Competitive antagonism of insect GABA receptors by iminopyridazine derivatives of GABA

Article abstract of DOI:10.1016/j.bmc.2012.07.049

A series of 4-(6-imino-3-aryl/heteroarylpyridazin-1-yl)butanoic acids were synthesized and examined for antagonism of GABA receptors from three insect species. When tested against small brown planthopper GABA receptors, the 3,4-methylenedioxyphenyl and the 2-naphthyl analogues showed complete inhibition of GABA-induced fluorescence changes at 100 μM in assays using a membrane potential probe. Against common cutworm GABA receptors, these analogues displayed approximately 86% and complete inhibition of GABA-induced fluorescence changes at 100 μM, respectively. The 4-biphenyl and 4-phenoxyphenyl analogues showed moderate inhibition at 10 μM in these receptors, although the inhibition at 100 μM was not complete. Against American cockroach GABA receptors, the 4-biphenyl analogue exhibited the greatest inhibition (approximately 92%) of GABA-induced currents, when tested at 500 μM using a patch-clamp technique. The second most active analogue was the 2-naphthyl analogue with approximately 85% inhibition. The 3-thienyl analogue demonstrated competitive inhibition of cockroach GABA receptors. Homology modeling and ligand docking studies predicted that hydrophobic 3-substituents could interact with an accessory binding site at the orthosteric binding site.

Full text of DOI:10.1016/j.bmc.2012.07.049

Bioorganic & Medicinal Chemistry 20 (2012) 5957–5964
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Competitive antagonism of insect GABA receptors by iminopyridazine
derivatives of GABA
Mohammad Mostafizur Rahman a, Yuki Akiyoshi b, Shogo Furutani c, Kazuhiko Matsuda c, Kenjiro Furuta b,
Izumi Ikeda a,b, Yoshihisa Ozoe a,b,
⇑
a Division of Bioscience and Biotechnology, United Graduate School of Agricultural Sciences, Tottori University, Tottori 680-8553, Japan
b Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-8504, Japan
c Department of Applied Biological Chemistry, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4-(6-imino-3-aryl/heteroarylpyridazin-1-yl)butanoic acids were synthesized and examined
for antagonism of GABA receptors from three insect species. When tested against small brown planthop-
per GABA receptors, the 3,4-methylenedioxyphenyl and the 2-naphthyl analogues showed complete inhi-
Received 24 April 2012
Revised 23 July 2012
Accepted 23 July 2012
Available online 8 August 2012
bition of GABA-induced fluorescence changes at 100
lM in assays using a membrane potential probe.
Against common cutworm GABA receptors, these analogues displayed approximately 86% and complete
inhibition of GABA-induced fluorescence changes at 100
oxyphenyl analogues showed moderate inhibition at 10
l
M, respectively. The 4-biphenyl and 4-phen-
Keywords:
lM in these receptors, although the inhibition
Iminopyridazine
Competitive antagonism
GABA receptor
GABA-gated chloride channel
Insect
at 100
exhibited the greatest inhibition (approximately 92%) of GABA-induced currents, when tested at
500 M using a patch-clamp technique. The second most active analogue was the 2-naphthyl analogue
lM was not complete. Against American cockroach GABA receptors, the 4-biphenyl analogue
l
with approximately 85% inhibition. The 3-thienyl analogue demonstrated competitive inhibition of cock-
roach GABA receptors. Homology modeling and ligand docking studies predicted that hydrophobic 3-sub-
stituents could interact with an accessory binding site at the orthosteric binding site.
Ó 2012 Elsevier Ltd. All rights reserved.
Insecticide
1. Introduction
ride ions, thereby suppressing the generation of action potentials.
GABAA receptors are also targets for drugs such as benzodiaze-
c
-Aminobutyric acid (GABA) is the major inhibitory neurotrans-
pines, barbiturates, and anesthetics.5 In addition to agonists and
drugs, two known types of antagonists exist for the Cys-loop recep-
tor: competitive antagonists, which bind to the same site as ago-
nists do, and noncompetitive antagonists, which bind to an
allosteric binding site in the channel domain.5,6 Both types of
antagonists stabilize the closed conformation of the channels and
block membrane conductance.
GABA receptors are widely distributed in both insect and mam-
malian nerve tissues. Insect GABA receptors exist not only in the
central nervous system but also in the peripheral nervous system.7
Inhibitory GABA receptor genes have been cloned from several in-
sect species to date. In Drosophila, molecular cloning and functional
expression of excitatory and metabotropic types of receptors have
also been reported.8–10 Insect inhibitory GABA receptors can be ex-
pressed as homo-pentamers, the subunits of which are encoded by
the single gene rdl, but four subunit variants are generated by
alternative splicing of the exons 3 and 6 of rdl.11 Homo-oligomeric
RDL receptors share distinctive characteristics with native GABA
receptors. Although insect GABA receptors are structurally similar
to mammalian GABA receptors, these two receptors have different
pharmacological characteristics, allowing GABA receptors to serve
mitter in the nervous system of animals. GABA released into the
synapse functions by binding to two types of membrane proteins:
ionotropic and metabotropic receptors.1,2 The ionotropic GABA
receptor belongs to the Cys-loop receptor family and mediates fast
synaptic inhibition.1,3 The receptors of this family are ligand-gated
ion channels that are formed by five subunits. There are two types
of ionotropic receptors in mammals: hetero-pentameric GABAA
receptors, which are composed of
subunits, and homo-pentameric GABAC receptors, which comprise
1-3 subunits.4 GABAA receptors with an
1-b2- 2 combination
are the most abundant subtype in the brain.4 When GABA or an
agonist binds to the orthosteric site in the -b subunit interface
a1-6, b1-3, c1-3, d, e, h, and p
q
a
c
a
of the extracellular domain of GABAA receptors, the integral chan-
nel rapidly opens to increase the membrane conductance of chlo-
Abbreviations: AC, American cockroach; CC, common cutworm; FLIPR, fluoro-
metric imaging plate reader; FMP, FLIPR membrane potential; GABA,
tyric acid; SBP, small brown planthopper.
c-aminobu-
⇑
Corresponding author. Tel./fax: +81 852 32 6573.
E-mail address: ozoe-y@life.shimane-u.ac.jp (Y. Ozoe).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.049

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M. M. Rahman et al. / Bioorg. Med. Chem. 20 (2012) 5957–5964
receptors from three insect species, we seek to find species differ-
ences in the receptors to utilize the information for future discov-
ery and the development of chemicals for insect pest control.
2. Results and discussion
2.1. Chemistry
In this study, we synthesized a series of iminopyridazine deriv-
atives of GABA, which consisted of 4-(6-iminopyridazin-1-yl)buta-
noic acids, by modifying the 3-position of the pyridazine ring
(Scheme 1). Wermuth et al. first described a seven-step synthetic
approach including pyridazinone intermediates for 4b, 4c (gab-
azine, SR 95531), 4k, and 4l.19 The synthesis of 4d was reported
by Melikian et al.,20 which also required seven synthetic steps. Re-
cently, Gavande et al. described a four-step synthesis procedure for
4c under microwave irradiation conditions starting with 3,6-
dichloropyridazine.21 Here, we have demonstrated an efficient
three-step synthesis of eleven 3-aryl/heteroaryl analogues starting
with 3-amino-6-chloropyridazine and a two-step synthesis of a 3-
unsubstituted analogue from 3-aminopyridazine (Scheme 1). The
first intermediates of nine analogues (1b–1i, 1l) were synthesized
in 19–78% yield using the Suzuki-Miyaura cross-coupling reaction
where aryl/heteroarylboronic acid was coupled with 3-amino-6-
chloropyridazine in presence of a palladium catalyst and a base
such as sodium carbonate. The first intermediates of two analogues
(1j, 1k) were synthesized in 73% and 75% yield, respectively, using
the Stille cross-coupling reaction between tributyl(heteroaryl)tin
and 3-amino-6-chloropyridazine in the presence of a palladium
catalyst. The N(2)-alkylated compounds 2a–2l in the second step
were synthesized in 11–74% yield by the reactions between 1a–
1l and ethyl 4-bromobutanoate. Free base esters of the alkylated
compounds 3a–3l were prepared in 77–93% yield from 2a–2l using
Figure 1. Structures of bicuculline and gabazine (SR 95531). Bold lines show the
GABA structural units.
as important targets for insecticides and parasiticides.11,12 For
example, the noncompetitive antagonist fipronil is used as an
insecticide.13 In contrast, no competitive antagonist has been
exploited in this respect. Although bicuculline and gabazine (SR
95531) (Fig. 1) are well known as competitive antagonists for
mammalian GABAA receptors,14,15 no potent competitive antago-
nist for insect GABA receptors is known. While bicuculline is inac-
tive for insect GABA receptors, gabazine was found to have
moderate antagonist activity against insect GABA receptors.16–18
In this study, we examined whether the antagonist activity of
gabazine against insect GABA receptors is increased by changing
the 3-substituent on the pyridazine ring. Here, we used GABA
receptors cloned from two insect species, small brown planthop-
pers (SBPs, Laodelphax striatella [striatellus] (Fallén)) and common
cutworms (CCs, Spodoptera litura (Fabricius)), which cause serious
damage on crops, and native GABA receptors expressed in the
abdominal ganglion neurons of American cockroaches (ACs, Peri-
planeta americana (Linnaeus)), a noxious insect species. With GABA
Scheme 1. Synthesis of 4-(6-iminopyridazin-1-yl)butanoic acid hydrochlorides.

M. M. Rahman et al. / Bioorg. Med. Chem. 20 (2012) 5957–5964
5959
K2CO3. Hydrolysis of the free base esters was carried out in acetic
acid and hydrochloric acid at 100 °C in 63–97% yield to give the tar-
get compounds 4a–4l.
3-substituent with 2-furyl and 2-thienyl groups to yield 4j and
4k nearly eliminated the antagonist activity. Substitution with a
3-thienyl group produced an analogue (4l) with moderate activity
in both receptors.
2.2. Antagonism of SBP and CC GABA receptors
Finally, we examined whether competitive GABA receptor
antagonism leads to insecticidal activity against SBP and CC larvae.
While showing significant antagonism against the GABA receptors
of these insects, the 4-biphenyl analogue (4f) did not exert insecti-
cidal effects (data not shown), indicating that higher in vitro po-
tency is necessary for insecticidal activity.
The application of GABA to membrane potential probe-loaded
cells expressing GABA receptors increases fluorescence, which is
detected by fluorometric imaging plate reader (FLIPR) membrane
potential (FMP) assays.22,23 Antagonist activity can be assessed
from a fluorescence reduction induced by a co-applied compound.
When tested at 100
lM, an analogue with no substituent at the 3-
2.3. Antagonism of AC GABA receptors
position of the pyridazine ring (4a) showed no or little antagonism
against SBP and CC GABA receptors (Figs. 2 and 3). The introduction
of a phenyl group into the 3-position of 4a led to 4b with weak
activity. The inclusion of a methoxy group at the 4-position of
the phenyl group of 4b to give gabazine (4c) resulted in a 2.7- and
3.3-fold increase in the inhibition of GABA-induced fluorescence
increases in SBP and CC receptors, respectively. The replacement
of the 4-methoxy group of 4c with a 3,4-methylenedioxy group,
yielding 4d, led to complete inhibition and 85.8% inhibition in
SBP and CC GABA receptors, respectively. The antagonist activity
of the 4-trifluoromethylphenyl analogue (4e) was comparable to
that of the 4-methoxyphenyl analogue (4c) in SBP receptors but
4e showed lower activity than 4c in CC receptors. The 4-biphenyl
analogue (4f) did not exceed 4c in terms of antagonist activity at
The antagonism of native AC GABA receptors by the synthesized
analogues was measured using the whole-cell patch-clamp tech-
nique. Figure 4A shows that GABA-induced currents are inhibited
by the 4-biphenyl analogue (4f). After confirmation of the constant
amplitude of GABA-induced currents, the neurons were perfused
with an external solution containing 500 lM 4f. During the perfu-
sion, GABA was repeatedly applied five times. This analogue atten-
uated the currents progressively, and a maximum inhibition of
92.0% was attained after the third application. The other analogues
were also tested in this fashion.
The phenyl (4b) and 4-methoxyphenyl (4c; gabazine) analogues
were not effective against the AC GABA receptor when tested at
500
lM (Fig. 4B). This finding is in agreement with a previous find-
100
of 4c in CC receptors, but it is notable that 4f showed a moderate
activity even at 10 M in both receptors. The 4-phenoxyphenyl
analogue (4g) was inferior to the 4-biphenyl analogue (4f) in SBP
receptors whereas they are comparable in CC receptors. While
the 1-naphthyl analogue (4h) was only moderately active or inac-
l
M in SBP receptors and showed inhibition comparable to that
ing with 4c tested at 10 l
M by a patch-clamp technique using AC
brain neurons.24 The analogue lacking a 3-substituent (4a) demon-
strated greater inhibition rather than 4c. The low activity of 4c is
unexpected when compared with the 3,4-methylenedioxyphenyl
(4d) and 4-trifluoromethylphenyl (4e) analogues, which showed
moderate activity. The replacement of the 3-substituent with a
3,4-methylenedioxyphenyl group to give 4d resulted in a 4-fold in-
crease in inhibition when compared with 4c. An analogue with a 4-
trifluoromethylphenyl group (4e) showed 3-fold greater inhibition
than 4c. Among the synthesized compounds, the 4-biphenyl ana-
logue (4f) showed the greatest activity, with 92.0% inhibition, an
approximately 10-fold increase compared to 4c. The 4-phenoxy-
phenyl analogue (4g) showed approximately 6-fold enhanced inhi-
bition when compared with 4c, but decreased inhibition compared
with 4f. The introduction of a 1-naphthyl group to yield 4h re-
sulted in a drop in inhibition. In contrast, the 2-naphthyl analogue
l
tive at 100
(4i) completely inhibited GABA-induced responses at 100
both receptors and showed approximately 41% and 29% inhibition
at 10 M in SBP and CC receptors, respectively. Thus, the 2-naph-
lM in SBP and CC receptors, the 2-naphthyl analogue
l
M in
l
thyl substitution is preferable to the 1-naphthyl substitution; the
naphthyl ring of 4h may provide a significant amount of steric hin-
drance for binding to the site. The 2-naphthyl analogue (4i) per se
showed agonist-like activity (i.e., caused an increase in fluores-
cence) at 100
lM, and co-application of GABA failed to induce an
increase in fluorescence (data not shown). The substitution of the
Figure 2. Inhibition of GABA-induced membrane potential changes in SBP GABA receptors. GABA at a concentration corresponding to the EC50 (1.0
lM) was used. Data
represent the means of two experiments with bars of the inhibition range, except for the data for 10 and 100
four experiments.
lM 4f, which are shown as the means standard deviations of

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M. M. Rahman et al. / Bioorg. Med. Chem. 20 (2012) 5957–5964
Figure 3. Inhibition of GABA-induced membrane potential changes in CC GABA receptors. GABA at a concentration corresponding to the EC50 (2.5
l
M) was used. Data
represent the means of two experiments with bars of the inhibition range, except for the data for 10 and 100
four experiments.
lM 4f, which are shown as the means standard deviations of
Figure 4. Inhibition of GABA-induced currents in AC GABA receptors. (A) Application protocol represented by current traces of inhibition by 500
lM 4f. (B) Inhibition by
gabazine and its analogues (500
experiments.
lM). GABA at a concentration corresponding to the EC50 (30 lM) was used. Data are the means standard deviations of three or four
(4i) displayed 9-fold greater inhibition than 4c. The high activity of
4f and 4i suggests that long aromatic substituents at the 3-position
of the pyridazine ring are tolerated. The replacement of the 4-
methoxyphenyl group of 4c with 5-membered heteroaromatic

M. M. Rahman et al. / Bioorg. Med. Chem. 20 (2012) 5957–5964
5961
Figure 5. Action of 4l on AC GABA receptors. The receptors were activated by 30
lM GABA. (A) Concentration-dependent inhibition of GABA-induced currents by 4l. (B) GABA
concentration–response curves in the presence (filled circles) and absence (open circles) of 326
l
M 4l. Imax (maximal magnitude of inward currents) = ꢀ569 30 pA,
EC50 = 49.2 10.0
l
M in the absence of 4l; Imax = -557 6 pA, EC50 = 496 64
l
M in the presence of 4l. Data represent the mean standard deviation of three experiments.
substituents to give 4j, 4k, and 4l increased the inhibition percent-
age by 3- to 6-fold. The 3-thienyl analogue (4l) showed a relatively
high activity among analogues with a heteroaromatic 3-substitu-
ent, indicating that the lone-pair electrons on the heteroatom of
the analogue at this position may provide some favorable electro-
negative effects toward receptor interaction. Overall, the inhibition
pattern of AC GABA receptors by the analogues differs from that of
SBP and CC GABA receptors, suggesting the existence of structural
differences in the binding site between insect species.
In the docking of GABA into the orthosteric site, which is formed
by six loops designated A–F,27 the side chain of Glu202 and the
backbone carbonyl oxygen atom of Ser203 in loop B were predicted
to function as hydrogen acceptors for the protonated amino group
of GABA (Fig. 6A). Arg109 in loop D and Ser174 in loop E serve as
hydrogen donors for the carboxylate anion of GABA. Phe204 in loop
B and Tyr252 in loop C likely exist near the protonated amino
group of GABA (Fig. 6A). These aromatic amino acids may have cat-
ion/
p interactions with it, as was recently suggested for the Dro-
sophila GABA receptor.28 Amino acids at the equivalent positions
2.4. Mode of antagonism
are implicated in GABA binding in the GABAA receptor.29
In the docking of the 4-biphenyl analogue (4f) into the orthos-
teric site, amino acid residues similar to those presumed to be in-
volved in GABA binding were predicted to interact with the
carboxyl group and the imino group (Fig. 6B). Hydrogen bonding
of the dissociated carboxyl and protonated imino groups of gab-
azine analogues with neighboring amino acid residues (Arg109,
Ser174, Ser203, and Phe204) appeared to play a major role in the
binding of gabazine analogues (Fig. 6B). The backbone carbonyl
group of Phe204 in loop B was predicted to function as an acceptor
for the imino hydrogen atom in place of Glu202, which interacts
To determine whether the synthesized compounds act as com-
petitive antagonists, we examined the GABA concentration–re-
sponse relationships in the presence and absence of a selected
analogue, 4l, in AC neurons. The IC50 value of 4l was determined
to be 346 18 (SD)
lM (n = 3) from its concentration–response
relationships (Fig. 5A). The GABA concentration–response curve
in the presence of 4l showed a parallel rightward shift relative to
that in the absence of 4l (Fig. 5B), indicating that 4l is a competitive
GABA receptor antagonist. Compound 4l was also examined for
antagonism of inhibitory glutamate receptors in AC neurons. This
analogue failed to inhibit glutamate-induced currents when tested
at 1 and 10 mM (data not shown).
with GABA. The amino acid of the rat GABAA receptor
a1 subunit
that is equivalent to Arg109 has been implicated in interacting
with gabazine.30 However, the location of bound gabazine seems
to differ from that in our predicted binding pocket.
2.5. Homology modeling and ligand docking
The docking studies of 4f predict that its large 3-substituent is
tolerable in the potential orthosteric binding site. Our model shows
that hydrophobic interactions predominate in the area that accom-
modates the 3-substituents of the analogues. The phenyl group at
one end of the 4f molecule is likely exposed outside the receptor.
Tyr88, Leu90, Tyr107, Val146, Val224, Arg254, Ile245, and
Leu247 are predicted to contribute to encompassing the 3-substi-
tuent of gabazine analogues. The aromatic 3-substituents may
The orthosteric binding site in Cys-loop receptors is located at
the extracellular interface between the principal and complemen-
tary faces of adjacent subunits.25 Loops A–C from the principal face
and loops D–F from the complementary face jointly form the GABA
binding site. To examine the mechanisms of the interaction of gab-
azine analogues with insect GABA receptors, we performed ligand
docking studies using a homology model. The zwitter ion forms of
GABA and the 4-biphenyl analogue (4f) were docked into a homol-
ogy model of the housefly GABA receptor constructed using the X-
ray crystal structure of the Caenorhabditis elegans glutamate-gated
chloride channel as a template.26 We used the housefly GABA
receptor for homology modeling in the present study, because
the full-length gene encoding the AC GABA receptor has not been
cloned and the amino acid sequence of the housefly GABA receptor
(GenBank accession AB177547) has a high shared identity (71.1%)
with a partial sequence of the AC GABA receptor (GenBank acces-
sion FJ612451).
form CH/p interactions with Val146 in loop A and Leu247 in loop
C. In the rat GABAA and GABAC receptors, amino acid residues
equivalent to Tyr107 and Phe111 in loop D and Phe144 in loop A
were reported to contribute to gabazine binding.31,32 Tyr107 and
Phe144 are found near the 4f molecule in our docking model, but
Phe111 is away from the binding pocket. It is of interest to note
that in the rat GABA receptors, gabazine sensitivity depended on
whether amino acids at the 107- and 144-positions are phenylala-
nine or tyrosine and that the three insect GABA receptors used in
the present study contain the same amino acids at the 107- and
144-positions as the GABAC receptor, which has low affinity for

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M. M. Rahman et al. / Bioorg. Med. Chem. 20 (2012) 5957–5964
Figure 6. Homology modeling and docking simulation. (A) Docking of GABA into the orthosteric site of a housefly GABA receptor homology model. (B) Docking of 4f.
gabazine. In addition to Phe144, Val146, a nearby amino acid in
loop A, faces the 4f molecule, which is consistent with the finding
that a homologous amino acid lines the GABA binding pocket of the
rat receptor.33 In the model, Val180 in loop F exists at the position
where the apex phenyl group of 4f interacts. This finding is consis-
tent with the finding that this region of loop F in the rat GABAA
receptor was identified as a region lining the GABA binding site.34
In this study, we synthesized gabazine analogues that are more
potent than gabazine against insect GABA receptors, but their po-
tency levels are still low compared with the levels reported with
GABAA receptors.30 Analogues with nanomolar affinity for GABAA
receptors and an analogue with low micromolar affinity for GABAC
receptors have recently been reported.35,36 Information from li-
gand docking studies with a homology model using the C. elegans
glutamate-gated chloride channel X-ray structure as a template
proved to be useful for studying the receptor-ligand interaction.
Analogues with higher potencies for insect GABA receptors may
be obtained by further modification of gabazine.
ture. In both cases, the reaction mixture was then heated under re-
flux with stirring under an argon atmosphere until completion of
the reaction. After cooling, the reaction mixture was evaporated
under reduced pressure to dryness. EtOAc (80 mL) was added to
the residue, and the flask containing the suspension was placed
in an ultrasonic bath for 5 min. The mixture was filtered, and the
filter paper was washed thoroughly with EtOAc (200 mL). The fil-
trate was evaporated under reduced pressure to dryness. The res-
idue was purified by silica gel column chromatography to yield
3-amino-6-aryl/heteroarylpyridazine (1b–1l).
3.3. General procedure for the synthesis of ethyl 4-(6-
iminopyridazin-1-yl)butanoates (3a–3l)
A
mixture of 3-amino-6-aryl/heteroarylpyridazine (1b–1l)
(1 mmol), ethyl 4-bromobutanoate (292 mg, 1.5 mmol), and N,N-
dimethylformamide (0.5 mL) was heated at 80 °C for 5 h. After
cooling, the precipitate was collected and recrystallized from
methanol and diethyl ether to give ethyl 4-(6-imino-3-aryl/hetero-
arylpyridazin-1-yl)butanoate hydrobromides (2b–2l). The hydro-
bromide (200 mg) was dissolved in a minimal amount of water.
A K2CO3 solution was used to make the solution alkaline, and then
the solution was extracted with a 1:1 mixture of EtOAc and Et2O.
The organic layer was dried with anhydrous Na2SO4 and was con-
centrated under reduced pressure to give ethyl 4-(6-imino-3-aryl/
heteroarylpyridazin-1-yl)butanoates (3b–3l). Ethyl 4-(6-iminopy-
ridazin-1-yl)butanoate (3a) was similarly prepared from 3-amino-
pyridazine (1a).
3. Experimental
3.1. General methods
The melting points of synthesized compounds were determined
using a Yanagimoto MP-500D apparatus and are uncorrected. 1H
NMR spectra were recorded on a JEOL JNM-A 400 spectrometer.
The chemical shifts (d values) are given in ppm relative to tetra-
methylsilane, and the J values are given in Hertz. The spin multi-
plicities are expressed as follows: s (singlet), d (doublet), t
(triplet), qn (quintet), and m (multiplet). High resolution mass
spectra were obtained with a Waters Synapt G2 spectrometer. Re-
agents were purchased from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan) and Tokyo Chemical Industry Co., Ltd. (Tokyo, Ja-
pan), unless otherwise noted.
3.4. General procedure for the synthesis of 4-(6-iminopyridazin-
1-yl)butanoic acid hydrochlorides (4a–4l)
A solution of ethyl 4-(6-imino-3-aryl/heteroarylpyridazin-1-
yl)butanoate (3b–3l) (0.5 mmol) in glacial acetic acid (10 mL)
and concentrated HCl (3 mL) was heated at 100 °C for approxi-
mately 12 h. After cooling, the reaction mixture was evaporated
to dryness under reduced pressure. The residue was recrystallized
with AcOH and EtOAc to afford 4-(6-imino-3-aryl/heteroarylpyri-
dazin-1-yl)butanoic acid hydrochlorides (4b–4l). 4-(6-Iminopyri-
dazin-1-yl)butanoic acid hydrochloride (4a) was similarly
obtained from ethyl 4-(6-iminopyridazin-1-yl)butanoate (3a).
3.2. General procedure for the synthesis of 3-amino-6-aryl/
heteroarylpyridazines (1b–1l)
In the case of 1b–1i and 1l, a mixture of 3-amino-6-chloropyrid-
azine (388 mg, 3.0 mmol), aryl/heteroarylboronic acid (4.5 mmol),
tetrakis(triphenylphosphine)palladium(0) (105 mg), and
a 2 M
Na2CO3 solution (3.3 mL) in toluene (20 mL) was stirred under an
argon atmosphere for 30 min at room temperature. In the case of
1j and 1k, a mixture of 3-amino-6-chloropyridazine (388 mg,
3.0 mmol), tributyl(heteroaryl)tin (4.1 mmol), and bis(triphenyl-
phosphine)palladium(II) dichloride (198 mg) in THF (10 mL) was
stirred under an argon atmosphere for 30 min at room tempera-
3.4.1. 4-(6-Iminopyridazin-1-yl)butanoic acid hydrochloride
(4a)
Yield 8% (from 1a), mp 144–145 °C. 1H NMR (CD3OD) d 2.15 (tt,
2H, J = 7.3, 6.8 Hz, –CO–CH2–CH2-CH2–), 2.50 (t, 2H, J = 6.8 Hz, –
CO–CH2–CH2–CH2–), 4.37 (t, 2H, J = 7.3 Hz, –CO–CH2–CH2–CH2–),

M. M. Rahman et al. / Bioorg. Med. Chem. 20 (2012) 5957–5964
5963
7.55 (dd, 1H, J = 9.4, 1.5 Hz, H-3), 7.73 (dd, 1H, J = 9.4, 4.2 Hz, H-4),
3.4.8. 4-[6-Imino-3-(1-naphthyl)pyridazin-1-yl]butanoic acid
hydrochloride (4h)
8.39 (dd, 1H, J = 4.2, 1.5 Hz, H-5). HRMS Calcd for C8H12N3O2
[M+ꢀCl] 182.0930. Found 182.0919.
Yield 23% (for three steps), mp 219–221 °C. 1H NMR (CD3OD) d
2.25 (tt, 2H, J = 7.3, 6.8 Hz, –CO–CH2–CH2–CH2–), 2.57 (t, 2H,
J = 6.8 Hz, –CO–CH2–CH2–CH2–), 4.47 (t, 2H, J = 7.3 Hz, –CO–CH2–
CH2–CH2–), 7.56–7.64 (m, 3H, H-5, H-40, H-50), 7.67–7.70 (m, 2H,
H-30, H-60), 8.00–8.08 (m, 4H, H-4, H-20, H-70, H-80). HRMS Calcd
for C18H18N3O2 [M+ꢀCl] 308.1399. Found 308.1405.
3.4.2. 4-(6-Imino-3-phenylpyridazin-1-yl)butanoic acid
hydrochloride (4b)
Yield 11% (for three steps), mp 265–267 °C (dec). 1H NMR
(CD3OD) d 2.23 (tt, 2H, J = 7.3, 6.8 Hz, –CO–CH2–CH2–CH2–), 2.56
(t, 2H, J = 6.8 Hz, –CO–CH2–CH2–CH2–), 4.46 (t, 2H, J = 7.3 Hz, –
CO–CH2–CH2–CH2–), 7.53–7.56 (m, 3H, H-30, H-40), 7.64 (d, 1H,
J = 9.6 Hz, H-5), 7.98–8.00 (m, 2H, H-20), 8.32 (d, 1H, J = 9.6 Hz, H-
3.4.9. 4-[6-Imino-3-(2-naphthyl)pyridazin-1-yl]butanoic acid
hydrochloride (4i)
Yield 5% (for three steps), mp 225–227 °C. 1H NMR (CD3OD) d
2.26 (qn, 2H, J = 6.8 Hz, –CO–CH2–CH2–CH2–), 2.59 (t, 2H,
J = 6.8 Hz, –CO–CH2–CH2–CH2–), 4.49 (t, 2H, J = 6.8 Hz, –CO–CH2–
CH2–CH2–), 7.57–7.62 (m, 2H, H-60, H-70), 7.66 (d, 1H, J = 9.8 Hz,
H-5), 7.93–7.95 (m, 1H, H-30), 8.02 (d, 2H, J = 8.8 Hz, H-50, H-80),
8.12 (dd, 1H, J = 8.8, 2.0 Hz, H-40), 8.50 (d, 2H, J = 9.8 Hz, H-4, H-
10). HRMS Calcd for C18H18N3O2 [M+ꢀCl] 308.1399. Found 308.1405.
4). HRMS Calcd for
258.1235.
C
14H16N3O2 [M+ꢀCl] 258.1243. Found
3.4.3. 4-[6-Imino-3-(4-methoxyphenyl)pyridazin-1-yl]butanoic
acid hydrochloride (4c) (Gabazine)
Yield 18% (for three steps), mp 199–201 °C; 1H NMR (CD3OD) d
2.22 (tt, 2H, J = 7.1, 6.9 Hz, –CO–CH2–CH2–CH2–), 2.55 (t, 2H,
J = 6.9 Hz, –CO–CH2–CH2–CH2–), 3.87 (s, 3H, –O–CH3), 4.43 (t, 2H,
J = 7.1 Hz, –CO–CH2–CH2–CH2–), 7.07 (dd, 2H, J = 6.9, 2.0 Hz, H-
30), 7.59 (d, 1H, J = 9.6 Hz, H-5), 7.95 (dd, 2H, J = 6.9, 2.0 Hz, H-20),
8.28 (d, 1H, J = 9.6 Hz, H-4). HRMS Calcd for C15H18N3O3 [M+ꢀCl]
288.1348. Found 288.1329.
3.4.10. 4-[3-(2-Furyl)-6-iminopyridazin-1-yl]butanoic acid
hydrochloride (4j)
Yield 34% (for three steps), mp 230–232 °C (dec). 1H NMR
(CD3OD) d 2.19 (tt, 2H, J = 7.1, 6.8 Hz, –CO–CH2–CH2–CH2–), 2.54
(t, 2H, J = 6.8 Hz, –CO–CH2–CH2–CH2–), 4.39 (t, 2H, J = 7.1 Hz, –
CO–CH2–CH2–CH2–), 6.68 (dd, 1H, J = 3.8, 1.8 Hz, H-40), 7.24 (dd,
1H, J = 3.8, 0.8 Hz, H-50), 7.59 (d, 1H, J = 9.6 Hz, H-5), 7.79 (dd, 1H,
J = 1.8, 0.8 Hz, H-30), 8.15 (d, 1H, J = 9.6 Hz, H-4). HRMS Calcd for
3.4.4. 4-[6-Imino-3-(3,4-methylenedioxyphenyl)pyridazin-1-
yl]butanoic acid hydrochloride (4d)
C
12H14N3O3 [M+ꢀCl] 248.1035. Found 248.1041.
Yield 19% (for three steps), mp 214–215 °C. 1H NMR (CD3OD) d
2.21 (tt, 2H, J = 7.3 Hz, 6.8 Hz, –CO–CH2–CH2–CH2–), 2.55 (t, 2H,
J = 6.8 Hz, –CO–CH2–CH2–CH2–), 4.42 (t, 2H, J = 7.3 Hz, –CO–CH2–
CH2–CH2–), 6.06 (s, 2H, –O–CH2–O–), 6.97 (m, 1H, H-60), 7.51–
7.53 (m, 2H, H-20, H-50), 7.57 (d, 1H, J = 9.8 Hz, H-5), 8.25 (d, 1H,
J = 9.8 Hz, H-4). HRMS Calcd for C15H16N3O4 [M+ꢀCl] 302.1141.
Found 302.1142.
3.4.11. 4-[6-Imino-3-(2-thienyl)pyridazin-1-yl]butanoic acid
hydrochloride (4k)
Yield 23% (for three steps), mp 225–227 °C. 1H NMR (CD3OD) d
2.20 (tt, 2H, J = 7.1, 6.8 Hz, –CO–CH2–CH2–CH2–), 2.55 (t, 2H,
J = 6.8 Hz, –CO–CH2–CH2–CH2–), 4.37 (t, 2H, J = 7.1 Hz, –CO–CH2–
CH2–CH2–), 7.20 (dd, 1H, J = 5.2, 3.7 Hz, H-40), 7.57 (d, 1H,
J = 9.6 Hz, H-5), 7.68 (dd, 1H, J = 5.2, 1.3 Hz, H-50), 7.81 (dd, 1H,
J = 3.7, 1.3 Hz, H-30), 8.27 (d, 1H, J = 9.6 Hz, H-4). HRMS Calcd for
3.4.5. 4-[6-Imino-3-(4-trifluoromethylphenyl)pyridazin-1-
yl]butanoic acid hydrochloride (4e)
C
12H14N3O2S [M+ꢀCl] 264.0807. Found 264.0802.
Yield 17% (for three steps), mp 257–260 °C (dec). 1H NMR
(CD3OD) d 2.25 (tt, 2H, J = 7.1, 6.9 Hz, –CO–CH2–CH2–CH2–), 2.57
(t, 2H, J = 6.9 Hz, –CO–CH2–CH2–CH2–), 4.49 (t, 2H, J = 7.1 Hz, –
CO–CH2–CH2–CH2–), 7.68 (d, 1H, J = 9.7 Hz, H-5), 7.86 (dd, 2H,
J = 8.8, 0.6 Hz, H-20), 8.19 (dd, 2H, J = 8.8, 0.6 Hz, H-30), 8.38 (d,
3.4.12. 4-[6-Imino-3-(3-thienyl)pyridazin-1-yl]butanoic acid
hydrochloride (4l)
Yield 21% (for three steps), mp 215–217 °C. 1H NMR (CD3OD) d
2.21 (tt, 2H, J = 7.1, 6.8 Hz, –CO–CH2–CH2–CH2–), 2.54 (t, 2H,
J = 6.8 Hz, –CO–CH2–CH2–CH2–), 4.41 (t, 2H, J = 7.1 Hz, –CO–CH2–
CH2–CH2–), 7.57–7.61 (m, 2H, H-5, H-50), 7.70 (dd, 1H, J = 5.2,
1.5 Hz, H-40), 8.19–8.20 (m, 1H, H-20), 8.27 (d, 1H, J = 9.3 Hz, H-4).
HRMS Calcd for C12H14N3O2S [M+ꢀCl] 264.0807. Found 264.0802.
1H, J = 9.7 Hz, H-4). HRMS Calcd for
326.1116. Found 326.1102.
C
15H15F3N3O2 [M+ꢀCl]
3.4.6. 4-[3-(4-Biphenyl)-6-iminopyridazin-1-yl]butanoic acid
hydrochloride (4f)
Yield 21% (for three steps), mp 234–236 °C. 1H NMR (CD3OD) d
2.24 (qn, 2H, J = 6.8 Hz, –CO–CH2–CH2–CH2–), 2.57 (t, 2H,
J = 6.8 Hz, –CO–CH2–CH2–CH2–), 4.47 (t, 2H, J = 6.8 Hz, –CO–CH2–
CH2–CH2–), 7.39 (t, 1H, J = 6.8 Hz, H-400), 7.47–7.50 (m, 2H, H-300),
7.64 (d, 1H, J = 9.6 Hz, H-5), 7.68–7.71 (m, 2H, H-200), 7.81 (dd,
2H, J = 6.8, 2.0 Hz, H-20), 8.08 (dd, 2H, J = 6.8, 2.0 Hz, H-30), 8.37
(d, 1H, J = 9.6 Hz, H-4). HRMS Calcd for C20H20N3O2 [M+ꢀCl]
334.1556. Found 334.1537.
3.5. FMP assays
A Drosophila S2 cell line expressing GABA receptors from SBPs
(Laodelphax striatella [striatellus] (Fallén)) (GenBank accession
AB253526.1) or CCs (Spodoptera litura (Fabricius)) (GenBank
DD171257.1) was used in this assay. These stable cell lines were
generated as previously reported.18 The cells were washed and dis-
persed in a saline buffer (120 mM NaCl, 5 mM KCl, 2 mM CaCl2,
8 mM MgCl2, 10 mM HEPES, and 32 mM sucrose, adjusted to pH
3.4.7. 4-[6-Imino-3-(4-phenoxyphenyl)pyridazin-1-yl]butanoic
acid hydrochloride (4g)
7.2 with an NaOH solution), and aliquots (100 lL each) of this cell
suspension (5 ꢁ 105 cells) were added to 96-well microplates for
the fluorescent assay. After 10 min, the cells were spun down at
1400 rpm for 5 min and loaded with the FMP blue dye (Molecular
Yield 50% (for three steps), mp 208–210 °C. 1H NMR (CD3OD) d
2.22 (tt, 2H, J = 7.3, 6.8 Hz, –CO–CH2–CH2–CH2–), 2.55 (t, 2H,
J = 6.8 Hz, –CO–CH2–CH2–CH2–), 4.44 (t, 2H, J = 7.3 Hz, –CO–CH2–
CH2–CH2–), 7.06–7.10 (m, 4H, H-200, H-300), 7.18–7.22 (m, 1H, H-
400), 7.39–7.44 (m, 2H, H-20), 7.61 (d, 1H, J = 9.6 Hz, H-5), 7.98–
8.00 (m, 2H, H-30), 8.30 (d, 1H, J = 9.6 Hz, H-4). HRMS Calcd for
Devices; 100 lL) at room temperature for 20 min. Gabazine and
gabazine analogues were first dissolved in DMSO and diluted with
a saline buffer. Gabazine or a gabazine analogue in a saline buffer
(25
lL) containing 1% DMSO was added to the cells in each well
C
20H20N3O3 [M+ꢀCl] 350.1505. Found 350.1479.
and incubated with for 74 s. Subsequently, GABA in a saline buffer

5964
M. M. Rahman et al. / Bioorg. Med. Chem. 20 (2012) 5957–5964
(25
lL) was added to each well. GABA concentrations correspond-
forms of GABA and 4f used in the docking studies were created
ing to the EC50s for SBP and CC receptors (1.0 and 2.5
l
M, respec-
using the Molecule Builder of MOE. Compound 4f was docked as
a protonated imino form, although it exists as resonance forms.37
The created ligands were docked into the potential binding site of
the generated model using ASEDock 2011.01.27 software (Chemical
Computing Group). The energy of the receptor and ligands was min-
imized using the MMFF94x force field. The potential docking site
was searched using the Site Finder of MOE. The stable conforma-
tions of ligands were obtained by the conformational search. Tether
weight was added to all receptor backbone atoms within 4.5 Å from
a ligand, while others were free. The binding mode with the highest
score was chosen for the final representation.
tively) were used for receptor activation. The fluorescent intensity
at 560 nm, upon excitation at 530 nm, was monitored using a Flex-
Station II plate reader (Molecular Devices). The inhibition percent-
age was determined based on changes in fluorescence before (the
average value for 20 s) and after (the maximal value after 10–
60 s) the addition of GABA. Each assay was repeated twice, unless
otherwise noted.
3.6. Electrophysiology
3.6.1. Isolation of neurons from ACs
The sixth abdominal ganglia were dissected from adult male
Acknowledgments
ACs (Periplaneta americana (Linnaeus)) and were placed in Ca2+
-
free saline containing 200 mM NaCl, 3.1 mM KCl, 4 mM MgCl2,
20 mM -glucose, and 10 mM HEPES, adjusted to pH 7.4 with an
NaOH solution. The ganglia were incubated for 20 min at 25 °C in
Ca2+-free saline containing collagenase (0.5 mg mLꢀ1) and trypsin
(0.2 mg mLꢀ1) and were rinsed with Ca2+-free saline twice. The
ganglia were then placed in Ca2+-containing saline (200 mM NaCl,
We thank Dr. T. Nakao (Mitsui Chemicals Agro, Inc.) for critical
reading of this manuscript. This work was supported in part by KA-
KENHI (a Grant-in-Aid for Scientific Research) (C).
D
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