Purification method of isepamicin sulfate crude product

Abstract

The invention relates to a purification method of an isepamicin sulfate crude product. Impurities comprise an impurity A, 3N-isepamicin, gentamicin B and the like. The method comprises the following steps: adding an isepamicin sulfate crude product aqueous solution into a water suspension generated by dialdehyde cellulose and sodium hydrogen sulfite, mixing and stirring while adjusting the pH value to 4-5 by using dilute sulphuric acid, generating Schiff base through a chemical reaction between amino groups of impurities and dialdehyde cellulose, forming impurity-loaded cellulose, and filtering to remove impurities, thereby obtaining the isepamicin sulfate crude product. The purity is greater than or equal to 99.2%, the yield is greater than or equal to 94.4%, and the dialdehyde cellulose can be regenerated and recycled. The method is simple to operate, high in yield and low in cost.

Description

Technical Field

[0001] This invention relates to the field of drug purification technology, specifically to a purification method for crude isapamicin sulfate; the impurities include impurity A, 3N-isopamidin, gentamicin B, etc., and is applicable to the purification of isapamicin sulfate raw material. Background Technology

[0002] Aminoglycoside antibiotics are glycoside antibiotics composed of amino sugars and aminocyclic alcohols linked by oxygen-bridged bonds. They are mostly polar compounds, readily soluble in water, and poorly absorbed by the gastrointestinal tract, generally requiring injection for administration. Aminoglycoside antibiotics work by binding to bacterial ribosomes, interfering with bacterial protein synthesis. They are a class of bactericidal agents that work during the stationary phase and are important drugs for treating severe infections caused by aerobic Gram-negative bacilli.

[0003] Isapamicin sulfate (CAS No.: 67814-76-0) is a semi-synthetic aminoglycoside drug, obtained by introducing a hydroxypropionyl group at the 1-position of the amino group of gentamicin B. It has advantages such as broad antibacterial spectrum, strong bactericidal activity, good enzyme stability and few adverse reactions, and has become a new generation of effective aminoglycoside drugs, widely used in the treatment of various bacterial infections. However, its synthesis is prone to producing structurally similar impurities, such as impurity A, 3N-isopamidin, gentamicin B, etc. [Wang Jian, Wang Qiaoqun, Sun Jin, et al. Isolation, identification and antibacterial activity of related substances in isapamicin sulfate [J]. Chinese Pharmaceutical Journal, 2007, 42(2):150-153], and the in vitro antibacterial activity of these impurities is lower than that of isapamicin sulfate, which will affect the efficacy and safety of the drug.

[0004] Existing purification techniques include chromatography and physical adsorption. Chromatography is complex to operate and costly, making it difficult to scale up production, while simple physical adsorption has poor selectivity and is prone to loss of main components.

[0005] Natural polysaccharides have wide applications in various fields such as health, environment, and energy, and occupy a prominent position in the development of human society. Cellulose is the most abundant natural resource on Earth, composed of linear chains of D-glucanose residues linked by β-(1→4)-glycosidic units, and has good biocompatibility. However, because cellulose is insoluble in water and other common organic solvents, its applicability is limited. Therefore, the derivatization of primitive cellulose has become a powerful and practical strategy, with oxidized cellulose being a prominent example.

[0006] Dialdehyde cellulose (CAS No.: 9032-52-4, abbreviated as DAC) is a natural polymer obtained by introducing aldehyde groups into the cellulose backbone. It is a water-insoluble solid, and its aldehyde functional groups can undergo various chemical reactions, such as Schiff base reactions, cationization, condensation reactions, sulfonation reactions, and silanization reactions. The oxidation of periodate ions has been widely used in polysaccharide chemistry, especially in cellulose. Dialdehyde cellulose is often prepared by oxidizing cellulose with periodate. Therefore, a purification method for crude isopamicin sulfate is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a simple, highly selective, and low-cost method for removing isopamicin sulfate impurities, thereby avoiding the loss of the main component.

[0008] The technical solution of this invention is: First, dialdehyde cellulose and sodium bisulfite are added to an aqueous solution, followed by an aqueous solution of isopamicin sulfate. Then, an appropriate amount of sulfuric acid solution is added to release the dialdehyde cellulose and protonate the aldehyde group to promote the reaction activity. The aldehyde group of dialdehyde cellulose reacts with the amino groups in impurities A, 3N-isopamircin, gentamicin B, etc., to form Schiff bases, generating cellulose-loaded insoluble impurities that are easier to remove. These impurities are then removed by filtration. After the filtrate is concentrated and crystallized, pure isopamicin sulfate is obtained.

[0009] Includes the following steps: S1: Add dialdehyde cellulose to an aqueous solution of sodium bisulfite and stir thoroughly for 6-8 hours to prepare a dialdehyde cellulose sodium bisulfite adduct suspension with a dialdehyde cellulose content of 0.05-0.15 g / mL; S2: Mix crude isopamicin sulfate with water and stir to prepare an aqueous solution of isopamicin sulfate, with an isopamicin sulfate content of 0.08-0.12 g / mL; S3: Mix the above suspension with the solution and stir. Add 0.1 mol / L sulfuric acid solution dropwise to adjust the pH to 4-5. Then, allow it to react fully for 10-12 hours at a water bath temperature of 20-60℃. S4: After the reaction is complete, filter to remove the insoluble solid dialdehyde cellulose that has adsorbed impurities; S5: Collect the filtrate in a distillation flask, and concentrate and crystallize it under reduced pressure in a rotary evaporator at 30~50℃ to obtain high-purity isapamicin sulfate.

[0010] A further preferred embodiment is characterized by the addition of a small amount of sulfuric acid solution to promote the reaction.

[0011] More preferably, the amount of dialdehyde cellulose used is 0.1-0.5 times that of isopamicin sulfate, preferably 0.1 times.

[0012] More preferably, the molar ratio of sodium bisulfite to dialdehyde cellulose is 0.3:1 to 0.8:1, preferably 0.5:1.

[0013] Further preferably, as a preferred method, the heating temperature in step S5 is 40°C.

[0014] The preparation steps of dialdehyde cellulose are as follows: (1): Weigh microcrystalline cellulose into an Erlenmeyer flask, add deionized water, and protect from light; (2): Add excess sodium periodate and adjust the pH value with sulfuric acid; (3): After the reaction is complete, ethylene glycol is added to remove unreacted sodium periodate, and the reaction is continued in a water bath shaker for 2-3 hours. (4): The product was filtered by suction filtration, washed with deionized water, dried in a constant temperature drying oven, ground and sieved to obtain dialdehyde cellulose.

[0015] Further preferred, in step (2), pH = 2 is preferred.

[0016] More preferably, the oven temperature in step (4) is 40~50℃.

[0017] Selective reaction principle: Due to steric hindrance, impurities preferentially react with dialdehyde cellulose. Subsequently, the cellulose that adsorbs impurities is filtered out, and the filtrate is obtained. After evaporation, concentration, and crystallization, high-purity isopamicin sulfate is obtained.

[0018] The beneficial effects of this invention are: the steric hindrance effect of the dialdehyde cellulose macromolecular carrier is used to achieve specific capture of impurities, and the dialdehyde cellulose can be recycled and regenerated, thus achieving green and efficient removal of impurities; and compared with traditional chromatographic separation methods, the method of this invention is simpler to operate, lower in cost, and has a purity > 99.2% and a yield > 94.4%. Attached Figure Description

[0019] Figure 1 This is the molecular structural formula of impurity A in this invention; impurity A is named: O-6-amino-6-deoxy-α-D-pyranoglucopyranosyl-(1→4)-2-deoxy-N1-[(S)-isoseryl]-D-streptoamine; Figure 2 This is the molecular structural formula of 3N-isopamirin in this invention; the name of 3N-isopamirin is: O-6-amino-6-deoxy-α-D-glucopyranosyl-(1→4)-O-[3-deoxy-4-C-methyl-3-(methylamino)-β-L-arabinopyranosyl-(1→6)]-2-deoxy]-N'-[(S)-isoseryl]-D-streptomycin; Figure 3This is the molecular structural formula of gentamicin B in this invention; Gentamicin B is named: O-6-amino-6-deoxy-α-D-glucopyranosyl-(1→4)-O-[3-deoxy-4-C-methyl-3-(methylamino)-β-L-arabinopyranosyl-(1→6)]-2-deoxy-D-streptoamine. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: Example 1 Preparation of dialdehyde cellulose Accurately weigh 5.0 g of microcrystalline cellulose (MCC) into a 250 mL Erlenmeyer flask, add 200 mL of deionized water, and wrap the Erlenmeyer flask with aluminum foil to protect it from light. Weigh 6.6 g of sodium periodate and add it to the MCC suspension. Adjust the pH to 2.0 with sulfuric acid solution and place the flask in a constant temperature water bath shaker (110 r / min) at 30 °C for 12 h in the dark. After the reaction, wash with 50 mL of ethylene glycol to remove unreacted sodium periodate, and continue the reaction in the water bath shaker for 2 h. After the reaction, filter the DAC obtained, wash it three times with deionized water, filter it, and then place the solid in a constant temperature drying oven at 45 °C for 10 h. After drying, grind it and pass it through a 100-mesh sieve to obtain 4.58 g of white powder. Prepare dialdehyde cellulose according to this method for the experiment.

[0021] The DAC was generated by oxidizing the MCC with sodium periodate. The aldehyde group was then reacted quantitatively with hydroxylamine hydrochloride solution to generate oxime. The released hydrochloric acid was titrated with standard sodium hydroxide solution to determine the aldehyde group content in the DAC. The aldehyde group content was determined to be 4.33 ± 0.23 (mmol / g).

[0022] Effect of raw material ratio on the purity and yield of isapamicin sulfate 1. Take 10.0g of crude isopamicin sulfate (excluding sulfuric acid, the isopamicin content is 95.6%, impurity A 2.2%, 3N-isopamidin 0.6%, gentamicin B 1.3%, and other impurities 0.3%) and place it in a beaker. Add 100mL of aqueous solution and stir on a magnetic stirrer to dissolve it completely, so as to prepare an aqueous solution containing 0.1g of isopamicin sulfate per 1mL of water.

[0023] 2. According to Table 1, weigh out different proportions of dialdehyde cellulose and place them in beakers. Weigh out sodium bisulfite and add it to 10 mL of water to prepare a sodium bisulfite solution. Add dialdehyde cellulose to the sodium bisulfite solution and stir at 30°C for 6 hours to prepare suspensions of dialdehyde cellulose sodium bisulfite adduct containing 0.05, 0.1, 0.3, and 0.5 g per mL of water, respectively.

[0024] 3. Mix the above isopamicin sulfate solution with the sodium bisulfite adduct of dialdehyde cellulose in a beaker, add 0.1 mol / L sulfuric acid solution dropwise to adjust the pH to 4.5, and then place the mixture on a magnetic stirrer and stir until fully reacted for 12 hours.

[0025] 4. After the reaction is complete, filter to remove the insoluble solid dialdehyde cellulose that has adsorbed impurities.

[0026] 5. Collect the filtrate in a distillation flask, and remove the aqueous solvent by vacuum distillation in a rotary evaporator at 40°C in a water bath until constant weight is achieved. Continue to maintain vacuum distillation for 0.5 hours, and collect high-purity isopamicin sulfate at the bottom of the flask.

[0027] Table 1. Ingredient Ratio (Cellulose dialdehyde / Isopamicin sulfate / Sodium bisulfite) The content of isapamicin sulfate was determined according to the detection method described in Part I of the main text of the 2020 edition of the Chinese Pharmacopoeia, and the yield was calculated. The purification results are shown in Table 2.

[0028] Yield = [(mass of pure isapamil sulfate × purity) / (mass of crude isapamil sulfate × purity)] × 100% Chromatographic conditions: Octadecylsilane-bonded silica gel (4.6 mm × 250 mm, 5 μm) was used as the stationary phase; 0.2 mol / L trifluoroacetic acid solution was used as mobile phase A, and methanol was used as mobile phase B, with gradient elution; the flow rate was 0.8 mL per minute; detection was performed using an evaporative light scattering detector (reference conditions: drift tube temperature 50 °C, carrier gas flow rate 1.5 L per minute); the injection volume was 10 μL.

[0029] Table 2. Purification results of Example 1 Example 2: The preparation of dialdehyde cellulose is as described in Example 1. Effect of different proportions of sodium bisulfite on impurity removal 1. Take 10g of crude isopamicin sulfate (excluding sulfuric acid, the isopamicin content is 95.6%, impurity A 2.2%, 3N-isopamidin 0.6%, gentamicin B 1.3%, and other impurities 0.3%) and place it in a beaker. Add 100mL of aqueous solution and stir on a magnetic stirrer to dissolve it completely, so as to prepare an aqueous solution containing 0.1g of isopamicin sulfate per 1mL of water.

[0030] 2. According to Table 3, weigh 1, 0.5, 0.3, and 0.1 grams of sodium bisulfite and add them to 10 mL of aqueous solution to prepare sodium bisulfite solutions of different concentrations. Add 1 gram of dialdehyde cellulose to each solution and stir at 30°C for 6 hours to prepare a suspension containing 0.1 g of dialdehyde cellulose per mL of water.

[0031] 3. Mix the above isopamicin sulfate solution with the sodium bisulfite adduct of dialdehyde cellulose in a beaker, add 0.1 mol / L sulfuric acid solution dropwise to adjust the pH to 4.5, and then place the mixture on a magnetic stirrer and stir until fully reacted for 12 hours.

[0032] 4. After the reaction is complete, filter to remove the insoluble solid dialdehyde cellulose that has adsorbed impurities.

[0033] 5. Collect the filtrate in a distillation flask, and remove the aqueous solvent by vacuum distillation in a rotary evaporator at 40°C in a water bath until constant weight is achieved. Continue to maintain vacuum distillation for 0.5 hours, and collect high-purity isopamicin sulfate at the bottom of the flask.

[0034] Table 3. Amount of sodium bisulfite in different proportions The content of isapamicin sulfate was determined according to the detection method described in Part I of the main text of the 2020 edition of the Chinese Pharmacopoeia, and the yield was calculated. The purification results are shown in Table 4.

[0035] Table 4. Purification results of Example 2 Comparative Example 1: The impact of not using sodium bisulfite in the purification process on impurity removal The preparation of dialdehyde cellulose is as described in Example 1. 1. Take 10.0g of crude isopamicin sulfate (excluding sulfuric acid, the isopamicin content is 95.6%, impurity A 2.2%, 3N-isopamidin 0.6%, gentamicin B 1.3%, and other impurities 0.3%) and place it in a beaker. Add 100mL of aqueous solution and stir on a magnetic stirrer to dissolve it completely, so as to prepare an aqueous solution containing 0.1g isopamicin sulfate per mL.

[0036] 2. Take a 50mL beaker, weigh out different proportions of dialdehyde cellulose according to Table 5, add 10mL of aqueous solution and stir at 30℃ for 6 hours to prepare a suspension containing 0.05~0.5g of dialdehyde cellulose per 1mL of water.

[0037] 3. Mix the above isopamicin sulfate solution and dialdehyde cellulose suspension in a beaker, add 0.1 mol / L sulfuric acid solution to adjust the pH to 4.5, and then place the beaker on a magnetic stirrer and stir for 12 hours to react completely.

[0038] 4. After the reaction is complete, filter to remove the insoluble solid dialdehyde cellulose that has adsorbed impurities.

[0039] 5. Collect the filtrate in a distillation flask, and remove the aqueous solvent by vacuum distillation in a rotary evaporator at 40°C in a water bath until constant weight is achieved. Continue to maintain vacuum distillation for 0.5 hours, and collect the purified isopamicin sulfate at the bottom of the flask.

[0040] Table 5. Formulations without sodium bisulfite The content of isapamicin sulfate was determined according to the detection method described in Part I of the main text of the 2020 edition of the Chinese Pharmacopoeia, and the yield was calculated. The purification results are shown in Table 6.

[0041] Table 6. Purification results of Comparative Example 1 in conclusion The purity and yield of the example were significantly higher than those of the comparative example.

[0042] When the ratio of DAC / raw material = 0.1 / 1 and the ratio of sodium bisulfite / DAC = 0.1 / 1, the purity is 99.2% and the yield is 94.4%. Under other conditions, the purity is >99.2% and the yield is >94.4%.

[0043] Recovery of dialdehyde cellulose The dialdehyde cellulose solids collected by filtration and adsorbing impurities undergo Schiff base hydrolysis under the action of dilute acid (such as dilute hydrochloric acid) or dilute alkali (such as sodium carbonate solution), releasing dialdehyde cellulose back into the solution. The filtrate is then discarded, and the white precipitate is collected, thus achieving the recovery of dialdehyde cellulose.

[0044] The scope of protection of this invention is not limited to the above embodiments and their variations. Conventional modifications and substitutions made by those skilled in the art based on the content of these embodiments are all within the scope of protection of this invention.

Claims

1. A method for purifying crude isapamicin sulfate, characterized in that: Includes the following steps: S1: Add dialdehyde cellulose to an aqueous solution of sodium bisulfite and stir thoroughly for 6-8 hours to prepare a dialdehyde cellulose sodium bisulfite adduct suspension with a dialdehyde cellulose content of 0.05-0.15 g / mL; S2: Mix crude isopamicin sulfate with water and stir to prepare an aqueous solution of isopamicin sulfate, with an isopamicin sulfate content of 0.08-0.12 g / mL; S3: Mix the above suspension with the solution and stir. Add 0.1 mol / L sulfuric acid solution dropwise to adjust the pH to 4-5. Then, allow it to react fully for 10-12 hours at a water bath temperature of 20-60℃. S4: After the reaction is complete, filter to remove the insoluble solid dialdehyde cellulose that has adsorbed impurities; S5: Collect the filtrate in a distillation flask, and concentrate and crystallize it under reduced pressure in a rotary evaporator at 30~50℃ to obtain high-purity isapamicin sulfate.

2. The purification method for crude isapamicin sulfate according to claim 1, characterized in that: The dosage of dialdehyde cellulose is 0.1-0.5 times that of isopamicin sulfate.

3. The purification method for crude isapamicin sulfate according to claim 1, characterized in that: In step S1, the molar ratio of sodium bisulfite to dialdehyde cellulose is 0.3:1-0.8:

1.

4. The purification method for crude isapamicin sulfate according to claim 1, characterized in that: The preferred water bath temperature in step S3 is 40°C.

5. The purification method for crude isapamicin sulfate according to claim 1, characterized in that: After removing sulfuric acid from crude isopamicin sulfate, the isopamicin content is 90-98%, including impurities A, 3N-isopamidin, and gentamicin B.

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