Biological screening and DNA nuclease activity of transition metal complexes of N2O2 type of Knoevenagel condensate Schiff base

Article abstract of DOI:10.1002/aoc.3466

A novel tetradentate N₂O₂ type of Knoevenagel condensate Schiff base, synthesized from 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one (4-aminoantipyrine) and 3-(cinnamyl)-pentane-2,4-dione, forms stable complexes with transition met

Full text of DOI:10.1002/aoc.3466

Full paper
Received: 3 December 2015
Revised: 5 January 2016
Accepted: 30 January 2016
Published online in Wiley Online Library: 17 March 2016
(wileyonlinelibrary.com) DOI 10.1002/aoc.3466
Biological screening and DNA nuclease activity
of transition metal complexes of N2O2 type of
Knoevenagel condensate Schiff base
Rajakkani Paul Pandiyan and Natarajan Raman*
A novel tetradentate N2O2 type of Knoevenagel condensate Schiff base, synthesized from 4-amino-2,3-dimethyl-1-phenyl-
3-pyrazolin-5-one (4-aminoantipyrine) and 3-(cinnamyl)-pentane-2,4-dione, forms stable complexes with transition metal ions
such as Cu(II), Co(II), Ni(II) and Zn(II). The structural features were derived from elemental analysis, molar conductance measure-
ments, infrared, UV–visible, 1H NMR, 13C NMR, mass and electron paramagnetic resonance spectroscopies. These complexes show
high conductance values, supporting their electrolytic nature. Spectroscopic and other analytical data of the complexes suggest
square planar geometry. In vitro calf thymus DNA binding studies were performed by employing UV–visible absorption spectros-
copy, viscometry and cyclic voltammetry. These techniques indicate that all the metal complexes bind to DNA via intercalation
mode. Antimicrobial screening of the synthesized ligand and complexes was conducted against Gram-positive bacteria, Gram-
negative bacteria and fungi. These complexes exhibit higher antimicrobial activities than the free Schiff base, as investigated
using the minimum inhibitory concentration method. Gel electrophoresis reveals that these complexes also promote the cleavage
of pUC18 plasmid DNA in the presence of activators. Copyright © 2016 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: Knoevenagel condensate Schiff base; cyclic voltammetry; intercalation mode; gel electrophoresis
Our previous reports exemplified a series of attempts to prepare
highly symmetric and conjugated assemblies generated by the
Introduction
DNA is the commonly known primary intracellular target of antitu-
mor drugs. The interaction between DNA and small molecules can
cause DNA damage in cancer cells, this damage inhibiting the
abundance of cancer cells and blocking their division.[1–3] Recently,
interactions of transition metal complexes with DNA have attracted
major attention due to the role of these complexes as the original
cancer therapeutic agents with photochemical properties, which
can function as potential probes of DNA structure and
conformation.[4,5] In particular, a variety of drugs and metal
complexes can interface with DNA via non-covalent approaches
such as electrostatic interaction, groove binding and intercalation.
Among these modes, binding to DNA through intercalation via
planar ligands can also intercalate with adjacent base pairs of
DNA with respect to planarity and donor sites of the ligand as well
as the coordination geometry of the metal centre.[6,7] These
features can assist in the determination of the potential of
chemotherapeutical agents. Several attempts are underway to
identify the most effective anticancer agent as a suggested
pertinent drug. Recent trends to control or inhibit nucleic acid
damage mainly involve the interaction of DNA with other
molecules. In recent years, various transition metal complexes have
been ventured as cleavage agents for DNA and also to function as
potential DNA-targeted antitumor drugs. Currently, transition metal
complexes derived from 4-aminoantipyrine have been used
extensively in various fields, including for biological, analytical and
therapeutic applications.[8,9]
condensation of β-diketones with primary amines but were not
able to afford favourable complexes. Because the above approach
is hampered with the starting material (enolic form of the
β-diketone) lacking conjugation in its structure, an alternative
precursor (3-substituted β-diketone) is chosen to investigate its
sequestration. A Knoevenagel-type β-diketone, which can never
undergo enolization due to the absence of a gamma proton, can
condense with the active methylene group of an aldehyde
substrate to afford a non-enolizable Knoevenagel condensate
ligand. This could further react efficiently with amines to form Schiff
base ligands, which could afford stable transition metal complexes
via coordination sites.[10] Moreover, the metal complexes of
nitrogen–oxygen chelating agents derived from 4-aminoantipyrine
Schiff base have been studied extensively due to their promising
applications in pharmacological areas[11] and being suitable
candidates to cleave DNA.[12]
In order to form a higher degree of conjugation of versatile
ligand systems, a Knoevenagel condensate Schiff base is designed
for the formation of stable complexes. Studies of the structure and
spectral and redox properties would be optimum models for
metalloproteins, which are essential to address the structure–redox
*
Correspondence to: Natarajan Raman, Research Department of Chemistry,
VHNSN College, Virudhunagar
ramchem1964@gmail.com
– 626 001, Tamil Nadu, India. E-mail:
It is our aim to design an appropriate ligand to obtain an
optimum metal complex to resolve the challenges arising earlier.
Research Department of Chemistry, VHNSN College, Virudhunagar – 626 001,
Tamil Nadu, India
Appl. Organometal. Chem. 2016, 30, 531–539
Copyright © 2016 John Wiley & Sons, Ltd.

R. P. Pandiyan and N. Raman
relationship. These aspects are intriguing for carrying out studies of
these model compounds, which can pave the way to identifying
the ligand environment as impacting on redox properties of the
central metal atom. Also, the spectral properties are essential for
exploring the DNA binding and DNA cleavage activity of the
synthesized metal complexes. The results from the complexes can
allow determination of the binding mode of the complexes to
DNA and inspire the development of new useful DNA probes and
effective inorganic complex nucleases.
Experimental
Materials and methods
The chemicals involved in this work were of AnalaR grade and were
used without further purification. The solvents were purified using
standard procedures. Acetylacetone and cinnmaldehyde were SD
fine products and used as supplied. 4-Aminoantipyrine and all
metal salts were obtained from E. Merck.
Scheme 1. Route for synthesis of Knoevenagel condensate Schiff base (L).
Yield 78%; yellow colour. Anal. Calcd for C36H36N6O2 (%): C, 73.9;
H, 6.2; N, 14.3. Found (%): C, 73.1; H, 5.9; N, 13.6. FT-IR (KBr, cmꢀ1):
1668 (C¼O), 1605 (C¼N), 1498 (C¼CH). 1H NMR (DMSO-d6, δ, ppm):
7.0–7.6 (m, Ar-H, 15H), 2.5 (s, ¼C–CH3, 6H), 3.1 (s, N¼C–CH3, 6H), 3.4
(s, N–CH3, 6H), 9.4 (s, HC¼CH, 2H). 13C NMR (DMSO-d6, δ, ppm):
125–132 (Ar–C, C1–C4, C16–C19), 158 (–C¼O, C13), 164 (–C¼N, C9),
9.2 (C–CH3, C10, C14), 40 (–N–CH3, C15), 122 (HC¼CH, C5, C6).
UV–visible (DMSO, cmꢀ1 (transition)): 34 482 (π–π*), 27 932 (n–π*).
Elemental analysis (C, H and N) data were obtained using a
Perkin-Elmer 240 elemental analyser. Vibrational spectra were
recorded with a Fourier transform infrared (FT-IR) Shimadzu model
1
IR-Affinity-1 spectrophotometer using KBr discs. H NMR and 13C
NMR spectra of the ligand and its Zn(II) complex were recorded
with a Bruker 400 MHz Avance III HD Nanobay NMR spectrometer
using deuterated dimethylsulfoxide (DMSO-d6) as internal
standard. Mass spectrometry experiments were performed using
a JEOL AccuTOF JMS-T100LC mass spectrometer equipped with a
custom-made electrospray interface (ESI). The room temperature
(RT) molar conductivity of the complexes in DMSO solution
(10ꢀ3 M) was measured using a deep vision 601 model digital
conductometer. X-band electron paramagnetic resonance (EPR)
spectra were obtained at liquid nitrogen temperature (LNT; 77 K)
and RT (300 K). Absorption spectra were recorded using a Shimadzu
model UV-1601 spectrophotometer at RT. Cyclic voltammetry
experiments were conducted using a CHI 620C electrochemical
analyser in freshly distilled DMSO solution.
Synthesis of metal complexes
A solution of metal(II) chloride in ethanol (1 mmol) was mixed with
an ethanolic solution of the Schiff base (1 mmol), and the resultant
mixture was refluxed for ca 3 h. The solid complex precipitated was
filtered off and washed thoroughly with ethanol and petroleum
ether and dried over anhydrous CaCl2 under vacuum. The metal
complexes were prepared according to the following equation:
Ethanol
Reflux 3 h
MCI2·xH2O þ L
½MLꢁCl2 þ xH2O
(1)
where M = Cu(II), x = 2; M = Co(II), x = 6; M = Ni(II), x = 6; and
M = Zn(II), x = 2.
[CuL]Cl2. Yield 65%; brown colour. Anal. Calcd for
C36H36N6O2Cl2Cu (%): C, 66.7; H, 5.6; N, 12.9; Cu, 9.8. Found (%): C,
66.1; H, 5.2; N, 12.0; Cu, 9.2. FT-IR (KBr, cmꢀ1): 1598 (–C¼N), 1654
(C¼O), 1494 (C¼CH), 501 (M–O), 457 (M–N). Λm (Ωꢀ1 molꢀ1 cm2):
142; μeff (BM): 1.84. UV–visible (DMSO, cmꢀ1 (transition)): 19 157 (d–d).
[CoL]Cl2. Yield 62%; green colour. Anal. Calcd for
C36H36N6O2Cl2Co (%): C, 61.1; H, 5.6; N, 13.0; Co., 9.1. Found (%): C,
66.5; H, 5.1; N, 12.5; Co., 8.7. FT-IR (KBr, cmꢀ1): 1581 (–C¼N), 1631 (–
C¼O), 1492 (C¼CH), 518 (M–O), 449 (M–N). Λm (Ωꢀ1 molꢀ1 cm2):
126; μeff (BM): 2.65. UV–visible (DMSO, cmꢀ1 (transition)): 18 756 (d–d).
[NiL]Cl2. Yield 68%; brownish yellow colour. Anal. Calcd for
C36H36N6O2Cl2Ni (%): C, 67.2; H, 5.6; N, 13.0; Ni, 9.1. Found (%): C,
66.5; H, 5.1; N, 12.6; Ni, 9.1. FT-IR (KBr, cmꢀ1): 1577 (–C¼N), 1645
(C¼O), 1496 (C¼CH), 513 (M–O), 451 (M–N). Λm (Ωꢀ1 molꢀ1 cm2):
130. UV–visible (DMSO, cmꢀ1 (transition)): 17 258 (d–d).
Synthesis of Knoevenagel condensate β-diketone ligand
The Knoevenagel condensate β-diketone ligand (3-(cinnamyl)pentane-
2,4-dione) was synthesized by modifying the procedure of Raman
and co-workers.[13] Acetylacetone (1.0 g, 10 mmol) was mixed
with cinnamaldehyde (1.3 g, 10 mmol) and piperidine (0.05 ml)
in ethanol (50 ml). The reaction mixture was stirred thoroughly
for a period of 4 h with occasional cooling. A yellow-coloured
crystalline solid was obtained after being kept in a refrigerator
for two days, which was filtered and washed with ethanol
followed by an excess of petroleum ether to remove any
unreacted reagents. Washing was repeated three times and the
compound was recrystallized from ethanol.
Synthesis of Knoevenagel condensate Schiff base (L)
[ZnL]Cl2. Yield 70%; greenish yellow colour. Anal. Calcd for
C36H36N6O2Cl2Zn (%): C, 66.5; H, 5.5; N, 12.9; Zn, 10.0. Found (%):
C, 66.5; H, 5.2; N, 12.2; Zn, 9.6. FT-IR (KBr, cmꢀ1): 1570 (–C¼N),
1640 (C¼O), 1494 (C¼CH), 509 (M–O), 468 (M–N). 1H NMR
(DMSO-d6, δ, ppm): 7.1–7.8 (m, Ar-H, 15H), 2.7 (s, ¼C–CH3, 6H), 3.2
(s, N¼C–CH3, 6H), 3.3 (s, N–CH3, 6H), 9.3 (s, HC¼CH, 2H). 13C NMR
(DMSO-d6, δ, ppm): 125–132 (Ar–C, C1–C4, C16–C19), 162 (–C¼O,
C13), 172 (–C¼N, C9), 9.2 (–C–CH3, C10, C14), 40 (–N–CH3, C15), 122
An ethanolic solution (50 ml) of 4-aminoantipyrine (2.0 g, 10 mmol)
was added to an ethanolic solution of 3-(cinnamyl)pentane-
2,4-dione (1.1 g, 5 mmol) and the solution was refluxed for ca 4 h
with vigorous stirring and allowed to cool. Then it was poured into
crushed ice. The yellow crystals formed were filtered and recrystal-
lized from ethanol. The route for the synthesis of L is shown in
Scheme 1.
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Appl. Organometal. Chem. 2016, 30, 531–539

Biological and DNA interactions of Knoevenagel Schiff base complexes
(HC¼CH, C5, C6). Λm (Ωꢀ1 molꢀ1 cm2): 132; μeff (BM): diamagnetic.
UV–visible (DMSO, cmꢀ1 (transition)): 32 112 (ligand-to-metal
charge transfer).
was measured with a digital stopwatch three times for each sample
and an average flow time was calculated. Data were presented as
(η/η0)1/3 versus concentration of metal(II) complexes/concentration
of DNA, where η and η0 are the viscosity of CT DNA solution in the
presence and absence of complexes, respectively. Viscosity values
were calculated after correcting for the flow time of buffer alone
(t0): η = (t ꢀ t0)/t0.[16]
DNA binding interaction
The interaction between metal complexes and DNA was studied
using electronic absorption, viscosity and electrochemical methods.
Disodium salt of calf thymus (CT) DNA was stored at 4 °C. All exper-
iments involving the interaction of the complexes with CT DNA
were carried out in Tris–HCl buffer (50 mM Tris–HCl, pH = 7.2)
containing 5% DMSO at RT. A solution of CT DNA in the buffer gave
a ratio of UV absorbance at 260 and 280 nm of about 1.89, indicat-
ing the CT DNA sufficiently free from protein.[14] The concentration
of DNA was measured using its extinction coefficient at 260 nm
(6600 Mꢀ1 cmꢀ1) after 1:100 dilutions. Stock solutions were stored
at 4 °C and used not more than 4 days. Doubly distilled water was
used to prepare solutions. Concentrated stock solutions of the
complexes were prepared by dissolving the complexes in DMSO
and diluting appropriately with the corresponding buffer to the
required concentration for all experiments.
Gel electrophoresis experiments
Interactions of the ligand and the complexes with pUC18 plasmid
DNA were studied using agarose gel electrophoresis. DNA cleavage
activities of these compounds were monitored using a reaction
mixture containing 1 μg of plasmid DNA, 250 μg of sample, 2 μl
of DMSO (1%) and 5 μl of hydrogen peroxide (40 mmol). The
reaction mixture was incubated at 37 °C for 3 h. After incubation,
2 μl of loading buffer (0.25% bromophenol blue, 0.25% xylene
cyanol and 30% glycerol) was poured to a platform fixed with a
comb to form slots. Electrophoresis was performed at 50 V for 1 h
in Tris borate EDTA (TBE) buffer using 1% agarose gel stained with
ethidium bromide (EB; 1 μg) and the bands were photographed.
Electronic absorption titrations
Antimicrobial screening
In order to compare the binding strength of the complexes with CT
DNA quantitatively, the intrinsic binding constants (Kb) were
obtained by monitoring the changes in the absorbance for the
complexes with increasing concentration of DNA. Absorption
titration was performed by keeping the concentration of the metal
complex constant at 50 μM while varying the concentration of CT
DNA within 30–180 μM. While measuring the absorption spectrum,
an equal quantity of CT DNA was added to both the complex
solution and the reference solution to eliminate the absorbance
of CT DNA itself. From the absorption data, Kb was determined from
the ratio of slope to intercept of a plot of [DNA]/(εa ꢀ εf) versus
[DNA] using the following equation:
The in vitro antimicrobial screening of L and the complexes was car-
ried out against certain human sensitive pathogenic Gram-positive
bacteria (Staphylococcus aureus and Bacillus subtilis) and Gram-
negative bacteria (Klebsiella pneumoniae, Salmonella typhi and
Escherichia coli) and fungi (Aspergillus niger, Fusarium solani,
Curvularia lunata, Rhizoctonia bataticola and Candida albicans)
using the dilution method.[17] Nutrient agar and dextrose agar
served as the medium for the growth of bacteria and fungi, and
kanamycin and fluconazole were chosen as standards for antibacte-
rial and antifungal activity, respectively. The samples were incu-
bated at 37 °C for 24 h (bacteria) and 48 h (fungi). The results
were recorded in terms of minimum inhibitory concentration
(MIC). MIC is the lowest concentration of an antimicrobial com-
pound that will inhibit the visible growth of a microorganism after
overnight incubation. These data are important in diagnostic labo-
ratories to confirm resistance of microorganisms to antimicrobial
agents and also to monitor the activity of new antimicrobial agents.
½DNAꢁ
½DNAꢁ
1
¼
þ
(2)
εa ꢀ εf εb ꢀ εf Kbðεb ꢀ εfÞ
where [DNA] is the concentration of CT DNA in base pairs. The
apparent absorption coefficients εa, εf and εb 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. Kb is given by the ratio of slope to
intercept.
Results and discussion
The synthesized metal complexes were characterized using
various physicochemical techniques. Samples are found to be
stable in air and moisture. These complexes are soluble freely in
dimethylformamide and DMSO. All the analytical data with respect
to these complexes are in agreement with the proposed general
formula [ML]Cl2. The magnetic susceptibility data indicate the
monomeric nature of these complexes. In addition, the high
conductance values of these metal complexes indicate their
electrolytic properties. The proposed structure of the Knoevenagel
condensate Schiff base complexes is shown Fig. 1.
Cyclic voltammetry studies
Cyclic voltammetry studies were performed using a CHI 620C
electrochemical analyser with a three-electrode system of glassy
carbon as working electrode, platinum wire as auxiliary electrode,
Ag/AgCl as reference electrode and 50 mM NaCl, 5 mM Tris buffer
(pH = 7.2). Solutions were deoxygenated by purging with nitrogen
for 30 min prior to measurements.
Viscosity experiments
FT-IR spectra
Viscosity experiments were carried out with an Ostwald viscometer,
immersed in a thermostatically controlled water-bath maintained
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.[15] Flow time
FT-IR spectra give essential information about the structure and
functional groups of the ligand as well as the ligation site involved
in coordination with metal atom. The spectra of the ligand and the
metal complexes were recorded in the region 400–4000 cmꢀ1 and
are shown in Fig. S1.
Appl. Organometal. Chem. 2016, 30, 531–539
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R. P. Pandiyan and N. Raman
analysis, the stoichiometry of this complex is concluded to be
tetra-coordinate, which could be of square planar geometry.
1H NMR and 13C NMR Spectra
The 1H NMR spectra of L and the diamagnetic zinc(II) complex were
recorded in DMSO-d6 using tetramethylsilane as internal standard.
1
The H NMR spectrum of L (Fig. S3) shows peaks at 7.0–7.6 ppm
(m) and 9.4 ppm (s) corresponding to the phenyl multiplet and (–
CH¼CH–) of cinnamaldehyde moiety of the Schiff base. L also
shows the following signals: ¼C–CH3 at 2.5 ppm (s), N¼C–CH3 at
3.1 ppm (s) and N–CH3 at 3.4 ppm (s). A slight downfield shift is
identified with these signals with respect to the coordination of
Zn at the centre with the ligand in the complex.
Figure 1. Proposed structure of Knoevenagel condensate Schiff base
complex.
The 13C NMR spectra of L and its Zn(II) complex are depicted in
Fig. S4. The peaks of aromatic carbons of L are noticed in the range
125–132 ppm. Moreover, the azomethine group (C¼N) and car-
bonyl (C¼O) carbons are appear at 158 and 164 ppm. In the Zn(II)
complex, it is seen that these peaks are shifted to the upfield region,
indicating the coordination of (C¼N) and (C¼O) groups towards
the metal centre.[25] There is no such significant difference noticed
with other signals for this complex.
The spectrum of the free ligand shows a band in the region
1605 cmꢀ1, characteristic of the stretching mode of ν(C¼N) indicat-
ing the formation of the Schiff base.[18–20] This band is found to shift
towards lower frequency in case of the metal complexes to
1570–1598 cmꢀ1 implying the role of the azomethine nitrogen in
coordination with metal ion.[21] The coordination of the nitrogen
to the metal ion would be expected to reduce the electron density
of the azomethine link and thus cause a shift in the ν(C¼N) band.[22]
Moreover the spectrum of the free ligand exhibits a band at
1668 cmꢀ1, assigned to vibrational frequency of ν(C¼O) group,
which is shifted to lower frequency around 1631–1654 cmꢀ1
indicating the coordination of the carbonyl oxygen atom of the
ligand to the metal centre. In addition, evidence of the bonding is
also shown by the observation of new bands which appear in the
spectra of all metal complexes in the low-frequency regions at
449–468 and 501–518 cmꢀ1, characteristic of M–N and M–O
vibrations, respectively, which are not identified in the spectrum
of the free ligand.[23]
Mass Spectra
The ESI mass spectra of L and the metal complexes were recorded
at RT. The obtained molecular ion peaks confirm the proposed
formulae for the synthesized compounds. The mass spectra of L
and [CuL]Cl2 complex are depicted in Fig. S5. The mass spectrum
of L shows the molecular ion peak at m/z 584 corresponding to
[C36H36N6O2]+. Also, the spectrum exhibits fragments at m/z 229,
188, 104 and 77 corresponding to [C13H15N3O]+, [C11H12N2O]+,
[C8H8]+ and [C6H5]+, respectively. The mass fragmentation pattern
of L is shown in Scheme 2. The mass spectrum of [CuL]Cl2 shows
a molecular ion peak at m/z 647, which is equivalent to its molecular
weight. The fragmentation of [CuL]Cl2 gives demetallation of
copper ion and the m/z value is 584, corresponding to the fragment
ion [C36H36N6O2]+. Moreover, the spectrum exhibits fragments at
m/z 229, 188, 104 and 77 corresponding to [C13H15N3O]+,
[C11H12N2O]+, [C8H8]+ and [C6H5]+, respectively. The m/z of all the
Electronic spectra and magnetic moments
The geometry of the metal complexes was deduced from electronic
spectra and magnetic data. Electronic spectra of the Schiff base
ligand and its metal(II) complexes were recorded at RT in DMSO
medium in the range 200–1100 nm. The absorption spectra of
the ligand and its Cu(II) complex are shown in Fig. S2. The spectrum
of the Schiff base ligand exhibits two bands at 34 482 and 27
932 cmꢀ1 which can be attributed to the intramolecular charge
transfer transitions of π → π* and n → π*, respectively. After
complex formation and coordination of free Schiff base ligand to
the metal ion, the transitions are shifted to either up or down in
frequency. In the case of the Cu(II) complex, d–d band is observed
2
at 19 157 cmꢀ1, assigned to B1g
→
2A1g transition, strongly
supporting the square planar geometry around the Cu(II) ion.
Also, d–d band at 18 756 cmꢀ1 is detected in the spectrum of the
1
Co.(II) complex, corresponding to A1g
→
1B1g transition, which
indicates the square planar geometry. The magnetic moment
value (2.65 BM) for this complex for the square planar geometry
reveals four unpaired electrons with the low-spin four-coordinated
square planar environment is observed around the Co.(II) ion.[24]
The Ni(II) complex shows d–d band at 17 258 cmꢀ1 corresponding
1
to A1g
→
1B1g transition. The square planar geometry is
established from the observed diamagnetism of the Ni(II) complex.
Similarly, the Zn(II) complex is also noted as exhibiting diamagne-
tism, which does not shown any d–d transition in the visible region.
Altogether, based on absorption spectra, magnetism and elemental
Scheme 2. Mass fragmentation pattern of L.
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Appl. Organometal. Chem. 2016, 30, 531–539

Biological and DNA interactions of Knoevenagel Schiff base complexes
fragments of ligand and complexes confirm the stoichiometry of
the complexes as [ML]Cl2. The observed peaks are in good
agreement with their formulae as deduced from microanalytical
data. Thus, the mass spectral data are in accordance with the
conclusion drawn from the analytical and conductance values.
overlap integral. From Table 1, the α2 and β2 values indicate that
there is a substantial interaction in the in-plane σ-bonding whereas
the in-plane π-bonding is almost ionic. The lower value of α2 (0.78)
compared with β2 (1.19) indicates that the in-plane σ-bonding is
more covalent than the in-plane π-bonding. These values are in
accordance with those reported by other authors.[28] Based on
these interpretations, a square planar geometry is proposed for this
complex. The EPR spectral data for the Cu(II) complex provide
supporting evidence for the conclusion derived on the basis of
electronic spectra and magnetic moment values.
EPR spectra
The X band EPR spectrum of the Cu(II) complex was recorded in
DMSO at LNT and at RT. The spectrum of the Cu(II) complex at RT
shows one major intense absorption band at high field and is
isotropic due to the tumbling motion of the molecules. However,
it shows four well-resolved peaks at LNT with low intensities around
the low-field region and one intense peak in the high-field region
(Fig. S6). The spin Hamiltonian parameters of the Cu(II) complex
have been calculated and are summarized in Table 1. From the
spectral data, it is found that A|| (114) > A⊥ (19); g|| (2.39) > g⊥
(2.08) > 2.0027, which supportdx2ꢀy2 as the ground state, character-
istic of square planar geometry and axial symmetry. Further, in axial
symmetry, the g values are related by the expression
DNA binding studies
Absorption spectra titrations
For the determination of the binding mode of metal complexes
with DNA, electronic absorption spectroscopy has been used
widely.[29] Thus the absorption spectra of the complexes in the
absence or presence of CT DNA at various concentrations were
measured. The intercalation or electrostatic interactions between
metal complex and DNA lead to hypochromic and bathochromic
shifts. This indicates the intercalative binding mode, due to the
strong intercalation between the metal complexes and the base
pairs of DNA, whereas the hyperchromic shift indicates the break-
down of the secondary structure of DNA.[30] With an increase of
[DNA], the absorption intensity of the complexes decreases
(hypochromic effect) and the λmax values shift to the red
(bathochromic shift).[31] This occurs while the binding induces a
strong stacking interaction between the planar aromatic chromo-
phoric groups of the ligand and DNA base pairs.
g∥ ꢀ 2:0027
G ¼
(3)
g⊥ ꢀ 2:0027
This measures the exchange interaction between the copper
centres in a polycrystalline solid. As reported earlier, if G > 4.0,
the local tetragonal axes are aligned in parallel or only slightly
distorted. While if G < 4.0, there is significant coupling exchange
and the distortion is high. The observed value for the exchange
interaction parameter for the Cu(II) complex is 4.8, which suggests
that the local tetragonal axes are aligned parallel or slightly
2
2
distorted and the unpaired electron occupies thedx ꢀy orbital. This
implies the absence of exchange coupling between Cu(II) centres in
the solid state.[26]
Figure 2 shows the absorption spectra of the Cu(II) and Ni(II)
complexes in the absence and presence of CT DNA in the UV
The degree of geometrical distortion is ascertained by the pa-
rameter g||/A|| (A|| in cmꢀ1), with values less than 140 cmꢀ1 associ-
ated with square planar structures, whereas higher values indicate
the distortion towards a tetrahedron.[27] For the copper complex,
the g||/A|| value is 209 cmꢀ1, being in good agreement with a signif-
icant deviation from planarity, which is further supported by the
bonding parameter (α2) value remaining less than unity.
The covalency parameters α2 (covalent in-plane σ-bonding) and
β2 (covalent in-plane π-bonding) were calculated using the follow-
ing equations:
À
Á
A∥
3
α2 ¼
þ g∥ ꢀ 2:0027 þ ðg⊥ ꢀ 2:0027Þ þ 0:04 (4)
0:0306
7
À
Á
E
β2 ¼ g∥ ꢀ 2:0027
where λ = ꢀ828 cmꢀ1 for the free copper ion and E is the
electronic transition energy. As we know, α2 = 1.0 indicates
complete ionic character, whereas α2 = 0.5 signifies 100% covalent
bonding, based on the assumption of negligibly small values of the
(5)
ꢀ8λα2
Table 1. Spin Hamiltonian parameters of Cu(II) complex in DMSO
solution at LNT
Complex
g-tensor
A (× 10ꢀ4 cmꢀ1
A|| A⊥ Aiso
)
g||/A||
G
g||
[CuL]Cl2 2.39
g⊥
giso
Figure 2. Absorption spectra of (a) [CuL]Cl2 and (b) [NiL]Cl2 complexes in
buffer (pH = 7.2) at 25 °C in presence of increasing amounts of DNA.
Arrows indicate the changes in absorbance upon increasing DNA
concentration.
2.08
2.35 114 19 54 209 4.8
Appl. Organometal. Chem. 2016, 30, 531–539
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R. P. Pandiyan and N. Raman
region. With an increase in DNA concentration, the metal-to-ligand
charge transfer transition band of the [CuL]Cl2 complex exhibits
8.7% hypochromism at 339 nm and bathochromism of 6 nm.
Further the intense absorption bands with maxima (λmax) at 342,
272 and 352 nm for [CoL]Cl2, [NiL]Cl2 and [ZnL]Cl2 exhibit
hypochromism of about 2.2, 7.1 and 3.2% and bathochromism of
2, 4 and 2 nm, respectively. Therefore, these spectral characteristics
suggest a mode of binding that involves a stacking interaction
between the aromatic chromophore and DNA base pairs. These
shifts confirm that the metal complexes interact with DNA by
intercalation via strong stacking interactions between the aromatic
rings of the complexes and base pairs of DNA.[32]
The intrinsic binding constants (Kb) of the metal complexes with
CT DNA have been estimated by monitoring the shift in the
intraligand band with increasing concentration of DNA using
equation (2). The Kb values are given in Table 2. The intrinsic
binding constants determined for [CuL]Cl2, [CoL]Cl2, [NiL]Cl2 and
Figure 3. Effect of increasing amounts of [EB] (*), [CuL]Cl2 (■), [ZnL]Cl2 (♦),
[NiL]Cl2 (▲) and [CoL]Cl2 (×) on the relative viscosity of CT DNA. Plot of
relative viscosity (η/η0)1/3 versus [complex]/[DNA].
[ZnL]Cl2 are 2.2 × 104, 1.1 × 104, 1.7 × 104 and 1.2 × 104 Mꢀ1
.
Obviously, the observed Kb values of the present complexes are
lower than that for a classical intercalator such as EB[33] and higher
than those for some Schiff base metal complexes,[34] indicating that
the present complexes strongly bind with DNA through an
intercalation mode into the double helix structure of DNA.
that all these complexes interact with the DNA through an
intercalation mode.[38]
Electrochemical behaviour
Viscosity measurements
Electrochemistry is another characteristic method for elucidating
the basic chemistry of biological systems. Substances with a
reduction potential above 0.5 V may be susceptible to undergoing
in vitro electron transfer.[39] The electrochemical behaviour of the
complexes can be evaluated using the cyclic voltammetric
technique, which provides a valuable complement to the
previously used studies like absorption spectral titration and
viscosity measurements. In general, the electrochemical potential
of a small molecule will shift positively when it intercalates with
the DNA double helix, and it will shift in the negative direction in
the case of electrostatic interaction with DNA.[40] In the present
study, cyclic voltammetry was used to determine the nature of
DNA binding mode of the metal complexes. The cyclic voltammo-
grams of the Cu(II) and Ni(II) complexes in the absence and
presence of varying amounts of DNA are shown in Fig. 4. The
quasi- reversible redox couple for each complex in DMSO solution
has been analysed upon addition of CT DNA and shifts of E1/2 and
ΔEp are given in Table 3.
The mode of interaction between the metal complexes and DNA
was clarified using viscosity measurements. Viscosity measure-
ments are sensitive to changes in the length of DNA molecules
and regarded as the least ambiguous and most critical test for the
binding mode, in the absence of crystallographic data.[35] In order
to verify the binding mode of complexes with CT DNA, the change
in DNA viscosity at RT was measured for various concentrations of
the complexes. The classical intercalator EB shows a significant
increase in relative viscosity of DNA solution on intercalation due
to an increase in overall DNA contour length on binding to
DNA.[36] In contrast, partial or non-classical intercalation of a
complex would bend or kink the DNA helix, shortening the effective
length of the DNA, and reducing DNA viscosity accordingly, while
the electrostatic and groove binding cause little or no effect on
the relative viscosity of DNA solution.[37]
The effects of the synthesized complexes on the viscosity of DNA
at 30 0.1 °C are shown in Fig. 3. Viscosity measurements clearly
show that all the complexes can intercalate between adjacent
DNA base pairs, causing an extension in the helix thereby increas-
ing the viscosity of DNA. When the concentration of the complexes
is increased, the complexes can intercalate strongly, leading to a
greater increase in viscosity of the DNA. Hence, based on spectro-
scopic studies along with viscosity measurements, we can conclude
In the absence of DNA, the cyclic voltammogram of the Cu(II)
complex reveals four characteristic peaks, which are assigned to
two anodic peaks Epa (ꢀ0.285 and 0.150 V) and two cathodic peaks
Epc (ꢀ0.329 and ꢀ0.711 V) at a scan rate of 0.1 V sꢀ1. The first redox
cathodic peak appears at ꢀ0.329 V for Cu(II) → Cu(I) (Epa = ꢀ0.285 V,
Epc = ꢀ0.329 V, ΔEp = 0.044 V and E1/2 = ꢀ0.449 V) and in the
second redox couple, the cathodic peak appears at ꢀ0.711 V for
Cu(I) → Cu(0) (Epa = 0.150 V, Epc = ꢀ0.711 V, ΔEp = 0.861 V and
E1/2 = ꢀ0.205 V).
Table 2. Electronic absorption parameters for interaction of DNA with
Cu(II), Co.(II), Ni(II) and Zn(II) complexes
In the case of the cyclic voltammogram of Co.(II) complex in the
absence of DNA, the first redox couple cathodic peak appears at
ꢀ1.251 V for Co.(II) → Co.(I) (Epa = ꢀ0.289 V, Epc = ꢀ1.251 V,
ΔEp = ꢀ0.962 V and E1/2 = 0.330 V). For the cyclic voltammogram
of the Ni(II) complex in the absence of DNA, the first redox couple
cathodic peak appears at ꢀ1.053 V for Ni(II) → Ni(I), (Epa = ꢀ0.007 V,
Compound
λ
max (nm)
Δλ
(nm)
H (%)a
Kb (× 104 Mꢀ1
)
Free
Bound
[CuL]Cl2
[CoL]Cl2
[NiL]Cl2
[ZnL]Cl2
339
340
268
350
345
342
272
352
6
2
4
2
8.7
2.2
7.1
3.2
2.2
1.1
1.7
1.2
E
pc = ꢀ1.053 V, ΔEp = 1.046 V and E1/2 = ꢀ0.530 V). Similar data are
noticed for the Zn(II) complex, for which the first redox couple
aH = [(Afree ꢀ Abound)/Afree] × 100%.
cathodic peak appears at 0.184 V for Zn(II) → Zn(0) (Epa = ꢀ0.357 V,
E
pc = 0.184 V, ΔEp = 0.541 V, and E1/2 = ꢀ0.173 V).
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Appl. Organometal. Chem. 2016, 30, 531–539

Biological and DNA interactions of Knoevenagel Schiff base complexes
this reveals the intercalation and electrostatic binding of the
complex to CT DNA or it may break the secondary structure of
DNA.[41]
DNA cleavage study
Gel electrophoresis is an extensively used technique to determine
the cleavage efficacy of complexes with nucleic acids. If a reaction
mixture is placed under the influence of an electric field, DNA, being
a negatively charged species, will migrate towards the anode and
this migration depends upon the gel concentration, size of DNA,
electric potential and buffer nature. DNA cleavage is controlled by
the relaxation of supercoiled circular form of pUC18 DNA into
nicked circular and linear forms. When circular plasmid DNA is
subjected to electrophoresis, the fastest migration will be observed
for the supercoiled conformation (Form I). In the case where one
strand of DNA is cleaved, the supercoil will relax to produce a
slower moving nicked conformation form (Form II). On the other
hand, the cleavage of both strands of DNA results in linear
conformation (Form III) where migration between Form I and Form
II will occur.[42]
The ability of the complexes in affecting DNA cleavage has been
investigated using gel electrophoresis with supercoiled pUC18 DNA
in TBE buffer in the presence of H2O2 under physiological
conditions. Control experiments suggest that untreated DNA does
not show any cleavage (lane I). Moreover, the free ligand does
not show any apparent cleavage (lane II). Obviously all the
complexes are found to exhibit nuclease activity as depicted in
Fig. 5. The supercoiled (Form I) DNA is cleaved to form a mixture
of Form II and Form III with the addition of the complexes. DNA
cleavage activities of the complexes are obviously concentration-
dependent. The DNA cleavage activities of the metal complexes
are greater in the presence of oxidant H2O2. This may be attributed
to the formation of hydroxyl free radicals. The OH• free radicals
participate in the oxidation of the deoxyribose moiety, followed
by hydrolytic cleavage of a sugar phosphate backbone. All the
complexes show pronounced nuclease activity in the presence of
oxidant H2O2 which may due to the increased production of
hydroxyl radicals.[43]
Figure 4. Cyclic voltammograms of (a) [CuL]Cl2 and (b) [NiL]Cl2 in buffer
(pH = 7.2) at 25 °C in the presence of increasing amounts of DNA.
Table 3. Electrochemical parameters for interaction of DNA with Cu(II),
Co.(II), Ni(II) and Zn(II) complexes (data from cyclic voltammetric
measurements)
Compound
E
1/2 (V)a
Bound
ΔEp (V)b
Ipa/Ipc
Free
Free
Bound
[CuL]Cl2
-0.449
-0.205
0.330
-0.353
-0.173
0.470
0.738
0.047
0.044
0.861
0.962
1.046
0.541
0.112
0.973
1.650
2.279
0.747
1.39
0.63
0.82
0.88
0.91
Antimicrobial screening
Antimicrobial screening of L, metal complexes and standard
samples (kanamycin and fluconazole) was conducted against
bacteria and fungi and the results are summarized in Tables 4, 5.
[CoL]Cl2
[NiL]Cl2
[ZnL]Cl2
-0.530
-0.173
aE1/2 is calculated as the average of anodic (Epa) and cathodic (Epc) peak
potentials: E1/2 = (Epa + Epc)/2.
bΔEp = Epa ꢀ Epc
.
The cyclic voltammogram of each complex is approximately
close to unity for peak current ratio, which is responsible for the
reaction of the complex on the glassy carbon as a quasi-reversible
redox process. The incremental addition of CT DNA to the
complexes causes a decrease in anodic and cathodic peak current
of the complexes. This result shows that the complexes stabilize
the duplex (GC pairs) in an intercalating manner. A gradual increase
in the addition of CT DNA to the metal complexes leads to a shift in
the potential of the peak in cyclic voltammograms. Both the
cathodic and anodic peaks exhibit positive or negative shift, which
indicates the intercalation of complex to DNA base pairs. If one of
the shifts occurs to be positive and the other one is negative, then
Figure 5. Gel electrophoresis pattern showing cleavage of pUC18 DNA
treated with metal complexes. Lane I, DNA control; lane II, DNA + L+ H2O2;
lane III, DNA + [CuL]Cl2 + H2O2; lane IV, DNA + [CoL]Cl2 + H2O2; lane V,
DNA + [NiL]Cl2 + H2O2; lane VI, DNA + [ZnL]Cl2 + H2O2.
Appl. Organometal. Chem. 2016, 30, 531–539
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R. P. Pandiyan and N. Raman
Table 4. Minimum inhibitory concentration (MIC) of synthesized ligand and metal complexes against growth of bacteria
Compound
MIC (× 104 μM)
Staphylococcus aureus
Bacillus subtilis
Escherichia coli
Klebsiella pneumoniae
Salmonella typhi
L
15.8
7.1
8.9
9.3
7.3
1.4
15.1
8.3
9.9
8.9
7.0
2.6
14.3
7.6
8.2
7.6
8.1
1.8
14.0
7.3
9.8
8.3
7.9
2.9
13.5
6.4
9.2
9.0
8.0
2.6
[CuL]Cl2
[CoL]Cl2
[NiL]Cl2
[ZnL]Cl2
Kanamycina
aUsed as standard.
Table 5. Minimum inhibitory concentration (MIC) of synthesized ligand and metal complexes against growth of fungi
Compound
MIC (× 104 μM)
Aspergillus niger
Fusarium solani
Curvularia lunata
Rhizoctonia bataticola
Candida albicans
L
16.8
7.1
9.6
8.1
7.4
1.2
15.8
7.4
8.7
9.7
7.3
1.5
15.3
7.9
8.9
8.4
7.8
1.2
13.7
8.4
9.0
8.6
8.4
1.5
16.0
8.1
7.2
8.2
8.1
1.8
[CuL]Cl2
[CoL]Cl2
[NiL]Cl2
[ZnL]Cl2
Fluconazolea
aUsed as standard.
As compared to free ligand, the metal(II) complexes exhibit higher
activity and their essential antimicrobial activity via metal chelate
arises due to the effect of metal ion on the normal cell process. Such
an enhancement of activity by metal chelates can be explained on
the basis of Overtone’s concept[44] and chelation theory.[45]
According to Overtone’s concept of cell permeability, the lipid
membrane that surrounds the cell favours the passage of only
lipid-soluble materials whose liposolubility essentially determines
the antimicrobial activity.
mode and have good binding ability. The antibacterial and antifun-
gal activities of the ligand and its complexes indicate that the com-
plexes have more potent biocidal and fungicidal activity than the
free ligand. Gel electrophoresis assay demonstrates that each metal
complex promotes the cleavage ability of pUC18 plasmid DNA via
hydrolytic cleavage mechanism in the presence of oxidizing agent.
These biological findings from our study would be helpful in the
investigation of DNA interaction with metal complexes and may
lead to the development of novel metal-based therapeutic drugs.
On chelation, the polarity of the metal ion is reduced to
maximum extent due to the overlap of the ligand orbital and partial
sharing of the positive charge of the metal ion with donor groups.
Further, it increases the delocalization of the π-electrons over the
whole chelate ring and enhances the lipophilicity of the complexes.
This increased lipophilicity favours the penetration of the complex
into the lipid membrane and blocking of the metal binding sites
in the enzymes of the microorganisms. The metal complexes can
also interfere with the respiration process of the cell and thus block
the synthesis of proteins, which restricts further growth of an
organism.
Acknowledgments
The authors express their heartfelt thanks to the College Managing
Board, Principal and Head of the Department of Chemistry, VHNSN
College (Autonomous), Virudhunagar for providing necessary
research facilities.
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