Binding activity of substituted benzyl derivatives of chloronicotinyl insecticides to housefly-head membranes, and its relationship to insecticidal activity against the housefly Musca domestica

Article abstract of DOI:10.1002/1526-4998(200010)56:10<875::AID-PS220>3.0.CO;2-A

Variously substituted benzyl derivatives of chloronlcotinyl insecticides were synthesized with a wide range of substituents including halogens, NO₂, CN, CF₃ and small alkyl and alkoxy groups at the ortho, meta and para positions, as well as multiple-substituted benzyl analogues. Their binding activity to the α-bungarotoxin binding site in housefly (Musca domestica) head membrane preparations was measured. Among the compounds tested, the activity of the meta-CN derivative was the highest, being 20-100 times higher than those of imidacloprid, acetamiprid and nitenpyram. The synergized insecticidal activity against houseflies was also measured for selected compounds with the metabolic inhibitor, NIA16388 (propargyl propyl phenylphosphonate). For the nitromethylene analogues, including both benzyl and pyridylmethyl analogues, higher binding activity usually resulted in higher insecticidal activity. (C) 2000 Society of Chemical Industry.

Full text of DOI:10.1002/1526-4998(200010)56:10<875::AID-PS220>3.0.CO;2-A

Pest Management Science
Pest Manag Sci 56:875±881 (2000)
Binding activity of substituted benzyl
derivatives of chloronicotinyl insecticides to
housefly-head membranes, and its relationship
to insecticidal activity against the housefly
Musca domestica
Hisashi Nishiwaki,1 Yoshiaki Nakagawa,1* David Y Takeda,1 Atsushi Okazawa,1
Miki Akamatsu,1 Hisashi Miyagawa,1 Tamio Ueno1 and Keiichiro Nishimura2
1Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
2Research Institute for Advanced Science and Technology, Osaka Prefecture University, Osaka 599-8570, Japan
Abstract: Variously substituted benzyl derivatives of chloronicotinyl insecticides were synthesized with
a wide range of substituents including halogens, NO2, CN, CF3 and small alkyl and alkoxy groups at the
ortho, meta and para positions, as well as multiple-substituted benzyl analogues. Their binding
activity to the a-bungarotoxin binding site in house¯y (Musca domestica) head membrane prepara-
tions was measured. Among the compounds tested, the activity of the meta-CN derivative was the
highest, being 20±100 times higher than those of imidacloprid, acetamiprid and nitenpyram. The
synergized insecticidal activity against house¯ies was also measured for selected compounds with the
metabolic inhibitor, NIA16388 (propargyl propyl phenylphosphonate). For the nitromethylene ana-
logues, including both benzyl and pyridylmethyl analogues, higher binding activity usually resulted in
higher insecticidal activity.
# 2000 Society of Chemical Industry
Keywords: chloronicotinyl insecticides; house¯y; Musca domestica; nicotinic acetylcholine receptor (nAChR);
a-bungarotoxin
1
INTRODUCTION
oxygen atom of the nitro group, but not the nitrogen
atom of the pyridine ring, plays the role of hydrogen
acceptor. Comparative molecular ®eld analysis
(CoMFA) is a method for the analysis of three-
dimensional quantitative structure-activity relation-
ships (3-D QSAR).16 Using this method, we have
found that the binding mode of a set of neonicotinoids
is more likely to be that proposed by Yamamoto et
al. 17 This CoMFA study also disclosed the occurrence
of other important steric and electrostatic interactions
encompassing the imidazolidine ring.17,18 However,
since the structural variation of the 6-chloropyridine
moiety was limited in the set of compounds examined,
the factors that affect the interaction of this moiety
with the receptor site remained unknown.
A series of recently developed insecticides, referred to
as chloronicotinyl or neonicotinoid insecticides, act at
the acetylcholine binding site of the nicotinic acet-
ylcholine receptor (nAChR) of insects.1±9 To date,
three compounds are available commercially; imida-
cloprid,10 acetamiprid11 and nitenpyram.12 These
insecticides are remarkably potent neurotoxic insecti-
cides, and are characterized by their persistent effects,
broad spectra, good systemic properties and low
toxicity to mammals and aquatic life. We have recently
shown that the activity of chloronicotinyl compounds,
including these commercial insecticides, is very weak
in the receptor binding assay using the rat membrane
fraction.13
Two different binding modes of neonicotinoids have
been proposed; by Yamamoto et al,14 and by
Kagabu.15 According to Yamamoto et al, the nitrogen
atoms of the pyridine and imidazolidine rings interact
with the hydrogen-donating and electron-rich sites,
respectively, of the receptor. According to Kagabu, the
In the present study, we have examined the
structure-activity relationships of chloronicotinyl com-
pounds to clarify the factors affecting the interaction of
the pyridine ring moiety and the receptor site.
Structural modi®cation of a benzene ring allows for
many more variations than that of a pyridine ring. We
* Correspondence to: Yoshiaki Nakagawa, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-
8502, Japan
E-mail: naka@kais.kyoto-u.ac.jp
(Received 29 October 1999; revised version received 25 April 2000; accepted 8 June 2000)
# 2000 Society of Chemical Industry. Pest Manag Sci 1526±498X/2000/$30.00
875

H Nishiwaki et al
Figure 1. Synthetic scheme for benzyl
derivatives of chloronicotinyl analogues.
therefore synthesized benzyl derivatives with various
substituents at the ortho, meta, and para positions of
the benzene ring in place of the 6-chloropyridine
moiety of the representative chloronicotinyl com-
pounds (I: Fig 1). In the imidazolidine moiety, a
nitromethylene group was introduced instead of the
nitroimino group of imidacloprid. This substitution
was made because the binding activity of nitromethy-
lene analogues having a pyridylmethyl group is higher
than that of the corresponding nitroimine ana-
logues.4,8,17,18 We measured the binding activity of
these compounds to a-bungarotoxin (a-BGTX) re-
ceptors of house¯y-head membrane preparations, and
also measured the insecticidal activity against house-
¯ies for selected compounds and compared it with the
binding activity.
on Wakogel C-200 or by recrystallization if a solid.
The structures of all compounds were con®rmed by
[1H]NMR. Some intermediates were used without
further puri®cation. The authenticity of the ®nal
compounds was also con®rmed by elemental analyses.
The analytical values forC, H and N agreed with the
calculated values within an error of Æ0.3%. The
[1H]NMR analyses were performed using a Bruker
AC-300 NMR spectrometer at 300MHz in deutero-
chloroform (CDCl3) with tetramethylsilane as the
internal standard. Melting points of the compounds
were measured with a Yanaco melting point apparatus
(Kyoto, Japan) and were uncorrected. Melting points
of the ®nal compounds are listed in Table 1. Typical
synthetic methods are described below.
2.1.1 3,5-Di-tert-butylbenzyl alcohol (Step A)
3,5-Di-tert-butylbenzoic acid (3.00g, 13mmol) dis-
solved in anhydrous tetrahydrofuran (THF; 100ml)
was added dropwise to lithium aluminium hydride
(0.97g, 26mmol) suspended in anhydrous THF
(5ml) while stirring in an ice-bath. The mixture was
removed from the ice-bath and stirred overnight at
room temperature. Water (2ml) followed by aqueous
sodium hydroxide solution (100g litre 1; 7ml) were
slowly added to the reaction mixture at 0°C. After
removing the precipitate by ®ltration, the ®ltrate was
dried over anhydrous magnesium sulfate and evapo-
rated to afford 3,5-di-tert-butylbenzyl alcohol as a
colourless oil (2.61g, 91% yield) for compound 38.
The necessary substituted benzyl alcohols to prepare
compounds 6, 34±37 and 39 were synthesized from the
corresponding benzoic acids using a similar procedure.
2
EXPERIMENTAL METHODS
2.1 Chemicals
Compounds 1±41 listed in Table 1 were synthesized
according to a published method.19 Compounds 42±
45 were kindly provided by Nihon Bayer Agrochem
KK (Ibaraki, Japan). Compound 46 and compound 47
were from Nippon Soda Co, Ltd (Tokyo, Japan) and
Takeda Chemical Industries, Ltd (Osaka, Japan),
respectively. The synthetic scheme for the preparation
of the benzyl compounds is shown in Fig 1. Reagents
used for the syntheses were purchased from Wako
Pure Chemical Industries, Ltd (Osaka, Japan), Naca-
lai Tesque, Inc (Kyoto, Japan), Tokyo Chemical
Industry Co, Ltd (Tokyo, Japan), and Aldrich
Chemical Co (Milwaukee, WI, USA). a-BGTX was
purchased from Sigma Chemical Co (St Louis, MI,
USA), and [125I]a-BGTX (c 74 TBqmmol 1) was
from Amersham Pharmacia Biotech (Buckingham-
shire, UK). The metabolic inhibitor, NIA 16388
(NIA; propargyl propyl phenylphosphonate), was
from our stock sample.20 Intermediates and the ®nal
compounds were puri®ed by column chromatography
2.1.2 2,5-Dichlorobenzyl bromide (Step B-1)
Triphenylphosphine (3.02g, 15mmol) was added
slowly to a solution of 2,5-dichlorobenzyl alcohol
(1.77g, 10mmol) and carbon tetrabromide (3.32g,
11mmol) in anhydrous dichloromethane (10ml), and
the mixture was re¯uxed for 2h. After evaporating the
876
Pest Manag Sci 56:875±881 (2000)

SAR of chloronicotinyl insecticides
Table 1. Binding and insecticidal activities of imidacloprid and its related compounds
Activity against house¯ies
Compound
no
Binding
Insecticidal
a,b
Xn
Y
pKi (M) (ÆSEM) (n)
pEC50
(M)
mp (°C)
1
2
3
4
5
6
7
8
9
H
2-F
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
4.65 (Æ0.18) (4)
3.97 (Æ0.16) (2)
3.43 (Æ0.18) (4)
3.53 (Æ0.15) (2)
3.51 (Æ0.14) (2)
2.94 (Æ0.06) (2)
3.01 (Æ0.05) (2)
5.66 (Æ0.13) (2)
5.24 (Æ0.09) (2)
4.38 (Æ0.00) (2)
4.72 (Æ0.25) (3)
4.04 (Æ0.15) (4)
3.35 (Æ0.26) (4)
6.73 (Æ0.28) (3)
3.54 (Æ0.08) (2)
4.89 (Æ0.10) (2)
5.40 (Æ0.30) (2)
4.90 (Æ0.01) (2)
4.11 (Æ0.18) (2)
2.78 (Æ0.22) (2)
2.87 (Æ0.09) (2)
2.32 (1)
3.61
2.52
2.09
<2.00 (13)
nd
145±149
169±170
197±199
195±197
209±211
147±150
165±167
149±152
176±178
167±170
198±200
182±184
164±168
182±185
154±155
142±144
169±172
166±169
118±119
122±125
136±138
210±213
224±227
118±120
150±152
188±192
205±208
167±170
161±164
198±200
208±211
251±258
181±184
184±186
186±189
120±124
124±126
192±196
159±163
191±195
174±176
Ð
2-Cl
2-CH3
2-NO2
2-CH3O
2-CF3
3-F
nd
nd
3.60
3.46
3.00
2.62
nd
3-Cl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
3-CH3
3-NO2
3-CH3O
3-CF3
3-CN
nd
3.98
<2.55 (6)
3.54
4.18
nd
3-C6H5O
4-F
4-Cl
4-Br
4-CH3
3.75
nd
4-C2H5
4-i-C3H7
4-t-C4H9
4-NO2
nd
nd
3.54 (Æ0.22) (4)
2.99 (Æ0.15) (2)
3.44 (Æ0.05) (2)
3.18 (Æ0.16) (2)
2.83 (Æ0.08) (2)
5.42 (Æ0.11) (2)
4.92 (Æ0.11) (2)
3.95 (Æ0.11) (2)
3.25 (Æ0.11) (2)
<3.48
nd
4-CH3O
4-CF3
4-CN
nd
nd
nd
2,3,4,5,6-F5
3,4-F2
nd
3.39
nd
3,5-F2
2,3-Cl2
2,5-Cl2
2,6-Cl2
3,4-Cl2
3,5-Cl2
2,3-(CH3)2
3,4-(CH3)2
3,5-(CH3)2
3,5-t-(C4H9)2
3,4-(CH3O)2
3-NO2,4-CH3
3,4-OCH2O-
H
nd
nd
nd
5.92 (Æ0.17) (2)
4.73 (Æ0.05) (2)
3.52 (Æ0.11) (2)
3.47 (Æ0.14) (2)
4.39 (Æ0.15) (2)
<3.57
3.23
<2.73 (40)
nd
nd
nd
nd
3.21 (Æ0.13) (2)
4.80 (Æ0.09) (6)
4.67 (Æ0.13) (2)
7.43 (Æ0.13) (2)
7.93 (Æ0.11) (2)
6.14 (1)
nd
2.69
nd
5.50
5.64
5.70
5.25
5.77
5.63
6-Cl
N
Ð
6-CH3
N
Ð
Imidacloprid
5.43 (Æ0.16) (2)
4.80 (Æ0.26) (2)
5.17c
Ð
Acetamiprid
Nitenpyram
Ð
Ð
a
The values in parentheses are percentage mortality at the dose indicated.
b
c
nd=not determined.
From Reference 13.
Pest Manag Sci 56:875±881 (2000)
877

H Nishiwaki et al
solvent, the residue was suspended in diethyl ether
(200ml) and ®ltered to remove insoluble materials.
The ®ltrate was evaporated and the residue was
puri®ed by silica-gel column chromatography with
hexane as the mobile phase to afford 2,5-dichloro-
benzyl bromide (2.22g, 93% yield) for compound 31.
Other substituted benzyl bromides were synthesized
from the corresponding alcohols to prepare com-
pounds 7, 30, 36, 37, 39 and 41 using a similar
procedure.
s), 7.11 (2H, d), 7.19 (2H, d), 8.71 (1H, br). Analysis:
calculate, C (61.79), H (6.48), N (18.01); found, C
(61.51), H (6.29), N (17.71). mp 118±119°C.
Compounds 1±18 and 20±41 were similarly prepared.
2.2 Bioassays
2.2.1 Insects
An insecticide-susceptible strain of the house¯y
(Musca domestica L, Takatsuki strain) was reared at
25°C. Three- to six-day-old ¯ies were used for the
binding and insecticidal tests. Among the insects,
females were selected for the insecticidal test.
2.1.3 2,3-Dimethylbenzyl chloride (Step B-2)
After dissolving 2,3-dimethylbenzyl alcohol (1.25g,
11mmol) in thionyl chloride (SOCl2; 2ml), the
solution was re¯uxed for 2h. Excess SOCl2 was
evaporated and the residue was puri®ed by silica-gel
column chromatography to afford 2,3-dimethylbenzyl
chloride as a colourless liquid (1.53g, 90% yield).
Other substituted benzyl chlorides were synthesized
from the corresponding alcohols to prepare com-
pounds 6, 34, 35 and 38 using a similar procedure.
2.2.2
[
125I] a-BGTX binding to membrane fraction
The procedures for the preparation of the house¯y
head membrane fraction and for the binding assay
were fundamentally the same as those described
previously.17,18 Brie¯y, house¯y heads (5ml) were
homogenized in sodium phosphate buffer (pH 7.4;
100mM; 5ml) containing sucrose (0.32M) and EDTA
(0.1mM). The homogenate was ®ltered through gauze
and centrifuged at 700g for 10min, and the super-
natant was further centrifuged at 125000g for 60min.
The ®nal precipitate was suspended in sodium
phosphate buffer (pH 7.4; 10mM; c 50ml) containing
sodium chloride (50mM) and Triton X-100
(1g litre 1). This suspension was used for the binding
assay. The protein concentration was measured by a
bicichoninic acid protein-assay kit (Pierce Chemical
Co, Rockford, IL, USA) with BSA as a standard. The
2.1.4 3-Phenoxybenzyl bromide (Step C)
N-Bromosuccinimide (8.10g, 50mmol) and benzoyl
peroxide (0.024g, 0.1mmol) were added to a solution
of 3-phenoxytoluene (9.20g, 50mmol) in anhydrous
carbon tetrabromide (30ml) and the mixture re¯uxed
for 2h. After removing the precipitate by ®ltration, the
®ltrate was evaporated. The residue was puri®ed by
silica-gel column chromatography with hexane ‡ ethyl
acetate (9‡1 by volume) as the mobile phase to afford
3-phenoxybenzyl bromide (1.76g, 78% yield). The
corresponding bromide for compound 14 was also
synthesized using this method.
®nal protein concentration of the membrane prepara-
1
tion was adjusted to 1.0mg ml
.
The membrane preparation was pre-incubated with
3
9
the test compounds (10
10 M) for at least 10min,
then [125I]a-BGTX (0.2nM) was added. After incu-
bating at 24°C for 60min, the reaction was terminated
by rapid ®ltration through a Uni®lter GF/B (Packard
Instrument Co, Meriden, CT, USA), which had been
treated with polyethyleneimine (1g litre 1). The ®lters
were rinsed three times with sodium phosphate buffer
(pH 7.4; 10mM) containing sodium chloride (50mM).
After washing with methanol, drying and adding
Microscinti-O (Packard Instrument Co, Meriden,
CT, USA) as scintillation cocktail, the radioactivity
was measured using a Topcount instrument (Packard
Instrument Co, Meriden; CT, USA). The speci®c
binding was determined as the difference of the
radioactivity in the presence and absence of non-
radioactive a-BGTX (10mM). The molar concentra-
tion required for 50% inhibition of the speci®c binding
of [125I]a-BGTX, IC50 (M), was determined by a non-
linear regression analysis using PRISM (Graphpad
Software Inc, San Diego, CA, USA). The binding
activity was expressed as the Ki value calculated by
2.1.5 N-(2-Methylbenzyl)ethylenediamine (Step D)
2-Methylbenzyl chloride (1.40g, 10mmol) dissolved
in acetonitrile (10ml) was added dropwise to ethyle-
nediamine (3.00g, 50mmol) while stirring in an ice-
bath. After removing from the ice-bath and stirring
overnight at room temperature, the solvent was
evaporated. Aqueous sodium hydroxide (1M; 50ml)
was added to the residue, which was then extracted
with dichloromethane. The organic layer was dried
over anhydrous magnesium sulfate and evaporated to
afford N-(2-methylbenzyl)ethylenediamine (1.30g,
79% yield). Diamines for other compounds were
similarly prepared.
2.1.6 1-(4-Methylbenzyl)-2-nitromethylene-
imidazolidine (Step E)
N-(4-Methylbenzyl)ethylenediamine (0.82g, 5mmol)
dissolved in ethanol (20ml) was added dropwise to
1,1-bis(methylthio)-2-nitroethylene (0.83g, 5mmol)
dissolved in ethanol (10ml). The mixture was re¯uxed
overnight. After evaporating the solvent, the residue
was recrystallized from ethanol to afford 1-(4-methyl-
benzyl)-2-nitromethylene-imidazolidine (0.66g, 57%
yield). [1H]NMR (CDCl3/TMS) d (ppm): 2.35 (3H,
s), 3.56 (2H, t), 3.75 (2H, t), 4.26 (2H, s), 6.70 (1H,
Ki ˆ IC50=ꢀ1 ‡ ‰LŠ=Kd†
where [L] is the concentration of the [125I]a-BGTX
and Kd is the dissociation constant of a-BGTX. The
Kd value was determined in each experiment by a non-
878
Pest Manag Sci 56:875±881 (2000)

SAR of chloronicotinyl insecticides
linear regression analysis from the saturation curve of
125I]a-BGTX to nAChR using PRISM. The recipro-
cal logarithm of the Ki value, pKi, was calculated for
greater than that of imidacloprid itself. Substituted
pyridine compounds 42±44 had higher activities than
the corresponding phenyl derivatives (1 vs 42; 17 vs 43;
19 vs 44). It is also interesting that these substituted-
pyridyl compounds had much greater activities than
the three standard compounds, imidacloprid (45),
acetamiprid (46) and nitenpyram (47).
[
each compound listed in Table 1.
2.2.3 Insecticidal activity
The method for the determination of insecticidal
activity was essentially the same as that reported by
Yamamoto et al,21 in which NIA16388 was topically
applied 1h before injection of the test compound.
Female house¯ies were anesthetized using carbon
dioxide. A methanol solution (1ml) containing NIA
(2g litre 1) was topically applied to the abdomen.
After 1h at 25°C, the ¯ies were again anesthetized
using carbon dioxide and solutions in ethanol ‡ water
(50‡50 v/v; 0.24ml) containing the test compounds at
various concentrations were injected into the dorsal
side of their thoraxes.
Introduction of any substituent at the ortho position
of the benzene ring was unfavourable to the binding
activity. Among meta-substituted derivatives, the
single introduction of substituents such as F (8), Cl
(9) and CN (14) signi®cantly enhanced activity,
whereas CH3 (10), OCH3 (12), CF3 (13) and
OC6H5 (15) groups decreased it. Compound 11 (3-
NO2) was equivalent to unsubstituted compound 1 in
terms of pKi. At the para position, introduction of Cl
(17) was favourable to activity, although other halo-
gens such as F (16) and Br (18) did not change the
activity signi®cantly. It was interesting that the activity
of the 4-CN compound (26) was 1/30 of that of the
unsubstituted compound 1, whereas that of the 3-CN
isomer (14) was 120 times higher than that of the
unsubstituted compound 1. Among the multiple-
substituted derivatives, compounds 28 (3,4-F2) and
33 (3,4-Cl2) had much higher binding activities than
compound 1.
At high concentrations, the ¯ies failed to regain
consciousness. Early symptoms at lower, but effective,
concentrations included hyperactivity and tremors of
the body and legs. Some house¯ies were unable to
recover or right themselves upon awakening. All
paralyzed insects were considered to be affected. In
the preliminary test, we examined the time-response
pro®les for several compounds (data not shown).
Since the most pronounced effect was seen 1h after
injection, we used 1h for the determination of 50%
effective concentration [EC50 (M)]. Control ¯ies
showed no adverse effects from carbon dioxide, the
synergist alone or the injection procedure. Fifteen ¯ies
were used for each preparation. Six concentrations
were generally used for each compound. The recipro-
cal logarithm of the EC50 value, pEC50, was calculated
for each compound using the probit method and
de®ned as the insecticidal activity. The pEC50 values
are listed in Table 1.
Previously, we analyzed the binding activity of
imidacloprid and related compounds to the house¯y
nAChR using CoMFA17,18 and agreed with the model
proposed by Yamamoto et al,14 in which the nitrogen
atom of the pyridine ring possibly interacts with the
positive site of the nAChR. In those analyses, two
benzyl analogues, one of which was the same as
compound 1 used in this study, were also included by
superimposing the meta-C atom onto the N atom of
the pyridine ring.17,18 For this purpose, electrostatic
charges were calculated using AM1, a semi-empirical
molecular orbital method. The electronic charge of the
C atom at the meta position of compound 1 was the
most negative, being consistent with that observed for
the corresponding pyridyl compound 42, in which the
nitrogen atom was the most negatively charged (Fig 2).
Previously, we showed that the nitromethylene (or
nitroimino) moiety and a portion of the imidazolidine
ring were mainly surrounded by a sterically and
electrostatically sensitive region of the receptor, but
no electrostatic ®eld appeared to surround the
pyridine ring in that CoMFA.17 To clarify the role of
the benzyl and pyridyl moieties, classical and 3-D
QSAR analyses are in progress in our laboratory using
an expanded set of compounds.
3
RESULTS AND DISCUSSION
3.1 Binding activity
The Kd value of a-BGTX was determined in each
membrane preparation in order to calculate the Ki
value for each compound. In this series of experi-
ments, the Kd values varied from 0.24 to 3.58nM. This
large deviation seemed not to be unusual, because the
Kd value has been found to vary over thirtyfold in other
receptor binding assays using [3H]apomorphine22 and
[3H]spiperon.23 In this study, the binding activity was
expressed as pKi as described above to minimize the
deviation depending on the preparation. In addition,
compound 40 was used as a reference compound for
each membrane preparation and its standard deviation
in the pKi value was Æ 0.09 for six determinations
(Table 1).
Among the benzyl compounds, the 3-CN derivative
(14) had the highest binding activity, being about 20
times greater than that of imidacloprid. It is interesting
that the activity of compound 43, which is the nitro-
methylene congener of imidaclopid, was 300 times
Figure 2. Charges of the carbon and nitrogen atoms on the aromatic rings
calculated by the AM1 method: (A) compound 1 and (B) compound 42.
Pest Manag Sci 56:875±881 (2000)
879

H Nishiwaki et al
Wollweber and Tietjen24 suggested that imidaclo-
prid and a-BGTX have different binding sites, because
the binding activities of a large range of compounds
evaluated using [3H]imidacloprid and [125I]a-BGTX
as radioligands could not be correlated. Nevertheless,
Liu et al8 showed that the binding activities of
chloronicotinyl compounds determined using
[3H]imidacloprid and [125I]a-BGTX were correlated.
Consequently, the binding activities evaluated with
and compounds 19 and 44, these two activity values
were linearly correlated (n =15, r =0.91, s =0.43). A
similar relationship between insecticidal activity
against house¯ies and binding activity evaluated using
[3H]imidacloprid was reported by Tomizawa et al. 25
Imidacloprid (45) has the nitroimine substructure, and
acetamiprid (46) and nitenpyram (47) do not carry the
imidazolidine ring structure, and so differ from the
mother structure of the present series of compounds.
The replacement of the nitromethylene group with
nitroimine on the imidazolidine ring and the opening
of the cyclic imidazolidine moiety might be favourable
to enhancing insecticidal activity. Structural simila-
rities between two other outliers (compounds 19 and
44) involve the position of the CH3 group. Com-
pounds 19 has a CH3 group at the para position of the
benzene ring and compound 44 has that at the
corresponding position on the pyridyl moiety. The
methyl group at these positions might have a role in
increasing insecticidal activity compared with that
expected from their binding activity.
[
study with the present set of compounds.
125I]a-BGTX have been used for a structure-activity
3.2 Insecticidal activity
The insecticidal activities of three standards, imida-
cloprid (45), acetamiprid (46) and nitenpyram (47),
were very high, as expected. The activities of the
substituted pyridyl compounds 42±44 were similar to
those of the standards. Among the benzyl analogues
tested, compound 17 (4-Cl) was the most potent,
although it was 10 times less potent than imidacloprid
(Table 1). The activity of compound 14 (3-CN), the
binding activity of which was the highest among the
benzyl analogues, was slightly less than that of
compound 17. Other substituted benzyl analogues
were of similar or less potency than the unsubstituted
compound 1. Compounds 5±7, 12, 13, 18, 20±27, 29±
32, 35±39 and 41 were not assayed, because their low
binding activity indicated that their insecticidal activity
would be less than that of the parent molecule
(compound 1).
When a compound arrives at the nAChR of insects,
it interacts with the receptor to cause excitation and/or
to block by competing with endogeneously released
ACh. To evaluate the binding activity, radiolabelled
nicotine26,27 and imidacloprid6±8,28 have also been
used as radioligands. The binding activity evaluated
with these and other ligands may re¯ect the behaviour
of these compounds in the insect nervous system more
directly than a-BGTX. We have only used the com-
petitive binding activity evaluated with a-BGTX as an
in vitro assay. De¯ection of the insecticidal activity of
compounds 19 and 44±47 from that expected from the
binding activity (Fig 3) may be related to these factors.
In our earlier studies, insecticidal activity was found
to increase with nerve excitatory activity for sets of
imidacloprid-related compounds.20,29 The insecticidal
activity of compounds is governed by pharmaco-
kinetic factors as well as pharmaco-dynamic ones.
Since the compounds were injected in both the
previous and the present experiments, the penetration
phase in which chemicals move through the cuticle (a
pharmaco-kinetic factor) was eliminated. Since the
insecticidal activity of the previous sets of compounds
against the American cockroach was enhanced by the
use of NIA as well as piperonyl butoxide (PB),20,29
another pharmaco-kinetic factor (detoxi®cation)
would be more-or-less eliminated under the condi-
tions. It may be better to additionally use PB to
determine the intrinsic insecticidal activity of the
present set of compounds and to correlate with the
binding activity.
The insecticidal activity against house¯ies was
plotted against binding activity (Fig 3). Except for
imidacloprid (45), acetamiprid (46), nitenpyram (47)
In summary, the insecticidal activity of substituted
benzyl and pyridyl derivatives of imidacloprid having a
nitromethylene group at the imidazolidine ring gen-
erally increased with the binding activity. Three
standard compounds showed a much higher insecti-
cidal activity than the benzyl derivatives, but their
binding activities were close to the average of the
tested compounds. Of the benzyl compounds, the
Figure 3. Relationship between the binding activity and the insecticidal
activity against houseflies: (*) benzyl analogues, (*) pyridylmethyl
analogues with a nitromethylene moiety, (~) imidacloprid, acetamiprid and
nitenpyram. The regression line (r =0.91 and s =0.43) was derived for 15
compounds designated by open and closed circles except for compounds
19 and 44.
880
Pest Manag Sci 56:875±881 (2000)

SAR of chloronicotinyl insecticides
14 Yamamoto I, Yabuta G, Tomizawa M, Saito T, Miyamoto T and
Kagabu S, Molecular mechanism for selective toxicity of
nicotinoids and neonicotinoids. Nihon Noyaku Gakkaishi (J
Pestic Sci) 20:33±40 (1995).
3-CN derivative had the highest binding activity, but
its insecticidal activity was only 1/13 that of imidaclo-
prid.
15 Kagabu S, Studies on the synthesis and insecticidal activity of
neonicotinoid compounds. Nihon Noyaku Gakkaishi (J Pestic
Sci) 21:231±239 (1996).
ACKNOWLEDGEMENTS
We are thankful to Mr Craig Wheelock of University of
California Davis for careful reviewing this manuscript,
and to Dr Yoshihisa Ozoe of Shimane University for
invaluable suggestions for the insecticidal test. We are
indebted to Dr Isao Ueyama of Nihon Bayer Agro-
chem KK, Dr Hideki Uneme of Takeda Chemical Ind,
Ltd and Dr Akira Nakayama of Nippon Soda Co, Ltd
for providing some test compounds. A part of this
study was performed in the RI Centre of Kyoto
University.
16 Cramer III RD, Patterson DE and Bunce JD, Comparative
molecular ®eld analysis (CoMFA). 1. Effect of shape on bind-
ing of steroids to carrier proteins. J Amer Chem Soc 110:5959±
5967 (1988).
17 Okazawa A, Akamatsu M, Ohoka A, Nishiwaki H, Cho W,
Nakagawa Y, Nishimura K and Ueno T, Prediction of the
binding mode of imidacloprid and related compounds to
house-¯y head acetylcholine receptors using three-dimensional
QSAR analysis. Pestic Sci 54:134±144 (1998).
18 Okazawa A, Akamatsu M, Nishiwaki H, Nakagawa Y, Miyagawa
H, Nishimura K and Ueno T, Three-dimensional quantitative
structure-activity relationship analysis of acyclic and cyclic
chloronicotinyl insecticides. Pest Manag Sci 56:509±515
(2000).
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