Receptor assay-guided isolation of anti-GABAergic insecticidal alkaloids from a fungal culture

The gamma-aminobutyric acid (GABA) receptor bears sites of action for insecticides. To discover GABA receptor-directed insecticides in natural products, fungal culture extracts were screened for their ability to inhibit specific binding of the radiolabeled noncompetitive antagonist [3H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane to housefly head membranes. The screening efforts led to the isolation of two alkaloids from Aspergillus terreus: PF1198A (alantrypinone) and PF1198B (serantrypinone), which had IC50 values of 0.34 and 2.1 microM, respectively, in this assay. These compounds were ca. 47-61-fold selective for housefly vs rat GABA receptors. Both compounds showed insecticidal activity against Myzus persicae in the range of 100-500 ppm. Binding assay-guided screening should provide significant opportunities for the identification of novel and selective insecticides.     

Phenylpyrazole insecticide photochemistry, metabolism, and GABAergic action: ethiprole compared with fipronil

Ethiprole differs from fipronil, the major phenylpyrazole insecticide, only in an ethylsulfinyl substituent replacing the trifluoromethylsulfinyl moiety. This study compares their photochemistry, metabolism, action at the gamma-aminobutyric acid (GABA) receptor, and insecticidal potency. On exposure to sunlight as a thin film, ethiprole undergoes oxidation (major), reduction, and desethylsulfinylation but not desulfinylation whereas the major photoreaction for fipronil is desulfinylation. Metabolic sulfone formation is more rapid with ethiprole than fipronil in human expressed CYP3A4 in vitro and mouse brain and liver in vivo. High biological activity is observed for the sulfide, sulfoxide, sulfone, and desulfinyl derivatives in both the ethiprole and the fipronil series in GABA receptor assays (human recombinant beta3 homomer and house fly head membranes) with [(3)H]EBOB and in topical toxicity to house flies with and without the P450-inhibiting synergist piperonyl butoxide. On an overall basis, the ethiprole series is very similar in potency to the fipronil series.     

Effects of tea components on the response of GABA(A) receptors expressed in Xenopus Oocytes

To study the effects of tea components on ionotropic gamma-aminobutyric acid (GABA) receptor response, ionotropic GABA receptors (GABA(A) receptors) were expressed in Xenopus oocytes by injecting cRNAs synthesized from cloned cDNAs of the alpha(1) and beta(1) subunits of the bovine receptors, and their electrical responses were measured by a voltage clamping method. Extracts of green tea, black tea, and oolong tea in an aqueous solution induced the GABA-elicited response, which showed that these teas contain GABA, whereas coffee does not. Caffeine weakly inhibited the response in a competitive manner (K(i) = 15 mM), and (+)-catechin inhibited it in a noncompetitive one (K(i) = 1.7 mM). Especially, two catechin derivatives, (-)-epicatechin gallate and (-)-epigallocatechin gallate, inhibited the response strongly. Alcohols such as leaf alcohol or linalool potentiated the response, possibly because their binding to the potentiation site enhances the GABA-binding affinity to GABA(A) receptors when they bind. Extracts of green tea made with ethyl ether, which must contain lipophilic components of green tea, inhibited the response elicited by GABA, possibly because the amounts of caffeine and catechin derivatives were much larger than fragrant alcohols in such extracts of tea.     

Synthesis of glucose-fipronil conjugate and its phloem mobility

Phloem-mobile insecticides are preferred to achieve economically useful activity. However, only a few phloem-mobile synthetic insecticides are available. One approach to converting nonmobile insecticides into phloem-mobile types is introducing sugar to the parent compound. To test whether the addition of a glucose group to a non-phloem-mobile insecticide enables conversion into phloem-mobile, N-[3-cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl]-1-(β-D-glucopyranosyl)-1H-1,2,3-triazole-4-methanamine (GTF) was prepared through click chemistry. A phloem-mobility test in Ricinus communis L. seedlings confirmed that GTF was mobile in the sieve tubes. Although GTF exhibited lower insecticidal activity against the third-instar larvae of Pzlutella xylostella than fipronil did, it can be reconverted into fipronil in adult plants of castor bean, thereby offsetting the decrease of insecticidal activity. Therefore, the presence of a glucose core confers phloem mobility to fipronil.     

Uptake and phloem transport of glucose-fipronil conjugate in Ricinus communis involve a carrier-mediated mechanism

Some compounds containing glucose are absorbed via the monosaccharide transporters of the plasma membrane. A glucose-fipronil conjugate, N-[3-cyano-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazol-5-yl]-1-(β-d-glucopyranosyl)-1H-1,2,3-triazole-4-methanamine (GTF), has been synthesized in our previous work. GTF exhibits moderate phloem mobility in Ricinus communis. In the current paper, we demonstrate that the uptake of GTF by Ricinus seedling cotyledon discs is partly mediated by an active carrier system (K(m)1 = 0.17 mM; V(max)1 = 2.2 nmol cm(-2) h(-1)). Four compounds [d-glucose, sucrose, phloridzin, and carbonyl cyanide m-chlorophenylhydrazone (CCCP)] were examined for their effect on GTF uptake. Phloridzin as well as CCCP markedly inhibit GTF uptake, and d-glucose weakly competes with it. The phloem transport of GTF in Ricinus seedlings is found to involve an active carrier-mediated mechanism that effectively contributes to the GTF phloem loading. The results prove that adding a glucose core is a reasonable and feasible approach to confer phloem mobility to fipronil by utilizing plant monosaccharide transporters.     

Effects of coffee components on the response of GABA(A) receptors expressed in Xenopus oocytes

The effects of both coffee components and coffee extract on the electrical responses of GABA(A) receptors expressed in Xenopus oocytes were studied by injecting cRNAs of the alpha(1) and beta(1) subunits of the bovine receptors. The aqueous extract of coffee dose-dependently inhibited the GABA-elicited responses, whereas the lipophilic extract of coffee by diethyl ether slightly potentiated it at low doses (0.1-0.4 microL/mL) but showed inhibition at high doses (0.5-0.8 microL/mL). Theophylline inhibited the response in a noncompetitive mechanism (K(i) = 0.55 mM), whereas theobromine and trigonelline hydrochloride inhibited it in a competitive manner, K(i) = 3.8 and 13 mM, respectively. Benzothiazole, catechol, 2,4-dimethylstyrene, guaiacol, 1-octen-3-ol, sotolone, and 2,3,5-trimethylphenol potentiated the responses significantly. Potentiation elicited by guaiacol and sotolone was independent of GABA concentrations, whereas that by 1-octen-3-ol was dependent. When 1-octen-3-ol (100 mg/kg) was orally administered to mice prior to intraperitoneal administration of pentobarbital, the sleeping time of mice induced by pentobarbital increased significantly.     

Synthesis and insecticidal activity of novel carbamate derivatives as potential dual-binding site acetylcholinesterase inhibitors

In biological systems, bivalent ligands often possess increased functional affinity for their receptors compared with monovalent ligands. On the basis of the structure of acetylcholinesterase (AChE), a series of novel carbamate heterodimetic derivatives were designed and synthesized with the aim of increasing the potency toward AChE inhibition. The AChE inhibitory ability of all the novel compounds was tested using AChE obtained from the brain of the housefly. The bioassay results showed that compounds 6j, 6k, 6m, 6n, 6p, and 6q had increased inhibitory activities in comparison with parent phenyl N-methylcarbamate (MH) at the concentration of 100 mg/L. Among them, the most potent AChE inhibitor of these compounds was 6q (IC(50) = 12 μM), which showed 62-fold greater AChE inhibitory activity than that of MH and 12-fold greater activity than metolcarb (MT), which suggested that the 3-nitrophenoxy moiety of compound 6q was able to interact with the aromatic amino acid residues lining the gorge and the phenyl N-methylcarbamate moiety was able to interact with the catalytic sites of AChE, simultaneously. The insecticidal activities of compounds 6j, 6k, 6m, 6n, 6p, and 6q were further evaluated. Consistent with the result in vitro bioassay, those compounds demonstrated better activities against Lipaphis erysimi than parent compound MH at the concentration of 300 mg/L, and compound 6q showed the best insecticidal activity, causing 98% mortality after 24 h of treatment.     

Effect of 3-O-octanoyl-(+)-catechin on the responses of GABA(A) receptors and Na+/glucose cotransporters expressed in xenopus oocytes and on the oocyte membrane potential

Recently, 3-O-octanoyl-(+)-catechin (OC) was synthesized from (+)-catechin (C) by incorporation of an octanoyl chain into C in the light of (-)-epicatechin gallate (ECg) and (-)-epigallocatechin gallate (EGCg), which are the major polyphenols found in green tea and have strong physiological activities. OC was found to inhibit the response of ionotropic gamma-aminobutyric acid (GABA) receptors (GABA(A) receptors) and Na+/glucose cotransporters expressed in Xenopus oocytes in a noncompetitive manner more strongly than does C. OC also induced a nonspecific membrane current and decreased the membrane potential of the oocyte, and thus death of the oocyte occurred even at lower concentrations than that induced by C or EGCg. Although EGCg produced H2O2 in aqueous solution, OC did not. This newly synthesized catechin derivative OC possibly binds to the lipid membrane more strongly than does C, Ecg, or EGCg and as a result perturbs the membrane structure.     

Potentiation of the ionotropic GABA receptor response by whiskey fragrance

It is well-known that the target of most mood-defining compounds is an ionotropic gamma-aminobutyric acid receptor (GABA(A) receptor). The potentiation of the response of these inhibitory neurotransmitter receptors induces anxiolytic, sedative, and anesthetic activity in the human brain. To study the effects of whiskey fragrance on the GABA(A) receptor-mediated response, GABA(A) receptors were expressed in Xenopus oocyte by injecting rat whole brain mRNA or cRNA prepared from the cloned cDNA for the alpha(1) and beta(1) subunits of the bovine receptors. Most whiskey components such as phenol, ethoxy, and lactone derivatives potentiated the electrical responses of GABA(A) receptors, especially ethyl phenylpropanoate (EPP), which strongly potentiated the response. When this compound was applied to mice through respiration, the convulsions induced by pentetrazole were delayed, suggesting that EPP was absorbed by the brain, where it could potentiate the GABA(A) receptor responses. The extract of other alcoholic drinks such as wine, sake, brandy, and shochu also potentiated the responses to varying degrees. Although these fragrant components are present in alcoholic drinks at low concentrations (extremely small quantities compared with ethanol), they may also modulate the mood or consciousness of the human through the potentiation of the GABA(A) receptor response after absorption into the brain, because these hydrophobic fragrant compounds are easily absorbed into the brain through the blood-brain barrier and are several thousands times as potent as ethanol in the potentiation of the GABA(A) receptor-mediated response.     

Aging of whiskey increases the potentiation of GABA(A) receptor response

It is known that the target of most mood-defining compounds such as ethanol is an ionotropic gamma-aminobutyric acid receptor (GABA(A) receptor). The potentiation of the response of these inhibitory neurotransmitter receptors induces anxiolytic, sedative, and anesthetic activities in the human brain. Because both extracts of whiskey by pentane and fragrant components in whiskey potentiate the GABA(A) receptor-mediated response, GABA(A) receptors were expressed in Xenopus oocyte by injecting cRNAs prepared from the cloned cDNA for the alpha(1) and beta(1) subunits of the bovine receptors in order to study the effects of whiskey itself on the GABA(A) receptor-mediated response. Whiskey itself also potentiated the electrical responses of GABA(A) receptors generally more than ethanol at the same concentration as that of the whiskey. The potentiation of the GABA(A) receptor-mediated response increased with the aging period of the whiskey. Inhalation of whiskey to mice increased the sleeping time induced by pentobarbital more than that of the same concentration of ethanol as the whiskey. These results suggest that not only ethanol but also minor components in whiskey play an important role in the potentiation of GABA(A) receptor-mediated response and possibly the sedative effect of whiskey. Although the minor components are present in extremely small quantities compared with ethanol in alcoholic beverages, they may modulate the mood or consciousness of humans through the potentiation of the GABA(A) receptor response after absorption into the brain, because these hydrophobic compounds are easily absorbed into the brain across the blood-brain barrier and are several thousands times as potent as ethanol in the potentiation of the GABA(A) receptor-mediated response.     

Design, synthesis, crystal structure analysis, and insecticidal evaluation of phenylazoneonicotinoids

On the basis of research of the proposed modes of action between neonicotinoids and insect nicotinic acetylcholine receptor (nAChR), a series of phenylazoneonicotinoids were designed and synthesized to further promote the π-π interaction between molecule and amino acid residues. The target compounds have been identified on the basis of satisfactory analytical and spectral ((1)H NMR, (13)C NMR, HRMS, and X-ray) data. The preliminary results revealed that tiny differences in substitutes resulted in different configurations and great bioactivity variations. Some compounds with electron-donating groups on positions 2 and 6 of the phenyl ring presented higher insecticidal activity than imidacloprid against cowpea aphids ( Aphis craccivora ). The impressive crystal structure of the excellent insecticidal activity compound 9q clearly proved that the functional electronegative pharmacophore was approximately vertical to the methyleneimidazolidine plane. The differences in the mode of interaction on nAChR of typical compounds 9h and 9q remain unclear.     

Structure-activity relationship development of dihaloaryl triazole compounds as insecticides and acaricides. 1. Phenyl thiophen-2-yl triazoles

An extended lipophilic system that incorporated some key elements of first-generation 2,6-dihaloaryl actives, such as 1, demonstrated desirable efficacy against chewing insects as well as sap-feeding insects. These four-ring systems, based on 2, were accessed primarily via Suzuki couplings of halothiophene derivatives with appropriately substituted boronic acids. In particular, phenylthiophene systems that incorporated haloxyether groups, such as those in 3, 4, and 5, had the broadest spectrum of activity across chewing and sap-feeding insect pests. Expansion of this structure-activity relationship to include compounds with differing substitution patterns on the thiophene-C-ring and aryl-D-rings was undertaken. The synthesis and insecticidal activity of 3-aryl-5-(thiophen-2-yl)-1-methyl-1H-[1,2,4]triazoles will be described.     

Molecular docking and three-dimensional quantitative structure-activity relationship studies on the binding modes of herbicidal 1-(substituted phenoxyacetoxy)alkylphosphonates to the E1 component of pyruvate dehydrogenase

Molecular docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies on the title compounds were performed to explore the possible inhibitory mechanism. To determine the probable binding conformations of the title phosphonate derivatives, the most potent compound 12 was chosen as a standard template and docked into the active site of PDHc E1. On the basis of the binding conformations, highly predictive 3D-QSAR models were developed with q2 values of 0.872 and 0.873 for comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), respectively. The predictive abilities of these models were validated by using a set of compounds that were not included in the training set. Both the CoMFA and the CoMSIA field distributions are in good agreement with the spatial and electronic structural characteristics of the binding groove of PDHc E1 selected in this work. Mapping the 3D-QSAR models to the active site of PDHc E1 provides new insight into the protein-inhibitor interaction mechanism, which is most likely valuable and applicable for designing highly active compounds in the future.     

Liquid chromatographic determination of fluridone aquatic herbicide and its metabolite in fish and crayfish

A residue method is described for determination of the aquatic herbicide fluridone (1-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]-4(1H)-pyridinone) and its metabolite (1-methyl-3-(4-hydroxyphenyl)-5-[3-(trifluoromethyl) phenyl]-4(1H)-pyridinone) in fish and crayfish tissues. Both compounds are extracted from tissues with methanol, and the extracts are subjected to acidic hydrolysis to release conjugated forms of fluridone and the metabolite. Sample extracts are purified by liquid-liquid partitioning and Florisil Sep-Pak column chromatography. Both compounds are separated and measured by reverse phase liquid chromatography with UV detection at 313 nm. In the absence of interfering peaks, the method has a detection limit of approximately 0.04 ppm of either compound. Overall, recoveries averaged 96% for fluridone and 78% for the metabolite for all tissue types combined.     

Synthesis and insecticidal activity of N-tert-butyl-N,N'-diacylhydrazines containing 1,2,3-thiadiazoles

N-tert-Butyl-N,N'-diacylhydrazines are nonsteroidal ecdysone agonists used as environmental benign pest regulators. In this paper, two series of new N-tert-butyl-N,N'-diacylhydrazine derivatives containing 1,2,3-thiadiazole were designed and synthesized. All structures of the synthesized compounds were confirmed by proton nuclear magnetic resonance ((1)H NMR), infrared spectroscopy (IR), and high-resolution mass spectrometry (HRMS). Bioasssay results indicated that most of the synthesized compounds possessed good insecticidal activities against Plutella xylostella L. and Culex pipiens pallens as compared with the positive control, tebufenozide. The results of this study indicated that 1,2,3-thiadiazoles, as an important active substructure, could improve or maintain the activity of the dicylhydrazines and favor novel pesticide development.     

Synthesis and insecticidal activity of some novel fraxinellone-based esters

In continuation of a program aimed at the discovery and development of natural products-based insecticidal agents, two series of novel fraxinellone-based esters were synthesized by modification at the C-4 or C-10 position of fraxinellone and evaluated for their insecticidal activity against the pre-third-instar larvae of Mythimna separata in vivo. An efficient method for the stereoselective synthesis of 4α-hydroxyfraxinellone from fraxinellonone was developed, and the steric configuration of 6h was unambiguously confirmed by X-ray crystallography. Among 37 compounds, some derivatives displayed potent insecticidal activity; especially compounds 6h, 6q, 6t, and 7q showed more promising insecticidal activity than toosendanin, a commercial botanical insecticide derived from Melia azedarach . This suggested that introduction of the fluorine atom on the phenyl ring could lead to a more potent compound than one possessing chlorine or bromine. Meanwhile, introduction of the heterocyclic fragments at the C-4 or C-10 position of fraxinellone was essential for their insecticidal activity. This will pave the way for further design, structural modification, and development of fraxinellone as an insecticidal agent.     

Syntheses and biological activities of octahydro-1H-cyclopenta[d]pyrimidine derivatives

Various nitromethylene derivatives were synthesized regioselectively. Compounds 8a-f were obtained by the reaction of 1-((5-chloropyridin-2-yl)methyl)-2-(nitromethylene)-octahydro-1H-cyclopenta[d]-pyrimidine (3) with primary amines and formaldehyde. The synthesized compounds were identified by 1H NMR, HRMS (EI), and IR, and preliminary bioassays indicated that most of them showed moderate insecticidal activities against Aphis craccivora. The relationship between hydrophobicity and biological activity was also discussed.     

Synthesis,fungicidal activity,and 3D-QSAR of pyridazinone-substituted 1,3,4-oxadiazoles and 1,3,4-thiadiazoles

A series of novel 5-[1-aryl-1,4-dihydro-6-methylpyridazin-4-one-3-yl] -2-arylamino-1,3,4-oxadiazoles, fungicidally active, were synthesized based on bioisosterism and tested in vivo against wheat leaf rust, Puccinia recondita. These compounds were shown to be fungicidally active, and their activity was influenced by the nature of the substituents. By using the three-dimensional quantitative structure-activity relationships (3D-QSAR) method of comparative molecular field analysis (CoMFA), we have studied the structure and activity relationship of the compounds containing both pyridazinone-substituted 1,3,4-thiadiazoles and pyridazinone-substituted 1,3,4-oxadiazoles. The 3D-QSAR modes gave good correlation between the variations on percent inhibition and the steric-electrostatic properties. The results are consistent with a common mode of action for the pyridazinone-substituted 1,3,4-thiadiazoles and the pyridazinone-substituted 1,3,4-oxadiazoles, which further confirms that the 1,3,4-oxadiazole ring is a bioisosteric analogue of the 1,3,4-thiadiazole ring. These offer important structural insights into designing highly active compounds prior to their synthesis.     

Synthesis, insecticidal, and acaricidal activities of novel 2-aryl-pyrrole derivatives containing ester groups

A series of novel 2-aryl-pyrrole derivatives containing ester groups were synthesized, and their structures were characterized by (1)H NMR spectroscopy and elemental analysis. The insecticidal activities against oriental armyworm, mosquito, diamondback moth, green rice leafhopper, and bean aphids and acaricidal activities against spider mite of these new compounds were evaluated. The results of bioassays indicated that some of these title compounds exhibited excellent insecticidal and acaricidal activities. The insecticidal activities against oriental armyworm of compounds IVa, IVd, IVe, IVf, IVg, IVi, IVk, and IVp were equal to commercialized Chlorfenapyr, and the insecticidal activities of most of compounds IVb, IVc, IVd, IVf, IVg, IVj, IVk, IVl, IVs, IVt, IVu, IVw, IVx, IVz, and Chlorfenapyr against mosquito at 0.10 mg kg (-1) were 100%, and the acaricidal activities of compounds IVd, IVe, IVf, IVg, IVh, IVi, and IVk were equal or superior to Chlorfenapyr. Especially, the results indicated that the acaricidal activity of [4-bromo-2-(4-chlorophenyl)-3-cyano-5-(trifluoromethyl)pyrrol-1-yl]methyl 3-methylbutanoate ( IVg) against spider mite was 2.65-fold as high as that of Chlorfenapyr from the value of LC 50.