Synthesis, structural characterization, fluorescence, antimicrobial, antioxidant and DNA cleavage studies of Cu(II) complexes of formyl chromone Schiff bases

Article abstract of DOI:10.1016/j.saa.2012.10.037

Cu(II) complexes have been synthesized from different Schiff bases, such as 3-((2-hydroxy phenylimino)methyl)-4H-chromen-4-one (HL₁), 2-((4-oxo-4H-chromen-3-yl)methylneamino) benzoicacid (HL₂), 3-((3-hydroxypyridin-2-ylimino)methyl)-4H-chromen-4-one (HL₃) and 3-((2-mercaptophenylimino)methyl)-4H-chromen-4-one (HL₄). The complexes were characterized by analytical, molar conductance, IR, electronic, magnetic, ESR, thermal, powder XRD and SEM studies. The analytical data reveal that metal to ligand molar ratio is 1:2 in all the complexes. Molar conductivity data indicates that all the Cu(II) complexes are neutral. On the basis of magnetic and electronic spectral data, distorted octahedral geometry is proposed for all the Cu(II) complexes. Thermal behaviour of the synthesized complexes illustrates the presence of lattice water molecules in the complexes. X-ray diffraction studies reveal that all the ligands and their Cu(II) complexes have triclinic system with different unit cell parameters. Antimicrobial, antioxidant and DNA cleavage activities indicate that metal complexes exhibited greater activity as compared with ligands.

Full text of DOI:10.1016/j.saa.2012.10.037

Accepted Manuscript
Synthesis, structural characterization, fluorescence, antimicrobial, antioxidant
and DNA cleavage studies of Cu(II) complexes of formyl chromone schiff bases
P. Kavitha, M. Saritha, K. Laxma Reddy
PII:
S1386-1425(12)01052-9
http://dx.doi.org/10.1016/j.saa.2012.10.037
SAA 9895
DOI:
Reference:
To appear in:
Spectrochimica Acta Part A: Molecular and Biomo‐
lecular Spectroscopy
Received Date:
Revised Date:
Accepted Date:
31 May 2012
28 September 2012
22 October 2012
Please cite this article as: P. Kavitha, M. Saritha, K. Laxma Reddy, Synthesis, structural characterization,
fluorescence, antimicrobial, antioxidant and DNA cleavage studies of Cu(II) complexes of formyl chromone schiff
bases, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2012), doi: http://dx.doi.org/
10.1016/j.saa.2012.10.037
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis, structural characterization, fluorescence, antimicrobial, antioxidant
and DNA cleavage studies of Cu(II) complexes of formyl chromone schiff bases
P. Kavitha a, M. Sarithab, K. Laxma Reddy a,*
a
Department of Chemistry, National Institute of Technology, Warangal 506004,Andhra Pradesh,
India
b Department of Biochemistry, Sri Venkateswara University, Tirupati 517502, Andhra Pradesh, India
Email: laxmareddychem12@gmail.com
Tel: +91870-2462650 Fax: +91-8702459547
ABSTRACT
Cu(II) complexes have been synthesized from different schiff bases, such as 3-((2-hydroxy
phenylimino)methyl)-4H-chromen-4-one
(HL1),
2-((4-oxo-4H-chromen-3-yl)methylneamino)
benzoicacid (HL2), 3-((3-hydroxypyridin-2-ylimino)methyl)-4H-chromen-4-one (HL3) and 3-((2-
mercaptophenylimino)methyl)-4H-chromen-4-one (HL4). The complexes were characterized by
analytical, molar conductance, IR, electronic, magnetic, ESR, thermal, powder XRD and SEM studies.
The analytical data revealed that metal to ligand molar ratio is 1:2 in all the complexes. Molar
conductivity data indicates that all the Cu(II) complexes are neutral. On the basis of magnetic and
electronic spectral data, distorted octahedral geometry is proposed for all the Cu(II) complexes.
Thermal behaviour of the synthesised complexes illustrates the presence of lattice water molecules in
the complexes. X-ray diffraction studies reveal that all the ligands and their Cu(II) complexes have
triclinic system with different unit cell parameters. Antimicrobial, antioxidant and DNA cleavage
activities indicate that metal complexes exhibited greater activity as compared with ligands.
Key words: Antimicrobial activity, Antioxidant, DNA cleavage, Fluorescence, Formylchromone
1. Introduction
Synthesis of chromone derivatives have been of special interest due to their biological activities
including antimycobacterial, anticonvulsant, antimicrobial, anticancer, antioxidant and mushroom
tyrosinase inhibition activities [1 4]. These derivatives also serve as intermediates to many products
of pharmaceuticals, agrochemicals and dyestuffs [5]. In addition, 3-formylchromone and its
derivatives inhibit a human protein phosphatase PTP1B [6]. Schiff bases are an important class of
ligands in catalysis and organic synthesis [7 10]. Furthermore, these ligands and their metal
complexes had a variety of biological, clinical, analytical and industrial applications. Schiff bases of
3-formyl chromone and its complexes also have a variety of applications in biological, clinical and
pharmacological areas [11 13]. A huge number of literature reports were available on DNA binding
1

studies of formyl chromone schiff bases and their metal complexes [14 16]. Knowledge of DNA
binding parameters of these molecules explores their potential applications, since their binding mode
could be associated with their ability to cause DNA damage [17 19]. This DNA damage is leading to
the inhibition of uncontrolled growth of cancerous cells. Complexes of essential metals like copper
and others are generally less toxic than that of non-essential ones and some of them even have
important cellular cytotoxic effects. In addition, high nucleobase affinity and comparatively strong
Lewis acidity of Cu(II) ion induce efficient DNA cleavage activity [20]. Prompted by the above
literature reports, we report here the synthesis, characterisation and biological activity studies of
Cu(II) complexes with 3-formyl chromone schiff bases such as 3-((2-hydroxy phenylimino)methyl)-
4H-chromen-4-one (HL1), 3-((3-hydroxypyridin-2-ylimino)methyl)-4H-chromen-4-one (HL3) and 3-
((2-mercaptophenylimino)methyl)-4H-chromen-4-one (HL4). The synthesis, characterisation and
antibacterial activity of 2-((4-oxo-4H-chromen-3-yl)methylneamino)benzoicacid (HL2) and its Cu(II)
complex was reported by C. Gyna Kumari et.al.[21]. However, we also report in this paper the
synthesis of Cu(II) complex of HL2 and its detailed characterisation, fluorescence and biological
activity studies (antifungal, antioxidant and DNA cleavage).
2. Experimental
2.1. Materials
AR grade 2-hydroxy acetophenone, phosphorous oxychloride (POCl3), 2-aminophenol, 2-amino-3-
hydroxy pyridine, 2-amino benzoic acid, 2-amino thiophenol, copper acetate, 1,1-diphenyl-2-picryl
hydrazyl (DPPH), butylated hydroxyl toluene (BHT) and organic solvents were used.
2.2. Synthesis of schiff bases
The ligands HL1, HL2 and HL3 were prepared as per the reported methods [11,22]. HL4 was prepared
as per the procedure given below.
The preparation of schiff base (HL4) was carried out by stirring the hot methanolic solutions of 3-
o
formyl chromone [23] (1 mM) and 2-amino thiophenol (1 mM) at 40 50 C up to 30 min. The
progress of reaction was monitored by TLC. The product thus obtained was recrystallized in
methanol. All these experimental details are given in Scheme 1.
2.3. Synthesis of Cu(II) complexes
A hot methanolic solution of schiff bases (2 mM) were added to a methanolic solution of
Cu(CH3COO)2·H2O (1 mM) and stirred for 1 2 h. After cooling, the green coloured complexes were
separated and washed several times with hot methanol and dried in vacuum.
2

2.4. Physical measurements
Elemental analysis for C, H, N, and S was performed using Perkin Elmer CHNS analyser. Molar
conductance of the complexes was measured in DMF using a Digisun conductivity meter. IR spectra
of the ligands and their Cu(II) complexes were recorded on Tensor 2 FTIR spectrophotometer within
the range 4000 400 cm-1 using KBr disc. The electronic spectra of the ligands and their Cu(II)
complexes were recorded on JASCO V-670 spectrophotometer in the range of 250–1400 nm.
Magnetic susceptibilities of the complexes were measured on a Guoy balance at room temperature, by
making diamagnetic corrections using Pascal’s constant. Thermo gravimetric analysis of the
complexes were carried out on a Perkin Elmer diamond TGA instrument at a heating rate of 20 oC and
nitrogen flow rate of 20 mL/min. The fluorescence spectra of ligands and their complexes were
recorded on FLUOROLOG-FL3-11 spectroflourimeter. The solid state ESR spectra of the complexes
were recorded using Bruker EMX EPR spectrometer at room temperature. The X-ray powder
diffraction analysis was carried out by using Xpert-Pro X-Ray diffractometer with Cu Kα radiation.
Scanning electron microscopy (SEM) of the complexes was obtained from Hitachi S-520 electron
1
microscope at an accelerated voltage of 10 kV. HNMR spectra of the ligands were recorded on
Bruker-300 MHz spectrometer using CDCl3 as a solvent and TMS as internal standard. Mass spectra
of the ligands were recorded on a Jeol JMSD-300 spectrometer.
2.5. Biological studies
2.5.1. Antimicrobial screening
In vitro antibacterial and antifungal activities of the ligands and their Cu(II) complexes were evaluated
against gram-negative (Proteus vulgaris, Klebsiella pnuemoniae) and gram-positive bacteria
(Staphylococcus aureus, Bacillus subtilis) and fungi strain (Candida albicans) using disc diffusion
method. Kanamycin (30 µg/disc) and Clotrimazole (10 µg/disc) are the standards for antibacterial and
antifungal activities. Sterile antibiotic discs (6 mm in diameter, prepared using Whatmann No. 1
paper) were placed over the nutrient agar medium. 100 µg of the compounds (initially dissolved in
DMSO) were transferred to each disc with the help of a micropipette. After overnight incubation at
37 °C for bacteria and 25 °C for fungi, the zone of inhibition was measured in mm and compared with
standard antibiotics. All the experiments were performed in triplicates and the average zones of
inhibition was recorded. Control measurements were carried out with DMSO. The compounds which
show significant activities were selected to determine the minimum inhibitory concentration (MIC)
using disc diffusion technique.
3

2.5.2. DPPH radical scavenging activity
The free radical scavenging activity of the ligands and their Cu(II) complexes were determined by
using DPPH according to the literature [24,25]. The principle for the reduction of DPPH free radicals
is that the antioxidant reacts with stable free radical DPPH and it converts into 1,1-diphenyl-2-
picrylhydrazine. The ability to scavenge the stable free radical DPPH is measured by decrease in the
absorbance at 517 nm.
2.5.2.1. Qualitative analysis
All the ligands and their Cu(II) complexes were applied on a TLC plate as a spot (100 µg/mL) using
mobile phase methanol:acetonitrile (7:1). It was allowed to develop the chromatogram for 30 min.
After the completion of the chromatogram, the plate was sprayed with DPPH (0.2% w/v). The colour
change (yellow spot on purple background on TLC plate) is an indication for the presence of
antioxidant activity.
2.5.2.2. Quantitative analysis
Each compound was dissolved in methanol 10 mg/10 mL and it was used as stock solution. From the
stock solutions 1 mL of each compound solution with different concentrations (0.25 µg 1.00 µg)
were added to the 3 mL of methanolic DPPH (0.004%) solution. After 30 min, the absorbance of the
test compounds was taken at 517 nm using UV-VIS spectrophotometer, which was compared with the
corresponding absorbance of standard BHT concentrations (0.25 µg 1.00 µg). DPPH solution was
used as control without the test compounds, whereas methanol was used as blank. The percentage of
scavenging activity of DPPH free radical was measured by using the following formula
Where, Ao is the absorbance of the control and Ai is the absorbance of the sample. IC50 values were
calculated for compounds, which exhibited the significant activity. IC50 is defined as concentration
sufficient to obtain 50% of maximum scavenging activity.
2.5.3. DNA cleavage studies
Agarose gel electrophoresis was used to study the DNA cleavage activity of ligands and their Cu(II)
complexes. pUC19 plasmid was cultured, isolated and used as DNA for the experiment. Test samples
(1 mg/mL) were prepared in DMF. 25 µg of the test samples and 5 µL H2O2 (500 µM) were added to
the isolated plasmid and incubated for 2 h at 37 oC. After incubation, 30 µL of plasmid DNA sample
mixed with bromophenol blue dye (1:1) was loaded into the electrophoresis chamber wells along with
the control DNA, 5M FeSO4 (treated with DNA) and standard DNA marker containing TAE buffer
(4.84 g Tris base, pH 8.0, 0.5 M EDTA/1 L). Finally it was loaded on to an agarose gel and
4

electrophoresed at a constant voltage of 50 V for 30 min. After the run, gel was removed and stained
with 10.01 µg/mL ethidium bromide and image was taken in Versadoc (Biorad) imaging system. The
extent of DNA cleavage and the results were compared with standard DNA marker.
3. Results and discussion
3.1. Characterisation of schiff base ligands
1
The schiff base ligands HL1, HL2 and HL3 were characterised using HNMR, 13CNMR and Mass
spectral data and were compared with the spectral data reported in the literature [11, 22]. However,
1
the ligand HL4 was characterized using analytical and HNMR, 13CNMR and Mass spectral data
(supplementary material). 1H NMR spectrum of HL4 ligand showed a signal at 4.75 ppm (S, 1H, SH)
is due to thiol proton. The methine proton of the azomethine group was observed as a singlet at 7.46
ppm (s, 1H, CH=N ). The aromatic protons are appeared as a set of multiplets in the range 8.25–
6.40 ppm (m, 9H, Ar CH). The 13CNMR spectrum of HL4 ligand showed a signal at 176.13 ppm is
due to the carbonyl carbon of the pyrone ring. The imine carbon and carbon adjacent to the oxygen of
the pyrone ring appear at 153.68 and 156.22 ppm, respectively. The aromatic carbons are observed in
the range of 147.27 110.51 ppm. The mass spectra of HL4 (Formula weight = 281) shows a
molecular ion peaks at m/z 282.04 and 304.02 corresponding to [M+1] and [M+23], respectively.
From all the spectral data, the proposed structures of the ligands are given in Fig. 1.
3.2. Characterisation of Cu(II) complexes
All the Cu(II) complexes were partially soluble in MeOH, CHCl3 but freely soluble in DMF and
DMSO. The analytical and physical data for the metal complexes are listed in Table 1. The analytical
data listed in Table 1 indicate that the metal to ligand molar ratio is 1:2 for all the complexes. The
conductivity data of all the metal complexes indicate that all are neutral in nature [26].
3.2.1. Determination of Cu(II) content in the complexes
Known amount (0.150 g) of the metal complexes were decomposed with concentrated nitric acid, the
excess nitric acid is being expelled by evaporation with concentrated sulphuric acid. This process is
repeated till the organic part of the complex got completely lost. The residue was cooled and dissolved
in dilute nitric acid and the determination of Cu(II) was carried out using spectrophotometric method
[27].
3.2.2. Infrared spectral studies
Infrared spectral data of ligands and their Cu(ІІ) complexes are listed in Table 2. All the schiff base
ligands have the most characteristic band of azomethine (C=N) group appeared in the range of
1600 1550 cm-1 [28]. In all their Cu(II) complexes this band was shifted about 10 25 cm-1 to lower
wave number. It indicates the involvement of nitrogen atom of the azomethine group in coordination
5

to the metal ion. A broad band appeared at 3250 cm-1 in HL1 and HL3 ligands is due to phenolic
υ(O H) group, which was disappeared in their complexes, confirms the participation of oxygen of
O H group in coordination to the metal ion. The stretching vibrations of S H have no significant
role, since its band is very weak in HL4 ligand. However, participation of the SH group in chelation is
ascertained from the shift of υ(C S) at 790 cm-1 in the ligand to lower frequencies by 40 cm-1 in the
complex [29]. In HL2 ligand, a strong band appeared at 1365 cm-1 is due the υ(C O) of carboxylic
group. In its Cu(II) complex it was shifted to 1351 cm-1, indicating the coordination of carboxylic
group through the oxygen atom to the metal ion [21]. In all the schiff base ligands, appearance of a
band in the range of 1650 1620 cm-1 assignable to the υ(C=O) of chromone moiety [30]. In their
Cu(II) complexes this band was shifted to lower wave number by about 10 50 cm-1, which confirms
the participation of oxygen atom of carbonyl group in coordination to the metal ion. Participation of
oxygen and nitrogen atoms in the coordination is further confirmed by far IR spectral bands at around
600 400 cm-1 range corresponding to υ(M N) and υ(M O), respectively. All complexes showed a
broad band around 3400 cm-1 due to υ(OH) from water molecules. The IR spectral data confirms, all
the schiff base ligands coordinated to a Cu(II) ion in a tridentate manner through ONO/ONS.
3.2.3. Electronic spectral and magnetic studies
The geometry of the Cu(ІІ) complexes was deducted from the electronic spectra. The electronic
spectra of HL1 and its Cu(II) complex are shown in Fig. 2. Electronic spectral data of all ligands and
their Cu(ІІ) complexes and magnetic moment values are depicted in Table 3. The electronic spectra of
all ligands show two bands. One band within the range of 25,000 26,000 cm-1 is attributed to the
n → π* transitions. Another band within the range of 31,000 34,000 cm-1 is due to the π → π*
transitions. All the Cu(ІІ) complexes show a band 18,000 19,000 cm-1 range and a shoulder on the
lower energy side at 13,000 15,000 cm-1 range indicates that these complexes are in distorted
2
2
octahedral structure [31]. The two bands observed in their electronic spectra due to B1g → B2g and
2
2B1g → Eg transitions, respectively. These transitions are characteristic of distorted octahedral
geometry [32].
The magnetic moment values (Table 3) of all Cu(II) complexes are observed in the range of
1.74 2.01 BM, which infer the complexes are in distorted octahedral structure [33]. The magnetic
moment values are higher than normal range of Cu(ІІ) complexes which may be referred to spin orbit
coupling.
3.2.4. ESR spectral studies
ESR spectra of all the Cu(II) complexes were recorded at room temperature in the solid state. The
spectral data of all the complexes are given in Table 4. The complexes, Cu(L3)2 and Cu(L4)2 showed a
6

broad band centred at g=2.09 and 2.06, respectively without resolved hyperfine structure.
[Cu(L2)2].H2O complex exhibits four well resolved hyperfine peaks and [Cu(L1)2].2H2O complex with
unresolved peaks (Fig. 3). The values of the complexes are g|| > gꢀ > 2.0023, indicating that the
unpaired electron in the ground state of Cu(ІІ) is predominately lies in
orbital [34] and also
suggesting a tetragonal distortion around Cu(II) ions [35]. Kivelson and Neiman showed that for an
ionic environment g|| is normally 2.3 or larger, but for covalent environment g|| is less than 2.3. In the
present study, g|| value for the Cu(II) complexes are in the range of 2.19 2.20, consequently the
environment is covalent. The (g||/A||) values also indicates the stereochemistry of the Cu(II)
complexes. Reported range for square planar complexes is 105 135 cm-1 and for tetragonal distorted
complexes is >135 cm-1 [36]. For the present Cu(II) complexes g||/A|| value is >135 cm-1, indicates the
tetragonal distortion. The metal-ligand σ bonding parameter α2 is calculated using the literature [37].
The value of exchange interaction term G was calculated from the following expression
According to Hathaway [38], if the value of G is greater than 4, the exchange interaction between
Cu(II) centres in the solid state is negligible. Whereas its values are less than 4, a considerable
exchange interaction exists in the solid complex. The G values obtained in the present Cu(ІІ)
complexes are greater than 4, indicating the absence of exchange interaction between Cu(ІІ) centres in
2
the solid state [39]. Further, orbital reduction parameters like K||2 = α2β2, Kꢀ = α2γ2 are calculated
using following expressions [40, 41].
,
7

Here α2 measures the in-plane σ bonding, β2 measures the in-plane π-bonding and γ2 is the out of plane
π-bonding. A number of conclusions are deduced from these parameters. α2 values for purely σ
bonding ligands such as ammonia and ethylenediamine lies in the range of 0.73 0.77. For completely
covalent bond α2 is 0.5 and for purely ionic bond α2 is 1. In the present complexes, α2 values fall in the
2
range 0.6 0.7 indicating appreciable covalency in the σ bond. Kꢀ > K||2 is for in-plane π-bonding and
Kꢀ2 < K||2 is for out of plane π-bonding. In the present complexes Kꢀ2 > K||2, this indicates that the
complexes are in plane π-bonded. The spin orbit coupling constant for the complexes was found to be
less than that of the free metal ion (-828 cm-1), suggesting considerable mixing of ground and excited
terms. The values obtained for hyperfine splitting and covalency parameters are well agreement with
literature reports [42].3.2.5. Thermal studies
TG graphs of all Cu(II) complexes are shown in Fig. 4. The decomposition stages, temperature range,
as well as the observed and calculated mass loss percentages of all the complexes are illustrated in
Table 5. Thermo gravimetric analysis (TGA) was used as a probe to establish the presence of water
molecules either in the coordination sphere or in the ionic sphere [43]. Generally in TG Analysis,
o
lattice water molecules were lost in the temperature range of 50 120 C and coordinated water
molecules at 120 250 oC range.
[Cu(L1)2].2H2O complex was decomposed in four steps. The first step is corresponding to the loss of
two lattice water molecules in the temperature range between 40 144 oC with a mass loss of 5.39%.
o
The second (145 289 oC) and third steps (290 460 C) are the decomposition of schiff base ligand
with the mass loss 23.09% and 16.93%, respectively. The final step found in the range of
(461 779 oC) with the mass loss of 41.82% can be regarded as the total loss of schiff base ligand. The
remaining mass 12.77% is due to the formation of CuO as residue.
[Cu(L2)2].H2O complex is decomposed in three steps. The first step is due to the loss of lattice water
o
molecule below the 120 C with the mass loss of 2.65%. The second step (121 532 oC) corresponds
to the loss of 61.76% which may be due to the removal of schiff base ligand. The third step (533 827
8

oC) is corresponding to the removal of a fragment of the schiff base ligand with the mass loss 23.54%.
The remaining mass 12.05% is corresponds to the formation of CuO as residue.
o
Cu(L3)2 complex is stable up to 137 C after that it is decomposed in two steps. In the first step
complex shows the mass loss of 18.04% in the temperature range (137 368 oC), corresponding to the
o
partial decomposition of schiff base ligand. And the second step found in the range (369 885 C)
with a mass loss of 68.45% was assigned to the complete removal of schiff base moiety. The residue
with a mass of 13.51% was regarded as CuO.
o
Cu(L4)2 complex is decomposed in two steps. The first (40 374 C) and second (375 852 oC) steps
are due to the loss of ligand molecules with the mass loss of 61.78% and 25.55% respectively. The
remaining mass 12.57% is regarded CuO as residue.
The overall weight loss and weights of residues are well in agreement with the experimental error.
On the basis of the above studies the proposed structures of the Cu(II) complexes are shown in Fig. 5.
3.2.6. Fluorescence studies
The emission spectra of all the ligands and their Cu(ІІ) complexes were recorded in the solid state.
The emission spectra of HL3 and its Cu(ІІ) complex are shown in Fig. 6. The HL1 ligand was
characterised by an emission band around 540 nm upon photo excitation at 398 nm. Cu(II) complex of
HL1 shows three emission bands at 541, 604 and 727 nm. The HL2 ligand exhibits a band at 492 nm
and their Cu(II) complex was characterised by the emission bands at 494, 540 and 652 nm. HL3 and
its Cu(II) complex exhibit emission bands at 524 and 552, 621, 748 nm respectively. HL4 and its
Cu(II) complex was characterised by an emission bands at 492 and (440, 490, 599) nm respectively
upon photo excitation at different wavelengths. There was a decrease in the intensities of the emission
bands of complexes compared to the respective ligands. Earlier reports showed that transition metals
decrease the fluorescence intensity very effectively [44 46].
3.2.7. Powder XRD and SEM studies
The Powder XRD patterns of HL3 and its Cu(ІІ) complex are shown in Fig. 7. The observed unit cell
parameters are given in Table 6. The Powder XRD patterns of all ligands and their Cu(ІІ) complexes
were recorded. Peak positions of all ligands were different from its Cu(II) complexes indicating the
formation of metal complexes. Unit cell parameters were found by using trial and error methods. All
compounds are triclinic with different unit cell parameters. The XRD patterns of all complexes are
similar and suggest that all the complexes possess similar structure. The average crystallite sizes for
all compounds is calculated using Debye Scherrer’s formula [47]
9

Where λ is the wavelength of Cu-Kα radiation (1.5406 Å), β is the full width at half maximum and θ
is the Braggs reflection angle. The crystallite sizes are found to be in the range of 10 100 nm for all
the compounds. The morphology and grain size of the metal complexes have been illustrated using the
scanning electron macrograph (SEM). The SEM micrographs of [Cu(L1)]2.2H2O and [Cu(L3)]2 are
shown in Fig. 8. SEM images of the metal complexes with uniform matrix imply their homogeneous
nature. [Cu(L1)]2.2H2O and [Cu(L3)]2 complexes show plate and semi needle like morphologies,
respectively.
3.2.8. Biological activity studies
3.2.8.1. Antimicrobial activity
The MIC values of the antimicrobial activity of all compounds are presented in Table 7. All the
complexes exhibited moderate to good activities when compared with the standard antibiotics. The
ligands hold less activity except HL2, against tested strains. All Cu(II) complexes show marked anti
microbial activity against all strains when compared to their corresponding ligands. The MIC values
were calculated, which have significant activity (Table 7). Higher activity of the complexes was
explained based on Overtones concept and chelation theory. According to Overtones concept of cell
permeability, the lipid membrane that surrounds the cell favours the passage of only lipid soluble
materials. Liposolubility is an important factor which controls the antimicrobial activity. On chelation,
the polarity of the metal ion is reduced to a greater extent due to the overlap of the ligand orbital and
partial sharing of positive charge of the metal ion with donor groups. Further, it increases the
delocalisation of π-electrons over the whole chelating ring and enhances the penetration of the
complexes into lipid membranes and blocking of the metal binding sites in the enzymes of
microorganisms. There are also other factors which increase the activity are solubility, conductivity
and bond length between the metal and ligand [48 54].
3.2.8.2. DPPH radical scavenging activity
The scavenging activity was increased by increasing the concentration of the compounds. DPPH
antioxidant assay is based on ability of decolourization of DPPH (a stable free radical) in the presence
of antioxidants. This radical contains an odd electron responsible for absorbance at 517 nm. When
DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized which can
be quantitatively measured by changes in absorbance. In the present study, based on the IC50 values,
the scavenging activities of ligands and their Cu(II) complexes are calculated and those values are
given in Table 8. From the results, it was found that the IC50 values of [Cu(L1)2].2H2O is 0.16 µg/mL.
[Cu(L1)2].2H2O show good activity compared to remaining complexes. HL1 and HL2 ligands do not
show any scavenging activity. The radical scavenging activity of the compound depends on the
10

structural factors such as the phenolic hydroxyl, carboxylic groups and other structural features [55].
The order of the scavenging activity of all the complexes according to their IC50 values is given
below.
[Cu(L1)2].2H2O > [Cu(L2)2].H2O >[Cu(L3)2] >[Cu(L4)2]
3.2.8.3. DNA cleavage activity
DNA cleavage activity was studied for all the ligands and their Cu(II) complexes using gel
electrophoresis method against pUC19 DNA in the presence and absence of H2O2 as an oxidant. The
results of the DNA cleavage studies are depicted in Fig. 9. When circular plasmid was subjected to gel
electrophoresis, the fastest migration will be observed for the supercoil form (Form I). If scission
occurs on one strand it is called as nicked circular (Form II). The supercoil will relax to generate a
slower moving nicked circular form (Form II). If both strands are cleaved, a linear form (Form III)
that migrates between Form I and Form II will be generated [56]. To investigate the mechanism of
nucleolytic activity, experiments were also performed in the presence of H2O2. All the ligands
exhibited significant activity in the presence of H2O2. In the absence of H2O2, all ligands do not show
cleavage activity but their Cu(II) complexes are able to convert supercoiled (Form I) to nicked circular
(Form II) and open circular form (Form III). Thus revealed that all Cu(II) complexes except [Cu(L3)2]
show good nuclease activity in the absence of any additives. Control (DNA alone) does not show any
cleavage. FeSO4 is used as standard, it shows complete DNA cleavage activity. In the presence of
H2O2, the intensity of the treated DNA samples has diminished because of the DNA cleavage [57].
Ligands HL1, HL2 and HL4 show cleavage activity except HL3. [Cu(L3)2] complex shows moderate
activity compared to the remaining complexes. In all the ligands (except HL3) and their Cu(II)
complexes, more pronounced activity was observed in the presence of oxidant compared to the
absence of oxidant, may be due to the increased production of hydroxyl radicals. These results
indicate that the Cu(II) ion also plays an important role in the cleavage activity. As the compound has
DNA cleavage activity, it can be concluded that the compound inhibits the growth of the pathogenic
organism by cleaving the genome [58].
4. Conclusions
In the present study, we synthesised 3-formyl chromone schiff bases and their Cu(II) complexes. All
synthesized complexes were characterized by using various spectroscopic techniques. The results
revealed that all the ligands were coordinated to the Cu(II) ions in tridentate manner and geometrical
structures of these complexes were found to be tetragonally distorted octahedral. From the
thermograms the number of lattice water molecules present in the complexes was calculated. The well
defined crystalline and homogeneous nature of the Cu(II) complexes was observed from PXRD and
11

SEM studies. The schiff bases and their Cu(II) complexes are fluorescent in nature. All the Cu(II)
complexes exhibited prominent antimicrobial activity than ligands. All the Cu(II) complexes show
good DPPH scavenging activity. DNA cleavage activity was observed in the presence of H2O2 for all
ligands (except HL3) and their Cu(II) complexes.
Acknowledgements
Authors wish to thank Prof.B.Viswanathan, NCCR, and IIT Madras for generously providing TG
facility. Authors would like to express thanks to the Director, CIF, Pondicherry University, for
recording Fluorescence spectra and Vimta Labs Ltd, Hyderabad for DNA cleavage studies.
References
[1] G. Athanasellis, G. Melagraki, A. Afantitis, K. Makridima, O. Igglessi-Markopoulou, Arkivoc
10 (2006) 28 34.
[2] G.R. Singh, N.K. Singh, M.P. Girdhar, S. Ishar, Tetrahedron 58 (2002) 2471 2480.
[3] L.Z. Piao, H.R. Park, Y.K. Park, S.K. Lee, J.H. Park, M.K. Park, Chem. Pharm. Bull. 50
(2002) 309 311.
[4] V. Barve, F. Ahmed, S. Adsule, S. Banerjee, J. Med. Chem. 49 (2006) 3800 3808.
[5] H. Harnisch, Liebigs Ann. Chem. 765 (1972) 8 14.
[6] Y.S. Shim, K.Ch. Kim, D.Y. Chi, K.H. Lee, H. Cho, Bioorg. Med. Chem. Lett. 13 (2003)
2561 2563.
[7] X.M. Ouyang, B.L. Fei, T.A. Okamuro, W.Y. Sun, W.X. Tang, N. Ueyama, Chem.
Lett.(2002) 362 363.
[8] A. Datta, N.K. Karan, S. Mitra, G. Rosair, Z. Naturforsch. 576 (2002) 999 1002.
[9] C. Jayabalakrishnan, K. Natarajan, Trans. Met. Chem. 27 (2002) 75 79.
[10] H. Sharghi, M.A. Nasseri, Bull. Chem. Soc. Jpn. 76 (2003) 137 142.
[11] K.M. Khan, N. Ambreen, U.R. Mughal, S. Jalil, S. Perveen, M.I. Choudhary, Eur. J. Med.
Chem. 45 (2010) 4058 4064.
[12] K.M. Khan, N. Ambreen, S. Hussain, S. Perveen, M.I. Choudhary, Bioorg. Med. Chem. 17
(2009) 2983 2988.
[13] T. Rosu, E. Pahontu, C. Maxim, R. Georgescu, N. Stanica, G.L. Almajan, A. Gulea,
Polyhedron 29 (2010) 757 766.
[14] Y. Li, Z.Y. Yang, Z.C. Liao, Z.C. Han, Z.C. Liu, Inorg. Chem. Commun. 13 (2010)
1213 1216.
[15] B.D. Wang, Z.Y. Yang, M. Hua Lu, J. Hai, Q. Wang, Z.N. Chen, J. Organomet. Chem. 694
(2009) 4069 4075.
12

[16] D.D. Qin, Z.Y. Yang, B.D. Wang, Spectrochim. Acta A 68 (2007) 912 917.
[17] C.H. Ng, K.C. Kong, S.T. Von, P. Balraj, P. Jensen, E. Thirthagiri, H. Hamada, M. Chikira,
Nucleic Acids Res. 28 (2000) 4856 4864.
[18] A. Sancar, G.B. Sancar, Annu. Rev. Biochem. 57 (1988) 29 67.
[19] S.S. David, S.D. Williams, Chem. Rev. 98 (1998) 1221 1261.
[20] P. Kumar, B. Baidya, S.K. Chaturvedi, R.H. Khan, D. Manna, B. Mondal, Inorg. Chim. Acta
376 (2011) 264 270.
[21] M. Padmaja, J. Pragathi, C. Gyana Kumari, J. Chem. Pharm. Res. 3 (2011) 602 - 613.
[22] I. Sigg, G. Haas, T. Winkler, Helv. Chim. Acta 65 (1982) 275 – 279.
[23] A. Nohara, T. Umetani, Y. Sanno, Tetrahedron Lett.14 (1973) 1995 1998.
[24] A. Braca, N.D. Tommasi, L.D Bari, C. Pizza, M. Politi, and I. Morelli, J. Nat. Prod. 64
(2001) 892–895.
[25] M.R. Saha, S.M.R. Hasan, R. Akter, M.M. Hossain, M.S. Alamb, M.A. Alam, M.E.H.
Mazumder, Bangl. J. Vet. Med. 6 (2008) 197–202.
[26] W.J. Geary, Coord. Chem. Rev. 7 (1971) 81 122.
[27] A.I. Vogel, Quantitative Inorganic Analysis, third ed., Longman, 1961, 824.
[28] M.S. Yadawe, S.A. Patil, Trans. Met. Chem. 22 (1997) 220 224.
[29] A.A. Solimana, W. Linert, Thermochim. Acta. 338 (1999) 67 75.
[30] O.E. Sherif, H.M. Abd El-Fattah, M.M.El. Ajily, J. Therm. Anal. Cal. 74 (2003) 181 200.
[31] N. Raman, S. Ravichandran, C. Thangaraja, J. Chem. Sci. 116 (2004) 215 219.
[32] A.B.P. Lever, Inorganic Electronic Spectroscopy, second ed., Elsevier, Amsterdam, 1984.
[33] S. Srivastava, A. Kalam, Synth. React. Inorg. Met. Org. Chem. 34 (2004) 1529 1541.
[34] D. Arish, M. Sivankaran Nair, J. Mol. Struct. 983 (2010) 112 121.
[35] S.M. Abdallah, G.G. Mohamed, M.A. Zayed, M.S. Abou El-Ela, Spectrochim. Acta A 73
(2009) 833 840.
[36] S.M. Emam, S.A. AbouEl-Enein, F.A. El-Saied, S.Y. Alshater, Spectrochim. Acta A 92
(2012) 96 104.
[37] M. Narayana, S.G. Sathyanarayana, Siva Ramasastry, Indian. J. Chem. 13 (1975) 611.
[38] (a) B.J. Hathaway, D.E. Billing, Coord. Chem. Rev. 5 (1970) 143 207.
(b) B.J. Hathaway, Struct. Bonding (Berlin) 57 (1984) 55 118.
[39] H. Montgomery, E.C. Lingefetter, Acta. Cryst. 20 (1966) 728 730.
[40] R.C. Chikate, A.R. Belapure, S.B. Padhye, D.X. West, Polyhedron 24 (2005) 889 899.
13

[41] B.R. McGarvey, "Transition Metal Chemistry" (R. L. Carlin, Ed.) Marcel Dekker, New
York, 3 (1966) 146.
[42] K. Laxma Reddy, S. Sri Hari, P. Lingaiah, Indian J. Chem. 23A (1984) 172-174.
[43] A.A.A. Emara, F.S.M. Abd El-Hameed, S.M.E. Khalil, Phosphor. Sulfur Silicon 114 (1996)
1 15.
[44] A.W. Czarnik, Chem. Biol. 2 (1995) 423 428.
[45] A.W. Varnes, R.B. Dodson, E.L. Wehry, J. Am. Chem. Soc. 94 (1972) 946 950.
[46] M. Kalanithi, D. Kodimunthiri, M. Rajarajan, P. Tharmaraj, Spectrochim. Acta A 82 (2011)
290 298.
[47] B.E. Warren, X-ray Diffraction. Dover, New York, 1990, 253.
[48] M.G. Wahed, H.A. Bayoumi, M.I. Mohamme, Bull. Korean Chem. Soc. 24 (2003)
1313 1318.
[49] J.T. Makode, A.R. Yaul, S.G. Bhadange, A.S. Aswar, Russ. J. Inorg. Chem. 54 (2009)
1372 1377.
[50] V.P. Singh, A. Katiyar, S. Singh, BioMetals. 21 (2008) 491 501.
[51] Z.H. Chohan, Met. Based Drugs 6 (1999) 75 80.
[52] R.R. Coombs, S.A. Westcott, A. Decken, F.J. Baerlocher, Trans. Met. Chem. 30 (2005)
411 418.
[53] Z.H. Chohan, A. Munawar, C.T. Supuran, Met. Based Drugs 8 (2001) 137 143.
[54] Z.H. Chohan, M.S. Iqbal, S.K. Aftab, Appl. Organometal. Chem. 24 (2010) 47-56.
[55] M.A.R. Bhuiyan, M.Z. Hoque, S.J. Hossain, World J. Agric. Sci., 5 (2009) 318-322.
[56] Q.L. Zhang, J.G. Liu, J.Z. Liu, H. Li, Y. Yang, H. Xu, H. Chao, L.N. Ji, Inorg. Chim. Acta
339 (2002) 34 40.
[57] M.B. Halli, Patil, B. Vijayalaxmi, Indian J. Chem. 50A (2011) 664 669.
[58] A. Kulakarni, S.A. Patil, P.S. Badami, Eur. J. Med. Chem. 44 (2009) 2904 2912.
14

FIGURES
O
O
O
O
H
H
C
O
N R
C
Anhydrous DMF,POCl3
R-NH2
MeOH
O
OH
OH
OH
C O
OH
SH
R=
N
HL1
HL2
HL3
HL4
Scheme 1. Synthesis of schiff base ligands
15

O
O
OH
H
N
C
3-(-(2-hydroxyphenylimino)methyl)-4H-chromen-4-one
(HL1)
HO
O
O
C O
H
N
C
2-((4-oxo-4H-chromen-3-yl)methyleneamino)benzoic acid
(HL2)
O
OH
H
N
C
N
O
3-((3-hydroxypyridin-2-ylimino)methyl)-4H-chromen-4-one
(HL3)
O
SH
H
C N
O
3-(-(2-mercaptophenylimino)methyl)-4H-chromen-4-one
(HL4)
Figure 1. Structures of schiff base ligands
16

(b)
(a)
Figure 2. Electronic spectra of (a) HL1 (b) [Cu(L1)2].2H2O
17

(a)
(b)
Figure 3. ESR spectra of (a) [Cu(L1)2].2H2O, (b) [Cu(L4)2].
18

(a)
(b)
(c)
(d)
Figure 4. TG graphs of (a) [Cu(L1)2].2H2O, (b) [Cu(L2)2].H2O, (c) [Cu(L3)2] ,
(d) [Cu(L4)2]
19

O
O
A
H
N C
H
X
Cu
.nH2O
O
O
X
C N
A
A=C and X=O/S/COO
A=N and X=O
Figure 5. Structure of Cu(II) complexes
20

(b)
(a)
Figure 6. Emission spectra of (a) HL3 (b) [Cu(L3)2]
21

(b)
(a)
Figure 7. PXRD patterns of (a) HL3 (b) [Cu(L3)2]
22

(b)
(a)
Figure 8. SEM micrographs of (a) [Cu(L1)2].2H2O (b) [Cu(L3)2]
23

a
b
Figure 9. DNA cleavage studies of ligands and
their Cu(II) complexes.
a. DNA cleavage activity in the absence of
H2O2. Lane 1: DNA + HL1, Lane 2: DNA +
[Cu(L1)2].2H2O, Lane 3: DNA + HL2, Lane 4:
DNA + [Cu(L2)2].H2O, Lane 5: DNA + HL3,
Lane 6: DNA + [Cu(L3)2], Lane 7: DNA + HL4,
Lane 8: DNA + [Cu(L4)2], Lane 9: DNA +
FeSO4, Lane control : DNA alone.
b. DNA cleavage in the presence of H2O2. Lane
1: DNA + HL1 + H2O2, Lane 2: DNA +
[Cu(L1)2].2H2O + H2O2, Lane 3: DNA + HL2+
H2O2, Lane 4: DNA + [Cu(L2)2].H2O + H2O2,
Lane 5: DNA + HL3 + H2O2, Lane 6: DNA +
[Cu(L3)2] + H2O2, Lane 7: DNA + HL4 + H2O2,
Lane 8: DNA + [Cu(L4)2] + H2O2, Lane 9: DNA
+ FeSO4 + H2O2, Lane C1: DNA + H2O2, Lane
C2: DNA alone.
24

Table1. Physical, analytical and molar conductivity data of Cu(ІІ) complexes
Mol.
Wt
Colour
(%yield)
% Found (cald)
N
Molar Conductivity
(mho−1mol−1cm2)
Molecular
Formula
C
H
S
Cu
[Cu(L1)2].2H2O
[Cu(C16O3NH10)2].2H2O
627.5
Green
(75)
61.19
3.75
4.39
10.09
(10.11)
20
18
(61.19) (3.82) (4.46)
[Cu(L2)2].H2O
[Cu(C17O4NH10)2].H2O
665.5 Dark green
(80)
61.59 3.49 4.15
(61.30) (3.30) (4.20)
9.30
(9.54)
[Cu(L3)2]
Cu[(C15O3N2H9)2]
593.5 Leaf green
(80)
60.55
3.04
9.51
10.82
(10.70)
14
15
(60.65) (3.03) (9.43)
[Cu(L4)2]
Cu(C16O2NSH10)2
623.5
Green
(76)
61.54
3.19
4.38
10.25
10.24
(10.19)
(61.58) (3.20) (4.49) (10.26)
26

Table 2. FTIR spectral data of ligands and their Cu(ІІ) complexes
Compound
υ(OH) υ(C═O) υ(C═N) υ(CO) υ(C-S) υ(M-N) υ(M-O)
HL1
HL2
HL3
HL4
3241
1643
1651
1647
1622
1603
1605
1605
1591
1597
1587
1365
3246
790
749
[Cu(C16O3NH10)2].2H2O
439
449
446
468
532
528
511
523
[Cu(C17O4NH10)2].H2O
[Cu(C15O3N2H9)2]
[Cu(C16O2NSH10)2]
1621
1599
1644
1589
1580
1572
1351
27

Table 3. Electronic spectral data of ligands and their Cu(ІІ) complexes
Band position ( cm-1) Assignment
µeff(B.M)
-
Compound
HL1
26000
33003
26315
32258
25316
37037
27027
32467
14598
19762
14405
18726
23075
13700
19840
13513
19607
n → π*
π → π*
n → π*
π → π*
n → π*
HL2
-
HL3
-
-
π → π*
n → π*
HL4
π → π*
[Cu(L1)2].2H2O
2B1g → 2B2g
2B1g → 2Eg
1.86
[Cu(L2)2].H2O
2B1g → 2B2g
1.92
2B1g → 2Eg
Charge transfer
2B1g → 2B2g
2B1g → 2Eg
[Cu(L3)2]
[Cu(L4)2]
1.74
2.01
2B1g → 2B2g
2B1g → 2Eg
28

Table 4. ESR spectral data of Cu(ІІ) complexes
ESR spectral data [Cu(L1)2].2H2O [Cu(L2)2].H2O [Cu(L3)2] [Cu(L4)2]
g||
2.20
2.04
2.19
2.05
-
-
-
-
gꢀ
│g│
2.09
157
65
2.09
137
49
2.09
-
-
2.06
-
-
A|| ×10-4 cm-1
Aꢀ×10-4 cm-1
│A│×10-4 cm-1
96
5.24
140
0.68
0.64
0.66
0.44
0.45
78
4.00
160
0.63
0.57
0.85
0.36
0.54
-
-
-
-
G
g||/A|| (cm-1)
α2
β2
-
-
-
-
-
-
-
-
-
-
γ2
K||2
2
Kꢀ
-λ( cm-1)
493
444
-
-
29

Table
data
5. TG
of
Compound
Temperature
range(oC)
Mass loss
Found (cald%)
Assignment
[Cu(L1)2].2H2O
[Cu(C16O3NH10)2].2H2O
28-144
145-289
290-460
461-779
>780
40-120
121-532
533-827
>828
5.39(5.74)
23.09(23.11)
16.93(16.41)
41.82(42.07)
12.77(12.67)
2.65(2.70)
61.76(61.61)
23.54(23.74)
12.05(11.95)
2H2O
C9O2H5
C7NH5
C16O3NH10
CuO(Residue)
H2O
C24O5N2H14
C10O2H6
[Cu(L2)2].H2O
[Cu(C17O4NH10)2].H2O
CuO (Residue)
[Cu(L3)2]
[Cu(C15O3N2H9)2]
137-368
369-885
>886
18.04(18.02)
68.45(68.57)
13.51(13.41)
C5ON2H3
C25O4N2H15
CuO(Residue)
[Cu(L4)2]
[Cu(C16O2NSH10)2]
40-374
375-852
>853
61.78(61.92)
25.55(25.33)
12.67(12.75)
C22ON2S2H14
C10O2H6
CuO(Residue)
Cu(ІI) complexes
30