Muscarinic Agonists as Insecticides and Acaricides

A series of known agonists of the mammalian muscarinic receptor were prepared and evaluated for their insecticidal potential. It was discovered that pests such as Nilaparvata lugens (brown planthopper), Nephotettix cincticeps (green leafhopper), Tetranychus urticae (two-spotted spider mite) and Aphis gossypii (cotton aphid) were particularly sensitive to most of these compounds. Several analogs proved to be extremely active, surpassing commercial standards in some of the laboratory bioassays. These compounds exhibited a range of potencies for the insect (Musca) muscarinic receptor. Addition of GTP significantly reduced the affinity of the most potent analog for the Musca mAChR, indicating the compound functions as an agonist in insect tissue. Regression analysis indicated that significant relationships exist between displacement of [³H]QNB at the Musca muscarinic receptor and whole organism toxicity to three insect and one mite species. The results suggested that the insect muscarinic receptor represents a viable target site for insecticidal action. © 1997 SCI.     

Receptor-binding affinity and larvicidal activity of tetrahydroquinoline-type ecdysone agonists against Aedes albopictus

Tetrahydroquinolines (THQs), a class of nonsteroidal ecdysone agonists, are good candidates for novel mosquito control agents because they specifically bind to mosquito ecdysone receptors (EcRs). We have recently performed quantitative structure–activity relationship (QSAR) analyses of THQs to elucidate the physicochemical properties important for the ligand–receptor interaction. Based on previous QSAR results, here, we newly synthesized 15 THQ analogs with a heteroaryl group at the acyl moiety and evaluated their binding affinity against Aedes albopictus EcRs. We also measured the larvicidal activity of the combined set of previously and newly synthesized compounds against A. albopictus to examine the contribution of receptor-binding to larvicidal activity. Multiple regression analyses showed that the binding affinity and the molecular hydrophobicity of THQs are the key determinants of their larvicidal activity.     

Identification of novel juvenile-hormone signaling activators via high-throughput screening with a chemical library

Juvenile hormone (JH) is an insect-specific hormone that regulates molting and metamorphosis. Hence, JH signaling inhibitors (JHSIs) and activators (JHSAs) can be used as effective insect growth regulators (IGRs) for pest management. In our previous study, we established a high-throughput screening (HTS) system for exploration of novel JHSIs and JHSAs using a Bombyx mori cell line (BmN_JF&AR cells) and succeeded in identifying novel JHSIs from a chemical library. Here, we searched for novel JHSAs using this system. The four-step HTS yielded 10 compounds as candidate JHSAs; some of these compounds showed novel basic structures, whereas the others were composed of a 4-phenoxyphenoxymethyl skeleton, the basic structure of several existing JH analogs (pyriproxyfen and fenoxycarb). Topical application of seven compounds to B. mori larvae significantly prolonged the larval period, suggesting that the identified JHSAs may be promising IGRs targeting the JH signaling pathway.     

Substituted thiotrifluoropropanones as potent selective inhibitors of juvenile hormone esterase

A series of 27 substituted thio-1,1,1-trifluoropropanones was synthesized by reacting the corresponding thiol with 1,1,1-trifluoro-3-bromopropanone. The resulting sulfides were screened as inhibitors of hemolymph juvenile hormone esterase and α-naphthyl acetate esterase activity of the cabbage looper, Trichoplusia ni, electric eel acetylcholinesterase, bovine trypsin, and bovine α-chymotrypsin. The presence of the sulfide bond increased the inhibitory potency on all of the enzymes tested when compared with compounds lacking the sulfide. In general, the compounds proved to be poor inhibitors of chymotrypsin and moderate inhibitors of trypsin. By varying the substituent on the sulfide, good inhibitory activity was obtained on α-naphthyl acetate esterase, acetylcholinesterase, while some of the compounds proved to be extremely powerful inhibitors of juvenile hormone esterase. The most powerful inhibitor tested was 3-octylthio-1,1,1-trifluoro-2-propanone, with an I₅₀ of 2.3 × 10^?9M on JH esterase. This compound showed a molar refractivity similar to that of the JH II backbone, was not toxic to T. ni, and was moderately toxic to mice, with a 48-hr LD₅₀ of >750 mg/kg. It effectively delayed pupation when applied to prewandering larvae of T. ni, as expected for a JH esterase inhibitor. Thus, some members of this series are promising for evaluating the role of JH esterase in insect development. The series also indicates that, by varying the substituent on the sulfide moiety, potent “transition-state” inhibitors can be developed for a wide variety of esterases and proteases.     

Ovicidal activity of juvenile hormone mimics in the bean bug,Riptortus pedestris

Insect juvenile hormone (JH) mimics (JHMs) are known to have ovicidal effects if applied to adult females or eggs. Here, we examined the effects of exogenous JHMs on embryonic development of the bean bug, Riptortus pedestris. The expression profiles of JH early response genes and JH biosynthetic enzymes indicated that JH titer was low for the first 3 days of the egg stage and increased thereafter. Application of JH III skipped bisepoxide (JHSB₃) or JHM on Day 0 eggs when JH titer was low caused reduced hatchability, and the embryos mainly arrested in mid- or late embryonic stage. Application of JHMs on Day 5 eggs also resulted in an arrest, but this was less effective compared with Day 0 treatment. Interestingly, ovicidal activity of synthetic JHMs was much lower than that of JHSB₃. This study will contribute to developing novel insecticides that are selective among insect species.     

Inhibition of juvenile hormone biosynthesis in the tobacco hornworm, Manduca sexta, by a bisthiolcarbamate juvenoid and related compounds

Biosynthesis of juvenile hormone in the tobacco hornworm, Manduca sexta, is inhibited by the bisthiolcarbamate juvenoid N-ethyl-1,2-bis(isobutylthiolcarbamoyl)ethane both in vitro and in vivo. In vitro an extremely steep dose-response curve was obtained with an ID₅₀ value of 6 × 10^?6M. However, in vivo topical treatment with the compound resulted in mild JH antagonistic symptoms, suggesting rapid metabolism of the compound. In agreement with results from metabolic studies performed on plants and in mammals, sulfoxidation of the thiocarbamate S-(4-chlorobenzyl)N,N-diethylthiocarbamate resulted in an enhanced inhibitory effect on JH biosynthesis in vitro. This suggests that the corresponding thiocarbamate sulfoxides may act as intermediates in carbomylating critical thiol sites important in the terpenoid biosynthesis pathway. Furthermore, this study shows that these prototype compounds are interesting tools for further investigation of chemical inhibition of JH biosynthesis in insects.     

Purification and partial characterization of a glutathione S-transferase from the two-spotted spider mite, Tetranychus urticae

Glutathione S-transferases (GSTs) are known to catalyze conjugations by facilitating the nucleophilic attack of the sulfhydryl group of endogenous reduced glutathione on electrophilic centers of a vast range of xenobiotic compounds, including insecticides and acaricides. Elevated levels of GSTs in the two-spotted spider mite, Tetranychus urticae Koch, have recently been associated with resistance to acaricides such as abamectin [Pestic. Biochem. Physiol. 72 (2002) 111]. GSTs from acaricide susceptible and resistant strains of T. urticae were purified by glutathione-agarose affinity chromatography and characterized by their Michaelis-Menten kinetics towards artificial substrates, i.e., 1-chloro-2,4-dinitrobenzene and monochlorobimane. The inhibitory potential of azocyclotin, dicumarol, and plumbagin was low (IC₅₀ values > 100 μM), whereas ethacrynic acid was much more effective, exhibiting an IC₅₀ value of 4.5 μM. GST activity is highest in 2-4-day-old female adults and dropped considerably with progressing age. Furthermore, molecular characteristics were determined for the first time of a GST from T. urticae, such as molecular weight (SDS-PAGE) and N-terminal amino acid sequencing (Edman degradation). Glutathione-agarose affinity purified GST from T. urticae strain WI has a molecular weight of 22.1 kDa. N-terminal amino acid sequencing revealed a homogeneity of ≈50% to insect GSTs closely related to insect class I GSTs (similar to mammalian Delta class GSTs).     

The kinetics of insecticide action. Part II: The relationship between the pharmacokinetics of substituted benzyl (1RS)-cis,trans-chrysanthemates and their relative toxicities to mustard beetles (Phaedon cochleariae fab.)

A mathematical model describing insect pharmacokinetics has been applied to data describing the penetration and elimination of a series of pyrethroid analogues [the methylbenzyl (1 RS)-cis, trans-chrysanthemates], applied to mustard beetles. The three parameters of the model (k_p, k_e and λ) have been estimated for each compound. The influence of chemical structure on pharmacokinetics, investigated using canonical correlation analysis, is discussed in relation to relative toxicity and knockdown action. In general, physicochemical properties which result in a slow flow of insecticide through the insect are associated with high toxicity. A large proportion (84%) of the variation in relative toxicity has been explained by resistance to elimination (1/k_e), implying that exposure of the site of action to insecticide is related to the total integral IM₂ for material (M₂) inside the insect. The remaining variation has been attributed to differences in behaviour at receptor sites. An optimum partition value (Δπ₀ ≈ 1.6) seems necessary for maximum pharmacodynamic activity in this series of compounds.     

Stage-specific action of juvenile hormone analogs

The discovery of juvenile hormones (JH) and their synthetic analogs (JHA) generated excitement and hope that these compounds will replace first- and second-generation insecticides that have not so desirable environmental and human safety profiles. However, JHAs used commercially during the past four decades did not meet these expectations. The recent availability of advanced molecular and histological methods and the discovery of key players involved in JH action provided some insights into the functioning of JHA in a stage and species-specific manner. In this review, we will summarize recent findings and stage-specific action of JHA, focusing on three commercially used JHA, methoprene, hydroprene and pyriproxyfen and economically important pests, the red flour beetle, Tribolium castaneum, and the tobacco budworm, Heliothis virescens, and disease vector, the yellow fever mosquito, Aedes aegypti.     

In vitro inhibition of acetylcholinesterase by O,O-dimethyl S-aryl phosphorothioates

In vitro inhibition of house cricket head, house fly head, and bovine erythrocyte acetylcholinesterase by O,O-dimethyl S-aryl phosphorothioates was studied by Main's kinetic treatment. The potency of the compounds as reflected by the bimolecular reaction constants (k_i) indicated that house fly head acetylcholinesterase was the most sensitive to the inhibition followed by house cricket head and bovine erythrocyte acetylcholinesterase. There are no linear relationships between the phosphorylation rate constants and the total binding energies for the inhibition of three enzymes by this series of compounds, suggesting that the initial binding and the phosphorylation rate are not related. The structure and activity relationships were analyzed by multiple regression analyses with the use of Hammett's sigma, alkaline hydrolysis rates of the compounds, and p_i constants. The hydrophobic bonding of the compound on the enzyme surface as reflected by the p_i constant played a significant role in the determination of the potency of the inhibition of house cricket head and house fly head acetylcholinesterase by those compounds. However, the alkaline hydrolysis rates of the compounds, or the Hammett's sigma values seems to play a more important role in the determination of the inhibition of bovine erythrocyte acetylcholinesterase. Moderate insecticidal activity toward house crickets, house flies, and mosquito larvae were found.     

Studies on a fluorescent insect growth regulator metabolite complex with house-fly microsomal cytochrome P-450

The fluorescent insect growth regulator 5[[[5-(dimethylamino)-1-naphthalenyl]amino]-1,3-benzodioxole (DNSAB) forms a metabolite complex with house-fly microsomal cytochrome P-450. Formation of the metabolite complex is dependent on the presence of NADPH and O₂; NADH supports the reaction at a reduced rate. The presence of antibodies to house-fly cytochrome c (P-450) reductase in reaction mixtures inhibits the complex formation, indicating that the reductase is necessary for transfer of electrons from NADPH to cytochrome P-450 to complete the reaction. In the oxidized form, the metabolite complex has a single absorbance maximum at 431 nm, whereas the reduced form has two absorbance maxima at 426 (major) and 455 nm (minor). The pH of the media affects the extinction of the 426- and 455-nm Soret bands; increased pH decreases the extinction of the 426-nm band and increases the extinction of 455-nm band. Formation of the DNSAB metabolite-cytochrome P-450 complex decreases the amount of CO-reactive cytochrome P-450 by 24%. The metabolite complex is not dissociable by treatment with ferricyanide or by using centrifugation techniques. Dissociation is accomplished by addition of DNSAB to the oxidized metabolite complex. Kinetic analysis of the complex formation gives apparent K_m and V_max values at 2.55 ± 1.0 μM and 1.1 ± 0.4 × 10^?2 ΔA min^?1 nmol^?1 cytochrome P-450, respectively. Addition of juvenile hormone [(E,E)-cis-methyl-10,11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate; JH] to the reaction medium competitively inhibits the formation of the metabolite complex giving an inhibition constant of 16 μM. DNSAB synergized the lethal effects of JH against Aedes aegypti larvae threefold; however, JH did not synergize DNSAB. These data suggest that DNSAB may acquire its hormonal qualities by complexing a species of cytochrome P-450 that metabolizes JH, thereby prolonging the in vivo lifetime of this hormone.     

Aminopyrifen,a novel 2-aminonicotinate fungicide with a unique effect and broad-spectrum activity against plant pathogenic fungi

Aminopyrifen is a novel 2-aminonicotinate fungicide with unique chemistry and a novel mode of action. The fungicide showed high antifungal activity mainly against Ascomycetes and its related anamorphic fungi under in vitro and pot conditions (EC₅₀ values: 0.0039–0.23 mg/L and 1.2–12 mg/L, respectively). The active ingredient strongly inhibited germ-tube elongation of Botrytis cinerea below 0.1 mg/L and invasion into a plant. The compound exhibited no cross-resistance to commercial fungicides in B. cinerea. The antifungal agent showed high preventive efficacy and translaminar action. In the field, aminopyrifen controlled gray mold and powdery mildew at 150 mg/L. Our findings suggest that aminopyrifen is useful for protecting crops from various plant pathogens.     

Inhibitors of choline acetyltransferase as potential insecticides

Choline acetyltransferase (E.C.2.3.1.6) catalyses the synthesis of acetylcholine and is therefore a target for a new insecticide. We have prepared a variety of compounds, mainly choline analogues, as inhibitors of this enzyme. One of these, 2-isothiocyanatoethyltrimethylammonium iodide, has a K_i of 0.06 μM (K_m for choline is 150 μM ) and is apparently the most powerful inhibitor known for this enzyme. Although some of our compounds are insecticidal we believe, on the basis of electrophysiological studies, that they act, not on choline acetyltransferase, but on the acetylcholine receptor of the insect.     

Selectivity, acetylcholinesterase inhibition kinetics, and quantitative structure-activity relationships of a series of N-(2-oxido-1,3,2-benzodioxa-phosphol-2-yl) amino acid ethyl or diethyl esters

The inhibitory effects of a recently introduced series of the titled compounds on insect and mammalian acetylcholinesterase (AChE) activity were examined, where the median inhibition concentration (I₅₀) and the inhibition kinetic parameters, bimolecular inhibition rate constant (k_i), affinity constant (K_a), and phosphorylation rate constant (k_p), were determined for each compound. Results indicated that all examined dioxaphospholenes had less inhibitory effects on mammalian AChE than fenitrothion, a commercial pesticide with moderate mammalian toxicity. The highest selectivity was obtained with compounds containing glutamic and leucine moieties (2.70 and 2.18, respectively) while selectivity of fenitrothion was 0.93. The low inhibitory effects of the examined dioxaphospholenes on mammalian AChE were attributed to their low phosphorylation rates (k_p < 2.2 min⁻¹) compared to that of fenitrothion (k_p = 4.84 min⁻¹). QSAR equations indicated that the inhibition process is controlled mainly by both the phosphorylation rate (direct effect) and the affinity of compounds toward the enzyme (inverse effect). Although the compounds’ hydrophobicity had no effects on the inhibition process, it affects the compounds’ toxicity since it affects the ability of compounds to penetrate insects to reach the enzyme active site.     

Comparison of the activity of non‐steroidal ecdysone agonists between dipteran and lepidopteran insects,using cell‐based EcR reporter assays

BACKGROUND: Diacylhydrazine (DAH) analogues have been developed successfully as a new group of insect growth regulators, called ecdysone agonists or moulting accelerating compounds. These DAHs have been shown to manifest their toxicity via interaction with the ecdysone receptor (EcR) in susceptible insects, as does the natural insect moulting hormone 20‐hydroxyecdysone (20E). A notable feature is their high activity and specificity, particularly against lepidopteran insects, raising the question as to whether non‐lepidopteran‐specific analogues can be isolated. However, for the discovery of ecdysone agonists that target other important insect groups such as Diptera, efficient screening systems that are based on the activation of the EcR are needed. RESULTS: In this study, a dipteran‐specific reporter‐based screening system with transfected S2 cells of Drosophila melanogaster Meig. was developed in order to discover and evaluate compounds that have ecdysone agonistic or antagonistic activity. A library of non‐steroidal ecdysone agonists containing different mother structures with DAH and other related analogues such as acylaminoketone (AAK) and tetrahydroquinoline (THQ) was tested. None of the compounds tested was as active as 20E. This is in contrast to the very high activity of several DAH and AAK congeners in lepidopteran cells (Bombyx mori L.‐derived Bm5 cells). The latter agrees with a successful docking of a DAH, tebufenozide, in the binding pocket of the lepidopteran EcR (B. mori), while this was not the case with the dipteran EcR (D. melanogaster). Of note was the identification of two THQ compounds with activity in S2 but not in Bm5 cells. Although marked differences in activity exist with respect to the activation of EcR between dipterans and lepidopterans, there exists a positive correlation (R = 0.724) between the pLC₅₀ values in S2 and Bm5 cells. In addition, it was found through protein modelling that a second lobe was present in the ligand‐binding pocket of lepidopteran BmEcR but was lacking in the dipteran DmEcR protein, suggesting that this difference in structure of the binding pocket is a major factor for preferential activation of the lepidopteran over the dipteran receptors by DAH ligands. CONCLUSIONS: The present study confirmed the marked specificity of DAH and AAK analogues towards EcRs from lepidopteran insects. THQ compounds did not show this specificity, indicating that dipteran‐specific ecdysone‐agonist‐based insecticides based on the THQ mother structure can be developed. The differences in activity of ecdysone agonists in dipteran and lepidopteran ecdysone‐reporter‐based screening systems are discussed. Copyright © 2010 Society of Chemical Industry     

Synthesis and herbicidal activity of 3-substituted toxoflavin analogs

We investigated the synthesis and herbicidal activity of 23 toxoflavin analogs, 1a–w, in which aromatic rings (R) were introduced into the C-3 position. In paddy field conditions, 1k (R=2-CF₃–C₆H₄) and 1w (R=2-thienyl) showed excellent herbicidal activity. Under upland field conditions, we found that toxoflavin analogs 1a (R=C₆H₅), 1n (R=2-CH₃O–C₆H₄), and 1p (R=4-CH₃O–C₆H₄) exhibited wide herbicidal spectrum against Echinochloa crus-galli (L) var. crus-galli (ECHCG), Chenopodium album, and Amaranthus viridis (AMAVI). The analog with the 2-fluoro group on benzene ring 1b also showed high herbicidal activity against both ECHCG and AMAVI.     

A cell-based reporter assay for screening for EcR agonist/antagonist activity of natural ecdysteroids in Lepidoptera (Bm5) and Diptera (S2) cell cultures,followed by modeling of ecdysteroid-EcR interactions and normal mode analysis

Ecdysteroid signal transduction is a key process in insect development and therefore an important target for insecticide development. We employed an in vitro cell-based reporter bioassay for the screening of potential ecdysone receptor (EcR) agonistic and antagonistic compounds. Natural ecdysteroids were assayed with ecdysteroid-responsive cell line cultures that were transiently transfected with the reporter plasmid ERE-b.act.luc. We used the dipteran Schneider S2 cells of Drosophila melanogaster and the lepidopteran Bm5 cells of Bombyx mori, representing important pest insects in medicine and agriculture. Measurements showed an EcR agonistic activity only for cyasterone both in S2 (EC₅₀ = 3.3 μM) and Bm5 cells (EC₅₀ = 5.3 μM), which was low compared to that of the commercial dibenzoylhydrazine-based insecticide tebufenozide (EC₅₀ = 0.71 μM and 0.00089 μM, respectively). Interestingly, a strong antagonistic activity was found for castasterone in S2 cells with an IC₅₀ of 0.039 μM; in Bm5 cells this effect only became visible at much higher concentrations (IC₅₀ = 18 μM). To gain more insight in the EcR interaction, three-dimensional modeling of dipteran and lepidopteran EcR-LBD was performed. In conclusion, we showed that the EcR cell-based reporter bioassay tested here is a useful and practical tool for the screening of candidate EcR agonists and antagonists. The docking experiments as well as the normal mode analysis provided evidence that the antagonist activity of castasterone may be through direct binding with the receptor with specific changes in protein flexibility. The search for new ecdysteroid-like compounds may be particularly relevant for dipterans because the activity of dibenzoylhydrazines appears to be correlated with an extension of the EcR-LBD binding pocket that is prominent in lepidopteran receptors but less so in the modeled dipteran structure.     

Structure-activity correlations of the insecticide Prolan and its analogs

Structure-activity correlations for 45 insecticidal diaryl nitropropanes (Prolan analogs) were analyzed by multiple regression analysis. Molecular bulk constants including van der Waal's radii, molar attraction constants, parachor, steric constants such as Taft's E₈ and Verloop's dimensional steric constants, hydrophobic constants such as II, and electronic parameters such as σ, F, and R were evaluated. It was concluded that the diaryl nitropropanes like the diaryl trichloroethanes fit into a receptor site which has an optimum volume for maximum interaction. The interaction between the insecticide and the receptor shows high correlation with steric constants for the aryl substituents and with intermolecular attractive forces. Highly asymmetrical compounds such as 1-(p-fluorophenyl)-1-(p-hexoxyphenyl)-2-nitropropane were surprisingly effective insecticides.