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To discover novel inhibitors of the complex I reduced nicotinamide adenine dinucleotide (NADH) oxidoreductase as fungicides, a series of 6-isothiazol-5-ylpyrimidin-4-amine-containing compounds were designed using a computer-aided pesticide design method and splicing of substructures from diflumetorim and isotianil. In vitro fungicidal bioassays indicated that compounds T17 - T24 showed high inhibitory activity against Rhizoctonia solani with an effective concentration (EC 50 ) value falling between 2.20 and 23.85 μg/mL, which were more active than or equivalent to the lead diflumetorim with its EC 50 of 19.80 μg/mL. In vivo antifungal bioassays demonstrated that, at a concentration of 200 μg/mL, T7 and T21 showed higher inhibition against Pseudoperonospora cubensis than all other compounds, while T23 exhibited the highest inhibition against Sphaerotheca fuliginea . T23 showed an approximately twofold lower inhibition potency against R. solani complex I NADH oxidoreductase than diflumetorim. Molecular docking and transcriptomic analyses indicated that T23 and diflumetorim both might share the same mode of action, targeting NADH oxidoreductase. T23 as a good fungicidal candidate against R. solani is worthy of further investigation.

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., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.

Succinate dehydrogenase inhibitors (SDHIs) are a class of fungicides targeting the pathogenic fungi mitochondrial SDH. Here, molecular docking, three-dimensional quantitative structure-activity relationship (3D-QSAR), and molecular dynamics (MD) simulations were used to guide SDHI innovation. Molecular docking was performed to explore the binding modes of SDH and its inhibitors. 3D-QSAR models were carried out on 33 compounds with activity against Rhizoctonia cerealis ( R. cerealis ); their structure-activity relationships were analyzed using comparative molecular field analysis and comparative molecular similarity indices analysis. MD simulations were used to assess the stability of the complexes under physiological conditions, and the results were consistent with molecular docking. Binding free energy was calculated through the molecular mechanics generalized born surface area method, and the binding free energy was decomposed. The results are consistent with the activity of bioassay and indicate that van der Waals and lipophilic interactions contribute the most in the molecular binding process. Afterward, we designed and synthesized 12 compounds under the guidance of the above-mentioned analyses, bioassay found that F9 was active against R. cerealis with the EC 50 value of 9.43 μg/mL, and F4 , F5 , and F9 were active against Botrytis cinerea with an EC 50 values of 5.80, 3.17, and 1.63 μg/mL, respectively. They all showed good activity between positive controls of pydiflumetofen and thifluzamide. Our study provides new considerations for effective SDHIs discovery.

Computer-aided molecular modeling was applied to design a series of Spodoptera frugiperda RyR agonists. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were used to generate 3D-QSAR models. MD simulations in the complex with S. frugiperda native, mutant RyR, and mammalian RyR1 under physiological conditions were used to validate the detailed binding mechanism. Binding free energy calculation by molecular mechanics generalized surface area (MM-GBSA) explained the role of key amino acid residues in ligand-receptor binding. Therefore, 14 new compounds were effectively designed and synthesized, and a bioassay indicated that compounds A-2 and A-3 showed comparable activity to that of chloranthraniliprole with LC 50 values of 0.27, 0.18, and 0.20 mg L -1 , respectively, against S. frugiperda . Most target compounds also displayed good activity against Mythinma separata at 0.1 mg L -1 . Molecular docking and MM-GBSA calculations demonstrated that A-3 had a better binding capacity with native and mutant S. frugiperda RyRs.

The development of new fungicides is vital for safeguarding crops and ensuring sustainable agriculture. Building on our previous finding that 4-(3,4-dichloroisothiazole)-7-hydroxy coumarins can be used as fungicidal leads, 44 novel coumarin ester derivatives were designed and synthesized to evaluate whether esterification could enhance their fungicidal activity. In vitro fungicidal bioassays indicated that compound 2ai displayed good activity against Alternaria solani , Botrytis cinereal , Cercospora arachidicola , Physalospora piricola and Sclerotinia sclerotiorum , with an EC 50 value ranging from 2.90 to 5.56 μg/mL, comparable to the lead compound 1a , with its EC 50 value ranging from 1.92 to 9.37 μg/mL. In vivo bioassays demonstrated that compounds 1a , 2ar and 2bg showed comparable, excellent efficacy against Pseudoperonospora cubensis at a dose of 25 µg/mL. Our research shows that the esterification of 4-(3,4-dichloroisothiazole) 7-hydroxycoumarins results in a fungicidal activity equivalent to that of its lead compounds. Furthermore, our density functional theory (DFT) calculations and 3D-QSAR modeling provide a rational explanation of the structure-activity relationship and offer valuable insights to guide further molecular design.

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.

To discover novel target-based fungicidal candidates, a molecular design model was established with a three-dimensional (3D) structure of Rhizoctonia solani pyruvate kinase (RsPK) simulated with the AlphaFold 2 and YZK-C22 as a fungicidal lead. A series of novel [1,2,4]triazolo[3,4- b ][1,3,4]thiadiazole derivatives were rationally designed, synthesized, evaluated for their fungicidal performance, and validated for their mode of action. The in vitro bioassays with R. solani indicated that compounds 5g , 5o , and 5z with an EC 50 value ranging from 1.01 to 1.54 μg/mL displayed higher fungicidal activity than the positive control YZK-C22 with its EC 50 of 3.14 μg/mL. Especially, 5o exhibited high potency and a broad spectrum against Alternaria solani , Botrytis cinerea , Cercospora arachidicola , Physalospora piricola , R. solani , and Sclerotinia sclerotiorum with its EC 50 value falling between 1.54 and 13.10 μg/mL. Like all positive controls, 5g , 5o , and 5z showed excellent in vivo growth inhibition against Pseudoperonospora cubensis at 200 μg/mL. Even though the PK enzymatic inhibition assay showed that 5o was approximately 2.6 times less active than YZK-C22 (IC 50 : 29.14 vs 11.15 μg/mL, respectively), the similar fluorescence quenching patterns of RsPK by 5o and YZK-C22 , and the docking results of interactions between RsPK and 5o or YZK-C22 implied that they might share the similar binding site in the RsPK active pocket. Our studies suggested that 5o could be used as a potent fungicidal lead for further optimization. The results of comparative molecular field analysis (CoMFA) provided a direction for further molecular design.

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.