Pyrazole bridged binuclear transition metal complexes: Synthesis, characterization, antimicrobial activity and DNA binding/cleavage studies

Article abstract of DOI:10.1016/j.molstruc.2011.10.008

A new ligand system having pyrazolato endogenous bridging component and N₄S₂ donating sites is synthesized by the condensation of 3,5-dichloroformyl-1H-pyrazole with phenylthiosemicarbazide. Both ligand and its binuclear Co^II

Full text of DOI:10.1016/j.molstruc.2011.10.008

Journal of Molecular Structure 1006 (2011) 580–588
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Pyrazole bridged binuclear transition metal complexes: Synthesis,
characterization, antimicrobial activity and DNA binding/cleavage studies
⇑
Naveen V. Kulkarni, Anupama Kamath, Srinivasa Budagumpi, Vidyanand K. Revankar
Department of Chemistry, Karnatak University, Pavate Nagar, Dharwad 580 003, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 June 2011
Received in revised form 7 October 2011
Accepted 7 October 2011
Available online 14 October 2011
A new ligand system having pyrazolato endogenous bridging component and N4S2 donating sites is syn-
thesized by the condensation of 3,5-dichloroformyl-1H-pyrazole with phenylthiosemicarbazide. Both
ligand and its binuclear CoII, NiII, CuII and ZnII complexes are characterized by the spectral and analytical
methods. All the complexes were found to be binuclear monomeric in nature with octahedral geometry
and nonelectrolytes. Electrochemical activity is observed only for the CuII complex in the applied poten-
tial range. Ligand and complexes were screened for antimicrobial activity and made to interact with
Escherichia coli DNA to investigate the binding/cleaving ability by absorption, hydrodynamic, thermal
denaturation and electrophoresis studies. The copper complex exhibited higher inhibition against a gram
negative bacterium, E. coli and shown good intercalating ability with E. coli DNA.
Keywords:
Pyrazole
Thiosemicarbazide
Antimicrobial activity
DNA binding study
Intercalation
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
metal ion. The inhibitory action of antimicrobial agents is also pre-
sumed to follow the same mechanism.
Pyrazole derivatives are the subject of many research studies
due to their widespread potential biological activities such as anti-
tumour [1], anti-inflammatory [2], antipyretic [3], antiviral [4],
antimicrobial [1], anticonvulsant [5], antihistaminic [6], antide-
pressant [7], insecticides [8] and fungicides [8]. Pyrazoles are also
used as antioxidant additives to fuels [9]. Pyrazole is very fascinat-
ing and significant to the coordination chemists, due to its 1,2-
diazine unit which can provide the endogenous bridging between
two similar or dissimilar transition or inner transition metal ions
[10,11]. The appropriate ring substitutions especially at the 3 and
5 positions are expected to supplement the nucleophilicity and ste-
ric accessibility of the pyrazole nitrogens [10,12]. With the aid of
other ligating functionalities incorporated through the ring substi-
tution, the pyrazole derivatives can act as polydentate chelates and
form stable coordination compounds of transition metal ions with
greater specificity.
As the presumed mechanism of action authenticates, the anti-
cancer agents interact through characteristic binding modes with
the DNA of cancer infected cell in such a way that, the cell cannot
replicate further. This inhibition of replication finally leads to the
death of the infected cell [13]. Especially the coordination com-
plexes are known to perturb the 3D-structure of DNA with the
interaction of N-7 nitrogen of nucleotide and hence inhibiting
the replication [13]. The detailed SAR studies have rationalized this
in terms of labiality and nucleophilicity of ligand and nature of the
In our laboratory we observed that the pyrazole based transition
metal complexes with thiosemicarbazide arms have shown good
antibacterial activity as well as strong intercalating interaction with
DNA of Escherichia coli [14]. It is also envisaged in the literature that
the presence of biologically active phenylthiosemicarbazide arms
and metal incorporation enhance the activity [15]. By the influence
of these results, in continuation, we have developed similar system
of different thiosemicarbazide with aromatic substitutions. In this
article we wish to report the complexes of a polydentate chelate,
architectured by incorporating the phenyl thiosemicarbazide frag-
ment at the 3 and 5 positions of a pyrazole core. The thiosemicarba-
zide arms are expected to provide versatile ligational properties
and hence to give stable and interesting transition metal com-
plexes. We made an interaction of this ligand with CoII, NiII, CuII
and ZnII ions in a 1:2 fashion to get dinuclear, monomeric com-
plexes. The resulted complexes are isolated and characterized
spectro-analytically. The prepared compounds are made to interact
with E. coli DNA and the interaction studies were monitored by
absorption, hydrodynamic, thermal denaturation and electrophore-
sis studies.
2. Materials and methods
2.1. Analysis and physical measurements
All chemicals used were of reagent grade and the solvents were
distilled prior to use. The metal estimation was done by standard
methods [16]. Carbon, hydrogen, nitrogen, and sulfur analyses
⇑
Corresponding author. Tel.: +91 836 4250821 (R), mobile: +91 9900485652.
E-mail address: vkrevankar@rediffmail.com (V.K. Revankar).
0022-2860/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2011.10.008

N.V. Kulkarni et al. / Journal of Molecular Structure 1006 (2011) 580–588
581
were carried out on a Thermo quest elemental analyzer and the re-
2.4. DNA binding/cleavage studies
2.4.1. Isolation of DNA E. coli
sults are represented in Table 1. The molar conductivity measure-
ments in DMF were made on ELICO-CM-82 conductivity bridge
with conductivity cell having cell constant 0.51 cmꢀ1. The mag-
netic susceptibility measurements were made at room tempera-
ture using Faraday balance by using Hg[Co(SCN)4] as calibrant.
The 1H NMR spectra were recorded in DMSO-d6 solvent on Bru-
ker-300 MHz spectrometer at room temperature using TMS as
internal reference. IR spectra were recorded in a KBr matrix using
Impact-410 Nicolet (USA) FT-IR spectrometer in 4000–400 cmꢀ1
range. The ESR spectrum of the copper complex was carried out
on Varian E-4X-band EPR spectrometer, using TCNE as the g-mar-
ker. The FAB mass spectra were drawn from JEOL SX 102/DA-6000
mass spectrometer using Argon/Xenon (6 kV, 10 mA) as the FAB
gas and 3-nitrobenzylalcohol as matrix. Thermal studies (Thermo
gravimetric and Differential Thermal analysis) were carried out
in nitrogen atmosphere on Universal V2.4F TA Instrument with
limiting temperature of 1000 °C and heating rate 10 °C/min. Cyclic
voltammetric studies were performed at room temperature in DMF
under oxygen free condition created by purging pure nitrogen gas,
with CHI1110A Electrochemical analyzer (USA) comprising three
electrode assembly of glassy carbon working electrode, platinum
auxiliary electrode and Ag+/AgCl reference electrode. Tetramethy-
lammoniumchloride (0.01 M) was used as supporting electrolyte
and instrument was standardized by ferrocene/ferrocenium redox
couple. The electronic spectra of the complexes were recorded on
Hitachi 150–20 spectrophotometer with scan range of 1000–
200 nm. The same, equipped with temperature controlling thermo-
stat is used for the DNA binding studies (absorption and thermal
denaturation studies). Bio-Rad Trans illuminator and a Polaroid
camera were used in the gel electrophoresis experiment and Os-
wald micro-viscometer is used for hydrodynamic measurements.
Nutrient broth peptone (10 g/L), Yeast extract (5 g/L) and (NaCl
10 g/L) were used for culturing of E. coli. The 50 mL media was pre-
pared, autoclaved for 15 min at 121 °C, 15 lb pressure. The auto-
claved media were inoculated with the seed culture and
incubated at 37 °C for 24 h for E. coli. The fresh bacterial culture
(1.5 mL) was centrifuged to obtain the pellet and was dissolved
in 0.5 mL of lysis buffer (100 mM tris PH 8.0, 50 mM EDTA, 10%
SDS) and to which 0.5 mL of saturated phenol was added and incu-
bated at 55 °C for 10 min. The incubated solution was centrifuged
at 10,000 rpm for 10 min and to the supernatant an equal volume
of chloroform, isoamyl alcohol (24:1) and 1/20th volume of 3 M so-
dium acetate (PH 4.8) were added. The mixture was again centri-
fuged at 10,000 rpm for 10 min and to the supernatant
3
volumes of chilled absolute alcohol was added. The precipitated
DNA was separated by centrifugation and dried and dissolved in
TE buffer (10 mM tris pH 8.0, 1 mM EDTA) and stored in cool.
The concentration of DNA per nucleotide [C(p)] was measured by
using its known extinction coefficient at 260 nm (ꢁ6600 Mꢀ1 cmꢀ1
)
[18]. The absorbance at 260 nm (A260) and at 280 nm (A280) for
DNA was measured to check its purity. The ratio A260/A280 was
found to be 1.88, indicating that DNA was satisfactorily free from
protein [19]. Tris buffer [5 mM tris (hydroxymethyl) aminometh-
ane, tris, PH 7.2, 50 mM NaCl] was used for the absorption, viscosity,
and thermal denaturation experiments.
2.4.2. Absorption studies
The complexes were dissolved in DMSO and then diluted to the
desired concentration with distilled water. The spectroscopic titra-
tions were carried out by adding increasing amounts of DNA to a
solution of the complex at a fixed concentration contained in a
quartz cell. After each addition, the UV–Visible spectra were re-
corded after equilibration at 20 °C for 10 min. The intrinsic binding
constant Kb was determined from the plot of A0/[A – A0] vs [DNA]ꢀ1
according to equation [20]
2.2. Preparation of ligand LH3 (1-H pyrazole-3,5-dicarboxybis(phenyl-
thiosemicarbazide))
The commercially obtained (Sigma Aldrich) 1H-pyrazole-3,5-
dicarboxylicacid (0.01 M) was refluxed with thionylchloride
(10 mL) in anhydrous condition for 4 h at about 110–120 °C. The
content was then evaporated to dryness to remove the excess thio-
nylchloride, thus obtained pasty mass was allowed to cool. Phenyl-
thiosemicarbazide [17] in dry ethanol (0.02 M) was added drop
wise and reflux was carried out for additional 3 h. Resultant solu-
tion was then cooled in ice bath to get a colorless amorphous solid,
which is separated by filtration and dried over anhydrous CaCl2
and recrystalised from ethanol. The reaction pathway is repre-
sented in Fig. 1.
A0=½A ꢀ A0ꢂ ¼
eG=½eHꢀG
ꢀ
eGꢂ þ G=½eHꢀG ꢀ eGꢂ ꢃ 1=K½DNAꢂ
e
where A0 – absorbance in the free complex; A – absorbance at given
DNA concentration; [DNA] – concentration of DNA in base pairs; eG
– the apparent absorption coefficients of DNA in free form; eH–G
the apparent absorption coefficients of DNA in bounded form of
complex. The data were fitted to above equation and graph was ob-
–
tained with a slope equal to
e
G/[eH–G
ꢀ
e
G] ꢃ 1/K and intercept equal
to G/[eH–G G]. Hence Kb was obtained from the ratio of the inter-
e
ꢀ
cept to the slope.
2.3. Preparation of complexes
2.4.3. Hydrodynamic measurements (viscosity measurements)
Viscosity measurements were carried out using an Oswald mi-
cro-viscometer maintained at constant temperature (20 °C) in a
thermostat bath. The complexes were dissolved in DMSO and then
diluted to the desired concentration with distilled water. The DNA
To the ethanolic solution of the ligand LH3 (0.454 g, 0.01 M),
corresponding metal(II) chloride(0.02 M) in ethanol was added
with stirring and refluxed on water bath for 4 h. So obtained solid
complex was separated by filtration under suction, washed with
hot ethanol and dried in vacuo.
concentration was kept constant in all samples (100 lM), but the
complex concentration was increased each time (from 20 to
Table 1
Elemental analysis and conductivity data of compounds.
Compound Empirical formula
Elemental analysis in% calculated (found)
Molar cond. KM in ohmꢀ1 cm2 molꢀ1
C
H
N
M
–
S
Cl
LH3
CoL
NiL
CuL
ZnL
[C19H18N8S2O2]
[Co2L(
[Ni2L(
[Cu2L(
[Zn2L(
50.21 (50.12) 3.99 (3.83) 24.65 (24.58)
OH)(H2O)4] 34.71 (34.82) 3.53 (3.62) 17.05 (17.14) 17.93 (18.12) 9.74 (9.63)
OH)(H2O)4] 34.75 (34.64) 3.51 (3.62) 17.07 (17.16) 17.83 (17.78) 9.75 (9.86)
14.12 (14.23)
–
–
–
–
15.2
11.4
l
l
l
Cl)(H2O)4]
33.32 (33.45) 3.21 (3.30) 16.37 (16.22) 18.56 (18.92) 9.35 (9.53)
33.14 (33.27) 2.61 (2.82) 16.28 (16.23) 19.02 (19.54) 9.30 (9.42)
5.11 (5.23) 14.0
5.08 (5.18) 12.9
lCl)(H2O)4]

582
N.V. Kulkarni et al. / Journal of Molecular Structure 1006 (2011) 580–588
Fig. 1. Schematic representation of preparation of ligand LH3.
80
l
M). Mixing of the solution was achieved by bubbling the nitro-
assigned to amide carbonyl stretching vibration and band around
1540 cmꢀ1 is assigned to pyrazole ring
(C@N). The thione (C@S)
gen gas through viscometer. The flow time was measured with a
m
digital stopwatch. The sample flow times were measured three
form of ligand is suggested by the thioamidic absorptions observed
times and the mean value was used. Data are presented in plot
around 1291 cmꢀ1 and 788 cmꢀ1 [25]. Upon complexation the peak
1/3
of (g/g0)
versus the ratio [complex]/[DNA], where
g
and g0 are
corresponding to
m
(C@O) disappears by providing a new band
(C@N), which suggests the possi-
the specific viscosity of DNA in presence and in absence of the
around 1620 cmꢀ1 assignable to
m
complex respectively. The values of
using equation [21]
g
and g0 were calculated by
ble imidolformation and coordination of ligandthrough azomethine
nitrogen. Appearance of a sharp band in the region 3400 cmꢀ1 attrib-
utable to the
m(AOH) supports the same. Pyrazole ring nitrogens are
g
¼ ðt ꢀ t0Þ=t0
involved in ligation as diazene bridge, which is suggested by the high
where t – the observed flow time of DNA containing solution; t0 –
the flow time of DNA solution alone.
frequency shift of ring m(C@N) and presence of m(MAN) around
465 cmꢀ1. It is seen that thioamidic bands that have major contribu-
Relative viscosities for DNA were calculated from the relation
tion from C@S group are disappeared in complexes suggesting the
(g/g0).
thioenolisation, which is supported by the appearance of
The absence of (ASH) allow us to conceive the sulfur coordination
upon deprotonation, which is further supported by the presence of
(MAS) band in the complexes. Sharp band at 3550 cmꢀ1 in the CoII
and NiII complexes evidences the
OH and presence of coordinated
m(CAS).
m
2.4.4. Thermal denaturation study
Thermal denaturation studies were carried out on UV-spec-
trometer equipped with temperature controlling thermostat. The
melting curves (Tm-curve) of both free E. coli DNA and DNA bound
complexes were obtained by measuring the hyperchromicity of
DNA at 260 nm as a function of temperature. The melting tempera-
m
l
water molecules broaden the higher wavelength side of spectra. The
numerical IR spectral data is tabulated (Table 2).
3.3. 1H NMR spectral studies
tures were measured with 60
lM DNA in phosphate buffer at pH
6.8 ( = 0.2 M NaCl). The temperature was scanned from 25 to
85 °C at a rate of 5 °C per min. The mid-point of the hyperchro-
mic transition was noted as the melting temperature (Tm) [22].
l
1H NMR study of the ligand and ZnII complex was carried out in
the scan range of 0–16 dppm. The ligand displayed a sharp singlet
peak at 12.03 dppm which is attributable to NAH proton of pyra-
zole [26]. Disappearance of the same in the complex reveals the
formation of pyrazolide ion and coordination of pyrazole as endog-
enous bridge. In ligand, amide protons are resonated at 10.63 dppm
and disappeared upon complexation evidencing the enolisation
process, which is attested by the peak due to AOH at 11.63 dppm.
The thioamidic protons assigned at 10.16 dppm were disappeared
in the complex suggesting the thioenolisation and subsequent
deprotonation. The aromatic and phenyl amine protons resonated
around 7–8 dppm and 8.3 dppm respectively experience a small
shift in complex due to the changed electronic environment
brought about by the chelation. Further the appearance of broad
peak at around 3.4 dppm reveals the presence of coordinated water
in the complex.
2.4.5. Electrophoresis
For the gel electrophoresis experiments [23], the solution of
complexes in DMF (1 mg/mL) was prepared and these test samples
(1 lg) were added to the genomic DNA samples of E. coli and incu-
bated for 2 h at 37 °C. Agarose gel was prepared in TAE buffer
(4.84 g Tris base, PH 8.0, 0.5 M EDTA/l. PH 7.3), the solidified gel at-
tained at ꢁ55 °C was placed in electrophoresis chamber flooded
with TAE buffer. After that 20 lL of each of the incubated com-
plex-DNA mixtures (mixed with bromophenol blue dye at 1:1 ra-
tio) was loaded on the gel along with standard DNA marker and
electrophoresis was carried out under TAE buffer system at 50 V
for 2 h. At the end of electrophoresis, the gel was carefully stained
with EtBr (ethidiumbromide) solution (10 lg/mL) for 10–15 min
and visualized under UV light using a Bio-Rad Trans illuminator.
The illuminated gel was photographed by using a Polaroid camera
(a red filter and Polaroid film were used).
3.4. UV–Visible spectral studies
The electronic spectra of ligand and complexes were recorded in
DMF solution in the scan range 200–1000 nm. Ligand exhibits a
band around 270 nm which is due to the intra ligand
3. Results and discussion
p ?
p⁄ tran-
3.1. Molar conductivity measurements
sition, which is unaltered in spectra of complexes. The peak at
320 nm is assigned for n ? p⁄ transition of imine group and the
transitions occurred around 275–300 are due to n ? p⁄ transitions
of carbonyl group [27]. In cobalt complex, the band around 390 nm
is assigned to LMCT, n ? p⁄ transition. The band at 450 and
644 nm are attributed to d ? d transitions, which are represented
The molar conductance values of all the complexes measured at
room temperature in DMF solution with 10ꢀ3 mol dmꢀ3 concentra-
tion fall in the range 11.4–15.2 mho cm2 molꢀ1, which indicate the
non-electrolytic nature of the complexes [24].
4
as 4T1g ? 4A2g and T1g (F) ? 4T1g (P) assigning the octahedral
3.2. Infrared spectral analysis
structure. In NiII complex the lowest energy band observed at
900 nm was due to 3A2g ? 3T2g
(
m
1) and bands at 600 and 440 nm
2) and 3A2g ? 3T2g(P) (
3) respec-
tively, suggesting the octahedral geometry. The copper complex
IR spectrum of ligand shows sharp absorptions around
were assigned to 3A2g ? 3T1g
(m
m
3200 cmꢀ1 attributed to (ANH). A strong band at 1665 cmꢀ1 is
m

N.V. Kulkarni et al. / Journal of Molecular Structure 1006 (2011) 580–588
583
Table 2
Infrared spectral data of ligand and its complexes in cmꢀ1 (s – sharp, m – medium and b – broad).
Compound
m
(lOH)
m
(OAH)
m(NAH)
m
(NAH) pyrazole
m(C@O) amide
m
(C@N)
m
(C@N) pyrazole
m(C@S) thioamide
m
(CAS)
m
(M AN)
m(MAS)
LH3
CoL
NiL
CuL
ZnL
–
–
3286m
3227m
3230m
3220m
3215m
3176s
1665s
–
1540
1291s
788s
–
–
–
3561m
3550m
–
–
3350b
3340b
3342b
3346b
–
–
–
–
–
–
–
–
1622s
1620s
1625s
1621s
1550s
1578s
1560m
1551s
–
–
–
–
–
–
–
–
658m
653m
649s
662s
468s
468s
469m
476s
420s
416s
418s
412s
exhibit band around 650 nm with low
e value, which is absent in
zinc complex, is assigned to d ? d transition. The intense band ob-
served at 480 nm is referred for ligand to metal charge transfer
transition (LMCT). The electronic spectrum of zinc complex shows
band at 350 nm, accounted for the intra ligand and LMCT
transitions.
3.5. Magnetic properties
The copper complex exhibits the effective magnetic moment leff
1.64 BM at 303 K. Fairly low value obtained evidences the existence
endogenous pyrazole bridge with possible spin–spin interaction.
The room temperature magnetic moment values of bis-complexes
of nickel and cobalt complexes were found to be 2.78 and 4.26 BM
respectively, suggesting the six coordinated distorted octahedral
geometry [28,29], which is in line with the spectral analysis data.
Fig. 2. Structures of complexes.
FAB mass spectra. The peaks at highest m/z value can be assigned
for molecular mass by certainty, with the aid of consistent isotopic
pattern. A peak at m/z 721 was observed, in the CuII complex which
is in consistent with the total molecular mass of the coordination
complex (Fig. 3). Apart from this, spectrum shows some other
peaks, which are due to molecular cations of various fragments
of complex. By comparing the analytical and spectral data of co-
balt, nickel and zinc complexes, it is evident that these are mono-
meric and binuclear complexes. The tentative structure assigned
for the complexes is represented as Fig. 2.
3.6. ESR spectral study
The X-band ESR spectrum of CuII complex in solid form scanned
in the region of 9000 MHz with corresponding field intensity of
ꢁ3000 Gauss exhibits isotropic intense broad signal with
giso = 2.075 with no hyperfine splitting. This type of unusual ESR
spectrum was assigned for complexes having large organic ligand
substitutions with exogenous chloro bridge. The broadening in sig-
nal is may be attributed to the existence of dipolar interaction in
the complex. Further broadening of signal suggests alike chemical
environment for both copper ions [26,30].
3.8. Thermal studies
3.7. FAB mass spectral studies
The thermal stability and decomposition pattern of the com-
plexes was analyzed by TG and DTA studies. Thermo gravimetric
analysis of the complexes has been carried out in nitrogen atmo-
sphere with heating rate of 10 °C/min.
The spectral and analytical data suggests the empirical formula
[M2L(lCl)(H2O)4] for the copper complex, this is supported by the
Fig. 3. FAB mass spectrum of the copper complex, [Cu2L(lCl)(H2O)4].

584
N.V. Kulkarni et al. / Journal of Molecular Structure 1006 (2011) 580–588
The high value of
DEp, separation between the cathodic and anodic
peak potentials (Epa–Epc) for the couple CuI/CuII which is greater
than 60 mV indicate the quasi-reversible nature of the redox process
[31]. The dependency of peak potentials on scan rate and almost
constant values of Ipc/Ipa, peak current ratio (ꢁ1), which are the
characteristic features for quasireversible process, are observed in
the present study supporting the presumed nature of redox process.
The electrochemical activity of copper complex and its quasirevers-
ible nature can be explained in terms of flexibility and the size of the
coordination cavity in the complexes and the geometric require-
ments and the size of the metal ions in the different oxidation states
[32]. Further detailed studies are required to understand the nature
of chemical reactions following the electron transfers which are
helpful in enzyme catalysis.
3.10. Anti biogram analysis of the compounds against fungi and
bacteria
Fig. 4. Cyclic voltammogram of [Cu2L(lCl)(H2O)4] with scan rate 0.1 V/s.
The comparative biological activities were studied by screening
the ligand and its complexes antibacterial and antifungal activity
against gram negative bacteria E. coli, Pseudomonas aeruginosa and
It is observed that in the present copper complex, decomposi-
tion takes place in two stages. The first step of decomposition cor-
responds to a mass loss of ꢁ9.98% in the range 120–150 °C is
attributed to the loss of four coordinated water molecule. The cor-
responding DTA peak at ꢁ137 °C signifies the endothermic process.
Second stage of mass loss of 13.57% taking place at the range 200–
250 °C with respective DTA curve at ꢁ220 °C representing the exo-
thermic process, corresponds to the decomposition of ligand. The
final product was analyzed to be metal oxide.
fungi Aspergillus niger, Cladosporium at 500 lg and 250 lg (in
DMF) concentrations using cup-plate method. Gentamycine and
Flucanzol were used as standards for bacteria and fungi respec-
tively. Out of above concentrations the later is treated as minimum
inhibitory concentration (MIC). The data is summarized in Tables 4
and 5 and the same is represented graphically in Figs. 5 and 6. Li-
gand is found to be moderately active against bacteriaE. coli, P. aeru-
ginosa and fungus A. niger and show mild inhibition in case of
fungus Cladosporium. A definite enhancement in the activity of
compound is observed upon complexation, which is illustrated
graphically. The cobalt and zinc complexes show moderate activity
against all microorganisms, which is appreciable as compared to
the ligand where as the nickel complex shows better activity
against bacteria and moderate activity against fungi. The copper
complex shows very good (as good as the internal standard) activity
against E. coli and comparatively good activity against P. aeruginosa
and both the fungi. This enhancement in antimicrobial property,
which is brought about upon complexation can be related to the in-
creased lipophilicity which powers the rate of entry of molecules
into the cell and inertness of certain metal ligand linkages which
protects the molecule against enzymatic degradation.
3.9. Cyclic voltammetric studies
The ligand and complexes (0.001 M in DMF) were scanned in the
potential range of ꢀ1.0 V to 1.0 V in deareated condition with differ-
ent scan rates (0.05, 0.1 and 0.15 V/s). The ligand and other com-
plexes do not show electrochemical response in the applied range
revealing that the redox property exhibited by the copper complex
is purely metal based. The voltammogram with scan rate 0.1 V/s is
given in Fig. 4 and numerical results are represented in Table 3. A
cathodic peak observed in the voltammograms in the range
Epc = ꢀ0.05 to ꢀ0.14 V evidences the reduction of metallic species,
CuII ? CuI. The reverse scan shows two anodic peaks with potentials
in the range, Epa1 = ꢀ0.35 to ꢀ0.45 V and Epa2 = 0.55 to 0.61 V
corresponding to the oxidation reactions, CuI ? CuII and CuII ? CuIII.
Table 3
Cyclic voltammetry results.
Complex
CuL
Scan rate (V/s)
Epc (V)
Epa (V)
D
Ep (V)
E1/2 (V)
Ipc/Ipa
0.15
0.1
0.05
ꢀ0.05
ꢀ0.1
ꢀ0.45
ꢀ0.40
ꢀ0.35
0.52
0.58
0.61
0.40
0.30
0.21
ꢀ0.25
ꢀ0.25
ꢀ0.24
1.06
1.08
1.10
ꢀ0.14
D
Ep = Epa1 – Epc; E1/2 = [Epc + Epa1]/2).
Table 4
Analysis of antibacterial activity.
Compound
500
E. coli
Zone of inhibition in % of
cm inhibition
Gentamycine 3.2
l
g
250
E. coli
Zone of inhibition in % of
lg
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Zone of inhibition in % of
Zone of inhibition in % of
cm
inhibition
cm
inhibition
cm
inhibition
100
2.9
0.8
1.0
1.6
2.5
1.2
–
100
3.0
0.6
0.9
1.8
3.0
1.2
–
100
2.4
0.5
0.7
1.3
2
100
LH3
CoL
NiL
CuL
ZnL
DMF
0.8
1.1
2.2
3.4
1.4
0.1
25.00
34.37
68.75
106.2
43.75
3.12
27.58
34.48
55.17
86.20
41.37
–
20.00
30.00
60.00
100
40.00
–
20.83
29.16
54.16
8.33
33.34
–
0.8
–

N.V. Kulkarni et al. / Journal of Molecular Structure 1006 (2011) 580–588
585
Table 5
Analysis of the antifungal activity.
Compound 500
lg
250 lg
A. niger
Cladosporium
A. niger
Cladosporium
Zone of inhibition in % of
Zone of inhibition in % of
Zone of inhibition in % of
Zone of inhibition in % of
cm
inhibition
cm
inhibition
cm
inhibition
cm
inhibition
Flucanzole 1.2
100
0.9
0.2
0.3
0.4
0.5
0.3
–
100
1.0
0.1
0.2
0.4
0.6
0.3
–
100
0.8
–
100
–
LH3
CoL
NiL
0.3
0.4
0.5
0.8
0.5
–
25.00
33.34
41.66
66.67
41.66
–
22.23
33.34
44.45
55.56
33.34
–
10.00
20.00
40.00
60.00
30.00
–
0.1
0.3
0.4
0.1
–
12.50
37.50
50.00
12.50
–
CuL
ZnL
DMF
The compounds have exhibited the higher inhibitory action
against bacterium, E. coli. Hence to investigate the applicability of
the said mechanism, the in vitro interaction studies of compounds
with E. coli DNA were carried out.
In the case of copper complex (Fig. 7) the broad intense band at
480 nm assigned to LMCT is monitored as a function of added DNA.
Observations made reveal that increase in amount of DNA results
the decrease in molar absorptivity (hypochromism) as well as
red shift of ꢁ16 nm (bathochromic shift) which indicate the inter-
calative mode of interaction of complex with DNA [33]. The intrin-
sic binding constant Kb determined by the plot of A0/[A – A0] vs
[DNA]ꢀ1 with value 1.25 ꢃ 104 Mꢀ1, which is good as compared
with the classical intercalators (Kb ꢁ 106) suggest the moderate
intercalative interaction [34]. In the case of ligand and other com-
plexes similar behavior is observed but with low hypochromism
and insignificant red shift. This suggests the low binding capacity
of the compounds which is evidenced by the magnitude of intrinsic
3.11. DNA binding/cleavage studies
3.11.1. Absorption studies
In the present investigation the binding of ligand and its metal
complexes (500
lM) with E. coli DNA, in different concentrations
(50–200 M) has been characterized classically through absorption
l
spectral titrations by following the changes in absorbance and shift
in the wavelength as a function of added concentration of DNA.
a. Escherichia coli
b. Pseudomonas aeruginosa
Fig. 5. Graphical representation of antibacterial analysis of the ligand and complexes.

586
N.V. Kulkarni et al. / Journal of Molecular Structure 1006 (2011) 580–588
a. Aspergillus Niger
b. Cladosporium
Fig. 6. Graphical representation of antifungal activity of ligand and complexes.
Fig. 7. Absorption spectrum of [Cu2L(
tion with respect to the increase in the DNA concentration.
l
Cl)(H2O)4] showing the variation of absorp-
Fig. 8. Effect of added compound with increasing concentration on the viscosity of
DNA at 20 °C.
binding constants obtained, i.e., Kb = 9.8 ꢃ 103 Mꢀ1, 6.8 ꢃ 103 Mꢀ1
,
7.3 ꢃ 103 Mꢀ1 and 4.5 ꢃ 103 Mꢀ1 for nickel, cobalt, zinc complexes
viz., chloride/hydroxyl group, which is responsible for the interac-
tion of the compounds with N-7 of nucleotide, which leads to the
perturbation of DNA structure and hence inhibiting the replication.
The higher DNA binding ability of copper complex, in particular
can be assigned to its biocompatibility, high nucleobase affinity
and capacity to posses biologically accessible redox potentials. It
can recognize nucleic acids, in a sequence-specific fashion and bind
to them in such a way that their functioning will be altered [35].
and ligand respectively.
A majority reports indicate that the presence of labile ligand
units, play a vital role in the DNA binding strategy of metal com-
plexes. Effectiveness of the DNA binding/cleavage depends on the
nucleophilic nature of the labile ligand attached to the metal cen-
ters and the nature of metal ion itself. In the present complexes the
architecture of ligand is so that it provides at least one labile ligand

N.V. Kulkarni et al. / Journal of Molecular Structure 1006 (2011) 580–588
587
increases, the double stranded DNA gradually dissociates to single
strands and generates a hyperchromic effect on the absorption
spectra of DNA bases (k max = 260 nm). In order to identify this
transition process, the melting temperature Tm, which is defined
as the temperature where half of the total base pairs are un-
bounded, is introduced. According to the literature the interaction
of natural or synthesized organics and metallointercalators gener-
ally results in a considerable increase in melting temperature.
Hence the DNA melting experiment is useful in establishing the ex-
tent of intercalation binding between the compound and DNA. The
Tm of E. coli DNA in the absence of any added complex was found
to be 58 1 °C in our experimental conditions. Under the same set
of conditions, the presence of all the complexes has increased the
Tm. The copper complex show hypochromacity with increased
Fig. 9. Effect of added complexes on the melting temperature (Tm) of E. coli DNA.
Tm of 64 1 °C (DTm = 5 °C) which is characteristic of intercalative
binding behavior (Fig. 9) [21]. The ligand though shows hypochro-
macity and shift of Tm for 2–3 °C, but the extent is less as com-
pared to the copper complex. Which suggest the mild
intercalation of ligand with DNA. All other complexes show little
variation in Tm and decrease in absorbance with values falling in
between the copper complex and the ligand. The results obtained
by the hydrodynamic and absorbance studies are well supported
by the thermal denaturation experiment.
3.11.4. Gel electrophoresis method
After binding to DNA, duly designed metal complexes can in-
duce several changes in DNA conformation. Metal complexes,
which could induce DNA deformations, such as bending, ‘local
denaturation’ (overwinding and underwinding), intercalation, mi-
cro loop formation and subsequent DNA shortening lead to de-
crease in molecular weight of DNA. Gel electrophoresis is an
extensively used technique for the study binding of compounds
with nucleic acids; in this method segregation of the molecules
will be on the basis of their relative rate of movement through a
gel under the influence of an electric field. DNA is negatively
charged and when it is placed in an electric field, it migrates to-
wards the anode; the extent of migration of DNA is decided by
the strength of electric field, buffer, density of agarose gel and size
of the DNA. Generally it is seen that mobility of DNA is inversely
proportional to its size. Gel electrophoresis picture is shown in
Fig. 10. The photograph shows the bands with different bandwidth
and brightness compared to the control. The difference observed in
the intensity and the band width is the criterion for the evaluation
of binding/cleavage ability of ligand and its transition metal com-
plexes with DNA of E. coli. Unaided DNA does not show any signif-
icant cleavage of DNA even after a longer exposure time in Lane-C,
representing Control experiment. There is no significant binding/
cleavage of DNA is taking place by means of ligand and CoII com-
plex which has been indicated by the intensity and the mobility
(tailing) of lane-I and II, which are found to be as same as the con-
trol band. In lane III and V width of the DNA band is enlarged due
to the tailing and shows little increase in intensity, which may be
attributed to the binding of NiII and ZnII complexes to the DNA for
small extent. The high intensity band with maximum tailing in
lane IV suggests the strong binding of CuII complex to the DNA.
From the above observation it has been concluded that the interca-
lative binding of the metal complexes caused a change in the con-
formation of DNA [36].
Fig. 10. Photograph showing the effects of metal complexes on DNA of E. coli. Lane
M: DNA marker, Lane C: Untreated DNA, Lane I: Ligand (LH3), Lane II:
[Co2L(lOH)(H2O)4] Lane III: [Ni2L(lOH)(H2O)4], Lane IV: [Cu2L(lCl)(H2O)4], Lane
V: [Zn2L(
l
Cl)(H2O)4].
3.11.2. Viscometric measurements
This is one of the most critical tests for inferring the binding
mode (intercalation or other binding modes) of DNA in solu-
tion and was regarded as the least ambiguous and the most critical
test of binding mode in solution in the absence of crystallographic
structural data. Intercalation of drugs like ethidium bromide causes
a significant increase in the viscosity of DNA solutions due to in-
crease in the separation of base pairs at intercalation sites and
hence increase in overall DNA contour length. By the other hand,
molecules that bind exclusively in the DNA grooves typically cause
less pronounced (positive or negative) or no changes in the DNA
solution viscosity. As a means for further exploring the binding of
present ligand and complexes, viscosity measurements were car-
ried out on solution of E. coli DNA (100
l
M) by varying the concen-
M) under appropriate
obtained, where and g0 are
tration of added compounds (20–80
l
1/3
conditions. The values of (
g/g0
)
g
the specific viscosity of DNA in presence and in absence of the com-
plex respectively were plotted against [compound]/[DNA] (Fig. 8).
The results reveal that all the complexes and ligand that are bound
to E. coli DNA show increase in relative viscosities with an increase
in the [compound]/[DNA] ratio suggesting the intercalative binding
mode of the compounds with DNA [20], which is in line with the re-
sults obtained from absorption studies. Change in viscosity caused
by the copper complex was more evident than by others, which
indicate the strong intercalating capacity of complex.
4. Conclusion
It is concluded from analytical and spectral data that, the ligand
acts as hexadentate tribasic chelate with N4S2 donating sites. All
the complexes are non-electrolytic in nature. Electronic and mag-
netic moment data witnessed the octahedral geometry around
the metal ions. Only CuII complex showed electrochemical activity
3.11.3. Thermal denaturation studies
Information about the interaction strength of complexes with
DNA is offered by thermal behaviors of DNA in the presence of
complexes. It is known that when the temperature of solution

588
N.V. Kulkarni et al. / Journal of Molecular Structure 1006 (2011) 580–588
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complexation. The observations made in DNA binding study of li-
gand and its complexes interacting with E. coli DNA reveal the
moderate intercalative mode of interaction of the compounds
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The authors thank Department of chemistry and USIC, Karnatak
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Recording of FAB mass spectra (CDRI Lucknow), ESR spectrum
(IIT Bombay) are gratefully acknowledged. The authors (NVK, AK
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