Synthesis and bio-evaluation of aryl hydrazono esters for oviposition responses in Aedes albopictus

Article abstract of DOI:10.1016/j.bmcl.2010.11.101

A novel series of aryl hydrazono esters (AHE) (1-13) were synthesized (yield 76-98%) to study the oviposition responses in Aedes albopictus (Skuse) mosquitoes for the first time. At a concentration of 10 μg ml^-1 in dual choice experiment, among the screened compounds, AHE-12 showed remarkable oviposition attractant activity with an oviposition activity index (OAI) of +0.299 (greater than 95% confidence limit) comparable to p-cresol (OAI +0.320) which is well-reported oviposition attractant for Aedes aegypti. Conversely, AHE-10 exhibited highest oviposition deterrent activity with OAI -0.247. The possible utilization of these compounds will be in integrated vector management strategies.

Full text of DOI:10.1016/j.bmcl.2010.11.101

Bioorganic & Medicinal Chemistry Letters 21 (2011) 794–797
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and bio-evaluation of aryl hydrazono esters for oviposition
responses in Aedes albopictus
Prabal Bandyopadhyay a, Lopamudra Guha a, T. Seenivasagan a, Manisha Sathe a, Pratibha Sharma b,
B. D. Parashar a, M. P. Kaushik a,
⇑
a Defence R & D Establishment, Jhansi Road, Gwalior 474 002 (M.P.), India
b School of Chemical Sciences, Devi Ahilya University, Khandwa Road, Indore 452 001 (M.P.), India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of aryl hydrazono esters (AHE) (1–13) were synthesized (yield 76–98%) to study the
Received 25 September 2010
Revised 12 November 2010
Accepted 20 November 2010
Available online 4 December 2010
oviposition responses in Aedes albopictus (Skuse) mosquitoes for the first time. At a concentration of
10 l
g mlÀ1 in dual choice experiment, among the screened compounds, AHE-12 showed remarkable
oviposition attractant activity with an oviposition activity index (OAI) of +0.299 (greater than 95%
confidence limit) comparable to p-cresol (OAI +0.320) which is well-reported oviposition attractant for
Aedes aegypti. Conversely, AHE-10 exhibited highest oviposition deterrent activity with OAI À0.247.
The possible utilization of these compounds will be in integrated vector management strategies.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Aryl hydrazono ester
Oviposition
Aedes albopictus
Dengue
Chikungunya
Aedes albopictus (Ae. albopictus), is a day biting mosquito, highly
anthropophilic and potential vector for dengue and chikungunya.
Identification of suitable oviposition sites by mosquitoes is a criti-
cal feature of their life history, because it ultimately influences the
survivorship of their progeny.1 Oviposition behavior in mosquitoes
is influenced by visual, tactile and olfactory factors, with the latter
considered being of primary importance. These cues include color
and optical density of the water, texture and moisture, tempera-
ture and reflectance of the oviposition substrate.2 The chemical
factors involved in oviposition site selection by mosquitoes have
gained much interest in recent years and considerable attention
has been paid to the chemical cues influencing mosquito oviposi-
tion.3 Most of the behaviors associated with reproduction/oviposi-
tion are mediated by chemical cues of different origin and
therefore such chemical identities can determine the ultimate sur-
vival of mosquito population.4 The most important aspect in any
organism’s life is to ensure successors after them. Therefore, in or-
der to abolish a vector like mosquito, killing of the adult as well as
its progeny is of the prime remedial importance. Earlier efforts
were made to identify a potential synthetic attractant or repellant
for mosquitoes using short-chain fatty acid esters against Aedes ae-
gypti (Ae. aegypti),5 role of larval water and pre-existing eggs in ovi-
position by Ae. aegypti and Ae. albopictus,6 certain fatty acids and
esters identified from egg extracts of Ae. aegypti as oviposition
attractant7 and oviposition responses of Ae. aegypti and Ae. albopic-
tus to certain fatty acid esters8 laid the foundation to explore syn-
thetic compounds of newer origin. To the best of our knowledge, a
finger-count literature is available regarding oviposition responses
of Ae. albopictus to chemical compounds.8,9 Pheromone based or
chemical mediated oviposition studies in mosquitoes are scanty.
Many such reports are available for agriculturally important pests.
One of the selected examples is the deterrent ovipositional activity
mediated by phenolic acids as compared to non phenolic ones in
Spruce Budworm.10 Floral phenyl esters attract many wide ranges
of insects including Dacus dorsalis.11 Methyl benzoate and some of
its derivatives mostly nitrogenous allied with host plant aromas
were used to study attraction of feral northern and western corn
rootworm beetles (Diabrotica barberi and D. virgifera virgifera).12
Formic acid 4-(3-oxobutyl)phenylester (raspberry ketone formate
[RKF]) was tested successfully against wild populations of male
melon flies, Bactrocera cucurbitae Coquillett (Diptera: Tephriti-
dae).13 tert-Butyl ester of methyl cyclohexane carboxylic acids
were used to lure male Mediterranean fruit flies Ceratitis capitata
(Weidman).14 Certain aromatic compounds (ligands) were found
to mediate receptor binding in odorant binding protein in Ae. ae-
gypti.15 Encouraged by these examples, in continuation of our stud-
ies on bioactive compounds,16 herein we report the synthesis of
aryl hydrazono esters (AHE)17 and the oviposition response studies
in Ae. albopictus. From our preliminary studies we found better
attractant properties in case of unsymmetrical aryl hydrazono es-
ters than that of the symmetrical ones. Hence, we focused our
⇑
Corresponding author. Tel.: +91 751 2343972; fax: +91 751 2347542.
E-mail address: mpkaushik@rediffmail.com (M.P. Kaushik).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.101

P. Bandyopadhyay et al. / Bioorg. Med. Chem. Lett. 21 (2011) 794–797
795
to form the desired aryl hydrazono esters.18 All the compounds
were purified by recrystallisation to get highly pure AHE.
The details of R1, R2, and R3 groups of the various aromatic
amines and b-keto esters were illustrated in Table 1.
(i)
R1
(ii)
N
N
Cl
R1 NH2
1
2
O
All the compounds (AHE-1 to AHE-13) were characterized by IR,
NMR (1H and 13C), ESI-MS and elemental analysis. The spectral
data and the elemental analysis of all the compounds reported in
this study correlate with the proposed structures. The IR spectrum
showed absorption at 3159 cmÀ1 and 1193, 1167 cmÀ1 which cor-
responds to NH stretching of hydrazono group and C–O stretching
of ester group. In case of 1H NMR the sharp singlet at d 2.6 ppm
indicated the presence of ketonic methyl group, while quartet at
d 4.4 and triplet at d 1.4 ppm confirmed the presence of CH2 and
CH3 of ethyl group of b-keto ester. The presence of ketonic carbonyl
and esteric carbonyl was observed at d 196 ppm and d 165 ppm,
respectively, in 13C NMR.
R2
H2C
O
O
R3
O
O
R1
N
R2
O
R1 NH
N
R2
O
Tautomerism
N
R3
O
R3
O
3
4
Scheme 1. Reagents and conditions: (i) NaNO2/HCl, 0–5 °C, (ii) CH3COONa/C2H5OH.
Laboratory bioassay19 on oviposition responses was carried out
in fine wire mesh fitted cages (750 Â 600 Â 600 mm) with a sleeve
opening on one side. The oviposition responses of AHE-1 to AHE-13
Table 1
were evaluated on gravid female mosquitoes at 10 l
g mlÀ1 (Fig. 1)
Synthesized aryl hydrazono ester (AHE) compounds
using standard procedure.20 HPLC-grade methanol was used as a
control. The basis for measuring the oviposition response was the
number of eggs received in both control and treatment bowls.
The oviposition activity was expressed as oviposition activity index
(OAI) and calculated by using the formula OAI = (Nt À Ns)/(Nt + Ns),
where, Nt is the number of eggs laid in test solution and Ns is the
number of eggs laid in control. As suggested by Kramer and Mul-
la,21 compounds with an OAI of +0.3 and above are considered as
attractants, while those with À0.3 and below are considered as
repellents. p-cresol is well-reported as oviposition attractant for
Ae. aegypti,22 we compared the OAI value with it. Out of 13 com-
pounds (AHE-1 to AHE-13), AHE-12 showed significant positive
oviposition response with OAI 0.299 (P <0.05) in Ae. albopictus.
Structurally, AHE-12 consists of naphthalene ring attached to ester
via hydrazono group. Interestingly, as such naphthalene was re-
ported as attractant in case of female Tabanidae (Diptera),23 and
also as repellent in Ae. aegypti.24 However, we found AHE-12 was
acting as positive stimulant for oviposition of Ae. albopictus. Addi-
tionally, when phenyl ring of AHE-1 was replaced by naphthalene
ring in AHE-12 the oviposition attractant activity was found to be
increased significantly with OAI +0.157 to +0.299. It was also evi-
dent from the Table 2 that few compounds (AHE-1, AHE-4, AHE-
6 and AHE-11) although displayed positive OAI which indicates
that more eggs occur in the treated than in the control, though they
were not significant (P >0.05). Conversely, AHE-3, AHE-5, AHE-9
Entry
Code
R1
R2
R3
Yield* (%)
4a
4b
4c
4d
4e
4f
4g
4h
4i
AHE-1
AHE-2
AHE-3
AHE-4
AHE-5
AHE-6
AHE-7
AHE-8
AHE-9
AHE-10
AHE-11
AHE-12
AHE-13
C6H5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CF3
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
CH2CH(CH3)2
CH3
C2H5
C2H5
C2H5
90
84
77
78
98
87
76
98
95
97
92
84
81
4-NO2C6H4
4-MeC6H4
4-HOC6H4
2-MeOCC6H4
2-HOH2CC6H4
4-MeOCC6H4
4-FC6H4
4-FC6H4
3-ClC6H4
4-MeOC6H4
Naphthalen-1-yl
4-EtOOCC6H4
4j
4k
4l
CH3
CH3
4m
*
Isolated yield.
studies only up to unsymmetrical aryl hydrazono esters. Results
from this study will be useful to identify a potential oviposition
stimulant or deterrent against dengue and chikungunya vector.
The synthetic pathway of the designed AHE was described in
Scheme 1. Various aromatic amines were reacted with aqueous so-
dium nitrite solution in acidic medium under ice-cold condition
(0–5 °C) to form the corresponding diazonium chloride salt inter-
mediate, which was further condensed with ethanolic solution of
b-keto ester in presence of sodium acetate under ice-cold condition
Figure 1. Oviposition response in different aryl hydrazono ester (AHE) compounds.

796
P. Bandyopadhyay et al. / Bioorg. Med. Chem. Lett. 21 (2011) 794–797
Table 2
Oviposition response of Aedes albopictus to Aryl hydrazono esters
Code
Aryl hydrazono estes
No. of eggs laid in different treatments,
(mean + SE)
P value* (significance) OAI value
Control
10 ppm
AHE-1
AHE-2
AHE-3
AHE-4
AHE-5
AHE-6
AHE-7
AHE-8
AHE-9
Ethyl 2-(2-phenylhydrazono)-3-oxobutanoate
Ethyl 2-(2-(4-nitrophenyl)hydrazono)-3-oxobutanoate
Ethyl 2-(2-p-tolylhydrazono)-3-oxobutanoate
Ethyl 2-(2-(4-hydroxyphenyl)hydrazono)-3-oxobutanoate
Ethyl 2-(2-(2-acetylphenyl)hydrazono)-3-oxobutanoate
Ethyl 2-(2-(2-(hydroxymethyl)phenyl)hydrazono)-3-oxobutanoate
Ethyl 2-(2-(4-acetylphenyl)hydrazono)-3-oxobutanoate
Ethyl 2-(2-(4-fluorophenyl)hydrazono)-3-oxobutanoate
Isobutyl 2-(2-(4-fluorophenyl)hydrazono)-3-oxobutanoate
65.70 14.67
51.71 17.10
71.43 23.81
90.86 23.02
94.71 26.37
92.86 32.23
43.57 13.86
39.71 5.60
73.57 19.67
71.71 24.41
90.20 24.51
52.0 13.50
47.86 7.37
105.86 28.52
73.86 20.43
116.71 34.87
40.86 6.08
38.57 9.95
49.14 17.89
43.29 14.62
76.43 17.37
110 23.17
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
<0.05
>0.05
+0.157
+0.003
À0.197
+0.076
À0.124
+0.114
À0.032
À0.015
À0.199
À0.247
+0.147
+0.299
+0.073
AHE-10 Methyl 2-(2-(3-chlorophenyl)hydrazono)-3-oxobutanoate
AHE-11 Ethyl 4,4,4-trifluoro-2-(2-(4-methoxyphenyl)hydrazono)-3-oxobutanoate 56.86 18.85
AHE-12 Ethyl 2-(2-(naphthalen-1-yl)hydrazono)-3-oxobutanoate
AHE-13 Ethyl 4-(2-(1-ethoxy-1,3-dioxobutan-2-ylidene)hydrazinyl)benzoate
59.29 18.58
27.0 8.16
31.29 11.98
*
P value indicates the level of significance, P <0.05 indicate greater than 95% confidence limit.
and AHE-10 displayed negative OAI which signifies more eggs laid
in the control than in the treated one, hence indicating the com-
pounds to be oviposition deterrent. AHE-10 showed highest ovipo-
sition deterrent activity with OAI À0.247 against Ae. albopictus. The
oviposition response was found to be neutral in case of AHE-2,
AHE-7, AHE-8 and AHE-13 as in these cases, there was no signifi-
cant difference (P >0.05) in egg laying between the treatment and
control. From this study, it was revealed that compounds which
contain hydroxyl group (AHE-4 and AHE-6) or its analog (AHE-
11) attached to phenyl ring or its side chain exhibited moderate
oviposition attractancy to Ae. albopictus females. This was sup-
ported by the fact that mosquitoes possess hygroreceptors in their
body (mainly tarsi25), hence can sense the (active ingredient) vapor
content in an oviposition substrate. On the other hand, compounds
(AHE-8, AHE-9 and AHE-10) containing halogen groups (Cl and F)
displayed moderate to good deterrent properties. In insects,
halogenated analogs were reported as inhibitors of chemical
communication.26
the help in maintenance of the mosquito culture. P.B is thankful to
DRDO, New Delhi, for financial support.
Supplementary data
Supplementary data (FT-IR, 1H, 13C NMR, ESI-MS and elemental
analysis) associated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2010.11.101.
References and notes
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In conclusion, a series of aryl hydrazono esters (AHE) (1–13)
were synthesized and evaluated for the oviposition responses
against Ae. albopictus for the first time. AHE-12 showed significant
oviposition attractant activity with an oviposition activity index
(OAI) of +0.299 (greater than 95% confidence limit) comparable
to well-reported oviposition attractant for Ae. aegypti, p-cresol
(OAI +0.320). Conversely, AHE-10 exhibited highest oviposition
deterrent activity with OAI À0.247. The present study shows the
potential application of these compounds as oviposition attrac-
tants which may have potential application in mosquito trapping
for identification, surveillance and control.
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18. The aromatic amine (1–13) (0.01 mol) was dissolved in a mixture of 4.0 mL of
concentrated hydrochloric acid (HCl) and 4.0 mL of distilled water. The amine
hydrochloride solution was kept at freezing temperature. To this, an aqueous
solution of sodium nitrite (NaNO2) 0.69 g (0.01 mol) in 5.0 mL of distilled water
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Acknowledgements
sodium acetate (CH3COONa) in
a solution of 1.53 mL (0.012 mol) ethyl
The authors are thankful to Dr. R. Vijayaraghavan, Director,
DRDE, Gwalior for his keen interest and encouragement. We thank
Mrs. Ruchi Yadav and all the members of Entomology division for
acetoacetate in 25.0 mL of ethyl alcohol was taken and cooled in an ice-bath.
Now the diazotized solution was added to this solution dropwise with
thorough stirring. The reaction mixture was kept overnight, filtered under

P. Bandyopadhyay et al. / Bioorg. Med. Chem. Lett. 21 (2011) 794–797
797
suction, washed thoroughly with cold water, dried and recrystallised from a
mixture of dimethylformamide (DMF) and ethyl alcohol (EtOH) (3:7, v/v) to
give the corresponding butanoate.
freeze after every experiment. The bowls were rinsed with tap water and
washed with methanol before commencing the experiment. The experiment
was completely randomized and in each bioassay females were allowed to
choose between the treated and untreated (control) substrates. The position of
substrate in dual choice experiment was changed in a clockwise rotation every
day. Each compound was evaluated separately in duplicates, and the
experiment was repeated for seven days with different batch of mosquitoes.
The number of eggs laid in control and treatment were counted manually to
assess the oviposition preference of the mosquito species.
19. Ae. albopictus used for the oviposition experiments were utilized from the
laboratory colony maintained since 1973 in our insectaria. The experiments
were performed at 27 2 °C, 75 5% RH, L10:D14 regime. The illumination
inside the oviposition cages was 80–100 lx. Four to seven-days old adults of Ae.
albopictus (25 pairs) were kept in separate standard-sized wooden cages
(750 Â 600 Â 600 mm) with a sleeve opening on one side. Sucrose (10%) was
provided to adults, and female mosquitoes were fed on rabbit blood for two
days initially and then every alternative day until the experiment was
completed. Laboratory bioassays were performed (duplicates) in two
separate cages, enamel bowls with 10 cm diameter filled with 500 mL of de-
chlorinated water was used as the oviposition substrate. One milliliter of the
stock solution in methanol containing the desired compound (to make 10 ppm
concentration) was added to the enamel bowls. All stock solution of the test
chemicals were dissolved in HPLC-grade methanol (Merck) and stored in deep
20. WHO—World Health Organization, Technical Report, 1963, Series No. 265.
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