DNA-binding, oxidative DNA cleavage, and coordination mode of later 3d transition metal complexes of a Schiff base derived from isatin as antimicrobial agents

Article abstract of DOI:10.1080/00958972.2011.634005

A Schiff base, obtained by the condensation of isatin monohydrazone with 2,3,5- trichlorobenzaldehyde, and its Co(II), Ni(II), Cu(II), and Zn(II) complexes have been synthesized and characterized. The interaction of these complexes with DNA is investigate

Full text of DOI:10.1080/00958972.2011.634005

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DNA-binding, oxidative DNA cleavage,
and coordination mode of later 3d
transition metal complexes of a
Schiff base derived from isatin as
antimicrobial agents
N. Raman a , K. Pothiraj a & T. Baskaran a
a Research Department of Chemistry , VHNSN College ,
Virudhunagar – 626001, India
Published online: 08 Nov 2011.
To cite this article: N. Raman , K. Pothiraj & T. Baskaran (2011) DNA-binding, oxidative DNA
cleavage, and coordination mode of later 3d transition metal complexes of a Schiff base derived
from isatin as antimicrobial agents, Journal of Coordination Chemistry, 64:22, 3900-3917, DOI:
10.1080/00958972.2011.634005
To link to this article: http://dx.doi.org/10.1080/00958972.2011.634005
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Journal of Coordination Chemistry
Vol. 64, No. 22, 20 November 2011, 3900–3917
DNA-binding, oxidative DNA cleavage, and coordination
mode of later 3d transition metal complexes of a Schiff base
derived from isatin as antimicrobial agents
N. RAMAN*, K. POTHIRAJ and T. BASKARAN
Research Department of Chemistry, VHNSN College, Virudhunagar – 626001, India
(Received 5 July 2011; in final form 11 October 2011)
A
Schiff base, obtained by the condensation of isatin monohydrazone with 2,3,5-
trichlorobenzaldehyde, and its Co(II), Ni(II), Cu(II), and Zn(II) complexes have been
synthesized and characterized. The interaction of these complexes with DNA is investigated
using viscosity, absorption titration, and electrochemical techniques. The results indicate that
the complexes bind to Calf thymus DNA through intercalation. Oxidative cleavage activities of
the complexes are studied using supercoiled pBR322 DNA by gel electrophoresis.
Antimicrobial study reveals that copper and zinc complexes are better antimicrobial agents
than the Schiff base and its other complexes.
Keywords: Schiff base; Metal complexes; DNA cleavage; Metal–DNA interaction;
Antimicrobial studies
1. Introduction
Various hydrazones which have applications such as biologically active compounds and
analytical reagents are obtained depending on the experimental conditions. In
analytical chemistry, hydrazones find application in detection, determination, and
isolation of compounds containing the carbonyl group [1]. As biologically active
compounds, hydrazones find applications in the treatment of diseases, such as
antitumor, tuberculosis, leprosy, and mental disorder [2]. Tuberculostatic activity is
attributed to the formation of stable chelates with transition metals present in the cell.
Recently, a number of attempts have been made to obtain Mn(III), Fe(III), and Co(III)
complexes with the bishydrazone formed by the condensation of isatin monohydrazone
with 2-hydroxy-1-naphthaldehyde [3].
Isatin, an endogenous indole and its derivatives exhibit a wide range of biological
activities [4]. Isatin-based Schiff base copper(II) complex is related to the antiviral drug,
methisazone. Significant interest in the design of metal compounds such as drugs and
diagnostic agents is termed medicinal inorganic chemistry [5, 6]. Application of
electroanalytical techniques includes determination of reaction mechanisms.
*Corresponding author. Email: drn_raman@yahoo.co.in
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ß 2011 Taylor & Francis
http://dx.doi.org/10.1080/00958972.2011.634005

Schiff base derived from isatin
3901
Redox properties of a drug can give insights into its metabolic fate or pharmaceutical
activity [7–9].
Interest in preparation of new metal complexes for interaction with DNA has arisen
for applications in biotechnology and medicine. Metal complexes bind to DNA by non-
covalent interactions such as electrostatic binding, groove binding, and intercalation
[10–12]. Among these interactions, intercalation is an important DNA-binding mode,
related to the antitumor activity of the compound [13]. The intercalating ability of the
complex depends on the planarity of ligands, the coordination geometry, ligand donor
type, and the metal ion type [14]. Copper is a biologically relevant element and many
enzymes that depend on copper for their activity have been identified. Because of its
biological relevance, a large number of copper(II) complexes have been explored for
their biological activities [15, 16]. Many techniques have been applied for investigation
of biological efficiencies of metal complexes. Compounds having ability to bind and
cleave double stranded DNA under physiological conditions are of importance for their
utility as diagnostic agents in medicinal applications and for genomic research [17].
DNA cleavage reactions generally proceed via oxidative or hydrolytic cleavage
pathways. The hydrolytic pathway involves phosphodiester bond hydrolysis leading
to the formation of fragments that could be relegated through enzymatic processes. The
oxidative process results in nucleobase oxidation and/or degradation of sugar by
abstraction of sugar hydrogen atoms [18].
Although much attention has been directed to metal complexes of the Schiff base
derived from isatin [19], no investigations have appeared describing metal complexes of
the Schiff base derived from isatin monohydrazone and 2,3,5-trichlorobenzaldehyde.
The free ligand and its complexes have been tested for in vitro antimicrobial activity
against four different bacteria and four different fungi by minimum inhibitory
concentration (MIC). The interaction of the complexes with DNA and the evaluation of
the binding constants have been explored. The probable binding mode of complexes
with DNA and their DNA-damaging properties have been deduced.
2. Experimental
2.1. Reagents and equipments
All chemicals were purchased from commercial sources and used as received without
purification. The isatin monohydrazone was prepared according to the literature
procedure [20]. Supercoiled plasmid (SC) pBR322 DNA and Tris-HCl buffer were
purchased from Bangalore Genei (India). Calf thymus DNA (CT-DNA), agarose
(molecular biology grade), and ethidium bromide (EB) were obtained from Sigma
(USA). Tris-HCl buffer was prepared using deionized and sonicated triply distilled
water. Solvents used for electrochemical and spectral measurements were purified by
reported procedures [21].
Carbon, hydrogen, and nitrogen analyses of the ligand and complexes were carried
out on a CHN analyzer Carlo Erba 1108, Heraeus. Infrared spectra (4000–400 cmꢀ1
KBr discs) of the samples were recorded on a Perkin-Elmer 783 series FT-IR
spectrophotometer. The electronic absorbance spectra from 200 to 1100 nm were
1
recorded on an UV-1601 spectrophotometer (Shimadzu). H NMR spectra of ligand

3902
N. Raman et al.
and its zinc complex (300 MHz) were recorded on a Bruker Avance DRX-300 FT-NMR
spectrometer using CDCl3 and DMSO-d6 as solvents, respectively. Tetramethylsilane
was used as internal standard. Chemical shifts were reported in ꢀ scale. EPR spectra of
complex in solid state at 300 K and in frozen DMSO at 77 K were recorded on a Varian
E-112 spectrometer at X-band, using TCNE as marker with 100 kHz modulation
frequency and 9.1 GHz microwave frequency. Mass spectrometry experiments were
performed on a JEOL-AccuTOF JMS-T100LC mass spectrometer equipped with a
custom-made electrospray interface (ESI). Molar conductances of 10ꢀ3 molLꢀ1 solution
of the complexes in DMF were measured at room temperature with a Deep Vision
Model-601 digital direct reading deluxe conductivity meter. Magnetic susceptibility
measurements were carried out by employing the Gouy method at room temperature on
powder sample of the complex. CuSO4 ꢁ 5H2O was used as calibrant. The metal contents
of the complexes were determined according to the literature method [22].
2.2. DNA-binding and cleavage studies
2.2.1. Electronic absorption spectral studies. DNA-binding experiments were per-
formed in Tris-HCl/NaCl buffer (5 mmol Lꢀ1 Tris–HCl/50 mmol Lꢀ1 NaCl buffer, pH
7.2) using DMF (10%) solution of metal complexes. The concentration of CT-DNA
was determined from the absorption intensity at 260 nm with a " value of 6600
(mol L )
ꢀ1 ꢀ1 cmꢀ1 [23]. Absorption titration experiments were made using different
concentrations of CT-DNA, while keeping the complex concentration constant.
Correction was made for the absorbance of the CT-DNA itself. Samples were
equilibrated before recording each spectrum. For metal complexes, the intrinsic binding
constant (Kb) was determined from the spectral titration data using the following
equation [24]:
½DNAꢂ=ð"a ꢀ "fÞ ¼ ½DNAꢂ=ð"b ꢀ "fÞ þ 1=Kbð"b ꢀ "fÞ,
where [DNA] is the concentration of DNA in base pairs, the apparent absorption
coefficients "a, "f, and "b correspond to Aobsd/[Complex], the extinction coefficient of
the free complex and the extinction coefficient of the complex when fully bound to
DNA, respectively. Kb is the equilibrium binding constant in (mol L )
ꢀ1 ꢀ1. Each sample
solution was scanned from 200 to 500 nm.
2.2.2. Electrochemical studies. Cyclic voltammetry was carried out on a CH instru-
ment electrochemical analyzer. All voltammetric experiments were performed in a single
compartment cell of volume 10–15 mL containing a three electrode system comprising a
glassy carbon working electrode, Pt-wire as auxiliary electrode, and an Ag/AgCl
electrode as reference electrode. The supporting electrolyte was 5 mmol Lꢀ1 Tris-HCl/
50 mmol Lꢀ1 NaCl buffer (pH 7.2). Deaerated solutions were used by purging N2 gas
for 15 min prior to measurements.
2.2.3. Viscosity measurements. Viscosity experiments were conducted on an
Ubbelodhe viscometer, immersed in a thermostated water-bath maintained at 37ꢃC.
DNA samples with an approximate average length of 200 base pairs were prepared by

Schiff base derived from isatin
3903
sonication in order to minimize complexities arising from DNA flexibility [25].
Titrations were performed for all four complexes (2–12 mmol Lꢀ1), and each complex
was introduced into the CT-DNA solution (10 ꢄ 10ꢀ5 mol Lꢀ1) present in the
viscometer. Data were presented as (ꢁ/ꢁ0)1/3 versus the ratio of the concentration of
the complex to CT-DNA, where ꢁ is the viscosity of CT-DNA in the presence of the
complex, and ꢁ0 is the viscosity of CT-DNA alone. Viscosity values were calculated
from the observed flow time of CT-DNA containing solutions corrected from the flow
time of buffer alone (t0), ꢁ ¼ (t ꢀ t0)/t0.
2.2.4. Cleavage of pBR322 DNA. The DNA cleavage activity of the complexes was
studied by using agarose gel electrophoresis. SC pBR322 DNA (0.3 mg), dissolved in
5 mmol Lꢀ1 Tris-HCl/50 mmol Lꢀ1 NaCl buffer (pH 7.2), was treated with the
complexes. The mixture was incubated at 37ꢃC for 24 h and then mixed with the
loading buffer (2 mL) containing 25% bromophenol blue, 0.25% xylene cyanol, and
30% glycerol. Each sample (5 mL) was loaded into 0.8%(w/v) agarose gel.
Electrophoresis was undertaken for 2 h at 50 V in Tris-acetate-EDTA (TAE) buffer
(pH 8.0). The gel was stained with EB for 5 min after electrophoresis, and then
photographed under UV light. To enhance the DNA cleaving ability by the complexes,
hydrogen peroxide (100 mmol Lꢀ1) was added into each sample. The cleavage
mechanism was further investigated by using the hydroxyl radical scavenger
ethanol (4 mL) and the superoxide anion radical scavenger, superoxide
dismutase (SOD, 4Units). All experiments were carried out in triplicate under the
same conditions.
2.3. Antimicrobial analyses
The synthesized ligand and corresponding metal(II) complexes were screened in vitro
for their antimicrobial activity against Staphylococcus aureus, Escherichia coli, Bacillus
subtilis, and Pseudomonas aeruginosa bacterial strains and Aspergillus niger, Rhizopus
stolonifer, Rhizoctonia bataicola, and Candida albicans fungal strains using the agar well
diffusion method [26]. The complexes were stored dry at room temperature and
dissolved in DMF (10 mgmLꢀ1). Each bacterium was suspended and diluted to
ca 106 cfu mLꢀ1. They were flood inoculated onto the surface of Mueller–Hinton agar
(MHA). For fungi, dilution was prepared as 106 cfu mLꢀ1 and they were flood
inoculated onto the surface of potato dextrose agar (PDA). The wells (7.5 mm in
diameter) were cut from the agar and 100 mL of each stock complex solution was
delivered into them. The plates were then incubated at 37ꢃC for 24 h and 48 h for
bacteria and fungi, respectively. Streptomycin and nystatin were used as standards for
antibacterial and antifungal drugs, respectively. Inhibitory activity of DMF was also
tested in the same way. The antimicrobial activities of the complexes were evaluated by
measuring the inhibition-zone diameter observed. MIC was determined by the broth
dilution technique according to the National Committee for Clinical Laboratory
Standard [27]. The lowest concentration (highest dilution) required to arrest the growth
of bacteria was regarded as MIC.

3904
N. Raman et al.
2.4. Preparation of Schiff base (L)
The synthesis of L is schematically presented in scheme 1. The Schiff base has been
synthesized by refluxing hot methanolic solution (50 mL) of 2,3,5-trichlorobenzalde-
hyde (2.09 g, 10 mmol) and hot methanolic solution (40 mL) of isatin monohydrazone
Scheme 1. The synthesis of the Schiff base ligand and its metal(II) complexes.

Schiff base derived from isatin
3905
(1.61 g, 10 mmol) for 5 h with a few drops of hydrochloric acid. The prod-
uct obtained after evaporation of the solvent was filtered and washed with cold
methanol.
C15H8N3OCl3: Yield (%): 75.38, dark red, m.p.: 258ꢃC, m.wt: 352.60. Anal. Calcd (%):
C, 51.10; H, 2.29; N, 11.92; Cl, 30.16. Found (%): C, 50.98; H, 2.25; N, 11.88; Cl, 30.11;
1H NMR (CDCl3, ꢀ/ppm): 6.91–7.94 (m, 6H, Ar–H), 8.91 (s, 1H, CH¼N), 9.83 (s, 1H,
NH); IR (KBr, ꢂ/cmꢀ1): 3283 ꢂ(NH), 3060 ꢂ(Ar–CH), 1722 ꢂ(C¼O), 1645 ꢂ(C¼N),
1614 ꢂ[(CH¼N)(azomethine)], 1543 ꢂ(Ar–C¼C), 993 ꢂ(NN). UV-Vis (DMF, ꢃmax/nm):
225, 339, and 384.
2.5. Preparation of Co(II), Ni(II), Cu(II), and Zn(II) complexes
Hot methanolic solution (25 mL) of Schiff base (0.705 g, 2 mmol) was mixed with hot
methanolic solution (15 mL) of CoCl2 ꢁ 6H2O (0.237 g, 1 mmol)/NiCl2 ꢁ 6H2O (0.237 g,
1 mmol)/CuCl2 ꢁ 2H2O (0.170 g, mmol)/ZnCl2 (0.136 g, 1 mmol) and refluxed on a water
bath for 3 h. The separated complex was filtered, washed thoroughly with water and
methanol, and dried in vacuum over fused CaCl2.
[CoC30H16N6O2Cl8]: Yield (%): 72.15, reddish brown, m.p.: 228ꢃC, m.wt: 835.05. Anal.
Calcd (%): Co, 7.06; C, 43.15; H, 1.93; N, 10.06; Cl, 33.96. Found (%): Co, 7.02;
C, 43.07; H, 1.91; N, 9.98; Cl, 33.67; IR (KBr, ꢂ/cmꢀ1): 3284 ꢂ(NH), 3058 ꢂ(Ar–CH),
1705 ꢂ(C¼O), 1644 ꢂ(C¼N), 1603 ꢂ[(CH¼N)(azomethine)], 1545 ꢂ(Ar–C¼C), 1003,
ꢂ(NN), 570 ꢂ(Co–O), 435 ꢂ(Co–N); UV-Vis (DMF, ꢃmax/nm): 229, 352, 401, 603, and
684; molar conductance (DMF): 7.68 ꢀꢀ1 cm2 molꢀ1; ꢄeff (BM): 4.82.
[Ni C30H16N6O2Cl8]: Yield (%): 70.20, light red, m.p.: 235ꢃC, m.wt: 834.81. Anal. Calcd
(%): Ni, 7.03; C, 43.16; H, 1.93; N, 10.07; Cl, 33.97. Found (%): Ni, 6.95; C, 43.12;
H, 1.88; N, 10.01; Cl, 33.85; IR (KBr, ꢂ/cmꢀ1): 3282 ꢂ(NH), 3056 ꢂ(Ar–CH), 1706
ꢂ(C¼O), 1643 ꢂ(C¼N), 1605 ꢂ[(CH¼N)(azomethine)], 1548 ꢂ(Ar–C¼C), 1001, ꢂ(NN),
560 ꢂ(Ni–O), 440 ꢂ(Ni ꢀ N); UV-Vis (DMF, ꢃmax/nm): 233, 351, 402, 485, 624, and 772;
molar conductance (DMF): 1.28 ꢀꢀ1 cm2 molꢀ1; ꢄeff (BM): 3.08.
[CuC30H16N6O2Cl8]: Yield (%): 71.85, brown, m.p.: 217ꢃC, m.wt: 839.66. Anal. Calcd
(%): Cu, 7.57; C, 42.91; H, 1.92; N, 10.01; Cl, 33.78. Found (%): Cu, 7.46; C, 42.83; H,
1.89; N, 9.95; Cl, 33.72. IR (KBr, ꢂ/cmꢀ1): 3282 ꢂ(NH), 3059 ꢂ(Ar–CH), 1710 ꢂ(C¼O),
1645 ꢂ(C¼N), 1599 ꢂ[(CH¼N)(azomethine)], 1544 ꢂ(Ar–C¼C), 1002 ꢂ(NN), 565 ꢂ(Cu–
O), 430 ꢂ(Cu–N); UV-Vis (DMF, ꢃmax/nm): 230, 354, 400, 538, 615, and 768; molar
conductance (DMF): 5.45 ꢀꢀ1 cm2 molꢀ1; ꢄeff (BM): 1.98.
[ZnC30H16N6O2Cl8]: Yield (%): 71.28, reddish yellow, m.p.: 240ꢃC, m.wt: 841.51.
Anal. Calcd (%): Zn, 7.77; C, 42.82; H, 1.92; N, 9.99; Cl, 33.70. Found (%): Zn,
1
7.71; C, 42.79; H, 1.85; N, 9.93; Cl, 33.65. H NMR (DMSO-d6, ꢀ/ppm): 6.89–7.93
(m, 12H, Ar–H), 8.51 (s, 2H, CH¼N), 9.82 (s, 2H, NH); IR (KBr, ꢂ/cmꢀ1): 3284
ꢂ(NH), 3058 ꢂ(Ar–CH), 1708 ꢂ(C¼O), 1644 ꢂ(C¼N), 1603 ꢂ[(CH¼N)(azomethine)],
1547 ꢂ(Ar–C¼C), 1005, ꢂ(NN), 562 ꢂ(Zn–O), 425 ꢂ(Zn–N); UV-Vis (DMF, ꢃmax
/
nm): 231, 348, and 402; molar conductance (DMF): 12.45 ꢀꢀ1 cm2 molꢀ1; ꢄeff (BM):
diamagnetic.

3906
N. Raman et al.
3. Results and discussion
Spectroscopic studies and analytical data support the proposed structure of the ligand
and its metal complexes (scheme 1). Ligand (L) is soluble in many organic solvents but
complexes are soluble only in DMSO and DMF. Molar conductance measurements in
DMF show that complexes are non-electrolytes.
3.1. IR spectra
The functional groups of the Schiff base and the metal complexes were detected by
infrared spectra. Spectral features of the complexes were strikingly similar, but were
different from those of the ligand. In spectra of the complexes, the band due to the
aldimine ꢂ(CH¼N) shifted to lower frequency by 9–15 cmꢀ1, indicating its involvement
in chelation with metal [28]. However, the band corresponding to ketimine remained
almost unaffected, indicating nonparticipation in bond formation with metal. The band
corresponding to ꢂ(C¼O) shifted to lower frequency by 12–17 cmꢀ1 in the metal
complexes, suggesting its coordination with metal. The high intensity band at 1643–
1645 cmꢀ1 in the IR spectra of the Schiff base and its metal complexes was assigned to
ꢂ(C¼N) for ketimine. The IR spectrum of the Schiff base exhibited band due to ꢂ(NH)
at 3282–3284 cmꢀ1. The unaltered positions of ꢂ(NH) and ꢂ(C¼N) in the metal
complexes indicated that these groups were not involved in coordination. New bands at
425–440 and 560–570 cmꢀ1 in spectra of the complexes were assigned to stretching
frequencies of M–N and M–O, respectively [29]. Thus, the IR spectral results provide
evidence for bidentate complexation of Schiff base with metal.
3.2. Electronic spectral analysis and magnetic properties
Electronic spectra of the Schiff base and its complexes were recorded in DMF. The
absorption maxima and magnetic moment data of the complexes are listed in table 1.
Table 1. Electronic absorption spectral data, magnetic moments, and molar conductances of metal(II)
complexes at room temperature.
Band
assignments
Magnetic
moment (BM)
Molar conductance
(ꢀꢀ1 cm2 molꢀ1
)
Complex
[CoL2Cl2]
ꢃmax (nm)
Geometry
4
603
684
4TIg(F) ! TIg(P)
4
4T1g(F) ! A2g(F)
Octahedral
Octahedral
4.82
3.08
7.68
1.28
3
[NiL2Cl2]
[CuL2Cl2]
[ZnL2Cl2]
485
624
772
3A2g(F) ! T1g(P)
3
3A2g(F) ! T1g(F)
3
3A2g(F) ! T2g(F)
2
538
615
768
2B1g ! B2g
2
2B1g ! Eg
Octahedral
–
1.98
5.45
2
2B1g ! A2g
231
348
402
INCT
INCT
MLCT
Diamagnetic
12.45

Schiff base derived from isatin
3907
The Schiff base showed strong bands at 225, 339, and 384 nm due to ꢅ–ꢅ* and n–ꢅ*
transitions [30].
The electronic spectrum of Co(II) complex exhibited bands at 603 (" ¼ 182 dm3
(mol L
)
ꢀ1 ꢀ1 cmꢀ1
)
and 684 nm (" ¼ 148 dm3 (mol L
)
ꢀ1 ꢀ1 cmꢀ1), assigned to
4
4
4
4TIg(F) ! TIg(P) and T1g(F) ! A2g(F) transitions, respectively, corresponding to
octahedral geometry around Co(II) [31]. The observed magnetic moment of 4.82 BM
for Co(II) complemented the electronic spectral findings [32].
The electronic spectrum of Ni(II) complex exhibited three bands at 485 (" ¼ 160 dm3
(mol L
(mol L
)
)
ꢀ1 ꢀ1 cmꢀ1), 624 (" ¼ 134 dm3 (mol L
3T2g(F) transitions, respectively, characteristic of an octahedral Ni(II) complex, which
was further supported by its magnetic moment of 3.08 BM [31, 32].
The electronic spectrum of the Cu(II) complex reported here showed three bands at
)
ꢀ1 ꢀ1 cmꢀ1) and 772 nm (" ¼ 102 dm3
ꢀ1 ꢀ1 cmꢀ1), attributed to A2g(F) ! T1g(P), A2g(F) ! T1g(F) and A2g(F) !
3
3
3
3
3
538 (" ¼ 98 dm3 (mol L
)
ꢀ1 ꢀ1 cmꢀ1), 615 (" ¼ 84 dm3 (mol L
)
ꢀ1 ꢀ1 cmꢀ1) and 768 nm
(" ¼ 63 dm3 (mol L
)
ꢀ1 ꢀ1 cmꢀ1), assigned to B1g ! B2g, B1g ! Eg, and B1g ! A2g
2
2
2
2
2
2
transitions, respectively. The magnetic moment of 1.98 BM was higher than the spin-
only value (1.73 BM) expected for one unpaired electron and offered the possibility of
an octahedral geometry. The Zn(II) complex was diamagnetic and from the empirical
formula of this complex, an octahedral geometry was proposed [33, 34].
3.3. Molar conductivity measurements
The molar conductivities of metal(II) complexes in DMF (l0ꢀ3 molLꢀ1) are given in
table 1. The conductance values measured (1.28–12.45 ꢀꢀ1 cm2 molꢀ1) were too low to
account for dissociation and hence the complexes were considered as non-electrolytes.
3.4. 1H NMR spectral analysis
The 1H NMR spectrum of the free ligand showed peaks at 9.83 and 8.91 ppm, assigned
to (NH) of the indole ring and the azomethine (CH¼N), respectively. The aromatic ring
protons of both the ligand and its zinc complex were multiplets in the range 6.89–
7.94 ppm. However, in the spectrum of zinc complex, the azomethine (CH¼N) protons
showed an upfield shift to 8.51 ppm, thereby suggesting coordination of imino nitrogen
to zinc [35].
3.5. Mass spectral studies
The ESI mass spectrum gave additional structural information about the stereochem-
istry of the studied compounds. The mass spectra were characterized by strong to
medium molecular ion peaks, in good accord with their suggested empirical formulae as
indicated from elemental analyses. The mass spectrum of the ligand showed a peak at
m/z 353 corresponding to [M]þ while the isotopic peaks of chlorine (37Cl) present in the
ligand were observed at m/z 355 (M þ 2), 357 (M þ 4), and 359 (M þ 6). The mass
spectra of [CuL2Cl2], [CoL2Cl2], [NiL2Cl2], and [ZnL2Cl2] complexes showed peaks at
m/z 840, 835, 836 (M þ 1), and 842 corresponding to the molecular ion peak of the
respective complexes and also the isotopic peaks of all the complexes were observed

3908
N. Raman et al.
Table 2. Spin Hamiltonian parameters of Cu(II) complex in DMSO solution.
g-tensors
gav
[CuL2Cl2] 2.26 2.05 2.15
Hyperfine constant ꢄ 10ꢀ4 cmꢀ1
Bonding parameters
ꢆ2 ꢇ2 ꢈ2
Complex
g
k
g
A
A
Aav
g /A
k
G
K
K
?
K
?
k
?
k
k
130
90
106.66
173.84 5.43 0.68 0.92 0.68 0.63 0.46 0.33
with the weak relative intensities at (M þ 2), (M þ 4), (M þ 6), (M þ 8), (M þ 10),
(M þ 12), (M þ 14), and (M þ 16). In all the complexes, the observed fragment ions
[C30H16N6Cl7O2M]þ. and [C30H16N6Cl6O2M]þ were due to elimination of two
coordinated chlorides one by one. All complexes gave a few unstable fragment ions,
namely [C15H8N3Cl3O]þ, [C7H3NCl3]þ, and [C6H2Cl3]þ. Mass spectra of the ligand and
its complexes are given in ‘‘Supplementary material’’ (figures S1–S5). The mass spectral
and elemental analyses reveal that the complexes are monomeric.
3.6. Electron paramagnetic resonance spectra
X-band EPR spectra of the polycrystalline copper complex recorded in DMSO at 300 K
(RT) and 77 K (LNT) are given in ‘‘Supplementary material’’ (figures S6 and S7). The
spin Hamiltonian parameters calculated are given in table 2. The spectrum of the
complex at 300 K showed one intense absorption at high field and was isotropic due to
tumbling motion of the molecules. The EPR spectrum of the Cu(II) complex in DMSO
at 77 K showed well-resolved hyperfine spectra giving g 4 g 4 2.0027, corresponding
k
?
to the presence of an unpaired electron in the dx2ꢀy2 orbital. For a Cu(II) complex, g is
k
a parameter sensitive enough to indicate covalence. The fact that g is less than 2.3 is an
indication of significant covalent character to the M–L bond [36].
k
The geometric parameter G, which is a measure of exchange interaction between the
copper centre in polycrystalline compounds, was calculated using the equation
G ¼ (g ꢀ 2.0027)/(g ꢀ 2.0027). According to Hathaway [37, 38], if the value of G is
k
?
greater than four, the exchange interaction between Cu(II) centers in the solid state
is negligible, whereas when it is less than four, a considerable exchange interaction is
indicated in the solid complex. The calculated G value is 5.43 for the present copper
complex indicating that magnetic interaction between Cu(II) ions is negligible in the
solid complex [39].
The EPR parameters g , g , gav, gav, A , and A for copper complex and the energies
k
?
k
?
of d–d transitions were used to evaluate the bonding parameters ꢆ2, ꢇ2, and ꢈ2, which
may be regarded as measure of the covalency of the in-plane ꢉ-bonding and the in-plane
ꢅ- and out-of-plane ꢅ-bonding, respectively. The value of in-plane ꢉ-bonding parameter
ꢆ2 was calculated using the expression [40].
ꢆ2 ¼ ꢀðA =0:036Þ þ ð g ꢀ 2:0027Þ þ ð3=7Þð g ꢀ 2:0027Þ þ 0:04:
jj
jj
?
The following simplified expressions were used to calculate the bonding
parameters [41].
K2 ¼ ð g ꢀ 2:0027ÞEdꢀd=8ꢃ0,
jj
jj
K2 ¼ ð g ꢀ 2:0027ÞEdꢀd=2ꢃ0,
?
?

Schiff base derived from isatin
3909
where K ¼ ꢆ2ꢇ2, K ¼ ꢆ2ꢈ2, and ꢃ0 represents the one electron spin-orbit coupling
k
?
constant for the free ion, equal to ꢀ828 cmꢀ1. Hathaway [42] has pointed out that for
pure ꢉ-bonding K ꢅ K ꢅ 0.77 and for in-plane ꢅ-bonding, K 5 K , while for out-of-
k
?
k
?
plane ꢅ-bonding K 5 K . In the present copper complex, K 4 K indicates the
?
k
k
?
presence of significant out-of-plane ꢅ-bonding. Furthermore ꢆ2, ꢇ2, and ꢈ2 have values
less than 1.0, which is expected for 100% ionic character of the bonds and become
smaller with increasing covalent bonding. Therefore, the evaluated values of ꢆ2, ꢇ2, and
ꢈ2 of the complex are consistent with both in-plane ꢉ and in-plane ꢅ-bonding.
The Fermi contact hyperfine interaction term K may be obtained from [43],
K ¼ Aav=Pꢇ2 þ ð gav ꢀ 2:0027Þ=ꢇ2,
where P is the free ion dipolar term and its value is 0.036. K is a dimensionless quantity,
which is a measure of the contribution of s electrons to the hyperfine interaction and is
generally found to have a value of 0.30. The K value obtained for the copper complex is
in agreement with those estimated by Assour [44]. The results of EPR parameters,
electronic absorption, and magnetic measurement data revealed that the prepared
copper complex has octahedral geometry.
3.7. Metal–DNA interaction
Transition metal complexes bind to DNA via both covalent and/or non-covalent
interactions. In covalent binding the labile ligand of the complexes is replaced by a
nitrogen base of DNA such as guanine N7. On the other hand, the non-covalent DNA
interactions include intercalation, electrostatic, and groove (surface) binding of metal
complexes, outside of the DNA helix, along major or minor grooves [45].
3.7.1. Electronic absorption titration study of the DNA-binding. Electronic absorption
spectroscopy was used to study the interaction of metal complexes with DNA [46]. The
change in absorbance and shift in wavelength upon addition of increasing concentration
of DNA solution in a fixed concentration of metal complexes gives information on the
mode of interaction. Metal complex binding with DNA through intercalation usually
results in hypochromism and bathochromism due to the strong stacking interaction
between the aromatic chromophore and the base pairs of DNA. The DNA-binding
constant of metal(II) complexes are usually determined by the MLCT band because no
light absorption for DNA is found in the range of wavelength over 395 nm and thus no
interference exists. The extent of the hypochromism in the visible MLCT band is
consistent with the strength of intercalative interaction [47].
To clarify interactions between the compound and DNA, electronic absorption
spectra of the metal complexes in the absence and presence of the CT-DNA (at a
constant concentration of the compounds) were obtained; the DNA-binding of copper
complex is shown in figure 1. With increasing DNA concentrations, the absorption
band at 398 nm of the Co(II) complex represented hypochromism of 12.65%; the
absorption band at 401.7 nm of the Ni(II) complex exhibited hypochromism of 19.93%;
the absorption band at 400.5 nm of the Cu(II) complex appeared hypochromism of
22.84% and the absorption band at 402.5 nm of the Zn(II) complex appeared
hypochromism of 16.71%. The bathochromism of Co(II), Ni(II), Cu(II), and Zn(II)

3910
N. Raman et al.
Figure 1. Absorption spectrum of copper(II) complex in the absence and presence of increasing amounts of
CT-DNA at room temperature in 50 mmol Lꢀ1 Tris-HCl/50 mmol Lꢀ1 NaCl buffer (pH 7.2). Arrow shows
the absorbance change upon increasing DNA concentration.
Table 3. Absorption properties of metal(II) complexes.
ꢃmax (nm)
Complex
Free
Bound
Dꢃ (nm)
Hypochromicity (%)
Kb ꢄ 104 ((mol Lꢀ1 ꢀ1
) )
[CoL2Cl2]
[NiL2Cl2]
[CuL2Cl2]
[ZnL2Cl2]
398.0
401.7
400.5
402.5
399.5
404
403.5
404.3
1.5
2.2
3.0
1.8
12.65
19.93
22.84
16.71
2.54 ꢆ 0.20
3.99 ꢆ 0.32
6.94 ꢆ 0.36
3.62 ꢆ 0.28
complexes was shifted to 1.5, 2.2, 3.0, and 1.8 nm, respectively. The hypochromism
observed for the ꢅ ! ꢅ* transition band indicated strong binding of the metal
complexes to DNA. The binding constant (Kb) values of the Co(II), Ni(II), Cu(II), and
Zn(II) complexes were 2.54 ꢆ 0.20 ꢄ 104 (mol L
)
ꢀ1 ꢀ1, 3.99 ꢆ 0.32 ꢄ 104 (mol Lꢀ1 ꢀ1
) ,
)
ꢀ1 ꢀ1, respectively, which
6.94 ꢆ 0.36 ꢄ 104 (mol L
)
were consistent with hypochromism degree (table 3).
ꢀ1 ꢀ1, and 3.62 ꢆ 0.28 ꢄ 104 (mol L
The Kb values obtained here are lower than those reported for classical intercalators
(EB and [Ru(phen)(dppz)] whose binding constants are on the order of
)
106–107 (mol Lꢀ1 ꢀ1) [48, 49] and also compared to other reported later 3d metal
complexes (Kb ¼ 0.88–3.83 ꢄ 104 (mol L
)
ꢀ1 ꢀ1). Thus, the synthesized complexes have
slightly superior intercalating properties due to containing more electron-releasing
substituents and coordinated chlorides [50, 51]. It is clear that the hypochromism and
Kb values are not enough evidence, but these results can suggest an intimate association
of the compounds with CT-DNA and indicate that the binding strength of complex is in
the order Cu(II) complex 4 Ni(II) complex 4 Zn(II) complex 4 Co(II) complex.
3.7.2. Cyclic voltammetry study of the DNA-binding. Electrochemical investigations of
metal–DNA interactions provide a useful complement to spectroscopic methods, for

Schiff base derived from isatin
3911
example, for non-absorbing species, and yield information about interactions with both
the reduced and oxidized forms of the metal [52]. In figure 2, the cyclic voltammograms
of copper complex in the presence of CT-DNA in various concentrations are shown. It
has been pointed out that the electrochemical potential of a small molecule will shift
positively when it intercalates into DNA double helix, and if it is bound to DNA by
electrostatic interaction, the potential would shift in a negative direction [53].
The two quasi-reversible and one irreversible redox couples for Cu(II) complex, one
quasi-reversible redox couple for Co(II) complex and only one irreversible redox couple
for Zn(II) complex in 1 : 2 DMF : buffer solution that has been studied upon addition of
CT-DNA and the shifts of the cathodic (Epc) and anodic (Epa) potentials are given in
table 4. No new redox peaks appeared after the addition of CT-DNA to each complex,
but the current intensity of all the peaks decreased significantly, suggesting the existence
of an interaction between each complex and CT-DNA. The decrease in current intensity
can be explained in terms of an equilibrium mixture of free and DNA-bound complexes
to the electrode surface [54].
Figure 2. Cyclic voltammogram of copper(II) complex in the absence and presence of increasing amounts of
CT-DNA at room temperature in DMF : buffer (1 : 2) mixture (pH 7.2) (scan rate 0.1 V sꢀ1). The arrows show
the current changing upon increasing DNA concentration.
Table 4. Electrochemical behavior of metal(II) complexes in the absence and presence of CT-DNA.
Epc (V)
Free Bound
Epa (V)
DEp (V)
E1/2 (V)
Free Bound KR/KO
Complex Redox couple
Free Bound Free Bound
[CoL2Cl2] Co(II)/Co(I) ꢀ0.668 ꢀ0.645 ꢀ0.305 ꢀ0.289 0.363 0.356 ꢀ0.973 ꢀ0.934
[CuL2Cl2] Cu(III)/Cu(II) 0.018 0.005 0.086 0.107 0.068 0.102 0.104 0.112
4.56
1.36
1.93
–
Cu(II)/Cu(I) ꢀ0.543 ꢀ0.535 ꢀ0.155 ꢀ0.146 0.388 0.389 ꢀ0.698 ꢀ0.681
Cu(I)/Cu(0) ꢀ0.684 ꢀ0.690
[ZnL2Cl2] Zn(II)/Zn(I) ꢀ0.415 ꢀ0.369
–
–
–
–
–
–
–
–
–
–
–
–
–

3912
N. Raman et al.
Cu(II), Co(II), and Zn(II) complexes exhibit different electrochemical behavior upon
the addition of CT-DNA at various concentrations. For increasing amounts of CT-
DNA, the cathodic potentials Epc show a positive shift except copper complex
(DEpc ¼ þ13, þ8, and ꢀ6 mV for copper complex, DEpc ¼ þ35 mV for cobalt complex
and DEpc ¼ þ23 mV for zinc complex) while the anodic potential Epc shifted to more
positive values (DEpa ¼ þ21 and þ9 mV for copper complex, DEpa ¼ þ16 mV for cobalt
complex). The shift in E1/2 values can be used to estimate the ratio of equilibrium
constants, KR/KO for binding of M(II) and M(I) forms, respectively, to DNA. For a
Nernstian electron transfer system in which both the oxidized and reduced forms are
associated with a third species such as DNA in solution can be applied. Here, Mnþ
-
DNA represents the metal complexes bound to DNA with nþ charge on the metal
centre. Thus for a one electron transfer,
Eꢃ ꢀ Eꢃ ¼ 0:0591 logðKR=KOÞ,
b
f
where Eꢃf and Eꢃb are the formal potentials of the M(II)/M(I) couple in the free and
bound forms, respectively. KR and KO are the corresponding binding constants for the
M2þ and Mþ species to DNA. For Co(II) and Cu(II) the complexes, KR is higher than
KO, suggesting that the interaction of metal complexes with DNA tends to stabilize the
M(II) over the M(I) state (table 4). Finally, the conclusion derived from the CV study is
that copper complex can bind to DNA by both intercalation and electrostatic
interaction, while cobalt and zinc complex can bind to DNA by intercalative binding
mode [55, 56].
3.7.3. Viscosity measurements. Viscosity titration measurements were carried out to
clarify the interaction between the investigated compounds and CT-DNA. It is
sensitive to length changes and regarded as the least ambiguous and the most critical
test of a binding model in solution in the absence of crystallographic structural
data [57]. A classical intercalation model demands that the DNA helix must lengthen
as base pairs are separated to accommodate the binding complexes, leading to
increase of DNA viscosity [58]. In contrast, a partial and/or non-classical
intercalation of the complex could bend (or kink) the DNA helix, reducing its
viscosity concomitantly. In addition, some compounds such as [Ru(bpy)3]2þ, which
interact with DNA by an electrostatic binding mode, have no influence on DNA
viscosity [59]. To further elucidate the binding mode of the present complexes,
viscosity measurements were carried out by keeping [DNA] ¼ 10 ꢄ 10ꢀ5 mol Lꢀ1 and
varying the concentration of the compounds. The effects of metal complexes on the
viscosities of CT-DNA are shown in figure 3. With the ratios of the investigated
compounds to CT-DNA increasing, the relative viscosities of DNA increased
steadily, indicating that there exist intercalation between the compounds and DNA
helix. The increased degree of viscosity, which may depend on the binding affinity of
compounds to DNA, follows the order Cu(II) complex 4 Ni(II) complex 4 Zn(II)
complex 4 Co(II) complex. On the basis of the electronic absorption titration and
voltammetric studies together with the viscosity measurements, we find that all four
metal complexes bind to CT-DNA via an intercalative binding mode and the Cu(II)
complex binds to CT-DNA more strongly.

Schiff base derived from isatin
3913
Figure 3. Change in relative specific viscosity of CT-DNA (10 ꢄ 10ꢀ5 molLꢀ1) on addition of [CuL2Cl2] (^),
[NiL2Cl2] (m), [ZnL2Cl2] (g) and [CoL2Cl2] (.) in 50 mmol Lꢀ1 Tris–HCl/50 mmol Lꢀ1 NaCl buffer (pH 7.2)
at 37ꢃC, R ¼ [complex]/[DNA].
Figure 4. Cleavage of SC pBR322 DNA (0.3 mg) by metal(II) complexes (0.30 mmol Lꢀ1) in the presence of
hydrogen peroxide (100 mmol Lꢀ1) in 50 mmol Lꢀ1 Tris–HCl/50 mmol Lꢀ1 NaCl buffer (pH 7.2). Lane 1,
DNA control; Lane 2, DNA þ H2O2; Lane 3, DNA þ [CuL2Cl2]; Lane 4, DNA þ ethanol (4 mL); Lanes 5–8,
DNA þ H2O2 þ [CoL2Cl2], [CuL2Cl2], [ZnL2Cl2], and [NiL2Cl2], respectively; Lane 9, DNA þ H2O2 þ ethanol
(4 mL) þ [CuL2Cl2]; Lane 10, DNA þ H2O2 þ SOD (4 Units) þ [CuL2Cl2].
3.8. DNA-cleavage studies
Antitumor agents can interact with DNA and cause DNA strand scission, for this
reason we have also studied the capacity to cleave DNA by agarose gel electrophoresis
using plasmid pBR322 DNA. The naturally occurring supercoiled form (Form I), when
nicked, gives an open circular relaxed form (Form II) and upon further cleavage, results
in the linear form (Form III). When subjected to gel electrophoresis, relatively fast
migration is observed for Form I. Form II migrates slowly and Form III migrates
between Forms I and II. As shown in figure 4, control experiments suggested that
untreated DNA, DNA incubated with either peroxide or copper complex alone and in
the presence of ethanol with DNA did not show significant DNA cleavage (Lanes 1–4).
In the presence of H2O2, Form I converted into Form II (nicked DNA) and Form III

3914
N. Raman et al.
Table 5. The in-vitro antimicrobial activity of ligand and its metal(II) complexes evaluated by MIC
(mg mLꢀ1).
Antibacterial activity
Antifungal activity
Complex
S. aureus B. subtilis E. coli P. aeruginosa A. niger R. bataicola R. stolonifer C. albicans
Ligand (L)
[CoL2Cl2]
[NiL2Cl2]
[CuL2Cl2]
[ZnL2Cl2]
Streptomycin
Nystatin
150
75
75
50
75
5
250
100
75
50
100
10
100
25
50
20
25
5
100
50
50
25
20
10
–
150
50
75
25
20
–
200
50
75
20
25
–
200
75
100
50
25
–
100
25
50
20
20
–
–
–
–
5
10
10
10
(linear DNA) (Lanes 5–8), which indicated that all four complexes are potent DNA
cleavage agents in the presence of H2O2 as an oxidizing agent under the present
experimental conditions. The addition of hydroxyl radical scavenger (ethanol)
completely inhibited DNA cleavage activity (Lane 9). Moreover, on addition of
superoxide anion radical scavenger (SOD), there is no apparent inhibition in the DNA
cleavage (Lane 10), suggesting that the cleavage mechanism involves oxidative reactions
via hydroxyl radical species [60].
3.9. Antimicrobial evaluation of ligand and its metal complexes
The ligand and metal complexes were screened for antibacterial activity and the results
are presented in table 5. The MIC was determined by assaying using the broth dilution
technique. The synthesized Schiff base has an inhibitory effect (MIC values of
100–250 mg mLꢀ1) on growth of the tested bacterial strains. All complexes showed
greater bactericidal activities against E. coli (MIC 20–50 mg mLꢀ1), S. aureus (MIC 50–
75 mg mLꢀ1), P. aeruginosa (MIC 20–50 mg mLꢀ1) and B. subtilis (MIC 50–100 mg mLꢀ1
)
than the ligand. In the fungal studies, the ligand had an inhibitory effect (MIC values in
range 100–200 mg mLꢀ1) on the growth of the tested strains and complexes again
showed greater fungicidal activities against A. niger (MIC 20–75 mg mLꢀ1), R. stolonifer
(MIC 25–100 mg mLꢀ1), R. bataicola (MIC 20–75 mg mLꢀ1), and C. albicans (MIC
20–50 mg mLꢀ1). Cu(II) and Zn(II) complexes had greater bacterial and fungal activities
than the ligand. The synthesized complexes, having more electron releasing substituents
and two coordinated chlorides, increased the microbial activity as compared to reported
isatin-based Schiff base metal complexes [50, 61].
Chelation [62, 63] reduces the polarity of the metal ion considerably, mainly because
of the partial sharing of its positive charge with donor groups and possible ꢅ-electron
delocalization on the whole chelate ring. Lipids and polysaccharides are important
constituents of cell walls and membranes, which are preferred for metal ion interaction.
The cell wall also contains several aminophosphates, carbonyl, and cysteinyl ligands,
which maintain the integrity of the membrane by acting as a diffusion barrier and also
provides suitable sites for binding. Chelation reduces not only the polarity of the metal
ion, but increases the lipophilic character of the chelate, and the interaction between
metal ion and the lipid is favored. This may lead to breakdown of the permeability

Schiff base derived from isatin
3915
barrier of the cell, resulting in interference with the normal cell processes. If the
geometry and charge distribution around the molecule are incompatible with the
geometry and charge distribution around the pores of the bacterial cell wall, penetration
through the wall by the toxic agent cannot take place and this will prevent the toxic
reaction within the pores.
Chelation is not the only criterion for antibacterial activity. Some important factors
such as the nature of the metal ion, nature of the ligand, coordinating sites, and
geometry of the complex, concentration, hydrophilicity, lipophilicity, and the presence
of co-ligands have considerable influence on antibacterial activity. Certainly, steric and
pharmacokinetic factors also play a decisive role in deciding the potency of an
antimicrobial agent. The presence of lipophilic and polar substituents is expected to
enhance antibacterial activity. Heterocyclic ligands with multifunctionality have a
greater chance of interaction either with nucleoside bases (even after complexation with
metal ion) or with biologically essential metal ions present in the biosystem can be
promising candidates as bactericides since they always tend to interact with enzymatic
functional groups, in order to achieve higher coordination numbers [64]. Thus
antibacterial property of metal complexes cannot be ascribed to chelation alone, but is
an intricate blend of several contributions.
4. Conclusion
A ligand and its complexes have been synthesized and characterized by electronic
absorption spectra, IR, 1H NMR, and mass spectral analysis. All the metal ions are six-
coordinate and the geometry can be described as octahedral. The molar conductances
reveal that all the complexes are non-electrolytes. DNA-binding properties of the
metal(II) complexes with DNA have been investigated by UV-Vis spectra, voltammetry,
and viscosity measurements. Results indicate that the metal(II) complexes bind to
CT-DNA via an intercalative mode. All the complexes effectively cleave plasmid DNA
in the presence of oxidizing agent. DNA cleavage mechanism studies show that the
complexes can promote DNA cleavage through oxidative pathway. They display higher
nuclease activity for the Cu(II) complex than other complexes, so the cooperative
interaction of metal ions is a factor to cleave DNA. The antimicrobial activities show
that the metal complexes are more active than the ligand and Cu(II) and Zn(II)
complexes are more active than the Ni(II) and Co(II) complexes.
Acknowledgments
The authors express their sincere thanks to the College Managing Board, Principal and
Head of the Department of Chemistry, VHNSN College, for providing necessary
research facilities. They also thank Sophisticated Analytical Instrumentation Facility,
Central Drug Research Institute, Lucknow, for providing CHN analysis data, ESI-
Mass, and Sophisticated Analytical Instrumentation Facility, Indian Institute of
Technology, Bombay, for EPR measurements.

3916
N. Raman et al.
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