A method for purifying 2'-fluoro-2'-deoxyguanosine
By employing a combined process of acetylation-deacetylation-pulping, the problem of low purity of 2'-fluoro-2'-deoxyguanosine in existing technologies has been solved, enabling efficient and low-cost industrial production of high-purity 2'-fluoro-2'-deoxyguanosine.
Patent Information
- Application Number
- CN202610346688.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-03-20
- Publication Date
- 2026-07-03
Smart Images
Figure CN122325519A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to a method for purifying 2'-fluoro-2'-deoxyguanosine. Background Technology
[0002] Unmodified small nucleic acids are easily degraded by nucleases in vivo under physiological conditions. To ensure their drug-like properties, nucleosides typically require multi-site chemical modification to enhance their resistance to nuclease degradation. Among these modifications, 2'-fluoromodification, compared to the unmodified 2'-hydroxyl structure, significantly improves the nucleoside's tolerance to nucleases, thereby increasing drug stability in plasma, prolonging its half-life in tissues, and ultimately achieving sustained drug release. Currently, 2'-fluoro-2'-deoxyguanosine can be prepared via chemical synthesis, but such methods often involve cumbersome protection and deprotection steps and require strict control of the regioselectivity and stereoselectivity of the glycosylation reaction. In contrast, enzymatic synthesis of this compound offers advantages such as mild reaction conditions and environmental friendliness, making it a more attractive alternative.
[0003] Existing literature reports a method for generating 2'-fluoro-2'-deoxy-2,6-diaminopurine nucleoside from a 2,6-diaminopurine substrate with high water solubility and a 2'-fluoro-2'-deoxyuridine substrate under the catalysis of thymidine phosphorylase (TP) and purine nucleoside phosphorylase (PNP), followed by the formation of 2'-fluoro-2'-deoxyguanosine under the catalysis of adenosine deaminase (ADA). However, since excess 2,6-diaminopurine and uracil are usually present in the reaction system, these impurities tend to remain in the product. Furthermore, 2,6-diaminopurine and uracil exhibit similar solubility to the target product, making conventional water washing ineffective in removing them, thus hindering the acquisition of high-purity 2'-fluoro-2'-deoxyguanosine. To improve product purity, existing techniques often employ column chromatography for separation and purification.
[0004] While the aforementioned methods can achieve purification to some extent, their procedures are cumbersome, and the purity of the resulting product remains unsatisfactory, failing to meet the combined requirements of efficiency and quality for industrial production. Therefore, developing a simple, high-purity purification method for 2'-fluoro-2'-deoxyguanosine suitable for large-scale production has become a pressing technical problem in this field. Summary of the Invention
[0005] The purpose of this invention is to solve the problems in the prior art by proposing a purification method for 2'-fluoro-2'-deoxyguanosine.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: A purification method for 2'-fluoro-2'-deoxyguanosine, the purification route of which is as follows: The purification steps are as follows: The crude 2'-fluoro-2'-deoxyguanosine containing 2,6-diaminopurine and uracil impurities was subjected to acetylation to generate diacetylated 2'-fluoro-2'-deoxyguanosine, which was then separated. The diacetylated 2'-fluoro-2'-deoxyguanosine was then subjected to a deacetylation reaction to obtain crude 2'-fluoro-2'-deoxyguanosine. Finally, it was purified by pulping to obtain high-purity 2'-fluoro-2'-deoxyguanosine with a purity of over 99%.
[0007] As an important aspect of this invention, high-purity 2'-fluoro-2'-deoxyguanosine directly prepared by the above-described purification method is also protected. This product is characterized by a chemical purity of not less than 98.6%, preferably not less than 99.0%, more preferably not less than 99.5%, and a single impurity content of less than 1.0%. Due to its extremely high purity, this product is particularly suitable as a key intermediate or active pharmaceutical ingredient in the preparation of small nucleic acid drugs.
[0008] Preferably, the following specific operating steps are included: Step 1: Dissolve the crude 2'-fluoro-2'-deoxyguanosine containing 2,6-diaminopurine and uracil impurities as shown in Formula I in an organic solvent, and acetylate it with an acetylation reagent under alkaline conditions. After separation and purification, diacetylated 2'-fluoro-2'-deoxyguanosine as shown in Formula II is obtained. Step 2: The diacetylated 2'-fluoro-2'-deoxyguanosine shown in Formula II is contacted with an ammonia reagent to carry out a deacetylation reaction, yielding the crude compound shown in Formula III. The crude compound is then purified by pulping to obtain the final high-purity 2'-fluoro-2'-deoxyguanosine product.
[0009] Preferably, the reagent is selected from the following compounds: The base mentioned in step one is selected from at least one of triethylamine, N,N-diisopropylethylamine, and pyridine.
[0010] The acetylation reagent mentioned in step one is selected from acetic anhydride, acetyl chloride, or a combination of both.
[0011] In the crude 2'-fluoro-2'-deoxyguanosine containing 2,6-diaminopurine and uracil impurities described in step one, the effective content of 2'-fluoro-2'-deoxyguanosine, the molar ratio of the base and the acetylation reagent is 1:(3-6):(1.8-3).
[0012] The acetylation reaction in step one is carried out at a temperature of 10-30°C for 3-9 hours.
[0013] The ammonia mentioned in step two is selected from at least one of ammonia water, a methanol solution of ammonia, and an ethanol solution of ammonia.
[0014] In step two, the reaction temperature of diacetylated 2'-fluoro-2'-deoxyguanosine with ammonia is 40-50℃, and the reaction time is 12-16 hours.
[0015] The solvent used for pulping and purification in step two is selected from one or a mixture of ethanol and isopropanol.
[0016] The solvent used in step one is N,N-dimethylformamide.
[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. High product purity and excellent quality: This invention utilizes a combined process of "acetylation-deacetylation-pulping" to effectively remove impurities such as 2,6-diaminopurine and uracil, which are difficult to separate during enzymatic synthesis. This results in a stable yield of 2'-fluoro-2'-deoxyguanosine with a purity exceeding 99%, reaching a maximum of 99.5%. Its purity is significantly higher than that of traditional column chromatography methods (comparative method only 96.8%), and the product quality fully meets the high standards of pharmaceutical R&D and production, providing a reliable guarantee for the safety and efficacy of downstream nucleic acid drugs.
[0018] 2. High purification efficiency and excellent recovery rate: This invention cleverly utilizes acetylation to convert the target product into a diacetylated derivative, while the main impurities, not participating in the reaction, can be effectively removed through simple filtration, significantly improving separation efficiency. Compared to column chromatography, the product recovery rate of this invention is generally above 80%, reaching a maximum of 91.5%, far exceeding the 67.5% of the comparative example, significantly reducing production costs.
[0019] 3. Simple process, suitable for industrial production: The purification method of this invention avoids the cumbersome and difficult-to-scale column chromatography operation. The entire process is simple, easy to operate, and the solvent is recyclable. The raw materials used are widely available and inexpensive. Therefore, this method can not only stably produce high-quality products, but also has good prospects for industrial scale-up, which is of great significance for promoting the industrialization of 2'-fluoronucleoside drugs. Attached Figure Description
[0020] Figure 1 This is a process flow diagram of a purification method for 2'-fluoro-2'-deoxyguanosine proposed in this invention. Detailed Implementation
[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0022] It should be further understood that the term "and / or" as used in this specification and the appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.
[0023] The specific implementation is as follows, please refer to... Figure 1 A method for purifying 2'-fluoro-2'-deoxyguanosine includes the following steps.
[0024] Step 1: The crude 2'-fluoro-2'-deoxyguanosine containing 2,6-diaminopurine and uracil impurities, as shown in Formula I, is dispersed in N,N-dimethylformamide solvent and reacted with an acetylation reagent in the presence of a base and the catalyst 4-dimethylaminopyridine. After the reaction is complete, the product is separated and purified to obtain diacetylated 2'-fluoro-2'-deoxyguanosine as shown in Formula II, thereby achieving the removal of 2,6-diaminopurine and uracil impurities. Step 2: The diacetylated 2'-fluoro-2'-deoxyguanosine (Formula II) obtained in Step 1 is contacted with an ammonia reagent to carry out a deacetylation reaction, yielding the crude compound shown in Formula III; the crude compound is then purified by pulping to obtain high-purity 2'-fluoro-2'-deoxyguanosine.
[0025] The above technical solution discloses a purification process for 2'-fluoro-2'-deoxyguanosine by derivatizing crude 2'-fluoro-2'-deoxyguanosine containing impurities, thereby achieving purification without column chromatography, stably obtaining ultra-high purity products, and possessing good prospects for large-scale production.
[0026] Furthermore, the reagent is selected from the following compounds: The base mentioned in step one is any one or a combination of triethylamine, N,N-diisopropylethylamine or pyridine.
[0027] The acetylation reagent used in step one is any one or a combination of acetic anhydride or acetyl chloride.
[0028] The ammonia mentioned in step two is any one or a combination of ammonia water, a methanol solution of ammonia, or an ethanol solution of ammonia.
[0029] In step two, the solvent used for pulping is any one or a combination of ethanol or isopropanol.
[0030] In step one, the effective content of 2'-fluoro-2'-deoxyguanosine, the molar ratio of the base and the acetylation reagent in the crude 2'-fluoro-2'-deoxyguanosine containing 2,6-diaminopurine and uracil impurities is 1:(3-6):(1.8-3).
[0031] The solvent used in step one is N,N-dimethylformamide, and the effective content of 2'-fluoro-2'-deoxyguanosine in the crude product to the mass-volume ratio of the solvent is 1:(3-6)g / mL.
[0032] The acetylation reaction in step one is carried out at a temperature of 10-30℃ for 3-9 hours.
[0033] The effective content of 2'-fluoro-2'-deoxyguanosine in the crude 2'-fluoro-2'-deoxyguanosine containing 2,6-diaminopurine and uracil impurities in step one, and the mass-to-volume ratio of 2'-fluoro-2'-deoxyguanosine to the ammonia used in step two, is 1:(3-7) g / mL.
[0034] In step two, the reaction temperature of the diacetylated 2'-fluoro-2'-deoxyguanosine with ammonia is 40-50℃, and the reaction time is 12-16 hours. Example 1:
[0035] Step 1: Synthesis of diacetylated 2'-fluoro-2'-deoxyguanosine; Take crude 2'-fluoro-2'-deoxyguanosine containing impurities of 2,6-diaminopurine and uracil (the effective content of 2'-fluoro-2'-deoxyguanosine was determined to be 100g), place it in a reaction flask, add 500mL of N,N-dimethylformamide, and stir to disperse evenly. Add 99.5g of triethylamine and 4.8g of 4-dimethylaminopyridine sequentially, and continue stirring for 10 minutes. Maintain the system temperature at 15℃, slowly add 88.3g of acetic anhydride dropwise, and after the addition is complete, raise the temperature to 25℃ and maintain the reaction for 5 hours. After the reaction is complete, add 1L of ethyl acetate, cool to 15℃, add 1L of water dropwise to the mixture, stir for 20 minutes, and then filter.
[0036] The resulting filter cake was washed with 500 mL of ethyl acetate, filtered to dryness, and the two filtrates were combined. After separation, the aqueous phase was extracted once with 500 mL of ethyl acetate, the organic phases were combined, and then washed twice with 500 mL of 10 wt% sodium chloride solution each time. The organic phase was concentrated under reduced pressure to about 200 mL, and then 500 mL of ethanol was added and evaporated to about 200 mL. The resulting suspension was used directly in the next reaction step.
[0037] Step 2: Preparation of pure 2'-fluoro-2'-deoxyguanosine; At a controlled temperature of 20°C, 500 mL of ethanol and 300 mL of concentrated ammonia were added to the yellow suspension obtained in the first step. The mixture was then heated to 45°C and stirred for 15 hours. After the reaction was completed, the mixture was concentrated under reduced pressure until no obvious fractions were observed. Then, 400 mL of ethanol was added and distilled once. 500 mL of isopropanol was added to the resulting residue, and the mixture was heated to reflux and stirred for 3 hours. The temperature was then slowly lowered to 20°C, and stirring was continued for another 3 hours. The mixture was filtered to obtain an off-white solid.
[0038] The wet filter cake was tested for purity, requiring a purity >98.0% and a single impurity <1.0%. If it failed, the pulping operation was repeated until it passed. The qualified wet filter cake was dried to obtain 80.0 g of white solid, with a recovery rate of 80.0% and a purity of 99.3%. Example 2:
[0039] The operation of this embodiment is basically the same as that of Example 1, except that the base used in the first step is N,N-diisopropylethylamine, and the amount used is 152.5g.
[0040] The above operations yielded 88.0 g of a white solid with a recovery rate of 88.0% and a purity of 99.2%. Example 3:
[0041] The operation of this embodiment is basically the same as that of Embodiment 1, except that the base used in the first step is pyridine, and the amount used is 124.4g.
[0042] The above operations yielded 84.3 g of white solid with a recovery rate of 84.3% and a purity of 98.6%. Example 4:
[0043] The operation of this embodiment is basically the same as that of Example 1, except that the acetylation reagent used in the first step is acetyl chloride, and the amount used is 92.3g.
[0044] The above operations yielded 86.5 g of a white solid with a recovery rate of 86.5% and a purity of 98.1%. Example 5:
[0045] The operation of this embodiment is basically the same as that of Embodiment 1, except that the ammonia reagent used in the second step is an ethanol solution of ammonia (concentration of 2 mol / L, purchased from Sinopharm).
[0046] The above operations yielded 87.9 g of white solid with a recovery rate of 87.9% and a purity of 99.3%. Example 6:
[0047] The operation of this embodiment is basically the same as that of Embodiment 1, except that the ammonia reagent used in the second step is a methanol solution of ammonia (concentration of 2 mol / L, purchased from Sinopharm).
[0048] The above operations yielded 88.3 g of a white solid with a recovery rate of 88.3% and a purity of 98.9%. Example 7:
[0049] The operation of this embodiment is basically the same as that of embodiment 1, except that the pulping solvent used in the second step is ethanol.
[0050] The above operations yielded 83.3 g of white solid with a recovery rate of 83.3% and a purity of 99.5%. Example 8:
[0051] The operation of this embodiment is basically the same as that of Example 1, except that: in the first step, the amount of DMF is 300 mL, the amount of triethylamine is 159.2 g, the amount of 4-dimethylaminopyridine is 2.4 g, and the amount of acetic anhydride is 120.4 g.
[0052] The above operations yielded 91.5 g of white solid with a recovery rate of 91.5% and a purity of 99.3%. Example 9:
[0053] The operation of this embodiment is basically the same as that of Example 1, except that: in the first step, the amount of DMF is 600 mL, the amount of triethylamine is 79.6 g, the amount of 4-dimethylaminopyridine is 3.8 g, and the amount of acetic anhydride is 72.3 g.
[0054] The above operations yielded 85.9 g of white solid with a recovery rate of 85.9% and a purity of 99.3%. Example 10:
[0055] The operation of this embodiment is basically the same as that of Example 2, except that: in the first step, the amount of DMF is 400 mL, the amount of 4-dimethylaminopyridine is 3.4 g, and the amount of acetic anhydride is 120.4 g.
[0056] The above operations yielded 89.4 g of white solid with a recovery rate of 89.4% and a purity of 99.0%. Example 11:
[0057] The operation of this embodiment is basically the same as that of Embodiment 1, except that the amount of concentrated ammonia water used in the second step is 200 mL.
[0058] The above operations yielded 79.9 g of white solid with a recovery rate of 79.9% and a purity of 98.6%. Example 12:
[0059] The operation of this embodiment is basically the same as that of embodiment 1, except that the amount of concentrated ammonia water used in the second step is 500 mL.
[0060] The above operations yielded 89.9 g of white solid with a recovery rate of 89.9% and a purity of 98.6%.
[0061] Comparative example: Experimental process and results using conventional purification methods: Crude 2'-fluoro-2'-deoxyguanosine (with an effective content of 10 g, determined by content analysis) was purified by column chromatography. 60 g of 200-300 mesh silica gel was used, with a column diameter-to-height ratio of 1:8. A mixed solvent of dichloromethane and methanol (volume ratio gradient from 20:1 to 10:1) was used as the eluent, with a total eluent volume of 15 L. The purified product yielded 6.75 g of a white solid, with a recovery rate of 67.5% and a purity of 96.8%.
[0062] During the experiment, it was found that due to the poor solubility of 2'-fluoro-2'-deoxyguanosine and guanosine impurities, a large amount of eluent was required, and the separation effect was not ideal. Therefore, the recovery rate and purity of the comparative example were significantly lower than those of the embodiments of the present invention.
[0063] Results analysis: The results of Examples 1-12 above show that the purification method provided by this invention can stably prepare high-quality 2'-fluoro-2'-deoxyguanosine products with a purity of over 98.6%, with most examples reaching over 99.0% and the highest reaching 99.5%, achieving a high recovery rate of over 80%. Compared with the comparative examples, this invention has achieved unexpected technical effects in both product quality (purity) and production efficiency (recovery rate), providing strong technical support for the industrial application of 2'-fluoronucleosides.
[0064] The results from Examples 1-3, 8, 9, and 10 show that when triethylamine or N,N-diisopropylethylamine was used as the base in the first step, the product recovery rate and purity were superior to those in the examples using pyridine. This is presumably because pyridine has a relatively weak base, which may lead to insufficient reactivity and a slightly lower conversion rate, thus affecting the purity and yield of the final product. Furthermore, pyridine has a strong odor, requiring more adequate ventilation in the experimental environment. Considering all factors, triethylamine or N,N-diisopropylethylamine is the preferred base reagent.
[0065] Based on the results of Examples 1 and 4, it can be seen that the recovery rate and product purity were relatively low when acetyl chloride was used as the acetylation reagent in the first step. This may be because acetyl chloride has higher reactivity than acetic anhydride, making it more prone to side reactions. Therefore, to ensure the purity of the final product, acetic anhydride is preferred as the acetylation reagent.
[0066] Based on the results of Examples 1, 5, and 6, it can be seen that when concentrated ammonia is used as the ammonia reagent in the second step, the recovery rate is slightly higher than when using an ethanol solution or a methanol solution of ammonia. At the same time, considering that concentrated ammonia is inexpensive, readily available, and easy to store, concentrated ammonia is the preferred ammonia reagent.
[0067] Combining the results of Examples 1 and 7, it can be seen that when isopropanol is used as the pulping solvent in the second step, the purity of the obtained product is higher (99.5%). This is because isopropanol has a lower solubility for the product than ethanol, resulting in less product loss in the mother liquor during pulping and more complete precipitation. This is beneficial for removing trace impurities while increasing the yield, thereby further improving the product purity.
[0068] The above description is merely a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art without creative effort within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. A method for purifying 2'-fluoro-2'-deoxyguanosine, characterized in that, Includes the following steps: The crude 2'-fluoro-2'-deoxyguanosine containing 2,6-diaminopurine and uracil impurities was subjected to acetylation to generate diacetylated 2'-fluoro-2'-deoxyguanosine, which was then separated. The diacetylated 2'-fluoro-2'-deoxyguanosine was then subjected to a deacetylation reaction to obtain crude 2'-fluoro-2'-deoxyguanosine, which was then purified by pulping to obtain high-purity 2'-fluoro-2'-deoxyguanosine.
2. The purification method for 2'-fluoro-2'-deoxyguanosine according to claim 1, characterized in that, The specific operating steps include the following: Step 1: Dissolve the crude 2'-fluoro-2'-deoxyguanosine containing 2,6-diaminopurine and uracil impurities as shown in Formula I in an organic solvent, and acetylate it with an acetylation reagent under alkaline conditions. After separation and purification, diacetylated 2'-fluoro-2'-deoxyguanosine as shown in Formula II is obtained. Step 2: The diacetylated 2'-fluoro-2'-deoxyguanosine shown in Formula II is contacted with an ammonia reagent to carry out a deacetylation reaction, yielding the crude compound shown in Formula III. The crude compound is then purified by pulping to obtain high-purity 2'-fluoro-2'-deoxyguanosine.
3. The purification method for 2'-fluoro-2'-deoxyguanosine according to claim 2, characterized in that, The base mentioned in step one is selected from at least one of triethylamine, N,N-diisopropylethylamine, and pyridine.
4. The purification method for 2'-fluoro-2'-deoxyguanosine according to claim 2, characterized in that, The acetylation reagent mentioned in step one is selected from acetic anhydride, acetyl chloride, or a combination of both.
5. The purification method for 2'-fluoro-2'-deoxyguanosine according to claim 2, characterized in that, In the crude 2'-fluoro-2'-deoxyguanosine containing 2,6-diaminopurine and uracil impurities described in step one, the effective content of 2'-fluoro-2'-deoxyguanosine, the molar ratio of the base and the acetylation reagent is 1:(3-6):(1.8-3).
6. The purification method for 2'-fluoro-2'-deoxyguanosine according to claim 2, characterized in that, The acetylation reaction in step one is carried out at a temperature of 10-30°C for 3-9 hours.
7. The purification method for 2'-fluoro-2'-deoxyguanosine according to claim 1, characterized in that, The ammonia mentioned in step two is selected from at least one of ammonia water, a methanol solution of ammonia, and an ethanol solution of ammonia.
8. The purification method for 2'-fluoro-2'-deoxyguanosine according to claim 1, characterized in that, In step two, the reaction temperature of diacetylated 2'-fluoro-2'-deoxyguanosine with ammonia is 40-50℃, and the reaction time is 12-16 hours.
9. The purification method for 2'-fluoro-2'-deoxyguanosine according to claim 1, characterized in that, The solvent used for pulping and purification in step two is selected from one or a mixture of ethanol and isopropanol.
10. The purification method for 2'-fluoro-2'-deoxyguanosine according to claim 1, characterized in that, The solvent used in step one is N,N-dimethylformamide.