DNA cleavage and antimicrobial investigation of Co(II), Ni(II), and Cu(II) complexes with triazole Schiff bases: Synthesis and spectral characterization

Article abstract of DOI:10.1007/s00044-010-9327-0

The Co(II), Ni(II), and Cu(II) complexes with Schiff bases derived from 3-substituted-4-amino-5-mercapto-1,2,4-triazole and fluvastatin have been synthesized. Schiff bases exhibited thiol-thione tautomerism and coordinated to metal ion through azomethine

Full text of DOI:10.1007/s00044-010-9327-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:346–354
DOI 10.1007/s00044-010-9327-0
ORIGINAL RESEARCH
DNA cleavage and antimicrobial investigation of Co(II), Ni(II),
and Cu(II) complexes with triazole Schiff bases: synthesis
and spectral characterization
•
Ajaykumar D. Kulkarni Sangamesh A. Patil
•
•
Vinod H. Naik Prema S. Badami
Received: 16 July 2009 / Accepted: 16 February 2010 / Published online: 7 March 2010
Ó Springer Science+Business Media, LLC 2011
Abstract The Co(II), Ni(II), and Cu(II) complexes with
Schiff bases derived from 3-substituted-4-amino-5-mer-
capto-1,2,4-triazole and fluvastatin have been synthesized.
Schiff bases exhibited thiol–thione tautomerism and coor-
dinated to metal ion through azomethine nitrogen and
thiolate sulphur atoms. Square planar geometry for all the
metal complexes of the type ML2 has been proposed in the
light of analytical, spectral (IR, UV–Vis., ESR, and FAB-
mass), magnetic, and thermal studies. The antimicrobial
studies of Schiff bases and their metal complexes against
various antibacterial (Escherichia coli, Staphylococcus
aureus, Pseudomonas aeruginosa and Bacillus subtilis) and
antifungal (Aspergillus niger, and Pencillium Chrysoge-
num) species by Minimum Inhibitory Concentration
method revealed that, the metal complexes possess more
healing antibacterial activity than the Schiff bases. Co(II),
Ni(II), and Cu(II) complexes cleave the DNA isolated from
A. niger.
Introduction
Metal complexes with Schiff base ligands have played
an important role since the early days of Coordination
Chemistry (Holm, 1974; Yamada, 1966; Sacconi, 1966).
Indeed, a great deal of study has been carried out on the
synthesis and characterization of transition metal com-
pounds with Schiff bases as ligands, mainly due to their
applications in the field of organic chemistry (Katsuki,
1995; Ito and Katsuki, 1999) and in catalytic processes
(Canali and Sherrington, 1999). Also, the last decade has
seen an upsurge of interest in metal ion therapeutics for
both diagnosis and treatment of diseases (Reichert et al.,
1999; Schwietert and McCue, 1999). For well over a
hundred years, the synthesis and functionalization of in-
doles has been a major area of focus for chemists. Indoles
are of great significance in view of their (i) occurrence in
nature as a prominent sub-structure of a large number of
alkaloids (Somei and Yamada, 2003; Hibino and Choshi,
2002) and (ii) wide-ranging biological activities (Gribble,
1995). In addition, 1,2,4-triazoles are an important class of
heterocyclic compounds which are well known for their
potential antimicrobial properties. The substituted 1,2,4-
triazoles are associated with diverse biological activities
such as, fungicidal, antimicrobial, anticonvulsant, and
antiviral activities (Walser et al., 1991; Ewiss et al., 1986;
Bhat et al., 2001; Kitazaki et al., 1996; Todoulou et al.,
1994; Xiang-Shu et al., 2009). A number of metal com-
plexes with 1,2,4-triazole ligands have been synthesized
and studied for their applicability in diverse fields (Heleen
et al., 1991; Joost et al., 1992). Such a vide spectrum of
biological applications of 1,2,4-triazoles prompted us to
synthesize Schiff bases, derived from triazoles and stitch
them with various metal ions. Very recently, a number of
metal complexes of 1,2,4-triazole Schiff bases, which
Keywords Antibacterial ꢀ Antifungal ꢀ DNA cleavage ꢀ
Spectroscopic studies ꢀ Schiff base complexes ꢀ
Transition metal complexes
A. D. Kulkarni ꢀ S. A. Patil (&) ꢀ V. H. Naik
P.G. Department of Chemistry, Karnatak University,
Dharwad 580003, Karnataka, India
e-mail: patil1956@rediffmail.com
P. S. Badami
Department of Chemistry, Shri Sharanabasaveswar College
of Science, Gulbarga 585102, Karnataka, India
123

Med Chem Res (2011) 20:346–354
347
possess very good antimicrobial properties have been
reported from this laboratory (Ajaykumar et al., 2009a, b;
Gangadhar et al., 2008; Gangadhar et al., 2009a, b).
In continuation of our earlier study, we report here the
synthesis, antimicrobial properties, and DNA cleavage
studies of Co(II), Ni(II), and Cu(II) metal complexes with
Schiff bases derived from 3-substituted-4-amino-5-mer-
capto-1,2,4-triazole and fluvastatin which is an indole
derivative. The structural features of Schiff bases and their
metal complexes have been elucidated by various spectral
and analytical techniques.
(SH), 1105 (C=S). FAB MS: m/z 405. Anal.: Obsd.(Calc.)
C, 65.11 (65.18); H, 4.81 (4.93); N, 17.19 (17.28).
1
Schiff base II: H NMR (d6-DMSO): 10.6 (s, 1H, SH),
10.04 (s, 1H, CH=N), 7.1–7.6 (m, 8H, Ar–H), 6.47–6.56 (d,
2H, –CH=CH–), 2.38 (s, 1H, –CH3), 6.47–6.56 (S, 6H,
isopropyl group). IR (KBr) cm-1: 1619 (C=N), 2753 (SH),
1102 (C=S). FAB MS: m/z 419. Anal.: Obsd.(Calc.) C,
65.94 (65.87); H, 5.19 (5.25); N, 16.66 (16.71).
Synthesis of Co(II), Ni(II), and Cu(II) complexes [1–6]
Ethanolic solution (40 ml) of Schiff bases (2 mmol) was
mixed with ethanolic solution (10 ml) of CoCl2ꢀ6H2O/
NiCl2ꢀ6H2O/CuCl2ꢀ2H2O (1 mmol) and refluxed on water
bath for 2 h. Then, to the reaction mixture, sodium acetate
(1 mmol) was added and reflux was continued for further
2 h. The separated complex was filtered, washed thor-
oughly with water and ethanol, and dried in vacuum over
fused CaCl2. Yield of all the metal complexes lie in the
range of 66–76%.
Materials and methods
Materials
All the chemicals used were of reagent grade. 3-sub-
stituted-4-amino-5-mercapto-1,2,4-triazole was synthe-
sized according to the literature (Dhaka et al., 1974;
Escobar-Valderrama et al., 1989; Palmer and Dines, 2004;
Ajaykumar et al., 2009a, b). Fluvastatin was recrystallized
before use.
Characterization
Carbon, hydrogen, and nitrogen were estimated by using
Elemental Analyzer Carlo Erba EA1108 analyzer. The IR
spectra of the Schiff bases and their Co(II), Ni(II), and
Cu(II) complexes were recorded on a HITACHI-270 IR
spectrophotometer in the 4000–400 cm-1 region in KBr
disc. The electronic spectra of the complexes were recor-
ded in HPLC grade DMSO on a VARIAN CARY 50-BIO
UV-spectrophotometer in the region of 200–1100 nm. The
1H-NMR spectra of the Schiff bases were recorded in
d6-DMSO on a BRUKER 300 MHz spectrometer at room
temperature using TMS as an internal reference. FAB-
Mass spectra were recorded on a JEOL SX 102/DA-6000
mass spectrometer/data system using Argon/Xenon (6 kV,
10A) as the FAB gass with accelerating voltage set as
10 kV, the spectra were recorded at room temperature, and
m-nitrobenzyl alcohol was used as the matrix. The mass
spectrometer, was operated in the positive ion mode. The
Synthesis of Schiff bases (I–II)
The synthesis of Schiff bases is schematically presented in
Scheme 1. The Schiff bases have been synthesized by re-
fluxing the reaction mixture of hot ethanolic solution
(30 ml) of 3-substituted-4-amino-5-mercapto-1,2,4-triazole
(0.01 mol) and hot ethanolic solution (30 ml) of fluvastatin
(0.01 mol) for 4-5 h with addition of 4–5 drops of conc.
hydrochloric acid. The product obtained after the evapo-
ration of the solvent was filtered, washed with cold ethanol
and recrystallized from hot ethanol. Yield (M.P.): 74%
(189°C) and 76% (196°C) of SB I and SB II, respectively.
1
Schiff base I: H NMR (d6-DMSO): 10.4 (s, 1H, SH),
10.01 (s, 1H, CH=N), 7.1–7.7 (m, 8H, Ar–H), 7.3 (s, 1H,
triazole-H), 6.47–6.55 (d, 2H, –CH=CH–), 6.47–6.56 (S,
6H, isopropyl group). IR (KBr) cm-1: 1616 (C=N), 2752
Scheme 1 Synthesis of Schiff
bases I and II
F
F
N
N
Reflux 4 - 5 h
HCl
+
N
SH
R
N
N
H
CH
N
NH2
H3C CH3
H3C CH3
O
Triazole
Fluvastatin
R
N
SH
N
N
R
H
CH3
Schiff base No.
SB I
Formula
C22H20FN5S
C23H22FN5S
SB II
123

348
Med Chem Res (2011) 20:346–354
ESR spectrum was recorded under liquid nitrogen tem-
perature (LNT) on Varian-E-4X-band EPR spectrometer,
and the field set is 3000 G at modulation frequency of
100 K Hz using TCNE as ‘g’ marker. Thermogravimetric
analyses data were measured from room temperature to
1000°C at a heating rate of 10°C/min. The data were
obtained by using a PERKIN–ELMER DIAMOND TG/
DTA instrument. Molar conductivity measurements were
recorded on ELICO-CM-82 T Conductivity Bridge with a
cell having cell constant 0.51, and magnetic moment
measurement was carried out using Faraday balance.
under Vilberlourmate Gel documentation system and
photographed to determine the extent of DNA cleavage.
Then, the results are compared with standard DNA marker.
In vitro antibacterial and antifungal studies
The biological activities of synthesized Schiff bases and
their Co(II), Ni(II), and Cu(II) complexes have been
studied for their antibacterial and antifungal activities by
agar and potato dextrose agar diffusion methods. The
antibacterial and antifungal activities were done at 100,
200, and 500 lg/ml concentrations in DMF solvent using
four bacteria (E. coli, S. aureus, P. aeruginosa, and
B. subtilis) and two fungi (A. niger and P. chrysogenum)
strains by the minimum inhibitory concentration (MIC)
method (Sadana et al., 2003). These bacterial strains were
incubated for 24 h at 37°C, and fungi strains were incu-
bated for 48 h at 37°C. Standard antibacterial (Streptomy-
cin) and antifungal drug (Nyastatin) were used for
comparison under similar conditions. Activity was deter-
mined by measuring the diameter of the zone of inhibition
(mm).
DNA cleavage experiment
Preparation of culture media
Potato dextrose broth [potato, 250; dextrose, 20; in (g/l)]
was used for the culture of A. niger. The 50-ml media was
prepared, autoclaved for 15 min at 121°C under 15-lb
pressure. The autoclaved media of A. niger was inoculated
with the seed culture and incubated for 48 h. at 37°C.
Isolation of DNA
The fresh bacterial culture (1.5 ml) is centrifuged to obtain
the pellet which is then dissolved in 0.5 ml of lysis buffer
(100 mM tris pH 8.0, 50 mM EDTA, 10% SDS). To this
mixture, 0.5 ml of saturated phenol was added, incubated
at 55°C for 10 min, and then centrifuged at 10,000 rpm for
10 min. To the supernatant, equal volume of chloro-
form:isoamyl alcohol (24:1) and 1/20th volume of 3 M
sodium acetate (pH 4.8) was added. Then, the solution was
centrifuged at 10,000 rpm for 10 min, and to the super-
natant, three volumes of chilled absolute alcohol was
added. The precipitated DNA was separated by centrifu-
gation, and the pellet was dried and dissolved in TAE
buffer (10 mM tris pH 8.0, 1 mM EDTA), and stored in
cold condition.
Results and discussion
The Schiff bases (I and II) form square–planar complexes
(1–6) with CoCl2ꢀ6H2O/NiCl2ꢀ6H2O/CuCl2ꢀ2H2O in etha-
nol (Scheme 2). All the Co(II), Ni(II), and Cu(II) com-
plexes are stable and non-hygroscopic in nature. The
complexes are insoluble in common organic solvents but
soluble in DMF and DMSO. The elemental analyses
showed that, the Co(II), Ni(II), and Cu(II) complexes have
1:2 stoichiometry of the type ML2 where L stands for a
singly deprotonated ligand, which exhibits thiol–thione
tautomerism (Fig. 1). The molar conductance values are
too low to account for any dissociation of the complexes in
DMF, indicating the non-electrolytic nature of the com-
plexes in DMF (Table 1). Several attempts were made to
develop the single crystal of the complexes. However,
those attempts failed due to insolubility of the complexes in
common organic solvents.
Agarose gel electrophoresis
Cleavage products were analyzed by agarose gel electro-
phoresis method. Test samples (1 mg/ml) were prepared in
DMF. The samples (25 lg) were added to the isolated
DNA of A. niger. The samples were incubated for 2 h at
37°C, and then, 20 ll of DNA sample (mixed with bro-
mophenol blue dye @ 1:1 ratio) was loaded carefully into
the electrophoresis chamber wells along with standard
DNA marker containing TAE buffer (4.84 g Tris base, pH
8.0, 0.5 M EDTA/1 l) and finally loaded on agarose gel.
Then, a constant potential of 50 V for around 30 min was
applied. The gel was removed and stained with 10.0 lg/ml
ethidium bromide for 10–15 min. The bands were observed
IR spectral studies
The IR spectral data results provide strong evidences for
the complexation of Schiff bases with metal (II) ions
(Table 2).
The IR spectra of the Schiff bases exhibited a character-
istic high intensity band in the region of 1619–1616 cm-1
,
assigned to m(HC=N). In comparison with the spectra of the
Schiff bases, all the Co(II), Ni(II) and Cu(II) complexes
exhibited the band of m(HC=N) in the region of
123

Med Chem Res (2011) 20:346–354
349
F
Scheme 2 Synthesis of metal
complexes
F
N
N
CoCl . H O
6
2
2
4
Reflux h
N
N
R
N
N
CH
N
CH
N
S
2
NiCl . H O
6
+
CH3
H3C
CH3
H3C
CuCl2 . H2O
M
2
H3C
2
2
CH3
HC
R
N
S
N
N
SH
R
N
N
N
N
F
R
M
Formula
Complex
No.
1
2
3
4
H
CH3
H
CH3
H
Co(II)
Co(II)
Ni(II)
Ni(II)
Cu(II)
Cu(II)
Co(C22H19FN5S)2
Co(C23H21FN5S)2
Ni(C22H19FN5S)2
Ni(C23H21FN5S)2
Cu(C22H19FN5S)2
Cu(C23H21FN5S)2
5
6
CH3
F
Fig. 1 Structure of Schiff bases
I and II: Thiol–thione
tautomerism
F
N
CH
N
N
CH
H3C CH3
H3C CH3
N
N
N
S
R
R
SH
N
N
H
N
N
Table 1 Elemental analyses of Schiff bases and their Co(II), Ni(II), and Cu(II) complexes along with molar conductance and magnetic moment
data
Compound number Empirical formula M%
C%
H%
N%
Molar conductance leff (BM)
Ohm-1 cm2 mol-1
Obsd. Calcd. Obsd. Calcd. Obsd. Calcd. Obsd. Calcd.
SB-I
C22H20FN5S
23H22FN5S
–
–
–
–
65.11 65.18 4.81
65.94 65.87 5.19
4.93
5.25
4.38
4.69
4.39
4.69
4.36
4.67
17.19 17.28
16.66 16.71
16.14 16.15
15.72 15.64
–
–
SB-II
C
–
–
1
2
3
4
5
6
Co(C22H19FN5S)2 6.791 6.805 60.96 60.89 4.36
Co(C23H21FN5S)2 6.610 6.592 61.58 61.67 4.72
Ni(C22H19FN5S)2 6.692 6.697 60.88 60.97 4.31
Ni(C23H21FN5S)2 6.442 6.487 61.72 61.74 4.59
Cu(C22H19FN5S)2 7.197 7.233 60.58 60.61 4.28
Cu(C23H21FN5S)2 7.102 7.007 61.57 61.40 4.65
8.2
6.9
3.57
3.58
D
16.08 16.16 10.1
15.71 15.66 9.5
D
16.13 16.07 11.6
15.52 15.57 11.2
1.82
1.83
1608–1607 cm-1 showing the shift of band to lower wave
numbers indicating that the azomethine nitrogen atom is
coordinated to the metal ion (Azza Abu-Hussen and Adel
Emara, 2004). A characteristic strong band in the region of
2753–2752 cm-1 and another band around 1100 cm-1 in
the spectra of the Schiff bases are ascribed to m(SH) of tri-
azole and to m(C=S) (Sen et al., 1998), respectively. These
observations suggest that, the Schiff bases exhibit thiol–
thione tautomerism. The deprotonation of the thiol group is
indicated by the absence of a band around 2753 cm-1 in all
the metal complexes indicating that the metal is coordinated
through sulphur atom. This is further supported by the band
around 747–742 cm-1 in the metal complexes due to m(C–
S). The new bands in the region of 470–462 and
419–406 cm-1 in the IR-spectra of the complexes are
assigned to stretching frequencies of (M–N) and metal-sul-
phur bond formation, respectively.
1H NMR spectral study of Schiff bases I and II
In the 1H-NMR spectrum of the Schiff base and II, the thiol
group exhibited proton signal at 10.6 ppm (s, 1H, SH). A
123

350
Med Chem Res (2011) 20:346–354
Table 2 The prominent IR frequencies (in cm-1) of Schiff bases and
their metal complexes
and 1.83 BM for the Cu(II) complexes (5 and 6), respec-
tively further supports the square planar geometry for these
complexes.
Compound number m(HC=N) m(SH) m(C=S) C–S M–N M–S
C22H20FN5S
23H22FN5S
1616
1619
2752 1105
2753 1102
–
–
–
–
–
FAB-mass spectral studies of Schiff bases and their
metal complexes
C
–
Co(C22H19FN5S)2 1608
Co(C23H21FN5S)2 1608
–
–
–
–
–
–
–
–
–
–
–
–
744 466
743 466
747 470
744 468
742 462
743 466
406
408
416
411
419
416
The FAB-mass spectrum of representative Schiff base-II,
and its metal complexes are discussed here. The spectrum
of Schiff base-II showed a molecular ion peak M? at m/z
419, equivalent to its molecular weight. In addition to this,
the fragmentation peaks observed at m/z 305, 248, and 183
are due to the cleavage of C3H4N3S, C3H7N, and C4H3N,
respectively. The results confirm the formation of Schiff
base-II supporting the IR and NMR results. The FAB-mass
spectra of Co(II) (2), Ni(II) (4)) and Cu(II) (6) complexes
of Schiff base-II showed a molecular ion peak M? at m/z
895, 894, and 899, respectively, equivalent their molecular
weight which supports the formation of respective metal
complexes of the type ML2. All the fragments of the spe-
cies [M2HL2]?, which underwent demetallation to form
[L ? H]? gave a fragment ion peak at m/z 418.
Ni(C22H19FN5S)2
Ni(C23H21FN5S)2
1607
1608
Cu(C22H19FN5S)2 1608
Cu(C23H21FN5S)2 1608
characteristic proton signal at 10.04 ppm (s, 1H, –CH=N)
is assigned to aldimine proton. A sharp signal at 2.38 ppm
(s, 3H, CH3) and signals in the region 7.1–7.6 ppm (m, 8H,
Ar–H) are due to methyl protons of triazole moiety and
aromatic protons, respectively. In addition to this, the
isopropyl attached to indole ring exhibited signals in the
region 1.65–1.78 ppm and the resonance observed at
6.47–6.56 ppm (m, 2H) are ascribed to –CH=CH– group,
respectively. In the case of Schiff base I, all the signals due
to various differently positioned protons were observed in
accordance with Schiff base II) The resonance due to
proton of triazole ring is observed in the aromatic region
and a signal due to CH3 is absent in the proton NMR
spectrum of Schiff base I. Thus, the NMR results support
the formation of Schiff bases in line with IR inferences.
ESR studies of Cu(C23H21FN5S)2 complex
ESR spectrum of Cu(II) complex has been studied under
liquid nitrogen temperature using TCNE as a ‘g’ marker.
The four coordinate Cu(II) complexes at LNT show well-
resolved four copper hyperfine lines, characteristic of
monomeric Cu(II) complexes and superhyperfine lines due
to azomethine nitrogen. The ‘g’ tensor values of Cu(II)
complex can be used to derive the ground state. In square
planar complexes, the unpaired electron lies in the dx2ꢁy2
Electronic spectral and magnetic studies
The electronic spectra of Co(II) complexes exhibited
absorption bands in the region of 16365–16380 cm-1
2
orbital giving B1g as the ground state with gk [ g?. For
1
1
corresponding to A1g ? B1g which indicate the square
planar geometry for the Co(II) complexes. The magnetic
moment values of the Co(II) complexes (1 and 2) are 3.57
and 3.58 BM, respectively, and it supports the square
planar geometry for the present Co(II) complexes (Duta
and Syamal, 1992). The Ni(II) complexes exhibited two
bands in the region 18800–18810 and 21635–31640 cm-1
attributed to the 1A1g ? B1g and 1A1g ? B2g transitions,
respectively, which indicate square planar geometry around
Ni(II) ion. This has been supported by the diamagnetic
nature of the complexes 3 and 4. The Electronic spectra of
the present Cu(II) complex, gk and g? values have been
found to be 2.5958 and 2.3777, respectively, and G value is
1.5774. Hence, it is clear from the results that, the
gk [ g? [ 2 and G \ 4 which is consistent with dx2ꢁy2
ground state (Ray and Kauffman, 1990; Anthonisamy and
Murugesan 1998) suggesting square planar geometry for
the Cu(II) complex. Thus, the ESR spectral results provide
further evidence to the magnetic and electronic spectral
results.
1
1
Thermal decomposition study of metal complexes
Cu(II) complexes display
a
broad band around
17475–17505 cm-1 assignable to B1g ? A1g transitions
(Kumar et al., 1999). In addition to this, two weak shoul-
ders appearing in the region 20765–20875 and
2
2
The thermal behavior of Co(II), Ni(II), and Cu(II) com-
plexes has been studied as a function of temperature. The
thermal behavior of all the complexes is almost same.
Hence, only the representative Co(II) (2), Ni(II) (4), and
Cu(II) (6) complexes have been discussed here. The ther-
mal analyses (TGA and DTA) of Co(C23H21FN5S)2 com-
plex showed that the two fluvastatin and triazole moieties
11350–11555 cm-1 may be ascribed to B1g ? Eg and
2
2
2
2B1g ? B2g transitions, respectively. These assignments
suggests the square planar geometry for the Cu(II) com-
plexes under investigation. The magnetic moments of 1.82
123

Med Chem Res (2011) 20:346–354
351
of Schiff base were lost in the regions of 215–325 centered
at 272°C and 400–475 centered at 451°C corresponding to
the mass losses of 64.92% (calc. 65.02%) and 28.26%
(calc. 28.37%), respectively (Fig. 2). Finally, the formation
of metal oxide took place at above 500°C. The
Ni(C23H21FN5S)2 complex loses its fluvastatin and triazole
moieties in the regions of 205–320 centered at 274°C and
355–405 centered at 379°C corresponding to mass losses of
64.98% (calc. 65.10%) and 28.34% (calc. 28.41%),
respectively. The formation of metal oxide took place at
425°C. In the case of Cu(C23H21FN5S)2 complex, the loss
of fluvastatin and triazole moieties were found to be in the
regions of 205–285 centered at 251°C and 300–370 cen-
tered at 327°C corresponding to the mass losses of 64.59%
(calc. 64.73%) and 28.12% (calc. 28.25%), respectively.
Finally, the metal oxide formed at 400°C. Thus, the TGA
and DTA analyses of this study support the ML2 ratio of
the metal complexes in the absence of coordination of
water molecule.
Fig. 3 DNA cleavage studies of Co(II) (2), Ni(II) (4), and Cu(II) (6)
complexes. M: Standard Molecular weight Marker; C—Control DNA
of A. niger; Lane 1: A. niger DNA treated with Co(II); Lane 2:
A. niger DNA treated with Ni(II); Lane 3: A. niger DNA treated with
Cu(II)
Cu(II) complexes, respectively, compared to the control
DNA of A. niger (Lane-C). This shows that the control
DNA alone does not show any apparent cleavage whereas
Co(II), Ni(II), and Cu(II) complexes have shown the same.
However, the nature of reactive intermediates involved in
the DNA cleavage by the complexes has not been clear.
The results indicated the important role of metal ions in the
isolated DNA cleavage reaction. From these results, we
infer that the Co(II), Ni(II), and Cu(II) complexes act as a
potent nuclease agents. As the compound was observed to
cleave the DNA, it can be concluded that the compound
inhibits the growth of the pathogenic organism by cleaving
the genome.
Biological results
DNA cleavage studies
The representative Co(II) 2, Ni(II) 4, and Cu(II) 6 com-
plexes were studied for their DNA cleavage activity by
agarose gel electrophoresis method.
Figure 3 shows the results of oxidative DNA cleavage
experiments carried out with the complexes of Co(II),
Ni(II), and Cu(II) by agarose gel electrophoresis method.
Control experiments clearly revealed that the untreated
DNA does not show any cleavage (Fig. 3; Lane-C)
whereas, all the metal complexes have exhibited cleavage
activity on DNA. The difference in the migration was
observed in the Lanes 1, 2, and 3 of Co(II), Ni(II), and
In vitro antibacterial and antifungal activity
The microbial results are systematized in Table 3. The
antibacterial and antifungal studies suggested that the
Fig. 2 TGA/DTA spectra of
Co(II) (2) complex
123

352
Med Chem Res (2011) 20:346–354
Schiff bases were found to be biologically active, and
some of their metal complexes showed significantly
enhanced antibacterial and antifungal activities. It is,
however, known (Chohan et al., 2004; Chohan and
Praveen, 2001) that the chelating tends to make the
Schiff bases act as more powerful and potent bactereo-
static agents, thus inhibiting the growth of bacteria and
fungi more than the parent Schiff bases. It is assumed
that factors, such as solubility, conductivity, dipole
moment, and cell permeability mechanism (influenced by
the presence of metal ions) may contribute to the
increase in the activities of the metal complexes relative
to Schiff bases.
Co(II) [ Ni(II) [ Cu(II) and that against bacterial species
are in the order of S. aureus [ P. auregenosa [ B.
subtitles.
From the antifungal studies, it is clear that the Co(II),
Ni(II), and Cu(II) complexes are found to possess higher
antifungal activities than the respective Schiff bases. The
activities of metal complexes are in the order of
Co(II) [ Ni(II) [ Cu(II), and that against the fungi species
are in the order of A. niger [ P. crysogenum.
The minimum inhibitory concentration (MIC) of some
selected compounds, which showed significant activity
against selected bacterial and fungi species, was deter-
mined, and the MIC values of these compounds indicated
that these compounds are the most active in inhibiting the
growth of the tested organisms at a 10 lg/ml concentration
(Table 4). It has been reported in past that the metal
complexes with Schiff bases possess a high antimicrobial
activity (Ajaykumar et al., 2009a, b; Gangadhar et al.,
2008; Rehman et al., 2005; Gangadhar et al., 2009a, b).
In the case of antibacterial studies, it is observed that
both the Schiff bases were found to be active against
S. aureus and B. subtitlis. All the Co(II), Ni(II), and
Cu(II) metal complexes exhibited much enhanced activity
against S. aureus, B. subtitlis, and P. aerugenosa. The
activities of metal complexes are in the order of
Table 3 Antimicrobial results of metal complexes
Compound
Conc. (lg ml-1
)
Growth inhibition against bacteria in mm
Growth inhibition against fungi in mm
S. aureus
B. subtilis
P. auregenosa
E. coli
A. Niger
P. Crysogenum
C22H20FN5S
100
200
500
100
200
500
100
200
500
100
200
500
100
200
500
100
200
500
100
200
500
100
200
500
500
500
8
10
13
12
12
14
14
14
17
14
16
18
12
12
15
10
14
18
8
8
10
12
8
7
10
12
8
7
8
10
10
13
12
12
14
14
14
18
14
16
20
13
13
15
14
16
20
11
11
15
14
14
17
–
8
8
12
8
12
8
C23H22FN5S
10
14
13
14
16
10
11
12
10
12
14
10
11
14
7
9
10
12
8
8
12
10
10
14
14
14
16
7
12
14
14
17
14
15
18
12
12
15
13
13
16
11
11
14
13
13
15
–
Co(C22H19FN5S)2
Co(C23H21FN5S)2
Ni(C22H19FN5S)2
Ni(C23H21FN5S)2
Cu(C22H19FN5S)2
Cu(C23H21FN5S)2
11
13
10
10
12
7
12
16
10
13
16
8
7
13
7
7
12
6
12
16
6
12
15
7
13
16
7
10
13
6
12
16
28
–
10
14
29
–
8
9
15
35
–
13
25
–
Streptomycin
Nyastatin
26
25
Less than 12 mm—inactive; 12–16 mm—moderately active; above 16 mm—highly active
123

Med Chem Res (2011) 20:346–354
353
Table 4 Results of minimum inhibitory concentration (lg/ml)
Compound
S. aureus
B. subtilis
P. auregenosa
E. coli
A. Niger
P. Crysogenum
Co(C22H19FN5S)2
Co(C23H21FN5S)2
Ni(C22H19FN5S)2
Ni(C23H21FN5S)2
Cu(C22H19FN5S)2
Cu(C23H21FN5S)2
10
10
15
10
15
15
15
20
15
15
20
20
15
10
10
10
15
15
20
25
20
20
25
25
10
10
15
10
15
10
15
10
15
10
15
10
3-substituted-4-(4-hexyloxyphenyl)-4H-1,2,4-triazoles.
Chem Res 18:49–58
Med
Conclusion
Dhaka KS, Mohan J, Chadha VK, Pujari HK (1974) Heterocyclic
systems containing a bridgehead nitrogen atom. Ind J Chem
12:288
Duta RL, Syamal A (1992) Elements of magnetochemistry, 2nd edn.
Elsevier, New Delhi
Escobar-Valderrama JL, Garcia-Tapia JH, Ramirez-Ortiz J, Rosales
MJ, Toscano RA, Valdes-Martinez J (1989) Crystal, molecular
and electronic structure of l-H-3-methyl-4-amine-5-thione-1,2,4-
triazol. Can J Chem 67:198–201
Ewiss NF, Bahajaj AA, Elsherbini EA (1986) Synthesis of hetero-
cycles, Part VI-synthesis and antimicrobial activity of some
4-amino-5-aryl-1,2,4-triazole-3-thions and their derivatives.
J Heterocycl Chem 23:1451–1458
On the basis of spectroscopic results, the square planar
geometry is proposed for all the newly synthesized com-
plexes. All these complexes cleaved the DNA of A. niger.
The results show that the metal complexes possess higher
antibacterial and antifungal activities than the Schiff bases.
Thus, the biological studies revealed the important role of
metal ions in the biological systems in line with other
studies.
Gribble GW (1995) Five-membered rings with one heteroatom and
fused carbocyclic derivatives. In: Bird CW, Katrizky AR, Rees
CW, Scriven EFV (eds) Comprehensive heterocyclic chemistry,
2nd edn, vol 2. Pergamon, Oxford. p 207
Gangadhar BB, Prakash GA, Prema SB, Sangamesh AP (2008)
Synthesis, spectral characterization, in vitro antibacterial, anti-
fungal and cytotoxic activity of Co(II), Ni(II) and Cu(II)
complexes with 1,2,4-triazole Schiff bases. Eur J Med Chem
43:2639–2649
Gangadhar BB, Prema SB, Sangamesh AP (2009a) Synthesis, spectral
characterization, fluorescence and in vitro biological studies of
Co(II), Ni(II) and Cu(II) complexes with 1,2,4-triazole Schiff
bases. J Enzyme Inhib Med Chem 24(2):381–394
Gangadhar BB, Sangamesh AP, Prema SB (2009b) Synthesis, spectral
characterization, in vitro microbial and cytotoxic studies of
lanthanum(III) and thorium(IV) complexes with 1,2,4-triazole
Schiff bases. J Enzyme Inhib Med Chem 24(03):730–741
Heleen AN, Jaap GH, Ronald H, Jan R, Theo LS, Derk JS, Johannes
GV (1991) pH Control of the photophysical properties of
ruthenium complexes containing 3-(Pyrazin-2-yl)-1,2,4-triazole
ligands. Inorg Chem 30(1):48–54
Hibino S, Choshi T (2002) Simple indole alkaloids and those with a
nonrearranged monoterpenoid unit. Nat Prod Rep 19(2):148–180
Holm RH (1974) Inorganic biochemistry. In: Eirchorn GL (ed) vol.
2;1137. Elsevier, Amsterdam
Ito YN, Katsuki T (1999) Asymmetric catalysis of new generation
chiral metallosalen complexes. Bull Chem Soc Jpn 72(4):603–
619
Joost PC, John HD, Lucas RG, Jaap GH, Anthony LS, Jan R (1992)
Synthesis and properties of isostructural transition-metal (cop-
per, nickel, cobalt and iron) compounds with 7,70,8,80-Tetra-
cyanoquinodimethanide(1-) in an unusual monodentate
coordination mode. crystal structure of Bis(3,5-bis(pyridin-2-
yl)-4-amino-1,2,4-triazole)bis(7,70,8,80
References
Ajaykumar DK, Sangamesh AP, Prema SB (2009a) SNO donor Schiff
bases and their Co(II), Ni(II) and Cu(II) complexes: synthesis,
characterization, electrochemical and antimicrobial studies.
J Sulfur Chem 30(2):145–159
Ajaykumar K, Sangamesh AP, Prema SB (2009b) Electrochemical
properties of some transition metal complexes: synthesis,
characterization and in vitro antimicrobial studies of Co(II),
Ni(II), Cu(II), Mn(II) and Fe(III) complexes. Int J Electrochem
Sci 4:717–729
Anthonisamy VSX, Murugesan R (1998) EPR of hexakis(1-propyl-
tetrazole) copper(II) tetrafluoroborate and Cu(II)-doped hexa-
kis(1-propyltetrazole)zinc(II) tetrafluoroborate. Dynamic to
static Jahn–Teller distortion. Chem Phys Lett 287(3–4):353–358
Azza Abu-Hussen AA, Adel Emara AA (2004) Metal complexes of
some thiocarbohydrazone ligands: synthesis and structure.
J Coord Chem 57(11):973–987
Bhat AR, Bhat GV, Shenoy GG (2001) Synthesis and in vitro
antimicrobial activity of new 1,2,4-triazoles. J Pharm Pharmacol
53:267–272
Canali L, Sherrington DC (1999) Utilisation of homogeneous and
supported chiral metal(salen) complexes in asymmetric catalysis.
Chem Soc Rev 28(2):85–93
Chohan ZH, Praveen M (2001) Synthesis, characterization, coordi-
nation and antibacterial properties of novel asymmetric 1,10-
disubstituted ferrocene-derived Schiff-base ligands and their
Co(II), Cu(II), Ni(II) and Zn(II) complexes. Appl Organomet
Chem 15(7):617–625
Chohan ZH, Supuran CT, Scozzafava A (2004) Metalloantibiotics:
synthesis and antibacterial activity of Cobalt(II), Copper(II),
Nickel(II) and Zinc(II) complexes of Kefzol. J Enzyme Inhib
Med Chem 19(1):79–84
Cui Xiang-Shu, Chen Jing, Chai Kyu-Yun, Lee JinSeok, Quan Zhe-
Shan (2009) Synthesis and anticonvulsant evaluation of
tetracyanoquinodimethanido)copper (II). Inorg Chem 31(2):198–
202
123

354
Med Chem Res (2011) 20:346–354
Katsuki T (1995) Catalytic asymmetric oxidations using optically
active (salen) manganese (III) complexes as catalysts. Coord
Chem Rev 140:189–214
Kitazaki T, Tamura N, Tasaka A, Matsushita Y, Hayashi R, Konogi
K, Itoh K (1996) Optically active antifungal azoles. VI.
Synthesis and antifungal activity of N-((1R, 2R)-2-(2,4-diflur-
ophenyl)-2-hydroxy-1-methyl-3-(1H-1,2,4-triazol-1-yl)propyl)-N0-
(4-substituted phenyl)-3-(2H, 4H)-1,2,4-triazolones and 5-(1H,
4H)-tetrazolones. Chem Pharm Bull 44:314–327
Kumar A, Singh G, Handa RN, Dubey SN (1999) Synthesis and
characterization of complexes of cobalt(II), nickel(II), copper(II)
and zinc(II) with Schiff bases derived from cinnamaldehyde and
4 amino-3-ethyl-5-mercapto-s-triazole and 4-amino-5-mercapto-
3-n-propyl-s-triazole. Ind J Chem 38(A):613–617
Palmer MH, Dines C (2004) An ab initio study of the structure,
tautomerism and molecular properties of the C- and N-amino-
1,2,4-triazoles. J Mol Struct 705:177–187
Ray RK, Kauffman GB (1990) EPR Spectra and covalency of bis
(amidinourea/O-alkyl-1-amidinourea)copper(II) complexes Part
II. Properties of the CuN4 2-chromophore. Inorg Chim Acta
173:207–214
Rehman SU, Chohan ZH, Gulnaz F, Supuran CT (2005) In vitro
antibacterial antifungal and cytotoxic activities of some couma-
rins and their metal complexes. J Enzyme Inhib Med Chem
20(4):333–340
Reichert DE, Lewis JS, Anderson CJ (1999) Metal complexes as
diagnostic tools. Coord Chem Rev 184(1):3–66
Sacconi L (1966) Four-, five- and six-coordinated complexes of 3D
metals with substituted salicylaldimines. Coord Chem Rev
1:192–204
Sadana AK, Mirza Y, Aneja KR, Prakash O (2003) Hypervalent
iodine mediated synthesis of 1-aryl/hetryl-1,2,4-triazolo[4, 3-a]
pyridines and 1-aryl/hetryl 5-methyl-1,2,4-triazolo[4,3-a]quino-
lines as antibacterial agents. Eur J Med Chem 38:533–536
Schwietert CW, McCue JP (1999) Coordination compounds in
medicinal chemistry. Coord Chem Rev 184(1):67–89
Sen AK, Singh G, Singh K, Handa RN, Dubey SN, Squattrito PJ
(1998) Synthesis and characterization of some monofunctional
bidentate Schiff bases derived from cinnamaldehyde and s-tri-
azoles, and their Co(II), Ni(II), Cu(II) and Zn(II) complexes.
Proc Ind Acad Sci: Chem Sci 110(2):75–81
Somei M, Yamada F (2003) Simple indole alkaloids and those with a
nonrearranged monoterpenoid unit. Nat Prod Rep 20(2):216–242
Todoulou OG, Papadaki-Valiraki AE, Filippatos EC, Ikeda S, De
Clercq E (1994) Synthesis and anti-myxovirus activity of some
novel N,N0-disubstituted thioureas. Eur J Med Chem 29:127–131
Walser A, Flynn T, Mason C (1991) Pentacyclic triazolodiazepines as
PAF-antagonists. J Heterocycl Chem 28:1121–1125
Yamada S (1966) Recent aspects of the stereochemistry of Schiff-
base-metal complexes. Coord Chem Rev 1:415–437
123