Plant vaccine lead compound based on carbohydrates and synthesis method thereof

Abstract

The invention relates to a plant vaccine lead compound based on carbohydrates and a synthesis method thereof, glucosamine groups are modified by an organic synthesis process, and a derivative with a tetrasaccharide structure, namely a compound 12, with induction activity is obtained by a glycosylation reaction. And then removing part of functional groups to obtain a chitosan oligosaccharide analogue with a clear structure and a single component, namely a compound 14. Through experimental research, optimization of a technology for inducing plant disease resistance by the chitosan oligosaccharide is realized. Results show that the plant vaccine lead compound provided by the invention can obviously promote the growth and development of plants at a lower concentration, so that the product provided by the invention has a good popularization and application prospect in the related field of agriculture.

Description

Technical Field

[0001] The present invention belongs to the field of carbohydrate chemistry, and particularly relates to a lead compound of a plant vaccine based on saccharide substances and a synthesis method thereof. Background Art

[0002] Plant vaccines, also known as plant immune inducers, belong to the emerging vaccine engineering technology field. The principle is to activate the immune defense system of plants using physical, chemical or biological factors, induce plants to produce resistance to the invasion of pathogenic bacteria, or defend against extreme weather such as cold and drought, which is similar to human or animal vaccines. Plant vaccines have many advantages such as lower use concentration, no pesticide residues, no environmental pollution, significant and long-lasting induced disease resistance effects, and at the same time, pathogenic bacteria do not develop drug resistance, have no impact on non-pathogenic microorganisms, and are conducive to maintaining ecological balance and protecting the environment. In addition, plant vaccines can be used in combination with conventional pesticides to reduce the amount of pesticide used.

[0003] Saccharide plant vaccines are a typical representative of plant vaccines. They are saccharide substances that can induce plant resistance and increase yield at the same time, and are usually considered to be able to mimic the cell wall structure of plants or pathogenic bacteria. Due to the unique mode of action of plants on them, saccharide plant vaccines can regulate the growth and development of plants or activate the plant's own immune system at extremely low concentrations. Saccharide plant vaccines are easily decomposed by microorganisms in the soil and will not leave residues, and the resistance components induced by them are all normal substances of plant metabolism and are safe for humans and animals. Plant vaccines induce a wide range of disease resistance spectra and long-lasting disease resistance. After being induced multiple times for a long time, plants will not produce specific resistance, and pathogenic bacteria will not develop resistance either, and such substances have the characteristics of environmental friendliness and no pollution. At present, the saccharide plant vaccines that have been studied more include chitosan oligosaccharide, chitin oligosaccharide, glucan oligosaccharide, oligogalacturonic acid, etc.

[0004] However, up to now, in the reports on the synthesis research of saccharide plant vaccines, the main raw materials used are naturally obtained oligosaccharides, which not only have relatively complex components but also inconsistent molecular weights, making it difficult to obtain a single compound with high purity and affecting the actual use effect of plant vaccines.

[0005] Based on this, using glucosamine as a raw material, we obtained an analogue of chitosan oligosaccharide with a clear structure and a single component, β-(1→3)-linked N-acetylglucosaminotetraose, that is, compound 14, through a reasonable and feasible synthesis route. The seed soaking experiment research of the product of the present invention on rape and cucumber shows that compound 14 has a significant promoting effect on the growth and development of plants. Therefore, compound 14 has potential application and promotion value in promoting the growth and development of plants. Summary of the Invention

[0006] The object of the present invention is to provide a preparation method of a lead compound of a saccharide plant vaccine with a single composition and good biological activity in view of the problems such as the relatively complex oligosaccharide components and inconsistent molecular weights existing in nature.

[0007] For the above object, the lead compound of the saccharide plant vaccine with good biological activity provided by the present invention is prepared by the following method: In this study, an innovative strategy of phthaloyl protecting amino groups is adopted, and thioglycoside is used as a glycosyl donor to achieve the functional modification of glucosamine by chemical synthesis. Through multiple key reactions, a 2-acetamido-2-deoxytetrasaccharide molecule with a β-(1→3) glycosidic bond is successfully prepared, providing a new method for the preparation of novel chitin analogues.

[0008] The technical route of the present invention is realized through the following steps:

[0009] Step 1: Synthesis of Compound 1 In a reaction flask, add D-glucosamine hydrochloride, sodium bicarbonate, phthalic anhydride and a certain amount of distilled water, and stir at a certain temperature until the reactants are completely dissolved. Add sodium bicarbonate in batches within a certain time, and continue to stir the reaction mixture at a certain temperature for a certain time. After the reaction is completed, acidify the reaction solution with a certain amount of hydrochloric acid solution at a certain temperature, then add a certain amount of toluene and absolute ethanol to the reaction flask, and recover the solvent by vacuum distillation. The residue is dried in vacuo to obtain the crude product of known compound 1.

[0010] Step 2: Synthesis of Compound 2 In a reaction flask, add the crude product of Compound 1, pyridine and acetic anhydride, and gradually add a certain amount of methanol dropwise with stirring. Stir the reaction at room temperature for a period of time, and then recover methanol and methyl acetate by vacuum distillation. Then add a certain amount of acetic anhydride and a certain amount of pyridine again, stir the reaction at room temperature for a certain time, and detect the completion of the reaction by TLC. Recover the solvent by vacuum distillation. The residue is dissolved in dichloromethane, and the organic phase is washed with hydrochloric acid solution, saturated sodium bicarbonate solution and saturated sodium chloride solution, and the organic phase is separated. The organic phase is dried with an appropriate amount of anhydrous magnesium sulfate for a certain time, filtered, and the organic phase is recovered by vacuum distillation to obtain a mixture. The mixture is separated by silica gel column chromatography, and n-hexane:ethyl acetate = 3:2 is used as the eluent. Through ultraviolet lamp irradiation, collect the colorless solution of the first band product, recover the solvent by vacuum distillation, and the residue is dried in vacuo to obtain known compound 2.

[0011] Step 3: Synthesis of Compound 3 In a reaction flask, dissolve Compound 2 in dichloromethane, add a certain amount of ethanethiol, mix well, then place the reaction flask in an ice-water bath, stir and dropwise add a certain amount of boron trifluoride diethyl ether at a certain temperature. Stir the reaction mixture at room temperature for a certain time. After detecting the completion of the reaction by TLC, add a certain amount of triethylamine, mix well, then recover the solvent by distillation under reduced pressure. Dissolve the residue in dichloromethane, wash the organic phase with deionized water, separate the organic phase, add an appropriate amount of anhydrous magnesium sulfate to dry for a certain time, filter, recover the solvent by distillation under reduced pressure to obtain the crude product. Add a certain amount of absolute ethanol to the crude product, fully dissolve it, then cool and crystallize, and obtain the known Compound 3 after suction filtration.

[0012] Step 4: Synthesis of Compound 4 In a reaction flask, add distilled water, acetone, Compound 3 and a certain amount of concentrated hydrochloric acid solution with a mass fraction of 37%. Stir to fully dissolve the reactants, then heat up to a certain temperature and stir the reaction at this temperature for a certain time. After detecting the completion of the reaction by TLC, recover the solvent by distillation under reduced pressure, and obtain the crude product of the known Compound 4 after vacuum drying the residue.

[0013] Step 5: Synthesis of Compound 5 In a reaction flask, dissolve the crude product of Compound 4 in a certain amount of N,N-dimethylformamide, add a certain amount of benzaldehyde dimethyl acetal and p-toluenesulfonamide, stir to fully dissolve the reactants, then heat up to a certain temperature and stir the reaction at this temperature for a certain time. After detecting the completion of the reaction by TLC, add a certain amount of triethylamine, recover the solvent by distillation under reduced pressure to obtain a mixture. Separate the mixture by column chromatography on silica gel, using n-hexane:ethyl acetate = 2:1 as the eluent. Through ultraviolet lamp irradiation, collect the colorless solution of the third band product. After recovering the solvent by distillation under reduced pressure, obtain the known Compound 5 after vacuum drying the residue.

[0014] Step 6: Synthesis of Compound 6 In a reaction flask, add Compound 5, acetic anhydride and pyridine, stir the reaction at room temperature for a certain time. After detecting the completion of the reaction by TLC, recover the solvent by distillation under reduced pressure. Dissolve the residue in dichloromethane, wash the organic phase with hydrochloric acid, saturated sodium bicarbonate solution and saturated sodium chloride solution, separate the organic phase, add an appropriate amount of anhydrous magnesium sulfate to dry for a certain time, filter, recover the solvent by distillation under reduced pressure for the organic phase to obtain a mixture. Separate the mixture by column chromatography on silica gel, using n-hexane:ethyl acetate = 3:2 as the eluent. Through ultraviolet lamp irradiation, collect the colorless solution of the first band product, recover the solvent by distillation under reduced pressure, and obtain the known Compound 6 after vacuum drying the residue.

[0015] Step 7: Synthesis of Compound 7 In a reaction flask, compound 5, methanol and N-iodosuccinimide were added, and dichloromethane was added. The reaction was stirred at room temperature for a certain time under N2 protection. The reaction flask was transferred to a low-temperature reactor, a certain amount of silver trifluoromethanesulfonate was added, and the reaction was stirred at a certain temperature for a certain time. After the reaction was detected to be complete by TLC, a certain amount of triethylamine was added, filtered through diatomaceous earth, the solvent was recovered by distillation under reduced pressure, the residue was dissolved in dichloromethane, and the organic phase was washed with deionized water, saturated aqueous sodium thiosulfate solution and saturated sodium chloride solution. The organic phase was separated, an appropriate amount of anhydrous magnesium sulfate was added and dried for a certain time, filtered, and the solvent was recovered by distillation under reduced pressure to obtain a mixture. The mixture was separated by column chromatography on silica gel, and n-hexane:ethyl acetate = 3:2 was used as the eluent. Through ultraviolet lamp irradiation, the colorless solution of the third band product was collected, the solvent was recovered by distillation under reduced pressure, and the residue was dried under vacuum to obtain the known compound 7.

[0016] Step 8: Synthesis of compound 8 In a reaction flask, compound 6, compound 7 and N-iodosuccinimide were added, and dichloromethane was added. The reaction was stirred at room temperature for a certain time under N2 protection. The reaction flask was transferred to a low-temperature reactor, a certain amount of silver trifluoromethanesulfonate was added, and the reaction was stirred at 0 °C for a certain time. After the reaction was detected to be complete by TLC, a certain amount of triethylamine was added, filtered through diatomaceous earth, the solvent was recovered by distillation under reduced pressure, the residue was dissolved in dichloromethane, and the organic phase was washed with deionized water, saturated aqueous sodium thiosulfate solution and saturated sodium chloride solution. The organic phase was separated, an appropriate amount of anhydrous magnesium sulfate was added and dried for a certain time, filtered, and the solvent was recovered by distillation under reduced pressure from the organic phase to obtain a mixture. The mixture was separated by column chromatography on silica gel, and n-hexane:ethyl acetate = 3:2 was used as the eluent. Through ultraviolet lamp irradiation, the colorless solution of the fourth band product was collected, the solvent was recovered by distillation under reduced pressure, and the residue was dried under vacuum to obtain the known compound 8.

[0017] Step 9: Synthesis of compound 9 In a reaction flask, compound 8, a methanol solution of magnesium methoxide and dichloromethane were added, and the reaction was stirred at room temperature for a certain time under N2 protection. After the reaction was detected to be complete by TLC, it was neutralized to pH = 7 with glacial acetic acid, the solvent was recovered by distillation under reduced pressure, the residue was dissolved in dichloromethane, the organic phase was washed with deionized water, the organic phase was separated, an appropriate amount of anhydrous magnesium sulfate was added and dried for a certain time, filtered, and the solvent was recovered by distillation under reduced pressure from the organic phase to obtain a mixture. The mixture was separated by column chromatography on silica gel, and n-hexane:ethyl acetate = 2:1 was used as the eluent. Through ultraviolet lamp irradiation, the colorless solution of the third band product was collected, the solvent was recovered by distillation under reduced pressure, and the residue was dried under vacuum to obtain the known compound 9.

[0018] Step 10: Synthesis of compound 10 In a reaction flask, compound 9, compound 6 and N-iodosuccinimide were added, and dichloromethane was added. The reaction was stirred at room temperature for a certain time under N2 protection. The reaction flask was transferred to a low-temperature reactor, a certain amount of trifluoromethanesulfonic acid was added, and the reaction was stirred at a certain temperature for a certain time. After the reaction was detected to be complete by TLC, it was neutralized with a certain amount of triethylamine, filtered through diatomaceous earth, the solvent was recovered by distillation under reduced pressure, the residue was dissolved in dichloromethane, and the organic phase was washed successively with deionized water, saturated sodium thiosulfate aqueous solution and saturated sodium chloride solution. The organic phase was separated, an appropriate amount of anhydrous magnesium sulfate was added and dried for a certain time, filtered, and the organic phase was recovered by distillation under reduced pressure to obtain a mixture. The mixture was separated by silica gel column chromatography, dichloromethane:ethyl acetate = 10:1 was used as the eluent, and the colorless solution of the third band product was collected by irradiating with an ultraviolet lamp. The solvent was recovered by distillation under reduced pressure, and the residue was dried under vacuum to obtain the known compound 10.

[0019] Step 11: Synthesis of compound 11 In a reaction flask, compound 10, a methanol solution of magnesium methoxide and dichloromethane were added, and the reaction was stirred at room temperature for a certain time under N2 protection. After the reaction was detected to be complete by TLC, it was neutralized with glacial acetic acid, the solvent was recovered by distillation under reduced pressure, the residue was dissolved in dichloromethane, the organic phase was washed with deionized water, the organic phase was separated, an appropriate amount of anhydrous magnesium sulfate was added and dried for a certain time, the desiccant was filtered off, and the organic phase was recovered by distillation under reduced pressure to obtain a mixture. Dichloromethane:ethyl acetate = 10:1 was used as the eluent, and the colorless solution of the first band product was collected by irradiating with an ultraviolet lamp. The solvent was recovered by distillation under reduced pressure, and the residue was dried under vacuum to obtain the known compound 11.

[0020] Step 12: Synthesis of compound 12 In a reaction flask, a certain amount of compound 11, compound 6 and N-iodosuccinimide were added, and dichloromethane was added. The reaction was stirred at room temperature for a certain time under N2 protection. The reaction flask was transferred to a low-temperature reactor, a certain amount of trifluoromethanesulfonic acid was added, and the reaction was stirred at a certain temperature for a certain time. After the reaction was detected to be complete by TLC, it was neutralized with a certain amount of triethylamine, filtered through diatomaceous earth, the solvent was recovered by distillation under reduced pressure, the residue was dissolved in dichloromethane, and the organic phase was washed successively with deionized water, saturated sodium thiosulfate aqueous solution and saturated sodium chloride solution. The organic phase was separated, an appropriate amount of anhydrous magnesium sulfate was added and dried for a certain time, filtered, and the organic phase was recovered by distillation under reduced pressure to obtain a mixture. The mixture was separated by silica gel column chromatography, dichloromethane:ethyl acetate = 10:1 was used as the eluent, and the colorless solution of the second band product was collected by irradiating with an ultraviolet lamp. The solvent was recovered by distillation under reduced pressure, and the residue was dried under vacuum to obtain compound 12.

[0021] Step 13: Synthesis of compounds 13-1 and 13-2 In a reaction flask, a certain amount of Compound 12 was dissolved in a mixed solvent of tetrahydrofuran and methanol. Subsequently, a certain amount of p-toluenesulfonic acid was added as a catalyst, and the reaction system was heated to a certain temperature and stirred for a certain period of time. After the reaction was completed, a certain amount of saturated aqueous sodium bicarbonate solution was added dropwise to the mixture to neutralize the excess acidic substances, and then deionized water was added for washing. The organic phase was separated, dried over anhydrous magnesium sulfate for a certain period of time. After filtration and recovery of the solvent by vacuum distillation, the residue was separated by column chromatography on silica gel, with dichloromethane:methanol = 10:1 as the eluent. By irradiating with an ultraviolet lamp, the colorless solution of the second band product was collected, and Compound 13-1 was obtained after recovery of the solvent by vacuum distillation; by irradiating with an ultraviolet lamp, the colorless solution of the third band product was collected, and Compound 13-2 was obtained after recovery of the solvent by vacuum distillation.

[0022] Step 14: Synthesis of Compound 14 Compound 13-1 and Compound 13-2 were placed in a reaction flask, a certain amount of 95% ethanol was added as a solvent, and then a certain amount of 80% hydrazine hydrate and a certain amount of water were added. The mixture was stirred and refluxed at a certain temperature for a certain period of time. After the reaction was completed, the reaction mixture was distilled under reduced pressure to recover the solvent, and the resulting residue was directly used for the next reaction. A certain amount of water, a certain amount of methanol and a certain amount of sodium bicarbonate were successively added to the above residue. The mixture was cooled to 0 °C, and a certain amount of acetic anhydride was added dropwise. After stirring and reacting at a certain temperature for a certain period of time, the solvent was recovered by vacuum distillation. The residue was chromatographed on a Sephadex G-25 column (distilled water), and by the sulfuric acid carbonization color development method, the colorless solution of the first band product was collected, and Compound 14 was obtained after freeze-drying at low temperature.

[0023] The advantages of the present invention are as follows: (1) A reasonable and feasible synthetic route was designed, and the synthesis conditions of Compound 12, Compound 13-1, Compound 13-2 and Compound 14 were obtained.

[0024] (2) The effect of Compound 14 on inducing crop disease resistance was studied, and the results showed that Compound 14 is expected to be popularized and applied in the agricultural related fields.

[0025] The substantial features of the present invention are: a reasonable and feasible route was designed in the present invention, the synthesis processes and conditions of 4 new compounds were obtained, and the research on the performance of Compound 14 in inducing crop disease resistance was carried out. The results showed that: Compound 14 has the effects of shortening the plant growth cycle and promoting plant growth, and is expected to be popularized and applied in crop planting. Description of the Drawings

[0026] Figure 1 is the 1 1H NMR spectrum of Compound 2; Figure 2 is the 1 H NMR spectrum of Compound 3; Figure 3 is the 1 H NMR spectrum of Compound 5; Figure 4 is the 1 H NMR spectrum of Compound 6; Figure 5 is the 1 H NMR spectrum of Compound 7; Figure 6 is the 1 H NMR spectrum of Compound 8; Figure 7 is the 1 H NMR spectrum of Compound 9; Figure 8 is the 1 H NMR spectrum of Compound 10; Figure 9 is the 1 H NMR spectrum of Compound 11; Figure 10 is the 1 H NMR spectrum of Compound 12; Figure 11 is the 13 C NMR spectrum of Compound 12; Figure 12 is the high-resolution mass spectrum of Compound 12; Figure 13 is the 1 H NMR spectrum of Compound 13-1; Figure 14 is the 1 H NMR spectrum of Compound 13-2; Figure 15 is the high-resolution mass spectrum of Compound 13-1; Figure 16 is the high-resolution mass spectrum of Compound 13-2; Figure 17 is the 1 H NMR spectrum of Compound 14; Figure 18 is the 13 C NMR spectrum of Compound 14; Figure 19 is the high-resolution mass spectrum of Compound 14; Figure 20It is the seed soaking diagrams of aqueous solution of Compound 14, aqueous solution of β-(1→3)-2-deoxy-2-acetamido-diglucose, and tap water on rape seeds, where: 20-1 is the seed soaking diagram of rape seeds soaked in aqueous solution of 0.001 mg / mL Compound 14, 20-2 is the seed soaking diagram of rape seeds soaked in aqueous solution of 0.001 mg / mL β-(1→3)-2-deoxy-2-acetamido-diglucose, and 20-3 is the seed soaking diagram of rape seeds soaked in tap water.

[0027] Figure 21 It is the seed soaking diagrams of aqueous solution of Compound 14, aqueous solution of β-(1→3)-2-deoxy-2-acetamido-diglucose, and tap water on cucumber seeds, where: 21-1 is the seed soaking diagram of cucumber seeds soaked in aqueous solution of 0.001 mg / mL Compound 14, 21-2 is the seed soaking diagram of cucumber seeds soaked in aqueous solution of 0.001 mg / mL β-(1→3)-2-deoxy-2-acetamido-diglucose, and 21-3 is the seed soaking diagram of cucumber seeds soaked in tap water. Detailed implementation mode Example

[0028] Step 1: Synthesis of Compound 1 In a 100 mL reaction flask, add 1.0 g of D-glucosamine hydrochloride, 0.4 g of sodium bicarbonate, 0.75 g of phthalic anhydride, and 15 mL of distilled water. Stir at 40 °C until the reactants are completely dissolved. Add 0.1 g of sodium bicarbonate every 20 minutes within 2 h, for a total of 0.5 g. The reaction mixture continues to stir at 40 °C for 1 h. After the reaction is completed, acidify the reaction solution to pH = 5 with 2 M hydrochloric acid solution at 0 °C. Then add 10 mL of toluene and 10 mL of absolute ethanol to the reaction flask, and recover the solvent by vacuum distillation. The residue is dried in vacuo to obtain 1.45 g of a yellow solid, which is the crude product of known compound 1.

[0029] Step 2: Synthesis of Compound 2 In a 100 mL reaction flask, 1.2 g of the crude product of compound 1, 4.0 mL of pyridine and 2.0 mL of acetic anhydride were added. 1.0 mL of methanol was added dropwise with stirring. After stirring at room temperature for 5 minutes, methanol and methyl acetate were recovered by distillation under reduced pressure. Then, 4.0 mL of acetic anhydride and 8.0 mL of pyridine were added again, and the reaction was stirred at room temperature for 12 h. The reaction was monitored by TLC and found to be complete. The solvent was recovered by distillation under reduced pressure. The residue was dissolved in 20 mL of dichloromethane. The organic phase was washed three times with 10 mL of 1 mol / L hydrochloric acid solution each time, then washed three times with 10 mL of saturated sodium bicarbonate aqueous solution each time, and then washed three times with 10 mL of saturated sodium chloride solution each time. The organic phase was separated, dried over an appropriate amount of anhydrous magnesium sulfate for 2 h, and the desiccant was removed by filtration. After the solvent was recovered from the organic phase by distillation under reduced pressure, a yellow-brown syrupy mixture was obtained. The mixture was separated by column chromatography on 300-mesh silica gel, using n-hexane:ethyl acetate = 3:2 as the eluent. By irradiation with an ultraviolet lamp, the colorless solution of the first band product was collected. The solvent was recovered by distillation under reduced pressure, and the residue was dried in vacuo to obtain 1.56 g of a white powdery substance, which was known compound 2, with a yield of 89%. f Rf was 0.42 (n-hexane:ethyl acetate = 1:1, TLC plate model: Huanghai brand HSGF254).

[0030] The results of nuclear magnetic resonance test: 1 H NMR (600 MHz, CDCl3) δ 7.86 (m, 2H), 7.75 – 7.73 (m, 2H), 6.51 (d, J J = 8.9 Hz, 1H,), 5.88 (t, J J = 9.8 Hz, 1H), 5.21(t, J J = 19.3 Hz, 1H), 4.46 (t, J J = 9.7 Hz, 1H), 4.38 (dd, 1H), 4.14 (dd, 1H),4.03 - 4.01 (m, 1H), 2.11,2.03,1.99,1.86 (4s, 9H ) ppm. Step 3: Synthesis of compound 3 In a 100 mL reaction flask, 1.0 g of compound 2 was dissolved in 20 mL of dichloromethane. 1.7 mL of ethanethiol was added and the mixture was stirred well. Then the reaction flask was placed in an ice - water bath, and 2.3 mL of boron trifluoride etherate with a purity of 98% was added dropwise under stirring at 0 °C. The reaction mixture was stirred at room temperature for 24 h. After the reaction was completed as detected by TLC, 3.0 mL of triethylamine was added. The mixture was stirred well and the solvent was recovered by distillation under reduced pressure. The residue was dissolved in 20 mL of dichloromethane. The organic phase was washed with 30 mL of deionized water each time for 3 times. The organic phase was separated, and an appropriate amount of anhydrous magnesium sulfate was added for drying for 2 h. The desiccant was removed by filtration, and the solvent was recovered by distillation under reduced pressure to obtain the crude product. 2 mL of anhydrous ethanol was added to the crude product. After complete dissolution, it was cooled for crystallization, filtered by suction, and dried in vacuo to obtain 0.90 g of a white powdery substance, which was known compound 3 with a yield of 90%. f The R

[0031] value was 0.51 (n - hexane:ethyl acetate = 1:1, TLC plate model: Huanghai brand HSGF254). 1 H NMR (600 MHz, CDCl3) δ 7.86 (s, 2H), 7.74 (s, 2H), 5.83 (t, J J = 9.7Hz, 1H), 5.49 (d, J J = 10.6 Hz, 1H), 5.18 (t, J J = 9.7 Hz, 1H), 4.40 (t, J J =10.4 Hz, 1H), 4.31 (dd, J J = 12.2, 4.7 Hz, 1H), 4.20 – 4.16 (m, 1H), 3.92 –3.88 (m, 1H), 2.68 (d, J J = 16.0 Hz, 2H), 2.11 (s, 3H), 2.04 (s, 3H), 1.86 (s,3H), 1.22 (t, J J = 7.4 Hz, 3H) ppm. Step 4: Synthesis of compound 4 In a 100 mL reaction flask, add 4.0 mL of distilled water, 8.0 mL of acetone, 0.5 g of compound 3, and 2.0 mL of a 37% hydrochloric acid solution by mass. Stir to fully dissolve the reactants, then heat up to 60 °C and stir the reaction for 12 h at this temperature. After detecting the completion of the reaction by TLC, recover the solvent by vacuum distillation. The residue is dried in vacuo to obtain 0.35 g of a white powdery substance, which is the crude product of the known compound 4.

[0032] Step 5: Synthesis of compound 5 In a 100 mL reaction flask, dissolve 0.5 g of the crude product of compound 4 in 15 mL of N,N-dimethylformamide. Add 1.0 mL of benzaldehyde dimethyl acetal and 0.03 g of p-toluenesulfonamide. Stir to fully dissolve the reactants, then heat up to 40 °C and stir the reaction for 5 h at this temperature. After detecting the completion of the reaction by TLC, add 3.0 mL of triethylamine. Recover the solvent by vacuum distillation to obtain a yellowish-brown syrupy mixture. The residue is separated by column chromatography on 300-mesh silica gel, using n-hexane:ethyl acetate = 2:1 as the eluent. Through irradiation with an ultraviolet lamp, collect the colorless solution of the third band product. After recovering the solvent by vacuum distillation, the residue is dried in vacuo to obtain 0.44 g of a white powdery substance, which is the known compound 5, with a yield of 70%, R f is 0.51 (n-hexane:ethyl acetate = 1:1, TLC plate model: Huanghai brand HSGF254).

[0033] Results of nuclear magnetic resonance test: 1 H NMR (600 MHz, CDCl3) δ 7.88 (d, J J = 19.7 Hz, 2H), 7.75 (dd, J J =5.5, 3.0 Hz, 2H), 7.52 – 7.46 (m, 2H), 7.39 (d, J J = 5.8 Hz, 3H), 5.58 (s,1H), 5.43 (d, J J = 10.6 Hz, 1H), 4.68 (td, J J = 9.5, 2.6 Hz, 1H), 4.41 (dd, J J =10.5, 4.9 Hz, 1H), 4.34 (t, J J = 10.3 Hz, 1H), 3.82 (t, J J = 10.2 Hz, 1H), 3.72(td, J= 9.7, 4.9 Hz, 1H), 3.63 (t, J = 9.2 Hz, 1H), 2.75 – 2.65 (m, 2H), 2.48 (s, 1H), 1.20 (t, J = 7.4 Hz, 3H) ppm. Step 6: Synthesis of Compound 6 In a 100 mL reaction flask, add 0.5 g of Compound 5, 0.24 mL of acetic anhydride and 10 mL of pyridine. Stir the reaction at room temperature for 12 h. After detecting the completion of the reaction by TLC, recover the solvent by distillation under reduced pressure. The residue is dissolved in 20 mL of dichloromethane. The organic phase is washed with 10 mL of 1 mol / L hydrochloric acid solution each time for three times, then the organic phase is separated. The organic phase is washed with 10 mL of saturated sodium bicarbonate aqueous solution each time for three times, then the organic phase is separated. The organic phase is washed with 10 mL of saturated sodium chloride aqueous solution each time for three times, then the organic phase is separated. Add an appropriate amount of anhydrous magnesium sulfate and dry for 2 h. Filter. After distilling off the solvent from the organic phase under reduced pressure, a yellowish-brown syrupy mixture is obtained. The mixture is separated by column chromatography on 300-mesh silica gel, with n-hexane:ethyl acetate = 3:2 as the eluent. By irradiating with an ultraviolet lamp, the colorless solution of the first band product is collected. After distilling off the solvent under reduced pressure, the residue is dried under vacuum to obtain 0.49 g of a white powdery substance, which is the known Compound 6 with a yield of 89%, R f is 0.53 (n-hexane:ethyl acetate = 1:1, TLC plate model: Huanghai brand HSGF254).

[0034] Results of nuclear magnetic resonance test: 1 1H NMR (600 MHz, CDCl3) δ 7.86 (dd, J = 15.9, 7.0 Hz, 2H), 7.74 (dt, J = 5.5, 2.5 Hz, 2H), 7.46 (dd, J = 7.4, 2.4 Hz, 2H), 7.38 – 7.33 (m, 3H), 5.93 (t, J = 9.3 Hz, 1H), 5.58 (d, J = 10.6 Hz, 1H), 5.55 (s, 1H), 4.44 – 4.35 (m, 2H), 3.85 – 3.77 (m, 3H), 2.69 (dd, J = 22.0, 7.4 Hz, 2H), 1.89 (s, 3H), 1.20 (t,J = 7.4 Hz, 3H) ppm. Step 7: Synthesis of Compound 7 In a 100 mL reaction flask, add 0.5 g of Compound 5, 0.9 mL of methanol and 1.0 g of N-iodosuccinimide. Then add 15 mL of dichloromethane and stir the reaction at room temperature for 1 h under N2 protection. Transfer the reaction flask to a low-temperature reactor, add 0.5 g of silver trifluoromethanesulfonate, and stir the reaction at 0 °C for 4 h. After detecting the completion of the reaction by TLC, add 3.0 mL of triethylamine, filter through diatomaceous earth, recover the solvent by distillation under reduced pressure. Dissolve the residue in 20 mL of dichloromethane. Wash the organic phase with 30 mL of deionized water each time for three times, then wash the organic phase with 30 mL of saturated sodium thiosulfate aqueous solution each time for three times, and then wash the organic phase with 30 mL of saturated sodium chloride solution each time for three times. Separate the organic phase, add an appropriate amount of anhydrous magnesium sulfate to dry for 2 h, filter, recover the solvent by distillation under reduced pressure to obtain a yellowish-brown syrupy mixture. Separate the mixture by column chromatography on 300-mesh silica gel, using n-hexane:ethyl acetate = 3:2 as the eluent. Under ultraviolet light irradiation, collect the colorless solution of the third band product, recover the solvent by distillation under reduced pressure, and obtain 0.37 g of a white powdery substance after vacuum drying, which is the known Compound 7 with a yield of 79%, R f is 0.52 (n-hexane:ethyl acetate = 1:1, TLC plate model: Huanghai brand HSGF254).

[0035] Nuclear magnetic resonance test results: 1 H NMR (600 MHz, CDCl3) δ 7.86 (s, 2H), 7.73 (d, J = 2.8 Hz, 2H), 7.50(d, J = 4.7 Hz, 2H), 7.38 (d, J = 5.7 Hz, 3H), 5.58 (s, 1H), 5.20 (d, J = 8.5Hz, 1H), 4.63 (t, J = 8.3 Hz, 1H), 4.41 (dd, J = 10.6, 4.7 Hz, 1H), 4.24 (t, J = 9.5 Hz, 1H), 3.85 (t, J = 10.1 Hz, 1H), 3.63 (d, J = 21.6 Hz, 2H), 3.45(s, 3H) ppm. Step 8: Synthesis of Compound 8 In a 100 mL reaction flask, add 0.57 g of Compound 6, 0.48 g of Compound 7 and 1.0 g of N-iodosuccinimide. Add 15 mL of dichloromethane and stir the reaction at room temperature for 1 h under N2 protection. Transfer the reaction flask to a low-temperature reactor, add 0.5 g of silver trifluoromethanesulfonate, and stir the reaction at 0 °C for 4 h. After detecting the completion of the reaction by TLC, add 3.0 mL of triethylamine, filter through diatomaceous earth, recover the solvent by distillation under reduced pressure. Dissolve the residue in 20 mL of dichloromethane. Wash the organic phase with 30 mL of deionized water each time for three times, separate the organic phase, then wash the organic phase with 30 mL of saturated sodium thiosulfate aqueous solution each time for three times, separate the organic phase, then wash the organic phase with 30 mL of saturated sodium chloride solution each time for three times, separate the organic phase, add an appropriate amount of anhydrous magnesium sulfate and dry for 2 h, filter off the desiccant. After recovering the solvent from the organic phase by distillation under reduced pressure, a yellow-brown syrupy residue is obtained. The residue is separated by column chromatography on 300-mesh silica gel, with n-hexane:ethyl acetate = 3:2 as the eluent. Through ultraviolet lamp irradiation, collect the colorless solution of the fourth band product, recover the solvent by distillation under reduced pressure, and the residue is dried under vacuum to obtain 0.78 g of a white powdery substance, which is the known Compound 8 with a yield of 80%, and the R f is 0.43 (n-hexane:ethyl acetate = 1:1, TLC plate model: Huanghai brand HSGF254).

[0036] Results of nuclear magnetic resonance test: 1 H NMR (600 MHz, CDCl3) δ 7.68 (s, 2H), 7.60 (s, 4H), 7.51 (d, J J = 6.9Hz, 3H), 7.42 – 7.36 (m, 6H), 7.33 – 7.31 (m, 3H), 5.61 (d, J J = 9.8 Hz, 1H),5.58 (d, J J = 6.3 Hz, 1H), 5.56 (s, 1H), 5.41 (s, 1H), 4.91 (d, J J = 8.5 Hz,1H), 4.87 – 4.83 (m, 1H), 4.36 (dd, J J = 10.5, 4.8 Hz, 1H), 4.19 – 4.16 (m,2H), 4.10 (dd, J J = 10.5, 4.9 Hz, 1H), 3.83 (d, J= 10.2 Hz, 1H), 3.73 (t, J = 9.1 Hz, 1H), 3.63 (d, J = 9.3 Hz, 3H), 3.49 – 3.45 (m, 1H), 3.31 (s, 3H), 1.72 (s, 3H) ppm. Step 9: Synthesis of Compound 9 In a 100 mL reaction flask, add 0.5 g of Compound 8, 10 mL of a methanol solution of magnesium methoxide with 7 - 8 wt.%, add 10 mL of dichloromethane, stir and react at room temperature for 24 h under N2 protection. After detecting the completion of the reaction by TLC, neutralize it to pH = 7 with glacial acetic acid, recover the solvent by distillation under reduced pressure. Dissolve the residue in 20 mL of dichloromethane, wash the organic phase with 30 mL of deionized water each time for a total of three times, separate the organic phase, add an appropriate amount of anhydrous magnesium sulfate and dry for 2 h, filter to remove the desiccant, recover the solvent from the organic phase by distillation under reduced pressure to obtain a pale yellow syrupy mixture. The residue is separated by column chromatography on 300 - mesh silica gel, using n - hexane:ethyl acetate = 2:1 as the eluent. Through ultraviolet lamp irradiation, collect the colorless solution of the third - band product, recover the solvent by distillation under reduced pressure, and the residue is dried in vacuo to obtain 0.40 g of a white powdery substance, which is the known Compound 9 with a yield of 84%, R f is 0.39 (n - hexane:ethyl acetate = 1:1, TLC plate model: Huanghai brand HSGF254).

[0037] 1H NMR test results: 1 1H NMR (600 MHz, CDCl3) δ 7.68 (s, 2H), 7.59 (s, 4H), 7.48 (dd, J = 30.6, 5.6 Hz, 4H), 7.39 (d, J = 7.6 Hz, 5H), 7.36 – 7.31 (m, 3H), 5.55 (s, 1H), 5.44 (s, 1H), 5.36 (d, J = 8.4 Hz, 1H), 4.90 (d, J = 8.5 Hz, 1H), 4.85 (d, J = 9.8 Hz, 1H), 4.34 (t, J = 12.9 Hz, 2H), 4.21 – 4.16 (m, 1H), 4.12 (d, J = 10.2 Hz, 2H), 3.83 (t,J = 10.2 Hz, 1H), 3.72 (t, J = 9.1 Hz, 1H), 3.65 – 3.61 (m, 1H), 3.59 (d, J = 4.7 Hz, 1H), 3.47 (t, J = 9.1 Hz, 1H), 3.38 (dt, J = 9.3, 4.7 Hz, 1H), 3.31 (s, 3H) ppm. Step 10: Synthesis of Compound 10 In a 100 mL reaction flask, 0.50 g of Compound 9, 0.35 g of Compound 6 and 1.0 g of N-iodosuccinimide were added. 15 mL of dichloromethane was added, and the reaction was stirred at room temperature for 1 h under N2 protection. The reaction flask was transferred to a low-temperature reactor, 0.01 mL of trifluoromethanesulfonic acid was added, and the reaction was stirred at 0 °C for 4 h. The reaction was detected by TLC to be complete, neutralized with 3.0 mL of triethylamine, filtered through diatomaceous earth, the solvent was recovered by vacuum distillation, the residue was dissolved in 20 mL of dichloromethane, and the organic phase was washed with 30 mL of deionized water each time for three times. The organic phase was separated, and then the organic phase was washed with 30 mL of saturated sodium thiosulfate aqueous solution each time for three times. The organic phase was separated, and then the organic phase was washed with 30 mL of saturated sodium chloride solution each time for three times. The organic phase was separated, an appropriate amount of anhydrous magnesium sulfate was added for drying for 2 h, the desiccant was filtered off, the solvent was recovered from the organic phase by vacuum distillation to obtain a yellow syrup-like mixture, and the residue was separated by column chromatography on 300-mesh silica gel. Dichloromethane:ethyl acetate = 10:1 was used as the eluent. Through ultraviolet lamp irradiation, the colorless solution of the third band product was collected, the solvent was recovered by vacuum distillation, and the residue was dried in vacuo to obtain 0.61 g of a white powdery substance, which was the known Compound 10 with a yield of 80%, R f was 0.43 (dichloromethane:ethyl acetate = 6:1, TLC plate model: Huanghai brand HSGF254).

[0038] 1H NMR test results: 1 H NMR (600 MHz, CDCl3) δ 7.72 (dd, J = 5.3, 3.0 Hz, 2H), 7.61 (d, J= 8.3 Hz, 2H), 7.60 – 7.53 (m, 4H), 7.52 – 7.43 (m, 3H), 7.43 – 7.28 (m, 14H), 7.20 (s, 2H), 5.52 – 5.46 (m, 2H), 5.44 (s, 1H), 5.37 (d, J = 8.3 Hz, 1H), 5.35 (s, 1H), 5.08 (d, J = 8.4 Hz, 1H), 4.81 (d, J = 8.5 Hz, 1H), 4.77 (d, J = 10.2 Hz, 1H), 4.64 – 4.59 (m, 1H), 4.31 (dd, J = 10.3, 4.8 Hz, 1H), 4.16(dd, J = 10.4, 4.8 Hz, 1H), 4.12 – 4.08 (m, 1H), 4.08 – 3.99 (m, 3H), 3.78 (t, J = 10.2 Hz, 1H), 3.68 (t, J = 10.2 Hz, 1H), 3.62 (td, J = 9.2, 3.0 Hz, 2H), 3.53 (qd, J = 10.3, 5.1 Hz, 3H), 3.38 – 3.29 (m, 2H), 3.26 (s, 3H), 1.67 (s, 3H). Step 11: Synthesis of Compound 11 In a 100 mL reaction flask, 0.4 g of Compound 10, 8 mL of a methanol solution of magnesium methoxide with 7 - 8 wt% and 10 mL of dichloromethane were added. The reaction was stirred at room temperature for 24 h under N2 protection. After the reaction was complete as detected by TLC, it was neutralized to pH = 7 with glacial acetic acid, and the solvent was recovered by distillation under reduced pressure. The residue was dissolved in 20 mL of dichloromethane, and the organic phase was washed with 30 mL of deionized water each time for a total of three times. The organic phase was separated, an appropriate amount of anhydrous magnesium sulfate was added for drying for 2 h, the desiccant was removed by filtration, and the solvent was recovered from the organic phase by distillation under reduced pressure to obtain a pale yellow syrupy mixture. Using dichloromethane:ethyl acetate = 10:1 as the eluent, under ultraviolet lamp irradiation, the colorless solution of the first band product was collected, the solvent was recovered by distillation under reduced pressure, and the residue was dried in vacuo to obtain 0.33 g of a white powdery substance, which was the known Compound 11 with a yield of 85%, R fIt is 0.39 (methylene chloride:ethyl acetate = 6:1, TLC plate model: Huanghai brand HSGF254).

[0039] Nuclear magnetic resonance test results: 1 H NMR (600 MHz, CDCl3) δ 7.73 (dd, J J = 5.3, 3.0 Hz, 2H), 7.64 – 7.60(m, 2H), 7.56 (dd, J J = 5.3, 2.9 Hz, 4H), 7.47 (d, J J = 7.6 Hz, 2H), 7.45 –7.30 (m, 15H), 7.21 (s, 2H), 5.49 (s, 1H), 5.44 (s, 1H), 5.38 (s, 1H), 5.17(d, J J = 8.4 Hz, 1H), 5.06 (d, J J = 8.4 Hz, 1H), 4.80 (d, J J = 8.7 Hz, 1H), 4.77(d, J J = 10.1 Hz, 1H), 4.65 – 4.60 (m, 1H), 4.31 (dd, J J = 10.3, 4.8 Hz, 1H),4.23 (t, J J = 9.7 Hz, 1H), 4.17 (dd, J J = 10.4, 4.8 Hz, 1H), 4.13 – 4.03 (m,3H), 4.01 – 3.97 (m, 1H), 3.77 (t, J J = 10.2 Hz, 1H), 3.68 (t, J J = 10.2 Hz,1H), 3.62 (t, J J = 9.1 Hz, 2H), 3.52 (dq, J J = 9.4, 5.7, 4.7 Hz, 2H), 3.35 (td, J J = 9.4, 6.5 Hz, 2H), 3.26 (s, 3H) ppm. Step 12: Synthesis of Compound 12 In a 100 mL reaction flask, 0.54 g of compound 11, 0.24 g of compound 6 and 0.4 g of N-iodosuccinimide were added. 15 mL of dichloromethane was added, and the reaction was stirred at room temperature for 1 h under N2 protection. The reaction flask was transferred to a low-temperature reactor, 0.014 mL of trifluoromethanesulfonic acid was added, and the reaction was stirred at 0 °C for 4 h. After the reaction was detected to be complete by TLC, it was neutralized with 1.0 mL of triethylamine, filtered through diatomaceous earth, the solvent was recovered by distillation under reduced pressure, the residue was dissolved in 20 mL of dichloromethane, and the organic phase was washed with 30 mL of deionized water each time for three times. The organic phase was separated, and then the organic phase was washed with 30 mL of saturated sodium thiosulfate aqueous solution each time for three times. The organic phase was separated, and then the organic phase was washed with 30 mL of saturated sodium chloride solution each time for three times. The organic phase was separated, an appropriate amount of anhydrous magnesium sulfate was added for drying for 2 h, the desiccant was filtered off, the solvent of the organic phase was distilled off under reduced pressure to obtain a yellow syrup-like mixture, the residue was separated by column chromatography on 300-mesh silica gel, dichloromethane:ethyl acetate = 10:1 was used as the eluent, and the colorless solution of the second band product was collected by irradiation with an ultraviolet lamp. The solvent was recovered by distillation under reduced pressure, and the residue was dried in vacuo to obtain 0.47 g of a white powdery substance, which was compound 12, and the yield was 64%. R f is 0.43 (dichloromethane:ethyl acetate = 3:1, TLC plate model: Huanghai brand HSGF254).

[0040] 1H NMR test results: 1 H NMR (600 MHz, CDCl3) δ 7.77 – 7.73 (m, 2H), 7.65 – 7.62 (m, 4H),7.59 – 7.54 (m, 4H), 7.48 – 7.45 (m, 2H), 7.36 (ddd, J J = 31.8, 16.0, 8.2 Hz,21H), 7.27 – 7.26 (m, 1H), 7.18 (s, 2H), 5.50 – 5.45 (m, 2H), 5.40 (s, 2H),5.35 (s, 1H), 5.33 (d, J J = 8.3 Hz, 1H), 4.99 (d, J J = 8.4 Hz, 1H), 4.87 (d, J J = 8.3 Hz, 1H), 4.80 (d, J J = 8.4 Hz, 1H), 4.77 (d, J J = 9.4 Hz, 1H), 4.55 (dt, J= 19.6, 9.6 Hz, 2H), 4.31 (dd, J = 10.2, 4.5 Hz, 1H), 4.15 (dq, J = 15.1,5.0, 4.3 Hz, 2H), 4.01 (q, J = 10.0 Hz, 5H), 3.79 – 3.74 (m, 1H), 3.64 – 3.50(m, 8H), 3.34 – 3.29 (m, 2H), 3.26 (s, 3H), 3.24 – 3.20 (m, 1H), 1.68 (s, 3H) ppm. 13 C NMR (150 MHz, CDCl3) δ 169.84, 167.23, 167.21, 167.13, 167.08,137.31, 137.23, 136.99, 133.90, 133.67, 133.59, 133.49, 131.48, 131.01,129.05, 129.03, 128.20, 128.17, 128.11, 126.24, 126.09, 126.05, 126.03,123.47, 123.22, 123.07, 101.53, 101.20, 101.16, 101.10, 99.73, 97.43, 96.95,96.93, 79.95, 79.59, 78.87, 74.25, 73.91, 73.40, 69.79, 68.62, 68.57, 66.39,66.14, 65.95, 65.78, 56.75, 55.82, 55.76, 55.63, 20.26 ppm. Step 13: Synthesis of Compounds 13-1 and 13-2 In a 25 mL reaction flask, 0.143 g of compound 12 was dissolved in a mixed solvent of 4 mL of tetrahydrofuran and 2 mL of methanol. Subsequently, 0.005 g of p-toluenesulfonic acid was added as a catalyst, and the reaction system was heated to 55 °C and stirred for 48 hours. After the reaction, 5 mL of saturated sodium bicarbonate aqueous solution was added dropwise to the mixture to neutralize the excess acidic substances. The organic phase was washed with 10 mL of deionized water each time for three times, and then the organic phase was separated. Subsequently, the organic phase was dried over anhydrous magnesium sulfate. After filtration and recovery of the solvent by vacuum distillation, the residue was separated by column chromatography on 300-mesh silica gel, with dichloromethane:methanol = 10:1 as the eluent. By irradiating with an ultraviolet lamp, the colorless solution of the second band product was collected. After recovery of the solvent by vacuum distillation, a white powdery product, compound 13-1 (67 mg, yield 60%), was obtained. R f value was 0.45 (dichloromethane:methanol = 5:1, TLC plate model: Huanghai brand HSGF254); by irradiating with an ultraviolet lamp, the colorless solution of the third band product was collected. After recovery of the solvent by vacuum distillation, a white powdery product, compound 13-2 (22 mg, yield 20%) was obtained. R f value was 0.40 (dichloromethane:methanol = 5:1, TLC plate model: Huanghai brand HSGF254).

[0041] 13-1 NMR test results: 1 H NMR (600 MHz, Methanol- d 4) δ 7.78-7.74 (m, 3H), 7.67 – 7.43 (m,13H), 7.22 – 7.01 (m, 8H), 5.21 (d, J = 8.4 Hz, 1H), 5.17 (d, J = 8.9 Hz,1H), 4.77 (d, J = 8.4 Hz, 1H), 4.68 (d, J = 8.5 Hz, 1H), 4.48 (dd, J = 10.6,7.9 Hz, 1H), 4.35 – 4.31 (m, 1H), 4.17 (dd, J = 10.4, 8.0 Hz, 1H), 3.87 (ddd, J= 24.8, 18.5, 12.4 Hz, 9H), 3.79 – 3.74 (m, 2H), 3.72 – 3.54 (m, 11H), 3.48– 3.34 (m, 10H), 3.20 (s, 3H), 1.66 (s, 3H) ppm. 13 - 2 NMR test results: 1 H NMR (600 MHz, Methanol - d 4) δ 7.77 (dt, J = 7.7, 3.8 Hz, 3H), 7.69 –7.49 (m, 10H), 7.13 (d, J = 76.4 Hz, 6H), 5.34 (t, J = 4.9 Hz, 1H), 4.94 (d, J = 7.8Hz, 1H), 4.85 (d, J = 8.4 Hz, 1H), 4.74 (d, J = 8.4 Hz, 1H), 4.68 (d, J = 8.4 Hz, 1H), 4.48 (dd, J = 10.8, 7.6 Hz, 1H), 4.34 (dd, J = 10.7, 7.6 Hz,1H), 4.16 (dd, J = 10.6, 7.9 Hz, 1H), 3.94 – 3.73 (m, 11H), 3.69 (s, 2H),3.65 (s, 1H), 3.35 (d, J = 2.9 Hz, 2H), 3.20 (s, 3H) ppm. Step 14: Synthesis of Compound 14 Place 0.10 g of Compound 13-1 and Compound 13-2 in a 25 mL reaction flask, add 3 mL of 95% ethanol solution as the solvent, then add 0.25 mL of 80% hydrazine hydrate and 0.25 mL of water. Reflux the mixture under stirring at 80 °C for 12 hours. After the reaction is completed, distill off the solvent under reduced pressure, and the resulting residue is directly used for the next reaction. Add 0.1 mL of water, 3 mL of methanol and 0.01 g of sodium bicarbonate to the above residue in sequence. Cool the mixture to 0 °C and add 1 mL of acetic anhydride dropwise. After stirring at 20 °C for 12 hours, distill off the solvent under reduced pressure. The residue is chromatographically separated through a Sephadex G-25 column (distilled water). By the sulfuric acid carbonization color development method, collect the colorless solution of the first band product. After freeze-drying at low temperature, 0.042 g of a white powdery product, Compound 14, is obtained with a yield of 60%, and the R f value is 0.21 (developing agent: ethyl acetate: isopropanol: water = 1:1:1, chromatography plate model: Huanghai brand HSGF254).

[0042] Results of nuclear magnetic resonance test: 1 H NMR (600 MHz, Deuterium Oxide) δ 4.54 (d, J = 7.8 Hz, 1H), 4.52(d, J = 8.9 Hz, 1H), 4.50 (d, J = 8.4 Hz, 1H), 4.32 (d, J = 7.8 Hz, 1H), 3.87(d, J = 11.5 Hz, 4H), 3.81 – 3.74 (m, 2H), 3.70 (d, J = 11.2 Hz, 9H), 3.62(s, 1H), 3.52 (d, J = 9.2 Hz, 1H), 3.49 – 3.45 (m, 3H), 3.43 (s, 3H), 3.41(s, 4H), 2.01 (s, 3H), 1.99 (s, 3H), 1.98 (s, 3H), 1.96 (s, 3H) ppm. 1313C NMR (150 MHz, Deuterium Oxide) δ 174.56, 174.19, 173.89, 173.83, 102.26, 101.00, 100.79, 100.52, 81.00, 80.56, 80.07, 75.86, 75.47, 75.37, 75.31, 73.31, 69.92, 68.62, 68.56, 68.44, 60.90, 60.78, 60.71, 57.24, 55.78, 54.95, 54.84, 54.54, 22.62, 22.54, 22.48, 22.39 ppm. The compound 14 synthesized by the present invention was used for seed soaking and seedling raising test experiments: A stock solution of the compound 14 was prepared at a ratio of 4 mg:20 mL of distilled water, with a concentration of 0.2 mg / mL. 0.2 mL of the stock solution was taken and added to 40 mL of distilled water to obtain a dilution solution with a concentration of 0.001 mg / mL for seed soaking test. β-(1→3)-2-deoxy-2-acetamido glucan disaccharide was used for seed soaking and seedling raising test experiments: A stock solution of β-(1→3)-2-deoxy-2-acetamido glucan disaccharide was prepared at a ratio of 4 mg:20 mL of distilled water, with a concentration of 0.2 mg / mL. 0.2 mL of the stock solution was taken and added to 40 mL of distilled water to obtain a dilution solution with a concentration of 0.001 mg / mL for seed soaking test.

[0043] Rapeseed seeds were soaked for 2 hours, drained, and then air-dried for half an hour, and their germination signs were observed and their germination rates were calculated. The seed soaking diagrams of rapeseed seeds are as Figure 20 , where: 20-1 is the seed soaking diagram of rapeseed seeds soaked in the above solution of compound 14, 20-2 is the seed soaking diagram of rapeseed seeds soaked in the above solution of β-(1→3)-2-deoxy-2-acetamido glucan disaccharide, and 20-3 is the seed soaking diagram of rapeseed seeds soaked in tap water. The experimental data show that under the temperature control condition of about 20 °C, the seed soaking treatment with the plant vaccine of the present invention has a significant promoting effect on the germination of rapeseed seeds. Specifically, the rapeseed seed group (20-1) soaked in the above solution of compound 14 for 2 hours and then drained and air-dried for half an hour achieved a germination rate of 80%, and some seeds had shown germination characteristics to varying degrees; the treatment group (20-2) with the above solution of β-(1→3)-2-deoxy-2-acetamido glucan disaccharide had a germination rate of 60%, but the germination phenomenon was 10 minutes later than the first group; while the control group (20-3) soaked only in tap water had a germination rate of only 10%, and no effective germination was observed.

[0044] Soak cucumber seeds in the same diluent as above for 7 hours, drain them, and then air-dry for 2 hours. Observe their germination signs and calculate their germination rate. The soaking diagram of cucumber seeds is as shown in Figure 21 , where: 21-1 is the soaking diagram of cucumber seeds soaked in the above solution of compound 14, 21-2 is the soaking diagram of cucumber seeds soaked in the above solution of β-(1→3)-2-deoxy-2-acetamido-diglucose, and 21-3 is the soaking diagram of cucumber seeds soaked in tap water. The experimental data show that under the temperature control condition of about 20 °C, the soaking treatment with the plant vaccine of the present invention has a significant promoting effect on the germination of cucumber seeds. Specifically, the seed group (21-1) soaked in the above solution of compound 14 for 7 hours and then drained and air-dried achieved a 100% germination rate, and some seeds had shown germination characteristics to varying degrees; the germination rate of the treatment group with the above solution of β-(1→3)-2-deoxy-2-acetamido-diglucose (21-2) reached 60%, but the germination phenomenon lagged behind the first group by 1 hour; while the control group (21-3) soaked only in tap water had a germination rate of only 14%, and no effective germination was observed.

Claims

1. A lead compound of a plant vaccine based on saccharide substances and its synthesis method, characterized in that: The structure of the new compound synthesized in the present invention is as follows:

2. A lead compound of a plant vaccine based on saccharide substances and a synthesis method thereof, characterized in that: The new compound as described in Claim 1 is synthesized by the following technical route:

3. A lead compound of a plant vaccine based on saccharide substances and a synthesis method thereof, characterized in that: The molar ratio of the raw materials for synthesizing each compound as described in Claim 1 is as follows: (1) In the preparation method of Compound 12, the molar ratio of the reactants is Compound 11: Compound 6: N-iodosuccinimide: trifluoromethanesulfonic acid = 1: 1.2: 4: 0.2; (2) In the preparation methods of Compounds 13-1 and 13-2, the molar ratio of the reactants is Compound 12: p-toluenesulfonic acid = 1: 0.32; (3) In the first-step reaction of the preparation method of Compound 14, the molar ratio of the reactants is Compound 13-1: 80% hydrazine hydrate = 1: 63.7; (4) In the first-step reaction of the preparation method of Compound 14, the molar ratio of the reactants is Compound 13-2: 80% hydrazine hydrate = 1: 61.5; (5) In the second-step reaction of the preparation method of Compound 14, the molar ratio of the reactants is Compound 13-1: acetic anhydride: sodium bicarbonate = 1: 131: 1.5; (6) In the second-step reaction of the preparation method of Compound 14, the molar ratio of the reactants is Compound 13-2: acetic anhydride: sodium bicarbonate = 1: 127: 1.

5.

4. A lead compound of a plant vaccine based on saccharide substances and a synthesis method thereof, characterized in that : The solvents used for synthesizing each compound as described in Claim 1 and their dosages are as follows: (1) In the preparation method of Compound 12, the solvent used for the reaction is dichloromethane, and the dosage is 27.8 mL of solvent per gram of Compound 11; (2) In the preparation methods of Compounds 13-1 and 13-2, the solvent used for the reaction is a mixed solvent of methanol and tetrahydrofuran with a volume ratio of 1:2, and the dosage is 42 mL of solvent per gram of Compound 12; (3) In the first-step reaction of the preparation method of Compound 14, the solvent used is 95% ethanol, and the dosage is 30 mL of solvent per gram of Compound 13-1 (or Compound 13-2); (4) In the second-step reaction of the preparation method of Compound 14, the solvent used is a mixed solvent of methanol and distilled water with a volume ratio of 1:0.03, and the dosage is 31 mL of solvent per gram of Compound 13-1 (or Compound 13-2).

5. A lead compound of a plant vaccine based on saccharide substances and a synthesis method thereof, characterized in that: The synthesis conditions of each compound as described in Claim 1 are as follows: (1) In the preparation method of Compound 12, the reaction conditions are: the reaction temperature is 0 °C, and the reaction time is 5 h; (2) In the preparation methods of Compound 13-1 and Compound 13-2, the reaction conditions are: the reaction temperature is 55 °C, and the reaction time is 48 h; (3) In the first-step reaction of the preparation method of Compound 14, the reaction conditions are: the reaction temperature is 80 °C, and the reaction time is 12 h; (4) In the second-step reaction of the preparation method of Compound 14, the reaction conditions are: the reaction temperature is 20 °C, and the reaction time is 12 h.

6. A lead compound of a plant vaccine based on saccharide substances and a synthesis method thereof, characterized in that: The preparation method of the seed soaking solution of Compound 14 synthesized in the present invention is as follows: The proportion for preparing the mother liquor is: Compound 14: distilled water = 4 mg: 20 mL, and the concentration is 0.2 mg / mL; The proportion for preparing the dilution solution is: mother liquor: distilled water = 0.2 mL: 40 mL, and the concentration is 0.001 mg / mL.

7. A lead compound of a plant vaccine based on saccharide substances and a synthesis method thereof, characterized in that: Application of the synthesized compound 14 in the presoaking of rapeseed and cucumber seeds. The usage method is as follows: Rapeseed seeds: Immerse the rapeseed seeds in the above-mentioned solution of compound 14 for 2 hours. After the seeds have fully absorbed water, take them out and drain, and then place them in a ventilated place to dry naturally for 0.5 hour; Cucumber seeds: Immerse the cucumber seeds in the above-mentioned solution of compound 14 for 7 hours to allow the seeds to fully absorb water. After draining the excess water, dry them in a cool and ventilated place for 2 hours.

8. A lead compound of a plant vaccine based on saccharide substances and a synthesis method thereof, characterized in that: Application of the synthesized compound 14 in related fields such as the presoaking of plant seeds.

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