Design, Synthesis, Herbicidal Evaluation, and Molecular Simulation of 2-(6-Phenylnicotinoyl)cyclohexane-1,3-dione Derivatives as Novel 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors

4-Hydroxyphenylpyruvate dioxygenase (HPPD) has been an essential target enzyme in recent studies on herbicide discovery. In this study, a series of 3-hydroxy-2-(6-phenylnicotinoyl)cyclohex-2-en-1-one derivatives were rationally designed following the active substructure combination principle, yielding potent novel HPPD inhibiting herbicides. The title compounds were characterized using infrared spectroscopy (IR), ¹H and ¹³C nuclear magnetic resonance spectroscopies (NMR), and high-resolution mass spectrometry (HRMS). Among the designed compounds, <b>IV-45</b> showed the highest potency against <i>Arabidopsis thaliana</i> HPPD (<i>At</i>HPPD), with a half-maximal inhibitory concentration (IC₅₀) of 0.21 μM, exceeding that of mesotrione (0.23 μM) <i>in vitro</i>. Bioassay results revealed that 150 g a.i./ha of compounds <b>IV-8</b> and <b>IV-45</b> effectively targeted <i>Setaria viridis</i> and <i>Echinochloa crus-galli</i>, inhibiting their growth by over 80%. Crop safety tests demonstrated that compound <b>IV-45</b> was safer than the commercial herbicide mesotrione for cotton and wheat. Moreover, compound <b>IV-45</b> was virtually harmless to corn, rice, and peanuts. Molecular docking confirmed that compound <b>IV-45</b> underwent a classical bidentate chelating interaction with Co²⁺ and a tight π-π stacking interaction with Phe381 and Phe424 in active pocket. Molecular dynamics simulations further verified that compound <b>IV-45</b> stably bonded to <i>At</i>HPPD, exerting a strong inhibitory effect. This work suggests the potential utility of 2-(6-phenylnicotinoyl)cyclohexane-1,3-dione as a herbicide skeleton for targeting HPPD.