2′,3′-cG of cyclic dinucleotides 4′-Me AMP, its preparation method and its application in the preparation of innate immune activators

Abstract

This invention relates to the fields of chemical synthesis of nucleosides and oligonucleotides and innate immunity. The invention discloses a cyclic dinucleotide 2′,3′-cG 4′‑Me AMP, its preparation method, and its application in the preparation of innate immune activators, wherein the cyclic dinucleotide 2′,3′-cG 4′‑Me The chemical structure of AMP is shown below. Compared with the natural cyclic dinucleotide 2′,3′-cGAMP, 2′,3′-cGAMP has different chemical structures. 4′‑Me AMP exhibits higher serum stability and significantly enhances innate immune activation in human cells carrying the inactivated STING-R232H mutant, demonstrating its potential therapeutic value in diseases related to STING dysfunction.

Description

Technical Field

[0001] This invention relates to the fields of chemical synthesis of nucleosides and oligonucleotides and the field of innate immunity, and specifically to a cyclic dinucleotide 2′,3′-cG 4′-Me AMP, its preparation method, and its application in the preparation of innate immune activators. Background Technology

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

[0003] However, 2′,3′-cGAMP is easily degraded by nucleases, has poor cell membrane permeability, and shows almost no activation effect on the widely distributed inactivated STING mutants (such as R232H), severely limiting its potential as a direct drug. To improve the druggability of 2′,3′-cGAMP, researchers have attempted to introduce structural modifications at sites such as its bases, phosphodiester bonds, and 2ʹ-H / OH, becoming a common optimization strategy in recent years (2′,3′-Cyclic GMP-AMP Dinucleotides for STING-Mediated Immune Modulation: Principles, ImmunotherapeuticPotential, and Synthesis, ChemMedChem, 2022, 17, e202100671). However, no cyclic dinucleotide analogues that can effectively activate inactivated STING mutants have yet been discovered. Summary of the Invention

[0004] To address the shortcomings of current natural cyclic dinucleotide 2′,3′-cGAMP, such as poor stability and weak immune activation against various hSTING mutants (e.g., hSTING-R232H mutant), this invention discloses a cyclic dinucleotide 2′,3′-cGAMP. 4′- Me AMP, its preparation method and its application in the preparation of innate immune activators, cyclic dinucleotide 2′,3′-cG 4′-Me AMP is a novel cyclic dinucleotide analogue, namely a 2′,3′-cG containing C4′-methylguanosine modification. 4′-Me AMP. Cell experiments showed that this molecule had a significantly enhanced activation effect on the inactivated STING-R232H mutant, demonstrating its potential therapeutic value in diseases related to STING dysfunction.

[0005] The technical solution of this invention is:

[0006] To achieve the above objectives, the first aspect of the present invention provides a C4′-methylguanylic acid modified cyclic dinucleotide 2′,3′-cG 4′-Me AMP, in which the 2′,3′-cG 4′-Me The chemical structural formula of AMP is:

[0007] .

[0008] A second aspect of the present invention provides the cyclic dinucleotide 2′,3′-cG 4′-Me The preparation method of AMP includes the following steps:

[0009] (1) A first mixed solution is formed by triazole, triethylamine, 2-chlorophenyl dichlorophosphate, and dichloromethane. After 1 hour, the first mixed solution is reacted with 2-N-(acetyl)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytriphenylmethyl)-β-D-guanosine. The molar ratio of triazole, triethylamine, 2-chlorophenyl dichlorophosphate, 2-N-(acetyl)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytriphenylmethyl)-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 solution obtained 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-(dimethoxytriphenylmethyl)-β-D-guanosine;

[0010] (2) The 2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytriphenylmethyl)-β-D-guanosine, 1-(trimethylmethyl-2-sulfonyl)-3-nitro-1,2,4-triazole and anhydrous pyridine obtained in step (1) are mixed a second time to form a second mixed solution; then 3-hydroxypropionitrile is added. The mixture is reacted with the second mixed solution, wherein the molar ratio of 2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytriphenylmethyl)-β-D-guanosine, 1-(mesotrimyl-2-sulfonyl)-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; the reacted solution is acidified with an appropriate amount of oxalic acid solution, then diluted with dichloromethane, washed, dried, and filtered to obtain the crude product;

[0011] The crude product was reacted with dichloroacetic acid and dichloromethane to remove dimethoxytriphenylmethyl groups. The molar ratio of the crude product to the volume ratio of dichloroacetic acid and dichloromethane was 1.0 mmol : 0.3-1 mL : 10-20 mL. The resulting solution was neutralized with an appropriate amount of saturated sodium bicarbonate solution. After removing the solvent by rotary evaporation, the solution was diluted with dichloromethane, washed, dried, filtered, and separated by column chromatography to obtain 2-N-(acetyl)-2′-O-(2-chlorophenyl-2-acrylonitrile phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine.

[0012] (3) Triazole, triethylamine, 2-chlorophenyl dichlorophosphate, and dichloromethane are mixed to form a third mixed solution. After 1 hour, the third mixed solution is reacted with 2′-O-(tert-butyldimethylsilyl)-5′-O-(dimethoxytriphenylmethyl)-6-N-(benzoyl)-β-D-adenosine. The molar ratio of triazole, triethylamine, 2-chlorophenyl dichlorophosphate, 2′-O-(tert-butyldimethylsilyl)-5′-O-(dimethoxytriphenylmethyl)-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 chromatography was performed to obtain 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-5′-O-(dimethoxytriphenylmethyl)-6-N-(benzoyl)-β-D-adenosine;

[0013] (4) The 2-N-(acetyl)-2′-O-(2-chlorophenyl-2-acrylonitrile 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-(dimethoxytriphenylmethyl)-6-N-(benzoyl)-β-D-adenosine obtained in step (3) are mixed with pyridine IV to form a fourth mixed solution; then the fourth mixed solution is mixed with 1-(trimethylmethyl-2-sulfonyl)-3 The reaction of nitro-1,2,4-triazole, wherein the molar ratio of 2-N-(acetyl)-2′-O-(2-chlorophenyl-2-acrylonitrile phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine, 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-5′-O-(dimethoxytriphenylmethyl)-6-N-(benzoyl)-β-D-adenosine, and 1-(mesotrimyl-2-sulfonyl)-3-nitro-1,2,4-triazole to pyridine is 0.17 mmol : 0.22 mmol : 0.51 mmol : 2 mL, the reaction solution is acidified with an appropriate amount of oxalic acid solution, then diluted with dichloromethane, washed, and dried to obtain the crude product;

[0014] The crude product was reacted with dichloroacetic acid and dichloromethane to remove dimethoxytriphenylmethyl groups. The molar ratio of the crude product to the volume ratio of dichloroacetic acid and dichloromethane was 3.0 mmol : 0.3 mL : 10 mL. The resulting solution was neutralized with an appropriate amount of saturated sodium bicarbonate solution. After removing the solvent by rotary evaporation, the solution was 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-acrylonitrile phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine dinucleotide.

[0015] (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-cyanoethyl 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. The 2-cyanoethyl group is removed by the reaction. After the reaction is completed, the solvent is evaporated. Then, it is mixed with 1-(trimethylmethyl-2-sulfonyl)-3-nitro The reaction involves the cyclization of 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-6-N-(benzoyl)-β-D-adenosine-2-N-(acetyl)-2′-O-(2-chlorophenyl-2-acrylonitrile phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine dinucleotide and 1-(trimethylmethyl-2-sulfonyl)-3-nitro-1,2,4-triazole in a volume ratio of 0.2 mmol : 1.1 mmol : 2 mL : 6 mL : 20 mL. A small amount of water is added to quench the reaction after completion. The solvent was removed by vortexing, and the mixture was acidified with an appropriate amount of oxalic acid solution. After dilution with dichloromethane, the mixture was 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.

[0016] (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 with tetramethylguanidine, pyridine-2-carboxaldehyde oxime, 1,4-dioxane and water to form the sixth mixture. In the 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-carboxaldehyde 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. After the reaction removes 2-chlorophenyl, part of the solvent is removed by vortexing. Then, the sixth mixed solution is reacted with a methylamine-ethanol solution to remove acetyl and benzoyl groups.

[0017] After the reaction was complete, part of the solution was evaporated, and a mixed solution of triethylamine-triethylamine hydrofluoric acid-pyridine was added back to remove the tert-butyldimethylsilyl group. After the reaction was complete, part of the solvent was removed by rotary evaporation. Acetone was added with stirring, and the precipitate was collected by centrifugation. The washed precipitate was dissolved in water, filtered through a liquid chromatography membrane, and purified by liquid chromatography to obtain cyclic dinucleotide 2′,3′-cG. 4′-Me AMP.

[0018] Preferably, in step (1), the reaction conditions include: argon protection, a temperature of -1 to 1°C, a time of 1 to 3 hours, a stirring speed of 250 to 500 rpm, and quenching with 2 mL of 1 M TEAB buffer solution after the reaction is completed. More preferably, the temperature is 0°C and the time is 2 hours.

[0019] Preferably, in step (2), the reaction conditions include: argon protection, temperature of 24-26℃, time of 1-3 hours, more preferably 2 hours, stirring speed of 250-500 rpm, and after the reaction, adding an appropriate amount of 5% oxalic acid solution to adjust the pH of the system to 3-4; the conditions for removing dimethoxytriphenylmethyl include: temperature of -1-1℃, time of 8-20 minutes, more preferably 0℃, time of 10 minutes, stirring speed of 250-500 rpm, and after the reaction, adding saturated sodium bicarbonate solution to neutralize.

[0020] Preferably, in step (3), the reaction conditions include: argon protection, temperature of -1 to 1℃, time of 1 to 3 hours, more preferably 0℃, time of 2 hours, stirring speed of 250 to 500 rpm, and quenching with 2 mL of 1M TEAB buffer solution after the reaction is completed.

[0021] Preferably, in step (4), the reaction conditions include: argon protection, temperature of 24-26℃, time of 1-3 hours, more preferably 2 hours, stirring speed of 250-500 rpm, and after the reaction, adding an appropriate amount of 5% oxalic acid solution to adjust the pH of the system to 3-4; the conditions for removing dimethoxytriphenylmethyl include: temperature of -1-1℃, time of 8-15 minutes, more preferably 0℃, time of 10 minutes, stirring speed of 250-500 rpm, and after the reaction, adding saturated sodium bicarbonate solution to neutralize. Preferably, in step (5), the conditions for the removal of 2-cyanoethyl reaction include: argon protection, temperature of 24-26℃, time of 15-30 minutes, more preferably 20 minutes, stirring speed of 250-500 rpm, and after the reaction, the mixture is co-rotated three times with anhydrous acetonitrile and thoroughly dried; the conditions for the cyclization reaction include: argon protection, temperature of 24-26℃, time of 5-8 hours, more preferably 6 hours, stirring speed of 250-500 rpm, and after the reaction, an appropriate amount of 5% oxalic acid solution is added to adjust the pH of the system to 3-4.

[0022] Preferably, in step (6), the conditions for the removal of 2-chlorophenyl reaction include: argon protection, temperature of 24-26°C, time of 16-19 hours, more preferably 17 hours, and stirring speed of 250-500 rpm;

[0023] The conditions for the deacetyl and benzoyl group removal reaction include: argon protection, temperature of 24-26℃, time of 2-4 hours, more preferably 3 hours, and stirring speed of 250-500 rpm;

[0024] The conditions for the removal of tert-butyldimethylsilane include: argon protection, temperature of 40-60℃, time of 4-6 hours (more preferably 5 hours), and stirring speed of 250-500 rpm. After the reaction, some solvent is removed by rotary evaporation, and 30 mL of pre-cooled acetone is added while the mixture is still hot and stirring, resulting in the precipitation of a large amount of solid. Stirring is continued at 24-26℃ for 20 min, and the precipitate is collected by centrifugation.

[0025] A third aspect of the present invention provides the cyclic dinucleotide 2′,3′-cG 4′-Me Application of AMP in the preparation of innate immune activators.

[0026] The fourth aspect of the present invention provides the cyclic dinucleotide 2′,3′-cG4′-Me Application of AMP in the preparation of drugs for treating diseases related to STING dysfunction.

[0027] The advantages and beneficial effects of this invention are:

[0028] This invention obtains cyclic dinucleotide 2′,3′-cGAMP by introducing a methyl group at the C4ʹ position of guanine G in the natural 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 dysfunction. Attached Figure Description

[0029] Figure 1 It is a natural cyclic dinucleotide 2′,3′-cGAMP and a cyclic dinucleotide 2′,3′-cG 4′-Me Graph showing the stability test of AMP in fetal bovine serum;

[0030] Figure 2 It is a natural cyclic dinucleotide 2′,3′-cGAMP and a cyclic dinucleotide 2′,3′-cG 4′-Me A comparative diagram showing the induction of interferon expression by AMP in HEK293T cells through STING activation, where A) represents 2′,3′-cGAMP and 2′,3′-cGAMP. 4 ′-Me AMP activates wild-type hSTING (B) for 2′,3′-cGAMP and 2′,3′-cG 4′-Me AMP activates the hSTING-R232H mutant. Detailed Implementation

[0031] The specific embodiments of the present invention will be described in detail below. The specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0032] Dichloromethane (purchased from Tianjin Binhai New Area Guangshunda Chemical Reagent Co., Ltd., item number A1040); Sodium bicarbonate (purchased from Tianjin Binhai New Area Guangshunda Chemical Reagent Co., Ltd., item number 2049); Dissolved acetonitrile (≤10 ppm) (purchased from Hebei Dinaxingke Biotechnology Co., Ltd., item number R1012-4); Anhydrous magnesium sulfate (purchased from Tianjin Binhai New Area Guangshunda Chemical Reagent Co., Ltd., item number S2509); Sodium chloride (purchased from Tianjin Chemical Reagent Supply and Marketing Co., Ltd., item number 017); Methanol (purchased from Beijing Bailingwei Technology Co., Ltd., item number 980290-500ML); Triethylamine (purchased from Beijing Inokai Co., Ltd., item number T9710); 1,2,4-Triazole (purchased from Beijing Inokai Co., Ltd., item number 45140A); 2-Chlorophenyl dichlorophosphate (purchased from Shanghai Haohong Biomedical Technology Co., Ltd., item number 1275469-5g); 1-(trimethylmethyl-2-sulfonyl)-3-nitro-1,2,4-triazole (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number M109334-5g); Pyridine (purchased from Tianjin Binhai New Area Guangshunda Chemical Reagent Co., Ltd., item number S2442); Dichloroacetic acid (purchased from Beijing Bailingwei Technology Co., Ltd., item number 140166); Oxalic acid (purchased from...) The following products were purchased from Beijing Kaiguo Technology Co., Ltd. (item number AP009571): 3-Hydroxypropionitrile (item number B040147) from Anhui Zesheng Technology Co., Ltd.; tert-butylamine (item number 237824) from Beijing Bailingwei Technology Co., Ltd.; pyridine-2-carboxaldehyde oxime (item number 529268) from Beijing Bailingwei Technology Co., Ltd.; tetramethylguanidine (item number 940257) from Beijing Bailingwei Technology Co., Ltd.; 1,4-dioxane (item number S2438) from Tianjin Binhai New Area Guangshunda Chemical Reagent Co., Ltd.; methylamine-ethanol solution (33% wt) (item number M433515-100ml) from Beijing Inokai Co., Ltd.; triethylamine trihydrofluoride (item number 433417) from Beijing Bailingwei Technology Co., Ltd.

[0033] Example 1

[0034] A cyclic dinucleotide 2′,3′-cG 4′-Me The preparation method and synthetic route of AMP are as follows:

[0035]

[0036] Compound 1 is 2-N-(acetyl)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytriphenylmethyl)-β-D-guanosine; Compound 1 was synthesized from D-ribose and guanine as starting materials according to the literature (Synthesis of 4′-C-Methylnucleosides, Bioscience, Biotechnology and Biochemistry, 1993, 57, 1433).

[0037] Compound 2 is 2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytriphenylmethyl)-β-D-guanosine;

[0038] Compound 3 is 2-N-(acetyl)-2′-O-(2-chlorophenyl-2-acrylonitrile phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine;

[0039] Compound 4 is 2′-O-(tert-butyldimethylsilyl)-5′-O-(dimethoxytriphenylmethyl)-6-N-(benzoyl)-β-D-adenosine; purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., catalog number BD215385.

[0040] Compound 5 is 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-5′-O-(dimethoxytriphenylmethyl)-6-N-(benzoyl)-β-D-adenosine;

[0041] 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-cyanoethyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine dinucleotide;

[0042] 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;

[0043] Compound 8 is a cyclic dinucleotide 2′,3′-cG 4′-Me AMP.

[0044] The specific steps are as follows:

[0045] (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, and dried triethylamine (0.25 mL, 1.60 mmol, 4.0 eq) and 1 mL dried DCM (dichloromethane) were added to dissolve it. The mixture was stirred at 0 °C at a stirring speed of 300 rpm. A dichloromethane solution (1 mL) of 2-chlorophenyl dichlorophosphate (157 mg, 0.64 mmol, 1.6 eq) was added dropwise to the reaction system at a rate of one drop per second to form the first mixed solution. After reacting at 0 °C for 1 h, compound 1 (300 mg, 0.4 mmol, 1.0 eq) was dissolved in 1 mL dried DCM (dichloromethane) and added dropwise to the first mixed solution at a rate of one drop per second. The mixture was reacted at 0 °C for 2 h. After the reaction was monitored by TLC, the temperature was lowered 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 separated, washed twice each with 1 M TEAB buffer solution and distilled water, collected, dried over anhydrous MgSO4, and purified by column chromatography after the solvent was evaporated to obtain a white solid product (compound 2).

[0046] (2) Preparation of compound 3: Under argon protection, compound 2 (328 mg, 0.36 mmol, 1.0 eq) and 1-(trimethylmethyl-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, and 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. After the solution was evaporated under reduced pressure, it was redissolved in 5 mL of DCM. An appropriate amount of 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 three times with DCM. The organic phases were combined, washed once with saturated NaCl solution, and the organic phase was collected and dried with anhydrous MgSO4. The solvent was evaporated to obtain the crude product.

[0047] Dissolve the crude product in 10 mL of DCM. oThe mixture was stirred at 300 rpm under temperature C. 0.3 mL of dichloroacetic acid was added, and the reaction was maintained in an ice bath for 10 min to remove dimethoxytriphenylmethyl groups. After the reaction was complete as monitored by TLC, a few drops of methanol were added to quench the reaction, and stirring was continued for another 10 min. Saturated NaHCO3 solution was added to neutralize the mixture until no more bubbles emerged. The organic phase was extracted and separated, and the aqueous phase was extracted multiple times with DCM. The organic phases were combined, washed twice with saturated NaCl solution, and collected 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). 3143 mg of the target compound was obtained, with a three-step yield of 76%.

[0048] The nuclear magnetic resonance (NMR) analysis results of compound 3 are as follows:

[0049] 31 P NMR (162 MHz, CDCl3) δ (ppm): -7.87, -9.27;

[0050] 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);

[0051] 13 C 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.

[0052] The NMR results confirm that compound 3 has the correct structure.

[0053] (3) Preparation of compound 5: Under argon protection, triazole (174 mg, 2.52 mmol, 4.0 eq) and dried triethylamine (0.35 mL, 2.52 mmol, 4.0 eq) were weighed into a 25 mL round-bottom flask, and 1 mL of dried DCM was added to dissolve them. 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 dried 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 by TLC monitoring, 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.

[0054] The reaction system was washed twice each with 1 M TEAB buffer solution and distilled water. The organic phase was collected, dried over anhydrous MgSO4, and the solvent was removed by vortexing to obtain a white solid product, which is compound 5. After drying, it was used directly in the next step.

[0055] (4) Preparation of Compound 6: Under argon protection, Compound 5 (210 mg, 0.22 mmol, 1.3 eq), Compound 3 (110 mg, 0.17 mmol, 1.0 eq), and 2 mL of pyridine were mixed to form a fourth mixed solution. The fourth mixed solution was then reacted with 1-(trimethylmethyl-2-sulfonyl)-3-nitro-1,2,4-triazole (150 mg, 0.51 mmol, 3.0 eq) at room temperature for 2 h with a stirring speed of 300 rpm. After the reaction was completed, 1 mL of water was added to quench the reaction. The pyridine was evaporated, and the residue was dissolved in 10 mL of DCM. The pH of the system was adjusted to 3-4 using 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 evaporated to obtain the crude product, which was directly used in the next step.

[0056] 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 and the reaction proceeded for 10 min to remove dimethoxytriphenylmethyl groups. After the reaction was complete as monitored by TLC, 2 mL of methanol was added to quench the reaction, and stirring was continued at low temperature for another 10 min. Then, saturated NaHCO3 solution was added to neutralize until no more bubbles emerged. The organic phase was washed twice with saturated NaCl solution, and the aqueous phase was extracted twice with DCM and discarded. The organic phases were combined, evaporated to dryness under reduced pressure, and separated by column chromatography (methanol / dichloromethane, v / v, 1 / 50–1 / 30) to give 6250 mg of the compound, in 70% yield.

[0057] The nuclear magnetic resonance (NMR) analysis results of compound 6 are as follows:

[0058] 31 P NMR (162 MHz, CDCl3) δ (ppm): -7.39, -8.29, -8.35, -9.75;

[0059] 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);

[0060] 13C 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

[0061] HRMS (ESI): C 57 H 72 Cl2N 11 O 16 P2Si2[M+H] + , calc.1354.3549, found 1354.3563

[0062] The results of NMR and mass spectrometry confirm that compound 6 has the correct structure and molecular weight.

[0063] (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). The mixture was stirred at room temperature for 20 min to form the fifth mixed solution, and the 2-acrylonitrile ethyl group was removed by the reaction. After the reaction was completed by TLC monitoring, the mixture was concentrated under reduced pressure, acetonitrile was added and vortexed three times, and the mixture was dried thoroughly. Under argon protection, 1-(trimethylmethyl-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 at 26 °C for 6 h with a stirring speed of 300 rpm. After the reaction was completed by TLC monitoring, a small amount of water was added to quench the reaction. The solvent was evaporated, and after dissolving in an appropriate amount of DCM, the pH of the system was adjusted to 3-4 by adding 5% oxalic acid solution. The organic phase was separated, and the aqueous phase was extracted twice with DCM. The organic phases were combined and washed twice with saturated NaCl solution. The organic phase was dried with anhydrous MgSO4 and purified by column chromatography (methanol / dichloromethane, v / v, 1 / 30) to give 7-8 mg of the compound, with a two-step yield of 65%.

[0064] (6) Preparation of compound 8:

[0065] 1) Compound 7 (300 mg, 0.26 mmol, 1.0 eq) was dissolved in a mixed solvent of 2 mL 1,4-dioxane and 2 mL water. Tetramethylguanidine (133 mg, 1.16 mmol, 4.5 eq) and pyridine-2-carboxaldehyde oxime (141 mg, 1.16 mmol, 4.5 eq) were added. The mixture was stirred at 26 °C for 17 h at a stirring speed of 300 rpm to form the sixth mixed solution.

[0066] 2) Dry the solvent by rotary evaporation, add 10 mL of methylamine-ethanol solution (33% wt), stir at 26℃ for 3 h at a stirring speed of 300 rpm, and the reaction removes acetyl and benzoyl groups.

[0067] 3) The solvent was evaporated, and the residue was co-evolved three times with a mixed solvent of dry pyridine (1.2 mL) and triethylamine (0.6 mL). The residue was then dissolved in a mixed solution of 1 mL anhydrous pyridine, 5 mL triethylamine, and 3 mL triethylamine-trihydrofluoride. The reaction was carried out at 50 °C with stirring at 300 rpm for 5 h to remove the tert-butyldimethylsilyl group. After the reaction was complete, some solvent was removed by rotary evaporation. While still hot and stirring, 30 mL of pre-cooled acetone was added, precipitating a large amount of solid. Stirring was continued at 26 °C for 20 min, and the precipitate was collected by centrifugation. The precipitate was washed once with acetone, dissolved in an appropriate amount of water, filtered through a liquid chromatography membrane, and purified by liquid chromatography to obtain compound 8, namely the cyclic dinucleotide 2′,3′-cG.4′-Me AMP.

[0068] Liquid chromatography purification conditions: Innoval ODS-2 C18 column (21.2 × 250 mm, 5 μm), UV 254 nm monitoring. Mobile phase gradient: Phase A was 50 mM TEAA aqueous solution, and Phase B was acetonitrile. 0-2 min: maintain 98% A - 2% B; 2-32 min: increase from 98% A - 2% B to 70% A - 30% B; 32-37 min: increase from 70% A - 30% B to 100% B; 37-43 min: maintain 100% B; flow rate: 10 mL / min.

[0069] The nuclear magnetic resonance (NMR) analysis results of compound 8 are as follows:

[0070] 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);

[0071] 31 P-decoupling 1H 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);

[0072] 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

[0073] 31 P NMR (162 MHz, D2O) δ (ppm): -1.23, -2.27

[0074] HRMS (ESI): C 21 H 25 N 10 O 13 P2[M-H] - , calc.687.1078, found 687.1034

[0075] The results of NMR and mass spectrometry confirm that compound 8 has the correct structure and molecular weight.

[0076] Example 2

[0077] cyclic dinucleotide 2ʹ,3ʹ-cG 4ʹ-Me AMP stability verification in serum

[0078] The experimental steps are as follows:

[0079] 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 dinucleotide were added to a total volume of 30 μL. The mixture was incubated at 37 °C, 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 the 30 μL sample system. After dilution with 50 μL of water, the sample was analyzed by HPLC in an injection volume of 30 μL.

[0080] Purification conditions: The instrument was a FLEXA Purification System HP-Q-100, the column was an InnovalODS-2 C18 column (21.2 × 250 mm, 5 μm), the column temperature was 25 ℃, and the detection wavelength was 254 nm. Mobile phase gradient: Phase A was 10 mM TEAA aqueous solution, and Phase B was acetonitrile. The mobile phase gradient was as follows: 0–2 min, 100% A; 2–12 min, from 100% A to 78% A / 22% B; 12–13.5 min, from 78% A / 22% B to 100% B; 13.5–15 min, 100% B; 15–17 min, from 100% B to 100% A; 17–19 min, 100% A. The flow rate was 1 mL / min. The remaining intact cyclic dinucleotide content was calculated based on peak area and analyzed graphically, as shown below. Figure 1 As shown.

[0081] from Figure 1 As can be seen from this, compared to the modified 2′,3′-cG 4′-Me For AMP, natural 2′,3′-cGAMP degrades faster, with a half-life of approximately 33 hours. 4′-Me AMP degradation is relatively slow, with half-lives greater than 90 hours. This corresponds to serum stability: 2′,3′-cG 4′-MeThe AMP level is much higher than that of 2′,3′-cGAMP. This is because the presence of the guanosine C4ʹ-methyl group creates steric hindrance near the adjacent phosphodiester bond, hindering the binding of the substrate to the exonuclease in serum, thus preventing the 2′,3′-cGAMP from being absorbed. 4′-Me AMP is more difficult for enzymes to cleave. Therefore, the introduction of a C4′-methyl group into guanosine significantly improves the serum stability of cyclic dinucleotides, which is expected to improve the pharmacokinetic properties of cyclic dinucleotide drugs and thus enhance their therapeutic potential.

[0082] Example 3

[0083] 2′,3′-cG of cyclic dinucleotides 4′-Me Assay for AMP-activated interferon expression in HEK293T cells:

[0084] Since HEK293T cells lack endogenous STING expression, they were transfected with hSTING-wild-type or hSTING-R232H expression vectors and two dual-luciferase reporter vectors. One vector encodes the IFN-β promoter controlling firefly luciferase, and the other encodes Renida luciferase. After 24 hours of culture, IFN-β promoter activity was quantified based on the ratio of firefly luciferase to Renida luciferase activity.

[0085] The experimental steps are as follows:

[0086] (1) HEK293T cells were cultured at a concentration of 1.5 × 10⁻⁶. 5 The cells were evenly distributed in 24-well cell culture plates and incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0087] (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).

[0088] (3) Cyclic dinucleotide transfection: 24 hours after plasmid transfection, small molecules are transfected. Two transfection methods are used for small molecules:

[0089] 1. Direct transfection: Aspirate the DMEM culture medium from the culture plate, dissolve the cyclic dinucleotide directly in preheated 37 ℃ opti-DMEM, add 500 μL to each well, for a final concentration of 1 μM per well. After culturing for 4 hours, add 1 mL of normal DMEM culture medium containing serum and continue culturing for 24 hours.

[0090] 2. Permeabilization solution treatment: Aspirate the DMEM culture medium from the culture plate and dissolve the cyclic dinucleotide directly in the preheated permeabilization solution at 37 ℃. Add 500 μL to each well, resulting in a final concentration of 1 μM per well. After 30 minutes, aspirate the permeabilization solution and replace it with 1 mL of normal DMEM culture medium containing serum, and continue culturing for 24 hours. The permeabilization solution components are: 50 mM 4-hydroxyethylpiperazine ethanesulfonic 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 digitalis saponins.

[0091] (4) Dual fluorescence reporter assay: 24 hours after transfection with cyclic dinucleotide, the culture medium was aspirated. Each well was washed with 1 mL of pre-cooled PBS buffer, and 100 μL of Passive Lysis lysis buffer was added. The mixture was incubated at 26°C on a horizontal shaker for 20 minutes. The mixture was then transferred to a prepared 1.5 mL EP tube, centrifuged at 13,000 rpm for 10 minutes, and the supernatant was aspirated into a black 96-well plate, 20 μL per well.

[0092] Add 20 μL of Luciferase Assay buffer to each well and read the first fluorescence data (Luc value) using a microplate reader. Then add 20 μL of Stop & Glo buffer and read the second fluorescence data (Ren value) using a microplate reader. Calculate the ratio of the two fluorescence intensities (Luc value / Ren value), and normalize it using the fluorescence intensity ratio of the blank group (normally transfected with three plasmids, but not transfected with cyclic dinucleotides) to obtain the final activity data. Each data point is repeated three times, and error analysis is performed. Figure 2 As shown.

[0093] In HEK293T (HEK293T / hSTING-WT) cells transfected with hSTING-wild-type, direct treatment with 2′,3′-cGAMP increased IFN-β expression activity by only 1.6-fold. However, treatment with permeabilization buffer increased it by 4-fold. Figure 2 (A). Conversely, using 2′,3′-cG 4′-Me Following AMP treatment, regardless of whether permeabilization buffer was used, IFN-β expression activity increased only slightly by 1.4–1.8-fold. These results further confirm that 2′,3′-cGAMP is a potent agonist of hSTING-wild-type cells, but exhibits poor membrane permeability in HEK293T cells; while 2′,3′-cGAMP... 4′-Me AMP has a weaker ability to activate hSTING-wild type.

[0094] In HEK293T (HEK293T / hSTING-R232H) cells transfected with hSTING-R232H, treatment with 2′,3′-cGAMP resulted in only a slight increase in IFN-β expression activity, independent of permeabilization treatment. Figure 2 (B). Conversely, even in the absence of a permeabilizing 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 2′,3′-cGAMP is significantly more potent than native 2′,3′-cGAMP in mammalian cells. 4′-Me AMP can more effectively activate the hSTING-R232H variant, inducing an innate immune response. This is related to 2′,3′-cG 4′-Me AMP is associated with better permeability and superior serum stability.

[0095] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope 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 as described in claim 1 4′-Me The method for preparing AMP is characterized by, Includes the following steps: (1) A first mixed solution was formed by triazole, triethylamine, 2-chlorophenyl dichlorophosphate, and dichloromethane. After 1 hour, the first mixed solution was reacted with 2-N-(acetyl)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytriphenylmethyl)-β-D-guanosine. The molar ratio of triazole, triethylamine, 2-chlorophenyl dichlorophosphate, 2-N-(acetyl)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytriphenylmethyl)-β-D-guanosine to the volume ratio of dichloromethane was 1.6 mmol : 1.6 mmol : 0.6 mmol : 0.4 mmol : 1-3 mL of the solution obtained 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-(dimethoxytriphenylmethyl)-β-D-guanosine; (2) The 2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytriphenylmethyl)-β-D-guanosine, 1-(mesodyl-2-sulfonyl)-3-nitro-1,2,4-triazole, and pyridine obtained in step (1) are mixed a second time to form a second mixed solution; then 3-hydroxypropionitrile is reacted with the second mixed solution, wherein the molar ratio of 2-N-(acetyl)-2′-O-(2-chlorophenyl phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-5′-O-(dimethoxytriphenylmethyl)-β-D-guanosine, 1-(mesodyl-2-sulfonyl)-3-nitro-1,2,4-triazole, and 3-hydroxypropionitrile to the volume ratio of pyridine is 1.

0. mmol : 3.5 mmol : 2.0 mmol : 1-3 mL, the solution after reaction was acidified with an appropriate amount of oxalic acid solution, then diluted with dichloromethane, washed, dried and filtered to obtain crude product; The crude product was reacted with dichloroacetic acid and dichloromethane to remove dimethoxytriphenylmethyl groups. The molar ratio of the crude product to the volume ratio of dichloroacetic acid and dichloromethane was 1.0 mmol : 0.3-1 mL : 10-20 mL. The resulting solution was neutralized with an appropriate amount of saturated sodium bicarbonate solution. After removing the solvent by rotary evaporation, the solution was diluted with dichloromethane, washed, dried, filtered, and separated by column chromatography to obtain 2-N-(acetyl)-2′-O-(2-chlorophenyl-2-acrylonitrile phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine. (3) Triazole, triethylamine, 2-chlorophenyl dichlorophosphate, and dichloromethane are mixed to form a third mixed solution. After 1 hour, the third mixed solution is reacted with 2′-O-(tert-butyldimethylsilyl)-5′-O-(dimethoxytriphenylmethyl)-6-N-(benzoyl)-β-D-adenosine. The molar ratio of triazole, triethylamine, 2-chlorophenyl dichlorophosphate, 2′-O-(tert-butyldimethylsilyl)-5′-O-(dimethoxytriphenylmethyl)-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-(dimethoxytriphenylmethyl)-6-N-(benzoyl)-β-D-adenosine; (4) The 2-N-(acetyl)-2′-O-(2-chlorophenyl-2-acrylonitrile 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-(dimethoxytriphenylmethyl)-6-N-(benzoyl)-β-D-adenosine obtained in step (3) are mixed with pyridine IV to form a fourth mixed solution; then the fourth mixed solution is mixed with 1-(trimethylmethyl-2-sulfonyl)-3 The reaction of nitro-1,2,4-triazole, wherein the molar ratio of 2-N-(acetyl)-2′-O-(2-chlorophenyl-2-acrylonitrile phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine, 2′-O-(tert-butyldimethylsilyl)-3′-O-(2-chlorophenyl phosphate)-5′-O-(dimethoxytriphenylmethyl)-6-N-(benzoyl)-β-D-adenosine, and 1-(mesotrimyl-2-sulfonyl)-3-nitro-1,2,4-triazole to the volume ratio of pyridine is 0.17 mmol : 0.22 mmol : 0.51 mmol : 2 mL, the reaction solution is acidified with an appropriate amount of oxalic acid solution, then diluted with dichloromethane, washed, dried, and filtered to obtain the crude product; The crude product was reacted with dichloroacetic acid and dichloromethane to remove dimethoxytriphenylmethyl groups. The molar ratio of the crude product to the volume ratio of dichloroacetic acid and dichloromethane was 3.0 mmol : 0.3 mL : 10 mL. The resulting solution was neutralized with an appropriate amount of saturated sodium bicarbonate solution. After rotary evaporation to remove the solvent, the solution was 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-acrylonitrile 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-cyanoethyl 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. The 2-cyanoethyl is removed by the reaction. After the reaction is completed, the solvent is evaporated. Then, it is mixed with 1-(trimethylmethyl-2-sulfonyl)-3-nitro 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-acrylonitrile phosphate)-3′-O-(tert-butyldimethylsilyl)-4′-C-(methyl)-β-D-guanosine dinucleotide, 1-(mesmetryl-2-sulfonyl)-3-nitro-1,2,4-triazole, and the volume ratio of tert-butylamine, acetonitrile, and pyridine is 0.

2. mmol : 1.1 mmol : 2 mL : 6 mL : 20 mL. After the reaction was completed, a small amount of water was added to quench the reaction, the solvent was removed by vortexing, and the mixture was acidified with an appropriate amount of oxalic acid solution. Then, it was 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 with tetramethylguanidine, pyridine-2-carboxaldehyde oxime, 1,4-dioxane and water to form the sixth mixture. The solution contains 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-carboxaldehyde oxime, and the volume ratio of 1,4-dioxane to water in a ratio of 0.

26. The reaction was carried out with mmol : 1.16 mmol : 1.16 mmol : 2 mL : 2 mL to remove 2-chlorophenyl groups. After the reaction, some of the solvent was removed by vortexing. The sixth mixed solution was then reacted with methylamine-ethanol solution to remove acetyl and benzoyl groups. After the reaction, some of the solution was removed by vortexing. A mixed solution of triethylamine-triethylamine hydrofluoric acid-pyridine was added again to remove tert-butyldimethylsilyl groups. After the reaction, some of the solvent was removed by rotary evaporation. Acetone was added with stirring, and the precipitate was collected by centrifugation. The precipitate was dissolved in water, filtered through a liquid chromatography membrane, and purified by liquid chromatography 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 to 1℃, time of 1 to 3 hours, and quenching with 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℃, time of 1-3 hours, and after the reaction, 5% oxalic acid solution is added to adjust the pH of the system to 3-4; the conditions for removing dimethoxytriphenylmethyl include: temperature of -1-1℃, 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 to 1℃, time of 1 to 3 hours, and quenching with 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℃, time of 1-3 hours, and after the reaction, an appropriate amount of 5% oxalic acid solution is added to adjust the pH of the system to 3-4; the conditions for removing dimethoxytriphenylmethyl include: temperature of -1-1℃, 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 2-cyanoethyl reaction include: argon protection, temperature of 24-26℃, time of 15-30 minutes, and after the reaction, anhydrous acetonitrile is used for co-rotation and thorough drying; the conditions for the cyclization reaction include: argon protection, temperature of 24-26℃, time of 5-8 hours, and after the reaction, an appropriate amount of 5% oxalic acid solution is added to adjust the pH of the system to 3-4.

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

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

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

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