A pentacyclic triterpene derivative, a preparation method thereof and application thereof in pine wood nematode control

Abstract

The present application relates to the technical field of forestry pest control, and discloses a pentacyclic triterpene derivative, a preparation method thereof and application of the pentacyclic triterpene derivative in pine wood nematode control. The pentacyclic triterpene derivative has a structure as shown in formula I and is prepared by the following steps: tea saponin is dissolved in ethanol, adsorbed by activated carbon, concentrated, adsorbed by macroporous resin, eluted, desorbed, hydrolyzed, crystallized, purified to obtain a pentacyclic triterpene compound, the hydroxyl group on the pentacyclic triterpene compound is protected to obtain an intermediate I, the double bond on the intermediate I is protected to obtain an intermediate II, 4-chlorobenzoyl hydrazine is added and reacted with the intermediate II to obtain an intermediate III, the double bond of the intermediate III is deprotected to obtain an intermediate IV, and the hydroxyl group of the intermediate IV is deprotected to finally obtain the pentacyclic triterpene derivative. The pentacyclic triterpene derivative has the effect of preventing and treating pine wood nematode disease and can be prepared into a pine wood nematode-killing pharmaceutical preparation.

Description

Technical Field

[0001] This invention relates to the field of forestry pest and disease control technology, and in particular to a pentacyclic triterpenoid derivative, its preparation method, and its application in the control of pine wilt disease. Background Technology

[0002] Pine wilt disease is a devastating forest disease caused by the pine wilt nematode (Bursaphelenchus xylophilus). Spread by the pine sawyer beetle (Monochamus alternatus), the disease infects pine trees, disrupting water transport and causing trees to wither and die within as little as 40 days. Due to its extremely high mortality rate and rapid spread, it is often referred to as "pine cancer." Traditional control methods, such as cleaning diseased trees and using traditional insecticides to control the vector insects, suffer from high costs, environmental pollution, and limited effectiveness. Therefore, developing efficient, green, and targeted new control technologies has become a crucial issue urgently needing to be addressed in the field of forestry conservation.

[0003] Recent studies have shown that pine trees activate terpenoid biosynthesis pathways within 5 days of pine wood nematode infection (Yu L, Wang Y, Wang X, Han S, Wang L, Wang X. Transcriptomic, metabonomic and proteomic analyses reveal that terpenoids and flavonoids are required for Pinus koraiensis early defense against Bursaphelenchus xylophilus infection[J]. BMC Plant Biol. 2025, 25(1):185), indicating that terpenoids are crucial for early defense against pine wood nematode infection in pine trees. Among terpenoids, pentacyclic triterpenes are an important class of plant secondary metabolites, composed of six isoprene units with a closed pentacyclic skeleton. They are widely found in various traditional Chinese medicines and natural plants, existing as free esters or glycosides, suggesting that structurally diverse pentacyclic triterpenes and their derivatives may also possess the potential to combat plant parasitic nematodes.

[0004] However, most existing research on the activity of pentacyclic triterpenes focuses on human disease-related models (such as cancer cells and inflammation models), and very few studies have systematically explored their activity against pine wilt disease, a specific forestry pathogen (Ding Wenxin, Du Bolin, Li Jiao, et al. Research progress on pentacyclic triterpenoid natural products [J]. Acta Pharmaceutica Sinica, 2024, 59(5):1163-1175). Research on the application of pentacyclic triterpenoid compounds in the control of pine wilt disease is expected to provide a new solution for controlling "pine cancer". Summary of the Invention

[0005] To address the aforementioned technical problems, this invention has discovered that pentacyclic triterpenes and their derivatives possess the potential to combat plant parasitic nematodes. This invention provides a pentacyclic triterpenoid derivative, its preparation method, and its application in the control of pine wilt disease.

[0006] To achieve the above objectives, the present invention provides a pentacyclic triterpenoid derivative, characterized in that it has a structure as shown in Formula I: (Formula I).

[0007] The present invention also provides a method for preparing the above-mentioned pentacyclic triterpenoid derivative, characterized by comprising the following steps: S1. Dissolve tea saponin in an ethanol-water solution at 65–75°C, add activated carbon to adsorb impurities for 30–60 min, collect the filtrate, concentrate the filtrate under reduced pressure to 1 / 3–1 / 4 of the filtrate volume, dilute the concentrate with deionized water to 2–3 times the concentrate volume, adsorb onto AB-8 macroporous adsorption resin for 18–36 h, elute once with a 30–50% (v / v) ethanol-water solution, and then elute a second time with a 70–80% (v / v) ethanol-water solution. Collect the second eluent, add sodium hydroxide to react, and react at 75°C. The reaction was carried out at 85°C for 2-4 hours. After the reaction solution was cooled to 25-35°C, the pH was adjusted to pH 4-5 with hydrochloric acid. The precipitate was allowed to stand for 1-2 hours. The precipitate was then dissolved in an 80-90% (v / v) ethanol aqueous solution. P-Toluenesulfonic acid was added and refluxed. The reaction temperature was 85-95°C for 6-12 hours. After the reaction was completed, the precipitate was poured into ice water and allowed to stand for 4-8 hours to crystallize. The crystals were separated by centrifugation for 10-15 minutes, and the precipitate was collected. The precipitate was dispersed in acetone, filtered, and the filtrate was dried under reduced pressure for 12-36 hours to obtain the pentacyclic triterpenoid compound. S2. Dissolve the pentacyclic triterpenoid compound obtained in step S1 in N,N-dimethylformamide, add tert-butyldimethylchlorosilane and imidazole to react, the reaction temperature is 25-30℃, the reaction time is 12-24h, the reaction is terminated with water after the reaction is completed, and the mixture is extracted with ethyl acetate. The extract is dried under reduced pressure for 12-36h to obtain intermediate I. S3. Dissolve intermediate I in dichloromethane, add bromine to react, the reaction temperature is 0-5℃, the reaction time is 1-2h, after the reaction is completed, terminate the reaction with sodium sulfite solution, and dry the reaction solution under reduced pressure for 12-36h to obtain intermediate II. S4. Dissolve intermediate II in anhydrous ethanol, add 4-chlorobenzoyl hydrazine and acetic acid to react, the reaction temperature is 65-70℃, the reaction time is 4-6h, and after the reaction is completed, dry the reaction solution under reduced pressure for 12-36h to obtain intermediate III. S5. Dissolve intermediate III in acetic acid, add zinc powder to react, the reaction temperature is 25-30℃, the reaction time is 2-3h, after the reaction is completed, filter, and dry the filtrate under reduced pressure for 12-36h to obtain intermediate IV. S6. Dissolve intermediate IV in tetrahydrofuran, add tetrabutylammonium fluoride and react at a temperature of 25-30°C for 6-8 hours. After the reaction is complete, dry the reaction solution under reduced pressure for 12-36 hours to obtain the pentacyclic triterpenoid derivative.

[0008] Preferably, in step S1, tea saponin is dissolved in an ethanol-water solution with a mass-to-volume ratio of 1:8-12 g / mL, and the volume fraction of ethanol in the ethanol-water solution is 60%-70%. The amount of activated carbon added is 0.05 to 0.1 times the mass of tea saponin, and the adsorption temperature is 65 to 75℃. The vacuum concentration temperature is 60–70℃; The AB-8 macroporous adsorption resin was added at a mass of 6 to 9 times that of tea soap, and the adsorption temperature was 25 to 35°C. The volume ratio of the ethanol-water solution used for each elution to the mass ratio of the macroporous resin was 3-5:1 mL / g, and the elution time was 2-4 h. The mass-to-volume ratio of sodium hydroxide added to the secondary eluent is 0.5–1.5:100 g / mL; The concentration of hydrochloric acid used to adjust the pH is 1–2.5 mol / L; The volume of the ethanol-water solution used to dissolve the precipitate is 3 to 5 times the mass of the precipitate. The volume ratio of p-toluenesulfonic acid to the solution is 0.3–0.5 g / mL; The temperature of the ice water is 4–10℃, and the volume of the ice water is 2–4 times the volume of the reaction liquid; Centrifugation speed: 8000–10000 rpm; The mass-to-volume ratio of the precipitate to acetone is 1:2-3 g / mL; The filtrate is dried under reduced pressure at a vacuum of 0.05–0.2 atmospheres and a temperature of 45–65°C.

[0009] Preferably, in step S2, the mass-to-volume ratio of the pentacyclic triterpenoid compound to the N,N-dimethylformamide solvent is 1:8-16 g / mL; The mass of tert-butyldimethylchlorosilane is 1.6 to 1.8 times that of the pentacyclic triterpenoid compound; The mass of imidazole is 1.5 to 1.7 times that of the pentacyclic triterpenoid compound; The volume of water and ethyl acetate used to terminate the reaction were 1 to 3 times the volume of N,N-dimethylformamide. The extract was dried under reduced pressure at a vacuum of 0.05–0.2 atmospheres and a temperature of 45–65°C.

[0010] Preferably, in step S3, the mass-to-volume ratio of intermediate I to dichloromethane solvent is 1:4 to 8 g / mL; The mass-to-volume ratio of intermediate I to bromine is 1:0.09–0.1 g / mL; The sodium sulfite solution has a mass fraction of 30-35%, and its volume is 1 / 2 to 1 times the volume of the dichloromethane solvent. The reaction solution is dried under reduced pressure at a vacuum of 0.05–0.2 atmospheres and a temperature of 45–65°C.

[0011] Preferably, in step S4, the mass-to-volume ratio of intermediate II to ethanol solvent is 1:3 to 6.5 g / mL; The mass of 4-chlorobenzoyl hydrazide is 0.15 to 0.25 times the mass of intermediate II; The mass-to-volume ratio of intermediate II to acetic acid is 1:0.3–0.5 g / mL; The reaction solution is dried under reduced pressure at a vacuum of 0.05–0.2 atmospheres and a temperature of 45–65°C.

[0012] Preferably, in step S5, the mass-to-volume ratio of intermediate III to acetic acid is 1:2.5-6 g / mL; The mass of zinc powder is 0.09 to 0.15 times the mass of intermediate III; The filtrate is dried under reduced pressure at a vacuum of 0.05–0.2 atmospheres and a temperature of 45–65°C.

[0013] Preferably, in step S6, the mass-to-volume ratio of intermediate IV to tetrahydrofuran is 1:2.5-6 g / mL; The mass of tetrabutylammonium fluoride is 1.1 to 1.3 times the mass of intermediate IV; The reaction solution is dried under reduced pressure at a vacuum of 0.05–0.2 atmospheres and a temperature of 45–65°C.

[0014] The present invention also provides the application of the above-mentioned pentacyclic triterpenoid derivatives or the pentacyclic triterpenoid derivatives prepared by the above preparation method in the control of pine wilt disease. The pentacyclic triterpenoid derivatives can be made into drug preparations for killing pine wilt disease. The drug preparations are single-ingredient or compound preparations containing pentacyclic triterpenoid derivatives.

[0015] The principle of this invention is as follows: Camellia oleifera saponin is purified by adsorption using activated carbon; the organic acid structure of Camellia oleifera saponin is removed by sodium hydroxide; and the sugar group is removed by hydrochloric acid to obtain a pentacyclic triterpenoid compound. Using imidazole as a catalyst, tert-butyldimethylchlorosilane is used to protect the hydroxyl groups on the pentacyclic triterpenoid compound to prevent hydroxyl oxidation in subsequent reactions, yielding intermediate I. The double bonds on intermediate I are protected with bromine to obtain intermediate II. Under acetic acid catalysis, 4-chlorobenzoyl hydrazine is added to react with intermediate II to obtain intermediate III. The double bond protecting group is removed by zinc to obtain intermediate IV. Finally, the pentacyclic triterpenoid derivative is obtained by removing the hydroxyl protecting group with tetrabutylammonium fluoride.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention uses tea saponin as raw material and after derivatization treatment, the pentacyclic triterpenoid derivative obtained has significantly improved activity against pine wood nematode compared with tea saponin, which is based on the synergistic effect of the pentacyclic triterpenoid derivative with multiple targets.

[0017] 2. In the preparation of the pentacyclic triterpenoid derivatives of the present invention, after protecting the hydroxyl groups on the pentacyclic triterpenoid compound with tert-butyldimethylchlorosilane and protecting the double bonds on the pentacyclic triterpenoid compound with bromine, only the carbonyl group on the pentacyclic triterpenoid compound can react with the amino group on 4-chlorobenzoylhydrazine, thus ensuring the consistency of the structure and the uniformity of the quality and activity of the pentacyclic triterpenoid derivatives.

[0018] 3. The preparation process of this invention is simple, the reaction conditions are mild, the product has high purity, and it can be mass-produced industrially. Attached Figure Description

[0019] Figure 1 The images show the infrared spectra of the pentacyclic triterpenoid compounds prepared in the embodiments of the present invention and the pentacyclic triterpenoid derivatives obtained in Example 1 of the present invention.

[0020] Figure 2 The image shows the 1H NMR spectrum of the pentacyclic triterpenoid compound obtained in an embodiment of the present invention.

[0021] Figure 3 The above is the 1H NMR spectrum of the pentacyclic triterpenoid derivative prepared in Example 1 of this invention. Detailed Implementation

[0022] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only a part of the embodiments of this application, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0023] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques described in the literature or reference books in this field or in accordance with the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products.

[0024] Preparation of pentacyclic triterpenoids: 50g of tea saponin (purchased from Jiangxi Xinzhongye Tea Industry Technology Co., Ltd.) was mixed with 500mL of 65% ethanol aqueous solution and dissolved at 70℃ for 1.5h. 5g of activated carbon was added, and the mixture was kept warm for another 45min. The mixture was filtered while hot, and the filtrate was collected. The filtrate was concentrated under reduced pressure to 180mL. The concentrate was diluted with deionized water to 450mL, and 400g of AB-8 macroporous resin was added. The mixture was adsorbed at 30℃ for 24h. The resin was eluted with 2000mL of 30% ethanol aqueous solution for 4h, and then desorbed with 2000mL of 75% ethanol aqueous solution for 4h. The eluent was collected. 20g of sodium hydroxide was added, and the mixture was reacted at 75℃ for 3h. The reaction solution was cooled to 30℃, and the pH was adjusted to 4 with 1.5 mol / L hydrochloric acid. The mixture was allowed to stand for 2h to obtain 35g of precipitate. The precipitate was redissolved in 140 mL of 90% ethanol aqueous solution; 70 g of p-toluenesulfonic acid was added, and the mixture was refluxed at 85 °C for 8 h; the reaction solution was poured into 420 mL of ice water at 4 °C and allowed to stand for 6 h to crystallize; then, 25 g of precipitate was collected by centrifugation at 10000 rpm for 10 min; the precipitate was dispersed in 50 mL of acetone, filtered, and the filtrate was vacuum dried at 0.05 atm and 65 °C for 12 h to obtain 18 g of white powdery pentacyclic triterpenoid compound.

[0025] Embodiment 1 of the present invention (1) Dissolve 1g of pentacyclic triterpenoid compound in 12mL of N,N-dimethylformamide, add 1.7g of tert-butyldimethylchlorosilane and 1.6g of imidazole, react at 25℃ for 18h, add 24mL of water to terminate the reaction, extract with 18mL of ethyl acetate, and dry the extract under vacuum at 0.05 atm and 65℃ for 12h to obtain 2.3g of intermediate I.

[0026] (2) Dissolve 2g of intermediate I in 8mL of dichloromethane, add 0.18mL of bromine, react at 5℃ for 2h, and after the reaction is completed, add 8mL of sodium sulfite solution (mass fraction of 32%) to terminate the reaction. Take the dichloromethane layer and dry it under vacuum at 0.05 atm and 65℃ for 12h to obtain 1.9g of intermediate II.

[0027] (3) Dissolve 1.2g of intermediate II in 3.6mL of ethanol, add 0.18g of 4-chlorobenzoyl hydrazine and 0.36mL of acetic acid, and reflux at 65℃ for 4h. After the reaction is completed, the reaction solution is vacuum dried at 0.05 atm and 65℃ for 12h to obtain 1.3g of intermediate III.

[0028] (4) Dissolve 1.2g of intermediate III in 3mL of acetic acid, add 0.11g of zinc powder and react at 25℃ for 2h. Filter and vacuum dry the filtrate at 0.05 atm and 65℃ for 12h to obtain 1.1g of intermediate IV.

[0029] (5) Dissolve 1g of intermediate IV in 2.5mL of tetrahydrofuran, add 1.1g of tetrabutylammonium fluoride and react at 25°C for 6h. After the reaction is completed, the reaction solution is vacuum dried at 0.05 atm and 65°C for 12h to obtain 0.8g of pentacyclic triterpenoid derivative.

[0030] Embodiment 2 of the present invention (1) Dissolve 1g of pentacyclic triterpenoid compound in 16mL of N,N-dimethylformamide, add 1.8g of tert-butyldimethylchlorosilane and 1.5g of imidazole, react at 30℃ for 16h, terminate the reaction with 16mL of water after the reaction is completed, extract with 16mL of ethyl acetate, and dry the extract at 0.2 atm and 45℃ for 36h to obtain 2.5g of intermediate I.

[0031] (2) Dissolve 2g of intermediate I in 12mL of dichloromethane, add 0.2mL of bromine, react at 0℃ for 1h, and terminate the reaction with 6mL of sodium sulfite solution (mass fraction of 32%) after the reaction is completed. Take the dichloromethane layer and dry it under vacuum at 0.2 atm and 45℃ for 36h to obtain 2.0g of intermediate II.

[0032] (3) Dissolve 1g of intermediate II in 6.5mL of ethanol, add 0.25g of 4-chlorobenzoyl hydrazine and 0.5mL of acetic acid, reflux at 70℃ for 6h, and after the reaction is completed, vacuum dry at 0.2 atm and 45℃ for 36h to obtain 1.2g of intermediate III.

[0033] (4) Dissolve 1.2g of intermediate III in 7.2mL of acetic acid, add 0.18g of zinc powder and react at 30℃ for 3h. Filter and vacuum dry the filtrate at 0.2 atm and 45℃ for 36h to obtain 1.0g of intermediate IV.

[0034] (5) Dissolve 1g of intermediate IV in 6mL of tetrahydrofuran, add 1.3g of tetrabutylammonium fluoride and react at 30°C for 6h. After the reaction is completed, the reaction solution is vacuum dried at 0.2 atm and 45°C for 36h to obtain 0.9g of pentacyclic triterpenoid derivative.

[0035] Embodiment 3 of the present invention (1) Dissolve 1g of pentacyclic triterpenoid compound in 8mL of N,N-dimethylformamide, add 1.6g of tert-butyldimethylchlorosilane and 1.5g of imidazole, react at 25℃ for 24h, terminate the reaction with 24mL of water, extract with 24mL of ethyl acetate, and dry the extract at 0.1 atm and 55℃ for 24h to obtain 2.1g of intermediate I.

[0036] (2) Dissolve 2g of intermediate I in 16mL of dichloromethane, add 0.2mL of bromine, stir at 5℃ for 2h, terminate the reaction with 10mL of sodium sulfite solution (mass fraction of 32%), take the dichloromethane layer and vacuum dry at 0.1 atm and 55℃ for 24h to obtain 2.0g of intermediate II.

[0037] (3) Dissolve 1g of intermediate II in 5mL of ethanol, add 0.2g of 4-chlorobenzoyl hydrazine and 0.4mL of acetic acid, reflux at 65℃ for 6h, and after the reaction is completed, the reaction solution is vacuum dried at 0.1 atm and 55℃ for 24h to obtain 1.1g of intermediate III.

[0038] (4) Dissolve 1.1g of intermediate III in 5.5mL of acetic acid, add 0.12g of zinc powder and react at 25℃ for 3h. Filter and vacuum dry the filtrate at 0.1 atm and 55℃ for 24h to obtain 1.0g of intermediate IV.

[0039] (5) Dissolve 1g of intermediate IV in 5mL of tetrahydrofuran, add 1.2g of tetrabutylammonium fluoride and react at 25°C for 7h. After the reaction is completed, the reaction solution is vacuum dried at 0.1 atm and 55°C for 24h to obtain 0.8g of pentacyclic triterpenoid derivative.

[0040] Comparative Example 1 The reaction was basically the same as in Example 1, except that the reaction did not go through step (3), other conditions remained unchanged, and the product of the previous step was used in the subsequent step to obtain 1g of comparative product 1.

[0041] Comparative Example 2 The reaction was basically the same as in Example 1, except that the reaction conditions in step (1) were changed to heating at 50°C for 5 h, while other conditions remained unchanged, and 0.9 g of comparative product 2 was obtained.

[0042] Comparative Example 3 The reaction was basically the same as in Example 1, except that the 4-chlorobenzoyl hydrazine added in step (3) was replaced with an equimolar mass of benzoyl hydrazine, and other conditions remained unchanged, resulting in 0.8 g of comparative product 3.

[0043] Performance testing: 1. The pentacyclic triterpenoid compounds prepared in the embodiments of the present invention and the pentacyclic triterpenoid derivatives prepared in Example 1 of the present invention were measured by matrix-assisted laser desorption / ionization flight mass spectrometry (Bruker MALDI-8020), nuclear magnetic resonance (Burker Avance III), and Fourier transform infrared spectroscopy (Thermo Scientific Nicolet iS10). The relevant information is as follows: The infrared spectrum and proton NMR spectrum of the pentacyclic triterpenoid compounds obtained in the embodiments of the present invention are shown in the figure. Figure 1 , Figure 2 White to off-white solid. IR (KBr pellet, cm⁻¹): ν(OH) = 3437.69, ν(CH) = 2949.75, ν(C=O) = 1722.61. 1H NMR (400MHz, CD₃OD): δ 9.32 (s, 1H), 5.49 (s, 1H), 5.36 (m, 2H), 5.32 (s, 1H), 5.28 (t, 1H, J = 6.3 Hz), 4.35 (s, 1H), 4.03 (m, 1H), 3.78 (d, 1H, J = 12.3 Hz), 3.70 (d, 1H, J = 9.7 Hz), 3.31 (m, 3H), 2.05 (t, 1H, J = 12.3 Hz). =12.1Hz), 1.96–1.25(m, 14H), 1.12(dd, 1H, J=6.5, 3.8Hz), 1.11(dd, 1H, J =8.2, 3.2Hz), 1.02(s, 3H), 0.99(s, 3H), 0.98-0.91(s, 12H).Anal.Calcd.for C30H48O6: C, 71.39; H, 9.59%; Found: C, 69.50; H, 9.07%.

[0044] The infrared spectrum and proton NMR spectrum of the pentacyclic triterpenoid derivatives obtained in Example 1 of this invention are shown below. Figure 1 , Figure 3Brownish-yellow solid. IR (KBr pellet, cm⁻¹): ν(C=O) = 1658.88, δ(CH) = 827.68, ν(C-Cl) = 732.46. 1H NMR (400 MHz, CDCl₃): δ 9.71 (s, 1H), 7.71 (d, 2H, J = 8.1 Hz), 7.69 (s, 1H), 7.51 (d, 1H, J = 8.0 Hz), 5.40 (m, 2H), 5.34 (s, 1H), 5.31 (t, 1H, J = 6.3 Hz), 5.19 (s, 1H), 4.38 (s, 1H), 3.82 (d, 1H, J = 12.3 Hz), 3.72 (d, 1H, J = 9.7 Hz). Hz), 3.67 (m, 1H), 3.34 (m, 3H), 2.08 (t, 1H, J =12.1Hz), 1.82–1.25 (m, 14H), 1.14 (dd, 1H, J =8.2, 3.2Hz), 1.07 (dd, 1H, J =6.5, 3.8 Hz), 1.00 (s, 3H), 0.98 (s, 3H), 0.94-0.78 (s, 12H). Anal.Calcd.for C38H57ClN2O6: C, 67.78; H, 8.53; Cl, ​​5.26; N, 4.16%; Found: C, 64.01; H, 8.11; Cl, ​​5.01; N, 3.92%.

[0045] Depend on Figure 1 It can be seen that the absorption peak of the secondary amide C=O stretching vibration of the pentacyclic triterpenoid derivative appears at 1658.88 cm⁻¹, the absorption peak of the out-of-plane bending vibration of CH on the para-substituted benzene ring appears at 827.68 cm⁻¹, and the absorption peak of the C-Cl stretching vibration is at 732.46 cm⁻¹, indicating that the amino group of 4-chlorobenzoylhydrazide undergoes a nucleophilic addition reaction with the aldehyde group of the pentacyclic triterpenoid compound. Figure 2 , Figure 3 It can be seen that the characteristic peak of the aldehyde group at δ 9.25 disappears in the pentacyclic triterpenoid derivative, while the characteristic peak of the hydrogen on the benzene ring appears at δ 7.70 and 7.52, indicating that the aldehyde group on the pentacyclic triterpenoid compound successfully undergoes a nucleophilic addition reaction with 4-chlorobenzoylhydrazine.

[0046] 2. Test the killing activity of the pentacyclic triterpenoid derivatives prepared in Examples 1-3 of the present invention and the products obtained in Comparative Examples 1-3 against pine wood nematodes.

[0047] Sample stock solution: Weigh 1 mg of each test compound and dissolve it in 100 μL of DMSO. Transfer the test compound solution to a centrifuge tube containing 1% Tween-80 aqueous solution to prepare a test solution with a concentration of 10 mg / mL.

[0048] Nematode suspension: Collect active pine wood nematodes from cultured Botrytis cinerea plates, wash with sterile water or culture medium, and adjust to a nematode suspension of 50 nematodes / 10 μL for later use.

[0049] Nematode activity assay: The sample stock solution was diluted with culture medium to the working concentration (200, 150, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 µg / mL), and nematode suspension was added. The samples were placed in a constant temperature incubator at 25℃, and the survival of the nematodes was observed under a microscope after 48 hours. Live nematodes were defined as those exhibiting "S", coiled, or spiral movements; dead nematodes were defined as those that did not move and exhibited "J" or "C" shapes, or those that were rigid and had no refractive properties in their body walls. The median lethal concentration (LC50) of each sample was obtained. A lower LC50 value indicates higher nematode activity. The background solution of each treatment was used as a negative control, and nematophene under the same treatment was used as a positive control. Each treatment was repeated in triplicate. The LC50 values ​​of each sample are shown in Table 1.

[0050] Table 1. LC50 of pentacyclic triterpenoid derivatives and their compounds against pine wood nematodes. 50 value Table 1 shows that the pentacyclic triterpenoid derivatives obtained in Examples 1-3 of this invention have good killing effects on pine wood nematodes. Compared with tea saponin, pentacyclic triterpenoid compounds, and thiophene, the pentacyclic triterpenoid derivatives of Examples 1-3 of this invention have significantly improved killing effects on pine wood nematodes. Comparative product 1, comparative product 2, and comparative product 3 all have weak insecticidal effects. The structures of comparative product 1 and comparative product 3 have changed. Although comparative product 2 only increased the reaction temperature, it led to the removal of the protecting group, reduced reaction selectivity and yield, and resulted in increased by-products and decreased activity. This indicates that the reaction materials and conditions of this invention are irreplaceable.

[0051] Example 4 of this invention describes the application of the pentacyclic triterpenoid derivative obtained in Example 1 in the control of pine wilt disease.

[0052] Take 30g of the pentacyclic triterpenoid derivative obtained in Example 1 of this invention, and mix it with 6g of sodium lignosulfonate, 2g of anhydrous sodium sulfate, 1g of sodium dodecyl sulfate, and 61g of kaolin as an excipient. After each material is pulverized by air jet milling to D90 < 10μm, water is added and kneaded in a mixer, followed by extrusion granulation, drying, and passing through a 40-60 mesh sieve to obtain the pentacyclic triterpenoid derivative water dispersible agent.

[0053] Physicochemical property testing: Moisture content ≤2.0%, suspension rate ≥90%, wetting time ≤60s, disintegration time ≤1min, and decomposition rate ≤5% after accelerated thermal storage stability (54±2℃, 14 days), meeting the quality technical indicators of water-dispersible granules.

[0054] Application methods for pine wilt disease control: Dilute the pentacyclic triterpenoid derivative water dispersible agent with water 800-1200 times and spray it evenly over the entire pine forest affected by pine wilt nematode, focusing on the trunk and branches. Apply at the early stage of disease occurrence or before the emergence of the longhorn beetle, and repeat every 10-15 days depending on the infestation level.

[0055] Example 5 illustrates the application of the pentacyclic triterpenoid derivative obtained in Example 2 of this invention in the control of pine wilt disease.

[0056] Take 10g of the pentacyclic triterpenoid derivative prepared in Example 2 of this invention, and pulverize it separately with 5g of polyvinylpyrrolidone K30 and 85g of anhydrous glucose through a 100-mesh sieve. Mix the above materials in a three-dimensional mixer for 30 minutes until homogeneous to obtain a soluble powder of the pentacyclic triterpenoid derivative.

[0057] Physicochemical properties test: The appearance is a uniform powder, the fineness (passing through a 75μm sieve) is ≥98%, the dissolution time (20°C static water) is ≤3min, and the moisture content is ≤3.0%.

[0058] Application method for pine wilt control: Use professional trunk injection equipment to prepare a 5-10% concentrated solution of pentacyclic triterpenoid derivative soluble powder with deionized water. Drill holes at the base of the pine trunk (hole diameter about 6mm, angled downwards to the xylem). Inject 1-2mL of the solution per centimeter of diameter at breast height, allowing the solution to be transported to the entire tree through the tree's vascular system.

[0059] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several improvements and substitutions without departing from the technical principles of the present invention, and these improvements and substitutions should also be considered within the scope of protection of the present invention.

Claims

1. A pentacyclic triterpenoid derivative, characterized in that, It has a structure as shown in Equation I: (Equation I).

2. The method for preparing a pentacyclic triterpenoid derivative as described in claim 1, characterized in that, Includes the following steps: S1. Dissolve tea saponin in an ethanol-water solution at 65–75°C, add activated carbon to adsorb impurities for 30–60 min, collect the filtrate, concentrate the filtrate under reduced pressure to 1 / 3–1 / 4 of the filtrate volume, dilute the concentrate with deionized water to 2–3 times the concentrate volume, adsorb onto AB-8 macroporous adsorption resin for 18–36 h, elute once with a 30–50% (v / v) ethanol-water solution, and then elute a second time with a 70–80% (v / v) ethanol-water solution. Collect the second eluent, add sodium hydroxide to react, and react at 75°C. The reaction was carried out at 85°C for 2-4 hours. After the reaction solution was cooled to 25-35°C, the pH was adjusted to pH 4-5 with hydrochloric acid. The precipitate was allowed to stand for 1-2 hours. The precipitate was then dissolved in an 80-90% (v / v) ethanol aqueous solution. P-Toluenesulfonic acid was added and refluxed. The reaction temperature was 85-95°C for 6-12 hours. After the reaction was completed, the precipitate was poured into ice water and allowed to stand for 4-8 hours to crystallize. The crystals were separated by centrifugation for 10-15 minutes, and the precipitate was collected. The precipitate was dispersed in acetone, filtered, and the filtrate was dried under reduced pressure for 12-36 hours to obtain the pentacyclic triterpenoid compound. S2. Dissolve the pentacyclic triterpenoid compound obtained in step S1 in N,N-dimethylformamide, add tert-butyldimethylchlorosilane and imidazole to react, the reaction temperature is 25-30℃, the reaction time is 12-24h, the reaction is terminated with water after the reaction is completed, and the mixture is extracted with ethyl acetate. The extract is dried under reduced pressure for 12-36h to obtain intermediate I. S3. Dissolve intermediate I in dichloromethane, add bromine to react, the reaction temperature is 0-5℃, the reaction time is 1-2h, after the reaction is completed, terminate the reaction with sodium sulfite solution, and dry the reaction solution under reduced pressure for 12-36h to obtain intermediate II. S4. Dissolve intermediate II in anhydrous ethanol, add 4-chlorobenzoyl hydrazine and acetic acid to react, the reaction temperature is 65-70℃, the reaction time is 4-6h, and after the reaction is completed, dry the reaction solution under reduced pressure for 12-36h to obtain intermediate III. S5. Dissolve intermediate III in acetic acid, add zinc powder to react, the reaction temperature is 25-30℃, the reaction time is 2-3h, after the reaction is completed, filter, and dry the filtrate under reduced pressure for 12-36h to obtain intermediate IV. S6. Dissolve intermediate IV in tetrahydrofuran, add tetrabutylammonium fluoride and react at a temperature of 25-30°C for 6-8 hours. After the reaction is complete, dry the reaction solution under reduced pressure for 12-36 hours to obtain the pentacyclic triterpenoid derivative.

3. The preparation method according to claim 2, characterized in that: In step S1, tea saponin is dissolved in an ethanol-water solution with a mass-to-volume ratio of 1:8-12 g / mL, and the volume fraction of ethanol in the ethanol-water solution is 60%-70%. The amount of activated carbon added is 0.05 to 0.1 times the mass of tea saponin, and the adsorption temperature is 65 to 75℃. The vacuum concentration temperature is 60–70℃; The AB-8 macroporous adsorption resin was added at a mass of 6 to 9 times that of tea soap, and the adsorption temperature was 25 to 35°C. The volume ratio of the ethanol-water solution used for each elution to the mass ratio of the macroporous resin was 3-5:1 mL / g, and the elution time was 2-4 h. The mass-to-volume ratio of sodium hydroxide added to the secondary eluent is 0.5–1.5:100 g / mL; The concentration of hydrochloric acid used to adjust the pH is 1–2.5 mol / L; The volume of the ethanol-water solution used to dissolve the precipitate is 3 to 5 times the mass of the precipitate. The volume ratio of p-toluenesulfonic acid to the solution is 0.3–0.5 g / mL; The temperature of the ice water is 4–10℃, and the volume of the ice water is 2–4 times the volume of the reaction liquid; Centrifugation speed: 8000–10000 rpm; The mass-to-volume ratio of the precipitate to acetone is 1:2-3 g / mL; The filtrate is dried under reduced pressure at a vacuum of 0.05–0.2 atmospheres and a temperature of 45–65°C.

4. The preparation method according to claim 2, characterized in that: In step S2, the mass-to-volume ratio of the pentacyclic triterpenoid compound to the N,N-dimethylformamide solvent is 1:8-16 g / mL; The mass of tert-butyldimethylchlorosilane is 1.6 to 1.8 times that of the pentacyclic triterpenoid compound; The mass of imidazole is 1.5 to 1.7 times that of the pentacyclic triterpenoid compound; The volume of water and ethyl acetate used to terminate the reaction were 1 to 3 times the volume of N,N-dimethylformamide. The extract was dried under reduced pressure at a vacuum of 0.05–0.2 atmospheres and a temperature of 45–65°C.

5. The preparation method according to claim 2, characterized in that: In step S3, the mass-to-volume ratio of intermediate I to dichloromethane solvent is 1:4-8 g / mL; The mass-to-volume ratio of intermediate I to bromine is 1:0.09–0.1 g / mL; The sodium sulfite solution has a mass fraction of 30-35%, and its volume is 1 / 2 to 1 times the volume of the dichloromethane solvent. The reaction solution is dried under reduced pressure at a vacuum of 0.05–0.2 atmospheres and a temperature of 45–65°C.

6. The preparation method according to claim 2, characterized in that: In step S4, the mass-to-volume ratio of intermediate II to ethanol solvent is 1:3 to 6.5 g / mL; The mass of 4-chlorobenzoyl hydrazide is 0.15 to 0.25 times the mass of intermediate II; The mass-to-volume ratio of intermediate II to acetic acid is 1:0.3–0.5 g / mL; The reaction solution is dried under reduced pressure at a vacuum of 0.05–0.2 atmospheres and a temperature of 45–65°C.

7. The preparation method according to claim 2, characterized in that: In step S5, the mass-to-volume ratio of intermediate III to acetic acid is 1:2.5-6 g / mL; The mass of zinc powder is 0.09 to 0.15 times the mass of intermediate III; The filtrate is dried under reduced pressure at a vacuum of 0.05–0.2 atmospheres and a temperature of 45–65°C.

8. The preparation method according to claim 2, characterized in that: In step S6, the mass-to-volume ratio of intermediate IV to tetrahydrofuran is 1:2.5-6 g / mL; The mass of tetrabutylammonium fluoride is 1.1 to 1.3 times the mass of intermediate IV; The reaction solution is dried under reduced pressure at a vacuum of 0.05–0.2 atmospheres and a temperature of 45–65°C.

9. The application of a pentacyclic triterpenoid derivative according to claim 1 or a pentacyclic triterpenoid derivative prepared by any one of claims 2 to 8 in the control of pine wilt disease, characterized in that, The pentacyclic triterpenoid derivative can be formulated into a drug preparation for killing pine wood nematodes, and the drug preparation is a single-ingredient or compound preparation containing the pentacyclic triterpenoid derivative.

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