Ethynyl-4-n-propylbicycloorthobenzoate Research Articles

Fluxametamide: A novel isoxazoline insecticide that acts via distinctive antagonism of insect ligand-gated chloride channels

Fluxametamide is a novel wide-spectrum insecticide that was discovered and synthesized by Nissan Chemical Industries, Ltd. To identify the mode of action of fluxametamide, we first performed [3H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) binding assays. Fluxametamide potently inhibited the specific binding of [3H]EBOB to housefly-head membranes, suggesting that fluxametamide affects insect γ-aminobutyric acid (GABA)-gated chloride channels (GABACls). Next, the antagonism of housefly GABACls and glutamate-gated chloride channels (GluCls) was examined using the two-electrode voltage clamp (TEVC) method. Fluxametamide inhibited agonist responses in both ion channels expressed in Xenopus oocytes in the nanomolar range, indicating that this insecticide is a ligand-gated chloride channel (LGCC) antagonist. The insecticidal and LGCC antagonist potencies of fluxametamide against fipronil-susceptible and fipronil-resistant strains of small brown planthoppers and two-spotted spider mites, which are insensitive to fipronil, were evaluated. Fluxametamide exhibited similar levels of both activities in these fipronil-susceptible and fipronil-resistant arthropod pests. These data indicate that fluxametamide exerts distinctive antagonism of arthropod GABACls by binding to a site different from those for existing antagonists. In contrast to its profound actions on the arthropod LGCCs, the antagonistic activity of fluxametamide against rat GABACls and human glycine-gated chloride channels was nearly insignificant, suggesting that fluxametamide has high target-site selectivity for arthropods over mammals. Overall, fluxametamide is a new type of LGCC antagonist insecticide with excellent safety for mammals at the target-site level.

Synthesis and structure-activity relationship analysis of bicyclophosphorothionate blockers with selectivity for housefly γ-aminobutyric acid receptor channels

Bicyclophosphorothionates (2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane-1-sulfides) are blockers (or non-competitive antagonists) of gamma-aminobutyric acid (GABA) receptor channels. Twenty-two bicyclophosphorothionates with different 3- and 4-substituents were synthesised, and [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) binding assays were performed to evaluate their affinities for housefly and rat GABA receptors. Introduction of an isopropyl group at the 3-position enhanced the affinity of bicyclophosphorothionates for housefly GABA receptors and reduced the affinity towards rat GABA receptors. The 4-isopentyl-3-isopropylbicyclophosphorothionate showed the highest affinity for housefly GABA receptors (IC(50) = 103 nM) among the analogues tested, while the 4-cyclohexylbicyclophosphorothionate showed the highest affinity for rat GABA receptors (IC(50) = 125 nM). Among the bicyclophosphorothionates synthesised to date, the former analogue exhibited the highest selectivity for housefly GABA receptors, with an IC(50)(rat)/IC(50)(fly) ratio of approximately 97. Three-dimensional GABA receptor models successfully explained the structure-activity relationships of the bicyclophosphorothionates. The results indicate that minor structural modifications of blockers can change their selectivity for insect versus mammalian GABA receptors. The substituent at the 3-position of the bicyclophosphorothionates dictates selectivity for housefly versus rat GABA receptors. This information should prove useful for the design of safer insecticides and parasiticides.

The antiparasitic isoxazoline A1443 is a potent blocker of insect ligand-gated chloride channels

A structurally unique isoxazoline class compound, A1443, exhibits antiparasitic activity against cat fleas and dog ticks comparable to that of the commercial ectoparasiticide fipronil. This isoxazoline compound inhibits specific binding of the γ-aminobutyric acid (GABA) receptor channel blocker [3H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) to housefly-head membranes, with an IC50 value of 455pM. In contrast, the IC50 value in rat-brain membranes is>10μM. To study the mode of action of this isoxazoline, we utilized MdGBCl and MdGluCl cDNAs, which encode the subunits of housefly GABA- and glutamate-gated chloride channels, respectively. Two-electrode voltage clamp electrophysiology was used to confirm that A1443 blocks GABA- and glutamate-induced chloride currents in Xenopus oocytes expressing MdGBCl or MdGluCl channels, with IC50 values of 5.32 and 79.9 nM, respectively. Blockade by A1443 was observed in A2′S-MdGBCl and S2′A-MdGluCl mutant channels at levels similar to those of the respective wild-types, and houseflies expressing A2′S-MdGBCl channels were as susceptible to A1443 as standard houseflies. These findings indicate that A1443 is a novel and specific blocker of insect ligand-gated chloride channels.

Studies on the mode of action and the selectivity of insecticides acting on ligand-gated ion channels in the nervous system

The mode of action and the mechanisms for the selectivity of neurotoxic insecticides were investigated using computational chemistry, molecular biology, homology modeling of the target site and X-ray crystallography. Whole-cell, patch-clamp electrophysiology has been used to show that non-competitive antagonists (NCAs), such as 4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) and γ-hexachlorocyclohexane (γ-HCH), are more effective blockers of native cockroach (Periplaneta americana) and recombinant house fly (Musca domestica) γ-aminobutyric acid receptors (GABARs) than glutamate-gated chloride channels (GluCls). Site-directed mutagenesis studies have been used to show that that Ser278 in the second transmembrane region is important for determining the less potent actions of these NCAs on GluCls. The structure-activity relationship and the mechanism of the selectivity of neonicotinoids targeting nicotinic acetylcholine receptors (nAChRs) have also been investigated. Electrophysiological studies show that loops C and D of insect nAChRs are critical in determining the selectivity of neonicotinoids, results consistent with the crystal structures of Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP) in complex with imidacloprid. Neonicotinoids show diverse actions on nAChRs as measured using voltage-clamp electrophysiology. Single channel recording and X-ray crystallography are helping to elucidate the mechanism of super agonist actions of clothianidin and an analog.

Molecular cloning of a GABA receptor subunit from Laodelphax striatella (Fallén) and patch clamp analysis of the homo-oligomeric receptors expressed in a Drosophila cell line

A cDNA encoding a gamma-aminobutyric acid (GABA) receptor subunit was cloned from the small brown planthopper Laodelphax striatella. The L. striatella GABA receptor subunit was found to have high amino acid sequence similarity to the bd-type splice variant of the Drosophila GABA receptor Rdl subunit and several other GABA receptor subunits, with identities of over 70%. The cDNA was inserted into the expression vector pAc5.1-lac-Hygro. Clonal cell lines stably expressing homo-oligomeric L. striatella GABA receptors were generated by transfecting the vector into D.mel-2 cells. Expression of functional GABA receptors in the cell lines was demonstrated by whole-cell patch clamp recordings. GABA induced inward currents with an EC(50) value of 29 microM and a Hill coefficient of 1.7. The GABA-evoked responses reversed close to the Nernst equilibrium potential for chloride ions. The amplitudes of agonist-induced currents were found to be in the order muscimol (100 microM) >/= GABA (100 microM) > isoguvacine (100 microM) > cis-4-aminocrotonic acid (CACA) (100 microM) > 5-(4-piperidyl)-3-isoxazolol (4-PIOL) (1 mM). Antagonists such as fipronil (100 nM), 4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) (100 nM), dieldrin (100 nM) and SR95531 (gabazine) (1 microM) suppressed GABA-induced currents. The functional expression of a GABA receptor from an agricultural pest presents a unique opportunity to discover new molecules active at this important target site.

Synthesis, 3D-QSAR, and docking studies of 1-phenyl-1 H-1,2,3-triazoles as selective antagonists for β3 over α1β2γ2 GABA receptors

A series of 16 1-phenyl-1 H-1,2,3-triazoles with substituents at both the 4- and 5-positions of the triazole ring were synthesized, and a total of 49 compounds, including previously reported 4- or 5-monosubstituted analogues, were examined for their ability to inhibit the specific binding of [ 3H]4′-ethynyl-4- n-propylbicycloorthobenzoate (EBOB), a non-competitive antagonist, to human homo-oligomeric β3 and hetero-oligomeric α1β2γ2 γ-aminobutyric acid (GABA) receptors. Among all tested compounds, the 4- n-propyl-5-chloromethyl analogue of 1-(2,6-dichloro-4-trifluoromethylphenyl)-1 H-1,2,3-triazole showed the highest level of affinity for both β3 and α1β2γ2 receptors, with K i values of 659 pM and 266 nM, respectively. Most of the tested compounds showed selectivity for β3 over α1β2γ2 receptors. Among all 1-phenyl-1 H-1,2,3-triazoles, the 4- n-propyl-5-ethyl analogue exhibited the highest (>1133-fold) selectivity, followed by the 4- n-propyl-5-methyl analogue of 1-(2,6-dibromo-4-trifluoromethylphenyl)-1 H-1,2,3-triazole with a >671-fold selectivity. The 2,6-dichloro plus 4-trifluoromethyl substitution pattern on the benzene ring was found to be important for the high affinity for both β3 and α1β2γ2 receptors. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) provided similar contour maps, revealing that an electronegative substituent at the 4-position of the benzene ring, a compact, hydrophobic substituent at the 4-position of the triazole ring, and a small, electronegative substituent at the 5-position of the triazole ring play significant roles for the high potency in β3 receptors. Molecular docking studies suggested that the putative binding sites for 1-phenyl-1 H-1,2,3-triazole antagonists are located in the channel-lining 2′-6′ region of the second transmembrane segment of β3 and α1β2γ2 receptors. A difference in the hydrophobic environment at the 2′ position might underlie the selectivity of 1-phenyl-1 H-1,2,3-triazoles for β3 over α1β2γ2 receptors. The compounds that had high affinity for β3 receptors with homology to insect GABA receptors showed insecticidal activity against houseflies with LD 50 values in the pmol/fly range. The information obtained in the present study should prove helpful for the discovery of selective insect control chemicals.

The channel-lining 6′ amino acid in the second membrane-spanning region of ionotropic GABA receptors has more profound effects on 4′-ethynyl-4-n-propylbicycloorthobenzoate binding than the 2′ amino acid

The noncompetitive antagonist of ionotropic gamma-aminobutyric acid (GABA) receptors 4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) is a useful tool to probe the antagonist-binding site. In the present study, four mutants of the human GABA(A) receptor beta3 subunit were stably expressed in S2 cells and examined for their abilities to bind [(3)H]EBOB to identify the binding site of EBOB. The homo-oligomeric beta3 GABA receptor was used as a housefly GABA receptor model, as the beta3 subunit has a high sequence similarity with the housefly Rdl subunit in the second membrane-spanning (M2) region. The A274S mutation at the -1' position in the M2 region had no effect on [(3)H]EBOB binding. The A277S mutation at the 2' position led to a decrease in the affinity of EBOB for the GABA receptor. The T281V mutant at the 6' position and the A277S/T281V double mutant completely abolished the binding ability. A beta3 GABA receptor homology model predicts these interactions between the receptor and EBOB. These results suggest that EBOB interacts with threonine 281 and alanine 277, and that threonine 281 plays a more critical role in interacting with EBOB than alanine 277.

Synthesis and Structure−Activity Relationships of 1-Phenyl-1H-1,2,3-triazoles as Selective Insect GABA Receptor Antagonists

To study the interaction of phenylheterocycles with gamma-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 antagonist, to the housefly and rat GABA receptors, as well as to the beta3 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 beta3 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 beta3 receptors. The human beta3 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.

Differential blocking actions of 4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) and γ-hexachlorocyclohexane (γ-HCH) on γ-aminobutyric acid- and glutamate-induced responses of American cockroach neurons

4'-Ethynyl-4-n-propylbicycloorthobenzoate (EBOB) has been employed extensively as a radioligand in binding assays to evaluate the pharmacology of gamma-aminobutyric acid (GABA)-gated Cl- channels (GABARs) of insects and mammals, and gamma-hexachlorocyclohexane (gamma-HCH) was used as an insecticide targeting insect GABARs. Since recent studies have shown that not only GABARs but also glutamate-gated chloride channels (GluCls) are blocked by picrotoxinin, dieldrin and fipronil, the actions of EBOB and gamma-HCH on native GABARs and GluCls of terminal abdominal ganglion neurons in American cockroach (Periplaneta americana) were tested using patch-clamp electrophysiology. A marked run-down of the GABA- and glutamate-induced responses of the cockroach neurons occurred, when a standard pipette solution was employed, but addition of pyruvate to the solution permitted stable recordings of these responses. With this solution, EBOB and gamma-HCH were found to block not only the GABA- but also glutamate-gated responses, with the actions augmented by repeated co-application with the agonists. It was also found that prolonged pre-application of EBOB and gamma-HCH prior to co-application with GABA and glutamate resulted in enhanced blocking actions, indicating resting-state actions of the blockers. The blocking actions of EBOB and gamma-HCH on the GABA- and glutamate-induced responses were compared by determining IC50 values under steady state condition. The IC50 values for the actions of EBOB on GABAR and GluCls differed less than those of gamma-HCH.

Novel 17β-Substituted Conformationally Constrained Neurosteroids that Modulate GABAA Receptors

The goal of this study was to develop a series of allopregnanolone analogues substituted by conformationally constrained 17beta side chains to obtain additional information about the structure-activity relationship of 5alpha-reduced steroids to modulate GABA(A) receptors. Specifically, we introduced alkynyl-substituted 17beta side chains in which the triple bond is either directly attached to the 17beta-position or to the 21-position of the steroid skeleton. Furthermore, we investigated the effects of C22 and C20 modification. The in vitro binding affinity for the GABA(A) receptor of the new analogues was measured by allosteric displacement of the specific binding of [(3)H]4'-ethynyl-4-n-propyl-bicycloorthobenzoate (EBOB) to GABA(A) receptors on synaptosomal membranes of rat cerebellum. An allosteric binding model that has been successfully applied to ionotropic glycine receptors was employed. The most active derivative is (20R)-17beta-(1-hydroxy-2,3-butadienyl)-5alpha-androstane-3-ol (20), which possesses low nanomolar potency to modulate cerebellar GABA(A) receptors and is 71 times more active than the control compound allopregnanolone. Theoretical conformational analysis was employed in an attempt to correlate the in vitro results with the active conformations of the most potent of the new analogues.

Structure–Activity Relationships of seco-Prezizaane Terpenoids in γ-Aminobutyric Acid Receptors of Houseflies and Rats

Thirteen seco-prezizaane terpenoids isolated from star anise species ( Illcium floridanum, Illcium parviflorum, and Illcium verum) were investigated for their ability to inhibit the specific binding of [ 3H]4′-ethynyl-4- n-propylbicycloorthobenzoate (EBOB), a non-competitive antagonist of γ-aminobutyric acid (GABA) receptors, to housefly-head and rat-brain membranes. Veranisatin A was found to be the most potent inhibitor in both membranes, with an IC 50 fly of 78.5 nM and an IC 50 rat of 271 nM, followed by anisatin (IC 50 fly=123 nM; IC 50 rat=282 nM). Six of the other 11 tested compounds were effective only in housefly-head membranes. Pseudoanisatin proved to display a high (>26-fold) selectivity for housefly versus rat GABA receptors (IC 50 fly=376 nM; IC 50 rat >10,000 nM). Although pseudoanisatin does not structurally resemble EBOB, Scatchard plots indicated that the two compounds bind to the same site in housefly receptors. Anisatin and pseudoanisatin exhibited moderate insecticidal activity against German cockroaches. Comparative molecular field analysis (CoMFA), a method of three-dimensional quantitative structure–activity relationship (3D-QSAR) analysis, demonstrated that seco-prezizaane terpenoids can bind to the same site as do picrotoxane terpenoids such as picrotoxinin and picrodendrins, and the CoMFA maps allowed us to identify the parts of the molecules essential to high activity in housefly GABA receptors.

5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid and its derivatives inhibit ionotropic γ-aminobutyric acid receptors by binding to the 4′-ethynyl-4-n-propylbicycloorthobenzoate site

Acyclic noncompetitive antagonists of ionotropic γ-aminobutyric acid (GABA) receptors, bearing an ester or ether linkage, were designed, synthesized, and assayed for their inhibition of the specific binding of [3H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a radiolabeled noncompetitive antagonist, to rat brain and housefly head membranes. 5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid (DBCPP), a butyl benzoate analogue, was found to competitively inhibit the binding of [3H]EBOB in rat brain membranes, with an IC50 of 88 nM. The potency conferred by the p-substituent decreased in the order CC(CH2)2COOH>CC(CH2)2COOCH3>CCH>Br. Pentyl phenyl ethers were equally potent compared with butyl benzoates, while phenyl pentanoates and benzyl butyl ethers were less potent. These compounds were generally less active in housefly head membranes than in rat brain membranes. The introduction of an isopropyl group into the 1-position of the 3,3-dimethylbutyl group of a butyl benzoate and two benzyl butyl ethers caused an increase in potency in housefly GABA receptors, whereas this modification at the corresponding position of other compounds led to an unchanged or decreased potency. In the case of rat receptors, this modification resulted in a decrease in potency except for a phenyl pentanoate. To confirm that DBCPP interferes with GABA receptor function, we performed whole-cell patch clamp experiments with rat dorsal root ganglion neurons in the primary culture. Repeated co-applications of GABA and DBCPP suppressed GABA-induced whole-cell currents with an IC50 of 0.54 μM and a Hill coefficient of 0.7. These findings indicate that DBCPP and its derivatives inhibit ionotropic GABA receptors by binding to the EBOB site and that there might be structural difference in the noncompetitive antagonist-binding site between rat and housefly GABA receptors.

Fipronil-Related Heterocyclic Compounds: Structure–Activity Relationships for Interaction with γ-Aminobutyric Acid- and Voltage-Gated Ion Channels and Insecticidal Action

To investigate the role of the heterocyclic moieties of nitrogen-containing phenyl heterocyclic compounds (PHCs) in interaction with γ-aminobutyric acid (GABA)-gated chloride channels, diverse classes of PHCs were examined for their ability to inhibit the specific binding of [3H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a noncompetitive GABA antagonist, to housefly head and rat brain membranes. PHCs that inhibited [3H]EBOB binding include pyrazoles; 1H-1,2,3-triazoles; a 1H-1,2,4-triazole; 1,3,4-oxadiazol-2(3H)-ones; a 1,3,4-oxadiazole-2(3H)-thione; 1,2,4-oxadiazoles; a 1,2,4-thiadiazole; thiazoles; a 4(3H)-pyrimidinone; and a 2,4(1H,3H)-pyrimidinedione. An analogue (1) of the pyrazole insecticide fipronil, bearing an SCF3 group in place of the S(O)CF3, was found to be the most potent inhibitor with IC50s of 7.55 and 177 nM in housefly head and rat brain membranes, respectively. 3-(2,6-Dichloro-4-trifluoromethyl phenyl)-5-t-butyl-1,3,4-oxadiazol-2(3H)-one exhibited the highest selectivity for housefly GABA receptors versus rat receptors (IC50 rat/IC50 fly >204). PHCs that exhibited a comparable selectivity include a pyrazole and a 1H-1,2,3-triazole. Interaction of 16 selected PHCs with rat brain GABA-gated channels was also examined using [3S]t-butylbicyclophosphorothionate (TBPS), a radioligand for the mammalian noncompetitive antagonist site. A plot of pIC50s of 12 PHCs revealed a close correlation (r = 0.93) between their potency in inhibiting [3H]EBOB and [35S]TBPS binding. Scatchard analyses of the inhibition of [35S]TBPS binding by 1 suggested a competitive-type inhibition. A plot of the potency of 14 PHCs in inhibiting [3H]EBOB binding to housefly head membranes against their piperonyl butoxide-synergized insecticidal effect on German cockroaches yielded a close correlation (r = 0.89), with the exception of five triazoles and a 2,4(1H,3H)-pyrimidinedione. Although several selected PHCs also inhibited the specific binding of [3H]batrachotoxinin A 20-α-benzoate, a tritiated analogue of the sodium channel activator batrachotoxinin, to synaptosomes and membranes prepared from mouse brains, housefly heads, and American cockroach nerve cords, the concentrations required were higher than those of standard compounds producing significant effects on this system. The results demonstrate that a variety of five- and six- membered, nitrogen-containing heterocyclic compounds, bearing a 2,6-dichloro-4-trifluoromethyphenyl or 2,4,6- trichlorophenyl group, interact with ionotropic GABA receptors. Several PHCs display higher affinities for housefly GABA receptors and high selectivity as compared to rat GABA receptors. PHCs' insecticidal activity is mediated by their interaction with GABA-gated chloride channels.

Bicyclophosphorothionate antagonists exhibiting selectivity for housefly GABA receptors

2,6,7-Trioxa-1-phosphabicyclo[2.2.2]octane 1-sulfides (bicyclophosphorothionates) with various C1–4 alkyl groups at the 3- and 4-positions were synthesized and tested for their ability to compete with [3H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a non-competitive antagonist of γ-aminobutyric acid (GABA) receptors, for specific binding to rat-brain and housefly-head membranes, and for their insecticidal activity against houseflies. Among the 3,4-substituted analogues, 20 compounds were selectively active for housefly GABA receptors versus rat GABA receptors. The 3-alkyl groups of C3 length and the 4-alkyl groups of C4 length were tolerated in housefly receptors, whereas such bulky substituents were deleterious in rat receptors. The 4-isobutyl-3-isopropyl analogue was the most potent in housefly receptors (IC50 = 45.2 nM), and tert-butylbicyclophosphorothionate (TBPS), with the 4-tert-butyl group and no 3-substituent, was the most potent in rat receptors (IC50 = 62.2 nM). Their receptor selectivities (rat IC50/housefly IC50) were 52 and 0.038, respectively. The insecticidal activity (LD50) of 20 active analogues was well correlated with their potency (IC50) in inhibiting [3H]EBOB binding to housefly-head membranes (r = 0.93). The results obtained in the present study indicate that the introduction of appropriate alkyl groups into the 3- and 4-positions of bicyclophosphorothionate leads to non-competitive antagonists with increased affinity and selectivity for housefly ionotropic GABA receptors versus rat GABAA receptors. © 1999 Society of Chemical Industry