Spectral characterization, electrochemical and anticancer studies on some metal(II) complexes containing tridentate quinoxaline Schiff base

Article abstract of DOI:10.1016/j.saa.2014.02.075

Co(II), Ni(II), Cu(II) and Zn(II) complexes of a tridentate ONO donor Schiff base ligand derived from 3-(2-aminoethylamino)quinoxalin-2(1H)-one were synthesized. The ligand and its metal complexes were characterized using elemental analysis, molar conductance, IR, ¹H NMR, mass, magnetic susceptibility, electronic spectra and ESR spectral studies. Electrochemical behavior of the synthesized compounds was studied using cyclic voltammetry. The grain size of the synthesized compounds was determined by powder XRD. The Schiff base and its complexes have been screened for their antimicrobial activities against the bacterial species E. coli, K. pneumoniae, P. aeruginosa and S. aureus; fungal species include, A. niger, and C. albicans by disc diffusion method. The results show that the complexes have higher activity than the free ligand. The interaction of the complexes with calf thymus DNA (CT DNA) has been investigated by electronic absorption method. Furthermore, the DNA cleavage activity of the complexes was studied using agarose gel electrophoresis. In vitro anticancer studies of the ligand and its complexes using MTT assay was also done.

Full text of DOI:10.1016/j.saa.2014.02.075

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 396–404
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spectral characterization, electrochemical and anticancer studies
on some metal(II) complexes containing tridentate quinoxaline
Schiff base
⇑
Justin Dhanaraj Chellaian , Jijo Johnson
Department of Chemistry, University College of Engineering Nagercoil, Anna University, Tirunelveli Region, Konam, Nagercoil 629004, Tamil Nadu, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
ꢀ
Tridentate ONO donor quinoxaline
Schiff base ligand.
Complexes have antimicrobial
activity.
Complexes show DNA binding and
DNA cleaving abilities.
The metal complexes possesses
anticancer activity.
N
O
NH
O
O
N
N
M
N
N
O
O
HN
O
N
(Where M = Co(II), Ni(II), Cu(II) and Zn(II))
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 January 2014
Received in revised form 6 February 2014
Accepted 13 February 2014
Available online 26 February 2014
Co(II), Ni(II), Cu(II) and Zn(II) complexes of a tridentate ONO donor Schiff base ligand derived from 3-(2-
aminoethylamino)quinoxalin-2(1H)-one were synthesized. The ligand and its metal complexes were
characterized using elemental analysis, molar conductance, IR, 1H NMR, mass, magnetic susceptibility,
electronic spectra and ESR spectral studies. Electrochemical behavior of the synthesized compounds
was studied using cyclic voltammetry. The grain size of the synthesized compounds was determined
by powder XRD. The Schiff base and its complexes have been screened for their antimicrobial activities
against the bacterial species E. coli, K. pneumoniae, P. aeruginosa and S. aureus; fungal species include,
A. niger, and C. albicans by disc diffusion method. The results show that the complexes have higher activ-
ity than the free ligand. The interaction of the complexes with calf thymus DNA (CT DNA) has been inves-
tigated by electronic absorption method. Furthermore, the DNA cleavage activity of the complexes was
studied using agarose gel electrophoresis. In vitro anticancer studies of the ligand and its complexes using
MTT assay was also done.
Keywords:
Schiff base
ONO donor
Metal complexes
Antimicrobial
DNA
MTT assay
Ó 2014 Elsevier B.V. All rights reserved.
Introduction
preparative accessibility, diversity and structural variability [1].
Metal based and metal binding agents are used for design of inor-
Metal chelate Schiff base complexes have continued to play the
role of one of the most important stereochemical models in main
group and transition metal coordination chemistry due to their
ganic drugs. The use and significance of inorganic compounds had
been important to the medical field [2]. Schiff base compounds are
well known to exhibit a wide range of applications in pharmaceu-
tical, antimicrobial, anticarcinogenic reagents, industrial and ana-
lytical fields [3]. Many transition metal complexes have been
utilized as probes of DNA structure, as agents for mediation of
⇑
Corresponding author. Tel.: +91 4651245765.
E-mail address: c.justindhanaraj@gmail.com (J.D. Chellaian).
http://dx.doi.org/10.1016/j.saa.2014.02.075
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

J.D. Chellaian, J. Johnson / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 396–404
397
strand scission of duplex DNA and as chemotherapeutic agents [4].
Coordination of organic compounds with metal ions causes drastic
change in the biological properties of the ligand and the metal ion
moieties. Nitrogen containing heterocyclic molecules constitutes
the largest portion of chemical entities, which are part of many
natural products, fine chemicals and biologically active pharma-
ceuticals vital for enhancing the quality of life [5]. Transition metal
complexes with their varied coordination environments and tun-
able redox and spectral properties are able to interact with duplex
DNA in various ways [6]. One of the potential approaches in anti-
cancer chemistry is focused on the design of new metal com-
pounds with different substituents and labile sites which may
increase their cytotoxicity [7]. Studies on synthesis of quinoxa-
line-based Schiff base complexes have considerable importance
because of their interesting chemical and biological properties.
Quinoxaline derivatives are widely distributed in nature and have
a variety of biological applications. Quinoxalines are also impor-
tant in material science, as magnetic materials, organic light emit-
ting diodes and non-linear materials [8].
pneumoniae, P. aeruginosa and S. aureus; also the fungal species,
A. niger, and C. albicans by disc diffusion method [10]. Chloram-
phenicol was used as standard antibacterial agent whereas Nysta-
tin was used the standard antifungal agent. The test organisms
were grown on nutrient agar (Muller Hinton agar for bacteria
and antimytotic agar for fungi) medium in petri plates. The com-
pounds were prepared in DMF and soaked in filter paper disc of
5 mm diameter and 1 mm thickness. The discs were placed on
the previously seeded plates and incubated at 37 °C and the diam-
eter of inhibition zone around each disc was measured after 24 h
for bacterial and 72 h for fungal species.
DNA binding experiment
Electronic absorption titrations were performed in Tris–HCl/
NaCl buffer (5 mmol Lꢁ1 Tris–HCl/50 mmol Lꢁ1 NaCl buffer pH
7.2) using DMF (10%) solution of metal complexes at room temper-
ature. The concentration of CT-DNA was determined from the
absorption intensity at 260 nm with
e ) -
value of 6600 (mol Lꢁ1 ꢁ1
The present study describes the synthesis of Schiff base derived
from 3-(2-aminoethylamino)quinoxalin-2(1H)-one and o-vanillin
(1). Subsequently, the complexes of Co(II), Ni(II), Cu(II) and Zn(II)
ions with 1 were synthesized and characterized.
cmꢁ1. Absorption titration experiments were made using different
concentrations of CT-DNA, keeping the complex concentration
constant. Correction was made for absorbance of the CT-DNA itself.
Metal-DNA solutions were allowed to incubate for 5 min before the
absorption spectra were recorded. For metal(II) complexes,
the intrinsic binding constant (Kb) was determined by monitoring
the changes of absorption in the MLCT band with increasing
concentration of DNA using the following equation [11,12].
Experimental
Materials
½DNAꢂ=ðea
ꢁ
ef Þ ¼ ½DNAꢂ=ðeb
ꢁ
ef Þ þ 1=Kbðeb
ꢁ
ef
Þ
The chemicals used were of AnalaR or synthesis grade, o-vanil-
lin was obtained from Spectrochem PVT. LTD., Mumbai, India and
o-phenylenediamine was purchased from Loba Chemie India and
was purified by recrystallization. Oxalic acid, ethylenediamine
and metal(II) chlorides were obtained from Merck and were used
as received without any further purification. Solvents were purified
and dried before use by standard procedures [9].
where [DNA] is the concentration of DNA in base pairs. The
apparent absorption coefficients ea ef and eb correspond to Aobsd
[complex], the extinction coefficient of the complex when fully
bound to equilibrium binding constant in (mol Lꢁ1 ꢁ1
respectively.
,
/
)
Each sample solution was scanned from 200 to 500 nm.
Cleavage of pUC18 DNA
Instruments
The extent of cleavage of super coiled pUC18 DNA to its nicked
circular form was studied using agarose gel electrophoresis. pUC
Elemental analysis of ligand and its metal complexes were car-
ried out using Perkin-Elmer elemental analyzer. Molar conduc-
tance of the complexes was measured using a coronation digital
conductivity meter. IR spectra were recorded using Jasco FTIR-
18 DNA (0.3 l
g) dissolved in 5 mmol Lꢁ1 Tris–HCl/50 mmol Lꢁ1
NaCl buffer (pH 7.2), was treated with the complexes. The mixture
was incubated at 37 °C for 2 h and then mixed with the loading
buffer containing 25% bromophenol blue, 0.25% xylene cyanol
410 spectrometer in KBr pellets from 400 to 4000 cmꢁ1 1H NMR
.
and 30% glycerol. Each sample (10ꢁ3 M, 0.5
lL) was loaded into
spectra were recorded with Brucker 300 MHz spectrometer using
CDCl3 solvent for ligand and its Zn(II) complex with TMS as inter-
nal standard. DART-MS spectrum was recorded by JEOL-Accu TOF
JMS mass spectrometer. Magnetic moments were measured by
Guoy method and corrected for diamagnetism of the component
using Pascal’s constants. Electronic spectra were recorded on Ther-
mo Scientific Evolution-200 UV–Visible spectrophotometer in the
range 190–1100 nm. ESR spectrum of the Cu(II) complex was
recorded at 300 and 77 K in DMSO solution using Varian, USA
E-112 ESR spectrometer using tetracyano etthylene (TCNE) as
g-marker. Cyclic voltammetry measurements were performed
using electrochemical analyzer CH instruments electrochemical
work station (Model 650 C) using a glassy carbon working
electrode (GCE) with Ag/AgCl reference electrode and platinum
counter electrode. Powder XRD studies were carried out using
XPERT-PRO, X-ray diffractometer system.
1% (w/v) agarose gel. Electrophoresis was undertaken for 2 h at
50 V in Tris–acetate–EDTA (TAE) buffer (pH 8.0). The gel was
stained with ethidium bromide for 5 min after electrophoresis
and then photographed under a UV transilluminator. To enhance
the DNA cleaving activity of the complexes, hydrogen peroxide
(100 l
mol Lꢁ1) was added to each sample. The DNA cleavage effi-
ciency of the complexes was measured by determining the ability
of the complexes to convert the super coiled DNA to nicked circular
form and linear form.
In vitro anticancer study
The human cervical cancer cell line (HeLa) was obtained from
National Centre for Cell Science (NCCS), Pune and grown in Eagles
Minimum Essential Medium (EMEM) containing 10% fetal bovine
serum (FBS). All cells were maintained at 37 °C, 5% CO2, 95% air
and 100% relative humidity. The monolayer cells were detached
with trypsin-ethylenediamine tetraacetic acid (EDTA) to make
single cell suspensions and viable cells were counted using a
hemocytometer and diluted with a medium containing 5% FBS to
Biological studies
In vitro antimicrobial activity
In vitro antibacterial and antifungal activities of the ligand and
its complexes were tested against the bacterial species E. coli, K.
give final density of 1 ꢃ 105 cells/mL. 100
lL per well of cell sus-
pension were seeded into 96-well plates at plating density of

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J.D. Chellaian, J. Johnson / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 396–404
10,000 cells per well and incubated at 37 °C, 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) and diluted to twice the desired
final maximum test concentration with serum free medium. Addi-
tional four, 2-fold serial dilutions were made to provide a total of
AOACH3), 3.95 (S, 1H, free ANH), 6.75–8.63 (m, Ar–H); DART-MS
(m/z): 339.
Synthesis of metal complexes
Methanolic solution of metal(II) chlorides (2 mmol) was added
in drops to a methanolic solution of the ligand (1) (4 mmol) and
the mixture was refluxed on a water bath for 3–4 h. The solid com-
plex obtained was filtered, washed with hot methanol and dried in
vacuum over anhydrous calcium chloride.
Co(II) complex (C36H32CoN8O6 (2)): Brownish pink solid; Yield
(%): 62; Anal. Calcd (%): C (59.10), H (4.41), N (15.32), O (13.12),
Co (8.06); Found (%): C (59.30), H (4.84), N (15.40), O (13.18), Co
(8.32); IR ˆ(cmꢁ1): 2945 (ring NH), 1605 (HC@NA), 1239 (CAO),
485 (MAN), 609 (MAO); DART-MS (m/z): 732; Molar conductance
(Oꢁ1 cm2 molꢁ1): 18.3.
five sample concentrations. Aliquots of 100
sample dilutions were added to the appropriate wells already
containing 100 L of medium, gave the required final sample con-
lL of these different
l
centrations. Following drug addition, the plates were incubated for
an additional 48 h at 37 °C, 5% CO2, 95% air and 100% relative
humidity. The medium without samples were served as control
and triplicate was maintained for all concentrations.
3-[4,5-Dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT)
assay
Ni(II) complex (C36H32N8NiO6 (3)): Pale green solid; Yield (%): 73;
Anal. Calcd. (%): C (59.12), H (4.41), N (15.32), O (13.13), Ni (8.02);
Found (%): C (59.34), H (4.84), N (15.04), O (13.28), Ni (8.17); IR
ˆ(cmꢁ1): 2947 (ring NH), 1608 (HC@NA), 1234 (CAO), 492
(MAN), 628 (MAO); DART-MS (m/z): 733; Molar conductance
(Oꢁ1 cm2 molꢁ1): 14.9.
Cu(II) complex (C36H32N8CuO6 (4)): Dark green solid; Yield (%):
67; Anal. Calcd. (%): C (58.73), H (4.38), N (15.22), O (13.04), Cu
(8.63); Found (%): C (58.79), H (4.25), N (15.35), O (13.43), Cu
(8.79); IR ˆ(cmꢁ1): 2940 (ring NH), 1603 (HC@NA), 1236 (CAO),
490 (MAN), 581 (MAO); DART-MS (m/z): 737; Molar conductance
(Oꢁ1 cm2 molꢁ1): 29.2.
3-[4,5-Dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide
(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,
15 ll of MTT (5 mg/ml) in phosphate buffered saline (PBS) was
added to each well and incubated at 37 °C for 4 h. The medium
with MTT was then flicked off and the formazan crystals formed
were dissolved in 100 lL of DMSO and absorbance at 570 nm
was measured using micro-plate reader. The % cell inhibition was
determined using the following formula [13,14].
Zn(II) complex (C36H32N8O6Zn (5)): Yellow solid; Yield: 59%, Anal.
Calcd. (%): C (58.58), H (4.37), N (15.18), O (13.01), Zn (8.86); Found
% Cell inhibition ¼ 100 ꢁ Abs ðsampleÞ=Abs ðcontrolÞ
(%): C (58.79), H (4.47), N (15.32), O (13.23), Zn (8.73); IR ˆ(cmꢁ1
)
ꢃ 100:
ð1Þ
2934 (ring NH), 1605 (HC@NA), 1231 (CAO), 496 (MAN), 590
(MAO), 1H NMR (CDCl3) d (ppm): 9.25 (S, 1H, ACH@N), 3.91 (S,
1H, AOACH3), 11.12 (S, 1H, ring ANH), 6.38–7.83 (m, Ar–H),
DART-MS (m/z): 739. Molar conductance (Oꢁ1 cm2 molꢁ1): 30.1.
Nonlinear regression graph was plotted between % cell inhibi-
tion and log concentration and IC50 was determined using Graph
Pad Prism software.
Results and discussion
Synthesis of ligand
The ligand 1 was prepared by the condensation of 3-(2-amino-
ethylamino)quinoxalin-2(1H)-one with o-vanillin (Scheme 1). The
solid complexes obtained by the reaction between 1 and metal(II)
chlorides were filtered, washed with hot methanol and dried in
vacou over anhydrous calcium chloride.
The metal(II) complexes of 1 are non-hygroscopic, soluble in
DMSO, DMF and sparingly soluble in chloroform. The complexes
are stable at room temperature. The elemental analysis data of
the ligand 1 and its metal complexes (2–5) indicate that the metal
to ligand ratio is 1:2.
3-(2-Aminoethylamino)quinoxalin-2(1H)-one was prepared by
the condensation of quinoxaline-2,3(1,4H)-dione and ethylenedia-
mine. To the hot aqueous solution of 3-(2-aminoethylamino)qui-
noxalin-2(1H)-one (2 mmol), methanolic solution of o-vanillin
(2 mmol) was added drop by drop. Immediately after the addition
of o-vanillin an orange red solid (1) was formed. The reaction mix-
ture was heated with stirring at 50–60 °C for 2 h. The solid sepa-
rated out was cooled to room temperature, filtered and washed
with petroleum ether. Then the compound was recrystallised from
methanol and dried in vacuum over anhydrous calcium chloride
(Scheme 1).C18H18N4O3: Orange red solid; Yield (%): 87; Anal. Calcd
(%): C (63.89), H (5.36), N (16.56), O (14.19); Found (%): C (63.68), H
(5.43), N (16.60), O (14.19); IR ˆ(cmꢁ1): 2847 (free NH), 3415
(o-vanillin AOH), 3041 (quinoxaline AOH), 1614 (HC@NA), 1247
(CAO); 1H NMR (CDCl3) d (ppm): 13.19 (S, 1H, o-vanillin AOH),
13.59 (S, 1H, quinoxaline AOH) 9.14 (S, 1H, ACH@N), 3.91 (S, 1H,
Molar conductance
The metal(II) complexes of ligand 1 (10ꢁ3 mol dmꢁ3) were dis-
solved in DMF and molar conductance of the solutions at room
temperature was measured. The molar conductance data indicate
H
H
N
N
N
N
N
O
+
N
NH2
OH
HO
o-vanillin
N
H
O
HO
O
3-(2-aminoethylamino)quinoxalin-
(1)
2(1H)-one
O
Scheme 1. Synthetic route of 1.

J.D. Chellaian, J. Johnson / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 396–404
399
that, all the metal complexes are having conductance values in
accordance with non-electrolytes [15,16].
complex, due to variation in electron density due to chelation.
Singlets appeared at 3.95 and 3.91 ppm in the spectrum of ligand
are attributed to the free >NH and AOACH3 group of o-vanillin
moiety respectively. The singlet peak due to free ANH proton is
absent in the spectrum of 5 and new singlet peak appeared at
11.12 ppm. This confirms the conversion of free ANHA group into
AN@C group and ring >C@NA group into ANHAC@N in complexes.
IR spectra
The IR spectrum of the ligand 1 exhibited characteristic azome-
thine band ˆ(AHC@NA) at 1614 cmꢁ1. The strong band observed at
1614 cmꢁ1 is shifted to lower wave number by 11–9 cmꢁ1 in the
spectra of metal(II) complexes. The lowering of wave number
may be attributed to the decrease in electron density on the nitro-
gen atom of the azomethine group [17]. This indicates the coordi-
nation of azomethine (AHC@NA) group to the metal ion in
complexes. The presence of bands observed at 3415 and
3041 cmꢁ1 in 1 is attributed to ˆ(OH) stretching frequencies of
o-vanillin and quinoxaline moiety respectively. The absence
of these bands in the complexes 2–5 indicates the coordination
of AOH group through deprotonation [18]. This is further
evidenced by the ˆ(CAO) band in these complexes (1236–
1231 cmꢁ1), appearing at lower frequencies compared to that of
the ligand 1 (1247 cmꢁ1) [19]. In the spectra of complexes the band
due to ring ˆ(NH) was observed at 2947–2934 cmꢁ1 region. The
free ˆ(NH) stretching frequency was observed in ligand 1 at
2847 cmꢁ1. This band is absent in the spectra of complexes due
to the conversion of free ANHA group into AN@C< group and ring
>C@NA group into ANHAC@N in complexes. In the spectra of the
complexes, appearance of new bands in the region 496–485 cmꢁ1
and 628–581 cmꢁ1 has been attributed to MAN and MAO bonds
respectively [20,21]. From the IR spectral data it is clear that the li-
gand 1 acts as a tridentate ligand, coordinated with metal ion via
two phenolic AOH groups through deprotonation and through azo-
methine (AHC@NA) nitrogen atom.
Mass spectra
Mass spectra of l and its complexes gave vital clues for elucidat-
ing the structure and to determine the stoichiometry of the com-
pounds. The DART mass spectrum of 1 (C18H18N4O3) shows a
well defined molecular ion peak at m/z = 339, which coincides with
the formula weight of the Schiff base. The molecular ion peaks of
complexes 2, 3, 4 and 5 were observed at 732, 733, 737 and
739 m/z respectively. Along with the molecular ion peaks, spectra
show some other peaks which are attributed to molecular cations
of various fragments. Elemental analysis and mass spectral studies
confirm the metal to ligand ratio as 1:2.
Magnetic susceptibility and electronic spectra
The UV–Vis. spectra of 1–5 (10ꢁ3 mol Lꢁ1) were recorded in
DMF in the wavelength range 190–1100 nm. The magnetic suscep-
tibility and UV–Vis. spectral data are presented in Table 1. Ligand 1
exhibits electronic transitions with strong bands at 250–270 nm,
assigned to
p
–
p
* transitions, which remain almost unchanged in
* tran-
the complexes. The transition at 306 nm is assigned to n–
p
sition of azomethine group. Due to the coordination of azomethine
nitrogen this band shows shift in the electronic spectra of all com-
plexes. The complexes 2, 3 and 4 show a shoulder like absorption
peak at 330–400 nm region, which is assigned to the charge-trans-
1H NMR spectra
fer transition from the filled p
atoms to vacant d orbital of the metal [24]. The complex 2 exhib-
p orbital of the phenolic oxygen
1H NMR spectra of 1 (Fig. 1a) and its Zn(II) complex 5 (Fig. 1b)
were recorded in CDCl3. The signal for azomethine proton
(ACH@NA) in the ligand 1 appears as a singlet at 9.14 ppm [22].
In the spectrum of the complex 5, this signal shows small shift,
indicates the coordination of azomethine nitrogen atom. The sin-
glet peak observed at 13.19 and 13.59 ppm in the spectrum of 1
is assigned to AOH protons of o-vanillin and quinoxaline moiety
[23] respectively. In the spectrum of the complex 5 these peaks
disappeared, indicating the participation of phenolic AOH groups
in chelation through deprotonation. The aromatic proton signals
observed in the region 6.75–8.63 ppm show small shifts in
ited an absorption band at 485 nm, for d–d transition correspond-
4
ing to T1g(F) ? 4T1g(P) transition, suggesting an octahedral
geometry. It has a magnetic moment of 5.18 BM indicating a high
spin octahedral structure for the complex. Complex 3 exhibits a leff
value of 3.21 BM. Presence of spin allowed electronic transition at
3
510 nm corresponding to A2g(F) ? 3T1g (F) transition suggests an
octahedral geometry for the complex. Complex 4 exhibits a mag-
netic moment of 1.89 BM corresponding to one unpaired electron.
The band observed at 464 nm is assigned to 2B1g ? 2E1g transition,
indicating the complex to have distorted octahedral geometry
Fig. 1a. 1H NMR spectrum of 1.

400
J.D. Chellaian, J. Johnson / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 396–404
Fig. 1b. 1H NMR spectrum of 5.
Table 1
Electronic spectral data and magnetic moment values of 2–5.
Complex
kmax (nm)
Band assignments
Magnetic moment (BM)
5.18
Geometry
2
485
338
4T1g (F) ? 4T1g (P)
INCT
Octahedral
3
4
5
510
390
3A2g (F) ? 3T1g (F)
INCT
3.21
Octahedral
464
385
2B1g
?
2E1g
1.89
Distorted octahedral
Octahedral
INCT
239
299
INCT
INCT
Diamagnetic
[25,26]. The complex 5 is diamagnetic and two intra ligand bands
were observed in the electronic spectrum at 239 and 299 nm. As
per the empirical formula, an octahedral geometry is proposed
for this complex.
ESR spectra
N
O
NH
The X-band ESR spectra of the present Cu(II) complex (4) was
recorded in DMSO at liquid nitrogen temperature (77 K) and at
room temperature (300 K). The X-band ESR spectrum of the com-
plex 4 at room temperature exhibits a single isotropic broad signal
in the high field. The ESR spectrum of the complex 4 at 77 K shows
a well resolved hyperfine splitting and exhibits two different g val-
ues indicating magnetic anisotropy in the complex. The ground
state of the complex 4 is derived from the g-tensor values. In the
present Cu(II) complex the g-tensor values are g|| (2.18) > g\
(2.05) > 2.0027, suggests that this complex has a distorted octahe-
dral geometry with axial symmetry and the unpaired electron lies
in the d2x ꢁ y2 orbital [27]. The small g|| value can be attributed to
the large covalent interaction in MAL bond. The g values are re-
lated to the exchange interaction coupling constant (G) by the
expression, G = (g|| ꢁ 2)/(g\ ꢁ 2). If G > 4.0 suggests that the local
tetragonal axes are only slightly misaligned and the exchange
interactions between Cu(II) centers in the solid state are negligible.
The absence of a half field signal in the spectrum at 1600 G due to
O
O
N
N
M
N
N
O
O
HN
O
N
D
ms = 2 transitions, ruled out any Cu–Cu interaction [28].
Based on the above results the proposed structure of the com-
(Where M = Co(II), Ni(II), Cu(II) and Zn(II))
Fig. 2. Proposed structure of metal complexes.
plexes is given in Fig. 2.
Electrochemical studies
of 1 is recorded in the potential range ꢁ0.8 to 0.6 V, for 2–5 it is
recorded in the potential range ꢁ1.0 to 1.0 V with scan rate
0.005 V/s.
The cyclic voltammograms of 1 and its metal complexes (2–5)
were recorded at 300 K in DMSO solution. The cyclic voltammogram

J.D. Chellaian, J. Johnson / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 396–404
401
The ligand shows one irreversible peak at Epc = 0.35 V vs Ag/
AgCl. The complex 2 exhibits two irreversible peaks one at
Epc = 0.6 V corresponding to Co(II)/(I) couple. The corresponding
oxidation peak at Epa = ꢁ0.7 V during the reverse scan corresponds
to Co(II)/(III) couple. The peak to peak separation
DE is 1.3 V indi-
cating that the process is irreversible. The complex 3 shows one
irreversible peak at Epc = 0.6 V. The complex 4 (Fig. 3) exhibited
two redox processes; each reduction is associated with a single
electron transfer at room temperature [29]. Among the two redox
processes, one is irreversible with reduction peak at Epc = 0.55 V
and the corresponding oxidation peak at Epa = ꢁ0.55 V. The peak
to peak separation
DE is 1.1 V. The second process is quasi revers-
ible, the peaks observed at Epc = ꢁ0.19 V and Epa = ꢁ0.79 with peak
separation value E = 0.6 V. The complex 5 shows irreversible peak
at Epc = 0.65 V vs Ag/AgCl and the associated anode peak at Epa
D
=
ꢁ0.70 V. The peak to peak separation value
DE = 1.35 V corre-
sponding to irreversible process.
Powder XRD
The powder XRD patterns of the compounds 1–5 were recorded
over the 2h = 0–80 Å range and are given in Fig. 4. From the ob-
served data, all the complexes except 2 show sharp peaks indicat-
ing their crystalline nature. The complex 2 is amorphous. The
average crystallite sizes of the complexes dXRD were calculated
using Scherrer’s formula [30]. The ligand 1 and the complexes 3,
4 and 5 have an average grain size of 21, 14, 08 and 16 nm, respec-
tively, suggesting that the ligand and complexes are in a nanocrys-
talline phase.
Fig. 4. Powder XRD pattern of (a) 2 and (b) 3.
process. Such increase activity of the metal chelates can be
explained on the basis of Overtone’s concept [32] and Chelation
theory [33].
In the present study, the complex 4 exhibited higher antibacte-
rial and antifungal activities. Though the Schiff base ligand and its
metal(II) complexes possess activity, it could not reach the
effectiveness of the standard drugs. The variation in the effective-
ness of the different compounds against different organisms
depends either on the impermeability of the cells of the microbes
or differences in ribosomes of microbial cells [34,35].
Biological studies
In vitro antimicrobial studies
The in vitro antimicrobial activity of the ligand and its com-
plexes are given in Table 2. The standard error for the experiment
is 0.001 mm and the experiment is repeated three times under
similar conditions. DMF is used as negative control and Chloram-
phenicol is used as positive standard for antibacterial and Nystatin
for antifungal activities. The variation in the effectiveness of the
different compounds against different organisms depends on their
impermeability of the microbial cells or on the difference in the
ribosome of the microbial cells [31]. The metal(II) complexes
exhibited higher activity. The increase in the antimicrobial activity
of the metal chelates is due to the effect of metal ion on normal cell
DNA binding studies
The interaction of the metal(II) complexes (2–5) with DNA was
investigated by electronic absorption titration to evaluate their
binding affinities. Electronic absorption spectroscopy was an effec-
tive method to examine the binding mode of DNA with metal
complexes. Intercalative mode of binding usually results in hypo-
chromism and red shift because of the strong stacking interaction
between an aromatic chromophore and the base pairs of DNA. The
extent of the spectral change is related to the strength of binding
[36]. The electronic absorption titration of the complex 3 is shown
in Fig. 5. The absorption band of the complex 2 at 396.8 nm exhib-
ited hypochromism of 4.1% and bathochromism of 4 nm. The
absorption band of 3 at 257.6 nm exhibited hypochromism of
18.9% and bathochromism of 4.8 nm. The absorption band of 4 at
389.6 nm exhibited hypochromism of 10.9% and bathochromism
of 2.4 nm. The complex 5 at 329.6 nm exhibited hypochromism
of 3.2% and bathochromism of 2.5 nm. These results suggest an
association of the complexes (2–5) with DNA and interacting with
DNA through intercalation. The intrinsic binding constant (Kb) val-
ues were also calculated (2.5 ꢃ 105, 3.2 ꢃ 105, 3.5 ꢃ 105 and
2.6 ꢃ 105 Mꢁ1 for the complexes 2, 3, 4 and 5 respectively).
DNA cleavage studies
The DNA cleavage activity of the compounds (1–5) was studied
in the presence of H2O2 as an oxidizing agent using pUC18 DNA by
agarose gel electrophoresis. The cleavage efficiency was noted by
Fig. 3. Cyclic voltammogram of 4.

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J.D. Chellaian, J. Johnson / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 396–404
Table 2
Antimicrobial activity of 1–5.
Compound
Inhibition zone (mm)
Bacterial species
Fungal species
K. pneumoniae
E. coli
P. aeruginosa
S. aureus
C. albicans
A. niger
1
6
6
6
8
6
6
2
3
4
5
10
6
12
8
24
–
8
12
6
6
22
–
6
6
11
6
23
–
6
6
10
6
24
–
13
6
10
10
–
6
8
14
6
–
Chloramphenicola
Nystatina
23
22
a
Standard.
Fig. 5. Electronic absorption spectrum of (a) complex 3 in the absence of CT DNA
and (b) in the presence of increasing amount of CT DNA.
Fig. 6. Changes in the agarose gel electrophoretic pattern of pUC18 DNA induced by
H2O2. (1) Marker DNA; (2) Control DNA; (3) pUC18 DNA + H2O2 + 1; (4) pUC18
DNA + H2O2 + 2; (5) pUC18 DNA + H2O2 + 3; (6) pUC18 DNA + H2O2 + 4; (7) pUC18
DNA + H2O2 + 5.
measuring the ability of the complex to convert the super coiled
DNA into nicked open circular form or linear form. When circular
plasmid DNA is subjected to electrophoresis, the fastest migration
will be observed for the supercoiled form (Form I). If one strand is
cleaved, the supercoils will relax to produce a slower-moving open
circular form (Form II). If both strands are cleaved, a linear form
(Form III) will be generated that migrates in between [37].
Control experiments with H2O2 as an oxidizing agent did not
show any cleavage of the supercoiled DNA under similar experi-
mental conditions. It is evident from Fig. 6, there is a considerable
increase in the intensity of bands for open circular form (Form II) in
the case of Cu(II) complex (4) (lane 6). This suggests that the com-
plex 4 has nicking activity [38]. There is no appreciable level of in-
crease in the intensity of bands of open circular form for the DNA
samples treated with the ligand 1 (lane 3), complexes 3 (lane 5)
and 5 (lane 7). Shearing of pUC18 DNA is evident in lane 4, where
the plasmid DNA was treated with the Co(II) complex (2). Oxida-
tive cleavage was observed in the presence of H2O2. The complexes
cleaved DNA more efficiently in the presence of an oxidant, due to
the formation of hydroxyl free radicals.
cervical carcinoma (HeLa) cells by MTT assay and the results are
expressed in terms of IC50 values. The MTT cell proliferation assay
has been widely accepted as a reliable way to measure the cell
proliferation rate [39]. The compounds were applied in the concen-
tration range 0.1–100 lM. The data obtained by the MTT assay
show that all complexes have very good inhibitory effect on the
growth of human cervical carcinoma (HeLa) cells. Table 3 illus-
trates the IC50 values for the compounds tested. The IC50 values
of the investigated compounds ranges from 17.67 to 69.59
The complex 5 exhibited higher activity with IC50 value 17.67
l
l
M.
M.
The complexes 3 and 4 were also exhibited good anticancer activity
with IC50 values 33.23 and 35.61 M respectively. The complex 2
gave IC50 value at 66.83 M. The ligand has lowest growth inhibi-
l
l
tory activity against HeLa cells (69.51 lM). From the results (Fig. 7)
Table 3
Anticancer activity of 1–5.
Compound
IC50 (l )
Mꢁ1
1
2
3
4
5
69.51
66.83
33.23
35.61
17.67
In vitro anticancer studies
Anticancer activity of newly synthesized ligand (1) as well as
corresponding metal complexes (2–5) was investigated on human

J.D. Chellaian, J. Johnson / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 396–404
403
Fig. 7. Cytotoxic profiles of (a) 1, (b) 2, (c) 3, (d) 4 and (e) 5 towards human cervical cancer cell line (HeLa).
it is clear that the metal chelates have higher anticancer activity
than their corresponding ligand.
metal chelates (2–5) have enhanced anticancer activity than the
ligand.
Acknowledgement
Conclusion
We are thankful to Council of Scientific and Industrial Research
(CSIR), New Delhi, India for the financial support in the form of a
research project (Scheme No: 01(2453)/11/EMR-II).
Co(II), Ni(II), Cu(II) and Zn(II) complexes with ligand 1 have
been synthesized and characterized based on elemental analysis,
molar conductance, magnetic moment and spectral data. The Schiff
bases acts as tridentate ONO donor ligand. The complexes are non-
electrolytes. All complexes have octahedral geometry except Cu(II)
complex (4), which has distorted octahedral geometry. The metal
complexes show redox behavior. All compounds except 2 were
found to be nanocrystalline. In vitro antimicrobial study indicates
that metal chelates have higher activity than ligand. DNA binding
studies revealed that all the complexes of 1 exhibits stronger bind-
ing affinity to DNA through intercalation mode. DNA cleavage
activity of the compounds (1–5) was performed by agarose gel
electrophoretic assay. The complex 4 exhibited effective DNA
cleavage. The complexes also showed oxidative hydroxyl radical
cleavage in the presence of H2O2 as an oxidant. The synthesized
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