Synthesis, crystal structure, antioxidant activity, and DNA-binding studies of a novel Ni(II) [2x2] grid complex with a rigid bistridentate Schiff base ligand

Article abstract of DOI:10.1248/cpb.58.1077

With a bistridentate Schiff-base ligand, N′,N′³- bis[(1E)-1-(2-pyridinyl)ethylidene)] isophthalohydrazide (H₂L), a [2x2]G grid complex, [Ni₄(HL)₄](ClO₄) ₄·4H₂O·0.5 CH₃OH (1) has been synthesized and characterized spectroscopically and crystallographically. Spectrometric titrations, ethidium bromide displacement experiments, circular dichroism spectral analysis and viscosity measurements indicate that the compound 1 strongly binds with calf-thymus DNA, presumably via intercalation mechanism. Furthermore, the antioxidant activity (superoxide and hydroxyl radical) of the ligand and its nickel(II) complex is determined by using spectrophotometer methods in vitro. Complex 1 is found to possess potent antioxidant activity and be better than standard antioxidants like mannitol.

Full text of DOI:10.1248/cpb.58.1077

August 2010
Note
Chem. Pharm. Bull. 58(8) 1077—1080 (2010)
1077
Synthesis, Crystal Structure, Antioxidant Activity, and DNA-Binding
Studies of a Novel Ni(II) [2؋2] Grid Complex with a Rigid Bistridentate
Schiff Base Ligand
Lei JIA,a,b Jun XU,a Xi-Ming XU,c Long-Hai CHEN,a Peng JIANG,c Fei-Xiang CHENG,a
Guang-Nong LU,a Qin WANG,*,c Jin-Cai WU,a and Ning TANG*,a
a Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, State Key Laboratory of
Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University; c School of Life
Science, Lanzhou University; Lanzhou 730000, P. R. China: and b Department of Physics and Chemistry, Henan
Polytechnic University; Jiaozuo 454000, P. R. China.
Received September 29, 2009; accepted May 12, 2010; published online May 13, 2010
With a bistridentate Schiff-base ligand, N؅,N؅3-bis[(1E)-1-(2-pyridinyl)ethylidene)] isophthalohydrazide
(H2L), a [2؋2]G grid complex, [Ni4(HL)4](ClO4)4·4H2O·0.5 CH3OH (1) has been synthesized and characterized
spectroscopically and crystallographically. Spectrometric titrations, ethidium bromide displacement experi-
ments, circular dichroism spectral analysis and viscosity measurements indicate that the compound 1 strongly
binds with calf-thymus DNA, presumably via intercalation mechanism. Furthermore, the antioxidant activity
(superoxide and hydroxyl radical) of the ligand and its nickel(II) complex is determined by using spectropho-
tometer methods in vitro. Complex 1 is found to possess potent antioxidant activity and be better than standard
antioxidants like mannitol.
Key words crystal structure; Ni(II) complex; intercalation binding; antioxidant activity
It is well known that deoxyribonucleic acid (DNA) plays
an important role in the life process since it contains all the
genetic information for cellular function.1) Investigations of
the interactions of DNA with transition metal complexes are
Fig. 1. The Structure of H2L
basis to design new types of the pharmaceutical molecules to
elucidate the mechanism involved in the site specific recogni-
tion of DNA and to determine the principles governing the over the whole experiment (24 h). It indicated that the Ni(II)
recognition.2,3) Schiff bases exhibit remarkable biological ac- complex is quite stable in solution. The molar conductivity
tivity and play an important role in bioinorganic chemistry. data is in accordance with the 1 : 4 type electrolyte.
For example, the Cr(III) complexes can lead to DNA dam-
Crystal Structure The crystal and experimental data is
age, plasmid cleavage, and protein cleavage.4,5) The acid presented in Table 1. The selected bond distances and angles
hydrazides possess R–CO–NH–NH2, a class of Schiff base, are presented in Table 2. Single-crystal X-ray analyses re-
their corresponding aroylhydrazones, R–CO–NH–NϭCH– vealed that complex 1 crystallized in the space group I4/m
RЈ, and the dependence of their mode of chelation with consisting of the cationic square, [Ni4H4L4]4ϩ. As shown in
transition metal ions present in the living system have been Fig. 2, in which the ligand H2L coordinates to metal ion, it
of significant interest.6) Various coordination compounds becomes negatively charged, HLϪ, and the ligands were di-
of aroylhydrazones have been reported to act as enzyme vided into pairs, one of which lies above and the other below
inhibitors and are useful due to their pharmacological the mean plane through the four metal ions, giving a novel
applications.7—9)
[2ϫ2] grids. The four Ni(II) atoms are distorted octahedral
Nickel is recognized as an essential trace element for coordinated by two acyloxy oxygen, two amide, and two
bacteria, plants, animals, and humans, though the role pyridine N atoms, evidenced by the angles O1–Ni1–N1
of this metal in animal biochemistry is still not well de- (154.3(2)°) and O1–Ni1–N2 (76.8(2)°). The distortion
fined.10) These facts encouraged us to synthesize a novel lig- mainly originates from the extremely rigid binding pockets
and NЈ,NЈ3-bis[(1E)-1-(2-pyridinyl)ethylidene)] isophthalo- present within each ligand, with two coplanar five-membered
hydrazide (H2L, Fig. 1) and their Ni(II) complex. In addition, chelate rings formed upon complexation, including one to
the DNA-binding properties of the complex [Ni4(HL)4] the smaller-sized pyrazolate heterocycle, and the resulting
(ClO4)4·4H2O·0.5 CH3OH (1) was discussed in detail.
proximity of the parallel ligand strands in the very compact
grid array. So, four Ni(II) atoms occupy the corners of a
[2ϫ2] grid with edge lengths Ni···Ni 8.486 Å. The
Results and Discussion
Synthesis and General Properties Complex 1 was pre- metal–metal diagonal distances are 11.465 Å for 1. The grids
pared in high yield from reactions of the Schiff base ligand are not perfectly square as can be seen by the dihedral angle
H2L in the presence of Ni(ClO4)2·6H2O. The UV–Vis inves- between the two chelating planes around each Ni(II) center
tigations revealed that the spectra of the Ni(II) complex in ranges from 87.01 to 92.69 Å for 1. The ligand molecules on
N,N-dimethylformamide (DMF) solution have remained un- opposite sides of the grids are also inclined to one another.
altered for the solutions and its molar conductance values This is shown by the dihedral angles involving the opposing
have no obvious change for very freshly prepared and for benzene rings, 9.14° for 1. Distances between some of the
∗ To whom correspondence should be addressed. e-mail: tangn@lzu.edu.cn; wangqin329@yahoo.com.cn
© 2010 Pharmaceutical Society of Japan

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Vol. 58, No. 8
Table 1. Crystal and Experimental Data
Empirical formula
Formula weight
C88.50H86Cl4N24Ni4O28.50
2318.47
Temperature
223(2) K
Wavelength
Crystal system
Space group
0.71073 Å
Tetragonal
I4/m
Unit cell dimensions (Å, °)
a
22.1357(5)
b
22.1357(5)
c
26.6683(12)
b
90
Volume (Å3)
Z
13067.2(7)
4
1.137
0.676
Dcalcd (g/cm3)
Absorption coefficient (mmϪ1
F(000)
)
Fig. 3. Packing in the Cationic Lattice of 1 Shows the Tetragonal Chan-
4596
nels
Range of h k l
Ϫ25/26, Ϫ26/24, Ϫ31/31
36384
5659/37/393
5659
0.9354, 0.6137
0.980
R1ϭ0.0742, wR2ϭ0.1139
0.530, Ϫ0.383
Reflections collected
Data/restraints/parameters
Independent reflections
Max. and min. transmission
Goodness-of-fit on F2
Final R indices [IϾ2s(I)]
Largest diff. peak and hole
Table 2. Selected Bond Distance and Angle (Å, °)
Ni(1)–N(5)a)
1.979(6)
1.981(6)
2.108(5)
2.067(4)
N(5)–Ni(1)b)
1.979(6)
2.108(5)
2.103(6)
2.067(4)
89.2(2)
154.3(2)
77.5(2)
99.2(2)
Ni(1)–N(2)
N(4)–Ni(1)b)
Ni(1)–N(4)a)
Ni(1)–N(1)
O(2)–Ni(1)b)
O(2)–Ni(1)b)
N(5)a)–Ni(1)–N(2)
N(5)a)–Ni(1)–O(2)a)
N(2)–Ni(1)–O(2)a)
N(5)a)–Ni(1)–O(1)
N(2)–Ni(1)–O(1)
O(2)a)–Ni(1)–O(1)
N(5)a)–Ni(1)–N(1)
N(2)–Ni(1)–N(1)
176.4(2)
O(2)a)–Ni(1)–N(1)
O(1)–Ni(1)–N(1)
N(5)a)–Ni(1)–N(4)a)
N(2)–Ni(1)–N(4)a)
77.6(2)
105.7(2)
104.6(2)
76.8(2)
92.0(2)
100.8(2)
78.1(2)
O(2)a)–Ni(1)–N(4)a) 155.0(2)
Fig. 4. Electronic Spectra of the Complex 1 in the Presence of 0—10 ml
1.0ϫ10Ϫ4 M CT-DNA
O(1)–Ni(1)–N(4)a)
N(1)–Ni(1)–N(4)a)
92.13(19)
97.6(2)
Arrow shows the emission intensities changes upon increasing DNA concentration.
Inset: plots of [DNA]/(eaϪef) vs. [DNA] for the titration of DNA to complex.
a) Ϫyϩ1/2, xϪ1/2, Ϫzϩ1/2.
aromatic rings are around 3.574 Å, indicative of p–p stack-
ing. The aromatic rings alongside one ligand “plane” show
some deviation from coplanarity, with dihedral angles be-
tween two pyridine rings of 9.15—21.79°. As shown in Fig.
3, the cationic squares in 1 are stacked in a parallel and over-
lapping fashion along the c direction to form large rhombic
channels which are filled with solvent molecules.
DNA-Binding Studies The ligand has no effect on the
absorption of DNA solutions. The absorption spectra of 1 in
the absence and presence of calf-thymus DNA (CT-DNA) is
given in Fig. 4. In the absence of CT-DNA, the UV–Vis
absorption spectra of the complex 1 has strong p–p* transi-
tions band at lmaxϭ296 nm and a strong n–p* transitions
band at lmaxϭ372 nm. With increasing DNA concentration,
the absorption bands of the compound show decreases in
molar absorptivity (hypochromism). Addition of DNA also
leads to changes in the position of absorption bands. The
296 nm band is red shifted by 6 nm in the presence of DNA.
These variations are strongly indicative of the intercalation
mode of the compounds with CT-DNA, involving a strong p-
stacking interaction between the compounds and DNA base
pairs.11) In order to study the binding ability of the com-
pounds with DNA quantitatively, the binding constant Kb was
Fig. 2. Cationic Molecular Square of Complex 1

August 2010
1079
determined using the equation in literature.12) In plots of
[DNA]/(eaϪef) versus [DNA], Kb is given by the ratio of
slope to the intercept. The binding constants Kb for 1 is found
to be 1.84ϫ106 MϪ1. The Kb value obtained here is higher
than the other mononuclear or dinuclear Ni(II) complexes
reported before.13)
In order to further investigate the interaction mode
between the complex 1 and CT-DNA, the fluorescence titra-
tion experiments are performed. The fluorescence intensity
of ethidium bromide (EB) at 584 nm show a remarkable
decreasing trend with the increasing concentration of the
complex 1, indicating that some EB molecules are released
from EB–DNA after an exchange with the complex 1 which
results in the fluorescence quenching of EB. We assume it
may be due to the metal complex competing with EB for the
DNA binding sites thus displacing the EB, which implies that
complex 1 binds more strongly to DNA than EB at 50 mM
NaCl concentration. The quenching plots determined by lit-
erature illustrate that the quenching of EB bound to DNA by
the complexes is in good agreement with the linear
Stern–Volmer equation.14) The Kq value of Ni(II) complex is
1.52ϫ105 MϪ1. This is in accordance with the above absorp-
tion titration result.
The circular dichroism (CD) spectrum of CT-DNA was
monitored in the presence of 1. On addition of the complex 1
to CT-DNA, it shows faint red shift with intensity increase in
the positive band. This observation is supportive of the inter-
calative mode of binding of the complex 1, where in the
stacking of the complex molecules between the base pairs of
DNA leads to an enhancement in the positive band and the
partial unwinding of the helix is reflected in the decreased in-
Fig. 5. (a) Effect of Tested Complex 1 and Ligand on OϪ2 ·, (b) Effect of
Tested Complex 1 and Ligand on OH·
Experiments were performed in triplicate. Values are expressed as meanϮstandard
deviation (nϭ3)
tensity of the negative band, which attributed to a strong con- also in a concentration-dependent manner (Fig. 5b). The
formational change in DNA helix. The results obtained here complex 1 shows highly active scavenging effect on OH·.
validate those obtained from the UV–Vis spectral studies.
Moreover, mannitol is a well-known natural antioxidant, so
The effects of the compounds together with EB on the vis- we also studied the scavenging activity of mannitol against
cosity of DNA at 25.0Ϯ0.1 °C are investigated. It is found hydroxyl radical using the same model. We find that when
that the viscosity of DNA increases steadily with the increase arrive at similar suppression ratio, concentration of complex
of the concentration of the compound, which is similar to 1 is far less than that of mannitol. The suppression ratio take
that of a classical intercalator EB.15) This result demonstrates the order of 1ϾH2L. The metal complex we studied in this
that the complex 1 and EB bind to DNA through the classical paper deserve to be further researched.
intercalation mode, which also parallels the pronounced
Experimental
hypochromism and spectral red shift of the complex in the
absorption spectrum experiment.
Materials Nitroblue tetrazolium (NBT), methionine (MET), vitamin B2
(VitB2) were purchased from Sigma Chemical Co. Calf thymus DNA (CT-
DNA) and ethidium bromide (EB) were obtained from Sigma Chemical Co.
All the experiments were carried out in doubly distilled water buffer contain-
ing 5 mM Tris[Tris(hydroxymethyl)-aminomethane] and 50 mM NaCl, and
adjusted to pH 7.2 with hydrochloric acid. The concentration of DNA solu-
tion was determined from UV absorption at 260 nm using a molar absorp-
Antioxidant Activity As can be seen in Fig. 5a, the
inhibitory effect of the tested complexes on OϪ2 · is concentra-
tion related. The antioxidant activities of the complex and
ligand are expressed as 50% inhibitory concentration (IC50 in
mM). IC50 values of 1 is 12.53Ϯ0.32 mM. The IC50 value of
the ligand can not be read in Fig. 5a. It is clear that the scav-
enger effect on OϪ2 · can be enhanced by the formation of
metal–ligand coordination complexes and the nature of the
metal ion also affects the ability. Some complexes are better
effective inhibitor for O2Ϫ· than that of the nitroxide Tempo
(IC50ϭ60Ϯ3.10 mM) which has been recently used in biologi-
cal system for its capacity to mimic superoxide dismutase.16)
tion coefficient e260ϭ6600 molϪ1 cmϪ1
.
Physical Measurements The UV–Vis absorption measurements were
conducted by using a Varian Cary 100 spectrophotometer equipped with
quartz cells. All fluorescence emission spectra were measured using a Hi-
tachi F-4500 spectrofluorophotometer equipped with a xenon lamp source
and a quartz cell of 1 cm path length. Viscosity experiments were carried out
on an Ubbelodhe viscometer. The CD spectra were recorded on a Jasco
J-810 spectropolarimeter. The elemental analyses were performed in the
microanalytical laboratory, Department of Chemistry, National University of
1
Although superoxide is a relatively weak oxidant, it decom- Singapore. The H-NMR spectra were recorded with a Bruker ACF300 FT-
NMR instrument using tetramethylsilane (TMS) as an internal reference in
poses to form stronger relative oxidative species, such as sin-
D2O for the ligand. Electrospray ionization (ESI) mass spectra were
recorded on a Finnigan MAT LCQ mass spectrometer using the syringe
pump method. All conductivity measurements were performed in N,NЈ-
dimethyl formamide (DMF) with a DDS-11A conductor at 25 °C. The
antioxidant activites were tested on a 721E spectrophotometer (Shanghai
gle oxygen and hydroxyl radicals, which initiate peroxidation
of lipids.17) These results show that complex 1 has significant
scavenging activity of superoxide radical.
We can find that the complex and ligand scavenge OH·

1080
Vol. 58, No. 8
Analytical Instrument Factory, China).
tained following reagents: safranin (11.4 mM), ethylenediaminetetraacetic
acid (EDTA)-Fe(II) (40 mM), H2O2 (1.76 mM), the tested compound (5—
30 mM) and a phosphate buffer (67 mM, pH 7.4). The assay mixtures were in-
cubated at 37 °C 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
meanϮstandard deviation (S.D.). The suppression ratio for OH· was calcu-
lated from the following expression:
DNA Binding Experiment Methods Absorption titration experiments
were performed by fixing concentration of 1 as constant at 10 mM while
varying the concentration of CT-DNA (10Ϫ4 mol/l). Fluorescence spectra of
the competitive binding experiments were carried out by maintaining the EB
and CT-DNA concentration at 3 mM and 30 mM, respectively, while increasing
the concentrations of the compound. Viscosity experiments were carried out
on an Ubbelodhe viscometer, immersed in a thermostated water-bath main-
tained at 25.0Ϯ0.1 °C. Titrations were performed for the compounds (1—
5 mM), and each compound was introduced into DNA solution (50 mM) pres-
ent in the viscometer. The CD spectra of DNA were recorded on a Jasco
J-810 spectropolarimeter at 25.0Ϯ0.1 °C. CT-DNA used were 200 mM in
concentration and compounds solutions was added to a ratio of 1 : 1 (DNA/
compound).
X-Ray Crystallography A suitable brown block-shaped single crystal
with dimensions of 0.80ϫ0.40ϫ0.10 mm3 was mounted in a sealed tube for
data collection which was performed on a Bruker AXS SMART CCD dif-
fractometer equipped with a graphite-monochromatic MoKa radiation
(lϭ0.71073 Å) at 223(2) K. Unit cell dimensions were obtained with least-
squares refinements, and all structures were solved by direct methods. The
program SMART was used to collect the intensity data,12) SAINT for inte-
gration of the intensity,18) SADABS for absorption correction and
SHELXTL for structure solution and refinements on F2.19,20) The phenyl ring
was disordered into two positions with occupancy ratio 63 : 37. There are lot
of residual peaks which were too weak and could not be characterised. H
atom of N3 was located from different map and refined with restraints in
bond length and parameters. The H atoms of the solvent molecules were not
located.
suppression ratio (%)ϭ[(AiϪA0)/(AcϪA0)]ϫ100
(2)
(Where Aiϭthe absorbance in the presence of the tested compound; A0ϭthe
absorbance in the absence of the tested compound; Acϭthe absorbance in the
absence of the tested compound, EDTA-Fe (II), H2O2.)
Acknowledgements This work was supported by the Fundamental Re-
search Funds for the Central Universities and the Ministry of Education,
Singapore through NUS (FRC Grant No. R143-000-371-112 ).
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Synthesis of the Ligand and the Complex The ligand H2L was pre-
pared as described previously.21) In short, H2L was obtained by the conden-
sation of 2-acetylpyridine and isophthalic hydrozide in ethanol solution with
the addition of several drops of conc. HCl. The Ni(II) complex 1,
[Ni4(HL)4](ClO4)4·4H2O·0.5 CH3OH, was prepared by the reaction of
Ni(ClO4)2·6H2O and H2L. To a solution of H2L (0.2 g, 0.5 mmol) in 5 ml
CH3OH and 5 ml CHCl3 was added a solution of Ni(ClO4)2·6H2O (0.18 g,
0.5 mmol) in 5 ml CH3OH. This reaction mixture was stirred for 1 h, a brown
precipitate was filtered off and washed with cold methanol. Yield: 0.23 g
(60.2 %). Anal. Calcd for C85.5H86Cl4Ni4N24O28.5: C, 44.85; H, 3.74; N,
14.50. Found: C, 44.52; H, 3.56; N, 14.68. Brown single crystals of complex
1 suitable for X-ray study was obtained by slow evaporation of the mother
solution. The complex is soluble at room temperature in methanol, DMF and
dimethyl sulfoxide (DMSO).
Superoxide Radical Scavenging Assay The superoxide radicals (O2Ϫ·
)
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.22) 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 compound (5—30 mM) and a phos-
phate buffer (67 mM, pH 7.8). After illuminating with a fluorescent lamp at
30 °C for 10 min, the absorbance of the samples (Ai) was measured at
560 nm. The suppression ratio for OϪ2 · was calculated from the following ex-
pression:
suppression ratio (%)ϭA0ϪAi/A0ϫ100
(1)
Drug activity was expressed as the 50% inhibitory concentration (IC50). IC50
values were calculated from regression lines where: x was the tested com-
pound concentration in mM and y was percent inhibition of the tested com-
pounds.
Hydroxyl Radical Scavenging Assay The hydroxyl radicals (OH·) in
aqueous media were generated through the Fenton system.23) The solution of
the tested compound was prepared with DMF. The 5 ml assay mixture con-