Synthesis, crystal structure, DNA interaction and antioxidant activities of two novel water-soluble Cu(2+) complexes derivated from 2-oxo-quinoline-3-carbaldehyde Schiff-bases

Article abstract of DOI:10.1016/j.ejmech.2009.06.009

Two novel 2-oxo-quinoline-3-carbaldehyde (4′-hydroxybenzoyl) hydrazone, thiosemicarbazone ligands and its corresponding Cu(2+) complexes were synthesized, and the two complexes' structures were determined by X-ray single crystal diffraction. The interaction of the two Cu(2+) complexes with calf thymus DNA (CT-DNA) was investigated by electronic absorption spectroscopy, fluorescence spectroscopy and viscosity measurement. The experimental evidences indicated that the two water-soluble Cu(2+) complexes could strongly bind to CT-DNA via an intercalation mechanism. The intrinsic binding constants of complexes 1 and 2 with CT-DNA were 7.31 × 10⁶ and 2.33 × 10⁶ M^-1, respectively. Furthermore, the antioxidant activities (hydroxyl radical and superoxide) of the two water-soluble metal complexes were determined by hydroxyl radical and superoxide scavenging method in vitro.

Full text of DOI:10.1016/j.ejmech.2009.06.009

European Journal of Medicinal Chemistry 44 (2009) 4477–4484
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, crystal structure, DNA interaction and antioxidant activities of two
novel water-soluble Cu(2þ) complexes derivated from 2-oxo-quinoline-3-
carbaldehyde Schiff-bases
*
Zeng-Chen Liu, Bao-Dui Wang, Zheng-Yin Yang , Yong Li, Dong-Dong Qin, Tian-Rong Li
College of Chemistry and Chemical Engineering, State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Two novel 2-oxo-quinoline-3-carbaldehyde (40-hydroxybenzoyl) hydrazone, thiosemicarbazone ligands
and its corresponding Cu(2þ) complexes were synthesized, and the two complexes’ structures were
determined by X-ray single crystal diffraction. The interaction of the two Cu(2þ) complexes with calf
thymus DNA (CT-DNA) was investigated by electronic absorption spectroscopy, fluorescence spectros-
copy and viscosity measurement. The experimental evidences indicated that the two water-soluble
Cu(2þ) complexes could strongly bind to CT-DNA via an intercalation mechanism. The intrinsic binding
Article history:
Received 3 March 2009
Received in revised form
4 June 2009
Accepted 5 June 2009
Available online 17 June 2009
constants of complexes
1
and
2
with CT-DNA were 7.31 ꢀ 106 and 2.33 ꢀ 106 Mꢁ1
, respectively.
Keywords:
2-Oxo-quinoline-3-carbaldehyde
Schiff-bases
Crystal structure
DNA binding
Furthermore, the antioxidant activities (hydroxyl radical and superoxide) of the two water-soluble metal
complexes were determined by hydroxyl radical and superoxide scavenging method in vitro.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Antioxidant activity
1. Introduction
retard the progress of many chronic diseases as well as lipid per-
oxidation. It is well known that SOD has been investigated for use in
Over the past decades, the metal complexes that can interact
with DNA have been extensively studied as DNA footprint, novel
chemotherapeutics and highly sensitive diagnostic agents [1–6].
Generally, the transition metals play a very important role in
organism and their complexes can interact non-covalently with
nucleic acid by intercalation, groove-binding or external electro-
static binding for cations [3,7–11]. And that many transition metal
complexes have been investigated to be utilized as probes of DNA
structure, agents for mediation of strand scission of duplex DNA
and chemotherapeutic agents [12–14]. Most notably, some Pt(2þ)
complexes (cisplatin and carboplatin) have found their way into the
pharmaceutical armamentarium as applied clinical antitumor
drugs [15]. Additionally, The importance of reactive oxygen species
and free radicals has attracted considerable attention over the past
protection against oxidative injury. However, the use of SOD
protein as a therapeutic is limited by its price, charge and rapid
clearance [16]. Recently study suggested that many transition metal
complexes also exhibited interesting antioxidant activities (OHꢂ,
ꢂ
Oꢁ2 , etc.) [17,18]. Therefore, it is significant to search for transition
metal complexes as potential antioxidants.
Nitrogen heterocyclic compounds have been used widely in the
pharmaceutical industry because of their perfect biologicalactivities
[19–23]. 2-Oxo-quinoline is a kind of alkaloid which exists in nature
extensively as same as quinoline. Moreover, some derivatives of 2-
oxo-quinoline have been also synthesized and investigated, because
they exhibit some preferable biological activities such as
antioxidation, antiproliferation, anti-inflammation and anticancer
[24–27]. Also, some Schiff-bases and their metal complexes often
exhibit diverse biological and pharmaceutical activities [28–32].
However, the studies on the antioxidant activities and DNA-binding
mechanism of Schiff-base transition metal complexes derivated
from 2-oxo-quinoline-3-carbaldehyde have not been explored. In
addition, although many Schiff-base complexes exhibit good bio-
logical activities, the water-solubility of the compounds is still
unsatisfactory, which restricts their actual application. In this paper,
two water-soluble Cu(2þ) complexes with two Schiff-base ligands
are synthesized and characterized by X-ray single crystal diffraction.
decades. Reactive oxygen species (ROS), which include free radicals,
ꢂ
such as hydroxyl radicals (OHꢂ) and superoxide anion radicals (O2ꢁ
)
are various forms of activated oxygen. And that they are exacer-
bating factors in cellular injury and aging process. Antioxidants can
protect the human body from free radicals and ROS effects, and
* Corresponding author. Tel.: þ86 9318913515; fax: þ86 9318912582.
E-mail address: yangzy@lzu.edu.cn (Z.-Y. Yang).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.06.009

4478
Z.-C. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 4477–4484
The DNA-binding modes of the two Cu(2þ) complexes with CT-DNA
are investigated by UV–vis, fluorescence and viscosity measure-
ments. The results show that the two water-soluble Cu(2þ)
complexes can interact with DNA through intercalation and the
DNA-binding ability of complex 1 is higher than that of complex 2. In
addition, the two water-soluble complexes are found to possess
potentially excellent antioxidant activities.
2. Results and discussion
The two water-soluble Cu(2þ) complexes were prepared by
direct reaction of ligand with appropriate mole ratios of Cu(2þ)
nitrate in methanol. The two complexes were air stable and soluble
in water, ethanol, methanol, DMF and DMSO. The single crystal X-
ray analyses showed that formula of the two Cu(2þ) complexes
were
Cu(H2L2)NO3 (complex 2).
[Cu(H2L1)(CH3OH)]$NO3$CH3OH
(complex
1)
and
Fig. 1. ORTEP view of [CuL1] showing the atom numbering of scheme and 50% thermal
Table 1 summarizes the crystal data, data collection and
ellipsoids probability for the non-hydrogen atoms.
refinement parameters for complexes 1 and 2.
and a six-membered (CuNCCCO) chelating rings and a nitrate
which is a monodentate, and the planar quadrangle may be a little
distorted as a result of the hydrogen bond between the O3 and
hydrogen atoms of other molecules. In addition, the C]S bond
(1.743 Å) has been found to be enolic and N1C1C9H gives a perfect
planar configuration as a consequence of sp2 hybridization. In
addition, the asymmetric unit cell of the complex consists of four
crystallographically independent molecules of the complex.
2.1. Crystal structure of complexes 1 and 2
The ORTEP representation of the structure of complex 1,
including atom numbering scheme, is shown in Fig. 1 and the
selected bond lengths and bond angles are listed in Table 2. The
coordination of the hydrazone with Cu(2þ) results in the formation
of a five-membered (CuONNC) and a six-membered (CuNCCCO)
chelating rings, otherwise, there is a methanol molecule which
takes part in coordination and also a non-coordinative methanol
molecule, on the contrary, the NOꢁ3 does not coordinate with
Cu(2þ), and the coordination of Cu(2þ) with N2O2O4O5 gives
a distorted quadrangular configuration. The perfect planar of
N3C10C11H is a consequence of sp2 hybridization rather than sp3
hybridization. In addition, the asymmetric unit cell of the complex
consists of four crystallographically independent molecules of the
complex.
2.2. DNA-binding studies
2.2.1. Electronic absorption titration
Electronic absorption spectroscopy is one of the most useful
techniques for studying binding mode of metal complexes to DNA
Table 2
Selected bond lengths (Å) and angles (ꢃ) for complex 1.
The ORTEP representation of the structure of complex 2,
including atom numbering scheme, is shown in Fig. 2 and the
selected bond lengths and bond angles are listed in Table 3. As can
be seen from Fig. 2, the coordination geometry of H2L2 with Cu(2þ)
is quadrangular, which is composed of a five-membered (CuNNCS)
Bond names
Bond lengths (Å)
Bond angles
Angle (ꢃ)
Cu(1)–O(2)
Cu(1)–O(4)
Cu(1)–N(2)
Cu(1)–O(5)
C(7)–O(2)
C(7)–N(1)
C(8)–N(2)
C(8)–C(9)
C(8)–H(8)
C(9)–C(19)
C(9)–C(10)
C(10)–O(4)
C(10)–N(3)
C(11)–C(12)
C(11)–C(16)
N(2)–N(1)
N(3)–H(3N)
0.78(3)
N(4)–O(9)
N(4)–O(7)
C(17)–O(5)
C(18)–O(6)
O(1)–H(1A)
O(5)–H(5O)
O(6)–H(6)
C(11)–N(3)
1.9123(15)
1.9204(15)
1.9235(19)
1.9557(18)
1.295(3)
1.320(3)
1.291(3)
1.436(3)
0.9300
1.368(3)
1.452(3)
1.270(3)
1.341(3)
1.390(3)
1.399(3)
1.384(3)
0.78(3)
1.215(3)
1.226(3)
1.253(3)
1.428(3)
1.395(3)
0.8200
0.66(3)
0.8200
1.379(3)
O(2)–Cu(1)–O(4)
O(2)–Cu(1)–N(2)
O(4)–Cu(1)–N(2)
O(2)–Cu(1)–O(5)
O(4)–Cu(1)–O(5)
N(2)–Cu(1)–O(5)
C(1)–C(6)–C(5)
174.76(6)
81.71(7)
93.32(7)
94.73(8)
90.45(7)
169.12(9)
118.0(2)
120.0(2)
122.0(2)
124.2(2)
117.8(2)
117.9(2)
124.2(2)
117.9
Table 1
Crystal data and structure refinement of complexes 1 and 2.
C(1)–C(6)–C(7)
C(5)–C(6)–C(7)
Complex 1
Complex 2
Empirical formula
FW
C11H9CuN5O4S
370.83
C19H20CuN4O8
495.93
Green
0.35 ꢀ 0.31 ꢀ 0.22 mm
Monoclinic
p21c
7.7826(4)
27.6765(14)
9.6798(5)
90
102.548(2)
90
2035.18(18)
4
1.619
1.130
O(2)–C(7)–N(1)
O(2)–C(7)–C(6)
N(1)–C(7)–C(6)
N(2)–C(8)–C(9)
N(2)–C(8)–H(8)
C(9)–C(8)–H(8)
O(4)–C(10)–N(3)
N(3)–C(10)–C(9)
C(7)–N(1)–N(2)
C(8)–N(2)–N(1)
C(8)–N(2)–Cu(1)
N(1)–N(2)–Cu(1)
C(10)–N(3)–C(11)
C(10)–N(3)–H(3N)
C(11)–N(3)–H(3N)
C(3)–O(1)–H(1A)
C(7)–O(2)–Cu(1)
C(10)–O(4)–Cu(1)
C(17)–O(5)–Cu(1)
C(17)–O(5)–H(5O)
Cu(1)–O(5)–H(5O)
C(18)–O(6)–H(6)
O(4)–C(10)–C(9)
Crystal color
Black
Crystal size
Crystal system
Space group
0.24 ꢀ 0.27 ꢀ 0.32 mm
Triclinic
p-1
117.9
a (Å)
b (Å)
c (Å)
a
8.3415(9)
8.7979(9)
10.6365(11)
94.747(2)
111.701(2)
110.179(2)
660.66(12)
2
117.54(19)
116.59(19)
108.89(19)
117.71(19)
127.69(16)
114.57(15)
125.7(2)
114.6(18)
119.6(18)
109.5
110.54(13)
126.85(14)
125.42(16)
119(3)
106(3)
109.5
b
g
Volume (Å3)
Z
Dcalc (Mg mꢁ3
)
1.864
1.838
374
Abs coeff. (mmꢁ1
F(000)
)
1020
1.47-27
qmin and max(deg)
2.12-26
Reflections collected/ 3626/2560 [R(int) ¼ 0.0122] 12365/4397 [R(int) ¼ 0.0313]
unique
Final R indices
[I > 2 (I)]
R indices (all data)
R1 ¼ 0.0311, wR2 ¼ 0.0938
R1 ¼ 0.0349, wR2 ¼ 0.0965
R1 ¼ 0.0364, wR2 ¼ 0.0951
R1 ¼ 0.0517, wR2 ¼ 0.1030
s
125.87(19)

Z.-C. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 4477–4484
4479
0.35
a
(complex 1)
0.30
0.25
0.20
0.15
0.10
0.05
200
250
300
350
400
Wavelength (nm)
450
500
Fig. 2. ORTEP view of [CuL2] showing the atom numbering of scheme and 50% thermal
ellipsoids probability for the non-hydrogen atoms.
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
b
(complex 2)
[33,34]. The UV–vis absorption spectra of complexes 1 and 2 in the
absence or presence of CT-DNA are shown in Fig. 3. The absorption
bands of complex 1 at 206 and 260 nm exhibit hypochromism of
about 30.76% and 72.00%, respectively. Moreover, complex 2 at 206
and 260 nm exhibit hypochromism of about 30.56% and 37.50%,
respectively. These results suggest that the complex 1 show
stronger hypochromicity than complex 2. Furthermore, it also likely
intimates that the two water-soluble Cu(2þ) complexes can bind to
the helix via intercalation in the Tris–HCl (pH ¼ 7.2) which do not
contain any organic solvent. After the complexes intercalate to the
base pair of DNA, the p* orbital of the intercalated ligand on the
complexes can couple with
p
orbital of the base pairs, thus
200
250
300
350
Wavelength (nm)
400
450
500
decreasing the
p–
p* transition energies. On the other hand, the
Fig. 3. (a) Electronic spectra of complex 1 (10
amounts of CT-DNA (0–10 M). Arrow shows the absorbance changes upon increasing
DNA concentration. (b) Electronic spectra of complex 2 (10 M) in the presence of
M). Arrow shows the absorbance changes upon
mM) in the presence of increasing
Table 3
m
Selected bond lengths (Å) and angles (ꢃ) for complex 2.
m
increasing amounts of CT-DNA (0–10
increasing DNA concentration.
m
Bond names
Bond lengths (Å)
Bond angles
Angle (ꢃ)
Cu(1)–O(1)
Cu(1)–N(2)
Cu(1)–O(2)
Cu(1)–S(1)
C(1)–N(1)
C(1)–C(2)
C(1)–C(6)
C(2)–C(3)
C(2)–H(2)
C(3)–C(4)
C(3)–H(3)
C(4)–C(5)
C(4)–H(4)
C(5)–C(6)
C(5)–H(5)
C(6)–C(7)
C(7)–C(8)
C(7)–H(7)
C(8)–C(10)
C(8)–C(9)
C(9)–O(1)
C(9)–N(1)
C(10)–N(2)
C(10)–H(10)
C(11)–N(3)
C(11)–N(4)
C(11)–S(1)
N(1)–H(1N)
N(2)–N(3)
N(4)–H(3N)
N(4)–H(4N)
N(5)–O(2)
N(5)–O(3)
N(5)–O(4)
1.9360(18)
1.9728(19)
2.0401(18)
2.2393(7)
1.373(3)
1.392(3)
1.404(3)
1.377(4)
0.9300
1.403(4)
0.9300
1.357(4)
0.9300
1.415(4)
0.9300
1.411(4)
1.365(4)
0.9300
1.441(3)
1.453(3)
1.246(3)
1.348(3)
1.287(3)
0.9300
1.311(3)
1.340(3)
1.743(2)
0.69(3)
O(1)–Cu(1)–N(2)
O(1)–Cu(1)–O(2)
N(2)–Cu(1)–O(2)
O(1)–Cu(1)–S(1)
N(2)–Cu(1)–S(1)
O(2)–Cu(1)–S(1)
C(9)–N(1)–C(1)
C(9)–N(1)–H(1N)
C(1)–N(1)–H(1N)
C(10)–N(2)–N(3)
C(10)–N(2)–Cu(1)
N(3)–N(2)–Cu(1)
C(11)–N(3)–N(2)
C(11)–N(4)–H(3N)
C(11)–N(4)–H(4N)
H(3N)–N(4)–H(4N)
O(2)–N(5)–O(3)
O(2)–N(5)–O(4)
O(3)–N(5)–O(4)
C(9)–O(1)–Cu(1)
N(5)–O(2)–Cu(1)
C(11)–S(1)–Cu(1)
93.08(8)
90.37(8)
168.06(8)
177.76(6)
85.71(6)
90.45(6)
126.5(2)
118(3)
coupling p* orbital are partially filled by electrons, thus decreasing
the transition probabilities [35,36].
2.2.2. Fluorescence spectra
The two Cu(2þ) complexes exhibit weak luminescence in Tris–
HCl buffer with a maximum wavelength of about 400 and 450 nm.
The results of the emission titration for the two complexes with
helix DNA (CT-DNA) that are illustrated in the titration curves are
shown in Fig. 4. Upon the addition of CT-DNA, the emission
intensities at about 450 nm of the two complexes increase by
around 1.41 and 1.20 times for complexes 1 and 2, respectively. In
addition, corresponding 5 and 6 nm blue shifts emerge with the
increase of emission intensities. The results of emission titration
suggest that the two water-soluble complexes are all protected
from solvent water molecules by the hydrophobic environment
inside the DNA helix; as a result, the accessibility of solvent water
molecules to these compounds is reduced. Compared to the
intensity enhancement of the two complexes in the presence of CT-
DNA, complex 1 has preferable DNA-binding ability than complex
2. In order to illustrate quantitatively the consequence, changes in
emission intensities at about 450 nm for the two Cu(2þ) complexes
have been plotted against the added DNA concentration per mole of
complexes. According to the Scatchard equation [37–39], a plot of
r/Cf versus r gives the binding constants of complexes 1 and 2. The
results imply that both the compounds can insert between DNA
base pairs and complex 1 (K ¼ 7.31 ꢀ106) can interact with DNA
more strongly than complex 2 (K ¼ 2.33 ꢀ 106).
115(3)
114.3(2)
125.04(16)
120.66(15)
113.1(2)
117(2)
117(3)
125(4)
122.3(3)
118.3(3)
119.3(3)
127.76(16)
107.74(16)
95.04(9)
1.394(3)
0.86(4)
0.74(4)
1.215(3)
1.228(3)
1.249(4)

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Z.-C. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 4477–4484
180
160
140
120
100
80
a
(complex 1)
9
8
7
6
5
4
3
2
1
60
40
20
0
380 400 420 440 460 480 500 520 540 560 580 600
Wavelength (nm)
0.46
0.48
0.50
0.52
0.54
0.56
0.58
r
180
160
140
120
100
80
b
6
(complex 2)
5
4
3
2
1
0
60
40
20
0
380 400 420 440 460 480 500 520 540 560 580 600
Wavelength (nm)
0.25 0.30 0.35 0.40 0.45 0.50 0.55
r
Fig. 4. (a) The emission enhancement spectra of complex 1 (10 mM) in the presence of 0, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5 and 20.0 mM CT-DNA. Arrow shows the emission intensity
changes upon increasing DNA concentration. Inset: Scatchard plot of the fluorescence titration data of complex 1, K ¼ 7.31 ꢀ106. (b) The emission enhancement spectra of complex 2
(10
m
M) in the presence of 0, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0 and 17.5
m
M CT-DNA. Arrow shows the emission intensity changes upon increasing DNA concentration. Inset: Scatchard plot
of the fluorescence titration data of complex 2, K ¼ 2.33 ꢀ 106.
2.2.3. EB-DNA experiment
varying amounts of the two water-soluble complexes 1 and 2. The
The DNA-binding modes of compound are further monitored by
fluorescent EB displacement assay. It is well known that EB can
emit intense fluorescence due to strong intercalation nonspecifi-
cally between DNA base pairs [40,41]. Competitive binding of other
drugs to DNA and EB will result in displacement of bound EB and
decrease in the fluorescence intensity. This fluorescence-based
competition technique can provide indirect evidence for the DNA-
binding mode. Fig. 5 shows the emission spectra of DNA-EB spectra
with increasing amounts of the two water-soluble Cu(2þ)
complexes. The emission intensity of the DNA-EB system
results indicate that the viscosity of DNA increase on addition of the
concentrations of complexes 1 and 2. As general viewpoint,
the viscosities of DNA increase steadily when the compounds
intercalate between adjacent DNA base pairs. This illustrate that the
two water-soluble Cu(2þ) complexes can interact with DNA
through an intercalative mode and complex 1 can bind to CT-DNA
more tightly than complex 2. In addition, the results obtained from
viscosity are consistent with that obtained from the spectroscopic
studies.
(lem ¼ 587 nm) decrease apparently with the increasing concen-
2.3. Antioxidant activity
tration of the Cu(2þ) complexes. The quenching plots illustrate that
the quenching of EB bound to DNA by the complexes are in good
agreement with the linear Stern–Volmer equation. The fluores-
cence quenching is caused by EB changing from a hydrophobic
environment into an aqueous environment. And the fluorescence
quenching phenomenon at 587 nm of the DNA-EB system indicates
that the two water-soluble Cu(2þ) complexes can displace EB from
the DNA-EB system. Such a characteristic change is often observed
in intercalative DNA interaction.
Fig. 7a depicts the inhibitory effect of the complexes on OHꢂ. The
inhibitory of the complexes is marked and suppression ratio
increases with increasing concentration in the range of tested
concentration. The sequence of the suppression ratio for OHꢂ is
complex 1 > complex 2 at different concentration. Moreover,
mannitol and Vitamin C are well-known natural antioxidant
compounds, so we compare the two water-soluble Cu(2þ)
complexes with them in the same way. As reported in previous
paper [36]. The 50% inhibitory concentration (IC50) value of
mannitol is about 9.6 mM. The antioxidant experiments testify that
2.2.4. Viscosity measurement
As a mean for further clarifying the binding mode of complex to
DNA, the viscosity measurements are also carried out [42,43]. Fig. 6
shows the relative viscosity change of DNA in the presence of
complex 1 (IC50 ¼ 4
m
M) and complex 2 (IC50 ¼ 5
mM) represent
very excellent antioxidant (OHꢂ) activities which are much better
than that of standard antioxidants like mannitol and Vitamin C [44].

Z.-C. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 4477–4484
4481
a
2.0
140
120
100
80
(complex 1)
1.8
1.6
1.4
1.2
1.0
0.8
60
40
20
0
580 600 620 640 660 680 700
Wavelength (nm)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
[complex 1] (10-5)
b
140
120
100
80
(complex 2)
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
60
40
20
0
580 600 620 640 660 680 700
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
[complex 2] (10-5)
Wavelength (nm)
Fig. 5. (a) The emission spectra of DNA-EB system, lem ¼ 587 nm, in the presence of 0, 5, 10, 15, 20, 25, 30, 35, 40 and 45
mM complex 1. Arrow shows the emission intensity changes
upon increasing complex concentration. Inset: Stern–Volmer plot of the fluorescence titration data of complex 1, Kq ¼ 5.91 ꢀ104. (b) The emission spectra of DNA-EB system,
lem ¼ 587 nm, in the presence of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60
mM complex 2. Arrow shows the emission intensity changes upon increasing complex concentration.
Inset: Stern–Volmer plot of the fluorescence titration data of complex 2, Kq ¼ 3.97 ꢀ 104.
ꢂ
Fig. 7b shows that the inhibitory of two complexes on super-
oxide radical is also related to concentration, at the range from 1.0
to 5.0 mM, the percentage scavenging effect valued from 48.56% to
perfect ability to suppress Oꢁ2 , and the suppression ratio of complex
1 (IC50 ¼ 1.0
m
M) is higher than that of complex 2 (IC50 ¼ 3.5
mM).
82.34% for complex 1 and from 38.20% to 72.56% for complex 2. It
illuminates that the two water-soluble Cu(2þ) complexes exhibit
3. Conclusions
In summary, two new water-soluble Cu(2þ) complexes of 2-
oxo-quinoline-3-carbaldehyde (40-hydroxybenzoyl) hydrazone and
thiosemicarbazone were evidenced to interact with DNA by inter-
calation and could be potential anticancer reagents. And that
compared with the Cu(2þ) complex of 2-oxo-quinoline-3-carbal-
dehyde thiosemicarbazone, complex 1 demonstrated that there
was preferable DNA-binding property as a result of the strong
stacking interaction between an aromatic chromophore and the
base pairs of DNA. Simultaneously, the water–solution activities of
the two complexes could enhance the biological compatibility in
vivo. In addition, the two water-soluble Cu(2þ) complexes of Schiff-
bases also exhibited excellent antioxidant (hydroxyl radical OHꢂ and
1.7
1.6
complex 1
complex 2
1.5
1.4
1.3
1.2
1.1
ꢂ
superoxide radical Oꢁ2 ) activities and the antioxidant activity of
complex 1 is better than that of complex 2 due to the existence of
hydroxyl of 2-oxo-quinoline-3-carbaldehyde (40-hydroxybenzoyl)
hydrazone [45].
4. Experimental
0.1
0.2
0.3
0.4
[M]/[DNA]
0.5
0.6
0.7
4.1. Instrumentation
1H NMR spectra were recorded on a Varian VR300-MHz spec-
trometer with TMS as an internal standard. The melting points of
Fig. 6. Effect of increasing amounts of complex 1 (-) and complex 2 (C) on the
relative viscosity of calf thymus DNA at 25(ꢄ0.1) ꢃC.

4482
Z.-C. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 4477–4484
spectrophotometrically by employing an extinction coefficient of
90
80
70
60
50
40
30
20
10
0
a
6600 Mꢁ1 cmꢁ1 at 260 nm.
4.3. Synthesis of ligands H2L1 and H2L2
As shown in Scheme 1 2-oxo-quinoline-3-carbaldehyde was
prepared according to the literature [46]. An ethanol solution
containing 4-hydroxybenzoyl hydrazine was added to another
ethanol solution containing 2-oxo-quinoline-3-carbaldehyde. The
mixture was refluxed for 12 h with stirring and a yellow precipitate
was separated out. The precipitation was filtrated under decom-
pression and washed with ethanol. Recrystallization from DMF/
H2O (V:V ¼ 1:1) gave the ligand H2L1, which was dried under
vacuum. Yield 85%; m.p. 298–300 ꢃC. 1H NMR (DMSO-d6, ppm):
d
12.066 (1H, s, –OH), 12.023 (1H, s, –N1H–), 11.866 (1H, s, –N3H–),
2
4
6
Concentration (µM)
8
10
12
8.715 (1H, s, –CH]N–), 8.496 (1H, s, 19-H), 7.863–7.922 (2H, s, 1,5-
H), 7.527–7.578 (1H, m, 12-H), 7.424–7.475 (1H, m, 13-H), 7.322–
7.359 (1H, m, 15-H), 7.201–7.250 (1H, m, 14-H), 6.931–6.990 (2H, m,
2,4-H).
90
80
70
60
50
40
30
20
10
0
b
The ligand H2L2 was synthesized according to the same proce-
dure as the synthesis method of ligand H2L1. Yield 76%; m.p. 314–
316 ꢃC. 1H NMR (DMSO-d6, ppm):
d
12.003 (1H, s, –N3H–), 11.643
(1H, s, –N1H–), 8.769 (1H, s, 7-H), 8.286–8.310 (2H, m, –NH2), 8.103
(1H, s, 7-H), 7.636–7.661 (1H, m, 2-H), 7.497–7.554 (1H, m, 3-H),
7.310–7.337 (1H, m, 5-H), 7.196–7.249 (1H, m, 4-H).
4.4. Preparation of Cu(2þ) complexes
The ligand H2L1 (0.2 mmol, 0.0492 g) and the Cu(2þ) nitrate
(0.2 mmol, 0.0483 g) were added to methanol. After 5 min, the
mixtures were filtered to remove any insoluble residues and then
were stirred for 10 h under reflux. A pitchy precipitate (complex 1)
was separated from the solution by suction filtration, purified by
washing several times with ethanol and dried for 24 h under
vacuum. Complex 2 was prepared by the same method. Complexes
1 and 2 were pitchy and green powers, respectively. But the crystal
of complex 1 was black.
1
2
3
Concentration (µM)
4
5
Fig. 7. (a) Scavenging effect of Cu(2þ) complexes on hydroxyl radicals. (-) complex 1;
(:) complex 2. Experiments were performed in triplicate. Values are expressed as
mean ꢄ standard deviation (n ¼ 1). (b) Scavenging ratio of the two Cu(2þ) complexes
on superoxide. (C) complex 1; (A) complex 2. Experiments were performed in
triplicate. Values are expressed as mean ꢄ standard deviation (n ¼ 1).
4.5. X-ray crystallography
the compound were determined on a Beijing XT4-100X micro-
scopic melting point apparatus. The UV–vis spectra were recorded
on a Perkin–Elmer Lambda-35 UV–vis spectrophotometer. Fluo-
rescence spectra were obtained on a Shimadzu RF-5301 spectro-
photometer at room temperature. The antioxidant activities were
performed in water with 721E spectrophotometer (Shanghai
Analytical Instrument factory China).
The single crystals of two water-soluble complexes were gained
in the methanol using diffusing method. A black crystal of complex
1 (0.24 ꢀ 0.27 ꢀ 0.32 mm) was measured on a Bruker Smart-1000
CCD diffractometer with graphite monochromatic Mo Ka radiation
(
l
¼ 0.71073 Å) at 296(2) K, 2.12ꢃ <
q
< 26ꢃ for hkl (ꢁ10 ꢅ h ꢅ 9,
ꢁ10 ꢅ k ꢅ 10, ꢁ10 ꢅ l ꢅ 13) in the triclinic. A green crystal of
complex 2 (0.35 ꢀ 0.31 ꢀ0.22 mm) was measured on a Bruker
4.2. Materials
H
CH
N
CT-DNA, ethidium bromide (EB), nitro-bluetetrazolium (NBT),
methionine (MET) and Vitamin B2 (VitB2) were purchased from
Sigma Chemical Co. Acetanilide was purchased from Guang Fu
Chemical Co., Tianjin, China. All materials and solvents were of
analytical reagent grade quality and were used without further
purification. M(NO3)2$nH2O (M ¼ Cu) were from China. Tris–HCl
buffer, KH2PO4–Na2HPO4 buffers and EDTA–Fe(2þ) were prepared
with twice distilled water.
3 POCl3
DMF
CHO
Cl
CHO
70%HAc
O
N
N
H
O
O
C
HO
NH NH2
OH
CH
N
NH
C
O
H2L1
C2H5OH
N
H
O
All the experiments involving the interaction of the two water-
soluble complexes with CT-DNA were carried out in doubly distilled
water containing 5 mM Tris [Tris(hydroxymethyl)-aminomethane]
and 50 mM NaCl and adjusted to pH ¼ 7.2 with hydrochloric acid.
Solution of CT-DNA gave ratios of absorbance at 260 and 280 nm of
about 1.8–1.9:1, indicating that the CT-DNA was sufficiently free of
protein. The CT-DNA concentration per nucleotide was determined
S
CH
N
NH
C
S
NH2
H2L2
H2N
NH NH2
C
C2H5OH
N
O
H
Scheme 1. Preparation of the ligands.

Z.-C. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 4477–4484
4483
Smart-1000 CCD diffractometer with graphite monochromatic Mo
values were calculated from the observed flow time of DNA con-
taining solution corrected from the flow time of buffer alone (t0)
Ka
radiation (
l
¼ 0.71073 Å) at 296(2) K, 1.47ꢃ <
q
< 27ꢃ for hkl
(ꢁ9 ꢅ h ꢅ 9, ꢁ35 ꢅ k ꢅ 33, ꢁ12 ꢅ l ꢅ 11) in the monoclinic. The
positions and anisotropic thermal parameters of all non-hydrogen
atoms were refined on F2 by full-matrix least-squares techniques
with the SHELX-97 program package [47]. Absorption correction
was employed using Semi-empirical methods from equivalents.
h
¼ t ꢁ t0.
4.7. Scavenger measurements of hydroxyl radical (OHꢂ) and
ꢂ
superoxide radical (O2ꢁ
)
The hydroxyl radical (OHꢂ) in aqueous media was generated by
the Fenton system. The solution of the tested complex was
prepared with two distilled water. The 5 mL assay mixture con-
4.6. DNA-binding study methods
tained following reagents: safranin (11.4
(40 M), H2O2 (1.76 mM), the tested complex (1–6
a phosphate buffer (67
incubated at 30 ꢃC for 10 min in a water bath. After that, the
absorbance was measured at 520 nm. All the tests were run in
triplicate and expressed as the mean and (ꢄ) standard deviation
(SD).
m
M), EDTA–Fe(2þ)
4.6.1. Electronic absorption spectroscopy
m
m
M) and
The Cu(2þ) complexes were dissolved in Tris–HCl buffer (5 mM
m
M, pH ¼ 7.4). The assay mixtures were
Tris–HCl; 50 mM NaCl, pH ¼ 7.2). The example of fixed amount
compounds (10 mM) were titrated with increasing amounts of DNA,
over a range of DNA concentrations from 0 to 10 mM.
4.6.2. Fluorescence spectroscopy
In order to affirm quantitatively the affinity of the two water-
soluble complexes binding to DNA, the intrinsic binding constant K
of the Cu(2þ) complexes was obtained by the electronic absorption
spectroscopy method. The two water-soluble complexes were
dissolved in Tris–HCl buffer (5 mM Tris–HCl; 50 mM NaCl, pH ¼ 7.2)
Scavenging ratioð%Þ ¼ ½ðAi ꢁ A0Þ=ðAc ꢁ A0Þꢆ ꢀ 100
where Ai ¼ the absorbance in the presence of the tested compound;
A0 ¼ the absorbance in the absence of the tested compound;
Ac ¼ the absorbance in the absence of the tested compound, EDTA–
Fe(2þ) and H2O2.
at concentration 10
m
M. The fluorescence titration experiments
M) while
gradually increasing the concentration of CT-DNA with the range
ꢂ
The superoxide radical (O2ꢁ ) was produced by the system of
were performed with fixed concentration of drugs (10
m
MET/VitB2/NBT and determined spectrophotometrically by NBT
phtotoreduction method with a little modification in the method
adopted elsewhere [48–50]. The amount of Oꢁ2 ꢂ could be calculated
by measuring the absorbance at 560 nm. Solution of VitB2 and NBT
were prepared under the condition of avoiding light. The tested
compounds were dissolved in water. The assay mixture, in a total
from 2.5 to 25
equation.
mM. K values were determined using the following
r=Cf ¼ Kbð1 ꢁ nrÞ
where r ¼ Cb/[DNA], Cf ¼ Ct [(F ꢁ F0)/(Fmax ꢁ F0)], Cb and Ct is the
concentration of free compound and the total compound, respec-
tively. F is the observed fluorescence emission intensity at a given
DNA concentration, F0 is the intensity in the absence of DNA, and
Fmax is the fluorescence intensity of the totally bound compound.
Binding data were casted into the form of a Scatchard plot of r/Cf
versus r. All experiments were conducted at 20 ꢃC in a buffer con-
taining 5 mM Tris–HCl (pH ¼ 7.2) and 50 mM NaCl.
volume of 5 mL, contained MET (10 mM), NBT (46
mM), VitB2
(3.3
(67
mM), the tested compound (1–6 mM) and a phosphate buffer
mM, pH ¼ 7.8). After illuminating with a fluorescent lamp at
30 ꢃC for 10 min, the absorbance of the samples (Ai) was measured
at 560 nm. The sample without the tested compound was used as
control and its absorbance was A0. All experimental results were
expressed as the mean and (ꢄ) standard deviation (SD) of triplicate
ꢂ
determinations. The suppression ratio for Oꢁ2 was calculated from
the following expression. The scavenging ratio ha ¼ (A0 ꢁ Ai)/
4.6.3. EB displacement experiment
A0 ꢀ 100% [51].
Further support for the two water-soluble Cu(2þ) complexes
binding to DNA via intercalation was given through the emission
5. Supplementary data
quenching experiment. A 2 mL solution of 5
m
M DNA and 0.4
mM EB
was titrated by complexes
slit ¼ 10 nm). According to the classical Stern–Volmer equation
(
lex ¼ 525 nm, lem ¼ 587 nm,
Crystallographic data for the structural analysis have been
deposited with the Cambridge Crystallographic Data Centre, CCDC
(719432, 719431). Copy of this information may be obtained free of
charge from the Director, CCDC, 12 Union Road, Cambridge, CB2
1EZ, UK (fax: þ44 1223 336033; e-mail: deposit@ccdc.cam.ac.uk or
http://www.ccdc.cam.ac.uk/deposit).
F0=F ¼ Kq½Qꢆ þ 1
where F0 is the emission intensity in the absence of quencher, F is
the emission intensity in the presence of quencher, Kq is the
quenching constant, and [Q] is the quencher concentration. The
plots can be used to characterize the quenching as being predom-
inantly dynamic or static. Plots of F0/F versus [Q] appear to be linear.
Acknowledgement
This work is supported by the National Natural Science Foun-
dation of China (20475023) and Gansu NSF (0710RJZA012).
4.6.4. Viscosity measurements
Hydrodynamic method, such as determination of viscosity,
which is exquisitely sensitive to the change of length of DNA, may
be the most effective means of studying the binding mode of
complexes with DNA. To further confirm the interaction mode of
the two water-soluble Cu(2þ) complexes with DNA, a viscosity
study was carried out. Viscosity measurements were conducted on
an Ubbelodhe viscometer, immersed in a thermostated water bath
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