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To increase the structural diversity of insecticides and meet the needs of effective integrated insect management, the structure of chlorantraniliprole was modified based on a previously established three-dimensional quantitative structure-activity relationship (3D-QSAR) model. The pyridinyl moiety in the structure of chlorantraniliprole was replaced with a 4-fluorophenyl group. Further modifications of this 4-fluorophenyl group by introducing a halogen atom at position 2 and an electron-withdrawing group (e.g., iodine, cyano, and trifluoromethyl) at position 5 led to 34 compounds with good insecticidal efficacy against Mythimna separata , Plutella xylostella , and Spodoptera frugiperda . Among them, compound IV f against M. separata showed potency comparable to that of chlorantraniliprole. IV p against P. xylostella displayed a 4.5 times higher potency than chlorantraniliprole. In addition, IV d and chlorantraniliprole exhibited comparable potencies against S. frugiperda . Transcriptome analysis showed that the molecular target of compound IV f is the ryanodine receptor. Molecular docking was further performed to verify the mode of action and insecticidal activity against resistant P. xylostella .

Pests represent an important impediment to efficient agricultural production and pose a threat to global food security. On the basis of our prior research focused on identifying insecticidal leads targeting insect ryanodine receptors (RyRs), we aimed to identify evodiamine scaffold-based novel insecticides. Thus, a variety of evodiamine-based derivatives were designed, synthesized, and assessed for their insecticidal activity against the larvae of Mythimna separata ( M. separata ) and Plutella xylostella ( P. xylostella ). The preliminary bioassay results revealed that more than half of the target compounds exhibited superior activity compared to evodiamine, matrine, and rotenone against M. separata . Among these, compound 21m displayed the most potent larvicidal efficiency, with a remarkable mortality rate of 93.3% at 2.5 mg/L, a substantial improvement over evodiamine (10.0% at 10 mg/L), matrine (10.0% at 200 mg/L), and rotenone (30.0% at 200 mg/L). In the case of P. xylostella , compounds 21m and 21o displayed heightened larvicidal activity, boasting LC 50 values of 9.37 × 10 -2 and 0.13 mg/L, respectively, surpassing that of evodiamine (13.41 mg/L), matrine (291.78 mg/L), and rotenone (18.39 mg/L). A structure-activity relationship analysis unveiled that evodiamine-based derivatives featuring a cyclopropyl sulfonyl group at the nitrogen atom of the B ring and a fluorine atom in the E ring exhibited more potent larvicidal effects. This finding was substantiated by calcium imaging experiments and molecular docking, which suggested that 21m could target insect RyRs, including resistant mutant RyRs of P. xylostella (G4946E and I4790M), with higher affinity than chlorantraniliprole (CHL). Additionally, cytotoxicity assays highlighted that the potent compounds 21i , 21m , and 21o displayed favorable selectivity and low toxicity toward nontarget organisms. Consequently, compound 21m emerges as a promising candidate for further development as an insecticide targeting insect RyRs.

Three-dimensional quantitative structure-activity relationship (3D-QSAR) is one of the most important and effective tools to direct molecular design in new pesticide development. Chlorantraniliprole is an anthranilic diamide ryanodine receptor (RyR) agonist with ultrahigh activity, high selectivity, and mammalian safety. To continue our studies on new insecticide development, here, we designed new insecticidal N -phenylpyrazoles by using 3D-QSAR of chlorantraniliprole analogues as a guide. Most of the target compounds synthesized exhibited medium to excellent activity against Mythimna separata , Plutella xylostella , and Spodoptera frugiperda . Compounds III b and III y showed similar activity against M. separata as chlorantraniliprole (LC 50 values: 0.21, 0.25, and 0.16 μg mL -1 respectively). Compounds III b exhibited a 3-fold higher potency against P. xylostella than chlorantraniliprole. For S. frugiperda , the potency of III a and III b was 2.9 and 2.0 times higher than that of the positive control, respectively. The mode of action of the title compounds was validated by calcium imaging experiments and molecular docking using their target RyRs. III b can dock well with mutated P. xylostella RyRs, implying a potentially lower cross-resistance risk as compared with commercial RyR agonists. Density functional theory calculations suggested the feasibility of higher potency with the structural modifications. Compound III b was found to be an ultrahigh active insecticidal candidate with a broad spectrum for integrated pest management.

Pests are one of the major factors causing crop damage and food security problems worldwide. Based on our previous studies on the discovery of insecticidal leads targeting the ryanodine receptors (RyRs), a three-dimensional quantitative structure-activity relationship (3D-QSAR) model was established to design and synthesize a series of anthranilic diamides containing a halogenated phenyl 3-trifluoroethoxypyrazole moiety. The preliminary bioassays disclosed that IIb , IIIb , and IIIf against Mythimna separata showed comparable activity to chloranthraniliprole (LC 50 : 0.16, 0.16, 0.14, and 0.13 mg·L -1 , respectively). More than half of the target compounds displayed good activity against Plutella xylostella, where IIIf was the most active compound, 25 times more active than chloranthraniliprole (LC 50 : 6.0 × 10 -6 versus 1.5 × 10 -4 mg·L -1 ). For Spodoptera frugiperda , IIIf displayed slightly inferior potency to chlorantraniliprole (LC 50 : 0.47 versus 0.31 mg·L -1 ). For RyR mutants of S. frugiperda (G4891E, I4734M), compound IIIf could show higher affinity than chlorantraniliprole according to the binding mode and energy in molecular docking experiments. Calcium imaging technique, molecular docking, density functional theory calculations, and electrostatic potential studies validated that the RyR was the target of the most active candidate IIIf , which deserves further development.

The diamide insecticides show exceptional activity against Lepidoptera insects via activation of ryanodine receptors (RyRs). In the present study, a series of anthranilic diamides containing a fluoroaniline moiety were designed, synthesized, and evaluated for insecticidal potency. Most titled compounds exerted moderate to remarkably high activity against Mythimna separata , Plutella xylostella , and Spodoptera frugiperda . The insecticidal activity of compound II l and II ac against M. separata was 26.7 and 26.7% at 0.1 mg L -1 , respectively, equivalent to that of chlorantraniliprole (0.1 mg L -1 , 30.0%). Compounds II l , II y , and II z exhibited 8.0-, 1.8-, and 4.7-fold higher potency than chlorantraniliprole against P. xylostella , respectively, as compared with their LC 50 s. Compounds II k and II aa showed good insecticidal activity against S. frugiperda with LC 50 of 0.56 and 0.46 mg L -1 , respectively, comparable to that of the commercial insecticide chlorantraniliprole with LC 50 of 0.31 mg L -1 . Calcium imaging experiments indicated RyRs as the action target. Molecular docking suggested a higher binding energy of 8.647 kcal/mol between II l and the M. separata RyR than the 7.820 kcal/mol between chlorantraniliprole and the M. separata RyR. Meanwhile, the docking results of II l with mutated P. xylostella RyR at site G4946E showed that II l could have a good inhibition effect on the resistant P. xylostella . The density functional theory calculations suggested the importance of the fluoroaniline moiety in potency. Those novel anthranilic diamides containing a fluorinated aniline moiety are good insecticidal candidates.

Insect ryanodine receptors (RyRs) are molecular targets of the anthranilic diamide insecticides. In the present study, a new series of anthranilic diamides containing a cyanophenyl pyrazole moiety were rationally designed by active-fragment assembly and computer-aided design using the 3D structure of Plutella xylostella RyRs as a receptor and chlorantraniliprole as a ligand. Most of the titled compounds showed good toxicity against Mythimna separate , P. xylostella , and Spodoptera frugiperda . Compounds CN06 , CN11 , and CN16 with corresponding LC 50 values of 0.15, 0.29, and 0.52 mg·L -1 , respectively, against M. separate showed comparable activity to that of chlorantraniliprole (0.13 mg·L -1 ). Surprisingly, CN06 , CN11 , and CN16 with corresponding LC 50 values of 1.6 × 10 -5 , 3.0 × 10 -5 , and 2.8 × 10 -5 mg·L -1 , respectively, against P. xylostella were at least 5-fold more active than chlorantraniliprole (1.5 × 10 -4 mg·L -1 ). In the case of S. frugiperda , CN06 , CN11 , and CN16 had good potency but lower than chlorantraniliprole in terms of LC 50 values (0.58, 0.54, and 0.56 mg·L -1 versus 0.31 mg·L -1 ). Molecular docking of CN06 and chlorantraniliprole to P. xylostella RyRs validated the molecular design, and the calcium imaging technique further proved the potential target of CN06 as RyRs. Compounds CN06 , CN11 , and CN16 could be more effective than chlorantraniliprole in targeting the resistant RyR mutants of S. frugiperda (G4891E, I4734M) through the binding mode and energy obtained by molecular docking. Density functional theory calculations (DFT) and electrostatic potential (ESP) studies gave the structure-activity relationship. Compounds CN06 , CN11 , and CN16 could be used as potent insecticide leads for further optimization.

The diamondback moth Plutella xylostella , a global insect pest of cruciferous vegetables, has evolved resistance to many classes of insecticides including diamides. Three point mutations (I4790M, I4790K, and G4946E) in the ryanodine receptor of P. xylostella (PxRyR) have been identified to associate with varying levels of resistance. In this study, we generated a knockin strain (I4790K-KI) of P. xylostella, using CRISPR/Cas9 to introduce the I4790K mutation into PxRyR of the susceptible IPP-S strain. Compared to IPP-S, the edited I4790K-KI strain exhibited high levels of resistance to both anthranilic diamides (chlorantraniliprole 1857-fold, cyantraniliprole 1433-fold) and the phthalic acid diamide flubendiamide (>2272-fold). Resistance to chlorantraniliprole in the I4790K-KI strain was inherited in an autosomal and recessive mode, and genetically linked with the I4790K knockin mutation. Computational modeling suggests the I4790K mutation reduces the binding of diamides to PxRyR by disrupting key hydrogen bonding interactions within the binding cavity. The approximate frequencies of the 4790M, 4790K, and 4946E alleles were assessed in ten geographical field populations of P. xylostella collected in China in 2021. The levels of chlorantraniliprole resistance (2.3- to 1444-fold) in these populations were significantly correlated with the frequencies (0.017–0.917) of the 4790K allele, but not with either 4790M (0–0.183) or 4946E (0.017–0.450) alleles. This demonstrates that the PxRyR I4790K mutation is currently the major contributing factor to chlorantraniliprole resistance in P. xylostella field populations within China. Our findings provide in vivo functional evidence for the causality of the I4790K mutation in PxRyR with high levels of diamide resistance in P. xylostella , and suggest that tracking the frequency of the I4790K allele is crucial for optimizing the monitoring and management of diamide resistance in this crop pest. [Display omitted] • I4790K mutation was knocked into the ryanodine receptor of Plutella xylostella. • PxRyR I4790K knockin strain exhibited high levels of resistance to diamides. • PxRyR I4790K is the most potent allele contributing to diamide resistance. • Tracking the frequency of I4790K allele is crucial for IRM of P. xylostella. [ABSTRACT FROM AUTHOR]