An Effective and Promising Strategy for Plant Protection: Synthesis of L-Carvone-Based Thiazolinone–Hydrazone/Nanochitosan Complexes with Antifungal Activity and Sustained Releasing Performance

The development of novel natural product-derived nano-pesticide systems with loading capacity and sustained releasing performance of bioactive compounds is considered an effective and promising plant protection strategy. In this work, 25 L-carvone-based thiazolinone–hydrazone compounds 4a~4y were synthesized by the multi-step modification of L-carvone and structurally confirmed. Compound 4h was found to show favorable and broad-spectrum antifungal activity through the in vitro antifungal activity evaluation of compounds 4a~4y against eight phytopathogenic fungi. Thus, it could serve as a leading compound for new antifungal agents in agriculture. Moreover, the L-carvone-based nanochitosan carrier 7 bearing the 1,3,4-thiadiazole-amide group was rationally designed for the loading and sustained releasing applications of compound 4h, synthesized, and characterized. It was proven that carrier 7 had good thermal stability below 200 °C, dispersed well in the aqueous phase to form numerous nanoparticles with a size of~20 nm, and exhibited an unconsolidated and multi-aperture micro-structure. Finally, L-carvone-based thiazolinone–hydrazone/nanochitosan complexes were fabricated and investigated for their sustained releasing behaviors. Among them, complex 7/4h-2 with a well-distributed, compact, and columnar micro-structure displayed the highest encapsulation efficiency and desirable sustained releasing property for compound 4h and thus showed great potential as an antifungal nano-pesticide for further studies.


Introduction
In agriculture, fungicides are usually applied to protect plants that are susceptible to fungal infection.Nevertheless, the long-term employment of the current commercial fungicides has caused the development of fungi resistance and the running-off of bioactive components into the surrounding environment (e.g., soil and air), which would further result in the low efficiency of the fungicides in protecting plants and irreversible environmental pollution [1,2].The development of nano-pesticide systems with high loading capacity for bioactive compounds and desirable sustained releasing performance is considered an effective, emerging, and promising strategy for improving the utilization efficiency of fungicides [3][4][5], and the construction of a complex containing a natural product-based bioactive compound and a corresponding polysaccharide carrier has also proven to be an ingenious design strategy for novel nano-pesticide systems [6][7][8].
Herein, a novel series of L-carvone-based thiazolinone-hydrazone compounds were synthesized by the multi-step modification of natural forest product L-carvone and structurally confirmed by 1 H/ 13 C NMR, HRMS, and FT-IR.Then, the antifungal activity of the target compounds against eight phytopathogenic fungi was preliminarily evaluated by the in vitro method.In addition, an L-carvone-based nanochitosan carrier bearing the 1,3,4-thiadiazole-amide group was designed, synthesized, and characterized by UV-vis, FT-IR, XRD, TGA, SEM, and TEM, along with the construction and sustained releasing behavior of L-carvone-based thiazolinone-hydrazone/nanochitosan complexes.

Discovery of Antifungal Compounds
As illustrated in Scheme 1, target compounds 4a~4y were synthesized by the multistep modification of the natural forest product L-carvone.At first, L-carvone 4-methylthiosemicarbazone 2 was prepared by the nucleophilic addition of L-carvone and 4-methylthiosemicarbazide with hydrochloric acid as a catalyst and subsequently converted into intermediate 3 under the treatment of ethyl bromoacetate under alkaline conditions.Then, target compounds 4a~4y were synthesized through the condensation of intermediate 3 and the corresponding aryl aldehydes.The chemical structures of the target compounds were confirmed by 1 H/ 13 C NMR, HRMS, and FT-IR.The related spectra and data can be found in the Supplementary Material (Figures S1-S108).

Design and Synthesis of an L-Carvone-Based Nanochitosan Carrier Bearing the 1,3,4-Thiadiazole-Amide Group
Compound 4h, consisting of a hydrophobic L-carvone moiety, thiazolinone-hydrazone, and fluorine-substituted phenyl groups, had been found to show desirable antifungal activity.For designing a carrier for the sustained release of the antifungal compound, L-carvone and 1,3,4-thiadiazole-amide moieties were both introduced into the skeleton of chitosan.In our previous work [8], the original skeleton of the L-carvone moiety has been proven to be effective for improving the dispersibility and hydrophobicity of chitosan.Furthermore, both of the 1,3,4-thiadiazole-amide and thiazolinone-hydrazone groups possessed a five-membered heterocycle containing N and S atoms, along with amido linkage, and thus we envisioned that the 1,3,4-thiadiazole-amide group could further increase the dispersibility of the carrier and its interaction with compound 4h because of the similarity of the 1,3,4-thiadiazole-amide and thiazolinone-hydrazone groups.In addition, the fluorine-substituted phenyl group could provide a hydrogen bond acceptor (HBA), which was able to interact with the hydrogen bond donors (HBD) from the NH 2 and OH of chitosan.
As illustrated in Scheme 2, an L-carvone-based nanochitosan carrier 7 bearing the 1,3,4thiadiazole-amide group was synthesized through the chemical modification of chitosan.Firstly, L-carvone chloride 5 was obtained by the method described in previously published papers [8,25]

Characterization of the L-Carvone-Based Nanochitosan Carrier Bearing the 1,3,4-Thiadiazole-Amide Group
The L-Carvone-based nanochitosan carrier 7 bearing the 1,3,4-thiadiazole-amide group was characterized by UV-vis, FT-IR, TG, and XRD, and the results are shown in Figure 2. Firstly, carrier 7 displayed a maximum absorption at λ = 245.5 nm in its UV-vis spectrum, as can be seen in Figure 2A, while there was no obvious UV-absorption for that of chitosan, which indicates that the incorporation of the L-carvone-based 1,3,4-thiadiazole-amide group with π-π conjugated structure into the original skeleton of chitosan occurred smoothly in the facile condition.

Characterization of the L-Carvone-Based Nanochitosan Carrier Bearing the 1,3,4-Thiadiazole-Amide Group
The L-Carvone-based nanochitosan carrier 7 bearing the 1,3,4-thiadiazole-amide group was characterized by UV-vis, FT-IR, TG, and XRD, and the results are shown in Figure 2. Firstly, carrier 7 displayed a maximum absorption at λ = 245.5 nm in its UV-vis spectrum, as can be seen in Figure 2A, while there was no obvious UV-absorption for that of chitosan, which indicates that the incorporation of the L-carvone-based 1,3,4-thiadiazole-amide group with π-π conjugated structure into the original skeleton of chitosan occurred smoothly in the facile condition.Subsequently, a FT-IR analysis of carrier 7 was carried out, and the resulting spectrum was compared with that of unmodified chitosan (Figure 2B).Both in the IR spectra of carrier 7 and chitosan, the peaks at 1156, 1079, 1028, and 895 cm −1 , attributed to the asymmetric ν(C-O-C), ν(C-O of secondary alcohol), ν(C-O of primary alcohol), and ν(pyranose ring), were found, demonstrating that carrier 7 reserved the natural structure of chitosan [40,41].It was also observed that there were three newly emerging absorption peaks at 2923, 1637, and 1385 cm −1 in the IR spectrum of carrier 7, assigned to the ν(Csp3-H) in the molecular skeleton of L-carvone, ν(C=O) in the newly constructed amide bond, and ν(C=N) in the 1,3,4-thiadiazole ring, respectively, which further confirmed the successful incorporation of the molecular skeletons of L-carvone, 1,3,4-thiadiazole-amide, and chitosan.
In addition, TGA curves of carrier 7 and chitosan were studied, as can be seen in Figure 2C, to study the thermal stabilities of carrier 7 and chitosan from 40 to 800 °C.Carrier 7 and chitosan showed similar TGA profiles, and they could both be divided into three stages.The first stage of weight loss was due to the desorption of unbonded water, with mass losses of 2.81% (7, 138 °C) and 2.30% (Cs, 133 °C).When the samples were heated to 209 °C and 245 °C, carrier 7 and chitosan started the second stage of weight loss, respectively, which was the main stage of the mass loss of the samples because of the decomposition of the polysaccharide unit and the L-carvone-based 1,3,4-thiadiazole-amide group.Therefore, it could be deduced that, compared with that of unmodified chitosan, the thermal stability of carrier 7 decreased slightly at a higher temperature because the introduction of the L-carvone-based 1,3,4-thiadiazole-amide group led to a reduction in the hydrogen bonds between the microparticles of carrier 7 [39], but it kept the good thermal stability of chitosan below 200 °C.
The crystallinities of carrier 7 and chitosan were investigated by powder XRD technology.As can be seen in Figure 2D, something different occurred in the two XRD patterns.For example, the main characteristic peak for chitosan at 2θ = 20.1°changed into that of carrier 7 at 2θ = 20.4°after the chemical modification of chitosan through intermediate 6.Moreover, two broad diffraction peaks emerged at 2θ angles of 12.4° and 35.3°, respectively.Hence, it could be inferred that the chemical modification of chitosan would change its crystal structure [40].Subsequently, a FT-IR analysis of carrier 7 was carried out, and the resulting spectrum was compared with that of unmodified chitosan (Figure 2B).Both in the IR spectra of carrier 7 and chitosan, the peaks at 1156, 1079, 1028, and 895 cm −1 , attributed to the asymmetric ν(C-O-C), ν(C-O of secondary alcohol), ν(C-O of primary alcohol), and ν(pyranose ring), were found, demonstrating that carrier 7 reserved the natural structure of chitosan [40,41].It was also observed that there were three newly emerging absorption peaks at 2923, 1637, and 1385 cm −1 in the IR spectrum of carrier 7, assigned to the ν(C sp3 -H) in the molecular skeleton of L-carvone, ν(C=O) in the newly constructed amide bond, and ν(C=N) in the 1,3,4-thiadiazole ring, respectively, which further confirmed the successful incorporation of the molecular skeletons of L-carvone, 1,3,4-thiadiazole-amide, and chitosan.
In addition, TGA curves of carrier 7 and chitosan were studied, as can be seen in Figure 2C, to study the thermal stabilities of carrier 7 and chitosan from 40 to 800 • C. Carrier 7 and chitosan showed similar TGA profiles, and they could both be divided into three stages.The first stage of weight loss was due to the desorption of unbonded water, with mass losses of 2.81% (7, 138 • C) and 2.30% (Cs, 133 • C).When the samples were heated to 209 • C and 245 • C, carrier 7 and chitosan started the second stage of weight loss, respectively, which was the main stage of the mass loss of the samples because of the decomposition of the polysaccharide unit and the L-carvone-based 1,3,4-thiadiazole-amide group.Therefore, it could be deduced that, compared with that of unmodified chitosan, the thermal stability of carrier 7 decreased slightly at a higher temperature because the introduction of the L-carvone-based 1,3,4-thiadiazole-amide group led to a reduction in the hydrogen bonds between the microparticles of carrier 7 [39], but it kept the good thermal stability of chitosan below 200 • C.
The crystallinities of carrier 7 and chitosan were investigated by powder XRD technology.As can be seen in Figure 2D, something different occurred in the two XRD patterns.For example, the main characteristic peak for chitosan at 2θ = 20.1 • changed into that of carrier 7 at 2θ = 20.4• after the chemical modification of chitosan through intermediate 6.
Moreover, two broad diffraction peaks emerged at 2θ angles of 12.4 • and 35.3 • , respectively.Hence, it could be inferred that the chemical modification of chitosan would change its crystal structure [40].
Furthermore, the micro-morphology of L-carvone-based nanochitosan carrier 7 bearing the 1,3,4-thiadiazole-amide group was visualized by its TEM and SEM images.The introduction of a bulky and hydrophobic L-carvone-based 1,3,4-thiadiazole-amide group into the original skeleton of chitosan would inhibit the formation of hydrogen bonds between the nanoparticles of carrier 7 and depress the aggregation of these nanoparticles.Thus, as shown in Figure 3, carrier 7 could disperse well in the aqueous phase to form numerous nanoparticles with a size of~20 nm and exhibited an unconsolidated and multiaperture micro-structure, which was beneficial for improving the loading capacity of the carrier for the antifungal compound.
Furthermore, the micro-morphology of L-carvone-based nanochitosan carrier 7 bearing the 1,3,4-thiadiazole-amide group was visualized by its TEM and SEM images.The introduction of a bulky and hydrophobic L-carvone-based 1,3,4-thiadiazole-amide group into the original skeleton of chitosan would inhibit the formation of hydrogen bonds between the nanoparticles of carrier 7 and depress the aggregation of these nanoparticles.Thus, as shown in Figure 3, carrier 7 could disperse well in the aqueous phase to form numerous nanoparticles with a size of~20 nm and exhibited an unconsolidated and multiaperture micro-structure, which was beneficial for improving the loading capacity of the carrier for the antifungal compound.

Fabrication and Sustained Releasing Behavior of the L-Carvone-Based Thiazolinone-Hydrazone/Nanochitosan Complexes
For exploring novel nano-pesticides with sustained releasing properties, L-carvonebased thiazolinone-hydrazone/nanochitosan complexes were fabricated by the reported method [8].Compound 4h with antifungal activity was loaded on L-carvone-based nanochitosan carrier 7 bearing the 1,3,4-thiadiazole-amide group in three different mass ratios of 7/4h to obtain three complexes: 7/4h-1, 7/4h-2, and 7/4h-3, respectively.Then, the EE values of these complexes were determined, and the results were listed in entries 1~3 of Table 1.It was observed that complex 7/4h-2 had the highest EE value, and thus the mass ratio (2:1) of carrier to compound 4h for fabricating complex 7/4h-2 was chosen as the optimized one.For comparison, unmodified chitosan and another reported L-carvonebased nanochitosan 9 were also employed as carriers to load compound 4h with antifungal activity in the optimized mass ratio (2:1) of carrier to compound 4h, along with the determination of the EE values of the resulting complexes Cs/4h and 9/4h (entries 4 and 5).According to the comparison of the results in entries 2, 4, and 5, the descending order for the EE values of complexes 7/4h-2, Cs/4h, and 9/4h was 7/4h-2 > 9/4h > Cs/4h, suggesting that the introduction of L-carvone and 1,3,4-thiadiazole-amide moieties could effectively enhance the loading capacity of chitosan-based carriers for compound 4h.To view the micro-structures of L-carvone-based thiazolinone-hydrazone/nanochitosan complexes, the SEM image of complex 7/4h-2 was taken as an example and can be seen in Figure 4. Obviously, a well-distributed, compact, and columnar complex

Fabrication and Sustained Releasing Behavior of the L-Carvone-Based Thiazolinone-Hydrazone/Nanochitosan Complexes
For exploring novel nano-pesticides with sustained releasing properties, L-carvonebased thiazolinone-hydrazone/nanochitosan complexes were fabricated by the reported method [8].Compound 4h with antifungal activity was loaded on L-carvone-based nanochitosan carrier 7 bearing the 1,3,4-thiadiazole-amide group in three different mass ratios of 7/4h to obtain three complexes: 7/4h-1, 7/4h-2, and 7/4h-3, respectively.Then, the EE values of these complexes were determined, and the results were listed in entries 1~3 of Table 1.It was observed that complex 7/4h-2 had the highest EE value, and thus the mass ratio (2:1) of carrier to compound 4h for fabricating complex 7/4h-2 was chosen as the optimized one.For comparison, unmodified chitosan and another reported L-carvone-based nanochitosan 9 were also employed as carriers to load compound 4h with antifungal activity in the optimized mass ratio (2:1) of carrier to compound 4h, along with the determination of the EE values of the resulting complexes Cs/4h and 9/4h (entries 4 and 5).According to the comparison of the results in entries 2, 4, and 5, the descending order for the EE values of complexes 7/4h-2, Cs/4h, and 9/4h was 7/4h-2 > 9/4h > Cs/4h, suggesting that the introduction of L-carvone and 1,3,4-thiadiazole-amide moieties could effectively enhance the loading capacity of chitosan-based carriers for compound 4h.To view the micro-structures of L-carvone-based thiazolinone-hydrazone/nanochitosan complexes, the SEM image of complex 7/4h-2 was taken as an example and can be seen in Figure 4. Obviously, a well-distributed, compact, and columnar complex was generated by carrier 7 and compound 4h, and there were a lot of tiny particles of compound 4h adhered to the surface of the complex.
was generated by carrier 7 and compound 4h, and there were a lot of tiny particles of compound 4h adhered to the surface of the complex.The sustained releasing behaviors of the fabricated complexes were investigated, and the results are shown in Figure 5.All the complexes exhibited different sustained releasing behaviors, and their sustained releasing curves could be divided into two or more sections.For complexes 7/4h-2, Cs/4h, and 9/4h, the sustained releasing behaviors of these complexes were similar, and at the initial period, the particles of compound 4h on the surfaces of the complexes passed into the aqueous phase.Subsequently, the complexes broke down with the entrance of moisture through small apertures, and the inner particles of compound 4h were released.Both complexes 7/4h-1 and 7/4h-3 displayed the multistage releasing property of compound 4h, though their burst-releasing amounts and ratios were relatively big.In terms of the total releasing ratio, the descending order of all the complexes was 7/4h-1 ≈ 7/4h-2 ≈ 7/4h-3 > 9/4h > Cs/4h, revealing that the introduction of L-carvone and 1,3,4-thiadiazole-amide moieties could significantly alter the interactions of the chitosan-based carrier with compound 4h and consequently facilitate the releaseout of the internal particles of compound 4h.Among these complexes, we found that complex 7/4h-2 showed great potential as an antifungal nano-pesticide and deserved further studies.The sustained releasing behaviors of the fabricated complexes were investigated, and the results are shown in Figure 5.All the complexes exhibited different sustained releasing behaviors, and their sustained releasing curves could be divided into two or more sections.For complexes 7/4h-2, Cs/4h, and 9/4h, the sustained releasing behaviors of these complexes were similar, and at the initial period, the particles of compound 4h on the surfaces of the complexes passed into the aqueous phase.Subsequently, the complexes broke down with the entrance of moisture through small apertures, and the inner particles of compound 4h were released.Both complexes 7/4h-1 and 7/4h-3 displayed the multi-stage releasing property of compound 4h, though their burst-releasing amounts and ratios were relatively big.In terms of the total releasing ratio, the descending order of all the complexes was 7/4h-1 ≈ 7/4h-2 ≈ 7/4h-3 > 9/4h > Cs/4h, revealing that the introduction of L-carvone and 1,3,4-thiadiazole-amide moieties could significantly alter the interactions of the chitosanbased carrier with compound 4h and consequently facilitate the release-out of the internal particles of compound 4h.Among these complexes, we found that complex 7/4h-2 showed great potential as an antifungal nano-pesticide and deserved further studies.
was generated by carrier 7 and compound 4h, and there were a lot of tiny particles of compound 4h adhered to the surface of the complex.The sustained releasing behaviors of the fabricated complexes were investigated, and the results are shown in Figure 5.All the complexes exhibited different sustained releasing behaviors, and their sustained releasing curves could be divided into two or more sections.For complexes 7/4h-2, Cs/4h, and 9/4h, the sustained releasing behaviors of these complexes were similar, and at the initial period, the particles of compound 4h on the surfaces of the complexes passed into the aqueous phase.Subsequently, the complexes broke down with the entrance of moisture through small apertures, and the inner particles of compound 4h were released.Both complexes 7/4h-1 and 7/4h-3 displayed the multistage releasing property of compound 4h, though their burst-releasing amounts and ratios were relatively big.In terms of the total releasing ratio, the descending order of all the complexes was 7/4h-1 ≈ 7/4h-2 ≈ 7/4h-3 > 9/4h > Cs/4h, revealing that the introduction of L-carvone and 1,3,4-thiadiazole-amide moieties could significantly alter the interactions of the chitosan-based carrier with compound 4h and consequently facilitate the releaseout of the internal particles of compound 4h.Among these complexes, we found that complex 7/4h-2 showed great potential as an antifungal nano-pesticide and deserved further studies.

Synthesis of L-Carvone-Based Intermediate 3
A mixture of L-carvone 4-methyl-thiosemicarbazone 2 (16.8 g, 70.8 mmol) and sodium ethoxide (4.87 g, 71.6 mmol) in anhydrous ethanol (150 mL) was stirred at room temperature, and then ethyl bromoacetate (11.9 g, 71.3 mmol) was poured into the solution under continuous stirring.The reaction mixture was heated to reflux and kept for 4 h.After that, the reaction mixture was cooled down to room temperature, and the resulting precipitate was filtered out to obtain L-carvone-based intermediate 3 as a white solid with a yield of 93.0%.

Synthesis of L-Carvone-Based Thiazolinone-Hydrazone Compounds 4a~4y
L-Carvone-based intermediate 3 (1.67 g, 6.00 mmol), aryl aldehyde (7.00 mmol), and potassium hydroxide (0.34 g, 6.06 mmol) were mixed and dissolved in anhydrous ethanol (20 mL).The reaction mixture was continuously stirred and refluxed for 6 h.Upon completion of the reaction, the mixture was left to cool, and a vast amount of yellow powder was precipitated.The precipitate was filtered and washed with anhydrous ethanol several times to afford target compounds 4a~4y as yellow powders with yields of 77.4~91.6%.

Antifungal Activity Evaluation of the Target Compounds
Antifungal activity evaluation of target compounds 4a~4y was conducted by the in vitro method, and the eight tested phytopathogenic fungi included Fusarium oxysporum f. sp.cucumerinum, Cercospora arachidicola, Physalospora piricola, Alternaria solani, Gibberella zeae, Rhzioeotnia solani, Bipolaris maydis, and Colleterichum orbicalare.The commercial antifungal agent chlorothalonil was used as a positive control (PC).At first, the tested compound was dissolved in acetone and diluted with sorporl-144 (200 µg/mL) to prepare a stock solution with a concentration of 500 µg/mL.Then, 1 mL of solution containing the tested compound and 9 mL of potato sucrose agar (PSA) substrate were mixed in a culture dish to obtain a medicated medium with a final concentration of 50 µg/mL, and subsequently, a circle mycelium disc with a diameter of 4 mm was also placed into the culture dish.All culture dishes were cultivated in an incubator at 24 ± 1 • C for 48 h, and then the expanded diameter of every mycelium was measured.The inhibitory rates of the tested compounds were calculated by comparing the expanded mycelium diameters in every treatment group and control check.L-Carvone chloride 5 was prepared according to the literature [8,25].To a mixture of 5-amino-1,3,4-thiadiazole-2-thiol (2.00 g, 15.0 mmol) and potassium hydroxide (0.89 g, 15.9 mmol) in ethanol (20 mL) and water (5 mL), a solution of L-carvone chloride 5 (2.59 g, 14.0 mmol) in ethanol (10 mL) was added dropwise, and then the reaction mixture was stirred at room temperature for 12 h.After the reaction was completed, the mixture was extracted with ethyl acetate (30 mL × 3), and the combined organic layer was washed with a saturated sodium chloride solution (30 mL).The organic layer was separated out and concentrated under reduced pressure to obtain L-carvone-based 1,3,4-thiadiazole-amine as a crude product.
The obtained crude product of L-carvone-based 1,3,4-thiadiazole-amine was re-dissolved in DCM (25 mL), and chloroacetyl chloride (1.12 g, 9.92 mmol) was injected into the solution.The reaction mixture was kept for stirring at room temperature and monitored by TLC until the reaction was completed.Subsequently, the reaction mixture was concentrated in vacuum, and the residue was further purified by column chromatography (PE:EA = 5:1, v/v) to obtain L-carvone-based 1,3,4-thiadiazole-amide intermediate 6 as a white powder with a yield of 71.6%.mass ratios (7/4h) of 1:1, 2:1, and 3:1 was added separately under magnetic stirring.The resulting mixture was continuously stirred until the full evaporation of the solvent, and then the residue was dried in an oven at a temperature of 60 • C to afford complexes 7/4h-1, 7/4h-2, and 7/4h-3.Similarly, samples Cs and 9 were employed as carriers for loading 4h with the optimized mass ratio of 7/4h to fabricate complexes Cs/4h and 9/4h, respectively.
The 4h-loading complex (approximately 3.0 mg) was placed into a centrifugal tube (10 mL), and then DCM (10.0 mL) was poured into it.The obtained suspension was treated with ultrasonic (75 W) for 150 s, followed by centrifugation, to obtain the supernatant for UV-vis spectroscopy detection.The EE values of the complexes were calculated by the following equation: EE (%) = loading amount of 4h in the complex/initial amount of 4h.

Sustained Releasing Behavior of L-Carvone-Based Thiazolinone-Hydrazone/Nanochitosan Complexes
The sustained releasing behaviors of complexes 7/4h-1, 7/4h-2, and 7/4h-3, 9/4h, and Cs/4h were investigated in an ethanol-water solution (1:9, v/v) at room temperature.Firstly, the complex (approximately 6.0 mg) was placed into an ethanol-water solution (50 mL, 1:9, v/v).At specific time points, 5 mL of the supernatant was sampled from the system and extracted with DCM (10 mL).Meanwhile, the same volume of the release medium was refilled into the system.The concentration of 4h in the combined organic layer was determined by UV-vis spectroscopy, and the releasing ratio was calculated by the following formula: releasing ratio (%) = total releasing amount of 4h/total loading amount of 4h in the complex.

Conclusions
In conclusion, twenty-five L-carvone-based thiazolinone-hydrazone compounds 4a~4y were synthesized by the multi-step modification of natural forest product L-carvone and structurally confirmed by 1 H/ 13 C NMR, HRMS, and FT-IR.The in vitro antifungal evaluation of the target compounds against eight phytopathogenic fungi suggested that compound 4h displayed desirable and broad-spectrum antifungal activity and could serve as a leading compound for novel antifungal agents in agriculture.In addition, the L-carvonebased nanochitosan carrier 7 bearing the 1,3,4-thiadiazole-amide group was rationally designed and synthesized for the loading and sustained releasing applications of compound 4h, along with several characterizations, including UV-vis, FT-IR, XRD, TGA, SEM, and TEM.We found that carrier 7 had good thermal stability below 200 • C, dispersed well in the aqueous phase to form numerous nanoparticles with a size of~20 nm, and exhibited an unconsolidated and multi-aperture micro-structure.Finally, L-carvone-based thiazolinone-hydrazone/nanochitosan complexes were fabricated and investigated for their sustained releasing behaviors.Among these complexes, complex 7/4h-2 with the highest encapsulation efficiency for compound 4h and a well-distributed, compact, and columnar micro-structure showed desirable sustained releasing performance and thus great potential as an antifungal nano-pesticide for further studies.Therefore, the introduction of L-carvone and 1,3,4-thiadiazole-amide moieties could effectively enhance the loading capacity and sustained releasing properties of the chitosan-based carrier for compound 4h by improving the interaction between the chitosan-based carrier and the hydrophobic compound 4h.