Synthesis and structure-activity relationships of 1-phenyl-1H-1,2,3- triazoles as selective insect GABA receptor antagonists

Article abstract of DOI:10.1021/jf052773i

To study the interaction of phenylheterocycles with γ-aminobutyric acid (GABA) receptors, 4- or 5-alkyl-(or phenyl)-1-phenyl-1H-1,2,3-triazoles were synthesized and examined for their ability to inhibit the specific binding of [³H]-4′-ethynyl-4

Full text of DOI:10.1021/jf052773i

J. Agric. Food Chem. 2006, 54, 1361−1372 1361
Synthesis and Structure−Activity Relationships of
1-Phenyl-1H-1,2,3-triazoles as Selective Insect GABA Receptor
Antagonists
MOHAMMAD SAYED ALAM,† RYU KAJIKI,† HIROMI HANATANI,† XIANGYU KONG,†
FUMIYO OZOE,† YOSHIHISA MATSUI,† FUMIO MATSUMURA,‡ AND
YOSHIHISA OZOE*,†
Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane
University, Matsue, Shimane 690-8504, Japan, and Department of Environmental Toxicology and the
Center for Environmental Health Sciences, University of California, Davis, California 95616
To study the interaction of phenylheterocycles with γ-aminobutyric acid (GABA) receptors, 4- or 5-alkyl-
(or phenyl)-1-phenyl-1H-1,2,3-triazoles were synthesized and examined for their ability to inhibit the
specific binding of [3H]-4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a noncompetitive antago-
nist, to the housefly and rat GABA receptors, as well as to the â3 subunit homo-oligomer of the
human GABA receptor investigated as a model receptor. 4-Substituted 1-phenyl-1H-1,2,3-triazoles
were found to be more potent competitive inhibitors than the 5-substituted regioisomers in the case
of all receptors. The 4-tert-butyl or 4-n-propyl analogue of 1-(2,6-dichloro-4-trifluoromethylphenyl)-
1H-1,2,3-triazole exhibited the highest level of inhibition of [3H]EBOB binding to all receptors. Most
of the synthesized analogues were more active in terms of the inhibition of EBOB binding to the
housefly and human â3 GABA receptors than to the rat receptor. The 4-cyclohexyl analogue showed
the highest (185-fold) housefly versus rat receptor selectivity. A three-dimensional quantitative
structure-activity relationship (3D-QSAR) analysis demonstrated that both the 4-trifluoromethyl-2,6-
dichloro substitution on the phenyl ring and a small, bulky, hydrophobic substituent at the 4-position
of the triazole ring played significant roles in conferring high potency in cases involving the housefly
and human â3 receptors. The human â3 receptor resembled the housefly receptor in terms of their
recognition of phenyltriazoles, whereas 3D-QSAR analysis revealed a slight difference between the
two receptors in terms of their mechanisms of recognition of the para-substituent on the phenyl moiety.
Some of the triazoles synthesized here exhibited insecticidal activity, which was correlated with their
ability to inhibit [3H]EBOB binding to the housefly receptor. Thus, 1-phenyl-1H-1,2,3-triazoles with
the appropriate substituents exert insecticidal activity by selectively acting at the site for noncompetitive
antagonism of insect GABA receptors.
KEYWORDS: 1-Phenyl-1,2,3-triazole; GABA receptor; antagonist; insecticide; fipronil
INTRODUCTION
receptor in the brain is composed of R1, â2, and γ2 subunits.
GABA influences the opening of the channels to increase the
membrane conductance of chloride ions, thereby suppressing
the generation of action potentials (2). Insects have similar, but
pharmacologically different, GABA receptors from those of
mammals, and insect receptors reside not only in the central
but also in the peripheral nervous system (3, 4); thus, the readily
accessible peripheral GABA receptors have been considered as
a promising target for insect control chemicals. In insects, the
Rdl subunit, encoded by a dieldrin resistance-associated gene,
is the only subunit known to form a chloride channel (3).
γ-Aminobutyric acid (GABA) performs a significant role as
a major inhibitory neurotransmitter in both vertebrates and
invertebrates, including insects. In mammals, GABA binds to
two major receptor types, ionotropic and G protein-coupled
receptors, which are crucial for the accurate and proper
functioning of the central nervous system (1). The ionotropic
receptors are heteropentameric ligand-gated ion channels that
are assembled from five of eight subunit classes (R, â, γ, δ, ꢀ,
θ, π, and F), four of which contain several variants. The major
Fipronil (Figure 1) is a phenylpyrazole insecticide that acts
as a noncompetitive antagonist on insect ionotropic GABA
receptors (5) while it blocks glutamate-gated chloride channels
(6). Electrophysiological studies have shown that fipronil
* Author to whom correspondence should be addressed (telephone +81
852 32 6573; fax +81 852 32 6092; e-mail ozoe-y@life.shimane-u.ac.jp).
† Shimane University.
‡ University of California.
10.1021/jf052773i CCC: $33.50
© 2006 American Chemical Society
Published on Web 01/19/2006

1362 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Alam et al.
was kept at 0-5 °C. After the mixture had been stirred for 30 min,
sodium azide (190 mg, 2.91 mmol) dissolved in water (5m L) was
added in a dropwise manner to the mixture, and the mixture was stirred
overnight at room temperature. After the reaction was complete, the
mixture was extracted with CH2Cl2 (50 mL × 2), and the extract was
dried with sodium sulfate overnight. Then the CH2Cl2 was evaporated,
and the residue was chromatographed on silica gel (n-hexane), giving
2,6-dichloro-4-trifluoromethylphenyl azide as a red oil. 2,6-Dichloro-
4-trifluoromethylphenyl azide (412 mg, 1.61 mmol), an alkyne (1.61
mmol), and toluene (3 mL) were placed in a sealed tube and heated at
110 °C for 16 h. After the reaction, the mixture was partitioned between
water and ether. The ether layer was dried with sodium sulfate
overnight. The ether was evaporated, and the residue was subjected to
silica gel column chromatography (n-hexane/EtOAc ) 30:1), thus
affording the 4-alkyl/phenyl and 5-alkyl/phenyl isomers of the desired
compound. Compounds 1-12 and 24-33 were synthesized according
to this method. The analytical data for the phenyltriazoles are available
as Supporting Information.
Procedure for the Synthesis of 4(or 5)-n-Butyl-1-[4-(4-methoxy-
carbonyl-1-butynyl)phenyl]-1H-1,2,3-triazoles (Scheme 1). A mixture
of 28 (120 mg, 0.43 mmol), methyl 4-pentynoate (48 mg, 0.43 mmol),
trans-dichlorobis(triphenylphosphine)palladium(II) (3.1 mg, 4.3 µmol),
copper(I) iodide (0.41 mg, 2.2 µmol), and diethylamine (2.6 mL) was
refluxed under a nitrogen atmosphere for 24 h. The diethylamine was
evaporated, and the residue was partitioned between water and EtOAc.
After the EtOAc layer had been dried with sodium sulfate, the solution
was concentrated. The residue was subjected to silica gel column
chromatography (n-hexane/EtOAc ) 30:1) to afford 18 (117 mg,
87.5%) as brown crystals. Compounds 16, 17, and 19 were synthesized
in a similar manner. The analytical data for 16-19 are available as
Supporting Information.
Procedure for the Synthesis of 4(or 5)-n-Butyl-1-(4-ethynylphe-
nyl)-1H-1,2,3-triazoles (Scheme 1). A mixture of 26 (114 mg, 0.33
mmol), triphenyl phosphine (4.5 mg, 0.017 mmol), palladium(II) acetate
(1.8 mg, 8.2 µmol), (trimethylsilyl)acetylene (30 mg, 0.33 mmol), and
triethylamine (5 mL) was heated at 90 °C under a nitrogen atmosphere
for 14 h. The triethylamine was evaporated, and dry THF (5 mL) was
added to the residue under a nitrogen atmosphere. After the solution
had cooled to 10 °C, 1.0 M tetrabutylammonium fluoride in THF (0.5
mL) was added in a dropwise manner to the solution, with stirring
under a nitrogen atmosphere. The solution was stirred at room
temperature for 2 h. Then, the THF was evaporated, and the residue
was partitioned between water and EtOAc. After the EtOAc layer had
been dried with sodium sulfate, the solution was concentrated. The
residue was subjected to silica gel column chromatography (n-hexane/
EtOAc ) 30:1) to give 20 (34.0 mg, 35.2%) as brown crystals.
Compounds 21-23 were synthesized in a similar manner. The analytical
data for 20-23 are available as Supporting Information.
Regioselective Synthesis of 1-(2,6-Dichloro-4-trifluoromethylphe-
nyl)-4-phenyl-1H-1,2,3-triazole. This reaction was carried out accord-
ing to the method of Rostovtsev et al. (20). Ethynylbenzene (240 mg,
2.3 mmol) and 2,6-dichloro-4-trifluoromethylphenyl azide (600 mg,
2.3 mmol) were suspended in a 1:1 mixture of water and tert-butyl
alcohol (12 mL). Sodium ascorbate (230 µL of freshly prepared 1 M
solution in water, 0.23 mmol) was added to the mixture, followed by
copper(II) sulfate pentahydrate (5.8 mg, 0.023 mmol) in 77 µL of water.
The resulting heterogeneous mixture was stirred vigorously overnight.
The reaction mixture was diluted with water and cooled in an ice bath,
and a light yellow precipitate was collected by filtration. The yellow
precipitate was purified by silica gel column chromatography (n-hexane/
EtOAc ) 3:1) to afford 595 mg (99%) of a pure product as white
crystals, which gave a 1H NMR spectrum identical to that of 1
synthesized according to the method described above.
Figure 1. Structures of fipronil and 1-phenyl-1H-1,2,3-triazoles.
blocked GABA-induced currents in Drosophila homo-oligo-
meric GABA-gated chloride channels (Rdl) expressed in Xe-
nopus oocytes (7, 8) and that an analogue of fipronil antagonized
the action of GABA in the ventral ganglia of Drosophila larvae
(9). In addition, two types of related phenyl heterocyclic
compounds (PHCs), that is, 1-phenyl- and 2-phenyl-1,2,3-
triazoles, have been reported to block GABA-activated Cl-
currents in cell bodies isolated from locust metathoracic ganglia
(10) and nematode somatic muscle cells (11), respectively.
Fipronil was found to more potently inhibit the specific binding
of [3H]-4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a
noncompetitive antagonist of ionotropic GABA receptors, to
housefly head membranes than to mouse brain membranes (12).
The low sensitivity of mammalian GABA receptors to fipronil
was shown to result from the difference in subunit compositions
between mammalian and insect receptors (13). 2-Phenylpyri-
midines and -1,3-thiazines are also recognized as inhibitors of
[3H]EBOB binding to housefly head and mouse brain mem-
branes (14). Moreover, 3-phenylpyrimidinones are thought to
function in a similar manner (15). We previously demonstrated
that a variety of five- and six-membered nitrogen-containing
heterocyclic compounds bearing a 2,4,6-trisubstituted phenyl
group interact with ionotropic GABA receptors (16). The
quantitative structure-activity relationships (QSARs) of anti-
GABAergic PHCs have not yet been completely elucidated,
although the dipole moment of PHCs was hypothesized to be
responsible for their insecticidal activity (17), and favorable and
unfavorable moieties of PHCs have been predicted by three-
dimensional (3D)-QSAR analysis (18).
The present study was undertaken to gain a better understand-
ing of the interaction of PHCs with GABA receptors with the
ultimate goal of contributing to the design of safe, novel
insecticides. To this end, 1-phenyl-1H-1,2,3-triazoles with
various substituents at the 4- and 5-positions of the triazole ring
and on the phenyl ring (Figure 1) were synthesized and
examined for their effects on [3H]EBOB binding to housefly,
rat, and human â3 GABA receptors. We report here the results
of the QSAR analysis of the phenyltriazoles. A preliminary
report of some of this work has been published previously in
abstract form (19).
MATERIALS AND METHODS
1
General. H NMR spectra were recorded on a JEOL JNM-A 400
spectrometer, and chemical shifts are given in parts per million relative
to tetramethylsilane. Mass spectra were obtained on a Hitachi M80-B
spectrometer. Melting points were determined using a Yanagimoto MP
500D apparatus and remain uncorrected. [propyl-2,3-3H]EBOB (30.0
or 47.5 Ci/mmol) was purchased from Perkin-Elmer Life Sciences
Japan, Co. Ltd. (Tokyo, Japan). Phenyltriazoles 13-15 were available
from our earlier study (16). General chemicals were obtained from
Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
General Procedure for the Synthesis of 4(or 5)-Alkyl/phenyl-1-
phenyl-1H-1,2,3-triazoles (Scheme 1). 2,6-Dichloro-4-trifluoromethyl-
aniline (670 mg, 2.91 mmol) was added to a mixture of water (50 mL)
and concentrated HCl (50 mL), and the mixture was stirred at room
temperature for 30 min. Sodium nitrite (200 mg, 2.91 mmol) dissolved
in water (5 mL) was added in a dropwise manner to the mixture, which
Generation of Drosophila Schneider 2 (S2) Cells Stably Express-
ing the Human GABA Receptor â3 Subunit Homo-oligomer. The
cDNA encoding the â3 subunit of the human GABA receptor (accession
number NM_000814) was amplified by PCR from first-strand cDNA
derived from normal adult human brain tissue (BioChain). A KpnI/
ApaI fragment containing the 1.4 kb open reading frame of the â3
subunit gene (hsâ3) was inserted into the Drosophila expression vector
pMT (Invitrogen), in which cDNA expression is driven by the

1-Phenyl-1H-1,2,3-triazoles as GABA Receptor Antagonists
J. Agric. Food Chem., Vol. 54, No. 4, 2006 1363
Scheme 1
metallothionein promoter and induced by copper sulfate. S2 cells were
co-transfected with pMThsâ3 and the selection vector pCoHygro by a
calcium phosphate transfection method, according to the manufacturer’s
instructions (Invitrogen, the DES kit). The transfected cells were
maintained for ∼3 weeks in selection medium containing hygromycin
B. Antibiotic-resistant polyclonal cells were used for the binding assays
described below.
[3H]EBOB binding assays were carried out according to the methods
of Deng et al. (21) and Cole and Casida (23). Housefly head membranes
(200 µg of protein), cell membranes expressing human â3 receptors
(50 µg of protein), or rat brain membranes (125 µg of protein) were
incubated with various concentrations of phenyltriazoles and 0.5 nM
[3H]EBOB in 1 mL of buffer B at 22 °C for 70 min (housefly and
human â3 receptors) or at 37 °C for 90 min (rat receptor). The protein
content was measured according to the method of Bradford (24). After
incubation, the mixture was filtered through Whatman GF/B filters and
rapidly rinsed twice with 5 mL of cold (10 °C) buffer B using a Brandel
M-24 cell harvester. The radioactivity of [3H]EBOB that had specifically
bound to the membranes on the filters was measured with a liquid
scintillation counter. Nonspecific binding was determined in the
presence of 5 µM unlabeled EBOB. Experiments were repeated at least
three times. The IC50 values were calculated from the mean values using
the standard probit method. Scatchard analysis was performed using
1.0 nM [3H]EBOB and various concentrations of cold EBOB. Experi-
ments were repeated five times.
Molecular Modeling and 3D-QSAR Studies. All computations
were carried out using the molecular modeling software SYBYL, ver.
6.9 (Tripos). The structures of all phenyltriazoles were constructed on
the basis of the X-ray crystal structure of 9 and were fully optimized
by following the semiempirical molecular orbital method AM1.
Comparative molecular field analysis (CoMFA) was used to study the
3D-QSAR of phenyltriazoles in the human â3 and housefly GABA
receptors. The molecules were superimposed at the 1-, 2-, 3-, 4-, and
5-positions of the triazole ring by the Fit Atoms menu of SYBYL for
CoMFA.
Membrane Preparation and [3H]EBOB Binding Assays. Housefly
head membranes were prepared from adult houseflies (Musca domestica
L.) of the WHO/SRS strain according to a modification of the method
of Deng et al. (21). Housefly heads were homogenized in 10 mM Tris-
HCl buffer, pH 7.5, containing 0.25 M sucrose (buffer A) with a glass-
Teflon homogenizer; then, the homogenate was filtered through four
layers of 64-µm mesh nylon screen and centrifuged at 500g for 5 min.
The supernatant was filtered through four layers of 64-µm mesh nylon
screen and centrifuged at 25000g for 30 min. The pellets were
suspended in buffer A and were kept on an ice bath for 30 min. The
suspension was centrifuged at 25000g for 30 min. The pellets were
resuspended in 10 mM sodium phosphate buffer, pH 7.5, containing
0.3 M NaCl (buffer B) and were used immediately for the binding
assays.
Rat brain membranes were prepared from 5-week-old male Wistar
rats according to a modification of the method of Squires et al. (22).
Forebrains stored at -80 °C were thawed and homogenized in ice-
cold 1 mM EDTA using a glass-Teflon homogenizer. The homogenate
was centrifuged at 1000g for 10 min. The supernatant was then
centrifuged at 25000g for 30 min. The resulting pellets were suspended
in 1 mM EDTA and dialyzed three times (2 h each) in cellophane tubing
against 2 L of distilled, deionized water at 4 °C. After dialysis, the
inner solution was centrifuged at 25000g for 30 min, and the pellets
were stored at -80 °C until use.
S2 cells expressing human homo-oligomeric â3 receptors were grown
at 28 °C in Schneider’s Drosophila medium (Gibco) containing 10%
fetal bovine serum (Gibco), using a shaking incubator at 50 rpm.
Schneider’s Drosophila medium was supplemented with hygromycin
B at a concentration of 300 µg/mL. S2 cells were grown at a viability
of >96% and a density of 6-20 × 106 cells/mL of culture medium
for ∼3-4 days. Copper sulfate was added to the medium at a
concentration of 500 µM/mL 24 h before the preparation of protein.
S2 cell-containing Schneider’s Drosophila medium was centrifuged at
1000g for 5 min, and after the supernatant had been discarded, the
cells were washed with buffer A. The pellets were resuspended in buffer
A and centrifuged at 1000g for 5 min, and after the supernatant had
been discarded, the pellets were washed with buffer A. The membranes
were prepared by homogenizing the pelleted cells in buffer A with a
glass-Teflon homogenizer. The supernatant was centrifuged at 25000g
for 20 min at 4 °C. The resulting pellets were superficially washed
with buffer B, suspended in buffer B, and used immediately for the
binding assays.
Insecticidal Assays. Insecticidal activity was determined by the
topical application of acetone (1 µL) containing various concentrations
of phenyltriazoles on the dorsal surface on the thorax of adult female
houseflies (M. domestica L., WHO/SRS strain, 3-5 days old after
emergence). Thirty flies were used for each dosage. An acetone solution
(1 µL) of piperonyl butoxide (10 µg), a cytochrome P450 inhibitor,
was applied topically to each fly 1 h before the application of the
phenyltriazoles. The flies were supplied with sugar and water and were
kept at 25 °C. The rate of mortality was determined after 24 h.
Experiments were repeated three times. The LD50 values were calculated
from the mean values using the standard probit method.
RESULTS
Synthesis of 1-Phenyl-1H-1,2,3-triazoles. A total of 30
1-phenyl-1H-1,2,3-triazoles (1-12 and 16-33 in Tables 1 and
2) were synthesized in the present study. The 1,3-dipolar
cycloaddition between an alkyne and a phenyl azide gave a
mixture of anti- and syn-triazole regioisomers, that is, 4-sub-
stituted- and 5-substituted-1-phenyl-1H-1,2,3-triazoles. The anti-

1364 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Alam et al.
Table 1. Potencies of 1-(2,6-Dichloro-4-trifluoromethylphenyl)-4(or 5)-(alkyl or phenyl)-1H-1,2,3-triazoles in Terms of the Inhibition of [3H]EBOB
Binding to Human
â
3, Housefly, and Rat GABA Receptors
[3H]EBOB binding, IC50 (nM)
insecticidal activity,
LD50 (pmol/fly)
compd
R1
R2
human
154
â
3
housefly
183
rat
RSa
>55
1
Ph
H
H
>10000
(22.7c)
>10000
(32.1c)
8500
7110
(101
570
−
242b)
(110−
309b)
(5490−
9290b)
2
Ph
1270
>8
8
(362
−
925b)
(734−
2420b)
3
n-Hex
H
H
470
1060
(340−
661b)
(616−
1980b)
(7020
−
10400b)
4
n-Hex
H
1040
>10000
(43.8c)
192
>10000
(29.5c)
4310
(780−
1410b)
5
n-Pen
H
33.4
22
>2
42
>6
65
3990
(22.4−
47.2b)
(115−
328b)
(3550−
5250b)
(3280−
4840b)
6
n-Pen
H
1670
5070
>10000
(19.1c)
1660
(1200−
2380b)
(3220−
9020b)
7
n-Bu
H
3.35
40.0
1930
(2.56−
4.37b)
(25.0−
66.0b)
(1230−
2220b)
(1280−
3050b)
8
n-Bu
H
>10000
(49.9c)
0.997
1630
>10000
(12.0c)
643
(1180−
2240b)
9
t-Bu
H
9.90
1.86
(0.777
−
1.297b)
(5.59−
16.40b)
(503−
816b)
(0.69−
3.95b)
10
11
12
13
14
15
t-Bu
H
>10000
(49.6c)
2.57
>10000
(46.0c)
9.04
>10000
(9.0c)
730
n-Pr
H
81
>9
8.66
(2.03−
3.24b)
(5.49−
14.01b)
(498
−
1198b)
(1.29−
11.67b)
n-Pr
H
909
1060
>10000
(12.1c)
6560d
(528−
1440b)
(640
−
1910b)
c-Hex
(CH2)3Cl
Ph
14.1
35.4d
185
64
76.9
(10.7
−
18.3b)
(20.9
−
60.0b)
(4490
−
9580b)
(57.7−
101.7b)
H
1.96
27.6d
1770d
(1.43
−
2.69b)
(17.0
−
44.9b)
(1360
−
2320b)
NH2
24.4
34.9d
1090d
31
(16.0
−
41.9b)
(25.9
−
47.0b)
(840−
1410b)
a Selectivity for housefly versus rat receptor (IC50rat/IC50fly). b Ninety-five percent confidence limits. c Inhibition (percent) at 10
µ
M. d Reference 16.
isomers were obtained in yields that were 1.1-3.2-fold higher
than those of the syn-isomers. The regioisomers were separated
by silica gel column chromatography. The regiochemistry of
1-(2,6-dichlorophenyl)-1H-1,2,3-triazoles was established on the
basis of an X-ray crystallographic analysis of 9 (Figure 2 and
Supporting Information). The regiochemistry of the 1-(2,6-
unsubstituted phenyl)-1H-1,2,3-triazoles was determined by
NOE observation between the ortho-protons of the phenyl group
and the 1-protons of the n-butyl group of the syn-isomers. The
1H NMR spectra showed that the triazole proton of the
4-substituted 1-(2,6-unsubstituted phenyl)triazoles was shifted
downfield compared to that of the corresponding 1,5-substituted
triazoles, as has been reported for other triazoles (25-28). In
contrast, the opposite relationship was observed in the case of
1-(2,6-dichlorophenyl)-1H-1,2,3-triazoles; that is, the 1,4-
substituted triazoles had a triazole signal that was upfield of
that of the corresponding 1,5-substituted triazoles. The only
exceptions were observed with 1 and 2, which produced the
triazole proton signals at almost the same chemical shift. The
regiochemistry of 1 (anti) and 2 (syn) was determined on the
basis of the fact that copper(I)-catalyzed regioselective cycload-
dition (20) gave 1 and that the ortho-protons of the 4-phenyl
group of 1 were shifted downfield compared to those of the
5-phenyl group of 2, as are the protons of the alkyl groups of
other 4- or 5-substituted triazoles. Phenyltriazoles with an
ethynyl or a 4-methoxycarbonyl-1-butynyl group at the 4-posi-
tion of the phenyl group were prepared by palladium-catalyzed
cross-coupling between the corresponding bromo analogues and
terminal alkynes.
Effects of the 4- and 5-Substituents of 1-Phenyl-1H-1,2,3-
triazoles on Interaction with GABA Receptors. Using [3H]-
EBOB, a specific radioligand that binds to the noncompetitive
antagonist binding site of GABA receptors (21, 23), a series of
phenyltriazoles were assayed to examine the effects of a phenyl
or an alkyl group at the 4- or 5-position of the triazole moiety
upon interaction with GABA-gated chloride channels. Table 1
lists the respective potencies of 1-(2,6-dichloro-4-trifluoro-
methylphenyl)-1H-1,2,3-triazoles in terms of their ability to
inhibit specific [3H]EBOB binding to human â3, housefly, and
rat GABA receptors. Comparison of the 4- and 5-substituted
isomers of the phenyltriazoles revealed that the potencies of
the former isomers were generally higher than those of the latter
isomers. Among this series of compounds (1-15), 11 and 9
were found to be the most potent in the nanomolar range in the
housefly and human â3 receptors, followed by 14, 13, 15, and
fly
7 and then by 5 and 1. On the basis of the IC50 values, the
rank order of potency with respect to the 4-substituent was as
follows: t-Bu, n-Pr > (CH2)3Cl, c-Hex, n-Bu > n-Pen, Ph >
n-Hex. Replacement of the terminal methyl group of the 4-n-
butyl group of 7 with a chlorine atom to produce 14 did not
result in a marked change in potency. The introduction of an
amino group into the 5-position of 1 led to a >5-fold increase

1-Phenyl-1H-1,2,3-triazoles as GABA Receptor Antagonists
J. Agric. Food Chem., Vol. 54, No. 4, 2006 1365
Table 2. Potencies of 1-(Substituted phenyl)-4(or 5)-n-butyl-1H-1,2,3-triazoles in Terms of the Inhibition of [3H]EBOB Binding to Human
and Rat GABA Receptors
â3, Housefly,
[3H]EBOB binding, IC50 (nM)
insecticidal activity,
LD50 (nmol/fly)
compd
R1
R2
R3/R4
Cl
R5
human
â
3
housefly
rat
RSb
16
n-Bu
H
MCBa
>10000
(19.3d)
>10000
(43.8d)
>10000
(34.7d)
>10000
(22.0d)
41.6
>10000
(8.9d)
>10000
(16.2d)
>10000
(9.4d)
>10000
(20.1d)
>10000
(3.2d)
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
H
n-Bu
H
Cl
H
MCB
MCB
MCB
n-Bu
H
>10000
(47.0d)
>10000
n-Bu
H
H
>10000
(−
0.6d)
(
−
1.3d)
n-Bu
H
Cl
Cl
H
C
C
C
C
tCH
tCH
tCH
tCH
670
>10000
(42.3d)
>10000
(10.6d)
4060
15
(28.6−
58.2c)
(500−
920c)
n-Bu
H
>10000
(12.0d)
>10000
(47.4d)
9100
>10000
(28.5d)
>10000
(29.4d)
>10000
(8.5d)
n-Bu
H
(2900−
6020c)
n-Bu
H
H
≈
10000
(7130
−
13540c)
(52.5d)
>10000
(48.2d)
>10000
(24.5d)
>10000
(48.1d)
>10000
(18.6d)
>10000
(32.5d)
>10000
(26.5d)
>10000
(32.2d)
>10000
(11.3d)
>10000
(18.1d)
>10000
(11.9d)
n-Bu
H
Cl
Cl
Cl
Cl
H
Cl
98.7
480
>20
>34
65.1
(65.0−
138.8c)
(340−
660c)
(41.1−
120.7c)
n-Bu
H
Cl
4900
>10000
(37.9d)
290
(4020−
6160c)
n-Bu
H
Br
Br
Br
Br
CF3
CF3
H
37.4
(25.7−
52.3c)
(200−
420c)
n-Bu
H
940
>10000
(49.5d)
>10000
(29.2d)
>10000
(22.1d)
>10000
(21.1d)
>10000
(12.2d)
>10000
(17.1d)
>10000
(3.4d)
(607
3830
−
1521c)
n-Bu
H
(2690−
5770c)
n-Bu
H
H
3280
(2720−
3970c)
n-Bu
H
H
≈
10000
(51.1d)
6360
n-Bu
H
H
(5230
−
7950c)
n-Bu
H
Cl
Cl
4220
(2540−
8030c)
n-Bu
H
2460
(1680−
3790c)
a MCB, C C(CH2)2CO2CH3. b Selectivity for housefly versus rat receptor (IC50rat/IC50fly). c Ninety-five percent confidence limits. d Inhibition (percent) at 10
t µM.
Figure 2. X-ray crystal structure of 9.
in potency in the case of all three receptors, as seen with 15.
Most compounds remained basically inactive (IC50 > 10 µM)
in the rat receptor or were much less active than in the case of
other receptors. Even the most potent inhibitors (11 and 9) had
IC50 values of several hundred nanomolar, and therefore it was
concluded that these compounds exhibited high selectivity
(IC50rat/IC50fly) for the housefly versus the rat GABA receptor
(Table 1). Figure 3 shows the concentration-inhibition curves
of these selectively active phenyltriazoles. Compound 13 was
found to be the most selective (185-fold) analogue in this regard,

1366 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Alam et al.
Figure 4. Scatchard plots of [3H]EBOB binding to the cloned human
receptor in the presence ( ) and absence ( ) of 1 nM 9. The amount of
specifically bound [3H]EBOB is expressed as fmol/0.05 mg of protein.
Bmax 3.26 pmol/mg of protein, Kd 5.00 nM in the absence of 9. Bmax
3.64 pmol/mg of protein, Kd 13.1 nM in the presence of 1 nM 9.
standard deviation for five experiments each
â3
b
9
)
)
)
)
The data are the mean
±
performed in duplicate.
Figure 5. Correlation between the inhibitory activity of selected 1-phenyl-
1H-1,2,3-triazoles in terms of the inhibition of [3H]EBOB binding to the
housefly GABA receptor and their insecticidal activity (A) and correlation
between the inhibitory potencies of selected 1-phenyl-1H-1,2,3-triazoles
Figure 3. Dose
[3H]EBOB binding to rat (
receptors. The data are the mean
−
response curves of 7 (A), 9 (B), and 11 (C) in inhibiting
), housefly ( ), and human 3 ( ) GABA
standard deviation for at least three
in the inhibition of [3H]EBOB binding to human
â3 and housefly GABA
9
b
â
2
receptors (B).
±
experiments, each performed in duplicate.
this activity was very low. Comparison of 20, 22, 24, 26, 28,
30, and 32 with 7 demonstrated the importance of both the p-CF3
group and the two o-chlorine atoms; the rank order of potency
regarding the phenyl substitution was 2,6-Cl2-4-CF3 > 2,6-Cl2-
4-Br > 2,4,6-Cl3 g 2,6-Cl2-4-CtCH. Compounds with a 4-(4-
methoxycarbonyl-1-butynyl) group on the phenyl group exhib-
ited no activity.
as previously reported (16). To determine whether phenyltri-
azoles bind to the same site as that for EBOB binding, a
Scatchard analysis of [3H]EBOB binding to the cloned human
â3 receptor was carried out in the presence of 9. The two lines
obtained in the presence and absence of 9 intersected near the
abscissa axis, thus indicating that the two compounds do indeed
bind to the same site (Figure 4).
Effects of Substituents of the Phenyl Moiety of 1-Phenyl-
1H-1,2,3-triazoles on Interaction with GABA Receptors. The
effects of the substituents of the 1-phenyl group on specific [3H]-
EBOB binding were examined. The substituents on the phenyl
ring exerted marked effects on the potency of triazoles in the
inhibition of EBOB binding to the human â3, housefly, and rat
receptors (Table 2). Among the 4(or 5)-n-butyl-1-phenyl-1H-
1,2,3-triazoles (16-33) with substitution patterns other than the
2,6-dichloro-4-trifluoromethyl substitution on the phenyl group,
20, 24, and 26 were the only compounds found to be active in
the case of both human â3 and housefly GABA receptors.
Compounds 23, 25, 27-29, and 31-33 showed marginal
activity in the case of the human â3 receptor, whereas they were
almost inactive in the case of the housefly and rat receptors.
1-(4-Monosubstituted phenyl)-4(or 5)-n-butyl-1H-1,2,3-triazoles
22, 18, and 23 exhibited higher activity in the case of the rat
GABA receptor than in case of the housefly receptor, although
Insecticidal Activity. To test the validity of [3H]EBOB
binding as an in vitro assay, 1-phenyl-1H-1,2,3-triazoles with
a wide range of potency were selected and examined for their
in vivo potency, that is, insecticidal activity against houseflies
pretreated with piperonyl butoxide. Compound 9 showed the
highest insecticidal activity among the tested compounds, with
an LD50 value of 1.86 pmol/fly. The plot of pLD50 values (log-
fly
fly
[1/LD50 (mol)]) versus pIC50 values (log[1/IC50 (M)]) of
seven compounds (1, 5, 7, 9, 11, 13, and 24) gave a high
correlation (r2 ) 0.91) (Figure 5A). This finding indicated that
the interaction of phenyltriazoles with the housefly GABA
receptor leads to significant insecticidal activity.
Relationship between the Ability of 1-Phenyl-1H-1,2,3-
triazoles To Inhibit the Binding of [3H]EBOB to Human â3
and Housefly GABA Receptors. To validate the relevance of
the human â3 receptor as a model of the housefly receptor, we
plotted the potencies (pIC50 or log[1/IC50 (M)]) of 15 triazoles
(1-3, 4, 6, 7, 9, 11-15, 20, 24, and 26) in inhibiting [3H]EBOB

1-Phenyl-1H-1,2,3-triazoles as GABA Receptor Antagonists
J. Agric. Food Chem., Vol. 54, No. 4, 2006 1367
Table 3. CoMFA Results of 1-Phenyl-1H-1,2,3-triazoles: log(1/IC50) ) a + [CoMFA Field Terms]
cross-validated
relative contribution
steric electrostatic
model
a
Ca
n
s
r 2
Fc,n
spress
q2
-c-1
model for housefly receptor
6.608
5.199
4
5
16
24
0.192
0.282
0.962
0.960
70.546
85.850
0.558
0.845
0.684
0.638
0.679
0.551
0.321
0.449
model for
â3 receptor
a Optimum component.
Figure 6. CoMFA contour maps showing the results of analyses of the human
orthogonal views of maps with 12.
â3 GABA receptor: (A, B) orthogonal views of maps with 3; (C, D)
binding obtained using membranes from human â3 receptor-
expressing S2 cells against those obtained using housefly head
membranes (Figure 5B). This plot yielded a good correlation
(r2 ) 0.87), indicating a similarity in ligand recognition between
the two receptors.
receptor affinity, and the red contours represent regions in which
negatively charged moieties of a ligand increase receptor
affinity. Both the human â3 and housefly receptor models
provided analogous contour maps, except that the para-sub-
stituent of the phenyl group was surrounded by a red contour
in the housefly receptor model, whereas in the case of the human
â3 receptor model, the red contour was replaced with a green
contour. That is, the housefly receptor probably recognizes the
electronegativity of the para-substituent, whereas the human â3
receptor might recognize it as a sterically fitting substituent.
The presence of the green and blue contours around the
4-position of the triazole ring in both human â3 and housefly
receptor models displays a binding site area in which a
substituent of a high-affinity ligand is accommodated. The blue
contours indicate that lipophilic interactions between the binding
site and ligands enhance the antagonist activity. These findings
are consistent with the results showing that the optimal
substituent of the triazole ring is a tert-butyl or n-propyl group
at the 4-position. The longer alkyl chains at the 4- and
5-positions of the triazole ring sterically hinder binding as
demonstrated by the yellow contours.
Molecular Modeling and 3D-QSAR. To obtain quantitative
information regarding the steric and electrostatic requirements
for the effects of 1-phenyl-1H-1,2,3-phenyltriazoles on the
human â3 and housefly receptors, 3D-QSAR analyses were
performed using the CoMFA module in the SYBYL program.
The results are summarized in Table 3. For the human â3 and
housefly receptors, respectively, 24 and 16 active phenyltriazoles
were successfully included in the CoMFA analysis. The squared
cross-validated correlation coefficient (q2) values were 0.638
and 0.684 for the human â3 and housefly receptor models,
respectively, and these q2 values indicate the validity of the
suggested mode of interaction [i.e., a q2 value of g0.25
corresponds to the conventional 95% confidence interval for
the probability of a chance correlation (29)].
Figures 6 and 7 show the contour maps of the CoMFA
analysis of the human â3 and housefly receptors, respectively.
The green contours indicate sterically favorable regions that
enhance the affinity for receptors, whereas the yellow contours
are sterically unfavorable regions. The blue contours indicate
regions in which positively charged moieties of a ligand increase
DISCUSSION
In the present study, we synthesized 30 1-phenyl-1H-1,2,3-
triazoles and performed [3H]EBOB binding assays to evaluate

1368 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Alam et al.
Figure 7. CoMFA contour maps showing the results of analyses of the housefly GABA receptor: (A, B) orthogonal views of maps with 3; (C, D)
orthogonal views of maps with 8.
the potency of synthesized compounds at GABA receptors. We
used three receptor sources, that is, housefly head and rat brain
membranes, and S2 cell membranes stably expressing the â3
subunit homo-oligomer of the human GABA receptor, to study
the structure-activity relationships of phenyltriazoles and to
probe the structure of the binding site. The human â3 receptor
was used as a model receptor, and it should be noted that no
such homo-oligomeric channel has been identified in the brain
(30). The rat â3 subunit expressed in HEK293 cells has been
reported to form a high-affinity site for the noncompetitive
GABA antagonist tert-butylbicyclophosphorothionate (31). The
murine â3 subunit was shown to form a functional but
spontaneously opening homo-oligomeric channel when ex-
pressed in Xenopus laeVis oocytes (32). Moreover, it has been
reported that the human â3 subunit homo-oligomer contained
a binding site for [3H]EBOB (13), and the human â3 subunit
homomer resembled the housefly GABA receptor in terms of
its sensitivity to and selectivity for insecticides (33). The present
study included the human â3 receptor to determine whether this
interpretation of the data is just as applicable in the case of
Figure 8. Amino acid residues of the second transmembrane segment
of housefly Rdl and human 3 subunits. Pore-facing amino acids are
underlined. Accession numbers: housefly Rdl subunit, AB177547; human
3 subunit, NM_000814.
â
â
be involved in the formation of the noncompetitive antagonist
binding site (34-36). The only difference between the housefly
Rdl and human â3 subunits in terms of the sequence of this
region is the 5′ amino acid (Val versus Ile) adjacent to the pore-
facing 6′ amino acid (Figure 8). The difference in ligand
recognition is very unlikely to be explained solely as a result
of this amino acid difference. Nevertheless, the good correlation
observed here between IC50hâ3 and IC50fly values suggested that
the â3 receptor would be a useful tool for screening compounds
acting at the housefly GABA receptor.
Some 4-substituted phenyltriazoles were found to be potent
inhibitors of [3H]EBOB binding to GABA receptors. Scatchard
analysis using 9 showed that the phenyltriazoles and EBOB bind
to the same site, at least in the case of the â3 receptor. The
compounds that most potently inhibited [3H]EBOB binding to
the housefly receptor were the 4-n-propyl (11) and 4-tert-butyl
(9) analogues of 1-(2,6-dichloro-4-trifluoromethylphenyl)-1H-
1,2,3-triazole. In addition to these compounds, 7 and 14 strongly
inhibited EBOB binding to the human â3 receptor. The
requirement of a small hydrophobic substituent at the 4-position
of the triazole moiety for high activity was clearly observed, as
shown by the green and blue contours in the CoMFA maps
(Figures 6 and 7). The requirement of a similar hydrophobic
moiety has been reported in several studies of different GABA
antagonists: 4-alkyl-1-phenylpyrazoles (37), 5-alkyl-2-(4-ethy-
nylphenyl)-1,3-dithianes (38), 4-alkyl-1-(4-ethynylphenyl)-2,6,7-
trioxabicyclo[2.2.2]octanes (39), and 4-alkyl-1-phospha-2,6,7-
trioxabicyclo[2.2.2]octane 1-sulfides (40). The bulky alkyl group
of C2-C3 in length gave high activity. The putative common
hâ3
fly
phenyltriazoles. The plot of IC50
versus IC50 values of
phenyltriazoles gave a good correlation, although the human
â3 receptor was more sensitive to phenyltriazoles than the
housefly receptor, thus supporting the structural similarity
between the binding sites of these two receptors. To further
confirm the finding, we performed a 3D-QSAR analysis based
hâ3
fly
on IC50 and IC50 values. The CoMFA analysis revealed
not only a similarity but also a difference between the two
receptors in terms of the mode of recognition of ligands. The
electronegativity of the para-substituent of the 1-phenyl group
is more important in the housefly receptor than in the human
â3 receptor; the para-substituent is preferably recognized as a
sterically favorable substituent in the case of the human â3
receptor. GABA receptors are known to be formed by five
4-transmembrane subunits. The 2′ amino acid residue in the
channel pore-lining R-helix domain (i.e., the second transmem-
brane domain, TM2) of GABA receptor subunits is thought to

1-Phenyl-1H-1,2,3-triazoles as GABA Receptor Antagonists
J. Agric. Food Chem., Vol. 54, No. 4, 2006 1369
hydrophobic moiety has been observed, even in naturally
occurring terpenoid GABA antagonists (41). The IC50 values
of 11 and 9 in housefly (9.04 and 9.90 nM, respectively) and
human â3 (2.57 and 0.997 nM, respectively) receptors were
comparable to the reported IC50 values of fipronil in the case
of the housefly (2.3 or 6.3 nM) and human â3 (2.4 or 3.1 nM)
receptors (12, 13, 42, 43) as well as those of the 4-tert-butyl
and 4-isopropyl analogues of fipronil in the housefly (2.4 and
5.3 nM, respectively) and human â3 (1.8 and 1.9 nM,
respectively) receptors (37). On the other hand, the IC50 values
of 11 and 9 in the case of the rat receptor (732 and 643 nM,
respectively) were smaller than the reported IC50 values of
fipronil for the mouse (4300 or 1010 nM) (12, 42) or native
human brain receptors (2470 nM) (13). In our similar [3H]EBOB
assay, the IC50 values of fipronil for the housefly and rat
receptors were 2.43 and 728 nM, respectively, and thus the
selectivity ratio was 299 (Ozoe, unpublished data). Conse-
quently, 11 and 9 resulted in lower housefly versus rat GABA
receptor selectivity (81- and 65-fold, respectively) than fipronil.
Compound 13 showed the highest selectivity (185-fold) among
the phenyltriazoles tested.
dihedral angles of 45.4 ( 4.1° (n ) 4); and those with the
1-(unsubstituted phenyl) and a 4-substituent have dihedral angles
of 23.1 ( 1.4° (n ) 4). Compound 15, bearing 4-amino and
4-phenyl groups, had a dihedral angle of 66.1°. In phenyltri-
azoles lacking the o-chlorine atoms on the phenyl group and
the 5-substituent, the two rings were almost coplanar, whereas
in phenyltriazoles containing those substituents, the rings were
almost perpendicular. A group of 4-substituted 1-(2,6-dichlo-
rophenyl)-1H-1,2,3-triazoles, to which most active triazoles
belong, adopted intermediate dihedral angles. In addition to an
appropriate 4-substituent of the triazole ring, the angle created
by the two rings may also play an important role in the potency
and selectivity of 4-substituted 1-phenyl-1H-1,2,3-triazoles.
The 4-ethynyl-2,6-dichloro substitution on the benzene ring
was not effective at increasing the activity of the 4-alkyl-1-
phenyl-1H-1,2,3-triazoles (i.e., a 17-fold reduction was observed,
fly
in comparison with the IC50 of the 2,6-dichloro-4-trifluoro-
methylphenyl analogue); however, the p-ethynyl substitution
conferred high activity in the case of the 4-alkyl-1-phenylpyra-
zoles (37). The activity of 4-alkyl-1-phenyl-2,6,7-trioxabicyclo-
[2.2.2]octane-type GABA antagonists is also known to be greatly
enhanced by the introduction of an ethynyl group into the para-
position of the phenyl group (44). The 4-(4-methoxycarbonyl-
1-butynyl) substitution on the benzene ring did not increase the
activity of 4(or 5-)-n-butyl-1-phenyl-1H-1,2,3-triazoles, although
the introduction of this substituent into the 4-position of the
phenyl group resulted in achieving high potency in the 5-alkyl-
2-phenyl-1,3-dithiane-type (45, 46) and straight-chain GABA
antagonists (47). Taken together, the present findings are
expected to provide insights into the orientation of noncompeti-
tive antagonists at the binding site of GABA receptors.
Akamatsu et al. (48) postulated that the housefly and rat GABA
receptors have four major binding subsites for noncompetitive
antagonists, as based on the 3D-QSAR analysis of picrotoxinin-
type antagonists including dioxatricyclododecenes, hexachlo-
rocyclohexanes, endosulfan, bicyclophosphates, and other related
compounds. After reviewing large amounts of data focusing on
three classes of antagonists (i.e., plant-derived terpene lactone
picrodendrins, fipronil-related heterocyclic compounds, and
bicyclophosphorothionates), Ozoe and Akamatsu (18) presented
a model of the noncompetitive antagonist binding site. Accord-
ing to this model, it was proposed that various types of
antagonists, all of which share common structural features, bind
to an identical site in the GABA receptor in different or
overlapping orientations. Recently, Sammelson et al. (37) also
reached the same conclusion in a study of 4-alkyl-1-phenylpyra-
zoles and 4-alkyl-1-phenyl-2,6,7-trioxabicyclo[2.2.2]octanes. A
sterically bulky alkyl group at the 4-position, as well as
electronegative atoms in both heterocyclic rings, significantly
affected binding to the receptor. The p-ethynyl group conferred
high activity in the case of 1-phenylpyrazoles and 1-phenyl-
2,6,7-trioxabicyclo[2.2.2]octanes. Figure 9 shows the super-
imposition of three representative types of heterocyclic GABA
antagonists overlaid with the emphasis on the heterocyclic rings.
As can be seen in this figure, the para-substituent of the dithiane
points to a different direction from those of the other two
molecules. The introduction of a 4-methoxycarbonyl-1-butynyl
group, which is known to be an effective substituent in dithiane-
type antagonists, into the 4-position of the phenyl group of
1-phenyl-1H-1,2,3-triazoles probably forces deflection of the
molecules away from the binding site, thereby rendering the
molecules inactive.
The activity of the 4-n-butyl-1-phenyl-1H-1,2,3-triazoles with
a variety of one, two, or three substituents on the benzene ring
was examined to gain a better understanding of the significance
of the 2,6-dichloro-4-trifluoromethylphenyl group in achieving
high levels of activity (Table 2). Most of the 1-(4-monosub-
stituted phenyl)triazoles, including the 4-trifluoromethylphenyl
analogue (30) (or the didechloro analogue of 7), exhibited almost
no activity in the case of the housefly and rat receptors, whereas
four of these triazoles exhibited low activity in the case of the
human â3 receptor. It was of note that the 4-ethynyl, 4-bromo,
and 4-(4-methoxycarbonyl-1-butynyl) substituents on the ben-
zene ring conferred high potency in the case of other types of
GABA antagonists such as 5-phenyl-1,3-dithianes (38) and
1-phenyl-2,6,7-trioxabicyclo[2.2.2]octanes (39, 44), but not in
the case of the 1-phenyl-1H-1,2,3-triazoles. It is also worth
noting that removal of the 4-trifluoromethyl group from 7 led
to an inactive analogue (32) in the case of the housefly and rat
receptors, although the resulting analogue retained low activity
in the case of the human â3 receptor. The combination of the
4-trifluoromethyl and 2,6-dichloro substitutions was found to
be essential for attaining high levels of activity. Likewise, 2,6-
dichlorophenyl analogues with the 4-bromo and 4-chloro
substituents were potent inhibitors in the housefly and human
â3 receptors; the potency of these inhibitors decreased in the
order CF3 > Br > Cl. From analyses based on a wide variety
of 4-alkyl-1-phenylpyrazoles (37), phenyltriazoles, and related
compounds seen in patents, it can be speculated that the 2,4,6-
trisubstituted phenyl pattern plays an important role in both the
in vivo and in vitro potency of this type of GABA antagonists
and insecticides. The 5-amino group of fipronil-type pyrazoles
interacts with the 2- or 6-substituent on the phenyl group to
force the rings twist out of plane, consequently rendering the
energetically favorable, orthogonal arrangement (37). According
to our calculation, fipronil had a dihedral angle of 100.6° formed
by the two rings. Moreover, in the case of the 1-phenyl-1H-
1,2,3-triazoles, the o-chlorine atoms on the phenyl group and
the 5-substituent on the triazole ring greatly affected the dihedral
angle between the planes of the aromatic rings. Molecular orbital
calculations showed that the analogues with a 1-(2,6-dichlo-
rophenyl) group and a 5-substituent have dihedral angles of 82.9
( 7.8° (n ) 11); those with a 1-(2,6-dichlorophenyl) group and
a 4-substituent have dihedral angles of 59.4 ( 1.3° (n ) 13);
those with the 1-(unsubstituted phenyl) and a 5-substituent have
In conclusion, the issue regarding whether phenyltriazoles
and fipronil bind to an identical site in the GABA receptor still

1370 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Alam et al.
Figure 9. Superimposition of three representative heterocyclic GABA antagonists: (A) orthogonal views of the superimposed molecules; (B) structures
of the superimposed molecules.
Figure 10. Superimposition of fipronil on 9. Numbers indicate the electric charges on the atoms.
ABBREVIATIONS USED
needs to be addressed. The 4-tert-butyl group of phenyltriazole
9 and the trifluoromethylsulfinyl group of fipronil are sterically
similar, as pointed out by Sammelson et al. (37), but they differ
greatly in terms of the electron density of the substituents; the
trifluoromethylsulfinyl group has highly negative charges on
its fluorine atoms and oxygen atom, whereas the 4-tert-butyl
group has positive charges on its hydrogen atoms (Figure 10).
Furthermore, fipronil and its closely related analogues exhibited
an almost equal capacity for binding to the GABA receptor of
dieldrin-resistant houseflies bearing the Rdl mutation (an Ala
to Ser mutation at the 2′-position in TM2) to that of susceptible
houseflies, whereas 9 showed less of an ability to bind to the
former receptor than to the latter receptor (49). Recently, a
fipronil-resistant Drosophila strain was shown to have a second
point mutation in the third transmembrane domain in addition
to the Rdl mutation in TM2 (50). These observations led us to
speculate that fipronil binds to a site or sites that differ from
the site for phenyltriazoles and other antagonists, or else it binds
in a quite different orientation. More studies will be needed to
clarify this point.
CoMFA, comparative molecular field analysis; 3D-QSAR,
three-dimensional quantitative structure-activity relationship;
EBOB, 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo-
[2.2.2]octane or 4′-ethynyl-4-n-propylbicycloorthobenzoate;
GABA, γ-aminobutyric acid; IC50fly, median inhibition concen-
tration in the housefly GABA receptor; IC50rat, median inhibition
concentration in the rat GABA receptor; IC50hâ3, median
inhibition concentration in the human â3 GABA receptor; PCR,
Polymerase Chain Reaction; PHC, phenyl heterocyclic com-
pound; QSAR, quantitative structure-activity relationship; Rdl
(resistant to dieldrin), a gene encoding an insect GABA receptor
subunit; Rdl, an insect GABA receptor subunit encoded by Rdl;
TM2, the second transmembrane domain.
ACKNOWLEDGMENT
Part of this work was conducted at the Department of Molecular
and Functional Genomics, Center for Integrated Research in
Science, Shimane University.

1-Phenyl-1H-1,2,3-triazoles as GABA Receptor Antagonists
J. Agric. Food Chem., Vol. 54, No. 4, 2006 1371
(18) Ozoe, Y.; Akamatsu, M. Non-competitve GABA antagonist:
probing the mechanisms of their selectivity for insect versus
mammalian receptors. Pest Manag. Sci. 2001, 57, 923-931.
(19) Ozoe, Y.; Kajiki, R. Potency and selectivity of phenyltriazoles
in GABA receptors: effects of substituents on the inhibition of
[3H]EBOB binding. Pestic. Sci. Soc. Jpn. Annu. Meeting Abstr.
2002, 116.
Supporting Information Available: Spectroscopic data for
phenyltriazoles and X-ray crystallographic data for 9. This
material is available free of charge via the Internet at http://
pubs.acs.org.
LITERATURE CITED
(20) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
A stepwise Huisgen cycloaddition process: copper(I)-catalyzed
regioselective “ligation” of azides and terminal alkynes. Angew.
Chem., Int. Ed. 2002, 41, 2596-2599.
(1) Bormann, J. The ‘ABC’ of GABA receptors. Trends Pharmacol.
Sci. 2000, 21, 16-19.
(2) Rabow, L. E.; Russek, S. J.; Farb, D. H. From ion currents to
genomic analysis: recent advances in GABAA receptor research.
Synapse 1995, 21, 189-274.
(3) Hosie, A. M.; Aronstein, K.; Sattelle, D. B.; ffrench-Constant,
R. H. Molecular biology of insect neuronal GABA receptors.
Trends Neurosci. 1997, 20, 578-583.
(4) Mezler, M.; Mu¨ller, T.; Raming, K. Cloning and functional
expression of GABAB receptors from Drosophila. Eur. J.
Neurosci. 2001, 13, 477-486.
(5) Gant, D. B.; Chalmers, A. E.; Wolff, M. A.; Hoffman, H. B.;
Bushey, D. F. Fipronil: action at the GABA receptor. ReV.
Toxicol. 1998, 2, 147-156.
(6) Zhao, X.; Yeh, J. Z.; Salgado, V. L.; Narahashi, T. Fipronil is a
potent open channel blocker of glutamate-gated chloride channels
in cockroach neurons. J. Pharmacol. Exp. Ther. 2004, 310, 192-
201.
(7) Buckingham, S. D.; Hosie, A. M.; Roush, R. L.; Sattelle D. B.
Actions of agonists and convulsant antagonists on a Drosophila
melanogaster GABA receptor (Rdl) homo-oligomer expressed
in Xenopus oocytes. Neurosci. Lett. 1994, 181, 137-140.
(8) Hosie, A. M.; Baylis, H. A.; Buckingham, S. D.; Sattelle, D. B.
Actions of the insecticide fipronil, on dieldrin-sensitive and
-resistant GABA receptors of Drosophila melanogaster. Br. J.
Pharmacol. 1995, 115, 909-912.
(21) Deng, Y.; Palmer, C. J.; Casida, J. E. House fly head GABA-
gated chloride channel: four putative insecticide binding sites
differentiated by [3H]EBOB and [35S]TBPS. Pestic. Biochem.
Physiol. 1993, 47, 98-112.
(22) Squires, R. F.; Casida, J. E.; Richardson, M.; Saederup, E. [35S]-
t-Butylbicyclophosphorothionate binds with high affinity to brain-
specific sites coupled to γ-aminobutyric acid-A and ion recog-
nition sites. Mol. Pharmacol. 1983, 23, 326-336.
(23) Cole, L. M.; Casida, J. E. GABA-gated chloride channel: binding
site for 4′-ethynyl-4-n-[2,3-3H2]propylbicycloorthobenzoate ([3H]-
EBOB) in vertebrate brain and insect head. Pestic. Biochem.
Physiol. 1992, 44, 1-8.
(24) Bradford, M. M. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem. 1976, 72, 248-254.
(25) Alonso, G.; Garc´ıa-Lo´pez, M. T.; Garuc´ıa-Mun˜oz, G.; Madron˜-
ero, R.; Rico, M. Heterocyclic N-glycosides. VI. The reaction
of glycosyl azides with propiolic acid and methyl propiolate. J.
Heterocycl. Chem. 1970, 7, 1269-1272.
(26) Raghavendra, M. S.; Lam, Y. Regiospecific solid-phase synthesis
of substituted 1,2,3-triazoles. Tetrahedron Lett. 2004, 45, 6129-
6132.
(27) Wedegaertner, D. K.; Kattak, R. K.; Harrison, I.; Cristie, S. K.
Aryl azide-allene cycloaddition. The contrasting behavior of two
simple allenes, 1,2-cyclononadiene and 1,2-propadiene. J. Org.
Chem. 1991, 56, 4463-4467.
(28) Tornoe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on
solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed
1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org.
Chem. 2002, 67, 3057-3064.
(29) Clark, M.; Cramer, R. D., III The probability of chance
correlation using partial least squares (PLS). Quantum Struct.-
Act. Relat. 1993, 12, 137-145.
(30) Sieghart, W.; Sperk G. Subunit composition, distribution and
function of GABAA receptor subtypes. Curr. Top. Med. Chem.
2002, 2, 795-816.
(31) Slany, A.; Zezula, J.; Tretter, V.; Sieghart, W. Rat â3 subunits
expressed in human embryonic kidney 293 cells form high
affinity [35S]-t-butylbicyclophosphorothionate binding sites modu-
lated by several allosteric ligands of γ-aminobutyric acid type
A receptors. Mol. Pharmacol. 1995, 48, 385-391.
(32) Wooltorton J. R. A.; Moss, S. J.; Smart, T. G. Pharmacological
and physiological characterization of murine homomeric â3
GABAA receptors. Eur. J. Neurosci. 1997, 9, 2225-2235.
(33) Ratra, G. S.; Casida, J. E. GABA receptor subunit composition
relative to insecticide potency and selectivity. Toxicol. Lett. 2001,
122, 215-222.
(34) ffrench-Constant, R. H.; Rocheleau, T. A.; Steichen, J. C.;
Chalmers, A. E. A point mutation in a Drosophila GABA
receptor confers insecticide resistance. Nature 1993, 363, 449-
451.
(35) Perret, P.; Sarda, X.; Wolff, M.; Wu, T.-T.; Bushey, D.; Goeldner,
M. Interaction of non-competitive blockers within the γ-ami-
nobutyric acid type A chloride channel using chemically reactive
probes as chemical sensors for cysteine mutants. J. Biol. Chem.
1999, 274, 25350-25354.
(9) Bloomquist, J. R. Cyclodiene resistance at the insect GABA
receptor/chloride channel complex confers broad cross resistance
to convulsants and experimental phenylpyrazole insecticides.
Arch. Insect Biochem. Physiol. 1994, 26, 69-79.
(10) von Keyserlingk, H. C.; Willis, R. J. The GABA activated Cl-
channel in insects as target for insecticidal action: a physiological
study. In Neurotox ’91sMolecular Basis of Drug and Pesticide
Action; Duce, I. R., Ed.; Elsevier: London, U.K., 1992; pp 79-
104.
(11) Bascal, Z.; Holden-Dye, L.; Willis, R. J.; Smith, S. W. G.;
Walker, R. J. Novel azole derivatives are antagonists at the
inhibitory GABA receptor on the somatic muscle cells of the
parasitic nematode Ascaris suum. Parasitology 1996, 112, 253-
259.
(12) Cole, L. M.; Nicholson, R. A.; Casida, J. E. Action of
phenylpyrazole insecticides at the GABA-gated chloride channel.
Pestic. Biochem. Physiol. 1993, 46, 47-54.
(13) Ratra, G. S.; Kamita, S. G.; Casida, J. E. Role of human GABAA
receptor â3 subunit in insecticide toxicity. Toxicol. Appl.
Pharmacol. 2001, 172, 233-240.
(14) Pullman, D. A.; Smith, I. H.; Larkin, J. P.; Casida, J. E.
Heterocyclic insecticides acting at the GABA-gated chloride
channel: 5-alkyl-2-arylpyrimidines and -1,3-thiazines. Pestic. Sci.
1996, 46, 237-245.
(15) Whittle, A. J. 3-Aryl pyrimidinones: a novel class of potent
insecticides. In AdVances in the Chemistry of Insect Control III;
Briggs, G. G., Ed.; Royal Society of Chemistry: Cambridge,
U.K., 1994; pp 156-170.
(16) Ozoe, Y.; Yagi, K.; Nakamura, M.; Akamatsu, M.; Miyake, T.;
Matsumura, F. Fipronil-related heterocyclic compounds: struc-
ture-activity relationships for interaction with γ-aminobutyric
acid- and voltage-gated ion channels and insecticidal action.
Pestic. Biochem. Physiol. 2000, 66, 92-104.
(17) Whittle, A. J.; Fitzjohn, S.; Mullier, G.; Pearson, D. P. J.; Perrior,
T. R.; Taylor, R.; Salmon, R. The use of computer-generated
electrostatic surface maps for the design of new ‘GABA-ergic’
insecticides. Pestic. Sci. 1995, 44, 29-31.

1372 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Alam et al.
(36) Jursky, F.; Fuchs, K.; Buhr, A.; Tretter, V.; Sigel, E.; Sieghart,
W. Identification of amino acid residues of GABAA receptor
subunits contributing to the formation and affinity of the tert-
butylbicyclophosphorothionate binding site. J. Neurochem. 2000,
74, 1310-1316.
(37) Sammelson, R. E.; Caboni, P.; Durkin, K. A.; Casida, J. E.
GABA receptor antagonists and insecticides: common structural
features of 4-alkyl-1-phenylpyrazoles and 4-alkyl-1-phenyltri-
oxabicyclooctanes. Bioorg. Med. Chem. 2004, 12, 3345-3355.
(38) Palmer, C. J.; Casida, J. E. Insecticidal 1,3-dithianes and 1,3-
dithiane 1,1-dioxides. J. Agric. Food Chem. 1992, 40, 492-496.
(39) Palmer, C. J.; Cole, L. M.; Larkin, J. P.; Smith, I. H.; Casida, J.
E. 1-(4-Ethynylphenyl)-4-substituted-2,6,7-trioxabicyclo[2.2.2]-
octanes: effect of 4-substitutent on toxicity to houseflies and
mice and potency at the GABA-gated chloride channel. J. Agric.
Food Chem. 1991, 39, 1329-1334.
(40) Ju, X.-L.; Ozoe, Y. Bicyclophosphorothionate antagonists ex-
hibiting selectivity for housefly GABA receptors. Pestic. Sci.
1999, 55, 971-982.
(41) Schmidt, T. J.; Gurrath, M.; Ozoe, Y. Structure-activity
relationships of seco-prezizaane and picrotoxane/picrodendrane
terpenoids by Quasar receptor-surface modeling. Bioorg. Med.
Chem. 2004, 12, 4159-4164.
(42) Hainzl, D.; Casida, J. E. Fipronil insecticide: novel photochemi-
cal desulfinylation with retention of neurotoxicity. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 12764-12767.
(43) Caboni, P.; Sammelson, R. E.; Casida, J. E. Phenylpyrazole
insecticide photochemistry, metabolism, and GABAergic ac-
tion: ethiprole compared with fipronil. J. Agric. Food Chem.
2003, 51, 7055-7061.
(44) 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.
(45) Li, Q. X.; Casida, J. E. Structure-activity studies leading to
potent chloride channel blockers: 5e-tert-butyl-2-[4-(substituted-
ethynyl)phenyl]-1,3-dithianes. Bioorg. Med. Chem. 1994, 2,
1423-1434.
(46) Li, Q. X.; Casida, J. E. Affinity probes for the GABA-gated
chloride channel: selection of 5e-tert-butyl-2e-[4-(substituted-
ethynyl)phenyl]-1,3-dithianes and optimization of linker moiety.
Bioorg. Med. Chem. 1995, 3, 1667-1674.
(47) Hamano, H.; Nagata, K.; Fukada, N.; Shimotahira, H.; Ju, X.-
L.; Ozoe, Y. 5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pen-
tynoic acid and its derivatives inhibit ionotropic γ-aminobutyric
acid receptors by binding to the 4′-ethynyl-4-n-propylbicy-
cloorthobenzoate site. Bioorg. Med. Chem. 2000, 8, 665-
674.
(48) Akamatsu, M.; Ozoe, Y.; Ueno, T.; Fujita, T.; Mochida, K.;
Nakamura, T.; Matsumura, F. Sites of action of noncompetitive
GABA antagonists in houseflies and rats: three-dimensional
QSAR analysis. Pestic. Sci. 1997, 49, 319-332.
(49) Ozoe, Y.; Ishikawa, K.; Tomiyama, S.; Ozoe, F.; Kozaki, T.;
Scott, J. G. Antagonism of the GABA receptor of dieldrin-
resistant houseflies by fipronil and its analogues. In Synthesis
and Chemistry of Agrochemicals Series VIII; Lyga, J. W.,
Theodoritis, G., Eds.; American Chemical Society: Washington,
DC, in press.
(50) Le Goff, G.; Hamon, A.; Berge´, J.-B.; Amichot, M. Resistance
to fipronil in Drosophila simulans: influence of two point
mutations in the RDL GABA receptor subunit. J. Neurochem.
2005, 92, 1295-1305.
Received for review November 9, 2005. Revised manuscript received
December 12, 2005. Accepted December 13, 2005.
JF052773I