Spectroscopic, DNA cleavage and antimicrobial studies of Co(II), Ni(II) and Cu(II) complexes of sulfur donor schiff bases

Article abstract of DOI:10.1080/17415991003668186

A series of Co(II), Ni(II) and Cu(II) complexes of the type ML₂ of Schiff bases derived from methylthiosemicarbazide and isatin/chloroisatin have been synthesized. Schiff bases exhibit thiol-thione tautomerism wherein sulfur plays an important role in the coordination. In view of analytical, spectral (IR, NMR, UV-vis, ESR, FAB-Mass) and magnetic studies, it has been concluded that all the metal complexes possess octahedral geometry. The measured molar conductance values in DMF indicate that the complexes are non-electrolytes in nature. The redox behaviors of the metal complexes are investigated by using cyclic voltammetry. The Schiff bases and their metal complexes have been screened for their antibacterial (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi) and antifungal activities (Aspergillus niger, Aspergillus flavus and Cladosporium) by the minimum inhibitory concentration method. The DNA cleavage studies showed the cleavage of DNA by the agarose gel electrophoresis method.

Full text of DOI:10.1080/17415991003668186

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Spectroscopic, DNA cleavage and
antimicrobial studies of Co(II), Ni(II)
and Cu(II) complexes of sulfur donor
Schiff bases
Sangamesh A. Patil a , Vinod H. Naik a , Ajaykumar D. Kulkarni a &
Prema S. Badami b
a Department of Chemistry , Karnatak University , Dharwad,
580003, Karnataka, India
b Department of Chemistry , Shri Sharanabasaveswar College of
Science , Gulbarga, 585102, Karnataka, India
Published online: 07 Apr 2010.
To cite this article: Sangamesh A. Patil , Vinod H. Naik , Ajaykumar D. Kulkarni & Prema S.
Badami (2010) Spectroscopic, DNA cleavage and antimicrobial studies of Co(II), Ni(II) and
Cu(II) complexes of sulfur donor Schiff bases, Journal of Sulfur Chemistry, 31:2, 109-121, DOI:
10.1080/17415991003668186
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Journal of Sulfur Chemistry
Vol. 31, No. 2, April 2010, 109–121
Spectroscopic, DNA cleavage and antimicrobial studies of
Co(II), Ni(II) and Cu(II) complexes of sulfur donor Schiff bases
Sangamesh A. Patila*, Vinod H. Naika, Ajaykumar D. Kulkarnia and Prema S. Badamib
aDepartment of Chemistry, Karnatak University, Dharwad 580003, Karnataka, India; bDepartment of
Chemistry, Shri Sharanabasaveswar College of Science, Gulbarga 585102, Karnataka, India
(Received 15 October 2009; final version received 1 February 2010)
A series of Co(II), Ni(II) and Cu(II) complexes of the type ML2 of Schiff bases derived from
methylthiosemicarbazide and isatin/chloroisatin have been synthesized. Schiff bases exhibit thiol–thione
tautomerism wherein sulfur plays an important role in the coordination. In view of analytical, spectral (IR,
NMR, UV-vis, ESR, FAB-Mass) and magnetic studies, it has been concluded that all the metal complexes
possess octahedral geometry. The measured molar conductance values in DMF indicate that the complexes
are non-electrolytes in nature. The redox behaviors of the metal complexes are investigated by using
cyclic voltammetry. The Schiff bases and their metal complexes have been screened for their antibacterial
(Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi) and antifungal
activities (Aspergillus niger, Aspergillus flavus and Cladosporium) by the minimum inhibitory concentra-
tion method. The DNA cleavage studies showed the cleavage of DNA by the agarose gel electrophoresis
method.
Keywords: antimicrobial; DNA; electrochemical; spectroscopy; sulfur donor Schiff bases
1. Introduction
Metal complexes have been gaining increasing importance in recent years, particularly in the
design of repository, slow-release or long-acting drugs in nutrition and in the study of metabolism
(1). Metal complexes of the Schiff bases have also been widely studied due to their relevance
in biological systems (2). Isatin (indole-2,3-dione) is a versatile lead molecule for designing
potential bioactive agents and its derivatives were reported to possess a broad spectrum of antiviral
activities (3, 4). Isatin is used to prepare a large variety of heterocyclic compounds such as
indoles and also as a raw material for drug synthesis (5). Schiff bases and Mannich bases of isatin
are known to possess a wide range of pharmacological properties that include antibacterial (6),
anticonvulsant (7), anti-HIV (8), antifungal (9) and antiviral activities (10). Ligands consisting
of sulfur donor atoms are the subject of interest in coordination chemistry. The chemistry of
thiosemicarbazides has received considerable attention in view of their variable bonding modes,
promising biological implications, structural diversity and ion-sensing ability (11–13).As regards
*Corresponding author. Email: patil1956@rediffmail.com
ISSN 1741-5993 print/ISSN 1741-6000 online
© 2010 Taylor & Francis
DOI: 10.1080/17415991003668186
http://www.informaworld.com

110 S.A. Patil et al.
biological implications, thiosemicarbazide metal complexes have intensively been investigated
for antiviral, anticancer, antitumoral, antimicrobial, antiamoebic and anti-inflammatory activities.
The interaction of transition metal complexes with DNA has extensively been studied for their
usage as probes for DNA structure and their potential application in chemotherapy. Very recently,
Cu(II) complexes have been reported to be active in DNA strand scission (14–16).
In continuation of our earlier work on Schiff’s base metal complexes (17–20), here we report
the antimicrobial and DNA cleavage activity studies of Co(II), Ni(II) and Cu(II) metal complexes,
with Schiff bases derived from methylthiosemicarbazide and isatin/chloroisatin. The structural
features of the Schiff bases and their metal complexes have been studied by various spectral and
analytical techniques.
2. Results and discussion
The Schiff bases (I and II) form octahedral complexes (1–6) with CoCl2· 6H2O/NiCl2·6H2O/
CuCl2·2H2O in ethanol. All the Co(II), Ni(II) and Cu(II) complexes are colored, stable and non-
hygroscopic in nature. These 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 deprotonated ligand. The molar
conductancevaluesaretoolowtoaccountforanydissociationofthecomplexesinDMF, indicating
the non-electrolytic nature of the complexes in DMF (Table 1).
2.1. IR spectral studies
The prominent infrared spectral data of Schiff bases and their Co(II), Ni(II) and Cu(II) complexes
are presented inTable 2. The IR spectra of the Schiff bases exhibited two characteristic bands in the
regions 3244–3195 and 3254–3234 cm−1, respectively, due to ν(NH) groups (21).A characteristic
strong band at 2672–2666 cm−1 is ascribed to ν(SH) and another characteristic strong band at
1199–1193 cm−1 is assigned to ν(C S) (22). These observations suggest that the Schiff bases
=
exhibit thiol–thione tautomerism (Figure 1). In addition to this, the characteristic high-intensity
bands in the regions 1617–1615 and 3202–3206 cm−1 and a strong band in the region 1705–
1700 cm−1 in the IR spectra of the Schiff bases are assigned to ν(C N) (23), ν(NH) of isatin
=
=
moiety and ν(C O) (20, 23, 24), respectively.
In comparison with the spectra of the Schiff bases, all the Co(II), Ni(II) and Cu(II) complexes
exhibited the band of ν(C N) in the region 1588–1574 cm−1; the shift to lower wave numbers
=
indicates that the nitrogen atom of the azomethine group is coordinated to the metal ion (25).
The deprotonation of the thiol group is indicated by the absence of a band around ∼2670 cm−1in
−
all the metal complexes, which is due to ν(S H) of the Schiff bases, indicating that the metal
ion is coordinated through the sulfur atom. This is further supported by the band around 772–
781 cm−1 in the metal complexes due to ν(C S). The band corresponding to ν(C O) was shifted
to lower wave frequency by about 45–40 cm−1 and appeared in the region 1666–1653 cm−1 in
the metal complexes, suggesting the coordination of carbonyl oxygen to the metal ion (26).
The new bands in the region 473–460 cm−1 in the spectra of the complexes are−a1ssigned to
−
=
−
stretching frequencies of M N bonds (27). The bands in the region 348–336 cm of far-IR
spectra are due to metal–sulfur bond formation (28). The unaltered position of a band due to
ν(NH) of isatin moiety in all the metal complexes indicates that the nitrogen atom is not involved
in coordination.
Thus the IR spectral data results provide strong evidences for the complexation of Schiff bases
with metal (II) ions.

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.
M%
C%
H%
N%
Molar
Comp.
no.
conductance
Empirical formula
Colour/yield %
78%
75%
Brown/70%
Obsd.
Calcd.
Obsd.
Calcd.
Obsd.
Calcd.
Obsd.
Calcd.
(ꢀ−1 cm2 mole−1
)
μeff (BM)
I
II
1
2
3
4
5
6
C10H10N4OS
C10H9N4O3SCl
–
–
–
–
51.51
44.52
45.72
40.22
45.29
45.43
46.14
40.15
51.28
44.77
45.21
40.40
45.80
45.80
46.36
40.13
4.56
3.12
3.75
2.13
3.27
3.17
3.77
3.42
4.27
3.35
3.42
2.69
3.43
3.43
3.40
3.01
23.25
20.70
21.62
18.43
21.17
18.24
21.45
18.61
23.93
20.89
21.33
18.85
21.37
18.88
21.17
18.72
–
–
–
–
Co(C10H9N4OS)2
Co(C10H8N4OSCl)2
Ni(C10H9N4OS)2
Ni(C10H9N4OSCl)2
Cu(C10H9N4OS)2
Cu(C10H9N4OSCl)2
11.46
9.22
11.66
11.66
11.59
9.56
11.23
9.93
11.06
11.06
11.90
9.79
27.8
19.25
25.88
26.64
28.4
24.11
4.32
4.84
2.92
3.32
1.76
1.78
Brown/66%
Yellowish green/68%
Yellowish green/69%
Dark green/65%
Dark green/69%

112 S.A. Patil et al.
Table 2. The important infrared frequencies (in cm−1) of Schiff bases and their metal complexes.
−
ν(N H)
−
=
=
=
−
−
−
Compound
ν(N H)
of isatin ν(C N) ν(C O) ν(C S) ν(SH) ν(C S) ν(M N) ν(M S)
C10H10N4OS
C10H9N4OSCl
3244–3254
3195–3234
3241
3193
3243
3192
3241
3191
3202
3206
3199
3204
3201
3204
3198
3207
1617
1615
1586
1575
1582
1579
1588
1574
1705
1700
1664
1655
1666
1653
1665
1658
1199
1193
2672
2666
–
–
–
–
–
–
Co(C10H9N4OS)2
Co(C10H8N4OSCl)2
Ni(C10H9N4OS)2
Ni(C10H8N4OSCl)2
Cu(C10H9N4OS)2
Cu(C10H8N4OSCl)2
–
–
–
–
–
–
–
–
–
–
–
–
773
777
772
780
776
781
471
465
460
473
463
469
347
336
345
341
348
339
H
O
N
N
S
NH
H3C
R
N
HN
C
N
C
R
HN
CH3
SH
N
O
H
Schiff base
R
Molecular formula
C10H10N4OS
I
H
II
Cl
C H9N4OSCl
10
Figure 1. Structure of Schiff bases: thiol–thione tautomerism.
Table 3. NMR data of Schiff bases.
Schiff base
1H NMR data (ppm)
C10H10N4OS
11.3 (s, 1H, NH of isatin moiety), 9.2 (s, 1H, NH of thiosemicarbazone), 8.46 (s, 1H, SH), 6.99–7.61
(m, 4H, Ar–H), 3.3 (s, 3H, CH3)
C10H9N4OSCl 11.1 (s, 1H, NH of isatin moiety), 9.18 (s, 1H, NH of thiosemicarbazone), 8.42 (s, 1H, SH), 6.01–7.87
(m, 3H, Ar–H), 3.16 (s, 3H, CH3)
2.2. 1H NMR spectral study of Schiff bases I and II
The 1H NMR data of both the Schiff bases are presented in Table 3 and the 1H NMR spectrum of
Schiff bases I and II are given in Supplementary material (Figures S1 and S2, respectively). In the
1H NMR spectrum of the Schiff base I, the NH protons of isatin and thiosemicarbazide moieties
exhibited signals at 11.3 ppm (s, 1H) and 9.2 ppm (s, 1H), respectively (29). A characteristic
−
proton signal at 8.46 ppm (s, 1H) is assigned to the SH proton. In addition to this, the multiplet
signals in the region 6.99–7.61 ppm (m, 4H) and a sharp singlet signal at 3.3 ppm (s, 3H) are due
to aromatic protons and CH3 protons, respectively (30). In the case of Schiff base II, the signals
observed at 11.1 ppm (s, 1H) and 9.18 ppm (s, 1H) are attributed to the NH protons of isatin and
−
thiosemicarbazide moieties, respectively. A signal at 8.42 ppm (s, 1H) is ascribed to the SH
proton. The signals observed as a multiplet in the region 7.01–7.87 (m, 3H) and as a singlet at
3.16 ppm are due to aromatic and CH3 protons, respectively.
2.3. Electronic spectral and magnetic studies
The octahedral Co(II) complexes exhibit electronic spectral bands in the region 8000–10,000,
4
4
4
14,000–16,000 and 18,000–20,000 cm−1 corresponding to T1g (F) → T2g (F) (ν1), T1g (F)

Journal of Sulfur Chemistry 113
Table 4. Ligand field parameters of Co(II) and Ni(II) complexes.
Transitions (cm−1
ν1 ν2 ν3
)
Cald. ν2
(cm−1
Cald.
Complex No.
)
Dq (cm−1
)
B1 (cm−1
)
% Distortion ν2/ν1 LSFE μeff (BM)
β
βo (%)
Co(C10H9N4OS)2
9411 14326 19965 12885
947.80
948.40
980.23
982.32
330.28
299.14
414.62
422.63
1.666
1.993
1.402
1.245
1.586 32.49
1.585 32.51
1.43 33.61
1.40 33.66
–
–
0.313 68.72
0.283 71.66
0.373 60.73
0.400 60.04
Co(C10H8N4OSCl)2 9543 15121 19952 12629
Ni(C10H9N4OS)2 9808 14128 21441 13889
Ni(C10H8N4OSCl)2 9820 14141 21830 13961
3.194
3.193
4
4
→ T2g (F) (ν2) and 4T1g (F) → T2g (P) (ν3) transitions, respectively. In the present study, the
brownish Co(II) complexes showed the absorption bands in the region 9411–9543, 14,326–15,121
and 19,665–19,952 cm−1 corresponding to ν1, ν2 and ν3 transitions, respectively. These bands are
the characteristic of high-spin octahedral Co(II) complexes (28). The ligand field parameters
calculated are presented in Table 4. The magnetic measurement of Co(II) complexes exhibited
magnetic moment values of 4.32–4.84, which agree well with the octahedral range of 4.3–5.7 BM
for Co(II) complexes (31).
The greenish Ni(C10H9N4OS)2 complex exhibited three bands at 9808, 14,128 and 21,441 cm−1
3
3
3
3
3
3
attributed to the A2g → T2g (ν1), A2g → T1g (F) (ν2) and A2g → T1g (P) (ν3) transitions,
respectively, which indicate an octahedral geometry around the Ni(II) ion (28). The ligand field
parameters are given in Table 4 (32). The value of ν2/ν1 is found to be around 1.44. The cal-
culated value of μeff is 3.194, which is within the range 2.8–3.5 BM, suggesting the octahedral
environment. The values of the nephelauxetic parameter, β, indicate low covalent character of
the metal–ligand σ bonds (33). Ni(II) complexes showed the magnetic moment values of 2.92–
3.32 BM, which are within the range 2.8–3.5 BM, suggesting consistency with their octahedral
environment (34). Hence, the ligand field parameters correlate with the electronic spectral and
magnetic properties.
The electronic spectra of Cu(II) complexes display two prominent bands: a low-intensity broad
band around 16,231 cm−1 is assignable to 2Eg → T2g transition and another high-intensity band
2
at 26,325 cm−1 is due to ligand → metal charge transfer. On the basis of electronic spectra, the dis-
torted octahedral geometry around the Cu(II) ion is suggested (35). The Cu(II) complexes showed
magnetic moment of 1.76–1.78 BM, which is slightly higher than the spin-only value (1.73 BM)
expected for one unpaired electron, which offers the possibility of an octahedral geometry (36).
2.4. FAB-Mass spectral studies
Representative FAB-Mass spectrum of Schiff base I is depicted in Figure 2. The spectrum showed
a molecular ion peak at m/z 234, which is equivalent to its molecular weight. In addition to this,
the fragment peaks observed at m/z 219 and 145 are due to the cleavage of CH3 and CN2SH2,
respectively. In the case of Schiff base II, the molecular ion peak is observed at m/z 268, which
is ascribed to C10H9N4SOCl.
The FAB-Mass spectrum of all the complexes exhibited molecular ion peak equivalent of
their molecular weight along with other fragmentation peaks. Hence, only the representative
Co(C10H9N4OS)2, Ni(C10H9N4OS)2 and Cu(C10H9N4OS)2 complexes are discussed here. The
spectrum of Co(C10H9N4OS)2 showed a molecular ion peak M+ at m/z 525, which is equiv-
alent of its molecular weight. The fragmentation peaks observed at 436, 391, 302 and 157
correspond to C2N2SH5, C8N2H5O, C2N2SH5 and C8N2H5O, respectively. In the case of the
Ni(C10H9N4OS)2 complex (Figure 3), the molecular ion peak M+ at m/z 524 corresponds to its
molecular weight. The complexes exhibiting fragmentation peaks at 435, 390, 301 and 156 corre-
spond to cleavages of C2N2SH5, C8N2H5O, C2N2SH5 and C8N2H5O, respectively. The spectrum

114 S.A. Patil et al.
100
50
234
145
219
107
95
154
136
0
100 110 120 130 140 150 160 170 180 190 200 210 220 230 240
m/z
100
50
0
250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400
m/z
Figure 2. FAB-Mass spectrum of Schiff base I.
100
156
160
50
301
136
140
105
92
329
129
120
199
261
307
320
289
280
258
343
210 224 245
0
100
180
200
220
240
260
300
340
m/z
5
100
362
50
0
524
435
390
401
451
460
501
407
420
361
360
380
400
440
480
500
520
540
560
580
600
620
m/z
Figure 3. FAB-Mass spectrum of Ni(C10H9N4OS)2.
of the Cu(C10H9N4OS)2 complex exhibited a molecular ion peak M+, which is equivalent of its
molecular weight, at m/z 529 with all other fragmentation peaks.
2.5. ESR spectral studies
The ESR spectral studies of the Cu(II) complexes provide information of the metal ion envi-
ronment. The ESR spectra of the Cu(II) complexes were recorded in DMSO at liquid nitrogen
temperature (LNT) and at RT. The ESR spectrum of one representative Cu(C10H9N4OS)2 com-
plex is discussed here. The spectrum at RT shows one intense absorption band in the high field
region and is isotropic due to the tumbling motion of the molecules. However, this complex at
LNT shows four well-resolved peaks in the low field region. The g and g values have been
|
−
found to be 2.113 and 2.034, respectively. These values indicate that the unpaired electron lies
2
2
predominantly in the dx −y orbital (37). The trend g > g > 2.0023 observed for the complex
ꢀ
⊥
2
2
indicates that the unpaired electron is localized in the dx −y orbital of the Cu(II) ion and is char-
acteristic for the axial symmetry. The value of gav was calculated to be 2.06. Thus, the results
confirmed that the Cu(II) complexes possess distorted octahedral geometry.
2.6. Electrochemistry
All the complexes were studied for their electrochemical behavior. Both the Cu(II) complexes
exhibited similar electrochemical properties. A cyclic voltammogram of Cu(C10H8N4OSCl)2

Journal of Sulfur Chemistry 115
Figure 4. Cyclic voltammogram of Cu(C10H8N4OSCl)2.
(Figure 4) displays a reduction peak at Epc = −0.5V with a corresponding oxidation peak at
Epa = −0.25V. The peak separation of this couple (ꢁEp) is 0.25 at 0.1V and increases with
increasing scan rate. The most significant feature of the Cu(II) complex is the Cu(II)/Cu(I) couple.
The difference between forward and backward peak potentials can provide a rough evaluation of
the degree of the reversibility of one electron transfer reaction. The analyses of cyclic voltametric
responses with the scan rate varying from 50 to 300 mV s−1 give the evidence for quasi-reversible
one electron transfer process. The ratio of cathodic to anodic peak height was less than 1. How-
ever, the peak current increases with increasing square root of the scan rates. This establishes
the electrode process as diffusion-controlled (38). The separation in peak potentials increases
at higher scan rates. These characteristic features are consistent with the quasi-reversibility of
Cu(II)/Cu(I) couple.
The cyclic voltammogram of the Co(C10H8N4OSCl2)2 complex is depicted in Figure 5. It
exhibits the first reduction peak at Epc = −0.65V with an associated oxidation peak at Epa
=
−0.13V and second reduction peak at Epc = 0.24V with an associated oxidation peak at Epa
=
Figure 5. Cyclic voltammogram of Co(C10H8N4OSCl)2.

116 S.A. Patil et al.
0.59V corresponding to Co(II)/Co(I) and Co(I)/Co(0), respectively. The peak separation of this
(ꢁEp) is 0.52 and 0.35V at a scan rate of 0.1V s−1 for the first and second redox couples,
respectively. This Co(II) complex also has a quasi-reversible character and the peak current rises
with increasing square root of the scan rates. The difference between forward and backward peak
potentials can provide a rough evaluation of the degree of the reversibility.
2.7. Pharmacological results
2.7.1. In vitro antibacterial and antifungal activity
The antibacterial studies inferred that the Schiff bases (I and II) were highly active against
Pseudomonas aeruginosa and Escherichia coli and moderately active against Salmonella typhi
and Staphylococcus aureus. All the Co(II), Ni(II) and Cu(II) metal complexes showed potential
antibacterial activity against all the bacterial strains (Table 5) than the respective Schiff bases.
In the case of antifungal activity, the Schiff bases and their Co(II), Ni(II) and Cu(II) complexes
Table 5. Antimicrobial results of Schiff bases and their metal complexes.
% Inhibition against bacteria
MIC
Concentration
(μg mL−1
)
Compound
E. coli S. aureus P. aeruginosa S. typhi A. flavus Cladosporium A. niger
C10H10N4OS
10
30
50
44
66
73
78
40
56
58
69
57
62
65
76
48
65
68
70
51
54
66
73
55
63
66
76
45
61
64
69
100
C10H9N4OSCl
10
30
50
52
59
61
75
44
64
69
73
51
55
63
78
45
60
68
70
58
59
69
77
56
66
74
78
57
66
78
85
100
Co(C10H9N4OS)2
Co (C10H8N4OSCl )2
Ni(C10H9N4OS )2
Ni (C10H9N4OSCl )2
Cu(C10H9N4OS)2
Cu(C10H8N4OSCl)2
10
30
50
67
76
83
94
60
73
82
89
68
78
80
85
57
68
79
90
78
84
91
97
75
85
89
94
71
84
91
95
100
10
30
50
65
77
84
93
57
64
81
91
75
85
84
96
68
79
79
93
76
80
87
98
74
79
88
95
80
85
90
96
100
10
30
50
63
78
79
94
56
63
75
86
65
75
79
94
56
68
78
86
90
91
95
80
93
94
99
90
93
97
100
100
100
10
30
50
65
73
79
88
57
58
64
79
60
70
88
89
55
69
73
85
88
95
97
99
82
86
92
95
86
88
90
95
100
10
30
50
61
74
79
90
71
78
80
82
71
76
84
90
51
65
78
80
84
90
95
98
74
79
91
93
77
87
96
98
100
10
30
50
65
73
88
98
63
69
84
97
73
75
81
94
65
66
79
85
85
87
89
98
76
81
95
96
76
79
84
94
100
Gentamycin
Flucanozole
100
100
100
–
100
–
100
–
100
–
–
100
–
100
–
100

Journal of Sulfur Chemistry 117
Table 6. MIC results (μg mL−1).
Compound
E. coli
P. aeruginosa
S. typhi
A. flavus
Cladosporium
A. niger
C10H10N4OS
C10H9N4OSCl
25
25
10
10
10
10
25
25
10
10
10
10
10
25
25
10
25
10
10
–
25
25
10
10
10
10
25
10
10
10
>100
>100
10
Co(C10H9N4OS)2
Co (C10H8N4OSCl)2
Ni( C10H9N4OS )2
Ni (C10H8N4OSCl)2
Cu(C10H9N4OS)2
Cu(C10H8N4OSCl)2
10
10
25
10
10
25
10
>100
>100
>100
>100
>100
>100
–
>100
were found to be highly active. All the metal complexes possess higher antifungal activity than
the Schiff bases. This higher antimicrobial activity of the metal complexes, compared with that
of Schiff bases, is perhaps due to the change in structure due to coordination, and chelating tends
to make metal complexes act as more powerful and potent bactereostatic agents, thus inhibiting
the growth of the microorganisms (39, 40). Moreover, coordination reduces the polarity of the
metal ion mainly because of the partial sharing of its positive charge with the donor groups
within the chelate ring system formed during the coordination. This process, in turn, increases the
lipophilic nature of the central metal atom, which favors its permeation more efficiently through
the lipid layer of the microorganism, thus destroying them more aggressively. The minimum
inhibitory concentration (MIC) of some selected compounds, which showed significant activity
against selected bacterial and fungi species, was determined. The results indicated that these
compounds are the most active in inhibiting the growth of the tested organisms at a concentration
of 10 μg mL−1 (Table 6).
2.7.2. DNA cleavage activity
The representative Schiff base C10H10N4OS and its Co(C10H9N4OS)2, Ni(C10H9N4OS)2 and
Cu(C10H9N4OS)2 complexes are studied for their DNA cleavage activity by the agarose gel
electrophoresis method against DNA of E. coli.
Figure 6. DNA cleavage on genomic DNA of E. coli. M, standard molecular weight marker; Control, control DNA
of E. coli; lane 1, E. coli DNA treated with C10H10N4OS; lane 2, E. coli DNA treated with [Co(C12H9N4OS)2]; lane 3,
E. coli DNA treated with [Ni(C12H9N4OS)2]; lane 4, E. coli DNA treated with [Cu(C12H9N4OS)2].

118 S.A. Patil et al.
DNA-binding studies are important for the rational design and construction of new and more
efficient drugs targeted to DNA (41). The electrophoresis analysis clearly revealed that the Schiff
base I and their metal complexes have acted on DNA as there was a difference in molecular weight
between the control and the treated DNA samples. The difference was observed in the bands of
lanes 1–4 compared with the control DNA of E. coli (Figure 6). This shows that the control DNA
alone does not show any apparent cleavage, whereas the Schiff base and its complexes do show.
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 coordination of sulfur to the metal
in these isolated DNA cleavage reactions. 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.
3. Experimental
3.1. Analysis and physical measurements
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 4000–250 cm−1 region in a KBr disc.
The electronic spectra of the complexes were recorded in HPLC-grade DMF and DMSO solvents
on a VARIAN CARY 50-BIO UV spectrophotometer in the region 200–1100 nm. The 1H NMR
spectra of ligands were recorded in D6-DMSO on a BRUKER 300 MHz spectrometer at RT 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 m) as the FAB gas. The accelerating
voltage was 10 kV, the spectra were recorded at RT and m-nitrobenzyl alcohol was used as the
matrix. The mass spectrometer was operated in the positive ion mode. The electrochemistry of
all the complexes was recorded on a CHI1110A-electrochemical analyzer (made in the USA)
in DMF containing 0.05 M n-Bu4NClO4 as the supporting electrolyte. The ESR spectrum was
recorded on a Varian-E-4X-band EPR spectrometer and the field is set at 3000 G at modulation
frequency of 100 kHz under LNT using TCNE as a ‘g’marker. Molar conductivity measurements
were recorded on an ELICO-CM-82 T Conductivity Bridge with a cell having a cell constant of
0.51 and the magnetic moment was carried out by using Faraday balance.
3.2. Methods
All the chemicals used were of reagent grade.
3.2.1. Synthesis of 3-methylthiosemicarbazone
Freshly distilled methyl amine (3.4 mL) was dissolved in ammonia (20 mL), and carbon disul-
fide (8 mL) was added to it gradually with stirring in an ice bath. Ethanol (30 mL) was added
to it gradually with constant stirring in an ice bath until the carbon disulfide was completely
dissolved. The reaction mixture was then allowed to stand for 2–3 h. An aqueous sodium
chloroacetate (0.1 m) solution was added, followed by hydrazine hydrate (10 mL). The reac-
tion mixture was stirred for 2–3 h and allowed to stand overnight. The crystals separated
were filtered, washed with cold ethanol and recrystallized from hot ethanol. Yield (m.p.): 81%
(130◦C).

Journal of Sulfur Chemistry 119
NH
H
H3C
R
O
N
N
O
O
NH
C
H3C
Reflux
+
NH2
C
S
NH
4
5
H
NH
R = H, Cl
N
R
Ethanol
H
S
Scheme 1. Synthesis of Schiff bases.
3.2.2. Synthesis of Schiff bases I and II
The synthesis of Schiff bases is presented in Scheme 1. The Schiff bases have been synthesized by
refluxing the reaction mixture of hot ethanolic solution (30 mL) of 3-methylthiosemicarbazone
(0.01 mol) and hot ethanolic solution (30 mL) of isatin/chloroisatin (0.01 mol) for 4–5 h with
addition of four to five drops of hydrochloric acid. The precipitate formed during reflux was
filtered, washed with cold EtOH and recrystallized from hot EtOH.
Yield (m.p.): 78% (234 ◦C) and 75% (252 ◦C) Schiff bases I and II, respectively.
3.2.3. Synthesis of Co(II), Ni(II) and Cu(II) complexes (1–6)
An alcoholic solution (45 mL) of Schiff bases (2 mmol) was mixed with an alcoholic solution
(5 mL) of 1 mmol of CoCl2·6H2O/NiCl2·6H2O/CuCl2·2H2O and refluxed on a water bath for
2 h. Then, to the reaction mixture, 2 mmol of sodium acetate was added, and reflux was contin-
ued for 3 h. The separated complex was filtered, washed thoroughly with water, ethanol, ether
and finally dried in a vacuum over fused CaCl2. The yield of the metal complexes lies in the
range 65–70%.
4. Pharmacology
4.1. DNA cleavage experiment
4.1.1. Preparation of culture media
DNA cleavage experiments were carried out according to the literature (42, 43). Nutrient broth
(peptone, 10 g l−1; yeast extract, 5 g l−1; NaCl, 10 g l−1) was used for the culturing of E. coli.
The 50 mL medium was prepared and autoclaved for 15 min at 121 ◦C under 15-lb pressure. The
autoclaved medium was inoculated with the seed culture. The E. coli was incubated for 24 h.
4.1.2. Isolation of DNA
The fresh bacterial culture (1.5 mL) was centrifuged to obtain the pellet, which was then dis-
solved in 0.5 mL of lysis buffer (100 mM Tris pH 8.0, 50 mM EDTA, 10% sodium dodecyl
sulphate (SDS)). To this, 0.5 mL of saturated phenol was added and incubated at 55 ◦C for 10 min.
It was then centrifuged at 10,000 rpm for 10 min, and to the supernatant, an equal volume of chlo-
roform:isoamyl alcohol (24:1) and 1/20th volume of 3 M sodium acetate (pH 4.8) were added.
Then, this solution was centrifuged at 10,000 rpm for 10 min and to the supernatant, 3 vol of
chilled absolute alcohol was added. The precipitated DNA was separated by centrifugation and
the pellet was dried and dissolved in a TAE buffer (10 mM Tris pH 8.0, 1 mM EDTA) and stored
in cold conditions.

120 S.A. Patil et al.
4.1.3. Agarose gel electrophoresis
Cleavage products were analyzed by the agarose gel electrophoresis method (42, 43). Test samples
(1 mg mL−1) were prepared in DMF. The samples (25 μg) were added to the isolated DNA of
E. coli. The samples were incubated for 2 h at 37 ◦C. Then 20 μL of DNA sample (mixed with
bromophenol blue dye at a 1:1 ratio) was loaded carefully into the electrophoresis chamber wells
along with a standard DNA marker containing TAE buffer (4.84 g Tris base, pH 8.0, 0.5 M EDTA
per 1 L) and finally loaded on agarose gel and a constant electricity of 50V was passed for around
30 min. The gel was removed and stained with 10.0 μg mL−1 ethidium bromide for 10–15 min
and the bands observed under Vilberlourmate Gel documentation system and photographed to
determine the extent of DNA cleavage. Then, the results were compared with that of a standard
DNA marker.
4.2. In vitro antibacterial and antifungal assay
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 method, respectively. The antibacterial and antifungal activities were done at 10, 30,
50 and 100 μg mL−1 concentrations in DMF solvent by using four bacteria (E. coli, S. aureus,
P. aeruginosa and S. typhi) and three fungi (Aspergillus niger, A. flavus and Cladosporium)
by the MIC method (44). These bacterial strains were incubated for 24 h at 37 ◦C and fungal
strains were incubated for 48 h at 37 ◦C. Standard antibacterial (Gentamycin) and antifungal
drugs (Flucanozole) were used for comparison under similar conditions.
CH3
HN
C
N
S
M
S
N
H
N
O
R
N
R
O
H
N
N
C
H3C
NH
R = H, Cl
M = Co(II), Ni(II) and Cu(II)
Complex no.
R
H
M
Molecular formula
Co(C10H9N4OS)2
Co(C10H8N4OSCl)2
Ni(C10H9N4OS)2
Ni(C10H8N4OSCl)2
Cu(C10H9N4OS )2
Cu(C10H9N4OSCl)2
1
2
3
4
5
6
Co(II)
Co(II)
Ni(II)
Ni(II)
Cu(II)
Cu(II)
Cl
H
Cl
H
Cl
Figure 7. Structure of metal complexes.

Journal of Sulfur Chemistry 121
5. Conclusion
The newly synthesized Schiff bases act as tridentate ligands and exhibit thiol–thione tautomerism.
The metal ion is coordinated through the azomethine nitrogen, lactonyl oxygen and sulfur atom
via deprotonation. On the basis of analytical, spectral and magnetic studies, the structure given
in Figure 7 has been proposed for the metal complexes.
The Cu(II) and Co(II) complexes exhibited the one electron transfer quasi-reversible redox
couple. The Schiff bases and their metal complexes were found to be highly active against some
of the bacterial and fungal species. The activity is significantly increased on coordination. The
DNA cleavage studies revealed that the complexes show non-specific cleavage of DNA.
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