Separation and purification method of amino polycaprolactone carboxylic acid

Abstract

The invention discloses a separation and purification method of amino polycaprolactone carboxylic acid, and relates to the field of purification of medical intermediates. For a 2000-10000 Da NH2-PCL-COOH crude product, pressurization gradient elution, molecular weight adaptive dialysis, dichloromethane extraction and ice methanol sedimentation are sequentially carried out on the NH2-PCL-COOH crude product through a weakly acidic cation exchange resin column activated at the constant temperature of 50-80 DEG C to complete purification. Through innovation of a resin constant-temperature activation process and construction of an ion exchange-dialysis-extraction sedimentation synergistic purification system, organic tin impurities and homolog impurities can be efficiently removed at the same time, the purity of the purified NH2-PCL-COOH product is larger than 97%, and the tin element is almost free of residues; the resin and the dialysis bag can be repeatedly used, the operation is simple and convenient, the batch consistency is good, the method is suitable for pilot plant test and industrial production, and the bottlenecks of low purification efficiency, substandard purity and the like in the prior art are effectively solved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical intermediate purification, specifically to a method for the separation and purification of aminopolycaprolactone carboxylic acid. Background Technology

[0002] Polycaprolactone (PCL) is a polyester material synthesized by ring-opening polymerization of ε-caprolactone under the catalysis of organometallic compounds. It exhibits good molecular chain flexibility, excellent thermal stability, and good biocompatibility and in vivo biodegradability, demonstrating great application potential in biomedicine, material modification, and drug modification. Among them, aminopolycaprolactone carboxylic acid (NH2-PCL-COOH), as an important functional derivative of PCL, possesses amino and carboxyl functional groups at both ends of its molecule, enabling precise material modification and targeted drug coupling. It has become a key intermediate in the biopharmaceutical field, and its purity directly determines the application safety and performance stability of downstream products.

[0003] Currently, the preparation of NH2-PCL-COOH still uses organotin compounds such as stannous octoate as polymerization initiators, and modifies the terminal functional groups of PCL through sulfonation and amination reactions. However, the existing preparation process has significant defects, resulting in two types of difficult-to-remove impurities in the crude product, which have become the core bottleneck restricting the pharmaceutical and industrial applications of NH2-PCL-COOH. The specific problems are as follows:

[0004] (1) Organotin initiator residues are difficult to completely remove: Organotin compounds such as stannous octoate used in the polymerization process have strong binding to the product, and conventional purification processes cannot completely remove them. However, the biomedical field has strict requirements on the metal residue limits of PCL derivatives. Trace amounts of organotin residues will cause the product to fail to meet pharmaceutical standards.

[0005] (2) The separation of homologue impurities is difficult: the amination modification reaction of NH2-PCL-COOH cannot achieve 100% conversion. The crude product is often mixed with homologue impurities such as unaminated PCL carboxyl derivatives. The structural difference between these impurities and the target product is limited to the terminal functional group. As the molecular weight of NH2-PCL-COOH increases, the difference in physicochemical properties between the two further narrows, and conventional separation methods are difficult to achieve effective separation.

[0006] (3) Existing purification processes have defects: Currently, the purification of NH2-PCL-COOH mainly adopts traditional methods such as silica gel column chromatography, ordinary ion exchange, and single dialysis. These methods not only have problems such as large solvent consumption, long purification cycle, and inability to reuse packing materials, but more importantly, the single purification process cannot simultaneously take into account the removal of organotin residues and the separation of homologue impurities, resulting in generally unsatisfactory product purity and poor batch consistency, which makes it difficult to meet the requirements of industrial production.

[0007] (4) Poor treatment effect of ion exchange resin: In the existing technology, conventional activation methods usually involve soaking the resin in acid, alkali or salt solutions at room temperature. Under this temperature condition, the synthetic oligomers, porogens and unreacted monomers remaining in the resin channels are difficult to fully swell and diffuse out. At the same time, room temperature activation results in slow elution kinetics for chemical impurities such as sulfonating agents and chloromethylating agents remaining on the resin skeleton, making it difficult to break their weak interactions with functional groups. In addition, as an end-functionalized polymer, NH2-PCL-COOH can form hydrogen bonds or electrostatic adsorption with resin functional groups with its amino and carboxyl groups. Conventional activation fails to occupy these active sites in advance, resulting in the resin's own impurities competing with the target product for adsorption during the purification process. This not only reduces the metal ion removal efficiency but also causes secondary pollution from impurities.

[0008] In summary, developing a separation and purification method for NH2-PCL-COOH that can simultaneously and efficiently remove organotin residues and separate homologue impurities, while being simple to operate, having low solvent consumption, and being industrially scalable, is a pressing technical problem in this field that needs to be solved to overcome the multiple bottlenecks of existing technologies. Summary of the Invention

[0009] To address the technical challenges of existing NH2-PCL-COOH separation and purification methods, such as the difficulty in completely removing organotin initiator residues, the difficulty in separating homologue impurities, and the poor treatment effect of ion exchange resins, this invention provides a method for the separation and purification of aminopolycaprolactone carboxylic acid. This method is simple and easy to implement, not only easily separating difficult-to-separate impurities and completely removing organometallic compounds, but also significantly reducing the amount of solvent used compared to ordinary silica gel column chromatography, thus greatly shortening the purification time. The packing material can be reused multiple times, eliminating the need to replace it with new packing material for each purification, saving column packing time.

[0010] The technical solution adopted in this invention is as follows:

[0011] A method for separating and purifying aminopolycaprolactone carboxylic acid includes the following steps:

[0012] S1. Resin activation and soaking: The weak acidic cation exchange resin is activated at a constant temperature, and then the activated resin is soaked several times with an equal volume mixture of deionized water and organic solvent. The soaking mixture is replaced every 8-16 hours.

[0013] S2, Ion exchange column pressure gradient elution: The crude aminopolycaprolactone carboxylic acid is dissolved in a mixed solution of organic solvent and deionized water. The solution is added in batches to the ion exchange column packed with the resin treated in step S1. Pressure gradient elution is performed using the eluent. The pure NH2-PCL-COOH eluent is collected by thin-layer chromatography.

[0014] S3. Dialysis purification: After evaporating and concentrating the eluent from step S2 to remove all solvent, pour it into a dialysis bag and place it in deionized water and organic solvent for dialysis. During the process, change the mixture in the external dialysis bag regularly.

[0015] S4. Extraction and precipitation: The mixture in the bag after dialysis in step S3 is concentrated under reduced pressure to remove the organic solvent. The remaining liquid is extracted with dichloromethane, the dichloromethane phase is dried and concentrated under reduced pressure to obtain a concentrate. 20-50 times the volume of ice-cold methanol is added to the concentrate for precipitation to obtain a white solid pure product, namely NH2-PCL-COOH pure product.

[0016] Furthermore, the molecular weight of aminopolycaprolactone carboxylic acid is 2000-10000 Da.

[0017] Further, in step S1, the isothermal activation method is as follows: under constant temperature conditions of 50~80℃, the solution is alternately rinsed with pure water, 0.2-2 mol / L hydrochloric acid solution, and 0.2-2 mol / L KOH solution, and the pH is adjusted to 6-7. The pure water, hydrochloric acid solution, and KOH solution are all preheated to 50~80℃ before use; the number of soaking times is 3-5 times, and the organic solvent in the soaking mixture is at least one of dioxane, DMF, and isopropanol.

[0018] Furthermore, before constant temperature activation, impurities are removed using ethanol. The specific method is as follows: the weakly acidic cation exchange resin is immersed in ethanol at a volume ratio of 1:1.5-3.5 between the resin and ethanol. The ethanol is replaced every 25-45 minutes until the ethanol phase is clear, colorless, and free of residue.

[0019] For example, take 1 L of weakly acidic cation exchange resin and soak it in 2 L of ethanol, changing the ethanol every half hour until the ethanol phase is clear, colorless, and residue-free. Pour the resin into the column and rinse it with 1-2 L of pure water at a constant temperature of 50-80℃. Prepare 4 L of 1 mol / L hydrochloric acid and 2 L of 1 mol / L KOH at a constant temperature of 50-80℃ in advance. Then rinse with 2 L of hydrochloric acid solution for 40 min, then rinse with pure water to pH 6, then rinse with 2 L of KOH solution for 40 min, then rinse with pure water to pH 7, then rinse with 2 L of hydrochloric acid solution, and finally rinse with pure water to pH 6. Finally, soak the activated weakly acidic cation exchange resin with an equal volume mixture of deionized water and any one of the following organic solvents: dioxane, DMF, or isopropanol; change the soaking solution every 12 hours, and repeat the soaking process 3 times. Then drain the soaking solution.

[0020] Furthermore, the weakly acidic cation exchange resin is any one of D152 macroporous weakly acidic cation exchange resin, D113 macroporous weakly acidic cation exchange resin, and D201 macroporous weakly acidic cation exchange resin. The weakly acidic cation exchange resin activated at a constant temperature of 50~80℃ is used to remove organotin impurities from NH2-PCL-COOH and separate homologue impurities, with significant effect. The organotin impurity is stannous octoate.

[0021] Furthermore, in step S2, the organic solvent used for dissolution is any one of acetonitrile, tetrahydrofuran, and acetone.

[0022] Furthermore, in step S2, the ratio of the crude NH2-PCL-COOH sample mass to the volume of the resin used is 50-100 g: 1 L.

[0023] Further, in step S2, the eluent is a mixture of any one of acetonitrile, tetrahydrofuran, and acetone with an equal volume of 10-50 mmol / L sodium dihydrogen phosphate.

[0024] Furthermore, in step S3, the molecular weight cutoff of the dialysis bag is 2000-5000 Da, the volume ratio of organic solvent to deionized water is 1-5:1, and the organic solvent is DMF.

[0025] Furthermore, in step S4, the dichloromethane phase is dried using a 4A molecular sieve.

[0026] Furthermore, in the pure NH2-PCL-COOH product, the tin content was not detected or was ≤0.66 μg·kg. -1 The product purity is >97%.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention can simultaneously and accurately remove homologue impurities and organotin impurities. Tin element was not detected in the target product of 2000-6000Da, and only trace amounts remained in the product of 8000-10000Da. The purity of the purified product is >97%, which fully meets the stringent purity and metal residue requirements of the biomedical field.

[0029] (2) The present invention is easy to operate and requires no special equipment. The amount of solvent is significantly reduced compared with traditional silica gel column chromatography, thus reducing the emission of organic solvents. The ion exchange resin and dialysis bag can be reused multiple times, saving the time of repeated column packing and pretreatment, and greatly improving the purification efficiency.

[0030] (3) The entire process parameters of this invention are precisely matched, and it can stably purify NH2-PCL-COOH with different molecular weights of 2000-10000Da. The batch consistency of the products is excellent, and it is easy to achieve standardization and large-scale scale-up.

[0031] (4) The purification process of this invention relies entirely on the principle of physical separation, without introducing new impurities, and ensuring the integrity of the product structure and performance; the purification yield of products with different molecular weights is consistently above 80%, taking into account the economic efficiency of industrial production on the basis of high purity. Attached Figure Description

[0032] Figure 1 This is the liquid phase spectrum of the soaking solution after routine activation of D152 macroporous weakly acidic cation exchange resin.

[0033] Figure 2 The liquid phase spectrum is shown for the soaking solution of D152 macroporous weakly acidic cation exchange resin after isothermal activation.

[0034] Figure 3 The liquid phase spectrum is shown before purification of NH2-PCL2000-COOH.

[0035] Figure 4 The liquid phase spectrum is shown after purification of NH2-PCL2000-COOH.

[0036] Figure 5 The liquid phase spectrum is shown before purification of NH2-PCL3000-COOH.

[0037] Figure 6 The liquid phase spectrum is shown after purification of NH2-PCL3000-COOH.

[0038] Figure 7 The liquid phase spectrum is shown before purification of NH2-PCL5000-COOH.

[0039] Figure 8 The liquid phase spectrum is shown after purification of NH2-PCL5000-COOH.

[0040] Figure 9 The liquid phase spectrum is shown before purification of NH2-PCL6000-COOH.

[0041] Figure 10 The liquid phase spectrum is shown after purification of NH2-PCL6000-COOH.

[0042] Figure 11 The liquid phase spectrum is shown before purification of NH2-PCL8000-COOH.

[0043] Figure 12 The liquid phase spectrum is shown after purification of NH2-PCL8000-COOH.

[0044] Figure 13 The liquid phase spectrum of NH2-PCL10000-COOH before purification.

[0045] Figure 14The liquid phase spectrum is shown after purification of NH2-PCL10000-COOH.

[0046] Figure 15 The liquid phase spectrum of NH2-PCL2000-COOH after purification by the method in Comparative Example 1.

[0047] Figure 16 The liquid chromatography spectrum of NH2-PCL5000-COOH after purification by the method in Comparative Example 2.

[0048] Figure 17 The liquid chromatography spectrum of NH2-PCL8000-COOH after purification by the method in Comparative Example 3.

[0049] Figure 18 This is a flowchart of the process flow of the present invention. Detailed Implementation

[0050] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited thereto.

[0051] Resin isothermal activation steps: Take 1 L of D152 macroporous weakly acidic cation exchange resin and soak it in 2 L of ethanol at room temperature. Replace the ethanol every half hour until the ethanol phase is clear, colorless, and free of residue. Pour the resin into the column and rinse it with 2 L of pure water at a constant temperature of 60°C. Prepare 4 L of 1 mol / L hydrochloric acid and 2 L of 1 mol / L KOH at a constant temperature of 60°C in advance. Then rinse with the prepared 2 L of 60°C hydrochloric acid solution for 40 min, then rinse with 60°C pure water to bring the pH to 6. Rinse with 2 L of 60°C KOH solution for 40 min, then rinse with 60°C pure water to bring the pH to 7. Rinse with 2 L of 60°C hydrochloric acid solution, and finally rinse with room temperature pure water until the pH is 6. Next, the activated weakly acidic cation exchange resin was soaked in a mixture of equal volumes of room temperature deionized water and isopropanol; the soaking solution was changed every 12 hours, and the soaking was repeated 3 times. Then the soaking solution was drained.

[0052] Routine activation: After the resin is fully swollen with pure water, it is washed with pure water to remove surface impurities; then it is soaked in 2M HCl solution to fully protonate the resin and dissolve metal ion impurities in the pores, and then washed with pure water until neutral; next, it is soaked in 2M NaOH solution to convert the resin to sodium form and further remove acidic impurities and residual oligomers, and washed with pure water again until neutral; finally, it is rinsed with ethanol to remove hydrophobic porogens and unreacted monomers, completing the activation.

[0053] The soaking solutions after conventional activation and those after isothermal activation were respectively sent for HPLC analysis, and the results are as follows: Figure 1 , 2 As shown. By Figure 1It can be seen that impurities remain in the conventional activation soaking solution. These impurities are those leached from the resin, indicating that conventional activation is unlikely to completely remove resin impurities. In contrast, the resin soaking solution after isothermal activation has a relatively clean baseline, and no characteristic peaks of impurities were found. Figure 2 As shown, the isothermal activation method is significantly better than the conventional activation method for removing resin impurities.

[0054] The process flow diagram of the method for separating and purifying aminopolycaprolactone carboxylic acid of the present invention is as follows: Figure 18 As shown in the following specific embodiment.

[0055] Example 1

[0056] S1. Activate 1 liter of D152 macroporous weak acid cation exchange resin at the above constant temperature of 50°C. Then, soak the activated resin in an equal volume mixture of deionized water and dioxane organic solvent. Replace the soaking solution every 12 hours and repeat the soaking process 3 times. Then, drain the soaking solution.

[0057] S2. Dissolve 50 g of crude NH2-PCL2000-COOH in an equal volume mixture of acetonitrile and deionized water. Slowly add the solution in batches to an ion exchange column packed with the above-treated resin. Continuously add sufficient eluent for pressure gradient elution and monitor the eluent using thin-layer chromatography until pure NH2-PCL2000-COOH is completely eluted. The eluent used is a mixture of acetonitrile and an equal volume of 10 mmol / L sodium dihydrogen phosphate; the volume of the eluent should be 18 times the volume of the resin.

[0058] S3. After evaporating and concentrating the eluent to remove all solvent, pour it into a dialysis bag with a molecular weight cutoff of 2000 Da. Place the bag in a mixed solvent of DMF and deionized water (DMF to deionized water volume ratio of 1:1) for dialysis. Replace the DMF and deionized water mixture on the outside of the dialysis bag periodically.

[0059] S4. After dialysis, the mixture in the dialysis bag is concentrated under reduced pressure to remove the organic solvent. The remaining residue is extracted with dichloromethane. The dichloromethane phase is dried and concentrated under reduced pressure, and then precipitated with 20 times the volume of ice-cold methanol to obtain a white solid NH2-PCL2000-COOH pure product; the yield is 88.8%.

[0060] Example 2

[0061] S1. Activate 1 liter of D113 macroporous weak acid cation exchange resin at 60°C using the above-mentioned constant temperature method; then soak the activated resin in an equal volume mixture of DMF and deionized water; replace the soaking solution every 12 hours, repeat the soaking process 3 times, and then drain the soaking solution.

[0062] S2. Dissolve 60 g of crude NH2-PCL3000-COOH in a mixed solution of tetrahydrofuran and deionized water of equal volume. Slowly add the solution in batches to an ion exchange column packed with the above-treated resin. Continuously add sufficient eluent for pressure gradient elution and monitor the eluent using thin-layer chromatography until pure NH2-PCL3000-COOH is completely eluted. The eluent used is a mixed solution of tetrahydrofuran and an equal volume of 25 mmol / L sodium dihydrogen phosphate; the volume of the eluent should be 22 times the volume of the resin.

[0063] S3. After evaporating and concentrating the eluent to remove all solvent, pour it into a dialysis bag with a molecular weight cutoff of 3000 Da. Place it in a mixed solvent of DMF and deionized water (DMF to deionized water volume ratio of 2:1) for dialysis. Replace the DMF and deionized water mixture on the outside of the dialysis bag regularly during the process.

[0064] S4. After dialysis, the mixture in the dialysis bag was concentrated under reduced pressure to remove the organic solvent. The remaining residue was extracted with dichloromethane. The dichloromethane phase was dried and concentrated under reduced pressure, and then precipitated with 35 times its volume of ice-cold methanol to obtain a white solid NH2-PCL3000-COOH pure product; the yield was 82.2%.

[0065] Example 3

[0066] S1. Activate 1 liter of D201 macroporous weak acid cation exchange resin at the above constant temperature, adjusting the activation temperature to 80℃; then soak the activated resin in an equal volume mixture of deionized water and isopropanol organic solvent; replace the soaking solution every 12 hours, and soak for 3 consecutive times, then drain the soaking solution.

[0067] S2. Dissolve 80 g of crude NH2-PCL5000-COOH in an equal volume mixture of acetone and deionized water. Slowly add the solution in batches to an ion exchange column packed with the above-treated resin. Continuously add sufficient eluent for pressure gradient elution and monitor the eluent using thin-layer chromatography until pure NH2-PCL5000-COOH is completely eluted. The eluent used is a mixture of acetone and an equal volume of 50 mmol / L sodium dihydrogen phosphate; the volume of the eluent is 20 times the volume of the resin.

[0068] S3. After evaporating and concentrating the eluent to remove all solvent, pour it into a dialysis bag with a molecular weight cutoff of 3000 Da. Place the bag in a mixed solvent of DMF and deionized water (DMF to deionized water volume ratio of 3:1) for dialysis. Replace the DMF and deionized water mixture on the outside of the dialysis bag periodically.

[0069] S4. After dialysis, the mixture in the dialysis bag was concentrated under reduced pressure to remove the organic solvent. The remaining residue was extracted with dichloromethane. The dichloromethane phase was dried and concentrated under reduced pressure, and then precipitated with 50 times its volume of ice-cold methanol to obtain a white solid NH2-PCL5000-COOH pure product; the yield was 84.1%.

[0070] Example 4

[0071] S1. Activate 1 liter of D152 macroporous weak acid cation exchange resin at the above constant temperature, adjusting the activation temperature to 65℃; then soak the activated resin in an equal volume mixture of DMF and deionized water; change the soaking solution every 12 hours, soaking for 3 consecutive times, and then drain the soaking solution.

[0072] S2. Dissolve 50 g of crude NH2-PCL6000-COOH in an equal volume mixture of tetrahydrofuran and deionized water. Slowly add the solution in batches to an ion exchange column packed with the above-treated resin. Continuously add sufficient eluent for pressure gradient elution and monitor the eluent using thin-layer chromatography until pure NH2-PCL-COOH is completely eluted. The eluent used is a mixture of acetone and an equal volume of 30 mmol / L sodium dihydrogen phosphate; the volume of the eluent is 25 times the volume of the resin.

[0073] S3. After evaporating and concentrating the eluent to remove all solvent, pour it into a dialysis bag with a molecular weight cutoff of 5000 Da. Place it in a mixed solvent of DMF and deionized water (DMF to deionized water volume ratio of 4:1) for dialysis. Replace the DMF and deionized water mixture on the outside of the dialysis bag regularly during the process.

[0074] S4. After dialysis, the mixture in the dialysis bag was concentrated under reduced pressure to remove the organic solvent. The remaining residue was extracted with dichloromethane. After drying the dichloromethane phase and concentrating it under reduced pressure, it was precipitated with 45 times its volume of ice-cold methanol to obtain a white solid NH2-PCL6000-COOH pure product; the yield was 81.7%.

[0075] Example 5

[0076] S1. Activate 1 liter of D113 macroporous weak acid cation exchange resin at 60°C using the above-mentioned constant temperature method; then soak the activated resin in an equal volume mixture of DMF and deionized water; replace the soaking solution every 12 hours, repeat the soaking process 3 times, and then drain the soaking solution.

[0077] S2. Dissolve 100 g of crude NH2-PCL8000-COOH in an equal volume mixture of tetrahydrofuran and deionized water. Slowly add the solution in batches to an ion exchange column packed with the above-treated resin. Continuously add sufficient eluent for pressure gradient elution and monitor the eluent using thin-layer chromatography until pure NH2-PCL8000-COOH is completely eluted. The eluent used is a mixture of tetrahydrofuran and an equal volume of 40 mmol / L sodium dihydrogen phosphate; the volume of the eluent is 40 times the volume of the resin.

[0078] S3. After evaporating and concentrating the eluent to remove all solvent, pour it into a dialysis bag with a molecular weight cutoff of 5000 Da. Place it in a mixed solvent of DMF and deionized water (DMF to deionized water volume ratio of 4:1) for dialysis. Replace the DMF and deionized water mixture on the outside of the dialysis bag regularly during the process.

[0079] S4. After dialysis, the mixture in the dialysis bag is concentrated under reduced pressure to remove the organic solvent. The remaining residue is extracted with dichloromethane. The dichloromethane phase is dried and concentrated under reduced pressure, and then precipitated with 35 times its volume of ice-cold methanol to obtain a white solid NH2-PCL8000-COOH pure product; the yield is 80.8%.

[0080] Example 6

[0081] S1. Activate 1 liter of D201 macroporous weak acid cation exchange resin at the above constant temperature, adjusting the activation temperature to 70℃; then soak the activated resin in an equal volume mixture of DMF and deionized water; replace the soaking solution every 12 hours, and soak continuously for 3 times, then drain the soaking solution.

[0082] S2. Dissolve 80 g of crude NH2-PCL10000-COOH in an equal volume mixture of tetrahydrofuran and deionized water. Slowly add the solution in batches to an ion exchange column packed with the above-treated resin. Continuously add sufficient eluent for pressure gradient elution and monitor the eluent using thin-layer chromatography until pure NH2-PCL10000-COOH is completely eluted. The eluent used is a mixture of acetonitrile and an equal volume of 25 mmol / L sodium dihydrogen phosphate, with the eluent volume being 30 times the resin volume.

[0083] S3. After evaporating and concentrating the eluent to remove all solvent, pour it into a dialysis bag with a molecular weight cutoff of 5000 Da for dialysis. The dialysis bag is placed in a mixed solvent of DMF and deionized water (DMF to deionized water volume ratio of 5:1) for dialysis. During dialysis, the DMF and deionized water mixture on the outside of the dialysis bag should be replaced regularly.

[0084] S4. After dialysis, the mixture in the dialysis bag is concentrated under reduced pressure to remove the organic solvent. The remaining residue is extracted with dichloromethane. The dichloromethane phase is dried and concentrated under reduced pressure, and then precipitated with 50 times its volume of ice-cold methanol to obtain a white solid NH2-PCL10000-COOH pure product; the yield is 85.5%.

[0085] Performance testing

[0086] Comparison of organometallic content detection before and after purification (the main heavy metal in NH2-PCL-COOH prepared by using stannous octoate as an initiator is tin; the content of stannous octoate can be calculated by measuring the content of the heavy metal tin).

[0087] Instruments and reagents

[0088] Thermo Fisher X-Series II ICP-MS inductively coupled plasma mass spectrometer, Thermo Fisher Scientific, Inc., USA; Milestone ETHOS microwave digestion system, Beijing LabTech Instruments Co., Ltd. Tin standard solution (1000 μg·mL) -1 The reagents used were from the National Nonferrous Metals and Electronic Materials Analysis and Testing Center; nitric acid (GR); self-made deionized water; and all other reagents were of analytical grade. A total of six batches of samples covering different molecular weights were selected for this evaluative sampling inspection.

[0089] Preparation of standard solutions

[0090] Accurately measure 1 mL of tin standard solution into a 100 mL volumetric flask, dilute to the mark with nitric acid solution to prepare a multi-element mixed stock solution, and then measure appropriate amounts of each solution and dilute with nitric acid solution to prepare a series of reference solutions: d1 (1 ng·mL) -1 ), d2( 8 ng·mL -1 ), d3( 20 ng·mL -1 ), d4 (40 ng·mL -1 ), d5( 60 ng·mL -1 ), d6(100 ng·mL -1 ), d7 (200 ng·mL -1 Preparation of internal standard solution: Accurately measure 50 μg of germanium standard solution into a 1000 mL volumetric flask, dilute to the mark with nitric acid solution to obtain germanium (50 ng / mL). -1 ) internal standard solution.

[0091] Preparation of test solution

[0092] Accurately weigh 0.5 g of each of the pure products obtained in Examples 1-6 above, place them in a polytetrafluoroethylene microwave digestion vessel, add 5 mL of lead-free nitric acid, soak overnight, seal, and digest using a microwave digestion furnace. After cooling, transfer the product to a 50 mL volumetric flask, dilute with water to the mark, and shake well to obtain the final product. Prepare a blank solution using the same method. The microwave digestion temperature program is as follows: raise to 80°C within 5 min, hold for 5 min; raise to 120°C within 5 min, hold for 5 min; raise to 180°C within 5 min, hold for 30 min; and finally lower to 40°C within 10 min. The power is set to 1200 W.

[0093] Determination methods

[0094] The isotope Ge was selected as the internal standard during the determination. The instrument's internal standard injection tube was always inserted into the internal standard solution during the instrument's analysis. The instrument's sample tube was sequentially inserted into standard solutions of varying concentrations for measurement (concentration increasing sequentially). A standard curve was plotted with the measured value (average of three readings) on the ordinate and the concentration on the abscissa. The instrument's sample tube was inserted into the test solution for measurement, and the average of three readings was taken. The corresponding concentration was calculated from the standard curve, and the concentration of the corresponding blank solution was subtracted to calculate the content of each element. The results of the impurity element tests for aminopolycaprolactone carboxylic acid at various molecular weights before and after purification are shown in Table 1.

[0095] Table 1. Test results of impurity elements in the samples (μg·kg) -1 )

[0096]

[0097] Comparative analysis of the changes in the content of stannous octoate, a heavy metal impurity, before and after purification shows that this purification method has a good removal effect on Sn, the main heavy metal impurity involved in NH2-PCL-COOH. Specifically, Sn was not detected in NH2-PCL2000-COOH, NH2-PCL3000-COOH, NH2-PCL5000-COOH, and NH2-PCL6000-COOH after purification, while only trace amounts of organotin impurities were detected in NH2-PCL8000-COOH and NH2-PCL10000-COOH after purification.

[0098] The samples before and after purification of aminopolycaprolactone carboxylic acid were compared by HPLC analysis. The HPLC analysis methods are shown in Table 2. Table 2 HPLC Analytical Methods

[0099]

[0100] The obtained HPLC chromatogram is as follows: Figures 3 to 14 As shown.

[0101] High-performance liquid chromatograms of crude NH2-PCL2000-COOH, NH2-PCL3000-COOH, etc. are shown below. Figure 3 , Figure 5 , Figure 7 , Figure 9 , Figure 11 , Figure 13 As shown, the spectra all show obvious impurity peaks, the main peak content is low, and the product purity is not high.

[0102] The high-performance liquid chromatograms of the corresponding products purified using the method of this invention are as follows: Figure 4 , Figure 6 , Figure 8 , Figure 10 , Figure 12 , Figure 14 As shown, the impurity peaks have basically disappeared, while the main peaks are significantly prominent and their contents are all greater than 97%, indicating that this method can efficiently remove homologue impurities and organotin residues, and the purification effect is excellent.

[0103] The HPLC analysis results are shown in Table 3: Table 3 HPLC Analysis Results

[0104]

[0105] Through analysis and comparison, it can be seen that the purity of NH2-PCL-COOH before purification is basically below 90%, while the purity of the products obtained after purification by the purification method involved in this invention reaches above 97%.

[0106] Comparative Example 1

[0107] Except for changing the resin activation method to conventional room temperature activation, all other operations, process parameters, and raw materials were completely consistent with Example 1: 1L of D152 macroporous weakly acidic cation exchange resin was soaked in 2L of ethanol at room temperature to remove impurities, then rinsed directly with room temperature pure water until pH=6, and then soaked in an equal volume mixture of deionized water and dioxane; subsequently, the crude NH2-PCL2000-COOH product was subjected to ion exchange column elution, dialysis, extraction, and precipitation. During ion exchange column elution, the eluent was a mixture of acetonitrile and an equal volume of 10mmol / L sodium dihydrogen phosphate, with the eluent volume reaching 90 times the resin volume. During dialysis, a dialysis bag with a molecular weight cutoff of 2000Da was used, and 20 times the volume of ice-cold methanol was used for precipitation.

[0108] The purified NH2-PCL2000-COOH contained 11.45 μg·kg⁻¹. -1 The calculated stannous octoate content is 39.30 μg·kg. -1The product purity is 75.861% (e.g., ...). Figure 15 (As shown in the figure); the product yield was only 62.45%, and the resin showed a significant decrease in adsorption capacity after one use, making it unusable.

[0109] The results above show that conventional room temperature activation cannot completely remove impurities from the resin itself and achieve efficient activation. Furthermore, the adsorption efficiency of the resin for organotin ions is significantly reduced, making it impossible to effectively remove organotin impurities or achieve efficient separation of homologue impurities, resulting in product purity far lower than that of this invention. At the same time, the resin activated by conventional methods has poor stability, cannot be reused, and consumes more solvent. This demonstrates the key role of the 50~80℃ isothermal activation process of this invention in improving purification effect and reducing cost.

[0110] Comparative Example 2

[0111] Purification was performed using a single process: ion exchange column pressure gradient elution (all other process parameters and raw materials were identical to those in Example 3 of this invention). D201 macroporous weakly acidic cation exchange resin was activated at 80°C and packed into a column. Crude NH2-PCL5000-COOH was dissolved in an equal volume mixture of acetone and deionized water and loaded onto the column. A pressure gradient elution was performed using an equal volume mixture of acetone and 50 mmol / L sodium dihydrogen phosphate. After collecting the eluent by thin-layer chromatography, the eluent was directly concentrated under reduced pressure and precipitated with ice-cold methanol, eliminating the need for dialysis purification.

[0112] Testing revealed that tin was not detected in the purified NH2-PCL5000-COOH, but the product purity was only 87.998% (e.g., ...). Figure 16 As shown in the figure, there are obvious homologue impurity peaks; and the product yield is only 38%, with some target products lost along with the impurities because they were not separated from them.

[0113] The above results indicate that single ion-exchange column chromatography can only remove organotin impurities, but cannot separate homologues with similar physicochemical properties to the target product, and easily leads to the loss of the target product, resulting in a significant decrease in both yield and purity. The synergistic purification method of ion exchange, dialysis, and extraction precipitation in this invention is the core guarantee for achieving efficient removal of dual impurities.

[0114] Comparative Example 3

[0115] The crude NH2-PCL8000-COOH product was purified using the existing silica gel column chromatography process: the column was packed with 200-300 mesh silica gel, and dichloromethane-methanol (9:1 volume ratio) was used as the eluent for atmospheric pressure column chromatography. The target eluent was collected by thin-layer chromatography, concentrated under reduced pressure, and recrystallized with methanol. The remaining raw materials were the same as those in Example 5 of this invention.

[0116] The purified NH2-PCL8000-COOH contained 26.16 μg·kg⁻¹. -1 The calculated stannous octoate content is 89.73 μg·kg. -1 The product purity is 90.534% (e.g., ...). Figure 17 (As shown); it has almost no separation effect on impurities, and the solvent consumption of silica gel column chromatography is 20 times that of the present invention, and the silica gel packing material cannot be reused.

[0117] Therefore, traditional silica gel column chromatography cannot effectively remove organotin impurities, has poor separation effect on homologue impurities, and the product purity does not meet biomedical standards; at the same time, it has problems such as large solvent volume, non-reusable packing material, and poor batch consistency.

[0118] Comparative Example 4 The D152 macroporous weakly acidic cation exchange resin in Example 1 was replaced with 200-300 mesh silica gel. All other operations, process parameters, raw materials, etc. were completely consistent with Example 1: the silica gel was treated according to all the conditions of Example 1, including constant temperature activation at 50℃, ethanol purification, and soaking in deionized water-dioxane; all process parameters, such as sample loading, eluent, dialysis, extraction, and precipitation with ice-cold methanol, were completely consistent with Example 1. The purification target was crude NH2-PCL2000-COOH.

[0119] Experimental results showed that the product purity was 71.58%, which did not meet the 97% pharmaceutical purity requirement; the residual tin content was 12.76 μg·kg. -1 The organotin impurities were not effectively removed; the product yield was 57.30%, resulting in significant loss of the target product; and the silica gel could not be reused.

[0120] Ordinary silica gel lacks ion exchange capabilities, making it unable to selectively adsorb and remove organotin impurities, nor can it separate homologues with minimal differences in terminal functional groups. The use of a weakly acidic cation exchange resin subjected to isothermal activation treatment is key to achieving high-efficiency purification in this invention.

[0121] Comparative Example 5 The purification process employed was: supercritical CO2 extraction (40℃, 15MPa, 60min), alkaline quartz sand filtration, and petroleum ether precipitation. The purified product was the same crude NH2-PCL2000-COOH as in Example 1. Weak acidic cation exchange resin was not used, pressure gradient elution was not performed, dialysis was not conducted, and ice-cold methanol precipitation was not used.

[0122] Experimental results showed that the product purity was 79.17%, and homologue impurities were not effectively separated; the residual tin content was 18.32 μg·kg⁻¹. -1 The residual amount was much higher than that of this invention; the product yield was 67.45%.

[0123] The supercritical fluid extraction combined with alkaline quartz sand process can only partially remove organotin impurities and cannot separate homologues of impurities that are highly similar in structure to NH2-PCL-COOH, thus failing to meet the purity requirements of pharmaceutical-grade aminopolycaprolactone carboxylic acid.

Claims

1. A method for separating and purifying aminopolycaprolactone carboxylic acid, characterized in that, Includes the following steps: S1. Resin activation and soaking: The weak acidic cation exchange resin is activated at a constant temperature, and then the activated resin is soaked several times with an equal volume mixture of deionized water and organic solvent. The soaking mixture is replaced every 8-16 hours. S2. Ion exchange column pressure gradient elution: The crude aminopolycaprolactone carboxylic acid is dissolved in a mixed solution of organic solvent and deionized water. The solution is added in batches to the ion exchange column packed with resin after step S1. Pressure gradient elution is performed using the eluent. The pure aminopolycaprolactone carboxylic acid eluent is collected by thin-layer chromatography. S3. Dialysis purification: After evaporating and concentrating the eluent from step S2 to remove all solvent, pour it into a dialysis bag and place it in deionized water and organic solvent for dialysis. During the process, change the mixture in the external dialysis bag regularly. S4. Extraction and precipitation: The mixture in the bag after dialysis in step S3 is concentrated under reduced pressure to remove the organic solvent. The remaining liquid is extracted with dichloromethane, the dichloromethane phase is dried and concentrated under reduced pressure to obtain a concentrate. 20-50 times the volume of ice-cold methanol is added to the concentrate for precipitation to obtain a white solid pure product, namely NH2-PCL-COOH pure product.

2. The method for separating and purifying aminopolycaprolactone carboxylic acid according to claim 1, characterized in that, The molecular weight of the aminopolycaprolactone carboxylic acid is 2000-10000 Da.

3. The method for separating and purifying aminopolycaprolactone carboxylic acid according to claim 1, characterized in that, In step S1, the constant temperature activation method is as follows: under constant temperature conditions of 50~80℃, rinse alternately with pure water, 0.2-2mol / L hydrochloric acid solution, and 0.2-2mol / L KOH solution, and adjust the pH to 6-7. The pure water, hydrochloric acid solution and KOH solution are all preheated to 50~80℃ before use.

4. The method for separating and purifying aminopolycaprolactone carboxylic acid according to claim 1, characterized in that, In step S1, the soaking is performed 3-5 times, and the organic solvent in the soaking mixture is at least one of dioxane, DMF, and isopropanol.

5. The method for separating and purifying aminopolycaprolactone carboxylic acid according to claim 1, characterized in that, In step S1, before constant temperature activation, impurities are removed by ethanol. The specific method is as follows: the weak acid cation exchange resin is immersed in ethanol at a volume ratio of 1:1.5-3.5 between the resin and ethanol. The ethanol is replaced every 25-45 minutes until the ethanol phase is clear, colorless and free of residue.

6. The method for separating and purifying aminopolycaprolactone carboxylic acid according to any one of claims 1 to 5, characterized in that, The weakly acidic cation exchange resin is any one of D152 macroporous weakly acidic cation exchange resin, D113 macroporous weakly acidic cation exchange resin, and D201 macroporous weakly acidic cation exchange resin.

7. The method for separating and purifying aminopolycaprolactone carboxylic acid according to claim 6, characterized in that, In step S2, the organic solvent used for dissolution is any one of acetonitrile, tetrahydrofuran, and acetone.

8. The method for separating and purifying aminopolycaprolactone carboxylic acid according to claim 6, characterized in that, In step S2, the eluent is a mixture of any one of acetonitrile, tetrahydrofuran, and acetone with an equal volume of 10-50 mmol / L sodium dihydrogen phosphate.

9. The method for separating and purifying aminopolycaprolactone carboxylic acid according to claim 6, characterized in that, In step S3, the molecular weight cutoff of the dialysis bag is 2000-5000 Da, and the volume ratio of organic solvent to deionized water is 1~5:

1.

10. The method for separating and purifying aminopolycaprolactone carboxylic acid according to claim 6, characterized in that, In step S4, the dichloromethane phase is dried using a 4A molecular sieve.

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