Synthesis of nalidixic acid based hydrazones as novel pesticides

Article abstract of DOI:10.1021/jf904144e

Thirty-one substituted hydrazones of nalidixic acid hydrazide were synthesized and characterized by spectral techniques. These compounds were evaluated for various biological activities, namely, fungicidal, insecticidal, and nitrification inhibitory activities. The antifungal activity was evaluated against five pathogenic fungi, namely, Rhizoctonia bataticola, Sclerotium rolfsii, Rhizoctonia solani, Fusarium oxysporum, and Alternaria porii. They showed maximum inihibition against A. porii with ED₅₀ = 34.2 151.3 μg/mL. The activity was comparable to that of a commercial fungicide, hexaconazole (ED₅₀ = 25.4 μg/mL). They were also screened for insecticidal activity against third-instar larvae of Spodoptera litura and adults of Callosobruchus maculatus and Tribollium castaneum. Most of them showed 70-100% mortality against S. litura through feeding method at 0.1% dose. These compounds were not found to be effective nitrification inhibitors.

Full text of DOI:10.1021/jf904144e

3056 J. Agric. Food Chem. 2010, 58, 3056–3061
DOI:10.1021/jf904144e
Synthesis of Nalidixic Acid Based Hydrazones as
Novel Pesticides
NISHA
A
GGARWAL
,
†,§ RAJESH
K
UMAR,§ CHITRA
S
*
RIVASTVA,# PREM
,†
D
UREJA,*,§ AND
J. M. KHURANA
†Department of Chemistry, University of Delhi, Delhi 110 007, India, §Division of Agricultural Chemicals,
and #Division of Entomology, Indian Agricultural Research Institute, New Delhi 110 012, India
Thirty-one substituted hydrazones of nalidixic acid hydrazide were synthesized and characterized by
spectral techniques. These compounds were evaluated for various biological activities, namely,
fungicidal, insecticidal, and nitrification inhibitory activities. The antifungal activity was evaluated
against five pathogenic fungi, namely, Rhizoctonia bataticola, Sclerotium rolfsii, Rhizoctonia solani,
Fusarium oxysporum, and Alternaria porii. They showed maximum inihibition against A. porii with
ED50 = 34.2-151.3 μg/mL. The activity was comparable to that of a commercial fungicide,
hexaconazole (ED50 = 25.4 μg/mL). They were also screened for insecticidal activity against
third-instar larvae of Spodoptera litura and adults of Callosobruchus maculatus and Tribollium
castaneum. Most of them showed 70-100% mortality against S. litura through feeding method at
0.1% dose. These compounds were not found to be effective nitrification inhibitors.
KEYWORDS: Nalidixic acid; hydrazones; antifungal activity; insecticidal activity; nitrification inhibitory
activity
INTRODUCTION
overcoming these losses. The use of nitrification inhibitors mini-
mizes these effects. Nitrapyrin, dicyandiamide, etridiazole, etc.,
are the common commercial nitrification inhibitors. The high
costs of development and subsequent registration of effective
inhibitors are serious issues in their extensive use (11), under-
scoring a need to develop simple, efficient, economical, and safe
nitrification inhibitors.
Nalidixic acid (1,8-naphthapyridine derivative) is the first
synthetic quinolone antibiotic introduced in the 1960s. Structure-
activity relationships (SAR) of compounds based on nalidixic
acid have led to a large group of synthetic bioactive molecules
known as the quinolones (12). Hydrazide-hydrazones have been
claimed to exhibit appreciable antimicrobial activity (13-15).
Furthermore, a number of hydrazide-hydrazone derivatives
have been claimed to possess interesting antibacterial, antifun-
gal (16, 17), anticonvulsant (18), anti-inflammatory (19), anti-
malarial (20), and antituberculosis activities (21). The hydrazone
derivatives are used as active ingredients in the method for
controlling agricultural and horticultural insect pests (22). There-
fore, to explore the biopotential of nalidixic acid derivatives, the
present study was undertaken to synthesize a series of novel
nalidixic acid based hydrazones and to evaluate them as fungi-
cides, insecticides, and nitrification inhibitors.
The major constraint limiting higher agricultural productivity
is the onslaught of insect pests and diseases. Over 30% yield losses
in major crops are caused by pathogenic fungi and insect pests (1).
These soil-inhabiting fungi cause wilt diseases, leaf wilting, dry
root rot, yellowing, and eventually plant death (2). Among these
Fusarium oxysporum and Alternariaporii are the most devastating
fungi, especially for banana plant and onion crop (3, 4). Insect
pests affect the growth of plants and reduce the photosynthetic
ability of crop. On most crops, damage arises from extensive
feeding by larvae, which leads to complete stripping of the
plants (5). Among all insect pests, Spodoptera litura is an
extremely serious polyphagous pest of several cultivated crops,
the larvae of which can defoliate many economically important
crops (6). This pest has developed resistance to many conven-
tional and currently available insecticides (7). Hence, there is a
need to search for an alternate pesticide that can fit into farmers’
budgets as well as an integrated pest management (IPM) pro-
gram.
Another major problem of global concern is the low yields of
crops due to low efficiency of fertilizers inputs, which results in
U.S. $16 billion annual loss of nitrogen (N) fertilizers worldwide (8).
The factors contributing to N losses are mainly ammonia
volatilization, nitrification, and denitrification and nitrate-leach-
ing. These processes contribute to various health and environ-
mental hazards (9, 10). Regulation of urea hydrolysis and
nitrification in agricultural systems has been a major strategy in
MATERIALS AND METHODS
Chemicals and Instruments. All of the chemicals usedwerepurchased
from Sigma-Aldrich and used without further purification. Reactions were
monitored by thin layer chromatography (TLC) on precoated Merck silica
gel 60F254, and the spots were visualized either under UV or by iodine
vapor. Melting points were determined on a JSW melting point apparatus
and are uncorrected. Infrared (IR) spectra were recorded on Perkin-Elmer
*Corresponding authors [e-mail (J.M.K.) jmkhurana1@yahoo.co.in,
(P.D.) pd_dureja@yahoo.com].
pubs.acs.org/JAFC
Published on Web 02/04/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 5, 2010 3057
model 2000 FT-IR spectrophotometer as KBr pellet, and values are
expressed as νmax cm-1. Mass spectra were recorded on a JEOL (Japan)
JMS-DX303 and micromass LCT, mass spectrometer/data system. The
1H NMR and 13C NMR spectra were recorded on a Bruker Spectrospin
spectrometer (400 and 75.5 MHz), using tetramethylsilane as an internal
standard. The chemical shift values were recorded on δ scale, and the
coupling constants (J) are in hertz. Elemental analysis for all compounds
was performed on a Carlo Erba model EA-1108 elemental analyzer and
data of C, H, and N were within (0.4% of calculated values.
Synthesis. Substituted Hydrazones (1-31). Nalidixic acid hydrazide
was prepared according to a reported procedure (12). To a stirred solution
of nalidixic acid hydrazide (5 mmol) in methanol (10 mL) were added
substituted benzaldehydes (5.5 mmol), and the mixture was stirred at room
temperature for 3 h. The precipitate thus obtained was filtered, washed
with a minimum amount of cold methanol, and recrystallized from ethanol
(Figure 1).
1-Ethyl-7-methyl-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic
acid (2-fluoro-benzylidene)-hydrazide (1): yield, 85%; white solid; melting
point, 280 °C; IR, 3449, 2989, 1676, 1608, 1568, 1231, 756; 1H NMR δ 1.53
(t, 3H, N-CH2CH3), 2.71 (s, 3H, 7-CH3), 4.60 (q, 2H, N-CH2),
7.10-7.17 (m, 1H, H-3-phenyl), 7.19-7.27 (m, 1H, H-5-phenyl),
7.33-7.35 (m, 1H, H-4-phenyl), 7.37 (d, J = 8.40 Hz, 1H, H-6-
naphthyridine), 7.49-7.56 (m, 1H, H-6-phenyl), 8.56 (s, 1H, H-2-
naphthyridine), 8.67 (d, J = 5.20 Hz, 1H, H-5-naphthyridine), 9.03 (s,
1H, NdCH), 13.23 (s, 1H, NH); HRMS calculated for C19H17FN4O2,
352.1336, found, 353.1308 (Mþ þ H).
In Vitro Antifungal Activity. The antifungal activity of the com-
pounds was evaluated in vitro using a food poison technique (23).
In Vitro Insecticidal Activity. The biological assay was conducted
against third-instar larvae of Spodoptera litura (7 ( 1 day old) and adults
of Callosobruchcus maculatus (1-2 days old) and Tribollium castaneum
(12 ( 1 days old). Adults of C. maculatus and T. castaneum were obtained
Figure 1. Synthesis of substituted nalidixic acid based hydrazones
(1-31).
Table 1. Antifungal Activity Data of Nalidixic Acid Based Hydrazones (1-31) against Five Pathogenic Fungia
antifungal activity
RS
RB
SR
FO
AP
compd
R
R
β
R
β
R
β
R
β
R
β
1
2-fluorophenyl
668.0
243.7
593.2
503.9
370.6
627.6
610.5
271.5
575.3
307.8
209.7
300.7
533.4
322.1
378.0
453.3
494.6
255.1
649.9
382.2
284.7
528.0
399.9
201.0
371.9
315.7
256.2
389.7
188.2
638.2
324.0
4.4
2.5
4.7
3.0
1.8
1.1
4.0
4.2
1.0
0.7
2.9
2.3
4.3
3.4
1.7
5.3
1.6
4.3
2.8
4.5
2.9
3.7
2.4
1.0
4.6
3.7
2.8
4.5
1.4
4.3
2.4
4.7
1.2
539.7
219.9
570.4
438.8
437.7
380.2
654.6
286.9
463.6
364.0
242.3
320.2
346.0
297.7
374.4
463.6
540.6
282.5
396.8
374.8
358.7
459.7
384.3
186.0
197.7
259.0
202.4
428.6
177.1
523.9
203.8
13.0
1.6
5.2
3.3
2.4
1.0
5.6
3.7
5.6
0.5
5.6
4.9
5.0
3.9
1.7
4.3
0.5
4.0
3.1
2.3
1.6
5.6
1.1
1.4
2.7
2.5
2.7
5.2
2.9
1.2
5.0
1.3
1.0
358.7
234.5
245.6
393.9
307.5
438.8
328.6
244.7
225.7
330.4
157.4
238.2
408.3
301.2
372.1
252.5
353.6
297.7
341.6
188.2
171.7
316.7
356.2
173.6
170.4
226.5
191.6
374.3
166.5
405.4
247.5
18.3
2.9
2.7
4.0
4.0
3.4
1.4
4.2
1.9
0.2
1.2
3.8
1.1
1.8
4.2
1.2
2.4
1.2
4.1
2.9
4.3
4.4
3.4
2.1
4.7
1.7
1.7
4.9
2.0
4.5
3.8
2.6
2.1
107.4
79.9
3.3
0.7
5.6
4.5
1.2
3.3
5.0
2.9
3.7
4.9
0.0
5.2
5.8
5.1
1.8
3.0
2.5
3.4
4.6
3.1
3.4
1.6
4.9
2.6
5.3
2.7
2.6
0.9
0.3
3.6
5.3
1.6
78.3
64.8
2.4
4.8
3.8
2.1
2.4
3.7
1.5
1.4
5.3
3.5
0.2
3.4
5.3
1.0
2.5
1.7
1.8
1.0
5.3
3.0
3.4
3.2
5.3
2.3
0.9
3.7
3.2
1.9
2.1
2.8
5.9
1.2
2
4-fluorophenyl
2-chlorophenyl
3-chlorophenyl
4-chlorophenyl
3-bromophenyl
2-nitrophenyl
3
143.3
131.9
118.7
209.2
168.8
212.2
125.3
181.2
63.1
90.1
98.8
4
5
87.2
6
125.3
71.3
7
8
3-nitrophenyl
4-nitrophenyl
89.0
70.1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
4-hydroxyphenyl
3-methoxyphenyl
4-methoxyphenyl
2-methylphenyl
3-methylphenyl
4-methylphenyl
4-isopropylphenyl
2,4-dichlorophenyl
2,6-dichlorophenyl
2,4-dihydroxyphenyl
2,4-dimethoxyphenyl
3,4-dimethoxyphenyl
4-hydroxy-3-methoxyphenyl
4-hydroxy-3-ethoxyphenyl
3,4,5-trimethoxyphenyl
2-pyridyl
145.9
57.6
151.5
128.6
103.1
195.1
277.1
121.1
114.2
196.5
176.3
136.6
376.8
184.8
102.5
79.1
121.1
109.2
66.9
87.3
124.5
119.4
87.2
143.8
131.1
102.1
128.2
92.1
79.4
77.3
1-naphthyl
2-naphthyl
134.5
89.5
115.3
75.6
9-anthryl
cyclohexyl
222.1
62.6
151.3
34.2
adamantyl
crotonyl
258.5
95.4
132.7
89.8
hexaconazole
20.6
25.5
a RB, Rhizoctonia bataticola; SR, Sclerotium rolfsii; RS, Rhizoctonia solani; FO, Fusarium oxysporum; AP, Alternaria porii; R, ED50 value (μg/mL), calculated from mean
percentage inhibition, which is an average of four replicates and its standard deviation (() ranging from (0.22 to (5.87; β, chi square for heterogeneity (tabular value at 0.05
level) = 5.99 (degrees of freedom = 3).

3058 J. Agric. Food Chem., Vol. 58, No. 5, 2010
Aggarwal et al.
Figure 2. Antifungal activity of halogen-substituted derivatives (1-6) against A. porii and F. oxysporum (error bars shows standard deviation).
from the Division of Entomology, Indian Agricultural Research Institute
(IARI), New Delhi. India. Insects were reared on green gram (Vigna
radiate) and wheat at 27 ( 1 °C and 70% relative humidity. Insecticidal
activity of the synthesized compounds was evaluated by film residue
method and residue method for contact and feeding toxicity, respectively.
The larvae of S. litura were collected from castor fields of IARI and were
reared in the laboratory at 27 ( 1 °C and 70% relative humidity according
to a reported method (24).
applied on the ventral side of the S. litura larvae. Ten treated larvae were
released in glass bottles, and fresh tender castor leaves were given as food.
Each treatment was kept in triplicate, and solvent was used as control.
Mortality was observed after 24 h.
Nitrification Inhibitory Activity. The above synthesized compounds
were also evaluated for their effect on nitrification inhibition at a 5% dose
according to the following reported method.
Soil. The soil for in vitro incubation experiments was collected from the
farm of the Institute. A composite soil sample was collected in bulk from
the cultivated fields of known history from a depth of 0-15 cm following a
standard sampling procedure. The physical and chemical characteristics of
the soil were as follows: sand, 60.8%; clay, 20.5%; and slit, 18.7%; pH 7.9
(soil/water 1:2.5); EC at 25 °C, 0.35 dSm-1; organic carbon, 0.50%;
available N, 55.72 mg kg-1 of soil; nitrate-N, 12.9 mg kg-1 of soil; nitrite-
N, traces; and ammonium-N, 5.6 mg kg-1 of soil. It was air-dried at room
temperature, ground, and passed through a 2 mm sieve. The soil was
thoroughly mixed before use.
Bioassays. Film Residue Method. All synthesized (1-31) hydra-
zones were weighed and dissolved in dichloromethane to prepare 0.1%
stock solution. One milliliter of a 0.1% solution of various compounds was
added to each Petri plate (90 mm). Petri plates with test solution were
rotated vigorously to prepare uniform film and were allowed to dry for
3-5 min. The experiments were performed in triplicates along with solvent
as a control and reference insecticide as malathion. Ten adults (2-5 days
old) were released in each Petri plate and were kept at 27 ( 1 °C with 70%
relative humidity. Mortality was observed after 24 h. Adults were
considered to be dead if they failed to respond to stimulus by touch.
Residue Method. Grains of green gram (V. radiate) were treated with
test compounds dissolved in dichloromethane (0.6 mg in 5 mL) at a dose of
20 mg of active ingredient kg-1 of grains (0.002% of active ingredient).
The treated grains (10 g) were kept in a plastic box, and 10 adults of
T. castaneum were released in it. The experiment was conducted in
triplicate with solvent as control. Mortality was observed after 48 h.
Feeding Method. The castor leaf was dipped in a 0.1% solution of
synthesized compounds (1-31) for 2 s and then air-dried. Moist filter
paper was placed in glass Petri plates (9 cm diameter) on which treated leaf
disks were kept. Larvae of S. litura prestarved for 4 h were released
individually into each Petri plates. Thirty replications were kept for each
treatment. Solvent was used as control. Mortality was observed after 24 h.
Topical Treatment. The 0.1% stock solution of various compounds was
prepared in dichloromethane. Two microliters of each compound was
Experiment. The experiments were conducted following a completely
randomized design (CRD) with three replicates. The test chemicals and
reference inhibitor were tested at a 5% dose of applied urea-N along with a
urea alone control. The samples were incubated in 100 mL capacity plastic
beakers (50 g of air-dried soil was taken per beaker). A calculated amount
of the test chemical (0.5 mg for 5% dose of applied urea-N, respectively) in
acetone was added to each beaker and mixed thoroughly. In all of the
treatments including control, the same volume of acetone was added. After
thorough mixing, 10 mg of urea-N (200 mg of urea-N kg-1 of soil) in
aqueous solution was added and mixed thoroughly. Distilled water was
added to each beaker to maintain the moisture at 50% water-holding
capacity of the soil. The controls were similarly processed with urea alone
at 200 mg kg-1 urea-N level without adding any test/reference inhibitor.
All of the beakers were accurately weighed, labeled, and kept at 28 ( 1 °C
with 98% relative humidity in an incubator. Soil moisture was maintained

Article
J. Agric. Food Chem., Vol. 58, No. 5, 2010 3059
by adding distilled water every alternate day after taking the difference of
weight if necessary (25).
extracting solution was shaken for an hour on a reciprocal shaker and
filtered. Ammonium-N, nitrite-N, and nitrate-N were estimated by
indophenol blue, sulfanilic acid, and phenol-disulfonic acid meth-
ods (26, 27), respectively. The contents of ammonium-N, nitrite-N, and
nitrate-N were obtained from the standard curves and expressed in
milligrams per kilogram. The nitrification rate (NR) and percent nitrifica-
tion inhibition (NI) were calculated using Sahrawat’s formulas (26).
Sampling and Estimation of Ammonium-N, Nitrite-N, and Nitrate-N
from the Soil Samples. The soil samples (5 g) were withdrawn on the 21st
day of incubation and extracted with aqueous sodium sulfate (50 mL, 1 M)
by shaking on a reciprocal shaker for 1 h. The soil samples were filtered
and estimated for ammonium-N, nitrite-N, and nitrate-N. The soil with
RESULTS AND DISCUSSION
Methyl ester of naldixic acid was synthesized (28), which was
further hydrazinolyxed to naldixic acid hydrazide (12). The
hydrazide was condensed with various aldehydes to afford 31
substituted hydrazones (Figure 1). All of the hydrazones were
characterized using spectral techniques and microanalysis, and
the respective data are provided as Supporting Information. The
spectral and analytical data for the four known hydrazones were
in agreement with that reported in the literature (29).
In Vitro Antifungal Activity. All of the synthesized hydrazones
(1-31) were screened for fungicidal activity against Rhizoctonia
bataticola, Sclerotium rolfsii, Rhizoctonia solani, Fusarium oxy-
sporum, and Alternaria porii by the poisoned food technique, and
their calculated ED50 values are reported in Table 1. Most of the
Figure 3. Plot of log ED50 values for all test fungi versus electronic
parameters (σo, σm, σp) for o-, m-, p-nitrophenyl-substituted nalidixic acid
hydrazones (7-9).
compounds showed maximum inhibition against A. porii (ED50
34.2-151.3 μg/mL) followed by F. oxysporum (ED50
=
=
62.6-376.8 μg/mL). Compound 29 (ED50 = 34.2-188.2 μg/
mL) showed maximum antifungal activity against all test fungi,
being effective against A. porii (ED50 = 34.2 μg/mL) comparable
with hexaconazole (ED50 = 25.5 μg/mL), a commercial fungi-
cide. Fluoro-substituted analogues were more effective than the
chloro and bromo substituents irrespective of their positions
(Figure 2). Sigma values for o-, m-, and p-nitrophenyl/methyl-
phenyl-substituted nalidixic acid hydrazones were plotted against
corresponding log ED50 values (Figures 3 and 4). The sigma
values were low to moderate for o-NO2 (σo = 0.8), m-NO2 (σm
=
0.71), and p-NO2 (σp = 0.78) and for o-CH3 (σo = -0.37), m-
CH3 (σm = -0.07), and p-CH3 (σp = -0.17) (30). In all cases a
quadratic relationship with a very high R2 (coefficient of
determination) value was observed. A perusal of the trend
equations revealed the significantly high antifungal activity of
hydrazone derivatives with an electron-withdrawing nitro group
Figure 4. Plot of log ED50 value for all test fungi versus electronic
parameters (σo, σm, σp) for o-, m-, p-methylphenyl-substituted nalidixic
acid hydrazones (13-15).
Figure 5. Antifungal activity profile of aryl derivatives (11, 12, 20, 21, 24) with varied number and position of methoxy group(s) against A. porii and
F. oxysporum.

3060 J. Agric. Food Chem., Vol. 58, No. 5, 2010
Aggarwal et al.
Table 3. Effect of Nalidixic Acid Based Hydrazones (1-31) on Ammonium-N,
Nitrite-N, and Nitrate-N Contents and Rate of Nitrification
ammonia-N nitrite-N
(mg/kg)
nitrate-N
(mg/kg)
nitrification
rate (%)
compd
R
(mg/kg)
1
2
3
4
5
6
7
8
9
2-fluorophenyl
4-fluorophenyl
2-chlorophenyl
3-chlorophenyl
4-chlorophenyl
3-bromophenyl
2-nitrophenyl
3-nitrophenyl
4-nitrophenyl
24
16
25
15
55
30
9
172
144
127
147
100
117
164
126
181
165
170
179
165
138
185
151
125
155
154
144
95
95
92
91
81
80
98
96
97
96
93
96
94
86
97
94
86
94
94
90
14
17
8
Figure 6. Effect of increase in size of ring on antifungal activity.
10 4-hydroxyphenyl
11 3-methoxyphenyl
12 4-methoxyphenyl
13 2-methylphenyl
14 3-methylphenyl
15 4-methylphenyl
16 4-isopropylphenyl
17 2,4-dichlorophenyl
18 2,6-dichlorophenyl
19 2,4-dihydroxyphenyl
20 2,4-
13
20
11
22
13
21
20
19
10
15
Table 2. Percent Mortality of Nalidixic Acid Based Hydrazones (1-31)
against Spodoptera litura, Callosobruchus maculatus, and Tribollium
castaneum
mortality (%)
S.
litura
T.
C.
castaneum maculatus
compd
R
feeding contact feeding contact contact
1
2-fluorophenyl
4-fluorophenyl
90
50
10
90
40
20
100
80
90
70
10
70
0
10
10
10
10
30
10
30
10
10
20
60
10
10
40
10
70
10
60
20
20
40
30
25
12
10
20
22
10
10
0
dimethoxyphenyl
21 3,4-
2
17
22
19
14
149
133
112
142
95
93
93
96
3
2-chlorophenyl
3-chlorophenyl
4-chlorophenyl
dimethoxyphenyl
22 4-hydroxy-3-
methoxyphenyl
4
5
6
3-bromophenyl
2-nitrophenyl
3-nitrophenyl
10
30
23 4-hydroxy-3-
ethoxyphenyl
24 3,4,5-
7
20
30
8
9
4-nitrophenyl
10
10
trimethoxyphenyl
25 2-pyridyl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
4-hydroxyphenyl
3-methoxyphenyl
4-methoxyphenyl
2-methylphenyl
3-methylphenyl
4-methylphenyl
4-isopropylphenyl
2,4-dichlorophenyl
2,6-dichlorophenyl
2,4-dihydroxyphenyl
2,4-dimethoxyphenyl
3,4-dimethoxyphenyl
11
26
2
159
133
195
174
175
151
184
93
84
99
96
95
92
94
26 1-naphthyl
27 2-naphthyl
28 9-anthryl
80
70
10
100
100
40
100
40
8
10
29 cyclohexyl
31 crotonyl
urea
9
13
11
2
10
10
mL) against F. oxysporum and A. porii. Compounds with a naphthyl
substituent (compounds 26 and 27) at the β-position (ED50
=
4-hydroxy-3-methoxyphenyl 100
20
10
10
75.5-256.2 μg/mL) were found to be more active as compared to
substitution at the R-position (ED50 = 115.2-315.7 μg/mL).
In Vitro Insecticidal Activity. All of these compounds were also
evaluated for in vitro insecticidal activity against the lepidopteran
insect pest of field crops and the coleopteran insect pest of stored
grains at 0.1% dose through contact as well as feeding method.
Bioassay of the compounds was conducted against third-instar
larvae of S. litura and adults of storage insects C. maculatus and
T. castaneum. Most of the compounds showed insecticidal activity
(mortality 80-100%) against S. litura when tested through the
feeding method. The insecticidal activity of these compounds was
comparable to that of cypermethrin, a commercial pyrethroid
insecticide when tested at a 0.1% dose. No mortality was observed
when these compounds were tested by topical treatment (i.e.,
through direct penetration on the insects) against S. litura Com-
pounds tested against adults of C. maculatus by contact method
showed adult mortality ranging from 20 to 70%. None of the
compounds showed mortality against T. castaneum when tested
either by contact or by feeding method. The results of bioassay
(Table 2) indicated that of the various tested compounds, com-
pound 7 showed the best toxicity against S. litura as well as
C. maculatus at 0.1% dosage. This compound showed complete
mortality against S. litura and 60 and 20% mortality against
C. maculatus and T. castaneum, respectively. Apart from compound
4-hydroxy-3-ethoxyphenyl
3,4,5-trimethoxyphenyl
2-pyridyl
90
90
100
90
1-naphthyl
2-naphthyl
10
10
10
70
10
15
15
10
0
9-anthryl
cyclohexyl
20
10
0
70
adamantyl
crotonyl
50
10
0
60
100
0
cypermethrin
malathion
100
100
100
at the para position and an electron-releasing methyl group at the
meta position. The above observation was further supported by
the activity data of the compounds having methoxy group(s) at
meta and/or para positions (Figure 5). An increase in activity was
observed when a hydroxyl group (compound 10) was replaced
with a methoxy group (compound 12) in the aromatic ring.
Increase in size of either saturated ring (compound 29, having a
6-membered ring, and compound30, having a 10-membered ring)
or unsaturated ring (compound 26, having a 10-membered ring,
and compound 28, having a 14-membered ring) resulted in a
decrease in fungicidal activity (Figure 6). The compounds with
unsaturated aliphatic and heterocyclic substitution showed higher
fungicidal activity (compounds 25 and 31, ED50 = 77.2-95.4 μg/

Article
J. Agric. Food Chem., Vol. 58, No. 5, 2010 3061
7, compounds 17, 18, 20, 22, and 25 were also found to be very
effective against S. litura as they also showed 100% mortality.
They showed moderate to good mortality against C. maculatus but
did not show any mortality against T. castaneum. Compounds 3,
11, 13, and 16 showed least toxicity against all of the insect pests. In
general, it was found that all of the compounds were effective
against S. litura larvae, the voracious feeding stage, when mixed in
the food, whereas these compounds were not effective against
hard-body storage insects such as C. maculatus and T. castaneum
through either contact or feeding method.
Nitrification Inhibitory Activity. Results obtained in the in vitro
soil incubation study are reported in Table 3. All of these
compounds are not significantly active as nitrification inhibitors.
Ammonium-N content was in the range of 10-55 mg/kg.
Compound 5 showed maximum ammonium-N content (55 mg/
kg) as compared to urea (11 mg/kg). The nitrite-N content was
below 0.5 mg/kg in all of the treatments, whereas urea showed
2 mg/kg. Nitrate-N content for the test chemicals varied from 100
to 195 mg/kg. The performance of compound 5 was best in
minimizing the production of nitrate-N.
In conclusion, antifungal data of hydrazones revealed that
compounds with a monosubstituted phenyl ring exhibit better
activity than those with a disubstituted phenyl ring. Most of them
were found to be potent insecticidal agents at a 0.1% dose against
S. litura. Compound 29 (ED50 = 34.2-188.2 μg/mL), comprising
a cyclohexyl ring, has emerged as a potent fungicide having
insecticidal properties with mortality range of 70% against
S. litura. The result obtained from bioassay indicates that this class
of compounds can be utilized for the design of new molecules with
good pesticidal activity. Thus, these molecules hold promise for
further detailed bioefficacy study, especially against insect pests.
(12) Gaurav, G.; Suvarna, G. K. Synthesis and evaluation of new
quinazolone derivatives of nalidixic acid as potential antibacterial
and antifungal agents. Eur. J. Med. Chem. 2006, 41, 256–262.
Kidwai, M.; Misra, P.; Kumar, R. The fluorinated quinolones. Curr.
Pharm. Des. 1998, 4, 101–118.
(13) Xiao-Dong, Y.; JinHua, Z. Synthesis and biological activity of
hydrazone derivatives containing pyrazole. J. Chem. Res. 2008, 9,
489–491.
(14) Mamolo, M. G.; Falagiani, V.; Zampieri, D.; Vio, L.; Banfi, E.
Synthesis and antimycobacterial activity of [5-(pyridin-2-yl)-1,3,4-
thiadiazol-2-ylthio]acetic acid arylidene-hydrazide derivatives.
Farmaco 2001, 56, 587–592.
(15) Rollas, S.; Gulerman, N.; Erdeniz, H. Synthesis and antimicrobial
activity of some new hydrazones of 4-fluorobenzoic acid hydrazide
and 3-acetyl-2,5-disubstituted-1,3,4-oxadiazolines. Farmaco 2002,
57, 171.
(16) Kamel, A. M.; Lobna, M. A; El-Sayed, M. L.; Mohamed, I. H.;
Rania, H. B. Hydrazones of 2-aryl-quinoline-4-carboxylic acid
hydrazides: synthesis and preliminary evaluation as antimicrobial
agents. Bio. Med. Chem. 2006, 14, 8675–8682.
(17) Ozdemir, A.; Turan-zitouni, G.; Kaplancikl, Z. A.; Demirci, F.;
Iscan, G. Studies on hydrazone derivatives as antifungal agents.
J. Enzym. Inhib. Med. Chem. 2008, 23, 470–475.
(18) Dimmock, J. R.; Vashishtha, S. C.; Stables, J. P. Anticonvulsant
properties of various acetylhydrazones, oxamoylhydrazones and
semicarbazones derived from aromatic and unsaturated carbonyl
compounds. Eur. J. Med. Chem. 2000, 35, 241–248.
(19) Kalsi, R.; Shrimali, M.; Bhalla, T. N.; Barthwal, J. P. Synthesis and
anti-inflammatory activity of indolyl azetidinones. Indian J. Pharm.
Sci. 2006, 41, 353–359.
(20) Melnyk, P.; Leroux, V.; Sergheraert, C.; Grellier, P. Design, synth-
esis and in vitro antimalarial activity of an acylhydrazone library.
Bioorg. Med. Chem. Lett. 2006, 16, 31–35.
(21) Patole, J.; Sandbhor, U.; Padhye, S.; Deobagkar, D. N.; Anson,
C. E.; Powell, A. Structural chemistry and in vitro antitubercular
activity of acetylpyridine benzoyl hydrazone and its copper complex
against Mycobacterium smegmatis. Bioorg. Med. Chem. Lett. 2003,
13, 51–55.
Supporting Information Available: MP, yield, IR, mass, and
NMR data of synthesized compounds. This information is avail-
able free of charge via the Internet at http://pubs.acs.org.
(22) Tomokazu, H. I.; Nobuharu, A. O.; Hiroshi, H. K.; Atsushi, K. K.
Hydrazone derivatives, processes for production thereof, and uses
thereof. U.S. Patent 5304573, 1994.
LITERATURE CITED
(23) Aggarwal, N.; Kumar, R.; Dureja, P.; Rawat, D. S. Schiff bases as
potential fungicides and nitrification inhibitors. J. Agric. Food Chem.
2009, 57, 8520–8525.
(24) Srivastva, C.; Reddy, D. S. Evaluation of antifeedent activity of
neem formulations against tobacco caterpillar Spodoptera litura.
Indian J. Plant Prot. 2006, 34, 126–128.
(25) Kumar, R.; Devakumar, C.; Kumar, D.; Panneerselvam, P.; Kak-
kar, G.; Arivalagan, T. Influence of edaphic factors on the miner-
alization of neem oil coated urea in four Indian soils. J. Agric. Food
Chem. 2008, 56, 10183–10191.
(26) Sahrawat, K. L. On the criteria for comparing the ability of
compounds for retardation of nitrification in soil. Plant Soil 1980,
55, 487–490.
(27) Keeney, D. R.; Nelson, D. W. Nitrogen inorganic forms. In Methods
of Soil Analysis, Part 2. Chemical and Microbiological Properties;
Page, A. L., Ed.; Soil Science Society of America and American Society
of Agronomy: Madison, WI, 1989; pp 643-698.
(28) Grundy, J. B.; James, G.; Pattenden, G. Esterification of sterically
hindered carboxylic acids using dimethyl sulphate. Tetrahedron Lett.
1972, 13, 757–758.
(29) Youssef, A. F.; Omar, F. A.; Elsherief, H. A.; Abuo-Rahma, A. A.
Design, synthesis and antidepressant activity of some N2-substituted
nalidixic acid hydrazides and their cyclized analogs. Bull. Pharm. Sci.
1998, 21, 15–26.
(30) Hansch, C.; Leo, A.; Unger, S. H.; Kim, K. H.; Nikaitani, D.; Lien,
E. J. Aromatic substituent constants for structure-activity correla-
tions. J. Med. Chem. 1973, 16, 1207.
(1) Bhargava, M. C.; Choudhary, R. K.; Jain, P. C. Genetic engineering
of plants for insect resistance. In Entomology: Novel Approaches;
Jain, P. C., Bhargava, M. C., Eds.; New India Publishing: New Delhi,
India, 2008; pp 133-144.
(2) Chattapadhyay, T. K.; Dureja, P. Antifungal activity of 4-methyl-6-
alkyl-2H-pyran-2-ones. J. Agric. Food Chem. 2006, 54, 2129–2133.
(3) Donnell, K.; Kistler, C.; Cigelnik, E.; Ploetz, R. C. Multiple
evolutionary origins of the fungus causing Panama disease of
banana: Concordant evidence from nuclear and mitochondrial gene
genealogies. Proc. Natl. Acad. Sci.U.S.A. 1998, 95, 2044–2049.
(4) Makelo, M. N.; Narla, R. D.; Mibey, R. K.; Makini, F. W. Effect of
plant extracts on growth of Alternaria porri (Ellis) Cif. and other fungal
pathogens of onion. J. Trop. Microbiol. Biotechnol. 2007, 3, 7–11.
(5) Ahmad, M.; Arif, M. I.; Ahmad, M. Occurrence of insecticide
resistance in field populations of Spodoptera litura (Lepidoptera:
Noctuidae) in Pakistan. Crop Prot. 2007, 26 (6), 809–817.
(6) Kumari, V.; Singh, N. P. Spodoptera litura nuclear polyhedrosis
virus (NPV-S) as a component in integrated pest management (IPM)
of Spodoptera litura (Fab.) on cabbage. J. Biopest. 2009, 2, 84–86.
(7) Rajendra Prasad, M. N.; Gowda, M. V. C Mechanisms of resistance
to tobacco cutworm (Spodoptera litura F.) and their implications to
screening for resistance in groundnut. Euphytica 2006, 149, 387–399.
(8) Raun, W. R.; Johnson, G. V. Improving nitrogen use efficiency for
cereal production. Agron. J. 1999, 91, 357–363.
(9) Prasad, R.; Power, J. F. Nitrification inhibitors for agriculture,
health and the environment. Adv. Agron. 1995, 54, 233–281.
(10) Prasad, R. Fertilizer urea, food security, health and the environment.
Curr. Sci. 1998, 75, 677–683.
Received for review November 25, 2009. Revised manuscript received
January 19, 2010. Accepted January 19, 2010.
(11) McCarty, G. W. Modes of action of nitrification inhibitors. Biol.
Fertil. Soils 1999, 29, 1–9.