Copper(II) and nickel(II) complexes of benzyloxybenzaldehyde-4-phenyl-3- thiosemicarbazone: Synthesis, characterization and biological activity

Article abstract of DOI:10.1016/j.saa.2010.05.016

Benzyloxybenzaldehyde-4-phenyl-3-thiosemicarbazone ligand (L) has been synthesized from benzyloxybenzaldehyde and 4-phenyl-3-thiosemicarbazide. Complexes of this ligand with chlorides of Cu(II) and Ni(II) have been prepared. The structure of the ligand (L

Full text of DOI:10.1016/j.saa.2010.05.016

Spectrochimica Acta Part A 77 (2010) 248–252
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Copper(II) and nickel(II) complexes of benzyloxybenzaldehyde-4-phenyl-
3-thiosemicarbazone: Synthesis, characterization and biological activity
B. Prathimaa, Y. Subba Raoa, S. Adinarayana Reddyb, Y.P. Reddyc, A. Varada Reddya,∗
a Analytical Division, Dept. of Chemistry, S.V. University, Tirupati 517502, India
b Dept. of Material Science and Nanotechnology, Y.V. University, Kadapa 516001, India
c Dept. of Physics, Sri Padhmavati Mahila Visva Vidyalayam (Women’s University), Tirupati 517502, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Benzyloxybenzaldehyde-4-phenyl-3-thiosemicarbazone ligand (L) has been synthesized from benzy-
loxybenzaldehyde and 4-phenyl-3-thiosemicarbazide. Complexes of this ligand with chlorides of Cu(II)
and Ni(II) have been prepared. The structure of the ligand (L) is proposed based on elemental analysis, IR
and 1H NMR spectra. Its complexes with Cu(II) and Ni(II) ions are characterized from the studies of elec-
tronic as well as EPR spectra. On the basis of electronic and EPR studies, rhombically distorted octahedral
structure has been proposed for Cu(II) complex while the Ni(II) complex has been found to acquire an
octahedral structure. The ligand and their metal complexes have been tested in vitro for their biological
effects. Their antibacterial activities against Gram-negative bacteria (Escherichia coli and Klebsiella pneu-
moniae) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) have been investigated.
The prepared metal complexes exhibit higher antibacterial activities than the parent ligand. The in vitro
antioxidant activity of free ligand and its metal(II) complexes have also been investigated and the results
however reveal that the ligand exhibits greater antioxidant activity than its complexes.
Received 30 December 2009
Received in revised form 28 April 2010
Accepted 15 May 2010
Keywords:
EPR spectra
In vitro antibacterial and antioxidant
activities
Electronic spectra
Benzyloxybenzaldehyde-4-phenyl-3-
thiosemicarbazone
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
of imine group(–N CH–) which imparts the biological activity
[19]. Collins et al. have reported the correlation between struc-
The chemistry of thiosemicarbazones has received consider-
able attention in view of their variable bonding modes, promising
biological implications, structural diversity and ion-sensing abil-
ity [1–3]. Thiosemicarbazones are now well established as an
important class of sulphur donor ligands particularly for transi-
tion metal ions [4–6]. This is due to remarkable biological activities
observed for these compounds, which have since been shown to
be related to their metal complexing ability. These compounds
present a great variety of biological activities ranging from antiviral
[7] to anticancer [8], antitumour [9,10], antibacterial [11–13], anti-
inflammatory and antiamoebic [14–16] activities. The inhibitory
action of these compounds is attributed to their chelating proper-
ties [17]. The activity of these compounds is strongly dependent
upon the nature of the hetero atomic ring and the position of
attachment of thiosemicarbazone group to the ring as well as
the form of thiosemicarbazone moiety [18]. These compounds
are studied extensively due to their flexibility, their selectivity
and sensitivity towards the central metal atom, their structural
similarities with natural biological substances and the presence
ture and anti-mycobacterial activity in a series of 2-acetylpyridine
thiosemicarbazones [20]. In many cases, due coordination to differ-
ent transition metal ions that can be found in biological systems, it
is possible to obtain complexes that are more efficient drugs than
the corresponding free ligands [21–24].
In the present work, as
a continuation of the research
program related to the coordination chemistry and biological
activity of thiosemicarbazones in our laboratory [25], the syn-
thesis and spectroscopic characterization of Cu(II) and Ni(II)
complexes of the ligand, benzyloxybenzaldehyde-4-phenyl-3-
thiosemicarbazone are reported. The in vitro antibacterial and
antioxidant activities of these compounds are evaluated.
2. Experimental
2.1. Materials
All the chemicals used are of analytical grade. Organic chemicals
such as thiobarbituric acid (TBA), trichloroaceticacid (TCA), ␣-
tocopherol, butylated hydroxytoluene (BHT), 1,1-diphenyl-2-picryl
hydrazyl (DPPH), 4-phenyl-3-thiosemicarbazide and dimethyl for-
mamide (DMF) are procured from Sigma–Aldrich and all metal salts
are procured from E-Merck chemical companies.
∗
Corresponding author. Tel.: +91 877 2249666x303; fax: +91 877 2249611.
E-mail address: ammireddyv@yahoo.co.in (A. Varada Reddy).
1386-1425/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2010.05.016

B. Prathima et al. / Spectrochimica Acta Part A 77 (2010) 248–252
249
2.2. General procedures
To each agar well, 100 ml of the compound reconstituted in DMF
in concentration of 1.0 mg/ml is added. DMF is used as a negative
control and antibiotics such as ampicillin and tetracycline are used
as positive control standards. All the plates are incubated at 37 ◦C
for 24 h and they are observed for the growth inhibition zones. The
presence of clear zones around the wells indicated that the com-
pound is active. The diameter of zone of inhibition is calculated in
millimeters. The well diameter is deducted from the zone diame-
ter to get the actual zone of inhibition diameter and the values are
tabulated.
The IR spectra of the compounds are recorded on a Nicolet FT-
IR 560 Magna spectrometer using KBr(neat). The Bruker 300 MHz
NMR spectrometer is used to obtain the 1H NMR spectrum of the
ligand. A mass spectrum was recorded in a Quattro LC-Micro mass.
Elemental analysis is obtained from vario-micro qub elementar
analyzer. The electronic spectra of the complexes are recorded on
a PerkinElmer UV/VIS Lambda 950. EPR spectra are recorded on
an EPR spectrometer (JEOL FE-1X) operating in the X-band fre-
quencies with a modulation frequency of 100 kHz. 100 mg of each
compound is taken in a quartz tube for EPR measurements. The
magnetic field is scanned from 2200 to 4200 G, with a scan speed
of 250 G min−1. Absorbance is measured using systronics UV–VIS
spectrometer-117. Centrifugation is done using REMI centrifuge. A
digital pH meter (model L1-10 Elico, India) is used for measuring
pH.
2.6. DPPH scavenging activity
The principle for the reduction in DPPH free radicals is that the
antioxidant reacts with stable free radical DPPH and converts it
to 1,1-diphenyl-2-picrylhydrazine. The ability to scavenge the sta-
ble free radical DPPH is measured by decrease in the absorbance
at 517 nm. Solutions of BBPTS and its Cu(II) and Ni(II) complexes
at 100 ␮M concentrations are added to 100 ␮M DPPH and kept
in ethanol tubes. The tubes are kept at ambient temperature for
20 min and absorbances are measured at 517 nm. ␣-Tocopherol is
used as a positive control [28]. These measurements are run in
triplicate. The percentage of scavenging activity is calculated as
follows:
2.3. Synthesis of
benzyloxybenzaldehyde-4-phenyl-3-thiosemicarbazone
Hot ethanolic solution containing 2.12 g of benzyloxybenzalde-
hyde is mixed with hot ethanolic solution containing 1.67 g of
4-phenyl-3-thiosemicarbazide. The mixture obtained is refluxed
for an hour, then stirred for 3.5 h at 60–70 ◦C and kept at room
temperature for a day. The resulting intense yellow colored pre-
cipitate is filtered, washed with ethanol and dried. The scheme is
shown below.
ꢀ
ꢁ
ADPPH − ATEST
Scavenging activity (%) =
× 100
ADPPH
where ADPPH is the absorbance of DPPH without test sample (con-
trol) and ATEST is the absorbance of DPPH in the presence of test
sample.
2.4. Synthesis of metal complexes
A hot ethanolic solution (25 ml) of free ligand (0.002 mol) and
a hot ethanolic solution (25 ml) of the corresponding metal salt
(0.001 mol) are refluxed for 3–4 h at 50 ◦C and on cooling the con-
tents, the colored complexes are precipitated out. They are filtered,
washed with 50% ethanol and dried. The purity of the complex is
checked by TLC.
2.7. Inhibition of lipid peroxidation in rat brain homogenate
2.7.1. Preparation of rat brain homogenate
Albino Wistar rats (180–200 g) are used for the study. Prior to
decapitation and removal of the brain, the animals are anesthetized
with ether and perfused transcardially with ice-cold normal saline
to prevent contamination of brain tissue with blood. Tissue was
weighed and its homogenate (10%, w/v) was prepared in 0.15 M
KCl and centrifuged at 800 rpm for 10 min. The supernatant is used
immediately for the study [29].
2.5. Antibacterial screening
In vitro antibacterial screening is performed by the agar disc
diffusion method [26,27]. The bacterial species used in the screen-
ing are Gram-negative bacteria such as Klebsiella pneumoniae and
Escherichia coli and Gram-positive bacteria such as Staphylococ-
cus aureus and Bacillus subtilis. Stock cultures of the test bacterial
species are maintained on nutrient agar media (Hi-media Lab-
oratories, Mumbai) by sub culturing in petri dishes. The media
are prepared by adding the components as per manufacturer’s
instructions and they are sterilized in the autoclave at 121 ◦C
and atmospheric pressure for 15 min. Each medium is cooled to
45–60 ◦C and 20 ml of it is poured into a Petri dish and allowed to
solidify. After solidification, Petri plates with media are spread with
1.0 ml of bacterial suspension prepared in sterile distilled water.
The wells are bored with cork borer and the agar plugs are removed.
2.7.2. Iron(III) induced lipid peroxidation
The incubation mixture contains in a final volume of 1.5 ml brain
homogenate (0.5 ml of 10%, w/v), KCl (0.15 M) and ethanol (10 ␮l)
or test compound dissolved in ethanol. Peroxidation is initiated
by adding ferric chloride (100 ␮M) to give the final concentration
stated. After incubation for 20 min at 37 ◦C, reactions were stopped
by adding 2 ml of ice-cold 0.25 M HCl containing 15% trichloroaceti-
cacid(TCA), 0.38%. thiobarbituric acid (TBA) and 0.05% butylated
hydroxy toluene (BHT). The samples are heated at 80 ◦C for 15 min,
cooled and centrifuged at 1000 rpm for 10 min. The absorbances

250
B. Prathima et al. / Spectrochimica Acta Part A 77 (2010) 248–252
Fig. 1. Mass spectrum of the benzyloxybenzaldehyde-4-phenyl-3-thiosemicarbazone.
of the supernatant solutions are measured at 532 nm. Percentage
inhibitionofthiobarbituricacid reactivesubstances(TBARS)formed
by test compounds are calculated by comparing with the control.
Iron(III) solutions are prepared fresh in distilled water and other
solutions in 0.15 M KCl. Since most buffers trap hydroxyl radical
or interfere with iron conversion, the reactions are carried out in
unbuffered 0.15 M KCl solution [30,31].
The inhibition percentages of the selected ligand and its metal
complexes are evaluated using lipid peroxidation method. The fol-
lowing formula is used in calculating inhibition percentages.
complexes Cu(II) and Ni(II) are stable in air and insoluble in most
of the common organic solvents. Thus the complexes may be
formulated as [M(L)2X2], where M = Cu(II) or Ni(II).
3.3. Electron paramagnetic spectrum of the Cu(II) complex
The EPR spectrum of the Cu(II) complex recorded at room
temperature with polycrystalline sample is shown in Fig. 2. The
spectrum of Cu(II) complex at room temperature reveals three sets
of resonances at low, mid and high fields corresponding to g1, g2
and g3 respectively. From the peak positions, the g values evalu-
ated are g1 = 2.285, g2 = 2.167 and g3 = 2.066. Hyperfine structure is
not resolved. The calculated g values provide valuable information
on the electronic ground state of the ion. For g values g1 > g2 > g3, if
the quantity of R {R = (g2 − g3)/(g1 − g2)} is greater than unity, the
ground state is 2A1(dz2 ) and R is less than unity, the ground state is
ꢀ
ꢁ
ACONT − ATEST
Inhibition percentage =
× 100
ACONT
where ACONT is the absorbance of the control reaction and ATEST is
the absorbance in the presence of the test sample.
3. Results and discussion
3.1. Characterization of
benzyloxybenzaldehyde-4-phenyl-3-thiosemicarbazone
Benzyloxybenzaldehyde-4-phenyl-3-thiosemicarbazone
is
analyzed by IR and 1H NMR spectroscopy. The IR spectrum of
ligand exhibits absorption bands around 1543.18 cm−1 (C N),
1253.18 cm−1(C S) and 3307.85 cm−1(–NH). The 1H NMR
(300 MHz, ı (ppm), CDCl3) investigations provide the follow-
ing information 8.0–7.15 (m, 14H, Ar–H), 11.66 (s, NH), 5.13 (s,
N
CH), 3.12 (s, OCH2). Elemental analysis (Found C 69.78; H 5.30;
N 11.63; S 8.87; Calcd: C 69.32; H 5.043; N 11.35; S 8.725%). The
mass spectrum of ligand (Fig. 1) shows a molecular ion (M+) peak
at m/z value is 362.1, corresponding to the species [C21H19N3OS]+,
which confirms the proposed formula. Benzyloxybenzaldehyde-
4-phenyl-3-thiosemicarbazone is a yellow crystalline solid with a
melting point of 150–152 ◦C.
3.2. Characterization of metal complexes
The
complexes
are
synthesized
by
reacting
(L)
benzyloxybenzaldehyde-4-phenyl-3-thiosemicarbazone
Fig. 2. Powder X-band EPR spectrum of [Cu(L)2Cl2] at room temperature
(ꢀ = 9.205 GHz).
with the metal ions in 2:1 molar ratio in ethanolic medium. The

B. Prathima et al. / Spectrochimica Acta Part A 77 (2010) 248–252
251
Fig. 4. Solid state electronic spectrum (nm) of [Ni(L)2Cl2] complex.
Fig. 3. Solid state electronic spectrum (nm) of [Cu(L)2Cl2] complex.
Table 1
Band head data and assignment for Ni(II) in ligand at room temperature.
2A1(dx 2 ). The data compare well with those of the other reported
rhombic octahedral complexes.
Transitions from
3A2g(F)
Band positions
Observed wave
2
−y
Calculated wave
number (cm−1
)
numbers (cm−1
)
3.4. Electronic spectrum of the Cu(II) complex
3T1g(P)
3T1g(F)
3T2g(F)
24,265
15,357
9521
24,310
15,140
9550
The electronic spectrum (optical absorption) of Cu(II) com-
plex displays four bands at 1238 nm, 1100 nm, 887 nm and
812 nm (Fig. 3) which indicates characteristic of rhombic sym-
metry with the general ordering of the energy levels as
2A1(dx 2 ) < 2A1(d 2 ) < 2A2(dxy) < 2B1(dxz) < 2B2(dyz). Accord-
the earlier findings that 6-coordinate paramagnetic Ni(II) complex
with two ligands do not inhibit the growth of test organisms[34].
From Table 2, it is clear that Cu(II) metal chelate exhibits effective
antibacterial activities. The increased activity of the metal chelate
can be explained on the basis of chelation theory. It is known that
chelation tends to make the ligand act as more powerful and potent
bactericidal agent, killing more of the bacteria than the ligand. It
is observed that in a complex, the positive charge of the metal
is partially shared with the donor atoms present in the ligands
and there may be ꢁ-electron delocalization over the whole chela-
tion. This increases the lipophilic character of the metal chelate
and favors its permeation through the lipoid layer of the bacterial
membranes. The other factors like solubility; conductivity and bond
length between the metal and ligand also increase the activity.
The synthesized ligand and its metal complexes have been
screened for reduction in DPPH free radicals and inhibition of
iron(III) induced lipid peroxidation at 100 ␮m concentration. The
free ligand shows good activity in DPPH scavenging (42%) and ferric
ion induced lipid peroxidation (60%) as seen in the case of standard
antioxidant ␣-tocopherol, but Cu(II) and Ni(II) complexes have not
shown any activity against DPPH scavenging and ferric ion induced
lipid peroxidation. Relevant data is given in Table 3.
2
−y
z
ingly the absorption bands observed at room temperature are
assigned as follows: 1238 nm (8075 cm−1
)
to 2A1(dx 2 ) →
2
−y
2A1(dz2 ), 1100 nm (9088 cm−1) to 2A1(dx 2 ) → 2A2(dxy), 887 nm
2
−y
(11,271 cm−1) to 2A1(dx 2 ) → 2B1(dxz) and 812 nm (12,312 cm−1
)
2
−y
to 2A1(dx 2 ) → 2B2(dyz) respectively. These observations are in
2
−y
tune with those reported earlier [32] and the bands are accord-
ingly ascribed to Cu(II) complex in octahedral coordination with
rhombic (C2v) distortion.
3.5. Electronic spectrum of the Ni(II) complex
The electronic spectrum of Ni(II) complex displays three absorp-
tion bands (Fig. 4) at 412 nm (24,265 cm−1), 651 nm (15,357 cm−1
)
and 1050 nm (9521 cm−1). All these bands are characteristic
of Ni(II) complex in octahedral symmetry. The broad bands
at 24,265 cm−1 (ꢀ1), 15,357 cm−1 (ꢀ2) and 9521 cm−1 (ꢀ3) are
assigned to the spin allowed transitions from the ground state
3
3
3
3
3
3A2g(F) → T1g(P), A2g(F) → T1g(F) and A2g(F) → T2g(F) states
respectively. The positions of the bands indicate that the complex
has six coordinated octahedral geometry [33]. The approximate
values of Dq = 955 cm−1 and B = 720 cm−1 give good fit of the exper-
imental and calculated values of band heads. The observed and the
calculated band partitions and their assignments are depicted in
Table 1.
Table 2
Antibacterial screening data of the ligand and its Ni(II) and Cu(II) complexes (diam-
eter of zone of inhibition in mm).
Compound
K. pnuemoniae
E. coli
B. subtilis
S. aureus
3.6. Antibacterial activity
Ligand (L)
[Ni(L)2Cl2]
[Cu(L)2Cl2]
Ampicillin
Tetracycline
–
–
28
43
32
–
–
19
40
33
–
–
20
43
30
–
–
24
42
32
The antibacterial screening data show that the ligand (L) does
not exhibit antibacterial activity. Ni(II) complex does not inhibit
the growth of test organisms. Our results are in agreement with

252
B. Prathima et al. / Spectrochimica Acta Part A 77 (2010) 248–252
Table 3
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Effect of ligand and its metal complexes on scavenging of DPPH and Fe3+ induced
lipid peroxidation at 100 ␮M concentration.
Compound
DPPH scavenging (%)
Fe3+ induced lipid peroxidation
Ligand(L)
42
–
60
–
[Ni(L)2Cl2]
[Cu(L)2Cl2]
␣-Tocopherol
–
–
53
65
4. Conclusions
In this paper coordination chemistry of ligand, obtained
from the reaction of benzyloxybenzaldehyde and 4-phenyl-3-
thiosemicarbazide is described. Cu(II) and Ni(II) complexes have
been synthesized using the ligand and characterized on the basis
of analytical and spectral data. The EPR and electronic spectral
studies suggested that Cu(II) complex has rhombically distorted
octahedron with 2A1g(dx 2 ) as the ground state, where as N(II)
2
−y
complex exhibits octahedral geometry. The antibacterial activity
of the ligand enhanced upon complexation with metal ions partic-
ularly for Cu(II), against four bacteria (B. subtilis, S. aureus, E. coli and
K. pneumonia), but Ni(II) complex did not show antibacterial activ-
ity. The synthesized ligand and its metal complexes were screened
for reduction of DPPH and inhibition of iron(III) induced lipid per
oxidation at 100 ␮M concentration. Among them, free ligand shows
good activity in DPPH scavenging (42%) and ferric ion induced lipid
per oxidation (60%) as seen in the case of standard antioxidant ␣-
tocopherol, but Cu(II) and Ni(II) complexes have not show activity
against DPPH scavenging and ferric ion induced lipid peroxidation.
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Acknowledgement
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We sincerely thank Prof. J. Lakshmana Rao, Department of
Physics, S. V. University Tirupati, for his help in EPR measurements.
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