Antimicrobial, antioxidant and SOD activities of copper(II) complexes derived from 2-aminobenzothiazole derivatives

Article abstract of DOI:10.1080/00958972.2016.1250153

A series of Cu(II) complexes have been synthesized from bidentate Schiff base ligands (by condensation of Knoevenagel condensate of acetoacetanilide (obtained from substituted benzaldehydes and acetoacetanilide) and 2-aminobenzothiazole). They were charac

Full text of DOI:10.1080/00958972.2016.1250153

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: http://www.tandfonline.com/loi/gcoo20
Antimicrobial, antioxidant and SOD activities
of copper(II) complexes derived from 2-
aminobenzothiazole derivatives
J. Joseph, G. Boomadevi Janaki, K. Nagashri & R. Selwin Joseyphus
To cite this article: J. Joseph, G. Boomadevi Janaki, K. Nagashri & R. Selwin Joseyphus
(2016): Antimicrobial, antioxidant and SOD activities of copper(II) complexes derived
from 2-aminobenzothiazole derivatives, Journal of Coordination Chemistry, DOI:
10.1080/00958972.2016.1250153
To link to this article: http://dx.doi.org/10.1080/00958972.2016.1250153
Accepted author version posted online: 25
Oct 2016.
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Date: 26 October 2016, At: 04:45

Publisher: Taylor & Francis
Journal: Journal of Coordination Chemistry
DOI: http://dx.doi.org/10.1080/00958972.2016.1250153
Antimicrobial, antioxidant and SOD activities of copper(II) complexes derived
from 2-aminobenzothiazole derivatives
J. JOSEPH*†, G. BOOMADEVI JANAKI†, K. NAGASHRI‡ and R. SELWIN JOSEYPHUS§
†Department of Chemistry, Noorul Islam Centre for Higher Education, Kumaracoil-629 180, Tamil Nadu, India
‡Department of Chemistry, Renganayagi Varatharaj College of Engineering, Sivakasi, Tamilnadu, India
§Department of Chemistry, Mar Ivanios College, Trivandrum, Kerala, India
A series of Cu(II) complexes have been synthesized from bidentate Schiff base ligands (by
condensation of Knoevenagel condensate of acetoacetanilide (obtained from substituted
benzaldehydes and acetoacetanilide) and 2-aminobenzothiazole). They were characterized by
elemental analysis, IR, 1H-NMR, 13C-NMR, UV-Vis., molar conductance, magnetic moment,
ESR spectra and electrochemical studies. Based on the magnetic moment, ESR and electronic
spectral data a distorted square planar geometry has been suggested for the complexes.
Antibacterial and antifungal screening of the ligands and their complexes reveal that all the
complexes show higher activities than the ligands. The antioxidant activities of the ligands and
complexes were determined by superoxide and hydroxyl radical scavenging methods in vitro,
indicating that the complexes exhibit more effective antioxidant activity than the ligands alone.
The results show that the Cu(II) complexes also have similar superoxide dismutase activity to
that of native Cu, Zn-SOD. All complexes exhibit suitable Cu(II)/Cu(I) redox potential (E1/2) to
act as synthetic antioxidant enzyme mimics.
Keywords: Bidentate; Geometry; ESR; Azomethine
Introduction
Most pharmaceutical agents contain transition metal as a constituent. Metals in therapeutic
agents have become increasingly important, resulting in a variety of exciting and valuable drugs
*Corresponding author. Email: chem_joseph@yahoo.co.in
1

such as cis-platin for cancer, bismuth for ulcers, Gd for MRI contrasting agents, etc. Transition
metal complexes of Schiff bases have attracted many researchers due to their biological activities
such as antifungal, antibacterial, anticancer and herbicidal [1-5]. The complexation of metal to
Schiff base ligand containing imine group improves the pharmacological activities of the ligand.
Investigations on the interaction between transition metal complexes and DNA have attracted
many researchers due to their importance in cancer therapy and molecular biology [6-9]. Schiff
base metal complex is an attractive metal-based drug due to its special activities in pharmacology
and physiology [10-12]. The coordination chemistry of copper(II) differs from other transition
metals due to its size and electronic configuration. Copper is an important element present in
several cell types with essential functions in the human body, participating in various
biochemical processes [13, 14]. Complexes of copper with nitrogen or oxygen ligands are of
considerable interest due to broad spectrum biological activities. Copper complexes, especially
mononuclear copper complexes, are used as antifungal and antibacterial drugs, DNA structural
probes, potential anticancer drugs and many neurodegenerative diseases, including
atherosclerosis (heart disease and strokes), degenerative brain disorders, arthritis, cancers, etc.
In continuation of our research work, the present study focuses on synthesis and
characterization of copper complexes of 2-aminobenzothiazole derivatives. The synthesized
ligands and their copper complexes were subjected to antimicrobial activities using serial
dilution technique.
Experimental
All chemicals used were of A.R. grade. Microanalytical data and FAB Mass spectra of the
compounds were recorded. The amount of copper present in the copper complexes was estimated
using ammonium oxalate method. NMR spectra of the ligands were recorded using TMS as
internal standard. Chemical shifts are expressed in parts per million relative to TMS. The FAB
mass spectra of the ligands and their complexes were recorded on a JEOL SX 102/DA-6000
mass spectrometer/data system using argon/xenon (6 kV, 10 mA) as the FAB gas. The
accelerating voltage was 10 kV and the spectra were recorded at room temperature using
m-nitrobenzylalcohol (NBA) as the matrix. IR spectra of the ligands and their copper complexes
were recorded on a Perkin-Elmer 783 spectrophotometer from 4000-200 cm-1 using KBr discs.
Electronic spectra were recorded with a Systronics 2201 Double beam UV-Vis.
2

spectrophotometer from 200-800 nm. The magnetic susceptibility values were calculated using
the relation μeff = 2.83 (χm.T)1/2. The diamagnetic corrections were made by Pascal’s constant
and Hg[Co(SCN)4] was used as a calibrant. ESR spectra of the copper complexes were recorded
at 300 and 77 K on a Varian E112 X-band spectrometer. Cyclic voltammetric measurements
were performed on CHI 604D using a glassy carbon working electrode, Pt wire auxiliary
electrode and a Ag/AgCl reference electrode. Tetrabutylammoniumperchlorate (TBAP) was used
as the supporting electrolyte. All solutions were purged with N2 for 30 min prior to each set of
experiments. The X-ray diffractometer system JEOL JDX 8030 was used to record powder data
for the copper complexes.
Preparation of Knoevenagel condensate β-ketoanilides
Ethanolic solution of 4-nitrobenzaldehyde (L1)/benzaldehyde (L2)/4-methoxybenzaldehyde (L3)
and 4-hydroxybenzaldehyde (L4) was added to acetoacetanilide in a beaker with continuous
stirring. The mixture was allowed to cool at room temperature and then piperidine was added to
it drop by drop with constant stirring. Stirring was continued for further 10 min. Then the
mixture was kept in a refrigerator for 48 h. After that the mixture was extracted with chloroform
and 5% hydrochloric acid. The mixture was vigorously shaken in a separating funnel. The
organic layer consists of Knoevenagel condensate β-ketoanilides and excess chloroform. The
bottom layer was eluted and dried by magnesium sulphate to remove water. The excess
chloroform was distilled out. The product was collected as orange yellow semi-solid and
preserved in a dessicator.
Synthesis of Schiff base
Hot ethanolic solution of Knoevenagel condensate β-ketoanilides was refluxed with an ethanolic
solution (30 mL) of 2-aminobenzothiazole and 1 g of anhydrous K2CO3 for about 12 h. The
solvent was reduced to one-third and the pasty mass so obtained was treated with hot water and
set aside in a refrigerator for 12 h. The solid material formed was removed by filtration and
recrystallized from ethanol. Yield: 72%.
L1: Yield: 72%. Anal. Calcd for C31H22N6S2O2: C, 64.79; H, 3.86; N, 14.63. Found: C, 64.85; H,
3.92; N, 14.66. FAB mass spectrometry (FAB-MS): m/z 575 [M+1]. 1H-NMR (400 MHz,
3

CDCl3, δ, ppm): 6.4—7.5 (17H, m, Ar-H), 1.5 (3H, s, H3C-C), 5.8 (1H, s, aldehydic CH proton)
and 11.2 (1H, s, N–H). 13C-NMR (400 MHz, CDCl3, ppm): 10.1 (H3C-C), 18.9 (HC=C), 142.2
(C=CH), 152.8 (C-1), 122.5 (C-2), 116.8 (C-3), 153.9 (C-4), 117.7 (C-5), 145.8 (C-6), 122.5
(C-12), 125.8 (C-13), 156.2 (C-14), 120.1 (C-15), 132.5 (C-16), 126.3 (C-17), 143.8 (C-19),
124.2 (C-20), 120.5 (C-21), 123.8 (C-22), 124.1 (C-23), 125.4 (C-24), 143.8 (C-19′), 124.2
(C-20′), 120.5 (C-21′), 123.8 (C-22′), 124.1 (C-23′), 125.4 (C-24’), 172.6 (C-18), 172.6 (C-18′).
L2: Yield: 58%. Anal. Calcd for C31H22N5S2: C, 66.01; H, 3.93; N, 12.42. Found: C, 66.05; H,
3.99; N, 12.48. FAB mass spectrometry (FAB-MS): m/z 576 [M+1]. 1H-NMR (400 MHz,
CDCl3, δ, ppm): 6.8—7.9 (17H, m, Ar-H), 1.5 (3H, s, H3C-C), 5.6 (1H, s, aldehydic CH proton)
and 11.8 (1H, s, N–H). 13C-NMR (400 MHz, CDCl3, ppm): 10.8 (H3C-C), 20.4 (HC=C), 146.4
(C=CH), 153.6 (C-1), 120.5. (C-2), 116.8 (C-3), 153.9 (C-4), 117.7 (C-5), 145.8 (C-6), 123.1
(C-12), 125.6 (C-13), 156.2 (C-14), 124.6 (C-15), 156.2 (C-16), 125.6 (C-17), 143.8 (C-19),
124.2 (C-20), 120.5 (C-21), 123.8 (C-22), 124.1 (C-23), 125.4 (C-24), 143.8 (C-19′), 124.2
(C-20′), 120.5 (C-21′), 123.8 (C-22′), 124.1 (C-23′), 125.4 (C-24’), 172.6 (C-18), 172.6(C-18′).
L3: Yield: 66%. Anal. Calcd for C31H23N5S2O: C, 68.24; H, 4.25; N, 12.52. Found: C, 68.32; H,
4.36; N, 12.60. FAB mass spectrometry (FAB-MS): m/z 546 [M+1]. 1H-NMR (400 MHz,
CDCl3, δ, ppm): 6.4—7.5 (9H, m, Ar-H), 1.5 (3H, s, H3C-C), 1.8 (3H, s, aldehydic CH proton)
and 11.2 (1H, s, N–H). 13C-NMR (400 MHz, CDCl3, ppm): 11.8 (H3C-C), 19.5 (HC=C), 144.4
(C=CH), 152.8 (C-1), 122.5 (C-2), 116.8 (C-3), 153.9 (C-4), 117.7 (C-5), 145.8 (C-6), 123.8
(C-12), 126.9 (C-13), 156.8 (C-14), 121.6 (C-15), 133.4 (C-16), 126.9 (C-17), 143.8 (C-19),
124.2 (C-20), 120.5 (C-21), 123.8 (C-22), 124.1 (C-23), 125.4 (C-24), 143.8 (C-19′), 124.2
(C-20′), 120.5 (C-21′), 123.8 (C-22′), 124.1 (C-23′), 125.4 (C-24’), 172.6 (C-18), 172.6 (C-18′).
L4: Yield: 62%. Anal. Calcd for C32H25N5S2O: C, 68.68; H, 4.51; N, 12.52. Found: C, 68.78; H,
4.65; N, 12.61. FAB mass spectrometry (FAB-MS): m/z 560 [M+1]. 1H-NMR (400 MHz,
CDCl3, δ, ppm): 6.4—7.5 (9H, m, Ar-H), 1.5 (3H, s, H3C-C), 1.8 (3H, s, aldehydic CH proton)
and 11.2 (1H, s, N–H). 13C-NMR (400 MHz, CDCl3, ppm): 10.9 (H3C-C), 19.9 (HC=C), 142.6
(C=CH), 152.8 (C-1), 122.5 (C-2), 116.8 (C-3), 153.9 (C-4), 117.7 (C-5), 145.8 (C-6), 123.9
4

(C-12), 126.2 (C-13), 156.7 (C-14), 120.8 (C-15), 133.9 (C-16), 127.0 (C-17), 143.8 (C-19),
124.2 (C-20), 120.5 (C-21), 123.8 (C-22), 124.1 (C-23), 125.4 (C-24), 143.8 (C-19′), 124.2
(C-20′), 120.5 (C-21′), 123.8 (C-22′), 124.1 (C-23′), 125.4 (C-24’), 172.6 (C-18), 172.6 (C-18′).
Synthesis of copper(II) complexes
An ethanolic solution of L1-L4 was added to a solution of copper acetate in ethanol (20 mL) and
the mixture was refluxed for 1 h, concentrated to one-third volume and kept at 0 °C for 2 h. The
solid product formed was filtered, washed several times with small amounts of ethanol and
diethyl ether and dried in vacuo. Yield: 64%.
Complex of L1: Yield: 62%. Anal. Calcd for CuC37H32N6O6: C, 56.65; H, 4.11; N, 10.72, Cu,
8.11. Found: C, 56.68; H, 4.17; N, 10.78; Cu, 8.17. FAB mass spectrometry (FAB-MS), m/z 785
[M+1]. μeff (BM) = 1.90; Λm (S cm2 mol-1) = 08.
Complex of L2: Yield: 66%. Anal. Calcd for CuC37H32N5O4: C, 57.42; H, 4.17; N, 9.06, Cu,
8.22. Found: C, 57.48; H,4.24; N, 9.15; Cu, 8.27. FAB mass spectrometry (FAB-MS), m/z 775
[M+1]. μeff (BM) = 1.92; Λm (S cm2 mol-1) = 12.
Complex of L3: Yield: 72%. Anal. Calcd for CuC37H33N5O5: C, 58.83; H, 4.41; N, 9.28, Cu,
8.42. Found: C, 58.92; H, 4.16; N, 9.34; Cu, 8.53. FAB mass spectrometry (FAB-MS), m/z 756
[M+1]. μeff (BM) = 2.02; Λm (S cm2 mol-1) = 06.
Complex of L4: Yield: 70%. Anal. Calcd for CuC38H35N5OS2: C, 59.32; H, 4.59; N, 9.11; Cu,
8.27. Found: C, 74.62; H, 4.63; N, 9.19; Cu, 8.31. FAB mass spectrometry (FAB-MS), m/z 770
[M+1]. μeff (BM) = 1.96; Λm (S cm2 mol-1) = 10.
Antimicrobial activity
The in vitro antimicrobial activities of the investigated compounds were tested against the
bacterial species, Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922),
Klebsiella pneumaniae (ATCC 2473), Proteus vulgaris (ATCC 8427) and Pseudomonas
aeruginosa (ATCC 27853) and fungal species Aspergillus niger (ATCC 16888), Rhizopus
5

stolonifer (ATCC 14037), Aspergillus flavus (ATCC 9643), Rhizoctonia bataicola (ATCC
14016) and Candida albicans (ATCC 10231) by disc diffusion method. MIC is the lowest
concentration of an antimicrobial compound that will inhibit the visible growth off a
microorganism after overnight incubation. MIC of the various compounds against bacterial
strains was tested through a modified agar well diffusion method.
Antioxidant assay
The samples were tested individually at a final concentration of 10 μM. The soluttion contained 1
mL of DPPH (diphenylpicrylhydrazyl radical, 60 μM) and different concentrations of the
antioxidant solutions of ligands and their complexes, resulting in a final concentraation of DPPH
of 30 μM. The mixtures were vigorously mixed and allowed to stand in the dark for 30 min at
25 °C. The absorbances of the resulting solutions were measured at 517 nm againnst a blank
sample containing only DPPH, the negative control; Rutin and Vitamin C served as positive
controls.
Scavenging effect (%) = ((A0-A1) / A0) × 100
(1)
where A0 is the absorbance of the control reaction and A1 is the absorbance in the presence of the
samples or standards.
Inhibition of superoxide radicals by riboflavin photoreduction method
Superoxide scavenging was determined by the NBT reduction method. The reactiion mixture
containing EDTA (0.1M), 0.0015% NaCN, riboflavin (0.12 mM), NBT (1.5 mM)) and various
concentrations of test sample and total volume was adjusted by adding phosphatee buffer
(6.7 mM, pH 7.8) to 3 mL in a test tube. The tubes were uniformly illuminated under an
incandescent lamp for 15 minutes, and thereafter the optical density was measureed at 530 nm.
The percentage inhibition of superoxide production was evaluated by comparing the absorbance
of the control and experimental tubes. The percentage inhibition was calculated bby using the
formula
Percentage inhibition = ((C-T) × 100) / C
6

where C and T are the optical density of the control and test samples, respectively.
DNA binding
Thermal denaturation (Tm) studies
Thermal denaturation studies were performed on a Systronics spectrophotometer equipped with a
thermostatic cell holder. The Tm value of CT-DNA was determined in the absence and presence
of Cu(II) complexes by keeping the concentration of DNA and [Cu(L10)(H2O)] (30 µM) in a 1:1
ratio. The DNA samples were continuously heated at 1 °C/min, while the absorption changes at
260 nm were monitored. The melting temperature (Tm), which is defined as the temperature
where half of the total base pairs are unbound, was determined from the midpoint of the melting
curves. ΔTm values were calculated by subtracting Tm of free DNA from Tm of DNA interacting
with the complex.
UV–Vis absorption spectroscopy
Absorption spectra were recorded on a Systronics 2201 Double beam UV-Vis.
spectrophotometer using 1 cm quartz micro-cuvettes. Absorption titrations were performed by
keeping the concentration of the complexes constant (10 µM), and by varying the concentration
of CT-DNA from 0 to 10 µM. In the reference cell, a DNA blank was placed so as to cancel any
absorbance due to DNA at the measured wavelength. For [Cu(L10)(H2O)] the binding constants
(Kb) were determined from the spectroscopic titration data using the following equation:
[DNA]/(εa-εf) = [DNA]/(εb-εf)+ 1/(εa-εf)
The ‘apparent’ extinction coefficient (εa) was obtained by calculating Aobsd/[Cu]. The terms (εf)
and (εb) correspond to the extinction coefficients of free (unbound) and the fully bound
complexes, respectively. A plot of [DNA]/(εa-εf) versus [DNA] will give a slope of 1/(εa-εf) and
an intercept of 1/Kb(εa-εf). Kb is the ratio of the slope to the intercept.
Electrochemical studies
Electrochemical behavior of copper complexes was recorded at room temperature in a CHI 604D
Electrochemical analyzer. Cyclic voltammetric measurements were performed using a glassy
7

carbon working electrode, Pt wire auxiliary electrode and a Ag–AgCl reference electrode.
Tetrabutylammonium perchlorate was used as the supporting electrolyte. All solutions were
purged with N2 for 30 min prior to each set of experiments. The electrochemical behavior of the
Cu(II) complexes in DMSO were performed in the presence and absence of CT DNA.
Partition coefficient determination
The lipophilicities of the complexes were determined by the shake flask method using phosphate
buffer (0.12 M NaCl) and n-octanol. Each compound was dissolved in the phase in which
duplicate determinations using three different solvent ratios were performed for each complex.
Following mixing and phase separation according to literature methods, each phase was analyzed
for solute content and the concentration was determined using spectrophotometric methods. All
n-octanol/water partition coefficients were determined by UV-visible spectroscopy. Octanol and
buffer solutions were pre-saturated with each other prior to use.
3. Results and discussion
The synthetic routes of the ligands (obtained from the condensation of Knoevenagel condensate
β-ketoanilides and 2-aminobenzothiazole) and their corresponding copper(II) complexes have
been outlined in scheme 1. They were thoroughly characterized by elemental analyses and
various analytical and spectroscopic techniques to identify their formulas to elucidate molecular
structures of ligands and their copper complexes. The purity of ligands and their copper
complexes has been checked by TLC. The analytical data provided in the Experimental section
for the ligands and their complexes were in agreement with the molecular formulas
[Cu(L)(OAc)2]. All the compounds are stable at room temperature in the solid state without
decomposition. The melting points of the complexes are higher than that of the ligands revealing
that the complexes are more stable than the ligands.
The ligands are more soluble in common polar organic solvents, such as CHCl3, EtOH
and THF, but partially soluble in nonpolar organic solvents, such as hexane, heptane and toluene.
The complexes are soluble only in polar organic solvents, such as DMF and DMSO. The
observed low molar conductance values of the copper complexes are consistent with
non electrolytes. Therefore, acetate is bonded to copper. The IR peak shift in νC=N of the ligand
in the complexes gave an idea about its coordination to copper(II). The geometry around Cu(II)
8

has been deduced from the absorption band observed in the UV–Vis spectra and magnetic
moment values.
Conductometric measurements in DMSO solution of the complexes showed no
significant change in the conductivity in comparison with that of the pure solvents. This behavior
is in agreement with the neutrality of the complexes, according to the formula assigned. The
conductivities of DMSO solutions were measured during a week and no major changes were
observed in solvents, showing the stability of the complexes in these solvents.
IR spectra
IR spectra provide information regarding the nature of the functional group attached to copper.
IR spectra of L1 and its copper complex are shown in figure 1. The spectrum of the ligand
showed two different -C=N bands from 1646-1681 cm-1, which are shifted to lower frequencies
in spectra of all the complexes (1580-1634 cm-1), indicating involvement of ν(-C=N) nitrogen in
coordination to copper. The present ligand systems are bidentate, bonded to copper ion via two
nitrogens (-C=N) of the Schiff base. New bands at 456 cm-1 and 514 cm-1 correspond to ν(M-N)
and ν(M-O) [15]. Also bands at 1386 cm-1 and 1290 cm-1 correspond to symmetric and
asymmetric ν(M-O), evidence for participation of COO- ion in the complexes. These facts are
further supported by appearance of bands between 1394-1458 cm-1 and 1282-1328 cm-1
attributed to υasy(COO-) and υsy(COO-), respectively, in all copper complexes. The difference in
Δυ between υasy(COO-) and υsy(COO-) in metal complexes was ∼100 cm-1 (110-135 cm-1)
suggesting coordination of carboxylate is monodentate. Thus copper complexes were
coordinated through azomethine nitrogens and acetates. The IR spectral features reinforced the
conclusion drawn from conductance measurements [16].
Electronic absorption spectra
Electronic absorption spectra of ligands and their complexes were recorded at 300 K in DMSO
and presented in the Experimental section. L1 shows two bands at 352 nm and 245 nm which
may be assigned to n→π* and π→π* transitions, respectively. The electronic spectrum of
[CuL1(OAc)2] shows a shoulder of CT-transition at 320 nm and a broad d–d absorption centered
at 584 nm, due to 2B1g→2A1g as reported for square planar Cu(II) complexes. The room
temperature magnetic moment for copper complexes (μeff = 1.75 BM) falls in the normal range
9

for copper(II) species with S = 1/2 and confirms that the copper(II) complex has square-planar
geometry with dx2–y2 ground state.
UV spectra of the complexes have been recorded in the pH range 6-8 (biological
experiments are performed at pH = 7) with the use of different buffer solutions (150 mM NaCl
and 15 mM trisodium citrate at pH values regulated by HCl solution) and no significant changes
(shift of the absorption or new absorption peaks) occurred, indicating that the complexes are
stable in the pH range 6-8. They showed similar UV-Vis spectral pattern in DMSO solution, in
the presence of the buffer solution (150 mM NaCl and 15 mM trisodium citrate in the pH = 7.0)
used in the biological experiments and in the pH range 6-8 suggesting that the compounds are
stable in solution.
NMR spectra
The 1H NMR spectra of ligands were recorded in DMSO-d6 and the data along with the
assignments are summarized in the Experimental section. NMR spectra of the Cu(II) complexes
are less resolved than that of the ligand due to broadening from paramagnetic properties of the
compound. The data obtained from the elemental analyses, IR, and 1H NMR spectral
measurements are in agreement with each other.
FAB mass spectra
The FAB mass spectra of the ligands and their copper complex were recorded and compared for
stoichiometric compositions. FAB mass spectrum of copper complex of L1 is shown in figure 2.
The molecular ion peak for L1 is observed at 575 m/z ratio which is also supported by the
"Nitrogen Rule", since the compound possesses an even number of nitrogens. The molecular ion
peak for the copper complex was observed at m/z = 785, confirming the stoichiometry of copper
chelates as [CuL1(OAc)2] type. It is also supported by FAB mass spectra of the other complexes.
The proposed path of the decomposition for the investigated Schiff base is presented in
scheme 2. Microanalytical data are also in agreement with values calculated from molecular
formulas assigned to these complexes, which is further supported by the FAB-mass studies of
representative complexes. The mass spectral features reinforced the conclusion drawn from
elemental analysis, copper estimation and molar conductance values.
10

ESR spectra
EPR spectra of the Cu(II) complexes have been recorded at room temperature as well as at liquid
nitrogen temperature and their gll and g⊥ values calculated. The data show that gll = 2.085–2.219
and g⊥ = 2.018–2.046 (table 1). The values of gll and g⊥ are close to 2 and gll > g⊥. This suggests
that the unpaired electron is in the d 2 orbital and hence, 2B1 is the ground state. For an ionic
2
x –y
environment gll > 2.3, while for a covalent environment gll < 2.3. The copper complexes show
gll < 2.3 indicating considerable covalent character. The g values are related by the expression
G = (gll − 2)/(g┴ − 2) which suggests an exchange interaction between copper centers in the
polycrystalline solid. If G > 4, the exchange interaction is negligible. In the present case, the
axial symmetry parameter, G, is 4.025–5.896, which indicates no significant exchange
interaction in these solid complexes.
The EPR spectra of copper(II) complexes were recorded in DMSO at 300 and 77 K. The
300 K spectrum shows an isotropic pattern, expected for Cu2+ in solution, but the spectra for the
frozen solutions show the usual anisotropic pattern expected for a powder sample. The absence
of half field signal at 1600 G, corresponding to ΔMs = 2 transition, rules out any Cu-Cu
interaction in the EPR spectra [17]. The spin Hamiltonian parameters of the complexes are given
in table 1. The frozen DMSO solution is axial with gII > g⊥ > 2.0023, indicating a d 2 ground
2
x –y
state [18] which is in agreement with the electronic absorption spectroscopic assignments. The
frozen solution spectrum of the complex shows four-line hyperfine splitting AII with signals from
63Cu and 65Cu slightly resolved at the low field component. The most remarkable feature is that
the gII value (2.18-2.24) is substantially higher than the majority of known copper(II) complexes.
A factor potentially contributing to increase of gII is distortion from square-planar geometry [19].
The electron spin resonance and optical spectra have been used to determine the covalent
bonding parameters for Cu(II) in various ligand field environments. Since there has been interest
in the nature of bonding parameters in the system, we adopted simplified molecular orbital
theory to calculate the bonding coefficients such as in-plane σ-bonding (α2), out-plane π-bonding
(β2) and in-plane π-bonding (γ2). The observed α2 (less than unity) and β2 (greater than 0.5)
values indicate that the copper(II) complexes have some covalent character.
The degree of geometrical distortion was described by a parameter; f = g///A// is an index
of tetragonal distortion. The f values less than 135 are associated with the square-planar
structures, whereas higher values indicate distortions towards tetrahedra [20]. The f values of
11

complexes were 126–141, indicating significant distortion from planarity. The nature of ligand
moieties and functional groups (in the ligands) influenced distortion from planarity. For the
present complexes, the gII/AII is 143-145, in agreement with significant deviation from planarity
and which is further confirmed by the bonding parameter α2 whose value is less than unity.
Redox behavior
The electrochemical behavior of the Cu(II) complexes in DMSO (0.1 M of TBAP as supporting
electrolyte, scan rate 100 mV s−1 at 300 K) were examined and tabulated (table 2). The cyclic
voltammogram of [CuL1(OAc)2] in DMSO solution at 300 K in the potential range +0.8 to -0.8
V at scan rate 0.1 Vs-1 is shown in figure 3. It shows a well-defined redox process corresponding
to the formation of the quasi-reversible Cu(II)/Cu(I) couple. The anodic peak at Epa =
-0.602 mV versus Ag/AgCl and the associated cathodic peak at Epc = -0.746 mV correspond to
the Cu(II)/Cu(I) couple. [CuL1(OAc)2] exhibits quasi-reversible behavior. It also shows one
irreversible peak at 0.416 mV which was assigned to Cu(II)/Cu(III). Similar electrochemical
behavior was observed for other complexes.
On comparing the cyclic voltammograms, we observed that the variation in oxidation and
reduction potential may be due to distortion in the geometry of the complexes which arises due
to different donors coordinated to copper. The present ligand systems stabilize the unusual
oxidation states of copper ion during electrolysis. The separation between the cathodic and
anodic peak current (I = Ipa/Ipc = 1.1) indicates a quasi reversible one electron process
assignable to Cu(II)/Cu(I) couple.
Substitution effects
Ligands containing unsaturated nitrogens and sulfur have been shown to facilitate reduction of
Cu(II) to Cu(I). Karlin suggested that copper sites in metalloproteins exhibit geometries that
favor Cu(I) [21]. The nature and position of substituents have also been shown to affect the value
of the redox potentials of the Cu(II)/Cu(I) couple. In the present study, the copper complexes of
substituted Knoevenagel condensates contain 2-aminobenzothiazole. The observed redox
potential of the Cu(II)/Cu(I) couple is controlled by the molecular structures of the complexes,
which depend on the substituents present in the ligands. The introduction of electron-
withdrawing substituent at the 4-position in the Knoevenagel condensates produced an increase
12

in the redox potential compared to that of the electron-releasing substituents. The
electrochemical behaviors of copper complexes in DMSO solutions were measured and are
summarized in table 2.
Powder XRD study
In absence of single crystal, X-ray powder data are useful to deduce accurate cell parameters.
The diffraction pattern reveals the crystalline nature of the complex. The crystallite sizes of the
copper complexes were calculated from Scherre’s formula. The appearance of crystallinity in the
Cu(II) complex is due to the inherent crystalline nature of the copper complexes (table 3). The
powder XRD pattern of the copper complex of L1 is shown in figure 4.
Solvatochromism
The electronic absorption spectra of copper complexes were performed in different solvents. The
structural characteristics of copper complexes are: (i) their high solubility in various organic
solvents and (ii) the change in the color of the solution on going from one solvent to another, i.e.,
strong solvatochromism of their solutions. The spectra showed one broad band which
corresponds to promotion of the electron in the lower energy orbital to the hole in dx2-y2 orbital of
copper(II) (d9). The position of this band shifted to longer wavelength (red shift) as the polarity
of solvent increases. Therefore, the copper complex structure depends on the polarity of solvents.
Antimicrobial study
The in vitro antimicrobial activities of the investigated compounds were tested against bacterial
and fungal species. The minimum inhibitory concentration (MIC) values of the compounds are
summarized in tables 4 and 5. A comparative study of the ligands and their complexes (MIC
values) indicates that complexes exhibit higher antimicrobial activity than the free ligands. The
activity of the Schiff base ligands may arise from hydroxyl groups, which play an important role
in antibacterial activity, as well as the presence of two imine groups important in elucidating the
mechanism of transformation in biological systems. The obtained results indicate that the
complexes have higher activity than the ligands against the same microorganisms under identical
experimental conditions, suggesting that chelation could facilitate a complex to cross a cell
membrane and can be explained by Tweedy’s chelation theory [22, 23]. The variation in the
13

effectiveness of different compounds against different organisms depends either on the
impermeability of the cells of the microbes or on differences in ribosome of microbial cells. The
activity of the Schiff base ligands and their metal complexes increases as the concentration
increases. The activity of complexes may be due to the effect of the acetate ions. These
complexes disturb the respiration process of the cell and thus block the synthesis of proteins
restricting further growth of the organism and as a result microorganisms die.
The prepared complexes were more active against Gram-positive than Gram-negative
bacteria, indicating that the antimicrobial activity of the compounds is related to cell wall
structure of the bacteria. The cell wall is essential to the survival of bacteria and some antibiotics
are able to kill bacteria by inhibiting a step in the synthesis of peptidoglycan. Gram-positive
bacteria possess a thick cell wall containing many layers of peptidoglycan and teichoic acids, but
Gram negative bacteria have a relatively thin cell wall consisting of a few layers of
peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and
lipoproteins. These differences in cell wall structure can produce differences in antibacterial
susceptibility and some antibiotics can kill only Gram-positive bacteria and are ineffective
against Gram-negative pathogens. This higher antimicrobial activity of the copper complexes
may be ascribed to electronic and/or structural changes of the ligated molecules due to
coordination, making the metal complexes more powerful bacteriostatic agents, thus inhibiting
the growth of the microorganisms.
Absorption spectral features of DNA binding
DNA binding studies of the complexes were studied using absorption spectra. A complex bound
to DNA through intercalation is characterized by change in absorbance (hypochromism) and red
shift in wavelength due to intercalation involving a strong stacking interaction between the
aromatic substituent and the DNA base pairs. The extent of hypochromism is consistent with the
strength of the intercalative interaction.
After interaction with the base pairs of DNA, the π→π* orbital of the bound ligand can
couple with the π orbital of the base pairs, due to decrease in π→π* transition energy, which
results in bathochromic shift. The results show that the absorbance (hypochromism) decreased
with successive addition of CT-DNA to the complex solution.
14

Hypochromism and bathochromic shift are observed for the complexes, suggesting that
binding is intercalative. The observed Kb values for copper complexes are equal to classical
intercalators bound to CT-DNA. The Kb values for the complexes are 4.4×106 M-1, 3.2×106 M-1,
2.8×106 M-1 and 3.6×106 M-1, respectively. The values of Kb have been described in the literature
for classical intercalators (ethidium bromide–DNA) whose binding constant have been found to
be in the order of 107 M-1. The results show that the present complexes are involved in
intercalative interactions with CT-DNA.
Viscosity measurements
The nature of the interaction between the complexes and CT-DNA were determined by viscosity
measurements. A classical intercalative mode was obtained on the basis of increase in viscosity
of DNA solution due to an increase in separation of base pairs at the intercalation sites and
increase in DNA length. Hydrodynamic measurements are sensitive to length change (i.e.
viscosity and sedimentation) are critical tests of a binding model in solution in the absence of
crystallographic structural data. A classical intercalation model demands that the DNA helix
must lengthen as base pairs separate. The plots of (η/ηo)1/3 vs [Complex]/[DNA] (η and ηo are the
relative viscosities of DNA in the presence and absence of compound, respectively) gives a
measure of the viscosity changes. On increasing amounts of complexes, the relative viscosity of
DNA increases steadily, similar to the behavior of ethidium bromide. The result shows that metal
complexes intercalate between the base pair of DNA, consistent with spectroscopic results.
Thermal denaturation studies
Thermal denaturation studies show evidence for the ability of the complexes to stabilize the
double-stranded DNA. Interactions between DNA and complexes were indicated by increase in
the thermal melting temperature (Tm). Thermal denaturation experiments also revealed
intercalation of these metal complexes with DNA. When the temperature increases in solution,
the double-stranded DNA dissociates to single strands and generates a hypochromic effect on the
absorption spectra of DNA bases (λmax = 260 nm). The extinction coefficient of the DNA base at
260 nm in the double-helical form is much less than that in the single strand form; hence, melting
of the helix leads to an increase in the absorption at this wavelength. Thus, the helix to coil
transition temperature can be determined by monitoring the absorbance of the DNA bases at λmax
15

= 260 nm as a function of temperature. This transition process, the melting temperature Tm,
defined as the temperature where half of the total base pairs are bonded, is introduced. The Tm of
calf thymus DNA is 65.8 0.2 °C. Under the same set of conditions, addition of metal
complexes increased Tm by 1 °C, 2 °C and 3 °C, respectively, which indicate that these
compounds stabilize the double helix of DNA. This method is easy to identify when more than
one transition occurs. This transition of double stranded DNA to single stranded DNA is termed
as the melting temperature of DNA (Tm). These variations in Tm of calf thymus DNA strongly
supported intercalation of metal complex into double helix DNA.
SOD activity
To correlate the structural aspects with the SOD activity, EPR spectra of both complexes were
recorded at 77 K in DMSO. Diaz et al. [24] have reported a good correlation between f factor
(gll/All, where All is expressed in cm-1) and SOD like activity, for a series of Cu(II) complexes.
An f factor value smaller than 135 cm is obtained for square planar Cu(II) complexes, and this
value increases with increasing square planar distortion. The f value for native SOD is 160 cm,
indicating a tetrahedral distortion from square planar geometry, which is one of the features that
enhances the catalytic activity of the enzyme. From the above EPR data, the f values for 1:1
complexes were determined to be 150, 145, 142 and 135 cm, respectively.
During SOD activity, there is change in the oxidation state from Cu(II) to Cu(I). Partially
filled orbitals get extra stabilization due to Crystal Field Stabilization Energy (CFSE) during the
complex formation process, whereas completely filled orbitals tend to have a tetrahedral
geometry which will have minimum ligand-ligand repulsion. Thus flexible ligands around the Cu
ion will help to shuttle between different oxidation states. The copper complexes exhibiting
appreciable square planar distortion are expected to show higher SOD activity.
Synthetic SOD mimics should be designed by taking into consideration longer shelf life,
higher lipid solubility and penetration into cells. It would be preferable to have SOD mimic
redox active metal complexes with antioxidants and other inflammatory drugs, because of added
pharmacological activity and reduced toxicity of the ligands. Curcumin has a remarkable
pharmacological activity including anti-inflammatory, anticarcinogenic and antioxidant activity
with almost no side effects. We hoped that the proposed copper complexes had higher biological
activity and behaved as synthetic enzyme mimic agents.
16

Antioxidant
Antioxidants that exhibit DPPH radical scavenging activity have received attention. Compounds
with antioxidant properties could be expected to offer protection in rheumatoid arthritis and
inflammation and lead to potentially effective drugs. Copper complexes of L3 showed good
antioxidant activity due to the presence of OH group and efficient hydrogen donors to stabilize
the unpaired electrons and thereby scavenge free radicals. The introduction of OH group in the
ligand system markedly increases the antioxidant efficiency of the complexes with careful
selection of the substituents on the ligand, the antioxidant behavior of the complexes can be
improved.
Partition coefficient determination
Partition coefficients, P, were measured to find the capacity of organic/inorganic based
compounds to cross lipid layers and inhibit microbial growth. The synthesized compounds were
tested for partition coefficients using the shake flask method. The synthesized compounds have
different P values due to conjugated system and different substituents present in the compounds.
The synthesized ligands have LogP values between 4.4-6.8, suggesting that ligands have
liphophilic characteristics.
The biosensitive copper complexes were subjected to the shake flask method to find the
partition coefficients, LogP in the range 3.4-4.2. Copper complex containing electron-
withdrawing substituent in L1 has low LogP value compared to other complexes. This liphophilic
behavior suggested that the complexes may penetrate more efficiently across the lipoidal
bacterial membrane than free ligands. These complexes may behave as effective
chemotherapeutic drugs after clinical trials.
Oxidative damage
The oxidative damage of HSA along with copper complexes in the presence of H2O2 was
performed at room temperature for 30 min (incubation). It was observed that copper complex
was able to cause extensive protein degradation after incubation with H2O2. In the incubation
period, the generation of hydroxyl radical is responsible for the observed degradation. The
hydroxyl radical mediated protein degradation in the presence of copper complex was confirmed
17

using Rhodamine B dye test. The solution containing hydroxyl radical which degrades
Rhodamine B dye during incubation with HAS and copper complex was monitored using
UV-Vis spectroscopy.
Conclusion
Bidentate Schiff base ligands derived from Knoevenagel condensate β-ketoanilides (obtained
from acetoacetanilide and benzaldehyde) and 2-aminobenzothiazole and their copper(II)
complexes were characterized by analytical and spectral techniques. From the electronic
absorption spectra and ESR spectral data, a distorted square-planar geometry was assigned for
the complexes. The lower conductivity values indicated that all the complexes are non-
electrolytes. Based on their magnetic susceptibility and ESR spectral data, monomeric
complexes were confirmed. The redox properties of copper ion, azomethine (>C=N) group,
hydroxyl moiety and also heterocyclic moieties are responsible for increased antimicrobial
activity. The new derivatives may provide choice and flexibility to change the structure in order
to find a less toxic derivative with enhanced activity. Copper complex of L1 presents a promising
pharmacological profile that could be an interesting starting point for future chemical
modifications in order to improve activity.
Acknowledgements
We express our sincere thanks to the Professor and Head, Department of Chemistry, and the
management of Noorul Islam Centre for Higher Education, Kumaracoil for their constant
encouragement and providing research facilities. JJ also thanks DST, New Delhi for financial
assistance (YSS/CS-029/2013).
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19

Scheme 1. Systematic route for synthesis of copper complex.
20

Scheme 2. Fragmentation pattern of the copper complex of L1.
21

Scheme 2 (continued)
22

Figure 1. UV-Visible spectrum of L1 and its copper complex in DMSO.
23

Figure 2. FAB mass spectra of L1 and its copper complex.
24

8.00E-05
6.00E-05
4.00E-05
2.00E-05
0.00E+00
-2.00E-05
-4.00E-05
2000
1500
1000
500
0
-500
-1000
-1500
-2000
E(MV)[Ag/AgCl]
Figure 3. Cyclic voltammogram of the copper complex of L1 in DMSO solution at room
temperature.
25

Figure 4. Powder XRD pattern of the copper complex of L1.
26

Table 1. ESR spectral parameters for the copper complexes.
α2
β2
γ2
Complex
g||
giso
A||
K||
F=(g||/A||)
g⊥
A⊥
K⊥
[CuL1(OAc)2]
at 300 K
2.06
[CuL1(OAc)2]
at 77 K
2.22 2.06 -
2.08
2.20 2.08 -
2.12
2.26 2.05 -
2.10
2.24 2.03 -
156 44 0.84 0.52 0.70 1.6 0.76 145
148 44 0.86 0.43 0.76 1.4 0.70 151
158 52 0.74 0.49 0.81 1.3 0.82 143
146 42 0.72 0.54 0.80 1.2 0.72 146
[CuL2(OAc)2]
at 300 K
[CuL2(OAc)2]
at 77 K
[CuL3(OAc)2]
at 300 K
[CuL3(OAc)2]
at 77 K
[CuL4(OAc)2]
at 300 K
[CuL4(OAc)2]
at 77 K
27

Table 2. Electrochemical parameters for the copper complexes.
Complex
Epa
Epc
Potential assignment
Cu(II)/Cu(I)
ΔEp
[CuL1(OAc)2]
-0.154
-0.272
118
[CuL2(OAc)2]
[CuL3(OAc)2]
[CuL4(OAc)2]
-0.580
1.96
-0.730
-
150
Cu(II)/Cu(I)
Ligand oxidation
-0.620
0.465
-0.752
-
132
-
Cu(II)/Cu(I)
Cu(II)/Cu(III)
-0.606
0.436
-0.778
-0.597
169
-
Cu(II)/Cu(I)
Cu(I)/Cu(0)
28

Table 3. Powder X-ray diffraction data for the copper complexes.
Complex
Grain size (nm)
[CuL1(OAc)2]
[CuL2(OAc)2]
[CuL3(OAc)2]
[CuL4(OAc)2]
60
66
78
52
29