Synthesis, structure and in vitro biological activity of pyridoxal N(4)-substituted thiosemicarbazone cobalt(III) complexes

Article abstract of DOI:10.1016/j.ica.2014.05.035

Cobalt(III) complexes containing pyridoxal N(4)-substituted thiosemicarbazone ligands with the composition [Co(HL^1-2·Cl) (HL^1-2·H₂O)] (1-2) have been synthesized from the reaction of [CoCl₂(PPh₃)

Full text of DOI:10.1016/j.ica.2014.05.035

Accepted Manuscript
Synthesis, structure and in vitro biological activity of pyridoxal N(4)-substituted
thiosemicarbazone cobalt(III) complexes
Rajendran Manikandan, Paranthaman Vijayan, Panneerselvam Anitha,
Govindan Prakash, Periasamy Viswanathamurthi, Ray Jay Butcher,
Krishnaswamy Velmurugan, Raju Nandhakumar
PII:
S0020-1693(14)00327-2
http://dx.doi.org/10.1016/j.ica.2014.05.035
ICA 16017
DOI:
Reference:
Inorganica Chimica Acta
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Revised Date:
Accepted Date:
18 April 2014
29 May 2014
30 May 2014
Please cite this article as: R. Manikandan, P. Vijayan, P. Anitha, G. Prakash, P. Viswanathamurthi, R.J. Butcher,
K. Velmurugan, R. Nandhakumar, Synthesis, structure and in vitro biological activity of pyridoxal N(4)-substituted
thiosemicarbazone cobalt(III) complexes, Inorganica Chimica Acta (2014), doi: http://dx.doi.org/10.1016/j.ica.
2014.05.035
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Synthesis, structure and in vitro biological activity of pyridoxal N(4)-
substituted thiosemicarbazone cobalt(III) complexes
Rajendran Manikandana, Paranthaman Vijayana, Panneerselvam Anithaa, Govindan Prakasha,
Periasamy Viswanathamurthia,*, Ray Jay Butcherb, Krishnaswamy Velmuruganc, Raju
Nandhakumarc
aDepartment of Chemistry, Periyar University, Salem-636 011, Tamil Nadu, India.
bDepartment of Chemistry, Howard University, 525 College Street NW, Washington, DC
20059, USA.
cDepartment of Chemistry, Karunya University, Karunya Nagar, Coimbatore - 641 114,
India.
* Corresponding author. Fax: +91 427 2345124.
E-mail address: viswanathamurthi72@gmail.com (P. Viswanathamurthi).
1

Abstract
Cobalt(III) complexes containing pyridoxal N(4)-substituted thiosemicarbazone ligands with
the composition [Co(HL1-2.Cl)(HL1-2.H2O)] (1-2) have been synthesized from the reaction of
[CoCl2(PPh3)2]
and
pyridoxal
N-methyl-thiosemicarbazone
hydrochloride
(H3L1.Cl)/pyridoxal N-phenyl-thiosemicarbazone hydrochloride (H3L2.Cl). The richness of
the coordination chemistry of this ligand is highlighted by the modulation of its charge from
neutral (H3L.Cl) (L) to dianionic (HL.Cl) (L2-) and monoanionic forms (HL.H2O) (L-) and
coordinated as tridentate binegative mode around cobalt(III) ion by forming neutral complex.
The new complexes were characterized by various analytical and spectroscopic techniques
1
(IR, electronic, H-NMR and ESI-Mass). The X-ray crystal structure of the complex 2,
demonstrated distorted octahedral coordination geometry around the metal centre. Further,
the investigation of effect of substitution (CH3 or C6H5) on terminal N(4)-nitrogen of
thiosemicarbazone exhibited its influence on the potential binding and cleavage ability with
DNA, BSA binding, free radical scavenging and cytotoxicity.
Keywords: Cobalt(III) complexes; DNA interaction; Protein binding; Antioxidant;
Cytotoxicity.
2

1. Introduction
Schiff base compounds are strong chelators obtained from the efficient condensation of
aldehydes with amines. Metal complexes of these chelate ligands have variety of potential
applications in diverse areas like medicine, agriculture, analytical chemistry, catalysis,
enzyme modeling, magneto-structural chemistry, miscellaneous studies, dye and polymer
industry etc. Aromatic Schiff bases derived from pyridine carbaldehyde and
thiosemicarbazides exhibit admirable yield with high purity compared to their aliphatic
counterpart, mainly because of the elevated strength and stability of their imine bonds
conferred by the electron-rich pyridinyl side groups. In aromatic Schiff bases, pyridoxal
based ones and especially, their metal complexes have drawn special interest in the past few
decades due to their powerful antitumor activities [1, 2].
Previous reports on metal complexes of pyridoxal thiosemicarbazone ligands always
suggests a terdentate binding mode via the S, N (iminic) and O (phenolic) donor atoms (Fig.
1a-b). Different types of stoichiometry are observed for this binding mode depending on the
nature of the inorganic anion, M(H2L)X2 (M = Mn, Cu or Zn; X = C1 or NO3), M(HL)X (M
= Co, Cu or Zn; X = C1 or CH3CO2), ML (M = Ni or Cu; from acetate salts),
.
[Co(HL)L].4.5H2O, [Co(HL)2]Cl.EtOH, [Ni(HL)py]NO3,
[Ni(H2L)2](NO3)2 H2O,
[{MoO2(HL)}Cl]n, [Fe(HL)2]Xn.nH2O (X = SO4 or NO3) and [Fe(HL)(L)].8H2O [3]. In some
cases the coordination via phenolic-oxygen, imine-nitrogen and azomethine nitrogen atoms
(Fig. 1c) [4] or through the hydroxymethyl group is also possible (Fig. 1d) [2]. In an AuIII
complex, the ligand coordinates bidentately through the N and S-atoms of the
thiosemicarbazide fragment (Fig. 1e) [5]. Unusual coordination of the HL- anion to the metal
only via its S-atom was also found in the pyridoxal thiosemicarbazonato complex of
dimethylthallium(III) (Fig. 1f) [6]. As shown in Scheme 1, the ligand pyridoxal
thiosemicarbazone can be found in different states from the neutral zwitterionic form L to the
dianionic form L2- and monoanionic form L-.
<Insert Figure 1 and Scheme 1>
Cobalt is an element of biological attention and its role is mainly focused on its presence
in the active center of vitamin B12, which regulates indirectly the synthesis of DNA [7].
Additionally, cobalt is an essential element present in co-enzyme of vitamin B12 and which
is used as a supplement of the vitamin [8] and at least eight cobalt dependent proteins have
been reported [7]. So far since the first reported studies on the biological activity of Co
complexes [9] in 1952, diverse structurally characterized cobalt complexes have been studied
as hydrolytic agents for DNA cleavage [10] and others showing antitumor-antiproliferative
3

[11-13], antimicrobial [14-16], antifungal [17,18], antiviral [19,20] and antioxidant [21,22]
activity have been reported. For the last decade, our group has been actively engaged in the
synthesis and characterization of N(4)-substituted thiosemicarbazone complexes with various
transition metals and studied their applications [23]. In the continuation of our efforts in
understanding
the
coordination
propensities
of
pyridoxal
N(4)-substituted
thiosemicarbazones, we have carried out the structural characterization of new cobalt(III)
complexes containing pyridoxal N(4)-substituted thiosemicarbazones (H3L1–2.Cl) and tested
their cytotoxicity on human breast carcinoma (MCF-7) and human cervix carcinoma (HeLa)
tumor cell lines. Hence, in this paper, we are reporting the synthesis, characterization,
crystallography, DNA binding/cleavage, protein binding ability and cytotoxic activities of
new Co(III) pyridoxal N(4)-substituted thiosemicarbazone complexes.
2. Experimental
2.1. Materials and methods
All the reagents used were chemically pure and AR grade. The solvents were purified and
.
dried according to standard procedures. CoCl2 6H2O, 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 Bangalore GeNei, Bangalore,
India. The human breast carcinoma cell line (MCF-7) and human cervix carcinoma cell line
(HeLa) 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).
0
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.
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 obtained as KBr pellets on a Nicolet Avatar model spectrophotometer at
4000 - 400 cm-1 range. Electronic spectra of the ligand and their complexes have been
recorded in methanol using a Shimadzu UV - 1650 PC spectrophotometer at 800 - 200 nm
1
range. Emission spectra were measured with a Jasco FP 6600 spectrofluorometer. H-NMR
spectra were recorded in Jeol GSX - 300 instrument at room temperature using
tetramethylsilane as the internal standard in dimethylsulfoxide (DMSO-d6) as solvent. The
4

ESI-MS spectra were recorded by LC-MS Q-ToF Micro analyzer (Shimadzu) in the SAIF,
Punjab University, Chandigarh. Melting points were checked on a Technico micro heating
table and are uncorrected. The requisite precursor metal complex [CoCl2(PPh3)2] were
prepared according to the literature method [24].
2.3. Preparation pyridoxal thiosemicarbazone hydrochloride ligands
The synthetic routes for ligands (H3L1-2.Cl) are presented in Scheme 2. Pyridoxal N(4)-
substituted thiosemicarbazone hydrochloride has been prepared according to a modification
of the procedure described in the literature [25]. Substituted thiosemicarbazide (2 mmol) was
dissolved in 10 mL of ethanol with constant stirring, and was added to pyridoxal
hydrochloride (2 mmol) dissolved in 10 mL of ethanol. The mixture was stirred at 80 ºC for
30 min. After the reaction mixture was cooled to room temperature, the yellow solid was
isolated by filtration, washed with ethanol and dried in vacuo.
2.3.1. Pydridoxal-4-methyl-thiosemicarbazone hydrochloride (H3L1.Cl)
H3L1.Cl was prepared from the reaction of pyridoxal hydrochloride (0.4072 g; 2 mmol) and
4-methyl-thiosemicarbazide (0.2103 g; 2 mmol). Yield: 0.4245 g, 73%; Colour: yellow solid;
M. p.: 226 ºC; Anal. Calc. for C10H15ClN4O2S (290.77 g, mol-1): C, 41.30; H, 5.19; N, 19.26;
S, 11.02%. Found: C, 41.12; H, 5.36; N, 19.57; S, 10.88%. IR (KBr, cm-1): 3380(m)
ν(CH2OH), 3300(m) ν(-NH-R), 3200(s) ν(OH), 3152(m) ν(NH), 2706(m) ν(NH+), 1615(s)
ν(C=N), 830(s) ν(C=S). UV-Vis (CH3OH, λ/nm (ε/M-1 cm-1)): 230 (11 467), 338 (11 567).
1H NMR (300 MHz, DMSO-d6, δ, ppm): 12.17 (s, 1H, OH), 8.93 (s, 1H, NH), 8.65 (s, 1H,
NH-R), 8.52 (s, 1H, CH=N), 8.17 (s, 1H, Py), 5.39 (s, 1H, OH), 4.78 (s, 2H, CH2), 3.02 (s,
3H, CH3), 2.62 (s, 3H, CH3).
2.3.2. Pydridoxal-4-phenyl-thiosemicarbazone hydrochloride (H3L2.Cl)
H3L2.Cl was prepared from the reaction of pyridoxal hydrochloride (0.4072 g; 2 mmol) and
4-phenyl-thiosemicarbazide (0.3344 g; 2 mmol). Yield: 0.5645 g, 80%; Colour: yellow solid;
M. p.: 226 ºC; Anal. Calc. for C15H17ClN4O2S (352.84 g, mol-1): C, 51.06; H, 4.85; N, 15.87;
S, 9.08%. Found: C, 50.79; H, 4.73; N, 15.58; S, 9.42%. IR (KBr, cm-1): 3388(s) ν(CH2OH),
3360(m) ν(-NH-R), 3240 ν(OH), 3157(m) ν(NH), 2822(m) ν(NH+), 1640(s) ν(C=N), 860(m)
ν(C=S). UV-Vis (CH3OH, λ/nm (ε/M-1 cm-1)): 233 (31 547), 324 (36 671), 349 (35 568). 1H
NMR (300 MHz, DMSO-d6, δ, ppm): 11.58 (s, 1H, OH), 9.97 (s, 1H, NH), 8.90 (s, 1H, NH-
5

R), 8.40 (s, 1H, CH=N), 7.52-7.75 (m, 5H, Ph), 6.97 (s, 1H, Py), 5.38 (s, 1H, OH), 4.78 (s,
2H, CH2), 1.56 (s, 3H, CH3).
2.4. Synthesis of cobalt(III) complexes
All the new metal complexes were prepared according to the following general procedure.
A warm ethanol solution (10 mL) containing H3L1-2.Cl (1 mmol) was added to ethanol
solution (10 mL) of [CoCl2(PPh3)2] (1 mmol). The resulting reddish solution was refluxed for
5 h. Dark red coloured crystalline powder was obtained on slow evaporation. They were
filtered off, washed with cold ethanol, and dried under vacuo.
2.4.1. Synthesis of [Co(HL1.Cl)(HL1.H2O)] (1)
The complex was synthesized from [CoCl2(PPh3)2] (0.654 g; 1 mmol) and H3L1.Cl (0.2907
g; 1 mmol). Yield: 0.4456 g, 72%; Colour: red solid; M.p.: 172 ºC; Anal. calcd for
C20H28ClCoN8O5S2 (619.00 g, mol-1): C, 38.81; H, 4.56; N, 18.10; S, 10.36%. Found: C,
38.53; H, 4.86; N, 18.49; S, 10.62%. IR (KBr, cm-1): 3422(b) ν(H2O), 3391(m) ν(CH2OH),
3286(s) ν(-NH-R), 2776(m) ν(NH+), 1611(s) ν(C=N), 1580(s) ν(C=N), 755(s) ν(C-S). UV-
Vis (CH3OH, λ/nm (ε/M-1 cm-1)): 223 (21 5711), 254 (15 1373), 358 (53 094), 438 (44 870)
1H NMR (300 MHz, DMSO-d6, δ, ppm): 8.69 (s, 1H, NH-R), 8.53 (s, 1H, CH=N), 7.60 (s,
1H, Py), 5.29 (s, 1H, OH), 4.72 (s, 2H, CH2), 2.86 (s, 3H, CH3), 2.16 (s, 3H, CH3). ESI-MS,
m/z (%): 565.0 (57) [M-Cl.H2O]+.
2.4.2. Synthesis of [Co(HL2.Cl)(HL2.H2O)] (2)
The complex was synthesized from [CoCl2(PPh3)2] (0.654 g; 1 mmol) and H3L2.Cl (0.3528
g; 1 mmol). Single crystals suitable for X-ray diffraction were grown at room temperature
from ethanol. Yield: 0.6019 g, 81%; Colour: red solid; M.p.: 196 ºC; Anal. calcd for
C30H32ClCoN8O5S2 (743.14 g, mol-1): C, 48.49; H, 4.34; N, 15.08; S, 8.63%. Found: C,
48.74; H, 4.66; N, 15.57; S, 8.42%. IR (KBr, cm-1): 3420(b) ν(H2O), 3396(m) ν(CH2OH),
3370(s) ν(-NH-R), 2751(s) ν(NH+), 1600(s) ν(C=N), 1586(s) ν(C=N), 750(s) ν(C-S). UV-Vis
(CH3OH, λ/nm (ε/M-1 cm-1)): 225 (10 74740), 263 (91 6151), 372 (29 0074), 454 (23 8945).
1H NMR (300 MHz, DMSO-d6, δ, ppm): 9.90 (s, 1H, NH-R), 8.85 (s, 1H, CH=N), 7.55-7.78
(m, 5H, Ph), 6.98 (s, 1H, Py), 5.28 (s, 1H, OH), 4.78 (s, 2H, CH2), 2.16 (s, 3H, CH3). ESI-
MS, m/z (%): 689.1 (68) [M-Cl.H2O]+.
6

2.5. X-ray Crystallography
Crystal data were collected on an Oxford/Agilent Gemini diffractometer. Structures were
solved using the direct methods program SHELXL [26]. All nonsolvent heavy atoms were
located using subsequent difference Fourier syntheses. The structures were refined against F2
with the program SHELXL [27], in which all data collected were used including negative
intensities. All nonsolvent heavy atoms were refined anisotropically. All nonsolvent
hydrogen atoms were idealized using the standard SHELXL idealization methods.
2.6. DNA binding and cleavage experiments
Luminescence measurement was performed to clarify the binding 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 oC 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. Emissive titration
experiments were performed with a fixed concentration of metal complexes (25 µM). While
gradually increasing the concentration of DNA (0 - 25 µM), the emission spectra were
monitored 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
and 684 nm, respectively.
DNA cleavage experiments were carried out according to reported procedure [28]. For the
gel electrophoresis experiment, supercoiled pBR322 DNA was treated with the cobalt(III)
complexes in TEA buffer (10 mM Tris acetate, 10 mM EDTA, pH = 8.0) and the solution
was then incubated at 37 ºC for 2 h. Loaded 20 µL of DNA sample (mixed with bromophenol
blue dye, 1:1 ratio), 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 µg/mL ethidium
bromide and observed the bands under illuminator.
2.7. Protein binding
The excitation wavelength of BSA at 280 nm and the emission at 344 nm were monitored
for the protein binding studies. The excitation and emission slit widths and scan rates were
7

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.
The concentrated stock solutions of the complexes were 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 a micropipet for the addition of
the complexes.
2.8. Antioxidant assays
.
2.8.1. DPPH scavenging assay
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the compounds
was measured according to the method of Blios [29]. 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 adjusted to 100 µL with methanol. About 5 mL of
a 0.1 mM methanolic 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
µL of methanol in 5 mL of 0.1 mM methanolic solution of DPPH. The tubes were allowed to
stand for 20 min at 27 ºC. The absorbance of the sample was measured at 517 nm against the
blank (methanol).
.
2.8.2. OH scavenging assay
The hydroxyl radical scavenging activity of the compounds has been investigated by using
the Nash method [30]. 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 spectrophotometrically at 412 nm. A mixture of 1.0 mL of iron-EDTA solution
(0.13% ferrous ammonium sulphate 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 ºC for 15 min in a water bath. After incubation, the
8

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 colour formed was measured
spectrophotometrically at 412 nm against the reagent blank.
.
2.8.3. NO scavenging assay
The assay of nitric oxide (NO) scavenging activity is based on a method [31] where
sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric
oxide, which interacts with oxygen to produce nitrite ions. These can be estimated 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% sulfanilamide, 2% H3PO4 and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride
were added. The absorbance of the chromophore formed was measured at 546 nm.
2.9. In vitro cytotoxic activity evaluation by MTT assays
Standard 3-(4,5-dimethylthiozole)-2,5-diphenyltetraazolium bromide (MTT) assay
procedures were used [32]. 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 1x105 cells/mL. One hundred microlitres 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 ºC, 5% CO2, 95% air and 100% relative humidity. After 24 h the cells
were treated with serial concentrations 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 frozen 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 µL of these different drug dilutions were added to the
appropriate wells already containing 100 µL of medium, resulted the required final drug
concentrations. Following drug addition the plates were incubated for an additional 48 h at 37
9

ºC, 5% CO2, 95% air and 100% relative humidity. The medium containing without samples
were served as control and triplicate were maintained for all concentrations.
MTT is a yellow water soluble tetrazolium salt. A mitochondrial enzyme in living cells,
succinate-dehydrogenase, cleaves the tetrazolium ring, converting the MTT to an insoluble
purple formazan. Therefore, the amount of formazan produced is directly proportional to the
number of viable cells. After 48 h of incubation, 15 µL of MTT (5 mg/mL) in phosphate
buffered saline (PBS) was added to each well and incubated at 37 ºC for 4 h. The medium
with MTT was then flicked off and the formed formazan crystals were solubilized in 100 µL
of DMSO and then measured the absorbance at 570 nm using a micro plate reader. The %
cell inhibition was determined using the following formula. % cell Inhibition = 100 - Abs
(sample)/Abs (control) x 100. Nonlinear regression graph was plotted between % Cell
inhibition and Log10 concentration and IC50 was determined using GraphPad Prism software.
3. Results and Discussion
Reaction in warm ethanol solution of H3L1-2.Cl and [CoCl2(PPh3)2] in 1:1 molar ratio
(Scheme 2) with the following expectations that these pyridoxal N(4)-substituted
thiosemicrazones could behave as
<Insert Scheme 2>
(i) A binegative tridentate ONS chelating system to yield a four coordinated complex
(product 1), or
(ii) A uninegative tridentate chelating system through ONS donor atoms to form yet
another type of four coordinated complex (product 2).
Unexpectedly, the pyridoxal N(4)-substituted thisemicarbazone ligands (H3L1-2.Cl)
involved in this work deceived all our expectations and formed stable six coordinated metal
chelates with 1:2 metal–ligand stoichiometry (product 3), yielded the red bis ligand
complexes of the general formula [Co(HL1-2.Cl)(HL1-2.H2O)] (1-2) (Scheme 2). The isolated
complexes are crystalline powder that are stable in air, in addition to being soluble in
common organic solvents such as dichloromethane, chloroform, benzene, acetonitrile,
ethanol, methanol, dimethylformamide, DMSO and also soluble in water. All the new
complexes were structurally characterized by elemental analyses, IR, electronic, 1H NMR and
ESI-Mass spectra. In addition single crystals of complex 2, used to determine its molecular
structure by X-ray crystallographic analysis, which is shown in Fig. 2.
10

3.1. IR spectra
IR spectroscopy has been used in order to confirm the bideprotonation and binding mode
of pyridoxal N(4)-substituted thiosemicarbazones and identify the tautomeric as well as the
ionic form of pyridoxal thiosemicarbazones in the solid state (Figs. S1-S4, Supporting
information). Evidence of the solid compound being the HCl adduct appears from the IR
spectrum in which a band at 2706-2822 cm-1 could be addressed as the ν(N–H+ Cl-)
stretching vibration confirming that the nitrogen atom of the pyridine ring bears a proton
[33]. There is no peak observed around 2500 cm-1 assignable to the SH group; absorptions
attributed at 830-860 cm-1 which could be assigned to the ν(C=S) vibration of the free ligand
indicating that it is present in the thione form. This band completely disappeared in the
complexes, and two new bands appeared at 1580-1586 cm−1 and 750-755 cm-1 corresponding
to a possible ν(C=N–N=C) and ν(C-S) stretching vibrations respectively, indicating coordination of
a thiolate sulfur atom after enolization followed by deprotonation. In the high-energy range
there appears a strong and broad band with the maximum at 3380-3388 cm-1 (ligand), i.e., at
3391-3396 (complexes) that can be unambiguously assigned to the ν(OH) (hydroxymethyl)
vibration [34]. In addition, the new broad band appeared at 3420-3422 cm-1 are assigned to
pyridine ring NH+ adduct H2O (ν(N–H+.H2O)) in complexes. Further, the stretching
vibrations of the ν(NHR) (aminic) group appear in the 3286-3370 cm-1 region; while the
νNH (hydrazinic) band occurring at 3152-3157 cm-1 in the free ligands disappears in the
complexes, suggesting deprotonation of the –NH group [34]. Coordination of the phenolate
group is indicated by the disappearance of the OH stretching vibration as observed for the
free ligands at 3200-3240 cm-1 in the IR spectra of the complexes [25]. The free ligands
showed a very strong absorption around 1615-1640 cm-1 characteristic of the azomethine
ν(>C=N) group. In all the complexes, the band due to azomethine was observed at a lower
region around 1600-1611 cm-1 indicating the coordination of azomethine nitrogen to cobalt.
3.2. Electronic spectra
The electronic spectral data for the investigated ligands and complexes in methanol,
recorded in the range of 200–800 nm. As can be seen (Figs. S5-S8, Supporting information),
the spectra display two to four electronic transitions. The strong peak at the blue end of the
spectra of the compounds at 230 -324 nm can be ascribed to π-π* pyridine and phenyl ring
11

absorptions [35]. The bands appeared in the region 338-372 nm have been assigned to n-π*
transitions of imine function of azomethine moiety. Metallation of ligands causes a red shift
in these electronic transitions for complexes. Also the absorption band at 438-454 nm is
attributed to ligand-to-metal charge transfer transitions (LMCT) [36].
3.3. 1H NMR spectra
1H NMR spectroscopy has also been used in order to confirm the bideprotonation of the
pyridoxal N(4)-substituted thiosemicarbazones and the stability of the complexes in solution
1
(Figs. S9-S12, Supporting information). The H NMR spectra of the ligands showed a broad
signal at δ 11.58-12.17 and 8.93-9.97 due to the phenolic OH and N–NH– protons, indicating
their existence in the hydroxy-thione tautomeric form possessing a –NH–(C=S)–NHR moiety
[37]. In the spectra of ligands, a singlet observed at δ 8.40-8.52 was assigned to azomethine
proton and the signal appeared at δ 6.97-8.17 due to the aromatic pyridine proton [3(k)] . The
presence of a singlet at δ 3.02 was assigned to N-methyl protons of H3L1.Cl ligand and
multiplet appeared at δ 7.52-7.75 assignable to N-phenyl protons of the H3L2.Cl ligand. A
sharp singlet at δ 8.65-8.90 confirmed the presence of terminal NH protons. The peak
appeared at δ 1.56-2.62 has been assigned to methyl group. The methylene protons and
aliphatic OH protons (CH2OH) appeared in the region at δ 4.78 and 5.38-5.39, respectively
for all the ligands. The 1H NMR spectra of the complexes exhibited a sharp singlet at δ 8.53-
8.85 and 8.69-9.90 corresponding to azomethine proton and terminal –NH protons of the
thiosemicarbazone, respectively. A singlet which appeared at δ 6.98-7.60 in all the new
complexes has been assigned to aromatic pyridine protons. The N-methyl group protons and
N-phenyl group protons appeared in the region at δ 2.86 and 7.55-7.78, respectively. The
methyl protons, methylene protons and aliphatic OH protons (CH2OH) also appeared in the
corresponding region for all the complexes.
3.4. Mass Spectra
ESI-Mass spectral analyses of the new complexes were studied in order to confirm the
molecular mass of the complexes (Figs. S13-S14, Supporting information). Complexes 1 and
2 showed an intense peak at m/z = 565.0 and 689.1 respectively, due to the [M-Cl.H2O]+ ion.
The obtained molecular masses are in good agreement with that of the calculated molecular
masses.
12

3.5. X-ray crystallography
Single-crystal X-ray analysis reveals that complex 2 crystallize in the triclinic system,
with space group P21/c. Crystal details, crystallographic data, and refinement were
summarized in Table 1. Selected bond lengths and bond angles have been reported in Table
2. The ORTEP view of the molecular structure with atom labelling scheme of the respective
complex 2 without hydrogen atom has been shown in Fig. 2. The [Co(HL3.Cl)(HL3.H2O)]
unit contains a Co(III) ion in an distorted octahedral environment of O2N2S2 donor atoms
from the two tridentate pyridoxalthiosemicarbazone ligands. The [Co(O2N2S2)] coordination
core consists of two oxygen atoms (phenolate), two nitrogen atoms (imine) in trans
disposition, and two sulfur atoms. The binding of the ligands involve the formation of four
metallocycles, comprised of two five-membered thiosemicarbazide S, N-chelate ring together
with two six-membered pyridoxilydene N, O-chelate ring. Though the donar atoms and
biding modes of the two ligands are same, they are not identical in nature. i.e, one ligand
bound with metal as bianoinic whiles the other as monoanionic and so the complex remains
neutral with cobalt ion in 3+ oxidation state. Bianionic form (form II) of the ligand
(HL2.Cl)(L2-) has bound via deprotonated phenolic oxygen and thiolic sulphur formed in the
rearrangement hydrazinic bonds. Pyridinium cation of this form has been satisfied by
chloride ion and hence as the whole the ligand act as bianionic in nature. Monoanioic form
(form III) of the ligand (HL2.H2O)(L-) is similar with form II in binding modes, however the
pyridinum ion has not been satisfied by negative ions and hence the ligand shows
monoanionic nature. Neverthless the pyridinium hydrogen create hydrogen bond with a water
molecule (Scheme 1) which is evident from the crystal structure of complex 2. The distances
to the ligand donor atoms are Co–S1A = 2.2151(16) Å, Co–S1B = 2.1961(13) Å; Co–O1A =
1.911(4) Å, Co–O1B = 1.9273(13) Å; and Co–N1A = 1.914(4) Å, Co–N1B = 1.911(4) Å
respectively and these values are equal to that of several cobalt(III) complexes previously
noted [3(c)]. The angles around the Co center deviate significantly from 90°, the N1B–Co–
S1A and O1A–Co–N1A angles have opened up to 95.97 and 94.64°, respectively and the
N1A–Co–S1A and O1A–Co–N1B angles have reduced to 86.97 and, 82.50° indicating
distortion from a regular octahedron [38, 39]. The packing of complex 2 is supported by
intermolecular hydrogen bonding between the H-atom of water and the N(3)-atom of the
hydrazinic group and Cl atom as well as between the oxygen atom of water and H-atom of
the pyridinyl group N(2). The observed molecular conformation in complex 2 is stabilized by
an intramolecular N-H...Cl and N-H...H2O interactions (Fig. 3).
13

<Insert Figure 2 and 3>
Table 1
Crystal data and structure refinement for the complex 2.
Complex
2
Empirical formula
Formula weight
Colour
C30H32ClCoN8O5S2
743.14
Red
Crystal dimensions (mm3)
Temperature (K)
Wavelength (Å)
Crystal system
0.3545x0.2683x0.2280
123(2)
1.54184
Monoclinic
P 21/c
Space group
Unit cell dimensions (Å, °)
a = 13.875(2)
b = 17.8123(15)
c = 13.7429(12)
β = 105.122(12)
3279.0(6)
Volume Å3
Z
4
Calculated density (Mg/m3)
Absorption coefficient (mm-1)
F(000)
1.505
6.490
1536
Theta range for data collection (°)
Absorption correction
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
R indices [I>2σ(I)]
3.30-74.16
Analytical
Full-matrix least squares on F2
6464 /3/413
1.040
R1 = 0.0803, wR2 = 0.1928
R1 = 0.1136, wR2 = 0.2286
0.914 and -0.653
R indices (all data)
Largest diff. peak and hole (e Å-3)
14

Table 2
Selected bond lengths (Å) and angles (°) for the complex 2.
Co-O(1A)
1.911(4)
1.914(4)
2.2151(16)
1.9273(13)
1.911(4)
2.1961(13)
1.280(6)
1.369(6)
1.289(7)
1.752(6)
1.291(6)
1.387(5)
1.291(7)
1.751(5)
1.292(7)
1.378(7)
1.285(7)
1.372(6)
0.8800
O(1A)-Co-S(1A)
O(1B)-Co-S(1B)
N(1B)-Co-N(1A)
O(1A)-Co-N(1A)
N(1A)-Co-S(1A)
O(1A)-Co-S(1B)
O(1A)-Co-N(1B)
O(1A)-Co-O(1B)
N(1B)-Co-S(1A)
S(1B)-Co-S(1A)
N(1B)-Co-O(1B)
N(1A)-Co-O(1B)
N(1B)-Co-S(1B)
N(1A)-Co-S(1B)
O(1B)-Co-S(1A)
176.81(12)
176.87(8)
176.58(19)
94.64(18)
86.97(14)
90.61(11)
82.50(17)
89.78(12)
95.97(14)
92.12(6)
Co-N(1A)
Co-S(1A)
Co-O(1B)
Co-N(1B)
Co-S(1B)
O(1A)-C(1A)
N(1A)-N(3A)
N(1A)-C(8A)
S(1A)-C(9A)
O(1B)-C(1B)
N(1B)-N(3B)
N(1B)-C(8B)
S(1B)-C(9B)
N(3A)-C(9A)
N(4A)-C(9A)
N(3B)-C(9B)
N(4B)-C(9B)
N(2A)-H(2AA)
N(2B)-H(2BA)
95.79(13)
86.06(13)
87.34(12)
90.81(13)
87.59(6)
0.8800
15

3.6. DNA binding properties
DNA is the primary pharmacological target of antitumor drugs and therefore, it is essential
to explore the interactions of metal complexes with DNA for the development of effective
chemotherapeutic agents. Particularly, metal complexes in a well-tailored ligand framework
are responsible for specific shape and surface features that complement the molecular target
site usually major/minor groove of the DNA double helix and metal complex-DNA
interactions are therefore, of paramount importance. The mode and propensity of binding of
the metal complexes to CT-DNA were examined by using emission spectra.
3.6.1. Fluorescence spectral studies
Fluorescence emission spectroscopy is an effective method to examine the binding mode
and binding extent of metal complexes with DNA. In general, hypochromism and red-shift
are associated with the intercalation of the metal complexes to the DNA helix, due to the
intercalative mode involving a strong stacking interaction between the aromatic chromophore
of the metal complexes and the base pairs of DNA [40]. The emission spectra of the two
cobalt(III) complexes in the absence and presence of CT-DNA are given in Fig. 4. It can be
seen from this figure, when titrated by CT-DNA, the emission bands of complexes 1 and 2 at
428 and 414 nm exhibit hypochromism of about 20.12% and 18.36% with slightly red shifts
of 3 nm. The marked decreases in the fluorescence intensity of the new complexes indicate
the intercalative binding mode of DNA. In order to quantitatively investigate the binding
affinity of the cobalt(III) complexes toward CT-DNA, the intrinsic binding constants Kb were
determined by monitoring the changes in emissions at 428 and 414 nm for complexes 1 and 2
using the following equation Scatchard equation.
CF = CT [(I/I0)–P]/[1–P]
where, CT is concentration of the probe (complex) added, CF is the concentration of the free
probe, and I0 and I are its emission intensities 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 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
concentration was equal to CT–CF. The scatchard plots of r/CF versus r for complexes 1 and 2
with incresing concentration of CT-DNA shown in Fig. S15 (Supporting information). 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 [41]. The K (emission binding constant) values are 1.69 x 105 M-1 and 1.76 x 105 M-
16

1
for complexes 1 and 2. These results revealed that the complexes bind to DNA via
intercalation mode. The order of the binding constants is 2 > 1, indicates that 2 is showing
stronger binding ability due to having N(4)-substitution of thiosemicarbazone for phenyl
enhances DNA binding affinity. The interaction of phenyl with DNA is favoured by the
larger extension of the aromatic moiety. This result is in accordance with other works on
cobalt complexes containing phenylthiosemicarbazone [42].
<Insert Figure 4>
3.6.2. Competitive studies with methylene blue
DNA binding study point out that the new cobalt(III) complexes effectively bind to DNA.
Further, this has been confirmed by methylene blue (MB) displacement experiments. In
general, the intrinsic fluorescence intensity of DNA is very low, and that of MB in Tris–HCl
buffer is also not high due to quenching by the solvent molecules. However, on addition of
DNA to MB, the fluorescence intensity of MB will be enhanced because of its intercalation
into the DNA. Thus, MB can be used to probe the interaction of complexes with DNA. The
fluorescence intensity of MB can be quenched by the addition of second DNA binding
molecule by either replacing the MB and/or by accepting the excited-state electron of the MB
through a photoelectron transfer mechanism. The fluorescence spectra of MB were measured
using an excitation wavelength of 605 nm, and the emission range was set between 630 and
850 nm. The fluorescence quenching spectra of DNA-bound MB for complexes 1 and 2 were
shown in Fig. S16 (Supporting information). This illustrate that, as the concentration of the
complexes increases, the emission band at 684 nm exhibited hypochromism up to 51.69%
and 76.24% of the initial fluorescence intensity. The observed decrease in the fluorescence
intensity clearly indicates that the MB molecules are displaced from their DNA binding sites
and are replaced by the complexes under investigation [43]. Quenching data were analyzed
according to the following Stern-Volmer equation:
F0/F = Kq [Q] + 1
where F0 is the emission intensity in the absence of complex, F is the emission intensity in
the presence of complex, Kq is the quenching constant, and [Q] is the concentration of the
complex. The Kq value is obtained as a slope from the plot of F0/F versus [Q]. In the Stern-
Volmer plot (Fig. S17, Supporting information) of F0/F versus [Q], the quenching constant
(Kq) is obtained from the slope which was 1.4 x 104 M-1 and 4.30 x 104 M-1 for the
complexes 1 and 2 respectively. Further the apparent DNA binding constant (Kapp) were
calculated using the following equation,
17

KMB [MB] = Kapp [complex]
(where the complex concentration has the value at a 50% reduction of the fluorescence
intensity of MB, KMB (1.0 × 10-7 M-1) is the DNA binding constant of MB to DNA, [MB] =
7.5 µM), where found to be 1.05 x 106 M-1 and 3.2 x 106 M-1 respectively for 1 and 2. The
experimental results data again suggest that the complexes bind to DNA via intercalation, but
the highest value of complex 2 suggesting its stronger ability to displace MB from the MB-
DNA system.
3.7. DNA cleavage activity
Since cobalt(III) complexes showed good binding propensity with CT DNA, therefore the
DNA cleavage activity of complexes was evaluated by agarose gel electrophoresis using
supercoiled pBR322 plasmid DNA in a medium of 10 mM Tris acetate/10 mM EDTA buffer
solution (pH 8.0). The reaction mixture was subjected to agarose gel electrophoresis with
increasing complex concentrations incubated at 310 K for 30 min. The activity of complexes
was assessed by the conversion of DNA from Form I (Supercoiled Form) to Form II (Nicked
Circular Form) and then to Form III (Linear Form). As shown in Fig. 5, it was observed that
the complexes was found to exhibits nuclease activity at different concentration (10, 25 & 50
µg/mL), followed by conversion of supercoiled DNA (Form I) into NC DNA (Form II)
without concurrent formation of Form III, suggesting single strand DNA cleavage. When the
concentration was increased from 10 µg/mL to 50 µg/mL, the production of NC form of
DNA was also increased. The figure shows that the complex 2 exhibits higher cleavage
activity than the complex 1.
<Insert Figure 5>
3.8. BSA binding studies
Serum albumin (SA) is the most abundant protein in plasma and is capable of binding,
transporting and delivering an extraordinarily diverse range of endogenous and exogenous
compounds like fatty acids, nutrients, steroids, certain metal ions, hormones and a variety of
therapeutic drugs [44-46] in the blood stream to their target organs [47]. Because of its
structural homology with human serum albumin (HSA), bovine serum albumin (BSA) has
been most extensively studied. Binding to these proteins may lead to loss or enhancement of
the biological properties of the original drug, or provide paths for drug transportation. In
order to get more information on the binding of the complexes with BSA, fluorescence
18

spectrum of BSA was studied upon the addition of the complexes. Though BSA contains
three fluorophores, namely, tryptophan, tyrosine, and phenylalanine, the intrinsic
fluorescence of BSA is mainly due to tryptophan alone, because phenylalanine has a very low
quantum yield and the fluorescence of tyrosine is almost quenched when it becomes ionized
or near to an amino group, a carbonyl group, or a tryptophan residue [48]. Changes in the
emission spectra of tryptophan are common in response to protein conformational transitions,
subunit associations, substrate binding, or denaturation. Hence, the interaction of BSA with
cobalt complexes was studied by fluorescence measurement at room temperature. A solution
of BSA (1 µM) was titrated with various concentrations of the complexes (0-50 µM) and
fluorescence spectra were recorded in the range of 290 − 500 nm upon excitation at 280 nm.
On increasing the concentration of complexes, a progressive decrease in the fluorescence
intensity was observed, accompanied with a blue shift are shown in Fig. S18 (Supporting
information).
It can be seen from Fig. S18, the fluorescence emission intensities of BSA at 344 nm show
moderate decreasing trend with increasing concentration of the two complexes, up to 62.69%
and 57.44% from the initial fluorescence intensity of BSA accompanied by a hypsochromic
shift of 2 and 3 nm indicating that the interaction of the complexes with BSA could cause
changes in protein secondary structure leading to changes in tryptophan environment of BSA
[49].
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. S19A, Supporting information). If it is
assumed that the binding of complexes with BSA occurs at equilibrium, the equilibrium
binding constant can be analyzed according to the Scatchard equation:
log [(I0 – I)/I] = log Kbin + n log [Q]
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 vs log [Q] (Fig. S19B, Supporting information). The calculated Kq,
Kbin, and n values are given in Table 3. The values of Kq and Kbin suggested that the complex
2 interact with BSA more strongly than the complex 1 could be due to the presence of
extended delocalization of electrons by the aromatic ring ie., phenyl group substituted at the
thiosemicarbazone which facilitate its BSA binding propensity as observed in the DNA
binding experiments discussed in the previous section.
Table 3
19

Quenching constant (Kq), binding constant (Kbin), and number of
binding sites (n) for the interactions of complexes with BSA.
Complex
1
2
Kq (M-1)
Kbin (M-1)
1.12 x 105
1.27 x 105
N
1.52
1.60
8.58 x 104
8.66 x 104
3.9. Evaluation of radical scavenging ability
The free radicals play an important role in the inflammatory process. Many
thiosemicarbazone derivatives with a broad spectrum have been reported to act either as
inhibitors of free radical production or as radical scavengers [23,42, 50-54]. Thus,
compounds with possible antioxidant properties could play a crucial role against
inflammation and can lead to potentially effective drugs. Antioxidants which exhibit radical
scavenging activity have been receiving increased attention since they show interesting
anticancer, anti-aging and anti-inflammatory activities [52-57]. The radical scavenging
activities of our compounds along with standards such as butylated hydroxytoluene (BHT)
and Vitamin C in a cell free system have been examined with reference to DPPH radicals
(DPPH), hydroxyl radicals (OH), and nitric oxide (NO) and their corresponding IC50 values
have been tabulated in Table 4. The power of radical scavenging ability of the ligands and
complexes were found to be higher than that of the standard antioxidants (Fig. S20,
Supporting information). By comparing the antioxidant activity of the ligands and with that
of the metal complexes it became clear that the metal complexes possess higher scavenging
activity towards various free radicals than the respective parent ligands. The NO scavenging
power of the tested complexes was found to be enhanced than that for the other radicals
studied.
Table 4
20

Antioxidant activity of ligands, cobalt(III) complexes, BHT and vitamin C against various
radicals.
IC50 (µM)
Compound
DPPH
OH
NO
H3L1.Cl
H3L2.Cl
1
65.32 0.5
61.21 0.2
48.73 0.3
45.27 0.4
86.53 0.6
147.20 0.8
47.45 0.3
43.74 0.4
39.83 0.1
33.68 0.6
163.80 0.5
232.20 0.7
33.92 0.2
32.29 0.4
27.39 0.2
26.53 0.3
154.30 0.8
215.72 0.9
2
BHT
Vitamin C
3.10. Cytotoxic activity evaluation by MTT assay
The positive results obtained from DNA binding, DNA cleavage, protein binding and
antioxidative studies of the new Co(III) pyridoxl N(4)-substituted thiosemicarbazone
complexes encouraged us to test its cytotoxicity against a panel of human cancer cell lines. In
vitro cytotoxicity of compounds was evaluated by means of the standard MTT-dye reduction
assay which is a widely used method in biological evaluation. The cytotoxic activity of
pyridoxal N(4)-substituted thiosemicarbazone ligands and their Co(III) complexes were
tested against human breast carcinoma (MCF-7) and human cervix carcinoma (HeLa) tumor
cell lines. Compounds were dissolved in DMSO and blank samples containing the same
volume of DMSO were taken as controls to identify the activity of solvent in this cytotoxicity
experiment. The exhibited IC50 values (the concentration needed to inhibit 50% of the cellular
proliferation) after 48 h of incubation are given in Table 5. The cell viability was
concentration-dependent (1-100 µM), with increasing the concentrations a decrease in cell
viability was observed (Fig. S21a-b, Supporting information).
Table 5
21

IC50 (µM) value of cobalt(III) complexes and cisplatin against human
breast carcinoma cell line (MCF-7) and human cervix carcinoma cell
line (HeLa).
IC50 (µM)a
MCF-7
>100
Compound
HeLa
>100
>100
H3L1.Cl
H3L2.Cl
1
>100
6.01 0.08
1.00 0.09
3.19 0.02
83.24 1.03
20.28 2.09
53.50 0.07
2
Cisplatin
aFifty percent inhibitory concentration after exposure
for 48 h in the MTT assay.
The IC50 value showed that the Co(III) complex exhibited significant activity against
MCF-7 and HeLa cell lines. Interestingly, on comparing the IC50 values of complex 2 with
well-known anticancer drug cisplatin against MCF-7 and HeLa cell lines the inhibitory
activity of the complex 2 is about two to three times higher than that of cisplatin. It was also
observed that 1 and 2 demonstrated a noticeable cytotoxicity against all cell lines when
compared with the free ligands, implying that the biological activity is largely ascribed by the
presence of the Co(III) metal centre. In addition, complex 2 exhibited a slightly higher
cytotoxicity than complex 1 against both cell lines. These results suggest that the terminal
phenyl substitution of the ligand increased the cytotoxic activity of the complex 2 in these
two cell lines, as observed in the screening of other previously reported antitumor agents
containing phenyl substitution [58,59]. Furthermore, a comparison of the biological activities
of new complexes with that of our previously reported cobalt complexes with other ligands
has revealed that the complex 2 exhibits superior activity [23d].
4. Conclusions
Two new trivalent cobalt complexes with pyridoxal N(4)-substituted thisemicarbazone
ligands have been synthesized and well characterized in detailed by elemental analysis and
1
spectral techniques (IR, electronic, H NMR and ESI-Mass). The molecular structure of the
complex 2 investigated through X-ray crystallography demonstrated distorted octahedral
geometry around the metal ion with two ligand molecules. From the X-ray analysis, it was
found that in complex 2, the two ligands coordinated as a dibasic tridentate donor by forming
a stable pairs of five-membered and six-membered rings through phenolic oxygen,
azomethine nitrogen, and thiolate sulfur atoms. The new complexes were subjected to studies
22

of their potential biological properties such as DNA binding/cleavage, protein binding,
antioxidant and cytotoxicity. The DNA interaction of the complexes has been evaluated by
fluorescence spectroscopy which revealed that both complexes can bind to DNA via
intercalation. The experimental results suggested that the complex 2 can bind to DNA more
strongly than the complex 1 due to phenyl substitution in thiosemicarbazone. Both the
complexes cleaved plasmid pBR322 DNA through gel electrophoresis assay. The protein
binding properties of the complexes examined by the fluorescence spectra suggested that the
binding affinity increases in the phenyl substitution at the terminal nitrogen of the
thiosemicarbazone moiety. In addition, the complexes also exhibited excellent radical
scavenging activities over the ligands, and standard antioxidants. The cytotoxicity of the
complexes in MTT assay showed that the complexes exhibited higher activity than their
parent ligands and the predictable standard cisplatin in both cell lines used for the
investigation. Correlating the activity of the complexes with their substitution of
thiosemicarbazone, we found that the substitution of the phenyl ring at the thiosemicarbzone
of the ligand increases the degree of cytotoxicity.
Acknowledgements
We are thankful to the IISC Bangalore, STIC Cochin, SAIF Panjab University,
Chandigarh, KMCH Coimbatore and Biogenics Hubli, India for providing instrumental
facilities. The author (R. N) thankful to DST for financial assistant in the form project
(No.SR/FT/CS-95/2010).
Supplementary material
CCDC 977570 contains the supplementary crystallographic data for the complex
[Co(HL3.Cl)(HL3.H2O)]. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:
deposit@ccdc.cam.ac.uk.
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23

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27

HO
HO
H
N
H
N
H
N
N
N
C
S
N
C
S
R
R
N
N
H
O
H
O
M
M
CH3
CH3
(a)
(b)
(R = H, CH3 or C6H5)
CH3
M
H
O
HO
N
S
C
H
N
N
CH2CH3
S
N
H
N
H
N
C
CH3
N
NH
HO
H
O
OH
M
CH3
H
H
N
N
(c)
H3CH2C
C
S
N
N
O
H
M
CH3
(d)
OH
HO
H
H
N
N
N
N
C
H
N
C
S
H
M
HO
CH3
N
N
N
H
O
S
M
M
CH3
CH3
(f)
(e)
Fig. 1. Modes of binding in pyridoxal thiosemicarbazone ligands.
28

HO
N
HO
HO
N
H
N
H
N
H
N
H
N
N
N
N
C
S
R
N
C
S
R
N
C
S
R
N
H
OH
H
O
H
O
Cl
Cl
H2O
CH3
CH3
CH3
Form II
(HL1-2.Cl) (L2-)
Form III
(HL1-2.H2O) (L-)
Form I
(H3L1-2.Cl) (L)
(R = CH3 or C6H5)
Scheme 1. Pyridaxol N(4)-substituted thiosemicarbazone ligand modulation.
29