Pesticidal and antifertility activities of triorganogermanium(IV) complexes synthesized using a green chemical approach

Article abstract of DOI:10.1002/aoc.2960

Microwave chemistry is a green chemical method that improves reaction conditions and product yields while reducing solvent amounts and reaction times. This paper deals with the synthesis, spectral and biological studies of germanium(IV) complexes with chelating hydrazones derived from 1-(pyridine-2-yl)ethanone (F₁) and 1-(furan-2-yl)ethanone (F ₂) with isonicotinohydrazide (INH). The complexes have been synthesized under a microwave-green chemical approach and investigated using a combination of microanalytical analysis, melting point, IR spectra, ¹H NMR spectra and ¹³C NMR spectra. Trimethylgermanium(IV) chloride and triphenylgermanium(IV)chloride interact with the hydrazones in a 1:1 molar ratio (metal:ligand), resulting in the formation of coloured products. On the basis of conductance and spectral evidence, a pentacoordinated structure for germanium(IV) complexes has been assigned for these products. The ligand is coordinated to the germanium(IV) via the azomethine nitrogen atom and the enolic oxygen atom. The free ligands and their metal complexes have been tested in male rats in order to assess their antifertility properties. Ligands and their metal complexes have also been tested in vitro against a number of pathogenic microorganisms in order to assess their antimicrobial and pesticidal properties. Both the ligands and their complexes were found to possess appreciable antifertility activity and other activities, which have been discussed in brief. Copyright 2013 John Wiley & Sons, Ltd. This paper deals with the synthesis, spectral, and biological studies of germanium(IV) complexes with chelating hydrazones derived from 1-(pyridine-2-yl)ethanone (F₁) and 1-(furan-2-yl)ethanone (F₂) with isonicotinohydrazide (INH) and have been synthesized under microwave - green chemical approach and investigated using a combination of microanalytical analysis, melting point, IR spectra, ¹H NMR spectra and ¹³C NMR spectra. Ligands and their metal complexes have also been tested in vitro against a number of pathogenic microorganisms in order to assess their antimicrobial and pesticidal properties. Copyright

Full text of DOI:10.1002/aoc.2960

Full Paper
Received: 2 October 2012
Revised: 7 December 2012
Accepted: 7 December 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/aoc.2960
Pesticidal and antifertility activities of
triorganogermanium(IV) complexes
synthesized using a green chemical approach
a
a
b
a
Nirmala Singh , Sheenam Watts , Suresh C. Joshi and Ran V. Singh *
Microwave chemistry is a green chemical method that improves reaction conditions and product yields while reducing solvent
amounts and reaction times. This paper deals with the synthesis, spectral and biological studies of germanium(IV) complexes
with chelating hydrazones derived from 1-(pyridine-2-yl)ethanone (F
1 2
) and 1-(furan-2-yl)ethanone (F ) with isonicotinohydra-
zide (INH). The complexes have been synthesized under a microwave–green chemical approach and investigated using a
1
13
combination of microanalytical analysis, melting point, IR spectra, H NMR spectra and C NMR spectra. Trimethylgermanium
IV)chloride and triphenylgermanium(IV)chloride interact with the hydrazones in a 1:1 molar ratio (metal:ligand), resulting in
(
the formation of coloured products. On the basis of conductance and spectral evidence, a pentacoordinated structure for
germanium(IV) complexes has been assigned for these products. The ligand is coordinated to the germanium(IV) via the
azomethine nitrogen atom and the enolic oxygen atom. The free ligands and their metal complexes have been tested in male rats
in order to assess their antifertility properties. Ligands and their metal complexes have also been tested in vitro against a number
of pathogenic microorganisms in order to assess their antimicrobial and pesticidal properties. Both the ligands and their
complexes were found to possess appreciable antifertility activity and other activities, which have been discussed in brief.
Copyright © 2013 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: 1-(pyridine-2-yl) ethanone; 1-(furan-2-yl)ethanone; isonicotinohydrazide; antimicrobial and pesticidal properties; antifertility
activity
bioavailability and acquired isoniazid resistance. Chemical modi-
fication of the hydrazine unit of isoniazid with a functional group
Introduction
Microwave dielectric effects are used increasingly in organometallic
that blocks acetylation while maintaining strong antimycobacter-
ial action has the potential to improve clinical outcome and to re-
duce the emergence in patients of acquired isoniazid resistance.
Bioinorganic compounds and biomimetics containing a C¼N
[1]
synthesis. When we compare this unconventional energy source
technology with classical heating we find that the conventional heat-
ing process usually starts at the wall of the reactor and, as a result, the
core takes a much longer time to achieve the target temperature.
Rapid and homogeneous heating by an unconventional method
[
13]
bond are widely represented.
Schiff bases have been inten-
sively investigated for their strong coordination capability and
[
14–16]
(
microwave method) has many benefits, such as reaction rate acceler-
diverse biological activities.
Isoniazid Schiff bases have
[2]
[3]
ation, milder reaction conditions and higher chemical yield; also, it
is a cost-effective technique and less equipment is required. The
shorter reaction time and expanded reaction range offered by micro-
wave-assisted synthesis are suited to increased demand in industry.
Hydrazones are a versatile class of ligands that have extensive
applications in various fields, possessing pronounced biological
been found to show better anticancer, antitubercular and antimi-
[
17,18]
crobial activity than isoniazid.
The present article was directed to the preparation of germa-
0
nium(IV) complexes with ligands N -(1-(pyridine-2-yl)ethylidene)
isonicotinohydrazone (F INH) and N-(1-(furan-2-yl)ethylidine)iso-
1
nicotinohydrazone (F INH) through a green chemical approach
2
[
4,5]
and pharmaceutical activities such as antitumour,
antimicro-
and their biological aspects have been studied.
[6]
[7]
[8]
bial, antituberculosis and antimalarial. Hydrazones play an
important role in improving the antitumour selectivity and toxic-
ity profile of antitumour agents by forming drug carrier systems
Experimental
[
9]
Chemicals
employing suitable carrier proteins. They also have anti-inflam-
[
10]
matory and analgesic activity equal or close to that of aspirin.
The remarkable biological activity of acid hydrazides R–CO–NH–
NH as well as their corresponding aroylhydrazones R–CO–NH–
Germanium(IV) compounds, Me
3 3
GeCl and Ph GeCl, as well as
2-acetylpyridine and 2-acetylfuran, were purchased from Alfa Aesar
2
N¼CH–R and the dependence of their mode of chelation with
the metal ions present in the living system have been of great im-
* Correspondence to: R. V. Singh, Department of Chemistry, University of
Rajasthan, Jaipur 302 004, India. E-mail: rvsjpr@hotmail.com
[11,12]
portance.
Enzymatic acetylation of the antitubercular isonia-
zid by N-acetyltransferase represents a major metabolic pathway
for isoniazid in humans. Acetylation greatly reduces the thera-
peutic activity of the drug, resulting in underdosing, decreased
a Department of Chemistry, University of Rajasthan, Jaipur 302 004, India
b Department of Zoology, University of Rajasthan, Jaipur 302 004, India
Appl. Organometal. Chem. 2013, 27, 269–276
Copyright © 2013 John Wiley & Sons, Ltd.

N. Singh et al.
3
2
O
and isoniazid was purchased from SD Fine Chemicals Ltd (Mumbai,
India). Solvents of AR grade were distilled from appropriate drying
agents prior to use.
N
7
4
15
N
N
NH
5
6
14
Physical Measurements and Analytical Methods
11 10
1
3
12
Molecular weights of the synthesized ligands and their metal
[
19]
complexes were determined by the Rast camphor method.
[
20]
2
Figure 2. Structure of ligand (F INH).
Nitrogen was estimated by the Kjeldahl method.
Germanium
(
IV) was estimated gravimetrically as GeO . IR spectra of the ligands
2
and their complexes were recorded with the help of a Nicolet
Magna FTIR-550 spectrophotometer using KBr pellets as well as
The white precipitate of NaCl formed during the course of the re-
action was recovered by filtration and the filtrate was dried under
reduced pressure. The resulting products were repeatedly
1
13
in Nujol mulls. H and C NMR spectra were recorded on a
JEOL-AL-300 FT NMR spectrometer in DMSO-d using tetramethyl-
6
washed with n-hexane and petroleum ether and finally dried at
silane (TMS) as the internal standard. Electronic spectra of the
complexes were recorded in DMF on a UV-160A Shimadzu spectro-
photometer in the range 200–600 nm.
ꢀ
6
0 C/0.5 mmHg for 3–4 h (Figs 3 and 4).
Thermal method
In the thermal method, instead of 5–7 min reactions were
completed in 7–8 h and the reaction mixture was refluxed over
a distillation assembly fitted with quick-fit interchangeable joints
and the white precipitation of sodium chloride obtained was
removed by filtration. The mother liquor was concentrated by
removing excess solvent. Compounds were dried under reduced
pressure for 3–4 h. These were purified by the same process as
described in the above method. Purity was further checked by
TLC on silica gel-G using anhydrous methanol and tetrahydrofu-
ran as a solvent. Physical properties and analytical data are given
in Table 1.
1 2
Preparation of the Hydrazones F INH and F INH
Two different routes were employed for synthesis of the ligands
for comparison purposes: eco-friendly microwave-assisted synthe-
sis and the conventional thermal method. The progress of the
reaction was monitored by thin-layer chromatography on silica gel-G.
1 2
Microwave-assisted synthesis of ligands F INH and F INH
F INH and F INH were prepared by the condensation of F and F
2
1
2
1
with isonicotinohydrazide (INH) in the presence of a few drops of
ethanol (~3 ml) and irradiated by microwave irradiation for ~4–5
min. The resulting coloured precipitates were recrystallized with
alcohol and dried under vacuum.
Antifertility Activity
Conventional thermal method for synthesis of ligands F
1
INH and F
2
INH.
A
Male albino rats (Rattus norvegicus) weighing 170–200 g were
similar procedure was followed for synthesis by the thermal
method where instead of a few drops of alcohol ~100 ml was
used and the contents were refluxed for nearly 5–6 h. The solu-
tion was then concentrated under reduced pressure, which on
cooling gave a coloured crystalline precipitate. These were
recrystallized twice in alcohol (Figs 1 and 2). Their physical prop-
erties and analytical data are given in Table 1.
used in the study. They were housed in separated polypropylene
ꢀ
cases under controlled conditions of temperature (22 ꢁ 3 C).
humidity (50 ꢁ 5%) and light (12 light:12 dark cycle), and fed with
pellet diet (Ashirwad Industries Ltd, Punjab, India) and water ad
libitum. The animals were divided into six groups containing six
animals of each. Group A served as vehicle-treated control. In
groups B and C ligands F INH and F INH and in groups D, E, F
1
2
and G compounds [Me Ge(F IN)], [Me Ge(F IN)], [Ph Ge(F IN)]
3
1
3
2
3
1
and [Ph Ge(F IN)] suspended in olive oil were given orally for a
3
2
Synthesis of the Complexes
ꢂ1
period of 60 days (30 mg kg body weight per day). All the ani-
mals were screened for fertility and autopsied for biochemical
examination. The sperm density in cauda epididymis and density
of testicular and cauda epididymis, protein, sialic acid, glycogen,
cholesterol, acid and alkaline phosphatase and serum testoster-
one were measured by standard laboratory techniques. The data
were analysed statistically using Student’s t-test and significance
of differences was set at P < 0.01 and P < 0.001.
Microwave method
For the preparation of [R
Ph), methanol solutions of Me
with the corresponding sodium salts of the ligands (F
INH) in equimolar ratio using 2–3 ml methanol as a solvent in
3
Ge(FIN)] complexes (where, R = Me or
GeCl and Ph GeCl were mixed
INH and
3
3
1
F
2
the microwave oven for 5–7 min. The products recovered from
the microwave oven dissolved in a few millilitres of dry methanol.
3
5
2
Pesticidal Activity
O
4
Pesticides can save farmers money by preventing crop losses to insects
and other pests. With this in view the experiment was conducted to
determine the pesticidal activities of the present compounds.
The pesticidal activity of the two ligands F INH and F INH and
N
7
O
N
NH
14
6
1
2
1
1
10
their germanium(IV) complexes were conducted on Helicoverpa
armigera and Spodoptera litura pests. A bioactive experiment
was conducted on their larvae. On the basis of the larval mortality
rate the activity of the ligands and their metal complexes was
13
12
1
Figure 1. Structure of ligand (F INH).
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Appl. Organometal. Chem. 2013, 27, 269–276

Pesticidal activities of triorganogermanium(Iv) complexes
2
3
R
N
6
O
4
R
Ge
R
7
5
N
N
O
14
10
11
13
12
Figure 3. Structure of Ge complex ([R
3
1
Ge(F IN)]).
2
3
R
N
O
4
6
R
Ge
R
7
5
N
N
1
4
5
N
10
1
1
1
1
2
13
3 2 3 6 5
Figure 4. Structure of Ge complex ([R Ge(F IN)]), where R = CH or C H .
determined. Helicoverpa armigera (cotton bollworm) is one of the
key pests causing severe yield losses infesting several crops such
as cereals, pulses, cotton, vegetables and fruit crops, as well as
[
21]
wild hosts.
This includes direct losses through shedding and
reduced quality (Figs 5 and 6).
Spodoptera litura is a noctuid moth and is considered an
agricultural pest. It is also known as the cluster caterpillar, cotton
leafworm, tobacco cutworm and tropical armyworm. It is an inter-
national pest and feeds on many herbaceous plants, including
lettuce, cabbage, beetroot, peanuts, geranium, cotton, banana,
fuchsias, strawberry and tomato.
Materials and methods
Larvae of Helicoverpa armigera and Spodoptera litura were used for
the experiments. The experiments were conducted during the
months of August to October, i.e. during the reproductive period
of the cotton crop. The equipment used in experiments, such as
rearing trays, moth chambers, forceps and brushes, were regularly
decontaminated in 5% sodium hypochlorite and rectified spirit.
Preparation of solutions
Solutions of different concentrations (0.02%, 0.05% and 0.1%) of
compounds were prepared by adding the required quantity of
the compounds to methanol. Distilled water was used as control.
Rearing technique of Helicoverpa armigera and Spodoptera litura
Field-collected larvae of both pests were reared separately in
round galvanized trays (35 cm ꢃ 11.5 cm) containing fresh, ten-
der leaves of cotton to raise the initial culture. Every day a suffi-
cient amount of fresh leaves of cotton were provided to larvae
Appl. Organometal. Chem. 2013, 27, 269–276
Copyright © 2013 John Wiley & Sons, Ltd.
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N. Singh et al.
Table 2. Pesticidal effect of ligands and their Ge(IV) complexes
against Helicoverpa armigera at 72 h after treatment
Treatment
Concentration (%)
0.05
0
.02
0.1
a
Mortality (%)
Mortality (%)
Mortality (%)
Control
4(2.40)
4 (2.40)
53.90 (80.65)
45.87 (68.52)
72.86 (98.85)
62.20 (90.52)
73.22 (98.61)
69.05 (96.39)
1.42 4.17
4 (2.40)
59.34 (90.80)
52.47 (78.64)
75.40 (99.54)
71.12 (97.60)
75.30 (99.33)
72.46 (97.99)
1.12 3.16
b
c
F
F
1
INH
INH
38.87 (56.73)
2
34.98 (49.97)
60.18 (88.50)
52.25 (78.93)
69.05 (96.39)
61.88 (90.86)
1.20 3.52
Me
3
Ge(F
Ge(F
Ge(F
Ge(F
1
IN)
IN)
Ph
3
1
Me
3
2
IN)
IN)
Ph
3
2
SEM ꢁ CD at 5%
Figure 5. Cotton boll damage by Helicoverpa armigera larva.
a
Mean of three replications.
b
Figures outside parentheses are arcsine-transformed values.
c
Figures in parentheses are retransformed values.
Table 3. Pesticidal effect of ligands and their Ge(IV) complexes
against Spodoptera litura at 72 h after treatment
Treatment
Concentration (%)
0.05
0
.02
0.10
a
Mortality (%)
Mortality (%)
Mortality (%)
Control
4(2.40)
4(2.40)
29.95(31.23)
20.28(20.42)
69.05(86.39)
61.88(80.66)
62.33(80.78)
54.95(70.69)
1.83 4.29
4(2.40)
52.76(69.66)
29.15(30.06)
75.35(89.33)
72.45(87.99)
73.12(88.61)
68.24(78.64)
2.17 5.32
b
c
F
F
1
INH
INH
26.29 (22.54)
22.40(16.07)
66.05(84.19)
58.75(72.00)
55.35(72.60)
45.73(55.11)
1.82 4.78
2
Figure 6. Cottonleaf damage by Spodoptera litura larva.
Me
3
Ge(F
Ge(F
Ge(F
Ge(F
1
IN)
IN)
Ph
3
1
Me
3
2
IN)
IN)
of both pests, separately. The petiole of each leaf was wrapped
with cotton wool and dipped in water to maintain leaf turgidity.
Freshly formed pupae were transferred to glass jars (15 cm ꢃ 10
cm), having moist sponge and blotting paper at the bottom
and covered with muslin, which was fastened with rubber bands.
Two pairs were released in each earthen pot. The pots were
placed in plastic trays containing water to maintain the desired
level of humidity (>70%) inside the pot. The temperature of lab-
Ph
3
2
SEM ꢁ CD at 5%
a
Mean of three replications.
b
Figures outside parentheses are arcsine-transformed values.
c
Figures in parentheses are retransformed values.
ꢀ
oratory was maintained up to 27 ꢁ 2 C. Eggs laid on the muslin
after 24 h treatment. Mortality counts were recorded at 72 h after
treatment. The mortality percentage was calculated and the data
thus obtained were statistically analysed.
were removed daily and kept in glass jars for hatching. The neo-
nate larvae were reared in glass jars for hatching. After changing
all the stages of life cycle of the pests, the neonate third instar lar-
vae were reared and used for experimental purposes.
Statistical analysis
The mortality of Helicoverpa armigera and Spodoptera litura larvae
in relation to the initial population was worked out in terms of
percentage mortality in larvae. The percentage values were trans-
formed to arcsine scale and statistically analysed following a
completely randomized design (CRD).
Bio-efficacy of the compounds
Ligands and their metal complexes were used at three concentra-
tions, i.e. 0.02%, 0.05% and 0.1%, against both the pests in
statistically designed experiments (Tables 2 and 3). 2 ml of each
concentration of the suspension of the respective treatment
was uniformly applied with the help of a Potter tower on leaves
of cotton placed in Petri dishes (15 cm diameter). Petri dishes
with treated leaves were kept under a ceiling fan to evaporate
water from the leaves. After complete evaporation of water they
were transferred to the glass jars (15 cm height ꢃ 10 cm diame-
ter). Then third instar larvae of both pests were released into each
glass jar separately and were allowed to feed on treated food
materials for 24 h. Three replications of each treatment were
maintained. Fresh untreated leaves of cotton were provided daily
Antifungal Activity
The complexes were screened against Fusarium oxysporum and
Macrophomia phaseolina fungi. The antifungal activity of the syn-
thesized compounds was evaluated by the ’radial growth method’.
Radial growth method
Then principle involved in this method is to ’poison’ the nutrient
medium with a fungi toxicant (compound) and then allowing a
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Pesticidal activities of triorganogermanium(Iv) complexes
O
HO
N
test fungus to grow on such medium. The fungi were grown in
potato dextrose agar (PDA) medium (glucose 20 g, starch 20 g,
agar agar 20 g, 1000 ml water) and the complexes, after being
dissolved in the requisite concentration in methanol, were mixed
with this medium. Several series of concentrations were
prepared. The medium was then poured into Petri plates and a
small disc (0.7 cm) of the fungal culture was cut into a sterile cork
borer and transferred aseptically to the centre of a Petri plate
containing the medium with a certain amount of the compound.
Suitable checks were kept of where the culture plates were
grown under the same conditions on PDA without the
compound. These Petri plates were wrapped in polythene bags
containing a few drops of alcohol and were placed in an incuba-
N
N
N
N
NH
N
N
Figure 7. Equilibrium forms of the ligand: left, amide form; right, imidol
form.
n(Ge–O) and n(Ge–N), respectively. One of the hydrazone bands
at 1480–1500 cm due to the pyridine ring nitrogen remain
unchanged on complexation, indicating non-involvement of
ꢂ1
ꢀ
tor at 25 ꢁ 2 C. Two replicates were used in each case. The colony
[23]
the ring nitrogen in complex formation.
The overall IR spec-
diameter, after 96 h, was compared with the check. The linear
growth of the fungus was obtained by measuring the diameter
of the colonies in the Petri plates, and the percentage inhibition
was calculated using the following formula:
tral evidence suggests that both the ligands are bidentate, coor-
dinating through amide-oxygen and azomethine-nitrogen,
forming a five-membered chelate ring.
1
H NMR Spectra
%
inhibition ¼ 100ðC ꢂ TÞ=C
1
H NMR spectral data of the ligands and their corresponding
where, C is diameter of the fungal colony in the check/control
plate and T is the diameter of the fungal colony in the test plate.
germanium complexes were recorded in DMSO-d with TMS as
an internal standard . Disappearance of the –NH proton signal
6
at d 10.04–11.02 ppm in the spectra of the complexes is consid-
Antibacterial Activity
ered additional evidence of enolization of the ligands during
1
complexation. In the H NMR spectra of ligand F INH, the pyri-
1
Various methods are available for evaluation of the antibacterial activ-
ity of different types of compounds. In the present work, activities of
synthesized compounds were evaluated by the ’paper disc plate
method’ using the inhibition zone technique. The complexes were
screened against Escherichia coli and Staphylococcus aureus bacteria.
dine ring protons (i.e. H , H ) appear as a doublet (d 8.27ppm,
2
6
J = 5.8Hz) and H , H proton signal appears as a doublet at d
3
5
8
.10 ppm, J = 5.8Hz and furan ring proton (12-H to 14-H) signal
1
as a multiplet at d 7.68–6.54 (m) ppm. In the H NMR spectra of
the ligand F INH pyridine ring protons H , H appear as a
2
2
6
Inhibition zone technique
doublet at d 8.23 ppm, J = 5.6 Hz and H
3
, H
5
proton signal as a
doublet at d 7.12 ppm, J = 5.6 Hz and the proton of the other pyr-
idine ring (12-H to 15-H) appeared as a multiplet at d 8.80–7.87
Activity against bacteria was evaluated by the paper disc plate
method. For this purpose, pure cultures of the organism were
dissolved in peptone–water and then uniformly seeded on nutrient
agar plates having the following composition: peptone 5 g, beef
extract 5 g, NaCl 5 g, agar agar 20 g and distilled water 1000 ml.
The reference drug used was streptomycin. All compounds were
dissolved in methanol in different concentrations. Paper discs of
Whatman filter paper with a diameter of 5 mm were soaked in
these solutions. These discs were placed on the medium previously
seeded with organisms in Petri dishes at suitable distances. These
(4H, m) ppm. In the spectra of germanium complexes the signal
due to the pyridine ring and furan ring were shifted downfield.
A sharp singlet at d 2.32–2.49 ppm due to methyl protons
attached to azomethine of the ligands underwent a downfield
shift due to coordination of the azomethine nitrogen (Table 4).
1
3
C NMR Spectra
13
ꢀ
C NMR spectral data recorded for both the ligands and their
discs were stored in an incubator at 35ꢁ 2 C. The inhibition zone
complexes support the proposed structures (Figs 1–4). Signals
due to carbons attached to the azomethine nitrogen and enolic
oxygen appear at d 158.54–162.72 and d 167.60–174.98 ppm,
respectively. However, in the spectra of corresponding metal
complexes, a considerable downfield shift is observed in these
signals. The shifts in the positions of carbons adjacent to the
around each disc was measured (in mm) after 24–30 h.
Spectroscopic Characterization
IR Spectra
coordinating atoms clearly indicate bonding of the azomethine
A comparison of the IR spectra of the complexes and ligands
1
nitrogen and enolic oxygen to the metal atoms. Thus the
and C NMR spectra also confirm the monobasic bidentate na-
H
F
1
1
INH and F
2
INH shows that n(C¼O) of the ligands at 1660–
13
ꢂ1
ꢂ1
670 cm and n(NH) at 3230–3235 cm were absent in the
ture of the ligands already established by the IR spectral studies.
spectra of the respective complexes . This is presumably due to
amide–imidol tautomerism (Fig. 7) and their subsequent coordina-
[
22]
tion through the imidol oxygen. This is supported by the appear-
Biological Activities
ance of a new band in the spectra of the complexes at 1525–1545
ꢂ1
cm attributed to the azine group, >C¼N–N¼C<, which is absent
Body and Organ Weights
ꢂ1
in the spectra of the hydrazones. Bands at 1600–1610 cm due to
n(C ;N) of the hydrazones shift to lower wavenumbers, indicating
No significant change was observed in the body weights of male
coordination of the azomethine nitrogen. Non-ligand bands at
rats after treatment with ligands F INH and F INH and their com-
plexes. However, the weights of testes, epididymis, seminal
1
2
ꢂ1
ꢂ1
8
80 ꢁ 10 cm
and 670 ꢁ 10 cm
have been assigned to
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N. Singh et al.
1
Table 4. H NMR spectral data of ligands and their complexes
1
Compound
H NMR spectral data (d, ppm)
–;NH
–;CH
3
Aromatic protons
Ph–Ge/Me–Ge
Pyridine ring protons
3-H and 5-H
Furan ring
protons 12-H
to 14-H
2
-H and 6-H
12-H to 15-H
F
F
1
2
INH
INH
10.04 (s)
11.02 (s)
—
2.49 (s)
2.32 (s)
2.35 (s)
2.16 (s)
2.28 (s)
2.10 (s)
8.27 (2H, d J = 5.8 Hz)
8.23 (2H, d J = 5.6 Hz)
8.25 (2H, d J = 5.8 Hz)
8.15 (2H, d J = 5.8 Hz)
8.18 (2H, d J = 5.6 Hz)
8.08 (2H, d J = 5.6 Hz)
8.10 (2H, d J = 5.8 Hz)
7.12 (2H, d J = 5.6 Hz)
8.06 (2H, d J = 5.8 Hz)
7.89 (2H, d J = 5.8 Hz)
6.97 (2H, d J = 5.6 Hz)
6.87 (2H, d J = 5.6 Hz)
—
7.68–6.54 (m)
—
8.80–7.87(4H, m)
—
—
7.32–6.25 (m)
7.12–6.12 (m)
—
—
Me
3
Ge(F
Ge(F
Ge(F
Ge(F
1
IN)
IN)
1.32 (9H, s)
7.32–7.47 (15H, m)
1.48 (9H, s)
7.50–7.59 (15H, m)
Ph
3
1
—
—
Me
3
2
IN)
IN)
—
8.78–7.69 (4H, m)
8.68–7.43(4H , m)
Ph
3
2
—
—
vesicle and ventral prostate were reduced after administration of
ligands F INH and F INH and their germanium(IV) complexes
Table 5).
resulted in decreases in the weight of testis and accessory sex
organs. The reduction in the weight of testis may be due to
reduced tubular size, decreased number of germ cells and
1
2
(
[
24]
elongated spermatids.
The observed reduction in weight of
Sperm motility, density, fertility test and serum testosterone
accessory sex organs may be due to several factors, such as
reduced bioavailability of oestrogen and/or anti-androgenic
Results showed a significant decrease (P ≤ 0.01–0.001) in sperm
[
25]
activities of these compounds.
level in the ligands F INH and F
The decreased testosterone
2
INH and their germanium(IV)
motility in cauda epididymis after treatment with ligands F
and F INH and their germanium(IV) complexes. Sperm density
in testes and epididymis were also reduced after various treat-
ments. A sharp decline in fertility of ligands F INH and F INH
and their germanium(IV) complexes was observed. The test was
2–95% negative. The serum level of testosterone was also
1
INH
1
2
complexes in treated animals supports this view. The decreased
mortality of sperm in cauda epididymis indicates lowered ability
1
2
[
26]
of sperm to interact with the oocyte plasma membrane.
Reduction in sperm density may be due to alteration in androgen
3
[
27]
gonadotropin.
The observed negative fertility in the present
reduced in all treated groups (Table 6).
study may be attributed to lack of forward progression, reduction
in density of spermatozoa and altered biochemical milieu of
Biochemical change
[
28]
cauda epididymis. A fall in testicular glycogen indicated inhibi-
A marked reduction in testicular sialic acid and glycogen was
tion of glycogen synthesis, eventually decreasing the spermato-
observed in the ligands F
IV) complexes, whereas testicular protein, cholesterol, acid and
alkaline phosphatase activity were increased significantly
Table 7).
1
INH and F
2
INH and their germanium
[29]
genic process.
Reduction in testicular sialic acid content may
(
be due to absence of spermatozoa or reduced androgen produc-
[30]
tion. Increased concentration of cholesterol in testes suggests
(
that impairment of spermatogenesis is due to decreased andro-
[
31]
gen concentration
. Further, increase in testicular acid and
Results and discussion
alkaline phosphatase activity may be the result of labialization
[
32]
The present study revealed that administration of ligands F
1
INH
IN)],
IN)] to male rats
of lysosomal system
and releasing the enzyme that
and F
2
INH and their germanium(IV) complexes [Me
IN)], [Ph Ge(F IN)] and [Ph Ge(F
3 1
Ge(F
degenerates the tissue resulting in reduction of various micro-
[
33]
[
Me Ge(F
3
2
3
1
3
2
molecules, including reduction in sperm count.
From the
Table 5. Body weight, testes and accessory sex organ weight analysis after administration of ligands and their germanium(IV) complexes
ꢂ1
Group
Treatment
Body weight
mg 100 g body weight
Initial
Final
Testes
Epididymis
Seminal vesicle
Ventral prostate
A
B
C
D
E
Control
180.0 ꢁ 15.4
190.0 ꢁ 10.3
188.0 ꢁ 9.2
179.0 ꢁ 8.7
178.0 ꢁ 7.3
185.0 ꢁ 7.9
184.0 ꢁ 8.6
190.0 ꢁ 7.8
205.0 ꢁ 9.6
200.0 ꢁ 8.7
198.0 ꢁ 9.3
194.0 ꢁ 8.4
208.0 ꢁ 10.3
202.0 ꢁ 9.5
1220.0 ꢁ 41.5
1010.0 ꢁ 40.2
1022.0 ꢁ 30.8
805.0 ꢁ 31.3
825.0 ꢁ 25.5
490.0 ꢁ 20.5
410.0 ꢁ 10.4
424.0 ꢁ 11.3
470.0 ꢁ 18.0
310.0 ꢁ 14.5
340.0 ꢁ 12.5
225.8 ꢁ 11.7
230.0 ꢁ 10.8
210.0 ꢁ 14.3
218.0 ꢁ 16.2
360.0 ꢁ 27.4
a
a
a
a
a
a
b
b
b
b
b
b
b
F
F
1
2
INH
INH
290.0 ꢁ 14.4
b
295.0 ꢁ 14.8
b
b
Me
Me
Ph
3
Ge(F
Ge(F
Ge(F
Ge(F
1
IN)
IN)
IN)
IN)
325.0 ꢁ 10.7
210.0 ꢁ 10.5
b
b
3
2
334.0 ꢁ 9.6
225.0 ꢁ 10.2
b
b
b
b
F
3
1
750.0 ꢁ 25.2
764.0 ꢁ 22.8
290.0 ꢁ 8.7
195.0 ꢁ 11.4
b
b
G
Ph
3
2
292.0 ꢁ 7.8
205.0 ꢁ 10.9
a
b
Mean values of six animals: P ≤ 0.01; P ≤ 0.001.
Groups B and C compared with group A.
Groups D and E compared with group B.
Groups F and G compared with group C.
wileyonlinelibrary.com/journal/aoc
Copyright © 2013 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2013, 27, 269–276

Pesticidal activities of triorganogermanium(Iv) complexes
Table 6. Sperm dynamics, fertility test and serum testosterone level after administration of ligands and their germanium(IV) complexes
ꢂ1
Group
Treatment
Sperm motility (%)
cauda epididymis
Sperm density (million ml
)
Fertility (%)
Sperm testosterone
ꢂ1
(mg ml
)
Testes
Cauda epididymis
A
B
C
D
E
Control
70.0 ꢁ 2.15
4.35 ꢁ 0.75
49.8 ꢁ 0.32
100% (+ve)
32% (ꢂve)
40% (ꢂve)
70% (ꢂve)
80% (ꢂve)
90% (ꢂve)
95% (ꢂve)
2.5 ꢁ 0.1
a
b
a
b
F
F
1
INH
INH
48.0 ꢁ 4.51
2.95 ꢁ 0.71
2.98 ꢁ 0.95
36.0 ꢁ 0.20
1.9 ꢁ 0.1
b
a
b
b
2
52.0 ꢁ 5.22
38.0 ꢁ 0.30
1.8 ꢁ 0.2
1.0 ꢁ 0.1
0.9 ꢁ 0.8
0.9 ꢁ 0.2
0.8 ꢁ 0.4
b
b
b
b
b
a
b
Me
Me
3
Ge(F
Ge(F
1
IN)
IN)
38.0 ꢁ 4.30
1.15 ꢁ 0.31
1.34 ꢁ 0.42
1.10 ꢁ 0.22
1.28 ꢁ 0.51
28.7 ꢁ 0.15
b
a
b
b
b
3
2
41.0 ꢁ 5.80
30.0 ꢁ 0.05
b
b
F
Ph
Ph
3
Ge(F
Ge(F
1
IN)
IN)
31.0 ꢁ 4.21
26.0 ꢁ 0.11
b
b
G
3
2
34.0 ꢁ 4.36
29.3 ꢁ 0.21
a
b
Mean values of six animals: P ≤ 0.01; P ≤ 0.001.
Groups B and C compared with group A.
Groups D and E compared with group B.
Groups F and G compared with group C.
1 2
Table 7. Biochemical changes in testes of rats after administration of ligands F INH and F INH and their germanium(IV) complexes
Group
Treatment
Testicular glycogen
Testicular sialic
Testicular
Testicular acid
phosphatase
(KA units)
Testicular alkaline
phosphatase
(KA units)
Testicular
ꢂ1
ꢂ1
(mg g
)
acid (mg g
)
protein
cholesterol
ꢂ1
ꢂ1
(
mg g
)
(mg g
)
A
B
C
D
E
Control
2.74 ꢁ 0.08
4.90 ꢁ 0.06
250.0 ꢁ 10.5
4.71 ꢁ 0.18
65.0 ꢁ 2.15
6.91 ꢁ 0.37
b
b
a
a
b
b
F
F
1
INH
INH
1.10 ꢁ 0.07
1.25 ꢁ 0.11
0.30 ꢁ 0.06
0.34 ꢁ 0.02
0.32 ꢁ 0.07
0.36 ꢁ 0.10
3.60 ꢁ 0.05
3.75 ꢁ 0.02
315.0 ꢁ 8.7
6.71 ꢁ 0.91
80.7 ꢁ 3.17
9.10 ꢁ 0.31
10.11 ꢁ 0.25
11.50 ꢁ 0.23
12.17 ꢁ 0.16
12.80 ꢁ 0.18
13.22 ꢁ 0.21
b
b
b
b
b
a
a
b
b
b
b
b
b
b
2
335.0 ꢁ 6.4
7.62 ꢁ 0.82
85.3 ꢁ 3.54
b
b
b
b
a
b
b
Me
Me
3
Ge(F
Ge(F
1
IN)
IN)
2.10 ꢁ 0.03
2.12 ꢁ 0.08
2.15 ꢁ 0.04
2.21 ꢁ 0.07
350.0 ꢁ 7.4
8.31 ꢁ 0.62
93.0 ꢁ 4.53
a
b
b
3
2
360.0 ꢁ 8.5
8.98 ꢁ 0.58
93.80 ꢁ 6.02
a
b
b
F
Ph
Ph
3
Ge(F
Ge(F
1
IN)
IN)
370.5 ꢁ 9.4
9.15 ꢁ 0.72
94.0 ꢁ 5.41
a
b
b
G
3
2
380.5 ꢁ 2.5
9.85 ꢁ 0.26
95.0 ꢁ 4.45
a
b
Mean values of six animals: P ≤ 0.01; P ≤ 0.001.
Groups B and C compared with group A.
Groups D and E compared with group B.
Groups F and G compared with group C.
above-mentioned findings, it has been concluded that ligands
INH and F INH and their germanium(IV) complexes are potent
fertility inhibitors, and addition of Me or Ph moiety to the
Table 8. Antifungal screening data of the ligands and their organo-
germanium complexes
F
1
2
3
3
Compound
Fusarium oxysporum
concentration, ppm)
Macrophomina
phaseolina
ligands enhances their activity in reducing fertility.
(
(
concentration, ppm)
5
0
100
200
50
100
200
Pesticidal Results
F
F
1
INH
INH
52
54
64
67
72
64
66
72
75
78
72
74
80
82
84
48
50
58
62
68
60
62
68
72
78
75
78
88
90
92
After 72 h treatment the results of pesticidal activity revealed that
there was a significant difference in percent mortality at different
concentrations; it was noted that as concentration of ligands
2
Me
3
Ge(F
Ge(F
Ge(F
1
IN)
IN)
Ph
3
1
1 2
F INH and F INH and their germanium(IV) complexes increases
Me
3
2
IN)
the larval mortality rate also increases. The chemical-treated
leaves were slowly or much less fed to the larvae of Helicoverpa
armigera and Spodoptera litura, which led to starvation of the
developing larvae.
Ph
3
Ge(F
2
IN)
76
92
80
86
72
84
82
94
94
Bavistin
100
100
100
Antifungal Screening
[
34]
inhibitory effect than do the parental ligands, due to chelation
of the metal atom with the ligand moieties.
The results of antifungal activities of germanium complexes and
their parent ligands against some pathogenic fungi are listed in
Table 8. The inferences drawn from these observations clearly
indicate that the ligands are active against various pathogenic
fungi. The results also show that the metal complexes have more
The enhanced activity of germanium complexes was ascribed
to the increased lipophilic nature of these complexes arising
[
35]
due to chelation.
Further, the results of bioactivity were
compared with the conventional fungicide Bavistin.
Appl. Organometal. Chem. 2013, 27, 269–276
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/aoc

N. Singh et al.
Table 9. Antibacterial screening data of the ligands and their orga-
nogermanium complexes
Acknowledgements
The authors are thankful to the Council of Scientific and Industrial
Research, New Delhi, for financial assistance through grant no. 01
Compound
Staphylococcus
aureus
Escherichia coli
(concentration,
ppm)
(2571)/12/EMR-II, and to Professor Marcel Gielen, Vrije Universiteit
(concentration,
Brussel, Faculty of Engineering HRNMR Unit, Building F&G, Plein-
laan 2, B-1050 Brussels, Belgium, for help in improving the
manuscript.
ppm)
5
00
1000
500
1000
F
F
1
2
INH
INH
12
14
15
17
16
17
18
13
16
17
18
17
19
20
7
10
12
16
16
17
18
10
14
16
18
17
18
20
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3
Ge(F
Ge(F
Ge(F
Ge(F
1
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IN)
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wileyonlinelibrary.com/journal/aoc
Copyright © 2013 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2013, 27, 269–276