Synthetic saponin compositions and methods of synthesis thereof

Abstract

Synthetic saponin compositions and methods of their synthesis are described. In particular, synthetic saponin compositions comprising at least 90% saponin QS-21-Api and less than 1.0% isomer B as determined by UV absorbance at 200 nm and methods of their synthesis are described:

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology. Specifically, this invention relates to synthetic saponin compositions, their synthesis methods, compositions containing the same, their high-performance liquid chromatography detection methods, and their uses. Background Technology

[0002] QS-21 (Quillaja Saponin-21) is an immunostimulatory saponin compound extracted from the natural plant *Quillajasaponaria molina*. As a highly effective vaccine adjuvant, QS-21 has been used in several approved vaccines.

[0003] In 2017, Shingrix, a shingles vaccine containing QS-21, was approved for marketing in the United States.

[0004] In 2021, the malaria vaccine RTS,S (Mosquirix), containing QS-21, was approved by the World Health Organization (WHO). This is the world's first vaccine against this deadly disease (malaria is a disease transmitted to humans through the bite of female Anopheles mosquitoes infected with the Plasmodium parasite).

[0005] In 2021, the COVID-19 vaccine Covevax, which contains QS-21, was launched.

[0006] In 2023, Arexvy, a respiratory syncytial virus vaccine containing QS-21, was launched.

[0007] In 2023, the malaria vaccine R21, which contains QS-21, was launched.

[0008] Naturally extracted QS-21 is a mixture containing two main components, QS-21-Api and QS-21-Xyl (in a ratio of approximately 65:35).

[0009]

[0010] Obtaining a single structure QS-21-Api from natural plants is extremely difficult, and this difficulty hinders the application of single-structure QS-21-Api.

[0011] In 2005, David Y. Gin et al. reported the chemical synthesis of a single structure QS-21-Api, but the amount of QS-21-Api obtained by their method was limited, and they used very expensive reagents (such as the glycosylation reagent 2,4,6-tri-tert-butylpyridine). Some reactions were not suitable for scale-up, so this method is not suitable for large-scale industrial production.

[0012] To date, all commercially available vaccines use QS-21 mixtures extracted from natural plants, and the field is still seeking a synthetic method suitable for the industrial production of a single-structure QS-21-Api. Invention Overview

[0014] On one hand, the present invention provides a synthetic saponin composition, wherein the synthetic saponin composition comprises at least 90% saponin QS-21-Api and less than 1.0% isomer B, as detected by ultraviolet absorbance at 200 nm.

[0015]

[0016] On the other hand, the present invention provides a pharmaceutical composition comprising the synthetic saponin composition of the present invention and a pharmaceutically acceptable excipient.

[0017] On the other hand, the present invention provides an adjuvant composition comprising the synthetic saponin composition of the present invention.

[0018] On the other hand, the present invention provides an immunogenic composition comprising the adjuvant composition of the present invention and an antigen.

[0019] On the other hand, the present invention provides the use of the synthetic saponin compositions of the present invention in the preparation of anticancer drugs, adjuvants or immunogenic compositions.

[0020] On the other hand, the present invention provides synthetic saponin compositions of the present invention for the treatment of cancer, or as adjuvants or immunogenic drugs.

[0021] On the other hand, the present invention provides a method for treating cancer, comprising administering a therapeutically effective amount of the synthetic saponin composition of the present invention.

[0022] On the other hand, the present invention provides an immunization method comprising administering a therapeutically effective amount of the synthetic saponin composition of the present invention.

[0023] On the other hand, the present invention provides an HPLC method for determining the main peak and isomer B content of saponin QS-21-Api in synthetic saponin compositions, the method comprising:

[0024] The stationary phase was AQ C18 with a particle size of 5 μm. Mobile phase A was water-phosphoric acid (1000:1), and mobile phase B was acetonitrile. The elution conditions were as follows:

[0025] 0 59 41 45 59 41

[0026] The detection wavelength is 200nm.

[0027] On the other hand, the present invention provides a method for synthesizing the synthetic saponin composition of the present invention, which includes the following steps:

[0028] (a) Compound I-2 is obtained by glycosylation of compound I-1 with thioglycoside SAP-2;

[0029]

[0030] (b) Deacetyl protection of compound I-2 is obtained to obtain compound I-3, and compound I-3 is glycosylated with thioglycoside SAP-5 to obtain compound I-4.

[0031]

[0032] (c) The synthetic saponin composition is obtained by reacting the compounds of formula I-4.

[0033] Each of R2 is an independent hydroxyl protecting group, preferably benzyl.

[0034] Or, two R2 atoms together with the oxygen atom they are attached to form... Structure, where R x and R y Each independently represents H and C. 1-6 Alkyl and phenyl, preferably H, methyl or phenyl;

[0035] R4 is selected from C 1-6 Alkyl groups and optionally C 1-6 Alkyl-substituted phenyl groups are preferably methyl, ethyl, propyl, phenyl, p-methylphenyl, o-methylphenyl, or m-methylphenyl; and

[0036] Pg1, Pg2, and Pg3 are each independently a hydroxyl protecting group. Preferably, Pg1 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, or tert-butyldiphenylsilyl; Pg2 is selected from benzyl, C 1-6 Alkylbenzyl, naphthylmethyl, or triphenylmethyl; Pg3 is selected from acetyl, pivaloyl, or benzoyl, more preferably, Pg1 is triisopropylsilyl, Pg2 is benzyl, and Pg3 is acetyl.

[0037] On the other hand, the present invention provides a method for synthesizing the synthetic saponin composition of the present invention, which includes the following steps:

[0038] (a') Protecting the hydroxyl group of the compound of formula II-1-1 to obtain the compound of formula II-1, and then removing the Pg4 protecting group from the compound of formula II-1 to obtain the compound of formula II-2;

[0039]

[0040] (b') React the compound of formula II-2 with a halogen-containing electrophilic reagent to generate an imine ester of formula II-2-1, and then glycosylate the imine ester of formula II-2-1 with compound SAP-18 to obtain compound II-3.

[0041]

[0042] (c') The synthetic saponin composition is obtained by reacting the compound of formula II-3.

[0043] R6 is an independent hydroxyl protecting group, preferably benzyl.

[0044] Or two R6 atoms together with the oxygen atom they are attached to form Structure, where R x and R y Each independently represents H and C. 1-6 Alkyl and phenyl, preferably H, methyl or phenyl;

[0045] Pg4 and Pg5 are each independently a hydroxyl protecting group. Preferably, Pg4 is selected from triisopropylsilyl, tert-butyldimethylsilyl or trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl; Pg5 is selected from benzyl, C 1-6 Alkylbenzyl, naphthylmethyl, or triphenylmethyl, more preferably, Pg4 is triisopropylsilyl and Pg5 is benzyl;

[0046] R3 is allyl, methylthiomethyl, 3,4-dimethoxybenzyl, p-methoxybenzyl, 2,3,4-trimethoxybenzyl, 3,4,5-trimethoxybenzyl, 2,5-dimethoxybenzyl, 2-methoxybenzyl, 3,5-dimethoxybenzyl, 2,3-dimethoxybenzyl, 3-methoxybenzyl, 2,6-dimethoxybenzyl, 1-naphthylmethyl, or 2-naphthylmethyl;

[0047] R5 is Where X is a halogen, preferably chlorine or fluorine; and R z R5 is H or phenyl, more preferably, R5 is

[0048] Each of Y1 is an independent hydroxyl protecting group. Preferably, each of Y1 is independently selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl or benzyl. More preferably, Y1 is benzyl.

[0049] Y2 is a hydroxyl protecting group. Preferably, Y2 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl or benzyl. More preferably, Y2 is selected from triethylsilyl or benzyl.

[0050] On the other hand, the present invention provides a method for synthesizing the intermediate SAP-18, which includes the following steps:

[0051] (a”) Selectively remove the TBS protecting group from the compound of formula III-1 to obtain compound III-2, and perform a glycosylation reaction between the compound of formula III-2 and compound III-3 to obtain compound III-4;

[0052]

[0053] (b”) React the compound of formula III-4 with a basic reagent or a reducing reagent to generate compound III-5, and react the compound of formula III-5 with a silyl ether reagent or benzyl bromide to obtain compound III-6;

[0054]

[0055] (c”) React the compound of formula III-6 with an acidic reagent to generate compound III-7, react the compound of formula III-7 with an oxidizing agent to obtain compound III-8, and react the compound of formula III-8 with a silyl ether reagent or benzyl bromide to obtain compound III-9.

[0056]

[0057] (d”) The compound of formula III-9 is reacted with a metal catalyst to remove the protecting group to obtain compound III-10, and the compound of formula III-10 is reacted with a halogen-containing electrophilic reagent to obtain compound III-11.

[0058]

[0059] (e”) The synthetic saponin composition is obtained by reacting the compound of formula III-11.

[0060] Wherein, each of Y1 is an independent hydroxyl protecting group, preferably, each of Y1 is independently selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl or benzyl, more preferably, Y1 is benzyl;

[0061] R9 is Where X is a halogen, preferably chlorine or fluorine; and R z R9 is H or phenyl, more preferably, R9 is

[0062] R8 is allyl or 2-butenyl, preferably allyl; and R 10 It is either acetyl or benzoyl.

[0063] The method of this invention can synthesize QS-21-Api products with low isomer B content (i.e., the synthetic saponin composition), avoiding the use of expensive reagents (for example, the glycosylation method using thioglycosides (SAP-2, SAP-5) as donors in the above reaction formula avoids the use of glycosylation reagent 2,4,6-tri-tert-butylpyridine), greatly reducing production costs), making it suitable for industrial production and achieving large-scale synthesis. Furthermore, the QS-21-Api product obtained thereby has high purity and low isomer B content, which helps to reduce safety risks. Attached Figure Description

[0064] Figure 1 This is the synthetic QS-21-Api product (i.e., the synthetic saponin composition) obtained in Preparation Example 10 of the present invention. 1 H-NMR spectrum.

[0065] Figure 2 This is the mass spectrometry of the synthetic QS-21-Api product (i.e., the synthetic saponin composition) obtained in Preparation Example 10 of the present invention.

[0066] Figure 3 This is a high-performance liquid chromatogram of the synthetic QS-21-Api product (i.e., the synthetic saponin composition) obtained in Preparation Example 10 of the present invention.

[0067] Figure 4 The results are from a mouse immune response test using the synthetic QS-21-Api product (i.e., the synthetic saponin composition) obtained in Preparation Example 10 of the present invention as an adjuvant. Invention Details

[0069] definition

[0070] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques that are obvious to one of ordinary skill in the art. While it is believed that the following terms will be well understood by one of ordinary skill in the art, the following definitions are set forth to better explain the invention.

[0071] In this invention, combinations of substituents and variables are permitted only if such combinations result in chemically stable compounds. When a substituent is replaced by two or more groups, these multiple groups can exist on the same or different carbon atoms, provided a stable structure is produced.

[0072] As used herein, the terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0073] In this invention, the subscript number of the carbon atom "C" indicates the number of carbon atoms. For example, C1 represents 1 carbon atom, C2 represents 2 carbon atoms, and C... p-q This represents pq (0 < p < q) carbon atoms. The group name following the carbon atom "C" indicates the type of group; for example, C1 alkyl represents methyl, C2 alkenyl represents vinyl, and C... p-q Alkyl groups are alkyl groups with a carbon number of pq.

[0074] As used in this article, the term "halogenated" or "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0075] As used in this article, the term "C" 1-6 "Alkyl" refers to a straight-chain or branched aliphatic saturated hydrocarbon group with 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, etc.

[0076] Solid lines may be used in this article. wavy lines solid wedge Or virtual wedge Depicting the chemical bonds of the compounds of the present invention. Solid lines are used to depict bonds to asymmetric atoms, indicating all possible stereoisomers at that atom (e.g., specific enantiomers, racemic mixtures, etc.). Wavy lines are used to depict bonds to asymmetric atoms, indicating that the bond is a solid wedge. Or virtual wedge Any type of bond. Using real or imaginary wedges to depict the bonds connecting to asymmetric atoms, it is intended to show the existence of the stereoisomers shown.

[0077] The term “about” means within ±10% of the stated value, preferably within ±5%, and more preferably within ±2%.

[0078] As used herein, the terms “synthetic saponin composition” and “synthetic QS-21-Api product” and “QS-21-Api product” are used interchangeably and all refer to the high-purity QS-21-Api product synthesized by the method of the present invention, which contains extremely low levels of isomer B, thus helping to reduce safety risks.

[0079] As used in this article, the term "glycosylation" refers to the chemical process in which a sugar molecule (donor) covalently bonds with a sugar or non-sugar molecule (acceptor, such as sugar, protein, lipid or other small molecule) through a glycosidic bond (CO, CN, CS, etc.).

[0080] As used herein, the term "hydroxyl protecting group" is a chemical group used in organic synthesis to temporarily shield the activity of the hydroxyl group (-OH) and prevent it from undergoing side reactions in multi-step reactions. Examples of protecting groups described, for instance, are those described in *Protective Groups in Organic Chemistry*, ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene & P. ​​G.W. Uts, *Protective Groups in Organic Synthesis*, John Wiley & Sons, 1991, which are incorporated herein by reference. Protecting groups can be removed at appropriate subsequent stages using methods known in the art. Such hydroxyl protecting groups include, but are not limited to, methyl, benzyl, triphenylmethyl, methoxymethyl, acetyl, benzoyl, p-toluenesulfonyl, trimethylsilyl, tert-butyldimethylsilyl, triisopropylsilyl, etc.

[0081] As used herein, the term "halogenated electrophilic reagent" includes trichloroacetonitrile, 2,2,2-trifluoro-N-phenylacetylimide chloride, etc.

[0082] As used herein, the term "silicone ether reagent" includes trimethylsilicon chloride, triethylsilicon chloride, triisopropylsilicon chloride, tert-butyldimethylsilicon chloride, tert-butyldiphenylsilicon chloride, etc.

[0083] Synthetic saponin compositions

[0084] One object of the present invention is to provide a synthetic saponin composition, as detected by ultraviolet absorbance at 200 nm, wherein the synthetic saponin composition comprises at least 90% saponin QS-21-Api and less than 1.0% isomer B.

[0085]

[0086] In some embodiments, the synthetic saponin composition comprises 90-99.8% of saponin QS-21-Api.

[0087] In some embodiments, the synthetic saponin composition comprises 93-99.5% saponin QS-21-Api.

[0088] In some embodiments, the synthetic saponin composition contains 95-99% saponin QS-21-Api.

[0089] In some embodiments, the synthetic saponin composition contains 95-98% saponin QS-21-Api.

[0090] In some embodiments, the synthetic saponin composition contains less than 0.5% isomer B.

[0091] In some embodiments, the synthetic saponin composition contains less than 0.1% isomer B.

[0092] In some embodiments, the synthetic saponin composition contains less than 0.05% isomer B.

[0093] Isomer B

[0094] Isomer B is an isomer of QS-21-Api that readily transforms in solution; the two are tautomers. Although literature reports (Journal of Pharmaceutical Sciences, 1996, 22-28) indicate that isomer B and QS-21 can elicit a comparable immune response, actual studies have shown that higher levels of isomer B can affect some properties of liposomes, such as causing instability and precipitation. Furthermore, controlling the content of isomer B can reduce uncertain safety risks; therefore, controlling the content of isomer B is crucial. The saponin synthesis provided by this invention contains at least 90% of the QS-21-Api main peak and less than 1.0% of isomer B.

[0095] Synthesis method of synthetic saponin composition

[0096] Synthesis Method 1

[0097] Another object of the present invention is to provide a method for synthesizing the synthetic saponin composition of the present invention (synthesis method 1), which includes the following steps:

[0098] (a) Compound I-2 is obtained by glycosylation of compound I-1 with thioglycoside SAP-2;

[0099]

[0100] (b) The Pg3 protecting group was removed from the compound of formula I-2 to obtain the compound of formula I-3, and the compound of formula I-3 was subjected to a glycosylation reaction with the thioglycoside SAP-5 to obtain the compound of formula I-4.

[0101]

[0102] (c) The synthetic saponin composition is obtained by reacting the compounds of formula I-4.

[0103] Each R2 group is an independent hydroxyl protecting group, or two R2 groups together with the oxygen atom they are attached to form a protective group. Structure, where R x and R y Each independently represents H and C. 1-6 Alkyl and phenyl;

[0104] R4 is selected from C 1-6 Alkyl groups and optionally C 1-6 Alkyl-substituted phenyl;

[0105] Pg1, Pg2, and Pg3 are each independently protected by a hydroxyl group.

[0106] In some implementations, R2 is benzyl.

[0107] In some implementations, the two R2 atoms together with the oxygen atoms they are attached to form Structure, where R x and R y Each can be independently H, methyl, or phenyl.

[0108] In some implementations, R4 is methyl, ethyl, propyl, phenyl, p-methylphenyl, o-methylphenyl, or m-methylphenyl.

[0109] In some embodiments, Pg1 is selected from triisopropylsilyl, tert-butyldimethylsilyl or trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl; Pg2 is selected from benzyl, C 1-6 Alkyl-substituted benzyl, naphthyl, or triphenylmethyl; Pg3 is selected from acetyl, pivaloyl, or benzoyl.

[0110] In some implementations, Pg1 is triisopropylsilyl, Pg2 is benzyl, and Pg3 is acetyl.

[0111] In some embodiments, step (a) is carried out in the presence of the following reagents: silver trifluoromethanesulfonate, silver oxide, silver carbonate, silver nitrate, silver perchlorate, mercuric chloride, mercuric bromide, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, trifluoromethanesulfonic anhydride, trifluoromethanesulfonic acid, boron trifluoride ether, scandium trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, copper trifluoromethanesulfonate, ferric trifluoromethanesulfonate, ferric chloride, aluminum trichloride, tin tetrachloride, stannous chloride, zinc chloride, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, N-iodosuccinimide, N-bromosuccinimide, or any combination thereof.

[0112] In some embodiments, step (a) is carried out in the presence of a combination of N-iodosuccinimide and silver trifluoromethanesulfonate.

[0113] In some embodiments, step (a) is performed at a temperature of -100 to 0°C, preferably -80 to -50°C, and more preferably -75 to -60°C.

[0114] In some embodiments, the deprotection reaction described in step (b) is carried out under alkaline conditions. For example, the deprotection reaction described in step (b) is carried out in the presence of sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium tert-butoxide, sodium aminoacetate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, barium hydroxide, calcium hydroxide, magnesium hydroxide, lithium hydroxide, hydrazine hydrate, ammonia methanol, ammonia ethanol, or ammonia solution.

[0115] In some embodiments, the deprotection reaction described in step (b) is carried out at a temperature of 0–80°C, preferably 10–40°C, and more preferably 20–30°C.

[0116] In some embodiments, the glycosylation reaction described in step (b) is carried out in the presence of the following reagents: silver trifluoromethanesulfonate, silver oxide, silver carbonate, silver nitrate, silver perchlorate, mercuric chloride, mercuric bromide, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, trifluoromethanesulfonic anhydride, trifluoromethanesulfonic acid, boron trifluoride ether, scandium trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, copper trifluoromethanesulfonate, ferric trifluoromethanesulfonate, ferric chloride, aluminum trichloride, tin tetrachloride, stannous chloride, zinc chloride, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, N-iodosuccinimide, N-bromosuccinimide, or any combination thereof.

[0117] In some embodiments, the glycosylation reaction described in step (b) is carried out in the presence of a combination of N-iodosuccinimide and trimethylsilyl trifluoromethanesulfonate.

[0118] In some embodiments, the glycosylation reaction described in step (b) is carried out at a temperature of -80 to 0°C, preferably -70 to -30°C, and more preferably -60 to -40°C.

[0119] According to some embodiments of the present invention, step (c) includes:

[0120] (c1) The Pg1 protecting group of compound I-4 is removed to obtain compound I-5. Compound I-5 is reacted with trichloroacetonitrile to generate trichloroacetylimine ester. The trichloroacetylimine ester is glycosylated with compound SAP-9 to obtain compound I-6. Compound I-6 is removed from the Pg3 protecting group to obtain compound I-7.

[0121]

[0122] In some implementations, step (c) further includes:

[0123] (c2) The compound of formula I-7 is subjected to an esterification reaction with the compound of formula I-9 to obtain the compound of formula I-10;

[0124]

[0125] In some implementations, step (c) further includes:

[0126] (c3) Remove the Pg1 protecting group from the compound of formula I-10 to obtain the compound of formula I-11, react the compound of formula I-11 with trichloroacetonitrile to generate trichloroacetylimine ester, and glycosylate the trichloroacetylimine ester with compound SAP-18 to obtain the compound of formula I-12.

[0127]

[0128] In some implementations, step (c) further includes:

[0129] (c4) Remove all protecting groups from the compound of formula I-12 to obtain the synthetic saponin composition;

[0130]

[0131] In steps (c1) to (c4) above,

[0132] R1 is an independent hydroxyl protecting group;

[0133] R2 can be an independent hydroxyl protecting group, or two R2 groups together with the oxygen atom they are attached to form a protective group. Structure, where R x and R y Each independently represents H and C. 1-6 Alkyl and phenyl;

[0134] Pg1, Pg2, and Pg3 are each independently protected by a hydroxyl group;

[0135] Y1 is an independent hydroxyl protecting group.

[0136] Y2 is a hydroxyl protecting group.

[0137] In some embodiments, R1 is independently selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, benzyl, C 1-6 Alkyl benzyl or naphthylmethyl.

[0138] In some embodiments, R1 is independently selected from tert-butyldimethylsilyl or benzyl.

[0139] In some implementations, R2 is benzyl.

[0140] In some implementations, the two R2 atoms together with the oxygen atoms they are attached to form Structure, where R x and R y Each can be independently H, methyl, or phenyl.

[0141] In some embodiments, Pg1 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, or tert-butyldiphenylsilyl; Pg2 is selected from benzyl, C 1-6 Alkylbenzyl, naphthylmethyl, or triphenylmethyl; Pg3 is selected from acetyl, pivaloyl, or benzoyl.

[0142] In some implementations, Pg1 is triisopropylsilyl, Pg2 is benzyl, and Pg3 is acetyl.

[0143] In some embodiments, Y1 is independently selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, or benzyl.

[0144] In some implementations, Y1 is benzyl.

[0145] In some embodiments, Y2 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, or benzyl.

[0146] In some embodiments, Y2 is selected from triethylsilyl or benzyl.

[0147] In some embodiments, the deprotection reaction of the Pg1 protecting group described in step (c1) is carried out in the presence of a fluoride ion reagent or a fluorine-containing reagent. The fluoride ion reagent is, for example, but not limited to, TBAF, hydrogen fluoride, triethylamine hydrogen fluoride, cesium fluoride, potassium fluoride, trifluoroacetic acid, and trifluoromethanesulfonic acid.

[0148] In some embodiments, the reaction to remove the Pg1 protecting group described in step (c1) is carried out at a temperature of 0–80°C, preferably 10–40°C, and more preferably 20–30°C.

[0149] In some embodiments, the glycosylation reaction described in step (c1) is carried out in the presence of silver trifluoromethanesulfonate, silver oxide, silver carbonate, silver nitrate, silver perchlorate, mercuric chloride, mercuric bromide, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, trifluoromethanesulfonic anhydride, trifluoromethanesulfonic acid, boron trifluoride ether, scandium trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, copper trifluoromethanesulfonate, ferric trifluoromethanesulfonate, ferric chloride, aluminum trichloride, tin tetrachloride, stannous chloride, zinc chloride, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, or any combination thereof.

[0150] In some embodiments, the glycosylation reaction described in step (c1) is carried out in the presence of trimethylsilyl trifluoromethanesulfonate.

[0151] In some embodiments, the glycosylation reaction described in step (c1) is carried out at a temperature of -20 to 20°C, preferably -10 to 10°C, and more preferably 0 to 5°C.

[0152] In some embodiments, the deprotection reaction of the Pg3 protecting group described in step (c1) is carried out under alkaline conditions. For example, the deprotection reaction of the Pg3 protecting group described in step (c1) is carried out in the presence of sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium tert-butoxide, sodium amide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, barium hydroxide, calcium hydroxide, magnesium hydroxide, lithium hydroxide, hydrazine hydrate, ammonia methanol, ammonia ethanol, or ammonia solution.

[0153] In some embodiments, the reaction to remove the Pg3 protecting group described in step (c1) is carried out at a temperature of 0–80°C, preferably 10–60°C, and more preferably 30–50°C.

[0154] In some embodiments, the esterification reaction described in step (c2) is carried out in the presence of 2,4,6-trichlorobenzoyl chloride and 4-dimethylaminopyridine.

[0155] In some embodiments, the esterification reaction described in step (c2) is carried out at a temperature of 0–80°C, preferably 10–40°C, and more preferably 25–35°C.

[0156] In some embodiments, the deprotection reaction of the Pg1 protecting group described in step (c3) is carried out in the presence of a fluoride ion reagent or a fluorine-containing reagent. The fluoride ion reagent is, for example, but not limited to, TBAF, hydrogen fluoride, triethylamine hydrogen fluoride, cesium fluoride, potassium fluoride, trifluoroacetic acid, and trifluoromethanesulfonic acid.

[0157] In some embodiments, the reaction to remove the Pg1 protecting group described in step (c3) is carried out at a temperature of -30 to 0°C, preferably -20 to 0°C, and more preferably -15 to -5°C.

[0158] In some embodiments, the reaction of the compound of formula I-11 described in step (c3) with trichloroacetonitrile is carried out at a temperature of 0–40°C, preferably 10–30°C, and more preferably 15–20°C.

[0159] In some embodiments, the glycosylation reaction described in step (c3) is carried out in the presence of the following reagents: silver trifluoromethanesulfonate, silver oxide, silver carbonate, silver nitrate, silver perchlorate, mercuric chloride, mercuric bromide, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, trifluoromethanesulfonic anhydride, trifluoromethanesulfonic acid, boron trifluoride ether, scandium trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, copper trifluoromethanesulfonate, ferric trifluoromethanesulfonate, ferric chloride, aluminum trichloride, tin tetrachloride, stannous chloride, zinc chloride, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, or any combination thereof.

[0160] In some embodiments, the glycosylation reaction described in step (c3) is carried out in the presence of boron trifluoride diethyl ether.

[0161] In some embodiments, the glycosylation reaction described in step (c3) is carried out at a temperature of -100 to -20°C, preferably -90 to -40°C, and more preferably -80 to -60°C.

[0162] In some embodiments, step (c4) is carried out under hydrogenation conditions. Preferably, step (c4) is carried out in the presence of a hydrogenation catalyst. Preferably, the hydrogenation catalyst is palladium on carbon or palladium hydroxide on carbon.

[0163] In some embodiments, step (c4) is carried out in the presence of an acid. Preferably, the acid is formic acid, acetic acid, propionic acid, benzoic acid, benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, citric acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, or trifluoroacetic acid. More preferably, the acid is trifluoroacetic acid.

[0164] In some embodiments, the hydrogenation in step (c4) is preceded by a step of treating the compound of formula I-12 with an acid. Preferably, the acid is trifluoroacetic acid. Preferably, this step is carried out at a temperature of -20 to 20°C, more preferably -10 to -10°C, and more preferably -5 to 5°C.

[0165] According to some embodiments of the present invention, the synthesis method 1 of the present invention includes the following steps:

[0166] (a) Compound I-2 is obtained by glycosylation of compound I-1 with thioglycoside SAP-2;

[0167]

[0168] (b) The Pg3 protecting group was removed from the compound of formula I-2 to obtain the compound of formula I-3, and the compound of formula I-3 was subjected to a glycosylation reaction with the thioglycoside SAP-5 to obtain the compound of formula I-4.

[0169]

[0170] (c1) The Pg1 protecting group of compound I-4 is removed to obtain compound I-5. Compound I-5 is reacted with trichloroacetonitrile to generate trichloroacetylimine ester. The trichloroacetylimine ester is glycosylated with compound SAP-9 to obtain compound I-6. Compound I-6 is deacetylated to obtain compound I-7.

[0171] (c2) The compound of formula I-7 is subjected to an esterification reaction with the compound of formula I-9 to obtain the compound of formula I-10;

[0172]

[0173] (c3) Remove the Pg1 protecting group from the compound of formula I-10 to obtain the compound of formula I-11, react the compound of formula I-11 with trichloroacetonitrile to generate trichloroacetylimine ester, and glycosylate the trichloroacetylimine ester with compound SAP-18 to obtain the compound of formula I-12.

[0174]

[0175] (c4) Remove all protecting groups from the compound of formula I-12 to obtain the synthetic saponin composition;

[0176] Each of R1 is an independent hydroxyl protecting group.

[0177] R2 can be an independent hydroxyl protecting group, or two R2 groups together with the oxygen atom they are attached to form a protective group. Structure, where R x and R y Each independently represents H and C. 1-6 Alkyl and phenyl;

[0178] R4 is selected from C 1-6 Alkyl groups and optionally C 1-6 Alkyl-substituted phenyl;

[0179] Pg1, Pg2, and Pg3 are each independently protected by a hydroxyl group;

[0180] Y1 is an independent hydroxyl protecting group;

[0181] Y2 is a hydroxyl protecting group.

[0182] In some embodiments, R1 is independently selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, benzyl, C 1-6Alkyl benzyl or naphthylmethyl.

[0183] In some embodiments, R1 is independently selected from tert-butyldimethylsilyl or benzyl.

[0184] In some implementations, R2 is benzyl.

[0185] In some implementations, the two R2 atoms together with the oxygen atoms they are attached to form Structure, where R x and R y Each can be H, methyl, or phenyl independently.

[0186] In some implementations, R4 is methyl, ethyl, propyl, phenyl, p-methylphenyl, o-methylphenyl, or m-methylphenyl.

[0187] In some embodiments, Pg1 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, or tert-butyldiphenylsilyl; Pg2 is selected from benzyl, C 1-6 Alkylbenzyl, naphthylmethyl, or triphenylmethyl; Pg3 is selected from acetyl, pivaloyl, or benzoyl.

[0188] In some implementations, Pg1 is triisopropylsilyl, Pg2 is benzyl, and Pg3 is acetyl.

[0189] In some embodiments, Y1 is independently selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, or benzyl.

[0190] In some implementations, Y1 is benzyl.

[0191] In some embodiments, Y2 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, or benzyl.

[0192] In some embodiments, Y2 is selected from triethylsilyl or benzyl.

[0193] In some embodiments, step (a) is carried out in the presence of the following reagents: silver trifluoromethanesulfonate, silver oxide, silver carbonate, silver nitrate, silver perchlorate, mercuric chloride, mercuric bromide, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, trifluoromethanesulfonic anhydride, trifluoromethanesulfonic acid, boron trifluoride ether, scandium trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, copper trifluoromethanesulfonate, ferric trifluoromethanesulfonate, ferric chloride, aluminum trichloride, tin tetrachloride, stannous chloride, zinc chloride, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, N-iodosuccinimide, N-bromosuccinimide, or any combination thereof.

[0194] In some embodiments, step (a) is carried out in the presence of a combination of N-iodosuccinimide and silver trifluoromethanesulfonate.

[0195] In some embodiments, step (a) is performed at a temperature of -100 to 0°C, preferably -80 to -50°C, and more preferably -75 to -60°C.

[0196] In some embodiments, the deprotection reaction described in step (b) is carried out under alkaline conditions. For example, the deprotection reaction described in step (b) is carried out in the presence of sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium tert-butoxide, sodium aminoacetate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, barium hydroxide, calcium hydroxide, magnesium hydroxide, lithium hydroxide, hydrazine hydrate, ammonia methanol, ammonia ethanol, or ammonia solution.

[0197] In some embodiments, the deprotection reaction described in step (b) is carried out at a temperature of 0–80°C, preferably 10–40°C, and more preferably 20–30°C.

[0198] In some embodiments, the glycosylation reaction described in step (b) is carried out in the presence of the following reagents: silver trifluoromethanesulfonate, silver oxide, silver carbonate, silver nitrate, silver perchlorate, mercuric chloride, mercuric bromide, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, trifluoromethanesulfonic anhydride, trifluoromethanesulfonic acid, boron trifluoride ether, scandium trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, copper trifluoromethanesulfonate, ferric trifluoromethanesulfonate, ferric chloride, aluminum trichloride, tin tetrachloride, stannous chloride, zinc chloride, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, N-iodosuccinimide, N-bromosuccinimide, or any combination thereof.

[0199] In some embodiments, the glycosylation reaction described in step (b) is carried out in the presence of a combination of N-iodosuccinimide and trimethylsilyl trifluoromethanesulfonate.

[0200] In some embodiments, the glycosylation reaction described in step (b) is carried out at a temperature of -80 to 0°C, preferably -70 to -30°C, and more preferably -60 to -40°C.

[0201] In some embodiments, the deprotection reaction of the Pg1 protecting group described in step (c1) is carried out in the presence of a fluoride ion reagent or a fluorine-containing reagent. The fluoride ion reagent is, for example, but not limited to, TBAF, hydrogen fluoride, triethylamine hydrogen fluoride, cesium fluoride, potassium fluoride, trifluoroacetic acid, and trifluoromethanesulfonic acid.

[0202] In some embodiments, the reaction to remove the Pg1 protecting group described in step (c1) is carried out at a temperature of 0–80°C, preferably 10–40°C, and more preferably 20–30°C.

[0203] In some embodiments, the glycosylation reaction described in step (c1) is carried out in the presence of silver trifluoromethanesulfonate, silver oxide, silver carbonate, silver nitrate, silver perchlorate, mercuric chloride, mercuric bromide, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, trifluoromethanesulfonic anhydride, trifluoromethanesulfonic acid, boron trifluoride ether, scandium trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, copper trifluoromethanesulfonate, ferric trifluoromethanesulfonate, ferric chloride, aluminum trichloride, tin tetrachloride, stannous chloride, zinc chloride, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, or any combination thereof.

[0204] In some embodiments, the glycosylation reaction described in step (c1) is carried out in the presence of trimethylsilyl trifluoromethanesulfonate.

[0205] In some embodiments, the glycosylation reaction described in step (c1) is carried out at a temperature of -20 to 20°C, preferably -10 to 10°C, and more preferably 0 to 5°C.

[0206] In some embodiments, the deprotection reaction of the Pg3 protecting group described in step (c1) is carried out under alkaline conditions. For example, the deprotection reaction of the Pg3 protecting group described in step (c1) is carried out in the presence of sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium tert-butoxide, sodium amide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, barium hydroxide, calcium hydroxide, magnesium hydroxide, lithium hydroxide, hydrazine hydrate, ammonia methanol, ammonia ethanol, or ammonia solution.

[0207] In some embodiments, the reaction to remove the Pg3 protecting group described in step (c1) is carried out at a temperature of 0–80°C, preferably 10–60°C, and more preferably 30–50°C.

[0208] In some embodiments, the esterification reaction described in step (c2) is carried out in the presence of 2,4,6-trichlorobenzoyl chloride and 4-dimethylaminopyridine.

[0209] In some embodiments, the esterification reaction described in step (c2) is carried out at a temperature of 0–80°C, preferably 10–40°C, and more preferably 25–35°C.

[0210] In some embodiments, the deprotection reaction of the Pg1 protecting group described in step (c3) is carried out in the presence of a fluoride ion reagent or a fluorine-containing reagent. The fluoride ion reagent is, for example, but not limited to, TBAF, hydrogen fluoride, triethylamine hydrogen fluoride, cesium fluoride, potassium fluoride, trifluoroacetic acid, and trifluoromethanesulfonic acid.

[0211] In some embodiments, the reaction to remove the Pg1 protecting group described in step (c3) is carried out at a temperature of -30 to 0°C, preferably -20 to 0°C, and more preferably -15 to -5°C.

[0212] In some embodiments, the reaction of the compound of formula I-11 described in step (c3) with trichloroacetonitrile is carried out at a temperature of 0–40°C, preferably 10–30°C, and more preferably 15–20°C.

[0213] In some embodiments, the glycosylation reaction described in step (c3) is carried out in the presence of the following reagents: silver trifluoromethanesulfonate, silver oxide, silver carbonate, silver nitrate, silver perchlorate, mercuric chloride, mercuric bromide, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, trifluoromethanesulfonic anhydride, trifluoromethanesulfonic acid, boron trifluoride ether, scandium trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, copper trifluoromethanesulfonate, ferric trifluoromethanesulfonate, ferric chloride, aluminum trichloride, tin tetrachloride, stannous chloride, zinc chloride, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, or any combination thereof.

[0214] In some embodiments, the glycosylation reaction described in step (c3) is carried out in the presence of boron trifluoride diethyl ether.

[0215] In some embodiments, the glycosylation reaction described in step (c3) is carried out at a temperature of -100 to -20°C, preferably -90 to -40°C, and more preferably -80 to -60°C.

[0216] In some embodiments, step (c4) is carried out under hydrogenation conditions. Preferably, step (c4) is carried out in the presence of a hydrogenation catalyst. Preferably, the hydrogenation catalyst is palladium on carbon or palladium hydroxide on carbon.

[0217] In some embodiments, step (c4) is carried out in the presence of an acid. Preferably, the acid is formic acid, acetic acid, propionic acid, benzoic acid, benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, citric acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, or trifluoroacetic acid. More preferably, the acid is trifluoroacetic acid.

[0218] In some embodiments, the hydrogenation in step (c4) is preceded by a step of treating the compound of formula I-12 with an acid. Preferably, the acid is trifluoroacetic acid. Preferably, this step is carried out at a temperature of -20 to 20°C, more preferably -10 to -10°C, and more preferably -5 to 5°C.

[0219] Synthesis Method 2

[0220] Another object of the present invention is to provide a method for synthesizing another synthetic saponin composition of the present invention (synthesis method 2), which includes the following steps:

[0221] (a') Protecting the hydroxyl group of the compound of formula II-1-1 to obtain the compound of formula II-1, and then removing the Pg4 protecting group from the compound of formula II-1 to obtain the compound of formula II-2;

[0222]

[0223] (b') React the compound of formula II-2 with a halogen-containing electrophilic reagent to generate an imine ester of formula II-2-1. The imine ester of formula II-2-1 was subjected to a glycosylation reaction with compound SAP-18 to obtain compound II-3.

[0224]

[0225] (c') The synthetic saponin composition is obtained by reacting the compound of formula II-3.

[0226] Each R6 group is an independent hydroxyl protecting group, or two R6 groups together with the oxygen atom they are attached to form a protective group. Structure, where R x and R y Each independently represents H and C. 1-6 Alkyl and phenyl;

[0227] Pg4 and Pg5 are each independently protected by hydroxyl groups;

[0228] R3 is allyl, methylthiomethyl, 3,4-dimethoxybenzyl, p-methoxybenzyl, 2,3,4-trimethoxybenzyl, 3,4,5-trimethoxybenzyl, 2,5-dimethoxybenzyl, 2-methoxybenzyl, 3,5-dimethoxybenzyl, 2,3-dimethoxybenzyl, 3-methoxybenzyl, 2,6-dimethoxybenzyl, 1-naphthylmethyl, or 2-naphthylmethyl;

[0229] R5 is Where X is a halogen; and R z It is H or phenyl;

[0230] Y1 is an independent hydroxyl protecting group;

[0231] Y2 is a hydroxyl protecting group.

[0232] In some implementations, R6 is benzyl.

[0233] In some implementations, the two R6 atoms together with the oxygen atoms they are attached to form Structure, where R x and R y Each can be independently H, methyl, or phenyl.

[0234] In some embodiments, Pg4 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, and tert-butyldiphenylsilyl; Pg5 is selected from benzyl, C 1-6 Alkyl benzyl, naphthylmethyl, or triphenylmethyl.

[0235] In some implementations, Pg4 is triisopropylsilyl and Pg5 is benzyl.

[0236] In some implementations, R5 is Where X is chlorine or fluorine; and R z It can be H or phenyl.

[0237] In some implementations, R5 is

[0238] In some embodiments, Y1 is independently selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl or benzyl.

[0239] In some implementations, Y1 is benzyl.

[0240] In some embodiments, Y2 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, or benzyl.

[0241] In some embodiments, Y2 is selected from triethylsilyl or benzyl.

[0242] In some embodiments, the hydroxyl protection reaction described in step (a') is carried out in the presence of a haloalkyl reagent and a base, such as, but not limited to, allyl bromide, allyl chloride, methylthiomethyl bromide, 3,4-dimethoxybenzyl bromide, p-methoxybenzyl bromide, p-methoxybenzyl chloride, 2,3,4-trimethoxybenzyl bromide, 3,4,5-trimethoxybenzyl bromide, 2,5-dimethoxybenzyl bromide, 2-methoxybenzyl bromide, 3,5-dimethoxybenzyl bromide, 2,3-dimethoxybenzyl bromide, 3-methoxybenzyl bromide, 2,6-dimethoxybenzyl bromide, 1-naphthylmethyl bromide, or 2-naphthylmethyl bromide. The base is, for example, but not limited to, sodium hydride, lithium hydride, potassium tert-butoxide, etc.

[0243] In some embodiments, the hydroxyl protection reaction described in step (a') is carried out in the presence of reagents such as allyl bromide, chloromethyl methyl sulfide, 2-(bromomethyl)naphthalene, 1-(bromomethyl)naphthalene, 4-methoxybenzyl chloride, 2,3,4-trimethoxybenzyl chloride, 3,4,5-trimethoxybenzyl chloride, etc.

[0244] In some embodiments, the hydroxyl protection reaction described in step (a') is carried out at a temperature of -30 to 30°C, preferably -10 to 10°C, and more preferably -5 to 5°C.

[0245] In some embodiments, the deprotection reaction of the Pg4 protecting group described in step (a') is carried out in the presence of a fluoride ion reagent or a fluorine-containing reagent. Such fluoride ion reagents or fluorine-containing reagents are, for example, but not limited to, TBAF, hydrogen fluoride, triethylamine hydrogen fluoride, cesium fluoride, potassium fluoride, trifluoroacetic acid, and trifluoromethanesulfonic acid.

[0246] In some embodiments, the reaction to remove the Pg4 protecting group described in step (a') is carried out at a temperature of -30 to 30°C, preferably -10 to 10°C, and more preferably -5 to 5°C.

[0247] In some embodiments, the glycosylation reaction described in step (b') is carried out in the presence of silver trifluoromethanesulfonate, silver oxide, silver carbonate, silver nitrate, silver perchlorate, mercuric chloride, mercuric bromide, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, trifluoromethanesulfonic anhydride, trifluoromethanesulfonic acid, boron trifluoride ether, scandium trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, copper trifluoromethanesulfonate, ferric trifluoromethanesulfonate, ferric chloride, aluminum trichloride, tin tetrachloride, stannous chloride, zinc chloride, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, or any combination thereof. Preferably, it is carried out in the presence of boron trifluoride ether.

[0248] In some implementations, step (c') includes:

[0249] (c1') Selectively remove the R3 protecting group from the compound of formula II-3 to obtain the compound of formula II-4;

[0250]

[0251] In some implementations, step (c') further includes:

[0252] (c2') The compound of formula II-4 is subjected to an esterification reaction with the compound of formula II-5 to obtain the compound of formula II-6;

[0253] In some implementations, step (c') further includes:

[0254] (c3') Remove all protecting groups from the compound of formula II-6 to obtain the synthetic saponin composition.

[0255]

[0256] In the above steps (c1') to (c3')

[0257] R1 is an independent hydroxyl protecting group;

[0258] R6 can be an independent hydroxyl protecting group, or two R6 groups together with the oxygen atom they are attached to form a protective group. Structure, where R x and R y Each independently represents H and C. 1-6 Alkyl and phenyl;

[0259] Pg4 and Pg5 are each independently protected by hydroxyl groups;

[0260] R3 is allyl, methylthiomethyl, 3,4-dimethoxybenzyl, p-methoxybenzyl, 2,3,4-trimethoxybenzyl, 3,4,5-trimethoxybenzyl, 2,5-dimethoxybenzyl, 2-methoxybenzyl, 3,5-dimethoxybenzyl, 2,3-dimethoxybenzyl, 3-methoxybenzyl, 2,6-dimethoxybenzyl, 1-naphthylmethyl, or 2-naphthylmethyl;

[0261] Y1 is an independent hydroxyl protecting group;

[0262] Y2 is a hydroxyl protecting group.

[0263] In some embodiments, R1 is independently selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, or benzyl.

[0264] In some embodiments, R1 is tert-butyldimethylsilyl or benzyl.

[0265] In some implementations, R6 is benzyl.

[0266] In some implementations, the two R6 atoms together with the oxygen atoms they are attached to form Structure, where R x and R y Each can be independently H, methyl, or phenyl.

[0267] In some embodiments, Pg4 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, and tert-butyldiphenylsilyl; Pg5 is selected from benzyl, C 1-6Alkyl benzyl, naphthylmethyl, or triphenylmethyl.

[0268] In some implementations, Pg4 is triisopropylsilyl and Pg5 is benzyl.

[0269] In some embodiments, Y1 is independently selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl or benzyl.

[0270] In some implementations, Y1 is benzyl.

[0271] In some embodiments, Y2 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, or benzyl.

[0272] In some embodiments, Y2 is selected from triethylsilyl or benzyl.

[0273] In some embodiments, step (c1') is carried out in the presence of a catalyst of palladium, iridium, rhodium, mercury, ruthenium, platinum, copper, silver, gold, or a combination thereof.

[0274] In some embodiments, step (c1') is carried out in the presence of 2,3-dichloro-5,6-dicyanobenzoquinone, cerium ammonium nitrate, silver oxide, iodine, bromine, bromosuccinimide, magnesium perchlorate, or any combination thereof.

[0275] In some embodiments, step (c2') is carried out in the presence of 2,4,6-trichlorobenzoyl chloride and 4-dimethylaminopyridine.

[0276] In some embodiments, step (c3') is carried out under hydrogenation conditions. Preferably, step (c3') is carried out in the presence of a hydrogenation catalyst. Preferably, the hydrogenation catalyst is palladium on carbon or palladium hydroxide on carbon.

[0277] In some embodiments, step (c3') is carried out in the presence of an acid. Preferably, the acid is formic acid, acetic acid, propionic acid, benzoic acid, benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, citric acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, or trifluoroacetic acid, more preferably trifluoroacetic acid.

[0278] In some embodiments, step (c3') further includes treating the compound of formula I-12 with an acid before the hydrogenation. Preferably, the acid is trifluoroacetic acid.

[0279] According to some embodiments of the present invention, the synthesis method 2 of the present invention includes the following steps:

[0280] (a') Protecting the hydroxyl group of the compound of formula II-1-1 to obtain the compound of formula II-1, and then removing the Pg4 protecting group from the compound of formula II-1 to obtain the compound of formula II-2;

[0281]

[0282] (b') React the compound of formula II-2 with a halogen-containing electrophilic reagent to generate an imine ester of formula II-2-1. The imine ester of formula II-2-1 was subjected to a glycosylation reaction with compound SAP-18 to obtain compound II-3.

[0283]

[0284] (c1') Selectively remove the R3 protecting group from the compound of formula II-3 to obtain the compound of formula II-4;

[0285]

[0286] (c2') The compound of formula II-4 is subjected to an esterification reaction with the compound of formula II-5 to obtain the compound of formula II-6;

[0287]

[0288] (c3') Remove all protecting groups from the compound of formula II-6 to obtain the synthetic saponin composition.

[0289]

[0290] R6 is an independent hydroxyl protecting group.

[0291] Or two R6 atoms together with the oxygen atom they are attached to form Structure, where R x and R y Each independently represents H and C. 1-6 Alkyl and phenyl;

[0292] Pg4 and Pg5 are each independently protected by hydroxyl groups;

[0293] R1 is an independent hydroxyl protecting group;

[0294] R3 is allyl, methylthiomethyl, 3,4-dimethoxybenzyl, p-methoxybenzyl, 2,3,4-trimethoxybenzyl, 3,4,5-trimethoxybenzyl, 2,5-dimethoxybenzyl, 2-methoxybenzyl, 3,5-dimethoxybenzyl, 2,3-dimethoxybenzyl, 3-methoxybenzyl, 2,6-dimethoxybenzyl, 1-naphthylmethyl, or 2-naphthylmethyl;

[0295] R5 is Where X is a halogen; and R z It is H or phenyl;

[0296] Y1 is an independent hydroxyl protecting group;

[0297] Y2 is a hydroxyl protecting group.

[0298] In some implementations, R6 is benzyl.

[0299] In some implementations, the two R6 atoms together with the oxygen atoms they are attached to form Structure, where R x and R y Each can be independently H, methyl, or phenyl.

[0300] In some embodiments, Pg4 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, and tert-butyldiphenylsilyl; Pg5 is selected from benzyl, C 1-6 Alkyl benzyl, naphthylmethyl, or triphenylmethyl.

[0301] In some implementations, Pg4 is triisopropylsilyl and Pg5 is benzyl.

[0302] In some embodiments, R1 is independently selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, or benzyl.

[0303] In some embodiments, R1 is independently selected from tert-butyldimethylsilyl or benzyl.

[0304] In some implementations, R5 is Where X is chlorine or fluorine, R z It can be H or phenyl.

[0305] In some implementations, R5 is

[0306] In some embodiments, Y1 is independently selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl or benzyl.

[0307] In some implementations, Y1 is benzyl.

[0308] In some embodiments, Y2 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, or benzyl.

[0309] In some embodiments, Y2 is selected from triethylsilyl or benzyl.

[0310] In some embodiments, the hydroxyl protection reaction described in step (a') is carried out in the presence of a haloalkyl reagent and a base, such as, but not limited to, allyl bromide, allyl chloride, methylthiomethyl bromide, 3,4-dimethoxybenzyl bromide, p-methoxybenzyl bromide, p-methoxybenzyl chloride, 2,3,4-trimethoxybenzyl bromide, 3,4,5-trimethoxybenzyl bromide, 2,5-dimethoxybenzyl bromide, 2-methoxybenzyl bromide, 3,5-dimethoxybenzyl bromide, 2,3-dimethoxybenzyl bromide, 3-methoxybenzyl bromide, 2,6-dimethoxybenzyl bromide, 1-naphthylmethyl bromide, or 2-naphthylmethyl bromide. The base is, for example, but not limited to, sodium hydride, lithium hydride, potassium tert-butoxide, etc.

[0311] In some embodiments, the hydroxyl protection reaction described in step (a') is carried out in the presence of 1-naphthylmethyl bromide, 2-naphthylmethyl bromide, or p-methoxybenzyl bromide.

[0312] In some embodiments, the hydroxyl protection reaction described in step (a') is carried out at a temperature of -30 to 30°C, preferably -10 to 10°C, and more preferably -5 to 5°C.

[0313] In some embodiments, the deprotection reaction of the Pg4 protecting group described in step (a') is carried out in the presence of a fluoride ion reagent or a fluorine-containing reagent. Such fluoride ion reagents or fluorine-containing reagents are, for example, but not limited to, TBAF, hydrogen fluoride, triethylamine hydrogen fluoride, cesium fluoride, potassium fluoride, trifluoroacetic acid, and trifluoromethanesulfonic acid.

[0314] In some embodiments, the reaction to remove the Pg4 protecting group described in step (a') is carried out at a temperature of -30 to 30°C, preferably -10 to 10°C, and more preferably -5 to 5°C.

[0315] In some embodiments, the glycosylation reaction described in step (b') is carried out in the presence of silver trifluoromethanesulfonate, silver oxide, silver carbonate, silver nitrate, silver perchlorate, mercuric chloride, mercuric bromide, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, trifluoromethanesulfonic anhydride, trifluoromethanesulfonic acid, boron trifluoride ether, scandium trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, copper trifluoromethanesulfonate, ferric trifluoromethanesulfonate, ferric chloride, aluminum trichloride, tin tetrachloride, stannous chloride, zinc chloride, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, or any combination thereof. Preferably, it is carried out in the presence of boron trifluoride ether.

[0316] In some embodiments, step (c1') is carried out in the presence of a catalyst of palladium, iridium, rhodium, mercury, ruthenium, platinum, copper, silver, gold, or a combination thereof.

[0317] In some embodiments, step (c1') is carried out in the presence of 2,3-dichloro-5,6-dicyanobenzoquinone, cerium ammonium nitrate, silver oxide, iodine, bromine, bromosuccinimide, magnesium perchlorate, or any combination thereof.

[0318] In some embodiments, step (c2') is carried out in the presence of 2,4,6-trichlorobenzoyl chloride and 4-dimethylaminopyridine.

[0319] In some embodiments, step (c3') is carried out under hydrogenation conditions. Preferably, step (c3') is carried out in the presence of a hydrogenation catalyst. Preferably, the hydrogenation catalyst is palladium on carbon or palladium hydroxide on carbon.

[0320] In some embodiments, step (c3') is performed in the presence of an acid. Preferably, the acid is formic acid, acetic acid, propionic acid, benzoic acid, benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, citric acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, or trifluoroacetic acid. More preferably, the acid is trifluoroacetic acid.

[0321] In some embodiments, step (c3') further includes treating the compound of formula I-12 with an acid before the hydrogenation. Preferably, the acid is trifluoroacetic acid.

[0322] Synthetic method of SAP-18, an intermediate for synthesizing saponin compositions

[0323] Another object of the present invention is to provide a method for synthesizing SAP-18, an intermediate in the synthetic saponin composition of the present invention, comprising the following steps:

[0324] (a”) Selectively remove the TBS protecting group from the compound of formula III-1 to obtain compound III-2, and then perform a glycosylation reaction between the compound of formula III-2 and compound III-3 to obtain compound III-4.

[0325]

[0326] (b”) React the compound of formula III-4 with a basic reagent or a reducing reagent to generate compound III-5, and react the compound of formula III-5 with a silyl ether reagent or benzyl bromide to obtain compound III-6;

[0327]

[0328] (c”) React the compound of formula III-6 with an acidic reagent to generate compound III-7, react the compound of formula III-7 with an oxidizing reagent to obtain compound III-8, and react the compound of formula III-8 with a silyl ether reagent or benzyl bromide to obtain compound III-9.

[0329]

[0330] (d”) The compound of formula III-9 is reacted with a metal catalyst to remove the protecting group to obtain compound III-10, and compound III-10 is reacted with a halogen-containing electrophilic reagent to obtain compound III-11.

[0331]

[0332] (e”) The intermediate SAP-18 is obtained by reacting the compound of formula III-11.

[0333] Each of Y1 is an independent hydroxyl protecting group;

[0334] R9 is Where X is a halogen; and R z It is H or phenyl;

[0335] R8 is allyl. or 2-butenyl

[0336] R 10 It is either acetyl or benzoyl.

[0337] In some embodiments, Y1 is independently selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl or benzyl.

[0338] In some implementations, Y1 is benzyl.

[0339] In some implementations, R9 is Where X is chlorine or fluorine, and R z It can be H or phenyl.

[0340] In some implementations, R9 is

[0341] In some implementations, R8 is allyl.

[0342] In some embodiments, the selective removal of the TBS protecting group described in step (a”) is carried out in the presence of a fluorinated reagent. The fluorinated reagent is, for example, but not limited to, TBAF, pyridine hydrofluoride, etc.

[0343] In some implementations, the selective removal of the TBS protecting group described in step (a”) is carried out in the presence of a TBAF reagent.

[0344] In some embodiments, the selective removal of the TBS protective base described in step (a”) is carried out at a temperature of -30 to 50°C, preferably -10 to 30°C, and more preferably 5 to 15°C.

[0345] In some embodiments, the glycosylation reaction described in step (a”) is carried out in the presence of silver trifluoromethanesulfonate, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, trifluoromethanesulfonic acid, boron trifluoride ether, N-iodosuccinimide, or any combination thereof.

[0346] In some embodiments, the glycosylation reaction described in step (a”) is carried out in the presence of a combination of N-iodosuccinimide and silver trifluoromethanesulfonate.

[0347] In some embodiments, the glycosylation reaction described in step (a”) is carried out at a temperature of -90 to -30°C, preferably -80 to -50°C, and more preferably -75 to -65°C.

[0348] In some embodiments, the alkaline reagent or reducing agent described in step (b”) is, for example, but not limited to: sodium hydroxide, lithium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, diisobutylaluminum hydride, sodium borohydride, lithium aluminum hydride, or any combination thereof.

[0349] In some implementations, the alkaline reagent or reducing agent described in step (b”) is potassium carbonate.

[0350] In some embodiments, the reaction of the compound of formula III-4 described in step (b”) with a basic or reducing agent is carried out at a temperature of 0–50°C, preferably 10–40°C, and more preferably 20–30°C.

[0351] In some embodiments, the reaction of formula III-5 described in step (b”) with the silyl ether reagent or benzyl bromide is carried out in the presence of sodium hydride.

[0352] In some embodiments, the reaction of formula III-5 described in step (b”) with the silyl ether reagent or benzyl bromide is carried out at a temperature of 0–50°C, preferably 10–40°C, more preferably 20–30°C.

[0353] In some implementations, the silane reagent described in step (b”) is trimethylsilicon chloride, triethylsilicon chloride, triisopropylsilicon chloride, tert-butyldimethylsilicon chloride, tert-butyldiphenylsilicon chloride, etc.

[0354] In some implementations, the acidic reagent described in step (c”) is hydrochloric acid, p-toluenesulfonic acid, methanesulfonic acid, or any combination thereof.

[0355] In some embodiments, the oxidizing agent described in step (c”) is 2,2,6,6-tetramethylpiperidine oxide, iodophenyl diacetic acid, Desmond-Martin oxidant, sodium hypochlorite, or any combination thereof.

[0356] In some implementations, the metal catalyst described in step (d”) is a palladium, iridium, or ruthenium catalyst.

[0357] According to some embodiments of the present invention, step (e”) includes:

[0358] (e1”) Perform a glycosylation reaction between the compound of formula III-11 and the compound of formula III-12 to obtain the compound of formula III-13;

[0359]

[0360] Preferably, step (e”) further includes:

[0361] (e2”) The compound of formula III-13 is subjected to a hydroxyl protection reaction to obtain compound III-14, and the compound III-14 is reacted with a metal catalyst to obtain intermediate SAP-18.

[0362]

[0363] Each of Y1 is an independent hydroxyl protecting group.

[0364] Y2 is a hydroxyl protecting group.

[0365] R9 is Where X is a halogen; and R z It can be H or phenyl.

[0366] R 11 Allyl or 2-butenyl

[0367] In some embodiments, Y1 is independently selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl or benzyl.

[0368] In some implementations, Y1 is benzyl.

[0369] In some embodiments, Y2 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl, or benzyl.

[0370] In some embodiments, Y2 is selected from triethylsilyl or benzyl.

[0371] In some implementations, R9 is Where X is chlorine or fluorine, Rz It can be H or phenyl.

[0372] In some implementations, R9 is

[0373] In some implementation schemes, R 11 Allyl

[0374] In some embodiments, the glycosylation reaction described in step (e1”) is carried out in the presence of silver trifluoromethanesulfonate, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, trifluoromethanesulfonic acid, boron trifluoride ether, tris(pentafluorophenyl)borane or any combination thereof.

[0375] In some embodiments, the glycosylation reaction described in step (e1”) is carried out in the presence of tris(pentafluorophenyl)borane.

[0376] In some embodiments, the glycosylation reaction described in step (e1”) is carried out at a temperature of 0–50°C, preferably 10–30°C, and more preferably 15–25°C.

[0377] In some implementations, the metal catalyst described in step (e2”) is a palladium, iridium, or ruthenium catalyst.

[0378] In some implementations, the metal catalyst described in step (e2”) is a palladium catalyst.

[0379] Composition and Use

[0380] Another object of the present invention is to provide a pharmaceutical composition comprising the synthetic saponin composition of the present invention and a pharmaceutically acceptable excipient.

[0381] The pharmaceutically acceptable excipients described in this invention refer to inactive ingredients in pharmaceutical compositions that do not cause significant irritation to the organism and do not interfere with the biological activity of the administered compound, such as, but not limited to, solubilizers, surfactants, stabilizers, antioxidants, excipients, etc., commonly found in the art.

[0382] Another object of the present invention is to provide an adjuvant composition comprising the synthetic saponin composition of the present invention.

[0383] In some embodiments, the adjuvant composition of the present invention further comprises a TLR agonist. The TLR agonist is, for example, but not limited to, MPLA, GLA, etc.

[0384] Another object of the present invention is to provide an immunogenic composition comprising the adjuvant composition of the present invention and an antigen.

[0385] In some implementations, the antigen is selected from antigens associated with varicella-zoster virus, herpesvirus, or herpes simplex virus.

[0386] In some embodiments, the antigen is selected from antigens associated with Plasmodium falciparum or Plasmodium vivax.

[0387] In some implementations, the antigen is selected from antigens associated with human respiratory first syncytial virus.

[0388] In some implementations, the antigen is selected from human papillomavirus-associated antigens.

[0389] In some embodiments, the antigen is selected from antigens associated with Streptococcus pneumoniae, Mycobacterium spp., and Mycobacterium tuberculosis.

[0390] In some implementations, the antigen is selected from antigens related to the novel coronavirus.

[0391] In some embodiments, the antigen is derived from HIV, untyped Haemophilus influenzae, Moraxella catarrhalis, influenza virus, rabies virus, FeLV, bovine LV, FeIV, canine distemper virus, canine infectious hepatitis virus, feline calicivirus, feline rhinotracheitis virus, TGE virus, foot-and-mouth disease virus, and combinations thereof.

[0392] In some implementations, the antigen is a tumor-associated antigen.

[0393] Another object of the present invention is to provide the use of the synthetic saponin compositions of the present invention in the preparation of anticancer drugs, adjuvants or immunogenic compositions.

[0394] Another object of the present invention is to provide the synthetic saponin compositions of the present invention for use in treating cancer, or as adjuvants or immunogenic drugs.

[0395] Another object of the present invention is to provide a method for treating cancer, comprising administering a therapeutically effective amount of the synthetic saponin composition of the present invention.

[0396] Another object of the present invention is to provide an immunization method comprising administering a therapeutically effective amount of the synthetic saponin composition of the present invention.

[0397] Determination methods

[0398] Another object of the present invention is to provide an HPLC method for determining the main peak and isomer B content of saponin QS-21-Api in synthetic saponin compositions, the method comprising:

[0399] The stationary phase was AQ C18 with a particle size of 5 μm. Mobile phase A was water-phosphoric acid (1000:1), and mobile phase B was acetonitrile. The elution conditions were as follows:

[0400]

[0401]

[0402] The detection wavelength is 200nm. Example

[0403] To make the objectives and technical solutions of this invention clearer, the invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, specific experimental methods not mentioned in the following embodiments were performed according to conventional experimental methods. The starting materials and reaction reagents used in the synthesis of this invention are commercially available or synthesized according to literature reports.

[0404] The abbreviations used in this article and their meanings are shown in Table 1.

[0405] Table 1

[0406]

[0407] Preparation Example

[0408] Preparation Example 1: Synthesis of QS21-SM13B-01

[0409]

[0410] 120 g of QS21-SM3-07 and 111 g of QS21-SM2-05B were added to a single-necked flask. Under nitrogen protection, 1200 mL of anhydrous DCM and 50 g of 4A molecular sieve were added, and the mixture was stirred for 30 min. The temperature was lowered to -75 °C, and 70 g of NIS and 20 g of AgOTf were added. The reaction was allowed to proceed for 2 h. The mixture was quenched with triethylamine, the organic phase was concentrated, and purified by silica gel column chromatography (elution gradient: EA:n-hexane = 1:20 → 1:10 → 1:5) to obtain 138.2 g of QS21-SM13B-01 as a white waxy solid, with a yield of 81%.

[0411] 1¹H NMR (400MHz, chloroform-d) δ 7.36–7.21 (m, 10H), 5.17 (t, J = 9.2Hz, 1H), 5.09 (d, J = 1.7Hz, 1H), 4.93 (d, J = 7.6Hz, 1H), 4.79 (d, J = 12.0Hz, 1H), 4.60–4.48 (m, 2H), 4.14–4.07 (m, 3H), 3 .96(dt,J=9.9,4.8Hz,2H),3.52(td,J=9.5,5.3Hz,1H),3.38(dd,J=9.6Hz,1H),3.31– 3.18(m,2H),1.92(s,3H),1.50(s,3H),1.31(t,J=3.1Hz,6H),1.09(d,J=5.7Hz,18H).

[0412] Preparation Example 2: Synthesis of QS21-SM13B-02

[0413]

[0414] 960 mL of methanol and 96 mL of water were added to a reaction flask and stirred until homogeneous. Then, 47.8 g of QS21-SM13B-01 and 33 g of potassium carbonate were added, and the mixture was reacted at room temperature for 4 h. The product was extracted with DCM, concentrated, and purified by silica gel column chromatography (mobile phase: ethyl acetate / n-heptane) to obtain 40.9 g of QS21-SM13B-02 as a white solid, with a yield of 91%.

[0415] 1H NMR (600MHz, chloroform-d) δ7.39–7.24(m,10H),5.30–5.26(s,1H),5.11–5.06(d,J=1.8Hz,1H),4.94–4.90(d,J=11.6Hz,1H),4.89–4.81(d, J=7.6Hz,1H),4.77–4.71(d,J=11.9Hz,1H),4.69–4.62(dd,J=17.6,11.7Hz,2H),4.12–4.06(d,J=5.2Hz,2H),3.96–3.90(dt,J=8.5,4 .0Hz,2H),3.73–3.67(t,J=8.9Hz,1H),3.54–3.48(td,J=9.2,5.3Hz,1H),3.40–3.32(dq,J=9.8,6.2Hz,1H),3.26–3.14(m,2H),1.53 –1.49(s,3H),1.36–1.32(s,3H),1.32–1.28(d,J=6.2Hz,3H),1.17–1.11(dt,J=12.6,6.8Hz,3H),1.11–1.06(dd,J=7.2,2.3Hz,18H).

[0416] Preparation Example 3: Synthesis of QS21-SM13B-05

[0417]

[0418] 1 g of QS21-SM13B-02 and 0.7 g of QS21-SM1-18 were added to a reaction flask, along with 30 mL of anhydrous DCM and 0.3 g of 4A molecular sieve. The mixture was stirred for 30 min. The temperature was lowered to -60 °C, and 0.4 g of NIS and 0.1 g of TMSOTf were added. The reaction was allowed to proceed for 15 min. The mixture was quenched with triethylamine, and the organic phase was concentrated. The solution was then purified by silica gel column chromatography (elution gradient: EA:n-heptane = 1:8 → 1:5) to obtain 1.23 g of QS21-SM13B-05 as a white solid, with a yield of 75.3%.

[0419] 1¹H NMR (600 MHz, chloroform-d) δ 7.62–7.28 (m, 20H), 6.08–6.00 (s, 1H), 5.81–5.77 (s, 1H), 5.30–5.25 (s, 1H), 5.16–5.13 (d, J = 2.3 Hz, 1H), 4.93–4.86 (m, 2H), 4.77–4.73 (d, J = 11.3 Hz, 1H), 4.67–4.60 (m, 2H), 4.58–4.50 (m, 3H), 4.16–4.09 (m, 2H), 4.0 3–4.02(s,1H),4.01–3.97(dd,J=9.6,6.8Hz,1H),3.95–3.87(m,2H),3.48–3.39(m,2H),3.37–3.32(m,1H),3.26–3.20 (dd,J=11.8,10.0Hz,1H),1.58–1.51(s,3H),1.42–1.31(m,9H),1.23–1.18(m,3H),1.17–1.11(dd,J=7.1,2.3Hz,18H).

[0420] Synthesis of Preparation Example 4: QS21-SM13B-06

[0421]

[0422] 37 g of QS21-SM13B-05 and 1.11 L of THF were added to a reaction flask. Under nitrogen protection, 100 mL of a THF solution containing 9.4 g of TBAF·3H2O was added, and the reaction was carried out at room temperature for 1 h. The organic phase was concentrated and purified by silica gel column chromatography (elution gradient: EA:n-heptane = 1:8 → 1:5) to obtain 25.69 g of QS21-SM13B-06 as a white solid, with a yield of 85.3%.

[0423] 1H NMR (600MHz, chloroform-d) δ7.59–7.17(m,20H),6.03–5.97(s,1H),5.77–5.72(s ,1H),5.41–5.35(d,J=3.6Hz,1H),5.32–5.25(s,1H),4.92–4.88(d,J=7. 5Hz,1H),4.87–4.83(d,J=11.0Hz,1H),4.71–4.66(dd,J=11.3,4.4Hz,1H ),4.64–4.58(m,2H),4.57–4.50(m,2H),4.50–4.45(m,1H),4.27–4.20(m, 1H),4.17–4.10(m,2H),4.02–3.96(m,2H),3.93–3.80(m,3H),3.68–3.60 (m,2H),3.42–3.38(td,J=9.3,5.3Hz,1H),3.31–3.26(m,2H),3.21–3.15 (ddd,J=13.7,10.0,4.0Hz,1H),2.08–2.03(s,2H),1.53–1.47(s,3H),1. 38–1.31(d,J=4.1Hz,3H),1.31–1.22(m,5H),1.10–1.04(d,J=2.2Hz,3H).

[0424] Synthesis of Preparation Example 5: QS21-SM13B-07

[0425]

[0426] 50.0 g of QS21-SM13B-06 and 500 mL of dichloromethane were added to a reaction flask. Under nitrogen protection, the mixture was cooled to 0 °C, and 87.0 g of trichloroacetonitrile and 39.1 g of cesium carbonate were added. The reaction was allowed to proceed for 4 h. The organic phase was concentrated and purified by silica gel column chromatography (elution gradient: EA:n-heptane = 1:8 → 1:5) to give 44.2 g of QS21-SM13B-07 as a white solid, with a yield of 75.1%.

[0427] Synthesis of Preparation Example 6: QS21-SM13B-08

[0428]

[0429] 34 g of QS21-SM13B-07, 19 g of QS21-SM4-25, and 900 mL of anhydrous DCM were added to a reaction flask, and the mixture was stirred for 30 min. The mixture was cooled to 0 °C, and 3.12 g of TMSOTf was added. The reaction was allowed to proceed for 15 min. The mixture was quenched with triethylamine, and the organic phase was concentrated. The solution was then purified by silica gel column chromatography (elution gradient: EA:n-heptane = 1:8 → 1:5) to obtain 32.2 g of QS21-SM13B-08 as a white solid, with a yield of 72.7%.

[0430] 1 H NMR (600MHz, chloroform-d) δ7.59–7.17(m,20H),6.03–5.97(s,1H),5.77–5.72(s,1H) ),5.41–5.35(d,J=3.6Hz,1H),5.32–5.25(s,1H),4.92–4.88(d,J=7.5Hz,1H) ,4.87–4.83(d,J=11.0Hz,1H),4.71–4.66(dd,J=11.3,4.4Hz,1H),4.64–4.5 8(m,2H),4.57–4.50(m,2H),4.50–4.45(d,J=11.5Hz,1H),4.27–4.20(m,1H), 4.17–4.10(m,2H),4.02–3.96(d,J=2.2Hz,2H),3.93–3.80(m,3H),3.68–3.6 0(d,J=13.2Hz,3H),3.42–3.38(td,J=9.3,5.3Hz,1H),3.31–3.26(m,2H),3.2 1–3.15(ddd,J=13.7,10.0,4.0Hz,1H),2.08–2.03(s,2H),1.53–1.47(s,3H) ,1.38–1.31(d,J=4.1Hz,4H),1.31–1.22(m,5H),1.10–1.04(d,J=2.2Hz,3H).

[0431] Synthesis of Preparation Example 7: QS21-SM13B-09

[0432]

[0433] 500 mL of methanol and 50 mL of water were added to a reaction flask and stirred until homogeneous. Then, 25.1 g of QS21-SM13B-08 and 16.5 g of potassium carbonate were added, and the mixture was reacted at 40 °C for 8 h. The mixture was extracted with DCM, dried, concentrated, and purified by silica gel column chromatography (eluent: EA / n-heptane) to obtain 20.3 g of QS21-SM13B-09 as a white solid, with a yield of 85.3%.

[0434] 1 ¹H NMR (600MHz, chloroform-d) δ 7.59–7.18 (m, 25H), 6.01–5.91 (s, 1H), 5.74–5.66 (d, J = 9.8Hz, 2H), 4.96–4.86 (dd, J = 18.2, 9.4Hz, 2H), 4.71–4.66 (m, 1H), 4.63–4.59 (m, 2H), 4.59–4.57 (d ,J=3.8Hz,1H),4.55–4.48(m,3H),4.47–4.45(d,J=5.7Hz,1H),4.43–4.41(s,1H),4.19 –4.15(dd,J=7.7,5.7Hz,1H),4.15–4.09(q,J=7.2Hz,3H),4.08–4.05(d,J=5.6Hz,1H),3 .99–3.94(dt,J=9.5,6.0Hz,1H),3.94–3.92(s,2H),3.89–3.80(m,2H),3.80–3.78(q,J =3.2,2.8Hz,1H),3.62–3.59(s,2H),3.53–3.47(dtd,J=13.5,6.4,2.9Hz,2H),2.05–2.0 3(s,4H),1.54–1.49(s,3H),1.38–1.33(d,J=4.3Hz,3H),1.33–1.30(dd,J=6.5,2.6Hz, 3H), 1.28–1.22 (q, J=7.0Hz, 8H), 1.11–1.06 (m, 3H), 1.05–0.99 (dt, J=8.7, 7.0Hz, 18H).

[0435] Preparation Example 8: Synthesis of QS21-SM12-19

[0436]

[0437] 24 g of QS21-SM12-17 (synthesized according to literature J.AM.CHEM.SOC.2006,128,11906-11915) was added to a reaction flask, and 200 mL of toluene was added and stirred until dissolved. 3.6 g of triethylamine was added at 30 °C, and the mixture was stirred for 5 minutes. Then, 6.6 g of 2,4,6-trichlorobenzoyl chloride was added, and the mixture was stirred at 30 °C for 90 minutes. 22 g of QS21-SM13B-09 and 1.64 g of DMAP were added at 30 °C, and the mixture was stirred continuously. TLC analysis showed that the reaction proceeds were complete. The reaction solution was concentrated to dryness to obtain crude QS21-SM12-18. Purification was performed by silica gel column chromatography (mobile phase: EA / n-heptane) to give 27.2 g of QS21-SM12-18 as a white solid, with a yield of 66.8%.

[0438] 27.2 g of QS21-SM12-18 was placed in a reaction flask, and 200 mL of tetrahydrofuran was added and stirred until dissolved. The mixture was then cooled to -10 °C. 500 mg of tetrabutylammonium fluoride trihydrate was added, and the mixture was stirred continuously. After the reaction was confirmed to be complete by TLC, 1.2 eq of acetic acid was added to quench the reaction mixture. The reaction solution was concentrated to dryness to obtain 25.1 g of crude QS21-SM12-19. Purification was performed by silica gel column chromatography (mobile phase: EA / n-heptane) to obtain 15.1 g of QS21-SM12-19 as a white solid, with a yield of 74%.

[0439] 1¹H NMR (600 MHz, chloroform-d) δ 7.49 (dd, J = 7.5, 1.9 Hz, 2H), 7.39–7.31 (m, 9H), 7.31–7.20 (m, 15H), 5.96 (s, 1H), 5.70 (s, 1H), 5.42 (dd, J = 3.6, 1.3 Hz, 1H), 5.30 (d, J = 2.7 Hz, 1H), 5.29 (s, 1H), 5.24 (s, 1H), 4.94 (dd, J = 7.9, 3.8 Hz, 1H), 4.89 (d, J = 3.3 Hz, 1H), 4.87 (t, J = 3.8 Hz, 1H), 4.85 (d, J = 3.2 Hz, 1H), 4.83 (d, J = 2.0 Hz, 1H), 4.71–4.66 (m, 1H), 4.65 (dd, J = 11.2, 3.7 Hz, 1H), 4.62–4.56 (m, 2H), 4.53 (d, J = 1.9 Hz, 1H), 4.52–4.38 (m, 4H), 4.27 (dd, J = 6.5, 1.3 Hz, 1H), 4.17 (td, J = 7.3, 5.4 Hz, 2H), 4.12 (d, J = 5.9 Hz, 1H), 4.07 (td, J = 7.3, 6.9, 3.3 Hz, 1H), 4.03 (dd, J = 4.0, 2.1 Hz, 1H), 3.99–3.92 (m, 5H), 3.90–3.82 (m, 2H), 3.80 (dd, J = 9.0, 1.9 Hz, 1H), 3.73–3.65 (m, 3H), 3.63 (s, 3H), 3.58 (td, J = 9.6, 6.7 Hz, 2H), 3.38 (td, J = 9.2, 5.2 Hz, 1H), 3.26 (dd, J = 9.1, 7.5 Hz, 1H), 3.15 (dd, J = 11.7, 9.8 Hz, 1H), 2.75 (d, J = 2.9 Hz, 1H), 2.64–2.54 (m, 2H), 2.52–2.42 (m, 2H), 1.77–1.70 (m, 2H), 1.67 (ddd, J = 15.8, 7.4, 4.9 Hz, 2H), 1.61–1.49 (m, 4H), 1.47 (d, J = 3.5 Hz, 3H), 1.42 (ddd, J = 12.9, 9.2, 5.1 Hz, 1H), 1.33 (d, J = 9.8 Hz, 4H), 1.31–1.19 (m, 8H), 1.15 (d, J = 6.5 Hz, 2H), 1.10–0.96 (m, 3H), 0.91–0.84 (m, 58H), 0.83 (dd, J = 7.2, 3.1 Hz, 9H), 0.11 (s, 3H), 0.06 (td, J = 6.5, 2.1 Hz, 29H).

[0440] Synthesis of Preparation Example 9QS21-02-C-01

[0441]

[0442] 50g of QS21-SM13B-09 was added to a reaction flask. Under nitrogen protection, 2.5L of DMF was added and stirred until dissolved. The mixture was then cooled to 0℃, and 3.0g of sodium hydride, 27.2g of 2-(bromomethyl)naphthalene, and 1.5g of TBAI were added. After 4 hours, TLC showed that the starting material had reacted completely. The reaction was quenched with methanol, water was added, and the mixture was extracted three times with EA. The extract was dried and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (elution gradient: n-heptane:EA = 30:1 → 10:1) to obtain 46.4g of QS21-02-C-01 as a white solid, with a yield of 83%.

[0443] Mass spectrometry: MALDI 1381.638 (+Na).

[0444] Synthesis of Preparation Example 10QS21-02-C-02

[0445]

[0446] 22.0 g of QS21-02-C-01 was added to a reaction flask, and 500 mL of THF was added under nitrogen protection. After stirring and dissolving, the mixture was cooled to 0 °C, and 10.2 g of TBAF·3H2O was added. After 2 hours, TLC showed that the reaction of the starting material was complete. After quenching with water, the mixture was extracted three times with DCM, dried, concentrated, and purified by silica gel column chromatography (elution gradient: n-heptane:EA = 20:1 → 8:1) to obtain 16.1 g of QS21-02-C-02 as a white solid, with a yield of 82.3%.

[0447] Mass spectrometry: HRMS: 1225.5186(+Na).

[0448] Synthesis of Preparation Example 11 QS21-02-C-03

[0449]

[0450] 1.60 g of QS21-02-C-02 was added to a reaction flask, followed by 48 mL of DCM under nitrogen protection. After stirring until dissolved, the mixture was cooled to 0 °C, and 7.68 g of trichloroacetonitrile and 0.81 g of DBU were added. After 4 hours, TLC showed that the reaction of the starting material was complete. The crude product was directly concentrated and purified by silica gel column chromatography (elution gradient: n-hexane:EA = 20:1 → 10:1) to obtain 1.08 g of QS21-02-C-03 as a pale yellow solid, with a yield of 60%.

[0451] Synthesis of Preparation Example 12 QS21-02-C-04

[0452]

[0453] In a 100 mL single-necked flask, 1.0 g of QS21-02-C-03, 1.26 g of QS21-SM17-04, 30 mL of DCM, and 1.0 g of 4A molecular sieve were added. The mixture was stirred at room temperature for 30 min under nitrogen protection, then cooled to -78 °C. 0.12 g of boron trifluoride diethyl ether was added, and the reaction was maintained at this temperature for 3 h. After adjusting the pH to 7-8 with triethylamine, the mixture was concentrated and purified by silica gel column chromatography (elution gradient: n-heptane:EA = 16:1 → 8:1) to obtain 1.42 g of QS21-02-C-04 as a white solid, with a yield of 69%.

[0454] Mass spectrometry: MALDI 3090.246(+Na).

[0455] Synthesis of Preparation Example 13 QS21-02-C-05

[0456]

[0457] 7.6 g of QS21-02-C-04, 380 mL of DCM, 38 mL of water, and 2.0 g of DDQ were added to a reaction flask. After reacting at 25 °C for 1 h, the reaction was quenched with 50 mL of saturated sodium bicarbonate aqueous solution. The mixture was separated, and the aqueous phase was extracted five times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain 5.9 g of QS21-02-C-05 as a white solid, with a yield of 82%.

[0458] Mass spectrometry: MALDI 2950.145(+Na).

[0459] Preparation Example 14: Synthesis of QS-21-Api-01

[0460]

[0461] 50 g of QS21-SM12-19 was added to a single-necked flask. Under nitrogen protection, 1500 mL of DCM was added, the mixture was cooled to 0 °C, 171 g of trichloroacetonitrile was added, and 36 g of DBU was added dropwise. The reaction was carried out at 15 °C for 3 h. The organic phase was concentrated and purified by column chromatography (elution buffer:elution buffer gradient: n-heptane:EA = 8:1) to obtain 43 g of QS21-SM12-20 as a pale yellow solid, with a yield of 80%.

[0462] 43g of the obtained QS21-SM12-20 was added to 2500mL of DCM and 45g of QS21-SM17-04. The mixture was cooled to -70℃, and 3.2g of boron trifluoride diethyl ether was added. The reaction was continued at -70℃. After the reaction was completed, triethylamine was added to quench the reaction, and the crude product was concentrated. The crude product was purified by silica gel column chromatography (elution gradient: n-heptane:EA = 15:1 → 10:1 → 5:1) to obtain 66.4g of QS-21-Api-01 as a white solid, with a yield of 87.2%.

[0463] 1H NMR(600MHz,CDCl3)δ9.53–9.49(s,1H),7.51–7.08(m,65H),5.97–5.93(s,1H),5.72–5.69(s,1H),5.40–5.35(d,J=7.9Hz,1H),5.34–5.31(d,J=3.6Hz,2H),5.19–5.17(d,J=1.7Hz,1H),5.17–5.15(d,J=2.4Hz,2H),4.99–4.95(td,J=5.9,3.5Hz,1H),4.93–4.90(d,J=11.0Hz,1H),4.89–4.88(d,J=5.2Hz,2H),4.86–4.84(s,2H),4.84–4.83(m,2H),4.82–4.79(d,J=7.5Hz,1H),4.79–4.78(d,J=2.0Hz,2H),4.77–4.75(dd,J=10.2,3.4Hz,2H),4.74–4.72(s,1H),4.68–4.64(d,J=11.1Hz,1H),4.64–4.61(d,J=11.2Hz,1H),4.59–4.56(q,J=4.3,3.5Hz,3H),4.51–4.48(d,J=4.9Hz,2H),4.47–4.46(d,J=5.2Hz,1H),4.46–4.45(s,1H),4.44–4.42(d,J=9.5Hz,1H),4.42–4.40(d,J=9.4Hz,1H),4.40–4.38(d,J=4.7Hz,1H),4.38–4.36(d,J=5.1Hz,1H),4.36–4.33(d,J=10.2Hz,1H),4.22–4.20(d,J=7.3Hz,1H),4.15–4.13(m,1H),4.13–4.11(d,J=7.1Hz,1H),4.11–4.08(m,2H),4.04–4.02(dd,J=4.1,2.2Hz,1H),3.98–3.95(m,1H),3.95–3.93(s,2H),3.93–3.86(m,2H),3.85–3.81(dd,J=11.2,4.9Hz,1H),3.81–3.78(d,J=9.3Hz,1H),3.78–3.75(m,1H),3.75–3.73(m,1H),3.72–3.70(m,1H),3.70–3.66(tt,J=6.0,3.1Hz,5H),3.67–3.64(m,2H),3.63–3.60(s,3H),3.60–3.57(m,1H),3.54–3.49(m,2H),3.49–3.43(m,2H),3.43–3.38(m,1H),3.37–3.32(ddt,J=9.4,7.4,4.6Hz,3H), 3.27–3.22(dd,J=9.2,7.6Hz,1H),3.20–3.11(m,2H),2.94–2.88(dd,J=14.1,4.5Hz,1H),2 0.70–2.62(dd,J=15.3,7.2Hz,1H), 2.59–2.53(td,J=8.5,7.9,3.7Hz,2H), 2.52–2.40(m,2H), 2.27–2.18(m,2H), 2.08–2.02(s,2H), 1.88–1.80(tt,J=6.4,2.6Hz,5H), 1.77–1.65(m, 6H), 1.64–1.58(d,J=6.5Hz,6H), 1.55–1.49(qd,J=7.5,4.6Hz,2H), 1.43–1.41(s,3H), 1.33–1.29(s,4H), 1.28–1.24(m,5H), 1.23–1.20(m,7H), 1.17–1.14(d,J=6.4Hz,3H), 1.09–1 .07(s,3H),0.99–0.94(m,12H),0.91–0.84(m,61H),0.84–0.82(d,J=6.8Hz,3H),0.76–0. 74(s,3H),0.69–0.61(m,7H),0.12–0.10(s,3H),0.09–0.04(m,26H),0.01–-0.01(s,1H)。.

[0464] 13C NMR(151MHz,CDCl3)δ213.26,175.25,170.89,170.59,168.34,143.42,139.16,139.13,139.11,138.83,138.72,138.66,138.55,138.33,138.07,137.99,137.96,137.34,137.22,136.77,135.28,129.59,128.51,128.50,128.42,128.41,128.37,128.35,128.34,128.32,128.27,128.24,128.22,128.04,128.01,127.97,127.94,127.92,127.84,127.79,127.76,127.69,127.64,127.62,127.58,127.52,127.50,127.47,127.44,127.38,127.23,127.16,121.61,109.35,109.32,107.25,107.20,106.36,103.88,102.18,101.72,101.33,98.07,93.93,93.87,91.56,87.06,85.86,84.07,83.69,82.66,81.67,81.25,80.94,80.06,79.06,78.42,78.38,78.10,75.73,75.64,75.17,75.12,74.94,74.77,74.49,74.38,74.23,74.02,73.56,73.35,73.22,72.90,72.88,72.65,72.26,71.76,71.64,71.02,69.77,69.63,68.97,68.77,68.15,67.05,67.02,66.99,66.78,66.70,66.18,65.83,63.66,63.16,63.10,62.91,53.72,50.64,48.94,48.93,46.81,46.22,43.44,43.20,42.69,41.47,41.46,40.81,39.72,39.40,39.06,38.68,38.48,38.08,37.27,36.83,36.18,35.26,34.74,32.75,32.31,31.99,31.91,31.88,31.76,30.79,30.57,30.46,29.69,29.36,29.01,27.61,27.58,26.30, 26.04, 26.00, 25.97, 25.95, 25.92, 25.91, 25.88, 25.83, 25.79, 25.75, 25.68, 25.16, 24.95, 24.34, 24.05, 23.81, 23.33, 22.79, 22.68, 22.56, 20.23, 19.18, 18.40, 17.98, 17.94, 17.89, 17.86, 17.85, 17.83, 17.03, 17.01 ,16.33,16.27,15.87,14.78,14.49,14.40,14.11,13.72,12.25,12.20,11.97,11.70,7.14,4.89,4.70,1.01,-0.01,-4.02,-4.22,-4.23,-4.44,-4.52,-4.54,-4.56,-4.59,-4.62,-4.72,-4.75,-4.93,-5.22,-5.25,-5.32。 .

[0465] Synthesis of Preparation Example 15QS-21Api-01

[0466]

[0467] Add 5.9 g of QS21-02-C-05 to a reaction flask, add 100 mL of toluene, and stir until dissolved. Add 500 mg of triethylamine at 30 °C, stir for 5 minutes, then add 800 mg of 2,4,6-trichlorobenzoyl chloride, and stir at 30 °C for 90 minutes. Add 2.6 g of QS21-SM12-17 and 200 mg of DMAP at 30 °C, and continue stirring. TLC analysis showed that the reaction was complete. The reaction solution was concentrated to dryness to obtain the crude product. Purification by silica gel column chromatography (elution gradient: n-heptane:EA = 15:1 → 10:1 → 5:1) yielded 5.1 g of QS-21-Api-01 as a white solid, with a yield of 85.5%. MS: 3998.109 (M+Na).

[0468] Preparation Example 16: Synthesis of QS-21-Api Product

[0469]

[0470] Add 40g of QS-21Api-01 to a flask, followed by 1200mL of DCM. Cool to 0℃, add 1600mL of TFA and 400mL of water, and react at 0℃ for 3h. Concentrate the organic phase to obtain a viscous solid. Add 600mL of a mixed solvent of THF:MeOH = 1:2 and 8g of palladium on carbon (10%) to the solid. After purging with hydrogen three times, perform a hydrogenation reaction. The reaction ends after about 10h. Filter and concentrate to obtain the crude product. Purify the crude product using preparative high-performance liquid chromatography (reversed-phase C18 packing, mobile phase: water:acetonitrile = 63:37), and lyophilize to obtain 2.1g of a white solid, containing 98.34% QS-21-Api and 0.37% isomer B, with a yield of 10%.

[0471] QS-21-Api: 1H NMR(600MHz,CD3CN:D2O=3:7)δ9.46(s,1H),5.36(d,J=7.8Hz,2H),5.33(d,J=3.7Hz,1H),5.25(d,J=2.9Hz,1H),5.18(dt,J=10.1,3.2Hz,1H),5.11(d,J=3.7Hz,1H),4.98(d,J=2.1Hz,1H),4.80(s,1H),4.59(d,J=7.7Hz,1H),4.47(d,J=7.4Hz,3H),4.29(dtd,J=9.5,6.6,2.6Hz,1H),4.16(d,J=9.6Hz,1H),4.06(d,J=2.9Hz,1H),4.02(td,J=5.7,3.1Hz,2H),3.97(dd,J=4.2,2.1Hz,1H),3.94–3.90(m,2H),3.91–3.87(m,3H),3.85(dd,J=6.5,4.0Hz,3H),3.82(d,J=3.6Hz,2H),3.81–3.68(m,10H),3.67(s,3H),3.64(d,J=5.3Hz,1H),3.62(d,J=5.2Hz,1H),3.57(d,J=6.4Hz,1H),3.55–3.47(m,5H),3.45(dd,J=9.2,6.1Hz,2H),3.40(t,J=8.9Hz,1H),3.28–3.17(m,3H),2.94(dd,J=14.3,4.6Hz,1H),2.69–2.57(m,2H),2.52(d,J=6.6Hz,2H),2.31(t,J=13.6Hz,1H),1.95(td,J=13.7,13.1,6.1Hz,5H),1.84(ddd,J=13.3,10.0,2.7Hz,2H),1.80–1.65(m,5H),1.62(qq,J=8.9,4.6Hz,6H),1.54–1.43(m,5H),1.40(s,3H),1.39–1.36(m,1H),1.33(d,J=6.2Hz,4H),1.17(s,3H),1.16–1.11(m,2H),1.11–1.04(m,3H),1.01(s,3H),0.98–0.92(m,12H),0.91(d,J=4.0Hz,3H),0.89(d,J=5.7Hz,5H),0.76(s,3H).

[0472] MS:1988.9266(-H)。

[0473] Isomer B: ¹H NMR (600MHz, CD₃CN:D₂O=3:7) δ 9.37(s,¹H), 5.39(d,J=8.0Hz,¹H), 5.34(s,¹H), 5.20(s,¹H), 5.07(s,¹H), 4.99(s,¹H), 4.97(s,¹H), 4.93(s,¹H), 4.69(d,J=8.1Hz,¹H), 4.19(s,¹H), 4.07(d,J=10.5Hz,¹H) ),3.97(d,J=10.0Hz,5H),3.89(s,3H),3.85(s,2H),3.79(d,J=9.4Hz,3H),3.72(d,J=13.5Hz,6H),3. 68(s,2H),3.62(d,J=7.0Hz,5H),3.57(s,2H),3.49(d,J=9.0Hz,5H),3.46(d,J=9.1Hz,2H),3.39(s,1H ),3.33(d,J=9.2Hz,1H),3.28(s,1H),3.21(d,J=16.0Hz,3H),2.89(d,J=14.3Hz,1H),2.63(d,J=16.3 Hz,1H),2.53(s,3H),2.16(s,1H),1.88(s,4H),1.80–1.63(m,7H),1.56(s,8H),1.40(d,J=13.9Hz,3H ),1.31(s,5H),1.25(d,J=5.5Hz,5H),1.13(d,J=6.2Hz,3H),1.09(s,3H),1.08(m,1H),1.06(m,1H),1 .03(s,3H),0.95(s,3H),0.91(s,3H),0.89–0.78(m,16H),0.70(d,J=14.3Hz,3H).MS:1988.9208(-H).

[0474] Synthesis of Preparation Example 17 QS21-SM9C-02

[0475]

[0476] Under ice bath conditions, 73.5 g of QS21-SM9C-01 and 735 mL of THF were added to a 3 L reaction flask, and the mixture was stirred until dissolved. Maintaining the temperature below 10 °C, 100 mL of a THF solution containing 46.1 g of TBAF·3H2O was added. The ice bath was removed, and the reaction was stirred for 15 h. The reaction was detected by TLC (developing solvent: n-heptane:EA = 2:1) to confirm completion. The reaction solution was quenched in 2 L of water, and the aqueous phase was extracted with 500 mL of ethyl acetate. The organic phases were combined, washed with 500 mL of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated at 45 °C. Purification was performed by silica gel column chromatography: the sample was dissolved and loaded with DCM; eluent: n-heptane:EA = 5:1, yielding 57.36 g of QS21-SM9C-02 as a white solid, yield: 92%.

[0477] MS:1988.9208(+Na)661.2114.

[0478] Synthesis of Preparation Example 18QS21-SM9C-03

[0479]

[0480] At room temperature, 35.5 g of QS21-SM9C-02, 34 g of QS21-SM5-07, 17.5 g of molecular sieve, and 710 g of DCM were added to a reaction flask. The mixture was cooled to -60°C, and 15.0 g of NIS and 4.28 g of AgOTf were added to the reaction system. The mixture was stirred for 30 min. After the starting material was completely eliminated as detected by TLC (eluent: n-heptane:EA = 2:1), triethylamine was slowly added dropwise to the reaction system to control the pH between 7 and 8. The mixture was filtered, and the filter cake was washed with dichloromethane and concentrated under reduced pressure at 45°C. The solution was purified by silica gel column chromatography (eluent: n-heptane:EA = 5:1), and after concentration, 50.98 g of viscous solid QS21-SM9C-03 was obtained, with a yield of 88%.

[0481] 1¹H NMR (600 MHz, chloroform-d) δ 7.48 (dd, J = 6.4, 3.0 Hz, 2H), 7.44–7.34 (m, 5H), 7.34–7.22 (m, 15H), 5.94–5.85 (m, 1H), 5.53 (s, 1H), 5.38–5.31 (m, 1H), 5.16 (s, 1H), 5.15–5.08 (m, 2H), 4.9 0–4.86(m,3H),4.85(d,J=7.6Hz,3H),4.79(dd,J=10.5,3.5Hz,1H),4.71(d,J=7.3Hz,1 H),4.65(s,1H),4.63(d,J=5.0Hz,1H),4.56(d,J=11.7Hz,1H),4.31(t,J=9.3Hz,1H),4. 26(dd,J=10.2,4.8Hz,1H), 4.15(dd,J=13.5,5.0Hz,1H), 4.05(dd,J=13.4,5.5Hz,1H), 3.92(ddd,J=23.5,10.7,5.9Hz,5H),3.74–3.66(m,2H),3.62(dd,J=8.8,5.2Hz,1H),3.5 8(t,J=8.6Hz,1H),3.50(t,J=9.5Hz,1H),3.42–3.36(m,1H),3.12(dd,J=11.7,9.2Hz,1 H), 2.96 (t, J = 6.6Hz, 1H), 2.10 (s, 3H), 2.05 (s, 3H), 2.02 (s, 3H), 1.96 (d, J = 2.6Hz, 3H).

[0482] 13 C NMR (151 MHz, chloroform-d) δ 170.18, 170.17, 169.92, 169.23, 133.86, 128.49, 128.40, 128.30, 128.10, 127.91, 127.75, 127.70, 127.56, 126.11, 117.08, 102.04, 10 1.26,100.71,98.56,83.48,82.37,79.91,79.56,75.16,74.81,73.76,72.84,70.91,70.18,69.31,68.82,67.14,62.35,61.01,21.23,20.67,20.61,20.53.

[0483] MS(+Na) 1063.3925.

[0484] Synthesis of Preparation Example 19QS21-SM9C-04

[0485]

[0486] Add 50.97 g of QS21-SM9C-03, 1000 mL of methanol, 100 mL of water, and 67.7 g of potassium carbonate to a 2 L reaction flask, and stir at 25 °C for 3 hours. After the starting material has completely disappeared as determined by TLC, filter the reaction mixture. Wash the filter cake with methanol, concentrate at 45 °C, and then purify by silica gel column chromatography (eluent: n-heptane:EA = 1:1, DCM:MeOH = 50:1). The concentrated product yields 28.20 g of QS21-SM9C-04 as a pale yellow paste, with a yield of 66%.

[0487] MS(+Na) 895.3497.

[0488] Synthesis of Preparation Example 20QS21-SM9C-05

[0489]

[0490] Add 17.09 g of QS21-SM9C-04 and 341 g of DMF to a 1 L reaction flask and cool to 0 °C. Slowly add 8.22 g of sodium hydride and 0.14 g of tetrabutylammonium iodide. After 10 min, add 33.5 g of benzyl bromide to the reaction system and continue stirring at 25 °C for 3 h. After the starting material has completely disappeared as detected by TLC, cool the reaction system to 0 °C and quench the reaction by adding 1 mL of methanol. Add 300 mL of water and extract to the aqueous phase with 500 mL of DCM. Combine the organic phases, wash the organic phase with saturated sodium chloride aqueous solution, dry with anhydrous sodium sulfate, and concentrate at 45 °C. Purify by silica gel column chromatography (eluent: n-heptane:EA = 5:1). The concentrate yielded 20.05 g of pure QS21-SM9C-05 as a pale yellow paste, with a yield of 83%.

[0491] 11H NMR (600 MHz, CDCl3) δ 7.59 (dd, J=6.8, 2.9 Hz, 2H), 7.49 (d, J=7.5 Hz, 2H), 7.47–7.13 (m, 36H), 5.97 (ddt, J=16.1, 10.4, 5.2 Hz, 1H), 5.60 (s, 1H), 5.45–5.38 (m, 1H), 5.17 (d, J=10.5 Hz, 1H), 5.07 (d, J=11.8 Hz, 1H), 5.02 (d, J=5.1 Hz, 2H), 4.96 (d, J=11.5 Hz, 1H), 4.92 (dd, J=11.7, 8.1 Hz, 2H), 4.86 (s, 2H), 4.84–4.78 (m, 2H), 4.78–4.72 (m, 2H), 4.64 (d, J=11.7 Hz, 1H), 4.58 (d, J=11.5 Hz, 1H), 4.54 (d, J=11.7 Hz, 1H), 4.53–4.46 (m, 3H), 4.36 (dd, J=10.2, 4.9 Hz, 1H), 4.25 (dd, J=13.3, 5.1 Hz, 1H), 4.12 (dd, J=13.3, 5.4 Hz, 1H), 4.04 (td, J=10.0, 4.8 Hz, 1H), 3.94 (dd, J=9.4, 3.8 Hz, 1H), 3.86–3.74 (m, 3H), 3.68 (d, J=3.0 Hz, 1H), 3.64–3.55 (m, 2H), 3.51 (dd, J=13.3, 7.6 Hz, 3H), 3.43–3.32 (m, 2H), 2.95 (t, J=6.5 Hz, 1H), 2.77 (dd, J=11.8, 9.3 Hz, 1H).

[0492] 13C NMR (151MHz, chloroform-d) δ 139.20, 139.11, 139.02, 138.93, 138.82, 138.69, 138.27, 137.69, 134.14, 128.78, 128.48, 128.42, 128.38, 128.37, 128.34, 128.25, 128.20, 128.17, 128.04, 127.87, 127.86, 127.76, 127.74, 127.68, 127.63, 127.55, 1 27.51,127.49,127.45,127.43,127.31,126.24,116.80,103.08,101.54,101.33,99.20,83.57,82.45,81.88,80.00,79.51,78.41,75.21,74.85,74.67,74.58,74.43,74.41,73.39,73.15,73.08,72.52,69.29,68.82,68.72,63.20,62.60.

[0493] Synthesis of Preparation Example 21QS21-SM9C-06

[0494]

[0495] Add 20.05 g of QS21-SM9C-05, 450 mL of DCM, and 150 mL of MeOH (DCM:MeOH = 3:1) to a 1 L reaction flask, then add 4.64 g of p-toluenesulfonic acid monohydrate, and heat to 45 °C for 4 h. After the starting material has completely disappeared as detected by TLC, cool the reaction system to 0 °C, add triethylamine to quench the reaction (controlling the pH to 7), filter, wash the filter cake with DCM, and concentrate the filtrate at 45 °C. Purify by silica gel column chromatography (eluent: n-heptane:EA = 5:1), and concentrate to obtain 9.68 g of QS21-SM9C-06 as a pale yellow paste, with a yield of 52%.

[0496] MS(+Na) 1167.5033.

[0497] Synthesis of Preparation Example 22 QS21-SM9C-07

[0498]

[0499] Add 9.68 g of QS21-SM9C-06, 200 mL of dichloromethane, and 100 mL of water to a reaction flask. Add 0.59 g of TEMPO and 6.80 g of BAIB, and stir the reaction for 14 h. After the starting material has completely disappeared as determined by TLC (eluent: dichloromethane:methanol = 10:1), quench the reaction by adding 1 M sodium thiosulfate aqueous solution. Wash the aqueous phase with DCM, dry with anhydrous sodium sulfate, and concentrate at 45 °C. Purify by silica gel column chromatography (eluent: n-heptane:EA = 1:1, DCM:MeOH = 20:1), concentrate, and obtain 7.74 g of QS21-SM9C-07 as a white solid, yield 79%. Proceed directly to the next step.

[0500] Synthesis of Preparation Example 23 QS21-SM9C-09

[0501]

[0502] 7.73 g of QS21-SM9C-07 and 150 mL of DMF were added to a reaction flask and stirred until dissolved. The mixture was cooled to 0 °C, and then 0.67 g of sodium hydride and 11.4 g of benzyl bromide were added. The ice bath was removed, and the mixture was stirred at room temperature for 16 h. TLC analysis (developing solvent: dichloromethane:methanol = 10:1, n-heptane:EA = 2:1) confirmed complete disappearance of the starting material. 200 mL of ethyl acetate was added to dilute the reaction mixture, and the pH was adjusted to 6 with dilute hydrochloric acid. 100 mL of water was added, and the mixture was extracted. The aqueous phase was washed with 200 mL of ethyl acetate, dried over anhydrous sodium sulfate, and concentrated at 45 °C. The solution was purified by silica gel column chromatography (eluent: n-heptane:EA = 5:1) to obtain a white powdery solid, QS21-SM9C-09, in 50% yield.

[0503] 1¹H NMR (600 MHz, chloroform-d) δ 7.53–7.48 (d, J = 7.6 Hz, 3H), 7.45–7.32 (tq, J = 20.1, 7.7 Hz, 33H), 7.30–7.22 (dt, J = 17.9, 7.4 Hz, 10H), 6.00–5.90 (ddt, J = 16.3, 10.6, 5.3 Hz, 1H), 5.42–5.36 (d, J = 17.2 Hz, 1H), 5.31–5.26 (d, J = 11.6 Hz, 2H), 5.25–5.21 (d, J = 12.4 Hz, 1H), 5.17–5.13 (d, J = 10.5 Hz, 1H), 5.05–4.99 (m, 3H), 4.99–4.91 (m, 5H), 4.91–4.88 (m, 4H), 4.86–4.75 (m, 3H), 4.73–4.70 (d, J = 7.8 Hz, 1H), 4.69–4.65 (d, J = 11.8 Hz, 1H), 4.60–4.56 (dd, J = 11.6, 4.7 Hz, 2H), 4.52–4.46 (m, 4H), 4.43–4.39 (d, J = 10.0 Hz, 1H), 4.27–4.22 (dd, J = 13.3, 5.1 Hz, 1H), 4.12–4.06 (dd, J = 13.3, 5.4 Hz, 1H), 3.93–3.87 (dd, J = 9.7, 3.6 Hz, 1H), 3.81–3.74 (q, J = 9.7 Hz, 2H), 3.69–3.63 (q, J = 6.7 Hz, 2H), 3.54–3.40 (tq, J = 22.4, 8.8, 7.2 Hz, 5H), 3.39–3.35 (dd, J = 9.8, 3.0 Hz, 1H), 3.31–3.26 (t, J = 8.5 Hz, 1H), 2.78–2.70 (t, J = 6.8 Hz, 1H), 2.42–2.34 (t, J = 11.0 Hz, 1H).

[0504] 13C NMR(151MHz,Chloroform-d)δ170.00,139.09,138.93,138.87,138.84,138.75,138.60,138.14,138.11,135.29,133.79,128.5 4,128.42,128.38,128.35,128.33,128.32,128.24,128.12,128.08,128.00,127.88,127.75,127.70,127.65,127.63,127.61, 127.57,127.55,127.53,127.51,127.42,127.37,127.29,116.91,102.47,101.74,98.80,83.79,82.94,81.79,79.60,78.45,78.13,77.31,76.71,75.62,74.98,74.93,74.90,74.67,74.30,73.32,72.85,72.75,72.68,70.27,68.82,68.22,67.12,63.07.

[0505] MS(+Na) 1361.5784.

[0506] Synthesis of Preparation Example 24QS21-SM9C-10

[0507]

[0508] 4.47 g of QS21-SM9C-09 and 90 mL of acetic acid were added to a 250 mL reaction flask. After purging with argon, 1.54 g of tetrakis(triphenylphosphine)palladium was added, and the reaction was carried out at 40 °C for 4 h. After the starting material was completely eliminated by TLC (n-heptane:EA = 3:1), the organic phase was concentrated and purified by silica gel column chromatography (eluent: n-heptane:EA = 10:1-8:1-5:1) to obtain 3.90 g of QS21-SM9C-103 as a white solid, with a yield of 90%.

[0509] MS(+Na)1321.5461.

[0510] Synthesis of Preparation Example 25QS21-SM9C-11

[0511]

[0512] 3.89 g of QS21-SM9C-10 and 100 mL of dichloromethane were added to a reaction flask, stirred until dissolved, purged with argon, and cooled to 0 °C. Then, 4.34 g of trichloroacetonitrile and 0.23 g of DBU were added, and the mixture was stirred at 0 °C for 3 h. TLC analysis (n-heptane:EA = 3:1) confirmed the complete disappearance of the starting material. The mixture was concentrated and purified by column chromatography (basic alumina as the stationary phase; eluent: n-heptane:EA = 10:1). Concentration yielded 2.17 g of QS21-SM9C-11 as a white solid, yield: 50%.

[0513] Synthesis of Preparation Example 26 QS21-SM17-01

[0514]

[0515] Add 0.1 g of QS21-SM9C-11 and 0.038 mg of QS21-C-SM1-02 (obtained from J.AM.CHEM.SOC.2005, 127, 3256-3257) to a 50 mL single-necked flask. Add 2 mL of DCM and stir at room temperature for 30 min. Cool to 5-10 °C, add 1.1 mg of tris(pentafluorophenyl)borane in dichloromethane solution, and react at 20 °C for 1 h. After the reaction of the starting material is complete as determined by TLC, triethylamine is added to quench the reaction until the pH of the system is 6-7. The system is concentrated to obtain the crude product, which is purified by silica gel column chromatography (n-heptane:EA = 15:1 → 10:1) to obtain 0.097 g of QS21-SM17-01 as a white solid, with a yield of 77.0%.

[0516] (MALDI)(+Na)1830.9019.

[0517] Synthesis of Preparation Example 27 QS21-SM17-03

[0518]

[0519] Add 2.04 g of QS21-SM17-01 to the reaction flask. Under nitrogen protection, add 40 mL of DCM and 1.46 g of 2,6-dimethylpyridine. Cool to 5 °C, add 2.4 g of TESOTf, and react at 15-20 °C for 3 h. After the starting material has completely disappeared as detected by TLC, concentrate the organic phase and purify by silica gel column chromatography (eluent: n-heptane: EA = 15:1 → 12:1 → 10:1) to obtain 1.69 g of QS21-SM17-03 as a white solid, with a yield of 78%.

[0520] (MALDI)(+Na)1944.9837.

[0521] Synthesis of Preparation Example 28 QS21-SM17-04

[0522]

[0523] 1.3 g of QS21-SM17-03 and 76 mL of acetic acid were added to a 250 mL single-necked flask. After purging with nitrogen three times, tetra(triphenyl)phosphine palladium was added, and the reaction was carried out at 30 °C for 2 h. TLC showed that the starting material had completely disappeared. The reaction system was concentrated under reduced pressure at 50 °C to obtain a crude product, which was purified by silica gel column chromatography (n-heptane:EA = 10:1 → 5:1 → 2:1) to obtain 1.02 g of QS21-SM17-04 as a white powder, with a yield of 81%.

[0524] 1¹H NMR (600 MHz, chloroform-d) δ 9.52–9.49 (s, 1H), 7.42–7.32 (m, 10H), 7.33–7.22 (m, 25H), 7.22–7.08 (m, 10H), 5.36–5.30 (t, J=3.7 Hz, 1H), 5.18–5.13 (d, J=2.6 Hz, 2H), 4.93–4.87 (m, 2H), 4.87–4.83 (m, 2H), 4.82–4.80 (d, J=11.8 Hz, 1H), 4.80–4.72 (dt, J=20.7, 6.8 Hz, 7H), 4.60–4.56 (d, J=11.8 Hz, 1H), 4.56–4.53 (d, J=3.7 Hz, 1H), 4.50–4.43 (m, 2H), 4.41–4.37 (d, J=11.1 Hz, 2H), 4.36–4.32 (d, J=10.2 Hz, 1H), 4.22–4.18 (d, J=7.5 Hz, 1H), 3.93–3.85 (m, 2H), 3.72–3.59 (m, 5H), 3.55–3.50 (t, J=8.5 Hz, 1H), 3.49–3.43 (m, 2H), 3.42–3.38 (dd, J=9.9, 6.0 Hz, 1H), 3.38–3.31 (qd, J=10.0, 3.4 Hz, 2H), 3.21–3.14 (t, J=8.4 Hz, 1H), 2.98–2.92 (dd, J=14.2, 4.4 Hz, 1H), 2.58–2.53 (t, J=6.9 Hz, 1H), 2.25–2.16 (td, J=13.7, 12.4, 6.6 Hz, 2H), 1.91–1.81 (m, 4H), 1.80–1.73 (m, 2H), 1.73–1.67 (d, J=14.5 Hz, 1H), 1.67–1.58 (m, 2H), 1.56–1.50 (d, J=13.1 Hz, 1H), 1.42–1.35 (s, 1H), 1.35–1.30 (s, 3H), 1.31–1.25 (m, 2H), 1.22–1.14 (d, J=20.1 Hz, 3H), 1.09–1.02 (s, 5H), 1.03–0.97 (t, J=7.9 Hz, 9H), 0.97–0.95 (s, 3H), 0.90–0.86 (d, J=15.0 Hz, 6H), 0.71–0.64 (m, 10H).

[0525] Found: 1904.9511 for (MALDI) (+Na).

[0526] Experimental Example

[0527] Experimental Example 1: Immune response in BALB / c mice (compared to a mixture of naturally extracted QS-21)

[0528] Female BALB / c mice aged 6-8 weeks were selected. An experimental group (n=6 / group) was prepared using lyophilized VZV-gE powder combined with BDS01 adjuvant (containing the QS-21-Api product of this invention and the TLR agonist MPLA). A control group (n=6 / group) was prepared using lyophilized VZV-gE antigen combined with A01 adjuvant (containing the naturally extracted QS-21 mixture already used in commercially available vaccines and the TLR agonist MPLA). Both groups were immunized twice via intramuscular injection, with an interval of 14 days between immunizations.

[0529] The dose level of BDS01 adjuvant in the experimental group vaccine was 5 μg QS-21-Api per animal, which is equivalent to 1 / 10 of the human dose.

[0530] Serum samples were collected on days 13 and 28 after the initial immunization for specific antibody detection. On day 28, spleen cells were extracted, ground, and isolated for cellular immunoassay.

[0531] ELISpot (Enzyme-Linked Immunosorbent Assay)

[0532] The separated mouse spleen cells from the previous step were placed in pre-chilled sterile PBS and dissociated using a Miltenyi tissue dissociator. The dissociated single-cell suspension was transferred to 50 ml polypropylene tubes. The cells were filtered through a 70 μm cell sieve, and the filtrate was treated with ACK lysis buffer and washed with PBS by centrifugation. The supernatant was discarded, and the cells were resuspended in complete culture medium and counted. The cell density was adjusted to 1 × 10⁻⁶ cells based on the count results. 7 Cells / ml

[0533] Pre-coated Mabtech 96-well plates (IFN-γ and IL-2) were conditioned in α-MEM complete medium for 1–4 hours. Cells were cultured at 2.5 × 10⁶ cells / well. 5 Seed 50 μL / well of T cells into a 96-well plate, then stimulated again with peptide gE or culture medium (negative control) overnight (20 h). Discard the liquid from the 96-well plate, wash 5 times with PBS, add biotinylated detection antibody (anti-mouse IFN-γ or IL-2), incubate at room temperature (25°C) for 2 h, wash 5 times with PBS, add Streptavidin-ALP and incubate again at room temperature for 1 h, wash 5 times with PBS, add the chromogenic substrate BCIP / NBT-plus, develop in the dark for 10 min, then slowly rinse with tap water to stop the development. Air dry the reaction strips at room temperature in the dark, and count the spots using an ELISpot reader.

[0534] The number of spots in the test sample is the number of spots in the peptide-stimulated wells minus the number of spots in the negative control wells. The final result is reported in ELISPOT detection units (SFC / Million, Spots Forming Cells per Million cells), which is the number of IFN-γ or IL-2 positive cells per million cells. The results are displayed on... Figure 1 middle.

[0535] ELISA (Enzyme-Linked Immunosorbent Assay)

[0536] Total anti-gE IgG was determined by ELISA. A 96-well microplate was coated with gE antigen overnight at 4°C. After washing twice with PBST (containing 0.05% Tween 20), 1% casein-PBS blocking buffer was added. The plate was incubated at 25°C for 1 hour, followed by washing twice with PBST. Then, 50 μl of serially diluted mouse serum or positive serum was added, and the plate was incubated at 25°C for 1 hour, followed by washing four times with PBS.

[0537] Then, add 50 μL of 1:5000 diluted Goat Anti-Mouse IgG H&L (HRP) working solution per well. After incubating at room temperature for 1 hour, wash 6 times with PBST. Next, add 100 μL of TMB chromogenic solution to each well, and incubate at room temperature in the dark for 10 minutes. Finally, add 100 μL of 1M HCl to each well to stop the incubation. Set the microplate reader to a master wavelength of 450 nm and a reference wavelength of 620 nm. The sample absorbance = OD0. 450 -OD 620 A four-parameter fit was performed with the sample dilution factor as the X-axis and the absorbance value as the Y-axis. Then, the threshold (OD = 0.15) was used as the Y-value for interpolation to obtain the sample dilution factor X-value, which is the antibody titer value of the sample. The results are shown in [the table / image / etc.]. Figure 4 .

[0538] in conclusion

[0539] The adjuvant BDS01 prepared from the synthetic QS-21-Api product of the present invention has superior cellular immunogenicity to adjuvant A01 (containing a mixture of naturally extracted QS-21), and its humoral immunogenicity is comparable to that of adjuvant A01. The humoral immune response after one immunization and after two immunizations is not inferior to that of adjuvant A01, and there is no statistically significant difference.

Claims

1. A synthetic saponin composition, as determined by ultraviolet absorbance at 200 nm, wherein the synthetic saponin composition comprises at least 90% saponin QS-21-Api and less than 1.0% isomer B:

2. The synthetic saponin composition according to claim 1, comprising 90-99.8%, preferably 93-99.5%, more preferably 95-99%, and even more preferably 95-98% of said saponin QS-21-Api.

3. The synthetic saponin composition according to claim 1 or 2, comprising less than 0.5%, less than 0.1%, and less than 0.05% of said isomer B.

4. A method for synthesizing the saponin composition according to any one of claims 1-3, comprising the following steps: (a) Compound I-2 is obtained by glycosylation of compound I-1 with thioglycoside SAP-2; (b) The Pg3 protecting group was removed from the compound of formula I-2 to obtain the compound of formula I-3, and the compound of formula I-3 was subjected to a glycosylation reaction with the thioglycoside SAP-5 to obtain the compound of formula I-4. (c) The synthetic saponin composition is obtained by reacting the compounds of formulas I-4, wherein... Each of R2 is an independent hydroxyl protecting group, preferably benzyl. Or, two R2 atoms together with the oxygen atom they are attached to form... Structure, where R x and R y Each independently represents H and C. 1-6 Alkyl and phenyl, preferably H, methyl or phenyl; R4 is selected from C 1-6 Alkyl groups and optionally C 1-6 Alkyl-substituted phenyl groups are preferably methyl, ethyl, propyl, phenyl, p-methylphenyl, o-methylphenyl, or m-methylphenyl. Pg1, Pg2, and Pg3 are each independently a hydroxyl protecting group. Preferably, Pg1 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, or tert-butyldiphenylsilyl; Pg2 is selected from benzyl, C 1-6 Alkylbenzyl, naphthylmethyl, or triphenylmethyl; Pg3 is selected from acetyl, pivaloyl, or benzoyl, more preferably, Pg1 is triisopropylsilyl, Pg2 is benzyl, and Pg3 is acetyl.

5. The synthesis method according to claim 4, wherein step (a) is carried out in the presence of the following reagents: silver trifluoromethanesulfonate, silver oxide, silver carbonate, silver nitrate, silver perchlorate, mercuric chloride, mercuric bromide, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, trifluoromethanesulfonic anhydride, trifluoromethanesulfonic acid, boron trifluoride ether, scandium trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, copper trifluoromethanesulfonate, ferric trifluoromethanesulfonate, ferric chloride, aluminum trichloride, tin tetrachloride, stannous chloride, zinc chloride, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, N-iodosuccinimide, N-bromosuccinimide, or any combination thereof, preferably a combination of N-iodosuccinimide and silver trifluoromethanesulfonate.

6. The synthesis method according to claim 4, wherein the removal of the Pg3 protecting group in step (b) is carried out in the presence of sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium tert-butoxide, sodium amino, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, barium hydroxide, calcium hydroxide, magnesium hydroxide, lithium hydroxide, hydrazine hydrate, ammonia methanol, ammonia ethanol, or ammonia water.

7. The synthesis method according to claim 4, wherein the glycosylation reaction in step (b) is carried out in the presence of the following reagents: silver trifluoromethanesulfonate, silver oxide, silver carbonate, silver nitrate, silver perchlorate, mercuric chloride, mercuric bromide, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, trifluoromethanesulfonic anhydride, trifluoromethanesulfonic acid, boron trifluoride ether, scandium trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, copper trifluoromethanesulfonate, ferric trifluoromethanesulfonate, ferric chloride, aluminum trichloride, tin tetrachloride, stannous chloride, zinc chloride, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, N-iodosuccinimide, N-bromosuccinimide, or any combination thereof, preferably a combination of N-iodosuccinimide and trimethylsilyl trifluoromethanesulfonate.

8. The synthesis method according to any one of claims 4-7, wherein step (c) comprises: (c1) The Pg1 protecting group of compound I-4 is removed to obtain compound I-5. Compound I-5 is reacted with trichloroacetonitrile to generate trichloroacetylimine ester. The trichloroacetylimine ester is glycosylated with compound SAP-9 to obtain compound I-6. Compound I-6 is removed from the Pg3 protecting group to obtain compound I-7. Preferably, step (c) further includes: (c2) The compound of formula I-7 is subjected to an esterification reaction with the compound of formula I-9 to obtain the compound of formula I-10; Preferably, step (c) further includes: (c3) Remove the Pg1 protecting group from the compound of formula I-10 to obtain the compound of formula I-11, react the compound of formula I-11 with trichloroacetonitrile to generate trichloroacetylimine ester, and glycosylate the trichloroacetylimine ester with compound SAP-18 to obtain the compound of formula I-12. Preferably, step (c) further includes: (c4) Remove all protecting groups from the compound of formula I-12 to obtain the synthetic saponin composition. Each of R1 is an independent hydroxyl protecting group. Preferably, each of R1 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl or benzyl. More preferably, each of R1 is tert-butyldimethylsilyl or benzyl. Each of Y1 is an independent hydroxyl protecting group. Preferably, each of Y1 is independently selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl or benzyl. More preferably, Y1 is benzyl. Y2 is a hydroxyl protecting group. Preferably, Y2 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl or benzyl. More preferably, Y2 is selected from triethylsilyl or benzyl.

9. The synthesis method according to claim 8, wherein the glycosylation reaction in step (c1) is carried out in the presence of the following reagents: silver trifluoromethanesulfonate, silver oxide, silver carbonate, silver nitrate, silver perchlorate, mercuric chloride, mercuric bromide, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, trifluoromethanesulfonic anhydride, trifluoromethanesulfonic acid, boron trifluoride ether, scandium trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, copper trifluoromethanesulfonate, ferric trifluoromethanesulfonate, ferric chloride, aluminum trichloride, tin tetrachloride, stannous chloride, zinc chloride, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, or any combination thereof, preferably trimethylsilyl trifluoromethanesulfonate.

10. The synthesis method according to claim 8, wherein the esterification reaction in step (c2) is carried out in the presence of 2,4,6-trichlorobenzoyl chloride and 4-dimethylaminopyridine.

11. The synthesis method according to claim 8, wherein the glycosylation reaction in step (c3) is carried out in the presence of the following reagents: silver trifluoromethanesulfonate, silver oxide, silver carbonate, silver nitrate, silver perchlorate, mercuric chloride, mercuric bromide, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, trifluoromethanesulfonic anhydride, trifluoromethanesulfonic acid, boron trifluoride ether, scandium trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, copper trifluoromethanesulfonate, ferric trifluoromethanesulfonate, ferric trichloride, aluminum trichloride, tin tetrachloride, stannous chloride, zinc chloride, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, or any combination thereof, preferably boron trifluoride ether.

12. The synthesis method according to claim 8, wherein step (c4) is carried out under hydrogenation conditions, preferably in the presence of a hydrogenation catalyst, wherein the hydrogenation catalyst is preferably palladium on carbon or palladium hydroxide on carbon.

13. The synthesis method according to claim 8 or 12, wherein step (c4) further comprises, prior to the hydrogenation, treating the compound of formula I-12 with an acid, preferably trifluoroacetic acid.

14. The synthesis method according to claim 8, wherein step (c4) is carried out in the presence of an acid, preferably formic acid, acetic acid, propionic acid, benzoic acid, benzenesulfonic acid, p-toluenesulfonic acid, or trifluoroacetic acid, more preferably trifluoroacetic acid.

15. A method for synthesizing the synthetic saponin composition according to any one of claims 1-3, comprising the following steps: (a') Protecting the hydroxyl group of the compound of formula II-1-1 to obtain the compound of formula II-1, and then removing the Pg4 protecting group from the compound of formula II-1 to obtain the compound of formula II-2; (b') React the compound of formula II-2 with a halogen-containing electrophilic reagent to generate an imine ester of formula II-2-1, and then glycosylate the imine ester of formula II-2-1 with compound SAP-18 to obtain compound II-3. (c') The synthetic saponin composition is obtained by reacting the compound of formula II-3. R6 is an independent hydroxyl protecting group, preferably benzyl. Or two R6 atoms together with the oxygen atom they are attached to form Structure, where R x and R y Each independently represents H and C. 1-6 Alkyl and phenyl, preferably H, methyl or phenyl; Pg4 and Pg5 are each independently a hydroxyl protecting group. Preferably, Pg4 is selected from triisopropylsilyl, tert-butyldimethylsilyl or trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl; Pg5 is selected from benzyl, C 1-6 Alkylbenzyl, naphthylmethyl, or triphenylmethyl, more preferably, Pg4 is triisopropylsilyl and Pg5 is benzyl; R3 is allyl, methylthiomethyl, 3,4-dimethoxybenzyl, p-methoxybenzyl, 2,3,4-trimethoxybenzyl, 3,4,5-trimethoxybenzyl, 2,5-dimethoxybenzyl, 2-methoxybenzyl, 3,5-dimethoxybenzyl, 2,3-dimethoxybenzyl, 3-methoxybenzyl, 2,6-dimethoxybenzyl, 1-naphthylmethyl, or 2-naphthylmethyl; R5 is Where X is a halogen, preferably chlorine or fluorine; and R z R5 is H or phenyl, more preferably, R5 is Each of Y1 is an independent hydroxyl protecting group. Preferably, each of Y1 is independently selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl or benzyl. More preferably, Y1 is benzyl. Y2 is a hydroxyl protecting group. Preferably, Y2 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl or benzyl. More preferably, Y2 is selected from triethylsilyl or benzyl.

16. The synthesis method according to claim 15, wherein the glycosylation reaction in step (b') is carried out in the presence of the following reagents: silver trifluoromethanesulfonate, silver oxide, silver carbonate, silver nitrate, silver perchlorate, mercuric chloride, mercuric bromide, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, methyl trifluoromethanesulfonate, trifluoromethanesulfonic anhydride, trifluoromethanesulfonic acid, boron trifluoride ether, scandium trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, copper trifluoromethanesulfonate, ferric trifluoromethanesulfonate, ferric chloride, aluminum trichloride, tin tetrachloride, stannous chloride, zinc chloride, zinc bromide, zinc iodide, zinc trifluoromethanesulfonate, or any combination thereof, preferably boron trifluoride ether.

17. The synthesis method according to claim 15, wherein step (c') comprises: (c1') Selectively remove the R3 protecting group from the compound of formula II-3 to obtain the compound of formula II-4; Preferably, step (c') further includes: (c2') The compound of formula II-4 is subjected to an esterification reaction with the compound of formula II-5 to obtain the compound of formula II-6; Preferably, step (c') further includes: (c3') Remove all protecting groups from the compound of formula II-6 to obtain the synthetic saponin composition. in R1 is a hydroxyl protecting group, preferably selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl or benzyl, more preferably, R1 is tert-butyldimethylsilyl or benzyl.

18. The synthesis method according to claim 17, wherein step (c1') is carried out in the presence of a catalyst of palladium, iridium, rhodium, mercury, ruthenium, platinum, copper, silver, gold or a combination thereof.

19. The synthesis method according to claim 17, wherein step (c1') is carried out in the presence of 2,3-dichloro-5,6-dicyanobenzoquinone, cerium ammonium nitrate, silver oxide, iodine, bromine, bromosuccinimide, magnesium perchlorate, or any combination thereof.

20. The synthesis method according to claim 17, wherein step (c2') is carried out in the presence of 2,4,6-trichlorobenzoyl chloride and 4-dimethylaminopyridine.

21. The synthesis method according to claim 17, wherein step (c3') is carried out under hydrogenation conditions, preferably in the presence of a hydrogenation catalyst, said hydrogenation catalyst being palladium on carbon or palladium hydroxide on carbon.

22. The synthesis method according to claim 21, wherein step (c3') is carried out in the presence of an acid, preferably formic acid, acetic acid, propionic acid, benzoic acid, benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, citric acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, or trifluoroacetic acid. More preferably, the acid is trifluoroacetic acid.

23. The synthesis method according to claim 21, wherein step (c3') further comprises, prior to the hydrogenation, treating the compound of formula I-12 with an acid, preferably trifluoroacetic acid.

24. A method for synthesizing intermediate SAP-18 as described in claim 8 or 15, comprising the following steps: (a”) Selectively remove the TBS protecting group from the compound of formula III-1 to obtain the compound of formula III-2, and then perform a glycosylation reaction between the compound of formula III-2 and the compound of formula III-3 to obtain the compound of formula III-4. (b”) React the compound of formula III-4 with a basic reagent or a reducing reagent to generate compound III-5, and react the compound of formula III-5 with a silyl ether reagent or benzyl bromide to obtain compound III-6; (c”) React the compound of formula III-6 with an acidic reagent to generate compound III-7, react the compound of formula III-7 with an oxidizing reagent to obtain compound III-8, and react the compound of formula III-8 with a silyl ether reagent or benzyl bromide to obtain compound III-9. (d”) The compound of formula III-9 is reacted with a metal catalyst to remove the protecting group to obtain compound III-10, and compound III-10 is reacted with a halogen-containing electrophilic reagent to obtain compound III-11. (e”) The intermediate SAP-18 is obtained by reacting the compound of formula III-11. Wherein, each of Y1 is an independent hydroxyl protecting group, preferably, each of Y1 is independently selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl or benzyl, more preferably, Y1 is benzyl; R9 is Where X is a halogen, preferably chlorine or fluorine; and R z R9 is H or phenyl, more preferably, R9 is R8 is allyl or 2-butenyl, preferably allyl; and R 10 It is either acetyl or benzoyl.

25. The synthesis method according to claim 24, wherein the glycosylation reaction in step (a”) is carried out in the presence of silver trifluoromethanesulfonate, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, trifluoromethanesulfonic acid, boron trifluoride ether, N-iodosuccinimide or any combination thereof, preferably a combination of N-iodosuccinimide and silver trifluoromethanesulfonate.

26. The synthesis method according to claim 24, wherein the alkaline reagent or reducing agent in step (b”) is sodium hydroxide, lithium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, diisobutylaluminum hydride, sodium borohydride, lithium aluminum hydride, or any combination thereof.

27. The synthesis method according to claim 24, wherein the acidic reagent in step (c”) is hydrochloric acid, p-toluenesulfonic acid, methanesulfonic acid, or any combination thereof.

28. The synthesis method according to claim 24, wherein the oxidizing agent in step (c”) is 2,2,6,6-tetramethylpiperidine oxide, iodophenyldiacetic acid, Desmond-Martin oxidant, sodium hypochlorite, or any combination thereof.

29. The synthesis method according to claim 24, wherein the metal catalyst in step (d”) is a palladium, iridium, or ruthenium catalyst.

30. The synthesis method according to claim 24, wherein step (e”) comprises: (e1”) Perform a glycosylation reaction between the compound of formula III-11 and the compound of formula III-12 to obtain the compound of formula III-13; Preferably, step (e”) further includes: (e2”) React the compound of formula III-13 with a silyl ether reagent or benzyl bromide to obtain the compound of formula III-14, and react the compound of formula III-14 with a metal catalyst to obtain intermediate SAP-18. Wherein, Y2 is a hydroxyl protecting group, preferably, Y2 is selected from triisopropylsilyl, tert-butyldimethylsilyl, trimethylsilyl, triethylsilyl, tert-butyldiphenylsilyl or benzyl, more preferably Y2 is selected from triethylsilyl or benzyl; R 11 It is allyl or 2-butenyl, preferably, R 11 It is allyl.

31. The synthesis method according to claim 30, wherein the glycosylation reaction in step (e1”) is carried out in the presence of silver trifluoromethanesulfonate, trimethylsilyl trifluoromethanesulfonate, triethylsilyl trifluoromethanesulfonate, tert-butyldimethylsilyl trifluoromethanesulfonate, trifluoromethanesulfonic acid, boron trifluoride ether, tris(pentafluorophenyl)borane or any combination thereof, preferably tris(pentafluorophenyl)borane.

32. The synthesis method according to claim 30, wherein the metal catalyst in step (e2”) is a palladium, iridium or ruthenium catalyst.

33. Use of the synthetic saponin composition according to any one of claims 1-3 in the preparation of anticancer drugs, adjuvants or immunogenic compositions.

34. A pharmaceutical composition comprising a synthetic saponin composition according to any one of claims 1-3 and a pharmaceutically acceptable excipient.

35. An adjuvant composition comprising a synthetic saponin composition according to any one of claims 1-3.

36. The adjuvant composition of claim 35, wherein it comprises a TLR agonist, such as MPLA.

37. An immunogenic composition comprising the adjuvant composition according to claim 35 or 36 and an antigen. in, Preferably, The antigen is selected from antigens associated with varicella-zoster virus, herpes virus, or herpes simplex virus; or The antigen is selected from antigens associated with Plasmodium falciparum or Plasmodium vivax; or The antigen is selected from antigens associated with human respiratory first synchronized virus; or The antigen is selected from human papillomavirus-associated antigens; or The antigen is selected from antigens related to Streptococcus pneumoniae, Mycobacterium spp., and Mycobacterium tuberculosis; or The antigen is selected from antigens related to the novel coronavirus; or The antigen is derived from HIV, untyped Haemophilus influenzae, Moraxella catarrhalis, influenza virus, rabies virus, FeLV, bovine LV, FeIV, canine distemper virus, canine infectious hepatitis virus, feline calicivirus, feline rhinotracheitis virus, TGE virus, foot-and-mouth disease virus, and combinations thereof; or The antigen is a tumor-associated antigen.

38. An HPLC method for determining the main peak and isomer B content of saponin QS-21-Api in synthetic saponin compositions, the method comprising: The stationary phase was AQ C18 with a particle size of 5 μm. Mobile phase A was water-phosphoric acid (1000:1), and mobile phase B was acetonitrile. The elution conditions were as follows: The detection wavelength is 200nm.

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