Characterization, nucleolytic, cytotoxic and antibacterial property of tetrakis(μ-4-chloro-2-nitrobenzoato-κ2O,O′)bis[aquacopper(II)], tetrakis(μ-2-chloro-6-fluorobenzoato-κ2O,O′)bis[aquacopper(II)] and tetrakis(μ-2-chlorobenzoato-κ<

Article abstract of DOI:10.1016/j.poly.2009.02.006

Three water soluble dicopper(II) complexes, tetrakis(μ-4-chloro-2-nitrobenzoato-κ²O,O′) bis[aquacopper(II)] (1), tetrakis(μ-2-chloro-6-fluorobenzoato-κ²O,O′)bis[aquacopper(II)] (2) and tetrakis(μ-2-chlorobenzoato-κ²O,O′)bi

Full text of DOI:10.1016/j.poly.2009.02.006

Polyhedron 28 (2009) 1320–1330
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Characterization, nucleolytic, cytotoxic and antibacterial property
of tetrakis(
tetrakis(
-2-chloro-6-fluorobenzoato-j2O,O0)bis[aquacopper(II)]
and tetrakis(
-2-chlorobenzoato-j2O,O0)bis[aquacopper(II)]
l
-4-chloro-2-nitrobenzoato-j2O,O0)bis[aquacopper(II)],
l
l
a
a
b
c
Eng-Khoon Lim a, Siang-Guan Teoh a, , Siu-Mun Goh , Cynn-Dee Ch’ng , Chew-Hee Ng , Hoong-Kun Fun ,
Mohd Mustaqim Rosli c, Nazalan Najimudin d, Hooi-Kheng Beh e, Lay-Jing Seow e, Norhayati Ismail e,
Zhari Ismail e, Mohd Zaini Asmawi e, Yew-Hoong Cheah f
*
a School of Chemical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia
b Faculty of Engineering and Science, Universiti Tunku Abdul Rahman, 53300 Kuala Lumpur, Malaysia
c X-ray Crystallography Unit, School of Physics, Universiti Sains Malaysia, 11800 Penang, Malaysia
d School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia
e School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia
f Bioassay Unit, Herbal Medicine Research Center, Institute for Medical Research, 50588 Jalan Pahang, Kuala Lumpur, Malaysia
a r t i c l e i n f o
a b s t r a c t
Three water soluble dicopper(II) complexes, tetrakis(l
-4-chloro-2-nitrobenzoato-j2O,O0) bis[aquacop-
Article history:
per(II)] (1), tetrakis(l l-2-chloro-
-2-chloro-6-fluorobenzoato-j2O,O0)bis[aquacopper(II)] (2) and tetrakis(
Received 2 October 2008
Accepted 7 February 2009
Available online 20 February 2009
benzoato-j2O,O0)bis[aquacopper(II)] (3), have been synthesized. Complex 1 crystallizes in monoclinic
system, space group C2/c with a = 28.813(3), b = 7.2368(6), c = 17.0053(14) Å, b = 105.395° and Z = 4,
ꢀ
while
2
crystallizes in triclinic system, space group P1 with a = 7.5638(2), b = 13.0494(4), c =
Keywords:
16.7079(5) Å,
a
= 106.003°, b = 90.388°, c = 91.855° and Z = 2. In addition, all the complexes have been
Dicopper(II) complexes
DNA cleavage
Cytotoxic
characterized by their elemental analysis, FT-IR, UV–Vis spectroscopy and cyclic voltammetry. All com-
plexes have been found to be redox active displaying two quasi-reversible redox couples corresponding
to CuIICuII to CuIICuI and CuIICuI to CuICuI redox processes, respectively. The results from the gel electro-
phoresis experiment of 1–3 on pBR322 DNA, have demonstrated the occurrence of oxidative cleavage in
the presence of hydrogen peroxide. The hydroxyl radical has been found to be the reactive oxygen species
that is responsible for the cleavage reaction. In the antiproliferation tests against MCF-7, DU 145, HeLa
and HepG2 cancer cell lines, all complexes show significant preference against HepG2 cell lines. All com-
plexes also show significant preference against HepG2 cancer cells line over Chang human normal liver
cells which means 1–3 are more potent against cancer cells than normal cells. In antibacterial tests
against nine pathogenic bacterial species, all complexes show a very selective trend whereby they exhibit
antibacterial activity against the Gram negative bacteria Enterobacter aerogenes.
Antibacterial
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
ence of thiol or hydrogen peroxide, research activities in this area
have blossomed [14,15]. Until now, a number of Cu(II) intercalat-
In the past decade, the synthesis of biologically active Cu(II)
complexes has attracted significant attention mostly due to their
role in biological systems as well as potential uses as pharmaceu-
tical agents. There are a number of papers that reported that Cu(II)
complexes exhibit anti-tumour, antimycobacterial and antimicro-
bial activities [1–13]. However, since the discovery of the ability
of bis(1,10-phenantroline)copper(II) to interact with DNA via the
intercalating mode and to further induce DNA cleavage in the pres-
ing complexes based on 1,10-phenantroline derivatives have been
synthesized and their DNA cleavage mechanisms well established.
[16–24]. Apart from oxidative cleavage, Cu(II) complexes also can
induce hydrolytic, photolytic and electrolytic cleavage of DNA
[25–28]. Recently, Ng et al. have demonstrated that neutral Cu(II)
amino acid complexes such as bis[N,N-di-(N0-methylacetamido)-
L
-alaninato]copper(II) and bis(N,N0-dimethylglycinato)copper(II)
can induce oxidative cleavage of DNA in the presence of hydrogen
peroxide [29,30]. In our laboratory, we have initiated a study of the
DNA cleavage activity of neutral Cu(II) complexes of benzoic acid
derivatives which are water soluble and redox active. Herein, the
* Corresponding author. Tel.: +60 46533565; fax: +60 46574854.
E-mail address: sgteoh@usm.my (S.-G. Teoh).
0277-5387/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2009.02.006

E.-K. Lim et al. / Polyhedron 28 (2009) 1320–1330
1321
grams were recorded using a standard three electrode arrange-
ment consisting of a platinum working electrode, a platinum
auxiliary electrode and a Ag/AgCl reference electrode. The com-
plexes were dissolved in Tris–NaCl (TN) pH 7.5 buffer prior to
measurement.
Cl
NO2
Cl
2.5. X-ray crystallography
F
Cl
COOH
ClFBzo
COOH
COOH
ClBzo
Diffraction data were collected using a Bruker APEX2 [30] CCD
area-detector, graphite monochromated Mo
K
a
radiation
ClNO2Bzo
(k = 0.71073 Å) at 100(1) K for 1 and 297(2) K for 2 and the Oxford
Cyrosystem Cobra low-temperature attachment. The data was re-
duced using SAINT [31]. A semi-empirical absorption correction
was applied to the data using SADABS [31]. The structure was solved
by direct methods and refined against F2 by full-matrix least
squares using SHELXTL [32]. Hydrogen atoms were placed in calcu-
lated positions. Crystal data and experimental details of the struc-
ture determinations are listed in Table 1.
Fig. 1. Chemical structure and abbreviation for the ligands.
synthesis, characterization and nuclease activity of 1–3, together
with their antiproliferation and antibacterial activity is reported.
The ligands in complexes 1–3 are shown in Fig. 1 and the abbrevi-
ated formula of the complexes are [Cu(II)(ClNO2Bzo)(H2O)]2,
[Cu(II)(ClFBzo)(H2O)]2 and [Cu(II)(ClBzo)(H2O)]2, respectively.
2.6. DNA cleavage experiments
2. Experimental
The electrophoresis experiments were performed on a horizon-
tal gel electrophoresis system. Agarose gel electrophoresis experi-
ments were carried out on plasmid pBR322 DNA. All samples in
Tris–NaCl buffer at pH 7.5 were incubated at 37 °C for 2 h, and then
electrophoresed on 1% agarose gel for 1 h at 80 V. Essentially sam-
ples with an increasing concentration of complexes with a given
2.1. Synthesis of tetrakis(l
-4-chloro-2-nitrobenzoato-j2O,O0)
bis[aquacopper(II)] (1)
Copper(II) chloride hydrate (3.00 g, 0.018 mol) was dissolved in
ethanol solution (50 ml). To this solution was added 50 ml ethanol
solution of 4-chloro-2-nitrobenzoic acid (7.26 g, 0.036 mol). The
resultant solution was stirred and refluxed for 2 h. The green solu-
tion was then filtered and allowed to cool down to room tempera-
ture. Crystals of the title compound were obtained after 3 days of
slow evaporation at room temperature. Yield 32.5%. Anal. Calc.
for 1: C, 34.84; H, 1.67; N, 5.80. Found: C, 35.19; H, 1.42; N, 5.52%.
concentration of DNA (0.25 lg/ll) for each sample were incubated
in the absence or presence of hydrogen peroxide. The resultant
bands for each set of experiments were photographed under UV
light by using a Polaroid camera, GelCam, fixed with an electropho-
resis hood.
2.7. Cell line and cell culture
2.2. Synthesis of tetrakis(l
-2-chloro-6-fluorobenzoato-j2O,O0)
bis[aquacopper(II)] (2)
Human cancer cell lines, namely MCF-7, DU 145, HeLa, HepG2
and Chang were obtained from the American Type Culture Collec-
tion (ATCC, Manassas, VA, USA). The cancer cells were maintained
in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Co.,
Carlsbad, California, USA) supplemented with 10% heat-inactivated
fetal calf serum (Invitrogen Co., Carlsbad, California, USA), 100 U/
ml penicillin and 100 lg/ml streptomycin (Flowlab, Sydney, Aus-
tralia). Cells were kept in the logarithmic growth phase by routine
passage every 2–3 days using 0.025% trypsin–EDTA treatment.
Copper(II) chloride hydrate (3.00 g, 0.018 mol) was dissolved in
ethanol solution (50 ml). To this solution was added 50 ml ethanol
solution of 2-chloro-6-fluorobenzoic acid (6.28 g, 0.036 mol). The
resultant solution was stirred and refluxed for 2 h. The green solu-
tion was then filtered and allowed to cool down to room tempera-
ture. Crystals of the title compound were obtained after 3 days of
slow evaporation at room temperature. Yield 34.2%. Anal. Calc.
for 2: C, 39.23; H, 1.89. Found: C, 39.02; H, 1.46%.
2.8. In vitro antiproliferation assay
2.3. Synthesis of tetrakis(l
-2-chlorobenzoato-j2O,O0)
bis[aquacopper(II)] (3)
The antiproliferative and cytotoxic activity of 1–3 towards MCF-
7, DU 145, HeLa, HepG2 and Chang cell lines was evaluated by
using the sulforhodamine B (SRB) method, as previously described
[33–37]. Cells were seeded 24 h prior to treatment in a 96-well
plate at plating densities ranging from 10000 to 20000 cells/well
depending on the doubling time of individual cell lines in order
to obtain semi-confluent cultures. One plate from each cell line
was fixed in situ with sodium trichloroacetate, TCA (Sigma Chem-
ical Co.), to represent a measurement of the cell population for
each cell line at the time of drug addition (Tz). Test agents were dis-
solved in DMSO (Sigma Chemical Co.) and followed by a 2ꢀ serial
Copper(II) chloride hydrate (3.00 g, 0.018 mol) was dissolved in
ethanol solution (50 ml). To this solution was added 50 ml ethanol
solution of 2-chlorobenzoic acid (5.64 g, 0.036 mol). The resultant
solution was stirred and refluxed for 2 h. The green solution was
then filtered and allowed to cool down to room temperature. Crys-
tals of the title compound were obtained after 3 days of slow evap-
oration at room temperature. Yield 35.2%. Anal. Calc. for 3: C,
42.82; H, 2.57. Found: C, 42.51; H, 2.17%.
2.4. Physical measurements
dilution for six points ranging from 6.25 to 200 lg/ml. The final
concentration of DMSO used in the corresponding wells did not ex-
ceed 1% (v/v), which affect cell viability. Control cultures received
the same concentration of solvent alone. Following drug addition,
the plates were incubated for an additional 48 h period.
Elemental analyses were performed using a Perkin–Elmer 2400
series-11 CHN/O analyzer. Infrared spectra (KBr pellets) were re-
corded using a Perkin–Elmer 2000 Infrared Spectrometer. The solu-
tion UV–Vis spectra were obtained from
a
Jasco V-530
At the end of incubation, cells were fixed in situ with 50 ll of
Spectrophotometer. The cyclic voltammetric experiments were
performed on a BAS-Epsilon electrochemical system. Voltammo-
cold 50% (w/v) TCA and incubated for 1 h at 4 °C. The plates were
then washed with tap water and air dried. SRB (Sigma Chemical

1322
E.-K. Lim et al. / Polyhedron 28 (2009) 1320–1330
Table 1
acetic acid and the bound stain was subsequently solubilised with
10 mM trizma base (Sigma Chemical Co.). The absorbance at
505 nm was read on a spectrophotometric plate reader.
Crystal data, data collection and structure refinement parameters of [Cu(II)(ClNO2
Bzo)(H2O)]2 and [Cu(II)(ClFBzo)(H2O)]2.
Complex
[Cu(II)(ClNO2Bzo)(H2O)]2 [Cu(II)(ClFBzo)(H2O)]2
Dose–response curves were constructed to obtain the response
parameters which were the GI50, TGI and LC50. Percentage growth
is calculated as: [(Ti ꢁ Tz)/(C ꢁ Tz)] ꢀ 100 where (Tz) is time zero, (C)
is control growth and (Ti) is test growth in the presence of the drug
at the six concentration levels. The GI50 value (growth inhibitory
activity) corresponds to the concentration of test agents causing
50% decrease in net cell growth which was calculated from [(Ti
ꢁ Tz)/(C ꢁ Tz)] ꢀ 100 = 50, the TGI value (cytostatic activity) is the
concentration of test agents resulting in total growth inhibition
which was calculated from Ti = Tz, and the LC50 value (cytotoxic
activity) is the concentration of the test agents causing net 50% loss
of initial cells at the end of the incubation period which was calcu-
lated from [(Ti ꢁ Tz)/Tz] ꢀ 100 = (ꢁ50). All data were derived from
three independent experiments.
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system, space group
C28H16Cl4Cu2N4O18
965.33
100(1)
0.71073
monoclinic, C2/c
C28H16Cl4Cu2F4O10
857.29
297(2)
0.71073
triclinic, P1
ꢀ
Unit cell dimensions
a (Å)
b (Å)
c (Å)
28.813(3)
7.2368(6)
17.0053(14)
90
105.395(6)
90
3418.6(5)
4
1.876
7.5638(2)
13.0494(4)
17.0053(14)
106.003(2)
90.388(2)
91.855(2)
1584.17(8)
2
a
(°)
b (°)
c
(°)
Volume (Åꢁ3
)
Z
Dcalc (Mg mꢁ3
)
1.797
1.759
Absorption coefficient (mmꢁ1
Crystal size (mm)
)
1.646
0.48 ꢀ 0.28 ꢀ 0.17
1.47–27.50
30039/3931 [0.0773]
0.43 ꢀ 0.37 ꢀ 0.35
1.27–35.00
48847/13817
[0.0321]
13817/0/437
1.080
R1 = 0.0358,
wR2 = 0.0915
R1 = 0.0602,
wR2 = 0.1131
0.936 and ꢁ0.647
2.9. Antibacterial screening (inhibition zone)
h range (°)
Reflections collected/unique
The antibacterial activity of 1–3 was determined by screening
against Gram negative bacteria: Enterobacter aerogenes, Klebsiella
pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Shigella son-
nei and Gram positive bacteria: Bacillus cereus, Bacillus subtilis and
Staphylococus aureus using the well diffusion method. Sensitivity
plates were inoculated with Gram negative bacteria and Gram po-
[Rint
]
Data/restraints/parameters
3931/0/261
1.112
R1 = 0.0491,
wR2 = 0.1304
R1 = 0.0608,
wR2 = 0.1479
Goodness-of-fit on F2
Final R indices [I > 2
r(I)]
R indices (all data)
Largest difference in peak and 2.161 and ꢁ1.164
sitive bacteria and the well was loaded with 75 ll of test com-
hole
pound solution using a sterilized micropipette. The incubation
was done for 24 h at 37 2 °C. Inhibition was recorded by measur-
ing the diameter of the inhibitory zone after the period of incuba-
tion. Three replicas were made for each treatment. To evaluate the
effect of concentration on antibacterial activity, three different
Co.) solution (100 ll) at 0.4% (w/v) in 1% acetic acid was added to
the corresponding wells and further incubated for 10 min at room
temperature. After staining, unbound dye was washed out by 1%
concentrations (125, 250 and 500 lg/ml) of the test compounds
Fig. 2. The molecular structure of 1, showing 50% probability displacement ellipsoids and the atomic numbering.

E.-K. Lim et al. / Polyhedron 28 (2009) 1320–1330
1323
Fig. 3. The crystal packing of 1.
were used. DMSO, which exhibited no antibacterial activity, was
used as a negative control. The stability of the complexes in DMSO
medium (2% v/v) were checked by using UV–Vis technique where-
by no apparent changes were observed in the absorption peaks of
the complexes for two weeks.
normal range of tetracarboxylatocopper(II) dimers such
as diamminetetrakis-
-(O,O0-indole-3-carboxylate)dicopper(II),
l
Cu(L)4(H2O)2 [LH = (1S, 3R)-3-carbamoyl-2,2,3-trimethylcyclopen-
tane-1-carboxylic acid] and [Cu2(taa)4 (dmf)]2 [taa = 2-thiophene-
acetate anion] [38–40]. The carboxylate bridging angles of O(2)–
C(7)–O(1) and O(5)–C(14)–O(6) are similar at 126.7(3)° and
127.3(3)°, respectively. The bond distances of Cu(1)–O(5), Cu(1)–
O(6A), Cu(1)–O(2A), Cu(1)–O(1) and Cu(1)–O(1W) are 1.964(2),
1.964(2), 1.967(2), 1.987(2) and 2.130(3) Å, respectively, while
the bond angles of O(5)–Cu(1)–O(1W), O(6A)–Cu(1)–O(1W),
2.10. Antibacterial screening (MIC)
The MIC (minimum inhibitory concentration) value for the
complexes which showed positive result towards antibacterial
activity was further determined using the microdilution broth
method. Sample solutions were added to the broth at different con-
centrations. Measured samples of each bacterial suspension were
added to the serial dilution of the test substances. The inoculated
test tubes were incubated at 37 2 °C. After 24 h, the turbidity
was evaluated. The MIC was defined as the lowest antibacterial
concentration of the test samples that inhibit more than 99% of
the bacterial population. The experiments were repeated three
times and the results were expressed as average values. The sol-
vent showed no antibacterial action.
Table 2
Selected bond lengths (Å) and bond angles (°) of [Cu(II)(ClNO2Bzo)(H2O)]2.
Bond lengths (Å)
Cu(1)–O(5)
Cu(1)–O(6A)
Cu(1)–O(2A)
Cu(1)–O(1)
Cu(1)–O(1W)
Cu(1)–Cu(1)#1
O(3)–N(1)
O(4)–N(1)
O(7)–N(2)
O(8)–N(2)
1.964(2)
1.964(2)
1.967(2)
1.987(2)
2.130(3)
2.6356(8)
1.235(4)
1.221(4)
1.227(4)
1.219(4)
3. Result and discussion
3.1. Characterization of complex 1
Bond angles (°)
O(5)–Cu(1)–O(6A)
O(5)–Cu(1)–O(2A)
O(6A)–Cu(1)–O(2A)
O(5)–Cu(1)–O(1)
O(6A)–Cu(1)–O(1)
O(2A)–Cu(1)–O(1)
O(5)–Cu(1)–O(1W)
O(6A)–Cu(1)–O(1W)
O(2A)–Cu(1)–O(1W)
O(1)–Cu(1)–O(1W)
O(2A)–C(7)–O(1)
O(5)–C(14)–O(6A)
168.78(10)
90.45(10)
89.81(10)
89.26(10)
88.29(10)
168.69(10)
94.79(10)
96.30(10)
97.28(10)
94.01(10)
126.7(3)
The crystal structure of 1 is depicted in Fig. 2 and the crystal
packing in the solid state is depicted in Fig. 3. Selected interatomic
distances and angles are given in Table 2. Complex 1 which is neu-
tral, centrosymmetric and dinuclear consists of two Cu(II) centers
bridged by four carboxylate groups of the four ligands. Each Cu(II)
center is coordinated by four O atoms of carboxylate groups in the
basal plane and the O atom of a water molecule in the apical posi-
tion, giving a square-pyramidal coordination environment. The
CuꢂꢂꢂO bond distance of water molecule is markedly longer than
the CuꢂꢂꢂO bond distance of carboxylate groups. The intramolecular
CuꢂꢂꢂCu distance in the dinuclear unit 2.6356(8) Å, which is in the
127.2(3)
#1: ꢁx + 1/2, ꢁy + 1/2, ꢁz.

1324
E.-K. Lim et al. / Polyhedron 28 (2009) 1320–1330
Fig. 4. The molecular structure of 2, showing 50% probability displacement ellipsoids and the atomic numbering.
O(2A)–Cu(1)–O(1W) and O(1)–Cu(1)–O(1W) are 94.79(10)°,
96.30(10)°, 97.28(10)° and 94.01(10)°, respectively, with a tau va-
with DNA through van der Waals interaction. The crystal structure
of 3 was earlier reported elsewhere [41].
lue
s
ꢃ 0, indicating a square-pyramidal coordination geometry.
The IR spectrum of 1 shows a broad absorption band at 3440
cmꢁ1 corresponding to HꢂꢂꢂO stretching of coordinated water mol-
ecules in the complex. The C@C–H absorption band can be ob-
The crystal structure of 2 is depicted in Fig. 4 and the crystal pack-
ing in the solid state is depicted in Fig. 5. Similar to complex 1,
complex 2 also is neutral, centrosymmetric and dinuclear in which
there are two crystallographically independent molecules, both sit-
uated over their centers of inversion. Each Cu(II) center displays a
square-pyramidal coordination environment where the four O
atoms of carboxylate groups from the ligands form the basal plane
and the O atom of a water molecule occupies the apical position.
The CuꢂꢂꢂO bond distance of the water molecule is clearly longer
than the CuꢂꢂꢂO bond distance of the carboxylate groups. The intra-
molecular CuꢂꢂꢂCu distance in the dinuclear unit is 2.6334(4) Å,
which is almost similar with 1 and also in normal range of tetra-
carboxylato copper(II) dimers [38–40]. The carboxylate bridging
angles of O(1)–C(7)–O(2) and O(4)–C(14)–O(3) show almost paral-
lel values at 126.46(18)° and 126.23(15)°, respectively. The bond
distances of Cu(1)–O(1), Cu(1)–O(2A), Cu(1)–O(4A), Cu(1)–O(3)
and Cu(1)–O(1W) are 1.9515(14), 1.9584(14), 1.9734(13),
1.9845(13) and 2.1177(14) Å, respectively, and the bond angles
of O(1)–Cu(1)–O(1W), O(2A)–Cu(1)–O(1W), O(4A)–Cu(1)–O(1W)
and O(3)–Cu(1)–O(1W) are 98.90(6)°, 92.85(7)°, 95.62(6)° and
served at 3095 cmꢁ1. The asym(COOꢁ) and sym(COOꢁ) absorption
m m
can be observed as strong bands at 1614 and 1411 cmꢁ1, respec-
tively [39,42,43].
The
D
[
m
asym(COOꢁ) ꢁ
m
sym(COOꢁ)] for 1 is 203 cmꢁ1 indicating
that the coordination of carboxylate groups are closer to bidentate
rather than to monodentate mode [39,42,43]. This result is in
agreement with the crystal structure of 1 which shows the carbox-
ylate groups forming bidentate bridging groups in the complex.
The strong NO2 absorption band is not much affected by the com-
plex formation, with ligand and complex showing the NO2 absorp-
tion at 1537 and 1536 cmꢁ1, respectively, indicating that the NO2
groups are not involved in the coordination to the metal. The IR
spectrum of 2 exhibits HꢂꢂꢂO stretching of coordinated water mole-
cules as a broad absorption band centered at 3419 cmꢁ1. The C@C–
H absorption bands can be observed at 3093 and 3065 cmꢁ1. The
strong antisymmetric and symmetric carboxylate stretching can
be observed as strong bands at 1617 and 1404 cmꢁ1, respectively.
The calculated
D
[
m
asym(COOꢁ) ꢁ
m
sym(COOꢁ)] for 2 is 213 cmꢁ1
95.96(6)°, respectively, with
a
tau value
s
ꢃ 0, indicating
a
indicating the coordination of the carboxylate groups are bidentate
in nature. This result concurs with the crystal structure of 2 which
shows the carboxylates group forming bidentate bridges in the
complex. For complex 3, the HꢂꢂꢂO stretching of coordinated water
molecules can be seen as a broad absorption band centered at
3416 cmꢁ1. The peaks at 3078 and 3035 cmꢁ1 can be assigned to
square-pyramidal coordination geometry. The hydrogen bondings
that exist in the crystal formation of [Cu(II)(NO2Bzo)(H2O)]2 and
[Cu(II)(ClFBzo)(H2O)]2 are shown in Tables 3 and 5. These hydro-
gen bonding sites could allow the complexes to interact with
DNA through hydrogen bonding. In addition, the benzene rings of
1
(interplanar distances = 3.758(2) Å) and
2
(interplanar dis-
the C@C–H stretching. The
m m
asym(COOꢁ) and sym(COOꢁ) stretching
tances = 3.7936(15) Å) could also allow the complexes to interact
can be observed as strong bands at 1599 and 1399 cmꢁ1, respec-

E.-K. Lim et al. / Polyhedron 28 (2009) 1320–1330
1325
Fig. 5. The crystal packing of 2.
tively. The calculated
D
[
m
asym(COOꢁ) ꢁ
m 3 is
sym(COOꢁ)] for
Table 3
200 cmꢁ1 indicating a bidentate mode of coordination for the car-
boxylate groups. This result is in agreements with crystal structure
of 3 which shows the carboxylate groups forming bidentate
bridges in the complex [41].
Hydrogen bonding distances and angles of [Cu(II)(ClNO2Bzo)(H2O)]2.
D–HꢂꢂꢂA
Distance (Å) Distance (Å) Distance (Å) Angle (°)
D–H
HꢂꢂꢂA
DꢂꢂꢂA
D–H–A
O(1W)ꢂꢂꢂH(11W)ꢂꢂꢂO(1) 0.88(5)
O(1W)ꢂꢂꢂH(21W)ꢂꢂꢂO(3) 0.69(5)
1.95(5)
2.28(5)
2.5077
2.822(3)
2.944(3)
3.231(4)
172(4)
163(5)
134.80
As this paper reports the nuclease property of the complexes in
the Tris–NaCl pH 7.5 buffer solution, the electronic spectral data of
the complexes was obtained in the Tris–NaCl pH 7.5 buffer solu-
tion. The UV–Vis spectrum of 1 displays a broad absorption band
at kmax = 698 nm which can be assigned to the d–d transition while
C(12)ꢂꢂꢂH(12A)ꢂꢂꢂO(4)
0.9297
D, donor atom; A, acceptor atom.
the bands at 263 and 218 nm in UV region can be assigned to the
Table 4
*
p–p
transitions. For complex 2, a broad d–d transition absorption
Selected bond lengths (Å) and bond angles (°) of [Cu(II)(ClFBzo)(H2O)]2.
*
band occurs at 736 nm while
p
–p
transitions occur at 263 and
Bond lengths (Å)
Cu(1)–O(1)
Cu(1)–O(2A)
Cu(1)–O(4A)
Cu(1)–O(3)
Cu(1)–O(1W)
Cu(1)–Cu(1)#1
Cl(1)–C(1)
212 nm. In complex 3, the d–d transition band can be observed
1.9515(14)
1.9584(14)
1.9734(13)
1.9845(13)
2.1177(14)
2.6334(4)
1.728(3)
*
at 732 nm while
212 nm.
p–p
transitions can be observed at 271 and
The cyclic voltammogram of 1–3 were recorded in the Tris–
NaCl (TN) pH 7.5 buffer solution whereby all the complexes show
redox activity. Complex 1 shows two cathodic reduction peaks at
ꢁ159 and ꢁ304 mV and two anodic oxidation peaks at ꢁ54 mV
and 100 mV which correspond to the CuIICuII to CuIICuI and CuIICuI
Cl(2)–C(8)
F(1)–C(5)
1.727(3)
1.350(3)
F(2)–C(12)
1.346(3)
Bond angles (°)
Table 5
O(1)–Cu(1)–O(2A)
O(1)–Cu(1)–O(4A)
O(2A)–Cu(1)–O(4A)
O(1)–Cu(1)–O(3)
O(2A)–Cu(1)–O(3)
O(4A)–Cu(1)–O(3)
O(1)–Cu(1)–O(1W)
O(2A)–Cu(1)–O(1W)
O(4A)–Cu(1)–O(1W)
O(3)–Cu(1)–O(1W)
O(1)–C(7)–O(2A)
O(4A)–C(14)–O(3)
168.02(6)
90.69(7)
90.48(6)
87.64(6)
88.83(6)
168.42(6)
98.90(6)
92.85(7)
95.62(6)
95.96(6)
126.46(18)
126.23(15)
Hydrogen bonding distances and angles of [Cu(II)(ClFBzo)(H2O)]2.
D–HꢂꢂꢂA
Distance (Å) Distance (Å) Distance (Å) Angle (°)
D–H
HꢂꢂꢂA
DꢂꢂꢂA
D–H–A
O(1W)ꢂꢂꢂH(11W)ꢂꢂꢂO(3) 0.8496
1.9436
2.4619
2.1610
2.2032
2.3473
1.9213
2.5761
2.7802(19)
2.4619
167.99
134.40
151.15
141.83
144.57
169.27
130.97
O(2W)ꢂꢂꢂH(12W)ꢂꢂꢂF(1)
O(2W)ꢂꢂꢂH(12W)ꢂꢂꢂF(2)
O(1W)ꢂꢂꢂH(21W)ꢂꢂꢂF(3)
O(1W)ꢂꢂꢂH(21W)ꢂꢂꢂF(4)
0.8507
0.8507
0.8495
0.8495
2.935(2)
2.919(3)
3.079(2)
2.761(2)
2.5761
O(2W)ꢂꢂꢂH(22W)ꢂꢂꢂO(5) 0.8501
C(11)ꢂꢂꢂH(11A)ꢂꢂꢂO(6)
0.9304
D, donor atom; A, acceptor atom.
#1: ꢁx + 1, ꢁy, ꢁz + 1.

1326
E.-K. Lim et al. / Polyhedron 28 (2009) 1320–1330
Fig. 6. Cleavage of supercoiled pBR322 by complex 1 at different concentrations in TN buffer pH 7.5. L1 Gene Ruler 1 kb DNA ladder, L2 DNA alone, L3 DNA + H2O2 alone, L4
DNA + 40 M complex 1 alone (without H2O2). Lanes 5–12, DNA with increasing concentration of complex 1 + H2O2: lane 5, 5 M; lane 6, 10 M; lane 7, 15 M; lane 8,
20 M; lane 9, 25 M; lane 10, 30 M; lane 11, 35 M; lane 12, 40 M.
l
l
l
l
l
l
l
l
l
to CuICuI redox process [44–46]. Similar to complex 1, complex 2
also exhibits two cathodic reduction peaks and two anodic oxida-
tion peaks at ꢁ92, ꢁ291, ꢁ42 and 115 mV, while for complex 3, the
two cathodic reduction peaks and two anodic oxidation peaks can
be observed at ꢁ77, ꢁ274, ꢁ26 and 120 mV. All complexes display
a quasi-reversible redox behaviour, as judged from the anodic to
1 is capable of inducing double strand scission. At 40 lM (lane 12,
Fig. 6), the supercoil was completely cleaved. The requirement of
H2O2 for DNA cleavage indicates an oxidative cleavage mechanism.
Similar oxidative cleavage mechanisms have been reported by var-
ious Cu(II) complexes with different ligands [20,29,30,47–49]. In
contrast, some Cu(II) complexes require a coreductant, such as
ascorbate and mercaptoethanol to trigger oxidative DNA cleavage
[15,50–53].
Fig. 7 shows the result of the plasmid pBR322 DNA cleavage by
an increasing concentration of 2 in the presence of H2O2. Once
again, no DNA cleavage is observed in the control lanes (lanes 3
and 4, Fig. 7), wherein pBR322 DNA was incubated with H2O2 alone
or 2 alone. DNA cleavage by 2 occurs in the presence of H2O2 and
cathodic peak separation value,
process CuIICuII to CuIICuI are 259, 207 and 197 mV, respectively,
and
Ep of 1–3 for process CuIICuI to CuICuI are 250, 249 and
248 mV, respectively ( Ep 6 59 mV for reversible process). The
D
Ep (=Epa ꢁ Epc);
DEp of 1–3 for
D
D
E1/2 redox potential, taken as the average of Epa and Epc, of 1–3
for process CuIICuII to CuIICuI are ꢁ130, ꢁ104 and ꢁ99 mV, respec-
tively, while E1/2 of 1–3 for process CuIICuI to CuICuI are ꢁ179,
ꢁ167 and ꢁ150 mV, respectively. The E1/2 values of 1 are at a more
negative potential compared to 2 and 3 indicating that 1 has a
more stable Cu(II) state compared to 2 and 3.
cleavage can be seen at complex concentration as low as 5
(lane 5, Fig. 7). On increasing the complex concentration from 10
to 40 M (lanes 6–12, Fig. 7), the nicked and linear bands build
up and the supercoil band diminishes, indicating single and double
strand scission. At 40 M (lane 12, Fig. 7), the supercoiled form was
lM
l
3.2. DNA cleavage studies
l
completely cleaved. The necessity of H2O2 for DNA cleavage indi-
cates oxidative cleavage. Complex 3 shows a similar DNA cleavage
pattern observed in 1 and 2, with total cleavage occurring at
Fig. 6 shows the result of the plasmid pBR322 DNA cleavage
reaction by using an increasing concentration of 1 in the presence
of H2O2. Control experiments in lanes 3 and 4 show that neither
40
l
M.
with H2O2 alone nor with complex alone (40 lM) can result in
From the DNA cleavage results of 1–3, it is obvious that 1–3 are
any DNA cleavage. However, DNA cleavage can be induced by
increasing complex concentration in the presence of H2O2, as
shown in lanes 5–12, Fig. 6. In general, if scission occurs on one
strand of the DNA, the supercoil (Form I) will relax to generate a
slow moving open circular or nicked form (Form II) while if scis-
sion occurs on both strands, a linear form (Form III) that migrates
in between Form I and Form II will be generated. At complex con-
effective DNA cleavers comparable to other Cu(II) compounds al-
ready described in the literature [20,29,30,47–49]. All complexes
exhibit similar DNA cleavage efficiency which means different
functional groups attached to the benzene ring in 1 (Cl and NO2),
2 (Cl and F) and 3 (Cl only) do not influence the DNA cleavage effi-
ciency. In addition the DNA cleavage experiment of CuCl2 in the
presence of H2O2 was carried out and the results show that no
centration as low as 5
lM (lane 5, Fig. 6), clear cleavage can be ob-
DNA cleavage was observed up to 40 lM. This shows that chelation
served wherein a small amount of supercoil was converted to the
nicked form. Further increases in the complex concentration from
10 to 40 lM (lanes 6–12, Fig. 6), builds up both the more intense
band of nicked form and the less intense linear band and dimin-
ishes the supercoil band. Formation of the linear band implies that
of the benzoate derivative ligands to Cu(II) can lead to the appear-
ance of DNA cleavage activity. Interestingly, research papers on
H2O2 oxidative DNA cleavage system of water soluble tetracarb-
oxylatocopper(II) dimers with benzoate derivative ligands appear
to be scarce.

E.-K. Lim et al. / Polyhedron 28 (2009) 1320–1330
1327
Fig. 7. Cleavage of supercoiled pBR322 by complex 2 at different concentrations in TN buffer pH 7.5. L1 Gene Ruler 1 kb DNA ladder, L2 DNA alone, L3 DNA + H2O2 alone, L4
DNA + 40 M complex 2 alone (without H2O2). Lanes 5–12, DNA with increasing concentration of complex 2 + H2O2: lane 5, 5 M; lane 6, 10 M; lane 7, 15 M; lane 8,
20 M; lane 9, 25 M; lane 10, 30 M; lane 11, 35 M; lane 12, 40 M.
l
l
l
l
l
l
l
l
l
A mechanistic study on the DNA cleavage by 1 and 2 with var-
is involved in the mechanism. Several Cu(II) complexes such as
ious inhibiting agents such as t-butanol, mannitol, DMSO and NaN3
has been carried out and the results are depicted in Figs. 8 and 9.
The results reveal that hydroxyl radical inhibitors such as t-butanol
and DMSO (lanes 4 and 6, Figs. 8 and 9) are able to quench the
cleavage induced by both complexes to some degree. This suggests
that the hydroxyl radical is the reactive oxygen species (ROS) that
bis(N,N0-dimethylglycinato)copper(II), bis[N-(N0-methylacetami-
do)-L-alaninato)copper(II) and Cu(II) chelated with 1,10-phenantr-
oline-derived ligands have shown the same mechanism in the
presence of H2O2 [19,29,30]. On the other hand, the cleavage reac-
tion by both complexes are not inhibited by the singlet oxygen
inhibitor NaN3 (lanes 7–12, Figs. 8 and 9) but instead shows an
Fig. 8. Effect of various scavengers on the cleavage of pBR322 by 40
1 + H2O2. Lanes 4–12 involve reaction of 40 M complex 1 + H2O2 with DNA in presence of various scavengers; lane 4, t-butyl alcohol (1 M); lane 5, 1 mM mannitol; lane 6,
DMSO (1 M); lane 7, 1 mM NaN3; lane 8, 10 mM NaN3; lane 9, 20 mM NaN3; lane 10, 30 mM NaN3; lane 11, 40 mM NaN3; lane 12, 50 mM NaN3.
lM complex 1. Lane 1, Gene Ruler 1 kb DNA ladder; lane 2, DNA alone; lane 3, DNA + 40 lM complex
l

1328
E.-K. Lim et al. / Polyhedron 28 (2009) 1320–1330
Fig. 9. Effect of various scavengers on the cleavage of pBR322 by 40
lM complex 2. Lane 1, Gene Ruler 1 kb DNA ladder; lane 2, DNA alone; lane 3, DNA + 40 lM complex
2 + H2O2. Lanes 4–12 involve reaction of 40 M complex 2 + H2O2 with DNA in presence of various scavengers; lane 4, t-butyl alcohol (1 M); lane 5, 1 mM mannitol; lane 6,
l
DMSO (1 M); lane 7, 1 mM NaN3; lane 8, 10 mM NaN3; lane 9, 20 mM NaN3; lane 10, 30 mM NaN3; lane 11, 40 mM NaN3; lane 12, 50 mM NaN3.
Table 6
enhancement. This clearly indicates that singlet oxygen is not the
Cytotoxic activity of [Cu(II)(ClNO2Bzo)(H2O)]2, [Cu(II)(ClFBzo)(H2O)]2 and [Cu(II)
(ClBzo)(H2O)]2 toward human cancer cell lines.
reactive oxygen species (ROS) that is responsible in the cleavage
reaction. Complex 3 shows similar results to that observed in 1
and 2.
Cancer cell lines
Cytotoxic activity (
lg/ml) of complexes
[Cu(II)(ClNO2Bzo)
(H2O)]2
[Cu(II)(ClFBzo)
(H2O)]2
[Cu(II)(ClBzo)
(H2O)]2
1. MCF-7
GI50 value
TGI value
LC50 value
174.00 10.51
>200
>200
157.63 10.72
>200
>200
150.72 7.64
194.8 6.97
>200
Table 8
Antibacterial activity of [Cu(II)(ClNO2Bzo)(H2O)]2, [Cu(II)(ClFBzo)(H2O)]2 and
[Cu(II)(ClBzo)(H2O)]2, results correspond to diameter of the inhibition zone (mm).
2. DU145
GI50 value
TGI value
LC50 value
Bacteria
Complexes
>200
>200
>200
165.5 31.02
>200
>200
159.5 12.35
>200
>200
[Cu(II)(ClNO2Bzo)
(H2O)]2
[Cu(II)(ClFBzo)
(H2O)]2
[Cu(II)(ClBzo)
(H2O)]2
3. Hela
Gram ꢁve bacteria
GI50 value
TGI value
LC50 value
143.47 8.46
190.77 9.84
>200
141.20 2.29
>200
>200
142.53 2.48
>200
>200
Enterobacter aerogenes (ꢁ)
500
250
125
lg/ml
lg/ml
lg/ml
10
7
12
9
10
8
R
8
R
4. HepG2
GI50 value
TGI value
LC50 value
68.36 11.32
122.85 14.96
194.22 3.67
81.18 9.60
144.80 21.92
194.1 8.35
63.50 9.80
126.26 21.75
192.32 10.85
Klebsiella pneumoniae (ꢁ)
500 g/ml
l
R
R
R
R
R
R
R
R
R
R
R
Proteus mirabilis (ꢁ)
500 g/ml
GI50, growth inhibition activity; TGI, cytostatic activity; LC50, cytotoxic activity.
l
R
Pseudomonas aeruginosa (ꢁ)
500
Shigella sonnei (ꢁ)
500 g/ml
Escherichia coli
500 g/ml
l
g/ml
R
R
R
l
Table 7
Cytotoxic activity of [Cu(II)(ClNO2Bzo)(H2O)]2, [Cu(II)(ClFBzo)(H2O)]2 and [Cu(II)(ClB-
zo)(H2O)]2 toward Chang human normal liver cell line.
l
Gram +ve bacteria
Normal cell line
Cytotoxic activity (
l
g/ml) of complexes
Bacillus cereus (+)
[Cu(II)(ClNO2Bzo)
(H2O)]2
[Cu(II)(ClFBzo)
(H2O)]2
[Cu(II)(ClBzo)
(H2O)]2
500
Bacillus subtilis (+)
500 g/ml
l
g/ml
R
R
R
R
R
R
R
R
Chang
l
GI50 value
TGI value
LC50 value
178.89 3.44
>200
>200
>200
>200
>200
185.88 16.21
>200
>200
Staphylococus aureus (+)
500 g/ml
l
R
R, resistant; well diameter = 5 mm.
GI50, growth inhibition activity; TGI, cytostatic activity; LC50, cytotoxic activity.

E.-K. Lim et al. / Polyhedron 28 (2009) 1320–1330
1329
Table 9
Antibacterial activity of [Cu(II)(ClNO2Bzo)(H2O)]2, [Cu(II)(ClFBzo)(H2O)]2, [Cu(II)(ClBzo)(H2O)]2 and chloramphenicol, the results are expressed as MIC (minimal inhibitory
concentration).
Gram ꢁve bacteria
Enterobacter aerogenes (ꢁ) (
[Cu(II)(ClNO2Bzo)(H2O)]2
250
[Cu(II)(ClFBzo)(H2O)]2
125
[Cu(II)(ClBzo)(H2O)]2
250
Chloramphenicol
3.0
lg/ml)
3.3. Cytotoxic studies
4. Conclusion
The antiproliferative activities of 1–3 on human cell lines have
been evaluated by obtaining GI50, TGI, and LC50 values as shown
in Table 4. MCF-7, DU 145, HeLa, HepG2 and Chang cells were ex-
posed to graded concentrations of 1–3 for 48 h. All the copper-con-
taining complexes exhibit 50% growth inhibition (GI50) in all
cancer cell types at concentration <200 lg/ml. The data reveals
an interesting finding in which 1–3 show the same selective trend
against the cancer cell lines whereby they exhibit higher cytotoxic
Three water soluble dicopper(II) complexes, tetrakis(
l
l
-4-
-2-
chloro-2-nitrobenzoato-O,O0)bis[aquacopper(II)] (1), tetrakis(
chloro-6-fluorobenzoato-O,O0)bis[aquacopper(II)] (2) and tetra-
kis(l
-2-chloro-benzoato-O,O0)bis[aquacopper(II)] (3), have been
synthesized. All the complexes have been tested for their nucleo-
lytic, antiproliferation and antibacterial activity. For nucleolytic
activity, all complexes can induce oxidative DNA cleavage in the
presence of H2O2. Complexes 1–3 show good DNA cleavage ability
comparable to other Cu(II) complexes described in the literature.
Different functional group attached at the benzene ring in 1 (Cl
and NO2), 2 (Cl and F) and 3 (Cl only) do not influence the DNA
cleavage efficiency. Hydroxyl radical is the reactive oxygen species
that responsible for the cleavage reactions. In antiproliferation
studies, all complexes show the same selective trend exhibiting
higher cytotoxic effect against HepG2 cancer cell lines compared
to MCF-7, DU 145 and HeLa cancer cell lines. It is interesting to
note that 1–3 show more than twofold cytotoxic effect against
HepG2 human liver cancer cells compared to Chang human normal
liver cells, which means 1–3 are more potent against cancer cells
than normal cells. The antibacterial activity of 1–3 exhibit a very
selective trend whereby they are active against Gram ꢁve bacteria
E. aerogenes while inactive against all other pathogenic bacteria.
However, 2 appears to be the most active against the Gram nega-
tive bacteria E. aerogenes compared to 1 and 3. The cleavage or
fragmentation of DNA by the ROS generated by the complexes
could be responsible for the antiproliferative activity exhibited
by the complexes.
effect against HepG2 cancer cell lines (GI50 = 63–82
lg/ml) com-
pared to MCF-7, DU 145, HeLa (GI50 = 140–180 g/ml) cancer cell
l
lines. Another interesting finding is that 1–3 show more than two-
fold selective cytotoxicity against HepG2 human liver cancer cells
compared to Chang human normal liver cells (GI50 = 178–
>200 lg/ml). This finding is in contrast with the compound
bis(phenanthroline)-4-methylcoumarin-6,7-dioxacetatocopper(II),
[Cu(4-Mecdoa)(phen)2] which shows selective cytotoxic effect
against Chang human normal liver cells compared to HepG2 hu-
man liver cancer cells [54]. Furthermore, 1–3 have almost similar
cytotoxic effect against Hela cells and 2–3 have slightly higher
cytotoxic effect against MCF-7 and DU 145 cells compared to 1.
In HepG2 cells, all GI50, TGI, and LC50 values of 1–3 can be obtained
within the testing concentration range.
3.4. Antibacterial studies
The antibacterial screening of 1–3 have been carried out
against nine pathogenic bacterial species using disc diffusion
method and the results are presented in Table 5. Referring to Table
5, it is noted that 1–3 exhibit very selective antibacterial activity,
being active against Gram negative E. aerogenes while inactive
against other tested pathogenic bacteria. This finding is interesting
because usually copper complexes which exhibit antibacterial
activity against Gram E. aerogenes will also exhibit antibacterial
activity against other bacteria. For example heterotrinuclear thio-
cyanato bridged Cu(II)–Hg(II)–Cu(II) complexes and valine-de-
rived Schiff base copper(II) complexes not only show
antibacterial activity against E. aerogenes but are also active
against Staphylococcus aureus, Escherichia coli, P. aeruginosa and
Proteus vulgaris [55,56]. This shows that the antibacterial activity
of 1–3 is very selective within the testing concentration range.
For comparison, 2 appears to be the most active complex against
Gram negative bacteria E. aerogenes compared to 1 and 3, as 2 has
the widest inhibition zone and can exhibit antibacterial activity at
the lowest concentration. Antibacterial tests of CuCl2 against the
nine pathogenic bacterial species have also been carried out. The
results show that the CuCl2 is inactive against all the tested path-
ogenic bacterial. From these results it may be inferred that the
benzoate derivatives chelated to Cu(II) can induce the antibacte-
rial activity against Gram negative bacteria E. aerogenes. The MIC
experiments of 1–3 against the Gram negative bacteria E. aeroge-
nes were further carried out and the results are presented in Table
Acknowledgements
We like to thank the RU Grant from Universiti Sains, Malaysia,
and FRGS Grant from Ministry of Higher Education (MOHE), Malay-
sia, for funding this research.
Appendix A. Supplementary data
CCDC 671169 and 671170 contains the supplementary crystal-
lographic data for complexes 1 and 2. These data can be obtained
free of charge via http://www.ccdc.cam.ac.uk/conts/retriev-
ing.html, or from the Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033;
or e-mail: deposit@ccdc.cam.ac.uk. Supplementary data associated
with this article can be found, in the online version, at doi:10.1016/
j.poly.2009.02.006.
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