Synthesis, characterization and biological studies of copper(II) complexes with 2-aminobenzimidazole derivatives

Article abstract of DOI:10.1016/j.molstruc.2017.02.021

Novel series of four copper(II) complexes with 2-aminobenzimidazole derivatives (obtained from the Knoevenagel condensate of acetylacetone (obtained from acetylacetone and halogen substituted benzaldehydes) and 2-aminobenzimidazole) were synthesized. They

Full text of DOI:10.1016/j.molstruc.2017.02.021

Accepted Manuscript
Synthesis, characterization and biological studies of copper(II) complexes with 2-
aminobenzimidazole derivatives
J. Joseph, A. Suman, K. Nagashri, R. Selwin Joseyphus, Nisha Balakrishnan
PII:
S0022-2860(17)30154-0
10.1016/j.molstruc.2017.02.021
MOLSTR 23415
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 8 July 2016
Revised Date: 14 August 2016
Accepted Date: 4 February 2017
Please cite this article as: J. Joseph, A. Suman, K. Nagashri, R.S. Joseyphus, N. Balakrishnan,
Synthesis, characterization and biological studies of copper(II) complexes with 2-aminobenzimidazole
derivatives, Journal of Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.02.021.
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Synthesis, characterization and biological studies of copper(II) complexes with
2-aminobenzimidazole derivatives
J. Joseph1 , A. Suman1, K. Nagashri2, R.Selwin Joseyphus3 and Nisha Balakrishnan1
Department of Chemistry, Noorul Islam Centre for Higher Education, Kumaracoil-629 180, Tamil Nadu
Department of Chemistry, Renganayagi Varatharaj College of Engineering, Salvarpatti, Sivakasi-
626128, Tamilnadu, India
Department of Chemistry, Mar Ivonis College, Trivandrum, Kerala, India
Abstract
Novel series of four copper(II) complexes with 2-aminobenzimidazole derivatives (obtained
from the Knoevenagel condensate of acetylacetone (obtained from acetylacetone and halogen
substituted benzaldehydes) and 2-aminobenzimidazole) were synthesized. They were structurally
1
characterized using elemental analysis, molar conductance, FAB mass, FT- IR, H &13C-NMR,
UV–Vis., and EPR techniques. On the basis of analytical and spectral studies, the distorted
square planar geometry was assigned for all the complexes. The antibacterial screening of the
ligands and their copper complexes indicated that all the complexes showed higher anti
microbial activities than the free ligands. Superoxide dismutase and antioxidant activities of the
copper complexes have also been performed. In the electrochemical technique, the shift in ∆Ep,
E1/2 and Ipc values were explored for the interaction of the complexes with CT-DNA. During the
electrolysis process, the present ligand system stabilizes unusual oxidation state of copper in the
complexes. It is believed that the copper complexes with curcumin analogs may enhance
chemotherapeutic behavior.
Keywords: DNA binding; Antioxidant; Complexes; Schiff base; Antimicrobial.
Corresponding author: Email: josephniche@gmail.com;Tel: 91+ 9489486295

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1.
Introduction
β-diketones have gained a lot of interest due to their potential importance as versatile
ligands for chelation with different metals and also as intermediate in the core of heterocyclic
systems such as flavones, benzodiazepine, pyrazole, isoxazole and pyrimidine. They are well
known molecule exhibited keto-enol tautomerism [1-9]. Transition metal complexes containing
β-diketone scaffolds are commonly found in biological systems and also play an important role
in biochemical processes such as catalysis of drug interaction with bio molecules. Metal
complexes with β-diketone derivatives were synthesized and showed broad spectrum of
biological activities. Recently, the Cu(II), Ni(II), Co(II) and Zn(II) complexes were exhibited
broad spectrum of biological and physic chemical properties and motivate researchers in the field
of coordination chemistry of bio essential metal ions [10-14]. Many biologically active
compounds were used as drugs possess modified pharmacological and toxicological potentials
when administered in the form of metal-based compounds [15-16]. The structural modulation
and coordination of ligands with different metal ions may significantly vary the pharmacological
activities. In the present study, we have also decided to synthesize a series of novel β-diketone
derivatives and copper complexes.
In view of the importance of metal complexes, the preparation, characterization and
in vitro antibacterial and antifungal properties of copper complexes were performed. The newly
synthesized β-diketone and their metal complexes were characterized by IR, 1H NMR, 13C NMR,
molar conductance, magnetic moments, electronic and elemental analyses data. The synthesized
ligands and their complexes have been screened for in vitro antibacterial activity against
Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and
antifungal activity .In vitro free radical scavenging activities of the ligands (L1–L4) and copper
chelates were evaluated by DNA binding and anti oxidant assay methods [17-22].

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2. Experimental
All chemicals and solvents were reagent grade and were purchased from Merck. All
supporting electrolyte solutions were prepared using analytical grade reagents and doubly
distilled water. Calf thymus DNA purchased from Genei Biolab, Bangalore, India.
Instrumentation
Elemental analysis of ligands and their copper complexes were carried out using Perkin-
Elmer elemental analyzer. Molar conductance of the complexes was measured using a
1
13
coronation digital conductivity meter. The H & C-NMR spectra of the ligands were recorded
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 spectrophotometer in 4000-200 cm-1 range using KBr disc. Electronic spectra
were recorded in a Systronics 2201 Double beam UV-Vis., spectrophotometer within the range
of 200-1100 nm region. Magnetic moments were measured by Guoy method and corrected for
diamagnetism of the component using Pascal’s constants. Cyclic voltammetry was performed 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 previous to measurements. The interactions
between metal complexes and DNA were studied using electrochemical and electronic
absorption techniques. The FAB-mass spectra of ligands and their copper complexes were
recorded on a JEOL SX 102/DA-6000 mass spectrometer/data system using Argon/Xenon (6 kV,
10 A) as the FAB gas. The Thermogravimetric analyses data were measured from 0ºC to 1000ºC
temperature at a heating rate of 10ºC/min. The data were obtained by using a Perkin Elmer
Diamond TG/DTA instrument.

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Synthesis of ligands and their copper complexes
Synthesis of Knoevenagel condensate β-diketones
A series of Knoevenagel condensate of β-diketone with different substituted aromatic
aldehydes [L1/ 3-bromobenzaldehyde, L2/3-fluorobenzaldehyde, L3/3-chlorobenzaldehyde, L4/3-
cyanobenzaldehyde] was refluxed in the presence of potassium carbonate as catalyst in the
ethanolic medium. The progress of reaction was monitored by TLC technique. After completion
of reaction, the reaction mixture was poured into crushed ice. The yellow coloured knoevenagel
condensate of β–diketone(s) was obtained. It was filtered, washed with ice cold water and dried
in vacuum.
Synthesis of Schiff base ligands
Hot ethanolic solutions of 2-aminobenzimidozole (2 M) was added dropwise to
Knoevenagel condensate of β-diketone (s) (1 M) was refluxed for 12 hrs in the presence of
potassium carbonate as catalyst. The progress of reaction was monitored by TLC. After
completion of reaction the solid material was removed by filtration and recrystallized.
Synthesis of copper complexes
Equimolar hot Ethanolic solutions of 2-aminobenzimidozole derivatives and copper
acetate were refluxed for 6 hrs. The progress of reaction was monitored by TLC. Then, it was
poured into crushed ice. The solid material was removed by filtration and recrystallized.
DNA Binding Studies
The binding interactions between metal complexes and DNA were studied using
electrochemical and electronic absorption methods by using different concentrations of CT-
DNA. Solutions of CT DNA in 50 mM NaCl/50 mM Tris-HCl (pH 7.2) gave the ratio of the UV
absorbance at 260 and 280 nm, A260/A280, of ca. 1.8–1.9, indicating that the DNA was
sufficiently free of protein contamination. The DNA concentration was determined by the UV
absorbance at 260 nm after 1:100 dilution. The molar absorption coefficient was taken as 660 m
2 mol-1. Stock solutions were kept at 4 °C and used after not more than 4 days. Concentrated

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stock solutions of the complexes were prepared by dissolving the complexes in DMSO and
diluting suitably with the corresponding buffer to the required concentration for all of the
experiments.
Absorption titration experiment
Absorption titration experiment was performed by maintaining a constant concentration
of the complex while varying the nucleic acid concentration. This was achieved by dissolving an
appropriate amount of the copper complex stock solution and by mixing various amounts of
DNA stock solutions while maintaining the total volume constant. This resulted in a series of
solutions with varying concentrations 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 constant, Kb, was determined from the plot of
[DNA]/(ea- ef) vs [DNA], where [DNA] is the concentration of DNA in base pairs, ea, the
apparent extinction coefficient which is obtained by calculating Aobs/[complex] and ef
corresponds to the extinction coefficient of the complex in its free form. The data were fitted to
the following equation where eb refers to the extinction coefficient of the complex in the fully
bound form.
[DNA]/(ea- ef) = [DNA]/( eb- ef) + 1/Kb(eb- ef) -------------- (1)
Each set of data, when fitted to the above equation, gave a straight line with a slope of 1/(eb- ef)
and a y-intercept of 1/Kb(eb- ef). Kb was determined from the ratio of the slope to intercept.
Antimicrobial activities
The in vitro antimicrobial activities of the investigated compounds were tested against the
bacterial species and fungal species. One day prior to the experiment, the bacterial and fungal
cultures were inoculated in broth (inoculation medium) and incubated overnight at 37°C.
Inoculation medium containing 24 hr grown culture was added aseptically to the nutrient
medium and mixed thoroughly to get the uniform distribution. This solution was poured (25 mL
in each dish) into petri dishes and then allowed to attain room temperature. Wells (6 mm in
diameter) were cut in the agar plates using proper sterile tubes. Then, wells were filled up to the

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surface of agar with 0.1 mL of the test compounds dissolved in DMSO (200 mM/mL). The plates
were allowed to stand for an hour in order to facilitate the diffusion of the drug solution. Then
◦
the plates were incubated at 37 C for 24 hr for bacteria and 48 hr for fungi and the diameter of
the inhibition zones were read. Minimum inhibitory concentrations (MICs) were determined by
using serial dilution method. The lowest concentration (mg/mL) of compound, which inhibits the
growth of bacteria after 24 hr incubation at 37°C, and of fungi after 48 hr incubation at 37°C was
taken as the MIC. The concentration of DMSO in the medium did not affect the growth of any of
the microorganisms tested.
Superoxide dismutase activity (SOD)
The superoxide dismutase activity (SOD) of the copper complexes were evaluated using
•-
alkaline DMSO as source of superoxide radicals (O2 ) generating system in association with
nitro blue tetrazolium chloride (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 different
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 a sample prepared under similar condition except NaOH was absent in DMSO.
Antioxidant Assay
The free radical scavenging activity of the Schiff base ligand (L) and its Cu(II), Co(II),
Ni(II) and Zn(II) complexes was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH)
method. Different concentrations of test compounds (12.5, 25, 50 and 100 mg) and standard
Vitamin C was taken in different test tubes and the volume of each test tube was adjusted to 100
µL by adding distilled DMF. To the tubes containing sample solutions in DMF, 5 mL methanolic
solution of DPPH (0.1 mM) was added to these tubes. The tubes were allowed to stand for 30
min. The control experiment was carried out as above without the test samples. The absorbance
of test solutions was measured at 517 nm. The reduction of DPPH was calculated relative to the
measured absorbance of the control. The radical scavenging activity was calculated using the
following formula:

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% Scavenging of DPPH = [(Control OD – Sample OD) /Control OD] X 100 --------- (2)
3.
Results and discussion
The analytical, physical properties and molar conductance data of the complexes are
given in Table1. 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 conductance values (6–
11 ꢀ-1cm2mol-1) of the complexes support their non-electrolytic nature. Thus, the present
complexes have non electrolytic nature as evidenced by the involvement of acetate ions in
coordination. This result was further confirmed from the chemical analysis of CH3COO- ion, not
precipitated by addition of FeCl3.The elemental analysis data of the complexes are in good
agreement with theoretical values presented in Table 1. The results obtained from micro
analytical measurements, metal estimation, conductivity and mass spectral data confirm the
stoichiometry of the copper complex as [CuL(OAc)2]. The magnetic moments of copper(II) in
any of its geometry lies around 1.85 B.M. which is very close to spin-only value i.e. 1.73 B.M.
The values which we found in our case lie in the range, 1.80–1.84 B.M. These values are typical
for mononuclear copper (II) compounds having d9-electronic configuration. The observed
magnetic moments of all the complexes correspond to distorted square planar Cu(II) complexes.
However, the values are slightly higher than the expected spin-only values due to spin orbit
coupling contribution.
IR Spectra
The IR spectrum of the ligand (L1, fig. 1) showed a band at 1680 cm-1 for the imine
ʋ(C=N) group which results from the Schiff base condensation of 2-aminobenzimidazole and
Knoevenagel condensate. It has shifted to a lower frequency of 1663 cm-1 after complexation
[23]. Moreover, the appearance of new bands at 466 cm-1 and 533 cm-1 corresponds to ʋ(M-N)
and (M-O) [24]. Also, the new bands at 1357cm-1 at 1255 cm-1 corresponds to symmetric and
asymmetric stretching for the ʋ(M-O) which evidence the participation of the COO- ion in the

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complexes. These facts are further supported by the appearance of bands between1380 cm-1-
1465 cm-1 and 1255 cm-1-1357cm-1 attributed to ʋasy(COO-) and ʋsy(COO-), respectively in all
copper complexes. The difference in ∆ʋ between ʋasy(coo-)and ʋsy(coo-) in metal complexes are
~100 cm-1 (105-125 cm-1) suggested the mode of coordination of carboxylate group in copper
complexes as monodentate manner [25]. Finally, it was concluded that the ligand(s) was
coordinated to copper atom through bidentate manner (imine nitrogen atoms). The coordination
modes are imine nitrogen atoms and acetate ions, respectively. The IR spectrum of ligand (L1)
and its copper complex was shown in figs.1 &2.
1H-NMR
The 1H NMR spectrum of the ligands were recorded and presented. The ligand (L1, fig. 3)
exhibits different signals as follows: the peak was observed at 5.6 ppm as singlet which
corresponds to –NH group (benzimidazole moiety) and D2O exchangeable peak. The aromatic
protons are observed in the range 7.32—7.80 ppm as multiplet peak. The signal was observed at
8.02 and 2.38-2.42 ppm which are assignable to the proton of the CH=C (Knoevenagel
condensate) and methyl group as singlet peaks. The other ligands showed similar NMR spectral
features (supplementary materials). There is a slight shift in aromatic proton peak was observed
due to the presence of substituents in the benzaldehyde moiety.
The 13C NMR spectra of ligands were recorded and presented. In the ligand L1(fig.4), the
aromatic ring carbons are appeared in the range 124.8-127.6 ppm. The methyl carbons are also
observed around 24.6 ppm. The peak was observed at 140.2 and 145.8 ppm which corresponds
to –
CH=C< and –CH=
C<, respectively. The peak was also observed at 192.4 ppm corresponds
to H3C-C. The other ligands showed similar carbon NMR spectral features. There is a slight shift
in aromatic ring carbon peaks were observed due to the presence of substituent’s in the
benzaldehyde moiety.

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Mass Spectra
The FAB mass spectra of the Schiff bases and their corresponding copper complexes were
recorded and compared their stoichiometry compositions. The [CuL1(OAc)2] complex shows the
molecular ion peak at m/z 757 and 759 (isotopic peaks of copper). This molecular ion undergoes
dissociation which yielded a fragment ion peak at m/z 638 and further decomposition leads to the
formation of demetallation to form the species [L]+ gave fragment ion peak at m/z 575. The
Copper complexes of [CuL2(OAc)2] shows molecular ion peak (M+) at m/z = 745, respectively.
Further, the fragmentation of two acetate ions was observed at m/z = 626. After losing their
metal, the corresponding ligands ([L]+) shows molecular ions peaks at m/z = 564.The mass
spectrum of Cu(II) complex of [CuL3(OAc)2] shows a molecular ion peak (M+) at m/z = 756.
The second fragmentation of two acetate ions leads to the formation of ligand moiety of 2-
aminobenzimidazole with molecular ion peak at m/z = 638, resulting from the elimination of
metal results ion peak ([L]+) at m/z = 574. The mass fragmentation pattern of copper complex .
In case of [CuL4(OAc)2] complex, the molecular ion peak observed at m/z = 737. This molecular
ion further by losing two acetate atoms gave a fragment ion peak at m/z = 618 and these
demetallation to form the species [L]+ gave fragment ion peak at m/z = 555. Elemental analysis
values are in close agreement with the values calculated from molecular formula of these
complexes which is further supported by the FAB-mass studies of representative complexes.
EPR Spectra
The X- band EPR Spectrum of copper complexes was recorded in the solution state. The
[CuL1(OAc)2] complex at 300 K shows an intense absorption at high field. In the complex at
frozen state shows four well-resolved peaks with different intensities in the low field region (Fig.
5) and one intense peak in the high field region. The magnetic susceptibility 1.8 B.M indicates
that the copper complex is mononuclear. This is also evident from the absence of a half field
signal observed in the spectrum at 1600 G due to the ms=±2 transition, ruling out Cu-Cu
interactions [26].

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In the square planer complexes, the unpaired electron is in the dx2-y2 orbital given 2B1g as
the ground state g11> g >2. The observed values (A11= 169>A =52.gll=2.34>g =2.05>2) are well
agreed with related square planer system [27]. In the axial spectrum, the “g” values are related
with the exchange interaction is negligible. If the value of G is less than 4, the exchange
interaction is considerable, For the present copper complexes, the G value of 6.8 suggest that the
tetragonal axes are aligned parallel or slightly misaligned, consistent with dx2-y2 ground state. The
in plane σ-bonding covalence parameters α2, is related to All, gll and g according to the
following equation,
α2=All/P+gll-2.0023+3/7 g -2.0023+0.04
If the value of α2=0.5 indicates covalent binding, while the values of α2=1.0 suggested
complete ionic bonding in complexes. The observed value of α2=0.56 indicates that the
complexes have some covalent character, the out of plane π bonding.γ2 and in-plane π bonding
(β2) parameters are calculated from the following expression.
β2 = (gll-2.0023E/-8λd2)
γ2 = g -2.0023 E/(-8λd2)
In these equations, λ= - 828cm-1 for the free metal ion. The observed β2 (1.72) and α2
(0.56) values indicate that there is interaction in the out of planeπ bonding where as in the in-
plane π bonding is complex ionic. This is also confirmed by the orbital reducing factors which
are estimated using the following reactions.
Kll =gll-2.0023∆E/8λ0
K = g -2.0027∆ E/2λ0
Where
λ0 is the spin orbital coupling constant for the copper(II) ion is -828cm-1
Kll and K are the parallel and perpendicular components of the orbital reduction
factor(K), respectively.

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The significant information about the nature of bonding in the copper(II) complexes can
be derived from the relative magnitudes of Kll<K . In case of pure of σ bonding
Kll≈K =0.77.where as Kll<K implies considerable in-planeπ bonding while her out of plane π
bonding Kll >K , For the present complexes.the observed order is Kll (0.96)>K (0.54) implying a
greater than contribuction from out of planeπ bonding than from in-plane π bonding.
The degree of geometrical distortion plays a significant role in the biological activities. It
was described as f = g///A// (index of tetragonal distortion). The “f” values are less than 135 cm
associated with the square-planar geometry, whereas higher values indicate distortions towards
tetrahedron. The f values of complexes were found to be in the range 126–141, indicating
significant distortion from planarity in the synthesized copper complexes. The nature of ligand
moieties and functional groups (in the ligands) were influenced on distortion from planarity. For
the present complexes, the gII/AII is 142-148 which is in agreement with significant deviation
from planarity and which is further confirmed by the bonding parameter α2 whose value is less
than unity.
TGA analysis
The thermal decomposition behavior of the complexes [CuL1(OAc)2] (fig.6),
[CuL2(OAc)2] was recorded upto 800°c in nitrogen atmosphere. The correlations between the
different decomposition steps of the complexes with the corresponding weight losses are
discussed in terms of the proposed formula of the complexes.
The thermal decomposition pattern of the [CuL1(OAc)2] complex displays three steps of
weight loss: (i) the coordinate acetate ions are dissociated from the complex relatively low
temperature range at 190-360°C, (ii) elimination of 2-aminobenzimidazole moiety was observed
with partial decomposition of the parent organic ligand in the temperature range 360-580°C and
(iii) the final decomposition step comprise the complete removal of the organic residual at the
temperature range 580-800°C and the formation of the metallic oxide CuO as a final thermal
decomposition product. The final decomposition products were identified by IR spectroscopy

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with corresponding spectra obtained under the same condition as the acetate and imine groups.
All other complexes showed similar thermogravimetric profiles.
DNA studies
DNA as a potential therapeutic target molecule has attracted by researchers towards
therapeutic agents. The binding studies between molecules and DNA are significant and fruitful
approach to develop efficient drug molecules. In the binding studies, calf thymus DNA was
selected as DNA molecule because of its medical importance, low cost and ready availability
properties to study the probable antimicrobial action mechanism of the effective compound at
molecular level by UV–Vis spectroscopic and electrochemical methods.
Hypochromism and hyperchromism are significant spectral terms to distinguish the
change of DNA double-helical structure in absorption spectroscopy. Due to the interaction
between the electronic states of intercalating chromophore and that of the DNA base, the
observed large hypochromism suggested a close proximity of the aromatic chromophore to the
DNA bases with a fixed concentration of DNA, UV–vis absorption spectra were recorded with
increasing amount of compound.
Electronic spectroscopy
The DNA binding efficiency of copper complexes were measured and presented. In the
absorption spectrum of copper complex with L1 showed an absorption band around 312.2 and
465 nm (due to π–π* and d-d transitions). In order to increase the concentration of DNA, there is
change in absorption and shift in wavelength was observed (i.e., hypochromicity with a red-
shifted). The observed hypochromicity was attributed to the π-stacking or hydrophobic inter-
actions of the aromatic phenyl rings with amino acids in the DNA [28]. As compared the results
with the standard intercalators, the copper complexes with electron withdrawing substituted
ligand has shown higher binding affinity than other complexes. Therefore, we concluded that the
planar nature of molecule, aromatic rings, conjugation effect and electron-withdrawing
substituted groups may greatly promote the DNA-binding ability of their Knoevenagel
condensate complexes.

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Cyclic voltammetric studies
The cyclic voltammogram of the glassy carbon electrode in solutions containing
[CuL1(OAc)2] in the absence and in the presence of varying amount of DNA were shown in
Fig.7. In the presence of DNA causes a considerable decrease in the voltammetric current of the
redox wave with a slight shift in E1/2 potential to positive region. The drop of the voltammetric
currents in the presence of DNA may be attributed to slow diffusion of the copper complex
bound to CT DNA. The net shift in E1/2 and decrease in current intensity can be explained in
terms of an equilibrium mixture of free and DNA-bound complexes to the electrode surface [29].
Finally, the conclusion arrived from the electrochemical behavior of copper complexes could
bound with DNA by intercalative binding mode.
Anti-microbial studies
The in vitro antimicrobial activities of the investigated compounds were tested against the
bacterial species, Acietobacter baumannil, Staphylococcus aureus, Escherichia coli, Klebsiella
pneumonia and Pseudomonas aeruginosa by disc diffusion method [30, 31] and summarized in
table 3. The inhibitions around the antibiotic discs were measured after incubation and
Streptomycin was used as standard drug.
The Schiff base and its complexes were screened for their anti microbial activity against
invitro anti bacterial of the Complexes have been carried out against the both Gram positive and
Gram negative bacteria using disc diffusion method. In the present study the results indicates that
all complexes have a higher anti microbial activity than the free ligand. This is due to the greater
lipophilic nature of the complexes. When concentration of the complexes increases, the activity
also increases. The concentration plays a vital role in increasing the degree of inhibition. The
higher anti microbial activity of the metal complexes compared to ligand may be due to the
change in structure or coordination and chelating tends to make metal complexes act more
powerful and potent bactereostatic agents, thus inhibiting the growth of the bacteria. This
increase in the activity of the complexes can be explained on the basis of chelation theory.

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Chelation considerably reduces the polarity of the metal ion because of partial sharing of its
positive charge with donor groups and possible electron delocalization over the whole chelate
ring. Such a chelation could enhance the lipophilic character of the central metal atoms.
Antibacterial activity of copper complexes was screened towards gram positive and gram
negative bacteria. Further, the ligand showed low activity and the complexes moderate to higher
activities as compared to standard drug towards the all organism tested due to the presence of
-NH group present in benziimidazole moiety plays an important role in the biological activity.
This group is believed to impart the transformation reaction in biological system. The
antimicrobial activity of four complexes can be referred to the increase of their lipophilic
character which in turn deactivates enzymes responsible for respiration processes and other
cellular enzymes, which is playing a vital role in various metabolic pathways of the tested
microorganisms.
Generally, metal complexes have shown higher activity against Gram-positive bacteria
than against Gram negative pathogens. In the present study indicated that the Gram-positive
bacteria were inhibited more strongly than Gram negative bacteria and explained on the basis of
complex structure of cell wall. The gram-negative bacteria possessing an extra outer layer on top
of the peptidoglycan and found to be highly impermeable. In the case of gram-positive bacteria,
the polysaccharides in their cell wall called teichoic acid (negatively charged) and facilitated the
passage of the positive metal ions.
The polysaccharides and lipids are prime important constituents of cell wall and
membranes and favorable for metal ion interaction. This cell wall also constitutes carbonyl,
phosphate and cystenyl ligands which help in maintaining the integrity of the membrane by
performing as a diffusion barrier and also provide suitable sites for binding interaction.
Moreover, the reduction in polarity enhances the lipophilic character of the copper chelates and
interaction between the lipid and copper ion is favoured. This may lead to the breakdown of the
permeability barrier of the cell resulting in intervention with the normal cell processes and

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blocking of the metal binding sites in the enzymes of the microorganisms. These complexes may
also disturb the respiration process of the cell and therefore block the protein synthesis, which
confines further growth of the organism.
In addition to this, mode of action of these complexes may involve formation of a
hydrogen bond through imine nitrogen atom with the active specific centers of cell constituents,
resulting in interference with the normal cell process. The relative difference in the activity of the
complexes against different kinds of microorganisms depend either on the difference in the
ribosomes of the microbial cells or on the impermeability of the cells of the microbes. The low
activity of some complexes may be ascribed due to low lipid solubility, consequently the metal
ion may not be able to reach the favorable site of action of the cell wall to get interfere with the
normal cell activity. Though chelation dominate in assessing the biological behavior of the
complexes but simultaneously other factors such as dipole moment, solubility, size, redox
potential of metal ions, coordinating sites, solubility, bond length between metal and the ligand,
geometry of complexes, steric, pharmacokinetic, concentration and hydrophobicity have
considerable influence on the antimicrobial potency. Hence, it may be concluded that the
antimicrobial activity of the complexes may not be due to chelation alone but it is convoluted
blend of numerous contributions.
SOD activity
Superoxide anions have a very short half life and produced continuously. In this
colorimetric based assay, Inhibition of the reduction of nitroblue tetrazolium (NBT) to formazan
(F) by the reported metal(II) complexes was used for detection of the SOD mimetic catalytic
activity of these chelates in the phosphate buffer under similar biological conditions. As the
reaction proceeding, the farmazan color is developed and the color changes from colorless to
blue, which was associated with an increase in the absorbance at 560 nm. SOD reduces the
superoxide ion concentration and thereby lowers the rate of formazan formation. In the SOD-like
activity test, the metal complexes compete with NBT for oxidation of the generated superoxide

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ions. The more efficient the complex, the lower the concentration that corresponds to 50%
inhibition of NBT reduction; this concentration is termed IC50 for comparative purposes.
The data in Table 4 also reports the scavenging efficiency of each complex, giving its
final concentration that produced efficient quenching of the superoxide anion radical. It also
reveals that there are difference between the values of scavenging effectiveness and the catalytic
constant of the complexes. This difference in the reactivates of these complexes can rationalized
by means of correlation between the redox potential of the couples CuII/CuI during the catalytic
cycle and the SOD-mimetic activity.
The higher biological activities of copper complexes may be attributed to the flexible
ligands, which are able to accommodate the geometrical change from CuII to CuI, specially the
labile water molecules, which are proposed to be easily substituted by the substrate O2 •- , in the
•-
catalytic process, just like the O2 , in place of H2O bound to copper site in the mechanism of
•-
dismutation of O2 by native SOD. The mechanism proposed for the dismutation of superoxide
anions by both superoxide dismutase and complexes 2 and 3 is thought to involve redox cycling
of metal(II) ions (eqs. 1 and 2):
-
M2+ + O2
→ M+ + O2
-
M+ + O2 + 2H+ → M+ + H2O2
It has been proposed that electron transfer between copper(II) and superoxide anion
radicals occurs through direct binding. Therefore, copper complexes exhibited higher SOD
activity than other metal complexes. This observation was confirmed by distortion of geometry
(“f” factor value). The synthesized copper complexes have higher distortion of geometry.
Comparative study
Researchers have synthesized different copper complexes and performed different
biological studies for chemotherapeutic agents. Mahdi Behzad et al [32] have performed
antibacterial activity for the synthesized copper and nickel complexes and compared. The
observations indicated that copper complexes with square planar geometry showed higher
activity. The nature of ligands, central metal and geometry will decide the biological activities.

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Mohamed M. Ibrahim [33] synthesized a series of albendazole-based copper(II) complexes and
structurally characterized. Authors have observed two different geometries for copper
complexes. Among the copper complexes, the complex with square planar environment
exhibited higher biological activities. Nasibeh Imani [34] have arrived conclusion that the
prepared copper complex with distorted square planar geometry have shown higher antibacterial
activity. Generally, Copper(II) complexes are appeared as potential drug candidates for
chemotherapeutic agents [35–37]. The DNA-interaction and cytotoxicity of curcumin analogs
[38–41] along with methylacetoacetate with copper(II) complexes as similar to cisplatin analogs.
Ali and coworkers have reported that the Knoevenagel’s condensate of a curcumin derivative
showed better pharmaceutical activity [42]. These Cu(II) complexes have two labile chlorides
similar to cisplatin, they may interact covalently with DNA bases and form stable complexes.
We inspired from the above facts, in the present study, the copper complex with electron
withdrawing substituted ligands (-Cl group) possessed higher biological activities. Here, the
complex geometry, ligand structural core and copper atom may enhance biological activities.
Conclusion
The design and synthetic approach was focused on the development of copper complexes
with curcumin analogs with enhanced biological activities. The lower molar conductance values
of the complexes which corresponds to non-electrolytic nature. The antibacterial screening of the
ligands and their copper complexes indicated that all the complexes showed higher anti
microbial activities than the free ligands. Superoxide dismutase and antioxidant activities of the
copper complexes have also been performed. In the electrochemical technique, the shift in ∆Ep,
E1/2 and Ipc values were explored for the interaction of the complexes with CT-DNA.
Acknowledgements
The authors articulate their heartfelt thanks to the Science & Engineering Research Board
(SERB), DST, New Delhi for financial Support (YSS/029/2013). They also express their
gratitude to the Dr. A.P.Majeed Khan, Chancellor, Noorul Islam Centre for Higher Education,
Department of Chemistry for providing research facilities.

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Figure captions
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
IR spectrum of ligand L1
IR spectrum of copper complex of L1
1H NMR spectrum of L1
13C NMR spectrum of L1
ESR spectra of copper complex of L1 at 77 K
TGA profile of copper complex of L1
The absorption spectra of complexes in the absence and presence of CT-DNA of
[CuL1(OAc)2] complex. Arrow shows the absorbance changing upon the increase
of DNA concentration.
Figure 8
The cyclic voltammogram of copper complex of L1 in the absence and presence
of DNA at various concentrations. Arrow indicates the changes in voltammetric
currents upon increasing the DNA concentration.
Scheme 1
Schematic presentation of synthesis of copper complexes

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8