Potency and selectivity of trifluoroacetylimino and pyrazinoylimino nicotinic insecticides and their fit at a unique binding site niche

Article abstract of DOI:10.1021/jm800191a

Neonicotinoid agonists with a nitroimino or cyanoimino pharmacophore are the newest of the four most important classes of insecticides. Our studies on the nicotinic receptor structure in the neonicotinoid-bound state revealed a unique niche of about 6 ? depth beyond the nitro oxygen or cyano nitrogen tip. The N-substituted imino pharmacophore was therefore extended to fill the gap. Excellent target site selectivity with high insecticidal activity and low toxicity to mammals were achieved rivaling those of the current neonicotinoid insecticides as illustrated here by 3-(6-chloropyridin-3-ylmethyl)-2- trifluoroacetyliminothiazoline and its pyrazinoylimino analogue.

Full text of DOI:10.1021/jm800191a

J. Med. Chem. 2008, 51, 4213–4218
4213
Potency and Selectivity of Trifluoroacetylimino and Pyrazinoylimino Nicotinic Insecticides and
Their Fit at a Unique Binding Site Niche
Motohiro Tomizawa,‡ Shinzo Kagabu,§ Ikuya Ohno,§ Kathleen A. Durkin,| and John E. Casida*,‡
EnVironmental Chemistry and Toxicology Laboratory, Department of EnVironmental Science, Policy, and Management, UniVersity of
California, Berkeley, California 94720-3112, Department of Chemistry, Faculty of Education, Gifu UniVersity, Gifu 501-1193, Japan, and
Molecular Graphics and Computational Facility, College of Chemistry, UniVersity of California, Berkeley, California 94720-1460
ReceiVed February 22, 2008
Neonicotinoid agonists with a nitroimino or cyanoimino pharmacophore are the newest of the four most
important classes of insecticides. Our studies on the nicotinic receptor structure in the neonicotinoid-bound
state revealed a unique niche of about 6 Å depth beyond the nitro oxygen or cyano nitrogen tip. The
N-substituted imino pharmacophore was therefore extended to fill the gap. Excellent target site selectivity
with high insecticidal activity and low toxicity to mammals were achieved rivaling those of the current
neonicotinoid insecticides as illustrated here by 3-(6-chloropyridin-3-ylmethyl)-2-trifluoroacetyliminothiazoline
and its pyrazinoylimino analogue.
Introduction
rivaling those of the present neonicotinoids and other chemo-
types of commercial insecticides.
The nicotinic acetylcholine (ACh) receptor (nAChRa) is an
important target of insecticides for crop protection and public
health and of therapeutic agents for neurological dysfunction.
Neonicotinoid insecticides such as imidacloprid (IMI) and
thiacloprid (THIA) (Chart 1) are increasingly replacing orga-
nophosphorus compounds and methylcarbamates because of
improved safety and effectiveness particularly due to the
selection of resistant pest strains.1,2 High neonicotinoid potency
and selectivity toward the insect nAChR are ultimately attribut-
able to the differential binding site interactions which have been
mapped with mollusk ACh binding proteins (AChBPs)3,4 as
excellent structural surrogates for the extracellular ligand-binding
domain of the nAChRs.5,6 The IMI binding site is located at an
interfacial region between the primary or (+)-face and the
complementary or (-)-face subunits of the AChBP (correspond-
ing to R and non-R subunits, respectively, of the nAChR) (Figure
1). The nitro oxygen or cyano nitrogen tip of IMI or THIA,
respectively, H-bonds to the backbone HN of C190 and/or S189
on loop C of the primary subunit of AChBP.3 The AChBP-IMI
structure, fascinatingly, provides a space of approximately 6 Å
depth that extends from the IMI nitro tip oxygen toward Q57
on loop D of the (-)-face subunit. Q57 of AChBP is spatially
equivalent to R, K, or N of the insect nAChR ꢀ subunits, while
T with a shorter side chain takes this position on the ꢀ2 subunit
of the vertebrate neuronal nAChR.
Results and Discussion
Structure-Activity Relationships (SAR) at the Insect
nAChR. Replacement of the nitro- or cyanoimino pharmacoph-
ore by extended substituents may provide point(s) for hydrogen
acceptance and/or van der Waals (VDW) contacts at the targeted
regional binding domain: i.e., loop D amino acid(s) on the
complementary or ꢀ subunit of the insect nAChR. According
to this hypothesis, phenyl, pyridine, and pyrazine derivatives
(1-7) were prepared and binding affinities evaluated with
Drosophila nAChR (Table 1). The affinity of the phenyl
analogue (1) was 9.3- to 25-fold lower than that of the pyridine
compounds (2-4). The 2,5-pyrazine analogue (5) showed the
highest affinity similar to a pyridinylidene-2-pyrazinecarboxa-
mide neonicotinoid derivative with an affinity 4-fold less than
IMI.7,8 The SAR clearly indicates that hydrogen-accepting
nitrogen atom(s) plays a crucial role in recognition by the amino
acid(s) at the binding domain. The pyrazine moiety with two
nitrogen atoms provides two hydrogen-accepting points. Nitro-
gen at the 5-position (equivalent to that of 3-pyridine) is
important, and one at the 2-position (corresponding to that of
2-pyridine) enhances the potency. The 4-chlorophenyl com-
pound (6) had >250-fold lower activity than unsubstituted 1,
and similarly, the 6-chloro-3-pyridinyl analogue (7) had an 8.5-
fold reduced affinity compared to the unsubstituted 3-pyridinyl
compound (3), suggesting that chlorine provides spatial hin-
drance at the binding domain. Most interestingly, compounds
8 and 9 with a trifluoroacetyl substituent, providing both VDW-
contacting and hydrogen-accepting abilities, gave high affinities.
Insecticidal Activities (Table 1). Adult female houseflies
(Musca domestica) were used for quantitative evaluation of
insecticidal activity. First, intrinsic activities [defined by in-
trathoracic injection into flies which are pretreated with a
cytochrome P450 inhibitor (synergist)] of test compounds were
compared with neonicotinoid insecticides IMI and THIA.
Analogues with phenyl (1), pyrazine (5), chloropyridine (7),
and trifluoroacetyl (8, 9) substituents had moderate to high
insecticidal activity, whereas the three pyridine isomers (2, 3,
4) with high nAChR affinity were not insecticidal, suggesting
metabolic instability. Interestingly chloropyridine analogue 7
The structural model for the AChBP-neonicotinoid complex3
prompted the present study of selected ligands proposed to fit
the loop D amino acid(s) in the insect nAChRs. We designed a
series of prototype compounds with extended N-substituted
imine substituents (pyrazinoylimine and trifluoroacetylimine)
(Chart 1 and Figure 1), finally leading to candidate nicotinic
insecticides with high potency and excellent target site selectivity
* To whom correspondence should be addressed. Phone: 510-642-5424.
Fax: 510-642-6497. E-mail: ectl@nature.berkeley.edu.
‡
Department of Environmental Science, Policy, and Management,
University of California.
§
Gifu University.
College of Chemistry, University of California.
|
a Abbreviations: AChBP, acetylcholine binding protein; IMI, imidaclo-
prid; MD, molecular dynamics; nAChR, nicotinic acetylcholine receptor;
THIA, thiacloprid; VDW, van der Waals.
10.1021/jm800191a CCC: $40.75
2008 American Chemical Society
Published on Web 06/21/2008

4214 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Tomizawa et al.
Chart 1. Structures of Two Major Neonicotinoid Insecticides Imidacloprid (IMI) and Thiacloprid (THIA) with Nitro- and Cyanoimine
Moieties, Respectively, and the N-Substituted Imino Analogues (1-9)
with moderate nAChR affinity was highly insecticidal, presum-
ably conferring improved hydrophobicity and metabolic stability
by introduction of the chlorine atom. In topical application,
compounds 1 and 5 were quite potent with synergist, whereas
7-9 were as active as or more so than IMI and THIA.
Remarkably, topically applied trifluoroacetyliminothiazoline 9
showed high insecticidal activity even in the absence of
synergist, conceivably due to higher hydrophobicity and meta-
bolic stability than those of IMI and THIA. More specifically,
the activity level of 9 rivals those of other types of commercial
insecticides (Table 1 footnote).
The common brown house mosquito (Culex quinquefasciatus)
provides an example of a major pest with increasing difficulty
to control because of selection of strains resistant to the major
pyrethroid, organophosphate, and methylcarbamate insecticides.
Neonicotinoids are possible replacements if they have suitable
potency and little or no cross-resistance.11,12 Neonicotinoid 9
was compared with permethrin, the standard mosquito control
agent, for contact toxicity to adults and potency (50% lethal
concentration or LC50) on fourth instar larvae. Permethrin was
far superior to 9 in contact adulticidal activity (data not shown).
Although 9 was only 2.4-fold less active than permethrin as a
larvicide on the susceptible colony, it was 6.7 times more potent
on the permethrin-resistant colony (Table 2). The target site
cross-resistance of pyrethroids and DDT or of organophosphates
andmethylcarbamatesdoesnotcarryovertotheneonicotinoids,11,12
making them candidates for further development as mosquito
control agents.
Selectivity. Selective toxicity is critical for insecticide use,
combining outstanding effectiveness for pests with safety for
humans and wildlife. The selectivity of representative com-
pounds 5 and 9 was evaluated by comparing the binding affinity
to the Drosophila and chick R4ꢀ2 nAChRs and toxicity to
houseflies and mice (Table 3). Relative to target site selectivity,
compounds 5 and 9 had low potency at the chick R4ꢀ2 nAChR
(600- and 161-fold less active, respectively, than the insect
Figure 1. Design of selective nicotinic agonists interacting with loop D amino acid(s) in the insect nAChR. (a) IMI nestled in the interfacial
agonist-binding pocket between the (+)-face (primary, yellow) and (-)-face (complementary, blue) subunits of the Aplysia californica AChBP3 as
a structural surrogate of the nAChR extracellular ligand-binding domain. (b) AChBP-IMI binding site interactions (zoomed-in) featuring hydrogen-
bonding between the IMI nitro tip oxygen and the backbone HN of the loop C C190 and/or S189 (not shown, since it would obscure the nitro tip
of IMI). An approximately 6 Å depth cavity exists beyond the nitro tip oxygen of IMI toward the loop D Q57 amide side chain. (c) Alignment of
the loop D amino acid sequences from two mollusk AChBP subtypes, five insect ꢀ subunits (represented by fruit fly Drosophila melanogaster,
peach-potato aphid Myzus persicae, and tobacco budworm Heliothis Virescens) and the vertebrate (human, rodent, and chick) ꢀ2 subunit of the
nAChR. (d) Selected prototype compounds with N-substituted imine moiety and extended pharmacophore replacing the regular nitro- or cyanoimine
moiety. The trifluoroacetylimine or pyrazinoylimine is expected to undergo hydrogen-bonding and/or van der Waals (VDW) contacts with the loop
D niche, whereas the nitro or cyano tip atom interacts primarily with the loop C region.

Imino Nicotinic Insecticides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4215
Table 1. SAR of Neonicotinoids in Terms of Insect nAChR Affinity
and Insecticidal Activity
and vertebrate (rat) ꢀ2 subunit were 720 and 280 nM,
respectively (Table 3 footnote). In sharp contrast, insecticides
IMI and THIA showed high affinities to the hybrid receptor
(3.6 and 11 nM, respectively),13 strongly indicating that the
insect R subunit plays a critical role in the recognition of IMI
and THIA with nitro- and cyanoimino moieties. However, the
insect ꢀ subunit is presumably important for embracing the
extended pyrazinoylimine or trifluoroacetylimine moiety of 5
or 9.
LD50 (µg/g)b,c
topical
injection
compda IC50 ( SD (nM)b,c synergistd
synergistd
alonee
Pyrazinoylimine and Analogues
1
2
3
4
5
6
7
121 ( 6
1.0
>1 (0%)
2.0
>10 (0%)
13 ( 1
>10 (0%) >10 (0%)
4.8 ( 0.2
10 ( 2
1.5 ( 0.04
>30000 (10%)
41 ( 2
>1 (13%) >10 (0%) >10 (0%)
>1 (13%) >10 (0%) >10 (0%)
0.035
>1 (0%)
0.080
Insect nAChR Binding Site Interactions. A structural model
for the interfacial agonist-binding domain of the insect nAChR
from M. persicae (R2ꢀ1) is established on the basis of the crystal
structure of Aplysia californica AChBP, which is sensitive to
neonicotinoids.4 This model is representative of insect nAChRs,
since the important amino acids forming the binding pockets
are fully conserved in all of the known insect nAChRs. The
AChBP molecular dynamics (MD) simulations defining the
binding site interactions with 5 and 9 are given in Supporting
Information as the foundation for those with the insect nAChR
structural model.
2.8
>10 (0%)
>10 (0%) >10 (0%)
0.12
>10 (0%)
Trifluoroacetylimine Analogues
8
9
7.7 ( 0.7
3.1 ( 1.0
0.064
0.027
0.071
0.035
∼10
0.75f
Nitro- and Cyanoimine Standards
IMI
THIA
4.3 ( 0.3
2.7 ( 0.4
0.021
0.032
0.20
0.14
>10 (0%)
>10 (19%)
a Calculated log P values by ALOGPS9 for compounds 1-9 are 3.56,
2.48, 2.21, 2.20, 1.61, 4.14, 3.35, 2.40, and 3.04, respectively, compared
with those of IMI and THIA of 0.65 and 1.91 (observed log P of 0.57 and
1.26),2 respectively. b Assayed as [3H]IMI binding to the Drosophila nAChR
and toxicity against adult female houseflies. c Percent inhibition or mortality
at the indicated concentration or dose is given in parentheses. d Pretreated
topically with the synergist O-propyl O-(2-propynyl)phenylphosphonate
(PPP) (100 µg/g) before administration of the test chemical. e Standard
insecticides chlorpyrifos and propoxur gave LD50 of 2.1 and 37 µg/g,
respectively (present study), compared with those of parathion, propoxur,
dieldrin, and DDT of 1.3, 23, 0.7, and 14 µg/g, respectively.10 f Contact
toxicity in the absence of metabolic inhibitor: dose for 50% knockdown,
0.04 µg/cm2 (0.5-8 h); LD50, 0.2 µg/cm2 (24 h).
In a MD snapshot for the insect nAChR liganded with
pyrazinoylimine 5 (Figure 2), the pyrazine nitrogen atom at the
5-position H-bonds with the loop D R81 guanidine NH2 (2.6
Å), although R81 may take multiple geometries because of its
flexible carbon units. The N at the 2-position H-bonds with loop
D W79 indole HN (2.2 Å), and the NC(O) oxygen makes
contact with the W79 indole HN (2.6 Å). As with IMI and
THIA,3,4 the amidine plane π-stacks with the loop C Y224
aromatic residue (3-4 Å) and also with the loop B W174 side
chain (4-5 Å). Similarly in a MD result for the complex with
trifluoroacetylimine 9, the three fluorine atoms variously interact
with loops C and D: i.e., H-bonding to the backbone HN of
C226 (2.4 Å) and V225 (3.3 Å) (not displayed), to R81
guanidine NH2 (directly or possibly via a water bridge) (3.5
Å), and to W79 indole HN (2.5 Å). VDW contacts with the
W79 indole ring. The NC(O) oxygen H-bonds to the W79 indole
HN (2.7 Å). The amidine plane of 9 is sandwiched via
π-stacking between the Y224 phenol ring (3-4 Å) and W174
indole residue (4-5 Å). For both compounds 5 and 9, the
chloropyridine Cl VDW-contacts to the backbone carbonyl
oxygens of loops E N131 and L141 (3.3-3.7 and 3.2-3.4 Å,
respectively) and the pyridine N undergoes H-bonding with the
backbone carbonyl oxygens of loop E I143 and loop B W174
(4.3-4.4 and 3.9 Å, respectively) possibly via water bridge(s).
Therefore, the pyrazinoyl (and possibly the pyridinoyl) moiety
embraces primarily the loop D niche on the partnering subunit
and the trifluoroacetyl substituent is nestled in an interfacial
region between loops C and D, whereas the nitro- or cyanoimino
pharmacophore of IMI or THIA principally interacts with the
loop C tip area on the primary subunit. The present structural
models are consistent with the observed SAR.
Table 2. Mosquito Larvicidal Activity of Neonicotinoid 9 and
Permethrin with Susceptible and Permethrin-Resistant Colonies of
Fourth Instar Culex quinquefasciatus
LC50 (ppm)
insecticide
susceptible
resistant
9
0.012
0.0050
0.024b
0.16
permethrina
a 1-RS-Permethrin (trans e 65% and cis g 35%). b Estimated from a
separate experiment showing that the resistant colony was 2-fold less
sensitive than the susceptible colony.
Table 3. Selectivity between Insects and Vertebrates in Terms of
nAChR Affinity and Organismal Toxicity
IC50 ( SD (nM)
LD50 (mg/kg)
compda Drosophilab chick R4ꢀ2c ratio houseflyb moused
ratio
5
9
IMI
1.5 ( 0.04
3.1 ( 1.0
4.3 ( 0.3
900 ( 96 600
500 ( 100 161
2600 ( 85 605
860 ( 31 319
0.035 >24 (0%)d >685
0.027 >36 (0%)d >1333
0.021 45
0.032 28
2142
875
THIA 2.7 ( 0.4
a IC50 values (nM) of compounds 5 and 9 as displacers of [3H]IMI
binding to the hybrid nAChR consisting of peach-potato aphid (M.
persicae) R2 and rat ꢀ2 subunits are 720 ( 46 and 280 ( 24, respectively,
compared with those of IMI and THIA (3.6 ( 0.1 and 11 ( 1 nM,
respectively).13 b Data from Table 1 (binding affinity assayed with [3H]IMI
and insecticidal activity with synergist via injection). c Assayed with
[3H]nicotine. d Intraperitoneal administration. Percent lethality at the
indicated dose (maximal dose administered because of the solubility
limitation in vehicle) with no poisoning signs.
Target Site Selectivity. When the loop D regions on the ꢀ
subunits from the insect (W79, L80, and R81) and vertebrate
(W75, L76, and T77) receptors are overlaid, the insect R81 more
intimately faces the pyrazine or CF3 moiety compared to the
vertebrate T77 (g4 Å difference), presumably serving as a
determinant for target site selectivity (Figure 3). Intriguingly,
target site selectivity for IMI/THIA depends on multiple bound
ligand conformations such that the final binding constant
represents a combination of individual constants specific to
different conformations; i.e., the weakly binding neonicotinoids
adopt two different binding orientations at the vertebrate nAChR
binding pocket, whereas a single tight binding conformation
reflects the high affinity to the insect nAChR.4 This unique
nAChR). Fascinatingly, at the organismal level, compounds 5
and 9 were >685- and >1300-fold less toxic to mice than
houseflies. These intrinsic selectivity ratios at target site and
toxicity levels were similar to those of IMI and THIA.1,2 In
addition, the binding affinities of 5 and 9 to a hybrid nAChR
consisting of peach-potato aphid Myzus persicae R2 subunit

4216 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Tomizawa et al.
Figure 2. Structural models for binding site interactions of pyrazinoylimino (5) and trifluoroacetylimino (9) nicotinic insecticides with the R-ꢀ
subunit interfacial agonist-binding domain of insect nAChR. (a) Relevant amino acids displayed are loop B W174 in pink; loop C Y224 in yellow;
loop D W79, L80, and R81 in aquamarine; loop E N131, L141, and I143 in green. (b) Superimposition of bound conformation of 5 or 9 overlaid
with that of IMI as observed in the insect receptor binding pocket.
1H and 13C NMR spectra were recorded for solutions in CDCl3
unless otherwise stated using a JEOL ECA-500 spectrometer
(Tokyo, Japan) at 500 and 125 MHz, respectively. Mass spectra
were determined at 70 eV with the JEOL JMS-700 instrument.
Combustion analyses were performed with the Yanaco CHN
CORDER MT-6 elemental analyzer (Kyoto, Japan).
2-Benzoylimino-3-(6-chloro-3-pyridylmethyl)thiazoline (1), 2-(4-
Chlorobenzoyl)imino-3-(6-chloro-3-pyridylmethyl)thiazoline (6),
and 2-(6-Chloro-3-pyridinoyl)imino-3-(6-chloro-3-pyridylmeth-
yl)thiazoline(7).Toanice-cooledsolutionof3-[(6-chloro-3-pyridinyl)-
methyl]-2-iminothiazoline15,16 (113 mg, 0.5 mmol) and triethy-
lamine (101 mg, 1 mmol) in pyridine (3 mL) was added slowly
Figure 3. Structural comparison of loop D amino acids from insect
and vertebrate ꢀ subunits with R81 and T77, respectively, suggesting
their dissimilar interactions with 5 and 9. Loop D amino acids W79,
L80, and R81 (aquamarine) on the ꢀ subunit of the insect agonist-
binding pocket (Myzus R2ꢀ1) interacting with 5 (left) or 9 (right) are
superimposed onto the equivalent region W75, L76, and T77 (yellow)
on the ꢀ subunit of the vertebrate (chick) R4ꢀ2 interface.14
benzoyl chloride (77 mg, 0.55 mmol) (for compound 1), 4-chlo-
robenzoyl chloride (180 mg, 1.03 mmol) (for compound 6), or
6-chloro-3-pyridinoyl chloride (175 mg, 0.99 mmol) (for compound
7). The reaction mixture was stirred at this temperature for 3.5 h,
poured onto 20 mL of cold water, and alkalized to about pH 10
with Na2CO3. The resulting precipitate was filtered off, washed with
water, and dried. Recrystallization from ethanol gave the products.
Compound 1: yield 45%; mp ) 130 °C. 1H NMR: δ 5.48 (s, 2H),
6.71 (d, 1H, J ) 5.1 Hz), 7.02 (d, 1H, J ) 5.1 Hz), 7.31 (d, 1H,
J ) 8.2 Hz), 7.43-7.52 (m, 3H), 7.70 (1H, dd, J ) 8.2 Hz, 2.3
Hz), 8.30 (m,2H), 8.48 (d, 1H, J ) 2.3 Hz). 13C NMR: δ 48.7,
110.2, 124.9, 125.1, 128.3, 129.3, 130.5, 131.8, 136.6, 138.8, 149.2,
151.8, 168.2, 174.2. EI-LRMS m/z (%): 329 (M+, 96), 293 (M+
- Cl, 8), 224 (M+ - COC6H5, 25), 188 (29), 126 (Cl - Py -
CH2, 36), 105 (COC6H5, 100). EI-HRMS calcd for C16H12ClN3OS,
309.0390; found 309.0390. Anal. (C16H12ClN3OS) calcd C, 58.27;
H, 3.67; N, 12.74; found C, 57.97; H, 3.90; N, 12.74. Compound
concept may also be applicable to the present pyrazinoylimine
and trifluoroacetylimine insecticides.
Concluding Remarks. Highly potent and selective nicotinic
insecticides were achieved by pharmacophore modification of
neonicotinoids with extended and hydrophobic substituents
fitting the loop D amino acid(s) in the insect nAChR. The
present investigation illustrates receptor structure-guided ligand
design for lead generation and discovery of novel insecticides
with excellent target site selectivity, high insecticidal activity,
and low toxicity to mammals.
1
6: yield, 61%; mp ) 191 °C. H NMR: δ 5.48 (s, 2H), 6.74 (d,
1H, J ) 4.8 Hz), 7.02 (d, 1H, J ) 4.8 Hz), 7.32 (d, 1H, J ) 8.2
Hz), 7.41 (d, 2H, J ) 8.9 Hz), 7.68 (1H, dd, J ) 8.2 Hz, 2.1 Hz),
8.23 (d, 2H, J ) 8.9 Hz), 8.48 (d, 1H, J ) 2.1 Hz). 13C NMR: δ
48.7, 110.3, 124.9, 125.0, 128.4, 130.3, 130.6, 135.0, 137.9, 138.6,
149.1, 151.9, 168.2, 173.2. EI-LRMS m/z (%): 363 (M+, 88), 293
(M+ - Cl, 6), 224 (M+ - COC6H5, 39), 188 (21), 139 (COC6H4Cl,
Experimental Section
General. 1-(6-Chloropyridin-3-ylmethyl)-2-trifluoroacetylimi-
noimidazoline (8) and its thiazoline analogue 9 were available from
our previous studies.15,16 All melting points (mp) are uncorrected.

Imino Nicotinic Insecticides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4217
100), 126 (Cl - Py - CH2, 46), 111 (C6H5Cl, 60). EI-HRMS calcd
for C16H11Cl2N3OS, 363.0000; found 363.0014. Anal.
(C16H11Cl2N3OS) calcd C, 52.76; H, 3.04; N, 11.54; found C, 52.56;
(C14H10ClN5OS) calcd C, 50.68; H, 3.04; N, 21.11; found C, 50.69;
H, 3.35; N, 20.90.
Biology. Radioligand binding experiments involved (1) [3H]IMI
with the native Drosophila brain nAChR and insect-vertebrate
hybrid recombinant receptor consisting of aphid Myzus R2 and rat
ꢀ2 subunits and (2) [3H]nicotine with chick R4ꢀ2 nAChR.13,16,17
Insecticidal activity was evaluated with adult female houseflies via
intrathoracic injection and topical application in the absence and
the presence of a cytochrome P450 inhibitor [O-propyl O-(2-
propynyl)phenylphosphonate (PPP)], which serves as a synergist
by reducing the oxidative detoxification rate.18 Mosquito larvicidal
activities against susceptible (CQ1) and permethrin-resistant (Marin)
colonies of Culex quinquefasciatus were examined with fourth
instars and 24 h of exposure according to McAbee et al.19
Mammalian toxicity was determined with male albino Swiss-
Webster mice (25-30 g) treated intraperitonealy with the test
compound dissolved in Me2SO.
1
H, 3.32; N, 11.34. Compound 7: yield, 76%; mp ) 176 °C. H
NMR: δ 5.48 (s, 2H), 6.80 (d, 1H, J ) 4.6 Hz), 7.07 (d, 1H, J )
4.6 Hz), 7.33 (d, 1H, J ) 8.2 Hz), 7.40(d, 1H, J ) 8.3 Hz), 7.66
(dd, 1H, J ) 8.2 Hz, 2.3 Hz), 8.44 (d, 1H, J ) 2.3 Hz), 8.46 (dd,
1H, J ) 8.3 Hz, 2.3 Hz), 9.24 (d, 1H, J ) 2.3 z). 13C NMR: δ
48.9, 110.7, 123.9, 124.8, 125.3, 129.9, 131.0, 138.5, 139.2, 149.0,
151.1, 152.0, 154.1, 168.2, 171.5. EI-LRMS m/z (%): 364 (M+,
100), 293 (M+ - Cl, 4), 224 (M+ - COPy - Cl, 41), 208 (15),
188 (15), 140 (COPy - Cl, 93), 126 (Cl - Py - CH2, 85), 112
(Py - Cl, 45). EI-HRMS calcd for C15H10Cl2N4OS, 363.9952; found
363.9948. Anal. (C15H10Cl2N4OS) calcd C, 49.33; H, 2.76; N, 15.34;
found C, 49.02; H, 2.94; N, 15.25.
3-(6-Chloro-3-pyridylmethyl)-2-(2-pyridinoyl)iminothiazoline (2),
3-(6-Chloro-3-pyridylmethyl)-2-(3-pyridinoyl)iminothiazoline (3),
3-(6-Chloro-3-pyridylmethyl)-2-(4-pyridinoyl)iminothiazoline (4),
and 3-(6-Chloro-3-pyridylmethyl)-2-(pyrazinoyl)iminothiazoline
(5). To a solution of 3-[(6-chloro-3-pyridinyl)methyl]-2-iminothia-
zoline (75 mg, 0.25 mmol) and triethylamine (202 mg, 2.0 mmol)
in acetonitrile (15 mL) was added slowly 2-pyridinoyl chloride
hydrochloride (56 mg, 0.31 mmol) (for compound 2), 3-pyridinoyl
chloride hydrochloride (37 mg, 0.21 mmol) (for compound 3),
4-pyridinoyl chloride hydrochloride (56 mg, 0.31 mmol) (for
compound 4), or pyrazinoyl chloride hydrochloride (50 mg, 0.28
mmol) (for compound 5). The reaction mixture was stirred at the
refluxing temperature for 4 h, and the solvent and excess triethy-
lamine were removed in vacuo. The residue was taken up with
ethyl acetate, washed successively with 10% aqueous Na2CO3 and
water, and dried. Chromatography on silica with ethyl acetate and
methanol (5:1) gave the products. Compound 2: yield, 18%; mp )
Modeling and Calculations. The X-ray crystal structure for
Aplysia californica AChBP6 as the EPI-bound form (PDB code
2BYQ) was used as the template for building the insect nAChR
homology model. The protein sequences of the R2 and ꢀ1 subunits
for M. persicae20,21 obtained from UniProt22 (accession numbers
P91764 for R2 and Q9NFX8 for ꢀ1) were aligned with 2BYQ using
the CLUSTALW23 Web server at the European Bioinformatics
Institute.24,25 Chain A of the PDB structure was chosen for sequence
alignment with the ligand binding domain (residues R2 1-240
versus 2BYQ:A and ꢀ1 1-240 versus 2BYQ:A). These alignments
were input to the SWISS-MODEL protein homology server.26 The
resulting model subunit structures were imported into Maestro 7.5
(Schro¨dinger, L.L.C., Portland, OR). The R2 and ꢀ1 model
structures were aligned via comparison of backbone atoms onto
the corresponding subunits in 2BYQ with further optimization
limited to a single R2/ꢀ1 subunit pair and hence a single binding
interface. This R2ꢀ1 model was subjected to cycles of energy
minimization using the OPLS_2005 force field implemented in
Macromodel 9.1.27,28 Up to 5000 steps per minimization were run
to achieve a gradient of 0.5 with respect to energy. In the initial
minimization cycles, the backbone was held constant. In subsequent
minimization cycles, the region within 15 Å of the binding pocket
was free to move with progressive constraints on the remainder of
the structure. Docking calculations were carried out using AutoDock
4.29,30 The receptor was treated as rigid, while flexible ligands were
docked in a 15 Å cubic grid centered on the active site. In each
case, a 200 step Lamarkian genetic algorithm search was performed.
Good quality hits were those with binding energies below -7 kcal/
mol. Selected hits were treated for further MD simulations. These
were minimized, then equilibrated for 10-100 ps and simulated
for 100 ps to 1 ns at 300 K with a 1 fs time step and SHAKE
applied to all bonds to hydrogen. These simulations were run using
Macromodel with the OPLS2005 force field and a water continuum
model.
1
207-208 °C. H NMR: δ 5.51 (s, 2H), 6.73 (d, 1H, J ) 4.1 Hz),
7.05 (d, 1H, J ) 4.1 Hz), 7.27 (d, 1H, J ) 8.3 Hz), 7.37 (m, 1H),
7.73(m, 1H), 7.79 (m, 1H), 8.29 (d, 2H, J ) 8.3 Hz), 8.48 (d, 1H,
J ) 2.1 Hz), 8.76 (d, 1H, J ) 4.2 Hz). 13C NMR: δ 49.0, 110.7,
124.5, 124.8, 125.4, 125.6, 130.3, 136.7, 139.3, 149.6, 149.8, 151.8,
153.8, 169.0, 173.0. EI-LRMS m/z (%): 330 (M+, 28), 252 (M+
- Py), 224 (M+ - COPy, 53), 204 (M+ - ClPyCH2, 5), 189 (5),
126 (Cl - Py - CH2, 100). EI-HRMS calcd for C15H11ClN4OS,
330.0342; found 330.0357. Anal. (C15H11ClN4OS) calcd C, 54.46;
H, 3.35; N, 16.94; found C, 54.08; H, 3.47; N, 16.86. Compound
1
3: yield, 83%; mp ) 155 °C. H NMR: δ 5.50 (s, 2H), 6.78 (d,
1H, J ) 4.8 Hz), 7.07 (d, 1H, J ) 4.8 Hz), 7.33 (d, 1H, J ) 8.3
Hz), 7.39 (m, 1H), 7.70 (d, 1H, J ) 8.3, 2.1 Hz), 8.48 (d, 1H, J )
2.1 Hz), 8.50 (m, 1H), 8.71 (dd, 1H, J ) 4.8, 2.0 Hz), 9.51 (d, 1H,
1.4 Hz). 13C NMR: δ 48.9, 110.6, 123.3, 124.9, 125.3, 130.2, 132.0,
136.7, 138.7, 149.1, 151.0, 152.0, 152.2, 168.3, 172.7. EI-LRMS
m/z (%): 330 (M+, 51), 293 (M+ - Cl, 44), 224 (M+ - COPy,
49), 204 (M+ - ClPyCH2, 44), 188 (16), 126 (Cl - Py - CH2,
100). EI-HRMS calcd for C15H11ClN4OS, 330.0342; found 330.0343.
Anal. (C15H11ClN4OS) calcd C, 54.46; H, 3.35; N, 16.94; found C,
54.19; H, 3.35; N, 16.67. Compound 4: yield 30%; mp ) 179-180
1
°C. H NMR: δ 5.51 (s, 2H), 6.81 (d, 1H, J ) 4.6 Hz), 7.10 (d,
Acknowledgment. J.E.C. was supported by the William
Muriece Hoskins Chair in Chemical and Molecular Entomology.
M.T. and J.E.C. were supported by National Institute of
Environmental Health Sciences Grant R01 ES08424, and K.A.D.
was supported by National Science Foundation Grant CHE-
0233882. The authors received technical assistance from
Berkeley colleague Daniel Nomura. Anthony Cornel and Roy
McAbee of the Mosquito Control Research Laboratory, Depart-
ment of Entomology, University of California at Davis, assayed
and provided the data for the mosquito larvicidal activity.
1H, J ) 4.6 Hz), 7.34 (d, 1H, J ) 8.5 Hz), 7.67 (dd, 1H, J ) 8.5
Hz, 2.3 Hz), 8.08 (d, 2H, J ) 6.3 Hz), 8.48 (d, 1H, J ) 2.3 Hz),
8.76 (d, 2H, J ) 6.3 Hz). 13C NMR: δ 48.9, 110.9, 122.8, 125.0,
125.5, 130.1, 138.6, 143.8, 149.2, 150.4, 152.1, 168.7, 172.5. EI-
LRMS m/z (%): 330 (M+, 53), 293 (M+ - Cl, 11), 224 (M+
-
COPy, 18), 204 (M+ - ClPyCH2, 12), 189 (15), 126 (Cl - Py -
CH2, 55). EI-HRMS calcd for C15H11ClN4OS, 330.0342; found
330.0334. Anal. (C15H11ClN4OS) calcd C, 54.46; H, 3.35; N, 16.94;
found C, 54.42; H, 3.52; N, 16.87. Compound 5: yield 35%; mp )
1
249-250 °C. H NMR: δ 5.54 (s, 2H), 6.83 (d, 1H, J ) 4.8 Hz),
7.10 (d, 1H, J ) 4.8 Hz), 7.33 (d, 1H, J ) 8.3 Hz), 7.76 (dd, 1H,
J ) 8.3, 2.2 Hz), 8.49 (d, 1H, J ) 2.2 Hz), 8.68 (m, 1H), 8.75 (m,
1H), 9.51 (m, 1H). 13C NMR (DMSO-d6): δ 48.7, 111.0, 125.0,
128.4, 132.0, 140.4, 145.1, 146.0, 146.8, 149.3, 150.4, 150.5, 168.1,
170.4. EI-LRMS m/z (%): 331 (M+, 28), 252 (M+ - pyrazyl, 55),
224 (M+ - COpyrazyl, 26), 126 (Cl-pyridyl - CH2, 100). EI-
HRMS calcd for C14H10ClN5OS, 331.0295; found 331.0295. Anal.
Supporting Information Available: Structural models for
binding site interactions of compounds 5 and 9 with the A.
californica AChBP and a table listing elemental analysis results of
target compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.

4218 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Tomizawa et al.
crucial role in the high affinity and selectivity for the Drosophila
nicotinic receptor: an anomaly for the nicotinoid cation-π interaction
model. Biochemistry 2003, 42, 7819–7827.
References
(1) Tomizawa, M.; Casida, J. E. Selective toxicity of neonicotinoids
attributable to specificity of insect and mammalian nicotinic receptors.
Annu. ReV. Entomol. 2003, 48, 339–364.
(2) Tomizawa, M.; Casida, J. E. Neonicotinoid insecticide toxicology:
mechanisms of selective action. Annu. ReV. Pharmacol. Toxicol. 2005,
45, 247–268.
(3) Tomizawa, M.; Talley, T. T.; Maltby, D.; Durkin, K. A.; Medzihrad-
szky, K. F.; Burlingame, A. L.; Taylor, P.; Casida, J. E. Mapping the
elusive neonicotinoid binding site. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 9075–9080.
(4) Tomizawa, M.; Maltby, D.; Talley, T. T.; Durkin, K. A.; Medzihrad-
szky, K. F.; Burlingame, A. L.; Taylor, P.; Casida, J. E. Atypical
nicotinic agonist bound conformations conferring subtype selectivity.
Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 1728–1732.
(5) Celie, P. H. N.; van Rossum-Fikkert, S. E.; van Dijk, W. J.; Brejc,
K.; Smit, A. B.; Sixma, T. K. Nicotine and carbamylcholine binding
to nicotinic acetylcholine receptors as studied in AChBP crystal
structures. Neuron 2004, 41, 907–914.
(6) Hansen, S. B.; Sulzenbacher, G.; Huxford, T.; Marchot, P.; Taylor,
P.; Bourne, Y. Structures of Aplysia AChBP complexes with nicotinic
agonists and antagonists reveal distinctive binding interfaces and
conformations. EMBO J. 2005, 24, 3635–3646.
(7) Ishimitsu, K.; Kishimoto, T.; Yamada, Y.; Yamada, T.; Takakusa, N.
Preparation of (N-Heterocyclylimino)heterocyclic Compounds as
Insecticides. PCT Int. Appl. WO 92/15564, 1992.
(8) Samaritoni, J. G.; Demeter, D. A.; Gifford, J. M.; Watson, G. B.;
Kempe, M. S.; Bruce, T. J. Dihydropiperazine neonicotinoid com-
pounds. Synthesis and insecticidal activity. J. Agric. Food Chem. 2003,
51, 3035–3042.
(9) Tetko, I. V.; Gasteiger, J.; Todeschini, R.; Mauri, A.; Livingstone,
D.; Ertl, P.; Palyulin, V. A.; Radchenko, E. V.; Zefirov, N. S.;
Makarenko, A. S.; Tanchuk, V. Y.; Prokopenko, V. V. Virtual
computational chemistry laboratory: design and description. J. Com-
put.-Aided Mol. Des. 2005, 19, 453–463.
(10) Palmer,C.J.;Casida,J.E.1-(4-Ethynylphenyl)-2,6,7-trioxabicyclo[2.2.2]octanes:
a new order of potency for insecticides acting at the GABA-gated chloride
channel. J. Agric. Food Chem. 1989, 37, 213–216.
(11) Corbel, V.; Duchon, S.; Zaim, M.; Hougard, J.-M. Dinotefuran: a
potential neonicotinoid insecticide against resistant mosquitoes. J. Med.
Entomol. 2004, 41, 712–717.
(12) Liu, H.; Cupp, E. W.; Guo, A.; Liu, N. Insecticide resistance in
Alabama and Florida mosquito strains of Aedes albopictus. J. Med.
Entomol. 2004, 41, 946–952.
(13) Tomizawa, M.; Millar, N. S.; Casida, J. E. Pharmacological profiles
of recombinant and native insect nicotinic acetylcholine receptors.
Insect Biochem. Mol. Biol. 2005, 35, 1347–1355.
(14) Tomizawa, M.; Maltby, D.; Medzihradszky, K. F.; Zhang, N.; Durkin,
K. A.; Presley, J.; Talley, T. T.; Taylor, P.; Burlingame, A. L.; Casida,
J. E. Defining nicotinic agonist binding surfaces through photoaffinity
labeling. Biochemistry 2007, 46, 8798–8806.
(15) Latli, B.; D’Amour, K.; Casida, J. E. Novel and potent 6-chloro-3-
pyridinyl ligands for the R4ꢀ2 neuronal nicotinic acetylcholine
receptor. J. Med. Chem. 1999, 42, 2227–2234.
(17) Zhang, N.; Tomizawa, M.; Casida, J. E. Structural features of
azidopyridinyl neonicotinoid probes conferring high affinity and
selectivity for mammalian R4ꢀ2 and Drosophila nicotinic receptors.
J. Med. Chem. 2002, 45, 2832–2840.
(18) Liu, M.-Y.; Lanford, J.; Casida, J. E. Relevance of [3H]imidacloprid
binding site in house fly head acetylcholine receptor to insecticidal
activity of 2-nitromethylene- and 2-nitroimino-imidazolidines. Pestic.
Biochem. Physiol. 1993, 46, 200–206.
(19) McAbee, R. D.; Kang, K.-D.; Stanich, M. A.; Christiansen, J. A.;
Wheelock, C. E.; Inman, A. D.; Hammock, B. D.; Cornel, A. J.
Pyrethroid tolerance in Culex pipiens pipiens Var molestus from Marin
County, California. Pest Manage. Sci. 2004, 60, 359–368.
(20) Sgard, F.; Fraser, S. P.; Katkowska, M. J.; Djamgoz, M. B. A.; Dunbar,
S. J.; Windass, J. D. Cloning and functional characterization of two
novel nicotinic acetylcholine receptor R subunits from the insect pest
Myzus persicae. J. Neurochem. 1998, 71, 903–912.
(21) Huang, Y.; Williamson, M. S.; Devonshire, A. L.; Windass, J. D.;
Lansdell, S. J.; Millar, N. S. Cloning, heterologous expression and
co-assembly of Mpꢀ1, a nicotinic acetylcholine receptor subunit
from the aphid Myzus persicae. Neurosci. Lett. 2000, 284, 116–120.
(22) Bairoch, A.; Apweiler, R.; Wu, C. H.; Barker, W. C.; Boeckmann,
B.; Ferro, S.; Gasteiger, E.; Huang, H.; Lopez, R.; Magrane, M.;
Martin, M. J.; Natale, D. A.; O’Donovan, C.; Redaschi, N.; Yeh, L.-
S. L. The universal protein resource (UniProt). Nucleic Acids Res.
2005, 33, D154-D159.
(23) Thompson, J. D.; Higgins, D. G.; Gibson, T. J. CLUSTALW:
improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties and weight
matrix choice. Nucleic Acids Res. 1994, 22, 4673–4680.
(24) Lassmann, T.; Sonnhammer, E. L. L. Kalign, an accurate and fast
multiple sequence alignment algorithm. BMC Bioinf. 2005, 6, 298.
(25) Lassmann, T.; Sonnhammer, E. L. L. Kalign, Kalignvu and Mumsa:
Web servers for multiple sequence alignment. Nucleic Acids Res. 2006,
34, W596-W599.
(26) Schwede, T.; Kopp, J.; Guex, N.; Peitsch, M. C. SWISS-MODEL: an
automated protein homology-modeling server. Nucleic Acids Res. 2003,
31, 3381–3385.
(27) Mohamadi, F.; Richard, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton,
M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. Macromodel,
an integrated software system for modeling organic and bioorganic
molecules using molecular mechanics. J. Comput. Chem. 1990, 11,
440–467.
(28) Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J. Development and
testing of the OPLS all-atom force field on conformational energetics
and properties of organic liquids. J. Am. Chem. Soc. 1996, 118, 11225–
11236.
(29) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.;
Belew, R. K.; Olson, A. J. Automated docking using a Lamarckian
genetic algorithm and an empirical binding free energy function.
J. Comput. Chem. 1998, 19, 1639–1662.
(30) Huey, R.; Morris, G. M.; Olson, A. J.; Goodsell, D. S. A semiempirical
free energy force field with charge-based desolvation. J. Comput.
Chem. 2007, 28, 1145–1152.
(16) Tomizawa, M.; Zhang, N.; Durkin, K. A.; Olmstead, M. M.; Casida,
J. E. The neonicotinoid electronegative pharmacophore plays the
JM800191A