Design and Synthesis of 3-Carene-Derived Amide-Thiourea/Nanochitosan Complexes with Excellent Laccase Inhibitory Activity and Sustained Releasing Performance for Crop Protection

The discovery of natural product-derived novel nanopesticide systems can effectively address the adverse effects caused by the improper use of traditional fungicides. In this research, 33 novel 3-carene-derived amide-thiourea derivatives <b>5a</b>-<b>5zg</b> were designed using laccase as the biological target, synthesized from natural renewable forest biomass resource 3-carene as the starting material, and structurally confirmed by Fourier-transform infrared spectroscopy, nuclear magnetic resonance, high-resolution mass spectrometry, and single crystal X-ray diffraction. The antifungal activity of the target compounds against eight plant pathogenic fungi was evaluated, and the results presented that target compound <b>5g</b> exhibited excellent and broad-spectrum antifungal activity against the eight tested phytopathogenic fungi. Furthermore, the important contribution of the <i>gem</i>-dimethylcyclopropane structure in the antifungal activity of compound <b>5g</b> was revealed through two negative controls without the <i>gem</i>-dimethylcyclopropane structure. Besides, compound <b>5g</b> also demonstrated a prominent laccase inhibitory activity. The fluorescence quenching of the laccase with compound <b>5g</b>, the chelating characteristics of compound <b>5g</b>, and the interaction mode between the laccase and compound <b>5g</b> presented that the target compound <b>5g</b> probably exhibited excellent antifungal activity by acting on the laccase target. Cytotoxicity assay revealed that compound <b>5g</b> had a low cytotoxicity for LO2 and HEK293T cell lines. On the other hand, to further improve the application potential of compound <b>5g</b>, the 3-carene molecular skeleton containing <i>gem</i>-dimethylcyclopropane ring was grafted onto chitosan, and two nanopesticide carriers CACS and CATCS with sustained releasing performance were synthesized for loading compound <b>5g</b>. 3-Carene-derived nanochitosan carrier CATCS showed a relatively regular, loose, and porous reticular structure, which displayed high dispersibility and good thermostability. In addition, this carrier had a higher drug-loading capacity and sustained releasing performance than that of the unmodified chitosan. This research identified that the target compound <b>5g</b> could be used as a promising lead compound for fungicide against the laccase target, meanwhile, the complex <b>5g</b>/CATCS deserved further study as a nanopesticide candidate.