DNA interaction, antimicrobial, electrochemical and spectroscopic studies of metal(II) complexes with tridentate heterocyclic Schiff base derived from 2′-methylacetoacetanilide

Article abstract of DOI:10.1016/j.molstruc.2011.06.006

A new Schiff base ligand (HL) was synthesized by the condensation reaction between 2′-methyleacetoacetanilide and 2-amino-3-hydroxypyridine. Its Co(II), Ni(II), Cu(II) and Zn(II) complexes were prepared by the interaction of the ligand with metal(II) chloride. They were characterized by elemental analysis, IR, ¹H NMR, EPR, UV-Vis, magnetic susceptibility measurements, conductivity measurements and FAB-mass spectra. The interaction of the complexes with calf thymus DNA (CT-DNA) has been investigated by UV absorption, viscosity and cyclic voltammetry methods, and the mode of CT-DNA binding to the complexes has been explored. Furthermore, the DNA cleavage activity by the complexes was performed. It was found to be oxidative hydroxyl radical cleavage in the presence of 3-mercaptopropionic acid (MPA). The Schiff base and its complexes have been screened for their antibacterial (Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa) and antifungal (Aspergillus niger, Rhizopus stolonifer, Rhizoctonia bataicola and Candida albicans) activities and the data reveal that the complexes have higher activity than the free ligand.

Full text of DOI:10.1016/j.molstruc.2011.06.006

Journal of Molecular Structure 1000 (2011) 135–144
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
DNA interaction, antimicrobial, electrochemical and spectroscopic studies
of metal(II) complexes with tridentate heterocyclic Schiff base derived
from 20-methylacetoacetanilide
⇑
Natarajan Raman , Krishnan Pothiraj, Thanasekaran Baskaran
Research Department of Chemistry, VHNSN College, Virudhunagar 626 001, India
a r t i c l e i n f o
a b s t r a c t
A new Schiff base ligand (HL) was synthesized by the condensation reaction between 20-methyleaceto-
acetanilide and 2-amino-3-hydroxypyridine. Its Co(II), Ni(II), Cu(II) and Zn(II) complexes were prepared
by the interaction of the ligand with metal(II) chloride. They were characterized by elemental analysis, IR,
1H NMR, EPR, UV–Vis, magnetic susceptibility measurements, conductivity measurements and FAB-mass
spectra. The interaction of the complexes with calf thymus DNA (CT-DNA) has been investigated by UV
absorption, viscosity and cyclic voltammetry methods, and the mode of CT-DNA binding to the com-
plexes has been explored. Furthermore, the DNA cleavage activity by the complexes was performed. It
was found to be oxidative hydroxyl radical cleavage in the presence of 3-mercaptopropionic acid
(MPA). The Schiff base and its complexes have been screened for their antibacterial (Staphylococcus aur-
eus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa) and antifungal (Aspergillus niger, Rhizo-
pus stolonifer, Rhizoctonia bataicola and Candida albicans) activities and the data reveal that the complexes
have higher activity than the free ligand.
Article history:
Received 28 December 2010
Received in revised form 7 June 2011
Accepted 9 June 2011
Available online 16 June 2011
Keywords:
Schiff base
Metal(II) complexes
DNA binding
Antimicrobial activity
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
electrostatic interactions, groove binding or intercalation [12,13].
Compounds showing the properties of effective binding as well
The heterocyclic Schiff base ligands are well known due to their
wide range of applications in pharmaceutical and industrial fields
[1]. Several Schiff base ligands derived from pyridine derivatives
and their copper(II) complexes have also been shown to inhibit tu-
mor growth [2]. These studies have shown that complexation of
metals to Schiff base ligands improves the biopotentials of the
ligands [3]. Many reports are available for the preparation and
properties of above modeled metal complexes as they mimic
metalloproteins [4]. In addition, the reports of quinolin-2(1H)-
one-derived Schiff bases and their metal complexes have emerged
more recently [5–8].
In recent years, the binding studies of transition metal com-
plexes have become an important field in the development of
DNA molecular probes and chemotherapeutics [9]. Metal
complexes such as cobalt, nickel, copper and zinc with N-contain-
ing ligands have shown excellent binding and cleavage activities
[10,11]. There is a substantial literature supporting the application
of artificial DNA cleaving agents in biotechnology; structural stud-
ies of nucleic acids or development of new drugs. Transition metal
complexes can interact with DNA through covalent binding,
as cleaving double stranded DNA under physiological conditions
are of great importance since these could be used as diagnostic
agents in medicinal and genomic research.
Although Schiff bases containing a heterocyclic nucleus have
efficient biological activity, the studies based on 2-amino-3-
hydroxypyridine Schiff base chemistry are less extensive. Our lab-
oratory has been currently involved in exploring this chemistry
[14–16]. Bearing the above facts in mind, and as a part of our
continuing research, herein the synthesis of tridentate heterocyclic
Schiff base ligand and its complexes has been reported. In order to
investigate the coordination mode of ligand, the complexes of
Co(II), Ni(II), Cu(II) and Zn(II) have been characterized by UV–Vis,
IR, 1H NMR, FAB-mass and EPR studies. The antimicrobial, DNA
binding and cleavage properties of the complexes have also been
investigated.
2. Experimental
2.1. Materials and measurements
The chemicals used in this work viz., 20-methylacetoacetanilide,
2-amino-3-hydroxypyridine, acetic acid and Tris–HCl buffer were
of Merck grade. The AR grade of 3-mercaptopropionic acid, sodium
⇑
Corresponding author. Tel.: +91 92451 65958; fax: +91 4562 281338.
E-mail address: drn_raman@yahoo.co.in (N. Raman).
0022-2860/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2011.06.006

136
N. Raman et al. / Journal of Molecular Structure 1000 (2011) 135–144
chloride, sodium hydroxide, CoCl2ꢀ6H2O, NiCl2ꢀ6H2O, CuCl2ꢀ2H2O
and ZnCl2 were used. Commercial solvents were distilled and then
used for the preparation of ligand and its complexes. CT-DNA and
pBR322 DNA were purchased from Bangalore Genei (India). Aga-
rose (molecular biology grade) and ethidium bromide were pur-
chased from Sigma–Aldrich (USA). Carbon, hydrogen and
nitrogen analyses of the complexes were carried out on a CHN ana-
lyzer Carlo Erba Model 1108. The infrared spectra (4000–400 cmꢁ1
KBr disks) of the samples were recorded on a Perkin–Elmer 783
series FT-IR spectrophotometer. The electronic absorbance spectra
in the 200–1100 nm were recorded on UV-1601 spectrophotome-
ter (Shimadzu). 1H NMR spectra of ligand and its zinc complex
(300 MHz) were recorded on a Bruker Avance DRX-300 FT-NMR
spectrometer using CDCl3 and DMSO-d6 as solvents respectively.
Tetramethylsilane was used as internal standard. Chemical shifts
were reported in d scale. EPR spectra of copper(II) and nickel(II)
complexes in DMSO were recorded on VARIAN EPR spectrometer
at the X-band frequency (9.1 GHz) at RT (300 K) and LNT (77 K)
using tetracyanoethylene (TCNE) as field marker. Fast atomic bom-
bardment mass spectra (FAB-MS) were obtained using a VGZAB-HS
spectrometer in a 3-nitrobenzylalcohol matrix. Molar conductance
of 10ꢁ3 M solutions of the complexes in N,N0-dimethylformamide
(DMF) were measured at room temperature with a Deep vision
Model-601 digital direct reading deluxe conductivity meter. Mag-
netic susceptibility measurements were carried out by employing
the Gouy method at room temperature on powder sample of the
complex using CuSO4ꢀ5H2O as calibrant. The metal contents of
the complexes were determined according to the literature method
[17].
repeated in triplicate to get the concurrent values. Data are pre-
1/3
sented as (
g/
g0)
versus the ratio [complex]/[DNA] = R, where g
and g0 are the specific viscosity of DNA in the presence and ab-
sence of the complex respectively. The values of
culated by the following equation:
g and g0 were cal-
g
¼ ðt ꢁ tbÞ=tb
where tb is the observed flow time of DNA containing solution and, t
is the flow time of buffer alone. Relative viscosities for DNA were
calculated from the relation, g/g0.
2.2.3. Methodology for DNA binding analysis using electrochemical
technique
Cyclic voltammetry was carried out at CH instrument electro-
chemical analyzer. All voltammetric experiments were performed
in single compartmental cell of volume 10–15 mL containing a
three-electrode system comprised of a glassy carbon working elec-
trode, Pt-wire as auxiliary electrode and an Ag/AgCl electrode as
reference electrode. The supporting electrolyte was 0.1 M Bu4N-
ClO4 in milli-Q water. Deaerated solutions were used by purging
N2 gas for 10 min prior to measurements.
2.3. DNA cleavage experiments
To know the DNA cleavage action of compounds, 200 mg of aga-
rose was dissolved in 25 mL of TAE buffer (4.84 g Tris base, pH 8.0,
0.5 M EDTA/1 L) by boiling and poured into the gel cassette fitted
with a comb. When the gel attained ꢃ55 °C, allowed to solidify
and then, comb was removed carefully. The solid gel thus obtained
was placed in the electrophoresis chamber flooded with Tris–base
buffer. Subsequently (0.2 lg, 30 lM) of DNA sample (mixed with
2.2. Spectroscopic studies on DNA interaction
bromophenol blue dye at 1:1 ratio) was loaded carefully into the
wells, along with standard DNA marker and the constant 50 V of
electricity was made to pass for around 30 min. Finally, the gel
was removed carefully and stained with EB (Ethidium bromide)
solution (10 mg/mL) for 10–15 min and the bands were observed
under UV transilluminator. The illuminated gel was photographed
using a polaroid camera.
To test for the presence of reactive oxygen species (ROS) gener-
ated during strand scission and for possible complex-DNA interac-
tion sites, various reactive oxygen intermediate scavengers were
added to the reaction mixture. The scavengers used were N,N0-
dimethylsulfoxide (DMSO), ethanol (EtOH), sodium azide (NaN3)
(0.5 mM) and superoxide dismutase (SOD) (4 units).
2.2.1. Methodology for DNA binding analysis using electronic spectra
All the experiments involving the interaction of the complexes
with DNA were carried out in Tris–HCl buffer (50 mM, pH 7.2). A
solution of calf thymus DNA in the buffer gave a ratio of UV absor-
bance at 260 and 280 nm of about 1.8–1.9:1, indicating that the
DNA was sufficiently free from protein. Absorption titration exper-
iment was performed by maintaining the metal complex concen-
tration constant (10
lM) and varying the concentration of DNA
in interaction medium by adding 0–100
l
M DNA. While measuring
the absorption spectra, equal amount of DNA was added to com-
plex solution and the reference solution to eliminate the absor-
bance of DNA itself. From the absorption data, the intrinsic
binding constant (Kb) was determined using the following equation
by plotting [DNA]/(ea
½DNAꢂ=ðea ef Þ ¼ ½DNAꢂ=ðeb
where [DNA] is the concentration of DNA in base pairs, the apparent
absorption coefficients ea ef and eb correspond to Aobsd/[complex],
ꢁ
e
f) versus [DNA].
2.4. In vitro antibacterial and antifungal assays
ꢁ
ꢁ
ef Þ þ 1=½Kbðeb
ꢁ ef Þꢂ
The biological activities of synthesized Schiff base and its com-
plexes have been studied for their antibacterial and antifungal
activities by agar and potato dextrose agar diffusion method
respectively in DMSO solvent against Staphylococcus aureus, Bacil-
lus subtilis, Escherichia coli and Pseudomonas aeruginosa bacterial
and Aspergillus niger, Rhizopus stolonifer, Rhizoctonia bataicola and
,
the extinction coefficient for the free complex and extinction coef-
ficient for the complex in fully bound form, respectively. The data
were fitted to the above equation with a slope equal to 1/(eb
ꢁ
ef)
and y-intercept equal to 1/[Kb(eb f)] and Kb was obtained from
ꢁ
e
Candida albicans fungi species [19]. The stock solution (1 mg mLꢁ1
)
the ratio of the slope to the intercept [18].
of the test chemical was prepared by dissolving 10 mg of the test
compound in 10 mL of N,N0-dimethylsulfoxide (DMSO) solvent.
The stock solution was suitably diluted with sterilized distilled
water to get serial dilution. Control for each dilution was prepared
by diluting 10 mL of solvent instead of stock solution with steril-
ized distilled water. The bacteria were subcultured in agar med-
ium. The petri disks were incubated for 24 h at 37 °C. Standard
antibacterial drug, Streptomycin was also screened under similar
conditions for comparison. The fungi were subcultured in potato
dextrose agar medium. Standard antifungal drug, Nystatin was
used for comparison. The petri disks were incubated for 48 h at
37 °C. The MIC (Minimum Inhibitory Concentration) for each
2.2.2. Methodology for DNA binding analysis using viscosity
measurement
Viscosity measurements were carried out using an Oswald
microviscometer, maintained at constant temperature (37 °C) in
a thermostat. The DNA concentration was kept constant in all sam-
ples, but the complex concentration was increased each time (from
50 to 500 lM). Mixing of the solution was achieved by bubbling
the nitrogen gas through viscometer. The mixture was left for
10 min at 37 °C after addition of each aliquot of complex. The flow
time was measured with a digital stopwatch. The experiment was

N. Raman et al. / Journal of Molecular Structure 1000 (2011) 135–144
137
tested substance was determined by microscopic observation of
microbial growth. It corresponds to the well with the lowest con-
centration of the tested substance where microbial growth was
clearly inhibited [19].
1550, 1455
m
(ArAC@C), 1311
m
(CAO), 550–575
m
(ZnAO), 430–455
m(ZnAN); UV–Vis (kmax, nm) (DMF): 346 and 370; molar conduc-
tance (DMF): 4.3 ohmꢁ1 cm2 molꢁ1
; leff (B.M.): Diamagnetic.
3. Results and discussion
2.5. Synthesis
3.1. Physicochemical properties of the synthesized compounds
2.5.1. Synthesis of Schiff base ligand (HL)
A mixture of 20-methylacetoacetoanilide (10 mmol 1.911 g),
2-amino-3-hydroxypyridine (10 mmol 1.110 g) and few drops of
acetic acid in 50 mL ethanolic solution was refluxed for 3 h. After
cooling to room temperature, the solution was neutralized with
concentrated aqueous ammonia. Brownish yellow color precipitate
was obtained, washed with water and ethanol, and dried in vacuo.
The resulting solid product was recrystallized from ethanol at
60 °C.
The ligand was prepared by refluxing an appropriate amount of
20-methylacetoacetoanilide with 2-amino-3-hydroxypyridine in
1:1 molar ratio and the complexes were formed with the metal
salts and ligand in the ratio of 1:2. Formation of the complexes
can be represented by the following general equation:
MX2 þ 2HL ! ½ML2ꢂ þ 2HX
where M = Co(II), Ni(II), Cu(II), Zn(II); X = Cl; HL = Schiff base ligand,
Formulation of the complexes has been done on the basis of their
elemental analysis data, molar conductance values, magnetic sus-
ceptibility, IR and 1H NMR data. All the complexes show 1:2 me-
tal–ligand stoichiometry. They are non-hygroscopic, decomposed
above 200 °C and possess good keeping qualities. The molar con-
ductance values in DMF (Table 1) reveal the non-electrolytic nature
of the complexes [20].
C
16H17N3O2: Yield (%): 77.78, brownish yellow, M.pt: 145 °C,
M.Wt: 283, Anal. Calc. (%): C, 67.82; H, 6.04; N, 14.83. Found (%):
C, 67.76; H, 5.92; N, 14.85; 1H NMR (CDCl3, d, ppm): 2.20 (s, 3H, ali-
phatic-CH3).), 2.75 (s, 3H, aromatic-CH3), 3.62 (s, 2H, CACH2AC),
6.85–7.82 (m, 7H, ArAH), 8.60 (s, 1H, CONH), 10.85 (s, 1H, ArAOH);
IR (KBr,
2920,
m
m
cmꢁ1): 3383
(aliphatic-CH), 1655
m
(OH), 3255
(CONH), 1612
(ArAC@C), 1255, m
m
(NH), 3051
m
(ArACH), 2982,
(C@N), 1575
(CAO). UV–
m
m
m
m
[(C@N)(pyridine)], 1548, 1460
Vis (kmax, nm) (DMF): 345, 362.
3.2. Electronic spectra and magnetic properties of metal(II) complexes
2.5.2. Synthesis of metal complexes
The electronic spectra were recorded in DMF solution and data
are given in the Table 1. The ligand exhibits absorption bands in
UV–Vis region around 345 and 362 nm. The first band below
Hot ethanolic solutions of appropriate metal chloride salts of
Co(II), Ni(II), Cu(II) and Zn(II) (0.237, 0.237, 0.170 and 0.136 g,
1 mmol) were added to the similar hot solution (60 °C) of Schiff
base ligand (HL, 0.556 g, 2 mmol, 40 mL). The resulting mixture
was stirred under reflux for 2 h whereupon the complexes precip-
itated. They were collected by filtration, washed several times with
ethanol and diethyl ether, and dried in a desiccator over anhydrous
CaCl2 at room temperature.
345 nm is assigned to
p ?
p⁄ transition. Another band observed
at 362 nm is assigned to n ? p⁄ transition originating from the
amide or imine function of the Schiff base ligand [21].
The magnetic moment value (1.91 B.M.) for [CuL2] as well as the
broad band in its electronic spectrum centered at 805 and 977 nm,
assigned to 2B1g ? 2E1g and 2B1g ? 2A1g transitions suggested a
distorted octahedral geometry around the copper(II) ion [22].
However, the band observed at 368 nm was probably due to a
ligand-metal charge transfer transition.
[CoC32H32N6O4]: Yield (%): 70.72, reddish brown, M.pt: 175 °C,
M.Wt: 624, Anal. Calc. (%): Co, 9.45; C, 61.63; H, 5.17; N, 13.4.
Found (%): Co, 9.40; C, 61.58; H, 5.13; N, 13.41; IR (KBr,
m
cmꢁ1):
(CONH),
(ArAC@C),
(CoAN); UV–Vis (kmax
3254
1593
1309
m
m
m
(NH), 3069
(C@N), 1575
(CAO), 550–580
m
(ArACH), 2938
[(C@N)(pyridine)], 1535, 1455
(CoAO), 430–450
m(aliphatic-CH), 1645 m
The Ni(II) complex reported herein is high spin with a room
temperature magnetic moment value of 3.10 B.M. which is in the
normal range observed for octahedral Ni(II) complexes [22]. This
indicates that, the Ni(II) complex is probably octahedral [22,23].
The electronic spectrum of Ni(II) complex displayed three bands
at 446, 578 and 925 nm, assigned to 3A2g(F) ? 3T2g(F),
m
m
m
m
,
nm) (DMF): 347, 366, 464, 697 and 898; molar conductance
(DMF): 7.8 ohmꢁ1 cm2 molꢁ1
;
leff (B.M.): 3.65.
[NiC32H32N6O4]: Yield (%): 71.60, red, M.pt: 168 °C, M.Wt: 624,
Anal. Calc. (%): Ni, 9.41; C, 61.66; H, 5.17; N, 13.48. Found (%):
3
3A2g(F) ? 3T1g(F) and A2g(F) ? 3T1g(P) d–d transitions, respec-
Ni, 9.36; C, 61.55; H, 5.18; N, 13.44; IR (KBr,
(NH), 3056 (ArACH), 2975, 2920 (aliphatic-CH), 1643
(C@N), 1575 [(C@N)(pyridine)], 1546, 1455 (ArAC@C),
(CAO), 550–575 (NiAO), 440–460 (NiAN); UV–Vis (kmax
m
cmꢁ1): 3260
tively. The spectrum also showed a band at 368 nm, attributed to
ligand to metal charge transfer transition.
m
m
m
m(CONH),
1595
m
m
m
The measured magnetic moment value (3.65 B.M.) of the Co(II)
complex was lower than spin-only value (3.87 B.M.) and also lower
than the value reported for complex having octahedral geometry
[24]. The electronic spectrum of [CoL2] showed broad bands at
1310
m
m
m
,
nm) (DMF): 346, 368, 446, 578 and 925. molar conductance
(DMF): 5.5 ohmꢁ1 cm2 molꢁ1
;
leff (B.M.): 3.10.
[CuC32H32N6O4]: Yield (%): 71.33, dark brown, M.pt: 182 °C,
M.Wt: 628, Anal. Calc. (%): Cu, 10.11; C, 61.18; H, 5.13; N, 13.37.
4
464, 697 and 898 nm which are assigned to T1g(F) ? 4T2g(F),
4
4T1g(F) ? 4A2g(F) and T1g(F) ? 4T2g(P) transitions, respectively, in
Found (%): Cu, 10.07; C, 61.12; H, 5.07; N, 13.31; IR (KBr,
m
cmꢁ1): 3257
m
(NH), 3055
m
(ArACH), 2978, 2920
m
(aliphatic-CH),
[(C@N)(pyridine)], 1548,
(CuAO), 430–460
around the cobalt(II) ion. A moderately intense peak observed at
366 nm is also due to ligand to metal charge transfer transition.
The electronic spectrum of Zn(II) complex showed a band at
370 nm which is accounted for the ligand to metal charge transfer
transition.
1645
1458
m
m
(CONH), 1595
(ArAC@C), 1315
m
(C@N), 1575 m
m(CAO), 555–585
m
m
(CuAN); UV–Vis (kmax, nm) (DMF): 349, 368, 805 and 977; molar
conductance (DMF): 6.4 ohmꢁ1 cm2 molꢁ1
;
leff (B.M.): 1.91.
[ZnC32H32N6O4]: Yield (%): 70.51, yellow, M.pt: 186 °C, M.Wt:
630, Anal. Calc. (%): Zn, 10.37; C, 61.00; H, 5.11; N, 13.33. Found
(%): Zn, 10.35; C, 60.98; H, 5.13; N, 13.30; 1H NMR (DMSO-d6, d,
ppm): 2.20 (s, 6H, aliphatic-CH3), 2.75 (s, 6H, aromati-CH3), 3.62
(s, 4H, CACH2AC), 6.85–7.82 (m, 14H, ArAH), 8.60 (s, 2H, CONH).
3.3. Molar conductivity
With a view to study the electrolytic nature of the mononuclear
metal complexes, their molar conductivities were measured in
DMF at 10ꢁ3 M. The molar conductivity values of all the complexes
are in the range 4.3–7.8 ohmꢁ1 cm2 molꢁ1 (shown in Table 1),
IR (KBr,
m
cmꢁ1): 3257
(CONH), 1597
m(NH), 3062
m(ArACH), 2984, 2925
m(ali-
phatic-CH), 1642
m
m(C@N), 1575 m[(C@N)(pyridine)],

138
N. Raman et al. / Journal of Molecular Structure 1000 (2011) 135–144
Table 1
Electronic absorption spectral data of metal(II) complexes at 300 K.
Complex
[CoL2]
kmax (nm)
Band assignments
Geometry
Magnetic moment (B.M.)
3.65
Molar conductance (ohmꢁ1 cm2 molꢁ1
)
347
366
464
697
898
INCT
LMCT
4T1g(F) ? 4T2g(F)
4T1g(F) ? 4A2g(F)
4T1g(F) ? 4T2g(P)
Octahedral
7.8
[NiL2]
346
368
446
578
925
INCT
LMCT
3A2g(F) ? 3T2g(F)
3A2g(F) ? 3T1g(F)
3A2g(F) ? 3T1g(P)
Octahedral
3.10
5.5
[CuL2]
[ZnL2]
349
368
805
977
346
370
INCT
LMCT
Distorted octahedral
Octahedral
2B1g
2B1g
?
?
2E1g
2A1g
1.91
6.4
4.3
INCT
LMCT
Diamagnetic
which is in agreement with the non-electrolytic nature of the
complexes.
matic ring protons. The sharp singlet proton signal at 10.85 ppm
is assigned to aromatic AOH for pyridine nucleus of the Schiff base
ligand. In the Zn(II) complex, the aromatic AOH protons were dis-
appeared indicating the coordination of these to Zn(II) ion by
deprotonation (Fig. 1).
3.4. Infrared spectra of the ligand and its metal(II) complexes
The IR spectra of the complexes are compared with those of the
free ligand in order to determine the coordination sites that may
involve in chelation. The position and/or the intensities of these
peaks are expected to be changed upon chelation. The IR spectra
3.6. FAB-mass spectral studies of Schiff base and their metal complexes
The FAB mass spectrum of Schiff base ligand (HL) showed a
molecular ion peak [Mꢁ1] at m/z 282 which is equivalent to its
molecular weight corresponding to its formula, C16H17N3O2. In
addition, the fragment peaks observed at m/z 189, 174, 107
(M + 1), 91 and 77 are due to the fragment ions [C11H13N2O]+ꢀ,
[C10H10N2O]+ꢀ, [C7H8N]+ꢀ, [C7H7]+ꢀ, and [C6H5]+ꢀ respectively. The
FAB-mass spectrum of Cu(II) complex showed a molecular ion peak
(M+) at m/z 628 that is equivalent to its molecular weight having
the formula CuC32H32N6O4. Thus, the mass spectral data results
along with elemental analyses agree with the formation of [ML2]
complexes of 1:2 stoichiometry. Therefore, the above spectral
studies confirmed the structure of ligand and its complexes as gi-
ven by Scheme 1. This is further confirmed by the mass spectra
of other complexes.
of the Schiff base exhibited a characteristic band due to m(OH) in
the region 3383 cmꢁ1. The sharp band at 3255 cmꢁ1 is due to the
stretching frequency for amide (NH) bond of the free ligand [25].
Furthermore, the characteristic high intensity band in the region
1612 cmꢁ1 is assigned to
m
(C@N) respectively [26]. In the IR spectra
of the Schiff base, a high intensity band in the region 1255 cmꢁ1
and a band around 1655 cmꢁ1 are assigned to
(CAO) for aromatic
pyridine nucleus and
(C@O) for amide carbonyl carbon of 20-
m
m
methylacetoacetanilide moiety [27], respectively.
In comparison with the spectra of the Schiff base and its all the
metal(II) complexes exhibited the band of m(C@N) in the region
1593–1597 cmꢁ1 showing the shift to lower wave numbers indicat-
ing that, the nitrogen of azomethine group is coordinated to the me-
tal ion [26,28]. A medium to high intensity band appeared in the
region 1309–1315 cmꢁ1 due to phenolic
m
(CAO) indicates the coor-
dination of phenolic oxygen via deprotonation [27]. The higher side
shift of (CAO) in the metal complexes may be due to the expected
high mesomeric interaction in the complex that is probably acti-
vated by the presence of the metal ion. The (C@O) band of the li-
3.7. EPR spectra of Cu(II) and Ni(II) complexes
m
EPR spectral studies of Cu(II) complex provides information of
the metal ion environment. The EPR spectrum of the Cu(II) com-
m
gand at 1655 cmꢁ1 for the amide carbonyl group was shifted by
10–13 cmꢁ1 to lower energies in the spectra of the complexes. This
indicates that the amide carbonyl oxygen is bonded to the metallic
ions [29,30]. The new bands in 550–585 and 430–460 cmꢁ1 region
observed for all the complexes are assigned to stretching vibrations
of (MAO) and (MAN) bonds, respectively [31].
3.5. 1H NMR spectral studies of Schiff base and its zinc(II) complex
The 1H NMR spectra of the Schiff base ligand (HL) and its dia-
magnetic Zn(II) complex recorded in CDCl3 and DMSO-d6 respec-
tively showed a sharp signal in the region 2.20 and 2.75 ppm
which may be assigned to the aliphatic and aromatic methyl pro-
tons (ACH3). However, the singlet at 8.60 ppm may be attributed
to the pendant secondary amide proton (CANHAC) of Schiff base
ligand and its Zn(II) complex. The characteristic multiplet at
3.62 ppm may be assigned to the methylene protons of (CACH2AC)
and a sharp multiplet observed in the range 6.85–7.82 for Schiff
base ligand and its Zn(II) complex may be attributed to the aro-
Fig. 1. 1H NMR spectrum of Zn(II) complex.

N. Raman et al. / Journal of Molecular Structure 1000 (2011) 135–144
139
sidered the existence of some exchange interactions between the
Cu(II) centers and if G > 4, the exchange interactions are neglected.
Thus, in case of Cu(II) complex, the geometric parameter G = 3.28
confirms the existence of some exchange interactions between
the Cu(II) centers.
It has been shown that g|| is a moderately sensitive function for
indicating covalency [34]. Relatively speaking g|| > 2.3 is character-
istic of an ionic environment and g|| < 2.3 of a covalent environ-
ment in MAL bonding. In copper complex, g|| < 2.3 was observed
which indicates a fair degree of covalent character in the CuAL
bonding.
The EPR parameters and d–d transition energies were used to
evaluate the bonding parameter
garded as measure of the covalency of the in-plane
and the in-plane - and out-of-plane bonding, respectively.
The in-plane
bonding parameter a2 was calculated by using the
expression [35].
a c
2, b2 and 2, which may be re-
r
bonding
p
p
r
a2 ¼ ꢁðA =0:036Þ þ ðg ꢁ 2:0027Þ þ ð3=7Þðg? ꢁ 2:0027Þ þ 0:04
k
k
The orbital reduction factors K|| and K\ were estimated from the
expression [36].
K2k ¼ ðg ꢁ 2:0027Þd—d transition=8k0
k
K2? ¼ ðg? ꢁ 2:0027Þd—d transition=2k0
where K|| = a2b2, K\
=
a2c2 and k0 represents the one electron spin-
orbit coupling constant for the free ion, equal to ꢁ828 cmꢁ1. Signif-
icant information about the nature of bonding in the copper(II)
complex can be derived from the magnitude of K|| and K\. In case
of pure
r
bonding K|| ꢄ K\ ꢄ 0.77 whereas K|| < K\ implies consider-
able in-plane bonding, while for out-of-plane bonding, K|| > K\. In
the present copper(II) complex, it is observed that K|| < K\ which
indicates the presence of significant in-plane bonding. K is dimen-
sionless quantity, which is a measure of the contribution of s elec-
trons to the hyperfine interaction and is generally found to have a
value of 0.30. The K value obtained for Cu(II) complex is in agree-
ment with those estimated by Assour [37] and Abragam and Pryce
[38].
The covalency of the in-plane
plete ionic character, whereas
r
a
-bonding,
a
2 = 1 indicates com-
2 = 0.5 denotes 100% covalent
bonding, with the assumption of negligibly small values of the
overlap integral. The b2 parameter gives an indication of the cova-
lency of the in-plane
covalency of the bonding. The values of a2 and b2 for the complex
indicate that the in-plane -bonding and in-plane -bonding are
appreciably covalent, and are consistent with very strong in-plane
-bonding in the complex. For the copper(II) complex, the high val-
ues of b2 compared to a2 indicate that the in-plane
-bonding is
less covalent than the in-plane -bonding. These data are well con-
p
-bonding. The smaller the b2 the larger is the
r
p
r
Scheme 1. Synthesis of Schiff base ligand and its metal(II) complexes.
p
r
sistent with other reported values [39,40].
plex was recorded in DMSO at liquid nitrogen temperature (LNT)
and at room temperature (RT). The EPR spectrum at RT showed
one intense absorption band in the high field region, isotropic
due to the tumbling motion of the molecules. However, the com-
plex at LNT showed four well resolved peaks in low field region.
The g|| and g\ values were found to be 2.22 and 2.07, respectively
(Table 2). The trend g|| > g\ > 2.0027 observed for the complex indi-
cates that the unpaired electron is localized in the dx2–y2 orbital of
the Cu(II) ion, characteristic for the axial symmetry [32]. The gav
and g||/A|| was calculated to be 2.12 and 261 respectively. Thus,
the results confirmed that, the Cu(II) complex exhibits distorted
octahedral geometry. From the values of the g factors the geomet-
ric parameter G, representing a measure of the exchange interac-
tion between the Cu(II) centers in the compound was determined
following the formula G = (g|| ꢁ 2)/(g\ ꢁ 2) [33]. If G < 4, it is con-
The EPR spectrum of nickel(II) complex recorded in DMSO solu-
tion at room temperature (RT) exhibits an isotropic and unresolved
signal centered at giso = 2.14 corresponding to an S = 1 state. This
giso value is close to that of typical nickel(II) complex (g ꢄ 2.13–
2.17) [41]. The EPR spectrum of Ni(II) complex recorded at liquid
nitrogen temperature (LNT) corresponds to two well resolved
hyperfine splitting and the observed g values are g\ = 2.18 and
g|| = 2.12 and gav = 2.16. In general, d8 low-spin nickel(II) (S = 1)
complex shows anisotropic signals at g\ = 2.2 and g|| = 2.0 for
tetragonally elongated octahedral distortion [42–44]. The pattern
of g values [g\ (2.18) > g|| (2.12) > ge] is indicative of a dz2 ground
state, where the z-axis is perpendicular to the plane of the mole-
cule. Thus, the EPR study of the copper(II) and nickel(II) complexes
provided supportive evidence to the conclusion obtained on the
basis of electronic spectrum and magnetic moment values.

140
N. Raman et al. / Journal of Molecular Structure 1000 (2011) 135–144
Table 2
Spin Hamiltonian parameters of Cu(II) complex in DMSO solution.
Complex
[CuL2]
g-tensors
g||
Hyperfine constant ꢅ 10ꢁ4 cmꢁ1
Bonding parameters
g\
gav
A||
A\
Aav
g||/A||
G
a2
b2
c2
K||
K\
K
2.22
2.07
2.12
85
140
121.66
261
3.28
0.52
0.79
0.97
0.34
0.50
0.22
3.8. DNA binding studies
3.8.1. Absorption spectral features of DNA binding
complexes are all accompanied by a small red shift by about 4, 4,
5 and 2 nm respectively. The spectroscopic changes suggest that
the intrinsic binding constants (Kb) of the Co(II), Ni(II) Cu(II) and
Titration with electronic absorption spectroscopy is an effective
method to investigate the binding mode of DNA with metal com-
plexes [45]. Transition metal complexes can bind to DNA via both
covalent and/or non-covalent interactions. In the case of covalent
binding, the labile ligand of the complexes can be replaced by a
nitrogen base of DNA such as guanine N7, while the non-covalent
DNA interactions include intercalative, electrostatic and groove
(surface) binding of metal complexes outside of DNA helix, along
major or minor groove. In the intercalative binding mode, the p⁄
Zn(II) complexes are 3.62 ꢅ 104 Mꢁ1
,
3.83 ꢅ 104 Mꢁ1
,
8.29 ꢅ
104 Mꢁ1 and 2.66 ꢅ 104 Mꢁ1, respectively (shown in Table 3). The
results indicate that the binding strength of complex is increasing
in the order of Cu(II) > Ni(II) > Co(II) > Zn(II). The Kb value obtained
here is lower than that reported for classical intercalators (for ethi-
dium bromide and [Ru(phen)(dppz)] whose binding constants have
been found to be in the order of 106–107 Mꢁ1 [49,50]. These results
suggest an intimate association of the compounds with CT-DNA and
it is also likely that compounds bind to the helix via intercalative
mode.
orbital of the intercalated ligand can couple with the
the DNA base pairs, thus, decreasing the
p⁄ transition energy
and resulting in the bathochromism. On the other hand, the cou-
pling orbital is partially filled by electrons, thus, decreasing the
p orbital of
p
?
3.8.2. DNA binding of electrochemical behavior
p
The application of cyclic voltammetry (CV) to the study of bind-
ing of metal complexes to DNA provides a useful complement to the
method for the investigation of electronic absorption spectra [51].
Cyclic voltammogram of Cu(II) complex in the absence and pres-
ence of CT-DNA is shown in Fig. 3. As seen in this figure, in the ab-
sence of CT-DNA, the cyclic voltammogram featured two anodic
peaks Epa (0.368 and ꢁ0.320 V) and two cathodic peaks Epc
(0.113 and ꢁ0:765 V) at 0.1 V sꢁ1. The first reduction and oxidation
potential observed at Epc = 0.113 V and Epa = 0.368 V is assigned to
the redox couple Cu(III)/Cu(II). The second reduction and oxidation
potential observed at Epc = ꢁ0:765 V and Epa = ꢁ0.320 V can be
attributed to the redox couple Cu(II)/Cu(I) (Table 4). The ratio of
Ipa/Ipc was less than unity for the above two redox couples. This also
indicates that two quasi-reversible one-electron transfer reduction
process are involved in copper(II) complex.
transition possibilities and concomitantly resulting in the hypo-
chromism [46]. Generally, the binding of an intercalative molecule
to DNA is accompanied by hypochromism and/or significant red-
shift (bathochromism) in the absorption spectra due to the strong
stacking interaction between the aromatic chromophore of the li-
gand and DNA base pairs with the extent of hypochromism and
red-shift commonly consistent with the strength of the intercala-
tive interaction [47]. Therefore, in order to obtain evidence for
the binding ability of each compound to CT DNA, spectroscopic
titration of compound solutions with CT DNA should be performed
[48].
The electronic absorption spectra of copper complex in the ab-
sence and presence of CT DNA are given in Fig. 2. With increasing
concentration of CT DNA, for the Co(II), Ni(II), Cu(II) and Zn(II) com-
plexes, the absorption bands at 366, 368, 368 and 370 nm respec-
tively also show hypochromism of 15.95%, 16.26%, 32.45% and
8.25%, respectively. The hypochromism observed for the metal
In the presence of CT-DNA, the cyclic voltammogram of the cop-
per(II) complex exhibited shifts in the anodic and cathodic peak
potentials followed by decrease in peak currents, indicating the
interaction existing between the copper(II) complex and CT-DNA.
The drop of the voltammetric currents in the presence of DNA
can be attributed to diffusion of the metal complex bound to the
large, slowly diffusing DNA molecule [52]. The E1/2 values in the
presence of DNA exhibit negative shifts of 0.216 and ꢁ0.516 V
for copper(II) complex. The shift in the value of the formal poten-
0
tial (D
E0 ) can be used to estimate the ratio of equilibrium binding
constants (KR/KO) according to the model of interaction described
by Carter and Bard [53]. From this model one can obtain
D
E00 ¼ E0b0 ꢁ Ef00 ¼ 0 : 059 log KR=KO
where E0b0 and E0f 0 are the formal potentials of the bound and free
complex forms, respectively, and KR and KO are the corresponding
binding constants for the binding of reduction and oxidation species
Table 3
Absorption properties of metal(II) complexes.
Complex kmax(nm)
D
k (nm) Hypocromocity (%) Kb ꢅ 104 (Mꢁ1
)
Free
Bound
[CoL2]
[NiL2]
[CuL2]
[ZnL2]
366.0 370.0
368.0 372.0
368.0 373.0
370.0 372.0
4.0
4.0
5.0
2.0
15.95
16.26
32.45
8.25
3.62
3.83
8.29
2.66
Fig. 2. Absorption spectra of copper(II) complex (10 lM) in the absence and
presence of increasing amounts of CT-DNA at room temperature in 50 mM Tris-HCl/
NaCl buffer (pH = 7.2). Arrow shows the absorbance changing upon increasing DNA
concentration.

N. Raman et al. / Journal of Molecular Structure 1000 (2011) 135–144
141
strongly to CT DNA and the experimental values for all the com-
plexes are tabulated in Table 4.
The above results of metal-DNA interaction by the cyclic vol-
tammogram studies confirm that Cu(II) complex bound to DNA
via intercalation as well as electrostatic binding mode whereas
Ni(II), Co(II) and Zn(II) complexes were bound through electro-
static binding mode.
3.8.3. Viscosity measurements
In order to further clarify the interaction nature between the
compounds and DNA, viscosity measurements were carried out.
Optical photophysical probes provide necessary but not sufficient
evidence to support a binding model. Hydrodynamic measure-
ments that are sensitive to length change (i.e. viscosity and sedi-
mentation) are regarded as the least ambiguous and the most
critical tests of a binding model in solution in the absence of crys-
tallographic structural data [54]. A classical intercalation model
demands that the DNA helix must lengthen as base pairs are sep-
arated to accommodate the binding complexes, leading to the in-
crease of DNA viscosity, as for the behaviors of the known DNA
intercalators [55]. In contrast, a partial and/or non-classical inter-
calation of the complex could bend (or kink) the DNA helix, reduc-
ing its viscosity concomitantly. In addition, some complexes such
as [Ru(bpy)3]2+, which interact with DNA by an electrostatic bind-
ing mode, have no influence on DNA viscosity [56]. The effects of
all the compounds on the viscosity of CT DNA are shown in
Fig. 4. The viscosity measurements clearly show that all the com-
pounds can intercalate between adjacent DNA base pairs, causing
an extension in the DNA helix and thus increasing the viscosity
of DNA with an increasing concentration of the complexes. On
the basis of all the spectroscopic studies together with the viscosity
measurements, we find that the metal complexes can bind to CT
DNA via an intercalative mode.
Fig. 3. Cyclic voltammagram of copper(II) complex in the absence and presence of
increasing amounts of CT-DNA at room temperature in DMF:buffer [mixture 50 mM
Tris–HCl/NaCl buffer (pH = 7.2)] (1:2) solution (Scan rate = 0.1 V sꢁ1) and 0.1 M
Bu4NClO4 as supporting electrolyte. Arrow shows the current changing upon
increasing DNA concentration.
to DNA, respectively. The KCu(II)/KCu(III) and KCu(I)/KCu(II) values for the
copper(II) complex are 0.25 and 4.2, suggesting the stronger binding
affinity in the Cu(II) species compared to the Cu(I) species for the
copper(II) complex.
In the absence of CT DNA, cyclic voltammogram of Ni(II) com-
plex exhibits a quasi-reversible redox wave corresponding to
Ni(II)/Ni(I) with Epc and Epa values ꢁ0.867 and ꢁ0.558 V, respec-
tively at the scan rate of 0.1 V sꢁ1. The ratio of anodic to cathodic
peak current value was less than one. The formal electrode poten-
tials E1/2, DEp (difference in cathodic Epc and anodic Epa peak poten-
3.9. Chemical nuclease activity
tials) were 0.309 and ꢁ0.712 V, respectively. The addition of CT
DNA to Ni(II) complex, the cathodic and anodic values were shifted
to ꢁ0.882 and ꢁ0.571 V, and also the formal electrode potential
There has been considerable interest in DNA cleavage reactions
that are activated by transition metal complexes. The delivery of
metal ion to the helix, in locally generating oxygen or hydroxide
radicals, yields an efficient DNA cleavage reaction. In order to as-
sess the chemical nuclease activities of the Co(II), Ni(II), Cu(II)
and Zn(II) complexes for DNA strand scission, pBR322 DNA was
incubated with all the above metal(II) complexes under the reac-
tion conditions. The cleavage reaction can be monitored by gel
electrophoresis. When circular pBR322 DNA is subjected to elec-
trophoresis, relatively fast migration will be observed for the intact
supercoiled form (Form I). If scission occurs on one strand (nick-
ing), the supercoiled form will relax to generate a slower-moving
nicked form (Form II). If both strands are cleaved, a linear form
(Form III) that migrates between Form I and Form II will be
generated.
values were shifted to E1/2 = 0.311 V and
D
Ep = ꢁ0.727 V, respec-
tively. The ratio of Ipa/Ipc was also less than one for CT DNA bound
Ni(II) complex. The shift in potentials and decrease in current ratio
suggest the binding of Ni(II) complex to CT DNA. The KNi(I)/KNi(II)
value for the complex is 0.55, suggesting that the stronger binding
affinity in the Ni(II) state compared to the Ni(I) state for the nick-
el(II) complex.
In the cyclic voltammograms of Co(II) and Zn(II) complexes, the
single irreversible cathodic peak values observed for each complex
are ꢁ0.484 and ꢁ0.625 V respectively in the absence CT DNA and
also in the presence of CT DNA, the shift in the irreversible cathodic
values are ꢁ0.509 and ꢁ0.652 V respectively, at the scan rate of
0.1 V sꢁ1. The ratio of equilibrium constants for Co(II) and Zn(II)
complexes are found to be 0.38 and 0.35, respectively. From the
voltammogram data, it is concluded that all the complexes bind
The chemical nuclease activities of the four complexes have
been studied using supercoiled pBR322 plasmid DNA as a substrate
Table 4
Electrochemical behavior of metal(II) complexes in the absence and presence of CT DNA.
Complex
Redox couple
Epc (V)
Epa (V)
D
Ep (V)
E1/2 (V)
KR/KO
Free
Bound
Free
Bound
Free
Bound
Free
Bound
[CoL2]
[NiL2]
[CuL2]
Co(II)/Co(I)
Ni(II)/Ni(I)
Cu(III)/Cu(II)
Cu(II)/Cu(I)
Zn(II)/Zn(I)
ꢁ0.484
ꢁ0.867
0.113
ꢁ0.509
ꢁ0.882
0.078
–
–
–
–
–
–
0.38
0.55
0.25
4.22
0.35
ꢁ0.558
0.368
ꢁ0.320
–
ꢁ0.571
0.354
ꢁ0.305
–
ꢁ0.712
0.255
0.445
–
ꢁ0.727
0.276
0.423
–
0.309
0.240
ꢁ0.543
–
0.311
0.216
ꢁ0.516
–
ꢁ0.765
ꢁ0.728
[ZnL2]
ꢁ0.625
ꢁ0.652
E1/2 = Epc + Epa and
DEp = Epa – Epc.

142
N. Raman et al. / Journal of Molecular Structure 1000 (2011) 135–144
Fig. 4. Change in relative specific viscosity of CT-DNA (100 lM) on addition of
Scheme 2. Mechanistic pathways proposed for the chemical nuclease activity by all
[CoL2] (ꢅ), [NiL2] (ꢁ), [CuL2] (N) and [ZnL2] (j) in 50 mM Tris–HCl/NaCl buffer
the four complexes.
(pH = 7.2) at 37 °C.
as the reactive species in the cleavage reaction. The DNA cleavage
reaction of the complexes in the presence of MPA probably pro-
ceeds through the oxidative hydroxyl radical pathway in a similar
way as proposed by Sigman and co-workers (Scheme 2) [58].
in a medium of 50 mM Tris–HCl/NaCl buffer (pH = 7.2) in the pres-
ence of 3-mercaptopropionic acid under physiological conditions.
There was no DNA-cleavage observed for the controls in which
either in the absence of MPA with DNA or in the presence of
MPA with DNA (Lanes 1 and 2). In Fig. 5, all the complexes were
found to exhibit nuclease activity in the presence of 3-mercapto-
propionic acid (MPA) as reducing agent (Lanes 3–6). The different
DNA-cleavage efficiency of the complexes may be due to the differ-
ent binding affinity of the complexes to DNA. The nuclease effi-
ciency of the complexes is known to depend on the reducing
agent used for initiating the DNA cleavage. Sigman and co-workers
have found that 3-mercaptopropionic acid is superior to ascorbic
acid as a reducing agent on the nuclease activity of a bis(o-phenan-
throline)copper(II) complex because it produces less background
cleavage [57].
In order to obtain information about the active oxygen species,
responsible for the DNA damage, we investigated the DNA cleavage
in the presence of hydroxyl radical scavengers (DMSO, EtOH), sin-
glet oxygen quenchers (NaN3) and superoxide scavenger (SOD) un-
der our experimental conditions. From Fig. 5, it is seen that no
obvious inhibitions are observed for the Cu(II) complex in the pres-
ence of SOD (Lane 7), the results rule out the possibility of DNA
cleavage by superoxide. Addition of singlet oxygen quencher
NaN3 (Lane 8) do not show any appreciable inhibitory effect on
the chemical nuclease activity by the complex, so there is no
involvement of singlet oxygen of DNA cleavage process. The addi-
tion of DMSO (Lane 9), EtOH (Lane 10) partly diminishes the nucle-
ase activity of the Cu(II) complex which is indicative of the
involvement of hydroxyl radical and/or ‘‘metal-oxo’’ intermediate
3.10. Antimicrobial activity
The biological activities of the newly synthesized Schiff base
and its metal complexes have been studied for their antibacterial
and antifungal activities by disk diffusion method [59]. The mini-
mum inhibitory concentration (MIC) was determined by assaying
at serial dilution of concentrations along with standards at the
same concentrations. The antimicrobial results of Schiff base and
its metal complexes are systematized in Table 5. The biological
activity of the Schiff base exhibited a considerable enhancement
on coordination with the metal ions against all bacterial strains
and fungal strains. In case of antibacterial studies it was observed
that, copper complex was found to be potentially much enhanced
activity active against S. aureus (MIC = 25
25 g/mL) and zinc complex was found to be higher activity
against S. aureus (MIC = 25 g/mL) as compared to the Schiff base
(HL). But copper and zinc complexes were found to have moderate
activity (MIC = 25–100 g/mL) and cobalt and nickel complexes
were found to have low activity (MIC = 50–200 g/mL) against
lg/mL), E. coli (MIC =
l
l
l
l
some of the microorganisms respectively, as compared to the stan-
dard drug (Streptomycin). The ligand and metal complexes showed
low antifungal activity against all the species, except copper com-
plex which was found to have moderate activity against A. niger
(MIC = 25
lg/mL), as compared to the standard (Nystatin).
However, they showed low antifungal activity against most of
Fig. 5. Cleavage of SC pBR322 DNA (0.2
Tris–HCl/NaCl buffer (pH = 7.2). Lane 1, DNA control; Lane 2, DNA + MPA; Lanes 3–6, DNA + MPA + [CuL2], [NiL2], [CoL2] and [ZnL2], respectively; Lane 7, DNA + MPA + SOD (4
units) + [CuL2]; Lane 8, DNA + MPA + NaN3 (0.5 mM); Lane 9, DNA + MPA + DMSO (4 L) + [CuL2]; Lane 10, DNA + MPA + ethanol (0.5 mM) + [CuL2].
lg, 30 lM) by four metal(II) complexes (0.30 mM) in the presence of reducing agent 3-mercaptopropionic acid (0.70 mM) in 50 mM
l

N. Raman et al. / Journal of Molecular Structure 1000 (2011) 135–144
143
Table 5
The in vitro antimicrobial activity of ligand and their metal(II) complexes evaluated by MIC (Minimum Inhibitory Concentration,
l
g/mL).
Complex
Antibacterial activity
Antifungal activity
S. aureus
B. subtilis
E. coli
P. aeruginosa
A. niger
R. bataicola
R. stolonifer
C. albicans
HL
[CoL2]
[NiL2]
[CuL2]
[ZnL2]
Streptomycin
Nystatin
200
100
100
25
25
5
400
100
200
50
100
10
200
50
100
25
50
5
400
200
100
50
100
10
200
50
100
25
50
–
400
100
200
100
100
–
400
200
200
50
100
–
600
400
400
200
100
–
–
–
–
–
5
10
10
10
the species. A comparative study of MIC values of the Schiff base
and its complexes indicate that, generally, the metal complexes
have a better activity than the free ligand. This is probably due
to the greater lipophilic nature of the complexes. It was evident
from the data that this activity significantly increased on coordina-
tion. This enhancement in the activity may be rationalized on the
basis that their structures mainly possess an additional C@N bond.
It has been suggested that the Schiff base with nitrogen and oxygen
donor systems inhibit enzyme activity, since the enzymes which
require these groups of their activity appear to be especially more
susceptible to deactivation by metal ions on coordination. More-
over, coordination reduces the polarity of the metal ion mainly be-
cause of the partial sharing of its positive charge with the donor
groups [60,61] within the chelate ring system formed during coor-
dination. This process, in turn, increases the lipophilic nature of the
central metal atom, which favors its permeation more efficiently
through the lipid layer of the microorganism [62] thus destroying
them more aggressively. The toxicity of the complexes can be re-
lated to the strength of the metal–ligand bond, besides other fac-
tors such as size of the cation, receptor sites, diffusion and a
combined effect of the metal and the ligands for inactivation of
the biomolecules.
Acknowledgements
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
also thank the Sophisticated Analytical and Instrumentation
Facility, Central Drug Research Institute, Lucknow, for providing
CHN-analysis data and FAB-Mass spectra and Sophisticated Analyt-
ical and Instrumentation Facility, Indian Institute of Technology,
Bombay, for EPR measurements.
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The present paper reports on the synthesis, characterization
and their electronic absorption spectra of Schiff base ligand and
its metal complexes. The synthetic procedure in this work resulted
in the formation of complexes in the molar ratio (1:2) (M:L),
respectively. In these complexes, the tridentate Schiff base ligand
is coordinated to metal ion through the azomethine-nitrogen and
phenolic-oxygen atoms forming stable five membered heterocyclic
rings. The present study reveals that octahedral geometry around
Co(II), Ni(II) and Zn(II) complexes whereas Cu(II) complex exhibits
distorted octahedral geometry. The conductance measurements
show that all the metal complexes are non-electrolytes in nature.
The DNA binding study takes place via an intercalative mode; cop-
per complex has stronger binding strength as compared to the
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supercoiled Form I to the open circular Form II or the linear Form
III in the presence of MPA. These findings clearly indicate that
transition metal based complexes have many potential practical
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