Triterpenoid saponin compounds, preparation method and application thereof

Abstract

The application discloses a triterpene glycoside saponin compound or a pharmaceutically acceptable salt, ester or solvate thereof, which is shown as formula I. The application further discloses a preparation method and application of the triterpene glycoside saponin compound shown as formula I.

Description

Technical Field

[0001] This invention relates to triterpenoid glycoside saponins, their preparation methods, and their application as adjuvants in vaccines. Background Technology

[0002] The prevention and control of infectious diseases is crucial to ensuring public health security. Vaccines are considered an effective tool against infectious diseases. However, modern vaccines, while highly safe, often have low immunogenicity, leading to the important concept of adjuvants. Adjuvants are substances that can stimulate the immune system and enhance the immunogenicity of vaccines, without having specific antigenic activity themselves. Saponins are a class of glycosides whose aglycones are triterpenoids or spirostane compounds, belonging to plant-derived adjuvants. However, because saponins can damage cell membranes and cause hemolysis of erythrocytes, their application as adjuvants is limited. To reduce saponin toxicity, more effective and less toxic saponins are being extensively studied, while other types of saponins and their derivatives and complexes are also being continuously explored.

[0003] QS (qS) are saponins extracted from the soap tree and are a class of effective adjuvants frequently used in vaccines. Saleh et al. prepared equine herpesvirus-1 (EHV-1) using three adjuvants: Motanide ISA-206, mineral oil, and QS, and inoculated them into mice to test the vaccine's effect in reducing viral dispersibility. The results showed that the conversion rate of CTLs continued to increase up to 30 days after a second vaccination. Furthermore, ELISA and cellular complement fixation assays demonstrated that QS was the optimal vaccine adjuvant. Oliveira et al. used inactivated rabies virus (RV) as the antigen and employed B. atropine inactivated spores (BA-IS), QS, and a combination of both as adjuvants to combat rabies. The results showed that both BAIS and QS increased antibody titers, while the combination of the two doubled the antibody titer.

[0004] Because QS has been found to be a mixture of at least nearly a hundred structurally related saponin glycosides, their isolation and separation are typically very difficult. QS-21 is currently the most widely reported adjuvant, used as a potent adjuvant in clinical vaccines. QS-21 is a triterpenoid saponin with a fatty chain, isolated in 1992 by American chemists Kensil et al. from plants in the Rosaceae family. It possesses extremely strong immunomodulatory activity, inducing the response of exogenous antigens in cellular T lymphocytes (CTLs) and the production of antigen-specific antibodies, and enhancing cellular immune responses. The structure of QS-21 is extremely complex; its sugar chain, fatty chain, and aglycone are among the most structurally complex of saponins to date. Furthermore, the absolute configuration of the chiral center of the fatty chain is not yet fully determined, and the fatty chain readily undergoes intramolecular transfer in aqueous solution, reaching a certain isomer equilibrium. Therefore, the synthesis of QS-21 presents a significant challenge. Simultaneously, QS-21 can induce hemolysis and has certain systemic and local toxic side effects. To reduce its toxicity, QS-21 is often combined with other components to form adjuvant systems, thereby improving vaccine reactivity. Studies have found that using ALF liposomes to bind MPLA and QS-21 as adjuvants against HIV gp 140 protein can effectively increase serum antibody titers. Other studies have used a subcutaneous delivery technology—nanopatch—to form an adjuvant complex with QS-21. Results showed that, compared to traditional intramuscular injection, nanopatch significantly reduces the dosage of antigen and QS-21 used and induces higher IgG titers. Summary of the Invention

[0005] Based on the above, the present invention provides a triterpenoid glycoside saponin compound, its preparation method, and its application in vaccines. The triterpenoid glycoside saponin compound has good adjuvant activity, high tolerability, and low toxicity. It can be used in combination with other adjuvant compounds in vaccines to enhance the immunogenicity of vaccines. At the same time, the preparation process of the triterpenoid glycoside saponin compound of the present invention is time-saving, low-cost, and high-purity, and can be used for stable and efficient production, thereby comprehensively improving production efficiency and product quality.

[0006] In a first aspect, the present invention provides a triterpenoid glycoside saponin compound or a pharmaceutically acceptable salt, ester, or solvate thereof.

[0007] The triterpenoid glycoside saponin compound is shown in Formula I:

[0008]

[0009] in,

[0010] R1 to R9 are selected from hydrogen, deuterium, alkyl, aryl, aralkyl, alkylaryl, aldehyde, and acyl.

[0011] R 11 To R18 Selected from hydrogen, deuterium, alkyl, aryl, aralkyl, alkylaryl, aldehyde, and acyl groups;

[0012] R 19 Selected from hydrogen, deuterium, alkyl, aryl, aralkyl, alkylaryl, aldehyde, acyl and R 10 ;

[0013] R 10 Selected from the following groups:

[0014]

[0015] Where R a R b R c R d R e R f Selected from hydrogen, deuterium, alkyl, halogen, cyano, carboxyl, and ester groups, R g Selected from hydrogen, deuterium, and alkyl groups;

[0016] n is an integer selected from 0 to 10.

[0017] In some embodiments, R1 to R9 are selected from hydrogen, deuterium, C1-C6 alkyl, C6-C10 aryl, C7-C10 aralkyl, C7-C10 alkylaryl, C1-C6 aldehyde and C1-C6 acyl.

[0018] In some implementation schemes, R 11 To R 18 It is selected from hydrogen, deuterium, C1-C6 alkyl, C6-C10 aryl, C7-C10 aralkyl, C7-C10 alkylaryl, C1-C6 aldehyde and C1-C6 acyl.

[0019] In some implementation schemes, R 19 Selected from hydrogen, deuterium, C1-C6 alkyl, C6-C10 aryl, C7-C10 aralkyl, C7-C10 alkylaryl, C1-C6 aldehyde, C1-C6 acyl and R 10 .

[0020] In some embodiments, R1 is selected from hydrogen, deuterium, C1-C6 alkyl, phenyl, and benzyl. In some embodiments, R1 is hydrogen or deuterium.

[0021] In some embodiments, R2 and R3 are selected from hydrogen, deuterium, C1-C6 alkyl, and C1-C6 aldehyde. In some embodiments, R2 is a C1-C6 alkyl group, such as methyl or ethyl; R3 is a C1-C6 aldehyde group, such as -CHO. In other embodiments, R2 is a C1-C6 aldehyde group, such as -CHO; R3 is a C1-C6 alkyl group, such as methyl or ethyl.

[0022] In some embodiments, R4 is selected from hydrogen, deuterium, C1-C6 alkyl, phenyl, and benzyl. In some embodiments, R1 is hydrogen or deuterium.

[0023] In some embodiments, R5 is selected from hydrogen, deuterium, and C1-C6 alkyl groups. In some embodiments, R5 is methyl or ethyl.

[0024] In some embodiments, R6 is selected from hydrogen, deuterium, and C1-C6 alkyl groups. In some embodiments, R6 is methyl or ethyl.

[0025] In some embodiments, R7 is selected from hydrogen, deuterium, and C1-C6 alkyl groups. In some embodiments, R7 is methyl or ethyl.

[0026] In some embodiments, R8 is selected from hydrogen, deuterium, and C1-C6 alkyl groups. In some embodiments, R8 is methyl or ethyl.

[0027] In some embodiments, R9 is selected from hydrogen, deuterium, and C1-C6 alkyl groups. In some embodiments, R9 is methyl or ethyl.

[0028] In some implementation schemes, R 10 Selected from the following groups:

[0029]

[0030] Where R a R b R c R d R e R f The group is selected from hydrogen, deuterium, alkyl, halogen, cyano, carboxyl, and ester, and n is selected from integers from 0 to 10.

[0031] In some implementation schemes, R a R b R c R d R e R f It is selected from hydrogen, deuterium, C1-C6 alkyl, halogen, cyano, C1-C6 carboxyl and C1-C6 ester.

[0032] In some implementation schemes, R f Selected from hydrogen or deuterium. In some embodiments, R f Selected from C1-C6 alkyl groups.

[0033] In some implementation schemes, R c It is selected from halogens (e.g., bromine or iodine), cyano, carboxyl (e.g., COOH) or ester (e.g., -COOCH, -CH2OOCH3).

[0034] In some implementation schemes, R a R b R d R e Selected from hydrogen or deuterium.

[0035] In some implementations, n is 0, 1, 2, 3, 4, 5, 6, or 7.

[0036] In some embodiments, the compound of formula I is shown as that of formula II, formula II-1, or formula II-2:

[0037]

[0038] Each substituent is defined as in formula I.

[0039] In some embodiments, the compound of formula I is selected from the following compounds:

[0040]

[0041]

[0042] In a second aspect, the present invention also provides a method for preparing the triterpenoid glycoside saponin compound as described above, comprising one or more of the following steps:

[0043] (1) The compound of formula I-1 is converted into the compound of formula I-2 through a reaction.

[0044]

[0045] (2) The compound of formula I-2 is converted into the compound of formula I-3 through a reaction.

[0046]

[0047] (3) The compounds of formula I-4 and I-5 are converted into the compound of formula I-6 through a reaction.

[0048]

[0049] (4) The compound of formula I-6 is converted into the compound of formula I-7 through a reaction.

[0050]

[0051] (5) The compounds of formula I-7 and I-3 are converted into the compound of formula I-8 through a reaction.

[0052]

[0053] (6) The compound of formula I-8 is converted into the compound of formula I-9 through a reaction.

[0054]

[0055] (7) The compound of formula I-9 is converted into the compound of formula I-10 through a reaction.

[0056]

[0057] (8) The compound of formula I-11 is converted into the compound of formula I-12 through a reaction.

[0058]

[0059] (9) The compounds of formula I-10 and I-12 are converted into the compound of formula I-13 by reaction.

[0060]

[0061]

[0062] (10) The compound of formula I-13 is converted into the compound of formula I-14 by reaction.

[0063]

[0064] (11) The compound of formula I-14 is converted into the compound of formula I-15 through a reaction.

[0065]

[0066]

[0067] (12) Compounds I-15 and I-16 are converted into compound II by reaction.

[0068]

[0069] R2, R3 and R 10 The limited form I.

[0070] According to a preferred embodiment of the present invention, in step (1), the oxidant used is osmium tetroxide / NMO, and the solvent used is acetone / water (i.e., a mixed solvent system of acetone and water). Using acetone / water as the solvent not only maintains a high yield (above 80%), but also accelerates the reaction rate, thereby shortening the reaction time (about 4 hours). When other solvents (e.g., tert-butanol / tetrahydrofuran / water) are used, the reaction time is more than 10 hours.

[0071] According to a preferred embodiment of the present invention, in step (2), TIPSCl is used in a solvent in the presence of a pyridine organic base, preferably DMAP. Preferably, the solvent is an aprotic polar solvent, preferably DMF. Step (2) can be carried out at 20°C-40°C.

[0072] According to a preferred embodiment of the present invention, in step (3), compounds of formula 1-4 are reacted with Ph2SO and Tf2O in dichloromethane in the presence of a pyridine organic base at a temperature of -50°C to -30°C for 0.5 to 1.5 hours; then compounds of formula 1-5 are added, and the reaction is first carried out at a temperature of -50°C to -30°C for, for example, 0.5 to 1.5 hours, and then carried out at a temperature of -5°C to 5°C for, for example, 20 to 40 minutes.

[0073] According to a preferred embodiment of the present invention, in step (3), the pyridine organic base includes 2,6-di-tert-butyl-4-methylpyridine or 2,4,6-tri-tert-butylpyridine. 2,6-di-tert-butyl-4-methylpyridine has a lower cost than 2,4,6-tri-tert-butylpyridine (the price difference is more than 30 times), and can achieve comparable or higher yields. Therefore, preferably, in step (3), the pyridine organic base is 2,6-di-tert-butyl-4-methylpyridine.

[0074] According to a preferred embodiment of the present invention, in step (4), the reagents used are hydrofluoric acid and triethylamine, the reaction temperature is 20-40°C, and the reaction time can be 10-20 hours.

[0075] According to a preferred embodiment of the present invention, in step (5), compounds of formulas 1-7 are reacted with Ph₂SO₄ and Tf₂O in dichloromethane in the presence of a pyridine organic base at a temperature of -78°C to -30°C for 20 to 40 minutes; then compounds of formulas 1-3 are added, and the reaction is carried out at a temperature of -50°C to -30°C for, for example, 1 to 3 hours. In this step, by adding a pyridine organic base first, the yield of the reaction product can be increased.

[0076] According to a preferred embodiment of the present invention, in step (5), the pyridine organic base includes 2,6-di-tert-butyl-4-methylpyridine or 2,4,6-tri-tert-butylpyridine. 2,6-di-tert-butyl-4-methylpyridine has a lower cost than 2,4,6-tri-tert-butylpyridine (the price difference is more than 30 times), and can achieve comparable or higher yields. Therefore, preferably, in step (5), the pyridine organic base is 2,6-di-tert-butyl-4-methylpyridine.

[0077] According to a preferred embodiment of the present invention, in step (6), the reagents used are TBAF and acetic acid; the solvent is an ether solvent, preferably THF; the reaction temperature can be 20-40°C; and the reaction time can be 1-4 hours, preferably 1.5 to 2.5 hours.

[0078] According to a preferred embodiment of the present invention, in step (7), the reagents used are Cl3CN and DBU; the solvent can be a halocarbon solvent, preferably dichloromethane; the reaction temperature can be -5°C to 5°C, preferably 0°C; and the reaction time can be 2 hours to 4 hours.

[0079] According to a preferred embodiment of the present invention, in step (8), the reagents used are TESOTf and pyridine organic bases (preferably 2,6-dimethylpyridine); the solvent can be a halocarbon solvent, preferably dichloromethane; the reaction temperature can be 20°C to 40°C; and the reaction time can be 0.5 hours to 1.5 hours.

[0080] According to a preferred embodiment of the present invention, in step (9), the reagent used is boron trifluoride diethyl ether solution; the solvent can be a halocarbon solvent, preferably dichloromethane; the reaction temperature can be -78°C to -40°C, preferably -55°C to -45°C; and the reaction time can be 1 hour to 3 hours.

[0081] According to a preferred embodiment of the present invention, in step (10), the reagents used are Pd / C and / or Pd(OH)2 / C, hydrogen gas; the reaction solvent can be tetrahydrofuran and / or methanol; the temperature can be 20°C to 40°C; and the reaction time can be 1 hour to 24 hours, preferably 10 hours to 20 hours.

[0082] According to a preferred embodiment of the present invention, in step (11), the reagent used is trifluoroacetic acid, the solvent can be alcohol or water, the reaction temperature can be -5°C to 5°C, and the reaction time can be 1 hour to 3 hours.

[0083] According to a preferred embodiment of the present invention, in step (12), the reagent used is a C1-C6 alkylamine, preferably triethylamine; the solvent is an aprotic polar solvent, preferably DMF; the reaction temperature can be from 20°C to 40°C; and the reaction time can be from 1 hour to 4 hours.

[0084] This invention provides a method for preparing a compound of formula I-2, comprising converting a compound of formula I-1 into a compound of formula I-2 through a reaction.

[0085]

[0086] The oxidant used is osmium tetroxide / NMO, and the solvent used is acetone / water.

[0087] This invention provides a method for preparing a compound of formula I-6, comprising converting a compound of formula I-4 and a compound of formula I-5 into a compound of formula I-6 through a reaction.

[0088]

[0089] The compound of formula I-4 is reacted with Ph2SO and Tf2O in dichloromethane in the presence of a pyridine organic base at a temperature of -50°C to -30°C for, for example, 0.5 to 1.5 hours; then the compound of formula I-5 is added, and the reaction is first carried out at a temperature of -50°C to -30°C for, for example, 0.5 to 1.5 hours, and then carried out at a temperature of -5°C to 5°C for, for example, 20 to 40 minutes.

[0090] This invention provides a method for preparing a compound of formula I-8, comprising converting a compound of formula I-7 and a compound of formula I-3 into a compound of formula I-8 through a reaction.

[0091]

[0092] The compound of formula I-7 is reacted in dichloromethane with Ph2SO and Tf2O in the presence of a pyridine organic base at a temperature of -78°C to -30°C for, for example, 20 to 40 minutes; then the compound of formula I-3 is added, and the reaction is carried out at a temperature of -50°C to -30°C for, for example, 1 to 3 hours, wherein the pyridine organic base includes 2,6-di-tert-butyl-4-methylpyridine.

[0093] The present invention also provides the use of the triterpenoid glycoside saponin compound represented by Formula I in vaccines. In the stated application, the triterpenoid glycoside saponin compound represented by Formula I serves as an adjuvant. The present invention also provides a pharmaceutical composition comprising the triterpenoid glycoside saponin compound represented by Formula I and an immunologically effective amount of an antigen associated with a disease-causing bacterium or virus.

[0094] The triterpenoid glycoside saponin compounds represented by Formula I can be used as adjuvants to enhance cellular absorption of toxins. The compounds of this invention are particularly suitable for the treatment or prevention of in vivo growths or other proliferative diseases. However, the compounds of this invention can also be used for in vitro research or clinical purposes. Attached Figure Description

[0095] Figure 1 The results of a flow cytometry experiment in the application example are shown. Detailed Implementation

[0096] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the specific embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention.

[0097] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0098] The "stereoisomers" described in this invention refer to compounds that have the same chemical structure but whose atoms or groups are arranged differently in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometrical isomers (cis / trans) isomers, and hindered isomers, etc.

[0099] Depending on the choice of starting materials and methods, the compounds of this invention can exist as one or a mixture of possible isomers, such as racemic mixtures and diastereomeric mixtures (depending on the number of asymmetric carbon atoms). Optically active (R)- or (S)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituents may be E or Z configurations; if the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituents may be cis or trans configurations.

[0100] Any mixture of stereoisomers obtained can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization.

[0101] Unless otherwise indicated, the structural formulas described in this invention include all isomers (e.g., enantiomers, diastereomers, and geometric isomers (or conformational isomers): for example, R and S configurations containing an asymmetric center, (Z) and (E) isomers of double bonds, and (Z) and (E) conformational isomers. Therefore, any single stereochemical isomer of the compounds of this invention, or a mixture of its enantiomers, diastereomers, or geometric isomers (or conformational isomers), is within the scope of this invention.

[0102] Racemic mixtures of any resulting end product or intermediate can be separated into optical enantiomers using known methods, such as by separating their diastereomeric salts. Racemic products can also be separated by chiral chromatography, such as high-performance liquid chromatography (HPLC) using chiral adsorbents. In particular, enantiomers can be prepared by asymmetric synthesis, for example, see Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2nd Ed. Robert E. Gawley, Jeffrey Aubé, Elsevier, Oxford, UK, 2012); Eliel, ELStereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, SH Tables of Resolving Agents and Optical Resolutions p. 268 (ELEliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972); Chiral Separation Techniques: A Practical Approach (Subramanian, G. Ed., Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2007).

[0103] The salts mentioned in this invention are pharmaceutically acceptable salts, and the term "pharmaceutically acceptable salts" is well known in the field, as described in the literature: Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmacol Sci, 1997, 66, 1-19. Pharmaceutically acceptable, non-limiting examples of salts include inorganic acid salts formed by reactions with amino groups, such as hydrochlorides, hydrobroms, phosphates, metaphosphates, sulfates, sulfites, nitrates, and perchlorates, and organic acid salts, such as carboxylates, sulfonates, sulfinates, and thiocarboxylates, specifically, but not limited to, methanesulfonates, ethanesulfonates, formates, acetates, succinates, benzoates, succinates, bis(hydroxynaphthyl) salts, salicylates, galactobionates, gluconates, mandelates, 1,2-ethanedisulfonates, 2-naphthalenesulfonates, carbonates, trifluoroacetates, glycolates, hydroxyethylsulfonates, oxalates, maleates, tartrates, citrates, succinates, malonates, benzenesulfonates, p-toluenesulfonates, malates, fumarates, lactates, lactobionates, or oxalic acid, or obtained by other methods described in the literature, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecyl sulfate, ethanesulfonate, glucono-heptahydrate, glycerophosphate, gluconate, hemisulfate, heptahydrate, hexanoate, hydroiodate, 2-hydroxy-ethanesulfonate, lacturonate, laurate, lauryl sulfate, nicotinate, nitrate, oleate, palmitate, pyrate, pectinate, persulfate, 3-phenylpropionate, picrate, pentanoate, propionate, stearate, thiocyanate, undecanoate, valerate, etc. Furthermore, pharmaceutically acceptable salts also include salts obtained by using suitable bases, such as alkali metals, alkaline earth metals, ammonium, and N+(C1-4 alkyl)4 salts. The present invention also conceives of quaternary ammonium salts formed from any compound containing an N group. Water-soluble or oil-soluble or dispersed products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Pharmaceutically acceptable salts further include suitable, non-toxic ammonium, quaternary ammonium salts, and amine cations that resist the formation of equilibrium ions, such as halides, carboxylates, sulfates, phosphates, nitrates, C1-8 sulfonates, and aromatic sulfonates.

[0104] Medicinal salts can form with inorganic and organic acids, such as acetates, aspartates, benzoates, benzenesulfonates, bromides / hydrobromoates, bicarbonates / carbonates, hydrogen sulfates / sulfates, camphor sulfonates, chlorides / hydrochlorides, theophylline salts, citrates, ethanedisulfonates, fumarates, gluconate, glucuronide, gluconate, glucuronide, hippurate, hydroiodide / iodide, hydroxyethyl sulfonate, lactate, lacturonide, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, methyl sulfate, naphthate, naphthalenesulfonate, nicotinate, nitrates, stearate, oleate, oxalate, palmitate, pyrate, phosphates / hydrogen phosphates / dihydrogen phosphates, polygalactonates, propionates, stearates, succinates, sulfosalicylate, tartrates, toluenesulfonates, and trifluoroacetates.

[0105] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid.

[0106] Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, sulfosalicylic acid, etc.

[0107] In this invention, "solvent" refers to an association formed by one or more solvent molecules and the compound of this invention. Solvents forming solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol. The term "hydrate" refers to an association formed when the solvent molecules are water.

[0108] "Pharmaceutical composition" means a mixture of one or more compounds, salts, or physiologically / pharmaceutically acceptable salts or prodrugs described herein with other chemical components, such as physiologically / pharmaceutical acceptable carriers or excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the compound to a living organism.

[0109] As used in this invention, the term "alkyl" refers to a saturated straight-chain or branched monovalent hydrocarbon group having 1-20 carbon atoms, or 1-10 carbon atoms, or 1-8 carbon atoms, or 1-6 carbon atoms, or 1-4 carbon atoms, or 1-3 carbon atoms, wherein the alkyl group may be independently and optionally substituted by one or more substituents described in this invention. Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), n-propyl (n-Pr, -CH2CH2CH3), isopropyl (i-Pr, -CH(CH3)2), n-butyl (n-Bu, -CH2CH2CH2CH3), isobutyl (i-Bu, -CH2CH(CH3)2), sec-butyl (s-Bu, -CH(CH3)CH2CH3), tert-butyl (t-Bu, -C(CH3)3), n-pentyl (-CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1- Butyl (-CH2CH(CH3)CH2CH3), n-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3) ), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), n-heptyl, n-octyl, etc. The term "alkyl" and its prefix "alkane" are used herein to refer to both straight-chain and branched saturated carbon chains. The term "alkane" is used herein to refer to a saturated divalent hydrocarbon group obtained by eliminating two hydrogen atoms from a straight-chain or branched saturated hydrocarbon; examples of such groups include, but are not limited to, methylene, methine, methinepropyl, etc.

[0110] The term "cycloalkyl" refers to a monovalent or polyvalent, non-aromatic, saturated or partially unsaturated ring that does not contain heteroatoms, including monocyclic rings of 3-12 carbon atoms or bicyclic rings of 7-12 carbon atoms. Bicyclic carbocyclic rings with 7-12 atoms can be bicyclic [4,5], [5,5], [5,6], or [6,6] systems, while bicyclic carbocyclic rings with 9 or 10 atoms can be bicyclic [5,6] or [6,6] systems. Suitable cyclic aliphatic groups include, but are not limited to, cycloalkyl, cycloalkenyl, and cycloynyl groups. Examples of cyclic aliphatic groups include, but are by no means limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopentyl-1-enyl, 1-cyclopentyl-2-enyl, 1-cyclopentyl-3-enyl, cyclohexyl, 1-cyclohexyl-1-enyl, 1-cyclohexyl-2-enyl, 1-cyclohexyl-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, etc. Furthermore, the "cyclic aliphatic group" or "carbocyclic", "carbocyclic group", and "cycloalkyl" may be substituted or unsubstituted, wherein the substituent may be, but is not limited to, hydroxyl, amino, halogen, cyano, aryl, heteroaryl, alkoxy, alkylamino, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, mercapto, nitro, aryloxy, hydroxy-substituted alkoxy, hydroxy-substituted alkyl-C(=O), alkyl-C(=O), alkyl-S(=O), alkyl-S(=O)2-, hydroxy-substituted alkyl-S(=O), hydroxy-substituted alkyl-S(=O)2, carboxyalkoxy, etc.

[0111] The terms “heterocyclic,” “heterocyclic group,” “heterocyclic alicyclic group,” or “heterocyclic” are used interchangeably herein to refer to monocyclic, bicyclic, or tricyclic systems in which one or more carbon atoms on the ring are independently and optionally substituted with heteroatoms, which have the meaning as described herein. The ring may be fully saturated or contain one or more unsaturations, but is by no means aromatic, and has only one connection point to another molecule. One or more hydrogen atoms on the ring are independently and optionally substituted with one or more substituents described herein. Some of these embodiments are that the "heterocycle", "heterocyclic group", "heterocyclic alicyclic group" or "heterocyclic" group is a 3-7 membered monocyclic ring (1-6 carbon atoms and 1-3 heteroatoms selected from N, O, P, S, wherein S or P is optionally replaced by one or more oxygen atoms to obtain a group such as SO, SO2, PO, PO2, and when the ring is a three membered ring, there is only one heteroatom), or a 7-10 membered bicyclic ring (4-9 carbon atoms and 1-3 heteroatoms selected from N, O, P, S, wherein S or P is optionally replaced by one or more oxygen atoms to obtain a group such as SO, SO2, PO, PO2).

[0112] Heterocyclic groups can be carbonyl or heteroatomyl. "Heterocyclic group" also includes groups formed by the fusion of a heterocyclic group with a saturated or partially unsaturated ring or heterocycle. Examples of heterocycles include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiophenyl, piperidinyl, morpholinyl, thiomorpholinyl, thiazolyl, thiazolyl, oxazolyl, piperazine, homopiperazine, aziridine, oxacyclobutyl, thiohexacyclobutyl, piperidinyl, homopiperidinyl, glycidyl, aziridineheptyl, oxacycloheptyl, thiohexacycloheptyl, 4-methoxy-piperidin-1-yl, 1,2,3,6-tetrahydropyridin-1-yl, oxacyclobutyl... 2-diazine Base, sulfur nitrogen 1-pyrrololin-1-yl, 2-pyrrololin-3-pyrrololin-1-yl, dihydroindolyl, 2H-pyranyl, 4H-pyranyl, dioxacyclohexyl, 1,3-dioxopentyl, pyrazolinyl, dithiaalkyl, dithiamonyl, dihydrothiophenyl, pyrazolinyl imidazolinyl, imidazolinyl, 1,2,3,4-tetrahydroisoquinolinyl, 1,2,6-thiadiazinane 1,1-dioxo-2-yl, 4-hydroxy-1,4-azaphosphane 4-oxide-1-yl, 2-hydroxy-1-(piperazin-1-yl)acetone-4-yl, 2-hydroxy-1-(5,6-dihydro-1,2,4-triazin-1(4H)-yl)acetone-4-yl, 5,6-dihydro-4 H-1,2,4-oxadiazine-4-yl, 2-hydroxy-1-(5,6-dihydropyridin-1(2H)-yl) acetone-4-yl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, azabicyclo[2.2.2]hexyl, 2-methyl-5,6,7,8-tetrahydro-[1,2,4]triazol[1,5-c]pyrimidin-6-yl, 4,5,6,7-tetrahydroisoxazol[4,3-c]pyridin-5-yl, 3H-indolyl-2-oxo-5-azabicyclo[2.2.1]heptane-5-yl, 2-oxo-5-azabicyclo[2.2.2]octane-5-yl, quinazinyl and N-pyridinyl urea. Examples of heterocyclic groups also include 1,1-dioxothiomorpholino, and those in which two carbon atoms on the ring are replaced by oxygen atoms, such as pyrimidinide groups. Furthermore, the heterocyclic group can be substituted or unsubstituted, wherein the substituent can be, but is not limited to, oxo(=O), hydroxyl, amino, halogen, cyano, heteroaryl, alkoxy, alkylamino, alkyl, alkenyl, alkynyl, heterocyclic, mercapto, nitro, aryloxy, hydroxy-substituted alkoxy, hydroxy-substituted alkyl-C(=O), alkyl-C(=O), alkyl-S(=O), alkyl-S(=O)2-, hydroxy-substituted alkyl-S(=O), hydroxy-substituted alkyl-S(=O)2, carboxyalkoxy, etc.

[0113] The term "aryl" can be used alone or as a part of "aranyl," "aranalkoxy," or "aranoxyalkyl," referring to a monocyclic, bicyclic, or tricyclic carbocyclic system containing 6-14 membered rings, wherein at least one ring system is aromatic, and each ring system contains 3-7 membered rings with only one attachment point connected to the rest of the molecule. The term "aryl" can be used interchangeably with the term "aromatic ring," as aromatic rings can include phenyl, naphthyl, and anthracene. Furthermore, the aryl group may be substituted or unsubstituted, wherein the substituent may be, but is not limited to, hydroxyl, amino, halogen, cyano, aryl, heteroaryl, alkoxy, alkylamino, alkyl, alkenyl, alkynyl, heterocyclic, mercapto, nitro, aryloxy, hydroxy-substituted alkoxy, hydroxy-substituted alkyl-C(=O), alkyl-C(=O), alkyl-S(=O), alkyl-S(=O)2-, hydroxy-substituted alkyl-S(=O), hydroxy-substituted alkyl-S(=O)2, carboxyalkoxy, etc.

[0114] The term "halogen" refers to F, Cl, Br, or I.

[0115] In this invention, "halogenated" means replacing the following group with a halogen, and the number of substitutions can be one or more. "TIPS" represents triisopropylsilyl group.

[0116] “TES” stands for triethylsilyl.

[0117] “Bn” represents benzyl.

[0118] Example 1 Compound 1 and its preparation method

[0119]

[0120]

[0121] Synthesis of Compound 01-1

[0122]

[0123] Under nitrogen protection, L-rhamnose (40 g, 243.9 mmol, 1.0 eq) and allyl alcohol (80 mL, 2 vol) were added to a 1 L three-necked flask, followed by the addition of Dowex resin (40 g, 100% wt, Dowex 50WX8, hydrogen form, 50-100 mesh) at room temperature. The reaction mixture was heated to 90 °C and stirred overnight. TLC analysis (2:1 = dichloromethane:methanol, anisaldehyde staining) showed the disappearance of the starting material. The reaction mixture was cooled to ambient temperature, filtered, and washed with acetone (2 × 50 mL, 2 × 1 vol). The filtrate was concentrated to give 01-1 as a black solid (80 g crude product), which was used directly in the next reaction without purification.

[0124] Synthesis of Compound 01-2

[0125]

[0126] Under nitrogen protection, 80 g of the 01-1 obtained in the previous step was dissolved in acetone (320 mL, 4 vol), and 2,2-dimethoxypropane (216 mL, 2.7 vol) and p-toluenesulfonic acid monohydrate (0.8 g, 0.01 wt) were added at room temperature, and the mixture was stirred overnight at room temperature. TLC analysis (1:1 petroleum ether / ethyl acetate, anisaldehyde staining) showed that the starting material disappeared. The reaction system was quenched with triethylamine (20 mL), then concentrated and evaporated to dryness to obtain the crude product, which was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 3:1) to obtain pure 01-2 as a yellow oil (40 g, total yield of 67% for both steps). 1 H NMR(300MHz,Chloroform-d)δ5.98–5.85(m,1H),5.35–5.20(m,2H),5.01(s,1H),4.23–4.16(m,2H),4.12-4.10(m ,1H),4.05-3.97(m,1H),3.72–3.66(m,1H),3.43–3.32(m,1H),1.53(s,3H),1.48–1.32(m,3H),1.33-1.26(m,3H).

[0127] Synthesis of Compound 01-3

[0128]

[0129] Under nitrogen protection, 01-2 (35 g, 143.44 mmol, 1 eq) was dissolved in N',N'-dimethylformamide (700 mL), cooled to 0 °C in an ice-water bath, and sodium hydride (8.6 g, 215.16 mmol, 1.5 eq, 60%, 60% dispersion in oil) was added. The mixture was stirred at room temperature for 30 minutes. The reaction mixture was again cooled to 0 °C in an ice-water bath, and benzyl bromide (36.6 g, 215.16 mmol, 1.5 eq) was added dropwise while maintaining the reaction temperature below 10 °C. The mixture was then stirred overnight at room temperature. The reaction mixture was cooled to 0-10 °C and quenched slowly with saturated ammonium chloride aqueous solution (10 mL), and extracted with ethyl acetate (3 x 500 mL). The combined organic layers were washed with saturated sodium chloride solution (1×400mL), dried over anhydrous sodium sulfate, concentrated to obtain crude product, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20:1) to obtain pure O1-3 as a yellow oil (35g, yield 73%). 1H NMR(300MHz,Chloroform-d)δ7.37–7.27(m,5H),5.90-5.86(m,1H),5.32-5. 27(m,1H),5.26-5.18(m,1H),5.01(s,1H),4.89(d,J=6.0Hz,1H),4.62(d,J=6 .0Hz,1H),4.28-4.27(m,1H),4.19-4.14(m,2H),4.01-3.96(m,1H),3.73-3.6 9(m,1H),3.24-3.20(m,1H),1.51(s,3H),1.37(s,3H),1.27(d,J=6.0Hz,3H).

[0130] Compound 01-4

[0131]

[0132] Under nitrogen atmosphere, dichloromethane (836 mL) and methanol (76 mL) were transferred to a 3-liter, 3-necked flask. Triphenylphosphine (35.77 g, 136.358 mmol, 1.2 eq), palladium acetate (5.10 g, 22.726 mmol, 0.2 eq), and diethylamine (116.35 g, 1590.848 mmol, 14 eq) were added at room temperature. O1-3 (38 g, 113.632 mmol, 1 eq) was dissolved in dichloromethane (836 mL) and added to the 3-liter, 3-necked flask. The mixture was heated to 30 °C and stirred for 18 h. The reaction mixture was concentrated to obtain a crude product, which was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 7:1) to give O1-4 as a yellow solid (28 g, 83% yield). MS m / z [MH]-(ESI): 293.85.

[0133] Compound 01-5

[0134]

[0135] O1-4 (8 g, 27.179 mmol, 1 eq) was dissolved in dichloromethane (100 mL) under nitrogen atmosphere, cooled to 0 °C in an ice-water bath, and 2,6-dimethylpyridine (10.19 g, 95.126 mmol, 3.5 eq) was added. Triisopropylsilyl trifluoromethanesulfonate (14.16 g, 46.204 mmol, 1.7 eq) was added dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 1 h, then heated to room temperature for 5 h. 10 mL of saturated sodium bicarbonate aqueous solution was added to the reaction system, the organic phase was separated, and the remaining aqueous phase was extracted with dichloromethane (3 x 30 mL). The combined organic phases were washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, concentrated to obtain a crude product, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20:1) to give O1-5 as a colorless oil (8 g, yield 65%). MS m / z[M+Na]+(ESI):473.35.

[0136] Compound 01-6

[0137]

[0138] O1-5 (35 g, 77.659 mmol, 1 eq) was dissolved in methanol (525 mL) under nitrogen atmosphere, and anhydrous palladium on carbon (10%, 7 g, 20% wt) was added. The reaction system was replaced with hydrogen (hydrogen package), and the reaction was carried out at room temperature for 8 h. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with methanol, the filtrates were combined and concentrated to obtain the crude product, which was then purified by silica gel column chromatography (petroleum ether / ethyl acetate = 8:1) to give O1-6 as a colorless oil (20 g, yield 71%). MS m / z [M+H]+ (ESI): 361.15

[0139] Compound 03-1

[0140]

[0141] D-glucose (30 g, 205.48 mmol, 1 eq) was dissolved in pyridine (750 mL) under nitrogen atmosphere, cooled to 0 °C in an ice-water bath, and benzoyl chloride (60.66 g, 431.50 mmol, 2.1 eq) was added dropwise. The reaction mixture was stirred at 0 °C for 2 h. Methanesulfonyl chloride (51.78 g, 452.05 mmol, 2.2 eq) was added dropwise to the reaction system at 0 °C, and the mixture was stirred at room temperature for 1 h after the addition was complete. The mixture was cooled to 0 °C in an ice-water bath, and the reaction was quenched by adding methanol (12 mL). The mixture was then diluted with ethyl acetate (1000 mL), washed with water (1 x 200 mL) of saturated sodium chloride solution (1 x 200 mL), dried over anhydrous sodium sulfate, concentrated to obtain a crude product, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1) to give 03-1 as a white solid (54 g, two-step yield 60%). 1H NMR(300MHz,DMSO-d6)δ8.59-7.94(m,4H),7.78–7.60(m,2H),7.58-7.48(m,4H),6.75-6.72(m,1H),5.6 2–5.59(m,1H),5.33-5.29(m,1H),5.08-5.04(m,1H),4.82-4.77(m,1H),4.74–4.62(m,2H),3.32(s,3H).

[0142] Compound 03-2

[0143]

[0144] O3-1 (5 g, 11.57 mmol, 1 eq) was added to a 1 L three-necked flask under nitrogen atmosphere, followed by toluene (55 mL, 11 vol). Then tetrabutylammonium chloride (7.07 g, 25.46 mmol, 2.2 eq) and sodium azide (2.78 g, 42.82 mmol, 3.7 eq) were added. The resulting mixture was then slowly heated to 105 °C and stirred at 100–110 °C for 20 hours. The reaction mixture was cooled to ambient temperature and transferred to a separatory funnel. The reaction flask was rinsed with toluene (200 mL) and water (150 mL), and the rinsing solution was also transferred to the separatory funnel. The organic layer was separated and washed with water (1 x 100 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated to obtain a crude product, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 12:1) to give O3-2 as a white solid (2.5 g, 52% yield). MS m / z[M+Na]+(ESI):402.00. 1 H NMR(400MHz,Chloroform-d)δ8.13-8.06(m,4H),7.62–7.60(m,2H),7.57-7.48(m,4H),6.55-6.53(m,1H),5. 4-5.91(m,1H),4.95-4.93(m,1H),4.71-4.66(m,1H),4.62-4.57(m,1H),4.50–4.42(m,1H),4.24-4.22m,1H).

[0145] Compound 03-20

[0146]

[0147] O3-2 (5 g, 13.180 mmol, 1 eq) was dissolved in methanol (70 mL, 12 vol) under nitrogen atmosphere. Sodium hydroxide (0.28 g, 7.117 mmol, 0.54 eq) was added at ambient temperature. After the addition was complete, the mixture was stirred at room temperature. After 14 hours, the pH of the reaction system was adjusted to pH 7 with acetic acid. The crude product was concentrated and diluted with ethyl acetate (200 mL), washed with water (3 x 50 mL), and the organic layer was dried over anhydrous sodium sulfate. The crude product O3-20 (2 g, 88% yield) was concentrated to obtain a pale yellow solid. The crude O3-20 was not purified and was used directly in the next reaction.

[0148] Compound 03-3

[0149]

[0150] O3-20 (5 g, 29.213 mmol, 1 eq) was dissolved in N',N'-dimethylformamide (125 mL, 25 vol) under nitrogen atmosphere. The resulting mixture was transferred to a 1 L three-necked flask and cooled to 0–5 °C. NaH (2.10 g, 87.639 mmol, 3.0 eq, 60% dispersion in oil) was added in portions over 10 minutes at 0–5 °C. The mixture was stirred at 0–10 °C for 30 minutes, followed by the slow addition of benzyl bromide (25.0 g, 146.20 mmol, 5 eq) over 20 minutes while keeping the batch temperature below 10 °C. The reaction was stirred at 0 °C for 3 hours. The reaction was quenched with water (10 mL) at 0 °C, and the mixture was extracted with ethyl acetate (3 x 100 mL). The combined organic phases were dried over anhydrous sodium sulfate, concentrated to obtain a crude product, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1) to give 03-3 as a yellow oil (3.8 g, yield 38%). MS m / z [M+Na]+ (ESI): 374.25.

[0151] Compound 03-4

[0152]

[0153] O3-3 (2.3 g, 6.545 mmol, 1 eq) was dissolved in N',N'-dimethylformamide (20.7 mL, 9 vol) and water (2.3 mL, 1 vol) under nitrogen atmosphere. Osmium tetroxide aqueous solution (166.40 mg, 0.655 mmol, 0.10 eq, 2.5 wt% in H2O) was added in one step to obtain a pale yellow solution. After stirring at room temperature for 30 minutes, N-methylmorpholine-N-oxide (2.30 g, 19.633 mmol, 3.00 eq, 50 wt% in water) was added, and the mixture was stirred at room temperature for 4 hours. The reaction solution was quenched with saturated sodium sulfite aqueous solution (30 mL), extracted with ethyl acetate (3 x 50 mL), and the combined organic phases were dried over anhydrous sodium sulfate. The crude product was concentrated and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1:1) to give O3-4 as a yellow oil (2.1 g, yield 83%). MS m / z[M+Na]+(ESI):408.20.

[0154] Compound 03-5

[0155]

[0156] O3-4 (4 g, 10.378 mmol, 1 eq) was dissolved in N',N'-dimethylformamide (20 mL, 5 vol) under nitrogen atmosphere. Imidazole (1695.70 mg, 24.907 mmol, 2.40 eq) and 4,4-dimethylaminopyridine (0.13 g, 1.038 mmol, 0.10 eq) were added. The mixture was cooled to 0-10 °C in a water ice bath, and triisopropylchlorosilane (3.00 g, 15.567 mmol, 1.5 eq) was added dropwise. The mixture was stirred at room temperature for 20 h. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated sodium chloride aqueous solution (3 x 20 mL), dried over anhydrous sodium sulfate, concentrated to obtain a crude product, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 15:1) to give O3-5 as a colorless oil (3.5 g, yield 62%). MS m / z[M+Na]+(ESI):564.20.

[0157] Compound 01-25

[0158]

[0159] Under nitrogen protection, D-xylose (20 g, 133.39 mmol, 1.0 eq) and methanol (40 mL, 2 vol) were added to a 1 L three-necked flask, followed by the addition of Dowex resin (20 g, 100% wt, Dowex 50WX8, hydrogen form, 50-100 mesh) at room temperature. The reaction mixture was heated to 65 °C and stirred overnight. The reaction mixture was cooled to ambient temperature, filtered, and washed with methanol (2 × 20 mL). The filtrate was concentrated and purified by silica gel column chromatography (dichloromethane / methanol = 10:1) to give 01-25 as a yellow solid (20 g, 92% yield).

[0160] Compound 01-26

[0161]

[0162] Under nitrogen protection, 01-25 (20 g, 121.9 mmol, 1.0 eq), tetrahydrofuran, and N',N'-dimethylformamide (400 mL, 1:1, 20 vol) were added to a 1 L three-necked flask. The reaction mixture was cooled to 0 °C in an ice-water bath. Sodium hydride (17.56 g, 439.02 mmol, 3.6 eq, 60% dispersed in oil) was added slowly in portions over 20 minutes. Tetrabutylammonium iodide (9 g, 24.39 mmol, 0.2 eq) was added to the reaction mixture. Benzyl bromide (83.41 g, 487.80 mmol, 4 eq) was slowly added to the flask over 10 minutes. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was cooled to 0 °C, and ice water (200 mL) was slowly added to quench the reaction. Extracted with ethyl acetate (3 × 500 mL), the combined organic phases were dried over anhydrous sodium sulfate, concentrated to obtain a crude product, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1) to give 01-26 a yellow oily substance (35 g, yield 66%). MS m / z [M+Na]+ (ESI): 457.10.

[0163] Compound 01-27

[0164]

[0165] Under nitrogen protection, 01-26 (35 g, 80.65 mmol, 1.0 eq) was dissolved in acetic acid (385 mL, 11 vol) and added to a 1 L three-necked flask. 2N sulfuric acid aqueous solution (52.5 mL, 1.5 vol) was added, and the mixture was reacted at 90 °C for 5 hours. The reaction mixture was then cooled to room temperature, concentrated to obtain a crude product, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1) to give 01-27 as a white solid (15 g, 45% yield). MS m / z [M+Na]+ (ESI): 443.00.

[0166] Compound 03-100

[0167]

[0168] Under nitrogen protection, O1-27 (6 g, 14.28 mmol, 1.0 eq), diphenyl sulfoxide (8.08 g, 40.0 mmol, 2.8 eq), and 2,6-di-tert-butyl-4-methylpyridine (8.78 g, 42.85 mmol, 3 eq) dissolved in dichloromethane (300 mL, 50 vol) were added to a 1 L three-necked flask. The mixture was cooled to -78 °C, and trifluoromethanesulfonic anhydride (5.63 g, 20.0 mmol, 1.4 eq) was added dropwise. The mixture was then heated to -40 °C and stirred for 1 h. O1-6 (10.2 g, 28.58 mmol, 2 eq) was added at -40 °C, and the mixture was stirred at -40 °C for 1 h, followed by stirring at 0 °C for 30 min. The reaction was quenched by adding triethylamine (14.4 g, 142.80 mmol, 10 eq) at -40 °C, diluted with dichloromethane (100 mL), washed with saturated sodium bicarbonate aqueous solution and saturated sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, concentrated to obtain a crude product, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 15:1) to give a colorless oily substance (7 g, yield 64%). MS m / z [M+Na]+ (ESI): 785.30

[0169] Compound 03-7

[0170]

[0171] Under nitrogen protection, 03-100 (7 g, 9.18 mmol, 1.0 eq) was dissolved in dichloromethane (70 mL, 10 vol), and triethylamine trifluoride (7.02 g, 45.93 mmol, 5 eq) was added at room temperature. The mixture was stirred at room temperature for 16 h. Triethylamine was added at 0 °C, and the pH of the reaction system was adjusted to pH 7. The mixture was diluted with dichloromethane (100 mL), washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was then purified by silica gel column chromatography (petroleum ether / ethyl acetate = 4:1) to give 03-7 as a white solid (4 g, yield 71%). MS m / z [M+Na]+ (ESI): 629.20

[0172] Compound 03-8

[0173]

[0174] Under nitrogen protection, O3-7 (4 g, 6.60 mmol, 1.0 eq) and diphenyl sulfoxide (3.73 g, 18.48 mmol, 2.8 eq) were added to a 1 L three-necked flask and dissolved in dichloromethane (200 mL, 50 vol). The mixture was cooled to -78 °C, and trifluoromethanesulfonic anhydride (2.60 g, 9.240 mmol, 1.4 eq) was added dropwise. The mixture was reacted at -78 °C for 15 min, and then heated to -40 °C and stirred for 15 min. 2,6-Di-tert-butyl-4-methylpyridine (4.06 g, 19.80 mmol, 3 eq) was added at -40 °C, and the mixture was reacted at -40 °C for 1 h. Then, O3-5 (10.71 g, 19.80 mmol, 3 eq) was added to the reaction system, and the mixture was stirred at -40 °C for 2 h. The reaction was quenched by adding triethylamine (6.67 g, 6.00 mmol, 10 eq) at -40 °C, diluted with dichloromethane (200 mL), and washed with saturated sodium bicarbonate aqueous solution and saturated sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, concentrated to obtain a crude product, and purified twice by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1) to give a pale yellow oil (6.5 g, yield 86%). MS m / z [M+Na]+ (ESI): 1152.35

[0175] Compound 03-9

[0176]

[0177] Under nitrogen protection, 03-8 (6.5 g, 5.75 mmol, 1.0 eq) was dissolved in tetrahydrofuran (65 mL, 10 vol), and tetrabutylammonium fluoride (6.9 mL, 6.90 mmol, 1.2 eq, 1 M in THF) and acetic acid (172.5 mg, 2.87 mmol, 0.5 eq) were added at room temperature. The reaction mixture was reacted for 2 h at room temperature. The reaction solution was concentrated to obtain a crude product, which was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 4:1) to give 03-8 as a white solid (3.5 g, yield 62%). MS m / z [M+Na]+ (ESI): 996.20

[0178] Compound 03-10

[0179]

[0180] Under nitrogen protection, 03-9 (100 mg, 0.103 mmol, 1 eq) was dissolved in dichloromethane (2 mL, 20 vol), cooled to 0 °C, and trichloroacetonitrile (170.45 mg, 1.184 mmol, 11.5 eq) was added. DBU (20.32 mg, 0.134 mmol, 1.3 eq) was added dropwise to the reaction system at 0 °C, and the reaction was continued at 0 °C for 3 h. The reaction system was diluted with dichloromethane (40 mL), washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated to obtain a crude product, and purified by silica gel column chromatography (petroleum ether / ethyl acetate / triethylamine = 15:5:1) to give 03-10 as a pale yellow oil (100 mg, yield 87%).

[0181] Compound 03-11

[0182]

[0183] Under nitrogen protection, saponin (100 mg, 0.205 mmol, 1 eq) was dissolved in dichloromethane (2 mL, 20 vol), cooled to 0 °C, and 2,6-dimethylpyridine (220.17 mg, 2.050 mmol, 10 eq) was added. Triethylsilyl trifluoromethanesulfonate (271.56 mg, 1.025 mmol, 5 eq) was added dropwise to the reaction system at 0 °C, and the reaction was continued at 0 °C for 3 h. The reaction system was diluted with dichloromethane (20 mL), washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated to obtain a crude product, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1:1) to give 03-11 as a white solid (100 mg, yield 68%). 1 H NMR(400MHz,Chloroform-d)δ9.32(s,1H),5.33(s,1H),4.54(s,1H),3.82-3.78(m,1H),2.98-2.93(m, 1H),2.25-2.18(m,1H),1.91-1.70(m,8H),1.48-1.20(m,10H),1.17-0.84(m,33H),0.75-0.47(m,15H).

[0184] Compound 03-12

[0185]

[0186] 03-11 Dried overnight in a vacuum drying oven after being treated with pyridine three times with water and then with toluene three times with water.

[0187] 03-10 Toluene with water was dried three times in a vacuum drying oven overnight.

[0188] Under nitrogen protection, 03-11 (14.07 mg, 0.020 mmol, 1.1 eq) and 03-10 (20 mg, 0.018 mmol, 1.00 eq) were dissolved in dichloromethane (1.6 mL, 80 vol). Freshly activated 4A molecular sieve (40 mg, powder) was added to the reaction system, and the mixture was stirred at room temperature for 2 h. The mixture was then cooled to -78 °C, and boron trifluoride diethyl ether (1.88 mg, 0.016 mmol, 0.8 eq) was added. The mixture was heated to -50 °C and reacted for 2 h. The reaction system was then cooled to -78 °C, quenched with triethylamine (0.1 mL), filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (toluene / ethyl acetate = 4:1) to give 03-12 as a white solid (20 mg, yield 67%). MS m / z [M-TES+NH4]+ (ESI): 1574.10.

[0189] Compound 03-13

[0190]

[0191] Under nitrogen protection, 03-12 (360 mg, 0.20 mmol, 1 eq) was dissolved in tetrahydrofuran / methanol (1:1, 10.8 mL, 30 vol). Aqueous Pd / C (216 mg, 60% wt) and anhydrous Pd(OH)₂ / C (288 mg, 80% wt) were added, and the reaction system was replaced with hydrogen gas. The mixture was stirred at room temperature for 16 h. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with methanol. The combined filtrates were concentrated to obtain 03-13 as a pale yellow solid (270 mg crude product, directly used in the next step).

[0192] Compound 03-14

[0193]

[0194] Under nitrogen protection, 03-13 (270 mg crude product) was dissolved in water / trifluoroacetic acid (2:1, 5.4 mL, 20 vol) and stirred at 0 °C for 2 h. The reaction solution was concentrated to obtain 200 mg crude product, which was purified by Prep-HPLC (ACN / water with 0.05% NH4HCO3). The eluent was directly lyophilized to give 03-14 as a pale yellow solid (50 mg, overall yield of 25%). MS m / z [M+H]+ (ESI): 926.60.

[0195] Compound 03-0

[0196]

[0197] Under nitrogen protection, 03-14 (20 mg, 0.022 mmol, 1 eq) was dissolved in DMF (0.4 mL, 20 vol). Triethylamine (6.67 mg, 0.066 mmol, 3 eq) was added at room temperature and stirred for 15 min. Then, 04-11 (12.09 mg, 0.026 mmol, 1.2 eq) was added, and the reaction was stirred at room temperature for 2 h. The reaction solution was directly purified by Prep-HPLC (ACN / water with 0.05% NH4HCO3). The eluent was directly lyophilized to obtain 03-0 (3.2 mg, yield 12%). MS m / z [M+H]+ (ESI): 1269.70.

[0198] Example 2 - Compound 2 and its preparation method

[0199]

[0200] Synthetic route

[0201]

[0202] Compound 04-1

[0203]

[0204] Under nitrogen protection, spiculic acid (100 mg, 0.21 mmol, 1 eq) was dissolved in dichloromethane (2 mL, 20 vol), cooled to 0 °C, and 2,6-dimethylpyridine (220.17 mg, 2.10 mmol, 10 eq) was added. Triethylsilyl trifluoromethanesulfonate (278.18 mg, 1.05 mmol, 5 eq) was added dropwise to the reaction system at 0 °C, and the reaction was carried out at 0 °C for 3 h. The reaction system was diluted with dichloromethane (20 mL), washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated to obtain a crude product, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1:1) to give 04-1 as a white solid (100 mg, yield 68%). 1H NMR (400MHz, Chloroform-d) δ 5.33 (s, 1H), 4.56–4.54 (s, 1H), 3.22–3.18 (m, 1H), 2.97–2.92 (m, 1H), 2.36 (s, 1H), 2.24–2.18 (t, J = 12.0Hz, 1H), 1.89–1.71 (m, 6H), 1.61–1.43 (m, 6H), 1.35 (s, 3H), 1.35–1.23 (m, 3H), 1.15–1.12 (m, 1H), 1.05–0.88 (m, 32H), 0.74 (s, 3H), 0.68–0.64 (,, 9H), 0.62–0.55 (m, 6H). 04-1 No MS signal was found in LCMS.

[0205] Compound 04-2

[0206]

[0207] 04-1 Dried with pyridine three times to remove water, then with toluene three times, and dried overnight in a vacuum drying oven. 03-10 Dried with toluene three times to remove water, and dried overnight in a vacuum drying oven.

[0208] Under nitrogen protection, 04-1 (40 mg, 0.057 mmol, 1.1 eq) and 03-10 (58.00 mg, 0.052 mmol, 1.00 eq) were dissolved in dichloromethane (1.6 mL, 80 vol). Freshly activated 4A molecular sieve (40 mg, powder) was added to the reaction system, and the mixture was stirred at room temperature for 2 h. The temperature was lowered to -78 °C, and boron trifluoride diethyl ether (5.89 mg, 0.041 mmol, 0.8 eq) was added. The temperature was raised to -50 °C, and the reaction was carried out for 2 h. The reaction system was then cooled to -78 °C, quenched with triethylamine (0.1 mL), filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (toluene / ethyl acetate = 4:1) to give 04-2 as a white solid (40 mg, yield 67%). MS m / z [M-TES+NH4]+ (ESI): 1560.10

[0209] Compound 04-8

[0210]

[0211] Under nitrogen protection, 04-2 (255 mg) was dissolved in methanol (7.6 mL, 30 vol), and aqueous Pd / C (76.5 mg, 30% wt) and anhydrous Pd(OH)2 / C (153 mg, 60% wt) were added. The reaction system was then replaced with hydrogen gas and stirred at room temperature for 16 h. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with methanol, and the filtrates were combined and concentrated to obtain crude 04-2. This crude product was purified by Prep-HPLC (ACN / water with 0.05% NH4HCO3), and the eluent was directly lyophilized to give 04-2 as a white solid (120 mg, 80% yield). MS m / z [M+H]+

[0212] (ESI): 912.45

[0213] Compound 04-9

[0214]

[0215] Under nitrogen protection, 4-iodobenzoic acid (9 g, 0.036 mol, 1 eq) was dissolved in DMF (90 mL, 10 vol), followed by the addition of TEA (7.33 g, 0.072 mol, 2 eq), EDCI (13.82 g, 0.072 mol, 2 eq), and 1-hydroxypyrrolidine-2,5- (4.14 g, 0.036 mol, 1 eq), stirred at room temperature for 3 h. Diluted with dichloromethane (500 mL), washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated to obtain crude product, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1) to give 04-9 as a white solid (7 g, yield 56%). MS m / z [M+H]+ (ESI): 345.90

[0216] Compound 04-10

[0217]

[0218] Under nitrogen protection, 04-9 (7 g, 0.02 mol, 1 eq) was dissolved in DMF (70 mL, 10 vol), and TEA (6.06 g, 0.06 mol, 3 eq) and SM2 (7.86 g, 0.02 mol, 1 eq) were added. The mixture was stirred at room temperature for 3 h. The solution was diluted with dichloromethane (500 mL), washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. This crude product was then purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1:1) to give 04-10 as a white solid (7 g, yield 68%). MS m / z [M+H]+ (ESI): 362.05

[0219] Compound 04-11

[0220]

[0221] Under nitrogen protection, 04-10 (1 g, 2.77 mmol, 1 eq) was dissolved in DMF (10 mL, 10 vol), and DIEA (714 mg, 5.54 mmol, 2 eq), EDCI (1.062 g, 5.542 mmol, 2 eq), and 1-hydroxypyrrolidine-2,5- (318 mg, 2.77 mmol, 1 eq), stirred at room temperature for 16 h. Diluted with dichloromethane (500 mL), washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated to obtain crude product, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 3:1) to give 04-11 as a white solid (0.6 g, yield 47%). MS m / z [M+H]+ (ESI): 459.05

[0222] Compound 04-0

[0223]

[0224] Under nitrogen protection, 04-8 (120 mg, 0.131 mmol, 1 eq) was dissolved in DMF (0.36 mL, 30 vol). Triethylamine (39.69 mg, 0.393 mmol, 3 eq) was added at room temperature and stirred for 15 min. Then, 04-11 (72.0 mg, 0.157 mmol, 1.2 eq) was added, and the reaction was stirred at room temperature for 2 h. The reaction solution was directly purified by Prep-HPLC (ACN / water with 0.05% NH4HCO3). The eluent was directly lyophilized to obtain 04-0 (7.6 mg, yield 10%). MS m / z [MH]-(ESI): 1253.65

[0225] Example 3 - Compound 3 and its preparation method

[0226]

[0227] Synthetic route of compound 3

[0228]

[0229] Compound 3 was synthesized following the above route.

[0230] Example 4 - Compound 4 and its preparation method

[0231] Synthetic route of compound 4

[0232] Compound 4 was synthesized following the above route.

[0233] Application Examples of Adjuvants with Herpes

[0234] I. Experimental Design

[0235] Six animal experiments were set up. Groups 1-3 were used to test the protective efficacy of mouse vaccines using liposome adjuvants combined with gE protein. The adjuvant dosage and protein injection dosage were both 5 μg. The composition of the liposomes was the same except for the use of saponins; all other excipients and preparation processes were identical. Group 4 was a 5 μg aluminum adjuvant combined with 5 μg protein experimental group. Group 5 was a 5 μg single injection group. Group 6 was a blank control group. The specific experimental group schemes are shown in Table 1-2 below. Among them, the adjuvant for Group 1 was prepared into a liposome adjuvant using a TLR4 receptor agonist and commercially available QS21 saponin; the adjuvant for Group 2 was prepared into a liposome adjuvant using a TLR4 receptor agonist and compound 1; and the adjuvant for Group 3 was prepared into a liposome adjuvant using a TLR4 receptor agonist and compound 2.

[0236] Table 1

[0237]

[0238] Table 2

[0239] animal Pre-immunization immunity mice Varicella virus infection Intramuscular injection, 0.5ml

[0240] II. Experimental Results:

[0241] Fourteen days after immunization, all animals were euthanized, and spleen immune cells were harvested. Flow cytometry was used to detect the expression of cytokines IFN-γ and IL-2. The results are as follows: Figure 1 As shown, in adjuvant groups 1-3, the expression of IFN-γ and IL-2 in CD4+ T cells was significantly enhanced.

[0242] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A method for preparing triterpenoid glycoside saponin compounds, wherein the triterpenoid glycoside saponin compound is: Compound 1 The method includes the following steps: （10） The reagents used are Pd / C and / or Pd(OH)2 / C, and hydrogen gas; the reaction solvent is tetrahydrofuran and / or methanol; the temperature is 20℃ to 40℃. (11) will ， （12） Compound 1.

2. The production method according to claim 1, wherein The method further includes the following steps: (1) The compound of formula I-1 is converted into the compound of formula I-2 through a reaction. Formula I-1 Formula I-2 (2) The compound of formula I-2 is converted into the compound of formula I-3 through a reaction. Formula I-2 Formula I-3 (3) Compounds of formula I-4 and I-5 are converted into compound I-6 through a reaction. Formula I-4 Formula I-5 Formula I-6 (4) The compound of formula I-6 is converted into the compound of formula I-7 through a reaction. Formula I-6 Formula I-7 (5) The compounds of formula I-7 and I-3 are converted into the compound of formula I-8 through a reaction. Formula I-7 Formula I-3 Formula I-8 (6) The compound of formula I-8 is converted into the compound of formula I-9 through a reaction. Formula I-8 Formula I-9 (7) The compound of formula I-9 is converted into the compound of formula I-10 through a reaction. Formula I-9 Formula I-10 （8） （9） 。 3. The preparation method according to claim 2, wherein, In step (1), the oxidant used is osmium tetroxide / NMO, and the solvent used is acetone / water; and / or In step (2), the reaction is carried out in a solvent using TIPSCl in the presence of a pyridine organic base; and / or In step (3), compound I-4 is reacted with Ph2SO and Tf2O in dichloromethane in the presence of a pyridine organic base at a temperature of -50°C to -30°C for 0.5 to 1.5 hours; then compound I-5 is added, and the reaction is first carried out at a temperature of -50°C to -30°C for 0.5 to 1.5 hours, followed by a reaction at a temperature of -5°C to 5°C for 20 to 40 minutes; and / or In step (3), the pyridine organic base is selected from 2,6-di-tert-butyl-4-methylpyridine or 2,4,6-tri-tert-butylpyridine, and / or In step (4), the reagents used are hydrofluoric acid and triethylamine, and the temperature is 20-40°C; and / or In step (5), compound I-7 is reacted with Ph2SO and Tf2O in dichloromethane in the presence of a pyridine organic base at a temperature of -78°C to -30°C for 20 to 40 minutes; then compound I-3 is added, and the reaction is carried out at a temperature of -50°C to -30°C for 1 to 3 hours; and / or In step (5), the pyridine organic base is selected from 2,6-di-tert-butyl-4-methylpyridine or 2,4,6-tri-tert-butylpyridine; and / or In step (6), the reagents used are TBAF and acetic acid; the solvent is an ether solvent; the reaction temperature is 20-40℃; and / or In step (7), the reagents used are Cl3CN and DBU; the solvent is a haloalkane solvent; the reaction temperature is -5°C to 5°C; and / or In step (8), the reagents used are TESOTf and pyridine organic bases; the solvent is a haloalkane solvent; the reaction temperature is 20°C to 40°C; and / or In step (9), the reagent used is boron trifluoride diethyl ether solution; the solvent is a halocarbon solvent; and the reaction temperature is -78°C to -40°C.

4. The preparation method according to claim 3, wherein, In step (2), TIPSCl is used to carry out the reaction in a solvent in the presence of DMAP.

5. The preparation method according to claim 3, wherein, In step (6), the solvent used is THF.

6. The preparation method according to claim 3, wherein, In step (7), the solvent used is dichloromethane.

7. The preparation method according to claim 3, wherein, In step (8), the pyridine organic base is 2,6-dimethylpyridine; The solvent used is dichloromethane.

8. The preparation method according to claim 3, wherein, In step (9), the solvent used is dichloromethane; the reaction temperature is -55°C to -45°C.

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