Cyclic dinucleotide 2 ', 3'-cG4 '-MeAMP, preparation method and application of cyclic dinucleotide 2', 3 '-cG4'-MeAMP in preparation of innate immune activator

Abstract

The invention relates to the field of chemical synthesis of nucleoside and oligonucleotide and the field of innate immunity, and discloses cyclic dinucleotide 2 ', 3'-cG4 '-MeAMP, a preparation method and application of the cyclic dinucleotide 2', 3 '-cG4'-MeAMP in preparation of innate immunity activators, the chemical structural formula of the cyclic dinucleotide 2 ', 3'-cG4 '-MeAMP is shown in the specification, and compared with natural cyclic dinucleotide 2', 3 '-cGAMP, the cyclic dinucleotide 2', 3 '-cG4'-MeAMP has the structural formula shown in the specification. The 3 '-cG4'-MeAMP has higher serum stability, has a remarkably enhanced innate immune activation effect on human cells carrying an inactivated STING-R232H mutant, and shows a potential treatment value in STING function defect related diseases.

Description

Technical Field

[0001] The present invention relates to the chemical synthesis field of nucleosides and oligonucleotides and the field of innate immunity, and relates to a cyclic dinucleotide 2', 3'-cG 4′-Me AMP, preparation method and use thereof in the preparation of innate immune activators. Background Art

[0002] Cyclic dinucleotides are key second messengers in the cGAS (cGAMP synthase)-STING (stimulator of interferon genes) innate immune pathway. In response to double-stranded DNA, cGAS synthesizes cyclic dinucleotides, which bind to and activate STING, inducing the secretion of type I interferons, inflammatory cytokines, and tumor necrosis factor, triggering antiviral, anti-tumor, and inflammatory immune responses (cGAS in action: Expanding roles in immunity and inflammation, Science, 2019, 363, eaat8657). Among them, 2′,3′-cyclic GMP-AMP dinucleotide (2′,3′-cGAMP) is an endogenous ligand for mammalian STING and exhibits potent immune activation in mammalian cells. Therefore, 2′,3′-cGAMP is considered a potential drug candidate for the treatment of infections, cancer, and autoimmune diseases.

[0003] However, 2′,3′-cGAMP itself is easily degraded by nucleases, has poor cell membrane permeability, and has little activating effect on the inactive STING mutants (such as R232H) that are prevalent in the human population, severely limiting its potential as a direct drug. To improve the drugability of 2′,3′-cGAMP, researchers have attempted to introduce structural modifications at sites such as the base, phosphodiester bond, and 2ʹ-H / OH, which has become a common optimization strategy in recent years (2′,3′-Cyclic GMP-AMP Dinucleotides for STING-Mediated Immune Modulation: Principles, Immunotherapeutic Potential, and Synthesis, ChemMedChem, 2022, 17, e202100671). However, no cyclic dinucleoside analogs that effectively activate inactive STING mutants have been identified. Summary of the Invention

[0004] In view of the poor stability of the natural cyclic dinucleotide 2',3'-cGAMP and the poor immune activation capability of the 2',3'-cGAMP for various hSTING mutants (such as the hSTING-R232H mutant), the application discloses a cyclic dinucleotide 2',3'-cG 4′- Me AMP, a preparation method thereof and application of the cyclic dinucleotide 2',3'-cG 4′-Me AMP is a novel cyclic dinucleotide analogue, that is, 2',3'-cG 4′-Me AMP containing a C4'-methylguanosine modification. The cell experiment results show that the molecule has a significantly enhanced activation effect on the inactivated STING-R232H mutant, and shows potential therapeutic value in diseases related to STING function defects.

[0005] The technical scheme of the application is as follows: In order to achieve the above-mentioned purpose, the application provides a C4'-methylguanosine acid modified cyclic dinucleotide 2',3'-cG 4′-Me AMP in a first aspect of the application, wherein the 2',3'-cG 4′-Me The chemical structural formula of the 2',3'-cG .

[0006] The application provides a preparation method of the cyclic dinucleotide 2',3'-cG 4′-Me AMP in a second aspect of the application, and the preparation method comprises the following steps: (1) a first mixed solution is formed by triazole, triethylamine, 2-chlorophenyl phosphorodichloridate and dichloromethane, and after 1 hour, the first mixed solution is reacted with 2-N-(acetyl)-3'-O-(tert-butyldimethylsilyl)-4'-C-(methyl)-5'-O-(dimethoxytrityl)-beta-D-guanosine, and the molar amount of the triazole, triethylamine, 2-chlorophenyl phosphorodichloridate, 2-N-(acetyl)-3'-O-(tert-butyldimethylsilyl)-4'-C-(methyl)-5'-O-(dimethoxytrityl)-beta-D-guanosine and the volume amount of the dichloromethane are in a ratio of 1.6 mmol: 1.6 mmol: 0.6 mmol: 0.4 mmol: 1-3 mL; after the reaction, the obtained solution is diluted with dichloromethane, and then washed, dried, filtered and column chromatography purified to obtain 2-N-(acetyl)-2'-O-(2-chlorophenyl phosphate)-3'-O-(tert-butyldimethylsilyl)-4'-C-(methyl)-5'-O-(dimethoxytrityl)-beta-D-guanosine; (2) The 2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytrityl)-β-D-guanosine, 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole and anhydrous pyridine obtained in step (1) are mixed for a second time to form a second mixed solution; and 3-hydroxypropionitrile is added. reacting with the second mixed solution, wherein the molar amounts of 2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytrityl)-β-D-guanosine, 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole, and 3-hydroxypropionitrile and the volume ratio of anhydrous pyridine are 1.0 mmol: 3.5 mmol: 2.0 mmol: 1-3 mL; acidifying the solution after the reaction by adding an appropriate amount of oxalic acid solution, then diluting with dichloromethane, washing, drying, and filtering to obtain a crude product; The crude product is reacted with dichloroacetic acid and dichloromethane to remove the dimethoxytrityl group, wherein the molar amount of the crude product, the volume ratio of dichloroacetic acid and dichloromethane is 1.0 mmol: 0.3-1 mL: 10-20 mL. The reaction solution is neutralized by adding an appropriate amount of saturated sodium bicarbonate solution, and the solvent is removed by rotary evaporation. The solution is diluted with dichloromethane, washed, dried, filtered, and separated by column chromatography to obtain 2-N-(acetyl)-2′-O-(2-chlorophenyl-2-nitrileethyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine; (3) triazole, triethylamine, 2-chlorophenyl dichlorophosphate and dichloromethane are used to form a third mixed solution. One hour later, the third mixed solution is reacted with 2′-O-(tert-butyldimethylsilyl)-5′-O-(dimethoxytrityl)-6-N-(benzoyl)-β-D-adenosine. The molar amount of triazole, triethylamine, 2-chlorophenyl dichlorophosphate, 2′-O-(tert-butyldimethylsilyl)-5′-O-(dimethoxytrityl)-6-N-(benzoyl)-β-D-adenosine and the volume ratio of dichloromethane are 1.6 mmol: 1.6 mmol: 0.6 mmol: 0.4 mmol: 1-3 mL, the solution obtained after the reaction was diluted with dichloromethane, washed, dried, filtered, and column chromatographed to obtain 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-5′-O-(dimethoxytrityl)-6-N-(benzoyl)-β-D-adenosine; (4) The 2-N-(acetyl)-2′-O-(2-chlorophenyl-2-nitrileethyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine obtained in step (2) and the 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-5′-O-(dimethoxytrityl)-6-N-(benzoyl)-β-D-adenosine obtained in step (3) are mixed with pyridine to form a fourth mixed solution; the fourth mixed solution is then mixed with 1-(mesityl-2-sulfone)-3 -nitro-1,2,4-triazole reaction, the molar amount of the 2-N-(acetyl)-2′-O-(2-chlorophenyl-2-nitrileethyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine, 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-5′-O-(dimethoxytrityl)-6-N-(benzoyl)-β-D-adenosine, 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole, and the volume ratio of pyridine are 0.17 mmol: 0.22 mmol: 0.51 mmol: 2 mL, and the solution after the reaction is acidified by adding an appropriate amount of oxalic acid solution, then diluted with dichloromethane, washed, and dried to obtain a crude product; The crude product is reacted with dichloroacetic acid and dichloromethane to remove the dimethoxytrityl group, wherein the molar amount of the crude product, the volume ratio of dichloroacetic acid and dichloromethane is 3.0 mmol: 0.3 mL: 10 mL. An appropriate amount of saturated sodium bicarbonate solution is added to the reaction solution for neutralization. The solvent is removed by rotary evaporation, and the solution is diluted with dichloromethane, washed, dried, and separated by column chromatography to obtain 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-6-N-(benzoyl)-β-D-adenosine-2-N-(acetyl)-2′-O-(2-chlorophenyl-2-nitrileethyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine dinucleotide; (5) The 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-6-N-(benzoyl)-β-D-adenosine-2-N-(acetyl)-2′-O-(2-chlorophenyl-2-nitrileethyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine dinucleotide obtained in step (4) is mixed with tert-butylamine and acetonitrile to form a fifth mixed solution, and the 2-nitrileethyl group is removed by reaction. After the reaction is completed, the solvent is dried by spin drying and then mixed with 1-(mesityl-2-sulfone)-3-nitrile The cyclization reaction of 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-6-N-(benzoyl)-β-D-adenosine-2-N-(acetyl)-2′-O-(2-chlorophenyl-2-nitrileethyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine dinucleotide and 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole, and the volume ratio of tert-butylamine, acetonitrile, and pyridine are 0.2 mmol: 1.1 mmol: 2 mL: 6 mL: 20 mL. After the reaction, a small amount of water is added to quench the reaction. The solvent was spun off, acidified with an appropriate amount of oxalic acid solution, and then diluted with dichloromethane, washed, dried, and purified by column chromatography to obtain 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-6-N-(benzoyl)-β-D-adenosine-2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine cyclic dinucleotide; (6) The 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-6-N-(benzoyl)-β-D-adenosine-2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine cyclic dinucleotide obtained in step (5) is mixed for the sixth time with tetramethylguanidine, pyridine-2-formaldehyde oxime, 1,4-dioxane and water to form a sixth A mixed solution, the molar amounts of 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-6-N-(benzoyl)-β-D-adenosine-2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine cyclic dinucleotide, tetramethylguanidine, pyridine-2-carbaldehyde oxime, and the volume ratio of 1,4-dioxane and water are 0.26 mmol: 1.16 mmol: 1.16 mmol: 2 mL: 2 mL, the reaction is carried out to remove the 2-chlorophenyl group, and after completion, part of the solvent is spun off, and the sixth mixed solution is reacted with a methylamine-ethanol solution to remove the acetyl group and the benzoyl group.

[0007] After the reaction is completed, part of the solution is removed by swirl, and a mixed solution of triethylamine-triethylamine hydrofluoride-pyridine is added again to remove the tert-butyldimethylsilyl group by reaction. After the reaction is completed, part of the solvent is removed by swirl evaporation, acetone is added under stirring, and the precipitate is collected by centrifugation. The washed precipitate is dissolved in water, filtered with a liquid filter membrane, and purified by liquid phase to obtain cyclic dinucleotide 2′,3′-cG 4′-Me AMP.

[0008] Preferably, in step (1), the reaction conditions include: argon protection, a temperature of -1-1°C, a reaction time of 1-3 hours, a stirring speed of 250-500 rpm, and quenching by adding 2 mL of 1 M TEAB buffer solution after the reaction is completed. More preferably, the temperature is 0°C and the reaction time is 2 hours.

[0009] Preferably, in step (2), the reaction conditions include: argon protection, temperature of 24-26°C, time of 1-3 hours, more preferably 2 hours, stirring speed of 250-500 rpm, and adding an appropriate amount of 5% oxalic acid solution to adjust the pH of the system to 3-4 after the reaction; the conditions for removing the dimethoxytrityl group include: temperature of -1-1°C, time of 8-20 minutes, more preferably temperature of 0°C, time of 10 minutes, stirring speed of 250-500 rpm, and adding saturated sodium bicarbonate solution for neutralization after the reaction.

[0010] Preferably, in step (3), the reaction conditions include: argon protection, temperature of -1-1°C, time of 1-3 hours, more preferably 0°C, time of 2 hours, stirring speed of 250-500 rpm, and quenching by adding 2 mL of 1M TEAB buffer solution after the reaction is completed.

[0011] Preferably, in step (4), the reaction conditions include: argon protection, temperature of 24-26°C, time of 1-3 hours, more preferably 2 hours, stirring speed of 250-500 rpm, and adding an appropriate amount of 5% oxalic acid solution after the reaction to adjust the pH of the system to 3-4; the conditions for removing the dimethoxytrityl group include: temperature of -1-1°C, time of 8-15 minutes, more preferably temperature of 0°C, time of 10 minutes, stirring speed of 250-500 rpm, and adding saturated sodium bicarbonate solution for neutralization after the reaction. Preferably, in step (5), the conditions for the removal of 2-nitroethyl group reaction include: argon protection, temperature of 24-26°C, time of 15-30 minutes, more preferably 20 minutes, stirring speed of 250-500 rpm, and after the reaction, using anhydrous acetonitrile to co-rotate three times and fully dry; the conditions for the cyclization reaction include: argon protection, temperature of 24-26°C, time of 5-8 hours, more preferably 6 hours, stirring speed of 250-500 rpm, and after the reaction, adding an appropriate amount of 5% oxalic acid solution to adjust the system pH to 3-4.

[0012] Preferably, in step (6), the conditions for removing the 2-chlorophenyl group include: argon protection, a temperature of 24-26°C, a time of 16-19 hours, more preferably 17 hours, and a stirring speed of 250-500 rpm; The conditions for the removal of acetyl and benzoyl reactions include: argon protection, a temperature of 24-26° C., a time of 2-4 hours, more preferably 3 hours, and a stirring speed of 250-500 rpm; The conditions for removing the tert-butyldimethylsilyl group reaction include: argon protection, temperature of 40-60°C, time of 4-6 hours, preferably 5 hours, and stirring at 250-500 rpm. After the reaction, the solvent is partially removed by rotary evaporation, and 30 mL of pre-cooled acetone is added while the reaction is hot and stirring, resulting in the precipitation of a large amount of solid. Stirring is continued at 24-26°C for 20 minutes, and the precipitate is collected by centrifugation.

[0013] The third aspect of the present invention provides a cyclic dinucleotide 2',3'-cG 4′-Me Application of AMP in the preparation of innate immunity activators.

[0014] The fourth aspect of the present invention provides a cyclic dinucleotide 2',3'-cG 4′-MeApplication of AMP in the preparation of drugs for treating diseases related to STING functional deficiency.

[0015] The advantages and beneficial effects of the present invention are: The present invention introduces a methyl group into the C4ʹ position of guanine G in the natural cyclic dinucleotide 2′,3′-cGAMP to obtain the cyclic dinucleotide 2′,3′-cGAMP. 4′-Me AMP can be used to prepare innate immune activators, significantly improving serum stability. In particular, it has a significantly enhanced innate immune activation effect on human cells carrying the inactivated STING mutant R232H, and has potential value in the preparation of drugs for treating diseases related to STING functional defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a natural cyclic dinucleotide 2′,3′-cGAMP and cyclic dinucleotide 2′,3′-cG 4′-Me Figure 1 shows the stability test of AMP in fetal bovine serum; Figure 2 It is a natural cyclic dinucleotide 2′,3′-cGAMP and cyclic dinucleotide 2′,3′-cG 4′-Me Comparison of AMP-induced expression of immune factor interferon in HEK293T cells by activating STING, where A) is 2′,3′-cGAMP and 2′,3′-cG 4 ′-Me AMP activates wild-type hSTING, B) 2′,3′-cGAMP and 2′,3′-cG 4′-Me AMP activates the hSTING-R232H mutant. DETAILED DESCRIPTION

[0017] The following describes the specific embodiments of the present invention in detail. The specific embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0018] Dichloromethane (purchased from Tianjin Binhai New Area Guangshun Da Chemical Reagent Co., Ltd., product number A1040); sodium bicarbonate (purchased from Tianjin Binhai New Area Guangshun Da Chemical Reagent Co., Ltd., product number 2049); dissolved acetonitrile (≤10 ppm) (purchased from Hebei Dina Xingke Biotechnology Co., Ltd., product number R1012-4); anhydrous magnesium sulfate (purchased from Tianjin Binhai New Area Guangshun Da Chemical Reagent Co., Ltd., product number S2509); sodium chloride (purchased from Tianjin Chemical Reagent Supply and Marketing Company, product number 017); methanol (purchased from Beijing Bailingwei Technology Co., Ltd., product number 980290-500ML); triethylamine (purchased from Beijing Yinuokai Company, product number T9710); 1,2,4-triazole (purchased from Beijing Yinuokai Company, product number 45140A); 2-chlorophenyl dichlorophosphate (purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd., product number 1275469-5g); 1-(mesityl-2-sulfonyl)-3-nitro-1,2,4-triazole (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number M109334-5g); pyridine (purchased from Tianjin Binhai New Area Guangshunda Chemical Reagent Co., Ltd., product number S2442); dichloroacetic acid (purchased from Beijing Bailingwei Technology Co., Ltd., product number 140166); oxalic acid (purchased from (purchased from Beijing Kaiguo Technology Co., Ltd., product number AP009571); 3-hydroxypropionitrile (purchased from Anhui Zesheng Technology Co., Ltd., product number B040147); tert-butylamine (purchased from Beijing J&K Technology Co., Ltd., product number 237824); pyridine-2-carbaldehyde oxime (purchased from Beijing J&K Technology Co., Ltd., product number 529268); tetramethylguanidine (purchased from Beijing J&K Technology Co., Ltd., product number 940257); 1,4-dioxane (purchased from Tianjin Binhai New Area Guangshunda Chemical Reagent Co., Ltd., product number S2438); methylamine-ethanol solution (33% wt) (purchased from Beijing Yinuokai Company, product number M433515-100 ml); and triethylamine trihydrofluoride (purchased from Beijing J&K Technology Co., Ltd., product number 433417).

[0019] Example 1 A cyclic dinucleotide 2′,3′-cG 4′-Me The preparation method and synthetic process of AMP are as follows:

[0020] Compound 1 is 2-N-(acetyl)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytrityl)-β-D-guanosine; Compound 1 was synthesized using D-ribose and guanine as starting materials according to the literature (Synthesis of 4′-C-Methylnucleosides, Bioscience, Biotechnology and Biochemistry, 1993, 57, 1433).

[0021] Compound 2 is 2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytrityl)-β-D-guanosine; Compound 3 is 2-N-(acetyl)-2′-O-(2-chlorophenyl-2-nitrileethyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine; Compound 4 is 2′-O-(tert-butyldimethylsilyl)-5′-O-(dimethoxytrityl)-6-N-(benzoyl)-β-D-adenosine; it was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., catalog number BD215385.

[0022] Compound 5 is 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-5′-O-(dimethoxytrityl)-6-N-(benzoyl)-β-D-adenosine; Compound 6 is 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-6-N-(benzoyl)-β-D-adenosine-2-N-(acetyl)-2′-O-(2-chlorophenyl-2-nitrileethyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine dinucleotide; Compound 7 is 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-6-N-(benzoyl)-β-D-adenosine-2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine cyclic dinucleotide; Compound 8 is a cyclic dinucleotide 2′,3′-cG 4′-Me AMP.

[0023] The specific steps are as follows: (1) Preparation of compound 2: under argon protection, triazole (115 mg, 1.60 mmol, 4.0 eq) was weighed into a 25 mL round-bottom flask, dry triethylamine (0.25 mL, 1.60 mmol, 4.0 eq) and 1 mL dry DCM (dichloromethane) were added, stirred at 0 °C, the stirring speed was 300 rpm, 2-chlorophenyldichlorophosphate (157 mg, 0.64 mmol, 1.6 eq) in dichloromethane (1 mL) was added to the reaction system at a rate of one drop per second to form a first mixed solution, after 1 h of reaction at 0 °C, compound 1 (300 mg, 0.4 mmol, 1.0 eq) was dissolved in 1 mL dry DCM (dichloromethane) and added to the first mixed solution at a rate of one drop per second, and reacted at 0 °C for 2 h. After the reaction was monitored by TLC, it was moved to 26 °C, 2 mL of 1 M TEAB buffer solution was added, and stirring was continued for half an hour at a stirring speed of 300 rpm. The organic phase was extracted and washed with 1 M TEAB buffer solution and distilled water twice respectively, and the organic phase was collected, dried over anhydrous MgSO4, and the solvent was rotary evaporated and purified by column chromatography to obtain white solid product (compound 2).

[0024] (2) Preparation of compound 3: under argon protection, compound 2 (328 mg, 0.36 mmol, 1.0 eq) and 1-(mesityl-2-sulfonyl)-3-nitro-1,2,4-triazole (373 mg, 1.26 mmol, 3.5 eq) were weighed into a 10 mL round-bottom flask, 1 mL of anhydrous pyridine was added to form a second mixed solution; 3-hydroxypropionitrile (0.05 mL, 0.72 mmol, 2.0 eq) was added to the second mixed solution, and the reaction was carried out at 26 °C for 2 h with a stirring speed of 300 rpm. After the reaction was monitored by TLC, a small amount of water was added to quench the reaction, and the rotary evaporation was carried out under reduced pressure. The crude product was dissolved in 5 mL DCM, and the pH of the system was adjusted to 3-4 by adding an appropriate amount of 5% oxalic acid solution. The organic phase was separated, the aqueous phase was extracted with DCM three times, the organic phase was combined, and the saturated NaCl solution was washed once. The organic phase was collected and dried over anhydrous MgSO4, and the solvent was rotary evaporated to obtain the crude product.

[0025] The above crude product was dissolved in 10 mL DCM, 0 oThe mixture was stirred at 300 rpm and 0.3 mL of dichloroacetic acid was added. The reaction was maintained in an ice bath for 10 minutes to remove the dimethoxytrityl group. After completion of the reaction, monitored by TLC, a few drops of methanol were added to quench the reaction and stirring was continued for 10 minutes. Saturated NaHCO₃ solution was then added to neutralize the mixture until no bubbles appeared. The organic phase was separated by extraction, and the aqueous phase was extracted multiple times with DCM. The combined organic phases were washed twice with saturated NaCl solution and dried over anhydrous MgSO₄. The solvent was evaporated and the mixture was separated by column chromatography (methanol / dichloromethane, v / v, 1 / 50-1 / 30). 143 mg of the target compound 3 was obtained with a three-step yield of 76%.

[0026] The nuclear magnetic resonance (NMR) analysis results of compound 3 are as follows: 31 P NMR (162 MHz, CDCl3) δ (ppm): -7.87, -9.27; 1 H NMR (400 MHz, CDCl3) δ (ppm): 9.65 (br, 1H, -NHCO-), 8.48 (s, 1H, -NHCO-), 7.78 (s, 1H, -N=CH), 7.14-7.20 (m, 1H, -ArH), 6.89-7.08 (m, 3H, -ArH), 5.86 (t, J = 6.8 Hz, 1H, -H1′), 5.75-5.82 (m, 1H, -P-CH2), 4.45 (t, J = 4.1Hz, 1H, -P-CH2), 4.12-4.23 (m, 1H, -H3′), 4.07 (dd, J = 6.4, 13.9 Hz, 1H, -H2′), 3.63 (d, J = 12.5 Hz, 1H, -H5′), 3.34-3.45 (m, 1H, -H5′′), 2.65 (t, J = 5.8Hz, 1H, -CH2-CN), 2.51 (dd, J = 5.7, 12.0 Hz, 1H, -CH2-CN), 2.12 (s, 3H, Ac-CH3), 1.12 (s, 3H, C4′-CH3), 0.82 (s, 9H, -tBu), 0.03 (s, 3H, Si-CH3), 0.00(s, 3H, Si-CH3); 13C NMR (100.4 MHz, CDCl3) δ (ppm): 172.7, 172.7, 155.7, 155.5, 148.0,147.9, 147.7, 147.7, 146.0, 145.6, 145.7, 139.6, 139.5, 130.7, 130.6, 128.1,128.0, 126.6, 125.0, 125.0, 124.3, 124.2, 121.9, 121.0, 120.0, 116.6, 116.2,88.8, 86.7, 86.4, 78.6, 78.2, 72.7, 72.6, 67.3, 67.1, 63.4, 63.3, 63.1, 63.1,24.3, 24.3, 19.7, 19.6, 19.5, 19.5, 19.1, 19.0, 18.2, -4.5, -4.6, -4.8. According to the NMR results, it can be confirmed that the structure of compound 3 is correct.

[0027] (3) Preparation of compound 5: Under argon protection, triazole (174 mg, 2.52 mmol, 4.0 eq) and dry triethylamine (0.35 mL, 2.52 mmol, 4.0 eq) were weighed into a 25 mL round-bottom flask, 1 mL of dry DCM was added to dissolve, and the mixture was stirred at 0°C at a stirring speed of 300 rpm. A DCM solution (1 mL) of 2-chlorophenyl dichlorophosphate (245 mg, 1.00 mmol, 1.6 eq) was added dropwise to the reaction system at a rate of one drop per second to form a third mixed solution. After reacting at 0°C for 1 h, compound 4 (500 mg, 0.63 mmol, 1.0 eq) was dissolved in 1 mL of dry DCM and added dropwise to the third mixed solution at a rate of one drop per second. The mixture was reacted at 0°C for 2 h. After the reaction was completed as monitored by TLC, the temperature was moved to 26°C, 2 mL of 1 M TEAB buffer solution was added, and stirring was continued for half an hour.

[0028] The reaction system was washed twice with 1 M TEAB buffer solution and distilled water, and the organic phase was collected and dried over anhydrous MgSO4. The solvent was removed by swirl to obtain a white solid product, namely compound 5, which was directly used in the next step after drying.

[0029] (4) Preparation of compound 6: Under argon protection, compound 5 (210 mg, 0.22 mmol, 1.3 eq) and compound 3 (110 mg, 0.17 mmol, 1.0 eq) were mixed with 2 mL of pyridine to form a fourth mixed solution; the fourth mixed solution was then reacted with 1-(mesityl-2-sulfonyl)-3-nitro-1,2,4-triazole (150 mg, 0.51 mmol, 3.0 eq) and stirred at room temperature for 2 h at a stirring speed of 300 rpm. After the reaction was completed, 1 mL of water was added to quench the reaction. The pyridine was spin-dried, and the residue was dissolved in 10 mL of DCM. The pH of the system was adjusted to 3-4 with 5% oxalic acid solution, and the organic phase was collected. The aqueous phase was extracted twice with DCM and discarded. The organic phases were combined and washed twice with saturated NaCl solution. The organic phase was collected and spin-dried to obtain a crude product, which was directly used in the next step.

[0030] The crude product (496 mg, 3.0 mmol, 1.0 eq) was dissolved in 10 mL of DCM. 0.3 mL of dichloroacetic acid was added with stirring at 0°C for 10 minutes to remove the dimethoxytrityl group. After completion of the reaction, monitored by TLC, 2 mL of methanol was added to quench the reaction. Stirring was continued at low temperature for 10 minutes, followed by neutralization with saturated NaHCO₃ solution until no bubbles formed. The organic phase was washed twice with saturated NaCl solution, and the aqueous phase was extracted twice with DCM and discarded. The combined organic phases were evaporated to dryness under reduced pressure and separated by column chromatography (methanol / dichloromethane, v / v, 1:50-1:30) to afford 250 mg of compound 6 in a 70% yield.

[0031] The nuclear magnetic resonance (NMR) analysis results of compound 6 are as follows: 31 P NMR (162 MHz, CDCl3) δ (ppm): -7.39, -8.29, -8.35, -9.75; 1 H NMR (400 MHz, CDCl3) δ (ppm): 11.52 (s, 1H), 10.18 (s, 1H), 9.26 (s, 1H), 8.81 (s, 1H), 8.15 (s, 1H), 8.04 (d, J = 7.5 Hz, 1H), 7.68 (d, J = 5.1Hz, 1H), 7.62 (t, J = 7.3 Hz, 1H), 7.53 (t, J= 7.5 Hz, 2H), 6.97-7.35 (m, 8H),6.73-6.85 (m, 1H), 6.52-6.61 (m, 1H), 6.01-6.21 (m, 3H), 5.91-5.95 (m, 1H),5.40-5.45 (m, 1H), 5.30 (s, 1H), 4.84 (s, 1H), 4.26-4.53 (m, 4H), 4.06 (d, J =11.5 Hz, 1H), 3.87 (t, J = 11.8 Hz, 1H), 2.72- 2.90 (m, 1H), 2.04 (s, 3H), 1.44(m, 3H), 1.03 (m, 9H), 0.74 (s, 9H), 0.27 (m, 3H), 0.21 (m, 3H), -0.12 (s,3H), -0.40 (m, 3H); 13 C NMR (100.4 MHz, CDCl3) δ (ppm): 172.4, 164.5, 155.2, 154.9, 152.4,150.6, 148.0, 147.7, 147.2, 147.0, 145.5, 145.4, 145.3, 145.2, 144.9, 144.8,144.7, 143.1, 143.0, 139.8, 133.4, 133.0, 130.8, 130.5, 130.3, 128.9, 128.2,128.1, 128.0, 127.5, 127.4, 126.9, 126.0, 125.9, 125.8, 124.4, 123.7, 123.6,123.0, 122.9, 122.8, 120.9, 119.0, 116.6, 115.8, 90.7, 90.6, 86.7, 86.4,86.2, 86.1, 86.0, 85.8, 85.7, 85.6, 79.9, 79.8, 73.2, 73.1, 72.7, 72.6, 63.4,63.3, 63.3, 62.8, 62.7, 53.5, 29.7, 25.9, 25.8, 25.4, 23.7, 19.8, 19.7, 19.5,19.4, 19.2, 19.1, 18.4, 17.8, -4.3, -4.4, -4.6, -4.6, -5.1, -5.7, -5.8 HRMS (ESI): C 57 H 72 Cl2N 11 O 16 P2Si2[M+H] + , calc.1354.3549, found 1354.3563 According to the results of NMR and mass spectrometry, it can be confirmed that the structure and molecular weight of compound 6 are correct.

[0032] (5) Preparation of compound 7: In a 25 mL round-bottom flask, compound 6 (30 mg, 0.022 mmol, 1.0 eq) was dissolved in a tert-butylamine-acetonitrile mixed solvent (1 / 3, V / V, 0.8 mL in total) and stirred at room temperature for 20 min to form a fifth mixed solution to remove the 2-nitroethyl group. After the reaction was completed, the solution was concentrated under reduced pressure, acetonitrile was added, and the mixture was vortexed three times and dried thoroughly. Under argon protection, 1-(mesityl-2-sulfonyl)-3-nitro-1,2,4-triazole (33 mg, 0.11 mmol, 5.0 eq) and 2 mL of anhydrous pyridine were added to the reaction system for cyclization reaction. The reaction was carried out at 26°C for 6 h with stirring at 300 rpm. After the reaction was completed, a small amount of water was added to quench the reaction. The solvent was evaporated to dryness, dissolved in an appropriate amount of DCM, and then 5% oxalic acid solution was added to adjust the pH of the system to 3-4. The organic phase was separated, and the aqueous phase was extracted twice with DCM. The combined organic phases were washed twice with saturated NaCl solution, dried over anhydrous MgSO4, and separated and purified by column chromatography (methanol / dichloromethane, v / v, 1 / 30) to obtain 8 mg of compound 7 with a two-step yield of 65%.

[0033] (6) Preparation of compound 8: 1) Compound 7 (300 mg, 0.26 mmol, 1.0 eq) was dissolved in a mixed solvent of 2 mL of 1,4-dioxane and 2 mL of water. Tetramethylguanidine (133 mg, 1.16 mmol, 4.5 eq) and pyridine-2-carbaldehyde oxime (141 mg, 1.16 mmol, 4.5 eq) were added, and the mixture was stirred at 26°C for 17 h at a stirring speed of 300 rpm to perform a sixth mixing to form a sixth mixed solution.

[0034] 2) The solvent was evaporated and 10 mL of methylamine-ethanol solution (33% wt) was added. The mixture was stirred at 26°C for 3 h at 300 rpm to remove the acetyl and benzoyl groups.

[0035] 3) The solvent was evaporated, and the residue was co-evaporated three times with a mixture of dry pyridine (1.2 mL) and triethylamine (0.6 mL), then dissolved in 1 mL of dry pyridine, 5 mL of triethylamine, and 3 mL of a mixture of triethylamine-trifluoride acid salt. The reaction was stirred at 50 °C for 5 h at a stirring speed of 300 rpm. The reaction was terminated, and the solvent was evaporated. While hot, 30 mL of pre-cooled acetone was added to the reaction mixture, and a large amount of solid was precipitated. The mixture was stirred at 26 °C for 20 min, and the precipitate was collected by centrifugation. The precipitate was washed once with acetone, dissolved in water, and filtered. Compound 8, a cyclic dinucleotide 2',3'-cG 4′-Me AMP.

[0036] Liquid chromatography conditions: Column type Innoval ODS-2 C18 column (21.2 x 250 mm, 5 μm), UV 254 nm monitoring. Mobile phase gradient: A phase is 50 mM TEAA aqueous solution, B phase is acetonitrile. 0-2 min, 98% A-2% B, 2-32 min, 98% A-2% B to 70% A-30% B, 32-37 min, 70% A-30% B to 100% B, 37-43 min, 100% B, flow rate 10 mL / min.

[0037] The nuclear magnetic resonance (NMR) analysis results of compound 8 are as follows: 1 H NMR (600 MHz, D2O) δ (ppm): 8.25 (s, 1H, A-H8), 8.20 (s, 1H, A-H2),7.79 (s, 1H, G-H8), 6.11 (s, 1H, A-H1′), 5.84 (d, 1H, J = 8.5 Hz, G-H1′), 5.73(td, 1H, J = 4.4, 8.1 Hz, G-H2′), 4.99 (dd, 1H, J = 3.9, 9.1 Hz, A-H3′), 4.70 (m,1H, A-H2′), 4.42-4.43 (m, 2H, A-H4′, G-H3′), 4.40 (d, J = 13.0 Hz, A-H5′), 4.27(dd, 1H, J = 3.0, 11.2 Hz, G-H5′), 4.06 (dd, 1H, J= 2.0 Hz, 12.1 Hz, A-H5′′),3.87 (dd, 1H, J = 4.4, 11.2 Hz, G-H5′′), 1.25 (s, 3H, -CH3); 31 P-decoupling 1 H NMR (600 MHz, D2O) δ (ppm): 8.25 (s, 1H, A-H8), 8.20(s, 1H, A-H2), 7.79 (s, 1H, G-H8), 6.11 (s, 1H, A-H1′), 5.84 (d, 1H, J = 8.5Hz, G-H1′), 5.73 (dd, 1H, J = 4.3, 8.5 Hz, G-H2′), 4.99 ( dd, 1H, J = 3.9, 9.1Hz, A-H3′), 4.70 (m, 1H, A-H2′), 4.42-4.43 (m, 2H, A-H4′, G-H3′), 4.40 (d, J =12.1 Hz, A-H5′), 4.27 (d, 1H, J = 11.3 Hz, G-H5′), 4.06 (d, 1H, J = 11.7 Hz, A-H5′′), 3.87 (d, 1H, J = 11.2 Hz, G-H5′′), 1.25 (s, 3H, -CH3); 13 C NMR (150.6 MHz, D2O) δ (ppm): 158.9, 155.4, 152.9, 152.6, 147.8,138.8, 118.7, 89.7, 86.2, 86.1, 86.0, 85.9, 80.0, 79.9, 79.8, 74.5, 74.4,73.9, 72.1, 72.0, 70.6, 70.5, 70.1, 70.1, 62.1, 62.0, 61.4, 46.7, 18.7, 8.22 31 P NMR (162 MHz, D2O) δ (ppm): -1.23, -2.27 HRMS (ESI): C 21 H25 N 10 O 13 P2[MH] - , calc.687.1078, found 687.1034 According to the results of NMR and mass spectrometry, it can be confirmed that the structure and molecular weight of compound 8 are correct.

[0038] Example 2 cyclic dinucleotide 2ʹ,3ʹ-cG 4ʹ-Me Verification of AMP stability in serum The experimental steps are as follows: In a 200 μL centrifuge tube, 20% fetal bovine serum (6 μL), 10 mM PBS buffer (pH 7.2–7.4), 5 mM MgCl₂, and 0.1 μg / μL cyclic dinucleoside were added to a total volume of 30 μL. The cells were incubated in a 37°C incubator, with samples taken at 0, 1, 2, 6, 12, 24, 36, and 48 hours. The reaction was terminated by adding 5 μL of 0.5 M EDTA (pH 8.0) buffer to each 30 μL sample. The sample was diluted with 50 μL of water and analyzed by HPLC injection with a 30 μL injection volume.

[0039] Purification conditions: The instrument was a FLEXA Purification System HP-Q-100, the chromatographic column was an InnovaLDS-2 C18 column (21.2 × 250 mm, 5 μm), the column temperature was 25 °C, and the detection wavelength was 254 nm. The mobile phase gradient was as follows: Phase A was 10 mM TEAA in water, Phase B was acetonitrile, 100% A from 0 to 2 minutes, 100% A to 78% A / 22% B from 2 to 12 minutes, 78% A / 22% B to 100% B from 12 to 13.5 minutes, 100% B from 13.5 to 15 minutes, 100% B to 100% A from 15 to 17 minutes, and 100% A from 17 to 19 minutes. The flow rate was 1 mL / min. The remaining intact cyclic dinucleoside content was calculated based on the peak area and analyzed graphically, as shown in Figure 2. Figure 1 shown.

[0040] from Figure 1 As can be seen, compared with the modified 2′,3′-cG 4′-Me For AMP, the degradation rate of natural 2′,3′-cGAMP is faster, with a half-life of about 33 hours. 4′-Me AMP degradation is relatively slow, with a half-life of more than 90 hours.4′-Me AMP is much larger than 2′,3′-cGAMP. This is because the presence of guanosine C4ʹ-methyl causes a certain amount of steric hindrance near the adjacent phosphodiester bond, hindering the binding of the substrate to the nuclease exonuclease in the serum, making 2′,3′-cG 4′-Me AMP is more difficult to be cleaved by enzymes. Therefore, the introduction of a C4′-methyl group into guanosine greatly improves the serum stability of cyclic dinucleotides, which is expected to improve the pharmacokinetic properties of cyclic dinucleoside drugs and thus enhance their therapeutic potential.

[0041] Example 3 cyclic dinucleotide 2′,3′-cG 4′-Me AMP activated interferon expression test in HEK293T cells: Because HEK293T cells lack endogenous STING expression, they were transfected with either hSTING-wild-type or hSTING-R232H expression vectors and two dual-luciferase reporter vectors: one encoding firefly luciferase under the control of the IFN-β promoter and the other encoding Renilla luciferase. After 24 hours of culture, IFN-β promoter activity was quantified based on the ratio of firefly to Renilla luciferase activity.

[0042] The experimental steps are as follows: (1) HEK293T cells were cultured at 1.5 × 10 5 The cells were plated at a density of 100 μg / ml in a 24-well cell culture plate and cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0043] (2) Polyjet reagent was used to transfect pcDNA3.1-hSTING plasmid (wild type or R232H mutant, 50 ng / well), pGL3-INFb plasmid (200 ng / well) and pGL4.74-Rluc-TK plasmid (50 ng / well).

[0044] (3) Cyclic dinucleotide transfection: 24 hours after plasmid transfection, small molecules are transfected. There are two ways to transfect small molecules: 1. Direct transfection: Aspirate the DMEM medium from the culture plate and dissolve the cyclic dinucleotide directly in 37°C pre-warmed Opti-DMEM. Add 500 μL per well to a final concentration of 1 μM per well. After incubation for 4 hours, add 1 mL of normal DMEM supplemented with serum and continue incubation for 24 hours.

[0045] 2. Permeabilization Solution: Aspirate the DMEM medium from the culture plate and dissolve the cyclic dinucleotide directly in preheated permeabilization solution at 37°C. Add 500 μL per well to a final concentration of 1 μM. After 30 minutes, aspirate the permeabilization solution and replace with 1 mL of normal DMEM supplemented with serum. Continue incubating for 24 hours. The permeabilization solution consists of: 50 mM 4-hydroxyethylpiperazineethanesulfonic acid, 100 mM potassium chloride, 3 mM magnesium chloride, 0.1 mM dithiothreitol, 85 mM sucrose, 0.2% fetal bovine serum, 1 mM ATP, 0.1 mM GTP, and 10 μg / mL digitonin.

[0046] (4) Dual fluorescence reporter assay: 24 hours after cyclic dinucleotide transfection, aspirate the culture medium. Wash each well with 1 mL of pre-chilled PBS buffer, add 100 μL of Passive Lysis Buffer, incubate at 26°C on a horizontal shaker for 20 minutes, transfer to a prepared 1.5 mL EP tube, centrifuge at 13,000 rpm for 10 minutes, and aspirate the supernatant into a black 96-well plate, 20 μL per well.

[0047] Add 20 μL of Luciferase Assay buffer mixture to each well, and read the first fluorescence data Luc value with a microplate reader. Then add 20 μL of Stop&Glo buffer mixture, and read the second fluorescence data Ren value with a microplate reader. Calculate the ratio of the two fluorescence intensities (Luc value / Ren value), and normalize the fluorescence intensity ratio of the blank group (normal transfection of three plasmids, no transfection of cyclic dinucleotides) to obtain the final activity data. Repeat each data three times and perform error analysis. Figure 2 shown.

[0048] In HEK293T cells transfected with hSTING-wild type (HEK293T / hSTING-WT), direct treatment with 2′,3′-cGAMP increased IFN-β expression by only 1.6-fold. However, treatment with permeabilization buffer increased IFN-β expression by 4-fold ( Figure 2 In contrast, the 2′,3′-cG 4′-Me After AMP treatment, IFN-β expression activity was only slightly increased by 1.4-1.8 fold, regardless of whether permeabilization solution was used. These results further confirmed that 2′,3′-cGAMP is a potent agonist of hSTING-wild type, but has poor membrane permeability in HEK293T cells; 4′-Me AMP has a weak ability to activate hSTING-wild type.

[0049] In HEK293T cells transfected with hSTING-R232H (HEK293T / hSTING-R232H), treatment with 2′,3′-cGAMP resulted in only a slight increase in IFN-β expression, independent of permeabilization ( Figure 2 In contrast, even in the absence of permeabilization fluid, 2′,3′-cG 4′-Me AMP-induced activation of IFN-β expression was also significantly enhanced by 2.3-fold. These results indicate that compared with native 2′,3′-cGAMP in mammalian cells, 2′,3′-cG 4′-Me AMP can more effectively activate the hSTING-R232H variant and induce innate immune responses. 4′-Me AMP has better permeability and better serum stability.

[0050] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A cyclic dinucleotide 2′,3′-cG 4′-Me AMP, characterized in that Its chemical structural formula is: 。 2. A cyclic dinucleotide 2′,3′-cG according to claim 1 4′-Me The preparation method of AMP is characterized in that: The following steps are involved: (1) Triazole, triethylamine, 2-chlorophenyl dichlorophosphate and dichloromethane are prepared into a first mixed solution, and after 1 hour, the first mixed solution is reacted with 2-N-(acetyl)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytrityl)-β-D-guanosine; the molar amount of the triazole, triethylamine, 2-chlorophenyl dichlorophosphate, 2-N-(acetyl)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytrityl)-β-D-guanosine and the volume ratio of dichloromethane are 1.6 mmol: 1.6 mmol: 0.6 mmol: 0.4 mmol: 1-3 mL, the resulting solution after the reaction was diluted with dichloromethane, washed, dried, filtered, and purified by column chromatography to obtain 2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytrityl)-β-D-guanosine; (2) The 2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytrityl)-β-D-guanosine, 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole and pyridine obtained in step (1) are mixed for a second time to form a second mixed solution; and 3-hydroxypropionitrile is reacted with the second mixed solution, wherein the molar amount of the 2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytrityl)-β-D-guanosine, 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole, 3-hydroxypropionitrile and the volume ratio of pyridine are 1.0 mmol: 3.5 mmol: 2.0mmol: 1-3 mL, add appropriate amount of oxalic acid solution to the solution after reaction to acidify it, then dilute it with dichloromethane, wash, dry and filter to obtain a crude product; The crude product is reacted with dichloroacetic acid and dichloromethane to remove the dimethoxytrityl group, wherein the molar amount of the crude product, the volume ratio of dichloroacetic acid and dichloromethane is 1.0 mmol: 0.3-1 mL: 10-20 mL. The reaction solution is neutralized by adding an appropriate amount of saturated sodium bicarbonate solution, and the solvent is removed by rotary evaporation. The solution is diluted with dichloromethane, washed, dried, filtered, and separated by column chromatography to obtain 2-N-(acetyl)-2′-O-(2-chlorophenyl-2-nitrileethyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine; (3) triazole, triethylamine, 2-chlorophenyl dichlorophosphate and dichloromethane are used to form a third mixed solution. One hour later, the third mixed solution is reacted with 2′-O-(tert-butyldimethylsilyl)-5′-O-(dimethoxytrityl)-6-N-(benzoyl)-β-D-adenosine. The molar amount of triazole, triethylamine, 2-chlorophenyl dichlorophosphate, 2′-O-(tert-butyldimethylsilyl)-5′-O-(dimethoxytrityl)-6-N-(benzoyl)-β-D-adenosine and the volume ratio of dichloromethane are 1.6 mmol: 1.6 mmol: 0.6 mmol: 0.4 mmol: 1-3 mL, the solution obtained after the reaction was diluted with dichloromethane, washed, dried, and filtered to obtain 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-5′-O-(dimethoxytrityl)-6-N-(benzoyl)-β-D-adenosine; (4) The 2-N-(acetyl)-2′-O-(2-chlorophenyl-2-nitrileethyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine obtained in step (2) and the 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-5′-O-(dimethoxytrityl)-6-N-(benzoyl)-β-D-adenosine obtained in step (3) are mixed with pyridine to form a fourth mixed solution; the fourth mixed solution is then mixed with 1-(mesityl-2-sulfone)-3 -nitro-1,2,4-triazole reaction, the molar amount of the 2-N-(acetyl)-2′-O-(2-chlorophenyl-2-nitrileethyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine, 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-5′-O-(dimethoxytrityl)-6-N-(benzoyl)-β-D-adenosine, 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole, and the volume ratio of pyridine are 0.17 mmol: 0.22 mmol: 0.51 mmol: 2 mL, and the solution after the reaction is acidified by adding an appropriate amount of oxalic acid solution, then diluted with dichloromethane, washed, dried, and filtered to obtain a crude product; The crude product is reacted with dichloroacetic acid and dichloromethane to remove the dimethoxytrityl group, wherein the molar amount of the crude product and the volume ratio of the dichloroacetic acid and dichloromethane are 3.0 mmol: 0.3 mL: 10 mL. An appropriate amount of saturated sodium bicarbonate solution is added to the reaction solution for neutralization. The solvent is removed by rotary evaporation, and the solution is diluted with dichloromethane, washed, and dried to obtain 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-6-N-(benzoyl)-β-D-adenosine-2-N-(acetyl)-2′-O-(2-chlorophenyl-2-nitrileethyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine dinucleotide; (5) The 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-6-N-(benzoyl)-β-D-adenosine-2-N-(acetyl)-2′-O-(2-chlorophenyl-2-nitrileethyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine dinucleotide obtained in step (4) is mixed with tert-butylamine and acetonitrile to form a fifth mixed solution, and the 2-nitrileethyl group is removed by reaction. After the reaction is completed, the solvent is dried by spin drying and then mixed with 1-(mesityl-2-sulfone)-3-nitrile The cyclization reaction of 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-6-N-(benzoyl)-β-D-adenosine-2-N-(acetyl)-2′-O-(2-chlorophenyl-2-nitrileethyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine dinucleotide, 1-(mesityl-2-sulfone)-3-nitro-1,2,4-triazole, and the volume ratio of the tert-butylamine, acetonitrile, and pyridine is 0.

2. mmol: 1.1mmol:2mL:6mL:20mL. After the reaction, a small amount of water was added to quench the reaction, the solvent was spun off, and an appropriate amount of oxalic acid solution was used for acidification. The product was then diluted with dichloromethane, washed, dried, and purified by column chromatography to obtain 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-6-N-(benzoyl)-β-D-adenosine-2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine cyclic dinucleotide; (6) The 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-6-N-(benzoyl)-β-D-adenosine-2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine cyclic dinucleotide obtained in step (5) is mixed for the sixth time with tetramethylguanidine, pyridine-2-formaldehyde oxime, 1,4-dioxane and water to form a sixth mixed product. The molar amount of 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-6-N-(benzoyl)-β-D-adenosine-2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine cyclic dinucleotide, tetramethylguanidine, pyridine-2-carbaldehyde oxime, and the volume ratio of 1,4-dioxane and water are 0.

26. mmol: 1.16 mmol: 1.16 mmol: 2 mL: 2 mL, react to remove 2-chlorophenyl, after completion, remove part of the solvent, then react the sixth mixed solution with methylamine-ethanol solution to remove acetyl and benzoyl groups, after completion, remove part of the solution, re-add a mixed solution of triethylamine-triethylamine hydrofluoride-pyridine to remove tert-butyldimethylsilyl, after completion, remove part of the solvent by rotary evaporation, add acetone under stirring, collect the precipitate by centrifugation, dissolve the precipitate in water, filter with liquid membrane, and purify by liquid phase to obtain cyclic dinucleotide 2′,3′-cG 4′-Me AMP.

3. The preparation method according to claim 2, characterized in that In step (1), the reaction conditions include: argon protection, temperature of -1-1°C, time of 1-3 hours, and quenching by adding TEAB buffer solution after the reaction is completed.

4. The preparation method according to claim 2, characterized in that In step (2), the reaction conditions include: argon protection, temperature of 24-26°C, time of 1-3 hours, and adding 5% oxalic acid solution after the reaction to adjust the pH of the system to 3-4; the conditions for removing the dimethoxytrityl group include: temperature of -1-1°C, time of 8-20 minutes.

5. The preparation method according to claim 2, characterized in that In step (3), the reaction conditions include: argon protection, temperature of -1-1°C, time of 1-3 hours, and quenching by adding TEAB buffer solution after the reaction is completed.

6. The preparation method according to claim 2, characterized in that In step (4), the reaction conditions include: argon protection, temperature of 24-26°C, time of 1-3 hours, adding an appropriate amount of 5% oxalic acid solution after the reaction to adjust the system pH to 3-4; the conditions for removing the dimethoxytrityl group include: temperature of -1-1°C, time of 8-15 minutes.

7. The preparation method according to claim 2, characterized in that In step (5), the conditions for the removal of the 2-nitroethyl group include: argon protection, a temperature of 24-26°C, a reaction time of 15-30 minutes, and after the reaction, anhydrous acetonitrile is used for co-rotation and sufficient drying; the conditions for the cyclization reaction include: argon protection, a temperature of 24-26°C, a reaction time of 5-8 hours, and after the reaction, an appropriate amount of 5% oxalic acid solution is added to adjust the system pH to 3-4.

8. The preparation method according to claim 2, characterized in that In step (6), the conditions for removing the 2-chlorophenyl group include: argon protection, temperature of 24-26°C, and time of 16-19 hours; the conditions for removing the acetyl group and the benzoyl group include: argon protection, temperature of 24-26°C, and time of 2-4 hours; the conditions for removing the tert-butyldimethylsilyl group include: argon protection, temperature of 40-60°C, and time of 4-6 hours.

9. A cyclic dinucleotide 2′,3′-cG according to claim 1 4′-Me AMP or the cyclic dinucleotide 2′,3′-cG prepared by the preparation method according to any one of claims 2 to 8 4′-Me Application of AMP in the preparation of innate immunity activators.

10. A cyclic dinucleotide 2′,3′-cG according to claim 1 4′-Me AMP or the cyclic dinucleotide 2′,3′-cG prepared by the preparation method according to any one of claims 2 to 8 4′-Me Application of AMP in the preparation of drugs for treating diseases related to STING functional deficiency.

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