Preparation method of pseudouridine

Abstract

The invention discloses a method for preparing high-purity houridine (formula 1) by adopting tert-butyl diphenyl silyl protection. According to the method, a formula 42 is used as a raw material, and the high-purity pseudouridine (formula 1) is prepared through hydroxyl protection, condensation, reduction, cyclic etherification and deprotection in sequence. The method has the advantages of high yield, high purity and the like.

Description

A method for preparing pseudouridine Technical Field

[0001] This invention belongs to the field of chemical synthesis. Specifically, this invention relates to a method for preparing the pseudouridine shown in Formula 1, the novel structural intermediate involved, and its uses. Background Technology

[0002] Pseudorabylidine (as shown in Formula 1) is a raw material for the synthesis of nucleic acid vaccines. As an exogenous substance, mRNA vaccines can trigger an immune response after entering the human body, causing the mRNA in the mRNA vaccine to be cleared by the immune system before it can exert its effects. Replacing uridine with pseudouridine reduces the problem of mRNA being easily recognized and cleared by the immune system, thus reducing the risk of adverse immune reactions.

[0003] 1 However, existing methods for synthesizing pseudouridine nucleosides often suffer from drawbacks such as high levels of isomer impurities and low yields. For example, during the research and development process, the inventors discovered that existing methods for synthesizing pseudouridine nucleosides often have various shortcomings, such as high impurity content, low purity of the obtained pseudouridine nucleosides, and insufficient yield.

[0004] For example, the existing technical literature Tetrahedron Letters (2003), 44(45), 8321-8323 reports a method in which compound 3 is protected on the hydroxyl group of DMOP under PPTS acid catalysis to obtain 4, compound 5 undergoes nucleophilic addition reaction with compound 4 under the action of n-butyllithium to obtain 6, 6 is chelated by ZnCl2 and then ring-opened by L-Selectride chiral reduction of furan ring to obtain compound 7, the two hydroxyl groups on compound 7 are ring-closed by Mitsunobu reaction to obtain compound 8, and 8 is dehydroetherified on furan, protected by acetone fork and tert-butyl protection of pyrimidine ring under acidic conditions to obtain 1 (pseudouracil nucleoside), with an overall yield of 46.3%. However, after studying the method disclosed in that document, the inventors found that the method has significant shortcomings. For example: 1. In the reaction from 3 to 4: compound 3 reacts with DMOP to generate 41, and 41 further reacts with DMOP to generate 4. Experiments showed that the reaction time for 41 to generate 4 is long. The 2,2-dimethoxypropane (DMOP) protecting group on the primary hydroxyl group of compound 4 is easily degraded and removed, resulting in a low actual yield of 4 (only 40.0%). 2. In the reaction that generates 7, it was found that 4.70% of impurity 7-α was generated (HPLC retention time, 7: 14.91 min, 7-α: 14.41 min). Impurity 7-α was converted to impurity 8-α by the subsequent Mitsunobu reaction with 7. Impurity 8-α was then converted to product 1 and impurity 1-α by the subsequent deprotection reaction with 8, affecting the product yield and purity. 3. Compound 7 undergoes a Mitsunobu reaction with triphenylphosphine (Ph3P) and diisopropyl azodicarbonate (DIAD) to generate compound 8. The two hydroxyl groups on compound 7 show poor selectivity. Experiments revealed that the Mitsunobu reaction cyclization method yields 25.66% of the isomer impurity 8-C (HPLC retention time of 8 is 9.60 min, and that of impurity 8-C is 11.56 min). (Literature reports a yield of 70%) Therefore, the existing methods for synthesizing pseudouridine nucleosides (Formula 1) contain a large number of 7-α, 8-α, 1-α, and 8-C isomers, resulting in low yields.

[0005] Therefore, there is an urgent need in this field for a method to synthesize pseudouridine nucleosides with high purity and high yield. Summary of the Invention

[0006] The purpose of this invention is to provide a method for synthesizing pseudouridine nucleosides with high purity and high yield, thereby overcoming the deficiencies in the prior art.

[0007] Another object of the present invention is to provide novel intermediates with entirely new structures for the synthesis of pseudouridine, methods for preparing these intermediates, and uses of these intermediates in the preparation of pseudouridine.

[0008] In a specific embodiment, the present invention provides a method for preparing the pseudouridine shown in Formula 1, wherein the reaction formula of the method is shown below: The method includes the following steps: 1) reacting compound 42 to obtain compound 62; 2) reacting compound 62 to obtain compound 72; 3) reacting compound 72 to obtain compound 82; 4) hydrolyzing compound 82 to obtain pseudouridine (formula 1).

[0009] In a specific implementation, the reaction formula of the method is as follows: In step 1), compound 42 is condensed with 2,4-di-tert-butoxy-5-bromopyrimidine (formula 5) to give compound 62; in step 2), compound 62 is reduced with lithium tri-sec-butylborohydride (L-Selectride) under zinc chloride chelation to give compound 72; in step 3), compound 72 is cyclized with Mitsunobu reaction to give compound 82; in step 4), compound 82 is hydrolyzed with trifluoroacetic acid (TFA) to give pseudouracil nucleoside (formula 1).

[0010] In a specific embodiment, in step 2), compound 62 is reduced with lithium trisec-butylborohydride (L-Selectride) under zinc chloride chelation to obtain compound 72 and isomer impurity 72-α. Preferably, the content of the isomer impurity 72-α is <4%.

[0011] In a specific embodiment, in step 3), compound 72 undergoes a Mitsunobu reaction to cyclize and yield compound 82 and isomer impurity 82-C. Preferably, the content of the isomer impurity 82-C is <1.0%.

[0012] In a specific implementation, the reagents used in the Mitsunobu reaction in step 3) are tributylphosphine (TBP) and N,N,N',N'-tetramethylazodicarbonamide (TMAD).

[0013] In a specific embodiment, the solvent for the Mitsunobu reaction in step 3) is toluene, and the reaction temperature is -5~5℃, preferably 0℃.

[0014] In a specific implementation, the reduction reaction temperature in step 2) is -80~0℃, preferably -10~0℃.

[0015] In a second aspect, the present invention provides a compound represented by Formula 62 as shown below. .

[0016] In a third aspect, the present invention provides a compound represented by Formula 72 as shown below. .

[0017] In a fourth aspect, the present invention provides a compound of formula 82 as shown below. .

[0018] In a fifth aspect, the present invention provides the use of compounds of formula 62, 72 or 82 in the preparation of pseudouridine nucleosides of formula 1. , , 1.

[0019] In a preferred embodiment, the pseudouridine shown in Formula 1 is used to prepare an mRNA vaccine.

[0020] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0021] Figure 1 shows the compound represented by Formula 62. 1 Figure 2 shows the 1H NMR spectrum of the compound represented by Formula 62; Figure 3 shows the MS spectrum of the compound represented by Formula 72. 1 Figure 4 shows the 1H NMR spectrum of the compound represented by Formula 72; Figure 5 shows the MS spectrum of the compound represented by Formula 82. 1 1H NMR spectrum; Figure 6 shows the MS spectrum of the compound represented by Formula 82. Detailed Implementation

[0022] To overcome the various shortcomings of existing technologies, the inventors, through extensive and in-depth research, unexpectedly discovered a new synthetic route involving novel intermediates. This route yields high purity and high yield of pseudouridine nucleosides. Based on this, the present invention was completed.

[0023] During the research and development of the method and the new intermediate of this invention, the inventors discovered that existing methods for synthesizing pseudouridine often have various shortcomings, such as high impurity content, low purity of the obtained pseudouridine, and insufficient yield.

[0024] To prepare synthetic pseudouridine with high purity and high yield, the inventors have creatively developed a method for preparing high-purity pseudouridine via tert-butyldiphenylsilyl protection. The pseudouridine prepared by this method not only has high purity but also a significantly improved yield.

[0025] In a specific embodiment, the present invention provides a method for preparing the pseudouridine shown in Formula 1, wherein the reaction formula of the method is shown below: The method includes the following steps: 1) reacting compound 42 to obtain compound 62; 2) reacting compound 62 to obtain compound 72; 3) reacting compound 72 to obtain compound 82; 4) hydrolyzing compound 82 to obtain pseudouridine (formula 1).

[0026] Based on the teachings of this invention and conventional techniques in the art, those skilled in the art will know the specific methods for implementing the above reaction steps. For example, in step 1), compound 42 is condensed with 2,4-di-tert-butoxy-5-bromopyrimidine (formula 5) to obtain compound 62; in step 2), compound 62 is reduced with lithium tri-sec-butylborohydride (L-Selectride) under zinc chloride chelation to obtain compound 72; in step 3), compound 72 is cyclized with Mitsunobu reaction to obtain compound 82; in step 4), compound 82 is hydrolyzed under trifluoroacetic acid (TFA) to obtain pseudouracil nucleoside (formula 1).

[0027] The method of this invention synthesizes pseudouridine nucleoside (Formula 1), significantly reducing the content of isomer impurities generated in each reaction step. In a specific embodiment, in step 2), compound 62 is reduced by lithium trisec-butylborohydride (L-Selectride) under zinc chloride chelation to obtain compound 72 and isomer impurity 72-α. The content of the isomer impurity 72-α is <4%.

[0028] In step 3), compound 72 undergoes a Mitsunobu reaction for cyclization to yield compound 82 and isomer impurity 82-C. Preferably, the content of the isomer impurity 82-C is <1.0%.

[0029] Based on the method for synthesizing pseudouridine (Formula 1), the inventors further optimized various process parameters. In a specific embodiment, the reagents for the Mitsunobu reaction in step 3) are tributylphosphine (TBP) and N,N,N',N'-tetramethylazodicarbonamide (TMAD). The solvent for the Mitsunobu reaction is toluene, and the reaction temperature is -5 to 5°C, preferably 0°C. In step 2), the reduction reaction temperature is -80 to 0°C, preferably -10 to 0°C.

[0030] The method for synthesizing pseudouridine (Formula 1) of the present invention involves several novel intermediates. These intermediates allow for the high-purity and high-yield preparation of synthetic pseudouridine, which can then be used to prepare mRNA vaccines. In specific embodiments, the present invention provides compounds shown in Formula 62, Formula 72, and Formula 82. , , .

[0031] The main advantages of this invention include: 1. After protecting the primary hydroxyl group with tert-butyldiphenylsilyl, the stability of the compound is significantly increased, and the phenomenon of de-tert-butyldiphenylsilyl protecting group is not easy to occur in subsequent condensation, reduction and etherification reactions, resulting in high yield.

[0032] 2. The yield of compound 72 of this invention is high when it is prepared into compound 82 by the Mitsunobu reaction. Because the tert-butyldiphenylsilyl group on the primary hydroxyl group of compound 72 is large, the selectivity between the two hydroxyl groups is good. Only product 82 is generated during the Mitsunobu reaction, and the positional isomer impurity 82-C is hardly generated (<1.0%).

[0033] 3. The method for preparing pseudouridine of the present invention has a high yield.

[0034] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts, and eq represents the equivalent amount of the reaction substrate.

[0035] Example 1 (Preparation of Formula 41) In a 500 ml four-necked flask, 29.98 g (202.42 mmol, 1.0 eq) of methyl methacrylate (MCA) and 300 ml of acetone were added and stirred. The mixture was then cooled to 5 °C, and concentrated sulfuric acid (3.98 g, 40.58 mmol, 0.2 eq) was added dropwise over 0.5 h. The reaction mixture was kept at 5 °C and stirred for 1 h. The reaction was continued at room temperature for 2 h. Sodium bicarbonate was slowly added to the reaction mixture to adjust the pH to 7. The mixture was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, yielding a yellow crude product. The crude product was purified by column chromatography (eluent: dichloromethane:methanol = 10:1) to obtain 36.38 g of 41, with a yield of 95.5%.

[0036] Compound 41 1 ¹H NMR: (400 MHz, Chloroform-d) δ 4.83 (d, 1H), 4.78 (d, 1H), 4.63 (t, 1H), 3.99 (dd, 1H), 3.82 (dd, 1H), 1.48 (s, 3H), 1.39 (s, 3H). Example 2 (Preparation of Formula 42) Under N2 protection, 41 (1.00 g, 5.31 mmol, 1.0 eq), imidazole (0.45 g, 6.61 mmol, 1.24 eq), and tetrahydrofuran (4 ml) were added sequentially to a 25 ml three-necked flask and stirred until dissolved. The reaction mixture was cooled to 5°C, and tert-butyldiphenylchlorosilane (TBDPSCl) (1.60 g, 5.82 mmol, 1.1 eq) was added dropwise over 1 h. The mixture was then stirred at 30°C for 5 h. The white imidazole hydrochloride was removed by filtration. The solvent was removed by rotary evaporation of the filtrate, yielding a pale yellow crude product. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 20:1 → 10:1) to give 2.17 g of 42, with a yield of 95.6%.

[0037] MS (ESI) of compound 42 + ):C 24 H 30 O5Si, m / z 444.22 [M+H2O] + .

[0038] Compound 42 1 ¹H NMR: (400 MHz, DMSO-d⁶) δ 7.69 – 7.56 (m, 4H), 7.56 – 7.41 (m, 6H), 4.94 (d, 1H), 4.80 (d, 1H), 4.73 (dd, 1H), 3.89 (dd, 1H), 3.78 (dd, 1H), 1.37 (d, 6H), 0.98 (s, 9H). Example 3 (Preparation of Formula 62) Under N2 protection, 5% tetrahydrofuran (4.71 g, 15.53 mmol, 1.2 eq) was added to a 200 mL three-necked flask, and stirred at room temperature until dissolved and clear. The reaction solution was cooled to -78 °C, and a 2.5 M n-butyllithium solution (8.80 mL, 22.00 mmol, 1.7 eq) reddish-brown solution was added dropwise over 0.5 h. 42% tetrahydrofuran (5.48 g, 12.85 mmol, 1.0 eq) was added dropwise over 0.5 h, and the reaction was stirred for 7 h. Saturated brine (40 mL) was added to the reaction solution to extract the organic phase. The aqueous phase was extracted twice with ethyl acetate (30 mL × 1 and 20 mL × 1). The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give a yellowish-green crude product. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 30:1 → 20:1) to obtain 7.61 g of 62, with a yield of 91.0%.

[0039] MS (APCI) of compound 62 + ):C 36H 50 N₂O₇Si, m / z 651.35 [M+H] + .

[0040] Compound 62 1 ¹H NMR: (400 MHz, DMSO-d6) δ 8.23 ​​(s, 1H), 7.68 – 7.63 (m, 4H), 7.51 – 7.44 (m, 6H), 6.32 (s, 1H), 4.88 – 4.78 (m, 2H), 4.19 – 4.10 (m, 1H), 3.87 (t, 1H), 3.70 (dd, 1H), 1.56 (d, 18H), 1.23 (d, 6H), 1.04 (s, 9H). Example 4 (Preparation of Formula 72) Under N2 protection, 62 (5.05 g, 7.76 mmol, 1.0 eq) and dichloromethane (249.0 ml) were added to a 500 ml three-necked flask and stirred at room temperature until dissolved and clear. The reaction solution was cooled to 0 °C, and a colorless and transparent 1 M zinc chloride THF solution (10.80 ml, 10.80 mmol, 1.4 eq) was added dropwise over 30 min. Then, 1 M L-selectride THF solution (28.00 ml, 28.00 mmol, 3.6 eq) was added dropwise over 30 min. The mixture was stirred at 0 °C for 8 h. Anhydrous ethanol was added to the reaction solution and stirred for 30 min. Water, 30% hydrogen peroxide aqueous solution, and 1 M sodium hydroxide aqueous solution were added, and the reaction was quenched by stirring for 30 min. The organic phase was extracted, and the aqueous phase was extracted three times (30 ml × 3) with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain a pale yellow crude product. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 15:1) to obtain 4.52 g of 72, with a yield of 89.2%, an HPLC purity of 98.51%, and an impurity of 1.28% 74-α.

[0041] MS (APCI) of compound 72 + ):C 36 H 52 N₂O₇Si, m / z 653.36 [M+H] + .

[0042] Compound 72 1¹H NMR: (400 MHz, Acetone-d6) δ 8.29 (d, 1H), 7.81 – 7.75 (m, 4H), 7.47 – 7.40 (m, 6H), 5.24 (d, 1H), 4.54 (d, 1H), 4.39 (d, 2H), 4.29 (dd, 1H), 4.02 (d, 1H), 3.97 – 3.87 (m, 2H), 1.56 (d, 18H), 1.28 (d, 6H), 1.06 (s, 9H). HPLC detection method: Vanquish Core high-performance liquid chromatograph (Thermo Fisher), column: Shimadzu ShimNex HE Sil 5um; detector: DAD (detection wavelength: 210 nm). nm); Column temperature: 25℃; Flow rate: 0.4 ml / min; Mobile phase A: n-hexane, Mobile phase B: ethanol; Mobile phase A: Mobile phase B = 9:1; Diluent: ethanol; Injection volume: 1 μl.

[0043] The retention time of 62 was 9.57 min, the retention time of 72 was 11.04 min, and the retention time of 72-α was 10.47 min.

[0044] Example 5 (Preparation of Formula 82) Under N2 protection, N,N,N',N'-tetramethylazodicarbonamide (0.540 g, 3.14 mmol, 2.0 eq) and toluene (83 ml) were added to a 100 ml three-necked flask and stirred until dissolved and clear. The reaction solution was cooled to 2°C, and tributylphosphine (0.630 g, 3.11 mmol, 2.0 eq) was added. The mixture was stirred in an ice-water bath for 30 min. 72 (1.017 g, 1.56 mmol, 1.0 eq) was then added. The reaction solution was stirred at 0°C for 8 h, and the solvent was removed by concentration under reduced pressure to obtain a yellow crude product. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 20:1) to obtain 0.93 g of 82, with a yield of 93.9% and an HPLC purity of 99.21%. Impurity 82-α was 0.08%, and impurity 82-c was not detected (<0.05%).

[0045] MS (APCI) of compound 82 + ):C 36 H 50 N₂O₆Si, m / z 635.35 [M+H] + .

[0046] Compound 82 1¹H NMR: (400 MHz, Acetone-d6) δ 8.32 (d, 1H), 7.84 – 7.71 (m, 4H), 7.55 – 7.39 (m, 6H), 4.96 (dd, 1H), 4.77 – 4.65 (m, 2H), 4.19 – 4.10 (m, 1H), 3.97 (dd, 1H), 3.89 (dd, 1H), 1.62 (d, 18H), 1.56 (s, 3H), 1.34 (s, 3H), 1.08 (s, 9H). The retention time of 72 was 10.47 min, that of 82 was 9.11 min, and that of 82-α was 8.49 min.

[0047] Example 6 (Preparation of Formula 1) Add 0.102 g (0.16 mmol, 1.0 eq) of 82, 0.2 ml of water, and 1.8 ml of trifluoroacetic acid to a 10 mL reaction tube. Heat the reaction mixture to 50 °C and stir for 6 h. Adjust the pH of the reaction mixture to 6-7 with sodium hydroxide. Concentrate under reduced pressure to remove the solvent, yielding a pale yellow crude product. Purify the crude product by column chromatography (eluent: dichloromethane:methanol = 10:1) to obtain 0.037 g of the product, with a yield of 94.9% and an HPLC purity of 99.52%.

[0048] MS (ESI) of compound 1 - ): C9H 12 N₂O₆, m / z 243.05 [MH] - .

[0049] Compound of Formula 1 1 ¹H NMR: (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 10.87 (s, 1H), 7.52 (s, 1H), 4.91 (d, 1H), 4.78 (dd, 1H), 4.69 (d, 1H), 4.49 – 4.43 (m, 1H), 3.95 – 3.81 (m, 2H), 3.73 – 3.65 (m, 1H), 3.64 – 3.54 (m, 1H), 3.49 – 3.39 (m, 1H). Example 7 (Preparation of impurity formula 72-α reference standard) Under N2 protection, 62 (1.10 g, 1.69 mmol, 1.0 eq) and dichloromethane (90 mL) were added to a 100 mL three-necked flask and stirred at room temperature until dissolved and clear. The reaction solution was cooled to 0 °C and 1 M L-selectride THF solution (6.00 mL, 6.00 mmol, 3.6 eq) was added dropwise over 30 min. The mixture was slowly heated to room temperature and stirred for 10 h. Anhydrous ethanol was added to the reaction solution and stirred for 30 min, followed by the addition of water, 30% hydrogen peroxide aqueous solution, and 1 M sodium hydroxide aqueous solution, and stirred for 30 min to quench the reaction. The organic phase was extracted, and the aqueous phase was extracted three times with ethyl acetate (10 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain a pale yellow crude product. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 15:1) to give 1.00 g of 72-α, with a yield of 90.9%.

[0050] Compound 72-α 1 ¹H NMR: (400 MHz, DMSO-d⁶+D₂O) δ 8.23 ​​(s, 1H), 7.70 (m, 4H), 7.49–7.42 (m, 6H), 4.84 (d, 1H), 4.42 (dd, 1H), 4.33 (dd, 1H), 3.98–3.91 (m, 1H), 3.88 (d, 1H), 3.80 (dd, 1H), 1.56 (s, 18H), 1.20 (d, 6H), 1.02 (s, 9H). The retention time of 72-α was 10.47 min.

[0051] Example 8 (Preparation of impurity formula 82-α reference standard) Under N2 protection, N,N,N',N'-tetramethylazodicarbonamide (0.53 g, 3.07 mmol, 2.0 eq) and toluene (81 ml) were added to a 100 ml three-necked flask and stirred until dissolved and clear. The reaction solution was cooled to 2°C, and tributylphosphine (0.62 g, 3.06 mmol, 2.0 eq) was added. The mixture was stirred in an ice-water bath for 30 min. 72-α (1.00 g, 1.53 mmol, 1.0 eq) was then added. The reaction solution was stirred at 0°C for 5 h. The solvent was removed by concentration under reduced pressure to obtain a yellow crude product. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 20:1) to give 0.83 g of 82-α, in 85.6% yield.

[0052] Compound 82-α 1¹H NMR: (400 MHz, DMSO-d⁶) δ 8.14 (d, 1H), 7.68 – 7.61 (m, 4H), 7.49 – 7.40 (m, 6H), 5.20 (d, 1H), 4.96 – 4.87 (m, 2H), 4.21 (t, 1H), 3.83 (dd, 1H), 3.76 (dd, 1H), 1.54 (d, 18H), 1.24 (s, 6H), 1.03 (s, 9H). The retention time of 8²⁻α was 8.49 min.

[0053] In Example 9, four 100 ml three-necked flasks were prepared. Under N2 protection, the following were added to flasks (1-4): Reaction solution 1 was treated with diisopropyl azodicarbonate (DIAD) (0.018 g, 0.089 mmol, 2.1 eq); reaction solution 2 was treated with diethyl azodicarbonate (DEAD) (0.016 g, 0.092 mmol, 2.1 eq); reaction solution 3 was treated with N,N,N',N'-tetramethylazodicarbonamide (TMAD) (0.017 g, 0.099 mmol, 2.3 eq); and reaction solution 4 was treated with azodicarbonyldipiperidine (ADDP) (0.023 g, 0.091 mmol, 2.1 eq). Toluene (2 ml) was first added to each of the four reaction solutions, followed by the addition of triphenylphosphine (TPP) (0.023 g, 0.088 mmol, 2.0 eq) dropwise to each. 1. Reaction solution 1 was prepared by adding triphenylphosphine (TPP) (0.023 g, 0.088 mmol, 2.0 eq) to reaction solution 2; tributylphosphine (TBP) (0.020 g, 0.099 mmol, 2.3 eq) was added to reaction solution 3; and tributylphosphine (TBP) (0.020 g, 0.099 mmol, 2.3 eq) was added to reaction solution 4. The mixtures were stirred at 0°C for 30 min, and then 72 (0.027 g, 0.043 mmol, 1.0 eq) was added to each solution. The reaction mixtures were reacted for 8 h. TLC analysis was performed on flasks 1-4, as shown in the table below. The preferred reaction is tributylphosphine (TBP) + N,N,N',N'-tetramethylazodicarbonamide (TMAD), which produces product 82.

[0054] In Example 10, under N2 protection, 62 (three portions of 0.100 g, 0.15 mmol, and 1.0 eq) was placed into three 10 ml reaction tubes, numbered 1-3. DCM was first added to reaction tubes 1, 2, and 3, and the mixture was stirred at room temperature (10°C) to dissolve it into a colorless and clear solution.

[0055] Reaction solution 1 was cooled to -78℃, reaction solution 2 to -40℃, and reaction solution 3 to 0℃. 1M ZnCl₂THF solution (0.18 ml, 0.18 mmol, 1.2 eq) was added dropwise to each of reaction solutions 1-3, with stirring for 30 min after the addition was complete. Then, 1M L-selectride THF solution (0.54 ml, 0.54 mmol, 3.6 eq) was added dropwise, with stirring for 30 min after the addition was complete. After the addition was complete, reaction solution 1 was reacted at -80℃ for 15 h; reaction solution 2 at -40℃ for 4 h; and reaction solution 3 at 0℃ for 4 h. The peak area percentages of 62, 72, and 72-α in the reaction solutions were monitored using HPLC. The preferred reaction temperature is 0℃, which results in a high yield of product 72 and fewer impurities 72-α.

[0056] In Example 11, four 25 ml three-necked flasks were prepared. Under N2 protection, 0.052 g (0.30 mmol, 2.0 eq) of yellow TMAD powder was added to flasks 1-4 respectively. The following amounts were added to flasks 1-4: dichloromethane (4.8 ml); acetonitrile (4.0 ml); tetrahydrofuran (6.2 ml); and toluene (8.0 ml). The reaction solutions were stirred and dissolved at 0°C until clear. Tributylphosphine solution (0.060 g, 0.30 mmol, 2.0 eq) was then added dropwise to each flask. 72 (0.10 g, 0.15 mmol, 1.0 eq) was added to each flask, and the mixture was stirred at 0°C for 8 h. The peak area percentages of 72, 82, and 82-C in the reaction solution were monitored using HPLC. Toluene is preferred as the reaction solvent, and the amount of impurity 82-C is relatively small.

[0057] In Example 12, three 25 ml three-necked flasks were prepared. Under N2 protection, 0.052 g of yellow TMAD powder (0.30 mmol, 2.0 eq) was added to flasks 1-3 respectively. Toluene (8.0 ml) was added and stirred until dissolved and clear. Reaction solution 1 was cooled to 0°C, reaction solution 2 was heated to 30°C, and reaction solution 3 was heated to 60°C. Tributylphosphine solution (0.060 g, 0.30 mmol, 2.0 eq) was added dropwise to each flask. While maintaining the respective temperatures, 0.10 g of 72 (0.15 mmol, 1.0 eq) was added to each flask, and the reactions were maintained for 8 h. The peak area percentages of 72, 82, and 82-C in the reaction solutions were monitored using HPLC. 0°C is preferred as the reaction solvent, and the amount of impurity 82-C is relatively small.

[0058] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing the pseudouridine shown in Formula 1, wherein the reaction formula of the method is shown below: The method includes the following steps: 1) Compound 42 reacts to give compound 62; 2) Compound 62 reacts to yield compound 72; 3) Compound 72 is reacted to give compound 82; 4) Compound 82 is hydrolyzed to give pseudouridine (Formula 1).

2. The preparation method according to claim 1, characterized in that, The reaction formula for the method is shown below: In step 1), compound 42 is condensed with 2,4-di-tert-butoxy-5-bromopyrimidine (formula 5) to give compound 62; in step 2), compound 62 is reduced with lithium tri-sec-butylborohydride (L-Selectride) under zinc chloride chelation to give compound 72; in step 3), compound 72 is cyclized with Mitsunobu reaction to give compound 82; in step 4), compound 82 is hydrolyzed with trifluoroacetic acid (TFA) to give pseudouracil nucleoside (formula 1).

3. The method as described in claim 2, characterized in that, In step 2), compound 62 is reduced with lithium trisec-butylborohydride (L-Selectride) under zinc chloride chelation to give compound 72 and isomer impurity 72-α. Preferably, the content of the isomer impurity 72-α is <4%.

4. The method as described in claim 2, characterized in that, In step 3), compound 72 undergoes a Mitsunobu reaction for cyclization to yield compound 82 and isomer impurity 82-C. Preferably, the content of the isomer impurity 82-C is <1.0%.

5. The method as described in claim 2, characterized in that, In step 3), the reagents for the Mitsunobu reaction are tributylphosphine (TBP) and N,N,N',N'-tetramethylazodicarbonamide (TMAD).

6. The method as described in claim 2, characterized in that, In step 3), the solvent for the Mitsunobu reaction is toluene, and the reaction temperature is -5~5℃, preferably 0℃.

7. The method as described in claim 6, characterized in that, In step 2), the reduction reaction temperature is -80~0℃, preferably -10~0℃.

8. The compound shown in Formula 62 below, 。 9. The compound shown in Formula 72 below. 。 10. The compound shown in Formula 82 below. 。 11. Use of the compounds shown in Formula 62, Formula 72 or 82 in the preparation of pseudouridine nucleosides shown in Formula 1. 、 、 1。

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