Preparation method of tumor-associated antigen DSGb5 glycolipid

Abstract

The invention relates to a preparation method of tumor-associated antigen DSGb5 glycolipid. Specifically, an effective chemical enzymatic preparation method is provided, so that the complex DSGb5 glycolipid antigen can be artificially prepared, and a material basis is provided for research and development of malignant kidney cancer vaccines and related tumor immunotherapy.

Description

Preparation method of tumor-associated antigen DSGb5 glycolipid Technical Field

[0001] The present invention relates to the field of medicine, and more particularly to a method for preparing a tumor-associated antigen DSGb5 glycolipid. As a sugar antigen, it can be used in the development of renal cancer vaccines and tumor immunity-related drugs. Background Art

[0002] Cancer is a major threat to human health, with both morbidity and mortality rates increasing in recent years, presenting a significant challenge in contemporary medical research. Current cancer treatments primarily aim to alleviate patient suffering, improve survival, and enhance quality of life. Furthermore, resistance to anticancer drugs is a frequent problem. Tumor vaccines are an effective approach for cancer prevention and treatment and have become a hot topic in drug development. Tumor-associated carbohydrate antigens are highly expressed on the surface of tumor cells and represent promising targets for tumor vaccine development. Disialylated heptasaccharide ganglioside (DSGb5) is a tumor-associated antigen overexpressed in renal cancer cells and is closely associated with their abnormal proliferation and metastasis. The development of DSGb5-specific antitumor vaccines would be beneficial for the prevention and treatment of malignant renal cancer. Due to the microheterogeneity and complexity of gangliosides, it is extremely difficult to directly isolate and obtain DSGb5 glycolipids with uniform composition, well-defined structure, and sufficient quantity from cancer tissue, severely limiting their application in vaccine development.

[0003] DSGb5 is composed of a hydrophilic heptasaccharide linked by a hydrophobic ceramide lipid, making its synthesis extremely challenging. Previous work has focused on the synthesis of the heptasaccharide moiety, and related biological activities have shown that the function of DSGb5 is synergistically exerted by the sugar and lipid chains. However, due to the complexity of DSGb5 glycolipids, there are currently no reports on their synthesis.

[0004] Therefore, there is an urgent need in the art to provide a preparation method that can efficiently synthesize disialylated heptasaccharide ganglioside.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a preparation method capable of efficiently synthesizing disialylated heptasaccharide ganglioside.

[0007] The present invention provides a method for preparing disialylated heptasaccharide ganglioside, comprising the steps of:

[0008] (xii) in the presence of α-2,3-sialyltransferase Cst-I, compound 2 reacts with 5'-adenosine monophosphate-sialic acid divalent salt to produce compound 3;

[0009] (xiii) Compound 3 and C 17 H 37 COR undergoes acylation reaction to generate compound 4, wherein R is Cl or -OH; and

[0010] (xiv) in the presence of cyclodextrin and α2,6-sialyltransferase ST6GalNAc5, compound 4 reacts with 5'-adenosine monophosphate-sialic acid divalent salt to obtain disialylated heptasaccharide ganglioside DSGb5 of compound 1;

[0011] In another preferred embodiment, the method further comprises the step: (ix) reacting compound 5, trimethylsilyl trifluoromethanesulfonate TMSOTf and compound 16 to produce compound 8.

[0012] In another preferred embodiment, the method further comprises the step: (x) partially deprotecting compound 8, and then reacting with an acetic acid reagent to obtain compound 17.

[0013] In another preferred embodiment, the method further comprises the step of: (xi) subjecting compound 17 to a deprotection reaction to obtain compound 2.

[0014] In another preferred embodiment, the method further comprises the step of: (iv) deprotecting compound 12, and then reacting with benzaldehyde dimethyl acetal to produce compound 13.

[0015] In another preferred embodiment, the method further comprises the step of: (v) reacting compound 13 with 2-(bromomethyl)naphthalene NapBr to produce compound 14.

[0016] In another preferred embodiment, the method further comprises the step of: (vi) partially deprotecting compound 14, and then reacting with benzoyl cyanide to generate compound 7.

[0017] In another preferred embodiment, the method further comprises the step: (vii) reacting compound 6 with compound 7 to produce compound 15.

[0018] In another preferred embodiment, the method further comprises the step: (viii) partially deprotecting compound 15 to generate compound 16.

[0019] In another preferred embodiment, the method further comprises the step of: (i) reacting compound 9 with an acetylating agent to produce compound 10; preferably, the acetylating agent is acetic anhydride or acetyl chloride.

[0020] In another preferred embodiment, the method further comprises the step of: (ii) reacting compound 10 with an acetic acid solution of HBr to produce compound 11.

[0021] In another preferred embodiment, the method further comprises the step of: (iii) reacting compound 11 in the presence of 2,6-lutidine to produce compound 5.

[0022] In another preferred embodiment, the method further includes the step of: the cyclodextrin is selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin sodium, methylated-β-cyclodextrin, or a combination thereof, preferably methylated-β-cyclodextrin.

[0023] In another preferred embodiment, step (xii) includes one or more of the following features:

[0024] The reaction is carried out in a buffer solution at pH 7-8, preferably, such as HEPES buffer or Tris-HCl buffer;

[0025] The 5'-adenosine monophosphate-sialic acid divalent salt is a sodium salt or a potassium salt;

[0026] The reaction temperature is 37±5°C, preferably 37±2°C, more preferably 37±1°C;

[0027] The concentration of α-2,3-sialyltransferase Cst-I is 50-200 μg / mL; preferably 80-120 μg / mL; and / or

[0028] The equivalent ratio of compound 2 to 5'-adenosine monophosphate-sialic acid disodium salt is 1:1-2, preferably 1:1.2-1.5.

[0029] In another preferred embodiment, step (xiii) includes one or more of the following features:

[0030] The reaction solvent is selected from the group consisting of THF, H2O, or a combination thereof;

[0031] The acylation reaction is carried out in the presence of an acid binding agent, such as an acid binding agent selected from the group consisting of NaHCO3, KHCO3, Na2CO3, K2CO3, or a combination thereof;

[0032] The reaction temperature is 25±15°C, preferably 25±10°C, more preferably 25±5°C; and / or

[0033] Compound 3 and C 17 H 37 The equivalent ratio of COR is 1:1-2; preferably 1:1.2-1.5.

[0034] In another preferred embodiment, step (xiv) includes one or more of the following features:

[0035] The reaction is carried out in a buffer solution of pH 6.5-8, preferably pH 6.8-7.4, such as HEPES buffer or Tris-HCl buffer;

[0036] The reaction temperature is 37±5°C, preferably 37±2°C, more preferably 37±1°C;

[0037] The concentration of α2,6-sialyltransferase ST6GalNAc5 is 5-100 μg / mL; preferably 10-50 μg / mL;

[0038] The equivalent ratio of compound 4 to 5'-adenosine monophosphate-sialic acid disodium salt is 1:1-2; preferably 1:1.2-1.5; and / or

[0039] The equivalent ratio of compound 4 to cyclodextrin is 1:1-4, preferably 1:1.5-2.5.

[0040] In another preferred embodiment, step (ix) includes one or more of the following features:

[0041] The reaction React in the presence of molecular sieves; such as Na2O·Al2O3·2 SiO2·9 / 2 H2O;

[0042] The reaction solvent is selected from the group consisting of toluene, dichloromethane, chloroform, or a combination thereof;

[0043] The reaction temperature is 0±10°C, preferably 0±5°C, more preferably 0±2°C; and / or

[0044] The equivalent ratio of compound 16 to compound 5 is 1:1-2; preferably 1:1.2-1.5; and / or

[0045] The equivalent ratio of compound 16 to TMSOTf is 1:0.1-1, preferably 1:0.2-0.5.

[0046] In another preferred embodiment, step (x) comprises the following steps:

[0047] (x-1) Compound 8 reacts in a hydrofluoric acid pyridine solution to obtain intermediate 8-1;

[0048] (x-2) reacting intermediate 8-1 in the presence of a base (such as NaOH, KOH, or a combination thereof) and neutralizing to produce intermediate 8-2; and

[0049] (x-3) Intermediate 8-2 is reacted with an acetylating agent to obtain compound 17.

[0050] In another preferred embodiment, step (xi) comprises the following steps:

[0051] (xi-1) In the presence of 2,3-dichloro-5,6-dicyano-p-benzoquinone DDQ, compound 17 reacts to obtain an intermediate 17-1; and

[0052] (xi-2) In the presence of 1,3-propanedithiol, pyridine and water, intermediate 17-1 reacts to give compound 2.

[0053] In another preferred embodiment, step (iv) comprises the following steps:

[0054] (iv-1) in the presence of a base (such as NaOH, KOH, or a combination thereof), deprotecting compound 12 to produce intermediate 12-1; and

[0055] (iv-1) In the presence of camphorsulfonic acid (CSA), intermediate 12-1 reacts with benzaldehyde dimethyl acetal to produce compound 13.

[0056] In another preferred embodiment, step (v) comprises the following step: reacting compound 13 with 2-(bromomethyl)naphthalene NapBr in the presence of NaH to produce compound 14.

[0057] In another preferred embodiment, step (vi) comprises the following steps:

[0058] (vi-1) dissolving compound 14 in a mixed solution of DCM, TFA, and H2O (volume ratio (8-12): (0.5-2): 1, preferably 10:1:1), and deprotecting compound 14 to produce intermediate 14-1; and

[0059] (vi-1) In the presence of a base (such as diethylamine, triethylamine, dimethylamine, trimethylamine, or a combination thereof), intermediate 14-1 reacts with benzoyl cyanide to produce compound 7.

[0060] In another preferred embodiment, step (vii) includes one or more of the following features:

[0061] The reaction React in the presence of molecular sieves; such as Na2O·Al2O3·2 SiO2·9 / 2 H2O;

[0062] The reaction solvent is selected from the group consisting of toluene, dichloromethane, chloroform, or a combination thereof;

[0063] The reaction temperature is 0±10°C, preferably 0±5°C, more preferably 0±2°C; and / or

[0064] The equivalent ratio of compound 7 to compound 6 is 1:1-3; preferably 1:1.5-2.5; and / or

[0065] The equivalent ratio of compound 7 to TMSOTf is 1:0.1-0.5, preferably 1:0.2-0.3.

[0066] In another preferred embodiment, step (viii) comprises: (viii) partially deprotecting compound 15 in the presence of hydrazine acetate to produce compound 16.

[0067] In another preferred embodiment, step (i) includes one or more of the following features:

[0068] The acetylating agent is acetic anhydride or acetyl chloride;

[0069] The reaction is carried out in the presence of 4-dimethylaminopyridine DMAP;

[0070] The reaction solvent is pyridine; and / or

[0071] The reaction temperature is 25±10°C, preferably 25±5°C, more preferably 25±2°C.

[0072] In another preferred embodiment, step (ii) includes one or more of the following features:

[0073] The concentration of hydrogen bromide in the hydrogen bromide acetic acid solution is 30-35 wt%;

[0074] The reaction solvent is selected from the group consisting of toluene, dichloromethane, or a combination thereof.

[0075] In another preferred embodiment, step (iii) includes one or more of the following features:

[0076] The reaction solvent is selected from the group consisting of acetonitrile, ethanol, or a combination thereof;

[0077] The reaction temperature is 25±10°C, preferably 25±5°C, more preferably 25±2°C; and / or

[0078] The equivalent ratio of compound 11 to 2,6-lutidine is 1:1-3, preferably 1:1.5-2.

[0079] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 is the hydrogen spectrum of DSGb5 glycolipid 1 ( 1H NMR, 600 MHz, CD3OD). DETAILED DESCRIPTION

[0081] After extensive and intensive research, screening, and testing, the present inventors have developed a method for preparing the tumor-associated antigen DSGb5 glycolipid. Specifically, the present invention provides, for the first time, an effective chemical-enzymatic preparation method capable of efficiently and artificially preparing the complex DSGb5 glycolipid antigen. This method will provide a material foundation for the development of vaccines for malignant renal cancer and related tumor immunotherapy. This is the basis for the present invention.

[0082] the term

[0083] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0084] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0085] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0086] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0087] Preparation method

[0088] The present invention provides a method for preparing disialylated heptasaccharide ganglioside, comprising the steps of:

[0089] (xii) in the presence of α-2,3-sialyltransferase Cst-I, compound 2 reacts with 5'-adenosine monophosphate-sialic acid divalent salt to produce compound 3;

[0090] (xiii) Compound 3 and C 17 H 37 COR undergoes acylation reaction to generate compound 4, wherein R is Cl or -OH; and

[0091] (xiv) in the presence of cyclodextrin and α2,6-sialyltransferase ST6GalNAc5, compound 4 reacts with 5'-adenosine monophosphate-sialic acid divalent salt to obtain disialylated heptasaccharide ganglioside DSGb5 of compound 1;

[0092] In particular, to address the problems of difficult and efficient installation of sialic acid and low enzymatic reaction efficiency of glycolipids as substrates in aqueous solution, the present invention developed α2,3-sialyltransferase Cst-I to catalyze the installation of α2,3 sialic acid, and cyclodextrin promoted α2,6-sialyltransferase ST6GalNAc5 to catalyze the installation of α2,6 sialic acid, thereby obtaining DSGb5 glycolipid.

[0093] Unexpectedly, the α2,3-sialyltransferase Cst-I can well recognize the Gb5 sphingosine glycolipid substrate 2 and catalyze the installation of α2,3-sialic acid to obtain compound 3; cyclodextrin can promote the α2,6-sialyltransferase ST6GalNAc5 to catalyze the glycolipid substrate 4 to obtain DSGb5.

[0094] Furthermore, the method further comprises the steps of:

[0095] (ix) reacting compound 5, trimethylsilyl trifluoromethanesulfonate (TMSOTf) and compound 16 to produce compound 8;

[0096] (x) partially deprotecting compound 8, and then reacting with an acetic acid reagent to obtain compound 17; and

[0097] (xi) Compound 17 is subjected to a deprotection reaction to obtain compound 2

[0098] Specifically, addressing the shortcomings of previous syntheses of the Gb5 pentasaccharide (Compound 8), which suffer from poor stereoselectivity and low yield, the oxazoline disaccharide donor (Compound 5) developed in this invention allows for stereoselective β-glycosylation, effectively preparing protected Gb5 pentasaccharide lipids. Furthermore, the use of a naphthylidene (Nap) group to protect the hydroxyl groups on the sugar ring avoids the issue of incompatibility with double bonds on the aliphatic chain during subsequent deprotection.

[0099] Preferably, the glycosylation coupling reaction between compound 5 and compound 16 is carried out under anhydrous and low temperature (eg, -10°C to 0°C) conditions.

[0100] Furthermore, the method further comprises the steps of:

[0101] (iv) deprotecting compound 12 and then reacting it with benzaldehyde dimethyl acetal to produce compound 13;

[0102] (v) reacting compound 13 with 2-(bromomethyl)naphthalene NapBr to produce compound 14;

[0103] (vi) partially deprotecting compound 14 and then reacting it with benzoyl cyanide to produce compound 7;

[0104] (vii) reacting compound 6 with compound 7 to produce compound 15; and

[0105] (viii) partially deprotecting compound 15 to generate compound 16;

[0106] In particular, to address the difficulty in stereoselective synthesis of Gb3 sphingosine glycolipid (Compound 16), this patent developed a large silicon-protected sugar donor (Compound 6) that can undergo stereoselective α-glycosylation, effectively preparing the protected Gb3 sphingosine glycolipid precursor (Compound 15). Subsequently, the Lev (3-levulinyl) protecting group is selectively removed to obtain the trisaccharide acceptor.

[0107] Preferably, the glycosylation coupling reaction between compound 6 and compound 7 is carried out under anhydrous and low temperature (eg, -10°C to 0°C) conditions.

[0108] Furthermore, the method further comprises the steps of:

[0109] (i) reacting compound 9 with an acetylating agent to produce compound 10; preferably, the acetylating agent is acetic anhydride or acetyl chloride;

[0110] (ii) reacting compound 10 with a solution of HBr in acetic acid to produce compound 11; and

[0111] (iii) Compound 11 reacts in the presence of 2,6-lutidine to produce compound 5;

[0112] In any reaction step of the present invention, the reaction solvent, reaction temperature, reaction time, catalyst, charging ratio, etc. can be selected according to the specific reactants.

[0113] For example, representative inert reaction solvents include, but are not limited to: acetonitrile, dichloromethane, benzene, toluene, xylene, trifluorotoluene, halogenated benzenes such as chlorobenzene, fluorobenzene, dichlorobenzene and difluorobenzene, chloroform, acetone, ethyl acetate, diethyl ether, tetrahydrofuran or a combination thereof. In some embodiments, the solvent can be toluene, dichloromethane, THF or a combination thereof.

[0114] For example, the reaction temperature can be from about -25°C to about 30°C, or from about -20°C to about 20°C, or from about -25°C to about 15°C, or from about -10°C to about 10°C, or from about 0°C to about 5°C. In some embodiments, the reaction temperature can be from about -20°C to about 25°C.

[0115] The main advantages of the present invention include:

[0116] The present invention provides for the first time an effective chemical-enzymatic preparation method for DSGb5, which can efficiently realize the artificial preparation of complex DSGb5 glycolipid antigens. This can solve the problem of the difficulty in obtaining DSGb5 with a clear structure and sufficient quantity, thereby providing a material basis for the development of malignant renal cancer vaccines and related tumor immunotherapy.

[0117] The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0118] Example 1

[0119] Step 1: First, compound 9 was dissolved in pyridine, followed by the addition of 14 equivalents of acetic anhydride and a catalytic amount of DMAP. The reaction system was stirred at room temperature overnight under an argon atmosphere. After the reaction was complete, an appropriate amount of methanol was added to quench the reaction. After concentration under reduced pressure, ethyl acetate was added to dissolve the mixture, and the mixture was washed with dilute hydrochloric acid and then water. The organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 10 in a yield of 95%. 1 H NMR (400MHz, CDCl3): δ6.63(d,J=7.5Hz,1H),6.34(d,J=3.5Hz,1H),5.42(d,J=2.7Hz,1H),5.37( d,J=3.3Hz,1H),5.19(dd,J=10.3,7.9Hz,1H),4.95(dd,J=10.3,3.4Hz,1H),4.70(d,J=8.0Hz,1H ),4.54(ddd,J=11.0,7.5,3.6Hz,1H),4.22(t,J=6.6Hz,1H),4.16-4.09(m,4H),4.03–3.93(m,2H ),2.17(s,3H),2.16(s,3H),2.14(s,3H),2.06(s,6H),2.05(s,3H),1.97(s,3H); HRMS(ESI)calcd for C 28 H 36 F3N 18 [M+Na] + 754.1777, found 754.1762.

[0120] Step 2: Compound 10 was spin-dried three times with toluene (3 × 3 mL) under reduced pressure, then dissolved in CH2Cl2. An appropriate volume of hydrogen bromide and acetic acid solution was then added. The reaction mixture was stirred at room temperature for 3 hours, then concentrated under reduced pressure and spin-dried three times with toluene to yield compound 11, which was immediately used in the next step. Step 3: Compound 11 was dissolved in CH3CN, 1.5 equivalents of 2,6-lutidine was added, and stirred at room temperature for 1.5 hours. After the reaction was complete, the mixture was concentrated under reduced pressure and purified by silica gel column chromatography (eluents: toluene and acetone). Oxazoline donor 5 was obtained in a 96% yield over two steps. 1 H NMR (400MHz, CDCl3): δ6.24(d,J=7.1Hz,1H),5.46–5.34(m,2H),5.17(dd,J=10.5,7.8Hz,1H),5.02(dd,J=10.5,3.4Hz,1H),4.77(d,J= 7.9Hz,1H),4.20–4.03(m,7H),3.90(t,J=6.6Hz,1H),2.14(s,3H),2.10(s,3H),2.07(s,6H),2.03(s,3H),1.97(s,3H); HRMS(ESI)calcd for C 26 H 32 F3NO 16 [M+H] + 672.1746, found 672.1747.

[0121] Example 2

[0122] Step 1: Compound 12 was dissolved in a mixed solution of 1,4-dioxane, methanol, and 1M sodium hydroxide (volume ratio: 3:1:1). After stirring at room temperature for 8 hours, the reaction solution was neutralized with an acidic resin to a pH of ~7, filtered, and the filtrate was concentrated under reduced pressure. The resulting intermediate was dissolved in DMF, and under argon protection, 1.2 equivalents of benzaldehyde dimethyl acetal and 0.3 equivalents of camphorsulfonic acid (CSA) were added. After stirring at room temperature overnight, triethylamine was added to quench the reaction. The reaction mixture was concentrated under reduced pressure and then purified by silica gel column chromatography (eluent: dichloromethane and methanol) to obtain compound 13 in a final yield of 70%. 1H NMR (500 MHz, CDCl3:CD3OD 1:1)δ7.52–7.48(m,2H),7.35–7.31(m,3H),5.75(dt,J=15.4,6.7Hz,1H),5. 58(s,1H),5.49(dd,J=15.4,7.5Hz,1H),4.45(d,J=7.7Hz,1H),4.30(d,J=7.8 Hz,1H),4.24–4.09(m,4H),3.94–3.85(m,3H),3.73–3.68(m,2H),3.64–3.56( m,5H),3.39(d,J=9.4Hz,1H),3.31(dd,J=3.2,1.6Hz,1H),2.08-2.02(m,2H), 1.41-1.35(m,2H),1.28-1.23(m,20H),0.86(t,J=6.9Hz,3H); HRMS(ESI)calcd for C 37 H 63 N4O 12 [M+NH4] + 755.4437, found 755.4434.

[0123] Step 2: Compound 13 was dissolved in DMF (5 mL) solution, and then 9 equivalents of NaH and 9 equivalents of 2-(bromomethyl)naphthalene were added at 0°C and stirred at room temperature overnight. The reaction mixture was then quenched with an appropriate amount of methanol. The reaction mixture was concentrated under reduced pressure, diluted with dichloromethane, and washed with saturated aqueous NH4Cl solution and saturated brine. The organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to obtain compound 14 in a yield of 76%. 1H NMR (500MHz, CDCl3): δ7.84–7.65(m,20H),7.63–7.53(m,7H),7.49–7.37(m,16H),7.32–7.27(m,4H),5.67(dt,J=15.4,6.7Hz,1H),5. 46-5.37(m,3H),5.02-4.95(m,2H),4.94–4.86(m,3H),4.83–4.73(m,2H),4.71-4.65(m,2H),4.50–4.39(m,4H),4.24–4.18(m,1H),4. 11–3.95(m,5H),3.86(dd,J=9.6,7.8Hz,1H),3.81–3.69(m,4H),3.65(dd,J=10.3,5.3Hz,1H),3.53(dd,J=9.2,7.8Hz,1H),3.41–3.36 (m,1H),3.29(dd,J=9.7,3.6Hz,1H),2.88-2.84(m,1H),2.06-2.00(m,2H),1.28-1.22(m,22H),0.88(t,J=6.9Hz,3H); HRMS(ESI)calcd for C 103 H 107 N3O 12 [M+Na] + 1600.7747,found 1600.7755.

[0124] Step 3: First, compound 14 was dissolved in a mixture of DCM:TFA:H₂O (volume ratio 10:1:1) and stirred at room temperature until TLC indicated the disappearance of the starting material. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane and transferred to a separatory funnel. It was then washed with saturated NaHCO₃ and then saturated brine. The aqueous layer was back-extracted once with dichloromethane. The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (eluents: acetone and toluene) to obtain the intermediate compound. The intermediate was dissolved in anhydrous DMF, triethylamine was added, and the mixture was cooled to -20°C. After that, 1 equivalent of benzoyl cyanide was added. The reaction mixture was stirred for 15 minutes, then the temperature was returned to 0°C and the reaction was continued for 15 minutes. Subsequently, the reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to obtain compound 7 in a two-step yield of 66%. 1H NMR (600MHz, CDCl3): δ8.01-7.95(m,2H),7.86–7.61(m,24H),7.58–7.30(m,21H),5.70(dt,J=14.8,6.5Hz,1H),5.45(dd,J=15.6,8.5Hz,1H),5.18( d,J=11.1Hz,1H),4.96-4.85(m,4H),4.83(d,J=11.5Hz,1H),4.77–4.67(m ,3H),4.64(d,J=11.9Hz,1H),4.56–4.50(m,1H),4.49-4.43(m,2H),4.41-4 .36(m,2H),4.32(dd,J=11.2,6.6Hz,1H),4.11(t,J=9.4Hz,1H),4.04(dd, J=10.1,6.6Hz,1H),4.01-3.93(m,2H),3.80-3.77(m,2H),3.74(d,J=10.8H z,1H),3.69-3.61(m,3H),3.49(t,J=8.6Hz,1H),3.41-3.34(m,2H),3.08– 3.04(m,1H),2.09-2.02(m,2H),1.33-1.23(m,22H),0.88(t,J=7.1Hz,3H); HRMS(ESI)calcd for C 103 H 107 N3NaO 13 [M+Na] + 1616.7696,found 1616.7695.

[0125] Step 4: 2 equivalents of trifluoroimidate donor 6 (Lev: 3-levulinyl) and equivalent trisaccharide acceptor 7 were spun twice with toluene under reduced pressure and mixed with fresh activated The mixture was mixed with 5% paraformaldehyde and 0.5% paraformaldehyde, and anhydrous toluene was slowly added. The mixture was stirred for half an hour at room temperature under an argon atmosphere. The reaction system was then transferred to an ice bath and 0.2 equivalents of TBSOTf Lewis acid catalyst were added. The mixture was stirred until TLC showed the complete disappearance of the receptor. An appropriate amount of pyridine was added to quench the reaction. The mixture was filtered through celite and the filtrate was dried under reduced pressure to obtain an oily mixture. The mixture was purified by silica gel column chromatography (eluents: toluene and acetone) to obtain the β-glycosidically linked trisaccharide product 15 in a yield of 76%. 1H NMR (500MHz, CDCl3): δ7.91–7.88(m,2H),7.84–7.72(m,15H),7.72–7.65(m,6H),7.60–7.55 (m,2H),7.52–7.33(m,20H),7.30–7.21(m,5H),7.02-6.99(m,2H),5.68–5.60(m,2H),5.46– 5.37(m,3H),5.06(d,J=12.3Hz,1H),4.96–4.87(m,4H),4.80-4.72(m,2H),4.70-4.66(m,2H ),4.54(d,J=12.4Hz,1H),4.49-4.45(m,3H),4.44-4.41(m,2H),4.35(d,J=7.7Hz,1H),4.12 –4.05(m,2H),4.04–3.98(m,3H),3.95(dd,J=8.6,5.5Hz,1H),3.84(d,J=3.0Hz,1H),3.78-3 .72(m,3H),3.71–3.65(m,2H),3.65–3.59(m,1H),3.50–3.45(m,2H),3.37-3.33(m,1H),3.2 9(t,J=7.2Hz,1H),2.94(dd,J=9.9,3.0Hz,1H),2.51–2.36(m,4H),2.05-1.98(m,2H),1.92( s,3H),1.29-1.23(m,22H),1.09(s,9H),0.92(s,9H),0.90(t,J=6.9Hz,3H); HRMS(ESI)calcd for C 129 H 143 N3NaO 21 Si[M+Na] + 2120.9876,found 2120.9875.

[0126] Step 5: Compound 15 was dissolved in a mixture of CH2Cl2-CH3OH (volume ratio 3:1), 5 equivalents of hydrazine acetate were added, and the mixture was stirred at room temperature for 20 minutes. The mixture was then diluted with dichloromethane and washed with water. The organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (eluent: petroleum ether-ethyl acetate) to obtain compound 16 in an 83% yield. 1H NMR (400MHz, CDCl3): δ7.90-7.83(m,4H),7.82-7.73(m,8H),7.72–7.57(m,16H),7 .56–7.51(m,2H),7.49–7.32(m,17H),7.31–7.27(m,1H),7.25–7.16(m,2H),7.03- 6.92(m,2H),5.68(dt,J=15.5,6.7Hz,1H),5.48–5.39(m,2H),5.29(d,J=3.5Hz,1H ),5.17(d,J=12.1Hz,1H),5.00-4.91(m,2H),4.89–4.81(m,3H),4.77–4.66(m,3H) ,4.51(d,J=11.9Hz,1H),4.49–4.43(m,4H),4.41(d,J=7.7Hz,1H),4.21-4.10(m,4 H),4.06(dd,J=10.2,6.4Hz,1H),4.02-3.93(m,3H),3.81–3.51(m,9H),3.44–3.36 (m,1H),3.28(t,J=7.2Hz,1H),2.92(dd,J=9.9,2.9Hz,1H),2.07-2.01(m,2H),1.3 1-1.23(m,22H),1.09(s,9H),0.95(s,9H),0.89(t,J=6.9Hz,3H); HRMS(ESI)calcd for C 124 H 137 N3NaO 19 Si[M+Na] + 2022.9508,found 2022.9510.

[0127] Example 3

[0128] Step 1: First, 1.5 equivalents of oxazoline donor 5, 1.0 equivalents of acceptor 16 and freshly activated The mixture was mixed with 4% paraformaldehyde and 2% paraformaldehyde, and anhydrous toluene was slowly added. The mixture was stirred at room temperature under an argon atmosphere for half an hour. The reaction system was then transferred to an ice bath, 0.2 equivalents of TMSOTf were added, and the mixture was stirred until TLC showed the complete disappearance of the receptor. An appropriate amount of pyridine was added to quench the reaction, and the mixture was filtered through celite. The filtrate was then dried under reduced pressure to obtain an oily mixture. The mixture was purified by silica gel column chromatography (eluents: petroleum ether and ethyl acetate) to obtain the β-glycosidically linked pentasaccharide product 8 in a yield of 75%. 1H NMR(400MHz,CDCl3):δ8.02–7.97(m,2H),7.86–7.52(m,30H),7.50–7.30(m,18H),7.25-7.19(m,2H),6.04(d,J=7.5Hz,1H),5.72–5.59(m,2H),5.42(dd,J=15.5,8.5Hz,1H),5.35(d,J=3.6Hz,1H),5.33–5.30(m,1H),5.14(d,J=11.4Hz,1H),5.02–4.93(m,2H),4.90-4.84(m,3H),4.79–4.67(m,6H),4.59-4.51(m,3H),4.49–4.34(m,5H),4.30–4.18(m,2H),4.11–4.10(m,1H),4.09–3.99(m,5H),3.98–3.86(m,3H),3.83–3.55(m,11H),3.49-3.39(m,2H),3.33–3.23(m,2H),2.88(dd,J=9.9,2.9Hz,1H),2.77(t,J=6.6Hz,1H),2.11(s,3H),2.05(m,5H),2.03(s,3H),1.94(s,6H),1.91(s,3H),1.28–1.21(m,22H),1.10(s,9H),0.93(s,9H),0.87((t,J=6.9Hz,3H);HRMS(ESI)calcd for C 150 H 170 F3N4NaO 35 Si[M+Na+H] 2+ 1347.5627,found 1347.8843.

[0129] Step 2: The synthesized pentasaccharide 8 was dissolved in pyridine. Under an argon atmosphere at 0°C, a defined volume of hydrofluoric acid-pyridine solution was added and stirred until TLC indicated complete disappearance of the starting material. The reaction was then quenched by addition of a saturated NaHCO₃ solution. The reaction mixture was diluted with dichloromethane, washed with saturated NaHCO₃, dried over anhydrous Na₂SO₄, filtered, and the filtrate concentrated under reduced pressure to yield an intermediate. This intermediate was dissolved in a mixture of 1,4-dioxane, methanol, and 1M sodium hydroxide (10:1:1 by volume) and stirred at room temperature. After completion of the reaction, the reaction mixture was neutralized with an acidic resin to a pH of ~7, filtered, and the filtrate concentrated under reduced pressure to yield an amino-containing intermediate. The above intermediate was dissolved in a mixed solvent of methanol and water (volume ratio 4:1), and 2.5 equivalents of triethylamine and 1.1 equivalents of acetic anhydride were added. The mixture was stirred at room temperature until TLC showed the disappearance of the starting material. The reaction mixture was concentrated under reduced pressure and purified by size exclusion chromatography on Sephadex LH 20 (eluent: dichloromethane and methanol) to obtain compound 17 in an 81% yield. 1 H NMR (500 MHz, CDCl3:CD3OD 1:1): δ7.85–7.59(m,23H),7.53–7.31(m,17H),7.27-7.22(m,2H),5.68–5.56 (m,1H),5.43–5.36(m,1H),5.21(d,J=11.1Hz,1H),5.10(d,J=3.8Hz,1H),4.99 (d,J=11.4Hz,1H),4.93–4.83(m,4H),4.68(d,J=5.5Hz,2H),4.59(m,2H),4.49 –4.42(m,2H),4.40–4.35(m,2H),4.28(d,J=8.4Hz,1H),4.19(t,J=5.7Hz,1H), 4.11(d,J=2.9Hz,1H),4.05(d,J=7.4Hz,1H),4.03–3.68(m,18H),3.67–3.59(m ,4H),3.54–3.43(m,5H),3.38(dd,J=9.2,7.7Hz,1H),3.31-3.28(m,1H),3.23( dd,J=10.0,2.8Hz,1H),3.11–3.05(m,1H),2.74(t,J=5.9Hz,1H),2.04-1.98(m ,2H),1.83(s,3H),1.30–1.18(m,22H),0.86(t,J=7.0Hz,3H); HRMS(ESI)calcd for C 116 H 136 N4O 27 [M+Na] +2039.9284,found 2039.9284.

[0130] Step 3: Compound 17 was dissolved in a mixed solution of dichloromethane and water (volume ratio 12:1), and 6.6 equivalents of DDQ were added under light-shielding conditions. After stirring at room temperature for 3 hours, the reaction was quenched with a saturated aqueous NaHCO3 solution and then purified using a C18 reverse-phase silica gel column (eluent: methanol and water) to obtain an intermediate. The obtained intermediate was concentrated and dissolved in a mixed solvent of pyridine and water (volume ratio 1 / 1). 30 equivalents of triethylamine and 10 equivalents of 1,3-propanedithiol were added. The reaction mixture was stirred at room temperature for 12 hours. ESI-MS showed the disappearance of the starting material. After the reaction mixture was concentrated under reduced pressure, it was purified using C18 reverse-phase silica gel (eluent: methanol and water) to obtain compound 2 in a final yield of 91%. 1 H NMR (800MHz, CD3OD): δ5.80–5.76(m,1H),5.51(ddt,J=15.4,7.6,1.5Hz,1H),4.96(d,J=4.0Hz,1H),4.7 3(d,J=8.4Hz,1H),4.45–4.42(m,1H),4.37(d,J=7.6Hz,1H),4.32(d,J=7.8Hz,1H),4.28(m,1H),4.19(dd ,J=3.1,1.3Hz,1H),4.13(d,J=3.1Hz,1H),4.09(dd,J=10.8,8.4Hz,1H),4.03–3.99(m,2H),3.96(dd,J= 10.2,3.9Hz,1H),3.94–3.85(m,6H),3.84–3.82(m,2H),3.81–3.69(m,8H),3.60–3.52(m,7H),3.47(dd,J =9.7,3.3Hz,1H),3.45-3.42(m,1H),3.30(dd,J=9.1,7.8Hz,1H),2.96-2.92(m,1H),2.13–2.08(m ,2H),2.01(s,3H),1.46-1.42(m,2H),1.35-1.29(m,20H),0.92(t,J=7.1Hz,3H); HRMS(ESI)calcd for C 50 H 91 N2O 27 [M+H] + 1151.5804,found 1151.5805.

[0131] Example 4

[0132] Step 1: Compound 2 was dissolved in HEPES buffer (100 mM, pH 7.2). CMP-Neu5Ac (1.5 equivalents), MnCl2 (10 mM), and sialyltransferase Cst-I (100 μg / mL) were added sequentially. The mixture was incubated at 37°C for 0.5 hours. ESI-MS analysis showed the disappearance of the starting material. After centrifugation of the reaction mixture, the supernatant was purified using a C18 reverse-phase silica gel column (eluent: methanol and water), ultimately obtaining the α2,3-sialylated product 3 in a 96% yield. 1 H NMR (800MHz, CD3OD): δ5.82(dt,J=14.4,6.8Hz,1H),5.49(dd,J=15.4,7.1Hz,1H),4.94(d,J=3.9Hz,1H),4.73(d,J =8.6Hz,1H),4.44-4.41(m,2H),4.35(d,J=7.9Hz,1H),4.26(t,J=6.3Hz,1H),4.18-4.16(m,2H),4.12(d,J=3.0Hz,1 H),4.05–3.98(m,3H),3.95-3.91(m,3H),3.90(d,J=3.5Hz,1H),3.89(d,J=4.5Hz,1H),3.87(d,J=4.4Hz,1H),3.86- 3.84(m,5H),3.81(dd,J=11.5,2.5Hz,1H),3.79–3.77(m,1H),3.76–3.66(m,10H),3.64–3.60(m,2H),3.59–3.55(m, 4H),3.53(d,J=8.9Hz,1H),3.50(d,J=9.9Hz,1H),3.45(d,J=8.8Hz,1H),3.30-3.29(m,1H),3.21(d,J=20.2Hz,1H),2.87(d,J=12.1Hz,1H ),2.11-2.09(m,2H),2.01(s,6H),1.72(d,J=13.3Hz,1H),1.44-1.41(m,2H),1.31-1.28(m,20H),0.90(t,J=7.1Hz,3H); HRMS(ESI)calcd for C 61 H 108 N3O 35 [M+H] + 1442.6758,found 1442.6756.

[0133] Step 2: Dissolve the above compound 3 sample in a mixed solution containing saturated NaHCO3, THF and H2O (volume ratio 1:1:3), add C 17H 37 A solution of COCl (1.5 equivalents) in THF was added, and the ice bath was subsequently removed. The reaction system was returned to room temperature and stirred for 30 minutes. TLC showed that the reaction was complete, and the reaction mixture was concentrated under reduced pressure and purified by size exclusion chromatography on Sephadex LH 20 (eluent: methanol) to obtain compound 4 in an 85% yield. 1 H NMR (500MHz, CD3OD): δ5.68(dt,J=14.1,6.7Hz,1H),5.48–5.41(m,1H),4.94(d, J=3.9Hz,1H),4.71(d,J=8.4Hz,1H),4.42(d,J=7.8Hz,1H),4.41-4.38(m,1H),4. 31(d,J=7.8Hz,1H),4.24(t,J=6.2Hz,1H),4.19-4.14(m,2H),4.11(t,J=2.0Hz,1 H),4.09-4.04(m,2H),4.02(dd,J=9.7,3.2Hz,1H),3.99–3.95(m,2H),3.94–3.91 (m,1H),3.90-3.87(m,3H),3.86–3.79(m,6H),3.77–3.64(m,10H),3.63–3.52(m, 10H),3.51-3.48(m,2H),3.43-3.40(m,1H),3.27(d,J=8.3Hz,1H),2.89–2.83(m, 1H),2.19-2.15(m,2H),2.02(d,J=7.6Hz,1H),2.00(s,6H),1.71(t,J=11.3Hz,1H ),1.61-1.55(m,2H),1.31-1.27(m,50H),0.89(t,J=6.8Hz,6H); HRMS(ESI)calcd for C 79 H 140 N3O 36 [MH] - 1706.9222,found 1706.9224.

[0134] Step 3: Compound 4 was dissolved in Tris-HCl buffer (100 mM, pH 7.0), and methyl-β-cyclodextrin (2 equivalents), CMP-Neu5Ac (1.5 equivalents), MgCl2 (10 mM), and ST6GalNAc5 (20 μg / mL) were added in sequence. The reaction system was incubated at 37 ° C for 12 hours. The reaction was monitored by ESI-MS every 8 hours, and ST6GalNAc5 (10 μg / mL) and CMP-Neu5Ac (0.5 equivalents) were added until the reaction was complete. The reaction mixture was centrifuged, and the supernatant was purified by HW-40F molecular exclusion chromatography (eluent: methanol) to obtain compound 1 in an 80% yield. 1 H NMR (600MHz, CD3OD): δ5.68(dt,J=14.2,6.7Hz,1H),5.45(dd,J=15.3,7.8Hz,1H),4.97-4.96(m,1H),4.94–4.93(m,1H),4.66(d,J=8.5Hz,1H),4.4 4-4.38(m,2H),4.35-4.31(m,2H),4.22-4.18(m,2H),4.14–4.11(m,1H), 4.10–4.05(m,2H),4.04–3.96(m,4H),3.95–3.92(m,1H),3.91-3.88(m,4H ),3.87-3.83(m,5H),3.80-3.79(m,2H),3.76-3.74(m,3H),3.73–3.69(m ,5H),3.67(d,J=5.4Hz,1H),3.66(d,J=4.7Hz,1H),3.65-3.62(m,2H),3.6 1–3.53(m,9H),3.52–3.41(m,5H),3.27(d,J=8.4Hz,1H),2.89–2.80(m,2 H),2.23-2.18(m,2H),2.05-2.01(m,2H),2.00(s,9H),1.76-1.67(m,1H), 1.63-1.56(m,3H),1.41-1.37(m,2H),1.32-1.28(m,48H),0.90(t,J=6.9Hz,6H); HRMS(ESI)calcd for C 90 H 156 N4O 44 [M-2H] 2- 998.5052, found 998.5054.

[0135] discuss

[0136] The main challenges in the synthesis of DSGb5 are: 1) stereoselective and regioselective installation of α2,3- and α2,6-linked sialic acids; 2) rational glycosylation sequence assembly of the sugar chain; and 3) rational introduction of sphingosine and ceramide fatty acid moieties into the sugar chain. This invention provides an efficient chemoenzymatic preparation method, enabling the first preparation of the complex DSGb5 glycolipid antigen. A key feature of this synthetic route is the use of an oxazoline disaccharide donor for stereoselective chemical glycosylation to prepare the Gb5 pentaglycolipid. This strategy overcomes the shortcomings of previous Gb5 syntheses, which have been characterized by poor stereoselectivity and low yield. Furthermore, the use of a naphthalene methylene group to protect the hydroxyl groups on the sugar ring avoids the incompatibility of the double bond on the aliphatic chain with subsequent deprotection. Furthermore, methyl-β-cyclodextrin promotes the enzymatic sialylation of the Gb5 pentaglycolipid, successfully site-specifically installing two sialic acids to yield the DSGb5 complex glycolipid. This overcomes the low efficiency of enzymatic reactions in aqueous solutions for glycolipids and avoids the isomer separation and low yield challenges associated with chemical sialylation. The effective synthesis of tumor-associated glycolipid DSGb5 antigen will provide a material basis for the development of malignant renal cancer vaccines and related tumor immunotherapy.

[0137] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for preparing disialylated heptasaccharide ganglioside, characterized in that: Including steps: (xii) in the presence of α-2,3-sialyltransferase Cst-I, compound 2 reacts with 5'-adenosine monophosphate-sialic acid divalent salt to produce compound 3; (xiii) Compound 3 and C 17 H 37 COR undergoes acylation reaction to generate compound 4, wherein R is Cl or -OH; and (xiv) in the presence of cyclodextrin and α2,6-sialyltransferase ST6GalNAc5, compound 4 reacts with 5'-adenosine monophosphate-sialic acid divalent salt to obtain disialylated heptasaccharide ganglioside DSGb5 of compound 1; 2. The preparation method according to claim 1, wherein The method further comprises the steps of: (ix) reacting compound 5, trimethylsilyl trifluoromethanesulfonate (TMSOTf) and compound 16 to produce compound 8; (x) partially deprotecting compound 8, and then reacting with an acetic acid reagent to obtain compound 17; and (xi) subjecting compound 17 to a deprotection reaction to obtain compound 2; 3. The preparation method according to claim 2, wherein The method further comprises the steps of: (iv) deprotecting compound 12 and then reacting it with benzaldehyde dimethyl acetal to produce compound 13; (v) reacting compound 13 with 2-(bromomethyl)naphthalene NapBr to produce compound 14; (vi) partially deprotecting compound 14 and then reacting it with benzoyl cyanide to generate compound 7; (vii) reacting compound 6 with compound 7 to produce compound 15; and (viii) partially deprotecting compound 15 to generate compound 16; 4. The preparation method according to claim 2 or 3, characterized in that The method further comprises the steps of: (i) reacting compound 9 with an acetylating agent to produce compound 10; preferably, the acetylating agent is acetic anhydride or acetyl chloride; (ii) reacting compound 10 with a solution of HBr in acetic acid to produce compound 11; and (iii) Compound 11 reacts in the presence of 2,6-lutidine to produce compound 5; 5. The preparation method according to claim 1, wherein The cyclodextrin is selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, sodium sulfobutyl-β-cyclodextrin, methylated-β-cyclodextrin, or a combination thereof, preferably methylated-β-cyclodextrin.

6. The preparation method according to claim 1, wherein Step (xii) includes one or more of the following features: The reaction is carried out in a buffer solution at pH 7-8, preferably, such as HEPES buffer or Tris-HCl buffer; The 5'-adenosine monophosphate-sialic acid divalent salt is a sodium salt or a potassium salt; The reaction temperature is 37±5°C, preferably 37±2°C, more preferably 37±1°C; The concentration of α-2,3-sialyltransferase Cst-I is 50-200 μg / mL; preferably 80-120 μg / mL; and / or The equivalent ratio of compound 2 to 5'-adenosine monophosphate-sialic acid divalent salt is 1:1-2, preferably 1:1.2-1.

5.

7. The preparation method according to claim 1, wherein Step (xiii) includes one or more of the following features: The reaction solvent is selected from the group consisting of THF, H2O, or a combination thereof; The acylation reaction is carried out in the presence of an acid binding agent, such as an acid binding agent selected from the group consisting of NaHCO3, KHCO3, Na2CO3, K2CO3, or a combination thereof; The reaction temperature is 25±15°C, preferably 25±10°C, more preferably 25±5°C; and / or Compound 3 and C 17 H 37 The equivalent ratio of COR is 1:1-2; preferably 1:1.2-1.

5.

8. The preparation method according to claim 1, wherein Step (xiv) includes one or more of the following features: The reaction is carried out in a buffer solution of pH 6.5-8, preferably pH 6.8-7.4, such as HEPES buffer or Tris-HCl buffer; The reaction temperature is 37±5°C, preferably 37±2°C, more preferably 37±1°C; The concentration of α2,6-sialyltransferase ST6GalNAc5 is 5-100 μg / mL; preferably 10-50 μg / mL; The equivalent ratio of compound 4 to 5'-adenosine monophosphate-sialic acid divalent salt is 1:1-2; preferably 1:1.2-1.5; and / or The equivalent ratio of compound 4 to cyclodextrin is 1:1-4, preferably 1:1.5-2.

5.

9. The preparation method according to claim 2, wherein Step (x) comprises the following steps: (x-1) Compound 8 reacts in a hydrofluoric acid pyridine solution to obtain intermediate 8-1; (x-2) intermediate 8-1 is reacted in the presence of a base (such as NaOH, KOH, or a combination thereof) and then neutralized to produce intermediate 8-2; and (x-3) Intermediate 8-2 is reacted with an acetylating agent to obtain compound 17.

10. The preparation method according to claim 2, characterized in that Step (xi) comprises the following steps: (xi-1) In the presence of 2,3-dichloro-5,6-dicyano-p-benzoquinone DDQ, compound 17 reacts to obtain an intermediate 17-1; and (xi-2) In the presence of 1,3-propanedithiol, pyridine and water, intermediate 17-1 reacts to give compound 2.

11. The preparation method according to claim 3, wherein Step (iv) comprises the following steps: (iv-1) in the presence of a base (such as NaOH, KOH, or a combination thereof), deprotecting compound 12 to produce intermediate 12-1; and (iv-1) In the presence of camphorsulfonic acid (CSA), intermediate 12-1 reacts with benzaldehyde dimethyl acetal to produce compound 13.

12. The preparation method according to claim 3, wherein Step (vi) comprises the following steps: (vi-1) dissolving compound 14 in a mixed solution of DCM, TFA, and H2O (volume ratio (8-12): (0.5-2): 1, preferably 10:1:1), and deprotecting compound 14 to produce intermediate 14-1; and (vi-1) In the presence of a base (such as diethylamine, triethylamine, dimethylamine, trimethylamine, or a combination thereof), intermediate 14-1 reacts with benzoyl cyanide to produce compound 7.

13. The preparation method according to claim 3, characterized in that Step (viii) comprises: (viii) partially deprotecting compound 15 in the presence of hydrazine acetate to generate compound 16.

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