Crystal structures, DNA-binding and cytotoxic activities studies of Cu(II) complexes with 2-oxo-quinoline-3-carbaldehyde Schiff-bases

Article abstract of DOI:10.1016/j.ejmech.2010.08.060

Three novel 2-oxo-quinoline-3-carbaldehyde Schiff-bases and their Cu(II) complexes were synthesized. The molecular structures of Cu(II) complexes were determined by X-ray crystal diffraction. The DNA-binding modes of the complexes were also investigated b

Full text of DOI:10.1016/j.ejmech.2010.08.060

European Journal of Medicinal Chemistry 45 (2010) 5353e5361
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Crystal structures, DNA-binding and cytotoxic activities studies of Cu(II)
complexes with 2-oxo-quinoline-3-carbaldehyde Schiff-bases
,
a
a
Zeng-Chen Liu a, Bao-Dui Wang a, Bo Li b, Qin Wang b, Zheng-Yin Yang a , Tian-Rong Li , Yong Li
*
a College of Chemistry and Chemical Engineering, State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, PR China
b College of Life Science, 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:
Three novel 2-oxo-quinoline-3-carbaldehyde Schiff-bases and their Cu(II) complexes were synthesized.
The molecular structures of Cu(II) complexes were determined by X-ray crystal diffraction. The DNA-
binding modes of the complexes were also investigated by UVevis absorption spectrum, fluorescence
spectrum, viscosity measurement and EBeDNA displacement experiment. The experimental evidences
indicated that the ligands and Cu(II) complexes could interact with CT-DNA (calf-thymus DNA) through
intercalation, respectively. Comparative cytotoxic activities of ligands and Cu(II) complexes were also
determined by MTT [3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide] and SRB (sul-
forhodamine B) methods. The results showed that the three Cu(II) complexes exhibited more effective
cytotoxic activity against HL60 cells and HeLa cells than corresponding ligands. Also, CuL3 showed higher
cytotoxic activity than CuL1 and CuL2.
Received 27 May 2010
Received in revised form
26 August 2010
Accepted 26 August 2010
Available online 15 September 2010
Keywords:
2-Oxo-quinoline-3-carbaldehyde Schiff-
base
Crystal structure
DNA-binding
Cytotoxic activity
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
In addition, as small molecules, a great many Schiff-base
complexes with transition metals have provoked wide interests
There has been an increasing focus on the binding study of small
molecules to DNA during the last decades, since deoxyribonucleic
acid (DNA) is an important genetic substance in organisms [1e3].
And errors in gene expression can often cause diseases and play
a secondary role in the outcome and severity of human diseases [4].
So a more complete understanding of DNAedrug binding is valuable
in the rational design of DNA structural probe, DNA footprinting,
sequence-specific cleaving agents and potential anti-cancer drugs
[5,6].
In order to develop new drugs which specifically target DNA, it
is necessary to understand the different binding modes which
a small molecule is capable of undergoing. The recognition modes
for the noncovalent binding of small molecules to DNA are inter-
calative binding, groove binding and external electrostatic binding
[7,8]. Among these interactions, intercalation and groove binding
are the most important DNA-binding modes as they invariably lead
to cellular degradation. Additionally, the metal ion type and
different functional groups of ligands, which are responsible for the
geometry of complexes, also affect the affinity of metal complexes
to DNA.
because of their diverse biological and pharmaceutical activities
[9,10]. In the previous literature, a number of Cu(II) complexes with
biological activities such as antibacterial and anti-cancer properties
have been also reported [11,12]. So the investigation on the interac-
tion of the Schiff-base transition metal complexes with DNA has
a great significance for disease defense, new medicine design and
filtration and clinical application of drugs. Quinoline and its deri-
vates, naturally occurring antibiotic, are one of the most widely
utilized as antibacterial and antimalarial drugs [13e15]. Also, 2-oxo-
quinoline appears frequently in the structure of various natural
compounds that have biological activities [16,17]. Simultaneously,
our previous work showed some Cu(II) Schiff-bases complexes
derived from 2-oxo-quinoline-carbaldehyde can bind to DNA by
intercalation [18]. With the increasing interest in the interaction of
transition metal complexes with DNA, in this paper, three novel
Schiff-base ligands derived from 2-oxo-quinoline-3-carbaldehyde
and benzoyl (2-hydroxybenzoyl and 3,4-dimethyl pyrryl-2-carbox-
ylic acid) hydrazine are designed andsynthesized, moreover, their Cu
(II) complexes are also characterized by X-ray crystal diffraction. And
the binding modes for the interaction of the three Cu(II) complexes
with CT-DNA are investigated by electronic absorption spectroscopy,
fluorescence spectroscopy and viscosity measurement. The experi-
mental results indicate that the three Cu(II) complexes can bind to
DNA through intercalative binding modes. On the other hand,
* Corresponding author. Tel.: þ86 931 891 3515; fax: þ86 931 891 2582.
E-mail address: yangzy@lzu.edu.cn (Z.-Y. Yang).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.060

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Z.-C. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 5353e5361
cytotoxic activities against HeLa cell and HL60 cell were also inves-
tigated in our work. As previous reports demonstrated that several
organic-copper complexes could inhibit some tumor cell growth in
vitro and in human tumor cell cultures [19e21]. So there is a need to
test the cytotoxic activities of the compounds. According to the
experiments, it has been found that the cytotoxic activities of Cu(II)
complexes are higher than that of ligands, moreover, compared with
CuL1 and CuL2, CuL3 exhibited more potent cytotoxic effect against
the two cell lines. The results should be valuable in designing novel
agents for targeting nucleic acids as well as setting the stage for the
synthesis of chemical anticarcinogenic drugs.
2. Chemistry
As shown in Scheme 1, 2-oxo-quinoline-3-carbaldehyde 5 was
prepared according to the literature [22]. And that the compound
was characterized by NMR. 3,4-Dimethyl-2-ethoxycarbonyl-
pyrrole 1 was synthesized by the reported papers [23,24]. The
compositions of Cu(II) complexes were confirmed by X-ray single
crystal diffraction. The three Cu(II) complexes were found to
possess five coordinative square-pyramidal configuration. DNA-
binding study and cytotoxic experiments were also carried out to
evaluate the binding modes and potential anti-cancer activities.
Scheme 1. The synthesis line of ligands (2-oxo-quinoline-3-carbaldehyde-benzoyl hydrazone 6a, 2-oxo-quinoline-3-carbaldehyde-2-hydroxybenzoyl hydrazone 6b, 2-oxo-quin-
oline-3-carbaldehyde-3,4-dimethylpyrryl-2-carboxylic acid hydrazone 6c).

Z.-C. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 5353e5361
5355
The results revealed that the Cu(II) complexes exhibited stronger
2.2. Cytotoxicity assay
affinity with CT-DNA.
Tumor cell lines used in this work were grown in RPMI-1640
medium supplemented with 10% (vol/vol) calf serum 2 mmolꢂ1
glutamine, 100 U mLꢂ1 penicillin (U ¼ 1 unit of activity), and
2.1. X-ray crystallography
100
under 5% CO2. Cells in 100
well plates (Falcon, CA), then treated with varied concentration (10,
20, 40, 80 and 160 M) of the compounds. The culture medium was
removed from the plates after 48 h of culture, and each well was
washed once with 200 phosphate-buffered saline (PBS,
pH ¼ 7.2). The reaction was stopped with the addition of 10 l of
m
g mLꢂ1 streptomycin (GIB-CO, Grand Island, NY) at 310 K
A green crystal of Cu(II) complex of H2L1 (0.35 ꢀ 0.33 ꢀ 0.29 mm)
mL culture medium were seeded into 96-
was measured on a Bruker APEX-II CCD diffractometer with graphite
ꢀ
m
monochromatic Mok
a
radiation (
l
¼ 0.71073 A) at 296(2) K. The
crystallographic data are given in Table 1. The intensity data were
collected by the
u
scan mode within 1.56ꢁ < < 25.50ꢁ for hkl
q
mL
m
(ꢂ8 ꢃ h ꢃ 8, ꢂ20 ꢃ k ꢃ 14, ꢂ24 ꢃ l ꢃ 25) 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 (G. M. Sheldrick, Bruker AXS,
Madison, WI, 2008). Absorption correction was employed using
Semi-empirical methods from equivalents.
1 M NaOH, and colour development was assayed at 405 nm using
a microplate reader (BIO-RAD 680). The nonenzymatic hydrolysis
of the p-NPP (p-nitrophenol phosphate ester) substract was
determined for each assay by including wells that did not contain
cells as blank wells. Cell survival was expressed as an absorbance
(A) percentage defined by (Adrug-blank/Acontrol-blank ꢀ 100).
A green crystal of Cu(II) complex of H2L2 (0.35 ꢀ 0.33 ꢀ 0.27 mm)
was measured on a Bruker APEX-II CCD diffractometer with graphite
ꢀ
3. Results and discussion
monochromatic Mok
a
radiation (
l
¼ 0.71073 A) at 296(2) K. The
crystallographic data are given in Table 1. The intensity data were
collected by the
u
scan mode within 2.74ꢁ < < 27.27ꢁ for hkl
q
3.1. Characterization of compounds
(ꢂ9 ꢃ h ꢃ 9, ꢂ11 ꢃ k ꢃ 12, ꢂ15 ꢃ l ꢃ 18) in the Triclinic. 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 (G. M. Sheldrick, Bruker AXS, Madison,
WI, 2008). Absorption correction was employed using Semi-
empirical methods from equivalents.
3.1.1. Properties and structures of the complexes
The complexes are soluble in ethanol, methanol, DMF (N,N-
dimethylformamide) and DMSO (dimethyl sulfoxide), insoluble in
diethyl ether. They are all air stable. The crystals of the three Cu(II)
complex are obtained by evaporating method. The structures of the
three complexes are shown in Fig. 1.
A browncrystalofCu(II)complexofH2L3 (0.25 ꢀ 0.20 ꢀ 0.18 mm)
was measured on a Bruker APEX-II CCD diffractometer with graphite
ꢀ
3.1.2. Crystal structures of the Cu(II) complexes
monochromatic Mok
a
radiation (
l
¼ 0.71073 A) at 296(2) K. The
The ORTEP representation of the structure of CuL1, including
atom numbering scheme, is shown in Fig. 1(a) and the selected
bond lengths and bond angles are listed in Table 2. The coordination
of H2L1 with Cu(II) ion results in the formation of a five membered
crystallographic data are given in Table 1. The intensity data were
collected by the
u
scan mode within 2.22ꢁ < < 25.17ꢁ for hkl
q
(ꢂ10 ꢃ h ꢃ 10, ꢂ11 ꢃ k ꢃ 13, ꢂ18 ꢃ l ꢃ 15) in the Triclinic. The posi-
tions 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 (G. M. Sheldrick, Bruker AXS,
Madison, WI, 2008). Absorption correction was employed using
Semi-empirical methods from equivalents.
(CuONNC) and
a six membered (CuNCCCO) chelating rings.
Furthermore, there is an ethanol molecule which takes part in
1
3
ꢀ
coordination (Cu eO , 1.9465(18) A) and another ethanol molecule
that is non-coordinative. On the contrary, the NO-3 does not coor-
dinate with Cu(II) ion, and the coordination of Cu1 with
N1O1O2O3O4 gives a square-pyramidal configuration, and the
Table 1
2
carbonyl group [C12-O , 1.278(3) A] of benzoyl hydrazine exists as
enolization to achieve the charge balance. In addition, in the X-ray
structural analysis, one unit cell of the crystal of CuL1 contains four
CuL1 molecules, but they are not independent crystallographically.
The ORTEP representation of the structure of CuL2, including
atom numbering scheme, is shown in Fig. 1(b) and the selected
bond lengths and bond angles are listed in Table 3. The coordination
of H2L2 with Cu(II) ion results in the formation of a five membered
ꢀ
Crystal data and structure refinement of the Cu(II) complex.
CuL1
CuL2
CuL3
Formula
FW
C21H29Cu2N4O9
545.02
C19H19CuN5O11
556.93
C19H24CuN5O8
513.97
Crystal colour
Green
Green
Brown
Crystal size (mm) 0.35 ꢀ 0.33 ꢀ 0.29 0.35 ꢀ 0.33 ꢀ 0.27 0.25 ꢀ 0.20 ꢀ 0.18
Crystal system
Space group
Monoclinic
P21/c
7.1988(3)
17.4054(6)
20.7016(6)
90.00
108.271(2)
90.00
2463.10(15)
4
Triclinic
P-1
Triclinic
P-1
ꢀ
a (A)
8.0506(7)
10.4305(9)
15.2937(13)
76.351(4)
77.907(4)
68.184(4)
1147.99(17)
2
8.433(3)
10.339(4)
14.451(7)
109.571(5)
92.259(8)
107.360(6)
1119.4(8)
2
(CuONNC) and
a six membered (CuNCCCO) chelating rings.
ꢀ
b (A)
Furthermore, there is two methanol molecules in the Cu(II) crystal
ꢀ
c (A)
1
4
ꢀ
(ꢁ)
(ꢁ)
(ꢁ)
structure, one takes part in coordination (Cu eO , 2.194 A) and the
other is non-coordinative. The nitrate has proved to be a very useful
ligand for the construction of coordination complex. There is
a nitrate which takes part in coordination with monodentate type
in the crystal. Also, there is a free nitrate in the lattice. In addition, in
the X-ray structural analysis, one unit cell of the crystal of CuL2
contains the two CuL2 molecules, and they are crystallographically
symmetrical in the lattice.
a
b
g
3
ꢀ
V (A )
Z
Dcalc (g/cm3)
Abs coeff (mmꢂ1
F(000)
1.470
0.944
1136
1.611
1.022
570
1.525
1.031
532
)
qmin and max (deg) 1.56e25.50
2.74e27.27
3688/4438
2.22e25.17
3708/4747
Reflections
collected
3757/4579
The ORTEP representation of the structure of CuL3, including
atom numbering scheme, is shown in Fig. 1(c) and the selected
bond lengths and bond angles are listed in Table 4. The coordinated
configuration (square-pyramidal) of Cu(II) for ion CuL3 is similar to
CuL1, where each Cu(II) ion is five-coordinated with two oxygen
atoms and one nitrogen atom from H2L3 and one oxygen atom of
Unique
[R(int) ¼ 0.0331]
R1 ¼ 0.0387
[R(int) ¼ 0.0146]
R1 ¼ 0.0477
[R(int) ¼ 0.0166]
R1 ¼ 0.0418
Final R indices
[I > 2sigma(I)]
R indices
wR2 ¼ 0.1233
R1 ¼ 0.0488,
wR2 ¼ 0.0977
R1 ¼ 0.0367,
wR2 ¼ 0.0932
wR2 ¼ 0.1056
R1 ¼ 0.0575,
(all data)
wR2 ¼ 0.1321
wR2 ¼ 0.1166

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Z.-C. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 5353e5361
Table 2
1
ꢁ
ꢀ
Selected bond lengths (A) and angles ( ) for the CuL .
Bond names
Bond lengths
Bond angles
Angle
Cu1eO2
Cu1eN1
Cu1eO1
Cu1eO3
Cu1eO4
O2eC12
N1eC11
N1eN2
O1eC1
1.9288(18)
1.926(2)
1.9276(17)
1.9465(18)
2.3647(18)
1.278(3)
1.287(3)
1.397(3)
1.271(3)
1.338(3)
1.385(3)
1.325(3)
1.417(4)
1.486(3)
1.439(3)
1.382(4)
1.379(4)
1.448(3)
1.367(3)
1.400(4)
1.406(4)
1.415(4)
1.393(4)
1.361(5)
O2eCu1eN1
O2eCu1eO1
N1eCu1eO1
O2eCu1eO3
N1eCu1eO3
O1eCu1eO3
O2eCu1eO4
N1eCu1eO4
O1eCu1eO4
O3eCu1eO4
C12eO2eCu1
C11eN1eN2
C11eN1eCu1
N2eN1eCu1
C1eO1eCu1
C1eN7eC3
C12eN2eN1
C19eO3eCu1
O2eC12eN2
O2eC12eC13
N2eC12eC13
N1eC11eC10
N1eC11eH11
O1eC1eN7
81.07(8)
172.68(7)
93.10(8)
95.76(8)
170.73(9)
89.31(8)
88.40(7)
96.86(8)
96.73(7)
91.74(8)
110.83(15)
117.1(2)
128.23(17)
114.68(15)
126.90(17)
125.1(2)
108.36(19)
125.40(19)
124.6(2)
117.8(2)
117.2(2)
123.4(2)
118.3
N7eC1
N7eC3
N2eC12
O3eC19
C12eC13
C11eC10
C13eC14
C13eC18
C1eC10
C10eC9
C8eC3
C8eC9
C8eC7
C3eC4
C7eC6
117.3(2)
the absorption of CuL1, CuL2 and CuL3 at 208 nm have 2 nm, 4 nm
and 5 nm redshifts, respectively, which are the characteristic of
intercalation in the presence of CT-DNA. According to the electronic
absorption spectrum, we can deduce initially that the three Cu(II)
complexes can bind to DNA by intercalation, but the binding mode
need to be proved through more experiments.
3.2.2. Fluorescence spectrum titration
As reported, the fluorescence emission spectrum has been found
to be able to distinguish the binding modes of drugs to DNA [28]. The
three Cu(II) complexes can emit weak luminescence in TriseHCl
buffer with a max emission wavelength of about 450 nm. The fluo-
rescence titrations with DNA are conducted by keeping the
concentrations of complexes constant, and varying the DNA
concentrations. Fig. 3 displays the titration curves of the compounds
with CT-DNA. The addition of the two Cu(II) complexes leads to the
conspicuous increase in the fluorescence emission which clearly
indicates that the binding mode of complexes with CT-DNA can be
Fig. 1. ORTEP view of (a) (CuL1), (b) (CuL2) and (c) (CuL3) showing the atom numbering
of scheme and 30% thermal ellipsoids probability for the non-hydrogen atoms.
H2O and ethanol molecules. The carbonyl radical [C1eO5, 1.290
ꢀ
(4) A] from 3,4-Dimethylpyrryl-2-carboxylic acid hydrazide also
take enolization which is same to that of CuL1. A free water mole-
cule and nitrate are not coordinated with the Cu(II) ion. Addition-
ally, there are two CuL3 molecules in one unite of the crystal of CuL3.
Table 3
2
ꢁ
ꢀ
Selected bond lengths (A) and angles ( ) for the CuL .
Bond names
Bond lengths
Bond angles
Angle
Cu1eO2
Cu1eN2
Cu1eO1
Cu1eO4
Cu1eO5
C1eO1
1.9739(18)
1.9539(19)
1.9354(17)
2.194(2)
1.9752(17)
1.263(3)
1.339(3)
1.390(3)
1.279(3)
1.340(3)
1.350(3)
1.255(3)
1.340(3)
1.425(4)
1.381(4)
1.372(3)
1.227(3)
1.243(3)
1.242(3)
O1eCu1eN2
O1eCueO2
N2eCu1eO2
O1eCu1eO5
N2eCu1eO5
O2eCu1eO5
O1eCu1eO4
N2eCu1eO4
O2eCu1eO4
O5eCu1eO4
O1eC1eN1
N1eC2eC7
90.41(7)
169.92(7)
80.84
3.2. DNA-binding mode and affinity
3.2.1. Electronic absorption spectrum titration
93.03(7)
167.35(8)
94.48(7)
92.64(8)
104.30(8)
94.37(8)
87.72(8)
117.4(2)
118.2(2)
125.5(2)
120.6(2)
122.5(2)
120.0(2)
122.3(2)
118.2(2)
111.17(15)
The binding modes of complexes to DNA are characterized
classically through electronic absorption titrations. After the
complexes intercalate to the base pair of DNA, the p* orbital of the
intercalated ligands on the complexes could couple of with
orbitals of the base pairs, thus decreasing the
energies. On the other hand, the coupling p* orbital are partially
filled by electrons, thus decreasing the transition probabilities [25].
Therefore, these interactions can result in obvious hypochromism
and redshift which were corresponded to what were reported
previously [26]. As shown in Fig. 2, the absorption bands of CuL1,
CuL2 and CuL3 at 208 nm exhibit hypochromism of about 44.0%,
80.77% and 52.2%, simultaneously, following the hypochromism
associated with binding of the two complexes to the DNA helix [27],
C1eN1
C2eN1
p
C10eN2
C11eN3
C13eO3
C11eO2
C11eO3
C18eO4
C19eO11
N2eN3
N5eO10
N5eO9
N5eO8
pe
p* transition
O1eC1eC9
C3eC2eN1
N2eC10eC9
N3eC11eC12
O3eC13eC14
O3eC13eC12
N4eO5eCu1

Z.-C. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 5353e5361
5357
Table 4
Selected bond lengths (A) and angles ( ) for the CuL .
observed phenomenon is consistent with that of a classical inter-
calation and can be caused by different terminal ligands.
3
ꢁ
ꢀ
Bond names
Bond lengths
Bond angles
Angle
3.2.4. DNAeEB competitive experiment
Cu1eO1
Cu1eO5
Cu1eO6
Cu1eN7
Cu1eO8
N1eO2
N1eO7
N1eO3
N6eN7
C1eO5
C1eN6
C5eO8
C5eN4
C22eN2
C25eO1
C3eC7
1.951(2)
1.916(2)
2.275(2)
1.940(2)
1.929(2)
1.209(4)
1.243(4)
1.255(4)
1.385(3)
1.290(4)
1.332(4)
1.267(3)
1.346(3)
1.352(4)
1.452(4)
1.450(4)
1.384(3)
1.389(4)
1.423(4)
O5eCu1eO8
O5eCu1eN7
O8eCu1eN7
O5eCu1eO1
O8eCu1eO1
N7eCu1eO1
O5eCu1eO6
O8eCu1eO6
N7eCu1eO6
O1eCu1eO6
O2eN1eO7
C5eN4eC6
C1eN6eN7
C3eN7eCu1
N6eN7eCu1
C25eO1eCu1
C5eO8eCu1
C1eO5eCu1
O8eC5eN4
167.75(9)
81.76(9)
93.18(9)
94.43(10)
88.58(9)
169.42(11)
96.78(10)
94.74(9)
94.61(9)
95.64(11)
120.9(3)
125.8(2)
109.4(2)
127.26(19)
113.87(17)
126.0(2)
125.93(17)
110.91(18)
117.2(2)
Ethidium bromide (EB) is one of the most sensitive fluorescent
probes which can bind to DNA through intercalation [34,35].
Competitive binding to DNA of the drugs with EB could provide rich
information with regard to the DNA-binding affinity. As shown in
Fig. 5, the fluorescence intensity decrease obviously with the
increasing concentration of the three Cu(II) complexes. The Kq values
for Cu(II) complexes are 6.75 ꢀ104 Mꢂ1 (CuL1),1.81 ꢀ105 Mꢂ1 (CuL2)
and 2.48 ꢀ 105 Mꢂ1 (CuL3), respectively. The quenching plots illus-
trate that the quenching of EB bound to DNA by the compounds are
in good agreement with the linear SterneVolmer equation and the
binding ability of three compounds follows the order
CuL3 > CuL2 > CuL1 which are in agreement with the fluorescence
and viscosity measurements. Furthermore, such quenching
constants of the Cu(II) complexes suggest that the interaction of all
the compounds with DNA should be of intercalation [36].
C6eN4
C6eC10
C9eC12
3.3. Cytotoxic activity
intercalation. Because the enhancement of fluorescence intensity
implied that these compounds can insert between DNA base pair, as
a result, these compounds are protected from solvent water mole-
cules by the hydrophobic environment inside the DNA helix [29].
The microenvironment of fluorophores residue change accordingly
and the accessibility of solvent water molecules to these compounds
is reduced. And that an enhanced fluorescence is believed to be one
of the criteria for intercalative binding [30,31]. According to the
Scatchard equation, a plot of r/Cf versus r gives the intrinsic binding
constants of the compounds. The results imply that all the
compounds can insert between DNA base pairs and CuL3
(Kb ¼ 3.3 ꢀ 106 Mꢂ1) can interact with DNA more strongly than CuL1
(Kb ¼ 1.27 ꢀ 106 Mꢂ1) and CuL2 (Kb ¼ 2.76 ꢀ 106 Mꢂ1). However, the
DNA-drugs binding modes and binding affinity need to be proved
further by viscosity studies and EBeDNA displacement experiment.
The cytotoxicity assays of ligands and Cu(II) complexes against
two kinds of tumor cells [human leukemia HL-60 and uterine
cervix carcinoma cell (Hela)] are shown in Fig. 6(a) and (b). The
biological assays of the ligands and Cu(II) complexes show Cu(II)
complexes exhibit more significant activities than corresponding
ligands against HL-60 and Hela cells. For HeLa cell line CuL3
(IC50 ¼ 38
mM) demonstrated a much higher inhibitory effect than
CuL2 (IC50 ¼ 43
m mM) shows nearly no
M), CuL1 (IC50 > 160
activity. Moreover, the inhibitory effects of compounds
(IC50CuL1 ¼19
m
M, IC50CuL2 ¼18
m
M, IC50CuL3 ¼ 8
mM) for HL-60
cell follow the order CuL3 > CuL2 > CuL1. The CuL3 whose IC50 is
8 mM is especially most active. The cytotoxic activity studies in vitro
indicate that the three Cu(II) complexes have rather activities than
corresponding ligands against the two tumor cells, the three Cu(II)
complexes which possess better cytotoxic activities than ligands
may be attributed to the extended planar structure induced by the
pep* conjugation resulting from the chelating of the metal ion with
ligand. In addition, the inhibitory rates of CuL3 against the two
tumor cells are higher than CuL1 and CuL2, which can be caused by
the 3,4-dimethylpyrryl heterocycle from CuL3.
3.2.3. Viscosity measurements
Measurements of DNA viscosity that is sensitive to DNA length
change are regarded as the least ambiguous and the most critical
tests of binding in solution in the absence of crystallographic
structural data [32]. An intercalative mode drug to DNA will lead to
obvious increase of DNA viscosity [33]. Fig. 4 shows that the three
Cu(II) complexes cause a remarkable increase of viscosity and the
increasing degree of relative viscosity, which may be depend on its
affinity to DNA, follows the order of CuL3 > CuL2 > CuL1. The
4. Conclusion
In this paper, three new 2-oxo-quinoline-3-carbaldehyde Schiff-
bases ligands and their Cu(II) complexes are synthesized and
Fig. 2. Absorption spectra of CuL1 (a), CuL2 (b) and CuL3 (c) (10
30 M; subsequent spectra). Arrows show the absorbance changes upon increasing DNA concentration.
mM) in the absence and presence of increasing amounts of CT-DNA (0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5,
m

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Z.-C. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 5353e5361
Fig. 3. Emission enhancement spectra of compounds [CuL1, CuL2 and CuL3 (10
mM)] in the absence and presence of increasing amounts of CT-DNA (2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20,
22.5, 25, 27.5, 30
m
M); lex ¼ 370 nm, lem ¼ 450 nm. Arrows show the emission intensity changes of upon the increasing CT-DNA concentration. Inset: Scatchard plot of the fluo-
rescence titration data of compounds, Kb ¼ 1.27 ꢀ 106 Mꢂ1 (CuL1). Kb ¼ 2.76 ꢀ 106 Mꢂ1 (CuL2). Kb ¼ 3.30 ꢀ 106 Mꢂ1 (CuL3).
characterized. And that the X-ray diffraction shows the three Cu(II)
complexes exhibit similar coordinative modes. In addition, the
binding modes of CT-DNA to the Cu(II) complexes are also studied.
The photophysical and viscosity measurements indicate that the
three Cu(II) complexes can interact with CT-DNA through inter-
calative binding modes, respectively. The cytotoxicities of the three
Cu(II) complexes are tested in vitro and the biological assays
suggest the Cu(II) complexes exhibit higher activity than corre-
sponding ligands against HeLa and HL-60 cells, which can be
attributed to the interaction between Cu(II) ion and ligands.
Moreover, the most active compound is CuL3 which may be
explained as resulting from a difference in the terminal functional
group (3,4-dimethylpyrryl heterocycle) relative to CuL1 and CuL2.
The finding are significant for us to explore the DNA-binding
studies and cytotoxic activities on the Cu(II) Schiff-bases complexes
of quinolone derivates.
5. Experimental protocols
5.1. DNA-binding study methods
5.1.1. Electronic spectral titration
Absorption titration experiment was performed by maintaining
the three Cu(II) complexes concentration constant (10 mM) and
gradually increasing the concentration of CT-DNA. The compounds
were dissolved in a mixed solvent of 1% CH3OH and 99% TriseHCl
buffer. The reference solution was the corresponding TriseHCl
buffer solution. While measuring the absorption spectra, equal
amount of CT-DNA was added to both compound solution and the
reference solution to eliminate the absorbance of CT-DNA itself. The
sample solution was scanned in the range 200e500 nm.
Fig. 4. Effect of increasing amounts of CuL1, CuL2 and CuL3 on the relative viscosity of
CT-DNA at 25.0 ꢄ 0.1 ꢁC, [DNA] ¼ 5.0
mM.

Z.-C. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 5353e5361
5359
Fig. 5. Emission spectra of DNAeEB system (10
m
M DNA and 0.33
m
M EB), lex ¼ 521 nm, lem ¼ 540e700 nm, in the presence of (0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, and
30 m
M) CuL1 (a), CuL2 (b) and CuL3 (c). Arrows show the emission intensity changes upon increasing compounds concentration. SterneVolmer plots of the fluorescence titration of
CuL1 (a), CuL2 (b) and CuL2 (c). Quenching constant Kq ¼ 6.75 ꢀ104 Mꢂ1 (CuL1), Kq ¼ 1.81 ꢀ105 Mꢂ1 (CuL2), Kq ¼2.48 ꢀ 105 Mꢂ1 (CuL3).
5.1.2. Fluorescence spectral titration
r=Cf ¼ Kbð1 ꢂ nrÞ
To investigate the binding modes and compare further the
where r ¼ Cb/[DNA], Cf ¼ Ct[(F ꢂ F0)/(Fmax ꢂ F0)], Cb and Ct is the
affinity of the compounds bound to CT-DNA, the fluorescence
titration spectrum was studied A fixed amount of the compound
(10 mM) was titrated with increasing amounts of CT-DNA. The
samples were excited at 370 nm and the total fluorescence emis-
sion intensity was monitored at 450 nm for the Cu(II) complexes. K
values were determined using the following equations [32].
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
Fig. 6. (a) Cytotoxic activity of compounds against HeLa. (b) Cytotoxic activity of compounds against HL-60.

5360
Z.-C. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 5353e5361
0
versus r. All experiments were conducted at 20 ꢁC in a buffer con-
taining 5 mM TriseHCl (pH ¼ 7.2) and 50 mM NaCl.
d
d
7.861e7.889 (1H, m, eC7 eH),
d
d
7.512e7.633 (4H, m, eC5,6,7,8eH),
0
0
7.330e7.357 (1H, m, eC9 eH),
7.199e7.250 (1H, m, eC8 eH).
2-Oxo-quinoline-3-carbaldehyde-2-hydroxybenzoyl hydrazone
5.1.3. Viscosity measurement experiment
(H2L2) 6b and 2-oxo-quinoline-3-carbaldehyde-3,4-dimethyl-
pyrryl-2-carboxylic acid hydrazone (H2L3) 6c were synthesized
according to the same procedure as the synthesis method of ligand
The viscosity titrations were conducted on an Ubbelohde
viscometer in a thermostatic water bath maintained at 298 (ꢄ0.1)
K. The DNA concentration was kept constant (5
mM) and gradually
H2L1. H2L2, Yield, 76%. Colour, yellow. m.p.:314e316 ꢁC. 1H-NMR
0
increased the concentration of tested compound (0.5e3.5
mM). The
(DMSO-d6, ppm):
eOH), 8.715 (1H, s, eCH]N),
(2H, m, eC6 ,9 eH),
(1H, m, eC7eH),
d
12.043 (2H, s, eN1eH, eN3 eH),
d
11.875 (1H, s,
time of the solution’s flowing through the capillary was determined
to the nearest 0.02 s by a stop-watch with the viscometer. Each
sample was measured three times and an average flow was
d
d
8.496 (1H, s, eC4eH),
d
7.861e7.922
0
0
d
7.527e7.578 (1H, m, eC5eH),
d 7.424e7.475
d
7.334e7.361 (1H, m, eC8eH),
d
7.201e7.252 (1H,
0
0
calculated at last. Data were presented as (
of the concentration of the compound to CT-DNA, where
h
/
h0
)
1/3 versus the ratio
was the
m, eC6eH), 6.928e6.986 (1H, m, eC7 ,8 eH). H2L3, Yield, 80%.
d
h
Colour, khaki. m.p.:317e319 ꢁC. 1H-NMR (DMSO-d6, ppm):
d
12.171
0
viscosity of CT-DNA in the presence of the compound and h0 was
the viscosity of CT-DNA alone. Viscosity values were calculated
from the observed flow time of CT-DNA containing solutions cor-
(1H, s, eN1eH,),
d
11.242 (1H, s, eN3 eH),
d
8.352 (1H, s, eCH]N),
7.532e7.574
7.217e7.254 (1H,
d
8.302 (1H, s, eC4eH), 7.800e7.802 (1H, m, eN5eH),
d d
(1H, m, eN7eH),
d
7.333e7.354 (1H, m, eN8eH),
d
0
0
rected from the flow time of buffer alone (t0),
h
¼ t ꢂ t0.
m, eN6eH),
d
6.750e6.756 (1H, m, eN6 eH),
d
3.337 (1H, s, eN7 eH),
0
0
d
2.252 (3H, s, eC11 eH), 1.970 (3H, s, C9 eH).
d
5.1.4. EBeDNA competition experiment
EBeDNA experiments were conducted by adding the
compounds solution to the TriseHCl buffer of EBeDNA. The change
of fluorescence intensity was recorded. The excitation and the
emission wavelength were 521 nm and 587 nm, respectively.
According to the classical SterneVolmer equation:
5.3. Preparation of Cu(II) complexes
The ligand H2L1 (0.2 mmol, 0.0582 g) and Cu(II) nitrate
(0.2 mmol, 0.0483 g) were added to ethanol (10 mL). After 5 min,
the mixtures were filtered to remove any insoluble residues and
then were stirred for 10 h under reflux. A green precipitation
(yield:85%) of the Cu(II) complex was separated from the solution
by suction filtration, purified by washing several times with
Ethanol and dried for 24 h under vacuum. The Cu(II) complex was
characterized by X-ray single crystal diffraction.
F0=F ¼ Kq½Qꢅ 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.
The Cu(II) complex of H2L2 and H2L3 was prepared by the same
method. The ligand H2L2 (0.2 mmol, 0.0614 g) and Cu(II) nitrate
(0.2 mmol, 0.0483 g) were added to methanol (10 mL). After 5 min,
the mixtures were filtered to remove any insoluble residues and
then were stirred for 10 h under reflux. A green precipitation
(yield:80%) of the Cu(II) complex was separated from the solution
by suction filtration, purified by washing several times with
methanol and dried for 24 h under vacuum. The Cu(II) complex was
characterized by X-ray single crystal diffraction. The ligand H2L3
(0.2 mmol, 0.061 g) and Cu(II) nitrate (0.2 mmol, 0.0483 g) were
added to ethanol (10 mL). After 5 min, the mixtures were filtered to
remove any insoluble residues and then were stirred for 10 h under
reflux. A brown precipitation (yield:75%) of the Cu(II) complex was
separated from the solution by suction filtration, purified by
washing several times with ethanol and dried for 24 h under
vacuum. The Cu(II) complex was characterized by X-ray single
crystal diffraction.
5.2. Preparation and characterization of compounds
As shown in Scheme 1, 3,4-dimethylpyrryl-2-carboxylic acid
hydrazine 2 was prepared easily by refluxing 3,4-dimethyl-2-
ethoxycarbonyl-pyrrole 1 (0.5 g, 3 mmol) with stirring in an
ethanol solution (30 mL) containing hydrazine hydrate (80%,
0.375 g, 6 mmol). After cooling to room temperature, a white
precipitation separated out. The precipitation was filtrated under
decompression and washed with ethanol. Recrystallization from
CH3OH/H2O (V:V ¼ 1:2) gave the white product in 70% yield.
m.p.:218e220 ꢁC. 1H-NMR (DMSO-d6 ppm):
d
10.62 (1H, s, pyrrole
6.59 (1H, d, pyrrole eCH), 4.27
2.13 (3H, s, eCH3),
1.90 (3H, s, eCH3). 13C-NMR
eNH),
(2H, s, eNH2),
(DMSO-d6 ppm):
d
8.51 (1H, s, eNHeC]O),
d
d
d
d
d
:162.34, 121.24, 121.00, 118.19, 117.98, 10.10, 9.79.
The 13C-NMR spectrum is shown in Fig. S1 (Supplementary
information).
2-Oxo-quinoline-3-carbaldehyde 5. Yield, 85%. Colour, yellow.
Acknowledgements
m.p.:303e305 ꢁC. 1H-NMR (DMSO-d6 ppm):
d
d
d
d
12.24 (1H, s, eN1H),
7.92 (1H, d, eC5eH),
7.35 (1H, d, eC8eH).
10.24 (1H, s, eCHO),
d
8.51 (1H, s, eC4eH),
7.25 (1H, t, eC7eH),
This work is supported by the National Natural Science Foun-
dation of China (20975046).
d
7.66 (1H, t, eC6eH),
d
2-Oxo-quinoline-3-carbaldehyde-benzoyl hydrazone (H2L1) 6a
was synthesized by the following method (Scheme 1). An ethanol
solution (10 mL) containing benzoyl hydrazine (0.48 g, 3.5 mmol)
was added to another ethanol solution (10 mL) containing 2-oxo-
quinoline-3-carbaldehyde 5 (0.6 g, 3.5 mmol). The mixture was
refluxed for twelve hours with stirring and a white precipitation
separated out. The precipitation was filtrated under decompression
and washed with ethanol. Recrystallization from DMF/H2O
(V:V ¼ 1:1) gave the yellow ligand H2L1, which was dried under
Appendix. Supplementary information
Crystallographic data for the structural analysis have been
deposited with the Cambridge Crystallographic Data Centre, CCDC
(737811, 746589, 763151). 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).
vacuum. Yield, 85%. m.p.:307e309 ꢁC. 1H-NMR (DMSO-d6 ppm):
0
d
12.055 (1H, s, eN1eH),
d
12.025 (1H, s, eN3 eH),
d
8.723 (1H, s,
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2010.08.060.
0
0
eCH]N), d d
8.487 (1H, s, eC4eH), 7.932e7.957 (2H, s, eC6 ,10 eH),

Z.-C. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 5353e5361
5361
[18] Z.C. Liu, B.D. Wang, Z.Y. Yang, Y. Li, D.D. Qin, T.R. Li, Eur. J. Med. Chem. 44
(2009) 4477e4484.
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