Synthesis and spectroscopy of CoII, NiII, CuII and ZnII complexes derived from 3,5-disubstituted-1H-pyrazole derivative: A special emphasis on DNA binding and cleavage studies

Article abstract of DOI:10.1016/j.ejmech.2009.10.026

A series of novel Co^II, Ni^II, Cu^II and Zn^II complexes of 1H-pyrazole-3,5-dicarboxylic(2′-hydroxy-3′-hydrazinequinoxaline) has been prepared and characterized by the spectral and analytical techniques. Cu^II ion reacts with the ligand LH₃ and forms the complex in one compartment of the ligand whereas, the other compartment remains free. In Co^II, Ni^II and Zn^II complexes both compartments of LH₃ are involved in the coordination. DNA binding/cleavage studies were revealed the stronger binding capability of the present Ni^II complex, confirmed by the absorbance, viscometric and gel-electrophoresis studies. Similarly, remaining complexes do the same in the ligand field with lesser binding constants, subsequently, no complex was found to cleave the DNA. Finally, Cu^II complex shows growth inhibitory activity against biogram.

Full text of DOI:10.1016/j.ejmech.2009.10.026

European Journal of Medicinal Chemistry 45 (2010) 455–462
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and spectroscopy of CoII, NiII, CuII and ZnII complexes derived from
3,5-disubstituted-1H-pyrazole derivative: A special emphasis on DNA binding
and cleavage studies
*
Srinivasa Budagumpi, Naveen V. Kulkarni, Gurunath S. Kurdekar, M.P. Sathisha, Vidyanand K. Revankar
Department of Studies in Chemistry, Karnatak University, Pavatenagar, Dharwad 580 003, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
A series of novel CoII, NiII, CuII and ZnII complexes of 1H-pyrazole-3,5-dicarboxylic(20-hydroxy-30-
hydrazinequinoxaline) has been prepared and characterized by the spectral and analytical techniques.
CuII ion reacts with the ligand LH3 and forms the complex in one compartment of the ligand whereas,
the other compartment remains free. In CoII, NiII and ZnII complexes both compartments of LH3 are
involved in the coordination. DNA binding/cleavage studies were revealed the stronger binding capa-
bility of the present NiII complex, confirmed by the absorbance, viscometric and gel-electrophoresis
studies. Similarly, remaining complexes do the same in the ligand field with lesser binding constants,
subsequently, no complex was found to cleave the DNA. Finally, CuII complex shows growth inhibitory
activity against biogram.
Article history:
Received 25 July 2009
Received in revised form
10 October 2009
Accepted 15 October 2009
Available online 28 October 2009
Keywords:
3,5-Dichloroformyl-1H-pyrazole
Quinoxaline
Ó 2009 Elsevier Masson SAS. All rights reserved.
Gel-electrophoresis
DNA
Biogram
1. Introduction
chemists to probe DNA and to develop highly sensitive and
appropriate diagnostic agents.
Stable, inert, and non-toxic metal complexes containing
spectroscopically active metal centers are exceptionally valuable
as probes of biological systems. Transition metal complexes of
pyrazole derivatives have been the subject of much recent notice
because of their beneficial effects as anticancer agents [1–3]. A
large body of evidences indicate the mechanism of action of
anticancer agents binds through distinctive binding modes to
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. The binding modes are responsible for
activity and the mechanism by which DNA replication is totally
inhibited in cancer cells. The classical coordination complex of
platinum, iproplatin is biologically more active especially as
antitumor drug because of its structural features, which leads to
the formation of sequence-specific adducts between iproplatin
and DNA, which eventually provide the clues for binding modes
with DNA. A more complete understanding of how to target DNA
sites with specificity, size and shape will lead not only to novel
chemotherapeutics but also to a greatly expanded ability for
In coordination chemistry point of view, pyrazole is an
important class of organic compounds with two nitrogen atoms
as hetero atoms attached neighboring to each other. Both the
nitrogen atoms can strongly coordinate to the two metal centers
by deprotonation; hence can act as endogenous bridge between
the metal ions and preferably form binucleating metal
complexes [4–6]. Some of the 3,5-disubstituted pyrazole deriv-
atives can be converted in to macroacyclic and macrocyclic
compounds as well [7,8] further these acts as polydentate
chelates and form the stable coordination compounds of tran-
sition metal ions with greater specificity. The diazine bridge and
the aided potential donor groups like hydrazide –NH and
phenolic –OH at 3- and 5- positions are the interesting struc-
tural peculiarities for designing the ligand which are as same as
in iproplatin. Hence the complexes derived from this type of
ligand systems are expected to bind with DNA as iproplatin can
do. In this connection, we have prepared a series of transition
metal complexes and investigated their DNA binding activity. In
the present research article we wish to report the synthesis,
structure, redox properties, anti-biogram and DNA binding and
cleavage studies of transition metal complexes of 3,5-disubsti-
tuted pyrazole derivative.
* Corresponding author. Tel.: þ91 836 4250821, þ91 09900485652 (mobile).
E-mail address: vkrevankar@rediffmail.com (V.K. Revankar).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.026

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S. Budagumpi et al. / European Journal of Medicinal Chemistry 45 (2010) 455–462
2. Results and discussion
were recorded in DMF solution in the scan range 200–1000 nm.
Ligand exhibits a band at 277 nm, due to the intra-ligand
transition, which remains unchanged in all the complexes. The
shoulder at 390 nm in ligand is assigned for n / * transition of >N–
p
/ p*
2.1. Chemistry
p
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.
H group [13]. This band is not found in complexes, suggesting the
coordination of nitrogen atoms. A shoulder at 425 nm was assigned
to n / p* transition originating from the amide functions [13].
Electronic spectrum of cobalt complex shows an asymmetric
band at 623 nm, assigned to d–d transition, and a sharp charge
transfer band at 410 nm, which is in consistent with the tetrahedral
geometry [13,14]. Nickel complex displays two distinct bands at 575
and 615 nm which are assigned as d–d transitions and a sharp
charge transfer transition occurs at 330 nm, collectively all these
offers the square pyramidal geometry [15]. Electronic spectrum of
copper complex exhibits charge transfer transition at 360 nm and
a broad band in the region 475–530 nm corresponds to the d–
d transition. Such broad asymmetric bands near 550 nm have been
observed for related copper complexes with square pyramidal
geometry [16,17]. Finally, the spectrum of zinc complex shows band
in the range 426–453 nm is accounted for the ligand to metal
charge transfer transition.
2.1.1. Infrared spectral studies
The IR spectral details are shown in Table 2. IR spectrum of
ligand shows a strong band at 1653 cmꢀ1, assigned to amide
carbonyl (>C]O) [9], remains unchanged on complexation sug-
gesting its non-involvement in the coordination. Broad band at
3447 cmꢀ1 shows the presence of hydroxyl (–OH) groups of qui-
noxaline moiety, which on complexation disappears and could be
due to coordination of oxygen to metal ions after deprotonation.
Intense bands at 3174, 3045 and 3000 cmꢀ1 in ligand are attributed
to hydrazide N–H, amide N–H and pyrazole ring N–H respectively
[9]. In complexes, hydrazide N–H shifts to the lower region which
suggests the coordination of hydrazide nitrogen and the same was
further supported by the 1H NMR spectrum of the ZnII complex.
In case of CoII, NiII and ZnII complexes a new band appeared in
the region 1520–1535 cmꢀ1 and the same was assigned to the
formation of new >C]N bond in pyrazole ring which is less than
that of the normal >C]N in pyrazole [10]. This observation
supports the coordination of both the nitrogen atoms of the pyr-
azole moiety to the metal centers. In CuII complex, the sharp
splitting of bands at 1528, 1503 cmꢀ1 and 3054, 3044 cmꢀ1
suggests that, the coordination occurs at one side of the ligand as
shown in the Fig. 4. CoII and NiII complexes show a sharp band at
3419 and 3426 cmꢀ1 which are due to the presence of –OH as
exogenous bridging unit between metal centers [11].
2.1.4. Magnetic properties
Magnetic moment data of the complexes were recorded at room
temperature and are shown in Table 1. Copper complex shows the
effective magnetic moment meff 1.62 B.M., which is less than that of
the spin only value (1.73 BM) and attributed to the weak antifer-
romagnetic interaction between copper centers through the
bridged quinoxaline oxygen atoms. Thus the magnetic moment
value and spectral data supports the square pyramidal geometry for
the complex. In addition to this, the magnetic moments of nickel
and cobalt complexes were found to be 2.82 and 4.05 BM respec-
tively supporting the assigned geometry. These magnetic moment
values are almost same as spin only values of CoII and NiII free ions
respectively. The lower magnetic moment values are due to the
very weak antiferromagnetic interaction between metal centers
through the exogenous –OH bridge. These magnetic moment value
and spectral data confirms the square pyramidal [18] and tetrahe-
dral [19] geometries around nickel and cobalt ions respectively.
2.1.2. 1H NMR spectral studies
1H NMR spectrum of the ligand and its ZnII complex was scanned
in the range 0–16
11.9, 10.9 and 4.3
d
ppm. The ligand displayed the signals at 14.1,
d
ppm which can be attributed to N–H of pyrazole
ring [12], –OH proton of quinoxaline, amide protons and hydrazide
protons respectively. In addition to this, a set of multiplet was
observed in the range 6.96–7.85
aromatic protons.
d ppm due to the presence of
2.1.5. ESR spectral study
The comparative study of 1H NMR data of ligand and its zinc
complex reveals about the ligational behavior of the ligand. In the
1H NMR spectrum of zinc complex, the signals due to pyrazole N–H
and quinoxaline –OH protons were absent indicating the involve-
ment of both the nitrogen atoms of pyrazole ring and oxygen atoms
of quinoxaline in the coordination after deprotonation respectively.
Further hydrazide N–H peak is shifted to 3.4 d ppm, which suggests
the coordination of nitrogen atoms and there is no significant
changes in the remaining peaks including in their intensities.
The powder state ESR spectrum of copper complex was oper-
ated in the region 9000 MHz with corresponding field intensity at
w3000 Gauss at room temperature. The spectrum exhibits
isotropic intense broad signal with giso 2.072 and no hyperfine
splitting was observed. ESR spectrum of this kind have been
reported earlier for complexes having large organic ligands [20],
and also the broad ESR signal indicates the presence of chloro
coordinated binuclear copper(II) complexes [21]. This broadening is
because of the dipolar interaction.
2.1.3. UV–visible spectral studies
2.1.6. Molar conductivity measurements
UV-visible spectral data of the ligand and its complexes are
tabulated in Table 3. The electronic spectra of ligand and complexes
The molar conductance value of complexes was obtained at
room temperature in DMF solution with 10ꢀ3 mol/dm3
Table 1
Analytical, magnetic moment and conductance data of CoII, NiII, CuII and ZnII complexes.a
Sl. No
Complex
Metal
Carbon
Hydrogen
Nitrogen
Chloride
meff in BM
Molar conductance
mho cm2 moleꢀ1
1)
2)
3)
4)
[Co2L(OH)]$3H2O
[Ni2LCl(H2O)2]$2H2O
[CuLH2Cl]2$4H2O
[Zn2L(OH)]$3H2O
17.45 (16.92)
17.00 (17.62)
10.49 (11.11)
19.10 (20.01)
32.27 (32.86)
36.31 (36.98)
41.65 (41.02)
36.46 (35.94)
3.25 (3.01)
3.45 (4.01)
2.97 (3.09)
2.31 (2.40)
20.71 (21.09)
20.17 (20.91)
23.14 (23.72)
20.26 (20.91)
5.71 (5.78)
5.04 (5.44)
5.78 (5.01)
5.06 (5.70)
4.05
2.82
1.62
–
3.5
3.8
3.3
2.4
a
Calculated values are given the parenthesis.

S. Budagumpi et al. / European Journal of Medicinal Chemistry 45 (2010) 455–462
457
Table 2
Infrared spectral data of ligand and complexes b: Broad band; s: Sharp band.
Compounds
>C ¼ O (amide)
N–H (hydrazide)
N–H (pyrazole)
–OH (quinoxaline)
>C ¼ N (pyrazole)
M-N
–OH (bridge)
LH3
1656
1653
1648
1657
1661
3219
w3150
w3100
w3200
w3100
3045
–
–
3044
–
b 3447
–
–
b 3435
–
1565
1520
1531
1531
1535
–
–
[Co2L(OH)]$3H2O
[Ni2LCl(H2O)2]$2H2O
[CuLH2Cl]2$4H2O
[Zn2L(OH)]$3H2O
472
475
476
473
s 3419
s 3426
–
–
concentration. The molar conductivity values of all the complexes
fall in the range 2.4–3.8 ohm cm2 molꢀ1 (shown in Table 1), which
is in agreement with non-electrolytic nature of the complexes [22].
The cyclic voltammogram of ligand and copper complex were
scanned in the potential range of þ1.0 to ꢀ1.0 V with different scan
rates viz., 0.05, 0.1 and 0.15 V/s. Ligand did not show any electro-
chemical response over the working potential range. The cyclic
voltammogram of copper complex is shown in Fig. 1. The voltam-
mogram shows one electron transfer process in anodic potential
scan, with oxidation peak at 0.31, 0.33 and 0.34 V at above
mentioned scan rates and the same is attributed to the oxidation of
CuII / CuIII. After the oxidation peak, cathodic peaks were observed
at 0.12, 0.14, 0.15 V and ꢀ0.36, ꢀ0.37, ꢀ0.39 V during cathodic scan
at above mentioned scan rates and were assigned to the reduction
of CuIII / CuII and finally CuII / CuI [12]. It is concluded from cyclic
voltammetric studies that, the copper complex exhibits one-elec-
tron transfer process in anodic scan and two-electron transfer
process in cathodic scan. The separation between anodic and the
2.1.7. FAB mass spectral studies
The elemental and analytical data of the copper complex
suggests the empirical formula [CuLH2Cl]2.4H2O and this is sup-
ported by the FAB mass spectrum. The peaks at highest m/z value
cannot always be assigned with certainty, but the isotopic pattern is
consistent with a binuclear complex. A peak at m/z 1216 was
observed, corresponding to the mass of the entire mononuclear,
dimeric complex including coordinated chloride ion and lattice
celled water molecules [23]. Apart from this, spectrum shows some
other peaks, which are due to molecular cations of various frag-
ments of complex. By comparing all the analytical and spectral data
of cobalt, nickel and zinc complexes, it is evident that these are
monomeric and binuclear complexes.
first cathodic peak potentials i.e.,
D
Ep ¼ Epa ꢀ Epc is 192 mV indi-
cates the quasireversible redox process.
2.1.8. Thermal studies
2.2. Biochemistry
The thermogravimetric analysis of the CuII complex has been
carried out in nitrogen environment over the temperature range
30–1000 ꢁC. TG curves of copper complex show weight losses in
three considerable steps. In the first step 4.7%, in the temperature
range 55–120 ꢁC attributed to the weight loss of four lattice celled
water molecules and this process is endothermic in nature, which is
evident by the DTA signal at 98 ꢁC. The weight loss at second step is
15.25% observed in the temperature range 130–280 ꢁC which may
be due to combined elimination of chloride as HCl and some part of
the ligand, the corresponding DTA signal is at 201 ꢁC. Partially
decomposed complexes are unstable and decompose completely in
the next step means, the remaining part of the ligand is completely
eliminated in third stage of weight loss in the temperature range
280–520 ꢁC, and thereafter the curve became plateau due to the
formation of stable CuO.
2.2.1. Anti-biogram analysis
In order to study the comparative biological activities, the
compounds were screened for antibacterial (against Escherichia
coli, Aeruginosa Pseudomonasa) and antifungal (against Aspergillus
Niger, Cladosporium) activities at 500 mg and 250 mg concentrations
in DMF, out of which the later was treated as minimum inhibitory
concentration (MIC).
The copper complex shows moderate activity i.e. 43.75 and
41.37% zone of inhibition against E. coli, A. Pseudomonasa bacteria
respectively at 500 mg concentration, which is more than that of
ligand activity. In MIC level, only copper complex is active against A.
Pseudomonasa with 12.5% zone of inhibition. In antifungal studies,
copper complex exhibits extremely high activity, 100% zone of
inhibition, against A. Niger which is as good as the internal standard
at both the concentrations. The moderate activity, 77.7 and 33.3%, at
both the concentration levels was shown against Cladosporium.
2.1.9. Cyclic voltammetric studies
The cyclic voltammograms of ligand and copper complex were
recorded at room temperature in DMF solution under oxygen free
conditions, which was created by purging pure nitrogen gas. An
assembly of three electrodes consists of glassy carbon working
electrode, a platinum auxiliary electrode and Ag/AgCl reference
electrode was used and tetramethyl ammonium chloride with
0.1 M concentration as supporting electrolyte.
Table 3
UV–visible spectral data of ligand and complexes.
Compounds
Peak values (in nm) (lmax in cmꢀ1
)
LH3
430 (23,256), 390 (25,641), 277 (36,101)
623–600 (16,051–16,667), 421 (23,753),
342 (29,250), 311 (32,154), 275 (36,364)
615 (16,260), 575 (17,391), 330 (30,303),
340 (29,412), 320 (31,250), 311 (32,154)
476–530 (21,008–18,868), 360 (27,778),
325 (30,769), 310 (32,258)
[Co2L(OH)]$3H2O
[Ni2LCl(H2O)2]$2H2O
[CuLH2Cl]2$4H2O
[Zn2L(OH)]$3H2O
453 (22,075), 426 (23,474), 400 (25,000),
275 (36,364)
Fig. 1. Cyclic voltammogram of copper (II) complex at scan rate of 0.1 V sꢀ1
.

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S. Budagumpi et al. / European Journal of Medicinal Chemistry 45 (2010) 455–462
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
DNA
Ni(II) complex
Cu(II) complex
0
20
40
Temperature
60
80
100
Fig. 4. Effects of increasing temperature on E. coli DNA alone, E. coli DNA with Ni-
complex and Cu-complex.
Fig. 2. Gel-electrophoresis picture of ligand and its complexes. Photograph showing
the effects of transition metal complexes on DNA of E. Coli. Lane M: DNA marker, Lane
type of binding of the complexes to DNA is found. With decrease in
the absorbance and appreciable red shift of the absorption
maximum at around 330 nm in all the complexes (the shift being
11, 23, 17 and 14 nm for [Co2L(OH)]$3H2O, [Ni2LCl(H2O)2]$2H2O,
[CuLH2Cl]2$4H2O and [Zn2L(OH)]$3H2O respectively) indicates the
intercalation. The binding constants of the complexes
[Co2L(OH)]$3H2O, [Ni2LCl-
C: Untreated DNA, Lane 1: Ligand (LH3), Lane 2:
[Ni2Lm(Cl)(H2O)2], Lane 4: [Cu2(LH2Cl)2].4H2O, Lane 5: [Zn2Lm(OH)].
[Co2Lm(OH)], Lane 3:
2.2.2. DNA cleavage studies by gel-electrophoresis method
Gel-electrophoresis technique works on the migration of DNA
under the influence of electric potential. Gel-electrophoresis
picture is shown in Fig. 2. The photograph shows the bands with
different bandwidth, compared to the control, is the differentiating
criteria for binding ability of complexes with E. coli in this study.
Control experiment using DNA alone does not show any significant
cleavage of DNA even after a longer exposure time. In lanes 2–4,
width of the DNA band is little bigger and intense in appearance
and shows tailing to a very lesser extent. This result revealed the
stronger binding of the complexes to DNA in [Co2L(OH)],
[Ni2L(Cl)(H2O)2], and [Cu2(LH2Cl)2]$4H2O systems and could be
attributed to the binding but not the cleavage. On the other hand,
lanes 1 and 5 do not show any significant changes even in the
intensity and bandwidth compared to the control which elucidates
the inactiveness of ligand and [Zn2L(OH)] complex towards the
binding or cleavage of E. Coli DNA.
(H2O)2]$2H2O, [CuLH2Cl]2$4H2O and [Zn2L(OH)]$3H2O are found to
be 6.1 ꢂ104, 1.34 ꢂ106, 1.87 ꢂ 105 and 3.25 ꢂ105 respectively.
2.2.4. DNA melting temperature (Tm) studies
The Tm of E. coli DNA is the temperature at which 50% of the
nucleotide and its perfect complement are in duplex. Typically,
annealing or hybridizations are performed at 5–10 ꢁC below the Tm
of a duplex. Stability of the DNA double helix influences the melting
temperature (Tm) of DNA, while the binding of compounds to DNA
alters the Tm depending on the strength of interactions. The inter-
calation of the complexes into the DNA base pairs causes stabili-
zation of base stacking and hence raises the melting temperature of
the double stranded DNA. The DNA melting experiment is useful in
establishing the extent of intercalation [27]. As shown in Fig. 4, the
Tm of DNA in the absence of any added complex was found to be
58 ꢃ 1 ꢁC, under our experimental conditions [28]. Under the same
set of conditions, the presence of [CuLH2Cl]2$4H2O and [Ni2LCl(-
H2O)2]$2H2O complexes increased the Tm of about 3 and 6 ꢁC
respectively, which is characteristic of an intercalative binding
behavior of the complexes with the DNA [29].
2.2.3. DNA binding analysis using electronic spectral method
The UV–visible spectral technique is the most commonly used
method to know the interaction of metal complexes with DNA
[24–26], since transition metal complexes often have abundant
spectroscopic properties. The representative absorption spectrum
of NiII complex in presence/absence of E. Coli DNA is shown in the
Fig. 3. The binding behavior of complexes to E. coli DNA helix has
been followed through absorption spectral titrations. With
increasing concentration of E. coli DNA, the absorption bands of the
complexes were affected, resulting in the tendency of hypo-
chromism and a remarkable red shift is observed in all the
complexes. There are two isosbestic points observed for all the four
complexes. This implies that 1: 1 complex: DNA stoichiometry is
maintained during the binding process and/or there is only one
2.2.5. DNA binding analysis using viscosity measurement
The hydrodynamic method (viscometric measurement) is also
a crucial tool to find the nature of binding of metal complexes to the
Fig. 3. Absorption spectra [Ni2Lm(Cl)(H2O)2] in the absence (very first line from
pinnacle) and in the presence of increasing amounts of E. coli DNA. Arrow mark shows
the absorption changes upon changing the E. coli DNA concentration.
Fig. 5. Effects of increasing amounts of Co-complex, Ni-complex, Cu-complex and Zn-
complex on the relative viscosities of E. coli DNA at 29 ꢁC.

S. Budagumpi et al. / European Journal of Medicinal Chemistry 45 (2010) 455–462
459
O
O
O
O
HN
NH
HN
NH
NH
OH2
N
N
NH
NH
N
N
N
N
NH
N
N
Ni
Ni
M
M
O
O
OH2
Cl
N
N
O
O
O
H
N
N
M: Co & Zn
O
O
H
N
N
NH
NH
N
H
N
N
H
N
OH
N
Cl
Cu
.
4H2O
N
O
O
N
HO
N
Cu
N
Cl
N
H
NH
HN
N
H
N
N
N
H
O
O
Fig. 6. Proposed structures of the complexes.
DNA, inwhich the solution viscosity of DNA is sensitive to the changes
intheeffectivelengthofDNAmoleculesisoneofthemostcriticaltests
for inferring the binding mode (intercalation or other binding modes)
of DNA. This study was regarded as the least ambiguous and the most
critical tests of binding mode in solution state in absence of
crystallographic structural data [30–32]. Under the appropriate
conditionsintercalationcausesnoteworthyincreaseintheviscosityof
DNA solution due tothe disjointingofbasepairs at intercalationspots.
The results of the viscosity measurements for all the complexes that
are bound to DNA show increase in relative viscosities with an
O
O
O
O
SOCl2
Reflux
HO
OH
Cl
Cl
N
N
N
N
H
H
Pyrazole-3,5-dicarboxylic acid
3,5-dichloroformyl pyrazole
H
N
N
N
NH2
+ 2
OH
2-hydroxy-3-hydrazinequinoxaline
Reflux in absolute Ethanol
O
O
HN
NH
NH
HN
N
N
H
N
N
OH
HO
N
N
LH3
Fig. 7. Scheme: preparation of the ligand.

460
S. Budagumpi et al. / European Journal of Medicinal Chemistry 45 (2010) 455–462
increaseinthe[complex]/[DNA]ratio(where[complex]is50,100,150
and 200 l) as shown in Fig. 5. Thus, the increase in the viscosity has
pressure. The resultant white pasty solid was cooled in an ice bath
for about 15 min and the hot ethanolic solution of 2-hydroxy-
3-hydrazinequinoxaline (0.04 M) was added and further refluxed
for about 4 h, where upon a clear pale yellow solution was
produced. The resultant solution was then cooled to get the yellow
colored ligand (LH3) (Fig. 7), which was separated by filtration
under suction and dried under lamp. The ligand is recrystallized
from hot ethanol. Yield: 78%, M.P.: >300 ꢁC.
m
been attributed to the enlargement of the separation between base
pairs, which are pushed apart to accommodate the intercalating
molecule [33–35]. These results suggested an intercalative binding
mode of the complexes with DNA and also hold up the results
obtained from electronic absorption studies.
3. Conclusion
4.1.2. Preparation of complexes
It is concluded from analytical and spectral data that, the ligand
acts as tridentate monobasic and hexadentate tribasic chelate with
donating sites N2O and N4O2 respectively. Electronic and magnetic
moment data support the square pyramidal geometry around
copper and nickel metal ions, whereas cobalt is tetrahedral. Zinc
complex is found to be tetrahedral and copper complex is mono-
nucleating and dimeric in nature, which are confirmed by the
spectral and elemental analysis (Fig. 6). ESR spectrum of copper
complex shows line broadening due to bulkier organic ligand and
magnetic moment confirms the weak antiferromagnetic interac-
tion between copper centers. Anti-biogram studies witnessed the
prominent activity of copper complex with compared to the
activity of tested compounds. The DNA binding experiments using
electronic spectral technique show the hypochromism at d–d
transition region and hypochromism and red shift at the charge
transfer region. Viscosity measurement confirms the intercalative
binding mode of complexes with the E. coli DNA. The same is
further witnessed by the DNA melting temperature studies.
The complexes of the ligand LH3 viz., CoII, NiII, CuII and ZnII were
prepared by refluxing the respective metal chlorides (0.002 mol) in
50 ml of ethanol with ligand (0.001 mol) for about 3–4 h at water
bath temperature. In case of cobalt and nickel complexes the pH of
the solution was raised by the addition of alcoholic NH3 in order to
separate the complexes in solid form. So obtained complexes were
filtered under suction and dried over anhydrous CaCl2.
4.2. Biochemistry
4.2.1. Methodology for anti-biogram analysis against bacteria
Media Used: Peptone-10 g, NaCl-10 g and Yeast extract 5 g, Agar
20 g in 1000 ml of distilled water.
Initially, the stock cultures of E. coli and Pseudomonas were
revived by inoculating in broth media and grown at 37 ꢁC for 18 h.
The agar plates of the above media were prepared and wells were
made in the plate. Each plate was inoculated with 18 h old cultures
(150
ml) and spread evenly on the plate. After 20 min, the wells
were filled with 100
ml of each compound (10 mg/ml in DMF). The
4. Experimental protocol
control plates with Gentamycin (10 mg/ml) and DMF were also
prepared. All the plates were incubated at 37 ꢁC for 24 h and the
diameter of inhibition zone was noted. The values were compared
with that of Gentamycine and the samples showing significant
inhibition were selected for the further calculation of minimum
inhibition concentration (MIC).
The MIC of compounds was determined by assaying at 500 and
250 mg concentrations along with standard gentamycin at the same
concentrations. The cultures were grown for 24 h and the zones
were compared with that of gentamycin and % of inhibition was
calculated.
All the chemicals used were of reagent grade and the solvents
were dried and distilled before use according to the standard
procedures. The preparation of 2-hydroxy-3-hydrazino quinoxaline
was prepared according to the earlier reports [36] and pyrazole-3,5-
dicarboxylic acid was purchased. The metal chlorides used were in
the hydrated form. All the compounds were analyzed for Carbon,
Hydrogen and Nitrogen by Thermo quest elemental analyzer. Esti-
mation of the metal(II) ions was done according to the standard
methods. The molar conductivity measurements were made on
ELICO-CM-82 conductivity bridge. The magnetic susceptibility
measurements were made on Faraday balance at room temperature
using Hg[Co(SCN)4] as calibrant. The 1H NMR spectra were recorded
in DMSO-d6 solvent on Bruker-300 MHz spectrometer at room
temperature using TMS as internal reference. IR spectra were
recorded in a KBr matrix using an Impact-410 Nicolet (USA) FT-IR
spectrometer in 4000–400 cmꢀ1 range. The electronic spectra of the
complexes were recorded on a Hitachi 150–20 spectrophotometer
in the range of 1000–200 nm. The cyclic voltammetric studies were
performed at room temperature in DMSO under O2 free condition
using CH instruments Electrochemical analyzer, CHI-1110 A (USA).
The ESR spectra of the copper complexes were scanned on a Varian
E-4X-band EPR spectrometer, using TCNE as the g-marker. TG and
DTA measurements of the complexes were recorded in nitrogen
atmosphere on Universal V2.4F TA Instrument keeping final
temperature at 800 ꢁC and heating rate was 10 ꢁC/min. The FAB
mass spectra were drawn from JEOL SX 102/DA-6000 mass spec-
trometer using Argon/Xenon (6 kV, 10 mA) as the FAB gas.
4.2.2. Methodology for anti-biogram analysis against fungi
Media Used: Potato Dextrose Agar (PDA). 250 g of peeled potato
were boiled for 20 min and squeezed and filtered. To this filtrate
20 g of dextrose was added and the volume was made up to
1000 ml by distilled water.
Initially, the stock cultures of A. niger and Cladosporium were
revived by inoculating in broth media and grown at 37 ꢁC for 48 h.
The agar plates of the above media were prepared and wells were
made in the plate. Each plate was inoculated with 48 h old cultures
(150
ml) and spread evenly on the plate. After 20 min, the wells
were filled with 500
ml of each compound (10 mg/ml in DMF). The
control plates with Flucanazole (10 mg/ml) and DMF were also
prepared. All the plates were incubated at 37 ꢁC for 48 h and the
diameter of inhibition zone was noted. The values were compared
with that of flucanazole and the samples showing significant
inhibition were selected for the further calculation of MIC.
The MIC of compounds was determined by assaying at 500 and
250 mg concentrations along with standard flucanazole at the same
4.1. Chemistry
concentrations. The cultures were grown for 48 h and the zones were
compared with that of flucanazole and % of inhibition was calculated.
4.1.1. Preparation of the ligand (LH3)
Pyrazole-3,5-dicarboxylic acid (0.02 M) was refluxed with 10 ml
of thionyl chloride for about 4 hrs at 110–115 ꢁC under anhydrous
conditions. Excess thionyl chloride was removed under reduced
4.2.3. Methodology for DNA cleavage analysis
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

S. Budagumpi et al. / European Journal of Medicinal Chemistry 45 (2010) 455–462
461
media was prepared, autoclaved for 15 min at 121 ꢁC, 15 lb pres-
sure. The autoclaved media were inoculated with the seed culture
and incubated at 37 ꢁC for 24 h.
Isolation of DNA: DNA was isolated using the procedure
mentioned below,
The fresh bacterial culture (1.5 ml) was centrifuged to obtain the
pellet and made to dissolve in 0.5 ml of lysis buffer (100 mM tris pH
8.0, 50 mM EDTA, 50 mM lysozyme) and to which added 0.5 ml of
saturated phenol followed by the incubation at 55 ꢁC for 10 min.
The resultant mixture was centrifuged at 10,000 rpm for 10 min
and to the supernatant, equal volume of chloroform and isoamyl
alcohol (24.1) and 1/20th volume of 3 M sodium acetate (pH 4.8)
were added and further, centrifuged at the same conditions. Soon
after, 3 volumes of chilled absolute alcohol were added and so
obtained DNA was separated by centrifugation. Finally the pellet
was dried and dissolved in TE buffer (10 mM tris pH 8.0, 1 mM
EDTA) and stored in cool.
4.2.5. Methodology for DNA binding analysis using viscosity
measurement
Viscosity measurements were carried out using an Oswald micro-
viscometer, maintained at constant temperature (29 ꢁC) in a thermo-
stat. The DNA concentration was kept constant in all samples, but the
complex concentration was increased each time (from 50 to
250 mM). Mixing of the solution was achieved by bubbling the
nitrogen gas through viscometer. The mixture was left for 10 min at
29 ꢁC after addition of each aliquot of complex. The flow time was
measured with a digital stopwatch. The experiment was repeated in
1/3
triplicate to get the concurrent values. Data are presented as (
versus the ratio [complex]/[DNA], where
viscosity of DNA in presence and in absence of the complex respec-
h/h0)
h
and h0 are the specific
tively. The values of
h
and h0 were calculated by using equation (2),
h
¼ ðt ꢀ t Þ t
(2)
=
b
b
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 (h/h0).
Sample preparation: The samples (10 mg/ml) were prepared in
DMSO.
Treatment of DNA with the samples: The synthetic compounds
(100 mg) were added separately to the DNA sample of E. coli. The
4.2.6. Methodology for thermal denaturation study
sample mixtures were incubated at 37 ꢁC for 2 h.
Thermal denaturation studies were carried out on UV–visible
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 hyper-
chromicity of E. coli DNA at 260 nm as a function of temperature.
Agarose gel-electrophoresis: To know the DNA cleavage action of
compounds, 200 mg of agarose 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 w55 ꢁC,
allowed to solidify and then, comb was removed carefully. So
obtained solid gel was placed in the electrophoresis chamber floo-
The melting temperatures were measured in 50 mM DNA in the
same buffer at pH 7.2. The 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.
ded with TAE buffer. Subsequently 20 mL 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 made to pass for around 30 min. Finally, the gel was
removed carefully and stained with ETBR (Ethidium bromide)
Acknowledgments
solution (10 mg/ml) for 10–15 min and the bands were observed
under UV transilluminator. The illuminated gel was photographed
by using a polaroid camera (a red filter and polaroid film were used).
The authors thank the USIC, Karnatak University, Dharwad for
providing spectral facility. Recording of FAB mass spectra (CDRI
Lucknow) and ESR spectra (IIT Bombay) are gratefully acknowl-
edged. The authors thank (Srinivasa Budagumpi) University Grants
Commission, New Delhi, India for awarding Research Fellowship in
Science for Meritorious Students and Karnatak University, Dhar-
wad, Karnataka, India (Naveen V. Kulkarni and Sathisha M.P.) for
providing research fellowships.
4.2.4. Methodology for DNA binding analysis using electronic
spectral method
The concentration of E. coli DNA per nucleotide [C(p)] was
measured by using its known extinction coefficient at 260 nm
(6600 Mꢀ1 cmꢀ1) [30]. The absorbance at 260 nm (A260) and at
280 nm (A280) for E. coli DNA was measured to check its purity. The
ratio A260/A280 was found to be 2.22, indicating that E. coli DNA is
satisfactorily free from protein. Buffer [5 mM tris(hydroxymethyl)
aminomethane, pH 7.2, 50 mM EDTA] was used for the absorption,
viscosity, and thermal denaturation experiments.
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