Synthesis, characterization and crystal structure of cobalt(III) complexes containing 2-acetylpyridine thiosemicarbazones: DNA/protein interaction, radical scavenging and cytotoxic activities

Article abstract of DOI:10.1016/j.jphotobiol.2013.11.008

The synthesis, structure and biological studies of cobalt(III) complexes supported by NNS-tridentate ligands are reported. Reactions of 2-acetylpyridine N-substituted thiosemicarbazone (HL^1-3) with [CoCl ₂(PPh₃)₂

Full text of DOI:10.1016/j.jphotobiol.2013.11.008

Journal of Photochemistry and Photobiology B: Biology 130 (2014) 205–216
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Synthesis, characterization and crystal structure of cobalt(III) complexes
containing 2-acetylpyridine thiosemicarbazones: DNA/protein
interaction, radical scavenging and cytotoxic activities
b
Rajendran Manikandan a, Periasamy Viswanathamurthi a, , Krishnaswamy Velmurugan ,
⇑
Raju Nandhakumar b, Takeshi Hashimoto c, Akira Endo c
a Department of Chemistry, Periyar University, Salem 636011, Tamil Nadu, India
b Department of Chemistry, Karunya University, Karunya Nagar, Coimbatore 641 114, India
c Department of Materials and Life Sciences, Sophia University, Tokyo 102-8554, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 October 2013
Received in revised form 6 November 2013
Accepted 11 November 2013
Available online 22 November 2013
The synthesis, structure and biological studies of cobalt(III) complexes supported by NNS-tridentate
ligands are reported. Reactions of 2-acetylpyridine N-substituted thiosemicarbazone (HL1–3) with
[CoCl2(PPh3)2] resulted [Co(L1–3)2]Cl (1–3) which were characterized by elemental analysis and various
spectral studies. The molecular structure of the complex 1 has been determined by single crystal X-ray
diffraction studies. In vitro DNA binding studies of complexes 1–3 carried out by fluorescence studies
and the results revealed the binding of complexes to DNA via intercalation. The binding constant (Kb) val-
ues of complexes 1–3 from fluorescence experiments showed that the complex 3 has greater binding pro-
pensity for DNA. The DNA cleavage activity of the complexes 1 and 3 were ascertained by gel
electrophoresis assay which revealed that the complexes are good DNA cleavage agents. Further, the
interactions of the complexes with bovine serum albumin (BSA) were also investigated using fluores-
cence spectroscopic method, which showed that the complexes 1–3 could bind strongly with BSA. The
antioxidant property of the complexes was evaluated to test their free-radical scavenging ability. Further-
more, in vitro cytotoxicity of the complexes against MCF-7 and A431 cell lines was assayed which showed
higher activity and efficiently vanished the cancer cells even at low concentrations.
Keywords:
Cobalt(III) complexes
DNA binding
DNA cleavage
Protein binding
Antioxidant activity
Cytotoxicity
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
very promising candidates for anticancer therapy; an idea sup-
ported by a considerable number of research articles describing
Cancer is undoubtedly one of the main health concerns facing
our society and one of the primary targets regarding medicinal
chemistry. Even though platinum-based complexes had been in
the primary focus of research on chemotherapy agents [1–3], the
interests in this field have shifted to non-platinum based agents
[4–11], in order to find different metal complexes with less side ef-
fects and similar, or better, cytotoxicity. Thus, a wide variety of me-
tal complexes based on titanium, gallium, germanium, palladium,
gold, copper, nickel, ruthenium and tin are being intensively stud-
ied as platinum replacements [4–13].
Cobalt is an essential trace element in human, exhibiting many
useful biological functions. Numerous compounds, naturally occur-
ring and man-made, contain the cobalt at two common oxidation
states Co(II) and Co(III). There is growing interest in investigating
the cobalt and other transition metal complexes for their interac-
tion with DNA [14–18]. Moreover, cobalt complexes appear to be
the synthesis and cytotoxic activities of numerous cobalt com-
plexes [19–26].
In addition, thiosemicarbazones are considered as an important
class of nitrogen–sulfur donor ligands because of their highly inter-
esting chemical, biological and medicinal properties, such as DNA
binding, antioxidant, antibacterial, antiproliferative, antimalarial,
anticancer and antitumor [27–33]. The presence of substituents
at the 4-position have been shown to affect the activity of thio-
semicarbazones and their metal complexes [34]. For example, me-
tal complexes of 2-acetylpyridine thiosemicarbazones are found to
exhibit increased antineoplastic activity when the N atom in the 4-
position is part of a hexamethyleneiminyl ring instead of being a
propyl- or dipropyl-carrying amine group [9]. The variable coordi-
nation behavior of thiosemicarbazone ligands towards transition
metals, as well as antitumor activity of the resulting complexes
are studied by West et al. [35–38]. We have previously reported
the synthesis and biological studies of cadmium(II) complexes
from 2-acetylpyridine thiosemicarbazones [39].
⇑
Corresponding author. Fax: +91 427 2345124.
E-mail address: viswanathamurthi72@gmail.com (P. Viswanathamurthi).
1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotobiol.2013.11.008

206
R. Manikandan et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 205–216
In this study, the complexes of 2-acetylpyridine N-substituted
dimethylsulfoxide (DMSO-d6) as solvent. The ESI-MS spectra were
recorded by using LC-MS Q-ToF Micro-analyzer (Shimadzu) in the
SAIF, Panjab University, Chandigarh. Melting points were checked
on a Technico micro-heating table and are uncorrected. The requi-
site precursor metal complex [CoCl2(PPh3)2] were prepared accord-
ing to the literature method [40].
thiosemicarbazone with Co(III) have been synthesized and charac-
terized. The interactions of the complexes with CT-DNA/protein
were investigated by using fluorescence spectroscopy. The DNA
cleavage ability has been monitored by using gel electrophoresis
experiment in the presence of the complexes using pBR322 DNA.
The free radical scavenging ability, assays by a series of antioxidant
assays involving DPPH, hydroxyl and nitric oxide radical. In vitro
cytotoxic activities of the complexes were tested by MTT assays
against human breast cancer cell line (MCF-7) and human skin car-
cinoma cell line (A431). The synthetic routes for the ligands and
complexes are shown in Scheme 1.
2.3. Synthesis of Schiff base ligands
The Schiff base ligands were prepared by condensation of equi-
molar amounts of 2-acetylpyridine (2 mmol) and substituted thio-
semicarbazide (2 mmol) in ethanol (20 mL) under reflux for 5 h.
Then the reaction mixture was cooled to room temperature, the
yellow solid was separated, filtered off, washed with ethanol and
dried in vacuo [41,42].
2. Experimental
2.1. Materials and methods
2.3.1. 2-Acetylpyridine-thiosemicarbazone (HL1)
All the reagents used were chemically pure and AR grade. The
solvents were purified and dried according to standard procedures.
CoCl2ꢀ6H2O, calf-thymus DNA (CT-DNA), methylene blue (MB) and
bovine serum albumin (BSA) were purchased from Sigma-Aldrich
and used as received. The pBR322 DNA was purchased from Banga-
lore GeNei, Bangalore, India. The human breast cancer cell line
(MCF-7) and human skin carcinoma cell line (A431) was obtained
from National Centre for Cell Science (NCCS), Pune, India and
grown in Dulbeccos Modified Eagles Medium (DMEM) containing
10% fetal bovine serum (FBS). Cells were maintained at 37 °C, 5%
CO2, 95% air and 100% relative humidity. Maintenance cultures
were passaged weekly, and the culture medium was changed twice
a week.
The ligand was obtained from the reaction of 2-acetylpyridine
(0.22 mL; 2 mmol) and thiosemicarbazide (0.1822 g; 2 mmol).
Yield: 0.3104 g, 80%; Color: yellow solid; M.p.: 180 °C; Anal. Calcd.
for C8H10N4S (194.25 g molꢁ1): C, 49.46; H, 5.18; N, 28.84; S,
16.50%. Found: C, 49.32; H, 5.26; N, 28.69; S, 16.32%. IR (KBr,
cmꢁ1): 3373, 3260(m)
(C@N), 1583(s) (C@N), 836(m)
/Mꢁ1 cmꢁ1)): 310 (46,064), 202 (32,588). 1H NMR (300 MHz,
m
(ANHAR), 3183(m)
m(NH), 1609(s)
m
m
m
(C@S). UV-Vis (CH3CN, k/nm
(e
DMSO-d6, d, ppm): 10.31 (s, 1H, NH); 8.19 (s, 2H, NH2); 8.59–
8.38 & 7.79–7.35 (m, 4H, aromatic); 2.38 (s, 3H, CH3).
2.3.2. 2-Acetylpyridine-4-methyl-thiosemicarbazone (HL2)
The ligand was obtained from the reaction of 2-acetylpyridine
(0.22 mL; 2 mmol) and 4-methyl-thiosemicarbazide (0.2103 g;
2 mmol). Yield: 0.3038 g, 73%; Color: yellow solid; M.p.: 170 °C;
Anal. Calcd. for C9H12N4S (208.28 g, molꢁ1): C, 51.90; H, 5.80; N,
26.90; S, 15.39%. Found: C, 51.78; H, 5.72; N, 26.29; S, 15.78%. IR
2.2. Physical measurements
Microanalyses of carbon, hydrogen, nitrogen and sulfur were
carried out using Vario EL III Elemental analyzer at SAIF – Cochin,
India. The IR spectra of the ligand and their complexes were ob-
tained as KBr pellets on a Nicolet Avatar model spectrophotometer
at 4000–400 cmꢁ1 range. Electronic spectra of the ligand and their
complexes have been recorded in acetonitrile using a Shimadzu
UV-1650 PC spectrophotometer at 800–200 nm range. Emission
spectra were measured with a Jasco FP 6600 spectrofluorometer.
1H NMR spectra were recorded in Jeol GSX-400 instrument at room
temperature using tetramethylsilane as the internal standard in
(KBr, cmꢁ1): 3289(m)
(C@N), 1578(s) (C@N), 834(m)
/Mꢁ1 cmꢁ1)): 310 (44,364), 203 (30,211). 1H NMR (300 MHz,
m
(ANHAR), 3240(m)
m(NH), 1613(s)
m
m
m(C@S). UV–Vis (CH3CN, k/nm
(e
DMSO-d6, d, ppm): 10.38 (s, 1H, NH); 8.65 (s, 1H, NH-R); 8.58–
8.41 & 7.81–7.35 (m, 4H, aromatic); 3.08 (s, 3H, CH3), 2.36 (s, 3H,
CH3).
2.3.3. 2-Acetylpyridine-4-phenyl-thiosemicarbazone (HL3)
The ligand was obtained from the reaction 2-acetylpyridine
(0.22 mL; 2 mmol) and 4-pheyl-thiosemicarbazide (0.3344 g;
2 mmol). Yield: 0.4324 g, 80%; Color: yellow solid; M.p.: 185 °C;
Anal. Calcd. for C14H14N4S (270.35 g, molꢁ1): C, 62.19; H, 5.21; N,
20.72; S, 11.85%. Found: C, 62.46; H, 5.56; N, 26.54; S, 11.63%. IR
H
N
H
N
CH3
CH3
H
N
H2N
C
S
R
H
N
R
N
C
S
O
N
N
R = H (HL1)
(KBr, cmꢁ1): 3301(m)
(C@N), 1580(s) (C@N), 801(m) m
m(ANHAR), 3241(m)
m(NH), 1598(s)
R = CH3 (HL2)
R = C6H5 (HL3)
m
m
(C@S). UV–Vis (CH3CN, k/nm
(e
/Mꢁ1 cmꢁ1)): 341 (18,249), 309 (15,890), 297 (16,657), 226
[CoCl2(PPh3)2]
EtOH
(22,190). 1H NMR (300 MHz, DMSO-d6, d, ppm): 10.65 (s, 1H,
NH); 10.25 (s, 1H, NH-R); 8.61–8.49 & 7.85–7.21 (m, 9H, aromatic);
2.40 (s, 3H, CH3).
reflux, 5 h
HN
R
H3C
N
C
2.4. Synthesis of cobalt(III) Schiff base complexes
N
S
N
All the new metal complexes were prepared according to the
following general procedure. A warm ethanolic solution (10 mL)
containing Schiff base ligand (1 mmol) was added to an ethanolic
solution of (10 mL) [CoCl2(PPh3)2] (1 mmol). The resulting red col-
or solution was refluxed for 5 h. Dark red colored crystalline pow-
der was obtained on slow evaporation. They were filtered off,
washed with ethanol, and dried under vacuo.
Cl
Co
[Co(L1)2]Cl (1)
[Co(L2)2]Cl (2)
[Co(L3)2]Cl (3)
S
C
N
R
N
N
N
H
CH3
Scheme 1. Preparation of ligands and corresponding cobalt(III) complexes.

R. Manikandan et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 205–216
207
2.4.1. Synthesis of [Co(L1)2]Cl (1)
Table 1
Crystal data and structure refinement for the complex 1.
The complex was synthesized from [CoCl2(PPh3)2] (0.654 g;
1 mmol) and HL1 (0.194 g; 1 mmol). Yield: 0.3991 g, 83%; Color:
red solid; M.p.: 268 °C; Anal. Calcd. for C16H18ClCoN8S2 (480.89 g,
molꢁ1): C, 39.96; H, 3.77; N, 23.30; S, 13.33%. Found: C, 39.62; H,
3.43; N, 23.49; S, 13.13%. IR (KBr, cmꢁ1): 3376, 3256(m)
Complex
1
Empirical formula
Formula weight
Color
C
16H18ClCoN8S2
480.89
Red
Crystal dimensions (mm)
Temperature (K)
Wavelength (Å)
Crystal system
0.18 ꢂ 0.14 ꢂ 0.03
m
(ANHAR), 1620(s)
m
(C@N), 1598(s)
m
e
(C@N), 1577(s)
m(C@N),
110
772(s) (CAS). UV–Vis (CH3CN, k/nm (
m
/Mꢁ1 cmꢁ1)): 402 (8465),
0.71070
Monoclinic
P21/c
354 (13,900), 296 (28,090), 226 (47,859). 1H NMR (300 MHz,
DMSO-d6, d, ppm): 8.10 (s, 2H, NH2); 8.09–7.44 (m, 4H, aromatic);
2.80 (s, 3H, CH3). ESI-MS, m/z (%): 445.1 (85) [MꢁCl]+.
Space group
Unit cell dimensions (Å, °)
a = 11.501(7)
b = 10.417(7)
c = 17.149(11)
2.4.2. Synthesis of [Co(L2)2]Cl (2)
a
= 90
b = 93.344(3)
= 90
The complex was synthesized from [CoCl2(PPh3)2] (0.654 g;
1 mmol) and HL2 (0.208 g; 1 mmol). Yield: 0.402 g, 79%; Color:
red solid; M.p.: 228 °C; Anal. Calcd. for C18H22ClCoN8S2 (508.94 g,
molꢁ1): C, 42.47; H, 4.36; N, 22.02; S, 12.60%. Found: C, 42.28; H,
c
Volume (Å3)
2051(2)
Z
4
Calculated density (g cmꢁ3
)
1.609
1.197
4.72; N, 22.37; S, 12.91%. IR (KBr, cmꢁ1): 3248(w)
m
(ANHAR),
(C@N), 773(s) (CAS).
/Mꢁ1 cmꢁ1)): 418 (7430), 357 (8091),
Absorption coefficient (mmꢁ1
)
1598(s)
m
(C@N), 1579(s)
m(C@N), 1560(s)
m
m
F(000)
1016
Theta range for data collection (°)
Absorption correction
Refinement method
No. of reflections measured
Data/restraints/parameters
Goodness-of-fit on F2
3.0–27.5
Lorentz-polarization
UV–Vis (CH3CN, k/nm (
e
302 (11,3486), 225 (21,882). 1H NMR (300 MHz, DMSO-d6, d,
ppm): 8.70 (s, 1H, NH-R); 7.82–7.78 & 7.61–7.41 (m, 4H, aromatic);
2.92 (s, 3H, CH3), 2.37 (s, 3H, CH3). ESI-MS, m/z (%): 473.1 (40)
[MꢁCl]+.
Full-matrix least squares on F2
Total: 15511, unique: 4654
3991/0/279
0.969
R indices [I > 2
r
(I)]
R1 = 0.1000, wR2 = 0.2659
3.71, ꢁ1.56
D
qmax, D
qmin (e Å3)
2.4.3. Synthesis of [Co(L3)2]Cl (3)
The complex was synthesized from [CoCl2(PPh3)2] (0.654 g;
1 mmol) and HL3 (0.270 g; 1 mmol). Yield: 0.5317 g, 84%; Color:
red solid; M.p.: 296 °C; Anal. Calcd. for C28H26ClCoN8S2 (633.08 g,
molꢁ1): C, 53.12; H, 4.14; N, 17.69; S, 10.13%. Found: C, 53.43; H,
DNA cleavage experiments were carried out according to re-
ported procedure [46]. For the gel electrophoresis experiment,
supercoiled pBR322 DNA was treated with the cobalt(III) com-
plexes in TEA buffer (10 mmol Tris acetate, 10 mmol EDTA, pH
8.0) and the solution was then incubated at 37 °C for 2 h. Loaded
4.38; N, 17.27; S, 10.54%. IR (KBr, cmꢁ1): 3389(m)
m
(ANHAR),
(C@N), 753(s) (CAS).
/Mꢁ1 cmꢁ1)): 420 (9873), 372 (15,696),
1630(m)
UV–Vis (CH3CN, k/nm (
m
(C@N), 1599(s)
m
(C@N), 1571(s)
m
m
e
254 (27,848), 227 (35,443). 1H NMR (300 MHz, DMSO-d6, d,
ppm): 10.23 (s, 1H, NH-R); 8.68–8.52 & 7.84–7.25 (m, 9H, aro-
matic); 2.20 (s, 3H, CH3). ESI-MS, m/z (%): 597.1 (60) [MꢁCl]+.
20 lL of DNA sample (mixed with bromophenol blue dye, 1:1 ra-
tio), carefully into the wells, along with standard DNA marker.
The samples were analyzed by electrophoresis for 30 min at 50 V
on a 0.8% agarose gel in TEA (4.84 g Tris–acetate, 0.5 mol EDTA/1
L, pH 8.0). The gel was stained with 10 lg/mL ethidium bromide
and observes the bands under illuminator.
2.5. X-ray structure determination of complex 1
All data were obtained using a Rigaku Mercury CCD diffractom-
2.7. Protein binding studies
eter with graphite monochromated Mo
K
a
radiation
(k = 0.71070 Å). The structure was solved by direct methods and
expanded using Fourier techniques [43,44]. All non-hydrogen
atoms were refined anisotropically, using full-matrix least squares
refinements on F2 with crystal structure crystallographic software
package [45]. Table 1 gives further details of data collection, refine-
ment, and the structural details of complex [Co(L1)2].
The excitation wavelength of BSA at 280 nm and the emission at
344 nm were monitored for the protein binding studies. The exci-
tation and emission slit widths and scan rates were maintained
constant for all of the experiments. A stock solution of BSA was
prepared in 5 mM Tris–HCl/50 mM NaCl buffer (pH = 7.2) and
stored in the dark at 4 °C for further use. A concentrated stock solu-
tion of the compounds was prepared as mentioned for the DNA
binding experiments, except that the phosphate buffer was used
instead of a Tris–HCl buffer for all of the experiments. Titrations
were manually done by using a micropipet for the addition of
the complexes.
2.6. DNA binding and cleavage experiments
Luminescence measurement was performed to clarify the bind-
ing affinity of cobalt(III) complexes by emissive titration at room
temperature. The complexes were dissolved in mixed solvent of
5% DMSO and 95% Tris–HCl buffer (5 mM Tris–HCl/50 mM NaCl
buffer for pH = 7.2) for all the experiments and stored at 4 °C for
further use and used within 4 days. Tris–HCl buffer was subtracted
through base line correction. The excitation wavelength was fixed
by the emission range and adjusted before measurements. Emis-
sive titration experiments were performed with a fixed concentra-
2.8. Antioxidant assays
2.8.1. DPPHÅ scavenging assay
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
activity of the compounds was measured according to the method
of Blois [47]. The DPPH radical is a stable free radical. Because of
the odd electron, DPPH shows a strong absorption band at
517 nm in the visible spectrum. As this electron becomes paired
off in the presence of a free radical scavenger, the absorption
vanishes and the resulting decolorization is stoichiometric with
respect to the number of electrons taken up. Various concentrations
of the experimental complexes were taken and the volume was
tion of metal complexes (25
concentration of DNA (0–25
l
M). While gradually increasing the
lM), the emission spectra were mon-
itored by keeping the excitation of the test compounds at 400 nm.
MB-DNA experiments were conducted by adding the complexes to
the Tris–HCl buffer of MB-DNA. The change in the fluorescence
intensity was recorded. The excitation and emission wavelengths
were 605 nm and 684 nm, respectively.

208
R. Manikandan et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 205–216
adjusted to 100
l
L with methanol. About 5 mL of a 0.1 mM metha-
100% relative humidity. The medium containing without samples
were served as control and triplicate were maintained for all
concentrations.
nolic solution of DPPH was added to the aliquots of samples and
standards (BHT and vitamin C) and shaken vigorously. A negative
control was prepared by adding 100
l
L of methanol in 5 mL of
MTT is a yellow water soluble tetrazolium salt. A mitochondrial
enzyme in living cells, succinate-dehydrogenase, cleaves the tetra-
zolium ring, converting the MTT to an insoluble purple formazan.
Therefore, the amount of formazan produced is directly propor-
tional to the number of viable cells. After 48 h of incubation,
0.1 mM methanolic solution of DPPH. The tubes were allowed to
stand for 20 min at 27 °C. The absorbance of the sample was mea-
sured at 517 nm against the blank (methanol).
2.8.2. OHÅ scavenging assay
15 lL of MTT (5 mg/mL) in phosphate buffered saline (PBS) was
The hydroxyl radical scavenging activity of the compounds has
been investigated by using the Nash method [48]. In vitro hydroxyl
radicals were generated by an Fe3+/ascorbic acid system. The
detection of hydroxyl radicals was carried out by measuring the
amount of formaldehyde formed from the oxidation reaction with
DMSO. The formaldehyde produced was detected spectrophoto-
metrically at 412 nm. A mixture of 1.0 mL of iron-EDTA solution
(0.13% ferrous ammonium sulfate and 0.26% EDTA), 0.5 mL of EDTA
solution (0.018%), and 1.0 mL of DMSO (0.85% DMSO (v/v) in 0.1 M
phosphate buffer, pH 7.4) were sequentially added in the test
tubes. The reaction was initiated by adding 0.5 mL of ascorbic acid
(0.22%) and 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 of ice-cold trichloroacetic acid (17.5% w/v). Subsequently,
3.0 mL of Nash reagent was added to each tube and left at room
temperature for 15 min. The intensity of the color formed was
measured spectrophotometrically at 412 nm against the reagent
blank.
added to each well and incubated at 37 °C for 4 h. The medium
with MTT was then flicked off and the formed formazan crystals
were solubilized in 100
bance at 570 nm using a micro-plate reader. The% cell inhibition
was determined using the following formula. Cell inhibi-
lL of DMSO and then measured the absor-
%
tion = 100 ꢁ Abs (sample)/Abs (control) ꢂ 100. Nonlinear regres-
sion graph was plotted between % cell inhibition and log10
concentration and IC50 was determined using GraphPad Prism
software.
3. Results and discussion
The analytical data of the ligands and their corresponding com-
plexes summarized in the experimental section agreed well with
the theoretical values within the limit of experimental error and
confirmed the general formulae [Co(Ln)2]Cl (n = 1–3) proposed for
the new complexes. All these complexes are stable in air at room
temperature and highly soluble in common organic solvents and
water. In addition, ESI-Mass spectra of 1, 2 and 3 were also con-
firmed the molecular weight (496.88, 524.94 and 649.08) of the
complexes. The molecular ion peaks (445.2, 473.4 and 597.2)
[MꢁCl]+ were in good agreement with the suggested empirical for-
mulae as indicated from elemental analyses.
2.8.3. NOÅ scavenging assay
The assay of nitric oxide (NO) scavenging activity is based on a
method [49] where sodium nitroprusside in aqueous solution at
physiological pH spontaneously generates nitric oxide, which
interacts with oxygen to produce nitrite ions. These can be esti-
mated using the Greiss reagent. Scavengers of nitric oxide compete
with oxygen leading to less production of nitrite ions. For the
experiment, sodium nitroprusside (10 mM) in phosphate buffered
saline was mixed with a fixed concentration of the complex and
standards were incubated at room temperature for 150 min. After
the incubation period, 0.5 mL of Griess reagent containing 1% sul-
fanilamide, 2% H3PO4 and 0.1% N-(1-naphthyl) ethylenediamine
dihydrochloride were added. The absorbance of the chromophore
formed was measured at 546 nm.
3.1. X-ray crystallography
The single crystal ORTEP diagram of complex 1 with atom num-
bering scheme is shown in Fig. 1. Selected bond lengths and bond
angles are given in Table 2. The coordination geometry around
Co(III) metal ion can be described as a distorted octahedron, the co-
balt atom being bonded with two tridentate NNS donor ligand
molecules in such a way that four 5-membered chelate rings are
2.9. In vitro cytotoxic activity evaluation by MTT assays
Standard
3-(4,5-dimethylthiozole)-2,5-diphenyltetraazolium
bromide (MTT) assay procedures were used [50]. The monolayer
cells were detached with trypsin–ethylenediaminetetraacetic acid
(EDTA) to make single cell suspensions and viable cells were
counted using
a hemocytometer and diluted with medium
containing 5% FBS to give final density of 1 ꢂ 105 cells/mL. One
hundred microliters per well of cell suspension were seeded into
96-well plates at plating density of 10,000 cells/well and incubated
to allow for cell attachment at 37 °C, 5% CO2, 95% air and 100% rel-
ative humidity. After 24 h the cells were treated with serial con-
centrations of the test samples. They were initially dissolved in
neat dimethylsulfoxide (DMSO) to prepare the stock (200 mM)
and stored frozen prior to use. At the time of drug addition, the fro-
zen concentrate was thawed and an aliquot was diluted to twice
the desired final maximum test concentration with serum free
medium. Additional three, 10 fold serial dilutions were made to
provide a total of four drug concentrations. Aliquots of 100
these different drug dilutions were added to the appropriate wells
already containing 100 L of medium, resulted the required final
lL of
l
drug concentrations. Following drug addition the plates were
incubated for an additional 48 h at 37 °C, 5% CO2, 95% air and
Fig. 1. ORTEP view of complex 1.

R. Manikandan et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 205–216
209
Table 2
nitrogen atom [56]. Further the pyridine ring m(C@N) group fre-
quency shifted to lower frequencies 1577–1560 cmꢁ1 in the com-
plexes indicating that the coordination of the pyridine nitrogen
also. Hence, from the infrared spectroscopic data, it is inferred that
the azomethine nitrogen, pyridine nitrogen and sulfur atoms are
involved in the coordination to metal ion in all the complexes.
Selected bond lengths (Å) and angles (°) for the complex 1.
Co(1)AS(1)
Co(1)AS(2)
Co(1)AN(1)
Co(1)AN(2)
Co(1)AN(5)
Co(1)AN(6)
S(1)AC(7)
S(2)AC(15)
N(1)AC(1)
N(1)AC(5)
N(2)AN(3)
N(2)AC(6)
N(3)AC(7)
N(4)AC(7)
N(5)AC(9)
N(5)AC(13)
N(6)AN(7)
N(6)AC(14)
N(7)AC(15)
N(8)AC(15)
2.2304(11)
2.2136(12)
1.952(3)
1.888(3)
1.962(3)
1.897(3)
1.753(4)
1.754(4)
1.345(5)
1.363(5)
1.382(5)
1.316(5)
1.327(5)
1.341(5)
1.348(5)
1.372(5)
1.385(5)
1.397(6)
1.329(5)
1.329(5)
S(1)ACo(1)AS(2)
S(1)ACo(1)AN(1)
S(1)ACo(1)AN(2)
S(1)ACo(1)AN(5)
S(1)ACo(1)AN(6)
S(2)ACo(1)AN(1)
S(2)ACo(1)AN(2)
S(2)ACo(1)AN(5)
S(2)ACo(1)AN(6)
N(1)ACo(1)AN(2)
N(1)ACo(1)AN(5)
N(1)ACo(1)AN(6)
N(2)ACo(1)AN(5)
N(2)ACo(1)AN(6)
N(5)ACo(1)AN(6)
90.61(4)
169.07(10)
86.23(11)
91.62(10)
94.54(12)
90.00(11)
92.80(12)
169.57(10)
86.93(12)
82.85(14)
89.74(14)
96.39(15)
97.50(15)
179.19(15)
82.74(15)
3.3. Electronic spectra
The electronic spectra of the free ligands exhibited two to three
bands in the region 341–202 nm corresponding to the intra ligand
n–pꢃ transition of the azomethine portion and pꢃ transitions of
p–
the aromatic ring. The bands are shifted to higher energy region
in the spectra of complexes and appeared in the region 372–
225 nm. In addition an intense absorption band observed in all
the complexes in the regions of 420–402 nm were assigned to
ligand to metal charge transfer transition [37].
3.4. 1H NMR Spectra
The 1H NMR spectra of free ligands showed a signal at d 10.65–
10.31 ppm, characteristics of hydrazinic NH proton [42]. But, the
NMR spectra of complexes 1–3 did not register any signals corre-
sponding to hydrazinic NH proton. This indicated that the ligands
adopted the thioenol form, followed by deprotonation prior to
coordination with the metal ion. The signals corresponding to the
protons of aromatic moieties of the ligands and their complexes
were observed as multiplets in the range of d 8.68–7.21 ppm. The
peak appeared at 3.08–2.20 ppm has been assigned to methyl
group [42,57]. In addition, ANHAR proton appeared in the region
at 10.25–8.10 ppm [57].
formed. The azomethinic nitrogens [N(2) and N(6)] are placed in a
trans position. The chlorine atom acts as a counterion.
The NNS planes of the ligands are practically orthogonals, with
angles near to 90°. The bond lengths are similar to those found for
other cobalt(III) compounds with pyridine-2-carbaldehyde thio-
semicarbazone ligand [28]. In the same way, there are not appre-
ciable differences in the bond lengths with respect to the free
thiosemicarbazone ligand [51], except for the N(3)AC(7)/
N(7)AC(15) and C(7)AS(1)/C(14)AS(2) distances. In the free ligand,
the N(3)AC(7) and C(7)AS bond lengths are 1.314(3) and
1.692(3) Å. The shortening of the N(3)AC(7) length with respect
to the free ligand suggests an increase in the double bond character
for this bonding under complexation. The C(7)AS bond shows the
opposite tendency decreasing in double bond character with re-
spect to the free ligand. In complex 1 the four nitrogen and two sul-
fur atoms exhibit ligand–metal–ligand bite angles and are found to
be S(1)ACo(1)AN(2), 86.23(11); S(2)ACo(1)AN(6), 86.93(12);
N(1)ACo(1)AN(2), 82.85(14); N(5)ACo(1)AN(6), 82.74(15) respec-
tively. The trans angles of the complex 1 [S(1)ACo(1)AN(1),
169.07(10); S(2)ACo(1)AN(5), 169.57(10); N(2)ACo(1)AN(6),
179.19(15), respectively] deviate slightly from the expected linear
trans geometry. The bond lengths involving the cobalt(III) ion and
the links in the thiosemicarbazone chain are close to those found in
NNS and ONS systems of alkyl- and arylthiosemicarbazones with
cobalt(III) ions [52–55]. The CoAS and CoANazo distances range be-
tween 2.20–2.23 and 1.86–1.96 Å, respectively, in all thiosemicar-
bazone-cobalt(III) compounds [34,36,38].
Based on the analytical, spectroscopic characterization (FT-IR,
UV-visible, 1H NMR and ESI-Mass) and single-crystal X-ray crystal-
lographic studies of complex 1, an octahedral structure has been
proposed for all the new cobalt(III) complexes (Scheme 1).
3.5. DNA binding properties
3.5.1. Fluorescence spectral studies
Luminescence measurement was performed to clarify the bind-
ing affinity of cobalt complexes to the CT-DNA. The results of the
fluorescence titration for complexes 1–3 with CT-DNA are illus-
trated in Fig. 2. Complexes bound to DNA through intercalation of-
ten show decreased intensities (hypochromism) and a red shift of
their emission bands due to the strong stacking interaction be-
tween the aromatic chromophores of the complex and base pairs
of DNA [58]. All these complexes could emit luminescence at ambi-
ent temperature with a maximum excitation wavelength at 400
and emission wavelength at 429 nm containing Tris–HCl buffer
(5 mM Tris–HCl/50 mM NaCl buffer for pH = 7.2). Upon increasing
concentration of CT-DNA the emission intensities of complex 1
exhibited hypochromism of 21% with red shift of 1 nm at 430 nm
respectively. However the emissive bands at 429 nm exhibited
hypochromism up to 20.8% and 20% of the initial fluorescence
intensity for complexes 2 and 3. Based on the emission behavior
of these DNA binding enhancement, the effect of intensity of bands
decreased from the original intensities. The intrinsic binding con-
stants were obtained according to the following Scatchard
equation.
3.2. Infrared spectra
The IR spectra of the ligands and their corresponding complexes
provided significant information about the metal ligand bonding.
The spectra of the ligands displayed characteristic absorption
bands at 3241–3183, 1613–1598, 1583–1578 and 836–801 cmꢁ1
due to hydrazinic
vibrations respectively. The bands due to hydrazinic
m
NAH, azomethine mC@N
,
m
C@N (pyridine) and mC@S
NAH and mC@S
m
vibrations of the free ligands were absent in the complexes, thus
indicating that thioenolisation and deprotonation had taken place
prior to coordination. This fact was further confirmed by the
appearance of two new bands in the range 1630–1598 cmꢁ1 and
CF ¼ CT ½ðI=I0Þ ꢁ Pꢄ=½1 ꢁ Pꢄ
773–753 cmꢁ1 that corresponds to
m(C@NAN@C) and
m(CAS) stretching
where CT is concentration of the probe (complex) added, CF is the
concentration of the free probe, and I0 and I are its emission inten-
sities in the absence and in the presence of DNA, respectively. P is
the ratio of the observed emission quantum yield of the bound
probe to the free probe. The value of P was obtained from a plot
vibrations respectively [33,35]. The bands attributed to azome-
thine m(C@N) stretching of the ligands were shifted to lower frequen-
cies (1599–1579 cmꢁ1) in the metal complexes indicating the
coordination of the ligands to metal through the azomethine

210
R. Manikandan et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 205–216
Fig. 2. Flourescence spectra of cobalt(III) complexes 1–3 in 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.2 and arrows an indicate absence and presence of incresing amounts of
CT-DNA concentration at 25 °C. [Complex = 25 M (—— lines)], DNA = 0–25 M.
l
l
of I/I0 versus 1/[DNA] such that the limiting emission yield is given
by the y-Intercept. The amount of bound probe (CB) at any concen-
tration was equal to CT ꢁ CF. The Scatchard plots of r/Cf versus r for
complexes 1–3 with increasing concentration of CT-DNA shown in
Fig. 3. A plot of r/CF versus r (=CB/[DNA]) gave the binding isotherm
and the best fit of the data resulted in the intrinsic binding constant
(Kb) values was calculated from the ratio of slope and the intercept
[59]. It has been found that the binding constant (Kb) for complexes
1, 2 & 3 were 1.2 ꢂ 105 Mꢁ1, 8.27 ꢂ 104 Mꢁ1 & 9.4 ꢂ 105 Mꢁ1
respectively. The observed values of Kb revealed that the complexes
bind to DNA via the intercalative mode [60]. From the results ob-
tained, it has been found that complex 3 strongly bound with CT-
DNA than other two complexes and the order of binding affinity
is 3 > 1 > 2. Even though the binding mode of the complexes needs
to be discussed furthermore experiments.
fluorescence resulting from the displacement of MB from a DNA se-
quence by a quencher, and the quenching is due to the reduction of
the number of binding sites on the DNA that are available to the
MB. The fluorescence quenching spectra of DNA-bound MB for
complexes 1, 2 and 3 were shown in Fig. 4. This illustrate that, as
the concentration of the complexes increases, the emission band
at 684 nm exhibited hypochromism up to 16.25%, 43.40% and
29.90% of the initial fluorescence intensity. The observed decrease
in the fluorescence intensity clearly indicates that the MB mole-
cules are displaced from their DNA binding sites and are replaced
by the complexes under investigation [61]. Quenching data were
analyzed according to the following Stern–Volmer equation:
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.
The Kq value is obtained as a slope from the plot of F0/F versus [Q]. In
the Stern–Volmer plot (Fig. 5) of F0/F versus [Q], the quenching con-
3.5.2. Competitive binding with methylene blue
The emission titration results indicate that the complexes effec-
tively bind to DNA. In order to confirm the binding mode and com-
pare their binding affinities, methylene blue (MB) displacement
experiments were carried out. Methylene Blue (MB) is a planar cat-
ionic dye which is widely used as a sensitive fluorescence probe for
native DNA. MB emits intense fluorescent light in the presence of
DNA due to its strong intercalation between the adjacent DNA base
pairs. The displacement technique is based on the decrease of
stant (Kq) is obtained from the slope which was 7.16 ꢂ 103 Mꢁ1
,
4.82 ꢂ 103 Mꢁ1 and 1.04 ꢂ 104 Mꢁ1 for the complexes 1, 2 and 3,
respectively. Further the apparent DNA binding constant (Kapp
)
were calculated using the following equation,
KMB ½MBꢄ ¼ Kapp ½complexꢄ
(where the complex concentration has the value at a 50% reduction
of the fluorescence intensity of MB, KMB (1.0 ꢂ 10ꢁ7 Mꢁ1) is the DNA
binding constant of MB to DNA, [MB] = 7.5 lM), where found to be
5.37 ꢂ 105 Mꢁ1, 3.61 ꢂ 105 Mꢁ1 and 7.5 ꢂ 105 Mꢁ1 respectively for
1, 2 and 3. The experimental results data again suggest that the
complexes bind to DNA via intercalation but the complex 3 binds
to DNA more strongly than the complexes 1 and 2, which is in
agreement with the results observed from the emission spectra.
Since these changes indicate only one kind of quenching process,
it may be concluded that all of the complexes can bind to DNA
via the same mode.
3.6. DNA cleavage activity
The ability of the metal complexes to perform DNA cleavage is
generally monitored by agarose gel electrophoresis and in the
present work pBR322 DNA was chosen to investigate its cleavage.
The DNA cleavage was controlled by relaxation of the supercoiled
circular form of CT-DNA into the open circular form and linear
form. When circular plasmid DNA is run on horizontal gel using
electrophoresis, the supercoiled form will migrate first (Form I).
Fig. 3. Scatchard plots of r/Cf versus r for complexes 1–3 with incresing concen-
tration of CT-DNA.

R. Manikandan et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 205–216
211
Fig. 4. Flourescence quenching curves of MB bound to CT-DNA in presence of complexes (1–3) in 5 mM Tris–HCl/50 mM NaCl buffer at pH 7.2. Arrow shows an indicate
emission intensity changes upon incresing concentration of complexes at 25 °C. [DNA = 7.5 M], [MB] = 7.5 M and complexes [0–50 M].
l
l
l
that the complex 1 exhibits more effective DNA cleavage activity
than the complex 3.
3.7. Fluorescence quenching of BSA with cobalt(III) complexes
Generally, the fluorescence of BSA is caused by two intrinsic
characteristics of the protein, namely tryptophan and tyrosine.
Changes in the emission spectra of tryptophan are common in re-
sponse to protein conformational transitions, subunit associations,
substrate binding, or denaturation. Therefore, the intrinsic fluores-
cence of BSA can provide considerable information on their struc-
ture and dynamics and is often utilized in the study of protein
folding and association reactions. The interaction of BSA with co-
balt complexes was studied by fluorescence measurement at room
temperature. A solution of BSA (1
concentrations of the complexes (0–50
l
M) was titrated with various
M). Fluorescence spectra
l
were recorded in the range of 290–450 nm upon excitation at
280 nm. The effects of the compound on the fluorescence emission
spectrum of BSA are shown in Fig. 7.
Fig. 5. Stern–Volmer plots of the MB–DNA fluorescence titration for complexes 1–
3.
The addition of the above complexes to the solution of BSA re-
sulted in a significant decrease of the fluorescence intensity of BSA
at 344 nm, up to 43.7%, 43.8% and 97.6% from the initial fluores-
cence intensity of BSA accompanied by a hypsochromic shift of 2,
2 and 4 nm for complexes 1, 2 and 3, respectively. The observed
blue shift is mainly due to the fact that the active site in protein
is buried in a hydrophobic environment. This result suggested a
definite interaction of all of the complexes with the BSA protein
[62,63].
Furthermore fluorescence quenching data were analyzed with
the Stern–Volmer equation and Scatchard equation. From the plot
of I0/I versus [Q] The quenching constant (Kq) can be calculated
using the plot of I0/I versus [Q] (Fig. 8A). If it is assumed that the
binding of compounds with BSA occurs at equilibrium, the equilib-
rium binding constant can be analyzed according to the Scatchard
equation:
If the strands (nicking) of the selected DNA is cleaved by interac-
tion with the metal complexes, the supercoils will relax to produce
an open circular form (Form II) that moves slower than form I,
whereas with cleavage of both strands to a linear form (Form III)
the resulting system will migrate in between the above two forms.
As seen in Fig. 6, control photoreactions with control DNA (lane C),
resulted in no obvious DNA cleavage was observed. In contrast,
photoreactions at 365 nm (lanes 1–6), resulted in significant pro-
duction of nicked DNA depending on the concentrations of com-
plexes used. As increasing concentrations of complexes 1 and 3,
the amount of Form I (supercoiled form) of pBR322 DNA dimin-
ishes gradually, whereas that of Form II (circular form) increases.
When concentration reached 40 lg/mL for complex 1, DNA was
completely converted from Form I to Form II. The results indicated
log½ðI0 ꢁ IÞ=Iꢄ ¼ log Kbin þ n log½Qꢄ
where Kbin is the binding constant of the compound with BSA and n
is the number of binding sites. The number of binding sites (n) and
the binding constant (Kbin) have been found from the plot of log
(I0 ꢁ I)/I versus log [Q] (Fig. 8B). The calculated Kq, Kbin, and n values
are given in Table 3. The given value of n is around two for all of the
compounds, indicating the two binding site of BSA hydrophobic
environment. The ability of complexes to quench the emission
intensity of BSA, as attributed from Kq and Kbin values. Among the
three complexes, the complex 3 has better interaction with BSA
than the other two complexes. The order of efficiency of the tested
complexes to bind with BSA was observed as 3 > 1 > 2.
Fig. 6. Agarose gel electrophoresis diagram showing the cleavage of pBR322 DNA
by Co(III) Schiff base complex in TAE Buffer (4.84 g Tris base, pH = 8, 0.5 M EDTA/
1 L). Lane C, Control DNA (untreated complex); Lanes 1–3, in the different
concentrations of complex 1: (1) 10; (2) 20; (3) 40
l
g/mL; Lanes 4–6, in the
g/mL.
different concentrations of complex 3: (1) 10; (2) 20; (3) 40
l

212
R. Manikandan et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 205–216
Fig. 7. The emission spectrum of bovine serum albumin (BSA) (1
lM; kex = 280 nm, kem = 344 nm) in the presence of increasing amounts of the complexes 1–3 (0–50 lM). The
arrow shows that the emission intensity decreases upon the increase in concentration of the compounds.
Fig. 8. Stern–Volmer plots (A) and Scatchard plots (B) of the fluorescence titration of the complexes 1–3 with BSA.
Table 3
effective drugs. Antioxidants that exhibit radical scavenging activ-
ity are receiving increased attention since they present interesting
anticancer, anti-ageing and anti-inflammatory activities [64–66].
Therefore, the compounds with antioxidant properties may offer
protection against rheumatoid arthritis and inflammation.
The radical scavenging activities of our compounds along with
standards, such as butylated hydroxyl toluene (BHT) and Vitamin
C in a cell free system, have been examined with reference to DPPH
radicals (DPPHÅ), hydroxyl radicals (OHÅ), and nitric oxide (NO), and
their corresponding IC50 values were shown in Table 4. From
Table 4, it can be concluded that a much less scavenging activity
Quenching constant (Kq), binding constant (Kbin), and number of binding sites (n) for
the interactions of complexes with BSA.
Complex
Kq (Mꢁ1
)
Kbin (Mꢁ1
)
N
1
2
3
1.96 ꢂ 105
1.90 ꢂ 105
3.66 ꢂ 105
2.38 ꢂ 105
2.27 ꢂ 105
5.61 ꢂ 105
1.83
1.85
2.02
Table 4
Antioxidant activity of ligands, cobalt(III) complexes, BHT and vitamin C against
various radicals.
Compound
IC50
(
l
M)
HL1
250
DPPH
OH
58.39 0.3
NO
38.23 0.2
HL2
HL3
1
2
HL1
63.03 0.2
53.45 0.3
65.56 0.2
15.43 0.4
14.44 0.1
16.53 0.3
86.53 0.6
147.20 0.8
200
HL2
52.00 0.1
70.03 0.4
35.96 0.2
31.86 0.3
39.19 0.3
163.80 0.5
232.20 0.7
36.52 0.5
43.06 0.3
36.85 0.1
32.80 0.3
40.39 0.3
154.30 0.8
215.72 0.9
HL3
3
150
100
50
1
2
3
BHT
BHT
Vit C
Vitamin C
3.8. Evaluation of radical scavenging ability
0
OH
NO
DPPH
Radical
Free radicals play an important role in inflammatory process.
Thus, the compounds with possible antioxidant properties could
play a crucial role against the inflammation and lead to potentially
Fig. 9. Radical scavenging activity of the compounds (HL1–3
& 1–3) and the
standards, BHT and Vitamin C.

R. Manikandan et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 205–216
213
Fig. 10a. The % growth inhibition against log10 concentrations of cobalt(III) complexes (1–3) on breast cancer cell line (MCF-7).
was exhibited by the free ligands when compared to that of their
corresponding cobalt complexes which is due to the chelation of
them with the cobalt ions. The overall scavenging activity of the
tested compounds was found to increase in the order of HL3 <
HL1 < HL2 < 3 < 1 < 2 (Fig. 9). The complex 2 showed better activity
compared to the other complexes, which may be due to the elec-
tron donating effect of methyl group present in HL2 ligand. The
IC50 values (Table 4) indicated that the various radical scavenging
activities of the complexes are in the order of NOÅ < OHÅ < DPPHÅ.
The lower IC50 values observed in antioxidant assays have demon-
strated that these complexes have a strong potential to be applied
as scavengers to eliminate the radicals. Further, it is significant to
mention that the metal complexes synthesized herein possess
superior antioxidant activity against the above said radicals than
that of the standard antioxidant butylated hydroxyl toluene
(BHT) and Vitamin C.
3.9. Cytotoxic activity evaluation by MTT assay
Since the DNA binding and antioxidant experiments conducted
so far revealed that the cobalt(III) complexes exhibit good binding
and antioxidant activity, it is considered worthwhile to study the
cytotoxic activity of these complexes. Cytotoxicity is a common
Fig. 10b. The % growth inhibition against log10 concentrations of cobalt(III) complexes (1–3) on skin carcinoma cell line (A431).
Fig. 11a. Cytotoxic effect of cobalt(III) complexes against MCF-7 at different concentrations (0.1 lM, 1.0 lM, 10 lM, 100 lM). Cell viability decreased with increasing
concentrations of complexes.

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R. Manikandan et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 205–216
Fig. 11b. Cytotoxic effect of cobalt(III) complexes against A431 at different concentrations (0.1 lM, 1.0 lM, 10 lM, 100 lM). Cell viability decreased with increasing
concentrations of complexes.
limitation in terms of the introduction of new compounds in the
pharmaceutical industry.
4. Conclusions
Cytotoxic potential of newly synthesized cobalt(III) complex
was investigated on two cell lines including human skin carcinoma
cell line (A431) and human breast cancer cell line (MCF-7). The
compounds were applied in range of concentrations from 0.1 to
2-Acetylpyridine N-substituted thiosemicarbazone ligands and
their corresponding cobalt(III) complexes have been synthesised
and characterized. The molecular structure of the complex 1 has
been determined by single crystal X-ray diffraction study and it
showed that cobalt(III) cation is coordinated with two ligand mol-
ecules. The two ligand molecules in this complex are deprotonated
at the imino nitrogen, resulting in two tridendate monoanionic
species coordinating to the central metal via the thiolato sulfur,
imine nitrogen and pyridine nitrogen, resulting in a distorted octa-
hedral moiety. The DNA binding properties of the complexes were
investigated by fluorescence measurements. The results supported
the fact that the complexes bind to CT-DNA via intercalation. The
binding constant values (Kb) showed that the DNA binding affinity
increased in the order 3 > 1 > 2. The protein binding properties of
the complexes examined by the fluorescence spectra suggested
that the binding affinity of the complex 3 was stronger than that
of the other complexes. In addition, the complex 1 can efficiently
cleave the pBR322 DNA. The antioxidant studies revealed that all
the complexes exhibited higher activities compared to the stan-
dards BHT and vitamin C. The complexes 1–3 exhibited good cyto-
toxic activity against MCF-7 and A431 cancer cell line. In particular,
among the three complexes, complex 3 exhibited higher cytotoxic-
ity against above cancer cell lines and the IC50 value of the com-
plexes showed that the activity is higher than that of cisplatin.
100 lM and cells left for 48 h. The activities of the compounds
were determined by means of a colorimetric assay (MTT assay)
which measures mitochondrial dehydrogenase activity as an indi-
cation of cell viability, and the results were expressed in terms of
IC50 values. Upon increasing the concentration of complexes from
0.1 lM to 100 lM, the % cell inhibition also increased. It is evident
from Figs. 10 and 11 that the number of cells decreased with an in-
crease in the concentration of the complexes. All the complexes
have cytotoxic potencies, with IC50 values generally in the low
micromolar range. The IC50 values of complexes and standard are
given in Table 5. As a general observation, complex 3 is more active
than the other complexes in the cell lines tested, and it exhibited
much better activity than cisplatin in the MCF-7 cell line. The cyto-
toxicity of the tested complexes against both cancer cell lines fol-
lows the order 3 > 2 > 1. The higher cytotoxic activity for the
complex 3 may be due to terminal phenyl substitution of the coor-
dinated HL3 ligand. By comparing the cytotoxicity with that of the
conventional standard cisplatin, we found that the complexes
exhibited excellent activity in both the cancer cell lines. However,
among the tested cell lines, the cytotoxic activity of complexes
against human breast cancer cell line (MCF-7) was higher than that
of skin carcinoma cell line (A431).
Acknowledgements
We are thankful to the IISC Bangalore, STIC Cochin, SAIF Panjab
University, Chandigarh, KMCH Coimbatore and Biogenics Hubli, In-
dia for providing instrumental facilities. The author (R.N) thankful
to DST for financial assistant in the form Project (No. SR/FT/CS-95/
2010).
Table 5
IC50
(lM) value of cobalt(III) complexes and cisplatin against human breast cancer
cell line (MCF-7) and skin carcinoma cell line (A431).
Complex
IC50
MCF-7
(l
M)a
A431
1
2
3
22.99 0.08
7.24 0.09
1.99 0.13
3.19 0.02
32.12 1.03
16.85 2.09
9.30 0.03
12.33 0.07
Appendix A. Supplementary material
CCDC 917524 contains the supplementary crystallographic data
for the complex [Co(L1)2]Cl. These data can be obtained free of
charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or
Cisplatin
a
Fifty percent inhibitory concentration after exposure for 48 h in the MTT assay.

R. Manikandan et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 205–216
215
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article can be found, in the online version, at http://dx.doi.org/
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