Effects of monoterpenoid insecticides on [H]-TBOB binding in house fly GABA receptor and Cl uptake in American cockroach ventral nerve cord

Monoterpenoids and their derivatives from plant essential oils showed good insecticidal activities in previous studies, but the mechanisms of their action as natural insecticides are not known yet. In the present work, we evaluated the pharmacological action of five monoterpenoids (α-terpineol, carvacrol, linalool, pulegone, and thymol) on native insect GABA receptors from house flies and American cockroaches using radiotracer methods. In the [³H]-TBOB binding assay, carvacrol, pulegone, and thymol all enhanced the [³H]-TBOB binding to membrane preparation of house fly heads with EC₅₀ values of 48 μM, 432 μM, and 6 mM, respectively. Moreover, these three monoterpenoids at concentrations of 500 μM and 1 mM also significantly increased the ³⁶Cl⁻ uptake induced by GABA in membrane microsacs prepared from American cockroach ventral nerve cords. These results revealed that carvacrol, pulegone, and thymol are all positive allosteric modulators at insect GABA receptors. The other two monoterpenoids that were tested, α-terpineol and linalool, showed little or no effect in both the [³H]-TBOB binding and ³⁶Cl⁻ uptake assays.     

Spirosultam LY219048: A new chemical class of insecticide acting upon the GABA receptor/chloride ionophore complex

This study assessed the toxicity and mode of action of a new experimental insecticide, LY219048 in insects and mammals. LY219048 produced rapid convulsions in mice and had LD₅₀ values of 0.7 mg kg^?1 and 4 mg kg^?1 after intracerebral and intraperitoneal injection, respectively. In initial screens against insects, LY219048 showed low activity against the German cockroach (Blatella germanica L.). Lethality from dietary exposure required one to two weeks, even at concentrations as high as 10000 mg kg^?1 (LC₅₀ = 485 mg kg^?1). In contrast, it had an LC₅₀ value of 8.3 mg kg^?1 against insecticide-susceptible Drosophila melanogaster (Meig.) when synergized with piperonyl butoxide. Significant resistance to LY219048 (> 12-fold) was detected in a cyclodiene-resistant strain of D. melanogaster possessing an altered target site resistance mechanism. This finding suggested that LY219048 blocked the 4-aminobutyric acid (GABA)-gated chloride channel in a manner similar to that of the cyclodienes. In physiological studies in larval D. melanogaster central neurons, LY219048 antagonized the reduction of firing caused by 1 mM GABA. Dose-response experiments showed that the ED₅₀ for blocking inhibition under these conditions was c. 1 μ. Studies of ³⁶CI uptake into bovine brain synaptosomes found that LY219048 was a potent antagonist. At 10 μ it completely blocked chloride flux stimulated by 50 μM GABA. LY219048 competitively displaced [³H]TBOB binding from bovine brain membranes, with an IC₅₀ of 42 nM, which was comparable to values determined for TBPS (35 nM) and picrotoxinin (267 nM). There was little or no displacement (<25%) of [³H]flunitrazepam or [³H]muscimol binding by 10 μM LY219048. Taken together, these results provide strong evidence that this new chemical class of insecticide manifests its acute toxicity by blocking the GABA-gated chloride channel.     

Biochemical identification of putative GABA/benzodiazepine receptors in house fly thorax muscles

[³H]Flunitrazepam ([³H]Flu) was used to identify benzodiazepine binding sites in house fly thorax muscle membranes using a filter assay. [³H]Flu bound to a finite number of sites in a concentration- and time-dependent manner, reaching equilibrium in 10 min. Scatchard plots of the binding indicated a high-affinity site at 0.2 pmol/mg protein (K_d 24.3 nM) and a low-affinity site at 8.2 pmol/mg protein (K_d994nM). Binding of [³H]Flu to the high-affinity binding site was inhibited by several benzodiazepine analogs, with Flu, diazepam, and Ro 5-4864 being more potent than β-CCE, Ro 5-3027, and Ro 5-2180. Clonazepam was least potent in inhibiting [³H]Flu binding. Thus, the drug specificity of these insect muscle benzodiazepine binding sites was quite different from both the mammalian central and peripheral benzodiazepine receptor sites, though closer to the peripheral ones. GABA (γ-aminobutyric acid) and its agonists enhanced the specific binding of [³H]Flu in a dose-dependent manner, and this effect was inhibited with the GABA antagonist bicuculline. The effect was biphasic since at high GABA concentrations this stimulation was reduced. The data suggest that house fly muscles have benzodiazepine receptors, which are coupled allosterically to GABA receptors, analogous to the GABA/benzodiazepine receptors of vertebrates, but with some differences in their drug specificities.     

Presence of Muscarinic Acetylcholine Receptors in the Cattle Tick Boophilus microplus and in Epithelial Tissue Culture Cells of Chironomus tentans

A muscarinic acetylcholine receptor (mAChR) has been demonstrated and partially characterized in larvae of the cattle tick Boophilus microplus. Its properties are compared with mAChR from an epithelial cell line from the dipteran insect Chironomus tentans. Competition studies with cholinergic ligands of different specificity revealed the muscarinic nature of the cholinergic receptors investigated in both species. In homogenates from tick larvae, specific binding sites for [³H]quinuclidinyl benzilate (QNB) with high affinity (1·2±(0·13) nM ; B_max 22·5 pmol mg protein⁻¹) were detected that do not bind nicotinic compounds specifically. The estimated IC₅₀ values for nicotine, imidacloprid and α-bungarotoxin were all in the mM range. Additionally, with tick larvae, high-affinity nicotinic binding sites were detected with [³H]nicotine which could be displaced by high concentrations of imidacloprid or QNB. The estimated IC₅₀ values for nicotine, α-bungarotoxin, imidacloprid and QNB were 43(±8) nM , 0·8(±0·2) μM , 2·8(±0·6) μM and 78(±1·9) μM , respectively. With homogenates of the non-neuronal insect cell line from C. tentans, only high-affinity binding sites for [³H]QNB were found. Muscarinic antagonists selectively displaced [³H]quinuclidinyl benzilate (QNB) binding to tick larvae homogenates. The mAChR of B. microplus preferred pirenzepine (IC₅₀ 2·13(±1·02) μM ) among different subtype-specific mAChR antagonists (4-DAMP had IC₅₀ 49·9(±9·13) μM and methoctramine had IC₅₀ 121(±14·2) μM ) indicating a type of binding site similar to the vertebrate M1 mAChR subtype. The tick muscarinic receptor seems to be a G-protein-coupled receptor, as concluded from the 4·8-fold reduction in receptor affinity for binding of the muscarinic agonist oxotremorine M upon treatment with the non-hydrolysable GTP-analogue γ-S-GTP. Binding data for the agonists oxotremorine M (IC₅₀ 71·3(±19·6) μM ) and carbachol (IC₅₀ 253(±87·1) μM ) parallel the biological efficacy of these compounds, in that, while oxotremorine M showed some activity against ticks, carbachol was ineffective.     

Binding Sites for Ca2+-Channel Effectors and Ryanodine in Periplaneta americana—Possible Targets for New Insecticides

The calcium channel and the ‘calcium release channel’ of muscle membrane of the cockroach Periplaneta americana have been characterized. Biological assays with calcium channel blockers and ryanodine on different insects and acari revealed pronounced insecticidal effects with ryanodine, but not with calcium channel blockers, at concentrations between 0·1 and 300 μg ml⁻¹. Skeletal muscle membranes derived either from the tubular network or from the sarcoplasmatic reticulum of P. americana were characterized with respect to the binding of the dihydropyridine (DHP) [³H]isradipine (PN 200-110), the phenyl-alkylamine [³H]verapamil and the alkaloid [³H]ryanodine. Preliminary binding studies with the benzothiazepine [³H]diltiazem suggest a low-affinity binding site with a IC₅₀ value of 3·3 μM . All binding sites tested were sensitive to treatment with proteinase K. Optimal conditions for binding of the radioligand ryanodine revealed the highest specific binding at pH 8 and at calcium chloride concentrations between 100 and 500 μM . EGTA at 10 μM abolished 95% of the ryanodine binding. Binding studies with calcium channel binding sites revealed a pronounced effect of low Ca²⁺ concentrations on specific isradipine binding, whereas verapamil and diltiazem binding were only reduced by the presence of 200 μM EGTA. With respect to high Ca²⁺ concentrations, specific binding of diltiazem, isradipine and verapamil was reduced by 73, 40 and 20%, respectively, at 5 mM Ca²⁺. Radioligand binding experiments showed high-affinity binding sites for ryanodine and isradipine. K_D values of 0·95 nM (B_max=550 fmol mg⁻¹ protein) and 0·75 nM (B_max=213 fmol mg⁻¹ protein) were determined respectively. A lower-affinity binding site was identified in binding studies with verapamil (K_D=7·4 nM and B_max=27 fmol mg⁻¹ protein). [³H]isradipine displacement studies with several dihydropyridines revealed the following ranking of affinity: nitrendipine>isradipine>Bay K8664≪nicardipine. Displacement of [³H]verapamil binding by effectors of the phenylalkylamine binding site showed that bepridil and S(-)verapamil had the highest affinities of the compounds tested followed by (±)verapamil, nor-methylverapamil and R(+)verapamil.     

GABA receptors of insects susceptible and resistant to cyclodiene insecticides

Toxicity tests revealed up to 40-fold resistance to a number of cyclodiene insecticides in a laboratory-reared, cyclodiene-resistant (CYW) housefly strain (Musca domestica L.). Using [³⁵S] TBPS as a probe for convulsant sites in insects, saturable specific binding was detected in thorax and abdomen membranes prepared from housefly strains susceptible (CSMA) and resistant (CYW) to cyclodienes. Scatchard analysis of[³⁵S] TBPS binding data to CSMA and CYW membranes failed to provide evidence for significant differences between the two strains in either the affinity (K_d) or density (B_max) of saturable binding sites. For several polychlorocycloalkane insecticides, the ligand displacement profile of [³⁵S] TBPS binding was almost identical for the CSMA and CYW houseflies. Therefore, using [³⁵S] TBPS as a probe for convulsant sites, a 40-fold resistance to cyclodienes in the CYW housefly strain cannot be accounted for only in terms of alterations in TBPS binding sites.     

Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors

γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [³H]4′-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [³H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [³H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [³H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [³H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [³H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B_1a, were potent inhibitors of [³H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides in insect GABARs.     

Structural Effects of N-Arylcarbamoylpyrazolines on Calcium Uptake in Rat Brain Synaptosomes

N-Arylcarbamoylpyrazolines with various substituents at the para position of the carbamoyl benzene ring inhibited ATP-dependent Ca²⁺-uptake in synaptosomes prepared from the rat brain. The activity of these compounds was evaluated as log(1/I₅₀), the reciprocal logarithm of half inhibitory concentration, I₅₀ (m ), from the concentration–response curve for the inhibition of Ca²⁺-uptake. Among the compounds tested, methyl 3-(4-chlorophenyl)-4-methyl-1-[N-(4-trifluoromethylphenyl)carbamoyl]-2-pyrazoline-4-carboxylate was the most potent, the I₅₀ value of which as 9·12×10⁻⁷ m . Variations in the activity in terms of log(1/I₅₀) were quantitatively analysed using a substituent parameter, showing that the higher the electron-withdrawing effect of the substituent, the higher was the activity. The substituent effects were similar to those on insecticidal activity against the Americal cockroach. The higher the inhibitory activity against Ca²⁺ uptake, the higher seemed to be the insecticidal activity. Methyl(4S) - 3 - (4 - chlorophenyl) - 4 - methyl - 1 - [N - (4 - chlorophenyl)carbamoyl] - 2 - pyrazoline -4-carboxylate had higher inhibitory activity against Ca²⁺-uptake and higher in-secticidal activity than the R-isomer, but the difference was greater in theCa²⁺-uptake system.     

Enhancement of neurotransmitter release from invertebrate synaptosomes by pyrethroids during pulsed-depolarization: A functional assay for effects on repolarization

The release of [³H]neurotransmitters was used as a functional assay to assess the actions of selected neurotoxins on the synaptosomal membranes prepared from the invertebrate nervous systems of squid and house fly. A reproducible release of [³]neurotransmitter was evoked by pulsed-depolarization in the presence of elevated K⁺ or of veratridine. Pretreatment with deltamethrin resulted in a substantial enhancement of [³H]neuro-transmitter release during pulsed-depolarization. This enhanced neurotransmitter release was greatly reduced or absent when synaptosomes of knockdown-resistant house flies were examined. No enhanced neurotransmitter release due to deltamethrin pretreatment was apparent from any synaptosomal preparation under non-depolarizing conditions. Under similar conditions, collaborative experiments demonstrated that deltamethrin causes a significant change in protein phosphorylation activities which follow depolarization. The most significant change caused by deltamethrin was the prolonged elevation of the level of phosphorylation on a number of key synaptic proteins beyond the normal time of their recovery to the dephosphorylated state. The most notable protein reacting to deltamethrin in this manner was calcium-cadlmodulin-dependent protein kinase.     

Structural modifications increase the insecticidal activity of ryanodine

The toxicity of ryanodine ( 1 ) and 9,21-didehydroryanodine ( 2 ) (the principal active ingredients of the botanical insecticide ryania) to adult female house flies (Musca domestica L.) is attributable to binding to the ryanodine receptor (ryr) and thereby disrupting the Ca²⁺-release channel. These ryanoids, assayed in house flies with piperonyl butoxide (PBO) to suppress cytochrome P450-dependent detoxification, give injected KD₅₀ values of 0·07–0·11 μg g^-1, injected LD₅₀ values of 0·39–0·45 μg g^-1 and topical LD₅₀ values of 12– 50 μg g^-1. They inhibit the [³H]ryanodine binding site of house fly and rabbit muscle with IC₅₀ values of 3–10 nM . This study examines the effect of structure on potency, with 15 variants of the cyclohexane substituents, two 4,6-cyclic boron and two methylated derivatives, and four modifications of the isopropyl and ester substituents. The most effective compound examined was 10-deoxy- 2 ( 3 ) which was more potent than 2 by 2–4-fold on injection and 29-fold applied topically following PBO (LD₅₀ 0·41 μg g^-1). Additional high-potency compounds were 10-oxo- 1 and the cyclohexane variants with lactam, 21-nor-9-oxo and 21-nor-10-deoxy substituents. Other modifications usually reduced toxicity. The injected knockdown potency of the ester ryanoids was generally related to their effectiveness in competing with [³H]ryanodine at the ryr of rabbit skeletal muscle. Two non-ester ryanoids, ryanodol and 9,21-didehydroryanodol, were found to be more toxic than predicted from their potency at the ryr and may therefore act in a different manner such as at a K⁺ channel, as suggested by Usherwood and Vais. Clearly ryanoids are challenging prototypes for a potential new generation of insecticides. © 1997 SCI.     

Interaction of formamidine pesticides with insect neural octopamine receptors: Effects on radioligand binding and cyclic AMP production

The formamidines chlordimeform, demethylchlordimeform (DCDM), amitraz and BTS-2727l were tested for their respective abilities to inhibit [³H]mianserin binding in membrane preparations of cockroach brain and, in the same tissue, to stimulate octopamine-sensitive receptors coupled to adenylate cyclase. Analysis of [³H]mianserin binding indicated negative cooperativity between two binding sites with respective K_d and B_max values of 3.82 mM and 0.886 pmol (mg protein)^?1 for a high-affinity site and 218 nM and 13.56pmol (mg protein)^?1 for a low-affinity site. DCDM, BTS-27271 and amitraz inhibit [³H]mianserin binding with IC₅₀ values similar to those obtained with octopamine and the μ-adrenergic antagonist, phentolamine, whereas chlordimeform is a poor competitor for the binding sites. Similarly DCDM, BTS-27271 and amitraz elevate cyclic AMP production in brain membrane preparations in a dose-dependent manner with K_a values of 0.32 uM, 1.5 uM and 3.4 uM respectively, whereas chlordimeform was again without effect. The formamidine-mediated responses were fully additive with the evaluation of cyclic AMP obtained using the maximal concentration of dopamine but not with octopamine-mediated increases; thus the formamidine effects appear to be expressed through partial agonism of octopamine receptors that are coupled to adenylate cyclase.     

Pyrethroid action on the nicotinic acetylcholine receptor/channel

The interactions of natural pyrethrins and nine pyrethroids with the nicotinic acetylcholine (ACh) receptor/channel complex of Torpedo electric organ membranes were studied. None caused significant reduction in [³H]ACh binding to the receptor sites, but all inhibited [³H]perhydrohistrionicotoxin ([³H]H₁₂-HTX) binding to the channel sites in presence of carbamylcholine. Allethrin inhibited [³H]H₁₂-HTX binding noncompetitively, but [³H]imipramine binding competitively, suggesting that allethrin binds to the receptor's channel sites that bind imipramine. The pyrethroids were divided into two types according to their actions: type I, which included pyrethrins, allethrin, bioallethrin, resmethrin, and tetramethrin, was more potent in inhibiting [³H]H₁₂-HTX binding and acted more rapidly (i.e., in <30 sec). Type II, which included permethrin, fluvalinate, cypermethrin and fenvalerate, was less potent and their potency increased slowly with time. Also, inhibition of the initial rate of [³H]H₁₂-HTX binding by type I compounds increased greatly by the presence of the agonist carbamylcholine, but this was not so with type II compounds. The receptor-regulated ⁴⁵Ca²⁺ flux into Torpedo microsacs was inhibited by pyrethrins and pyrethroids, suggesting that their action on this receptor function is inhibitory. There was very poor correlation between the potencies of pyrethrins and pyrethroids in inhibiting [³H]H₁₂-HTX binding and their toxicities to house flies, mosquitoes, and the American cockroach. However, the high affinities that several pyrethroids have for this nicotinic ACh receptor suggest that pyrethroids may have a synaptic site of action in addition to their well known effects on the axonal channels.     

Quantitative autoradiography of GABA receptors in locust (Schistocerca americana) brain

Conventional film autoradiography was used at the light microscopic level for the localization and quantization of 4-aminobutyric acid (GABA) receptors in the locust brain (Schistocerca americana). Localization of the receptor site was achieved via binding with the receptor-ligand probe [³ H]muscimol. Frozen sections were cut and subsequently incubated either in 40 nM [³H]muscimol or by coincubating sections with [³H]muscimol and one of the following: GABA (50 μM)], a receptor specific agonist [muscimol (1 μM) or isoguvacine (1 μM)], an uptake inhibitor [nipecotic acid (50 μM)], or a noncompetitive channel modulator [avermectin B_1a, (1 μM) or aldrin (50 μM)]. Through computer image enhancement and densitometric analysis of the optical density of [³H]muscimol binding sites, the interaction of the above compounds with the putative GABA receptor was determined for various anatomical regions of the locust brain. By comparing the differently treated, but adjacent sections, GABA receptor distribution was quantitated and mapped. Receptor sites were found distributed in the antennal lobes, central body, β-lobe and β-lobe of the corpus pedunculatum, protocerebral bridge, and calyx as well as the optic lobe regions.     

The metabolism of fenvalerate in plants: The conjugation of the acid moiety

The metabolism of the pyrethroid insecticide fenvalerate [(RS)-α-cyano-3-phenoxybenzyl (RS)-2-(4-chlorophenyl)-3-methylbutyrate] ( I ), and of its most insecticidal (αS,2S) isomer ( II ), has been examined in cabbage plants grown and treated under laboratory conditions with [¹⁴C]chlorophenyl- and [ring-¹⁴C]benzyllabelled preparations of the two compounds. Both insecticides disappeared from the treated leaves with similar half-lives of approximately 12–14 days; they underwent ester cleavage to a significant extent, together with some hydroxylation at the 2- or 4-position of the phenoxy ring, and hydrolysis of the nitrile group to amide and carboxyl groups. Most of the carboxylic acids and phenols thus produced occurred as glycoside conjugates. In separate experiments, the uptake and metabolism of 2-(4-chlorophenyl)-3-methylbutyric acid ( X ), the acidic half of the molecule, were examined in the laboratory, using abscised leaves of kidney bean, cabbage, cotton, cucumber and tomato plants. The acid X was found to be readily converted, mainly into glucose and 6-O-malonylglucose esters in kidney bean, cabbage and cucumber plants, into glucosylxylose, sophorose and gentiobiose esters in cotton, and into two types of triglucose esters with differing isomerism in tomato. One of the acetyl derivatives of the trisaccharide conjugates was identical with the synthetic deca-acetyl derivative of the [1 → 6]-triglucose ester.     

Temperature effects on the action of acylurea insecticides against tobacco hornworm (Manduca sexta) larvae

The toxic effects of six acylurea insecticides on larvae of the tobacco hornworm were investigated at each of four environmental temperatures (20, 25, 30 and 35°C). This spans the range of temperatures which the insects can tolerate. For all the acylureas tested, mortality increased with temperature when either newly hatched or fourth-instar larvae were given insecticide in their food. Sub-lethal growth inhibition also became more pronounced at progressively higher environmental temperatures. This temperature dependence of acylurea action was not due to altered uptake of the insecticide, since there was no significant variation with temperature in the amount of [¹⁴C]flufenoxuron taken up by fifth-instar larvae when given a single meal containing labelled insecticide. Additionally, mortality of fourth-instar larvae given a single intra-haemocoelic injection of flufenoxuron was significantly greater at higher temperatures, implying that temperature affects a process that occurs after insecticide uptake. The intrinsic ability of acylureas to inhibit chitin synthesis is temperaturesensitive, since flufenoxuron inhibited the incorporation of [¹⁴C]N-acetylalucosamine into chitin by proleg epidermis in vitro significantly less well at 20°C than at the higher temperatures tested. However, there was no significant variation between the effectiveness of in-vitro chitin synthesis inhibition at 25, 30 and 35°C. These data show that the effectiveness of acylurea insecticides is subject to strong temperature effects in the range of temperatures likely to be experienced in the field.     

Effect of pyridazinone herbicides on lipid metabolism in groundnut (Arachis hypogaea) leaves

The effect of five substituted pyridazinones (pyrazon, San 133-410H, San 9774, norflurazon, and San 6706) on lipid metabolism in groundnut (Arachis hypogaea) leaves was investigated under nonphotosynthetic conditions. In experiments with leaf disks, the uptake of [1-¹⁴C]acetate, [³²P]orthophosphate, and [³⁵S]sulfate was significantly inhibited by these herbicides and the magnitude of inhibition varied, depending on the substituents. When the incorporation of these precursors into lipids was measured and expressed as percentage of total uptake, no effect was observed in the case of [1-¹⁴C]acetate but there was significant inhibition in the incorporation of the other two precursors, suggesting that pyridazinones interfere with the metabolism of the phospholipids and the sulfolipid. None of these compounds affected the uptake of [methyl-¹⁴C]choline but all inhibited its incorporation into phosphatidylcholine indicating that phosphatidylcholine metabolism is vulnerable to pyridazinones. The fatty acid synthetase of isolated chloroplasts assayed in the absence of light was inhibited 20–50% by the pyridazinones at 0.1–0.5 mM concentrations. San 9774 showed the most potent inhibition. In addition, the pyridazinone herbicides significantly inhibited sn-glycerol-3-phosphate acyltransferase(s) in both chloroplast and microsomal fractions but showed no effect on phosphatidic acid phosphatase. The magnitude of inhibition of fatty acid synthetase and acyltransferase(s) is related to the nature of the substituent groups on the herbicide. Trifluorophenyl substitution at position 2 or amino substitution at position 5 of the pyridazinone molecule caused the maximum inhibitory effect.     

Structure-Activity Relationships for the Action of Dihydropyrazole Insecticides on Mouse Brain Sodium Channels

Assays of radiosodium uptake into mouse brain vesicles and the binding of [³H]batrachotoxinin A 20-α-benzoate (BTX-B) were used to compare the actions of six dihydropyrazole (3-aryl-1-arylcarbamoyl-2-pyrazoline) insecticides on mouse brain sodium channels. The relative potencies of the six dihydropyrazoles as inhibitors of either pumiliotoxin B-stimulated sodium uptake measured in the presence of scorpion (Leiurus quinquestriatus) venom or veratridine-stimulated sodium uptake were closely correlated with the relative potencies of these compounds as inhibitors of the binding of BTX-B to mouse brain sodium channels. A comparison of the enantiomers of the most potent dihydropyrazole, RH 3421, as inhibitors of radiosodium uptake showed that the (−) enantiomer of RH 3421 was approximately six-fold more potent than the (+) enantiomer. The potencies of these dihydropyrazoles in these assays and the stereoselectivity observed in the action of enantiomers of RH 3421 are in good agreement with available information on insecticidal activity in this group of compounds. Assays of the combined effects of RH 3421 and dibucaine as inhibitors of BTX-B binding revealed mutually competitive interactions between these compounds. This finding is consistent with the existence of a common site of action for dihydropyrazoles and local anesthetics on the sodium channel. The results of these studies provide further evidence for the toxicological relevance of the effects of dihydropyrazoles on sodium channels.     

Protective effect of dimercaptosuccinic acid on methylmercury and mercuric chloride inhibition of rat brain muscarinic acetylcholine receptors

The reactivation of the rat brain muscarinic acetylcholine receptor (mACh-R) binding with dimercaptosuccinic acid (DMSA) after in vitro and in vivo inhibition by mercuric chloride (HgCl₂) and methylmercuric chloride (MeHg) was investigated. Receptor binding was estimated by the potent and specific antagonist l-[³H]quinuclidinyl benzilate ([³H]QNB). Rat brain synaptosomal membranes were exposed to HgCl₂ and MeHg. At 1 × 10^?4M. HgCl₂ caused complete inhibition of the [³H]QNB binding. The inhibition of [³H]QNB binding by HgCl₂ was still higher than 50% at 1 × 10^?8M. MeHg caused less inhibition of [³H]QNB binding than HgCl₂. The inhibited receptors showed a significant degree of reactivation when treated with DMSA. The recovery was almost complete after MeHg inhibition or with the lower HgCl₂ concentrations. Generally, the reactivation was dependent on the concentration of DMSA. When rats injected with either early or delayed doses of DMSA following administration with five consecutive daily doses (8 mg/kg body wt, Gavage method) of MeHg or HgCl₂, the inhibition of [³H]QNB binding was less than untreated ones. The early treatment with DMSA decreased the inhibition of [³H]QNB binding due to MeHg or HgCl₂ intoxication. However, DMSA was more effective in reducing HgCl₂ inhibition than MeHg either in vitro or in vivo treatment. The ability of DMSA to reactivate the mACh-R after inhibition with the mercurials emphasizes the involvement of essential sulfhydryl groups in [³H]QNB binding sites, and also shows that these sulfhydryl groups are the primary target for the MeHg and HgCl₂ inhibition of the rat brain muscarinic receptors.     

Studies on the mechanism of action of cymoxanil in Phytophthora infestans

Colony growth and germ tube emergence of sporangia and encysted zoospores of Phytophthora infestans were highly sensitive to cymoxanil (ED₅₀ 0.5–1.5 μg/ml), whereas differentiation of sporangia and zoospore release were insensitive at concentrations up to 100 μg/ml. Treated sporangia did not show distorted germ tubes. Oxygen consumption for glucose oxidation by germinating sporangia and zoospore motility were not inhibited at concentrations up to 100 μg/ml. Cymoxanil hardly affected the uptake of radiolabeled precursors of DNA, RNA, and protein at concentrations up to 100 μg/ml. Incorporation of [¹⁴C]phenylalanine into protein was completely insensitive. RNA synthesis as measured by [³H]uridine incorporation was differentially inhibited in the various developmental stages of the fungus. Inhibition did not occur at differentiation of sporangia, whereas at cyst and sporangial germination and mycelial growth this process was inhibited 20–45% at a concentration of 100 μg cymoxanil/ml. Endogenous RNA polymerase activity of isolated nuclei was not inhibited by cymoxanil. DNA synthesis as measured by [methyl-³H]thymidine incorporation was inhibited 20–80% at the various stages of development at cymoxanil concentrations higher than 10 μg/ml. Metalaxyl, a specific inhibitor of ribosomal RNA synthesis, inhibited [³H]uridine incorporation 40–60% at all developmental stages. The data suggest that although DNA synthesis is affected more than RNA synthesis, inhibition of both biosynthetic processes is a secondary effect. The primary mode of action of cymoxanil thus remains unknown.     

Design, synthesis, insecticidal activity and structure-activity relationship of 3,3-dichloro-2-propenyloxy-containing phthalic acid diamide structures

BACKGROUND: Phthalic acid diamide derivatives are among the most important classes of synthetic insecticides. In this study, a 3,3‐dichloro‐2‐propenyloxy group, the essential active group of pyridalyl derivatives, was incorporated into phthalic acid diamide derivatives with the aim of combining the active groups to generate more potent insecticides. RESULTS: Thirty‐one new phthalic acid diamides were obtained, and these were characterised by ¹H and ¹³C NMR. The structure of N²‐[1,1‐dimethyl‐2‐(methoxy)ethyl]‐3‐iodo‐N¹‐[4‐(3,3‐dichloro‐2‐propenyloxy)‐3‐(trifluoromethyl)phenyl]‐1,2‐benzenedicarboxamide was determined by X‐ray diffraction crystallography. The insecticidal activities of the compounds against Plutella xylostella were evaluated. The title compounds exhibited excellent larvicidal activities against P. xylostella. Structure‐activity relationships revealed that varying the combination of aliphatic amide and aromatic amide moieties, or the nature and position of substituent Y on the aniline ring, could aid the design of structures with superior performance. CONCLUSION: A series of novel phthalic acid diamides containing a 3,3‐dichloro‐2‐propenyloxy group at the 4‐position of the aniline ring were designed and synthesised. Structure‐activity relationships with the parent structure provided information that could direct further investigation on structure modification. Copyright © 2012 Society of Chemical Industry