Transition metal complexes of 2, 6-di ((phenazonyl-4-imino) methyl)-4-methylphenol: Structure and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2011.02.012

A symmetric ligand 2, 6-di ((phenazonyl-4-imino)methyl)-4-methylphenol (Dpmp) and its cobalt dinuclear complex (Co₂(Dpmp) ₂(NO3)₂(H₂O)₂·NO3· EtOH, (1) and zinc mononuclear complex Zn(Dpmp)(NO₃)₂, (2) have been prepared. The crystal structures were determined by single-crystal X-ray diffraction. The biological activity has been evaluated by examining their anti-oxidative activity and ability to bind to bovine serum albumin (BSA) and calf-thymus DNA (CT DNA) with UV-vis absorption, fluorescence, viscosity measurements and circular dichroism (CD) spectroscopies. The complexes exhibit good binding propensity to BSA and CT DNA. Both 1 and 2 have been found to promote cleavage of pUC19 DNA in the absence of any reducing agent. Antioxidant tests in vitro show the compounds possess significant antioxidant activity against superoxide and hydroxyl radicals.

Full text of DOI:10.1016/j.ejmech.2011.02.012

European Journal of Medicinal Chemistry 46 (2011) 1638e1647
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Transition metal complexes of 2, 6-di ((phenazonyl-4-imino)
methyl)-4-methylphenol: Structure and biological evaluation
,
Hongyan Liu a, Xiaoyan Shi b, Min Xu a, Zhengpeng Li a, Liang Huang a, Decheng Bai c, ZhengZhi Zeng a
*
a Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering,
Lanzhou University, Lanzhou 730000, PR China
b Department of Prosthodontics, School of Stomatology, Lanzhou University, Lanzhou 730000, PR China
c Key Laboratory of Pre-clinical Study for New Drugs of Gansu Province, Basic Medical College, Lanzhou University, Lanzhou 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A symmetric ligand 2, 6-di ((phenazonyl-4-imino)methyl)-4-methylphenol (Dpmp) and its cobalt
dinuclear complex (Co2(Dpmp)2(NO3)2(H2O)2$NO3$EtOH, (1) and zinc mononuclear complex Zn(Dpmp)
(NO3)2, (2) have been prepared. The crystal structures were determined by single-crystal X-ray diffrac-
tion. The biological activity has been evaluated by examining their anti-oxidative activity and ability to
bind to bovine serum albumin (BSA) and calf-thymus DNA (CT DNA) with UVevis absorption, fluores-
cence, viscosity measurements and circular dichroism (CD) spectroscopies. The complexes exhibit good
binding propensity to BSA and CT DNA. Both 1 and 2 have been found to promote cleavage of pUC19 DNA
in the absence of any reducing agent. Antioxidant tests in vitro show the compounds possess significant
antioxidant activity against superoxide and hydroxyl radicals.
Received 4 November 2010
Received in revised form
26 December 2010
Accepted 8 February 2011
Available online 15 February 2011
Keywords:
Transition metal complexes
Bovine serum albumin binding
DNA interaction
Ó 2011 Elsevier Masson SAS. All rights reserved.
Cleavage
1. Introduction
which have different coordination geometry contrast to the
reported structures. This may result in new biological activities. As
The phenazone-type pharmaceuticals dimethylaminophena-
zone (DMAA), metamizole, phenazone and propyphenazone have
widely been used in medical care [1]. For example, phenazone is
used as an analgesic either alone or in combination with other
drugs. Furthermore, a great many phenazone complexes have also
provoked a great interest in their diverse spectra of biological and
pharmaceutical activities, such as antibacterial and antineoplastic
activities [2]. In addition, metal complexes that bind and cleave
DNA or proteins under physiological conditions are of current
interests for their varied applications in nucleic acid and protein
chemistry [3e7]. Transition metal complexes are being used at the
forefront of many of these efforts. Stable, inert, and water-soluble
complexes containing spectroscopically active metal centers are
extremely valuable as probes for biological systems. Previous study
revealed that transition metal complexes of the Schiff base of
4-amino-3-antipyrine show significant antibacterial activity [8].
However, the crystal structures and interactions with DNA or BSA of
these complexes were not studied. At present, we synthesized the
dinuclear (Co(II)) and mononuclear complex (Zn(II)) of the ligand 2,
6-di ((phenazonyl-4-imino) methyl)-4-methylphenol (Dpmp)
we all know, serum albumins are proteins involved in the transport
of metal ions and metal complexes with drugs through the blood
stream [9,10]. Many drugs, including anticoagulants, tranquilizers,
anti-inflammatory, and general anesthetics, are transported in the
blood via combination with albumin [11]. The nature and magni-
tude of drugealbumin interactions significantly influence the
pharmacokinetics of drugs, and the binding parameters are useful
in studying proteinedrug binding as they greatly influence
absorption, distribution, metabolism, and excretion properties of
typical drugs [11,12]. On the other hand, DNA is the material of
inheritance and controls the structure and function of cells [13].
Binding studies of small molecules to DNA are very important in the
development of DNA molecular probes and new therapeutic
reagents. The study on the cleavage capacity of complex to DNA is
considerably interesting as it can contribute to understanding their
toxicity mechanism and to develop novel artificial nuclease. All
these aroused our interest in the study of the new complexes of
Dpmp with a view towards evaluating the binding behaviors of
these compounds with CT DNA, BSA and exploring their anti-
oxidative abilities.
In this paper, the ligand Dpmp and its dinuclear (Co(II)) and
mononuclear (Zn(II)) complexes have been synthesized and char-
acterized. The affinity of the complexes for BSA and CT DNA has
been investigated. The DNA cleavage study was carried out at
* Corresponding author. Tel./fax: þ86 931 8912582.
E-mail address: zengzhzh@yahoo.com.cn (ZhengZhi Zeng).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.012

H. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 1638e1647
1639
physiological pH and temperature. The anti-oxidative activity of the
complexes has been evaluated by determining the IC50 values
against the superoxide radicals (Oꢀ2 :) and Hydroxyl radicals (OH$).
2. Results and discussion
2.1. Synthesis and characterization of the complexes
The complexes were prepared by direct reaction of the ligand
(Dpmp) with appropriate mole ratios of M(II) (M ¼ Co and Zn)
nitrate in ethanol. The transition metal complexes were stable
in atmospheric conditions for extended periods, and are easily
soluble in dimethylsulfoxide (DMSO), chloroform and N, N-dime-
thylformamide (DMF), partially in ethanol and methanol, and
insoluble in water, benzene and ethylacetate. The IR spectra of the
complexes are similar to the ligand. The bands at the 3456 cmꢀ1 is
(eOH) vibration in the ligand. In the complexes these bands are
presented at 3430 and 3414 cmꢀ1
Dn (ligandecomplexes) is at 16
and 42 cmꢀ1. The
(C]N) vibration of the free ligand Dpmp is at
1587 cmꢀ1; for the complexes these peaks appear at 1535 and
n
,
n
1538 cmꢀ1. The (C]O) vibration of the free ligand is at 1657 cmꢀ1
n ;
for the complexes these peaks appear at 1627 and 1635 cmꢀ1. These
shifts demonstrate that the ketone oxygen and N atom of the C]N
have formed a coordination bond with the metal ion. The band at
539 cmꢀ1 for both complexes 1 and 2 is assigned to
n(MeO).
2.2. X-ray structure characterization
Fig. 1. The molecular structure and partially labeling of 1 (a); the coordination poly-
hedron of 1 (b).
The X-ray crystallography data of complexes 1 and 2 were
collected by the
u
scan mode within 1.72ꢁ <
q
< 25.80ꢁ for hkl
< 25.48ꢁ for
(ꢀ12 ꢂ h ꢂ 9, ꢀ14 ꢂ k ꢂ 14, ꢀ15 ꢂ l ꢂ 17) and 1.66ꢁ <
q
hkl (ꢀ9 ꢂ h ꢂ 12, ꢀ17 ꢂ k ꢂ 18, ꢀ24 ꢂ l ꢂ 24), respectively. The
complex 1 crystallized in a triclinic lattice with space groups P ꢀ 1
while 2 in a monoclinic lattice with space group P2(1)/n. Each unit
cell contains two or four molecules (Fig. S1).
For complex 1, as Fig. 1a shows, it consists of [Co2(Dpmp)2
(NO3)2(H2O)2]þ, a nitrate ion and an ethanol molecule. Single-
crystal X-ray diffraction studies shows that the two Co atoms
exhibit identical coordination environment, the metal centre
allows the formation of a hexa-coordination environment with
three oxygen atoms and one nitrogen atom from two Dpmp
ligands, one oxygen atom of water molecule and the remaining one
oxygen atom from the nitrate ion. The geometry can be described as
a distorted octahedron (Fig. 1b). For O(1), O(2), N(3), O(7) and Co(1)
atoms form the basal plane with Co(1)-O(1) 2.092(2), Co(1)-O(2)
2.019(2), Co(1)-N(3) 2.100(3) and Co(1)-O(7) 2.108(3) Å, the apical
positions are occupied by O(4) with a Co(1)-O(4) distance of 2.169
(3) Å and O(3)A with a Co(1)-O(3)A distance of 2.194(2) Å.
For complex 2, as Fig. 2a shows, it consists of a neutral [Zn
(Dpmp)(NO3)2] unit. The zinc atoms in complex 2 have a hexa-
coordinated geometry (Fig. 2b) with two oxygen atoms and one
nitrogen atom of Dpmp and three oxygen atoms of two nitrate ions.
The geometry can be described as a distorted octahedron. For O(1),
O(2), O(4), O(7) and Zn(1) atoms form the basal plane with Zn(1)-O
(1) 2.151(2), Zn(1)-O(2) 2.013(2), Zn(1)-O(4) 2.365(3) and Zn(1)-O
(7) 2.032(3) Å, the apical positions are occupied by O(5) with a Zn
(1)-O(5) distance of 2.050(2) Å and N(3) with a Zn(1)-N(3) distance
of 2.194(2) Å.
2.3. Binding of serum albumins
2.3.1. Quenching mechanism of BSA fluorescence by 1 and 2
Serum albumins, BSA and HSA, are most widely studied abun-
dant proteins in plasma [14,15]. As the major soluble protein
constituents of the circulatory system, they contribute to colloid
Fig. 2. The molecular structure and partially labeling of 2 (a); the coordination poly-
hedron of 2 (b).

1640
H. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 1638e1647
osmotic blood pressure and are chiefly responsible for the main-
tenance of blood pH [16]. BSA is the most extensively studied serum
albumin, due to its structural homology with HSA [17]. A useful
feature of the intrinsic fluorescence of proteins is the high sensi-
tivity of tryptophan and its local environment. Changes in the
emission spectra of tryptophan are common in response to protein
conformational transitions, subunit associations, substrate binding,
or denaturation [17,18]. Therefore, the intrinsic fluorescence of
proteins can provide considerable information on their structure
and dynamics and is often utilized in the study of protein folding
and association reactions. The interaction of the complexes with
BSA has been studied from tryptophan emissionequenching
experiments.
Addition of the complexes to BSA results in a significant
decrease of the fluorescence intensity of BSA at 340 nm (Fig. 3), up
to 72% of the initial fluorescence intensity of BSA for 1, 55% for 2,
which indicates that there were interactions between the
complexes and BSA. UVevis spectra of BSA in the absence and
presence of complexes 1 and 2 (Fig. S2) show that the absorption
intensity of BSA was enhanced as the complexes increased, and
there was a little blue shift. As is well known, dynamic quenching
only affected the excited state of fluorophores but did not change
the absorption spectrum. However, the formation of a non-
fluorescence ground state complex induced a change in the
absorption spectrum of fluorophores, therefore, the possible
quenching mechanism of BSA by both complexes 1 and 2 are static
quenching processes [19].
To elucidate further the quenching mechanism, fluorescence
quenching data were analyzed with the SterneVolmer equation. All
of the SterneVolmer plots of BSA-1 and BSA-2 systems at different
temperatures follow a linear relation (inset in Fig. 3), this suggests
that the quenching type was probably single quenching (static or
dynamic quenching). The values for kq (Table 1) are three orders of
magnitude greater than the maximum diffusion collision quench-
ing rate constant (2.0 ꢃ 1010 L molꢀ1 sꢀ1) for a variety of quenchers
with biopolymers [20]. These results indicate that the quenching
was not initiated from dynamic collision but static quenching. The
binding constants KA are increased with the rising temperature,
which indicated that the stability of the state complexes increases
with increasing temperature [21]. The Ksv value of 1 is larger than
Ksv of 2 at any temperature, indicating that the interaction between
1 and BSA is stronger than 2.
2.3.2. The binding parameters
Based on the plot of log(F0 ꢀ F)/F versus log(1/([Dt] e (F0 ꢀ F)
[Pt]/F0)) (Fig. 4), the number of binding sites n and binding constant
KA can be obtained, as presented in Table 1. The values of n
approximately equal to 1 indicate the existence of just a single
binding site in BSA for the complexes.
Thermodynamic parameters for a binding interaction can be
used as major evidence for the nature of intermolecular forces [22].
Among these parameters, the free energy change
possibility of reaction, and the enthalpy change
change S are the main evidence for determining acting forces. To
obtain such information, the thermodynamic parameters were
calculated from the Van’t Hoff equation. If H z 0, S > 0, the main
force is a hydrophobic interaction; if H < 0, S > 0, the main force
is an electrostatic effect; and if H < 0, S < 0, Van der Waals and
D
G reflects the
D
H and entropy
D
D
D
D
D
D
D
hydrogen bond interactions play major roles in the reaction [23].
The values of thermodynamic parameters are shown in Table 1,
where the negative sign for
binding of 1 and 2 with BSA.
D
D
G indicates the spontaneity of the
H is small positive value and S is
D
positive value, indicating that the main binding force between the
complexes and BSA is hydrophobic interaction.
According to the Förster nonradiative energy-transfer theory
[24], the efficiency of energy transfer is E, the critical distance for
50% energy transfer is R0, and the actual distance of separation is r.
These values were calculated using Eqs. (1)e(3):
R06
R60 þ r6
F
F0
E ¼ 1 ꢀ
¼
(1)
ꢀ4
R60 ¼ 8:78 ꢃ 10ꢀ25K2n
f
J
Trp
(2)
where K2 is the orientation factor, fTrp is the quantum yield of the
donor tryptophan in the absence of acceptor, n is the refractive
index of the medium intervening between the donor and acceptor,
and J is the spectral overlap integral defined by Eq. (3):
X
X
J ¼
FðvÞ
3
ðvÞvꢀ4Dv=
FðvÞ
D
v
(3)
where F(
n
) is the fluorescence intensity of the donor,
3
(n
) is the
molar extinction coefficient of the acceptor in units of Mꢀ1 cmꢀ1
,
Fig. 3. Fluorescence spectra of BSA after excitation at 280 nm at pH 7.4 and 288 K at
and n
is the frequency in cmꢀ1. The fluorescence emission spectrum
increasing amounts (0e6.25 mM) of the complexes (a) 1 and (b) 2. The arrows show the
changes upon increasing amounts of complex. Inset: plots of F0/F for BSA against [Q] of
of BSA and the UV absorption spectrum of 1 and 2 are shown
the complex at different temperatures.
in Fig. 5, which shows that they have some overlap. The value of

H. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 1638e1647
1641
Table 1
Binding parameters for the BSA-complex system.
Complex
1
2
T(K)
288 ꢄ 0.2
3.60 ꢄ 0.23
3.60 ꢄ 0.23
4.49 ꢄ 0.13
0.95
0.997
ꢀ31.16
298 ꢄ 0.2
4.34 ꢄ 0.13
4.34 ꢄ 0.13
5.28 ꢄ 0.07
1.04
10.986
ꢀ32.65
9.135
310 ꢄ 0.2
5.47 ꢄ 0.46
5.47 ꢄ 0.46
6.09 ꢄ 0.21
0.99
0.993
ꢀ34.43
288 ꢄ 0.2
1.94 ꢄ 0.04
1.94 ꢄ 0.04
2.21 ꢄ 0.06
0.92
0.996
ꢀ29.47
298 ꢄ 0.2
2.19 ꢄ 0.10
2.19 ꢄ 0.10
2.34 ꢄ 0.02
0.87
310 ꢄ 0.2
Ksv (ꢃ105 L molꢀ1
)
2.33 ꢄ 0.01
2.33 ꢄ 0.01
2.69 ꢄ 0.13
0.79
Kq (ꢃ1013 L molꢀ1 sꢀ1
)
KA (ꢃ105 L molꢀ1
)
n
Ra
0.998
0.999
ꢀ32.22
D
D
D
E
G(kJ molꢀ1
)
)
ꢀ30.63
H(kJ molꢀ1
S(J molꢀ1 Kꢀ1
8.921
)
139.91
140.22
0.2933
3.27
140.21
134.20
132.72
0.2471
2.81
132.71
R0 (nm)
r (nm)
3.78
3.39
a
R is the correlation coefficient.
J for 1 was 4.4064 ꢃ10ꢀ14 cm3L molꢀ1 and for 2 was 1.791 ꢃ
10ꢀ14 cm3L molꢀ1. K2 was taken as 2/3. n Was taken as 1.36 [24].
fTrp was determined in the study to be 0.15 [22]. With use of the
values of J, K2, n, and fTrp, the R0, E and the actual distance r-value
was calculated (Table 1). BSA has two tryptophan residues: Trp-212
is located in a hydrophobic fold and the additional tryptophan
(Trp-134) is located on the surface of the molecule. In this study, the
compounds were probably bound to the Trp-212 residue mainly
through the hydrophobic interaction according to the thermody-
namic results and the number of binding sites n. The actual distance
r is 3.78 nm for 1 and 3.39 nm for 2, indicating the energy transfer
from BSA to 1 and 2 occurs with very high probability.
to 55.0% for 1 and to 61.6% for 2 at the molar ratio complex/BSA of
1:1. The marked decrease probably occurred because of the strong
interaction between the complexes and BSA, with the order 1 > 2,
which can be confirmed by the value of the binding constant KA.
This decreased helicity suggests that the binding of complexes with
BSA induces a slight unfolding of the constitutive polypeptides of
the protein, resulting in a conformational change in the protein
which increased the exposure of some hydrophobic regions that
were previously covered (Fig. 6b).
2.4. Interaction with DNA
2.4.1. Fluorescence spectra
2.3.3. Effects of 1 and 2 on BSA conformation
The enhancements in the emission intensity of 1, 2 and free
ligand (Dpmp) with increasing CT DNA concentrations are shown in
Fig. S3. In the absence of DNA, 1, 2 and Dpmp emit weak lumi-
nescence in Tris buffer at ambient temperature, with a maximum
appearing at 529 nm. When DNA is present the intensity of the
emission for 1, 2 and Dpmp all increase with respect to DNA
concentration. This phenomenon is related to the extent to which
the complexes penetrate into the hydrophobic environment inside
the DNA, thereby avoiding the quenching effect of solvent water
molecules. The marked increase in the emission intensity agrees
with those observed for other intercalators [26].
Further experiments were carried out on the CD spectra to verify
the binding process. As Fig. 6a shows, the CD spectra of BSA
exhibited two negative bands in the UV region at 208 and 222 nm,
characteristic of the
binding of complexes to BSA caused a decrease in band intensity in
the ultra-UV CD, clearly indicating a decrease in the -helical
content in the protein. The average fractions of secondary structure
estimated from the CD data are given in Table 2. The regular -helix
aR) content was 42.3% and the distorted -helix (aD) content was
a-helical structure of the protein [25]. The
a
a
(
a
22.9% in the BSA, while the aR content was reduced to 37.4% and the
distorted aD content decreased to 17.6%, when the complexes bind
to the complex 1. For complex 2, aR and aD contents are reduced to
40.1% and 21.5%, respectively. Moreover, the binding of the two
2.4.2. Electronic absorption titration
UV spectroscopic titration is an effective method to examine the
binding mode of DNA with metal complexes [27] since the
observed changes of the spectra may give evidence of the existing
interaction and its mode [28]. In general, hyperchromism and
complexes to BSA arouses the increase of the
turn (T), and unordered (U) structure. The calculated results
exhibited a reduction of -helix (aR and aD) structures from 65.2%
b-strand (bR and bD),
a
Fig. 4. Plots of log(F0 ꢀ F)/F vs. log(1/([Dt] ꢀ (F0 ꢀ F)[Pt]/F0)) for the BSA-complex system at different temperatures. (a) 1 and (b) 2.

1642
H. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 1638e1647
Table 2
Average estimates of the secondary structure fractions of BSA obtained with the
CDPro software package after reaction with complexes 1 and 2.
Compound
Fraction of secondary structure, %
aR
aD
bR
bD
T
U
BSA
42.3
37.4
40.1
22.9
17.6
21.5
5.5
6.1
5.6
4.2
6.4
4.4
7.1
10.5
9.1
18.1
22.6
19.3
BSA þ 1
BSA þ 2
hypochromism are the spectral features of DNA concerning changes
of its double helix structure; hyperchromism means the breakage
of the secondary structure of DNA and hypochromism shows that
binding of complex to DNA can be due to electrostatic effect or
intercalation which may stabilize the DNA duplex. Additionally, the
existence of a red-shift is indicative of stabilization of DNA duplex
[17,29]. The electronic absorption spectra of the ligand and its
complexes 1, 2 in the absence and presence of the CT DNA (at
a constant concentration of the compounds) were obtained (Fig. 7).
In the UV spectrum of Dpmp, 1 and 2, the band centered at 335 nm
exhibits a hypochromism of 13.4%, 30.1%, 49.4%, respectively. This
indicates tight binding possibly by intercalation. Kb values of Dpmp,
1 and 2 were 1.60(ꢄ0.27) ꢃ 105 Mꢀ1, 8.02(ꢄ0.18) ꢃ 105 Mꢀ1 and
4.51(ꢄ0.31) ꢃ 105 Mꢀ1, respectively, (inset in Fig. 7). The Kb values
of complexes are higher than that of ligand suggesting that the
affinity of CT DNA is enhanced, when it is coordinated to Co(II) and
Zn(II). It is noteworthy that the Kb values are higher than the
binding affinity of EB for DNA, (Kb ¼ 1.23(ꢄ0.07) ꢃ 105 Mꢀ1) [30],
suggesting that the existing interactions may cause EB displace-
ment from its complex with DNA [30]. The competitive studies with
EB are not carried since the ligand and complexes exhibit fluores-
cence emission at 545 nm when excited at 500 nm, which is close
to the emission of DNAeEB system (lem ¼ 594 nm). The results
derived from the fluorescence and UV titration experiments
suggest that both the ligand and complexes can bind tightly to CT
DNA by intercalation.
Fig. 5. Overlap of fluorescence spectra of BSA and UV absorption spectrum of (a) 1 and
(b) 2. F: Fluorescence spectrum of BSA (1.50
complexes (1.50 M).
mM); U: UV absorption spectrum of the
m
2.4.3. Viscosity titration measurements
To further clarify the interactions between the studied
compounds and CT DNA, viscosity measurements were carried out.
Measurements of DNA viscosity that is sensitive to DNA length are
regarded as the least ambiguous and the most critical tests of
binding in solution in the absence of crystallographic structural
data [31,32]. A classical intercalation mode demands that the DNA
helix must lengthen as base pairs are separated to accommodate
the binding complexes, leading to the increase of DNA viscosity, as
for the behaviors of the known DNA intercalators [33]. In contrast,
a partial and/or non-classical intercalation of the complex could
bend (or kink) the DNA helix, reducing its viscosity concomitantly
[34]. The effects of all the compounds on the viscosity of CT DNA are
shown in Fig. S4. The viscosities of the DNA increase steadily with
increasing concentrations of ligand and complexes 1 and 2, and the
extent of the increase observed for the ligand is smaller than that
for complexes. Viscosity measurements clearly show that all the
compounds can intercalate between adjacent DNA base pairs,
causing an extension in the helix and thus increase the viscosity of
DNA, and that the complexes can intercalate a little more strongly
and deeply than the free ligand. The results obtained from the
viscosity experiments validate those obtained from the spectro-
scopic studies.
2.4.4. Cleavage of pUC19 DNA
Fig. 6. (a) CD spectra of the BSA-complex system at room temperature (in cells of
1 mm path length). a: 1.50 BSA; b: 1.50 2; c: 1.50
BSA þ 1.5
BSA þ 1.5 M 1. (b) Schematic model showing the effect of ligands on the tryptophan
environment in protein.
The study on the cleavage capacity of transition metal complex
to DNA is considerably interesting as it can contribute to under-
standing the toxicity mechanism of them and to develop novel
m
M
mM
mM
mM
m

H. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 1638e1647
1643
Fig. 8. Cleavage of pUC19 DNA (12
m
M) by the complexes 1 and 2 in TriseHCl/NaCl
buffer (pH 7.2) at 37 ꢁC for 5 h: lane 1: DNA control; lane 2: DNA þ 10
mM 1; lane 3:
DNA þ 20
m
M 1; lane 4: DNA þ 30
m
M 1; lane 5: DNA control; lane 6: DNA þ 10
mM 2;
lane 7: DNA þ 20
m
M 2; lane 8: DNA þ 30
mM 2.
SC DNA to its nicked circular (NC) form, while 30 mM of 2 cleaved
>60% of SC, the cleaved amount was enhanced with the increase of
the concentration of the complex, showing the potential chemical
nuclease activity of the complexes. Furthermore, 1 showed higher
cleavage activity than 2 at the same concentration, which probably
determined by 1 is a dinuclear complex but 2 is mononuclear.
Therefore, hydrolytic cleavage of DNA because of high Lewis acidity
of these metal ions probably occurred.
2.5. Anti-oxidative activity
Generation of reactive oxygen species (ROS) is a normal process
in the life of aerobic organisms. Oxidative stress or excessive
production of ROS is being implicated in many diseases [35]. (O2ꢀ:
)
and (OH$) are two clinically important ROS in the human body [34].
They are produced in most organ systems and participate in various
physiological and pathophysiological processes such as carcino-
genesis, aging, viral infection, inflammation, and others [36].
Consequently, in this paper, the M (M ¼ Cu and Zn) salts, the ligand
and its complexes 1 and 2 were studied for their antioxidant
activity by comparing their scavenging effects on Oꢀ2 : and OH$. Fig. 9
shows the plots of suppression ratio (%) for Oꢀ2 : and OH$, indicating
that the average suppression ratio increases with increasing
concentration of the compounds. The value of IC50 of Dpmp, 1 and 2
for Oꢀ2 : are 49.2
order of 2 > Dpmp > 1. Complex 1 (IC50 ¼ 39.1
effective of the three compounds for scavenging Oꢀ2 :. The order of
IC50 for OH$ was 2 (50.9
M) > 1 (43.1 M) > Dpmp (30.1 M). It is
mM, 39.1
mM and 55.8
mM, respectively, with the
mM) is the most
m
m
m
proved that the hydroxyl radical scavenging effects of ligand are
higher than that of the complexes. Complex 1 was found to be
a higher effective inhibitor for both Oꢀ2 : and OH$ than 2. This indi-
cates a considerable potential for using them as inhibitor of the
radical.
3. Conclusion
The synthesis and characterization of 2, 6-di ((phenazonyl-4-
imino) methyl)-4-methylphenol and its Co(II) and Zn(II) complexes
have been achieved with physicochemical and spectroscopic
methods. Firstly, the binding behavior of the complexes with BSA
was investigated under simulated physiologic conditions. The
results showed that the intrinsic fluorescence of BSA was quenched
by a static quenching mechanism and the complexes bound to BSA
via hydrophobic interaction. The average binding distance between
the complexes and BSA was obtained. The CD data indicate that the
binding of 1 and 2 to BSA induces a conformational change in BSA.
The complexes show good binding affinity to BSA giving relatively
high binding constants. Therefore, the complexes can be deposited
and transported by albumin. Secondly, the interaction of the
complexes with CT DNA revealed that they can bind tightly to DNA
probably via the intercalative mode. Complex 1 exhibits the highest
Kb value, which is comparable to the Kb value of EB. Noticeably, both
the two complexes have been found to promote cleavage ability of
pUC19 DNA with the order 1 > 2. The ligand and complexes showed
Fig. 7. UV spectra of (a) 1, (b) 2 and (c) Dpmp ([compounds] ¼ 10
in the absence and presence of CT DNA ([CT DNA] ¼ 0e5 M) at increasing amounts.
The arrows show the changes upon increasing amounts of CT DNA. Inset: plot of
[DNA]/(3a 3f) versus [DNA].
mM) in DMF solution
m
ꢀ
artificial nuclease. The cleavage ability of complexes 1 and 2 to
pUC19 DNA was investigated by gel electrophoresis in TriseHCl/
NaCl buffer (pH 7.2) at 37 ꢁC for 5 h. Both 1 and 2 show efficient
DNA cleavage activity (Fig. 8). A 30 mM of 1 showed 95% cleavage of

1644
H. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 1638e1647
hydrochloride) (pH 7.2)) followed by exhaustive stirring at 4 ꢁC for
three days, and kept at 4 ꢁC for no longer than a week. BSA stock
solution (1.50 ꢃ 10ꢀ5 mol Lꢀ1) was kept in the dark at 0e4 ꢁC. Tris/
HCl buffer solution (pH 7.4) and NaCl solution (0.1 M) were
prepared to maintain the ionic strength.
Carbon, hydrogen, and nitrogen were analyzed on an Elemental
Vario EL analyzer. Infrared spectra (4000e400 cmꢀ1) were deter-
mined with KBr disks on a Thermo Mattson FTIR spectrometer. The
UVevis spectra were recorded on a Varian Cary 100 UVevis
spectrophotometer. The fluorescence spectra were recorded on
a Hitachi RF-4500 spectrofluorophotometer. 1H NMR spectra were
measured on a Varian VR 300-MHz spectrometer, using TMS
(tetramethylsilane) as a reference. Mass spectra were performed on
a VG ZAB-HS Fast-atom bombardment (FAB) instrument and elec-
trospray mass spectra (ESI-MS) were recorded on a LQC system
(Finnigan MAT, USA) using DMF as mobile phase. CD measurements
were performed on
a Jasco-20 automatic recording spec-
tropolarimeter (Japan) in cells of 1 mm path length at room
temperature.
4.2. Synthesis of the ligand and complexes
4.2.1. Preparation of the ligand (L)
2, 6-Diformyl-4-methylphenol was prepared with the literature
methods [37]. Then an ethanol solution containing 4-amino-
phenazone (2.03 g, 10 mmol) was added dropwise to a solution of 2,
6-diformyl-4-methylphenol (0.82 g, 5 mmol) in ethanol (150 ml).
The mixture was stirred and heated to reflux for 3 h. The yellow
precipitate was collected by filtration and washed with ethanol.
The resulting Schiff base, 2, 6-di ((phenazonyl-4-imino) methyl)-
4-methylphenol (Dpmp) was obtained (Scheme 1), which was dried
under vacuum. Yield, 86%. Anal. Calcd. (%) for C31H30N6O3: C, 69.66;
H, 5.62; N, 15.73. Found: C, 69.65; H, 5.37; N, 15.42. 1H NMR (CDCl3,
300 MHz, br, broad; s, singlet; m, multiplet): d(ppm) 9.81 (2H, s, CH]
N), 3.13 (6H, s, CH3), 2.44 (6H, s, CH3), 2.33 (3H, s, CH3), 7.26e7.48
(12H, m, PheH). FAB MS: m/z ¼ 535.4 (M þ H). IR: nmax(cmꢀ1):
n
(C]
O): 1657 cmꢀ1 (C]N): 1587 cmꢀ1 (CeN): 1295 cmꢀ1
, n , n , n(eOH):
3456 cmꢀ1. UV (DMF): lmax (nm): 248, 337, 394, 420.
Fig. 9. Plots of the (a) superoxide radical scavenging effects (%) and (b) hydroxyl
radical scavenging effects (%) for M (M ¼ Co, Zn) salts Dpmp (L), 1 and 2.
4.2.2. Preparations of the complexes
A 5 mL ethanol solution of Co(NO3)2$6H2O (0.146 g, 0.5 mmol)
was added slowly to a magnetically stirred 50 mL ethanol solution
of the ligand (0.267 g, 0.5 mmol). The mixture was stirred in air for
4 h whereby a deep red solution was formed. It was filtered and
kept in air. Red prismatic single crystals of Co(II) complex (1)
suitable for X-ray crystallography were obtained on slow evapo-
ration of the filtrate. The Zn(II) complex (2) was prepared by the
same method using Zn(NO3)2$6H2O instead ((Scheme 1)).
Elemental Anal. Found (calculated) (%) for 1 C33H38CoN8O11 (%): C,
considerable anti-oxidative activity to Oꢀ2 : and OH$, the suppression
rate of 1 is greater than that of 2. These findings clearly indicate that
the complexes may have potential practical applications. Informa-
tion obtained from our study would be helpful to understand the
mechanism of interactions of phenazone-type pharmaceuticals and
their complexes with serum albumin and nucleic acid and should
be useful in the development of potential probes for BSA and DNA
structure and conformation, or new therapeutic reagents for some
certain diseases.
50.74 (50.66); H, 4.79 (4.96); N, 14.21 (14.33). IR for 1 (cmꢀ1):
n
(C]
O): 1623 cmꢀ1
,
n
(C ¼ N): 1535 cmꢀ1
, n , n(eOH):
(CeN): 1293 cmꢀ1
3430 cmꢀ1. UV for 1 (DMF): lmax (nm): 247, 332, 379. Elemental
Anal. Found (calculated) for 2 C31H29ZnN8O9 (%): C, 51.29 (51.45); H,
4. Experimental
4.12 (4.01); N, 15.63 (15.49). IR for complex 2 (cmꢀ1):
n
(C]O):
4.1. Materials and instrumentation
1635 cmꢀ1 (C]N): 1538 cmꢀ1 (CeN): 1293 cmꢀ1
, n , n , n(eOH):
3414 cmꢀ1. UV for 2 (DMF): lmax (nm): 244, 273, 334, 377.
All starting materials were of analytical grade and double-
distilled water was used throughout the experiments. The reagents
and solvents were purchased commercially and used without
further purification unless otherwise noted. CT DNA, pUC19 DNA
and BSA were obtained from Sigma Chemicals Co. (USA). Agarose
was purchased from Promega Co. (German), ethidium bromide
(EB) were obtained from Huamei Chemical Co. (Beijing, China).
DNA stock solution was prepared by dilution of CT DNA to buffer
(50 mM NaCl, 5 mM TriseHCl (tris (hydroxymethyl) aminomethane
4.3. Crystallography
Single-crystal X-ray diffraction collection was performed on
a Rigaku RAXIS-RAPID diffractometer using a MoK radiation
a
(
l
¼ 0.71073 Ǻ). The structure 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

H. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 1638e1647
1645
4.4. Albumin binding studies
HCHO
MnO2
In fluorometric titration experiments, 2.0 mL of operating
solution (BSA 1.50 ꢃ 10ꢀ6 mol Lꢀ1, NaCl 0.10 mol Lꢀ1, pH 7.4) was
titrated by successive additions of a 5.00 ꢃ 10ꢀ4 mol Lꢀ1 methanol
stock solution of 1 and 2. Titrations were done using a micro-
injector, and the fluorescence intensity was measured (excitation at
280 nm and 5 nm slit widths) at three temperatures (288, 298, and
310 K). The temperature of samples was maintained with recycled
water. The UVevis spectrophotometer equipped with 1.0 cm quartz
cells was used for scanning the UV spectrum in the wavelength
range from 200 to 500 nm. The Tris/HCl buffer solution was used as
a reference solution.
H2O, 50oC
Acetone
OH
O
OH
N
O
OH OH OH
4-aminophenazone
C2H5OH
N
OH
N
N
N
N
O
O
Dpmp
Fluorescence quenching data were analyzed with the Sterne
Volmer Eq. (4) [39]
H
O
3
2
2
5
5
F0=F ¼ 1 þ Kqs0½Qꢅ ¼ 1 þ Ksv½Qꢅ
(4)
H
2
2
C
where F and F0 are the relative fluorescence intensities in the
presence and absence of quencher, respectively, [Q] is the
concentration of quencher, Ksv is the SterneVolmer quenching
constant, kq is the bimolecular quenching rate constant, s0 is the
average bimolecular lifetime in the absence of quencher evaluated
at about 10ꢀ8 s [40], f is the fraction of the initial fluorescence which
is accessible to the quencher.
Η2Ο
N
O
N
N
N
N
N
N
N
Co
O
O
O
O
NO3
O
N
O
N
O3N
Zn
The apparent binding constant, KA and binding sites n were
evaluated using the following equation (Eq. (5)) [41]:
O
NO3
N
O
N
N
Co
N
N
O
ꢀ
ꢁ
O
ðF0 ꢀ FÞ
1
log
¼ nlog KA ꢀ nlog
(5)
N
O
N
N
F
½Dtꢅ ꢀ ðF0 ꢀ FÞ½Ptꢅ=F0
H2O
N
where F0 and F are the fluorescence intensities before and after the
addition of the quencher, respectively, [Dt] and [Pt] are the total
quencher concentration and the total protein concentration,
respectively.
1
2
Scheme 1. A schematic depiction of the syntheses of the ligand and complexes.
The values of
DG, DH, and DS were calculated according to the
data on the binding constant KA at 288 K, 298 K, and 310 K using
Eqs. (6)e(8).
thermal parameters of all non-hydrogen atoms were refined on F2
by full-matrix least-squares techniques with the SHELX-97
program package [38]. Crystal data and details of the structure
determination for 1 and 2 are summarized in Table 3. CCDC 787905
and CCDC 787906 contain the supplementary crystallographic data
for this paper.
D
G ¼ ꢀRTln K
(6)
(7)
(8)
ꢂ
ꢃ
KA2
KA1
1
1
D
H
R
ln
¼
ꢀ
T1 T2
D
D
D
G ¼ H ꢀ T
S
Table 3
Crystal data and structure refinement for complexes 1 and 2.
CD spectra (200e300 nm) were recorded at a BSA concentration
of 1.50 ꢃ 10ꢀ6 mol Lꢀ1, and the results were taken as molar ellip-
1
2
ticity ([q
]) in deg cm2 dmolꢀ1. In order to obtain the secondary
Empirical formula
Formula weight
Temperature
Crystal system, space group
a (Å)
C33H38CoN8O11
781.64
C31H29ZnN8O9
722.99
structure fractions of BSA, the CDPro software package was used.
This consists of the three programs, SELCON3, CONTIN, CDSSTR,
and a program for determining tertiary structure class (CLUSTER).
One of the major advantages of the CDPro software package is that
the programs have been modified to accept any given set of refer-
ence proteins (CD spectra and secondary structure fractions), and
seven such reference sets are provided. Moreover, input data files
for these three programs are identical. More information about
CDPro is available at the following website: http://lamar.colostate.
edu/sreeram/CDPro [25].
296(2) K
Triclinic, P ꢀ 1
10.5167(18)
11.975(2)
14.552(3)
88.532(3)
78.160(3)
80.733(3)
1770.3(5)
2
296(2) K
Monoclinic, P2(1)/n
10.136(3)
15.372(4)
20.568(6)
90
93.757(15)
90
3197.9(14)
4
1.502
0.836
1492
0.29 ꢃ 0.28 ꢃ 0.23
1.66e25.48
0.995
b (Å)
c (Å)
a
b
g
(ꢁ)
(ꢁ)
(ꢁ)
Volume (Å3)
Z
Calculated density (g/cm3)
Absorption coefficient (mmꢀ1
F(000)
1.446
0.557
814
)
4.5. Spectroscopic studies on DNA interaction
Crystal size (mm)
0.30 ꢃ 0.25 ꢃ 0.20
1.72e25.80
1.015
q
range for data collection (ꢁ)
Goodness-of-fit on F2
4.5.1. Electronic absorption spectra
Final R indices [I > 2
s
(I)]
R1 ¼ 0.0545,
wR2 ¼ 0.1335
787905
R1 ¼ 0.0458,
wR2 ¼ 0.0779
787906
The UVevis absorbance at 260 and 280 nm of the CT DNA
solution in 50 mM NaCl/5 mM TriseHCl buffer (pH 7.2) give a ratio
of w1.9, indicating that the DNA was sufficiently free of protein
CCDC number

1646
H. Liu et al. / European Journal of Medicinal Chemistry 46 (2011) 1638e1647
[42]. The DNA concentration was determined by measuring the UV
A0. The suppression ratio for Oꢀ2 : was calculated from the following
absorption at 260 nm, taking the molar absorption coefficient (e260
)
Eq. (10):
of CT DNA as 6600 Mꢀ1 cmꢀ1 [43]. The intrinsic binding constant Kb
for the interaction of the studied complex with CT DNA was
calculated by absorption spectral titration data using the following
equation [44]:
Suppression ratio ¼ ðA0 ꢀ AiÞ=A0
(10)
where Ai is the absorbance in the presence of the ligand or its
complexes and A0 is the absorbance in the absence of the ligand or
its complexes.
ꢄ
ꢅ
ꢄ
ꢅ
ꢄ
ꢅ
Hydroxyl radical (OH$) scavenging activity through the Fenton
reaction [48]. The solution of the compound to be tested was
prepared in DMF. The tested samples contained 1 mL of 0.15 M
½DNAꢅ= 3a
ꢀ
3f ¼ ½DNAꢅ= 3b
ꢀ
3f þ 1=Kb 3b
ꢀ
3f
(9)
where [DNA] is the concentration of DNA in base pairs, 3a corre-
sponds to the extinction coefficient observed (Aobsd/[complex]), 3f
corresponds to the extinction coefficient of the free compound
which was calculated from the LamberteBeer equation, 3b is the
extinction coefficient of the compound when fully bound to DNA,
and Kb is the intrinsic binding constant. The ratio of slope to
phosphate buffer (pH ¼ 7.4), 1 mL of 40
mg/mL safranin, 1 mL of
1.0 mM EDTA-Fe(II), 1 mL of 3% H2O2, and 0.5 mL of the solution of
the tested compound (prepared as a series dilutions of the tested
compound). The reaction mixtures were incubated at 37 ꢁC for
60 min in a water-bath. The absorbance of the samples and
a control were measured at 520 nm. The suppression ratio for OH$
was calculated from the following Eq. (11) [49].
intercept in the plot of [DNA]/(3a
of Kb. 3f can be obtained from the intercept 1/Kb(3b
ꢀ
3f) versus [DNA] gives the values
ꢀ
3f).
ꢆ
ꢇꢈ
4.5.2. Fluorescence spectra
Suppression ratio ¼ Asample ꢀ Ablank ðAcontrol ꢀ Ablank
Þ
The complexes at a fixed concentration (10 mM) were titrated
(11)
with increasing amounts of CT DNA. Excitation wavelength of the
samples were 437 nm, scan speed ¼ 240 nm/min, slit width 10/
10 nm. All experiments were conducted at 20 ꢁC in a buffer con-
taining 5 mM TriseHCl (pH 7.2) and 50 mM NaCl concentrations.
where Asample is the absorbance of the sample in the presence of the
tested compound, Ablank is the absorbance of the blank in the
absence of the tested compound and Acontrol is the absorbance in
the absence of the tested compound and EDTA-Fe(II). IC50 value was
introduced to denote the molar concentration of the tested
compound which caused good inhibitory or scavenging effect on
radicals.
4.5.3. Viscosity experiments
Viscosity experiments were conducted on an Ubbelohde
viscometer, immersed in a thermostated water-bath maintained at
25.0 ꢁC. Titrations were performed for the complexes (1e5
m
m
M),
M)
versus
is
and each compound was introduced into DNA solution (5
present in the viscometer. Data were presented as (h/h0)
the ratio of the concentration of the compound and DNA, where
the viscosity of DNA in the presence of compound and h0 is the
viscosity of DNA alone. Viscosity values were calculated from
the observed flow time of DNA containing solution corrected from
Acknowledgment
1/3
h
This study was supported by the Foundation of Key Laboratory
of Nonferrous Metals Chemistry and Resources Utilization of Gansu
Province and State Key Laboratory of Applied Organic Chemistry
and the NSFC (20171019).
the flow time of buffer alone (t0),
h
¼ t ꢀ t0 [45,46].
Appendix. Supplementary material
4.6. DNA cleavage
Supplementary material can be found, in the online version, at
doi: 10.1016/j.ejmech.2011.02.012.
The cleavage of supercoiled (SC) pUC19 DNA was studied by
agarose gel electrophoresis, carried out in a dark room at 37 ꢁC
using SC DNA (2
mL, 12 mM) in TriseHCl/NaCl buffer (pH 7.2) and the
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