Synthesis of 1,3-di- and 1,3,4-trisubstituted 1,6-dihydro-6-iminopyridazines as competitive antagonists of insect GABA receptors

Abstract

Insect GABA receptors (GABARs) represent important targets for insecticides. Thirteen iminopyridazine GABA analogs were synthesized and evaluated for their antagonism of three cloned insect GABARs. Of the synthesized analogs, 4-[4-cyclobutyl-1,6-dihydro-6-imino-3-(2-naphthyl)pyridazin-1-yl]butyronitrile greatly reduced GABA-activated responses in small brown planthopper (SBP) and common cutworm (CC) GABARs at 100 µM. Removal of the cyclobutyl group did not affect the levels of inhibition in both GABARs, whereas it increased the inhibition levels in housefly (HF) GABARs to afford an analog with an IC₅₀ of 75.5 µM. 4-[3-(4-Biphenylyl)-1,6-dihydro-6-iminopyridazin-1-yl]butyronitrile and the 3-(2-fluoro-4-biphenylyl) congener exhibited >80% inhibition in SBP and CC GABARs at 100 µM. These analogs showed relatively potent antagonism of HF GABARs, with IC₅₀s of 37.9 µM and 42.3 µM, respectively. Ethyl 3-[3-(4-biphenylyl)-1,6-dihydro-6-iminopyridazin-1-yl]propylphosphonate was the most potent inhibitor of GABA-induced currents in HF GABARs, with an IC₅₀ of 18.8 µM. These findings suggest that the iminopyridazine analogs could be leads for the development of insecticides.

Introduction

γ-Aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the central nervous system of mammals, mediates inhibitory neurotransmission through two types of receptors: ionotropic and metabotropic.^1–4) The ionotropic GABA receptor (GABAR) belongs to the Cys-loop receptor family of ligand-gated ion channels, including nicotinic acetylcholine, glycine, and serotonin type 3 receptors.^5,6) Ionotropic GABARs are categorized into two types: hetero-pentameric type A receptors (GABA_ARs), which consist of α1-6, β1-3, γ1-3, δ, ε, θ, and π subunits, and homo-pentameric type C receptors (GABA_CRs), which include ρ1-3 subunits.^7,8) Each subunit consists of a large extracellular GABA-binding domain, a transmembrane domain containing four α-helical segments, and a large intracellular loop. The second transmembrane segments of five subunits form a central chloride channel pore.⁹⁾ The GABA_ARs that contain two α subunits, two β subunits, and one γ subunit are the most abundant subtype in the adult brain.^7,10) GABA or an agonist binds to the orthosteric binding site in the extracellular interface between the α and β subunits of GABA_ARs, which triggers the opening of the channel, thus enhancing chloride permeability through the neuronal membrane and inhibiting the generation of action potentials. The orthosteric agonist binding site is composed of six discontinuous loops, A–F, located in adjacent subunits; loops A–C are in the principal subunit, and loops D–F are in the complementary subunit.⁹⁾ Competitive antagonists, which share a common binding site with the agonist GABA, stabilize the closed conformation of the channel. GABA_ARs represent targets for clinically important drugs such as benzodiazepines, barbiturates, neurosteroids, and anesthetics.¹¹⁾

In insects, GABARs play important physiological roles not only in the nervous system but also in the peripheral tissues.^12,13) The insect ionotropic GABAR consists of five Rdl subunits.^12–14) The Rdl transcript is alternatively spliced at exons 3 and 6 to produce four variant subunits (Rdl_ac, Rdl_ad, Rdl_bc, and Rdl_bd). Insect GABARs represent important targets for insecticides and parasiticides.^12,13,15) Phenylpyrazole insecticides, such as fipronil, exert insecticidal effects by acting as noncompetitive antagonists.¹⁶⁾ However, the development of resistance to fipronil due to its extensive use has been reported in several insect species.^17–19) Although two novel classes of insecticidal GABAR antagonists, isoxazolines and 3-benzamido-N-phenylbenzamides, have recently been reported,^20–23) there are continuing efforts to develop novel insecticides that inhibit GABAR functions. High-affinity competitive antagonists for insect GABARs could also be potential insecticides. Bicuculline and gabazine (SR 95531) (Fig. 1) represent competitive antagonists of mammalian GABA_ARs.^24,25) However, a potent competitive antagonist for insect GABARs is not available. Whereas bicuculline is inactive against most insect GABARs, gabazine shows weak or moderate antagonistic activity against insect GABARs.^26–28) It has been previously reported that the introduction of substituents at the 5-position of the pyridazine ring of gabazine produces competitive antagonists with micromolar-affinity against the parasitic nematode Ascaris suum, whereas the 5-substitutent is detrimental to rat GABARs.^29,30) This selectivity is informative in terms of the design of insecticides. In a previous study, we synthesized 1,6-dihydro-6-iminopyridazines (IPs) in which the 3-position of the pyridazine ring of gabazine was modified (Fig. 1), and we observed enhanced antagonism of insect GABARs.³¹⁾ These findings prompted the synthesis of other analogs. Here, we report the synthesis of 1,3-di- and 1,3,4-trisubstituted IPs and their antagonism of GABARs cloned from two agricultural insect pest species and a sanitary insect species. In this new synthesis, we examined the effects of the 4-substituent and the carboxylate bioisosteres at the 1-position on the antagonist potency of IP derivatives in insect GABARs.

Fig. 1. Chemical structures of GABA, the GABAR antagonists bicuculline and gabazine (SR 95531), and 1,6-dihydro-6-iminopyridazines (IPs). Bold lines indicate the GABA structural scaffold. The numbers of the carbon atoms of IPs are indicated at their sides.

Materials and Methods

1. General methods

Reagents were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), unless otherwise noted. Namgel NAM-200H (Nagara Science) was used for the purification of 8 and 9. The melting points of synthesized compounds were determined on a cover glass with a Yanagimoto MP-500D apparatus and are uncorrected. ¹H NMR spectra were recorded on a JEOL JNM-A 400 spectrometer. Chemical shifts (δ_H values) are given in ppm relative to tetramethylsilane (TMS) and the J values are given in Hertz. Spin multiplicities are expressed as follows: s (singlet), d (doublet), t (triplet), q (quartet), qn (quintet), and m (multiplet). High-resolution mass spectrometry (HRMS) analyses were performed on a Waters SYNAPT G2 spectrometer using the positive electrospray ionization mode.

2. Synthesis of 3-amino-6-chloro-5-cyclobutylpyridazine (1)

3. General procedure for the synthesis of 3-amino-6-aryl-5-cyclobutylpyridazine (2a–2c)

A mixture of 1 (183.5 mg, 1.0 mmol), arylboronic acid (1.5 mmol), tetrakis(triphenylphosphine)palladium(0) (35 mg), and an aqueous 2 M Na₂CO₃ solution (1.1 mL) in toluene (10 mL) was stirred under an argon atmosphere for 30 min at room temperature. The reaction mixture was then heated under reflux with stirring in an argon atmosphere until the starting material disappeared (ca. 48 hr). After cooling, the reaction mixture was concentrated to dryness under reduced pressure. EtOAc (30 mL) was added to the flask containing the residue, and the flask was placed in an ultrasonic bath for 5 min. The mixture was filtered and the filter paper was washed thoroughly with EtOAc (100 mL). The combined filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography to yield 3-amino-6-aryl-5-cyclobutylpyridazine (2a–2c).

3.1. 3-Amino-5-cyclobutyl-6-phenylpyridazine (2a)

Yield 13% (for two steps), mp 166–168°C. ¹H NMR δ_H (CD₃OD): 1.88–1.98 (m, 1H), 2.09–2.26 (m, 3H), 2.45–2.52 (m, 2H), 3.48–3.56 (m, 1H), 7.39–7.49 (m, 3H), 7.60 (d, 1H, J=1.5 Hz), 7.87–7.90 (m, 2H).

3.2. 3-Amino-6-(4-biphenylyl)-5-cyclobutylpyridazine (2b)

Yield 13% (for two steps), mp 175–177°C. ¹H NMR δ_H (CD₃OD): 1.92–1.96 (m, 1H), 2.13–2.27 (m, 3H), 2.46–2.53 (m, 2H), 3.49–3.55 (m, 1H), 7.32–7.37 (m, 1H), 7.43–7.47 (m, 2H), 7.65–7.68 (m, 3H), 7.73–7.75 (m, 2H), 7.98–8.00 (m, 2H).

3.3. 3-Amino-5-cyclobutyl-6-(2-naphthyl)pyridazine (2c)

Yield 16% (for two steps), mp 172–174°C. ¹H NMR δ_H (DMSO-d₆): 1.84–1.88 (m, 1H), 1.98–2.18 (m, 1H), 2.21–2.23 (m, 2H), 2.41–2.46 (m, 2H), 3.48–3.54 (m, 1H), 6.19 (s, 2H), 7.50–7.56 (m, 2H), 7.79 (s, 1H), 7.93 (d, 1H, J=7.6 Hz), 7.99 (d, 1H, J=8.8 Hz), 8.03 (d, 1H, J=7.6 Hz), 8.27 (d, 1H, J=8.8 Hz), 8.55 (s, 1H).

4. General procedure for the synthesis of ethyl 4-(3-aryl-4-cyclobutyl-1,6-dihydro-6-iminopyridazin-1-yl)butanoate hydrobromide (3a–3c)

A mixture of 2a, 2b, or 2c (1 mmol), ethyl 4-bromobutanoate (292 mg, 1.5 mmol), and DMF (0.5 mL) was heated at 80°C until the starting material disappeared (ca. 40 hr). After cooling, the precipitate was collected and washed with EtOAc (5 mL). The residue was recrystallized from MeOH and EtOAc to give ethyl 4-(3-aryl-4-cyclobutyl-1,6-dihydro-6-iminopyridazin-1-yl)butanoate hydrobromide (3a–3c).

4.1. Ethyl 4-(4-cyclobutyl-1,6-dihydro-6-imino-3-phenylpyridazin-1-yl)butanoate hydrobromide (3a)

Yield 8% (for three steps), mp 208–210°C. ¹H NMR δ_H (CD₃OD): 1.12 (t, 3H, J=7.1 Hz), 1.95–2.01 (m, 1H), 2.19–2.38 (m, 5H), 2.52–2.60 (m, 4H), 3.66–3.71 (m, 1H), 3.99 (q, 2H, J=7.1 Hz), 4.51 (t, 2H, J=7.1 Hz), 7.55–7.56 (m, 3H), 8.01–8.04 (m, 2H), 8.08 (d, 1H, J=1.5 Hz).

4.2. Ethyl 4-[3-(4-biphenylyl)-4-cyclobutyl-1,6-dihydro-6-iminopyridazin-1-yl]butanoate hydrobromide (3b)

Yield 9% (for three steps), mp 204–206°C. ¹H NMR δ_H (CD₃OD): 1.13 (t, 3H, J=7.1 Hz), 1.97–2.02 (m, 1H), 2.17–2.37 (m, 5H), 2.54–2.62 (m, 4H), 3.68–3.72 (m, 1H), 4.01 (q, 2H, J=7.1 Hz), 4.52 (t, 2H, J=6.8 Hz), 7.37–7.41 (m, 1H), 7.46–7.50 (m, 2H), 7.69–7.71 (m, 2H), 7.81–7.83 (m, 2H), 8.11–8.13 (m, 3H).

4.3. Ethyl 4-[4-cyclobutyl-1,6-dihydro-6-imino-3-(2-naphthyl)pyridazin-1-yl]butanoate hydrobromide (3c)

Yield 5% (for three steps), mp 212–214°C. ¹H NMR δ_H (CD₃OD): 1.09 (t, 3H, J=7.1 Hz), 1.98–2.03 (m, 1H), 2.18–2.36 (m, 3H), 2.38–2.43 (m, 2H), 2.55–2.63 (m, 4H), 3.71 (t, 1H, J=8.3 Hz), 3.99 (q, 2H, J=7.1 Hz), 4.54 (t, 2H, J=6.8 Hz), 7.57–7.60 (m, 2H), 7.93–7.95 (m, 1H), 8.01–8.05 (m, 2H), 8.13 (dd, 1H, J=6.8, 2.0 Hz), 8.26 (d, 1H, J=1.0 Hz), 8.58 (s, 1H).

5. General procedure for the synthesis of 4-(3-aryl-4-cyclobutyl-1,6-dihydro-6-iminopyridazin-1-yl)butanoic acid hydrobromide (4a–4c)

Compound 3a, 3b, or 3c (200 mg) was dissolved in a minimal amount of aqueous K₂CO₃ solution to yield an alkaline solution. The solution was then extracted with a 1 : 1 mixture of EtOAc and Et₂O. The organic layer was dried over anhydrous Na₂SO₄ and filtered. The filtrate was concentrated under reduced pressure to give free base ester. Hydrobromic acid in AcOH (10 mL, 30%) was added to the free base ester and heated at 100°C for ca. 12 hr. After cooling, the reaction mixture was concentrated to dryness under reduced pressure. The residue was recrystallized with MeOH and EtOAc to afford 4-(3-aryl-4-cyclobutyl-1,6-dihydro-6-iminopyridazin-1-yl)butanoic acid hydrobromide (4a–4c).

5.1. 4-(4-Cyclobutyl-1,6-dihydro-6-imino-3-phenylpyridazin-1-yl)butanoic acid hydrobromide (4a)

Yield 7% (for four steps), mp 181–183°C. ¹H NMR δ_H (CD₃OD): 1.95–2.10 (m, 1H), 2.09–2.23 (m, 3H), 2.27–2.37 (m, 4H), 2.53–2.60 (m, 2H), 3.63–3.71 (m, 1H), 4.48 (t, 2H, J=7.8 Hz), 7.53–7.55 (m, 3H), 8.00–8.03 (m, 3H). HRMS (ESI) m/z ([M–Br]⁺): Calcd. for C₁₈H₂₂N₃O₂: 312.1712, Found: 312.1727.

5.2. 4-[3-(4-Biphenylyl)-4-cyclobutyl-1,6-dihydro-6-iminopyridazin-1-yl]butanoic acid hydrobromide (4b)

Yield 6% (for four steps), mp 217–219°C. ¹H NMR δ_H (CD₃OD): 1.98–2.02 (m, 1H), 2.15–2.40 (m, 5H), 2.54–2.60 (m, 4H), 3.66–3.75 (m, 1H), 4.52 (t, 2H, J=7.3 Hz), 7.37–7.41 (m, 1H), 7.46–7.49 (m, 2H), 7.69 (d, 2H, J=8.8 Hz), 7.80–7.83 (m, 2H), 8.11–8.13 (m, 3H). HRMS (ESI) m/z ([M–Br]⁺): Calcd. for C₂₄H₂₆N₃O₂: 388.2025, Found: 388.2024.

5.3. 4-[4-Cyclobutyl-1,6-dihydro-6-imino-3-(2-naphthyl)pyridazin-1-yl]butanoic acid hydrobromide (4c)

Yield 4% (for four steps), mp 218–220°C. ¹H NMR δ_H (CD₃OD): 1.95–2.02 (m, 1H), 2.18–2.33 (m, 3H), 2.35–2.42 (m, 2H), 2.57–2.61 (m, 4H), 3.66–3.75 (m, 1H), 4.53 (t, 2H, J=7.1 Hz), 7.56–7.61 (m, 2H), 7.92–7.94 (m, 1H), 8.00–8.05 (m, 2H), 8.12–8.15 (m, 1H), 8.24 (d, 1H, J=1.0 Hz), 8.58 (s, 1H). HRMS (ESI) m/z ([M–Br]⁺): Calcd. for C₂₂H₂₄N₃O₂: 362.1869, Found: 362.1893.

6. Synthesis of 4-[4-cyclobutyl-1,6-dihydro-6-imino-3-(2-naphthyl)pyridazin-1-yl]butyronitrile hydrobromide (5)

A mixture of 2c (275 mg, 1.0 mmol), 4-bromobutyronitrile (178 mg, 1.2 mmol), and DMF (0.5 mL) was heated at 80°C until the starting material disappeared (ca. 20 hr). After cooling, the precipitate was collected and washed with EtOAc (5 mL). The residue was recrystallized from MeOH and EtOAc to give 4-[4-cyclobutyl-1,6-dihydro-6-imino-3-(2-naphthyl)pyridazin-1-yl]butyronitrile hydrobromide (5). Yield 10% (for three steps), mp 228–230°C. ¹H NMR δ_H (DMSO-d₆): 1.87–1.90 (m, 1H), 2.03–2.10 (m, 1H), 2.17–2.23 (qn, 2H, J=6.3 Hz), 2.28–2.35 (m, 2H), 2.49–2.50 (m, 2H), 2.67–2.70 (m, 2H), 3.63–3.67 (m, 1H), 4.43 (t, 2H, J=6.3 Hz), 7.59–7.62 (m, 2H), 7.98–8.00 (m, 1H), 8.05–8.07 (m, 3H), 8.11–8.17 (m, 1H), 8.69 (s, 1H). HRMS (ESI) m/z ([M–Br]⁺): Calcd. for C₂₂H₂₃N₄: 343.1923, Found: 343.1944.

7. General procedure for the synthesis of 3-amino-6-(aryl/heteroaryl)pyridazine (6a–6g)

A mixture of 3-amino-6-chloropyridazine (388 mg, 3.0 mmol), aryl/heteroarylboronic acid (4.5 mmol), tetrakis(triphenylphosphine)palladium(0) (105 mg), and an aqueous 2 M Na₂CO₃ solution (3.3 mL) in toluene (30 mL) was stirred in an argon atmosphere at room temperature for 30 min. The reaction mixture was then heated under reflux with stirring in an argon atmosphere until the starting material disappeared (ca. 70 hr). After cooling, the reaction mixture was concentrated to dryness under reduced pressure. 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 combined filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography to yield 3-amino-6-aryl/heteroarylpyridazine (6a–6g).

7.1. 3-Amino-6-(4-methoxyphenyl)pyridazine (6a)

Yield 42%, mp 162–164°C. ¹H NMR δ_H (CDCl₃): 3.86 (s, 3H), 4.81 (s, 2H), 6.81 (d, 1H, J=9.2 Hz), 6.99 (d, 2H, J=8.5 Hz), 7.57 (d, 1H, J=9.2 Hz), 7.89 (d, 2H, J=8.5 Hz).

7.2. 3-Amino-6-(4-ethoxyphenyl)pyridazine (6b)

Yield 59%, mp 151–153°C. ¹H NMR δ_H (DMSO-d₆): 1.34 (t, 3H, J=7.1 Hz), 4.07 (q, 2H, J=7.1 Hz), 6.33 (s, 2H), 6.82 (d, 1H, J=9.3 Hz), 6.99 (dd, 2H, J=6.8, 2.0 Hz), 7.73 (d, 1H, J=9.3 Hz), 7.87 (dd, 2H, J=6.8, 2.0 Hz).

7.3. 3-Amino-6-(4-trifluoromethoxyphenyl)pyridazine (6c)

Yield 42%, mp 107–109°C. ¹H NMR δ_H (DMSO-d₆): 6.55 (s, 2H), 6.87 (d, 1H, J=9.3 Hz), 7.45 (d, 2H, J=8.8 Hz), 7.84 (d, 1H, J=9.3 Hz), 8.08 (d, 2H, J=8.8 Hz).

7.4. 3-Amino-6-(4-biphenylyl)pyridazine (6d)

Yield 36%, mp 221–223°C. ¹H NMR δ_H (DMSO-d₆): 6.56 (s, 2H), 6.89 (d, 1H, J=9.3 Hz), 7.38 (t, 1H, J=7.3 Hz), 7.49 (t, 2H, J=7.3 Hz), 7.72–7.78 (m, 4H), 7.88 (d, 1H, J=9.3 Hz), 8.07 (d, 2H, J=7.3 Hz).

7.5. 3-Amino-6-(2-fluoro-4-biphenylyl)pyridazine (6e)

Yield 64%, mp 154–156°C. ¹H NMR δ_H (DMSO-d₆): 6.57 (s, 2H), 6.88 (d, 1H, J=9.3 Hz), 7.42 (t, 1H, J=6.8 Hz), 7.48–7.56 (m, 2H), 7.60–7.64 (m, 4H), 7.88–7.92 (m, 2H).

7.6. 3-Amino-6-(2-naphthyl)pyridazine (6f)

Yield 19%, mp 212–214°C. ¹H NMR δ_H (DMSO-d₆): 6.54 (s, 2H), 6.91 (d, 1H, J=9.6 Hz), 7.51–7.56 (m, 2H), 7.93–8.01 (m, 4H), 8.21 (d, 1H, J=9.6 Hz), 8.47 (s, 1H).

7.7. 3-Amino-6-(3-thienyl)pyridazine (6g)

Yield 49%, mp 155–157°C. ¹H NMR δ_H (DMSO-d₆): 6.39 (s, 2H), 6.82 (d, 1H, J=9.3 Hz), 7.61–7.63 (m, 1H), 7.70–7.71 (m, 1H), 7.74 (d, 1H, J=9.3 Hz), 7.97–7.98 (m, 1H).

8. General procedure for the synthesis of 4-[3-(aryl/hetero­aryl)-1,6-dihydro-6-iminopyridazin-1-yl]butyronitrile hydrobromide (7a–7g)

A mixture of 6a, 6b, 6c, 6d, 6e, 6f, or 6g (1 mmol), 4-bromobutyronitrile (178 mg, 1.2 mmol), and DMF (0.5 mL) was heated at 80°C until the starting material disappeared (ca. 15 hr). After cooling, the precipitate was collected and washed with EtOAc (5 mL). The residue was recrystallized from MeOH and EtOAc to give 4-[3-(aryl/heteroaryl)-1,6-dihydro-6-iminopyridazin-1-yl]butyronitrile hydrobromide (7a–7g).

8.1. 4-[1,6-Dihydro-6-imino-3-(4-methoxyphenyl)pyridazin-1-yl]butyronitrile hydrobromide (7a)

Yield 33% (for two steps), mp 247–249°C. ¹H NMR δ_H (DMSO-d₆): 2.19 (qn, 2H, J=7.0 Hz), 2.67 (t, 2H, J=7.0 Hz), 3.84 (s, 3H), 4.38 (t, 2H, J=7.0 Hz), 7.11 (d, 2H, J=8.8 Hz), 7.62 (d, 1H, J=9.5 Hz), 7.95 (d, 2H, J=8.8 Hz), 8.38 (d, 1H, J=9.5 Hz), 9.02 (broad s, 2H). HRMS (ESI) m/z ([M–Br]⁺): Calcd. for C₁₅H₁₇N₄O: 269.1402, Found: 269.1431.

8.2. 4-[1,6-Dihydro-3-(4-ethoxyphenyl)-6-iminopyridazin-1-yl]butyronitrile hydrobromide (7b)

Yield 48% (for two steps), mp 257–259°C. ¹H NMR δ_H (DMSO-d₆): 1.36 (t, 3H, J=7.0 Hz), 2.18 (qn, 2H, J=7.0 Hz), 2.69 (t, 2H, J=7.0 Hz), 4.12 (q, 2H, J=7.0 Hz), 4.39 (t, 2H, J=7.0 Hz), 7.10 (d, 2H, J=8.8 Hz), 7.65 (d, 1H, J=9.8 Hz), 7.95 (d, 2H, J=8.8 Hz), 8.40 (d, 1H, J=9.8 Hz), 9.09 (broad s, 2H). HRMS (ESI) m/z ([M–Br]⁺): Calcd. for C₁₆H₁₉N₄O: 283.1559, Found: 283.1577.

8.3. 4-[1,6-Dihydro-6-imino-3-(4-trifluoromethoxyphenyl)pyridazin-1-yl]butyronitrile hydrobromide (7c)

Yield 29% (for two steps), mp 223–225°C. ¹H NMR δ_H (DMSO-d₆): 2.20 (qn, 2H, J=6.9 Hz), 2.69 (t, 2H, J=6.9 Hz), 4.42 (t, 2H, J=6.9 Hz), 7.58 (d, 2H, J=7.8 Hz), 7.71 (d, 1H, J=9.5 Hz), 8.13 (d, 2H, J=7.8 Hz), 8.44 (d, 1H, J=9.5 Hz), 9.22 (broad s, 2H). HRMS (ESI) m/z ([M–Br]⁺): Calcd. for C₁₅H₁₄F₃N₄O: 323.1120, Found: 323.1116.

8.4. 4-[3-(4-Biphenylyl)-1,6-dihydro-6-iminopyridazin-1-yl]butyronitrile hydrobromide (7d)

Yield 28% (for two steps), mp 267–269°C. ¹H NMR δ_H (DMSO-d₆): 2.21 (qn, 2H, J=6.9 Hz), 2.72 (t, 2H, J=6.9 Hz), 4.44 (t, 2H, J=6.9 Hz), 7.43 (t, 1H, J=7.4 Hz), 7.51 (t, 2H, J=7.4 Hz), 7.72–7.78 (m, 3H), 7.89 (d, 2H, J=8.3 Hz), 8.10 (d, 2H, J=8.3 Hz), 8.50 (d, 1H, J=9.3 Hz), 9.18 (broad s, 2H). HRMS (ESI) m/z ([M–Br]⁺): Calcd. for C₂₀H₁₉N₄: 315.1610, Found: 315.1617.

8.5. 4-[1,6-Dihydro-3-(2-fluoro-4-biphenylyl)-6-iminopyridazin-1-yl]butyronitrile hydrobromide (7e)

Yield 37% (for two steps), mp 223–225°C. ¹H NMR δ_H (DMSO-d₆): 2.21 (qn, 2H, J=6.9 Hz), 2.71 (t, 2H, J=6.9 Hz), 4.44 (t, 2H, J=6.9 Hz), 7.46 (t, 1H, J=7.2 Hz), 7.53 (t, 2H, J=7.2 Hz), 7.63 (d, 2H, J=7.2 Hz), 7.71–7.78 (m, 2H), 7.95–8.01 (m, 2H), 8.52 (d, 1H, J=9.8 Hz), 9.27 (broad s, 2H). HRMS (ESI) m/z ([M–Br]⁺): Calcd. for C₂₀H₁₈FN₄: 333.1515, Found: 333.1552.

8.6. 4-[1,6-Dihydro-6-imino-3-(2-naphthyl)pyridazin-1-yl]butyronitrile hydrobromide (7f)

Yield 12% (for two steps), mp 261–263°C. ¹H NMR δ_H (DMSO-d₆): 2.24 (qn, 2H, J=7.0 Hz), 2.72 (t, 2H, J=7.0 Hz), 4.45 (t, 2H, J=7.0 Hz), 7.63–7.66 (m, 2H), 7.71 (d, 1H, J=9.3 Hz), 8.00–8.15 (m, 4H), 8.60 (d, 1H, J=9.3 Hz), 8.63 (s, 1H), 9.17 (broad s, 2H). HRMS (ESI) m/z ([M–Br]⁺): Calcd. for C₁₈H₁₇N₄: 289.1453, Found: 289.1489.

8.7. 4-[1,6-Dihydro-6-imino-3-(3-thienyl)pyridazin-1-yl]butyronitrile hydrobromide (7g)

Yield 35% (for two steps), mp 246–248°C. ¹H NMR δ_H (DMSO-d₆): 2.17 (qn, 2H, J=6.9 Hz), 2.68 (t, 2H, J=6.9 Hz), 4.37 (t, 2H, J=6.9 Hz), 7.66–7.69 (m, 2H), 7.76–7.78 (m, 1H), 8.37–8.40 (m, 2H), 9.14 (broad s, 2H). HRMS (ESI) m/z ([M–Br]⁺): Calcd. for C₁₂H₁₃N₄S: 245.0861, Found: 245.0886.

9. Synthesis of ethyl 3-[3-(4-biphenylyl)-1,6-dihydro-6-iminopyridazin-1-yl]propylphosphonate hydrobromide (8)

A mixture of 6d (247 mg, 1 mmol), diethyl (3-bromopropyl)phosphonate (328 mg (95%), 1.2 mmol), and DMF (0.5 mL) was heated at 80°C until the starting material disappeared (72 hr). After cooling, the precipitate was collected and washed with EtOAc (5 mL). The residue was purified by silica gel column chromatography (MeOH:EtOAc=1 : 1) to yield ethyl 3-[3-(4-biphenylyl)-1,6-dihydro-6-iminopyridazin-1-yl]propylphosphonate hydrobromide (8). Yield 19% (for two steps), mp 161–163°C. ¹H NMR δ_H (CD₃OD): 1.26 (t, 3H, J=7.1 Hz), 1.66–1.74 (m, 2H), 2.16–2.28 (m, 2H), 3.94 (q, 2H, J=7.1 Hz), 4.50 (t, 2H, J=7.3 Hz), 7.38 (t, 1H, J=7.4 Hz), 7.46 (t, 2H, J=7.4 Hz), 7.59 (d, 1H, J=9.6 Hz), 7.66 (d, 2H, J=8.3 Hz), 7.77 (d, 2H, J=8.3 Hz), 8.03 (d, 2H, J=8.3 Hz), 8.28 (d, 1H, J=9.6 Hz). HRMS (ESI) m/z ([M–Br]⁺): Calcd. for C₂₁H₂₅N₃O₃P: 398.1634, Found: 398.1637.

10. Synthesis of 3-[3-(4-biphenylyl)-1,6-dihydro-6-iminopyridazin-1-yl]propylphosphonic acid hydrobromide (9)

A mixture of 8 (239 mg, 0.5 mmol) and 2 M hydrobromic acid (10 mL) was refluxed for 40 hr. After cooling, the reaction mixture was concentrated to dryness under reduced pressure and washed with EtOAc (5 mL). The residue was purified by silica gel column chromatography (MeOH : EtOAc=1 : 1) to yield 3-[3-(4-biphenylyl)-1,6-dihydro-6-iminopyridazin-1-yl]propylphosphonic acid hydrobromide (9). Yield 18% (for three steps), mp 290–293°C. ¹H NMR δ_H (DMSO-d₆): 1.64–1.73 (m, 2H), 2.06–2.11 (m, 2H), 4.41 (t, 2H, J=7.1 Hz), 7.43 (d, 1H, J=7.4 Hz), 7.51 (t, 2H, J=7.4 Hz), 7.67 (d, 1H, J=9.3 Hz), 7.76 (d, 2H, J=7.4 Hz), 7.89 (d, 2H, J=8.3 Hz), 8.09 (d, 2H, J=8.3 Hz), 8.47 (d, 1H, J=9.3 Hz), 9.24 (broad s, 2H). HRMS (ESI) m/z ([M–Br]⁺): Calcd. for C₁₉H₂₁N₃O₃P: 370.1321, Found: 370.1352.

11. FMP assays

Drosophila S2 cell lines stably expressing small brown planthopper (SBP, Laodelphax striatellus) and common cutworm (CC, Spodoptera litura) GABARs were created by transfecting respective Rdl_bd subunit cDNAs [DDBJ accession Nos. AB253526 (SBP) and DD171257 (CC)] as previously described.^28,32) FMP assays were performed using these cell lines as described in a previous report.³¹⁾ In these assays, changes in membrane potential were determined based on changes in the fluorescence of an FMP dye. Briefly, the cells were washed and suspended in a buffered saline (120 mM of NaCl, 5 mM of KCl, 2 mM of CaCl₂, 8 mM of MgCl₂, 10 mM of HEPES, and 32 mM of sucrose, adjusted pH to 7.2 with NaOH). The cell suspension (100 µL; 5×10⁵ cells) was added to 96-well microplates and incubated for 10 min at room temperature. The cells were then centrifuged at 1,400 rpm for 5 min at 4°C and loaded with the FMP blue dye reagent (Molecular Devices) diluted 10-fold with the buffered saline (100 µL) for 20 min at room temperature. Synthesized IPs were dissolved in DMSO and then diluted with a saline buffer. IPs dissolved in a saline buffer (25 µL) containing 1% DMSO were added to the cells in each well and incubated for 74 sec. After incubation, GABA in a saline buffer (25 µL) was added to each well. The half-maximal effective concentrations (EC₅₀s) of GABA for SBP and CC GABARs (1.0 µM and 2.5 µM, respectively) were used to activate the receptors. Fluorescence (emission, 560 nm; excitation, 530 nm) was measured using a FlexStation II plate reader (Molecular Devices). The inhibition percentage was determined based on changes in fluorescence before (average over 20 sec) and after (maximum after 10–60 sec) the addition of GABA. The assay was replicated twice.

12. Expression of housefly (HF, Musca domestica) GABARs in Xenopus oocytes and two-electrode voltage clamp (TEVC) recordings

TEVC experiments were performed as previously reported.³³⁾ After a mature female African clawed frog (Xenopus laevis) was anesthetized with 0.1% (w/v) ethyl m-aminobenzoate methanesulfonate, the ovarian lobes were surgically removed. The ovarian lobes were treated with collagenase (2 mg/mL; Sigma-Aldrich) in Ca²⁺-free standard oocyte solution (SOS) (100 mM of NaCl, 2 mM of KCl, 1 mM of MgCl₂, 5 mM of HEPES, pH 7.6) for 90–120 min at room temperature. The oocytes were then gently washed with a sterile SOS (100 mM of NaCl, 2 mM of KCl, 1.8 mM of CaCl₂, 1 mM of MgCl₂, 5 mM of HEPES, pH 7.6) supplemented with 2.5 mM of sodium pyruvate, 50 µg of gentamycin/mL (Gibco), 100 U of penicillin/mL (Invitrogen) and 100 µg of streptomycin/mL (Invitrogen) and were incubated at 16°C overnight in the buffer. The oocytes were injected with 5 ng of cRNA encoding the HF Rdl_ac subunit and were incubated under the same conditions for 2 days. The capped cRNA was synthesized using T7 polymerase (Ambion mMESSAGE mMACHINE T7 Ultra Kit), the primer pcDNA3-cRNAF (5′-CTCTCTGGCTAACTAGAGAACC-3′), and cDNA encoding the ac variant of the HF GABAR subunit [DDBJ accession Nos. AB177547 (RDL_bd, complete cds), AB824728 (exon 3 a version, partial cds), and AB824729 (exon 6 c version, partial cds)].

GABA-induced currents were recorded using an OC-725C Oocyte Clamp amplifier (Warner Instruments) at a holding potential of −80 mV. The recorded currents were analyzed by Data-Trax2™ software (World Precision Instruments). The glass capillary electrodes were filled with 2 M of KCl and had a resistance of 0.5–1.5 MΩ at 18–22°C. GABA was dissolved in SOS, and the IPs were dissolved in DMSO and then diluted by SOS to the final concentration (0.1% DMSO, v/v). After 10 µM (EC₅₀) of GABA was added to the oocytes a few times for 3 sec to detect a control response, the IP solution was applied over 1 min before the following applications of GABA and during the remainder of the experiments. The EC₅₀ of GABA was applied repeatedly in the presence of an IP solution for 3 sec at 1-min intervals until a maximum inhibition of GABA response was reached. The inhibition percentage was calculated from the ratio of the average of two minimum responses during the application of IP to the average of a few responses induced by 10 µM of GABA. EC₅₀s, Hill coefficients (n_Hs), and half-maximal inhibitory concentrations (IC₅₀s) were obtained from concentration-response relationships by nonlinear regression analysis using OriginPro 8J software (OriginLab).

13. Homology modeling and docking simulation

A homology model of a homomeric HF GABAR containing Rdl_ac subunits was built using the X-ray crystal structure of the Caenorhabditis elegans glutamate-gated chloride channel (GluCl) (PDB: 3RIF) as a template.³⁴⁾ The model was created using MOE 2011.10 software (Chemical Computing Group). The alignment of the C. elegans GluCl α subunit and HF Rdl_ac sequences was carried out using ClustalW software. Geometry optimization was performed using the AMBER99 force field. The structures of GABA and 7e were created in the zwitterionic and protonated imino forms, respectively, using the MOE Builder. The stable conformations of ligands were obtained by a conformational search. The receptor and ligands were energy minimized using the MMFF94x force field. Docking of ligands into the potential binding site of the GABAR homology model was carried out using ASEDock 2011.01.27 software (Chemical Computing Group). The docking site was identified using the MOE Site Finder. The docking pose with the highest score was chosen for the final representation.

Results and Discussion

1. Chemistry

In the present study, we synthesized thirteen IPs with various R¹, R², and R³ substituents on the pyridazine ring, starting from 3-amino-6-chloropyridazine (Fig. 1, Schemes 1 and 2). The synthesized IPs are divided into three types. The first type consists of the 3-substituted 1-(3-carboxypropyl)-4-cyclobutyl-IPs (4a–4c). The second type consists of a 3-substituted 1-cyanopropyl-4-cyclobutyl-IP (5) and the 3-substituted 1-cyanopropyl-IPs (7a–7g). The third type consists of a 3-substituted 1-(ethyl phosphonopropyl)-IP (8) and a 3-substituted 1-phosphonopropyl-IP (9).

Scheme 1. Synthesis of 4-(3-aryl-4-cyclobutyl-1,6-dihydro-6-iminopyridazin-1-yl)butanoic acid hydrobromide salts (4a–4c) and 4-[4-cyclobutyl-1,6-dihydro-6-imino-3-(2-naphthyl)pyridazin-1-yl]butyronitrile hydrobromide (5).

Scheme 2. Synthesis of 4-(3-aryl/heteroaryl-1,6-dihydro-6-iminopyridazin-1-yl)butyronitrile hydrobromide salts (7a–7g), ethyl 3-[3-(4-biphenylyl)-1,6-dihydro-6-iminopyridazin-1-yl]propylphosphonate hydrobromide (8), and 3-[3-(4-biphenylyl)-1,6-dihydro-6-iminopyridazin-1-yl]propylphosphonic acid hydrobromide (9).

Introduction of a cyclobutyl group onto the 5-position of 3-amino-6-chloropyridazine to afford 1 was achieved by the Minisci reaction with cyclobutanecarboxylic acid, albeit in a low (18%) yield (Scheme 1). The Suzuki–Miyaura cross-coupling reaction between 1 and arylboronic acid in the presence of a palladium catalyst afforded 2a–2c in 70–88% yields. N(2)-Alkylation of 2a–2c with ethyl 4-bromobutanoate provided 3a–3c in 33–66% yields. Compounds 3a–3c were then converted to free base esters by treatment with K₂CO₃, followed by hydrolysis in acetic acid and hydrobromic acid at 100°C to give IPs 4a–4c in 71–85% yields. The Suzuki–Miyaura cross-coupling reaction between 3-amino-6-chloropyridazine and aryl/heteroarylboronic acid gave 6a–6g in 19–64% yields (Scheme 2). N(2)-Alkylation of 6a–6g with 4-bromobutyronitrile afforded IPs 7a–7g in 58–82% yields. N(2)-Alkylation of 2c with 4-bromobutyronitrile produced IP 5 in a 65% yield (Scheme 1). N(2)-Alkylation of 6d with diethyl (3-bromopropyl)phosphonate generated IP 8 in a 52% yield. The hydrolysis of 8 in 2 M hydrobromic acid under reflux gave IP 9 in a 95% yield (Scheme 2).

2. Antagonism of SBP and CC GABARs

We first examined the IP antagonism of GABARs constituted from Rdl_bd subunits cloned from two agricultural insect pests, the SBP and the CC, that cause serious damage to crops. In these tests, we used fluorometric imaging plate reader (FLIPR) membrane potential (FMP) technology.^35,36) Application of GABA to Drosophila cell lines stably expressing SBP and CC GABARs induces chloride ion efflux and membrane depolarization, which is then recorded with a fluorescent dye loaded in the cells. The maximum increase in fluorescence was measured after the application of the EC₅₀ of GABA to cells expressing GABARs. The levels of antagonism by IPs were evaluated as a decrease in fluorescence when IPs were applied simultaneously with GABA. When tested at 100 µM, IPs 4a, 4b, and 4c, in which R²=phenyl, 4-biphenylyl, and 2-naphthyl, respectively, R¹=COOH, and R³=cyclobutyl, showed less than 50% inhibition of the GABA response in SBP and CC GABARs (Figs. 2 and 3). Our previous study showed that gabazine (R¹=COOH, R²=4-methoxyphenyl, and R³=H) exhibited 76.4% and 60.2% inhibition of the GABA response in SBP and CC GABARs, respectively, at 100 µM.³¹⁾ Therefore, the cyclobutyl group is detrimental in this case.

Fig. 2. Inhibition of GABA-induced responses by IPs in SBP GABARs. Receptors were activated by 1.0 µM (EC₅₀) of GABA. Data represent the means of two experiments with bars indicating the range of duplicates. Inhibition percentages at 10 µM are shown for IPs with >50% inhibition at 100 µM. Inhibition data of gabazine (Gz) are taken from our previous report.³¹⁾

Fig. 3. Inhibition of GABA-induced responses by IPs in CC GABARs. Receptors were activated by 2.5 µM (EC₅₀) of GABA. Greater than 100% inhibition was observed for 5, 7d, 7e, and 7f due to their hyperpolarizing effects on cells. The negative value for 4b and 4c is due to their agonistic or potentiating effects on cells. Data represent the means of two experiments with bars indicating the range of duplicates. Inhibition percentages at 10 µM are shown for IPs with >50% inhibition at 100 µM. Inhibition data of gabazine (Gz) are taken from our previous report.³¹⁾

Replacement of the carboxyl group of gabazine with a cyano group, affording 7a, resulted in a 45.6% and a 73.1% inhibition of the GABA response in SBP and CC GABARs, respectively, at the same concentration, indicating that the carboxyl and cyano groups are bioisosteric in this case. While a dramatic change in activity was not observed with the carboxyl/cyano group substitution in insect GABARs, the conversion of gabazine to 7a led to an ca. 8-fold increase in potency in human ρ1 GABARs.³⁷⁾ IPs 7b and 7c, in which R²=4-ethoxyphenyl and 4-trifluoromethoxyphenyl, respectively, R¹=CN, and R³=H, displayed less than a 60% inhibition of the GABA response in SBP and CC GABARs. IPs 7d, 7e, and 7f, in which R²=4-biphenylyl, 2-fluoro-4-biphenylyl, and 2-naphthyl, respectively, R¹=CN, and R³=H, showed 83.4%, 86.7%, and 93.0% inhibitions, respectively, of the GABA response in SBP GABARs. The GABA-induced responses in CC GABARs were completely inhibited by 7d, 7e, and 7f when tested at 100 µM. IP 7g, in which R¹=CN, R²=3-thienyl, and R³=H, showed a 66.2% and a 73.6% inhibition of the GABA response in SBP and CC GABARs, respectively. Replacement of the carboxyl group of 4c, a trisubstituted IP, with a cyano group to give 5 led to a 79.7% and a complete inhibition of the GABA response in SBP and CC GABARs, respectively. 4-Cyclobutyl substitution on 5, a 1-(3-cyanopropyl) analog, was not detrimental as compared with 7f. This result is in contrast to that of 4c, a 1-(3-carboxypropyl) analog, in which a reduction in the percentage of inhibition was observed by the introduction of a cyclobutyl group at the 4-position.

An IP with R¹=P(=O)(OH)(OC₂H₅), R²=4-biphenylyl, and R³=H (8) displayed a 57.6% and a 50.7% inhibition of the GABA response in SBP and CC GABARs, respectively, at 100 µM. Replacement of P(=O)(OH)(OC₂H₅) with P(=O)(OH)₂ on 8, yielding 9, showed ca. 3-fold lower inhibition of the GABA response in SBP and CC GABARs than did 8.

3. Antagonism of HF GABARs

The IP antagonism of HF GABARs was subsequently examined using the Xenopus oocyte expression system and the TEVC technique. HF GABARs were transiently expressed in oocytes by injecting cRNA encoding the HF Rdl_ac subunit as previously described.²³⁾ The inhibition percentages at 100 µM of each compound are shown in Fig. 4A. Compounds 4a–4c, which have a cyclobutyl group at the 4-position, showed weak or no activity. However, removal of the cyclobutyl group of 4b and 4c increased inhibition percentages to 67.6±5.1% and 47.8±2.9%, respectively. In this case, the cyclobutyl group is detrimental to IP activity. The disadvantage of the introduction of the 4-cyclobutyl group can also be seen in 5 as compared with 7f.

Fig. 4. Inhibition of GABA-induced currents by IPs in HF GABARs expressed in Xenopus oocytes. (A) Inhibition of GABA-induced currents by 100 µM of synthesized analogs. Data represent the means±SEM of three to six independent assays from at least two frogs. (B) Application protocol of GABA and 7d. (C) Examples of current traces showing antagonistic effects of the IPs 7d, 7e, 7f, and 8 on HF GABARs. The receptors were activated by 10 µM (EC₅₀) of GABA.

In HF GABARs, gabazine had little activity in inhibiting GABA-induced currents (Fig. 4A). Substitution of a cyano group for the carboxyl group of gabazine to give 7a did not result in an increase in activity. Replacement of the methoxyphenyl group of 7a with an ethoxyphenyl or trifluoromethoxyphenyl group to give 7b or 7c, respectively, resulted in slight increases in activity. Replacement of the 4-methoxyphenyl group of 7a with 4-biphenylyl, 2-fluoro-4-biphenylyl, and 2-naphthyl groups to give 7d, 7e, and 7f, respectively, led to ca. 20-, ca. 21-, and ca. 15-fold greater inhibition percentages, respectively, as compared to 7a, indicating that the bulky aromatic rings at this position are favorable. The IP inhibition of GABA-activated currents in HF GABARs gradually proceeded with repeated application of GABA, and the maximum inhibition of currents at each concentration of the IPs was recorded, as shown by 7d (Fig. 4B). The inhibition of currents by 7d, 7e, 7f, and 8 is presented in Fig. 4C. The IC₅₀s of 7d, 7e, and 7f were calculated to be 37.9±3.0 µM, 42.3±1.8 µM, and 75.5±7.9 µM, respectively, from concentration-current inhibition relationships (Fig. 5A).

Fig. 5. Potencies and modes of antagonism of IPs in HF GABARs expressed in Xenopus oocytes. (A) Concentration-current inhibition curves of 7d (black squares), 7e (red circles), 7f (blue triangles), and 8 (dark cyan inverted triangles). (B) GABA concentration–response curves in the absence (black squares) and presence of 40 µM of 7d (red circles) and 20 µM of 8 (blue triangles). Data are the means±SEM of three to four independent assays from at least two frogs.

IP 8, in which the cyano group of 7d is replaced with an ethyl phosphonate, exhibited the greatest inhibition among the IPs tested in the present study, with an 87.3% inhibition at 100 µM and an IC₅₀ of 18.8±3.8 µM (Fig. 5A). Removal of the ethyl group of 8 to give 9 resulted in a lower inhibition (39.5%).

4. Mode of antagonism

Gabazine-type IPs with a carboxypropyl side chain were previously reported to act as competitive antagonists of American cockroach GABARs.³¹⁾ To determine the mode of antagonism of synthesized IPs, in particular, IPs with cyano and phosphonate functionalities at the end of the side chain, GABA concentration–response relationships were examined in HF GABARs expressed in Xenopus oocytes in the presence and absence of 7d and 8. A rightward shift was observed in the concentration-response curves in the presence of the cyanide 7d (40 µM) and the ethyl phosphonate 8 (20 µM), indicating the competitive antagonism of these compounds (Fig. 5B). The EC₅₀ and the n_H of GABA were 7.42±0.56 µM and 1.80±0.16, respectively, in the absence of IPs. The EC₅₀s were 16.1±2.1 µM and 16.6±1.2 µM in the presence of 7d and 8, respectively, and n_Hs were 1.49±0.29 and 1.72±0.26 in the presence of 7d and 8, respectively. The maximum current amplitudes did not change in the presence of both compounds. These findings indicate that IPs with the cyano and ethyl phosphonate functionalities at the end of the side chain bind to the orthosteric site.

5. Homology modeling and ligand docking

To understand the interaction between synthesized IPs and the amino acid residues in the orthosteric site of insect GABARs, ligand-docking studies were performed using an HF GABAR homology model. The model was built using the X-ray crystal structure of the C. elegans GluCl as a template.³⁴⁾ The zwitterionic form of GABA and the protonated form of IP 7e were docked into the constructed model. The binding poses of GABA and 7e with the highest docking score were selected for presentation (Fig. 6).

Fig. 6. Homology modeling and docking simulation. (A) Docking of GABA into the orthosteric site of an HF GABAR homology model. (B) Docking of 7e. The principal and complementary subunits are shown in orange and magenta, respectively. Hydrogen bonds are indicated by cyan dotted lines. The orthosteric site is located in the interface of both subunits.

The docking studies of GABA showed that the side chain of Glu202 and the backbone carbonyl oxygen atom of Ser203 in loop B function as hydrogen acceptors for the protonated amino group of GABA and that Arg109 in loop D and Ser174 in loop E serve as hydrogen donors for the carboxylate anion of GABA (Fig. 6A). Both experimental evidence and molecular dynamics simulation indicated that the amino acid residue corresponding to Arg109 plays a critical role in the interaction with GABA in Drosophila Rdl GABAR.³⁸⁾ The two aromatic amino acids, Phe204 in loop B and Tyr252 in loop C, surround the protonated amino group of GABA and may produce cation-π interactions, as proposed for Drosophila Rdl GABARs.^38–40) The bound GABA is in an extended conformation that is similar to that of the Drosophila Rdl GABAR and GABA_CR but not to that of the GABA_AR.^38,40)

Several amino acids located in loops D, E, and F of the α1 and ρ1 subunits and those in loop A and C of the β2 and ρ1 subunits in GABA_ARs and GABA_CRs have been identified as gabazine-interacting residues by site-directed mutagenesis and functional analysis of the mutants.^41–47) The docking studies of 7e using an HF GABAR homology model showed that the side chain carboxylate of Glu202 in loop B functions as an acceptor of the protonated imino hydrogen and that the guanidino group of Arg109 in loop D serves as a hydrogen donor for the lone pair of electrons of the nitrile nitrogen (Fig. 6B). The amino acid equivalent to Arg109 in the α1 subunit of GABA_ARs was reported to be involved in the interaction with gabazine.⁴³⁾ Tyr252 in loop C could form an aromatic π–π interaction with the pyridazine ring of 7e. Because 7e does not possess a carboxyl group, interaction with Ser174 was not observed in the binding of this ligand. The present docking studies of 7e predict that its bulky aromatic substituent at the 3-position is tolerable in the orthosteric binding site. However, when an IP analog with R¹=COOH, R²=4-biphenylyl, and R³=H was docked into an HF Rdl_bd GABAR model in a previous study, the biphenylyl group was oriented in a direction opposite to that observed in the present study.³¹⁾ It is necessary to determine the orientation of the 3-substituent, as this is prerequisite for the development of IP derivatives with high potency against insect GABARs.

Conclusion

We synthesized IP competitive antagonists with low-micromolar IC₅₀ through the bioisosteric replacement of the carboxyl group of gabazine. Ligand-docking studies using an HF GABAR homology model predicted that an aromatic substituent at the 3-position of the pyridazine ring is tolerable in the orthosteric site of insect GABARs. The iminopyridazine analogs could be a lead for the development of insecticides.

Acknowledgment

We thank Dr. T. Nakao for helpful collaborative work. We also thank Dr. T. Kita, Mr. K. Nomura, and Ms. M. Takashima for technical assistance. This work was supported in part by JSPS KAKENHI (Grant Number 26292031).

References

• 1) M. Chebib and G. A. R. Johnston: J. Med. Chem. 43, 1427–1447 (2000).
• 2) N. G. Bowery and S. J. Enna: J. Pharmacol. Exp. Ther. 292, 2–7 (2000).
• 3) B. Bettler, K. Kaupmann, J. Mosbacher and M. Gassmann: Physiol. Rev. 84, 835–867 (2004).
• 4) R. W. Oslen and W. Sieghart: Neuropharmacology 56, 141–148 (2009).
• 5) S. M. Sine and A. G. Engel: Nature 440, 448–455 (2006).
• 6) A. J. Thompson, H. A. Lester and S. C. R. Lummis: Q. Rev. Biophys. 43, 449–499 (2010).
• 7) P. J. Whiting, T. P. Bonnert, R. M. McKernan, S. Farrar, B. Le Bourdellès, R. P. Heavens, D. W. Smith, L. Hewson, M. R. Rigby, D. J. Sirinathsinghji, S. A. Thompson and K. A. Wafford: Ann. N. Y. Acad. Sci. 868, 645–653 (1999).
• 8) D. Zhang, Z.-H. Pan, M. Awobuluyi and S. A. Lipton: Trends Pharmacol. Sci. 22, 121–132 (2001).
• 9) P. S. Miller and T. G. Smart: Trends Pharmacol. Sci. 31, 161–174 (2010).
• 10) R. M. Mckernan and P. J. Whiting: Trends Neurosci. 19, 139–143 (1996).
• 11) G. A. R. Johnston: Pharmacol. Ther. 69, 173–198 (1996).
• 12) S. D. Buckingham and D. B. Sattelle: “Insect Pharmacology: Channels, Receptors, Toxins and Enzymes,” ed. by L. I. Gilbert and S. S. Gill, Elsevier, Amsterdam, pp. 29–64, 2010.
• 13) Y. Ozoe: Adv. Insect Physiol. 44, 211–286 (2013).
• 14) R. H. ffrench-Constant, T. A. Rocheleau, J. C. Steichen and A. E. Chalmers: Nature 363, 449–451 (1993).
• 15) S. D. Buckingham, P. C. Biggin, B. M. Sattelle, L. A. Brown and D. B. Sattelle: Mol. Pharmacol. 68, 942–951 (2005).
• 16) P. Perret, X. Sarda, M. Wolff, T.-T. Wu, D. Bushey and M. Goeldner: J. Biol. Chem. 274, 25350–25354 (1999).
• 17) T. Nakao, A. Naoi, N. Kawahara and K. Hirase: Pestic. Biochem. Physiol. 97, 262–266 (2010).
• 18) T. Nakao, A. Kawase, A. Kinoshita, R. Abe, M. Hama, N. Kawahara and K. Hirase: J. Econ. Entomol. 104, 646–652 (2011).
• 19) T. Nakao, M. Hama, N. Kawahara and K. Hirase: J. Pestic. Sci. 37, 37–44 (2012).
• 20) Y. Ozoe, M. Asahi, F. Ozoe, K. Nakahira and T. Mita: Biochem. Biophys. Res. Commun. 391, 744–749 (2010).
• 21) G. P. Lahm, D. Cordova, J. D. Barry, T. F. Pahutski, B. K. Smith, J. K. Long, E. A. Benner, C. W. Holyoke, K. Joraski, M. Xu, M. E. Schroeder, T. Wagerle, M. J. Mahaffey, R. M. Smith and M.-H. Tong: Bioorg. Med. Chem. Lett. 23, 3001–3006 (2013).
• 22) T. Nakao, S. Banba, M. Nomura and K. Hirase: Insect Biochem. Mol. Biol. 43, 366–375 (2013).
• 23) Y. Ozoe, T. Kita, F. Ozoe, T. Nakao, K. Sato and K. Hirase: Pestic. Biochem. Physiol. 107, 285–292 (2013).
• 24) C.-G. Wermuth, J.-J. Bourguignon, G. Schlewer, J.-P. Gies, A. Schoenfelder, A. Melikian, M.-J. Bouchet, D. Chantreux, J.-C. Molimard, M. Heaulme, J.-P. Chambon and K. Biziere: J. Med. Chem. 30, 239–249 (1987).
• 25) S. Ueno, J. Bracamontes, C. Zorumski, D. S. Weiss and J. H. Steinbach: J. Neurosci. 17, 625–634 (1997).
• 26) A. M. Hosie and D. B. Sattelle: Br. J. Pharmacol. 119, 1577–1585 (1996).
• 27) H. Satoh, H. Daido and T. Nakamura: Neurosci. Lett. 381, 125–130 (2005).
• 28) K. Narusuye, T. Nakao, R. Abe, Y. Nagatomi, K. Hirase and Y. Ozoe: Insect Mol. Biol. 16, 723–733 (2007).
• 29) A. H. Duittoz and R. J. Martin: J. Exp. Biol. 159, 149–164 (1991).
• 30) R. J. Martin, J.-M. Sitamze, A. H. Duittoz and C. G. Wermuth: Eur. J. Pharmacol. 276, 9–19 (1995).
• 31) M. M. Rahman, Y. Akiyoshi, S. Furutani, K. Matsuda, K. Furuta, I. Ikeda and Y. Ozoe: Bioorg. Med. Chem. 20, 5957–5964 (2012).
• 32) T. Nakao and K. Hirase: J. Pestic. Sci. 38, 123–128 (2013).
• 33) Y. Eguchi, M. Ihara, E. Ochi, Y. Shibata, K. Matsuda, S. Fushiki, H. Sugama, Y. Hamasaki, H. Niwa, M. Wada, F. Ozoe and Y. Ozoe: Insect Mol. Biol. 15, 773–783 (2006).
• 34) R. E. Hibbs and E. Gouaux: Nature 474, 54–60 (2011).
• 35) K. S. Schroeder and B. D. Neagle: J. Biomol. Screen. 1, 75–80 (1996).
• 36) C. Joesch, E. Guevarra, S. P. Parel, A. Bergner, P. Zbinden, D. Konrad and H. Albrecht: J. Biomol. Screen. 13, 218–228 (2008).
• 37) I. Yamamoto, J. E. Carland, K. Locock, N. Gavande, N. Absalom, J. R. Hanrahan, R. D. Allan, G. A. R. Johnston and M. Chebib: ACS Chem. Neurosci. 3, 293–301 (2012).
• 38) J. A. Ashby, I. V. McGonigle, K. L. Price, N. Cohen, F. Comitani, D. A. Dougherty, C. Molteni and S. C. R. Lummis: Biophys. J. 103, 2071–2081 (2012).
• 39) I. McGonigle and S. C. R. Lummis: Biochemistry 49, 2897–2902 (2010).
• 40) F. Comitani, N. Cohen, J. Ashby, D. Botten, S. C. R. Lummis and C. Molteni: J. Comput. Aided Mol. Des. 28, 35–48 (2014).
• 41) E. Sigel, R. Baur, S. Kellenberger and P. Malherbe: EMBO J. 11, 2017–2023 (1992).
• 42) D. A. Wagner and C. Czajkowski: J. Neurosci. 21, 67–74 (2001).
• 43) J. H. Holden and C. Czajkowski: J. Biol. Chem. 277, 18785–18792 (2002).
• 44) J. G. Newell and C. Czajkowski: J. Biol. Chem. 278, 13166–13172 (2003).
• 45) D. A. Wagner, C. Czajkowski and M. W. Jones: J. Neurosci. 24, 2733–2741 (2004).
• 46) J. H. Kloda and C. Czajkowski: Mol. Pharmacol. 71, 483–493 (2007).
• 47) J. Zhang, F. Xue and Y. Chang: Mol. Pharmacol. 74, 941–951 (2008).