A method for purifying coenzyme A cysteine ​​impurities and a method for synthesizing the same

Abstract

The present invention discloses a purification method and a synthesis method of a coenzyme A cysteine ​​impurity. The purification method of the present invention comprises the following steps: using a high performance liquid chromatography method to gradiently elute the test object on a chromatographic column; the test object comprises a coenzyme A cysteine ​​impurity; the mobile phases used in the gradient elution are mobile phase A and mobile phase B; the mobile phase A is an ammonium acetate aqueous solution and methanol; the volume ratio of the ammonium acetate aqueous solution to the methanol is (8-12):1; the molar concentration of the ammonium acetate aqueous solution is 10-60 mM; the mobile phase B is methanol or acetonitrile. The purification method of the present invention can obtain coenzyme A cysteine ​​impurities with high HPLC purity and high yield.

Description

Technical Field

[0001] The present invention relates to a method for purifying coenzyme A cysteamine impurities and a method for synthesizing the same. Background Art

[0002] Coenzyme A molecules are composed of pantothenic acid, β-mercaptoethylamine and 3',5'-adenosine diphosphate. Molecular formula C 21 H 36 N7O 16 P3S, relative molecular mass is 767.54, pure product is 413U / mg, the structural formula is as follows:

[0003]

[0004] Coenzyme A is a coenzyme of acetylase in organisms. It participates in the tricarboxylic acid cycle, plays the role of transferring acetyl groups, and plays an important role in the metabolism of sugars, proteins, and lipids. It is widely present in animals, plants, microbial tissues and cells. The highest content is in the liver of animals, followed by the heart and adrenal glands. It is mainly used clinically in the auxiliary treatment of fatty liver, hepatitis, hepatic coma, coronary atherosclerosis, myocardial infarction, nephrotic syndrome, uremia, neonatal hypoxia, diabetes, acidosis and other diseases. It can also be used to protect against radiation damage and slow down the development of muscle atrophy.

[0005] For a drug, the small amount of impurities it contains is the most important cause of drug side effects. Therefore, the inspection of its purity is one of the important foundations for ensuring the safety and effectiveness of drugs. The content of purity inspection varies according to the properties and characteristics of each drug, but basically involves the inspection and research of their respective "related substances". Various related substances of coenzymes mainly come from process impurities in the production process and degradation products caused by its instability. Although the purification process of coenzyme A has made great progress, process impurities are still an important source of related substances of coenzyme A. This is mainly because the manufacturing process of coenzyme A mainly relies on microbial synthesis methods, the composition of bacteria after fermentation metabolism is complex, and the thiol group in the coenzyme A molecule is easily oxidized by air, hydrogen peroxide, iodine, etc. into inactive disulfides. These products may be very similar to the properties of the drug itself, which makes purification difficult. At present, the only coenzyme A impurities available on the market are oxidized coenzyme A and 3'-dephosphorus coenzyme A, while coenzyme A cysteamine impurities have not yet been produced. This impurity is due to the addition of cysteine ​​during the process. At the same time, due to the presence of cysteine ​​in the biosynthesis pathway of coenzyme A, the accumulation of cysteine ​​will also increase. The decarboxylation product cysteamine reacts with coenzyme A to generate related substances. This impurity belongs to the process impurity and will increase under the destruction of oxygen, heat and alkali. This impurity may exist in coenzyme A raw materials and coenzyme A for injection. At present, there is no research on the analysis of the synthesis method of this impurity in coenzyme A raw materials and their preparations. There is a lack of detection methods and judgment basis for the production and drug safety of coenzyme A. Therefore, this is of great significance for the quality research of coenzyme A related products. Summary of the invention

[0006] The present invention provides a method for refining a coenzyme A cysteine ​​impurity and a method for synthesizing the same. The present invention adopts low-cost and simple process reaction conditions for the first time to quickly prepare a product with high purity and high yield; the refining method of the present invention can obtain a coenzyme A cysteine ​​impurity with high HPLC purity and high yield.

[0007] The present invention solves the above technical problems through the following technical solutions:

[0008] The first aspect of the present invention provides a method for purifying coenzyme A cysteine ​​impurities, which comprises the following steps: using a high performance liquid chromatography method to perform gradient elution on a chromatographic column on the substance to be tested;

[0009] The analyte includes coenzyme A cysteamine impurities;

[0010] The mobile phases used in the gradient elution are mobile phase A and mobile phase B;

[0011] The mobile phase A is ammonium acetate aqueous solution and methanol;

[0012] The volume ratio of the ammonium acetate aqueous solution to the methanol is (8-12):1;

[0013] The molar concentration of the ammonium acetate aqueous solution is 10-60 mM;

[0014] The mobile phase B is methanol or acetonitrile;

[0015]

[0016] In the first aspect of the present invention, the HPLC purity of the coenzyme A cysteamine impurity may be 60% to 90%, preferably 70% to 85%, more preferably 85% to 90%.

[0017] The coenzyme A cysteine ​​impurity is preferably injected as a solution with a concentration of ≤10 mg / mL. When the concentration of the coenzyme A cysteine ​​impurity is >10 mg / mL, it can be diluted before injection. The dilution solvent is water, such as water for injection.

[0018] In the first aspect of the present invention, the analyte may further include one or more of coenzyme A, cysteamine and coenzyme A-related substances.

[0019] The coenzyme A related substance can be selected from one or more of the following compounds:

[0020]

[0021] After the gradient elution, the removal rate of impurities in the analyte can be above 10%.

[0022] In the first aspect of the present invention, the coenzyme A cysteine ​​impurity is preferably prepared by the following synthesis method:

[0023] The synthesis method of the coenzyme A cysteamine impurity comprises the following steps: reacting coenzyme A with cysteamine in a solvent in the presence of a catalyst;

[0024]

[0025] In the method for preparing the coenzyme A cysteine ​​impurity, the coenzyme A can be conventional coenzyme A, preferably coenzyme A with a purity of more than 50%.

[0026] The solvent may be a conventional solvent for such reactions, preferably water, such as purified water or water for injection.

[0027] The amount of the solvent used can be any amount as long as it does not affect the reaction. The mass volume ratio of the coenzyme A to the solvent can be 1.2 to 5 mg / mL, preferably 1.3 to 3.5 mg / mL, for example 2.5 mg / mL.

[0028] The mass volume ratio of the cysteamine to the solvent may be 1.5-2.5 mg / mL, for example 2.5 mg / mL.

[0029] The molar ratio of the cysteamine to the coenzyme A can be a conventional molar ratio for such reactions, preferably (8-12):1, such as 10:1.

[0030] The catalyst may be a conventional catalyst for such reactions, preferably hydrogen peroxide, more preferably hydrogen peroxide at a concentration of 1%.

[0031] The molar ratio of the catalyst to the coenzyme A can be a conventional molar ratio for such reactions, preferably (18-22):1, such as 20:1.

[0032] The reaction temperature can be a conventional temperature for such reactions, preferably 37°C to 60°C, such as 38°C.

[0033] The reaction is preferably carried out in an oscillating manner. The frequency of the oscillation may be 100 to 150 times / min, for example, 150 times / min. The stroke of the oscillation may be 15 mm. The instrument used for the reaction is preferably a water bath reciprocating shaker. The progress of the reaction can be monitored in a conventional manner in the art (e.g., TLC, HPLC), and the disappearance or no-reaction of the raw material coenzyme A is generally used as the end point of the reaction. The reaction time may be more than 20 minutes, for example, 1 hour.

[0034] In the first aspect of the present invention, the gradient elution may be a five-stage gradient elution.

[0035] When the gradient elution is a five-stage gradient elution, in the first gradient, the volume percentage of the mobile phase A may be 100% to 100%, and the volume percentage of the mobile phase B may be 0% to 0%;

[0036] In the second gradient, the volume percentage of the mobile phase A may be 100% to 94%, and the volume percentage of the mobile phase B may be 0% to 6%;

[0037] In the third gradient, the volume percentage of the mobile phase A may be 94% to 94%, and the volume percentage of the mobile phase B may be 6% to 6%;

[0038] In the fourth gradient, the volume percentage of the mobile phase A may be 94% to 75%, and the volume percentage of the mobile phase B may be 6% to 25%;

[0039] In the fifth gradient, the volume percentage of the mobile phase A may be 75% to 50%, and the volume percentage of the mobile phase B may be 25% to 50%.

[0040] When the gradient elution is a five-stage gradient elution, in the first gradient, the elution time may be 4-8 minutes, for example, 6 minutes;

[0041] In the second gradient, the elution time may be 3 to 7 minutes, for example 5 minutes;

[0042] In the third gradient, the elution time may be 4 to 8 minutes, for example 6 minutes;

[0043] In the fourth gradient, the elution time may be 15 to 22 minutes, for example 18 minutes;

[0044] In the fifth gradient, the elution time may be 3 to 7 minutes, for example, 5 minutes.

[0045] The elution time refers to the difference between the starting point and the end point of the elution time in the gradient elution section.

[0046] The gradient elution procedure is preferably the following procedure:

[0047]

[0048] In the first aspect of the present invention, in the mobile phase A, the volume ratio of the aqueous ammonium acetate solution to the methanol can be (8-11):1, for example, 10:1.

[0049] In the first aspect of the present invention, the molar concentration of the aqueous solution of ammonium acetate may be 10-30 mM, for example 20 mM.

[0050] In the first aspect of the present invention, the pH of the mobile phase A may be 6.0 to 7.5, for example 6.5.

[0051] When the pH of the mobile phase A described in the present invention is 6.0-7.5, the peak shape of the coenzyme A cysteamine impurity is a symmetrical peak shape.

[0052] In the first aspect of the present invention, the mobile phase B may be methanol.

[0053] In the first aspect of the present invention, preferably, before the gradient elution procedure is started, the equilibrated chromatographic column is firstly flushed with a 10% methanol aqueous solution, and then flushed with mobile phase A.

[0054] The time for washing with the 10% methanol aqueous solution can be 5 to 10 minutes; the time for washing the equilibrated chromatographic column with the 10% methanol aqueous solution can be adjusted accordingly according to the different manufacturers and models of the high performance liquid chromatograph.

[0055] In the gradient elution, it is preferred to collect the eluate with a retention time of 15 to 25 minutes; more preferably, it is preferred to collect the eluate with a retention time of 10 to 13 minutes.

[0056] After the gradient elution procedure is completed, the method further includes steps of cleaning and preserving the chromatographic column.

[0057] In the first aspect of the present invention, before the analyte is subjected to the gradient elution, the chromatographic column is preferably flushed with 10% methanol for 10 to 15 minutes at a flow rate of 1 to 2 mL / min; and then flushed with 85% methanol for 10 minutes at a flow rate of 2 mL / min.

[0058] In the first aspect of the present invention, the chromatographic column may be a preparative chromatographic column.

[0059] In the first aspect of the present invention, the instrument used for the high performance liquid chromatography is preferably a Varian SD-1 high pressure liquid preparation system.

[0060] Wherein, the filler of the chromatographic column can be a water-resistant filler.

[0061] The water-resistant filler is preferably octadecylsilane bonded silica gel.

[0062] The pore size of the filler of the chromatographic column can be 7-10 nm.

[0063] The particle size of the filler of the chromatographic column can be 10 μm.

[0064] The length of the chromatographic column can be 25 cm.

[0065] The diameter of the chromatographic column may be 20 mm.

[0066] The chromatographic column preferably has octadecylsilane bonded silica gel as filler, the particle size of the filler is 10 μm, the length is 25 cm, and the diameter is 20 mm; for example, SHIM-PACK PRC-ODS (H) (20 mm*25 cm, 10 μm).

[0067] The flow rate of the mobile phase may be 8-12 mL / min, for example 10 mL / min.

[0068] The detector used in the high performance liquid chromatography can be an ultraviolet detector. The detection wavelength of the ultraviolet detector can be 259nm.

[0069] The second aspect of the present invention provides a method for synthesizing a coenzyme A cysteamine impurity, which comprises the following steps: reacting coenzyme A with cysteamine in a solvent in the presence of a catalyst;

[0070]

[0071] In the preparation method described in the second aspect of the present invention, the conditions and operations in the synthesis method of the coenzyme A cysteine ​​impurity are the same as those described in the synthesis method of the coenzyme A cysteine ​​impurity in the first aspect of the present invention.

[0072] In the present invention, "preparative chromatography column" refers to a chromatography column with a length of 50 mm to 500 mm and a diameter of 10 mm to 100 mm.

[0073] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0074] The reagents and raw materials used in the present invention are commercially available.

[0075] The positive and progressive effects of the present invention are: the present invention adopts low-cost and simple process reaction conditions for the first time to quickly prepare coenzyme A cysteine ​​impurities with high purity and yield; the purification method of the present invention can obtain coenzyme A cysteine ​​impurities with high HPLC purity and high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 This is the liquid phase diagram of the crude solution of coenzyme A cysteine ​​impurity.

[0077] Figure 2 Liquid phase diagram after preparation and purification of Coenzyme A cysteine ​​impurity.

[0078] Figure 3 This is the primary mass spectrum of the coenzyme A cysteine ​​impurity.

[0079] Figure 4 This is the secondary mass spectrum of the coenzyme A cysteine ​​impurity. DETAILED DESCRIPTION

[0080] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0081] In the embodiment, the sources and specifications of the products are as follows:

[0082] Coenzyme A hydrate (Sigma, ≥85% HPLC); methanol (HPLC, Fisher); ammonium acetate (HPLC grade, Fluka); 30% hydrogen peroxide (AR, Sinopharm); cysteamine (Sigma TCI, >95.0%).

[0083] In the embodiment, the instrument models and sources used are as follows:

[0084] High performance liquid chromatography (LC2030C, Shimadzu Technology Co., Ltd., Japan); preparative liquid phase system (PrepStarSD-1); Waters ultra-high performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry (G2-XS); WNE water bath shaker; SHIM-PACK PRC-ODS (H) preparative column (20mm*25cm, 10μm); Dikma chromatographic column (250*4.6mm, 5μm); pH meter (FE28, Mettler Toledo); electronic balance (T1000Y, Changshu Shuangjie Testing Instrument Factory); ultrasonic cleaning machine (Shenzhen Yujie Cleaning Equipment Co., Ltd.); vacuum freeze dryer (Shanghai Dongfulong Technology Co., Ltd.).

[0085] In the examples, the data processing software used is LabSolutions Chemistry Workstation.

[0086] Example 1

[0087] Preparation method of coenzyme A cysteine ​​impurity:

[0088] The starting material is coenzyme A hydrate, purity ≥ 85%, HPLC grade, 10mL reaction system: 25mg coenzyme A and 25mg cysteamine in 8mL water, then add 2mL 1% hydrogen peroxide solution to mix and fully dissolve. The water bath reciprocating shaker is set at 38°C, the oscillation frequency is 150 times / min, and it is stopped after 1 hour.

[0089]

[0090] Example 2

[0091] HPLC method to detect the purity of cysteine ​​impurities in crude solution of coenzyme A cysteine ​​impurities and purified product solution:

[0092] The experimental conditions are as follows:

[0093] Instrument: LC2030C High Performance Liquid Chromatograph

[0094] Chromatographic column: Dikma C18 (250*4.6mm, 5μm)

[0095] Mobile phase: Mobile phase A is 20mM ammonium acetate: methanol = 94:6, mobile phase B is methanol

[0096] The flow rate was 1.0 mL / min, the detection wavelength was 259 nm, the column temperature was 25°C, the injection volume was 10 μL, and the detector was a UV detector. The elution gradient is shown in the table below, and the percentages are by volume.

[0097]

[0098] The purity of the crude solution of coenzyme A cysteine ​​impurities determined by the above method is more than 84%. Figure 1 shown.

[0099] Example 3

[0100] The crude solution of coenzyme A cysteamine impurities obtained in Example 1 was purified by the following specific steps:

[0101] Instrument: Varian SD-1 High Pressure Liquid Preparation System

[0102] Chromatographic column: SHIM-PACK PRC-ODS (H) (20mm*25cm, 10μm)

[0103] Mobile phase: Mobile phase A is 20 mM ammonium acetate: methanol = 91:9, pH = 6.5, mobile phase B is methanol, and phase C is a crude solution of coenzyme A cysteine ​​impurities (containing unreacted raw materials).

[0104] The flow rate was 10 mL / min and the detection wavelength was 259 nm. The elution gradient is shown in the table below, and the percentages are by volume.

[0105]

[0106]

[0107] The eluate with a retention time of 10 to 13 minutes was collected, and the purity of the coenzyme A cysteine ​​impurity HPLC was measured by the method of Example 2 to be 97%. After two freeze-dryings, the yield was 95.8%. Figure 2 shown.

[0108] Example 4

[0109] Mass spectrometry detection of Coenzyme A cysteamyl impurities:

[0110] The obtained coenzyme A cysteine ​​impurities were subjected to primary and secondary mass spectrometric analysis using Waters ultra performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry (G2-XS). The measurement conditions were: an electrospray ionization (ESI) source, positive ionization mode, a quadrupole scanning range of 50-1000 m / z, and a secondary mass spectrometry collision energy of 25-30 eV.

[0111] The detection results are: primary mass spectrometry molecular ion peak [M+H] + The mass-to-charge ratio (m / z) is 843.1364, such as Figure 3 As shown; the mass-to-charge ratios (m / z) of the main ions in the secondary mass spectrum are 330.0586 and 336.1398, as shown Figure 4As shown, all of them are consistent with the theoretical value, that is, the molecular weight of the coenzyme A cysteine ​​impurity is 843.67.

Claims

1. A method for purifying coenzyme A cysteine ​​impurities, characterized in that: The method comprises the following steps: using a high performance liquid chromatography method to perform gradient elution on a chromatographic column on the substance to be tested; The analyte includes coenzyme A cysteamine impurities; The mobile phases used in the gradient elution are mobile phase A and mobile phase B; The mobile phase A is ammonium acetate aqueous solution and methanol; The pH of the mobile phase A is 6.5; The volume ratio of the ammonium acetate aqueous solution to the methanol is 10:1; The molar concentration of the ammonium acetate aqueous solution is 20 mM; The mobile phase B is methanol; The flow rate of the mobile phase is 10 mL / min; The structure of the coenzyme A cysteine ​​impurity is as follows: The procedure of the gradient elution is as follows: The coenzyme A cysteine ​​impurity is prepared by the following synthesis method: The synthesis method of the coenzyme A cysteamine impurity comprises the following steps: reacting coenzyme A with cysteamine in a solvent in the presence of a catalyst; The analyte further comprises one or more of coenzyme A, cysteamine and coenzyme A related substances; The coenzyme A related substance is selected from one or more of the following compounds: The chromatographic column is SHIM-PACK PRC-ODS; The particle size of the chromatographic column filler is 10 μm, the length is 25 cm, and the diameter is 20 mm; In the gradient elution, the eluate with a retention time of 10 to 13 minutes is collected.

2. The method for purifying coenzyme A cysteaminated impurities according to claim 1, characterized in that: The refining method meets one or both of the following conditions: (1) The HPLC purity of the coenzyme A cysteine ​​impurity is 60% to 90%; (2) The coenzyme A cysteine ​​impurity is injected as a solution with a concentration of ≤10 mg / mL; when the concentration of the coenzyme A cysteine ​​impurity is >10 mg / mL, it is diluted before injection.

3. The method for purifying coenzyme A cysteaminated impurities according to claim 2, characterized in that: The refining method meets one or more of the following conditions: (1) The HPLC purity of the coenzyme A cysteine ​​impurity is 70% to 85%; (2) The coenzyme A cysteine ​​impurity is injected as a solution with a concentration of ≤10 mg / mL; when the concentration of the coenzyme A cysteine ​​impurity is >10 mg / mL, it is diluted before injection; the dilution solvent is water.

4. The method for purifying coenzyme A cysteaminated impurities according to claim 2, characterized in that: The refining method meets one or more of the following conditions: (1) The HPLC purity of the coenzyme A cysteine ​​impurity is 85% to 90%; (2) The coenzyme A cysteine ​​impurity is injected as a solution with a concentration of ≤10 mg / mL; when the concentration of the coenzyme A cysteine ​​impurity is >10 mg / mL, it is diluted before injection; the dilution solvent is water for injection.

5. The method for purifying coenzyme A cysteamated impurities according to claim 2, characterized in that: The synthesis method meets one or more of the following conditions: (1) The coenzyme A is coenzyme A with a purity of 50% or more; (2) The solvent is water; (3) The mass volume ratio of the coenzyme A to the solvent is 1.2 to 5 mg / mL; (4) The mass volume ratio of the cysteamine to the solvent is 1.5 to 2.5 mg / mL; (5) The molar ratio of cysteamine to coenzyme A is (8-12):1; (6) The catalyst is hydrogen peroxide; (7) The molar ratio of the catalyst to the coenzyme A is (18-22):1; (8) The reaction temperature is 37°C to 60°C; (9) The reaction time is more than 20 minutes; The reaction described in (10) was carried out in a shaking manner.

6. The method for purifying coenzyme A cysteaminated impurities according to claim 5, characterized in that: The synthesis method meets one or more of the following conditions: (1) The mass volume ratio of the coenzyme A to the solvent is 1.3-3.5 mg / mL; (2) the mass volume ratio of the cysteamine to the solvent is 2.5 mg / mL; (3) The molar ratio of cysteamine to coenzyme A is 10:1; (4) The catalyst is hydrogen peroxide with a concentration of 1%; (5) The molar ratio of the catalyst to the coenzyme A is 20:1; (6) The reaction temperature is 38°C; (7) The reaction time is 1 hour; (8) The reaction is carried out in an oscillating manner; the frequency of the oscillation is 100 to 150 times / min; (9) The reaction is carried out in an oscillating manner; the oscillation stroke is 15 mm.

7. The method for purifying coenzyme A cysteamated impurities according to claim 6, characterized in that: The mass volume ratio of the coenzyme A to the solvent is 2.5 mg / mL; Or, the solvent is water for injection.

8. The method for purifying coenzyme A cysteaminated impurities according to claim 1, characterized in that: The refining method meets the following conditions: After the gradient elution procedure is completed, the method further includes steps of cleaning and preserving the chromatographic column.

9. The method for purifying coenzyme A cysteamated impurities according to claim 1, characterized in that: The refining method meets the following conditions: The detector used in the high performance liquid chromatography is an ultraviolet detector; the detection wavelength of the ultraviolet detector is 259nm.

Images