4-Azolylphenyl isoxazoline insecticides acting at the GABA gated chloride channel

Article abstract of DOI:10.1016/j.bmcl.2013.03.031

Isoxazoline insecticides have been shown to be potent blockers of insect GABA receptors with excellent activity on a broad pest range, including Lepidoptera and Hemiptera. Herein we report on the synthesis, biological activity and mode-of-action for a class of 4-heterocyclic aryl isoxazoline insecticides.

Full text of DOI:10.1016/j.bmcl.2013.03.031

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3001–3006
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4-Azolylphenyl isoxazoline insecticides acting at the GABA gated
chloride channel
⇑
George P. Lahm , Daniel Cordova, James D. Barry, Thomas F. Pahutski, Ben K. Smith, Jeffrey K. Long,
Eric A. Benner, Caleb W. Holyoke, Kathleen Joraski, Ming Xu, Mark E. Schroeder, Ty Wagerle,
Michael J. Mahaffey, Rejane M. Smith, My-Hahn Tong
DuPont Crop Protection, Stine-Haskell Research Center, 1094 Elkton Road, Newark, DE 19711, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Isoxazoline insecticides have been shown to be potent blockers of insect GABA receptors with excellent
activity on a broad pest range, including Lepidoptera and Hemiptera. Herein we report on the synthesis,
biological activity and mode-of-action for a class of 4-heterocyclic aryl isoxazoline insecticides.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 2 January 2013
Revised 28 February 2013
Accepted 7 March 2013
Available online 21 March 2013
Keywords:
Isoxazolines
Insecticides
Effective pest management aims to limit the onset of resistance
through reduction of selection pressure. This is accomplished
through the use of pesticide rotation strategies. Therefore, the dis-
covery of new classes of chemistry that work either by new bio-
chemical mechanisms or known mechanisms that display
minimal cross resistance is of great importance in the defense of
crops.
New isoxazoline insecticides of type 1, characterized by a 4-
carboxamide group on the phenyl, were recently reported by
Nissan, along with work concurrent to our studies on 4-heterocy-
clic phenyl isoxazolines (see Fig. 1).1 Here we describe a new class
of 4-azolylphenyl isoxazoline insecticides as potent blockers of
insect GABA receptors culminating in the identification of excep-
tionally potent 3-cyano-4-triazolyl analogs such as 2 (DP-12).2
The isoxazolines appear to trace their history through the dis-
covery of the insecticidal phthalic diamides 3 from Nihon Nohyaku
(1999) and the anthranilic diamides from DuPont (2001) (see
Fig. 2).3,4 The diamides as a class exhibit their action through selec-
tive activation of insect ryanodine receptors (RyRs).5
the amide could be found in anthranilic diamides of type 6, where
the oxime residue was explored as a replacement for the trifluoro-
methyl substituent. (Nissan, 2003).7 The combination of these sub-
stituent modifications by Nissan in 2003–4 appears to have set the
stage for the functionality leading to the discovery of the 4-phenyl
carboxamide isoxazoline insecticides 1 in 2005. Perhaps the most
surprising aspect of this new class is identification of the site of ac-
tion as blockers of the GABA gated chloride channel in contrast to
their diamide precursors that work as activators of the ryanodine
receptor.8
The compounds of Tables 1–3 were prepared as described in
Schemes 1–4. Isoxazolines 11 containing a range of ortho groups
(R1) were prepared as outlined in Scheme 1. Treatment of alde-
hydes 7 with hydroxyl amine produced oximes 8. Cycloaddition
to produce the isoxazoline 10 was accomplished by treatment of
8 with sodium hypochlorite in the presence of the 1-trifluoro-
methyl-1-aryl styrenes 9 in yields of 39–56%.2,9 While the azole
group could be introduced in the first step of the synthesis we
found the most expeditious route to be displacement of fluorine
from the 4-fluorophenyl-1-isoxazolines 10 with azoles affording
the 4-azolylisoxazolines 11 in good yield.10 The styrenes 9 were
prepared by Suzuki cross-coupling of arylboronic acids with 2-bro-
mo-3,3,3-trifluoropropene.11
Incorporation of a 3-trifluoromethylisoxazoline group into the
phthalic diamides 3 as a replacement for the trifluoromethyl sub-
stituent produced the first trifluoromethyl isoxazoline 5 (Nissan,
2004).6 Compound 5 contained an inverted orientation of the isox-
azoline ring with respect to those found in type 1 along with an in-
verted orientation of the amide group. A similar orientation of the
embedded oxime in 1 along with the corresponding orientation for
Isoxazolines 16 containing a 4-pyridyl group at the 5-position of
the isoxazoline could be prepared from the pyridylstyrene
derivatives 15 as outlined in Scheme 2. Direct C–H borylation by
treatment of 2,6-disubstituted pyridines with bis(pinacolato)dibo-
rane, [IrCl(COD)]2, and bipyridyl ligand provided exclusively the
4-pridylboronates 13 in good yield.12 Palladium catalyzed coupling
⇑
Corresponding author.
E-mail address: george.p.lahm@dupont.com (G.P. Lahm).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.031

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G. P. Lahm et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3001–3006
yield of 18.13 Formation of the oxamyl chloride, cycloaddition with
the styrene and triazole displacement, as in Scheme 1, afforded the
target isoxazoline 20.
F3C
O
N
Cl
H
N
CF3
N
Isoxazolines 24 containing a 5-cyano-3-pyridyl group could be
prepared as outlined in Scheme 4. The required 3-pyridinecarbox-
aldhyde 22 was available in a single step from 2-cyano crotonit-
rile 21 by a Vilsmeier reaction.14 The yield of 22 could be
improved over literature methods by reduction in the amount
of phosphorus oxychloride to 2.2 equivalents and use of dichloro-
ethane as solvent (vs neat) at 80 °C affording 22 in 33% yield. For-
mation of the oxamyl chloride, cycloaddition with the styrene
and triazole displacement afforded low yields of the target
isoxazoline 24, although in sufficient quantity for biological
evaluation.
1
Cl
Me
O
F3C
O
Br
N
N
2
Br
CN
N
DP-12
Figure 1. Isoxazoline insecticides.
Insecticidal activity of the isoxazolines is summarized in Tables
1–3. Compounds were tested against a series of insects under stan-
dard laboratory procedures.14 Potency was evaluated on fall army-
worm (Spodoptera frugiperda, Sf), corn earworm (Helicoverpa zea,
Hz), potato leafhopper (Empoasca fabae, Ef), and western flower
thrips (Frankliniella occidentalis, Fo). Insecticidal activity is reported
as an LC50 in ppm, the lethal concentration required for 50% mor-
tality. As a general rule all new compounds showed higher potency
against the two species of Lepidoptera.
of 13 with 2-bromo-3,3,3-trifluoropropene afforded fair yields of
the styrenes 15. Cycloaddition with oximes 8, followed by azole
displacement as described in Scheme 1, provided the 4-pyridyl
isoxazolines 16.
Isoxazolines 20 containing a 5-Cl-3-pyridyl group could be pre-
pared as outlined in Scheme 3. Reduction of 5,6-dichloronicotinic
acid to the corresponding aldehyde by treatment of the acid chlo-
ride with lithum tri-t-butoxyaluminum hydride afforded a modest
H
H
N
O
N
O
F3C
F3C
R
I
R
O
H
N
N
H
Me
O
Me
Me
4
3
1999
2001
MeO
N
O
N
H
N
H
N
O
O
R
R
I
O
Me
F3C
H
N
N
H
Me
O
Me
5
2004
6
2003
Figure 2. Isoxazolines trace their history through the diamide insecticides.
Table 1
Insecticidal potency of isoxazolines. Insect LC50 (ppm) are on fall armyworm (Sf, Spodoptera frugiperda), corn earworm (Hz, Helicoverpa zea), and potato leafhopper (Ef, Empoasca
fabae), and western flower thrips (Fo, Frankliniella occidentalis)
R1
N
O
A1
Cl
N
A2
CF3
Cl
Entry
R1
A1
A2
Sfa LC50 ppm
Hza LC50 ppm
Efa LC50 ppm
Foa LC50 ppm
DP-1
DP-2
DP-3
DP-4
DP-5
DP-6
DP-7
DP-8
DP-9
DP-10
DP-11
H
H
H
Me
CF3
Cl
CH
N
N
N
N
N
N
N
N
N
N
N
CH
N
N
N
N
N
N
N
N
N
>50
45.8
1.0
1.2
>10
4.0
2.6
2.5
0.2
26.9
>100
>50
31.2
4.5
>10
>10
9.6
9.6
5.5
8.9
>100
>100
>250
>250
7.1
7.5
15.8
7.5
>250
>250
26.3
33.6
11.9
9.1
9.1
8.0
1.4
>250
8.4
Br
6.0
NO2
CN
OMe
SO2Me
15.4
0.68
>250
>250
a
LC50 values were obtained for multiple test rates, each tested in replicate (n P3). LC50 calculations were determined by Probit analysis using a maximum quasi-likelihood
curve fitting algorithm.

G. P. Lahm et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3001–3006
3003
Table 2
Insecticidal potency of isoxazolines. Insect LC50 (ppm) are on fall armyworm (Sf, Spodoptera frugiperda), corn earworm (Hz, Helicoverpa zea), potato leafhopper (Ef, Empoasca fabae),
and western flower thrips (Fo, Frankliniella occidentalis)
CN
N
O
R2
R3
N
N
N
CF3
R4
Entry
R2
R3
R4
Sfa LC50 ppm
Hza LC50 ppm
Efa LC50 ppm
Foa LC50 ppm
DP-12
DP-13
DP-14
DP-15
DP-16
DP-17
DP-18
DP-19
Br
CF3
Cl
Cl
Cl
Cl
Cl
Cl
H
H
Br
CF3
Cl
0.25
0.3
0.11
0.08
>100
2.8
0.87
0.4
2.0
2.2
0.7
0.8
>50
>50
6.6
0.8
3.5
0.5
Cl
F
Cl
Cl
CN
Me
1.41
1.20
>100
>10
>10
>10
Cl
0.6
CN
OMe
Cl
60.0
29.2
4.7
9.2
4.9
3.0
Cl
17.2
a
LC50 values were obtained for multiple test rates, each tested in replicate (n P3). LC50 calculations were determined by Probit analysis using a maximum quasi-likelihood
curve fitting algorithm.
Table 3
Insecticidal potency of isoxazolines. Insect LC50 (ppm) are on fall armyworm (Sf, Spodoptera frugiperda), corn earworm (Hz, Helicoverpa zea), and potato leafhopper (Ef, Empoasca
fabae), and western flower thrips (Fo, Frankliniella occidentalis)
CN
N
O
R5
X
N
N
Q
N
CF3
Entry
R5
X
Q
Sfa EC50 ppm
Hza EC50 ppm
Efa EC50 ppm
Foa EC50 ppm
DP-20
DP-21
DP-22
DP-23
DP-24
DP-25
DP-26
3-Cl
3,5-diCl
N
N
N
CH
CH
CH
CH
CH
CH
CH
N
N
N
>100
30.0
2.7
1.1
0.7
>100
>100
1.7
>10
3.3
>250
>250
1.9
19.8
4.5
13.1
>250
3.6
33.0
2.3
3,5-di-CF3
3,5-diCl
3,5-diBr
3,4,5-triCl
3-Cl, 4-F, 5-Cl
0.7
0.5
4.3
1.1
45.9
5.7
142.5
5.1
N
a
LC50 values were obtained for multiple test rates, each tested in replicate (n P3). LC50 calculations were determined by Probit analysis using a maximum quasi-likelihood
curve fitting algorithm.
HO
H
F3C
O
N
O
N
R1
F
a
b
Cl
R1
F
R1
F
H
Cl
Cl
CF3
8
7
10
9
Cl
F3C
O
N
Cl
R1
N
c
A
Cl
A
11
Scheme 1. Reagents and conditions: (a) NH2OH, MeOH, 25 °C, 87–95%; (b) 6–13% NaOCl, THF, 0–25 °C, 39–56%; (c) K2CO3, CH3CN, azole, 80 °C, 27–80%.
DP-1 through DP-3 were initially prepared to investigate insec-
ticidal activity for the azole groups pyrazole, imidazole and triazole
at the 4-position of the aryl ring. While both the pyrazole DP-1 and
imidazole DP-2 displayed only weak levels of activity, the triazole
DP-3 was quite active on both lepidopteran pests and demon-
strated fair activity on Ef and Fo. Based on the activity of the tria-
zole DP-3 we turned our attention to an investigation of R1
substituent groups (compounds DP-4 to DP-11). The ortho-methyl
group was of particular interest as this substituent appeared to
maximize activity for the Nissan isoxazoline 4-carboxamides (i.e.,
1). For the triazoles, however, we observed a modest reduction in
activity when methyl was introduced at R1 as in DP-4. On the other
hand, compound DP-9, containing a cyano group at R1 demon-
strated exceptionally high levels of insecticidal activity and for

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G. P. Lahm et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3001–3006
O
B
R2
R2
R2
b
a
O
CF3
+
N
N
Br
CF3
N
R4
12
R4
R4
13
14
15
X = CR3, N
F3C
O
N
R2
R1
N
N
A
R4
A
16
Scheme 2. Reagents and conditions: (a) Ir2(COD)2Cl2 (10 mol %), bipyidyl (10 mol %,), bis(pinacolato)diborane (0.6 equiv), heptanes, 98 °C, 82–93%; (b) Pd(Ph3P)4 (10 mol %),
4 N aq KOH, DME/THF, 75 °C, 49–79%.
Cl
O
N
O
Cl
Cl
Cl
CO2H
Cl
Cl
c, d
a, b
H
N
Cl
CF3
N
N
Cl
17
18
19
Cl
N
N
O
N
e
N
N
Cl
CF3
20
Cl
Scheme 3. Reagents and conditions: (a) oxalyl chloride, DMF (cat) CH2Cl2; (b) LiAlH(O-tBu)3, CuI, THF/acetonitrile, À78 °C, 15% (steps a–b); (c) NH2OHÁH2O, MeOH, 25 °C,
76%; (d) 6% NaOCl, THF, 25 °C, 39%; (e) K2CO3, CH3CN, triazole, 80 °C, 24%.
600
Table 4
DMSO
DP-20 (10 µM)
Mortality of beet armyworm exposed to treated soybean leaf material collected at
different intervals after sprayinga
500
400
300
200
100
0
Treatment
Mortality (%)
g ai/hab 0 DATb 10 DATb 17 DATb
DP-9 (10SE)c
DP-12 (10SE)c
25
50
100
25
50
100
25
100
100
100
100
100
100
100
91
97
58
97
100
94
98
100
100
100
100
100
100
100
Chlorantraniliprole (coragen
20SC)c
50
73
n/a
100
100
0
100
100
0
100
97
0
Indoxacarb (steward 1.25SC)c
Untreated check
0
10
20
30
40
50
60
70
Time (min)
a
b
c
A total of 64 larvae were used to assess each treatment on each date.
g ai/ha = grams active per hectare; DAT = days after treatment.
SE = suspo-emulsion; SC = suspension concentrate.
Figure 3. Nerve activity of DP-20 in the P. americana cercal-reflex-N5 assay. A
suction electrode attached to motor nerve, N5, recorded the air puff-induced
efferent signals. In the presence of saline or DMSO air puffs induced action
potentials with a spike frequency below 150 Hz. Following application of DP-20
(10 lM) a strong increase in the air-puff response is observed.
active, although somewhat less than the unsubstituted parent DP-
3. The trifluoromethyl substituent of compound DP-5 showed a
significant reduction in activity, somewhat surprising based on
analogy with halogen substituents. Other R1 groups including
methoxy DP-10 and methylsulfonyl DP-11 were very weak.
certain insect species, including Sf, Ef and Fo, activity was equal or
superior to commercial standards.
Compounds containing ortho halogen groups, chloro DP-6 and
bromo DP-7, as well as the nitro derivative DP-8 were moderately

G. P. Lahm et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3001–3006
3005
Table 5
Based on the activity of the 2-cyanophenyl triazole DP-9, a ser-
ies of analogs containing a range of terminal aryl substituents was
investigated (Table 2). Compounds DP-12 through DP-15 contain-
ing halogen and/or trifluoromethyl groups in positions R2–R4
showed an increase in activity versus DP-9 and were among the
most active compounds explored comparing favorably with stan-
dards on many key pest species. Substituent groups such as cyano
and methoxy (DP-16 and DP-17) each resulted in a dramatic loss
in activity. Replacement of the central substituent R3 with groups
such as methyl and cyano (i.e., DP-18, DP-19) provided moderately
active compounds although somewhat less than their trihalo
counterparts.
Control of Caliothrips indicus nymphs and adults 15 days after treatment
Treatment
Rate (g ai/ha)
Number of insects
Nymphs Adults
DP-9 (10SE)
DP-12 (10SE)
25
50
150
25
21cd
20cd
15d
6b
7ab
6b
16cd
22cd
8d
8ab
6b
4b
50
150
250
410
n/a
Acephate (orthene 90S)
Aldicarb (temik 15G)
Untreated check
14d
5b
56bc
142a
7ab
8ab
Based on an insecticidal spectrum that included hemipteran
pests there was significant interest in lower logP analogs to im-
prove systemic properties and increase availability to pests that
feed on plant juices. Pyridine derivatives incorporating nitrogen
on either of the phenyl rings were explored as one approach to re-
duce logP. These compounds were, however, less active than their
carbon analogs. The terminal 4-pyridyl derivatives DP-20 through
DP-22 showed a dramatic loss in activity for the chloro derivatives
DP-20 and DP-21 and a more modest loss in activity for the bis-tri-
fluoromethyl derivative DP-22. Specifically, for DP-22 there was
observed an approximate order of magnitude reduction in activity
on the lepidopteran pests Sf and Hz, while the activity was compa-
rable on Ef and Fo. Incorporating nitrogen ortho to the azole in DP-
23 through DP-26 resulted in a similar although less severe, loss in
activity. Of this group DP-22 and DP-24 were most active across
the four insect species, although with less activity than standards
on major hemipteran pests.
Compounds DP-9 and DP-12 were evaluated in several trials to
determine if laboratory efficacy translated to acceptable levels of
control in the field. In one trial, soybean was sprayed in the field
and leaf samples were periodically collected, brought into the
laboratory, and exposed to larvae of beet armyworm (Spodoptera
exigua). Both compounds resulted in control equivalent to commer-
cial standards indoxacarb and chlorantraniliprole (RynaxypyrÒ) at
17 days after treatment; although, rates of at least 25 and 50 g ai/
ha were needed for DP-12 and DP-9, respectively (Table 4).
In a cotton trial in Louisiana, DP-9 and DP-12 provided effective
control of cotton thrips (Caliothrips indicus) nymphs with equivalent
efficacy to acephate and better control than aldicarb at much lower
rates (Table 5). None of the treatments were effective at controlling
adult thrips, although numbers of adults were quite low.
Means within columns followed by the same letter are not significantly different
(fisher’s least significant difference test, P = 0.05).
g ai/ha = grams active per hectare.
SE = suspo-emulsion, S = solution, G = granular.
100
80
60
Control
GABA
DP-20 (1 µM)
GABA
Rinse 20”
GABA
40
20
0
5 sec
1
10
100
1000
10000
DP-20 Concentration (nM)
Figure 4. The isoxazoline, DP-20 inhibits GABA gated currents in P. americana
neurons. Dissociated neurons were voltage clamped at holding potential of
M GABA (inset left trace).
a
À60 mV and repeatedly stimulated with pulses of 100
l
Perfusion of DP-20 inhibited the GABA response (inset middle trace) in a dose-
dependent manner with an IC50 = 10.8 nM. Following saline rinse a partial recovery
of the GABA response was observed (inset right trace).
CN
O
N
O
Cl
NC
Cl
a
b, c
H
N
Cl
CF3
NC
CN
N
Cl
CF3
21
22
23
Cl
Cl
CN
N
N
O
d
N
N
N
Cl
N
CF3
HN
N
24
Cl
Scheme 4. Reagents and conditions: (a) POCl3, DMF, ClCH2CH2Cl, 80 °C, 33%; (b) NH2OHÁHCl, NaOAc, EtOH, 25 °C, 81%; (c) NCS, DMF, 25 °C; (d) K2CO3, CH3CN, DMF, 80 °C, 4%
(steps c–d).

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G. P. Lahm et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3001–3006
Table 6
The D. melanogaster strain, RDL (resistance-to-dieldrin), is a
strain of laboratory-selected flies with resistance to dieldrin and
other cyclodienes. Resistance is associated with a single amino acid
substitution of serine for alanine at residue 302 of the rdl gene
which encodes for a GABA receptor subunit.17,18 To evaluate for
cyclodiene cross-resistance, DP-20 was tested on Xenopus laevis
oocytes expressing the D. melanogaster rdl GABA receptor. Perfu-
sion with DP-20 produced a dose-dependent inhibition in the
GABA response with IC50 values of 75 and 97 nM for wild type
and mutant (A302S) receptors, respectively (Fig. 5). Studies using
expressed GABA receptors from the housefly, Musca domestica,
found the antiparasitic isoxazoline, A1443, to similarly block
wild-type and the A299S homologs of the rdl receptor.8 The ab-
sence of differential potency indicates that cross-resistance be-
tween isoxazoline and cyclodienes would not be expected.
In summary, a series of 4-azolylphenyl isoxazoline insecticides
have been discovered which exert their action as potent blockers of
the GABA gated chloride channel and importantly lack evidence of
cyclodiene cross-resistance. Optimization of this class has identi-
fied 4-cyano triazoles, such as DP-9 and DP-12, to be highly effec-
tive in field trials and competitive with standards, offering the
potential for a new class of chemistry in the defense of crops.
Activity of select isoxazolines on toxicity and GABA response of thoracic neurons from
adult P. americana
Cpd
P. americana LD50
(
l
g/roach)
GABAR IC50 (nM)
DP-1
DP-5
DP-20
16.7
4.4
0.4
850
49.2
10.8
100
80
60
40
20
0
wild type
A302S
0.01 µM
DP-20
0.1 µM
DP-20
1 µM
DP-20
2 min
References and notes
0.01
0.1
1
10
DP-20 Concentration (µM)
1. (a) Mita, T.; Furukawa, H.; Masuzawa, Y.; Komoda, M. JP 07/016017, 2007.; (b)
Mita, T.; Furukawa, H.; Masuzawa, Y.; Komoda, M. JP 07/016017, 2007; Chem.
Abstr. 2007, 146, 163097.
Figure 5. Dose-dependent inhibitory effect of DP-20 on the GABA-induced currents
of Xenopus oocytes expressing wild type (circle) and A302S (square) RDL GABA
receptors. The inset shows a typical response of DP-20 on oocytes expressing the
A302S RDL GABA receptor. For all oocytes the membrane potential held at À60 mV.
2. (a) Lahm, G. P.; Patel, K. M.; Pahutski, T. F.; Smith, B. K.; PCT Intl. Appl. WO 07/
075459, 2007; Chem. Abstr. 2007, 147, 111908.; (b) Lahm, G. P.; Long, J. K.;
Pahutski, T. F.; Smith, B. K.; Mahaffey, M. J.; Rauh, J. R.; Cordova, D.; Smith, R.
M.; Barry, J. D.; Presented at the 239th National Meeting of the American
Chemical Society: San Francisco, CA, March 2010; paper AGRO 8.
3. (a) Tohnishi, M.; Nakao, H.; Kohno, E.; Nishida, T.; Furuya, T.; Shimizu, T.; Seo,
A.; Sakata, K.; Fujioka, S.; Kanno, H. Eur. Patent 919,542, 1999; Chem. Abstr.
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Tsubata, K.; Fujioka, S.; Kodama, H.; Hirooka, T.; Nishimatsu, T. J. Pestic. Sci.
2005, 30, 354.
4. (a) Lahm, G. P.; Selby, T. P.; Freudenberger, J. H.; Stevenson, T. M.; Myers, B. J.;
Seburyamo, G.; Smith, B. K.; Flexner, L.; Clark, C. E.; Cordova, D. Bioorg. Med.
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S. frugiperda Larvae injected with isoxazolines exhibited symp-
toms which included periodic body wall contractions and hyper-
sensitivity to mechanical stimulation. This was indicative of
neuro/muscular poisoning though distinct from those observed
with diamide insecticides.15 When adult cockroach, Periplaneta
americana, were injected, poisoning began as brief periodic wing
fluttering that progressed until insects became uncoordinated with
difficulty remaining upright. Once prostrate insects displayed peri-
odic volleys of leg tremors and eventually died. The KD50 (50%
knockdown concentration) values were determined for DP-1, DP-
5 and DP-20 having values of 16.7, 4.4 and 0.4 lg/roach, respec-
tively. These analogs were selected for in vitro studies as they
provided a range of insecticidal potency and their increased aque-
ous solubility allowed for testing at micromolar concentrations.
To determine whether isoxazoline insecticides were active
against insect RyRs, analogs were tested against native P. ameri-
cana and recombinant Drosophila melanogaster RyRs as described
previously, and were found to be inactive at concentrations up to
30 l
M (data not shown).15 As poisoning was neuro/muscular in
nature, extracellular recordings were conducted on nerve 5 (N5)
of P. americana metathoracic ganglia. Bath perfusion of DP-20
(10 lM) induced a strong increase in the air puff-induced (cercus
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stimulation) nerve frequency while spontaneous nerve activity re-
mained unaffected (Fig. 3). This suggested that the target was more
likely a ligand-gated receptor than a voltage-gated ion channel.
Neurotransmission in the cercal reflex-N5 pathway entails both
excitatory nicotinic acetylcholine receptors (nAChRs) and inhibi-
tory GABA gated chloride channels16; therefore action of DP-20
was investigated on both systems. No effect was observed on nAC-
hRs (data not shown); however, GABA-induced currents in P. amer-
icana thoracic neurons were potently blocked. The isoxazoline, DP-
20, inhibited GABA-induced chloride currents with an IC50 value of
10.8 nM (Fig. 4). Further evaluation showed that roach toxicity for
DP-1, DP-5 and DP-20 correlates well with GABA gated chloride
channel inhibition (Table 6).
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