CN117106004A

CN117106004A - Triterpene saponin compounds and their preparation methods and applications

Info

Publication number: CN117106004A
Application number: CN202210535692.4A
Authority: CN
Inventors: 刘泽群; 吴杰; 贺伟; 胡雅灵
Original assignee: Sinovac Research & Development Co ltd
Current assignee: Sinovac Research & Development Co ltd
Priority date: 2022-05-17
Filing date: 2022-05-17
Publication date: 2023-11-24

Abstract

本发明公开了一种三萜糖苷皂苷化合物或其药学上可接受的盐、酯或者溶剂化物，所述三萜糖苷皂苷化合物如式I所示。本发明还公开了式I所示的三萜糖苷皂苷化合物的制备方法及应用。 The invention discloses a triterpene glycoside saponin compound or a pharmaceutically acceptable salt, ester or solvate thereof. The triterpene glycoside saponin compound is represented by Formula I. The invention also discloses the preparation method and application of the triterpene glycoside saponin compound represented by formula I.

Description

Triterpene saponin compound and preparation method and application thereof

Technical Field

The invention relates to a triterpene glycoside saponin compound, a preparation method thereof and application thereof as an adjuvant in vaccines.

Background

The prevention and control of infectious diseases is the key to ensuring public health safety, vaccines are considered as effective tools against infectious diseases, and aiming at the defects of high safety but poor immunogenicity of modern vaccines, an important concept of an adjuvant is proposed. Adjuvants are substances that stimulate the immune system, enhance the immunogenicity of a vaccine, and do not themselves have specific antigenic effects. The saponin is a kind of glycoside with triterpene or spirostane compound, and belongs to plant source adjuvant. However, since saponins can damage cell membranes, which causes hemolysis of erythrocytes, their use in adjuvants is limited. In order to reduce the toxicity of saponins, more effective and less toxic saponins have been widely studied, and other kinds of saponins, their derivatives and complexes have been continuously explored.

Quillaja saponaria saponin (QS) is a saponin extracted from Quillaja saponaria, and is an effective adjuvant often used in vaccines. Saleh et al prepared equine herpes virus-1 (EHV-1) using three adjuvants, motanide ISA-206, mineral oil and QS, and inoculated into mice to examine the effect of the vaccine in reducing viral dispersion. The results show that CTLs conversion increased continuously to 30d after the second vaccination, and furthermore ELISA and cell complement binding assays demonstrated that QS was the best vaccine adjuvant. Oliveira et al use inactivated Rabies Virus (RV) as antigen and B. Atropine inactivated spore (BA-IS), QS and their combination as adjuvants to combat rabies. The results indicate that both BAIS and QS increase antibody titres, while both binding antibody titres will increase doubly.

Since QS is found to be a mixture of at least nearly hundred structurally related saponins, their isolation and isolation is often very difficult. QS-21 is the most widely reported adjuvant at present and is used as a powerful adjuvant in clinical vaccines. QS-21 is a fatty chain-containing disaccharide triterpenoid saponin isolated from Rosaceae plants by American chemist Kensil et al in 1992, has extremely strong immune auxiliary activity, can induce the generation of corresponding antigen-specific antibodies of antigen-added Cell T Lymphocytes (CTL), and can enhance the immune response of cells. The structure of QS-21 and its complexity, whether it is sugar chain, fatty chain or aglycone is the most complex structure type in saponin at present, and the absolute configuration of the chiral center of fatty chain has not been completely determined, and the fatty chain is easy to undergo intramolecular transfer in aqueous solution to reach a certain isomer balance, so the synthesis of QS-21 molecule has great challenges. Meanwhile, QS-21 can induce cell hemolysis, and has certain system and local toxic and side effects. In order to reduce its toxicity, QS-21 often constitutes an adjuvant system with other components to increase the reactogenicity of the vaccine. It has been found that the use of ALF liposomes conjugated MPLA and QS-21 as adjuvants against HIVgp 140 protein is effective in increasing antibody titers in serum. There has also been studies using a subcutaneous delivery technique, nano-patches, to form an adjuvant complex with QS-21. The results indicate that the nanosheets can significantly reduce the dose of antigen and QS-21 used and induce higher IgG titers compared to conventional intramuscular injection.

Disclosure of Invention

Based on the above, the invention provides a triterpene glycoside saponin compound and a preparation method and application thereof in vaccines, wherein the triterpene glycoside saponin compound has better adjuvant activity, higher tolerance and smaller toxic and side effects, can be used in combination with other adjuvant compounds in the vaccines to enhance the immunogenicity of the vaccines, and meanwhile, the preparation process of the triterpene glycoside saponin compound has the advantages of less time consumption, low cost and high purity, can be used for stable and efficient production, and can comprehensively improve the production efficiency and the product quality.

In a first aspect, the present invention provides a triterpene glycoside saponin compound or a pharmaceutically acceptable salt, ester or solvate thereof,

the triterpene glycoside saponin compound is shown as a formula I:

wherein,

R ₁ to R ₉ Selected from the group consisting of hydrogen, deuterium, alkyl, aryl, aralkyl, alkaryl, aldehyde, and acyl;

R ₁₁ to R ₁₈ Selected from the group consisting of hydrogen, deuterium, alkyl, aryl, aralkyl, alkaryl, aldehyde, and acyl;

R ₁₉ selected from hydrogen, deuterium, alkyl, aryl, aralkyl, alkaryl, aldehyde, acyl and R ₁₀ ；

R ₁₀ Selected from the following groups:

wherein R is _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;

n is an integer from 0 to 10.

In some embodiments, R ₁ To R ₉ Selected from the group consisting of hydrogen, deuterium, C1-C6 alkyl, C6-C10 aryl, C7-C10 aralkyl, C7-C10 alkaryl, C1-C6 aldehyde, and C1-C6 acyl.

In some embodiments, R ₁₁ To R ₁₈ Selected from the group consisting of hydrogen, deuterium, C1-C6 alkyl, C6-C10 aryl, C7-C10 aralkyl, C7-C10 alkaryl, C1-C6 aldehyde, and C1-C6 acyl.

In some embodiments, 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 。

In some embodiments, R ₁ Selected from hydrogen, deuterium, C1-C6 alkyl, phenyl and benzyl. In some embodiments, R ₁ Hydrogen or deuterium.

In some embodiments, R ₂ And R is ₃ Selected from hydrogen, deuterium, C1-C6 alkyl and C1-C6 aldehyde groups. In some embodiments, R ₂ And R is ₃ Selected from C1-C6 alkyl and C1-C6 aldehyde groups. In some embodiments, R ₂ C1-C6 alkyl, such as methyl or ethyl; r is R ₃ Is a C1-C6 aldehyde group, such as-CHO. In other embodiments, R ₂ C1-C6 aldehyde groups, such as-CHO; r is R ₃ Is a C1-C6 alkyl group, such as methyl or ethyl.

In some embodiments, R ₄ Selected from hydrogen, deuterium, C1-C6 alkyl, phenyl And benzyl. In some embodiments, R ₁ Hydrogen or deuterium.

In some embodiments, R ₅ Selected from hydrogen, deuterium and C1-C6 alkyl. In some embodiments, R ₅ Is methyl or ethyl.

In some embodiments, R ₆ Selected from hydrogen, deuterium and C1-C6 alkyl. In some embodiments, R ₆ Is methyl or ethyl.

In some embodiments, R ₇ Selected from hydrogen, deuterium and C1-C6 alkyl. In some embodiments, R ₇ Is methyl or ethyl.

In some embodiments, R ₈ Selected from hydrogen, deuterium and C1-C6 alkyl. In some embodiments, R ₈ Is methyl or ethyl.

In some embodiments, R ₉ Selected from hydrogen, deuterium and C1-C6 alkyl. In some embodiments, R ₉ Is methyl or ethyl.

In some embodiments, R ₁₀ Selected from the following groups:

wherein R is _a 、R _b 、R _c 、R _d 、R _e 、R _f Selected from hydrogen, deuterium, alkyl, halogen, cyano, carboxyl and ester groups, n is selected from integers from 0 to 10.

In some embodiments, R _a 、R _b 、R _c 、R _d 、R _e 、R _f Selected from hydrogen, deuterium, C1-C6 alkyl, halogen, cyano, C1-C6 carboxyl and C1-C6 ester groups.

In some embodiments, R _f Selected from hydrogen or deuterium. In some embodiments, R _f Selected from C1-C6 alkyl.

In some embodiments, R _c Selected from halogen (e.g. bromine or iodine), cyano, carboxyl (e.g. COOH) or ester (e.g. -COOCH, -CH) ₂ OOCH ₃ )。

In some embodiments, R _a 、R _b 、R _d 、R _e Selected from hydrogen or deuterium.

In some embodiments, n is 0, 1, 2, 3, 4, 5, 6, or 7.

In some embodiments, the compound of formula I is represented by formula II, formula II-1, or formula II-2:

the definition of each substituent is the same as that of the formula I.

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

in a second aspect, the present invention also provides a process for preparing a triterpene glycoside saponin compound as described above comprising one or more of the following steps:

(1) Converting the compound of formula I-1 into a compound of formula I-2 by reaction,

(2) Converting the compound of formula I-2 into a compound of formula I-3 by reaction,

(3) Converting the compound of formula I-4 and the compound of formula I-5 into a compound of formula I-6 by reaction,

(4) Converting the compound of formula I-6 to a compound of formula I-7 by reaction,

(5) Converting the compound of formula I-7 and the compound of formula I-3 into a compound of formula I-8 by reaction,

(6) Converting the compound of formula I-8 into a compound of formula I-9 by reaction,

(7) Converting the compound of formula I-9 into a compound of formula I-10 by reaction,

(8) Converting the compound of formula I-11 to a compound of formula I-12 by reaction,

(9) Converting the compound of formula I-10 and the compound of formula I-12 into a compound of formula I-13 by reaction,

(10) Converting the compound of formula I-13 to a compound of formula I-14 by reaction,

(11) Converting the compound of formula I-14 to a compound of formula I-15 by reaction,

(12) Converting the compound of formula I-15 and the compound of formula I-16 by reaction to a compound of formula II,

R ₂ 、R ₃ and R is ₁₀ Is defined as in formula I.

According to a preferred embodiment of the invention, in step (1), the oxidizing agent used is osmium tetroxide/NMO and the solvent used is acetone/water (i.e. a mixed solvent system of acetone and water). By using acetone/water as the solvent, not only can the higher yield (more than 80%) be maintained, but also the reaction speed can be increased, thereby shortening the reaction time (about 4 hours). When other solvents (such as t-butanol/tetrahydrofuran/water) are used, the reaction time of the reaction is 10 hours or longer.

According to a preferred embodiment of the invention, in step (2), the step is carried out with TIPSCl in the presence of a pyridine-based organic base, preferably DMAP, in a solvent. Preferably, the solvent is an aprotic polar solvent, preferably DMF. Step (2) may be carried out at 20℃to 40 ℃.

According to a preferred embodiment of the present invention, in step (3), the compound of formula 1-4 is reacted with in dichloromethane in the presence of a pyridine-based organic basePh ₂ SO and Tf ₂ O is reacted for 0.5 to 1.5 hours at a temperature of-50 ℃ to-30 ℃; the compound of formula I-5 is then added, first at a temperature of-50℃to-30℃for, for example, 0.5 to 1.5 hours, followed by a reaction at a temperature of-5℃to 5℃for, for example, 20 to 40 minutes.

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

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 ℃, and the reaction time may be 10-20 hours.

According to a preferred embodiment of the present invention, in step (5), the compound of formula 1-7 is reacted with Ph in methylene chloride in the presence of a pyridine-based organic base ₂ SO and Tf ₂ O is reacted for 20 minutes to 40 minutes at the temperature of-78 ℃ to-30 ℃; the compound of formula I-3 is then added and reacted at a temperature of from-50℃to-30℃for, for example, 1 hour to 3 hours. In this step, the yield of the reaction product can be improved by adding the pyridine-based organic base first.

According to a preferred embodiment of the present invention, in step (5), the pyridine-based organic base includes 2, 6-di-t-butyl-4-methylpyridine or 2,4, 6-tri-t-butylpyridine, 2, 6-di-t-butyl-4-methylpyridine has a lower cost (the price of the two is 30 times or more different) than 2,4, 6-tri-t-butylpyridine, and a comparable or higher yield can be achieved. Thus, preferably, in step (5), the pyridine-based organic base is 2, 6-di-tert-butyl-4-methylpyridine.

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 ℃; the reaction time may be 1-4 hours, preferably 1.5 to 2.5 hours.

According to a preferred embodiment of the present invention, in step (7), the reagent used is Cl ₃ CN and DBU; the solvent may be a halogenated hydrocarbon solvent, preferably dichloromethane; the reaction temperature may be-5 ℃ to 5 ℃, preferably 0 ℃; the reaction time may be 2 hours to 4 hours.

According to a preferred embodiment of the invention, in step (8), the reagents used are TESOTf and a pyridine-based organic base (preferably 2, 6-lutidine); the solvent may be a halogenated hydrocarbon solvent, preferably dichloromethane; the reaction temperature may be 20 ℃ to 40 ℃; the reaction time may be 0.5 hours to 1.5 hours.

According to a preferred embodiment of the present invention, in step (9), the reagent used is boron trifluoride etherate; the solvent may be a halogenated hydrocarbon solvent, preferably dichloromethane; the reaction temperature may be-78 ℃ to-40 ℃, preferably-55 ℃ to-45 ℃; the reaction time may be 1 hour to 3 hours.

According to a preferred embodiment of the present invention, in step (10), the reagent used is Pd/C and/or Pd (OH) ₂ Hydrogen; the reaction solvent may be tetrahydrofuran and/or methanol; the temperature may be 20 ℃ to 40 ℃; the reaction time may be 1 to 24 hours, preferably 10 to 20 hours.

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

According to a preferred embodiment of the 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 may be 20 ℃ to 40 ℃; the reaction time may be 1 hour to 4 hours.

The invention provides a preparation method of a compound of formula I-2, which comprises the steps of converting a compound of formula I-1 into the compound of formula I-2 through reaction,

Wherein the oxidant is osmium tetroxide/NMO, and the solvent is acetone/water.

The invention provides a preparation method of a compound of formula I-6, which comprises the steps of converting a compound of formula I-4 and a compound of formula I-5 into the compound of formula I-6 through reaction,

wherein a compound of formula I-4 is reacted with Ph in methylene chloride in the presence of a pyridine organic base ₂ SO and Tf ₂ O is reacted at a temperature of-50℃to-30℃for, for example, 0.5 to 1.5 hours; the compound of formula I-5 is then added, first at a temperature of-50℃to-30℃for, for example, 0.5 to 1.5 hours, followed by a reaction at a temperature of-5℃to 5℃for, for example, 20 to 40 minutes.

The invention provides a preparation method of a compound of formula I-8, which comprises the steps of converting a compound of formula I-7 and a compound of formula I-3 into the compound of formula I-8 through reaction,

wherein a compound of formula I-7 is reacted with Ph in methylene chloride in the presence of a pyridine organic base ₂ SO and Tf ₂ O is reacted at a temperature of-78 ℃ to-30 ℃ for, for example, 20 minutes to 40 minutes; the compound of formula I-3 is then added and reacted at a temperature of-50℃to-30℃for, for example, 1 to 3 hours, the pyridine-based organic base comprising 2, 6-di-tert-butyl-4-methylpyridine.

The invention also provides application of the triterpenoid glycoside saponin compound shown in the formula I in a vaccine. In such applications, the triterpene glycoside saponin compounds of formula I are used as adjuvants. The invention also provides a pharmaceutical composition comprising a triterpene glycoside saponin compound represented by the formula I and an immunologically effective amount of an antigen associated with a disease-causing bacterium or virus.

The triterpene glycoside saponin compounds of formula I are useful as adjuvants to enhance cellular uptake of toxins. The compounds of the invention may be particularly useful in the treatment or prevention of neoplasms or other proliferative diseases in vivo. However, the compounds of the invention described above may also be used to achieve in vitro research or clinical purposes.

Drawings

FIG. 1 shows the results of flow cytometry in the application example.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It should be apparent to those skilled in the art that the detailed description is merely provided to aid in understanding the invention and should not be taken as limiting the invention in any way.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

"stereoisomers" as used herein refers to compounds having the same chemical structure but different arrangements of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis/trans), atropisomers, and the like.

Depending on the choice of starting materials and methods, the compounds of the invention may be present in the form of one of the possible isomers or mixtures thereof, for example racemates 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 substituent may be in the E or Z configuration; if the compound contains a disubstituted cycloalkyl group, the substituents of the cycloalkyl group may have cis or trans configuration.

The resulting mixture of any stereoisomers may be separated into pure or substantially pure geometric isomers, enantiomers, diastereomers, e.g., by chromatography and/or fractional crystallization, depending on the differences in the physicochemical properties of the components.

Unless otherwise indicated, the structural formulae described herein include all isomeric forms (e.g., enantiomers, diastereomers, and geometric isomers (or conformations), such as R, S configuration containing an asymmetric center, the (Z), (E) isomers of double bonds, and the conformational isomers of (Z), (E).

Any of the resulting racemates of the end products or intermediates can be resolved into the optical enantiomers by methods familiar to those skilled in the art, e.g., by separation of the diastereoisomeric salts thereof obtained, using known methods. The racemic product can also be separated by chiral chromatography, e.g., high Performance Liquid Chromatography (HPLC) using chiral adsorbents. In particular, enantiomers may be prepared by asymmetric synthesis, for example, reference may be made to Jacques, et al, encomers, racemates and Resolutions (Wiley Interscience, new York, 1981); principles of Asymmetric Synthesis (2nd Ed.Robert E.Gawley,Jeffrey Aub e, elsevier, oxford, UK, 2012); eliel, e.l. stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); wilen, S.H.tables of Resolving Agents and Optical Resolutions p.268 (E.L.Eliel, ed., univ.ofNotre Dame Press, notre Dame, IN 1972); chiral Separation Techniques: A Practical Approach (Subramannian, G.ed., wiley-VCH Verlag GmbH & Co.KGaA, weinheim, germany, 2007).

The salts mentioned in the present invention are pharmaceutically acceptable salts, wherein "pharmaceutically acceptable salts" are well known in the art, as in the literature: berge et al describe pharmaceutically acceptable salts in detail in J. Pharmacol Sci,1997,66,1-19. Examples of pharmaceutically acceptable non-limiting salts include inorganic salts formed by reaction with amino groups such as hydrochloride, hydrobromide, phosphate, metaphosphate, sulfate, sulfite, nitrate, perchlorate, and organic salts such as carboxylate, sulfonate, sulfinate, thiocarboxylate, and the like, specifically such as, but not limited to, methanesulfonate, ethanesulfonate, formate, acetate, succinate, benzoate, succinate, pamoate, salicylate, galactarate, glucoheptonate, mandelate, 1, 2-ethanedisulfonate, 2-naphthalenesulfonate, carbonate, trifluoroacetate, glycolate, isethionate, oxalate, maleate, tartrate, citrate, succinate, malonate, benzenesulfonate, p-toluenesulfonate, malate, fumarate, lactate, lactobionate, or oxalic acid, or by other methods described in the book literature such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartate, benzenesulfonate, bisulfate, borate, butyrate, camphoric acid, camphorsulfonate, cyclopentylpropionate, digluconate, dodecylsulfate, ethanesulfonate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, caproate, hydroiodite, 2-hydroxy-ethanesulfonate, lactobionic aldehyde, laurate, lauryl sulfate, nicotinate, nitrate, oleate, palmitate, pamoate, pectate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, undecanoate, valerate, and the like. In addition, pharmaceutically acceptable salts also include salts obtained with suitable bases, such as alkali metal, alkaline earth metal, ammonium and N+ (C1-4 alkyl) 4 salts. The present invention also contemplates quaternary ammonium salts formed from any compound containing a group of N. The water-soluble or oil-soluble or dispersible product may be obtained by quaternization. Alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Pharmaceutically acceptable salts further include suitable, non-toxic ammonium, quaternary ammonium salts and counter-ion forming amine cations such as halides, carboxylates, sulphates, phosphates, nitrates, C1-8 sulphonates and aromatic sulphonates.

Pharmaceutically acceptable salts may be formed with inorganic and organic acids such as acetate, aspartate, benzoate, benzenesulfonate, bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, chloride/hydrochloride, clenchine salts, citrate, ethanedisulfonate, fumarate, glucoheptonate, gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate, lactate, lactoaldehyde, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, methylsulfate, naphthoate, naphthalenesulfonate, nicotinate, nitrate, stearate, oleate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, polygalactoate, propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate and trifluoroacetate.

Inorganic acids from which salts may be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

Organic acids from which salts may 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 and the like.

"solvate" according to the present invention refers to an association of one or more solvent molecules with a compound according to the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethylsulfoxide, ethyl acetate, acetic acid, aminoethanol. The term "hydrate" refers to an association of solvent molecules that are water.

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

The term "alkyl" as used herein means a saturated straight or branched monovalent hydrocarbon radical of 1 to 20 carbon atoms, or 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms, wherein the alkyl radical may be independently and optionally substituted with one or more substituents as described herein. Examples of alkyl groups include, but are not limited to, methyl (Me, -CH 3), ethyl (Et, -CH2CH 3), n-propyl (n-Pr, -CH2CH2CH 3), isopropyl (i-Pr, -CH (CH 3) 2), n-butyl (n-Bu, -CH2CH2CH 3), isobutyl (i-Bu), -CH2CH (CH 3) 2), sec-butyl (s-Bu, -CH (CH 3) CH2CH 3), tert-butyl (t-Bu, -C (CH 3) 3), n-pentyl (-CH 2CH2CH2CH2CH 3), 2-pentyl (-CH (CH 3) CH2CH2CH 3), 3-pentyl (-CH (CH 2CH 3) 2), 2-methyl-2-butyl (-C (CH 3) 2CH 3), 3-methyl-2-butyl (-CH (CH 3) 2), 3-methyl-1-butyl (-CH 2CH (CH 3) 2), 2-methyl-1-butyl (-CH 2CH 3) 2 (-CH 2), 2-pentyl (-CH 2CH2CH 3), 2-hexyl (CH 2) CH2CH2CH 3), 3-methyl-2-pentyl (-CH (CH 3) CH (CH 3) CH2CH 3), 4-methyl-2-pentyl (-CH (CH 3) CH2CH (CH 3) 2), 3-methyl-3-pentyl (-C (CH 3) (CH 2CH 3) 2), 2-methyl-3-pentyl (-CH (CH 2CH 3) CH (CH 3) 2), 2, 3-dimethyl-2-butyl (-C (CH 3) 2CH (CH 3) 2), 3-dimethyl-2-butyl (-CH (CH 3) C (CH 3) 3), n-heptyl, n-octyl, and the like. The term "alkyl" and its prefix "alkane" are used herein to encompass both straight and branched saturated carbon chains. The term "alkylene" is used herein to denote a saturated divalent hydrocarbon group resulting from elimination of two hydrogen atoms from a linear or branched saturated hydrocarbon, examples of which include, but are not limited to, methylene, ethylene, isopropylidene, and the like.

The term "cycloalkyl" refers to a monovalent or polyvalent, non-aromatic, saturated or partially unsaturated ring, and does not contain heteroatoms, including single rings of 3-12 carbon atoms or bicyclic rings of 7-12 carbon atoms. The bicyclic carbocycle having 7-12 atoms may be a bicyclo [4,5], [5,6] or [6,6] system, while the bicyclic carbocycle having 9 or 10 atoms may be a bicyclo [5,6] or [6,6] system. Suitable cycloaliphatic groups include, but are not limited to, cycloalkyl, cycloalkenyl, and cycloalkynyl. Examples of the cyclic aliphatic group include, but are in no way limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopentyl-1-alkenyl, 1-cyclopentyl-2-alkenyl, 1-cyclopentyl-3-alkenyl, cyclohexyl, 1-cyclohexyl-1-alkenyl, 1-cyclohexyl-2-alkenyl, 1-cyclohexyl-3-alkenyl, cyclohexanedienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl and the like. And the "cycloaliphatic" or "carbocycle", "carbocyclyl", "cycloalkyl" may be substituted or unsubstituted, wherein the substituents may be, but are not limited to, hydroxy, amino, halogen, cyano, aryl, heteroaryl, alkoxy, alkylamino, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, mercapto, nitro, aryloxy, hydroxy-substituted alkoxy, hydroxy-substituted alkyl-C (=o), alkyl-S (=o) 2-, hydroxy-substituted alkyl-S (=o) 2, carboxyalkoxy, and the like.

The terms "heterocycle", "heterocyclyl", "heteroalicyclic" or "heterocyclic" are used interchangeably herein to refer to a monocyclic, bicyclic, or tricyclic ring system in which one or more carbon atoms in the ring are independently and optionally substituted with heteroatoms having the meanings as described herein, and the ring may be fully saturated or contain one or more unsaturations, but is in no way aromatic, with only one point of attachment to other molecules. The hydrogen atoms on one or more rings are independently and optionally substituted with one or more substituents described herein. Some embodiments are "heterocycle", "heterocyclyl", "heteroalicyclic" or "heterocyclic" groups which are monocyclic (1-6 carbon atoms and 1-3 heteroatoms selected from N, O, P, S where S or P is optionally substituted with one or more oxygen atoms to give a group such as SO, SO2, PO2, where the ring is a three membered ring, with only one heteroatom), or 7-10 membered bicyclic (4-9 carbon atoms and 1-3 heteroatoms selected from N, O, P, S where S or P is optionally substituted with one or more oxygen atoms to give a group such as SO, SO2, PO 2).

The heterocyclic group may be a carbon group or a heteroatom group. "heterocyclyl" also includes groups formed by the merging 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, tetrahydrothiopyranyl, piperidinyl, morpholinyl,thiomorpholinyl, thiazalkyl, thiazolidinyl, oxazolidinyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, piperidinyl, homopiperidinyl, epoxypropyl, azepanyl, oxepinyl, thiepanyl, 4-methoxy-piperidin-1-yl, 1,2,3, 6-tetrahydropyridin-1-yl, oxazepineRadical, diaza->Radical, thiazal->Pyrrolin-1-yl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxacyclohexyl, 1, 3-dioxanyl, pyrazolinyl, dithianyl, dithiadienyl, dihydrothienyl, pyrazolidinimidazolinyl, imidazolidinyl, 1,2,3, 4-tetrahydroisoquinolinyl, 1,2, 6-thiadiazinon-1, 1-dioxo-2-yl, 4-hydroxy-1, 4-azaphosphan-4-oxide-1-yl, 2-hydroxy-1- (piperazin-1-yl) ethanon-4-yl, 2-hydroxy-1- (5, 6-dihydro-1, 2, 4-triazin-1 (4H) -yl) ethanon-4-yl, 5, 6-dihydro-4H-1, 2, 4-oxadiazin-4-yl, 2-hydroxy-1- (5, 6-dihydropyridin-1 (2H) -ethanon-4-yl, 3.0.bicyclo [ 3.0. ]Hexyl, 3-azabicyclo [4.1.0]Heptyl, azabicyclo [2.2.2]Hexyl, 2-methyl-5, 6,7, 8-tetrahydro- [1,2,4]Triazole [1,5-c ]]Pyrimidin-6-yl, 4,5,6, 7-tetrahydroisoxazole [4,3-c]Pyridin-5-yl, 3H-indolyl 2-oxo-5-azabicyclo [2.2.1]Heptan-5-yl, 2-oxo-5-azabicyclo [2.2.2]Octane-5-yl, quinolizinyl and N-pyridyl urea. Examples of heterocyclic groups also include 1, 1-dioxothiomorpholinyl, and wherein two carbon atoms in the ring are replaced by oxygen atoms such as pyrimidinedione groups. And the heterocyclic group may be substituted or unsubstituted, wherein the substituents may be, but are not limited to, oxo (=o), hydroxy, amino, halogen, cyano, heteroaryl, alkoxy, alkylamino, alkyl, alkenyl, alkynyl, heterocyclic, mercapto, nitro, aryloxy, hydroxy-substituted alkoxyalkyl-C (=o), alkyl-S (=o) 2-, hydroxy-substituted alkyl-S (=o) 2, carboxyalkoxy, and the like.

The term "aryl" may be used alone or as part of an "aralkyl", "aralkoxy" or "aryloxyalkyl" group, to denote monocyclic, bicyclic, and tricyclic carbocyclic ring systems containing a total of 6-14 membered rings, wherein at least one ring system is aromatic, wherein each ring system contains 3-7 membered rings, and only one attachment point is attached to the remainder of the molecule. The term "aryl" may be used interchangeably with the term "aromatic ring", e.g., aromatic rings may include phenyl, naphthyl and anthracenyl. And the aryl group may be substituted or unsubstituted, wherein the substituents may be, but are not limited to, hydroxy, amino, halogen, cyano, aryl, heteroaryl, alkoxy, alkylamino, alkyl, alkenyl, alkynyl, heterocyclyl, mercapto, nitro, aryloxy, hydroxy-substituted alkoxy, hydroxy-substituted alkyl-C (=o), alkyl-S (=o) 2-, hydroxy-substituted alkyl-S (=o) 2, carboxyalkoxy, and the like.

The term "halogen" refers to F, cl, br or I.

"halo" as used herein means a halogen substituted for a subsequent group, and the number of substitutions may be one or more. "TIPS" means triisopropylsilyl.

"TES" means triethylsilyl.

"Bn" means benzyl.

Example 1 Compound 1 and method for preparing the same

Synthesis of Compound 01-1

L-rhamnose (40 g,243.9mmol,1.0 eq) allyl alcohol (80 mL,2 vol) was added to a 1L 3-neck flask under nitrogen and Dowex resin (40g,100%wt,Dowex 50WX8, hydrogen form, 50-100 mesh) was added at room temperature. The reaction mixture was heated to 90 ℃ and stirred overnight. TLC analysis (2:1=dichloromethane: methanol, anisaldehyde staining) showed the disappearance of starting material. The reaction mixture was cooled to ambient temperature, filtered, and washed with acetone (2×50ml,2×1 vol.). The filtrate was concentrated to give 01-1 as a black solid (80 g crude), which was used directly in the next reaction without purification.

Synthesis of Compound 01-2

Under nitrogen, 01-1 (80 g) obtained in the above step was dissolved in acetone (320 mL,4 vol), 2-dimethoxypropane (216 mL,2.7 vol) and p-toluenesulfonic acid monohydrate (0.8 g,0.01 wt) were added at room temperature, and stirred at room temperature overnight. TLC analysis (1:1 petroleum ether/ethyl acetate, anisaldehyde staining) showed the disappearance of starting material. The reaction was quenched with triethylamine (20 mL) and then concentrated to dryness to give the crude product which was purified by column on silica gel (petroleum ether/ethyl acetate=3:1) to give pure 01-2 as a yellow oil (40 g, 67% overall yield in two 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).

Synthesis of Compound 01-3

01-2 (35 g,143.44mmol,1 eq) was dissolved in N ', N' -dimethylformamide (700 mL), the ice-water bath was cooled to 0℃and sodium hydride (8.6 g,215.16mmol,1.5eq,60% dispersion in oil) was added and stirred at room temperature for 30 minutes under nitrogen. The reaction was cooled to 0℃again in an ice-water bath, benzyl bromide (36.6 g,215.16mmol,1.5 eq) was added dropwise while keeping the reaction temperature below 10℃and then stirred overnight at room temperature. The reaction mixture was cooled to 0-10 ℃ and quenched by slow addition of saturated aqueous ammonium chloride (10 mL) and extracted with ethyl acetate (3 x500 mL). The combined organic layers were washed with saturated sodium chloride solution (1×400 mL), dried over anhydrous sodium sulfate, concentrated to give the crude product, and purified by column on silica gel (petroleum ether/ethyl acetate=20:1) to give pure 01-3 as a yellow oil (35 g, 73% yield). ¹ H 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),3.73-3.69(m,1H),3.24-3.20(m,1H),1.51(s,3H),1.37(s,3H),1.27(d,J＝6.0Hz,3H).

Compounds 01-4

Dichloromethane (836 mL) and methanol (76 mL) were charged into a 3 liter, 3-necked flask under nitrogen, and triphenylphosphine (35.77 g, 136.356 mmol,1.2 eq), palladium acetate (5.10 g,22.726mmol,0.2 eq) and diethylamine (116.35 g,1590.848mmol,14 eq) were added at room temperature. 01-3 (38 g, 113.630 mmol,1 eq) was dissolved in dichloromethane (836 mL) and added to a 3L 3 neck flask. The mixture was heated to 30℃and stirred for 18h. The reaction mixture was concentrated to give crude product, and purified by silica gel column (petroleum ether/ethyl acetate=7:1) to give 01-4 as yellow solid (28 g, yield 83%). MS M/z [ M-H ] - (ESI): 293.85.

Compounds 01-5

01-4 (8 g, 27.178 mmol,1 eq) was dissolved in dichloromethane (100 mL) under nitrogen, the ice-water bath was cooled to 0deg.C, and 2, 6-lutidine (10.19 g,95.126mmol,3.5 eq) was added. Triisopropyl silicone triflate (14.16 g,46.204mmol,1.7 eq) was added dropwise at 0deg.C. The reaction solution was stirred at 0℃for 1h, and then warmed to room temperature for 5h. To the reaction was added 10mL of saturated aqueous sodium bicarbonate solution, the organic phase was separated, and the remaining aqueous phase was extracted with dichloromethane (3×30 mL). The combined organic phases were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, concentrated to give the crude product, and purified by column on silica gel (petroleum ether/ethyl acetate=20:1) to give 01-5 as a colorless oil (8 g, yield 65%). MS M/z [ M+Na ] + (ESI): 473.35.

Compounds 01-6

01-5 (35 g,77.659mmol,1 eq) was dissolved in methanol (525 mL) under nitrogen, and anhydrous palladium on carbon (10%, 7g,20% wt) was added. The reaction system was replaced with hydrogen (hydrogen bag) and reacted at room temperature for 8 hours. The reaction solution was filtered through celite, the filter cake was rinsed with methanol, the filtrates were concentrated to give the crude product, and purified by column on silica gel (petroleum ether/ethyl acetate=8:1) to give 01-6 as a colorless oil (20 g, yield 71%). MS M/z [ M+H ] + (ESI): 361.15

Compound 03-1

D-glucose (30 g,205.48mmol,1 eq) was dissolved in pyridine (750 mL) under nitrogen, the ice-water bath was cooled to 0deg.C, and benzoyl chloride (60.66 g,431.50mmol,2.1 eq) was added dropwise. The reaction solution was stirred at 0℃for 2h. Methanesulfonyl chloride (51.78 g,452.05mmol,2.2 eq) was added dropwise to the reaction system at 0℃and after the addition was completed, the mixture was stirred at room temperature for 1h. The temperature was reduced to 0℃in an ice-water bath, and methanol (12 m) was added to the reaction systemL) quenched and diluted with ethyl acetate (1000 mL), washed with water (1 x200 mL) saturated aqueous sodium chloride (1 x200 mL), dried over anhydrous sodium sulfate, concentrated to give the crude product, and purified by column on silica gel (petroleum ether/ethyl acetate=10:1) to give 03-1 as a white solid (54 g, 60% yield in two steps). ¹ H NMR(300MHz,DMSO-d ₆ )δ8.59-7.94(m,4H),7.78–7.60(m,2H),7.58-7.48(m,4H),6.75-6.72(m,1H),5.62–5.59(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).

Compound 03-2

To a 1 liter 3-necked flask was added 03-1 (5 g,11.57mmol,1 eq) under nitrogen followed by toluene (55 mL,11 vol). Tetrabutylammonium chloride (7.07 g,25.46mmol,2.2 eq) and sodium azide (2.78 g,42.82mmol,3.7 eq) were then added. The resulting mixture was then slowly heated to 105℃and stirred at 100-110℃for 20 hours. The reaction mixture was cooled to ambient temperature and transferred to a separatory funnel. The reaction flask was rinsed with toluene dilution (200 mL) and water (150 mL) and the rinse was also transferred to a separation funnel. The organic layer was separated and washed with water (1×100 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated to give crude product, and purified by silica gel column (petroleum ether/ethyl acetate=12:1) to give 03-2 as a white solid (2.5 g, yield 52%). 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).

Compounds 03-20

03-2 (5 g,13.180mmol,1 eq) was dissolved in methanol (70 mL,12 vol) under nitrogen and sodium hydroxide (0.28 g,7.117mmol,0.54 eq) was added at ambient temperature. After the addition was completed, the resulting mixture was stirred at room temperature. After 14 hours, the reaction system was adjusted to ph=7 with acetic acid, concentrated to give a crude product, diluted with ethyl acetate (200 mL), washed with water (3×50 mL), and the organic layer was dried over anhydrous sodium sulfate and concentrated to give a crude product 03-20 (2 g, yield 88%) as a pale yellow solid. The crude 03-20 was used directly in the next reaction without purification.

Compound 03-3

03-20 (5 g,29.213mmol,1 eq) was dissolved in N ', N' -dimethylformamide (125 mL,25 vol) under nitrogen. The resulting mixture was charged into a 1 liter 3-neck flask and cooled to 0-5 ℃. NaH (2.10 g,87.639mmol,3.0eq, 60% dispersion in oil) was added in portions over 10 min at 0-5 c the mixture was stirred at 0-10 c for 30 min, then benzyl bromide (25.0 g,146.20mmol,5 eq) was added slowly over 20 min while keeping the batch temperature below 10 c. The reaction was stirred for 3h at 0deg.C. The reaction was quenched with water (10 mL) at 0 ℃, extracted with ethyl acetate (3×100 mL), the combined organic phases dried over anhydrous sodium sulfate, concentrated to give the crude product, and purified by column on silica gel (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.

Compounds 03-4

03-3 (2.3 g,6.545mmol,1 eq) was dissolved in N ', N' -dimethylformamide ((20.7 mL,9 vol) and water (2.3 mL,1 vol) under nitrogen, an aqueous osmium tetroxide solution (166.40mg,0.655mmol,0.10eq,2.5wt%in H2O) was added in one portion to give a pale yellow solution, after stirring at room temperature for 30 minutes, N-methylmorpholine-N-oxide (2.30g,19.633mmol,3.00eq,50wt%in water) was added, stirring at room temperature for 4h, the reaction solution was quenched with saturated aqueous sodium sulfite (30 mL), extracted with ethyl acetate (3X 50 mL), the combined organic phases were dried over anhydrous sodium sulfate, concentrated to give crude product, and purified by column on silica gel (petroleum ether/ethyl acetate=1:1) to give 03-4 as a yellow oil (2.1 g, yield 83%). MS M/z [ M+Na ] + (ESI): 408.20).

Compounds 03-5

03-4 (4 g,10.378mmol,1 eq) was dissolved in N ', N' -dimethylformamide ((20 mL,5 vol)) and imidazole (1695.70 mg, 24.307 mmol,2.40 eq) and 4, 4-dimethylaminopyridine (0.13 g,1.038mmol,0.10 eq) were added under nitrogen, the mixture was cooled to 0-10℃with an aqueous ice bath and triisopropylchlorosilane (3.00 g,15.567mmol,1.5 eq) was added dropwise and stirred at room temperature for 20h the reaction solution was diluted with ethyl acetate (100 mL), the saturated aqueous sodium chloride solution was washed (3X 20 mL), the organic phase was dried over anhydrous sodium sulfate, concentrated to give the crude product, and purified by silica gel column (petroleum ether/ethyl acetate=15:1) to give 03-5 as a colorless oil (3.5 g, yield 62%). MS/z [ M+Na ] + (ESI): 564.20.

Compounds 01-25

To a 1L 3-necked flask was added D-xylose (20 g,133.39mmol,1.0 eq), methanol (40 mL,2 vol) under nitrogen, and Dowex resin (20g,100%wt,Dowex 50WX8, hydrogen form, 50-100 mesh) at room temperature. The reaction mixture was heated to 65 ℃ 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 (dichloromethane/methanol=10:1) to give 01-25 as yellow solid (20 g, yield 92%).

Compounds 01-26

To a 1L 3-necked flask was added 01-25 (20 g,121.9mmol,1.0 eq) tetrahydrofuran and N ', N' -dimethylformamide (400 mL,1:1, 20 vol) under nitrogen, the reaction mixture was cooled to 0deg.C in an ice-water bath and sodium hydride (17.56 g,439.02mmol,3.6 eq) was added slowly in portions over 20 minutes as a 60% dispersion in oil. Tetrabutylammonium iodide (9 g,24.39mmol,0.2 eq) was added to the reaction mixture. Benzyl bromide (83.41 g,487.80mmol,4 eq) was slowly added to the flask over 10 minutes and the reaction mixture was stirred at room temperature for 16h. The reaction solution was cooled to 0℃and ice water (200 mL) was slowly added to quench the reaction. Extraction with ethyl acetate (3×500 mL), drying of the combined organic phases over anhydrous sodium sulfate, concentration gave crude product, and purification by silica gel column (petroleum ether/ethyl acetate=5:1) gave 01-26 as yellow oil (35 g, 66% yield). MS M/z [ M+Na ] + (ESI): 457.10.

Compounds 01-27

To a 1L 3-necked flask was added 01-26 (35 g,80.65mmol,1.0 eq) dissolved in acetic acid (385 mL,11 vol) under nitrogen protection, 2N aqueous sulfuric acid (52.5 mL,1.5 vol) was added, after reacting at 90℃for 5 hours, and the reaction mixture was cooled to room temperature, concentrated to give crude product, and purified by silica gel column (petroleum ether/ethyl acetate=5:1) to give 01-27 as white solid (15 g, yield 45%). MS M/z [ M+Na ] + (ESI): 443.00.

Compounds 03-100

To a 1L 3-necked flask was added 01-27 (6 g,14.28mmol,1.0 eq) diphenyl sulfoxide (8.08 g,40.0mmol,2.8 eq), 2, 6-di-tert-butyl-4-methylpyridine (8.78 g,42.85mmol,3 eq) dissolved in methylene chloride (300 mL,50 vol), cooled to-78deg.C, and trifluoromethanesulfonic anhydride (5.63 g,20.0mmol,1.4 eq) was added dropwise thereto under nitrogen atmosphere, and the mixture was stirred for 1 hour at-40deg.C. 01-6 (10.2 g,28.58mmol,2 eq) was added at-40℃and stirred for 1h at 40℃and 30min at 0 ℃. The reaction was quenched by the addition of triethylamine (14.4 g,142.80mmol,10 eq) at-40 ℃, diluted with dichloromethane (100 mL), washed with saturated aqueous sodium bicarbonate and saturated sodium chloride, respectively, the organic phase dried over anhydrous sodium sulfate, concentrated to give the crude product, and purified by column on silica gel (petroleum ether/ethyl acetate=15:1) to give 03-100 as a colorless oil (7 g, 64% yield). MS M/z [ M+Na ] + (ESI): 785.30

Compounds 03-7

03-100 (7 g,9.18mmol,1.0 eq) was dissolved in dichloromethane (70 mL,10 vol) under nitrogen, triethylamine (7.02 g,45.93mmol,5 eq) was added at room temperature and stirred at room temperature for 16h. Triethylamine was added at 0 ℃ to adjust the PH of the reaction system to ph=7, dichloromethane was added to dilute (100 mL), washed with saturated sodium chloride, the organic phase was dried over anhydrous sodium sulfate, concentrated to give the crude product, and purified by silica gel column (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

Compounds 03-8

To a 1L 3-necked flask was added 03-7 (4 g,6.60mmol,1.0 eq) under nitrogen, diphenyl sulfoxide (3.73 g,18.48mmol,2.8 eq) was dissolved in methylene chloride (200 mL,50 vol), cooled to-78 ℃, and trifluoromethanesulfonic anhydride (2.60 g,9.240mmol,1.4 eq) was added dropwise thereto under nitrogen, followed by reaction at-78℃for 15min and stirring at-40℃for 15min. 2, 6-Di-tert-butyl 4-methylpyridine (4.06 g,19.80mmol,3 eq) was added at-40℃and reacted at-40℃for 1h, then 03-5 (10.71 g,19.80mmol,3 eq) was added to the reaction system and stirred at-40℃for 2h. The reaction was quenched by the addition of triethylamine (6.67 g,6.00mmol,10 eq) at-40 ℃, diluted with dichloromethane (200 mL), washed with saturated aqueous sodium bicarbonate and saturated sodium chloride, respectively, the organic phase dried over anhydrous sodium sulfate, concentrated to give the crude product, and purified 2 times over a silica gel column (petroleum ether/ethyl acetate=10:1) to give 03-8 as a pale yellow oil (6.5 g, 86% yield). MS M/z [ M+Na ] + (ESI): 1152.35

Compounds 03-9

03-8 (6.5 g,5.75mmol,1.0 eq) was dissolved in tetrahydrofuran (65 mL,10 vol) under nitrogen, tetrabutylammonium fluoride (6.9mL,6.90mmol,1.2eq,1M in THF) and acetic acid (172.5 mg,2.87mmol,0.5 eq) were added at room temperature, and the reaction was carried out at room temperature for 2h. The reaction solution was concentrated to give a crude product, which was purified by silica gel column (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

Compounds 03-10

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

Compounds 03-11

Soap-skin acid (100 mg,0.205mmol,1 eq) was dissolved in dichloromethane (2 mL,20 vol) under nitrogen, cooled to 0deg.C, and 2.6-lutidine (220.17 mg,2.050mmol,10 eq) was added. Triethylsilyl triflate (271.56 mg,1.025mmol,5 eq) was added dropwise to the reaction system at 0deg.C and reacted at 0deg.C for 3h. The reaction was diluted with dichloromethane (20 mL), washed with saturated sodium chloride, the organic phase was dried over anhydrous sodium sulfate, Concentration gave the crude product and purification by silica gel column (petroleum ether/ethyl acetate=1:1) gave 03-11 as a white solid (100 mg, 68% yield). 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).

Compounds 03-12

03-11 was dried overnight with pyridine and toluene 3 times and vacuum oven.

03-10 toluene was taken up with water 3 times and dried overnight in a vacuum oven.

03-11 (14.07 mg,0.020mmol,1.1 eq) and 03-10 (20 mg,0.018mmol,1.00 eq) were dissolved in dichloromethane (1.6 mL,80 vol) under nitrogen, and the reaction system was stirred at room temperature for 2h with the addition of freshly activated 4A molecular sieves (40 mg, powder). Cooled to-78 ℃, boron trifluoride diethyl etherate (1.88 mg,0.016mmol,0.8 eq) was added. Heating to-50 ℃ to react for 2h. The reaction was cooled to-78 ℃, quenched with triethylamine (0.1 mL), filtered, and the filtrate concentrated to give the crude product, which was purified by column on silica gel (toluene/ethyl acetate=4:1) to give 03-12 as a white solid (20 mg, yield 67%). MS M/z [ M-TES+NH ₄ ]+(ESI):1574.10.

Compounds 03-13

Under nitrogen, 03-12 (360 mg,0.20mmol,1. Eq) was dissolved in tetrahydrofuran/methanol (1:1, 10.8mL,30 vol) and aqueous Pd/C (216 mg,60% w.t) and anhydrous Pd (OH) were added ₂ C (288 mg,80% w.t.), the reaction was replaced by hydrogen and stirred at room temperature for 16h. The reaction solution was filtered through celite, the filter cake was washed with methanol, and the filtrates were concentrated to give 03-13 as pale yellow solid (270 mg crude, directly used in the next step).

Compounds 03-14

03-13 (270 mg crude) was dissolved in water/trifluoroacetic acid (2:1, 5.4mL,20 vol) under nitrogen and stirred at 0deg.C for 2h. The reaction mixture was concentrated to give 200mg of crude product, which was purified by prep-HPLC (ACN/water with 0.05% NH) ₄ HCO ₃ ) The eluate was directly lyophilized to give 03-14 as a pale yellow solid (50 mg, 25% of total yield in two steps). MS M/z [ M+H ]]+(ESI):926.60.

Compounds 03-0

Under the protection of nitrogen, 03-14 (20 mg,0.022mmol,1 eq) was dissolved in DMF (0.4 mL,20 vol), triethylamine (6.67 mg,0.066mmol,3 eq) was added at room temperature and stirred for 15min, then 04-11 (12.09 mg,0.026mmol,1.2 eq) was added and stirred at room temperature for 2h, and the reaction solution was directly sent to Prep-HPLC for purification (ACN/water with 0.05% NH) ₄ HCO ₃ ) The eluate was directly lyophilized to give 03-0 (3.2 mg, yield 12%). MS M/z [ M+H ]]+(ESI):1269.70.

Example 2 Compound 2 and method for preparing the same

Synthetic route

Compound 04-1

Nitrogen protectionWith the aid of this, echinocystic acid (100 mg,0.21mmol,1 eq) was dissolved in dichloromethane (2 mL,20 vol), cooled to 0deg.C, and 2.6-lutidine (220.17 mg,2.10mmol,10 eq) was added. Triethylsilyl triflate (278.18 mg,1.05mmol,5 eq) was added dropwise to the reaction system at 0deg.C and reacted for 3h at 0deg.C. The reaction system was diluted with dichloromethane (20 mL), washed with saturated sodium chloride, the organic phase was dried over anhydrous sodium sulfate, concentrated to give the crude product, and purified by silica gel column (petroleum ether/ethyl acetate=1:1) to give 04-1 as a white solid (100 mg, yield 68%). ¹ HNMR (400 mhz, chloroform-d) delta 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=12.0 hz, 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 has no MS signal in s.

Compound 04-2

04-1 was dried overnight with pyridine and toluene 3 times and vacuum oven. 03-10 toluene was taken up with water 3 times and dried overnight in a vacuum oven.

Under nitrogen, 04-1 (40 mg,0.057mmol,1.1 eq) and 03-10 (58.00 mg,0.052mmol,1.00 eq) were dissolved in dichloromethane (1.6 mL,80 vol) and the reaction was stirred at room temperature for 2h with the addition of freshly activated 4A molecular sieve (40 mg, powder). Cooled to-78 ℃, boron trifluoride diethyl etherate (5.89 mg,0.041mmol,0.8 eq) was added. Heating to-50 ℃ to react for 2h. The reaction was cooled to-78 ℃, quenched with triethylamine (0.1 mL), filtered, and the filtrate concentrated to give the crude product, which was purified by column on silica gel (toluene/ethyl acetate=4:1) to give 04-2 as a white solid (40 mg, yield 67%). MS M/z [ M-TES+NH ₄ ]+(ESI):1560.10

Compound 04-8

Under the protection of nitrogen, the air conditioner is provided with a nitrogen inlet,04-2 (255 mg) was dissolved in methanol (7.6 mL,30 vol) and aqueous Pd/C (76.5 mg,30% w.t) and anhydrous Pd (OH) were added ₂ and/C (153 mg,60% w.t.), the reaction was replaced with hydrogen and stirred at room temperature for 16h. The reaction mixture was filtered through celite, the filter cake was washed with methanol, the filtrates were concentrated to give a crude 04-2, and Prep-HPLC was performed to purify (ACN/water with0.05% NH) ₄ HCO ₃ ) The eluate was directly lyophilized to give 04-2 as a white solid (120 mg, yield 80%). MS M/z [ M+H]+

(ESI):912.45

Compound 04-9

4-iodobenzoic acid (9 g,0.036mol,1 eq) was dissolved in DMF (90 mL,10 vol) under nitrogen, TEA (7.33 g,0.072mol,2 eq), EDCI (13.82 g,0.072mol,2 eq), 1-hydroxypyrrolidine-2, 5-(4.14 g,0.036mol,1 eq) and stirred at room temperature for 3h. Dilute with dichloromethane (500 mL), wash with saturated sodium chloride, dry the organic phase over anhydrous sodium sulfate, concentrate to give crude product, and purify on silica gel column (petroleum ether/ethyl acetate=5:1) to give 04-9 as a white solid (7 g, 56% yield.) MS M/z [ M+H]+(ESI):345.90

Compounds 04-10

04-9 (7 g,0.02mol,1 eq) was dissolved in DMF (70 mL,10 vol) under nitrogen, TEA (6.06 g,0.06mol,3 eq) was added, SM2 (7.86 g,0.02mol,1 eq) and stirred at room temperature for 3h. Dilute with dichloromethane (500 mL), wash with saturated sodium chloride, dry the organic phase over anhydrous sodium sulfate, concentrate to give the crude product, and purify on silica gel column (petroleum ether/ethyl acetate=1:1) to give 04-10 as a white solid (7 g, 68% yield.) MS M/z [ M+H ] + (ESI): 362.05

Compounds 04-11

Under nitrogen, 04-10 (1 g,2.77mmol,1 eq) was dissolved in DMF (10 mL,10 vol) and DIEA (714mg,5.54m mol,2eq), EDCI (1.062 g, 5.552 mmol,2 eq), 1-hydroxypyrrolidine-2, 5 eq)(318 mg,2.77mmol,1 eq) was stirred at room temperature for 16h. Dilute with dichloromethane (500 mL), wash with saturated sodium chloride, dry the organic phase over anhydrous sodium sulfate, concentrate to give the crude product, and purify on silica gel column (petroleum ether/ethyl acetate=3:1) to give 04-11 as a white solid (0.6 g, 47% yield.) MS M/z [ M+H]+(ESI):459.05

Compound 04-0

Under nitrogen, 04-8 (120 mg,0.131mmol,1 eq) was dissolved in DMF (0.36 mL,30 vol), triethylamine (39.69 mg,0.393mmol,3 eq) was added at room temperature and stirred for 15min, then 04-11 (72.0 mg,0.157mmol,1.2 eq) was added and stirred at room temperature for 2h, and the reaction solution was directly sent to Prep-HPLC purification (ACN/water with 0.05% NH) ₄ HCO ₃ ) The eluate was directly lyophilized to give 04-0 (7.6 mg, yield 10%). MS M/z [ M-H ]]-(ESI):1253.65

Example 3 Compound 3 and method for preparing the same

Synthetic route for compound 3

Compound 3 was synthesized by reference to the above route.

Example 4 Compound 4 and method for preparing the same

Synthetic route for compound 4

Compound 4 was synthesized by reference to the above route.

Application example evaluation of adjuvant with blister

1. Experimental protocol

Setting 6 groups of animal experiments, wherein the group 1-group 3 are liposome adjuvants matched with gE protein for carrying out the protection efficacy test of the pestilence vaccine, the dose of the adjuvants is 5 mug, the dose of the protein injection is 5 mug, the liposome composition is different except that the saponin is used, and the other auxiliary materials and the preparation process are consistent; group 4 was a 5 μg aluminum adjuvant with 5 μg protein experimental group; group 5 was a 5 μg single injection group; group 6 is a blank control group. The specific experimental set up schemes are shown in tables 1-2 below. Wherein the group 1 adjuvant: preparing a liposome adjuvant by using a TLR4 receptor agonist and commercial QS21 saponin; group 2 adjuvant: preparing a liposome adjuvant by using a TLR4 receptor agonist and the compound 1; group 3 adjuvant: the TLR4 receptor agonist and the compound 2 are used for preparing liposome adjuvant.

TABLE 1

TABLE 2

Animals	Pretreatment for immunization	Immunization
			A mouse	Varicella live virus infection	Intramuscular injection, 0.5ml

2. Experimental results:

after 14 days of immunization, each group of animals was euthanized, spleen immunocytes were taken and the expression of cytokines IFN-gamma, IL-2 was detected by flow cytometry. The detection results are shown in FIG. 1, and IFN-gamma and IL-2 expression of the adjuvant group 1-3, CD4+T cells is obviously enhanced.

It should be noted that the above-described embodiments are only for explaining the present invention and do not constitute any limitation of the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.

Claims

1. A triterpene glycoside saponin compound or pharmaceutically acceptable salt, ester or solvate thereof,

the triterpene glycoside saponin compound is shown as a formula I:

wherein,

R ₁₀ Selected from the following groups:

n is an integer from 0 to 10.

2. The triterpene glycoside saponin compound according to claim 1, wherein R ₁ To R ₉ Selected from the group consisting of hydrogen, deuterium, C1-C6 alkyl, C6-C10 aryl, C7-C10 aralkyl, C7-C10 alkaryl, C1-C6 aldehyde, and C1-C6 acyl; r is R ₁₁ To R ₁₈ Selected from the group consisting of hydrogen, deuterium, C1-C6 alkyl, C6-C10 aryl, C7-C10 aralkyl, C7-C10 alkaryl, C1-C6 aldehyde, and C1-C6 acyl;

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 ；

R ₁ Selected from hydrogen, deuterium, C1-C6 alkyl, phenyl and benzyl;

R ₂ and R is ₃ Selected from hydrogen, deuterium, C1-C6 alkyl and C1-C6 aldehyde groups;

R ₄ selected from hydrogen, deuterium, C1-C6 alkyl, phenyl and benzyl;

R ₅ selected from hydrogen, deuterium, and C1-C6 alkyl;

R ₆ selected from hydrogen, deuterium, and C1-C6 alkyl;

R ₇ selected from hydrogen, deuterium, and C1-C6 alkyl;

R ₈ selected from hydrogen, deuterium, and C1-C6 alkyl;

R ₉ selected from hydrogen, deuterium, and C1-C6 alkyl;

n is 0, 1, 2, 3, 4, 5, 6 or 7.

3. The triterpene glycoside saponin compound according to claim 1 or 2, wherein R ₁₀ Selected from the following groups:

wherein R is _a 、R _b 、R _c 、R _d 、R _e 、R _f Selected from hydrogen, deuterium, C1-C6 alkyl, halogen, cyano, C1-C6 carboxyl and C1-C6 ester groups, n is selected from integers from 0 to 10.

4. A triterpene glycoside saponin compound according to any of claims 1-3, wherein R _c Selected from halogen (e.g. bromine or iodine), cyano, carboxyl (e.g. COOH) or ester (e.g. -COOCH, -CH) ₂ OOCH ₃ )。

5. The triterpene glycoside saponin compound according to any one of claims 1 to 4, wherein the compound of formula I is represented by formula II, formula II-1 or formula II-2:

the definition of each substituent is the same as that of the formula I.

6. The triterpene glycoside compound according to claim 1, wherein the compound of formula I is selected from the group consisting of:

7. a process for preparing the triterpene glycoside saponin compound according to any one of claims 1 to 6, comprising one or more of the following steps:

R ₂ 、R ₃ and R is ₁₀ Is defined as in formula I.

8. The preparation method according to claim 7, wherein,

in the step (1), the oxidant is osmium tetroxide/NMO, and the solvent is acetone/water; and/or

In step (2), TIPSCl is adopted to react in a solvent in the presence of pyridine organic base, preferably DMAP; and/or

In step (3), the compound of formula I-4 is reacted with Ph in methylene chloride in the presence of a pyridine-based organic base ₂ SO and Tf ₂ O is reacted at a temperature of-50℃to-30℃for, for example, 0.5 to 1.5 hours; then adding a compound of formula I-5, first reacting at a temperature of-50 ℃ to-30 ℃ for, for example, 0.5 hours to 1.5 hours, followed by a reaction at a temperature of-5 ℃ to 5 ℃ for, for example, 20 minutes to 40 minutes; and/or

In step (3), the pyridine-based organic base comprises 2, 6-di-tert-butyl-4-methylpyridine or 2,4, 6-tri-tert-butylpyridine, and/or

In the step (4), the reagents used are hydrofluoric acid and triethylamine, and the temperature is 20-40 ℃; and/or

In step (5), the compound of formula I-7 is reacted with Ph in methylene chloride in the presence of a pyridine-based organic base ₂ SO and Tf ₂ O is reacted at a temperature of-78 ℃ to-30 ℃ for, for example, 20 minutes to 40 minutes; then adding a compound of formula I-3, reacting at a temperature of-50 ℃ to-30 ℃ for, for example, 1 hour to 3 hours; and/or

In step (5), the pyridine-based organic base includes 2, 6-di-t-butyl-4-methylpyridine or 2,4, 6-tri-t-butylpyridine; and/or

In step (6), the reagents used are TBAF and acetic acid; the solvent is an ether solvent, preferably THF; the reaction temperature is 20-40 ℃; and/or

In step (7), the reagent used is Cl ₃ CN and DBU; the solvent is a halogenated hydrocarbon solvent, preferably dichloromethane; the reaction temperature is-5 ℃ to 5 ℃; and/or

In step (8), the reagents used are TESOTf and pyridine-based organic bases (preferably 2, 6-lutidine); the solvent is a halogenated hydrocarbon solvent, preferably dichloromethane; the reaction temperature is 20 ℃ to 40 ℃; and/or

In the step (9), the reagent used is boron trifluoride diethyl etherate solution; the solvent is a halogenated hydrocarbon solvent, preferably dichloromethane; the reaction temperature is-78 ℃ to-40 ℃, preferably-55 ℃ to-45 ℃; and/or

In step (10), the reagent used is Pd/C and/or Pd (OH) ₂ Hydrogen; the reaction solvent is tetrahydrofuran and/or methanol; the temperature is 20 ℃ to 40 ℃; and/or

In the step (11), the reagent used is trifluoroacetic acid, the solvent can be alcohol or water, and the reaction temperature is-5 ℃ to 5 ℃; and/or

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 is 20℃to 40 ℃.

9. A process for the preparation of a compound of formula I-2 comprising converting a compound of formula I-1 to a compound of formula I-2 by reaction

Wherein the oxidant is osmium tetroxide/NMO, and the solvent is acetone/water.

10. A process for the preparation of a compound of formula I-6 comprising converting a compound of formula I-4 and a compound of formula I-5 by reaction to a compound of formula I-6

11. A process for the preparation of a compound of formula I-8 comprising converting a compound of formula I-7 and a compound of formula I-3 by reaction to a compound of formula I-8,

12. Use of the triterpenoid glycoside saponin compound according to any one of claims 1-6 in a vaccine, preferably as an adjuvant.