Synthesis, crystal structure, DNA-binding properties, cytotoxic and antioxidation activities of several ternary copper(II) complexes with a new reduced Schiff base ligand

Article abstract of DOI:10.1016/j.ica.2012.05.014

Three new ternary copper(II) complexes, [Cu₂(phen) ₂(PDIMAla)(H₂O)₂](ClO₄) ₂·CH₃OH (1), [Cu₂(dpq) ₂(PDIMAla)(H₂O)₂](ClO₄) ₂·CH₃OH (2) and [Cu₂(dppq) ₂(PDIMAla)(H₂O)₂](ClO₄) ₂·CH₃OH (3) (phen = 1,10-phenanthroline, dpq = dipyrido[3,2:2′,3′-f]quinoxaline, dppz = dipyrido[3,2-a:2′, 3′-c]phenazine, H₂PDIMAla = N,N′-(p-xylylene)di-alanine acid) have been synthesized and the complex 1 has been structurally characterized by single-crystal X-ray crystallography, spectrometric titrations, ethidium bromide displacement experiments, CD (circular dichroism) spectral analysis and viscosity measurements. The results indicate that the three compounds, especially the complex 3, can strongly bind to calf-thymus DNA (CT-DNA). The intrinsic binding constants K_b of the ternary copper(II) complexes with CT-DNA are 0.38 × 10⁵, 1.4 × 10⁵ and 7.8 × 10⁵ 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 human hepatic (HepG2), human promyelocytic leukemia (HL60) and human prostate (PC3) cell lines. Investigation of antioxidation property show that all the copper(II) complexes have strong scavenging effects for hydroxyl radicals.

Full text of DOI:10.1016/j.ica.2012.05.014

Inorganica Chimica Acta 391 (2012) 121–129
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis, crystal structure, DNA-binding properties, cytotoxic and antioxidation
activities of several ternary copper(II) complexes with a new reduced
Schiff base ligand
b
b
a
a
a
b,
Lei Jia a, , Jie Shi , Zhi-hong Sun , Fei-fei Li , Yuan Wang , Wei-na Wu , Qin Wang
⇑
⇑
a Department of Physics and Chemistry, Henan Polytechnic University, Jiaozuo 454000, PR China
b Institute of Cancer Biology and Drug Screening, 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:
Received 8 July 2011
Received in revised form 13 March 2012
Accepted 20 May 2012
Available online 30 May 2012
Three new ternary copper(II) complexes, [Cu2(phen)2(PDIMAla)(H2O)2](ClO4)2ꢀCH3OH (1), [Cu2(dpq)2
(PDIMAla)(H2O)2](ClO4)2ꢀCH3OH (2) and [Cu2(dppq)2(PDIMAla)(H2O)2](ClO4)2ꢀCH3OH (3) (phen = 1,10-
phenanthroline, dpq = dipyrido[3,2:20,30-f]quinoxaline, dppz = dipyrido[3,2-a:20,30-c]phenazine, H2PDI-
MAla = N,N0-(p-xylylene)di-alanine acid) have been synthesized and the complex 1 has been structurally
characterized by single-crystal X-ray crystallography, spectrometric titrations, ethidium bromide dis-
placement experiments, CD (circular dichroism) spectral analysis and viscosity measurements. The
results indicate that the three compounds, especially the complex 3, can strongly bind to calf-thymus
DNA (CT–DNA). The intrinsic binding constants Kb of the ternary copper(II) complexes with CT–DNA
are 0.38 ꢁ 105, 1.4 ꢁ 105 and 7.8 ꢁ 105 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 ter-
nary copper(II) complexes have significant cytotoxic activity against the human hepatic (HepG2), human
promyelocytic leukemia (HL60) and human prostate (PC3) cell lines. Investigation of antioxidation prop-
erty show that all the copper(II) complexes have strong scavenging effects for hydroxyl radicals.
Ó 2012 Elsevier B.V. All rights reserved.
Keywords:
Ternary copper(II) complexes
Cytotoxicity
DNA-binding properties
Reduce Schiff base
X-ray crystallography
Phenanthroline base
1. Introduction
medicinal chemistry and concentrate much attention as an ap-
proach to new drug development [6].
Currently, cancer is the second cause of death, accounting for
about a quarter of all deaths [1]. Nearly one half of all cancers that
are diagnosed result in the death of the patient, so that the devel-
opment of new anticancer therapies is one of the fundamental
goals in medicinal chemistry.
It is well known that copper is an essential element in human
normal metabolism. In biological systems, copper exists as a variety
of complexes which due to that the coordinated forms of copper are
more stable than the corresponding ionic species [2,3]. Therefore,
the copper complexes of non-steroidal anti inflammatory com-
pounds such as aspirin and niflumic acid have been studied as they
show good antiarthritic activity [4]. These complexes have been
found to be more active and desirable drugs than the ligands them-
selves, suggesting that the activity of such metal based drugs may
be due to the in vivo formation of metallic complexes [5]. So that
the synthesis and studies of metal complexes with active drugs as
ligands increase the interest for inorganic, pharmaceutical and
1,10-Phenanthroline (Phen) and its substituted derivatives,
both in the metal-free state and as ligands coordinated to transi-
tion metals, disturb the functioning of a wide variety of biological
systems [7]. Furthermore, when metal-free N,N0-chelating bases
are found to be bioactive, it is usually assumed that sequestering
of trace metals in situ is involved, and that the resulting metal com-
plexes are the active species [8–11]. One of the most biologically
active of the metal–phen complexes is [Cu(phen)2]2+. Tsang and
coworkers [12] reported that incubation of a human hepatic cell
line (Hep-G2) with [Cu(phen)2]2+ resulted in internucleosomal
DNA fragmentation. Transition metal cations, such as Cu(II) and
Fe(II), can bind to negatively charged DNA and have been shown
to play an important role in the local formation of OHÅ radicals
[13–15].
Further more, ternary complexes of Cu(II) with substituted
phenanthrolines (phens) have been reported and their cytotoxic
properties are evaluated recently [16–18]. The most potent com-
plex is bis(diisopropylsalicylato)(2,9-dimethylphenanthroline)cop-
per(II), which exhibits cytotoxicity comparable with the anticancer
drug cisplatin [PtCl2(NH3)2] [19]. Their cytotoxicity may be due to
chelation of transition metals such as copper or iron for example in
the test media. These biological activities are improved by the
⇑
Corresponding authors. Tel.: +86 391 3987818 (L. Jia), +86 931 8915350
(Q. Wang).
E-mail addresses: jlxj@hpu.edu.cn (L. Jia), qwang@lzu.edu.cn (Q. Wang).
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.05.014

122
L. Jia et al. / Inorganica Chimica Acta 391 (2012) 121–129
complexation with some divalent transition metals. Thus, antiviral
activity is found for divalent transition metals chelates with substi-
tuted phenanthrolines [20]. Moreover, such complexes are used as
DNA intercalating agents and have been found useful for examin-
ing distinctive conformations along the DNA helix [21].
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 was 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 activites were tested on a 721E spectrophotometer
(Shanghai Analytical Instrument Factory, China).
Herein we report the synthesis, crystal structure, DNA-binding,
cytotoxic and antioxidation activities of a series of ternary cop-
per(II) complexes [Cu2(D)2(PDIMAla)(H2O)2](ClO4)2ꢀCH3OH (1–3)
containing
D as 1,10-phenanthroline (phen, 1), dipyrido[3,2-
d:20,30-f]quinoxaline (dpq, 2), dipyrido[3,2-a:20,30-c]phenazine
(dppz, 3) (Scheme 1). The primary aim of the current study is to
determine the cancer chemotherapeutic potential of metal-free
phen and its substituted derivatives by using three human-derived
cancer model cell lines of human hepatic (HepG2), human promy-
elocytic leukemia (HL60) and human prostate (PC3). In order to
illustrate that the effect observed is due to the complexes rather
than the free metal ions, the anti-tumour activities of the simple
Cu(II) salt is also determined. The 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.
2.2. Synthesis of the ligand (H2PDIMAla)
H2PDIMAla: N,N’-(p-xylylene)di-(2-methyl) alanine acid. To a
solution of 2-amino-2-methyl propanoic acid (1.03 g, 10 mmol)
and NaOH (0.4 g, 10 mmol) in 20 mL of MeOH and 10 mL of water
was added terephthalaldehyde (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 excess 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 col-
orless solid was filtered off, washed with dry MeOH and Et2O, and
recrystallized from H2O/EtOH (1:3) after drying in air. Yield: 1.37 g
(89%). Anal. Calc. for C16H24N2O4: C, 62.32; H, 7.84; N, 9.08. Found:
C, 62.51; H, 7.63; N, 9.26%. ESI–MS Calc. for C14H20N2O4: 308.37.
Found: 307.3%. 1H NMR (D2O) d 1.38 (s, 12H), 3.85 (s, 4H), 7.42
2. Experimental
2.1. Materials and instrumentation
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
(d, 4H). IR (KBr, cmꢂ1):
1354.
m(NH) 3071, m(CH2) 2986, m
(COOꢂ) 1612,
2.3. Synthesis of the complex
[Cu2(phen)2(PDIMAla)(H2O)2](ClO4)2ꢀCH3OH (1)
(e
= 6600 Mꢂ1 cmꢂ1 at 260 nm) in the buffer. The ternary copper(II)
complexes were dissolved in a mixture solvent of 5% CH3OH and
95% Tris–HCl buffer (5 mM Tris–HCl, 50 mM NaCl, pH 7.1) at con-
centration 1 ꢁ 10ꢂ3 M.
To the green solution formed from Cu(ClO4)2ꢀ6H2O (0.371 g,
1 mmol) in MeOH (8 mL) and phen (0.181 g, 1 mmol) in MeOH
(8 mL) was added a filtered solution of H2PDIMAla (0.154 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:
0.651 g, 92 %. Anal. Calc. for C41H46Cl2Cu2N6O15: C, 46.62; H, 4.37;
The UV–Vis absorption spectra were recorded using a Varian
Cary 100 spectrophotometer and fluorescence emission spectra
N, 7.92. Found: C, 46.81; H, 4.56; N, 8.17%. IR (KBr, cmꢂ1):
2987, (CH2) 2927, (Cl-O) 1089.
(COOꢂ) 1595, 1386.
m(NH)
m
m
m
2.4. Synthesis of the complex [Cu2(dpq)2(PDIMAla)(H2O)2](ClO4)2ꢀCH3
OH (2)
To the green solution formed from Cu(ClO4)2ꢀ6H2O (0.371 g,
1 mmol) in MeOH (8 mL) and dpq (0.232 g, 1 mmol) in MeOH
(8 mL) was added a filtered solution of H2PDIMAla (0.154 g,
0.5 mmol) in H2O (20 mL) containing LiOH. The resulting blue solu-
tion was stirred for 6 h, the blue precipitate were isolated by filtra-
tion. Yield: 0.469 g, 62%. Anal. Calc. for C45H46Cl2Cu2N10O15: C,
46.40; H, 3.98; N, 12.02. Found: C, 46.31; H, 4.01; N, 12.31%. IR
(KBr, cmꢂ1):
O) 1083.
m(NH) 2973, m(CH2) 2912, m m(Cl-
(COOꢂ) 1631, 1388.
2.5. Synthesis of the complex [Cu2(dppz)2(PDIMAla)(H2O)2](ClO4)2CH3
OH (3)
To the green solution formed from Cu(ClO4)2ꢀ6H2O (0.371 g,
1 mmol) in MeOH (8 mL) and dpz (0.282 g, 1 mmol) in MeOH
(8 mL) was added a filtered solution of H2PDIMAla (0.154 g,
0.5 mmol) in H2O (20 mL) containing LiOH. The resulting blue
Scheme 1. Complexes 1–3 and the heterocyclic bases.

L. Jia et al. / Inorganica Chimica Acta 391 (2012) 121–129
123
Table 2
solution was stirred for 6 h, the blue precipitate were isolated by
filtration. Yield: 0.532 g, 66%. Anal. Calc. for C53H50Cl2Cu2N10O15
Selected bond lengths (Å) and bond angles (°) for complex 1.
:
C, 50.32; H, 3.98; N, 11.07. Found: C, 50.55; H, 4.32; N, 11.29%. IR
Cu(1)–O(1)
Cu(1)–N(3)
Cu(1)–O(1W)
O(1)–Cu(1)–N(2)
N(2)–Cu(1)–N(3)
N(2)–Cu(1)–N(1)
O(1)–Cu(1)–O(1W)
1.928(2)
2.012(2)
2.272(3)
178.50(9)
98.51(9)
82.76(10)
87.15(11)
Cu(1)–N(2)
Cu(1)–N(1)
1.998(2)
2.033 (2)
(KBr, cmꢂ1):
O) 1080.
m(NH) 3094, m(CH2) 2920, m m(Cl-
(COOꢂ) 1636, 1357.
O(1)–Cu(1)–N(3)
O(1)–Cu(1)–N(1)
N(3)–Cu(1)–N(1)
N(2)–Cu(1)–O(1W)
82.58(9)
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.
95.75(10)
143.83(9)
93.13(11)
2.6. X-ray crystallography
Table 3
Main intra and intermolecular hydrogen bonds for the complex 1 (Å, °).
Details of the crystal parameters, data collection and refine-
ments of complex 1 were listed in Table 1. Selected bond lengths
and bond angles were listed in Table 2. Main intra and intermolec-
ular hydrogen bonds were listed in Table 3. Single crystal X-ray dif-
fraction measurements were made on a Bruker AXS SMART CCD
D–HꢀꢀꢀA
d(D–H)
d(HꢀꢀꢀA)
d(DꢀꢀꢀA)
<(DHA)
O1w–H1wꢀꢀꢀO5
O1w–H2wꢀꢀꢀO6
N3–H3NꢀꢀꢀO2
0.85(2)
0.85(2)
0.85(2)
2.22(5)
2.16(4)
2.03(3)
2.933(10)
2.906(5)
2.783(3)
178(5)
173(4)
163(3)
diffractometer with Mo K
a (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
SMART41 was used to collect the intensity data, SAINT [22] for inte-
gration of the intensity [22], SADABS [23] for absorption correction
and SHELXTL [24] for structure solution and refinements on F2. All
non-H atoms were located from successive Fourier maps, and
hydrogen atoms were refined using a riding model. Anisotropic
thermal parameters were used for all non-H atoms, and fixed iso-
tropic parameters were used for H atoms. H atoms attached to N
and O were located from difference Fourier maps and refined with
N–H distances fixed at 0.85 (2) Å (Uiso(H) = 1.2 Ueq(N)) and O–H
distances fixed at 0.85 (2) Å (Uiso(H) = 1.2 Ueq(O)). The asymmetric
unit contains one half of the dinuclear molecule and the symmetry
operation relates the two halves is 1ꢂx, ꢂy, ꢂz. Three oxygen
atoms of the perchloride anion was disordered into two positions
with occupancy ratio 56:44.
½DNAꢃ=ðeb
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–ef) versus [DNA], Kb is given by the ratio of slope
,
to the intercept.
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 0–80
l
M DNA and 1.5
lM EB (at
2.7. Spectroscopic studies on DNA interaction
lM ternary cop-
per(II) complexes and the ligand (kex = 500 nm, kem = 520.0–
650.0 nm). According to the classical Stern–Volmer Eq. (2) [26]:
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
F0=F ¼ Kq½Qꢃ þ 1
ð2Þ
(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) [25],
where F0 is the emission intensity in the absence of quencher, F is
the emission intensity in the presence of quencher, Kq is the
Table 1
Selected crystallographic data for 1.
Compound
1
Empirical formula
Formula weight
Temperature (K)
Crystal system Triclinic
Space group
a (Å)
C
41H46Cl2Cu2N6O15
1060.82
223(2)
Monoclinic
P2(1)/c
10.5259(6)
18.6643(10)
11.6372(7)
90
b (Å)
c (Å)
a
(°)
b (°)
95.7630(10)°
90
2274.7(2)
2
1.549
1.127
c
(°)
Volume (Å3)
Z
Calculated density (Mg/m3)
Absorption coefficient (mmꢂ1
)
Number of parameters/restraints
Measured reflections
340/76
15,394
Independent reflections
5231
Final R indices [I > 2
Goodness-of-fit on F2
Largest difference peak and hole (e Åꢂ3
r
(I)]
0.0491, 0.1330
1.042
0.623 and ꢂ0.423
Fig. 1. ORTEP view of the cation in complex 1 showing 30% probability thermal
ellipsoids.
)

124
L. Jia et al. / Inorganica Chimica Acta 391 (2012) 121–129
quenching 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.
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) [31]:
Suppression ratio ð%Þ ¼ ½ðAi ꢂ A0Þ=ðAc ꢂ A0Þꢃ ꢁ 100
ð3Þ
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-
plexes were 120 and 60 lM. Each sample solution was scanned in
the range of 220–370 nm. CD spectrum was generated which repre-
sented the average of three scans from which the buffer background
had been subtracted.
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
(1–5
l
M), and each complex was introduced into DNA solution
(50
lM) 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,
DNA, where
g
and g0 is the viscosity of DNA alone. Viscosity values were calcu-
lated from the observed flow time of DNA-containing solution cor-
rected from the flow time of buffer alone (t0),
g = t–t0 [27,28].
2.9. Cytotoxicity assay
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. Human hepatic (HepG2) and
human prostate (PC3) cell lines were maintained in DMEM med-
ium, human promyelocytic leukemia (HL60) were maintained in
RPMI 1640 medium. Cells were cultured at 37 °C in a humidified
atmosphere of 5% CO2 in air. All media were supplemented with
10% fetal bovine serum and contained penicillin G (100 U/mL)
and 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
morphological 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 were washed with 200 lL PBS per well. For HL-60 cells,
after removing the medium and PBS, the plates were centrifuged
at 2500 rpm for 10 min. One hundred microliter buffer containing
0.1 M sodium acetate (pH 5.0), 0.1% Triton X-100, and 5 mM p-
nitrophenyl phosphate 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 Vic-
tor3 (PerkinElmer). Experiments were conducted in triplicate
(three independent experiments).
2.10. Antioxidant measurements
The hydroxyl radicals in aqueous media were generated
through the Fenton-type reaction [29,30]. 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.
Fig. 2. 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 absorbance changes upon increasing DNA concentration.
Inset: plots of [DNA]/(ea–ef) versus [DNA] for the titration of complex 1–3 with CT–
DNA.
l
l

L. Jia et al. / Inorganica Chimica Acta 391 (2012) 121–129
125
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).
longer than the intermolecular distance, 6.920 Å. The N–HꢀꢀꢀO
hydrogen bondings between aqua ligand and carbonyl oxygen of
the PDIMAla ligand along a-axis produce a layer structure. The
ꢂ
ClO4 anions, lattice water molecules are found between the
grooves and cavities with the layers. The interlayers are essentially
3. Results and discussion
3.1. Synthesis and general properties
Ternary copper(II) complexes containing heterocyclic bases are
prepared in high yield from reactions of the reduce Schiff base li-
gand H2PDIMAla in the presence of LiOH with copper(II) perchlo-
rate and N,N-donor heterocyclic bases. The complexes have been
characterized by elemental analysis and spectroscopic data. The
stability of copper(II) complexes 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 copper(II) complexes are prepared in DMSO
solution and for experiments freshly diluted in phosphate buffer
system (at pH 7.4, 7.8). Then, the UV–Vis spectrums and molar con-
ductivities are researched at different time intervals. The investiga-
tions reveal that the UV–Vis spectra have remained unaltered for
the solutions and the molar conductance values have no obvious
change for very freshly prepared and for over the whole experi-
ment (72 h). It indicates that the copper(II) complexes are quite
stable in solution. The characteristic bands of the carboxylate
groups are observed in the range 1595–1640 cmꢂ1 for asymmetric
stretching and 1320–1390 cmꢂ1 for symmetric stretching, respec-
tively [32]. The difference between as(COOꢂ)and the sym(COOꢂ)
stretching frequencies in complexes is 200 cmꢂ1, thus suggesting
a terminal coordination mode for the carboxylate group [33]. Weak
absorptions observed in the range of 2930–2990 cmꢂ1 can be
attributed to the
of the reduce Schiff base ligand and the
CH2
vibration observed in the range of 2970–3120 cmꢂ1 can be attrib-
uted to the NH of the ligand. The broad bands at 3430 cmꢂ1 are as-
cribed to the vibration of the O–H stretching of the water ligands.
Two topical bands in the region near 830 and 720 cmꢂ1 are as-
signed to (C@N) of heterocyclic bases. And the topical bands in
the region near 1090 cmꢂ1 are assigned to (Cl–O) of anion. The con-
ductivity measurements indicate 1:2 electrolytic nature of the
complexes.
3.2. Structure of the ternary copper complexes
The molecular structure of complex [Cu2(phen)2(PDIMAl-
a)(H2O)2](ClO4)2ꢀCH3OH (1) consists of dinuclear complex cations
as shown in Fig. 1. 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 PDIMAla li-
gand is sandwiched between the phen ligands through – interac-
tions, and thus the PDIMAla ligand is ‘‘S’’ shaped. The bond
lengths of Cu(1)–O(1) (1.928(2) Å) and Cu(1)–N(3) (2.012(2) Å)
are similar to those found in Cu(L-Thr)2ꢀH2O, where the mean
Cu–O and Cu–N (O and N atoms of L-threonine) distances are
1.95(1) and 1.98(1) Å, respectively [34]. The Cu–N(phen) bond
lengths of 1.998(2) and 2.033(2) Å and the bite angle N(1)–
Cu(1)–N(2) of 82.76° are close to the corresponding values for
some copper–phenanthroline complexes reported [35,36]. The
closest distances, 3.331 and 3.666 Å are between the centre of
the phenyl ring and the middle of the carbon atoms next to the
nitrogen atoms of the phen ligand. The interplanar angle between
the planes is 4.42°. The CuꢀꢀꢀCu distance in the cation, 7.819 Å, is
Fig. 3. The emission spectra of DNA–EB system (15
ex = 500 nm, em = 520–720 nm, in the presence of the complex 1 (a), complex 2
(b), complex (c). [DNA] = 10 M, [Compound] = 0–35 M. Arrow shows the
emission intensity changes upon increasing complex concentration. Inset: Stern–
Volmer plot of the fluorescence titration data of the complexes 1–3.
lM and 1.5 lM EB),
3
l
l

126
L. Jia et al. / Inorganica Chimica Acta 391 (2012) 121–129
Table 4
The anti-cancer activity of the free ligand, free metal salt and complexes 1–3 against
human hepatic cell line (HepG2), human promyelocytic leukemia cell line (HL60) and
human prostate cancer cell line (PC3) expressed as IC50 M).
(l
Test compound
Toxicities (IC50)
HepG2
HL60
PC3
Cu(ClO4)26H2O
H2PDIMAla
phen
dpz
dppz
1
>160
>160
4.1
40
85
>160
>160
6.2
10
50
>160
>160
>160
>160
>160
2.4
2.2
2.7
2
2.7
2.2
2.7
3
1.4
1.5
1.4
absence and presence of CT–DNA. Addition of increasing amounts
of CT–DNA result in hypochromism and red shift. With increasing
DNA concentration, the absorption bands of the three complexes
show decreases in molar absorptivity (hypochromism) as well as
slight bathochromism. The observed red-shift is an evidence of
the stabilization of the CT DNA duplex and the hypochromism
may be attributed to interaction between the aromatic rings of
the complexes and DNA base pairs [38].
Fig. 4. CD spectra of CT–DNA (120
l
M) in the absence and presence of the
copper(II) complexes 1, 2 and 3 (60
lM).
The Kb values of ternary copper(II) complexes with different
numbers of copper chelates are 0.38 ꢁ 105, 1.4 ꢁ 105, and
7.8 ꢁ 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 extended planar
structure of the dpq and dppz ligands greatly facilitating non-cova-
lent interactions with the DNA molecule. Compounds binding to
DNA through intercalation usually results in hypochromism and
bathochromism due to intercalative mode involving a strong stack-
ing interaction between an aromatic rings and DNA base pairs [39].
All these results suggest that metal complexes can interact with CT
DNA quite probably by intercalating the compounds into DNA base
pairs [40].
It is noteworthy that the Kb values are higher than the binding
affinity of EB for DNA, (Kb = 1.23 ( 0.07) ꢁ 105 Mꢂ1) [41], suggest-
ing that the existing interactions may cause EB displacement from
its complex with DNA [41]. Interestingly, 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-phenanthroline ([Cu(phen)(L-Thr)(H2O)](ClO4)
Fig. 5. Effect of increasing amounts of complexes 1, 2 and 3 on the relative viscosity
of CT–DNA at 25.0 °C.
(Kb, 6.35 ꢁ 103 Mꢂ1) [11], [Cu2(phen)2Cl4] (Kb, 4.75 ꢁ 104 Mꢂ1
)
held by ꢀꢀꢀ interactions as the complex 1 shows chemically signifi-
cant face to face ꢀꢀꢀ aromatic stacking inter-planar distance of
3.357 Å.
[42], [(phen)Cu(bipp)Cu(phen)](ClO4)4 (Kb, 1.4 ꢁ 104 Mꢂ1) [43].
This showed that comparatively the ternary copper(II) complex
samples can bind to CT–DNA very strongly.
3.3. Interactions with CT-DNA
3.3.1. Electronic absorption spectra
3.3.2. Competitive DNA-binding studies with EB
The interactions of metal complexes with DNA have been the
subject of interest for the development of effective chemothera-
peutic agents. Transition metal centers 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 [37].
In the UV region of the electronic spectra, the absorption bands
around 300 nm observed can be attributed to the –⁄ transition of
the coordinated ligands, while the intense electronic bands
observed in the spectral range 600–700 nm are assignable to the
d–d band, which is in agreement with five-coordinate geometry.
The binding of ternary copper(II) complexes to DNA helix have
been characterized through absorption spectral titrations, by fol-
lowing changes in absorbance and shift in wavelength. Fig. 2 shows
the absorption spectra of ternary copper(II) complexes in the
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 [44]. In our
experiments, as depicted in Fig. 3, for complex 1, 2 and 3, the fluo-
rescence intensity of EB at 584 nm show a remarkable decreasing
trend with the increasing concentration of the complex 1, 2 or 3,

L. Jia et al. / Inorganica Chimica Acta 391 (2012) 121–129
127
Fig. 6. Phase-contrast micrographs of cells treated with complexs 1, 2 and 3.
increasing the complex concentration could be caused due to the
displacement of the DNA bound EB by the ternary copper(II) com-
plexes. The Kq values of ternary copper(II) complexes are
2.81 ꢁ 104 Mꢂ1, 3.56 ꢁ 104 Mꢂ1, 6.16 ꢁ 104 Mꢂ1 for 1, 2 and 3,
respectively. The order of Kq for the complexes was very similar
to the absorption titration methods and a similar explanation can
be cited as stated earlier.
3.3.3. Circular dichroic spectral studies
Circular dichroic spectral techniques may give us useful infor-
mation on how the conformation of the CT–DNA chain is influ-
enced by the bound complex. The CD spectrum of CT–DNA
consists of a positive band at 275 nm that can be due to base stack-
ing 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 [45]. The
changes in CD signals of DNA observed on interaction with drugs
may often be assigned to the corresponding changes in DNA struc-
ture [46]. 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 [47,48].
Fig. 7. Effect (%) of hydroxyl radical scavenging for ligand and Cu(II) complexes.
indicating 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
The CD spectrum of CT–DNA is monitored in the presence of 1, 2
and 3, the changes observed in the three cases are shown in Fig. 4.
On addition of all the complexes to CT–DNA, faint red shift with
intensity increase in the positive or negative bands are observed.
When addition of
3 to CT–DNA, it is observed that the

128
L. Jia et al. / Inorganica Chimica Acta 391 (2012) 121–129
negative-band position 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. This phenomenon may be due to the
intercalation of the complex through -stacking which stabilizes
the right-handed B form DNA [49].
3.5. Hydroxyl radical scavenging activity
Fig. 7 shows the plots of hydroxyl radical scavenging effect (%)
for ligand and Cu(II) complexes, respectively, which are concentra-
tion-dependant. The values of IC50 of ligand for hydroxyl radical
scavenging effect cannot be read, while the values of IC50 of Cu(II)
complexes for hydroxyl radical scavenging effect are 4.5–7.5 lM
with the order of 1 < 2 < 3. These orders of IC50 are opposite to the
abilities of scavenging effects for hydroxyl radicals. It is marked that
the hydroxyl radical scavenging effects of Cu(II) complexes are
much higher than those of their ligand, possibly in that the larger
conjugated metal complexes can react with OHÅ to form larger sta-
ble macromolecular radicals than ligand [52]. Moreover, mannitol
is a well-known natural antioxidant, so we also investigate the scav-
enging activity of mannitol against hydroxyl radical using the same
model. We find that when arriving at similar suppression ratio, con-
centration of the two complexes is far less than that of mannitol.
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 [50,51]. Fig. 5 depicts the effect of complexes 1–3 on the
DNA viscosity. The change in relative viscosity for the dppz com-
plexes are more than that for the phen or dpq analogues suggesting
greater DNA binding propensity of the dppz complexes in compar-
ison to the phen or bpq analogues. This is consistent with the
observed trend shown by other optical methods and suggests pri-
marily an intercalation binding nature of the complex.
4. Conclusions
A new reduced Schiff base ligand H2PDIMAla and its 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-
ing effect on OHÅ. Although the mechanism between scavenging
hydroxyl radicals should be also studied further, these findings
clearly indicate that copper(II)-based complexes have many poten-
tial 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 three cell lines, and the IC50 values of all the metal
complexes are lower than that of phen and its substituted deriva-
tives, H2PDIMAla and Cu(ClO4)2ꢀ6H2O. Studies are currently under-
way in our laboratory to investigate more fully the mechanisms by
which phen and its substituted derivatives, H2PDIMAla 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 facilitating the development of
highly effective anti-cancer therapies.
3.4. Cytotoxic activity
To evaluate their cancer chemotherapeutic potential the ability
of the DMSO soluble copper complexes 1–3 to kill human derived
cancer cells was investigated using the human hepatic (HepG2),
human promyelocytic leukemia (HL60) and human prostate
(PC3) cell lines and was determined by calculation of IC50 (the drug
concentration causing a 50% reduction in cellular viability). Cells
were continuously exposed to test agent for 72 h, and their effects
on cellular viability was evaluated. It was intended that the results
from these studies would allow the identification of those ternary
derivatives with cancer chemotherapeutic potential. Comparison
of IC50 values, allowed the relative potency of each of the test com-
pounds to be determined and ranked. The IC50 values obtained for
all tested compounds are presented in Table 4. The three metal
complexes displayed a concentration-dependent cytotoxic profile
in all cell lines. Since the IC50 values for complexes 1–3 are statis-
tically lower than that for metal-free heterocyclic bases and H2PDI-
MAla in all of the tested cells, it suggests that coordinated
copper(II) ion plays a major role in mediating potency of the com-
plex. The IC50 values for phen on all the cells are not statistically
different, it displayed a greater effect against HL60 and HepG2 cell
lines, which means phen is more sensitive than dpq and dppz for
this two cell lines. All the heterocyclic bases show low IC50 values
against PC3 cell line. The results also suggest that 3 had more sig-
nificant cytotoxic activities than 1 and 2 against the three human
cancer cell lines, which is consistent with the result of DNA binding
studies above. This may be due to the complexes inducing DNA
damage in cancer cells and the nature of the compound itself.
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.
Acknowledgment
We thank the Prof. Jagadese J. Vittal and Tan Geok Kheng for
solving the crystal, the financial support of the Ministry of Educa-
tion grant through National University of Singapore (Grant No.
R143-000-371-112) and the National Natural Science Foundation
of China (21001040).
Appendix A. Supplementary material
CCDC 698877 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif. Supplementary data associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.ica.2012.05.014.
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