Structural effects of dinotefuran and analogues in insecticidal and neural activities

The insecticidal potencies of dinotefuran and analogues against the adult male American cockroach, Periplaneta americana (L) were measured by injection with or without metabolic inhibitors. The potency of dinotefuran was close to those of clothianidin and imidacloprid under the conditions used. The nerve-excitatory and nerve-blocking activities were measured with central nerve cords of P americana. The nerve-excitatory activity of dinotefuran was lower than that of imidacloprid, but was comparable with that of clothianidin. The nerve-blocking activity of dinotefuran was comparable with that of imidacloprid and slightly higher than that of clothianidin. Quantitative analyses showed that variations in the insecticidal activity were better correlated with variations in the nerve-blocking activity than with those in the nerve-excitatory activity when the contribution of the hydrophobic factor was allowed for.     

Identification of a high-affinity binding site for dinotefuran in the nerve cord of the American cockroach

The binding of the neonicotinoid insecticide dinotefuran to insect nicotinic acetylcholine receptors (nAChRs) was examined by a centrifugation method using the nerve cord membranes of American cockroaches and [3H]dinotefuran (78 Ci mmol-1). The Kd and Bmax values of [3H]dinotefuran binding were estimated to be 13.7 nM and 14.8 fmol 40 microg-1 protein respectively by Scatchard analysis. Epibatidine, an nAChR agonist, showed a rather lower affinity to the dinotefuran binding site (IC50=991 nM) than dinotefuran (IC50=5.02 nM). Imidacloprid and nereistoxin displayed lower potencies than dinotefuran but higher potencies than epibatidine. The potencies of five dinotefuran analogues in inhibiting the specific binding of [3H]dinotefuran to nerve cord membranes were determined. A good correlation (r2=0.970) was observed between the -log IC50 values of the tested compounds and their piperonyl butoxide-synergised insecticidal activities (-log LD50 values) against German cockroaches. The results indicate that a high-affinity binding site for dinotefuran is present in the nerve cord of the American cockroach and that the binding of ligands to the site leads to the manifestation of insecticidal activity.     

Nicotinic acetylcholine receptor binding of imidacloprid-related diaza compounds with various ring sizes and their insecticidal activity against Musca domestica

Fifteen 5-substituted 1-(6-chloro-3-pyridylmethyl)-2-nitromethylene-1,3- diazacyclohexanes and three other related compounds having a five- or seven-membered ring were synthesized and their biological activities were measured in vivo and in vitro. The insecticidal (in vivo) activity was evaluated against houseflies Musca domestica L under synergistic conditions with propargyl propyl phenyl phosphonate and piperonyl butoxide. The binding activity of each compound to nicotinic acetylcholine receptor in vitro was measured using [125I] alpha-bungarotoxin. The insecticidal activities of the unsubstituted diazacyclohexane analogues were slightly higher than those of the imidazolidine analogues, but the enlargement of ring size to diazacycloheptane lowered the activity. Substitution of 1,3-diazacyclohexane or imidazolidine rings was not generally favourable for the activity, but the unsubstituted 1,3-diazacyclohexane analogue showed the highest binding activity. Ring substitutions and ring enlargement decreased the activity 100-30,000-fold.     

Interaction of dinotefuran and its analogues with nicotinic acetylcholine receptors of cockroach nerve cords

To investigate the action of dinotefuran (MTI-446, 1-methyl-2-nitro-3-(tetrahydro-3-furylmethyl)guanidine), a recently developed insecticide, on insect nicotinic acetylcholine receptors (nAChRs), we determined the potencies of the compound and 15 analogues in inhibiting the specific binding of [3H]epibatidine (EPI), a nAChR agonist, and [3H]alpha-bungarotoxin (alpha-BGT), a competitive nAChR antagonist, to the nerve cord membranes of American cockroaches (Periplaneta americana). Racemic dinotefuran inhibited [3H]EPI binding with an IC50 of 890 nM and [3H]alpha-BGT binding with an IC50 of 36.1 microM. Scatchard analysis indicated that the dinotefuran inhibition of [3H]EPI binding was a competitive one. Slight structural modification caused a drastic reduction in potency; only four analogues were found to be equipotent to or more potent than dinotefuran. Chloropyridinyl and chlorothiazolyl neonicotinoid insecticides displayed two or three orders of magnitude higher potency than dinotefuran. There was a good correlation between the IC50 values of tested compounds obtained with [3H]EPI and those obtained with [3H]alpha-BGT. A better correlation was observed between 3-h knockdown activities (KD50) against German cockroaches (Blattella germanica) and IC50 values obtained from [3H]EPI assays than between 24-h lethal activities (LD50) and IC50 values. While the results indicate that dinotefuran and its analogues interact with the ACh-binding site in cockroach nAChRs, it remains to be elucidated why they displayed lower potencies than those expected based on their insecticidal activities.     

Correlations of the electrophysiological activity of neonicotinoids with their binding and insecticidal activities

The electrophysiological actions of various neonicotinoids, including substituted benzyl derivatives, against recombinant Drosophila SAD/chicken beta2 hybrid nicotinic acetylcholine receptor (nAChR) were measured to analyze the relationships between the in vivo (insecticidal) and in vitro (binding and agonist) activities. Most of the neonicotinoids tested were capable of inducing inward currents by activating the hybrid nAChRs expressed in Xenopus laevis oocytes, whereas some compounds had no agonist activity and only blocked the acetylcholine-induced currents. Variations in the agonist activity were well correlated with those in the binding potency evaluated using [3H]imidacloprid as well as insecticidal activities.     

Insecticidal and binding activities of N3-substituted imidacloprid derivatives against the housefly Musca domestica and the alpha-bungarotoxin binding sites of nicotinic acetylcholine receptors

N3-substituted imidacloprid congeners containing C1-C6 alkyl groups or various analogous groups, and their corresponding nitromethylene analogues, were used in this study. Their insecticidal activity against the housefly, Musca domestica, and their binding activity toward the nicotinic acetylcholine receptor were determined. The insecticidal test was conducted using the synergists piperonyl butoxide and propargyl propyl phenylphosphonate. The binding assay was performed with housefly head membrane preparations using radio-labelled alpha-bungarotoxin. Both insecticidal and binding activities were drastically lowered by the introduction of alkyl/allyl groups at the imidazolidine NH sites of both nitroimino and nitromethylene compounds. The binding activity of N3-substituted nitromethylene analogues was much higher than that of the corresponding nitroimino analogues. However, the insecticidal activity of both series of compounds with a given substituent was nearly identical. The insecticidal activity correlated positively with the binding activity after taking into account the structural difference of the nitroimino and nitromethylene moieties and a structural feature of the N3-substituents.     

Actions of quinolizidine alkaloids on Periplaneta americana nicotinic acetylcholine receptors

BACKGROUND: Botanical insecticides do not play a major role as crop protectants, but they are beneficial in some applications. The authors investigated the actions of naturally occurring alkaloids on insect nicotinic acetylcholine (ACh) receptors (nAChRs) by evaluating their abilities to inhibit specific binding of [³H]imidacloprid (IMI) to nerve‐cord membranes from Periplaneta americana L. Two alkaloids were also tested for their actions on nAChRs expressed by cockroach neurons using patch‐clamp electrophysiology. RESULTS: Four natural quinolizidine alkaloids (matrine, sophocarpine, cytisine and aloperine) exhibited more than 50% inhibition of [³H]IMI binding at 10 µM , although other compounds were found to have no or low inhibitory activity. The rank order of potency based on concentration–inhibition curves was cytisine > sophocarpine ≥ aloperine ≥ matrine. Patch‐clamp analysis indicated that sophocarpine and aloperine were not agonists of nAChRs expressed in P. americana neurons, yet, at 10 µM , aloperine, but not sophocarpine, suppressed ACh‐induced inward currents significantly. CONCLUSION: Three of the four natural alkaloids tested possess structural moieties that are necessary for interaction with P. americana nAChRs. Aloperine, which possesses a unique structure and showed a distinctive dose–response curve, was found to act as an antagonist. Appropriate modifications of these alkaloids might result in novel insecticidal nAChR ligands. Copyright © 2008 Society of Chemical Industry     

Binding activity of substituted benzyl derivatives of chloronicotinyl insecticides to housefly‐head membranes,and its relationship to insecticidal activity against the housefly Musca domestica

Variously substituted benzyl derivatives of chloronicotinyl insecticides were synthesized with a wide range of substituents including halogens, NO₂, CN, CF₃ and small alkyl and alkoxy groups at the ortho, meta and para positions, as well as multiple‐substituted benzyl analogues. Their binding activity to the α‐bungarotoxin binding site in housefly (Musca domestica) head membrane preparations was measured. Among the compounds tested, the activity of the meta‐CN derivative was the highest, being 20–100 times higher than those of imidacloprid, acetamiprid and nitenpyram. The synergized insecticidal activity against houseflies was also measured for selected compounds with the metabolic inhibitor, NIA16388 (propargyl propyl phenylphosphonate). For the nitromethylene analogues, including both benzyl and pyridylmethyl analogues, higher binding activity usually resulted in higher insecticidal activity. © 2000 Society of Chemical Industry     

Application of computational methods in the analysis of pesticide target-site and resistance mechanisms

Meta-diamide insecticides including broflanilide have a high insecticidal activity by acting on RDL GABA receptors. Both membrane potential assays and docking studies suggest that the target site of meta-diamides is different from that of conventional noncompetitive inhibitors, such as fipronil. In fact, meta-diamides are effective against cyclodiene- and fipronil-resistant pests that carry target-site mutations. Dinotefuran uniquely possesses a tetrahydrofuran ring, whereas other neonicotinoids possess aromatic rings. Moreover, dinotefuran has been reported to be effective against imidacloprid-resistant strains. A docking study predicted the weak binding of dinotefuran to cytochrome P450s which are associated with imidacloprid resistance. Metabolic assays revealed that dinotefuran was not metabolized by these cytochrome P450s. These findings suggest that the lack of metabolic activity of P450s against dinotefuran causes a low level of cross-resistance.     

Characterisation of the relationship between binding sites for imidacloprid and other nicotinic ligands in insects

Radioligand binding studies using the neurotoxins α-bungarotoxin, epibatidine, imidacloprid (IMI) and methyllycaconitine reveal heterogeneity at the level of the nicotinic acetylcholine receptor (nAChR) in membranes from the peach potato aphid Myzus persicae (Sulzer) and further suggest the presence of more than one ligand binding site per nAChR. These sites are able to interact allosterically with each other. Of particular interest, [³H]IMI has over an order of magnitude higher affinity in membranes of hemipteran pest species than in non-hemipteran insects, which may help explain why IMI is particularly effective for the control of sucking pests.     

Sites of Action of Noncompetitive GABA Antagonists in Houseflies and Rats: Three-Dimensional QSAR Analysis

Quantitative structure–activity relationships for insecticidal activity (against houseflies) and competitive activity against a specific [³⁵S]tert-butylbicyclophosphorothionate binding (to rat brain membranes) of some picrotoxinin-type 4-aminobutyric acid antagonists, including γ-BHC, endosulfan, bicyclophosphates, dioxatricyclododecenes and related compounds, were examined three-dimensionally using comparative molecular field analysis (CoMFA). The antagonists were classified into two series according to their molecular shapes: i.e. whether their structure was ‘linearly’ extended beyond the ‘mast-head’ position of the ‘boat-like’ skeletons (series 1) or not (series 2). The CoMFA showed that the slopes in steric and electrostatic fields around the molecule were significant for both series in governing the potency variations in insecticidal and binding activities. Hydrophobicity, a possible factor controlling transport behaviour of compounds, was significant in governing variations in insecticidal activity, but not for the case of the rat membrane binding. Assuming that there is a slight topological difference between series 1 and 2 compounds in terms of the mode of binding with the housefly receptor site, the insecticidal activity was analysable with a single equation for the combined set of compounds, but the rat membrane binding was not. The sterically and electrostatically favourable regions surrounding the molecular series indicated by CoMFA were roughly located at positions so as to interact with the binding subsites on the receptors proposed previously. © of SCI.     

Novel mode of action of spinosad: Receptor binding studies demonstrating lack of interaction with known insecticidal target sites

The ability of spinosyn A to either enhance or displace binding to selected insecticidally-relevant receptors was investigated using a number of radioligands including, [³H]imidacloprid and [³H]ivermectin in tissues from the ventral nerve cord (VNC) membranes of the American cockroach, Periplaneta americana and head membranes from the housefly, Musca domestica. In these insect neural tissues, spinosyn A does not appear to alter the binding of a number of radioligands suggesting that spinosyn A does not interact directly with a variety of known receptors, including nicotinic or γ-aminobutyric acid (GABA)-based insecticidal target sites. However, available data are consistent with spinosyn A interacting with a site distinct from currently known insecticidal target sites, thus supporting a novel insecticidal mechanism of action for the spinosyns.     

Comparative analysis of neonicotinoid binding to insect membranes: I. A structure-activity study of the mode of [3H]imidacloprid displacement in Myzus persicae and Aphis craccivora

Neonicotinoids bind selectively to insect nicotinic acetylcholine receptors with nanomolar affinity to act as potent insecticides. While the members of the neonicotinoid class have many structural features in common, it is not known whether they also share the same mode of binding to the target receptor. Previous competition studies with [3H]imidacloprid, the first commercialised neonicotinoid, indicated that thiamethoxam, representing a novel structural sub-class, may bind in a different way from that of other neonicotinoids. In the present work we analysed the mode of [3H]imidacloprid displacement by established neonicotinoids and newly synthesized analogues in the aphids Myzus persicae Sulzer and Aphis craccivora Koch. We found two classes of neonicotinoids with distinct modes of interference with [3H]imidacloprid, described as direct competitive inhibition and non-competitive inhibition, respectively. Competitive neonicotinoids were acetamiprid, nitenpyram, thiacloprid, clothianidin and nithiazine, whereas thiamethoxam and the N-methyl analogues of imidacloprid and clothianidin showed non-competitive inhibition. The chloropyridine or chlorothiazole heterocycles, the polar pharmacophore parts, such as nitroimino, cyanoimino and nitromethylene, and the cyclic or acyclic structure of the pharmacophore were not relevant for the mode of inhibition. Consensus structural features of the neonicotinoids were defined for the two mechanisms of interaction with [3H]imidacloprid binding. Furthermore, two sub-classes of non-competitive inhibitors can be discriminated on the basis of their Hill coefficients for imidacloprid displacement. We conclude from the present data that the direct competitors share the binding site with imidacloprid, whereas non-competitive compounds, like thiamethoxam, bind to a different site or in a different mode.     

Comparative analysis of neonicotinoid binding to insect membranes: II. An unusual high affinity site for [3H]thiamethoxam in Myzus persicae and Aphis craccivora

Neonicotinoids represent a class of insect-selective ligands of nicotinic acetylcholine receptors. Imidacloprid, the first commercially used neonicotinoid insecticide, has been studied on neuronal preparations from many insects to date. Here we report first intrinsic binding data of thiamethoxam, using membranes from Myzus persicae Sulzer and Aphis craccivora Koch. In both aphids, specific binding of [3H]thiamethoxam was sensitive to temperature, while the absolute level of non-specific binding was not affected. In M persicae, binding capacity (Bmax) for [3H]thiamethoxam was ca 450 fmol mg(-1) of protein at 22 degrees C and ca 700 fmol mg(-1) of protein at 2 degrees C. The negative effect of increased temperature was reversible and hence not due to some destructive process. The affinity for [3H]thiamethoxam was less affected by temperature: Kd was ca 11 nM at 2 degrees C and ca 15 nM at 22 degrees C. The membranes also lost binding sites for [3H]thiamethoxam during prolonged storage at room temperature, and upon freezing and thawing. In A craccivora, [3H]thiamethoxam was bound with a capacity of ca 1000 fmol mg(-1) protein and an affinity of ca 90 nM, as measured at 2 degrees C. Overall, the in vitro temperature sensitivity of [3H]thiamethoxam binding was in obvious contrast to the behaviour of [3H]imidacloprid studied in parallel. Moreover, the binding of [3H]thiamethoxam was inhibited by imidacloprid in a non-competitive mode, as shown with M persicae. In our view, these differences demonstrate that thiamethoxam and imidacloprid, which represent different structural sub-classes of neonicotinoids, do not share the same binding site or mode. This holds also for other neonicotinoids, as we report in a companion article.     

七种新烟碱类杀虫剂对韭菜迟眼蕈蚊幼虫及蚯蚓的选择毒力

为筛选出高效安全的韭蛆防治药剂,室内采用胃毒触杀联合毒力法比较了吡虫啉、啶虫脒、噻虫嗪、噻虫胺、呋虫胺、烯啶虫胺、噻虫啉与毒死蜱和高效氯氟氰菊酯等6种对照药剂对韭菜迟眼蕈蚊幼虫的毒力,同时用人工土壤法测定了13种药剂对蚯蚓的急性毒性,并通过盆栽试验验证了其对韭蛆和蚯蚓的选择毒力。结果表明,吡虫啉、噻虫胺、呋虫胺、噻虫啉、噻虫嗪对韭菜迟眼蕈蚊4龄幼虫的毒力明显高于6种对照药剂,对虫酰肼的相对毒力倍数分别为101.6、55.0、32.9、27.2、13.6;13种供试药剂中,除吡虫啉、啶虫脒、噻虫胺、呋虫胺对蚯蚓中等毒性外,其余均为低毒;盆栽试验中,吡虫啉、噻虫嗪、毒死蜱、噻唑膦、高效氯氟氰菊酯的防虫效果和保苗效果均分别高于其它药剂,但其中只有噻虫嗪对蚯蚓没有明显致死作用。     

新烟碱类簇合物的合成及其生物活性

为寻找高活性的新烟碱类杀虫剂,根据簇合效应原理,以吡虫啉为母体,合成了几类酰基连接的吡虫啉二效价簇合物。其化学结构经1H NMR和元素分析确证。初步的生物活性测试结果表明,化合物 4a、4c 在100 mg/L时对麦长管蚜Macrosiphum avenae的平均致死率分别达78.4%和77.3％,但活性均低于对照药剂吡虫啉。     

Characterisation of imidacloprid resistance mechanisms in the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae)

Effective control of the brown planthopper, Nilaparvata lugens Stål, across rice-growing regions of Asia has been seriously compromised over the last 2 years by the appearance of widespread resistance to the neonicotinoid insecticide, imidacloprid. Sequence analysis of the ligand-binding domain of the nicotinic acetylcholine receptor α1 subunit from two field-collected resistant strains (CHN-2 and IND-11) did not reveal the Y151S point mutation previously implicated in conferring target-site resistance in this species. This result was supported by ligand-binding studies with [³H]-imidacloprid that showed no significant change in insecticide binding to isolated membranes from susceptible and resistant strains. In contrast, there was an approximate 5-fold increase in the mixed function oxidase activity for the two resistant strains suggesting that imidacloprid metabolism by increased cytochrome P450 monooxygenase activity is the major mechanism of resistance in these strains.     

嘧啶酮类衍生物的合成及杀虫活性

为了创制高效广谱的绿色杀虫剂,以(E)-4,5-二氢-6-甲基-4-(3-吡啶亚甲基氨基)-1,2,4-三嗪-3(2H)-酮(吡蚜酮)为先导,用带有不同电荷密度的五元、六元取代苯环或杂环取代其结构中的吡啶环部分,合成了10个全新的嘧啶酮类衍生物,特别对其中所包含的三嗪环和二氢喹唑啉酮合成部分进行了重点研究。所有目标化合物的结构均经过核磁共振氢谱、高分辨质谱及红外光谱的确认。初步杀虫活性测试结果表明,目标化合物对蚜虫Aphis craccivora未表现出明显的杀虫活性,初步暗示了先导化合物吡蚜酮结构中所含的吡啶环部分可能对其杀虫活性起了重要作用。     

Toxicity and nicotinic acetylcholine receptor interaction of imidacloprid and its metabolites in Apis mellifera (Hymenoptera: Apidae)

Acute oral and contact toxicity tests of imidacloprid, an insecticide acting agonistically on nicotinic acetylcholine receptors (nAChR), to adult honeybees, Apis mellifera L var carnica, were carried out by seven different European research facilities. Results indicated that the 48-h oral LD50 of imidacloprid is between 41 and > 81 ng per bee, and the contact LD50 between 49 and 102 ng per bee. The ingested amount of imidacloprid-containing sucrose solution decreased with increasing imidacloprid concentrations and may be attributed to dose-related sub-lethal intoxication symptoms or to antifeedant responses. Some previously reported imidacloprid metabolites occurring at low levels in planta after seed dressing, i.e. olefine-, 5-OH- and 4,5-OH-imidacloprid, showed lower oral LD50 values (> 36, > 49 and 159 ng per bee, respectively) compared with the concurrently tested parent molecule (41 ng per bee). The urea metabolite and 6-chloronicotinic acid (6-CNA) exhibited LD50 values of > 99,500 and > 121,500 ng per bee, respectively. The pharmacological profile of the [3H]imidacloprid binding site in honeybee head membrane preparations is consistent with that anticipated for a nAChR. IC50 values for the displacement of [3H]imidacloprid by several metabolites such as olefine, 5-OH-, 4,5-OH-imidacloprid, urea and 6-CNA were 0.45, 24, 6600, > 100,000, and > 100,000 nM, respectively. Displacement of [3H]imidacloprid by imidacloprid revealed an IC50 value of 2.9 nM, thus correlating well with the observed acute oral toxicity of the compounds in honeybees. Neurons isolated from the antennal lobe of A mellifera and subjected to whole-cell voltage clamp electrophysiology responded to the application of 100 microM acetylcholine with a fast inward current of between 30 and 1600 pA at -70 mV clamp potential. Imidacloprid and two of the metabolites (olefine- and 5-OH-imidacloprid) acted agonistically on these neurons, whereas the others did not induce currents at test concentrations up to 3 mM. The electrophysiological data revealed Hill coefficients of approximately 1, indicating a single binding site responsible for an activation of the receptor and no direct cooperativity or allosteric interaction with a second binding site.     

Effects of mutations in Drosophila nicotinic acetylcholine receptor subunits on sensitivity to insecticides targeting nicotinic acetylcholine receptors

Several strains of Drosophila melanogaster possess mutant alleles in nicotinic acetylcholine receptor (nAChR) subunits, Dα1 and Dβ2 that confer resistance to neonicotinoids such as imidacloprid and nitenpyram, and Dα6, that confers resistance to spinosyns. These mutant strains were bioassayed with a selected set of nAChR active insecticides including neonicotinoids, spinosad, and sulfoxaflor, a new sulfoximine insecticide. All of the neonicotinoids examined, except dinotefuran showed reduced insecticidal efficacy on larvae of the Dα1 mutant, suggesting that this subunit may be important in the action of these insecticides. All of the neonicotinoids, including dinotefuran, showed reduced insecticidal efficacy on larvae possessing the Dβ2 mutation. A similar pattern of broad neonicotinoid resistance to that of Dβ2 alone was also observed for larvae with both the mutations (Dα1 + Dβ2). The Dβ2 mutation exhibited a lower level of cross-resistance to sulfoxaflor (<3-fold) than to any of the neonicotinoids (>13-fold). In contrast, there was no cross-resistance for any of the neonicotinoids or sulfoxaflor in adult flies with the Dα6 mutation, which confers high levels of resistance to spinosad. Thus in the D. melanogaster strains studied, target site resistance observed for the neonicotinoids and the spinosyns does not translate directly to resistance towards sulfoxaflor.