DNA binding propensity and nuclease efficacy of biosensitive Schiff base complexes containing pyrazolone moiety: Synthesis and characterization

Article abstract of DOI:10.1016/j.molstruc.2016.07.003

A series of novel Co(II), Cu(II), Ni(II) and Zn(II) complexes (1–8) were synthesized from pyrazolone precursor Schiff base(s), obtained by the condensation of 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one (4–aminoantipyrine) with cinnamaldehyde/benzalde

Full text of DOI:10.1016/j.molstruc.2016.07.003

Accepted Manuscript
DNA binding propensity and nuclease efficacy of biosensitive Schiff base complexes
containing pyrazolone moiety: Synthesis and characterization
Rajakkani Paulpandiyan, Natarajan Raman
PII:
S0022-2860(16)30674-3
10.1016/j.molstruc.2016.07.003
MOLSTR 22706
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 13 May 2016
Revised Date: 30 June 2016
Accepted Date: 1 July 2016
Please cite this article as: R. Paulpandiyan, N. Raman, DNA binding propensity and nuclease efficacy
of biosensitive Schiff base complexes containing pyrazolone moiety: Synthesis and characterization,
Journal of Molecular Structure (2016), doi: 10.1016/j.molstruc.2016.07.003.
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ACCEPTED MANUSCRIPT
DNA binding propensity and nuclease efficacy of biosensitive Schiff base
complexes containing pyrazolone moiety: Synthesis and characterization
Rajakkani Paulpandiyan and Natarajan Raman*
*Research Department of Chemistry, VHNSN College, Virudhunagar-626 001, India
E-mail: ramchem1964@gmail.com; Tel.: +091-092451-65958; Fax: +091-4562-28133

ACCEPTED MANUSCRIPT
DNA binding propensity and nuclease efficacy of biosensitive Schiff base
complexes containing pyrazolone moiety: Synthesis and characterization
Rajakkani Paulpandiyan and Natarajan Raman*
*Research Department of Chemistry, VHNSN College, Virudhunagar-626 001, India
E-mail: ramchem1964@gmail.com; Tel.: +091-092451-65958; Fax: +091-4562-281338
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ABSTRACT
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A series of novel Co(II), Cu(II), Ni(II) and Zn(II) complexes (1-8) were synthesized from
pyrazolone precursor Schiff base(s), obtained by the condensation of 4-amino-2,3-dimethyl-1-
phenyl-3-pyrazolin-5-one (4–aminoantipyrine) with cinnamaldehyde/benzaldehyde and
respective metal(II) chloride. They have been characterized by elemental analysis, magnetic
susceptibility, molar conductance measurements, UV-Vis., IR, NMR, ESI mass spectra and EPR
studies. These complexes show lower conductance values, supporting their non-electrolytic
nature. Spectroscopic and other analytical data of the complexes suggest octahedral geometry.
The binding properties of these complexes with DNA have been explored by electronic
absorption spectra, cyclic voltammetry and viscosity measurements which reveal that the
complexes have the ability to interact with calf thymus DNA (CT DNA) by intercalative mode.
The binding constant (Kb) values clearly signify that the complex 1 has more intercalating ability
than other complexes. DNA cleavage efficacy of these complexes with pUC18 DNA has been
investigated by gel electrophoresis technique. All the complexes have been found to promote
cleavage of pUC18 DNA from the super coiled form I to the open circular form II in presence of
hydrogen peroxide. The in vitro antibacterial and antifungal assay, investigated by Minimum
Inhibitory Concentration (MIC) method indicates that these complexes are good antimicrobial
agents against various pathogens.
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Keywords: Pyrazolone precursor Schiff base; non-electrolytic nature; octahedral geometry;
DNA cleavage efficacy; supercoiled form; antimicrobial agents.
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1. Introduction
Schiff bases intrigue attention of coordination chemists as versatile spacers due to their
viable accessibilities and structural diversity [1-2]. These are essential class of ligands based on
their synthetic feasibility, selectivity and coordinative ability towards the central metal atom as
well as the structural feature resemble to biological moiety. In particular, any ligand possess
azomethine group (-HC=N-) reveal significance as ‘privileged ligands’. The availability of lone
pair of electrons in sp2 hybridized orbital of nitrogen atom in azomethine group extensively
participate to excel its chelation effect, while exploring via cooperation of one or more donor
centers adjacent to azomethine group. Schiff bases afford to form stable complexes with majority
of transition metal ions and sequester in various oxidation states. Contemporarily, many groups
engaged with the synthesis and characterization of transition metal chelates of pyrazolone
derivatives. Amidst the pyrazolone derivatives, 4-aminoantipyrine occupies a prominent role due
to its importance in biological, pharmacological, clinical and analytical applications [3-6]. It has
been ventured as an analgesic, anti-inflammatory and anti-pyretic agent, later replaced due to its
influence of agranulocytosis side effect which is an acute condition associated with insensitive
and precarious leukopenia (lowered white blood cell count), most commonly the neutrophils
caused by neutropenia in the circulating blood. It leads to the deficiency of major class of
infection-fighting white blood cells. Human beings undergo this condition by experiencing the
risk of infections due to the suppressed immunity [7]. Alternatively, research is initiated with
derivatives of 4-aminoantipyrine to reduce the toxicity of those derivatives as drug candidates.
Recently, 4-aminoantipyrine derivatives are employing to exhibit remarkable properties like
multi-site coordination [8], anti-oxidant [9] anti-putrefactive [10] and optical characteristics [11]
in chemical and material science. On this basis, it is essential to choose 4-aminoantipyrine as a
precursor in this research field.
Since the microbes are developing resistance to antibiotics available in practice, demands
the search for the improved compound with potential effects against pathogenic bacteria. In this
context, impressive progress in medicinal chemistry has been accomplished using aromatic
aldehydes which occupied as critical entity in regulating biological activities. Parekh and co-
workers reported the synthesis of Schiff bases derived from 4-aminobenzoic acid and
cinnamaldehyde [12]. These were identified as potential antibacterial agents against a number of

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medically important bacterial strains. The above results prompted us to choose cinnamaldehyde
and benzaldehyde as reagents for preparing the Schiff bases.
Towards the objective of achieving a promising results from the above substrates, we
have succeeded with the synthesis of two different Schiff base ligands from 4-aminoantipyrine
with cinnamaldehyde/benzaldehyde (L1/L2). These Schiff base ligands are explored to coordinate
with Cu(II), Co(II), Ni(II) and Zn(II) metal ions to afford a series of metal complexes (1-8). Both
free ligands and the corresponding complexes were characterized by physicochemical and
variety of spectroscopic techniques. Ultimately, their collective and functional biological
properties were analyzed by in vitro antimicrobial bustle, DNA binding and DNA cleavage using
gel electrophoresis.
2. Materials and Methods
The chemicals and reagents used were of Analar grade and used as such without further
purification. However, the solvents were purified by the standard procedure. Cinnmaldehyde and
benzaldehyde were procured and used as received from SD fine products. 4- aminoantipyrine
and all other metal salts were supplied by Merck Chemicals.
Elemental analysis (C, H and N) data were carried out in Perkin-Elmyer 240 elemental
analyzer. Vibrational spectral data were collected from FT IR–Shimadzu model IR-Affinity-1
1
spectrophotometer using KBr discs. H NMR and 13C NMR of ligands and their Zn(II)
complexes were recorded with Bruker 400MHz Avance III HD Nanobay NMR spectrometer
using DMSO-d6 as the deuterated solvent. Mass spectrometry experiments were performed in
JEOL-AccuTOF JMS-T100LC mass spectrometer equipped with a custom-made electrospray
interface (ESI). The room temperature molar conductivity of complexes in DMSO solution
(10-3 M) was measured using a deep vision 601 model digital conductivity meter. The X-band
EPR spectrum was recorded at LNT (77 K) and room temperature (300 K). Absorption spectra
were recorded using UV-Vis spectrophotometer, Shimadzu model UV-1601 at room
temperature. Cyclic voltammetric (CV) experiments were conducted in CHI 620C
electrochemical analyzer in freshly distilled DMSO solution.

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2.1 Synthesis
2.1.1 Synthesis of Schiff base ligand (L1/L2)
Schiff bases (L1/L2) were synthesized by adopting the synthetic procedure reported in literature from our
research group [13]. 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one
(4-aminoantipyrine) (2.03 g,
10 mmol) in 40 mL of ethanol was stirred with cinnamaldehyde (1.32 g, 10 mmol, L1) / benzaldehyde
(1.06 g, 10 mmol, L2) in 1:1 molar ratio at ambient temperature by magnetic stirrer for ca. 1 h. The Schiff
base 4-cinnamylimino-2, 3-dimethyl-1-phenyl-3-pyrazal-5-one (L1)/ 4-benzylimino-2,3-dimethyl-1-
phenyl-3-pyrazal-5-one (L2) formed as orange solid, was isolated by filtration and washed with ethanol
followed by an excess of petroleum-ether to eliminate any unreacted reagent. After washing the product
for three times, compound was recrystallized from ethanol to obtain a pure solid and dried over anhydrous
calcium chloride in desiccator under mild vacuum. Thus, the formation of Schiff base ligands L1 and L2 is
displayed in Scheme 1.
(Kindly insert Scheme 1 here)
[L1] Yield: 80%; orange colour; Anal.Calc.for C20H19N3O (%) C (75.6), H (5.8), N (13.0);
Found (%): C (74.1), H (5.1) and N (12.6); FT-IR (KBr) (cm-1): 1595 (-CH=N), 492 (-CH=CH-)
and 1674 (C=O); 1H NMR (DMSO-d6) δ ppm: 7.0-7.6 (m,Ar-H), 2.4 (s,-C-CH3), 3.2 (s,-N-CH3),
6.6 (s,-CH=CH-), 9.6 (s,-CH=N); 13C NMR (DMSO-d6) δ ppm: 125-130 (Ar-C), 135 (-HC=CH),
160 (-CH=N), 152 (C=O) 10.2 (-C-CH3), 35.8 (-N-CH3); UV-Vis in DMSO, cm-1 (transition):
35714 (π-π*) and 29498 (n-π*)
[L2] Yield: 68%; Pale orange colour; Anal.Calc.for C18H17N3O (%) C (74.2), H (5.8), N (14.4);
Found (%): C (73.6), H (5.2) and N (14.0); FT-IR (KBr) (cm-1): 1590 (-CH=N), and 1678(C=O);
1H NMR (DMSO-d6) δ ppm: 7.0-7.6 (m,Ar-H), 2.4 (s,-C-CH3), 3.2 (s,-N-CH3), 9.6 (s,-CH=N);
13C NMR (DMSO-d6) δ ppm: 125-130 (Ar-C), 160 (-CH=N), 152 (C=O) 10.2 (-C-CH3),
35.8 (-N-CH3); UV-Vis in DMSO, cm-1 (transition): 35622 (π-π*) and 28952 (n-π*)
2.1.2 Synthesis of metal complexes
Cu(II), Co(II), Ni(II) and Zn(II) complexes were prepared by the addition of molar
quantity of ligand with respect to stoichiometry of metal salts using the following procedure.
A solution of metal(II) chloride in ethanol (2 mmol) was stirred with an ethanolic solution of
4-cinnamylimino-2,3-dimethyl-1-phenyl-3-pyrazal-5-one
phenyl-3-pyrazal-5-one (L2) (4 mmol) in 1:2 molar ratio, using magnetic stirrer at ambient
(L1)/4-Benzylimino-2,3-dimethyl-1-

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temperature for 30 min and then refluxed for ca. 3 h. Then the volume of the reaction mixture
was reduced to one-third using water bath and cooled. The solid product formed was filtered,
recrystallized from ethanol and dried in vacuo. Schematic route for the synthesis of metal
complexes (1-8) using the above Schiff base ligands (L1/L2) are exemplified in Scheme 2.
(Kindly insert Scheme 2 here)
[CuL1 Cl2] (1): Yield: 65 %; Brown colour; Anal. Calc. for CuC40H38N6O2Cl2 (%): C (62.4),
2
H (4.9), N (10.9) and Cu (8.2); Found (%): C (61.1), H (4.2), N (10.0) and Cu (8.0); FT-IR
(KBr) (cm-1): 1580 (-CH=N), 1654 (C=O), 1492 (-CH=CH-), 365 (M-Cl), 507 (M-O) and
464 (M-N);
14,836 cm-1 (d-d).
(Ω-1mol-1cm2) 14.0; ꢀeff (BM) 1.84; UV-Vis in DMSO, cm-1 (transition):
m
[CoL1 Cl2] (2): Yield: 62%; Green colour; Anal.Calc for CoC40H38N6O2Cl2 (%): C (62.8),
2
H (4.9), N (10.9) and Co (7.7); Found (%): C (62.0), H (4.2), N (10.1) and Co (7.1); FT-IR
(KBr) (cm-1): 1575 (-CH=N), 1645 (-C=O), 1492 (-CH=CH-), 360 (M-Cl), 505 (M-O) and
455 (M-N);
13,247 cm-1 (d-d).
(Ω-1mol-1cm2) 18.0; ꢀeff (BM) 4.65; UV-Vis in DMSO, cm-1(transition):
m
[NiL1 Cl2] (3): Yield: 68%; Brownish yellow colour; Anal.Calc for NiC40H38N6O2Cl2 (%):
2
C (62.8), H (4.9), N (10.9) and Ni (7.6); Found (%): C (62.1), H (4.2), N (10.1) and Ni (7.0);
FT-IR (KBr) (cm-1) 1578 (-CH=N), 1664 (C=O), 1492 (-CH=CH-), 372 (M-Cl), 512 (M-O) and
458 (M-N);
12,826 cm-1 (d-d).
(Ω-1mol-1cm2) 12.0; ꢀeff (BM) 3.12; UV-Vis in DMSO, cm-1 (transition):
m
[ZnL1 Cl2] (4): Yield: 70%; Greenish yellow colour; Anal.Calc for ZnC40H38N6O2Cl2 (%):
2
C (62.3), H (4.9), N (10.9) and Zn (8.5); Found (%) : C (61.5), H (4.2), N (10.2) and Zn (8.0);
FT-IR (KBr) (cm-1): 1580 (-CH=N), 1652 (C=O), 1492 (-CH=CH-), 374 (M-Cl), 515 (M-O) and
1
460 (M-N); H NMR (DMSO-d6) δ ppm: 7.0-7.6 (m, Ar-H), 2.4 (s,-C-CH3), 3.2 (s,-N-CH3),
13
6.6 (s,-CH=CH-), 9.4 (s,-CH=N); C NMR (DMSO-d6) δ ppm: 124-130 (Ar-C), 157 (-CH=N),
144 (C=O), 135 (-HC=CH-), 10.2 (-C-CH3) and 35.8 (-N-CH3);
ꢀeff (BM) diamagnetic; UV-Vis in DMSO, cm-1 (transition): 27,654 (LMCT).
(Ω -1mol-1cm2) 10.0;
m

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[CuL2 Cl2] (5): Yield: 68%; Dark brown colour; Anal. Calc. for CuC36H34N6O2Cl2 (%):
2
C (60.4), H (4.7), N (11.7) and Cu (9.9); Found (%): C (60.1), H (4.2), N (11.0) and Cu (9.2);
FT-IR (KBr) (cm-1): 1576 (-CH=N), 1652 (C=O), 360 (M-Cl), 509 (M-O) and 468 (M-N);
m (Ω-1mol-1cm2) 12.0; ꢀeff (BM) 1.86; UV-Vis in DMSO, cm-1 (transition): 14,928 cm-1 (d-d).
[CoL2 Cl2] (6): Yield: 66%; Green colour; Anal. Calc. for CoC36H34N6O2Cl2 (%): C (60.8),
2
H (4.8), N (11.7) and Co (8.3); Found (%): C (60.2), H (4.2), N (11.1) and Co (8.1); FT-IR
(KBr) (cm-1): 1580 (-CH=N), 1664 (-C=O), 368 (M-Cl), 512 (M-O) and 464 (M-N);
m (Ω-1mol-1cm2) 20.0; ꢀeff (BM) 4.62; UV-Vis in DMSO, cm-1 (transition): 13,456 cm-1 (d-d).
[NiL2 Cl2] (7): Yield: 60%; yellow colour; Anal.Calc for NiC36H34N6O2Cl2 (%): C (60.8),
2
H (4.9), N (11.7) and Ni (8.2); Found (%): C (60.1), H (4.2), N (11.1) and Ni (8.0); FT-IR (KBr)
(cm-1) 1572 (-CH=N), 1678 (C=O), 368 (M-Cl), 510 (M-O) and 452 (M-N);
(Ω-1mol-1cm2)
m
13.0; ꢀeff (BM) 3.35; UV-Vis in DMSO, cm-1 (transition): 12,654 cm-1 (d-d).
[ZnL2 Cl2] (8): Yield: 74%; Pale yellow colour; Anal.Calc for ZnC36H34N6O2Cl2 (%):
2
C (60.1), H (4.7), N (11.7) and Zn (9.1); Found (%) : C (59.5), H (4.2), N (11.2) and Zn (9.0);
FT-IR (KBr) (cm-1): 1570 (-CH=N), 1656 (C=O), 370 (M-Cl), 510 (M-O) and 462 (M-N);
1H NMR (DMSO-d6) δ ppm: 7.0-7.6 (m, Ar-H), 2.4 (s,-C-CH3), 3.2 (s,-N-CH3), 9.4 (s,-CH=N);
13C NMR (DMSO-d6) δ ppm: 124-130 (Ar-C), 157 (-CH=N), 144 (C=O), 10.2 (-C-CH3) and
35.8 (-N-CH3);
(Ω-1mol-1cm2) 16.0; ꢀeff (BM) diamagnetic; UV-Vis in DMSO, cm-1
m
(transition): 27,892 (LMCT).
2.3. DNA binding studies
The DNA-binding experiments were conducted at ambient temperature. Electronic
absorption spectroscopic method has been adopted to investigate the relative bindings of these
complexes to CT DNA in the medium of 5 mM Tris–HCl/NaCl buffer (pH = 7.2). As such the
solution of CT DNA exhibited a ratio of UV absorbance at 260 and 280 nm, A260/A280 = 1.8–1.9,
indicating the existence of DNA, adequately free of protein [14]. The CT DNA stock solution
was prepared in 5 mM Tris–HCl/ 50 mM NaCl buffer (pH = 7.2, stored at 4 ºC and used within 4

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days of preparation). The concentration of CT DNA was determined from its absorption intensity
at 260 nm with a molar extinction coefficient of 6600 M-1 cm-1 [15, 16].
2.3.1. Electronic absorption spectroscopic studies
The electronic absorption spectra of these complexes were recorded before and after the
addition of DNA in the medium of 5 mM Tris-HCl/50 mM NaCl buffer (pH 7.2). The intrinsic
binding constant for the interaction of each complex with DNA was obtained from the absorption
data. The absorption titrations were carried out by the addition of variable amount of DNA into
the solution of the complex (keeping its concentration as constant) taken in a quartz cell. The
titration process was repeated until the disappearance of change in the spectra, suggesting the
binding saturation. The intrinsic binding constant Kb was determined from the plot of
[DNA]/(εa- εf) vs. [DNA] according to equation (3).
[DNA]/ (εa - εf) = [DNA]/ (εb - εf) + 1/[Kb(εb - εf)] ------------ (3)
where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficients εa,
εf and εb correspond to Aobs/[complex], extinction coefficient for the free complex and the
extinction coefficient of the complex in the absolutely bound form respectively. These data were
fitted to Eq. (3), with a slope equals to 1/(εb - εf) and y-intercept equals to 1/[Kb (εb - εf)] and Kb
was obtained from the ratio of the slope to the intercept.
2.3.2. Cyclic voltammetric experiments
Cyclic voltammetry studies were carried out on a CHI 620C electrochemical analyzer
with triple electrode system, glassy carbon as the working electrode, platinum wire as auxiliary
electrode and Ag/AgCl as the reference electrode in the medium of 50 mM NaCl, 5 mM Tris
buffer (pH 7.2). Solution was deoxygenated by purging with N2 for 30 min prior to
measurements.
2.3.3. Viscosity measurements
Viscosity experiments were performed with Ostwald viscometer, immersed in a
thermostatic water-bath maintained at a constant temperature at 30.0 ± 0.1 °C. CT DNA samples
of approximately 0.5 mM were prepared by sonication in order to minimize complexities arising

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from CT DNA flexibility [17]. An average flow time was calculated by counting the flow time
using a digital stopwatch three times for each sample. Data were used to plot as (η/η°)1/3 versus
the concentration of the respective metal(II) complex, where η is the viscosity of CT DNA
solution in the presence of complex and η° is the viscosity of CT DNA solution in the absence of
complex. The relative specific viscosity value was estimated by the equation η = (t-t0)/t0, where
t0 is the flow time for the buffer and t is the observed flow time for DNA in the presence and
absence of metal complex [18].
2.4. Antimicrobial screening
The in vitro antimicrobial bustle of L1, L2 and respective complexes was examined
against certain human sensitive pathogenic Gram-positive bacteria (S.aureus and
B.subtilis), Gram-negative bacteria (K.pneumoniae, S.typhi and E.coli) and fungi (A.niger,
F.solani, C.lunata, R.bataticola and C.albicans) using the dilution method [19]. Nutrients like
agar and dextrose agar were used as the medium for the growth of bacteria and fungi, whereas
kanamycin and fluconazole were chosen as standards for antibacterial and antifungal activity,
respectively. These samples were incubated at 37 °C for 24 h (bacteria) and 48 h (fungi),
respectively. Each data were collected in terms of MIC (Minimum, Inhibitory Concentration).
2.5. DNA cleavage efficacy
Interaction between the ligands and the complexes with pUC18 plasmid DNA was
analyzed by agarose gel electrophoresis. Each sample was incubated for 3 h at 37 °C, before the
addition of bromophenol blue (0. 25 %), xylene cyanol and glycerol (30 %, 3 ꢀL). The DNA
cleavage mixture was subjected to electrophoresis on 1.0 % agarose gel containing 0.3 µM
ethidium bromide (EtBr). The gels were run at 50 V for 1 h in TBE (Tris-Borate EDTA) buffer
using 1% agarose gel stained with ethidium bromide (1 ꢀg) and the bands were photographed.
3. Results and discussion
A series of metal complexes were synthesized and characterized by various
physicochemical studies and samples were identified to be stable in air and moisture. These
complexes have been characterized by the micro analytical data, FT-IR, UV-Vis, NMR, EPR and
mass spectra. These complexes are readily soluble in DMF and DMSO. All the analytical data of

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these complexes suggest the proposed general formula [ML1 Cl2]/ [ML2 Cl2]. Their magnetic
2
2
susceptibility values of the complexes at room temperature are consistent with octahedral
geometry around the central metal ion. The low conductance (10-20 ohm−1 cm−2 mol−1) of the
complexes supports their non-electrolytic nature.
3.1. FT-IR spectroscopy
The coordination mode and sites of the ligands to the metal ions were investigated
by comparing the infrared spectra of the free ligands (L1 and L2) with their metal complexes. The
IR spectra of L1 and L2 and their complexes have been recorded in the region 350-4000 cm-1.
The spectra of free ligands show a band in the region 1595-1600 cm-1, a characteristic feature of
the υ(CH=N) (azomethine) stretching mode. This band is shifted towards lower frequency in the
spectra of metal complexes 1560-1580 cm-1 indicating the involvement of the imine nitrogen in
chelation with the metal ion and the lowering of this wave number may be attributed to the
decrease in electron density on the nitrogen atom of the azomethine group [20]. Coupled with
this, free ligands also reveal a band in the region 1674-1680 cm-1 allocated to υ(C=O) stretching
mode which is shifted to lower frequency around 1645-1664 cm-1 indicating the coordination of
the carbonyl oxygen atom of the ligands to metal ion. The coordination of the azomethine
nitrogen and carbonyl oxygen is further supported by the appearance of new bands around
505-515 cm-1 and 452-464 cm-1 which are due to υ(M–O) and υ(M–N) respectively. Furthermore, the
IR spectra of all the complexes show an additional band in the region 360 - 374 cm-1, which
could be perceived due to υ(M-Cl) [21]. The IR spectra of L1 and its Cu(II) complex (1) are shown
in Fig.S1.
3.2. Electronic spectra and magnetic moments
The free ligands exhibited two intense bands in 35,622–35,714 and 28,952-29,498 cm–1
regions due to π → π* and n → π* transitions, respectively [22]. In case of metal complexes,
these transitions are shifted to longer wavelengths due to the coordination of the ligand with
metal ion. The electronic spectra of Cu(II) complexes (1 and 5) displayed d–d transition bands in
2
the region 14,836-14,928 cm–1 which can be assigned to 2Eg → T2g transition, characteristic of d9
distorted octahedral geometry [23]. The observed magnetic moment of the Cu(II) complexes
(1.84-1.86 BM) at room temperature specify the non-coupled mononuclear complexes of

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magnetically diluted d9 system with s = 1/2 spin state of distorted octahedral geometry [24]. In
Co(II) complexes (2 and 6), this d-d band emerges at the region 13,247-13,456 cm–1 because of
4
4T1g(F) → T2g(F) transition which can be the evidence for octahedral geometry of d7 Co(II)
system. The observed magnetic moment values of these complexes (4.62-4.65 BM) indicate the
presence of three unpaired electrons, which suggests an octahedral geometry. The Ni(II)
complexes (3 and 7) reveal d-d bands in the region 12,654-12,826 cm–1, assigned to
3
3A2g(F) → T1g(F) transition. The examined magnetic moment values of these complexes
(3.12-3.35 BM) indicate the presence of two unpaired electrons and octahedral geometry is
assigned [25]. In contrast, Zn(II) complexes (4 and 8) do not exhibit any d-d band because of
their completely filled d10 configuration. The electronic absorption spectra of L1 and its Cu(II)
complex are depicted in Fig.S2.
1
3.3. H and 13C NMR spectra
1
Both H and 13C NMR spectra assist to characterize the structure of the prepared
ligands and their diamagnetic metal complexes. 1H NMR spectra of free Schiff base ligands and
1
their Zn(II) complexes have been recorded in DMSO-d6. The H NMR spectrum of L1 shows
peaks at 7.0–7.6 ppm (m) and 6.6 ppm (s), assigned to the phenyl ring protons and (-CH=CH-)
of cinnamaldehyde unit respectively. L1/L2 also shows the following signals: C-CH3 2.4 ppm
(s), N-CH3 3.2 ppm (s) and azomethine proton (-CH=N) signal at 9.6 ppm (s). The azomethine
proton (-CH=N) signal in zinc(II) complexes (4 and 8) are found to shift to upfield at 9.4 ppm
(s), compared to the free ligands, indicative of the shielding of azomethine group involved in
coordination with metal ion. There is no significant change noticed with the remaining signals of
complexes, 4 and 8. The 1H NMR spectra recorded for the Schiff base ligand (L1) and its Zn (II)
complex (4) are shown in Fig.S3.
The 13C NMR spectra of free ligands show aromatic carbon peaks in the range of
125-130 ppm. Moreover, the carbon signals of azomethine (-HC=N) and carbonyl (C=O) groups
are observed at 160 and 152 ppm for the free ligands. In case of Zn(II) complexes (4 and 8), it is
found that the above peaks are shifted to upfield region, 157 ppm and 144 ppm respectively,
supporting the coordination of (-HC=N) and (C=O) groups to the metal center. There is no much
difference in other signals of the complexes. The 13C NMR spectra of the Schiff base ligand (L1)
and its Zn(II) complex (4) are given in Fig.S4.

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3.4. EPR spectra
EPR data of the paramagnetic Cu(II) complex provide evidence on distribution of the
unpaired electrons, apparently elucidate the nature of bonding between the metal ion with its
ligand. EPR spectra of Cu(II) complexes, dissolved in DMSO were recorded at liquid nitrogen
temperature (LNT) and at room temperature (RT). The EPR spectrum of complex 1 is depicted
in Fig.S5.
The EPR spectra of Cu(II) complexes (1 and 5) exhibit parameters like axially symmetric
g-tensor with g׀׀ (2.13-2.32) > g⊥ (2.03-2.06) > ge (2.0023); A|| (138-152) > A (58-85) indicating
2
that the copper site possesses dx -y2 ground state, characteristic of an octahedral geometry [26].
The geometric parameter G refers to a measure of the exchange interaction among the multiple
copper centers in the polycrystalline compound. It is calculated by the following equation:
G = (g׀׀− 2) / (g⊥ −2) ……………………….(3)
As per earlier report, if value of G > 4.0, the local tetragonal axes exist in parallel
alignment or slightly misaligned. In case value of G < 4.0, significant exchange coupling occurs
and there is significant deviation. While the observed values for the exchange interaction
parameter of
Cu(II) complexes fall within the range of 4.3-5.3 G which suggests that the
local tetragonal axes are aligned in parallel or slightly deviated and the unpaired electron
2
occupies in the dx - y2 orbital. At this state, it infers the absence of exchange coupling among the
Cu(II) centers in solid state [27].
The empirical ratio of g|| /A|| is often used to detect the distortion in Cu(II) complexes. If
this ratio is close to 100, it indicates approximately a square-planar assembly around the Cu(II)
ion and if the values around 170 – 250 which support the distorted tetrahedral geometry. In case
if the ratio is existing within 110-170, it specifies almost an octahedral coordination around
Cu(II) ion with minor distortion [28]. For the synthesized Cu(II) complexes, the values of g|| /A||
are found to be 152 cm–1 (1) and 154 cm–1 (5) which infer that Cu(II) complexes reveal the
distorted octahedral geometry. The calculated spin Hamiltonian parameters for the Cu(II)
complexes are listed in Table 1.
(Kindly insert Table 1 here)

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3.5. Mass spectra
The electrospray ionization - mass spectra (ESI-MS) were recorded at room temperature
for the synthesized Schiff base ligands and the corresponding metal complexes. The molecular
ion peak and fragmentation observed in ESI-MS support the proposed formulae for the
synthesized metal complexes. ESI-MS of the free ligand (L1) and complex 1 are depicted in
Fig.S6. The mass spectrum of L1 shows the molecular ion peak at m/z 317 corresponding to
[C20H19N3O]+, in addition to the fragmentation peaks at m/z 214, 187, 103, 77 and 65 which are
attributed to the species, [C12H12N3O]+, [C11H11N2O]+, [C8H7]+, [C6H5]+ and [C5H5]+
respectively. The mass spectrum of complex 1 shows molecular ion peaks at m/z 770 [M+1],
which corresponds to its molecular weight. The fragmentation of [CuC40H38N6O2Cl2]+1 leads to
the elimination of two chloride ions followed by the demetallation based on evidence of m/z
values, 699 and 606 with respect to the fragmented ions [CuC40H38N6O2]+ and [C40H38N6O2]+
respectively. The intense peak (base peak) at m/z 317 represents the stable species [C20H19N3O]+
from the ligand moiety. Moreover, the fragmentation peaks in the spectrum at m/z 214, 187 and
103 support the existence of [C12H12N3O]+, [C11H11N2O]+ and [C8H7]+ respectively. Also, m/z
values of all the fragments of L1 and its copper(II) complex determine the stoichiometry of the
complexes which is found to be [ML1 Cl2]. Thus, all the observed peaks reveal a good agreement
2
with the formula estimated from micro analytical data. Altogether, ESI-MS data support the
conclusion drawn from the analytical and conductance values.
3.6. DNA binding studies
3.6.1 Absorption spectral titration
Absorption titration is universally employed to monitor the binding mode and propensity
of drugs with nucleic acid [29]. The change in the absorbance and shift in the wavelength at
UV-visible region due to the addition of calf thymus DNA (CT DNA) to metal complex solution
indicates the nature of interaction of the complexes with DNA. Thus the absorption spectra of
these complexes in the absence or presence of CT DNA at different concentrations were
measured. ‘‘Hyperchromic effect’’ and ‘‘hypochromic effect’’ are two types of spectral features
of DNA, concerning its double-helix structure. This effect of spectral feature reveals the
respective alternation of DNA in its conformation and structure while the drug binds to DNA.
Hypochromism results from the contraction of DNA in the helix axis as well as from the change

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in conformation on DNA, while hyperchromism results from the damage of the DNA double
helix structure [30]. With the increase of [DNA], the absorption intensity of the complex
decreases (hypochromic effect) and the λmax values shift to red region (bathochromic shift) [31].
This occurs while the binding induces a strong stacking interaction between the planar aromatic
chromophoric groups of the ligand with DNA base pairs.
The absorption spectra of all the complexes showed an intensive absorption band in the
region 336-348 nm, in 5 mM Tris-HCl/50 mM NaCl (pH 7.2) buffer solution. The observed
hypochromicity values in the presence of DNA were in the range 10.2–15.8 % for MLCT band
and their red shifts were observed in the region 3.0-8.0 nm which indicates the binding of DNA
with the complexes is through intercalative mode [32]. The absorption spectra of the complexes
1 and 3 in the absence and presence of CT DNA in the UV region are depicted in Fig.1.
(Kindly insert Fig.1 here)
The intrinsic binding constant Kb values calculated from the slope to the intercept ratio
from the plots between [DNA]/ (εb- ε f) and [DNA] are given in Table 2 and used to gather the
information concerning the binding affinity of the complexes with CT DNA. The binding
strength of the complexes is shown as in the following order: 1>3>4>2>5>6>8>7>. Complex 1
reveals strong hypochromism and a slight red shift as compared to other complexes highlighting
its higher DNA binding propensity. These results imply that the intercalative ligands with
extended aromatic plane and good conjugation effect can greatly support the DNA binding
facility. The determined intrinsic binding constants for all the complexes are recline within the
range 1.2-8.6 × 104 M-1. Comparing the values of Kb of the metal complexes with the classical
intercalators, these complexes have lesser binding affinity than the classical intercalators such as
EB [33] and higher than those of 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.
(Kindly insert Table 2 here)
3.6.2 Viscosity measurements
Hydrodynamic method, such as determination of viscosity, which is exquisitely sensitive
to the change of length of DNA, may be the most effective means studying the binding mode of
complexes to DNA in the absence of X-ray crystallographic data [35]. The relative specific
viscosity of DNA is determined by varying the concentration of the added metal complexes.

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Measuring the viscosity of DNA is a classical technique used to analyze the DNA binding mode
in solution. Under appropriate conditions, intercalation of drugs such as ethidium bromide [EB]
causes a significant increase in the viscosity of a DNA solution due to the increase in the
separation of the base pairs of the intercalation sites and hence, results in an increase in the
overall DNA contour length [36], as shown in Fig.2. In contrast, partial or non-classical
intercalation of 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].
(Kindly insert Fig.2 here)
Viscosity measurements results clearly show that all the complexes can intercalate
between adjacent DNA base pairs, causing an extension in the helix thereby increasing the
viscosity of DNA. Increasing tendency in the viscosity curve upon addition of various
concentrations of complex to DNA provided stronger evidence for the intercalation and further
verified intercalative mode of binding as investigated through spectroscopic and electrochemical
studies in the present work.
3.6.3 Electrochemical studies
Electrochemical investigation of drug–DNA interactions can provide a useful
complement to other methods and yield information about the mechanism of interaction and the
conformation of adduct [38]. In general, the electrochemical potential of a small molecule will
shift positively while it intercalates with DNA double helix, and it will shift to negative
direction, if the molecule is bound to DNA by groove binding [39]. In the present study, cyclic
voltammetry technique has been used to find out the nature of DNA binding mode of the metal
complex. The cyclic voltammograms of the complexes 1 and 4 in the absence and presence of
varying amount of DNA are shown in Fig.3.
(Kindly insert Fig. 3 here)
The cyclic voltammogram of each complex shows approximately close to unity for peak
current ratio, which is responsible for the reaction of the complex on the glassy carbon as the
quasi-reversible redox process. The incremental addition of CT DNA to the complex causes
decrease in anodic and cathodic peak current of the complex. This result shows that complex
stabilizes the duplex (GC pairs) as an intercalating manner. The drop of the voltammetric current
in the presence of CT DNA is due to slow diffusion of the copper complex bound to CT DNA.

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The formal potential, ̿1/2, taken as the average of Ep͗ and Ep͕ shifts slightly towards the
positive side on binding to DNA which suggests that copper complex binds intercalative to
CT DNA [40]. The quasi- reversible redox couple for each complex in DMSO solution has been
analyzed upon addition of CT DNA. The E1/2 and ∆Ep values are given in Table 3.
(Kindly insert Table 3 here)
3.7. Antimicrobial screening
The in vitro antimicrobial activity of the synthesized free ligands and their metal
complexes were tested against perceptive organisms such as two Gram-positive bacteria
(Staphylococcus aureus and Bacillus subtilis) and three Gram-negative bacteria (Escherichia
coli, Klebsiella pneumoniae and Salmonella typhi) and five fungi (Aspergillus niger, Fusarium
solani, Curvularia lunata, Rhizoctonia bataticola and Candida albicans) by well dilution
method. Kanamycin and fluconazole were used as standards for bacteria and fungi respectively.
The minimum inhibitory concentration (MIC) values, measured in antibacterial and antifungal
studies of the complexes are given in Tables 4 and 5.
(Kindly insert Tables 4 and 5 here)
It is observed that the activity of imine ligand is enhanced when coordinated to the metal
ions. The presence of imine group (-C=N) and chelation effect with central metal enhance the
antimicrobial action [41]. Moreover, it has been already proved that the ligand containing
nitrogen and oxygen donor systems might restrain enzyme production, since the enzymes which
require these groups for their activity appear to be especially more susceptible to deactivation by
the metal ion upon chelation. The presence of nitrogen and oxygen atoms may also be the higher
biocidal actions of the metal complexes. This enhancement of antimicrobial activity of these
metal complexes may be explained by Overtone concept and Tweedy′s chelation theory [42].
According to the Overtones concept, chelation reduces the polarity of the metal ion mainly
because of partial sharing of its positive charge with donor groups within the whole chelating
system. This process of chelation thus increases the lipophilic nature of the central metal atom,
which in turn, favors its permeation through the lipoid layer of the membrane thus causing the
metal complex to cross the bacterial membrane more effectively, which in turn increases the
activity of the complexes. These complexes also disturb the respiration process of the cell and
thus block the synthesis of proteins, which restricts further growth of organisms [43]. The

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complex 1 showed highest antimicrobial activity among all the other complexes. The variation in
the effectiveness of different compounds against different organisms depends either on the
impermeability of the cells of the microbes or on differences in ribosome of microbial cells [44].
Besides this, many other factors such as solubility, dipole moment and conductivity of
complexes may be the reason for remarkable antimicrobial activities of the complexes.
3.8. DNA cleavage study
DNA cleavage is controlled by the relaxation of supercoiled circular form of pUC18
DNA into nicked circular and linear forms. When circular plasmid DNA in the presence of metal
complex is subjected to electrophoresis, relatively fast migration will be viewed for the intact
supercoiled form (Form I). If scission occurs on one strand, the super coil will relax to turn out a
slower moving open circular form (Form II). If both the strands are cleaved, a linear form
(Form III) that migrates in between Form I and II will be generated [45].
The DNA cleavage ability of the complexes (1-4) has been investigated by gel
electrophoresis using supercoiled pUC18 DNA in TBE buffer in the presence of H2O2 at 35°C
for 5h. As seen from Fig.4, the control experiments with DNA alone (lane 1) and DNA in
presence of L1 (lane 2) do not show any cleavage activity. Obviously the complexes (1-4) are
found to exhibit nuclease activity. Thus the cleavage of super coiled form-I to the open circular
form-II is induced by all the metal complexes, in which the complexes 1, 3 and 4 exhibit
prominent cleavage activity than complex 2. The cleavage mechanism is explained as follows.
When the redox-active metal complexes interact with biomolecules in the presence of H2O2 as an
oxidizing agent, it is believed to produce different oxygen intermediates (reactive oxygen
species) depending on different metal complexes and conditions. The metal complexes in the
presence of H2O2 may produce reactive hydroxyl radical (OH*) that can cleave the deoxyribose
moiety, followed by hydrolytic cleavage of a sugar phosphate back bone [46]. It may also be
concluded that complexes which cleave the DNA may also inhibit the growth of the pathogenic
organism.
(Kindly insert Fig.4 here)

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Conclusion
Synthesis and characterization of a new set of pyrazolone precursor Schiff bases and a
series of transition metal(II) complexes of Cu, Zn, Co and Ni are reported as potential candidates
to study their DNA binding/cleavage and antimicrobial effects. The physicochemical and
spectral data infer that all these metal chelates (1-8) exist as mononuclear and adopted octahedral
geometry around the metal center. DNA binding properties of all the above complexes were
studied by electronic absorption spectra, cyclic voltammetry and viscosity measurement, which
deduce their association as intercalative DNA interaction with different binding affinities. The
binding strength of these complexes with DNA varies by the following order,
1>3>4>2>5>6>8>7. Gel electrophoresis experiments infer that DNA cleavage is enhanced to
significant extent in the presence of hydrogen peroxide. Active oxygen intermediate like
hydroxyl radicals generated from hydrogen peroxide upon cleavage mechanism could play an
important role. Compared to antibacterial and antifungal activities of the free ligands, their
complexes show efficient biocidal and fungicidal activity. Based on the summary of results,
promising candidates are identified for further investigation towards the direction of exploring
new and potential drug for cancer therapeutics.
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. IIT Bombay (SAIF) and IIT Chennai, India are gratefully
acknowledged for providing instrumental facilities.

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Figure captions
Scheme 1. The formation of Schiff base ligands L1 and L2
Scheme 2. Schematic route for the synthesis of metal complexes using Schiff base ligands
(L1/L2)
Fig.1 The absorption spectra of the complexes (1) and (3) in buffer pH =7.2 at 250C in presence
of increasing amount of DNA. Arrow indicates the changes in absorbance upon increasing the
DNA concentration.
Fig.2 Effect of increasing amount of [EB] ( ), Complexes 1 (♦), 2 (■), 3(▲) and 4 (×) on the
*
relative viscosity of CT DNA. Plot of relative viscosity (η/ηo)1/3 vs [Complex]/[DNA].
Fig.3 The cyclic voltammograms of the complexes 1 and 4 in buffer (pH = 7.2) at 250C in
presence of increasing amount of DNA.
Fig.4 Gel electrophoresis pattern showing cleavage of pUC18 DNA treated with metal
complexes. Lane I: DNA control; Lane II: DNA + L1+ H2O2; Lane III: DNA + 1 + H2O2; Lane
IV: DNA + 2 + H2O2; Lane V: DNA + 3 + H2O2 and Lane VI: DNA + 4 + H2O2.

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Scheme 1. The formation of Schiff base ligands L1 and L2

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Scheme 2. Schematic route for the synthesis of metal complexes using Schiff base ligands
(L1/L2)

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Fig.1 The absorption spectra of the complexes (1) and (3) in buffer pH =7.2 at 250C in presence
of increasing amount of DNA. Arrow indicates the changes in absorbance upon increasing the
DNA concentration.

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Fig.2 Effect of increasing amount of [EB] ( ), Complexes 1 (♦), 2 (■), 3(▲) and 4 (×) on the
*
relative viscosity of CT DNA. Plot of relative viscosity (η/ηo)1/3 vs [Complex]/[DNA].

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Fig.3 The cyclic voltammograms of the complexes 1 and 4 in buffer (pH = 7.2) at 250C in
presence of increasing amount of DNA.

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Fig.4 Gel electrophoresis pattern showing cleavage of pUC18 DNA treated with metal
complexes. Lane I: DNA control; Lane II: DNA + L1+ H2O2; Lane III: DNA + 1 + H2O2; Lane
IV: DNA + 2 + H2O2; Lane V: DNA + 3 + H2O2 and Lane VI: DNA + 4 + H2O2.

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Table Captions
Table 1. The spin Hamiltonian parameters of the Cu(II) complexes (1 and 5) in DMSO solution
at LNT.
Table 2. Electronic absorption parameters for the interaction of DNA with synthesized metal
complexes.
Table 3. Electrochemical parameters for the interaction of DNA with synthesized metal
complexes.
Table 4. Minimum inhibitory concentration of the synthesized free ligands and metal complexes
against the growth of bacteria (ꢀM).
Table 5. Minimum inhibitory concentration of the synthesized free ligands and metal complexes
against the growth of fungi (ꢀM).