Method for synthesizing pseudouridine

Abstract

The invention discloses a method for preparing high-purity houridine (formula 1). According to the method, a formula 44 is used as a raw material, and high-purity pseudouridine (formula 1) is prepared through condensation, reduction, cyclic etherification and deprotection in sequence. The method has the excellent effects of high yield, high purity and the like. ,

Description

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 trigger an immune response upon 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 a high number of isomer impurities and low yields.

[0004] 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

[0005] 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.

[0006] 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.

[0007] In a first aspect, the present invention provides a method for preparing pseudouracil nucleoside of Formula 1, wherein the reaction formula of the method is shown below: The method includes the following steps: 1) Compound 44 reacts to give compound 64; 2) Compound 64 reacts to yield compound 74; 3) Compound 74 reacts to give compound 84; and 4) Hydrolysis of compound 84 yields pseudouridine (Formula 1).

[0008] In a specific implementation, the reaction formula of the method is as follows: In step 1), compound 44 is condensed with 2,4-di-tert-butoxy-5-bromopyrimidine (formula 5) to give compound 64; In step 2), compound 64 is reduced with lithium trisec-butylborohydride (L-Selectride) under zinc chloride chelation to obtain compound 74; In step 3), compound 74 is cyclized by the Mitsunobu reaction to obtain compound 84; In step 4), compound 84 is hydrolyzed in the presence of acetic acid to obtain pseudouracil nucleoside (formula 1).

[0009] In a specific implementation, in step 2), compound 64 is reduced with lithium trisec-butylborohydride (L-Selectride) under zinc chloride chelation to obtain compound 74 and isomer impurity 74-α. ; Preferably, the content of the isomer impurity 74-α is <4%.

[0010] In a specific embodiment, in step 3), compound 74 undergoes a Mitsunobu reaction to cyclize and yield compound 84 and isomer impurity 84-C. ; Preferably, the content of the isomer impurity 84-C is <1.0%.

[0011] In a specific implementation, in step 3), the reagents for the Mitsunobu reaction are tributylphosphine (TBP) and N,N,N',N'-tetramethylazodicarbonamide (TMAD).

[0012] In a specific embodiment, in step 3), the solvent for the Mitsunobu reaction is tetrahydrofuran (THF), and the reaction temperature is 25-35°C, preferably 30°C.

[0013] In a specific implementation, in step 2), the reduction reaction temperature is -80~0℃; preferably -10~0℃.

[0014] In a second aspect, the present invention provides a compound of formula 64. .

[0015] In a third aspect, the present invention provides the compound shown in Formula 74. .

[0016] In a fourth aspect, the present invention provides a compound of formula 84. .

[0017] In a fifth aspect, the present invention provides the preparation of compounds of formula 64, formula 74 or 84 and their use in pseudouridine of formula 1. 1.

[0018] In a preferred embodiment, the pseudouridine shown in Formula 1 is used to prepare an mRNA vaccine.

[0019] 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

[0020] Figure 1 The compound shown in Formula 64 is displayed. 1 H NMR spectrum; Figure 2 The MS spectrum of the compound shown in Formula 64 is displayed; Figure 3 The compound shown in Formula 74 is displayed. 1 H NMR spectrum; Figure 4 The MS spectrum of the compound shown in Formula 74 is displayed; Figure 5 The single-crystal X-ray diffraction pattern of the compound represented by impurity formula 74-α is shown. Figure 6 The compound shown in Formula 84 is displayed. 1 H NMR spectrum; Figure 7 The MS spectrum of the compound shown in Formula 84 is displayed; Figure 8 The single-crystal X-ray diffraction pattern of the compound shown in Formula 84 is displayed. Detailed Implementation

[0021] During the research and development process, the inventors discovered that existing methods for synthesizing pseudouridine nucleosides often suffer from high impurity content, resulting in low purity and insufficient yield of the obtained pseudouridine nucleosides.

[0022] For example, Tetrahedron Letters (2003), 44(45), 8321-8323 reported a method for synthesizing pseudouridine nucleoside: compound 3 was protected on the hydroxyl group of DMOP under PPTS acid catalysis to obtain 4; compound 5 underwent a nucleophilic addition reaction with compound 4 in the presence of n-butyllithium to obtain 6; 6 was chelated with ZnCl2 and then ring-opened by chiral reduction of furan ring with L-Selectride to obtain compound 7; the two hydroxyl groups on compound 7 were ring-closed by Mitsunobu reaction to obtain compound 8; 8 was dehydroetherified from furan, protected with acetone fork and tert-butyl of pyrimidine ring under acidic conditions to obtain 1 (pseudouridine nucleoside), with an overall yield of 46.3%. After studying the method disclosed in this document, the inventors discovered that: 1. In the reaction from compound 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 of 41 with DMOP to generate 4 was 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. 7 undergoes a Mitsunobu reaction in the presence of triphenylphosphine (Ph3P) and diisopropyl azodicarbonate (DIAD) to generate 8. The two hydroxyl groups on compound 7 show poor selectivity. Experiments show that the cyclization of compound 8 via the Mitsunobu reaction using the literature method generates 25.66% of the isomer impurity 8-C (HPLC retention time of 8 is 9.60 min, and HPLC retention time of impurity 8-C is 11.56 min). Therefore, the existing methods for synthesizing pseudouridine (Formula 1) contain a large number of 7-α, 8-α, 1-α, and 8-C isomers, resulting in low yields.

[0023] To overcome this deficiency in the prior art, the inventors, through extensive and in-depth research, unexpectedly discovered a new synthetic route involving novel intermediates. This route yields pseudouridine nucleosides with high purity and high yield. Based on this, the present invention was completed.

[0024] The method and novel intermediate of the present invention In order to prepare synthetic pseudouracil nucleosides with high purity and high yield, the inventors have creatively developed a method for preparing high-purity pseudouracil nucleosides through triphenyl protection.

[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) Compound 44 reacts to give compound 64; 2) Compound 64 reacts to yield compound 74; 3) Compound 74 reacts to give compound 84; and 4) Hydrolysis of compound 84 yields 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 44 is condensed with 2,4-di-tert-butoxy-5-bromopyrimidine (Formula 5) to obtain compound 64; in step 2), compound 64 is reduced with lithium tri-sec-butylborohydride (L-Selectride) under zinc chloride chelation to obtain compound 74; in step 3), compound 74 is cyclized with Mitsunobu reaction to obtain compound 84; in step 4), compound 84 is hydrolyzed in the presence of acetic acid to obtain pseudouracil nucleoside (Formula 1).

[0027] The inventors have discovered that, in the process of synthesizing pseudouridine (Formula 1) using the method of this invention, the content of isomer impurities is significantly reduced at each step. In a specific embodiment, in step 2) above, compound 64 is reduced with lithium trisec-butylborohydride (L-Selectride) under zinc chloride chelation to obtain compound 74 and isomer impurity 74-α. ; The content of the isomer impurity 74-α is <4%.

[0028] In step 3) above, compound 74 undergoes Mitsunobu cyclization to yield compound 84 and isomer impurity 84-C. ; The content of the isomer impurity 84-C is <1.0%.

[0029] The inventors further optimized various process parameters of the above-described method for synthesizing pseudouridine (Formula 1). In a specific embodiment, in step 3), the reagents for the Mitsunobu reaction are tributylphosphine (TBP) and N,N,N',N'-tetramethylazodicarbonamide (TMAD). The solvent for the Mitsunobu reaction is tetrahydrofuran (THF), and the reaction temperature is 25-35°C, preferably 30°C. In step 2), the reduction reaction temperature is -80~0°C; preferably -10~0°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 a specific embodiment, the present invention provides the compound shown in Formula 64. ; The compound shown in Formula 74, ;and The compound shown in Formula 84, .

[0031] The main advantages of this invention include: 1. In this invention, the stability of the compound is significantly increased after the primary hydroxyl group is protected with triphenylmethyl groups. The compound is less likely to undergo detriphenylmethyl protection in subsequent condensation, reduction, and etherification reactions, resulting in a high yield.

[0032] 2. When the compound of formula 64 of the present invention is reduced with lithium trisec-butylborohydride (L-Selectride) under zinc chloride chelation to obtain compound of formula 74, the amount of α-configuration isomer impurity 74-α in the reaction solution is small (<4%).

[0033] 3. The yield of compound 74 of this invention is high when it is prepared into compound 84 by the Mitsunobu reaction. Because the triphenylmethyl protecting group on the primary hydroxyl group of compound 74 is large, the selectivity between the two hydroxyl groups is good. Only product 84 is generated during the Mitsunobu reaction, and the positional isomer impurity 84-C is hardly generated (<1.0%).

[0034] 4. The method for preparing pseudouridine of the present invention has a high yield.

[0035] 5. This invention uses single-crystal X-ray diffraction patterns to demonstrate the chiral configuration of 84 and impurity 74-α.

[0036] 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.

[0037] 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. When the reaction solution was cooled to 5°C, concentrated sulfuric acid (3.98 g, 40.58 mmol, 0.2 eq) was added dropwise, completing the addition in 0.5 h. The reaction solution was then stirred at 5°C for 1 h, and then allowed to react at room temperature for another 2 h. Sodium bicarbonate was slowly added to the reaction solution to adjust the pH to 7. The solution was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, yielding the 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%.

[0038] Compound 41 1 H 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 44) Under N2 protection, 41 (0.98 g, 5.21 mmol, 1 eq), triphenylchloromethane (2.09 g, 7.50 mmol, 1.4 eq), and dichloromethane (5 ml) were added to a 25 ml three-necked flask. The reaction solution was cooled to 0 °C. Triethylamine (0.87 g, 8.59 mmol, 1.6 eq) was added dropwise over 30 min. The reaction was carried out at 30 °C for 13 h. The reaction solution was filtered to remove triethylamine hydrochloride. The pH of the filtrate was adjusted to 7.0 with hydrochloric acid, and the organic phase was extracted. The organic phase was washed with saturated sodium chloride, and the aqueous phase was extracted with dichloromethane (5 ml × 2 times). The organic phases were combined and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 10:1) to obtain 2.15 g of 44, with a yield of 96.0%.

[0039] MS (ESI) of compound 44 + ):C 27 H 26 O5, m / z 453.17 [M+Na] + .

[0040] Compound 44 1 H NMR: (400 MHz, DMSO-d6) δ 7.43 – 7.25 (m, 15H), 4.93(d, 1H), 4.72 (t, 1H), 4.66 (d, 1H), 3.44 (dd, 1H), 3.20 (dd, 1H), 1.35 (d,6H). Example 3 (Preparation of Formula 64) Under N2 protection, 5 (2.54 g, 8.38 mmol, 1.2 eq) tetrahydrofuran (30 ml) was added to a 250 ml three-necked flask and stirred at room temperature until dissolved and clear. The reaction solution was cooled to below -78 °C, and a 2.5 M n-butyllithium (4.5 ml, 11.25 mmol, 1.6 eq) reddish-brown solution was added dropwise over 0.5 h. The reaction solution was stirred at -78 °C for 30 min. 44 (2.99 g, 6.95 mmol, 1.0 eq) was added dropwise, and the reaction was continued for 5 h after the addition was complete. The reaction solution was extracted with saturated brine (15 ml) at -78 °C, and the organic phase was extracted twice with ethyl acetate (10 ml × 2). 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 the crude product. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 50:1 → 10:1) to obtain 4.10 g 64, with a yield of 90.1%.

[0041] MS (APCI) of compound 64 + ):C 39 H 46 N₂O₇, m / z 655.34 [M+H] + .

[0042] Compound 64 1 H NMR: (400 MHz, DMSO-d6) δ 8.23 ​​(s, 1H), 7.42 – 7.30 (m,15H), 6.27 (s, 1H), 4.76 (d, 1H), 4.71 (d, 1H), 4.19 (m, 1H), 3.25 (d, 2H),1.55 (s, 18H), 1.22 (d, 6H). Example 4 (Preparation of Formula 74) Under N2 protection, 64 (1.00 g, 1.53 mmol, 1.0 eq) and dichloromethane (50 ml) were added to a 100 ml three-necked flask and stirred at room temperature (10 °C) until dissolved and clear. The reaction solution was cooled to 0 °C, and a colorless and transparent 1 M zinc chloride THF solution (1.8 ml, 1.80 mmol, 1.2 eq) was added dropwise over 30 min. The reaction solution was stirred at 0 °C for 30 min, and a 1 M L-selectride THF solution (5.4 ml, 5.40 mmol, 3.5 eq) was added dropwise over 30 min. The reaction was then stirred for another 5 h. Anhydrous ethanol was added to the reaction solution and stirred for 30 min. Then, 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 twice with dichloromethane (10 ml × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 20:1 → 15:1) to obtain 0.91 g 74, with a yield of 91.0%, HPLC purity of 99.02%, and impurity 74-α of 0.75%.

[0043] MS (APCI) of compound 74 + ):C 39 H 48 N₂O₇, m / z 657.36 [M+H] + .

[0044] Compound 74 1H NMR: (400 MHz, DMSO-d6) δ 8.19 (s, 1H), 7.51 – 7.45 (m,6H), 7.39 – 7.22 (m, 9H), 5.47 (d, 1H), 5.07 (d, 1H), 4.87 (d, 1H), 4.33 –4.22 (m, 1H), 4.17 (dd, 1H), 4.07 (d, 1H), 3.15 – 3.08 (m, 2H), 1.54 (d,18H), 1.41 (s, 3H), 1.19 (s, 3H). HPLC detection method: Vanquish Core high performance liquid chromatograph (Thermo Fisher); column: Shimadzu ShimNex HE Sil 5um; detector: DAD (detection wavelength: 210 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.

[0045] The retention time for 64 was 10.02 min. The retention time for 74 was 12.12 min. The retention time of 74-α was 11.40 min.

[0046] Example 5 (Preparation of Formula 84) Under N2 protection, N,N,N',N'-tetramethylazodicarbonamide (0.540 g, 3.14 mmol, 2.1 eq) and tetrahydrofuran (60 ml) were added to a 100 ml three-necked flask and stirred until dissolved and clear. Tributylphosphine (0.621 g, 3.07 mmol, 2.0 eq) was added and stirred for 30 min. 74 (0.998 g, 1.52 mmol, 1.0 eq) was added, and the reaction mixture was stirred at 30 °C for 5 h. The solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 20:1) to obtain 0.93 g of 84, with a yield of 95.9% and an HPLC purity of 99.50%. Impurities 84-α and 84-c were not detected (<0.05%).

[0047] MS (APCI) of compound 84 + ):C 39 H 46 N₂O₆, m / z 639.34 [M+H] +.

[0048] Compound 84 1 H NMR: (400 MHz, Acetone-d6) δ 8.30 (d, 1H), 7.60 – 7.49(m, 6H), 7.42 – 7.24 (m, 9H), 5.00 – 4.93 (m, 1H), 4.73 (dd, 1H), 4.64 (dd,1H), 4.24 – 4.15 (m, 1H), 3.42 – 3.27 (m, 2H), 1.62 (d, 18H), 1.55 (s, 3H), 1.32 (s, 3H). The retention time for 74 was 12.12 min. The retention time for 84 was 8.77 min. The retention time of 84-α was 9.46 min.

[0049] The structure of the new compound shown in Equation 84 was determined by single-crystal X-ray diffraction pattern as follows: .

[0050] Example 6 (Preparation of Formula 1) Add 1.70 g (2.66 mmol, 1.0 eq) of 84, 1.0 ml of water, and 2.4 ml of acetic acid to a 25 mL single-necked flask. Heat the reaction mixture to 50 °C and stir for 3 h. Add a small amount of sodium hydroxide solid to adjust the pH to 6-7. Concentrate under reduced pressure to remove the solvent, yielding a crude yellow oily product. Purify the crude product by column chromatography (eluent: dichloromethane:methanol = 20:1 → 5:1) to obtain 0.62 g of off-white solid 1, yield 95.4%, HPLC purity 99.82%.

[0051] MS (ESI) of compound 1 - ): C9H 12 N₂O₆, m / z 243.05 [MH] - .

[0052] Compound of Formula 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 74-α reference standard) Under N2 protection, 64 (1.10 g, 1.68 mmol, 1.0 eq) and tetrahydrofuran (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 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. 10 mL of saturated sodium chloride solution was added to extract the organic phase, and the aqueous phase was extracted with ethyl acetate (10 mL × 2). 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 the crude product. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 50:1 → 15:1) to give 0.97 g 74-α, with a yield of 88.1%.

[0053] Compound 74-α 1 H NMR: (400 MHz, DMSO-d6) δ 8.19 (s, 1H), 7.48 – 7.41 (m, 6H), 7.35 – 7.29 (m, 6H), 7.27 – 7.21 (m, 3H), 5.81 (d, 1H), 5.35 (d,1H), 4.78 (dd, 1H), 4.40 (dd, 1H), 4.24 (dd, 1H), 4.06 – 3.96 (m, 1H), 3.19 (dd, 1H), 3.12 (dd, 1H), 1.54 (d, 18H), 1.12 (d, 6H). The retention time of 74-α was 11.40 min.

[0054] The structure of Equation 74-α was determined by single-crystal X-ray diffraction pattern as follows: .

[0055] Example 8 (Preparation of impurity formula 84-α reference standard) Under N2 protection, N,N,N',N'-tetramethylazodicarbonamide (0.16 g, 0.93 mmol, 2.0 eq) and tetrahydrofuran (20 ml) were added to a 100 ml three-necked flask and stirred until dissolved. Tributylphosphine (0.19 g, 0.94 mmol, 2.0 eq) was added and stirred for 30 min. Then 74 (0.30 g, 0.46 mmol, 1.0 eq) was added. The reaction mixture was stirred at 30 °C for 5 h. The solvent was removed by concentration under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 50:1 → 20:1) to give 0.26 g of 84-α, with a yield of 88.5%.

[0056] Compound 84-α 1 H NMR: (400 MHz, DMSO-d6) δ 8.16 (d, 1H), 7.44 – 7.31 (m, 12H), 7.31 – 7.23 (m, 3H), 5.25 (d, 1H), 4.95 (dd, 1H), 4.81 (d, 1H), 4.24 (t, 1H), 3.25 (dd, 1H), 3.18 (dd, 1H), 1.54 (d, 18H), 1.24 (d, 6H). The retention time of 84-α was 9.46 min.

[0057] Example 9 Under N2 protection, take 64 (three portions of 0.100 g, 0.15 mmol, and 1.0 eq) and divide them into three 10 ml reaction tubes, labeled 1-3. Add DCM to each of reaction tubes 1, 2, and 3, and stir at room temperature (10°C) to dissolve into a colorless and clear solution.

[0058] Reaction solution No. 1 was cooled to -78°C. Reaction solution No. 2 was cooled to -40°C. Reaction solution No. 3 was cooled to 0°C. Add 1 M ZnCl₂ tetrahydrofuran solution (0.18 ml, 0.18 mmol, 1.2 eq) dropwise to reaction solutions 1-3, completing the addition over 30 min, and stir for 30 min. Then add 1 M L-selectride tetrahydrofuran solution (0.54 ml, 0.54 mmol, 3.6 eq) dropwise, completing the addition over 30 min. After the additions, 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. Monitor the peak area percentages of 64, 74, and 74-α in the reaction solutions using HPLC. The preferred reaction temperature is 0℃, which results in a high yield of product 74 and fewer impurities 74-α.

[0059] Example 10 Take four 100 ml three-necked flasks and, under N2 protection, add the following to flasks 1-4 respectively: Diisopropyl azodicarbonate (DIAD) (0.018 g, 0.089 mmol, 1.9 eq) was added to reaction solution 1. Diethyl azodicarbonate (DEAD) (0.016 g, 0.092 mmol, 2.0 eq) was added to reaction solution 2. Add N,N,N',N'-tetramethylazodicarbonamide (TMAD) (0.017 g, 0.099 mmol, 2.2 eq) to reaction solution No. 3. Add azodicarbonyl dipiperidine (ADDP) (0.023 g, 0.091 mmol, 2.0 eq) to reaction solution No. 4. Add tetrahydrofuran (2 ml) to reaction solutions 1-4 first, then add dropwise the following: Triphenylphosphine (TPP) (0.024 g, 0.092 mmol, 2.0 eq) was added to reaction solution 1. Triphenylphosphine (TPP) (0.024 g, 0.092 mmol, 2.0 eq) was added to reaction solution No. 2. Tributylphosphine (TBP) (0.020 g, 0.099 mmol, 2.2 eq) was added to reaction solution No. 3. Tributylphosphine (TBP) (0.020 g, 0.099 mmol, 2.2 eq) was added to reaction solution No. 4. The mixture was first stirred at 30°C for 30 min, then 74 (0.030 g, 0.046 mmol, 1.0 eq) was added to each flask, and the reaction was allowed to proceed for 5 h. Reaction solutions from flasks 1-4 were collected and analyzed by TLC, as shown in the table below: The preferred reaction is tributylphosphine (TBP) + N,N,N',N'-tetramethylazodicarbonamide (TMAD), which produces product 84.

[0060] Example 11 Take four 25 ml three-necked flasks. Under N2 protection, add 0.052 g, 0.30 mmol, and 2.0 eq of TMAD yellow powder to flasks 1-4 respectively. Add the following to flasks 1-4 respectively: Add dichloromethane (4.8 ml) to reaction flask No. 1; Add acetonitrile (4.0 ml) to reaction flask No. 2; Add tetrahydrofuran (6.2 ml) to reaction flask No. 3; Add toluene (8.0 ml) to reaction flask No. 4.

[0061] Reaction solutions 1-4 were dissolved and clarified by stirring at 30°C. A colorless, transparent solution of tributylphosphine (0.060 g, 0.30 mmol, 2.0 eq) was then added dropwise to each solution, and the mixture was stirred at 30°C for 5 h. The peak area percentages of 74, 84, and 84-C in the reaction solution were monitored by HPLC. Tetrahydrofuran is preferred as the reaction solvent, and the amount of impurity 84-C is relatively small.

[0062] Example 12 Take three 25 ml three-necked flasks, and under N2 protection, add 0.052 g (0.30 mmol, 2.0 eq) of TMAD yellow powder to flasks 1-3 respectively. Add 6.2 ml of tetrahydrofuran, stir to dissolve and clarify. Reaction solution No. 1 was cooled to 0°C. The temperature of reaction solution No. 2 was raised to 30°C. The temperature of reaction solution No. 3 was raised to 60℃. Add colorless and transparent tributylphosphine solution dropwise to each of the solutions (0.060 g, 0.30 mmol, 2.0 eq for solutions 1-3). Maintain each solution at its designated temperature, then add 74 (0.10 g, 0.15 mmol, 1.0 eq) to each solution and incubate for 8 h. Monitor the peak area percentages of 74, 84, and 84-C in the reaction solution using HPLC. 30°C is preferred as the reaction solvent, and the amount of impurity 84-C is relatively small.

[0063] 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 44 reacts to give compound 64; 2) Compound 64 reacts to yield compound 74; 3) Compound 74 reacts to give compound 84; and 4) Hydrolysis of compound 84 yields 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 44 is condensed with 2,4-di-tert-butoxy-5-bromopyrimidine (formula 5) to give compound 64; In step 2), compound 64 is reduced with lithium trisec-butylborohydride (L-Selectride) under zinc chloride chelation to obtain compound 74; In step 3), compound 74 is cyclized by the Mitsunobu reaction to obtain compound 84; In step 4), compound 84 is hydrolyzed in the presence of acetic acid to obtain pseudouracil nucleoside (formula 1).

3. The preparation method according to claim 2, characterized in that, In step 2), compound 64 is reduced with lithium trisec-butylborohydride (L-Selectride) under zinc chloride chelation to give compound 74 and isomer impurity 74-α. ； Preferably, the content of the isomer impurity 74-α is <4%.

4. The preparation method according to claim 2, characterized in that, In step 3), compound 74 undergoes Mitsunobu cyclization to yield compound 84 and isomer impurity 84-C. ； Preferably, the content of the isomer impurity 84-C is <1.0%.

5. The preparation method according to 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 preparation method according to claim 2, characterized in that, In step 3), the solvent for the Mitsunobu reaction is tetrahydrofuran (THF), and the reaction temperature is 25-35°C, preferably 30°C.

7. The preparation method according to claim 2, characterized in that, In step 2), the reduction reaction temperature is -80~0℃; preferably -10~0℃.

8. The compound shown in Formula 64, 。 9. The compound shown in Formula 74 。 10. The compound shown in Formula 84 。 11. Preparation of compounds shown in Formula 64, Formula 74 or 84 and their use in pseudouridines shown in Formula 1. 1。

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