Design and Synthesis of 3‑Carene-Derived Amide-Thiourea/Nanochitosan\nComplexes with Excellent Laccase Inhibitory Activity and Sustained\nReleasing Performance for Crop Protection

The\ndiscovery of natural product-derived novel nanopesticide systems\ncan effectively address the adverse effects caused by the improper\nuse of traditional fungicides. In this research, 33 novel 3-carene-derived\namide-thiourea derivatives <b>5a</b>–<b>5zg</b> were designed using laccase as the biological target, synthesized\nfrom natural renewable forest biomass resource 3-carene as the starting\nmaterial, and structurally confirmed by Fourier-transform infrared\nspectroscopy, nuclear magnetic resonance, high-resolution mass spectrometry,\nand single crystal X-ray diffraction. The antifungal activity of the\ntarget compounds against eight plant pathogenic fungi was evaluated,\nand the results presented that target compound <b>5g</b> exhibited\nexcellent and broad-spectrum antifungal activity against the eight\ntested phytopathogenic fungi. Furthermore, the important contribution\nof the <i>gem</i>-dimethylcyclopropane structure in the\nantifungal activity of compound <b>5g</b> was revealed through\ntwo negative controls without the <i>gem</i>-dimethylcyclopropane\nstructure. Besides, compound <b>5g</b> also demonstrated a prominent\nlaccase inhibitory activity. The fluorescence quenching of the laccase\nwith 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\nexhibited excellent antifungal activity by acting on the laccase target.\nCytotoxicity assay revealed that compound <b>5g</b> had a low\ncytotoxicity for LO2 and HEK293T cell lines. On the other hand, to\nfurther 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\ncarriers CACS and CATCS with sustained releasing performance were\nsynthesized for loading compound <b>5g</b>. 3-Carene-derived\nnanochitosan carrier CATCS showed a relatively regular, loose, and\nporous reticular structure, which displayed high dispersibility and\ngood thermostability. In addition, this carrier had a higher drug-loading\ncapacity and sustained releasing performance than that of the unmodified\nchitosan. This research identified that the target compound <b>5g</b> could be used as a promising lead compound for fungicide\nagainst the laccase target, meanwhile, the complex <b>5g</b>/CATCS deserved further study as a nanopesticide candidate.