Preparation method of decitabine impurity III
Through technical means such as alkali degradation and high-pressure liquid chromatography separation and purification, the problems of complex and low yield of decitabine impurity III preparation process in the prior art were solved, and high purity and high yield were achieved, and the quality of drugs and drug safety were improved.
Patent Information
- Application Number
- CN202311185414.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-14
- Publication Date
- 2025-03-21
AI Technical Summary
It is difficult to effectively prepare high-purity decitabine impurity III in the prior art, and the preparation process is complex and the yield is not high, which affects the quality of the drug and the safety of the drug.
Using alkali degradation, high-pressure liquid chromatography separation and purification, desalination and lyophilization, high-purity decitabine impurity III was successfully prepared by optimizing reaction conditions and separation methods.
The high yield and high purity preparation of decitabine impurity III is achieved, the process flow is simplified, and the product stability and drug safety are improved.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of chemical pharmacy, and specifically relates to a method for preparing decitabine impurity III. Background Art
[0002] Decitabine is a 2′-deoxycytidine analogue, called 5-aza-2′-deoxycytidine, chemical name 4-amino-1-(2-deoxy-β-D-erythro-ribofuranosyl)-l,3,5-triazine-2(1H)-one, molecular formula C 8 H 12 N 4 O 4 , molecular weight 228.21, decitabine can inhibit DNA methyltransferase and reduce DNA methylation, thereby inhibiting tumor cell proliferation and preventing the occurrence of drug resistance. It is the strongest known DNA methylation specific inhibitor and belongs to the S phase cell cycle specific drug. It is suitable for the treatment of myelodysplastic syndrome (MDS), sickle cell anemia, myeloid leukemia and some solid tumors. It can also be used to treat non-small cell lung cancer. The chemical structure of decitabine is as follows:
[0003]
[0004] Structure 1 4-amino-1-(2-deoxy-β-D-erythro-ribofuranose)-1,3,5-triazine-2(1H)-one
[0005] Decitabine has multiple chiral centers, and various impurities such as oxidation products, hydrolysis products, and degradation products may be generated during the synthesis, transportation, and storage process. The generation of these impurities poses a potential threat to the safety of clinical medication. During the long-term stability study, it was found that Decitabine impurity III (structural formula Figure 2 ) is unstable, its content gradually decreases, and it can be converted into other impurities. In order to better realize the economic and social benefits of the drug, its quality standards must strictly control various impurities. Therefore, it is necessary to prepare the impurity components in decitabine to control the quality of raw materials and preparations.
[0006]
[0007] Structure 2 Decitabine impurity III
[0008] Decitabine impurity III can be obtained by chemical synthesis. Various organic solvents are required during the synthesis process, and a variety of other irrelevant substances will be produced, which wastes raw materials and has a low yield. Since decitabine is unstable and easily hydrolyzed, it can be prepared by hydrolysis under alkaline conditions such as ammonia water, but due to spontaneous isomerization and ring rearrangement reactions, the hydrolysis product is relatively complex and not conducive to separation and purification. At present, the quality standards of decitabine have not been included in domestic and foreign pharmacopoeias, and there has been no systematic study on the preparation of decitabine impurity III in domestic and foreign literature. Therefore, obtaining high-purity decitabine impurity III is particularly important for studying the internal relationship of its impurity changes, and can also provide a reliable impurity reference for decitabine quality research to ensure product quality and improve drug safety. Summary of the invention
[0009] In view of the shortcomings of the current preparation process of decitabine impurity III, the present invention provides a preparation method of decitabine impurity III. The decitabine impurity III obtained by forced degradation, separation and purification, desalting and freeze-drying has the advantages of large yield, high purity and simple preparation method, and can provide a reliable impurity reference substance for decitabine quality research, so as to ensure product quality and improve the safety of medication.
[0010] The specific technical scheme of the preparation method of decitabine impurity III of the present invention is as follows:
[0011] (1) Alkaline degradation
[0012] Weigh decitabine into a beaker, add alkali solution, sonicate to dissolve, add catalyst DMAP (4-dimethylaminopyridine), place in an ice-water bath, adjust the pH to neutral, and obtain test solution 1;
[0013] (2) Separation and purification
[0014] Take the above test solution, use high pressure to prepare the liquid phase, the preparation column is DAC-50 dynamic axial compression column system, the filler is octadecylsilane bonded silica gel, the particle size is 10μm, the pore size is Wavelength 220nm, column temperature 30℃, mobile phase A: sodium dihydrogen phosphate aqueous solution, mobile phase B: acetonitrile, flow rate 40-50mL / min, collect the target eluate;
[0015] (3) Desalting and freeze-drying
[0016] The eluate obtained in step (2) was concentrated under reduced pressure, reconstituted with ethanol, filtered through a 0.2 μm filter membrane, desalted, and the solvent was extracted with a freeze dryer. After reconstituted with water, it was pre-frozen and freeze-dried again under the condition of a partition temperature controlled at 20° C. to obtain a high-purity sample of decitabine impurity III.
[0017] In the technical solution, the alkali solution used in the degradation step (1) is one or more of triethylamine, sodium hydroxide and potassium hydroxide.
[0018] The DMAP concentration used in step (1) is 3% of the molar number of the substrate.
[0019] Preferably, the ice water bath time in step (1) is 30-60 min.
[0020] Preferably, during the freeze-drying again in step (3), the vacuum degree is set to ≤10 mbar and the freeze-drying time is 12-16 h.
[0021] The ratio of decitabine to the hydrolysis product impurity III in the prior art depends on the reaction conditions, including reaction time, alkali concentration, etc. 5-azacytidine can be separated from the hydrolysis product carbamoylguanidine by silica gel column chromatography, ion exchange resin or silica gel thin layer chromatography, but carbamoylguanidine is extremely unstable. Due to spontaneous isomerization and ring rearrangement reactions, a mixture of α- and β-isomers of furanone and pyranone derivatives can be formed, and the monomer structure of decitabine impurity III cannot be successfully separated. In view of the shortcomings of the prior art, the present invention experimentally optimizes the degradation method, the high-pressure preparation liquid phase separation method and the post-treatment method in the preparation process of decitabine impurity III, determines its structural formula by mass spectrometry and nuclear magnetic resonance detection, and obtains a high-purity impurity III product.
[0022] Compared with the prior art, the present invention achieves the following beneficial technical effects:
[0023] (1) Decitabine of the present invention undergoes hydrolysis reaction in an ice-water bath under the catalysis of DMAP, and the preparation process is simple, the conditions are mild, the reaction time is short, the side reactions are few, and the yield of the hydrolysis product is high;
[0024] (2) The present invention adopts a high pressure liquid chromatography separation method, the preparative column is a DAC-50 dynamic axial compression column system, the filler is octadecylsilane bonded silica gel, the mobile phase A is a sodium dihydrogen phosphate aqueous solution, and the mobile phase B is acetonitrile, to achieve chromatographic separation of decitabine impurity III, and the purity reaches more than 99%;
[0025] (3) The post-treatment desalting of the present invention is simple, and the sample content is stable and the properties are good after two freeze-dryings. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is the HPLC spectrum of the crude impurity obtained by hydrolyzing decitabine with sodium hydroxide. The retention times of decitabine impurities 2, 3, 4, and 5 are 4.512, 5.838, 7.597, and 11.008 min, respectively;
[0027] Figure 2 This is the curve of the change of the sum of the peak areas of impurities 2, 3, 4, and 5 in the decitabine impurity III sample over time;
[0028] Figure 3This is the curve showing the change of peak area of impurities 2, 3, 4 and 5 in the decitabine impurity III sample over time. DETAILED DESCRIPTION
[0029] The present invention is fully described below by specific embodiments. The following embodiments are only a part of the present invention, rather than all embodiments, and should not be used to limit the protection scope of the present invention. This is obvious to ordinary technicians in the technical field described in the present invention.
[0030] The decitabine sample used in the present invention is provided by Shandong New Era Pharmaceutical Co., Ltd., and other raw materials / fillers used, unless otherwise specified, can be obtained through commercial channels. The high performance liquid phase preparative chromatographic column used in the decitabine impurity preparation process of the present invention adopts a 50 mm × 600 mm dynamic axial compression preparative column, and the HPLC instrument used in the analysis and detection process is a Thermo Ultimate 3000 high performance liquid chromatograph (Thermo Fisher Scientific, USA).
[0031] Example 1 Preparation of Decitabine Impurity III
[0032] (1) Alkaline degradation
[0033] Weigh 50 mg of decitabine into a beaker, add 50 ml of 100 mmol / L sodium hydroxide, dissolve by ultrasonication, add DMAP at a concentration of 3% of the molar number of the substrate, bathe in ice water for 60 min, adjust to neutrality with concentrated hydrochloric acid to obtain the test solution.
[0034] (2) Separation and purification
[0035] The test solution obtained above was taken and the liquid phase was prepared by high pressure. The preparation column was a DAC-50 dynamic axial compression column system, the filler was octadecylsilane bonded silica gel, the particle size was 10 μm, and the pore size was Wavelength 220nm, column temperature 30℃, mobile phase A: 50mmol / L disodium hydrogen phosphate aqueous solution, mobile phase B: acetonitrile, mobile phase A: mobile phase B: 95:5, flow rate 50mL / min, collect the target eluate.
[0036] (3) Desalting and freeze-drying
[0037] The eluate obtained in step (2) was concentrated under reduced pressure, reconstituted with ethanol, filtered through a 0.2 μm filter membrane, desalted, and the solvent was extracted with a freeze dryer. After reconstituted with water, it was pre-frozen, and the vacuum degree was set to ≤10 mbar. The partition was temperature-controlled at 20°C and freeze-dried again for 12 hours to obtain a high-purity sample of decitabine impurity III.
[0038] Example 2 Preparation of Decitabine Impurity III
[0039] (1) Alkaline degradation
[0040] Weigh 50 mg of decitabine into a beaker, add 25 mL of triethylamine, dissolve by ultrasonication, add DMAP at a concentration of 3% of the molar number of the substrate, bathe in ice water for 45 min, dilute with 1 times the amount of water, adjust to neutrality with acetic acid, and obtain the test solution.
[0041] (2) Separation and purification
[0042] The test solution obtained above was taken and the liquid phase was prepared by high pressure. The preparation column was a DAC-50 dynamic axial compression column system, the filler was octadecylsilane bonded silica gel, the particle size was 10 μm, and the pore size was Wavelength 220nm, column temperature 30℃, mobile phase A: 50mmol / L disodium hydrogen phosphate solution (pH adjusted to 8.0 with sodium hydroxide), mobile phase B: acetonitrile, mobile phase A: mobile phase B = 97:3, flow rate 40mL / min, collect the target eluate.
[0043] (3) Desalting and freeze-drying
[0044] The eluate obtained in step (2) was concentrated under reduced pressure, reconstituted with ethanol, filtered through a 0.2 μm filter membrane, desalted, and the solvent was extracted with a freeze dryer. After reconstituted with water, it was pre-frozen, and the vacuum degree was set to ≤10 mbar. The partition was temperature-controlled at 20°C and freeze-dried again for 16 hours to obtain a high-purity sample of decitabine impurity III.
[0045] Example 3 Preparation of Decitabine Impurity III
[0046] (1) Alkaline degradation
[0047] Weigh 50 mg of decitabine into a beaker, add 10 mL of concentrated aqueous ammonia, dissolve by ultrasonication, add DMAP at a concentration of 3% of the molar number of the substrate, bathe in ice water for 30 min, dilute with 2 times the amount of water, adjust to neutrality with acetic acid, and obtain the test solution.
[0048] (2) Separation and purification
[0049] The test solution obtained above was taken and the liquid phase was prepared by high pressure. The preparation column was a DAC-50 dynamic axial compression column system, the filler was octadecylsilane bonded silica gel, the particle size was 10 μm, and the pore size was Wavelength 220nm, column temperature 30℃, mobile phase A: 100mmol / L disodium hydrogen phosphate solution (pH adjusted to 8.0 with sodium hydroxide), mobile phase B: acetonitrile, mobile phase A: mobile phase B: 97:3, flow rate 40ml / min, collect the target eluate.
[0050] (3) Desalting and freeze-drying
[0051] The eluate obtained in step (2) was concentrated under reduced pressure, reconstituted with ethanol, filtered through a 0.2 μm filter membrane, desalted, and the solvent was extracted with a freeze dryer. After reconstituted with water, it was pre-frozen, and the vacuum degree was set to ≤10 mbar. The partition was temperature-controlled at 20°C and freeze-dried again for 14 hours to obtain a high-purity sample of decitabine impurity III.
[0052] Example 4 Preparation of Decitabine Impurity III
[0053] (1) Alkaline degradation
[0054] Weigh 50 mg of decitabine into a beaker, add 25 ml of 100 mmol / L potassium hydroxide, dissolve by ultrasonic, bathe in ice water for 30 min, adjust to neutrality with hydrochloric acid to obtain the test solution.
[0055] (2) Separation and purification
[0056] The test solution obtained above was taken and the liquid phase was prepared by high pressure. The preparation column was a DAC-50 dynamic axial compression column system, the filler was octadecylsilane bonded silica gel, the particle size was 10 μm, and the pore size was Wavelength 220nm, column temperature 30℃, mobile phase A: 50mmol / L potassium dihydrogen phosphate solution, mobile phase B: ethanol, mobile phase A: mobile phase B: 96:4, flow rate 50mL / min, collect the target eluate.
[0057] (3) Desalting and freeze-drying
[0058] The eluate obtained in step (2) was concentrated under reduced pressure, reconstituted with ethanol, filtered through a 0.2 μm filter membrane, desalted, and the solvent was extracted with a freeze dryer. After reconstituted with water, it was pre-frozen, and the vacuum degree was set to ≤10 mbar. The partition was temperature-controlled at 20°C and freeze-dried again for 16 hours to obtain a high-purity sample of decitabine impurity III.
[0059] Example 5 Conversion relationship between decitabine impurity III and other impurities
[0060] Weigh 50 mg of the decitabine impurity III sample obtained in Example 2, dissolve it in purified water, and dilute it to 100 mL. After filtering through a 0.45 μm filter membrane, place it at 22° C. and take samples for HPLC detection at regular intervals. The results are shown in Table 1. The sum of the peak areas of the impurities remains essentially unchanged ( Figure 2 ), the peak area of Decitabine impurity III decreases with time, the peak areas of impurities 2 and 4 increase with time, and the peak area of impurity 5 increases to a certain extent and then begins to decrease, indicating that Decitabine impurity III can be gradually converted into impurities 2, 4, and 5 ( Figure 3 ).
[0061] Table 1 Peak areas of impurities 2, 3, 4, and 5
[0062]
[0063]
Claims
1. A method for preparing decitabine impurity III, characterized in that: The structural formula is as follows:
2. The method for preparing decitabine impurity III according to claim 1, characterized in that: The specific steps include: (1) Alkaline degradation Weigh decitabine into a beaker, add alkali solution, sonicate to dissolve, add DMAP catalyst, place in an ice-water bath, adjust the pH to neutral, and obtain test solution 1; (2) Separation and purification Take the above test solution, use high pressure to prepare the liquid phase, the preparation column is DAC-50 dynamic axial compression column system, the filler is octadecylsilane bonded silica gel, the particle size is 10μm, the pore size is Wavelength 220nm, column temperature 30℃, mobile phase A: sodium dihydrogen phosphate aqueous solution, mobile phase B: acetonitrile, flow rate 40-50mL / min, collect the target eluate; (3) Desalting and freeze-drying The eluate obtained in step (2) was concentrated under reduced pressure, reconstituted with ethanol, filtered through a 0.2 μm filter membrane, desalted, and the solvent was extracted with a freeze dryer. After reconstituted with water, it was pre-frozen and freeze-dried again under the condition of a partition temperature controlled at 20° C. to obtain a high-purity sample of decitabine impurity III.
3. The preparation method according to claim 2, characterized in that: The alkali solution used in step (1) is one or more of triethylamine, sodium hydroxide and potassium hydroxide.
4. The preparation method according to claim 2, characterized in that: The DMAP concentration used in step (1) is 3% of the molar number of the substrate.
5. The preparation method according to claim 2, characterized in that: The ice water bath time in step (1) is 30-60 min.
6. The preparation method according to claim 2, characterized in that: When freeze-drying again in step (3), the vacuum degree is set to ≤10 mbar and the freeze-drying time is 12-16 hours.