Larvicidal and structure-activity studies of natural phenylpropanoids and their semisynthetic derivatives against the tobacco armyworm Spodoptera litura (Fab.) (Lepidoptera: Noctuidae)

Article abstract of DOI:10.1002/cbdv.200800345

The larvicidal activity of 18 phenylpropanoids, 1-18, including phenylpropenoate, phenylpropenal, phenylpropene, and their semisynthetic analogues, were evaluated against the tobacco armyworm, Spodoptera litura (FAB.), to identify promising structures with insecticidal activity. Amongst various phenylpropanoids, isosafrole, a phenylpropene, showed the best activity, with an LC₅₀ value of 0.6 μg/leaf cm², followed by its hydrogenated derivative dihydrosafrole (LC₅₀=2.7 μg/leaf cm ²). The overall larvicidal activity of various phenylpropene derivatives was observed in the following order: isosafrole (6) >dihydrosafrole (16)>safrole (12)>anethole (4)>methyl eugenol (11)>eugenol (13)>β-asarone (8)>dihydroasarone (18)>dihydroanethole (15). Dihydrosafrole might be a promising compound, although presenting a lower larvicidal activity than isosafrole, because of its better stability and resistance to oxidative degradation (due to the removal of the extremely reactive olefinic bond) in comparison to isosafrole. Such structure-activity relationship studies promote the identification of lead structures from natural sources for the development of larvicidal products against S. litura and related insect pests.

Full text of DOI:10.1002/cbdv.200800345

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Larvicidal and Structure–Activity Studies of Natural Phenylpropanoids and
Their Semisynthetic Derivatives against the Tobacco Armyworm Spodoptera
litura (Fab.) (Lepidoptera: Noctuidae)1)
by Anu Bhardwaja), Dhananjay Kumar Tewarya), Rakesh Kumarb), Vinod Kumarb),
Arun Kumar Sinha*b), and Adarsh Shankera)
a) Hill Area Tea Sciences Division, Institute of Himalayan Bioresource Technology, Palampur
(H.P.)-176061, India
(phone: þ91-1894-230426; fax: þ91-1894-230433; e-mail: aksinha08@rediffmail.com)
b) Natural Plant Products Division, Institute of Himalayan Bioresource Technology, Palampur
(H.P.)-176061, India
The larvicidal activity of 18 phenylpropanoids, 1–18, including phenylpropenoate, phenylpropenal,
phenylpropene, and their semisynthetic analogues, were evaluated against the tobacco armyworm,
Spodoptera litura (Fab.), to identify promising structures with insecticidal activity. Amongst various
phenylpropanoids, isosafrole, a phenylpropene, showed the best activity, with an LC50 value of 0.6 mg/leaf
cm2, followed by its hydrogenated derivative dihydrosafrole (LC50 ¼2.7 mg/leaf cm2). The overall
larvicidal activity of various phenylpropene derivatives was observed in the following order: isosafrole
(6)>dihydrosafrole (16)>safrole (12)>anethole (4)>methyl eugenol (11)>eugenol (13)>b-asarone
(8)>dihydroasarone (18)>dihydroanethole (15). Dihydrosafrole might be a promising compound,
although presenting a lower larvicidal activity than isosafrole, because of its better stability and resistance
to oxidative degradation (due to the removal of the extremely reactive olefinic bond) in comparison to
isosafrole. Such structure–activity relationship studies promote the identification of lead structures from
natural sources for the development of larvicidal products against S. litura and related insect pests.
Introduction. – One of the important concerns in pest management strategies is to
reduce the use of conventional chemical pesticides, due to their inherent potency to
induce resistance among the target insect species [1][2] and their potential harmful
effects on human health, environment, and non-target organisms [3]. In this context,
there have been increasing efforts in exploring the possibilities of developing plant-
based insecticides [4–6] or their semisynthetic derivatives [7]. Research approaches in
this direction include: i) screening programs to explore new potential flora, ii)
exploiting the published information on the insecticidal potential of plants for
developing viable formulations of active principles or blends from the abundantly
available source, and iii) exploiting the lead compounds from promising chemical
families, either to use them for developing insecticide formulations, or as a prelude for
structure–activity relationship (SAR) studies to obtain synthetic or semisynthetic
leads with superior performance. The knowledge on lead compounds is generally
gained by bioassay-guided fractionations of promising plant sources, followed by the
identification of marker compounds responsible for the desired activity.
1
)
IHBT Communication No. 0688
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
169
Our effort in this direction is primarily focused on exploring the potential of
phenylpropanoids as insecticidal agents. Phenylpropanoids (C6 –C3 unit), a major
group of naturally occurring phenolic compounds in plants, have attracted our interest
to evaluate their insecticidal properties, because of their involvement in plant defense
against pests [8–10]. The main objective of the present study is to identify the
promising insecticidal structures of natural phenylpropanoids, followed by under-
standing the SAR by modifying the structures to obtain leads with superior perform-
ance. The insecticidal potential of abundantly available natural phenylpropanoids was
evaluated against Spodoptera litura, one of the most widespread and economically
important crop pests [11].
Results and Discussion. – Preliminary Screening to Identify a Lead Structure.
Phenylpropanoids were tested against larvae of a lepidopteron insect, Spodoptera
litura. Host plant (castor oil plant, Ricinus communis) leaves were treated with a
formulation containing the desired compounds at different dosages and allowed to feed
by the starved larvae. The effect of these compounds on the larval mortality was
observed and compared with the controls, as explained in details in the Exper. Part. The
preliminary screening for insecticidal activity of phenylpropanoids with four different
functional groups, viz., an ester (methyl 4-methoxycinnamate; 1), an aldehyde (4-

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
methoxycinnamaldeyde; 2), an acid (4-methoxycinnamic acid; 3), and an olefin (4-
methoxyphenylpropene or anethole; 4), against the larvae was carried out at 15 mg/leaf
cm2 (Table 1). Compound 4, a phenylpropene with a C¼C bond adjacent to C(1),
showed a promising toxic activity against the larvae. The other three compounds
included in the preliminary screening did not show appreciable activities. Hence,
compound 4 was selected as starting point for further SAR investigations, because of its
larvicidal activity and the abounding availability of 4 and its related phenylpropenes in
nature [8].
Table 1. Larvicidal Activity of Phenylpropanoids against Spodoptera litura at 15 mg/leaf cm2
Compound
Mortality [%]a)
24 h
Compound
Mortality [%]a)
24 h
48 h
48 h
1
2
–b)
–
–
–
3
4
–
–
86.6ꢁ6.0
100ꢁ0.0
a) Mortality corrected for the mortality of the untreated control insects using Abbottꢂs formula; values
are means and standard-deviations of three replicates. b) Activity <10%.
Semisynthetic Derivatives of Phenylpropenes and SAR. A series of compounds
related to 4-methoxyphenylpropene (4), obtained either from natural sources or by
semisynthetic routes (5–18), were evaluated for their larvicidal activities, to under-
stand the effect of various substitution patterns on their performance. The insecticidal
potential of these compounds is compiled in Table 2. Some of the compounds, i.e.,
anethole (4), isosafrole (6), b-asarone (8), methyl eugenol (11), safrole (12), eugenol
(13), and dihydrosafrole (16) induced appreciable larval mortality (ꢀ60%) after 48 h
of exposure time. Increasing the exposure time had a significant (at the 5% level) effect
on the larval mortality for compounds 4, 8, 11, and 13, while no significant difference in
activity was observed for compounds 6, 12, and 16. Based on the larvicidal potential of
the compounds (Table 2), promising lead structures were identified and their toxicities
were quantified by means of dose–response lines.
Table 2. Larvicidal Activity of Phenylpropenes against Spodoptera litura at 15 mg/leaf cm2
Compound
Mortality [%]a)
24 h
Compound
Mortality [%]a)
24 h
48 h
48 h
5
6
7
8
9
–b)
–
12
13
14
15
16
17
18
96.0ꢁ6.0
26.0ꢁ6.0
–
53.0ꢁ6.0
100.0ꢁ0.0
10.0ꢁ0.0
–
100.0ꢁ0.0
66.0ꢁ0.0
16.0ꢁ15.3
53.0ꢁ6.0
100.0ꢁ0.0
23.0ꢁ6.0
56.0ꢁ6.0
100.0ꢁ0.0
100.0ꢁ0.0
–
–
16.0ꢁ11.5
60.0ꢁ0.0
–
–
–
–
10
11
36.0ꢁ15.3
76.6ꢁ11.5
a) Mortality corrected for the mortality of the untreated control insects using Abbottꢂs formula; values
are means and standard-deviations of three replicates. b) Activity <10%.

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
171
LC50 Values and other statistical parameters, generated by linear regression
analysis, are listed in Table 3. Isosafrole (6) was the most active compound, with an
LC50 value of 0.6 mg/leaf cm2 after 48 h of exposure, whereas dihydroanethole (15) was
the least toxic among the compounds tested in the series. Overall, the activity of the
compounds after 48 h of exposure decreased in the order: isosafrole (6)>dihydrosa-
frole (16)>safrole (12)>trans-anethole (4)>methyl eugenol (11)>eugenol (13)>b-
asarone (8)>dihydroasarone (18)>dihydroanethole (15). It can also be noted that, in
general, the LC50 value of compounds was negatively correlated with the exposure
time. Interestingly, there was not much difference in the activity between 24 and 48 h of
exposure for compounds 4, 6, 12, 15, and 16. It was also observed that compounds 8, 11,
and 13 showed delayed effects with very low mortalities at 24 h contact time in the
tested concentration range. Hence, LC50 values (12.7, 9.9, and 11.4 mg/leaf cm2, resp.)
for these compounds could be calculated only after 48 h of exposure. The activity of 6,
the most active phenylpropene, was comparable with that of the biological (NeemAzal)
and the chemical (methyl parathion) reference insecticides at 48 h contact time
(Table 3).
Table 3. Larvicidal Activity (LC50 values and regression parameters of probit analysis) of the Promising
Compounds against Spodoptera litura
Compound
24 h
48 h
LC50 [mg/leaf cm2]
c2
SlopeꢁSE
LC50 [mg/leaf cm2]
c2
SlopeꢁSE
4
6
8
11
12
13
15
16
9.4 (7.5–11.3)
2.1 (1.5–2.7)
–
–
4.1
0.8
–
–
0.2
–
0.0
4.1
–
1.7
2.8
1.4ꢁ0.2
1.4ꢁ0.2
–
8.0 (6.9–9.2)
0.6 (0.2–1.0)
5.2
0.3
1.1
0.5
0.5
0.8
0.1
4.8
0.2
0.6
4.3
2.4ꢁ0.4
1.0ꢁ0.3
1.3ꢁ0.2
1.4ꢁ0.2
3.7ꢁ0.6
1.3ꢁ0.2
1.3ꢁ0.2
1.3ꢁ0.1
1.3ꢁ0.2
0.4ꢁ0.1
1.2ꢁ1.3
12.7 (10.4–15.5)
9.9 (8.1–11.9)
7.6 (6.7–8.6)
11.4 (9.3–14.0)
14.2 (11.7–17.6)
2.7 (1.9–3.5)
13.9 (11.5–17.2)
2.6 (1.6–4.8)
0.3 (0.3–0.3)
–
8.5 (7.4–9.5)
3.3ꢁ0.5
–
14.5 (12.0–17.9)
3.2 (2.3–4.1)
–
7.0 (5.2–15.0)
0.6 (0.5–0.6)
1.4ꢁ0.2
1.3ꢁ0.2
–
18
NeemAzal
Methyl parathion
1.0ꢁ0.3
1.1ꢁ0.5
a) LC50 value was not determined due to the low mortality in the tested concentration range.
Most of the studies on phenylpropanoids pertaining to insecticidal activities
[12][13] are limited to and aiming at evaluating the activities of the individual
compounds against different target species of interest. In the present study, our effort
was to identify and select the most promising structure among the basic phenyl-
propanoids, based on preliminary screening results and source availability of the
compounds, followed by understanding the SAR of the promising compounds and their
related derivatives to obtain leads with enhanced activity. Though many of these
phenylpropanoids occur in plants, a lot of them are not available in sufficient quantity
and purity for their bioactivity evaluation [14]. It is also worth mentioning that some of
the dihydrophenylpropanoids have not been previously tested against S. litura.
In the first part of the study, the larvicidal activity of various phenylpropanoids
containing different functional groups was evaluated and compared (Table 1). None of

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the compounds having a functional group, such as an ester, aldehyde, or acid group,
attached at C(3) of the alkyl chain showed encouraging activity. However, the
phenylpropene 4, with a C¼C bond adjacent to C(1) of the alkyl chain, demonstrated
promising activity. Thus, it was observed that functional groups attached to the alkyl
side chain played a major role in contributing to the activity, which is in agreement with
earlier studies [8][9]. Considering the availability and economics, 4 and the related
phenlypropenes 5–18 were further investigated.
Antifeedant Effect. We also observed the antifeedant activities for compounds 5–
18, and the following antifeedant effects were found: >90% for anethole (4), isosafrole
(6), safrole (12), methyleugenol (11), b-asarone (8), and dihydrosafrole (16), between
60 and 80% for eugenol (13) and dihydroeugenol (17), and between 40 and 60% for
dihydroasarone (18), dihydroanethole (15), and isoeugenol (7). However, possible
physiological effects of these compounds on the larvae and thereby on their subsequent
feeding behavior could have affected the measurement and judgment of the
antifeedant activities.
Larvicidal Effect. Phenylpropenes occur in three isomeric forms, a, b, and g, in
nature. In a- or b-isomers, the C¼C bond is adjacent to C(1), where as in g-isomers, the
C¼C bond is adjacent to C(2) of the alkyl side chain (Scheme). It is known that the
activity of phenylpropenes is greatly influenced by the variations in the isomeric forms
and/or functionalities attached to either the aryl ring or the alkyl side chain of C6 –C3
systems. In the present study, during the initial screening of phenylpropenes with
different functionalities, anethole (4), isosafrole (6), b-asarone (8), methyl eugenol
(11), safrole (12), eugenol (13), dihydroanethole (15), and dihydrosafrole (16) showed
promising activity (Table 2) and were subjected to the dose–response bioassay to
quantify their toxicity (Table 3). Compound 4, with one MeO group at C(4) of the
aromatic ring, in trans-form, was active (LC50 value of 8.0 mg/leaf cm2), whereas its g-
counterpart, i.e., methyl chavicol (10), did not show activity. In contrast, isoeugenol (7),
with OH and MeO groups at C(4) and C(3) of the aromatic ring, respectively, was
inactive (0% mortality), whereas the g-isomer with the same substitution pattern (13)
showed good insecticidal activity with an LC50 value of 11.4 mg/leaf cm2 after 48 h of
exposure. Reports on the enhanced activity of phenylpropenes by non-polar
substitutions at the aromatic ring, (e.g., MeO vs. OH groups) in general [15], and
sensitivity of pests to MeO substituted compounds in particular, prompted us to screen
methylisoeugenol (5) and methyleugenol (11) with two MeO groups at C(3) and C(4).
It was observed that only the g-isomer 11 was active (Table 2). On altering the polarity
of 11 by replacing the MeO group at C(4) by an AcO group, i.e., compound 14, caused a
considerable decrease in activity, which suggests the dominant role of polar groups in
influencing the activity. Harmatha and Nawrot [15] have also reported similar
observations of reduced activity on substitution of polar groups in phenylpropanoids
against some stored grain pests. In another report on acaricidal activity of phenyl-
propenes [16], it was shown that bioefficacy increased linearly with molecular
lipophilicity. The activity of compounds 4 and 11 turned our attention to compound 8,
another closely related phenylpropene. Comparison of the activity of 8 with that of the
two related structures 4 and 5 (Table 3), with the only difference being the MeO group
substitution on the aromatic ring, shows that the effect of multiple apolar substituents is
additive and the presence of the C¼C bond at C(2) of the alkyl chain is important in

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
173
governing the larvicidal activity of such phenylpropenes. Della Greca et al. [17], in their
SAR studies of phenylpropanoids, have also observed that the presence of a MeO
group at the aromatic ring enhanced the effectiveness of phenylpropanoids.
Scheme. Comparative Benefits of Dihydrosafrole (16) over an Isomeric Mixture of Safrole
In earlier studies, it was observed that OCH2O substitution greatly enhanced the
pesticidal activity of phenylpropenes [10][15]. This OCH2O moiety is found in many
natural compounds, e.g., piperine alkaloids, from which the commercial synergist
piperonyl butoxide (PBO) is derived, which interacts with the cytochrome P450 and
inhibits the activity of the polysubstrate mono oxygenases (PSMOs) responsible for the
metabolism of toxins in insects. In the present study, substitution with an OCH2O group
also enhanced the activity of phenylpropenes against the tested larvae. Interestingly,
both isomeric forms, isosafrole (6) and safrole (12), demonstrated good activity
(Table 3). In this case, the b-isomer 6 was 12.6 times more active than the g-isomer 12
after 48 h of exposure, which is in accordance with various reports on higher activity of
b-isomers [15][18][19]. The inconclusive observation regarding the trend of activity of
phenylpropenes with respect to b- or g-isoforms prompted us to reduce the C¼C bond
present in the alkyl side chains of phenylpropenes and to compare the activity of the
resulting hydrogenated compounds, dihydroanethole (15), dihydrosafrole (16),
dihydroeugenol (17), and dihydroasarone (18) with either of their isomeric counter-
parts. Among the tested hydrogenated compounds, only compound 16 showed
promising activity with an LC50 value of 2.7 mg/leaf cm2 after 48 h of exposure
(Tables 2 and 3). It is pertinent to mention that phenylpropenes are vulnerable to
oxidation at the C¼C bond of the alkyl side chain when kept for a prolonged period of
time [20]. However, the hydrogenated products 15–18, devoid of oxidation suscept-
ibility, with substantial retention of activity, have the advantage of an extended shelf life
and storage potential (Scheme) and may be considered for their use in formulation with
other promising phytochemicals.
Phenylpropenes can act in different ways against insects [18]. Although the
mechanisms of toxicity of phenylpropenes against the lepidopteran larvae are not well
known, several observations point to some physiological effects at the endocrine,
enzymatic, and digestive level [18][21]. Some of the compounds under study, i.e., b-

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
asarone (8), methyleugenol (11), and eugenol (13), showed delayed mortality, which
could be attributed to several factors acting separately or together, viz., the uptake of
the active moiety of the compound could be time dependent, leading to a progressive
increase in the concentration of the compounds tested and their effect on the larval
body, or the active moiety of the compound could be converted into more toxic
metabolites in the larval integument and alimentary canal, resulting in time-dependent
effects [22].
Several phenylpropenes, such as eugenol (13) [4][13][23], isoeugenol (7) [13], b-
asarone (8) [24], anethole (4) [11][25], have been reported active against insects/mites.
SAR of plant-derived compounds against arthropod pests have been well studied [26].
However, only limited studies on SAR of phenylpropenes against insects/mites have
been reported [15][18][24][27][28], and no systematic studies are available on various
structures of phenylpropanoids, particularly phenylpropenes, against S. litura. It has to
be mentioned that a quantitative comparison of the toxicity of related phenyl-
propanoids is difficult with the phenylpropenes and some of their dihydroderivatives
tested in the present study, due to different test insects and bioassay methods.
Conclusions. – The larvicidal activity and SAR of natural phenylpropanoids and
their semisynthetic derivatives against S. litura were investigated, which has not been
reported earlier. It was shown that OCH2O substituted phenylpropenes have a
promising insecticidal potential against S. litura. Isosafrole showed the highest activity
(LC50 value of 0.6 mg/leaf cm2 after 48 h) while its isomer safrole showed to be 12.6
times less active (LC50 value of 7.6 mg/leaf cm2 after 48 h).
The problems associated with the separation of isomeric mixtures of phenyl-
propenes and, furthermore, susceptiblitity of the C¼C bond of, e.g., safrole/isosafrole,
with time towards air oxidation, resulting in the reduction of larvicidal activities, may
be solved by hydrogenation of the isomeric mixtures. In this context, the activity shown
by dihydrosafrole (LC50 value of 2.7 mg/leaf cm2 after 48 h) is of interest, in particular in
combination of its better stability and resistance to oxidative degradation in
comparison to isosafrole/safrole. This observation may prove a pivotal point for
further studies, and work in this direction is in progress. The incorporation of
dihydrosafrole within insecticidal formulations could increase the number of bio-
chemical targets in the insects, limiting the prospects for the onset of resistance and
offering the means of reducing pesticide dosage due to possible synergistic or additive
action.
A. B. and V. K. are indebted to CSIR (A. B.) and UGC (V. K. ), Government of India, Delhi, for
providing financial assistance in terms of senior research fellowships. We thank the director of the IHBT
for providing necessary facilities to succeed the research work.
Experimental Part
General. 2,3-Dichloro-5,6-dicyanobenzoquinone (DDQ) was purchased from Merck (India).
Column chromatography (CC): silica gel (SiO2; 60–120 mesh). 1H- and 13C-NMR Spectra: Bruker
Avance-300 spectrometer at 300 and 75.4 MHz, resp., in CDCl3; d in ppm, J in Hz. CEM Discover focused
microwave (2450 MHz, 300 W) was used wherever mentioned.
Test Compounds and Their Synthesis. Compounds 3–7 and 10–13 were procured from commercial
sources (Merck and Sigma Aldrich) and were used without any further purification. Compounds 1, 2, 9,

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
175
14, and 15–18 were synthesized by the reported methods [29][30]. Compound 8 was isolated and
purified from the essential oil of the rhizome of Acorus calamus by hydrodistillation using the method
reported by Sinha et al. [31].
Methyl 4-Methoxycinnamate (1). A mixture of 4-methoxycinnamaldehyde (2; 18.75 mmol), DDQ
(28.15 mmol), Amberlyst-15 (0.1–0.2 g), MeOH (5 ml), and toluene (15 ml) in a round-bottom flask was
irradiated in a focused monomode microwave system (100 W, 1108), fitted with a condenser and a Dean–
Stark apparatus, for 40 min. The precipitated DDQH2 was filtered, and the filtrate was passed over a bed
of neutral alumina column and eluted with a 2–5% mixture of MeOH in toluene. The obtained org. layer,
after evaporation under vacuum, provided 1 (90 % yield). White solid. M.p. 958. 1H-NMR: 7.67 (d, J¼
16.15, 1 H); 7.40 (d, J¼8.48, 2 H); 6.83 (d, J¼8.48, 2 H); 6.26 (d, J¼16.15, 1 H); 3.75 (s, 3 H); 3.71 (s,
3 H). 13C-NMR: 167.7; 161.4; 144.5; 129.7; 127.1; 115.2; 114.3; 55.3; 51.5. The spectral data matched well
with the reported values [30].
4-Methoxycinnamaldehyde (2). A mixture of 15 (8.5 mmol), dry dioxane (50 ml), AcOH (2–4
drops), and DDQ (26.5 mmol) in a beaker was sonicated for 2 h or till disappearance of the starting
material on the TLC plate. After completion of the reaction, the precipitated, solid DDQH2 was removed
by filtration, and the filtrate was evaporated. The residue was taken in AcOEt (50 ml) and washed with
H2O (2ꢂ10 ml), 2% NaHCO3 (2ꢂ5 ml), and brine (2ꢂ10 ml), dried (Na2SO4), and filtered. The
filtrate was evaporated to afford a crude yellow liquid, which was chromatographed on neutral alumina
using hexane/AcOEt mixtures with increasing proportions of AcOEt up to 40% to provide 2 (70%
1
yield). White solid. M.p. 58–598. H-NMR: 9.68 (d, J¼7.8, 1 H); 7.58 (d, J¼15.8, 1 H); 7.19–7.17 (m,
2 H); 6.72–6.70 (m, 2 H); 6.63 (dd, J¼15.8, 7.8, 1 H); 3.73 (s, 3 H). 13C-NMR: 190.0; 161.2; 150.3; 129.6;
127.6; 127.2; 114.0; 56.0. The spectral data matched well with the reported values [29].
2,4,5-Trimethoxyphenylpropene (9). A mixture of 18 (4.8 mmol), DDQ (6.0 mmol), and SiO2 (0.5–
0.6 g) in anh. dioxane (50 ml) was stirred at r.t. for 14 h under N2 till completion of the reaction. The
precipitated DDQH2 was filtered and the filtrate was evaporated and subsequently chromatographed on
SiO2 (hexane/AcOEt 7:3) to provide 9 (72% yield). White solid. M.p. 44–458. 1H-NMR: 6.91 (s, 1 H);
6.64 (dd, J¼1.5, 16.0, 1 H); 6.51 (s, 1 H); 6.02 (dq, J¼6.2, 16.0, 1 H); 3.84, 3.81, 3.77 (3s, 3 H each); 1.87
(dd, J¼6.2, 1.5, 3 H). 13C-NMR: 151.0; 149.0; 142.6; 124.4; 123.4; 118.3; 109.2; 97.3; 56.1; 55.7; 55.1; 18.7.
The spectral data matched well with the reported values [29].
Synthesis of 14. A mixture of 13 (1.0 mmol), AcONa (4.0 mmol) and Ac2O (4.0 mmol) was irradiated
by microwave (100 W, 1308) for 40 s. After work up, 14 was obtained (98% yield). The spectral data
matched well with the reported values [10].
Synthesis of 15–18. Compounds 15–18 were prepared according to the method reported by Joshi
et al. [29]. Compounds 4, 6, 7, or 8, resp., (15 mmol) were adsorbed on the powdered mixture of SiO2
(4 g), palladium chloride (0.2 g), and ammonium formate (0.5 g), and 4 ml of formic acid (85%) and 5 ml
of H2O were added. The mixture was irradiated in a monomode microwave (100 W, 1208), fitted with a
reflux condenser, for 8–10 min. Column purification of the crude mixture provided the corresponding
hydrogenated derivatives (88–94% yield). The spectral data of the compounds matched well with
reported values [29].
Test Insects. Insects, Spodoptera litura (Fab.) (Lepidoptera: Noctuidae), were obtained from infested
field crops, reared in the laboratory on Ricinus communis (Castor) leaves, and maintained at 23ꢁ18, 65ꢁ
5% rel. humidity, and a photoperiod of 16 :8 (L :D). Second instar larvae, obtained from the established
colony maintained for >20 generations in the laboratory, were used in the experiments.
Bioassays. Preliminary Screening. The larvicidal activity of 1–18 was evaluated by the leaf dip
method [32] against 2nd instar larvae. Larvicidal activity was evaluated on castor leaves (34 cm2) as a
preferred host, since it shows strong attractiveness and palatability for S. litura. In addition, preference
for castor was not overcome by exposure to novel deleterious chemicals [33]. It has also been shown that
the effect of the host on the mid gut carboxyl esterase activity, compared to the one of S. litura fed on
artificial diet, was not significant [34]. However, any possible effect of the induction of detoxification
enzymes on the alteration of the toxicity of the test compounds were taken into account, as the data were
corrected for the mortality of the untreated control insects using Abbottꢂs formula [35] whenever
required.

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
A known amount of test compounds was dissolved in acetone and then diluted with H2O to obtain
the desired concentrations. Test compounds were suspended in distilled H2O using Triton X-100 LR
spreader (S. D. Fine-Chem. Ltd, India) at 0.1 ml/l. Preliminary screening of the test compounds was
carried out at two high test dosages, i.e., 10000 and 5000 ppm, corresponding to a concentration of ca. 30
and 15 mg/leaf cm2, resp. Three leaf disks were separately dipped in each test soln. for 30 s and dried under
the gentle stream of air under laboratory conditions. Second instar larvae (10 larvae in each replicate),
starved for 3–4 h prior to the bioassay, were transferred individually on treated and control (disks treated
with H2O mixed with acetone/Triton only) leaf disks placed in Petri plates. All treated samples were
maintained at 23ꢁ18, 65ꢁ5% rel. humidity, and a photoperiod of 16 :8 (L :D) in the laboratory.
Mortality was determined 24 and 48 h after the larvae were placed on disks. Larvae not showing
movements when probed with a camel hairbrush were considered dead.
Dose–Response Experiment. Based on the preliminary screening results, promising test compounds
were selected and subjected to a dose-response bioassay. Test compound solns. (six concentrations each)
were prepared as described above to provide dosage in the range of 0 to 30 mg/leaf cm2. Commercial
pesticides, a biological (NeemAzal, 0–6.0 mg /leaf cm2) and a chemical (methyl parathion, 0–1.2 mg/leaf
cm2), commonly applied as S. litura controls, were used as positive controls.
Data Analysis. Mortality data were corrected, whenever required, for control mortality by using
Abbottꢂs formula [35]. The concentration that killed 50% of the population relative to the control (LC50
value) was determined using SPSS 10.0 for Windows 2000 and Excel Microsoft program 3.
REFERENCES
[1] W. J. Hayes, E. R. Laws, ꢃ Handbook of Pesticide Toxicologyꢂ, Academic Press, San Diego, 1991,
Vol. 1.
[2] S. E. Lee, J. E. Kim, H. S. Lee, Agric. Chem. Biotechnol. 2001, 44, 105.
[3] N. J. Armes, J. A. Wightman, D. R. Jadhav, G. V. Ranga Rao, Pestic. Sci. 1997, 50, 240.
[4] M. B. Isman, Crop. Prot. 2000, 19, 603.
[5] M. B. Isman, Annu. Rev. Entomol. 2006, 51, 45.
[6] S. A. M. Abdelgaleil, M. A. Abbassy, A.-S. H. Belal, M. A. A. Abdel Rasoul, Bioresour. Technol.
2008, 99, 5947.
[7] Y.-Q. Liu, L. Yang, Y. Liu, X. Di, H. Xiao, X. Tian, R. Gao, Nat. Prod. Res. 2007, 21, 967.
[8] J. B. Harborne, H. Baxter, ꢃPhytochemical Dictionary, A Handbook of Bioactive Compounds from
Plantsꢂ, Taylor & Francis Ltd, Washington, DC, 1993.
[9] J. L. Torres y Torres, J. P. N. Rosazza, J. Nat. Prod. 2001, 64, 1408.
[10] D. K. Tewary, A. Bhardwaj, A. Sharma, A. K. Sinha, A. Shanker, J. Pest Sci. 2006, 79, 209.
[11] L. A. Hummelbrunner, M. B. Isman, J. Agric. Food Chem. 2001, 49, 715.
[12] S. P. Ngoh, L. E. W. Choo, F. Y. Pang, Y. Huang, M. R. Kini, S. H. Ho, Pestic. Sci., 1998, 54, 261.
[13] D.-H. Kim, Y.-J. Ahn, Pest. Manage. Sci. 2001, 57, 301.
[14] D. Maes, M. E. Riveiro, C. Shayo, C. Davio, S. Debenedetti, N. De Kimpe, Tetrahedron 2008, 64,
4438.
[15] J. Harmatha, J. Nawrot, Entomol. Exp. Appl. 2002, 104, 51.
[16] E.-H. Kim, H.-K. Kim, Y.-J. Ahn, J. Agric. Food Chem. 2003, 51, 885.
[17] M. Della Greca, P. Monaco, A. Pollio, L. Previtera, Phytochemistry 1992, 31, 4119.
[18] Y. Huang, S. H. Ho, R. M. Kini, J. Econ. Entomol. 1999, 92, 676.
[19] J. Poplawski, B. Lozowicka, A. T. Dubis, B. Lachowska, Z. Winiecki, J. Nawrot, Pest Manage. Sci.
2000, 56, 560.
[20] V. Lander, P. Schreier, Flavour Fragrance J. 1991, 6, 21.
[21] F. Camps, A. Conchillo, A. Messeguer, Tetrahedron 1987, 43, 3067.
[22] K. E. Kabir, A. R. Khan, M. A. Mosaddik, J. Appl. Entomol. 2003, 127, 112.
[23] M. B. Isman, A. J. Wan, C. M. Passreiter, Fitoterapia 2001, 72, 65.
[24] C. Park, S.-I. Kim, Y.-J. Ahn, J. Stored Prod. Res. 2003, 39, 333.
[25] C. M. Passreiter, J. Wilson, R. Andersen, M. B. Isman, J. Agric. Food Chem. 2004, 52, 2549.

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
177
[26] C. Regnault-Roger, A. Hamraoui, J. Stored Prod. Res. 1995, 31, 291.
[27] S. G. Lee, S. I. Kim, Y. J. Ahn, J. B. Kim, B. Y. Lee, Pestic. Sci. 1997, 49, 119.
[28] D. Obeng-Ofori, C. Reichmuth, Int. J. Pest Manage. 1997, 43, 89.
[29] B. P. Joshi, A. Sharma, A. K. Sinha, Tetrahedron 2005, 61, 3075.
[30] A. K. Sinha, A. Sharma, A. Swaroop, V. Kumar, Tetrahedron 2007, 63, 1000.
[31] A. K. Sinha, R. Acharya, B. P. Joshi, J. Nat. Prod. 2002, 65, 764.
[32] D. K. Tewary, A. Bhardwaj, A. Shanker, Ind. Crop. Prod. 2005, 22, 241.
[33] S. S. Ghumare, S. N. Mukherjee, J. Insect Behav. 2005, 18, 105.
[34] S. S. Ghumare, R. N. Sharma, S. N. Mukherjee, Zool. Anz. 1993, 230, 95.
[35] W. S. Abbott, J. Econ. Entomol. 1925, 18, 265.
Received December 16, 2008