Synthesis, characterization and biological approach of metal chelates of some first row transition metal ions with halogenated bidentate coumarin Schiff bases containing N and O donor atoms

Article abstract of DOI:10.1016/j.jphotobiol.2016.02.004

The impregnation of halogen atoms in a molecule is an emerging trend in pharmaceutical chemistry. The presence of halogens (Cl, Br, I and F) increases the lipophilic nature of molecule and improves the penetration of lipid membrane. The presence of electronegative halogen atoms increases the bio- activity of core moiety. In the present study, Co(II), Ni(II) and Cu(II) complexes are synthesised using Schiff bases (HL^I and HL^II), derived from 8-formyl-7-hydroxy-4-methylcoumarin/3-chloro-8-formyl-7-hydroxy-4-methylcoumarin with 2,4-difluoroaniline/o-toluidine respectively. The synthesized compounds were characterized by spectral (IR, NMR, UV-visible, Mass, ESI-MS, ESR), thermal, fluorescence and molar conductivity studies. All the synthesized metal complexes are completely soluble in DMF and DMSO. The non-electrolytic nature of the metal complexes was confirmed by molar conductance studies. Elemental analysis study suggest [ML₂(H₂O)₂] stoichiometry, here M = Co(II), Ni(II) and Cu(II), L = deprotonated ligand. The obtained IR data supports the binding of metal ion to Schiff base. Thermal study suggests the presence of coordinated water molecules. Electronic spectral results reveal six coordinated geometry for the synthesized metal complexes. The Schiff bases and their metal complexes were evaluated for antibacterial (Pseudomonas aureginosa and Proteus mirabilis), antifungal (Aspergillus niger and Rhizopus oryzae), anthelmintic (Pheretima posthuma) and DNA cleavage (Calf Thymus DNA) activities.

Full text of DOI:10.1016/j.jphotobiol.2016.02.004

Synthesis, characterization and biological approach of metal chelates of some
first row transition metal ions with halogenated bidentate coumarin Schiff
bases containing N and O donor atoms
Chetan T. Prabhakara, Sangamesh A. Patil, Shivakumar S. Toragalmath,
Shivashankar M. Kinnal, Prema S. Badami
PII:
DOI:
Reference:
S1011-1344(15)30092-0
doi: 10.1016/j.jphotobiol.2016.02.004
JPB 10241
To appear in:
Received date:
Revised date:
Accepted date:
12 October 2015
31 January 2016
1 February 2016
Please cite this article as: Chetan T. Prabhakara, Sangamesh A. Patil, Shivakumar S.
Toragalmath, Shivashankar M. Kinnal, Prema S. Badami, Synthesis, characterization
and biological approach of metal chelates of some first row transition metal ions with
halogenated bidentate coumarin Schiff bases containing N and O donor atoms, (2016),
doi: 10.1016/j.jphotobiol.2016.02.004
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ACCEPTED MANUSCRIPT
Synthesis, characterization and biological approach of metal chelates
of some first row transition metal ions with halogenated bidentate
coumarin Schiff bases containing N and O donor atoms
Chetan T. Prabhakara a, Sangamesh A. Patil a, *, Shivakumar S. Toragalmath a,
Shivashankar M. Kinnal a, Prema S. Badami b
a
P.G. Department of Chemistry, Karnatak University, Dharwad -560003, Karnataka, India
b
Department of Chemistry, Shri Sharanabasaveshwar college of Science, Gulbarga-585102, Karnataka, India
Abstract
The impregnation of halogen atoms in a molecule is an emerging trend in pharmaceutical chemistry.
The presence of halogens (Cl, Br, I and F) increases the lipophilic nature of molecule and improves the
penetration of lipid membrane. The presence of electronegative halogen atoms increases the bio- activity of
core moiety. In the present study, Co(II), Ni(II) and Cu(II) complexes are synthesised using Schiff bases
(HLI and HLII), derived from 8-formyl-7-hydroxy-4-methylcoumarin/3-chloro-8-formyl-7-hydroxy-4-
methylcoumarin with 2,4-difluoroaniline/o-toluidine respectively. The synthesized compounds were
characterized by spectral (IR, NMR, UV-visible, Mass, ESI-MS, ESR), thermal, fluorescence and molar
conductivity studies. All the synthesized metal complexes are completely soluble in DMF and DMSO. The
non-electrolytic nature of the metal complexes was confirmed by molar conductance studies. Elemental
analysis study suggest [ML2(H2O)2] stoichiometry, here M = Co(II), Ni(II) and Cu(II), L = deprotonated ligand.
The obtained IR data supports the binding of metal ion to Schiff base. Thermal study suggests the presence of
coordinated water molecules. Electronic spectral results reveal six coordinated geometry for the synthesized
metal complexes. The Schiff bases and their metal complexes were evaluated for antibacterial (Pseudomonas
aureginosa and Proteus mirabilis), antifungal (Aspergillus niger and Rhizopus oryzae), anthelmintic
(Pheretima posthuma) and DNA cleavage (Calf Thymus DNA) activities.
Keywords: Halogenated Coumarin Schiff bases; Metal complexes; Fluorescence; DNA cleavage; Anthelmintic
1
* Author for correspondence: P.G. Department of Chemistry, Karnatak University, Dharwad-580003. Karnataka, India.
E-mail: patil1956@rediffmail.com Phone: 09066596583

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1. Introduction
Coumarin is a white crystalline compound having typical benzopyrone framework, occurs naturally in
plants and it can be synthesized chemically. Plant species of Compositae, Rutaceae and Umbelliferae families
contain higher level of coumarin and it can be isolated using various extraction procedures [1]. Chemically
coumarin can be synthesized by different methods [2-7]. The simple and common method used for synthesis of
coumarin is Pechmann condensation, in which, different phenols are used. 7-Hydroxycoumarin
(Umbelliferone), on its structural and biogenetic aspects is regarded as the parent coumarin among the
structurally more complex coumarins [8]. Coumarin derivatives have the ability to express numerous
pharmacological properties such as antimicrobial [9], antioxidant [10,11], antiplasmodial [12], anticancer [13],
cytotoxic [14], anthelmintic [15], inhibition of platelet aggregation [16], antineurodegenerative [17], anti HIV
[18,19] and anti-inflammatory [20] activities.
In recent years, many structural modifications have been made at various positions of the coumarin ring
system to get more pharmaceutically active coumarin derivatives. In an aromatic ring, the presence and position
of the electron withdrawing group may enhance its reactivity. The best pharmaceutical results can be expected
by replacing a hydrogen atom of a molecule by halogen. Many of such derivatives are used as antibiotics,
antineoplastic agents, CNS depressant, CNS stimulant and antipsychotic etc. Recently, K.N. Venugopala et al.,
showed that, halogenated coumarin derivatives express anti-malarial activity [21]. These compounds act as
inhibitors against various protein kinases [22]. Recent studies on halogen-bonding or X-bonding show the
increased affinity of ligand as potential anticancer drugs [23]. The presence of halogen atoms in a molecule
enhances the lipophilic nature and is less soluble in water, which also improves the penetration of lipid
membrane. Fluorine atom being less bulky among all the halogens (Cl, Br, I and F) has the ability to block
metabolically active positions.
In the light of all these observations, we have planned to synthesize new transition metal complexes of
halogen substituted coumarin Schiff bases and study their pharmacological activity. In this part of research
work, we have synthesized Co(II), Ni(II) and Cu(II) complexes of coumarin Schiff bases derived from 8-
formyl-7-hydroxy-4-methylcoumarin and 3-chloro-8-formyl-7-hydroxy-4-methylcoumarin with 2,4-
difluoroaniline and o-toluidine respectively. The synthesized compounds were characterized by spectral (IR,

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NMR, UV-visible, Mass, ESI-MS, ESR), thermal, fluorescence and molar conductivity studies. The bioassay of
these synthesized compounds was also carried out.
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. The IR spectra of the
Schiff base and their Co(II), Ni(II) and Cu(II) complexes were recorded on a HITACHI-270 IR
spectrophotometer in the region 4000-400 cm-1 using KBr disc. The electronic spectra of the metal complexes
were recorded in HPLC grade DMF solvent on a VARIAN CARY 50-BIO UV-spectrophotometer in the region
of 200-1100 nm. The 1H and 13C NMR spectra of Schiff bases were recorded in D6-DMSO on a BRUKER 400
MHz spectrometer at room temperature using TMS as an internal reference. The fluorescence studies of Schiff
base and their metal complexes were carried out on HITACHI F-7000 Fluorescence Spectrophotometer. Mass
spectrum of the Schiff bases and electro spray mass spectra of metal complexes were recorded using QP 2010S
and THERMO Finnegan LCQ Advantage max ion trap mass spectrometer respectively. The ESR spectrum of
Cu(II) complex (5) was recorded on Varian-E-4X-band EPR spectrometer and the field set was 3000 G at
modulation frequency of 100 KHz under liquid nitrogen temperature using TCNE as „g‟ marker. Thermo
gravimetric analyses data measured from a range of room temperature to 1000 ºC at a heating rate of 10 ºC/min.
The data obtained by using a PERKIN-ELMER DIAMOND TG/DTA instrument. Molar conductivity
measurements recorded on ELICO-CM-82 T Conductivity Bridge with a cell having cell constant 0.51 and
magnetic moment was carried out using faraday balance for all the synthesized metal complexes.
2.3 Synthesis
2.3.1 Synthesis of 7-hydroxy-4-methylcoumarin
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) added gradually with constant shaking. The reaction mixture was then kept in a
3

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refrigerator for 24 h. and poured into crushed ice with stirring. The separated solid was filtered washed with
water and recrystallized from ethanol as cream colored needles. Yield: 82%; Melting Point: 180-182 ºC.
2.3.2 Synthesis of 3-chloro-7-hydroxy-4-methylcoumarin
o
A mixture of dry resorcinol (0.2 mol) and ethyl-2-chloroacetoacetate (0.2 mol) was cooled to 0-5 C
and Conc. sulphuric acid (25 mL) 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 and washed
with water. Finally, recrystallized using ethanol to get a pure compound. Yield: 79%; Melting Point:
240-243 º C.
2.3.3 Synthesis of 8-formyl-7-hydroxy-4-methylcoumarin
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, 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: 20.0 % ; Melting Point: 176-178 ºC.
2.3.4 Synthesis of 3-chloro-8-formyl-7-hydroxy-4-methylcoumarin
A mixture of 0.03 mol of 3-Chloro-7-hydroxy-4-methylcoumarin and 0.07 mol of hexamine in 50 ml of
glacial acetic acid were heated for 4-5 hr and then dilute hydrochloric acid was added, heating was continued
for 20 min. Cooled the resulting mixture and extracted with ether. Pale yellow solid of 3-Chloro-8-formyl-7-
hydroxy-4-methylcoumarin was obtained after evaporating the ether. Yield: 25.0 %; Melting Point: 163-166 oC.
2.3.5 Synthesis of Schiff bases HLI and HLII
The steps involved in the synthesis of Schiff bases HLI and HLII are depicted in the Scheme 1 (a and
b). By refluxing the reaction mixture of hot ethanolic solutions of 2,4-difluoroaniline/o-toluidine with hot
ethanolic solutions of 8-formyl-7-hydroxy-4-methylcoumarin/3-chloro-8-formyl-7-hydroxy-4-methylcoumarin
respectively for about 6 h with addition of 1-2 drops of glacial acetic acid. The precipitate formed during
refluxion was filtered, washed with cold ethanol and recrystallized with acetic acid.

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Acetic acid,
CH3
CH3
Conc. HCl +Water
Hexamine
+
70 - 80 0C
O
O
OH
O
O
OH
CHO
8-Formyl-7-hydroxy-4-methylcoumarin
7-Hydroxy-4-methylcoumarin
2,4-Difluoroaniline
Reflux 6 h
CH3
O
O
OH
N
F
F
Schiff Base HLI
Scheme 1(a). Synthesis of Schiff base HLI.
CH3
O
CH3
O
Acetic acid,
Conc. HCl +Water
Cl
Cl
Hexamine
+
At 70 - 80 0C
O
OH
O
OH
CHO
3-Chloro-8-formyl-7-hydroxy-4-methylcoumarin
3-Chloro-7-hydroxy-4-methylcoumarin
Reflux 6 h
o-Toluidine
CH3
Cl
O
O
OH
N
CH3
Schiff Base HLII
Scheme 1(b). Synthesis of Schiff base HLII.
5

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2.3.5.1 NMR spectral study
1
The Schiff bases have been characterized by H and 13C NMR spectra to elucidate the differently
1
positioned proton and carbon respectively. The H NMR spectra of both the Schiff bases HLI and HLII are
shown in Figs. 1 and 2 respectively. The representative 13C NMR spectrum of Schiff base HLI is reproduced in
Fig. 3.
1
In H NMR spectrum of the HLI, a singlet signal at 14.68 ppm is ascribed to phenolic -OH. A
characteristic singlet proton signals at 9.35 ppm and 2.43 ppm are assigned to -CH=N and -CH3 respectively. In
addition to these, signals in the 6.29-7.90 ppm region are due to the aromatic protons. In case of HLII, the
singlet at 15.32, 9.21, 2.56 and 2.36 ppm are attributed to phenolic -OH, -CH=N, -CH3 of o-toluidine, -CH3 of
coumarin respectively. The signals in the region 6.98-7.91 ppm are of aromatic protons.
13
The C NMR spectrum of Schiff base HLI shows a signal observed at 164.51 ppm is ascribed
to lactonyl carbon (C=O) and a signal at 162.30 ppm is assigned to (C=N) group. Signal observed at
18.28 ppm is due to -CH3 group. The characteristic signals in the region 104.63-162.19 ppm are
assigned to aromatic carbons.
Fig. 1. 1H NMR Spectrum of Schiff base HLI.

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Fig. 2. 1H NMR Spectrum of Schiff base HLII.
Fig. 3. 13C NMR Spectrum of Schiff base HLI.
7

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2.3.5.2 Mass spectral study
The mass spectrum HLI of showed a molecular ion peak at m/z 315 which is equivalent to its molecular
weight. In case of the molecular ion peak at m/z 327 is ascribed to C18H14ClNO3. The fragmented peak at m/z
310 is assigned to the cleavage of -OH group. The representative mass spectrum of HLII has been depicted in
Fig. 4.
Fig. 4. Mass spectrum of Schiff base HLII.
2.3.6 Synthesis of Co(II), Ni(II) and Cu(II) complexes (1-6)
An alcoholic solution of Schiff bases HLI and HLII (2 mmol) was refluxed with (1 mmol) of
CoCl2.6H2O/NiCl2.6H2O/CuCl2.2H2O on water bath for 1h. Then, to the reaction mixture 1mmol of sodium
acetate was added and refluxion was continued for 3 h. The separated metal complex was filtered, washed
thoroughly with water, ethanol and ether and then 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 their metal complexes were studied
for their antibacterial and antifungal activities by disc diffusion method [24].
The antibacterial and antifungal activities were carried out at 200 μg/mL concentrations of metal
complexes dissolved in DMF solvent, against four bacterial species (Pseudomonas auregenosa and Proteus

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mirabilis) and two fungal species (Aspergillus niger and Rhizopus oryzae). 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 their metal complexes were incubated for 24 h and 48 h at 30 ºC
respectively. The standard Antibacterial drug (Gentamicin) and antifungal drug (Fluconazole) were also
screened as explained above. The zone of inhibition for each fungi against respective organism was recorded in
triplicate sets.
2.4.2 Anthelmintic activity
The assay was executed on adult Indian earthworm, Pheretima posthuma due to its anatomical and
physiological similarity with the intestinal roundworm parasite of human beings [25]. The anthelmintic assay
was studied as per the method given in the literature [26] 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 the anthelmintic study. The earthworms of 2-4 cm in length were used for all the
experimental protocol. All the synthesized compounds were subjected to study anthelmintic activity against the
earthworms at 2 and 10 µg/mL concentrations. DMSO in distilled water was used as control and Albendazole
as a reference drug. The paralyzing and death times were noted and their mean was calculated for triplicate sets.
The death time was recorded by placing the earthworms in warm water (50 ºC) and observed for stimulated
movement, if the worm was alive.
2.4.3 DNA Cleavage activity
The DNA cleavage activity was studied for the synthesised metal complexes of coumarin Schiff bases
using Calf-thymus DNA (Purchased from Bangalore Genei, Bengaluru, Cat. No. 105850). The synthetic
compounds were added separately to the Calf- thymus DNA, sample mixtures were incubated at 37 ºC for 2h.
The cleaved product were run on agarose gel electrophoresis according to the procedure [27] and observed
under UV transilluminator for DNA cleavage.
2.4.3.1 Agarose gel electrophoresis
In brief, 250mg 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 the gel cassette fitted with comb. Later comb was
9

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removed carefully after the gel solidified and gel placed in electrophoresis chamber flooded with TAE buffer.
20μL of sample mixture, Calf-thymus 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 calf-thymus DNA and standard DNA marker
( λ DNA/ HindIII digest, purchased from Merck, Bengaluru).
3. Results and discussion
The Co(II), Ni(II) and Cu(II) complexes are non-hygroscopic coloured solids, stable at room
temperature and soluble in DMF and DMSO. Carbon, hydrogen and nitrogen were estimated by using a C, H, N
analyzer. The low conductance values confirm the non-electrolytic nature of all the metal complexes. Elemental
analysis and molar conductance values are listed in Table 1.
3.1 Infrared studies
Infrared spectra of both the Schiff bases were studied. Characteristic high intense bands at 1623 and
1617 cm-1 for HLI and HLII respectively are attributed to the ν(C=N) vibrations [28]. The broad band at 3020
and 3026 cm-1 and a strong band at 1324 and 1320 cm-1 in the infrared spectra of HLI and HLII Schiff bases are
assigned to -OH stretching and phenolic ν(C-O) vibrations respectively. Another strong band at 1720 and
1724 cm-1 is assigned to ν(C=O), lactonyl carbon of the coumarin moiety of HLI and HLII respectively [29].
Typical infrared spectrum of Schiff base HLII is reproduced in Fig. 5.
In order to study the binding mode of Schiff base to metal ion in complexes, IR spectra of free ligands
were compared with the spectra of metal complexes. Characteristic IR spectra assignments of all the
synthesized metal complexes are compared with the corresponding free ligands. On comparison, the band
positions of various vibrations are ascertained.

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Table 1
Elemental analyses of Schiff bases [HLI and HLII] and their Co(II), Ni(II) and Cu(II) complexes along with their molar conductance and magnetic moment data.
Metal
Empirical
Formula
Yield
(%)
M%
Calcd. Obsd.
C%
Calcd. Obsd.
H%
Calcd. Obsd.
N%
Calcd.
Molar
Conductance
(Ohm-1 cm2
mole-1)
µeff
(BM)
Complex
Obsd.
HLI
HLII
1
C17H11F2NO3
C18H14ClNO3
82
78
--
--
--
--
64.73
65.95
64.76
65.96
3.50
4.33
3.52
4.31
4.43
4.28
4.44
4.27
--
--
--
--
C34H24CoF4N2O8
C36H30Cl2CoN2O8
C34H24NiF4N2O8
56
50
8.13
7.90
8.15
7.87
54.39
57.73
54.44
57.77
3.40
4.02
3.34
4.04
3.81
3.72
3.87
3.74
18.10
22.32
4.32
4.54
2
3
65
49
47
69
8.14
7.88
8.69
7.40
8.12
7.84
8.73
7.38
54.45
57.80
56.13
57.47
54.46
57.79
56.09
57.42
3.35
4.06
3.33
4.05
3.34
4.04
3.32
4.02
3.85
3.70
3.83
3.74
3.87
3.74
3.85
3.72
18.36
12.57
28.45
27.34
3.19
3.15
1.79
1.77
C36H30Cl2NiN2O8
C34H24CuF4N2O8
C36H30Cl2CuN2O8
4
5
6
11

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Fig. 5. Infrared spectrum of Schiff base HLII.
The important infrared spectral data are presented in the Table 2 and the representative IR spectrum of
Cu(II) complex (6) is reproduced in Fig. 6.
The main features in infrared spectra of the complexes are as follows. Shift in the stretching frequencies
of azomethine (-CH=N) groups to lower values is observed after the formation of transition metal complexes in
the range 1602-1590 cm-1 compared with free ligand due to the coordination of azomethine nitrogen atom.
Further, evidence of bonding was given by the new bands observed in the spectra of the metal complexes as a
medium or weak intense in the region 467-415 cm-1 due to (M-N) stretching vibrations supporting the
involvement of nitrogen atom of the azomethine group via coordination [30,31]. The new weak intensity bands
of (M-O) stretching vibrations occur at 591-575 cm-1 [32]. The presence of coordinated water molecules in
metal complexes are confirmed by a broad band in the region 3433-3449 cm-1 and two weak bands in the region
800-763 and 721-710 cm-1 due to ν(OH) rocking and wagging modes of vibrations, respectively [33,34]. High
intensity bands observed around 1323 cm-1 in the Schiff bases are due to phenolic C-O, appeared as a medium
to high intensity band in the region 1343-1326 cm-1 of the metal complexes. These observations suggest that, -
OH groups have been replaced by the metal ion forming M-O bonds via deprotonation. Slightly altered position
of ν(C=O) (lactonyl carbon) in all the metal complexes confirms its non involvement in coordination.

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3.2 Fluorescence study of Schiff bases
The emission spectra of Schiff bases are studied in DMSO. Schiff base HLI was characterized by an
emission band around 480 nm is due to formation of phenoxide anion. Upon addition of 2-3 drops of aqueous
alkali (2% NaOH) to Schiff base solution, we observed λmax value at 476 nm in DMSO. The emission spectrum
of HLII in DMSO shows an emission band around 513 nm. On addition of 2-3 drops of aqueous alkali (2%
NaOH) to the Schiff base solutions, we observed the λmax value at 503 nm. The shift in λmax values after
addition of 2-3 drops of aqueous alkali (2% NaOH) to both the Schiff bases clearly indicates that, the proton
transferred (H-bonded ion pair) species exist in equilibrium [35,36]. Fig.7, showing emission spectra of both
the Schiff bases individually and overlapping after adding alkali.
3.3 Solution electronic spectra study
In order to obtain the information regarding stereochemistry of metal ions, UV-Visible spectra of the
ligands and their metal complexes were recorded in DMF. The electronic absorption spectra are often very
helpful in the evaluation of results furnished by other methods of structural investigation. The electronic
spectral measurements were used to assign the stereochemistry of metal ions in complexes based on the
positions and number of d–d transition peaks.
Fig.6. Infrared spectrum of Cu (II) complex (6).
13

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Table 2
Prominent infrared frequencies (in cm-1) of Schiff bases and their metal complexes.
Schiff Base /
Metal
Coordinated
Water
Lactonyl
ν(C=O)
v(OH)
Phenolic
ν(C=N) Stretching ν(C-O) ν(M-N) ν(M-O)
Complex
v(OH)
HLI
--
--
1720
1724
1723
1722
1719
1723
1721
1724
1617
1623
1599
1590
1592
1597
1591
1602
3020
3026
1326
1320
1343
1335
1333
1331
1336
1326
-
-
-
-
HLII
1
2
3
4
5
6
3443
3445
3439
3449
3433
3436
-
-
-
-
-
-
419
423
467
445
459
415
583
589
587
575
582
591

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Fig. 7. Emission spectra of the Schiff bases HLI and HLII in DMSO.
The Schiff bases exhibited two absorption bands around 275 and 330 nm. Spectral data below 350 nm
are ligand centred transitions (intraligand π-π* and n-π*) of aromatic ring and non-bonding electrons present on
nitrogen of the azomethine group in the Schiff bases [37,38]. In the metal complexes imino π-π* transitions
were shifted to longer wavelengths as a consequence of coordination to metal, conforming the formation of
Schiff base metal complexes [39].
Solution electronic absorption spectra of all the synthesized metal complexes were recorded in DMF
(Table 3). The electronic spectra of Co(II) complexes exhibited absorption bands around 1005 and 517 nm are
corresponding to 4T1g (F) → 4T2g (F) (ν1) and 4T1g (F) → 4T1g (P) (ν3) transitions respectively. These bands are
characteristic of high spin octahedral Co(II) complexes [40]. Observed magnetic moment values of Co(II)
complexes were 4.32 and 4.54 BM and these results supports octahedral geometry for Co(II) complexes [41,
42].
15

The electronic spectraAofCpreCseEnt PlighTt EgreDen NMi(IAI) NcomUplSexeCs eRxhIiPbiteTd three bands around 920, 601
and 372 nm, presumably due to the three spin-allowed transitions 3A2g (F) → 3T2g (F) (ν1), 3A2g (F) → 3T1g (F)
3
3
(ν2) and A2g (F) → T1g (P) (ν3) respectively, which indicate octahedral geometry around Ni(II) ion [43].
Magnetic moment values obtained were 3.15 and 3.19 BM, which are agreeable with the octahedral
environment around Ni(II) ion [44,45].
The electronic spectra of Cu(II) complexes showed two absorption bands. A low intensity broad band
2
2
around 627 nm is assignable to Eg → T2g transition in support of distorted octahedral configuration of metal
complex [46]. Another high intensity band around 370 nm are due to symmetry forbidden ligand → metal
charge transfer. Obtained magnetic moment value for Cu(II) complexes were 1.77 and 1.79 BM, further these
values support the electronic spectral results [47,48]. Fig. 8 shows the representative solution electronic
spectrum of Cu(II) complex (6).
Table 3
Solution electronic spectral results of the synthesized metal complexes
Metal
Complex
λmax in cm-1
Band Assignments
Geometry
(nm)
9984 (1001)
18970 (527)
4T1g(F)→ 4T2g(F) (ν1)
4T1g (F)→ 4T1g (P) (ν3)
octahedral
octahedral
1
2
9907 (1009)
19709 (507)
4T1g(F)→ 4T2g(F) (ν1)
4T1g (F)→ 4T1g (P) (ν3)
10910 (916)
16740 (597)
26860 (372)
3A2g(F)→3T2g(F) (ν1)
3A2g(F)→3T1g(F) (ν2)
3A2g(F)→3T1g(P) (ν3)
3A2g(F)→3T2g(F) (ν1)
3A2g(F)→3T1g(F) (ν2)
3A2g(F)→3T1g(P) (ν3)
octahedral
octahedral
3
4
10810 (925 )
16500 (606)
26750 (373)
16021 (624)
26960 (370)
2T2g← 2Eg
L→M
2T2g← 2Eg
L→M
distorted
octahedral
5
6
16105 (631)
26901 (371)
distorted
octahedral

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Fig. 8. Solution electronic absorption spectrum of Cu(II) complex (6).
3.4 ESI-MS studies of metal complexes
ESI mass spectra of all the synthesized complexes showed M+1 peaks, which are equivalent to their
molecular weight, these facts support the formation of respective metal complexes of the type ML2(H2O)2. The
representative Co(C17H10F2NO3)2(H2O)2 is shown in Fig. 9. Here, a spectrum showed M+1 peak at m/z 724
equivalent of its molecular weight, it is an evidence that supports the formation of respective metal complex of
the type ML2(H2O)2. All fragments of the species undergo demetallation to form „L‟ gave a base peak at
m/z 314.
Fig. 9. ESI-MS of Co(II) complex (1).
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3.5 Electron spin resonance
ESR spectra of metal chelates provide the important information, regarding the environment around
metal ions. Fig. 10, shows the ESR spectrum of Cu(II) complex (5) at liquid nitrogen temperature using TCNE
as g-marker (2.00277). From the figure, g|| and g⊥ values have been found to be 2.1230 and 2.0370 respectively.
2
2
Here, g|| > g⊥ indicate that, the unpaired electron predominantly lies in dx y orbital. The gav and G value
-
calculated to be 2.0656 and 3.4783 respectively. G < 4 indicates there is an interaction in the metal complex.
Observed g|| value of the metal complex is less than 2.3 which is in agreement with the covalent character of
metal-ligand bond.
Fig. 10. ESR spectrum of Cu(II) complex (5).
3.6 Thermal studies of the metal complexes.
As a function of temperature, thermal behaviour of the synthesized Co(II), Ni(II) and Cu(II) complexes
of Schiff base HLII were studied. The nature of proposed chemical change, the percentage weight loss and the
percentage of metal oxide obtained are tabulated in Table 4. It shows that, thermal features of all the metal
complexes were almost same. Here, the representative TG/DTA spectrum (Fig. 11) of
Cu(C18H13ClNO3)2.(H2O)2 is explained. First decomposition occured in the region 225-247 ºC centred at 236 ºC
is due to loss of coordinated water molecules. Decomposition in the regions 310-440 ºC centred at 385 ºC and
440-800 ºC centred at 472 ºC corresponding to loss of organic moieties. Finally metal oxide was formed above
880 ºC.

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Fig. 11. TG/DTA spectrum of Cu(II) complex (6).
3.7 Electrochemical study
The electrochemical property of synthesized Cu(II) complex (5) was studied in order to monitor spectral
and structural changes accompanying electron transfer. Here, cyclic voltammogram (Fig. 12) of the
representative Cu(C17H10F2NO3)2.(H2O)2 is discussed. It displays an oxidation peak at Epa= 0.2101V with a
corresponding reduction peak at Epc= -0.0401V. The peak separation of this couple (∆Ep) is 0.17V at
100 mVs-1 suggested that Cu(II) / Cu(I) couple which is a one electron transfer. So, copper complex had a large
separation between the cathodic to anodic peak, it increases with the scan rates, indicating a quasi-reversible
character [49,50].
Fig. 12. Cyclic voltamogram of Cu(II) complex (5).
19

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Table 4
Thermo gravimetric data of the metal complexes (2, 4 and 6) of Schiff base HLII.
Metal
Complex
Decomposition
Temperature (ºC)
Weight loss (%)
Obsd. Calcd.
Metal oxide (%)
Inference
Obsd.
Calcd.
215-238
325-789
4.83
4.82
10.00
10.04
Loss of coordinated water molecules
Loss of organic moieties
2
4
85.17
85.14
220-248
318-782
4.84
4.83
9.89
9.92
Loss of coordinated water molecules
Loss of organic moieties
85.27
85.25
225-247
310-800
4.82
4.79
10.51
10.52
Loss of coordinated water molecules
Loss of organic moieties
6
84.67
84.69

I
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3.8 Fluorescence study of Schiff bases HL and its metal complexes
Fluorescence is a spectrochemical method of analysis where molecules of an analyte get excited by
irradiation at a certain wavelength and emit the radiation of a different wavelength.
In this investigation, we have studied the fluorescent nature of the Schiff base HLI and its metal
complexes with same concentration of 10-3 M in DMF solution at room temperature. The overlapping spectra of
Schiff base HLI and its Co(II), Ni(II) and Cu(II) complexes in DMF is shown in Fig. 13. The Co(II), Ni(II) and
Cu(II) complexes showed strong emission bands at 486, 481 and 484 nm respectively. Schiff base exhibits a
strong fluorescence intensity compared to other three metal complexes, it shows an emission band at 480 nm.
The weak intensity bands of metal complexes are due to decrease in electron density on ligand [51, 52]. The
decreased fluorescence intensities of synthesized metal complexes were compared with HLI is in the order
Co(II) > Ni(II) > Cu(II). Here, Co(II) complex (1) exhibits the weakest quenching whereas, strongest quenching
has been obtained for Cu(II) complex (5).
Fig. 13. Fluorescence study of the HLI and its Co(II) (1), Ni(II) (3) and Cu(II) (5) complexes.
21

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4.0 Bio-assay investigations
4.1 In-vitro antibacterial and antifungal activity
All the synthesized Schiff bases (HLI and HLII) and their corresponding Co(II), Ni(II) and Cu(II)
complexes were screened for their antibacterial and antifungal activity by using two species of bacteria, namely
Pseudomonas aeruginosa, Proteus mirabilis and two fungi species namely Aspergillus niger, Rhizopus oryzae
using Gentamicin and Fluconazole as standard drugs respectively at 200 µg/mL concentration by disc diffusion
method.
M.A. Phaniband et al., in the year 2011, synthesised similar compounds and studied antimicrobial
activities against two bacteria (S. aureus and E. coli) and two fungi (A. niger and C. albicans) species at 100
μg/mL concentration using Norfloxacin and Griseofulvin as standard drugs respectively. The results shows that,
halogen substituted Cu(II) more complex showed zone of inhibition against A. niger than non halogenated one
[53].
Copper has antimicrobial property. The importance of copper in biological system is reported with the
use of Cu(II) Schiff base complexes. Copper(II) forms stable complexes with nitrogen and oxygen donor
ligands [54-56]. Many of the other authors have showed that, Cu(II) complexes of the coumarin Schiff bases
express more antimicrobial activity against various pathogens [57,58].
The obtained results are given in Figs. 14 and 15. A comparative study of ligands and their Co(II),
Ni(II) and Cu(II) complexes indicates that, the metal complexes exhibit higher bio-activity than their free
ligands. Such an increased activity of metal complexes can be explained on the basis of Overtone‟s and
Tweedy‟s concepts [59,60]. According to the chelate hypothesis higher activity of metal chelates is due to the
increased lipophilicity from inclusion of metal ion with Schiff base.
By observing the results, all synthesised metal complexes are comparatively active against four
microbial species. The Cu(II) complexes showed higher bioactivity compare to Co(II) and Ni(II) complexes.
While comparing the antifungal results of the newly synthesised Cu(II) complexes of both halogenated
coumarin Schiff bases with previous studies, the present results have showed a significant activity against
Aspergillus niger at 200 µg/mL.

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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.
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4.2 Anthelmintic activity
Anthelmintic activity data of the newly synthesized compounds are shown in Table 5. The obtained
data shows that, the metal complexes have better anthelmintic activity compared to their respective Schiff
bases HLI and HLII using Albendazole as standard drug at 10 µg/mL concentration.
In the previous study, M. Manjunatha et al.,[61] showed that, all the synthesized Schiff bases and their
respective Co(II), Ni(II) and Cu(II) complexes have shown prominent anthelmintic activity at 10 µg/mL
concentration using Albendazole as standard drug. Whereas, in our present study, both the copper (II)
complexes have exhibited faster paralysis and death time of worms when compared to other synthesized
compounds at 10 µg/mL concentration using the same standard drug.
Table 5. Anthelmintic studies of Schiff bases and their metal complexes
Schiff Base/
Metal
Complex
Concn.
(μg/mL)
Time of Paralysis
(min)
Time of Death
(min)
2
10
2
19.06±0.04
7.03±0.03
18.19±0.08
8.15±0.04
21.40±0.12
10.19±0.01
19.20±0.10
9.10±0.10
HLI
HLII
10
2
10
2
10
2
12.14±0.04
6.00±0.03
14.20±0.05
7.23±0.11
13.03±0.03
6.29±0.01
15.40±0.04
9.22±0.12
15.10±0.13
9.41±0.20
15.10±0.02
9.11±0.04
1
2
3
4
5
6
10
2
10
11.20±0.01
5.14±0.03
14.21±0.02
8.45±0.12
2
10
9.10±0.01
4.45±0.01
10.05±0.03
8.23±0.10
2
10
10.04±0.03
4.20±0.04
12.20±0.09
7.25±0.12
Albendazole
(Std.)
10
--
3.38±0.03
--
6.35±0.05
--
DMSO*
* Used as control

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4.3 DNA Cleavage activity
DNA cleavage may be considered as an enzymatic reaction which comprises various biological
processes as well as biotechnological manipulation of genetic material. It has wide applications in molecular
biology, bioorganic chemistry and drug design [62,63]. Metal containing reagents that induce chemical DNA
scission are often referred to as artificial metallonucleases [64] and the attention focused towards the
development of new metallonucleases, which bind and cleave DNA at physiological conditions, have gained
momentum.
The metal ions play a crucial role in DNA cleavage activity because nature of the metal complexes may
be attributed to additional π-π interaction through the aromatic phenyl rings [65]. The presence of different
substituent‟s in the intercalative ligand could affect changes in space configuration and electron density
distribution around transition metal complexes.
All the synthesized Co(II), Ni(II) and Cu(II) complexes of bidentate ligands were subjected to their
DNA cleavage activity by gel electrophoresis method. Lane 1 and 6 i.e. metal complexes 1 and 6 showed
complete DNA cleavage whereas metal complexes 2, 3, 4 and 5 cleaved partially as shown in the Fig. 16. The
cleavage efficiency of metal complexes compared with control is due to their efficient DNA-binding ability
[66]. In the year 2012, M.B Halli et al., have studied DNA cleavage activity for their newly synthesized M(II)
compounds by agarose gel electrophoresis method against Calf-thymus DNA and obtained the similar kind of
results [67].
Fig. 16. DNA Cleavage activity of the synthesized metal complexes (1-6).
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5.0 Conclusion
The newly synthesized Schiff bases act as bidentate ligands. The metal ion is coordinated through the
azomethine nitrogen and oxygen atoms. The bonding of ligand to metal ion was confirmed by various spectral
studies. Metal complexes exhibited promising antibacterial, antifungal and anthelmintic activities. The
bioactivity significantly increased upon coordination. All these observations lead us to propose the following
structures (Figs. 17 and 18) in which metal complexes having stoichiometry of the type ML2(H2O)2
[M = Co(II), Ni(II) and Cu(II)].
F
CH3
F
OH2
O
O
O
N
M
N
O
O
O
OH2
F
CH3
F
Fig. 17. Proposed structure of metal complexes (1, 3 and 5) of
Schiff base HLI.
CH3
Cl
H3C
OH2
O
O
O
N
M
N
O
O
O
OH2
CH3
Cl
CH3
Fig.18. Proposed structure of metal complexes (2, 4 and 6) of
Schiff base HLII.

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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
UGC, New Delhi for the financial support through Major Research Project. [No:F.36-137/2008(SR) dated 27th
March 2009].
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