)
0.71073
˚
Wavelength (A)
Radiation
Crystal system
Space group
Z
Mo-Kꢃ
Orthorhombic
Pbca
4
8
ꢃ
˚
Unit cell dimensions (A, )
a
b
c
ꢃ
ꢅ
ꢆ
8.4100(3)
12.3782(4)
18.7118(6)
90.00
95.204(2)
90.00
1939.88(11)
1.327
0.088
15.7620(13)
22.5619(18)
28.864(2)
90.00
90.00
90.00
10264.7(15)
1.428
1.523
3
˚
Volume (A )
Calculated density (g cmꢁ3
)
Absorption coefficient (mmꢁ1
F(000)
)
816
4472
ꢂ range for data collection (ꢃ) 1.98–25.16
1.72–28.24
ꢁ20 ꢄ h ꢄ 20
ꢁ20 ꢄ k ꢄ 29
ꢁ38 ꢄ l ꢄ 21
12,255
Index ranges
ꢁ9 ꢄ h ꢄ 10
ꢁ14 ꢄ k ꢄ 14
ꢁ22 ꢄ l ꢄ 20
3461
Reflections collected
Independent reflections
Refinement method
2228 (R(int) ¼ 0.0427)
6245 (R(int) ¼ 0.0584)
Full matrix least-squares on F2 Full matrix least-squares on F2
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2ꢇ(I)]
R indices (all data)
3461/0/269
1.014
12,255/0/631
0.982
R1 ¼ 0.0427, wR2 ¼ 0.0943
R1 ¼ 0.0584, wR2 ¼ 0.1531
R1 ¼ 0.0792, wR2 ¼ 0.1108
R1 ¼ 0.1372, wR2 ¼ 0.1892
˚
Table 2. Selected bond lengths (A) for the ligand.
Bond names
Bond lengths
O1–C12
C12–C13
C10–C13
O2–C17
C16–C17
N1–C30
1.229(2)
1.430(2)
1.350(2)
1.250(2)
1.429(2)
1.320(2)
All these results suggest that the formula of the complexes is
[ML2(CH3OH)(H2O)] ꢀ [NO3(CH3OH)2].
3.3. DNA-binding studies
3.3.1. UV absorption measurement. Electronic absorption spectra of the ligand and its
lanthanide complexes in the absence and the presence of DNA (at a constant

3812
M.-F. Wang et al.
ꢃ
˚
Table 3. Selected bond lengths (A) and angles ( ) for Dy(III) complex.
Bond names
Bond lengths
Bond names
Bond angles
Dy1–O5
Dy1–O39
Dy1–O3
Dy1–O1
Dy1–O91
Dy1–O12
Dy1–O10
Dy1–N4
Dy1–N7
C10–O39
C10–C20
C9–O12
C9–C54
C22–O5
C22–C92
C67–O3
C67–C72
2.251(5)
2.270(5)
2.384(5)
2.388(5)
2.393(6)
2.395(5)
2.425(4)
2.560(6)
2.581(6)
1.253(9)
1.401(11)
1.249(9)
1.409(10)
1.272(9)
1.403(11)
1.260(8)
1.434(10)
O5–Dy1–O39
O5–Dy1–O3
O39–Dy1–O3
O5–Dy1–O1
O39–Dy1–O1
O3–Dy1–O1
O5–Dy1–O91
O39–Dy1–O91
O3–Dy1–O91
O1–Dy1–O91
O5–Dy1–N7
O39–Dy1–N7
O3–Dy1–N7
O1–Dy1–N7
O91–Dy1–N7
O5–Dy1–N7
N4–Dy1–N7
90.8(2)
144.58(18)
76.81(19)
144.90(19)
105.09(18)
70.40(17)
93.4(2)
79.1(2)
91.7(2)
74.63(19)
75.50(19)
73.83(19)
69.21(18)
138.66(18)
72.5(2)
131.97(18)
137.55(19)
Figure 2. ORTEP view of the ligand (left) and Dy(III) complex (right) showing atom numbering and 30%
probability thermal ellipsoids for non-hydrogen atoms.
concentration of the compounds) are shown in figure 3. With increasing DNA
concentrations the absorption bands at about 255 nm and 365 nm of the compounds
show hypochromism and a red shift of 5 nm. These spectral characteristics indicate
strong stacking interaction between an aromatic chromophore and base pairs of DNA,
suggesting that the compounds might bind to DNA by intercalation [38]. After
intercalating the base pairs of DNA, the ꢈ* orbital of the intercalated ligand could
couple with a ꢈ orbital of base pairs, thus decreasing the ꢈ–ꢈ* transition energy, and
further resulting in the bathochromism. The coupling ꢈ* orbital was partially filled by
electrons, decreasing the transition probabilities, and concomitantly resulting in
hypochromism [39].
In order to further test if the compounds could bind to DNA by intercalation, EB (a
typical indicator of intercalation) is employed [40]. Figure 4 shows that the maximal

Rare earth complexes
3813
Figure 3. Electronic absorption spectral change of the free ligand (a), Tb(III) (b), Dy(III) (c) complexes
(10 mmol Lꢁ1) in the absence and the presence of increasing amounts of ct-DNA (2.5, 5.0, 7.5, 10.0, 12.5, 15.0,
17.5, and 20.0 mmol Lꢁ1). Arrows show the absorbance changes upon increasing DNA concentration.
Figure 4. Visible absorption spectra of 1 ꢂ 10ꢁ5 mol Lꢁ1 EB (curve A); (A) þ 2.5 ꢂ 10ꢁ5 mol Lꢁ1 DNA
(curve B); (B) þ 2.5 ꢂ 10ꢁ5 mol Lꢁ1 Dy(III) complex (curve C) in Tris-HCl buffer (5 mmol Lꢁ1 Tris-HCl,
50 mmol Lꢁ1 NaCl, pH ¼ 7.20) solution.
absorption of EB at 475 nm (curve A) decreased and shifted to 480 nm (curve B) in the
presence of DNA, which is the characteristic of intercalation. Curve C is the absorption
of a mixture solution of EB, DNA, and the Dy(III) complex. The absorption at 488 nm
increased compared with curve B. This could result from two reasons: (1) there may

3814
M.-F. Wang et al.
exist competitive intercalation between Dy(III) complex and EB with DNA, releasing
some free EB from the DNA–EB system and (2) EB binds to Dy(III) complex strongly,
resulting in a decreased amount of EB intercalated into DNA. However, no new
absorption peaks were observed, so the second reason could be ruled out.
3.3.2. Fluorescence spectra titration. The steady-state emission spectra of 10 mmol Lꢁ1
solutions of the free ligand and its lanthanide complexes in Tris-HCl buffer show an
increase in the emission intensity with successive addition of DNA at room temperature
(figure 5). According to the Scatchard equation, a plot of r/Cf versus r gave Kb values of
(2.44 ꢆ 0.24) ꢂ 105, (3.63 ꢆ 0.26) ꢂ 105, and (5.32 ꢆ 0.76) ꢂ 105 (mol Lꢁ1 ꢁ1
)
from the
fluorescence data for the ligand, Tb(III), and Dy(III) complexes, respectively. In the
same calculation method and experimental conditions, the Kb values of two rare
earth complexes were compared with other known DNA-intercalative complexes, like
[EuL ꢀ (NO3)2] ꢀ NO3, 2.48 ꢂ 105 (mol Lꢁ1 ꢁ1
[41], [LaL2 ꢀ (NO3)2] ꢀ NO3, (2.2 ꢆ 0.3) ꢂ
)
105 (mol L
)
ꢁ1 ꢁ1, [SmL2 ꢀ (NO3)2] ꢀ NO3, (4.0 ꢆ 0.6) ꢂ 105 (mol L
)
ꢁ1 ꢁ1, [NdL2 ꢀ (NO3)2] ꢀ
NO3, (7.6 ꢆ 0.9) ꢂ 105 (mol Lꢁ1 ꢁ1
)
,
and [YbL2 ꢀ (NO3)2] ꢀ NO3, (3.1 ꢆ 0.5) ꢂ 105
(mol Lꢁ1 ꢁ1
[42]. The Kb values have no notable difference between the two rare
)
earth complexes and the above known DNA-intercalative complexes. The three
compounds have good ability to bind to DNA. Results obtained from the
Figure 5. Emission enhancement spectra of free ligand (a), Tb(III) (b) and Dy(III) (c) complexes
(10 mmol Lꢁ1) in the presence of 0, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20, 22.5, 25, 27.5, and 30.0 mmol Lꢁ1
DNA. Arrows show the emission intensity changes upon increasing DNA concentration. Scatchard plot
of the fluorescence titration data of ligand and metal complexes (d), Kb (a): (2.44 ꢆ 0.24) ꢂ 105, Kb (b):
(3.63 ꢆ 0.26) ꢂ 105, Kb (c): (5.32 ꢆ 0.76) ꢂ 105 (mol Lꢁ1 ꢁ1
) .

Rare earth complexes
3815
fluorescence spectra suggest that all the compounds are protected from water by the
hydrophobic environment inside the DNA helix and that the metal complexes can be
protected more efficiently than the ligand alone. Since the hydrophobic environment
inside the DNA helix reduces the accessibility of solvent water to the compound and
the compound mobility is restricted at the binding site, a decrease of the vibrational
modes of relaxation results. The binding affinities of the complexes are attributed to
the extension of the ꢈ system of the intercalated ligand and the coordination of rare
earth ion. However, the DNA binding modes and binding affinity need to be proved
further by viscosity studies and EB-DNA competitive experiment.
3.3.3. Effect of the ligand and complexes on the binding of EB to DNA. It is well known
that EB is one of the most sensitive fluorescence probes that can intercalate
nonspecifically into DNA effectively increasing the fluorescence of EB [43].
Competitive binding to DNA of the compounds with EB provides indirect evidence
for DNA binding mode. The emission spectra of EB bound to DNA in the absence and
the presence of each compound have been recorded for [EB] ¼ 4 ꢂ 10ꢁ7 mol Lꢁ1
,
[DNA] ¼ 5 ꢂ 10ꢁ6 mol Lꢁ1 with increasing amounts for each compound (figure 6). The
Kq values for the ligand, Tb(III) and Dy(III) complexes are (0.96 ꢆ 0.01) ꢂ 104,
Figure 6. Emission spectra of DNA-EB system ꢁex ¼ 525 nm, ꢁem ¼ 540–700 nm, in the presence of the
ligand (a), Tb(III) (b) and Dy(III) complexes (c) (0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 32.5,
35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, and 60 mmol Lꢁ1). Arrows show the emission intensity changes
upon increasing the ligand and the complexes. Stern-Volmer plot of the fluorescence titration data of ligand
and complexes (d), Kq (a): (0.96 ꢆ 0.01) ꢂ 104, Kq (b): (3.09 ꢆ 0.02) ꢂ 104, Kq (c): (3.61 ꢆ 0.05) ꢂ 104
(mol Lꢁ1 ꢁ1
) .

3816
M.-F. Wang et al.
(3.09 ꢆ 0.02) ꢂ 104, and (3.61 ꢆ 0.05) ꢂ 104 mol Lꢁ1, respectively. The quenching plots
illustrate that quenching of EB bound to DNA by the compounds is in agreement with
the linear Stern–Volmer equation and the binding ability follows the order Dy(III)
complex > Tb(III) complex > L, which is in agreement with the fluorescence titration
and viscosity measurements.
3.3.4. Effect of ionic strength on the fluorescence spectra. DNA is an anionic
polyelectrolyte with phosphate groups. Monitoring the spectral change with different
ionic strength is an efficient method for distinguishing the binding modes between metal
complex and DNA. NaCl is used to control the ionic strength of the solutions. The
addition of Naþ can neutralize the negatively charged phosphate groups, so Naþ would
weaken the electrostatic interaction between DNA and molecules [44]. If the compound
binds to DNA through an electrostatic interaction mode, the surface of DNA will be
surrounded by the sodium ions with the increasing ionic strength. Then the compound
is difficult to approach DNA molecules and the strength of interaction with DNA
decreases, and then the degree of fluorescence quenching also decreases [45, 46]. As seen
from figure 7, addition of NaCl to the Dy (III) complex in the presence of DNA has a
little increase on the fluorescence intensity, showing that the interaction of the Dy(III)
complex with DNA is not an electrostatic interaction.
Iodide and ferrocyanide anions quench fluorescence of complexes very efficiently in
aqueous solution; we used potassium iodide as the quencher to determine the relative
accessibilities of free and bound Dy(III) complex. Emission spectra for Dy(III) complex
and potassium iodide with the absence of DNA are illustrated in the titration curves
(figure 8a). Compared to Dy(III) complex alone, emission intensity decreases with
increasing concentrations of potassium iodide regardless of the buffer solution
containing DNA. The quenching plots illustrate that the quenching studies of the
Dy(III) complex are in good agreement with the linear Stern–Volmer equation and the
slopes were calculated by the linear least-squares method. The observed quenching
constants (Ksv) were 7.42 and 12.68 (mol Lꢁ1 ꢁ1
with and without DNA, respectively.
)
Figure 7. Fluorescence spectra of Dy(III) complex (10 mmol Lꢁ1)-DNA (10 mmol Lꢁ1
) in different
concentrations of NaCl (0, 0.04, 0.08, 0.12, 0.16, 0.20, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 mol Lꢁ1). Arrow
shows the increase of NaCl concentrations.

Rare earth complexes
3817
When the complex was bound to the DNA helix, the Ksv was smaller. Similar results can
be obtained for the ligand and Tb(III) complex and we conclude that the compounds
interact with the DNA helix and they are protected from the anionic quencher, owing to
the base pairs above and below the compounds [47].
3.3.5. Viscosity measurements. Hydrodynamic measurements (i.e., viscosity and
sedimentation), which are sensitive to DNA length changes, are regarded as the least
ambiguous and most critical test of binding in solution in the absence of crystallo-
graphic structural data [48]. A classical intercalation model demands that the DNA
helix lengthens as base pairs are separated to accommodate the bound ligand, leading to
an increase of DNA viscosity. In contrast, partial, non-classical intercalation of ligand
could bend (or kink) the DNA helix reducing its effective length and, concomitantly, its
viscosity [49]. Hence, viscosity study is applied to clarify the binding of these
compounds with DNA. Figure 9 shows that the compounds cause significant increase in
the viscosity of DNA. Viscosity measurement clearly shows that the compounds can
Figure 8. (a) Fluorescence spectra of Dy(III) complex (10 mmol Lꢁ1) with increasing concentration of KI (0,
2.5, 5, 7.5, 10, 12.5, 15, 17.5, and 20 mmol Lꢁ1). (b) Stern–Volmer plot of the fluorescence titration data of
Dy(III) complex. Effect of KI concentration (1: Dy(III) complex þ KI; 2: Dy(III) complex þ KI þ DNA).
Ksv (1) ¼ 12.68 (mol L
)
ꢁ1 ꢁ1, Ksv (2) ¼ 7.42 (mol Lꢁ1 ꢁ1
) .
Figure 9. Effects of increasing amounts of the ligand, Tb(III) and Dy(III) complexes on the relative viscosity
of DNA at 25.0 ꢆ 0.1ꢃC.

3818
M.-F. Wang et al.
Figure 10. Scavenging effects of the ligand, Tb(III), and Dy(III) complexes and manitol on hydroxyl radical.
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 stronger than the
free ligand.
On the basis of all the spectroscopic studies and the viscosity measurement, the
ligand, Tb(III) and Dy(III) complexes proved that they can bind to DNA in an
intercalative mode and the Dy(III) complex exhibits strongest binding affinity to DNA.
3.4. Scavenger measurements of OH.
Since the synthesized ligand and its lanthanide complexes exhibit good DNA binding
affinity, we investigate their antioxidant activity. Reactive oxygen species (ROS), such
as superoxide anion and hydroxyl radical, are usually generated by all aerobic cells
during normal oxygen metabolism, and the oxidation induced by ROS is involved in the
pathogenesis of various diseases through direct effects on DNA and by acting as a
tumor promoter [50]. Consequently, the antioxidant activity of the ligand and its
lanthanide complexes is studied by comparing their scavenging effects on hydroxyl
radical (OH. ) in KH2PO4–Na2HPO4 buffer solution.
The inhibitory effect of the compounds is marked and the average suppression ratio
for OH. increases with increasing compound concentration (figure 10). The values of
IC50 of the ligand, Tb(III), Dy(III) complexes and mannitol for hydroxyl radical
scavenging effects are 10.12, 1.699, 3.012, and 10.19 mmol Lꢁ1, respectively (table 4),
which indicates that formation of metal-ligand coordination complex enhances the
scavenger effect.
4. Conclusion
A Schiff base 4-(10-phenyl-30-methyl-50-hydroxypyrazol-40-yl) methyleneiminophena-
zone and its Ln(III) complexes have been prepared and characterized. Structures of
the ligand and Dy(III) complex were determined by X-ray single-crystal diffraction.

Rare earth complexes
3819
.
Table 4. Scavenging effects of the ligand, Tb(III), and Dy(III) complexes and mannitol on OH .
ꢁ1
.
Average inhibition (%) for OH (mmol L
)
IC50
Compound
1.0
2.0
3.0
4.0
5.0
6.0
Equation
(mmol Lꢁ1
)
R2
HL
Tb(III) complex 42.86 52.00 57.71 62.29 65.14 70.28 y ¼ 34.06x þ 42.16
21.85 25.21 30.25 36.13 40.34 47.06 y ¼ 31.26x þ 18.58 10.12
0.946
1.699 0.995
3.012 0.998
Dy(III) complex 32.57 42.86 50.28 53.71 58.29 61.71 y ¼ 37.19x þ 32.19
Mannitol
2.30 12.52 24.63 32.19 43.09 43.83 y ¼ 57.26x ꢁ 0.73
10.19
0.958
DNA binding properties and antioxidant activities were studied systematically. Results
indicate that the ligand and its complexes bind to DNA via intercalation and the
complexes have better DNA binding affinity than the free ligand. Furthermore, the
complexes have more active scavenging effects on OH. . Our work clearly indicates that
rare earth-based complexes have practical applications, such as understanding the
mechanisms of interaction between small molecules and DNA, the development of
potential anticancer drugs and new antioxidants.
Supplementary material
Crystallographic data for the structural analysis have been deposited with the
Cambridge Crystallographic Data Centre, CCDC (842956). 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).
Acknowledgments
This work is supported by the National Natural Science Foundation of China
(20975046, 81 171 337).
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