Affinity to the nicotinic acetylcholine receptor and insecticidal activity of chiral imidacloprid derivatives with a methylated imidazolidine ring

Article abstract of DOI:10.1271/bbb.100846

Four imidacloprid derivatives with an asymmetrically methylated imidazolidine ring were synthesized. Their affinity to the nicotinic acetylcholine receptor of housefly Musca domestica and insecticidal activity against the housefly were measured. The compound with a 5R-methylated imidazolidine ring demonstrated intrinsic activity comparable to that of the unsubstituted compound. Most of the compounds were synergized by oxygenase inhibitors.

Full text of DOI:10.1271/bbb.100846

Biosci. Biotechnol. Biochem., 75 (4), 780–782, 2011
Note
Affinity to the Nicotinic Acetylcholine Receptor and Insecticidal Activity
of Chiral Imidacloprid Derivatives with a Methylated Imidazolidine Ring
y
Hisashi NISHIWAKI, Hikaru NAGAOKA, Mituhiro KURIYAMA,
Satoshi YAMAUCHI, and Yoshihiro SHUTO
Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan
Received November 30, 2010; Accepted January 6, 2011; Online Publication, April 22, 2011
[doi:10.1271/bbb.100846]
Four imidacloprid derivatives with an asymmetri-
affinity, suggesting that the configuration of this position
would be accurately recognized by the receptor. On the
other hand, the introduction of a methyl group to
position 4 of the imidazolidine ring induced over 50-fold
less potency, and compound 5 (4R-CH3) was about
2-fold more potent than its enantiomer (4S-CH3), i.e.,
compound 6.
cally methylated imidazolidine ring were synthesized.
Their affinity to the nicotinic acetylcholine receptor
of housefly Musca domestica and insecticidal activity
against the housefly were measured. The compound
with a 5R-methylated imidazolidine ring demonstrated
intrinsic activity comparable to that of the unsubstituted
compound. Most of the compounds were synergized by
oxygenase inhibitors.
The insecticidal activity against female adult house-
flies of compounds 2–6 was measured with and without
such synergists as piperonyl butoxide (PBO) and
propargyl propyl phenylphosphonate (NIA) which sup-
pressed oxidative metabolic degradation and whose syn-
Key words: imidacloprid; Musca domestica; insecticidal
activity; synergist; nicotinic acetylcholine
receptor
7
–10)
ergistic effects on imidacloprid have been reported.
In the absence of a synergist, the S-isomers (compounds
4 and 6) were approximately 4-fold less potent than
compound 2, whereas their enantiomers were equipotent
to compound 2. PBO synergized the insecticidal activity
of unsubstituted compound 2 and compounds 3 and 4,
which have an imidazolidine ring methylated at position
5. The methylated compounds other than compound 3
were 4–11-fold less potent than compound 2 in the
presence of PBO. In the presence of NIA, all compounds
other than 5 were synergized over 10-fold, and com-
pound 3 showed 4-fold less potency than compound 2,
whereas compounds 4–6 were approximately 10-fold
less potent than compound 2. The insecticidal activity of
all compounds increased markedly in the presence of
both synergists. The insecticidal activity of the four
methylated compounds in the presence or absence
of a synergist was affected to varying degrees, suggest-
ing that these synergists had unknown and differing
influence on the metabolism of the compounds in
houseflies.
Imidacloprid (1, Fig. 1)1,2) is one of the typical
neonicotinoids marketed for pest control in many
countries. It acts on insect nicotinic acetylcholine
receptors (nAChRs) to kill pests. Various structure-
activity relationship (SAR) analyses have been per-
formed during the last two decades to understand the
structural factors influencing the insecticidal activity and
2
,3)
affinity to the receptor.
We have quantitatively
analyzed SAR for the nitromethylene analogue (2,
Fig. 1) and its derivatives, and demonstrated that some
steric and electrostatic factors around the pyridine ring
and the N3 atom of the imidazolidine ring influenced
4
–6)
receptor binding.
However, the effect on receptor
binding of the steric structure around the ethylene
moiety remains unknown. This ethylene moiety is
7
)
hydroxylated in the housefly, Musca domestica. The
stereoselective introduction of a substituent to the
ethylene moiety of the imidazolidine ring would
probably influence the metabolism of the compounds
in insects to induce changes in the insecticidal activity.
We synthesized in this study four methylated derivatives
with an asymmetric carbon atom in the imidazolidine
ring (3–6, Fig. 2) to evaluate their affinity to housefly
nAChRs and insecticidal activity against female adult
houseflies.
We have reported a positive correlation between their
affinity to the receptor of some imidacloprid derivatives
3
that was evaluated by using [ H] imidacloprid and the
insecticidal activity measured in the presence of both
1
1)
synergists. In this study, a positive correlation was
2
also observed between these activities (r ¼ 0:809,
1
1)
The inhibition constant, Ki (nM), was evaluated as an
indicator of the affinity to the receptor (Table 1).
Compound 3 (5R-CH3) was 7-fold more potent than
its enantiomer (5S-CH3), i.e., compound 4, and was
equipotent to unsubstituted compound 2. Stereoisomers
at position 5 of the imidazolidine ring had differing
n ¼ 21, including data previously reported ), suggest-
ing receptor binding to be an important factor even if the
ethylene moiety was modified. This study demonstrated
the possibility of improving the bioactivity of imidaclo-
prid derivatives by synthesizing asymmetrically modi-
fied compounds.
y
To whom correspondence should be addressed. Tel: +81-89-946-9973; Fax: +81-89-977-4364; E-mail: nhisa@agr.ehime-u.ac.jp
Abbreviations: CH-IMI, nitromethylene analogue of imidacloprid; IMI, imidacloprid; nAChR, nicotinic acetylcholine receptor; NIA, propargyl
propyl phenylphosphonate (Niagara 16388); PBO, piperonyl butoxide; SAR, structure-activity relationship

Biological Activities of Chiral Imidacloprid Derivatives
781
0
0
Experimental
The synthetic scheme for the preparation of compounds 3–6 is
(2 R)-N-(2 -t-Butoxycarbonylamino)propylphthalimide (8, step A).
To a reaction mixture of anhydrous tetrahydrofuran (THF, 100 mL)
containing 3.37 g (12.9 mmol) of triphenylphosphine, 2.52 g (17.1
mmol) of phthalimide, and 1.50 g (8.6 mmol) of N-t-butoxycarbonyl-D-
alaninol (7), 6.71 g (40% in toluene, 15.3 mmol) of diethylazodicar-
boxylate dissolved in anhydrous THF (50 mL) was added dropwise
while stirring in an ice bath. After 6 h at ambient temperature, the
solvent was evaporated, and the resulting residue was purified by
column chromatography (hexane:ethyl acetate ¼ 3:1) to afford 8
shown in Fig. 2. The metabolic inhibitor, NIA16388, was synthesized
according to a previously published method.12) NMR data were
measured by using a Jeol JNM-EX400 NMR spectrometer. The
authenticity of each final compound was confirmed by an elemental
analysis with a Yanako MT-5 CHN recorder. Melting point (mp) data
for the compounds were measured by Yanaco melting point apparatus
(Yanaco, Kyoto, Japan) and are uncorrected. Optical rotation values
were evaluated by using a P-2100 polarimeter (Jasco, Tokyo, Japan).
ꢀ
25
(2.23 g, 85%). Mp 122–123 C, ½ꢀꢁD ꢂ20:5 (c 0.27, CHCl3). NMR
ꢁ
H (CDCl3): 1.21 (3H, d, J ¼ 7 Hz, CH3), 1.25 (9H, s, (CH3)3C), 3.68
(
2H, d, J ¼ 9 Hz, CH2), 4.10 (1H, m, CH3CH), 4.71 (1H, br, NH), 7.71
(
2H, m, Ph), 7.84 (2H, m, Ph).
0
0
(
10, steps B-i and -ii). A concentrated HCl solution (15 mL) was added
2 R)-N-(2 -(6-Chloro-3-pyridyl)methylamino)propylphthalimide
(
dropwise to THF (50 mL) containing 8 (1.79 g, 5.9 mmol) while
stirring at ambient temperature. After continuing stirring overnight, the
solvent was evaporated to afford 9 which was used in the subsequent
reaction without further purification. The product was dissolved in
2
0 mL of acetonitrile, to which 1.68 mL of triethylamine and 0.97 g
6 mmol) of 2-chloro-5-chloromethylpyridine hydrochloride were
added, and the mixture was refluxed overnight. The solvent was
(
Fig. 1. Chemical Structures of the Imidacloprid Derivatives.
Fig. 2. Synthetic Route to the Imidacloprid Derivatives, i.e., Compounds 3 ((5R)-CH3-CH-IMI) and 5 ((4R)-CH3-CH-IMI).
Their enantiomers, i.e., compounds 4 ((5S)-CH3-CH-IMI) and 6 ((4S)-CH3-CH-IMI), were synthesized by the same method, using N-t-
butoxycarbonyl-L-alaninol as the starting material. A, Phthalimide, diethyl azodicarboxylate, triphenylphosphine/anhydrous THF; B-I,
concentrated HCl/THF; B-ii, 2-chloro-5-chloromethylpyridine, Et3N/acetonitrile; C-i, hydrazine hydrate/EtOH; C-ii, 1,1-bis(methylthio)-2-
nitroethylene, K2CO3/EtOH; D-i, hydrazine hydrate/EtOH; D-ii, 2-chloro-5-chloromethylpyridine, Et3N/acetonitrile; E-i, concentrated HCl/
THF; E-ii, 1,1-bis(methylthio)-2-nitroethylene, K2CO3/EtOH.
3
Table 1. Inhibition Constants for the Imidacloprid Derivatives for [ H]imidacloprid Binding to Housefly Head Membranes and Their Insecticidal
Activity against Houseflies
ED50 (pmol/insect)c
Compound
Config.a
Ki (nM)b
None
A)
PBO
(B)
Ratio
(A)/(B)
NIA
(C)
Ratio
(A)/(C)
PBO þ NIA
Ratio
(A)/(D)
no.
(
(D)
2
3
4
5
6
H
0:0367 ꢃ 0:0063
4.18d
1:00 ꢃ 0:22 (3)
4.2
3.4
4.3
0.8
1.4
0:128 ꢃ 0:013 (3) 33
0:474 ꢃ 0:032 (3) 12
0:829 ꢃ 0:406 (3) 25
0:136 ꢃ 0:019 (3)
0:0854 ꢃ 0:0228 (3)
0:643 ꢃ 0:306 (3)
0:774 ꢃ 0:348 (3)
1:00 ꢃ 0:46 (3)
31
68
33
8
5R
5S
4R
4S
0:0428 ꢃ 0:0079 5:82 ꢃ 1:06 (4) 1:72 ꢃ 0:35 (3)
0:313 ꢃ 0:190 21:0 ꢃ 4:02 (3) 4:85 ꢃ 1:75 (3)
2:07 ꢃ 0:28
6:30 ꢃ 2:35 (3) 7:99 ꢃ 1:59 (3)
1:12 ꢃ 0:55 (3)
5.6
5:64 ꢃ 1:07
16:1 ꢃ 3:15 (3) 11:4 ꢃ 6:44 (3)
0:943 ꢃ 0:494 (3) 17
16
aPosition of the imidazolidine ring attached by a methyl group and its configuration for compounds 3–6. Compound 2 is unsubstituted.
b
3
Inhibition constant of each test compound for [ H]imidacloprid binding to nAChR. The Ki value was calculated according to the following equation by using PRISM
3
software (Graphpad): Ki ¼ IC50=1 þ ð½Lꢁ=KdÞ, where [L] is the final concentration of the [ H]imidacloprid (0.63 TBq/mmol, 20 nM (GE Healthcare, UK)) and Kd
3
(
2.65 nM) is the dissociation constant of [ H]imidacloprid for the receptor fraction, which was newly measured in this study and is consistent with previous
studies.11,13) Ki values for the test compounds were obtained in three separate experiments. Data are presented as the mean and standard error of the mean. See detailed
method in Ref. 11.
cEffective dose for inducing paralysis or death 1 h after injection in 50% of the female adult houseflies. The value was calculated by using the probit transformation.14)
See details of the method in Ref. 11. Data are presented as the mean and standard error of the mean (the figure in parentheses is the number of experiments).
dCalculated from the data cited in Ref. 15.

782
H. NISHIWAKI et al.
evaporated, and the resulting residue was purified by column
chromatography (hexane:ethyl acetate ¼ 1:1) to afford 10 (0.39 g,
analysis. Calcd. for C11H13N4O2Cl; C, 49.17; H, 4.88; N, 20.85.
2
5
Found: C, 49.11; H, 4.58; N, 20.76. ½ꢀꢁD ꢂ7:5 (c 1.18, CHCl3). NMR
ꢁH (CDCl3): 1.36 (3H, d, J ¼ 6 Hz, CH3), 3.12 (1H, dd, J1 ¼ 10 Hz,
J2 ¼ 7 Hz, CH), 3.67 (1H, t, J ¼ 10 Hz, CH), 4.21 (1H, m, CH), 4.31
(2H, d, J ¼ 4 Hz, PyCH2), 6.64 (1H, s, CHNO2), 7.36 (1H, d,
J ¼ 8 Hz, Py), 7.56 (1H, dd, J1 ¼ 8 Hz, J2 ¼ 3 Hz, Py), 8.29 (1H,
d, J ¼ 3 Hz, Py), 8.77 (1H, br, NH). NMR ꢁC (CDCl3): 21, 47, 50, 51,
55, 96, 125, 129, 138, 149, 152, 159. >99% ee (DAICEL chiral
column OD-H, 254 nm, mobile phase of hexane:2-propanol ¼ 1:1,
tR ¼ 94 min).
25
2
0% in 2 steps). ½ꢀꢁD ꢂ29:1 (c 1.1, CHCl3). NMR ꢁH (CDCl3): 1.04
(
3H, d, J ¼ 7 Hz, CH3), 2.94 (1H, m, CH3CH), 3.52 (2H, m,
PhthalCH2), 3.67 (1H, d, J ¼ 12 Hz, PyCH2), 3.76 (1H, d,
J ¼ 12 Hz, PyCH2), 7.01 (1H, d, J ¼ 8 Hz, Py), 7.46 (1H, dd,
J1 ¼ 8 Hz, J2 ¼ 2 Hz, Py), 7.60 (2H, m, Ph), 7.69 (2H, m, Ph), 8.13
(1H, d, J ¼ 2 Hz, Py).
(
5R)-1-(6-Chloro-3-pyridylmethyl)-5-methyl-2-nitromethylene-imi-
dazolidine (3, steps C-i and -ii). To an ethanol solution containing
.39 g (1.2 mmol) of 10, 0.33 mL (5.8 mmol) of hydrazine monohy-
0
(4S)-1-(6-Chloro-3-pyridylmethyl)-4-methyl-2-nitromethylene-imi-
ꢀ
drate was added, and the mixture was refluxed for 6 h while stirring.
After the insoluble residue had been filtered, the resulting filtrate was
evaporated to afford 11 which was used for the subsequent reaction
without further purification. After diaminopropane 11 had been
dissolved in 20 mL of ethanol, 0.20 g (1.2 mmol) of 1,1-bis(methylth-
io)-2-nitroethylene and 0.16 g (1.2 mmol) of K2CO3 were added to the
solution, and the 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
dazolidine (6). Mp 139–141 C. Elemental analysis. Calcd. for
C11H13N4O2Cl: C, 49.17; H, 4.88; N, 20.85. Found: C, 49.09; H,
2
5
4.83; N, 20.83. ½ꢀꢁD þ7:8 (c 1.14, CHCl3). >99% ee (DAICEL
OD-H, 254nm, mobile phase hexane:2-propanol ¼ 1:1, tR ¼ 102 min).
Acknowledgments
We are grateful to Professor Toru Miyamoto of Tokyo
Agriculture University and to Professor Hisashi
Miyagawa and Professor Yoshiaki Nakagawa of Kyoto
University for respectively presenting the houseflies and
radiolabeled imidacloprid. Part of this study was
performed at INCS (Johoku and Tarumi stations) of
Ehime University.
ꢀ
acetate) to afford 3 (0.22 g, 67% in two steps). Mp 139–141 C.
Elemental analysis. Calcd. for C11H13N4O2Cl: C, 49.17; H, 4.88; N,
2
5
2
0.85. Found: C, 48.94; H, 4.91; N, 20.66. ½ꢀꢁD þ9:9 (c 1.54,
CHCl3). NMR ꢁH (CDCl3): 1.33 (3H, d, J ¼ 6 Hz, CH3), 3.39 (1H, m,
CH), 3.93 (2H, m, CH2), 4.30 (1H, d, J ¼ 17 Hz, PyCH2), 4.37 (1H, d,
J ¼ 17 Hz, PyCH2), 6.56 (1H, s, CHNO2), 7.35 (1H, d, J ¼ 8 Hz, Py),
7
.55 (1H, dd, J ¼ 8 Hz, J ¼ 2 Hz, Py), 8.29 (1H, d, J ¼ 2 Hz, Py), 8.70
(
1H, br, NH). NMR ꢁC (CDCl3): 18, 44, 50, 56, 97, 125, 130, 138, 148,
52, 159. >99% ee [confirmed by a 1H-NMR chemical shift of the
CHNO2 group by the shift reagent, Chirabite].
1
References
(
5S)-1-(6-Chloro-3-pyridylmethyl)-5-methyl-2-nitromethylene-imi-
ꢀ
1) Elbert A, Overbeck H, Iwaya K, and Tsuboi S, ‘‘Proc Brighton
Crop Protect Conf—Pests & Diseases,’’ BCPC, Farnham,
pp. 21–28 (1990).
dazolidine (4). Mp 140–142 C. Elemental analysis. Calcd. for
C11H13N4O2Cl: C, 49.17; H, 4.88; N, 20.85. Found: C, 49.12; H,
2
5
4
.86; N, 20.85. ½ꢀꢁD ꢂ9:5 (c 1.58, CHCl3). The NMR spectral data
2) Kagabu S, J. Agric. Food Chem., 59, 2887–2896 (2011).
3) Sun C, Yang D, Xing J, Wang H, Jin J, and Zhu J, J. Agric.
Food Chem., 58, 3415–3421 (2010).
agreed with those of its enantiomer. >99% ee (confirmed by the same
procedure as that for determining the chirality of 3).
0
(2R)-N-(6-Chloro-3-pyridylmethyl)-N -t-butoxycarbonyl-1,2-diami-
nopropane (13, steps D-i and -ii). Hydrazine monohydrate (3 mL,
4) Nishiwaki H, Nakagawa Y, Takeda DY, Okazawa A, Akamatsu
M, Miyagawa H, Ueno T, and Nishimura K, Pest Manag. Sci.,
56, 875–881 (2000).
5
1
3
3 mmol) was added dropwise to ethanol containing 8 (3.23 g,
0 mmol) while stirring at ambient temperature. After refluxing for
h, the reactant was filtered, and the resulting filtrate was evaporated
5) Nishiwaki H, Nakagawa Y, Ueno T, Kagabu S, and Nishimura
K, Pest Manag. Sci., 57, 810–814 (2001).
to afford 12. The residue was dissolved in 20 mL of acetonitrile, to
which 5 mL of triethylamine and 1.26 g (8 mmol) of 2-chloro-5-
chloromethylpyridine hydrochloride was added, and the mixture was
refluxed overnight. After the solvent had been evaporated, the residue
was dissolved in distilled water, and the pH was adjusted to 8.0 with
6) Kagabu S, Nishiwaki H, Sato K, Hibi M, Yamaoka N, and
Nakagawa Y, Pest Manag. Sci., 58, 483–490 (2002).
7) Nishiwaki H, Sato K, Nakagawa Y, Miyashita M, and
Miyagawa H, J. Pestic. Sci., 29, 110–116 (2004).
8) Liu M, Lanford J, and Casida JE, Pestic. Biochem. Physiol., 46,
200–206 (1993).
1
M of aqueous NaOH. The solution was extracted three times with
dichloromethane, and the organic phase was dried over sodium sulfate.
The solvent was evaporated, and the resulting residue was purified by
column chromatography (ethyl acetate:ethanol ¼ 9:1) to afford 13
9) Nishimura K, Kanda Y, Okazawa A, and Ueno T, Pestic.
Biochem. Physiol., 50, 51–59 (1994).
10) Yamamoto I, Tomizawa M, Saito T, Miyamoto T, Walcott EC,
and Sumikawa K, Arch. Insect Biochem. Physiol., 37, 24–32
(1998).
25
(
(
(
1.07 g, 36% in two steps). ½ꢀꢁD ꢂ61:1 (c 1.8, CHCl3). NMR ꢁH
CDCl3): 1.08 (3H, d, J ¼ 7 Hz, CH3), 1.39 (9H, s, (CH3)3C), 2.12
1H, m, CH), 3.69 (2H, m, CH2), 4.74 (1H, br, NH), 7.22 (1H, d,
11) Nishiwaki H, Nakagawa Y, Kuwamura M, Sato K, Akamatsu
M, Matsuda K, Komai K, and Miyagawa H, Pest Manag. Sci.,
59, 1023–1030 (2003).
J ¼ 8 Hz, Py), 7.62 (1H, d, J ¼ 8 Hz, Py), 8.27 (1H, s, Py).
4R)-1-(6-Chloro-3-pyridylmethyl)-4-methyl-2-nitromethylene-imi-
dazolidine (5, steps E-i and -ii). After deprotecting the Boc group of
.07 g (3.6 mmol) of 13 by concentrated HCl to obtain 14, 2.31 g
14 mmol) of 1,1-bis(methylthio)-2-nitroethylene and 1.93 g (14 mmol)
(
12) Soliman SA, J. Toxicol. Environ. Health, 10, 907–920 (1982).
13) Liu M, Latli B, and Casida JE, Pestic. Biochem. Physiol., 50,
171–182 (1994).
1
(
of K2CO3 were added to 20 mL of an ethanol solution containing 14,
and the mixture was refluxed overnight. After filtering to remove
K2CO3, the resulting filtrate was evaporated, and the residue was
purified by column chromatography (ethyl acetate:ethanol ¼ 9:1) to
14) Finney DJ, ‘‘Probit Analysis’’ 2nd ed., Cambridge University
Press, London (1952).
15) Nishiwaki H, Nakagawa Y, Ueno T, and Nishimura K, J. Pestic.
Sci., 26, 91–92 (2001).
ꢀ
afford 5 (0.13 g, 13% in two steps). Mp 139–141 C. Elemental