Synthesis method of guanosine gamma-thiophosphoric acid and derivatives thereof

Abstract

The invention relates to a synthesis method of guanosine gamma-thiophosphoric acid and derivatives thereof. The invention relates to a synthesis method of GTPYS (guanosine gamma-thiophosphoric acid) and a derivative MANT-GTP-gamma-S. The synthesis method comprises the following steps: preparing GTPYS; specifically, the method takes guanosine as an initial raw material and obtains MANT-GTP-gamma-S through three-step reaction: step 1, guanosine reacts with phosphorus oxychloride and tributylamine phosphate to generate guanosine diphosphate; 2, the guanosine diphosphate reacts with triethylamine, imidazole, triphenylphosphine and dithiopyridine for activation, guanosine diphosphate imidazole is generated, and guanosine gamma-thiophosphoric acid (GTPYS) is generated through a reaction between the guanosine diphosphate imidazole and tributylamine thiophosphate; and step 3, GTPYS is taken as a raw material and reacts with 2-(methylamino) benzoyl chloride under the condition that the pH value is 7-10, and MANT-GTP-gamma-S is generated. Compared with the prior art, the preparation method disclosed by the invention is simple in steps, beneficial to amplified production and capable of meeting actual production research requirements.

Description

Technical Field

[0001] The present application relates to a method for synthesizing guanosine γ-phosphorothioate and its derivatives, and specifically to a method for synthesizing GTPYS (guanosine γ-phosphorothioate) and its derivative MANT-GTP-γ-S. Background Art

[0002] GTPYS (guanosine gamma-thiophosphate) is a non-hydrolyzable GTP analog with extensive applications in biochemistry and cell biology. As a GTP substitute, it is used to study biochemical processes that require GTP but where its hydrolysis is undesirable. This is particularly true for investigating the intracellular functions and mechanisms of GTP-binding proteins, signal transduction, and cytoskeletal dynamics. Furthermore, GTPYS can be used as a pharmacological probe to investigate the regulatory mechanisms of microtubule-based organelle movement. For example, in certain studies, GTPYS has been used to investigate its effects on the transport velocity of organelles during rapid axonal transport. As an important biochemical reagent, GTPYS has broad applications in scientific research.

[0003] MANT-GTP-γ-S is a derivative of GTPYS (guanosine γ-thiophosphate) with diverse biological activities and applications. As a potent competitive adenylate cyclase (AC) inhibitor, it inhibits adenylate cyclase activity, thereby regulating intracellular cAMP levels. MANT-GTP-γ-S is also a potent YdeH inhibitor, binding to the active site of YdeH and thereby preventing it from performing its normal biological functions. It has important applications in biochemistry and molecular biology research.

[0004] MANT-GTP-γ-S also exhibits fluorescent properties, making it a fluorescent GTP derivative with unique application value in biochemical research. It can be used as an eIF5B substrate to track and detect biochemical processes such as protein activation and signal transduction that are bound to or regulated by GTP. It can stimulate insulin release from pancreatic β cells, inhibit organelle transport along axonal microtubules, and induce exocytosis in vivo. It plays an important role in studying G protein-coupled receptor (GPCR) and G protein signaling mechanisms. MANT-GTP-γ-S has broad application prospects in biochemistry and molecular biology for drug approval and scientific research.

[0005] The prior art CN116284189A provides a synthesis method for preparing MANTGTPγ-S through a ten-step reaction. This method uses compound 1 as a raw material, protects its hydroxyl groups and amino groups other than the 2-position, and then uses sodium hydride as a strong base to obtain an intermediate product (compound 4) with a MANT fluorescent group in high yield. On this basis, its phosphate side chain is further synthesized to obtain MANTGTP-γ-S in a relatively high yield.

[0006] However, it is well known to those skilled in the art that the ten-step reaction route is lengthy, not conducive to scale-up production, and far from meeting the needs of actual production research. Summary of the Invention

[0007] Based on the above needs for GTPYS (guanosine γ-thiophosphate) and its derivative MANT-GTP-γ-S, the present application provides a method for synthesizing GTPYS (guanosine γ-thiophosphate) and its derivative MANT-GTP-γ-S. The specific synthesis route scheme uses guanosine as the starting material and obtains MANT-GTP-γ-S through a three-step reaction: Step 1, guanosine reacts with phosphorus oxychloride to generate an intermediate 5'-monophosphate guanosine (intermediate 1A), and intermediate 1A reacts with tributylamine phosphate. , generating guanosine diphosphate (Formula 2, intermediate 2); Step 2, guanosine diphosphate reacts with triethylamine, imidazole, triphenylphosphine, and dithiodipyridine to generate guanosine diphosphate imidazole (Formula 3), and the compound of Formula 3 reacts with tributylamine thiophosphate under Lewis acid catalysis to generate guanosine γ-thiophosphate (Formula 4, GTPYS); Step 3, using GTPYS as a raw material, reacts with 2-(methylamino)benzoyl chloride at pH 7-10 to generate the MANT-GTP-γ-S product. The entire synthetic route is as follows:

[0008] Specifically, step 1, guanosine reacts with phosphorus oxychloride to generate 5'-monophosphate guanosine (intermediate 1A), and intermediate 1A reacts with tributylamine phosphate to generate diphosphate guanosine (Formula 2, intermediate 2). The reaction equation is as follows:

[0009] In one embodiment, in step 1, guanosine is reacted with phosphorus oxychloride at a molar ratio of 1:1.8 to produce guanosine 5'-monophosphate (intermediate 1A), and intermediate 1A is reacted with tributylamine phosphate at a molar ratio of 1:10 to produce a guanosine diphosphate intermediate; In one embodiment, in step 1, guanosine and phosphorus oxychloride are reacted at a molar ratio of 1:2.8 to generate an intermediate 5'-monophosphate guanosine (intermediate 1A), and intermediate 1A is reacted with tributylamine phosphate at a molar ratio of 1:7 to generate a guanosine diphosphate intermediate; In one embodiment, in step 1, guanosine and phosphorus oxychloride are reacted at a molar ratio of 1:2.4 to produce the intermediate 5'-monophosphate guanosine (Intermediate 1A). Intermediate 1A is then reacted with tributylamine phosphate at a molar ratio of 1:8.4 to produce guanosine diphosphate (Formula 2, Intermediate 2). Below this molar ratio, some raw materials remain, while above this molar ratio, there is no optimal change and the results are similar to those at this molar ratio.

[0010] Step 2: Activation of guanosine diphosphate: The activated guanosine diphosphate reacts with tributylamine thiophosphate to generate guanosine γ-thiophosphate (GTPYS, Formula 4); the activation of guanosine diphosphate refers to the reaction of guanosine diphosphate with triethylamine, imidazole, triphenylphosphine, and dithiodipyridine to generate guanosine diphosphate imidazole (Formula 3); the activated guanosine diphosphate is guanosine diphosphate imidazole, and the specific reaction equation is: The reaction solvent can be a polar aprotic solvent such as DMF, DMA, NMP, DMSO, etc. In some embodiments the imidazole is replaced by morpholine; In some embodiments, the triphenylphosphine is replaced by other phosphorous reagents such as tricyclohexylphosphine or tributylphosphine; In some embodiments, dithiodipyridine is replaced by diphenylacetyl disulfide, DDTT; In some embodiments, the molar ratio of guanosine diphosphate to triethylamine, imidazole (or morpholine), triphenylphosphine (or triphenylphosphine is replaced by other phosphorous reagents such as tricyclohexylphosphine or tributylphosphine), dithiodipyridine (or diphenylacetyl disulfide or DDTT) is: 1: (2.1-5.7): (3.0-5.1): (1.8-2.4): (1.2-2.1).

[0011] The molar ratio of the guanosine diphosphate imidazole to tributylamine thiophosphate is 1:1.10 to 1:1.27; If the molar ratio of the raw materials used exceeds the above range, the system will become impure, new impurities will be generated, or raw materials will be wasted; The Lewis acid refers to cadmium chloride, manganese chloride, and zinc chloride, and the molar ratio of the Lewis acid to guanosine diphosphate imidazole is 1.5:1 to 2.9:1.

[0012] Step 3: Using GTPYS as raw material, controlling the system pH to 7-10 and adding 2-(methylamino)benzoyl chloride, after preparative separation and purification, MANT-GTP-γ-S product is obtained. The specific reaction equation is as follows: In step 3, a phosphate buffer is used as a reactive solvent. The phosphate buffer is a solution of phosphoric acid and its salts or alkali metal hydroxides known to those skilled in the art. The pH of the reaction system is in the range of 7 to 10, including endpoint values. The molar ratio of the reaction raw material intermediate 4 to 2-(methylamino)benzoyl chloride is in the range of 1:1.5 to 1:5, which is excellent in terms of system purity and material utilization. When the molar ratio is lower than this, the purity of the system is low. When the molar ratio is higher than this, there is no positive effect on the system, but it will cause waste of raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Attachment Figure 1 LCMS detection spectrum of 5'-guanosine monophosphate; Attachment Figure 2 LCMS detection spectrum of guanosine diphosphate (intermediate 2); Attachment Figure 3 LCMS detection spectrum of guanosine diphosphate imidazole (intermediate 3, GDP-imidazolide); Attachment Figure 4 LCMS detection spectrum of guanosine γ-thiophosphate (GTPYS); Attachment Figure 5 LCMS detection spectrum of MANT-GTP-γ-S; Attachment Figure 6 HNMR detection spectrum of MANT-GTP-γ-S; Attachment Figure 7 HPLC detection spectrum of MANT-GTP-γ-S; DETAILED DESCRIPTION

[0014] The specific embodiments are merely explanations of the present application and are not limitations of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the embodiments as needed, but as long as they are within the scope of the claims of the present application, they are protected by patent law.

[0015] Unless otherwise specified, the raw materials and reagents used in this application are commercially available. The definitions or English abbreviations of each substance correspond to their Chinese names as follows: POCl3: phosphorus oxychloride; TMP: trimethyl phosphate; PPh3: triphenylphosphine; TEA: triethylamine; 1.PDS: 2,2'-dipyridyldithioate; DMF: N,N-dimethylformamide; DMA: dimethylacetamide; NMP: N-methylpyrrolidone; DMSO: dimethyl sulfoxide; Imidazole: imidazole; DDTT: N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formamidine, CAS number 1192027-04-5.

[0016] Tributylamine phosphate: Prepared by yourself. The preparation method is: phosphoric acid reacts with n-butylamine to produce tributylamine phosphate. The reaction equation is: The specific reaction conditions are as follows: under nitrogen, add 500g of water and 100g of phosphoric acid to a three-necked flask, cool to 5-10°C, dropwise add 566g of tri-n-butylamine, and stir for 1 hour. The reaction solution is directly freeze-dried to obtain tributylamine phosphate with a yield of 100%.

[0017] Tributylamine thiophosphate: self-prepared. The preparation method is: phosphoric acid thiophosphate reacts with n-butylamine to generate tributylamine thiophosphate. The reaction equation is: The specific reaction conditions are as follows: 50g of sodium thiophosphate is dissolved in 100g of water, loaded onto a pretreated cation exchange resin (the cation exchange resin is loaded into a glass silica gel column and activated with 2N aqueous hydrochloric acid. Activation is complete when the pH of the effluent fraction is less than 2, and then rinsed with pure water until neutral). Ion exchange is performed, followed by continuous rinsing with pure water, and the fraction with the lowest pH and highest conductivity is collected. 243g of tri-n-butylamine is added to this fraction, and the mixture is stirred for 1 hour. The fraction is then directly lyophilized to obtain tributylamine thiophosphate in a 100% yield.

[0018] LCMC detection method for 5'-monophosphate guanosine (intermediate 1A) and diphosphate guanosine (intermediate 2): Column: Waters Prep Nova-Pak HRC18 column, 60, 19X300 mm preparative column, eluent A = 25 mM TEAB (pH 7), eluent B = acetonitrile, eluent gradient is to add eluent B to eluent A from 0% to 50% over 10 minutes, and then eluent B is increased to 85% in the next 8 minutes, eluent flow rate = 5 ml / min, under such conditions, retention time (RT) = 17 minutes.

[0019] LCMS detection method for guanosine imidazole diphosphate (Formula 3) (GDP-imidazolide): chromatographic column: Waters Prep Nova-Pak HRC18 column, 60, 19×300 mm preparative column, eluent A = 25 mM TEAB (pH 7), eluent B = acetonitrile, eluent gradient: eluent B added to eluent A from 0% to 50% over 10 minutes, eluent B increased to 85% over the next 8 minutes, eluent flow rate = 5 ml / min. Under these conditions, retention time (RT) = 16 minutes.

[0020] LCMS detection method for guanosine γ-phosphorothioate (intermediate 4): Waters Prep Nova-Pak HRC18 column, 60, 19×300 mm preparative column, eluent A = 25 mM TEAB (pH 7), eluent B = acetonitrile, eluent gradient: eluent B increased from 0% to 50% in eluent A over 10 minutes, eluent B increased to 85% in the next 8 minutes, eluent flow rate = 5 ml / min, under these conditions, retention time (RT) = 15 minutes, and the product was obtained after freeze-drying.

[0021] In order to clearly and briefly illustrate the embodiments of the present application, the present application uses one or two examples of a certain type having similar results as representatives, for example: In step 1, trimethyl phosphate and triethyl phosphate can replace each other, and this application takes trimethyl phosphate as an example for explanation; in step 2, the solvent used can be a polar aprotic solvent such as DMF, DMA, NMP, DMSO, etc., with DMF being taken as an example; imidazole can be replaced by morpholine, with imidazole being taken as an example; the inventors of the present invention have tested triphenylphosphine, tricyclohexylphosphine or tributylphosphine, etc. as the phosphorous reagent, and only triphenylphosphine is taken as an example in the embodiment; the inventors of the present invention have used a variety of Lewis acid catalysts, such as cadmium chloride, manganese chloride, zinc chloride, etc. The usage ratio of guanosine imidazole diphosphate and manganese chloride is their molar ratio, and the molar ratio of guanosine imidazole diphosphate and manganese chloride is significantly better in the range of 1:1.5 to 1:2.9. Specifically, in some embodiments, the molar ratio of guanosine imidazole diphosphate and manganese chloride is 1:1.5, 1:2.0, 1:2.5, 1:2.9. During the experiment, the inventors also tried other ratios within the above range. The study found that when the dosage ratio of guanosine imidazole diphosphate and manganese chloride was lower than the molar ratio, the purity of the system was low, and the dosage ratio of guanosine imidazole diphosphate and manganese chloride was higher than the molar ratio. There was no positive effect on the reaction. The no positive effect means that as their dosage ratio increases beyond this range, the purity of the system will not further increase or decrease.

[0022] In step 3, the molar ratio of guanosine γ-phosphorothioate to 2-(methylamino)benzoyl chloride is 1:1.5 to 1:5. In some embodiments, the molar ratio of guanosine γ-phosphorothioate to 2-(methylamino)benzoyl chloride is 1:1, 1:1.5, 1:3, or 1:5. During the experiment, the inventors also tried other ratios within the above range and found that when the molar ratio of guanosine γ-phosphorothioate to 2-(methylamino)benzoyl chloride is lower than the range of 1:1.5 to 1:5, the purity of the system is low, and when it is higher than this range, there is no positive effect on the system.

[0023] Each example or comparative example in the present application has at least 3 parallel samples, and the average value is taken, that is, each data has statistical significance.

[0024] Example 1 Step 1 Example 1.1 Under nitrogen protection, add 200g of trimethyl phosphate and 10g of guanosine to a three-necked flask, cool to -5~0℃, and control the temperature at -5~0℃. Add 10g of phosphorus oxychloride. Stir for 10min after the addition is complete, and control the temperature at -5~0℃. React for 2h to generate 5'-monophosphate guanosine. LCMS detection shows that the raw material is completely converted. The LCMS detection spectrum of 5'-monophosphate guanosine is attached. Figure 1 50 g of tributylamine phosphate solution (50% content) was added dropwise to the system. After the addition was completed, the mixture was reacted at 0°C for 1 hour to obtain guanosine diphosphate. The reaction was confirmed to be complete by LCMS detection. 200 g of water was added dropwise to the system at a temperature of 0-5°C and stirred to quench the reaction. The intermediate 2 was obtained by preparative liquid phase purification. The LCMS detection spectrum of guanosine diphosphate is shown in the attached figure. Figure 2 The purity of guanosine diphosphate was 98.1% and the yield was 62%.

[0025] Example 1.2 Under nitrogen protection, 100g of trimethyl phosphate and 5g of guanosine were added to a three-necked flask, the temperature was cooled to -5~0°C, and the temperature was controlled at -5~0°C. 2.7g of phosphorus oxychloride was added, and the addition was completed. Stir for 10min and the temperature was controlled at -5~0°C. The reaction was carried out for 2h. LCMS detected that the raw material was completely converted. 50g of tributylamine phosphate solution (50% content) was added dropwise to the system. After the addition was completed, the reaction was carried out at 0°C for 1h. LCMS detection showed that a large amount of raw material remained and the reaction was incomplete.

[0026] Example 1.3 Under nitrogen protection, 100g of trimethyl phosphate and 5g of guanosine were added to a three-necked flask, cooled to -5~0°C and controlled at -5~0°C, 5.0g of phosphorus oxychloride was added, stirred for 10min after the addition was completed, and the temperature was controlled at -5~0°C. The reaction was allowed to proceed for 2h. LCMS detected that the raw material conversion was complete. 25g of tributylamine phosphate solution (50% content) was added dropwise to the system. After the addition was completed, the reaction was allowed to proceed at 0°C for 1h to obtain guanosine diphosphate. The reaction was confirmed to be complete by LCMS detection, and 200g of water was added dropwise to the system under control of 0~5°C and stirred to quench the reaction. The intermediate 2 obtained was purified by preparative liquid phase. The purity of guanosine diphosphate was 98.3% and the yield was 53%.

[0027] During the experimental research process, the applicant tried multiple mass ratios of guanosine to phosphorus oxychloride, such as 1:0.8, 1:1, 1:1.3, 1:1.5, 1:1.8, etc.; the mass ratios of 5'-guanosine monophosphate to tributylammonium phosphate were 1:3, 1:5, 1:7, 1:9, etc. When the mass ratio of guanosine to phosphorus oxychloride was in the range of 1:1 to 1.5, the mass ratio of 5'-guanosine monophosphate to tributylammonium phosphate was better in the range of 1:5 to 1:7.

[0028] Example 2 Step 2 Example 2.1 Under nitrogen protection, 100 g of DMF and 8 g of intermediate 2 of Example 1.1 were weighed and added to a three-necked flask. The reaction temperature was controlled at 10-20 ° C. 4 g of triethylamine and 5 g of imidazole were added to the system and stirred for 0.5 h. 9 g of triphenylphosphine and 5 g of disulfide dipyridine were added to the system. The system was stirred at 10-20 ° C. for 2 h to generate guanosine imidazole diphosphate. LCMS detection confirmed the completion of the reaction. The LCMS detection spectrum of guanosine imidazole diphosphate (intermediate 3, GDP-imidazolide) is shown in the attached Figure 3 .

[0029] The reaction system was transferred to a concentration flask and concentrated until no solvent was distilled off to obtain a yellow oil. 80 g of anhydrous acetonitrile was added, the temperature was lowered to -15 to -10 ° C, and the mixture was beaten for 2 h. The supernatant was removed, 40 g of DMF was added to the remaining solid, and the mixture was transferred to a three-necked flask. The mixture was protected by nitrogen and the temperature was controlled within the range of 10 to 20 ° C. 6.5 g of anhydrous manganese chloride was added to the system, and 6 g of tributylamine thiophosphate was added to the system. The system was kept at 10 to 20 ° C and stirred for 24 h. LCMS detection confirmed that the reaction was complete. The reaction system was transferred to a concentration flask and concentrated until no solvent was distilled off. The product was separated by preparative liquid phase separation. After concentration, lyophilization was performed to obtain guanosine γ-thiophosphate (GTPYS, intermediate 4). The LCMS detection spectrum of guanosine γ-thiophosphate is shown in FIG. Figure 4 , the purity was 99.1% and the yield was 78%.

[0030] Example 2.1.1 Under nitrogen protection, 100 g of DMF and 8 g of intermediate 2 in Example 1.1 were weighed and added to a three-necked flask. The reaction temperature was controlled at 10-20°C. 4 g of triethylamine and 5 g of imidazole were added to the system, and the mixture was stirred for 0.5 h. 9 g of triphenylphosphine and 5 g of disulfide dipyridine were added to the system, and the system was stirred at 10-20°C for 2 h. The reaction was confirmed to be complete by LCMS to obtain guanosine diphosphate imidazole.

[0031] The reaction system was transferred to a concentration flask and concentrated until no solvent was distilled off to obtain a yellow oil. 80 g of anhydrous acetonitrile was added, the temperature was lowered to -15 to -10 ° C, and the mixture was beaten for 2 h. The supernatant was removed, 40 g of DMF was added to the remaining solid, and the mixture was transferred to a three-necked flask. Under nitrogen protection, the temperature was controlled within the range of 10 to 20 ° C. 3.5 g of anhydrous manganese chloride was added to the system, and 6 g of tributylamine thiophosphate was added to the system. The system was kept at 10 to 20 ° C and stirred for 24 h. LCMS detection confirmed the completion of the reaction. The reaction system was transferred to a concentration flask and concentrated until no solvent was distilled off. The product was separated by preparative liquid phase separation. After concentration, lyophilization was performed to obtain intermediate 4 with a purity of 98.9% and a yield of 65%.

[0032] In this reaction step, the inventors of the present application tested multiple dosage ratios of guanosine imidazole diphosphate and manganese chloride, among which their molar ratio within the range of 1:1.5 to 1:2.9 showed significantly better effects. When the molar ratio of guanosine imidazole diphosphate to Lewis acid was lower than this molar ratio, the purity of the system was low. When the molar ratio of guanosine imidazole diphosphate to Lewis acid was higher than this molar ratio, the purity of the system neither increased nor decreased, that is, there was no positive effect on the reaction.

[0033] Example 2.1.2 Under nitrogen protection, 100 g of DMF and 8 g of intermediate 2 in Example 1.1 were weighed and added to a three-necked flask. The reaction temperature was controlled at 10-20°C. 10.4 g of triethylamine and 8 g of imidazole were added to the system, and the mixture was stirred for 0.5 h. 11.2 g of triphenylphosphine and 8 g of disulfide dipyridine were added to the system, and the system was stirred at 10-20°C for 2 h. The reaction was confirmed to be complete by LCMS to obtain guanosine diphosphate imidazole.

[0034] The reaction system was transferred to a concentration flask and concentrated until no solvent was distilled off to obtain a yellow oil. 80 g of anhydrous acetonitrile was added, the temperature was lowered to -15 to -10 ° C, and the mixture was beaten for 2 h. The supernatant was removed, 40 g of DMF was added to the remaining solid, and the mixture was transferred to a three-necked flask. Under nitrogen protection, the temperature was controlled within the range of 10 to 20 ° C. 6.5 g of anhydrous manganese chloride was added to the system, and 6 g of tributylamine thiophosphate was added to the system. The system was kept at 10 to 20 ° C and stirred for 24 h. LCMS detection confirmed the completion of the reaction. The reaction system was transferred to a concentration flask and concentrated until no solvent was distilled off. The product was separated by preparative liquid phase separation. After concentration, lyophilization was performed to obtain intermediate 4 with a purity of 98.2% and a yield of 57%.

[0035] The mass ratio of intermediate 2, triethylamine, imidazole, triphenylphosphine and disulfide dipyridine is 1:0.5:0.6:1.1:0.6 to 1:1.3:1:1.4:1. If the mass ratio exceeds this range, the system becomes impure and new impurities are generated or raw materials are wasted.

[0036] Example 2.2 At room temperature and under nitrogen, 100 g of DMF and 8 g of intermediate 2 from Example 1.1 were weighed and added to a reaction flask. The system dissolved and the reaction temperature was controlled within the range of 10-20°C. 4 g of triethylamine and 5 g of imidazole were added to the system. At room temperature of 20°C, 9 g of triphenylphosphine and 5 g of disulfide dipyridine were added to the system. The system was stirred at 15-20°C for 12 h. A sample was taken and sent to LCMS for reaction detection. The reaction showed completion. 9.459 g of anhydrous cadmium chloride was added to the system. The system was slightly warmed to room temperature of 20°C. 1.03 g of compound 11 was added to the system. The system was stirred at 15-20°C for 12 h. LCMS detection confirmed the completion of the reaction and complete consumption of the starting materials. Post-treatment: 5 ml of water was added to the solution, filtered, and the product was separated by preparative liquid phase separation. The product was concentrated and lyophilized to obtain intermediate 4 with a purity of 98.7% and a yield of 18%.

[0037] Example 2.3 At room temperature and under nitrogen, 100 g of DMF and 8 g of intermediate 2 from Example 1.1 were weighed and added to a reaction flask. The system dissolved and the reaction temperature was controlled at 10-20°C. 4 g of triethylamine and 5 g of imidazole were added to the system. At room temperature of 20°C, 9 g of triphenylphosphine and 5 g of disulfide dipyridine were added to the system. The system was stirred at 15-20°C for 12 h. A sample was taken and sent to LCMS for reaction detection. The reaction showed completion. 7.02 g of anhydrous zinc chloride was added to the system. The system was slightly warmed to room temperature of 20°C. 1.03 g of compound 11 was added to the system. The system was stirred at 15-20°C for 12 h. LCMS detection confirmed the completion of the reaction and complete consumption of the starting materials. Post-treatment: 5 ml of water was added to the solution, filtered, and the product was separated by preparative liquid phase separation. The product was concentrated and lyophilized to obtain intermediate 4 with a purity of 98.5% and a yield of 24%.

[0038] Example 3 Step 3 Example 3.1 At room temperature (25°C), 400 g of phosphate buffer solution with a pH of 9-10 was weighed and the system was cooled to 0-10°C. 2 g of intermediate 4 from Example 2.1 was added to the reaction flask. The system dissolved and the pH was adjusted to 9.0 with 1 M sodium hydroxide aqueous solution. 0.943 g of 2-(methylamino)benzoyl chloride was added to the system in batches. The system was maintained at 0-10°C and the pH was maintained at 7-8, and the reaction was stirred for 16 hours. The reaction solution was directly subjected to high-pressure preparative liquid phase purification, and the fraction was lyophilized to obtain MANT-GTP-γ-S with a purity of 96.6% and a yield of 62%. The LCMS detection spectrum of MANT-GTP-γ-S is shown in the attached figure. Figure 5 ; The HNMR detection spectrum of MANT-GTP-γ-S is shown in Figure 6 HPLC detection spectrum of MANT-GTP-γ-S is shown in Figure 7 .

[0039] Example 3.2 At room temperature (25°C), 200 g of phosphate buffer solution (pH 9-10) was weighed. At 15-20°C, 1 g of intermediate 4 from Example 2.1 was added to a reaction flask. The solution dissolved and the pH was adjusted to 10 with 1 M aqueous sodium hydroxide solution. 0.472 g of 2-(methylamino)benzoyl chloride was added in batches. The reaction was stirred for 16 hours while maintaining the pH at 15-20°C and 8-9. The reaction solution was directly subjected to high-pressure preparative liquid phase purification, and the fraction was lyophilized to obtain MANT-GTP-γ-S with a purity of 96.2% and a yield of 33%.

[0040] Example 3.3 At room temperature (25°C), 200 g of phosphate buffer solution (pH 7-8) was weighed and the system was maintained at 0-10°C. 1 g of intermediate 4 from Example 2.1 was added to a reaction flask. The system dissolved and the pH was adjusted to 7.5 with 1M sodium hydroxide solution. 0.472 g of 2-(methylamino)benzoyl chloride was added batchwise. The system was maintained at 0-10°C and the pH at 9-10, and the reaction was stirred for 16 hours. The reaction solution was directly subjected to high-pressure preparative liquid phase purification, and the fraction was lyophilized to obtain MANT-GTP-γ-S with a purity of 96.5% and a yield of 41%.

[0041] In each of the above specific examples 3, maintaining the pH at 7-8, 8-9, or 9-10 means that the pH control does not need to be so strict and only needs to be within the above range.

[0042] During the experiment, the applicant also tried multiple mass ratios of guanosine γ-phosphorothioate and 2-(methylamino)benzoyl chloride, such as 1:1, 1:1.5, 1:3, 1:5, etc., and found that the molar ratio of guanosine γ-phosphorothioate to 2-(methylamino)benzoyl chloride was within the range of 1:1.5 to 1:5.0. The system purity and material cost were better. When the molar ratio was lower than this, the system purity was low. When the molar ratio was higher than this, there was no positive effect on the system, but it would cause waste of raw materials.

[0043] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing guanosine γ-phosphorothioate, characterized in that: The method for preparing guanosine gamma-phosphorothioate comprises the following steps: guanosine reacts with phosphorus oxychloride to generate guanosine 5'-monophosphate; guanosine 5'-monophosphate reacts with tributylammonium phosphate to generate guanosine diphosphate; guanosine diphosphate reacts with triethylamine, imidazole, a phosphorous reagent, and dithiodipyridine to generate guanosine diphosphate imidazole; and guanosine diphosphate imidazole reacts with tributylammonium thiophosphate under the catalysis of Lewis acid to generate guanosine gamma-phosphorothioate.

2. The method for preparing guanosine gamma-phosphorothioate according to claim 1, wherein The molar ratio of guanosine to phosphorus oxychloride is 1:1.8 to 1:2.8, and the molar ratio of 5'-guanosine monophosphate to tributylamine phosphate is 1:7 to 1:

10.

3. The method for preparing guanosine gamma-phosphorothioate according to claim 1, characterized in that: In the step of reacting guanosine diphosphate with triethylamine, imidazole, a phosphorous reagent, and dithiodipyridine to generate guanosine diphosphate imidazole, the imidazole and morpholine can replace each other; the phosphorous reagent is triphenylphosphine, tricyclohexylphosphine, or tributylphosphine; and the dithiodipyridine and diphenylacetyl disulfide or DDTT can replace each other.

4. The method for preparing guanosine gamma-phosphorothioate according to claim 3, characterized in that: The molar ratio of the guanosine diphosphate to triethylamine, imidazole or morpholine, phosphorous reagent, dithiodipyridine or diphenylacetyl disulfide or DDTT is: 1: (2.1-5.7): (3.0-5.1): (1.8-2.4): (1.2-2.1).

5. The method for preparing guanosine gamma-phosphorothioate according to claim 1, wherein In the step of reacting guanosine imidazole diphosphate and tributylamine thiophosphate under the catalysis of Lewis acid to generate guanosine γ-thiophosphate, the Lewis acid is cadmium chloride, manganese chloride or zinc chloride; and the molar ratio of the Lewis acid to guanosine imidazole diphosphate is 1.5:1 to 2.9:

1.

6. The method for preparing guanosine gamma-phosphorothioate according to claim 5, characterized in that: The molar ratio of the guanosine diphosphate imidazole to tributylamine thiophosphate is 1:1.10 to 1:1.

27.

7. Guanosine gamma-phosphorothioate prepared by the method for preparing guanosine gamma-phosphorothioate according to any one of claims 1 to 6.

8. A method for preparing a guanosine γ-phosphorothioate derivative MANT-GTP-γ-S, characterized in that: The method for preparing the guanosine γ-phosphorothioate derivative MANT-GTP-γ-S comprises the step of reacting the guanosine γ-phosphorothioate according to claim 7 with 2-(methylamino)benzoyl chloride at pH 7-10 to generate MANT-GTP-γ-S.

9. The method for preparing the guanosine γ-phosphorothioate derivative MANT-GTP-γ-S according to claim 8, characterized in that: The molar ratio of the guanosine gamma-phosphorothioate to 2-(methylamino)benzoyl chloride is 1:1.5 to 1:

5.

10. The guanosine γ-phosphorothioate derivative MANT-GTP-γ-S prepared by the method for preparing the guanosine γ-phosphorothioate derivative MANT-GTP-γ-S according to any one of claims 8 to 9.

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