Evaluation of DNA binding, antioxidant and cytotoxic activity of mononuclear Co(III) complexes of 2-oxo-1,2-dihydrobenzo[h]quinoline-3- carbaldehyde thiosemicarbazones

Article abstract of DOI:10.1016/j.ejmech.2012.02.026

Four new 2-oxo-1,2-dihydrobenzo[h]quinoline-3-carbaldehyde N-substituted thiosemicarbazone ligands (H₂-LR, where R = H, Me, Et or Ph) and their corresponding new cobalt(III) complexes have been synthesized and characterized. The structures of t

Full text of DOI:10.1016/j.ejmech.2012.02.026

European Journal of Medicinal Chemistry 50 (2012) 405e415
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European Journal of Medicinal Chemistry
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Original article
Evaluation of DNA binding, antioxidant and cytotoxic activity of mononuclear
Co(III) complexes of 2-oxo-1,2-dihydrobenzo[h]quinoline-3-carbaldehyde
thiosemicarbazones
Eswaran Ramachandran a, Sajesh P. Thomas b, Paramasivan Poornima c, Palaniappan Kalaivani a,
,
Rathinasabapathi Prabhakaran a, Viswanadha Vijaya Padma c, Karuppannan Natarajan a
*
a Department of Chemistry, Bharathiar University, Coimbatore 641046, India
b Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore 560 012, India
c Department of Biotechnology, Bharathiar University, Coimbatore 641 046, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Four new 2-oxo-1,2-dihydrobenzo[h]quinoline-3-carbaldehyde N-substituted thiosemicarbazone ligands
(H2-LR, where R ¼ H, Me, Et or Ph) and their corresponding new cobalt(III) complexes have been
synthesized and characterized. The structures of the complexes 2 and 3 were determined by single
crystal X-ray diffraction analysis. The interactions of the new complexes with DNA were investigated by
absorption, emission and viscosity studies which indicated that the complexes bind to DNA via inter-
calation. Antioxidant studies of the new complexes showed that the significant antioxidant activity
against DPPH radical. In addition, the in vitro cytotoxicity of complexes 1e4 against A549 cell line was
assayed which showed higher cytotoxic activity with lower IC50 values indicating their efficiency in
killing the cancer cells even at very low concentrations.
Received 20 January 2012
Received in revised form
10 February 2012
Accepted 13 February 2012
Available online 18 February 2012
Keywords:
Cobalt(III) complexes
Benzo[h]quinoline
Thiosemicarbazones
DNA binding
Ó 2012 Elsevier Masson SAS. All rights reserved.
Antioxidant
1. Introduction
diversity. Among the transition metals, cobalt is an element of
biological interest and its role is mainly focused on its presence in
Cancer is one of the fatal diseases, which claims over 6 million
people each year worldwide and is still increasing. The majority of
drugs used for the treatment of cancer today are not ‘cancer cell-
specific’ and potently cytotoxic against normal cells. So, the most
rapidly developing area of pharmaceutical research is the discovery
of new drugs for cancer therapy. In this regard, cisplatin is widely
used and well-known metal-based drug for cancer therapy, but it
possesses inherent limitations such as side effects and low
administration dosage [1e4]. Therefore, attempts are being made
to replace this drug with suitable alternatives, and numerous
transition-metal complexes have been synthesized and screened
for their anticancer activities. Metals, in particular transition metals
offer potential advantages over the more common organic-based
drugs, including a wide range of coordination numbers and
geometries, accessible redox states, ‘tune-ability’ of the thermo-
dynamics and kinetics of ligand substitution, and a wide structural
the active centre of vitamin B12, which regulates indirectly the
synthesis of DNA [5]. In addition, among the metal complexes that
have been studied, cobalt complex containing nitrogen heterocyclic
thiosemicarbazone is of interest because of its biological activities
and ability to bind to DNA [6e10]. In the case of pharmaceuticals, in
addition to thiosemicarbazones, nitrogen heterocycles have also
exhibited remarkable biological activities [11e13]. In particular,
some derivatives of 2-oxoquinoline have shown biological activities
such as antioxidation, antiproliferation, anti-inflammation, and
anticancer [14e17]. We have recently reported the reactions of
thiosemicarbazones and heterocycles of thiosemicarbazones with
transition metal ions in an attempt to examine their mode of
binding and possible biological relevance of the resulting
complexes [18e20]. In addition, we have also reported the effect of
substitution on terminal N of thiosemicarbazones on the biological
properties of the complexes formed [21,22]. Though an extensive
amount of work has been done on various thiosemicarbazones, no
work seems to have been done on thiosemicarbazones prepared
from 2-oxo-1,2-dihydro-benzo[h]quinoline-3-carbaldehyde. This
aroused our interest in the syntheses of 2-oxo-1,2-dihydro-benzo
* Corresponding author. Tel.: þ91 422 2428319; fax: þ91 422 2422387.
E-mail address: k_natraj6@yahoo.com (K. Natarajan).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.026

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E. Ramachandran et al. / European Journal of Medicinal Chemistry 50 (2012) 405e415
[h]quinoline-3-carbaldehyde thiosemicarbazones, and their cobal-
t(III) complexes with a viewpoint towards evaluating their effect of
substitution on terminal nitrogen of thiosemicarbazones on bio-
logical properties such as DNA binding, antioxidative and cytotoxic
activity.
imine portion and p /
p* transitions of the aromatic ring,
respectively. The intense absorption bands observed for all the
complexes in the regions of 279e282 and 431e441, 455e463 nm
are attributed to the intra ligand
p /
p* transitions within the
coordinated ligand moiety and imines and ligand-to-metal charge-
transfer (LMCT) transitions respectively. The single crystal X-ray
analysis showed that formula of the complexes are [Co(HL)2]
NO3$4H2O. Table 1 summarizes the crystal data, data collection and
refinement parameters for the new Co(III) complexes.
Herein, we report the synthesis, characterization, DNA binding,
antioxidative and cytotoxicity studies of new cobalt(III) complexes
of
2-oxo-1,2-dihydro-benzo[h]quinoline-3-carbaldehyde
N-
substituted thiosemicarbazones. The crystal structures of 2 and 3
have been determined by X-ray crystallography. The investigation of
the biological properties of the cobalt(III) complexes has been
focused on the binding properties with calf thymus (CT-DNA) per-
formed by UV spectroscopy, DNA solution viscosity measurements,
and competitive binding studies with ethidium bromide (EB).
2.2. Crystal structure of the complexes 2 and 3
The ORTEP diagram of the complex 2 and 3 including the atom
numbering scheme are shown in Figs. 1 and 2, respectively and the
selected bond lengths and bond angles are listed in Table 2. X-ray
crystallographic data of all complexes revealed that mononuclear
cation complexes neutralized by one nitrate anion. The coordina-
tion geometry around Co(III) cation can be described as a distorted
octahedron, the cobalt atom being bonded to two tridentate ONS
donor ligand molecules in such a way that two five member and
two six member chelate rings are formed. And in complexes 2 and 3
the two oxygen, two nitrogen and two sulphur atoms exhibit
ligand-metal-ligand bite angles are found to be S1eCo1eN2
2. Results and discussion
2.1. Synthesis and characterization
The synthetic routes for the ligands and its corresponding
cobalt(III) complexes are shown in Scheme 1. The ligands (H2L1),
(H2L2), (H2L3) and (H2L4) were prepared by the condensation
reaction of 2-oxo-1,2-dihydro-benzo[h]quinoline-3-carbaldehyde
with corresponding 4-N-substituted thiosemicarbazide in meth-
anol medium. They were characterized by elemental analysis, IR
spectroscopy and 1H NMR spectroscopy. The assignments for the IR
and 1H NMR spectra are given in the Experimental section. The new
cobalt(III) complexes were prepared by the direct reaction of the
appropriate ligand with Co(NO3)∙6H2O in methanol medium in
good yields. The single crystals of new cobalt(III) complexes (2 and
3) were isolated by slow evaporation of the reaction mixture over
85.1(3)ꢀ;
S2eCo1eN6,
85.8(3)ꢀ;
O1eCo1eN2,
94.5(3)ꢀ;
O2eCo1eN6, 94.0(3)ꢀ and S1eCo1eN2, 86.88(15)ꢀ; S2eCo1eN6,
86.35(12)ꢀ; O1eCo1eN2, 94.41(18)ꢀ; O2eCo1eN6, 93.74(16)ꢀ
respectively. The trans angles of the complex 2 and 3 [O1eCo1eS1,
177.0 (2)ꢀ, O2eCo1eS2, 178.5(2)ꢀ; N2eCo1eN6, 175.6(4)ꢀ and
O1eCo1eS1, 177.25(11)ꢀ O2eCo1eS2, 176.91(13)ꢀ; N2eCo1eN6,
177.81(18)ꢀ respectively] deviate slightly from the expected linear
trans geometry. In complex 2, two crystallograhically independent
molecules present in the asymmetric unit. The CeS bond distance
increased from the reported value of C]S 1.6750(14) Å in the free
ligand to 1.738(3) Å [S(2)eC(6)] and 1.743(2) Å [S(4)eC(12)] in the
cobalt complexes [23]. Likewise, there was a diminution of the
carbon-hydrazinic nitrogen bond length from 1.365(3) Å [N(3)e
C(6)] in the free ligands to 1.339(3) Å [N(3)eC(6)] and 1.325(3) Å
[N(7)eC(12)] in the complexes. These facts indicate that the ligand
is deprotonated and shifts from the thione to the thiolate form, in
such a way that the negative charge is delocalized between the two
bonds [24].
a period of few days. The IR peak shift in
n(C]O) and n(C]N) and
the peak disappearance of (C]S) of the ligand in the complex gave
n
evidence for coordination of the ligands to cobalt ion in the
complexes. The 1H-NMR spectra of the new cobalt complexes when
compared with those of free ligands, did not show the signal for
N(3)H indicating that ligand coordinated to the Co(III) ion in all the
complexes in the thiolate form. These observations have also been
confirmed by X-ray single crystal structure analysis.
The electronic spectra of the free ligands exhibit bands at
291 nm and 335 nm corresponding to the n / p* transition of the
H
N
H
H
N
H
N
N
H2N
R
O
N
R
S
S
N
H
O
N
H
O
MeOH
R= H (H2L1)
R= Me (H2L2)
R= Et (H2L3)
R= Ph (H2L4)
MeOH Co(NO3)2.6H2O
H
.NO3
R
.4H2O
N
N
N
R=H [Co(HL1)2]NO3·4H2O (1)
R=Me [Co(HL2)2]NO3·4H2O (2)
R=Et [Co(HL3)2]NO3·4H2O (3)
R=Ph [Co(HL4)2]NO3·4H2O (4)
S
H
N
Co
N
O
O
H
S
R
N
N
H
N
Scheme 1. Preparation of the ligands and it corresponding cobalt(III) complexes.

E. Ramachandran et al. / European Journal of Medicinal Chemistry 50 (2012) 405e415
407
Table 1
Experimental data for crystallographic analyses.
Complex 2
Complex 3
CCDC number
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
a(Å)
861,100
C32H34CoN9O9S2
809.74
100(2)
Triclinic
861,101
C34H34CoN9O9S2
831.79
100(2)
Monoclinic
C2/c
19.1566(17)
29.8145(19)
15.1055(13)
90.00
120.731(12)
90.00
7415.9(10)
8
1.490
Pꢁ1
14.8718(16)
16.1264(17)
17.647(3)
112.450(11)
104.033(11)
103.809(9)
3526.2(7)
4
b(Å)
c(Å)
a
b
g
(ꢀ)
(ꢀ)
(ꢀ)
Volume/Å3
Z
rcalcmg/mm3
1.841
1.890
m/mmꢁ1
0.642
F(000)
1988
3424
Crystal size/mm3
Theta range for
data collection
Index ranges
0.25 ꢂ 0.20 ꢂ 0.20
0.20 ꢂ 0.20 ꢂ 0.20
2.4e26.0ꢀ
2.4e25.0ꢀ
Fig. 2. ORTEP views of Complex 3. The nitrate ion and water molecules have been
omitted for clarity.
ꢁ18 ꢃ h ꢃ 18
ꢁ19 ꢃ k ꢃ 19
ꢁ21 ꢃ l ꢃ 21
29,812
ꢁ22 ꢃ h ꢃ 22
ꢁ35 ꢃ k ꢃ 35
ꢁ17 ꢃ l ꢃ 17
35,969
Reflections collected
Based on the analytical, spectroscopic characterization (FTeIR,
UVevisible and NMR) and single-crystal X-ray crystallographic
studies for complexes 2 and 3, the octahedral structure has been
proposed for all the new Co(III) complexes (Scheme 1).
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
13,832[R(int) ¼ 0.1281] 6531[R(int) ¼ 0.1325]
13,832/18/995
1.015
6531/0/502
1.116
Final R indexes [I > 2
s
(I)]
R1 ¼ 0.1528,
wR2 ¼ 0.3534
R1 ¼ 0.2645,
wR2 ¼ 0.4188
R1 ¼ 0.0857,
wR2 ¼ 0.2206
R1 ¼ 0.1360,
wR2 ¼ 0.2640
1.29/ꢁ0.67
Final R indexes [all data]
2.3. DNA binding studies
Largest diff. peak/hole/e Åꢁ3 2.349/ꢁ0.800
It is a well-known fact that DNA binding studies is the prelim-
inary pharmacological target of many antitumor compounds, and
hence, the interaction between DNA and metal complex is of
paramount importance in understanding the mechanism. Thus, the
mode and tendency for binding of complexes 1e4 to CT-DNA were
studied with the aid of different techniques.
The dihedral angle between the mean planes of the five-
member chelation ring and the six member one is 11.92ꢀ, 10.22ꢀ
and 13.03ꢀ, 6.57ꢀfor 2 and 3 respectively. The crystal packing in 2
and 3 is stabilized by OeH···O and NeH···O hydrogen bonds. In
complex 2, these hydrogen bonds are facilitated by the amino
nitrogen atom N(4) and aromatic nitrogen atom N(1) which act as
hydrogen bond donors and N(3) acting as acceptor. In complex 3,
aromatic nitrogen N(1) acts as hydrogen bond donor and 2-oxo
oxygen atom acts as acceptor. Further, solvent water molecules
play an important role in stabilizing the supra molecular assemblies
in the crystal structures of 2 and 3.
2.3.1. Electronic absorption titration
Of all the techniques used, electronic absorption spectroscopy is
one of the most common techniques for the investigation of the
mode of interaction of metal complexes with DNA [25,26]. A
compound binding to DNA through intercalation usually results in
Table 2
Selected bond lengths (Å) and bond angles (ꢀ) for the complexes 2 and 3.
Complex 2
Complex 3
Co1eS1
Co1e S2
Co1eO1
Co1e O2
Co1eN6
Co1e N2
2.228(3)
2.201(3)
2.024(8)
1.954(7)
1.904(9)
1.903(9)
177.03(2)
92.3(2)
90.9(2)
178.5(2)
86.2(3)
85.1(3)
91.0(3)
94.5(3)
89.2(3)
91.9(3)
85.8(3)
88.8(3)
94.0(3)
175.6(4)
90.62(12)
2.1934(17)
2.2028(15)
1.964(4)
1.960(3)
1.902(4)
1.918(4)
O1eCo1eS1
O1eCo1eS2
O2eCo1eS1
O2eCo1eS2
O2eCo1eO1
N2eCo1eS1
N2eCo1eS2
N2eCo1eO1
N2eCo1eO2
N6eCo1eS1
N6eCo1eS2
N6eCo1eO1
N6eCo1eO2
N6eCo1eN2
S1eCo1eS2
177.25(11)
89.79(11)
90.50(12)
176.91(13)
87.13(16)
86.88(15)
92.47(13)
94.41(18)
87.54(16)
91.34(14)
86.35(12)
87.43(17)
93.74(16)
177.81(18)
92.59(6)
Fig. 1. ORTEP views of Complex 2. The nitrate ion and water molecules have been
omitted for clarity.

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E. Ramachandran et al. / European Journal of Medicinal Chemistry 50 (2012) 405e415
hypochromism with or without a small red or blue shift, due to the
intercalative mode involving a strong stacking interaction between
the planar aromatic chromophore and the base pairs of DNA
[27,28]. The absorption spectra of complexes in the absence and
presence of CT-DNA are given in Fig. 3. Upon increasing the
concentration of DNA, the absorption band of complex 1 at 309, 440
and 463 nm exhibit hypochromism of 38.17%, 27.18% and 25.52%,
with red shift of 5, 3, and 5 nm respectively, whereas the absorption
bands of complex 2 at 309 and 463 nm, exhibits hypochromism of
74.50% and 66.79%, with red shift of 11 and 6 nm, respectively.
Complex 3 at 279, 447 and 463 nm, and complex 4 at 300, 437 and
458 nm exhibited a hypochromism of about 75.30, 56.29%, and
54.80% and 76.45%, 62.17% and 61.38% with a red shift of 32, 4 and
9 nm and 6, 3 and 5 nm respectively. These results suggested that
complexes 1e4 are likely to bind to the DNA helix via intercalation,
due to stacking interaction between the planar aromatic chromo-
phore and the base pairs of DNA. The complex 4 showed more
hypochromicity than the remaining complexes, indicating that the
binding strength of the complex 4 is much stronger than the other
three complexes. In order to compare the binding strength of the
complexes, their intrinsic binding constants (Kb) can be determined
from the following equation [29].
extinction coefficient of the compound when fully bound to DNA.
From the plot of [DNA]/(εa ꢁ εf) versus [DNA] (Fig. 4), Kb is calcu-
lated from the ratio of the slope to the intercept. The intrinsic
binding constant (Kb) values were 2.90(ꢅ0.09)
ꢂ
104 Mꢁ1
,
6.03(ꢅ0.05)
ꢂ
104 Mꢁ1 104 Mꢁ1 and
,
6.16(ꢅ0.05)
ꢂ
8.79(ꢅ0.03) ꢂ 104 Mꢁ1 for complexes 1, 2, 3 and 4 respectively. The
observed values of Kb revealed that new cobalt complexes bind to
DNA via the intercalative mode. From the results obtained, it has
been found that complex 4 strongly bind with CT-DNA compared to
other complexes and the order of binding affinity is 1 < 2 z 3 < 4.
From the electronic absorption studies, though it has been found
that the four complexes can bind to DNA by intercalation, the
binding mode need to be proved through some more experiments.
2.3.2. Fluorescence spectral studies
The emission experiment has been widely used to further
confirm the interaction between the test complexes and CT-DNA.
The results of fluorescence titration spectra have also been
confirmed to be effective for characterizing the binding mode of the
metal complexes to DNA. Since all the four new complexes emitted
weak luminescence in the phosphate buffered saline at room
temperature, fluorescence spectral studies were carried out. Fig. 5
shows the results of the emission titration curve of the
complexes with CT-DNA. An increase in DNA concentration resul-
ted an increase in the emission intensity of the Co(III) complexes.
Upon increasing the concentration of CT-DNA, the emission
intensity of complexes 1e4 at 526, 525, 526 and 523 nm increased
by around 1.33, 1.49, 1.59 and 1.62 times, respectively, in compar-
ison to the same in the absence of DNA. This phenomenon is related
.ꢀ
ꢁ
.ꢀ
ꢁ
ꢀ
ꢁ
½DNAꢄ εa ꢁ εf ¼ ½DNAꢄ εb ꢁ εf þ 1=Kb εb ꢁ εf
where, [DNA] is the concentration of DNA in the base pairs, εa is the
apparent absorption coefficient corresponding to Aobs/[compound],
εf is the extinction coefficient of the free compound and εb is the
Fig. 3. Electronic spectra of the complex 1(A), 2(B), 3(C) and 4(D) in TriseHCl buffer upon addition of CT-DNA. [Compound] ¼ 25
mM, [DNA] ¼ 0e25
mM. Arrow shows the absorption
intensities decrease upon increasing DNA concentration.

E. Ramachandran et al. / European Journal of Medicinal Chemistry 50 (2012) 405e415
409
2.3.3. Ethidium bromide (EB) displacement studies
The absorption titration results indicate that the complexes
effectively bind to DNA. In order to confirm the binding mode and
compare their binding affinities, ethidium bromide displacement
experiments were carried out. EB is a planar cationic dye which is
widely used as a sensitive fluorescence probe for native DNA. EB
emits intense fluorescent light in the presence of DNA due to its
strong intercalation between the adjacent DNA base pairs [31,32].
Hence, EB displacement technique can provide indirect evidence
for the DNA binding mode. The displacement technique is based on
the decrease of fluorescence resulting from the displacement of EB
from a DNA sequence by a quencher and the quenching is due to the
reduction of the number of binding sites on the DNA that is avail-
able to the EB. Fig. 6 shows the fluorescence quenching spectra of
DNA-bound EB by complexes and illustrate that as the concentra-
tion of the complexes increases, the emission band at 603 nm
exhibited hypochromism up to 55.86, 60.72, 64.53 and 70.05% of
the initial fluorescence intensity for 1, 2, 3 and 4 respectively. The
observed decrease in the fluorescence intensity clearly indicates
that the EB molecules are displaced from their DNA binding sites
and are replaced by the compounds under investigation [33]. The
quenching parameter can be analyzed according to the
SterneVolmer equation,
Fig. 4. Plots of [DNA]/(εa ꢁ εf) versus [DNA] for the complexes 1e4 with CT-DNA.
to the extent to which the compound penetrates into the hydro-
phobic environment inside the DNA, thereby avoiding the
quenching effect of solvent water molecules. The binding of
complexes to CT-DNA leads to a marked increase in the emission
intensity, which also agrees with those observed for other inter-
calators [27,28,30]. These results revealed that the complexes bind
to DNA via intercalation mode.
F0=F ¼ Kq½Qꢄ þ 1
where F0 is the emission intensity in the absence of compound, F is
the emission intensity in the presence of compound, Kq is the
quenching constant, and [Q] is the concentration of the compound.
Fig. 5. Emission enhancement spectra of complexes 1(A), 2(B), 3(C) and 4(D) (25 mM) in the presence of increasing amounts of CT-DNA (0e25 mM; subsequent spectra). The arrow
shows the emission intensity increases upon increasing the DNA concentration.

410
E. Ramachandran et al. / European Journal of Medicinal Chemistry 50 (2012) 405e415
Fig. 6. Fluorescence quenching curves of ethidium bromide bound to DNA: 1(A), 2(B), 3(C) and 4(D). [DNA] ¼ 12
SternꢁVolmer plots of the fluorescence titrations of 1, 2, 3 and 4.
m
M, [EB] ¼ 12
mM, and [compound] ¼ 0e75
mM. Inset:
The Kq value is obtained as a slope from the plot of F0/F versus [Q].
In the SterneVolmer plot (inset in Fig. 6) of F0/F versus [Q], the
quenching constant (Kq) is obtained from the slope which was
1.65 ꢂ 104 Mꢁ1, 1.99 ꢂ 104 Mꢁ1, 2.49 ꢂ 104 Mꢁ1 and 3.10 ꢂ 104 Mꢁ1
for the complexes 1, 2, 3 and 4 respectively. Further, the apparent
DNA binding constant (Kapp) were calculated using the following
equation,
amounts of complexes 1, 2, 3 and 4 respectively. These results
suggest that the viscosity of DNA increases on addition of the
cobalt(III) complexes. In general, the viscosity of DNA increases
steadily when complexes intercalate between adjacent DNA base
pairs. These experimental results indicate that complexes 1, 2, 3
and 4 bind to CT-DNA by the intercalation mode.
KEB½EBꢄ ¼ Kapp½complexꢄ
(where the compound concentration is the value at a 50% reduction
in the fluorescence intensity of EB, KEB (1.0 ꢂ 107
M
ꢁ1) is the DNA
binding constant of EB, [EB] is the concentration of EB ¼ 12
mM),
and it was found to be 1.98 ꢂ 106
M
ꢁ1, 2.38 ꢂ 106 Mꢁ1
,
2.99 ꢂ 106 Mꢁ1 and 3.72 ꢂ 106 Mꢁ1 respectively for 1, 2, 3 and 4.
From these experimental data, it is seen that the order of binding
affinities of the complexes is in the order 4 > 3 > 2 > 1, which is in
agreement with the results observed from the electronic absorp-
tion and emission spectra. Furthermore, the observed quenching
constants and binding constants of the cobalt(III) complexes
suggest that the interaction of all the complexes with DNA should
be of intercalation [34].
2.3.4. Viscosity studies
Optical photo physical titration generally provides necessary
information, but not satisfactory evidence for the binding mode of
the complexes with DNA. For further clarification of the binding
mode, viscosity measurements need to be carried out on CT-DNA by
varying the concentration of added complexes [35,36]. Fig. 7 shows
the relative viscosity change of DNA in the presence of increasing
Fig. 7. Effect of increasing amount of the complexes (1e4) on the relative viscosity of
CT-DNA at 25 ꢀC.

E. Ramachandran et al. / European Journal of Medicinal Chemistry 50 (2012) 405e415
411
Fig. 8. 2-20-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the
complexes (1e4) and the standards, Rutin (Ru) and Quercetin (Qu).
Fig. 9. Treatment of complexes that exert an antiproliferative effect on lung cancer cell
line. A549 cells were treated with complexes (1e4) for 48 h. Control received appro-
priate carriers. Cell viability was assessed by MTT cell proliferation assay. Results
shown are mean ꢅ SD (n ¼ 9) of the respective IC50 values, which are from three
separate experiments performed in triplicate.
On the basis of absorption, emission, EB displacement and
viscosity studies, we concluded that all the cobalt complexes can
bind to CT-DNA in an intercalative mode and that the order of
complexes binding to CT-DNA is 4 > 3 > 2 > 1.
2.5. Cytotoxic activity studies
2.5.1. MTT assay
2.4. Antioxidant activity
The cytotoxicity assay for the new Co(III) complexes was
assessed using the method of MTT reduction. Cisplatin was used as
a positive control. All the complexes were found to be cytotoxic to
lung cancer cell line (A549). The IC50 values (50% inhibition of cell
Since the DNA binding experiments conducted so far revealed
that the Co(III) complexes exhibit good DNA binding affinity, it is
considered worthwhile to study the antioxidant activity of these
compounds. 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) assay is widely
used for assessing the ability of radical scavenging activity and it is
measured in terms of IC50 values. Because of the presence of odd
electron, DPPH shows a strong absorption band at 517 nm in the
visible spectrum. As this electron becomes paired off in the pres-
ence of a free radical scavenger, this absorption vanishes, and the
resulting decolorization is stoichiometric with respect to the
number of electrons taken up. The DPPH assay done for our
complexes is shown in Fig. 8. It is seen from the results that the all
the complexes exhibited moderate activities compared to the
standards Rutin (Ru) and Quercetin (Qu). Statistically the scav-
enging effect of the compounds with DPPH radical is in the order
4 > 1 > 2 > 3. The complex 4 showed better activity compared to
the other three complexes, which may be due to the electron-
withdrawing effect on terminal nitrogen atom of the ligands.
Ferric reducing antioxidant power (FRAP) is associated with the
capacity to prevent peroxide formation, so that they can act as
primary and secondary antioxidants. From the experimental data
(Table 3) we suggest that the new Co(III) complexes exhibited
strong reducing power. As expected, complexes 2 and 1 showed
strong reducing power comparable with that of positive controls
BHA and BHT. Since, our synthesised cobalt complexes are electron
donors; they can terminate the oxidation chain reactions thereby
reducing the oxidised intermediates into the stable form.
growth for 48 h) for complexes 1, 2, 3 and 4 are 20
mM (1),18 mM (2),
25 M (3) and 25 M (4) respectively (Fig. 9). The complexes
m
m
exhibited higher cytotoxic effects on lung cancer cells with lower
IC50 values indicating their efficiency in killing the cancer cells even
at low concentrations. The cytotoxic effectiveness of these
compounds with an IC50 of 20
mM (1) and 18 mM (2) were higher
than that of cisplatin (25 M). There are reports in the literature on
m
the cytotoxic effects of the complexes with longer incubation time
periods. The longer incubation period may result in the develop-
ment of cellular resistance for that particular complex. Beckford
et al have reported 50% inhibitory concentration of different
complexes after an exposure for 72 h at mM concentrations. But, the
data obtained for our complexes showed higher cytotoxicity with
short incubation period (48 h). Hence, our data are highly signifi-
cant when compared to the results of Beckford et al. [37]. Moreover,
the IC50 values of our complexes are comparable with the reported
IC50 values of standard anticancer drugs such as cisplatin.
Table 3
Ferric Reducing Antioxidant Power (FRAP) of new Co(III)
complexes.
Compound
FRAP (mM Fe (II) gꢁ1
)
1
2
3
4
BHA
BHT
2398.89 ꢅ 277.82
4521.11 ꢅ 38.92
1122.22 ꢅ 93.95
1148.89 ꢅ 48.34
1342.20 ꢅ 157.20
7486.70 ꢅ 162.30
Fig. 10. Lactate dehydrogenase release by A549 cell line: Treatment with the
complexes resulted in the release of LDH from the cell line. Results shown are
mean ꢅ SD (n ¼ 9) of the percentage of LDH released, which are from three separate
experiments performed in triplicate.

412
E. Ramachandran et al. / European Journal of Medicinal Chemistry 50 (2012) 405e415
that the complexes bind to DNA via intercalation. The binding
strength of the complexes with CT-DNA calculated with UV spec-
troscopic titrations have shown that the complex 4 has higher
binding affinity to CT-DNA than the rest of the complexes. Ethidium
bromide competitive binding studies have revealed that the facility
of the complexes to displace EB from the EB-DNA complex. The
antioxidant studies revealed that all the complexes exhibited
moderate activities compared to the standards Rutin (Ru) and
Quercetin (Qu). In addition, the in vitro cytotoxicity of complexes
1e4 against A549 cell line was assayed. The cytotoxicity of the
complexes are affected by the various functional group attached to
the terminal nitrogen atom, and the new Co(III) complexes showed
considerable cytotoxic activity against A549 cell line. In our studies,
we found that the antitumor property of the complexes was
enhanced with the change in substitution at terminal nitrogen
atom of the ligand.
Fig. 11. Nitric oxide assay: treatment with the complexes resulted in the release of
high levels of nitrite from both A549 cell line. Results shown are mean ꢅ SD (n ¼ 9) of
the n moles of nitrite released, which are from three separate experiments performed
in triplicate.
4. Experimental section
4.1. Materials and instrumentation
2.5.2. Lactate dehydrogenase release (LDH)
To further evaluate the toxicity, activity of LDH, a cytoplasmic
enzyme released into surrounding media during cell death, was
measured. Cell death by apoptosis leads to the release of cyto-
plasmic enzymes such as alkaline and acid phosphatase, glutamate-
oxalacetate transaminase, glutamate pyruvate transaminase and
arginosuccinatelyase. However, their use has been limited by the
presence of low amount in many cells and hence an elaborate
kinetic assay is required to measure the most enzyme activities.
LDH is a stable cytoplasmic enzyme which is released into the
culture medium following loss of membrane integrity resulting
from apoptosis. LDH activity, therefore, can be used as an indicator
of cell membrane integrity and serves as a general means to assess
cytotoxicity resulting from chemical compounds or environmental
toxic factors [38,39]. As can be seen from Fig. 10, a significant
difference was observed between the control cells and cells treated
with complexes 1e4 after 48 h of drug treatment. The four new
Co(III) complexes lead to the release of significant level of lactate
dehydrogenase by A549 cell line into the culture medium.
All starting materials used throughout the experiments were of
analytical or chemically pure grade. 2-Oxo-1,2-dihydrobenzo[h]
quinoline-3-carbaldehyde and H2L1-L4 were prepared according
to the literature procedures [27,41]. Solvents were purified and
dried according to standard procedures [42]. The reagents were
purchased commercially and used without further purification
unless otherwise noted. CT-DNA, Agarose and EB were obtained
from SigmaeAldrich and used as received. Elemental analyses (C,
H, N, S) were performed on Vario EL III Elemental analyzer
instrument. IR spectra (4000e400 cmꢁ1) for KBr disks were
recorded on a Nicolet Avatar Model FT-IR spectrophotometer. 1H
NMR spectra were recorded on Bruker AMX 500 at 500 MHz.
Melting points were determined with a Lab India instrument.
Electronic absorption spectra were recorded using Jasco V-630
spectrophotometer. Emission spectra were measured with Jasco FP
6600 spectrofluorometer.
4.2. Preparation of the compounds
2.5.3. Nitric oxide assay
4.2.1. 2-Oxo-1,2-dihydrobenzo[h]quinoline-3-carbaldehyde
thiosemicarbazone (H2L1)
Nitric oxide (NO) has been assumed to have an important
functional role in a variety of physiological systems. NO is derived
from L-arginine by the action of NO synthase (NOS), an enzyme
existing in three isoforms. Among them, two isoforms exist
constitutively and the third isoform is an inducible form (iNOS)
which is expressed in response to stress. NO is a gaseous signaling
molecule, the stable product of which is nitrite. Nitric oxide is
a well-known short-lived free radical produced non-enzymatically
by iNOS which causes damage in most of the biomolecules,
including DNA and protein [40]. The level of nitrite was found to
increase significantly in complex treated cells compared to control
(Fig. 11). The increased level of nitrite in the cell culture medium
further confirms the cytotoxic effects of the presently studied
complexes.
A methanolic solution of thiosemicarbazide (0.911 g, 0.01 mol)
dissolved in warm methanol (50 mL) was added to a methanol
solution (50 mL) containing 2-oxo-1,2-dihydro-benzo[h]quinoline-
3-carbaldehyde (2.23 g, 0.01 mol). The mixture was refluxed for an
hour during which period an yellow colour precipitate was formed.
The reaction mixture was then cooled to room temperature and the
solid compound was filtered. It was then washed with methanol
and dried under vacuum. Yield: 87%. MP: 262e264 ꢀC, Anal. calcd.
for C15H12N4OS (%): C, 60.79; H, 4.08; N,18.91; S,10.82. Found (%): C,
60.93; H, 4.13; N, 18.86; S,10.77. IR (KBr, cmꢁ1): 3241(ms)
1643(s) (C]O); 1588, 1546(s) (C¼ C); 835(m)
(C]N) þ
UVevis (DMSO), lmax (nm): 274, 283, 373 (m / m*, n / m*). 1H NMR
(DMSO-D6); 12.03 (s, N(1)H); 11.53 (s, N(3)H); 8.73 (s, 1H, C(3)
H); 8.54(s, 1H, C(14)H); 8.23 (s, 2H, N(4)H); 7.21e8.15 (m, 6H,
n
(NH);
n
n
n
n(C]S).
d
d
d
d
3. Conclusion
aromatic).
Four new 2-oxo-benzo[h]quinoline-3-carbaldehyde thio-
semicarbazones ligands and their corresponding Co(III) complexes
have been synthesized and characterized. The molecular structures
of the complexes 2 and 3 have been determined by single crystal X-
ray diffraction studies. The UVevisible, fluorescence spectroscopy
and DNA solution viscosity measurements studies have revealed
the ability of the complexes bind to DNA and supported the fact
4.2.2. 2-Oxo-1,2-dihydrobenzo[h]quinoline-3-carbaldehyde N-
methylthiosemicarbazone (H2L2)
It was prepared using the same procedure as described for H2L1
with 4-methyl-3-thiosemicarbazide (1.05 g, 0.01 mol) and 2-oxo-
1,2-dihydrobenzo[h]quinoline-3-carbaldehyde (2.23 g, 0.01 mol).
An yellow colour product was obtained. Yield: 85%. MP:
274e276 ꢀC, Anal. calcd. for C16H14N4OS (%): C, 61.92; H, 4.55; N,

E. Ramachandran et al. / European Journal of Medicinal Chemistry 50 (2012) 405e415
413
18.05; S, 10.33. Found (%): C, 61.87; H, 4.49; N, 18.09; S, 10.37. IR
(KBr, cmꢁ1): 3317(ms)
(NH); 1649(s) (C]O); 1550(s) (C]
N) þ (C]C); 811(m) (C]S). UVevis (DMSO), lmax (nm): 279, 373
(m / m*, n / m*). 1H NMR (DMSO-D6);
12.16 (s, N(1)H); 11.76 (s,
N(3)H); 8.61 (s, 1H, C(3)H); 8.21(s, 1H, C(14)H); 8.15 (s, 1H, N(4)
0.05 mmol). Dark red coloured crystals obtained were found to be
suitable for X-ray diffraction. Yield: 84%. MP: 286e288 ꢀC, Anal.
calcd. for C34H38CoN9O9S2 (%): C, 48.62; H, 4.56; N, 15.02; S, 7.64.
Found (%): C, 48.57; H, 4.61; N, 15.23; S, 7.68 IR (KBr, cmꢁ1):
n
n
n
n
n
d
d
d
d
3321(ms)
UVevisible (solvent MeOH, nm): 278 (ILCT); 433,456 (LMCT). 1H
NMR (DMSO-D6); 13.91(s, N(1)H); 8.98(s, 1H, C(3)H); 8.96(s,
n(NH); 1625(s) n(C]O); 1503(s) n(C]N); 717(m) n(CeS).
H); 7.67e7.98 (m, 6H, aromatic); 3.03 (s, 3H, C(17)H).
d
d
d
4.2.3. 2-Oxo-1,2-dihydrobenzo[h]quinoline-3-carbaldehyde N-
ethylthiosemicarbazone (H2L3)
2H, N(4)H); 8.95(s, 1H, C(14)H); 7.61e8.03 (m, 6H, aromatic); 3.37
(q, 2H, C(16)H); 1.15(t, 3H, C(17)H).
It was prepared using the same procedure as described for H2L1
with 4-ethyl- 3-thiosemicarbazide (1.19 g, 0.01 mol) and 2-oxo-1,2-
dihydro-benzo[h]quinoline-3-carbaldehyde (2.23 g, 0.01 mol). An
yellow colour product was obtained. Yield: 82%. MP: 259e260 ꢀC,
Anal. calcd. for C17H16N4OS (%): C, 62.94; H, 4.97; N, 17.27; S, 9.89.
Found(%): C, 62.85; H, 4.87; N,17.21; S, 9.78. IR (KBr, cmꢁ1): 3327(ms)
4.2.8. [Co(HL4)2]NO3$4H2O (4)
It was prepared using the same procedure as described for 1
with H2L4 (186 mg, 0.05 mmol) and Co(NO3)2$6H2O (0.146 g,
0.05 mmol). Dark red coloured crystalline powder obtained. Yield:
89%. MP: 299e300 ꢀC, Anal. calcd. for C42H38CoN9O9S2 (%): C, 53.90;
H, 4.09; N, 13.47; S, 6.85. Found (%):C, 53.82; H, 4.17; N, 13.36; S,
n
(NH); 1648(s)
UVevis (DMSO), lmax (nm): 295, 359 (m / m*, n / m*). 1H NMR
(DMSO-D6); 12.43 (s, N(1)H); 11.67 (s, N(3)H); 8.91 (s,1H, C(3)H);
8.81(s,1H,C(14)H);8.35(s,1H,N(4)H);7.65e8.01(m, 6H,aromatic);
3.34 (q, 2H, C(16)H); 1.21 (t, 3H, C(17)H).
n
(C]O); 1535(s)
n
(C]N) þ
n(C]C); 840(m) n(C]S).
6.73. IR (KBr, cmꢁ1): 3255(ms)
(C]N); 745(m)
(ILCT); 441,463 (LMCT). 1H NMR (DMSO-D6);
n
(NH); 1622(s)
(CeS). UVevisible (solvent MeOH, nm): 277
13.7212.05 (s,
n(C]O); 1490(s)
d
d
d
n
n
d
d
1H,N(1)H); 9.87 (s, 1H, N(4)H); 8.84 (s, 1H,C(1)H); 8.46 (s, 1H, C(6)
H); 7.98e7.95 (m, 11H, aromatic).
4.2.4. 2-Oxo-1,2-dihydro-benzo[h]quinoline-3-carbaldehyde N-
phenylthiosemicarbazone (H2L4)
4.3. Single-crystal X-ray diffraction studies
It was prepared using the same procedure as described for H2L1
with 4-phenyl-3-thiosemicarbazide (1.67 g, 0.01 mol) and 2-oxo-
1,2-dihydro-benzo[h]quinoline-3-carbaldehyde (2.23 g, 0.01 mol).
An yellow colour product was obtained. Yield: 91%. MP:
245e246 ꢀC, Anal. calcd. for C21H16N4OS (%): C, 67.72; H, 4.33; N,
15.04; S, 8.61. Found (%): C, 67.64; H, 4.47; N, 15.19; S, 8.58. IR (KBr,
Single crystal X-ray diffraction data of 2 and 3 were collected at
100 K on an Oxford Xcalibur Eos (Mova) Diffractometer with X-ray
generator operating at 50 kV and 1 mA, using Mo K
a radiation
(l
¼ 0.7107 Å) [43]. The structures were solved and refined using
SHELX97 module in the program suite WinGX [44,45]. The
molecular diagrams were generated using ORTEP-3 and the
packing diagrams were generated using Mercury 2.3. The
geometric calculations were carried out by PARST95 and PLATON
[46,47].
cmꢁ1): 3293(ms)
C); 845(m) (C]S). UVevis (DMSO), lmax (nm): 279, 373, 392
(m / m*, n / m*). 1H NMR (DMSO-D6);
12.01 (s, N(1)H); 11.87 (s,
N(3)H); 10.03 (s, 1H, C(3)H); 8.85(s, 1H, C(14)H); 8.39 (s, 1H, N(4)
n
(NH); 1652(s)
n(C]O); 1530(s)
n
(C]N) þ
n(C]
n
d
d
d
d
H); 7.35e7.98 (m, 11H, aromatic).
4.4. DNA binding studies
4.2.5. [Co(HL1)2]NO3$4H2O (1)
A warm methanolic solution (20 mL) containing H2L1 (0.148 g,
0.05 mmol) was added to a methanolic solution (20 mL) of Co(N-
O3)2$6H2O (0.146 g, 0.05 mmol). The resulting red colour solution
was refluxed for an hour. Dark red coloured crystalline powder was
obtained on slow evaporation. They were filtered off, washed with
cold methanol, and dried under vacuum. Yield: 83%. MP:
280e281 ꢀC, Anal. calcd. for C30H30CoN9O9S2 (%): C, 45.98; H, 3.86;
N, 16.09; S, 8.18. Found (%): C, 45.83; H, 3.92; N, 16.01; S, 8.07. IR
4.4.1. Titration experiments
All of the experiments involving the binding of compounds with
CT-DNA were carried out in double distilled water with
tris(hydroxymethyl)-aminomethane (Tris,
5 mM) and sodium
chloride (50 mM) and adjusted to pH 7.2 with hydrochloric acid. A
solution of CT-DNA in the buffer gave a ratio of UV absorbance of
about 1.9 at 260 and 280 nm, indicating that the DNA was suffi-
ciently free of protein. The DNA concentration per nucleotide was
determined by absorption spectroscopy using the molar extinction
coefficient value of 6600 Mꢁ1 cmꢁ1 at 260 nm. The compounds
were dissolved in a mixed solvent of 5% DMSO and 95% TriseHCl
buffer for all of the experiments. Absorption titration experi-
(KBr, cmꢁ1): 3293(ms)
754(m) (CeS). UVevisible (solvent MeOH, nm): 279 (ILCT);
431,455 (LMCT). 1H NMR (DMSO-D6);
13.94(s, N(1)H); 8.99 (s,
2H, N(4)H); 8.97(s, 1H, C(3)H); 8.86(s, 1H, C(14)H); 7.43e8.04 (m,
6H, aromatic).
n(NH); 1628(s) n(C]O); 1482(s) n(C]N);
n
d
d
d
ments were performed with
compounds (25 M) while gradually increasing the concentration
of DNA (5e25 M). While measuring the absorption spectra, an
a fixed concentration of the
m
m
4.2.6. [Co(HL2)2]NO3$4H2O (2)
It was prepared using the same procedure as described for 1 with
H2L2 (0.155 g, 0.05 mmol) and Co(NO3)2$6H2O (146 mg, 0.05 mmol).
Dark red coloured crystals obtained were found to be suitable for X-
ray diffraction. Yield: 79%. MP: 292e293 ꢀC, Anal. calcd. for
C32H34CoN9O9S2 (%): C, 47.35; H, 4.22; N, 15.53; S, 7.91. Found (%): C,
equal amount of DNA was added to both the test solution and the
reference solution to eliminate the absorbance of DNA itself. The
same experimental procedure was followed for emission studies
also. Further support for the complexes binding to DNA via inter-
calation is given through emission quenching experiments. DNA
was pretreated with ethidium bromide for 30 min. Then the test
solutions were added to this mixture of EB-DNA, and the change in
the fluorescence intensity was measured. The excitation and the
emission wavelength were 515 nm and 603e607 nm, respectively.
47.49; H, 4.27; N, 15.62; S, 7.85. IR (KBr, cmꢁ1): 3359(ms)
1624(s) (C]O); 1511(s) (C]N); 729(m)
(solvent MeOH, nm): 282 (ILCT); 433,456 (LMCT). 1H NMR (DMSO-
D6); 13.92(s, N(1)H); 8.99(s, 1H, C(3)H); 8.97(s, 2H, N(4)H);
n(NH);
n
n
n(CeS).UVevisible
d
d
d
8.95(s, 1H, C(14)H); 7.62e8.04 (m, 6H, aromatic);
4.4.2. Viscosity measurements
4.2.7. [Co(HL3)2]NO3$4H2O (3)
It was prepared using the same procedure as described for 1
with H2L3 (0.162 g, 0.05 mmol) and Co(NO3)2$6H2O (146 mg,
Viscosity experiments were carried out using a Schott Gerate
AVS 310 automated viscometer that was thermo stated at 25 ꢀC in
a constant temperature bath. The lengthening of the DNA helix has

414
E. Ramachandran et al. / European Journal of Medicinal Chemistry 50 (2012) 405e415
been examined in the absence and presence of increasing amounts
of complexes 1e4. Flow time was measured with a digital stop-
watch and for each sample the measurement was made in tripli-
arrested by adding 0.1 cm3 of DNPH reagent and the tubes were
incubated for further period of 15 min at 37 ꢀC. After this 0.1 cm3 of
media or cell extract was added to blank tubes after arresting the
reaction with DNPH. Then 3.5 cm3 of 0.4 N sodium hydroxide was
added to all the tubes. The colour developed was measured at
420 nm with a UVevisible spectrophotometer. The amount of LDH
released was expressed in percentage.
cates and the mean value of flow time was recorded. The obtained
1/3
data are presented as (
h
/h0)
versus the binding ratio r [48],
where
h
is the viscosity of CT-DNA in the presence of complex, and
h0 is the viscosity of CT-DNA alone in buffer solution.
4.5. Antioxidant assays
4.6.3. Nitric oxide (NO) assay
The amount of nitrite was determined by the literature method
[53]. Nitrite reacts with Griess Reagent to give a coloured complex
4.5.1. 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) assay
The DPPH radical scavenging activity of the complexes was
measured according to the method of Blois [49]. The concentration
of complexes necessary to decrease initial concentration of DPPH
by 50% (IC50) under the specified experimental condition was
calculated. The test solution was incubated at 37 ꢀC for 30 min in
dark. The decrease in absorbance of DPPH was measured at 517 nm.
The same experiment carried out without the test compounds
served as control.
measured at 540 nm. To 100
reagent I was added, mixed and allowed to react for 10 min. This
was followed by the addition of 50 L of Griess reagent II and the
reaction mixture was mixed well and incubated for another 10 min
at room temperature. The pink colour developed was measured at
540 nm in a microquant plate reader (Biotek Instruments).
mL of the medium, 50 mL of Griess
m
Acknowledgments
4.5.2. Ferric reducing antioxidant power (FRAP)
The antioxidant capacities of compounds and crude fractions
were estimated according to the procedure described by Pulido
Council of Scientific and Industrial Research, New Delhi, India
[Grant No. 01(2216)/08/EMR-II & 21(0745)/09/EMR-II] for the
financial assistance received, and the University Grants Commis-
sion, New Delhi, for the award of a Fellowship in Science for
Meritorious Students (RFSMS) to E. R under the UGC-SAP-DRS
programme, are gratefully acknowledged.
et al. [50]. FRAP reagent (900
m
L), prepared freshly and incubated at
37 ꢀC, was mixed with 90
m
L of distilled water and 30 L of test
m
sample or methanol (for the reagentꢀ blank). The test samples and
reagent blank were incubated at 37 C for 30 min in a water bath
and the FRAP reagent contained 2.5 mL of 20 mmol/L TPTZ solution
in 40 mmol/L HCl, 2.5 mL of 20 mmol/L FeCl3 $ 6H2O and 25 mLof
0.3 mol/L acetate buffer (pH 3.6) were added at the end of incu-
bation, the absorbance readings were taken immediately at 593 nm
using a spectrophotometer. Methanolic solutions of known Fe (II)
Appendix. Supplementary material
Crystallographic data for the structures reported in this paper
have been deposited with the Cambridge Crystallographic Data
Centre (CCDC) as supplementary publication number CCDC-
861100, and CCDC-861101 for complexes 2, and 3 respectively.
Copies of the data can be obtained free of charge from the CCDC (12
Union Road, CambridgeCB2 1EZ, UK; Tel.: þ44 1223 336408;
Fax: þ44 1223 336003; e-mail: deposit@ccdc.cam.ac.uk; Web site
http://www.ccdc.cam.ac.uk).
concentration, ranging from 100 to 2000
mmol/L, (FeSO4.7H2O)
were used for the preparation of the calibration curve. The
parameter Equivalent Concentration (EC1) is defined as the
concentration of antioxidant having a ferric-TPTZ reducing ability
equivalent to that of 1 mmol/L FeSO4$7H2O. EC1 was calculated as
the concentration of antioxidant giving an absorbance increase in
the FRAP assay equivalent to the theoretical absorbance value of
a 1 mmol/L concentration of Fe(II) solution, determined using the
corresponding regression equation.
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4.6. Cytotoxicity assays
4.6.1. 3-(4,5-Dimethylthiazol-2-yl)- 2,5- diphenyltetrazolium
bromide (MTT) assay
Cytotoxic effect of the four new complexes on human lung
cancer cells (A549) were assayed by the 3-(4,5-dimethylthiazol-2-
yl)- 2,5- diphenyltetrazolium bromide (MTT) assay [51]. The cells
were seeded at a density of 10,000 cells per well, in 200
the samples 1, 2, 3 and 4 were added to the cells at a final
concentration of 1, 10, 25 and 50 M in the cell culture media. After
48 h, the wells were treated with 20 l MTT (5 mg/mL PBS) and
incubated at 37 ꢀC for 4 h. The purple formazan crystals formed
ml and then
m
m
were dissolved in 200
reader.
mL DMSO and read at 570 nm in a micro plate
4.6.2. Release of lactate dehydrogenase (LDH)
LDH activity was determined by the linear region of a pyruvate
standard graph using regression analysis and expressed as
percentage leakage as described previously [52]. Briefly, to a set of
tubes, 1 cm3 of buffered substrate (lithium lactate) and 0.1 cm3 of
the media or cell extract were added and tubes were incubated at
37 ꢀC for 30 min. After adding 0.2 cm3 of NAD solution, the incu-
bation was continued for another 30 min. The reaction was then

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