Structural features of phenoxycarbonylimino neonicotinoids acting at the insect nicotinic receptor

Article abstract of DOI:10.1016/j.bmcl.2010.07.049

Substituted-phenoxycarbonylimino neonicotinoid ligands with an electron-donating group showed significantly higher affinity to the insect nicotinic receptor relative to that of the analogue with an electron-withdrawing substituent, thereby establishing in silico binding site interaction model featuring that the phenoxy ring of neonicotinoids and the receptor loop D tryptophan indole plane form a face-to-edge aromatic interaction.

Full text of DOI:10.1016/j.bmcl.2010.07.049

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5933–5935
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural features of phenoxycarbonylimino neonicotinoids acting
at the insect nicotinic receptor
Ikuya Ohno a, Motohiro Tomizawa b, Nozomi Miyazu b, Gohito Kushibiki b, Kumiko Noda b,
Yasunori Hasebe b, Kathleen A. Durkin c, Taiji Miyake d, Shinzo Kagabu b,
*
a The United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan
b Department of Chemistry, Faculty of Education, Gifu University, Gifu 501-1193, Japan
c Molecular Graphics and Computational Facility, College of Chemistry, University of California, Berkeley, CA 94720-1460, USA
d Research Center, Kureha Corporation, 16 Ochiai, Nishiki-machi, Iwaki, Fukushima 974-8686, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Substituted-phenoxycarbonylimino neonicotinoid ligands with an electron-donating group showed sig-
nificantly higher affinity to the insect nicotinic receptor relative to that of the analogue with an elec-
tron-withdrawing substituent, thereby establishing in silico binding site interaction model featuring
that the phenoxy ring of neonicotinoids and the receptor loop D tryptophan indole plane form a face-
to-edge aromatic interaction.
Received 6 May 2010
Revised 12 July 2010
Accepted 13 July 2010
Available online 16 July 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Imidacloprid
Neonicotinoid insecticides
Nicotinic receptor
Phenoxycarbonylimino pharmacophore
Neonicotinoid insecticides, represented by imidacloprid (IMI)
(Fig. 1), are agonists of the nicotinic acetylcholine receptor (nAChR)
and are broadly used for crop protection accounting for more than
20% of global insecticide market.1–4 High neonicotinoid potency
and selectivity toward the insect nAChR are ultimately attributable
to the disparate binding site interactions which have been deci-
phered in chemical or atomic scale using mollusc acetylcholine
binding protein (AChBP) as structural homologue of the extracellu-
lar ligand-binding domain of the nAChR.5–9 Our illustrative studies
in the nAChR structure-guided insecticide design led to identify
some highly potent compounds with acylimino and aryloxycar-
bonylimino pharmacophores (Fig. 1), which may confer dissimilar
binding mechanisms to that of IMI with the nitroimino functional
group.10–12 The present investigation discusses extensive struc-
ture–activity relationships (SARs) of the substituted-phenoxycar-
bonylimino neonicotinoid analogues in terms of insect nAChR
binding affinity and insecticidal activity. The observed SAR findings
consequently lead to predict the unique binding site interaction of
the phenoxycarbonylimino pharmacophore with the loop D sub-
site of the receptor.
Compounds 1 and 16 were prepared according to the published
procedure.11 The other compounds were available by adding of 3-
(6-chloropyridin-3-ylmethyl)-2-imino-1,3-thiazolidine to the
in situ prepared carbonate by reaction of substituted phenol with
4-nitrophenyl chloroformate or di-3-pyridinyl carbonate in the
presence of triethylamine (Scheme 1 and Supplementary data).
The 1,3-thiazoline derivatives were similarly synthesized.
Binding affinities of phenoxycarbonylimino neonicotinoid ana-
logues were evaluated with housefly (Musca domestica) brain
nAChR using [3H]IMI radioligand.13,14 Among the thiazolidine ana-
logues, unsubstituted phenoxy compound 1 had a binding affinity
(Ki) 46 nM (Table 1). However, the pyridin-3-oxycarbonylimino
compound (2) was a threefold less potent (140 nM), conceivably
IMI
O
N
Cl
C
R
N
N NH
O
N
acylimino
N
O
O
N
nitroimino
C
OAr
aryloxycarbonylimino
neonicotinoid pharmacophore
Figure 1. Neonicotinoid insecticide IMI and the pharmacophore modifications.
due to the decreased
the aromatic ring -electron density may play an important role
on the interaction with the regional subsite.
p-electron density of the pyridine. Hence
p
* Corresponding author. Tel.: +81 58 293 2253; fax: +81 58 293 2207.
E-mail address: kagabus@gifu-u.ac.jp (S. Kagabu).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.049

5934
I. Ohno et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5933–5935
4-position by an electron-donating methyl group (9 or 11) had
O
Cl
similar potency to that of unsubstituted 1. Interestingly, 3-methyl-
phenoxy analogue (10) showed a 2.6-fold higher potency than that
of 1. The 2,4-dimethyl analogue (12) had similar potency. The three
methoxy isomers (13–15) showed the identical activity pattern to
that of methyl analogues (9–11). Substitution at the 4-position
and/or possibly also at the 2-position appears to affect activity per-
haps due to steric clashes. On the other hand, the thiazoline ana-
logue with an unsubstituted phenoxy (16) was 5.2-fold more
potent relative to the thiazolidine compound (1), indicating that
O
OH O2N
O
O
O
R
R
Et3N
CH2Cl2
NO2
A
Cl
A
NH
S
Cl
N
N S
or
O
N
N
N
O
O
O
O
N
N
or
an additional
p-electron system on the heterocyclic ring possibly
N
R
makes a favourable interaction to the subsite. Alternatively, aro-
matisation of thiazoline ring may affect the conjugated electron
system of the carbonylamidine moiety, leading to a better hydro-
gen-accepting ability of the carbonyl oxygen. The 3-methyl or 3-
methoxy analogue (17 or 18) gave optimal affinity, rivalling that
of control neonicotinoid IMI. As expected, compound 19 with a
strong electron-withdrawing 4-trifluoromethyl group had a largely
Scheme 1. Preparations of N-aryloxycarbonylimino neonicotinoid analogues.
Table 1
SARs of phenoxycarbonylimino neonicotinoid analogues in terms of insect nAChR
affinity and insecticidal activity
reduced potency. Therefore, the p-electron density on the benzene
ring is decisively responsible for the interaction with a regional
subsite of the receptor.
Cl
or
N
N
S
N S
N
O
Subsequently insecticidal activity of phenoxycarbonylimino
neonicotinoids to adult female flies was determined by intratho-
racic injection route (Table 1).15,16 Knockdown activity of com-
O
or
R
N
pound is
a sign of the intrinsic neurotoxicity triggered by
receptor interactions, yet lethal potency is largely influenced by
metabolic detoxification phase. Compounds (1, 9–14 and 16–18)
with high nAChR affinity showed apparent knockdown effect
(KD50). However, the phenoxycarbonylimino analogues generally
Compound
Musca nAChR
Toxicity to Muscab
(lg/g
female)
No.
Substituent (R) Ki SDa (nM, n = 3) KD50 (6 h)
LD50 (24 h)
Thiazolidines
1
2
3
4
5
6
7
8
H
46 5.5
140 23
150 10
150 14
210 22
240 45
320 27
520 25
40 2.2
18 2.3
44 4.6
32 4.0
33 3.7
19 1.1
57 3.0
1.0
>1 (7%)c
>1 (20%)c
>1 (13%)c
>1 (13%)c
>1 (20%)c
>1 (7%)c
>1 (0%)c
>1 (0%)c
0.63
Pyridin-3-yl
2-F
3-F
4-F
4-Cl
4-NO2
4-CF3
2-CH3
3-CH3
4-CH3
2,4-(CH3)2
2-OCH3
3-OCH3
4-OCH3
>1 (13%)c
>1 (20%)c
>1 (0%)c
>1 (20%)c
>1 (0%)c
>1 (0%)c
>1 (0%)c
0.12
9
10
11
12
13
14
15
0.60
0.90
0.16
0.11
>1 (20%)c
>1 (13%)c
1.0
>1 (40%)c
>1 (7%)c
>1 (18%)c
1.0
>1 (20%)c
Thiazolines
16
17
18
19
H
8.9 0.1
4.7 0.2
5.6 0.6
180 7.2
4.5 0.2
0.08
0.08
0.52
0.22
3-CH3
3-OCH3
4-CF3
0.08
1.0
>1 (0%)c
0.01
>1 (20%)c
0.02
Control IMI
a
Assayed with [3H]IMI. Ki = IC50/(1 + [L]/Kd) with [L] 5 nM and Kd 5.4 nM.13,14
Fifty percent knockdown (KD50) and lethality (LD50) doses were evaluated for 6
b
and 24 h, respectively, after toxicant administration via intrathoracic injection into
flies, which were pretreated with a cytochrome P450 inhibitor [O-propyl O-(2-
propynyl) phenylphosphonate].15,16
c
Knockdown or mortality % at the indicated dose.
This hypothesis is then examined by the present SAR approach
with phenoxylcarbonylimino probes with electron-withdrawing
and electron-donating substituents clarifying the function of the
aromatic
p-electron system. First, an electron-withdrawing fluo-
rine atom (similar in size to hydrogen atom) was introduced on
2-, 3- or 4-position, and expectedly the fluorophenoxy compounds
3–5 clearly lost the affinity of compound 1 by 3.3- to 4.6-fold. Com-
pounds with 4-chloro, 4-nitro and 4-trifluoromethyl groups (6–8)
also resulted in diminished affinity to a large extent presumably
associated with electronic circumstance and/or possibly bulk of
substituent on the phenyl ring. In contrast, substitution on 2- or
Figure 2. Structural model for binding site interactions of compound 17 with the
a
–b subunit interfacial agonist-binding domain of insect nAChR. The compound 17
is embraced, with an energy of À9.34 kcal/mol, by the ligand-binding pocket.
Relevant amino acids in green (loop B W174; loop C Y224) are from subunit and
a
in yellow (loop D W79; loop E N131, L141, and I143) are from b subunit. The
pyridine nitrogen atom forms a water bridge to the backbone NH of I143 and the
carbonyl oxygen of N131. The neonicotinoid phenyl plane undergoes a T-shape
aromatic interaction with the loop D W79 indole.

I. Ohno et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5933–5935
5935
5. Tomizawa, M.; Talley, T. T.; Maltby, D.; Durkin, K. A.; Medzihradszky, K. F.;
Burlingame, A. L.; Taylor, P.; Casida, J. E. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
9075.
6. Tomizawa, M.; Maltby, D.; Talley, T. T.; Durkin, K. A.; Medzihradszky, K. F.;
Burlingame, A. L.; Taylor, P.; Casida, J. E. Proc. Natl. Acad. Sci. U.S.A. 2008, 105,
1728.
had inferior insecticidal potency (LD50) relative to that of the
nitroimino IMI. These observations suggest that the phenoxylcar-
bonylimino neonicotinoids may be intrinsically active but are more
metabolically labile, conceivably due to the hydrolysis, relative to
the analogue with nitroimino or acylimino group.10,12
´
7. Talley, T. T.; Harel, M.; Hibbs, R. E.; Radic, Z.; Tomizawa, M.; Casida, J. E.; Taylor,
A docking simulation for the most active compound 17, 3-meth-
ylphenoxycarbonyliminothiazoline analogue, was performed with
P. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 7606.
8. Tomizawa, M.; Talley, T. T.; Park, J. F.; Maltby, D.; Medzihradszky, K. F.; Durkin,
K. A.; Cornejo-Bravo, J. M.; Burlingame, A. L.; Casida, J. E.; Taylor, P. J. Med. Chem.
2009, 52, 3735.
9. Tomizawa, M.; Casida, J. E. Acc. Chem. Res. 2009, 42, 260.
10. Tomizawa, M.; Kagabu, S.; Ohno, I.; Durkin, K. A.; Casida, J. E. J. Med. Chem.
2008, 51, 4213.
11. Ohno, I.; Tomizawa, M.; Durkin, K. A.; Naruse, Y.; Casida, J. E.; Kagabu, S. Chem.
Res. Toxicol. 2009, 22, 476.
12. Ohno, I.; Tomizawa, M.; Aoshima, A.; Kumazawa, S.; Kagabu, S. J. Agric. Food
Chem. 2010, 58, 4999.
13. Tomizawa, M.; Latli, B.; Casida, J. E. J. Neurochem. 1996, 67, 1669.
14. Zhang, A.; Kayser, H.; Maienfisch, P.; Casida, J. E. J. Neurochem. 2000, 75, 1294.
15. Liu, M.-Y.; Lanford, J.; Casida, J. E. Pestic. Biochem. Physiol. 1993, 46, 200.
16. Yamamoto, I.; Tomizawa, M.; Saito, T.; Miyamoto, T.; Walcott, E. C.; Sumikawa,
K. Arch. Insect Biochem. Physiol. 1998, 37, 24.
17. We performed preliminary docking of compound 17 to an Aplysia californica
AChBP crystal structure (protein data bank code 2WNJ, see Ref. 18) complexed
with 3-(2,4-dimethyoxybenzylidene)-anabaseine which is similar in size to
compound 17. We then carried out docking of this ligand to a homology model
an aphid (Myzus persicae)
a
2b1 nAChR structural model6,10,11 to
examine the binding site interactions (Fig. 2).17–24 The compound
17 was calculated to be docked favourably by the interfacial ago-
nist-binding pocket between the Myzus
a2 and b1 subunits. The
chloropyridinyl chlorine atom can have favourable van der Waals
interactions with the backbone of loop E N131 and L141. The pyr-
idine nitrogen forms a water bridge to the backbone of loop E N131
and I143. The electronically conjugate amidine plane primarily p-
stacks with the loop C Y224 aromatic side chain and also interacts
via stacking or hydrophobic interactions with other aromatic resi-
dues like the loop B W174 indole moiety. Olefinic
p-electrons of
the thiazoline compound are sandwiched between Y224 and
W174, conferring the enhanced affinity compared with that of sat-
urated thiazolidine compound. The carbonyl and phenoxy oxygen
atoms make hydrogen-bonds with the loop D W79 aromatic NH
and/or loop C C226 backbone NH (not displayed). The neonicoti-
noid phenyl ring and the W79 indole side chain undergo a T-
shaped aromatic interaction reinforced by 3-methyl substituent
on the phenyl ring. The face-to-edge aromatic interaction can pro-
of the Myzus
a2b1 receptor (see Refs. 6,10,11), after first allowing the active
site of the homology model to optimise and adjust to ligands of similar size. It
is well known that a water molecule forms a critical hydrogen-bonding bridge
between the pyridinyl nitrogen of the neonicitinoid and the receptor (see Refs.
7,19), so we placed a water molecule in the active site prior to optimisation and
docking. Geometry optimisations included all residues within 5 Å of the active
site while residues beyond that were either partially constrained (5 Å beyond
inner shell) or frozen (remainder of the structure). Up to 5000 steps per
minimisation were run to achieve a gradient of 0.5 with respect to energy
using the OPLS forcefield in Macromodel (see Refs. 20,21). Docking calculations
were done using Glide as implemented in Maestro (Glide 5.5, Maestro 9.0,
Schrödinger, LLC, New York, NY, 2010) (see Ref. 22). The docking includes a
spatial fit of the ligand to the receptor grid, followed by minimisation and
scoring of hits based on a discretized ChemScore function (see Refs. 23,24).
Ligands were flexibly docked using standard precision and the top hits were
examined.
vide about as much stabilization as the more standard
p-stack-
ing.25 Accordingly, the present calculation result is fully reflective
of the observed SARs.
In summary, this investigation exemplifies a ligand molecular
design reconciling the chemorational SAR and the receptor struc-
ture-aided approaches, stimulating further discovery of novel nic-
otinic insecticides with unique biological properties.
18. Hibbs, R. E.; Sulzenbacher, G.; Shi, J.; Talley, T. T.; Conrod, S.; Kem, W. R.; Taylor,
P.; Marchot, P.; Bourne, Y. EBMO J. 2009, 28, 3040.
Supplementary data
19. Ohno, I.; Tomizawa, M.; Durkin, K. A.; Casida, J. E.; Kagabu, S. J. Agric. Food
Chem. 2009, 57, 2436.
20. Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J. J. Am. Chem. Soc. 1996, 118,
11225.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.07.049.
21. Mohamadi, F.; Richard, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.; Caufield,
C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11, 440.
22. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.;
Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.;
Shenkin, P. S. J. Med. Chem. 2004, 47, 1739.
23. Eldridge, M. D.; Murray, C. W.; Auton, T. R.; Paolini, G. V.; Mee, R. P. J. Comput.
Aided Mol. Des. 1997, 11, 425.
24. Baxter, C. A.; Murray, C. W.; Clark, D. E.; Westhead, D. R.; Eldridge, M. D.
Proteins 1998, 33, 367.
References and notes
1. Kagabu, S. In Chemistry of Crop Protection; Voss, A., Ramos, G., Eds.; Wiley-VCH:
Weinheim, 2003; pp 193–212.
2. Matsuda, K.; Shimomura, M.; Ihara, M.; Akamatsu, M.; Sattelle, D. B. Biosci.
Biotechnol. Biochem. 2005, 69, 1442.
3. Tomizawa, M.; Casida, J. E. Annu. Rev. Pharmacol. Toxicol. 2005, 45, 247.
4. Jeschke, P.; Nauen, R. Pest Manag. Sci. 2008, 64, 1084.
25. McGaughey, G. B.; Gagné, M.; Rappé, A. K. J. Biol. Chem. 1998, 273, 15458.