A method for producing adenosylcobalamin

Abstract

This invention provides a method for producing adenosylcobalamin. First, cyanocobalamin, cobalt chloride, adenine, nucleoside triphosphate, and water are mixed and added to a reaction vessel to prepare an adenosylcobalamin mixture. High-purity nitrogen and a NaBH4-NaOH aqueous solution are added during the reaction. Then, the adenosylcobalamin filtrate is purified by passing it through a purification column. The purified solution is then subjected to adsorption and elution on a resin column, and the eluent is crystallized to obtain pure adenosylcobalamin. This purification process effectively removes impurities introduced during the synthesis of adenosylcobalamin, with total impurities ≤0.5%, ensuring the safety and controllability of product quality.

Description

Technical Field

[0001] This invention relates to the field of vitamin B12 series product manufacturing technology, and in particular to a method for synthesizing adenosylcobalamin. Background Technology

[0002] Adenosylcobalamin is a product in the vitamin B12 series. Its chemical name is 5,6-dimethylbenzimidazolyl-5'-deoxyadenine nucleoside cobalamin, with the molecular formula C72H100CoN18O17P and a molecular weight of 1579. Adenosylcobalamin is a dark red crystalline or non-crystalline powder, highly hygroscopic, and readily decomposes upon exposure to light. It is slightly soluble in water, almost insoluble in ethanol, and insoluble in acetone, ether, and chloroform. Adenosylcobalamin is mainly used for megaloblastic anemia, nutritional anemia, and anemia during pregnancy. It is also used for neurological disorders such as polyneuritis, radiculitis, trigeminal neuralgia, sciatica, nerve paralysis, nutritional neurological disorders, and leukopenia caused by radiation and drugs.

[0003] Currently, the synthesis process of adenosylcobalamin includes the following steps:

[0004] (1) Dissolve cyanocobalamin in water to make a solution;

[0005] (2) Sodium borohydride is poured into anhydrous ethanol to make a solution;

[0006] (3) Add the prepared cyanocobalamin solution and the prepared sodium borohydride solution to the reaction vessel, and make the weight ratio of cyanocobalamin to sodium borohydride in the solution 2.2 to 2.6:1. Then slowly add adenine nucleoside triphosphate to the reaction vessel, while controlling the pH of the solution in the reaction vessel between 3.0 and 3.5. React for more than 90 minutes, and then centrifuge and filter.

[0007] (4) The filtrate is adsorbed through a macroporous resin column at a flow rate of 160-300 L / h, then a developing agent is introduced into the macroporous resin column to remove impurities, and finally a descaling agent is introduced into the macroporous resin column, and the concentrated eluted material is collected into the crystallization stock tank.

[0008] While stirring, acetone is added to the crystallization stock solution for crystallization, followed by filtration and drying to obtain the adenosylcobalamin product.

[0009] However, this process produces products with higher levels of related substances, which affects the purity of the final product. Summary of the Invention

[0010] To overcome the shortcomings of existing technologies, the present invention aims to provide an optimized synthesis method that can effectively reduce impurities in adenosylcobalamin raw materials and is suitable for industrial production.

[0011] To achieve the above objectives, the present invention adopts the following technical solution: a method for producing adenosylcobalamin, comprising the following steps: (1) preparing adenosylcobalamin mixture by mixing cyanocobalamin, cobalt chloride, adenine, nucleoside triphosphate and water and adding them to a reaction vessel. During the reaction process, high-purity nitrogen gas is added first, followed by NaBH4-NaOH aqueous solution; (2) purifying the adenosylcobalamin filtrate by passing it through a purification column; (3) adsorbing and desorbing the purified solution onto a resin column; (4) crystallizing and refining the desorbed solution to obtain pure adenosylcobalamin.

[0012] Preferably, in step (1), the mass ratio of cyanocobalamin to sodium borohydride is 1:0.3.

[0013] More preferably, the reaction temperature in step (1) is controlled at 20-30℃ and the reaction time is 40-60min.

[0014] More preferably, the reaction temperature in step (1) is controlled at 20-25℃.

[0015] More preferably, the pH of the reaction solution in step (1) is 4.5-5.5.

[0016] Preferably, the purification column in step (2) is an alumina column, and the medium is water-acetone.

[0017] More preferably, in step (2), the adsorption liquid is 0.890±0.002g / ml, the adsorption flow rate is 50-60ml / min, the expansion flow rate is 100-120ml / min, and the desorption flow rate is 60-80ml / min.

[0018] Preferably, in step (3), the resin is a macroporous adsorption chromatography resin and the eluent is an acetone-water solution.

[0019] More preferably, the specific gravity of the acetone-water solution in step (3) is 0.965±0.01g / ml.

[0020] Preferably, in step (4), the eluent is crystallized with acetone, filtered, and dried to obtain pure adenosylcobalamin.

[0021] This invention selects an optimized synthesis process and employs a purification column for separation and purification. This method offers advantages such as simple preparation process, good compatibility, high column efficiency, long column life, wide range of material selection, and good reproducibility, effectively removing impurities from the crude product. The purification process of this invention can effectively remove impurities introduced during the synthesis of adenosylcobalamin, with total related substances ≤0.5%, ensuring the safety and controllability of product quality. The eluent obtained by resin column separation in this invention, after crystallization and drying, does not affect the stability of product quality. Attached Figure Description

[0022] Figure 1Example 1: Liquid Chromatography of Adenosylcobalamin

[0023] Figure 2 Example 2: Liquid Chromatography of Adenosylcobalamin

[0024] Figure 3 Example 3: Liquid Chromatography of Adenosylcobalamin

[0025] Figure 4 Liquid chromatography spectrum of control example 1, adenosylcobalamin

[0026] Figure 5 Liquid chromatography spectrum of control example 2, adenosylcobalamin

[0027] Figure 6 Liquid chromatography spectrum of control example 3, adenosylcobalamin Detailed Implementation

[0028] To better understand the content of this invention, further description will be provided below with reference to specific embodiments. However, these embodiments should not be construed as limiting the scope of protection of this invention. It should be noted that those skilled in the art can make various improvements and modifications to this invention without departing from the principles thereof, and these improvements and modifications also fall within the scope of protection of the claims of this invention.

[0029] Example 1

[0030] (1) Weigh 20g of vitamin B12, cobalt chloride hexahydrate, and adenine nucleoside triphosphate, add them to 500ml of water in a 2L reactor, and stir at room temperature under nitrogen protection. Add an aqueous solution of sodium borohydride (dissolve 6g of sodium borohydride in 50ml of an aqueous solution of sodium hydroxide) to the reaction solution, controlling the temperature at 20-30℃ during the dropwise addition, and keep the temperature at 20-30℃ and stir for 40-60min after the dropwise addition is complete.

[0031] (2) Add 30% glacial acetic acid to the reaction solution to adjust the pH of the reaction solution to 4.5-5.5, remove insoluble matter by vacuum filtration, and transfer the filtrate to the next process.

[0032] (3) Add acetone to the filtrate from step (2) to control the specific gravity of the water-acetone solution at 0.880±0.002g / ml. Stir and mix thoroughly, then filter for purification. The sample loading flow rate is 50-60ml / min. Then perform layer development with water-acetone 0.890±0.002g / ml as the developing agent and 100-120ml / min as the developing flow rate. Finally, pass water-acetone 0.930±0.005g / ml as the eluent and collect the purified solution.

[0033] (4) Weigh 1L of LX-1600 resin, add purified water to pack the column, and pass 2BV of 50% acetone aqueous solution through it. Then rinse the resin column with purified water until the specific gravity of the waste liquid is 1.0g / ml. Pass 2BV of 6.3% sodium hydroxide aqueous solution through it, and then rinse the resin column with purified water until the pH of the waste liquid is about 8.0. Pass 2BV of 6.3% hydrogen chloride aqueous solution through it, and then rinse the resin column with purified water until the pH of the waste liquid is about 5.0.

[0034] (5) Pass the purified solution obtained in step (3) into a resin column for adsorption. After adsorption is complete, wash with deionized water at a volume of 5 BV. Develop the column with an acetone aqueous solution, and finally elute with an acetone aqueous solution with a specific gravity of 0.965 ± 0.01 g / ml. Start collecting the resin column waste liquid when it is colored, and stop collecting when the color turns light pink, thus obtaining the adenosylcobalamin eluent.

[0035] (6) Add acetone to the eluent to crystallize. Stir for 30 minutes and let stand for 8 hours.

[0036] (7) After standing, filter, rinse the filter cake with a small amount of acetone, and vacuum dry at 60°C for 4 hours. Liquid chromatography analysis showed a purity of 99.62%.

[0037] Table 1. Integration results of the liquid phase spectrum of adenosylcobalamin in this embodiment.

[0038] Peak Retention time high area area% 1 4.139 2802 11437 0.083 2 5.906 338 7315 0.053 3 7.060 575 6694 0.049 4 8.560 241 3910 0.029 5 9.715 252 4329 0.032 6 11.127 200 3662 0.027 7 15.597 568932 13658633 99.617 8 18.632 475 15104 0.110 total 573814 13711084 100.000

[0039] Example 2

[0040] (1) Weigh 20g of vitamin B12, cobalt chloride hexahydrate, and adenine nucleoside triphosphate, add them to 500ml of water in a 2L reactor, and stir at room temperature under nitrogen protection. Add an aqueous solution of sodium borohydride (dissolve 6g of sodium borohydride in 50ml of an aqueous solution of sodium hydroxide) to the reaction solution, controlling the temperature at 20-30℃ during the dropwise addition, and keep the temperature at 20-30℃ and stir for 40-60min after the dropwise addition is complete.

[0041] (2) Add 30% glacial acetic acid to the reaction solution to adjust the pH of the reaction solution to 4.5-5.5, remove insoluble matter by vacuum filtration, and transfer the filtrate to the next process.

[0042] (3) Add acetone to the filtrate from step (2) to control the specific gravity of the water-acetone solution at 0.880±0.002g / ml. Stir and mix thoroughly, then filter for purification. The sample loading flow rate is 50-60ml / min. Then perform layer development with water-acetone 0.890±0.002g / ml as the developing agent and 100-120ml / min as the developing flow rate. Finally, pass water-acetone 0.930±0.005g / ml as the eluent and collect the purified solution.

[0043] (4) Weigh 1L of LX-1600 resin, add purified water to pack the column, and pass 2BV of 50% acetone aqueous solution through it. Then rinse the resin column with purified water until the specific gravity of the waste liquid is 1.0g / ml. Pass 2BV of 6.3% sodium hydroxide aqueous solution through it, and then rinse the resin column with purified water until the pH of the waste liquid is about 8.0. Pass 2BV of 6.3% hydrogen chloride aqueous solution through it, and then rinse the resin column with purified water until the pH of the waste liquid is about 5.0.

[0044] (5) Pass the purified solution obtained in step (3) into a resin column for adsorption. After adsorption is complete, wash with deionized water at a volume of 5 BV. Develop the column with an acetone aqueous solution, and finally elute with an acetone aqueous solution with a specific gravity of 0.965 ± 0.01 g / ml. Start collecting the resin column waste liquid when it is colored, and stop collecting when the color turns light pink, thus obtaining the adenosylcobalamin eluent.

[0045] (6) Add acetone to the eluent to crystallize. Stir for 30 minutes and let stand for 8 hours.

[0046] (7) After standing, filter, rinse the filter cake with a small amount of acetone, and vacuum dry at 60°C for 4 hours. Liquid chromatography analysis showed a purity of 99.60%.

[0047] Table 2. Integration results of the liquid phase spectrum of adenosylcobalamin in this embodiment.

[0048] Peak Retention time high area area% 1 4.141 1935 7669 0.056 2 5.934 694 13414 0.099 3 7.136 374 4397 0.032 4 8.670 383 6422 0.047 5 9.856 399 7308 0.054 6 11.320 242 4278 0.031 7 15.908 553589 13554726 99.602 8 19.022 345 10621 0.078 total 557962 13608836 100.000

[0049] Example 3

[0050] (1) Weigh 20g of vitamin B12, cobalt chloride hexahydrate, and adenine nucleoside triphosphate, add them to 500ml of water in a 2L reactor, and stir at room temperature under nitrogen protection. Add an aqueous solution of sodium borohydride (dissolve 6g of sodium borohydride in 50ml of an aqueous solution of sodium hydroxide) to the reaction solution, controlling the temperature at 20-30℃ during the dropwise addition, and keep the temperature at 20-30℃ and stir for 40-60min after the dropwise addition is complete.

[0051] (2) Add 30% glacial acetic acid to the reaction solution to adjust the pH of the reaction solution to 4.5-5.5, remove insoluble matter by vacuum filtration, and transfer the filtrate to the next process.

[0052] (3) Add acetone to the filtrate from step (2) to control the specific gravity of the water-acetone solution at 0.880±0.002g / ml. Stir and mix thoroughly, then filter for purification. The sample loading flow rate is 50-60ml / min. Then perform layer development with water-acetone 0.890±0.002g / ml as the developing agent and 100-120ml / min as the developing flow rate. Finally, pass water-acetone 0.930±0.005g / ml as the eluent and collect the purified solution.

[0053] (4) Weigh 1L of LX-1600 resin, add purified water to pack the column, and pass 2BV of 50% acetone aqueous solution through it. Then rinse the resin column with purified water until the specific gravity of the waste liquid is 1.0g / ml. Pass 2BV of 6.3% sodium hydroxide aqueous solution through it, and then rinse the resin column with purified water until the pH of the waste liquid is about 8.0. Pass 2BV of 6.3% hydrogen chloride aqueous solution through it, and then rinse the resin column with purified water until the pH of the waste liquid is about 5.0.

[0054] (5) Pass the purified solution obtained in step (3) into a resin column for adsorption. After adsorption is complete, wash with deionized water at a volume of 5 BV. Develop the column with an acetone aqueous solution, and finally elute with an acetone aqueous solution with a specific gravity of 0.965 ± 0.01 g / ml. Start collecting the resin column waste liquid when it is colored, and stop collecting when the color turns light pink, thus obtaining the adenosylcobalamin eluent.

[0055] (6) Add acetone to the eluent to crystallize. Stir for 30 minutes and let stand for 8 hours.

[0056] (7) After standing, filter, rinse the filter cake with a small amount of acetone, and vacuum dry at 60°C for 4 hours. Liquid chromatography analysis showed a purity of 99.55%.

[0057] Table 3. Integration results of the liquid phase spectrum of adenosylcobalamin in this embodiment.

[0058]

[0059]

[0060] Compare with Example 1

[0061] (1) Dissolve cyanocobalamin in water to make a solution;

[0062] (2) Sodium borohydride is poured into anhydrous ethanol to make a solution;

[0063] (3) Add the cyanocobalamin solution and sodium borohydride solution obtained in step (1) to the reaction vessel, and make the weight ratio of cyanocobalamin to sodium borohydride in the solution 2.2 to 2.6:1. Then slowly add adenine nucleoside triphosphate to the reaction vessel, while controlling the pH of the solution in the reaction vessel between 3.0 and 3.5. React for more than 90 minutes, and then centrifuge and filter.

[0064] (4) The filtrate obtained in step (3) is adsorbed through a macroporous resin column at a flow rate of 160-300 L / h. Then, a developing agent is introduced into the macroporous resin column to remove impurities. Finally, a desiccant is introduced into the macroporous resin column, and the eluted concentrate is collected into the crystallization stock solution tank.

[0065] (5) Add acetone to the crystallization solution to allow crystallization. Stir for 3 hours and let stand for more than 2 hours.

[0066] Hour.

[0067] (6) After settling, filter the crystals and wash them sequentially with three different acetone aqueous solutions with densities greater than, equal to, and less than that of the mother liquor, then dry them under vacuum. Wash the crystals with crystalline acetone, and finally remove the washing liquid by vacuum filtration. Dry the crystals under vacuum at 60°C for 4 hours. The purity was determined to be 99.05% by liquid chromatography.

[0068] Table 4. Integration results of the liquid chromatography spectra of adenosylcobalamin in this control example.

[0069]

[0070]

[0071] Compare with Example 2

[0072] (1) Dissolve cyanocobalamin in water to make a solution;

[0073] (2) Sodium borohydride is poured into anhydrous ethanol to make a solution;

[0074] (3) Add the cyanocobalamin solution and sodium borohydride solution obtained in step (1) to the reaction vessel, and make the weight ratio of cyanocobalamin to sodium borohydride in the solution 2.2 to 2.6:1. Then slowly add adenine nucleoside triphosphate to the reaction vessel, while controlling the pH of the solution in the reaction vessel between 3.0 and 3.5. React for more than 90 minutes, and then centrifuge and filter.

[0075] (4) The filtrate obtained in step (3) is adsorbed through a macroporous resin column at a flow rate of 160-300 L / h. Then, a developing agent is introduced into the macroporous resin column to remove impurities. Finally, a desiccant is introduced into the macroporous resin column, and the eluted concentrate is collected into the crystallization stock solution tank.

[0076] (5) Add acetone to the crystallization solution to allow crystallization. Stir for 3 hours and let stand for more than 2 hours.

[0077] Hour.

[0078] (6) After settling, filter the crystals and wash them sequentially with three different acetone aqueous solutions with densities greater than, equal to, and less than that of the mother liquor, then dry them under vacuum. Wash the crystals with crystalline acetone, and finally remove the washing liquid by vacuum filtration. Dry the crystals under vacuum at 60°C for 4 hours. Liquid chromatography showed a purity of 99.10%.

[0079] Table 5. Integration results of the liquid chromatography spectra of adenosylcobalamin in this control example.

[0080]

[0081]

[0082] Compare with Example 3

[0083] (1) Dissolve cyanocobalamin in water to make a solution;

[0084] (2) Sodium borohydride is poured into anhydrous ethanol to make a solution;

[0085] (3) Add the cyanocobalamin solution and sodium borohydride solution obtained in step (1) to the reaction vessel, and make the weight ratio of cyanocobalamin to sodium borohydride in the solution 2.2 to 2.6:1. Then slowly add adenine nucleoside triphosphate to the reaction vessel, while controlling the pH of the solution in the reaction vessel between 3.0 and 3.5. React for more than 90 minutes, and then centrifuge and filter.

[0086] (4) The filtrate obtained in step (3) is adsorbed through a macroporous resin column at a flow rate of 160-300 L / h. Then, a developing agent is introduced into the macroporous resin column to remove impurities. Finally, a desiccant is introduced into the macroporous resin column, and the eluted concentrate is collected into the crystallization stock solution tank.

[0087] (5) Add acetone to the crystallization solution to allow crystallization. Stir for 3 hours and let stand for more than 2 hours.

[0088] Hour.

[0089] (6) After settling, filter the crystals and wash them sequentially with three different acetone aqueous solutions with densities greater than, equal to, and less than that of the mother liquor, then dry them under vacuum. Wash the crystals with crystalline acetone, and finally remove the washing liquid by vacuum filtration. Dry the crystals under vacuum at 60°C for 4 hours. The purity was determined to be 99.12% by liquid chromatography.

[0090] Table 6. Integration results of the liquid chromatography spectra of adenosylcobalamin in this control example.

[0091] Peak Retention time high area area% 1 4.161 6138 29169 0.216 2 4.585 448 9348 0.069 3 5.302 260 4709 0.035 4 5.635 337 3603 0.027 5 6.030 1064 22032 0.163 6 7.238 528 6996 0.052 7 8.380 254 3907 0.029 8 8.823 386 7145 0.053 9 10.001 373 7266 0.054 10 11.463 350 6142 0.046 11 12.184 201 3378 0.025 12 16.180 538348 13360129 99.116 13 19.349 382 12176 0.090 14 24.399 75 3281 0.024 total 549143 13479280 100.00

[0092] Effect Comparison

[0093] Example Comparison Example Comparison The first batch Purity: 99.62 Purity: 99.05% The purity increased by 0.57%. Second batch Purity: 99.60 Purity: 99.10 The purity increased by 0.50%. Third batch Purity: 99.55 Purity: 99.12 The purity increased by 0.43%. average Purity: 99.59 Purity: 99.09 The purity increased by 0.50%.

Claims

1. A method for producing adenosylcobalamin, characterized in that, Includes the following steps: (1) Prepare adenosylcobalamin mixture by mixing cyanocobalamin, cobalt chloride, adenine, nucleoside triphosphate and water and adding them into a reaction vessel. High-purity nitrogen gas is added first, followed by NaBH4-NaOH aqueous solution. (2) The reaction filtrate from step (1) is purified by passing it through a purification column; (3) The purified solution was subjected to adsorption and desorption on a resin column; (4) The solution was crystallized and purified to obtain pure adenosylcobalamin.

2. The method for producing adenosylcobalamin according to claim 1, characterized in that: In step (1), the mass ratio of cyanocobalamin to sodium borohydride is 1:0.

3.

3. The method for producing adenosylcobalamin according to claim 1, characterized in that: The reaction temperature in step (1) is controlled at 20-30℃ and the reaction time is 40-60min.

4. The method for producing adenosylcobalamin according to claim 3, characterized in that: The reaction temperature in step (1) is controlled at 20-25℃.

5. The method for producing adenosylcobalamin according to claim 1, characterized in that: The pH of the reaction solution in step (1) is 4.5-5.

5.

6. The method for producing adenosylcobalamin according to claim 1, characterized in that: The purification column in step (2) is an alumina column, and the medium is water-acetone.

7. The method for producing adenosylcobalamin according to claim 6, characterized in that: In step (2), the adsorption solution is 0.890±0.002g / ml, the adsorption flow rate is 50-60ml / min, the expansion flow rate is 100-120ml / min, and the desorption flow rate is 60-80ml / min.

8. The method for producing adenosylcobalamin according to claim 1, characterized in that: In step (3), the resin is a macroporous adsorption chromatography resin and the eluent is an acetone-water solution.

9. The method for producing adenosylcobalamin according to claim 8, characterized in that: The specific gravity of the acetone-water solution in step (3) is 0.965±0.01g / ml.

10. The method for producing adenosylcobalamin according to claim 1, characterized in that: In step (4), the eluent is crystallized with acetone, filtered, and dried to obtain pure adenosylcobalamin.

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