A method for purifying 2'-fluoro-2'-deoxyadenosine
The purification of 2'-fluoro-2'-deoxyadenosine by chemical derivatization and pulping method solves the problems of complex purification methods and low purity in the existing technology, and realizes the industrial production of high-purity 2'-fluoro-2'-deoxyadenosine.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGSU SYNTHGENE BIOTECHNOLOGY CO LTD
- Filing Date
- 2023-10-25
- Publication Date
- 2026-05-12
AI Technical Summary
The existing purification methods for 2'-fluoro-2'-deoxyadenosine are complex and produce low purity, which cannot meet the needs of industrial production.
A chemical derivatization method was used to remove adenine impurities through acetylation and ammonolysis, and then high-purity 2'-fluoro-2'-deoxyadenosine was obtained by pulping, avoiding column chromatography.
A simple and easy purification method was developed to obtain high-purity 2'-fluoro-2'-deoxyadenosine, which is suitable for industrial-scale production.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological drugs, in particular to a purification method of 2'-fluoro-2'-deoxyadenosine. BACKGROUND
[0002] Small nucleic acid structures in natural state are easily degraded by nucleases in vivo. In order to ensure the drugability of nucleic acid drugs and improve their anti-enzymatic ability, etc., nucleosides need to be chemically modified at multiple sites. 2'-fluorine modification has better nuclease resistance than 2'-OH, improves the stability of the drug in plasma, increases the half-life in tissues, and can prolong the drug efficacy time. In the prior art, 2'-fluoro-2'-deoxyadenosine can be prepared by chemical synthesis method, but generally needs redundant steps of protection and deprotection, and needs to control the region and stereoselectivity of glycosylation reaction. In contrast, enzyme synthesis of 2'-fluoro-2'-deoxyadenosine has mild conditions and is environmentally friendly, which is an attractive alternative approach.
[0003] In the prior art, a method for catalyzing 2'-fluoro-2'-deoxyuridine and adenine to perform transglycosylation reaction by nucleoside phosphorylase to generate 2'-fluoro-2'-deoxyadenosine is disclosed, and excess adenine remains in the product; because the solubility of adenine and 2'-fluoro-2'-deoxyadenosine is similar, it is difficult to wash off with water, and high-purity 2'-fluoro-2'-deoxyadenosine cannot be obtained. In order to obtain high-purity 2'-fluoro-2'-deoxyadenosine, purification is carried out by column chromatography in the prior art, so as to improve the purity.
[0004] Although the above method can purify 2'-fluoro-2'-deoxyadenosine, the process is complex and the purity is relatively low, which cannot meet the needs of industrial production.
[0005] Therefore, it is urgent to develop a purification method with simple synthesis process and high purity. SUMMARY
[0006] In view of the deficiencies in the prior art, the present application provides a purification method of 2'-fluoro-2'-deoxyadenosine.
[0007] The purification method of 2'-fluoro-2'-deoxyadenosine provided by the present application adopts the following technical scheme:
[0008] A purification method of 2'-fluoro-2'-deoxyadenosine, the steps of purification are as follows:
[0009]
[0010]
[0011] Preferably, the steps of purification are specifically as follows:
[0012] (1) The crude 2'-fluoro-2'-deoxyadenosine containing adenine shown in Formula I was dissolved in a solvent and reacted with an acetylation reagent in the presence of a base. After purification, diacetylated 2'-fluoro-2'-deoxyadenosine shown in Formula II was obtained.
[0013] (2) The diacetylated 2'-fluoro-2'-deoxyadenosine of Formula II was reacted with ammonia to obtain the compound of Formula III. The compound of Formula III was then pulped to obtain pure 2'-fluoro-2'-deoxyadenosine.
[0014] Preferably, the base in step (1) is at least one of triethylamine, N,N-diisopropylethylamine, and pyridine.
[0015] Preferably, the acetylation reagent in step (1) is one or both of acetic anhydride and acetyl chloride.
[0016] Preferably, in step (2), the ammonia is at least one of ammonia water, a methanol solution of ammonia, or an ethanol solution of ammonia.
[0017] Preferably, the solvent used for pulping in step (2) is one or both of ethanol and isopropanol.
[0018] Preferably, in step (1), the molar ratio of 2'-fluoro-2'-deoxyadenosine content, alkali, and acetylation reagent in the crude 2'-fluoro-2'-deoxyadenosine containing adenine is 1:(2-4):(1.8-3).
[0019] Preferably, the solvent used in step (1) is N,N-dimethylformamide (DMF), and the mass-volume ratio of 2'-fluoro-2'-deoxyadenosine in the crude product containing adenine to DMF is 1:(3-6).
[0020] Preferably, the reaction temperature in step (1) is 10-30℃ and the reaction time is 3-8h.
[0021] Preferably, the mass-volume ratio of 2'-fluoro-2'-deoxyadenosine in the crude 2'-fluoro-2'-deoxyadenosine containing adenine in step (1) to that in ammonia in step (2) is 1:(2-5).
[0022] Preferably, in step (2), the temperature at which diacetylated 2'-fluoro-2'-deoxyadenosine reacts with ammonia is 40-50°C and the reaction time is 12-16 h.
[0023] This application discloses a purification method for 2'-fluoro-2'-deoxyadenosine. First, diacetylated 2'-fluoro-2'-deoxyadenosine is obtained by chemical derivatization. The main impurity, adenine, does not participate in the reaction and can be removed by filtration. Then, the diacetylated 2'-fluoro-2'-deoxyadenosine is subjected to ammonolysis, and pure 2'-fluoro-2'-deoxyadenosine is obtained by hot slurrying.
[0024] In one specific feasible embodiment, a method for purifying 2'-fluoro-2'-deoxyadenosine includes the following steps:
[0025] (1) The crude 2'-fluoro-2'-deoxyadenosine containing adenine, as shown in Formula I, is dispersed in a solvent (DMF) and reacted with a catalyst (4-dimethylaminopyridine) and an acetylation reagent in the presence of a base. After purification, diacetylated 2'-fluoro-2'-deoxyadenosine as shown in Formula II is obtained, thus removing adenine.
[0026]
[0027] (2) The diacetylated 2'-fluoro-2'-deoxyadenosine of Formula II was deacetylated under the action of ammonia to obtain the compound shown in Formula III (crude product). The compound shown in Formula III (crude product) was pulped to obtain the pure 2'-fluoro-2'-deoxyadenosine.
[0028]
[0029] The above technical solution purifies crude 2'-fluoro-2'-deoxyadenosine containing adenine impurities through derivatization, providing a purification method for 2'-fluoro-2'-deoxyadenosine that avoids column chromatography and is easy to scale up.
[0030] Preferably, the base in step (1) is at least one of triethylamine, N,N-diisopropylethylamine, and pyridine.
[0031] Preferably, the acetylation reagent in step (1) is one or both of acetic anhydride and acetyl chloride.
[0032] Preferably, in step (2), the ammonia is at least one of ammonia water, a methanol solution of ammonia, or an ethanol solution of ammonia.
[0033] Preferably, the solvent used for pulping in step (2) is one or both of ethanol and isopropanol.
[0034] Preferably, in step (1), the molar ratio of 2'-fluoro-2'-deoxyadenosine content, alkali, and acetylation reagent in the crude 2'-fluoro-2'-deoxyadenosine containing adenine is 1:(2-4):(1.8-3).
[0035] Preferably, the solvent used in step (1) is DMF, and the mass-volume ratio of the content of 2'-fluoro-2'-deoxyadenosine in the crude product containing adenine to the solvent (DMF) is 1:(3-6).
[0036] Preferably, the mass-volume ratio of 2'-fluoro-2'-deoxyadenosine in the crude 2'-fluoro-2'-deoxyadenosine containing adenine in step (1) to that in ammonia in step (2) is 1:(2-5).
[0037] Preferably, the reaction temperature in step (1) is 10-30℃ and the reaction time is 3-8h.
[0038] Preferably, in step (2), the temperature at which diacetylated 2'-fluoro-2'-deoxyadenosine reacts with ammonia is 40-50°C and the reaction time is 12-16 h.
[0039] In summary, this application has the following beneficial effects:
[0040] This application discloses a purification method for 2'-fluoro-2'-deoxyadenosine, using inexpensive and readily available raw materials, and the purification method is simple and easy to scale up. Detailed Implementation
[0041] The following embodiments further illustrate the content of this application, but should not be construed as limiting the application. Any modifications or substitutions made to the methods, steps, or conditions of this application without departing from the spirit and substance of this application are within the scope of this application.
[0042] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the embodiments are all commercially available.
[0043] Example 1:
[0044] Step 1: Synthesis of Diacetylated 2'-Fluoro-2'-Deoxyadenosine (Compound shown in Formula II) Crude 2'-fluoro-2'-deoxyadenosine containing adenine (content determined to be 100 g of 2'-fluoro-2'-deoxyadenosine) was added to a reaction flask, followed by 500 mL of DMF and stirring. 93.9 g of triethylamine and 4.5 g of 4-dimethylaminopyridine were added, and the mixture was stirred for 10 min. The temperature was maintained at 15 °C, and 83.3 g of acetic anhydride was added dropwise. The reaction was then maintained at 25 °C for 5 h. After the reaction was complete, 1 L of ethyl acetate was added, and the temperature was maintained at 15 °C. 1 L of water was added dropwise to the mixture, and the mixture was stirred for 20 min. The mixture was filtered, and the filter cake was washed with 500 mL of ethyl acetate. The filtered liquid was dried, and the two filtrates were combined. The aqueous phase was extracted once with 500 mL of ethyl acetate. The organic phases were combined and washed twice with 500 mL of 10 wt% sodium chloride solution each time. The organic phase was desolventized under reduced pressure to a volume of 200 mL, and then 500 mL of ethanol was added and evaporated to a volume of 200 mL. This solution was then used directly for the next feeding step.
[0045] Step 2: Synthesis of Compound III and Preparation of Pure 2'-Fluoro-2'-Deoxyadenosine. Under controlled temperature of 20°C, 500 mL of ethanol and 300 mL of concentrated ammonia were added to the yellow suspension obtained in Step 1. The mixture was stirred at 45°C for 15 hours. After the reaction was complete, the solvent was removed until no obvious fractions remained. Then, 400 mL of ethanol was added and distilled once. 500 mL of isopropanol was added to the residue, and the mixture was heated to reflux and pulped for 3 hours. The temperature was then slowly lowered to 20°C, and pulped for another 3 hours. The mixture was filtered to obtain a white solid. The purity of the wet filter cake was measured. The purity was required to be >98.0% and the single impurity <1.0%. If the purity was not met, the pulping operation was repeated until it met the requirements. The wet filter cake with acceptable purity was dried to obtain 85.0 g of white solid, with a recovery rate of 85.0% and a purity of 99.3%.
[0046] Example 2:
[0047] The difference between this embodiment and Example 1 is that the alkali used in the first step is N,N-diisopropylethylamine, and the amount used is 143.9g.
[0048] The preparation yielded 83.0 g of white solid, with a recovery rate of 83.0% and a purity of 99.1%.
[0049] Example 3:
[0050] The difference between this embodiment and Example 1 is that the base used in the first step is pyridine, and the amount used is 117.4g.
[0051] 79.5g of white solid was obtained after preparation, with a recovery rate of 79.5% and a purity of 98.2%.
[0052] Example 4:
[0053] The difference between this embodiment and Example 1 is that the acetylation reagent used in the first step is acetyl chloride, and the amount used is 87.1g.
[0054] The preparation yielded 81.6 g of white solid, with a recovery rate of 81.6% and a purity of 98.3%.
[0055] Example 5:
[0056] The difference between this embodiment and Embodiment 1 is that the ammonia used in the second step is an ethanol solution of ammonia.
[0057] The concentration of the ammonia ethanol solution is 2 mol / L, product number: W420202-0010, manufacturer: Anaiji Chemical.
[0058] The preparation yielded 82.9g of white solid, with a recovery rate of 82.9% and a purity of 99.1%.
[0059] Example 6:
[0060] The difference between this embodiment and Embodiment 1 is that the ammonia used in the second step is a methanol solution of ammonia.
[0061] The methanol solution of ammonia has a concentration of 2 mol / L, product number: W330163-0010, manufacturer: Anaiji Chemical.
[0062] The preparation yielded 83.3g of white solid, with a recovery rate of 83.3% and a purity of 98.8%.
[0063] Example 7:
[0064] The difference between this embodiment and Embodiment 1 is that the pulping solvent used in the second step is ethanol.
[0065] 78.6 g of white solid was obtained after preparation, with a recovery rate of 78.6% and a purity of 99.6%.
[0066] Example 8:
[0067] The difference between this embodiment and Example 1 is that in the first step, the amount of DMF is 300 mL, triethylamine is 150.2 g, 4-dimethylaminopyridine is 2.3 g, and acetic anhydride is 113.6 g.
[0068] The preparation yielded 86.3g of white solid, with a recovery rate of 86.3% and a purity of 99.3%.
[0069] Example 9:
[0070] The difference between this embodiment and Example 1 is that in the first step, the amount of DMF is 600 mL, triethylamine is 75.1 g, 4-dimethylaminopyridine is 3.6 g, and acetic anhydride is 68.2 g.
[0071] The preparation yielded 81.0 g of white solid, with a recovery rate of 81.0% and a purity of 99.1%.
[0072] Example 10:
[0073] The difference between this embodiment and Example 2 is that in the first step, the amount of DMF is 400 mL, 4-dimethylaminopyridine is 3.2 g, and acetic anhydride is 113.6 g.
[0074] The preparation yielded 84.3g of white solid, with a recovery rate of 84.3% and a purity of 98.7%.
[0075] Example 11:
[0076] The difference between this embodiment and Embodiment 1 is that the amount of concentrated ammonia used in the second step is 200 mL.
[0077] 75.4 g of white solid was obtained after preparation, with a recovery rate of 75.4% and a purity of 98.2%.
[0078] Example 12:
[0079] The difference between this embodiment and Embodiment 1 is that the amount of concentrated ammonia used in the second step is 500 mL.
[0080] The preparation yielded 84.8g of white solid, with a recovery rate of 84.8% and a purity of 98.5%.
[0081] Comparative Example: Experimental Procedure and Results Using Conventional Purification Methods
[0082] Crude 2'-fluoro-2'-deoxyadenosine (containing 10g of 2'-fluoro-2'-deoxyadenosine, as determined by content analysis) was purified by column chromatography using 60g of 200-300 mesh silica gel with a column diameter-to-height ratio of 1 / 8. The eluent was dichloromethane and methanol in a volume ratio of 20 / 1 to 10 / 1, and the eluent volume was 15L. 6.43g of white solid was obtained, with a recovery rate of 64.3% and a purity of 96.5%.
[0083] During the experiment, it was found that due to the poor solubility of 2'-fluoro-2'-deoxyadenosine and adenosine impurities, a large amount of eluent was required, and the separation and purification effect was not good. Therefore, the recovery rate and purity of the comparative example were much lower than those of the examples in this application.
[0084] Based on the results of Examples 1-3, 8, 9, and 10, it can be seen that when triethylamine or N,N-diisopropylethylamine is used as the base in the first step, the recovery rate is higher than when pyridine is used. This may be because pyridine has a lower basicity than the former two, resulting in insufficient reactivity and a slightly lower conversion rate. In addition, pyridine has a stronger odor, which requires higher ventilation in the experimental environment. Therefore, triethylamine or N,N-diisopropylethylamine is preferred as the base.
[0085] Based on the results of Examples 1 and 4, it can be seen that the recovery rate is lower when acetyl chloride is used as the acetylation reagent in the first step; this may be because acetyl chloride has higher reactivity than acetic anhydride, making the side reactions more obvious; therefore, acetic anhydride is preferred as the acetylation reagent.
[0086] Based on the results of Examples 1, 5, and 6, it can be seen that when concentrated ammonia solution is used in the second step, the recovery rate is slightly higher than when ethanol solution or methanol solution of ammonia is used. Considering that concentrated ammonia solution is cheaper, more readily available, and easier to store, concentrated ammonia solution is preferred.
[0087] Combining the results of Examples 1 and 7, it can be seen that the recovery rate is higher when isopropanol is used as the pulping solvent in the second step. This is because isopropanol has a lower solubility for the product compared to ethanol, resulting in less loss in the mother liquor and more precipitated product, which is beneficial to improving the yield.
[0088] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.
Claims
1. A method for purifying 2'-fluoro-2'-deoxyadenosine, characterized in that, The purification steps are as follows: ； ； The purification steps are as follows: (1) The crude 2'-fluoro-2'-deoxyadenosine containing adenine shown in Formula I is dissolved in a solvent and reacted with an acetylation reagent in the presence of a base to obtain diacetylated 2'-fluoro-2'-deoxyadenosine shown in Formula II. (2) The diacetylated 2'-fluoro-2'-deoxyadenosine of Formula II is reacted with ammonia to obtain the compound of Formula III. The compound of Formula III is then pulped to obtain pure 2'-fluoro-2'-deoxyadenosine.
2. The purification method for 2'-fluoro-2'-deoxyadenosine according to claim 1, characterized in that: The base mentioned in step (1) is at least one of triethylamine, N,N-diisopropylethylamine, and pyridine.
3. The purification method for 2'-fluoro-2'-deoxyadenosine according to claim 1, characterized in that: The acetylation reagent mentioned in step (1) is one or both of acetic anhydride and acetyl chloride.
4. The purification method for 2'-fluoro-2'-deoxyadenosine according to claim 1, characterized in that: The ammonia mentioned in step (2) is at least one of ammonia water, a methanol solution of ammonia, and an ethanol solution of ammonia.
5. The purification method for 2'-fluoro-2'-deoxyadenosine according to claim 1, characterized in that: The solvent used for pulping in step (2) is one or both of ethanol and isopropanol.
6. The purification method for 2'-fluoro-2'-deoxyadenosine according to claim 1, characterized in that: In step (1), the molar ratio of 2'-fluoro-2'-deoxyadenosine content, alkali, and acetylation reagent in the crude 2'-fluoro-2'-deoxyadenosine containing adenine is 1:(2-4):(1.8-3). The solvent used in step (1) is N,N-dimethylformamide. The mass-volume ratio of 2'-fluoro-2'-deoxyadenosine in the crude product containing adenine to N,N-dimethylformamide is 1:(3-6).
7. The purification method for 2'-fluoro-2'-deoxyadenosine according to claim 1, characterized in that: Step (1) The reaction temperature is 10-30℃ and the reaction time is 3-8h.
8. The purification method for 2'-fluoro-2'-deoxyadenosine according to claim 1, characterized in that: The content of 2'-fluoro-2'-deoxyadenosine in the crude product containing adenine in step (1) is in a mass-volume ratio of 1:(2-5) to that of ammonia in step (2).
9. The purification method for 2'-fluoro-2'-deoxyadenosine according to claim 1, characterized in that: In step (2), the reaction temperature of diacetylated 2'-fluoro-2'-deoxyadenosine with ammonia is 40-50℃ and the reaction time is 12-16h.