Identification of Isoxazoline Compounds as Potential Insecticidal Agents through High-Throughput Screening and 3D-QSAR Analysis

<i>Plutella xylostella</i> poses a significant global agricultural threat. To identify potential insecticide candidates, we conducted high-throughput screening and constructed three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis. Screening our in-house isoxazoline database revealed a series of compounds exhibiting insecticidal activity against <i>P. xylostella</i>. Subsequently, a 3D-QSAR model was developed using the screened compounds to facilitate the optimization of compound <b>D36</b>. Bioassays indicated that <b>D36</b> exhibited superior activity against <i>P. xylostella</i>, with an LC₅₀ value of 0.025 mg/L, surpassing control insecticides indoxacarb (LC₅₀ = 1.45 mg/L), ethiprole (LC₅₀ = 3.81 mg/L), and fluxametamide (LC₅₀ = 0.679 mg/L). Moreover, <b>D36</b> exhibited LC₅₀ values of 0.934 and 0.601 mg/L against <i>Spodoptera frugiperda</i> and <i>Ostrinia furnacalis</i>, respectively. The results of the density functional theory and molecular docking explain the excellent activity of <b>D36</b>. Additionally, transcriptomic analysis, enzyme-linked immunosorbent assay, and molecular docking studies suggested that <b>D36</b> may affect the central nervous system of target insects and interact with GABA receptors. These results highlight the potential of <b>D36</b> as a novel isoxazoline candidate for the effective control of <i>P. xylostella</i> and other Lepidoptera pests.