DNA interaction studies of pyrazolone- and diimine-incorporated Mn(II), Co(II), Ni(II), Cu(II), and Zn(II) complexes: Synthesis, spectroscopic characterization, and antimicrobial study

Article abstract of DOI:10.1007/s00706-012-0857-7

Mn(II), Co(II), Ni(II), Cu(II), and Zn(II) mixed-ligand complexes were synthesized using 4-[(furan-2-ylmethylene)amino]-1,2-dihydro-1,5-dimethyl-2- phenyl-3H-pyrazol-3-one as the main ligand and 1,10-phenanthroline/2,2′- bipyridine as co-ligand(s). They w

Full text of DOI:10.1007/s00706-012-0857-7

Monatsh Chem (2013) 144:605–620
DOI 10.1007/s00706-012-0857-7
ORIGINAL PAPER
DNA interaction studies of pyrazolone- and diimine-incorporated
Mn(II), Co(II), Ni(II), Cu(II), and Zn(II) complexes: synthesis,
spectroscopic characterization, and antimicrobial study
•
Arunagiri Sakthivel Natarajan Raman
•
Liviu Mitu
Received: 20 April 2012 / Accepted: 29 August 2012 / Published online: 23 October 2012
Ó Springer-Verlag 2012
Abstract Mn(II), Co(II), Ni(II), Cu(II), and Zn(II)
mixed-ligand complexes were synthesized using 4-[(furan-2-
ylmethylene)amino]-1,2-dihydro-1,5-dimethyl-2-phenyl-3H-
pyrazol-3-one as the main ligand and 1,10-phenanthroline/
2,20-bipyridine as co-ligand(s). They were characterized by
the usual analytical and spectral techniques. The data
obtained reveal that the complexes adopt an octahedral
geometry around the central metal ions. These complexes
were found to be better antimicrobial agents than the free
ligands. The DNA (CT) binding properties of the com-
plexes were explored by UV–Vis, viscosity measurements,
cyclic voltammetry, differential pulse voltammetry, and
molecular docking studies. Binding constants for the above
complexes were found to be on the order of 104, indicating
that most of the synthesized complexes are partial inter-
calators. The DNA-cleavage activities of the complexes
were also assessed using supercoiled pUC19 DNA and gel
electrophoresis, and the results revealed that the hydroxyl
radical is likely to be the reactive species responsible
for the cleavage of pUC19 DNA by the synthesized
complexes.
Keywords Metal complex ꢀ DNA binding ꢀ
Molecular docking ꢀ DNA cleavage ꢀ
Antimicrobial activity
Introduction
Schiff bases are very attractive and have achieved recog-
nition for their roles in biological processes such as
mutagenesis and carcinogenesis. In addition to this, in
recent years, lots of research have been done on the
coordination chemistry of transition metal complexes with
Schiff base ligands in order to mimic the physical and
chemical behavior seen in biological processes [1, 2]. The
interactions of transition metal complexes with DNA have
been the focus of recent investigations. Metal complexes
that exhibit interactions with DNA have been studied with
the aim of developing probes for nucleic acid structures
and chemotherapy agents [3]. By changing the ligand
environment, it is possible to study the DNA binding and
cleavage abilities of metal complexes. Such studies are also
important for determining the mechanism of metal ion
toxicity [4, 5].
The transition metal complexes of 1,2-dihydro-1,
5-dimethyl-2-phenyl-3H-pyrazol-3-one (4-aminoantipyrine)
and its derivatives have been extensively examined due to
their wide range of applications in biological, analytical,
and therapeutic fields [6]. Furfural is an important nontoxic
compound. It is used as a fungicide and nematicide [7].
Hence, we elected to use this heterocyclic compound when
attempting to design a Schiff base ligand with 4-amino-
antipyrine. This ligand is useful as it contains aromatic
rings that are able to stack the bases in DNA, oxygen and
nitrogen atoms which can establish hydrogen bonds with
DNA, and there is the possibility that the metal complexes
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-012-0857-7) contains supplementary
material, which is available to authorized users.
A. Sakthivel ꢀ N. Raman (&)
Research Department of Chemistry, VHNSN College,
Virudhunagar, Tamil Nadu 626 001, India
e-mail: drn_raman@yahoo.co.in
L. Mitu
Department of Physics and Chemistry, University of Pitetsi,
Pitetsi 110040, Romania
123

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A. Sakthivel et al.
display a positive charge in order to interact electrostati-
cally with the phosphate groups of DNA. In general, metal
complexes that are capable of abstracting ribosyl hydrogen
are expected to oxidize guanine or other nucleobases
[8–10]. Current efforts are being made to design transition
metal complexes that can be used as chemical nucleases in
plasmid nicking or in direct strand scission. The practical
use of transition metal complexes as chemical nucleases
has also been documented [11, 12]. DNA binding, cleav-
age, and the antimicrobial activities of Mn(II), Co(II),
Ni(II), Cu(II), and Zn(II) complexes that are derived from
furfural with 4-aminoantipyrine and 1,10-phenanthroline
(phen)/2,20-bipyridine (bpy) are of interest to us in our
studies, as discussed in this present paper.
Results and discussion
Synthetic routes for the preparation of different compounds
are depicted in Scheme 1. The ligand L and the complexes
were found to be stable in air. L is soluble in common
organic solvents but the complexes are soluble only in
DMF and DMSO. The elemental analysis data on L and the
complexes are in good agreement with the presented
Scheme 1
123

DNA interaction studies of pyrazolone- and diimine-incorporated complexes
607
formulae. Physicochemical and spectroscopic studies
indicate the formation of monomeric complexes of
stoichiometry [M(L)(phen/bpy)2]Cl2 [13].
which are due to p–p* and n–p* transitions, exhibit blueshifts
or redshifts due to the coordination of the ligand with metal
ions. The electronic absorption spectral data for the ligand
and all of the complexes are given in Table 1. The electronic
spectra of the complexes [Cu(L)(phen)2]Cl2 and [Cu(L)
Infrared spectra
(bpy)2]Cl2 exhibitonebroadbandat15,760and13,755 cm-1
,
The IR spectra of the complexes were compared with the
ligands in order to ascertain the changes that might have
taken place. Although the Schiff base L and bpy/phen
mixed-ligand complexes contain different types of C=N
bonds arising from the bpy/phen ring and azomethine link-
age, they are not well resolved in the spectra of the
complexes [14, 15]. Furthermore, in the spectrum of L,
although the azomethine –CH=N band is superimposed on
that of the C=O group of the imide, it is only a weak band.
The peaks that appear in the range 1,639–1,600 cm-1 may
be due to stretching of the azomethine –CH=N group,
whereas the bands in the range 1,552–1541 cm-1 could be
due to the m(C=N) stretches of bpy/phen. The m(C=O) band
from L at 1655 cm-1 is shifted in frequency by *10 cm-1
on complexation [16, 17]. A shift in the frequency of the
band due to HC=N cannot be observed because it is not well
resolved in the infrared spectra of the ligand and the com-
plexes. However, the HC=N present in L is also involved in
the formation of a bond with the metal atom during com-
plexation, as clearly confirmed by the NMR data. The
change in the frequency of the C=N band by 10 cm-1
indicates that bpy/phen rings are involved in the formation
of the complex. Moreover, after coordination, the bands
observed at around 881–892 cm-1 in bpy, 847–871 cm-1 in
phen, 735–750 cm-1 (C–Hdef), and 1,471–1,480 cm-1 and
1,315–1,325 cm-1 (C–N stretching bands) prove that the
bpy and phen rings are coordinated to the metal ion through
nitrogen. The new bands observed in the regions 418–442
and 478–490 cm-1 for all of the complexes were assigned to
M–N and M–O stretching vibrations, respectively, con-
firming the metal coordination [18, 19].
respectively, with a low-intensity hump assigned to the
2Eg?2T2g transition. The above data reveal that the Cu(II)
complexes adopt a distorted octahedral geometry around the
central metal ion. The observed magnetic moments of the
complexes (1.93 and 1.82 BM, respectively) at room tem-
perature indicate uncoupled mononuclear complexes of a
magnetically diluted d9 system with an s = 1/2 spin state of
distorted octahedral geometry [20]. The monomeric nature of
the complexes is further supported by microanalytical data
and mass spectral data. The electronic spectra of the com-
plexes [Co(L)(phen)2]Cl2 and [Co(L)(bpy)2]Cl2 show three
broad bands in the visible region (10,813, 10,989, 27,778 and
10,111, 11,248, 24937 cm-1, respectively), which were
assigned to 4T1g(F)?4T2g(F), 4T1g(F)?4A2g(F), and
4T1g(F)?4T2g(P). These data reveal that the complexes have
octahedral geometries. The observed magnetic moments of
the cobalt(II) complexes at room temperature indicate that
these complexes are monomeric. This is further supported by
microanalytical data. The electronic spectra of the complexes
[Ni(L)(phen)2]Cl2 and [Ni(L)(bpy)2]Cl2 show three low-
intensity bands in the visible region around 10,977, 10,040,
27,732 and 11,248, 10,060, 25,125 cm-1, which were
assigned to 3A2g(F)?3T2g(F), 3A2g(F)?3T1g(F), and
3A2g(F)?3T1g(P) transitions, respectively, suggesting an
octahedral geometry around the Ni(II) ion. The observed
magnetic moments of the Ni(II) complexes (3.09 and
3.14 BM, respectively) at room temperature indicate uncou-
pled mononuclear complexes of a diluted d8 system with an
s = 1 spin state of octahedral geometry [21, 22]. The
monomeric nature of the nickel complexes is further proven
by their microanalytical data. The electronic spectra of the
complexes [Mn(L)(phen)2]Cl2 and [Mn(L)(bpy)2]Cl2 show
four broad, low-intensity bands in the visible region at around
15,478, 24,331, 27,732, 29,700 and 14,705, 26,954, 27,397,
Molar conductivity
The high molar conductivities of the complexes in 10-3
M
29,325 cm-1
,
respectively, which were assigned to
DMF solution reveal that they are electrolytic in nature,
implying that the chloride anion is not coordinated to the
central metal ion. The presence of a counter chloride ion
was confirmed by Volhard’s test.
6A1g?4T1g, 6A1g?4T2g(G), 6A1g?4Eg, 6A1g?4T2g(D) transi-
tions. The electronic spectral data suggest an octahedral
geometry around the Mn(II) ion. The observed magnetic
moments of the Mn(II) complexes (5.1–5.4 BM) at room
temperature indicate that these are monomeric complexes
with octahedral geometries [23]. This is further supported by
microanalytical data. The electronic absorption spectra for
the diamagnetic Zn(II) complexes show bands in the region
34,602–29,498 cm-1 which were assigned to intraligand
charge-transfer transitions [24].
Magnetic moments and electronic spectra
The free Schiff base ligand L exhibits two intense bands
at 35,211 and 27,777 cm-1 which are due to p–p* and
n–p* transitions, respectively. In all metal complexes, the
absorption bands at 31,055–34,602 and 27,778–24,937 cm-1
,
123

608
A. Sakthivel et al.
Table 1 Electronic absorption
spectral data for the synthesized
compounds at 300 K
Compound
L
Solvent
DMF
Absorption/cm-1
Band assignment
Geometry
–
35,211
27,777
15,760
33,898
13,755
31,055
10,813
10,989
27,778
10,111
11,248
24,937
10,977
10,040
27,732
11,248
10,060
25,125
15,748
24,331
27,732
29,700
14,705
26,954
27,397
29,325
33,783
29,498
34,602
INCT
INCT
E2g?T2g
[CuL(phen)2]Cl2
[CuL(bipy)2]Cl2
[CoL(phen)2]Cl2
DMF
DMF
DMF
Distorted octahedral
Distorted octahedral
Octahedral
INCT
2E2g?T2g
INCT
4T1g(F)?4T2g(F)
4T1g(F)?4A2g(F)
4T1g(F)?4T2g(P)
4T1g(F)?4T2g(F)
4T1g(F)?4A2g(F)
4T1g(F)?4T2g(P)
3A2g(F)?3T2g(F)
3A2g(F)?3T1g(F)
3A2g(F)?3T1g(P)
3A2g(F)?3T2g(F)
3A2g(F)?3T1g(F)
3A2g(F)?3T1g(P)
6A1g?4T1g
6A1g?4T2g(G)
6A1g?4Eg
6A1g?4T2g(D)
6A1g?4T1g
6A1g?4T2g(G)
6A1g?4Eg
6A1g?4T2g(D)
[CoL(bpy)2]Cl2
[NiL(phen)2]Cl2
[NiL(bpy)2]Cl2
[MnL(phen)2]Cl2
DMF
DMF
DMF
DMF
Octahedral
Octahedral
Octahedral
Octahedral
[MnL(bpy)2]Cl2
DMF
Octahedral
[ZnL(phen)2]Cl2
[ZnL(bpy)2]Cl2
DMF
DMF
INCT
Octahedral
Octahedral
INCT
INCT
Nuclear magnetic resonance spectra
observed at 8.0, 8.25, 8.45, and 8.86 ppm in the spectrum
of the complex [Zn(L)(phen)2]Cl2 (see the ESM) were
assigned to phen protons. There is no appreciable change in
the other signals of the complex [18].
The 13C NMR spectrum of L shows aromatic carbons at
112.06–143.19 ppm. The signals from the C=O and C=N
carbons of ligand L observed at 159 and 163 ppm are
shifted upfield to 153 and 160 ppm for the bpy mixed-
ligand Zn(II) complex and to 152.9 and 159.5 ppm for the
phen mixed-ligand Zn(II) complex (see the ESM). There
are no appreciable changes in the other peaks.
1
The H NMR spectra of the ligand L and the zinc com-
plexes were recorded in DMSO-d6. The 1H NMR spectrum
of the Schiff base ligand shows signals at d = 2.4 and
3.1 ppm for C–CH3 and N–CH3 protons of the 4-amino-
antipyrine moiety. It also shows signals at 6.63, 6.95, and
7.84 ppm for the furfuryl moiety and at 6.96, 7.35, and
7.38 ppm for the phenyl group of the 4-aminoantipyrine
moiety. The azomethine proton (CH=N) is observed at
9.54 ppm. The 1H NMR spectrum of the Schiff base ligand
is given in the Electronic supplementary material (ESM).
The signals for the azomethine proton (–CH=N) of the bpy
and phen mixed-ligand zinc complexes are shifted upfield,
to 9.34 and 9.41 ppm, respectively, compared to the free
ligand (9.54 ppm), suggesting the coordination of the
azomethine group with the metal ion. The zinc complex
[Zn(L)(bpy)2]Cl2 (Fig. 1) shows additional resonance
peaks at 7.67, 8.20, and 8.62 ppm, which were assigned to
bpy protons. Similarly, the additional resonance peaks
Mass spectra
The FAB mass spectra of synthesized L and the
[Mn(L)(bpy)2]Cl2 complexes were recorded and the
molecular ion peaks obtained confirm the proposed for-
mulae. The mass spectrum of L shows an M ? 1 peak at
m/z = 282 (91.71 %) corresponding to the (C16H15N3O2)?
ion. Also, the spectrum exhibits fragments at m/z = 214,
123

DNA interaction studies of pyrazolone- and diimine-incorporated complexes
609
Electron paramagnetic resonance spectra
To obtain further information about the stereochemistry
and the site of the metal–ligand bonding, and to determine
the magnetic interactions in the metal complexes, EPR
studies of all the copper(II) complexes were carried out.
The spectra of the complexes were recorded in powder and
also in DMSO solution at 300 K (room temperature) and
77 K (liquid nitrogen temperature, LNT). However, the
[Cu(L)(bpy)2]Cl2 complex in the frozen state shows four
well-resolved peaks with low intensities in the low-field
region and one intense peak in the high-field region
(Fig. 3). The spin Hamiltonian parameters and bonding
parameters were calculated and are summarized in
Tables 2 and 3, respectively. The EPR spectrum of
[Cu(L)(phen)2]Cl2 at both 300 and 77 K shows one intense
absorption band in the high-field region, and is isotropic
due to dumpling motion of the molecules (see the ESM) as
well as dipolar exchange and unresolved hyperfine inter-
action in the solid state. The magnetic moment calculated
from the equation leff = g[s(s ? 1)]1/2 using the experi-
mental giso value (2.14) agrees very well with the measured
magnetic susceptibility range of 1.93 BM, indicating
that the solid structure is retained in DMSO solution. The
EPR spectrum of the copper complex at 77 K indicates
a poorly resolved nitrogen super hyperfine structure
(Fig. 3) in the perpendicular region due to the interac-
tion of the odd Cu(II) electron with nitrogen atoms. The
magnetic susceptibility value reveals that the copper com-
plex has a magnetic moment of 1.93 BM, corresponding
to one unpaired electron, indicating that the complex is
mononuclear.
Fig. 1 1H NMR spectrum of the [Zn(L)(bpy)2]Cl2 complex in
DMSO-d6 (hash and astereisk indicate peaks from the DMSO solvent
and trace water, respectively)
202, 188, 173, and 157 corresponding to [C12H12N3O]?,
[C11H11N3O]?, [C11H11N2O]?, [C10H8N2O]?, and [C10H8
N2]?, respectively (Fig. 2a). The mass spectrum of
[Mn(L)(bpy)2]2? shows peaks at m/z = 648 and 649 with
abundances of 20 and 15 %, respectively. The peak at
m/z = 648 may represent the molecular ion peak of the
complex, and the other peak is an isotopic species
(Fig. 2b). The strongest peaks (base peaks) at m/z = 281,
157, and 95 represent the stable species (C16H15N3O2)?,
(C10H8N2)?, and (C5H4NO)?, respectively. The m/z of the
molecular ion peak confirms that the stoichiometry of the
complexes is of the [ML(bpy)2]Cl2 type. This is further
supported by the mass spectra of all the complexes.
The observed peaks are in good agreement with their
empirical formulae as indicated by microanalytical data.
Similarly, the DART-MS spectrum was recorded for the
[Co(L)(phen)2]Cl2 complex (see the ESM). The strongest
peak (base peak) at m/z = 181 represents the stable
[C12H8N2]? moiety (i.e., the phen ligand). Moreover, the
spectrum exhibits fragments at m/z = 702, 422, and 282
corresponding to [CoC40H31O2N7]?, [Co(C12H8N2)2]?, and
[C16H15N3O2]?, respectively. These represent the [Co(L)
(phen)2]Cl2 complex after the removal of the two chloride
ions, the Co(phen)2 moiety, and L, respectively. Similarly,
the other fragments are also in good agreement with the
proposed structure of the complex. Thus, the mass spectral
data confirm the stoichiometry of the complex as
[CoL(X)2]Cl2 (where L = ligand, X = phen). The mass
spectral data of other complexes also support the above
stoichiometry. This stoichiometry is further supported by the
elemental data, which are in close agreement with the values
calculated from molecular formulae assigned to these
complexes. Thus, the mass spectral data reinforce the con-
clusion drawn from the analytical and conductance values.
In the present case, Cu(II) complexes measured in
DMSO at LNT gave the following values: g = 2.2728,
k
g\ = 2.0665 for [Cu(L)(phen)2]Cl2; g = 2.2721,
k
g\ = 2.0536 for [Cu(L)(bpy)2]Cl2. For both complexes,
the g and g\ values were greater than 2.04, consistent with
k
copper(II) in axial symmetry with all the principal axes
aligned parallel. These g values indicate an elongated
tetragonally distorted octahedral stereochemistry [25].
However, the observation g [ g\ [ ge shows that the
k
unpaired electron is in the dx2–y2 orbital of the Cu(II) ion
[26]. The values of the g factors are used to determine
the geometric parameter G, representing a measure of
the exchange interaction between Cu(II) centers in a
polycrystalline compound, using the formula G = (gII
- 2.0023)/(g\ - 2.0023) [27]. If G \ 4, there is an
exchange interaction between the Cu(II) centers, while if
G [ 4, the exchange interaction is negligible. The present
copper complexes have G values greater than 4 indicating
exchange interaction is either absent or very little in the
solid complexes.
123

610
A. Sakthivel et al.
Fig. 2 FAB mass spectra of ligand L (a) and the complex [Mn(L)(bpy)2]Cl2 (b)
The EPR parameters g , g\, AII and the d–d transition
for [Cu(L)(bpy)2]Cl2, which indicate that both of these
complexes have some covalent character. The out-
of-plane p-bonding (c2) and in plane p-bonding
(b2) parameters were calculated using the following
expressions:
k
energies are used to evaluate the bonding parameters a2,
b2, and c2, which may be regarded as measures of the
covalency of the in-plane r bonds, in-plane p bonds, and
out-of-plane p bonds. a2 can be calculated using the
equation:
À
Á
À
Á
À
Á
À
Á
b2 ¼ g ꢁ 2:0023 E= ꢁ8ka2
k
a2 ¼ A =P þ g ꢁ 2:0023 þ 3=7ðg ꢁ 2:0023Þ
?
k
k
À
Á
c2 ¼ ðg ꢁ 2:0023ÞE= ꢁ2ca2 :
þ 0:04:
?
In these equations, k = -828 cm-1 for the free metal
ion, and the E values for the complexes [Cu(L)(phen)2]Cl2
and (Cu(L)(bpy)2)Cl2 are 15,760 and 13,755 cm-1
If a2 is 0.5, it indicates pure covalent bonding, while
a2 = 1.0 suggests pure ionic bonding. In the present
case, a2 is 0.6430 for [Cu(L)(phen)2]Cl2 and 0.6506
,
123

DNA interaction studies of pyrazolone- and diimine-incorporated complexes
611
p-bonding, while K [ k for out-of-plane p-bonding. For
k
?
the present case, the observed order is K (0.6426) [ k
?
?
k
(0.1518) for [Cu(L)(phen)2]Cl2 and K (0.5594) [ k
k
(0.1057) for [Cu(L)(bpy)2]Cl2, implying a greater contri-
bution from out-of-plane p-bonding than from in-plane
p-bonding to metal–ligand p-bonding. Thus, the spectral
data discussed above confirm the proposed structures of the
ligand L and the complexes (Scheme 1).
DNA binding studies: electronic spectra
Adding increasing amounts of calf thymus (CT) DNA to all
complexes results in a decrease in the molar absorption
(hypochromism) of the p–p* absorption band as well as a
redshift of a few nm (1–2 nm), indicating the binding of
the complexes to DNA in different modes in different
extents. The binding of an intercalative complex molecule
to DNA has been well characterized to produce notable
hypochromism and a significant redshift due to a strong
stacking interaction between the aromatic chromophore of
the ligand and DNA base pairs, with the degree of hypo-
chromism and the size of the redshift linked to the strength
of the intercalative interaction [28–30]. The magnitudes of
the hypochromism and the redshift observed for all of the
phen mixed-ligand complexes are higher than those seen
for the bpy mixed-ligand complexes (shown in Table 4).
The observed binding is typical of classical intercalators
and partially intercalating metal complexes bound to CT
DNA. A quantitative comparison of the DNA-binding
affinities of the complexes was performed using the fol-
lowing equation:
Fig. 3 The X-band EPR spectrum of the [Cu(L)(bpy)2]Cl2 complex
in DMSO at 77 K
respectively. The observed values for [Cu(L)(phen)2]Cl2
(b2 = 0.999 and c2 = 0.944) and for [Cu(L)(bpy)2]Cl2
(b2 = 0.8629 and c2 = 0.6498) indicate that there is an
interaction in the out-of-plane p-bonding whereas the in-
plane p-bonding is completely ionic. This is further
confirmed by orbital reduction factors, which can be
estimated using the following relations:
ÂÀ
Á
Ã
2
K
K
¼
g ꢁ 2:0023 DE =8k0
k
k
2
¼ ½ðg ꢁ 2:0023ÞDEꢂ=8k0;
?
?
where ko is the spin orbit coupling constant for the cop-
per(II) ion (-828 cm-1). K and K\ are the parallel and
k
½DNAꢂ=ðea ꢁ efÞ ¼ ½DNAꢂ=ðeb ꢁ efÞ þ 1=Kbðeb ꢁ efÞ;
perpendicular components of the orbital reduction factor
(K). Significant information about the nature of bonding in
the copper(II) complex can be derived from the relative
where [DNA] is the concentration of DNA in base pairs,
ea is the apparent extinction coefficient obtained by cal-
culating Aobs/[complex], ef corresponds to the extinction
coefficient of the complex in its free form, and eb refers to
magnitudes of K and k . In the case of pure r-bonding,
?
k
K & k , whereas K \ k implies considerable in-plane
?
?
k
k
Table 2 The spin Hamiltonian parameters of copper complexes in DMSO solution at 300 and 77 K
Complex
Hyperfine constant/10-4 cm-1
g|
g\
giso
A\
A|
Aiso
[CuL(phen)2]Cl2
[CuL(bpy)2]Cl2
125
85
110
120
120
98
2.2728
2.2721
2.0665
2.0536
2.1353
2.1264
Table 3 The EPR bonding parameters of copper complexes in DMSO solution
Complex
a2
b2
c2
G
K||
K\
K
[CuL(phen)2]Cl2
[CuL(bpy)2]Cl2
0.6430
0.6506
0.9994
0.8629
0.9443
0.6498
4.1023
5.0765
0.6426
0.5594
0.1518
0.1057
0.3154
0.2569
123

612
A. Sakthivel et al.
Table 4 Absorption spectral properties of the synthesized com-
plexes with DNA
Complexes
kmax/nm
Dk/nm H/%
Kb/104 M-1 a
Free
Bound
[CuL(bpy)2]Cl2
311.5 311.4
378.0
341.0 338.7
[CoL(phen)2]Cl2 338.5 339.8
[NiL(bpy)2]Cl2 340.1 339.5
[NiL(phen)2]Cl2 339.0 339.7
[ZnL(bpy)2]Cl2 339.5 339.7
[ZnL(phen)2]Cl2 365.4 360.6
[MnL(bpy)2]Cl2 342.0 340.9
0.1
6.0
2.3
1.3
0.6
0.7
0.2
4.8
1.1
0.4
9.06 1.84
7.40 2.50
[CuL(phen)2]Cl2 384
[CoL(bpy)2]Cl2
20.40 1.85
24.80 3.78
8.30 4.79
29.14 9.52
12.70 2.38
26.0
22.0
2.93
0.94
[MnL(phen)2]Cl2 340.5 340.9
18.85 2.31
a
Error limit: 5 %
Fig. 4 Absorption spectra of [Mn(L)(bpy)2]Cl2 in the presence of
increasing amounts of DNA ([complex] = 50 lM, [DNA] = 25–
125 lM from top to bottom) in Tris–HCl buffer. The arrow indicates
the change in absorbance upon increasing the DNA concentration
the extinction coefficient of the complex in the bound form.
A straight line is obtained when [DNA]/(ea – ef) versus
[DNA] is plotted. The slope of the straight line is equiva-
lent to 1/(eb – ef) and the y intercept is equivalent to 1/Kb(eb
– ef). The value of Kb (the binding constant) was deter-
mined from the ratio of the slope to the intercept.
From Table 4, we can infer that the Ni(II) complexes
interact with DNA to the greatest degree, with binding
constants Kb of 4.79 9 104 M-1 and 9.52 9 104 M-1
,
The intrinsic binding constants Kb for [M(L)(phen)2]2?
are greater than those for [M(L)(bpy)2]2?, suggesting that
the diimine rather than the 4-(furfurylideneamino)antipy-
rine face of each complex is involved in DNA binding, and
that coordinated phen and bpy rings are engaged in partial
insertion in between the base pairs of DNA. The observed
Kb values are much lower than those observed for typical
classical intercalators (EthBr, Kb = 1.4 9 106 M-1 in
25 mM Tris–HCl/40 mM NaCl buffer, pH 7.2). Hence, it
is obvious that the present complexes are involved in
weaker intercalative interactions, in spite of the steric clash
between the 4-(furfurylideneamino)antipyrine ligand and
the DNA double helix. Generally, the mixed-ligand com-
plex offers the variation in geometry, size, hydrophobicity,
and hydrogen-bonding ability which are the major factors
to determine the DNA binding affinity [31]. Here, the
ligand is not expected to be flat with the phenyl group; it
may be twisted out of the plane of phenanthroline. Further,
its lack of planarity diminishes the favorability of
the ligand for intercalation, while the diimine part of the
complex enhances the stacking of the diimine with the
DNA base pairs. Further, the small binding constant values
may indicate surface aggregation and groove binding rather
than any interaction with the DNA base pairs. It is also
notable that compounds exhibiting low binding constants
have been identified in the literature as intercalating agents
[32]. The electronic absorption spectrum of [Mn(L)(bpy)2]Cl2
in the absence and presence of increasing amounts of DNA is
given in Fig. 4, and the phen complex is shown in the ESM.
respectively. These values are very distinctive of metal-
based compounds that bind to DNA via intercalation. This
is because the nickel complexes enhance the oxidation of
guanine residues in DNA [33–35]. This has been experi-
mentally proven through DNA cleavage studies, as
described later in this manuscript. Moreover, this distinct
mechanism causes the nickel complexes to tend to act as
effective intercalators and artificial nucleases when com-
pared to other members.
Viscosity measurements
Measuring the viscosity of DNA is regarded as the least
ambiguous and most critical test of a DNA binding model
in solution, and affords stronger arguments for an interca-
lative DNA binding mode [36, 37]. The viscosity of DNA
is significantly enhanced by complete or partial intercala-
tion of complexes into the DNA base-pair stack, but it is
slightly disturbed by the electrostatic or covalent binding of
molecules [38]. To investigate the nature of DNA binding
of the mixed-ligand complexes, viscosity measurements of
CT DNA were carried out by varying the concentrations of
the added complexes. The changes in the relative specific
viscosity of CT DNA in the presence and absence of the
complex were plotted against 1/R (=[complex]/[DNA]).
A classical intercalation mode causes a significant
increase in the viscosity of DNA due to an increase in base-
pair separation at intercalation sites and hence an increase
123

DNA interaction studies of pyrazolone- and diimine-incorporated complexes
613
in the overall DNA length. In our study, the ability of each
complex to increase the viscosity of DNA depended upon
the diimine ligand. Phen insertion produces greater vis-
cosity than insertion of the bpy ligand (as shown in the
ESM). The results of this viscosity study agree well with
decrease in current intensity with increasing DNA con-
centration is observed; see Fig. 6), the shift in potential is
related to the ratio of the binding constants by the fol-
lowing equation:
À
Á
Eb ꢁ Ef ¼ 0:0591 log K =K
;
½redꢂ
½oxꢂ
the hypochromism and Kb values observed for the above
complexes. Lengthening of the DNA duplex occurs upon
the partial insertion of phen and the deeper insertion of bpy
between the DNA base pairs.
where Eb and Ef are the peak potentials of the complex in
its bound and free forms, respectively. In the present study,
both the bpy and the phen mixed-ligand complexes
exhibited one-electron transfer during the redox process,
and the Ipc/Ipa value was less than unity, indicating that the
reaction of the complex on the glassy carbon electrode
surface was quasi-reversible. Other complexes—those
including Co(II), Ni(II), Mn(II), and Zn(II)—show con-
siderable shifts in both their cathodic and anodic peak
potentials when CT DNA is added incrementally. Most of
the complexes we synthesized exhibited both anodic and
cathodic peak potential shifts which were either positive or
negative, except for manganese complexes (Table 5). This
indicates the mode of intercalation that occurs when the
DNA binds with bpy/phen mixed-ligand Schiff base com-
plexes. It is interesting to note that phen mixed-ligand
complexes show a greater decrease in current intensity than
bpy mixed-ligand complexes (Fig. 5 and the ESM). This
observation suggests that the intercalative mode that occurs
when DNA binds to phen mixed-ligand complexes is
stronger than that involved when DNA binds to bpy mixed-
ligand complexes. Another interesting aspect of our study
is that manganese complexes exhibit positive and negative
shifts in peak potential. This indicates that there is an in-
tercalative and electrostatic binding mode between DNA
and these complexes, or that they break up the secondary
structure of DNA.
Electrochemical studies
Cyclic and differential pulse voltammetric techniques are
extremely useful for probing the nature and mode of the
binding of metal complexes to DNA. Typical cyclic vol-
tammograms obtained for the complex [Cu(L)(bpy)2]Cl2 in
the absence and presence of varying amounts of DNA are
shown in Fig. 5. The incremental addition of CT DNA to
the complex causes the anodic and cathodic peak currents
of the complex to decrease. This result shows that the
complex stabilizes the duplex (GC pairs) by intercalation.
The incremental addition of CT DNA to the complex
causes a shift in the potential of the peaks in the cyclic
voltammograms. The cathodic and anodic peaks both show
positive or negative shifts, which indicates the intercalation
of the complex into the base pairs of the DNA. If one of the
peak shifts was positive and the other was negative, it
would indicate that the complex intercalates into and
electrostatically binds to the CT DNA, or that it breaks the
secondary structure of the DNA.
In the differential pulse voltammogram of the complex
[Cu(L)(bpy)2]Cl2 obtained in the absence and the presence
of varying amounts of DNA (in which a significant
Fig. 6 Differential pulse voltammogram (scan rate of 10 mV/s) of
100 lM of the complex [Cu(L)(bpy)2]Cl2 in the absence and presence
of increasing amounts of DNA: 10, 20, 40, 100, 150, 200, 250 lM.
Supporting electrolyte: 5 mM Tris–HCl ? 50 mM NaCl in water
(pH 7.2)
Fig. 5 Cyclic voltammogram (scan rate of 10 mV/s) of [Cu(L)(b-
py)2]Cl2 in the absence and presence of increasing amounts of DNA:
10, 20, 40, 100, 150, 200, 250 lM. Supporting electrolyte: 5 mM
Tris–HCl ? 50 mM NaCl in water (pH 7.2)
123

614
A. Sakthivel et al.
Table 5 Electrochemical parameters for the interaction of DNA with metal(II) complexes
Complex
E
1/2/V
DEp/V
K[red]/K[ox]
Ipc/Ipaa
Free
Bound
Free
Bound
[CuL(phen)Cl2]
[CuL(bpy)Cl2]
[CoL(phen)2]Cl2
[CoL(bpy)2]Cl2
[NiL(phen)2]Cl2
[NiL(bpy)2]Cl2
[ZnL(phen)2]Cl2
[ZnL(bpy)2]Cl2
[MnL(phen)2]Cl2
[MnL(bpy)2]Cl2
-0.2303
-0.1725
-0.7663
0.1624
-0.1068
-0.1795
-0.7128
0.1887
-0.1205
-0.1110
-0.0435
0.0413
-0.0031
-0.1050
-0.1264
0.0706
3.2900
1.5959
1.2294
0.7321
1.3658
1.3658
2.1797
3.1930
4.0813
0.5362
0.6782
0.6900
4.0317
0.1660
0.3615
0.4660
0.4050
6.3402
0.8319
0.8895
-0.5878
0.35025
-0.0438
-0.8503
-0.6267
0.3150
-0.5625
0.3705
0.0613
0.0326
-0.0645
-0.1136
0.1177
-0.0910
0.2562
-0.1289
-0.9411
-0.4882
0.3320
0.1978
0.1098
-0.1985
-0.0780
–0.0300
a
Error limit: 5 %
Antimicrobial activity
ligands because the metal complexes can serve as a vehicle
for the activation of ligands—the principal cytotoxic spe-
cies [41]. The 4-(furfurylideneamino)antipyrine Schiff base
ligand may play an important role in this antimicrobial
activity, as well as the presence of four imine groups brings
the transformation reaction in biological systems. The
activities of ligands and their complexes increase as the
concentration increases, because it is a well-known fact
that concentration plays a vital role in increasing the degree
of inhibition. The results indicate that the complexes show
more activity than the ligands against the same microor-
ganisms under identical conditions (Tables 6, 7). These
data are in good agreement with literature values [42].
It is well known that chelation is not the only criterion for
antibacterial activity. Some important factors such as the
nature of the metal ion, the nature of the ligand, the coordi-
nating sites, the geometry of the complex, the concentration,
hydrophilicity, lipophilicity, and the presence of co-ligands
have a considerable influence on the antibacterial activity.
Steric and pharmacokinetic factors undoubtedly also play a
The investigated compounds were tested for antimicrobial
activity against bacteria and fungi. The minimum inhibi-
tory concentration (MIC) values of the investigated
compounds are summarized in Table 6 and 7. A compar-
ative study of L and the complexes (in terms of MIC
values) indicates that the complexes exhibit higher anti-
microbial activities than the free ligand. From the MIC
values, it is apparent that the phen mixed-ligand complexes
are more potent than the other investigated complexes,
which can be explained via Overtone’s concept and
Tweedy’s chelation theory [39, 40]. These complexes also
disturb the respiration process of the cell and thus block the
synthesis of proteins that restrict further growth of the
organism. Furthermore, the mode of action of the com-
pound may involve the formation of a hydrogen bond
through the azomethine group with the active center of the
cell, resulting in interference with the normal cell process,
and the metal complexes are generally more active than the
Table 6 Minimum inhibitory
Compound
Aspergillus niger
Fusarium solani
Curvularia lunata
Rhizoctonla bataicola
concentrations (mg cm-3) of
the synthesized compounds
against the growth of four fungi
L
26.2
13.7
19.7
16.0
21.0
17.1
20.3
16.5
20.5
16.9
19.8
1.0
27.4
15.9
20.1
15.3
21.2
18.7
20.9
16.8
19.6
16.0
19.7
1.7
25.5
13.5
20.3
16.5
21.3
16.2
20.7
16.9
19.0
17.0
19.5
0.9
24.9
16.5
19.7
15.9
20.4
17.5
20.3
17.7
19.6
15.9
19.3
1.5
[CuL(phen)2]Cl2
[CuL(bpy)2]Cl2
[CoL(phen)2]Cl2
[CoL(bpy)2]Cl2
[NiL(phen)2]Cl2
[NiL(bpy)2]Cl2
[ZnL(phen)2]Cl2
[ZnL(bpy)2]Cl2
[MnL(phen)2]Cl2
[MnL(bpy)2]Cl2
Nystatin
123

DNA interaction studies of pyrazolone- and diimine-incorporated complexes
615
Table 7 Minimum inhibitory concentrations (mg cm-3) of the
synthesized compounds against the growth of four bacteria
not show any DNA-cleavage activity (lane 4 in Fig. 7b). In
this case, the cleavage is inhibited by free-radical scav-
engers (DMSO), implying that hydroxyl radicals mediate
the cleavage. This provides clear evidence for the
involvement of hydroxyl radicals in the DNA strand scis-
sion performed by M(II) complexes. Here, the toxicity of
metal is expressed through the formation of hydroxyl
radical, which involves redox process with metal center
and H2O2. Moreover, the mechanism commences with the
oxidation of deoxyribose units by the free radicals, leading
to the fragmentation of the sugar-phosphate backbone. The
loaded (L(phen/bpy)2)M2? complexes damage DNA more
efficiently in the presence of an oxidant and form DNA
adducts that migrate at different rates. This can be attrib-
uted to the formation of hydroxyl free radicals. The
production of a hydroxyl free radical in the reaction
between the complex and the oxidant may occur as shown
below:
Compound
S. aureus B. subtilis E. coli K. pneumoniae
L
21.7
20.9
20.9
16.1
13.3
18.2
18.0
18.9
14.8
19.6
16.7
18.7
2.3
28.0
10.5
15.3
11.5
16.3
13.2
18.7
15.9
17.0
17.1
16.5
1.3
29.6
12.5
14.7
13.9
16.4
17.7
18.3
17.1
17.6
14.9
17.3
2.0
[CuL(phen)2]Cl2 18.7
[CuL(bpy)2]Cl2 16.7
[CoL(phen)2]Cl2 12.0
[CoL(bpy)2]Cl2 17.0
[NiL(phen)2]Cl2 17.5
[NiL(bpy)2]Cl2 19.3
[ZnL(phen)2]Cl2 16.5
[ZnL(bpy)2]Cl2 18.5
[MnL(phen)2]Cl2 17.9
[MnL(bpy)2]Cl2
Streptomycin
19.8
1.7
À
Á
À
Á
decisive role in deciding the potency of an antimicrobial
agent. The presence of lipophilic and polar substituents is
expected to enhance antibacterial activity. Heterocyclic
ligands with multifunctionality which have a greater chance
of interacting with either nucleoside bases (even after com-
plexation with a metal ion) or with biologically essential
metal ions present in the biosystem may be promising can-
didates for bactericides since they always tend to interact
(especially with a few enzymatic functional groups) in order
to achieve higher coordination numbers [43]. Thus, the anti-
bacterial property of metal complexes cannot be ascribed
to chelation alone; it is a complicated blend of several
contributions.
Lðphen=bpyÞ2 M2þ þ H2O2 ! Lðphen=bpyÞ2 M3þ
þ OH ꢀ þOHꢁ
These OH free radicals participate in the oxidation of the
deoxyribose moiety, followed by the hydrolytic cleavage of
the sugar-phosphate backbone. Here, the most significant
shifts are observed for Co(II) and Ni(II) (Fig. 7b, lanes 7
and 5), whereas the DNA adducts with Cu(II) and Zn(II)
complexes (lanes 3 and 8) migrate to a lesser extent. This
difference in the migration patterns of the adducts points to
a difference in the binding modes of these compounds
compared to the other members of the series. Control
experiments using DNA alone did not show any significant
cleavage of pUC19 DNA, even after longer exposure times.
Based on these results, we can conclude that the Ni(II)
complexes (Fig. 7b, lane 5) loaded with H2O2 are most
effective at cleaving DNA, and can thus be considered
better artificial nucleases than the other members and the
control. Hence, it is evident from the interactions of the
complexes with CT DNA and pUC19 DNA that the cobalt
and nickel complexes are more effective DNA-damaging
agents than the other members. This is because the cobalt
and nickel complexes enhance the oxidation of guanine
residues in DNA [33–35]. This mechanism means that the
cobalt and nickel complexes are more effective intercala-
tors and artificial nucleases than the copper and zinc
complexes.
Cleavage of pUC19 DNA
The nuclease activity exhibited by certain transition metal
complexes in the presence of hydrogen peroxide can be
attributed to the participation of the hydroxyl radical in
DNA cleavage. When nicked, the naturally occurring
supercoiled circular form of pUC19 DNA (form I) gives
rise to an open circular form (form II). Relatively fast
migration is observed for form I, but rather slow migration
for form II. In the present study, a DNA cleavage experi-
ment was conducted at 35 °C in the presence and absence
of H2O2. As can be seen from the results shown in Fig. 7a,
the gel electrophoretic separation of plasmid pUC19 DNA
occurs when treated with [ML(bpy)2]Cl2 complexes.
Control experiments suggest that untreated DNA does not
show any significant DNA cleavage (lane 1). However, in
the presence of peroxide, most of the [ML(bpy)2]Cl2 and
[ML(phen)2]Cl2 complexes convert the supercoiled plas-
mid DNA into a mixture of nicked (form II) and linear
(form III) DNA (Fig. 7b and Fig. 7c). Further, in the
presence of DMSO (4 mm3), [CuL(bpy)2]Cl2 ? H2O2 does
Molecular docking analysis
The results of spectroscopic titration, viscosity, and cyclic
voltammetry experiments suggest that Cu(II), Ni(II),
Co(II), and Zn(II) complexes interact with DNA via
intercalation. The binding constant values are in the range
of 104 per mole for all of the complexes, which indicates
123

616
A. Sakthivel et al.
Fig. 8 Interaction of the complexes a [Cu(L)(bpy)2]Cl2 and b
[Cu(L)(phen)2]Cl2 with d(CGCGAATTCGCG) strands of DNA via
intercalation binding
docked metal complexes [CuL(phen)2]Cl2, [CuL(bpy)2]
Cl2, [CoL(phen)2]Cl2, [CoL(bpy)2]Cl2, [NiL(phen)2]Cl2,
[NiL(bpy)2]Cl2, [ZnL(phen)2]Cl2, [Zn(bpy)2]Cl2, [MnL
(phen)2]Cl2, and [MnL(bpy)2]Cl2 are -557.32, -555.19,
-557.33, -527.94, -547.01, -534.19, -528.81, -527.08,
-530.04, and -529.36 kJ mol-1, respectively, which
correlate well with the corresponding experimental DNA
binding values. It was found that the binding between the
DNA and target molecules was stronger when the relative
binding energy was more negative.
Fig. 7 a The gel electrophoretic separation of plasmid pUC19 DNA
treated with [M(L)(bpy)2]Cl2 complexes. Lane 1 DNA marker, lane 2
DNA ? ligand, lane 3 DNA ? [Ni(L)(bpy)2]Cl2, lane 4 DNA ?
[Co(L)(bpy)2]Cl2, lane 5 DNA ? [Cu(L)(bpy)2]Cl2, lane 6 DNA ?
[Mn(L)(bpy)2]Cl2, lane 7 DNA ? [Zn(L)(bpy)2]Cl2. b The gel
electrophoretic separation of plasmid pUC19 DNA treated with
[M(L)(bpy)2]Cl2 complexes. Lane 1 DNA marker, lane 2 DNA ? ligand,
lane
(bpy)2]Cl2 ? H2O2 ? DMSO, lane 5 DNA ? [Ni(L)(bpy)2]Cl2 ? H2O2,
lane DNA ? [Mn(L)(bpy)2]Cl2 ? H2O2, lane DNA ? [Co(L)
3 DNA ? [Cu(L)(bpy)2]Cl2 ? H2O2, lane 4 DNA ? [Cu(L)
Conclusion
6
7
(bpy)2]Cl2 ? H2O2, lane 8 DNA ? [Zn(L)(bpy)2]Cl2 ? H2O2. c The
gel electrophoretic separation of plasmid pUC19 DNA treated with
[M(L)(phen)2]Cl2 complexes. Lane 1 DNA marker, lane 2 DNA ?
The synthesized L and the complexes were characterized
1
by microanalytical data, IR, UV–Vis, H NMR, EPR, and
[Cu(L)(phen)2]Cl2 ? H2O2, lane
4
3
DNA ? [Co(L)(phen)2]Cl2 ? H2O2, lane
DNA ? [Cu(L)(phen)2]Cl2, lane
DNA ? [Ni(L)
mass spectra. The data show that the complexes are of type
[M(L)(phen/bpy)2]Cl2. The UV–Vis, magnetic suscepti-
bility, and EPR spectral data for the complexes suggest an
octahedral geometry around the central metal ion. The
complexes show high electrical conductance, indicating
that these chelates are electrolytes. Their magnetic sus-
ceptibility values provide evidence for their monomeric
nature. The planarity and flexible extended conjugation of
the ligands have a strong effect on the DNA-binding and
DNA-cleavage activities of these complexes. The interca-
lative binding of these complexes with DNA is supported
by their electronic absorption spectra as well as cyclic
voltammetry, difference pulse voltammetry, and visco-
metric studies. The planarity of the complexes increases
their DNA-binding and DNA-cleavage activities. The
5
(phen)2]Cl2 ? H2O2, lane 6 DNA ? [Zn(L)(phen)2]Cl2 ? H2O2, lane 7
DNA ? [Mn(L)(phen)2]Cl2 ? H2O2, lane 8 DNA ? ligand ? H2O2
that these complexes may intercalate between the DNA
base pairs. This is mainly due to effective stacking forces
between the aromatic ligands and DNA bases. Docking
experiments were performed in order to find the perferred
binding site and orientation of the ligand inside the DNA.
Docking experiments using the energy-minimized docked
structures suggest that the best possible geometries of the
metal complexes occur when they act as partial intercala-
tors (as shown in Fig. 8). Structural analysis of the docked
structures provides significant information on the binding
patterns of these complexes. The binding energies of the
123

DNA interaction studies of pyrazolone- and diimine-incorporated complexes
617
results obtained from in vitro antifungal and antibacterial
tests show that all of these complexes possess potent
antimicrobial activities toward bacteria and fungi. It was
found that the activities of the complexes are higher than
that of the free ligand.
according to a method described previously [45]. Chloride
ion was determined gravimetrically as silver chloride
[46]. The ligand L was synthesized by following the
previously reported procedure [47]. The ligand was
purified by column chromatography eluted with alco-
hol:DMF mixture and the purity was checked by TLC.
The complexes were purified by repeated washing with
ethanol.
Experimental
All reagents and chemicals were procured from Merck
(Darmstadt, Germany). Solvents used for electrochemical
and spectroscopic studies were purified by standard pro-
cedures [44]. DNA was purchased from Bangalore Genei
(Bangalore, India). Agarose (molecular biology grade) and
ethidium bromide (EB) were obtained from Sigma (St.
Louis, MO, USA). Tris(hydroxymethyl)aminomethane-
HCl (Tris–HCl) buffer solution was prepared using
deionized, doubly distilled water. Carbon, hydrogen, and
nitrogen analyses of the complexes were carried out on a
CHN analyzer (1108, Heraeus Carlo Erba, Hanau, Ger-
many). The IR spectra (KBr discs) of the samples were
recorded in the range of 400–4,000 cm-1 on a PerkinElmer
(Waltham, MA, USA) 783 series FTIR spectrophotometer.
Electronic absorption spectra in the 200–1,100 nm range
were obtained on a Shimadzu (Kyoto, Japan) UV-1601
spectrophotometer. N,N-dimethylformamide (DMF) was
used as solvent when obtaining electronic spectra for both
the ligand L and its complexes. The NMR spectra of L and
the zinc complexes were recorded on a Bruker (Billerica,
Synthesis of [M(L)(phen)2]Cl2 and [M(L)(bpy)2]Cl2
The metal(II) complexes were prepared by mixing the
appropriate molar quantities of the ligands and metal salts
using the following procedure. An ethanolic solution of L
(10 mmol) was heated under reflux with an ethanolic
solution of the metal(II) chloride (10 mmol) for ca. 3 h. To
the above mixture, an ethanolic solution of 1,10-phenan-
throline/2,20-bipyridine (20 mmol) was added, and the
reflux was continued for ca. 1 h. The resulting solid
product was filtered, washed with ethanol, and dried in
vacuo.
[4-[(Furan-2-ylmethylene)amino]-1,2-dihydro-1,5-
dimethyl-2-phenyl-3H-pyrazol-3-one]bis(1,10-
phenanthroline)copper(II) chloride (C40H31Cl2CuN7O2)
ꢀ
Yield: 62 %; IR (KBr): m = 1,647 (C=O), 1,601 (–CH=N),
1,545 (C=N) cm-1; Km = 157.50 S cm2 mol-1; leff
1.93 BM; UV–Vis (DMF): kmax = 15,760, 33,898 cm-1
MS: m/z = 741 (M?).
=
;
MA, USA) Avance DRX 300 FT-NMR spectrometer, using
DMSO-d6 as solvent, at the University Science Instru-
mentation Centre (USIC), Madurai Kamaraj University,
Madurai, India. Tetramethylsilane was used as the internal
standard. Fast atom bombardment mass spectra of the
complexes were recorded on a JEOL (Tokyo, Japan) SX
102/DA-6000 mass spectrometer/data system using argon/
xenon (6 kV, 10 mA) as the FAB gas. X-band EPR spectra
of the copper complexes were recorded at RT (300 K) and
LNT (77 K) using DPPH as the g-marker.
The molar conductances of the complexes (10-3 M) in
DMF were measured at room temperature with a Deep
Vision (Tamilnadu, India) model 601 digital direct reading
deluxe conductivity meter. Magnetic susceptibility mea-
surements were carried out using the Gouy method at room
temperature on powdered samples of the complexes.
CuSO4ꢀ5H2O was used as calibrant. Electrochemical
measurements were performed on a CHI (Austin, TX,
USA) 620C electrochemical analyzer with a three-elec-
trode system comprising a glassy carbon electrode as the
working electrode, a platinum wire as the auxiliary elec-
trode, and Ag/AgCl as the reference electrode. Solutions
were deoxygenated by purging with N2 prior to measure-
ments. The metal content of each complex was determined
[4-[(Furan-2-ylmethylene)amino]-1,2-dihydro-1,5-
dimethyl-2-phenyl-3H-pyrazol-3-one]bis(1,10-
phenanthroline)cobalt(II) chloride (C40H31Cl2CoN7O2)
ꢀ
Yield: 54 %; IR (KBr): m = 1,645 (C=O), 1,597 (–CH=N),
1,547 (C=N) cm-1; Km = 138.96 S cm2 mol-1; leff
=
4.81 BM; UV–Vis (DMF): kmax = 27,778, 10,989, 10,813
cm-1; MS: m/z = 736 (M?).
[4-[(Furan-2-ylmethylene)amino]-1,2-dihydro-1,5-dime-
thyl-2-phenyl-3H-pyrazol-3-one]bis(1,10-phenanthro-
line)nickel(II) chloride (C40H31Cl2N7NiO2)
ꢀ
Yield: 59 %; IR (KBr): m = 1,647 (C=O), 1,599 (–CH=N),
1,545 (C=N) cm-1; Km = 131.47 S cm2 mol-1; leff = 3.09
BM; UV–Vis (DMF): kmax = 27,732, 10,040, 10,977 cm-1
MS: m/z = 736 (M?).
;
[4-[(Furan-2-ylmethylene)amino]-1,2-dihydro-1,5-
dimethyl-2-phenyl-3H-pyrazol-3-one]bis(1,10-
phenanthroline)manganese(II) chloride
(C40H31Cl2MnN7O2)
ꢀ
Yield: 60 %; IR (KBr): m = 1,648 (C=O), 1,591 (–CH=N),
1,547 (C=N) cm-1; Km = 144.46 S cm2 mol-1; leff
=
5.10 BM; UV–Vis (DMF): kmax = 29,700, 27,732,
24,331, 15,748 cm-1; MS: m/z = 732 (M?).
123

618
A. Sakthivel et al.
[4-[(Furan-2-ylmethylene)amino]-1,2-dihydro-1,5-
dimethyl-2-phenyl-3H-pyrazol-3-one]bis(1,10-
112.1, 113.3, 116.1, 124.6, 126.8, 129.0, 134.4, 139.9,
143.2, 144.9, 149.1, 151.6, 153.0 (C=O), 160.0 (HC=N)
ppm; Km = 123.18 S cm2 mol-1; leff = 0 BM; MS: m/z
= 659 (M?).
phenanthroline)zinc(II) chloride (C40H31Cl2N7O2Zn)
Yield: 57 %; IR (KBr): ꢀm = 1,646 (C=O), 1,597 (–CH=N),
1,548 (C=N) cm-1; 1H NMR (DMSO-d6): d = 2.40 (s, 3H,
C–CH3), 3.10 (s, 3H, N–CH3), 6.63, 6.95 (m, 2H, furfuryl),
6.96–7.38 (m, 5H, phenyl), 7.84 (m, 1H, furfuryl), 8.00,
8.25, 8.45, 8.86 (m, 8H, phen), 9.41 (s, CH=N) ppm; 13C
NMR (DMSO-d6): d = 9.7 (C–CH3), 35.3 (N–CH3),
112.2, 113.6, 116.2, 124.7, 125.5, 126.9, 128.8, 129.1,
134.4, 139.5, 139.8, 143.2, 145.1, 148.8, 151.7, 152.9
DNA-binding and DNA-cleavage experiments
All experiments involving the interaction of the complexes
with calf thymus (CT) DNA were carried out in Tris–HCl
buffer (50 mM Tris–HCl, pH 7.2) containing 5 % DMF at
room temperature. A solution of CT DNA in the buffer
gave a ratio of UV absorbances at 260 and 280 nm of about
1.89:1, indicating that the CT DNA was sufficiently free
from protein [48]. The CT DNA concentration per nucle-
otide was determined by absorption spectroscopy using a
molar absorption coefficient of 6,600 M-1 cm-1 at 260 nm
[49].
(C=O), 159.5 (HC=N) ppm; Km = 136.86 S cm2 mol-1
;
leff = 0 BM; MS: m/z = 743 (M?).
[4-[(Furan-2-ylmethylene)amino]-1,2-dihydro-1,5-
dimethyl-2-phenyl-3H-pyrazol-3-one]bis(2,20-
bipyridine)copper(II) chloride (C36H31Cl2CuN7O2)
ꢀ
Yield: 64 %; IR (KBr): m = 1,647 (C=O), 1,601 (–CH=N),
1,545 (C=N) cm-1; Km = 112.69 S cm2 mol-1; leff
=
An absorption titration experiment was performed by
maintaining the concentration of the metal complex at
50 lM while varying the concentration of CT DNA within
40–400 lM. While measuring the absorption spectrum,
equal quantities of CT DNA were added to both the
complex solution and the reference solution to eliminate
the absorbance of CT DNA itself. Using the absorption
data, the intrinsic binding constant Kb was determined from
a plot of [DNA]/(ea – ef) versus [DNA] and the equation
1.82 BM; UV–Vis (DMF): kmax = 31,055, 13,755 cm-1
;
MS: m/z = 657 (M?).
[4-[(Furan-2-ylmethylene)amino]-1,2-dihydro-1,5-
dimethyl-2-phenyl-3H-pyrazol-3-one]bis(2,20-
bipyridine)cobalt(II) chloride (C36H31Cl2CoN7O2)
ꢀ
Yield: 61 %; IR (KBr): m = 1,648 (C=O), 1,599 (–CH=N),
1,545 (C=N) cm-1; Km = 132.45 S cm2 mol-1; leff
=
4.93 BM; UV–Vis (DMF): kmax = 24,937, 11,248, 10,111
cm-1; MS: m/z = 653 (M?).
À
Á
À
Á
Â
À
ÁÃꢁ1
½DNAꢂ = ea ꢁ ef ¼ ½DNAꢂ= eb ꢁ ef þ Kb eb ꢁ ef
;
ð1Þ
[4-[(Furan-2-ylmethylene)amino]-1,2-dihydro-1,5-
dimethyl-2-phenyl-3H-pyrazol-3-one]bis(2,20-
bipyridine)nickel(II) chloride (C36H31Cl2N7NiO2)
where [DNA] is the concentration of CT DNA in base
pairs. The apparent absorption coefficients ea, ef, and eb
correspond to Aobs/[M], the extinction coefficient for the
free metal(II) complex, and the extinction coefficient for
the metal(II) complex in the fully bound form, respectively
[50]. Kb is given by the ratio of the slope to the intercept.
Cyclic voltammetry and differential pulse voltammogram
studies were performed on a CHI 620C electrochemical
analyzer with a three-electrode system comprising glassy
carbon as the working electrode, a platinum wire as the
auxiliary electrode, and Ag/AgCl as the reference electrode.
Solutions were deoxygenated by purging with N2 prior to
measurements.
ꢀ
Yield: 60 %; IR (KBr): m = 1,649 (C=O), 1,592 (–CH=N),
1,546 (C=N) cm-1; Km = 130.09 S cm2 mol-1; leff
=
3.14 BM; UV–Vis (DMF): kmax = 25,125, 11,248, 10,060
cm-1; MS: m/z = 652 (M?).
[4-[(Furan-2-ylmethylene)amino]-1,2-dihydro-1,5-
dimethyl-2-phenyl-3H-pyrazol-3-one]bis(2,20-
bipyridine)manganese(II) chloride (C36H31Cl2MnN7O2)
ꢀ
Yield: 58 %; IR (KBr): m = 1,647 (C=O), 1,602 (–CH=N),
1,543 (C=N) cm-1; Km = 152.70 S cm2 mol-1; leff
=
5.40 BM; UV–Vis (DMF): kmax = 29,325, 27,397, 26,954,
14,705 cm-1; MS: m/z = 649 (M?).
Viscosity experiments were carried out on an Ostwald
viscometer immersed in a thermostated water bath main-
tained at a constant temperature of 30.0 0.1 °C. CT
DNA samples of approximately 0.5 mM were prepared by
sonicating in order to minimize complexities arising from
CT DNA flexibility [51]. The flow time was measured with
a digital stopwatch three times for each sample, and an
average flow time was then calculated. Data are presented
as (g/g0)1/3 versus the concentration of the metal(II) com-
plex, where g is the viscosity of the CT DNA solution in
[4-[(Furan-2-ylmethylene)amino]-1,2-dihydro-1,5-
dimethyl-2-phenyl-3H-pyrazol-3-one]bis(2,20-
bipyridine)zinc(II) chloride (C36H31Cl2N7O2Zn)
ꢀ
Yield: 56 %; IR (KBr): m = 1,647 (C=O), 1,601 (–CH=N),
1,545 (C=N) cm-1; 1H NMR (DMSO-d6): d = 2.40 (s, 3H,
C–CH3), 3.10 (s, 3H, N–CH3), 6.63, 6.95 (m, 2H, furfuryl),
6.96–7.38 (m, 5H, phenyl), 7.84 (m, 1H, furfuryl), 7.67,
8.20, 8.62 (m, 8H, bpy), 9.34 (s, 1H, CH=N) ppm; 13C
NMR (DMSO-d6): d = 9.6 (C–CH3), 35.1 (N–CH3),
123

DNA interaction studies of pyrazolone- and diimine-incorporated complexes
619
the presence of the complex, and g0 is the viscosity of the
CT DNA solution in the absence of the complex. Viscosity
values were calculated after correcting the flow time of the
buffer alone (t0), g = (t – t0)/t0 [52].
structure of the complex of netropsin with B-DNA dode-
camer d(CGCGAATTCGCG)2 (NDB code GDLB05) was
downloaded from the Protein Data Bank. During the
docking analysis, the binding site of netropsin was used as
the binding site for our metal complexes across the entire
DNA molecule. The docked poses were generated by the
exhaustive search and optimization step. FRED selects the
single best pose from among a set of candidates. This pose
is then scored and the score is used to rank ligands in the
output hit list. The consensus structure step allows multiple
scoring functions to vote for the best docked structure in a
rank-by-vote approach.
The degree of cleavage of the supercoiled (SC) pUC19
DNA (33.3 lM, 0.2 lg) to its nicked circular (NC) form
was determined by agarose gel electrophoresis in 5 mM
Tris–HCl buffer (pH 7.2) containing 50 mM NaCl. After
they had been incubated for 2 h at 37 °C in a dark cham-
ber, the samples were added to a loading buffer containing
25 % bromophenol blue, 0.25 % xylene cyanol, and 30 %
glycerol (3 mm3), and the solution was finally loaded onto
0.8 % agarose gel containing 1 lg/cm3 ethidium bromide.
Electrophoresis was carried out in a dark chamber for 3 h
at 50 V in Tris–HCl–EDTA buffer. Bands were visualized
by UV light and photographed.
Statistical analysis
All of the experiments conducted in this work were done in
triplicate, and all of the data are reported here as the
mean standard deviation.
Antimicrobial activity
Acknowledgments The authors express their sincere thanks to the
Managing Board, the Principal, and the Head of the Department of
Chemistry for providing research facilities and moral support. AS
thanks the University Grants Commission (UGC) in Hyderabad for
financial support.
The in vitro biological screening effects of the investigated
compounds were tested against the Gram-positive bacteria
Staphylococcus aureus and Bacillus subtilis and the Gram-
negative bacteria Escherichia coli and Klebsiella pneu-
moniae by the well diffusion method [53], using agar
nutrient as the medium and streptomycin as the standard.
The antifungal activities of the compounds against the
fungi Aspergillus niger, Fusarium solani, Curvularia lu-
nata, and Rhizoctonia bataicola (cultured on potato
dextrose agar as medium, and with nystatin used as the
standard) were evaluated by the well diffusion method.
The stock solution (10-2 M) was prepared by dissolving
the compounds in DMSO, and the solutions were serially
diluted in order to determine the minimum inhibitory
concentration (MIC) values. In a typical procedure [54], a
well was made in the agar medium inoculated with
microorganisms. The well was filled with the test solution
using a micropipette, and the plate was incubated for 24 h
for bacteria and for 72 h for fungi at 35 °C. During this
period, the test solution diffused and the growth of the
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