4,5-Substituted 3-Isoxazolols with Insecticidal Activity Act as Competitive Antagonists of Housefly GABA Receptors

Article abstract of DOI:10.1021/acs.jafc.5b01843

The insect GABA receptor (GABAR), which is composed of five RDL subunits, represents an important target for insecticides. A series of 4,5-disubstituted 3-isoxazolols, including muscimol analogues, were synthesized and examined for their activities agains

Full text of DOI:10.1021/acs.jafc.5b01843

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Article
4,5-Substituted 3-Isoxazolols with Insecticidal Activity Act
as Competitive Antagonists of Housefly GABA Receptors
Genyan Liu, Fumiyo Ozoe, Kenjiro Furuta, and Yoshihisa Ozoe
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01843 • Publication Date (Web): 29 Jun 2015
Downloaded from http://pubs.acs.org on July 5, 2015
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4,5-Substituted 3-Isoxazolols with Insecticidal Activity Act as Competitive
Antagonists of Housefly GABA Receptors
Genyan Liu,† Fumiyo Ozoe,‡ Kenjiro Furuta,†,‡ and Yoshihisa Ozoe*,†,‡
†Division of Bioscience and Biotechnology, The United Graduate School of Agricultural
Sciences, Tottori University, Tottori 680ꢀ8553, Japan
‡Department of Life Science and Biotechnology, Faculty of Life and Environmental Science,
Shimane University, Matsue, Shimane 690ꢀ8504, Japan
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ABSTRACT: The insect GABA receptor (GABAR), which is composed of five RDL
subunits, represents an important target for insecticides. A series of 4,5ꢀdisubstituted
3ꢀisoxazolols, including muscimol analogs, were synthesized and examined for their
activities against four splice variants (ac, ad, bc, and bd) of housefly GABARs expressed in
Xenopus oocytes. Muscimol was a more potent agonist than GABA in all four splice variants,
whereas synthesized analogs did not exhibit agonism but rather antagonism in housefly
GABARs. The introduction of bicyclic aromatic groups at the 4ꢀposition of muscimol and the
simultaneous replacement of the aminomethyl group with a carbamoyl group at the 5ꢀposition
to afford six 4ꢀarylꢀ5ꢀcarbamoylꢀ3ꢀisoxazolols resulted in compounds that exhibited
significantly enhanced antagonism with IC50 values in the low micromolar range in the ac
variant. The inhibition of GABAꢀinduced currents by 100 ꢁΜ analogs was approximately
1.5ꢀ to 4ꢀfold greater in the ac and bc variants than in the ad and bd variants.
4ꢀ(3ꢀBiphenylyl)ꢀ5ꢀcarbamoylꢀ3ꢀisoxazolol displayed competitive antagonism, with IC50
values of 30, 34, 107, and 96 ꢁΜ in the ac, bc, ad, and bd variants, respectively, and exhibited
moderate insecticidal activity against houseflies, with an LD50 value of 5.6 nmol/fly. These
findings suggest that these 3ꢀisoxazolol analogs are novel lead compounds for the design and
development of insecticides that target the orthosteric site of housefly GABARs.
KEYWORDS: insect GABA receptors, competitive antagonists, 3ꢀisoxazolols, RDL,
twoꢀelectrode voltage clamp, insecticidal activity
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INTRODUCTION
γꢀAminobutyric acid (GABA) is a major neurotransmitter in both vertebrates and
invertebrates. Ionotropic GABA receptors (GABARs) mediate fast inhibitory synaptic
transmission by enhancing membrane permeability to chloride ions in response to GABA,
which is released from the presynaptic neuron.1 In insects, GABARs are predominantly
expressed in the central nervous system and play important physiological roles in sleep,
olfaction, and learning/memory.2,3 Because vertebrate and invertebrate GABARs have
different pharmacological properties, insect GABARs are an important target for safe
insecticides, such as phenylpyrazoles.2–4
Ionotropic GABARs are pentameric ligandꢀgated chloride channels, which belong to the
family of Cysꢀloop receptors. Whereas 19 constitutive subunits are present in mammalian
GABARs, RDL is the only subunit that constitutes inhibitory GABARs in insects.2–4
However, the RDLꢀencoding gene Rdl undergoes alternative splicing of exons 3 and 6 to
generate four variants (RDLac, RDLad, RDLbc, and RDLbd) in the fruit fly (Drosophila
melanogaster Meigen) and other insect species.5 The Drosophila variants of GABARs have
been reported to exhibit differential agonist sensitivity when expressed in Xenopus oocytes.6–8
The alternative splicing may also increase the pharmacological diversity of insect GABARs.
However, the physiology and pharmacology of these variants of GABARs have yet to be
characterized.
Efforts focused on GABAR pharmacology recently led to the discovery of two novel
chemotypes of insecticides, isoxazolines and benzamides, which act as noncompetitive
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antagonists at a unique allosteric site(s) in insect GABARs.9–13 These insecticides overcome
the emerging resistance to conventional GABAR antagonist insecticides (e.g., fipronil)
because of their different sites of action. In addition, competitive antagonists, which act at the
orthosteric agonistꢀbinding site of GABARs, have the potential to become novel insecticides.
In our recent studies, thioꢀ4ꢀPIOL and gabazine analogs (Figure 1) were synthesized and
examined for their antagonism of GABARs cloned from three insect species.14–16
Subsequently, we found that introducing bicyclic aromatic groups into the 4ꢀposition of the
isothiazole ring of thioꢀ4ꢀPIOL or into the 3ꢀposition of the dihydropyridazine ring of
gabazine enhances the antagonism of insect GABARs by these analogs. These analogs also
showed insecticidal activity, though this activity was moderate. These findings prompted
further exploration of potentially novel insecticides acting as competitive GABAR
antagonists. Here, we report the synthesis of 4,5ꢀdisubstituted 3ꢀisoxazolols (Figure 1) and
their differential antagonism of four splice variants of housefly (Musca domestica Linnaeus)
GABARs.
MATERIALS AND METHODS
Chemistry. Reagents and solvents were purchased from Wako Pure Chemical Industries,
Ltd. (Osaka, Japan), Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and SigmaꢀAldrich
Co., LLC. (Tokyo, Japan), unless otherwise noted. All airꢀ and moistureꢀsensitive reactions
were performed under an argon atmosphere using ovenꢀdried glassware. Reactions were
monitored by thin layer chromatography (TLC Silica gel 60 F254 plates, Merck KGaA,
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Darmstadt, Germany) using UV light or a KMnO4 spray reagent. Column chromatography
was performed using silica gel (Wakogel® Cꢀ200, 75−150 ꢁm, Wako). Melting points were
determined using a YANACO MPꢀ500D micro melting point apparatus and are uncorrected.
1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on a JEOL JNM Aꢀ400
spectrometer. Chemical shifts are reported in parts per million (ppm) on the δ scale, and
coupling constants (J) are in Hertz (Hz). Spin multiplicities are abbreviated as follows: s
(singlet), bs (broad singlet), d (doublet), t (triplet), and m (multiplet). Mass spectra (MS) were
measured using the positive electrospray ionization mode (ESI) on a Waters XEVO mass
spectrometer, and highꢀresolution mass spectra (HRMS) were obtained using ESI on a Waters
SYNAPT G2 spectrometer.
Synthesis
of
Methyl
3-Hydroxy-5-isoxazolecarboxylate
(2).17
Dimethyl
acetylenedicarboxylate (1.4 g, 10.0 mmol) was added to
a
solution of
1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene (1.8 g, 12.0 mmol) and Nꢀhydroxyurea (760 mg, 10.0
mmol) in methanol (15 mL) at 0 °C under argon. The solution was stirred at 0 °C for 30 min
and then at room temperature for 12 h. After the solvent was evaporated, the residue was
dissolved in water (25 mL) and acidified to pH 1 with conc. HCl. The product was extracted
with Et2O (3 × 30 mL), and the combined organic phases were washed with brine, dried over
Na2SO4, and evaporated under reduced pressure. The obtained solid was recrystallized from
1
chloroform to give 2 as a light yellow solid (840 mg, 59% yield). H NMR (400 MHz,
DMSOꢀd6): δ 11.97 (1H, s, OH), 6.76 (1H, s, ArꢀH), 3.87 (3H, s, COOCH3).
Synthesis of 3-Benzyloxy-5-methoxycarbonylisoxazole (3).18 A mixture of 2 (2.1 g, 15
mmol) and K2CO3 (3.1 g, 22.5 mmol) in acetone (30 mL) was heated at 70 °C for 1 h. Benzyl
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bromide (3.8 g, 22.5 mmol) was added dropwise, and the mixture was stirred at 50 °C for 12
h. The mixture was filtered, and the filtrate was concentrated under reduced pressure. Water
(30 mL) was added to the filtrate, and the product was extracted with Et2O (3 × 30 mL). The
combined organic phases were washed with brine, dried over Na2SO4, and evaporated under
reduced pressure. Column chromatography (hexane/EtOAc 15:1) gave 3 as a colorless oil
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(3.1 g, 87% yield). H NMR (400 MHz, CDCl3): δ 7.54–7.28 (5H, m, ArꢀH), 6.57 (1H, s,
ArꢀH), 5.32 (2H, s, OCH2), 3.95 (3H, s, COOCH3). MS: m/z 255.9 [M+Na]+.
Synthesis of 3-Benzyloxy-5-carbamoylisoxazole (4). A mixture of 3 (466 mg, 2.0 mmol)
and aqueous ammonia (5 mL, 28%) was stirred at room temperature for 12 h. The mixture
was concentrated to dryness under reduced pressure. Water (15 mL) was added to the residue,
and the mixture was extracted with EtOAc (3 × 15 mL). The combined organic phases were
washed with brine, dried over Na2SO4, and evaporated under reduced pressure to afford 4 as a
1
white solid (395 mg, 91% yield). mp 162–164 °C. H NMR (400 MHz, CDCl3): δ 7.48–7.32
(5H, m, ArꢀH), 6.59 (1H, s, ArꢀH), 6.43 (1H, bs, CONH2), 5.87 (1H, bs, CONH2), 5.31 (2H, s,
OCH2). MS: m/z 241.1 [M+Na]+.
Synthesis of Muscimol Hydrobromide (5a).19 Borane in THF (1 M, 5 mL, 5.0 mmol)
was slowly added to a solution of 4 (480 mg, 2.2 mmol) in dry THF (15 mL) at 0 °C. The
solution was stirred at room temperature for 16 h. After acidification (pH 1) with 4 M HCl,
the solution was stirred for 1 h and concentrated under reduced pressure. The residue was
suspended in water, and the solution was made basic (pH 10) with 4 M NaOH. The product
was extracted with EtOAc (3 × 30 mL); the combined organic phases were washed with brine,
dried over Na2SO4, and evaporated under reduced pressure to afford a light yellow oil. The oil
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was dissolved in a solution of HBr in AcOH (10 mL, 30%), and the mixture was stirred at
room temperature for 24 h. The reaction mixture was concentrated with a subsequent
azeotropic treatment of MeOH/toluene (1:1) three times, and the residue was recrystallized
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(MeOH/Et2O) to give 5a as a light brown solid (179 mg, 42% yield). H NMR (400 MHz,
DMSOꢀd6): δ 11.25 (1H, bs, OH), 8.45 (3H, bs, NH2·HBr), 6.18 (1H, s, ArꢀH), 4.14 (2H, s,
CH2). 13C NMR (100 MHz, DMSOꢀd6): δ 170.14, 165.22, 96.04, 34.29. HRMS: m/z calcd for
C4H7N2O2 [M–Br]+ 115.0508, found 115.0502.
Synthesis of 5-Carbamoyl-3-isoxazolol (6a). Compound 4 (349 mg, 1.6 mmol) was
suspended in a solution of HBr in AcOH (10 mL, 30%), and the mixture was stirred at room
temperature for 24 h. The reaction mixture was concentrated with a subsequent azeotropic
treatment of MeOH/toluene (1:1) three times, and the residue was recrystallized
1
(MeOH/EtOAc) to give 6a as a white solid (152 mg, 74% yield). mp 236–239 °C (dec.). H
NMR (400 MHz, DMSOꢀd6): δ 11.61 (1H, bs, OH), 8.15 (1H, bs, CONH2), 7.81 (1H, bs,
CONH2), 6.54 (1H, s, ArꢀH). 13C NMR (100 MHz, DMSOꢀd6): δ 170.40, 163.58, 157.39,
97.69. HRMS: m/z calcd for C4H5N2O3 [M+H]+ 129.0295, found 129.0290.
Synthesis of 3-Benzyloxy-5-carbamoyl-4-iodoisoxazole (7). A mixture of 4 (1.1 g, 5
mmol), Pd(OAc)2 (112 mg, 0.5 mmol), CsOAc (2.3 g, 12 mmol), NaHCO3 (420 mg, 5 mmol),
I2 (3.8 g, 15 mmol), 4 Å molecular sieves (150 mg), and Nꢀmethylformamide (30 mL) was
stirred at 75 °C for 16 h. After cooling, the reaction mixture was added to Na2S2O4 (solid)
until the color stopped changing, and then it was filtered through Celite®, which was
thoroughly washed with EtOAc. The combined organic phases were washed with water twice
and then with brine, dried over Na2SO4, and evaporated under reduced pressure. Column
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chromatography (hexane/EtOAc 10:1) gave 7 as a light yellow solid (980 mg, 57% yield). mp
179–182 °C. 1H NMR (400 MHz, CDCl3): δ 7.50–7.35 (5H, m, ArꢀH), 6.43 (1H, bs, CONH2),
5.94 (1H, bs, CONH2), 5.37 (2H, s, OCH2). MS: m/z 366.9 [M+Na]+.
General Procedure for the Synthesis of 4-Aryl-3-benzyloxy-5-carbamoylisoxazole
(8b–8g). A mixture of 7 (1 mmol), an arylboronic acid (1.5 mmol), Pd(PPh3)2Cl2 (0.08 mmol),
DMF (5 mL), and aqueous K2CO3 (0.5 mL, 3 M, 1.5 mmol) was stirred at 80 °C for 24 h.
After cooling, the reaction mixture was filtered through Celite® and diluted using Et2O. The
organic phase was washed with water and brine, dried over Na2SO4, and evaporated under
reduced pressure. Column chromatography (hexane/EtOAc) gave 8b–g.
3-Benzyloxy-4-(3-biphenylyl)-5-carbamoylisoxazole (8b). A white solid, yield 82%,
mp 145–147 °C. 1H NMR (400 MHz, CDCl3): δ 7.93 (1H, s, ArꢀH), 7.70–7.26 (13H, m,
ArꢀH), 6.40 (1H, bs, CONH2), 5.78 (1H, bs, CONH2), 5.42 (2H, s, OCH2). 13C NMR (100
MHz, CDCl3): δ 169.88, 157.95, 156.60, 141.09, 140.78, 135.39, 129.03, 128.83, 128.74,
128.60, 128.56, 128.09, 127.58, 127.37, 127.20, 126.59, 114.21, 72.27. MS: m/z 371.0
[M+H]+, 393.0 [M+Na]+.
3-Benzyloxy-5-carbamoyl-4-(2-naphthyl)isoxazole (8c). A white solid, yield 69%, mp
1
138–140 °C. H NMR (400 MHz, CDCl3): δ 8.14 (1H, s, ArꢀH), 7.88–7.73 (4H, m, ArꢀH),
7.72–7.26 (7H, m, ArꢀH), 6.39 (1H, bs, CONH2), 5.84 (1H, bs, CONH2), 5.43 (2H, s, OCH2).
13C NMR (100 MHz, CDCl3): δ 169.90, 157.95, 156.60, 135.33, 133.21, 132.90, 129.75,
129.58, 128.49, 128.37, 127.90, 127.74, 127.63, 127.26, 126.74, 126.23, 123.56, 114.31,
72.17. MS: m/z 367.0 [M+Na]+.
3-Benzyloxy-4-(4-biphenylyl)-5-carbamoylisoxazole (8d). A white solid, yield 90%,
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mp 164–167 °C. H NMR (400 MHz, CDCl3): δ 7.80–7.28 (14H, m, ArꢀH), 6.39 (1H, bs,
CONH2), 5.75 (1H, bs, CONH2), 5.42 (2H, s, OCH2). 13C NMR (100 MHz, CDCl3): δ 169.98,
158.03, 156.59, 141.72, 140.66, 135.48, 132.23, 132.13, 130.49, 128.84, 128.67, 128.60,
128.47, 128.08, 127.59, 127.19, 126.95, 125.21, 114.11, 72.34. MS: m/z 371.0 [M+H]+.
3-Benzyloxy-5-carbamoyl-4-(1-naphthyl)isoxazole (8e). A white solid, yield 77%, mp
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154–156 °C. H NMR (400 MHz, CDCl3): δ 7.95–7.25 (12H, m, ArꢀH), 6.05 (1H, bs,
CONH2), 5.72 (1H, bs, CONH2), 5.34 (2H, s, OCH2). 13C NMR (100 MHz, CDCl3): δ 170.35,
158.09, 157.37, 135.23, 133.66, 131.57, 129.83, 128.97, 128.52, 128.41, 128.05, 126.56,
126.25, 125.26, 125.19, 123.84, 112.32, 72.02. MS: m/z 367.1 [M+Na]+.
3-Benzyloxy-5-carbamoyl-4-(6-methoxy-2-naphthyl)isoxazole (8f). A light yellow
1
solid, yield 67%, mp 124–126 °C. H NMR (400 MHz, CDCl3): δ 8.07 (1H, s, ArꢀH),
7.78–7.65 (3H, m, ArꢀH), 7.46–7.30 (5H, m, ArꢀH), 7.18–7.10 (2H, m, ArꢀH), 6.40 (1H, bs,
13
CONH2), 6.06 (1H, bs, CONH2), 5.42 (2H, s, OCH2), 3.91 (3H, s, OCH3). C NMR (100
MHz, CDCl3): δ 169.92, 158.40, 158.21, 156.39, 135.41, 134.54, 129.90, 129.50, 128.55,
128.45, 127.86, 127.79, 126.57, 121.23, 119.11, 114.35, 105.61, 72.12, 55.32. MS: m/z 397.1
[M+Na]+.
3-Benzyloxy-5-carbamoyl-4-(6-hydroxy-2-naphthyl)isoxazole (8g). A light yellow
1
solid, yield 43%, mp 177–179 °C. H NMR (400 MHz, DMSOꢀd6): δ 9.73 (1H, s, ArꢀOH),
8.11 (1H, bs, CONH2), 7.97 (1H, s, ArꢀH), 7.85 (1H, bs, CONH2), 7.75–7.05 (10H, m, ArꢀH),
5.41 (2H, s, OCH2). 13C NMR (100 MHz, DMSOꢀd6): δ 169.11, 158.67, 158.25, 155.96,
135.67, 134.12, 129.56, 128.60, 128.37, 128.24, 127.93, 127.31, 127.06, 125.49, 120.77,
118.98, 111.59, 108.54, 71.63. MS: m/z 383.1 [M+Na]+.
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Synthesis of 5-Aminomethyl-4-(3-biphenylyl)-3-isoxazolol Hydrobromide (5b).
Compound 5b was prepared from 8b (185 mg, 0.5 mmol) according to the procedure
described for 5a. Recrystallization (MeOH/Et2O) gave 5b as a white solid (69 mg, 40%). mp
1
220–222 °C. H NMR (400 MHz, CD3OD): δ 7.75–7.30 (9H, m, ArꢀH), 4.36 (2H, s, CH2).
13C NMR (100 MHz, CD3OD): δ 163.30, 160.88, 143.29, 141.79, 130.50, 129.95, 129.33,
128.72, 128.66, 128.38, 128.07, 128.02, 111.90, 35.40. HRMS: m/z calcd for C16H15N2O2
[M–Br]+ 267.1134, found 267.1143.
Synthesis of 5-Aminomethyl-4-(2-naphthyl)-3-isoxazolol Hydrobromide (5c).
Compound 5c was prepared from 8c (196 mg, 0.57 mmol) according to the procedure
described for 5a. Recrystallization (MeOH/Et2O) gave 5c as a brown solid (85 mg, 47%). mp
1
226–228 °C. H NMR (400 MHz, CD3OD): δ 8.01–7.86 (4H, m, ArꢀH), 7.65–7.50 (3H, m,
13
ArꢀH), 4.41 (2H, s, CH2). C NMR (100 MHz, CD3OD): δ 170.22, 160.92, 134.85, 134.37,
129.63, 129.09, 128.77, 127.69, 127.20, 126.21, 112.05, 35.56. HRMS: m/z calcd for
C14H13N2O2 [M–Br]+ 241.0977, found 241.0989.
Synthesis of 4-(3-Biphenylyl)-5-carbamoyl-3-isoxazolol (6b). Compound 6b was
prepared from 8b (333 mg, 0.9 mmol) according to the procedure described for 6a.
Recrystallization (MeOH/EtOAc) gave 6b as a white solid (210 mg, 83%). mp 193–195 °C.
1H NMR (400 MHz, DMSOꢀd6): δ 12.03 (1H, bs, OH), 8.09 (1H, bs, CONH2), 7.90 (1H, s,
ArꢀH), 7.82 (1H, bs, CONH2), 7.71–7.27 (8H, m, ArꢀH). 13C NMR (100 MHz, DMSOꢀd6): δ
168.62, 158.59, 158.30, 140.04, 139.77, 128.87, 128.42, 128.35, 128.05, 127.43, 126.62,
126.19, 111.31. HRMS: m/z calcd for C16H13N2O3 [M+H]+ 281.0921, found 281.0914.
Synthesis of 5-Carbamoyl-4-(2-naphthyl)-3-isoxazolol (6c). Compound 6c was
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prepared from 8c (189 mg, 0.55 mmol) according to the procedure described for 6a.
Recrystallization (MeOH/EtOAc) gave 6c as a light yellow solid (95 mg, 68%). mp
221–223 °C. 1H NMR (400 MHz, DMSOꢀd6): δ 12.02 (1H, bs, OH), 8.10 (1H, s, ArꢀH), 8.06
(1H, bs, CONH2), 7.95–7.88 (3H, m, ArꢀH), 7.79 (1H, bs, CONH2), 7.66 (1H, d, J = 8.3 Hz,
ArꢀH), 7.58–7.47 (2H, m, ArꢀH). 13C NMR (100 MHz, DMSOꢀd6): δ 168.58, 158.39, 158.28,
132.33, 132.19, 128.35, 127.80, 127.25, 126.92, 126.23, 126.02, 124.96, 111.33. HRMS: m/z
calcd for C14H11N2O3 [M+H]+ 255.0770, found 255.0785.
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Synthesis of 4-(4-Biphenylyl)-5-carbamoyl-3-isoxazolol (6d). Compound 6d was
prepared from 8d (222 mg, 0.6 mmol) according to the procedure described for 6a.
Recrystallization (MeOH/EtOAc) gave 6d as a white solid (112 mg, 67%). mp 278–280 °C
1
(dec.). H NMR (400 MHz, DMSOꢀd6): δ 12.03 (1H, bs, OH), 8.09 (1H, bs, CONH2), 7.82
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(1H, bs, CONH2), 7.80–7.50 (6H, m, ArꢀH), 7.48–7.34 (3H, m, ArꢀH). C NMR (100 MHz,
DMSOꢀd6): δ 168.61, 158.56, 158.20, 139.65, 139.58, 129.96, 128.86, 127.48, 126.61,
126.54, 126.04, 110.98. HRMS: m/z calcd for C16H12N2O3Na [M+Na]+ 303.0740, found
303.0738.
Synthesis of 5-Carbamoyl-4-(1-naphthyl)-3-isoxazolol (6e). Compound 6e was
prepared from 8e (330 mg, 0.96 mmol) according to the procedure described for 6a.
Recrystallization (MeOH/EtOAc) gave 6e as a white solid (128 mg, 52%). mp 189–191 °C.
1H NMR (400 MHz, DMSOꢀd6): δ 11.77 (1H, bs, OH), 7.96 (2H, d, J = 7.8 Hz, ArꢀH), 7.92
(1H, bs, CONH2), 7.68 (1H, bs, CONH2), 7.59–7.41 (5H, m, ArꢀH). 13C NMR (100 MHz,
DMSOꢀd6): δ 169.34, 159.28, 157.69, 133.03, 131.46, 128.66, 128.44, 128.07, 126.08,
125.76, 125.39, 125.15, 110.58. HRMS: m/z calcd for C14H11N2O3 [M+H]+ 255.0770, found
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255.0785.
Synthesis of 5-Carbamoyl-4-(6-methoxy-2-naphthyl)-3-isoxazolol (6f). Compound 6f
was prepared from 8f (243 mg, 0.65 mmol) according to the procedure described for 6a.
Recrystallization (MeOH/EtOAc) gave 6f as a white solid (115 mg, 62%). mp 242–244 °C
1
(dec.). H NMR (400 MHz, DMSOꢀd6): δ 11.99 (1H, bs, OH), 8.03 (2H, bs, CONH2),
7.85–7.70 (4H, m, ArꢀH), 7.63 (1H, dd, J = 8.8, 2.0 Hz, ArꢀH), 7.32 (1H, d, J = 2.4 Hz, ArꢀH),
7.17 (1H, dd, J = 8.8, 2.4 Hz, ArꢀH), 3.89 (3H, s, OCH3). 13C NMR (100 MHz, DMSOꢀd6): δ
168.72, 158.58, 158.05, 157.71, 133.70, 129.52, 128.31, 127.89, 127.83, 125.99, 122.60,
118.64, 111.58, 105.90, 55.21. HRMS: m/z calcd for C15H12N2O4Na [M+Na]+ 307.0695,
found 307.0691.
Synthesis of 5-Carbamoyl-4-(6-hydroxy-2-naphthyl)-3-isoxazolol (6g). Compound 6g
was prepared from 8g (90 mg, 0.25 mmol) according to the procedure described for 6a.
Recrystallization (MeOH/EtOAc) gave 6g as a white solid (52 mg, 77%). mp 210–213 °C. 1H
NMR (400 MHz, DMSOꢀd6): δ 11.94 (1H, bs, OH), 9.70 (1H, bs, OH), 8.05–7.50 (6H, m,
ArꢀH and CONH2), 7.36–7.05 (2H, m, ArꢀH). 13C NMR (100 MHz, DMSOꢀd6): δ 168.73,
158.61, 157.91, 155.78, 129.56, 128.37, 128.12, 127.56, 127.11, 125.29, 121.64, 118.81,
111.69, 108.51. HRMS: m/z calcd for C14H11N2O4 [M+H]+ 271.0719, found 271.0714.
Preparation of cRNAs of Housefly RDL and Their Expression in Xenopus Oocytes.
The cRNAs of four housefly RDL variants (DDBJ accession Nos.: AB177547, complete cds
of RDLbd; AB824728, partial cds of exon 3a version; AB824729, partial cds of exon 6c
version) were prepared as previously described.20 In brief, the cDNA templates of four Rdl
variants (ac, bc, ad, and bd) including the upstream RNA polymerase promoter site were
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obtained by PCR amplification from pBluescript KS(ꢀ)ꢀMdRdlac, ꢀMdRdlbc, ꢀMdRdlad, and
ꢀMdRdlbd using KOD ꢀPlusꢀ Ver. 2 (Toyobo, Tokyo, Japan), a forward primer,
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5’ꢀTGTAAAACGACGGCCAGTꢀ3’,
and
a
reverse
primer
5’ꢀCAGGAAACAGCTATGACCꢀ3’. The PCR products were purified using the illustraTM
GFXTM PCR DNA and Gel Band Purification Kit (GE Healthcare UK, Ltd., Little Chalfont,
UK), and the integrity of amplified cDNAs was confirmed by sequence analysis. The capped
cRNAs were synthesized using T7 polymerase (mMessage mMachine® T7 Ultra Kit; Ambion,
Austin, TX). The cRNAs were precipitated with LiCl, dissolved in sterile RNaseꢀfree water,
and stored at ꢀ20 °C until use.
Mature female African clawed frogs (Xenopus laevis) were anesthetized with 0.1% (w/v)
ethyl mꢀaminobenzoate methanesulfonate in water. The ovarian lobes were dissected out and
then treated with collagenase (2 mg/mL, SigmaꢀAldrich) in a Ca2+ꢀfree standard oocyte
solution (Ca2+ꢀfree SOS) (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.6)
at room temperature for 90–120 min. The oocytes were then gently washed with sterile SOS
(100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.6) containing
gentamycin (50 µg/mL, Life TechnologiesTM, Thermo Fisher Scientific Inc., Waltham, MA),
penicillin (100 U/mL, Life TechnologiesTM), streptomycin (100 µg/mL, Life TechnologiesTM),
and sodium pyruvate (2.5 mM, SigmaꢀAldrich). Each oocyte was injected cytoplasmically
with 5 ng of cRNA dissolved in RNaseꢀfree water (9.2 nL), and then the oocytes were
incubated in sterile SOS for 48 h at 16 °C.
Two-electrode Voltage Clamp (TEVC) Recordings. Electrophysiological experiments
were performed as previously described.14 Briefly, GABAꢀinduced currents were recorded at
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a holding potential of ꢀ80 mV using an Oocyte Clamp OCꢀ725C amplifier (Warner
Instruments, Hamden, CT) and DataꢀTrax2TM software (World Precision Instruments Inc.,
Sarasota, FL). The glass capillary electrodes were fabricated using a pipette puller (Pꢀ97,
Sutter Instrument, Novato, CA) and filled with 2 M KCl (resistance ranging from 0.5 to 2.0
Mꢂ). Oocytes were placed in a recording bath that was continuously perfused with SOS at
18–22 °C.
For agonist assays, GABA dissolved in SOS was applied to oocytes for 3 s, at intervals
of 30–60 s to ensure a full recovery from desensitization. Concentration–response curves
were generated by sequential applications of increasing concentrations of GABA. Muscimol
hydrobromide was dissolved in SOS and tested using the same procedure as GABA. For
antagonist assays, 3ꢀisoxazolols were dissolved in DMSO and then diluted with SOS to the
desired concentrations [DMSO, ≤ 0.1% (v/v)]. The test compound solution was added to the
perfusate after two successive control applications of GABA and was then applied
consecutively for the remainder of the experiments. Antagonist solutions were perfused alone
for 30 s before their coꢀapplication with GABA. GABA, at a concentration corresponding to
the EC50 for each variant, was then coꢀapplied with an antagonist for 3 s, and the
coꢀapplication was repeated at 30ꢀ60 s intervals to obtain the highest constant inhibition. All
experiments were performed using at least four different oocytes obtained from at least two
different frogs.
EC50 and IC50 values were obtained from concentration–response data by nonlinear
regression analysis using OriginPro 8J (OriginLab, Northampton, MA). Statistical
significance was assessed using oneꢀway analysis of variance (ANOVA) followed by
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Duncan’s multiple range test.
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Insecticidal Assays. The WHO/SRS strain of houseflies (M. domestica) was used to
examine the insecticidal activity of 6b. Compound 6b dissolved in DMSO (0.1 µL) at various
concentrations was injected into the dorsal side of the thorax of adult female houseflies (3–5
days after eclosion). This volume of DMSO solution alone did not affect the viability of
houseflies. Twelve to fifteen houseflies were used at each concentration. The injected
houseflies were maintained with sugar and water at 25 °C. The number of dead and/or
paralyzed flies was counted after 24 h. The experiments were replicated five times. The LD50
value was calculated from the mean values of mortality at three dosages using the Probit
method.
Molecular Modeling and Ligand Docking Studies. A recently published Xꢀray crystal
structure of the homopentameric human β3 GABAR (PDB ID: 4COF)21 was used as a
template to construct a housefly RDLac GABAR homology model. A sequence alignment of
the RDLac and β subunits was carried out using ClustalW software, and this was used to build
all five subunits simultaneously using MOE 2011.10 software (Chemical Computing Group,
Montreal, Canada). The obtained pentamer model was optimized geometrically using the
AMBER99 force field. GABA and muscimol were created in the zwitterionic forms, and
compound 6b was created in a deprotonated hydroxyl form using MOE Builder. Potential
docking sites were searched using SiteFinder of MOE. The created ligands were docked into
the potential binding site of the generated model using the ASEDock program (2011.01.27,
Chemical Computing Group) with default parameters. The energies of the receptor and
ligands were minimized using the MMFF94x force field. The stable conformations of ligands
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were obtained by a conformational search. The binding mode with the highest score was
chosen for the final representation. Structural images were visualized using PyMOL Ver. 1.3
(Schrӧdinger, Tokyo, Japan).
RESULTS AND DISCUSSION
Chemistry. A new, convenient strategy to synthesize 4ꢀarylꢀsubstituted muscimols and
4ꢀarylꢀ5ꢀcarbamoylꢀ3ꢀisoxazolols starting from dimethyl acetylenedicarboxylate (1) was
established and is outlined in Scheme 1. 3ꢀIsoxazolol 2 was synthesized from 1 according to a
reported method,17 and the hydroxyl group was protected to give 3ꢀbenzyloxyisoxazole 3.18
Ammonolysis of ester 3 to amide 4 was followed by reduction with borane and deprotection
with hydrobromic acid to afford muscimol hydrobromide (5a). Treatment of 4 with 30%
hydrobromic acid in acetic acid afforded 5ꢀcarbamoylꢀ3ꢀisoxazolol (6a). To introduce
aromatic groups into the 4ꢀposition of the isoxazole ring, 4 was first iodinated at this position
using iodine and Pd(OAc)2 as a catalyst to give 7.22 The SuzukiꢀMiyaura crossꢀcoupling
reaction of 7 with the appropriate arylboronic acids in the presence of a palladium catalyst
afforded analogs 8b–g in 43–90% yields, the hydroxyl groups of which were deprotected
with hydrobromic acid to give 6b–g. Compounds 5b–c were obtained by reduction and
subsequent deprotection of 8b–c, as described for 5a.
Differential Sensitivity of Housefly GABAR Variants to GABA and Muscimol.
Similar to the case of D. melanogaster,5 four splice variants (ac, ad, bc, and bd) of RDL are
endogenously generated by alternatively splicing exons 3 (a and b) and 6 (c and d) of Rdl in
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houseflies. The two amino acid residues that differ between the sequences encoded by exons
3a and 3b are located upstream of the agonistꢀinteracting region of the RDL subunit, and the
ten residues that differ between the sequences encoded by exons 6c and 6d are located in a
region including loops F and C (Figure S1), which are predicted to be generally involved in
the interaction with agonists in Cysꢀloop receptors.23 In the present study, the four variants of
housefly RDL were expressed in Xenopus oocytes, and their responses to the agonists GABA
and muscimol were first investigated using a TEVC method.
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Application of GABA to the oocytes expressing housefly RDL GABARs induced
concentrationꢀdependent inward currents in all four variants when the voltage was clamped at
–80 mV. The GABA concentration–response curves show that the order of variants giving
higher sensitivity to GABA is RDLac ≈ RDLbc > RDLad ≈ RDLbd (Figure 2A); the EC50
values are given in Table 1. The finding that the alternative splicing of exon 3 did not affect
the sensitivity to GABA suggests that the two variable residues in exon 3 may not be
involved in the interaction with GABA. RDLac GABARs showed ~6ꢀfold higher sensitivity to
GABA than RDLad GABARs, and RDLbc GABARs had ~5ꢀfold higher sensitivity than
RDLbd GABARs. This finding indicates that the alternative splicing of exon 6 significantly
influences GABA potency in the RDL variants of housefly GABARs. Similarly, muscimol
(5a) was a more potent full agonist in the ac and bc variants than in the ad and bd variants
(Figure 2, Table 1). These different potencies of GABA and muscimol in the four variants are
similar to those in Drosophila RDL variants.6–8 The changes of agonist potencies may be due
to the residue difference in loop(s) F and/or C in the orthosteric binding site.
Antagonism of Housefly GABAR Variants by Synthesized Analogs. We examined the
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activity of synthesized compounds against RDL GABARs from three insect species. The
compounds were first tested against common cutworm (Spodoptera litura (Fabricius)) and
small brown planthopper (Laodelphax striatellus (Fallén)) RDLbd GABARs expressed in
Drosophila S2 cells using the fluorescent imaging plate reader (FLIPR®) membrane potential
(FMP) assays. Unexpectedly, synthesized analogs other than muscimol failed to show
significant activity at 100 ꢁM in both insect receptors (data not shown). In contrast to these
results, significant results were obtained against housefly RDL GABARs, prompting further
investigation described below. These contrasting results suggest that structural or functional
differences in the orthosteric binding sites of RDL GABARs might exist between insect
species.
As agonist potencies vary by the variant, synthesized analogs were assessed for their
functional characteristics in the four RDL variants of housefly GABARs expressed in
Xenopus oocytes using a TEVC method. Analogs were first tested at 100 ꢁM in the absence
and in the presence of the EC50 of GABA to determine if they are agonists or antagonists.
Unlike muscimol, all synthesized analogs showed no agonism but exhibited antagonism at
100 ꢁM in the four RDL variants.
In a previous study, we found that the 4ꢀ(3ꢀbiphenylyl)ꢀthioꢀ4ꢀPIOL and
4ꢀ(2ꢀnaphthyl)ꢀthioꢀ4ꢀPIOL analogs showed competitive antagonism in housefly RDLac
GABARs and common cutworm RDLbd GABARs, indicating that the 3ꢀbiphenyl and
2ꢀnaphthyl groups are beneficial for competitive antagonists of insect GABARs.14 Thus,
these groups were introduced into the 4ꢀposition of muscimol (5a) to afford 5b and 5c. Both
compounds showed antagonism at 100 ꢁM in all four RDL variants of housefly GABARs,
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albeit with less than 40% inhibition of GABAꢀinduced currents (Figure 3A). Although the
potencies of these analogs were low, these results indicate that the 3ꢀisoxazolol scaffold may
be useful for developing antagonists of housefly GABARs and that the bicyclic aromatic
system at the 4ꢀposition of the isoxazole ring may be beneficial for antagonistic activity as it
was in thioꢀ4ꢀPIOL analogs.
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Replacement of the aminomethyl group of muscimol (5a) with a carbamoyl group to
give 6a changed the function of muscimol from an agonist to an antagonist in the four
variants, with ~20% inhibition of GABAꢀinduced currents at 100 ꢁM in RDLac and RDLbc
GABARs and ~6% inhibition in RDLad and RDLbd GABARs. These findings indicate that the
protonated amino group is needed for agonist activity and that the carbamoyl group at the
5ꢀposition favors antagonism rather than agonism. The introduction of a 3ꢀbiphenylyl group
at the 4ꢀpoisiton of 6a to give 6b markedly increased the antagonistic activity against the four
variants, leading to 75.5, 76.1, 46.9, and 52.4% inhibition of GABAꢀinduced currents in
RDLac, RDLbc, RDLad, and RDLbd GABARs, respectively (Figure 3A). Figure 3B shows that
GABAꢀinduced currents were inhibited by the 3ꢀbiphenylyl analog (6b) at 100 ꢁM in the four
RDL variants. Compound 6c, with a 2ꢀnaphthyl substitution at the 4ꢀposition of the isoxazole
ring, also exhibited higher inhibition than 6a in all four variants, but relatively lower
inhibition compared with 6b. Additionally, two different aromatic groups, 4ꢀbiphenylyl and
1ꢀnaphthyl, were introduced into the 4ꢀposition to yield 6d and 6e, respectively. The
inhibition of GABAꢀinduced currents by 6d and 6e in the four variants was comparable to the
inhibition by 6b. These findings indicate that analogs with bicyclic aromatic groups at the
4ꢀposition of the isoxazole ring are well tolerated at the binding site and that they are
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effective in inhibiting GABAꢀinduced current in housefly GABARs. To investigate whether
the electronꢀdonating groups on the aromatic group at the 4ꢀposition increase activity, a
methoxyl and a hydroxyl group were introduced to the 6ꢀposition of the 2ꢀnaphthyl group of
6c to afford 6f and 6g, respectively. Compounds 6f and 6g were comparable in activity to 6c
in the four variants, although inhibition by 6f in the ac variant was higher than that of 6c
(Figure 3A). Overall, the synthesized analogs 5b–c and 6b–g were more potent against
RDLac and RDLbc GABARs than RDLad and RDLbd GABARs (Figure 3A), with ~1.5ꢀ to
~4ꢀfold higher inhibition in the former than in the latter. The different sensitivities of the four
variants to the synthesized analogs are similar to those of the agonists GABA and muscimol,
implying that the synthesized analogs bind to the same site as agonists.
Compounds 6b–g, which exerted relatively greater inhibitory effects, were further
evaluated in RDLac GABARs with the generation of antagonist concentration–response
curves in the presence of 10 ꢁM (EC50) GABA (Figure 4A). The 3ꢀbiphenylyl analog (6b),
with an IC50 value of 30.0 ꢁM, is among the analogs that displayed the greatest antagonism in
the ac variant (Table 2). The replacement of the 3ꢀbiphenylyl group with a 2ꢀnaphthyl and a
4ꢀbiphenylyl group to yield 6c and 6d resulted in 2.3ꢀ and 1.8ꢀfold increases in the IC50 value,
respectively. The 1ꢀnaphthyl analog (6e) had a potency similar to that of 6b in the ac variant.
Thus, the 3ꢀbiphenyl group is advantageous compared with the 4ꢀbiphenyl group; the
1ꢀnaphthyl group is preferable to the 2ꢀnaphthyl group. The introduction of a methoxy group
into the naphthyl group of 6c to yield 6f led to a 1.9ꢀfold increased potency in the ac variant,
whereas the potency of 6g, in which a hydroxyl group was introduced, was similar to that of
6c in the ac variant.
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Compound 6b was further examined for its potencies in other RDL variants of housefly
GABARs. Figure 4B shows concentration–response curves for 6b in the presence of the EC50
of GABA in the four variants. No significant differences were observed in the IC50 values of
6b between the ac and bc variants or between the ad and bd variants, whereas the IC50 values
of 6b in the ad and bd variants were ~3ꢀfold greater compared with those in the ac and bc
variants (Table 2). These findings were analogous to those in the EC50 values of GABA and
muscimol in the four variants, indicating that 6b most likely acts on the same site as agonists.
The different amino acid residues in the region encoded by exon 6 in different variants may
cause the difference in the sensitivity to competitive antagonists and agonists.
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Mode of Antagonism. To determine whether synthesized 3ꢀisoxazolols act as
competitive antagonists, the GABA concentration–response relationships in the presence and
absence of 6b were examined in the ac variant. The GABA concentration–response curves
made a parallel rightward shift with increasing 6b concentrations, indicating a competitive
mechanism (Figure 5). The EC50 values of GABA in the absence and presence of 30 and 100
ꢁM 6b were 10.6, 27.2, and 55.6 ꢁM, respectively. The potency of GABA was decreased 2.6ꢀ
and 5.2ꢀfold in the presence of 30 and 100 ꢁM 6b, respectively, whereas the efficacy of
GABA remained unchanged. These results indicate that 6b competes with GABA for the
orthosteric site to stabilize the closed conformation of chloride channels.
Insecticidal Activity. Competitive antagonists stabilize the closed conformation of
GABAR channels and should thus exert insecticidal effects when they act at insect GABARs.
There is no information about the insecticidal action of competitive antagonists. We therefore
investigated whether 6b, which showed the highest antagonism, has intrinsic insecticidal
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activity by injection into adult female houseflies. As a result, 6b proved to be insecticidal,
with an LD50 value of 5.6 (4.9–6.3) nmol/fly (95% confidence interval in parenthesis). The
finding that 6b shows insecticidal activity by definition is somewhat encouraging, although
the activity was not prominent and was observed by injection but not topical application.
Molecular Interaction between Ligands and Housefly RDL GABAR. To understand
the molecular mechanisms of the interaction between 3ꢀisoxazolols and insect GABARs,
GABA, muscimol, and the 3ꢀbiphenylyl analog (6b) were docked into the orthosteric binding
site of a housefly RDLac GABAR homology model constructed based on the Xꢀray crystal
structure of the human homopentameric β3 GABAR. The β3 subunit shares 38.7% amino
acid identity with the RDLac subunit. The most likely binding poses were selected based on
the score and shown in Figure 6. The orthosteric site of Cysꢀloop receptors, which is located
in the extracellular interface of two adjacent subunits (Figure S2), is formed by loops A–C
from the principal subunit and loops D–F from the complementary subunit.24 Similarly, the
orthosteric binding pocket of the constructed model is basically formed by Phe144, Val146,
Glu202, Ser203, Phe204, Gly205, Ile245, Leu247, and Arg254 from the principal subunit and
by Tyr88, Leu90, Tyr107, Arg109, and Met224 from the complementary subunit. The
distance between two key residues, Glu202 and Arg109, is approximately 9.0 Å.
The docking simulation showed that the protonated amino groups of GABA and
muscimol are located close to Glu202 of loop B and that they form an electrostatic interaction
and hydrogen bonds with this residue (Figures 6A, B). Arg109 of loop D electrostatically
interacts with the deprotonated carboxyl group of GABA and the deprotonated hydroxyl
group (or the isoxazole ring) of muscimol. In addition, Arg109 serves as a hydrogen bond
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donor for the carboxylate of GABA and the hydroxyl oxygen or the nitrogen atom of
muscimol. These important interactions were observed in the docking simulation results
using another housefly RDL GABAR homology model in our previous studies and are
apparently conserved in Drosophila RDL GABARs.14,15,25–27 Tyr252 of loop C, which
surrounds the protonated amino group of GABA and muscimol, may produce a cationꢀπ
interaction, as proposed for Drosophila RDL GABARs.25–27 Similar orientations and
interactions of GABA and muscimol in the binding site indicate that these two agonists
interact with housefly RDL GABARs in an identical mode.
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Docking of the 4ꢀ(3ꢀbiphenylyl) analog (6b) to the homology model of the housefly
RDLac GABAR predicts that the 5ꢀcarbamoylꢀ3ꢀisoxazolol scaffold of 6b lies between
Glu202 (loop B) and Arg109 (loop D) in the same orientation as muscimol. Similar to the
cases of GABA and muscimol, the side chain of Arg109 electrostatically interacts with the
deprotonated hydroxyl group of 6b and forms a hydrogen bond with the same group; the side
chain of Glu202 functions as a hydrogen acceptor for the carbamoyl group of 6b.
Furthermore, the two amino acid residues, Arg254 of loop C and Tyr107 of loop D, surround
the 3ꢀbiphenylyl group of 6b. The present docking studies of 6b predict that the 3ꢀbiphenylyl
group points out of the binding site and may form a πꢀcation interaction with Arg254 and a
πꢀπ interaction with Tyr107. The orientation of the 3ꢀbiphenylyl group is in contrast with that
of the 4ꢀsubstitution of thioꢀ4ꢀPIOL analogs in our previous study.14 This difference may be
due to the different templates used in the homology modeling. It has yet to be elucidated
which orientation is feasible.
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In conclusion, we synthesized a novel class of housefly competitive GABAR antagonists
(6b–g) by replacing the aminomethyl group of muscimol with a carbamoyl group and
simultaneously introducing bicyclic aromatic groups at the 4ꢀposition. All of the analogs
exhibited antagonism of the four splice variants of housefly GABARs, the most potent
compound being 4ꢀ(3ꢀbiphenylyl)ꢀ5ꢀcarbamoylꢀ3ꢀisoxazolol (6b). The potencies of 6b in
RDLac and RDLbc GABARs were ~3ꢀfold greater than those in RDLad and RDLbd GABARs,
and this potency difference in these variants is similar to the potency difference of agonists.
The identification of a novel series of competitive GABAR antagonists serves to widen the
current scope for insecticidal chemicals competitively acting at the orthosteric sites beyond
those of gabazine and thioꢀ4ꢀPIOL derivatives.
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Supporting Information
Alignments of amino acid sequences encoded by exons 3a, 3b, 6c, and 6d of housefly Rdl
(Figure S1). View of the orthosteric binding pocket in a housefly RDLac GABAR homology
model (Figure S2). The Supporting Information is available free of charge on the ACS
Publications website at http://pubs.acs.org/.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: ozoeꢀy@life.shimaneꢀu.ac.jp. Phone: +81ꢀ852ꢀ32ꢀ6573. Fax: +81ꢀ852ꢀ32ꢀ6573.
Funding
This work was supported in part by JSPS KAKENHI Grant Number 26292031.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Dr. I. Ikeda, and Mr. K. Nomura (Faculty of Life and Environmental Science,
Shimane University) for their useful advice and technical assistance.
ABBREVIATIONS USED
DMSO, dimethyl sulfoxide; EC50, half maximal effective concentration; GABA,
γꢀaminobutyric acid; GABAR, GABA receptor; IC50, half maximal inhibitory concentration;
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LD50, median lethal dose; PCR, polymerase chain reaction; RDL, name for an insect GABAR
subunit; SOS, standard oocyte solution; TEVC, twoꢀelectrode voltage clamp; Thioꢀ4ꢀPIOL,
5ꢀ(4ꢀpiperidyl)ꢀ3ꢀisothiazolol
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