Synthesis, characterization, antibiogram and DNA binding studies of novel Co(II), Ni(II), Cu(II), and Zn(II) complexes of Schiff base ligands with quinoline core

Article abstract of DOI:10.1007/s00044-010-9330-5

A series of Co(II), Ni(II), Cu(II), and Zn(II) complexes of Schiff base ligands L¹H₃ and L²H have been prepared. The ligands are synthesized by the condensation of 2-hydroxy-3-formylquinoline with salicyloylhydrazide and 2-hydrazinobenzothiazole in absolute ethanol. The prepared complexes were characterized by the analytical and spectral techniques. The stoichiometry of the complexes is found to be 1:1. The presence of coordinated and lattice water is confirmed by the TG and DTA studies. Subsequently all the prepared complexes were screened for antimicrobial activity against bacteria and fungi. The Cu(II) complexes have been found to be more active than the ligand. In addition the DNA binding/cleaving capacity of the compounds was analyzed by absorption spectroscopy, viscosity measurement, thermal denaturation, and gel electrophoresis methods.

Full text of DOI:10.1007/s00044-010-9330-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:421–429
DOI 10.1007/s00044-010-9330-5
ORIGINAL RESEARCH
Synthesis, characterization, antibiogram and DNA binding studies
of novel Co(II), Ni(II), Cu(II), and Zn(II) complexes of Schiff base
ligands with quinoline core
•
Gurunath S. Kurdekar Sathisha Mudigoudar Puttanagouda
•
•
Naveen V. Kulkarni Srinivasa Budagumpi
•
Vidyanand K. Revankar
Received: 13 August 2009 / Accepted: 16 February 2010 / Published online: 12 March 2010
Ó Springer Science+Business Media, LLC 2011
Abstract A series of Co(II), Ni(II), Cu(II), and Zn(II)
complexes of Schiff base ligands L1H3 and L2H have been
prepared. The ligands are synthesized by the condensation
of 2-hydroxy-3-formylquinoline with salicyloylhydrazide
and 2-hydrazinobenzothiazole in absolute ethanol. The
prepared complexes were characterized by the analytical
and spectral techniques. The stoichiometry of the com-
plexes is found to be 1:1. The presence of coordinated and
lattice water is confirmed by the TG and DTA studies.
Subsequently all the prepared complexes were screened for
antimicrobial activity against bacteria and fungi. The
Cu(II) complexes have been found to be more active than
the ligand. In addition the DNA binding/cleaving capacity
of the compounds was analyzed by absorption spectros-
copy, viscosity measurement, thermal denaturation, and gel
electrophoresis methods.
sites in heterocyclic rings. Among the ligand systems,
quinoline derivatives of hydrazone occupy special place
because, these ligands developed due to their diverse
chelating capability, structural flexibility (Rao et al., 1997)
and pharmacological activities like antibacterial, anti-
fungal, antitumoural, antiviral, antimalarial, antituberculo-
sis (West et al., 1993). In addition to this, the interaction of
these Schiff base metal complexes with DNA has been
extensively studied in the past decades. Due to the site-
specific binding properties and many fold applications in
cancer therapy, these coordination compounds were suit-
able candidates as DNA secondary structure probes, photo
cleavers, and antitumor drugs (Chan and Wong, 1995;
Pratviel et al., 1998; Liang et al., 2004). The design of
small complexes that bind/react at site-specific sequences
of DNA becomes important as they can draw significant
structural perturbations in the host. DNA is an important
cellular receptor, many chemicals exert their antitumor
effects through binding to DNA thereby changing the
replication of DNA and inhibiting the growth of the tumor
cells. This is the fundamental facet of designing new and
more efficient antitumor drugs and their effectiveness
depends on the mode and affinity of the binding. A more
complete understanding of how to target DNA sites with
specificity will lead not only to novel chemotherapeutics
but also to a greatly expanding ability for chemists to probe
DNA and to develop highly sensitive diagnostic agents
(Erkkila et al., 1999).
Keywords Quinoline ꢀ Salicyloylhydrazide ꢀ
2-Hydrazinobenzothiazole ꢀ Antimicrobial activity
DNA binding study
Introduction
Schiff base ligands have been studied extensively for years,
due to the synthetic flexibilities, selectivity as well as
sensitivity toward the transition metal ions. The architec-
tural beauty of these coordination complexes arises due to
the interesting ligand systems containing different donor
The coordination of quinoline has gained increasing
attention as antibacterials (Freixas and Span, 1991; Ryoichi
et al., 1991), monoamine oxidase inhibitors (Gracheva
et al., 1988), and herbicides (Hagen et al., 1988). On the
other hand, substituted quinolines are prominent building
blocks in both organic and inorganic molecular chemistry
with their p-stacking ability and coordination properties.
G. S. Kurdekar ꢀ S. Mudigoudar Puttanagouda ꢀ
N. V. Kulkarni ꢀ S. Budagumpi ꢀ V. K. Revankar (&)
Department of Chemistry, Karnatak University, Pavate Nagar,
Dharwad 580 003, Karnataka, India
e-mail: vkrevankar@rediffmail.com
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Med Chem Res (2011) 20:421–429
Specially, the 3-substituted 2-hydroxyquinolines show
tautomeric forms at 2- position of the heterocycle and
easily form a lactum tautomer. The interesting thing in the
study of these compounds is the existence of lactum tau-
tomer in presence of a transition metal ion. Furthermore,
the ligands designed in the present investigation, in such a
way, the complexes derives are associated at least with one
labile chloride to interact with the N-7 of nucleotide after
deprotonation. These observations prompted us to prepare
a series of quinoline Schiff base later first row transition
metal (II) complexes with the 3-substituted 2-hydroxy-
quinoline derivatives.
complex in solid form. The product was filtered off,
washed several times with ethanol, and dried in vacuum
over P2O5.
Biochemistry
Anti-biogram analysis against bacteria and fungi is done as
per the well reported method (Seeley 1991).
The samples showing significant inhibition were selec-
ted for the further determination of (Minimum Inhibitory
Concentrations) MICs.
DNA binding studies
Experimental
Isolation of DNA DNA was isolated using the procedure
mentioned below:
Chemistry
The fresh bacterial culture (1.5 ml) was centrifuged to
obtain the pellet. So obtained pellet was dissolved in 0.5 ml
of lysis buffer (100 mM tris pH 8.0, 50 mM EDTA, 50 mM
lysozyme) and 0.5 ml of saturated phenol was added to it
and incubated at 55°C for 10 min, and then centrifuged at
10,000 rpm for 10 min. To the supernatant liquid equal
volume of chloroform: isoamyl alcohol (24:1) mixture and
1/20th volume of 3 M sodium acetate (pH 4.8) was added.
Again it was centrifuged at 10,000 rpm for 10 min and to
the supernatant 3 volumes of chilled absolute alcohol
added. The precipitated DNA was separated by centrifu-
gation. The pellet was dried and dissolved in TE buffer
(10 mM tris pH 8.0, 1 mM EDTA) and stored in cool place.
Materials and methods
All the chemicals used were of reagent grade and the sol-
vents were distilled before use according to the standard
procedure. The preparation of 2-hydroxy-3-formylquino-
line (Meth-Cohn and Narine, 1978), salicyloylhydrazide
(Vogel, 1968), and 2-hydrazinobenzothiazole (Revankar
et al., 1990) were prepared according to the reported
methods. The metal salts used were in the hydrated form.
Preparation of the Schiff base ligands
A hot ethanolic solution of 2-hydroxy-3-formylquinoline
(0.005 mol, 25 ml), salicyloylhydrazide (0.005 mol, 25 ml)
was added in ethanolic solution, which was refluxed for
about 3-4 h on a water bath. The resulting solid product was
filtered in hot condition [yield- 80%].
Concentration measurement The concentration of E. coli
DNA per nucleotide [C(p)] was measured by using its
known extinction coefficient at 260 nm (6600 M–1 cm-1
)
(Sarma, 1980). The absorbance at 260 nm (A260) and at
280 nm (A280) for E. coli was measured to check its
purity. The ratio A260/A280 was found to be 1.84, indi-
cating that E. coli was satisfactorily free from protein.
Buffer [5 mM tris(hydroxymethyl) amino methane, tris, pH
7.2, 50 mM NaCl] was used for the absorption, viscosity,
and thermal denaturation experiments.
The same procedure was followed for the preparation of
the ligand L2H by using 2-hydrazinobenzothiazole, [yield-
70%] (The structure of both the ligands shown in Fig. 1).
Preparation of complexes
To the hot solution of ligand in ethanol (0.004 mol in
25 ml), an ethanolic solution of the metal chloride
(0.004 mol in 25 ml) was added drop wise and stirred for
an hour. The mixture was heated to reflux for 3–5 h to get
Absorption studies
In absorption studies the complex was dissolved in DMSO
and then diluted to the desired concentration with water.
Fig. 1 Structure of the ligands
H
O
H
H
S
N
N
N
N
H
N
N
OH
N
OH
(L1H3 )
HO
(L2H)
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Med Chem Res (2011) 20:421–429
423
The spectroscopic titrations were carried out by adding
increasing amounts of E. coli DNA to a solution of the
complex at a fixed concentration contained in a quartz cell,
UV-Vis spectra were recorded after equilibration at 23°C
for 10 min after each addition. The intrinsic binding con-
stant Kb was determined from the plot of A0/[A - A0] vs.
[DNA]-1 according to equation (Dang et al., 1998).
temperature was scanned from 25 to 80°C at a speed of 5°C
per min. The melting temperature (Tm) was taken as the
mid-point of the hyperchromic transition.
DNA cleavage experiment
Culture media: Potato dextrose broth (Peptone 10, NaCl 10
and yeast extract 5 g/l) was used for the growth of the
E. coli. The 50 ml media was prepared, autoclaved for
15 min at 121°C and 15 lb pressure. The autoclaved media
was inoculated with the seed culture and incubated at 37°C
for 24 h.
A0=½A ꢁ A0ꢂ ¼ eG=½eHꢁG ꢁ eGꢂ þ eG=½eHꢁG ꢁ eGꢂ
ꢃ 1=K½DNAꢂ
ð1Þ
where A0 and A are the absorbance observed for MLCT
absorption band for the free complex and absorbance
observed for MLCT absorption band at given DNA con-
centration, respectively. [DNA] is the concentration of
DNA in base pairs, eG and eH-G are the apparent absorption
coefficients in free and DNA bounded form of complex,
respectively. The data were fitted into the above equation
to obtain a graph, with a slope equal to eG=½eHꢁG ꢁ eGꢂ ꢃ
1=K and intercept equal to eG=½eHꢁG ꢁ eGꢂ hence Kb was
obtained from the ratio of the intercept to the slope.
Treatment of DNA with the samples: The samples
(10 mg/ml) were prepared in DMSO. The synthesized
compounds (100 lg) were added separately to the DNA
sample of E. coli. The samples mixtures were incubated at
37°C for 2 h.
Agarose gel electrophoresis
The 200 mg of agarose was weighed and dissolved it in
25 ml of TAE buffer (4.84 g Tris base, pH 8.0, 0.5 M
EDTA/l) by boiling. When the gel attained the temperature
of *55°C, it was poured into the gel cassette fitted with
comb. Then the comb was removed carefully and the
solidified gel was placed in the electrophoresis chamber
flooded with TAE buffer. To this electrophoresis chamber
20 ll of DNA sample (mixed with 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 supplied to it for around 30 min. After 30 min the gel
was removed carefully and stained with Ethidium Bromide
(ETBR) solution (10 lg/ml) and the bands were observed
under UV trans-illuminator for 10–15 min.
Viscosity measurements
Viscosity measurements were carried out using an Ostwald
micro-viscometer, maintained at constant temperature
(23°C) in a thermostat bath. The DNA concentration was
kept constant in all samples, but the complex concentration
was increased each time (from 20 to 80 lM). Mixing of the
solution was achieved by bubbling the nitrogen gas through
viscometer. The flow time was measured with a digital
stopwatch. The sample flow times were measured three
times and the mean value was used. Data are presented as
(g/g0)1/3 versus the ratio [complex]/[DNA], where g and g0
are the specific viscosity of DNA in presence and in
absence of the complex, respectively. The values of g and
g0 were calculated by using Eq. 2,
Analysis and physical measurement
À
Á
g ¼ t ꢁ tb =tb
ð2Þ
The metal estimation was done by standard methods.
Carbon, hydrogen, and nitrogen analysis were carried out
on a Thermo quest elemental analyser. The molar con-
ductance measurements were made on an ELICO-CM-82
conductivity bridge with a cell having cell constant of
0.51 cm-1. The magnetic measurements were made with
Faraday Balance at room temperature by using
Hg[Co(SCN)6] as calibrant. The electronic spectra of
compounds in DMSO were recorded using VARIAN
CARY 50 Bio UV-visible spectrophotometer. The IR
spectra of ligands and their complexes were recorded as
KBr pellets in the region 4000–400 cm-1 on Nicolet 170
where t is the observed flow time of DNA containing
solution upon the addition of compounds and tb is the flow
time of DNA containing solution alone. Relative viscosities
for DNA solution were calculated from the relation (g/g0)
(Satyanarayana et al., 1992).
Thermal denaturation studies
These were carried out on UV-spectrometer, equipped with
temperature controlling thermostat. The melting curves
(Tm) of both free E. coli DNA and E. coli DNA bound
complexes were obtained by measuring the hyperchrom-
icity of E. coli DNA at 260 nm as a function of tempera-
ture. The melting temperatures were measured with 45 lM
DNA in phosphate buffer at pH 6.8 (l = 0.2 M NaCl). The
1
SX FT-IR spectrometer. The H NMR spectra of ligands
and zinc(II) complexes were recorded in DMSO-d6 on
Bruker 300 MHz spectrometer using TMS as an internal
standard. The EPR spectra of copper(II) complexes were
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Med Chem Res (2011) 20:421–429
Table 1 Analytical, conductivity, and magnetic data for the ligands and their complexes
Compounds Compounds
abbreviations
Found (Calculated)
Conductivity Magnetic
moment leff BM
C
H
N
S
M
–
Cl
–
L1H3
C1
C17H13O3N3
65.7 (66.4) 4.0 (4.2) 13.1 (13.6)
–
–
–
–
–
–
–
[Co L1Clꢀ2H2O]ꢀH2O 44.2 (44.9) 3.4 (3.9) 8.9 (9.2)
12.6 (12.9) 7.4 (7.8) 15
12.4 (12.9) 7.4 (7.8) 18.4
14.1 (14.4) 7.7 (7.9) 22.3
13.7 (14.1) 7.2 (7.6) 12.1
4.43
C2
[Ni L1Clꢀ2H2O]ꢀH2O 44.5 (44.9) 3.3 (3.9) 9.0 (9.2)
46.1 (46.3) 2.3 (2.7) 9.4 (9.5)
2.95
C3
[Cu L1Cl]2ꢀ4H2O
1.69
C4
L2H
[Zn L1Clꢀ2H2O]ꢀH2O 43.7 (44.1) 3.5 (3.8) 8.6 (9.0)
Diamagnetic
C17H12ON4S
63.3 (63.7) 3.4 (3.7) 17.2 (17.5) 9.6 (10)
–
–
–
–
C5
[Co L2Clꢀ2H2O]
[Ni L2Clꢀ2H2O]
[Cu L2Clꢀ2H2O]
[Zn L2Clꢀ2H2O]
45.1 (45.4) 2.9 (3.3) 12.1 (12.4) 6.8 (7.1) 12.8 (13.1) 6.5 (7.9) 17.5
45.0 (45.4) 3.1 (3.3) 11.9 (12.4) 6.7 (7.1) 12.7 (13) 7.6 (7.9) 16.3
4.45
C6
3.1
C7
44.4 (44.9) 2.9 (3.3) 8.9 (9.2) 6.7 (7.0) 13.5 (13.9) 7.5 (7.8) 21.5
44.4 (44.7) 3.0 (3.2) 11.9 (12.2) 6.8 (7.0) 14.0 (14.3) 7.3 (7.7) 14.4
1.73
C8
Diamagnetic
recorded at room temperature on Varian E-4 X-band
spectrometer using TCNE as g-marker. The Ostwald vis-
cometer is used for hydrodynamic measurements.
in both the ligands was assigned to azomethine m(C=N).
The band at 1596 cm-1 in L2H is assigned to the benzo-
thiazole ring vibrations. Upon complexation azomethine
m(C=N) shows negative shift in (C1–C4) complexes, but in
case of (C5–C8) complexes positive shift was observed.
Collectively these observations signify the coordination of
azomethine nitrogen (Guofa et al., 1990). Similarly ben-
zothiazole ring vibrations {m(C=N)} also show the negative
shift in (C5–C8) complexes, which emphasize the
involvement of benzothiazole ring nitrogen in the coordi-
nation. The negative shift of m(C=O) group in all the
complexes of L1H3 ligand suggests the coordination of
amide carbonyl to the metal centre. The phenolic m(OH)
band is merged with stretching band of coordinated/lattice
celled water molecules in the range 3350–3410 cm-1 in the
complexes derived from L1H3. The complexes show a non-
ligand band in the region 430–455 cm-1 ascribable to the
m(M–N) (Thomas et al., 1995).
Results and discussion
Chemistry
The analytical and physicochemical data of the complexes
are summarized in Table 1. All complexes are soluble in
organic solvents like DMF, DMSO and acetonitrile.
IR spectral studies
The IR spectra of the free ligands were compared with the
spectra of the complexes. The IR spectral data and their
assignments are depicted in the Table 2. The strong band at
1662 cm-1 in the ligand L1H3 is assigned to the amide
carbonyl m(C=O). The strong band in the region 1606 cm-1
1H NMR spectral studies
Table 2 IR spectral data of the ligands and their complexes
The 1H NMR spectra of the ligands and their Zn(II)
complexes were recorded in DMSO-d6 solvent over the
range of 0–16 ppm. Singlets in L1H3 and L2H ligands at
8.7 and 8.3 ppm are assignable to the azomethine protons,
which are shifted to down field region upon complexation
with Zn(II) indicating the coordination of these function-
alities (Chohan et al., 2004). In addition to this, a set of
multiplet observed in the range 6.93–7.91 ppm is ascribed
to the aromatic protons in all the compounds. The peak
observed at 11.1 and 10.1 ppm in ligands is attributable to
the –OH of quinoline molecule in L1H3 and L2H, respec-
tively. A sharp singlet at 12 ppm observed in the ligand
L1H3 is due to –NH of amide carbonyl, which remains
unaltered in the corresponding Zn(II) complex. The
Compounds m(C=N)
Azomethine
m(C=N)
Benzothiazole
m(C=O) m(H2O)
L1H3
L2H
C1
1606
–
1662
–
3422
3437
3445
3442
3431
3447
3421
3448
3444
3447
1606
1560
1555
1555
1561
1635
1630
1637
1664
1596
–
1637
1634
1610
1610
–
C2
–
C3
–
C4
–
C5
1549
1555
1545
1568
C6
–
C7
–
C8
–
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Med Chem Res (2011) 20:421–429
425
absence of signal corresponding to quinoline –OH in (C4
and C8) complexes confirms the coordination of –OH
group to the metal ion via deprotonation. The peak
observed at 12.1 ppm in ligand L1H3 is assigned to the
phenolic –OH which is retained in the C4 complex.
The complex C3 exhibits a broad absorption signal with
giso = 2.02, with no hyperfine splitting is observed in the
spectrum of the complex.
The complex (C7) exhibits anisotropic signals with
gII = 2.05 and g\ = 2.04 values. From the observed trend,
gII [ g\, it is evident that the unpaired electron lies pre-
dominantly in the dx2ꢁy2 orbital with the possibility of some
Molar conductivity measurements
2
dz character being mixed with it because of low symmetry
(Kivelson and Neiman, 1961). The gII \ 2.3 indicates the
larger percentage of covalency in metal to ligand bonds
(Ray and Kuffman, 1989).
Molar conductivities of the complexes were measured in
DMSO solution with 10-3 M concentrations. All these
complexes show molar conductance in the range 12–
23 mho cm2 mol-1 (Table 1). These low values of molar
conductance of the complexes show that they are non-
electrolytes (Geary, 1971).
FAB mass studies
In the present investigation the FAB mass spectra of the
C2, C3, and C7 complexes have been recorded and show a
molecular ion peak at m/z 461, 906, and 459, respectively,
which corresponds to the molecular weight of the respec-
tive complexes. Apart from this, the spectra show some
other peaks correspond to the molecular cations of the
complexes. The conclusion drawn from FAB mass spectra
is that, the complex C3 is dimeric in nature where as the
complexes C2 and C7 are monomeric.
Electronic spectral studies
The electronic spectra of the ligands and their complexes
were recorded in DMSO solution. All the ligands exhibit
similar features in UV-Visible region with the bands around
280 and 386 nm. The broad intense band around 280 nm in
both the ligands can be assigned to intra ligand p–p* tran-
sition. This band is almost unchanged in the spectra of all
the complexes. The band at 385 nm with a shoulder on low
energy side is due to n–p* transition associated with the
azomethine linkage. This band experiences red shift in all
the complexes (Bhardwaj and Singh, 1994; Ainscough
et al., 1997). The bands at around 420–460 nm are attrib-
uted to the ligand to metal charge transfer transitions.
Thermal analysis
The TG-DTA experiments were carried out to explore the
thermal stability of the complexes. The thermal behaviors
of all the metal complexes were studied in temperature
range of 25–800°C. The TG-DTA studies of complexes
C3 and C6 reveal that the decomposition proceeds in three
steps. In the first stage weight loss at 98°C corresponds to
the presence of the lattice cell water in both the com-
plexes. The weight losses in the temperature range
110–200°C are due to the presence of the coordinated
water in both the complexes. This is marked by endo-
thermic peaks in DTA curves. A plateau was obtained
after heating above 600°C, which corresponds to the for-
mation of stable metal oxide.
Magnetic studies
The magnetic moment was recorded at room temperature
and is shown in Table 1. The leff values of (C1 and C5)
complexes are found to be 4.43 and 4.45 BM suggesting
octahedral geometry for both the C1 and C5 cobalt(II)
complexes (Thakkar and Bootwala, 1995). The nickel(II)
complexes (C2 and C6) have magnetic moment values of
2.95 and 3.1 BM which reveal the spin free octahedral
configuration (Thakkar and Bootwala, 1995). The cop-
per(II) complexes (C3 and C7) have leff value less than the
spin only value (1.69 and 1.73 BM) which accounts for
metal-metal interaction. In support of other spectral data,
magnetic moment values suggest square pyramidal geom-
etry for C3 and octahedral geometry for C7.
Biochemistry
The ligands and their complexes C1–C8 were screened for
antibacterial activity against E. coli and Pseudomo-
nas aeruginosa and antifungal activity against Aspergil-
lus niger and Cladosporium. The data are shown in
Table 3. The compounds which show the promising anti-
bacterial and antifungal activity were selected for mini-
mum inhibitory concentration studies, which are
determined by assaying at 250 lg concentrations along
with standards at the same concentrations. Even at the MIC
level the complexes C3, C4, and C7 were found to be more
active against bacteria and fungi than the Schiff base
EPR spectral studies
EPR studies of paramagnetic transition metal(II) com-
plexes give information about the distribution of the
unpaired electrons and hence about the nature of the
bonding between the metal ion and its ligands.
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Table 3 Screening of compounds against bacteria and fungi
Compounds E. coli
Pseudomonas aeruginosa Compound name Aspergillus niger
Cladosporium
Zone of
inhibition
in cm
% Inhibition Zone of
inhibition
in cm
% Inhibition
Zone of
inhibition
in cm
% Inhibition Zone of
inhibition
in cm
% Inhibition
Gentamycin 3.2
100
37.5
2.9
0.5
0.4
1.1
1.5
1.2
0.5
0.2
0
100
Flucanozole
L1H3
C1
1.2
0
100
0
0.9
0.2
0.1
0.1
0.4
0.6
0.5
0.2
0
100
22.2
11.1
11.1
44.4
66.6
55.5
22.2
0
L1H3
1.2
0.7
1.1
2.8
1.4
1.8
0.2
0.2
0.3
1.2
0
17.24
13.79
37.9
51.72
41.39
17.24
6.89
0
C1
21.87
34.37
87.5
43.75
56.25
6.2
0.2
0.4
0.6
1
16.6
C2
C2
33.3
66.6
83.3
8.3
0
C3
C3
C4
L2H
C4
L2H
0.1
0
C5
C5
C6
6.2
C6
0
0
C7
9.37
37.5
0
0.4
0.3
0
13.79
10.3
0
C7
0.7
0.6
0
58.3
50
1.1
0.4
0
122.2
44.4
0
C8
C8
DMF
DMF
0
ligands (Tables 4 and 5). This enhancement in the activity
can be explained on the basis of chelation theory (Thim-
maiah et al., 1985; Franklin and Snow, 1971). Such a
chelation could enhance the lipophilic character of the
central metal atom, which subsequently favors it
permeation through the lipid layers of cell membrane (Ja-
don et al., 1996) and blocking the metal binding sites on
enzymes of microorganism. The variation in the effec-
tiveness of different compound against different organisms
also depends on a nature of the metal ion, nature of the
ligand, geometry of the complexes, and impermeability of
the cell of the microbes or differences in ribosomes of
microbial cells (Aliye et al., 2009; Sengupta et al., 1998;
Kurtoglu et al., 2006).
Table 4 Antibacterial studies of compounds in MIC (250 lg) level
Compounds E. coli
Pseudomonas aeruginosa
Zone of
inhibition
in cm
% Inhibition Zone of
inhibition
in cm
% Inhibition
Absorption studies
Electronic absorption spectroscopy is universally employed
to determine the binding of complexes with DNA. The
interaction of complexes with DNA causes electronic
perturbations in the transition metal complexes at the
maxima. The data obtained from this experiment shows a
significant hypochromism with an insignificant blue shift at
the maxima when increasing amounts of E. coli-DNA were
added to the solution of complex. The extent of spectral
change is related to the strength of binding of the com-
plexes to E. coli DNA. These spectral characteristics sug-
gest that, the complexes bound to E. coli DNA by an
intercalative mode. Both the ligands have no effect on the
absorption of DNA solution. The representative absorption
spectrum of the nickel (C6) complex is given in Fig. 2. The
intrinsic binding constant Kb obtained for C1–C8 com-
plexes are given in the Table 6. By observing the intrinsic
binding constant of all the complexes it has been suggested
that the C6 and C7 complexes show more binding property
than the remaining complexes.
Gentamycin 3.2
100
21.87
2.9
0.2
1.1
0.8
–
100
6.89
37.93
27.5
–
L1H3
0.7
2.1
0.9
0.6
0
C3
65.62
28.1
18.75
0
C4
L2H
DMF
0
0
Table 5 Antifungal studies of compounds in MIC (250 lg) level
Compounds Aspergillus niger Cladosporium
% Inhibition Zone of % Inhibition
Zone of
inhibition
in cm
inhibition
in cm
Flucanozole 1.2
100
75
25
0
0.9
0.1
0.9
0
100
11.1
C4
0.9
0.3
0
C7
100
0
DMF
123

Med Chem Res (2011) 20:421–429
427
1.5
1.25
1
0.75
0.5
0.25
0
0
0.5
1
[Complex]/ [DNA]
Fig. 2 Absorption spectrum of Nickel (C6) complex in Tris buffer
upon addition of E. coli-DNA with complex
Fig. 3 Effect of increasing amounts of the ligand (L2H) and its
complexes (C5–C8) on the relative viscosities of E. coli DNA
0.65
0.55
0.45
0.35
0.25
Table 6 DNA binding constant of the complexes
Compounds
abbreviations
Complexes
Binding constant
Kb in M-1
C1
C2
C3
C4
C5
C6
C7
C8
[Co L1Clꢀ2H2O]ꢀH2O
[Ni L1Clꢀ2H2O]ꢀH2O
[Cu L1Cl]2ꢀ4H2O
[Zn L1Clꢀ2H2O]ꢀH2O
[Co L2Clꢀ2H2O]
1.08 9 103
1.63 9 103
1.53 9 103
1.31 9 103
1.26 9 103
2.06 9 104
1.43 9 104
1.07 9 104
0.15
[Ni L2Clꢀ2H2O]
20 30 40 50 60 70 80 90
[Cu L2Clꢀ2H2O]
Temperature (°C)
[Zn L2Clꢀ2H2O]
Fig. 4 Melting curves of E. coli-DNA in the absence and presence of
complexes Nickel (C6) and Copper (C7)
Viscosity measurement study
To further clarify the binding modes of the complexes with
DNA, viscosity measurements were carried out. Spectro-
scopic experiments provide necessary data, but not suffi-
cient clues to support
a binding mode. Viscosity
measurements that are sensitive to length change are
regarded as the least ambiguous and the most critical tests
of the binding model in solution in the absence of crys-
tallographic structural data (Satyanarayana et al., 1992).
The viscosity of DNA increases as the increase in the ratio
of complexes to DNA indicates the intercalative mode of
binding (Satyanarayana et al., 1992). The representative
graph of the ligand L2H and its complexes is shown in
Fig. 3. The extent of intercalation observed is more in the
nickel (C6) and copper (C7) complexes.
Fig. 5 Agarose gel showing the results of electrophoresis of E. coli-
DNA with the Schiff base ligand L2H and its complexes (C5–C8)
[M standard molecular weight marker, C- E. coli control DNA of
E. coli, 6 ligand (L2H), 7 Cobalt (C5), 8 Nickel (C6), 9 Copper(C7),
and 10 Zinc (C8)]
Thermal denaturation study
complexes is found to be 58 1°C (Tselepi-Kalouli and
Katsaros, 1989). (as shown in Fig. 4). On the bases of
viscometric measurements the complexes C6 and C7 have
chosen for the thermal denaturation experiment, because of
their stronger binding capability with the E. coli DNA than
any other complexes. However, with addition of nickel
Thermal behaviors of DNA in the presence of complexes
can give insight into their conformational changes when
temperature is raised, and provide the information about
the interaction strength of complexes with DNA (Kelly
et al., 1985). The Tm of E. coli DNA in absence of the
123

428
Med Chem Res (2011) 20:421–429
Fig. 6 The proposed structures
of the complexes are shown
below
HO
HO
H
H
N
H
H
N
OH2
O
N
.
H2O
.
O
M
N
.
O
N
Cl
4H2O
Cu
Cu
Cl
OH2
M: Co, Ni and Zn
N
O
N
O
Cl
H
O
N
O
H
N
S
N
N
OH2
H
H
N
M
OH
N
H2O
Cl
M: Co, Ni, Cu and Zn
Acknowledgments The authors thanks the staff of department of
chemistry and USIC, Karnatak University, Dharwad for providing
research and spectral facilities. Recording of FAB mass spectra
(CDRI Lucknow) and EPR spectra (IIT Bombay) is gratefully
acknowledged. Further, the authors thank UGC for providing RFSMS
and Karnatak University, Dharwad for providing research
fellowships.
(C6) and copper (C7) complexes, the Tm of the DNA
increases dramatically to 65 1°C. This is characteristic
of an intercalative binding behavior of the complexes
´
(Lopez et al., 1996).
DNA cleavage studies using gel electrophoresis
The cleavage efficiency of the metal complexes compared
to that of control is due to their efficient DNA binding
ability. Both the ligands and complexes were allowed to
interact with the E. coli DNA for cleavage study. In the
present investigation, the nickel (C6) and copper (C7)
complexes have shown increase in the intensity and tailing
compared to remaining compounds which illustrates the
higher binding ability of the said complexes with E. coli
DNA (shown in a Fig. 5).
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