Synthesis, characterization, antioxidant activity, and DNA-binding studies of 1-cyclohexyl-3-tosylurea and its Nd(III), Eu(III) complexes

Article abstract of DOI:10.1248/cpb.56.541

A new ligand, 1-cyclohexyl-3-tosylurea (H₂L), was prepared by condensation ethyl N-(3-tossulfonyl) carbamate and cyclohexanamine. Its two lanthanide(III) complexes, Ln(H₂L)₃·3NO₃ [Ln=Nd (1), and Eu (2)], have been synthesized and characterized on the base of element analyses, ESI-MS, molar conductivities, IR spectra and thermogravimetry/differential thermal analysis (TG-DTA). In addition, the DNA-binding properties of the ligand and its complexes have been investigated by electronic absorption spectroscopy, fluorescence spectroscopy, circular dichroic (CD) spectroscopy and viscosity measurements. The experiment results suggest that the ligand and its two complexes bind to DNA via a groove binding mode, and the binding affinity of the complex 2 is higher than that of the complex 1 and the ligand. Furthermore, the antioxidant activity (superoxide and hydroxyl radical) of the ligand and its metal complexes was determined by using spectrophotometer methods in vitro. These complexes were found to possess potent antioxidant activity and be better than standard antioxidants like vitamin C and mannitol. In particular, complex 2 displayed excellent activity on the superoxide and hydroxyl radical.

Full text of DOI:10.1248/cpb.56.541

April 2008
Chem. Pharm. Bull. 56(4) 541—546 (2008)
541
Synthesis, Characterization, Antioxidant Activity, and DNA-Binding
Studies of 1-Cyclohexyl-3-tosylurea and Its Nd(III), Eu(III) Complexes
Pin-Xian XI, Zhi-Hong XU, Xiao-Hui LIU, Feng-Juan CHEN, Liang HUANG, and Zheng-Zhi ZENG*
State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical Engineering, Lanzhou
University; Lanzhou 730000, P. R. China.
Received December 10, 2007; accepted January 22, 2008; published online January 25, 2008
A new ligand, 1-cyclohexyl-3-tosylurea (H2L), was prepared by condensation ethyl N-(3-tossulfonyl) carba-
mate and cyclohexanamine. Its two lanthanide(III) complexes, Ln(H2L)3·3NO3 [LnꢀNd (1), and Eu (2)], have
been synthesized and characterized on the base of element analyses, ESI-MS, molar conductivities, IR spectra
and thermogravimetry/differential thermal analysis (TG-DTA). In addition, the DNA-binding properties of the
ligand and its complexes have been investigated by electronic absorption spectroscopy, fluorescence spectroscopy,
circular dichroic (CD) spectroscopy and viscosity measurements. The experiment results suggest that the ligand
and its two complexes bind to DNA via a groove binding mode, and the binding affinity of the complex 2 is higher
than that of the complex 1 and the ligand. Furthermore, the antioxidant activity (superoxide and hydroxyl radi-
cal) of the ligand and its metal complexes was determined by using spectrophotometer methods in vitro. These
complexes were found to possess potent antioxidant activity and be better than standard antioxidants like vita-
min C and mannitol. In particular, complex 2 displayed excellent activity on the superoxide and hydroxyl radical.
Key words 1-cyclohexyl-3-tosylurea; rare earth complex; groove binding; antioxidant activity
dissolved in a solvent mixture of 1% CH3OH and 99% Tris–HCl buffer
(5 mM Tris–HCl, 50 mM NaCl, pH 7.2) at concentration 1.0ꢂ10ꢁ5 M. An ab-
sorption titration experiment was performed by maintaining 10 mM com-
pounds and varying the concentration of nucleic acid. While measuring the
absorption spectra, an equal amount of CT-DNA was added to both the com-
pound solution and the reference solution to eliminate the absorbance of CT-
DNA itself. EDTA–Fe(II) and Na2HPO4–KH2PO4 buffers were prepared
with twice distilled water.
The sulfonylureas have a wide range use in biology, and
many of their ramifications have been used as anti-diabetic
medicine and herbicide.1,2) Sulfonylurea regulating biological
functions of pancreatic islets, which has been used for the
treatment of type 2 diabetes mellitus because of its insulin
tropic activity on pancreatic islets.3) Subsequent interests
were focused on the redox properties, structures and biologi-
cal activity of their transition metal complexes.4) More re-
cently, the lanthanide(III) complexes of sulfonylurea increas-
ingly attracted attention, because of their certain anti-diabetic
activities. Yet it is noticed that the DNA-binding investiga-
tion of such complexes have been done relatively few. Be-
cause the interaction between metal complexes and DNA is
in close relationship with their potential biological and phar-
Physical Measurements The melting points of the compounds were de-
termined on a Beijing XT4-100x microscopic melting point apparatus (the
thermometer was not corrected). Carbon, hydrogen, and nitrogen were ana-
lyzed on an Elemental Vario EL analyzer. The metal contents of the com-
plexes were determined by titration with EDTA (xylenol orange tetrasodium
salt used as an indicator and hexamethylidynetetraimine as buffer). Infrared
spectra (4000—400 cmꢁ1) were determined with KBr disks on a Therrno
Mattson FTIR spectrometer. The UV–visible spectra were recorded on a
Varian Cary 100 UV–Vis spectrophotometer. The fluorescence spectra were
maceutical activities,5,6) studies on DNA-binding of metal recorded on a Hitachi RF-4500 spectrofluorophotometer. 1H-NMR spectra
were measured on a Varian VR 300-MHz spectrometer, using tetramethyl
silicon (TMS) as a reference in dimethyl sulfoxide (DMSO)-d6. The thermal
behaviour was monitored on a PCT-2 differential thermal analyzer. Mass
spectra were performed on a VG ZAB-HS (FAB) instrument and electro-
complexes are very important in the development of new
therapeutic reagents and DNA molecular probes. In order to
find more effective and less toxic anti-diabetic drugs, thou-
sands of analogues of sulfonlyureas were synthesized.6,7)
spray mass spectra (ESI-MS) were recorded on a LQC system (Finngan
MAT, U.S.A.) using CH3OH as mobile phase. All conductivity measure-
ments were performed in N,Nꢃ-dimethyl formamide (DMF) with a DDS-
11A conductor at 25 °C. The antioxidant activites were tested on a 721E
spectrophotometer (Shanghai Analytical Instrument Factory, China).
Preparation of the Ligand (H2L) The preparation of the ligand is
shown in Fig. 1. A mixture of p-toluene sulfonamide (5.14 g, 0.03 mol) and
finely pulverized K2CO3 (11.00 g, 0.078 mol) in 35 ml of acetone was stirred
and heated to reflux for 30 min. Acetone solution (10 ml) of ethyl chlorofor-
mate (3.80 ml, 0.04 mol) was added to the refluxing mixture and heating
continued for another 4 h. The mixture of the results were poured into
100 ml of H2O, then the aqueous phase was acidified with 10 ml of 1.0 N
aqueous HCl to get the solid, washed with 80 ml H2O for several times. Re-
crystallization from 20 ml ethanol provided 6.35 g (87%) of ethyl N-(3-
Here, we synthesized 1-cyclohexyl-3-tosylurea (H2L) and its
two lanthanide(III) complexes. In addition DNA-binding
property of two lanthanide(III) complexes with 1-cyclohexyl-
3-tosylurea is reported in this paper. These lanthanide com-
pounds possibly represent a new class of potential anti-dia-
betic agent.
Experimental
Materials Nitroblue tetrazolium (NBT), methionine (MET), vitamin B2
(VitB2) were purchased from Sigma Chemical Co. Calf thymus DNA (CT-
DNA) was purchased from Sigma without further purification. EDTA and
Ln2O3 (LnꢀNd, Eu) were produced in China. All chemicals used were of
analytical grade. The lanthanide(III) nitrates were prepared by dissolving
Ln2O3 in HNO3, and then crystallizing to get the products. All the experi-
ments involving interaction of the ligand and the 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 7.2 with hydrochloric acid. A solution of CT-DNA gave a ratio of UV
absorbance at 260 and 280 nm of about 1.8—1.9, indicating that the CT-
DNA was sufficiently free of protein.8) The CT-DNA concentration per nu-
cleotide was determined spectrophotometrically by employing an extinction
coefficient of 6600 Mꢁ1 cmꢁ1 at 260 nm.9) The ligand and the complexes were
Fig. 1. The Preparation of the Ligand
∗ To whom correspondence should be addressed. e-mail: zengzhzh@yahoo.com.cn
© 2008 Pharmaceutical Society of Japan

542
Vol. 56, No. 4
toluenesulfonyl) carbamate as a white crystalline solid: mp 76—78 °C.
tests were run in triplicate and expressed as the meanꢅstandard deviation
A solution of ethyl N-(3-toluenesulfonyl) carbamate (0.73 g, 3.00 mmol) (S.D.).
·
and cyclohexanamine (0.45 ml, 3.90 mmol) in 40 ml PhMe was heated to re-
flux for 6 h. After the solution stood overnight at room temperature, the re-
sulting precipitate was collected, washed twice time with PhMe, and dried in
vacuum to afford 0.67 g (75%) of a white solid: mp 156—158 °C; H2L
(DMSO-d6) d (ppm): 0.96—1.86 (m, 10H), 2.31 (s, 3H), 2.89 (bm, 1H),
3.56 (bm, 1H), 7.16—7.19 (d, 2H, Jꢀ8.1 Hz), 7.59—7.62 (d, 2H,
Jꢀ8.1 Hz). IR nmax (cmꢁ1): nNH (CONH): 3340, nNH (SOONH): 3047, n
(CꢀO): 1704, n (SꢀO): 1130. Umax: (CH3OH) nm (log e) 205 (4.61),
227(4.66). FAB-MS: m/zꢀ297 [MꢄH]ꢄ. Anal. Calcd for C14H20N2O3S: C,
56.73; H, 6.80; N, 9.45. Found: C, 56.80; H, 6.75; N, 9.56.
The suppression ratio for OH was calculated from the following expres-
sion:
suppression ratio (%)ꢀ[(AiꢁA0)/(AcꢁA0)]ꢂ100
(2)
(Where Aiꢀthe absorbance in the presence of the tested compound;
A0ꢀthe absorbance in the absence of the tested compound; Acꢀthe ab-
sorbance in the absence of the tested compound, EDTA–Fe(II), H2O2.)
Results and Discussion
Preparation of the Complexes The complex 1 was prepared as follows.
The solution of Nd(NO3)3·6H2O (438.25 mg, 1 mmol) in anhydrous
methanol (10 ml) was added dropwise to the solution of the ligand
(296.38 mg, 1 mmol) in warm (60 °C) anhydrous methanol (10 ml) with stir-
ring magnetically over 2 h. The clear solution obtained was reduced under
vacuum to 8 ml, water (40 ml) was added to the residue with stirring magnet-
ically and the resulting solid was collected by filtration. The product was re-
crystallized from mixture solvent of methanol and water (1 : 3), and dried in
a vacuum desiccator. Yield: 75%. The complex 2 was synthesized by the
same way. Anal. Calcd for complex 1 C42H60N9O18S3Nd: C, 41.37; H, 4.96;
N, 10.34; Nd, 11.83. Found: C, 41.41; H, 4.90; N, 10.31; Nd, 11.89. Lm
(s cm2 molꢁ1): 21.7. ESI-MS [CH3OH, m/z]: 1217.2 ({[Nd(H2L)3·
3NO3]ꢄH}ꢄ), 1154.3 ({[Nd(H2L)3·3NO3]ꢁNO3}ꢄ), 1091.3 ({[Nd(H2L)3·
All of the complexes are air stable for extended periods
and soluble in DMSO (dimethylsulphoxide) and DMF;
slightly soluble in methanol and ethanol; insoluble in ben-
zene, water and diethyl ether. The molar conductivities in
DMF solution indicate that the complex 1 and complex 2
(21.7, 33.6 s cm2 molꢁ1) are in the range expected for no
electrolytes.15) The elemental analyses, ESI-MS and molar
conductivities show that the formulas of the complexes con-
form to Ln(H2L)3·3NO3 [where LnꢀNd(III) and Eu(III)].
IR Spectra The IR spectra of the complexes are similar.
The band of n (CꢀO) appeared at 1704 cmꢁ1 in the ligand
while the band of the complexes was exhibited at 1641 cmꢁ1;
Dn(ligand-complexes) is equal to 63 cmꢁ1. This shift confirms that
the group loses its original characteristics and forms coordi-
native bonds with the metal.16) The band at the 1130 cmꢁ1
was n (SꢀO) vibration in the ligand. In the complexes these
bands are presented at 1090 cmꢁ1. Dn(ligand-complexes) is equal to
40 cmꢁ1. The spectra of the ligand exhibit nNH (CONH) vi-
bration bands at the 3340 cmꢁ1 and nNH (SOONH) vibrations
at the 3047 cmꢁ1, in the complexes the nNH (CONH) and the
nNH (SOONH) vibration were blue shifted to 3473 cmꢁ1 and
3278 cmꢁ1 respectively. In the complexes, the band at
560 cmꢁ1 or so is assigned to n (Ln–O). These shifts demon-
strate that the ligand coordinated Nd3ꢄ and Eu3ꢄ ions
through the oxygen of carbonyl and sulphanilamide. Weak
bands at 425 cmꢁ1 are assigned to n (Ln–N). These shifts and
the new band further confirm that the nitrogen of the imino-
group bonds to the rare earth ions. The absorption bands of
3NO3]ꢁ2NO3ꢁH}ꢄ), IR nmax (cmꢁ1): n(aminoantipyrine) (CꢀO): 1700 cmꢁ1
,
n(carbonyl) (CꢀO): 1615 cmꢁ1, n(–SOONH–) (N–H): 3727 cmꢁ1, n (NO3): 1479,
840. Umax: (CH3OH) nm (log e): 206 (4.61), 228 (4.66), 272 (4.74). Anal.
Calcd for complex 2 C42H60N9O18S3Eu: C, 41.11; H, 4.93; N, 10.27; Eu,
12.38. Found: C, 41.16; H, 4.86; N, 10.23; Eu, 12.30. Lm (s cm2 molꢁ1):
33.6. ESI-MS [CH3OH, m/z]: 1227.2 ({[Eu(H2L)3·3NO3]ꢄH}ꢄ), 1165.3
({[Eu(H2L)3·3NO3]ꢁNO3}ꢄ), 1102.3 ({[Eu(H2L)3·3NO3]ꢁ2NO3ꢁH}ꢄ);
IR nmax (cmꢁ1): n(aminoantipyrine) (CꢀO): 1700 cmꢁ1
, n(carbonyl) (CꢀO):
1615 cmꢁ1, n(–SOONH–) (N–H): 3727 cmꢁ1, n (NO3): 1479, 840. Umax
:
(CH3OH) nm (log e) 206 (4.61), 228 (4.66), 272 (4.74).
Superoxide Radical Scavenging Assay The superoxide radicals (O2ꢁ
)
·
were generated in vitro by non-enzymatic system and determined spec-
trophotometrically by NBT photoreduction method with a little modification
in the method adopted elsewhere.10—13) The amount of Oꢁ2 and suppression
·
ratio for Oꢁ2 can be calculated by measuring the absorbance at 560 nm. So-
·
lution of MET, VitB2 and NBT were prepared at avoiding light. The tested
compounds were dissolved in DMF. The assay mixture, in a total volume of
5 ml, contained MET (10 mM), NBT (46 mM), VitB2 (3.3 mM), the tested com-
pound (10—30 mM) and a phosphate buffer (67 mM, pH 7.8). After illuminat-
ing with a fluorescent lamp at 30 °C for 10 min, the absorbance of the sam-
ples (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 the coordinated nitrates were observed at about 1479 (nas)
and 840 (ns) cmꢁ1.17)
expressed as the meanꢅstandard deviation (S.D.) of triplicate determina-
tions. The suppression ratio for Oꢁ2 · was calculated from the following ex-
UV Spectra The study of the electronic spectra in the
pression:
ultraviolet and visible ranges for the complexes and the lig-
and were carried out in a solvent mixture of 1% CH3OH and
99% Tris–HCl buffer. The electronic spectra of ligand had
a strong band at lmaxꢀ203 nm, a medium band at
suppression ratio (%)ꢀA0ꢁAi/A0ꢂ100
(1)
Drug activity was expressed as the 50% inhibitory concentration (IC50).
IC50 values were calculated from regression lines where: x was the tested
compound concentration in mM and y was percent inhibition of the tested
compounds.
l
maxꢀ227 nm. The complexes also yield two bands at about
207 and 231. A new band appeared at 275 nm for complex 1
and 2. These changes indicate that complexes are formed.
Thermal Analyses Some data of thermal analyses are
listed in Table 1. The DTA curves of the complexes have an
endothermic peak between 90 and 96 °C, the corresponding
TG curves show that the weight loss is equal to around three
·
Hydroxyl Radical Scavenging Assay The hydroxyl radicals (OH ) in
aqueous media were generated through the Fenton system.14) The solution of
the tested compound was prepared with DMF. The 5 ml assay mixture con-
tained following reagents: safranin (11.4 mM), EDTA–Fe(II) (40 mM), H2O2
(1.76 mM), the tested compound (10—30 mM) and a phosphate buffer
(67 mM, pH 7.4). The assay mixtures were incubated at 37 °C for 30 min in a
waterbath. After which, the absorbance was measured at 520 nm. All the water molecules. These results are in accordance with the
Table 1. Thermal Analyses Data of the Complexes
%H2O loss
Decomposed
T2
Complex
Da T °C
Process
Process
Residue
Calc.
Found
T1
T3
1
2
96
90
Endothermic
Endothermic
4.98
4.94
5.21
5.17
341
337
453
448
556
538
Exothermic
Exothermic
Nd2O3
Eu2O3

April 2008
543
compositions of the complex determined by elemental analy- given in Figs. 3a, b and c, respectively. There exist in Fig. 3a
ses. Exothermic peaks appear around 331—363, 445—458 two well-resolved bands at about 202 and 230 nm for the lig-
and 536—558 °C, respectively. Initial temperature decom- and, and in Figs. 3b and c also have two well-resolved bands
posed is greater than 330 °C, this indicates that thermal sta- at about 204 and 230 nm for the complexes. With increasing
bility of the complexes are higher than that of the free ligand, DNA concentrations, the hypochromisms increased up to
whose stability shows that there may be a large conjugation 9.20% at 202 nm and 8.13% at 230 nm for ligand; 35.40% at
chelate ring exists in the complex.
203 nm and 8.10% at 230 nm for complex 1; 51.61% at
Since the crystal structures of the complexes have not been 204 nm and 32.46% at 230 nm for complex 2. The lmax for
obtained yet, we characterized the complexes and determined the ligand increased from 202 to 203, for complex 1 in-
their possible structure by elemental analyses, ESI-MS, creased from 203 to 205 nm and that for complex 2 increased
molar conductivities, IR data, TG-DTA, and UV–vis meas- from 204 to 206 nm. Such a small change in lmax is more in
urements. The likely structure of the complexes is shown in keeping with groove binding, leading to small perturbations.
Fig. 2.
Such small increases in the lmax and the hypochromicity
DNA-Binding Studies. Electronic Absorption Titration have been observed in the case of some porphyrin and cop-
Electronic absorption spectroscopy is universally employed per complexes on their interaction with DNA.19,20)
to determine the binding characteristics of metal complex
The absorption data were analyzed to evaluate the intrinsic
with DNA.18) The absorption spectra of the ligand, complex 1 binding constant Kb, which can be determined from Eq. 321)
and complex 2 in the absence and presence of CT-DNA are
[DNA]/(eaꢁef)ꢀ[DNA]/(ebꢁef)ꢄ1/Kb(ebꢁef)
(3)
where [DNA] is the concentration of DNA in base pairs, the
apparent absorption coefficient ea, ef and eb correspond to
Aobsd/[M], the extinction coefficient of the free compound and
the extinction coefficient of the compound when fully bound
to DNA, respectively. The binding constants, Kb for the com-
plexes 1 and 2 have been determined from the plot
ꢁ1
of [DNA]/(eaꢁef) vs. [DNA] and found to be 2.6ꢂ104 M
and 7.8ꢂ104 respectively, and the Kb for the ligand
(0.31ꢂ104 Mꢁ1) is very small. The Kb value obtained here is
lower than that reported for classical intercalator (for ethid-
ium bromide and [Ru(phen)DPPZ] whose binding constants
have been found to be in the order of 106—107 M).22,23) The
Fig. 2. The Suggested Structure of the Complexes
Fig. 3. Electronic Spectra of the Ligand (a), Complex 1 (b), Complex 2 (c) (10 mM) in the Presence of 0—70 ml 1.0ꢂ10ꢁ3 M CT-DNA
Arrow shows the emission intensities changes upon increasing DNA concentration. Inset: plots of [DNA]/(eaꢁef) vs. [DNA] for the titration of compound.

544
Vol. 56, No. 4
Fig. 4. The Emission Enhancement Spectra of the Ligand (a), Complex 1 (b), Complex 2 (c) (10 mM) in the Presence of 0—100 ml 1.0ꢂ10ꢁ3 M CT-DNA
Arrow shows the emission intensities changes upon increasing DNA concentration.
observed binding constant is more in keeping with the
groove binding with DNA, as observed in the literature.24,25)
The results indicate that the binding strength of complex 2 is
stronger than that of 1 and the ligand.
Fluorescence Spectra Fixed amounts (10 mM) of the
complexes were titrated with increasing amounts of CT-
DNA. Excitation and emission wavelengths of the samples
were 256 and 370 nm, slit width 5/10 nm. All experiments
were conducted at 20 °C in a solvent mixture of 1% CH3OH
and 99% Tris–HCl buffer.
The ligand and complexes emit luminescence in Tris
buffer with a maximum appearing at 375 nm. The fluores-
cence titrations spectra of the ligand, complex 1 and complex
2 in the absence and presence of CT-DNA are given in Figs.
4a, b and c. Compared to the ligand and complexes alone, the
Fig. 5. CD Spectrum of CT-DNA Adduct with Compound
riꢀmolar ratio, compound: CT-DNA.
fluorescence intensity decrease with the increase of CT-DNA
concentration. As shown in Fig. 4, the fluorescence intensity which is characteristic of DNA in the right-handed B form.
of the complex 1 and 2 are quenched steadily with the in- While groove binding interaction of small molecules with
creasing concentration of the CT-DNA. This phenomenon of DNA show little or no perturbations on the base stacking and
the quenching of luminescence of the complex by DNA may helicity bands, intercalation enhances the intensities of both
be attributed to the photoelectron transfer from the guanine the bands, stabilizing the right-handed B conformation of
base of DNA to the excited metal-to-ligand charge transfer CT-DNA. In all two cases, the intensities of both the negative
31)
(MLCT) state of the complex.24,26—
and positive bands decrease significantly. This suggests that
Circular Dichroic (CD) Spectroscopy CD spectral vari- the DNA binding of the complexes induces certain confor-
ations of CT-DNA were recorded by the respective addition mational changes, such as the conversion from a more B-like
of the ligand and complex 1 and 2 to CT-DNA. Figure 5 to a more C-like structure within the DNA molecule.32)
shows the CD spectra of CT-DNA which was added with the These changes are indicative of a non-intercalative mode of
ligand, complex 1 and 2. The observed CD spectrum of calf binding of these complexes and offer support to their groove
thymus DNA consists of a positive band at 277 nm due to binding nature.33,34)
base stacking and a negative band at 245 nm due to helicity,
Viscosity Studies Hydrodynamic measurements that are

April 2008
545
sensitive to the length change (i.e., viscosity and sedimenta-
tion) are regarded as the least ambiguous and the most criti-
cal tests of a binding model in solution in the absence of
crystallographic structural data.8,35) As a means for further
clarifying the binding of these compounds with DNA, vis-
cosity studies were carried out. Data are presented as
(h/h0)1/3 versus 1/R, where Rꢀ[DNA]/[compound]; h and
h0 are the relative viscosities of DNA in the presence and ab-
sence of compound, respectively. The relative viscosity val-
ues were calculated from the flow time of DNA-containing
solution (t) and the flow time of buffer alone (t0), using the
following expression36):
hꢀ(tꢁt0)/t0
(4)
Fig. 6. Effect of Increasing Amounts of the Complexes on the Relative
Viscosity of CT-DNA at 25.0ꢅ0.1 °C
Intercalating agents are expected to elongate the double helix
to accommodate the ligands in between the base leading to
an increase in the viscosity of DNA. In contrast, complexes
that bind exclusively in the DNA grooves by partial and/or
non-classical intercalation, under the same conditions, typi-
cally cause less pronounced (positive or negative) or no
change in DNA solution viscosity.37) Figure 6 shows the rela-
tive viscosity of DNA (50 mM) in the presence of varying
amounts of the ligand, complexes 1 and 2. The results reveal
that the complexes 1 and 2 show relatively small changes in
DNA viscosity, indicating that they bind weakly to DNA
which is consistent with DNA groove binding suggested
above.38,39) The increased degree of viscosity, which may de-
pend on its affinity to DNA follows the order of 2ꢆ1ꢆlig-
and, which is consistent with our foregoing hypothesis.
Antioxidant Activity It can be seen that the inhibitory
effect of the tested complexes on Oꢁ2 are concentration re-
·
lated and the suppression ratio increases with the increasing
of sample concentration in the range of the tested concentra-
tion (Fig. 7). The antioxidant activities of these compounds
are expressed as 50% inhibitory concentration (IC50 in mM).
IC50 values of 1 and 2 are 9.81ꢅ0.87 and 14.36ꢅ0.68 mM, re-
spectively. Although the IC50 value of the ligand can not be
read in Fig. 7, the compound 1 shows better inhibitory effect
than 2 and the ligand.
Fig. 7. (a) Effect of Tested Compounds on Oꢁ2 and (b) Effect of Tested
·
Compounds on OH
·
ꢀ, (ligand); ꢁ, (complex 1); ꢂ, (complex 2); experiments were performed in tripli-
cate. Values are expressed as meanꢅstandard deviation (nꢀ3).
·
We can find that all compounds scavenge OH also in a
concentration-dependent manner. The complexes show
·
highly active scavenging effect on OH . Moreover, mannitol ical and clearly suggested that the antioxidant activity of the
is a well-known natural antioxidant, so we also studied the complexes was also related to its ability to scavenge superox-
scavenging activity of mannitol against hydroxyl radical ide radical.
using the same model. We find that when arriving at similar
It is clearly shown that metal complexes exhibit consider-
suppression ratio, concentration of the two complexes is far able scavenging activity due to the chelation of organic mole-
less than that of mannitol. The suppression ratio take the cule to rare earth ions and rare earth ions such as La(III),
order of 1ꢆ2ꢆligand.
Sm(III), Eu(III) and Dy(III) exert differential and selective
It is clear that the scavenger effect on Oꢁ2 can be enhanced effects on scavenging radicals of the biological system.
by the formation of metal-ligand coordination complexes and Moreover, we find that the two complexes are better effective
·
·
the nature of the rare earth ions also affects the ability. Some inhibitor for OH than that of mannitol which is usually used
complexes are better effective inhibitor for Oꢁ2 than that of as special scavenger for OH . Therefore, the metal complexes
the nitroxide Tempo (IC50ꢀ60ꢅ3.10 mM) which has been re- we studied in this paper deserve to be further researched.
cently used in biological system for its capacity to mimic su-
·
·
peroxide dismutase.40) Although superoxide is a relatively Conclusion
weak oxidant, it decomposes to form stronger relative oxida-
In this paper, we have investigated and characterized H2L
tive species, such as single oxygen and hydroxyl radicals, and its two lanthanide(III) complexes Ln(H2L)3·3NO3
which initiate peroxidation of lipids.41) In the present study, [LnꢀNd (1), and Eu (2)]. In addition, the DNA-binding
the complexes effectively scavenged superoxide in a concen- properties were investigated by electronic absorption, fluo-
tration-dependent manner. These results showed the com- rescence, CD spectroscopy and viscosity measurement. The
plexes have significant scavenging activity of superoxide rad- results support the fact that complex 1, 2 and the ligand can

546
Vol. 56, No. 4
Biochem., 99, 2240—2247 (2005).
19) Pasternack R. F., Gibbs E. J., Villafranca J. J., Biochem. J., 22, 2406—
2414 (1983).
20) Vaidyanathan V. G., Nair B. U., Eur. J. Inorg. Chem., 2004, 1840—
1846 (2004).
21) Wolf A., Shimer G. H., Meehan T., Biochem. J., 26, 6392—6396
(1987).
22) Cory M., McKee D. D., Kagan J., Henry D. W., Miller J. A., J. Am.
Chem. Soc., 107, 2528—2536 (1985).
23) Waring M. J., J. Mol. Biol., 13, 269—282 (1965).
24) Vaidyanathan V. G., Nair B. U., J. Inorg. Biochem., 94, 121—126
(2003).
bond to CT-DNA by the mode of groove binding, and com-
plex 2 has stronger binding affinity than 1 and the ligand.
Furthermore, the two rare earth complexes have active scav-
enging effect on Oꢁ2 and OH . According to the IC50 values
obtained, complex 2 was found to exert superior activity in
comparison to the remaining compounds. These findings
clearly indicate that lanthanide-based complexes have many
potential practical applications, like the development of nu-
cleic acid molecular probes and new therapeutic reagents for
diseases.
·
·
25) Vijayalakshmi R., Unni Nair B., Biochim. Biophys. Acta, 1475, 157—
162 (2000).
Acknowledgment This project was supported by the National Natural
Science Foundation in China (20171019) and Zhide Foundation.
26) Xu Z. H., Chen F. J., Xi P. X., Liu X. H., Zeng Z. Z., J. Photochem.
Photobiol. A: Chem. (2007), doi:10.1016/j.jphotochem.2007.11.017
27) Mesmaeker A. K., Orellana G., Barton J. K., Turro N. J., J. Photochem.
Photobiol., 52, 461—472 (1990).
28) Chaires J. B., Dattagupta N., Crothers D. M., Biochem. J., 21, 3933—
3940 (1982).
29) Wu J. Z., Yuan L., Wu J. F., J. Inorg. Biochem., 99, 2211—2216
(2005).
30) Xi P. X., Liu X. H., Lu H. L., Zeng Z. Z., Transition Met. Chem., 32,
757—761 (2007).
31) Peng B., Chao H., Sun B., Li H., Gao F., Ji L. N., J. Inorg. Biochem.,
101, 404—411 (2007).
32) Mahadevan S., Palaniandavar M., Inorg. Chem., 37, 693—700 (1998).
33) Maheswari P. U., Palaniandavar M., J. Inorg. Biochem., 98, 219—230
(2004).
References
1) Howbert J. J., Grossman C. S., Crowell T. A., Rieder B. J., J. Med.
Chem., 33, 2393—2407 (1990).
2) Williams D. R., Chem. Rev., 72, 203—213 (1972).
3) Groop L. C., Diabetes Care, 15, 737—754 (1992).
4) Kessissoglou D. P., Manoussakis G. E., Hatzidimitriou A. G.,
Kanatzidis M. G., Inorg. Chem., 26, 1395—1402 (1987).
5) Xu H., Liang Y., Zhang P., Pu F., Zhou B. R., Wu J., Liu J. H., Liu Z.
G., Ji L. N., J. Biol. Inorg. Chem., 10, 529—538 (2005).
6) Song Y. M., Wu Q., J. Inorg. Biochem., 100, 1685—1691 (2006).
7) Xu H., Zheng K. C., Deng H., Lin L. J., Zhang Q. L., Ji L. N., Dalton
Trans., 3, 2260—2268 (2003).
8) Marmur J., J. Mol. Biol., 3, 208—218 (1961).
9) Kumar C. V., Asuncion E. H., J. Am. Chem. Soc., 115, 8547—8553 34) Zhang Z., Qian X. H., Int. J. Biol. Macromol., 38, 59—64 (2006).
(1993).
35) Satyanarayana S., Dabrowiak J. C., Chaires J. B., Biochem. J., 31,
9319—9324 (1992).
36) Cohen G., Eisenberg H., Biopolymers, 8, 45—55 (1969).
37) Liu C. S., Zhang H., Chen R., Shi X. S., Bu X. H., Yang M., Chem.
Pharm. Bull., 55, 996—1001 (2007).
10) Marta G. A., Gloria A., Joaquín B., Lucas D. C. A., Santiago G. G.,
José M. M. B., Inorg. Chem., 44, 9424—9433 (2005).
11) Winterbourn C. C., Biochem. J., 182, 625—628 (1979).
12) Sharma S. D., Rajor H. K., Chopra S., Sharma R. K., BioMetals, 18,
143—154 (2005).
38) Lerman L., J. Mol. Biol., 3, 18—30 (1961).
13) Li T. R., Yang Z. Y., Wang B. D., Chem. Pharm. Bull., 55, 26—28 39) Mahadevan S., Palaniandavar M., Inorg. Chim. Acta, 254, 291—302
(2007).
(1997).
14) Winterbourn C. C., Biochem. J., 198, 125—131 (1981).
15) Geary W. J., Coord. Chem. Rev., 7, 81—122 (1971).
16) Zeng Y. B., Tang N., J. Inorg. Biochem., 97, 258—264 (2003).
17) Nakamato K., “Infrared and Raman Spectra of Inorganic and Coordi-
nation Compound,” 3rd ed., Wiley-Interscience, New York, 1978.
18) Li H., Le X. Y., Pang D. W., Deng H., Xu Z. H., Lin Z. H., J. Inorg.
40) Samuni A., Krisna M. C., “Handbook of Synthetic Antioxidants,” ed.
by Packer L., Cadenas E., Marcel Dekker, New York, 1997, pp. 351—
373.
41) Zhong Z. M., Xing R. G., Liu S., Li P. C., Eur. J. Med. Chem., (2007).
doi:10.1016/j.ejmech.2007.10.018.