Synthesis, characterization, antimicrobial, pesticidal and DNA cleavage activity of germanium(IV) derivatives of 3-(2-methyl-2,3-dihydro-benzthiazo-2- yl)-chromen-2-one and N′-[1-2-oxo-2H-chrome-3yl-ethylidene]- hydrazinecarbodithionic acid benzyl ester ligands

Article abstract of DOI:10.1515/mgmc-2012-0902

The new ligands 3-(2-methyl-2,3-dihydro-benzthiazo-2-yl)-chromen-2one (AcBzH) and N′-[1-2-oxo-2H-chrome-3yl-ethylidene]-hydrazinecarbodithionic acid benzyl ester (AcBDTZH) were prepared by the reaction of 3-acetyl-2H-chromen-2-one with 2-aminothiophenol and S-benzyl dithiocarbazate, respectively. The germanium(IV) complexes have been prepared by reacting Ph ₃GeCl and Me₃GeCl in 1:1 molar ratio with these monofunctional bidentate AcBzH and AcBDTZH ligands by using microwave as well as conventional heating methods for comparison purposes. All the synthesized compounds were characterized by elemental analyses, melting point, IR, ¹H-NMR ¹³C-NMR, mass and X-ray powder diffraction techniques. These studies showed that the ligands coordinated to the germanium atom in a monobasic bidentate manner and trigonal bipyramidal environment around the germanium atom have been established for the complexes. To evaluate the effect of metal ion upon chelation, both ligands and their complexes have been screened for their antimicrobial activity against the various pathogenic bacterial and fungal strains. The metal complexes have shown antimicrobial activity as compared to the free ligands. The pesticidal activity and DNA cleavage activity of both ligands and their metal complexes have been tested and discussed. 2012 by Walter de Gruyter Berlin Boston.

Full text of DOI:10.1515/mgmc-2012-0902

Main Group Met. Chem., 2012;34(5-6):139–146 © 2012 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/mgmc-2012-0902
Synthesis, characterization, antimicrobial, pesticidal and DNA
cleavage activity of germanium(IV) derivatives of
3-(2-methyl-2,3-dihydro-benzthiazo-2-yl)-chromen-2-one and
N′-[1-2-oxo-2H-chrome-3yl-ethylidene]-hydrazinecarbodithionic
acid benzyl ester ligands
their biological activity (Mitra et al., 1997). Metal chelates
of dithiocarbazic acid, its S-alkyl/aryl esters and their Schiff
bases have been studied, mainly due to their potential anti-
cancer (Tarafdar et al., 2000), antibacterial (Crouse et al.,
2004), antifungal (Bi and Li, 1999), antiamoebic (Shailendra
et al., 2002) and insecticidal (Tampouris et al., 2007) activi-
ties. Various methods which generally involved condensation
reactions have been used to yield substituted dithiocarbazate
compounds, which behave as bidentate (Zhou et al., 2007),
tridentate (Wang et al., 2002) or multidentate (Ali et al., 2001)
chelating agents with hetero donor atoms. Benzothiazolines
have been the focus of much attention with regard to their
vast potentiality as versatile coordinating agents, variety of
biological activities and numerous applications in different
fields of chemistry. The group N-C-S of benzothiazolines is of
considerable chemotherapeutic interest and is responsible for
pharmacological activity. Benzothiazolines generate strong
interest stemming from their great physiological and biologi-
cal activities (Sharma et al., 2006). Advances in organoger-
manium chemistry particularly, as regards organogermanium
derivatives with extended pentacoordinaion spheres, most of
which are biologically active compounds (Seifullina et al.,
2004), have prompted our systematic studies in this area. The
organic germanium has been clinically used in many parts of
the world to treat a wide spectrum of illnesses and has been
the subject of extensive research in many disciplines: pathol-
ogy, biochemistry, pharmacology, immunology, oncology
and neurochemistry. Organic germanium has been used in a
broad spectrum of regimes – on its own, with diet and stress
counseling – and as a drug in clinical trials of cancer therapy,
in conjunction with chemotherapy, radiation therapy and
surgery (Swami et al., 2008).
Latika Dawara, Nighat Fahmi and R.V. Singh*
Department of Chemistry, University of Rajasthan, Jaipur
302004, India
*Corresponding author
e-mail: rvsjpr@hotmail.com
Abstract
The new ligands 3-(2-methyl-2,3-dihydro-benzthiazo-2-yl)-
chromen-2one (AcBzH) and N′-[1-2-oxo-2H-chrome-3yl-
ethylidene]-hydrazinecarbodithionic acid benzyl ester
(AcBDTZH) were prepared by the reaction of 3-acetyl-2H-
chromen-2-one with 2-aminothiophenol and S-benzyl dithio-
carbazate, respectively. The germanium(IV) complexes have
been prepared by reacting Ph3GeCl and Me3GeCl in 1:1
molar ratio with these monofunctional bidentate AcBzH
and AcBDTZH ligands by using microwave as well as con-
ventional heating methods for comparison purposes. All
the synthesized compounds were characterized by elemen-
1
tal analyses, melting point, IR, H-NMR 13C-NMR, mass
and X-ray powder diffraction techniques. These studies
showed that the ligands coordinated to the germanium atom
in a monobasic bidentate manner and trigonal bipyramidal
environment around the germanium atom have been estab-
lished for the complexes. To evaluate the effect of metal ion
upon chelation, both ligands and their complexes have been
screened for their antimicrobial activity against the various
pathogenic bacterial and fungal strains. The metal complexes
have shown antimicrobial activity as compared to the free
ligands. The pesticidal activity and DNA cleavage activity
of both ligands and their metal complexes have been tested
and discussed.
Our attempt to synthesize benzothioazoline and carbodi-
thioic acid ligands and new substituted germanium(IV) com-
plexes, along with the relationship between the structure and
antimicrobial activities, is presented in this paper.
Keywords: antimicrobial; germanium(IV) complexes;
pesticidal and DNA cleavage activity; S-benzyl
dithiocarbazate.
Results and discussion
The triphenylgermanium chloride and trimethylgermanium
chloridereactwithsodiumsaltofligandsAcBzHandAcBDZH
in unimolar ratio and led to the isolation of Ph3Ge(AcBz),
Me3Ge(AcBz), Ph3Ge(AcBDZ) and Me3Ge(AcBDZ) solids.
These reactions can be represented by the following general
equation:
Introduction
Dithiocarbazate and its substituted compounds remain of
interest to researchers because of their wide variation in
structure and properties. The structural nature of the metal ion
complexes of S-alkyl dithiocarbazates has been correlated to
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140 L. Dawara et al.
SH
C
H3C
SNa
C
H3C
H2C
+
CH3ONa
C
N
H2C
C
N
N
S
S
N
O
O
O
O
O
O
H3C
SNa
H2C
C
H3C
Methanol
NaCl
+
C
C
N
N
S
N
Ge
R
N
R3GeCl
+
C
S
C
S
R
H2
O
O
R
where R=Ph or Me.
the NH2 group, respectively. These bands remain unchanged
in the germanium(IV) complexes. Furthermore, strong bands
at 3250 cm-1 due to ν(NH) vibrations are observed. These
ν(N-H) absorption bands are absent in the complexes. A sharp
and strong band at 1622 cm-1 is due to the azomethine group
of the ligand. In the IR spectra of the complexes this showed
a lower shift of the order 15–25 cm-1. One strong band located
at 1050 cm-1 in the ligand was attributed to ν(C=S) moiety,
which disappears in the case of complexes. These data on
comparison with the spectrum of the ligand suggested that
the azomethine nitrogen and thiolic sulfur atom of the ligand
are involved in coordination with the metal ion. A doublet at
∼2960 and ∼2900 cm-1 is assigned to symmetric and asym-
metric vibrations of S-CH2-C6H5 grouping. The far IR spectra
of these metal complexes exhibited new bands, which are not
present in the spectrum of the ligand. These bands are located
at 680–660 cm-1 and 410–415 cm-1 and are due to the Ge←N
and Ge-S stretching vibrations, respectively, and this further
lends support to the proposed coordination.
These are soluble in DMF and DMSO. The reactions were
carried out in perfectly dry methanolic medium and proceeded
smoothly with the precipitation of NaCl. All the resulting
complexes have been obtained as monomers and non-elec-
trolytes as revealed by their molecular weight determinations
and conductance measurements, respectively.
Electronic spectra
The electronic spectrum of 3-(2-methyl-2,3-dihydro-benz-
thiazo-2-yl)-chromen-2one consists of two bands at 266 nm
and 320 nm attributable to φ-φ* and π-π* transitions which
remain unchanged in the complexes. An additional band is
also observed around 386 nm due to n-π* electronic transi-
tions of the azomethine, which indicates isomerization of the
ligand on complexation. The bands in the electronic spec-
trum of the ligand N′-[1-2-oxo-2H-chrome-3yl-ethylidene]-
hydrazinecarbodithionic acid benzyl ester and its complexes
appear at 266 and 325 nm, assigned to π-π* electronic transi-
tions within the benzene ring. Another band observed at 365
nm in the spectrum of the ligand is due to the n-π* transitions
of the azomethine (>C=N) group. However, in the spectra
of the complexes, this band appears in the lower wavelength
region. This is due to the polarization within the >C=N chro-
mophore resulting after chelation.
1H NMR spectra
The 1H NMR spectra further support the bonding pattern as
1
discussed above. The H NMR spectral data of the ligands
and their corresponding organogermanium(IV) complexes
were recorded in DMSO-d6 with TMS as an internal stan-
dard. The -NH proton signal at δ 4.60 ppm in the spectrum
of benzothiazoline disappears in the corresponding germa-
nium complexes indicating deprotonation of this functional
group. The free ligand shows a complex multiplet at δ 6.20–
8.00 ppm for the aromatic protons and this remains more or
less at the same position in the spectrum of the complexes.
IR spectra
The IR spectral data of the synthesized ligands and their com-
pounds are shown in Table 1. In the IR spectrum of the ligand
(AcBzH), absence of the ν(SH) mode at 2610–2540 cm-1 and
the presence of ν(NH) mode at 3310 cm-1 indicates the pres-
ence of the benzothiazoline ring structure in the ligand. In
the spectra of metal complexes the disappearance of ν(NH)
band shows its deprotonation and chelation of nitrogen of the
ligand to the central metal atom. The IR spectrum of the ligand
(AcBDZH) displays two sharp bands around 3350–3300 cm-1
and 3450–3350 cm-1 assignable to νsym and νasym vibrations of
1
The H NMR spectrum of the ligand (AcBDZH) exhibits
-CH2-proton signals at δ 4.15–4.16 ppm and aromatic pro-
ton signals at δ 6.38–7.50 ppm, and these remain at the
same position in the spectra of the metal complexes. The
proton of the -NH group of the ligand (AcBDZH) gives a
signal at δ 10.05–10.10 ppm, which is absent in the spectra
of metal complexes indicating the chelation of the ligand
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Germanium(IV) complexes of AcBzH and AcBDTZH 141
Table 1 IR (cm-1) and 1H NMR (δ, ppm) spectral data of the ligands and their corresponding complexes.
Compound
IR spectral data (cm-1)
1H NMR spectral data (δ, ppm)
(>C=N)
(Ge←N)
ν (-NH)
ν (C=S)
ν (Ge-S)
-NH
Aromatic
-S-CH2
-CH3
(AcBzH)
–
–
3310
–
–
3250
–
–
–
–
–
1050
9 4 5
9 5 6
–
4.60
–
–
8.30
–
–
8.00–6.20
8.15–6.80
8.10–6.80
7.50–6.38
8 .1 0 – 6 .8 5
7 .3 0 – 6 .4 0
–
–
–
4.16
4 .1 5
4 .1 6
1.08
1.10
1.10
1.09
1 .1 1
1 .1 2
Ph3Ge(AcBz)
Me3Ge(AcBz)
(AcBDTZH)
Ph3G e ( A c B D T Z )
Me3G e ( A c B D T Z )
1600
1605
1622
1 6 0 9
1 6 0 5
680
665
–
6 7 5
6 6 0
412
410
–
4 1 5
4 1 3
Bioassay
moiety to germanium with the sulfur atom. The signals at
δ 10.52–10.65 ppm observed in the ligands, AcBzH and
AcBDZH, were assigned to Ar-NH protons. The proton sig-
nals of the methyl groups appear at δ 1.08–1.12 ppm in the
Antimicrobial assay The antimicrobial screening data
show that both the ligands and their complexes individually
exhibited varying degrees of inhibitory effects on the growth
of the tested bacterial and fungal species. The antibacterial
(Figure 2) and antifungal (Figure 3) results evidently show
that the activity of the ligands became more pronounced when
coordinated to the metal. The increased activity of the metal
chelates can be explained on the basis of the chelation theory
(Geeta et al., 2010), according to which the polarities of the
ligand and the central metal atom are reduced through charge
equilibration over the whole chelate ring. This increases
the lipophilic character of the metal chelate and favors its
permeation through the lipoid layer of the bacterial membranes.
The variation in the effectiveness of different compounds
against different organisms depends either on the permeability
of the cells of the microbes or the difference in ribosomes of
microbial cells. It has also been proposed that concentration
plays a vital role in increasing the degree of inhibition: as the
concentration increases, the activity increases.
1
organogermanium(IV) complexes. The H NMR values for
all the compounds are given in Table 1.
13C NMR spectra
13C NMR spectral data have been recorded for the two
ligands and their complexes and these spectra also support
the authenticity of the proposed structures. The considerable
shifts in the positions of carbons of the germanium com-
plexes attached to N and S, respectively, clearly indicate that
the nitrogen and sulfur of the ligands group participate in the
complexation reaction. The signals due to the carbon atoms
attached to the thionic and azomethine groups in ligands
appear at 180.10–178.24 ppm and 162.90–160.75 ppm,
respectively. However, in the spectra of the corresponding
germanium(IV) complexes, these appear at ∼163 ppm (thi-
onic group) and at ∼164 ppm (azomethine group), respec-
tively. The considerable shifts in carbons attached to S and
N indicate the involvement of sulfur and nitrogen atoms in
coordination.
Pesticidal activity Both the ligands and their germanium
complexes were also evaluated for pesticidal activity and they
Mass spectra
Table 2 X-Ray diffraction data of the complex Ph3Ge(AcBDZ).
The EI mass spectrum of the [Ph3Ge(AcBDZ)] complex was
studied as a representative case. The molecular ion peak for
the complex [Ph3Ge(AcBDZ)] was observed at m/z 671.01
and this is in good agreement with its molecular weight,
which suggests the monomeric nature of the complex.
h
k
l
2-θ
2-θ
2-θ
d
d
Intensity
(exp.)
(exp.) (calc.) (diff.)
(exp.)
(calc.)
-1
-1
1
-1
1
-4
0
0
-3
-3
2
-5
-3
-5
-3
0
3
3
2
2
0
3
2
4
2
2
2
2
3
0
6
5
3
2
1
3
2
2
2
2
0
0
2
1
3
1
1
2
53.638 53.594 0.044 1.70733 1.70862 3.02
41.781 41.764 0.018 2.16021 2.16108 5.84
33.027 33.058 -0.031 2.71002 2.70752 5.15
48.023 48.002 0.021 1.89300 1.89377 7.39
30.728 30.736 -0.008 2.90729 2.90660 27.26
50.554 50.507 0.047 1.80400 1.80557 4.80
36.618 36.617 0.001 2.45206 2.45213 8.37
54.822 54.809 0.014 1.67320 1.67359 2.60
44.698 44.673 0.024 2.02580 2.02685 6.37
44.707 44673 0.034 2.02538 2.02685 7.81
49.188 49.185 0.002 1.85087 1.85095 17.25
X-ray structure determination
The possible lattice dynamics of the finely powdered product,
[Ph3Ge(AcBDZ)], has been deduced on the basis of X-ray
powder diffraction studies. The observed interplanar spac-
ing values (‘d’ in Å) have been measured from the diffracto-
gram of the compound and the Miller indices h, k and l have
been assigned to each d-value and 2-θ angles are reported.
The results show that the compound belongs to ‘mono-
clinic’ crystal system with unit cell parameters as a=9.2356,
b=9.1593, c=6.2398, maximum deviation of 2-θ=0.044 and
α=90, β=90, γ=120 at the wavelength of 1.540598 (Table 2
and Figure 1).
51.867 51.876 -0.009 1.76137 1.76109
4.75
56.084 56.129 -0.045 1.63852 1.63732 6.92
59.649 59.618 0.031 1.54882 1.54954 1.61
62.617 62.596 0.022 1.48234 1.48280 10.63
66.025 66.011 0.014 1.41386 1.41412
7.65
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142 L. Dawara et al.
65
52
39
26
13
0
r
20
25
30
35
40
45
50
55
60
65
70
2θ
Figure 1 X-Ray diffraction screening of the complex [Ph3Ge(AcBDZ)].
have a potent inhibitory effect on growth and development of
Corcyra cephalonica larva. The LC50 values in mg/l are shown
in Table 3. The data indicate that all germanium(IV) complexes
exhibit greater pesticidal activity than the respective ligands but
compound Ph3Ge(AcBDZ) was highly effective as a pesticide
with a LC50 value of 210 mg/l against C. cephalonica. A
possible explanation is that the compound inhibits the molting
hormone of pest larva, i.e., ecdysis disruption.
cleavageactivitybytheagarosegelelectrophoresismethodand
is presented in Figure 4. The gel after electrophoresis clearly
revealed that the ligand AcBDZH and its complexes acted
on DNA as there was a molecular weight difference between
the control and the treated DNA samples. The difference was
observed in the bands (Figure 4, lanes A–C) compared to the
control DNA of Escherichia coli. This shows that the control
DNA alone does not show any apparent cleavage where its
complexes are shown. The results indicated the important
role of 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.
Electrophoresis analysis result The ligand AcBDZH
and its germanium(IV) complexes were studied for their DNA
25
20
15
10
5
Experimental
The reagents Ph3GeCl, Me3GeCl and o-aminothiophenol as well
as 3-acetylcoumarin were purchased from Alfa Aesar (MA, USA)
and used as such. Solvents of analytical grade were distilled from
appropriate drying agents immediately prior to use. Molecular
weights were determined by the Rast Camphor method (Vogel,
2004). Germanium was determined gravimetrically as GeO2.
Nitrogen was estimated by the Kjeldahl’s method and sulfur was
estimated by the Messenger’s method (Makode and Aswar, 2004).
Infrared spectra of the ligands and their complexes were record-
ed with the aid of a Nicolet Megna FTIR-550 spectrophotometer
0
(Shimadzu, USA) on KBr pellets. H and 13C NMR spectra were
1
recorded on a JEOL-AL-300 FT NMR spectrometer (Japan) in
DMSO-d6 using TMS as the internal standard. A mass spectrum of
the complex was carried out at IIT Chennai. X-Ray powder diffrac-
tion was recorded with the aid of a PANalytical system with CuKα
as a radiation source of wavelength 1.542 Å with 2-θ 10–70°.
Escherichia coli 500 ppm
Escherichia coli 1000 ppm
Staphylococcus aureus
500 ppm
Staphylococcus aureus
1000 ppm
Preparation of the ligands
Two different methods, microwave-assisted synthesis and a conven-
tional method, were employed for the synthesis of the ligands.
Figure 2 Antibacterial screening of the ligands and their complexes.
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Germanium(IV) complexes of AcBzH and AcBDTZH 143
120
100
80
60
40
20
0
Aspergillius niger 100 ppm
Aspergillius niger 200 ppm
Fusarium oxysporum 100 ppm
Fusarium oxysporum 100 ppm
Figure 3 Antifungal screening of the ligands and their complexes.
Preparation of 3-(2-methyl-2,3-dihydro-benzothiazo-2-yl)-
chromen-2-one (AcBzH) 3-(2-Methyl-2,3-dihydro-benzothiazo-
2-yl)-chromen-2-one (AcBzH) was prepared by the condensation
of 3-acetylcoumarin (0.01 mol) with 2-mercaptoaniline in 1:1 molar
ratio in ethanol. The reaction mixture was stirred for 3–4 h and the
resulting product was filtered off, recrystallized from ethanol and
dried in vacuum. The analytical results yielded good consistence
with the proposed formula as follows (Scheme 1):
between the thermal method and the microwave method is given in
Table 4. Synthesis of AcBDZH is shown in Scheme 2.
Preparation of the metal complexes
For the synthesis of the complexes, Ph3GeCl and Me3GeCl and
sodium salt of the ligands (prepared by adding the corresponding
weight of sodium metal to the ligands) in 5 ml of dry methanol in
1:1 molar ratio were irradiated inside a microwave oven for approxi-
mately 5–8 min. The products were recovered from the microwave
oven and dissolved in few milliliters of dry methanol. The white
precipitate of sodium chloride formed during the course of the re-
action was removed by filtration, and the filtrate was dried under
reduced pressure. The resulting product was repeatedly washed with
petroleum ether and then finally dried at 40–60° C/0.5 mm Hg for
3–4 h. The purity was further checked by thin layer chromatography
using silica gel-G.
Preparation of N′-[1-2-oxo-2H-chrome-3yl-ethylidene]-
hydrazinecarbodithionic acid benzyl ester (AcBDZH) N ′-
[1-2-oxo-2H-chrome-3yl-ethylidene]-hydrazinecarbodithionic
acid benzyl ester (AcBDZH) was prepared by the condensation of
3-acetylcoumarin (0.01 mol) with, S-benzyldithiocarbazate (0.01
mol) in 1:1 molar ratio. The reaction mixture was irradiated in the
microwave oven by taking 2–3 ml solvent. The reactions were
completed in a short period (5–7 min). The resulting precipitate was
then recrystallized with alcohol and dried under vacuum. These were
characterized and analyzed before use. Elemental analyses (N and S)
were conducted using the methods mentioned above and their results
were found to be in good agreement with the calculated values.
The above ligand was also synthesized by a thermal method, where
instead of a few drops of alcohol the starting materials of the ligand
was dissolved in ∼100 ml of alcohol and the contents were refluxed
for nearly 4–5 h. The solution was then concentrated under reduced
pressure, which on cooling gave reddish brown crystalline precipi-
tates. These were recrystallized twice in alcohol. A comparison
Thermal method
These organogermanium(IV) complexes were also synthesized by
the thermal method. The reaction mixtures were heated under reflux
for 13–17 h and filtered to remove NaCl and the solvent was removed
C E. coli
M
A
B
C
Table 3 Pesticidal data of ligands and their metal complexes.
2
Compound
Correct
motility (%)
χ -test
LC50
(mg/l)
(AcBzH)
50
0.156
0.285
0.452
0.236
0.540
0.456
1.142
520
230
285
460
210
296
–
Ph3Ge(AcBz)
Me3Ge(AcBz)
(AcBDTZH)
Ph3Ge(AcBDTZ)
Me3Ge(AcBDTZ)
Control
66.66
61.11
55.55
88.88
72.22
–
Figure 4 DNA cleavage gel diagram of synthesized compounds.
M, standard molecular weight marker; (lane C) E. coli, control DNA
of E. coli; (lanes A–C), E. coli DNA treated with the ligand AcBDZH
and its Ge(IV) complexes, respectively.
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144 L. Dawara et al.
CH3
C
CH3
C
S
O
SH
+
N
-H2O
O
O
O
O
H
NH2
Scheme 1 Synthesis of 3-(2-methyl-2,3-dihydro-benzothiazo-2-yl)-chromen-2-one (AcBzH).
Table 4 Comparison between microwave and thermal methods.
Compound
Yield (%)
Microwave
Solvent (ml)
Time
Microwave (min)
Thermal
Thermal
Microwave
Thermal (h)
(AcBzH)
75
72
74
77
69
70
83
80
82
86
82
85
100
45
40
100
40
50
5
3
3
3
3
2
4
–
7
6
6
7
6
Ph3Ge(AcBz)
Me3Ge(AcBz)
(AcBDTZH)
Ph3Ge(AcBDTZ)
Me3Ge(AcBDTZ)
16
17
4
15
13
by the same procedure mentioned above, which was adopted to ob-
tain the complexes. The physicochemical properties and analytical
data of these complexes are listed in Table 5.
C-T
% of inhibition=
×100
C
where C and T are the diameters of the fungal colony in the control
and the test plates, respectively (Chaudhary and Singh, 2004).
Microbiological studies
In vitro antibacterial activity Antibacterial activity was tested
against E. coli and Staphylococcus aureus using the paper disc plate
method (Gaur et al., 2007). Each of the compounds was dissolved in
DMSO and solutions of the concentrations (500 and 1000 ppm) were
prepared separately. Paper discs of Whatman filter paper (No. 42) of
uniform diameter (2 cm) were cut and sterilized in an autoclave. The
paper discs soaked in the desired concentration of the complex solu-
tions were placed aseptically in the Petri dishes containing nutrient
agar media (agar 20 g+beef extract 3 g+peptone 5 g) seeded with E.
coli and S. aureus bacteria separately. The Petri dishes were incu-
bated at 37°C and the inhibition zones were recorded after 24 h of
incubation. The antibacterial activity of common standard antibiotic
Streptomycin was also recorded using the same procedure as above
at the same concentrations and solvent.
In vitro antifungal activity The antifungal activity of the stan-
dard fungicide (Flucanazone), ligands and complexes were tested for
their effect on the growth of microbial cultures and studied for their
interaction with Aspergillus niger and Fusarium oxysporum using
Czapek’s agar medium with the composition: glucose 20 g, starch
20 g, agar-agar 20 g and distilled water 1000 ml. To this medium
was added requisite amount of the compounds after being dissolved
in methanol to obtain certain concentrations (50, 100 and 200 ppm).
The medium was then poured into Petri plates and the spores of fungi
were placed on the medium with the aid of an inoculation needle.
These Petri plates were wrapped in polythene bags containing a few
drops of alcohol and were placed in an incubator at 30 2°C. The
controls were also run and three replicates were used in each case.
The linear growth of the fungus was recorded by measuring the di-
ameter of the fungal colony after 96 h and the percentage inhibition
was calculated by the equation:
Pesticidal activity Larvae of Corcyra cephalonica were obtained
from stock culture maintained at the storage section of the Division
S
CH3
S
C
CH3
C
Ethanol
H2C
C
N
C
O
H2C
H2N
+
N
H
S
-H2O
O
O
N
H
O
O
S
SH
CH3
C
H2C
N
C
N
S
O
O
Scheme 2 Synthesis of N′-[1-2-oxo-2H-chrome-3yl-ethylidene]-hydrazinecarbodithionic acid benzyl ester (AcBDZH).
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Germanium(IV) complexes of AcBzH and AcBDTZH 145
Table 5 Analytical data and physical properties of ligands and their metal complexes.
Compound
Color
Melting point (°C)
Found (calcd.) (%)
S
Mol. wt.
found (calcd.)
N
M
(AcBzH)
Yellow
77
4.74
2.34
3.40
7.60
4.17
5.77
10.86
5.36
7.78
17.40
9.55
13.22
–
293.12 (295.36)
596.56 (598.3)
410.86 (412.09)
366.85 (368.47)
670.25 (671.42)
481.96 (485.21)
Ph3Ge(AcBz)
Me3Ge(AcBz)
(AcBDTZH)
Ph3Ge(AcBDTZ)
Me3Ge(AcBDTZ)
Dark brown
Light brown
Red
Maroon
Orange
105
130
155
125
148
12.14
17.63
–
10.82
14.97
of Entomology, Agricultural Research Institute (Durgapura, Jaipur,
India). Insects were reared on grains of wheat at 27 1°C and 70%
relative humidity. Glass jars containing 500 g of wheat grains were
labeled to indicate the date of introduction of larvae and new emer-
gence. At alternate days, larvae were shifted to fresh jars so that
successive rearing jars could be maintained and insects of known
age could be obtained regularly. Insecticidal activity of the synthe-
sized compounds was tested by dipping and spray methods. The
synthetic compounds were weighed and dissolved in methanol to
prepare 1000 mg/l stock solution. Further concentrations, viz., 900,
800, 700, 600, 500, 400, 300, 200 and 100 mg/l were prepared by
serial dilution. Then, 1 ml of each concentration of various com-
pounds was directly poured in each Petri plate (90 mm) with the
aid of a micropipette. Petri plates with test solution were rotated
vigorously to prepare uniformly and were allowed to dry for 3–5
min. Each concentrations as well as control in methanol were repli-
cated thrice. In total, 20 adults (2–5 days old) were released in each
Petri plate and were kept at 27 1°C and 70% relative humidity.
Mortality was observed after 96 h. Adults were considered dead if
they failed to respond to stimulus by touch. Control mortality was
corrected by using Abbott’s formula (Shaki et al., 2009) and LC50
Agarose gel electrophoresis (Kapoor et al., 2011) Cleavage
products were analyzed by the agarose gel electrophoresis meth-
od. Test samples (1 mg/ml) 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 and then 20 μl of DNA sample (mixed
with bromophenol blue dye at 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/l) and
finally loaded on agarose gel and passed the constant 50 V of elec-
tricity for around 30 min. The gel was removed and stained with 10.0
g/ml ethidium bromide for 10–15 min, and the bands were observed
under a UV transilluminator and photographed to determine the
extent of DNA cleavage, and the results were compared with stan-
dard DNA marker.
Conclusions
Comparison between conventional and microwave synthe-
sis revealed that microwave irradiation is an efficient and
environmentally-benign method to accomplish various inor-
ganic syntheses to afford products in higher yields in shorter
reaction periods. On the basis of analytical data it has been
observed that the ligands coordinated to the metal atoms in
a monobasic bidentate manner and thus possess octahedral
geometry. Antimicrobial and pesticidal activity of the com-
plexes and the ligands showed that the former are more active
than the parent ligands. Furthermore, DNA cleavage studies
revealed that metal complexes cleave DNA more efficiently
in comparison to the ligands. The germanium complexes pos-
sess better biological activities than the ligand because of the
chelating effects between the ligand and metal ions. Results
obtained from this work would be useful in the development
of potential applications in the biological, pharmaceutical,
and physiological fields in the future.
2
was obtained by a graphical method. The χ -test was calculated by
statistical analysis.
% mortality observed-% mortality in control
Corrected % mortality=
100-% mortality in control
×100
DNA cleavage activity
Preparation of culture media Nutrient broth (peptone, 10; yeast
extract, 5; NaCl, 10 in g/l) was used for culturing of E. coli. The
50-ml medium was prepared and autoclaved for 15 min at 121°C
under 15 lb pressures. The autoclaved media was inoculated with the
seed culture and E. coli was incubated for 24 h.
Isolation of DNA The fresh bacterial culture (1.5 ml) was cen-
trifuged to obtain the pellet, which was then dissolved in 0.5 ml of
lysis buffer (100 mm Tris pH 8.0, 50 mm EDTA, 10% SDS). To this
0.5 ml of saturated phenol was added and incubated at 55°C for 10
min. Then, it was centrifuged at 10 000 rpm for 10 min and equal
volume of chloroform:isoamyl alcohol (24:1) and 1/20 volume of 3
m sodium acetate (pH 4.8) was added to this supernatant and centri-
fuged at 10 000 rpm for 10 min. To this supernatant three volumes
of chilled absolute alcohol was added. The precipitated DNA was
separated by centrifugation. The pellet was dried and dissolved in
TE buffer (10 mm Tris pH 8.0, 1 mm EDTA) and stored in cold
conditions.
Acknowledgments
The authors are thankful to U.G.C. New Delhi for financial assis-
tance through grant no. 34-324/08/(SR-1).
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