Synthesis, spectral, thermal, fluorescence, antimicrobial, anthelmintic and DNA cleavage studies of mononuclear metal chelates of bi-dentate 2H-chromene-2-one Schiff base

Article abstract of DOI:10.1016/j.jphotobiol.2015.03.033

The Co(II), Ni(II) and Cu(II) complexes have been synthesized with Schiff base (HL), derived from 8-formyl-7-hydroxy-4-methylcoumarin with benzylamine. The Schiff base and its metal complexes were structurally characterized based on IR, ¹H NMR,

Full text of DOI:10.1016/j.jphotobiol.2015.03.033

Accepted Manuscript
Synthesis, spectral, thermal, fluorescence, antimicrobial, anthelmintic and DNA
cleavage studies of mononuclear metal chelates of bi-dentate 2H-chromene-2-
one Schiff base
Chetan T. Prabhakara, Sangamesh A. Patil, Ajaykumar D. Kulkarni, Vinod H.
Naik, Manjunatha M., Shivshankar M. Kinnal, Prema S. Badami
PII:
S1011-1344(15)00117-7
DOI:
http://dx.doi.org/10.1016/j.jphotobiol.2015.03.033
JPB 9997
Reference:
To appear in:
Journal of Photochemistry and Photobiology B: Bi-
ology
Received Date:
Revised Date:
Accepted Date:
23 January 2015
16 March 2015
31 March 2015
Please cite this article as: C.T. Prabhakara, S.A. Patil, A.D. Kulkarni, V.H. Naik, M. M., S.M. Kinnal, P.S. Badami,
Synthesis, spectral, thermal, fluorescence, antimicrobial, anthelmintic and DNA cleavage studies of mononuclear
metal chelates of bi-dentate 2H-chromene-2-one Schiff base, Journal of Photochemistry and Photobiology B:
Biology (2015), doi: http://dx.doi.org/10.1016/j.jphotobiol.2015.03.033
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Synthesis, spectral, thermal, fluorescence, antimicrobial, anthelmintic
and DNA cleavage studies of mononuclear metal chelates of bi-
dentate 2H-chromene-2-one Schiff base
a
a,
b
c
Chetan T. Prabhakara , Sangamesh A. Patil *, Ajaykumar D. Kulkarni , Vinod H. Naik ,
d
a
e
Manjunatha M. , Shivshankar M. Kinnal , Prema S. Badami
a. P.G. Department of Chemistry, Karnatak University, Dharwad -560003, Karnataka, India.
.
b Department of Chemistry, K.L.S. Gogte Institute of Technology, Belgaum-590008, Karnataka, India.
c. Department of Chemistry, V.D.R. Institute of Technology, Haliyal-581329, Karnataka, India.
d. Department of Chemistry, C.M.R. Institute of Technology, Bangalore-560037, Karnataka, India.
e. Department of Chemistry, Shri Sharanabasaveshwar college of Science, Gulbarga-585102, Karnataka, India.
Abstract
The Co(II), Ni(II) and Cu(II) complexes have been synthesized with Schiff base (HL), derived from 8-
formyl-7-hydroxy-4-methylcoumarin with benzylamine. The Schiff base and its metal complexes were
1
13
structurally characterised based on IR, H NMR, C NMR, UV-visible, ESR, magnetic, thermal, fluorescence,
mass and ESI-MS studies. The complexes are completely soluble in DMF and DMSO. The molar conductance
values indicate that, all synthesized metal complexes are non-electrolytic in nature. Elemental analysis reveals
[
ML2(H2O)2] stoichiometry, here M = Co(II), Ni(II) and Cu(II), L = deprotonated ligand. The coordination
between metal ion and Schiff base was supported by IR data, through deprotonation of phenolic oxygen of
coumarin and azomethine nitrogen atoms. Solution electronic spectral results unveiled that all the synthesized
complexes posses six coordinated geometry around metal ion. Thermal studies suggest the presence of
coordinated water molecules. The Schiff base and its metal complexes have been screened for their antibacterial
(Escherichia coli, Pseudomonas aureginosa, Klebsiella pneumoniae and Staphylococcus aureus) and antifungal
(Penicillium chrysogenum and Aspergillus niger), anthelmintic (Pheretima posthuma) and DNA cleavage (Calf
Thymus DNA) activities.
Keywords: Fluorescence; Coumarin Schiff base; DNA Cleavage; Anthelmintic; Metal comple
*
Author for correspondence: P.G. Department of Chemistry, Karnatak University, Dharwad-580003. Karnataka, India.
1
E-mail: patil1956@rediffmail.com Phone: +91 9448341414

1
. Introduction
Coumarin (5,6-Benzo-alpha-pyrone) is a white crystalline oxygen heterocyclic compound, having
fluorescence emission properties. It occurs naturally throughout plant kingdom in the form of its derivatives
and can be synthesised chemically. Coumarin was first isolated from Tonka bean (Dipteryx odorata) in 1820
by Vogel [1] and synthesised by Perkin in 1868 [2]. Coumarin, a secondary metabolite is reported in plant
families such as Umbelliferae, Rutaceae etc. However, higher concentrations are found in fruits followed by
roots, stems and leaves. Few chief coumarin compounds have also been reported in microorganisms e.g.
novobiocin and coumermycin from Streptomyces and aflatoxins from Aspergillus species [3, 4]. Since many
years, especially organic and medicinal chemists have been carry out their investigation on coumarin, because
of its remarkable uses like antimicrobial [5], antioxidant [6], hypotensive [7], anthelmintic [8], inhibition of
platelet aggregation [9], cytotoxic activity [10], optical brighteners [11] and fluorescent probes [12]. Since,
last few decades, inorganic chemists have paid their considerable attention towards the synthesis of coumarin
derivatives because of their enhanced bioactivity upon complexation. Several metal complexes with various
Schiff bases, which are well characterized by spectral techniques and bioactivity assays conducted, have been
reported from our laboratory [13-16]. 7-Hydroxycoumarin (Umbelliferone) on its structural and biogenetic
sense, is regarded as the parent coumarin among structurally more complex coumarins [17]. Generally,
bioactivity of various coumarin derivatives depends on the presence and position of hydroxyl groups [18]. A
recent study has shown that 7-hydroxycoumarin inhibits the release of Cyclin D1, which is over expressed in
many types of cancer. Literature survey revealed that no work has been done on Schiff base metal complexes
of aldehydo hydroxy coumarin with benzylamine. Hence, in view of their significant usage, synthesis of new
Schiff base derivative of hydroxy coumarin would be worthwhile. All these developments in the synthesis of
Schiff base with metal ions are most fascinating. Here, in this investigation we have synthesized the Co(II),
Ni(II) and Cu(II) complexes with newly synthesized ON donor Schiff base derived from 8-formyl-7-hydroxy-
4
-methylcoumarin and benzylamine, which are characterized by spectral (IR, NMR, UV-Vis, Mass, ESI-MS,
ESR), fluorescence, thermal and molar conductivities. The synthesised, Schiff base and its metal complexes
were studied for their antibacterial, antifungal, anthelmintic and DNA cleavage activities.
2

2
. Experimental
2
.1 Reagents
All chemicals are of reagent grade, used without further purification.
2
.2 Physical measurements
Carbon, hydrogen and nitrogen were estimated using TRUSPEC CHN analyzer. IR spectra of Schiff
base and its Co(II), Ni(II) and Cu(II) complexes were recorded on a HITACHI-270 IR spectrophotometer in
-1
the region 4000 - 400 cm using KBr disc. Electronic spectra of metal complexes were recorded in HPLC
grade DMF solvent on a VARIAN CARY 50-BIO UV-spectrophotometer in the region of 200 - 1100 nm.
1
13
The H NMR and C NMR spectra of Schiff base were recorded in D -DMSO on a BRUKER 400 MHz
6
spectrometer at room temperature using TMS as an internal reference. The fluorescence studies of Schiff base
and its metal complexes were carried out on HITACHI F-7000 Fluorescence Spectrophotometer. Mass
spectrum of the Schiff base and electro spray mass spectra of the metal complexes were recorded using QP
2
010S and THERMO Finnegan LCQ Advantage max ion trap mass spectrometer respectively. The ESR
spectrum of Cu(II) complex was recorded on Varian-E-4X-band EPR spectrometer and the field set was
000 G at modulation frequency of 100 KHz under liquid nitrogen temperature using TCNE as „g‟ marker.
The electrochemistry of the synthesized complexes was recorded on CHI1110A-electrochemical analyzer
Made in U.S.A.) using dimethyl sulphoxide (DMSO) containing 0.05M n-Bu NClO as the supporting
3
(
4
4
electrolyte. Thermo gravimetric analyses data measured for synthesized metal complexes by using a
PERKIN-ELMER DIAMOND TG/DTG instrument. Platinum crucible was used for thermal study under
º
nitrogen atmosphere with flow rate of 100 mL/min from a range of room temperature to 1000 C at a heating
º
rate of 10 C/min. Molar conductivity measurements were recorded at room temperature on ELICO-CM-82 T
Conductivity Bridge with a cell having cell constant 0.51 and magnetic susceptibility was determined using
Faraday balance for all the synthesized metal complexes.
2
.3 Synthesis
2
.3.1 Synthesis of 7-hydroxy-4-methylcoumarin [19]
3

º
A mixture of dry resorcinol (0.2 mol) and ethylacetoacetate (0.2 mol) was cooled to 0 - 5 C and conc.
sulphuric acid (25 mL) was added gradually with constant shaking. The reaction mixture was then kept in a
refrigerator for 24 h. and poured into crushed ice with stirring. The separated solid was filtered washed with
water and recrystallized using ethanol as cream colored needles. Yield: 81%; Melting point: 180-182 ºC.
2
.3.2 Synthesis of 8-formyl-7-hydroxy-4-methylcoumarin [20]
A mixture of 0.03 mol of 7-hydroxy-4-methylcoumarin and 0.07 mol of hexamine in 50 ml of glacial
acetic acid were heated for 4-5 h and then 75 mL of 20% hydrochloric acid was added and heating was
continued for 20 min. Resulting mixture was cooled and extracted with ether. Pale yellow solid of 8-formyl-7-
hydroxy-4-methylcoumarin was obtained after evaporating the ether. Yield: 21.0 %; Melting point: 176-178 ºC.
2
.3.3 Synthesis of Schiff base HL
The Schiff base (HL) has been synthesized (Scheme 1) by refluxing a reaction mixture of hot ethanolic
solution (30 mL) of benzylamine and hot ethanolic solution (30 mL) of 8-formyl-7-hydroxy-4-methylcoumarin
0.01mol) for 5-6 h. with the addition of 2-3 drops of glacial acetic acid. The precipitate formed during reflux
(
was filtered, washed with cold ethanol and recrystallized using ethanol. Melting point 189-193 ºC.
CH3
CH3
Acetic acid,
Conc. HCl + Water
+
Hexamine
0
At 70 - 80 C
O
O
OH
O
O
OH
CHO
7-Hydroxy-4-methylcoumarin
8-Formyl-7-hydroxy-4-methylcoumarin
Reflux 6 h
Benzylamine
Ethanol
CH3
O
O
OH
N
(E)-8-((benzylimino)methyl)-7-hydroxy-4-methyl-2H-chromen-2-one
Scheme 1. Synthesis of Schiff base HL.
4

2
.3.3.1 NMR spectral studies
1
13
The Schiff base has been characterized by H NMR and C NMR to elucidate differently positioned
1
13
proton and carbon atoms. The H NMR and C NMR spectrum of HL are reproduced in Figs. 1 and 2
respectively.
1
In the H NMR spectrum of HL, a singlet signal at 14.86 ppm is ascribed to phenolic -OH. A
characteristic singlet proton signals at 9.09, 4.95 and 2.49 ppm are assigned to -CH=N, -CH and -CH
2
3
respectively. In addition to this, the multiplet signals in the 6.05-7.63 ppm range are due to aromatic protons.
1
3
In the C NMR spectrum of HL, signals at 173.22, 160.45, 56.84 and 18.33 ppm are due to C=N, C=O,
-
CH and -CH respectively. Signals appeared in the region 104.039 - 159.39 ppm are assigned to aromatic
2
3
carbons.
2
.3.3.2 Mass spectrum of the Schiff base
The formation of ligand was further investigated by its mass spectrum analysis. The mass spectrum of
HL is depicted in Fig. 3, exhibited a molecular ion peak at m/z 293; which is equivalent to its molecular weight.
The fragmentation peak at m/z 276 is due to cleavage of -OH group.
1
Fig. 1. H NMR Spectrum of HL.
5

1
3
Fig. 2. C NMR Spectrum of HL.
Fig. 3. Mass spectrum of HL.
6

2
.3.4 Synthesis of Co(II), Ni(II) and Cu(II) complexes [1-3]
An ethanolic solution of Schiff base (2mmol) (30 mL) was refluxed with
CoCl .6H O/NiCl .6H O/CuCl .2H O (1mmol) in ethanol (30 mL) on water bath for 1h. Then, to the reaction
2
2
2
2
2
2
mixture 1 mmol of sodium acetate was added and reflux was continued for 3 h. The separated complex was
filtered, washed thoroughly with water, ethanol and ether. Finally dried in vacuum over fused CaCl2.
2
.4 Pharmacology
2
.4.1 In vitro antibacterial and antifungal activities
The biological activities of the newly synthesized Schiff base and its metal complexes were studied for
their antibacterial and antifungal activities by disc diffusion method [21]. The antibacterial and antifungal
activities were carried out at 200 μg/mL concentrations of metal complexes dissolved in DMF solvent, against
four bacterial species {Escherichia coli (MTCC-1687), Pseudomonas aureginosa (MTCC-1688), Klebsiella
pneumoniae (MTCC-3384) and Staphylococcus aureus (MTCC-3160)} and two fungal species {Penicillium
chrysogenum (MTCC-5108) and Aspergillus niger (MTCC-1344)}. Bacteria and fungi cultures were sub
cultured on nutrient agar and potato dextrose agar medium respectively. Bacteria and fungi culture petridishes
treated with synthesised Schiff base and its metal complexes were incubated for 24 h and 48 h at 37 ºC
respectively. The standard antibacterial drug (Gentamicin) and antifungal drug (Fluconazole) were also
screened as explained above.
2
.4.2 Anthelmintic activity
The assay was executed on adult Indian earthworm, Pheretima posthuma due to its anatomical and
physiological similarity with intestinal roundworm parasite of human beings [22]. Anthelmintic assay was
studied as per the method given in literature [23] with minor modifications. The earthworms, Pheretima
posthuma collected from moist soil, washed with normal saline (0.5%) for about 30 s to remove all faecal
matter, were used for anthelmintic study. Earthworms of 2-4 cm in length were used in this experiment. All the
synthesized compounds were subjected to study anthelmintic activity against earthworms at 2 and 10 µg/mL
concentrations. DMSO in distilled water was used as control and Albendazole as a reference drug. The
7

paralyzing and death times were noted and their mean was calculated for triplicate sets. Death time was
recorded by placing earthworms in warm water (50 ºC) and observed for stimulated movement, if the worm
was alive.
2
.4.3 DNA Cleavage activity
DNA cleavage activity was studied for the synthesised Schiff base and its metal complexes using Calf-
thymus DNA (CT-DNA, Purchased from Bangalore Genei, Bengaluru, Cat. No. 105850). The synthetic
compounds were added separately to the CT-DNA, sample mixtures were then incubated at 37 ºC for 2h. The
cleaved product were run on agarose gel electrophoresis according to the procedure [24] and observed under
UV transilluminator for DNA cleavage.
2
.4.3.1 Agarose gel electrophoresis
In brief, 250 mg of agarose was dissolved in 25 mL of TAE buffer (4.84 g Tris base, pH 8.0, 0.5 M
EDTA/1 L) by boiling. The warmed agarose was poured into gel cassette fitted with comb. Later comb was
removed carefully after the gel solidified and gel placed in electrophoresis chamber flooded with TAE buffer.
2
0 μL of sample mixture, CT-DNA (mixed with bromophenol blue dye @ 1:1 ratio), were loaded carefully into
individual wells, along with standard DNA marker and gel was run in electrophoresis chamber by passing
constant 50 V of electricity for 45 min. Gel was then immersed in ETBR solution (10 μg/mL) for 10-15 min to
stain and then observed for bands under UV transilluminator and gel band pictures were recorded. The cleavage
ability of samples were compared with the CT- DNA and standard DNA marker ( λ DNA/ HindIII digest,
purchased from Merck, Bengaluru).
3
. Results and Discussion
All synthesized Co(II), Ni(II) and Cu(II) complexes of Schiff base are non-hygroscopic coloured solids,
stable at room temperature and remarkably soluble in DMF and DMSO. Elemental analysis was carried out
using CHN analyzer. Synthesized metal complexes (1-3) having stoichiometry of the type [ML2(H2O)2], here
-3
M= Co(II), Ni(II) and Cu(II). Molar conductivity of the metal complexes at 10 M concentrations were carried
out using DMF solvent at room temperature. Results obtained are given in Table 1, which indicate that all
synthesized metal complexes have conductivity values in the range, agreeable for non-electrolytic nature.
8

3
.1 Infrared spectra
IR spectra of Schiff base and its metal complexes are tabulated (Table 2) in order to confirm the
-1
coordination mode of Schiff base. In Schiff base spectrum, broad band in the region 3062-3052 cm assigned
to v (O-H) Absence of this band in its respective metal complexes spectrum, confirms the deprotonation of
.
-1
hydroxyl group and formation of the M-O bond [25]. The characteristic high intensity band at 1622 cm is
-1
attributed to v (C=N) [26, 27]. The strong bands at 1318 and 1717 cm are assigned to phenolic v (C-O) and v
(C=O) to lactonyl respectively.
In all synthesized metal complexes, the band assigned to azomethine group is shifted to lower frequency
-1
in the region 1605-1601 cm which indicates that chelation takes place through the nitrogen of the azomethine
group [28]. The position of the v (C=O) band remained largely unchanged, suggesting that lactone carbonyl
-1
oxygen is not involved in coordination to the metal. The strong band in the region 1337-1332 cm of metal
complexes is assigned to phenolic v (C-O), these observations supports the formation of ν (M-O) bond.
Presence of coordinated water molecules in the metal complexes were confirmed by a broad band around 3450
-1
-1
-1
cm and two bands in the region 745-735 cm and 780-770 cm due to v (-OH), rocking and wagging modes
-1
of vibrations respectively. The bands observed for metal complexes between 538-522 and 496-484 cm were
metal sensitive and are assigned to ν (M-O) and ν (M-N) [29] respectively. In Figs. 4 and 5 are presented the IR
Spectra of the Schiff base HL and its Ni(II) complex respectively.
Fig. 4. IR spectrum of the Schiff base HL.
9

Table 1. Elemental analyses of Schiff base (HL) and its metal complexes (1-3) along with molar conductance data.
Compound Empirical Formula
Name / No.
C %
H %
N %
Colour / % Yield
Molar
Conductance
-
1
Calcd. Found Calcd. Found Calcd. Found
value Ohm
2
-1
cm mole
HL
1
C
18
36
36
36
H
15 NO
3
73.71
63.63
63.65
63.20
73.69
63.61
63.60
63.21
5.15
4.75
4.75
4.71
5.13
4.78
4.73
4.70
4.78
4.12
4.12
4.09
4.76
4.10
4.11
4.11
Light orange / 88%
Brown / 61%
--
C
H32CoN
O
11.4
14.4
17.3
2
8
2
C
H32NiN
2
O
8
Light green / 66%
Pale brown / 62%
3
C
H32CuN
O
2 8
-1
Table 2. The prominent infrared frequencies (in cm ) of Schiff base (HL) and its metal complexes (1-3).
Compound
Name / No.
Compounds
Coordinated Lactonyl
Phenolic
s
s
s
m
s
water
ν(C=O)
ν(C=N)
ν(C-O)
ν(M-O)
ν(M-N)
br
ν(O-H)
--
HL
1
C
18
H
15 NO
3
1717
1715
1716
1717
1622
1602
1601
1605
1318
1332
1337
1337
--
--
Co(C18
H14NO
)
. 2H
. 2H
. 2H
O
3448
3449
3452
522
527
538
496
485
484
3
2
2
2
Ni(C18
H14NO
3
)
2
O
2
3
Cu(C18
H14NO
3
)
2
O
2
s
m
br
=
strong, = medium, = broad.
1
0

Fig. 5. IR spectrum of Ni(C H NO ) 2H O.
1
8
14
3 2.
2
3
.2 Solution electronic absorption spectra and magnetic studies
In order to obtain information concerning the stereochemistry of the metal ions, the UV–Vis spectra of
the ligand and complexes were recorded in DMF. The Schiff base (HL) exhibited two absorption bands around
55 and 330 nm. The spectral data below 350 nm are similar and are ligand centred transitions (intraligand π-π*
2
and n-π*) of aromatic ring and non-bonding electrons present on the nitrogen of the azomethine group in the
Schiff base [30, 31].
Solution electronic absorption spectra of the synthesized metal complexes are recorded in DMF
(Table 3). The electronic spectrum of Co(II) complex exhibited absorption band in the region at 568 and 980
4
4
4
4
nm, corresponding to T (F) → T (F) (ν ) and T (F) → T (P) (ν ) transitions respectively. These bands
1
g
2g
1
1g
1g
3
are characteristic of high spin octahedral Co(II) complexes [32]. Observed magnetic moment of Co(II) complex
was 4.90 BM and it supports octahedral geometry for Co(II) complex [33].
The spectrum of light green Ni(II) complex exhibited three bands at 989, 674 and 365 nm are attributed
3
3
3
3
3
3
to the A (F) → T (F) (ν ), A (F) → T (F) (ν ) and A (F) → T (P) (ν ) transitions respectively,
2
g
2g
1
2g
1g
2
2g
1g
3
which indicate octahedral geometry around Ni(II) ion [34]. Magnetic moment value obtained was 3.15 BM,
which is agreeable with the octahedral environment around Ni(II) ion [35, 36].
1
1

The electronic spectrum of Cu(II) complex (Fig. 6) showed two absorption bands. A low intensity broad
2
2
band in the region 690 nm is assignable to E → T transition in support of distorted octahedral configuration
g
2g
of metal complex [37]. Another high intensity band at 391 nm is due to symmetry forbidden ligand → metal
charge transfer. Obtained magnetic moment value for Cu(II) complex is 1.77 BM, further it supports the
electronic spectral data [38, 39].
Fig. 6. Electronic absorption spectrum of Cu(II) complex.
Table 3. Solution electronic spectral results along with magnetic susceptibility data of metal complexes (1-3).
Complex
No.
µ
eff
(BM)
-
1
λmax in cm (nm)
Band assignments
Geometry
4
4
T (F)→ T (F) (ν )
1
1
0204 (980)
7605 (568)
1g
2g
1
1
2
3
4
4
octahedral
4.90
T (F)→ T (P) (ν )
1g
1g
3
3
3
3
3
A (F)→ T (F) (ν )
1
1
2
0111 (989)
4836(674)
7397(365)
2g
2g
1
3
A (F)→ T (F) (ν )
octahedral
3.15
1.77
2
g
1g
2
3
A (F)→ T (P) (ν )
2
g
1g
2
3
2
14492 (690)
5575 (391)
T ← E
distorted
octahedral
2
g
g
2
L→M
3
.3 Electron spin resonance
Fig. 7 shows the ESR spectrum of Cu(C H NO ) 2H O was recorded in X band spectrometer
18
14
3 2.
2
(microwave frequency of 9.1GHz and field set was 3000 G at modulation frequency of 100 KHz) under liquid
nitrogen temperature in DMSO with g factor quoted relative to standard marker TCNE (g = 2.0027). ESR
1
2

spectra of the metal chelates provide important information regarding the environment around the metal ion in
the complexes.
|
|
⊥
The ESR spectrum of Cu(II) complex show typical axial behaviour with slightly different g and g
|
|
⊥
values. The g , g , g and G values of Cu(II) complex calculated from ESR spectrum was 2.0692, 2.0402,
av
|
|
⊥
2
.0498 and 1.7651 respectively. The trend g (2.0692) > g (2.0402) > g (2.0023) observed for copper complex
e
9
suggest that, unpaired electron is localized in dx2–y2 orbital of Cu(II) ion with 3d configuration [40].
||
Here, g < 2.3 is characteristic of a covalent nature of the M-L bonding. The ESR data provide further
evidence to magnetic and solution electronic spectral results.
Fig.7. ESR spectrum of Cu(II) complex (3).
3
.4 Fluorescence Spectra
As we know that coumarin itself shows good fluorescent material, we studied fluorescent effect of
synthesized coumarin Schiff base and its metal (II) complexes at room temperature. The Schiff base showed
λ max value at 502 and 507 nm in DMSO and DMF respectively (Fig 8). Upon addition of 2-3 drops of 2%
NaOH to the Schiff base solution, we observed λ max at 499 nm in DMSO and 501 nm in DMF (Fig. 9). The
shift of λ max to lower wavelength (i.e. blue shift) after addition of 2-3 drops of aqueous alkali indicates that,
proton transferred (H-bonded ion pair) species exist in equilibrium [41, 42]. The spectra of ligand and its metal
-3
complexes have been recorded in 10 M DMF solution. Fig. 10 shows the overlapping spectra of ligand and its
1
3

Co(II), Ni(II) and Cu(II) complexes in DMF. Here, ligand exhibits strong fluorescence intensity compared to
other three metal complexes. The weak intensity of band corresponding to complexes is due to decrease in
electron density on ligand [43, 44]. The decreased fluorescence intensity of synthesized metal complexes
(1-3) compared with free ligand (HL) is in the order Ni(II) > Co(II) > Cu(II).
Fig. 8. Emission spectra of Schiff base (HL) in DMSO and DMF.
DMSO
DMF
Fig. 9. Fluorescence spectra of Schiff base (HL): before and after adding 2% NaOH in DMSO and DMF.
1
4

Fig. 10. Fluorescence spectra of Schiff base (HL) and its metal complexes (1-3).
3
6
.5 ESI-Mass studies of metal complexes
The ESI-Mass spectra of Co(II) (1), Ni(II) (2) and Cu(II) (3) complexes showed a M+1 peak at m/z 681,
80 and 685 respectively, which are equivalent of their molecular weight, supports the formation of respective
metal complexes of type ML2(H2O)2 and a base peak at m/z 292 is due to the formation of deprotonated
ligand. The representative Ni(C H NO ) 2H O is shown in Fig. 11.
1
8
14
3 2.
2
Fig. 11. Mass spectrum of Ni(C H NO ) 2H O.
1
8
14
3 2.
2
1
5

3
.6 Thermo gravimetric analysis
As a function of temperature, thermal features of the synthesized Co(II), Ni(II) and Cu(II) complexes
are studied. The obtained results are tabulated in Table 4. Peaks in the regions 170-225 and 330-850 ºC are
corresponding to mass loss of coordinated water molecules and organic moieties respectively. Finally, the metal
complexes decompose gradually with the formation of metal oxide above 850 ºC. The representative TG/DTG
spectrum of Cu(C H NO ) 2H O is shown in Fig. 12. Here, decomposition of coordinated water molecules
1
8
14
3 2.
2
occurred in the region 170-225 ºC centred at 203 ºC and loss of organic moieties occurred in between 330-850
ºC. Thus, TG and DTG data provide useful information on the presence coordinated water molecules and
stability of metal complexes.
Table 4. Thermo gravimetric data of metal complexes (1-3).
Compound
name
Decomposition
temperature (ºC)
Weight loss (%) Metal oxide (%)
Found Calcd. Found Calcd.
Inference
1
2
3
173-183
5.31
5.30
11.02
10.91
11.54
11.04 Loss of coordinated water
molecules
Loss of organic moieties
10.92 Loss of coordinated water
molecules
Loss of organic moieties
11.57 Loss of coordinated water
molecules
3
45-433
83.67
5.34
83.66
5.30
187- 198
3
40-459
83.79
5.28
83.78
5.27
170-225
3
30-850
83.18
83.16
Loss of organic moieties
Fig. 12. TG/DTG spectrum of Cu(C H NO ) 2H O.
18
14
3 2.
2
3
.7 Electrochemistry
1
6

The redox behavior of the synthesized Cu(II) complex was investigated in DMSO by cyclic
voltammetric studies using a glassy carbon working electrode. Fig. 13, illustrate a cyclic volammogram of
Cu(C H NO ) 2H O, it shows a oxidation peak at E = 0.3270V with a corresponding reduction peak at E =
18
14
3 2.
2
pa
pc
0
.1450V. The ratio of the anodic and cathodic peak currents (Ipc/Ipa≈1), provide a rough evaluation of the
degree of the reversibility of one electron transfer reaction. Copper complex had a large separation between the
cathodic and anodic peak of 182 mV and it increase with the scan rates, indicating a quasi-reversible character
[
45, 46].
Fig. 13. Cyclic voltammogram of Cu(II) complex.
3
.8 Antimicrobial activity
Figs. 14 and 15, shows the results of antibacterial and antifungal activities of all synthesized
compounds. By observing the results it was found that, metal complexes exhibited higher lethal effect on
bacteria and fungi growth than their parent ligand. The Cu(II) complex showed promising antibacterial and
antifungal activity against all strains. It is suspected that, factors such as solubility, conductivity, dipole moment
and cell permeability mechanism (influenced by the presence of metal ions) may be the possible reasons for
increase in activity.
1
7

Fig. 14. Bacterial growth inhibition in millimetres (mm) for different compounds at 200 μg/mL concentration,
Gentamicin used as reference compound.
Fig. 15. Fungal growth inhibition in millimetres (mm) for different compounds at 200 μg/mL concentration,
Fluconazole used as reference compound.
3
.09 Anthelmintic activity
Table 5 shows, anthelmintic activity results of all the synthesized compounds. The obtained results
clearly indicate that metal complexes are more active than their parent ligand. In particularly Cu(II) complex
i.e. compound (3) showed prominent activity, compared with the standard drug Albendazole at 10 μg/mL
concentration.
1
8

Table 5. Anthelmintic studies of Schiff base (HL) and its metal complexes (1-3).
Time of
Time of death
(min)
Compound Conc.(μg/mL) paralysis (min)
Albendazole
10
3.48 ± 0.06
7.25±0.14
a
--
--
--
DMSO
HL
2
20.18±0.02
9.28±0.21
24.30±0.12
14.29±0.11
1
0
2
12.14±0.14
7.40±0.04
16.10± 0.25
9.13±0.01
10.27±0.03
5.39±0.22
19.50±0.02
10.12±0.03
1
2
3
1
0
2
21.20±0.09
15.51±0.00
1
0
2
16.41±0.06
9.31±0.01
1
0
a
DMSO as control.
3
.10 DNA Cleavage Activity
The DNA cleavage activity of Schiff base and its metal complexes were studied by agarose gel
electrophoresis method using calf thymus DNA(CT-DNA). The CT- DNA cleavage activity of Schiff base HL
and its complexes are shown in Fig. 16. M- is standard DNA molecular weight marker, C- is Control CT-DNA
i.e. untreated CT-DNA and lanes 1-3 and HL are CT-DNA treated with different metal complexes and Schiff
base respectively. Gel electrophoresis works on, migration of DNA under the influence of electric potential.
From the gel picture, we found that compound Cu(C H NO ) .2H O shown complete cleavage,
18
14
3 2
2
whereas Co(C H NO ) .2H O, Ni(C H NO ) .2H O and Schiff base HL shown partial cleavage. As Cu(II)
1
8
14
3 2
2
18 14
3 2
2
complex cleaved CT-DNA completely, this implies that Cu(II) complex is more effective than the other metal
complexes.
Fig. 16. DNA Cleavage activity of Schiff base (HL) and its metal complexes (1-3).
1
9

4
. Conclusion
The newly synthesized Schiff base act as bidentate ligand through azomethine nitrogen and phenolic
oxygen atoms of coumarin moiety. The bonding of ligand to metal ion was confirmed by various spectral
studies. Antimicrobial study reveals that, Cu(II) complex was found to be active against all the bacterial and
fungal species. Cu(II) complex (3) also showed prominent anthelmintic and DNA cleavage activities. It reveals
that, the Cu(II) complex is more effective than other synthesized compounds. All these observations put
together lead us to propose the following structure (Fig. 17) in which, metal complexes having stoichiometry of
type [ML2(H2O)2] (M=Co(II), Ni(II) and Cu(II), L= deprotonated Schiff base).
CH3
OH2
O
O
O
N
M
N
O
O
O
OH2
CH3
Fig. 17. Proposed structures of metal complexes (1-3).
Acknowledgment
The authors are grateful to the Chairman, Department of Chemistry, Karnatak University, Dharwad for
the laboratory facilities. Authors are thankful to University Scientific Instrumentation Centre, Karnatak
University, Dharwad, IISc Bengaluru, IIT Bombay, SAIF CDRI Lucknow, CMRIT Bengaluru and Biogenics
Hubballi, India for providing instrumental facilities. One of the author, Chetan T. Prabhakara is thankful to
th
UGC, New Delhi for the financial support through Major Research Project. [No:F.36-137/2008(SR) dated 27
March 2009].
2
0

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2
4

Highlights



Characterization of metal complexes using various spectroscopic techniques.
Fluorescence emission study reveal the quenching of Schiff base fluorescence.
Cu(II) complex of coumarin Schiff base has shown promising biological activities.

Graphical abstract : Synthesis of Schiff base HL.
CH3
CH3
Acetic acid,
Conc. HCl + Water
+
Hexamine
0
At 70 - 80 C
O
O
OH
O
O
OH
CHO
7-Hydroxy-4-methylcoumarin
8-Formyl-7-hydroxy-4-methylcoumarin
Reflux 6 h
Benzylamine
Ethanol
CH3
O
O
OH
N
(E)-8-((benzylimino)methyl)-7-hydroxy-4-methyl-2H-chromen-2-one

High resolution figures (3,4,11 and 11) of the paper [JPB_9997]
Fig. 3. Mass spectrum of HL.

Fig. 4. IR spectrum of the Schiff base HL.

Fig.7. ESR spectrum of Cu(II) complex (3).