Synthesis, structure elucidation, DNA interaction, biological evaluation, and molecular docking of an isatin-derived tyramine bidentate Schiff base and its metal complexes

Article abstract of DOI:10.1007/s00706-011-0699-8

Three new transition metal complexes [CuL₂Cl₂], [NiL₂Cl₂], and [ZnL₂Cl2], where L = Schiff base derived from isatin (1H-indole-2,3-dione) and tyra-mine (4-(2-aminoethyl) phenol), have been synthesized and characterized spectroscopically. The binding properties of these complexes with calf thymus DNA have been investigated by use of electronic absorption spectroscopy, viscosity measurement, cyclic voltammetry, and molecular docking analysis. Moreover, these complexes have been found to promote the cleavage of pUC19 DNA from the supercoiled form I to the open circular form II in the presence of hydrogen peroxide. The newly synthesized compounds were screened for antibacterial activity and subjected to molecular docking studies for inhibition of the enzyme Staphylococcus aureus sortase-A, which is a novel target for antibacterials.

Full text of DOI:10.1007/s00706-011-0699-8

Monatsh Chem (2012) 143:1019–1030
DOI 10.1007/s00706-011-0699-8
ORIGINAL PAPER
Synthesis, structure elucidation, DNA interaction, biological
evaluation, and molecular docking of an isatin-derived tyramine
bidentate Schiff base and its metal complexes
•
Natarajan Raman Sivasangu Sobha
•
Liviu Mitu
Received: 30 June 2011 / Accepted: 29 November 2011 / Published online: 3 January 2012
Ó Springer-Verlag 2011
Abstract Three new transition metal complexes
[CuL2Cl2], [NiL2Cl2], and [ZnL2Cl2], where L = Schiff
base derived from isatin (1H-indole-2,3-dione) and tyra-
mine (4-(2-aminoethyl)phenol), have been synthesized and
characterized spectroscopically. The binding properties of
these complexes with calf thymus DNA have been inves-
tigated by use of electronic absorption spectroscopy,
viscosity measurement, cyclic voltammetry, and molecular
docking analysis. Moreover, these complexes have been
found to promote the cleavage of pUC19 DNA from the
supercoiled form I to the open circular form II in the
presence of hydrogen peroxide. The newly synthesized
compounds were screened for antibacterial activity and
subjected to molecular docking studies for inhibition of the
enzyme Staphylococcus aureus sortase-A, which is a novel
target for antibacterials.
an urgent need to develop new antibacterials with a novel
mechanism of action to overcome the problem of resistance
[1]. Therefore, researchers are increasingly turning their
attention to folk medicine, looking for new leads to
develop better drugs against microbial infections. Staphy-
lococcus aureus is the most dangerous among the common
staphylococcal bacteria causing a wide variety of infec-
tions, from simple abscesses to fatal sepsis, and
endocarditis, meningitis, and toxinoses including food
poisoning and toxic shock syndrome. Staphylococcus
pathogenic versatility is compounded by its ability to
develop resistance to new antibiotics almost as fast as they
are introduced. However, nosocomial infections caused by
S. aureus are clinically serious and control of such infec-
tion requires strain typing to identify the extent of
virulence, the source of contamination, and resistance to
commonly used antibiotics. Hospital strains of S. aureus
are usually resistant to a variety of different antibiotics.
Very few strains are resistant to all clinically useful anti-
biotics except vancomycin and, hence, vancomycin-
resistant strains are increasingly reported nowadays [2–4].
Gram-positive pathogenic bacteria have proteins
on their surface that are important during infection. In
S. aureus, these surface proteins are anchored to the cell
wall by two sortase enzymes, sortase-A and sortase-B, that
recognize specific surface protein sorting signals. Because
of their universal presence in Gram-positive bacteria, sor-
tases are attractive targets for development of novel
antimicrobial agents [5].
Keywords Isatin-derived Schiff base ꢀ Metal complexes ꢀ
DNA interaction ꢀ Molecular docking ꢀ
Biological evaluation
Introduction
Bacterial resistance is a major worldwide health problem,
with multidrug resistance of pathogenic species. There is
N. Raman (&) ꢀ S. Sobha
Isatin (1H-indol-2,3-dione) analogues have a variety of
biological activity, for example antimicrobial, anticonvulsant,
analgesic, anti-inflammatory, anticancer, antitubercular, anti-
viral, and anti-HIV, because of the presence of the indole
backbone [6]. Tyramine (4-(2-aminoethyl)phenol) is a bio-
genic amine which is widely used as a pharmacological tool
Research Department of Chemistry, VHNSN College,
Virudhunagar 626 001, India
e-mail: drn_raman@yahoo.co.in
L. Mitu
Department of Chemistry, University of
Pitesti, 110040 Pitesti, Romania
123

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N. Raman et al.
to evaluate the role of the sympathetic nervous system in
human physiology and pathology [7].
by UV–visible spectroscopy, cyclic voltammetry, viscosity
measurement, and molecular docking analysis.
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 bind preferentially with negatively
charged biomolecules, for example the amino acid residues
of protein constituents, ATP, and nucleic acids. Moreover,
the ligands can interact with biomolecules by coordination
or hydrogen bonding and by dipole–dipole interactions [8].
DNA is also an important cellular receptor; many chemi-
cals exert their antitumor effects by binding to DNA,
thereby changing its replication and inhibiting the growth
of the tumor cells, which is the basis of designing new and
more efficient antitumor drugs, the effectiveness of which
depends on the mode and affinity of the binding [9].
Computational intelligence drug-discovery is innovative
technology designed to accelerate high-throughput drug
screening for general protein-targeted drug discovery. This
technology enables prediction of the binding modes and
molecular interactions of novel small-molecule drugs that
bind to protein targets [10]. The objectives of the work dis-
cussed in this paper were to overcome multi drug resistance
by design and synthesis of novel Cu(II), Ni(II), and Zn(II)
complexes and to evaluate these by use of in silico docking
studies in order to predict and confirm their molecular
interactions with biomolecules. In this work, molecular
docking was researched to analyze the binding properties of
the enzyme Staphylococcus aureus sortase-A with an isatin-
derived Schiff base and its metal complexes. The DNA-
binding properties of the complexes were also investigated
Results and discussion
The synthesized ligand and its complexes were found to be
stable in air at room temperature. The ligand was soluble in
methanol. The complexes were insoluble in water, alcohol,
and acetonitrile but soluble in DMF, CHCl3, and DMSO.
The proposed reaction of the Schiff base and the synthesis
of the complexes are given in Scheme 1.
The low conductance of all the complexes in DMSO
showed that the chloride ions were present inside the coor-
dination sphere; this was confirmed by a silver nitrate test.
The low molar conductance of these metal complexes sup-
ported their non-electrolytic nature. Results from elemental
analysis of 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 ML2Cl2,
where L is a bidentate ligand. The formation of these com-
plexes may proceed in accordance with to the equation:
MCl2 ꢀ nH2O þ 2L ! ½ML2Cl2ꢁ þ nH2O
where M = Cu(II), Ni(II), or Zn(II).
Mass spectra
The mass spectrum of the Schiff base ligand had a peak at
m/z = 266 corresponding to the [C16H14N2O2] ion. The spec-
trum also contained peaks for the fragments at m/z = 145,
122, 92, and 77 corresponding to [C8H5N2O] (M?), [C8H9O]
Scheme 1
HO
OH
O
MeOH
+
O
Reflux, 6 h
N
N
H
H2N
O
N
H
HO
H
N
O
MCl2, MeOH
Reflux, 2 h
Cl
N
M
N
Cl
O
N
H
M = Cu, Ni, Zn
OH
123

Isatin-derived tyramine bidentate Schiff base
1021
Table 1 Analytical and physical data of the Schiff base ligand and its complexes
Compound
Yield (%)
Color
Found (Calc)%
Formula weight
Km/S cm2 mol-1
M
C
H
N
Ligand
86
71
54
67
Dark brown
Pale green
Pale blue
-
72.8 (72.1)
57.6 (57.9)
58.0 (58.3)
57.4 (57.9)
5.4 (5.3)
4.2 (4.3)
4.2 (4.2)
4.2 (4.2)
10.8 (10.5)
8.4 (8.7)
8.4 (8.8)
8.3 (8.6)
266
667
662
669
-
[CuL2Cl2]
[NiL2Cl2]
[ZnL2Cl2]
9.5 (9.9)
8.8 (9.1)
9.7 (9.8)
1.9
2.1
1.4
Pale orange
([M ? 1]?), [C6H6N] (M?), and [C6H5] (M?), respectively.
The spectra of the Cu(II), Ni(II), and Zn(II) complexes had
molecular ion peaks at m/z = 667 (M?), 663 ([M ? 1]?), and
671 ([M ? 2]?), respectively. The Cu(II) complex gave a
fragment ion peak with loss of two chlorine atoms at
m/z = 597. All these fragments are leading to the formation of
the species [ML2] (M?), which undergoes demetallation to
yield the species [L] (M?), affording the fragment ion peak at
m/z = 266. Thus, the mass spectral data confirm the stoichi-
ometry of the complexes as being of [ML2Cl2] type. This
stoichiometry is further supported by the results from ele-
mental analysis, which are in close agreement with the values
calculated from molecular formulae assigned to these com-
plexes. Thus, the mass spectral data reinforce the conclusion
drawn from the analytical and conductance values.
which was attributed to the phenolic OH group present in
tyramine. The presence of this peak in the spectrum of the
zinc complex confirmed that the –OH proton was free from
complexation. The spectrum of the Schiff base also con-
tained a signal at 10.8 ppm assigned to the NH proton of
isatin. This peak remained substantially unchanged on
complexation. The signals around the region 6.8–7.9 ppm
were attributed to aromatic protons. Peaks observed at 1.5
and 2.4 ppm were assigned to two methylenic protons of
1
tyramine. The H NMR spectrum of the Zn(II) complex
contained a multiplet at 6.5–7.8 ppm, assigned to aromatic
protons. The 13C NMR spectrum of the ligand contained
aromatic carbons at 115.17–135.23 ppm and indole carbons
at 111.17–125.52 ppm. The spectrum of the ligand also
contained a signal for C=O carbon at 156.74 and a signal for
C=N carbon at 164.55 ppm, which were shifted downfield on
coordination, indicating the C=O (154.65 ppm) and C=N
(162.88 ppm) groups are participating in complex formation.
There were no appreciable changes of other peaks.
IR spectra
The IR spectra of the metal complexes were clearly indic-
ative of bonding of the ligand with the metal ions. The IR
spectrum of the Schiff base ligand had a broad band in the
region 3,570 cm-1 which was assigned to the OH group.
The appearance of this band in the spectra of the metal
complexes indicated that the phenolic OH group was free
from complexation. The three bands appearing at 3,248,
1,706, and 1,616 cm-1 in the ligand spectrum were assigned
to stretching vibration modes mNH, mC=O, and mC=N, respec-
tively. In the spectra of the metal complexes the band with a
maximum at 1,706 cm-1 in the free ligand was shifted
downwards to 1,687, 1,683, and 1,666 cm-1, indicating
coordination of the carbonyl oxygen with the metal ion. The
band appearing at 1,616 cm-1 in the free ligand, assigned to
the mC=N vibration mode, was shifted to the lower frequency
of 1,556–1,541 cm-1. This shift was indicative of coordi-
nation of an azomethinic nitrogen atom [11–13]. New bands
were found in the spectrum of the complex in the regions
511–515, 479–465, and 356–362 cm-1. These new bands
were assigned to mM=O, mM=N [14], and mM=Cl, respectively.
Electronic spectra
The electronic absorption spectrum of the ligand contained
absorption bands at ca 39,215 and 27,510 cm-1. The
spectrum of the copper complex contained two intra-ligand
charge-transfer bands at ca 43,478 and 37,593 cm-1 and a
d–d band at ca 13,262 cm-1 due to the Eg ? T2g transi-
tion. This d–d band was strongly indicative of distorted
octahedral geometry around the metal ion. This is further
supported by the magnetic susceptibility value of 1.84 lB.
The absorption spectrum of the nickel(II) complex contains
three d–d bands at 14,836, 16,806, and 23,255 cm-1. These
bands correspond to the 3A2g(F) ? 3T2g(F)(m1), 3A2g(F) ?
2
2
3
3
3T1g(F)(m2), and A2g(F) ? T1g(P)(m3) transitions, respec-
tively, and are characteristic of octahedral geometry. This
geometry is further supported by the magnetic susceptibility
(3.27 BM).The absorption spectrum of the Zn(II) complex
contained intra-ligand charge-transfer bands at ca. 38,461
and 28,575 cm-1 only.
NMR spectra
EPR spectral studies
1H NMR (300 MHz) and 13C NMR spectra of the Schiff base
and its zinc complex were recorded in CDCl3. The 1H NMR
spectrum of the Schiff base had a singlet at d = 5.5 ppm
The EPR spectrum of the Cu(II) complex was recorded in
DMSO solution at 300 K (room temperature) and 77 K
123

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N. Raman et al.
(liquid nitrogen temperature). The EPR spectra of the metal
chelates give information about hyperfine and super
hyperfine structures, which are important in studying the
metal ion environment in the complexes. The Cu(II)
complex at 300 K gave one intense absorption band in the
high field region and was isotropic because of the tumbling
motion of the molecules. However, this complex in the
frozen state contained four well-resolved peaks of low
intensity in the low-field region and one intense peak in the
high-field region. The spin Hamiltonians of the complex
complex is mononuclear. Electron paramagnetic resonance
and optical spectra have been used to determine covalent
bonding data for the Cu(II) ion in
a variety of
environments. Because of the wide interest in the nature
of the bonding in this system, we used simplified molecular
orbital theory [16] to calculate the bonding coefficients a2
(covalent in-plane r-bonding) and b2 (covalent in-plane
p-bonding). The in-plane r-bonding coefficient a2 is related
to g and g\ by the equation:
k
a2 ¼ ðA =0:036Þ þ ðg ꢂ 2:0027Þ þ 3=7ðg ꢂ 2:0023Þ
?
jj
jj
were calculated. The g and g\ values were computed from
k
þ 0:04
the spectrum by using the tetracyanoethylene (TCNE) free
radical as the ‘‘g’’ marker. From the values obtained it is
clear that A (154) [ A\ (32) and g (2.30) [ g\ (2.07) [
An a2 value of 0.5 is indicative of completely covalent
bonding whereas a value of a2 = 1.0 suggests completely
ionic bonding. The observed value of a2 (0.79) indicates that
the complex has some covalent character. The in-plane
p-bonding(b2) coefficientiscalculated byuse ofthe equation:
k
k
2.0023, suggesting that the complex has axially elongated
octahedral geometry and the unpaired electron lies pre-
dominantly in the dx2 -y2 orbital [15]. The axial symmetry
coefficient G is defined as:
À
Á
b2 ¼ g ꢂ 2:0027 E= ꢂ 8ka2
jj
g ꢂ 2:0023
jj
where k = -828 cm-1 for the free ion and E is the elec-
G ¼
:
g ꢂ 2:0023
?
tronic transition energy for 2Eg ? T2g. The observed
2
value of b2 (0.75) indicates that the r-bonding is more
covalent than the in-plane p-bonding. On the basis of the
above spectral and analytical data, the model proposed for
the copper complex is given in Fig. 1.
The calculated G (4.3) values for the complex indicate
there is no interaction between the copper centers in
DMSO. The magnetic moment of the complex is 1.84 BM,
corresponding to one unpaired electron, indicating the
Fig. 1 Model proposed for the
copper complex
123

Isatin-derived tyramine bidentate Schiff base
1023
Table 2 Electronic absorption spectral properties of the Cu(II),
Ni(II), and Zn(II) complexes
Compound
k
max/nm
Dk/nm
H%a
Kb/M-1 b
Free
Bound
[CuL2Cl2]
[NiL2Cl2]
[ZnL2Cl2]
321.0
305.5
305.3
285.5
323.1
307.1
306.4
286.5
2.1
1.6
1.1
1.0
23
19
13
11
2.3 9 104
1.9 9 104
1.3 9 104
a
H% = [Afree - Abound)/Afree] 9 100%
b
Kb = Intrinsic DNA binding constant determined from the UV–
visible absorption spectral titration
and presence of calf thymus DNA (CT DNA) are given in
Figs. 2 and 3.
The absorption spectra of the metal complexes con-
tained an intense absorption band at 321 nm for copper,
305.5 nm for nickel, and 305.3 and 285.5 nm for zinc in
5 mM Tris–HCl and 50 mM NaCl in pH 7.2 buffer solu-
tion. Increasing the concentration of CT DNA resulted in a
minor bathochromic shift in the range *2.1–1.0 nm and
significant hypochromicity in the range *23–11% for all
the complexes, indicating substantial groove–surface
binding propensity of the complexes to the CT DNA [17].
The equilibrium binding constant (Kb) values were 2.3 9
104 M-1, 1.9 9 104 M-1, and 1.1 9 104 M-1 for the
copper, nickel, and zinc complexes. These small binding
constants also indicate more surface aggregation and/or
groove binding than any interaction with the DNA base
pairs. To compare the binding strength of the complexes
with CT DNA, the intrinsic binding constants (Kb) were
obtained by monitoring the changes in the absorbance of
the complexes with increasing concentration of DNA. Kb
was obtained from the ratio of the slope to the intercept
from a plot of [DNA]/(ea – ef) versus [DNA]. The Kb
values are shown in Table 2.
Fig. 2 Absorption spectrum of the [CuL2Cl2] complex in buffer of
pH 7.2 at 25°C in the presence of increasing amounts of DNA. The
arrow indicates the changes in absorbance on increasing the DNA
concentration
Viscosity measurements
To clarify further the interaction between the metal com-
plexes and DNA, viscosity measurements were carried out.
As is well known, optical photophysical probes are nec-
essary, but not sufficient, clues to support a binding mode
between DNA and a small molecule. Hydrodynamic
measurements (i.e. viscosity and sedimentation), which are
sensitive to length change, are regarded as the least
ambiguous and the most critical tests of binding mode in
solution in the absence of crystallographic structural data
[18]. A classical intercalation model results in lengthening
of the DNA helix, as base pairs are separated to accom-
modate the binding ligand, leading to increased DNA
viscosity. In contrast, groove binding or partial intercala-
tion leads to only a minor change in the viscosity. A plot of
relative specific viscosity (g/go)1/3 versus the [complex]/
Fig. 3 Absorption spectrum of the [ZnL2Cl2] complex in buffer of
pH 7.2 at 25°C in the presence of increasing amounts of DNA. The
arrow indicates the changes in absorbance on increasing the DNA
concentration
DNA binding studies
Absorption titration
Absorption titration can be used to monitor the interaction
of a metal complex with DNA. In general, a complex
bound to DNA by intercalation usually results in hypo-
chromism and red shift (bathochromism), because of the
strong stacking interaction between aromatic chromophore
of the complex and the base pairs of DNA. The absorption
spectra of the Cu(II) and Zn(II) complexes in the absence
123

1024
N. Raman et al.
Fig. 6 Cyclic voltammogram of [NiL2Cl2] in buffer of pH 7.2 at
25°C in the presence of increasing amounts of DNA. The arrow
indicates the changes in voltammetric current on increasing the DNA
concentration
Fig. 4 Effect of increasing amounts of [CuL2Cl2] (diamonds),
[NiL2Cl2] (circles), [ZnL2Cl2] (triangles), and [EB] (squares) on
the relative viscosity of DNA. R = [complex]/[DNA] or [EB]/[DNA]
Table 3 Electrochemical data for interaction of DNA with the Cu(II),
Ni(II), and Zn(II) complexes
Compound Redox couple
E
1/2/V
DEp/V
Ipa/
Ipc
Free
Bound Free Bound
[CuL2Cl2] Cu(III) ? Cu(II) 0.140 0.127 0.58 0.61
Cu(II) ? Cu(I) -0.130 -0.147 0.56 0.58
1.60
1.30
1.20
Cu(I) ? Cu(0)
-0.410 -0.421 0.84 0.87
[NiL2Cl2] Ni(II) ? Ni(I)
-0.301 -0.345 0.475 0.526 0.94
[ZnL2Cl2] Zn(II) ? Zn(0) -0.298 -0.360 0.467 0.378 1.05
Data from cyclic voltammetric measurements: E1/2 is calculated as
the average of anodic (Epa) and cathodic (Epc) peak potentials
(E1/2 = (Epa ? Epc)/2); DEp = Epa - Epc
for example UV–visible spectroscopy, viscosity measure-
ment, and molecular docking analysis. The cyclic
voltammograms of the copper and nickel complexes in the
absence and presence of different amounts of DNA are
shown in Figs. 5 and 6.
Fig. 5 Cyclic voltammogram of [CuL2Cl2] in buffer of pH 7.2 at
25°C in the presence of increasing amounts of DNA. The arrow
indicates the changes in voltammetric current on increasing the DNA
concentration
In the absence of CT DNA, the first redox cathodic peak
appeared at -0.156 V for Cu(III) ? Cu(II) (Epa =
[DNA] ratio (R, Fig. 4) shows only a minor change in the
viscosity in comparison with classical DNA intercalators,
for example ethidium bromide. The results reveal the
partial intercalation and/or groove binding nature of the
complexes [19].
0.426 V, Epc = -0.156 V, DEp = 0.58 V, and E1/2
=
0.14 V); in the second redox couple, the cathodic peak
appeared at -0.408 V for Cu(II) ? Cu(I) (Epa = 0.154 V,
Epc = -0.408 V, DEp = 0.56 V, and E1/2 = -0.13 V);
and in the third redox couple, the cathodic peak appeared at
-0.836 V for Cu(I) ? Cu(0) (Epa = 0.008 V, Epc =
-0.836 V, DEp = 0.84 V, and E1/2 = -0.41 V). The
Ipa/Ipc ratios for these three redox couples were 1.6, 1.3,
and 1.2, respectively, which indicate that reaction of the
complex on the glassy carbon electrode surface is a quasi-
reversible redox process. During the incremental addition
Electrochemistry
Electrochemistry has been increasingly helpful in elucidat-
ing the basic chemistry of biological systems. Use of
electrochemical methods to study interactions of organic
compounds and metal compounds with DNA is a useful
complement to the previously used methods of investigation,
123

Isatin-derived tyramine bidentate Schiff base
1025
of CT DNA to the complex, the redox couples caused a less
negative shift in E1/2 and decrease of DEp (Table 3).The
Ipa/Ipc values also decreased in the presence of DNA. For
Ni(II) ? Ni(I) the redox couple cathodic peak appeared at
-0.539 V in the absence of CT DNA (Epa = -0.064 V,
Epc = -0.539 V, DEp = 0.475 V, and E1/2 = -0.301 V).
The Ipa/Ipc ratio was approximately unity. This indicates
the quasi-reversible redox process of the metal complex.
During incremental addition of CT DNA to the complex,
the redox couple caused a negative shift in E1/2 and a
decrease in DEp.
Finally a quasi-reversible transfer process with the redox
couple [Zn(II) ? Zn(0)] was observed for the Zn(II)
complex. The cathodic peak appeared at -0.532 V in the
absence of DNA (Epa = -0.065 V, Epc = -0.532 V,
DEp = 0.467 V, and E1/2 = -0.298 V). The Ipa/Ipc ratio
was 1.05. This indicates the quasi-reversible redox process
of the metal complex. Incremental addition of DNA to the
Zn(II) complex resulted in a slight decrease in the current
intensity and negative shift of the oxidation peak potential.
The resulting minor changes in the current and potential are
indicative of diffusion of the metal complexes bound to the
large, slowly diffusing DNA molecule [20]. Electrochem-
ical data for the Cu(II), Ni(II), and Zn(II) complexes are
shown in Table 3.
was reported for [Cu(L-arg)2](NO3)2 binding to DNA [21].
Thus, the narrower AT regions seemed to facilitate better
fit of these molecules into the minor groove than the GC
regions, probably as a result of van der Waals’ interactions
with the functional groups defining the groove. Thus, the
complexes bound preferentially at the AT region of the
dodecamer.
Hydrogen bonding between the C-2 carbonyl oxygen of
T and the N-3 nitrogen of A with the minor groove binder
seemed to be equally important [22]. The results suggested
binding of copper and zinc to the phosphate backbone of
the polynucleotide involving the terminal positively
charged guanidinium groups of arginine via H-bonding and
ionic contacts [23]. The optimum binding conformation in
the AT stretches of the minor groove was indicative of an
extensive H-bonding network. Structural analysis of the
docking positions gave significant details of the binding
pattern of the complexes.
This finding is consistent with binding data obtained
from spectroscopic titration, cyclic voltammetry, and vis-
cosity measurement. The binding energy for docking of the
copper, nickel, and zinc complexes with DNA were
-120.1, -119.0, and -118.1 kJ mol-1, respectively,
which correlated well with the experimental DNA binding
values. The larger the negative value of the binding energy
the greater the binding potential of the metal complexes
with DNA.
Docking of DNA–metal complexes
The docking studies further substantiated the existence of
strong interactions between the metal complexes and DNA.
The complexes could fit well into the minor groove of the
DNA with a binding site of three base pairs, as revealed by
the docked structures, preferentially involving the AT
residues, as shown in Fig. 7. A similar type of interaction
Chemical nuclease activity
Study of the DNA cleavage capacity of the transition metal
complexes is extremely interesting because it can con-
tribute to understanding of their mechanism of toxicity and
to the development of novel artificial nucleases. DNA
cleavage is controlled by relaxation of the supercoiled
circular form of pUC19 DNA into the nicked circular form
and the linear form. In electrophoresis of circular plasmid
DNA the fastest migration will be observed for the
supercoiled form (form I). If one strand is cleaved, the
supercoils will relax to produce a slower-moving open
circular form (form II). DNA cleavage was analyzed by
monitoring the conversion of supercoiled DNA (form I) to
nicked DNA (form II). If both strands are cleaved, a linear
form (form III) will be generated that migrates in between.
DNA cleavage was analyzed by monitoring the con-
version of supercoiled DNA (form I) to nicked DNA (form
II) and linear DNA (form III) in the presence of H2O2.
From Fig. 8, it is evident that the complexes cleave DNA
more efficiently in the presence of H2O2. This may be
attributed to the formation of hydroxyl free radicals.
Production of hydroxyl radicals by reaction of the metal
complex with hydrogen peroxide may be explained as
shown below:
Fig. 7 Interaction of the complexes [CuL2Cl2] and [ZnL2Cl2] with
d(CGCGAATTCGCG) strands of DNA by the minor groove binding
approach
123

1026
N. Raman et al.
1
2
3
4
5
6
Form II
Form I
Fig. 8 Changes in the agarose gel electrophoretic pattern of pUC19
DNA induced by H2O2 and metal complexes; lane 1, DNA alone;
lane 2, DNA ? H2O2; lane 3, DNA ? ligand ? H2O2; lane 4,
DNA ? [CuL2Cl2] complex ? H2O2; lane 5, DNA ? [NiL2Cl2]
complex ? H2O2; lane 6, DNA ? [ZnL2Cl2] complex ? H2O2
Tweedy’s chelation theory [24]. On chelation, the polarity
of the metal ion is reduced because of overlap of the ligand
orbital and partial sharing of positive charge of the metal
ion with the donor groups. Further, chelation reduces the
delocalization of p-electrons over the whole chelate ring
and enhances the lipophilicity of the complexes. This
increased lipophilicity enhances the penetration of the
complexes into the lipid membrane and blocking of the
metal-binding sites in the enzymes of the microorganisms.
The metal complexes also disturb the respiration process of
the cell and thus block the synthesis of proteins, which
restricts further growth of the organisms. The results
obtained from antibacterial tests showed that the metal
complexes are more active against bacteria. The copper
complex has greater antibacterial activity (16–27 mm)
against the tested microorganisms than the nickel and zinc
complexes, for which the zones of inhibition are in the
range of 11–24 mm. This may be attributed to the atomic
radius and the electronegativity of the Cu(II) ion. Studies
have revealed that complexes of electronegative metal ions
with high atomic radius have high antimicrobial activity.
Higher electronegativity and large atomic radius reduce the
effective positive charge on the metal complex, which
facilitates its interaction with the highly sensitive cellular
membranes [25].
Table 4 In-vitro antibacterial activity of Schiff base and its metal
complexes (zone of inhibition/mm)
Compound E. coli B. subtilis S. aureus S. typhi K. pneumoniae
L
8
6
21
23
19
16
6
17
15
10
12
10
16
14
12
14
16
27
24
16
23
[CuL2Cl2] 19
[NiL2Cl2] 15
[ZnL2Cl2] 11
Ampicillina 12
a
Ampicillin was used as the standard
ðLigandÞM2þ þ H2O2 ! ðLigandÞM3þ þ OHꢂ þ OH:
The OHꢀ free radicals participate in the oxidation of the
deoxyribose moiety; this is followed by hydrolytic cleav-
age of a sugar phosphate backbone. All the complexes have
pronounced nuclease activity in the presence of H2O2; this
may be because of the increased production of hydroxyl
radicals. Control experiments using DNA alone do not
result in significant cleavage of pUC19 DNA, even when
exposure time is longer. From these results it is concluded
that all the complexes effectively cleave DNA.
Antimicrobial activity
The synthesized Schiff base and its complexes were tested
for their in-vitro antibacterial activity. They were tested
against the bacteria Klebsiella pneumoniae, Staphylococ-
cus aureus, Bacillus subtilis, Escherichia coli, and
Salmonella typhi by the paper disc method. Comparative
study of the ligand and its complexes indicated the com-
plexes were more active than the free ligand and the
control; the results obtained are summarized in Table 4.
These results indicate the ligand has moderate antibac-
terial activity compared with the complexes against the
same microorganisms under identical experimental condi-
tions. The ligand has antimicrobial activity in the range
6–16 mm against all the pathogens. Such increased activity
of the complexes can be explained on the basis of
Docking of enzyme–Schiff base and its metal complexes
The results from in-vitro antimicrobial property screening
revealed specific antimicrobial activity of the metal com-
plexes. In-silico studies were conducted to support the in-
vitro activity. Computer-docking techniques are important
in the drug design and discovery. Mechanistic studies
which place a molecule into the binding site of the target
macromolecule in a non-covalent fashion [26, 27] can be
used to predict the correct binding geometry for each
ligand at the active site, to reveal binding energy values,
and to reveal hydrogen bonds formed with the surrounding
amino acids. In this work the bacterial surface enzyme
123

Isatin-derived tyramine bidentate Schiff base
1027
Fig. 11 The binding model of the Schiff base complex [ZnL2Cl2] in
the enzyme active site of Staphylococcus aureus sortase-A
Fig. 9 Crystal structure of Staphylococcus aureus sortase-A (PDB
ID1T2P)
Fig. 12 The binding model of the Schiff base complex [NiL2Cl2] in
the enzyme active site of Staphylococcus aureus sortase-A
Fig. 10 The binding model of the Schiff base complex [CuL2Cl2] in
the enzyme active site of Staphylococcus aureus sortase-A
models for the zinc and nickel complexes in the enzyme
active site of Staphylococcus aureus sortase-A are shown
in Figs. 11 and 12. The copper and zinc complexes have
the highest docked scores with the bacterial surface
enzyme Staphylococcus aureus sortase A. The copper
complex has a docked score of -480.64 and forms two H
bonds with Gly79 and Ser80. The zinc and nickel com-
plexes have docked scores of -480.21 and -460.53 and
form two H bonds with same amino acid residues. The
ligand has a docked score of -333.90 and forms one
hydrogen bond with Ser80. The docked scores and the
hydrogen bonds formed with the surrounding amino acids,
i.e. the binding affinities as predicted by molecular docking
studies, are in good agreement with results from antibac-
terial screening.
Staphylococcus aureus sortase-A (Fig. 9), the Schiff base,
and its metal complexes were docked by Open Eye with
Fast Rigid Exhaustive Docking using the FRED docking
software package.
As a result of docking one-hundred different positions
were generated for each compound, but only the top ranked
docked score was taken. A larger negative value of the
docked score indicates stronger receptor–ligand binding
affinity. Figure 10 shows the docking position of the cop-
per complex which seems to be important in binding to
Staphylococcus aureus sortase-A. The metal complexes
were the most active in this study, because of good
hydrogen-bonding interaction with the amino acid residues
of the active site of the sortase A enzyme. The binding
123

1028
N. Raman et al.
Conclusion
FT IR-8400S instrument and were recorded at the University
Science Instrumentation Centre (USIC), Madurai Kamaraj
University, Madurai, India. NMR spectra were recorded on a
Bruker Avance 300 FT-NMR spectrometer in CDCl3 using
TMS as internal reference. FAB-MS spectra were recorded
with a VGZAB-HS spectrometer 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 at liquid nitrogen temperature
(77 K) using tetracyanoethylene as the g-marker. Absorp-
tion spectra were recorded at room temperature using a
Shimadzu model UV-1601 spectrophotometer.
In the work discussed in this paper, a new Schiff base
ligand, 3-[2-(4-hydroxyphenyl)-ethylimino]-1,3-dihydroin-
dole-2-one, obtained by reaction of 1H-indole-2,3-dione
(isatin) and 4-(2-aminoethyl)phenol (tyramine), and its
Cu(II), Ni(II), and Zn(II) complexes were been character-
ized by use of spectral and analytical data. IR, electron
transition, and g tensor data led to the conclusion that the
Cu(II) ion assumes distorted octahedral geometry and
the other complexes are octahedral in nature. Interaction of
the metal complexes with DNA was investigated by
spectroscopic and molecular docking methods. The results
showed that all the complexes interact with DNA by minor
groove binding. The docked structures reveal the com-
plexes can fit well into the minor groove of the DNA with a
binding site of three base pairs preferentially involving the
AT residues. The complexes have enhanced chemical
nuclease activity in the presence of hydrogen peroxide.
Antimicrobial activity of the metal complexes was evalu-
ated against the bacterial strains Klebsiella pneumoniae,
Staphylococcus aureus, Bacillus subtilis, Escherichia coli,
and Salmonella typhi. This investigation concludes that the
metal complexes described may be potential new drugs for
management of bacterial diseases after evaluating the in-
vivo effect of the metal complexes. Results from molecular
docking experiments should extend knowledge of the nat-
ure of the binding of these compounds to the enzyme.
Hence this paper has widened the scope of development of
the tyramine-derived isatin-based Schiff base and its metal
complexes as promising DNA binding and antibacterial
agents. The research has led to the discovery of a series of
compounds for further pharmacological investigation.
3-[2-(4-Hydroxyphenyl)ethylimino]-1,3-dihydro-2H-
indole-2-one (C16H14N2O2)
The Schiff base ligand was prepared by addition of 25 cm3
methanolic solution of 1.36 g tyramine (0.01 mol) to
25 cm3 methanolic solution of 1.47 g isatin (0.01 mol)
and the reaction mixture was heated under reflux for ca.
6 h. The dark brown product formed was isolated by
filtration, washed with methanol, and dried in vacuo. Yield:
86%; m.p.: 213°C; FT-IR (KBr):vꢀ = 3,570 (OH), 3,248
(NH), 1,706 (C=O), 1,616 (C=N) cm-1; 1H NMR (CDCl3):
d = 10.8 (s, 1H, NH), 6.8–7.9 (m, 8H, phenyl), 5.5 (s, 1H,
OH), 2.4 (t, 2H, CH2Ar), 1.5 (t, 2H, CH2ArOH) ppm; 13C
NMR (CDCl3): d = 39.81 (–CH2Ar), 40.64 (CH2ArOH),
115.17, 116.25, 121.96, 122.41, 135.23 (Ar–C), 111.55,
115.17, 121.97, 125.32 (indole–C), 156.74 (C=O), 164.55
(C = N) ppm; UV–Vis (DMSO): kmax = 39,215, 27,510
cm-1; MS: m/z = 266.
Synthesis of metal complexes
The complexes were prepared by mixing the appropriate
molar quantity of the ligand and the metal salts by use of
the following procedure: 20 cm3 of a methanolic solution
of 0.532 g Schiff base (2 mmol) was stirred with 10 cm3
methanolic solution of anhydrous metal(II) chloride
(1 mmol) and heated under reflux for ca. 2 h. The solid
product obtained was isolated by filtration, washed with
methanol, and dried in vacuo.
Experimental
Reagents and instruments
All reagents, isatin, tyramine, and metal(II) chlorides were
Merck products and used as supplied. Commercial solvents
were distilled and then used for preparation of ligand and its
complexes. DNA was purchased from Bangalore Genei
(India). Microanalysis (C, H, and N) was performed with a
Carlo Erba 1108 analyzer at Sophisticated Analytical
Instrument Facility (SAIF), Central Drug Research Institute
(CDRI), Lucknow, India. Molar conductivities were mea-
sured for 10-3 M solutions in DMSO at room temperature
using a Systronic model-304 digital conductivity meter.
Magnetic susceptibility of the complexes were measured by
use of a Gouy balance using copper sulfate pentahydrate as
the calibrant. IR spectra of the ligand and its complexes, as
KBr discs, were obtained in the range 350–4,500 cm-1 on an
Bis[3-[2-(4-hydroxyphenyl)ethylimino]-1,3-dihydro-2H-
indole-2-one]dichlorocopper(II) (C32H28Cl2CuN4O4)
Yield: 71%; m.p.: [237°C; FT-IR (KBr):vꢀ = 3,570 (OH),
3,249 (NH), 1,687 (C=O), 1,556 (C=N), 514 (M=O), 479
(M=N), 362 (M–Cl) cm-1
; ;
Km = 1.9 S cm2 mol-1
leff = 1.84 BM; UV–Vis (DMSO): kmax = 43,478,
37,593, 13,262 cm-1; MS: m/z = 667 (M?).
Bis[3-[2-(4-hydroxyphenyl)ethylimino]-1,3-dihydro-2H-
indole-2-one]dichloronickel(II) (C32H28Cl2N4NiO4)
Yield: 54%; m.p.: [237°C; FT-IR (KBr):vꢀ = 3,570 (OH),
3,250 (NH), 1,683 (C=O), 1,553 (C=N), 513 (M=O), 471
123

Isatin-derived tyramine bidentate Schiff base
1029
(M=N), 358 (M–Cl) cm-1
; ;
Km = 2.1 S cm2 mol-1
binding ratio, where g is the viscosity of DNA in the
presence of complex and g0 is the viscosity of DNA alone.
leff = 1.86 BM; UV–Vis (DMSO): kmax = 14,836,
16,806, 23,255 cm-1; MS: m/z = 663 ([M ? 1]?).
Molecular modeling
Bis[3-[2-(4-hydroxyphenyl)ethylimino]-1,3-dihydro-2H-
indole-2-one]dichlorozinc(II) (C32H28Cl2N4O4Zn)
DNA–metal complex docking
Yield: 67%; m.p.: [237°C; FT-IR (KBr):vꢀ = 3,572 (OH),
3,249 (NH), 1,666 (C=O), 1,541 (C=N), 511 (M=O), 465
(M=N), 356 (M–Cl) cm-1; 1H NMR (CDCl3): d = 10.9 (s,
1H, NH), 6.5–7.8 (m, 8H, phenyl), 5.5 (s, 1H, OH), 2.4 (t,
2H, CH2Ar), 1.5 (t, 2H, CH2ArOH) ppm;13C NMR
(CDCl3): d = 39.81 (–CH2Ar), 40.64 (CH2ArOH),
115.17, 116.25, 121.96, 122.41, 135.23 (Ar–C), 111.55,
115.83, 121.96, 126.22 (indole–C), 154.65 (C=O), 162.88
(C=N) ppm; Km = 1.4 S cm2 mol-1; UV–Vis (DMSO):
kmax = 38,461, 28,575 cm-1; MS: m/z = 671 ([M ? 2]?).
Interaction of the metal complexes with DNA was also
studied by molecular modeling with special reference to
docking. All calculations were performed in Open Eye with
Fast Rigid Exhaustive Docking using the FRED docking
software package. The main function used for the calcula-
tion was Chemgauss 2. Before docking, the structures of the
metal complexes were constructed using ChemDrawUltra
11.0, geometry optimized by MM2 force field, and saved as
Pdb format. The crystal structure of the complex of ne-
tropsin with B-DNA dodecamer d(CGCGAATTCGCG)2
(NDB code GDLB05) was downloaded from the Protein
Data Bank. During the docking analysis, the binding site of
the netropsin drug in the minor groove of the above com-
plex was assigned as the binding site for our metal
complexes across the entire DNA molecule. The docked
positions were generated by use of exhaustive search and
optimization steps. FRED selected the single best position
from the set of candidates. This position was then scored
and the score was used to rank ligands in the output hit list.
The consensus structure step enabled multiple scoring
functions to vote for the best docked structure in a rank-by-
vote approach.
DNA binding experiments
The interaction between the metal complexes and DNA
was studied using electronic absorption, viscosity, and
electrochemical methods. The disodium salt of calf thymus
DNA was stored at 4°C. A solution of DNA in aqueous
buffer (50 mM NaCl, 5 mM Tris HCl, pH 7.2) gave a ratio
1.9 for UV absorbance at 260 and 280 nm (A260/A280),
indicating the DNA was sufficiently free from protein [28].
The concentration of DNA was measured by use of its
extinction coefficient at 260 nm (6,600 dm3 mol-1 cm-1
)
after 1:100 dilution. Stock solutions were stored at 4°C for
no more than 4 days. Doubly distilled water was used to
prepare solutions. In all experiments concentrated stock
solutions of the complexes were prepared by dissolving the
complexes in DMSO and diluting suitably with the corre-
sponding buffer to the required concentration.
Docking of the enzyme–Schiff base and its metal
complexes
Docking of the enzyme–Schiff base and its metal com-
plexes was also carried out by using FRED (version VIDA-
2.1.2, Open Eye Structure software). The crystal structure
of the Staphylococcus aureus sortase-A (PDB code 1T2P)
was downloaded from Protein Data Bank. Crystallographic
water molecules were removed from the protein. The main
function used for calculation was Shapegauss, which pre-
dicted favorable protein–ligand complex interactions.
Absorption titration experiments were carried out vary-
ing the DNA concentration and maintaining the complex
concentration constant. Absorbance values were recorded
after each successive addition of DNA solution and equil-
ibration (ca. 10 min). The absorption data were analyzed for
evaluation of the intrinsic binding constant Kb, by use of a
procedure reported elsewhere [29].
pUC19 DNA cleavage study
Electrochemical studies were carried out using a CHI
electrochemical analyzer, controlled by CHI620C software.
CV measurements were performed using a glassy carbon
working electrode and an Ag/AgCl reference electrode; the
supporting electrolyte was 50 mM NaCl, 5 mM Tris buffer
(pH 7.2). Before measurements, all solutions were deoxy-
genated by purging with N2 for 30 min.
Cleavage of pUC19 DNA was determined by agarose gel
electrophoresis. The gel-electrophoresis experiments were
performed by incubation of samples containing 30 mM
pUC19 DNA, 50 mMcopper complex, and 50 mMhydrogen
peroxide (H2O2) in Tris–HCl/NaCl buffer (pH 7.2) at 37°C
for 2 h. After incubation, the samples were electrophoresed
for 2 h at 50 V on 1% agarose gel using Tris–acetic acid–
EDTA buffer (pH 7.2). The gel was then stained using
1 lg cm-3 ethidium bromide (EB) and photographed under
ultraviolet light at 360 nm. All the experiments were per-
formed at room temperature unless otherwise stated.
Viscosity measurements at room temperature were car-
ried out using a semi-micro dilution capillary viscometer.
Each experiment was performed three times and an average
flow time was calculated. Data are reported as (g/g0) versus
123

1030
N. Raman et al.
6. Jarrahpour A, Khalili D, Clercq ED, Salmi C, Brunel JM (2007)
Molecules 12:1720
Antimicrobial activity studies
7. Jacob G, Gamboa A, Diedrich A, Shibao C, Robertson D, Bia-
ggioni I (2005) Hypertension 46:355
The in-vitro antibacterial activity of the ligand and its
complexes were tested against the bacteria Klebsiella
pneumoniae, Staphylococcus aureus, Bacillus subtilis,
Escherichia coli, and Salmonella typhi, by use of the paper
disc method using nutrient agar as the medium. The solu-
tion was prepared by dissolving the compound in DMSO
and entire blank discs were moistened with the solvent. For
disc assays, paper (6 mm) containing the compounds was
placed on nutrient agar plates previously spread with
0.1 cm3 of overnight cultures of microorganisms. After
incubation at 37°C for 36 h the diameter of the inhibition
zone was measured.
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Acknowledgments The authors express their sincere thanks to the
College Managing Board, Principal and Head of the Department of
Chemistry, VHNSN College, Virudhunagar, India, for providing the
necessary research facilities and financial support. They also wish to
acknowledge the help rendered by Thigarajar College with regard to
computational facilities.
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