Developing biologically active compounds having efficient DNA binding and cleavage activity: Spectroscopic investigation

Article abstract of DOI:10.1016/j.saa.2012.03.024

A new series of novel o-acetoacetotoluidide derived Schiff base and its metal complexes have been synthesized and structurally characterized. Elemental analysis, magnetic and spectroscopic data suggest that the complexes have octahedral geometry. Binding of these complexes with CT-DNA has been analyzed by absorption spectra, viscosity, cyclicvoltammetry and molecular docking analysis. Detailed analysis reveals that the metal complexes intercalate into the DNA base stack as intercalators. All the metal complexes cleave the pBR322 DNA upon irradiation. Antibacterial and antifungal activities of the ligand and its metal complexes are also explored and it has been observed that the complexes exhibit excellent activity against all types of bacteria and fungi than the ligand.

Full text of DOI:10.1016/j.saa.2012.03.024

Spectrochimica Acta Part A 93 (2012) 250–259
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Developing biologically active compounds having efficient DNA binding and
cleavage activity: Spectroscopic investigation
Natarajan Raman∗, Sivasangu Sobha
Research Department of Chemistry, VHNSN College, Virudhunagar-626 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of novel o-acetoacetotoluidide derived Schiff base and its metal complexes have been syn-
thesized and structurally characterized. Elemental analysis, magnetic and spectroscopic data suggest that
the complexes have octahedral geometry. Binding of these complexes with CT-DNA has been analyzed
by absorption spectra, viscosity, cyclicvoltammetry and molecular docking analysis. Detailed analysis
reveals that the metal complexes intercalate into the DNA base stack as intercalators. All the metal com-
plexes cleave the pBR322 DNA upon irradiation. Antibacterial and antifungal activities of the ligand and
its metal complexes are also explored and it has been observed that the complexes exhibit excellent
activity against all types of bacteria and fungi than the ligand.
Received 19 September 2011
Received in revised form 5 March 2012
Accepted 9 March 2012
Keywords:
Schiff base metal complexes
DNA interaction
Molecular docking
Biological evaluation
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
nucleic acids. Moreover, the ligands can interact with biomolecules
through coordinative or hydrogen bonds as well as dipole-dipole
Recently, the development of novel metal based biologically
active compounds as potential antitumor drugs is of great urgency
to overcome the drug-induced cellular resistance and the effi-
cacy of each drug against certain cancers. It is revealed from the
literature that the transition metal complexes can interact non-
covalently with DNA by intercalation, groove binding or external
electrostatic binding [1]. Many important applications of these
complexes require that they could bind to DNA in an intercala-
tive mode. In fact, the activity of many anticancer, antimalarial and
antibacterical agents finds its origin in intercalative interactions
with DNA. The significance of DNA cleavage in pharmaceuti-
cal and biotechnological applications has inspired researchers to
keep developing compounds that effectively cleave nucleic acid
molecule.
Schiff bases play an important role in inorganic chemistry as
they easily form stable complexes with most transition metal
ions. The development in the field of bioinorganic chemistry has
increased the interest in Schiff base complexes since it has been
recognized that many of these complexes may serve as models for
biologically important species [2]. The involvement of some metal
ions in regulation of physiological processes has stimulated the
development of metal-based therapeutics. The pharmaceutical use
of complexes arises from the fact that the positively charged metal
centers are favored to bind to negatively charged biomolecules
such as the amino acid residues of protein constituents, ATP and
interactions [3].
The metal complexes of o-acetoacetotoluidide and its deriva-
tives have been reported to show interesting biochemical
properties such as antitumor, antioxidant, antifungal and antimi-
crobial activities [4] and laboratory uses and many industrial
applications [5]. The important criteria for the development of
metallodrugs as chemotherapeutic agents are the ability of the
metallodrug to bring about DNA cleavage. A large number of transi-
tion metal complexes because of their redox properties, have been
found to promote DNA cleavage. Transition metal complexes have
been reported to bring about DNA cleavage either oxidatively or
hydrolytically or photolytically [6]. The pharmacological efficien-
cies of metal complexes depend on the nature of the metal ions
and the ligands [7]. It is declared in the literature that different lig-
ands and different complexes synthesized from same ligands with
different metal ions possess different biological properties [8]. So,
there is an increasing requirement for the discovery of new com-
pounds having antimicrobial activities. These facts prompted us
to design novel transition metal complexes having DNA binding
and DNA cleavage properties using o-acetoacetotoluitide derived
Schiff base. Hence, in the present work, we report the synthesis and
characterization of novel tridentate Schiff base derived from the
mono-condensation of o-acetoacetotoluidide, hydrazine hydrate
and salicylaldehyde and its transition metal(II) complexes. Infor-
mation obtained from our study would be helpful in understanding
the mechanism of interactions of the complexes with nucleic acid
and might also be useful in the development of potential probes of
DNA structure and conformation and new therapeutic reagents for
some uncommon diseases.
∗
Corresponding author. Tel.: +91 092451 65958; fax: +91 4562 281338.
E-mail address: drn raman@yahoo.co.in (N. Raman).
1386-1425/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2012.03.024

N. Raman, S. Sobha / Spectrochimica Acta Part A 93 (2012) 250–259
251
2. Experimental
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 DMF and diluting
suitably with the corresponding buffer to the required concentra-
tion for all the experiment. Absorption titration experiments were
carried out by varying the DNA concentration and maintaining the
complex concentration constant. Absorbance values were recorded
after each successive addition of DNA solution and equilibration (ca.
10 min). The absorption data were analyzed for an evaluation of the
intrinsic binding constant Kb using reported procedure [10].
Electrochemical studies were carried out using CHI Elec-
trochemical analyzer, controlled by CHI620 C software. CV
measurements were performed using a glassy carbon working
electrode and an Ag/AgCl reference electrode and supporting elec-
trolyte was 50 mM NaCl, 5 mM Tris buffer (pH 7.2). All solutions
were deoxygenated by purging with N2 for 30 min prior to mea-
surements.
2.1. Reagents and instruments
All reagents o-acetoacetotoluidide, hydrazine hydrate, salicy-
laldehyde and metal(II) chlorides were of Merck products and used
as supplied. Commercial solvents were distilled and then used for
the preparation of ligand and its complexes. pBR322 DNA was pur-
chased from Bangalore Genei (India). Microanalyses (C, H and N)
were performed in Carlo Erba 1108 analyzer at Sophisticated Ana-
lytical Instrument Facility (SAIF), Central Drug Research Institute
(CDRI), Lucknow, India. Molar conductivities in DMF (10−3 M) at
room temperature were measured using Systronic model-304 dig-
ital conductivity meter. Magnetic susceptibility measurements of
the complexes were carried out by Gouy balance using copper sul-
fate pentahydrate as the calibrant. The Infra-red spectra of the
ligand and its complexes were obtained as KBr discs in the range
350–4500 cm−1 on a FT IR-8400S instrument recorded at University
Science Instrumentation Center (USIC), Madurai Kamaraj Univer-
sity, Madurai. NMR spectra were recorded on a Bruker Avance
Dry 300 FT-NMR spectrometer in CDCl3 using TMS as the internal
reference. FAB-MS spectra were recorded with a VGZAB-HS spec-
trometer at room temperature in a 3-nitrobenzylalcohol matrix.
EPR spectra were recorded on a Varian E 112 EPR spectrometer in
DMSO solution both at room temperature (300 K) and liquid nitro-
gen temperature (77 K) using TCNE (tetracyanoethylene) as the
g-marker. The absorption spectra were recorded using Shimadzu
model UV-1601 spectrophotometer at room temperature.
Viscosity measurements at room temperature were carried out
using a semi-micro dilution capillary viscometer. Each experiment
was performed three times and an average flow time was calcu-
lated. Data were presented as (ꢀ/ꢀ0) versus binding ratio, where ꢀ
is the viscosity of DNA in presence of complex and ꢀ0 is the viscosity
of DNA alone.
2.4. Molecular modeling studies
2.4.1. Docking of metal complex with DNA
The interaction of the metal complexes with DNA was also
studied by molecular modeling with special reference to docking.
Extremely Fast Rigid Exhaustive Docking (FRED) version 2.1 was
used for docking studies (open eye Scientific Software, Santa Fe,
NM). This program generates an ensemble of different rigid body
orientations (poses) for each compound conformer within the bind-
ing pocket and then passes each molecule against a negative image
of the binding site. Poses clashing with this ‘bump map’ are elim-
inated. Poses surviving the bump test are then scored and ranked
with a Gaussian shape function. Prior to docking, the structures of
the metal complexes were constructed and geometry optimized
by MM2 force field. The crystal structure of B-DNA dodecamer
d(CGCGAATTCGCG)2 (NDB code GDLB05), was downloaded from
Protein Data Bank. Crystallographic water molecules were removed
from the B-DNA. The docked poses were generated by the exhaus-
tive search and optimization step FRED selects the single best pose
from the 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).
2.2. Synthesis of Schiff base ligand and its metal complexes
2.2.1. Synthesis of o-acetoacetotoluidide monohydrazone
Acetoacetotoluidide (1.91 g, 10 mmol) was dissolved in
methanol (40 mL) and was added to a solution of hydrazine
hydrate (0.05 g, 10 mmol) dissolved in hot methanol (10 mL). The
resulting mixture was refluxed for 3 h on a water-bath. On cooling,
the pale yellow compound formed was filtered, washed, dried and
recrystallized from methanol.
2.2.2. Synthesis of Schiff base ligand (L)
Schematic route for synthesis of Schiff base ligand and its metal
complexes is given in Scheme 1. The Schiff base has been synthe-
sized by refluxing the reaction mixture of hot methanolic solution
(30 mL) of salicylaldehyde (0.01 mol) and hot methanolic solution
(30 mL) of acetoacetotoluidide monohydrazone (0.01 mol) for ca.
5 h with the addition of 3–4 drops of hydrochloric acid. The dark
yellow color product separated was filtered and recrystallized from
methanol.
2.5. DNA photocleavage experiments
2.2.3. Synthesis of metal complexes
A solution of metal(II) chloride in ethanol (2 mmol) was refluxed
with an ethanol solution of the Schiff base (4 mmol) for ca. 2 h. Then
the solution was reduced to one-third on a water bath. The solid
complex precipitated was filtered off and washed thoroughly with
ethanol and dried in vacuo.
The DNA photocleavage of supercoiled pBR322 DNA (0.2 ␮g) to
its nicked and linear form was determined by agarose gel elec-
trophoresis in 50 mM Tris–HCl buffer (pH 7.2) containing 50 mM
NaCl. For photo-induced DNA cleavage studies, the reactions were
carried out under illuminated conditions using UV sources at
360 nm. After exposure to light, each sample was incubated for
1 h at 37 ◦C and analyzed for the photo-cleaved products using gel
electrophoresis as discussed below. The inhibition reactions for the
‘chemical nuclease’ reactions were carried out under dark condi-
tions by adding reagents (distamycin, 50 ␮M; DMSO 4 ␮L) prior
to the addition of each complex. The inhibition reactions for the
photo-induced DNA were also carried out at 360 nm using reagents
sodium azide (NaN3 100 ␮M;) as singlet oxygen scavenger and
superoxide dismutase (10 U) prior to the addition of the complex.
The samples after incubation for 1 h at 37 ◦C in a dark chamber
were added to the loading buffer containing 25% bromophenol blue,
2.3. DNA binding experiments
The interaction between metal complexes and DNA was studied
using electronic absorption, viscosity and electrochemical meth-
ods. Disodium salt of calf thymus DNA was stored at 4 ◦C. Solution of
DNA in the buffer 50 mM NaCl, 5 mM Tris–HCl (pH 7.2) in water gave
a ratio 1.9 of UV absorbance at 260 and 280 nm, A260/A280, indicating
that the DNA was sufficiently free from protein [9]. The concentra-
tion of DNA was measured using its extinction coefficient at 260 nm
(6600 M−1) after 1:100 dilution. Stock solutions were stored at 4 ◦C

252
N. Raman, S. Sobha / Spectrochimica Acta Part A 93 (2012) 250–259
Scheme 1. Schematic route for synthesis of Schiff base ligand and its metal complexes.
0.25% xylene cyanol, 30% glycerol (3 ␮L) and the solution was finally
loaded on 0.8% agarose gel containing 1 ␮g/mL ethidium bromide.
Electrophoresis was carried out in a dark chamber for 3 h at 50 V
in Tris-acetate-EDTA buffer. Bands were visualized by UV light and
photographed.
medium in petri plates. Disks were prepared and applied over the
long culture. The compounds were prepared in DMF and soaked in
filter paper disk of 5 mm diameter and 1 mm thickness. The con-
centrations of ligand and the complexes used in this study were
0.01 ␮g/mL. The disks were placed on the previously seeded plates
and incubated at 37 ◦C and the diameter of inhibition zone around
each disk was measured after 24 h for antibacterial activity and
after 74 h for antifungal activity. Growth inhibition was calculated
according to Ref. [11].
2.6. Antimicrobial studies
Antibacterial and antifungal activity of the ligand and its metal
complexes were tested in vitro against the bacterial species viz.
Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli,
Staphylococcus epidermidis, Klebsiella pneumoniae and the fungal
species Aspergillus niger, Fusarium solani, Culvularia lunata, Rhizoc-
tonia bataicola and Candida albicans by the paper disk method using
nutrient agar as the medium. Streptomycin was used as the stan-
dard antibacterial agent whereas nystatin was used as the standard
antifungal agent. The test organisms were grown on nutrient agar
3. Results and discussion
The Schiff base ligand (HL) and its Cu(II), Ni(II),Co(II) and Zn(II)
complexes were synthesized and characterized by spectral and ele-
mental analysis data. The complexes were found to be air stable. The
ligand was soluble in common organic solvents but their complexes
were soluble only in CHCl3, DMF and DMSO.

N. Raman, S. Sobha / Spectrochimica Acta Part A 93 (2012) 250–259
253
Table 1
Analytical and physical data of Schiff base ligand and its complexes.
a
Compound
Yield (%)
Color
Found (calc.)%
M
Formula weight
(∧m
)
ꢁeff (BM)
C
H
N
[C18H19O2N3]
81
71
65
62
59
Dark yellow
Dark green
Pale blue
Pink
–
69.3 (69.9)
63.8 (64.0)
63.8 (64.0)
63.7 (64.0)
62.7 (63.3)
6.1 (6.1)
5.2 (5.3)
5.1 (5.3)
5.2 (5.3)
5.3 (5.3)
12.9 (13.5)
12.2 (12.4)
12.1 (12.4)
12.1 (12.4)
11.8 (12.3)
309
680
675
676
682
–
–
[CuC36H36N6O4]
[NiC36H36N6O4]
[CoC36H36N6O4]
[ZnC36H36N6O4]
9.1 (9.3)
8.2 (8.6)
8.3 (8.7)
9.1 (9.5)
16.05
11.32
9.37
1.87
3.16
4.92
Yellow
18.75
Diamagnetic
a
The unit of the molar conductance is ꢂ −1 mol−1 cm2.
3.1. Elemental analysis and molar conductivity measurements
gave a fragment ion peak with loss of one fragment of ligand peak at
m/z 372. All these fragments leading to the formation of the species
[ML2]+ undergo de-metallation to yield the species [L+] giving the
fragment ion peak at m/z 309.
The results of elemental analysis for the metal complexes were
in good agreement with the calculated values (Table 1) showing
that the complexes have 1:2 metal–ligand stoichiometry of the type
ML2 wherein L acts as a tridentate ligand. The formation of these
complexes may proceed according to the equation given below:
3.4. IR spectra
The IR spectra of the complexes were compared with that of the
free ligand to determine the changes that might have taken place
during the complexation. The IR spectra of the complexes were
very similar to each other, except some slight shifts and inten-
sity change of a few vibration bands caused by different metal
ions which indicate that the complexes have similar structures.
The infrared spectra of the ligand and its metal(II) complexes and
important characteristic absorbance bands along with their pro-
posed assignments are summarized in Table 2. The characteristic
phenolic ꢃ(OH) mode due to the hydroxyl group of salicylalde-
hyde moiety presented in the Schiff base ligand was observed at
3371 cm−1. The disappearance of this band in all the complexes
indicated that phenolic ꢃ(OH) group was deprotonated upon com-
plexation. The ligand showed its characteristic ꢃ(C O) band at ca.
1722 cm−1, which was shifted to lower frequencies in the spectra of
the complexes (1656–1647 cm−1). In the IR spectrum of the Schiff
base ligand, the band observed at 1604 cm−1 was shifted to lower
frequency by 21–33 cm−1 on complexation, suggesting the coordi-
nation of the azomethine nitrogen [20]. The unaltered position of
bands due to ꢃ(NH) and ꢃ(C N) moiety of the acetoacetotoluidide
monohydrazone in all the metal complexes indicated that these
groups were not involved in coordination. The new bands in the
region of 480–502 and 428–435 cm−1 in the spectra of the com-
MCl2·nH2O + 2HL → [ML2] + 2HCl + nH2O
where, M = Cu(II), Ni (II), Co(II) and Zn(II)
The metal complexes were dissolved in DMSO and the molar
conductivities of 10−3 M of their solution at 25 ◦C were measured.
Table 1 shows the molar conductance values of the complexes. It is
evident from the results that the chelates are found to have molar
conductance values of 9.37–8.75 ꢂ−1 cm2 mol−1 supporting their
non-electrolytic nature.
3.2. Magnetic susceptibility and ultraviolet spectral
measurements
The electronic absorbance spectrum of the ligand in ethanol
showed two prominent peaks in the region 32,102 and
33,388 cm−1 which were assigned to intraligand charge trans-
fer (INCT) bands. The copper(II) complex exhibited intraligand
charge transfer bands at ca. 35,453 and 37,786 cm−1 and a
d–d band at 14,792 cm−1 which was due to 2Eg → T2g transi-
2
tion [12–15]. This d–d band transition band strongly favored a
distorted octahedral geometry around the metal ion. Its mag-
netic moment (1.87 BM) indicates that the complex exits in
monomeric nature. The electronic spectrum of the cobalt com-
plex gave three bands at 15,060, 16,528 and 20,080 cm−1, which
plexes were assigned to stretching frequencies of M O and M
bonds.
N
4
4
4
4
are assigned to T1g(F) → T2g(F)(ꢃ1), T1g(F) → A2g(F)(ꢃ2), and
4T1g(F) → T2g(P)(ꢃ3) transitions respectively [16–18]. This indi-
4
3.5. 1H-NMR spectra
cates that the complex of Co(II) is six coordinate and probably
an octahedral geometry which is also supported by its mag-
netic susceptibility value (4.92 BM). The absorption spectrum
of nickel(II) complex displayed three d–d bands at 14,636,
The 1H-NMR spectra of the Schiff base and its zinc complex were
recorded at room temperature in CDCl3. 1H-NMR spectrum of the
ligand showed a singlet at ␦ 5.3, attributed to the phenolic OH
group of salicylaldehyde moiety presented in the Schiff base ligand.
The absence of this peak noted for the zinc complex confirmed the
OH proton upon complexation. The Ph NH group gave singlet
at ␦ 11.2 in the free ligand and remained unchanged in the zinc
complex. It showed that the Ph NH group was not taking part
in complexation. The ligand showed the following signals: phenyl
multiplet at 6.8–8.2 ␦, CH N at 9.8 ␦, CH3 at 1.2-2.4 ␦, CH2 at
15,332 and 23,402 cm−1
,
attributed to A2g(F) → T2g(F)(ꢃ1),
3
3
3A2g(F) → T1g(F)(ꢃ2) and 3A2g(F) → T1g(P)(ꢃ3) transitions respec-
tively, being characteristic of an octahedral geometry [19]. This
geometry was further supported by its magnetic susceptibility
value (3.16 BM). The complex of Zn(II) is diamagnetic. According
to the empirical formula, an octahedral geometry is proposed for
this complex.
3
3
3.3. Mass spectra
Table 2
IR data of Schiff base ligand and its metal complexes.
The mass spectrum of Schiff base ligand showed peak at m/z 309
corresponding to [C18H19O2N3] ion. Also the spectrum exhibited
peaks for the fragments at m/z 189, 120, 106 and 77 corresponding
to [C11H13N2O]+, [C7H6NO]+, [C7H8N]+ and [C6H5]+1 respectively.
The spectra of Cu(II), Co(II), Ni(II) and Zn(II) complexes showed a
molecular ion peak [M+] at m/z 680, 676, 675 and 682 respectively
that are equivalent to their molecular weights. The Cu(II) complex
Compound
ꢃN
ꢃC
ꢃHC
ꢃC
ꢃ M
ꢃM
O
H
O
N
N
N
[C18H19O2N3]
3279
3279
3283
3279
3279
1722
1656
1651
1657
1647
1604
1571
1583
1575
1579
1639
1641
1639
1639
1639
–
428
430
432
435
–
[CuC36H36N6O4]
[NiC36H36N6O4]
[CoC36H36N6O4]
[ZnC36H36N6O4]
480
502
489
495

254
N. Raman, S. Sobha / Spectrochimica Acta Part A 93 (2012) 250–259
Table 3
3.1 ␦. The azomethine proton ( CH N) signal in the spectrum of the
zinc complex was shifted down field compared to the free ligand,
suggesting deshielding of azomethine group due to the coordina-
tion with metal ion. There was no appreciable change in all other
signals of the complex.
Electronic absorption spectral properties of Cu(II), Ni(II), Co(II) and Zn(II) complexes.
Compound
ꢄmax
ꢅꢄ (nm)
aH%
bKb × 105 (M−1
)
Free
Bound
[CuC36H36N6O4]
[NiC36H36N6O4]
[CoC36H36N6O4]
[ZnC36H36N6O4]
353.2
381.4
320.8
342.1
356.8
385.4
324.1
344.8
3.6
4.0
3.3
2.7
29
31
26
22
2.36
3.70
1.28
1.17
3.6. EPR spectra
The EPR spectrum of copper complex provides information
which is important in studying the metal ion environment. The EPR
spectra were recorded in DMSO at LNT and at RT. The spectrum of
the copper complex at RT showed one intense absorption band in
the high field and was isotropic due to the tumbling motion of the
molecules. However, this complex at LNT showed well resolved
a
H% = [Afree − Abound)/Afree] × 100%.
b
Kb = Intrinsic DNA binding constant determined from the UV–vis absorption
spectral titration.
extent of hypochromism is commonly consistent with the strength
of the intercalative interaction [22].
peaks with low field region. The copper complex exhibited the g
||
The absorption spectra of the copper and nickel complexes in
the absence and presence of CT-DNA are given in Figs. 1 and 2.
In the UV region, the intense absorption bands observed in the
metal complexes are attributed to the intra ligand ␲–␲* transi-
tion of the coordinated groups. Addition of increasing amounts of
CT-DNA resulted in hypochromism and bathochromic shift in the
UV spectra of [CuC36H36N6O4], [NiC36H36N6O4], [CoC36H36N6O4],
and [ZnC36H36N6O4]. The hypochromism in the intraligand (IL)
band reached as high as 29% at 353.2 nm with 3.6 nm red shift
for [CuC36H36N6O4]. The absorption spectrum of [NiC36H36N6O4],
complex showed an intensive absorption band at 381.4 nm exhibit-
ing hypochromism of about 31% and a bathocromic shift of
4.0 nm in 5 mM Tris–HCl, 50 mM NaCl, at pH 7.2 buffer solution,
further the intense absorption band with maxima of 320.8 nm
for [CoC36H36N6O4] showed hypochromism of about 26% and a
bathocromic shift of 3.3 nm, [Zn36H36N6O4] showed a band at
342.1 nm, attributed to intraligand (IL) ␲–␲* transition and result-
ing in the hypochromism of about 22% with 2.7 nm bathochromic
shift. The spectral characteristics obviously suggested that the
metal complexes interacted with DNA most likely through a mode
that involved a stacking interaction between the aromatic chro-
mophore and the base pairs of DNA.
value of 2.280 and g value of 2.046. These values indicated that
⊥
the Cu(II) lies predominantly in the dX
orbital, as was evident
2
2
from the value of the exchange interacti−oYn term G, estimated from
the expression:
(g|| − 2)
G =
(g⊥ − 2)
If G > 4.0, the local tetragonal axes are aligned parallel or only
slightly misaligned. If G < 4.0, significant exchange coupling is
present and the misaligned is appreciable. The observed value
for the exchange interaction parameter for the copper complex
(G = 6.35) suggests that the local tetragonal axes are aligned parallel
or slightly misaligned and the unpaired electron is present in the
dX
orbital. This result also indicates that the exchange coupling
2
2
−Y
effects are not operative in the present complex [21].
Electron paramagnetic resonance and optical spectra have been
used to determine the covalent bonding parameters for the Cu(II)
ion in various environments. Since there has been wide interest
in the nature of bonding parameters in the system, we adopted
the simplified molecular orbital theory [9] to calculate the bond-
ing coefficients such as ˛2 (covalent in-plane ␴-bonding) and ˇ2
(covalent in-plane ␲-bonding). The in-plane ␴-bonding bonding
parameter, ˛2 is related to g and g according to the following
The intrinsic binding constants (Kb) of the metal complexes with
CT DNA were obtained by monitoring the changes in the intraligand
band with increasing concentration of DNA using the following
equation
||
⊥
equation:
ꢀ
ꢁ
A
3
||
2
˛
=
+ (g|| − 2.0027) + (g⊥ − 2.0027) + 0.04
7
0.036
[DNA]
(εa − εf)
[DNA]
(εb − εf)
1
=
+
If the ˛2 value is 0.5, it indicates a complete covalent bonding,
[Kb(εb − εf)]
while the value of ˛2 = 1.0 suggests a complete ionic bonding. The
observed value of ˛2 (0.80) indicates that the complex has some
covalent character. The in-plane ␲-bonding (ˇ2) parameter was
calculated from the following equation:
where [DNA] is the concentration of DNA in base pairs, the
apparent absorption coefficients εa, εf, and εb are the appar-
ent, free and bound metal complex extinction coefficients,
respectively.
A
plot of [DNA]/(εb − εf) versus [DNA], gave
a
,
E
ˇ2 = (g|| − 2.0027)
−8ꢄ˛2
slope of 1/(εb − εf) and a Y-intercept equal to [Kb/(εb − εf)]−1
Kb is the ratio of the slope to the Y-intercept. Intrinsic binding
constants of 2.36 × 105 M−1
,
3.70 × 105 M−1
,
1.28 × 105 M−1
where ꢄ = −828 cm−1 for free ion and E is the electronic transition
and 1.17 × 105 M−1 were determined for [CuC36H36N6O4],
[NiC36H36N6O4], [CoC36H36N6O4] and [CuC36H36N6O4], respec-
tively. These values (Table 3) are comparable to the classical
DNA intercalators EB (ethidium bromide, 1.4 × 106 M−1 [23] and
many analogs, such as [Ru(phen)2(phehat)]2+ (phehat = 1,10-
phenanthrolino[5,6-b]1,4,5,8,9,12-hexaazatriphenylene,
energy of 2Eg → T2g. The observed value of ˇ2 (0.77) indicates that
2
the ␴-bonding is more covalent than the in-plane ␲-bonding.
Based on the above spectral and analytical data, the pro-
posed model of Schiff base and its metal complexes is given in
Supplementary file (Fig. S1).
2.5 × 106 M−1 and Ru(bpy)2(ppd)]2+ (1.1 × 106 M−1
) [24]. The
3.7. DNA binding of the metal complexes
result suggests that the interaction of four metal complexes with
DNA is a moderate intercalative mode.
3.7.1. Absorption spectral aspects of DNA binding
Electronic absorption spectroscopy is usually employed to
determine the binding of complexes with the DNA helix. A complex
bound to DNA through intercalation is characterized by the change
in absorbance (hypochromism) and red shift in wavelength, due
to the intercalative mode involving a strong stacking interaction
between the aromatic chromophore and the DNA base pairs. The
3.7.2. Viscosity measurements
To clarify the binding mode between the metal complexes
and DNA, viscosity of DNA solution containing varying amount
of added complex was measured. The results indicated that the
presence of the metal complex increased the viscosity of the DNA

N. Raman, S. Sobha / Spectrochimica Acta Part A 93 (2012) 250–259
255
Fig. 1. Absorption spectrum of [CuC36H36N6O4] complex in buffer (pH 7.2) at 25 ◦C upon addition of CT-DNA. Arrow indicates the changes in absorbance upon increasing the
DNA concentration. Inset: plot of [DNA]/(εa − εf) versus [DNA].
solution, as illustrated in Fig. 3. As general rule metal complexes
can increase the viscosity of DNA when they intercalate into
the double-stranded DNA (or) bind to the phosphate group of
DNA backbone [25]. The effect of metal complexes together with
ethidium bromide [EB] on the viscosity of CT-DNA is shown in
Fig. 3. On increasing the amounts of metal complexes, the relative
viscosity of DNA moderately increased steadily, which is similar to
the behavior of ethidium bromide. The results suggest that metal
complexes bind DNA with a moderate intercalative mode.
Fig. 2. Absorption spectrum of [NiC36H36N6O4] complex in buffer (pH 7.2) at 25 ◦
C
Fig. 3. Effect of increasing amounts of [CuC36H36N6O4] ( ), [NiC36H36N6O4] (
)
upon addition of CT-DNA. Arrow indicates the changes in absorbance upon increas-
[CoC36H36N6O4] ( ), [ZnC36H36N6O4] ( ) and [EB] ( ) on the relative viscosity of
ing the DNA concentration. Inset: plot of [DNA]/(εa − εf) versus [DNA].
DNA. R = [complex]/[DNA] or [EB]/[DNA].

256
N. Raman, S. Sobha / Spectrochimica Acta Part A 93 (2012) 250–259
spectroscopic and viscosity data of the complexes in the presence
of DNA. The cyclic voltammogram of [NiC36H36N6O4] complex in
the absence and in presence of varying amount of DNA is shown in
Supplementary file (Fig. S2).
For Co(III) → Co(II), the redox couple cathodic peak appeared
at 0.105 V in the absence of CT DNA (Ep = 0.382 V, Ep = 0.105 V,
a
ꢅEp = 0.487 V and E = −0.138 V). The ratio of ip /ip cis approxi-
1/2
a
mately unity. It indicated that the reaction of theccomplex on the
glassy carbon electrode surface is quasi-reversible redox process.
The incremental addition of CT DNA to the complex caused a posi-
tive shift in E1/2 and a decrease in ꢅEp value. The ip /ip values also
c
a
decreased in the presence of DNA.
Finally the zinc complex showed redox cathodic peak was
appearing at −0.304 V and anodic peak appearing at −0.549 V.
The separation of the anodic and cathodic peak potentials is
ꢅEp = −0.245 V. The formal potential E1/2 = −0.342 V. The incre-
mental addition of CT DNA to the complexes revealed that the redox
couples caused a less positive shift in E1/2 and decrease of ꢅEp .The
ip /ip values also decreased in the presence of DNA. The electro-
chemcical parameters of the Cu(II), Ni(II), Co(II) and Zn(II) complexes
are shown in Table 4. From these data it is understood that all the
synthesized complexes interact with DNA through intercalating
way.
a
Fig. 4. Cyclic voltammogram of [CuC36H36N6O4] in buffer (pH 7.2) at 25 ◦C in the
presence of increasing amount of DNA. Arrow indicates the changes in voltammetric
currents upon increasing the DNA concentration.
3.7.3. Cyclic voltammetry
The applications of electrochemical methods to the study of
metallointercation and coordination of metal ion and chelates to
DNA provide a useful complement to the previously used methods
of investigation, such as UV–vis spectroscopy [26]. The interactions
of some anticancer drugs with DNA have been studied with a vari-
ety of techniques, currently much attention have been focus to the
electrochemical investigations of interactions between anticancer
drugs and other DNA-targeted molecules and DNA. The cyclic
voltammograms of [CuC36H36N6O4] complex in the absence and in
presence of varying amount of DNA is shown in Fig. 4. In the absence
of CT DNA, the first redox cathodic peak appeared at −0.093 V
3.7.4. DNA docking
The discovery and development of novel therapeutic intercala-
tor agents for the treatment of malignancy are some of the most
important goals in modern medicinal chemistry. A very interesting
group in cancer therapy comprises molecules that target directly
DNA. Intercalation into DNA (insertion between a pair of base pairs)
is a very important process, especially with regards to the func-
tion of many anticancer drugs. The combination of spectroscopic
and electrochemical methods is helpful in understanding of the
interaction between DNA. In addition to this, the experiments are
very useful to determine the recognition of DNA sites, and also
to provide novel rational design of drugs for chemotherapy appli-
cations. Intercalation results from insertion of a planar aromatic
substituent between DNA base pairs, with concomitant unwind-
ing and lengthening of the DNA helix. The results of spectroscopic
and electrochemical studies reveal that all the metal complexes
are moderate intercalative compound and have good affinity for
DNA. For copper, nickel, cobalt and zinc complexes, the intrinsic
for Cu(III) → Cu(II) [Ep = 0.0259 V, Ep = −0.093 V, ꢅEp = 0.352. V
and E1/2 = 0.176 V] and in the secondcredox couple, the cathodic
a
peak appeared at −0.590 V for Cu(II) → Cu(I) [Ep = 0.034 V, Ep
=
a
c
−0.590 V, ꢅEp = 0.556 V and E1/2 = 0.312 V]. The ip /ipc ratio of these
a
two redox couples is 0.92 and 1.31 respectively which indicate that
the reaction of the complex on the glassy carbon electrode sur-
face is quasi-reversible redox process. The increment addition of
CT-DNA to the complex causes a positive shift in potential and a
decrease in the current intensity. Among the three kinds of binding
modes for small molecules to DNA, Bard has reported [27] that if
E1/2 is shifted to more negative value when small molecules inter-
act with DNA, then the interaction mode is electrostatic binding.
On the contrary, if E1/2 is shifted to more positive value, the inter-
action mode is intercalative binding. Hence, it is concluded that
the [CuC36H36N6O4] complex could bind to DNA by intercalative
mode.
binding constant values (Kb) are 2.36 × 105 M−1, 3.70 × 105 M−1
,
1.28 × 105 M−1 and 1.17 × 105 M−1 respectively, determined by
ultraviolet spectroscopy.
The binding constant values were in the range of 105 per mole for
all the complexes which indicated the possibility of intercalation of
these complexes between the DNA base pairs. It was mainly due to
more effective stacking forces between the aromatic nucleus and
DNA bases. Docking experiments in the energy minimized docked
structures suggested that the best possible geometry of the cop-
per and nickel complexes was partially intercalate into the major
groove as shown in Fig. 5. The intercalates were oriented parallel
to the base pairs, commonly ␲-stacking in the major groove.
The structural analysis of docked structures gave signifi-
cant details about the binding pattern of these complexes.
Binding energy of docked metal complexes [CuC36H36N6O4],
[NiC36H36N6O4], [CoC36H36N6O4] and [ZnC36H36N6O4] were found
to be −588.70, −590.45, −585.70.6 and −573.67 kJ mol−1 respec-
tively, correlating well with the experimental DNA binding values.
The more negative the relative binding, the more potent the binding
in between DNA and target molecules. To the best of our knowledge,
the present investigation should contribute to exploring more in
this area in the future.
For the CV behavior of [NiC36H36N6O4] in the absence of DNA,
the first redox couple cathodic peak appeared at −0.116 V for
Ni(III) → Ni(II), (Ep = 0.592 V, Ep = 0.116 V, ꢅEp = −0.476 V, and
a
c
E1/2 = −0.354 V). ip /ip ratio of these two redox couples is approxi-
c
mately unity. Thisaindicates that the reaction of the complex on the
working electrode surface is quasi-reversible redox process. In the
incremental addition of DNA to the complex the second cathodic
peak caused a positive shift in the potential and a decrease in the
current intensity. The changes of the voltammetric currents in the
presence of CT DNA can be attributed to diffusion of the metal
complex bound to the large, slowly diffusion DNA molecule. The
changes of the peak currents observed for the complexes upon addi-
tion of CT DNA may indicate that complexes possess higher DNA
binding affinity with complex. The results are similar to the above

N. Raman, S. Sobha / Spectrochimica Acta Part A 93 (2012) 250–259
257
Table 4
Electrochemical parameters for the interaction of DNA with Cu(II), Ni(II), Co(II) and Zn(II) complexes.
Compound
Redox couple
aE1/2 (V)
bꢅEp (V)
ip /ip
a
c
Free
Bound
Free
Bound
Cu(III) → Cu(II)
Cu(II) → Cu(I)
Ni(II) → Ni(I)
Co(III) → Co(II)
Zn(II) → Zn(0)
0.176
0.312
−0.354
−0.138
−0.342
0.366
−0.320
−0.319
−0.120
−0.401
0.352
0.556
−0.476
−0.487
−0.245
0.428
0.487
−0.469
−0.490
−0.085
0.92
1.31
1.07
1.30
0.91
[CuC36H36N6O4]
[NiC36H36N6O4]
[CoC36H36N6O4]
[ZnC36H36N6O4]
a
Data from cyclic voltammetric measurements; E1/2 is calculated as average of anodic (Ep ) and (Ep ) peak potential E1/2 = Ep + Epc /2.
a
c
a
b
ꢅEp = Ep − Ep
.
a
c
Fig. 5. Fast Rigid Exhaustive Docking molecular model of (a) copper and (b) nickel complexes partially intercalated from the major groove.
3.8. DNA photocleavage
relax to generate a slower-moving open circular form (Form II).
If both strands are cleaved, a linear form (Form III) that migrates
between Form I and Form II will be generated, even in the absence
of inhibitors on irradiation with UV light at 360 nm.
The presence of bioactive ligand and DNA binder in the
metal(II)complexes is essential for observing efficient photo-
induced DNA cleavage activity. Fig. 6 shows gel electrophoresis
pattern of pBR322 DNA after incubation with the four metal com-
plexes and irradiation at 360 nm. No DNA cleavage was observed
for controls in which the complex was absent (lane 0). The result
indicated the importance of the metal complexes for observing the
photo induced-DNA cleavage activity. All the complexes cleaved
the pBR322 DNA from its supercoil form SC. The supercoil will
The mechanistic aspects of the DNA cleavage reactions involv-
ing copper and nickel complexes have been investigated at
360 nm wavelength using different inhibitors which are shown in
Fig. 7a and b; the other complexes (cobalt and zinc) are given in
Supplementary file (Fig. S3 and Fig. S4). In the presence of sin-
glet oxygen quencher sodium azide the cleavage was inhibited. The
cleavage of the plasmid DNA was not inhibited in the presence of
hydroxyl radical scavenger like DMSO which has no apparent effect
on the photo-induced DNA cleavage activity. When pBR322 DNA
reacted with distamycin, a minor groove binder, the cleavage reac-
tion was not quenched. The addition of distamycin did not inhibit
the cleavage of all the complexes. While in presence of−superoxide
dismutase (SOD), a facile superoxide anion radical O2 quencher,
−
the cleavage was improved which indicated that O2 might be an
inhibitor in the photoactivated cleavage of the plasmid and the
−
reducing the amount of O2 could improve the cleavage effect.
3.9. Antimicrobial studies
Fig. 6. Gel electrophoresis diagram showing the photocleavage of pBR322 DNA by
the synthesized complexes in DMSO-Tris buffer medium on irradiation with UV light
of 360 nm for 60 min: lane 1, DNA control (60 min); lane 2, DNA + ligand (60 min);
lane 3; DNA + [CuC36H36N6O4]; DNA + [NiC36H36N6O4]; DNA + [CoC36H36N6O4];
DNA + [ZnC36H36N6O4].
3.9.1. Antibacterial activity
The Schiff base complexes have provoked wide interest because
they possess a diverse spectrum of biological and pharmaceutical

258
N. Raman, S. Sobha / Spectrochimica Acta Part A 93 (2012) 250–259
Table 6
The in vitro antifungal activity of Schiff base and its metal complexes (zone of inhi-
bition in mm).
Compound
A. niger
F. solani
C. lunata
R. bataicola
C. albicans
[C18H19N3O2]
[CuC36H36N6O4]
[NiC36H36N6O4]
[CoC36H36N6O4]
[ZnC36H36N6O4]
Nystatin
++
+
+
+
+
+++
++++
+++
++
++
++
++
+++
++
++
++
+++
++
+++
++
++
+++
++
+++
++
+++
++
+
++++
+++
Nystatin is used as the standard. DMF was used as antimicrobial inert solvent.
+++++ = Excellent activity (100% inhibition).
+++ = Good activity (60-70% inhibition).
++ = Moderate activity (30-50% inhibition).
+ = Less activity (10-20% inhibition).
Fig. 7. (a) Photoactivated cleavage of pBR322 DNA in the presence of
DNA + [CuC36H36N6O4] (60 min) and different inhibitors after irradiation at
360 nm for 60 min: lane 1 DNA control (60 min); lane 2, DNA + [CuC36H36N6O4]
(50 ␮M); lane 3, DNA + [CuC36H36N6O4] + sodium azide (100 ␮M); lane 4,
antifungal actions. The antifungal tests were carried out using the
disk diffusion method. The Schiff base ligand and its metal com-
plexes were also screened in vitro in order to find out the antifungal
activity against A. niger, F. solani, C. lunata, R. bataicola and C. albi-
cans. The results of the antifungal studies are presented in Table 6
which reveals that the metal complexes are toxic than the free
ligand against the same organisms. The increase in the antifungal
activity of the metal complexes inhibited multiplication process of
the microbes by blocking their active sites. Such increased activity
on metal chelation can be explained on the basis of Tweedy’s chela-
tion theory [30]. The chelation also increases the lipophilic nature
and an interaction between the metal ion and the lipid is favored.
This may be lead to the breakdown of the permeability barrier of
the cell resulting in interference with the normal cell processes.
While chelation is not the only factor for antimicrobial activity, it
is an intricate blend of several aspects such as nature of the metal
ion and the ligand, geometry of the metal complexes, lipophilicity,
steric and pharmacokinetic factors [31].
DNA + [CuC36H36N6O4] + SOD (1 U); lane 5, DNA + [CuC36H36N6O4] + DMSO (4 ␮L);
lane 6, DNA + [CuC36H36N6O4] + distamycin (100 ␮M). (b) Photoactivated cleavage
of pBR322 DNA in the presence of DNA + [NiC36H38N6O4] (60 min) and different
inhibitors after irradiation at 360 nm for 60 min: lane 1 DNA control (60 min); lane
2, DNA + [NiC36H36N6O4] (50 ␮M); lane 3, DNA + [NiC36H36N6O4] + sodium
azide (100 ␮M); lane 4, DNA + [NiC36H36N6O4] + SOD (1 U); lane 5,
DNA + [NiC36H36N6O4] + DMSO (4 ␮L); lane 6, DNA + [NiC36H36N6O4] + distamycin
(100 ␮M).
activities. The antibacterial activity of the Schiff base ligand, its
metal complexes and streptomycin (as a standard compound) were
tested against bacteria because bacteria could achieve resistance to
antibiotics through biochemical and morphological modifications
[28,29]. The organisms used in the present investigations included
S. aureus, P. aeruginosa, E. coli, S. epidermidis, K. pneumoniae. The
diffusion agar method was used to evaluate the antibacterial
activity of the synthesized metal complexes.
The results of the bactericidal study of the synthesized com-
pounds are displayed in Table 5. The zone of inhibition values
obtained reflects that the Schiff base ligand has moderate activity
against S. aureus and E. coli and less active against P. aeruginosa and
S. epidermidis. Ligand also showed a moderate activity towards K.
pneumoniae. The remarkable activity of the Schiff base ligand may
be arise from the two imine groups which import in elucidating the
mechanism of transformation reaction in biological system. All the
metal complexes were found to have higher antibacterial activity
against Schiff base ligand.
4. Conclusion
In this paper, a novel o-acetoacetotoluidide derived Schiff base
and its Cu(II), Ni(II), Co(II) and Zn(II) complexes have been syn-
thesized and characterized by spectral and analytical data. The IR,
electronic transition and g tensor data lead to the conclusion that
the Cu(II) ion assumes a distorted octahedral geometry and the
other complexes are octahedral in nature. Binding of these com-
plexes to CT-DNA has been investigated in detail by electronic
absorbance titrations, viscosity, cyclic voltammetry and molecu-
lar docking analysis. In addition, all of these metal complexes are
found to promote the photocleavage of pBR322 DNA under irradi-
ation. The singlet oxygen is suggested to be an important factor
responsible for the cleavage. Hence this paper has widened the
scope of developing the o-acetoacetotoluidide derived Schiff base
and its metal complexes as promising metal based antitumor drugs.
These complexes possess good biocidal (antibacterial and antifun-
gal) activity.
3.9.2. Antifungal activity
To provide in the field of bioinorganic chemistry, consequently,
the metal complexes synthesized have been evaluated for their
Table 5
The in vitro antibacterial activity of Schiff base and its metal complexes (zone of
inhibition in mm).
Compound
S.
P.
E. coli S.
epidermidis
K.
aureus
aeruginosa
pneumoniae
[C18
H
19N3O2]
++
+
++
+
+++
++
+++
++
+++
++
Acknowledgments
[CuC36H36N6O4] ++++
[NiC36H36N6O4] ++++
[CoC36H36N6O4] +++
[ZnC36H36N6O4] ++
+++
+++
++
++
++++
+++
+++
+++
+++
+++
++++
+++
++
++
+++
The authors express their sincere thanks to the College Man-
aging Board, Principal and Head of the Department of Chemistry,
VHNSN College, Virudhunagar, India for providing necessary
research facilities. They also wish to acknowledge the help ren-
dered by Thigarajar College, Madurai with regard to computational
facilities. NR thanks University Grants Commission (UGC) for
financial support.
Streptomycin
++++
Streptomycin is used as the standard. DMF was used as antimicrobial inert solvent.
+++++ = Excellent activity (100% inhibition).
+++ = Good activity (60–70% inhibition).
++ = Moderate activity (30–50% inhibition).
+ = Less activity (10–20% inhibition).

N. Raman, S. Sobha / Spectrochimica Acta Part A 93 (2012) 250–259
259
Appendix A. Supplementary data
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