Structure-activity relationship study of copper(II) complexes with 2-oxo-1,2-dihydroquinoline-3-carbaldehyde (4'-methylbenzoyl) hydrazone: Synthesis, structures, DNA and protein interaction studies, antioxidative and cytotoxic activity

Article abstract of DOI:10.1007/s00775-011-0844-1

Novel 2-oxo-1,2-dihydroquinoline-3-carbaldehyde (4'-methylbenzoyl) hydrazone (H₂L) (1) and its two copper(II) complexes have been synthesized. Single-crystal X-ray diffraction studies revealed that the structure of the new copper(II) chloride complex, [Cu(H₂L)Cl ₂] ·2H_2O (2), is square pyramidal and that of the copper(II) nitrate complex, [Cu(HL)NO₃]· DMF (3), is square planar. In 2, the copper atom is coordinated by the ligand with ONO donor atoms, one chloride ion in the apical position, and the other chloride in the basal plane. In 3, the ligand coordinates as a uninegative tridentate ONO^- species and with one nitrate ion in the basal plane. DNA binding experiments indicated that the ligand and copper(II) complexes can interact with DNA through intercalation. Bovine serum albumin binding studies revealed that the compounds strongly quench the intrinsic fluorescence of bovine serum albumin through a static quenching process. Antioxidative activity tests showed that 1 and its copper(II) complexes have significant radical scavenging activity against free radicals. Cytotoxic activities of the ligand and copper(II) complexes showed that the two copper(II) complexes exhibited more effective cytotoxic activity against HeLa and HEp-2 cells than the corresponding ligand. The entire biological activity results showed that the activity order was 1 < 2 < 3. SBIC 2011.

Full text of DOI:10.1007/s00775-011-0844-1

J Biol Inorg Chem (2012) 17:223–237
DOI 10.1007/s00775-011-0844-1
ORIGINAL PAPER
Structure–activity relationship study of copper(II)
complexes with 2-oxo-1,2-dihydroquinoline-3-carbaldehyde
(40-methylbenzoyl) hydrazone: synthesis, structures, DNA
and protein interaction studies, antioxidative and cytotoxic
activity
•
Duraisamy Senthil Raja Nattamai S. P. Bhuvanesh
•
Karuppannan Natarajan
Received: 28 April 2011 / Accepted: 3 September 2011 / Published online: 20 September 2011
Ó SBIC 2011
Abstract Novel 2-oxo-1,2-dihydroquinoline-3-carbalde-
hyde (40-methylbenzoyl) hydrazone (H2L) (1) and its two
copper(II) complexes have been synthesized. Single-crystal
X-ray diffraction studies revealed that the structure of the
new copper(II) chloride complex, [Cu(H2L)Cl2]ꢀ2H2O (2),
is square pyramidal and that of the copper(II) nitrate
complex, [Cu(HL)NO3]ꢀDMF (3), is square planar. In 2,
the copper atom is coordinated by the ligand with ONO
donor atoms, one chloride ion in the apical position, and
the other chloride in the basal plane. In 3, the ligand
coordinates as a uninegative tridentate ONO- species and
with one nitrate ion in the basal plane. DNA binding
experiments indicated that the ligand and copper(II) com-
plexes can interact with DNA through intercalation. Bovine
serum albumin binding studies revealed that the com-
pounds strongly quench the intrinsic fluorescence of bovine
serum albumin through a static quenching process. Anti-
oxidative activity tests showed that 1 and its copper(II)
complexes have significant radical scavenging activity
against free radicals. Cytotoxic activities of the ligand and
copper(II) complexes showed that the two copper(II)
complexes exhibited more effective cytotoxic activity
against HeLa and HEp-2 cells than the corresponding
ligand. The entire biological activity results showed that
the activity order was 1 \ 2 \ 3.
Keywords Copper(II) complexes ꢀ DNA binding ꢀ
Protein binding ꢀ Antioxidant ꢀ Cytotoxicity
Introduction
Metal complexes exert their anticancer effects through
binding to DNA, thereby changing the replication of DNA
and inhibiting the growth of the tumor cells, which is the
basis for designing new and more efficient antitumor drugs
[1, 2]. So, the design of small complexes that bind and
react at specific sequences of DNA has become quite
essential, and understanding the DNA binding properties of
transition metal complexes is important because of their
potential uses, such as drugs, regulators of gene expression,
and tools for molecular biology [3–6]. Since the discovery
of cisplatin, the synthesis of platinum complexes and their
applications as antineoplastic agents have gained a lot of
attention, whereas the inspiring clinical efficacy of cis-
platin and related platinum-based drugs, as anticancer
agents that bind covalently to DNA, is restricted by sig-
nificant side effects such as nephrotoxicity, emetogenesis,
neurotoxicity, and the emergence of drug resistance [7–10].
As a result, it is essential to design metal complexes of that
are less toxic, target-specific, and preferably with nonco-
valent binding to DNA. To develop new anticancer drugs
which specifically target DNA, it is necessary to under-
stand the different noncovalent binding modes of metal
complexes with DNA. Noncovalent binding modes are
mainly classified as intercalation, groove binding, and
electrostatic binding. Intercalation is one of the most
important DNA binding modes because it invariably leads
to cellular degradation. The planarity, coordination geom-
etry, and type of donor atom present in ligands play key
roles in determining the intercalating ability of complexes
D. Senthil Raja ꢀ K. Natarajan (&)
Department of Chemistry,
Bharathiar University,
Coimbatore 641046, Tamil Nadu, India
e-mail: k_natraj6@yahoo.com
N. S. P. Bhuvanesh
Department of Chemistry,
Texas A&M University,
College Station, TX 77842, USA
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J Biol Inorg Chem (2012) 17:223–237
with DNA [11–14]. The metal ion type and its valence,
which are responsible for the geometry of the complexes,
also influence the intercalating ability of metal complexes
[15, 16]. In this regard, copper(II) complexes having
square-planar and square-pyramidal geometry showed a
remarkable intercalative binding affinity for DNA [17–20].
Moreover, the selective permeability of cancer cell mem-
branes to copper complexes and their compact regulations
of the intracellular concentration have encouraged the
synthesis of copper-based drugs as potential anticancer
agents that are projected to have less severe side effects
[21]. On the other hand, many drugs, including anticoag-
ulants, tranquilizers, anti-inflammatory drugs, and general
anesthetics, are transported in the blood by combining with
albumin [22]. Therefore, it is also worthwhile to study the
interaction behaviors of the complexes with bovine serum
albumin (BSA) before we move on to studying their radical
scavenging and anticancer properties.
III Elementar analyzer. IR spectra (4,000–400 cm-1
)
obtained with KBr disks were recorded with a Nicolet
1
Avatar Fourier transform IR spectrophotometer. H NMR
spectra were recorded with Bruker AMX 500 instrument at
500 MHz using tetramethylsilane as an internal standard.
Melting points were determined with a Lab India instru-
ment. Electronic absorption spectra were recorded using a
JASCO V-630 spectrophotometer. Emission spectra were
measured with a JASCO FP 6600 spectrofluorometer. Mass
spectra were obtained with a VG ZAB-HS fast-atom
bombardment (FAB) instrument. EPR spectra were recor-
ded with a Bruker spectrometer operating at the X-band
and using 100-kHz magnetic field modulation.
Solid-state magnetic susceptibility measurements were
conducted at room temperature using a Faraday balance
calibrated using mercury(II) tetrathiocyanatocobalt(II) as a
calibrant. Molar conductivity was measured with a Sys-
tronic conductivity bridge with a dip-type cell, using a
5 9 10-3 M solution of complexes in 10% aqueous
dimethyl sulfoxide (DMSO).
Quinolines are a broadly distributed class of compounds
in nature with potentially advantageous effects in the field
of medicine. Some derivatives of quinoline and 2-oxo-
quinoline have been shown to have biological activities
such as antioxidation, antiproliferation, and anti-inflam-
mation [23–27]. Furthermore, a great many hydrazones and
their copper(II) complexes have also provoked immense
interest in their diverse spectra of biological and pharma-
ceutical activities, such as anticancer, antitumor, and anti-
oxidative activities [28–31]. In this regard, we have
recently reported copper(II) complexes derived from
2-oxo-1,2-dihydroquinoline-3-carbaldehyde N-substituted
thiosemicarbazones and their structure–activity relation-
ship for biological properties such as protein binding and
antioxidative and cytotoxic activity [32]. However, the
structural and biological behaviors of hydrazone transition
metal complexes derived from 2-oxo-1,2-dihydroquino-
line-3-carbaldehyde have not been explored well. This
aroused our interest in the synthesis of a new ligand, 2-oxo-
1,2-dihydroquinoline-3-carbaldehyde (40-methylbenzoyl)
hydrazone (H2L) (1), and its copper(II) complexes with a
view towards evaluating the interaction behaviors of these
compounds with DNA and BSA, and to explore their
antioxidative and cytotoxic abilities.
Synthesis of 1
4-Methylbenzohydrazide (1.50 g, 0.01 mol) dissolved in
warm methanol (50 mL) was added to a methanol solution
(50 mL) containing 2-oxo-1,2-dihydroquinoline-3-carbal-
dehyde (1.73 g, 0.01 mol). The mixture was refluxed for
1 h, during which a light-yellow precipitate was formed.
The reaction mixture was then cooled to room temperature
and the solid compound (1) was filtered off. It was then
washed with methanol and dried under a vacuum. Yield
91%. Melting point: 315–317 °C. Elemental analysis:
found (calculated) (%) for C18H15N3O2: C, 70.73 (70.81);
H, 4.99 (4.95); N, 13.72 (13.76). FAB mass spectrometry
(MS): m/z = 306 (M ? H). UV: kmax (nm): 327, 377. IR:
1
mmax (cm-1): mC=O: 1,657, mC=N: 1,558, mN–H: 3,280. H-
NMR (DMSO-d6 500 MHz, s, singlet; d, doublet; t, triplet;
m, multiplet): d 11.96 (s, 1H, N(3)H); 11.87 (s, 1H,
N(2)H); 8.71 (s, 1H, C(1)H); 8.46 (s, 1H, C(6)H);
7.84–7.86 (d, 2H, C(7,10)H); 7.51–7.55 (t, 1H, C(9)H);
7.32–7.40 (m, 4H, C(13,14,16,17)H); 7.19–7.23 (t, 1H,
C(8)H); 2.38 (s, 3H, C(18)H).
Synthesis of [Cu(H2L)Cl2]ꢀ2H2O
Materials and methods
A warm dimethylformamide (DMF) solution (10 mL)
containing 1 (153 mg, 0.5 mmol) was added to a metha-
nolic solution (20 mL) of CuCl2ꢀ2H2O (85 mg, 0.5 mmol).
The resulting greenish solution was refluxed for 30 min.
Green single crystals of [Cu(H2L)Cl2]ꢀ2H2O (2) suitable
for X-ray studies were obtained on slow evaporation. They
were filtered off, washed with cold methanol, and dried
under vacuum. Yield: 85%. Melting point: 328–331 °C.
All chemicals were reagent grade and used without puri-
fication. 2-Oxo-1,2-dihydroquinoline-3-carbaldehyde was
prepared according to the literature procedure [33]. Doubly
distilled water was used to prepare buffers. Ethidium bro-
mide (EB), BSA, and calf-thymus DNA (CT-DNA) were
purchased from Sigma-Aldrich and used as received. Ele-
mental analyses (C, H, N) were performed with a Vario EL
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225
Crystallography
Elemental analysis: found (calculated) (%) for
C18H19Cl2CuN3O4: C, 45.18 (45.44); H, 4.11 (4.02); N,
8.71 (8.83). UV: kmax (nm): 350, 380. IR: mmax (cm-1):
Single-crystal X-ray diffraction data of 2 and 3 were col-
lected with a Bruker three-circle platform diffractometer
equipped with a SMART 1000 CCD detector. Integrated
intensity information for each reflection was obtained by
reduction of the data frames with the program APEX2 [34].
All non-hydrogen atoms were refined with anisotropic
thermal parameters. The hydrogen atoms bound to carbon
were placed in idealized positions and refined using a
riding model. Hydrogen atoms attached to nitrogen and
oxygen were located from the Fourier difference maps and
were set riding on the respective parent atom. The struc-
tures were refined (weighted least-squares refinement on
F2) to convergence [35]. Relevant data concerning data
collection and details of structure refinement are summa-
rized in Table 1. Crystallographic data (without structure
factors) for the structures reported in this article have been
deposited with the Cambridge Crystallographic Data
Centre (CCDC) as supplementary publication numbers
m
C=O: 1,653, mC=N: 1,547. leff (27 °C): 1.71 lB. EPR (at
room temperature): g = 2.16 (solid), 2.14 (10% aqueous
DMSO). Molar conductivity: KM (S mol-1 cm2): 0.41.
Synthesis of [Cu(HL)NO3]ꢀDMF
It was prepared by the same procedure as described for 2
using H2L (153 mg (0.5 mmol) and Cu(NO3)2ꢀ3H2O
(121 mg, 0.5 mmol). Green single crystals of [Cu(HL)-
NO3]ꢀDMF (3) were obtained. Yield 87%. Melting point:
325–327 °C. Elemental analysis: found (calculated) (%)
for C21H21CuN5O6: C, 50.07 (50.15); H, 4.22 (4.21); N,
13.87 (13.92). UV: kmax (nm): 263, 368, 403, 423. IR: mmax
(cm-1): mC=O: 1,638, mC=N: 1,549. leff (27 °C): 1.73 lB.
EPR (at room temperature): g = 2.13 (solid), 2.17 (10%
aqueous DMSO). Molar conductivity: KM (S mol-1 cm2):
0.38.
Table 1 Experimental data for crystallographic analyses
2
3
Empirical formula
Formula weight
Temperature (K)
C18H19Cl2CuN3O4
475.80
C21H21CuN5O6
502.97
110(2)
110(2)
˚
Wavelength (A)
0.71073
0.71073
Triclinic
P-1
Crystal system
Space group
Monoclinic
P2(1)/c
Cell dimensions
˚
a (A)
7.643(6)
7.672(3)
10.865(4)
12.670(5)
80.502(6)
83.745(6)
84.813(6)
1,032.6(7)
2
˚
b (A)
9.759(8)
˚
c (A)
25.60(2)
a (°)
b (°)
c (°)
90
94.115(10)
90
3
˚
Volume (A )
1,905(3)
Z
4
Density (Mg/m3)
1.659
1.618
F(000)
Crystal size (mm3)
972
518
0.50 9 0.50 9 0.08
0.71 9 0.27 9 0.17
Index ranges
-9 B h B 9, -11 B k B 12, -32 B l B 31
15,101
-9 B h B 9, -14 B k B 14, -16 B l B 16
11,779
Reflections collected
Independent reflections
Absorption correction
Maximum and minimum transmission
Refinement method
Data/restraints/parameters
Goodness of fit on F2
Final R indices [I [ 2r(I)]
R indices (all data)
4,292 [R(int) = 0.0892]
Semiempirical from equivalents
0.8923 and 0.5292
4,659 [R(int) = 0.0305]
Semiempirical from equivalents
0.8338 and 0.5064
Full-matrix least squares on F2
Full-matrix least squares on F2
4,292/0/253
4,659/0/300
1.069
1.047
R1 = 0.0516, wR2 = 0.1271
R1 = 0.0601, wR2 = 0.1311
R1 = 0.0265, wR2 = 0.0713
R1 = 0.0298, wR2 = 0.0730
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CCDC-783730 and CCDC-787359 for 2 and 3, respectively.
Copies of the data can be obtained free of charge from the
CCDC (12 Union Road, Cambridge CB2 1EZ, UK; Tel:
?44-1223-336408; Fax: ?44-1223-336003; e-mail: depos-
it@ccdc.cam.ac.uk; Web site: http://www.ccdc.cam.ac.uk).
degassing. Stock solution of BSA was prepared in 50 mM
phosphate buffer (pH 7.2) and stored in the dark at 4 °C for
further use. Concentrated stock solution of compounds were
prepared by dissolving the compounds in DMSO–phosphate
buffer (1:50) and were diluted suitably with phosphate buffer
to required concentrations for all the experiments. Titrations
were done manually by using a micropipette for the addition
of compounds. If it is assumed that the binding of compounds
to BSA occurs at equilibrium, the equilibrium binding con-
stant can be analyzed according to the Scatchard equation:
DNA interaction experiments
All the experiments involving the binding of compounds 1, 2,
and 3 with CT-DNA were conducted in a doubly distilled
water buffer with 5 mM tris(hydroxymethyl)aminomethane
(Tris) and 50 mM sodium chloride and adjusted to pH 7.2
with hydrochloric acid. A solution of CT-DNA in the buffer
gave a UV absorbance ratio of about 1.9 at 260 and 280 nm,
indicating that the DNA was sufficiently free of protein. The
DNA concentration per nucleotide was determined by
absorption spectroscopy using the molar extinction coeffi-
cient value of 6,600 dm3 mol-1 cm-1 at 260 nm. The
compounds were dissolved in a mixed solvent of 5% DMSO
and 95% Tris–HCl buffer. Absorption titration experiments
were performed with fixed concentrations of the compounds
(25 lM) while gradually increasing the concentration of
DNA (2.5–25 lM). While measuring the absorption spectra,
we added an equal amount of DNA to both the compound
solution and the reference solution to eliminate the absor-
bance of DNA itself. The same experimental procedure was
also followed for emission studies. All the compounds were
excited at 375 nm and their corresponding emission spectra
was monitored in the range from 400 to 540 nm.
log ½ðI0 ꢂ IÞ=Iꢁ ¼ log Kbin þ n log½Qꢁ;
where Kbin is the binding constant of the compound with
BSA and n is the number of binding sites. The value of Kbin
can be determined from the slope of the plot of log[(I0 -
I)/I] versus log [Q]. For synchronous fluorescence spectra,
the same concentrations of BSA and compounds were used
and the spectra were measured at two different Dk (dif-
ference between the excitation and emission wavelengths
of BSA) values, such as 15 and 60 nm.
Antioxidant assays
The 2,20-diphenyl-1-picrylhydrazyl (DPPH) radical scav-
enging activity of the compounds was measured according
to the method of Blios [36]. The DPPH radical is a stable
free radical having kmax at 517 nm. A fixed concentration
of the experimental compound was added to solution of
DPPH in methanol (125 lM, 2 mL) and the final volume
was made up to 4 mL with doubly distilled water. The
solution was incubated at 37 °C for 30 min in the dark.
The decrease in absorbance of DPPH was measured at
517 nm.
EB-DNA experiments were conducted by adding the
solution of the compounds to the Tris–HCl buffer of EB-
DNA. The change of fluorescence intensity was recorded.
The excitation and the emission wavelengths were 515 and
602 nm, respectively. According to the classical Stern–
Volmer equation,
The hydroxyl (OH) radical scavenging activities of the
complexes have been investigated using the Nash method
[37]. In vitro hydroxyl radicals were generated by an Fe3?
–
I0=I ¼ Kq½Qꢁ þ 1;
ascorbic acid system. The detection of hydroxyl radicals
was performed by measuring the amount of formaldehyde
formed from the oxidation reaction with DMSO. The
formaldehyde produced was detected spectrophotometri-
cally at 412 nm. A mixture of 1.0 mL iron–EDTA solution
(0.13% ferrous ammonium sulfate and 0.26% EDTA),
0.5 mL EDTA solution (0.018%), and 1.0 mL DMSO
(0.85% v/v DMSO in 0.1 M phosphate buffer, pH 7.4)
were sequentially added to the test tubes. The reaction was
initiated by adding 0.5 mL ascorbic acid (0.22%) and the
mixture was incubated at 80–90 °C for 15 min in a water
bath. After incubation, the reaction was terminated by the
addition of 1.0 mL ice-cold trichloroacetic acid (17.5%
w/v). Subsequently, 3.0 mL Nash reagent was added to
each tube and the tubes were left at room temperature for
15 min. The intensity of the color formed was measured
spectrophotometrically at 412 nm against a reagent blank.
where I0 is the emission intensity in the absence of the
quencher, I is the emission intensity in the presence of
the quencher, Kq is the quenching constant, and [Q] is the
quencher concentration. The Kq value is obtained as a slope
from the plot of I0/I versus [Q].
Protein binding studies
An excitation wavelength of 280 nm was chosen since it
provides excitation of both tryptophan and tyrosine residues,
and the emission of both residues at 346 nm was monitored
for the protein binding studies. The excitation and emission
slit widths and scan rates were maintained constant for all the
experiments. Samples were carefully degassed using pure
nitrogen gas for 15 min. Quartz cells (4 cm 9 1 cm
9 1 cm) with high-vacuum Teflon stopcocks were used for
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J Biol Inorg Chem (2012) 17:223–237
227
Assay of nitric oxide (NO) scavenging activity is based
on the method in [38], where sodium nitroprusside in
aqueous solution at physiological pH spontaneously gen-
erates nitric oxide, which interacts with oxygen to produce
nitrite ions that can be estimated using Greiss reagent.
Scavengers of nitric oxide compete with oxygen, leading to
reduced production of nitrite ions. For the experiment,
sodium nitroprusside (10 mM) in phosphate-buffered sal-
ine was mixed with a fixed concentration of the compound
and the mixture was incubated at room temperature for
150 min. After the incubation period, 0.5 mL of Griess
reagent containing 1% sulfanilamide, 2% H3PO4, and 0.1%
N-(1-naphthyl) ethylenediamine dihydrochloride was
added. The absorbance of the chromophore formed was
measured at 546 nm.
The superoxide (O2-) radical scavenging assay was
based on the capacity of the compounds to inhibit formazan
formation by scavenging the superoxide radicals generated
in a riboflavin–light–nitroblue tetrazolium system [39].
Each 3 mL reaction mixture contained 50 mM sodium
phosphate buffer (pH 7.6), 20 lg riboflavin, 12 mM
EDTA, 0.1 mg nitroblue tetrazolium, and 1 mL complex
solution (20–100 lg/mL). The reaction was started by
illuminating the reaction mixture with different concen-
trations of the complex for 90 s. Immediately after illu-
mination, the absorbance was measured at 590 nm. The
entire reaction assembly was enclosed in a box lined with
aluminum foil. Identical tubes with reaction mixture kept
in the dark served as blanks.
bovine serum (FBS). NIH 3T3 fibroblasts were grown in
Dulbecco’s modified Eagle’s medium containing with 10%
FBS. For screening experiments, the cells were seeded into
96-well plates in 100 lL of the respective medium con-
taining 10% FBS, at a plating density of 10,000 cells per
well, and were incubated at 37 °C, 5% CO2, 95% air, and
100% relative humidity for 24 h prior to addition of the
compounds. The compounds were dissolved in DMSO and
diluted in the respective medium containing 1% FBS. After
24 h, the medium was replaced with the respective medium
with 1% FBS containing the compounds at various con-
centrations and the mixture was incubated at 37 °C, 5%
CO2, 95% air, and 100% relative humidity for 48 h.
Triplicate analyses were performed and the medium with-
out the compounds served as a control. After 48 h, 10 lL
MTT (5 mg/mL) in phosphate-buffered saline was added to
each well and the wells were incubated at 37 °C for 4 h.
The medium with MTT was then flicked off and the for-
mazan crystals that had formed were dissolved in 100 lL
DMSO. Then the absorbance at 570 nm was measured
using a microplate reader. The percentage of cell inhibition
was determined using the following formula: percentage of
inhibition = [mean optical density of untreated cells
(control)/mean optical density of treated cells (con-
trol)] 9 100. A graph of the percentage of cell inhibition
versus concentration was plotted and from this the IC50
value was calculated.
For the four assays described above, all the tests were
run in triplicate and various concentrations of the com-
pounds were used to fix a concentration at which the
compounds showed around 50% of activity. In addition, the
percentage of activity was calculated using the following
formula: percentage of activity = [(A0 - AC)/A0] 9 100
(A0 and AC are the absorbance in the absence and presence
of the compound tested, respectively). The 50% of activity
(IC50) can be calculated using the percentage of activity
results.
Results and discussion
Synthesis and characterization
The synthetic routes for the ligand (1) and its copper(II)
complexes are shown in Scheme 1. The ligand was prepared
by the condensation reaction of 2-oxo-1,2-dihydroquinoline-
3-carbaldehyde with 4-methylbenzohydrazide in methanol
medium. It was characterized by elemental analysis, IR
1
spectroscopy, H NMR spectroscopy, and FAB-MS. The
FAB mass spectra of the ligand showed a peak at m/z = 306
for (M ? H) and the measured molecular weight was con-
sistent with the expected value. The assignments for the IR
and 1H NMR spectra are given in ‘‘Materials and methods.’’
The copper(II) complexes were prepared by the direct
reaction of the ligand with copper(II) salts in
DMF and methanol medium.
In vitro anticancer activity evaluation by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assays
Cytotoxicity studies of the compounds and cisplatin were
conducted on human cervical cancer cells (HeLa), human
laryngeal epithelial carcinoma cells (HEp-2), and NIH 3T3
mouse embryonic fibroblasts, which were obtained from
the National Centre for Cell Science, Pune, India. Cell
viability was investigated using the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
method [40]. The HeLa and HEp-2 cells were grown in
Eagle’s minimum essential medium containing 10% fetal
The single crystals of new copper(II) complexes were
isolated by slow evaporation of the reaction mixture over a
period of 2–3 months. The IR peak shift of mC=O and mC=N
of the ligand in the complex give an idea of its coordination
to copper. The experimental leff values of 1.71 and 1.73 for
the two complexes confirmed the ?2 oxidation state of
copper in the complexes. All the compounds are soluble in
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J Biol Inorg Chem (2012) 17:223–237
Scheme 1 The synthetic route
for the ligand and its copper(II)
complexes. DMF
O
CHO
O
H
N
H2N
MeOH
N
+
N
H
dimethylformamide
N
O
H
N
O
H
(1)
MeOH
DMF
MeOH
DMF
CuCl2·2H2O
Cu(NO3)2·3H2O
H
N
Cl
N
N
N
O
Cu
O
N
O
·DMF
ONO2
Cu
N
O
H
·2H2O
H
Cl
(3)
(2)
methanol, ethanol, DMF, and DMSO and are stable in air.
X-band EPR spectra of the complexes at room tempera-
ture showed a single isotropic resonance with g values of
2.16 and 2.14 for 2 and 2.13 and 2.17 for 3 for the
powdered and 10% aqueous DMSO solution of the
complexes, respectively, indicating that the two copper(II)
complexes retained their solid-state structure in aqueous
solution. Moreover, the molar conductivities of copper(II)
complexes in 10% aqueous DMSO were measured to find
out the labile nature of the coordinated chloride and
nitrate in aqueous solution. The conductance values
measured (KM of 0.41 and 0.38 S mol-1 cm2 for 2 and 3,
respectively) were too low to account for any dissocia-
tion; hence the two copper(II) complexes were considered
to be nonelectrolytes and were quite stable in aqueous
solution. The structures of the new copper(II) complexes
were finally confirmed by single-crystal X-ray crystallo-
graphic studies.
X-ray structural characterization
The ORTEP view of the neutral complex [Cu(H2L)Cl2]
ꢀ2H2O (2) is given in Fig. 1. The ligand is coordinated as a
neutral moiety in an ONO fashion and the other two coor-
dination sites are filled by chloride ions completing a square-
pyramidal geometry around the copper ion in complex 2. The
˚
copper atom lies about 0.166 A above the average basal
plane towards the axial Cl2 atom in 2.
The pyramid is fairly regular and the axial Cu–Cl bond
length is longer than that of the basal one, which can be
ascribed to Jahn–Teller distortion. The dihedral angle
between the mean planes of the five-membered chelate ring
Fig. 1 ORTEP view of the molecular structure and atom-labeling scheme of 2. Thermal ellipsoids are drawn at the 50% probability level
123

J Biol Inorg Chem (2012) 17:223–237
229
˚
and the six-membered one is 4.49° for 2. Since the
hydrazone moieties have both hydrogen-bond donors and
hydrogen-bond acceptors, the species provide the possi-
bility of forming hydrogen bonds in the crystal. In fact, the
crystal lattice of the complex showed a three-dimensional
array in which each unit of the complex is hydrogen-bon-
ded to the other involving N2 and N3 nitrogen atoms, the
O30 and O40 oxygen atoms of water molecules, and the
Cl1 and Cl2 chlorine atoms.
with chloride. A very short distance (0.019 A) between the
copper atom and the average basal plane and a small
dihedral angle (3.72°) between the mean planes of the five-
membered chelation ring and the six-membered one
ensures that the planarity of the square should be appre-
ciable. The molecular packing suggests that the stabiliza-
tion of the lattice must have been due to several hydrogen
bonds, mainly involving the N3 and O34 atoms. Selected
bond distances and bond angles of 2 and 3 are given in
Table 2 and agree well with those found in related copper
complexes [41, 42].
An ORTEP view of [Cu(HL)NO3]ꢀDMF (3) is given in
Fig. 2. Complex 3 contains a tetracoordinated copper(II)
ion with one ONO tridentate uninegative ligand and one
nitrate ion coordinated through oxygen with one DMF
molecule in the lattice.
DNA binding activity
The N1–Cu–O2 bond angle [81.67(6)°] is significantly
smaller than that of other three [bond angles of N1–Cu–O1,
O2–Cu–O3, and O1–Cu–O3 are 92.47(6)°, 95.00(5)°, and
92.15(5)°, respectively], indicating a slightly distorted
square-planar geometry around the copper atom in the
complex. Unlike in 2, O2 is coordinated as a negative
donor (enolate and not keto form) in 3. This may be due to
the strong electron-withdrawing nature of nitrate compared
Electronic absorption spectroscopy is one of the most
useful techniques for DNA binding studies of small mol-
ecules. The absorption spectra of copper(II) complexes 2
and 3 and the ligand (1) in the absence and presence of
CT-DNA are given in Fig. 3. In the presence of CT-DNA,
the absorption bands of the ligand at 326 and 367 nm
exhibited hypochromism of about 24 and 25% respectively.
Square-pyramidal complex 2 exhibited hypochromism of
Fig. 2 ORTEP view of the molecular structure and atom-labeling scheme of 3. Thermal ellipsoids are drawn at the 50% probability level
123

230
J Biol Inorg Chem (2012) 17:223–237
˚
about 49% at 378 nm. On the other hand, the square-planar
complex (3) exhibited hypochromism of about 79, 81, and
80% at 368, 403, and 423 nm, respectively. To illustrate
quantitatively the consequence, the absorption data were
analyzed to evaluate the intrinsic binding constant (Kb),
which can be determined from the following equation:
Table 2 Selected bond lengths (A) and angles (°) for 2 and 3
2
3
Cu(1)–O(1)
1.927(3)
1.950(2)
1.967(3)
2.2232(16)
2.733(2)
1.9474(12)
1.9359(12)
1.9243(14)
Cu(1)–O(2)
Cu(1)–N(1)
Cu(1)–Cl(1)
½DNAꢁ=ðea ꢂ efÞ ¼ ½DNAꢁ=ðeb ꢂ efÞ þ 1=Kbðeb ꢂ efÞ;
Cu(1)–Cl(2)
where [DNA] is the concentration of DNA in base pairs,
and the apparent absorption coefficients ea, ef, and eb cor-
respond to Aobs/[compound], the extinction coefficient of
the free compound, and the extinction coefficient of the
compound when fully bound to DNA, respectively. From
the plot of [DNA]/(ea - ef) versus [DNA] (Fig. 3d), Kb is
calculated from the ratio of the slope to the intercept. The
Cu(1)–O(3)
1.9599(12)
1.2946(18)
1.326(2)
C(11)–O(2)
1.260(4)
1.337(4)
1.380(4)
169.93(10)
91.67(11)
80.89(11)
93.48(8)
92.49(8)
167.24(8)
94.10(9)
91.86(8)
83.93(8)
107.31(4)
C(11)–N(2)
N(1)–N(2)
1.3831(18)
170.01(5)
92.47(6)
O(1)–Cu(1)–O(2)
O(1)–Cu(1)–N(1)
O(2)–Cu(1)–N(1)
O(1)–Cu(1)–Cl(1)
O(2)–Cu(1)–Cl(1)
N(1)–Cu(1)–Cl(1)
O(1)–Cu(1)–Cl(2)
O(2)–Cu(1)–Cl(2)
N(1)–Cu(1)–Cl(2)
Cl(1)–Cu(1)–Cl(2)
N(1)–Cu(1)–O(3)
O(2)–Cu(1)–O(3)
O(1)–Cu(1)–O(3)
81.67(6)
calculated Kb values of 2.02( 0.07) 9 105 M-1
,
4.59( 0.17) 9 105 M-1, and 9.08( 0.27) 9 105 M-1 for
compounds 1, 2, and 3, respectively, suggested an intimate
association of the compounds with CT-DNA, and it is also
likely that these compounds bind to the helix via interca-
lation [43]. After the compounds have intercalated them-
selves among the base pairs of DNA, the p* orbital of the
intercalated compounds can couple with p orbitals of the
base pairs, thus decreasing the p ? p* transition energies.
Therefore, these interactions resulted in the observed
hypochromism [44]. Complexes 2 and 3 showed more
169.45(5)
95.00(5)
92.15(5)
Fig. 3 Absorption spectra of
compounds 1 (a), 2 (b), and 3
(c) (25 lM) in the presence of
increasing amounts of calf-
thymus DNA (CT-DNA) (0,
2.5, 5, 7.5, 10, 12.5, 15, 17.5,
20, 22.5, 25 lM). Arrows show
the absorbance changes upon
increasing DNA concentration.
d Plots of [DNA]/(ea - ef)
versus [DNA] for the titration of
compounds with CT-DNA
a
b
Ligand (1)
Complex 2
0.5
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0.4
0.3
0.2
0.1
0.0
300
350
400
450
300
350
400
450
Wavelength (nm)
Wavelength (nm)
c
d
Complex 3
0.30
0.25
0.20
0.15
0.10
0.05
0.00
18
15
12
9
1
2
3
6
3
250
300
350
400
450
500
0
5
10
15
20
25
[DNA] (μM)
Wavelength (nm)
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J Biol Inorg Chem (2012) 17:223–237
231
hypochromicity and higher Kb values than the ligand,
indicating that the binding strength of the copper(II)
complexes is much greater than that of the free ligand.
From the electronic absorption studies, although it was
found that the three compounds can bind to DNA by
intercalation, the binding mode need to be proved through
some more experiments. Hence, to start with we conducted
emission experiments to investigate the interactions
between the compounds studied and CT-DNA.
In the second investigation, emission quenching exper-
iments were conducted.
EB is one of the most sensitive fluorescent probes which
can bind to DNA through intercalation [46, 47]. Compet-
itive binding to DNA of drugs with EB could provide rich
information with regard to the DNA binding affinity. Fig-
ure 5 shows the emission spectra of the DNA–EB system
with increasing amounts of 1, 2, and 3.
The emission intensity of the DNA-EB system decreased,
apparently as the concentration of complexes 2 and 3 and free
ligand increased. This is because EB is expelled from the
hydrophobic environment into the water solution [48].
Quenching data were analyzed according to the Stern–Vol-
mer equation, which could be used to determine the fluo-
rescence quenching mechanism. The quenching plots
illustrate that the quenching of EB bound to CT-DNA by 2, 3,
and the free ligand (1) is in good agreement with the linear
Stern–Volmer equation, which also proves that they bind to
DNA. In the Stern–Volmer plots (Fig. 5d) of I0/I versus [Q],
the quenching constant (Kq) is given by the ratio of the slope
to the intercept. Further, the value of the binding constant
(Kapp) for the compounds can be obtained by using the fol-
lowing equation:
The enhancements in the emission intensity of the
compounds with increasing CT-DNA concentrations are
shown in Fig. 4. In the absence of DNA, compounds 1, 2,
and 3 weakly luminescence in Tris buffer when excited at
375 nm. The intensity of the emission for compounds 1,
2, and 3 increases with the increase of DNA concentra-
tion. Upon the addition of CT-DNA, the emission inten-
sities at 450 nm were increased by around 2 and 3 times
for complexes 2 and 3, respectively. But, the emission
intensity of the ligand (1) at 442 nm increased only
around 1.5 times. This phenomenon is related to the
extent to which the compound penetrates into the
hydrophobic environment inside the DNA, thereby
avoiding the quenching effect of solvent water molecules.
The binding of complexes 2 and 3 and free ligand to CT-
DNA leads to a marked increase in the emission intensity,
which also agrees with the increases observed for other
intercalators [45]. These results show that the complexes
bind more strongly than the free ligand. From the
observed intensity enhancement of the two complexes in
the presence of CT-DNA, it can be seen that 3 has more
DNA binding ability than 2. The higher binding affinity
of the copper(II) complexes is attributed to the extension
of the p system of the intercalated ligand due to the
coordination to the copper(II) ion. Since the complexes
have planar area greater than that of the free ligand,
leading the complexes to penetrate more deeply into and
stack more strongly with the base pairs of the DNA.
KEB½EBꢁ ¼ Kapp½compoundꢁ;
where the compound concentration is the value at a 50%
reduction of the fluorescence intensity of EB,
KEB = 1.0 9 107 M-1, and [EB] = 0.25 lM. The calcu-
lated Kq and Kapp values are given in Table 3.
These data suggest that the interaction of the copper(II)
complexes with CT-DNA is stronger than that of the free
ligand, which is consistent with the above absorption and
emission spectral observations. Since these changes indi-
cate only one kind of quenching process, it may be con-
cluded that 1, 2, and 3 bind to CT-DNA via the same mode.
Furthermore, such quenching constants and binding
constants of the ligand and copper(II) complexes suggest
a
b
c
Ligand (1)
Complex 3
180
Complex 2
175
150
125
100
75
160
140
120
100
80
120
100
80
60
40
20
0
60
50
40
25
20
0
0
400 420 440 460 480 500 520 540
400
425
450
475
500
525
400 420 440 460 480 500 520 540
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Fig. 4 Emission enhancement spectra of compounds 1 (a), 2 (b), and
3 (c) (25 lM) in the presence of increasing amounts of CT-DNA (0,
2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25 lM) (kex = 375 nm,
kem = 400–540 nm). Arrows show the emission intensity changes
upon increasing DNA concentration
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232
J Biol Inorg Chem (2012) 17:223–237
Fig. 5 Emission spectra of the
DNA–ethidium bromide (EB)
system (25 lM DNA and
0.25 lM EB), kex = 510 nm,
kem = 530–700 nm, in the
presence of 0, 1, 2, 3, 4, and
5 lM 1 (a), 2 (b), and 3 (c).
Arrows show the emission
intensity changes upon
a
b
Complex 2
Ligand (1)
750
750
600
450
300
150
0
600
450
300
150
0
increasing concentration of the
compounds. d Stern–Volmer
plots of the fluorescence
titration of 1, 2, and 3
550
600
650
700
550
600
650
700
Wavelength (nm)
Wavelength (nm)
c
750
d
Complex 3
1
2
3
3.0
2.5
2.0
1.5
1.0
600
450
300
150
0
0
1
2
3
4
5
550
600
650
700
Wavelength (nm)
[Q] (10-6 M)
loss or enhancement of the biological properties of the
original compounds, or may provide paths for their trans-
portation. BSA is the most extensively studied serum
albumin, owing to its structural homology with human
serum albumin. The interaction of BSA with our com-
pounds was studied by fluorescence measurements at room
temperature. A solution of BSA (1 lM) was titrated with
various concentrations of the complex (0–5 lM).
Table 3 Quenching constant (Kq) and binding constant (Kapp) for the
interactions of compounds with DNA
Compound
Kq (M-1
)
Kapp (M-1
)
1
2
3
8.93 ( 0.25) 9 104
1.73 ( 0.17) 9 105
3.97 ( 0.12) 9 105
2.23 ( 0.15) 9 105
4.33 ( 0.10) 9 105
9.93 ( 0.18) 9 105
Fluorescence spectra were recorded in the range from
290 to 450 nm upon excitation at 280 nm. The effect of the
compounds on the fluorescence spectrum of BSA is shown
in Fig. 6. Addition of the compounds to a solution of BSA
resulted in a significant decrease of the fluorescence
intensity of BSA at 346 nm, up to 45.7, 54.5, and 59.1% of
the initial fluorescence intensity of BSA, accompanied by a
small blueshift of 3, 5, and 6 nm for 1, 2, and 3, respec-
tively. The observed blueshift is mainly due to the fact that
the active site in the protein is buried in a hydrophobic
environment. This result suggested a definite interaction of
all three compounds with the BSA protein. The fluores-
cence quenching mechanisms are usually classified as
either static or dynamic quenching. A simple method to
explore the type of quenching is UV–vis absorption spec-
troscopy. UV–vis spectra of BSA in the absence and
presence of the compounds (Fig. 7) show that the absorp-
tion intensity of BSA was enhanced as the compounds
were added, and there was a small blueshift.
that the interaction of all the compounds with DNA should be
by intercalation [49]. On the basis of all the spectroscopic
studies, we come to the conclusion that the copper(II) com-
plexes and the free ligand can bind to CT-DNA in an inter-
calative mode and that the copper(II) complexes bind to
CT-DNA more strongly than the free ligand. Among the two
copper(II) complexes, the square-planar complex (3) has
greater binding ability than the square-pyramidal complex
(2) because the square-planar geometry allows it to more
easily intercalate itself among the base pairs of DNA in
comparison with the square-pyramidal geometry.
BSA binding activity
Serum albumin is the most abundant protein in plasma.
Therefore, it is important to consider the interactions of
bioactive compounds with plasma proteins, particularly
with serum albumin. Binding to these proteins may lead to
123

J Biol Inorg Chem (2012) 17:223–237
233
a
b
600
c
600
500
400
300
200
100
0
600
Ligand (1)
Complex 2
Complex 3
500
400
300
200
100
0
500
400
300
200
100
0
300
350
400
450
320
360
400
440
300
330
360
390
420
450
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Fig. 6 The emission spectrum of bovine serum albumin (BSA)
(1 lM; kex = 280 nm, kem = 346 nm) in the presence of increasing
amounts of compounds 1 (a), 2 (b), and 3 (c) (0, 1, 2, 3, 4, and 5 lM).
Arrows show the fluorescence quenching upon increasing the
concentration of the compounds
nonfluorescent ground-state complex induces a change in
the absorption spectrum of fluorophores, suggesting the
possible quenching mechanism of BSA by the compounds
is a static quenching process [50]. To elucidate the
quenching mechanism further, fluorescence quenching data
were analyzed with the Stern–Volmer equation and the
Scatchard equation. The quenching constant (Kq) can be
calculated using the plot of I0/I versus [Q] (Fig. 8a).
From the plot of log (I0 - I)/I versus log [Q] (Fig. 8b),
the number of binding sites (n) and the binding constant
(Kbin) were obtained. The calculated Kq, Kbin, and n values
are given in Table 4. The calculated value of n is around 1
for all the compounds, indicating the existence of just a
single binding site in BSA for all the compounds. The
values of Kq and Kbin for 1, 2, and 3 suggested that the
complexes interact with BSA more strongly than does the
ligand. In particular, the square-planar complex (3) has
better interaction with BSA than the square-pyramidal
complex (2). The binding constants are very comparable to
the binding constants of many biologically valuable drugs
found in the literature [51–53]. Moreover, the stronger
binding ability of the compounds with BSA clearly
0.6
BSA
BSA + 1
0.5
BSA + 2
BSA + 3
0.4
0.3
0.2
0.1
260
280
300
320
340
Wavelength (nm)
Fig. 7 Absorption spectra of BSA (10 lM), and with compounds 1,
2, and 3 (5 lM)
As is well known, dynamic quenching only affects the
excited state of fluorophores but does not change the
absorption spectrum. However, the formation of
a
Fig. 8 a Stern–Volmer plots of
the fluorescence titration of 1, 2,
and 3 with BSA. b Scatchard
plots of the fluorescence
titration of 1, 2, and 3 with BSA
a
2.5
b
0.2
1
2
3
0.0
2.0
1.5
1.0
0.5
0.0
-0.2
1
2
3
-0.4
-0.6
-0.8
0
1
2
3
4
5
-5.4
-5.6
log [Q]
-5.8
-6.0
[Q] (µM)
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234
J Biol Inorg Chem (2012) 17:223–237
conformation of the tryptophan microregion. It further
revealed that the hydrophobicity around tryptophan residues
is strengthened. The hydrophobicity observed in fluores-
cence and synchronous measurements confirmed the effec-
tive binding of the compounds with the BSA protein. Hence,
the strong interaction of these compounds with BSA protein
suggested that the compounds are potential candidates for
further biological studies such as antioxidative and anti-
cancer studies.
Table 4 Quenching constant (Kq), binding constant (Kbin), and
number of binding sites (n) for the interactions of compounds with
bovine serum albumin
Compound
Kq (M-1
)
Kbin (M-1
)
n
1
2
3
1.74 ( 0.12) 9 105
2.44 ( 0.09) 9 105
2.88 ( 0.15) 9 105
6.71 ( 0.25) 9 104
2.82 ( 0.11) 9 105
3.45 ( 0.08) 9 105
0.88
1.02
1.03
indicated that these compounds can be stored and released
by the protein.
Evaluation of radical scavenging ability
To further establish the point of interaction of BSA and
the compounds, synchronous fluorescence experiments were
conducted; these would provide information on the molec-
ular microenvironment, particularly near the fluorophore
functional groups [54]. The fluorescence of BSA is due to the
presence of tyrosine and tryptophan residues. Of them,
tryptophan is the most dominant fluorophore, located at the
substrate binding sites. Most of the compounds bind to the
protein in the active binding sites. Hence, the synchronous
method is usually applied to find out the conformational
changes around the tryptophan and tyrosine region. In syn-
chronous fluorescence spectroscopy, according to Miller
[55], the difference between the excitation wavelength and
the emission wavelength (Dk = kem - kex) indicates the
type of chromophores. A higher Dk value, such as 60 nm, is
indicative of the characteristic of a tryptophan residue,
whereas a lower Dk value, such as 15 nm, is characteristic of
a tyrosine residue [56]. The synchronous fluorescence
spectra of BSA with various concentrations of the com-
pounds were recorded at Dk = 15 nm (Fig. 9) and
Dk = 60 nm (Fig. 10). The fluorescence intensity of tyro-
sine is very slightly decreased, but the fluorescence intensity
of tryptophan is decreased drastically with increasing con-
centration of the compounds. This suggested that the inter-
action of the compounds with BSA protein affects the
Since the experiments reported so far revealed that the
ligand and its copper(II) complexes exhibit good DNA and
protein binding affinity, it was considered worthwhile to
study the antioxidant activity of these compounds. The
antioxidant properties of quinoline derivatives have
attracted a lot of interest and have been extensively
investigated, mainly in in vitro systems [57, 58]. The
radical scavenging activities of our compounds along with
the standards butylated hydroxyanisole and butylated
hydroxytoluene in a cell-free system were examined with
reference to hydroxyl radicals, DPPH radicals, nitric oxide,
superoxide anion radicals, and the determination of IC50
values. No significant radical scavenging activities were
observed in all the experiments performed with CuCl2 and
Cu(NO3)2, even up to 1.0 mM concentration under the
same experimental conditions. The IC50 values (Table 5)
indicated that the three compounds showed antioxidant
activity in the order 3 [ 2 [ 1 in all the experiments. The
superoxide anion radical scavenging power of the com-
pounds tested was the greatest and the nitric oxide scav-
enging power was the least.
These results are much better than the result observed
for standard antioxidants, butylated hydroxyanisole and
butylated hydroxytoluene, except in the DPPH radical
b
a
c
Complex 3
350
300
250
200
150
100
50
Ligand (1)
350
300
250
200
150
100
50
350
300
250
200
150
100
50
Complex 2
0
0
0
270
300
330
360
280
300
Wavelength (nm)
320
280
300
320
340
360
Wavelength (nm)
Wavelength (nm)
Fig. 9 Synchronous spectra of BSA (1 lM) in the presence of increasing amounts of compounds 1 (a), 2 (b), and 3 (c) (0, 1, 2, 3, 4, and 5 lM)
for a wavelength difference of Dk = 15 nm. Arrows show the emission intensity decrease upon increasing the concentration of the compounds
123

J Biol Inorg Chem (2012) 17:223–237
235
a
500
b
c
500
Ligand (1)
Complex 2
Complex 3
400
300
200
100
0
400
300
200
100
0
400
300
200
100
0
300
320
340
360
380
400
300
320
340
360
380
400
300
320
340
360
380
400
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Fig. 10 Synchronous spectra of BSA (1 lM) in the presence of increasing amounts of compounds 1 (a), 2 (b), and 3 (c) (0, 1, 2, 3, 4 and 5 lM),
for a wavelength difference of Dk = 60 nm. Arrows show the emission intensity decrease upon increasing the concentration of the compounds
Table 5 IC50 values (lM) calculated from various radical scavenging
assays of compounds 1, 2, and 3 and the standards butylated
hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)
Cytotoxic activity evaluation by MTT assay
The positive results obtained from DNA binding, BSA
binding, and antioxidation studies of 1, 2, and 3 encouraged
us to test their cytotoxicity against some cancer cells. The
results of cytotoxicity assays of the activity of the ligand
and the copper(II) complexes against the human cervical
cancer cell line (HeLa) and human laryngeal epithelial
carcinoma cells (HEp-2) are shown in Fig. 11. The bio-
logical assays of the ligand and the copper(II) complexes
show that the copper(II) complexes exhibit greater activi-
ties than the corresponding ligand against HeLa and HEp-2
cells. The IC50 value of 3 demonstrated a much higher
inhibitory effect than for 2 and 1 (Table 6).
ꢀ-
Compound OHꢀ
NO
DPPHꢀ
O2
1
128
6
181
5
161
4
91.3 4.5
2
17.2 1.5
68.6 4.3
33.1 2.2
8.16 1.24
3
9.18 0.93 27.4 2.3 18.92 1.40 3.12 0.53
BHA
BHT
312
278
6
8
621 11
726
9.79 0.92 297
9.87 0.75 278
8
5
8
DPPH 2,20-diphenyl-1-picrylhydrazyl
assay. From the results obtained for the two copper(II)
complexes, it can be inferred that the difference in the
planarity of the structures and counterions present in the
complexes is likely to induce variations in antioxidant
activities. The enhanced planarity of the complexes over
the free ligand is the reason for the enhanced antioxidant
activity of the two copper(II) complexes over the ligand.
Among the two complexes, the highest activity for 3 is due
to the greater planarity of the square-planar geometry
compared with the square-pyramidal geometry of 2.
The compounds are more active on HeLa cells than on
HEp-2 cells. The cytotoxic activity studies in vitro indicate
that the two copper(II) complexes have better activities
than the corresponding ligand but showed significantly less
activity than cisplatin. In addition, the IC50 values of all the
compounds for NIH 3T3 mouse embryonic fibroblasts
(normal cells) were above 300 lM, which confirmed that
the compounds act very specifically on cancer cells. But,
copper(II) chloride and copper(II) nitrate did not show any
Fig. 11 Cytotoxic activity of
compounds 1, 2, and 3 against
HeLa (a) and HEp-2 (b) cells
a
b
70
90
3
2
1
80
70
60
50
40
30
20
10
1
2
3
60
50
40
30
20
10
0
10
20
30
40
50
60
20
40
60
80
100 120 140 160
Concentration of compounds (10-6 M)
Concentration of the compounds (10-6 M)
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J Biol Inorg Chem (2012) 17:223–237
activity was observed and was well explained on the basis of
structural planarity. The findings are significant for us to
explore further the DNA and protein interaction and anti-
oxidative and cytotoxic activities of the transition metal
complexes containing different 2-oxo-1,2-dihydroquino-
line-3-carbaldehyde Schiff bases.
Table 6 IC50 values (lM) of compounds 1, 2, and 3 and cisplatin for
activity against HeLa and HEp-2 cancer cells
Compound
HeLa
HEp-2
1
127
5
145
105
4
3
2
28.3 1.6
19.3 1.2
3.73 0.17
3
95.4 2.1
Cisplatin
2.32 0.24
Acknowledgments Financial assistance received from the Council
of Scientific and Industrial Research, New Delhi, India [grants
01(2216)/08/EMR-II and 21(0745)/09/EMR-II], is gratefully
acknowledged. We would also like to thank G. Paramaguru and
R. Ranganathan (School of Chemistry, Bharathidasan University,
India) for their help in experimental work regarding EB–DNA
displacement.
significant activity on the cancer cells even up to 500 lM
concentration, which confirmed that the copper(II) chelation
with the ligand is the only factor responsible for the observed
cytotoxic properties of the new complexes. The better
cytotoxic activities of the two copper(II) complexes in
comparison with the ligand may be attributed to the extended
planar structure induced by the p ? p* conjugation result-
ing from the chelating of the metal ion with the ligand. In
addition, the inhibitory rate of 3 against HeLa and HEp-2
cancer cells is higher than that of 2 (Fig. 11), which may be
due to the square planarity and the presence of a nitrate
coligand in 3. These findings of cytotoxic activities in vitro
are further evidence to show that the complexes and the free
ligand bind to DNA, leading to cell death.
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