Complexes of Cu(II) and Ni(II) with bis(phenylthiosemicarbazone): Synthesis, spectral, EPR and in vitro - Antibacterial and antioxidant activity

Article abstract of DOI:10.1002/jccs.201000094

The synthesis of Cu(II) and Ni(II) complexes with 2,6-diacetylpyridine bis(4-phenyl-3-thiosemicarbazone) (L) ligand have been reported. The ligand was characterized by elemental analysis and spectral (IR and 1H NMR) studies and its complexes were characte

Full text of DOI:10.1002/jccs.201000094

Journal of the Chinese Chemical Society, 2010, 57, 677-682
677
Complexes of Cu(II) and Ni(II) with Bis(phenylthiosemicarbazone):
Synthesis, Spectral, EPR and in vitro - Antibacterial and Antioxidant
Activity
Y. Subba Rao,a B. Prathima,a S. Adinarayana Reddy,b K. Madhavic and A. Varada Reddya,*
aAnalytical Division, Dept. of Chemistry, S. V. University, Tirupati-517502, India
bDept. of Material Science and Nanotechnology, Y. V. University, Kadapa-516001, India
cInstitute of Pharmaceutical Technology, Sri Padhmavati Mahila Visva Vidyalayam,
(Women’s University), Tirupati-517502, India
The synthesis of Cu(II) and Ni(II) complexes with 2,6-diacetylpyridine bis(4-phenyl-3-thiosemicar-
bazone) (L) ligand have been reported. The ligand was characterized by elemental analysis and spectral
(IR and 1H NMR) studies and its complexes were characterized by electronic, UV/VIS, EPR and IR stud-
ies. The room-temperature EPR spectrum of Cu(II) complex was recorded and the analysis of its spectrum
gave {g} tensor with g1 = 2.25, g2 = 2.15, and g3 = 2.07 which were of distorted octahedral structure. The
electronic spectrum of Ni(II) complex indicated the octahedral geometry around the Ni(II) ion. Their anti-
bacterial activities were evaluated by the diameter of zone of inhibition (mm) against four bacteria such as
B. subtilis, S. aureus, E. coli and K. pneumonia. The ligand and the 6-coordinate Ni(II) complex did not in-
hibit the growth of the test organisms, while the Cu(II) complex showed good antibacterial activity than
parent ligand against the same bacteria. The free ligand and its metal complexes have been tested for in vi-
tro - antioxidant activity by reduction of 1,1-diphenyl-2-picryl hydrazyl (DPPH) and iron induced lipid
peroxidation using rat brain homogenate. The ligand exhibited potent in vitro - antioxidant activity than its
Ni(II) and Cu(II)complexes.
Keywords: Spectral studies; In vitro - antibacterial; Antioxidant activity; Cu(II) and Ni(II)
complexes; Bis(phenylthiosemicarbazone).
INTRODUCTION
Thiosemicarbazone ligands, derived from the combi-
ties with natural biological substances and the presence of
imine group (-N=CH-) which imparts the biological activ-
ity.9 (a)-Heterocyclic thiosemicarbazones derived from
pyridine have shown to present an interesting pharmaco-
logical profile and it has been demonstrated that the pres-
ence of a bulky group at terminals strongly improves their
biological activity,10 probably due to increase in lipophilic-
ity.
nation of a thisemicarbazide and an aldehyde or ketone, are
a useful ligand type for obtaining coordination spheres
with mixed N/S donors. Interest in these ligands has been
driven, in part, by potentially beneficial biological activity
of the ligands and their metal complexes, including anti-
tumor,1 fungicidal,2 bacterial3 and antiviral activity.4 Bio-
logical activities of metal complexes differ from those of
either ligands or the metal ions and increased and/or de-
creased biological activities have been reported for several
transition metal complexes, like Cu(II) and Ni(II).5-7 The
activity of these compounds is strongly dependent upon the
nature of the heteroatom ring and the position of attach-
ment of thiosemicarbazone to the ring as well as the form of
the thiosemicarbazone moiety.8 These have been studied
extensively due to their flexibility, selectivity and sensitiv-
ity towards the central metal atom, structural and similari-
In the present work, report is made regarding the syn-
thesis and spectroscopic characterization of Cu(II) and
Ni(II) complexes of the ligand, 2,6-diacetylpyridine bis(4-
phenyl-3-thiosemicarbazone) and in vitro - antibacterial
and antioxidant activity of these compounds that were
extensively studied.
EXPERIMENTAL
Materials and general procedures
All the chemicals were commercially available and
* Corresponding author. Tel: +91-877-2249666(303); Fax: +91-877-2249611; E-mail: ammireddyv@yahoo.co.in

678 J. Chin. Chem. Soc., Vol. 57, No. 4A, 2010
Rao et al.
used as received. The IR spectra of the compounds were re-
corded on a Nicolet FT-IR 560 Magna spectrometer using
KBr. The Bruker 300 MHz NMR spectrometer was used to
obtain the 1H NMR spectrum of the ligand. Elemental anal-
ysis was obtained from vario-micro qub elementar ana-
lyzer. The electronic spectra of the complexes were re-
corded on a Perkin Elmer UV/VIS Lambda 950. EPR spec-
tra were recorded on an EPR spectrometer (JEOL FE - 1X)
operating in the X-band frequencies with a modulation fre-
quency of 100 kHz. The magnetic field was scanned from
2200 to 4200 G, with a scan speed of 250G min-1. 100 mg of
powdered metal complex was taken in a quartz tube for
EPR measurements. Copper(II) chloride and nickel(II)
chloride salts were obtained from BDH AR-grade and were
used as such. Thiobarbituric acid (TBA), trichloroacetic
acid (TCA), a-tocopherol, butylatehydroxyl toluene (BHT),
1,1-diphenyl-2-picrylhydrazyl (DPPH), 4-phenyl-3-thio-
semicarbazide and 2,6-diacetylpyridene were procured
from Sigma Aldrich Chemical Company. All the solvents
obtained commercially were distilled before use.
10.654 (s, 1H, -NH-ph), 10.184 (s, 1H, -NH-ph), 8.538 (d,
2H, py-H, 3J = 8 Hz), 7.819 (d, 1H, py-H, 3J = 8 Hz), 7.552
(d, 4H, Ar-H, 3J = 8 Hz), 7.371 (t, 4H, Ar-H, 3J = 7.6 Hz),
7.213 (t, 2H, Ar-H, 3J = 7.2 Hz), 2.484 (s, 6H, -CH3) were
obtained. Anal. calc for C23H23N7S2: C 59.79; H 5.018; N
2.122; S 13.88. Found C 59.61; H 5.130; N 2.216; S 13.92.
Preparation of Cu(II) complex (1)
A hot ethanolic (20 mL) solution of ligand (0.001 M)
and a hot ethanolic (20 mL) solution of copper(II) chloride
(0.01 M) were mixed together with constant stirring and the
resulting mixture was refluxed for 4-5 hours at 60-65 °C.
On cooling, yellow orange colored complex was formed,
which was filtered off, washed with cold ethanol and dried
in vacuum. Yield was 40-50%. UV/VIS spectrum (nm)
1256, 1160, 948 and 845 were determined. EPR spectrum
with {g} tensor was found to be g1 = 2.25, g2 = 2.15, and g3
= 2.07, and effective magnetic momentum was found to be
1.81-1.87 BM.
Preparation of Ni(II) complex (2)
The ligand (0.50 mmol) was dissolved in absolute
ethanol (20 mL), then the corresponding nickel(II) chloride
(0.6 mmol) in absolute ethanol was added, and the resultant
mixture was refluxed by heating for about 3 hours until the
volume is reduced to 10 mL. The precipitate, thus formed
was filtered and washed with absolute ethanol twice, and
then dried in vacuum. Yield was 50-60%. UV/VIS spec-
trum (nm) 951, 630-653 and 462.
Synthesis of 2,6-diacetylpyridine bis-4-phenyl-3-thio-
semicarbazone
2,6-Diacetylpyridine bis(4-phenyl-3-thiosemicarba-
zone) (L) was prepared as per the procedure reported in lit-
erature.11 1.60 g of 2,6-diacetylpyridine was dissolved in
30 mL of absolute ethanol and mixed in a round bottomed
flask with 30 mL of absolute ethanolic solution containing
2.20 g of 4-phenyl-3-thiosemicarbazide. The mixture was
heated under reflux for 3 hours and then allowed to cool to
room temperature and kept for 12 hours. The yellow crys-
tals obtained were filtered and washed with cold ethanol
and then recrystalized from absolute ethanol (Scheme I).
The melting point was 152 °C and the yield was 65-70%.
BIOLOGICAL STUDIES
Antibacterial screening
The antibacterial activities of both ligand (L) and its
metal complexes 1 and 2 were studied by usual cup-plate-
agar diffusion method.12-14 The bacterial species used in the
screening were Klebsiella pneumonia, Escherichia coli,
1
The characterization of L was carried out by IR and H
1
NMR spectroscopy. The H NMR (400 Hz, DMSO, d):
Scheme I

Cu(II) and Ni(II) with Bis(phenylthiosemicarbazone)
J. Chin. Chem. Soc., Vol. 57, No. 4A, 2010 679
(gram negative) and Staphylococcus aureus Bacillus
subtilis (gram-positive). Stock cultures of the test bacterial
species were maintained on Nutrient Agar media (Hi-me-
dia laboratories, Mumbai) by sub culturing on petri dishes.
The media were prepared by adding the components as per
manufacturer’s instructions and sterilized in the autoclave
at 121 °C temperature and 15 lbs pressure for 15 minutes
and then cooled to 45-60 °C. 20 mL of each medium was
poured in a Petri dish and allowed to solidify. After solidifi-
cation, Petri plates with media were spread with 1.0 mL of
bacterial suspension, which is prepared in sterile distilled
water. The wells were bored with cork borer and the agar
plugs were removed. 100 ml of the compound reconstituted
in DMF (Dimethyl formamide) in concentrations of 1.0
mg/mL was added to the agar wells. DMF was used as a
negative control and antibiotics such as ampicillin and tet-
racycline were used as positive control standards. The
plates were incubated at 37 °C for 24 hours and then the
plates were observed for the growth inhibition zones. The
presence of clear zones around the wells indicated that the
compound is active. The diameter of the zone of inhibi-
tion was calculated in millimetres. The well diameter was
deducted from the zone diameter to get the actual zone of
the inhibition diameter and the values have been tabulated.
Iron induced lipid peroxidation
Inhibition of TBARS formation in rat brain homoge-
nate.17,18
Male Albino rats (180-200 g) were used for the study.
Prior to decapitation and removal of brain, the animals
were anesthetized with ether and perfused transcardially
with ice-cold normal saline to prevent contamination of
brain tissue with blood. Tissue was weighed and its homog-
enate (10% w/v) was prepared in 150 mmol KCl and centri-
fuged at 800 rpm for minutes. The supernatant was used
immediately for the study. The incubation mixture con-
tained in a final volume of 1 mL, brain homogenate (500
ml), KCl (150 mmol) and ethanol (10 ml) or the ethanolic
solutions of ligand (L) and its Cu(II) and Ni(II) complexes
at 100 mM. Peroxidation was initiated by adding Fe+3 (100
mM) to give the final concentration stated. After incubation
for 20 minutes at 37 °C reactions were stopped by adding 2
mL of ice-cold 0.25 M HCl containing 15% trichloro acetic
acid (TCA), 0.38% thiobarbituric acid (TBA) and 0.05%
butylated hydroxyl toluene (BHT). The samples were heated
at 80 °C for 15 minutes, cooled and centrifuged at 1000 rpm
for 10 minutes. The absorbance percentage inhibition of
thiobarbituric acid reactive substances (TBARS) formed
by test compounds were measured at 532 nm by using
systronics UV/VIS spectrometer-117 instrument and cal-
culated by comparing with the control. a-tocopherol was
used as a positive control. The percentage inhibition of
lipid peroxidation with selected ligand and its metal com-
plexes and standard compounds were calculated using the
following formula,
IN VITRO - ANTIOXIDANTS STUDIES
DPPH free radical scavenging activity
1,1-Diphenyl-2-picrylhydrazyl (DPPH) is a stable
free radical which has maximum optical absorbance at 517
nm. The reaction of DPPH with free radical scavenger
causes decline in the absorbance value at 517 nm.15,16 The
ethanolic solutions of ligand (L) and its Cu(II) and Ni(II)
complexes at 100 mM concentration were added to 100 mM
DPPH in ethanol. The test tubes were kept at an ambient
temperature for 20 minutes and the absorbances were mea-
sured at 517 nm, against control. a-tocopherol was used as
a positive control. These measurements were run in tripli-
cate. The percentage of scavenging activity was calculated
as follows:
Inhibition percentage
= [(ACONT – ATEST)/ACONT] ´ 100
where ACONT is the absorbance of the control without test
sample and ATEST is the absorbance in the presence of the
test sample.
RESULTS AND DISCUSSION
The Cu(II) and Ni(II) ions forms the 1:1 (metal :
ligand) complexes with ligand has been determined on the
basis of Job’s continuous variation method.19
Scavenging activity (%)
= [(ADPPH - ATEST)/ADPPH] ´ 100
Electronic and EPR spectra of Cu(II) complex
The powdered X-band EPR spectrum of 1 at room
temperature is shown in Fig. 1. The spectrum of Cu(II)
complex reveals three sets of resonances at low, mid and
where ADPPH is the absorbance of DPPH without test sam-
ple (control) and ATEST is the absorbance of DPPH in the
presence of test sample.

680 J. Chin. Chem. Soc., Vol. 57, No. 4A, 2010
Rao et al.
high fields corresponding to g1, g2 and g3 respectively.
From the peak positions g-values evaluated are g, g1 = 2.25,
g2 = 2.15, and g3 = 2.07 (Table 1). Hyperfine structure is not
resolved. The calculated g values provide valuable infor-
mation on the electronic ground state of the ion.20 For g val-
ues g1 > g2 > g3, the quantity R = g2-g3/g1-g2. If R is greater
than unity, then the ground state is 2A1 (dz2) and if R is less
than unity, then the ground state is 2A1(dx2-y2). In the pres-
ent study, the calculated R value is less than unity (R = 0.8)
and hence the ground state is 2A1 (dx2-y2). The room tem-
perature solid state electronic spectrum of 1 exhibits four
absorption bands as shown in Fig. 2, at 1256, 1160, 948 and
Table 1. Experimental EPR parameters
Compound Temperature g1 g2
27 °C
g3
gave meff (BM)
1
2.25 2.15 2.07 2.15 1.81-1.87
in the complex.22 Thus, these bands can be assigned to the
three spin-allowed transitions 3A2g(F) ® 3T2g(F), 3A2g(F)
® 3T1g(F) and 3A2g(F) ® 3T1g(P), respectively.
IR studies
The main vibrational bands of free ligand and of its
complexes are shown in Table 2. The high frequency bands
of the uncomplexed ligand centred at 3369 and 3300 cm-1,
2
2
2
845 nm which are attributed to A1(dx2-y2) ® A1(dz ),
2A1(dx2-y2) ® 2A2(dxy), 2A1(dx2-y2) ® 2B1(dxz) and
2A1(dx2-y2) ® 2B2(dyz), respectively. From the results and
analysis of electronic and EPR spectra, the site symmetry
of Cu(II) ion in Cu(II) complex is ascertained to be a
rhombically distorted octahedron with 2A1(dx2-y2) as the
ground state.21
Electronic spectrum of Ni(II) complex
The solid state electronic spectrum of 2 exhibits three
bands as shown in Fig. 3, at 951, 630-653 and 462 nm char-
acteristic of an octahedral geometry around the Ni(II) ion
Fig. 2. Solid state electronic spectrum of complex 1.
Fig. 1. Powder X-band EPR spectrum of complex 1 at
room temperature (n = 9.205 GHz).
Fig. 3. Solid state electronic spectrum of complex 2.

Cu(II) and Ni(II) with Bis(phenylthiosemicarbazone)
J. Chin. Chem. Soc., Vol. 57, No. 4A, 2010 681
Table 2. The main IR (cm-1) of the ligand and its complexes
Compounds
n (NH)
n (C=N, C=C)
n (C-S) + n (C-N)
n (C-S)
Ligand
3369, 3300
3403
1596, 1485, 1540
1598, 1500, 1443
1355, 1321
1317, 1254
1317, 1253
820
694
753
1
2
3297
1596, 1529, 1493, 1493
are attributed to n(N-H) stretching vibrations. The disap-
pearance of the latter one absorption upon complexation is
a consequence of the double deprotonation of the ligand.
Significant changes in the ligand bands upon complexation
include variation in the n(C=N, C=C) vibration energies
and systematic shifts of the n(C-S) absorption bands to
lower frequencies shown in Table 2. These data indicate
coordination through the azomethine nitrogen, the pyridine
nitrogen and the sulfur atoms.23
Table 3. Antibacterial screening data of ligand (L) and its Cu(II)
and Ni(II) complexes at the concentration of 1 mg/mL
Compound K. pnuemoniae E. coli B. subtilis S. aureus
Ligand (L)
-
9
-
-
-
1
12
-
11
-
10
-
2
-
Ampicillin
Tetracycline
43
32
40
33
43
30
42
32
Table 4. Antioxidant scavenging activity data of ligand (L) and
Antibacterial and antioxidants activity
its Cu(II) and Ni(II) complexes at concentration of 100
The results of antibacterial activity show that the
ligand (L) and Ni(II) themselves do not exhibit antibacte-
rial activities. However it is important to note that Cu(II)
complex exhibits antimicrobial activities. The Cu(II) com-
plex shows more activity than the Ni(II) complex. This may
be due to the higher stability of Cu(II) complex than the
Ni(II) complex. It is interesting to note that 6-coordinate
paramagnetic Ni(II) complex does not exhibit antibacterial
activities.24 Data has been demonstrated in Table 3.
The synthesized ligand and its metal complexes were
screened for reduction of DPPH and inhibition of iron in-
duced lipid peroxidation at 100 mM concentration. Among
them, free ligand showed good activity in DPPH scaveng-
ing (42%) and ferric ion induced lipid peroxidation (59%)
comparable with as seen in the case of standard antioxidant
a-tocopherol, but Cu(II) and Ni(II) complexes have no ac-
tivity against DPPH scavenging and ferric ion induced
lipid peroxidation. Data are given in Table 4.
mM
Fe3+ induced lipid
Compound
DPPH scavenging (%)
peroxidation (%)
Ligand (L)
42
5.2
2.7
53
59
-
1
2
-
a-tocopherol
65
as comparable with parent ligand and Ni(II) complex. The
synthesized ligand and its metal complexes were screened
for reduction of DPPH and inhibition of iron induced lipid
peroxidation at 100 mM concentration. Based on the results
obtained, the free ligand was found to be good antioxidant,
as comparable with standard a-tocopherol. However, its
antioxidant activity can be further confirmed by in vivo
methods.
ACKNOWLEDGEMENTS
We sincerely thank Prof. J. Lakshmana Rao, Depart-
ment of Physics, S. V. University, Tirupati, for his help in
EPR studies. We also sincerely thank Prof. M. R. Ashok,
Department of English, S. V. University, Tirupati, for his
help in correcting grammatical mistakes in the manuscript.
CONCLUSIONS
The Cu(II) and Ni(II) complexes with 2,6-diacetyl-
pyridine bis(4-phenyl-3-thiosemicarbazone) have been
synthesized and their structures are presented. The EPR,
magnetic and electronic spectral studies suggested that the
Cu(II) complex has a rhombically distorted octahedron
with 2A1(dx2-y2) as the ground state. Where as in the Ni(II)
complex the Ni(II) ion has a octahedral geomentry. The
Cu(II) complex showed good antibacterial activity against
four bacteria (B. subtilis, S. aureus, E. coli and P. aeruginosa)
Received December 3, 2009.
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