Docking model of the nicotinic acetylcholine receptor and nitromethylene neonicotinoid derivatives with a longer chiral substituent and their biological activities

Article abstract of DOI:10.1016/j.bmc.2014.12.058

In the present study, nitromethylene neonicotinoid derivatives possessing substituents that contain a sulfur atom, oxygen atom or aromatic ring at position 5 on the imidazolidine ring were synthesized to evaluate their affinity for the nicotinic acetylcholine receptor (nAChR) and their insecticidal activity against adult female houseflies. Comparing the receptor affinity of the alkylated derivative with the receptor affinity of compounds possessing either ether or thioether groups revealed that conversion of the carbon atom to a sulfur atom did not influence the receptor affinity, whereas conversion to an oxygen atom was disadvantageous for the receptor affinity. The receptor affinity of compounds possessing a benzyl or phenyl group was lower than that of the unsubstituted compound. Analysis of the three-dimensional quantitative structure-activity relationship using comparative molecular field analysis demonstrated that steric hindrance of the receptor should exist around the C3 of an n-butyl group attached at position 5 on the imidazolidine ring. A docking study of the nAChR-ligand model suggested that the ligand-binding region expands as the length of the substituent increases by brushing against the amino acids that form the binding region. The insecticidal activity of the compounds was positively correlated with the receptor affinity by considering log P and the number of heteroatoms, including sulfur and oxygen atoms, in the substituents, suggesting that the insecticidal activity is influenced by the receptor affinity, hydrophobicity, and metabolic stability of the compounds.

Full text of DOI:10.1016/j.bmc.2014.12.058

Accepted Manuscript
Docking model of the nicotinic acetylcholine receptor and nitromethylene ne-
onicotinoid derivatives with a longer chiral substituent and their biological
activities
Hikaru Nagaoka, Hisashi Nishiwaki, Takuya Kubo, Miki Akamatsu, Satoshi
Yamauchi, Yoshihiro Shuto
PII:
S0968-0896(14)00916-X
http://dx.doi.org/10.1016/j.bmc.2014.12.058
BMC 11990
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 October 2014
8 December 2014
24 December 2014
Please cite this article as: Nagaoka, H., Nishiwaki, H., Kubo, T., Akamatsu, M., Yamauchi, S., Shuto, Y., Docking
model of the nicotinic acetylcholine receptor and nitromethylene neonicotinoid derivatives with a longer chiral
substituent and their biological activities, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/
j.bmc.2014.12.058
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1
2
3
Docking model of the nicotinic acetylcholine receptor and nitromethylene neonicotinoid
derivatives with a longer chiral substituent and their biological activities
4
5
6
Hikaru Nagaoka,† Hisashi Nishiwaki,*† Takuya Kubo,† Miki Akamatsu,‡ Satoshi
Yamauchi,† Yoshihiro Shuto†
7
8
†
9
Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Japan
‡
Graduate School of Agriculture, Kyoto University, Kita-Shirakawa, Sakyo-Ku, Kyoto
606-8502, Japan
* To whom correspondence should be addressed.
Tel: +81-89-946-9973, Fax: +81-89-977-4364
E-mail: nishiwaki.hisashi.mg@ehime-u.ac.jp
Keywords
Nitromethylene neonicotinoids; QSAR; docking model; Musca domestica; nicotinic
acetylcholine receptor
Abbreviations
Ac, Aplysia californica; CH-IMI, nitromethylene analogue of imidacloprid; IMI,
imidacloprid; LBD, ligand-binding domain; Ls, Lymnaea stagnalis; nAChR, nicotinic
acetylcholine receptor; NIA, propargyl propyl phenylphosphonate (Niagara 16388); QSAR,
quantitative structure-activity relationship; SAR, structure-activity relationship
1

1
2
ABSTRACT
In the present study, nitromethylene neonicotinoid derivatives possessing substituents that
contain a sulfur atom, oxygen atom or aromatic ring at position 5 on the imidazolidine ring
were synthesized to evaluate their affinity for the nicotinic acetylcholine receptor (nAChR)
and their insecticidal activity against adult female houseflies. Comparing the receptor
affinity of the alkylated derivative with the receptor affinity of compounds possessing
either ether or thioether groups revealed that conversion of the carbon atom to a sulfur atom
did not influence the receptor affinity, whereas conversion to an oxygen atom was
disadvantageous for the receptor affinity. The receptor affinity of compounds possessing a
benzyl or phenyl group was lower than that of the unsubstituted compound. Analysis of the
three-dimensional quantitative structure–activity relationship using comparative molecular
field analysis demonstrated that steric hindrance of the receptor should exist around the C3
of an n-butyl group attached at position 5 on the imidazolidine ring. A docking study of the
nAChR-ligand model suggested that the ligand-binding region expands as the length of the
substituent increases by brushing against the amino acids that form the binding region. The
insecticidal activity of the compounds was positively correlated with the receptor affinity
by considering log P and the number of heteroatoms, including sulfur and oxygen atoms, in
the substituents, suggesting that the insecticidal activity is influenced by the receptor
affinity, hydrophobicity, and metabolic stability of the compounds.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
2

1
2
1. INTRODUCTION
Neonicotinoids are neuroactive insecticides that act on the nicotinic acetylcholine
3
receptor (nAChR) at the postsynaptic membrane in insects. They are widely used as
agricultural insecticides and for residential pest control. The affinity of neonicotinoids for
nAChR is lower in mammals than in target pests, which is why these insecticides exert
highly selective toxicity against pests over mammals. Imidacloprid (IMI, 1 in Fig. 1) was
developed as the first neonicotinoid insecticide that contained pyridine and imidazolidine
rings.1 Many structure–activity relationship analyses have been reported, and some
important neonicotinoid pharmacophores have been suggested.1-3 We focused on the
ethylene moiety of the imidazolidine ring, which is considered to be at an important
metabolic position in the housefly Musca domestica.4 The ethylene moieties of IMI and its
nitromethylene analogue (CH-IMI, 2 in Fig. 1) have not been recognized as important
pharmacophores because acyclic neonicotinoids, including nitenpyram, acetamiprid, and
dinotefuran, have been developed. We eventually determined that 5R-methylated and
5R-ethylated imidacloprid derivatives (3 and 4 in Fig. 1) were equipotent to the
unsubstituted compound 2, suggesting that this region could be considered a
pharmacophore.5,6 In addition, based on quantitative structure–activity relationship (QSAR)
analyses of the receptor affinity and on a docking study using the receptor model of
houseflies combined with a synthesized alkylated derivative, it was hypothesized that a
space that can accept a certain sized substituent (up to n-propyl group, 5 in Fig. 1) should
exist around position 5 of the imidazolidine ring in the ligand-binding region of the
receptor.6 Because the ligand-binding pocket space in the receptor is constructed of several
aromatic amino acid residues, such as tyrosine and tryptophan,6 it is expected that a
substituent that interacts with these amino acid residues and backbone peptide bonds in the
ligand-binding region would have a high affinity for the receptor. The oxygen atom can
interact with peptide bonds via hydrogen bonding, and the sulfur atom and benzyl and
phenyl groups are also expected to interact with aromatic amino acid residues through
sulfur-π and π-π interactions.7
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
3

1
2
In this study, we synthesized various CH-IMI derivatives with substituents that
possessed an oxygen atom, sulfur atom, or aromatic ring, which are expected to interact
with the ligand-binding region of nAChR, to evaluate their receptor affinity and insecticidal
activity (6–18 in Fig. 1). To elucidate the physicochemical properties of ligands interacting
with the receptor, three-dimensional QSARs were analyzed using the comparative
molecular field analysis (CoMFA) method. The docking model was also reconstructed to
discuss the mode of binding to the receptor. Furthermore, the relationship between receptor
affinity and insecticidal activity against houseflies was quantitatively analyzed to determine
the factors other than receptor affinity that influence insecticidal activity.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
2. MATERIALS AND METHODS
2.1. Insects
An insecticide-susceptible strain of the housefly (Musca domestica L, Takatsuki
strain) was reared at 25°C in our laboratory.
2.2. Chemicals
Compounds 6-18 were newly synthesized using the synthetic scheme shown in Fig.
2. The reagents used for the syntheses were purchased from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan), Nacalai Tesque, Inc. (Kyoto, Japan), Tokyo Chemical
Industry Co, Ltd. (Tokyo, Japan), and Aldrich Chemical Co. (Milwaukee, WI, USA). The
metabolic inhibitor NIA 16388 (NIA; propargyl propyl phenylphosphonate) was our stock
sample.5,6 1H and 13C NMR analyses were performed using a JEOL ECS-400 NMR
spectrometer in deuterochloroform (CDCl3), deuteromethanol (CD3OD) or deuterium oxide
(D2O)
with
tetramethylsilane
(for
CDCl3
and
CD3OD)
or
sodium
3-(trimethylsilyl)-1-propanesulfonate (for D2O) as the internal standard. The authenticity of
the final compounds was also confirmed by HRMS using a Xevo Q-TOFMS (Waters, UK).
The melting points of the compounds were measured using a Yanaco melting point
apparatus (Kyoto, Japan) and were uncorrected. Optical rotation values were determined
4

1
2
using a P-2100 polarimeter (Jasco, Tokyo, Japan).
3
(S)-Methyl-3-tert-butoxycarbonyl-2,2-dimethyloxazolidine-4-formate (20, step a)
4
One hundred milliliters of a methanol solution containing D-serine (4.5 g, 43 mmol, 19)
was cooled in a salt-ice bath at 0°C, and SOCl2 (18.6 mL, 258 mmol) was added dropwise.
The resulting mixture was stirred for 12 h at ambient temperature and then concentrated in
vacuo. After coevaporating the solvent with diethyl ether multiple times to remove excess
SOCl2, the residue was dissolved in 100 mL of CH2Cl2, to which Et3N (15.7 mL, 113
mmol) was added at 0°C. To this solution was added di-tert-butyl dicarbonate (11.3 g, 52
mmol) under stirring, and the resulting mixture was refluxed until the starting material was
consumed, as determined by TLC (methanol). The reaction mixture was concentrated in
vacuo, and the residue was dissolved in ethyl acetate (100 mL) and then washed with
saturated NaHCO3 followed by washing with brine (×3). The organic layer was dried over
Na2SO4, filtered, and concentrated to afford N-tert-butoxycarbonyl serine methyl ester
(Boc-Ser-OMe) as the intermediate as an oil, which was used in the subsequent reaction
without further purification. The crude Boc-NH-Ser-OMe was dissolved in a mixture of
acetone (100 mL) and 2,2-dimethoxypropane (81.0 mL, 659 mmol). To the resulting
mixture was added BF3-Et2O complex (1.1 mL, 9 mmol) at ambient temperature, and the
reaction mixture was stirred for 12 h. After determining that the reaction was complete by
TLC, 1.1 mL of Et3N was added to the mixture to quench the reaction, and the solvent was
removed in vacuo. The brown oil was then partitioned between Et2O and saturated
NaHCO3 (aq.). The aqueous layer was extracted with Et2O (×5), and the organic layers
were combined, dried over Na2SO4, and concentrated. The resulting brown oil was purified
by column chromatography (hexane:ethyl acetate = 10:1) to afford 20 as a yellow oil (9.6 g,
84%). The 1H and 13C NMR spectra were consistent with previously reported spectra.8
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
(R)-3-tert-Butoxycarbonyl-2,2-dimethyl-4-phthalimidomethyloxazolidine (21, step b)
After a mixture of NaBH4 (5.2 g, 139 mmol) and LiCl (5.9 g, 139 mmol) in dry EtOH (30
5

1
2
mL) was stirred for 30 min at 0°C, compound 20 (6.0 g, 23 mmol) dissolved in 15 mL of
dry THF was added. The resulting mixture was warmed to room temperature and stirred for
12 h. The precipitate was filtered over Celite and washed with EtOH. The filtrate was then
evaporated and partitioned between ethyl acetate and brine. After the aqueous layer was
extracted with ethyl acetate (×5), the organic layer was dried over Na2SO4 and concentrated
in vacuo to afford the intermediate alcohol as a yellow oil. To a reaction mixture of dry
THF (100 mL) containing 11 g (42 mmol) of PPh3, 8.2 g (56 mmol) of phthalimide, and 6.6
g (28 mmol) of the yellow oil, 22.7 mL (40% in toluene, 50 mmol) of diethyl
azodicarboxylate dissolved in dry THF (20 mL) was added dropwise while stirring in an ice
bath. After 12 h at ambient temperature, the solvent was evaporated, and the resulting
residue was purified by column chromatography (hexane:ethyl acetate = 3:1) to afford 21
as white crystals (5.4 g, 53%).
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
25
Mp 124-126. [α]D -32.4 (c 1.0, CHCl3). NMR δH (CDCl3): 7.85 (2H, m), 7.72 (2H, m),
4.36 (1H, m), 4.00-3.81 (4H, m), 1.64 (3H, s), 1.46 (3H, s), 1.33 (9H, s). NMR δC (CDCl3):
Major rotamer, 168.5, 152.6, 134.1, 132.3, 123.3, 94.4, 80.2, 65.8, 55.8, 40.7, 28.2, 27.2,
24.3; Minor rotamer, 168.3, 151.7, 133.6, 132.1, 123.1, 94.1, 80.2, 65.5, 55.2, 40.2, 28.0,
26.7, 22.9.
(2'S)-N-(2'-(6-Chloro-3pyridyl)methylamino)-3'-hydroxypropyl-phthalimide (22, step c)
A concentrated HCl solution (20 mL) was added to THF (30 mL) containing 21 (2.0 g, 6
mmol) while stirring at ambient temperature to remove the Boc group. After stirring for 30
min, the solvent was evaporated to afford a deprotected amine hydrochloride salt as a white
solid, which was used in the subsequent reaction without further purification. The crystal
was dissolved in 30 mL of CH3CN, to which 5.3 mL (38 mmol) of Et3N and 0.9 g (6 mmol)
of 2-chloro-5-chloromethylpyridine hydrochloride were added. After refluxing overnight,
the solvent was evaporated, and the resulting residue was purified by column
chromatography (hexane:ethyl acetate = 1:5) to afford 22 as white crystals (0.4 g, 19%).
25
Mp 103-105. [α]D -15.7 (c 0.36, CHCl3). NMR δH (CDCl3): 8.33 (1H, d, J = 2.8 Hz),
6

1
2
7.87-7.85 (2H, m), 7.77-7.75 (2H, m), 7.66 (1H, dd, J = 8.4, 2.4 Hz), 7.23 (1H, d, J = 8 Hz),
3.96-3.79 (4H, m), 3.60 (1H, dd, J = 11.6, 5.2 Hz), 3.49 (1H, dd, J = 12.0, 4.4 Hz), 2.96
(1H, m), 2.85 (1H, br). NMR δC (CDCl3): 169.1, 150.2, 149.4, 138.8, 134.5, 134.4, 131.7,
124.0, 123.5, 61.4, 57.7, 47.8, 38.1.
3
4
5
6
(R)-3-tert-Butoxycarbonyl-2,2-dimethyloxazolidine-4-methoxyethane (24, step d)
7
To a dry THF solution containing compound 23 (2.5 g, 11 mmol), which was prepared
from L-serine via steps a and b, 0.5 g (20 mmol) of NaH was added. After stirring for 5 min,
3.4 g (22 mmol) of iodoethane was added. After stirring for 10 h at ambient temperature, 1
mL of H2O was added to the reaction mixture, the solvent was removed in vacuo, and the
resulting residue was purified by column chromatography (hexane:ethyl acetate = 4:1) to
afford 24 as an oil (1.3 g, 47%). [α]D25 -18.6 (c 1.27, CHCl3). NMR δH (CDCl3): 4.06 (1H,
m), 3.95 (2H, m), 3.51 (2H, m), 3.46 (2H, m), 1.55 (6H, m), 1.48 (9H, s), 1.19 (3H, m).
NMR δC (CDCl3): Major rotamer, 151.6, 93.6, 79.6, 69.8, 66.5, 65.6, 56.3, 28.3, 26.7,
23.0,15.1; Minor rotamer, 152.1, 93.1, 80.1, 69.0, 66.5, 65.3, 56.4, 28.3, 27.4, 24.3, 15.1.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
(S)-tert-Butyl (1-ethoxy-3-hydroxypropane-2-yl)carbamate (25, step e)
To 20 mL of a MeOH solution containing compound 24 (1.3 g, 5 mmol), 2.0 g (10 mmol)
p-toluenesulfonic acid was added, and the reaction mixture was stirred at ambient
temperature for 18 h. The solvent was evaporated, and the resulting residue was dissolved
in CHCl3. The organic layer was washed with saturated NaHCO3 (aq.) (×3). The organic
25
layer was dried over Na2SO4 and concentrated to afford 25 as an oil (1.4 g, 71%). [α]D
-4.2 (c 1.02, CHCl3). NMR δH (CDCl3): 3.50 (3H, m), 2.92 (2H, m), 1.26 (9H, s), 0.87 (5H,
m). NMR δC (CDCl3): 130.8, 95.2, 72.5, 66.9, 31.9, 30.0, 22.7, 14.1.
(S)-2-Amino-3-(ethylthio)propanoic acid (27, step f)
To a 0.2 M Ba(OH)2 solution (70 mL) of D-cysteine (26, 2.0 g, 13 mmol), 2.8 mL (15
mmol) of diethyl sulfate was added while stirring at ambient temperature. After 3 h, 0.6 mL
7

1
2
of conc. H2SO4 was added to stop the reaction, and the precipitate was filtered. After
evaporation of the resulting filtrate, the pH was adjusted to 5.0 with NH3 aq., and the
solution was recrystallized with EtOH to afford 27 as white crystals (1.6 g, 85%). Mp
3
25
174-176. [α]D +36.6 (c 0.1, MeOH). NMR δH (D2O): 3.93 (1H, q, J = 4.0 Hz), 3.14 (1H,
4
5
dd, J = 15.2, 4.0 Hz), 3.04 (1H, dd, J = 14.4, 7.2 Hz), 2.63 (2H, dd, J = 14.4, 6.4 Hz), 1.25
(3H, t, J = 7.4 Hz). NMR δC (D2O): 176.0, 56.4, 34.4, 28.3, 16.7.
6
7
8
N-tert-Butoxycarbonyl-D-norleucinol (28, step g)
9
LiAlH4 (1.8 g, 46 mmol) was suspended in 50 mL of dry THF in an ice bath, and then
D-norleucine (3.0 g, 23 mmol) was slowly added. The reaction mixture was warmed to
room temperature and refluxed for 5 h. Three milliliters of H2O and 1.5 mL of 2 M NaOH
aq. were added to the reaction mixture to quench the reaction, and the quenched solution
was used in the subsequent reaction without further purification. To the mixture solution,
5.5 g (23 mmol) of di-tert-butyl dicarbonate was added, and the reaction mixture was
refluxed for 5 h. The resulting precipitate was filtered over Celite, and the precipitate was
washed with 150 mL of THF. The filtrate was then concentrated in vacuo to afford 28 as an
oil, which was used in the subsequent reaction without further purification because no
by-products were observed in the NMR spectra. (5.8 g, quant) [α]D25 +51.5 (c 1.0, CHCl3).
NMR δH (CDCl3): 4.85 (1H, br), 3.62-3.54 (2H, m), 3.20 (1H, m), 1.54-1.52 (2H, m), 1.45
(9H, s), 1.39-1.29 (4H, m), 0.90 (3H, t, J = 2.6 Hz). NMR δC (CDCl3): 156.5, 79.3, 65.5,
52.7, 31.1, 28.3, 27.3, 22.5, 13.9.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
(2'R)-N-(2'-tert-Butoxycarbonylamino)hexylphthalimide (29, step h)
To a reaction mixture of dry THF (80 mL) containing 9.0 g (34 mmol) of PPh3, 6.7 g (46
mmol) of phthalimide, and 5.0 g (23 mmol) of compound 28, 9.7 mL (40% in toluene, 41
mmol) of diethyl azodicarboxylate dissolved in dry THF (20 mL) was added dropwise
while stirring in an ice bath. After stirring for 12 h at ambient temperature, the solvent was
evaporated, and the resulting residue was purified by column chromatography
8

25
1
2
(hexane:ethyl acetate = 3:1) to afford 29 as white crystals (5.4 g, 68%). Mp 115-118. [α]D
-20.7 (c 1.0, CHCl3). NMR δH (CDCl3): 7.84-7.83 (2H, m), 7.70-7.68 (2H, m), 4.59 (1H,
br), 3.97 (1H, m), 3.74-3.63 (2H, m), 1.56 (1H, m), 1.47-1.30 (5H, m), 1.21 (9H, s),
0.91-0.90 (3H, m). NMR δC (CDCl3): 168.4, 155.6, 133.7, 132.0, 123.1, 78.9, 49.6, 42.4,
32.5, 28.0, 27.8, 22.4, 13.9.
3
4
5
6
7
(2'R)-N-(2'-(6-Chloro-3-pyridyl)methylamino)hexylphthalimide (30, step i)
8
To 50 mL of a THF solution containing 5.2 g (15 mmol) of compound 29, a conc. HCl
solution (20 mL) was added dropwise while stirring at ambient temperature. After stirring
overnight, the solvent was distilled to afford the deprotected amine as a white solid, which
was used in the subsequent reaction without further purification. The product was dissolved
in 30 mL of CH3CN, to which 10.7 mL (77 mmol) of Et3N and 2.5 g (15 mmol) of
2-chloro-5-chloromethylpyridine hydrochloride were added. After the reaction mixture was
refluxed overnight, the solvent was evaporated, and the resulting residue was purified by
column chromatography (hexane:ethyl acetate = 2:1) to afford 30 as a yellow oil (1.5 g,
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
25
27%). [α]D -23.3 (c 1.0, CHCl3). NMR δH (CDCl3): 8.25 (1H, d, J = 2.4 Hz), 7.84-7.82
(2H, m), 7.75-7.72 (2H, m), 7.57 (1H, dd, J = 8.4, 2.4 Hz), 7.12 (1H, d, J = 8.0 Hz), 3.86
(1H, d, J = 13.6 Hz), 3.78 (1H, d, J = 13.6 Hz), 3.72-3.69 (2H, m), 2.90 (1H, m), 1.34 (6H,
m), 0.91 (3H, t, J = 6.7 Hz). NMR δc (CDCl3): 168.6, 149.9, 149.3, 138.8, 135.0, 134.0,
131.8, 123.7, 123.2, 56.1, 47.1, 41.3, 32.4, 27.8, 22.7, 13.9.
(2'S)-N-(2'-(6-Chloro-3pyridyl)methylamino)-3'-(methoxymethoxy)propyl-phthalimide (31,
step j)
To a reaction mixture of dry CH2Cl2 (2 mL) containing 0.2 g (0.6 mmol) of compound 22
and 0.4 mL (2 mmol) of N,N-diisopropylethylamine, 0.09 mL (1 mmol) of methoxymethyl
chloride was added while stirring at ambient temperature. After 1 h, 0.5 mL of MeOH was
added to the reaction mixture, which was then stirred for an additional 30 min. The reaction
mixture was washed with saturated NaHCO3 (×3). The organic layer was dried over
9

25
Na2SO4, filtered, and concentrated to afford 31 as a red oil (0.28 g, quant.). [α]D -62.1 (c
1
2
2.0, CHCl3). NMR δH (CDCl3): 7.99 (1H, d, J = 2.0 Hz), 7.73-7.64 (4H, m), 7.30 (1H, dd, J
= 8.2, 2.6 Hz), 6.67 (1H, d, J = 8.4 Hz), 5.23 (2H, s), 4.54 (2H, d, J = 2.4 Hz), 4.42 (1H, d,
J = 6.4 Hz), 4.35 (1H, d, J = 6.4 Hz), 4.01 (1H, m), 3.56 (2H, d, J = 2.4 Hz), 2.90 (3H, s).
NMR δC (CDCl3): 167.9, 149.8, 149.6, 139.0, 133.9, 133.1, 131.4, 123.4, 122.9, 96.5, 66.1,
60.8, 56.1, 54.3, 39.2.
3
4
5
6
7
8
(R)-5-Butyl-1-(6-chloro-3-pyridylmethyl)-2-(nitromethylene)imidazolidine (6, step k).
To an EtOH solution containing 1.5 g (4 mmol) of compound 30, 1.0 mL (21 mmol) of
hydrazine monohydrate was added, and the mixture was refluxed for 3 h while stirring.
After the insoluble residue was removed by filtration, the resulting filtrate was evaporated
to afford the crude deprotected amine as an oil, which was used for the subsequent reaction
without further purification. After the oil was dissolved in 25 mL of ethanol, 0.6 g (3
mmol) of 1,1-bis(methylthio)-2-nitroethylene and 0.5 g (3 mmol) of K2CO3 were added,
and the reaction mixture was refluxed overnight. After removing K2CO3 by filtration, the
filtrate was evaporated in vacuo, and the resulting residue was purified by column
chromatography (ethyl acetate) to afford 6 as white crystals (0.8 g, 76%). Mp 102-103.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
25
[α]D +96.1 (c 1.15, CHCl3). NMR δH (CDCl3): 8.73 (1H, br), 8.29 (1H, d, J = 2.4 Hz),
7.60 (1H, dd, J = 8.0, 2.0 Hz), 7.35 (1H, d, J = 8.0 Hz), 6.57 (1H, s), 4.38 (2H, q, J = 16.8
Hz), 3.92 (1H, q, J = 10.0 Hz), 3.85 (1H, q, J = 8.7 Hz), 3.50 (1H, t, J = 9.0 Hz), 1.74 (1H,
m), 1.54 (1H, m), 1.31-1.24 (4H, m), 0.88 (3H, t, J =7.0 Hz). NMR δC (CDCl3): 159.1,
151.0, 148.1, 137.5, 129.9, 124.5, 96.4, 60.2, 47.7, 44.0, 31.4, 26.5, 22.3, 13.7. ESIMS m/z
[M+H]+: calcd for C14H20N4O2Cl, 311.1262; found, 311.1275.
S-1-(6-Chloro-3-pyridylmethyl)-5-hydroxymethyl-2-(nitromethylene)imidazolidine (7)
25
Mp 189-192. [α]D +10.1 (c 1.09, MeOH). NMR δH (CD3OD): 8.36 (1H, d, J =
2.4Hz), 7.80 (1H, dd, J = 8.6, 2.6 Hz), 7.48 (1H, d, J = 8.4 Hz), 6.70 (1H, s), 4.62 (2H, d, J
= 12.8 Hz), 4.02 (1H, m), 3.90 (1H, t, J = 10.8 Hz), 3.78 (1H, dd, J = 12.2, 3.4 Hz), 3.69
10

1
2
(1H, dd, J = 10.6, 7.4 Hz), 3.63 (1H, dd, J = 12.0, 4.0 Hz). NMR δC (CD3OD): 151.8, 149.7,
139.9, 133.0, 125.8, 97.6, 62.7, 61.8, 46.2, 45.4, 30.7. ESIMS m/z [M+H]+: calcd for
C11H14N4O3Cl, 285.0749; found, 285.0754.
3
4
5
S-1-(6-Chloro-3-pyridylmethyl)-5-methoxymethyl-2-(nitromethylene)imidazolidine (8)
Mp 148-151. [α]D25 +28.5 (c 0.12, CHCl3). NMR δH (CDCl3): 8.70 (1H, br), 8.31 (1H, d, J
= 2.4Hz), 7.60 (1H, dd, J = 8.4, 2.4 Hz), 7.35 (1H, d, J = 8.0 Hz), 6.54 (1H, s), 4.46 (2H, q,
J = 16.8 Hz), 4.04 (1H, m), 3.91 (1H, t, J = 10.2 Hz), 3.57 (1H, m), 3.52-3.45 (2H, m), 3.27
(3H, s). NMR δC (CDCl3): 159.4, 151.3, 148.4, 137.6, 130.2, 124.5, 96.6, 72.6, 59.8, 59.1,
45.4, 45.1. ESIMS m/z [M+H]+: calcd for C12H16N4O3Cl, 299.0902; found, 299.0911.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
S-1-(6-Chloro-3-pyridylmethyl)-5-ethoxymethyl-2-(nitromethylene)imidazolidine (9)
Mp 123-125. [α]D25 +105.6 (c 0.51, CHCl3). NMR δH (CDCl3): 8.70 (1H, br), 8.32 (1H, d, J
= 2.4Hz), 7.60 (1H, dd, J = 8.2, 2.6 Hz), 7.35 (1H, d, J = 8.0 Hz), 6.54 (1H, s), 4.47 (2H, q,
J = 18.5 Hz), 4.04 (1H, m), 3.89 (1H, t, J = 10.4 Hz), 3.57 (1H, m), 3.55-3.50 (2H, m), 3.41
(2H, q, J = 7.0 Hz), 1.14 (3H, t, J = 7.0 Hz). NMR δC (CDCl3): 159.4, 151.4, 148.5, 137.6,
130.1, 124.5, 96.6, 70.8, 67.1, 59.8, 45.5, 45.2, 14.9. ESIMS m/z [M+H]+: calcd for
C13H18N4O3Cl, 313.1056; found, 313.1067.
S-1-(6-Chloro-3-pyridylmethyl)-2-(nitromethylene)-5-propoxymethylimidazolidine (10)
Mp 111-114. [α]D25 +101.7 (c 1.05, CHCl3). NMR δH (CDCl3): 8.69 (1H, br), 8.31 (1H, d, J
= 2.4Hz), 7.60 (1H, dd, J = 8.2, 2.0 Hz), 7.34 (1H, d, J = 8.0 Hz), 6.53 (1H, s), 4.47 (2H, q,
J = 17.5 Hz), 4.05 (1H, m), 3.90 (1H, t, J = 10.0 Hz), 3.59-3.50 (3H, m), 3.32 (2H, t, J =
6.6 Hz), 1.57-1.48 (2H, m), 0.87 (3H, t, J = 7.4 Hz). NMR δC (CDCl3): 159.4, 151.3, 148.5,
137.6, 130.2, 124.5, 96.6, 73.4, 71.0, 59.9, 45.5, 45.2, 22.6, 10.5. ESIMS m/z [M+H]+:
calcd for C14H20N4O3Cl, 327.1229; found, 327.1224.
S-1-(6-Chloro-3-pyridylmethyl)-5-methylthiomethyl-2-(nitromethylene)imidazolidine (11)
11

1
2
Mp 115-117. [α]D25 +102.9 (c 0.13, CHCl3). NMR δH (CDCl3): 8.71 (1H, br), 8.31 (1H, d, J
= 2.4Hz), 7.60 (1H, dd, J = 8.4, 2.4 Hz), 7.37 (1H, d, J = 8.4 Hz), 6.55 (1H, s), 4.42 (2H, d,
J = 4.0 Hz), 4.02 (1H, m), 3.98 (1H, q, J = 9.4 Hz), 3.72 (1H, dd, J = 9.6, 6.0 Hz), 2.79 (1H,
dd, J = 13.4, 3.8 Hz), 2.68 (1H, dd, J = 13.2, 7.6 Hz), 2.10 (3H, s). NMR δC (CDCl3): 159.2,
151.6, 148.3, 137.6, 129.6, 124.8, 96.7, 59.2, 47.7, 44.8, 36.3, 16.1. ESIMS m/z [M+H]+:
calcd for C12H16N4O2SCl, 315.0680; found, 315.0683.
3
4
5
6
7
8
S-1-(6-Chloro-3-pyridylmethyl)-5-ethylthiomethyl-2-(nitromethylene)imidazolidine (12)
Mp 114-117. [α]D25 +57.1 (c 0.92, CHCl3). NMR δH (CDCl3): 8.71 (1H, br), 8.31 (1H, d, J
= 2.8Hz), 7.59 (1H, dd, J = 8.2, 2.6 Hz), 7.37 (1H, d, J = 8.0 Hz), 6.55 (1H, s), 4.41 (2H, d,
J = 4.8 Hz), 3.97 (2H, m), 3.72 (1H, m), 2.81 (1H, dd, J = 13.4, 3.8 Hz), 2.69 (1H, dd, J =
13.4, 7.4 Hz), 2.52 (2H, q, J = 7.3 Hz), 1.23 (3H, t, J = 7.4 Hz). NMR δC (CDCl3): 159.2,
151.7, 148.4, 137.6, 129.5, 124.8, 96.7, 59.5, 47.7, 44.8, 33.8, 26.8, 14.7. ESIMS m/z
[M+H]+: calcd for C13H18N4O2SCl, 329.0825; found, 329.0839.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
S-1-(6-Chloro-3-pyridilmethyl)-2-(nitromethylene)-5-propylthiomethylimidazolidine (13)
Mp 90-93. [α]D25 +101.7 (c 1.05, CHCl3). NMR δH (CDCl3): 8.70 (1H, br), 8.31 (1H, d, J =
2.4Hz), 7.59 (1H, dd, J = 8.0, 2.4 Hz), 7.37 (1H, d, J = 8.4 Hz), 6.54 (1H, s), 4.41 (2H, d, J
= 5.2 Hz), 3.97 (2H, m), 3.72 (1H, m), 2.80 (1H, dd, J = 13.0, 3.8 Hz), 2.68 (1H, dd, J =
13.4, 7.4 Hz), 2.46 (2H, t, J = 7.2 Hz), 1.56 (2H, m), 0.97 (3H, t, J = 7.2 Hz). NMR δC
(CDCl3): 159.2, 151.7, 148.4, 137.6, 129.6, 124.8, 96.6, 59.5, 47.7, 44.8, 34.9, 34.2, 22.8,
13.3. ESIMS m/z [M+H]+: calcd for C14H20N4O2SCl, 343.0989; found, 343.0996.
R-1-(6-Chloro-3-pyridylmethyl)-5-methoxyethyl-2-(nitromethylene)imidazolidine (14)
Mp 69-72. [α]D25 +8.32 (c 0.75, CHCl3). NMR δH (CDCl3): 8.71 (1H, br), 8.29 (1H, d, J =
2.4Hz), 7.57 (1H, dd, J = 8.2, 2.2 Hz), 7.35 (1H, d, J = 8.0 Hz), 6.56 (1H, s), 4.36 (2H, d, J
= 7.2 Hz), 3.95 (2H, m), 3.58 (1H, m), 3.42 (2H, t, J = 5.8 Hz), 3.29 (3H, s), 2.01 (1H, m),
1.81 (1H, m). NMR δC (CDCl3): 159.3, 151.5, 148.3, 137.5, 129.8, 124.7, 96.7, 68.3, 58.9,
12

1
2
58.7, 48.3, 44.4, 32.2. ESIMS m/z [M+H]+: calcd for C13H18N4O3Cl, 313.1055; found,
313.1067.
3
4
S-1-(6-Chloro-3-pyridylmethyl)-5-(methoxymethoxy)methyl-2-(nitromethylene)imidazolid
ine (15)
5
6
Mp 129-130. [α]D25 +142.8 (c 0.16, CHCl3). NMR δH (CDCl3): 8.71 (1H, br), 8.32
(1H, d, J = 2.8Hz), 7.59 (1H, dd, J = 8.4, 2.8 Hz), 7.35 (1H, d, J = 8.4 Hz), 6.55 (1H, s),
4.56 (2H, q, J = 6.7 Hz), 4.46 (2H, d, J = 2.8 Hz), 4.06 (1H, m), 3.93 (1H, t, J = 10.2 Hz),
3.66 (2H, d, J = 5.2 Hz), 3.62 (1H, m), 3.32 (3H, s). NMR δC (CDCl3): 159.4, 151.5, 148.5,
137.6, 129.9, 124.6, 96.7, 96.6, 67.4, 59.5, 55.8, 45.3, 45.2. ESIMS m/z [M+H]+: calcd for
C13H18N4O4Cl, 329.1004; found, 329.1017.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
S-1-(6-Chloro-3-pyridilmethyl)-5-methylthioethyl-2-(nitromethylene)imidazolidine (16)
Mp 138-140. [α]D25 +43.7 (c 0.2, CHCl3). NMR δH (CDCl3): 8.72 (1H, br), 8.30 (1H, d, J =
2.8 Hz), 7.59 (1H, dd, J = 8.2, 2.8 Hz), 7.37 (1H, d, J = 8.4 Hz), 6.56 (1H, s), 4.38 (2H, q, J
= 17.7 Hz), 4.00 (1H, m), 3.96 (1H, m), 3.55 (1H, m), 2.48 (2H, m), 2.08 (3H, s), 2.02 (1H,
m),1.87(1H, m). NMR δC (CDCl3): 159.2, 151.5, 148.3, 137.6, 129.7, 124.7, 96.7, 59.2,
47.8, 44.5, 31.2, 29.2, 15.7. ESIMS m/z [M+H]+: calcd for C13H18N4O2SCl, 329.0833;
found, 329.0839.
R-1-(6-Chloro-3-pyridylmethyl)-2-(nitromethylene)-5-phenylimidazolidine (17)
Mp 237-239. [α]D25 +88.1 (c 0.5, CHCl3). NMR δH (CDCl3): 8.81 (1H, br), 8.05 (1H, d, J =
2.4 Hz), 7.45 (4H, m), 7.32 (1H, d, J = 8.4 Hz), 7.27 (1H, d, J = 2.8 Hz), 7.26 (1H, d, J =
2.4 Hz), 6.73 (1H, s), 4.71 (1H, t, J = 9.0 Hz), 4.32 (1H, d, J = 16.4 Hz), 4.17 (1H, t, J =
10.2 Hz), 3.94 (1H, d, J = 16.0 Hz), 3.74 (1H, t, J = 9.4 Hz). NMR δC (CDCl3): 159.0,
151.7, 149.0, 138.1, 136.4, 129.8, 129.7, 128.9, 127.5, 124.8, 96.8, 63.7, 51.1, 44.0. ESIMS
m/z [M+H]+: calcd for C16H16N4O2Cl, 331.0953; found, 331.0962.
13

1
2
R-5-Benzyl-1-(6-chloro-3-pyridylmethyl)-2-(nitromethylene)imidazolidine (18)
Mp 137-139. [α]D25 +57.4 (c 1.4, CHCl3). NMR δH (CDCl3): 8.68 (1H, br), 8.18 (1H, d, J =
2.0 Hz), 7.51 (1H, dd, J = 8.0, 2.8 Hz), 7.31 (4H, m), 7.10 (1H, d, J = 8.4 Hz), 7.09 (1H, d,
J = 7.6 Hz), 6.58 (1H, s), 4.30 (2H, d, 16.6 Hz), 4.05 (1H, m), 3.73 (1H, t, J = 9.8 Hz), 3.54
(1H, dd, J = 9.6, 6.8 Hz), 3.10 (1H, dd, J = 14.0, 5.6 Hz), 2.79 (1H, dd, J = 13.4, 8.6 Hz).
NMR δC (CDCl3): 159.2, 151.8, 148.4, 137.6, 135.1, 129.4, 129.2, 128.9, 127.6, 124.8,
96.7, 60.9, 47.7, 44.7, 38.7. ESIMS m/z [M+H]+: calcd for C17H18N4O2Cl, 345.1114; found,
345.1118.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
2.3. Evaluation of Receptor Affinity
The assay method was essentially the same as that in our previous reports.5,6 From
the concentration–response curve, the molar concentration for 50% inhibition (IC50) of
[3H]imidacloprid binding to the receptor was calculated. The Ki value was calculated
according to the following equation using PRISM ver 5.0:
Ki = IC50/1 + ([L]/Kd)
where [L] is the final concentration of the radioligand (10 nM) and Kd (3.66 nM) is the
dissociation constant of [3H]imidacloprid for the receptor fraction. The Ki values of the test
compounds were obtained from three separate assays performed in duplicate and are listed in
Table 1.
2.4. 3D-QSAR Analysis Using CoMFA
For CoMFA, compounds 1–18, which were synthesized in this study, and
compounds 19–50, whose pKi values have previously been reported,6 were included in the
data set. The calculations for the optimization and superposition of the test chemicals were
performed using the molecular modeling software package SYBYL ver. 7.1 (Tripos
Associates, Inc., St Louis, MO). Following the previous report, the initial conformations of
the compounds were constructed and their structures were optimized.6 For the
superposition of these compounds, four atoms were selected on the basis of our previous
14

1
2
report,6,9,10 i.e., the nitrogen atoms of the pyridine ring and at the 1 position of the
imidazolidine ring, the carbon atom at the 2 position of the imidazolidine ring, and the
bridging carbon atom between the aromatic and imidazolidine rings. The potential energy
fields of each superposed conformer were calculated at the lattice intersections. The
electrostatic (Coulomb potential) and steric (Lennard-Jones potential) field energies were
calculated at each lattice point using a sp3-carbon atom with a charge of +1.0 as a probe.
The data for the receptor affinities of compounds 1–50 were correlated with these
parameters using the partial least-squares method. The steric and electrostatic potential
contour maps with n-propylthiomethyl CH-IMI 13 determined using Eq. 1 presented in
section 3.2 of the Results are shown in Fig. 3. The pKi values calculated using Eq. 1 are
shown in Table 2.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
2.5. Construction of Ligand-binding Domain Model of Housefly nAChR Combined
with a CH-IMI Derivative Using PDFAMS Software
Based on the crystallographic data of Ac-AChBP bound with IMI (PDB, 3C79), the
homology model of the ligand-binding domain (LBD) in the housefly nAChR was
constructed using the homology modeling software PDFAMS Pro 2.0 (In-Silico Sciences,
Inc., Tokyo, Japan) according to previous reports.6,12 The amino acid sequence of subunit 6
(isoform II) of M. domestica AChR (GenBank ID ABJ09669), which was the most common
among the six splicing variants in subunit 6 of the housefly receptor,13 was aligned with the
sequence of Ac-AChBP using PDFAMS, as shown in Fig. 4. In our previous study, we
confirmed that employing the sequence of α2 (GenBank ID ABD37617) or α5 (GenBank ID
ABY40460) subunit rather than the sequence of subunit 6 to construct the ligand-binding
domain model did not affect the results.6 Thus, only subunit 6 was considered in the present
study. The LBD was constructed using two of the same subunits because the Ac-AChBP
template was constructed as a homopentamer, and it is currently unknown whether the
nAChR of houseflies is constructed as a homopentamer or heteropentamer. Using the
simulated annealing method,14 the three-dimensional structure of the LBD was constructed.
15

1
2
The coordinate of IMI was fixed during the simulated annealing. The constructed model was
then energy-minimized using the force field and partial charges of the molecular mechanics
MMFF94.15,16 A graphical image of the surface of the ligand-binding pocket was created
using the multichannel surface tool of the MOLCAD module in the SYBYL software (Fig.
5).
3
4
5
6
7
2.6. Docking Study Using FRED Software
8
For the docking studies, MAKE RECEPTOR, OMEGA, FRED, and VIDA of OpenEye
Scientific Software Inc. (Santa Fe, NM) were employed. The CH-IMI-binding region of the
housefly nAChR model constructed in section 2.5., which was adopted as a template
receptor, was calculated using the “MAKE RECEPTOR” tool (ver. 3.0.0). The mol2 file of
the three-dimensional structure of the compound possessing an n-propylthiomethyl group
(13), which was constructed using the Sybyl software for CoMFA analysis, was submitted
to OMEGA (ver. 2.5.1.4) to generate possible conformers. Docking of compound 13 was
performed using FRED (Fast Rigid Exhaustive Docking; ver. 3.0.0) against up to 200
energy-stable conformers generated by OMEGA. VIDA (ver. 4.2.1) was used to view the
models, as shown in Fig. 6.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
2.7. Evaluation of Insecticidal Activity
The assay method was essentially the same as that used in our previous report.5,6
To evaluate the insecticidal activity, female houseflies anesthetized using carbon dioxide
were topically treated with methanol containing synergists piperonyl butoxide (PBO) and
NIA16388 (NIA) [0.2% (w/v)]. After 1 h, 0.22 µL of a 50% ethanol solution containing a
test chemical at various concentrations was injected into the dorsal side of the thorax of
reanesthetized flies. Insecticidal activity was evaluated 1 h after injection. The ED50 values
(effective dose for inducing paralysis or death in 50% of the houseflies) were calculated
using a probit transformation and are listed in Table 1.
16

1
2
2.8. Analysis of the Relationship between Receptor Affinity and Insecticidal Activity
Linear regression analysis between the receptor affinity and insecticidal activity
was performed using the QREG 2.05 software.17 The hydrophobicities, log P, of
compounds 3 – 18, 28 and 32 were measured using the shake flask method in a
water/n-octanol system.18 The log P values of compounds 19 - 21, 22 - 24, 25 - 27, 31, and
33 – 35 were referred to as the values of compounds 3, 4, 5, 28 and 32, respectively,
because the corresponding compound has the same substituent. The numbers of sulfur and
oxygen atoms were defined as the indicator valuables, such as Ithioether and Iether, respectively.
Compounds 29 and 30 were omitted from the analysis because their insecticidal activity
could not be measured even when applied at the high dose. The physicochemical
parameters employed are listed in Table 3.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
3. RESULTS
3.1. Receptor Affinity
The inhibition constant Ki (nM) was employed as an indicator of receptor affinity
(Table 1). The receptor affinity decreased as the number of carbon atoms in the side chain
increased from a methyl group to a propyl group (compound 3 vs. 5), whereas the affinity
of compound 6, which possessed a butyl group, was 4.6-fold higher than the affinity of
compound 5, suggesting that the increase in the number of carbon atoms in the linear
direction is not always disadvantageous in terms of affinity. Comparing the affinity of
compound 7 with the affinity of compound 3, the introduction of a hydroxyl group was
found to be disadvantageous for affinity. The receptor affinities of compounds 8, 9, and 10,
in which the hydroxyl group of compound 7 was replaced with a methoxy, ethoxy, and
propoxy group, respectively, were 169-, 37-, and 2.4-fold lower than the receptor affinity of
compound 7, respectively. The introduction of oxygen atoms was disadvantageous for
affinity (for example, propyl 5 vs. methoxymethyl 8), but elongation of the carbon chains
(from 8 to 10) increased the binding affinity. The affinities of compounds 11, 12, and 13, in
which the oxygen atoms of compounds 8, 9, and 10 were changed to sulfur atoms, were 46-,
17

1
2
58-, and 4.6-fold higher than the affinities of the corresponding ether compounds,
respectively, suggesting that the introduction of sulfur atoms was more advantageous for
affinity than oxygen atoms. In addition, the affinities of compounds 11 and 12 were 2-fold
lower than the affinities of compounds 5 and 6, demonstrating that conversion of carbon
atoms to sulfur atoms did not remarkably influence the receptor affinity. The conversion of
the carbon atom to a sulfur atom did not influence the receptor affinity, whereas the
conversion to an oxygen atom decreased the affinity, suggesting that the atom at this
position of the substituent would interact with the receptor. Compounds 9 and 14, in which
the carbon atoms at the 2- and 3-positions of the n-butyl group were converted to oxygen
atoms, respectively, exhibited similar receptor affinities, demonstrating that the position of
the oxygen atom introduced into the n-butyl group did not affect affinity. The affinity of
compound 15, which possessed two oxygen atoms on the side chain, was 38-fold lower
than the affinity of compound 10, demonstrating that an increase in the number of oxygen
atoms was disadvantageous for receptor affinity. The affinity of the methylthioethylated
compound 16 was 5.4-fold lower than the affinity of the ethylthiomethylated compound 12,
demonstrating that the position of the sulfur atom influenced receptor affinity. The affinities
of compounds 17 and 18, which possessed a benzene ring, were 2684- and 5.4-fold lower
than the affinity of compound 2, respectively, demonstrating that the introduction of a
benzene ring was disadvantageous for receptor affinity.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
3.2. CoMFAAnalysis
Statistical analysis of the receptor affinities of 50 compounds using CoMFA was
performed using Eq. 1, as follows:
pKi = 5.79 + [CoMFA field terms] (Eq. 1)
n = 50, s = 0.47, r2 = 0.93,
CN =5, Cross-validated [scv =1.03, q2 = 0.68], RC [Steric = 0.64, Electrostatic = 0.36]
18

1
2
In this and the following equations, n is the number of compounds, s is the standard deviation,
and r is the correlation coefficient. CN indicates the number of latent variables, and scv and q
are the standard deviation and correlation coefficient obtained from the leave-one-out
cross-validation, respectively. RC refers to the relative contribution of steric and electrostatic
effects to variations in receptor affinity. The pKi values calculated using Eq. 1 are shown in
Table 2. The major steric and electrostatic potential contour maps with the
3
4
5
6
7
n-propylthiomethyl CH-IMI analogue 13 were drawn according to Eq. 1 (Fig. 3). The blue
areas in Fig. 3A indicate regions in which the more positive electrostatic features of the
compounds increased activity, whereas the red area shows the region in which negative
electrostatic features were favorable. A positive electrostatic potential region appeared
around the chlorine atom of the pyridine ring and the hydrogen atom attached at N3 of the
imidazolidine ring, consistent with earlier reports.6,9,10 In addition, a negative electrostatic
region appeared around the nitrogen atom of the pyridine ring, also consistent with previous
research.6 In this study, a blue region newly appeared around the C3 and C4 positions of the
n-propylthiomethyl group attached at position 5 of the imidazolidine ring. Oxygen atoms are
more electronegative than carbon and sulfur atoms, and the receptor affinities of substituents
with an oxygen atom at this position were lower than the receptor affinities of the other
compounds, which explains why the blue region appeared (relatively positive charges would
be favorable for receptor affinity). In Fig. 3B, the green and yellow regions denote sterically
favorable and unfavorable moieties for receptor affinity, respectively. Both green and yellow
regions appeared around the C2–C4 positions of the n-propylthiomethyl group attached at
position 5 of the imidazolidine ring, suggesting that steric hindrance should exist around
these regions.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
3.3. Construction of a Ligand-binding Domain Model of the Housefly nAChR Bound
by a CH-IMI Derivative and Docking
The housefly nAChR model constructed based on the crystallographic data of the
AChBP of Lymnaea stagnalis (PDB code, 2ZJU)19 suggested the presence of a sterically
19

1
2
permissible region that could accept a substituent up to the length of an n-propyl group (Fig.
5A). However, the docking model of the housefly nAChR bound to a CH-IMI derivative,
which was reconstructed on the basis of Ac-AChBP (PDB code, 3C79) using PDFAMS,
demonstrated the presence of a sterically permissible region that could accept a substituent
longer than an n-propyl group (Fig. 5B). Four amino acid residues, namely, isoleucine,
tyrosine and two tryptophans, were found to construct part of the ligand-binding region
(yellow amino acid residues in Fig. 5A). Comparing these residues in Fig. 5A (housefly
receptor model constructed based on Ls-AChBP) to the ones in Fig. 5B (housefly receptor
model constructed based on Ac-AChBP) indicated that two tryptophan residues move
outside the ligand-binding region and that the orientation of the isoleucine and tyrosine
residues is reversed, thus explaining why the ligand-binding region expanded (Fig. 5C).
In the docking model constructed on the basis of Ac-AChBP bound by the
n-propylthiomethyl CH-IMI 13 (with comparatively higher receptor affinity), sulfur-π and
van der Waals interactions between the ligand and receptor were observed (Fig. 6). The
hydrophobic amino acid residues, aligned in the expanded pocket, should interact with the
hydrophobic side chain of the compound.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
3.4. Insecticidal Activity and Correlation of Structural Changes with Biological
Activities
The insecticidal activity of the synthesized derivatives is presented in terms of
their ED50 values in Table 1. Although the receptor affinity of compound 6 was 4.6-fold
higher than that of compound 5, the insecticidal activity of compound 6 was 15-fold lower
than that of compound 5, demonstrating that a linear increase in the number of carbon
atoms was disadvantageous regarding insecticidal activity. The insecticidal activity of
compound 7 was 120-fold lower than that of the methylated compound 3, suggesting that
the introduction of a hydroxyl group would be disadvantageous. Among compounds 8–10,
ethoxymethyl CH-IMI 9 had the lowest insecticidal activity. The insecticidal activities of
compounds 8 and 9 were 62- and 25-fold lower than those of the corresponding alkylated
20

1
2
compounds 5 and 6, demonstrating that the introduction of oxygen atoms was
disadvantageous for insecticidal activity, as observed for receptor affinity. The ED50 values
of compounds 11–13, which possessed sulfur atoms, were not high and ranged from 11–74
pmol/fly, although their Ki values were lower than 1 nM, indicating that these compounds
possessed higher receptor affinity. The insecticidal activity of compounds 11 and 12 were
234- and 20-fold lower than those of compounds 5 and 6, respectively, suggesting that
converting the carbon atom to a sulfur atom was disadvantageous for insecticidal activity.
Comparing the insecticidal activities of compounds 14 and 15 with the activity of
compound 9 revealed that converting carbon atoms to oxygen atoms and the number of
oxygen atoms did not influence insecticidal activity. In the case of a sulfur atom rather than
an oxygen atom, the insecticidal activity of compound 16 was 2.3-fold lower than that of
compound 12, suggesting that the substituted position does not influence insecticidal
activity, as observed for oxygen atoms. The insecticidal activities of compounds 17 and 18
were 1615- and 91-fold lower than that of compound 2, demonstrating that the introduction
of a bulky aromatic ring reduced insecticidal activity. Thus, a compound with higher
insecticidal activity was not observed among the synthesized compounds, although
compounds 6 (n-butyl CH-IMI) and 13 (propylthiomethyl CH-IMI) exhibited nearly
identical receptor affinities to the unsubstituted, methylated, and ethylated compounds.
The relationship between insecticidal activity and receptor affinity was
quantitatively analyzed considering the other factors of the test compounds using the
Hansch-Fujita method, which is one of the conventional QSAR methods, as follows:
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
pED50 = 0.62 ( 0.08) pKi – 0.73 ( 0.22) logP − 1.43 ( 0.33) Ithioether
– 0.77 ( 0.23) Iether ＋ 6.36 ( 0.77)
Eq. 2
n = 32, s = 0.56, r = 0.88, F (4 27) = 24.312 > 0.999
where F is the ratio between the regression and residual variances and the number in
parentheses is the 95% confidence interval. This equation suggested that higher receptor
21

1
2
affinity should be advantageous for insecticidal activity, whereas higher hydrophobicity
and the introduction of heteroatoms, particularly sulfur atoms, are disadvantageous for
insecticidal activity.
3
4
5
4. DISCUSSION
6
In our previous study,6 we constructed a docking model of the housefly nAChR
bound by a CH-IMI derivative based on the crystallographic data of the AChBP of L.
stagnalis bound by IMI (PDB code, 2ZJU),19 which suggested the presence of a sterically
permissible region that could accept a substituent up to the length of an n-propyl group
attached to the imidazolidine ring (Fig. 5A). In this study, however, the receptor affinity of
compound 6, which possessed an n-butyl group, was higher than that of compound 5, and
we could not explain this result based on the previous docking model. In fact, a FRED
docking simulation indicated that compound 6 was not able to settle in the ligand-binding
region of the previous model in the same manner reported earlier (data not shown). In this
case, we observed that 2-propanol and IMI bind to one of five agonist-binding regions in
Ac-AChBP (PDB, 3C79; Fig. S1A in Supplementary data). In the electron density map of
3C79, there was a meshed region over the ethylene moiety of imidacloprid that was
assigned as 2-propanol (data not shown). Both IMI and 2-propanol were registered as
ligands in the database (PDB, 3C79), and we hypothesized that the ligand-permissible
region should expand if these compounds are recognized as ligands interacting with the
receptor for the calculation. The “MAKE RECEPTOR” software supported this hypothesis
(Fig. S1B in Supplementary data), and a docking study employing FRED suggested that
this region could accept n-propylthiomethyl CH-IMI 13 (Fig. S1C). The binding model of
the housefly nAChR, which was reconstructed on the basis of Ac-AChBP using PDFAMS,
demonstrated the presence of a sterically permissible region that could accept a substituent
longer than an n-propyl group (Fig. 5B). Comparing the model in this study with the model
in the previous study, Tyr73 was notably moved (Fig. 5C). A previous QSAR analysis of
trypsin inhibitors such as benzamidines suggested that larger substituents appear to have
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
22

1
2
pushed an amino acid aside and interact positively with the enzyme surface,20 which was
potentially consistent with our findings. In the X-ray crystallographic data of Ac-AChBP,
the relative positions of Tyr73 and IMI were different among the ligand-binding regions
(Fig. S1D), suggesting that this tyrosine residue might be flexible. This study suggests the
expansion of the ligand-binding region according to the size of a substituent attached to the
compound, but it should be considered that the binding mode would change. The surface of
the ligand-binding region is not rigid, and the introduction of a bulky group might influence
the conformation of the receptor or the binding modes of the compounds. In addition, we
constructed the receptor model employing a homodimer. If the subunit compositions of the
housefly receptors are resolved, another binding mode might be suggested.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Compound 13, which has an n-propylthiomethyl group, exhibited high receptor
affinity, and the docking study suggested that a longer substituent could be acceptable in
the expanded ligand-binding region. In the CoMFA study, however, the sterically favorable
region (as green in Fig. 3B) did not appear around the tip of the substituents, likely due to
the lack of variation of the substituents. More substituents should be employed to clarify
the contribution of this region to the receptor affinity.
The analysis of the relationship between insecticidal activity and receptor affinity
suggested that higher receptor affinity should be advantageous for insecticidal activity,
whereas higher hydrophobicity and the introduction of heteroatoms, particularly sulfur
atoms, are disadvantageous for insecticidal activity. Compounds that possess heteroatoms,
such as sulfur and oxygen atoms, might be metabolized (for example, cleavage of ether
bonds and oxidation of sulfur atoms) before they reach the target site, although synergists
were applied before the insecticidal test. Any metabolic pathway that cannot be suppressed
by synergists such as NIA and PBO might be implicated.
In this study, we identified some CH-IMI derivatives possessing larger substituents
that exhibited high receptor affinity, although their insecticidal activity was not high. In
addition, a docking model of the housefly nAChR bound to CH-IMI derivatives suggested
that the ligand-binding region expands as the size of the substituent increases. It is clear
23

1
2
that receptor affinity primarily influences insecticidal activity. Based on the receptor model
constructed in this study, compounds that exhibit higher receptor affinity should be
designed, and the metabolic pathway targeted in insects should be considered, leading to
the development of novel neonicotinoid insecticides.
3
4
5
6
ASSOCIATED CONTENT
7
Supporting Information
8
1H and 13C NMR spectra of compounds 6-18. Supplementary Figure S1. The atomic
coordinate file of the housefly nAChR model constructed in the present study. These
materials are available free of charge via the Internet at http://pubs.acs.org.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
AUTHOR INFORMATION
Corresponding Author
* Phone: 81-89-946-9973. Fax: 81-89-977-4364.
E-mail: nishiwaki.hisashi.mg@ehime-u.ac.jp
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are grateful to Profs. Hisashi Miyagawa and Yoshiaki Nakagawa of Kyoto University
for gifting radiolabeled imidacloprid. A part of this study was performed at the INCS
(Johoku and Tarumi stations) of Ehime University.
24

1
2
REFERENCES
1. Kagabu, S. J. Agric. Food Chem. 2011, 59, 2887.
3
2. Ohno, I.; Tomizawa, M.; Miyazu, N.; Kushibiki, G.; Noda, K.; Hasebe, Y.; Durkin, K.
A.; Miyake, T.; Kagabu, S. Bioorg. Med. Chem. Lett. 2010, 20, 5933.
3. Tomizawa, M.; Kagabu, S.; Casida, J. E. J. Agric. Food Chem. 2011, 59, 2918.
4. Nishiwaki, H.; Sato, K.; Nakagawa, Y.; Miyashita, M.; Miyagawa, H. J. Pestic. Sci. 2004,
29, 110.
4
5
6
7
8
5. Nishiwaki, H.; Nagaoka, H.; Kuriyama, M.; Yamauchi, S.; Shuto, Y. Biosci. Biotechnol.
Biochem. 2011, 75, 780.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
6. Nishiwaki, H.; Kuriyama, M.; Nagaoka, H.; Kato, A.; Akamatsu, M.; Yamauchi, S.;
Shuto, Y. Bioorg. Med. Chem. 2012, 20, 6305.
7. Bissantz, C.; Kuhn, B.; Stahl, M. J. Med. Chem. 2010, 53, 5061.
8. Foss, F. W. Jr.; Snyder, A. H.; Davis, M. D.; Rouse, M.; Okusa, M. D.; Lynch, K. R.;
Macdonald, T. L. Bioorg. Med. Chem. 2007, 15, 663.
9. Okazawa, A.; Akamatsu, M.; Ohoka, A.; Nishiwaki, H.; Cho, W-J.; Nakagawa, Y.;
Nishimura, K.; Ueno, T. Pestic. Sci. 1998, 54, 134.
10. Okazawa, A,; Akamatsu, M.; Nishiwaki, H.; Nakagawa, Y.; Miyagawa, H.; Nishimura,
K.; Ueno, T. Pest manag. Sci. 2000, 56, 509.
11. Talley, T. T.; Harel, M.; Hibbs, R. E.; Radic, Z.; Tomizawa, M.; Casida, J. E.; Taylor, P.
Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 7606.
12. Matsuda, K.; Kanaoka, S.; Akamatsu, M.; Sattelle, D. B. Mol. Pharmacol. 2009, 76, 1.
13. Gao, J-R.; Deacutis, J. M.; Scott, J. G. Insect Mol. Biol. 2007, 16, 325.
14. Kirkpatrick, S.; Gelatt, C. D. Jr.; Vecchi, M. P. Science 1983, 220, 671.
15. Halgren, T. A. J. Comput. Chem. 1999, 20, 720.
16. Halgren, T. A. J. Comput. Chem. 1999, 20, 730.
17. Asao, M.; Shimizu, R.; Nakao, K.; Fujita, T. 1997, QREG 2.05. Japan Chemistry
Program Exchange, Society of Computer Chemistry, Japan.
18. OECD guideline for the testing of chemicals 107, Partition coefficient
25

1
2
3
4
5
6
7
(n-octanol/water): shake flask method. 1995.
19. Ihara, M.; Okajima, T.; Yamashita, A.; Oda, T.; Hirata, K.; Nishiwaki, H.; Morimoto,
T.; Akamatsu, M.; Ashikawa, Y.; Kuroda, S.; Mega, R.; Kuramitsu, S.; Sattelle, D. B.;
Matsuda, K. Invert. Neurosci. 2008, 8, 71.
20. Hansch, C.; Leo, A. In Exploring QSAR; Heller, S. R., Ed.; ACS: Washington, DC, 1995,
Chapter 7.
26

1
2
Figure legends
FIGURE 1 Chemical structures of imidacloprid (1) and its nitromethylene derivatives 2 -
18.
3
4
5
FIGURE 2 Synthetic route for imidacloprid derivatives 6 - 18. (a) SOCl2/MeOH;
6
(Boc)2O/THF; 2,2-dimethoxypropane, BF3-Et2O complex/acetone; NaBH4, LiCl/dry
7
THF;
(b)
phthalimide,
DEAD,
PPh3/dry
THF;
(c)
conc.
HCl/THF;
8
2-chloro-5-chloromethylpyridine, Et3N/acetone; (d) EtI/dry THF; (e) p-TsOH/MeOH; (f)
Me2SO4, 0.2 M Ba(OH)2/dry CH2Cl2; (g) LiAlH4/dry THF; (Boc)2O/THF; (h) phthalimide,
DEAD, PPh3/dry THF; (i) conc. HCl/THF; 2-chloro-5-chloromethylpyridine, Et3N/acetone;
(j) DIEA, MOMCl/dry CH2Cl2; (k) N2H4-H2O/EtOH; 1,1-bis(methylthio)-2-nitroethylene,
K2CO3/EtOH.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
FIGURE 3 Contour diagrams of electrostatic (A) and steric (B) fields with
n-propylthiomethyl CH-IMI 13 according to Eq. 1. (A) Red and blue areas indicate the
regions in which negative and positive electrostatic interactions with the receptor-binding
site increase receptor affinity, respectively. (B) Green and yellow areas indicate the
sterically favorable and unfavorable regions for receptor affinity, respectively.
FIGURE 4 Pairwise sequence alignment of subunit 6 (subtype II) of the nicotinic
acetylcholine receptor of Musca domestica (MdS6II, GenBank ID ABJ09669) with the
acetylcholine binding protein of Aplysia californica (Ac-AChBP, PDB, 3C79). An asterisk
indicates perfect identity, a colon indicates similar amino acids with conservation, and a
period indicates weakly similar amino acids with conservation. Trp35, Tyr73, Trp129, and
Ile169 are presented in red.
FIGURE 5 The molecular surface of the ligand-binding site of the housefly receptor model.
(A) Ligand-binding domain (LBD) constructed on the basis of the X-ray crystallographic
27

1
2
data of Ls-AChBP. Four amino acids (isoleucine, tyrosine and two tryptophans)
surrounding the ligand-binding pocket are presented in yellow. (B) LBD constructed on the
basis of the X-ray crystallographic data of the acetylcholine binding protein of Aplysia
californica. The corresponding four amino acids in Fig. 5A are presented in green. (C)
Merged image of Figs. 5A-B. In Figs. 5A-B, the color of the contour map indicates the
hydrophobicity (brown, hydrophobic; blue, hydrophilic).
3
4
5
6
7
8
FIGURE 6 The amino acid residues for constructing the expanded ligand-binding region
of the housefly receptor model and the n-propylthiomethyl CH-IMI 13 (drawn using
yellow-green bonds).
9
10
11
12
28

1
2
Table 1 Receptor affinity Ki (nM), insecticidal activity ED50 (pmol/fly) and hydrophobicity
log P of CH-IMI 2 and its synthesized derivatives 3-18.a
Receptor affinity
Ki (nM)
Insecticidal activity
ED50 (pmol/fly)
0.117b
No.
R
Log P
H
Me
0.0367b
-0.20
0.09
0.47
0.96
1.34
-0.55
-0.03
0.45
1.06
0.48
0.95
1.35
0.16
-0.11
0.75
1.23
1.56
2
3
0.0428b
0.0626b
Et
0.0597c
0.914c
4
n-Propyl
0.258c
0.253c
5
n-Butyl
0.0564
0.157
26.6
0.00466
3.77
7.49
15.7
94.3
11.0
59.2
73.7
11.3
68.9
63.1
171
0.441
0.615
3.80
19.4
1.71
16.3
1.92
3.50
7.77
17.9
28.5
13.0
3.07
6
Hydroxymethyl
Methoxymethyl
Ethoxymethyl
Propoxymethyl
Methylthiomethyl
Ethylthiomethyl
Propylthiomethyl
Methoxyethyl
Methoxymethoxymethyl
Methylthioethyl
Phenyl
0.00602
5.31
7
8
5.76
0.868
0.196
0.0393
0.0170
0.0153
0.178
3.65
9
0.373
0.581
0.100
0.0807
5.26
10
11
12
13
14
15
16
17
18
14.0
0.542
98.5
0.233
25.8
189
Benzyl
0.198
0.0483
10.7
3
4
5
6
7
8
a The biological activities of compounds 6-18 are presented as the mean standard error of
the mean (n=3).
b The values of the biological activities of compounds are cited from Ref. 5.
c The values of the biological activities of compounds are cited from Ref. 6.
29