Screening of dialkoxybenzenes and disubstituted cyclopentene derivatives against the cabbage looper, Trichoplusia ni, for the discovery of new feeding and oviposition deterrents

Article abstract of DOI:10.1021/jf071636d

The antifeedant, oviposition deterrent, and toxic effects of dialkoxybenzene minilibraries and of disubstituted cyclopentene minilibraries (i.e., consisting of four to five compounds) along with their pure constituent compounds were assessed against third instar larvae and adults of the cabbage looper, Trichoplusia ni, in laboratory bioassays in a search for new insect control agents. These compounds mimic naturally occurring bioactive odorants and tastants and are relatively easily prepared from commodity chemicals. Most of these libraries strongly deterred larval feeding, with some exhibiting strong toxic and oviposition deterrent effects as well. Our results suggest some structure-function relationships within these libraries. Replacement of a methyl group with larger alkyl substituents increased the feeding deterrent effects in most cases. The presence of a free hydroxyl group, irrespective of the carbon framework or alkyl substituent, served to reduce feeding deterrent effects in all series of compounds. Further, exceeding a certain group size also generally had a detrimental effect. This information will be useful in designing new insect control agents for agriculture. Some of these libraries and compounds may have potential for development as commercial insecticides.

Full text of DOI:10.1021/jf071636d

J. Agric. Food Chem. 2007, 55, 10323–10330 10323
Screening of Dialkoxybenzenes and Disubstituted
Cyclopentene Derivatives against the Cabbage
Looper, Trichoplusia ni, for the Discovery of New
Feeding and Oviposition Deterrents
YASMIN AKHTAR,‡ MURRAY B. ISMAN,*,‡ PEGGY M. PADURARU,§
†,§
SRINIVAS NAGABANDI,§ RANJEET NAIR,§ AND ERIKA PLETTNER
Faculty of Land and Food Systems, University of British Columbia, Vancouver,
British Columbia, Canada V6T 1Z4, and Department of Chemistry, Simon Fraser University, Burnaby,
British Columbia, Canada V5A 1S6
The antifeedant, oviposition deterrent, and toxic effects of dialkoxybenzene minilibraries and of
disubstituted cyclopentene minilibraries (i.e., consisting of four to five compounds) along with their
pure constituent compounds were assessed against third instar larvae and adults of the cabbage
looper, Trichoplusia ni, in laboratory bioassays in a search for new insect control agents. These
compounds mimic naturally occurring bioactive odorants and tastants and are relatively easily prepared
from commodity chemicals. Most of these libraries strongly deterred larval feeding, with some exhibiting
strong toxic and oviposition deterrent effects as well. Our results suggest some structure–function
relationships within these libraries. Replacement of a methyl group with larger alkyl substituents
increased the feeding deterrent effects in most cases. The presence of a free hydroxyl group,
irrespective of the carbon framework or alkyl substituent, served to reduce feeding deterrent effects
in all series of compounds. Further, exceeding a certain group size also generally had a detrimental
effect. This information will be useful in designing new insect control agents for agriculture. Some of
these libraries and compounds may have potential for development as commercial insecticides.
KEYWORDS: Trichoplusia ni; feeding deterrents; oviposition deterrents; toxicity; dialkoxybenzenes;
disubstituted cyclopentenes
INTRODUCTION
All phytophagous insects that have been investigated respond
behaviorally to some of these compounds, most of which
produce a deterrent response in the insects (3). Reduction or
complete inhibition of feeding has been demonstrated in
Orthoptera, Hemiptera, Coleoptera, larval Lepidoptera, and
larval Hymenoptera (4, 5).
Our knowledge of insect-plant chemical interactions indi-
cates that chemical signals are important behavioral guides for
insects, enabling them to find appropriate host plants for feeding
and oviposition, locate mates, sense the presence of predators,
and even assess the suitability of a host. Insect-plant chemical
interactions in nature are usually very subtle. Most plant
defensive chemicals discourage insect herbivory, either by
deterring feeding and oviposition or by impairing larval growth,
rather than by killing insects outright (1).
Among polyphagous insects, the balance of phagostimulatory
and deterrent inputs is probably the sole determinant of
acceptance or rejection of food, as shown clearly in many
investigations (6, 7) and demonstrated in simplified models (8, 9).
Antifeedants are described as substances that reduce feeding by
an insect acting either peripherally (on gustatory chemoreceptors)
or centrally. They can be found among all of the major classes of
secondary metabolites: alkaloids, phenolics, and terpenoids (2). It
is in the last category that the greatest number and diversity of
antifeedants and the most potent ones have been found (1).
Plant compounds also constitute important sensory cues
mediating oviposition in phytophagous insects. Plant compounds
may act as oviposition stimulants (10) or deterrents (11, 12).
Host plant acceptance by an ovipositing female is mediated by
a balance of sensory inputs from both positive and negative
stimuli received from these compounds (13). The relative
balance between these opposing cues is weighted by the internal
physiological state of the insect, such as egg load (14).
* Author for correspondence. Tel: 604-822-2329. Fax: 604-822-8640.
E-mail: murray.isman@ubc.ca.
The search for insect control agents that have potential use
as crop protectants (insecticides, antifeedants, and growth
inhibitors) often begins with the screening of compounds.
Initially, the test insects can be fed the compound either
† Author for correspondence regarding the chemistry of the com-
pounds presented.
‡ University of British Columbia.
§ Simon Fraser University.
10.1021/jf071636d CCC: $37.00
2007 American Chemical Society
Published on Web 11/20/2007

10324 J. Agric. Food Chem., Vol. 55, No. 25, 2007
Akhtar et al.
Scheme 1. Structures of the Target Compounds Tested in This Study
Scheme 2. Natural Products That Contain Cyclopentane or Cyclopentene
Units
example, at a dietary concentration of 3.0 mM/kg fresh weight,
parthenin reduced larval growth of Heliothis zea by 59% relative
to controls in a chronic feeding bioassay (22) and the related
compound, tetraneurin-A, acted as an antifeedant against sixth
instar larvae of Spodoptera litura (23).
This study will serve as the foundation for more detailed
investigations of the effects of individual lead compounds on
feeding and oviposition behaviors as well as the toxic effects
against T. ni and other agricultural pests. The cabbage looper
is an important pest of cruciferous plants but also attacks several
other crops including lettuce, beets, peas, celery, tomatoes,
certain ornamentals, and many weedy plants (24). Because T.
ni has evolved resistance against many synthetic insecticides
(25) and the microbial insecticide Bacillus thuringiensis (26, 27),
it is very important to develop new methods that could be used
to protect crops in integrated pest management schemes. Here
we have chosen to investigate compounds that mimic naturally
occurring bioactive odorants and tastants and that are relatively
easily prepared from commodity chemicals.
MATERIALS AND METHODS
incorporated into an artificial diet or sprayed on a host plant,
and the effects on insect behavior and development can be
monitored. Once a promising compound has been discovered,
the next step is often to investigate the mode of action. This
kind of information is needed to ensure safety to nontarget
organisms (humans, wildlife, beneficial insects).
Chemicals that inhibit feeding of phytophagous insects may
be an integral part of plant defense itself, conferring some
measure of resistance to insect attack, or they may be applied
to the plant in the same way as other agricultural chemicals
(15), serving as exogenous crop protectants (16). Interest in the
feeding and oviposition deterrent properties of compounds has
arisen both because deterrence is an important mediator of
plant-insect interactions and because it is potentially useful for
manipulating the behavior of crop pests (17).
Plant Material. Cabbage plants (Brassica oleraceae var. Stonehead)
used in the bioassays were routinely grown in plastic pots with a mixture
of sandy loam soil and peat moss (4:1) in a greenhouse at the University
of British Columbia, Vancouver, BC, Canada. Leaves were collected
from cabbage plants that were 5–6 weeks old.
Test Insects. T. ni larvae and moths were obtained from a long
established colony (>50 generations) maintained on an artificial diet,
Velvetbean Caterpillar Diet No. F9796 [Bio-Serv Inc. (Frenchtown,
NJ.)] in the insectary of the University of British Columbia (UBC).
The diet was supplemented with finely ground alfalfa, to improve
acceptability, and vitamins [No. 8045; Bioserv Inc. (Frenchtown,
NJ.)].
Test Compounds. Dialkoxybenzene minilibraries (consisting of four
to five compounds) and pure compounds were synthesized as described
elsewhere (28). Briefly, dialkoxybenzenes were synthesized from the
corresponding dihydroxybenzenes (11a, 11b, or 11c) by monoalkylation
(Scheme 3). The pure monoalkylated compounds were mixed in
equimolar amounts, for the synthesis of minilibraries, and subjected to
a second round of alkylation. Thus, the minilibraries have one alkyl
group constant and the other one variable. Testing the minilibraries of
increasing constant group size and the pure compounds for which R1
) R2, we can infer which general group size and regiochemistry (para,
a; meta, b; ortho, c) is best for a particular bioactivity.
One of the limitations of using plant-derived botanicals is
resource availability. In the present study we have used synthetic
libraries of compounds that can be generated rapidly, in large
amounts and in high purity.
The purpose of the present study was to assess the antifeedant,
oviposition deterrent, and toxic effects of dialkoxybenzene
minilibraries (Scheme 1, 1, 2, and 3) and of disubstituted
cyclopentenes (Scheme 1, 4) against the cabbage looper,
Trichoplusia ni. Low molecular weight phenol derivatives are
important constituents of smoke (18, 19), which is known to
have insect repellent and insecticidal properties (20). Substituted
cyclopentenes can be found in plants, in several contexts. For
example, chaulmoogric acid (Scheme 2, 5a) and its homologue
hydnocarpic acid (5b) are antibacterials from the seeds of
Hydnocarpus wightiana. Iridanes (Scheme 2, 6a), iridoids (6b),
and secoiridoids (6c) feature cyclopentane and cyclopentene
units prominently. Also, many plant sesquiterpenes, such as
valerenic acid (Scheme 2, 7), parthenin (8), tetraneurin-A (9),
and matricin (10) contain substituted cyclopentene units (21).
Some of these compounds possess insecticidal properties. For
The cyclopentene compounds were synthesized as shown in Scheme
4 and as described in detail elsewhere (29). Briefly, diol 15 can be
obtained from cyclopentadiene 13 and glyoxylic acid 14 in a four-step
procedure. For compounds with R1 ) R2, the diol is dialkylated by
treatment with KH in dimethylformamide, followed by the appropriate
alkyl halide. Production of compounds with different R groups
necessitated protection of the primary alcohol with a tert-butyldimeth-
ylsilyl (TBDMS) group. The resulting secondary alcohol 16 was
alkylated to 17, which was deprotected to furnish alcohol 18. This
compound was then alkylated to give a variant of 4, with R1 ) isopentyl
and R2 ) n-nonyl. This higher molecular mass compound was included
in the present study as a representative of a more hydrophobic, sterically
hindered and less volatile compound.
General Testing Procedure. Initially, all compounds were tested
at 50 µg/cm2 in feeding deterrent bioassays. Those compounds that

New Insect Control Agents
J. Agric. Food Chem., Vol. 55, No. 25, 2007 10325
Scheme 3. Optimized Synthesis of Dialkoxybenzenes
Scheme 4. Overview of the Synthesis of Cis-Substituted Racemic Cyclopentene Odorants for This Studya
a Abbreviations in order of appearance: TBDMSCl ) tert-butyl dimethyldiyl chloride; NEt3 ) triethylamine; TBAF ) tert-butylammonium fluoride; THF )
tetrahydrofuran; R-X ) alkyl halide (bromide or iodide); DMF ) dimethylformamide.
exhibited >50% feeding deterrence at this concentration were subjected
to further testing for oviposition deterrent effects and contact toxicity
at 0.25% of the test substance. For compounds exhibiting g50% values
for feeding deterrence and g70% mortality by contact, DC50 (concen-
tration causing 50% feeding deterrence compared with the control) and
LC50 (concentration causing 50% mortality compared with the control)
were determined, respectively, based on bioassays involving a minimum
of four concentrations (3.12–25 µg/cm2).
Comparison of Toxicity, Oviposition, and Feeding Deterrence
Values. The mortality of each test material was plotted against its respective
oviposition deterrence value (determined at 0.25%) to explore the relationship
between the two bioassays using correlation analysis. Similarly, feeding
deterrence was plotted against oviposition deterrence and mortality.
Data Analysis. Feeding deterrence data (percent) for initial screening
concentration were analyzed by analysis of variance (ANOVA) after
arcsin transformation using statistics software (35). Where significant
F values were found, Tukey’s HSD multiple comparison tests were
used to test for significant differences between individual treatments.
Feeding Deterrent Bioassays. Leaf disk choice bioassays (30, 31)
were conducted to determine feeding deterrent effects of the synthetic
compounds using freshly molted third instar larvae starved for 4–5 h
prior to each bioassay. Larvae were given the choice of feeding on
two leaf disks, one treated with 10 µL of a solution of the test substance
painted on each side and the other treated with a carrier solvent alone.
The number of larvae was 25 per treatment. Bioassays were terminated
when ∼50% of the control disk had been eaten (normally 3–5 h).
RESULTS
p-Dialkoxybenzene Libraries, Pure Compounds, and 1-Hy-
droxy-4-alkoxy Compounds. All six of the p-dialkoxybenzene
libraries and five individual compounds exhibited >50% feeding
deterrence at the initial screening concentration (50 µg/cm2) and,
therefore, were subjected to further testing (Table 1) against
third instar T. ni larvae for toxic and oviposition deterrent effects.
The response of the larvae to the initial screening concentration
Areas of control and treated leaf disks consumed by the larvae were
measured using Scion Image software, and feeding deterrence was
calculated (31) using the formula [(C - T)/(C + T)] × 100, where C
and T are areas consumed of the control and treated leaf disks,
respectively.
varied significantly in most cases (one-way ANOVA; F16405
9.6, p < 0.0001).
)
Oviposition Deterrent Bioassays. Oviposition response of T. ni
moths was measured according to our previously described oviposition
choice bioassay (32, 33). T. ni larvae were reared on normal diet from
neonates (<24 h old) until pupation. Pupae were sexed and put in
separate plastic containers until emergence. After eclosion, pairs of
moths (one male and one female) were introduced into each cage with
a control and a treated cabbage leaf. Pairs of moths (n ) 25) were
used per treatment. Each leaf (approximately 100–110 cm2) was sprayed
with 0.5 mL of MeOH or a methanolic solution of the test chemical
on each side. Eggs were counted on each cabbage leaf after 48 h. ODI
(oviposition deterrence index) was calculated using the formula ODI
) [(C - T)/(C + T)] × 100, where C and T are the numbers of eggs
laid on the control and treated leaf disks, respectively (32, 33).
Contact Toxicity Bioassays. Mortality was determined 24 h after
spraying larvae directly with test solutions (34). Third instar T. ni larvae
were sprayed in 90 mm × 15 mm Petri dishes (Falcon) lined with
Fisher Scientific filter paper (90 mm diameter). Small plastic hand
spraying bottles (50 mL capacity) were used. Larvae were then
transferred to Petri dishes (90 mm × 15 mm) with a small piece of
artificial diet. Each Petri dish contained 10 larvae. Three replicates,
each consisting of 10 larvae, were used per treatment.
Feeding Deterrent Effects. 1-Isopentyloxy-4-alkoxybenzene
had the lowest DC50 value (8.5 µg/cm2) followed by 1-butyloxy-
4-alkoxybenzene (14.5 µg/cm2) and 1-allyloxy-4-isopentoxy-
benzene (15.7 µg/cm2) (Table 1). 1-Hydroxy-4-methoxybenzene
and 1-hydroxy-4-propoxybenzene acted as feeding stimulants
at the screening concentration. 1-Hydroxy-4-ethoxybenzene (a
precursor to diethyl and the ethyl minilibrary) was a weak
feeding deterrent.
Toxic Effects. 1,4-Diethoxybenzene and the 1-ethoxy-4-
alkoxybenzene library were the most toxic (Table 1) at 0.25%
(LC50 values were 0.03% for both) followed by 1-butyloxy-4-
alkoxybenzene and 1-propoxy-4-alkoxybenzene. This high
specificity for the ethyl-substituted compounds is remarkable,
because mortality was <25% for other members in the p-
dialkoxybenzene series.
OViposition Deterrent Effects. 1,4-Diethoxybenzene, the
1-ethoxy-4-alkoxybenzene library, and the 1-butyloxy-4-alkoxy-
benzene library showed the strongest oviposition deterrent

10326 J. Agric. Food Chem., Vol. 55, No. 25, 2007
Akhtar et al.
Table 1. Bioactivities of 1,4-Dialkoxybenzene Libraries and Analogues against Third Instar T. ni Larvaea
DC50, µg/cm2
FD (%), mean ( SE
mortality (%) OD (%), mean ( SE
compound
1,4-dimethoxybenzene
1,4-diethoxybenzene
1,4-dipropoxybenzene
1,4-diisopentoxybenzene
1,4-diallyloxybenzene
Me library (1-methoxy-4-alkoxybenzene)
Et library (1-ethoxy-4-alkoxybenzene)
Pr library (1-propoxy-4-alkoxybenzene)
Bu library (1-butyloxy-4-alkoxybenzene)
iPent library (1-isopentyloxy-4-alkoxybenzene C5H11
allyl small library (1-allyloxy-4-alkoxybenzene) C3H7
1-allyloxy-4-butoxybenzene
1-allyloxy-4-isopentoxybenzene
1-hydroxy-4-methoxybenzene
1-hydroxy-4-ethoxybenzenef
1-hydroxy-4-propoxybenzene
1-hydroxy-4-isopentoxybenzene
R1
R2
(n ) 25)
(r2)b (n ) 25) (n ) 3 × 10)
(n ) 25–33)
c
-
CH3
CH3
9.9 ( 18.0cd
80.8 ( 11.3b
96.0 ( 2.8a
-
-
C2H5
C3H7
C5H11
C3H5
CH3
C2H5
C3H7
C4H9
C2H5
C3H7
C5H11
C3H5
25.9 (0.91)
20.3 (0.99)
-
100.0d
22.6
-
74.7 ( 13.3a
11.8 ( 13.1b
-
44.0 ( 18.3abc
96.9 ( 3.6a
23.9 (0.97)
34.6 (0.90)
23.4 (0.99)
39.7 (0.94)
14.5 (0.83)
5.8 (0.85)
27.9 (0.93)
22.6 (0.90)
15.7 (0.90)
-
16.0
23.3
96.8e
53.1
58.1
18.8
10.0
10.0
7.0
14.1 ( 13.1b
6.0 ( 5.1b
56.6 ( 16.9ab
9.6 ( 13.8b
50.1 ( 14.5ab
22.9 ( 14.2ab
5.4 ( 13.9b
16.8 ( 13.8ab
18.8 ( 14.3ab
-
CH3, C2H5, C3H7, C5H11
CH3, C2H5, C3H7, C5H11
CH3, C2H5, C3H7, C5H11
CH3, C2H5, C3H7, C5H11
CH3, C2H5, C3H7, C5H11
CH3, C2H5C3H7
C4H9
C5H11
80.2 ( 9.8b
90.7 ( 10.2a
53.4 ( 15.8abc
83.4 ( 9.7b
100.0 ( 0.0a
82.4 ( 10.7b
84.0 ( 10.7b
75.1 ( 12.7abc
-26.5 ( 18.6d
29.8 ( 18.2bcd
-28.0 ( 18.4d
29.5 ( 16.6bcd
C3H7
C3H7
H
CH3
C2H5
C3H7
C5H11
-
H
H
H
-
-
-
-
-
-
-
-
-
a Feeding deterrent (FD) effects (mean ( SE) at 50 µg/cm2 are expressed in %. DC50s (concentrations causing 50% feeding deterrence compared with the control)
were calculated for samples showing >50% feeding deterrence in initial screening (g50 µg/cm2) using Excel; linear regression analysis was conducted for all dose-response
experimental data. Mortality and oviposition deterrent effects were determined at 0.25% for samples showing >50% feeding deterrence in initial screening. LC50 (concentrations
causing 50% mortality compared with the control) was calculated for test compounds exhibiting g70% mortality at 0.25%. Means followed by the same letters within a
column do not differ significantly (Tukey’s test, p < 0.05). b Coefficient of determination. c Not tested. d LC50 ) 0.03%. e LC50 ) 0.03%. f Precursor to diethyl and the ethyl
minilibrary.
Table 2. Bioactivities of 1,3-Dialkoxybenzene Libraries and Analogues against Third Instar T. ni Larvaea
DC50, µg/cm2
FD (%), mean ( SE
(n ) 25)
mortality (%)
OD (%), mean ( SE
compound
1,3-dimethoxybenzene
1,3-diethoxybenzene
1,3-dipropoxybenzene
1,3-diisopentoxybenzene
Me library (1-methoxy-3-alkoxybenzene)
Et library (1-ethoxy-3-alkoxybenzene)
Pr library (1-propoxy-3-alkoxybenzene)
Bu library (1-butyloxy-3-alkoxybenzene)
R1
R2
(r2)b (n ) 25) (n ) 3 × 10) (n ) 25–33)
c
-
CH3
CH3
20.4 ( 19.1bcde
-
-
C2H5
C3H7
C5H11
CH3
C2H5
C3H7,
C4H9,
C2H5
C3H7
C5H11
89.4 ( 8.3ab
28.7 (0.85)
26.8 (0.79)
-
36.7
76.7d
-
3.4 ( 14.1b
33.0 ( 14.6ab
-
96.9 ( 3.1a
-12.2 ( 18.8de
54.8 ( 16.6abcd
69.8 ( 13.8abc
98.0 ( 1.5a
CH3, C2H5, C3H7, C5H11
CH3, C2H5, C3H7, C5H11
CH3, C2H5, C3H7, C5H11
CH3, C2H5, C3H7, C5H11
CH3, C2H5, C3H7, C5H11
CH3
61.5 (0.94)
26.5 (0.98)
21.5 (0.96)
14.4 (0.85)
19.8 (0.96)
-
16.7
20.0
50.0
30.0
20.8
-
70.2 ( 17.3a
14.3 ( 13.5ab
2.7 ( 14.2b
25.9 ( 13.9ab
35.7 ( 13.4ab
-
84.1 ( 8.9ab
iPent library (1-isopentyloxy-3-alkoxybenzene C5H11
82.6 ( 12.0ab
44.7 ( 17.5abcde
-18.7 ( 17.8e
4.0 ( 20.4cde
-5.8 ( 18.7de
1-hydroxy-3-methoxybenzene
1-hydroxy-3-ethoxybenzene
1-hydroxy-3-propoxybenzene
1-hydroxy-3-isopentoxybenzene
H
H
H
H
C2H5
C3H7
C5H11
-
-
-
-
-
-
-
-
-
a Feeding deterrent (FD) effects (mean ( SE) at 50 µg/cm2 are expressed in %. DC50s (concentrations causing 50% feeding deterrence compared with the control)
were calculated for samples showing >50% feeding deterrence in initial screening concentration (g50 µg/cm2) using Excel; linear regression analysis was conducted for
all dose-response experimental data. Mortality and oviposition deterrent effects were determined at 0.25% for samples showing >50% feeding deterrence in initial screening.
LC50 (concentrations causing 50% mortality compared with the control) was calculated for test compounds exhibiting g70% mortality at 0.25%. Means followed by the same
letters within a column do not differ significantly (Tukey’s test, p < 0.05). b Coefficient of determination. c Not tested. d LC50 ) 0.16%.
effects (74.7%, 56.6%, and 50.1%, respectively) when tested
at 0.25% (Table 1). Other members in the group demonstrated
weak oviposition deterrent effects (<23%). Responses of moths
varied significantly in most cases (one-way ANOVA; F10304
2.8, p < 0.003).
m-Dialkoxybenzene Libraries, Pure Compounds, and
1-Hydroxy-3-alkoxy Compounds. All five of the m-dialkoxy-
benzene libraries and two pure compounds exhibited >50%
feeding deterrence in initial screening (50 µg/cm2) and therefore
were subjected to further testing (Table 2). The response of
the larvae to initial screening concentration varied significantly
in most cases (one-way ANOVA; F12307 ) 8.6, p < 0.0001).
Feeding Deterrent Effects. The 1-butoxy-3-alkoxybenzene
library had the lowest DC50 value (14.4 µg/cm2) followed by
the 1-isopentoxy-3-alkoxybenzene library (DC50 ) 19.8 µg/
cm2). Three compounds acted as feeding stimulants to third
instar T. ni larvae.
Toxic Effects. 1,3-Dipropoxybenzene was the most toxic
(Table 2) at 0.25% and had a LC50 value of 0.16% followed
by the 1-propoxy-3-alkoxybenzene library (50% mortality).
)

New Insect Control Agents
J. Agric. Food Chem., Vol. 55, No. 25, 2007 10327
Table 3. Bioactivities of 1,2-Dialkoxybenzene Libraries and Analogues against Third Instar T. ni Larvaea
DC50, µg/cm2
FD (%), mean ( SE
mortality (%) OD (%), mean ( SE
compound
1,2-dimethoxybenzene
1,2-diethoxybenzene
1,2-dipropoxybenzene
1,2-dibutoxybenzene
1,2-diisopentoxybenzene
1,2-diallyloxybenzene
Me library (1-methoxy-2-alkoxybenzene)
Et library (1-ethoxy-2-alkoxybenzene)
Pr library (1-propoxy-2-alkoxybenzene)
Bu library (1-butoxy-2-alkoxybenzene)
R1
R2
(n ) 25)
(r2)b (n ) 25) (n ) 3 × 10)
(n ) 25–33)
c
CH3
CH3
26.0 ( 17.6abcd
-2.1 ( 18.6bcd
26.2 ( 17.6abcd
56.4 ( 13.8abcd
69.9 ( 11.5abc
70.4 ( 13.2abc
23.5 ( 15.9abcd
71.0 ( 10.6ab
100.0 ( 0.0a
-
-
-
-
-
-
C2H5
C3H7
C4H9
C5H11
C3H5
CH3
C2H5
C3H7
C4H9
C2H5
C3H7
C4H9
C5H11
C3H5
43.8 (0.90)
19.6 (0.96)
22.4 (0.99)
-
10.0
40.0
20.0
-
19.6 ( 14.4a
11.5 ( 13.8a
15.0 ( 16.6a
-
CH3, C2H5, C3H7, C4H9, C5H11
CH3, C2H5, C3H7, C4H9, C5H11
CH3, C2H5, C3H7, C4H9, C5H11
CH3, C2H5, C3H7, C4H9, C5H11
CH3, C2H5, C3H7, C4H9, C5H11
CH3, C2H5, C3H7, C4H9, C5H11
24.1 (0.95)
19.4 (0.89)
16.8 (0.90)
32.5 (0.90)
30.0 (0.90)
-
6.7
13.8
6.7
10.0
23.3
-
28.7 ( 16.4a
31.7 ( 17.4a
28.7 ( 16.7a
29.4 ( 16.7a
66.7 ( 16.9a
-
98.0 ( 1.9a
iPent library (1-isopentoxy-2-alkoxybenzene) C5H11
66.9 ( 12.7abc
67.8 ( 13.5abc
31.1 ( 15.5abcd
-7.5 ( 19.8cd
12.0 ( 20.3bcd
-5.3 ( 19.8bcd
-12.0 ( 20.0e
allyl library (1-allyloxy-2-alkoxybenzene)
1-hydroxy-2-allyloxybenzene
1-hydroxy-2-methoxybenzene
1-hydroxy-2-butoxybenzene
1-hydroxy-2-ethoxybenzene
1-hydroxy-2-propoxybenzene
C3H7
H
H
H
H
H
CH3
-
-
-
C4H9
C2H5
C3H7
-
-
-
-
-
-
-
-
-
a Feeding deterrent (FD) effects (mean ( SE) at 50 µg/cm2 are expressed in %. DC50s (concentrations causing 50% feeding deterrence compared with the control)
were calculated for samples showing >50% feeding deterrency in initial screening (g50 µg/cm2) using Excel; linear regression analysis was conducted for all dose-response
experimental data. Mortality and oviposition deterrent effects were determined at 0.25% for samples showing >50% feeding deterrency in initial screening. Means followed
by the same letters within a column do not differ significantly (Tukey’s test, p < 0.05). b Coefficient of determination. c Not tested.
Table 4. Bioactivities of Cyclopentene Ether Libraries and Analogues
OViposition Deterrent Effects. The 1-methoxy-3-alkoxyben-
against Third Instar T. ni Larvaea
zene library demonstrated the strongest oviposition deterrent
effect (70.2%) followed by the 1-isopentyl-3-alkoxybenzene
library (35.7%) (Table 2) when tested at 0.25%. Responses of
moths varied significantly in most cases (one-way ANOVA;
F6199 ) 2.19, p < 0.04).
o-Dialkoxybenzene Libraries, Pure Compounds, and 1-Hy-
droxy-2-alkoxy Compounds. Six o-dialkoxybenzene libraries
and three individual compounds exhibited >50% feeding
deterrence in initial testing and therefore were subjected to
further testing (Table 3) as explained above. The response of
the larvae to initial screening concentration varied significantly
in most cases (one-way ANOVA; F16401 ) 5.4, p < 0.0001).
Feeding Deterrent Effects. The 1-butoxy-2-alkoxybenzene
library had the lowest DC50 value (16.8 µg/cm2) followed by
the 1-propoxy-2-alkoxybenzene library (DC50 ) 19.4 µg/
a Feeding deterrent (FD) effects (mean ( SE) at 50 µg/cm2 are expressed in
%. DC50s (concentrations causing 50% feeding deterrence compared with the
control) were calculated for samples showing >50% feeding deterrence in initial
screening concentration (g50 µg/cm2) using Excel; linear regression analysis was
conducted for all dose-response experimental data. Mortality and oviposition
deterrent effects were determined at 0.25% for samples showing >50% feeding
deterrence in initial screening. Means followed by the same letters within a column
do not differ significantly (Tukey’s test, p < 0.05). *, coefficient of determination.
-, not tested.
cm2).
Toxic Effects. None of the o-dialkoxybenzene libraries or pure
compounds caused >40% mortality at 0.25% (Table 3).
OViposition Deterrent Effects. The 1-allyloxy-2-alkoxyben-
zene library demonstrated strong oviposition deterrent activity
(66.7%) at 0.25%. All other libraries and compounds had only
modest oviposition deterrent activities (Table 3) that were not
statistically significant (one-way ANOVA; F7205 ) 1.07, p )
0.38).
Cyclopentene Ethers 4. Three of six cyclopentene ethers
exhibited >50% feeding deterrence in initial screening and
therefore were subjected to further testing (Table 4). The
response of the larvae to initial screening concentration varied
significantly in most cases (one-way ANOVA; F8216 ) 4.6, p
< 0.0001).
Feeding Deterrent Effects. The dibutyl-, dipropyl-, and
diisopentylcyclopentene ethers demonstrated similar feeding
deterrent effects with DC50 values <35 µg/cm2. Interestingly,
the most volatile (smallest) congeners (dimethyl and diethyl)
were not deterrent. Deterrence was high in the medium
molecular size range and dropped off for the high molecular
mass nonyl/isopentyl ether.
Toxic Effects. The diisopentylcyclopentene ether was the most
toxic, exhibiting 44.8% mortality at 0.25% (Table 4).
OViposition Deterrent Effects. Although the diisopentylcy-
clopentene ether demonstrated the strongest oviposition deterrent
effects (47%) followed by the dipropylcyclopentene ether
(41.8%) and the dibutylcyclopentene ether (30%) when tested
at 0.25% (Table 4), they were not statistically significant (one-
way ANOVA; F275 ) 0.69, p ) 0.69).
Comparison of Toxicity, Oviposition, and Feeding Deter-
rence Values. Toxicity and oviposition deterrence: There was
a very slight positive correlation (y ) 0.33x + 18.0, R2 ) 0.18)
although there were some strong deterrents that were not toxic
in the data set.

10328 J. Agric. Food Chem., Vol. 55, No. 25, 2007
Akhtar et al.
Table 5. Summary of the Strongest Feeding and Oviposition Deterrents,
Grouped According to Their Contact Toxicity to Third Instar T. ni Larvaea
the para libraries and two of the meta libraries. There are two
phenomena that contribute to the deleterious effects of a
compound on an insect. A compound may have adverse effects
because it is not acceptable (deterrent) and the insect is
essentially deprived of food, or it may have postingestive or
toxic effects (36). Deterrence is usually associated with toxicity
under ecological conditions, meaning that either the deterrent
itself is toxic or that it is associated with a toxin (37). However,
an unequivocal link between deterrence and toxicity has rarely
been demonstrated (38). Azadirachtin is one example of a
compound possessing both deterrent and toxic properties
simultaneously. Our study suggests a lack of a lack of correlation
between feeding deterrence and toxic properties for the minili-
braries and compounds.
In addition to having feeding deterrent and toxic properties
some of these libraries (three para libraries, one meta library,
one ortho library, and one cyclopentene ether library) also
possess strong oviposition deterrent effects against T. ni moths.
All others possess medium to low oviposition deterrent effects.
Our study indicates a lack of correlation between feeding deterrence
and oviposition deterrence as reported by others (39, 40). However,
some plant extracts and pure allelochemicals may possess both
feeding and oviposition deterrent effects (32, 33, 41, 42). There
was a positive correlation between toxicity and oviposition
deterrence in the minilibraries and compounds. Seed and leaf
extracts of Virex negundo possess both toxic and oviposition
deterrence effects against Plutella xylostella (41). Therefore, it
can be assumed that toxicity plays an important role in predicting
host-plant choice and not the deterrent properties of chemicals
in a plant. The presence of toxic compounds usually signals
unsuitability of a plant, resulting in rejection behavior that
prevents the female moth from ovipositing and protecting her
offspring from the toxins that are likely to be encountered in
the plant.
a Compounds with >80% mortality were considered toxic, and compounds with
<25% mortality were considered of low toxicity. See Tables 1-3 for the activity
data. Strong feeding deterrency, >80%; moderate feeding deterrency, >60%. Strong
oviposition deterrency, >50%; moderate, >25%; weak, >10%; none, <10%.
Feeding deterrence and oviposition deterrence: There was no
correlation (y ) -0.26x + 48.0, R2 ) 0.04) within the data
set.
Feeding deterrence and toxicity: There was no correlation (y
Using minilibraries in insect behavioral screening studies has
two advantages. First, minilibraries would prevent the insects
from developing resistance or habituation as they contain
mixtures of compounds (43). Plant defense chemicals that
exhibit more than one mode of action are most suitable for crop
protection (44), constituting a “multichemical defense” against
a variety of potential herbivores. Second, using some individual
compounds and minilibraries that have been systematically
varied and have some overlapping compounds, we can obtain
some structure–function data, without having to test every
compound individually. This approach minimizes assay times
while still allowing the exploration of many compounds.
) 0.07x + 77.0, R2 ) 0.01) within the data set.
DISCUSSION
Our results have demonstrated that p-dialkoxybenzene librar-
ies and related pure compounds were the most active, with 65%
of the members exhibiting feeding deterrence in the range of
53–100% at 50 µg/cm2. This was followed by the m-dialkoxy-
benzene libraries and related individual compounds. Over half
of the members exhibited feeding deterrence in the range of
55–98%. Finally, half of the members in the o-dialkoxybenzene
libraries and related individual compounds exhibited feeding
deterrence in the range of 57–100%. The cyclopentene ethers
and analogues were the least active group, with one-third of
the members exhibiting feeding deterrence in the range of 59–76%
at the initial screening concentration. The presence of a free
hydroxyl group, irrespective of the carbon framework or alkyl
substituent, served to reduce feeding deterrent effects in all series
of compounds. Similarly, the presence of methyl groups,
irrespective of the carbon framework or position, also reduced
feeding deterrent effects in all libraries. Replacement of a methyl
group with larger alkyl substituents increased the feeding
deterrent effects in most cases. Further, exceeding a certain
group size (butyl, isopentyl) also generally had a detrimental
effect. Thus, our best oviposition and feeding deterrent lead
compounds from this study tend to be compounds with
intermediate (propyl) group sizes.
Our results suggest both antifeedant activity, causing a
reduction in food consumption, for most of the libraries tested
and contact toxicity for some. Both actions can reduce growth
and increase development time and likely expose herbivores to
increased mortality in the field as a result of biotic and abiotic
factors (45). Our study strongly suggests that some of these
libraries and individual compounds have potential for develop-
ment as commercial insecticides. However, their impact on
beneficial organisms and environmental fates needs to be
determined.
Future studies will focus on detailed investigations of the
effects of selected libraries and analogues on feeding and
oviposition behaviors as well as the toxic effects against other
agricultural pests in the laboratory and greenhouse. Selected
libraries might also be useful in “push-pull” or stimulodeterrent
diversionary strategies (46) for crop protection based on their
feeding/oviposition deterrent and feeding stimulant properties.
In this case the “push” can come from an antifeedant (Table 5)
It is interesting to note that most of these libraries have strong
feeding deterrent effects, but only a few have strong deterrent
as well as toxic properties. This is especially true for four of

New Insect Control Agents
J. Agric. Food Chem., Vol. 55, No. 25, 2007 10329
(13) Huang, X. P.; Renwick, J. A. A. Differential selection of host
plants by two Pieris species: The role of oviposition stimulants
and deterrents. Entomol. Exp. Appl. 1993, 68, 59–69.
(14) Minkenberg, O. P. J. M.; Tatar, M.; Rossenheim, J. A. Egg load
as a major source of variability in insect foraging and oviposition
behavior. Oikos 1992, 65, 134–142.
(15) Chapman, R. F. Chemical inhibition of feeding by phytophagous
insects, a review. Bull. Entomol. Res. 1974, 64, 339–363.
(16) Jermy, T. The role of rejective stimuli in the host selection of
phytophagous insects. Proc. Int. Congr. Entomol., 12th 1965.
(17) Usher, B. F.; Bernays, E. F.; Berbehenn, R. V. Antifeedant tests
with larvae of Pseudaletia unipuncta: variability of behavioral
response. Entomol. Exp. Appl. 1988, 48, 203–212.
(18) Guillen, M. D.; Manzanos, M. J. Characterization of smoke
flavourings obtained from mixtures of oak (Quercus sp.) wood
and aromatic plants (Thymus Vulgaris L. and SalVia laVandulifolia
Vahl.). FlaVour Fragrance J. 2005, 20, 676–685.
(19) Bortolomeazzi, R.; Sebastianutto, N.; Toniolo, R.; Pizzariello, A.
Comparative evaluation of the antioxidant capacity of smoke
flavouring phenols by crocin bleaching inhibition, DPPH radical
scavenging and oxidation potential. Food Chem. 2006, 100, 1481–
1489.
or an oviposition deterrent [1,4-diethoxybenzene (Table 1) and
many others (Tables 2, 3, and 4)] applied to the crop needing
protection, while the “pull” can come from an attractant
[1-hydroxy-4-propoxybenzene, 1,3-diisopentoxybenzene (Tables
1 and 5), and many others (Tables 2, 3, and 4)] applied to an
adjacent trap crop or trap rows of the main crop. As a result of
this behavioral manipulation, a mobile adult insect would likely
abandon an otherwise suitable host plant for feeding and
oviposition and move to another potential host plant some
distance away.
Since our minilibraries and compounds possess one or more
deterrence or toxicity activities, we could eventually utilize them
in a more targeted manner than conventional insecticides,
rendering our lead compounds more effective as crop pro-
tectants. To protect the tender, more valuable upper leaves of a
crop, these can be sprayed with an antifeedant (Table 5) and
the lower parts, where leaf damage can be tolerated, with a toxic
compound (Table 5) (47). Under this treatment regime, the
target insect will be driven to the lower leaves and suffer
mortality after coming in contact with the toxic compound. We
can choose a highly toxic compound or a less toxic compound
depending upon the situation.
(20) Murugan, K.; Murugan, P.; Noortheen, A. Larvicidal and repellent
potential of Albizzia amara Boivin and Ocimum basilicum Linn
against dengue vector, Aedes aegypti (Insecta: Diptera: Culicidae).
Bioresour. Technol. 2006, 98, 198–201.
ACKNOWLEDGMENT
(21) Dewick, P. M. Medicinal Natural Products: A Biosynthetic
Approach, 2nd ed.; John Wiley & Sons: Chichester, U.K., 2002.
(22) Isman, M. B.; Rodriguez, E. Larval growth inhibitors from species
of Parthenium (Asteraceae). Phytochemistry 1983, 22, 2709–2713.
(23) Datta, S.; Saxena, D. B. Pesticidal properties of parthenin (from
Parthenium hysterophorus) and related compounds. Pest Manage.
Sci. 2001, 57, 95–101.
We thank Nancy Brard for insect rearing and Richard Popoff
and Johanna Griscti for technical assistance. We also thank
Krilen Ramanaidu for assistance with bioassays.
LITERATURE CITED
(24) Davidson, R. H.; Lyon, W. F. Insect Pests of Farm, Garden, and
Orchard; John Wiley & Sons: New York, 1999.
(25) Mota-Sanchez, D.; Bills, P. S.; Whalon, M. E. Arthropod
Resistance to Pesticides: Status and Overview. In Pesticides in
Agriculture and the EnVironment; Marcel Dekker: New York,
2002; pp 241–272.
(26) Janmaat, A. F.; Myers, J. H. The cost of resistance to Bacillus
thuringiensis varies with the host plant of Trichoplusia ni. Proc.
R. Soc. London, Ser. B 2005, 272, 1031–1038.
(27) Wang, P.; Zhao, J.-Z.; Rodrigo-Sim´ on, A.; Kain, W.; Janmaat,
A. F.; Shelton, A. M.; Ferré, J.; Myers, J. Mechanism of resistance
to Bacillus thuringiensis toxin Cry1Ac in a Greenhouse population
of the cabbage looper Trichoplusia ni. Appl. EnViron. Microbiol.
2007, 73, 1199–1207.
(1) Isman, M. B. Insect antifeedants. Pestic. Outlook 2002, 13, 129–
176.
(2) Frazier, J. L. The perception of plant allelochemicals that inhibit
feeding. In Molecular Aspects of Insect-Plant Associations;
Brattsten, L. B., Ahmad, S. Eds.; Plenum Press: New York, 1986;
pp 1–42.
(3) Chapman, R. F. Contact chemoreception in feeding by phytopha-
gous insects. Annu. ReV. Entomol. 2003, 48, 455–484.
(4) Chyb, S.; Eichenseer, H.; Mullin, C. A.; Frazier, J. L. Identification
of sensilla involved in taste mediation in adult western corn
rootworm. J. Chem. Ecol. 1995, 21, 313–329.
(5) Schoonhoven, L. M.; van Loon, J. J. A. An inventory of taste in
caterpillars, each species its own key. Acta Zool. Acad. Sci. Hung.
2002, 48, 215–263.
(6) Simmonds, M. S. J.; Blaney, W. M. Gustatory codes in lepi-
dopterous larvae. Ym. Biol. Hung. 1990, 39, 17–27.
(7) Dethier, V. G. Electrophysiological studies of gustation in
lepidopterous larvae 11. Taste spectra in relation to food-plant
discrimination. Z. Vgl. Physiol. 1973, 82, 103–134.
(8) Blom, F. Sensory activity and food intake, a study of input-output
relationships in two phytophagous insects. Neth. J. Zool. 1978,
28, 277–340.
(9) Hirao, T.; Arai, N. Electrophysiological studies on gustatory
responses in common cutworm larvae, Spodoptera litura. J. Appl.
Entomol. Zool. 1993, 37, 129–136.
(10) Renwick, J. A. A.; Radke, C. D.; Sachdev-Gupta, K.; Stadler, E.
Leaf surface chemicals stimulating oviposition by Pieris rapae
(Lepidoptera, Pieridae) on cabbage. Chemoecology 1992, 3, 33–
38.
(28) Paduraru, P. M.; Popoff, R. T. W.; Nair, R.; Gries, R.; Gries, G.;
Plettner, E. Synthsis of substituted alkoxy benzene mini-libraries,
for the discovery of new insect olfaction or gustation inhibitors.
J. Comb. Chem. (in press).
(29) Nagabandi, S.; Plettner, E. (unpublished data).
(30) Akhtar, Y.; Rankin, C. A.; Isman, M. B. Decreased response to
feeding deterrents following prolonged exposure in the larvae of
a generalist herbivore Trichoplusia ni (Lepidoptera: Noctuidae).
J. Insect BehaV. 2003, 16, 811–831.
(31) Akhtar, Y.; Isman, M. B. Comparative growth inhibitory and
antifeedant effects of plant extracts and pure allelochemicals on
four phytophagous insect species. J. Appl. Entomol. 2004, 128,
32–38.
(32) Akhtar, Y.; Isman, M. B. Larval exposure to oviposition deterrents
alters subsequent oviposition behaviour in generalist Trichoplusia
ni and specialist, Plutella xylostella moths. J. Chem. Ecol. 2003,
29, 1853–1870.
(33) Chow, J. K.; Akhtar, Y.; Isman, M. B. The effects of larval
experience with a complex plant latex on subsequent feeding and
oviposition by the cabbage looper moth Trichoplusia ni (Lepi-
doptera: Noctuidae). Chemoecology 2005, 15, 129–133.
(34) Isman, M. B. Problems and opportunities for the commercialization
of insecticides. In Biopesticides of Plant Origin; Regnault-Roger,
(11) Sachdev-Gupta, K.; Renwick, J. A. A.; Radke, C. D. Isolation
and identification of oviposition deterrents to the cabbage butterfly,
Pieris rapae, from Erysimum cheiranthoides. J. Chem. Ecol. 1990,
1059, 1067.
(12) Dimock, M. B.; Renwick, J. A. A.; Radke, C. D.; Sachdev-Gupta,
K. Chemical constituents of an unacceptable crucifer, Erysimum
cheiranthoides, deter feeding by Pieris rapae. J. Chem. Ecol. 1991,
17, 525–533.

10330 J. Agric. Food Chem., Vol. 55, No. 25, 2007
Akhtar et al.
C., Philogene, B. J. R., Vincent, R., Eds.; Lavoisier: Paris, 2005;
pp 283–291.
lowtail butterfly, Papilio xuthus L. J. Chem. Ecol. 2004, 30, 287–
301.
(35) Statistix 7. Analytical Software, Statistix 7 for Windows 95, 98,
NT, 2000. Analytical Software, Tallahassee.
(36) Bernays, E. A.; Chapman, R. F. Plant secondary compounds and
Grasshoppers: Beyond plant defenses. J. Chem. Ecol. 2000, 26,
1773–1794.
(37) Berenbaum, M. R. Postingestive effects of phytochemicals on
insects: On Paracelsus and plant products. In Insect-Plant
Interactions; Miller, T. A., Miller, J., Eds.; Springer-Verlag: New
York, 1986; pp 123–153.
(38) Bernays, E. A.; Chapman, R. F. The evolution of deterrent
responses in plant feeding insects. In PerspectiVes in Chemore-
ceptions and BehaVior; Chapman, R. F., Bernays, E. A., Stoffol-
ano, J. G., Jr., Eds.; Springer-Verlag: New York, 1987; pp 159–
173.
(39) Singh, D. V.; Gupta, M. M.; Tripathi, A. K.; Prajapati, V.; Kumar,
S. Arjunetin from Terminalia arjuna as an insect feeding-deterrent
and growth inhibitor. Phytother. Res. 2004, 18, 131–134.
(40) van Loon, J. J. A.; Schoonhoven, L. M. Specialist deterrent
chemoreceptors enable Pieris caterpillars to discriminate between
chemically different deterrents. Entomol. Exp. Appl. 1999, 91, 29–
35.
(41) Yuan, L.; Xue, M.; Liu, Y.; Wang, H. Toxicity and oviposition
deterrence of Vitex negundo extracts to Plutella xylostella. Ying
Yong Sheng Tai Xue Bao 2006, 17, 695–698.
(43) Akhtar, Y.; Isman, M. B. Mitigating decreased feeding deterrent
response to antifeedants following prolonged exposure in Tri-
choplusia ni by binary mixtures of antifeedants. Chemoecology
2003, 13, 177–182.
(44) Raffa, K. F.; Frazier, J. L. A general model for quantifying
behavioral desensitization to antifeedants. Entomol. Exp. Appl.
1988, 46, 93–100.
(45) Akhtar, Y.; Isman, M. B. Decreased feeding deterrent response
to antifeedants in specialist herbivores: Effects of prolonged
exposure. Entomol. Exp. Appl. 2004, 111, 201–208.
(46) Miller, J. R.; Cowles, R. S. Stimulo-deterrent diversion: A concept
and its possible application to onion maggot control. J. Chem.
Ecol. 1990, 16, 3197–3212.
(47) Griffiths, D. C.; Maniar, S. P.; Merritt, L. A.; Mudd, A.; Pickett,
J. A.; Pye, B. J.; Smart, L. E.; Wadhams, L. J. Laboratory
evaluation of pest management strategies combining antifeedants
with insect growth regulator insecticides. Crop Prot. 1991, 10,
145–151.
Received for review June 4, 2007. Revised manuscript received
September 28, 2007. Accepted October 22, 2007. This research was
supported by NSERC Strategic Grant STPGP 307515 (to E.P. and
M.B.I.) and NSERC Discovery Grants 222923-05 (to E.P.) and 2729-
06 (to M.B.I.).
(42) Hajime, O.; Yasumasa, K.; Ritsuo, N. Hydroxybenzoic Acid
derivatives in a nonhost rutaceous plant, Orixa japonica, deter
both oviposition and larval feeding in a rutaceae-feeding swal-
JF071636D