Synthesis, crystal structures, DNA-binding properties, cytotoxic and antioxidation activities of several new ternary copper(II) complexes of N,N′-(p-xylylene)di-alanine acid and 1,10-phenanthroline

Article abstract of DOI:10.1016/j.ica.2009.12.047

Three novel ternary copper(II) complexes, [Cu₂(phen)₂(l-PDIAla)(H₂O)₂](ClO₄)₂·2.5H₂O (1), [Cu₄(phen)₆(d,l-PDIAla)(H₂O)₂](ClO₄)₆·3H₂O (2) and [Cu₂(phen)₂(d,l-PDIAla)(H₂O)](ClO₄)₂·0.5H₂O (3) (phen = 1,10-phenanthroline, H₂PDIAla = N,N'-(p-xylylene)di-alanine acid) have been synthesized and structurally characterized by single-crystal X-ray crystallography and other structural analysis. Spectrometric titrations, ethidium bromide displacement experiments, CD (circular dichroism) spectral analysis and viscosity measurements indicate that the three compounds, especially the complex 3, strongly bind to calf-thymus DNA (CT-DNA). The intrinsic binding constants of the ternary copper(II) complexes with CT-DNA are 0.89 × 10⁵, 1.14 × 10⁵ and 1.72 × 10⁵ M^-1, for 1, 2 and 3, respectively. Comparative cytotoxic activities of the copper(II) complexes are also determined by acid phosphatase assay. The results show that the ternary copper(II) complexes have significant cytotoxic activity against the HeLa (Cervical cancer), HepG2 (hepatocarcinoma), HL-60 cells (myeloid leukemia), A-549 cells (pulmonary carcinoma) and L02 (liver cells). Investigations of antioxidation properties show that all the copper(II) complexes have strong scavenging effects for hydroxyl radicals and superoxide radicals.

Full text of DOI:10.1016/j.ica.2009.12.047

Inorganica Chimica Acta 363 (2010) 855–865
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis, crystal structures, DNA-binding properties, cytotoxic and
antioxidation activities of several new ternary copper(II) complexes
of N,N0-(p-xylylene)di-alanine acid and 1,10-phenanthroline
c
a
a
c
a
a
Lei Jia a, , Peng Jiang , Jun Xu , Zhong-yan Hao , Xi-ming Xu , Long-hai Chen , Jin-cai Wu ,
*
Ning Tang a, , Qin Wang , Jagadese J. Vittal
c
b,
*
*
a Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, State Key Laboratory of Applied Organic Chemistry,
College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China
b Department of Chemistry, National University of Singapore, Singapore 117543, Singapore
c School 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 ternary copper(II) complexes, [Cu2(phen)2(
L
-PDIAla)(H2O)2](ClO4)2ꢀ2.5H2O (1),
Received 5 November 2009
Accepted 21 December 2009
Available online 4 January 2010
[Cu4(phen)6(
D
,L
-PDIAla)(H2O)2](ClO4)6ꢀ3H2O (2) and [Cu2(phen)2(
D,L
-PDIAla)(H2O)](ClO4)2ꢀ0.5H2O (3)
(phen = 1,10-phenanthroline, H2PDIAla = N,N’-(p-xylylene)di-alanine acid) have been synthesized and
structurally characterized by single-crystal X-ray crystallography and other structural analysis. Spectro-
metric titrations, ethidium bromide displacement experiments, CD (circular dichroism) spectral analysis
and viscosity measurements indicate that the three compounds, especially the complex 3, strongly bind
to calf-thymus DNA (CT-DNA). The intrinsic binding constants of the ternary copper(II) complexes with
CT-DNA are 0.89 ꢁ 105, 1.14 ꢁ 105 and 1.72 ꢁ 105 Mꢂ1, for 1, 2 and 3, respectively. Comparative cytotoxic
activities of the copper(II) complexes are also determined by acid phosphatase assay. The results show
that the ternary copper(II) complexes have significant cytotoxic activity against the HeLa (Cervical can-
cer), HepG2 (hepatocarcinoma), HL-60 cells (myeloid leukemia), A-549 cells (pulmonary carcinoma) and
L02 (liver cells). Investigations of antioxidation properties show that all the copper(II) complexes have
strong scavenging effects for hydroxyl radicals and superoxide radicals.
Keywords:
Ternary copper(II) complexes
Cytotoxicity
DNA-binding properties
Reduce Schiff base
X-ray crystallography
Phenanthroline base
Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction
interactions include intercalative, electrostatic and groove (sur-
face) binding of cationic metal complexes along outside of DNA he-
It is well known that deoxyribonucleic acid (DNA) plays an
important role in the life process since it contains all the genetic
information for cellular function. However, DNA molecules are
prone to be damaged under various conditions like interactions
with some molecules. This damage may cause various pathological
changes in living organisms, which is due to their possible applica-
tion as new therapeutic agents and their photochemical properties
which make them potential probes of DNA structure and confor-
mation [1–3]. The binding interaction of transition metal com-
plexes with DNA is of interest for both therapeutic and scientific
reasons [4]. Many transition metal complexes are known to bind
to DNA via both covalent and non-covalent interactions. In cova-
lent binding the labile ligand of the complexes is replaced by a
nitrogen base of DNA. On the other hand, the non-covalent DNA
lix, major or minor groove.
Copper is a biologically relevant element and many enzymes
that depend on copper for their activity have been identified. Cop-
per complexes of 1,10-phenanthroline and its derivatives are of
great interests since they exhibit numerous biological activities
such as anti-tumor [5], anti-Candida [6], antimycobacterial [7],
and antimicrobial activity [8]. Moreover, considerable attention
has been focused on the use of phenanthroline complexes as inter-
calating agents of DNA and as artificial nucleases [9].
Amino acids are the basic structural units of proteins that recog-
nize a specific base sequence of DNA. Copper complexes containing
amino acids have been studied as models for the behavior of cop-
per enzymes and some copper complexes with amino acid ligands
were reported to exhibit potent anti-tumor and artificial nuclease
activities [10,11]. Recent reports have also shown that amino
acid/peptide based copper(II) complexes show efficient DNA cleav-
age activity [12–14].
* Corresponding authors. Tel.: +86 931 8911218 (N. Tang), tel.: +65 6516 2975
(J.J. Vittal).
E-mail addresses: jialeikfc@gmail.com (L. Jia), tangn@lzu.edu.cn (N. Tang),
chmjjv@nus.edu.sg (J.J. Vittal).
Herein we report the synthesis, crystal structure, DNA-binding,
cytotoxic and antioxidation activities of three ternary copper(II)
0020-1693/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2009.12.047

856
L. Jia et al. / Inorganica Chimica Acta 363 (2010) 855–865
complexes [Cu2(phen)2(
L
-PDIAla)(H2O)2](ClO4)2ꢀ2.5H2O (1), [Cu4-
-PDI-
OH
(phen)6(
D,L-PDIAla)(H2O)2](ClO4)6ꢀ3H2O (2), [Cu2(phen)2(
D,L
O
N
H
H
N
Ala)(H2O)](ClO4)2ꢀ0.5 H2O (3) (Fig. 1). The primary aim of the
current study is to determine the cancer chemotherapeutic poten-
tial of metal-free phen, ternary copper(II) complexes 1-3, using five
human-derived cancer model cell lines of HeLa (Cervical cancer),
HepG2 (hepatocarcinoma), L02 (liver cells), HL-60 (myeloid leuke-
mia) and A-549 (pulmonary carcinoma). In order to illustrate that
the effect observed is due to the complexes rather than the free
metal ion, the anti-tumor activities of the simple Cu(II) salt, Cu-
(ClO4)2 is also determined. This work represents the first assess-
ment of the potential application of the three copper(II) complexes
1–3 as novel therapeutic agents for the treatment of cancer.
O
HO
Fig. 1. The chemical structure of the ligand.
2.3. Synthesis of the complex [Cu2(phen)2(
L-PDIAla)(H2O)2]
(ClO4)2ꢀ2.5H2O (1)
To the green solution formed from Cu(ClO4)2ꢀ6H2O (0.37 g,
1 mmol) in MeOH (8 mL) and phen (0.18 g, 1 mmol) in MeOH
(8 mL) was added
a filtered solution of L-H2PDIAla (0.14 g,
0.5 mmol) in H2O (20 mL) containing LiOH. The resulting blue solu-
tion was stirred for 1 h and then filtered and left for a week, after
which time the blue crystals were isolated by filtration. Yield:
0.6 g, 87%. Anal. Calc. for C38H43Cl2Cu2N6O16.5: C, 43.64; H, 4.11;
2. Experimental
2.1. Materials and instrumentation
N, 8.04. Found: C, 43.89; H, 4.02; N, 8.25%. IR (KBr, cmꢂ1):
t
(NH)
(COOꢂ) 1612, 1320;
All starting materials were obtained commercially and used as
received. Calf-thymus DNA (CT-DNA) and ethidium bromide (EB)
were obtained from Sigma Chemical Co. All the measurements
involving the interactions of the three metal complexes with CT-
DNA were carried out in doubly distilled water buffer containing
5 mM Tris and 50 mM NaCl, and adjusted to pH 7.1 with hydro-
chloric acid. UV–Vis spectrometer was employed to check the solu-
tion of CT-DNA purity (A260:A280 > 1.80) and the concentration
2979;
phen) 843, 734.
t(CH2) 2930; t t(Cl–O) 1089; t(C–H,
2.4. Synthesis of the complex [Cu4(phen)6(D,L-PDIAla)(H2O)2]
(ClO4)6ꢀ3H2O (2)
To the green solution formed from Cu(ClO4)2ꢀ6H2O (0.74 g,
2 mmol) in MeOH (16 mL) and phen (0.54 g, 3 mmol) in MeOH
(e
= 6600 Mꢂ1 cmꢂ1 at 260 nm) in the buffer. The ternary copper(II)
complexes were dissolved in a mixture solvent of 1% CH3OH or 1%
DMSO and 99% Tris–HCl buffer (5 mM Tris–HCl, 50 mM NaCl, pH
7.1) at concentration 1 ꢁ 10ꢂ3 M.
(16 mL) was added a filtered solution of
D,L-H2PDIAla (0.14 g,
0.5 mmol) in H2O (30 mL) containing LiOH. The resulting blue solu-
tion was stirred for 1 h and then filtered and left for a week, after
which time the dark blue crystals were isolated by filtration. Yield:
0.85 g, 60%. Anal. Calc. for C86H76Cl6Cu4N14O33: C, 44.90; H, 3.33; N,
The UV–Vis absorption spectra were recorded using a Varian
Cary 100 spectrophotometer and fluorescence emission spectra
were recorded using a Hitachi F-4500 spectrofluorophotometer.
The elemental analyses were performed in the microanalytical lab-
oratory, Department of Chemistry, National University of Singa-
pore. The 1H NMR spectra were recorded with a Bruker ACF300
FT-NMR instrument using TMS as an internal reference in D2O
for the ligand. The infrared spectra (KBr pellet) were recorded
using an FTS165 Bio-Rad FTIR spectrophotometer in the range of
4000–400 cmꢂ1. ESI mass spectra were recorded on a Finnigan
MAT LCQ mass spectrometer using the syringe pump method.
The CD spectra were recorded on a Jasco J-810 spectropolarimeter.
The antioxidant activities were tested on a 721E spectrophotome-
ter (Shanghai Analytical Instrument Factory, China).
8.52. Found: C, 44.72; H, 3.56; N, 8.35%. IR (KBr, cmꢂ1):
t
(NH) 2990,
t(CH2) 3120,
t t(Cl–O) 1085. t(C–H, phen) 845,
(COOꢂ) 1575, 1386.
736.
2.5. Synthesis of the complex [Cu2(phen)2(D,L-PDIAla)(H2O)]
(ClO4)2ꢀ0.5H2O (3)
To the green solution formed from Cu(ClO4)2ꢀ6H2O (0.37 g,
1 mmol) in MeOH (8 mL) and phen (0.18 g, 1 mmol) in MeOH
(8 mL) was added a filtered solution of
D,L-H2PDIAla (0.14 g,
0.5 mmol) in H2O (20 mL) containing LiOH. The resulting blue solu-
tion was stirred for 1 h and then filtered and left for a week, after
which time the dark blue crystals were isolated by filtration. Yield:
0.64 g, 92%. Anal. Calc. for C38H37Cl2Cu2N6O13.5: C, 46.01; H, 3.73; N,
8.48. Found: C, 46.15; H, 3.99; N, 8.62%. IR (KBr, cmꢂ1):
t
(NH) 2990,
t(CH2) 2998,
t t(Cl–O) 1089. t(C–H, phen) 843,
(COOꢂ) 1588, 1400.
2.2. Synthesis of the ligand (H2PDIAla)
733.
Caution. Perchlorate salts of metal complexes containing organ-
ic ligands are potentially explosive. Only small quantity of material
should be prepared and handled with suitable safety measures.
H2PDIAla: N,N’-(p-xylylene)di-alanine acid. To a solution of
L-
alanine or -alanine (0.89 g, 10 mmol) and NaOH (0.4 g, 10 mmol)
D,L
in 20 mL of MeOH and 10 mL of water was added terephthalalde-
hyde (0.67 g, 5 mmol) in 20 mL of MeOH. The yellow solution was
stirred for 3 h at room temperature prior to cooling in an ice bath.
The intermediate Schiff base that formed was reduced with an ex-
cess of NaBH4 (0.46 g, 12 mmol) in MeOH containing a few drops of
NaOH solution. The yellowish color was slowly discharged, after
stirring for another 1 h, the solution was acidified with acetic acid
to a pH of 5.0–6.0. The resulting colorless solid was filtered off,
washed with dry MeOH and Et2O, and recrystallized from H2O/
EtOH (1:3) after drying in air. Yield: 1.12 g (80%). Anal. Calc. for
C14H20N2O4: C, 59.99; H, 7.19; N, 9.99. Found: C, 59.63; H, 6.97;
N, 9.72%. ESI-MS Calcd. for C14H20N2O4: 280.32. Found: 279.3. 1H
NMR (D2O) d 1.54 (d, 3H), 3.74 (t, 1H), 4.29 (s, 2H), 7.57 (s, 2H).
2.6. X-ray crystallography
Details of the crystal parameters, data collection and refine-
ments were listed in Table 1. Selected bond lengths and bond an-
gles were listed in Table 2. Main intra and intermolecular
hydrogen bonds for complexes 1 and 3 were listed in Table 3. Sin-
gle-crystal X-ray diffraction measurements were made on a Bruker
AXS SMART CCD diffractometer with Mo Ka (k = 0.71073 Å) in a
sealed tube. Unit cell dimensions were obtained with least-squares
refinements, and all structures were solved by direct methods. The
program SMART [15] was used to collect the intensity data, SAINT
for integration of the intensity [15], SADABS [16] for absorption
IR (KBr, cmꢂ1): (COOꢂ) 1611, 1392.
t(NH) 2977; t(CH2) 2931; t

L. Jia et al. / Inorganica Chimica Acta 363 (2010) 855–865
857
Table 1
Selected crystallographic data for 1, 2 and 3.
Compound
1
2
3
Empirical formula
Formula weight
T (K)
C38H43Cl2Cu2N6O16.5
1045.76
223(2)
C86H76Cl6Cu4N14O33
2290.39
223(2)
C38H36Cl2Cu2N6O13.5
970.70
223(2)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
monoclinic
P2(1)
10.7337(5)
19.1792(8)
10.9753(5)
90
100.8550(10)°
90
2218.99(17)
2
triclinic
P1
monoclinic
P2(1)/n
21.2153(15)
9.3378(7)
22.0024(16)
90
113.541(2)
90
3996.0(5)
4
ꢀ
10.8999(6)
14.1203(8)
16.7903(9)
87.4460(10)
80.188(2)
80.6970(10)
2512.5(2)
1
a
(°)
b (°)
c
(°)
V (Å3)
Z
Calculated density (Mg/m3)
1.56
1.157
1.514
1.081
1.647
1.274
Absorption coefficient (mmꢂ1
)
Number of parameters/restraints
Measured reflections
613/107
12 908
670/65
27 417
628/126
22 305
Independent reflections
6414
8826
7045
Final R indices [I > 2
r
(I)]
0.0376, 0.0994
1.057
0.735 and ꢂ0.243
0.1178, 0.2880
1.080
0.992 and ꢂ0.721
0.0783, 0.1688
1.090
0.778 and ꢂ0.975
Goodness-of-fit (GOF) on F2
Largest difference peak and hole (e Åꢂ3
)
Table 2
Selected bond lengths (Å) and bond angles (°) for complexes 1–3.
Complex 1
Cu(1)–O(1)
Cu(1)–N(1)
Cu(1)–N(2)
Cu(1)–N(5)
1.935(2)
2.012(2)
2.027(2)
2.046(2)
2.1682(13)
89.26(8)
109.11(8)
109.85(7)
149.77(9)
100.63(9)
Cu(2)–O(3)
Cu(2)–N(4)
Cu(2)–N(3)
Cu(2)–N(6)
1.943(2)
2.002(2)
2.011(2)
2.036(2)
2.2148(13)
92.64(9)
173.68(9)
82.34(10)
93.64(7)
110.95(8)
O(1)–Cu(1)–N(1)
176.31(10)
93.99(9)
82.36(10)
82.11(9)
100.34(9)
140.97(9)
92.51(8)
81.78(8)
91.75(7)
99.08(7)
O(1)–Cu(1)–N(2)
N(1)–Cu(1)–N(2)
O(1)–Cu(1)–N(5)
N(1)–Cu(1)–N(5)
N(2)–Cu(1)–N(5)
O(1)–Cu(1)–O(5)
O(3)–Cu(2)–N(6)
N(3)–Cu(2)–O(6)
N(6)–Cu(2)–O(6)
Cu(1)–O(5)
Cu(2)–O(6)
N(1)–Cu(1)–O(5)
N(2)–Cu(1)–O(5)
N(5)–Cu(1)–O(5)
N(4)–Cu(2)–N(6)
N(3)–Cu(2)–N(6)
O(3)–Cu(2)–N(4)
O(3)–Cu(2)–N(3)
N(4)–Cu(2)–N(3)
O(3)–Cu(2)–O(6)
N(4)–Cu(2)–O(6)
Complex 2
Cu(1)–N(4)
Cu(1)–N(2)
Cu(1)–O(2)
Cu(1)–N(3)
1.968(9)
1.974(9)
2.010(7)
2.086(10)
2.132(8)
138.8(3)
100.4(3)
81.0(3)
Cu(2)–O(1)
Cu(2)–N(7)
Cu(2)–N(6)
Cu(2)–N(5)
1.969(7)
1.977(10)
2.002(10)
2.016(9)
2.192(9)
175.9(4)
94.3(3)
82.6(5)
81.3(3)
99.9(4)
N(4)–Cu(1)–N(2)
N(4)–Cu(1)–O(2)
N(2)–Cu(1)–O(2)
N(4)–Cu(1)–N(3)
N(2)–Cu(1)–N(3)
N(6)-Cu(2)–N(5)
O(1)–Cu(2)–O(3)
N(7)–Cu(2)–O(3)
N(6)–Cu(2)–O(3)
N(5)–Cu(2)–O(3)
174.8(4)
91.0(3)
92.7(3)
81.5(4)
93.3(4)
146.6(4)
91.3(4)
92.1(4)
105.9(4)
107.3(4)
Cu(1)–N(1)
Cu(2)–O(3)
O(2)–Cu(1)–N(3)
N(4)–Cu(1)–N(1)
N(2)–Cu(1)–N(1)
O(2)–Cu(1)–N(1)
N(3)–Cu(1)–N(1)
O(1)–Cu(2)–N(7)
O(1)–Cu(2)–N(6)
N(7)–Cu(2)–N(6)
O(1)–Cu(2)–N(5)
N(7)–Cu(2)–N(5)
118.8(3)
102.4(3)
Complex 3
Cu(1)–O(1)
Cu(1)–N(2)
Cu(1)–N(1
Cu(1)–N(5)
1.934(5)
2.002(6)
2.027(6)
2.079(6)
2.090(5)
124.6(2)
89.9(2)
90.5(2)
126.2(2)
109.0(3)
Cu(2)–O(4)
Cu(2)–N(4)
Cu(2)–N(6)
Cu(2)–N(3)
1.912(5)
1.996(5)
2.024(6)
2.088(6)
2.098(5)
175.1(3)
82.6(2)
99.9(2)
93.8(2)
81.3(2)
O(1)–Cu(1)–N(2)
O(1)–Cu(1)–N(1)
N(2)–Cu(1)–N(1)
O(1)–Cu(1)–N(5)
N(2)–Cu(1)–N(5)
N(6)–Cu(2)–N(3)
O(4)–Cu(2)–O(2)
N(4)–Cu(2)–O(2)
N(6)–Cu(2)–O(2)
N(3)–Cu(2)–O(2)
173.5(2)
92.7(2)
81.8(2)
82.4(2)
Cu(1)–O(5)
Cu(2)–O(2)
103.6(2)
125.2(3)
95.0(2)
86.52(19)
131.6(3)
103.21(19)
N(1)–Cu(1)–N(5)
O(1)–Cu(1)–O(5)
N(2)–Cu(1)–O(5)
N(1)–Cu(1)–O(5)
N(5)–Cu(1)–O(5)
O(4)–Cu(2)–N(4)
O(4)–Cu(2)–N(6)
N(4)–Cu(2)–N(6)
O(4)–Cu(2)–N(3)
N(4)–Cu(2)–N(3)
correction and SHELXTL [17] for structure solution and refinements
on F2. The positional and common isotropic thermal parameters
of all the hydrogen atoms associated with the water molecules (ex-
cept O3s which has half occupancy) were refined for 1. In com-
pound 2 the oxygen atoms of the lattice waters were assigned
occupancy of 0.5 and no hydrogen atoms were located. One of
the ClO4ꢂ anion was disordered in 3 and their occupancies were re-
fined. Further the methyl groups of the alanine side chains were
disordered in 2 and 3.
2.7. Spectroscopic studies on DNA interaction
2.7.1. Electronic absorption spectra
Electronic absorption titration of the ternary copper(II) complex
samples in the aqueous buffer solution (50 mM NaCl–5 mM Tris–
HCl, pH 7.1) were performed at a fixed complex concentration
(10 lM) while gradually increasing the concentration of CT-DNA.
The absorption data were analyzed to evaluate the intrinsic bind-
ing constant Kb, which can be determined from Eq. (1) [18],

858
L. Jia et al. / Inorganica Chimica Acta 363 (2010) 855–865
Table 3
lated from the observed flow time of DNA-containing solution cor-
Main intra and intermolecular hydrogen bonds for the title complex in this study
(Å, °).
rected from the flow time of buffer alone (t0),
g
= t ꢂ t0 [20,21].
D–Hꢀ ꢀ ꢀA
d(D–H)
d(Hꢀ ꢀ ꢀA)
d(Dꢀ ꢀ ꢀA)
<(DHA)
2.9. Cytotoxicity assay
Complex 1
O1S–H1SAꢀ ꢀ ꢀO9
O2S–H2SAꢀ ꢀ ꢀO13
O2S–H2SBꢀ ꢀ ꢀO6
N5–H5ꢀ ꢀ ꢀO12
O5–H5Aꢀ ꢀ ꢀO4
N6–H6ꢀ ꢀ ꢀO8
1.14
1.12
1.12
0.92
0.90
0.92
0.90
0.90
2.45
1.92
2.31
1.90
1.90
2.36
2.08
1.95
2.929(5)
2.947(5)
2.828(3)
2.719(2)
2.719(2)
3.161(3)
2.751(3)
2.828(3)
104
151
106
170
150
145
130
162
The reagent, p-nitrophenyl phosphate (p-NPP), was obtained
from Amresco. The compounds synthesized were dissolved in di-
methyl sulphoxide and diluted in culture medium. The final con-
centration of DMSO in cultures was always not exceeding 0.5%
(v/v), which did not cause toxicity. HeLa (Cervical cancer), HepG2
(hepatocarcinoma) and L02 (liver cells) were maintained in DMEM
medium, HL-60 cells (myeloid leukemia) were maintained in RPMI
1640 medium and A-549 cells (pulmonary carcinoma) in Ham’s
F12 medium. Cells were cultured at 37 °C in a humidified atmo-
sphere of 5% CO2 in air. All media were supplemented with 10% fe-
tal bovine serum and contained penicillin G (100 U/mL) and
O6–H6Aꢀ ꢀ ꢀO2
O6–H6Bꢀ ꢀ ꢀO2S
Complex 3
N5–H5Nꢀ ꢀ ꢀO11A
N6–H6Aꢀ ꢀ ꢀO6
O5–H51ꢀ ꢀ ꢀO3
O5–H52ꢀ ꢀ ꢀO6
0.72
0.92
0.91
0.89
2.45
2.57
1.83
2.18
3.11(2)
155
151
170
146
3.401(11)
2.725(8)
2.952(10)
streptomycin (100 lg/mL). After the cells were seeded in 96-well
plates at 4000 cells per well 12 h, they were exposed to all the
tested complexes with different concentrations for 72 h. The mor-
phological examination was performed with a Nikon ECLIPSE Ti
and phase contrast images were obtained by Nikon Digital Sightds
Fi1 (Nikon Corporation). The medium was removed and the wells
½DNAꢃ=ðea
ꢂ
ef Þ ¼ ½DNAꢃ=ðeb
ꢂ
ef Þ þ 1=Kbðeb
ꢂ
ef
Þ
ð1Þ
where [DNA] is the concentration of DNA in base pairs, the apparent
absorption coefficient ea ef and eb correspond to Aobsd/[M], the
extinction coefficient of the free compound and the extinction coef-
ficient of the compound when fully bound to DNA, respectively. In
plots of [DNA]/(ea
,
were washed with 200
removing the medium and PBS, the plates were centrifuged at
2500 rpm for 10 min. Buffer (100 L) containing 0.1 M sodium ace-
lL PBS per well. For HL-60 cells, after
ꢂ
ef) versus [DNA], Kb is given by the ratio of
l
slope to the intercept.
tate (pH 5.0), 0.1% Triton X-100, and 5 mM p-nitrophenyl phos-
phate was added to each well, and then the plates were
incubated for 1.5 h at 37 °C. After the reactions were stopped by
adding 1 M NaOH, the absorbance was read at 405 nm by Victor3
(Perkin–Elmer). Experiments were conducted in triplicate (three
independent experiments).
2.7.2. Fluorescence spectra
Further support for the ternary copper(II) complexes binding to
DNA is given through the emission quenching experiment. EB is a
common fluorescent probe for DNA structure and has been em-
ployed in examinations of the mode and process of metal complex
binding to DNA. A 2 mL solution of 15
saturating binding levels) was titrated by 0–60
l
M DNA and 1.5
lM EB (at
2.10. Antioxidant measurements
lM ternary cop-
per(II) complexes and the ligand (kex = 500 nm, kem = 520.0–
650.0 nm). According to the classical Stern–Volmer Equation (2)
[19]:
The hydroxyl radicals in aqueous media were generated
through the Fenton-type reaction [22,23]. The 5 ml reaction mix-
tures contained 2.0 ml of 100 mmol phosphate buffer (pH 7.4),
1.0 ml of 0.10 mol aqueous safranin, 1 ml of 1.0 mmol aqueous
EDTA–Fe(II), 1 ml of 3% aqueous H2O2, and a series of quantita-
tively microadding solutions of the tested compounds. The sample
without the tested compounds was used as the control. The reac-
tion mixtures were incubated at 37 °C for 60 min in a water-bath.
Absorbance at 520 nm was measured and the solvent effect was
corrected throughout. The scavenging effect for OHÅ was calculated
from the following expression (3) [24]:
F0=F ¼ Kq½Qꢃ þ 1
ð2Þ
where F0 is the emission intensity in the absence of quencher, F is
the emission intensity in the presence of quencher, Kq is the quench-
ing constant, and [Q] is the quencher concentration. The shape of
Stern–Volmer plots can be used to characterize the quenching as
being predominantly dynamic or static. Plots of F0/F versus [Q]
appear to be linear and Kq depends on temperature.
2.7.3. Circular dichroic spectral studies
The CD spectra of CT-DNA were recorded on a Jasco J-810 spec-
tropolarimeter at 25 °C. The concentration of CT-DNA and the com-
Suppression ratio ð%Þ ¼ ½ðAi ꢂ A0Þ=ðAc ꢂ A0Þꢃ ꢁ 100
ð3Þ
where Ai is the absorbance of the sample in the presence of the
tested compound, A0 is the absorbance of the blank in the absence
of the tested compounds and Ac is the absorbance in the absence of
the tested compounds and EDTA–Fe(II).
plexes were 120 and 60 lM. Each sample solution was scanned in
the range of 220–370 nm. CD spectrum was generated which rep-
resented the average of three scans from which the buffer back-
ground had been subtracted.
The superoxide radicals (O2ꢂÅ) were produced by the MET–
VitB2–NBT system [25]. The solution of MET (methionine), VitB2
(vitamin B2) and NBT (nitroblue tetrazolium) were prepared with
deionized double distilled water under lightproof conditions. The
5 ml reaction mixtures contained 2.5 ml of 100 mmol phosphate
buffer (pH 7.8), 1.0 ml of 50 mmol MET, 1.0 ml of 0.23 mmol
2.8. Viscosity measurements
Viscosity experiments were carried out on an Ubbelodhe vis-
cometer, immersed in a thermostated water-bath maintained to
25 °C. Titrations were performed for the ternary copper complexes
NBT, 0.50 ml of 33 lM VitB2, and a series of quantitatively mic-
(1–5
l
M), and each complex was introduced into DNA solution
roadding solutions of the tested compounds. After incubated at
30 °C for 10 min in a water-bath and then illuminated with a
721E spectrophotometer, the absorbance of the sample was mea-
sured at 560 nm and the solvent effect was corrected throughout.
The sample reaction mixtures without the tested compounds were
(50
l
M) present in the viscometer. Flow time was measured with
a digital stopwatch and each sample was measured three times
and an average flow time was calculated. Data were presented as
1/3
(g/g0
)
versus the ratio of the concentration of the complex and
is the viscosity of DNA in the presence of complex,
and g0 is the viscosity of DNA alone. Viscosity values were calcu-
ꢂÅ
DNA, where
g
used as the control. The scavenging effect for O2 was calculated
from the following expression (4):

L. Jia et al. / Inorganica Chimica Acta 363 (2010) 855–865
859
Suppression ratio ð%Þ ¼ ½ðA0 ꢂ AiÞ=A0ꢃ ꢁ 100
ð4Þ
where Ai is the absorbance in the presence of the tested compounds
and A0 is the absorbance in the absence of the tested compounds.
IC50 value was introduced to denote the molar concentration of
the tested compounds which caused a 50% inhibitory or scavenging
effect on hydroxyl radicals or superoxide radicals.
3. Results and discussion
3.1. Synthesis and general properties
Ternary copper(II) complexes containing H2PDIAla and phen are
prepared in high yield in the presence of LiOH with copper(II) per-
chlorate. The complexes have been characterized by elemental
analysis and spectroscopic data. The stability of copper(II) com-
plexes in an aqueous solution have been studied by observing
the UV–Vis spectrums and estimating the molar conductivities at
different time intervals for any possible change. The tested cop-
per(II) complexes are prepared in DMSO solution and for experi-
ments freshly diluted in phosphate buffer system (at pH 7.4, 7.8).
Then, the UV–Vis spectrums and molar conductivities are re-
searched at different time intervals. The investigations reveal that
the UV–Vis spectra have remained unaltered for the solutions and
its molar conductance values have no obvious change for very
freshly prepared and for over the whole experiment (72 h). It indi-
cates that the copper(II) complexes are quite stable in solution. The
C–H stretchings observed at 853 cmꢂ1, 737 cmꢂ1 are red shifted to
843 and 734 cmꢂ1, which due to the coordination of the two nitro-
gen atoms of phen ligands [26]. The characteristic bands of the car-
boxylate groups are observed in the range 1570–1640 cmꢂ1 for
asymmetric stretching and 1320–1400 cmꢂ1 for symmetric
Fig. 2. Molecular structure of 1. The C–H hydrogen atoms are not shown for clarity.
planes are 7.5° and 8.9°. The Cuꢀ ꢀ ꢀCu distance in the cation, 7.61 Å,
is longer than the intermolecular distance, 7.07 Å.
The O–Hꢀ ꢀ ꢀO hydrogen-bonding between aqua ligand and
carbonyl oxygen of the PDIAla ligand along a-axis produce hydro-
gen-bonded helices as shown in Fig. 3a and b. These hydrogen-
bonded helices are further assembled along the b-axis through
similar O–Hꢀ ꢀ ꢀO interactions to form a layer structure in the ab-
plane as shown in Fig. 3c. The ClO4ꢂ anions, lattice water molecules
are found between the grooves and cavities with the layers shown
in Fig. 3d. The interlayers are essentially held by hydrophobic
interactions.
stretching, respectively [27]. The difference between
and the
sym(COOꢂ) stretching frequencies in complex
>200 cmꢂ1, thus suggesting a terminal coordination mode for the
carboxylate group. While the difference between
as(COOꢂ) and
t
as(COOꢂ)
is
t
1
A perspective view of [Cu4(phen)6(
3H2O (2) is shown in Fig. 4. For the space P1 with Z = 1, there is
D
,
L
-PDIAla)(H2O)2](ClO4)6ꢀ
ꢀ
t
a centre of inversion present at the centre of the phenyl ring of
the D,L-PDIAla ligand. The basic connectivity of the cation is very
the
t
sym(COOꢂ) stretching frequencies in complexes 2 and 3 is
<200 cmꢂ1, which is due to the bridging coordination mode for
the carboxylate group [28]. Weak absorptions observed in the
similar to that found in 1 but the carbonyl oxygen atoms on both
sides of the ligand are bonded to [Cu(phen)2]2+ to provide a tetra-
meric cation. There are crystallographically two distinct Cu(II) cen-
tres: Cu1 has trigonal pyramidal geometry with nitrogen atoms of
the phen rings occupying the axial positions while Cu2 has dis-
torted square pyramidal geometry with the aqua ligand at the
range of 2930–2990 cmꢂ1 can be attributed to the mCH of the re-
2
duce Schiff base ligand and the vibration observed in the range
of 2970–3120 cmꢂ1 can be attributed to the mNH of the ligand.
The broad bands at 3430 cmꢂ1 are ascribed to the vibration of
the O–H stretching of the water ligands. And the topical bands in
apex. The ligand has ‘‘S” conformation stabilized by
p p interac-
ꢀ ꢀ ꢀ
the region ca. 1145 and ca. 1090 cmꢂ1 are assigned to
t(Cl–O) of
tions of the phen ring with a phen ring on each side. The distance
between the centre of the phenyl ring to the middle of the carbon
atoms close to the N atoms of the phen ligand is 3.42 Å. The
Cu1ꢀ ꢀ ꢀCu2 distance is 4.864 Å while the Cu2ꢀ ꢀ ꢀCu2a is 7.603 Å.
Due to the presence of several phen ligands, there are interest-
anion. PXRD are used to check the phase purity of the bulky sam-
ples in the solid state. For complexes 1, 2 and 3, the measured
PXRD patterns closely match the simulated patterns generated
from the results of single crystal diffraction data, indicative of pure
products.
ing
p p stacking observed. In addition to the triple p-sandwichs
ꢀ ꢀ ꢀ
formed by the phen ligands bonded to Cu2, they stack further
along c-direction to form 1D polymer. Here the single phenyl rings
in phen ligands with no nitrogen atoms attached form face-to-face
3.2. Structure of the ternary copper complexes
The molecular structure of complex [Cu2(phen)2(PDI-
Ala)(H2O)2](ClO4)2ꢀ2.5H2O (1) consists of dinuclear complex cat-
ions as shown in Fig. 2. Each of the Cu2+ ions in the dimeric
complex with N3O2 donor set has a distorted square pyramidal
geometry. The four basal positions are occupied by two N atoms
of the phen ligand, and the amine N atom and the carboxylate O
atom of the ligand. The coordination sphere at the apical position
is completed by O atom of the aqua ligand. The phenyl ring of
the PDIAla ligand is sandwiched between the phen ligands through
p p stacking along the a-axis with a distance of 3.418 Å as shown
ꢀ ꢀ ꢀ
in Fig. 5. The two phen ligands bonded to the terminal copper (II)
atoms are also engage in interactions with interplanar dis-
ꢀ ꢀ ꢀ
tances 3.502 and 3.565 Å. Fig. 5b shows face-to-face interac-
p
p
pꢀ ꢀ ꢀp
tions present in phen ligand attached to Cu1 although not all the
rings are involved.
Compound [Cu2(phen)2(
D
,
L
-PDIAla)(H2O)](ClO4)2ꢀ0.5H2O (3) is a
1D coordination polymer and the repeating unit comprises of a
dinuclear copper (II) unit as shown in Fig. 6. Both Cu1 and Cu2
are crystallographically distinct but having the same trigonal pyra-
midal. However Cu1 has an aqua ligand in the equatorial position
while Cu2 has the carbonyl oxygen atom from the neighboring
PDIAla ligand. One nitrogen of the phen ligand and carboxylate
p p interactions, and thus the PDIAla ligand is ‘‘S” shaped. The
ꢀ ꢀ ꢀ
closest distances, 3.35 and 3.41 Å are between the centre of the
phenyl ring and the middle of the carbon atoms next to the nitro-
gen atoms of the phen ligand. The interplanar angles between the

860
L. Jia et al. / Inorganica Chimica Acta 363 (2010) 855–865
Fig. 3. (a) A view of the hydrogen-bonded helical chain and (b) its CPK space filling model. (c) Assembly of helices forming a sheet and (d) packing viewed along a-axis.
Fig. 4. The ball and stick diagram of the cation in 2.
Fig. 5. Different types of pi–pi interactions in the cations of 2 with phen ligand (a) bonded to Cu2 and (b) bonded to Cu1.

L. Jia et al. / Inorganica Chimica Acta 363 (2010) 855–865
861
600–700 nm are assignable to the d–d band, which is in agreement
with five-coordinate geometry. The binding of complexes 1–3 to
DNA helix have been characterized through absorption spectral
titrations, by following changes in absorbance and shift in wave-
length. Fig. 8 shows the absorption spectra of ternary copper(II)
complexes in the absence and presence of CT-DNA. Addition of
increasing amounts of CT-DNA result in hyperchromism and blue
shift. As DNA double helix possesses many hydrogen-bonding sites,
it is likely that the amine groups of the amino acid chain forms
hydrogen bonds with DNA, which may contribute to the hyper-
chromism observed in the absorption spectra. A similar hyperchro-
mism has been observed also for a Cu(II) complex bearing N–H and
methyl groups [29,30].
The Kb values of ternary copper(II) complexes with different
numbers of copper chelates are 0.89 ꢁ 105, 1.14 ꢁ 105, and
1.72 ꢁ 105 for 1, 2 and 3, respectively, the significant difference
in DNA-binding affinity of the three ternary copper(II) complexes
can be understood as a result of the fact that the complex with
higher numbers of copper chelates shows stronger binding affinity
with DNA. This may be due to the high numbers of planar phen li-
gand and large number of copper(II) complex moieties present in
the complex which cooperatively act to increase the overall bind-
ing ability of the ternary copper(II) complexes with DNA. Interest-
ingly, the Kb values obtained for the above ternary copper(II)
complexes are higher than those for the other known mononuclear
and binuclear copper(II) complexes containing 1,10-phenanthro-
Fig. 6. Perspective view of the cation in 3 is shown and all the C–H hydrogen atoms
are omitted for clarity.
oxygen atom of the chelating PDIAla ligand are occupying the axial
positions in these metal centres. The conformation of the PDIAla li-
gand is also the same as observed in 1 and 2.
The carbonyl oxygen atom O2 is bonded to the neighboring Cu2
to generate 1D coordination polymeric structure. All the coordina-
tion polymers are aligned in parallel and interact with each other
through O–Hꢀ ꢀ ꢀO hydrogen-bonding as shown in Fig. 7.
line ([Cu(phen)(
L
-Thr)(H2O)](ClO4) (Kb, 6.35 ꢁ 103 Mꢂ1) [11], [Cu2-
(phen)2Cl4] (Kb, 4.75 ꢁ 104 Mꢂ1) [31], [(phen)Cu(bipp)Cu(phen)]
(ClO4)4 (Kb, 1.4 ꢁ 104 Mꢂ1) [32]. This showed that comparatively
the ternary copper(II) complex samples can bind to CT-DNA very
strongly.
3.3.2. Competitive DNA-binding studies with EB
3.3. Interactions with CT-DNA
In order to further investigate the interaction mode between
the binuclear complexes and CT-DNA, the fluorescence titration
experiments are performed. The fluorescence titration experi-
ments, especially the EB fluorescence displacement experiment,
have been widely used to characterize the interaction of complexes
with DNA by following the changes in fluorescence intensity of the
complexes. The intrinsic fluorescence intensities of DNA and that
of EB in Tris–HCl buffer are low, while the fluorescence intensity
of EB will be enhanced on addition of DNA as its intercalation into
the DNA. Therefore, EB can be used to probe the interaction of com-
plexes with DNA. If the complexes can intercalate into DNA, the
binding sites of DNA available for EB will be decreased, and hence
the fluorescence intensity of EB will be quenched [33]. In our
experiments, as depicted in Fig. 9, for complexes 1, 2 and 3, the
3.3.1. Electronic absorption spectra
The interactions of metal complexes with DNA have been the
subject of interest for the development of effective chemothera-
peutic agents. Transition metal centres are particularly attractive
moieties for such research since they exhibit well-defined coordi-
nation geometries and also often possess distinctive electrochem-
ical or photophysical properties, thus enhancing the functionality
of the binding agent [2].
In the UV–Vis region of the electronic spectra, the absorption
bands around 202 nm, 245 nm, 272 nm and 294 nm observed can
be attributed to the
while the intense electronic bands observed in the spectral range
p–
p* transition of the coordinated ligands,
Fig. 7. A segment of the crystal structure of 3 showing the inter-strand interactions between the coordination polymers. All the C–H hydrogen atoms expect coordination
waters are not shown for clarity.

862
L. Jia et al. / Inorganica Chimica Acta 363 (2010) 855–865
Fig. 9. The emission spectra of DNA-EB system (15
kex = 500 nm, kem = 520–720 nm, in the presence of the complex 1 (a), complex 2
(b), complex (c). [DNA] = 10 M, [Compound] = 0–60 M. Arrow shows the
emission intensity changes upon increasing ligand concentration. Inset: Stern–
Volmer plot of the fluorescence titration data of the complexes 1–3.
lM and 1.5 lM EB),
Fig. 8. Electronic spectra of the complex 1 (a), complex 2 (b), complex 3 (c) in Tris–
HCl buffer upon addition of calf-thymus DNA. [Compound] = 10 M, [DNA] = 0–
20 M. Arrow shows the emission intensities changes upon increasing DNA
concentration. Inset: plots of [DNA]/(ea f) vs.[DNA] for the titration of complex
l
3
l
l
l
ꢂ
e
1 (a), complex 2 (b), complex 3 (c) with CT-DNA.
fluorescence intensity of EB at 584 nm show a remarkable decreas-
ing trend with the increasing concentration of the complexes, indi-
cating that some EB molecules are released from EB-DNA after an
exchange with the complex 1, 2 or 3 which result in the fluores-
cence quenching of EB. This may be due either to the metal com-
plex competing with EB for the DNA-binding sites thus
displacing the EB (whose fluorescence is enhanced upon DNA-
binding) or it should be a more direct quenching interaction on
the DNA itself. We assume the reduction of the emission intensity
of EB on increasing the complex concentration could be caused due
to the displacement of the DNA bound EB by the ternary copper(II)
complexes. Such a quenched fluorescence behavior of EB bound to

L. Jia et al. / Inorganica Chimica Acta 363 (2010) 855–865
863
Fig. 10. CD spectra of CT-DNA (120
(II) complexes 1, 2, and 3 (60 M).
lM) in the absence and presence of the copper
Fig. 11. Effect of increasing amounts of complexes 1, 2 and 3 on the relative
viscosity of calf thymus DNA at 25.0 °C.
l
DNA caused by the interaction between the binuclear copper(II)
complexes and DNA is also found in other ternary copper com-
plexes [34,35]. The Kq values of ternary copper(II) complexes with
ture [37]. Thus simple groove binding and electrostatic interaction
of small molecules show less or no perturbation on the base-stack-
ing and helicity bands, whereas intercalation enhances the intensi-
ties of both the bands stabilizing the right-handed B conformation
of CT-DNA as observed for the classical intercalator methylene blue
[38].
different numbers of copper chelates are 7.17 ꢁ 103 Mꢂ1
,
1.44 ꢁ 104 Mꢂ1, 1.64 ꢁ 104 Mꢂ1 for 1, 2 and 3, respectively.
3.3.3. Circular dichroic spectral studies
The CD spectrum of CT-DNA was monitored in the presence of
1, 2 and 3, the changes observed in the three cases are shown in
Fig. 10. On addition of all the complexes to CT-DNA, faint red shift
with intensity increase in the positive bands are observed. When
addition of 3 to CT-DNA, it is observed that the negative-band po-
sition was shifted to 246 nm with more evident increase than 1
and 2 in molar ellipticity, while the intensity of the positive band
in the CD spectrum of DNA is perturbed remarkably with no shift.
Circular dichroic spectral techniques may give us useful infor-
mation on how the conformation of the CT-DNA chain is influenced
by the bound complex. The CD spectrum of CT-DNA consists of a
positive band at 275 nm that can be due to base-stacking and a
negative-band at 245 nm that can be due to helicity and it is also
characteristic of DNA in a right-handed B form [36]. The changes
in CD signals of CT-DNA observed on interaction with drugs may
often be assigned to the corresponding changes in CT-DNA struc-
Fig. 12. Phase-contrast micrographs of cells treated with complexes 1, 2, and 3.

864
L. Jia et al. / Inorganica Chimica Acta 363 (2010) 855–865
Table 4
lines, this result suggests that in general, HL-60 may be more sen-
sitive than other cells.
IC50 values of the tested compounds.
In order to prove that the cytotoxicity observed is due to the
ternary copper complexes, rather than to simple aquated metal
ions [Cu(H2O)x]y+, the ligand-free metal salts Cu(ClO4)2ꢀ6H2O, is
screened against all the model cell lines. The data suggests that
Cu(ClO4)2ꢀ6H2O displayed significantly less effective than phen,
H2PDIAla and their metal complexes.
The morphology examination also show that the proliferation of
the cells are significant inhibited and the cells exhibit morpholog-
ical change such as cell shrinkage and cell detachment (Fig. 12).
Cell line
IC50 (lM)
Cu(ClO4)2ꢀ6H2O
97
>160
>160
>160
>160
H2PDIAla
Phen
1
2
3
HeLa
HepG2
L02
HL60
A549
114
140
132
>160
>160
48
4.1
35
6.2
3.0
0.42
0.31
0.20
0.08
0.56
0.44
0.55
0.40
0.25
0.72
0.48
0.65
0.58
0.15
0.57
These observations are supportive of the intercalative mode of
binding of the complexes, where in the stacking of the complex
molecules between the base pairs of DNA led to an enhancement
in the positive band and the partial unwinding of the helix is re-
flected in the decreased intensity of the negative-band, which attri-
butes to a strong conformational change in DNA helix [39,40]. It is
similar to that observed for [Ru(NH3)4(qdppz)]2+ and [Co(NH3)6]3+
bound to DNA of short lengths with 160 base pairs [41,42].
3.5. Hydroxyl radical scavenging activity
Fig. 13a shows the plots of hydroxyl radical scavenging effect
(%) for ligands and Cu(II) complexes, respectively, which are con-
centration-dependant. The values of IC50 of ligands for hydroxyl
radical scavenging effect can not be read, while the values of IC50
of Cu(II) complexes for hydroxyl radical scavenging effect are
4.6–5.5 lM with the order of 1 < 3 < 2. It is marked that the hydro-
xyl radical scavenging effects of Cu(II) complexes are much higher
than those of their ligands, possibly in that the larger conjugated
metal complexes can react with OHÅ to form larger stable macro-
molecular radicals than ligands [47].
3.3.4. Viscosity measurements
Optical photophysical probes provide necessary, but not suffi-
cient clues to support a binding model. Hydrodynamic measure-
ments (i.e., viscosity and sedimentation) that are sensitive to
length change are regarded as the least ambiguous and the most
critical tests of a binding model in solution in the absence of crys-
tallographic structural data. A classical intercalation model results
in lengthening the DNA helix as base pairs are separated to accom-
modate the bound ligand, leading to the increase of DNA viscosity.
In contrast, a semi-intercalation of ligand could bend (or kink) the
DNA helix, reduce its effective length, and concomitantly, its vis-
cosity [43,44]. Fig. 11 depicts the effect of complexes 1–3 on the
DNA viscosity. The plots reveal that, at the first instance, the rela-
tive solution viscosity decreased with increasing concentration of
the complexes, indicating that the DNA becomes more compact,
resulting in DNA aggregation. The aggregation reduces the number
of independently moving DNA molecules in solution and hence
leads to a decrease in viscosity [45]. These results support that
complexes 1–3 binds to CT-DNA by partial intercalation and vali-
date those obtained from the spectral studies [46].
3.4. Cytotoxic activity
To evaluate their cancer chemotherapeutic potential, the inhibi-
tion ability of the copper complexes 1–3 against the HeLa (Cervical
cancer), HepG2 (hepatocarcinoma), L02 (liver cells), HL-60 (mye-
loid leukemia) and A-549 (pulmonary carcinoma) cell lines are
determined by calculation of IC50 (the drug concentration causing
a 50% reduction in cellular viability). All test agents are incubated
with the five model cell lines for a period of 72 h, after which cel-
lular viability is determined using different methods. Results ob-
tained for phen, H2PDIAla, the copper complexes 1–3 and simple
metal salt are determined and the IC50 values are presented in Ta-
ble 4. Phen and the three metal complexes displayed a concentra-
tion-dependent cytotoxic profile in all cell lines. Since the IC50
values for [Cu2(phen)2(
L
-PDIAla)(H2O)2](ClO4)2ꢀ2.5H2O (1), [Cu4-
(phen)6(
D,L-PDIAla)(H2O)2](ClO4)6ꢀ3H2O (2) and [Cu2(phen)2(
D,L-
PDIAla)(H2O)](ClO4)2ꢀ0.5H2O (3) are statistically lower than that
for metal-free phen and H2PDIAla in all of the tested cells, it sug-
gests that coordinated copper(II) ion plays a major role in mediat-
ing potency of the complex. The IC50 values for phen on all the cells
are not statistically different, it displayed a greater effect against
HL-60 and A-549 cell line, while the IC50 values for the metal com-
plexes on HL-60 cell are significant lower than other tested cell
Fig. 13. Plots of antioxidation properties for ligands and Cu(II) complexes. a and b
represents the hydroxyl radical scavenging effect (%) and the superoxide radical
scavenging effect (%) for ligands and Cu(II) complexes, respectively.

L. Jia et al. / Inorganica Chimica Acta 363 (2010) 855–865
865
3.6. Superoxide radical scavenging activity
via www.ccdc.cam.ac.uk/data_request/cif. Supplementary data
associated with this article can be found, in the online version, at
doi:10.1016/j.ica.2009.12.047.
Fig. 13b shows the plots of superoxide radical scavenging effect
(%) for ligands and Cu(II) complexes, respectively, which are also
concentration-dependant. The values of IC50 of ligands for superox-
ide radical scavenging effects can not be read, but the values of IC50
of Cu(II) complexes for superoxide radical scavenging effects are
References
[1] N. Maribel, Biol. Inorg. Chem. 4 (2003) 401.
[2] C. Metcalfe, J.A. Thomas, Chem. Soc. Rev. 32 (2003) 215.
[3] S. Arturo, B. Giampaolo, R. Giuseppe, L.G. Maria, T.J. Salvatore, J. Inorg.
Biochem. 98 (2004) 589.
9.32–12.55
l
M with an order of 1 < 3 < 2. It is clear that the scav-
ꢂÅ
enger effect on O2 can be by the formation of metal–ligand coor-
dination complexes and the nature of the transition metal ions also
affects the ability. Some complexes we have synthesized are better
[4] L.R. Kelland, Eur. J. Cancer. 41 (2005) 971.
[5] J.D. Ranford, P.J. Sadler, D.A. Tocher, Dalton Trans. (1993) 3393.
[6] G. Majella, S. Vivienne, M. Malachy, D. Michael, M. Vickie, Polyhedron 18
(1999) 2931.
[7] D.K. Saha, U. Sandbhor, K. Shirisha, S. Padhye, D. Deobagkar, C.E. Ansond, A.K.
Powelld, Bioorg. Med. Chem. Lett. 14 (2004) 3027.
[8] M.A. Zoroddu, S. Zanetti, R. Pogni, R. Basosi, J. Inorg. Biochem. 63 (1996) 291.
[9] W.K. Pogozelski, T.D. Tullius, Chem. Rev. 98 (1998) 1089.
[10] F.A. Cotton, G. Wilkinson, Advanced Inorganic Chemistry, Wiley, New York,
1972.
[11] S. Zhang, Y. Zhu, C. Tu, H. Wei, Z. Yang, L. Lin, J. Ding, J. Zhang, Z. Guo, J. Inorg.
Biochem. 98 (2004) 2099.
[12] A.K. Patra, S. Dhar, M. Nethaji, A.R. Chakravarty, Chem. Commun. (2003) 1562.
[13] P.K. Sasmal, A.K. Patra, A.R. Chakravarty, J. Inorg. Biochem. 102 (2008) 1463.
[14] R. Rao, A.K. Patra, P.R. Chetana, Polyhedron 27 (2008) 1343.
[15] SMART and SAINT Software Reference Manuals, Versions 5.6 and 6.4, Bruker AXS,
Madison, WI, 2003.
[16] G.M. Sheldrick, SADABS Software for Empirical Absorption Correction, University
of Göttingen, 2003.
[17] SHELXTL Reference Manual, Version 6.14, Siemens Energy & Automation, Inc.,
Analytical Instrumentation, Madison, WI, 2003.
ꢂÅ
effective inhibitor for O2 than that of the nitroxide Tempo
(IC50 = 60 3.11 M) which has been recently used in biological sys-
tem for its capacity to mimic superoxide dismutase [48,49].
Although superoxide is a relatively weak oxidant, it decomposes
to form stronger relative oxidative species, such as single oxygen
and hydroxyl radicals, which initiate peroxidation of lipids [25].
In the present study, the complexes effectively scavenged superox-
ide in a concentration-dependent manner. These results show the
complexes have significant scavenging activity of superoxide radi-
cal and clearly suggested that the antioxidant activity of the com-
plexes is also related to its ability to scavenge superoxide radical.
Therefore, the metal complexes we studied in this paper deserve
to be further researched.
[18] A. Wolf, G.H. Shimer, T. Meehan, Biochem 26 (1987) 6392.
[19] M.R. Efink, C.A. Ghiron, Anal. Biochem. 114 (1981) 199.
[20] M. Eriksson, M. Leijon, C. Hiort, B. Norden, A. Gradslund, Biochem 33 (1994)
5031.
[21] Y. Xiong, X.F. He, X.H. Zou, J.Z. Wu, X.M. Chen, L.N. Ji, R.H. Li, J.Y. Zhou, K.B. Yu, J.
Chem. Soc., Dalton Trans. (1999) 19.
[22] C.C. Winterbourn, Biochem. J. 182 (1979) 625.
[23] C.C. Winterbourn, Biochem. J. 198 (1981) 125.
[24] Z.Y. Guo, R.E. Xing, S. Liu, H.H. Yu, P.B. Wang, C.P. Li, P.C. Li, Bioorg. Med. Chem.
Lett. 15 (2005) 4600.
[25] B.D. Wang, Z.Y. Yang, P. Crewdson, D.Q. Wang, J. Inorg. Biochem. 101 (2007)
1492.
4. Conclusions
A new reduced Schiff base ligand H2PDIAla and three ter-
nary copper(II) complexes are prepared and characterized, the
DNA-binding properties of the complexes are investigated by
absorption, fluorescence, circular dichroic spectral and viscosity
measurements. The binding constant shows that the DNA-binding
affinity increases in the order: complex 1 < complex 2 < complex 3.
Furthermore, the three copper(II) complexes have active scaveng-
Å
ꢂÅ
[26] L. Jin, P. Yang, Polyhedron 16 (1997) 3395.
ing effect on O2 and OH . Although the mechanism between scav-
enging hydroxyl radicals and superoxide radicals should be also
studied further, these findings clearly indicate that copper-based
complexes have many potential practical applications, like the
development of nucleic acid molecular probes and new therapeutic
reagents for diseases. On the other hand, the three complexes show
considerable cytotoxic activity against five cell lines, and the IC50
values of all the metal complexes are lower than that of phen,
H2PDIAla and Cu(ClO4)2ꢀ6H2O. Studies are currently underway in
our laboratory to investigate more fully the mechanisms by which
phen, H2PDIAla and the three metal complexes control cancer cell
viability. It is intended that the results from these studies will
allow identification of key molecular targets, and in doing so will
assist in elucidating their mechanisms of action, along with facili-
tating the development of highly effective anti-cancer therapies.
[27] L. Yang, C.A. La, O.P. Anderson, D.C. Crans, Inorg. Chem. 41 (2002) 6322.
[28] M. Tsaramyrsi, M. Kaliva, A. Salifoglou, C.P. Raptopoulou, A. Terzis, V. Tangorlis,
J. Giapintzakis, Inorg. Chem. 40 (2001) 5772.
[29] H. González-Díaz, Á. Sánchez-González, Y. González-Díaz, J. Inorg. Biochem.
100 (2006) 1290.
[30] Y.N. Xiao, C.X. Zhan, J. Appl. Polym. Sci. 84 (2002) 887.
[31] Q.G. Zhang, F. Zhang, W.G. Wang, X.L. Wang, J. Inorg. Biochem. 100 (2006)
1344.
[32] J.Z. Wu, L. Yuan, J.F. Wu, J. Inorg. Biochem. 99 (2005) 2211.
[33] R. Indumathy, S. Radhika, M. Kanthimathi, T. Weyhermuller, B.U. Nair, J. Inorg.
Biochem. 101 (2007) 434.
[34] Z.Q. Liu, Y.T. Li, Z.Y. Wu, Y.L. Song, Inorg. Chim. Acta 361 (2008) 226.
[35] U. McDonnell, M.R. Hicks, M.J. Hannon, A. Rodger, J. Inorg. Biochem. 102 (2008)
2052.
[36] V.I. Ivanov, L.E. Minchenkova, A.K. Schyolkina, A.I. Poletayer, Biopolym 12
(1973) 89.
[37] P. Lincoln, E. Tuite, B. Norden, J. Am. Chem. Soc. 119 (1997) 1454.
[38] B. Norden, F. Tjerneld, Biopolym 21 (1982) 1713.
[39] V.G. Vaidyanathan, R. Vijayalakshmi, V. Subramanain, B.U. Nair, Bull. Chem.
Soc. Jpn. 75 (2002) 1143.
[40] M. Chauhan, F. Arjmand, Chem. Biodivers. 3 (2006) 660.
[41] P.U. Maheswari, M. Palaniandavar, J. Inorg. Biochem. 98 (2004) 219.
[42] B.I. Kankia, V. Buckin, V.A. Bloomfield, Nucleic Acids Res. 29 (2001) 2795.
[43] C.R. Brodie, J. Collins, J.R.A. Wright, Dalton Trans. (2004) 1145.
[44] C.S. Allardyce, P.J. Dyson, D.J. Ellis, S.L. Heath, Chem. Commun. (2001) 1396.
[45] J. Liu, J.B. Lu, H. Li, Q.L. Zhang, L.N. Ji, J.X. Zhang, L.H. Qu, H. Zhou, Transition
Met. Chem. 27 (2002) 686.
[46] J. Liu, H. Zhang, C. Chen, H. Deng, T. Lu, L. Ji, Dalton Trans. (2003) 114.
[47] J. Ueda, N. Saito, Y. Shimazu, T. Ozawa, Arch. Biochem. Biophys. 333 (1996)
377.
Acknowledgements
This work was supported by the National Science Foundation of
China (20571035) and the Ministry of Education, Singapore
through NUS (FRC Grant No. R143-000-371-112).
Appendix A. Supplementary material
[48] A. Samuni, M.C. Krisna, Antioxidant properties of nitroxides and nitroxide SOD
mimics, in: L. Packer, E. Cadenas (Eds.), Handbook of Synthetic Antioxidants,
Marcel Dekker, New York, 1997, pp. 351–373.
[49] P.X. Xi, Z.H. Xu, X.H. Liu, F.J. Chen, L. Huang, Z.Z. Zeng, Chem. Pharm. Bull. 56
(2008) 541.
CCDC 698880, 698876 and 746204 contain the supplementary
crystallographic data for 1, 2, and 3. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre