A pseudouridine intermediate and its preparation method and application
A simplified synthesis method using Red-Al and imidazole/TBSCl reactions with flash chromatography and silica gel purification addresses the inefficiencies of existing pseudouridine synthesis, enabling cost-effective and scalable production.
Patent Information
- Application Number
- CN202211594064.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-13
- Publication Date
- 2024-12-31
- Estimated Expiration
- 2042-12-13
AI Technical Summary
Existing pseudouridine intermediate synthesis methods are cumbersome, costly, and inefficient, limiting large-scale production due to complex operations and high costs of reagents like L-selectride.
A simplified synthesis method involving substitution reactions with Red-Al and subsequent reactions with imidazole and TBSCl, followed by purification steps such as flash chromatography and silica gel column purification, using relatively inexpensive reagents and solvents.
The method achieves a few-step, cost-effective, and efficient production of pseudouridine intermediates suitable for large-scale production with high yield and purity.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of drug synthesis, and specifically relates to a pseudouridine intermediate and a preparation method and application thereof. Background Art
[0002] Pseudouridine (1), as a metabolite of RNA, is excreted only from the kidneys. Therefore, it can be used clinically to monitor the occurrence and development of tumors and the diagnosis and treatment of kidney disease. As the study of pseudouridine deepens, researchers have also found that the level of pseudouridine in the urine of patients with gout and psoriasis is significantly increased.
[0003] Moreover, pseudouridine is an important part of the mRNA COVID-19 vaccine. It can protect synthetic mRNA from attacks by the immune system and improve molecular stability and protein production. That is why researchers added pseudouridine and its analogs to mRNA instead of uridine to solve the problem that mRNA drugs are easily recognized and cleared by the immune system, resulting in immune side effects.
[0004] One of the key intermediates in the synthesis of pseudouridine is (R)-2-((tert-butyldimethylsilyl)oxy)-1-((4S,5R)-5-((R)-(2,4-di-tert-butylpyrimidin-5-yl)(hydroxy)methyl)-2,2-dimethyl-1,3-dioxolane-4-ethyl)-1-ol (2):
[0005]
[0006] Compound (2) can generally be synthesized by the following steps, and pseudouridine (1) can be obtained through two steps of reaction:
[0007]
[0008] Step 1) shows that compound (3) is added with a reducing agent L-selectride and an ether solution of zinc chloride at -78°C. After a reaction time of 10 hours, the raw material reaction is complete. The subsequent treatment process requires the addition of EtOH, H2O, NaOH (6 mol / L), and 30% H2O2 in sequence to achieve the purpose of quenching the reaction. The operation is complicated, and the product is prone to contain impurities and affect subsequent reactions. In addition, L-selectride is expensive, which limits large-scale production.
[0009] The Chinese patent application document with publication number CN114702481A discloses a new synthesis method of pseudouridine, comprising the following steps: step 1) using D-ribose as a reaction initiator, dripping acetyl bromide in an organic solvent to obtain intermediate I; step 2) using uracil as a reaction initiator, using a strong Lewis base in an organic solvent to eliminate active hydrogen, and adding (Boc)2O in batches to obtain the corresponding intermediate II; step 3) compound I and compound II in an organic solvent, using a strong Lewis base, undergo condensation reaction to obtain intermediate III. Then trifluoroacetic acid is used to remove the protecting group to obtain intermediate IV, and then acetic anhydride is added, and compound V is obtained by chiral separation by lipase, and finally the protecting group is removed under alkaline conditions to obtain the final product VI, i.e., pseudouridine product. This method uses mild and safe chemical reagents and enzyme chiral separation, which shortens the reaction steps and reduces the difficulty of the reaction, thereby realizing the bottleneck of green synthesis technology. However, this method still has the problems of cumbersome operation steps, high cost, and low efficiency, and needs to be further improved. Summary of the invention
[0010] The technical problem to be solved by the present invention is how to solve the problems of complicated preparation process, cumbersome operation, high cost and low efficiency of the existing pseudouridine intermediate.
[0011] The present invention solves the above technical problems through the following technical means:
[0012] A method for preparing a pseudouridine intermediate comprises the following steps:
[0013] (1) Compound 3 is dissolved in an organic solvent, and Red-Al is added in an ice bath to carry out a substitution reaction; after the reaction is completed, a diluent is added to dilute, and alkaline water is added in an ice bath to quench the reaction, and the mixture is filtered and washed with a washing agent, and the filtrate is vacuum concentrated and purified by flash chromatography to obtain Compound 5; the structural formula of Compound 3 is The structural formula of the compound 5 is
[0014] (2) Compound 5 obtained in step (1) is dissolved in an organic solvent, and imidazole and TBSCl are added, stirred, and reacted. After the reaction is completed, a diluent is added to dilute the mixture, and the mixture is extracted with an extraction solvent. The organic phases are then combined, concentrated, and purified by a silica gel column to obtain a compound having the structural formula: Compound 2, a pseudouridine intermediate.
[0015] The technical route is as follows:
[0016]
[0017] Beneficial effects: The preparation method of the present invention has few steps, simple operation, relatively cheap raw materials and reagents, low cost, short reaction time and high efficiency for preparing pseudouridine intermediates.
[0018] Preferably, in step (1), the molar ratio of compound 3 to Red-Al is 1:1.5-2.5.
[0019] Preferably, the organic solvent in step (1) is selected from a mixture of one or more of dichloromethane, THF, DMF and methanol.
[0020] Preferably, the diluting solvent in step (1) is selected from a mixture of one or more of dichloromethane, THF, DMF and methanol.
[0021] Preferably, in step (1), the alkali is selected from sodium hydroxide and potassium hydroxide.
[0022] Preferably, the alkaline water in step (1) is one of 15% NaOH (aq) and 10% KOH (aq) by mass fraction.
[0023] Preferably, the washing reagent in step (1) is selected from one of dichloromethane, THF, DMF and methanol.
[0024] Preferably, the temperature of the substitution reaction in step (1) is 0-5°C and the time is 1-2h.
[0025] Preferably, in step (2), the molar ratio of compound 5, imidazole and TBSCl is 1:2.5-3.5:1.0-1.2.
[0026] Preferably, the organic solvent in step (2) is selected from a mixture of one or more of THF, DMF, DMAc and methyl tert-butyl ether.
[0027] Preferably, the dilution solvent in step (2) is selected from water and ethanol.
[0028] Preferably, the extraction solvent in step (2) is selected from one of ethyl acetate, dichloromethane and methyl tert-butyl ether.
[0029] Preferably, the reaction temperature in step (2) is 20-25°C.
[0030] The present invention also provides a pseudouridine intermediate prepared by the method.
[0031] The present invention also provides an application of the pseudouridine intermediate prepared by the method in preparing pseudouridine.
[0032] The advantages of the present invention are:
[0033] (1) The preparation method of the present invention is used to prepare pseudouridine intermediates, which has few steps, simple operation, relatively cheap raw materials and reagents, low cost, short reaction time and high efficiency.
[0034] (2) The post-processing operation of the method of the present invention is simple.
[0035] (3) The method of the present invention has a short reaction time and is easy to industrialize and can be used for large-scale production of pseudouridine. DETAILED DESCRIPTION
[0036] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0037] Embodiment 1:
[0038] The technical route of this embodiment is:
[0039]
[0040] A method for preparing a pseudouridine intermediate comprises the following steps:
[0041] (1) Compound 3 (1.0 g, 1.0 eq) was dissolved in 5 mL CH2Cl2 (organic solvent), replaced with N2 three times, and Red-Al (70 wt.%, 2.0 eq) toluene solution (1.1 g) was added in an ice bath to carry out a substitution reaction at a temperature of 3°C for 1.5 h. After the reaction was completed, 20 mL of THF (diluent) was added for dilution, cooled to 0°C in an ice bath, and 15% NaOH aqueous solution (0.05 mL) was added, and stirred at 0-5°C for 20-30 minutes to quench the reaction. The reaction mixture was filtered through diatomaceous earth, then washed with 30 mL of THF (washing agent), and the filtrate was concentrated in vacuo and purified by flash chromatography to obtain Compound 5. The structural formula of Compound 3 is The structural formula of compound 5 is
[0042] (2) Compound 5 (200 mg, 1.0 eq) obtained in step (1) was dissolved in 5 mL of DMF (organic solvent), and imidazole (98 mg, 3.0 eq) and TBSCl (80 mg, 1.1 eq) were added. The mixture was stirred at 25° C. for 2 h to allow for sufficient reaction (TLC monitoring showed that the raw material disappeared and new spots were generated). After the reaction was completed, 5 mL of water (diluent) was added for dilution, and the product was extracted with ethyl acetate (10 mL) (10 mL of ethyl acetate solution was used each time, for a total of 3 extractions), followed by washing with water (5 mL) (5 mL of water was used each time, for a total of 3 washings). The organic phases were combined, concentrated at 35-45° C., and purified by silica gel column to obtain a product having the structural formula: Compound 2, a pseudouridine intermediate (208 mg, yield 82%).
[0043] The yield is calculated as follows: 208 (product weight) × 414.50 (raw material molecular weight) ÷ 526.75 (product molecular weight) ÷ 200 (raw material weight) × 100% = 82%.
[0044] The pseudouridine intermediate prepared in this example was subjected to high-resolution mass spectrometry HRMS detection and analysis: the M+H+ molecular formula was C26H49N2O7Si+, the calculated value was 529.3304, and the measured value was 529.3315.
[0045] Embodiment 2:
[0046] The technical route of this embodiment is the same as that of embodiment 1.
[0047] A method for preparing a pseudouridine intermediate comprises the following steps:
[0048] (1) Compound 3 (1.0 g, 1.0 eq) was dissolved in 5 mL CH2Cl2 (organic solvent), replaced with N2 three times, and Red-Al (70 wt.%, 1.5 eq) toluene solution (1.65 g) was added in an ice bath to carry out a substitution reaction at 0°C for 2 h. After the reaction, 25 mL of THF (diluent) was added for dilution, cooled to 0°C in an ice bath, and 10% KOH aqueous solution (0.065 mL) was added. The reaction mixture was stirred at 0-5°C for 20-30 minutes to quench the reaction. The reaction mixture was filtered through diatomaceous earth, then washed with 30 mL of methanol (washing agent), and the filtrate was concentrated in vacuo and purified by flash chromatography to obtain Compound 5. The structural formula of Compound 3 is The structural formula of compound 5 is
[0049] (2) Compound 5 (200 mg, 1.0 eq) obtained in step (1) was dissolved in 5 mL of DMF (organic solvent), and imidazole (81.7 mg, 2.5 eq) and TBSCl (72.7 mg, 1.0 eq) were added, and stirred at 22°C for 2 h to allow for sufficient reaction (TLC monitoring showed that the raw material disappeared and new spots were generated). After the reaction was completed, 5 mL of water (diluent) was added for dilution, and the product was extracted with methyl tert-butyl ether (10 mL) (each time with 10 mL of ethyl acetate solution, for a total of 3 extractions), followed by washing with water (5 mL) (each time with 5 mL of water, for a total of 3 washings), the organic phases were combined, the organic phases were concentrated at 35-45°C, and purified by silica gel column to obtain a product having the structural formula: Compound 2, a pseudouridine intermediate (190 mg, yield 75%).
[0050] The yield calculation process: 190×414.50÷526.75÷200×100%=75%.
[0051] The pseudouridine intermediate prepared in this example was subjected to high resolution mass spectrometry HRMS detection and analysis, and the analysis data were consistent with those in Example 1.
[0052] Embodiment 3:
[0053] The technical route of this embodiment is the same as that of embodiment 1.
[0054] A method for preparing a pseudouridine intermediate comprises the following steps:
[0055] (1) Compound 3 (1.0 g, 1.0 eq) was dissolved in 5 mL CH2Cl2 (organic solvent), replaced with N2 three times, and Red-Al (70 wt.%, 2.5 eq) toluene solution (2.75 g) was added in an ice bath to carry out a substitution reaction at a temperature of 5°C for 1 h. After the reaction was completed, 30 mL of THF (diluent) was added for dilution, cooled to 0°C in an ice bath, and 15% NaOH aqueous solution (0.12 mL) was added, and stirred at 0-5°C for 20-30 minutes to quench the reaction. The reaction mixture was filtered through diatomaceous earth, then washed with 30 mL of THF (washing agent), and the filtrate was concentrated in vacuo and purified by flash chromatography to obtain Compound 5. The structural formula of Compound 3 is The structural formula of compound 5 is
[0056] (2) Compound 5 (200 mg, 1.0 eq) obtained in step (1) was dissolved in 5 mL of DMF (organic solvent), and imidazole (114.3 mg, 3.5 eq) and TBSCl (87.3 mg, 1.2 eq) were added. The mixture was stirred at 20° C. for 2 h to allow for sufficient reaction (TLC monitoring indicated that the raw material disappeared and new spots were generated). After the reaction was completed, 5 mL of water (diluent) was added for dilution, and the product was extracted with dichloromethane (10 mL) (10 mL of ethyl acetate solution was used each time, for a total of 3 extractions), followed by washing with ethanol (5 mL) (5 mL of water was used each time, for a total of 3 washings). The organic phases were combined, concentrated at 35-45° C., and purified by silica gel column to obtain a product having the structural formula: Compound 2, a pseudouridine intermediate (200 mg, yield 79%).
[0057] The yield calculation process: 200×414.50÷526.75÷200×100%=79%.
[0058] The pseudouridine intermediate prepared in this example was subjected to high resolution mass spectrometry HRMS detection and analysis, and the analysis data was consistent with that in Example 1.
[0059] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for preparing a pseudouridine intermediate, characterized in that: The following steps are included (1) Compound 3 is dissolved in an organic solvent, and Red-Al is added in an ice bath to carry out a substitution reaction; after the reaction is completed, a diluent is added to dilute it, and alkaline water is added in an ice bath to quench the reaction, and the mixture is filtered and washed with a washing agent, and the filtrate is vacuum concentrated and purified by flash chromatography to obtain compound 5; the structural formula of compound 3 is ; The structural formula of the compound 5 is ; (2) The compound prepared in step (1) is dissolved in an organic solvent, and imidazole and TBSCl are added, and the mixture is stirred to react. After the reaction is completed, a diluent is added to dilute the mixture, and the mixture is extracted with an extraction solvent. The organic phases are then combined, concentrated, and purified by a silica gel column to obtain a structure of Compound 2, a pseudouridine intermediate.
2. The method for preparing a pseudouridine intermediate according to claim 1, characterized in that: In the step (1), the molar ratio of compound 3 to Red-Al is 1:1.5-2.
5.
3. The method for preparing a pseudouridine intermediate according to claim 1 or 2, characterized in that: The organic solvent in step (1) is selected from a mixture of one or more of dichloromethane, THF, DMF, and methanol; the diluting solvent in step (1) is selected from a mixture of one or more of dichloromethane, THF, DMF, and methanol.
4. The method for preparing a pseudouridine intermediate according to claim 3, characterized in that: In the step (1), the base is selected from sodium hydroxide and potassium hydroxide; and in the step (1), the washing agent is selected from dichloromethane, THF, DMF and methanol.
5. The method for preparing a pseudouridine intermediate according to claim 4, characterized in that: The temperature of the substitution reaction in step (1) is 0-5°C and the time is 1-2h.
6. The method for preparing a pseudouridine intermediate according to claim 1, characterized in that: The organic solvent in step (2) is selected from a mixture of one or more of THF, DMF, DMAc, and methyl tert-butyl ether; the dilution solvent in step (2) is selected from one of water and ethanol; the extraction solvent in step (2) is selected from one of ethyl acetate, dichloromethane, and methyl tert-butyl ether.
7. The method for preparing a pseudouridine intermediate according to claim 1, characterized in that: In the step (2), the molar ratio of compound 5, imidazole and TBSCl is 1:2.5-3.5:1.0-1.
2.
8. The method for preparing a pseudouridine intermediate according to claim 1, characterized in that: The reaction temperature in step (2) is 20-25°C.
9. The method for preparing a pseudouridine intermediate according to claim 7, characterized in that: In the step (2), the molar ratio of compound 5, imidazole and TBSCl is 1:3:1.
1.
10. The method for preparing a pseudouridine intermediate according to claim 8, characterized in that: The reaction temperature in step (2) is 25°C.