Bio-important antipyrine derived Schiff bases and their transition metal complexes: Synthesis, spectroscopic characterization, antimicrobial, anthelmintic and DNA cleavage investigation

Article abstract of DOI:10.1016/j.molstruc.2016.07.123

Spectroscopic (IR, NMR, UV–vis, ESR, ESI-mass), magnetic and TGA studies suggests octahedral geometry for all the Co^II, Ni^II and Cu^II complexes of the Schiff bases, derived from 4-aminoantipyrine and 8-formyl-7-Hydroxy-4-m

Full text of DOI:10.1016/j.molstruc.2016.07.123

Accepted Manuscript
Bio-important antipyrine derived Schiff bases and their transition metal complexes:
Synthesis, spectroscopic characterization, antimicrobial, anthelmintic and DNA
cleavage investigation
M. Manjunath, Ajaykumar D. Kulkarni, Gangadhar B. Bagihalli, Shridhar Malladi,
Sangamesh A. Patil
PII:
S0022-2860(16)30817-1
10.1016/j.molstruc.2016.07.123
MOLSTR 22826
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 8 November 2015
Revised Date: 28 July 2016
Accepted Date: 31 July 2016
Please cite this article as: M. Manjunath, A.D. Kulkarni, G.B. Bagihalli, S. Malladi, S.A. Patil, Bio-
important antipyrine derived Schiff bases and their transition metal complexes: Synthesis, spectroscopic
characterization, antimicrobial, anthelmintic and DNA cleavage investigation, Journal of Molecular
Structure (2016), doi: 10.1016/j.molstruc.2016.07.123.
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Graphical Abstract (GA)
A series of metal complexes CoII, NiII and CuII have been synthesized
with the Schiff base derived from4-Aminoantipyrine and 8-formyl-7-
Hydroxy-4-methylcoumarin/5-formyl-6-hydroxycoumarin,
coordinated
through ONO donor sites. The syntheised compounds have tested their
antimicrobial, anthelmintic and DNA cleavage properties.

ACCEPTED MANUSCRIPT
Bio-important antipyrine derived Schiff bases and their transition metal
complexes: Synthesis, Spectroscopic characterization, Antimicrobial,
Anthelmintic and DNA cleavage investigation
Manjunath Ma, Ajaykumar D. Kulkarnib*, Gangadhar B. Bagihallic*, Shridhar Malladid,
Sangamesh A. Patile
aDepartment of Chemistry, CMR Institute of Technology, Bangalore, 560037. Karnataka, India
bDepartment of Applied Sciences, MIT Academy of Engineering (An Autonomous Institute Affiliated
to Savitribai Phule Pune University), Dehu Phata, Alandi (D), Pune-412105, India
cDepartment of Chemistry, K.L.E. Institute of Technology, Gokul Hubballi 580030. Karnataka,
India
dDepartment of Chemistry, KLS’s Gogte Institute of Technology, Udyambag, Belagavi-590006.
Karnataka, India.
eP.G. Department of Chemistry, Karnatak University, Pawate Nagar, Dharwad 580 003.
Karnataka, India
*ajaykumar.kulkarni@gmail.com
*g.bagihalli@gmail.com

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Abstract
Spectroscopic (IR, NMR, UV–vis, ESR, ESI-mass), magnetic and TGA studies suggests
octahedral geometry for all the CoII, NiII and CuII complexes of the Schiff bases, derived from 4-
aminoantipyrine
and
8-formyl-7-Hydroxy-4-methylcoumarin/5-formyl-6-hydroxycoumarin,
coordinated through ONO donor sites. Antibacterial (Escherichia coli, Staphylococcus aureus,
Pseudomonas aeruginosa and Salmonella typhi), antifungal (Aspergillus niger, Aspergillus flavus
and Cladosporium) and DNA cleavage properties of the metal complexes are investigated.The
results suggested that some of the synthesized compounds are potential antimicrobials. The
synthesized compounds tested for their anthelmintic activities and it was found that CoII and NiII
complexes exhibited good anthelmintic properties.
Keywords:Azomethine, Anthelmintic, DNA, Schiff base, Transition metal complexes,
Spectroscopy

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Introduction
Due to their wide spectrum of biological activities, heterocyclic compounds dominated
medicinal chemistry. Antipyrine and its derivatives have wide applications in biological activities.1,2
Along with biological activities, their interesting structural features have attracted many researchers
to work on the metal complexes of antipyrine derivatives.3,4 Antipyrine alone forms stable
coordination bond with metal ions in monodentate fashion through carbonyl oxygen atom.5,6
Subsequently it is reported that, 4-aminoantipyrine Schiff bases and their metal complexes possess
numerous biological applications that include antifungal, antibacterial, analgesic, antipyretic and
anti-inflammatory.7-9 Transition metal complexes of pyrazole derivatives have received more
attention as beneficial anticancer agents.10, 11 On the other side coumarin derivatives, well-known
fluorescent molecules, also possess wide range of biological activities viz. antimicrobial,
antiallergic, anti-inflammatory, antitumor etc.12-14 Coumarin derivatives have been found useful in
the treatment of human immunodeficiency virus.15,16 In vitro and in vivo studies have suggested the
possible use of coumarins in the treatment of cancer as well.17
Formation of coordination bond brings extensive changes in biological properties of ligand
and also in metal ions18 worth to mention chelation can cure many diseases. Very recently we have
reported biological importance of different structural groups including coumarins and their metal
complexes.19 A review on Schiff bases and their antimicrobial activities elaborated the importance
of this functional group in medicinal chemistry.20 Spectroscopically active metal centers with stable,
inert, and non-toxic nature are exceptionally valuable in biological systems.21 Here, in the present
work, primary amino group of antipyrine is made to react with an aldehyde group of formyl
coumarin thereby incorporating an azomethine group in the molecule which were further reacted
with CoII, NiII and CuII metal ions to form respective metal complexes. The newly synthesized
Schiff bases and their metal complexes are characterized by various spectroscopic techniques.
Selected newly synthesized compounds have been evaluated for their various biological properties.

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Experimental
Analysis and physical measurements
Carbon, hydrogen and nitrogen were estimated by using Elemental Analyzer Carlo Erba
EA1108 analyzer. Molar conductivity measurements were recorded on ELICO-CM-82 T
Conductivity Bridge with a cell having cell constant 0.51 and magnetic moment was carried out by
using faraday balance. The IR spectra of the Schiff bases and their CoII, NiII and CuII complexes
1
13
were recorded on a HITACHI-270 IR spectrophotometer. The H NMR and C NMR spectra of
ligands were recorded in DMSO-d6 on a BRUKER 300MHz spectrometer at room temperature
using TMS as an internal reference. Mass spectra were recorded on a JEOL SX 102/DA-6000 mass
spectrometer/data system. The electronic spectra of the complexes were recorded in HPLC grade
DMF and DMSO solvent on a VARIAN CARY 50-BIO UV-spectrophotometer in the region of
200–1100 nm. The ESR spectrum was recorded on Varian-E-4X-band EPR spectrometer and the
field set is 3000 G at modulation frequency of 100 kHz under room temperature using TCNE as g
marker. Thermo gravimetric analyses were done on PerkinElmer Diamond TG/DTA instrument and
measured from room temperature to 1000 OC at a heating rate of 10 OC min-1.
Methods
All the chemicals used were of reagent grade. 7-hydroxy- 4-methylcoumarin,22 6-hydroxy
coumarin,22 8-formyl- 7-hydroxy-4-methyl coumarin,23,24 and 5-formyl-6-hydroxy coumarin24,25
have been synthesized according to the published procedure.
Synthesis of Schiff bases [SB-I, SB-II]
Figure 1 represents the synthesis of Schiff bases. The Schiff base SBI has synthesized as
given below. To the hot ethanolic solution (30 mL) of 4-aminoantipyrine (0.01 mol) was added hot
ethanolic solution (30 mL) of 8-formyl-7-hydroxy-4- methylcoumarin. Then catalytic amount of
hydrochloric acid (2-3 drops) was added. The reaction mixture was refluxed for 4-5 h. The progress

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was checked by TLC. The precipitate formed during reflux was filtered, washed with cold EtOH
and recrystallized from hot EtOH.
In similar way Schiff base SB-II was synthesized with 5-formyl-6-hydroxycoumarin (0.01
mol).
CH3
H3C
N
N
O
H2N
O
CH3
CH3
Reflux,
4-5
N
N
N
CH3
+
EtOH
2 - 3 Drops
HO
O
O
CH
CHO
of Conc.HCl
OH
O
O
8-formyl-7-hydroxy-4-methyl coumarin
4-Aminoantipyrine
CH3
Schiff base - I
CH3
H3C
N
CH3
H2N
N
O
CHO
HO
N
N
O
N
CH3
+
Reflux,
EtOH
4-5 h
O
O
CH
2 - 3 Drops
of Conc.HCl
HO
5-Formyl-6-hydroxy coumarin
4-Aminoantipyrine
O
Schiff base-II
O
Figure 1. Synthesis scheme and structure of Schiff Bases SB-I & SB-II
Synthesis of CoII, NiII and CuII complexes (1–6)
To an aq. alcoholic solution (15 mL) of 1 m mol of CoCl2·6H2O/NiCl2·6H2O/CuCl2·2H2O
was added an alcoholic solution (40 mL) of Schiff bases (2 m mol) and refluxed on water bath for 2
h. Then, to the reaction mixture was added 2 m mol of sodium acetate and reflux was continued for
3 h. The separated complex was filtered, washed thoroughly with water, ethanol, ether and finally
dried in vacuum over fused CaCl2.
Biological application studies
In vitro antibacterial and antifungal assay
The Schiff bases and their CoII, NiII and CuII complexes have been studied for their
antibacterial and antifungal activities by agar and potato dextrose agar diffusion method. The
antibacterial and antifungal activities were done at 100, 50 & 25 mg mL-1 concentrations in DMF

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by using bacteria (E. coli, Staphylococcus aureus and Pseudomonas aeruginosa) and fungi (A.
niger, Aspergillus flavus and Cladosporium) by the MIC method.26 These bacterial stains were
O
O
incubated for 24 h at 37 C and fungal stains were incubated for 48 h at 37 C. Standard
antibacterial (Gentamycin) and antifungal drug (Fluconazole) was used for comparison under
similar conditions.
DNA cleavage experiment
DNA cleavage experiments were done according to the method reported in literature.
Isolation of DNA, preparation of culture media were followed as mentioned in the standard reported
method and the cleavage products wre analysed by agarose gel electrophoresis method.27,28 To
determine the extent of DNA cleavage, the results are compared with standard DNA marker.
Anthelmintic Activity assay
Anthelmintic activity assay was performed on adult Indian earthworms, Pheretima posthuma,
due to its anatomical and physiological resemblance with the intestinal roundworm parasite of
human beings done by the method given in the literature.29,30 Earthworms (Pheretima posthuma) of
3-4 cm in length and 0.1-0.2 cm in width were collected from moist soil and washed with normal
saline to remove all fecal matter were used for the experimental protocol. The compounds were
subjected against earthworms at 10 mg mL-1 by using albendazole as a standard drug. The
paralyzing and death times were noted and their mean was calculated for triplicate sets. The death
O
time was ascertained by placing the earthworms in warm water (50 C) which stimulated the
movement, if the worm was alive.
Results and discussion
4-amino antipyrine and formyl coumarins form Schiff bases (SB-I and SB-II) in ethanol
with ONO donor sites. These Schiff bases coordinated to CoCl2·6H2O/NiCl2·6H2O/CuCl2·2H2O

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forming stable and non-hygroscopic octahedral complexes (1–6) (Table 1) as presented in Figure 2.
Several efforts made to develop single crystal of complexes suitable for crystal analysis were failed
due to their poor solubility in common organic solvents. The elemental analyses showed that, the
CoII, NiII and CuII complexes have 1:2 stoichiometry of the type ML2, where L stands for a
deprotonated ligands SB-I and SB-II. The molar conductance values are too low to account for any
dissociation of the complexes in DMSO, indicating the non-electrolytic nature of the complexes in
DMSO.
Figure 2. Structure of Metal (II) Complexes
Table 1. Elemental analyses of Schiff bases and their CoII, NiII and CuII complexes along with
molar conductance and magnetic moment data.
M%
C%
H%
N%
Molar
Complex.
No.
Yield
%
Conductance µeff
Empirical Formula
Ohm-1 cm2
mole-1
(BM)
Obsd. Calcd. Obsd. Calcd. Obsd. Calcd. Obsd. Calcd.
I
C22H19N3O4
74% --
--
67.85 67.87 4.87
67.34 67.38 4.27
31.48 31.54 4.33
31.22 31.23 3.78
31.47 31.55 4.35
31.22 31.24 3.76
31.28 31.30 4.30
30.88 30.98 3.72
4.88
4.28
4.33
3.79
4.31
3.67
4.29
3.69
10.75 10.79 --
11.20 11.23 --
--
II
1
2
3
4
5
6
C21H16N3O4
72% --
--
--
Co(C22H18N3O4)2
Co(C21H15N3O4)2
Ni(C22H18N3O4)2
Ni(C21H15N3O4)2
Cu(C22H18N3O4)2
Cu(C21H15N3O4)2
55% 7.01
51% 7.29
60% 6.98
57% 7.27
56% 7.73
61% 8.00
7.04
7.30
7.01
7.26
7.75
8.03
5.01
5.18
5.00
5.17
4.96
5.16
5.02
5.20
5.01
5.20
4.98
5.17
28
22
23
16
19
27
4.66
4.25
3.12
2.90
1.73
1.76

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IR spectral studies
Table 2 represents the prominent infrared spectral data of Schiff bases and their CoII, NiII
and CuII complexes. The IR spectra of the Schiff bases SB-I (Figure S1) and SB-II exhibited strong
stretching vibrations at 1609 and 1615 cm-1 respectively which is ascribed to azomethine ν(HC=N)
group. All the CoII, NiII and CuII complexes exhibited the band of ν(HC=N) in the region 1566-
1560 cm-1, showing the shift to lower wave numbers indicates that, the nitrogen of imine group is
coordinated to the metal ion.31 The band corresponding to ν(C=O) at 1715 cm-1 and 1710 cm-1 of
antipyrine in SB-I and SB-II respectively was shifted to lower frequency by about 50-60 cm-1 in the
metal complexes, suggesting the coordination to the metal ions.32 The oxygen atom of carbonyl
group of antipyrine has large dipole moment, strong basic character and hence serves as potential
donor. The higher side shift of phenolic v(C-O) stretching frequency in the region 1350-1385 cm-1
in all the metal complexes compared to ligands indicates the coordination of phenolic oxygen atom
via deprotonation.33 The unaltered position of bands due to lactonyl ν(C=O) of the coumarin moiety
around 1735-1720 cm-1 in all the metal complexes suggests its non involvement in coordination to
the metal ions. The IR spectra of complexes show new bands at 370-386 cm-1 and 472-485 cm-1
assigned to v(M-O) and v(M-N) respectively.34 Thus, IR results give evidence for the bonding mode
of Schiff bases to metal ions (Figure S2 & S3).
Table 2. The important infrared frequencies (in cm−1) of Schiff bases and their metal
υ(C=O)
Antipyrine
cm-1
υ(C-O)
Phenolic
cm-1
Complex
No.
υ(HC=N)
υ(M-N)
cm-1
υ(M-O)
cm-1
cm-1
SB-I
SB-II
1
1609
1615
1560
1715
1710
1665
1309
1316
1355
---
---
----
---
472
379
2
3
4
1562
1565
1561
1660
1650
1662
1350
1350
1370
476
480
485
376
370
386
1566
1563
1648
1650
1380
1385
475
473
383
384
5
6

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1H NMR and 13C NMR study of Schiff bases
1H NMR spectrum of the SB-I (Figure S4), a single signal at 11.7 ppm is ascribed to
phenolic -OH. A characteristic singlet proton signals at 9.44 ppm, 2.4 ppm, and around 1.6 to 1.8
ppm are assigned to -HC=N, N-CH3 and C-CH3 groups of coumarin and pyrazoline moiety
respectively. In addition to this, the multiplet signals in the 6.3-7.9 ppm range are due to aromatic
protons. In case of SB-II, the singlet at 14.3 ppm, 9.1 ppm and 2.42 ppm are attributed to phenolic –
OH, -HC=N and N-CH3 respectively. The singlet at 1.5 and 1.8 ppm are ascribed to -CH3 of
pyrazoline moiety, multiplet signals in 6.1-7.7 ppm range are due to aromatic protons. In both the
Schiff bases phenolic -OH proton signals disappeared after the addition of D2O.35 The CHN
analysis results are in good agreement with the number of protons that are calculated from the
integration curves.
In the 13C NMR spectra of the Schiff bases I (Figure S5) and II, the signals appeared in
region
142.0 and 152 ppm. The signals due to methyl carbon were observed around 22-19 ppm. The
signals at 144.2-145.2 ppm are assigned to carbon of C=N and carbon of (C=O) at 160.0-162.2
and 162.2-169.7 ppm respectively. Thus conclusion of NMR studies further supports the IR results
δ
117.6-156.7 ppm are assigned to aromatic carbons. The pyrazoline ring carbon appeared at
δ
δ
δ
δ
and bonding mode of ligands.
Mass spectral studies of Schiff bases and their metal complexes
Representative mass spectrum of SBI is depicted in Figure S6. The spectrum showed a
molecular ion peak at m/z 389 which is equivalent to its molecular weight. In addition to this the
fragment ion peaks observed at m/z 327 and 104 are due to the cleavage of -OH and C10H4O5,
respectively. In case of Schiff base SBII the molecular ion peak is observed at m/z 374 which is
ascribed to C21H16N3O4.
The ESI-mass spectrum of CoII (1) (Figure S7) and NiII (3) (Figure S8) complexes were
discussed in supplementary file. The spectrum of CoII complex showed a molecular ion peak M+ at

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m/z 835, which is equivalent of its molecular weight [ML2]+. Fragments leading to the formation of
the species [ML]+ which undergoes demetallation to form the species [L+H]+ gave fragment ion at
m/z 389. The spectrum of Ni(II) complex showed a molecular ion peak M+ at m/z 834, which is
equivalent of its molecular weight [ML2]+. The other fragments leading to the formation of the
species [ML]+ which undergoes demetallation to form the species [L+H]+ gave fragment ion at m/z
389. All these fragmentation patterns are well observed in the ESI-mass spectrum.
Electronic spectral and magnetic studies
The octahedral CoII complexes exhibits absorption bands in the region 8000-10000 cm-1 and
18000-20000 cm-1 corresponding to 4T1g (F) → 4T2g (F) (ν1), 4T1g (F) → 4T1g (P) (ν3)
transitions respectively. In the present study, the CoII complexes exhibited two distinct absorption
bands in the region 9025-9900 and 19230-20161 cm-1 corresponding to ν1 and ν3 transitions
respectively suggesting high spin octahedral geometry around the CoII ion.36-37 The ν2 band that
involves two-electron transition was not observed in spectra because of its proximity to strong ν3
transition. The CoII complexes exhibited magnetic moment values µeff 4.66-4.25BM; it suggests an
octahedral geometry, further supporting the electronic spectral results.
The spectrum of NiII complexes exhibited three bands at 9810-9826, 15847-15562 and
3
3
3
25110-26544 cm-1 attributed to the 3A2g → T2g (ν1), 3A2g → T1g (F) (ν2) and 3A2g → T1g (P) (ν3)
transitions respectively. This suggests an octahedral geometry for the present NiII complexes. The
calculated ligand field parameters are given in Table 3. The values of ν2/ν1 are found to be 1.615
&1.584 (Complex 3 & 4 ) and the calculated ꢀeff value is around 3.194-3.193BM.
The values of the nephelauxetic parameters and β, indicate low covalent character of the
metal-ligand σ bonds.38-39 The magnetic measurement of NiII complexes showed the ꢀeff value 3.12
and 2.90 BM which are within the range of 2.80-3.50 BM, suggesting the octahedral environment.
Hence, the ligand field parameters correlate the electronic spectral and magnetic properties.
The electronic spectrum of CuII complexes showed the absorption band in the region 14044-
2
14192 cm-1, which is attributed to T2g ꢀ2Eg transition indicative of distorted octahedral

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geometry40-41 around CuII ion. The magnetic moment value observed for CuII complexes lies
slightly above the spin-only value 1.73 BM. This offers possibility of distorted octahedral geometry
around the CuII ion in line with the electronic spectral studies.42
Table 3. Ligand Field Parameters of NiII Complexes
Complex
No.
Transitions (cm-1)
ν1 ν2 ν3
ν2
Calcd.
cm-1
%
µeff
Distortion
BM
Dq
cm-1
B1 cm-1
ν2/ ν1 LFSE
β
βº %
Calcd.
Ni(C22H18N3O4)2 9810 15847 26544 15925 981.00 869.27
Ni(C21H15N3O4)2 9826 15562 25110 15605 985.60 749.18
0.497
0.280
1.615 33.634 3.194 0.823 17.68
1.584 33.689 3.193 0.709 29.05
ESR spectrum of CuII (5) complex
The ESR spectrum of representative CuII (5) (Figure S9) complex has been done to
characterize the metal ion environment. One intense absorption band in the high field region was
observed at RT which is isotropic due to the tumbling motion of the molecules (Supplementary file
Figure S4). The g|| is 2.131 and g⊥ is 2.016. The trend g||> g⊥> 2.0023 indicates that, the unpaired
2
2
electron is localized in the dx –y orbital of the CuII ion which is characteristic for the axial
symmetry.43 The gav was calculated to be 2.055. Hence, the conclusion obtained here further
ascertain that, the CuII complexes possess distorted octahedral geometry as it is observed in case of
electronic and magnetic studies.
Thermal studies of metal complexes
The thermal behaviour of representative CoII (1) (Figure S10), NiII (3) and CuII (5)
complexes gives an useful insight into coordination of water molecules. The TG/DTA curve of CoII
(1) complex showed that, the two coumarin and two antipyrine moieties were lost in the region 215-
O
245, 310- 345, 350-395 and 420-475 C respectively. Finally, the formation of metal oxide took
O
place above 500 C. The NiII (3) complex looses all its coumarin and antipyrine moieties in the
region 240-265, 330-340, 350-385 and 430- 475 OC respectively. The formation of metal oxide took

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O
place at 520 C. In case of CuII (5) complex, the loss of coumarin and antipyrine moieties were
O
found to be in the 220-240, 315- 345, 355-375 and 440-460 C region. Finally, the metal oxide
O
formed at 515 C. Hence, TG/DTA curves do not give any evidence for the coordination of water
molecules. This supports the bonding mode of Schiff bases to metal ions obtained from IR
inferences.
Biological application studies
In vitro antibacterial and antifungal activity
Table 4 & 5 represents the antimicrobial activity results of Schiff bases and their metal
complexes. The minimum inhibitory concentration (MIC) of some selected compounds, which
showed significant activity against selected bacterial and fungi strains were studied. The results
indicated that, these compounds were most active in inhibiting the growth of the tested organisms at
100, 50 & 25 mg mL-1.
In case of bacteriological and antifungal studies, the results were compared with the
standard drugs Gentamycine and Flucanazole respectively. It was observed that, SB-I is highly
active against P. aeruginosa,and A. niger whereas SB-II shown activity against A. flavus and A.
niger fungi. Both the Schiff bases showed these activities at a conc. level of 100 and 50 mL-1.
In case of antibacterial activity it was observed that, the CoII, NiII and CuII complexes show
good activity towards some organisms. It is evident from the results that all the metal complexes
possess higher antimicrobial activity than the Schiff bases against E. coli and S. aureus. In
particular CoII (1) and NiII (3) shown very promising activity. In case of antifungal activity, the
metal complexes of CoII and NiII show high activity towards Cladosporium and A. flavus and
against A. niger also in some cases.CuII complexes exhibited good activity against A. niger. The
higher antimicrobial activity of the metal complexes compared to Schiff bases may be regarded as
the change in structure because of coordination bond formation. Coordination reduces the polarity
of the metal ion by partial sharing of its positive charge with the donor groups within the chelate

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ring system. Chelating tends to make metal complexes act as more powerful and potent
bactereostatic agents, thus inhibiting the growth of the microorganisms.44-45 This process increases
the lipophilicity of the central metal ion, favouring its permeation through the lipid layer of the
microorganism, thus destroying them more aggressively.
Table 4. Antimicrobial Results of SB-I and its metal complexes
Antibacterial Activity
Antifungal Activity
(Zone of inhibition in %)
(Zone of inhibition in %)
Conc.
Compd.
SB-I
(ꢀg mL-1)
S.
Cladosp
orium
E.c
oli
P.aeruginosa
aureus
A. flavus
A. niger
100
50
25
20
22
56
33
26
39
23
21
25
52
42
59
83
50
82
90
75
66
72
53
64
71
51
76
32
42
36
58
39
71
79
52
82
89
81
35
57
40
70
83
55
83
90
85
76
71
65
65
80
50
46
44
35
25
1
100
50
25
3
5
100
50
25
100
50
55
50
55
47
69
50
52
41
54
46
81
87
25
45
50
56
48
48
80
*standard
100
100
100
100
100
100
100
* Gentamycine for Bacteria
*Fluconozole for Fungi.

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Table 5. Antimicrobial results of SB -II and its metal Complexes.
Antibacterial Activity
Antifungal Activity
(Zone of inhibition in %)
(Zone of inhibition in %)
Conc.
Compd.
(ꢀg mL-1)
Cladosp
orium
A.
flavus
E.
coli
S. aureus P.aeruginosa
A. niger
100
50
13
35
23
33
20
15
41
45
52
72
67
51
100
23
11
09
56
51
68
55
43
36
63
58
26
100
20
12
09
54
55
60
51
45
35
65
57
25
100
10
38
21
38
21
13
42
46
51
71
68
50
100
75
62
51
66
60
54
85
80
75
72
71
45
100
74
70
63
50
14
10
47
40
-
SB-II
25
100
50
2
4
6
25
100
50
25
100
50
88
69
36
100
25
100
*Standar
ds
* Gentamycine for Bacteria
* Fluconozole for Fungi
DNA cleavage activity
DNA-binding studies helps in the rational design and construction of new and more efficient
drugs targeted to DNA.46 The SB-I and its complexes were studied for their DNA cleavage activity
against DNA of E. Coli. From Figure 3 it can be seen that, the SB-I and their metal complexes have
acted on DNA as there was difference in molecular marker between the control and the treated
DNA samples.
The difference was observed in the lanes D1–D4 compared to the control DNA of E.Coli.
This infers that, Schiff base and its complexes shown DNA cleavage whereas the control DNA

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alone does not. Although the nature of reactive intermediates involved in the DNA cleavage is not
clear, results gives hint on the role of synthesized compounds on isolated DNA cleavage. Thus, we
can draw a conclusion that the compounds inhibits the growth of the pathogenic organism by
cleaving the genome.
Marker: Standard Molecular weight Marker; Control- DNA of E. coli; Lane-1: E. coli DNA treated
with Schiff base I; Lane-2: E. coli DNA treated with CoII (1); Lane D3: E. coli DNA treated with
NiII (3); Lane D4: E. coli DNA treated with CuII (5).
Figure 3. DNA Cleavage on Genomic DNA of E.coli.
Anthelmintic Activity
Table 6 represents the results of anthelmintic studies on adult Indian earthworms, Pheretima
posthuma, are tabulated in. It was selected due to its anatomical and physiological resemblance with
the intestinal roundworm parasite of human beings.
Figure 4 represents the results of anthelmintic activity of the synthesized compounds. Some
of the synthesized compounds exhibited good anthelmintic activity. The Schiff bases showed
pronounced activity. The CoII and NiII metal complexes have exhibited promising results. The
presented compounds showed interesting anthelmintic activity which can be potentially used for in-
vivo studies as in comparison with standard drug used.

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Figure 4. Anthelmintic Activity of Schiff bases and their metal complexes

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Table 6. Anthelmintic Activity of the Schiff Bases & CoII, NiII and CuII Complexes.
Time TakenforParalysis and Death of Worms
Ligand / Complexes
Paralysis (min)
Death (min)
16
SB-I
10
8
16
13
SB-II
1
2
3
4
5
6
7
9
10
13
7
12
15
16
10
10
8
Blank
Std(Albendazole)
No effects for 10 hours
10
17
Conclusion
The Schiff bases SB-I & SB-II acted as versatile tridentate ligands with ONO donor
sequence. The bonding mode of ligands to metal ion was deduced by IR studies with the support of
mass spectroscopic and TG/DTA studies. The octahedral geometry was corroborated with the
electronic, magnetic and ESR spectroscopic studies in which metal ion is coordinated through
carbonyl oxygen, azomethine nitrogen and phenolic oxygen atoms via deprotonation. In-vitro
studies of some of the synthesized compounds showed promising antimicrobial, DNA cleavage and
anthelmintic results.
Acknowledgements
The authors, Ajaykumar D. Kulkarni and Gangadhar B. Bagihalli are thankful to the
principal and management of MIT Academy of Engineering pune, K.L.E. Institute of Technology,
Hubballi for their support. Authors express their thanks to USIC, KUD for spectral analysis
support.

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Supplementary Information
Bio-important antipyrine derived Schiff bases and their transition metal
complexes: Synthesis, Spectroscopic characterization, Antimicrobial,
Anthelmintic and DNA cleavage investigation
Manjunath Ma, Ajaykumar D. Kulkarnib*, Gangadhar B. Bagihallic*, Shridhar Malladid,
Sangamesh A. Patile
aDepartment of Chemistry, CMR Institute of Technology, Bangalore, 560037. Karnataka, India
bDepartment of Applied Sciences, MIT Academy of Engineering (An Autonomous Institute Affiliated
to Savitribai Phule Pune University), Dehu Phata, Alandi (D), Pune-412105, India
cDepartment of Chemistry, K.L.E. Institute of Technology, Gokul Hubballi 580030. Karnataka,
India
dDepartment of Chemistry, KLS’s Gogte Institute of Technology, Udyambag, Belagavi-590006.
Karnataka, India.
eP.G. Department of Chemistry, Karnatak University, Pawate Nagar, Dharwad 580 003.
Karnataka, India
Table of Contents.
Figure 1. Synthesis scheme and structure of Schiff Bases SB-I & SB-II
Figure 2. Structure of Metal (II) Complexes
Figure 3. DNA Cleavage on Genomic DNA of E.coli.

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Figure 4. Anthelmintic Activity of Schiff bases and their metal complexes.
SB-I. Schiff base I derived from 8-formyl-7-hydroxy-4- methylcoumarin and 4-aminoantipyrine
SB-II. Schiff base I derived from 5-formyl-6-hydroxycoumarin and 4-aminoantipyrine
Table 1 Elemental analyses of Schiff bases and their Co(II), Ni(II) and Cu(II) complexes along
with molar conductance and magnetic moment data.
Table 2 The important infrared frequencies (in cm−1) of Schiff bases and their metal complexes.
Table 3. Ligand Field Parameters of Ni(II) Complexes.
Table 4. Antimicrobial Results of SB-I and its metal complexes
Table 5. Antimicrobial results of SB -II and its metal Complexes.
Table 6. Anthelmintic Activity of the Schiff Bases & CoII, NiII and CuII Complexes.
Spectroscopic information
SB-I: IR (KBr) ν/cm-11609, 1715, 1735, 1309; 1H NMR (300 MHz, DMSO-d6) δ 11.7
(-
13
OH), 9.44 (-HC=N), 2.4 (-CH3 of coumarin), 1.6 and 1.8 (-CH3of pyrazoline); 6.3-7.9 (Ar-H); C
NMR 117.6-156.7 (Ar-C), 142 & 152 (pyrazoline ring C), 22-19 (Methyl C), 144.2 (C=N), 160.0-
162.2 (C=O); Mass m/z,389.
δ
1
SB-II: IR (KBr) ν/cm-11615, 1710, 1720, 1316; H NMR (300MHz, DMSO-d6) δ 14.3
13
(-OH), 9.1 (-HC=N), 1.5 and 1.8 (-CH3of pyrazoline); 6.1-7.7(Ar-H); C NMR
δ
117.6-156.7 (Ar-
C), 142 & 152 (pyrazoline ring C), 22-19 (Methyl C), 145.2 (C=N), 162.2-169.7(C=O); Mass
m/z,374.
Complex 1: IR (KBr) ν/cm-11735, 1665, 1560, 1355, 472, 379; UV-Vis (DMSO) cm-1 9025,
19230; ESI-Mass m/z, M+ at 835.
Complex 2: IR (KBr) ν/cm-11720, 1660, 1562, 1350, 476, 376; UV-Vis (DMSO) cm-1 9900,
20161.
Complex 3: IR (KBr) ν/cm-11735, 1650, 1565, 1350, 480, 370; UV-Vis (DMSO) cm-1 9810,

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15847,26544; ESI-Mass m/z, M+ at 834.
Complex 4: IR (KBr) ν/cm-11720, 1662, 1561, 1370, 485, 386; UV-Vis (DMSO) cm-1 9826,
15562, 25110.
Complex 5: IR (KBr) ν/cm-11735, 1648, 1566, 1380, 475, 383; UV-Vis (DMSO)
cm-114044.
Complex 6: IR (KBr) ν/cm-11720, 1650, 1563, 1385, 473, 384; UV-Vis (DMSO)
cm-114192.
Figure S1: IR (KBr) spectrum of Schiff base I

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Figure S2: IR (KBr) spectrum of CoII complex 1.
Figure S3: IR (KBr) spectrum of NiII complex 3.

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Figure S4: 1H NMR (300 MHz, DMSO-d6) spectrum of Schiff base I
Figure S5: 13C NMR (300 MHz, DMSO-d6) spectrum of Schiff base I

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Figure S6. Mass Spectrum of Schiff Base SBI.
Figure S7. ESI mass Spectrum of CoII Complex (1)

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Figure S8. ESI mass Spectrum of NiII Complex (3)
Figure S9. ESR Spectrum of CuII Complex (
5) at RT

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Figure S10: TG/DTA curve of CoII complex (1)
Figure S1. IR (KBr) spectrum of Schiff base I
Figure S2. IR (KBr) spectrum of CoII complex (1).
Figure S3. IR (KBr) spectrum of NiII complex (3).
Figure S4. 1H NMR (300 MHz, DMSO-d6) spectrum of Schiff base I
Figure S5. 13C NMR (300 MHz, DMSO-d6) spectrum of Schiff base I
Figure S6. Mass Spectrum of Schiff Base SBI.
Figure S7. ESI mass Spectrum of CoII Complex (1)
Figure S8. ESI mass Spectrum of NiII Complex (3)
Figure S9. ESR Spectrum of CuII Complex (5) at RT
Figure S10. TG/DTA curve of CoII complex (1)