Copper complexes bearing 2-aminobenzothiazole derivatives as potential antioxidant: Synthesis, characterization

Article abstract of DOI:10.1016/j.jphotobiol.2016.06.030

Novel copper complexes of Schiff base ligands of 2-aminobenzothiazole derivatives were synthesized by the condensation of Knoevenagel condensate of acetoacetanilide (obtained from substituted benzaldehydes and acetoacetanilide) and 2-aminobenzothiazole. T

Full text of DOI:10.1016/j.jphotobiol.2016.06.030

Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 86–92
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Journal of Photochemistry & Photobiology, B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Copper complexes bearing 2-aminobenzothiazole derivatives as
potential antioxidant: Synthesis, characterization
⁎
J. Joseph , G. Boomadevi Janaki
Department of Chemistry, Noorul Islam Centre for Higher Education, Kumaracoil-629 180, Tamil Nadu, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel copper complexes of Schiff base ligands of 2-aminobenzothiazole derivatives were synthesized by the con-
densation of Knoevenagel condensate of acetoacetanilide (obtained from substituted benzaldehydes and
acetoacetanilide) and 2-aminobenzothiazole. They were characterized by elemental analysis, IR, 1H NMR, UV–
Vis., molar conductance, magnetic susceptibility measurements and electrochemical studies. Based on the mag-
netic moment and electronic spectral data, square planar geometry has been suggested for all the complexes. An-
tibacterial and antifungal screening of the ligands and their complexes reveal that all the complexes show higher
activities than the ligands. The binding behaviour of the complexes with calf thymus DNA has been investigated
by electronic absorption spectra, viscosity measurements and cyclic voltammetry. The DNA binding constants re-
veal that all these complexes interact with DNA through intercalation binding mode. Superoxide dismutase and
antioxidant activities of the copper complexes have also been studied. The antioxidant activities of the complexes
showed higher activities. Thermal denaturation studies suggested the nature binding affinity of copper com-
plexes with CT-DNA. All complexes exhibit suitable Cu(II)/Cu(I) redox potential to act as antioxidant enzymes
mimic. Further, the copper complexes also showed catalase activity. It is hope that copper complexes were capa-
ble of decrease ROS levels or reduce oxidative stress in Alzheimer's patients.
Received 14 April 2016
Accepted 17 June 2016
Available online xxxx
Keywords:
Acetoacetanilide
Knoevenagel
Antioxidant
Screening
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
The biological activity greatly depends on the nature of the metal ion
and the donor atoms of the ligands [11–12]. Metal complexes of Schiff
A natural products of β-diketones of siphonarienedione and
cyclopiatalantin possessing medicinal activity as well as β-diketones
are used as a powerful intermediates for the organic synthesis. 1,3-
diketone system of the polyphenol diferuloylmethane (curcumin)
bases derived from substituted aldehydes and heterocyclic compounds
containing nitrogen, sulphur and/or oxygen as ligand atoms are of inter-
est as simple structural models of more complicated biological systems
[13–15]. Many of the aldehydes tested were found to have highly potent
antimicrobial activity. These aldehydes may act as good candidates for
practical applications as well as interesting lead compounds for the de-
velopment of novel antimicrobial agents. Schiff bases of 2-
aminobenzothiazole derivatives have been of great importance due to
their synthetic flexibility and biological activity of their metal com-
plexes [16]. Diazotized 2-aminobenzothiazole with 1,3-dicarbonyl com-
pounds (benzoylacetone, methyl acetoacetate and acetoacetanilide)
obtained a new series of tridentate compounds exist in the intramolec-
ularly hydrogen bonded azo-enol tautomeric form in which one of the
carbonyl groups of the dicarbonyl moiety had enolised and hydrogen
bonded to one of the azo nitrogen atoms. The stable complexes showed
a normal paramagnetic moment [17].
The interaction of transition metal complexes with nucleic acids is a
major area of research due to the utility of these complexes in the design
and development of synthetic restriction enzymes, spectroscopic
probes, site specific cleavers and molecular photoswitches [18]. The
transition metal complexes are currently used as artificial nucleases
are because of their diverse structural feature, and the possibility to
tune their redox potential through the choice of proper ligands [19].
exhibits
a
variety of pharmacological activities including
antiinflammatory, anticarcinogenic, antibacterial and antifungal activi-
ties, hepato- and nephro-protective [1–2], thrombosis suppressing [3],
myocardial infarction protective [4] most of which are accredited to its
antioxidant and radical scavenging properties. Curcumin based analogs
of difluro Knoevenagel condensate and their Schiff base copper com-
plexes exhibited more effective in protease inhibition and apoptosis in-
ducers in cancer cells [5] (Scheme 1).
Schiff bases and their complexes have been studied for their variety
of biological actions by virtue of the azomethine linkage, due to its abil-
ity to reversibly bind oxygen, catalytic activity in hydrogenation of ole-
fins, transfer of an amino group, photochromic properties and
complexing ability towards some toxic metals. This high affinity for che-
lation of the Schiff bases towards the transition metal ions is utilized in
preparing their solid complexes [6–10].
⁎
Corresponding author.
E-mail address: chem_joseph@yahoo.co.in (J. Joseph).
http://dx.doi.org/10.1016/j.jphotobiol.2016.06.030
1011-1344/© 2016 Elsevier B.V. All rights reserved.

J. Joseph, G.B. Janaki / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 86–92
87
Scheme 1. Schematic outline of synthesis of ligands and their complexes.
Copper(II) complex is probably the most extensively studied among
the transition metal ions This is due to their lability, their high affinity
with different ligands and the wide variety of ligands geometries that
can accommodate significant role either in naturally occurring biologi-
cal systems or as pharmacological agents like (CuZn–SOD) superoxide
dismutase and it's disproportionate the toxic O•2− radical into molecular
oxygen and hydrogen peroxide [20–22]. In the present study we de-
scribe the synthesis and characterization of new series of curcumin an-
alog copper complexes of Knoevenagel condensate of Schiff base of 2-
aminobenzothiazole.
spectrophotometer in 4000–200 cm−1 range using KBr disc. Electronic
spectra were recorded in a Systronics 2201 Double beam UV–Vis., spec-
trophotometer within the range of 200–800 nm regions. Magnetic mo-
ments were measured by Guoy method and corrected for diamagnetism
of the component using Pascal's constants. Cyclic voltammetry was per-
formed on a CHI 604D electrochemical analyzer with three electrode
system of glassy carbon as the working electrode, a platinum wire as
auxiliary electrode and Ag/AgCl as the reference electrode.
Tetrabutylammoniumperchlorate (TBAP) was used as the supporting
electrolyte. Solutions were deoxygenated by eradication with N2 previ-
ous to measurements. The interactions between metal complexes and
DNA were studied using electrochemical and electronic absorption
techniques.
2. Experimental
2.1. Material
2.3. Preparation
All chemicals and solvents were analaR grade and were purchased
from Merck. All supporting electrolyte solutions were prepared using
analytical grade reagents. Calf thymus DNA purchased from Genie
Biolab, Bangalore, India.
2.3.1. Synthesis of β-Ketoanilides
An ethanolic solution of acetoacetanilide (1 M), was added drop
wise in the ethanolic solution of substituted benzaldehydes (L1-
anisaldehyde, L2-salicylaldehyde, L3-2-bromo benzaldehyde, L4-3,4-
dimethoxy benzaldehyde) (1 M) in the presence of anhydrous potassi-
um carbonate (1 M) in the mixture with stirring. The resulting mixture
was refluxed for about 8 h. The solid thus obtained was filtered, dried
and recrystallized from ethanol to obtain yellow colored β-
ketoanilide(s).
2.2. Instrumentation
The amount of copper present in the copper complexes was estimat-
ed using ammonium oxalate method. Elemental analysis of ligands and
their copper complexes were carried out using Elementar Vario EL III.
Molar conductance of the complexes was measured using a coronation
digital conductivity meter. The 1H NMR spectra of the ligands were re-
corded using TMS as internal standard. Chemical shifts are expressed
in units of parts per million relative to TMS. The IR spectra of the ligands
and their copper complexes were recorded on a Perkin-Elmer 783
2.3.2. Synthesis of Schiff Bases
Yellow colored β-ketoanilide(s) (1 M) was dissolved in ethanol
added drop wise to 2-aminobenzothiazole (2 M) and stirring at room
temperature. The resulting solution was refluxed about 8 h and the

88
J. Joseph, G.B. Janaki / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 86–92
product formed was poured into ice. The solid mass was obtained un-
dergo filtered and dried. Then, it was washed with petroleum ether in
order to remove nonpolar impurities. The reaction product was reduced
to one third of volume and yield brown colored substance.
a sample prepared under similar condition except NaOH was absent in
DMSO.
2.7. Hydrogen Peroxide Assay
2.3.3. Synthesis of Complexes
A solution of hydrogen peroxide (2.0 mM) was prepared in phos-
phate buffer (0.2 M, 7.4 pH) and its concentration was determined spec-
trophotometrically from absorption at 230 nm. The complexes of
different concentration and vitamin C (100 μg/mL) were added to
3.4 mL of phosphate buffer together with hydrogen peroxide solution
(0.6 mL). An identical reaction mixture without the sample was taken
as negative control. The absorbance of hydrogen peroxide at 230 nm
was determined after 10 min against the blank (phosphate buffer).
An ethanolic solution of Schiff base (1 M) was mixed with copper ac-
etate (1 M) in ethanol solution with continuous stirring. The mixture
was then refluxed for 7 h till the volume of the solution was reduced
to 10 mL. The complexes were precipitated in dry diethylether. The
solid product obtained was filtered, washed with distilled water and
cold ethanol and then dried in vacuum.
2.4. DNA Binding Studies
2.7.1. Catalase Activity
The binding interactions between metal complexes and DNA were
studied using electrochemical and electronic absorption methods by
using different concentrations of CT-DNA. Calf thymus DNA was stored
at 4 °C. The DNA stock solutions were prepared with buffer solution
(50 mM Tris-HCl at pH 7.2). The stock solutions of the complexes
were prepared by dissolving copper complexes in DMSO and diluting
with the corresponding buffer to the required concentration for all ex-
periments. This resulted in a series of solutions with varying concentra-
tions of DNA but with a constant concentration of the complex. The
absorbance (A) of the most red-shifted band of complex was recorded
after each successive additions of CT DNA. The intrinsic binding con-
stant, Kb, was determined from the plot of [DNA] / (εa − εf) vs [DNA],
where [DNA] is the concentration of DNA in base pairs, εa, the apparent
extinction coefficient which is obtained by calculating Aobs / [complex]
and εf corresponds to the extinction coefficient of the complex in its
free form. The data were fitted to the following equation where εb refers
to the extinction coefficient of the complex in the fully bound form.
CAT activity in erythrocytes was determined according to spectro-
photometric procedure by Beers and Sizer [23] and expressed in
Bergmeyer units (BU/g Hb). CAT activity was measured at 25 °C by re-
cording H2O2 decomposition at 240 nm. One BU of CAT activity is de-
fined as the amount of enzyme decomposing 1 g of H2O2/min.
2.7.2. Antimicrobial Activities
The antibacterical activity of samples was determined using a well
diffusion method. The antibacterial activities were performed by using
Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus
vulgaris and Pseudomonas aeruginosa, respectively.
The nutrient agar medium was boiled to dissolve completely and
sterilized at 15 lbs pressure (120 °C). After sterilization, 20 mL of
media was poured into the sterilized petri plates. These plates were
kept at room temperature and the medium got solidified in the plates.
Then, it was inoculated with microorganisms using sterile swabs. The
stock solutions were prepared by dissolving the compounds in appro-
priate solvents. The sample solutions were filled in the incubated plates
using a micropipette and incubated for 24 h at 37 °C. During incubation
period, the sample solution was diffused into the gel and inhibited the
growth of the microorganism. The zone of inhibition was developed
on the plate and measured.
½DNAꢀ=ðεa−εf Þ ¼ ½DNAꢀ=ðεb−εf Þ þ 1=Kbðεb−εf Þ
ð1Þ
Each set of data, when fitted to the above equation, gave a straight
line with a slope of 1/(εb − εf) and a y-intercept of 1/Kb(εb − εf). Kb
was determined from the ratio of the slope to intercept.
3. Results and Discussions
2.5. Thermal Denaturation
The metal complexes are soluble in CHCl3, DMSO, DMF and insoluble
in water. The elemental analysis data of the Schiff bases and their metal
complexes are equivalent with the calculated results from the empirical
formula of each compound (Table 1). The Cu(II) complexes were
dissolved in DMSO and the molar conductivities of 10−3 M of their
solution at room temperature were measured. The lower molar
conductivity values of copper complexes were found in the range of
(2–6) ohm−1 cm mol−1 suggesting them to be non-electrolytes
which is evidenced the presence of acetate ions in coordination sphere.
The analytical data are in a good agreement with the proposed stoichi-
ometry [CuL(OAc)2] of all the complexes. Furthermore, the magnetic
moment measurements were recorded at room temperature lie in be-
tween 1.74 and 1.79 B.M. corresponding to the presence of one unpaired
electron and it supports the square planar geometry around central
copper ion.
In order to identify the thermal behaviour of DNA, the melting tem-
perature Tm which is defined as the temperature where half of the total
base pairs get non-bonded was studied. Intercalation of synthesized or-
ganics and metallointercalators generally results in considerable in-
crease in melting temperature (Tm). Thermal denaturation
experiments were carried out by monitoring the absorption of CT DNA
in 50 μM concentration for the nucleotides at 260 nm with different
temperature in the presence (10 μM complex) and the absence of
each complex. The melting temperature (Tm, the temperature at
which 50% of double stranded DNA becomes single stranded) and the
curve width (σT, the temperature range between which 20 and 80% of
the absorption increases occurred) were recorded.
2.6. Antioxidant Assay
3.1. NMR Spectral Features
2.6.1. Superoxide Dismutase Activity (SOD)
The superoxide dismutase activity (SOD) of the copper complexes
were evaluated using alkaline DMSO as source of superoxide radicals
(O•2−) generating system in association with nitro blue tetrazolium chlo-
ride (NBT) as a scavenger of superoxide. Add 2.1 mL of 0.2 M potassium
phosphate buffer (pH 8.6) and 1 mL of 56 μL of NBT solutions to the dif-
ferent concentration of copper complex solution. The mixtures were
kept in ice for 15 min and then 1.5 mL of alkaline DMSO solution was
added while stirring. The absorbance was monitored at 540 nm against
The 1H NMR spectra of ligands were recorded in DMSO solution at
room temperature. All the protons were found to be in their expected
region. The ligand L2 showed the following spectral features for
Knoevenagel condensate of acetoacetaanilide moiety: the peak arises
at 6.5–8.4 ppm (m, 5H) corresponds to aromatic protons of
acetoacetanilide ring, phenyl multiplet of salicylaldehyde was observed
at 7.2–7.6 (m, 4H), methyl protons at 2.1 ppm (s, 3H), and –OH at 10.2
(s, H), respectively. In addition, peak appeared at 7.3 ppm, which is

J. Joseph, G.B. Janaki / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 86–92
89
Table 1
Physical characterization, analytical, molar conductance and magnetic susceptibility data of the ligands and their complexes.
Compound
Yield (%)
Color
(Found) calc
(ohm−1 cm mol −1
)
μeff (BM)
Cu
C
H
N
L1
67
76
68
74
56
78
72
59
Pale brown
Dark yellow
Brown
–
68.68
(68.62)
68.24
(68.21)
61.18
(61.24)
67.10
(67.15)
55.36
(55.28)
54.74
(54.77)
53.19
(53.13)
57.53
4.51
(4.56)
4.25
(4.29)
3.65
(3.59)
4.78
(4.72)
3.63
(3.78)
3.41
(3.48)
3.57
(3.53)
4.44
12.52
(12.50)
12.84
(12.89)
13.14
(13.21)
11.86
(11.92)
10.22
(10.19)
10.33
(10.31)
8.87
(8.72)
9.07
–
–
–
–
3
6
4
2
–
L2
–
–
–
–
L3
–
L4
Dark brown
Reddish brown
Yellowish brown
Black
–
[CuL1(OAc)2]
[CuL2(OAc)2]
[CuL3(OAc)2]
[CuL4(OAc)2]
9.16
(9.21)
9.35
(9.28)
8.05
(8.18)
8.23
1.74
1.79
1.77
1.76
Black
(8.27)
(57.48)
(4.48)
(9.14)
assigned to free –NH group of acetoacetanilide moiety. Moreover, the
multiplets within the range 7.8–8.2 ppm (m, 8H) were assigned to the
aromatic protons of benzothiazole ring [24]. It was concluded that the
absence of amino group of 2-aminobenzothiazole indicated the forma-
tion of Schiff base ligand system.
transitions within the Schiff base molecule. The electronic spectrum of
the corresponding complex [CuL1(OAc)2] in DMSO reveals a broad
band at 427 nm assigned to 2B1g → 2A1g transition which is characteris-
tic of square planar environment around the copper(II) ion. Similar
spectral features were observed for other copper complexes [28].
3.2. FT-IR
3.4. DNA Binding Experiments
The characteristic IR bands for the synthesized ligands and copper
complexes were listed in the given Table 2. The ligand L2 exhibits two
IR bands assigned to imine υ(C_N) groups of 2-aminobenzothiazoles
moiety at 1647 cm−1 and 1622 cm−1 are shifted to a lower frequency
of 1626 cm−1 and 1608 cm−1 after complexation [25]. Also, the new
IR bands near at 435 cm−1 and 505 cm−1 were assigned to (M-N)
and ν(M-O) [26]. In addition the appearance of new bands at
1368 cm−1 and 1270 cm−1 corresponds to symmetric and asymmetric
stretching for ν(M-O). It is also further supported by the appearance of
bands between 1370 cm−1–1374 cm−1 and 1268–1274 cm−1 attribut-
ed to υasym(COO–) and υsym(COO–) respectively for two copper com-
plexes. The difference in Δυ between υasym(COO–) and υsym(COO–) in
metal complexes was ~100 cm−1 suggests the mode of coordination
of carboxylate group (i.e., acetate ion) in copper complexes in a
monodentate manner. It was reported that the copper complexes
were behaved as bidentate and coordinate through azomethine nitro-
gen atoms. The IR Spectral features were reinforced the conclusion
drawn from conductance measurements [27].
3.4.1. Cyclic Voltammetric Studies
Cyclic voltometric technique is the versatile technique useful for the
DNA binding ability of electro active species. In the absence of DNA, Cy-
clic voltammogram of [CuL1(OAc)2] exhibited cathodic and anodic
peaks were observed at −0.241 V and −0.080 V which are assigned
to Cu(II)/Cu(I) conversion. Cyclic voltammogram of [CuL2(OAc)2]
showed two segments of cathodic and anodic peaks. The first segment,
cathodic and anodic peaks were observed at 0.120 V and 0.231 V, re-
spectively. This showed oxidation from +1 to +2 form at a cathodic
peak potential. This showed unusual oxidation state of ligand system.
This showed reduction occurs from +2 to +1 form at a cathodic peak
potential.
When addition of various concentration of CT-DNA in the same con-
centration of complex indicated decreases voltometric peak current
which is shown in the Fig. 1 & Fig. 2 for [CuL1(OAc)2] & [CuL2(OAc)2].
This is highly due to diffusion of the equilibrium mixture of free and
DNA-bound metal complex to the electrode surface, in which the peak
potentials both Epa and Epc as well as E1/2 have a shift to negative poten-
tial. The observed separation of cathodic and anodic potentials,
ΔEp = −0.215 and −0.250 for [CuL1(OAc)2] & [CuL2(OAc)2] indicated
that the reaction of copper complexes on the working electrode was
quasi reversible redox process.
3.3. Electronic Spectral Features
The electronic absorption spectra of the Schiff base ligands and their
copper complexes in DMSO solvent were recorded at room tempera-
ture. The band positions of the absorption maxima; band assignments
and the proposed geometry mentioned in Table 3. The absorption spec-
trum for ligand L1 showed a band at 270 nm attributed to n–π*
The peak currents observed for all copper complexes showed chang-
es in current indicated that both metal complex interact with CT-DNA
through partial intercalative mode [29]. It was reported that higher
Table 3
Table 2
Electronic spectra of Schiff base ligands and their copper complexes (nm).
Characteristic IR bands of the Schiff base ligands and its copper complexes (in cm−1).
Compound
Wavelength Band
Geometry
K
b (M−1
)
ΔG (cal)
Compound
ν(C_N) ν(C_N) ν(M-O) ν(M-N)
υ
asym(COO–)
υ
sym(COO–)
(nm)
assignments
L1
L2
L3
L4
1628
1622
1632
1627
1642
1647
1641
1643
1628
1624
1621
1627
–
–
–
–
512
505
514
509
–
–
–
–
442
435
447
444
–
–
–
–
1374
1368
1370
1371
–
–
–
–
L1
L2
L3
L4
270
285
309
319
π–π*
π–π*
n–π*
n–π*
–
–
–
–
–
–
–
–
–
–
–
–
[CuL1(OAc)2] 1610
[CuL2(OAc)2] 1608
[CuL3(OAc)2] 1603
[CuL4(OAc)2] 1607
1272
1270
1268
1274
[CuL1(OAc)2] 437
[CuL2(OAc)2] 464
[CuL3(OAc)2] 467
[CuL4(OAc)2] 439
2B1g
2B1g
2B1g
2B1g
→
2A1g Square planar 3.23 × 105
2A1g Square planar 3.18 × 105
2A1g Square planar 3.27 × 105
2A1g Square planar 3.24 × 105
−31,452
−30,527
−31,381
−31,309
→
→
→

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J. Joseph, G.B. Janaki / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 86–92
Table 4
Electrochemical parameters for the interaction of DNA with copper complexes.
Compound
Redox couple
E1/2 (V)
Free
ΔEp (V)
ipa/ipc
Bound
Free
Bound
−0.215 1.02
[CuL1(OAc)2] Cu(II) → Cu(I) −0.160 −0.517 −0.321
[CuL2(OAc)2] Cu(I) → Cu(II) −0.175 −1.475 −0. 351 −0.250 1.18
[CuL3(OAc)2] Cu(II) → Cu(I) −0.265 −0.421 −0.142
[CuL4(OAc)2] Cu(II) → Cu(I) −1.312 −1.562 −0.144
−0.268 1.15
−0.210 1.12
increase in molar absorptivity (hypochromism) with hypochromic
(blue) shift by 2–3 nm [31] indicating intercalative mode of binding
seen between aromatic chromophore of metal complexes with CT-
DNA in Fig. 4. The d-d transition for complexes at 464 nm indicates
that the copper complexes interacts with N7 of guanine of CT-DNA. All
these data were then fitted to Eq. (1) to obtain the intrinsic binding con-
stant (Kb) values and resulted that [CuL1(OAc)2] complexes bind with
DNA by partial intercalation mode [32–34]. The Intrinsic binding con-
stants (Kb) for copper complexes were in the range of
3.18 × 105 M−1–3.27 × 105 M−1 are less when compared with the re-
Fig. 1. Cyclic voltammogram of [CuL1(OAc)2] in the presence and absence of DNA.
decrease of current observed for the complex [CuL1(OAc)2] had stronger
binding affinity with CT-DNA and indicated all other copper complexes
were bind via partial intercalative binding mode [30] and their electro-
chemical parameters are given in Table 4.
ported
few
intercalating
complexes
[Ru(bpy)2(dppz)]2+
(4.90 × 106 M−1) [33] and [Ru(bpy)2(HBT)]2+ (5.71 × 106 M−1). Bind-
ing energy is the measure of stability of complexes. The binding energy
between DNA base pairs and complexes were calculated according to
the equation ΔG = −RT ln Kb and the values are tabulated in the
Table 3.
3.4.2. Absorption Spectral Titrations
In the DNA binding studies, Hyper and hypochromic effects are two
important spectral features concerns about the influence of molecules
with DNA either expansion or contraction of DNA structure. In general,
hypochromism was observed in the spectrum indicates the molecule
binding with DNA through intercalation mode. The extent of
hypochromism corresponds to the strength of intercalative binding in-
teractions. The ansence of any isobestic points in the spectrum indicates
more than one type of DNA-molecule interactions was observed. The
metal ion, charge of chelate and nature of ligand surrounded on the
metal center decides the nature binding with DNA. The DNA possesses
many hydrogen bonding sites which are available both in the minor
and major grooves and leads to groove binding of molecules.
3.4.3. Thermal Denaturation
The thermal behaviour of CT-DNA in the presence of complexes gave
insight into their conformational changes when temperature is raised
when temperature is raised and information about the interaction
strength of the complexes with DNA. The double- stranded DNA tends
to gradually dissociate to single strands on increase in the solution tem-
perature and generates a hyperchromic effect on the absorption spectra
of DNA bases (at 316 nm). In the present study melting temperature
(Tm) of DNA in the absence of copper complexes was found to be
54 1 °C. Under the same set of experimental conditions, addition of
The binding properties of complexes to the CT-DNA helix have been
determined by electronic absorption spectral methods. The magnitude
of hypochromism and red shift depends upon the strength of interac-
tion between metal complexes and CT-DNA. As the DNA concentration
is increased, the following changes to be observed in the absorbance
and shift in wavelength of ligand as well as metal complexes. On the ad-
dition of CT-DNA, the copper complex of [CuL1(OAc)2] showed broad
peak at 437 nm which shoulder at 270 nm results in decrease in
molar absorptivity (hypsochromism) blue shift of 2–7 nm shown in
the Fig. 3. These data suggested that metal complex clearly bind to par-
tial intercalative binding mode. The same binding features were ob-
served for the [CuL3(OAc)2] and [CuL4(OAc)2] complexes.
complexes increased the melting temperature Tm
(
1 °C) from 10 °C
to 15.3 °C, for all copper complexes respectively. This experimental
data indicates that the all Cu(II) complexes of peptides has interaction
with double helix CT-DNA. The intercalation of small molecules into
the double helix has as a result an increase of melting temperature at
which the double helix denaturates into single helix DNA, The signifi-
cant increase of Tm (ΔTm = 15.3 °C) suggests that the interaction of
the all copper complexes with DNA is performed through partial inter-
calation shown in the Fig. 5 [35,36].
On the addition of CT-DNA, The [CuL2(OAc)2] complex showed
broad peak at 464 nm which shoulder at 285 nm which results in
3.4.4. Antioxidant Activity
In this study radical scavenging properties of copper complexes
were determined by the NBT assay method and hydrogen peroxide
assay [37,38]. The generation of Superoxide radicals is determined by
Fig. 2. Cyclic voltammogram of [CuL2(OAc)2] in the presence and absence of DNA.
Fig. 3. Electronic spectra of [CuL1(OAc)2] in the presence and absence of DNA.

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91
Fig. 4. Electronic spectra of [CuL2(OAc)2] in the presence and absence of DNA.
Fig. 6. Hydrogen peroxide activity of Cu(II) complexes in (μmol dm−3).
the percentage of inhibition of the reduction of NBT (IC50). The signifi-
cant IC50 values are determined by UV–Vis. Spectroscopy. It has been re-
vealed that the synthesized copper complexes exhibited a quite strong
antioxidant activity in the range of 30–47 μmol dm−3. The reactivity
of free radicals can be neutralized by the donation of electron or hydro-
gen, from the above fact it has been suggested that the presence of hy-
droxy ligand system of [CuL2(OAc)2] act as the efficient hydrogen
donors to stabilize the unpaired electrons and scavenging free radicals.
Further it was supported by the fact, lower the IC50 values higher
ability of donating hydrogen. So scavenging activity of [CuL2(OAc)2]
copper complex is in the order of [CuL2(OAc)2] N [CuL3(OAc)2] N
[CuL1(OAc)2] N [CuL4(OAc)2] which are graphically represented in
the Figs. 6 & 7 and the values are tabulated in the Table 5. Curcumin pre-
vents the oxidation of hemoglobin and inhibits lipid peroxidation. The
antioxidant activity of curcumin could be mediated through antioxidant
enzymes such as superoxide dismutase, catalase, and glutathione
peroxidase [39]. Curcumin acts as a superoxide radical scavenger report
describes the H-atom donation from the β-diketone moiety to a lipid
alkyl or a lipid peroxyl radical as a potentially more important antioxi-
dant action of curcumin [40]. So the curcumin analog system of present
system also posses higher antioxidant activity.
stabilized by the proposed ligand systems. It is also disproportionate
H2O2 by converting it into the H2O and O2.
In Alzheimer's disease, the metal contamination may produce oxida-
tive stress and protein aggregation into Aβ-peptides. In general, antiox-
idant and enzyme mimetic compounds may act as good therapeutic
candidates for Alzheimer's disease for reduction of ROS levels and pep-
tide cleavage. In the present study, all the copper complexes showed en-
zyme mimetic activities. The prepared copper complexes act as good
candidates for antialzheimer's agents.
3.4.5. Antimicrobial Activity
The in vitro antimicrobial activities of the investigated compounds
were tested against the five bacterial species, Staphylococcus aureus,
Escherichia coli, Klebsiella pneumaniae, Proteus vulgaris, and Pseudomo-
nas aeruginosa by disc diffusion method [43]. The MIC values of all the
compounds against the microorganism are summarized in the Table 6.
The comparative study (MIC) of ligands with their copper complexes
indicated that the complexes have higher activities than the synthesized
ligands. In general, the synthesized metal complexes have higher bio-
logical activities compared to the free ligands. The increased inhibition
activity of the metal complexes can be explained on the basis of
Tweedy's chelation theory [44]. In metal complexes, on chelation the
polarity of the metal ion will be reduced to a greater extent due to the
overlap of the ligand orbital and partial sharing of the positive charge
of the metal ion with donor groups. Further, it increases the delocaliza-
tion of π- electrons over the whole chelate ring.
Cu(II) complexes containing small bioligands are considered as effi-
cient therapeutic agents because they mimic SOD activity [41,42]. In
these Cu(II) complexes, Cu-center is involved in a redox cyclic process
by successive encounters with superoxide and producing and H2O2
at the reactive site and can easily dismutase the excess of intracellular
O•2− and H2O2 and increasing the concentration which is act as excellent
SOD mimetics due to its effective bioligand system of 2-
aminobenzothiazole.
The redox property of copper complexes plays a key role in radical
neutralization and disproportionation of toxic species into nontoxic
species. All the synthesized copper complexes showed redox behaviour
(Cu2+/Cu+) of negative potentials. It indicates that copper complexes
act as good antioxidant systems. The unusual oxidation state was
3.4.6. Highly Conjugated System
The enhanced activity of the complexes may also be explained on
the basis of conjugation. Copper complexes have higher activity than li-
gands due to the presence of highly conjugated curcumin analog sys-
tem. Since The whole ligand system could act as highly conjugated
Fig. 5. Thermal Denaturarion Curve for all Cu(II) complexes.
Fig. 7. Superoxide dismutase activity of Cu(II) complexes in (μmol dm−3).

92
J. Joseph, G.B. Janaki / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 86–92
Table 5
constitutions, resulting in interfering with the normal cell process
possessing superior the pharmacological efficiencies when coordinating
with copper ion.
Antioxidant activity of Schiff base copper complexes in (μmol dm−3).
Compound
IC50 (μmol dm−3
)
IC50 (μmol dm−3
)
H2O2
O•2−
[CuL1(OAc)2]
[CuL2(OAc)2]
[CuL3(OAc)2]
[CuL4(OAc)2]
Vitamin C
40
36
38
47
40
30
37
46
Acknowledgement
We express our sincere thanks to the Chancellor, Noorul Islam Cen-
tre for Higher Education, Kumaracoil for providing research facilities.
This work was financially supported by DST, New Delhi under Young
scientist scheme 002/2014.
0.02
0.05
system may also responsible for higher antimicrobial activities. In the
present ligand system, the Knoevengel condensate of β-ketoanilide
has active carbonyl group that makes high liphophilic behaviour. It
enhances the pharmacological efficiencies when coordinating with
copper ion.
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aureus
coli
pneumaniae aeruginosa
vulgaris
L1
L2
L3
L4
28
31
22
24
26
29
21
25
12
11
6
32
36
27
26
11
13
8
27
31
25
22
14
12
7
30
33
24
29
12
0.11
8
[45] N. Raman, J. Joseph, Russ J Inorg Chem 55 (2010) 1064.
[CuL1(OAc)2] 13
[CuL2(OAc)2] 12
[CuL3(OAc)2]
9
[CuL4(OAc)2] 11
Streptomycin
8
9
9
1.9
10
1.8
2
1.8
2.5