Exploring DNA binding and nucleolytic activity of few 4-aminoantipyrine based amino acid Schiff base complexes: A comparative approach

Article abstract of DOI:10.1016/j.saa.2014.01.108

A series of novel Co(II), Cu(II), Ni(II) and Zn(II) complexes were synthesized from Schiff base(s), obtained by the condensation of 4-aminoantipyrine with furfural and amino acid (glycine(L1)/alanine(L2)/ valine(L3)) and respective metal(II) chloride. The

Full text of DOI:10.1016/j.saa.2014.01.108

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 404–413
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Exploring DNA binding and nucleolytic activity of few
4-aminoantipyrine based amino acid Schiff base complexes:
A comparative approach
⇑
N. Raman , A. Sakthivel, N. Pravin
Research Department of Chemistry, VHNSN College, Virudhunagar 626 001, Tamil Nadu, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
4-Aminoantipyrine derived Schiff bases and their metal complexes are valuable in designing novel agents
for targeting nucleic acids as well as setting the stage for the synthesis of chemical anticarcinogenic
drugs.
ꢀ
ꢀ
Designing and synthesis of effective
chemical nucleases.
4-Aminoantipyrine based metal
complexes are superior DNA binding
agents.
ꢀ
ꢀ
Valine Schiff base complexes are
better intercalators.
Synthesis of better antimicrobial
agents against pathogens.
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel Co(II), Cu(II), Ni(II) and Zn(II) complexes were synthesized from Schiff base(s), obtained
by the condensation of 4-aminoantipyrine with furfural and amino acid (glycine(L1)/alanine(L2)/vali-
ne(L3)) and respective metal(II) chloride. Their structural features and other properties were explored
from the analytical and spectral methods. The binding behaviors of the complexes to calf thymus DNA
were investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The intrinsic
binding constants for the above synthesized complexes are found to be in the order of 102 to 105 indicat-
ing that most of the synthesized complexes are good intercalators. The binding constant values (Kb)
clearly indicate that valine Schiff-base complexes have more intercalating ability than alanine and gly-
cine Schiff-base complexes. The results indicate that the complexes bind to DNA through intercalation
and act as efficient cleaving agents. The in vitro antibacterial and antifungal assay indicates that these
complexes are good antimicrobial agents against various pathogens. The IC50 values of [Ni(L1)2] and
[Zn(L1)2] complexes imply that these complexes have preferable ability to scavenge hydroxyl radical.
Ó 2014 Elsevier B.V. All rights reserved.
Received 28 October 2013
Received in revised form 20 January 2014
Accepted 22 January 2014
Available online 6 February 2014
Keywords:
4-Aminoantipyrine
Schiff-base complexes
Calf thymus DNA
Antimicrobial studies
Intercalative mode
⇑
Corresponding author. Tel.: +91 092451 65958; fax: +91 4562 281338.
E-mail address: ramchem1964@gmail.com (N. Raman).
http://dx.doi.org/10.1016/j.saa.2014.01.108
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 404–413
405
solution both at room temperature (300 K) and liquid nitrogen tem-
perature (77 K) using TCNE (tetracyanoethylene) as the g-marker.
The absorption spectra were recorded by using Shimadzu model
UV-1601 spectrophotometer at room temperature.
Introduction
The increasing microbial resistance to antibiotics is a very
important necessity which inculcates the search for new com-
pounds with potential effects against pathogenic bacteria and fun-
gi [1,2]. The most spectacular advances in medicinal chemistry
have been made when heterocyclic compounds played an impor-
tant role in regulating biological activities. Heterocyclic moieties
can be found in a large number of compounds which display bio-
logical activity [3]. Antipyrine (N-heterocyclic compound) and its
derivatives exhibit a wide range of biological activities and applica-
tions. Because of their interesting structural features as well as the
biological activity, a wide range of metal complexes derived from
antipyrine derivatives have been reported [4,5]. Pyrazolone-based
ligands display variable complexing behavior and a variety of coor-
dination possibilities to metal centers.
The coordination chemistry of Schiff base complexes involving
oxygen and nitrogen donor ligands has excited great interest
among chemists in recent years due to their applications in catal-
ysis and their relevance to bio-inorganic systems [6]. Inorganic
complexes can be used in foot-printing studies, as sequence spe-
cific DNA binding agents, as diagnostic agents in medicinal applica-
Synthesis of Schiff base ligands
An ethanolic solution of 4-aminoantipyrine (0.02 M) was added
to an ethanolic solution of furfural (0.02 mol) and amino acid (gly-
cine (L1)/alanine (L2)/valine (L2) (0.02 M)) which was dissolved in
alcoholic potash. The resultant mixture was refluxed for 4 h. The
solid product formed was filtered and recrystallized from ethanol.
L1. Yield: 74%. M.F. C18H17N4O3. IR (KBr pellet, cmꢁ1): 1652
m
(AC@N); 1602
m
(AHC@N); 3435, 1427, 1390
m
((ACOOꢁ),
(ACOOꢁ) asy, (ACOOꢁ)
m
m
sy). 1H NMR (d): (aromatic) 6.6–7.1 (m);
(COOH) 11(s); (CACH3) 2.3 (s); (NACH3) 3.1(s); (CH@N) 9.3(s).
MS m/z (%): 338 [M + 1]+. kmax (cmꢁ1) in DMF, 29,240, 37,075.
L2. Yield: 70%. M.F. C19H19N4O3. IR (KBr pellet, cmꢁ1): 1650
m
(AC@N); 1602
m
(AHC@N); 3431, 1427, 1389
m
((ACOOꢁ),
(ACOOꢁ) asy, (ACOOꢁ)
m
m
sy). 1H NMR (d): (aromatic) 6.6–7.4 (m);
(COOH) 11(s); (CACH3) 2.4 (s); (NACH3) 3.3 (s); (CH@N) 9.3(s).
MS m/z (%): 352 [M + 1]+. kmax (cmꢁ1) in DMF, 29,411, 35,335.
L3. Yield: 76%. M.F. C21H23N4O3. IR (KBr pellet, cmꢁ1): 1652
tions, and for genomic research.
A large number of metal
complexes have been used as cleavage agents for DNA and also
for novel potential DNA-targeted antitumor drugs. This is essential
for further expected applications in many areas like biological and
medicinal significance as potential artificial gene regulators (or)
cancer chemotherapeutic agents [7].
m
(AC@N); 1602
m
(AHC@N); 3441, 1419, 1390
m
((ACOOꢁ),
(ACOOꢁ) asy, (ACOOꢁ)
m
m
sy). 1H NMR (d): (aromatic) 6.5–7.3 (m);
(COOH) 11(s); (CACH3) 2.3 (s); (NACH3) 3.2 (s); (CH@N) 9.3(s).
MS m/z (%): 380 [M + 1]+. kmax (cmꢁ1) in DMF, 29,450, 35,575.
In view of biological importance of Schiff base derived from the
condensation of 4-aminoantipyrine with furfural and amino acids
(glycine/alanine/valine), its applications in various fields, in the
present investigation it is thought to synthesize the metal com-
plexes with transition metal ions such as Co(II), Ni(II), Cu(II) and
Zn(II). It is therefore of interest to carryout investigations to under-
stand how a ligand environment could affect the redox properties
of the central metal and thereby the spectral properties and also
interested to explore the DNA binding and DNA cleavage activity
of synthesized complexes. This result would be helpful for under-
standing the binding mode of the complex to DNA and it also lays
a foundation for developing new useful DNA probes and effective
inorganic complex nucleases.
Synthesis of metal complexes
A solution of metal(II) chloride in ethanol (0.02 M) was mixed
with an ethanolic solution of the Schiff base (0.04 M), and the
resultant mixture was refluxed for 3 h. The solid complex precipi-
tated was filtered off and washed thoroughly with ethanol and
petroleum ether and dried over anhydrous CaCl2 under vacuum.
Schematic diagram for the synthesis of ligand and metal complexes
is given in Scheme 1.
[Cu(L1)2]. Yield: 69%. M.F. C36H34N8O6Cu. IR (KBr pellet, cmꢁ1):
1647
m(AC@N); 1593 m(AHC@N); 3417, 1417, 1379 m
((ACOOꢁ),
(ACOOꢁ)
m
asy, (ACOOꢁ)
msy); 501 m(MAO); 449 m(MAN). MS m/z
(%): 739 [M + 1]+. kmax (cmꢁ1) in DMF, 11,050, 37,453.
[Cu(L2)2]. Yield: 72%. M.F.C38H38N8O6Cu. IR (KBr pellet, cmꢁ1):
Experimental
1645
m(AC@N); 1593 m(AHC@N); 3429, 1419, 1378 m
((ACOOꢁ),
(ACOOꢁ)
m
asy, (ACOOꢁ)
msy); 500 m(MAO); 435 m(MAN). MS m/z
Reagents and instruments
(%): 767 [M + 1]+. kmax (cmꢁ1) in DMF, 11,049, 37,878.
[Cu(L3)2]. Yield: 68%. M.F. C42H46N8O6Cu. IR (KBr pellet, cmꢁ1):
All reagents, 4-aminoantipyrine, furfural, glycine, alanine, valine
and metal(II) chlorides were of Merck products and they were used
as supplied. Commercial solvents were distilled and then used for
the preparation of ligands and their complexes. DNA was purchased
from Bangalore Genei (India). Microanalyses (C, H and N) were per-
formed in Carlo Erba 1108 analyzer at Sophisticated Analytical
Instrument Facility (SAIF), Central Drug Research Institute (CDRI),
Lucknow, India. Molar conductivities in DMF (10ꢁ3 M) at room tem-
perature were measured by using Systronic model-304 digital con-
ductivity meter. Magnetic susceptibility measurement of the
complexes was carried out by Gouy balance using copper sulfate
pentahydrate as the calibrant. Infrared spectra (4000–350 cmꢁ1
KBr disc) of the samples were recorded on an IR Affinity-1 FT-IR
Shimadzu spectrophotometer. NMR spectra were recorded on a
Bruker Avance Dry 300 FT-NMR spectrometer in CDCl3, using TMS
as the internal reference. Mass spectrometry experiments were per-
1647
m(AC@N); 1593 m(AHC@N); 3417, 1417, 1379 m
((ACOOꢁ),
(ACOOꢁ)
m
asy, (ACOOꢁ)
msy); 501 m(MAO); 449 m(MAN). MS m/z
(%): 823 [M + 1]+. kmax (cmꢁ1) in DMF, 12,254, 29,586.
[Co(L1)2]. Yield: 63%. M.F. C36H34N8O6Co. IR (KBr pellet, cmꢁ1):
1645
m(AC@N); 1593 m(AHC@N); 3408, 1415, 1380 m
((ACOOꢁ),
(ACOOꢁ)
m
asy, (ACOOꢁ)
msy); 503 m(MAO); 437 m(MAN). MS m/z
(%): 734 [M + 1]+. kmax (cmꢁ1) in DMF, 11,025, 14,903, 29,280.
[Co(L2)2]. Yield: 83%. M.F. C38H38N8O6Co. IR (KBr pellet, cmꢁ1):
1645
m(AC@N); 1597 m(AHC@N); 3429, 1419, 1382 m
((ACOOꢁ),
(ACOOꢁ)
m
asy, (ACOOꢁ)
msy); 500 m(MAO); 428 m(MAN). MS m/z
(%): 914 [M + 1]+. kmax (cmꢁ1) in DMF, 12,150, 13,774, 29,325.
[Co(L3)2]. Yield: 78%. M.F. C42H46N8O6Co. IR (KBr pellet, cmꢁ1):
1647
m(AC@N); 1597 m(AHC@N); 1417, 1379 m ,
((ACOOꢁ) masy
(ACOOꢁ)
msy); 570 m(MAO); 435 m(MAN). MS m/z (%): 818
[M + 1]+. kmax (cmꢁ1) in DMF, 11,049, 12,500, 29,761.
[Ni(L1)2]. Yield: 59%. M.F. C36H34N8O6Ni. IR (KBr pellet, cmꢁ1):
formed on
a
JEOL-AccuTOF JMS-T100LC mass spectrometer
1645
(ACOOꢁ)
(%): 734 [M + 1]+. kmax (cmꢁ1) in DMF, 11,695, 12,820, 24,937.
m
(AC@N); 1591
m
m
(AHC@N); 3410, 1409, 1382
m
((ACOOꢁ),
equipped with a custom-made electrospray interface (ESI). EPR
spectra were recorded on a Varian E 112 EPR spectrometer in DMSO
asy, (ACOOꢁ)
m
sy); 503 (MAO); 433 (MAN). MS m/z
m
m

406
N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 404–413
Scheme 1. Schematic route for the synthesis of Schiff base ligands and their metal complexes.
[Ni(L2)2]. Yield: 79%. M.F. C38H38N8O6Ni. IR (KBr pellet, cmꢁ1):
1637 (AC@N); 1595 (AHC@N); 1419, 1381
((ACOOꢁ) masy
(ACOOꢁ)
(CH@N) 9.6(s). MS m/z (%): 741 [M + 1]+. kmax (cmꢁ1) in DMF,
29,411, 35,842.
[Zn(L2)2]. Yield: 72%. M.F. C38H38N8O6Zn. IR (KBr pellet, cmꢁ1):
1647 (AC@N); 1597 (AHC@N); 1419, 1381
((ACOOꢁ) masy
(ACOOꢁ) (MAN). 1H NMR (d): (aromatic)
sy); 501 (MAO); 435
m
m
m
,
m
sy); 501 m(MAO); 435 m(MAN). MS m/z (%): 762
[M + 1]+. kmax (cmꢁ1) in DMF, 11,049, 12,468, 29,411.
m
m
m
,
[Ni(L3)2]. Yield: 63%. M.F. C42H46N8O6Ni. IR (KBr pellet, cmꢁ1):
m
m
m
1647
m
(AC@N); 1587
m
(AHC@N); 1415, 1380
m
((ACOOꢁ) masy
,
6.6–7.3 (m); (CACH3) 2.3 (s); (NACH3) 3.1(s); (CH@N) 9.6(s). MS
m/z (%): 769 [M + 1]+. kmax (cmꢁ1) in DMF, 29,498, 35,460.
[Zn(L3)2]. Yield: 82%. M.F. C42H46N8O6Zn. IR (KBr pellet, cmꢁ1):
(ACOOꢁ)
m
sy); 584 m(MAO); 420 m(MAN). MS m/z (%): 734
[M + 1]+. kmax (cmꢁ1) in DMF, 11,061, 12,642, 29,498.
[Zn(L1)2]. Yield: 72%. M.F. C36H34N8O6Zn. IR (KBr pellet, cmꢁ1):
1645
m
(AC@N); 1587
m
(AHC@N); 1416, 1386
m ,
((ACOOꢁ) masy
1647
(ACOOꢁ)
(d): (aromatic) 6.6–7.1 (m); (CACH3) 2.3 (s); (NACH3) 3.1(s);
m(AC@N); 1595
m(AHC@N); 3410, 1411, 1382
m
((ACOOꢁ),
(ACOOꢁ)
m
sy); 542 (MAO); 420 m
m
(MAN). 1H NMR (d): (aromatic)
m
asy, (ACOOꢁ)
m
sy); 501 (MAO); 433
m
m
(MAN). 1H NMR
6.6–7.4 (m); (CACH3) 2.3 (s); (NACH3) 3.1(s); (CH@N) 9.6(s). MS
m/z (%): 825 [M + 1]+. kmax (cmꢁ1) in DMF, 29,325, 35,971.

N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 404–413
407
DNA binding experiments
performed at 40 V for each hour in Tris–Acetate-EDTA (TAE) buffer
using 1% agarose gel containing 1.0 g/mL ethidium bromide. The
l
The interaction between metal complexes and DNA was studied
by electronic absorption, viscosity and electrochemical methods.
Disodium salt of calf thymus DNA was stored at 4 °C. All the exper-
iments involving the interaction of the complexes with calf thymus
(CT) DNA were carried out in Tris–HCl buffer (50 mM Tris–HCl, pH
7.2) containing 5% DMF at room temperature. Solution of CT DNA
in the buffer gave a ratio of UV absorbance at 260 and 280 nm,
cleavage efficiency was measured by determining the ability of the
complex to convert the super coiled (SC) DNA to nicked circular
form (NC) and linear form (LC). Inhibition reactions were carried
out by prior incubation of the pUC19 DNA (10 mM) with DMSO
(4 lL).
A
260/A280 of ca1.89:1, indicating that the CT DNA was sufficiently
Antimicrobial studies
free from protein [8]. The concentration of DNA was measured
by using its extinction coefficient at 260 nm (6600 Mꢁ1cmꢁ1) after
1:100 dilution. Stock solutions were stored at 4 °C and used not
more than 4 days. Doubly distilled water was used to prepare solu-
tions. Concentrated stock solutions of the complexes were pre-
pared by dissolving the complexes in DMF and diluting properly
with the corresponding buffer to the required concentration for
all the experiments.
The electronic spectra of the complexes were recorded before
and after the addition of DNA in the presence of 5 mM Tris–HCl/
50 mM NaCl buffer (pH 7.2). The intrinsic binding constant for
the interaction of complex with DNA was obtained from absorp-
Antibacterial activity
National Committee for Clinical Laboratory Standard (NCCLS)
approved standard nutrient agar was used as medium for testing
the activity of microorganisms as antibacterial agents [11]. For pre-
paring the agar media, 3 g of beef extract, 5 g of peptone, 5 g of
yeast extract and 5 g of sodium chloride were dissolved in
1000 mL of distilled water in a clean conical flask. The pH of the
solution was maintained at 7. The solution was boiled to dissolve
the medium completely and sterilized by autoclaving at 15 lbs
pressure (120 °C) for 30 min. After sterilization, 20 mL of media
was poured into the sterilized petri plates. These petri plates were
kept at room temperature for sometime. After few minutes the
medium got solidified in the plates. Then, it was inoculated with
microorganisms using sterile swabs. The stock solutions were pre-
pared by dissolving the compounds in appropriate solvents.
In a typical procedure [8], the antibacterial activities of the
compounds were evaluated by the disc diffusion method against
the bacterial microorganisms. The 5 mm diameter and 1 mm thick-
ness of the disc was filled with the test solutions using a micropi-
pette and the plates were incubated at 37 °C for 24 h. During this
period, the test solution was diffused and affected the growth of
the inoculated bacteria. The zone of inhibition, developed on the
plate was measured.
tion data. A fixed concentration value of complex (10
titrated with increasing amounts of DNA over the range of
20–150 M. The equilibrium binding constant (Kb) values for the
lM) was
l
interaction of the complexes with DNA were obtained from the
absorption spectral titration data using the following equation:
½DNAꢂ=ðea
ꢁ
ef Þ ¼ ½DNA=ðeb
ꢁ
ef Þꢂ þ 1=Kbðeb
ꢁ
ef
Þ
where ea the extinction coefficient observed for the charge transfer
absorption at a given DNA concentration, ef, the extinction coeffi-
cient of the complex free in solution, eb, the extinction coefficient
of the complex when fully bound to DNA, Kb, the intrinsic binding
constant and [DNA], the concentration in nucleotides. A plot of
[DNA]/(ea ꢁ ef) versus [DNA], gives Kb as the ratio of the slope to
the intercept.
Cyclic voltammetry studies were performed on a CHI 620C elec-
trochemical analyzer with three electrode system of glassy carbon
as the working electrode, a platinum wire as auxiliary electrode
and Ag/AgCl as the reference electrode. Solutions were deoxygen-
ated by purging with N2 prior to measurements. Viscosity experi-
ments were carried on an Ostwald viscometer, immersed in a
thermostated water-bath maintained at a constant temperature
at 30.0 0.1 °C. CT DNA samples of approximately 0.5 mM were
prepared by sonicating in order to minimize complexities arising
from CT DNA flexibility [9]. Flow time was measured with a digital
stopwatch three times for each sample and an average flow time
Antifungal activity
NCCLS approved standard potato dextrose agar was used as
medium for antifungal activity by disc diffusion method [8]. For
preparing the agar media, 200 g of potato extract, 20 g of agar
and 20 g of dextrose were dissolved in 1000 mL of distilled water
in a clean conical flask. The solution was boiled to dissolve the
medium completely and sterilized by autoclaving at 15 lbs pres-
sure (120 °C) for 30 min. After sterilization, 20 mL of media was
poured into the sterilized petri plates. These petri plates were kept
at room temperature for sometime. After few minutes the medium
got solidified in the plates. Then, it was inoculated with microor-
ganisms using sterile swabs. In a typical procedure [8], the anti-
fungal activities of the compounds were evaluated by the disc
diffusion method against the fungal microorganisms. The 5 mm
diameter and 1 mm thickness of the disc was filled with the test
solution using a micropipette and the plates were incubated at
37 °C for 72 h. During this period, the test solution was diffused
and affected the growth of the inoculated fungi. After 36 h of incu-
bation at 37 °C, the diameter of the inhibition was measured. Com-
pounds showing promising antibacterial/antifungal activity were
selected for minimum inhibitory concentration studies. The mini-
mum inhibitory concentration was determined by assaying at con-
centration of compounds along with standards at the same
concentration. Minimum inhibitory concentration (MIC) is the
lowest concentration of an antimicrobial compound, inhibiting
the visible growth of microorganisms after overnight incubation.
Minimum inhibitory concentrations are important in diagnostic
laboratories to confirm resistance of microorganisms to antimicro-
bial agents and also to monitor the activity of new antimicrobial
agents.
was calculated. Data were presented as (
g
/g0)
1/3 versus the concen-
is the viscosity of CT
tration of the metal(II) complexes, where
g
DNA solution in the presence of complex, and g0 is the viscosity
of CT DNA solution in the absence of complex. Viscosity values
were calculated after correcting the flow time of buffer alone (t0),
g
= (t ꢁ t0)/t0 [10].
Interaction with pUC19 plasmid DNA
The extent of pUC19 DNA cleavage in presence of activating
agent AH2 (ascorbic acid), and two radical scavengers DMSO (hy-
droxyl radical scavenger) and NaN3 (singlet oxygen scavenger)
was monitored using agarose gel electrophoresis. In reactions
using super coiled pUC19 plasmid DNA Form I (10
HCl buffer (50 mM) with 50 mM NaCl (pH 7.2) which was treated
with the metal complex and ascorbic acid (10 M) followed by
dilution with the Tris–HCl buffer to a total volume of 20 L. The
lM) in Tris–
l
l
samples were incubated for 1 h at 37 °C. A loading buffer contain-
ing 0.25% bromophenol blue was added and electrophoresis was

408
N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 404–413
Antioxidant property
spectrum of [Cu(L1)2]+Å complex shows molecular ion peak at m/z
739[M + 1] which is equivalent to its molecular weight. The
[Cu(L1)2] complex gives fragment ion peaks at m/z 681, 617,
537, 508, 493 and 416 corresponding to [C34H32N8O4Cu]+Å,
[C34H32N8O4]+Å, [C29H28N8O3]+Å, [C28H25N7O3]+Å, [C27H22N7O3]+Å and
[C21H17N7O3]+Å ions respectively. The mass spectrum of Schiff base
ligand (L2) shows molecular ion peak at m/z 352 for [C19H19N4O3]+Å
and its Co(II) complex shows molecular ion peak at m/z 763 [M + 1]
for the ion [CoC38H38N8O6]+Å and its fragmentation peaks at m/z
are 704, 646, 566, 537, 522, 444 and 363 corresponding to
[C38H38N8O6]+Å, [C36H36N8O4]+Å, [C31H32N8O3]+Å, [C30H29N7O3]+Å,
[C29H26N7O3]+Å, [C23H21N7O3]+Å, and [C18H17N7O2]+Å ions respec-
tively. In the similar manner the mass spectrum of Schiff base
ligand (L3) shows the molecular ion peak at m/z 380 corresponding
to [C21H23N4O3]+Å ion and also the spectrum exhibits peaks for the
fragments at m/z 365, 288, 209 corresponding to [C20H20N4O3]+Å,
[C14H15N4O3]+Å and [C9H11N4O2]+Å ions respectively. The spectrum
of [Zn(L3)2]+Å complex shows molecular ion peak at m/z 825
[M + 1] which is equivalent to its molecular weight. The m/z values
of all the fragments of the ligands and their complexes confirm the
stoichiometry of the complexes as [ML2].
Hydroxyl radicals were generated in aqueous media through
the Fenton-type reaction [12,13]. The aliquots of reaction mixture
(3 mL) contained 1.0 mL of 0.10 mmol aqueous safranin, 1 mL of
1.0 mmol aqueous EDTA–Fe(II), 1 mL of 3% aqueous H2O2, and a
series of quantitative microaddition of solutions of the test com-
pound. A sample without the tested compound was used as the
control. The reaction mixtures were incubated at 37 °C for
30 min in a water bath. The absorbance was then measured at
520 nm. All the tests were run in triplicate and are expressed as
the mean and standard deviation (SD) [14]. The scavenging effect
for OHÅ was calculated from the following expression:
Scavenging ratio ð%Þ ¼ ½ðAi ꢁ AoÞ=ðAc ꢁ AoÞꢂ ꢃ 100%
where Ai is the absorbance in the presence of the test compound; Ao
is the absorbance of the blank in the absence of the test compound;
Ac is the absorbance in the absence of the test compound, EDTA–
Fe(II) and H2O2.
Results and discussion
The Schiff base ligands (L1), (L2) and (L3) and their Co(II), Cu(II),
Ni(II) and Zn(II) complexes have been synthesized and character-
ized by spectral and elemental analysis data. The complexes are
found to be air stable. The ligands are soluble in common organic
solvents and all the complexes are freely soluble in CHCl3, DMF
and DMSO.
Infrared spectra of the ligands and their metal complexes
The coordination mode and sites of the ligands to the metal ions
were investigated by comparing the infrared spectra of the free li-
gands with their metal complexes. The IR spectra of the Schiff base
ligands (L1, L2 and L3) show a band at 1602 cmꢁ1 which is assigned
to azomethine m(CH=N) linkage, originating from amino and car-
bonyl groups of the starting reagents (4-aminoantipyrine and fur-
fural). These bands are shifted towards lower frequencies in the
spectra of their metal complexes (1587–1597 cmꢁ1). The compar-
ison of the IR spectra of the complexes with the above Schiff bases
indicates the involvement of the azomethine nitrogen in chelation
with the metal ion. The coordination of nitrogen to the metal ion
could be expected to reduce the electron density of the azomethine
Elemental analysis and molar conductivity measurements
The results of elemental analysis for the metal complexes are in
good agreement with the calculated values (Table 1) showing that
the complexes have 1:2 metal–ligand stoichiometry of the type
M(L)2 wherein L acts as a tridentate ligand. The metal(II) com-
plexes were dissolved in DMF and the molar conductivities of
10ꢁ3 M of their solution at room temperature were measured.
The lower conductance values (18–34 ohmꢁ1 cmꢁ2 molꢁ1) of the
complexes support their non-electrolytic nature.
link and thus causes a shift in the
m
(CH@N) group. In addition, the li-
(C@N) vibration,
gand also exhibits a band at 1652 cmꢁ1 due to the
m
originating from the condensation of amino group of amino acid
(glycine, alanine and valine) and furfurlyidine-4-aminoantipyrine
which is shifted by 13 cmꢁ1 on complexation. Moreover, the coor-
dination of the ligand to the metal centre via the carboxylic group
can also be obvious from the difference of maxima positions as ob-
Mass spectra
The mass spectrum of Schiff base ligand (L1) shows the molec-
ular ion peak at m/z 338 corresponding to [C18H17N4O3]+Å ion. Also
the spectrum exhibits peaks for the fragments at m/z 294, 280, 265,
236 and 156 corresponding to [C17H17ON4]+Å, [C16H15N4O]+Å,
[C15H12ON4]+Å, [C14H9ON3]+Å and [C9H5N3]+Å respectively. The
ꢁ
served for tsy(COO ) and tasy(COOꢁ). The bands are observed at 1390
and 14ꢁ27 cmꢁ1 respectively, for a free ligand. For comparison, the
ꢁ
tsy(COO
and tasy(COO
are displayed at 1363–1389 and
)
)
Table 1
Analytical and physical data of Schiff base ligands and their metal complexes.
S.No
Compound
Found (calc)%
M
Km (mho cm2 molꢁ1
)
Magnetic moment (BM)
C
H
N
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
L1
–
63.9 (64.1)
53.3 (53.4)
53.6 (53.7)
53.6 (53.8)
53.3 (53.3)
64.8 (64.9)
54.6 (54.5)
54.7 (54.8)
54.8 (54.8)
54.4 (54.7)
66.3 (66.5)
56.4 (56.5)
56.6 (56.8)
56.7 (56.8)
56.2 (56.4)
4.9 (5.1)
4.1 (4.2)
4.1 (4.3)
4.1 (4.3)
4.1 (4.2)
5.4 (5.5)
4.5 (4.6)
4.5 (4.6)
4.5 (4.6)
4.5 (4.6)
6.1 (6.1)
5.1 (5.2)
5.2 (5.2)
5.1 (5.2)
5.1 (5.2)
16.5 (16.6)
13.2 (13.9)
13.8 (13.9)
13.9 (13.9)
13.7 (13.8)
15.8 (15.9)
13.2 (13.4)
13.4 (13.5)
13.3 (13.5)
13.3 (13.4)
14.7 (14.8)
12.5 (12.5)
12.5 (12.6)
12.5 (12.6)
12.4 (12.5)
–
–
1.87
4.94
3.08
Diamagnetic
–
1.85
4.83
3.16
Diamagnetic
–
1.86
4.91
[Cu(L3)2]
[Co(L3)2]
[Ni(L3)2]
[Zn(L3)2]
L2
[Cu(L2)2]
[Co(L2)2]
[Ni(L2)2]
[Zn(L2)2]
L3
7.8 (7.9)
7.3 (7.3)
7.2 (7.3)
7.9 (8.1)
–
7.4 (7.6)
6.9 (7.1)
6.9 (7.1)
7.7 (7.8)
–
23.4
18.8
29.4
21.1
–
18.0
19.1
32.4
20.9
–
[Cu(L3)2]
[Co(L3)2]
[Ni(L3)2]
[Zn(L3)2]
7.1 (7.1)
6.5 (6.6)
6.5 (6.6)
7.2 (7.3)
34.0
31.5
26.5
28.6
3.07
Diamagnetic

N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 404–413
409
1409–1419 cmꢁ1, respectively for all the metal complexes. These
results reveal that the organic ligand is involved in the coordina-
tion through the carboxyl group. Conclusive evidence of the bond-
ing is also shown by the observation that new bands in the spectra
of all metal complexes appearing in the low frequency regions at
indicate the non-coupled mononuclear complexes of diluted d8
system with s = 1 spin state of octahedral geometry [16,17]. Mono-
meric nature of nickel complexes is further proved by their micro
analytical data. The electronic absorption spectra of the diamag-
netic [Zn(L1)2], [Zn(L2)2] and [Zn(L3)2] show that the bands at
29,411, 35,842, 29,498, 35,460, 29,325 and 35,971 cmꢁ1 are
assigned to intraligand charge transfer transitions [18].
500–584 cmꢁ1 and 420–449 cmꢁ1 characteristic to
t(MAO) and
t(MAN) stretching vibrations respectively, that are not observed
in the spectra of free ligands.
EPR spectra
1H NMR spectra
EPR studies of paramagnetic transition metal(II) complexes
yield information about the distribution of the unpaired electrons
and hence about the nature of the bonding between the metal
ion and its ligands. The X-band EPR spectra of all copper(II) com-
plexes were recorded in DMSO at liquid nitrogen temperature
and at room temperature. The spin Hamiltonian parameters of
the complexes have been calculated and summarized in Table 2.
The EPR spectra of the copper(II) complexes at room temperature
show one intense absorption band in the high field and is isotropic
due to the tumbling motion of the molecules [19]. However, these
complexes at liquid nitrogen temperature show three well-
resolved peaks with low intensities in the low field region
(Fig. S2) and one intense peak in the high field region. From this
spectral data, it is found that A|| 150 for [Cu(L1)2], 150 for
[Cu(L2)2] and 160 for [Cu(L3)2] > A\105 for [Cu(L1)2], 90
for [Cu(L2)2] and 85 for [Cu(L3)2], g|| (2.3407, 2.3051 and 2.3147
for [Cu(L1)2], [Cu(L2)2] and [Cu(L3)2]) > g\ (2.0601, 2.0472 and
2.0591 for [[Cu(L1)2], [Cu(L1)2] and [Cu(L3)2]) > 2.0027, In all the
three, g|| and g\ values are greater than 2.04, consistent with cop-
per(II) in axial symmetry with all the principal axes aligned paral-
lel. These g values indicate an elongated tetragonally distorted
octahedral stereochemistry [20]. From the values of g factors, it
is determined the geometric parameter G, representing a measure
of exchange interaction between Cu(II) centres in polycrystalline
compound, using the formula [21]
The 1H NMR spectra of the Schiff bases (L1), (L2) and (L3) and
their zinc complexes were recorded at room temperature in CDCl3.
The 1H NMR spectra of ligand (L1) and its [Zn(L1)2]Cl2 are given in
Figs. S1a and S1b (Supplementary file). 1H NMR spectrum of the
Schiff base ligand shows peaks at 6.6–7.4 d which are attributed
to phenyl multiplet of Schiff base ligand (obtained from the con-
densation of 4-aminoantipyrine and furfuraldehyde). The ligand
also shows the following signals: CACH3 2.3–2.4 d(s), NACH3
3.1–3.3 d(s); CH@N 9.3 d(s) and 6.62, 6.94 and 7.83 d for furfurylid-
ene moiety. The azomethine proton ACH@N signals in the spectra
of the zinc complexes are shifted to down field (9.6 d) compared to
the free ligands, suggesting deshielding of azomethine group due
to the coordination with metal ion. There is no appreciable change
in all other signals of the complexes. The peak at 11 d is attributed
to the ACOOH of amino acid (glycine/alanine/valine) moiety pres-
ent in Schiff bases. The absence of this peak noted for the zinc(II)
complexes confirms the loss of the COOH proton of amino acid
moiety due to complexation.
Magnetic moments and electronic spectra
The free Schiff base ligands (L1/L2/L3) exhibit two intense
bands in 29,240 & 37,075 (L1), 29,411 & 35,335 (L2) and 29,450
& 35,575 cmꢁ1 (L3) which are due to
p–
p⁄ and n–p⁄ transitions
respectively. In all metal complexes, the absorption bands are ob-
G ¼ gk ꢁ 2:0027=g? ꢁ 2:0027
served in the regions 35,842–37,453 and 29,375–29,761 cmꢁ1
which are due to p–
p⁄ and n–p⁄ transitions. These transitions ex-
If G < 4, it is considered the existence of some exchange interaction
between Cu(II) centres and if G > 4, the exchange interaction is neg-
ligible. The present copper complexes have G values greater than 4
indicating exchange interaction is either absent or very little in the
solid complexes. The empirical ratio of g||/A|| is frequently used to
evaluate distortion in copper(II) complexes. If this ratio is close to
100, it indicates roughly a square-planar structure around the cop-
per(II) ion and the values from 170 to 250 are indicative of distorted
tetrahedral geometry. If the ratio is in between 110 and 170, it indi-
cates nearly an octahedral environment around copper(II) ion with
small distortion [22]. For the present copper complexes, the g||/A||
values are 156, 153 and 144 cmꢁ1 which indicate that all the three
complexes have distorted octahedral geometry.
hibit blue or red shift due to the coordination of the ligand with
metal ions. The electronic spectra of [Cu(L1)2], [Cu(L2)2] and
[Cu(L3)2] complexes exhibit one broad band at 11,050, 11,049
and 12,254 cmꢁ1 respectively with low intensity hump which is
assigned to 2Eg ? 2T2g transition. The above data reveal that the
Cu(II) complexes adopt distorted octahedral geometry around the
central metal ion. The observed magnetic moments of the com-
plexes (1.87, 1.85 and 1.86 BM respectively) at room temperature
indicate the non-coupled mononuclear complexes of magnetically
diluted d9 system with s = 1/2 spin state of distorted octahedral
geometry [15]. The monomeric nature of the complexes is further
supported by micro analytical and mass spectral data. The elec-
tronic spectra of [Co(L1)2], [Co(L2)2] and [Co(L3)2] complexes show
three broad bands in the regions 11,025, 14,903, 29,280, 12,150,
13,774, 29,325 and 11,049, 12,500, 29,761 cmꢁ1 respectively
The covalency parameters a2 (covalent in-plane
and b2 (covalent in-plane
-bonding) have been calculated using
the following equations. If
2 = 1.0, it indicates complete ionic
character whereas
2 = 0.5 denotes 100% covalent bonding, with
r-bonding)
p
a
4
4
4
which are assigned to T1g(F) ? 4T2g(F), T1g(F) ? 4A2g(F) and T1g
(F) ? 4T2g(P). These data reveal that complexes have octahedral
geometry. The observed magnetic moments of cobalt(II) complexes
(4.94, 4.83 and 4.91 BM) at room temperature indicate the mono-
meric nature of the complexes. It is further supported by micro
analytical data. The electronic spectra of [Ni(L1)2], [Ni(L2)2] and
[Ni(L3)2] complexes show three bands of low intensity in the visi-
ble region around 11,695, 12,820 and 24,937 cmꢁ1, 11,049, 12,468
and 29,411 cmꢁ1 and 11,061, 12,642, and 29,498 cmꢁ1 which are
a
assumption of negligible small values of the overlap integral.
a2 ¼ ðA =0:036Þ þ ðg ꢁ 2:0027Þ þ 3=7ðg? ꢁ 2:0027Þ þ 0:04
k
k
b2 ¼ ðgk ꢁ 2:0027ÞE=ðꢁ8ka2
Þ
c2 ¼ ðg? ꢁ 2:0027ÞE=ðꢁ2ka2
Þ
assigned to A2g(F) ? 3T2g(F), A2g(F) ? 3T1g(F) and A2g(F) ? 3T1g
(P) transitions respectively suggesting an octahedral geometry
around Ni(II) ion. The observed magnetic moments of Ni(II) com-
plexes (3.08, 3.16 and 3.07 BM respectively) at room temperature
where k ¼ ꢁ828 cmꢁ1 for free copper ion and E is electronic transi-
3
3
3
tion energy. From Table 1, the observed b2 (0.7732) and 2 (0.5214)
c
values for [Cu(L1)2], b2 (0.6730) and
c
2 (0.3923) values for [Cu(L2)2]
and for [Cu(L3)2] b2(0.7804) and c2 (0.5682) indicate that there is

410
N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 404–413
Table 2
The spin Hamiltonian parameters of the copper complexes in DMSO solution at 300 and 77 K.
Kk2
K?2
Complex
A||
A\
Aiso
g||
g\
giso
G
g||/g\
a2
b2
c2
[Cu(L1)2]
[Cu(L2)2]
[Cu(L3)2]
150
150
160
105
90
85
120
110
110
2.3407
2.3051
2.3147
2.0601
2.0472
2.0591
2.1536
2.1351
2.1448
5.9931
6.8614
5.4938
156
153
144
0.8186
0.7775
0.8202
0.7732
0.6730
0.7804
0.5214
0.3923
0.5682
0.6329
0.5233
0.6401
0.4268
0.0763
0.1165
Table 3
interaction in the out-of-plane
p-bonding whereas the in-plane p-
Electronic absorption spectral properties of Cu(II), Co(II), Ni(II) and Zn(II) complexes.
bonding is completely ionic. This is further confirmed by orbital
reduction factors which are estimated using the following relations:
b
No
Compound
kmax
Dk (nm)
H%a
Kb (Mꢁ1
)
Hathaway [22] pointed out that for pure
r
-bonding, K|| ꢄ K\ and for
-bonding
Free
Bound
in-plane -bonding K|| < K\, while for out-of-plane
p
p
1
2
3
4
5
6
7
8
9
[Cu(L1)2]
[Co(L1)2]
[Ni(L1)2]
[Zn(L1)2]
[Cu(L2)2]
[Co(L2)2]
[Ni(L2)2]
[Zn(L2)2]
[Cu(L3)2]
[Co(L3)2]
[Ni(L3)2]
[Zn(L3)2]
260.1
339.8
339.4
339.8
339.4
342.0
339.0
341.0
341.6
257.7
340.0
339.0
260.9
339.6
337.4
339.4
337.02
340.0
341.0
339.0
339.6
259.3
339.2
342.0
0.8
0.2
2.0
0.4
2.38
2.00
2.00
2.00
2.0
20.66
13.47
15.3
5.520 ꢃ 103
6.600 ꢃ 102
1.438 ꢃ 104
1.029 ꢃ 105
5.570 ꢃ 103
5.813 ꢃ 104
7.039 ꢃ 104
5.969 ꢃ 104
4.181 ꢃ 105
1.113 ꢃ 105
4.527 ꢃ 105
6.248 ꢃ 104
K\ < K|| the following simplified expressions were used to calculate
K|| and K\:
8.33
K
K
¼ ðgk ꢁ 2:0027ÞE=8k0
¼ ðg? ꢁ 2:0027ÞE=2k0
k
20.97
11.97
12.73
10.24
28.20
21.71
29.56
10.25
?
The observed K|| values are (0.6329. 0.5233 and 0.6401) greater than
the values of K\ (0.4268, 0.0763 and 0.1165) implying a greater
10
11
12
1.6
0.8
3.0
contribution from out-of-plane
p-bonding than from in-plane p-
bonding in metal ligand -bonding.
p
a
H% = [Afree ꢁ Abound)/Afree] ꢃ 100%.
Thus, the EPR study of the copper(II) complex has provided
supportive evidence to the conclusion obtained on the basis of
electronic spectrum and magnetic moment value.
b
Kb = Intrinsic DNA binding constant determined from the UV–Vis absorption
spectral titration.
binding mode. From the binding constant values (Kb), it is under-
stood that the valine Schiff-base complexes have more intercalat-
ing ability than alanine and glycine Schiff-base complexes. In
addition to this, nickel, copper and cobalt complexes are having
higher binding constant values than and zinc complexes. In this
study, for glycine Schiff base complexes, the OD is increased when
DNA is added to the complexes i.e. hyperchromism is observed,
which indicates that added complexes break the secondary struc-
ture of the DNA during binding. In general binding constant (Kb)
values of these synthesized complexes are lower in comparison
to those observed for typical intercalators, ethidium bromide and
[Ru(bpy)2(dppz)]2+ whose binding constants are in the order of
1.4 ꢃ 106 and >106 Mꢁ1 [25,26].
DNA binding studies
Absorption titration
There has been an increasing focus on the interaction between
DNA and other molecules which is hopeful to understand the ac-
tion mechanism of some DNA-targeted drugs and toxic agents as
well as the origin of few diseases like gene mutation [23]. The
binding behavior of the metal complexes to DNA helix is often
investigated using absorption spectral titration followed by the
changes in the absorbance and shift in the wavelength. The repre-
sentative absorption spectra of [Cu(L3)2], [Zn(L2)2] and [Cu(L1)2]
complexes in the presence and absence of CT-DNA are shown in
Figs. S3a–c.The binding mode of complexes to CT-DNA helix has
been followed through absorption spectral titrations. With increas-
ing concentration of CT-DNA the absorption bands of the
complexes are affected resulting in the tendency of hypochromism
and a minor red shift is observed in all the complexes.
Hyperchromic effect and hypochromic effect are the spectral
features of DNA concerning its double helix structure. Hypochro-
mism results from the contraction of DNA in the helix axis as well
as from the change in conformation on DNA while hyperchromism
results from the damage of the DNA double helix structure [24].
The absorption spectrum of [Cu(L1)2] complex shows an intensive
absorption band at 260.1 nm, [Cu(L2)2] and [Cu(L3)2] complexes at
339.4 and 341.6 nm respectively in 5 mM Tris–HCl and 50 mM
NaCl buffer solution (pH 7.2). Further the intense absorption bands
with maxima of 339.8 nm, 342.0 nm and 257.69 nm for [Co(L1)2],
[Co(L2)2] and [Co(L3)2], 339.4 nm, 339.0 nm and 340.0 nm for
[Ni(L1)2], [Ni(L2)2] and [Ni(L3)2], 339.8 nm, 341.0 nm and
339.0 nm for [Zn(L1)2], [Zn(L2)2] and [Zn(L3)2] respectively have
Viscosity measurements
The viscometric measurement is also an important tool to find
the nature of binding of metal complexes to the DNA. The relative
specific viscosity of DNA is determined by varying the concentration
of the added metal complexes. In the absence of crystallographic
structural data, hydrodynamic methods that are sensitive to DNA
lengthchange are regarded as the least ambiguousand themost crit-
ical tests of binding in solution. A classical intercalation model re-
sults in the lengthening of the DNA helix as the base pairs is
separated to accommodate the binding molecule, leading to an in-
crease in the DNA viscosity. However, a partial and non-classical
intercalation of ligand may bend (or kink) DNA helix, resulting in
the decrease of its effective length and concomitantly its viscosity
[27]. The plots of (
and o are the relative viscosities of DNA in the presence and absence
g/go) g
1/3 versus R, (where R = [Complex]/[DNA];
g
of complex, respectively) give a measure of the viscosity changes.
The effects of all the complexes on the viscosity of CT-DNA are
shown in Fig. S4. As expected, the known DNA-intercalator ethidium
bromide increases the relative viscosity of DNA double helix result-
ing from intercalation. In contrast, groove (or) surface binding can
cause an increase in the effective length of DNA leading to a minor
increase in the effective length of DNA solution [28]. All the com-
plexes exhibit minor increase in the relative viscosity of CT-DNA
compared to ethidium bromide suggesting primarily groove binding
nature of the complexes.
been obtained. These are due to intra ligand
p–
p⁄ transitions.
The increase of the concentration of CT-DNA results in the minor
bathochromic shift in the range 0.2–3.0 nm and significant hypo-
chromicity lying in the range 8.33–29.56%. The spectroscopic
changes suggest that the complexes show appreciable binding
property to the CT-DNA. The Kb values are shown in Table 3.
These results suggest that the interaction of synthesized
metal(II) complexes with DNA is a significant weak intercalation

N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 404–413
411
Electrochemical studies
supercoiled circular form of pUC19 DNA into nicked circular form
and linear form. When circular plasmid DNA is conducted by
electrophoresis the fastest migration will be observed for the super
coiled form (Form I). If one strand is cleaved, the supercoils will
relax to produce a slow moving open circular form (Form II). If both
strands are cleaved a linear form (Form III) will be generated that
migrates in between I and II [29,30].
The ability of the complexes in affecting DNA cleavage has been
investigated by gel electrophoresis using super coiled pUC19 DNA
in Tris–HCl/50 mM buffer medium. When this experiment is
carried out to all the three series of complexes, it has been ob-
served that valine (L3) complexes have little bit higher cleavage
abilities than alanine (L2) and glysine (L1) complexes. Among the
four valine Schiff base complexes, nickel and copper complexes
are having higher cleaving ability. All the complexes are found to
exhibit nuclease activity. Fig. S6a shows the result of gel electro-
phoretic separations of plasmid pUC19 DNA induced by an addi-
tion of pUC19 DNA, [M(L3)2] in presence of AH2 (ascorbic acid).
Under the same conditions, free AH2 produced no cleavage of
pUC19 DNA. All super coiled (Form I) DNA was cleaved to form
the mixture of form II and form III with the addition of the complex
concentration. These phenomena imply that Cu(II), Co(II), Ni(II)
and Zn(II) complexes induce intensively the cleavage of plasmid
pUC19 DNA in the presence of AH2.
In order to clarify the cleavage mechanism of pUC19 DNA intro-
duced by [M(L3)2], the investigation has been carried out further on
adding DMSO (hydroxyl radical scavenger). It is found that no inhi-
bition of DNA cleavage has been observed indicating that hydroxyl
radical is not involved in the cleavage process (Fig. S6b). On the other
hand, addition of sodium azide (singlet oxygen scavenger) does not
show any apparent inhibition in the DNA which reveals that the sin-
glet oxygen lO2 is not responsible for the cleavage reaction (Fig. S6c).
These observations suggest that all the complexes mediated cleav-
age reaction do not proceed via radical cleavage.
Cyclic and differential pulse voltammetric techniques are extre-
mely useful in probing the nature and mode of DNA binding of me-
tal complexes. Typical cyclic voltammogram of complex, [Cu(L3)2]
in the absence and in the presence of varying amount of [DNA] is
shown in Fig. S5a. The incremental addition of CT-DNA to the
complex causes decrease of anodic and cathodic peak current of
the complex. This result shows that complex stabilizes the duplex
(GC pairs) by intercalating way. The incremental addition of CT-
DNA to the complex causes shift in the potential of peak in cyclic
voltammogram. Both the cathodic and anodic peak show positive
or negative shift which indicates intercalation of complex to DNA
of base pairs. If one of the shifts is positive and another one is neg-
ative, it reveals the intercalation and electrostatic binding of the
complex to CT-DNA or it may break the secondary structure of
DNA.
In differential pulse voltammogram of the complex, [Cu(L3)2] in
the absence and presence of varying amount of [DNA] with signif-
icant decrease of current intensity (Fig. S5b), the shift in potential
is related to the ratio of binding constant by the following
equation:
Eb ꢁ Ef ¼ 0:0591 logðK =K½oxdꢂÞ
½redꢂ
where Eb and Ef are peak potentials of the complex in the bound and
free form respectively. In the present study, [M(L1)2], [M(L2)2] and
[M(L3)2] complexes show one electron transfer during the redox
process and their ipc/ipa value is less than unity which indicates
the reaction of the complex on the glassy carbon electrode surface
is quasi-reversible. Other complexes [Co(II), Ni(II), Mn(II) and Zn(II)]
show considerable shift in both cathodic and anodic peak potentials
in the presence of incremental addition of CT-DNA.
Most of our synthesized complexes give both the anodic and
cathodic peak potential shifts which are either positive or negative
(Table 4). It indicates the intercalating mode of DNA binding with
glycine/alanine/valine ligand Schiff base complexes. It is interest-
ing to note that valine Schiff base ligand complexes give greater
decrease in current intensity than alanine and glycine Schiff base
ligand complexes. From this observation, it is concluded that inter-
calative mode of DNA binding to valine ligand complexes is stron-
ger than ala/gly ligand complexes.
Antimicrobial screening
Antibacterial activity
The Schiff base complexes have provoked wide interest because
they possess a diverse spectrum of biological and pharmaceutical
activities. The antibacterial activity of the Schiff base ligands and
their metal complexes and streptomycin (as a standard compound)
were tested against bacteria because bacteria can achieve resis-
tance to antibiotics through biochemical and morphological mod-
ifications [31]. The organisms used in the present investigation
included Staphylococcus aureus, Pseudomonas aeruginosa,
Nucleolytic activity
The study on the cleavage capacity of transition metal complex
to DNA is considerably interesting as it can contribute to under-
standing the toxicity mechanism of them and to develop novel
artificial nuclease. DNA cleavage is controlled by relaxation of
Table 4
Electrochemical parameters for the interaction of DNA with metal(II) complexes.
a
S. No.
Complexes
E1/2 (V)a
D
Ep (V)b
k[red]/k[oxd]
Ipc/Ipa
Free
Bound
Free
Bound
1
2
3
4
5
6
7
8
9
[Cu(L1)2]
[Co(L1)2]
[Ni(L1)2]
[Zn(L1)2]
[Cu(L2)2]
[Co(L2)2]
[Ni(L2)2]
[Zn(L2)2]
[Cu(L3)2]
[Co(L3)2]
[Ni(L3)2]
[Zn(L3)2]
ꢁ0.1035
ꢁ0.1760
ꢁ0.7623
+0.1624
ꢁ0.1490
ꢁ0.2065
ꢁ0.1347
ꢁ0.4923
ꢁ0.6267
ꢁ0.5035
ꢁ0.1034
ꢁ0.1656
ꢁ0.0785
ꢁ0.1615
ꢁ0.6128
+0.1887
ꢁ0.1461
ꢁ0.2265
ꢁ0.1166
ꢁ0.4320
ꢁ0.4882
ꢁ0.4810
ꢁ0.0811
ꢁ0.1636
+0.0470
+0.0360
ꢁ0.0325
+0.0523
ꢁ0.0700
+0.0756
ꢁ0.0647
+0.0670
ꢁ0.2298
+0.0620
+0.0320
+0.0724
+0.0750
+0.0312
ꢁ0.1064
+0.0816
ꢁ0.0910
+0.0656
ꢁ0.0626
ꢁ0.0220
ꢁ0.3185
+0.0230
+0.0610
+0.0644
0.8190
0.6640
1.0294
0.7321
0.4980
0.1841
0.7589
0.8357
1.0115
1.8450
0.8064
0.6816
0.7782
0.5900
0.5432
0.6560
0.9312
0.5260
0.8534
1.0116
0.8619
0.9312
0.8126
0.8745
10
11
12
Data from cyclic voltammetric measurements.
a
E1/2 is calculated as the average of anodic (Epa) and cathodic (Epc) peak potentials. E1/2 = EPa + Epc/2.
b
D
Ep = Epa ꢁ Epc
.

412
N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 404–413
Table 5
The in vitro antibacterial activity of ligands and their metal(II) complexes evaluated by MIC (Minimum Inhibitory Concentration,
lg/mL).
Compound
S. aureus
P. aeruginosa
E. coli
S. epidermidis
K. pneumoniae
L1
20.8
8.8
7.2
11.9
9.0
10.2
8.4
32
9.9
5.6
4.0
2.3
4.6
10.9
4.0
2.8
2.4
3.2
1.9
22.3
3.6
2.1
1.8
1.9
17.3
4.5
4.0
2.4
3.8
23.3
4.8
2.0
4.0
2.2
1.8
24.7
6.4
8.8
6.4
6.0
18.7
5.2
5.9
4.4
2.8
25.7
4.6
3.2
3.0
4.0
1.3
22.4
6.4
8.8
8.4
9.2
21.4
6.8
5.3
7.1
8.3
26.4
5.4
6.2
5.1
7.3
2.3
[Cu(L1)2]
[Co(L1)2]
[Ni(L1)2]
[Zn(L1)2]
L2
[Cu(L2)2]
[Co(L2)2]
[Ni(L2)2]
[Zn(L2)2]
L3
[Cu(L3)2]
[Co(L3)2]
[Ni(L3)2]
[Zn(L3)2]
Streptomycina
8.6
10.4
18.8
6.7
5.2
6.0
5.1
24.8
6.3
4.4
5.9
7.1
1.7
a
Streptomycin is used as the standard.
Escherichia coli, Staphylococcus epidermidis and Klebsiella pneumo-
niae. The disc diffusion method was used to evaluate the antibac-
terial activity of the synthesized metal complexes. The minimum
inhibitory concentration (MIC) was determined by assaying at se-
rial dilution of concentrations along with standards at the same
concentration.
Antifungal activity
To provide a medicinal scope in the field of bioinorganic chem-
istry, consequently, the metal complexes synthesized have been
evaluated for their antifungal actions. The antifungal tests were
carried out using the disc diffusion method. The Schiff base ligands
and their metal complexes were screened in vitro in order to find
out the antifungal activity against Aspergillus niger, Aspergillus fla-
vus, Culvularia lunata, Rhizoctonia bataicola and Candida albicans.
The results of the antifungal studies are presented in Table 6 which
reveal that the metal complexes are toxic than the free ligands
against the same organisms. Moreover, all the glycine complexes
are having higher antifungal activities than alanine and valine
complexes. As the number of alkyl group in the ligand increases,
it will decrease antifungicidal activity of the complexes. The in-
crease in the antifungal activity of the metal complexes inhibits
multiplication process of the microbes by blocking their active
sites. Such increased activity on metal chelation can be explained
on the basis of Tweedy’s chelation theory [32]. The chelation also
increases the lipophilic nature and the interaction between the
metal ion and the lipid is favored. This may lead to the breakdown
of the permeability barrier of the cell resulting in interference with
the normal cell processes. While chelation is not the only factor for
antimicrobial activity, it is an intricate blend of several aspects
such as nature of the metal ion and the ligand, the geometry of
the metal complexes, the lipophilicity, the presence of co-ligands,
The results of the bactericidal study of the synthesized com-
pounds are displayed in Table 5. All the complexes are more potent
than the ligand towards the specified bacteria species. Moreover,
all the glysine Schiff base complexes are having higher antimicro-
bial activities than alanine and valine complexes. As the number of
alkyl group in the ligand increases, it will decrease antibactericidal
activity of the complexes. The remarkable activity of the Schiff base
ligand may be due to the presence of azomethine group which im-
ports in elucidating the mechanism of transformation reaction in
biological system. All the metal complexes are found to have high-
er antibacterial activity against Schiff base ligands. The antibacte-
rial results evidently show that the activity of the Schiff base
compounds becomes more pronounced when coordinated to the
metal ions. The studies reveal that a significant antibacterial activ-
ity can be induced for complexes by using either a biocation or a
metallic ion exhibiting inhibitory properties as well as a ligand that
displays such activity. This enhancement in the activity may be
rationalized on the basis that their structures mainly possess an
additional C@N bond along with azomethine group.
Table 6
The in vitro antifungal activity of ligands and their metal(II) complexes evaluated by MIC (Minimum Inhibitory Concentration,
lg/mL).
Compound
Aspergillus niger
Aspergillus flavus
Culvularia lunata
Rhizoctonia bataicola
Candida albicans
L1
22.3
9.4
10.2
8.6
11.4
7.2
9.8
4.2
5.3
8.4
5.9
5.6
5.8
6.5
12.4
5.1
4.6
4.6
4.12
1.7
20.3
5.4
4.6
3.3
3.4
18.3
4.2
4.6
5.7
4.5
21.3
4.1
3.3
2.8
3.1
1.2
22.7
9.3
5.4
7.3
7.2
21.7
5.8
5.3
7.0
7.2
23.7
4.0
3.8
3.4
6.4
1.0
24.4
6.4
16.5
6.1
4.9
26.4
9.3
[Cu(L1)2]
[Co(L1)2]
[Ni(L1)2]
[Zn(L1)2]
L2
[Cu(L2)2]
[Co(L2)2]
[Ni(L2)2]
[Zn(L2)2]
L3
[Cu(L3)2]
[Co(L3)2]
[Ni(L3)2]
[Zn(L3)2]
Nystatina
8.4
25.3
11.2
9.4
7.8
9.6
9.1
12.2
22.3
12.32
15.44
13.32
16.25
1.1
10.8
24.4
10.2
11.32
9.35
12.35
1.5
a
Nystatin is used as the standard.

N. Raman et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 125 (2014) 404–413
413
the steric and pharmacokinetic factors [33]. The synthesized com-
pounds having amino acid moieties also show good activity.
also thank the Sophisticated Analytical and Instrumentation Facil-
ity, Central Drug Research Institute, Lucknow, for providing CHN-
analysis data and FAB-Mass spectra and Sophisticated Analytical
and Instrumentation Facility, Indian Institute of Technology, Bom-
bay, for EPR measurements.
Antioxidant property
According to relevant reports in the literature [34–37], few tran-
sition metal complexes may exhibit antioxidant activity. Hence, it is
tried to explore whether the synthesized complexes have the
hydroxyl radical scavenging property. The scavenging property of
the hydroxyl radicals of the synthesized complexes has been com-
pared to the well-known natural antioxidants mannitol and vitamin
C, using the same method as reported by Wu et al. [38]. The 50%
inhibitory concentration (IC50) values of mannitol and vitamin C
are about 9.6 ꢃ 10ꢁ3 and 8.7 ꢃ 10ꢁ3Mꢁ1 respectively. According to
the antioxidant experiments, the IC50 values of [Ni(L1)2] and
[Zn(L1)2] complexes are 1 ꢃ 10ꢁ7 Mꢁ1 and 1 ꢃ 10ꢁ8 Mꢁ1 respec-
tively which imply that above zinc and nickel complexes have pref-
erable ability to scavenge hydroxyl radical. The antioxidant
behavior is also observed in all valine Shiff base ligand complexes.
The IC50 value of [Cu(L3)2] complex is 1 ꢃ 10ꢁ4 Mꢁ1 .This value also
shows that [Cu(L3)2] complex has preferable ability to scavenge
hydroxyl radical. Based on the view of the observed IC50 values,
the [Ni(L1)2] and [Zn(L1)2] complexes can be considered as potential
drugs to eliminate the hydroxyl radicals.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2014.01.108.
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Conclusion
This work describes the synthesis and characterization of 4-
aminoantipyrine derived Schiff bases and their metal(II) com-
plexes. The Schiff base ligands are behaving as nitrogen and oxygen
donor tridentate ligands. The structure of the complexes has been
determined on the basis of elemental analyses, molar conductivi-
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The authors express their sincere thanks to the College Manag-
ing Board, Principal and Head of the Department of Chemistry,
VHNSN College for providing necessary research facilities. They